WO2006125105A2 - Methods and compositions for treating and diagnosing multiple sclerosis - Google Patents

Methods and compositions for treating and diagnosing multiple sclerosis Download PDF

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Publication number
WO2006125105A2
WO2006125105A2 PCT/US2006/019292 US2006019292W WO2006125105A2 WO 2006125105 A2 WO2006125105 A2 WO 2006125105A2 US 2006019292 W US2006019292 W US 2006019292W WO 2006125105 A2 WO2006125105 A2 WO 2006125105A2
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gene
expression
genes
subject
antigen
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PCT/US2006/019292
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French (fr)
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WO2006125105A3 (en
Inventor
Scott Jelinsky
Suzana Marusic
John Leonard
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Wyeth
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Publication of WO2006125105A2 publication Critical patent/WO2006125105A2/en
Publication of WO2006125105A3 publication Critical patent/WO2006125105A3/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/0004—Screening or testing of compounds for diagnosis of disorders, assessment of conditions, e.g. renal clearance, gastric emptying, testing for diabetes, allergy, rheuma, pancreas functions
    • A61K49/0008—Screening agents using (non-human) animal models or transgenic animal models or chimeric hosts, e.g. Alzheimer disease animal model, transgenic model for heart failure
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/502—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects
    • G01N33/5023—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects on expression patterns
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/564—Immunoassay; Biospecific binding assay; Materials therefor for pre-existing immune complex or autoimmune disease, i.e. systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, rheumatoid factors or complement components C1-C9
    • 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
    • A01K2217/07—Animals genetically altered by homologous recombination
    • A01K2217/075—Animals genetically altered by homologous recombination inducing loss of function, i.e. knock out
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2267/00—Animals characterised by purpose
    • A01K2267/03—Animal model, e.g. for test or diseases
    • A01K2267/0306—Animal model for genetic diseases
    • A01K2267/0325—Animal model for autoimmune diseases
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/28—Neurological disorders
    • G01N2800/285—Demyelinating diseases; Multipel sclerosis
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis

Definitions

  • the invention features, inter alia, methods and compositions for treating and diagnosing inflammatory, autoimmune and/or neurologic disorders (e.g., multiple sclerosis (MS)), as well as methods for identifying agents that can be used in such therapeutic and diagnostic methods.
  • the invention features a method of evaluating a compound. The method includes: contacting a test compound to a target protein that includes a polypeptide encoded by a gene selected from Table 1, or a fragment or functional domain thereof; evaluating an interaction between the test compound and the target protein; and, optionally, evaluating the test compound in an animal, or cell-based, model, e.g., a model of an inflammatory, autoimmune and/or neurologic disorder.
  • the step of evaluating an interaction includes evaluating a function of the target protein (e.g., a binding or enzymatic function), e.g., detecting binding between the test compound and the target protein, or evaluating an enzymatic activity of the protein, in the presence of the test compound.
  • the evaluating step can be performed using an in vitro assay.
  • the method of evaluating a compound is repeated for one or more of a plurality of compounds from a chemical library.
  • the invention features a method of evaluating a compound.
  • the method includes: contacting a test compound to a mammalian cell (e.g., a lymphocyte or a neuronal cell (e.g., a neuron or glial cell)); evaluating expression of a gene chosen from one or more genes in Table 1 in the mammalian cell; and, optionally, evaluating the test compound in an animal or cell-based model of a disorder, e.g., an inflammatory, autoimmune and/or neurologic disorder.
  • a mammalian cell e.g., a lymphocyte or a neuronal cell (e.g., a neuron or glial cell)
  • evaluating expression of a gene chosen from one or more genes in Table 1 in the mammalian cell e.g., an inflammatory, autoimmune and/or neurologic disorder.
  • test compounds that reduce or prevent increased expression of the gene are identified as candidate therapeutics or lead compounds for a therapeutic.
  • the mammalian cell includes a recombinant reporter construct.
  • the construct can include a regulatory sequence of a gene listed in Table 1 (e.g., all or a functional part of a promoter region) operably linked to a sequence encoding a reporter protein. Expression of the gene is evaluated by evaluating expression of the reporter construct.
  • the agent is an immunoglobulin (e.g., antibody or functional fragment thereof).
  • the agent is an inhibitor of the protein, e.g., an inhibitor of an enzymatic or other biological activity of the protein.
  • the agent is an enzyme that modifies the protein.
  • the agent is a polypeptide that sequesters the protein. In one embodiment, the agent decreases expression of the gene or protein.
  • the invention features a method of evaluating a subject by evaluating the expression or activity of at least one gene chosen from Table 1 or a polypeptide encoded by a gene selected from Table 1.
  • the method can be used, e.g., to obtain a value for a parameter indicative of the expression or activity, or to obtain a profile (e.g., a profile that includes a plurality of values for respective parameters indicative of the expression or activity of a plurality of genes or polypeptides selected from Table 1).
  • nucleic acid (e.g., mRNA or cDNA) levels are quantitated.
  • protein levels are quantitated.
  • the subject is a human.
  • the subject has been diagnosed with a disorder, e.g., an inflammatory, autoimmune and/or neurologic disorder (e.g., multiple sclerosis).
  • a disorder e.g., an inflammatory, autoimmune and/or neurologic disorder (e.g., multiple sclerosis).
  • the subject is at risk for a disorder, e.g., an inflammatory, autoimmune and/or neurologic disorder, e.g., multiple sclerosis.
  • the subject can be identified as at risk or having the disorder, e.g., multiple sclerosis, if the value has a difference, e.g., a statistically significant difference, relative to a normal or other reference value, e.g., obtained by similar methods for a reference cohort of subjects (e.g., normal subjects of similar gender, age, and so forth).
  • At least one address of the plurality includes a capture probe that binds specifically to a polypeptide chosen from the polypeptides encoded by the genes of Table 1.
  • the capture probe is an antibody or derivative thereof
  • the plurality of addresses includes addresses having polypeptide capture probes for all the genes of Table 1 (i.e., 100% of the genes) or a fraction of the genes of Table 1, e.g., at least 20%, 40%, 50%, 60%, 80%, or 90% of the genes of Table 1.
  • the array has no more than 4 000, 3 000, 2 000, 1 000, 500, or 250 addresses.
  • the subject expression profile can be determined in a subject during treatment.
  • the subject expression profile can be compared to a reference profile or to a profile obtained from the subject, prior to treatment, or prior to onset, of the disorder.
  • the subject expression profile is determined at intervals (e.g., regular intervals) during treatment.
  • the method further includes selecting a therapy based on said assignment.
  • the decision includes selecting a first course of action if the subject is an enhanced responder and a second course of action if the subject in a non-enhanced responder.
  • the is assigning the subject to a first class. In some embodiments, assignment to the first class will enable payment for a treatment provided to the subject. In some embodiments, payment is by a first party to a second party. In some embodiments, the first party is other than the patient. In some embodiments, the first party is selected from a third party payor, an insurance company, employer, employer sponsored health plan, HMO, or governmental entity. In some embodiments, the second party is selected from the subject, a healthcare provider, a treating physician, an HMO, a hospital, a governmental entity, or an entity which sells or supplies the drug.
  • the invention features a method of making a data record.
  • the method includes entering the result of a method described herein into a record, e.g., a computer readable record.
  • the record is available on the world wide web.
  • the record is evaluated by a third party payor, an insurance company, employer, employer sponsored health plan, HMO, or governmental entity, or a healthcare provider, a treating physician, an HMO, a hospital, a governmental entity, or an entity which sells or supplies the drug, or is otherwise relied on in a method described herein.
  • the data is provided by a first party to a second party, hi some embodiments, the first party is selected from the subject, a healthcare provider, a treating physician, an HMO, a hospital, a governmental entity, or an entity which sells or supplies the drug, hi some embodiments, the second party is a third party payor, an insurance company, employer, employer sponsored health plan, HMO, or governmental entity. In some embodiments, the first party is selected from the subject, a healthcare provider, a treating physician, an HMO, a hospital, an insurance company, or an entity which sells or supplies the drug and the second party is a governmental entity. In some embodiments, the first party is selected from the subject, a healthcare provider, a treating physician, an HMO, a hospital, an insurance company, or an entity which sells or supplies the drug and the second party is an insurance company.
  • the disclosure features a method of transmitting a record described herein.
  • the method includes a first party transmitting the record to a second party, e.g., by computer, compact disc, telephone, facsimile, email, or letter, hi some embodiments, the second party is selected from the subject, a healthcare provider, a treating physician, an HMO, a hospital, a governmental entity, or an entity which sells or supplies the drug, hi some embodiments, the first party is an insurance company or government entity and the second party is selected from the subject, a healthcare provider, a treating physician, an HMO, a hospital, a governmental entity, or an entity which sells or supplies the drug, hi some embodiments, the first party is a governmental entity or insurance company and the second party is selected from the subject, a healthcare provider, a treating physician, an HMO 3 a hospital, an insurance company, or an entity which sells or supplies the drug.
  • the invention features a method of providing (e.g., communicating, e.g., electronically communicating), information about the subject's level of expression of at least one gene selected from Table l,e.g., at least one gene described herein, e.g., the result of evaluating expression of a at least one gene selected from Table 1, to a third party, e.g., a hospital, clinic, a government entity, reimbursing party or insurance company (e.g., a life insurance company).
  • a third party e.g., a hospital, clinic, a government entity, reimbursing party or insurance company (e.g., a life insurance company).
  • a third party e.g., a hospital, clinic, a government entity, reimbursing party or insurance company (e.g., a life insurance company).
  • a third party e.g., a hospital, clinic, a government entity, reimbursing party or insurance company (e.g.
  • premiums can be increased (e.g., by a certain percentage) if expression of at least one gene selected from Table 1 is present in the candidate insured, or decreased if no gene selected from Table 1 is expressed.
  • Premiums can also be scaled depending on the level of expression of at least one gene from Table 1, or on the number of expressed genes of Table 1.
  • premiums can be assessed to distribute risk, e.g., commensurate with distribution of genes selected from Table 1.
  • premiums are assessed as a function of actuarial data that is obtained from individuals with one or more polymorphisms of at least one gene selected from Table 1.
  • information about the expression of at least one gene selected from Table 1, can be used, e.g., in an underwriting process for life insurance.
  • the invention features a method of providing (e.g., communicating, e.g., electronically communicating), information about the subject's gene expression levels, e.g., the result of evaluating at least one gene selected from Table 1, e.g., at least one gene described herein, to a third party, e.g., a hospital, clinic, a government entity, reimbursing party or insurance company (e.g., a life insurance company).
  • a third party e.g., a hospital, clinic, a government entity, reimbursing party or insurance company (e.g., a life insurance company).
  • a third party e.g., a hospital, clinic, a government entity, reimbursing party or insurance company (e.g., a life insurance company).
  • a third party e.g., a hospital, clinic, a government entity, reimbursing party or insurance company (e.g., a life insurance company).
  • a premium for insurance (e.g., life or medical) is evaluated as a function of information about one or more gene expression levels, e.g., expression levels of at least one gene selected from Table 1 (e.g., a level of expression associated with a gene encoding, e.g., a polypeptide associated with antigen processing and/or presentation, e.g., proteasome 28 subunit alpha (PSMEl); a component of complement; a component of the extracellular matrix; a polypeptide related to hematopoiesis, e.g., CD53 antigen, CD68 antigen, lysosomal-associated protein transmembrane 5 (LAPTM5), lymphocyte antigen 86 (LY86), macrophage expressed gene 1 (MPEGl), or small inducible cytokine A6 (SC YA6); a polypeptide related to lipid metabolism, e.g., lipocalin 2 (LCN2); a regulator of transcription; or a plast
  • premiums can be assessed to distribute risk, e.g., as a function of gene expression levels, e.g., the result of evaluating at least one gene of Table 1.
  • premiums are assessed as a function of actuarial data that is obtained from subjects that are enhanced or non-enhanced responders.
  • information about gene expression levels e.g., the result of evaluating at least one gene selected from Table 1, can be used, e.g., in an underwriting process for life insurance.
  • the information can be incorporated into a profile about a subject. Other information in the profile can include, for example, date of birth, gender, marital status, banking information, credit information, children, and so forth.
  • An insurance policy can be recommended as a function of the information on gene expression levels, e.g., the result of evaluating at least one gene selected from Table 1, along with one or more other items of information in the profile.
  • An insurance premium or risk assessment can also be evaluated as function of information about the expression of at least one gene selected from Table 1.
  • points are assigned on the basis of having a high expression of at least one gene selected from Table 1.
  • information about gene expression levels is analyzed by a function that determines whether to authorize the transfer of funds to pay for a service or treatment provided to a subject (or make another decision referred to herein).
  • the results of analyzing a expression of at least one gene selected from Table 1 may indicate that a subject has an inflammatory, autoimmune and/or neurologic disorder, e.g., multiple sclerosis, suggesting that a first treatment course is needed, thereby triggering an outcome that indicates or causes authorization to pay for a service or treatment provided to a subject.
  • an entity e.g., a hospital, care giver, government entity, or an insurance company or other entity which pays for, or reimburses medical expenses
  • an entity can use the outcome of a method described herein to determine whether a party, e.g., a party other than the subject patient, will pay for services (e.g., a particular therapy) or treatment provided to the patient.
  • services e.g., a particular therapy
  • a first entity e.g., an insurance company
  • a first entity e.g., an insurance company
  • the articles “a” and “an” refer to one or to more than one (e.g., to at least one) of the grammatical object of the article.
  • proteins and “polypeptides” are used interchangeably herein.
  • FIGS. 2A-2B are Venn diagrams depicting the classification of disease regulated genes in CNS tissue from, p35 -/-, IFN ⁇ -/- and wild type animals based on microarray analysis.
  • FIGS. 3A-3F are bar graphs depicting microarray results for six genes significantly regulated in all three genetic backgrounds tested. The first set of graphs in each figure depicts data for wild type mice. The second set of graphs in each figure depicts data for IFN ⁇ -/- mice. The last set of graphs in each figure depicts data for IL-12p35 -/-mice.
  • FIG 3A depicts data for TYRO protein tyrosine kinase binding protein.
  • FIG 3B depicts data for SlOO calcium binding protein A8 (calgranulin A).
  • FIG 3C depicts data for lipocalin 2.
  • FIG 3D depicts data for CD53 antigen.
  • FIG 3E depicts data for complement component 1, q subcomponent, beta polypeptide.
  • FIG 3F depicts data for SlOO calcium binding protein All (calgizzarin).
  • FIG 5 depicts Table 1, which lists exemplary genes with descriptions (encoded polypeptides), the expression of which is significantly regulated in spinal cord in EAE mice.
  • FIG 6 depicts Table 2, which lists exemplary genes with descriptions
  • Modulators of expression and/or activity of the genes and associated polypeptides described herein can be used to treat autoimmune, inflammatory and/or neurologic disorders, e.g., multiple sclerosis (MS).
  • the genes and proteins disclosed herein can also be useful screening assays for compounds that can be used to treat such disorders, e.g., MS.
  • the genes and proteins disclosed herein can also be used in methods of diagnosing such disorders, e.g., MS diagnosis or a predisposition for MS.
  • Applicants have shown that a plurality of genes are co- regulated in three mouse models of multiple sclerosis, each with a different genetic background. Examples of these genes are disclosed in Table 1 and the appended
  • genes and proteins disclosed herein can be used as targets in screens for compounds that can be useful for treatment or diagnosis of MS.
  • modulators such as inhibitors
  • Such inhibitors can be used to treat MS, or ameliorate one or more symptoms condition associated with MS.
  • the screens can be used to identify both direct and indirect interactions.
  • modulators can directly bind to a target protein or inhibit an activity of a target protein.
  • Other modulators can alter expression, stability, and/or localization of the target protein in a cell.
  • Protein fragments can be produced, e.g., using recombinant or biochemical means (e.g., from a nucleic acid sequence that codes for the target protein, or through proteolysis of the full-length target protein, e.g., using proteolytic enzymes).
  • exemplary proteolytic enzymes are enzymes that cleave peptides at specific sequences, e.g., trypsin, chymotrypsin, thermolysin, and clostripain.
  • a functional protein fragment is a fragment that retains at least part of an activity of the full-length protein, e.g., a binding or enzymatic activity.
  • the methods can be designed to identify a compound that binds to and/or modulates an activity of a target protein described herein. For example, it is frequently useful to inhibit the activity or availability of a target protein that is up- regulated in a mouse model of MS, relative to a normal counterpart, or to increase the activity or availability of a protein that is down-regulated in a mouse model of MS, relative to a normal counterpart.
  • a variety of methods can be used to evaluate activity of a target protein described herein in the presence of a test compound, e.g., a candidate therapeutic or a lead for a candidate therapeutic. These methods include in vitro assays, cell-based assays, and organismal assays.
  • Cell-based assays can include evaluating an activity by cell-associated proteins or can include evaluating mRNA or protein expression, e.g., directly or indirectly (e.g., using a reporter gene).
  • macromolecules are proteins, protein complexes, and glycoproteins, nucleic acids, e.g., DNA, RNA (e.g., double stranded RNA or RNAi), and PNA (peptide nucleic acid).
  • nucleic acids e.g., DNA, RNA (e.g., double stranded RNA or RNAi), and PNA (peptide nucleic acid).
  • small molecules are peptides, peptidomimetics (e.g., peptoids), amino acids, amino acid analogs, polynucleotides, polynucleotide analogs, nucleotides, nucleotide analogs, organic or inorganic compounds, e.g., heteroorganic or organometallic compounds.
  • One exemplary type of protein compound is an antibody or a modified scaffold domain protein.
  • a test compound can be the only substance assayed by the method described herein.
  • test compounds can be assayed either consecutively or concurrently by the methods described herein.
  • high throughput screening methods involve providing a combinatorial chemical or biopolymer library containing a large number of potential therapeutic compounds (e.g., potential modulators).
  • potential therapeutic compounds e.g., potential modulators.
  • Such "combinatorial chemical libraries” are screened in one or more assays, as described herein, to identify those library members (particular chemical species or subclasses) that have a desired characteristic activity.
  • the compounds thus identified can serve as conventional "lead compounds" or can themselves be used as potential or actual therapeutics.
  • a combinatorial chemical library is a collection of diverse chemical compounds generated by either chemical synthesis or biological synthesis, by combining a number of chemical "building blocks” such as reagents.
  • a linear combinatorial chemical library such as a polypeptide library is formed by combining a set of chemical building blocks (amino acids) in every possible way for a given compound length (i.e., the number of amino acids in a polypeptide compound). Millions of chemical compounds can be synthesized through such combinatorial mixing of chemical building blocks.
  • combinatorial chemical libraries include, but are not limited to, peptide libraries ⁇ see, e.g., U.S. Patent 5,010,175; Furka, Int. J. Pept. Prot. Res. 37:487-493 (1991);and Houghton et al, Nature 354:84-88 (1991)).
  • Other chemistries for generating chemical diversity libraries can also be used. Such chemistries include, but are not limited to: peptoids (e.g., PCT Publication No. WO 91/19735), encoded peptides (e.g., PCT Publication No.
  • Patent 5,539,083) antibody libraries ⁇ see, e.g., Vaughn et al, Nature Biotechnology, 14:309-314 (1996) and PCT/US96/10287), carbohydrate libraries (see, e.g. , Liang et al , Science, 21 A: 1520- 1522 (1996) and U.S. Patent 5,593,853), small organic molecule libraries (see, e.g., benzodiazepines, Baum C&EN, Jan 18, page 33 (1993); isoprenoids, U.S. Patent No. 5,569,588; thiazolidinones and metathiazanones, U.S. Patent No. 5,549,974; pyrrolidines, U.S.
  • Some exemplary libraries are used to generate variants from a particular lead compound.
  • One method includes generating a combinatorial library in which one or more functional groups of the lead compound are varied, e.g., by derivatization.
  • the combinatorial library can include a class of compounds which have a common structural feature (e.g., framework).
  • Test compounds can also be obtained from: biological libraries; peptoid libraries (libraries of molecules having the functionalities of peptides, but with a novel, non-peptide backbone which are resistant to enzymatic degradation but which nevertheless remain bioactive; see, e.g., Zuckermann et al. (1994) J. Med. Chem. 37:2678-85); spatially addressable parallel solid phase or solution phase libraries; synthetic library methods requiring deconvolution; the "one-bead one-compound” library method; and synthetic library methods using affinity chromatography selection.
  • the biological libraries include libraries of nucleic acids and libraries of proteins.
  • nucleic acid libraries encode a diverse set of proteins (e.g., natural and artificial proteins; others provide, for example, functional RNA and DNA molecules such as nucleic acid aptamers or ribozymes.
  • a peptoid library can be made to include structures similar to a peptide library. (See also Lam (1997) Anticancer DrugDes. 12:145).
  • Alibrary of proteins maybe produced by an expression library or a display library (e.g., a phage display library). Libraries of compounds may be presented in solution (e.g., Houghten (1992)
  • Biotechniques 13:412-421 or on beads (Lam (1991) Nature 354:82-84), chips (Fodor (1993) Nature 364:555-556), bacteria (Ladner, U.S. Patent No. 5,223,409), spores (Ladner U.S. Patent No. 5,223,409), plasmids (Cull et al. (1992) Proc. Natl. Acad. Sci. USA 89:1865-1869) or on phage (Scott and Smith (1990) Science 249:386-390; Devlin (1990) Science 249:404-406; Cwirla et al. (1990) Proc. Natl. Acad. Sci. USA 87:6378-6382; Felici (1991) J. MoI. Biol. 222:301-310).
  • Binding Assays Interaction with, e.g., binding to a target protein described herein or fragment thereof, e.g., a functional fragment thereof can be assayed in vitro, e.g., in a cell free system.
  • the reaction mixture can include a cognate binding partner, e.g., in an in vitro assay, to evaluate the ability of a test compound to modulate interaction between the target protein described herein and a cognate binding partner.
  • This type of assay can be accomplished, for example, by coupling one of the components, with a label (e.g., a radioisotope or enzymatic label) such that binding of the labeled component to the other can be determined by detecting the labeled compound in a complex.
  • a label e.g., a radioisotope or enzymatic label
  • a component can be labeled with 125 1, 35 S, 14 C, or 3 H, either directly or indirectly, and the radioisotope detected by direct counting of radioemmission or by scintillation counting.
  • a component can be enzymatically labeled with, for example, horseradish peroxidase, alkaline phosphatase, or luciferase, and the enzymatic label detected by determination of conversion of an appropriate substrate to product.
  • the test compound and/or the target protein described herein (or a fragment thereof) itself can be labeled.
  • Competition assays can also be used to evaluate
  • Cell-free assays involve preparing a reaction mixture of the target protein described herein and the test compound under conditions and for a time sufficient to allow the two components to interact and bind, thus forming a complex that can be removed and/or detected.
  • the interaction between two molecules can also be detected, e.g., using a fluorescence assay in which at least one molecule is fluorescently labeled.
  • a fluorescence assay in which at least one molecule is fluorescently labeled.
  • FET fluorescence energy transfer
  • a fluorophore label on the first or donor molecule is selected such that its emitted fluorescent energy will be absorbed by a fluorescent label on a second or acceptor molecule, which in turn is able to fluoresce due to the absorbed energy.
  • the donor molecule if a protein may simply utilize the natural fluorescent energy of tryptophan residues.
  • Labels can be chosen that emit different wavelengths of light, such that the acceptor molecule label may be differentiated from that of the donor. Since the efficiency of energy transfer between the labels is related to the distance separating the molecules, the spatial relationship between the molecules can be assessed. In a situation in which binding occurs between the molecules, the fluorescent emission of the acceptor molecule label in the assay should be maximal.
  • a FET binding event can be conveniently measured through standard fluorometric detection means well known in the art (e.g., using a fluorimeter).
  • fluorescence assay is fluorescence polarization (FP).
  • FP For FP, only one component needs to be labeled, typically the component that undergoes the larger change in molecular weight on binding. A binding interaction is detected by a change in molecular size of the labeled component. The size change alters the tumbling rate of the component in solution and is detected as a change in FP. See, e.g., Nasir et al. (1999) Comb. Chem. HTS 2: 177-190; Jameson et al. (1995) Methods Enzymol 246:283; Seethala et al. (1998) Anal. Biochem. 255:257. Fluorescence polarization can be monitored in multiwell plates, e.g., using the Tecan POLARIONTM reader.
  • determining the ability of a protein described herein to bind to a target molecule can be accomplished using real-time Biomolecular Interaction Analysis (BIA) (see, e.g., Sjolander and Urbaniczky (1991) Anal. Chem. 63:2338-2345 and Szabo et al. (1995) Curr. Opin. Struct. Biol. 5:699-705).
  • BIOA Biomolecular Interaction Analysis
  • a target protein described herein or fragment thereof e.g., a functional fragment thereof is anchored onto a solid phase.
  • Protein/test compound complexes anchored on the solid phase can be detected at the end of the reaction, e.g., the binding reaction.
  • a protein fragment can be anchored onto a solid surface, and the test compound, (which is not anchored), can be labeled, either directly or indirectly, with detectable labels discussed herein.
  • a target protein described herein or fragment thereof e.g., a functional fragment thereof or its binding partner to facilitate separation of complexed from uncomplexed forms of one or both of the proteins, as well as to accommodate automation of the assay.
  • Binding of a test compound to a target protein described herein, or interaction of such a protein with a second component in the presence and absence of a candidate compound can be accomplished in any vessel suitable for containing the reactants. Examples of such vessels include microtiter plates, test tubes, and micro-centrifuge tubes.
  • a fusion protein can be provided which adds a domain that allows one or both of the proteins to be bound to a matrix.
  • glutathione-S-transferase protein fragment fusion proteins can be adsorbed onto glutathione SEPHAROSE ® beads (Sigma Chemical, St. Louis, MO) or glutathione derivatized microtiter plates, which are then combined with the test compound or the test compound.
  • the mixture is incubated under conditions conducive to complex formation (e.g., at physiological conditions for salt and pH).
  • the beads or microtiter plate wells are washed to remove any unbound components, the matrix immobilized in the case of beads, complex determined either directly or indirectly, for example, as described above.
  • the complexes can be dissociated from the matrix, and the level of protein binding or activity determined using standard techniques.
  • Biotinylated proteins or target molecules can be prepared from biotin-NHS (N-hydroxy-succinimide) using techniques known in the art (e.g., biotinylation kit, Pierce Chemicals, Rockford, IL), and immobilized in the wells of streptavidin-coated 96 well plates (Pierce Chemical). To conduct the assay, the non-immobilized component is added to the coated surface containing the anchored component.
  • any complexes formed will remain immobilized on the solid surface.
  • the detection of complexes anchored on the solid surface can be accomplished in a number of ways. Where the previously non-immobilized component is pre-labeled, the detection of label immobilized on the surface indicates that complexes were formed. Where the previously non-immobilized component is not pre-labeled, an indirect label can be used to detect complexes anchored on the surface, e.g., using a labeled antibody specific for the immobilized component (the antibody, in turn, can be directly labeled or indirectly labeled with, e.g., a labeled anti-Ig antibody).
  • this assay is performed utilizing antibodies reactive with a target protein described herein or target molecules but which do not interfere with binding of the target protein to its target molecule or with an activity of the target protein.
  • Such antibodies can be derivatized to the wells of the plate, and unbound target or protein trapped in the wells by antibody conjugation.
  • Methods for detecting such complexes include immunodetection of complexes using antibodies reactive with the target protein, as well as enzyme-linked assays which rely on detecting an enzymatic activity associated with the target protein.
  • cell free assays can be conducted in a liquid phase.
  • the reaction products are separated from unreacted components, by any of a number of standard techniques, including but not limited to: differential centrifugation (see, for example, Rivas and Minton, (1993) Trends Biochem. Sci. 18:284-7); chromatography (gel filtration chromatography, ion-exchange chromatography); electrophoresis (see, e.g., Ausubel et ah, eds. Current Protocols in Molecular Biology 1999, J. Wiley: New York.); and immunoprecipitation (see, for example, Ausubel et ah, eds. (1999) Current Protocols in Molecular Biology, J. Wiley: New York).
  • differential centrifugation see, for example, Rivas and Minton, (1993) Trends Biochem. Sci. 18:284-7
  • chromatography gel filtration chromatography, ion-exchange chromatography
  • electrophoresis see, e.g., Ausubel et ah
  • the assay includes contacting a target protein described herein or fragment thereof, e.g., a functional fragment thereof with a cognate binding partner to form an assay mixture, contacting the assay mixture with a test compound, and determining the ability of the test compound to interact with the protein, wherein determining the ability of the test compound to interact with the protein includes determining the ability of the test compound to preferentially bind to the protein or biologically active fragment thereof, or to modulate the activity of a target molecule, as compared to the known compound.
  • the assay can include combining the compounds in a different order, e.g., to determine if the test compound can interfere with the interaction between the protein and its binding partner.
  • Assays can be conducted in a heterogeneous or homogeneous format.
  • a typical heterogeneous assays includes anchoring either the target product or the binding partner onto a solid phase, and detecting complexes anchored on the solid phase at the end of the reaction.
  • the reaction is carried out in a liquid phase, hi either approach, the order of addition of reactants can be varied to obtain different information about the compounds being tested.
  • test compounds that interfere with the interaction between the target products and the binding partners e.g., by competition, can be identified by conducting the reaction in the presence of the test substance.
  • test compounds that disrupt preformed complexes e.g., compounds with higher binding constants that displace one of the components from the complex, can be tested by adding the test compound to the reaction mixture after complexes have been formed.
  • a protein described herein or fragment thereof e.g., a functional fragment thereof can be used as a "bait protein" in a two-hybrid assay or similar assay (see, e.g., U.S. Patent No. 5,283,317; Zervos et al. (1993) Cell 72:223-232; Madura et al. (1993) J. Biol. Chem. 268:12046-12054; Bartel et al.
  • binding partners can be activators or inhibitors of an activity of the protein.
  • modulators of expression of genes of Table 1 are identified.
  • a cell is contacted with a candidate compound and mRNA or protein expression of the gene is evaluated, e.g., relative to the level of expression in the absence of the candidate compound.
  • the candidate compound is identified as a stimulator of a expression of the gene.
  • expression is less (e.g., statistically significantly less) in the presence of the candidate compound than in its absence, the candidate compound is identified as an inhibitor of expression of the gene.
  • Methods for detecting gene expression in a sample include detecting mRNA or cDNA and detecting protein, e.g., using an antibody or other binding protein, or using an activity assay. It is also possible to detect mRNA or cDNA using any of a variety of molecular techniques, including RT-PCR and microarray analysis.
  • Examples of methods of gene expression analysis known in the art include DNA arrays or microarrays (Brazma and ViIo, FEBS Lett, 2000, 480, 17-24; Celis et al., FEBS Lett., 2000, 480, 2-16), SAGE (serial analysis of gene expression) (Madden et al., Drug Discov. Today, 2000, 5, 415-425), READS (restriction enzyme amplification of digested cDNAs) (Prashar and Weissman, Methods EnzymoL, 1999, 303, 258-72), TOGA (total gene expression analysis) (Sutcliffe et al., Proc. Natl. Acad. Sd.
  • Reporter genes can be used to evaluate changes in expression of the genes described herein.
  • Exemplary regulatory sequences of the genes described herein include those located within 100, 200, 500, 700, or 1600 base pairs of the mRNA start site.
  • Reporter genes can be made by operably linking a regulatory sequence to a sequence encoding a reporter gene.
  • a number of methods are available for designing reporter genes.
  • the sequence encoding the reporter protein can be linked in frame to all or part of the sequence that is normally regulated by the regulatory sequence. Such constructs can be referred to as translational fusions. It is also possible to link the sequence encoding the reporter protein to only regulatory sequences, e.g., the 5' untranslated region, TATA box, and/or sequences upstream of the rnRNA start site. Such constructs can be referred to as transcriptional fusions. Still other reporter genes can be constructed by inserting one or more copies (e.g., a multimer of three, four, or six copies) of a regulatory sequence into a neutral or characterized promoter.
  • Reporter genes can be introduced into germline cells of non-human mammals, e.g., to produce transgenic animals, or into stem cells, into pluripotent stem cells or . embryonic stem cells. Reporter genes can also be introduced into culture cells, e.g., tissue culture cells. Typically the cell is a mammalian, e.g., human cell or a cell derived from a human cell.
  • Arrays can be fabricated by a variety of methods, e.g., photolithographic methods (see, e.g., U.S. Patent Nos. 5,143,854; 5,510,270; and 5,527,681), mechanical methods (e.g., directed-flow methods as described in U.S. Patent No. 5,384,261), pin based methods (e.g., as described in U.S. Pat. No. 5,288,514), and bead based techniques (e.g., as described in PCT US/93/04145).
  • photolithographic methods see, e.g., U.S. Patent Nos. 5,143,854; 5,510,270; and 5,527,681
  • mechanical methods e.g., directed-flow methods as described in U.S. Patent No. 5,384,261
  • pin based methods e.g., as described in U.S. Pat. No. 5,288,514
  • bead based techniques e.g., as described in PC
  • the T m of all capture probes on the array is similar, e.g., within 20, 10, 5, 3, or 2 0 C of one another.
  • a database scan of available sequence information for a species can be used to determine potential cross- hybridization and specificity problems.
  • the isolated nucleic acid is preferably mRNA that can be isolated by routine methods, e.g., including DNase treatment to remove genomic DNA and hybridization to an oligo-dT coupled solid substrate (e.g., as described in Current Protocols in Molecular Biology, John Wiley & Sons, N. Y). The substrate is washed, and the mRNA is eluted.
  • Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy- terminus in the following order: FRl 5 CDRl, FR2, CDR2, FR3, CDR3, FR4.
  • Camelid antibodies can include a single variable immunoglobulin domain.
  • Full-length immunoglobulin "light chains” (about 25 kDa or 214 amino acids) are encoded by a variable region gene at the NH 2 -terminus (about 110 amino acids) and a kappa or lambda constant region gene at the COOH—terminus.
  • Full- length immunoglobulin "heavy chains” (about 50 kDa or 446 amino acids) are similarly encoded by a variable region gene (about 116 amino acids) and one of the other aforementioned constant region genes, e.g., gamma (encoding about 330 amino acids).
  • isotype refers to the antibody class (e.g., IgM, IgGl, IgG2, IgG3, IgG4) that is encoded by heavy chain constant region genes.
  • the two domains of the Fv fragment, VL and VH are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VL and VH domains pair to form monovalent molecules (known as single chain Fv (scFv); see, e.g., Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. ScL USA 85:5879-5883).
  • single chain Fv single chain Fv
  • Such single chain antibodies are also intended to be encompassed within the term "antigen-binding fragment" of an antibody.
  • Human monoclonal antibodies (mAbs) directed against target proteins can be generated using transgenic mice carrying the human immunoglobulin genes rather than the mouse system. Splenocytes from these transgenic mice immunized with the antigen of interest are used to produce hybridomas that secrete human mAbs with specific affinities for epitopes from a human protein (see, e.g., WO 91/00906, WO 91/10741; WO 92/03918; WO 92/03917; Lonberg et al. 1994 Nature 368:856-859; Green et al. 1994 Nature Genet. 7: 13-21; Morrison et al. 1994 Proc. Natl. Acad.
  • Monoclonal antibodies can also be generated by other methods.
  • An exemplary alternative method referred to as the "combinatorial antibody display” method, has been developed to identify and isolate antibody fragments having a particular antigen specificity, and can be utilized to produce monoclonal antibodies (for descriptions of combinatorial antibody display see e.g., Sastry et al. 1989 PNAS 86:5728; Huse et al. 1989 Science 246:1275; and Orlandi et al. 1989 PNAS 86:3833 and phage display methods, e.g., US 2002-0102613). After immunizing an animal with an immunogen as described above, the antibody repertoire of the resulting B-cell pool is cloned.
  • All or part of the CDRs of a particular human antibody may be replaced with at least a portion of a non-human CDR or only some of the CDRs may be replaced with non- human CDRs. It is only necessary to replace the number of CDRs, or portions thereof required for binding of the humanized antibody to the target antigen, e.g., a protein disclosed herein.
  • Antibodies with altered function e.g. altered affinity for an agonist ligand, such as FcR on a cell, or the Cl component of complement can be produced by replacing at least one amino acid residue in the constant portion of the antibody with a different residue (see e.g., EP 388 151, US 5,624,821 and US 5,648,260). Similar type of alterations could be described which if applied to the murine, or other species immunoglobulin would reduce or eliminate these functions.
  • Hybridization of antisense oligonucleotides with niRNAcan interferes with one or more of the normal functions of mRNA.
  • the functions of mRNA to be interfered with include all vital functions such as, for example, translocation of the RNA to the site of protein translation, translation of protein from the RNA, splicing of the RNA to yield one or more mRNA species, and catalytic activity which may be engaged in by the RNA. Binding of specific protein(s) to the RNA may also be interfered with by antisense oligonucleotide hybridization to the RNA.
  • Exemplary antisense compounds include DNA or RNA sequences that specifically hybridize to the target nucleic acid.
  • the complementary region can extend for between about 8 to about 80 nucleobases.
  • the compounds can include one or more modified nucleobases.
  • Modified nucleobases may include, e.g., 5-substituted pyrimidines such as 5-iodouracil, 5-iodocytosine, and C5-propynyl pyrimidines such as C5- ⁇ ropynylcytosine and C5-propynyluracil.
  • modified nucleobases include N 4 -(C] -C ]2 ) alkylaminocytosines and N ⁇ N 4 -(Ci -C 12 ) dialkylaminocytosines. Modified nucleobases may also include 7-substituted-8-aza- 7-deazapurines and 7-substituted-7-deazapurines such as, for example, 7-iodo-7- deazapurines, 7-cyano-7-deazapurines, 7-aminocarbonyl-7-deazapurines.
  • N 6 -(C 1 -C 12 ) alkylaminopurines and N 6 ,N 6 -(C 1 -C 12 ) dialkylaminopurines are also suitable modified nucleobases.
  • other 6- substituted purines including, for example, 6-thioguanine may constitute appropriate modified nucleobases.
  • suitable nucleobases include 2-thiouracil, 8- bromoadenine, 8-bromoguanine, 2-fluoroadenine, and 2-fluoroguanine. Derivatives of any of the aforementioned modified nucleobases are also appropriate.
  • Artificial transcription factors can also be used to regulate a gene of Table 1 and/or a protein encoded thereby.
  • the artificial transcription factor can be designed or selected from a library.
  • the artificial transcription factor can be prepared by selection in vitro (e.g., using phage display, U.S. Pat. No. 6,534,261) or in vivo, or by design based on a recognition code (see, e.g., WO 00/42219 and U.S. Pat. No. 6,511,808). See, e.g., Rebar et al. (1996) Methods Enzymol 267:129; Greisman and Pabo (1997) Science 275:657; Isalan et al. (2001) Nat. Biotechnol.
  • nucleic acids encoding proteins that function as agents for the methods described herein or that are target proteins or fragments thereof may be operably linked to an expression control sequence in a vector in order to produce the protein recombinantly.
  • expression control sequences are known.
  • General methods of expressing recombinant proteins are also known and are exemplified in Kaufman, Methods in Enzymology 185, 537-566 (1990), Sambrook & Russell, Molecular Cloning: A Laboratory Manual, 3 rd Edition, Cold Spring Harbor
  • certain vectors are capable of directing the expression of genes to which they are operatively linked.
  • Exemplary viral vectors include replication defective retroviruses, adenoviruses and adeno-associated viruses.
  • Preferred selectable marker genes include the dihydrofolate reductase (DHFR) gene (for use in dhfr- host cells with methotrexate selection/amplification) and the neo gene (for G418 selection).
  • DHFR dihydrofolate reductase
  • neo gene for G418 selection.
  • a number of types of cells may act as suitable host cells for expression of a protein therapeutic. Any cell type capable of expressing the protein therapeutic may be used.
  • Exemplary mammalian host cells include, for example, monkey COS cells, Chinese Hamster Ovary (CHO) cells, human kidney 293 cells, human epidermal A431 cells, human Colo205 cells, 3T3 cells, CV-I cells, other transformed primate cell lines, normal diploid cells, cell strains derived from in vitro culture of primary tissue, primary explants, HeLa cells, mouse L cells, BHK, HL-60, U937, HaK, Rat2, BaF3,
  • a protein therapeutic or target protein may be produced by operably linking a polynucleotide encoding such a protein to suitable control sequences in one or more insect expression vectors, and employing an insect expression system.
  • Materials and methods for baculovirus/insect cell expression systems are commercially available, e.g., in kit form from, e.g., Invitrogen, San Diego, CA (the MAXBAC ® kit), e.g., as described in Summers and Smith, Texas Agricultural Experiment Station Bulletin No.
  • a protein therapeutic or target protein may be produced in lower eukaryotes such as yeast or in prokaryotes such as bacteria.
  • yeast strains include Saccharomyces cerevisiae, Schizosaccharomyces pombe, Kluyveromyces strains,
  • a protein therapeutic or target protein (e.g., a polypeptide described herein or fragment thereof) is produced in a bacterial cell, e.g., with or without a signal sequence (e.g., without either a prokaryotic or eukaryotic signal sequence).
  • a signal sequence e.g., without either a prokaryotic or eukaryotic signal sequence.
  • Expression in bacteria may result in formation of inclusion bodies incorporating the recombinant protein.
  • refolding of the recombinant protein may be required in order to produce active or more active material.
  • Several methods for obtaining correctly folded heterologous proteins from bacterial inclusion bodies are known in the art. These methods generally involve solubilizing the protein from the inclusion bodies, then denaturing the protein completely using a chaotropic agent.
  • the protein When cysteine residues are present in the primary amino acid sequence of the protein, the protein can be refolded in an environment which facilitates correct formation of disulfide bonds (e.g., a redox system).
  • a redox system e.g., a redox system
  • a protein described herein (e.g., a target protein, therapeutic, or immunoglobulin) thereof may also be expressed as a product of transgenic animals, e.g., as a component of the milk of transgenic cows, goats, pigs, or sheep which are characterized by somatic or germ cells containing a polynucleotide sequence encoding the protein.
  • An agent that modulates a gene of Table 1 or a protein encoded thereby can be used to treat a subject, e.g., a human subject.
  • the agent can be provided as a component of a pharmaceutical composition.
  • the pharmaceutical composition may include a therapeutically effective amount of an agent described herein.
  • a therapeutically effective amount is an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic result or to prevent or delay onset of a disorder.
  • a therapeutically effective amount of the composition may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the compound to elicit a desired response in the individual.
  • a therapeutically effective amount is also one in which any toxic or detrimental effects of the composition is outweighed by the therapeutically beneficial effects.
  • a therapeutically effective amount preferably modulates a measurable parameter, e.g., a measurable symptom of an inflammatory or autoimmune disorder, e.g., multiple sclerosis, relative to untreated subjects, e.g., to a statistically significant degree.
  • a measurable parameter e.g., a measurable symptom of an inflammatory or autoimmune disorder, e.g., multiple sclerosis
  • the ability of a compound to inhibit a measurable parameter can be evaluated in an animal model system predictive of efficacy in a human disorder, using in vitro assays, e.g., an assay described herein, or using appropriate human trials.
  • a compound discovered by the methods described herein can be formulated as a pharmaceutical composition compatible with an intended route of administration.
  • routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (topical), transmucosal, and rectal administration.
  • Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose.
  • a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycer
  • pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide.
  • the parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.
  • Pharmaceutical formulation is a well-established art, and is further described, e.g., in Gennaro (ed.), Remington: The Science and Practice of Pharmacy, 20 th ed., Lippincott, Williams & Wilkins (2000) (ISBN: 0683306472); Ansel et ah, Pharmaceutical Dosage Forms and Drug Delivery Systems, 7 th Ed., Lippincott Williams & Wilkins Publishers (1999) (ISBN: 0683305727); and Kibbe (ed.), Handbook of Pharmaceutical Excipients American Pharmaceutical Association, 3 rd ed.
  • a pharmaceutical composition typically includes a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable carrier includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible.
  • the composition can include a pharmaceutically acceptable salt, e.g., an acid addition salt or a base addition salt (see e.g., Berge et a (1977) J. Pharm. Sd. 66:1-19).
  • Particular effects mediated by an agent may show a difference that is statistically significant (e.g., P value ⁇ 0.05 or 0.02).
  • Statistical significance can be determined by any art known method. Exemplary statistical tests include: the Students T-test, Mann Whitney U non-parametric test, and Wilcoxon non-parametric statistical test. Some statistically significant relationships have a P value of less than 0.05 or 0.02.
  • Dosage regimens are adjusted to provide the optimum desired response ⁇ e.g., a therapeutic response). For example, a single bolus may be administered, several divided doses may be administered over time or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. It is possible to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage.
  • Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subjects to be treated; each unit contains a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.
  • An exemplary, non-limiting range for a therapeutically effective amount of an agent described herein is about 0.1 -20 mg/kg, more preferably about 1 - 10 mg/kg. Dosage values may vary with the type and severity of the condition to be alleviated. For any individual subject, specific dosage regimens can be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the compositions. Accordingly, the dosage ranges set forth herein are only exemplary.
  • Subjects who can be treated include human and non-human animals, e.g., non- mammals (such as chickens, amphibians, reptiles) and mammals, such as non-human primates, mice, sheep, dogs, cows, pigs, etc.
  • non- mammals such as chickens, amphibians, reptiles
  • mammals such as non-human primates, mice, sheep, dogs, cows, pigs, etc.
  • An agent described herein may be used as a pharmaceutical composition when combined with a pharmaceutically acceptable carrier.
  • a pharmaceutically acceptable carrier may contain, in addition to the agent and carrier, various diluents, fillers, salts, buffers, stabilizers, solubilizers, and other materials well known in the art.
  • Pharmaceutically acceptable carriers are non-toxic materials that does not interfere with the effectiveness of the biological activity of the active ingredient(s). The characteristics of the carrier typically depend on the route of administration.
  • a therapeutically effective amount of an agent is administered to a subject, e.g., mammal (e.g., a human).
  • the agent may be administered either alone or in combination with other therapies such as other treatments for atopic disorders.
  • the agent may be administered either simultaneously with the second agent, or sequentially. If administered sequentially, the attending physician can decide on the appropriate sequence of administering the agent described herein with other agents.
  • Administration of an agent described herein can be carried out in a variety of ways, including, for example, oral ingestion, inhalation, or cutaneous, subcutaneous, or intravenous injection or administration.
  • the agent can be in the form of a tablet, capsule, powder, solution, or elixir.
  • the pharmaceutical composition may additionally contain a solid carrier such as a gelatin or an adjuvant.
  • the tablet, capsule, and powder contain from about 5 to 95% of the agent or from about 25 to 90% of the agent.
  • a liquid carrier such as water, petroleum, oils of animal or plant origin such as peanut oil, mineral oil, soybean oil, or sesame oil, or synthetic oils may be added.
  • the liquid form of the pharmaceutical composition may further contain physiological saline solution, dextrose or other saccharide solution, or glycols such as ethylene glycol, propylene glycol or polyethylene glycol.
  • the pharmaceutical composition When administered in liquid form, the pharmaceutical composition contains from about 0.5 to 90% by weight of the agent, and preferably from about 1 to 50% the agent.
  • the agent can be in the form of a pyrogen-free, parenterally acceptable aqueous solution.
  • parenterally acceptable protein solutions having due regard to pH, isotonicity, stability, and the like, is within the skill in the art.
  • An exemplary pharmaceutical composition for intravenous, cutaneous, or subcutaneous injection can contain, in addition to the agent an isotonic vehicle such as sodium chloride injection, Ringer's injection, dextrose injection, dextrose and sodium chloride injection, lactated Ringer's injection, or other vehicle as known in the art.
  • the pharmaceutical composition may also contain stabilizers, preservatives, buffers, antioxidants, or other additive known to those of skill in the art.
  • the amount of an agent to be delivered can depend upon the nature and severity of the condition being treated, and on the nature of prior treatments that the patient has undergone.
  • the attending physician can decide the amount of agent with which to treat each individual patient. Initially, for example, the attending physician can administer low doses of the agent and observe the patient's response.
  • an exemplary pharmaceutical compositions may contain about 0.1 ⁇ g to about 10 mg of the immunoglobulin agent per kg body weight.
  • useful dosages can include between about 10 ⁇ g-1 mg, 0.1-5 mg, and 3-50 mg of the agent per kg body weight.
  • the duration of therapy using the pharmaceutical composition can vary, depending on the severity of the disease being treated and the condition and potential idiosyncratic response of each individual patient.
  • the duration of each application of the agent can be, e.g., in the range of 12 to 24 hours of continuous intravenous administration.
  • the attending physician can decide on the appropriate duration of intravenous therapy using a pharmaceutical composition described herein.
  • the disease or disorder can also be treated or prevented by administration or use of polynucleotides encoding such proteins (such as, for example, in gene therapies or vectors suitable for introduction of DNA).
  • the polynucleotides that encode an agent or that provide a nucleic acid agent activity can be inserted into vectors and used as gene therapy vectors.
  • Gene therapy vectors can be delivered to a subject by, for example, intravenous injection, local administration (see U.S. Patent 5,328,470), injection (e.g., US 20040030250 or 20030212022) or stereotactic injection (e.g., Chen et al. Proc. Natl. Acad. ScL USA 91:3054-3057, 1994).
  • the pharmaceutical preparation of the gene therapy vector can include the gene therapy vector in an acceptable diluent, or can comprise a slow release matrix in which the gene delivery vehicle is imbedded.
  • the pharmaceutical preparation can include one or more cells which produce the gene delivery system.
  • compositions can be administered using a medical device.
  • a pharmaceutical composition described herein can be administered with a needle-less hypodermic injection device, such as the devices disclosed in U.S. Patent Nos. 5,399,163; 5,383,851; 5,312,335; 5,064,413; 4,941,880; 4,790,824; or 4,596,556.
  • a needle-less hypodermic injection device such as the devices disclosed in U.S. Patent Nos. 5,399,163; 5,383,851; 5,312,335; 5,064,413; 4,941,880; 4,790,824; or 4,596,556.
  • Examples of well-known implants and modules that can be used include: U.S. Patent No. 4,487,603, which discloses an implantable micro- infusion pump for dispensing medication at a controlled rate; U.S. Patent No. 4.,486,194, which discloses a therapeutic device for administering agents through the skin; U.S. Patent No.
  • Modulators of the genes of Table 1 and proteins encoded thereby can be used to treat or prevent multiple sclerosis or one or more symptoms of multiple sclerosis (MS).
  • MS is a chronic disease characterized by the inflammation and scarring (sclerosis) of the myelin sheath and the underlying nerve.
  • exemplary symptoms associated with multiple sclerosis include: optic neuritis, diplopia, nystagmus, ocular dysmetria, internuclear ophthalmoplegia, movement and sound phosphenes, afferent pupillary defect, paresis, monoparesis, paraparesis, hemiparesis, quadriparesis, plegia, paraplegia, hemiplegia, tetraplegia, quadriplegia, spasticity, dysarthria, muscle atrophy, spasms, cramps, hypotonia, clonus, myoclonus, myokymia, restless leg syndrome, footdrop, dysfunctional reflexes, Babinski's reflex, paraesthesia, anaesthesia, neuralgia, neuropathic and neurogenic pain, Lhermitte's
  • Patients suitable for treatment using the methods described herein may be identified by criteria establishing a diagnosis of clinically definite MS as defined by the workshop on the diagnosis of MS (Poser et al., Ami. Neurol. 13:227, 1983). Briefly, an individual with clinically definite MS has had two attacks and clinical evidence of either two lesions or clinical evidence of one lesion and paraclinical evidence of another, separate lesion. Definite MS may also be diagnosed by evidence of two attacks and oligoclonal bands of IgG in cerebrospinal fluid or by combination of an attack, clinical evidence of two lesions and oligoclonal band of IgG in cerebrospinal fluid. Slightly lower criteria are used for a diagnosis of clinically probable MS.
  • Patients suitable for treatment can also be evaluated for expression and/or activity of one or more polypeptide encoded by a gene of Table 1, and can be identified as suitable for treatment if the expression and/or activity for one or more such polypeptides is elevated relative to a reference (provide such polypeptide is elevated in the mouse models).
  • Candidate patients for prevention can be identified by the presence of genetic factors. For example, a majority of MS patients have HLA-type DR2a and DR2b.
  • the MS patients having genetic dispositions to MS who can be suitable for treatment fall within two groups. First are patients with early disease of the relapsing remitting type. Entry criteria can include disease duration of more than one year, EDSS score of 1.0 to 3.5, exacerbation rate of more than 0.5 per year, and free of clinical exacerbations for 2 months prior to study.
  • the second group includes people with disease progression greater than 1.0 EDSS unit/year over the past two years.
  • Candidate patients for prevention may be identified by evaluating cytokine parameters, e.g., an IL-10 or IL-21 parameter (see, e.g., U.S. Application No. 10/806,611).
  • PSMEl encodes proteasome 28 subunit alpha (PA28 ⁇ ), which is a part of the HS regulator of the immunoproteasome.
  • PA28 ⁇ proteasome 28 subunit alpha
  • Two transcripts of PSMEl encoding different isoforms have been identified.
  • Exemplary human PSME amino acid sequences are listed at GenBank ® accession numbers NP_006254, NP_788955. LAPTM5.
  • LAPTM5 is a pentaspanner transmembrane protein that is conserved across evolution.
  • An exemplary human LAPTM5 amino acid sequence is listed at GenBank ® accession number NP_006753.
  • Macrosialin/CD68 is a transmembrane glycoprotein expressed in cytoplasmic granules of macrophages/monocytes, DC, granulocytes and myeloid progenitor cells.
  • An exemplary human CD68 amino acid sequence is listed at GenBank ® accession number NP 001242.
  • CSFlR Macrophage colony-stimulating factor (M-CSF) is one of several hematologic growth factors capable of regulating the survival, proliferation, and differentiation of macrophages.
  • An exemplary human M-CSF amino acid sequence is listed at GenBank ® accession number NP_005202.
  • SCYA6 SCYA6 encodes the chemokine ClO.
  • An exemplary human amino acid sequence is listed at GenBank ® accession number NP_116741.
  • CD53 is a member of tetraspanin family.
  • An exemplary human CD53 amino acid sequence is listed at GenBank ® accession number NP_000551.
  • Mpg-1 shows lineage-restricted and stage-specific expression in mature macrophages.
  • An exemplary human Mpg-1 amino acid sequence is listed at GenBank ® accession number XEM66227. ,
  • LY86/MD-1 is a 162 amino acid secreted protein that binds to and positively regulates the expression of RP105/CD180, a transmembrane protein with similarity to TLR4.
  • An exemplary human MD-I amino acid sequence is listed at GenBank ® accession number NP_004262.
  • LCN2 An exemplary human lipocalin-2 sequence is listed at GenBank ® accession number NP_005555.
  • EAE Experimental autoimmune encephalomyelitis
  • CNS central nervous system
  • MS multiple sclerosis
  • EAE is generally believed to be a Thl-induced disease because of the increased expression of ThI cytokines in the CNS. Furthermore injection of ThI but not Th2 T cells into immunocompetent mice is sufficient to induce EAE. Baron et al, J Exp Med 1993;177(l):57-68; Kuchroo et al, J Immunol 1993;151(8):4371-82 ;
  • ThI cells produce interferon (IFN)- ⁇ , together with other Thl-type cytokines.
  • IFN- ⁇ is a potent activator of macrophages, stimulator of expression of MHC class I and II molecules, and activator of adhesion molecules and inflammatory mediators, such as nitric oxide (NO) and TNF.
  • NO nitric oxide
  • TNF nitric oxide
  • mice deficient in IFN- ⁇ or IFN- ⁇ R often develop EAE with higher incidence and severity than wild-type (WT) mice.
  • IL-12 is a cytokine, composed of two disulfide- linked subunits, designated p40 and p35. It is produced by activated antigen presenting cells and it can induce differentiation of recently activated CD4 cells into ThI type. Trinchieri, Nat Rev Immunol 2003;3(2): ⁇ 33-46; Szabo et al, Annu Rev Immunol 2003;21:713-58.
  • IL-12 can induce production of IFN- ⁇ , granulocyte- macrophage colony-stimulatory factor (GM-CSF) and TNF, which all play an important role in EAE development.
  • GM-CSF granulocyte- macrophage colony-stimulatory factor
  • mice deficient in the p35 subunit of IL-12 still develop EAE.
  • IL- 12p35 -/— mice that develop EAE have reduced levels of IFN- ⁇ in draining lymph nodes early in disease but these levels increase later during the immune response. Furthermore, the expression of IFN- ⁇ mRNA in the CNS of IL-12p35 -/- mice with EAE is not significantly different from WT controls (Becher et al., J CHn Invest
  • mice Female IFN ⁇ -/-mice on C57B1/6 background and appropriate C57B1/6 WT control animals were obtained from Jackson Laboratories (Bar Harbor, ME) and used at 6-10 weeks of age. IL-12p35 -/- mice, backcrossed on C57B1/6 background for 5 generations, and appropriate C57B1/6 control WT mice were bred at Taconic Farms (Germantown, NY) and 6-10 week old females were used for experiments.
  • EAE Experimental autoimmune encephalomyelitis
  • tissue collection For EAE induction, all mice were injected subcutaneously with 200 ⁇ g of myelin oligodendrocytes glycoprotein (MOG) peptide 35-55 in complete Freund's adjuvant containing 5 mg/ml killed Mycobacterium tuberculosis. On the same day, the mice received 200 ng pertussis toxin intraperitoneally. Paralysis (EAE) was assessed, starting on day 5 after immunization, when all the mice were still symptom-free.
  • MOG myelin oligodendrocytes glycoprotein
  • EAE was scored as follows: 1- limp tail, 2- partial hind leg paralysis, 3-complete hind leg paralysis or partial hind and front leg paralysis, 4- complete hind and partial front leg paralysis, 5-moribund.
  • CNS and spleen tissues were collected from the mice at onset, peak and recovery stage of EAE.
  • CNS tissue was a pool of spinal cord and brain stem tissue of each individual mouse. Onset was defined as within 24 hours of the first clinical signs of EAE, with EAE score of 2 or less. Peak was defined as 4-6 days after the first signs of EAE, with the score of 3-4. The entire experimental design was repeated over three time-separated intervals. No systematic differences were observed and therefore data for individually analyzed mice from the three experiment were combined.
  • RNA RNA was collected from at least 10 mice/group/time point and processed individually.
  • CNS tissue from MS lesion.
  • CNS tissue was collected and analyzed by oil red O and hematoxylin staining of 10 ⁇ m snap frozen sections cut from each tissue block before and after tissue collected for gene expression profiling and scored for the degree of ongoing and recent demyelination and perivascular cuffing.
  • Acute MS lesions with ongoing or recent demyelination were identified on the basis of the presence of substantial numbers (graded as >3 on a 0-5 scale) of oil red O-positive macrophages containing neutral lipids resulting from myelin breakdown Li et al., Neuropathol Appl Neurobiol 1993;19(3):214-23.
  • Murine U74Av2 GeneChip ® (Affymetrix, Santa Clara, CA) as recommended by the manufacturer.
  • the chips were scanned using a Hewlett Packard GeneArray ® Scanner and raw data generated using Affymetrix ® MAS 4.0 software.
  • Hybridization intensities on each array were further normalized to a standard curve created from a set of eleven bacterial transcripts spiked in at defined concentrations. This standard curve was used to convert signal values for each qualifier on each array to frequency units expressed as parts per million. Use of the bacterial transcripts allowed for sensitivity for each array to be determined. Hill et al., Genome Biol 2001;2(12):RESEARCH0055.
  • RNA m 85% of the arrays we were able to detect transcripts expressed at 2.3 parts per million (ppm) and in 15% of the arrays were able to detect transcripts expressed at 5.7 ppm.
  • ppm parts per million
  • ⁇ -actin glyceraldehyde-3-phosphate dehydrogenase
  • ⁇ -actin ⁇ -actin are expressed at 400 ppm and 70 ppm respectively.
  • GAPDH glyceraldehyde-3-phosphate dehydrogenase
  • ⁇ -actin are expressed at 400 ppm and 70 ppm respectively.
  • Quality of RNA was verified by the 573' ratio for GAPDH and for ⁇ -actin as measured by the arrays. Ratios ranged from 0.8-1.1.
  • CS confidence score
  • Gene-specific primers and probes were used with the TaqMan Universal PCR Master Mix (PE Applied Biosystems) to amplify the equivalent of 50 ng of RNA generated from the cDNA. Reactions were incubated at 50 0 C for 2 minutes followed by 10 minutes at 95 °C then 40 cycles of PCR as follows: 95 °C for 15 seconds then 60 °C for 1 minute in an ABI 7900. The data were analyzed using SequenceDetectorTM version 2.0 software (PE Applied Biosystems) and were normalized to GAPDH primer set (PE Applied Biosystems).
  • EAE EAE. Mice were immunized with MOG 35-5S peptide in complete Freund's adjuvant, and the development of clinical signs of EAE were monitored. In at least three independent experiments, wild type, IL-12p35 -/- and IFN- ⁇ -/- animals showed comparable severity and time of disease onset (FIG. 1). Since the two gene-deficient mice were bred at two separate facilities, two sets of wild type B6 mice were examined. We found no significant differences in EAE development or gene expression profiles in the two sets of WT mice and therefore they were treated as a single control group.
  • DAP12 -/- mice were shown to be resistant to EAE induced by immunization with myelin oligodendrocyte glycoprotein (MOG) peptide Bakker et al, Immunity 2000;13(3):345-53. Resistance was associated with a strongly diminished production of IFN- ⁇ by myelin-reactive CD4+ T cells due to inadequate T cell priming in vivo. Bakker et al., 2000, supra. CD52 is highly expressed on lymphocytes and monocytes and antibody treatments have resulted in depletion of lymphocytes and in suppression of clinical and MRI inflammatory activity in MS patients Confavreux et al., Clin Neurol Neurosurg 2004;106(3):263-9.
  • MOG myelin oligodendrocyte glycoprotein
  • statins which are inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A reductase, and cholesterol-lowering drugs have immunomodulatory effects.
  • statins which are inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A reductase
  • cholesterol-lowering drugs have immunomodulatory effects.
  • Methallothioneins are low molecular weight, cysteine-rich, stress response proteins that can act as immunosuppressive agents in antigen-dependent adaptive immunity. Coyle et al,

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Abstract

Methods and compositions for treating and diagnosing inflammatory, autoimmune and/or neurologic disorders, e.g., multiple sclerosis, are provided. In addition, methods for identifying agents that can be used in such therapeutic and diagnostic methods are disclosed.

Description

METHODS AND COMPOSITIONS FOR TREATING AND DIAGNOSING MULTIPLE SCLEROSIS
TECHNICAL FIELD
This invention relates to methods and compositions for treating and diagnosing inflammatory, autoimmune and/or neurologic disorders, as well as methods for identifying agents that can be used in such therapeutic and diagnostic methods.
BACKGROUND
Multiple sclerosis (MS) is a central nervous system disease that is characterized by inflammation and loss of myelin sheaths, the fatty material that insulates nerves and facilitates nerve function.
SUMMARY
This invention features, inter alia, methods and compositions for treating and diagnosing inflammatory, autoimmune and/or neurologic disorders (e.g., multiple sclerosis (MS)), as well as methods for identifying agents that can be used in such therapeutic and diagnostic methods. In one aspect, the invention features a method of evaluating a compound. The method includes: contacting a test compound to a target protein that includes a polypeptide encoded by a gene selected from Table 1, or a fragment or functional domain thereof; evaluating an interaction between the test compound and the target protein; and, optionally, evaluating the test compound in an animal, or cell-based, model, e.g., a model of an inflammatory, autoimmune and/or neurologic disorder. In one embodiment, the test compound is a polypeptide, e.g., a peptide, or a small molecule (e.g., an organic molecule having a molecular weight less than 3000 Daltons). In one embodiment, the test compound is an immunoglobulin. In one embodiment, the target protein is at least 50% purified (e.g., at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 99% purified). In one embodiment, the test compound is contacted to the target protein in a cell-free system. The target protein can be a recombinant protein. In some embodiments, the gene encodes: a polypeptide associated with antigen processing and/or presentation, e.g., proteasome 28 subunit alpha (PSMEl); a component of complement; a component of the extracellular matrix; a polypeptide related to hematopoiesis, e.g., CD53 antigen, CD68 antigen, lysosomal-associated protein transmembrane 5 (LAPTM5), lymphocyte antigen 86 (LY86), macrophage expressed gene 1 (MPEGl), or small inducible cytokine A6 (SCYA6); a polypeptide related to lipid metabolism, e.g., lipocalin 2 (LCN2); a regulator of transcription; or a plastin, e.g., plastin 2 (PLS2). hi one embodiment, the step of evaluating an interaction includes evaluating a function of the target protein (e.g., a binding or enzymatic function), e.g., detecting binding between the test compound and the target protein, or evaluating an enzymatic activity of the protein, in the presence of the test compound. The evaluating step can be performed using an in vitro assay.
The method can further include evaluating the test compound in an animal or cell based model, e.g., a model of an inflammatory, autoimmune and/or neurologic disorder (e.g., multiple sclerosis). The animal model can be evaluated for one or more symptoms associated with the disorder.
In one embodiment, the test compound is selected and then evaluated in an animal or cell-based model, hi some embodiment, the evaluation step is carried out if an interaction (e.g., binding) satisfying a threshold value (e.g., at least one-, two-, or three-fold above background or above a control value) is detected, hi one embodiment, the cell-based model is a cell that includes a recombinant reporter construct, e.g., a reporter construct whose transcription is operably linked to a regulatory sequence that regulates a gene encoding a polypeptide in Table 1. hi one embodiment, the disorder is multiple sclerosis. For example, the animal model is an experimental autoimmune encephalomyelitis (EAE) mouse.
In one embodiment, the method of evaluating a compound is repeated for one or more of a plurality of compounds from a chemical library. hi another aspect, the invention features a method of evaluating a compound. The method includes: contacting a test compound to a mammalian cell (e.g., a lymphocyte or a neuronal cell (e.g., a neuron or glial cell)); evaluating expression of a gene chosen from one or more genes in Table 1 in the mammalian cell; and, optionally, evaluating the test compound in an animal or cell-based model of a disorder, e.g., an inflammatory, autoimmune and/or neurologic disorder. For example, test compounds that reduce or prevent increased expression of the gene are identified as candidate therapeutics or lead compounds for a therapeutic. In one embodiment, the mammalian cell includes a recombinant reporter construct. The construct can include a regulatory sequence of a gene listed in Table 1 (e.g., all or a functional part of a promoter region) operably linked to a sequence encoding a reporter protein. Expression of the gene is evaluated by evaluating expression of the reporter construct.
In some embodiments, the gene encodes: a polypeptide associated with antigen processing and/or presentation, e.g., proteasome 28 subunit alpha (PSMEl); a component of complement; a component of the extracellular matrix; a polypeptide related to hematopoiesis, e.g., CD53 antigen, CD68 antigen, lysosomal-associated protein transmembrane 5 (LAPTM5), lymphocyte antigen 86 (LY86), macrophage expressed gene 1 (MPEGl), or small inducible cytokine A6 (SCYA6); a polypeptide related to lipid metabolism, e.g., lipocalin 2 (LCN2); a regulator of transcription; or a plastin, e.g., plastin 2 (PLS2).
In one embodiment, evaluating expression of the gene includes evaluating expression of a plurality of genes listed in Table 1.
In one embodiment, evaluating expression of the gene includes isolating a nucleic acid (e.g., mRNA) from the cell, and evaluating the nucleic acid (e.g., mRNA) levels, e.g., by amplification (e.g., RT-PCR, quantitative RT-PCR), or by contacting the nucleic acid (e.g., mRNA or a cDNA thereof) to an array of capture probes.
In one embodiment, evaluating expression of the gene includes evaluating presence or abundance of one or more polypeptides encoded by one or more genes, or an activity of the one or more polypeptides. In some embodiments, the one or more polypeptides is encoded by one or more genes listed in Table 1.
In another aspect, the invention features a method of evaluating and using a compound by contacting a test compound to a target protein that includes a polypeptide encoded by a gene selected from Table 1, or a fragment or functional domain thereof; evaluating an interaction between the test compound and the target protein; and administering the test compound to a subject that has a disorder, e.g., an inflammatory, autoimmune and/or neurologic disorder (e.g., multiple sclerosis), hi one embodiment, the test compound is a polypeptide, an immunoglobulin or a small molecule.
In some embodiments, the gene encodes: a polypeptide associated with antigen processing and/or presentation, e.g., proteasome 28 subunit alpha (PSMEl); a component of complement; a component of the extracellular matrix; a polypeptide related to hematopoiesis, e.g., CD53 antigen, CD68 antigen, lysosomal-associated protein transmembrane 5 (LAPTM5), lymphocyte antigen 86 (LY86), macrophage expressed gene 1 (MPEGl), or small inducible cytokine A6 (SCYA6); a polypeptide related to lipid metabolism, e.g., lipocalin 2 (LCN2); a regulator of transcription; or a plastin, e.g., plastin 2 (PLS2). hi one embodiment, the test subject is an animal model, e.g., a model of an inflammatory autoimmune and/or neurologic disorder. In one embodiment, the animal model is an experimental autoimmune encephalomyelitis (EAE) mouse, hi one embodiment, the disorder is multiple sclerosis. In another aspect, the invention features a method of treating or preventing a disorder (e.g., an inflammatory, autoimmune and/or neurologic disorder) in a subject by administering to the subject an effective amount of an agent that modulates the expression or activity of one or more genes chosen from Table 1, or a protein comprising a polypeptide encoded by one or more genes selected from Table 1. hi one embodiment, the disorder is multiple sclerosis. In one embodiment, the subject is a human. In one embodiment, the agent is an immunoglobulin (e.g., antibody or functional fragment thereof). In one embodiment, the agent is an inhibitor of the protein, e.g., an inhibitor of an enzymatic or other biological activity of the protein. In another embodiment, the agent is an enzyme that modifies the protein. In one embodiment, the agent is a polypeptide that sequesters the protein. In one embodiment, the agent decreases expression of the gene or protein.
In some embodiments, the gene encodes: a polypeptide associated with in antigen processing and/or presentation, e.g., proteasome 28 subunit alpha (PSMEl); a component of complement; a component of the extracellular matrix; a polypeptide related to hematopoiesis, e.g., CD53 antigen, CD68 antigen, lysosomal-associated protein transmembrane 5 (LAPTM5), lymphocyte antigen 86 (LY86), macrophage expressed gene 1 (MPEGl), or small inducible cytokine A6 (SCYA6); a polypeptide related to lipid metabolism, e.g., lipocalin 2 (LCN2); a regulator of transcription; or a plastin, e.g., plastin 2 (PLS2).
In another aspect, the invention features a method of evaluating a subject by evaluating the expression or activity of at least one gene chosen from Table 1 or a polypeptide encoded by a gene selected from Table 1. The method can be used, e.g., to obtain a value for a parameter indicative of the expression or activity, or to obtain a profile (e.g., a profile that includes a plurality of values for respective parameters indicative of the expression or activity of a plurality of genes or polypeptides selected from Table 1). In one embodiment, nucleic acid (e.g., mRNA or cDNA) levels are quantitated. In other embodiments, protein levels are quantitated. In one embodiment, the subject is a human. For example, the subject has been diagnosed with a disorder, e.g., an inflammatory, autoimmune and/or neurologic disorder (e.g., multiple sclerosis). In one embodiment, the subject is at risk for a disorder, e.g., an inflammatory, autoimmune and/or neurologic disorder, e.g., multiple sclerosis. The subject can be identified as at risk or having the disorder, e.g., multiple sclerosis, if the value has a difference, e.g., a statistically significant difference, relative to a normal or other reference value, e.g., obtained by similar methods for a reference cohort of subjects (e.g., normal subjects of similar gender, age, and so forth).
In some embodiments, the gene encodes: a polypeptide associated with antigen processing and/or presentation, e.g., proteasome 28 subunit alpha (PSMEl); a component of complement; a component of the extracellular matrix; a polypeptide related to hematopoiesis, e.g., CD53 antigen, CD68 antigen, lysosomal-associated protein transmembrane 5 (LAPTM5), lymphocyte antigen 86 (LY86), macrophage expressed gene 1 (MPEGl), or small inducible cytokine A6 (SCYA6); a polypeptide related to lipid metabolism, e.g., lipocalin 2 (LCN2); a regulator of transcription; or a plastin, e.g., plastin 2 (PLS2).
In yet another aspect, the invention features a method of identifying a subject for treatment of an autoimmune, inflammatory and/or neurologic disorder, by evaluating the expression of one or more genes selected from Table 1 in cells of the subject, and identifying the subject as a subject suited for treatment of the disorder, as a function of a comparison between the expression and/or activity of the one or more genes for the subject and a corresponding reference, e.g., a reference subject, sample, or cohort. In one embodiment, the comparison includes evaluating one or more reference values for a parameter associated with a normal (e.g., a non-autoimmune) subject or a population of subjects. In one embodiment, the disorder is multiple sclerosis. In another embodiment, one or more nucleotides of a gene from Table 1 are evaluated and compared to a corresponding reference sequence for the gene. The nucleotide can be in a promoter region, other regulatory region, or a coding region. The method can be used to evaluate if the subject has one or more polymorphisms, e.g., single nucleotide polymorphisms, in a gene from Table 1.
Li some embodiments, the gene encodes: a polypeptide associated with antigen processing and/or presentation, e.g., proteasome 28 subunit alpha (PSMEl); a component of complement; a component of the extracellular matrix; a polypeptide related to hematopoiesis, e.g., CD53 antigen, CD68 antigen, lysosomal-associated protein transmembrane 5 (LAPTM5), lymphocyte antigen 86 (LY86), macrophage expressed gene 1 (MPEG1), or small inducible cytokine A6 (SCYA6); a polypeptide related to lipid metabolism, e.g., lipocalin 2 (LCN2); a regulator of transcription; or a plastin, e.g., plastin 2 (PLS2).
In one aspect, the invention features a method of monitoring efficacy of a treatment for a disorder (e.g., an inflammatory, autoimmune and/or neurologic disorder) by treating a subject having a disorder (e.g., an inflammatory, autoimmune and/or neurologic disorder) and evaluating the expression or activity of one or more genes selected from Table 1 in cells of the subject. In one embodiment, the disorder is multiple sclerosis. The method can include evaluating such expression or activity at one or more instances, e.g., over the course of a therapy, e.g., over the course of one, two, four, six weeks, six months, or longer after initiating a therapeutic regimen. In some embodiments, the gene encodes: a polypeptide associated with antigen processing and/or presentation, e.g., proteasome 28 subunit alpha (PSMEl); a component of complement; a component of the extracellular matrix; a polypeptide related to hematopoiesis, e.g., CD53 antigen, CD68 antigen, lysosomal-associated protein transmembrane 5 (LAPTM5), lymphocyte antigen 86 (LY86), macrophage expressed gene 1 (MPEGl), or small inducible cytokine A6 (SC YA6); a polypeptide related to lipid metabolism, e.g., lipocalin 2 (LCN2); a regulator of transcription; or a plastin, e.g., plastin 2 (PLS2).
In one aspect, the invention features a method of selecting a patient population for treatment of a disorder, e.g., an inflammatory, autoimmune and/or neurologic disorder, by evaluating the expression of one or more genes selected from Table 1 in cells of one or more subjects, and selecting a subject that has altered (e.g., elevated) expression of one or more genes selected from Table 1 relative to a reference. In one embodiment, the step of evaluating includes evaluating nucleic acid (e.g., mRNAor cDNA), or protein levels. In one embodiment, the selected subjects are administered an agent that modulates expression or activity of a gene selected from Table 1. In one embodiment, the selected subjects are administered a treatment for an inflammatory, autoimmune and/or neurologic disorder (e.g., multiple sclerosis).
In some embodiments, the gene encodes: a polypeptide associated with antigen processing and/or presentation, e.g., proteasome 28 subunit alpha (PSMEl); a component of complement; a component of the extracellular matrix; a polypeptide related to hematopoiesis, e.g., CD53 antigen, CD68 antigen, lysosomal-associated protein transmembrane 5 (LAPTM5), lymphocyte antigen 86 (LY86), macrophage expressed gene 1 (MPEGl), or small inducible cytokine A6 (SCYA6); a polypeptide related to lipid metabolism, e.g., lipocalin 2 (LCN2); a regulator of transcription or a plastin, e.g., plastin 2, (PLS2).
In one aspect, the invention features a method of identifying a target gene involved in a disorder by providing a plurality of animal models of the disorder, evaluating gene expression of one or a plurality of genes in the animal models, and evaluating if the one or more genes are similarly regulated, e.g., co-regulated, in each of the plurality of models. Li one embodiment, the plurality of animal models includes animals of different genotypes. In one embodiment, the animal models are mouse models of an autoimmune, inflammatory and/or neurologic disorder. In one embodiment, the disorder is multiple sclerosis. In one embodiment, the mouse model is an experimental autoimmune encephalomyelitis mouse.
In one embodiment, the method includes one or more comparisons, e.g., pair- wise comparisons. The comparisons can be made using frequency values, e.g., log transformed frequency values. The method can include applying a scoring function, e.g., that evaluates one or more of the following: the fold expression change ratio, a value based on a statistical test (e.g., the P value based on the Student's T test), the number of present cells, and the expression level for each comparison. A set of similarly regulated genes can be identified by selecting genes that have a value determined by the scoring function within a particular range. In another aspect, the invention features an array. The array includes a substrate having a plurality of addresses. Each address of the plurality includes a capture probe, e.g., a unique capture probe. In one embodiment, at least one address of the plurality includes a capture probe that hybridizes specifically to a gene selected from the Table 1. In one embodiment, the plurality of addresses includes addresses having nucleic acid capture probes for all the genes of Table 1 (i.e., 100% of the genes) or a fraction of the genes of Table 1, e.g., at least 20%, 40%, 50%, 60%, 80%, or 90% of the genes of Table 1. Preferably, the array has no more than 4 000, 3 000, 2 000, 1 000, 500, or 250 addresses. In another embodiment, at least one address of the plurality includes a capture probe that binds specifically to a polypeptide chosen from the polypeptides encoded by the genes of Table 1. Preferably, the capture probe, is an antibody or derivative thereof, m one embodiment, the plurality of addresses includes addresses having polypeptide capture probes for all the genes of Table 1 (i.e., 100% of the genes) or a fraction of the genes of Table 1, e.g., at least 20%, 40%, 50%, 60%, 80%, or 90% of the genes of Table 1. Preferably, the array has no more than 4 000, 3 000, 2 000, 1 000, 500, or 250 addresses.
Typically, an address has a single species of capture probe, e.g., each address recognizes a single species (e.g., a nucleic acid or polypeptide species). The addresses can be disposed on the substrate in a two-dimensional or three-dimensional configuration.
The array can be used in a method described herein, e.g., to evaluate a sample from a subject or a sample of cells. Information from evaluating the array can be used to determine risk, therapeutic efficacy, and/or diagnosis for a disorder, e.g., multiple sclerosis.
In another aspect, the invention features a method of evaluating a subject. The method includes providing a sample from the subject and determining a sample expression profile, wherein the profile includes one or more values representing the level of expression of one or more genes selected from Table 1. In one embodiment, the profile includes multiple values for the level of expression of genes from Table 1 , e.g., all the genes of Table 1 (i.e., 100% of the genes) or a fraction of the genes of Table 1, e.g., at least 20%, 40%, 50%, 60%, 80%, or 90% of the genes of Table 1. The method can further include comparing the value or the profile (e.g., a representation that can include one or more values, typically multiple values) to a reference value or a reference profile.
An alteration in the expression of one or more genes of the profile is an indication that the subject has or is disposed to having a disorder, e.g., an autoimmune disorder (e.g., multiple sclerosis). Typically, expression of a plurality of genes of the profile (e.g., at least about 5%, 10%, 15%, 20%, 40%, 50%, 60%, 70%, 80%, or 90%) is altered.
The method can be used to a) diagnose a disorder, e.g., an autoimmune disorder in a subject; and b) monitor a treatment for a disorder, e.g., an autoimmune disorder (e.g., multiple sclerosis) in a subject.
The subject expression profile can be determined in a subject during treatment. The subject expression profile can be compared to a reference profile or to a profile obtained from the subject, prior to treatment, or prior to onset, of the disorder. In a preferred embodiment, the subject expression profile is determined at intervals (e.g., regular intervals) during treatment.
In another aspect, the invention features a method of making a decision, e.g., whether to treat, or pay for the treatment of, an inflammatory, autoimmune and/or neurologic disorder (e.g., multiple sclerosis), which includes the following steps: providing a value which is a function of the expression of at least one gene selected from Table 1, e.g., at least one gene described herein; if the value meets a predetermined criterion, e.g., if it has a predetermined relationship with a reference value, assigning a subject to a first class, thereby making a decision. In a preferred embodiment the decision is recorded, e.g., in a computer readable medium.
In a preferred embodiment the method further includes selecting a therapy based on said assignment.
In a preferred embodiment the method further includes administering a therapy based on said assignment.
In a preferred embodiment the method further includes issuing, transmitting and/or receiving a prescription based on said assignment. In a preferred embodiment the method further includes issuing, transmitting and/or receiving a prescription based on said assignment.
In a preferred embodiment the method further includes paying for or causing a transfer of funds to pay for a therapy based on said assignment. In another aspect, the invention features a method of making a decision, e.g., selecting a payment class, for a course of treatment with a drug for a subject having an inflammatory, autoimmune and/or neurologic disorder (e.g., multiple sclerosis). The method includes providing (e.g., receiving) an evaluation of whether the patient is an enhanced responder (e.g., has increased expression level of at least one gene selected from Table 1, e.g., at least one gene described herein) or a non-enhanced responder (e.g., has stable or decreased expression level of at least one gene selected from Table 1, e.g., at least one gene described herein); and performing at least one of (1) if the subject is an enhanced responder selecting a first outcome, e.g., selecting a first payment class, and (2) if the subject is a non-enhanced responder selecting a second outcome, e.g., selecting a second payment class.
In some embodiments, assignment of the patient is to the first class and the assignment authorizes payment for a first treatment course.
In some embodiments, assignment of the patient is to the second class and the assignment authorizes payment for a course of treatment for a second treatment course.
In another aspect, the invention features a method of providing information on which to make a decision about a subject, or making such a decision. The method includes providing (e.g., by receiving) an evaluation of a subject, wherein the evaluation was made by a method described herein, e.g., by determining the level of expression of at least one gene selected from Table 1, e.g., at least one gene described herein, in the subject, thereby providing a value; providing a comparison of the value with a reference value, thereby, providing information on which to make a decision about a subject, or making such a decision.
In some embodiments, the method includes making the decision. In some embodiments, the method also includes communicating the information to another party (e.g., by computer, compact disc, telephone, facsimile, email, or letter). In some embodiments, the decision includes selecting a subject for payment, making or authorizing payment for a first course of action if the subject has an increased level or expression and a second course of action if the subject has .
In some embodiments, the decision includes selecting a first course of action if the subject is an enhanced responder and a second course of action if the subject in a non-enhanced responder.
In some embodiments, the is assigning the subject to a first class. In some embodiments, assignment to the first class will enable payment for a treatment provided to the subject. In some embodiments, payment is by a first party to a second party. In some embodiments, the first party is other than the patient. In some embodiments, the first party is selected from a third party payor, an insurance company, employer, employer sponsored health plan, HMO, or governmental entity. In some embodiments, the second party is selected from the subject, a healthcare provider, a treating physician, an HMO, a hospital, a governmental entity, or an entity which sells or supplies the drug. In some embodiments, the first party is an insurance company and the second party is selected from the subject, a healthcare provider, a treating physician, an HMO, a hospital, a governmental entity, or an entity which sells or supplies the drug. In some embodiments, the first party is a governmental entity and the second party is selected from the subject, a healthcare provider, a treating physician, an HMO, a hospital, an insurance company, or an entity which sells or supplies the drug.
In some embodiments, the subject is a human, e.g., a human diagnosed with an inflammatory, autoimmune and/or neurologic disorder (e.g., multiple sclerosis).
In another aspect, the invention features a method of making a data record. The method includes entering the result of a method described herein into a record, e.g., a computer readable record. In some embodiments, the record is available on the world wide web. In some embodiments, the record is evaluated by a third party payor, an insurance company, employer, employer sponsored health plan, HMO, or governmental entity, or a healthcare provider, a treating physician, an HMO, a hospital, a governmental entity, or an entity which sells or supplies the drug, or is otherwise relied on in a method described herein.
In another aspect, the invention features a data record (e.g., computer readable record), wherein the record includes results from a method described herein, hi some embodiments, the record is available on the world wide web. Li some embodiments, the record is evaluated and/or transmitted to a third party payor, an insurance company, employer, employer sponsored health plan, HMO, or governmental entity, or a healthcare provider, a treating physician, an HMO, a hospital, a governmental entity, or an entity which sells or supplies the drug.
In another aspect, the invention features a method of providing data. The method includes providing data described herein, e.g., generated by a method described herein, to provide a record, e.g., a record described herein, for determining if a payment will be provided, hi some embodiments, the data is provided by computer, compact disc, telephone, facsimile, email, or letter. In some embodiments, the data is provided by a first party to a second party, hi some embodiments, the first party is selected from the subject, a healthcare provider, a treating physician, an HMO, a hospital, a governmental entity, or an entity which sells or supplies the drug, hi some embodiments, the second party is a third party payor, an insurance company, employer, employer sponsored health plan, HMO, or governmental entity. In some embodiments, the first party is selected from the subject, a healthcare provider, a treating physician, an HMO, a hospital, an insurance company, or an entity which sells or supplies the drug and the second party is a governmental entity. In some embodiments, the first party is selected from the subject, a healthcare provider, a treating physician, an HMO, a hospital, an insurance company, or an entity which sells or supplies the drug and the second party is an insurance company.
In another aspect, the invention features a record (e.g., computer readable record) which includes a list and value of expression for at least one gene selected from Table 1, e.g., at least one gene described herein . hi some embodiments, the record includes more than one value for each gene.
In another aspect, the disclosure features a method of transmitting a record described herein. The method includes a first party transmitting the record to a second party, e.g., by computer, compact disc, telephone, facsimile, email, or letter, hi some embodiments, the second party is selected from the subject, a healthcare provider, a treating physician, an HMO, a hospital, a governmental entity, or an entity which sells or supplies the drug, hi some embodiments, the first party is an insurance company or government entity and the second party is selected from the subject, a healthcare provider, a treating physician, an HMO, a hospital, a governmental entity, or an entity which sells or supplies the drug, hi some embodiments, the first party is a governmental entity or insurance company and the second party is selected from the subject, a healthcare provider, a treating physician, an HMO3 a hospital, an insurance company, or an entity which sells or supplies the drug. In another aspect, the disclosure features a method of providing data. The method includes providing hybridization data from contacting an array including a plurality of spatially distinguishable regions described herein with a nucleic acid sample derived from a subject (e.g., a subject described herein), and providing a record of such data. In some embodiments, the subject has an inflammatory, autoimmune and/or neurologic disorder (e.g., multiple sclerosis). hi one embodiment, information about the expression of at least one gene selected from Table 1, e.g., at least one gene described herein (e.g., a gene encoding e.g., a polypeptide associated with antigen processing and/or presentation, e.g., proteasome 28 subunit alpha (PSMEl); a component of complement; a component of the extracellular matrix; a polypeptide related to hematopoiesis, e.g., CD53 antigen, CD68 antigen, lysosomal-associated protein transmembrane 5 (LAPTM5), lymphocyte antigen 86 (LY86), macrophage expressed gene 1 (MPEGl), or small inducible cytokine A6 (SCYA6); a polypeptide related to lipid metabolism, e.g., lipocalin 2 (LCN2); a regulator of transcription; or a plastin, e.g., plastin 2 (PLS2)), is analyzed by an entity that determines whether to authorize or transfer of funds to pay for a service or treatment provided to a subject (or make another decision referred to herein). For example, an expression that is not associated with an inflammatory, autoimmune and/or neurologic disorder (e.g., multiple sclerosis), can trigger an outcome that indicates or causes a refusal to pay for a service or treatment provided to a subject. For example, an entity, e.g., a hospital, care giver, government entity, or an insurance company or other entity which pays for, or reimburses medical expenses, can use the outcome of a method described herein to determine whether a party, e.g., a party other than the subject patient, will pay for services or treatment provided to the patient. For example, a first entity, e.g., an insurance company, can use the outcome of a method described herein to determine whether to provide financial payment to, or on behalf of, a patient, e.g., whether to reimburse a third party, e.g., a vendor of goods or services, a hospital, physician, or other care-giver, for a service or treatment provided to a patient. For example, a first entity, e.g., an insurance company, can use the outcome of a method described herein to determine whether to continue, discontinue, enroll an individual in an insurance plan or program, e.g., a health insurance or life insurance plan or program. In another aspect, the invention features a method of providing (e.g., communicating, e.g., electronically communicating), information about the subject's level of expression of at least one gene selected from Table l,e.g., at least one gene described herein, e.g., the result of evaluating expression of a at least one gene selected from Table 1, to a third party, e.g., a hospital, clinic, a government entity, reimbursing party or insurance company (e.g., a life insurance company). For example, choice of medical procedure, payment for a medical procedure, payment by a reimbursing party, or cost for a service or insurance can be function of the information. E.g., the third party receives the information, makes a determination based at least in part on the information, and optionally communicates the information or makes a choice of procedure, payment, level of payment, coverage etc.
In one embodiment, a premium for insurance (e.g., life or medical) is evaluated as a function of information about expression of one or more genes selected from Table 1, e.g., a gene encoding e.g., a polypeptide associated with antigen processing and/or presentation, e.g., proteasome 28 subunit alpha (PSMEl); a component of complement; a component of the extracellular matrix; a polypeptide related to hematopoiesis, e.g., CD53 antigen, CD68 antigen, lysosomal-associated protein transmembrane 5 (LAPTM5), lymphocyte antigen 86 (LY86), macrophage expressed gene 1 (MPEGl), or small inducible cytokine A6 (SCY A6); a polypeptide related to lipid metabolism, e.g., lipocalin 2 (LCN2); a regulator of transcription; or a plastin, e.g., plastin 2 (PLS2). For example, premiums can be increased (e.g., by a certain percentage) if expression of at least one gene selected from Table 1 is present in the candidate insured, or decreased if no gene selected from Table 1 is expressed. Premiums can also be scaled depending on the level of expression of at least one gene from Table 1, or on the number of expressed genes of Table 1. For example, premiums can be assessed to distribute risk, e.g., commensurate with distribution of genes selected from Table 1. In another embodiment, premiums are assessed as a function of actuarial data that is obtained from individuals with one or more polymorphisms of at least one gene selected from Table 1. In one embodiment, information about the expression of at least one gene selected from Table 1, can be used, e.g., in an underwriting process for life insurance. The information can be incorporated into a profile about a subject. Other information in the profile can include, for example, date of birth, gender, marital status, banking information, credit information, children and so forth. An insurance policy can be recommended as a function of the information about expression of at least one gene selected from Table 1 along with one or more other items of information in the profile. An insurance premium or risk assessment can also be evaluated as function of the information about the expression of at least one gene selected from Table 1. In one implementation, points are assigned for presence or absence of expression of at least one gene selected from Table 1.
In another aspect, the invention features a method of providing (e.g., communicating, e.g., electronically communicating), information about the subject's gene expression levels, e.g., the result of evaluating at least one gene selected from Table 1, e.g., at least one gene described herein, to a third party, e.g., a hospital, clinic, a government entity, reimbursing party or insurance company (e.g., a life insurance company). For example, choice of medical procedure, payment for a medical procedure, payment by a reimbursing party, or cost for a service or insurance can be function of the information. E.g., the third party receives the information, makes a determination based at least in part on the information, and optionally communicates the information or makes a choice of procedure, payment, level of payment, coverage, etc. based on the information.
In one embodiment, a premium for insurance (e.g., life or medical) is evaluated as a function of information about one or more gene expression levels, e.g., expression levels of at least one gene selected from Table 1 (e.g., a level of expression associated with a gene encoding, e.g., a polypeptide associated with antigen processing and/or presentation, e.g., proteasome 28 subunit alpha (PSMEl); a component of complement; a component of the extracellular matrix; a polypeptide related to hematopoiesis, e.g., CD53 antigen, CD68 antigen, lysosomal-associated protein transmembrane 5 (LAPTM5), lymphocyte antigen 86 (LY86), macrophage expressed gene 1 (MPEGl), or small inducible cytokine A6 (SC YA6); a polypeptide related to lipid metabolism, e.g., lipocalin 2 (LCN2); a regulator of transcription; or a plastin, e.g., plastin 2 (PLS2)). For example, premiums can be increased (e.g., by a certain percentage) if at least one gene selected from Table 1 is differentially expressed between an insured candidate (or a candidate seeking insurance coverage) and a reference value (e.g., a healthy or average person). As another example, premiums can be decreased if levels of at least one gene selected from Table 1 are sustained (as described herein) after treatment for an inflammatory, autoimmune and/or neurologic disorder, e.g., multiple sclerosis. Premiums can also be scaled depending on gene expression levels, e.g., the result of evaluating at least one gene of Table 1. For example, premiums can be assessed to distribute risk, e.g., as a function of gene expression levels, e.g., the result of evaluating at least one gene of Table 1. hi another example, premiums are assessed as a function of actuarial data that is obtained from subjects that are enhanced or non-enhanced responders. hi another embodiment, information about gene expression levels, e.g., the result of evaluating at least one gene selected from Table 1, can be used, e.g., in an underwriting process for life insurance. The information can be incorporated into a profile about a subject. Other information in the profile can include, for example, date of birth, gender, marital status, banking information, credit information, children, and so forth. An insurance policy can be recommended as a function of the information on gene expression levels, e.g., the result of evaluating at least one gene selected from Table 1, along with one or more other items of information in the profile. An insurance premium or risk assessment can also be evaluated as function of information about the expression of at least one gene selected from Table 1. In one implementation, points are assigned on the basis of having a high expression of at least one gene selected from Table 1.
In one embodiment, information about gene expression levels, e.g., the result of evaluating at least one gene selected from Table 1 , is analyzed by a function that determines whether to authorize the transfer of funds to pay for a service or treatment provided to a subject (or make another decision referred to herein). For example, the results of analyzing a expression of at least one gene selected from Table 1 may indicate that a subject has an inflammatory, autoimmune and/or neurologic disorder, e.g., multiple sclerosis, suggesting that a first treatment course is needed, thereby triggering an outcome that indicates or causes authorization to pay for a service or treatment provided to a subject. For example, an entity, e.g., a hospital, care giver, government entity, or an insurance company or other entity which pays for, or reimburses medical expenses, can use the outcome of a method described herein to determine whether a party, e.g., a party other than the subject patient, will pay for services (e.g., a particular therapy) or treatment provided to the patient. For example, a first entity, e.g., an insurance company, can use the outcome of a method described herein to determine whether to provide financial payment to, or on behalf of, a patient, e.g., whether to reimburse a third party, e.g., a vendor of goods or services, a hospital, physician, or other care-giver, for a service or treatment provided to a patient. For example, a first entity, e.g., an insurance company, can use the outcome of a method described herein to determine whether to continue, discontinue, enroll an individual in an insurance plan or program, e.g., a health insurance or life insurance plan or program.
As used herein, the articles "a" and "an" refer to one or to more than one (e.g., to at least one) of the grammatical object of the article.
The term "or" is used herein to mean, and is used interchangeably with, the term "and/or", unless context clearly indicates otherwise.
The term "proteins" and "polypeptides" are used interchangeably herein.
Other features, objects and advantages of the invention will be apparent from the description and drawings, and from the claims.
AU references, patent applications, and patents cited herein are hereby incorporated by reference in their entireties.
DESCRIPTION OF DRAWINGS
FIG 1 is a line graph depicting the mean clinical score of mice at various time intervals following scores for experimental autoimmune encephalomyelitis (EAE) induction with MOG35-55 peptide. Mice lacking interferon γ are presented by the filled squares ("IFNγ -/-") (■); mice lacking p35 are represented by the filled triangles ("p35 -/-") (A); and wild-type mice are represented by the filled diamonds (♦)•
FIGS. 2A-2B are Venn diagrams depicting the classification of disease regulated genes in CNS tissue from, p35 -/-, IFNγ -/- and wild type animals based on microarray analysis. FIGS. 3A-3F are bar graphs depicting microarray results for six genes significantly regulated in all three genetic backgrounds tested. The first set of graphs in each figure depicts data for wild type mice. The second set of graphs in each figure depicts data for IFNγ -/- mice. The last set of graphs in each figure depicts data for IL-12p35 -/-mice. FIG 3A depicts data for TYRO protein tyrosine kinase binding protein. FIG 3B depicts data for SlOO calcium binding protein A8 (calgranulin A). FIG 3C depicts data for lipocalin 2. FIG 3D depicts data for CD53 antigen. FIG 3E depicts data for complement component 1, q subcomponent, beta polypeptide. FIG 3F depicts data for SlOO calcium binding protein All (calgizzarin).
FIGS. 4A-F are bar graphs depicting data from verification of microarray results for six genes using TAQMAN® quantitative RT-PCR. The first set of graphs in each figure depicts data for wild type mice. The second set of graphs in each figure depicts data for IFNγ -/- mice. The last set of graphs in each figure depicts data for IL- 12p35 -/- mice. FIG 4A depicts data for TYRO protein tyrosine kinase binding protein. FIG 4B depicts data for SlOO calcium binding protein A8 (calgranulin A). FIG 4C depicts data for lipocalin 2. FIG 4D depicts data for CD53 antigen. FIG 4E depicts data for complement component 1, q subcomponent, beta polypeptide. FIG 4F depicts data for SlOO calcium binding protein All (calgizzarin).
FIG 5 depicts Table 1, which lists exemplary genes with descriptions (encoded polypeptides), the expression of which is significantly regulated in spinal cord in EAE mice.
FIG 6 depicts Table 2, which lists exemplary genes with descriptions
(encoded polypeptides), the expression of which is commonly regulated in the central nervous system and spleens of EAE mice.
DETAILED DESCRIPTION
Modulators of expression and/or activity of the genes and associated polypeptides described herein can be used to treat autoimmune, inflammatory and/or neurologic disorders, e.g., multiple sclerosis (MS). The genes and proteins disclosed herein can also be useful screening assays for compounds that can be used to treat such disorders, e.g., MS. The genes and proteins disclosed herein can also be used in methods of diagnosing such disorders, e.g., MS diagnosis or a predisposition for MS.
In one embodiment, Applicants have shown that a plurality of genes are co- regulated in three mouse models of multiple sclerosis, each with a different genetic background. Examples of these genes are disclosed in Table 1 and the appended
Example. Accordingly, the genes disclosed herein, and the proteins they encode, can be used in screening, treatment and diagnosis methods for MS.
Screening Methods
The genes and proteins disclosed herein can be used as targets in screens for compounds that can be useful for treatment or diagnosis of MS. For example, modulators, such as inhibitors, can be useful for reducing expression and/or activity of gene products that are up-regulated in multiple sclerosis. Such inhibitors can be used to treat MS, or ameliorate one or more symptoms condition associated with MS. The screens can be used to identify both direct and indirect interactions. For example, as described herein, modulators can directly bind to a target protein or inhibit an activity of a target protein. Other modulators can alter expression, stability, and/or localization of the target protein in a cell.
Numerous methods are available for evaluating an interaction between a test compound and a target protein described herein or fragment thereof, e.g., a functional fragment thereof. Protein fragments can be produced, e.g., using recombinant or biochemical means (e.g., from a nucleic acid sequence that codes for the target protein, or through proteolysis of the full-length target protein, e.g., using proteolytic enzymes). Exemplary proteolytic enzymes are enzymes that cleave peptides at specific sequences, e.g., trypsin, chymotrypsin, thermolysin, and clostripain. A functional protein fragment is a fragment that retains at least part of an activity of the full-length protein, e.g., a binding or enzymatic activity.
The methods can be designed to identify a compound that binds to and/or modulates an activity of a target protein described herein. For example, it is frequently useful to inhibit the activity or availability of a target protein that is up- regulated in a mouse model of MS, relative to a normal counterpart, or to increase the activity or availability of a protein that is down-regulated in a mouse model of MS, relative to a normal counterpart. A variety of methods can be used to evaluate activity of a target protein described herein in the presence of a test compound, e.g., a candidate therapeutic or a lead for a candidate therapeutic. These methods include in vitro assays, cell-based assays, and organismal assays. Cell-based assays can include evaluating an activity by cell-associated proteins or can include evaluating mRNA or protein expression, e.g., directly or indirectly (e.g., using a reporter gene).
These methods can be used to evaluate known modulators of the target proteins described herein or other compounds. A "compound" or "test compound" can be any chemical compound, for example, a macromolecule (e.g., a polypeptide, a protein complex, or a nucleic acid) or a small molecule (e.g., an amino acid, a nucleotide, an organic or inorganic compound). The test compound can have a formula weight of less than about 10 000, 5000, 2000, 1000, or 500 Daltons. A small molecule is generally considered to have a formula weight of less than 2000 Daltons. The test compound can be naturally occurring (e.g., a herb or a natural product), synthetic, or both. Examples of macromolecules are proteins, protein complexes, and glycoproteins, nucleic acids, e.g., DNA, RNA (e.g., double stranded RNA or RNAi), and PNA (peptide nucleic acid). Examples of small molecules are peptides, peptidomimetics (e.g., peptoids), amino acids, amino acid analogs, polynucleotides, polynucleotide analogs, nucleotides, nucleotide analogs, organic or inorganic compounds, e.g., heteroorganic or organometallic compounds. One exemplary type of protein compound is an antibody or a modified scaffold domain protein. A test compound can be the only substance assayed by the method described herein. Alternatively, a collection of test compounds can be assayed either consecutively or concurrently by the methods described herein. In one embodiment, high throughput screening methods involve providing a combinatorial chemical or biopolymer library containing a large number of potential therapeutic compounds (e.g., potential modulators). Such "combinatorial chemical libraries" are screened in one or more assays, as described herein, to identify those library members (particular chemical species or subclasses) that have a desired characteristic activity. The compounds thus identified can serve as conventional "lead compounds" or can themselves be used as potential or actual therapeutics.
A combinatorial chemical library is a collection of diverse chemical compounds generated by either chemical synthesis or biological synthesis, by combining a number of chemical "building blocks" such as reagents. For example, a linear combinatorial chemical library such as a polypeptide library is formed by combining a set of chemical building blocks (amino acids) in every possible way for a given compound length (i.e., the number of amino acids in a polypeptide compound). Millions of chemical compounds can be synthesized through such combinatorial mixing of chemical building blocks.
Preparation and screening of combinatorial chemical libraries is well known to those of skill in the art. Such combinatorial chemical libraries include, but are not limited to, peptide libraries {see, e.g., U.S. Patent 5,010,175; Furka, Int. J. Pept. Prot. Res. 37:487-493 (1991);and Houghton et al, Nature 354:84-88 (1991)). Other chemistries for generating chemical diversity libraries can also be used. Such chemistries include, but are not limited to: peptoids (e.g., PCT Publication No. WO 91/19735), encoded peptides (e.g., PCT Publication No. WO 93/20242), random bio- oligomers (e.g., PCT Publication No. WO 92/00091), benzodiazepines (e.g., U.S. Pat. No. 5,288,514), diversomers such as hydantoins, benzodiazepines and dipeptides (Hobbs et al, Proc. Nat. Acad. Sd. USA 90:6909-6913 (1993)), vinylogous polypeptides (Hagihara et al, J. Amer. Chem. Soc. 114:6568 (1992)), nonpeptidyl peptidomimetics with glucose scaffolding (Hirschmann et al, J. Amer. Chem. Soc. 114:9217-9218 (1992)), analogous organic syntheses of small compound libraries (Chen et al, J. Amer. Chem. Soc. 116:2661 (1994)), oligocarbamates (Cho et al, Science 261:1303 (1993)), and/or peptidyl phosphonates (Campbell et al, J. Org. Chem. 59:658 (1994)), nucleic acid libraries {see Ausubel, Berger and Sambrook, all supra), peptide nucleic acid libraries (see, e.g., U.S. Patent 5,539,083), antibody libraries {see, e.g., Vaughn et al, Nature Biotechnology, 14:309-314 (1996) and PCT/US96/10287), carbohydrate libraries (see, e.g. , Liang et al , Science, 21 A: 1520- 1522 (1996) and U.S. Patent 5,593,853), small organic molecule libraries (see, e.g., benzodiazepines, Baum C&EN, Jan 18, page 33 (1993); isoprenoids, U.S. Patent No. 5,569,588; thiazolidinones and metathiazanones, U.S. Patent No. 5,549,974; pyrrolidines, U.S. Patents 5,525,735 and 5,519,134; morpholino compounds, U.S. Patent 5,506,337; benzodiazepines, 5,288,514, and the like). Additional examples of methods for the synthesis of molecular libraries can be found in the art, for example in: DeWitt et al (1993) Proc. Natl. Acad. Sd. U.S.A. 90:6909; Erb et al (1994) Proc. Natl Acad. ScL USA 91:11422; Zuckermann et al (1994) J. Med. Chem. 37:2678; Cho et al. (1993) Science 261:1303; Carrell et al. (1994) Angew. Chem. Int. Ed. Engl. 33:2059; Carell et al. (1994) Angew. Chem. Int. Ed. Engl. 33:2061; and Gallop et al. (1994) J. Med. Chem. 37:1233.
Some exemplary libraries are used to generate variants from a particular lead compound. One method includes generating a combinatorial library in which one or more functional groups of the lead compound are varied, e.g., by derivatization. Thus, the combinatorial library can include a class of compounds which have a common structural feature (e.g., framework).
Devices for the preparation of combinatorial libraries are commercially available (see, e.g., 357 MPS, 390 MPS, Advanced ChemTech, Louisville KY;
SYMPHONY™, Rainin, Woburn, MA; 433A Applied Biosystems, Foster City, CA; 9050 Plus, Millipore, Bedford, MA). In addition, numerous combinatorial libraries are themselves commercially available (see, e.g., ComGenex, Princeton, NJ.; Asinex, Moscow, RU; Tripos, Inc., St. Louis, MO; ChemStar, Ltd, Moscow, RU; 3D Pharmaceuticals, Exton, PA; Martek Biosciences, Columbia, MD; etc.).
Test compounds can also be obtained from: biological libraries; peptoid libraries (libraries of molecules having the functionalities of peptides, but with a novel, non-peptide backbone which are resistant to enzymatic degradation but which nevertheless remain bioactive; see, e.g., Zuckermann et al. (1994) J. Med. Chem. 37:2678-85); spatially addressable parallel solid phase or solution phase libraries; synthetic library methods requiring deconvolution; the "one-bead one-compound" library method; and synthetic library methods using affinity chromatography selection. The biological libraries include libraries of nucleic acids and libraries of proteins. Some nucleic acid libraries encode a diverse set of proteins (e.g., natural and artificial proteins; others provide, for example, functional RNA and DNA molecules such as nucleic acid aptamers or ribozymes. A peptoid library can be made to include structures similar to a peptide library. (See also Lam (1997) Anticancer DrugDes. 12:145). Alibrary of proteins maybe produced by an expression library or a display library (e.g., a phage display library). Libraries of compounds may be presented in solution (e.g., Houghten (1992)
Biotechniques 13:412-421), or on beads (Lam (1991) Nature 354:82-84), chips (Fodor (1993) Nature 364:555-556), bacteria (Ladner, U.S. Patent No. 5,223,409), spores (Ladner U.S. Patent No. 5,223,409), plasmids (Cull et al. (1992) Proc. Natl. Acad. Sci. USA 89:1865-1869) or on phage (Scott and Smith (1990) Science 249:386-390; Devlin (1990) Science 249:404-406; Cwirla et al. (1990) Proc. Natl. Acad. Sci. USA 87:6378-6382; Felici (1991) J. MoI. Biol. 222:301-310).
Binding Assays Interaction with, e.g., binding to a target protein described herein or fragment thereof, e.g., a functional fragment thereof can be assayed in vitro, e.g., in a cell free system. In one embodiment, the reaction mixture can include a cognate binding partner, e.g., in an in vitro assay, to evaluate the ability of a test compound to modulate interaction between the target protein described herein and a cognate binding partner. This type of assay can be accomplished, for example, by coupling one of the components, with a label (e.g., a radioisotope or enzymatic label) such that binding of the labeled component to the other can be determined by detecting the labeled compound in a complex. A component can be labeled with 1251, 35S, 14C, or 3H, either directly or indirectly, and the radioisotope detected by direct counting of radioemmission or by scintillation counting. Alternatively, a component can be enzymatically labeled with, for example, horseradish peroxidase, alkaline phosphatase, or luciferase, and the enzymatic label detected by determination of conversion of an appropriate substrate to product. The test compound and/or the target protein described herein (or a fragment thereof) itself can be labeled. Competition assays can also be used to evaluate a physical interaction between a test compound and a target.
Cell-free assays involve preparing a reaction mixture of the target protein described herein and the test compound under conditions and for a time sufficient to allow the two components to interact and bind, thus forming a complex that can be removed and/or detected.
The interaction between two molecules can also be detected, e.g., using a fluorescence assay in which at least one molecule is fluorescently labeled. One example of such an assay includes fluorescence energy transfer (FET or FRET for fluorescence resonance energy transfer) (see, for example, U.S. Patent No. 5,631,169 and U.S. Patent No. 4,868,103). A fluorophore label on the first or donor molecule is selected such that its emitted fluorescent energy will be absorbed by a fluorescent label on a second or acceptor molecule, which in turn is able to fluoresce due to the absorbed energy. Alternately, the donor molecule (if a protein) may simply utilize the natural fluorescent energy of tryptophan residues. Labels can be chosen that emit different wavelengths of light, such that the acceptor molecule label may be differentiated from that of the donor. Since the efficiency of energy transfer between the labels is related to the distance separating the molecules, the spatial relationship between the molecules can be assessed. In a situation in which binding occurs between the molecules, the fluorescent emission of the acceptor molecule label in the assay should be maximal. A FET binding event can be conveniently measured through standard fluorometric detection means well known in the art (e.g., using a fluorimeter). Another example of a fluorescence assay is fluorescence polarization (FP).
For FP, only one component needs to be labeled, typically the component that undergoes the larger change in molecular weight on binding. A binding interaction is detected by a change in molecular size of the labeled component. The size change alters the tumbling rate of the component in solution and is detected as a change in FP. See, e.g., Nasir et al. (1999) Comb. Chem. HTS 2: 177-190; Jameson et al. (1995) Methods Enzymol 246:283; Seethala et al. (1998) Anal. Biochem. 255:257. Fluorescence polarization can be monitored in multiwell plates, e.g., using the Tecan POLARION™ reader. See, e.g., Parker et al. (2000) Journal ofBiomolecular Screening 5:77-88; and Shoeman et al. (1999) 38, 16802-16809. In another embodiment, determining the ability of a protein described herein to bind to a target molecule can be accomplished using real-time Biomolecular Interaction Analysis (BIA) (see, e.g., Sjolander and Urbaniczky (1991) Anal. Chem. 63:2338-2345 and Szabo et al. (1995) Curr. Opin. Struct. Biol. 5:699-705). "Surface plasmon resonance" or "BIA" detects biospecific interactions in real time, without labeling any of the interactants (e.g., BIAcore). Changes in the mass at the binding surface (indicative of a binding event) result in alterations of the refractive index of light near the surface (the optical phenomenon of surface plasmon resonance (SPR)), resulting in a detectable signal which can be used as an indication of real-time reactions between biological molecules. In one embodiment, a target protein described herein or fragment thereof, e.g., a functional fragment thereof is anchored onto a solid phase. Protein/test compound complexes anchored on the solid phase can be detected at the end of the reaction, e.g., the binding reaction. For example, a protein fragment can be anchored onto a solid surface, and the test compound, (which is not anchored), can be labeled, either directly or indirectly, with detectable labels discussed herein.
It may be desirable to immobilize either a target protein described herein or fragment thereof, e.g., a functional fragment thereof or its binding partner to facilitate separation of complexed from uncomplexed forms of one or both of the proteins, as well as to accommodate automation of the assay. Binding of a test compound to a target protein described herein, or interaction of such a protein with a second component in the presence and absence of a candidate compound, can be accomplished in any vessel suitable for containing the reactants. Examples of such vessels include microtiter plates, test tubes, and micro-centrifuge tubes. In one embodiment, a fusion protein can be provided which adds a domain that allows one or both of the proteins to be bound to a matrix. For example, glutathione-S-transferase protein fragment fusion proteins can be adsorbed onto glutathione SEPHAROSE® beads (Sigma Chemical, St. Louis, MO) or glutathione derivatized microtiter plates, which are then combined with the test compound or the test compound. The mixture is incubated under conditions conducive to complex formation (e.g., at physiological conditions for salt and pH). Following incubation, the beads or microtiter plate wells are washed to remove any unbound components, the matrix immobilized in the case of beads, complex determined either directly or indirectly, for example, as described above. Alternatively, the complexes can be dissociated from the matrix, and the level of protein binding or activity determined using standard techniques.
Other techniques for immobilizing either a target protein described herein or fragment thereof, e.g., a functional fragment thereof, or a target molecule on matrices include using conjugation of biotin and streptavidin. Biotinylated proteins or target molecules can be prepared from biotin-NHS (N-hydroxy-succinimide) using techniques known in the art (e.g., biotinylation kit, Pierce Chemicals, Rockford, IL), and immobilized in the wells of streptavidin-coated 96 well plates (Pierce Chemical). To conduct the assay, the non-immobilized component is added to the coated surface containing the anchored component. After the reaction is complete, unreacted components are removed (e.g., by washing) under conditions such that any complexes formed will remain immobilized on the solid surface. The detection of complexes anchored on the solid surface can be accomplished in a number of ways. Where the previously non-immobilized component is pre-labeled, the detection of label immobilized on the surface indicates that complexes were formed. Where the previously non-immobilized component is not pre-labeled, an indirect label can be used to detect complexes anchored on the surface, e.g., using a labeled antibody specific for the immobilized component (the antibody, in turn, can be directly labeled or indirectly labeled with, e.g., a labeled anti-Ig antibody).
In one embodiment, this assay is performed utilizing antibodies reactive with a target protein described herein or target molecules but which do not interfere with binding of the target protein to its target molecule or with an activity of the target protein. Such antibodies can be derivatized to the wells of the plate, and unbound target or protein trapped in the wells by antibody conjugation. Methods for detecting such complexes, in addition to those described above for the GST-immobilized complexes, include immunodetection of complexes using antibodies reactive with the target protein, as well as enzyme-linked assays which rely on detecting an enzymatic activity associated with the target protein. Alternatively, cell free assays can be conducted in a liquid phase. In such an assay, the reaction products are separated from unreacted components, by any of a number of standard techniques, including but not limited to: differential centrifugation (see, for example, Rivas and Minton, (1993) Trends Biochem. Sci. 18:284-7); chromatography (gel filtration chromatography, ion-exchange chromatography); electrophoresis (see, e.g., Ausubel et ah, eds. Current Protocols in Molecular Biology 1999, J. Wiley: New York.); and immunoprecipitation (see, for example, Ausubel et ah, eds. (1999) Current Protocols in Molecular Biology, J. Wiley: New York). Such resins and chromatographic techniques are known to one skilled in the art (see, e.g., Heegaard (1998) JMoI Recognit 11:141-8; Hage and Tweed (1997) J Chromatogr B Biomed Sci Appl. 699:499-525). Further, fluorescence energy transfer may also be conveniently utilized, as described herein, to detect binding without further purification of the complex from solution.
In one embodiment, the assay includes contacting a target protein described herein or fragment thereof, e.g., a functional fragment thereof with a cognate binding partner to form an assay mixture, contacting the assay mixture with a test compound, and determining the ability of the test compound to interact with the protein, wherein determining the ability of the test compound to interact with the protein includes determining the ability of the test compound to preferentially bind to the protein or biologically active fragment thereof, or to modulate the activity of a target molecule, as compared to the known compound. The assay can include combining the compounds in a different order, e.g., to determine if the test compound can interfere with the interaction between the protein and its binding partner. Assays can be conducted in a heterogeneous or homogeneous format. A typical heterogeneous assays includes anchoring either the target product or the binding partner onto a solid phase, and detecting complexes anchored on the solid phase at the end of the reaction. In a typical homogeneous assay, the reaction is carried out in a liquid phase, hi either approach, the order of addition of reactants can be varied to obtain different information about the compounds being tested. For example, test compounds that interfere with the interaction between the target products and the binding partners, e.g., by competition, can be identified by conducting the reaction in the presence of the test substance. Alternatively, test compounds that disrupt preformed complexes, e.g., compounds with higher binding constants that displace one of the components from the complex, can be tested by adding the test compound to the reaction mixture after complexes have been formed.
In still another embodiment, a protein described herein or fragment thereof, e.g., a functional fragment thereof can be used as a "bait protein" in a two-hybrid assay or similar assay (see, e.g., U.S. Patent No. 5,283,317; Zervos et al. (1993) Cell 72:223-232; Madura et al. (1993) J. Biol. Chem. 268:12046-12054; Bartel et al.
(1993) Biotechniques 14:920-924; Iwabuchi et al. (1993) Oncogene 8:1693-1696; and Brent WO94/ 10300), to identify other proteins, which bind to or interact with the protein. Such binding partners can be activators or inhibitors of an activity of the protein.
Expression Monitoring
In another embodiment, modulators of expression of genes of Table 1 are identified. For example, a cell is contacted with a candidate compound and mRNA or protein expression of the gene is evaluated, e.g., relative to the level of expression in the absence of the candidate compound. When expression is greater in the presence of the candidate compound than in its absence, the candidate compound is identified as a stimulator of a expression of the gene. Alternatively, when expression is less (e.g., statistically significantly less) in the presence of the candidate compound than in its absence, the candidate compound is identified as an inhibitor of expression of the gene.
Methods for detecting gene expression in a sample include detecting mRNA or cDNA and detecting protein, e.g., using an antibody or other binding protein, or using an activity assay. It is also possible to detect mRNA or cDNA using any of a variety of molecular techniques, including RT-PCR and microarray analysis.
Examples of methods of gene expression analysis known in the art include DNA arrays or microarrays (Brazma and ViIo, FEBS Lett, 2000, 480, 17-24; Celis et al., FEBS Lett., 2000, 480, 2-16), SAGE (serial analysis of gene expression) (Madden et al., Drug Discov. Today, 2000, 5, 415-425), READS (restriction enzyme amplification of digested cDNAs) (Prashar and Weissman, Methods EnzymoL, 1999, 303, 258-72), TOGA (total gene expression analysis) (Sutcliffe et al., Proc. Natl. Acad. Sd. USA, 2000, 97, 1976-81), protein arrays and proteomics (Celis et al., FEBS Lett., 2000, 480, 2-16; Jungblut et al., Electrophoresis, 1999, 20, 2100-10), expressed sequence tag (EST) sequencing (Celis et al., FEBS Lett., 2000, 480, 2-16; Larsson et al., J. Biotechnol, 2000, 80, 143-57), subtractive RNA fingerprinting (SuRF) (Fuchs et &l.,Anal. Biochem., 2000, 286, 91-98; Larson et al., Cytometry, 2000, 41, 203-208), subtractive cloning, differential display (DD) (Jurecic and Belmont, Curr. Opin. Microbiol., 2000, 3, 316-21), comparative genomic hybridization (Carulli et al., J. Cell Biochem. Suppl, 1998, 31 , 286-96), FISH (fluorescent in situ hybridization) techniques (Going and Gusterson, Eur. J. Cancer, 1999, 35, 1895-904) and mass spectrometry methods (reviewed in To, Comb. Chem. High Throughput Screen, 2000, 3, 235-41).
Reporter genes can be used to evaluate changes in expression of the genes described herein. Exemplary regulatory sequences of the genes described herein include those located within 100, 200, 500, 700, or 1600 base pairs of the mRNA start site.
Reporter genes can be made by operably linking a regulatory sequence to a sequence encoding a reporter gene. A number of methods are available for designing reporter genes. For example, the sequence encoding the reporter protein can be linked in frame to all or part of the sequence that is normally regulated by the regulatory sequence. Such constructs can be referred to as translational fusions. It is also possible to link the sequence encoding the reporter protein to only regulatory sequences, e.g., the 5' untranslated region, TATA box, and/or sequences upstream of the rnRNA start site. Such constructs can be referred to as transcriptional fusions. Still other reporter genes can be constructed by inserting one or more copies (e.g., a multimer of three, four, or six copies) of a regulatory sequence into a neutral or characterized promoter.
Reporter genes can be introduced into germline cells of non-human mammals, e.g., to produce transgenic animals, or into stem cells, into pluripotent stem cells or . embryonic stem cells. Reporter genes can also be introduced into culture cells, e.g., tissue culture cells. Typically the cell is a mammalian, e.g., human cell or a cell derived from a human cell.
Exemplary reporter proteins include chloramphenicol acetyltransferase, green fluorescent protein and other fluorescent proteins (e.g., artificial variants of GFP), beta-lactamase, beta-galactosidase, luciferase, and so forth. The reporter protein can be any protein other than the protein encoded by the endogenous gene that is subject to analysis. Epitope tags can also be used.
Exemplary methods can include evaluating nervous tissue of a transgenic mammal for altered expression of a reporter gene (e.g., a GFP or variant protein). The transgenic mammal can be administered a test compound, and, if the compound modulates expression of the reporter gene, the test compound is selected. This method can be used to identify compounds that reduce expression of the genes described herein.
Similarly, compounds can be screened using a cell-based assay, e.g., using cultures of cells that contain a reporter whose expression is operably linked to a regulatory sequence from the genes described herein (e.g., from a promoter, enhancer, untranslated region, upstream or downstream of the coding sequence).
Nucleic Acid Arrays
Arrays are useful molecular tools for characterizing a sample by multiple criteria. For example, an array having a capture probes for one or more genes of Table 1 can be used to assess a subject. Arrays can have many addresses, e.g., locatable sites, on a substrate. The featured arrays can be configured in a variety of formats, non-limiting examples of which are described below.
The substrate can be opaque, translucent, or transparent. The addresses can be distributed, on the substrate in one dimension, e.g., a linear array; in two dimensions, e.g., a planar array; or in three dimensions, e.g., a three dimensional array. The solid substrate may be of any convenient shape or form, e.g., square, rectangular, ovoid, or circular.
Arrays can be fabricated by a variety of methods, e.g., photolithographic methods (see, e.g., U.S. Patent Nos. 5,143,854; 5,510,270; and 5,527,681), mechanical methods (e.g., directed-flow methods as described in U.S. Patent No. 5,384,261), pin based methods (e.g., as described in U.S. Pat. No. 5,288,514), and bead based techniques (e.g., as described in PCT US/93/04145). The capture probe can be a single-stranded nucleic acid, a double-stranded nucleic acid (e.g., which is denatured prior to or during hybridization), or a nucleic acid having a single-stranded region and a double-stranded region. Preferably, the capture probe is single-stranded. The capture probe can be selected by a variety of criteria, and preferably is designed by a computer program with optimization parameters. The capture probe can be selected to hybridize to a sequence rich (e.g., non-homopolymeric) region of the gene. The Tm of the capture probe can be optimized by prudent selection of the complementarity region and length. Ideally, the Tm of all capture probes on the array is similar, e.g., within 20, 10, 5, 3, or 2 0C of one another. A database scan of available sequence information for a species can be used to determine potential cross- hybridization and specificity problems. The isolated nucleic acid is preferably mRNA that can be isolated by routine methods, e.g., including DNase treatment to remove genomic DNA and hybridization to an oligo-dT coupled solid substrate (e.g., as described in Current Protocols in Molecular Biology, John Wiley & Sons, N. Y). The substrate is washed, and the mRNA is eluted. The isolated mRNA can be reversed transcribed and optionally amplified, e.g., by rtPCR, e.g., as described in (U.S. Patent No. 4,683,202). The nucleic acid can be an amplification product, e.g., from PCR (U.S. Patent Nos. 4,683,196 and 4,683,202); rolling circle amplification ("RCA," U.S. Patent No. 5,714,320), isothermal RNA amplification or NASBA (U.S. Patent Nos. 5,130,238; 5,409,818; and 5,554,517), and strand displacement amplification (U.S. Patent No. 5,455,166). The nucleic acid can be labeled during amplification, e.g., by the incorporation of a labeled nucleotide. Examples of preferred labels include fluorescent labels, e.g., red-fluorescent dye Cy5 (Amersham) or green-fluorescent dye Cy3 (Amersham), and chemiluminescent labels, e.g., as described in U.S. Patent No. 4,277,437. Alternatively, the nucleic acid can be labeled with biotin, and detected after hybridization with labeled streptavidin, e.g., streptavidin-phycoerythrin (Molecular Probes).
The labeled nucleic acid can be contacted to the array. In addition, a control nucleic acid or a reference nucleic acid can be contacted to the same array. The control nucleic acid or reference nucleic acid can be labeled with a label other than the sample nucleic acid, e.g., one with a different emission maximum. Labeled nucleic acids can be contacted to an array under hybridization conditions. The array can be washed, and then imaged to detect fluorescence at each address of the array. The expression data can be stored in a database, e.g., a relational database such as a SQL database (e.g., Oracle or Sybase database environments). The database can have multiple tables. For example, raw expression data can be stored in one table, wherein each column corresponds to a gene being assayed, e.g., an address or an array, and each row corresponds to a sample. A separate table can store identifiers and sample information, e.g., the batch number of the array used, date, and other quality control information.
Expression profiles obtained from gene expression analysis on an array can be used to compare samples and/or cells in a variety of states as described in Golub et al. ((1999) Science 286:531). In one embodiment, multiple expression profiles from different conditions and including replicates or like samples from similar conditions are compared to identify genes whose expression level is predictive of the sample and/or condition. Each candidate gene can be given a weighted "voting" factor dependent on the degree of correlation of the gene's expression and the sample identity. A correlation can be measured using a Euclidean distance or the Pearson correlation coefficient.
The similarity of a sample expression profile to a predictor expression profile (e.g., a reference expression profile that has associated weighting factors for each gene) can then be determined, e.g., by comparing the log of the expression level of the sample to the log of the predictor or reference expression value and adjusting the comparison by the weighting factor for all genes of predictive value in the profile. Polypeptide Arrays
The expression level of a polypeptide encoded by a gene of Table 1 can be determined using an antibody specific for the polypeptide (e.g., using a western blot or an ELISA assay). Moreover, the expression levels of multiple polypeptides encoded by these genes can be rapidly determined in parallel using a polypeptide array having antibody capture probes for each of the polypeptides. Antibodies specific for a polypeptide can be generated by a method described herein (see "Immunoglobulins").
A low-density (96 well format) protein array has been developed in which proteins are spotted onto a nitrocellulose membrane (Ge (2000) Nucleic Acids Res. 28, e3, 1-VII). A high-density protein array (100,000 samples within 222 x 222 mm) used for antibody screening was formed by spotting proteins onto polyvinylidene difluoride (PVDF) (Lueking et al. (1999) Anal. Biochem. 270, 103-111). Polypeptides can be printed on a flat glass plate that contained wells formed by an enclosing hydrophobic Teflon mask (Mendoza et al. (1999) Biotechniques 27, 778-788.). Also, polypeptide can be covalently linked to chemically derivatized flat glass slides in a high-density array (1600 spots per square centimeter) (MacBeath and Schreiber (2000) Science 289, 1760-1763). De Wildt et al., describe a high-density array of 18,342 bacterial clones, each expressing a different single-chain antibody, in order to screening antibody-antigen interactions (De Wildt et al. (2000) Nature Biotech. 18, 989-994).
These art-known methods and other can be used to generate an array of antibodies for detecting the abundance of polypeptides in a sample. The sample can be labeled, e.g., biotinylated, for subsequent detection with streptavidin coupled to a fluorescent label. The array can then be scanned to measure binding at each address. The nucleic acid and polypeptide arrays described herein can be used in wide variety of applications. For example, the arrays can be used to analyze a patient sample. The sample is compared to data obtained previously, e.g., known clinical specimens or other patient samples. Further, the arrays can be used to characterize a cell culture sample, e.g., to determine a cellular state after varying a parameter, e.g., exposing the cell culture to an antigen, a transgene, or a test compound. Animal models
An animal model is any non-human animal that exhibits characteristic of a human disorder. Many such animals have been established by scientific methods as useful systems for testing potential therapeutics that can used for treating the human disorder. Frequently, the animal model has one or more symptoms of the human disorder and/or molecular indicators characteristic of the human disorder.
The Example below provides an exemplary animal model for MS, the EAE mouse. Other animal models are available, e.g., Theiler's murine encephalomyelitis virus-induced demyelinating disease (TMEV-IDD), tumor necrosis factor (TNF) transgenic mice TgK21 and Tg6074, and demyelination by mouse hepatitis virus (MHV).
Immunoglobulins
Immunoglobulin molecules can be used to modulate an activity of a protein encoded by a gene of Table 1. For example, one class of immunoglobulin molecules includes molecules that bind to a protein described herein and reduce an activity of the protein. Another class of immunoglobulin molecules includes molecules that bind to the proteins or interaction partners and reduce or prevent binding. Still other immunoglobulin molecules can be used to increase activity or stabilize a protein.
A typical immunoglobulin is an antibody. As used herein, the term "antibody" refers to a protein comprising at least one, and preferably two, heavy (H) chain variable domains (abbreviated herein as VH), and at least one and preferably two light (L) chain variable domains (abbreviated herein as VL). The VH and VL domains can be further subdivided into regions of hypervariability, termed "complementarity determining regions" ("CDR"), interspersed with regions that are more conserved, termed "framework regions" (FR). The extent of the framework region and CDRs has been precisely defined (see, Kabat et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242, and Chothia et al. (1987) J. MoI. Biol. 196:901-917, which are incorporated herein by reference). Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy- terminus in the following order: FRl5 CDRl, FR2, CDR2, FR3, CDR3, FR4. Camelid antibodies can include a single variable immunoglobulin domain. The antibody can further include a heavy and light chain constant region, to thereby form a heavy and light immunoglobulin chain, respectively, hi one embodiment, the antibody is a tetramer of two heavy immunoglobulin chains and two light immunoglobulin chains, wherein the heavy and light immunoglobulin chains are inter-connected by, e.g., disulfide bonds. The heavy chain constant region is comprised of three domains, CHl, CH2 and CH3. The light chain constant region is comprised of one domain, CL. The variable domain of the heavy and light chains contains a binding domain that interacts with an antigen. The constant regions of the antibodies typically mediate the binding of the antibody to host tissues or factors, including various cells of the immune system (e.g., agonist cells) and the first component (CIq) of the classical complement system.
As used herein, the term "immunoglobulin" refers to a protein that includes one or more polypeptides that have a domain that forms an immunoglobulin fold. The term "immunoglobulin" includes an antigen-binding fragment. An immunoglobulin can include a region encoded by an immunoglobulin gene. The recognized human immunoglobulin genes include the kappa, lambda, alpha (IgAl and IgA2), gamma (IgGl, IgG2, IgG3, IgG4), delta, epsilon and mu constant region genes, as well as the myriad immunoglobulin genes and gene segments. Full-length immunoglobulin "light chains" (about 25 kDa or 214 amino acids) are encoded by a variable region gene at the NH2-terminus (about 110 amino acids) and a kappa or lambda constant region gene at the COOH—terminus. Full- length immunoglobulin "heavy chains" (about 50 kDa or 446 amino acids) are similarly encoded by a variable region gene (about 116 amino acids) and one of the other aforementioned constant region genes, e.g., gamma (encoding about 330 amino acids). As used herein, "isotype" refers to the antibody class (e.g., IgM, IgGl, IgG2, IgG3, IgG4) that is encoded by heavy chain constant region genes.
The term "antigen-binding fragment" of an antibody (or simply "antibody portion," or "fragment"), as used herein, refers to one or more fragments of a full- length antibody that retain the ability to specifically bind to an antigen. Examples of binding fragments encompassed within the term "antigen-binding fragment" of an antibody include (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CHl domains; (ii) a F(ab')2 fragment, a bivalent fragment comprising two
Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VH and CHl domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (v) a dAb fragment (Ward et al, (1989) Nature 341:544-546), which consists of a VH domain; and (vi) an isolated complementarity determining region (CDR). Furthermore, although the two domains of the Fv fragment, VL and VH, are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VL and VH domains pair to form monovalent molecules (known as single chain Fv (scFv); see, e.g., Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. ScL USA 85:5879-5883). Such single chain antibodies are also intended to be encompassed within the term "antigen-binding fragment" of an antibody. These antibody fragments are obtained using conventional techniques known to those with skill in the art, and the fragments are screened for utility in the same manner as are intact antibodies. An "effectively human" immunoglobulin variable domain is an immunoglobulin variable domain that includes a sufficient number of human framework amino acid positions such that the immunoglobulin variable domain does not elicit an immunogenic response in a normal human. An "effectively human" antibody is an antibody that includes a sufficient number of human amino acid positions such that the antibody does not elicit an immunogenic response in a normal human. Human and effectively human immunoglobulin variable domains and antibodies can be used.
Antibodies can be made by immunizing an animal (e.g., non-human animals and non-human animals include human immunoglobulin genes) with the relevant antigen. Such antibodies may be obtained using the entire mature protein as an immunogen, or by using fragments (e.g., soluble fragments and small peptides). The peptide immunogens additionally may contain a cysteine residue at the carboxyl terminus, and are conjugated to a hapten such as keyhole limpet hemocyanin (KXH). Additional peptide immunogens may be generated by replacing tyrosine residues with sulfated tyrosine residues. Methods for synthesizing such peptides are known in the art, for example, as in Merrifield, J. Amer. Chem. Soc. 85, 2149-2154 (1963); Krstenansky et al., FEBS Lett. 211, 10 (1987). Antibodies can also be made by selecting ) antibodies from a protein expression library, e.g., a phage display library.
Human monoclonal antibodies (mAbs) directed against target proteins can be generated using transgenic mice carrying the human immunoglobulin genes rather than the mouse system. Splenocytes from these transgenic mice immunized with the antigen of interest are used to produce hybridomas that secrete human mAbs with specific affinities for epitopes from a human protein (see, e.g., WO 91/00906, WO 91/10741; WO 92/03918; WO 92/03917; Lonberg et al. 1994 Nature 368:856-859; Green et al. 1994 Nature Genet. 7: 13-21; Morrison et al. 1994 Proc. Natl. Acad. ScL USA 81:6851-6855; Bruggeman et al. 1993 Year Immunol 7:33-40; Tuaillon et al. 1993 PNAS 90:3720-3724; Bruggeman et al. 1991 Eur J Immunol 21:1323-1326).
Monoclonal antibodies can also be generated by other methods. An exemplary alternative method, referred to as the "combinatorial antibody display" method, has been developed to identify and isolate antibody fragments having a particular antigen specificity, and can be utilized to produce monoclonal antibodies (for descriptions of combinatorial antibody display see e.g., Sastry et al. 1989 PNAS 86:5728; Huse et al. 1989 Science 246:1275; and Orlandi et al. 1989 PNAS 86:3833 and phage display methods, e.g., US 2002-0102613). After immunizing an animal with an immunogen as described above, the antibody repertoire of the resulting B-cell pool is cloned. Methods are generally known for obtaining the DNA sequence of the variable domains of a diverse population of immunoglobulin molecules by using a mixture of oligomer primers and PCR. For instance, mixed oligonucleotide primers corresponding to the 5' leader (signal peptide) sequences and/or framework 1 (FRl) sequences, as well as primer to a conserved 3' constant region primer can be used for PCR amplification of the heavy and light chain variable domains from a number of murine antibodies (Larrick et al., 1991, Biotechniques 11:152-156). A similar strategy can also been used to amplify human heavy and light chain variable domains from human antibodies (Larrick et al., 1991, Methods: Companion to Methods in Enzymology 2 : 106- 110).
Chimeric antibodies, including chimeric immunoglobulin chains, can be produced by recombinant DNA techniques known in the art. For example, a gene encoding the Fc constant region of a murine (or other species) monoclonal antibody molecule is digested with restriction enzymes to remove the region encoding the murine Fc, and the equivalent portion of a gene encoding a human Fc constant region is substituted (see Robinson et al., PCT/US86/02269; EP 184 187; EP 171,496; EP 173,494; Neuberger et al., International Application WO 86/01533; Cabilly et al. U.S. Patent No. 4,816,567; Cabilly et al., EP 125,023; Better et al. (1988 Science 240:1041-1043); Liu et al. (1987) PNAS 84:3439-3443; Liu et al., 1987, J. Immunol. 139:3521-3526; Sun et al. (1987) PNAS 84:214-218; Nishimura et al., 1987, Cane. Res. 47:999-1005; Wood et al. (1985) Nature 314:446-449; and Shaw et al., 1988, J. Natl Cancer Inst. 80:1553-1559). An antibody or an immunoglobulin chain can be humanized by methods known in the art. Humanized antibodies, including humanized immunoglobulin chains, can be generated by replacing sequences of the Fv variable domain which are not directly involved in antigen binding with equivalent sequences from human Fv variable domains. General methods for generating humanized antibodies are provided by Morrison, 1985, Science 229:1202-1207, by Oi et al., 1986, BioTechniques 4:214, and by Queen et al. US 5,585,089, US 5,693,761 and US 5^693,762, the contents of all of which are hereby incorporated by reference. Those methods include isolating, manipulating, and expressing the nucleic acid sequences that encode all or part of immunoglobulin Fv variable domains from at least one of a heavy or light chain. Sources of such nucleic acid are well known to those skilled in the art and, for example, may be obtained from a hybridoma producing an antibody against a predetermined target. The recombinant DNA encoding the humanized antibody, or fragment thereof, can then be cloned into an appropriate expression vector.
Humanized or CDR-grafted antibody molecules or immunoglobulins can be produced by CDR-grafting or CDR substitution, wherein one, two, or all CDRs of an immunoglobulin chain can be replaced. See, e.g., U.S. Patent 5,225,539; Jones et al. 1986 Nature 321:552-525; Verhoeyan et al. 1988 Science 239:1534; Beidler et al. 1988 J. Immunol. 141:4053-4060; Winter US 5,225,539. Winter describes a CDR- grafting method which may be used to prepare the humanized antibodies (UK Patent Application GB 2188638A, filed on March 26, 1987; Winter US 5,225,539). All or part of the CDRs of a particular human antibody may be replaced with at least a portion of a non-human CDR or only some of the CDRs may be replaced with non- human CDRs. It is only necessary to replace the number of CDRs, or portions thereof required for binding of the humanized antibody to the target antigen, e.g., a protein disclosed herein.
In some implementations, monoclonal, chimeric and humanized antibodies can be modified by, e.g., deleting, adding, or substituting other portions of the antibody, e.g., the constant region. For example, an antibody can be modified as follows: (i) by deleting the constant region; (ii) by replacing the constant region with another constant region, e.g., a constant region meant to increase half-life, stability or affinity of the antibody, or a constant region from another species or antibody class; or (iii) by modifying one or more amino acids in the constant region to alter, for example, the number of glycosylation sites, agonist cell function, Fc receptor (FcR) binding, complement fixation, among others.
Methods for altering antibody constant regions are known. Antibodies with altered function, e.g. altered affinity for an agonist ligand, such as FcR on a cell, or the Cl component of complement can be produced by replacing at least one amino acid residue in the constant portion of the antibody with a different residue (see e.g., EP 388 151, US 5,624,821 and US 5,648,260). Similar type of alterations could be described which if applied to the murine, or other species immunoglobulin would reduce or eliminate these functions.
To identify an antibody that not only binds, but also has a particular function (e.g., inhibits), an antibody can be evaluated in a functional assay. For example, a plurality of antibodies that bind to a target (e.g., a protein described herein) can be evaluated in this manner. Antibodies that inhibit an activity of a protein described herein, e.g., an enzymatic activity, can be selected.
Nucleic Acid Antagonists In certain implementations, e.g., therapeutic implementations, nucleic acid antagonists are used to decrease expression of a gene of Table 1 or a protein encoded thereby. In one embodiment, the nucleic acid antagonist is an siRNAthat targets mRNA encoding a protein described herein. Other types of antagonistic nucleic acids can also be used, e.g., a nucleic acid aptamer, a dsRNA, a ribozyme, a triple-helix former, or an antisense nucleic acid. siRNAs are small double stranded RNAs (dsRNAs) that optionally include overhangs. For example, the duplex region of an siRNAis about 18 to 25 nucleotides in length, e.g., about 19, 20, 21, 22, 23, or 24 nucleotides in length. Typically the siRNA sequences are exactly complementary to the target mRNA. dsRNAs and siRNAs in particular can be used to silence gene expression in mammalian cells (e.g., human cells). See, e.g., Clemens et al. (2000) Proc. Natl. Sd. USA 97, 6499-6503; Billy et al. (2001) Proc. Natl. Sd. USA 98, 14428-14433; Elbashir et al. (2001) Nature. 411(6836):494-8; Yang et al. (2002) Proc. Natl. Acad. Sd. USA 99, 9942- 9947, U.S. 20030166282, 20030143204, 20040038278, and 20030224432. Anti-sense agents can include, for example, from about 8 to about 80 nucleobases (i.e. from about 8 to about 80 nucleotides), e.g., about 8 to about 50 nucleobases, or about 12 to about 30 nucleobases. Anti-sense compounds include ribozymes, external guide sequence (EGS) oligonucleotides (oligozymes), and other short catalytic RNAs or catalytic oligonucleotides which hybridize to the target nucleic acid and modulate its expression. Anti-sense compounds can include a stretch of at least eight consecutive nucleobases that are complementary to a sequence in the target gene. An oligonucleotide need not be 100% complementary to its target nucleic acid sequence to be specifically hybridizable. An oligonucleotide is specifically hybridizable when binding of the oligonucleotide to the target interferes with the normal function of the target molecule to cause a loss of utility, and there is a sufficient degree of complementarity to avoid non-specific binding of the oligonucleotide to non-target sequences under conditions in which specific binding is desired, i.e., under physiological conditions in the case of in vivo assays or therapeutic treatment or, in the case of in vitro assays, under conditions in which the assays are conducted.
Hybridization of antisense oligonucleotides with niRNAcan interferes with one or more of the normal functions of mRNA. The functions of mRNA to be interfered with include all vital functions such as, for example, translocation of the RNA to the site of protein translation, translation of protein from the RNA, splicing of the RNA to yield one or more mRNA species, and catalytic activity which may be engaged in by the RNA. Binding of specific protein(s) to the RNA may also be interfered with by antisense oligonucleotide hybridization to the RNA.
Exemplary antisense compounds include DNA or RNA sequences that specifically hybridize to the target nucleic acid. The complementary region can extend for between about 8 to about 80 nucleobases. The compounds can include one or more modified nucleobases. Modified nucleobases may include, e.g., 5-substituted pyrimidines such as 5-iodouracil, 5-iodocytosine, and C5-propynyl pyrimidines such as C5-ρropynylcytosine and C5-propynyluracil. Other suitable modified nucleobases include N4 -(C] -C]2) alkylaminocytosines and N^N4 -(Ci -C12) dialkylaminocytosines. Modified nucleobases may also include 7-substituted-8-aza- 7-deazapurines and 7-substituted-7-deazapurines such as, for example, 7-iodo-7- deazapurines, 7-cyano-7-deazapurines, 7-aminocarbonyl-7-deazapurines. Examples of these include 6-amino-7-iodo-7-deazapurines, 6-amino-7-cyano-7-deazapurines, 6- amino-7-aminocarbonyl-7-deazapurines, 2-amino-6-hydroxy-7-iodo-7-deazapurines, 2-amino-6-hydroxy-7-cyano-7-deazapurines, and 2-amino-6-hydroxy-7- aminocarbonyl-7-deazapurines. Furthermore, N6 -(C1 -C12) alkylaminopurines and N6,N6 -(C1 -C12) dialkylaminopurines, including N6 -methylaminoadenine andN6,N6 -dimethylaminoadenine, are also suitable modified nucleobases. Similarly, other 6- substituted purines including, for example, 6-thioguanine may constitute appropriate modified nucleobases. Other suitable nucleobases include 2-thiouracil, 8- bromoadenine, 8-bromoguanine, 2-fluoroadenine, and 2-fluoroguanine. Derivatives of any of the aforementioned modified nucleobases are also appropriate. Substituents of any of the preceding compounds may include C1 -C30 alkyl, C2 -C30 alkenyl, C2 - C30 alkynyl, aryl, aralkyl, heteroaryl, halo, amino, amido, nitro, thio, sulfonyl, carboxyl, alkoxy, alkylcarbonyl, alkoxycarbonyl, and the like.
Descriptions of other types of nucleic acid agents are also available. See, e.g., U.S. Patent No. 4,987,071; U.S. Patent No. 5,116,742; U.S. Pat. No. 5,093,246; Woolf et al. (1992) Proc Natl Acad Sd USA; Antisense RNA and DNA, D. A. Melton, Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, N. Y. (1988); 89:7305-9; Haselhoff and Gerlach (1988) Nature 334:585-59; Helene, C. (1991) Anticancer DrugDes. 6:569-84; Helene (1992) Ann. N.Y. Acad. Sd. 660:27-36; and Maher (1992) Bioassays 14:807-15.
Artificial Transcription Factors
Artificial transcription factors can also be used to regulate a gene of Table 1 and/or a protein encoded thereby. The artificial transcription factor can be designed or selected from a library. For example, the artificial transcription factor can be prepared by selection in vitro (e.g., using phage display, U.S. Pat. No. 6,534,261) or in vivo, or by design based on a recognition code (see, e.g., WO 00/42219 and U.S. Pat. No. 6,511,808). See, e.g., Rebar et al. (1996) Methods Enzymol 267:129; Greisman and Pabo (1997) Science 275:657; Isalan et al. (2001) Nat. Biotechnol. 19:656; and Wu et al. (1995) Proc. Natl. Acad. Sd. USA 92:344 for, among other things, methods for creating libraries of varied zinc finger domains. Optionally, a zinc finger protein can be fused to a transcriptional regulatory domain, e.g., an activation domain to activate transcription or a repression domain to repress transcription. The zinc finger protein can itself be encoded by a heterologous nucleic acid that is delivered to a cell or the protein itself can be delivered to a cell (see, e.g., U.S. Pat. No. 6,534,261. The heterologous nucleic acid that includes a sequence encoding the zinc finger protein can be operably linked to an inducible promoter, e.g., to enable fine control of the level of the zinc finger protein in the cell.
Recombinant Protein Production
The nucleic acids encoding proteins that function as agents for the methods described herein or that are target proteins or fragments thereof may be operably linked to an expression control sequence in a vector in order to produce the protein recombinantly. Many suitable expression control sequences are known. General methods of expressing recombinant proteins are also known and are exemplified in Kaufman, Methods in Enzymology 185, 537-566 (1990), Sambrook & Russell, Molecular Cloning: A Laboratory Manual, 3rd Edition, Cold Spring Harbor
Laboratory, N. Y. (2001) and Ausubel et ah, Current Protocols in Molecular Biology (Greene Publishing Associates and Wiley hiterscience, N. Y. (1989). As defined herein "operably linked" means enzymatically or chemically ligated to form a covalent bond between a particular polynucleotide encoding a protein of interest and the expression control sequence, in such a way that the protein of interest is expressed by a host cell which has been transformed (transfected) with the ligated polynucleotide/expression control sequence.
The term "vector," as used herein, refers to a nucleic acid molecule capable of transporting, or sustaining maintenance or replication of, another nucleic acid to which it has been linked. One type of vector is a "plasmid", which refers to a circular double stranded DNA loop into which additional DNA segments may be ligated. Another type of vector is a viral vector, wherein additional DNA segments may be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced {e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non- episomal mammalian vectors) can be integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome.
Moreover, certain vectors are capable of directing the expression of genes to which they are operatively linked. Exemplary viral vectors include replication defective retroviruses, adenoviruses and adeno-associated viruses.
The vector can include one or more regulatory sequences, e.g., promoters, enhancers and other expression control elements (e.g., polyadenylation signals) that control the transcription or translation of the genes. Examples of regulatory sequences are described, for example, in Goeddel; Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA (1990). The selection of regulatory sequences may depend on such factors as the choice of the host cell to be transformed, the level of expression of protein desired, etc. Exemplary regulatory sequences for mammalian host cell expression include viral elements that direct high levels of protein expression in mammalian cells, such as promoters and/or enhancers derived from FF-Ia promoter and BGH poly A, cytomegalovirus (CMV) (such as the CMV promoter/enhancer), Simian Virus 40 (SV40) (such as the SV40 promoter/enhancer), adenovirus, (e.g., the adenovirus major late promoter (AdMLP)) and polyoma. For further exemplary descriptions of viral regulatory elements, and sequences thereof, see e.g., U.S. Patent No. 5,168,062, U.S. Patent No. 4,510,245, and U.S. Patent No. 4,968,615.
The recombinant expression vectors may carry additional sequences, such as sequences that regulate replication of the vector in host cells (e.g., origins of replication) and selectable marker genes. The selectable marker gene facilitates selection of host cells into which the vector has been introduced (see e.g., U.S. Patents Nos. 4,399,216, 4,634,665 and 5,179,017). For example, typically the selectable marker gene confers resistance to drugs, such as G418, hygromycin or methotrexate, on a host cell into which the vector has been introduced. Preferred selectable marker genes include the dihydrofolate reductase (DHFR) gene (for use in dhfr- host cells with methotrexate selection/amplification) and the neo gene (for G418 selection). A number of types of cells may act as suitable host cells for expression of a protein therapeutic. Any cell type capable of expressing the protein therapeutic may be used. Exemplary mammalian host cells include, for example, monkey COS cells, Chinese Hamster Ovary (CHO) cells, human kidney 293 cells, human epidermal A431 cells, human Colo205 cells, 3T3 cells, CV-I cells, other transformed primate cell lines, normal diploid cells, cell strains derived from in vitro culture of primary tissue, primary explants, HeLa cells, mouse L cells, BHK, HL-60, U937, HaK, Rat2, BaF3,
32D, FDCP-I, PC12, Mix, or C2C12 cells.
A protein therapeutic or target protein may be produced by operably linking a polynucleotide encoding such a protein to suitable control sequences in one or more insect expression vectors, and employing an insect expression system. Materials and methods for baculovirus/insect cell expression systems are commercially available, e.g., in kit form from, e.g., Invitrogen, San Diego, CA (the MAXBAC® kit), e.g., as described in Summers and Smith, Texas Agricultural Experiment Station Bulletin No.
1555 (1987). Soluble forms of the polypeptides described herein and other proteins may also be produced in insect cells using appropriate isolated polynucleotides, e.g., forms in which the region encoding one or more, or sufficient segments, of the transmembrane domain and the cytoplasmic domain are removed.
A protein therapeutic or target protein may be produced in lower eukaryotes such as yeast or in prokaryotes such as bacteria. Suitable yeast strains include Saccharomyces cerevisiae, Schizosaccharomyces pombe, Kluyveromyces strains,
Pichia, Candida, or any yeast strain capable of expressing heterologous proteins.
Suitable bacterial strains include Escherichia coli, Bacillus subtilis, Salmonella typhimurium, or any bacterial strain capable of expressing heterologous proteins.
In one embodiment, a protein therapeutic or target protein (e.g., a polypeptide described herein or fragment thereof) is produced in a bacterial cell, e.g., with or without a signal sequence (e.g., without either a prokaryotic or eukaryotic signal sequence). Expression in bacteria may result in formation of inclusion bodies incorporating the recombinant protein. Thus, refolding of the recombinant protein may be required in order to produce active or more active material. Several methods for obtaining correctly folded heterologous proteins from bacterial inclusion bodies are known in the art. These methods generally involve solubilizing the protein from the inclusion bodies, then denaturing the protein completely using a chaotropic agent. When cysteine residues are present in the primary amino acid sequence of the protein, the protein can be refolded in an environment which facilitates correct formation of disulfide bonds (e.g., a redox system). General methods of refolding are disclosed in Kohno, Meth. Enzym., 185:187-195 (1990), EP 0433225 and U.S.
5,399,677. A protein described herein (e.g., a target protein, therapeutic, or immunoglobulin) thereof may also be expressed as a product of transgenic animals, e.g., as a component of the milk of transgenic cows, goats, pigs, or sheep which are characterized by somatic or germ cells containing a polynucleotide sequence encoding the protein.
Treatments and Pharmaceutical Compositions
An agent that modulates a gene of Table 1 or a protein encoded thereby can be used to treat a subject, e.g., a human subject. The agent can be provided as a component of a pharmaceutical composition. The pharmaceutical composition may include a therapeutically effective amount of an agent described herein. A therapeutically effective amount is an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic result or to prevent or delay onset of a disorder. A therapeutically effective amount of the composition may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the compound to elicit a desired response in the individual. A therapeutically effective amount is also one in which any toxic or detrimental effects of the composition is outweighed by the therapeutically beneficial effects. A therapeutically effective amount preferably modulates a measurable parameter, e.g., a measurable symptom of an inflammatory or autoimmune disorder, e.g., multiple sclerosis, relative to untreated subjects, e.g., to a statistically significant degree. The ability of a compound to inhibit a measurable parameter can be evaluated in an animal model system predictive of efficacy in a human disorder, using in vitro assays, e.g., an assay described herein, or using appropriate human trials. A compound discovered by the methods described herein can be formulated as a pharmaceutical composition compatible with an intended route of administration. Examples of routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (topical), transmucosal, and rectal administration. Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic. Pharmaceutical formulation is a well-established art, and is further described, e.g., in Gennaro (ed.), Remington: The Science and Practice of Pharmacy, 20th ed., Lippincott, Williams & Wilkins (2000) (ISBN: 0683306472); Ansel et ah, Pharmaceutical Dosage Forms and Drug Delivery Systems, 7th Ed., Lippincott Williams & Wilkins Publishers (1999) (ISBN: 0683305727); and Kibbe (ed.), Handbook of Pharmaceutical Excipients American Pharmaceutical Association, 3rd ed. (2000) (ISBN: 091733096X). Typically, a pharmaceutical composition includes a pharmaceutically acceptable carrier. As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. The composition can include a pharmaceutically acceptable salt, e.g., an acid addition salt or a base addition salt (see e.g., Berge et a (1977) J. Pharm. Sd. 66:1-19).
Particular effects mediated by an agent may show a difference that is statistically significant (e.g., P value < 0.05 or 0.02). Statistical significance can be determined by any art known method. Exemplary statistical tests include: the Students T-test, Mann Whitney U non-parametric test, and Wilcoxon non-parametric statistical test. Some statistically significant relationships have a P value of less than 0.05 or 0.02. The terms "induce", "inhibit", "potentiate", "elevate", "increase", "decrease" or the like, e.g., which denote distinguishable qualitative or quantitative differences between two states, and may refer to a difference, e.g., a statistically significant difference (e.g., P value < 0.05 or 0.02), between the two states.
Dosage regimens are adjusted to provide the optimum desired response {e.g., a therapeutic response). For example, a single bolus may be administered, several divided doses may be administered over time or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. It is possible to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subjects to be treated; each unit contains a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.
An exemplary, non-limiting range for a therapeutically effective amount of an agent described herein is about 0.1 -20 mg/kg, more preferably about 1 - 10 mg/kg. Dosage values may vary with the type and severity of the condition to be alleviated. For any individual subject, specific dosage regimens can be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the compositions. Accordingly, the dosage ranges set forth herein are only exemplary.
Subjects who can be treated include human and non-human animals, e.g., non- mammals (such as chickens, amphibians, reptiles) and mammals, such as non-human primates, mice, sheep, dogs, cows, pigs, etc.
An agent described herein may be used as a pharmaceutical composition when combined with a pharmaceutically acceptable carrier. Such a composition may contain, in addition to the agent and carrier, various diluents, fillers, salts, buffers, stabilizers, solubilizers, and other materials well known in the art. Pharmaceutically acceptable carriers are non-toxic materials that does not interfere with the effectiveness of the biological activity of the active ingredient(s). The characteristics of the carrier typically depend on the route of administration.
In practicing the method of treatment or use, a therapeutically effective amount of an agent is administered to a subject, e.g., mammal (e.g., a human). The agent may be administered either alone or in combination with other therapies such as other treatments for atopic disorders. When co-administered with one or more agents, the agent may be administered either simultaneously with the second agent, or sequentially. If administered sequentially, the attending physician can decide on the appropriate sequence of administering the agent described herein with other agents.
Administration of an agent described herein can be carried out in a variety of ways, including, for example, oral ingestion, inhalation, or cutaneous, subcutaneous, or intravenous injection or administration.
For oral administration, the agent can be in the form of a tablet, capsule, powder, solution, or elixir. When administered in tablet form, the pharmaceutical composition may additionally contain a solid carrier such as a gelatin or an adjuvant. The tablet, capsule, and powder contain from about 5 to 95% of the agent or from about 25 to 90% of the agent. When administered in liquid form, a liquid carrier such as water, petroleum, oils of animal or plant origin such as peanut oil, mineral oil, soybean oil, or sesame oil, or synthetic oils may be added. The liquid form of the pharmaceutical composition may further contain physiological saline solution, dextrose or other saccharide solution, or glycols such as ethylene glycol, propylene glycol or polyethylene glycol. When administered in liquid form, the pharmaceutical composition contains from about 0.5 to 90% by weight of the agent, and preferably from about 1 to 50% the agent. To administer an agent, e.g., by intravenous, cutaneous, or subcutaneous injection, the agent can be in the form of a pyrogen-free, parenterally acceptable aqueous solution. The preparation of such parenterally acceptable protein solutions, having due regard to pH, isotonicity, stability, and the like, is within the skill in the art. An exemplary pharmaceutical composition for intravenous, cutaneous, or subcutaneous injection can contain, in addition to the agent an isotonic vehicle such as sodium chloride injection, Ringer's injection, dextrose injection, dextrose and sodium chloride injection, lactated Ringer's injection, or other vehicle as known in the art. The pharmaceutical composition may also contain stabilizers, preservatives, buffers, antioxidants, or other additive known to those of skill in the art. The amount of an agent to be delivered can depend upon the nature and severity of the condition being treated, and on the nature of prior treatments that the patient has undergone. The attending physician can decide the amount of agent with which to treat each individual patient. Initially, for example, the attending physician can administer low doses of the agent and observe the patient's response. Larger doses of the agent may be administered until the optimal therapeutic effect is obtained for the patient, and at that point the dosage is not generally increased further, or by monitoring immunoglobulin levels (e.g., IgG or IgE levels) or one or more symptoms. In the case of an agent that is an immunoglobulin (e.g., a full length antibody, an exemplary pharmaceutical compositions may contain about 0.1 μg to about 10 mg of the immunoglobulin agent per kg body weight. For example, useful dosages can include between about 10 μg-1 mg, 0.1-5 mg, and 3-50 mg of the agent per kg body weight. The duration of therapy using the pharmaceutical composition can vary, depending on the severity of the disease being treated and the condition and potential idiosyncratic response of each individual patient. The duration of each application of the agent can be, e.g., in the range of 12 to 24 hours of continuous intravenous administration. The attending physician can decide on the appropriate duration of intravenous therapy using a pharmaceutical composition described herein.
With respect to agents that are proteins or nucleic acids, the disease or disorder can also be treated or prevented by administration or use of polynucleotides encoding such proteins (such as, for example, in gene therapies or vectors suitable for introduction of DNA). The polynucleotides that encode an agent or that provide a nucleic acid agent activity can be inserted into vectors and used as gene therapy vectors. Gene therapy vectors can be delivered to a subject by, for example, intravenous injection, local administration (see U.S. Patent 5,328,470), injection (e.g., US 20040030250 or 20030212022) or stereotactic injection (e.g., Chen et al. Proc. Natl. Acad. ScL USA 91:3054-3057, 1994). The pharmaceutical preparation of the gene therapy vector can include the gene therapy vector in an acceptable diluent, or can comprise a slow release matrix in which the gene delivery vehicle is imbedded. Alternatively, where the complete gene delivery vector can be produced intact from recombinant cells, e.g., retroviral vectors, the pharmaceutical preparation can include one or more cells which produce the gene delivery system.
Pharmaceutical compositions can be administered using a medical device. For example, in one embodiment, a pharmaceutical composition described herein can be administered with a needle-less hypodermic injection device, such as the devices disclosed in U.S. Patent Nos. 5,399,163; 5,383,851; 5,312,335; 5,064,413; 4,941,880; 4,790,824; or 4,596,556. Examples of well-known implants and modules that can be used include: U.S. Patent No. 4,487,603, which discloses an implantable micro- infusion pump for dispensing medication at a controlled rate; U.S. Patent No. 4.,486,194, which discloses a therapeutic device for administering agents through the skin; U.S. Patent No. 4,447,233, which discloses a medication infusion pump for delivering medication at a precise infusion rate; U.S. Patent No. 4,447,224, which discloses a variable flow implantable infusion apparatus for continuous drug delivery; U.S. Patent No. 4,439,196, which discloses an osmotic drug delivery system having multi-chamber compartments; and U.S. Patent No. 4,475,196, which discloses an osmotic drug delivery system. Of course, many other such implants, delivery systems, and modules are also known.
Multiple Sclerosis and Symptoms Thereof
Modulators of the genes of Table 1 and proteins encoded thereby can be used to treat or prevent multiple sclerosis or one or more symptoms of multiple sclerosis (MS).
MS is a chronic disease characterized by the inflammation and scarring (sclerosis) of the myelin sheath and the underlying nerve. Exemplary symptoms associated with multiple sclerosis include: optic neuritis, diplopia, nystagmus, ocular dysmetria, internuclear ophthalmoplegia, movement and sound phosphenes, afferent pupillary defect, paresis, monoparesis, paraparesis, hemiparesis, quadriparesis, plegia, paraplegia, hemiplegia, tetraplegia, quadriplegia, spasticity, dysarthria, muscle atrophy, spasms, cramps, hypotonia, clonus, myoclonus, myokymia, restless leg syndrome, footdrop, dysfunctional reflexes, Babinski's reflex, paraesthesia, anaesthesia, neuralgia, neuropathic and neurogenic pain, Lhermitte's symptom, proprioceptive dysfunction, trigeminal neuralgia, ataxia, intention tremor, dysmetria, vestibular ataxia, vertigo, speech ataxia, dystonia, dysdiadochokinesia, frequent micturation, bladder spasticity, flaccid bladder, detrusor-sphincter dyssynergia, erectile dysfunction, anorgasmy, frigidity, constipation, fecal urgency, fecal incontinence, depression, cognitive dysfunction, dementia, mood swings, emotional lability, euphoria, bipolar syndrome, anxiety, aphasia, dysphasia, fatigue, Uhthoff 's symptom, gastroesophageal reflux, and sleeping disorders.
Patients suitable for treatment using the methods described herein may be identified by criteria establishing a diagnosis of clinically definite MS as defined by the workshop on the diagnosis of MS (Poser et al., Ami. Neurol. 13:227, 1983). Briefly, an individual with clinically definite MS has had two attacks and clinical evidence of either two lesions or clinical evidence of one lesion and paraclinical evidence of another, separate lesion. Definite MS may also be diagnosed by evidence of two attacks and oligoclonal bands of IgG in cerebrospinal fluid or by combination of an attack, clinical evidence of two lesions and oligoclonal band of IgG in cerebrospinal fluid. Slightly lower criteria are used for a diagnosis of clinically probable MS. Patients suitable for treatment can also be evaluated for expression and/or activity of one or more polypeptide encoded by a gene of Table 1, and can be identified as suitable for treatment if the expression and/or activity for one or more such polypeptides is elevated relative to a reference (provide such polypeptide is elevated in the mouse models).
Candidate patients for prevention can be identified by the presence of genetic factors. For example, a majority of MS patients have HLA-type DR2a and DR2b. The MS patients having genetic dispositions to MS who can be suitable for treatment fall within two groups. First are patients with early disease of the relapsing remitting type. Entry criteria can include disease duration of more than one year, EDSS score of 1.0 to 3.5, exacerbation rate of more than 0.5 per year, and free of clinical exacerbations for 2 months prior to study. The second group includes people with disease progression greater than 1.0 EDSS unit/year over the past two years. Candidate patients for prevention may be identified by evaluating cytokine parameters, e.g., an IL-10 or IL-21 parameter (see, e.g., U.S. Application No. 10/806,611).
Exemplary Genes and Proteins from Table 1
Information about many of the genes and encoded proteins are publicly available, e.g., using the GenBank® database (U.S. Department of Health and Human Services). Some specifics for selected proteins are as follows:
PSMEl. PSMEl encodes proteasome 28 subunit alpha (PA28α), which is a part of the HS regulator of the immunoproteasome. Two transcripts of PSMEl encoding different isoforms have been identified. Exemplary human PSME amino acid sequences are listed at GenBank® accession numbers NP_006254, NP_788955. LAPTM5. NP_006753 Lysosomal-associated protein transmembrane 5
(LAPTM5) is a pentaspanner transmembrane protein that is conserved across evolution. An exemplary human LAPTM5 amino acid sequence is listed at GenBank® accession number NP_006753.
CD68. Macrosialin/CD68 is a transmembrane glycoprotein expressed in cytoplasmic granules of macrophages/monocytes, DC, granulocytes and myeloid progenitor cells. An exemplary human CD68 amino acid sequence is listed at GenBank® accession number NP 001242. CSFlR. Macrophage colony-stimulating factor (M-CSF) is one of several hematologic growth factors capable of regulating the survival, proliferation, and differentiation of macrophages. An exemplary human M-CSF amino acid sequence is listed at GenBank® accession number NP_005202. SCYA6. SCYA6 encodes the chemokine ClO. An exemplary human amino acid sequence is listed at GenBank® accession number NP_116741.
CD53. CD53 is a member of tetraspanin family. An exemplary human CD53 amino acid sequence is listed at GenBank® accession number NP_000551.
MPEGl. Mpg-1 shows lineage-restricted and stage-specific expression in mature macrophages. An exemplary human Mpg-1 amino acid sequence is listed at GenBank® accession number XEM66227. ,
LY86. LY86/MD-1 is a 162 amino acid secreted protein that binds to and positively regulates the expression of RP105/CD180, a transmembrane protein with similarity to TLR4. An exemplary human MD-I amino acid sequence is listed at GenBank® accession number NP_004262.
PLS2. An exemplary human plastin-2 amino acid sequence is listed at GenBank® accession number NP_002289.
LCN2. An exemplary human lipocalin-2 sequence is listed at GenBank® accession number NP_005555.
EXAMPLE
Experimental autoimmune encephalomyelitis (EAE) is a T cell-mediated inflammatory disease of the central nervous system (CNS), which clinically manifests as ascending paralysis. It can be induced in susceptible animals by immunizing them with myelin proteins or by injecting them with myelin protein-specific CD4 cells. EAE shares many clinical and pathological features with multiple sclerosis (MS), and is the commonly used animal model of this human disease. Martin et al., Crit Rev Clin Lab Sd 1995;32(2):121-82; Zamvil et al, Annu Rev Immunol 1990;8:579-621 ; Steinman, Neuron 1999;24(3):511-4.
EAE is generally believed to be a Thl-induced disease because of the increased expression of ThI cytokines in the CNS. Furthermore injection of ThI but not Th2 T cells into immunocompetent mice is sufficient to induce EAE. Baron et al, J Exp Med 1993;177(l):57-68; Kuchroo et al, J Immunol 1993;151(8):4371-82 ;
Miller et al., Immunol Today 1994;15(8):356-61 ; Kennedy et al., J Immunol 1992;149(7):2496-505. ThI cells produce interferon (IFN)-γ, together with other Thl-type cytokines. IFN-γ is a potent activator of macrophages, stimulator of expression of MHC class I and II molecules, and activator of adhesion molecules and inflammatory mediators, such as nitric oxide (NO) and TNF. Boehm et al., Annu Rev Immunol 1997;15:749-95. Despite this, mice deficient in IFN-γ or IFN-γR often develop EAE with higher incidence and severity than wild-type (WT) mice. Ferber et al., J Immunol 1996;156(l):5-7; Krakowski et al, Eur J Immunol 1996;26(7): 1641-6; Tran et al, J Immunol 2000;164(5):2759-68; Willenborg et al, J Immunol 1996;157(8):3223-7; Willenborg et al, J Immunol 1999;163(10):5278-86. Members of the ThI -inducing family of cytokines, IL- 12, IL-23 and IL-27 all appear to be important in ThI differentiation and EAE development. Goldberg et al., J Immunol 2004;173(10):6465-71; Cua et al., Nature 2003;421(6924):744-8; Leonard et al., J Exp Med 1995;181(l):381-6. IL-12 is a cytokine, composed of two disulfide- linked subunits, designated p40 and p35. It is produced by activated antigen presenting cells and it can induce differentiation of recently activated CD4 cells into ThI type. Trinchieri, Nat Rev Immunol 2003;3(2):\33-46; Szabo et al, Annu Rev Immunol 2003;21:713-58. IL-12 can induce production of IFN-γ, granulocyte- macrophage colony-stimulatory factor (GM-CSF) and TNF, which all play an important role in EAE development. Ferber et al, J Immunol 1996;156(l):5-7; Krakowski et al, Eur J Immunol 1996;26(7):1641-6; Tran et al, J Immunol
2000;164(5):2759-68; Willenborg et al, J Immunol 1996;157(8):3223-7; Willenborg et al, J Immunol 1999; 163(10):5278-86; McQualter et al, J Exp Med 2001;194(7):873-82; Marusic et al, Neurosci Lett 2002;332(3): 185-9; Ruddle et al, J Exp Med 1990;172(4):l 193-200; Selmaj et al, Ann Neurol 1991;30(5):694-700. However, mice deficient in the p35 subunit of IL-12 still develop EAE. Becher et al, JCHn Invest 2002;110(4):493-7; Gran et al, J Immunol 2002;169(12):7104-10. IL- 12p35 -/— mice that develop EAE have reduced levels of IFN- γ in draining lymph nodes early in disease but these levels increase later during the immune response. Furthermore, the expression of IFN-γ mRNA in the CNS of IL-12p35 -/- mice with EAE is not significantly different from WT controls (Becher et al., J CHn Invest
2002;l 10(4):493-7; Gran et al., J Immunol 2002;169(12):7104-10) but levels of IL-10 mRNA are increased. Becher et al., J CHn Invest 2002; 110(4):493-7. This finding may be significant because IL-10 has been suggested to play an important regulatory role in EAE. Kennedy et ah, J Immunol 1992;149(7):2496-505; Segal et a\., J Exp Med 1998;187(4):537-46. Differences have also been observed between WT and IFN-γ -/- mice during EAE. While WT mice have increased mRNA expression of RANTES and MCP-I, the mRNA for these chemokines are not detectable in CNS of IFN-γ -/- mice. In addition, mRNA for MIP-2 and TCA-3 are upregulated in CNS of IFN-γ -/-, but not WT mice. Tran et al, J Immunol 2000;164(5):2759-68. Collectively, these results clearly indicate that, while IFN- γ and IL- 12 are not essential for EAE induction, mice deficient in these genes have altered expression of various mediators of inflammation during the course of EAE.
Material and Methods
Mice. Female IFNγ -/-mice on C57B1/6 background and appropriate C57B1/6 WT control animals were obtained from Jackson Laboratories (Bar Harbor, ME) and used at 6-10 weeks of age. IL-12p35 -/- mice, backcrossed on C57B1/6 background for 5 generations, and appropriate C57B1/6 control WT mice were bred at Taconic Farms (Germantown, NY) and 6-10 week old females were used for experiments.
Experimental autoimmune encephalomyelitis (EAE) induction and tissue collection. For EAE induction, all mice were injected subcutaneously with 200 μg of myelin oligodendrocytes glycoprotein (MOG) peptide 35-55 in complete Freund's adjuvant containing 5 mg/ml killed Mycobacterium tuberculosis. On the same day, the mice received 200 ng pertussis toxin intraperitoneally. Paralysis (EAE) was assessed, starting on day 5 after immunization, when all the mice were still symptom-free. EAE was scored as follows: 1- limp tail, 2- partial hind leg paralysis, 3-complete hind leg paralysis or partial hind and front leg paralysis, 4- complete hind and partial front leg paralysis, 5-moribund. CNS and spleen tissues were collected from the mice at onset, peak and recovery stage of EAE. CNS tissue was a pool of spinal cord and brain stem tissue of each individual mouse. Onset was defined as within 24 hours of the first clinical signs of EAE, with EAE score of 2 or less. Peak was defined as 4-6 days after the first signs of EAE, with the score of 3-4. The entire experimental design was repeated over three time-separated intervals. No systematic differences were observed and therefore data for individually analyzed mice from the three experiment were combined. To isolate RNA, spleens and CNS tissues (spinal cord and brain stem) were collected from at least 10 mice/group/time point and processed individually. CNS tissue from MS lesion. CNS tissue was collected and analyzed by oil red O and hematoxylin staining of 10 μm snap frozen sections cut from each tissue block before and after tissue collected for gene expression profiling and scored for the degree of ongoing and recent demyelination and perivascular cuffing. Acute MS lesions with ongoing or recent demyelination were identified on the basis of the presence of substantial numbers (graded as >3 on a 0-5 scale) of oil red O-positive macrophages containing neutral lipids resulting from myelin breakdown Li et al., Neuropathol Appl Neurobiol 1993;19(3):214-23.
Array hybridization. RNA was quantified spectrophotometrically at 260 nm and the quality and integrity of the samples were verified by running them on 1% agarose gel and confirming the ribosomal RNA bands. Double-stranded cDNA was synthesized from 10 μg total RNA using the Superscript System (frwitrogen, Carlsbad, CA). The cDNA was purified and transcribed in vitro using T7 RNA polymerase. Biotinylated cRNA was generated using the labeled biotin labeled UTP and CTP (Perkin Elmer, Boston, MA). Fragmented cRNA were hybridized to a
Murine U74Av2 GeneChip® (Affymetrix, Santa Clara, CA) as recommended by the manufacturer. The chips were scanned using a Hewlett Packard GeneArray® Scanner and raw data generated using Affymetrix® MAS 4.0 software. Hybridization intensities on each array were further normalized to a standard curve created from a set of eleven bacterial transcripts spiked in at defined concentrations. This standard curve was used to convert signal values for each qualifier on each array to frequency units expressed as parts per million. Use of the bacterial transcripts allowed for sensitivity for each array to be determined. Hill et al., Genome Biol 2001;2(12):RESEARCH0055. m 85% of the arrays we were able to detect transcripts expressed at 2.3 parts per million (ppm) and in 15% of the arrays were able to detect transcripts expressed at 5.7 ppm. For reference, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and β-actin are expressed at 400 ppm and 70 ppm respectively. To be sure that we were looking at real signal, we chose to look at genes expressed at least two fold above the chip sensitivity as described below. Quality of RNA was verified by the 573' ratio for GAPDH and for β-actin as measured by the arrays. Ratios ranged from 0.8-1.1.
Data Analysis. We performed pair wise comparison on logio transformed frequency values for each disease state to naϊve animals for each genotype. We calculated the fold change ratio, the P value based on Student's t test, the number of present calls, and the expression level for each comparison. A confidence score (CS) was defined as CS(x) = FC(x) + PV(x) + PC(x) + EL(x), where FC, PV, PC, and EL are scores assigned to the fold change, P-value, number of present calls, and the expression level, respectively.
For each fold change ratio fc(x), FC(x) was assigned a value based on the following rules. If fc(x) is greater then 2.95 then FC(x) = 6; if fc(x) is greater then 1.95 then FC(x) = 6-(3-fc(x); if fc(x) is greater then 1.5 then FC(x) = 5-((2-fc(x)) X 6). For each pvalue (pv(x)), PV(x) was assigned a values based on the following rules. PV(x) was assigned 4 if the P- value was less then 0.01; If pv(x) is less then
0.05 then PV(x) = 3- (pv(x)-O.Ol) X 25). PC(x) was assigned3 if at least 100% of the samples are called P by the Affymetrix™ algorithm, assigned 2.5 if 50% - 99% of the samples are called P, assigned 1 if only 25%-49% of the samples were called P. EL(x) was assigned 3 if the average frequency of any group had a value of 10 or greater. Penalty points were assigned if the fold change was less then 1.5, the P- value was greater than 0.05, or the frequency values were less than 10 ppm. CS(x) ranged from -16 to 16, with qualifiers having a score of 16 considered the most significant changes. Genes with 11 or more points in any one pairwise comparison were considered to be significant and were included for further analysis. Using this paradigm, we have selected genes that have at least 1.5 fold change with a p value <=0.05 based on student test. We have included additional filtering criteria above a statistically derived p value to give greater confidence that the changes observed were real and not related to assay noise. These metrics also allowed us to rank order genes and select those with most significant changes for further interpretation.
TAQMAN™ Quantitative RT-PCR Assays. Total RNA was DNase treated and purified on a RNeasy prep column (Qiagen, Valencia,CA). Selected regulated genes identified via GeneChip® were verified by real-time quantitative RT-PCR. Briefly, TaqMan primers and probes were obtained from Applied Biosystems as part of the pre-designed, gene-specific TaqMan™ probe and primer sets. All primers and probes were used at 0.3 mM concentrations in the PCR reactions. Quantified RNA (2μg) was converted to cDNA using ABI High-Capacity cDNA archive Kit (PE Applied Biosystems). Gene-specific primers and probes were used with the TaqMan Universal PCR Master Mix (PE Applied Biosystems) to amplify the equivalent of 50 ng of RNA generated from the cDNA. Reactions were incubated at 50 0C for 2 minutes followed by 10 minutes at 95 °C then 40 cycles of PCR as follows: 95 °C for 15 seconds then 60 °C for 1 minute in an ABI 7900. The data were analyzed using SequenceDetector™ version 2.0 software (PE Applied Biosystems) and were normalized to GAPDH primer set (PE Applied Biosystems).
Results
Clinically similar EAE develops in wild-type, IL-I 2p35 -I- and IFNy -I- mice. We have examined the susceptibility of IL-12p35 -/-mice and IFN-γ -/- mice to
EAE. Mice were immunized with MOG35-5S peptide in complete Freund's adjuvant, and the development of clinical signs of EAE were monitored. In at least three independent experiments, wild type, IL-12p35 -/- and IFN-γ -/- animals showed comparable severity and time of disease onset (FIG. 1). Since the two gene-deficient mice were bred at two separate facilities, two sets of wild type B6 mice were examined. We found no significant differences in EAE development or gene expression profiles in the two sets of WT mice and therefore they were treated as a single control group.
Gene expression analysis. Oligonucleotide arrays were used to identify genes regulated during EAE development. We determined the gene expression profiles in CNS and spleen at onset and peak clinical disease in wild type, IFN-γ -/- and IL- 12p35 —/-mice and compared them to CNS and spleen tissue from naive animals. Onset was defined as within 24 hours of the first clinical signs of EAE with a disease severity score between 1 and 2. Peak was defined as disease lasting for 4-6 days with a clinical score between grades 3 and 4. RNA from CNS and spleen from at least 10 animals of each genotype at each time point was used for the analysis. Expression profiling, monitoring over 12,000 qualifiers using Affymetrix® MG-U74Av2 arrays, was performed on the RNA from individual animals. Because of the large number of replicas, we had statistical confidence that fold changes as low as 1.5 could readily be detected.
We first explored whether large-scale difference in expression existed among the different strains. Only eleven (0.09 % of the genes monitored) genes showed a significant difference (>1.5 fold change, pvalues<=0.05) between baseline samples from WT and IL-12p35 -/- while 122 (1.02%) qualifiers differed between WT and IFN-γ -/- naive mice. The relatively low number of differently expressed genes suggests the underlying baseline expression profiling is similar in all strains. Therefore any diseased-induced differences between the strains are likely due to the effect of the specific gene deletion on EAE development.
Expression analysis of CNS tissue collected during the course of EAE identified a large number of genes that were upregulated both at the onset and peak of clinical disease (FIG. 2A-2B). When all 3 genotypes were combined a total of 449 unique qualifiers were upregulated in CNS tissue (FIG. 2A), while 288 unique qualifiers were downregulated (FIG. 2B). Analysis of individual genotypes identified 233, 180 and 346 qualifiers that were upregulated in WT, IFN-γ -/- and IL-12p35 -/- animals, respectively (FIG. 2A). Of this subset, 86 (19%) genes were upregulated in all three genotypes, while only 8 (2.5%) genes were downregulated in all three genotypes. Not surprisingly, unique expression profiles were obtained for both IFN-γ -/- and IL-12p35 -/-mice during the course of disease with 48% and 41% of the genes regulated in IFN-γ -/- and IL-12p35 -/- mice respectively not regulated in WT animals. These gene sets may provide valuable information about immunoregulatory pathways under the direct control of IFN-γ and IL-12. However, as the frequency of null mutations in IFN-γ or IL-12p35 in MS patients is likely to be low, we would not expect a significant role for these genes in the pathogenesis of MS. We have therefore concentrated further analysis on genes that were similarly regulated in all three genotypes. Of the 233 genes that were upregulated in CNS tissue from WT mice with EAE, 203 (87%) were also upregulated in IL-12p35 -/-mice and 94 (40%) were also upregulated in IFN-γ -/- mice. Thus, sixty percent of genes regulated in WT animals during the course of disease are dependent on IFN-γ, confirming the importance of this pleiotropic cytokine in the local immune response within the CNS. There were 116 genes that were dependent on IFN-γ for induction. Of these, at least 22 of the 55 most highly regulated genes (>5 fold increase in expression) encompassed members of the MHC complex as well as genes reported in the literature to be regulated by IFN-γ. These genes showed similar expression patterns in both IL-12p35 -/- and wild-type mice consistent with previous observation that
IFN-γ levels in the CNS of mice with clinical EAE are similar in both genotypes. Becher et al., J Clin Invest 2002;110(4):493-7; Gran et al., J Immunol 2002;169(12):7104-10.
The expression patterns for 6 representative genes (LCN2, S100A8, CD53, TYROBP, SlOOAIl and ClQB) (FIGs. 3A-3G) that were significantly induced in all 3 genotypes were independently confirmed by quantitative RT-PCR (FIGs. 4A-4G). The high correlation between microarray and Taqman analysis confirms that these genes are differentially expressed, corroborating the quality of the data.
Identification of disease regulated genes in CNS and spleen. Data from experimental models of MS have highlighted the critical role of lymphocytes in the pathogenesis of disease. In EAE, CD4+ T cells found in secondary lymphoid organs migrate to the CNS (Ransohoff et al., Nat Rev Immunol 2003;3(7):569-81 ; Flugel et al., Immunity 2001;14(5):547-60), where they undergo re-activation following recognition of antigen. Ransohoff et al., Nat Rev Immunol 2003;3(7):569-81; Flugel et al., Immunity 2001;14(5):547-60. This re-activation results in the production of multiple inflammatory mediators, which facilitate further influx of inflammatory cells including mononuclear cells, predominately CD4+, CD8+ T cells and macrophages. Steinman et al., Annu Rev Neurosci 2002;25:491-505; Raine et al., Lab Invest 1990;63(4):476-89; Hickey et al., J Neurosci Res 1991;28(2):254-60; Krakowski et al., Eur J Immunol 1997;27(11):2840-7. In an attempt to determine the fraction of genes regulated in the CNS that could be attributed to infiltration of activated immune cells additional expression analysis was performed on spleen samples taken from the same animals during the course of disease. A significant fraction (17/86) of the genes, upregulated in CNS during EAE, were also upregulated in spleens of the same animals. These genes may be related to the influx of the inflammatory cells to the CNS or related to inflammatory processes, which occur both in spleen and in CNS (Table 2). Further experiments will determine if potential therapeutic targets are specifically enriched among these genes.
Expression analysis in chronic and acute MS lesions. EAE is a widely used model for studying the pathogenesis of MS and as such has been extensively used for target identification and validation. We wished to further validate the potential importance of the 86 genes selected in our analysis and determine if transcriptional regulation in EAE is comparable to transcriptional regulation in MS. The MS lesions in this study fell into two categories; 12 samples were from chronic MS lesions characterized by demyelination and absence of ORO+ macrophages and 2 samples were from active MS lesions characterized by demyelination accompanied by ORO+ macrophages. We have performed transcriptional profiling using Affymetrix U95Av2 arrays and compared it to the profile obtained from 4 normal CNS tissues (Manuscript in preparation). In order to determine if the 86 EAE regulated genes were also regulated in MS we needed to identify the homologous human U95 Av2 qualifiers. We identified human homologs for 71/86 EAE upregulated genes of which 60 were represented on the Affymetrix U95Av2 arrays. A significant fraction, 24/60, of these human genes is regulated in the CNS tissues from MS patients and 23 of these were upregulated (Table 1). Given the differences between EAE and MS, it is extremely encouraging that such a high fraction of overlapping regulated genes exist and it is suggestive that our murine model accurately reflect MS disease and that the gene targets identified will likely translate into MS. Future studies using larger number of samples with acute lesions will help generate further information. Finally, identifying genes that do overlap between EAE and MS may help prioritize genes for potential drug targets.
Discussion
Using gene expression profiling of CNS of mice with three different genotypes, wild-type, IFN-γ -/- and IL-12p35 -/-, we have identified a set of genes whose transcripts are increased in all three mouse strains during EAE development. Significant differences in gene expression exist between the three mouse strains during EAE development, but not between naive animals. This suggests that the differences in gene expression are due to different modes of induction of EAE. However, the genes regulated in all three mouse strains are likely to play the most critical role in EAE development. A significant fraction of these genes are also upregulated in MS lesions, further confirming their importance.
Our observation that IL-12p35 -/-mice develop EAE with similar severity and incidence confirms previously published findings indicating that IL-12p35 is dispensable for EAE induction. Becher et al, J Clin Invest 2002; 110(4):493-7; Gran et al, J Immunol 2002;169(12):7104-10. However, our observation that IFN-γ -I- mice develop EAE similar to WT differs from some previous reports. Ferber et al, J Immunol 1996;156(l):5-7; Krakowski et al, Eur J Immunol 1996;26(7):1641-6; Tran et al, J Immunol 2000;164(5):2759-68; Willenborg et al, J Immunol 1996;157(8):3223-7; Willenborg et al., J Immunol 1999;163(10):5278-86. The differences may be attributed to different genetic backgrounds and antigens used to induce EAE in different studies. Most studies that report increased severity of EAE in IFN-γ -/- and IFN-γR -/- mice use mouse strains (BALB/c and 129/Sv) that are normally not susceptible to EAE. Ferber et al., J Immunol 1996;156(l):5-7; Krakowski et al., Eur J Immunol 1996;26(7):1641-6; Tran et al, 2000, supra; Willenborg et al., 1996 supra; Willenborg et al., 1999, supra. In these mice EAE is characterized by a fulminant clinical course and the lesions are dominated by neutrophils. Limited data are reported on clinical course of EAE in IFN-γ -/- mice in EAE susceptible strains like B6 or B 10.PL. Ferber et al., 1996, supra; Chu et al., J Exp Med 2000;192(l):123-8. EAE induced with MBP in IFN-γ -/- B10.PL mice, a strain similar to B6, develop similar histological and clinical severity as WT animals. However, an increase in mortality was observed in IFN-γ -/- mice during a prolonged course of EAE. Ferber et al, 1996, supra. We have not observed an increase in mortality in the IFN-γ -/- animals used in these studies. In our experiments, EAE was relatively mild. As a result, most of the mice with an EAE score of 3 or 4 were euthanized to collect CNS and spleen samples during peak EAE. Some were allowed time to partially recover but were euthanized 9-12 days after onset (FIG. 1). Therefore, most animals that developed relatively severe EAE with a score of 3 or more were not permitted time to develop lethal EAE. hi an independent experiment, in which a more severe EAE was induced and the animals were followed through full disease development, a higher mortality in the IFN-γ -/- animals was observed. In that experiment, 40% of the IFN-γ -/- animals died or needed to be euthanized while none of the WT mice developed lethal EAE. hi none of the experiments was the fulminant course of disease observed, which is consistent with other reports on EAE development in IFN-γ -/- mice on B6 or BlO background. Ferber et al., 1996 supra; Chu et al., 2000, supra. Our finding indicates that IFN-γ may not always be protective in EAE. It is more likely that the role of IFN-γ in EAE depends on the mouse strain, severity of EAE, as well as antigen used to induce the disease.
The mechanism of altered course and severity of EAE in IFN-γ -/- and IFN- γR -/- mice is not completely understood. IFN-γ reduces T cell proliferation and
IFN-γ -/- B6 mice accumulate increased numbers of activated CD4+ T cells in their CNS during EAE. Chu et al, 2000, supra. In addition, IFN-γ -/- Balb/c mice have increased antigen-specific T cell proliferation during EAE. Tran et al, 2000; supra. IFN-γ -/- Balb/c mice also have increased neutrophil attracting chemokines, MIP-2 and TCA-3, in diseased CNS but have no increase in mononuclear cells chemoattractants, RANTES and MCP-I . Tran et al, 2000; supra. The differences in chemokine expression and numbers of infiltrating leukocytes may account for changes in the inflammatory effector functions during EAE development in IFN-γ -/- mice.
After determining that the genetically modified mouse strains, IFN-γ -/- and IL-12p35 -/- develop clinically similar EAE to WT mice, we used gene expression profiling to identify genes upregulated in all three strains. We expected that by focusing on genes which are upregulated in all three strains, we will be able to identify genes of the greatest importance for EAE development. There were 86 genes upregulated in all three genotypes. We first tried to determine if genes already identified as important in EAE are enriched in this subset. As expected, our 86-gene list did not include many genes identified in other microarray studies of EAE. Previous studies identified many genes regulated by IFN-γ (Ibrahim et al, Brain 2001;124(Pt 10):1927-38; Carmody et al., J Neuroimmunol 2002;133(l-2):95-107), most of which would have been eliminated from our analysis. Furthermore, our large sample size allowed statistical significant detection of subtle changes (1.5-fold) and the use of a more complete microarray allowed many additional genes to be detected. Since EAE is an inflammatory disease, it is not surprising that the majority of regulated genes have a role in the inflammatory response. A large fraction of these regulated genes have been reported to be involved in antigen processing and presentation (11/86), or are members of complement pathway (8/86). MHC molecules, which are involved in antigen presentation, and members of complement pathway have been shown in multiple studies to play an important role in EAE development. Sriram et al, J Immunol 1987; 139(5): 1485-9; Sriram et al, J Exp Med 1983;158(4):1362-7; Jonker et al, JAutoimmxui 1988;1(5):399-414; Morariu et al, Ann Neurol 1978;4(5):427-30; Linington et al, Brain 1989;112 ( Pt 4):895-911; Davoust et al, J Immunol 1999;163(12):6551-6; Boos et al, J Immunol 2004;173(7):4708-14. We have therefore further analyzed the set of 86 upregulated genes in an attempt to identify additional genes, which may be important in EAE development.
Several of the 86 regulated genes, including PSMEl, IFI30, and cathepsin family members, have been suggested to play a role in antigen processing and presentation. PSMEl is a part of the 1 IS regulator of the immunoproteasome, which is required for efficient antigen processing. Yamano et al, J Exp Med 2002;196(2):185-96. IFI30, also known as GILT (gamma-interferon inducible lysosomal thiol reductase) reduces disulfide bonds in antigens in preparation for further proteolysis and presentation by antigen presenting cells (APC). It is expressed constitutively in APC and inducible by IFN-γ in other cell types. Arunachalam et al, Proc Natl Acad Sd U S A 2000;97(2):745-50. Mice lacking IFBO are defective in processing antigens containing multiple disulfide bonds such as hen egg lysozyme. Marie et al, Science 2001;294(5545):1361-5. Five members of the cathepsin cysteine proteinases (A, S, C, H and Z) are all regulated in EAE-affected CNS, of which cathepsin S has been shown to be important for processing of the MHC class II invariant chain and trafficking and maturation of MHC class II molecules. Driessen et al, J Cell Biol 1999;147(4):775-90. It is possible that at least some of these genes play an important role in processing myelin proteins, thus allowing their presentation to the encephalitogenic T cells. Besides many genes associated with MHC and antigen processing, at least 14 additional genes (Table 1) seem to have their expression restricted mainly to the cells of hematopoietic origin. Several of these have already been shown to be important in EAE and/or immune response development. DAP- 12, CD52 and PNP can be considered potential therapeutic targets for EAE. Banti et ah, hit Immunopharmacol 2002;2(7):913-23; Bakker et al, Immunity 2000;13(3):345-53; Confavreux et al., Clin Neurol Neurosurg 2004;106(3):263-9. DAP12 -/- mice were shown to be resistant to EAE induced by immunization with myelin oligodendrocyte glycoprotein (MOG) peptide Bakker et al, Immunity 2000;13(3):345-53. Resistance was associated with a strongly diminished production of IFN-γ by myelin-reactive CD4+ T cells due to inadequate T cell priming in vivo. Bakker et al., 2000, supra. CD52 is highly expressed on lymphocytes and monocytes and antibody treatments have resulted in depletion of lymphocytes and in suppression of clinical and MRI inflammatory activity in MS patients Confavreux et al., Clin Neurol Neurosurg 2004;106(3):263-9. Humanized monoclonal antibodies against CD52 are currently being developed for treatment of MS. Id. PNP (purine-nucleoside phosphorylase) deficient mice have defective T cell development and functions Arpaia E et ah, J Exp Med 2000;191(12):2197-208. Patients with PNP deficiency suffer from recurring infections and lymphopenia associated with reduced T-cell counts. Inhibitors of PNP may thus act as T-cell selective immunosuppressive agents. Banti et ah, Int Immunopharrnacol 2002;2(7):913-23; Giblett et al., Lancet 1975;l(7914):1010-3.
CD53 and LCPl are also genes primarily expressed in cells of hematopoietic origin and have been associated with defects in immune functions, strongly suggesting that their expression may play a role in regulating immune responses in EAE. CD53 deficiency has been associated with a familiar syndrome of recurrent heterogeneous infectious diseases, caused by bacteria, fungi, and viruses. Mollinedo et al., Clin Diagn Lab Immunol 1997;4(2):229-31. Defect in LCPl (lymphocyte cytosolic protein 1) in mice results in impaired neutrophil functions. Neutrophils in these mice are unable to generate an adhesion-dependent respiratory response burst because of a markedly diminished integrin-dependent syk activation. Chen et al., 2003, supra. Since neutrophils have been shown to be important in effector phase of EAE, and depletion of neutrophils results in a reduction of EAE symptoms (McCoIl et ah, J Immunol 1998;161(ll):6421-6), therapeutics that affect neutrophil functions would be beneficial in treatment of EAE and MS. Because of their association with impaired immune responses in vivo, we believe that both CD53 and LCPl represent potential targets for manipulation of immune responses. In addition, CSFlR or its ligand, CSF-I (M-CSF, macrophage colony-stimulating factor) can be therapeutic targets in EAE based on their role in survival and proliferation of macrophages, an important effector cell in EAE and MS. Also, CSF-I expression has been shown to be upregulated in CNS of rats with EAE. Hulkower et ah, J Immunol 1993;150(6):2525-33. Finally, it has recently been shown that CSF-I deficient mice of MRL-Faslpr background have dramatically reduced autoimmune pathology, suggesting a role for CSF-I in autoimmune lupus. Lenda et ah, J Immunol 2004;173(7):4744-54.
Among the 86 genes identified in this study were also several genes related to lipid metabolism and/or binding of the serum lipoproteins and cholesterol (SAA3, ApoD, LCN2 and ADRP). Vogt et ah, JMoI Recognit 2001;14(l):79-86; Nilsen- Hamilton et al, Ann N Y Acad Sci 2003;995:94-108; Goessling et al, Am J Physiol Gastrointest Liver Physiol 2000;279(2):G356-65; Imamura et al, Am J Physiol Endocrinol Metab 2002;283(4):E775-83. The potential immunoregulatory role of genes encoding molecules involved in lipid metabolism has been suggested by recent findings that statins, which are inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A reductase, and cholesterol-lowering drugs have immunomodulatory effects. Neuhaus et al, Lancet Neurol 2004;3(6):369-71. These molecules involved in lipid metabolism can also play immunoregulatory roles in EAE.
Several genes (S100A6, S100A8 and SlOOAIl, ANXA2) encoding SlOO proteins or SlOO interacting proteins are upregulated in EAE. SlOO are small, acidic . proteins (10-12kDa), which mostly act intracellularly as Ca2+ signaling or Ca2+ buffering proteins. Marenholz et al, Biochem Biophys Res Commun 2004;322(4): 1111-22. Annexin A2 (ANXA2, lipocortin II) forms tetramers with S 100Al 0. Most S 100 proteins form homo- and heterodimers in solution upon Ca - binding. Heterodimers are formed with a variety of other proteins including other SlOO family members, annexin, annexin π, or peptides derived from p53, CapZ or Ndr-kinase. Id. Interestingly, some SlOO proteins are secreted and can act as cytokines. The S100A8/A9 heterodimer acts as chemokine in inflammation, probably binding the receptor for advanced glycation end products (RAGE). Id. In vivo blockade of RAGE has been shown to suppress disease in several EAE models. Yan et al, Nat Med 2003;9(3):287-93.
At least two additional genes, identified in this study, metallothionein-I and - II, have already been shown to have a protective role in EAE. Methallothioneins are low molecular weight, cysteine-rich, stress response proteins that can act as immunosuppressive agents in antigen-dependent adaptive immunity. Coyle et al,
Cell MoI Life Sci 2002;59(4):627-47. Metallothionein-I and -II can interact with cells of the immune system and modify their functional activities. Id. Elevated levels of metallothionein-I and -II have been found to be associated with rheumatoid arthritis (RA) (Youn et al, Clin Exp Immunol 2002;129(2):232-9), EAE (Penkowa et al, GHa 2000;32(3):247-63), and MS lesions (Penkowa et al, Cell MoI Life Sci
2003;60(6):1258-66). Metallothionein treatment reduces the incidence and severity of EAE as well as collagen-induced arthritis. Youn et al., 2002, supra; Penkowa et al., 2000, supra. On the other hand, mice with targeted disruptions of metallothionein-I and -II genes have increased EAE incidence and severity after immunization with MOG. Penkowa et al, J Neuroimmunol 2001;l 19(2):248-60.
The finding that a large fraction of the 86 identified genes have already been shown to play a role in EAE or to be potential therapeutic targets for EAE, strongly suggests that these genes are additional therapeutic targets for demyelinating autoimmune diseases such as EAE and MS.
A number of embodiments of the invention have been described. Nevertheless, it will be understood that various modifications may be made without departing from the spirit and scope of the invention. Accordingly, other embodiments are within the scope of the following claims.

Claims

WHAT IS CLAIMED IS:
1. A method of evaluating a test compound, comprising: contacting the test compound to a target protein comprising a polypeptide encoded by a gene selected from Table 1, or a fragment or functional domain thereof; evaluating an interaction between the test compound and the target protein; and evaluating the test compound in an animal or cell-based model.
2. The method of claim 1 , wherein the test compound is a polypeptide.
3. The method of claim 1 , wherein the test compound is an immunoglobulin.
4. The method of claim 1 , wherein the target protein is at least 50% purified.
5. The method of claim 1 , wherein the polypeptide is selected from the group consisting of: proteasome 28 subunit alpha (PSMEl), a component of complement, a component of the extracellular matrix, a polypeptide related to hematopoiesis, a polypeptide related to lipid metabolism, a regulator of transcription, and a plastin.
6. The method of claim 5, wherein the polypeptide related to hematopoiesis is CD53 antigen, CD68 antigen, lysosomal-associated protein transmembrane 5 (LAPTM5), lymphocyte antigen 86 (LY86), macrophage expressed gene 1 (MPEGl), or small inducible cytokine A6 (SCYA6).
7. The method of claim 5, wherein the polypeptide related to lipid metabolism is lipocalin 2 (LCN2).
8. The method of claim 5, wherein the plastin is plastin 2 (PLS2).
9. The method of claim 1 , wherein the contacting is effected in a cell-free system.
10. The method of claim 1 , wherein the step of evaluating an interaction comprises detecting binding between the test compound and the target protein.
11. The method of claim 1 , wherein the step of evaluating an interaction comprises evaluating an enzymatic activity of the target protein in the presence of the test compound.
12. The method of claim 10, wherein the test compound is selected for evaluation in an animal or cell-based model.
13. The method of claim 1 , wherein the cell-based model is a cell comprising a recombinant reporter construct.
14. The method of claim 1, wherein the animal model is an experimental autoimmune encephalomyelitis (EAE) mouse.
15. The method of claim 1 , wherein the disorder is multiple sclerosis.
16. The method of claim 1 , wherein the step of evaluating the test compound in an animal model comprises evaluating the animal model for one or more symptoms associated with the disorder.
17. The method of claim 1 , wherein the method is repeated for one or more of a plurality of compounds from a chemical library.
18. A method of evaluating a test compound, the method comprising: contacting the test compound to a mammalian cell; and evaluating expression of a gene chosen from one or more genes in Table 1 in the mammalian cell.
19. The method of claim 18, further comprising evaluating the test compound in an animal or cell-based model of a disorder.
20. The method of claim 18, wherein the chosen gene is selected from the group consisting of: proteasome 28 subunit alpha (PSMEl), a component of complement, a component of the extracellular matrix, a polypeptide related to hematopoiesis, a polypeptide related to lipid metabolism, a regulator of transcription, and a plastin.
21. The method of claim 20, wherein the polypeptide related to hematopoiesis is CD53 antigen, CD68 antigen, lysosomal-associated protein transmembrane 5 (LAPTM5), lymphocyte antigen 86 (LY86), macrophage expressed gene 1 (MPEGl), or small inducible cytokine A6 (SCYA6).
22. The method of claim 20, wherein the polypeptide related to lipid metabolism is lipocalin 2 (LCN2).
23. The method of claim 20, wherein the plastin is plastin 2 (PLS2).
24. The method of claim 19, wherein the disorder is multiple sclerosis.
25. A method of evaluating and using a test compound, the method comprising: contacting the test compound to a target protein comprising a polypeptide encoded by a gene selected from Table 1, or a fragment or functional domain thereof; evaluating an interaction between the test compound and the target protein; and administering the test compound to a subject that has a disorder.
26. The method of claim 25, wherein the gene encodes a polypeptide selected from the group consisting of: proteasome 28 subunit alpha (PSMEl), a component of complement, a component of the extracellular matrix, a polypeptide related to hematopoiesis, a polypeptide related to lipid metabolism, a regulator of transcription, and a plastin.
27. The method of claim 26, wherein the polypeptide related to hematopoiesis is CD53 antigen, CD68 antigen, lysosomal-associated protein transmembrane 5 (LAPTM5), lymphocyte antigen 86 (LY86), macrophage expressed gene 1 (MPEGl), or small inducible cytokine A6 (SCYA6).
28. The method of claim 26, wherein the polypeptide related to lipid metabolism is lipocalin 2 (LCN2).
29. The method of claim 26, wherein the plastin is plastin 2 (PLS2).
30. The method of claim 25, wherein the disorder is multiple sclerosis.
31. A method of treating or preventing a disorder in a subj ect, the method comprising administering to the subject an effective amount of an agent that modulates expression or activity of one or more genes selected from Table 1.
32. The method of claim 31, wherein the one or more genes are selected from the group consisting of: proteasome 28 subunit alpha (PSMEl), a component of complement, a component of the extracellular matrix, a polypeptide related to hematopoiesis, a polypeptide related to lipid metabolism, a regulator of transcription, and a plastin.
33. The method of claim 32, wherein the polypeptide related to hematopoiesis is CD53 antigen, CD68 antigen, lysosomal-associated protein transmembrane 5 (LAPTM5), lymphocyte antigen 86 (LY86), macrophage expressed gene 1 (MPEGl), or small inducible cytokine A6 (SCYA6).
34. The method of claim 32, wherein the polypeptide related to lipid metabolism is lipocalin 2 (LCN2).
35. The method of claim 32, wherein the plastin is plastin 2 (PLS2).
36. The method of claim 31 , wherein the disorder is multiple sclerosis.
37. A method of evaluating a subject, comprising evaluating expression or activity of at least one gene chosen from Table 1.
38. The method of claim 37, wherein the gene is selected from the group consisting of: proteasome 28 subunit alpha (PSMEl), a component of complement, a component of the extracellular matrix, a polypeptide related to hematopoiesis, a polypeptide related to lipid metabolism, a regulator of transcription, and a plastin.
39. The method of claim 38, wherein the polypeptide related to hematopoiesis is CD53 antigen, CD68 antigen, lysosomal-associated protein transmembrane 5 (LAPTM5), lymphocyte antigen 86 (LY86), macrophage expressed gene 1 (MPEGl), or small inducible cytokine A6 (SCYA6).
40. The method of claim 38, wherein the polypeptide related to lipid metabolism is lipocalin 2 (LCN2).
41. The method of claim 38, wherein the plastin is plastin 2 (PLS2).
42. The method of claim 37, wherein the subject has been diagnosed with multiple sclerosis.
43. A method of identifying a subj ect for treatment of an autoimmune disorder, the method comprising: evaluating the expression of one or more genes chosen from Table 1; and identifying the subject as a subject suited for treatment of an autoimmune disorder.
44. The method of claim 43, wherein the one or more chosen genes are selected from the group consisting of: proteasome 28 subunit alpha (PSMEl), a component of complement, a component of the extracellular matrix, a polypeptide related to hematopoiesis, a polypeptide related to lipid metabolism, a regulator of transcription, and a plastin.
45. The method of claim 44, wherein the polypeptide related to hematopoiesis is CD53 antigen, CD68 antigen, lysosomal-associated protein transmembrane 5 (LAPTM5), lymphocyte antigen 86 (LY86), macrophage expressed gene 1 (MPEGl), or small inducible cytokine A6 (SCYA6).
46. The method of claim 44, wherein the polypeptide related to lipid metabolism is lipocalin 2 (LCN2).
47. The method of claim 44, wherein the plastin is plastin 2 (PLS2).
48. The method of claim 43, wherein the autoimmune disorder is multiple sclerosis.
49. A method of monitoring efficacy of a treatment for a disorder, the method comprising: treating a subject having the disorder; and evaluating expression or activity of one or more genes chosen from Table 1 in cells of the subject.
50. The method of claim 49, wherein one or more chosen genes are selected from the group consisting of: proteasome 28 subunit alpha (PSMEl), a component of complement, a component of the extracellular matrix, a polypeptide related to hematopoiesis, a polypeptide related to lipid metabolism, a regulator of transcription, and a plastin.
51. The method of claim 50, wherein the polypeptide related to hematopoiesis is CD53 antigen, CD68 antigen, lysosomal-associated protein transmembrane 5 (LAPTM5), lymphocyte antigen 86 (LY86), macrophage expressed gene 1 (MPEGl), or small inducible cytokine A6 (SCYA6).
52. The method of claim 50, wherein the polypeptide related to lipid metabolism is lipocalin 2 (LCN2).
53. The method of claim 50, wherein the plastin is plastin 2 (PLS2).
54. The method of claim 49, wherein the disorder is multiple sclerosis.
55. A method of identifying a target gene involved in a disorder, the method comprising: providing a plurality of animal models of the disorder; evaluating gene expression of one or a plurality of genes in the animal models; and evaluating whether one or more genes are similarly regulated.
56. The method of claim 55, wherein the disorder is multiple sclerosis.
57. The method of claim 55, wherein one animal model is experimental autoimmune encephalomyelitis.
58. An array comprising a substrate having a plurality of addresses, wherein each address comprises a capture probe that hybridizes specifically to a gene chosen from Table 1.
59. The array of claim 58, wherein the plurality of addresses comprises addresses having nucleic acid capture probes for all genes of Table 1.
60. The array of claim 58, wherein the plurality of addresses comprises addresses having nucleic acid capture probes for a fraction of the genes of Table 1.
61. A method of evaluating a subj ect, comprising: providing a sample from the subject; and determining the sample expression profile, wherein the profile comprises one or more values representing the level of expression of one or more genes chosen from Table 1.
62. The method of claim 61 , wherein the subj ect has, or is disposed to having, multiple sclerosis.
63. A method of making a decision, comprising: providing a value which is a function of the expression of at least one gene selected from Table 1; and if the value meets a predetermined criterion, assigning a subject to a first class, thereby making a decision.
64. A method of making a decision, the method comprising: providing an evaluation of whether a subject is an enhanced responder or a non-enhanced responder; and performing at least one of: (a) if the subject is an enhanced responder, selecting a first outcome, and (b) if the subject is a non-enhanced responder, selecting a second outcome, thereby making a decision.
65. A method of providing information on which to make a decision about a subject or making such a decision, the method comprising: providing an evaluation of a subject, wherein the evaluation was made by determining the level of expression of at least one gene selected from Table 1 in the subject, thereby providing a value; and providing a comparison of the value with a reference value, thereby, providing information on which to make a decision about the subject, or making such a decision.
66. A method of making a data record, the method comprising entering the result of a method of claims 63-65 into a record.
67. A data record, wherein the record includes results from a method of claims 63-65.
68. A method of providing data, the method comprising providing data generated by a method of claims 63-66 to provide a record for determining if a payment will be provided.
69. A record comprising a list and value of expression for at least one gene selected from Table 1.
70. A method of transmitting the record of claim 67 or 69, the method comprising a first party transmitting the record to a second party.
71. A method of providing data, the method providing hybridization data from contacting an array of claims 58-60 with a nucleic acid sample derived from a subject, and providing a record of such data.
72. A method of providing information about a subject's level of expression of at least one gene selected from Table 1 to a third party, the method comprising communicating information about the subject's level of expression of at least one gene selected from Table 1 to the third party.
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