WO2005124342A2 - Methods and means for treatment of osteoarthritis - Google Patents

Methods and means for treatment of osteoarthritis Download PDF

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Publication number
WO2005124342A2
WO2005124342A2 PCT/EP2005/052875 EP2005052875W WO2005124342A2 WO 2005124342 A2 WO2005124342 A2 WO 2005124342A2 EP 2005052875 W EP2005052875 W EP 2005052875W WO 2005124342 A2 WO2005124342 A2 WO 2005124342A2
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polypeptide
seq
cells
acid sequence
chondrocytes
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PCT/EP2005/052875
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French (fr)
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WO2005124342A3 (en
Inventor
Nick Vandeghinste
Peter Herwig Maria Tomme
Frits Michiels
Libin Ma
Blandine Mille-Baker
Helmuth H.G. Van Es
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Galapagos N.V.
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Priority to JP2007515968A priority Critical patent/JP2008503712A/en
Priority to EP05760933A priority patent/EP1759203A2/en
Publication of WO2005124342A2 publication Critical patent/WO2005124342A2/en
Publication of WO2005124342A3 publication Critical patent/WO2005124342A3/en

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    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
    • C07H21/04—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids with deoxyribosyl as saccharide radical
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
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    • A61P19/00—Drugs for skeletal disorders
    • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
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    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
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    • 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
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    • 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
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    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6887—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids from muscle, cartilage or connective tissue
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/14—Type of nucleic acid interfering N.A.
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2320/00—Applications; Uses
    • C12N2320/10—Applications; Uses in screening processes
    • C12N2320/12—Applications; Uses in screening processes in functional genomics, i.e. for the determination of gene function
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    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/136—Screening for pharmacological compounds
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    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/10—Musculoskeletal or connective tissue disorders
    • G01N2800/105—Osteoarthritis, e.g. cartilage alteration, hypertrophy of bone
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10T—TECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION
    • Y10T436/00—Chemistry: analytical and immunological testing
    • Y10T436/14—Heterocyclic carbon compound [i.e., O, S, N, Se, Te, as only ring hetero atom]
    • Y10T436/142222—Hetero-O [e.g., ascorbic acid, etc.]
    • Y10T436/143333—Saccharide [e.g., DNA, etc.]

Definitions

  • the invention relates to the field of medicinal research, cartilage physiology and diseases involving the degeneration of cartilage tissue. More specifically, the invention relates to methods and means for identifying compounds that stimulate anabolic processes in chondrocytes and that typically induce the synthesis of cartilage. The invention also relates to the compounds that are useful in the treatment of osteoarthritis.
  • Cartilage is an avascular tissue made up largely of cartilage-specific cells, the chondrocytes, proteoglycans and collagen proteins, which are structural proteins that provide structural strength to connective tissue, such as skin, bone and cartilage.
  • Collagen ⁇ together with the protein collagen IX, forms a "biological alloy", which is molded into a fibril-like structure and is arranged in a precise network, providing cartilage with great mechanical strength.
  • the chondrocytes in normal articular cartilage occupy approximately 5% of the tissue volume, while the extra-cellular matrix makes up the remaining 95% of the tissue.
  • the chondrocytes secrete the components of the matrix, which in turn supplies the chondrocytes with an environment suitable for their survival under mechanical stress. Breakdown of articular cartilage, which is part of joints and which cushions the ends of the bones, causes the bones to rub against each other leading to pain and loss of movement.
  • Cartilage degradation may also be the result of an imbalance in cartilage synthesizing (anabolic) and cartilage degrading (catabolic) processes. Unlike most tissues, cartilage does not self-repair following injury. The inability of cartilage to self-repair after injury, disease, or surgery is a major limiting factor in rehabilitation of degrading joint surfaces and injury to meniscal cartilage. There are many diseases involving the degeneration of cartilage. Rheumatoid arthritis and osteoarthritis are among the most prominent. Osteoarthritis (also referred to as OA, or as wear-and-tear arthritis) is the most common form of arthritis and is characterized by loss of articular cartilage, often associated with hypertrophy of the bone.
  • the disease mainly affects hands and weight-bearing joints such as knees, hips and spines. This process thins the cartilage through a phenomenon called apoptosis, or programmed cell death.
  • apoptosis or programmed cell death.
  • the surface area has disappeared due to the thinning, there is a grade I osteoarthritis; when the tangential surface area has disappeared, there is a grade two osteoarthritis.
  • the clinical manifestations of the development of the osteoarthritis condition are: increased volume of the joint, pain, crepitation and functional disability that, gradually and steadily, first hinders the performance of lengthy walks and forced flexion and extension movements, depending on the affected joint, and then pain and limitation of minimum efforts emerge as well as pain at rest which interrupts sleeping. If the condition persists without correction and/or therapy, the joint is destroyed, leading the patient to major replacement surgery with total prosthesis, or to disability.
  • Therapeutic methods for the correction of the articular cartilage lesions that appear during the osteoarfhritic disease have been developed, but so far none of them have been able to achieve the regeneration of articular cartilage in situ and in vivo.
  • Osteoarthritis is difficult to treat. At present, no cure is available and treatment focuses on relieving pain and preventing the affected joint from becoming deformed. Common treatments include the use of non-steroidal anti-inflammatory drugs (NSAID's), which are often used to relieve pain, while specific COX-2 inhibitors are used to relieve severe pain. Medicines such as chondroitin and glucosamine are thought to improve the cartilage itself. These treatments may be relatively successful, but not a substantive amount of research data is available. In severe cases, joint replacement may be necessary. This is especially true for hips and knees. If a joint is extremely painful and cannot be replaced, it may be fused. This procedure stops the pain, but results in the permanent loss of joint function, making walking and bending difficult.
  • NSAID's non-steroidal anti-inflammatory drugs
  • COX-2 inhibitors are used to relieve severe pain.
  • Medicines such as chondroitin and glucosamine are thought to improve the cartilage itself.
  • the treatment that has 74% to 90% effectiveness and produces excellent results is the transplantation of cultured autologous chondrocytes, by taking chondral cellular material from the patient, sending it to a laboratory where it is seeded in a proper medium for its proliferation, and, once enough volume is achieved after a variable period that may last from weeks to months, transporting it in a special container and implanting it in the damaged tissues to cover the tissue's defects.
  • Another treatment includes the intraarticular instillation of Hylan G-F 20 (Synvisc, Hyalgan, Artz etc.), a substance that improves temporarily the rheology of the synovial fluid, producing an almost immediate sensation of free movement and a marked reduction of pain.
  • IGF-I Insulin-like growth factor-I
  • BMP bone morphogenetic protein
  • TGF.-b human trarisforrning growth factor-b
  • the present invention relates to a method for identifying compounds that induce cartilage-synthesizing processes, which lead to anabolic stimulation of chondrocytes, comprising contacting the compound with a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 101-128 and 401-594, or a functional fragment or derivative thereof, under conditions that allow said polypeptide to bind to the compound, and measuring a compound-polypeptide property related to the anabolic stimulation of chondrocytes.
  • the present invention also relates to expression inhibitory agents, pharmaceutical compositions comprising the same, methods for the in vitro production of cartilage tissue, and host cells expressing said agents.
  • aspects of the present method include the in vitro assay of compounds using polypeptide of a TARGET, and cellular assays wherein TARGET inhibition is followed by observing indicators of efficacy, mcluding collagen type ⁇ , alpha-1 (col2 ⁇ l) and aggrecan levels.
  • Another aspect of the invention is a method of treatment or prevention of a condition involving de-differentiation of chondrocytes and/or loss of cartilage thickness, in a subject suffering or susceptible thereto, by administering a pharmaceutical composition comprising an effective cartilage formation-enhancing amount of a TARGET inhibitor.
  • a further aspect of the present invention is a pharmaceutical composition for use in said method wherein said inhibitor comprises a polynucleotide selected from the group of an antisense polynucleotide, a ribozyme, and a small interfering RNA (siRNA), wherein said inhibitor comprises a nucleic acid sequence complementary to, or engineered from, a naturally occurring polynucleotide sequence encoding a polypeptide, comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 101-128 and 401-594, or a fragment thereof.
  • siRNA small interfering RNA
  • Another further aspect of the present invention is a pharmaceutical composition
  • a pharmaceutical composition comprising a therapeutically effective cartilage formation-enhancing amount of a TARGET inhibitor or its pharmaceutically acceptable salt, hydrate, solvate, or prodrug thereof in admixture with a pharmaceutically acceptable carrier.
  • the present polynucleotides and TARGET inhibitor compounds are also useful for the manufacturing of a medicament for the treatment of conditions involving de-differentiation of chondrocytes and/or cartilage 5 thickness loss.
  • the invention relates also to diagnostic methods.
  • Figure 1 Quantification of Col2 ⁇ l expression in primary human chondrocytes 12 days post 10 infection with the indicated viruses.
  • Figure 2. Example of duplicate screening results for Col2 ⁇ l expression of part of the SilenceSelect library.
  • Figure 3 Quantification of Col2 ⁇ l expression in primary human chondrocytes 12 days post infection with the indicated viruses.
  • Figure 4. Quantification of alcian blue staining on human primary chondrocytes, 12 days after infection with the indicated viruses, compared to uninfected cells.
  • Figure 5 (A-N): Quantification of aggrecan expression in primary human chondrocytes 12 days post infection with the indicated viruses.
  • Figure 6 On target analysis with different constructs targeting the indicated genes. 20. The on target analysis is assessed through detection of the Col2 ⁇ l expression in primary human chondrocytes 12 days post infection with the indicated viruses. Data are represented as luminescence units. The different thresholds corresponding to the different infection volumes are indicated as a line. Increasing the infection volumes leads to an increased threshold. 25
  • Figure 7 GAG analysis on chondrocytes in alginate cell culture 10 days post infection. The data are represented as relative GAG levels to the average of KD control 1 and KD control 2, being Ad-siRNA targeting PTGER4 and GRM7 respectively. Two individual data points are shown for every condition.
  • Figure 8 Hydroxyproline analysis on chondrocytes in alginate cell culture 10 days 30 post infection. The data are represented as relative hydroxyproline levels to the average of KD control 1 and KD control 2, being Ad-siRNA targeting PTGER4 and GRM7 respectively. Two individual data points are shown for every condition.
  • Figure 9 mRNA marker analysis on chondrocytes in alginate cell culture 10 days post infection. The cells are infected with either Ad5/ALPL and Ad5/BMP2 overexpressing viruses, and with an Ad-siRNA targeting the indicated gene. The data are represented as relative mRNA levels to the ALPL control.
  • the term "agent” means any molecule, including polypeptides, polynucleotides and small molecules.
  • anabolic stimulation of chondrocytes should be understood as inducing chondrogenesis or as inducing or enhancing the anabolic activity of chondrocytes. Anabolic stimulation takes place for instance by stimulating the synthesis of cartilage components, or inducing synthesis of components that are required for cartilage synthesis. "Anabolic stimulation of chondrocytes” may also be understood as a process in which the expression of the matrix Gla protein (MGP) is induced.
  • MGP matrix Gla protein
  • Anabolic stimulation of chondrocytes may furthermore be understood as inducing the expression of cartilage derived retinoic acid sensitive protein (CD-RAP), as inducing the expression of cartilage oligomeric matrix protein (COMP), as inducing the expression of aggrecan 1 (agcl, also termed chondroitin sulfate proteoglycan core protein 1, or CSPG1), or as inducing synthesis of collagen II, also known as collagen, type II, alpha ⁇ l (col2 ⁇ l), collagen of cartilage, chondrocalcin, and collagen, typg xi, alpha-3 (colll ⁇ 3).
  • CD-RAP cartilage derived retinoic acid sensitive protein
  • CAP cartilage oligomeric matrix protein
  • aggrecan 1 agcl, also termed chondroitin sulfate proteoglycan core protein 1, or CSPG1
  • collagen II also known as collagen, type II, alpha ⁇ l (col2 ⁇ l), collagen of cartilage,
  • the term 'antisense nucleic acid' refers to an oligonucleotide that has a nucleotide sequence that interacts through base pairing with a specific complementary nucleic acid sequence involved in the expression of the target such that the expression of the gene is reduced.
  • the specific nucleic acid sequence involved in the expression of the gene is a genomic DNA molecule or mRNA molecule that encodes (a part of) the gene. This genomic DNA molecule can comprise regulatory regions of the gene, or the coding sequence for the mature gene.
  • the term “assay” means any process used to measure a specific property of a compound.
  • a "screening assay” means a process used to characterize or select compounds based upon their activity from a collection of compounds.
  • binding affinity is a property that describes how strongly two or more compounds associate with each other in a non-covalent relationship. Binding affinities can be characterized qualitatively, (such as “strong”, “weak”, “high”, or “low”) or quantitatively (such as measuring the Kb).
  • carrier means a non-toxic material used in the formulation of pharmaceutical compositions to provide a medium, bulk and/or useable form to a pharmaceutical composition.
  • a carrier may comprise one or more of such materials such as an excipient, stabilizer, or an aqueous pH buffered solution.
  • physiologically acceptable carriers include aqueous or solid buffer ingredients including phosphate, citrate, and other organic acids; antioxidants including ascorbic acid; low molecular weight (less than about 10 residues) polypeptide; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and/or nonionic surfactants such as TWEENTM, polyethylene glycol (PEG), and PLURONICSTM.
  • aqueous or solid buffer ingredients including phosphate, citrate, and other organic acids; antioxidants including ascorbic acid; low molecular weight (less than about 10 residues) polypeptide
  • complex means the entity created when two or more compounds bind to each other.
  • compound is used herein in the context of a "test compound” or a "drug candidate compound” described in connection with the assays of the present invention.
  • these compounds comprise organic or inorganic compounds, derived synthetically or from natural sources.
  • the compounds include inorganic or organic compounds such is polynucleotides, lipids or hormone analogs that are characterized by relatively low molecular weights.
  • Other biopolymeric organic test compounds include peptides comprising from about 2 to about 40 amino acids and larger polypeptides comprising from about 40 to about 500 amino acids, such as antibodies or antibody conjugates.
  • condition or “disease” means the overt presentation of symptoms (i.e., illness) or the manifestation of abnormal clinical indicators (e.g., biochemical indicators). Alternatively, the term “disease” refers to a genetic or environmental risk of or propensity for developing such symptoms or abnormal clinical indicators.
  • contact or “contacting” means bringing at least two moieties together, whether in an in vitro system or an in vivo system.
  • de-differentiation refers to a general process wherein chondrocytes differentiate away from a cell phenotype that synthesizes cartilage components.
  • Such components include, but are not limited to, collagen II, aggrecan 1, versican, link protein, perlecan, SZP/lubricin, biglycan (DS-PGI), decorin (DS-PGII), epiphycan (DS-PGITQ, fibromodulin, lumican, CILP, C-type lectin, fibronectin, PRELP, COMP (thrombospondin- 5), thrombospondin-1 and -3, CMP (matrilin-1), matrilin-3, C-type lectin, fibronectin, condroadherin, tenascin-C, fibrillin, elastin, gp-39/YKL-40, matrix Gla protein/ MGP, pleiotrophin, chondromodulin-I/SCGP, chondromodulin-II, CD-RAP
  • the term "effective amount” or "therapeutically effective amounf ' means that amount of a compound or agent that will elicit the biological or medical response of a subject that is being sought by a medical doctor or other clinician.
  • the term “effective amount” is intended to mean an effective differentiation-promoting amount of an compound or agent that will bring about a biologically meaningful increase in the levels of chondrocyte markers, representative for the process of an increase in chondrocyte anabolism.
  • the term “expressible nucleic acid” means a nucleic acid coding for a proteinaceous molecule, an RNA molecule, or a DNA molecule.
  • endogenous shall mean a material that a mammal naturally produces.
  • Endogenous in reference to the term “protease”, “kjnase”, or G-Protein Coupled Receptor ("GPCR”) shall mean that which is naturally produced by a mammal (for example, and not limitation, a human).
  • GPCR G-Protein Coupled Receptor
  • non-endogenous in this context shall mean that which is not naturally produced by a mammal (for example, and not limitation, a human). Both terms can be utilized to describe both “in vivo” and “in vitro” systems.
  • the endogenous or non-endogenous TARGET may be in reference to an in vitro screening system.
  • expression inhibitory agent means a polynucleotide designed to interfere selectively with the transcription, translation and/or expression of a specific polypeptide or protein normally expressed within a cell. More particularly, “expression inhibitory agent” comprises a DNA or RNA molecule that contains a nucleotide sequence identical to or complementary to at least about 17 sequential nucleotides within the polyribonucleotide sequence coding for a specific polypeptide or protein.
  • Exemplary expression inhibitory molecules include ribozymes, double stranded siRNA molecules, self-complementary single- stranded siRNA molecules, genetic antisense constructs, and synthetic RNA antisense molecules with modified stabilized backbones.
  • expressible nucleic acid means a nucleic acid coding for a proteinaceous molecule, an RNA molecule, or a DNA molecule.
  • 'genetic antisense' as used herein refers to the incorporation of antisense constructs complementary to sequences of genes into the genome of a cell. Such inco ⁇ oration allows for the continued synthesis of the antisense molecule.
  • hybridization means any process by which a strand of nucleic acid binds with a complementary strand through base pairing.
  • hybridization complex refers to a complex formed between two nucleic acid sequences by virtue of the formation of hydrogen bonds between complementary bases.
  • a hybridization complex may be formed in solution (e.g., Co t or Ro t analysis) or formed between one nucleic acid sequence present in solution and another nucleic acid sequence immobilized on a solid support (e.g., paper, membranes, filters, chips, pins or glass slides, or any other appropriate substrate to which cells or their nucleic acids have been fixed).
  • stringent conditions refers to conditions that permit hybridization between polynucleotides and the claimed polynucleotides.
  • Stringent conditions can be defined by salt concentration, the concentration of organic solvent, e.g., formamide, temperature, and other conditions well known in the art. In particular, reducing the concentration of salt, increasing the concentration of formamide, or raising the hybridization temperature can increase stringency.
  • inhibitor or “inhibiting”, in relationship to the term “response” means that a response is decreased or prevented in the presence of a compound as opposed to in the absence of the compound.
  • inhibitor refers to the reduction, down regulation of a process or the elimination of a stimulus for a process that results in the absence or minimization of the expression of a protein or polypeptide.
  • induction refers to the inducing, up-regulation, or stimulation of a process that results in the expression of a protein or polypeptide, and that may also result in a phenotypical cellular change.
  • ligand means an endogenous, naturally occurring molecule specific for an endogenous, naturally occurring receptor.
  • pharmaceutically acceptable salts refers to the non-toxic, inorganic and organic acid addition salts, and base addition salts, of compounds of the present invention.
  • polynucleotide means a polynucleic acid, in single or double stranded form, and in the sense or antisense orientation, complementary polynucleic acids that hybridize to a particular polynucleic acid under stringent conditions, and polynucleotides that are homologous in at least about 60 percent of its base pairs, and more preferably 70 percent of its base pairs are in common, most preferably 90 per cent, and in a special embodiment 100 percent of its base pairs.
  • the polynucleotides include polyribonucleic acids, polydeoxyribonucleic acids, and synthetic analogues thereof.
  • polynucleotides are described by sequences that vary in length, that range from about 10 to about 5000 bases, preferably about 100 to about 4000 bases, more preferably about 250 to about 2500 bases.
  • a preferred polynucleotide embodiment comprises from about 10 to about 30 bases in length.
  • a special embodiment of polynucleotide is the polyribonucleotide of from about 10 to about 22 nucleotides, more commonly described as small interfering RNAs (siRNAs).
  • nucleic acids with modified backcartilages such as peptide nucleic acid (PNA), polysiloxane, and 2'-O-(2-methoxy)ethylphosphorothioate, or including non- naturally occurring nucleic acid residues, or one or more nucleic acid substituents, such as methyl-, thio-, sulphate, benzoyl-, phenyl-, amino-, propyl-, chloro-, and methanocarbanucleosides, or a reporter molecule to facilitate its detection.
  • polypeptide relates to proteins (such as TARGETS), proteinaceous molecules, fractions of proteins peptides and oligopeptides.
  • solvate means a physical association of a compound useful in this invention with one or more solvent molecules. This physical association includes hydrogen bonding. In certain instances the solvate will be capable of isolation, for example when one or more solvent molecules are incorporated in the crystal lattice of the crystalline solid.
  • Solvate encompasses both solution-phase and isolable solvates. Representative solvates include hydrates, ethanolates and methanolates.
  • subject includes humans and other mammals.
  • treating means an intervention performed with the intention of preventing the development or altering the pathology of, and thereby alleviating a disorder, disease or condition, including one or more symptoms of such disorder or condition. Accordingly, “treating” refers to both therapeutic treatment and prophylactic or preventative measures. Those in need of treating include those already with the disorder as well as those in which the disorder is to be prevented.
  • treatment refers to the act of treating a disorder, symptom, disease or condition, as the term “treating” is defined above.
  • the term 'vectors' also relates to plasmids as well as to viral vectors, such as recombinant viruses, or the nucleic acid encoding the recombinant virus.
  • vertebrate cells means cells derived from animals having vertera structure, including fish, avian, reptilian, amphibian, marsupial, and mammalian species. Preferred cells are derived from mammalian species, and most preferred cells are human cells. Mammalian cells include feline, canine, bovine, equine, caprine, ovine, porcine murine, such as mice and rats, and rabbits.
  • TARGETS are factors in the up-regulation and/or induction of anabolic processes of chondrocytes.
  • the term 'TARGET” or "TARGETS” means the proteins identified in accordance with the assay described below to be involved in the induction of the anabolic stimulation of chondrocytes.
  • the preferred TARGETS ⁇ are identified as SEQ TD NOS. 101- 128 and 401-594 in Table 1A.
  • Table 1A lists the polypeptides, polynucleotides and knockdown target sequences of the present invention.
  • Table IB lists exemplary fragments of the transmembrane protein TARGETS.
  • Table 1C lists exemplary KD target sequences useful in the practice of the present expression-inhibitory agent invention.
  • the present invention relates to a method for assaying for compounds that induce anabolic stimulation of chondrocytes, comprising contacting the compound with a polypeptide comprising an amino acid sequence of the polypeptides of SEQ ID NO: 101-128 ("TARGETS") or a functional fragment thereof under conditions that allow said polypeptide to bind to the compound, and detecting the formation of a complex between the polypeptide and the compound.
  • a polypeptide comprising an amino acid sequence of the polypeptides of SEQ ID NO: 101-128 (“TARGETS”) or a functional fragment thereof under conditions that allow said polypeptide to bind to the compound, and detecting the formation of a complex between the polypeptide and the compound.
  • TARGETS amino acid sequence of the polypeptides of SEQ ID NO: 101-128
  • One preferred means of measuring the complex formation is to determine the binding affinity of said compound to said polypeptide.
  • the invention relates to a method for identifying an agent that induces anabolic stimulation of chondrocytes, the method comprising further: (a) contacting a population of chondrocyte cells with one or more of said compound that exhibits binding affinity for said TARGETS, and (b) measuring a compound-polypeptide property related to the anabolic stimulation of chondrocytes.
  • the compound-polypeptide property referred to above is related to the anabolic stimulation of chondrocytes, and is a measurable phenomenon chosen by the person of ordinary skill in the art.
  • the measurable property may e.g.
  • chondrocytes can be the binding affinity for a peptide domain of the polypeptide TARGET or the level of any one of a number of biochemical marker levels of increased chondrocyte anabolism.
  • Anabolic stimulation of chondrocytes can e.g. be measured by measuring the level of proteins and other molecules that are induced during the differentiation process, such as key components of normal cartilage. In particular, the induction of the major protein component of cartilage, collagen ⁇ , is measured.
  • compound-polypeptide properties related to the anabolic stimulation of chondrocytes are measured in C20/A4; T/C-28a2; T/C-28a4; C-28/12; Ch-4,8,N; Ch-8-OA; TC6; MCT;MC615; IRC; RCS2; Hig82; and DI ORL UVA (DI) cells.
  • DI ORL UVA (DI) cells such porterties are also measured in non-chondrocyte cell systems.
  • in situ binding assays that determine the affinity of compounds to bind to polypeptides of the invention are performed using any cell type that expresses the polypeptide. Expression of the polypeptide is exogenous or endogenous.
  • any cell that contains the pathway cellular components is used to measure the compound-polypeptide property.
  • induction of col2 ⁇ l or aggrecan in chondrocytes is indicative of anabolic stimulation of chondrocytes.
  • non-chondrocyte cells can be engineered to contain a reporter molecule activated by the col2 ⁇ l or aggrecan promoters. In this way a non-chondrocyte can be used to measure a property indicative of anabolic stimulation of chondrocytes.
  • the invention relates to a method for identifying a compound that induces and/or increases anabolic stimulation of chondrocytes, said method comprising the steps of: culturing a population of cells expressing a polypeptide of any one of those listed in Table 1A, or a functional fragment or derivative thereof; determining a first level of chondrogenic differentiation in said population of cells; exposing said population of cells to a compound, or a mixture of compounds; deterrnining the level of chondrogenic differentiation in said population of cells during or after exposure of said population of cells to the compound, or the mixture of compounds; and identifying the compound that induces and/or increases chondrogenic differentiation.
  • the invention also relates to a method for identifying a compound that decreases the expression and/or activity of any one of the polypeptides listed in Table 1 A, said method comprising the steps of: culturing a population of cells expressing said polypeptide, or a fragment, or a derivative thereof; determining a first level of expression and/or activity of said polypeptide; exposing said population of cells to a compound, or a mixture of compounds; determining the level of expression and/or activity of said polypeptide during or after exposure of said population of cells to the compound, or the mixture of compounds; and identifying the compound that decreases the expression and/or activity of said polypeptide.
  • the identification of the compound may benefit from an extra step comprising exposing said population of cells to an agonist of said polypeptide.
  • the methods of the present invention may comprise the step of introducing a gene encoding any one of the polypeptides listed in Table 1A, in said population of cells.
  • the level of chondrocyte (re-)differentiation is determined by measuring the expression level of a marker gene, wherein a preferred marker gene encodes collagen type IT, alpha- 1 (col2 ⁇ l) or aggrecan.
  • the expression and/or activity of col2 ⁇ l or aggrecan is increased.
  • the present invention provides in one particular embodiment methods for identifying novel compounds, wherein the polypeptide is a GPCR. If so, the expression and/or activity of said GPCR is preferably determined by measuring the level of a second messenger.
  • Preferred second messengers are cyclic AMP, Ca 2+ or both.
  • the level of the second messenger is determined with a reporter gene under the control of a promoter that is responsive to the second messenger, wherein it is preferred that the promoter is a cyclic AMP-responsive promoter, an NF-KB responsive promoter, or a NF-AT responsive promoter, and wherein the reporter gene is selected from the group consisting of: alkaline phosphatase, GFP, eGFP, dGFP, luciferase and ⁇ - ga ⁇ tosidase.
  • the invention provides methods for identifying novel compounds, wherein the polypeptide is a kinase or a phosphatase.
  • the activity of said kinase or phosphatase is determined by measuring the level of phosphorylation of a substrate of said kinase or phosphatase.
  • the invention provides methods for identifying novel compounds, wherein the polypeptide is a protease.
  • the activity of said protease is measured by determining the level of cleavage of a substrate of said protease.
  • the compound that inhibits the polypeptide exhibits a binding affinity to the polypeptide of at most 10 micromolar.
  • the polypeptide TARGET comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 101-128 and 401- 594 (Tables 1A and IB).
  • the polypeptide TARGET comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 101-128 (Table 1A).
  • the present assay method may be designed to function as a series of measurements, each of which is designed to determine whether the drug candidate compound is indeed acting on the polypeptide to thereby induce the anabolic stimulation of chondrocytes.
  • an assay designed to determine the binding affinity of a compound to the polypeptide, or fragment thereof may be necessary, but not sufficient, to ascertain whether the test compound would be useful for increasing mean cartilage thickness when administered to a subject. Nonetheless, such binding information would be useful in identifying a set of test compounds for use in an assay that would measure a different property, further up the biochemical pathway, such as cartilage component synthesis, assayed by measuring the amount of collagen II.
  • Such second assay may be designed to confirm that the test compound, having binding affinity for the polypeptide, actually induces the anabolic stimulation of chondrocytes. Suitable controls should always be in place to insure against false positive readings.
  • the order of taking these measurements is not believed to be critical to the practice of the present invention, which may be practiced in any order. For example, one may first perform a screening assay of a set of compounds for which no information is known respecting the ⁇ compounds' binding affinity for the polypeptide. Alternatively, one may screen a set of compounds identified as having binding affinity for a polypeptide domain, or a class of compounds identified as being an inhibitor of the polypeptide. However, for the present assay to be meaningful to the ultimate use of the drug candidate compounds, a measurement of collagen II levels or aggrecan is necessary. Validation studies including controls, and measurements of binding affinity to the polypeptides of the invention are nonetheless useful in identifying a compound useful in any therapeutic or diagnostic application.
  • the binding affinity of the compound with the polypeptide TARGET can be measured by methods known in the art, such as using surface plasmon resonance biosensors (Biacore), by saturation binding analysis with a labeled compound (e.g. Scatchard and Lindmo analysis), by differential UV spectrophotometer, fluorescence polarization assay, Fluorometric Imaging Plate Reader (FLIPR®) system, Fluorescence resonance energy transfer, and Bioluminescence resonance energy transfer.
  • the bmding affinity of compounds can also be expressed in dissociation constant (Kd) or as IC50 or EC50.
  • Kd dissociation constant
  • IC50 represents the concentration of a compound that is required for 50% inhibition of bmding of another ligand to the polypeptide.
  • the EC50 represents the concentration required for obtaining 50% of the maximum effect in any assay that measures TARGET function.
  • the dissociation constant, Kd is a measure of how well a ligand binds to the polypeptide, it is equivalent to the ligand concentration required to saturate exactly half of the binding-sites on the polypeptide.
  • Compounds with a high affinity binding have low Kd, IC50 and EC50 values, i.e. in the range of 100 nM to 1 pM; a moderate to low affinity binding relates to a high Kd, IC50 and EC50 values, i.e. in the micromolar range.
  • the present assay method may also be practiced in a cellular assay
  • a host cell expressing TARGET can be a cell with endogenous expression or a cell over-expressing the TARGET e.g. by transduction.
  • the endogenous expression of the polypeptide is not sufficient to determine a baseline that can easily be measured, one may use using host cells that over-express TARGET.
  • Over-expression has the advantage that the level of the TARGET substrate end products is higher than the activity level by endogenous expression. Accordingly, measuring such levels using presently available techniques is easier.
  • the biological activity of TARGET may be measured by following the production of cartilage component synthesis.
  • the present invention further relates to a method for identifying a compound that induces anabolic stimulation of chondrocytes, comprising: (a) contacting a compound with a.polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 101-128 and 401-594; (b) determining the binding affinity of the compound to the polypeptide; (c) contacting a population of mammalian cells expressing said polypeptide with the compound that exhibits a binding affinity of at least 10 micromolar; and (d) identifying the compound that induces the synthesis of proteins that are a constituent of normal cartilage and/or that are required for the formation of cartilage.
  • libraries of compounds may be used such as antibody fragment libraries, peptide phage display libraries, peptide libraries (e.g. LOPAPTM, Sigma Aldrich), lipid libraries (BioMol), synthetic compound libraries (e.g. LOPACTM, Sigma
  • Preferred drug candidate compounds are low molecular weight compounds. Low molecular weight compounds, i.e. with a molecular weight of 500 Dalton or less, are likely to have good absorption and permeation in biological systems and are consequently more likely to be successful drug candidates than compounds with a molecular weight above 500 Dalton (Lipinski et al. (1997)). Peptides comprise another preferred class of drug candidate compounds. Peptides may be excellent drug candidates and there are multiple examples of commercially valuable peptides such as fertility hormones and platelet aggregation inhibitors. Natural compounds are another preferred class of drug candidate compound. Such compounds are found in and extracted from natural sources, and which may thereafter be synthesized.
  • the lipids are another preferred class of drug candidate compound.
  • Another preferred class of drug candidate compounds is an antibody.
  • the present invention also provides antibodies directed against a TARGET. These antibodies may be endogenously produced to bind to the TARGET within the cell, or added to the tissue to bind to TARGET polypeptide present outside the cell. These antibodies may be monoclonal antibodies or polyclonal antibodies.
  • the present invention includes chimeric, single chain, and humanized antibodies, as well as FAb fragments and the products of a FAb expression library, and Fv fragments and the products of an Fv expression library.
  • polyclonal antibodies may be used in the practice of the invention. The skilled artisan knows methods of preparing polyclonal antibodies.
  • Polyclonal antibodies can be raised in a mammal, for example, by one or more injections of an immunizing agent and, if desired, an adjuvant.
  • the immunizing agent and/or adjuvant will be injected in the mammal by multiple subcutaneous or intraperitoneal injections, ⁇
  • Antibodies may also be generated against the intact TARGET protein or polypeptide, or against a fragment, derivatives including conjugates, or other epitope of the TARGET protein or polypeptide, such as the TARGET embedded in a cellular membrane, or a library of antibody variable regions, such as a phage display library. It may be useful to conjugate the immunizing agent to a protein known to be immunogenic in the mammal being immunized.
  • the antibodies may be monoclonal antibodies. Monoclonal antibodies may be prepared using methods known in the art. The monoclonal antibodies of the present invention may be "humanized" to prevent the host from mounting an immune response to the antibodies.
  • a “humanized antibody” is one in which the complementarity deterrnining regions (CDRs) and/or other portions of the light and/or heavy variable domain framework are derived from a non-human immunoglobulin, but the remaining portions of the molecule are derived from one or more human immunoglobulins.
  • Humanized antibodies also include antibodies characterized by a humanized heavy chain associated with a donor or acceptor unmodified light chain or a chimeric light chain, or vice versa. The humanization of antibodies may be accomplished by methods known in the art (see, e.g. Mark and Padlan, (1994) "Chapter 4. Humanization of Monoclonal Antibodies", The Handbook of Experimental Pharmacology Vol. 113, Springer- Verlag, New York).
  • Transgenic animals may be used to express humanized antibodies.
  • Human antibodies can also be produced using various techniques known in the art, including phage display libraries (Hoogenboom and Winter, (1991) J. Mol. Biol. 227:381- 8; Marks et al. (1991). J. Mol. Biol. 222:581-97).
  • the techniques of Cole, et al. and Boerner, et al. are also available for the preparation of human monoclonal antibodies (Cole, et al. (1985) Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77; Boerner, et al (1991). J. Immunol., 147(l):86-95).
  • the antibodies may be monovalent antibodies.
  • Methods for preparing monovalent antibodies are well known in the art. For example, one method involves recombinant expression of immunoglobulin light chain and modified heavy chain. The heavy chain is truncated generally at any point in the Fc region so as to prevent heavy chain cross-linking. Alternatively; the relevant cysteine residues are substituted with another amino acid residue or are deleted so as to prevent cross-linking.
  • Bispecific antibodies are monoclonal, preferably human or humanized, antibodies that have binding specificities for at least two different antigens and preferably for a cell-surface protein or receptor or receptor subunit.
  • one of the binding specificities is for one domain of the TARGET; the other one is for another domain of the same or different TARGET.
  • Methods for making bispecific antibodies are known in the art. Traditionally, the recombinant production of bispecific antibodies is based on the co-expression of two immunoglobulin heavy-chain/light-chain pairs, where the two heavy chains have different specificities (Milstein and Cuello, (1983) Nature 305:537-9). Because of the random assortment of immunoglobulin heavy and light chains, these hybridomas (quadromas) produce a potential mixture of ten different antibody molecules, of which only one has the correct bispecific structure. Affinity chromatography steps usually accomplish the purification of the correct molecule.
  • the assay method uses a drug candidate compound identified as having a binding affinity for a TARGET, and/or has already been identified as having down-regulating activity such as antagonist activity vis-a-vis one or more TARGET.
  • the present invention further relates to a method for inducing anabolic stimulation of chondrocytes comprising contacting said cells with an expression inhibitory agent comprising a polynucleotide sequence that complements at least about 17 nucleotides of the polyribonucleotide comprising a nucleotide sequence selected from the group consisting of SEQ D NO: 1-28.
  • the expression-inhibitory agent comprises a polynucleotide sequence that complements a nucleotide sequence selected from the group consisting of SEQ TD NO: 202-315.
  • Another aspect of the present invention relates to a method for inducing the anabolic stimulation of chondrocytes, comprising by contacting said cell with an expression-inhibiting agent that inhibits the translation in the cell of a polyribonucleotide encoding a TARGET polypeptide.
  • a particular embodiment relates to a composition
  • a composition comprising a polynucleotide including at least one antisense strand that functions to pair the agent with the TARGET TARGET mRNA, and thereby down-regulate or block the expression of TARGET polypeptide.
  • the inhibitory agent preferably comprises antisense polynucleotide, a ribozyme, and a small interfering RNA (siRNA), wherein said agent comprises a nucleic acid sequence complementary to, or engineered from, a nat ⁇ ally-occurring polynucleotide sequence encoding a portion of a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ TD NO: 101-128.
  • the expression-inhibiting agent is complementary to a polynucleotide sequence selected from the group consisting of SEQ ID NO: 1-28. In an especially preferred embodiment the expression-inhibiting agent is complementary to a polynucleotide sequence selected from the group consisting of SEQ ED NO: 202-315.
  • An embodiment of the present invention relates to a method wherein the expression- inhibiting agent is selected from the group consisting of antisense RNA, antisense oligodeoxynucleotide (ODN), a ribozyme that cleaves the polyribonucleotide coding for SEQ ID NO: 101-128, a small interfering RNA (siRNA, preferably shRNA,) that is sufficiently complementary to a portion of the polyribonucleotide coding for SEQ ID NO: 101-128, such that the siRNA, preferably sbRNA, interferes with the translation of the TARGET polyribonucleotide to the TARGET polypeptide.
  • the expression- inhibiting agent is selected from the group consisting of antisense RNA, antisense oligodeoxynucleotide (ODN), a ribozyme that cleaves the polyribonucleotide coding for SEQ ID NO: 101-128, a small interfering RNA (siRNA
  • the expression-inhibiting agent is an antisense RNA, ribozyme, antisense oligodeoxynucleotide, or siRNA, preferably shRNA, complementary to a nucleotide sequence selected from the group consisting of SEQ ID NO: 1-28.
  • the expression-inhibiting agent is complementary to a polynucleotide sequence selected from the group consisting of SEQ ID NO: 202-315.
  • a special embodiment of the present invention relates to a method wherein the expression-inhibiting agent is a nucleic acid expressing the antisense RNA, antisense oligodeoxynucleotide (ODN), a ribozyme that cleaves the polyribonucleotide coding for SEQ ED NO: 101-128, a small interfering RNA (siRNA, preferably shRNA,) that is sufficiently complementary to a portion of the polyribonucleotide coding for SEQ ID NO: 101-128, such that the siRNA, preferably shRNA, interferes with the translation of the TARGET polyribonucleotide to the TARGET polypeptide.
  • the expression-inhibiting agent is a nucleic acid expressing the antisense RNA, antisense oligodeoxynucleotide (ODN), a ribozyme that cleaves the polyribonucleotide coding for SEQ ED NO: 101-128, a small interfer
  • nucleotide sequence is complementary to a polynucleotide selected from the group consisting of SEQ ID NO: 1-28.
  • nucleotide sequence is complementary to a polynucleotide selected from the group consisting of SEQ ID NO: 202-315.
  • the down regulation of gene expression using antisense nucleic acids can be achieved at the translational or transcriptional level.
  • Antisense nucleic acids of the invention are preferably nucleic acid fragments capable of specifically hybridizing with all or part of a nucleic acid encoding a TARGET polypeptide or the corresponding messenger RNA.
  • antisense nucleic acids may be designed which decrease expression of the nucleic acid sequence capable of encoding a TARGET polypeptide by inhibiting splicing of its primary transcript.
  • Any length of antisense sequence is suitable for practice of the invention so long as it is capable of down-regulating or blocking expression of a nucleic acid coding for a TARGET.
  • the antisense sequence is at least about 17 nucleotides in length.
  • the preparation and use of antisense nucleic acids, DNA encoding antisense RNAs and the use of oligo and genetic antisense is known in the art.
  • expression-inhibitory agent is a nucleic acid that is antisense to a nucleic acid comprising SEQ ID NO: 1-28.
  • an antisense nucleic acid e.g. DNA
  • an antisense nucleic acid may be introduced into cells in vitro, or administered to a subject in vivo, as gene therapy to inhibit cellular expression of nucleic acids comprising SEQ ID NO: 1-28.
  • Antisense oligonucleotides preferably comprise a sequence containing from about 17 to about 100 nucleotides and more preferably the antisense oligonucleotides comprise from about 18 to about 30 nucleotides.
  • Antisense nucleic acids may be prepared from about 10 to about 30 contiguous nucleotides complementary to a nucleic acid sequence selected from the sequences of SEQ ID NO: 1-28.
  • the antisense nucleic acids are preferably oligonucleotides and may consist entirely of deoxyribo-nucleotides, modified deoxyribonucleotides, or some combination of both.
  • the antisense nucleic acids can be synthetic oligonucleotides.
  • the oligonucleotides may be chemically modified, if desired, to improve stability and/or selectivity. Since oligonucleotides are susceptible to degradation by intracellular nucleases, the modifications can include, for example, the use of a sulfur group to replace the free oxygen of the phosphodiester bond. This modification is called a phosphorothioate linkage.
  • Phosphorothioate antisense oligonucleotides are water soluble, polyanionic, and resistant to endogenous nucleases.
  • the RN202-315NA duplex activates the endogenous enzyme ribonuclease (RNase) H, which cleaves the mRNA component of the hybrid molecule.
  • RNase ribonuclease
  • antisense oligonucleotides with phosphoramidite and polyamide (peptide) linkages can be synthesized. These molecules should be very resistant to nuclease degradation.
  • RNA enzymes catalytic RNA molecules (RNA enzymes) that have separate catalytic and substrate binding domains.
  • the substrate binding sequence combines by nucleotide complementarity and, possibly, non-hydrogen bond interactions with its TARGET sequence.
  • the catalytic portion cleaves the TARGET RNA at a specific site.
  • the substrate domain of a ribozyme can be engineered to direct it to a specified mRNA sequence.
  • the ribozyme recognizes and then binds a TARGET mRNA through complementary base pairing. Once it is bound to the correct TARGET site, the ribozyme acts enzymatically to cut the TARGET mRNA. Cleavage of the mRNA by a ribozyme destroys its ability to direct synthesis of the corresponding polypeptide.
  • Ribozyme forms include a hammerhead motif, a hairpin motif, a hepatitis delta virus, group I intron or RNaseP RNA (in association with an RNA guide sequence) motif or Neurospora VS RNA motif. Ribozymes possessing a hammerhead or hairpin structure are readily prepared since these catalytic RNA molecules can be expressed within cells from eukaryotic promoters (Chen, et al. (1992) Nucleic Acids Res. 20:4581-9).
  • a ribozyme of the present invention can be expressed in eukaryotic cells from the appropriate DNA vector. If desired, the activity of the ribozyme may be augmented by its release from the primary transcript by a second ribozyme (Ventura, et al. (1993) Nucleic Acids Res. 21 :3249-55). Ribozymes may be chemically synthesized by combining an oligodeoxyribonucleotide with a ribozyme catalytic domain (20 nucleotides) flanked by sequences that hybridize to the TARGET mRNA after transcription. The oligodeoxyribonucleotide is amplified by using the substrate binding sequences as primers.
  • the amplification product is cloned into a eukaryotic expression vector.
  • Ribozymes are expressed from transcription units inserted into DNA, RNA, or viral vectors. Transcription of the ribozyme sequences are driven from a promoter for eukaryotic RNA polymerase I (pol (I), RNA polymerase II (pol IT), or RNA polymerase IH (pol IH). Transcripts from pol II or pol DI promoters will be expressed at high levels in all cells; the levels of a given pol II promoter in a given cell type will depend on nearby gene regulatory sequences.
  • Prokaryotic RNA polymerase promoters are also used, providing that the prokaryotic RNA polymerase enzyme is expressed in the appropriate cells (Gao and Huang, (1993) Nucleic Acids Res. 21:2867-72). It has been demonstrated that ribozymes expressed from these promoters can function in mammalian cells (Kashani-Sabet, et al. (1992) Antisense Res. Dev. 2:3-15).
  • a particularly preferred inhibitory agent is a small interfering RNA (siRNA, preferably shRNA).
  • siRNA preferably shRNA, mediate the post-transcriptional process of gene silencing by double stranded RNA (dsRNA) that is homologous in sequence to the silenced RNA.
  • siRNA according to the present invention comprises a sense strand of 17-25 nucleotides complementary or homologous to a contiguous 17-25 nucleotide sequence selected from the group of sequences described in SEQ ID NO: 1-28, preferably from the group of sequences described in SEQ ID No: 202-315, and an antisense strand of 17-23 nucleotides complementary to the sense strand. Exemplary sequences are described as sequences complementary to SEQ ID NO: 202-315.
  • the most preferred siRNA comprises sense and anti-sense strands that are 100 per cent complementary to each other and the TARGET polynucleotide sequence.
  • the siRNA further comprises a loop region linking the sense and the antisense strand.
  • a self-complementing single stranded siRNA molecule polynucleotide according to the present invention comprises a sense portion and an antisense portion connected by a loop region linker.
  • the loop region sequence is 4-30 nucleotides long, more preferably 5-15 nucleotides long and most preferably 8 nucleotides long.
  • the linker sequence is UUGCUAUA (SEQ ID NO: 201).
  • Self-complementary single stranded siRNAs form hairpin loops and are more stable than ordinary dsRNA. In addition, they are more easily produced from vectors.
  • the siRNA can be modified to confirm resistance to nucleolytic degradation, or to enhance activity, or to enhance cellular distribution, or to enhance cellular uptake, such modifications may consist of modified intemucleoside linkages, modified nucleic acid bases, modified sugars and or chemical linkage the siRNA to one or more moieties or conjugates.
  • the nucleotide sequences are selected according to siRNA designing rules that give an improved reduction of the TARGET sequences compared to nucleotide sequences that do not comply with these siRNA designing rules (For a discussion of these rules and examples of the preparation of siRNA, WO2004094636, published November 4, 2004, and UA20030198627, are hereby incorporated by reference).
  • the present invention also relates to compositions, and methods using said compositions, comprising a DNA expression vector capable of expressing a polynucleotide capable of inducing anabolic stimulation of chondrocytes and described hereinabove as an expression inhibition agent.
  • a special aspect of these compositions and methods relates to the down-regulation or blocking of the expression of a TARGET polypeptide by the induced expression of a polynucleotide encoding an intracellular binding protein that is capable of selectively interacting with the TARGET polypeptide.
  • An intracellular binding protein includes any protein capable of selectively interacting, or binding, with the polypeptide in the cell in which it is expressed and neutralizing the function of the polypeptide.
  • the intracellular binding protein is a neutralizing antibody or a fragment of a neutralizing antibody having binding affinity to an epitope of the TARGET polypeptide of SEQ ID NO: 101-128 and 401- 594. More preferably, the intracellular binding protein is a single chain antibody.
  • a special embodiment of this composition comprises the expression-inhibiting agent selected from the group consisting of antisense RNA, antisense oligodeoxynucleotide (ODN), a ribozyme that cleaves the polyribonucleotide coding for SEQ ID NO: 101-128, and a small interfering RNA (siRNA) that is sufficiently homologous to a portion of the polyribonucleotide coding for SEQ ID NO: 101-128, such that the siRNA interferes with the translation of the TARGET polyribonucleotide to the TARGET polypeptide,
  • the polynucleotide expressing the expression-inhibiting agent is preferably included within a vector.
  • the polynucleic acid is operably linked to signals enabling expression of the nucleic acid sequence and is introduced into a cell utilizing, preferably, recombinant vector constructs, which will express the antisense nucleic acid once the vector is introduced into the cell.
  • recombinant vector constructs which will express the antisense nucleic acid once the vector is introduced into the cell.
  • viral-based systems including adenoviral, retroviral, adeno-associated viral, lentiviral, herpes simplex viral or a sendaviral vector systems, and all may be used to introduce and express polynucleotide sequence for the expression-inhibiting agents in TARGET cells.
  • the viral vectors used in the methods of the present invention are replication defective.
  • Such replication defective vectors will usually pack at least one region that is necessary for the replication of the virus in the infected cell. These regions can either be eliminated .(in whole or in part), or be rendered non-functional by any technique known to a person skilled in the art. These techniques include the total removal, substitution, partial deletion or addition of one or more bases to an essential (for replication) region. Such techniques may be performed in vitro (on the isolated DNA) or in situ, using the techniques of genetic manipulation or by treatment with mutagenic agents.
  • the replication defective virus retains the sequences of its genome, which are necessary for encapsidating, the viral particles.
  • the viral element is derived from an adenovirus.
  • the vehicle includes an adenoviral vector packaged into an adenoviral capsid, or a functional part, derivative, and/or analogue thereof.
  • Adenovirus biology is also comparatively well known on the molecular level. Many tools for adenoviral vectors have been and continue to be developed, thus making an adenoviral capsid a preferred vehicle for incorporating in a library of the invention.
  • An adenovirus is capable of infecting a wide variety of cells. However, different adenoviral serotypes have different preferences for cells.
  • the vehicle includes adenoviral fiber proteins from at least two adenoviruses.
  • Preferred adenoviral fiber protein sequences are serotype 17, 45 and 51. Techniques or construction and expression of these chimeric vectors are disclosed in US Published Patent Applications 20030180258 and 20040071660, hereby incorporated by reference.
  • the nucleic acid derived from an adenovirus includes the nucleic acid encoding an adenoviral late protein or a functional part, derivative, and/or analogue thereof.
  • an adenoviral late protein for instance an adenoviral fiber protein, may be favorably used to TARGET the vehicle to a certain cell or to induce enhanced delivery of the vehicle to the cell.
  • the nucleic acid derived from an adenovirus encodes for essentially all adenoviral late proteins, enabling the formation of entire adenoviral capsids or functional parts, analogues, and/or derivatives thereof.
  • the nucleic acid derived from an adenovirus includes the nucleic acid encoding adenovirus E2A or a functional part, derivative, and or analogue thereof.
  • the nucleic acid derived from an adenovirus includes the nucleic acid encoding at least one E4-region protein or a functional part, derivative, and or analogue thereof, which facilitates, at least in part, replication of an adenoviral derived nucleic acid in a cell.
  • the adenoviral vectors used in the examples of this application are exemplary of the vectors useful in the present method of treatment invention. Certain embodiments of the present invention use retroviral vector systems. Retroviruses are integrating viruses that infect dividing cells, and their construction is known in the art.
  • Retroviral vectors can be constructed from different types of retrovirus, such as, MoMuLV ("murine Moloney leukemia virus” MSV ("murine Moloney sarcoma virus”), HaSV ("Harvey sarcoma virus”); SNV ("spleen necrosis virus”); RSV ("Rous sarcoma virus”) and Friend virus.
  • Lentiviral vector systems may also be used in the practice of the present invention.
  • adeno-associated viruses are utilized.
  • the AAV viruses are DNA viruses of relatively small size that integrate, in a stable and site-specific manner, into the genome of the infected cells.
  • the polynucleotide agents of the present invention may be linked to one or more regulatory regions. Selection of the appropriate regulatory region or regions is a routine matter, within the level of ordinary skill in the art. Regulatory regions include promoters, and may include enhancers, suppressors, etc. Promoters that may be used in the expression vectors of the present invention include both constitutive promoters and regulated (inducible) promoters. The promoters may be prokaryotic or eukaryotic depending on the host.
  • prokaryotic (mcluding bacteriophage) promoters useful for practice of this invention are lac, lacZ, T3, 17, lambda P r , Pi, and tip promoters.
  • eukaryotic (including viral) promoters useful for practice of this invention are ubiquitous promoters (e.g. HPRT, vhnentin, actin, tubulin), intermediate filament promoters (e.g. desmin, neurofilaments, keratin, GFAP), therapeutic gene promoters (e.g. MDR type, CFTR, factor VTfi), tissue-specific promoters (e.g.
  • actin promoter in smooth muscle cells or Fit and Flk promoters active in endothelial cells
  • animal transcriptional control regions which exhibit tissue specificity and have been utilized in transgenic animals: elastase I gene control region which is active in pancreatic acinar cells (Swift, et al. (1984) Cell 38:639-46; Ornitz, et al. (1986) Cold Spring Harbor Symp. Quant. Biol.
  • promoters which may be used in the practice of the invention include promoters which are preferentially activated in dividing cells, promoters which respond to a stimulus (e.g.
  • Additional vector systems include the non-viral systems that facilitate introduction of polynucleotide agents into a patient.
  • a DNA vector encoding a desired sequence can be introduced in vivo by lipofection.
  • Synthetic cationic lipids designed to limit the difficulties encountered with liposome-mediated transfection can be used to prepare liposomes for in vivo transfection of a gene encoding a marker (Feigner, et. al. (1987) Proc. Natl. Acad Sci.
  • cationic lipids may promote encapsulation of negatively charged nucleic acids, and also promote fusion with negatively charged cell membranes (Feigner and Ringold, (1989) Nature 337:387-8).
  • Particularly useful lipid compounds and compositions for transfer of nucleic acids are described in International Patent Publications WO 95/18863 and WO 96/17823, and in U.S. Pat. No. 5,459,127.
  • lipofection to introduce exogenous genes into the specific organs in vivo has certain practical advantages and directing transfection to particular cell types would be particularly advantageous in a tissue with cellular heterogeneity, for example, pancreas, liver, kidney, and the brain.
  • Lipids may be chemically coupled to other molecules for the purpose of targeting.
  • Targeted peptides e.g., hormones or neurotransmitters, and proteins for example, antibodies, or non-peptide molecules could be coupled to liposomes chemically.
  • a cationic oligopepti.de e.g., International Patent Publication WO 95/21931
  • peptides derived from DNA binding proteins e.g., International Patent Publication WO 96/25508
  • a cationic polymer e.g., International Patent Publication WO 95/21931
  • It is also possible to introduce a DNA vector in vivo as a naked DNA plasmid see U.S. Pat. Nos. 5,693,622, 5,589,466 and 5,580,859).
  • Naked DNA vectors for therapeutic purposes can be introduced into the desired host cells by methods known in the art, e.g., transfection, electroporation, microinjection, transduction, cell fusion, DEAE dextran, calcium phosphate precipitation, use of a gene gun, or use of a DNA vector transporter (see, e.g., Wilson, et al. (1992) J. Biol. Chem 267:963-7; Wu and Wu, (1988) J. Biol. Chem. 263:14621-4; Hartmut, et al. Canadian Patent Application No. 2,012,311, filed Mar. 15, 1990; Williams, et al (1991). Proc. Natl. Acad. Sci. USA 88:2726-30).
  • a biologically compatible composition is a composition, that may be solid, liquid, gel, or other form, in which the compound, polynucleotide, vector, and antibody of the invention is maintained in an active form, e.g., in a form able to effect a biological activity.
  • a compound of the invention would have inverse agonist or antagonist activity on the TARGET; a nucleic acid would be able to replicate, translate a message, or hybridize to a complementary mRNA of a TARGET; a vector would be able to transfect a TARGET cell and expression the antisense, antibody, ribozyme or siRNA as described hereinabove; an antibody would bind a TARGET polypeptide domain.
  • a preferred biologically compatible composition is an aqueous solution that is buffered using, e.g., Tris, phosphate, or HEPES buffer, containing salt ions. Usually the concentration of salt ions will be similar to physiological levels.
  • Biologically compatible solutions may include stabilizing agents and preservatives.
  • the biocompatible composition is a pharmaceutically acceptable composition.
  • Such compositions can be formulated for administration by topical, oral, parenteral, intranasal, subcutaneous, and intraocular, routes.
  • Parenteral administration is meant to include intravenous injection, intramuscular injection, intraarterial injection or infusion techniques.
  • the composition may be administered parenterally in dosage unit formulations containing standard, well-known non-toxic physiologically acceptable carriers, adjuvants and vehicles as desired.
  • a particularly preferred embodiment of the present composition invention is a cartilage formation-enhancing pharmaceutical composition comprising a therapeutically effective amount of an expression-inhibiting agent as described hereinabove, in admixture with a pharmaceutically acceptable carrier.
  • compositions for the treatment or prevention of a condition a systemic or local decrease in mean cartilage thickness, or a susceptibility to the condition comprising an effective cartilage formation-enhancing amount of a TARGET antagonist or inverse agonist, its pharmaceutically acceptable salts, hydrates, solvates, or prodrugs thereof in admixture with a pharmaceutically acceptable carrier.
  • Pharmaceutical compositions for oral administration can be formulated using pharmaceutically acceptable carriers well known in the art in dosages suitable for oral administration. Such carriers enable the pharmaceutical compositions to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, and the like, for ingestion by the patient.
  • compositions for oral use can be prepared by combining active compounds with solid excipient, optionally grinding a resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries, if desired, to obtain tablets or dragee cores.
  • suitable excipients are carbohydrate or protein fillers, such as sugars, including lactose, sucrose, mannitol, or sorbitol; starch from corn, wheat, rice, potato, or other plants; cellulose, such as methyl cellulose, hydroxypropylmethyl-cellulose, or sodium carboxymethyl-cellulose; gums including arabic and tragacanth; and proteins such as gelatin and collagen.
  • disintegrating or solubilizing agents may be added, such as the cross-linked polyvinyl pyrrolidone, agar, alginic acid, or a salt thereof, such as sodium alginate.
  • Dragee cores may be used in conjunction with suitable coatings, such as concentrated sugar solutions, which may also contain gum arabic, talc, polyvinylpyrrolidone, carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures.
  • Dyestuffs or pigments may be added to the tablets or dragee coatings for product identification or to characterize the quantity of active compound, i.e., dosage.
  • compositions that can be used orally include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a coating, such as glycerol or sorbitol.
  • Push-fit capsules can contain active ingredients mixed with filler or binders, such as lactose or starches, lubricants, such as talc or magnesium stearate, and, optionally, stabilizers.
  • the active compounds may be dissolved or suspended in suitable liquids, such as fatty oils, liquid, or liquid polyethylene glycol with or without stabilizers.
  • Preferred sterile injectable preparations can be a solution or suspension in a non-toxic parenterally acceptable solvent or diluent.
  • Examples of pharmaceutically acceptable carriers are saline, buffered saline, isotonic saline (e.g. monosodium or disodium phosphate, sodium, potassium; calcium or magnesium chloride, or mixtures of such salts), Ringer's solution, dextrose, water, sterile water, glycerol ethanol, and combinations thereof 1,3-butanediol and sterile fixed oils are conveniently employed as solvents or suspending media. Any bland fixed oil can be employed including synthetic mono- or di-glycerides. Fatty acids such as oleic acid also find use in the preparation of injectables.
  • the composition medium can also be a hydrogel, which is prepared from any biocompatible or non-cytotoxic homo- or hetero-polymer, such as a hydrophilic polyacrylic acid polymer that can act as a drug absorbing sponge. Certain of them, such as, in particular, those obtained from ethylene and/or propylene oxide are commercially available.
  • a hydrogel can be deposited directly onto the surface of the tissue to be treated, for example during surgical intervention.
  • Embodiments of pharmaceutical compositions of the present invention comprise a replication defective recombinant viral vector encoding the polynucleotide inhibitory agent of the present invention and a transfection enhancer, such as poloxamer.
  • Poloxamer 407 An example of a poloxamer is Poloxamer 407, which is commercially available (BASF, Parsippany, N. J.) and is a non-toxic, biocompatible polyol.
  • a poloxamer impregnated with recombinant viruses may be deposited directly on the surface of the tissue to be treated, for example during a surgical intervention. Poloxamer possesses essentially the same advantages as hydrogel while having a lower viscosity.
  • the active expression-inhibiting agents may also be entrapped in microcapsules prepared, for example, by interfacial polymerization, for example, hydroxymethylcellulose or gelatin-microcapsules and poly-(methyhnethacylate) microcapsules, respectively, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules) or in macroemulsions.
  • colloidal drug delivery systems for example, liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules
  • Sustained-release preparations may be prepared.
  • sustained-release preparations include semi-permeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e.g. films, or microcapsules.
  • sustained-release matrices include polyesters, hydrogels (for example, poly(2-hydroxyethyl-methacrylate), or ⁇ oly(vinylalcohol)), polylactides (U.S. Pat. No.
  • copolymers of L-glutamic acid and gamma-ethyl-L-glutamate non- degradable ethylene-vinyl acetate
  • degradable lactic acid-glycolic acid copolymers such as the LUPRON DEPOTTM, (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate)
  • poly-D-(-)-3-hydroxybutyric acid While polymers such as ethylene-vinyl acetate and lactic acid-glycolic acid enable release of molecules for over 100 days, certain hydrogels release proteins for shorter time periods.
  • encapsulated antibodies When encapsulated antibodies remain in the body for a long time, they may denature or aggregate as a result of exposure to moisture at 37°C, resulting in a loss of biological activity and possible changes in immunogenicity. Rational strategies can be devised for stabilization depending on the mechanism involved. For example, if the aggregation mechamsm is discovered to be intermolecular S-S bond formation through thio-disulfide interchange, stabilization may be achieved by modifying sulfhydryl residues, lyophilizing from acidic solutions, controlling moisture content, using appropriate additives, and developing specific polymer matrix compositions.
  • therapeutically effective dose means that amount of protein, polynucleotide, peptide, or its antibodies, agonists or antagonists, which ameliorate the symptoms or condition.
  • Therapeutic efficacy and toxicity of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., ED50 (the dose therapeutically effective in 50% of the population) and LD50 (the dose lethal to 50% of the population).
  • the dose ratio of toxic to therapeutic effects is the therapeutic index, and it can be expressed as the ratio, LD50/ED50.
  • Pharmaceutical compositions that exhibit large therapeutic indices are preferred. The data obtained from cell culture assays and animal studies is used in formulating a range of dosage for human use.
  • the dosage of such compounds lies preferably within a range of circulating concentrations that include the ED50 with little or no toxicity.
  • the dosage varies within this range depending upon the dosage form employed, sensitivity of the patient, and the route of administration.
  • the therapeutically effective dose can be estimated initially either in cell culture assays or in animal models, usually mice, rabbits, dogs, or pigs. The animal model is also used to achieve a desirable concentration range and route of administration. Such information can then be used to determine useful doses and routes for administration in humans.
  • the exact dosage is chosen by the individual physician in view of the patient to be treated. Dosage and administration are adjusted to provide sufficient levels of the active moiety or to maintain the desired effect.
  • compositions according to this invention may be administered to a subject by a variety of methods. They may be added directly to TARGET tissues, complexed with cationic lipids, packaged within liposomes, or delivered to TARGET cells by other methods known in the art.
  • Localized administration to the desired tissues may be done by direct injection, transdermal abso ⁇ tion, catheter, infusion pump or stent.
  • the DNA, DNA/vehicle complexes, or the recombinant virus particles are locally administered to the site of treatment.
  • Alternative routes of delivery include, but are not limited to, intravenous injection, intramuscular injection, subcutaneous injection, aerosol inhalation, oral (tablet or pill form), topical, systemic, ocular, intraperitoneal and/or intrathecal delivery. Examples of ribozyme delivery and aciministration are provided in Sullivan et al. WO 94/02595.
  • Antibodies according to the invention may be delivered as a bolus only, infused over time or both administered as a bolus and infused over time.
  • Those skilled in the art may employ different formulations for polynucleotides than for proteins.
  • delivery of polynucleotides or polypeptides will be specific to particular cells, conditions, locations, etc.
  • recombinant viruses may be used to introduce DNA encoding polynucleotide agents useful in the present invention.
  • Recombinant viruses according to the invention are generally formulated and administered in the form of doses of between about 10 4 and about 10 14 pfu.
  • pfu plaque-forming unit
  • the present invention also provides methods of enhancing cartilage formation, which comprise the administration to said subject a therapeutically effective amount of an expression-inhibiting agent of the invention.
  • a further aspect of the invention relates to a method of treating or preventing a disease involving chondrocyte anabolic stimulation, comprising a ⁇ niinistering to said subject a cartilage formation-enhancing pharmaceutical composition as described herein.
  • diseases involving anabolic stimulation of chondrocytes include, but are not limited to osteoarthritis, rheumatoid arthritis, psoriatic arthritis, juvenile rheumatoid arthritis, gouty arthritis, septic or infectious arthritis, reactive arthritis, reflex sympathetic dystrophy, algodystrophy, Tietze syndrome or costal chondritis, fibromyalgia, osteochondritis, neurogenic or neuropathic arthritis, arthropathy, endemic forms of arthritis like osteoarthritis deformans endemica, Mseleni disease, and Handigodu disease; degeneration resulting from fibromyalgia, systemic lupus erythematos
  • congenital cartilage malformations including hereditary chondrolysis, chondrodysplasias and pseudoachondrodysplasias, are likely to benefit from programs that result in anabolic stimulation of chondrocytes, and these diseases therefore may also be treated by using the methods and means of the present invention.
  • congenital cartilage malformation related diseases are microtia, anotia, and metaphyseal chondrodysplasia.
  • the polypeptides or the polynucleotides employed in the methods of the present invention may be free in solution, affixed to a solid support, borne on a cell surface, or located intracellularly.
  • the polypeptide of the present invention or the compound to facilitate separation of complexes from uncomplexed forms of the polypeptide, as well as to accommodate automation of the assay.
  • Interaction (e.g., binding of) of the polypeptide of the present invention with a compound can be accomplished in any vessel suitable for containing the reactants. Examples of such vessels include microtitre plates, test tubes, and microcentrifuge tubes.
  • a fusion protein can be provided which adds a domain that allows the polypeptide to be bound to a matrix.
  • the polypeptide of the present invention can be "His" tagged, and subsequently adsorbed onto Ni-NTA microtitre plates, or ProtA fusions with the polypeptides of the present invention can be adsorbed to IgG, which are then combined with the cell lysates (e.g., ( 35 S-labelled) and the candidate compound, and the mixture incubated under conditions favorable for complex formation (e.g., at physiological conditions for salt and pH). Following incubation, the plates are washed to remove any unbound label, and the matrix is immobilized. The amount of radioactivity can be determined directly, or in the supernatant after dissociation of the complexes.
  • the complexes can be dissociated from the matrix, separated by SDS-PAGE, and the level of the protein binding to the protein of the present invention quantitated from the gel using standard electrophoretic techniques.
  • Other techniques for immobilizing protein on matrices can also be used in the method of identifying compounds.
  • either the polypeptide of the present invention or the compound can be immobilized utilizing conjugation of biotin and streptavidin.
  • Biotinylated protein molecules of the present invention can be prepared from biotin-NHS (N-hydroxy- succinimide) using techniques well known in the art (e.g., biotinylation kit, Pierce Chemicals, Rockford, 111.), and immobilized in the wells of streptavidin-coated 96 well plates (Pierce Chemical).
  • biotinylation kit N-hydroxy- succinimide
  • Pierce Chemicals Pierce Chemicals, Rockford, 111.
  • streptavidin-coated 96 well plates Piereptavidin-coated 96 well plates
  • antibodies reactive with the polypeptides of the present invention but which do not interfere with binding of the polypeptide to the compound can be derivatized to the wells of the plate, and the polypeptide of the present invention can be trapped in the wells by antibody conjugation.
  • preparations of a labeled candidate compound are incubated in the wells of the plate presenting the polypeptide of the present invention, and the amount of complex trapped in the well can be quantitated.
  • Another embodiment of the present invention relates to a method for in vitro production of cartilage tissue, comprising the steps of contacting chondrocyte cells with a polynucleotide sequence comprising a sequence selected from the group consisting of sequences complementary to SEQ ID No: 1-28, preferably selected from the group consisting of sequences complementary to SEQ ID NO: 202-315 for a time sufficient to re-differentiate the chondrocytes thereby producing a cartilaginous matrix.
  • the method comprises the steps of: (a) applying chondrocyte cells on a substrate to form a cellular substrate, (b) introducing a polynucleotide comprising a nucleotide sequence selected from the group consisting of sequences complementary to SEQ ID No: 1-28, preferably selected from the group consisting of sequences complementary to SEQ TD NO: 202-315, for a time sufficient to re-differentiate the chondrocyte cells, thereby producing a cartilaginous matrix.
  • the invention thus provides a method for producing a substrate with a matrix grown thereon, which matrix may be used for the provision of load-bearing implants, including joint prostheses, such as artificial knee joints and finger joints, and maxillofacial implants.
  • the present invention can also be used for special surgery devices, such as spacers, or cartilage fillers, and for use in augmentation, obliteration or reconstitution of cartilage defects and damaged or lost cartilage.
  • the present invention also relates to a combination of a load-bearing implant (preferably coated with a matrix as described above) with a cartilage filler comprising a matrix as described.
  • the method of the invention is also very suitable in relation to revision surgery, i.e., when previous surgical devices require replacement.
  • Suitable cells are stem cells cells, including mesenchymal stem cells cells and in particular chondrosyte precursor cells. The mesenchymal stem cells, and especially the chondrosyte precursor cells are found to be very effective in the cartilage producing process when taken from their original environment.
  • cells derived from cartilage biopsies of a subject may be cultured and utilized with the present invention.
  • the mesenchymal stem cells can be directly applied on the substrate or they can advantageously be multiplied in the absence of the substrate before being applied on the substrate. In the latter mode, the cells are still largely multipotent after multiplication and, for the pu ⁇ ose of the invention, they are still referred to as undifferentiated. Subsequently, the cells are allowed to differentiate. Differentiation can be induced or enhanced by the presence of suitable inductors, - such as bone morphogenic proteins (BMP2; BM4; BMP7), tiansforming growth factor beta (TGFbeta), CDMP1 and CDMP2.
  • suitable inductors - such as bone morphogenic proteins (BMP2; BM4; BMP7), tiansforming growth factor beta (TGFbeta), CDMP1 and CDMP2.
  • Especially suitable inductors of differentiation are the expression inhibitory agents of the present invention.
  • mesenchymal stem cells provides several advantages. Firstly, their lower differentiation implies a higher proUferation rate and allows the eventual functionality to be better directed and controlled. Moreover, culturing these cells not only produces the required cartilage matrix containing organic and inorganic components, but also results in the presence, in the culture medium and in the matrix, of several factors which are essential for growth of the tissue and for adaptation to existing living tissue.
  • the culture medium can be a source of active factors such as growth factors, to be used in connection with the implanting process.
  • undifferentiated cells are often available in large quantities and more conveniently than e.g., mature cartilage cells, and exhibit a lower morbidity during recovery.
  • the undifferentiated cells can be obtained from the patient for whom the implant is intended.
  • the cartilage resulting from these cells is autologous to the patient and thus no immune response will be induced.
  • Matrices as thick as 100 ⁇ m can be produced as a result of the use of undifferentiated cells.
  • the substrate on which the undifferentiated cells can be applied and cultured can be a metal, such as titanium, cobalt chromium alloy or stainless steel, a bioactive surface such as a calcium phosphate, polymer surfaces such as polyethylene, and the like.
  • siliceous material such as glass ceramics, can also be used as a substrate.
  • the substrate may be porous or non-porous.
  • the cells can be applied at a rate of e.g., 10 3 -10 6 per cm 2 , in particular 10 4 - 2 X 10 5 cells per cm 2 .
  • the culture medium to be used in the method according to the invention can be a commonly known culture medium such as MEM (minimum essential medium).
  • the medium can be a conditioned medium.
  • a conditioned medium is understood to be a medium wherein similar cells have previously been incubated, causing the medium to contain factors such as polypeptides, secreted by the cells which are important for cell growth and cell differentiation.
  • the cells are cultured for a time sufficient to produce a matrix layer, e.g, a matrix layer having a thickness of at least 0.5 ⁇ m, in particular from 1 up to 100 ⁇ m, more in particular of 10-50 ⁇ m.
  • the cells may be contacted with the culture medium for e.g. 2-15 weeks, in particular 4-10 weeks.
  • the production of the matrix when applied on a substrate, results in a continuous or quasi-continuous coating covering the substrate for at least 50%, in particular at least 80% of its surface area.
  • the invention provides a method for diagnosing a pathological condition involving chondrocyte de-differentiation, said method comprising the steps of: determining the nucleic acid sequence of any one of the genes encoding the polypeptides listed in Table 1A in a genomic DNA sample; comparing the sequence from step (a) with the nucleic acid sequence of a healthy subject; and identifying any difference(s) related to the pathological condition. Such differences may be further checked in in vitro assays applying similar marker genes as disclosed herein. Such assays will reveal the role of the gene or its encoded polypeptide in anabolic stimulation processes of chondrocytes.
  • Still another aspect or the invention relates to a method for diagnosing a pathological condition involving chondrocyte anabolic stimulation or a susceptibility to the condition in a subject, comprising deterrnining the amount of polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 101-128 and 401-594 in a biological sample, and comparing the amount with the amount of the polypeptide in a healthy subject, wherein an increase of the amount of polypeptide compared to the healthy subject is indicative of the presence of the pathological condition.
  • the activity and/or expression levels of the target genes as disclosed herein may have an effect on anabolic stimulation of chondrocytes. It remains to be determined to what level the activity should be elevated to diagnose for the disease. However, by comparing levels found in patients, individuals without symptoms and clearly healthy individuals the skilled person may easily determine these relevant levels. Since the skilled person is now aware which polypeptides should be monitored, the present invention provides novel tools for test assays for such diagnostics.
  • a prominent disease that may be controlled, checked and diagnosed by using the knowledge provided by the present invention is osteoarthritis.
  • the rate of chondrocyte anabolic stimulation can typically be measured by determining the deposition of cartilage, or cartilage components, or cartilage-containing extra-cellular matrix produced by the chondrocytes, in the medium.
  • a cell-based ELISA, enzymatic assays, or other general techniques known in the art can be used to measure cartilage components, like the ones described in Walsh G., Proteins: Biotechnology and Biochemistry. John Wiley and Sons, 2001.
  • the invention is further illustrated in the following figures and examples.
  • Example 1 Development of a high-throughput screening method for the detection of endogenous collagen type IL alpha- 1 (col2 ⁇ l) Principle of the assay: Normal human articular chondrocytes (NHAC's) that are grown in two-dimensional cultures become dedifferentiated and gradually cease to synthesize cartilage. They can be re- differentiated into anabolic, active chondrocytes in the presence of appropriate factors (e.g. BMP2). An assay to screen for such factors was developed by monitoring the levels of collagen type ⁇ , alpha-1 (col2 ⁇ l), a major constituent of normal cartilage.
  • NHAC's are seeded in 384 well plates and 1 day after plating infected with individual siRNA adenoviruses (Ad-siRNA) from the SilenceSelect collection (see WO03/020931). Col2 ⁇ l deposition is determined at 14 days after the start of the infection (14 dpi).
  • Ad-BMP2 described in WO 03/018799
  • BMP4 Ad5 dEl/E2A adenoviruses that mediate the expression of full length bone mo ⁇ hogenetic protein 4 pre-protein (see NP_570912).
  • Ad-LacZ referred to as pIPspAdApt6-lacZ in WO 02/070744
  • Ad-eGFP referred to as ⁇ IPspAdApt6-eGFP in WO 02/070744
  • BMP2 or Ad-eGFP BMP2 or Ad-eGFP
  • duration of infection duration of infection
  • toxicity using Ad-eGFP
  • infection efficiency using Ad-eGFP
  • day of readout Using Ad-BMP2 (BMP2 over-expression) as a positive control for assay development, the following protocol resulted in the highest dynamic range for the assay with the lowest standard deviation on the background signal: NHAC's are seeded on day 0 at 1500 cells/well of a 384- well plate in 60 ⁇ l of
  • DMEM/F12 (InVitrogen), containing 10% heat-inactivated fetal calf serum (FBS-HI) and Pen/Strep) and infected the next day with 2.5 ⁇ l of Ad-control- virus (Ad-BMP2 or Ad-eGFP; this corresponds to an assumed MOI of 2000). After 7 days, 10 ⁇ l of a 50 ⁇ g/ml 2-Phospho-
  • L-ascorbic acid in assay culture medium is added to each well.
  • Up-regulation of Col2al is read at 10 dpi: The medium is removed with a VacuSafe; 50 ⁇ l ice-cold MeOH is added with a multidrop and removed immediately with a VacuSafe; 80 ⁇ l of ice-cold MeOH is added with a multidrop to fixate the cells, and plates are incubated for 20 min at -20°C; MeOH is removed with a VacuSafe; plates are air-dried for 20 min, followed by 2x washing with 80 ⁇ l of phosphate buffered saline (PBS); 75 ⁇ l of blocking buffer (0.1% casein in PBS) is added and plates are incubated for at least 2 h at room temperature (RT); blocking buffer is removed; cells are washed with 25 ⁇ l of EC buffer (20 mM sodium phosphate, 2mM EDTA, 400 mM NaCl, 0.2% BSA, 0.05% CHAPS, 0.4% case
  • Example 2 Screening of 9216 adenoviral siRNA vectors in the chondrogenesis assay
  • the optimized protocol for screening the SilenceSelect library runs as follows: on day 0, propagated human primary chondrocytes are seeded in Greiner white, flat bottom, TC- treated 384 well plates with clear bottom (Catalogue number 781080) in 60 ⁇ l medium at a density of 1500 cells per well.
  • Ad-siRNA virus from each well of the SilenceSelectTM collection (WO 03/020931), stored in 384 well plates (estimated titer of 1 x 10 9 viral particles per ml) is transferred with the aid of a 96/384 channel dispenser (Tecan Freedom 200 equipped with TeMO96, TeMO384 and RoMa, Tecan AG, Switzerland) to individual wells of the 384 well plates containing chondrocytes.
  • the control plate is run under the same conditions as the aliquot plates from the SilenceSelect collection. All Ad- siRNA viruses are screened in duplicate on independent assay plates. After infection, plates are incubated at 37°C.
  • the threshold (2.5 times standard deviation) is indicated by dotted lines. Negative values indicate data points that scored below average. The results for some of the genes are shown in figure 3. A clear induction of the collagen II levels is observed upon infection of the Ad-siRNA targeting the indicated gene. The data are represented as relative light units (rlu) correlating to collagen II levels.
  • Example 3 Propagation of hits The 282 Ad-siRNA hits are subjected to further analysis to establish their therapeutic potential to induce chondrocyte anabolic stimulation.
  • a first step entails a quality control on the Ad-siRNA selected for further analysis (this example).
  • Next steps are the screening of the targets in other assays to validate their role in chondrocyte anabolic stimulation such as the induction of aggrecan, another main constituent of cartilage besides collagen IT (Example 4), the ability to induce chondrocyte anabolic stimulation in chondrocytes from other donors (Example 5), the induction of a correct marker profile in tluee-dimensional chondrocyte cultures (example 11), the presence of posttranslational modifications on aggrecan (example 9) and collagen II (example 10) in three-dimensional chondrocyte cultures, the development of additional Ad-siRNAs targeting the identified transcripts (example 7), and confirmation that the corresponding genes are indeed expressed in residing chondrocytes (example 12).
  • chondrocyte anabolic stimulation such as the induction of aggrecan, another main constituent of cartilage besides collagen IT (Example 4), the ability to induce chondrocyte anabolic stimulation in chondrocytes from
  • the propagated Ad-siRNAs are re-screened at three MOFs in the chondrogenesis assay in duplicate (see Example 1).
  • the Ad-siRNAs have to score at least once above threshold (average + 2.5 times standard deviation) to pass this quality control step.
  • Example 4 Aggrecan induction A second assay to screen for chondrocyte anabolic factors is developed by monitoring the levels of aggrecan, another major constituent of cartilage.
  • aggrecan glycosaminoglycans on aggrecan are stained by Alcian blue.
  • NHACs are seeded in 384 well plates and 1 day after plating infected with individual Ad-siRNA from the SilenceSelectTM collection. Aggrecan deposition is determined at 14 days post infection.
  • Ad-BMP2 as a positive control, we confirm in a series of experiments that several parameters optimized for the Coin cELISA assay are also applicable for the Alcian blue stain assay for aggrecan.
  • NHAC's are seeded on day 0 at 1500 cells/well of a 384 well black-plate with clear bottom in 60 ⁇ l of DMEM/F12, containing 10% FBS-HI and Pen/Strep and infected the next day with 2.5 ⁇ l of Ad-BMP2 or Ad-eGFP; at an MOI of 2000. After 7 days, 10 ⁇ l of a 50 ⁇ g/ml 2-Phospho-L-ascorbic acid in assay culture medium is added to each well.
  • Up- regulation of aggrecan is read at 10 dpi with Alcian blue staining: The medium is removed with a VacuSafe; 50 ⁇ l ice-cold MeOH is added with a multidrop and removed immediately with a VacuSafe; 80 ⁇ l of ice-cold MeOH is added with a multidrop to fixate the cells, and plates are incubated for 20 min at -20°C; MeOH is removed with a VacuSafe; plates are air dried for 20 min.
  • Donor dependency The 282 hits identified by the ColII cELISA assay are further subject to a donor dependency test to demonstrate that the induction of Coin production by a given hit is not restricted to a single donor.
  • donor I 11 -year-old donor
  • NHAC's from multiple donors with ages of 24 (donor II), 41 (donor DI), and 50 (donor TV) are obtained after informed consent (Cambrex, Venders, Belgium). Cells are seeded as described in Examples 1 and 3.
  • the propagated Ad-siRNAs are used to transduce NHAc cells from these different donors at three MOI's in duplicate in the chondrogenesis assay (see Examples 1 and 3).
  • the Ad-siRNAs have to score at least once above threshold (average + 2.5 times standard deviation) to pass this donor dependency test. Out of 101 hits that passed the Alcian blue assay for aggrecan, 97 score positive in Donor TV. Of the remaining 4, 1 score positive in Donor II. The other 3 did not score in the other donors tested. In addition, 40 of the 101 hits score positive in all three additional donors. These results demonstrate that 98 out of these 101 hits function in a non-donor- dependent manner. Exceptions are H33-145; H33-182; and H33-263 (see Table 1). Indicated in this Table 1 are the Target Gene Symbol, Gene Bank Accession Number, and drugability class of the genes that correspond to the target sequences. The results for some of the genes are shown in Table 2.
  • Target Ad-siRNAs are propagated using derivatives of PER.C6 ® cells (Crucell, Leiden, The Netherlands) at a 96-well plate level, followed by re-screening these viruses at several MOFs in the primary assay (see Example 1) and by sequencing the siRNAs encoded by the target Ad-siRNA viruses.
  • PER.E2A cells are seeded in 96 well plates at a density of 40,000 cells per well in 180 ⁇ l PER.E2A medium. Cells are then incubated overnight at 39°C in a 10% C0 2 humidified incubator.
  • cells are infected with 1 ⁇ l of crude cell lysate from SilenceSelect stocks containing target Ad-siRNAs. Cells are incubated further at 34°C, 10% CO 2 until appearance of cytopathic effect (as revealed by the swelling and rounding up of the cells, typically 7 days post infection). The supernatant is collected and the virus crude lysate is treated with proteinase K: 12 ⁇ l crude lysate is added to 4 ⁇ l Lysis buffer (lx Expand High Fidelity buffer with MgCl 2 (Roche Molecular Biochemicals, Cat.
  • 1 ⁇ l lysate is added to a PCR master mix composed of 5 ⁇ l lOx Expand High Fidelity buffer with MgCl 2 , 0.5 ⁇ l of dNTP mix (10 mM for each dNTP), 1 ⁇ l of 'Forward primer' (10 mM stock, sequence: 5' CCG TTT ACG TGG AGA CTC GCC, SEQ ID NO: 29), 1 ⁇ l of 'Reverse Primer' (10 mM stock, sequence: 5' CCC CCA CCT TAT ATA TAT TCT TTC C, SEQ ID NO: 30), 0.2 ⁇ l of Expand High Fidelity DNA polymerase (3.5 U/ ⁇ l, Roche Molecular Biochemicals) and 41.3 ⁇ l of H 2 O.
  • PCR is performed in a PE Biosystems GeneAmp PCR system 9700 as follows: the PCR mixture (50 ⁇ l in total) is incubated at 95°C for 5 min; each cycle runs at 95°C for 15 sec, 55°C for 30 sec, 68°C for 4 min, and is repeated for 35 cycles. A final incubation at 68°C is performed for 7 min. 5 ⁇ l of the PCR mixture is mixed with 2 ⁇ l of 6 x gel loading buffer, loaded on a 0.8% agarose gel containing 0.5 ⁇ g/ ⁇ l ethidium bromide to resolve the amplification products. The size of the amplified fragments is estimated from a standard DNA ladder loaded on the same gel. The expected size is ⁇ 500 bp.
  • siRNA constructs expressed by the target adenoviruses are amplified by PCR using primers complementary to vector sequences flanking the Sapl site of the pIPspAdapt6-U6 plasmid.
  • the sequence of the PCR fragments is determined and compared with the expected sequence. All sequences are, found to be identical to the expected sequence.
  • Example 7 Evaluation of the on target effect of the identified siRNA sequences. To evaluate whether the identified siRNA sequences really increase collagen ⁇ and aggrecan levels through the knock down of the target mRNA, a second siRNA sequence is identified that exerts the same effect. A number of additional siRNA sequences targeting the HIT sequences are designed and incorporated in adenoviruses according to WO 03/020931. After production of these adenoviruses, the Ad-siRNAs are infected at different volumes (1.5 ⁇ L 5 ⁇ l and 15 ⁇ l) in the chondrocytes and their effect on collagen ⁇ is assessed as described in example 1. The threshold (average 4- 2.5 standard deviation) is calculated for every volume.
  • Example 8 Development of a tlnee-dimensional-alginate culture system for a quantitative marker analysis for stable cartilage and the assessment of glycosaminoglycans (GAG) and hvdroxyprolines (Hyp) synthesis.
  • GAG glycosaminoglycans
  • Hyp hvdroxyprolines
  • Ad-siRNA siRNA adenoviruses
  • Ad-siRNA siRNA adenoviruses
  • mRNA expression pattern that correlates with anabolic active chondrocytes
  • aggrecan glycosaminoglycans
  • Normal cartilage NHAC's are cultured in a two-dimensional culture system for two or three passages for cell expansion purposes.
  • Cells are transduced with individual siRNA adenoviruses (Ad-siRNA) in the two-dimensional culture system and three days later are transferred into the three-dimensional alginate culture system. After 10 days in the alginate culture, various parameters can be assessed (e.g. mRNA marker analysis and protein modifications).
  • cells are trypsinised using the chondrocyte reagent pack (Cambrex) and washed once with 155 mM sodium chloride/20 mM Hepes ph 7.4 (Cambrex). Cells are re-suspended at a density of 2x10 6 cells/ml in 1.2% sodium alginate (Cambrex). The cell suspension is transferred into a syringe attached to a 21-22 gauge needle and expelled in a drop-wise fashion into 102 mM calcium chloride/5 mM Hepes pH 7.4 (1 ml and 5 ml respectively in 24-well and 6-well plate) Five and 50 beads per well are produced respectively in the 24-well and 6-well plates.
  • Alginate beads are finally re-suspended into 0.5 ml and 3 ml (respectively in 24-well and 6-well plate) of DMEMF/12 supplemented with 10% heat inactivated fetal calf serum (FBS-HI), 1% penicillin/streptomycin and 25 ⁇ g/ml of ascorbic acid (Fluka, SigmlOl-128 and 401- 5941drich).
  • FBS-HI heat inactivated fetal calf serum
  • penicillin/streptomycin 1% penicillin/streptomycin
  • 25 ⁇ g/ml of ascorbic acid Fluka, SigmlOl-128 and 401- 5941drich.
  • alginate beads For each adenoviral transduction, 60 alginate beads are generated: 2x5 beads were cultured in 24-well plates for the GAGs/Hyps assessments and 50 beads are cultured in a single well of a 6-well plate for the mRNA expression pattern determination. Control viruses
  • Ad-BMP2 described in WO 03/018799
  • Ad-ALPL Ad5 dEl/E2A adenoviruses that mediate the expression of full length liver/bone/kidney alkaline phosphatase (NP_000469).
  • Example 9 Effect of knock down of target genes in chondrocytes embedded in alginate beads, on the glycosaminoglycans fGAGs levels. Chondrocytes are infected and embedded in alginate beads according to example 8.
  • the Blyscan Assay is a quantitative dye-binding method for the analysis of sulfated
  • GAGs The dye label used in the assay is 1,9-dimethylmethylene blue employed under conditions that produce a specific label for the sulfated polysaccharides component of proteoglycans and/or the protein-free sulfated glycosaminoglycan chains.
  • Aggrecan is the predomninant proteoglycan in articular cartilage, representing ⁇ 90% of the cartilage proteoglycans. It is composed of a central core protein attached to ⁇ 50 keratan sulfate and ⁇ 100 chondroitin sulfate chains known as GAGs required for the biological function and the stability of aggrecan.
  • the papain digests generated after culturing the chondrocytes for 10 days in alginate beads are diluted in water 1:100, 1:200 if cells were originally transduced with the positives controls and 1:50, 1:100 if transduced with the negative controls. This dilution step allows readout values within the standard range of the assay.
  • the GAG standard provided by the manufacturer contains 100 ⁇ g/ml of chondroitin 4-sulfate purified from bovine trachea. This standard is run in duplicate at four concentrations corresponding to 1, 2, 3 and 5 ⁇ g of GAGs. Standard and controls are individually diluted in lOO ⁇ l final volume in eppendorf tubes.
  • One millihter of the Blyscan Dye Reagent is added to each tube and incubated for 30 minutes at room temperature with continuous shaking. When formed, the GAG-dye complex becomes insoluble and is then separated from the remaining excess soluble unbound dye by centrifugation (10000 x g for 10 minutes). Supernatant are discarded by inverting and careful draining of the tube contents.
  • One milliliter of Blyscan Dissociation Reagent is added to each tube and incubated for one hour to one and a half hour with continuous shaking. This reagent brings the GAG-bound dye back into solution. The GAG content of the assayed samples is spectrophotometrically determined by the amount of dye recovered from the GAGs in the test sample.
  • Two hundred microhters of the dissociation dye solutions are added to the wells of a 96-well plate and reading is performed on an automatic plate reader set at a dual wavelength (656 and 450 nm).
  • the GAGs concentrations measured in the Blyscan assay are normalized to DNA content by performing a fluorimetric Hoechst assay on the same papain digests.
  • Hoechst 33342 dye reagent Molecular Probes
  • Papain digests were diluted 1:1.7 and 1:3.3 in TE buffer, this dilution step allows readout values within the standard range of the assay.
  • Purified calf thymus DNA (SigmlOl-128 and 401-5941drich) is used as standard DNA: the initial stock solution (2 ⁇ g/ml) is sequentially diluted in TE buffer in order to obtain the following concentration range: 1.5, 1.0, 0.75, 0.50, 0.2 ⁇ g/ml. Standard and test samples are diluted in TE to a final volume of 100 ⁇ l and added to a 96-well plate. One hundred microliters of Hoechst 33342 dye reagent are added to the wells, reading is performed on a multifunctional microplate reader (Fluostar Galaxy, BMG Labtechnologies GmbH) with an excitation wavelength set at 360nm and an emission wavelength set at 440 nm.
  • a multifunctional microplate reader Feluostar Galaxy, BMG Labtechnologies GmbH
  • Ad-BMP2 described in WO 03/018799
  • Ad-BMP4 Ad5 dEl E2A adenoviruses that mediate the expression of full length bone morphogenetic protein 4 pre-protein (see NP_570912).
  • Ad-ALPL Ad5 dEl/E2A adenoviruses that mediate the expression of full length liver/bone/kidney alkaline phosphatase (NP_000469).
  • Ad-PTGER4 Ad5 dEl/E2A adenoviruses that comprise the siRNA sequence CCATGCCTATTTCTACAGC (SEQ ID NO: 31) to knock down the prostaglandin E receptor 4 mRNA.
  • Ad-GRM7 Ad5 dEl/E2A adenoviruses that comprise the siRNA sequence TCAGTAACAGCTCCCAGAC (SEQ ID NO: 32) to knock down the metabotropic glutamate receptor 7 mRNA.
  • Example 10 Quantitative analysis of Hvdroxyprolines (Hyps).
  • Hvdroxyprolines Hvdroxyprolines
  • articular cartilage approximately 95% of the collagen is type II collagen. Its polymers are the fibrils that form the basic cohesive framework of the tissue.
  • the collagen biosynthesis involves several unique posttranslational modifications including hydroxylation of proline and lysine residues. These modifications are crucial for collagen stability and resistance to proteolytic enzymes. Chondrocytes are infected and embedded in alginate beads according to example 8.
  • Ad-ALPL Ad5 dEl/E2A adenoviruses that mediate the expression of full length liver/bone/kidney alkaline phosphatase (NP 000469).
  • Ad-PTGER4 Ad5 dEl/E2A adenoviruses that comprise the siRNA sequence CCATGCCTATTTCTACAGC (SEQ ID NO: 33) to knock down the prostaglandin E receptor 4 mRNA.
  • Ad-GRM7 Ad5 dEl/E2A adenoviruses that comprise the siRNA sequence TCAGTAACAGCTCCCAGAC (SEQ ID NO: 34) to knock down the metabotropic glutamate receptor 7 rnRNA.
  • Example 11 Quantitative analysis of markers for stable cartilage- Assay general principle: Chondrocyte phenotypes can be categorized by characteristic patterns of gene expression. Quantitative RT-PCR techniques are used to monitor the expression pattern of a set of key marker molecules to define which phenotype is induced on the chondrocytes. Positive markers included collagen type II and FGFR3, typically expressed by cartilage chondrocytes.
  • the alginate beads are treated with 55 mM sodium citrate (Cambrex) in order to recover the chondrocytes from the alginate beads and harvest RNA: Incubation medium was removed from the 6-well plates with the Vacusafe and 5 ml of 55 mM sodium citrate are added to each well and incubated for 15 rninutes at room temperature. The partially solubilised beads are gently mixed and transferred to a FALCON tube, wells are rinsed once with 2 ml of sodium citrate solution to collect the remaining beads and released cells. Tubes are laid on their side and gently mixed every 2-3 minutes until beads have completely solubilised (+/- 15 minutes). Tubes are centrifuged at 1000 rpm for 10 minutes.
  • RNA is quantified using ribogreen reagent (Molecular Probes) and yeast RNA (Ambion) as standard RNA.
  • RNA is first diluted in water (Life Technologies-Invitrogen, Breda, The Netherlands) depending on the initial concentration of the sample: For RNA concentration below 25 ng/ml, samples are used undiluted, for RNA concentration between 25 and 50 ng/ml samples are diluted 1:2.5, For RNA concentration between 50 and 100 ng/ml samples are diluted 1:5 and for RNA concentration between 100 and 160 ng/ml samples are diluted 1:10.
  • RNAse inhibitor 1 ⁇ M of RNAse inhibitor
  • PCR reaction is performed in a Peltier Thermal Cycler-200 (BlOzym, Landgraaf, The Netherlands) as followed: the PCR total mixture (60 ⁇ l) is incubated 10 minutes at 25°C followed by 30 minutes at 48°C, followed by 5 minutes at 95°C.
  • Reverse transcription is followed by a quantitative PCR for specific amplification of the selected positive and negative marker genes, GAPDH is included as the endogenous control.
  • cDNA Five microliters of cDNA (from RT reaction) are added to 20 ⁇ l of a PCR reaction mix consisting of: IX Brilliant ® SYBR ® Green QPCR Master Mix (Sratagene Europe, Amsterdam, The Netherlands), 300 ⁇ M of each forward and reverse primers (table 3) (except for GAPDH and ALK-1, used at 100 ⁇ M)(Life Technologies-rnvitrogen, Breda, The Netherlands) and 300 nM of reference dye (Stratagene) (diluted 1:100 in H2O).
  • the PCR mixture (25 ⁇ l) is incubated for 10 minutes at 95°C followed by 40 cycles: 15 seconds at 95°C followed by 1 minute at 60°C in the ABI PRISM ® 7000 Sequence Detection System (Applied Biosystems, Nieuwerkerk AID Ijssel, The Netherlands).
  • Table 3 primer sequences for the positive and negative markers:
  • the results for the 14 genes are shown in figure 9.
  • the knock down of the mRNA of the respective genes results in expression of the positive markers, while the levels of the negative markers are either low or not detectable.
  • Ad-ALPL Ad5 dEl/E2A adenoviruses that mediate the expression of full length liver/bone/kidney alkaline phosphatase (NP_000469).
  • Example 12 Expression of cDNA's in human cartilage Upon identification of a modulator of cartilage synthesis, it is of the highest importance to evaluate whether the modulator is expressed in the tissue and the cells of interest. This can be achieved by measuring the RNA and/or protein levels. In recent years, RNA levels are being quantified through real time PCR technologies, whereby the RNA is first transcribed to cDNA and then the amplification of the cDNA of interest is monitored during a quantitative PCR reaction.
  • the amplification plot and the resulting Ct value are indicators for the amount of a specific RNA transcript present in the sample.
  • Ct values are determined in the presence or absence of the reverse transcriptase step (+RT versus -RT).
  • An amplification signal in the -RT condition indicates the occurrence of non-specific PCR products originating from the genomic DNA. If the +RT Ct value is 3 Ct values higher than the -RT Ct value, then the investigated RNA is present in the sample.
  • real time PCR with specific primers for the polynucleotides (“Assay on Demand" Applied Biosystems) is performed on human cartilage total RNA (Clinomics Biosciences).
  • RNA isolated from human cartilage is analyzed for the presence of transcripts listed in Table 4 via quantitative real time PCR. For the genes listed in Table 5 the obtained Ct values indicate that they are detected in all RNA samples. ELA1 RNA is not detected in the real time PCR analysis, underscoring the need for additional patient analysis.
  • Example 13 Identification Of Small Molecules That Inhibit TARGET Kinase Activity
  • Compounds are screened for inhibition of the activity of the TARGETS that are kinase polypeptides.
  • the affinity of the compounds to the polypeptides is determined in an experiment detecting changed reaction conditions after phosphorylation.
  • the TARGET kinase polypeptides are incubated with its substrate and ATP in an appropriate buffer. The combination of these components results in the in vitro phosphorylation of the substrate.
  • Sources of compounds include commercially available screening library, peptides in a phage display library or an antibody fragment library, and compounds that have been demonstrated to have binding affinity for a TARGET kinase.
  • the TARGET kinase polypeptides can be prepared in a number of ways depending on whether the assay will be run using cells, cell fractions or biochemically, on purified proteins.
  • the polypeptides can be applied as complete polypeptides or as polypeptide fragments, which still comprise TARGET kinase catalytic activity.
  • Identification of small molecules inhibiting the activity of the TARGET kinase polypeptides is performed by measuring changes in levels of phosphorylated substrate or ATP. Since ATP is consumed during the phosphorylation of the substrate, its levels correlate with the kinase activity. Measuring ATP levels via chemiluminescent reactions therefore represents a method to measure kinase activity in vitro (Perkin Elmer).
  • phosphorylated substrate changes in the levels of phosphorylated substrate are detected with phosphospecific agents and are correlated to kinase activity. These levels are detected in solution or after immobilization of the substrate on a microtiter plate or other carrier.
  • the phosphorylated substrate is detected via fluorescence resonance energy transfer (FRET) between the Eu labeled substrate and an APC labeled phosphospecific antibody (Perkin Elmer), via fluorescence polarization (FP) after binding of a phosphospecific antibody to the fluorescently labeled phosphorylated substrate (Panvera), via an Amplified Luminescent Proximity Homogeneous Assay (ALPHA) using the phosphorylated substrate and phosphospecific antibody, both coupled to ALPHA beads (Perkin Elmer) or using the IMAP binding reagent that specifically detects phosphate groups and thus alleviates the use of the phosphospecific antibody (Molecular Devices).
  • FRET fluorescence resonance energy transfer
  • FP fluorescence polarization
  • APHA Amplified Luminescent Proximity Homogeneous Assay
  • the substrate is immobilized directly or by using biotm-stieptavidin on a microtiter plate.
  • the level of phosphorylated substrate is detected using a classical ELISA where binding of the phosphospecific antibody is either monitored via an enzyme such as horseradish peroxidase (HRP) or alkaline phospahtase (AP) which are either directly coupled to the phosphospecific antibody or are coupled to a secondary antibody. Enzymatic activity correlates to phosphorylated substrate levels.
  • binding of the Eu-labeled phosphospecific antibody to the immobilized phosphorylated substrate is determined via time resolved fluorescence energy (TRF) (Perkin Elmer).
  • TRF time resolved fluorescence energy
  • the substrate can be coated on FLASH plates (Perkin Elmer) and phosphorylation of the substrate is detected using 33 P labeled ATP or 5 l labeled phosphospecific antibody.
  • Small molecules are randomly screened or are preselected based upon drug class, (i.e. known kinase inhibitors), or upon virtual ligand screening (VLS) results. VLS uses virtual docking technology to test large numbers of small molecules in silico for their binding to the polypeptide of the invention. Small molecules are added to the kinase reaction and their effect on levels of phosphorylated substrate is measured with one or more of the above- described technologies. Small molecules that inhibit the kinase activity are identified and are subsequently tested at different concentrations.
  • IC 5 0 values are calculated from these dose response curves. Strong binders have an IC 50 in the nanomolar and even picomolar range. Compounds that have an IC 50 of at least 10 micromol or better (nmol to pmol) are applied in collagen II assay to check for their effect on the induction of chondrocyte anabolic stimulation.
  • Example 14 Ligand Screens For TARGET GPCRs.
  • Example 14 A Reporter Gene Screen.
  • Mammalian cells such as Hek293 or CHO-K1 cells are either stably transfected with a plasmid harboring the luciferase gene under the control of a cAMP dependent promoter (CRE elements) or transduced with an adenovirus harboring a luciferase gene under the control of a cAMP dependent promoter.
  • CRE elements cAMP dependent promoter
  • reporter constructs can be used with the luciferase gene under the control of a Ca 2+ dependent promoter (NF-AT elements) or a promoter that is controlled by activated NF- ⁇ B.
  • NF-AT elements Ca 2+ dependent promoter
  • NF-AT elements promoter that is controlled by activated NF- ⁇ B.
  • adenovirus harboring the cDNA of a TARGET GPCR Forty (40) hours after transduction the cells are treated with the following: a) an agonist for the receptor and screened against a large collection of reference compounds comprising peptides (LOPAP, Sigma Aldrich), hpids (Biomol, TimTech), carbohydrates (Specs), natural compounds (Specs, TimTech), small chemical compounds (Tocris), commercially available screening libraries, and compounds that have been demonstrated to have binding affinity for a polypeptide comprising an amino acid sequence selected from the group consisting of the SEQ ID NOs of the TARGET GPCRs; or b) a large collection of reference compounds comprising peptides (LOPAP, Sigma Aldrich), hpids (Biomol, TimTech), carbohydrates (Specs), natural compounds (Specs, TimTech), small chemical compounds (Tocris), commercially available screening libraries, and compounds that have been demonstrated to have binding affinity for a polypeptide comprising an amino acid sequence selected from the group consisting of the SEQ ID
  • lipids BiomoL TimTech
  • carbohydrates Specs
  • natural compounds Specs, TimTech
  • small chemical compounds Tocris
  • commercially available screening libraries and compounds that have been demonstrated to have binding affinity for a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs of the TARGET GPCRs.
  • Compounds, which decrease the agonist induced increase in luciferase activity or the constitutive activity, are considered to be antagonists or inverse agonists for a TARGET GPCR. These compounds are screened again for verification and screened against their effect on anabolic stimulation of chondrocytes. The compounds are also screened to verify binding to the GPCR.
  • the binding and reporter activity assays can be performed in essentially any order to screen compounds.
  • cells expressing the NF-AT reporter gene can be transduced with an adenovirus harboring the cDNA encoding the ⁇ -subunit of G 15 or chimerical Got subunits.
  • Gi 5 is a promiscuous G protein of the G q class that couples to many different GPCRs and as such re-directs their signaling towards the release of intracellular Ca 2+ stores.
  • the chimerical G alpha subunits are members of the G s and Gj/ 0 family by which the last 5 C-terminal residues are replaced by those of G ⁇ , these chimerical G-proteins also redirect cAMP signaling to Ca 2+ signaling.
  • Example 14 B FLIPR screen.
  • Mammalian cells such as Hek293 or CHO-K1 cells are stably transfected with an expression plasmid construct harboring the cDNA of a TARGET GPCR. Cells are seeded, grown, and selected until sufficient stable cells can be obtained. Cells are loaded with a Ca 2+ dependent fluorophore such as Fura3 or Fura4.
  • the cells are screened against a large collection of reference compounds comprising peptides (LOPAP, Sigma Aldrich), lipids (BiomoL TimTech), carbohydrates (Specs), natural compounds (Specs, TimTech), small chemical compounds (Tocris), commercially available screening libraries, and compounds that have been demonstrated to have binding affinity for a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ TD NOs of the TARGET GPCRs, by simultaneously adding an agonist (alternatively no agonist need be added if the constitutive activity of the receptor is used) and a compound to the cells.
  • Activation of the receptor is measured as an almost instantaneously increase in fluorescence due to the interaction of the fluorophore and the Ca 2+ that is released.
  • Compounds that reduce or inhibit the agonist induced increase in fluorescence are considered to be antagonists or inverse agonists for the receptor they are screened against. These compounds are screened again to measure the amount of anabolic stimulation of chondrocytes as well as binding to a TARGET GPCR.
  • Example 14 C AequoScreen. CHO cells, stably expressing Apoaequorin are stably transfected with a plasmid construct harboring the cDNA of a TARGET GPCR.
  • Cells are seeded, grown, and selected until sufficient stable cells can be obtained.
  • the cells are loaded with coelenterazine, a cofactor for apoaequorin.
  • coelenterazine a cofactor for apoaequorin.
  • intracellular Ca 2+ stores are emptied and the aequorin will react with the coelenterazine in a light emitting process.
  • the emitted light is a measure for receptor activation.
  • the CHO, stable expressing both the apoaequorin and the receptor are screened against a large collection of reference compounds comprising peptides (LOPAP, Sigma Aldrich), lipids (Biomol, TimTech), carbohydrates (Specs), natural compounds (Specs, TimTech), small chemical compounds (Tocris), commercially available screening libraries, and compounds that have been demonstrated to have binding affinity for a polypeptide comprising an an ino acid sequence selected from the group consisting of SEQ ID NOS of the TARGET GPCRs, by simultaneously adding an agonist (alternatively no agonist need be added if the constitutive activity of the receptor is used) and a compound to the cells.
  • Activation of the receptor is measured as an almost instantaneously light flash due to the interaction of the apoaequorin, coelenterazine, and the Ca 2+ that is released.
  • Compounds that reduce or inhibit the agonist induced increase in light or the constitutive activity are considered to be antagonists or inverse agonists for the receptor they are screened against. These compounds are screened again to measure the amount of anabolic stimulation of chondrocytes as well as binding to a TARGET GPCR.
  • CHO cells stable expressing the apoaequorin gene are stably transfected with a plasmid construct harboring the cDNA encoding the ⁇ -subunit of G1 5 or chimerical G ⁇ subunits.
  • G 15 is a promiscuous G protein of the G q class that couples to many different GPCRs and as such redirects their signaling towards the release of intracellular Ca 2+ stores.
  • the chimerical G alpha subunits are members of the G s and Gy 0 family by which the last 5 C- terminal residues are replaced by those of G ⁇ q, these chimerical G-proteins also redirect cAMP signaling to Ca 2+ signaling.
  • Example 14 D Screening for compounds that bind to the GPCR polypeptides (displacement experiment) Compounds are screened for binding to the TARGET, GPCR polypeptides. The affinity of the compounds to the polypeptides is determined in a displacement experiment.
  • the GPCR polypeptides are incubated with a labeled (radiolabeled, fluorescent labeled) hgand that is known to bind to the polypeptide and with an unlabeled compound.
  • the displacement of the labeled ligand from the polypeptide is determined by measuring the amount of labeled hgand that is still associated with the polypeptide.
  • the amount associated with the polypeptide is plotted against the concentration of the compound to calculate IC50 values.
  • This value reflects the binding affinity of the compound to its TARGET, i.e. the TARGET GPCR polypeptides.
  • Strong binders have an IC50 in the nanomolar and even picomolar range.
  • Compounds that have an IC 50 of at least 10 micromol or better (nmol to pmol) are applied an anabohc stimulation of chondrocytes assay to check for their effect on osteogenesis.
  • the TARGET GPCR polypeptides can be prepared in a number of ways depending on whether the assay are run on cells, cell fractions or biochemically, on purified proteins.
  • Example 14 E Screening for compounds that bind to a TARGET GPCR (generic GPCR screening assay)
  • a G protein receptor When a G protein receptor becomes constitutively active, it binds to a G protein (G q , G s , Gi, G 0 ) and stimulates the binding of GTP to the G protein.
  • the G protein then acts as a GTPase and slowly hydrolyses the GTP to GDP, whereby the receptor, under normal conditions, becomes deactivated.
  • constitutively activated receptors continue to exchange GDP to GTP.
  • a non-hydrolyzable analog of GTP [ 35 S]GTP ⁇ S, can be used to monitor enhanced binding to membranes which express constitutively activated receptors.
  • [ 35 S]GTP ⁇ S can be used to monitor G protein coupling to membranes in the absence and presence of ligand.
  • a preferred approach is the use of a GPCR-G protein fusion protein.
  • the strategy to generate a TARGET GPCR-G protein fusion protein is well known for those known in the art.
  • Membranes expressing TARGET GPCR-G protein fusion protein are prepared for use in the direct identification of candidate compounds such as inverse agonist. Homogenized membranes with TARGET GPCR-G protein fusion protein are transferred in a 96-well plate. A pin-tool is used to transfer a candidate compound in each well plus [ 35 S]GTP ⁇ S, followed by incubation on a shaker for 60 minutes at room temperature.
  • Example 14 F Receptor Ligand Binding Study On Cell Surface
  • the receptor is expressed in mammalian cells (Hek293, CHO, COS7) by adenoviral transducing the cells (see US 6,340,595).
  • the cells are incubated with both labeled ligand (iodinated, tritiated, or fluorescent) and the unlabeled compound at various concentrations, ranging from 10 pM to 10 ⁇ M (3 hours at 4°C: 25 mM HEPES, 140 mM NaCl, 1 mM CaCb, 5 mM MgCl 2 and 0.2% BSA, adjusted to pH 7.4).
  • Reactions mixtures are aspirated onto PEI-treated GF/B glass filters using a cell harvester (Packard). The filters are washed twice with ice cold wash buffer (25 mM HEPES, 500 mM NaCl, 1 mM CaCl 2 , 5 mM MgCl 2 , adjusted to pH 7.4).
  • Scintillant (MicroScint-10; 35 ⁇ l) is added to dried filters and the filters counted in a (Packard Topcount) scintillation counter. Data are analyzed and plotted using Prism software (GraphPad Software, San Diego, Calif.). Competition curves are analyzed and IC 50 values calculated. If one or more data points do not fall within the sigmoidal range of the competition curve or close to the sigmoidal range the assay is repeated and concentrations of labeled hgand and unlabeled compound adapted to have more data points close to or in the sigmoidal range of the curve.
  • Example 14 G Receptor Ligand Binding Studies On Membrane Preparations
  • Membranes preparations are isolated from mammalian cells (Hek293, CHO, COS7) cells over expressing the receptor is done as follows: Medium is aspirated from the transduced cells and cells are harvested in 1 x PBS by gentle scraping. Cells are pelleted (2500 rpm 5 min) and resuspended in 50 mM Tris pH 7.4 (10 x 10 6 cells/ml). The cell pellet is homogenized by sonicating 3 x 5 sec (UP50H; sonotrode MSI; max amplitude: 140 ⁇ m; max Sonic Power Thickness: 125W/cm 2 ).
  • Membrane fractions are prepared by centrifuging 20 min at maximal speed (13,000 rpm ⁇ 15,000 to 20,000g or rcf). The resulting pellet is resuspended in 500 ⁇ l 50 mM Tris pH 7.4 and sonicated again for 3 x 5 sec. The membrane fraction is isolated by centrifugation and finally resuspended in PBS. Binding competition and derivation of IC 50 values are determined as described above.
  • Example 14 H Internalization screen (1) Activation of a GPCR-associated signal transduction pathway commonly leads to translocation of specific signal transduction molecules from the cytoplasm to the plasma membrane or from the cytoplasm to the nucleus.
  • Norak has developed their transfluor assay based on agonist-induced translocation of receptor- ⁇ -arrestin-GFP complex from the cytosol to the plasma membrane and subsequent internalization of this complex, which occurs during receptor desensitization.
  • a similar assay uses GFP tagged receptor instead of ⁇ -arrestin.
  • Hek293 cells are transduced with a TARGET GPCR vector that translates for a TARGET GPCR-eGFP fusion protein. 48 hours after transduction, the cells are set to fresh serum-free medium for 60 minutes and treated with a hgand for 15, 30, 60 or 120 minutes at 37°C and 5% CO 2 . After indicated exposure times, cells are washed with PBS and fixed with 5% paraformaldehyde for 20 minutes at RT. GFP fluorescence is visualized with a Zeiss microscope with a digital camera. This method aims for the identification of compounds that inhibit a ligand-mediated (constitutive activity-mediated) translocation of the fusion protein to intra
  • Example 141 Internalization screen 2
  • ⁇ -arrestin and ⁇ -galactosidase enzyme complementation and BRET based assays with receptor as energy donor and ⁇ - arrestin as energy acceptor.
  • specific receptor antibodies labeled with pH sensitive dyes are used to detect agonist induced receptor translocation to acidic lysosomes. All of the translocation assays are used for screening for both agonistic and antagonistic acting ligands.
  • Example 14 J Melanophore assay f Arena Pharmaceutical
  • the melanophore assay is based on the ability of GPCRs to alter the distribution of melanin containing melanosomes in Xenopus melanophores.
  • the distribution of the melanosomes depends on the exogenous receptor that is either Gi/ 0 or G s / q coupled.
  • the distribution of the melanosomes (dispersed or aggregated) is easily detected by measuring light absorption. This type of assay is used for both agonist as well as antagonist compound screens.
  • Example 15 Identification of small molecules that inhibit protease activity.
  • Compounds are screened for inhibition of the activity of the polypeptides of the present invention.
  • the affinity of the compounds to the polypeptides is determined in an experiment detecting changes in levels of cleaved substrate.
  • the polypeptides of the present invention are incubated with its substrate in an appropriate buffer. The combination of these components results in the cleavage of the substrate.
  • the polypeptides can be applied as complete polypeptides or as polypeptide fragments, which still comprise the catalytic activity of the polypeptide of the invention. Cleavage of the substrate can be followed in several ways.
  • the substrate protein is heavily labeled with a fluorescent dye, like fluorescent, resulting in a complete quenching of the fluorescent signal. Cleavage of the substrate however, releases individual fragments, which contain less fluorescent labels. This results in the loss of quenching and the generation of a fluorescent signal, which correlates to the levels of cleaved substrate. Cleavage of the protein, which results in smaller peptide fragments, can also be measured using fluorescent polarization (FP). Alternatively, cleavage of the substrate can also be detected using fluorescence resonance energy transfer (FRET): a peptide substrate is labeled on both sides with either a quencher and fluorescent molecule, like DABCYL and ED ANS.
  • FRET fluorescence resonance energy transfer
  • cleavage of the substrate both molecules are separated resulting in fluorescent signal correlating to. the levels of cleaved substrate.
  • cleavage of a peptide substrate can also generate a new substrate for another enzymatic reaction, which is then detected via a fluorescent, chermuminescent or colorimetric method.
  • Small molecules are randomly screened or are preselected based upon drug class, i.e. protease, or upon virtual ligand screening (VLS) results. VLS uses virtual docking technology to test large numbers of small molecules in silico for their binding to the polypeptide of the invention. Small molecules are added to the proteolytic reaction and their effect on levels of cleaved substrate is measured with the described technologies.
  • IC50 values are calculated from these dose response curves. Strong binders have an IC50 in the nanomolar and even picomolar range. Compounds that have an IC50 of at least 10 micromol or better (nmol to pmol) are applied in amyloid beta secretion assay to check for their effect on the beta amyloid secretion and processing.
  • EXAMPLE 16 Identification of small molecules that inhibit phosphodiesterase activity. Compounds are screened for inhibition of the activity of the polypeptides of the present invention. The affinity of the compounds to the polypeptides is determined in an experiment detecting changes in levels of substrate or product. In brief, the polypeptides of the present invention are incubated with its substrate in an appropriate buffer. The combination of these components results in the conversion of the substrate into its product. The polypeptides can be applied as complete polypeptides or as polypeptide fragments, which still comprise the catalytic activity of the polypeptide of the invention. Conversion of cAMP or cGMP in AMP or GMP can be followed 1) by determining the cAMP or cGMP levels using e.g. ELISA.
  • Alpha screen technology, Time resolved fluorescent technology, IMAP 2) by deterrnining the levels of the products AMP and GMP using a colorimetric assay The basis for the latter assay is the cleavage of cAMP or cGMP by a cyclic nucleotide phosphodiesterase. The 5 '-nucleotide released is further cleaved into the nucleoside and phosphate by the enzyme 5'-nucleotidase. The phosphate released due to enzymatic cleavage is quantified using BIOMOL GREENTM reagent in a modified Malachite Green assay. Small molecules are randomly screened or are preselected based upon drug class, i.e.
  • VLS virtual ligand screening

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Abstract

The present invention relates to in vivo and in vitro methods, agents and compound screening assays for inducing anabolic stimulation of chondrocytes, including cartilage formation enhancing pharmaceutical compositions, and the use thereof in treating and/or preventing a disease involving a systemic or local decrease in mean cartilage thickness in a subject.

Description

Methods and Means for Treatment of Osteoarthritis
FIELD OF THE INVENTION The invention relates to the field of medicinal research, cartilage physiology and diseases involving the degeneration of cartilage tissue. More specifically, the invention relates to methods and means for identifying compounds that stimulate anabolic processes in chondrocytes and that typically induce the synthesis of cartilage. The invention also relates to the compounds that are useful in the treatment of osteoarthritis. Cartilage is an avascular tissue made up largely of cartilage-specific cells, the chondrocytes, proteoglycans and collagen proteins, which are structural proteins that provide structural strength to connective tissue, such as skin, bone and cartilage. Collagen π, together with the protein collagen IX, forms a "biological alloy", which is molded into a fibril-like structure and is arranged in a precise network, providing cartilage with great mechanical strength. The chondrocytes in normal articular cartilage occupy approximately 5% of the tissue volume, while the extra-cellular matrix makes up the remaining 95% of the tissue. The chondrocytes secrete the components of the matrix, which in turn supplies the chondrocytes with an environment suitable for their survival under mechanical stress. Breakdown of articular cartilage, which is part of joints and which cushions the ends of the bones, causes the bones to rub against each other leading to pain and loss of movement. Cartilage degradation may also be the result of an imbalance in cartilage synthesizing (anabolic) and cartilage degrading (catabolic) processes. Unlike most tissues, cartilage does not self-repair following injury. The inability of cartilage to self-repair after injury, disease, or surgery is a major limiting factor in rehabilitation of degrading joint surfaces and injury to meniscal cartilage. There are many diseases involving the degeneration of cartilage. Rheumatoid arthritis and osteoarthritis are among the most prominent. Osteoarthritis (also referred to as OA, or as wear-and-tear arthritis) is the most common form of arthritis and is characterized by loss of articular cartilage, often associated with hypertrophy of the bone. The disease mainly affects hands and weight-bearing joints such as knees, hips and spines. This process thins the cartilage through a phenomenon called apoptosis, or programmed cell death. When the surface area has disappeared due to the thinning, there is a grade I osteoarthritis; when the tangential surface area has disappeared, there is a grade two osteoarthritis. There are other levels of degeneration and destruction, which affect the deep and the calcified layers that border with the subchondral bone. The clinical manifestations of the development of the osteoarthritis condition are: increased volume of the joint, pain, crepitation and functional disability that, gradually and steadily, first hinders the performance of lengthy walks and forced flexion and extension movements, depending on the affected joint, and then pain and limitation of minimum efforts emerge as well as pain at rest which interrupts sleeping. If the condition persists without correction and/or therapy, the joint is destroyed, leading the patient to major replacement surgery with total prosthesis, or to disability. Therapeutic methods for the correction of the articular cartilage lesions that appear during the osteoarfhritic disease have been developed, but so far none of them have been able to achieve the regeneration of articular cartilage in situ and in vivo.
REPORTED DEVELOPMENTS Osteoarthritis is difficult to treat. At present, no cure is available and treatment focuses on relieving pain and preventing the affected joint from becoming deformed. Common treatments include the use of non-steroidal anti-inflammatory drugs (NSAID's), which are often used to relieve pain, while specific COX-2 inhibitors are used to relieve severe pain. Medicines such as chondroitin and glucosamine are thought to improve the cartilage itself. These treatments may be relatively successful, but not a substantive amount of research data is available. In severe cases, joint replacement may be necessary. This is especially true for hips and knees. If a joint is extremely painful and cannot be replaced, it may be fused. This procedure stops the pain, but results in the permanent loss of joint function, making walking and bending difficult. The treatment that has 74% to 90% effectiveness and produces excellent results is the transplantation of cultured autologous chondrocytes, by taking chondral cellular material from the patient, sending it to a laboratory where it is seeded in a proper medium for its proliferation, and, once enough volume is achieved after a variable period that may last from weeks to months, transporting it in a special container and implanting it in the damaged tissues to cover the tissue's defects. Another treatment includes the intraarticular instillation of Hylan G-F 20 (Synvisc, Hyalgan, Artz etc.), a substance that improves temporarily the rheology of the synovial fluid, producing an almost immediate sensation of free movement and a marked reduction of pain. The residual effects of this substance act on the synovial receptors causing a pain reduction that lasts several weeks and even months. However, this isolated effect is counterproductive for the course of the disease and for the viability of the cartilage because, as it masks the symptoms, the joint is used with more intensity and its destruction is accelerated as the original problem is not corrected and the damaged articular cartilage is not restored. Other reported methods include application of tendinous, periosteal, fascial, muscular or perichondral grafts; implantation of fibrin or cultured chondrocytes; implantation of synthetic matrices, such as collagen, carbon fiber; administration of electromagnetic fields. All of these have reported minimal and incomplete results with formation of repair, but not regenerative tissue, resulting in a poor quality tissue that can neither support the weighted load nor allow the restoration of an articular function with normal movement. Stimulation of the anabolic processes, blocking catabolic processes, or a combination of these two, may result in stabilization of the cartilage, and perhaps even reversion of the damage, and therefore prevent further progression of the disease. Various triggers may stimulate anabolic stimulation of chondrocytes. Insulin-like growth factor-I (IGF-I) is the predominant anabolic growth factor in synovial fluid and stimulates the synthesis of both proteoglycans and collagen. It has also been shown that members of the bone morphogenetic protein (BMP) family, notably BMP2, BMP4, BMP6, and BMP7, and members of the human trarisforrning growth factor-b (TGF.-b) family can induce chondrocyte anabolic stimulation (Chubinskaya and Kuettner, 2003). A compound has recently been identified that induces anabolic stimulation of chondrocytes (US 6,500,854; EP 1.391211). However, most of these compounds show severe side effects and, consequently, there is a strong need for compounds that stimulate chondrocyte differentiation without severe side effects. The present invention relates to the relationship between the function of selected proteins identified by the present inventors (hereinafter referred to as "TARGETS") and anabolic stimulation of chondrocytes. SUMMARY OF THE INVENTION
The present invention relates to a method for identifying compounds that induce cartilage-synthesizing processes, which lead to anabolic stimulation of chondrocytes, comprising contacting the compound with a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 101-128 and 401-594, or a functional fragment or derivative thereof, under conditions that allow said polypeptide to bind to the compound, and measuring a compound-polypeptide property related to the anabolic stimulation of chondrocytes. The present invention also relates to expression inhibitory agents, pharmaceutical compositions comprising the same, methods for the in vitro production of cartilage tissue, and host cells expressing said agents. Aspects of the present method include the in vitro assay of compounds using polypeptide of a TARGET, and cellular assays wherein TARGET inhibition is followed by observing indicators of efficacy, mcluding collagen type π, alpha-1 (col2αl) and aggrecan levels. Another aspect of the invention is a method of treatment or prevention of a condition involving de-differentiation of chondrocytes and/or loss of cartilage thickness, in a subject suffering or susceptible thereto, by administering a pharmaceutical composition comprising an effective cartilage formation-enhancing amount of a TARGET inhibitor. A further aspect of the present invention is a pharmaceutical composition for use in said method wherein said inhibitor comprises a polynucleotide selected from the group of an antisense polynucleotide, a ribozyme, and a small interfering RNA (siRNA), wherein said inhibitor comprises a nucleic acid sequence complementary to, or engineered from, a naturally occurring polynucleotide sequence encoding a polypeptide, comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 101-128 and 401-594, or a fragment thereof. Another further aspect of the present invention is a pharmaceutical composition comprising a therapeutically effective cartilage formation-enhancing amount of a TARGET inhibitor or its pharmaceutically acceptable salt, hydrate, solvate, or prodrug thereof in admixture with a pharmaceutically acceptable carrier. The present polynucleotides and TARGET inhibitor compounds are also useful for the manufacturing of a medicament for the treatment of conditions involving de-differentiation of chondrocytes and/or cartilage 5 thickness loss. Furthermore, the invention relates also to diagnostic methods.
BRIEF DESCRIPTION OF THE DRAWINGS Figure 1. Quantification of Col2αl expression in primary human chondrocytes 12 days post 10 infection with the indicated viruses. Figure 2. Example of duplicate screening results for Col2αl expression of part of the SilenceSelect library. Figure 3 (A-N): Quantification of Col2αl expression in primary human chondrocytes 12 days post infection with the indicated viruses. 15 Figure 4. Quantification of alcian blue staining on human primary chondrocytes, 12 days after infection with the indicated viruses, compared to uninfected cells. Figure 5 (A-N): Quantification of aggrecan expression in primary human chondrocytes 12 days post infection with the indicated viruses. Figure 6 (A-K): On target analysis with different constructs targeting the indicated genes. 20. The on target analysis is assessed through detection of the Col2αl expression in primary human chondrocytes 12 days post infection with the indicated viruses. Data are represented as luminescence units. The different thresholds corresponding to the different infection volumes are indicated as a line. Increasing the infection volumes leads to an increased threshold. 25 Figure 7 (A-L): GAG analysis on chondrocytes in alginate cell culture 10 days post infection. The data are represented as relative GAG levels to the average of KD control 1 and KD control 2, being Ad-siRNA targeting PTGER4 and GRM7 respectively. Two individual data points are shown for every condition. Figure 8 (A-L): Hydroxyproline analysis on chondrocytes in alginate cell culture 10 days 30 post infection. The data are represented as relative hydroxyproline levels to the average of KD control 1 and KD control 2, being Ad-siRNA targeting PTGER4 and GRM7 respectively. Two individual data points are shown for every condition. Figure 9 (A-L): mRNA marker analysis on chondrocytes in alginate cell culture 10 days post infection. The cells are infected with either Ad5/ALPL and Ad5/BMP2 overexpressing viruses, and with an Ad-siRNA targeting the indicated gene. The data are represented as relative mRNA levels to the ALPL control.
DETAILED DESCRIPTION The following terms are used herein in accordance with the following definitions: The term "agent" means any molecule, including polypeptides, polynucleotides and small molecules. The term "anabolic stimulation of chondrocytes" should be understood as inducing chondrogenesis or as inducing or enhancing the anabolic activity of chondrocytes. Anabolic stimulation takes place for instance by stimulating the synthesis of cartilage components, or inducing synthesis of components that are required for cartilage synthesis. "Anabolic stimulation of chondrocytes" may also be understood as a process in which the expression of the matrix Gla protein (MGP) is induced. Anabolic stimulation of chondrocytes may furthermore be understood as inducing the expression of cartilage derived retinoic acid sensitive protein (CD-RAP), as inducing the expression of cartilage oligomeric matrix protein (COMP), as inducing the expression of aggrecan 1 (agcl, also termed chondroitin sulfate proteoglycan core protein 1, or CSPG1), or as inducing synthesis of collagen II, also known as collagen, type II, alpha^l (col2αl), collagen of cartilage, chondrocalcin, and collagen, typg xi, alpha-3 (colllα3). The term 'antisense nucleic acid' refers to an oligonucleotide that has a nucleotide sequence that interacts through base pairing with a specific complementary nucleic acid sequence involved in the expression of the target such that the expression of the gene is reduced. Preferably, the specific nucleic acid sequence involved in the expression of the gene is a genomic DNA molecule or mRNA molecule that encodes (a part of) the gene. This genomic DNA molecule can comprise regulatory regions of the gene, or the coding sequence for the mature gene. The term "assay" means any process used to measure a specific property of a compound. A "screening assay" means a process used to characterize or select compounds based upon their activity from a collection of compounds. The term "binding affinity" is a property that describes how strongly two or more compounds associate with each other in a non-covalent relationship. Binding affinities can be characterized qualitatively, (such as "strong", "weak", "high", or "low") or quantitatively (such as measuring the Kb). The term "carrier" means a non-toxic material used in the formulation of pharmaceutical compositions to provide a medium, bulk and/or useable form to a pharmaceutical composition. A carrier may comprise one or more of such materials such as an excipient, stabilizer, or an aqueous pH buffered solution. Examples of physiologically acceptable carriers include aqueous or solid buffer ingredients including phosphate, citrate, and other organic acids; antioxidants including ascorbic acid; low molecular weight (less than about 10 residues) polypeptide; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and/or nonionic surfactants such as TWEEN™, polyethylene glycol (PEG), and PLURONICS™. The term "complex" means the entity created when two or more compounds bind to each other. The term "compound" is used herein in the context of a "test compound" or a "drug candidate compound" described in connection with the assays of the present invention. As such, these compounds comprise organic or inorganic compounds, derived synthetically or from natural sources. .The compounds include inorganic or organic compounds such is polynucleotides, lipids or hormone analogs that are characterized by relatively low molecular weights. Other biopolymeric organic test compounds include peptides comprising from about 2 to about 40 amino acids and larger polypeptides comprising from about 40 to about 500 amino acids, such as antibodies or antibody conjugates. The term 'complementary to a nucleotide sequence' in the context of antisense oligonucleotides and methods should be understood as sufficiently complementary to such a sequence as to allow hybridization to that sequence in a cell, i.e., under physiological conditions. The term "condition" or "disease" means the overt presentation of symptoms (i.e., illness) or the manifestation of abnormal clinical indicators (e.g., biochemical indicators). Alternatively, the term "disease" refers to a genetic or environmental risk of or propensity for developing such symptoms or abnormal clinical indicators. The term "contact" or "contacting" means bringing at least two moieties together, whether in an in vitro system or an in vivo system. The term "de-differentiation" refers to a general process wherein chondrocytes differentiate away from a cell phenotype that synthesizes cartilage components. Such components include, but are not limited to, collagen II, aggrecan 1, versican, link protein, perlecan, SZP/lubricin, biglycan (DS-PGI), decorin (DS-PGII), epiphycan (DS-PGITQ, fibromodulin, lumican, CILP, C-type lectin, fibronectin, PRELP, COMP (thrombospondin- 5), thrombospondin-1 and -3, CMP (matrilin-1), matrilin-3, C-type lectin, fibronectin, condroadherin, tenascin-C, fibrillin, elastin, gp-39/YKL-40, matrix Gla protein/ MGP, pleiotrophin, chondromodulin-I/SCGP, chondromodulin-II, CD-RAP, chondrocalcin, PARP, lysozyme, and phospholipase A2. The term "effective amount" or "therapeutically effective amounf ' means that amount of a compound or agent that will elicit the biological or medical response of a subject that is being sought by a medical doctor or other clinician. In particular, with regard to inducing anabolic stimulation of chondrocytes, the term "effective amount" is intended to mean an effective differentiation-promoting amount of an compound or agent that will bring about a biologically meaningful increase in the levels of chondrocyte markers, representative for the process of an increase in chondrocyte anabolism. The term "expressible nucleic acid" means a nucleic acid coding for a proteinaceous molecule, an RNA molecule, or a DNA molecule. The term "endogenous" shall mean a material that a mammal naturally produces. Endogenous in reference to the term "protease", "kjnase", or G-Protein Coupled Receptor ("GPCR") shall mean that which is naturally produced by a mammal (for example, and not limitation, a human). In contrast, the term non-endogenous in this context shall mean that which is not naturally produced by a mammal (for example, and not limitation, a human). Both terms can be utilized to describe both "in vivo" and "in vitro" systems. For example, and not a limitation, in a screening approach, the endogenous or non-endogenous TARGET may be in reference to an in vitro screening system. As a further example and not limitation, where the genome of a mammal has been manipulated to include a non-endogenous TARGET, screening of a candidate compound by means of an in vivo system is viable. The term "expression" comprises both endogenous expression and overexpression by transduction. The term "expression inhibitory agent" means a polynucleotide designed to interfere selectively with the transcription, translation and/or expression of a specific polypeptide or protein normally expressed within a cell. More particularly, "expression inhibitory agent" comprises a DNA or RNA molecule that contains a nucleotide sequence identical to or complementary to at least about 17 sequential nucleotides within the polyribonucleotide sequence coding for a specific polypeptide or protein. Exemplary expression inhibitory molecules include ribozymes, double stranded siRNA molecules, self-complementary single- stranded siRNA molecules, genetic antisense constructs, and synthetic RNA antisense molecules with modified stabilized backbones. The term "expressible nucleic acid" means a nucleic acid coding for a proteinaceous molecule, an RNA molecule, or a DNA molecule. The term 'genetic antisense' as used herein refers to the incorporation of antisense constructs complementary to sequences of genes into the genome of a cell. Such incoφoration allows for the continued synthesis of the antisense molecule. The term "hybridization" means any process by which a strand of nucleic acid binds with a complementary strand through base pairing. The term "hybridization complex" refers to a complex formed between two nucleic acid sequences by virtue of the formation of hydrogen bonds between complementary bases. A hybridization complex may be formed in solution (e.g., Cot or Rot analysis) or formed between one nucleic acid sequence present in solution and another nucleic acid sequence immobilized on a solid support (e.g., paper, membranes, filters, chips, pins or glass slides, or any other appropriate substrate to which cells or their nucleic acids have been fixed). The term "stringent conditions" refers to conditions that permit hybridization between polynucleotides and the claimed polynucleotides. Stringent conditions can be defined by salt concentration, the concentration of organic solvent, e.g., formamide, temperature, and other conditions well known in the art. In particular, reducing the concentration of salt, increasing the concentration of formamide, or raising the hybridization temperature can increase stringency. The term "inhibit" or "inhibiting", in relationship to the term "response" means that a response is decreased or prevented in the presence of a compound as opposed to in the absence of the compound. The term "inhibition" refers to the reduction, down regulation of a process or the elimination of a stimulus for a process that results in the absence or minimization of the expression of a protein or polypeptide. The term "induction" refers to the inducing, up-regulation, or stimulation of a process that results in the expression of a protein or polypeptide, and that may also result in a phenotypical cellular change. The term "ligand" means an endogenous, naturally occurring molecule specific for an endogenous, naturally occurring receptor. The term "pharmaceutically acceptable salts" refers to the non-toxic, inorganic and organic acid addition salts, and base addition salts, of compounds of the present invention. These salts can be prepared in situ during the final isolation and purification of compounds useful in the present invention. The term "polynucleotide" means a polynucleic acid, in single or double stranded form, and in the sense or antisense orientation, complementary polynucleic acids that hybridize to a particular polynucleic acid under stringent conditions, and polynucleotides that are homologous in at least about 60 percent of its base pairs, and more preferably 70 percent of its base pairs are in common, most preferably 90 per cent, and in a special embodiment 100 percent of its base pairs. The polynucleotides include polyribonucleic acids, polydeoxyribonucleic acids, and synthetic analogues thereof. The polynucleotides are described by sequences that vary in length, that range from about 10 to about 5000 bases, preferably about 100 to about 4000 bases, more preferably about 250 to about 2500 bases. A preferred polynucleotide embodiment comprises from about 10 to about 30 bases in length. A special embodiment of polynucleotide is the polyribonucleotide of from about 10 to about 22 nucleotides, more commonly described as small interfering RNAs (siRNAs). Another special embodiment are nucleic acids with modified backcartilages such as peptide nucleic acid (PNA), polysiloxane, and 2'-O-(2-methoxy)ethylphosphorothioate, or including non- naturally occurring nucleic acid residues, or one or more nucleic acid substituents, such as methyl-, thio-, sulphate, benzoyl-, phenyl-, amino-, propyl-, chloro-, and methanocarbanucleosides, or a reporter molecule to facilitate its detection. The term "polypeptide" relates to proteins (such as TARGETS), proteinaceous molecules, fractions of proteins peptides and oligopeptides. The term 'ribozymes' as used herein relates to catalytic RNA molecules capable of cleaving other RNA molecules at phosphodiester bonds in a manner specific to the sequence. The term "solvate" means a physical association of a compound useful in this invention with one or more solvent molecules. This physical association includes hydrogen bonding. In certain instances the solvate will be capable of isolation, for example when one or more solvent molecules are incorporated in the crystal lattice of the crystalline solid. "Solvate" encompasses both solution-phase and isolable solvates. Representative solvates include hydrates, ethanolates and methanolates. The term "subject" includes humans and other mammals. The term "treating" means an intervention performed with the intention of preventing the development or altering the pathology of, and thereby alleviating a disorder, disease or condition, including one or more symptoms of such disorder or condition. Accordingly, "treating" refers to both therapeutic treatment and prophylactic or preventative measures. Those in need of treating include those already with the disorder as well as those in which the disorder is to be prevented. The related term "treatment," as used herein, refers to the act of treating a disorder, symptom, disease or condition, as the term "treating" is defined above. The term 'vectors' also relates to plasmids as well as to viral vectors, such as recombinant viruses, or the nucleic acid encoding the recombinant virus. The term "vertebrate cells" means cells derived from animals having vertera structure, including fish, avian, reptilian, amphibian, marsupial, and mammalian species. Preferred cells are derived from mammalian species, and most preferred cells are human cells. Mammalian cells include feline, canine, bovine, equine, caprine, ovine, porcine murine, such as mice and rats, and rabbits.
Applicants' Invention Based on TARGET Relationship to Anabolic Stimulation of Chondrocytes As noted above, the present invention is based on the present inventors' discovery that TARGETS are factors in the up-regulation and/or induction of anabolic processes of chondrocytes. The term 'TARGET" or "TARGETS" means the proteins identified in accordance with the assay described below to be involved in the induction of the anabolic stimulation of chondrocytes. The preferred TARGETS^ are identified as SEQ TD NOS. 101- 128 and 401-594 in Table 1A. Table 1A lists the polypeptides, polynucleotides and knockdown target sequences of the present invention. Table IB lists exemplary fragments of the transmembrane protein TARGETS. Table 1C lists exemplary KD target sequences useful in the practice of the present expression-inhibitory agent invention.
Table 1A
Figure imgf000012_0001
Figure imgf000013_0001
Table IB
Figure imgf000013_0002
Figure imgf000014_0001
Figure imgf000015_0001
Figure imgf000016_0001
Figure imgf000017_0001
Table 1C
Figure imgf000017_0002
Figure imgf000018_0001
Figure imgf000019_0001
Figure imgf000020_0001
The present invention relates to a method for assaying for compounds that induce anabolic stimulation of chondrocytes, comprising contacting the compound with a polypeptide comprising an amino acid sequence of the polypeptides of SEQ ID NO: 101-128 ("TARGETS") or a functional fragment thereof under conditions that allow said polypeptide to bind to the compound, and detecting the formation of a complex between the polypeptide and the compound. One preferred means of measuring the complex formation is to determine the binding affinity of said compound to said polypeptide. More particularly, the invention relates to a method for identifying an agent that induces anabolic stimulation of chondrocytes, the method comprising further: (a) contacting a population of chondrocyte cells with one or more of said compound that exhibits binding affinity for said TARGETS, and (b) measuring a compound-polypeptide property related to the anabolic stimulation of chondrocytes. The compound-polypeptide property referred to above is related to the anabolic stimulation of chondrocytes, and is a measurable phenomenon chosen by the person of ordinary skill in the art. The measurable property may e.g. be the binding affinity for a peptide domain of the polypeptide TARGET or the level of any one of a number of biochemical marker levels of increased chondrocyte anabolism. Anabolic stimulation of chondrocytes can e.g. be measured by measuring the level of proteins and other molecules that are induced during the differentiation process, such as key components of normal cartilage. In particular, the induction of the major protein component of cartilage, collagen π, is measured. In addition, compound-polypeptide properties related to the anabolic stimulation of chondrocytes are measured in C20/A4; T/C-28a2; T/C-28a4; C-28/12; Ch-4,8,N; Ch-8-OA; TC6; MCT;MC615; IRC; RCS2; Hig82; and DI ORL UVA (DI) cells. However, such porterties are also measured in non-chondrocyte cell systems. For example, in situ binding assays that determine the affinity of compounds to bind to polypeptides of the invention are performed using any cell type that expresses the polypeptide. Expression of the polypeptide is exogenous or endogenous. Furthermore, when the compound-polypeptide property is activation of a biological pathway, any cell that contains the pathway cellular components is used to measure the compound-polypeptide property. For example, induction of col2αl or aggrecan in chondrocytes is indicative of anabolic stimulation of chondrocytes. Specifically, non-chondrocyte cells can be engineered to contain a reporter molecule activated by the col2αl or aggrecan promoters. In this way a non-chondrocyte can be used to measure a property indicative of anabolic stimulation of chondrocytes. The invention relates to a method for identifying a compound that induces and/or increases anabolic stimulation of chondrocytes, said method comprising the steps of: culturing a population of cells expressing a polypeptide of any one of those listed in Table 1A, or a functional fragment or derivative thereof; determining a first level of chondrogenic differentiation in said population of cells; exposing said population of cells to a compound, or a mixture of compounds; deterrnining the level of chondrogenic differentiation in said population of cells during or after exposure of said population of cells to the compound, or the mixture of compounds; and identifying the compound that induces and/or increases chondrogenic differentiation. The invention also relates to a method for identifying a compound that decreases the expression and/or activity of any one of the polypeptides listed in Table 1 A, said method comprising the steps of: culturing a population of cells expressing said polypeptide, or a fragment, or a derivative thereof; determining a first level of expression and/or activity of said polypeptide; exposing said population of cells to a compound, or a mixture of compounds; determining the level of expression and/or activity of said polypeptide during or after exposure of said population of cells to the compound, or the mixture of compounds; and identifying the compound that decreases the expression and/or activity of said polypeptide. If the polypeptide activity is not readily measurable, the identification of the compound may benefit from an extra step comprising exposing said population of cells to an agonist of said polypeptide. Furthermore, the methods of the present invention may comprise the step of introducing a gene encoding any one of the polypeptides listed in Table 1A, in said population of cells. For high-throughput purposes it may be beneficial to have the gene stably integrated in the genome of said cells. In a preferred embodiment, the level of chondrocyte (re-)differentiation is determined by measuring the expression level of a marker gene, wherein a preferred marker gene encodes collagen type IT, alpha- 1 (col2αl) or aggrecan. For proper anabolic stimulation it is preferred that the expression and/or activity of col2αl or aggrecan is increased. The present invention provides in one particular embodiment methods for identifying novel compounds, wherein the polypeptide is a GPCR. If so, the expression and/or activity of said GPCR is preferably determined by measuring the level of a second messenger. Preferred second messengers are cyclic AMP, Ca2+ or both. Typically, the level of the second messenger is determined with a reporter gene under the control of a promoter that is responsive to the second messenger, wherein it is preferred that the promoter is a cyclic AMP-responsive promoter, an NF-KB responsive promoter, or a NF-AT responsive promoter, and wherein the reporter gene is selected from the group consisting of: alkaline phosphatase, GFP, eGFP, dGFP, luciferase and β- gaέtosidase. In another particular embodiment, the invention provides methods for identifying novel compounds, wherein the polypeptide is a kinase or a phosphatase. Preferably, the activity of said kinase or phosphatase is determined by measuring the level of phosphorylation of a substrate of said kinase or phosphatase. In yet another particular embodiment, the invention provides methods for identifying novel compounds, wherein the polypeptide is a protease. Preferably, the activity of said protease is measured by determining the level of cleavage of a substrate of said protease. Methods for determining second messenger levels, use of the reporter genes and second-messenger responsive promoters as well as phosphatase assays and protease assays are well known in the art and not further elaborated upon herein. In a preferred embodiment, the compound that inhibits the polypeptide exhibits a binding affinity to the polypeptide of at most 10 micromolar. In a preferred embodiment of the invention, the polypeptide TARGET comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 101-128 and 401- 594 (Tables 1A and IB). In an especially preferred embodiment of the invention, the polypeptide TARGET comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 101-128 (Table 1A). Depending on the choice of the skilled artisan, the present assay method may be designed to function as a series of measurements, each of which is designed to determine whether the drug candidate compound is indeed acting on the polypeptide to thereby induce the anabolic stimulation of chondrocytes. For example, an assay designed to determine the binding affinity of a compound to the polypeptide, or fragment thereof, may be necessary, but not sufficient, to ascertain whether the test compound would be useful for increasing mean cartilage thickness when administered to a subject. Nonetheless, such binding information would be useful in identifying a set of test compounds for use in an assay that would measure a different property, further up the biochemical pathway, such as cartilage component synthesis, assayed by measuring the amount of collagen II. Such second assay may be designed to confirm that the test compound, having binding affinity for the polypeptide, actually induces the anabolic stimulation of chondrocytes. Suitable controls should always be in place to insure against false positive readings. The order of taking these measurements is not believed to be critical to the practice of the present invention, which may be practiced in any order. For example, one may first perform a screening assay of a set of compounds for which no information is known respecting the^compounds' binding affinity for the polypeptide. Alternatively, one may screen a set of compounds identified as having binding affinity for a polypeptide domain, or a class of compounds identified as being an inhibitor of the polypeptide. However, for the present assay to be meaningful to the ultimate use of the drug candidate compounds, a measurement of collagen II levels or aggrecan is necessary. Validation studies including controls, and measurements of binding affinity to the polypeptides of the invention are nonetheless useful in identifying a compound useful in any therapeutic or diagnostic application. The binding affinity of the compound with the polypeptide TARGET can be measured by methods known in the art, such as using surface plasmon resonance biosensors (Biacore), by saturation binding analysis with a labeled compound (e.g. Scatchard and Lindmo analysis), by differential UV spectrophotometer, fluorescence polarization assay, Fluorometric Imaging Plate Reader (FLIPR®) system, Fluorescence resonance energy transfer, and Bioluminescence resonance energy transfer. The bmding affinity of compounds can also be expressed in dissociation constant (Kd) or as IC50 or EC50. The IC50 represents the concentration of a compound that is required for 50% inhibition of bmding of another ligand to the polypeptide. The EC50 represents the concentration required for obtaining 50% of the maximum effect in any assay that measures TARGET function. The dissociation constant, Kd, is a measure of how well a ligand binds to the polypeptide, it is equivalent to the ligand concentration required to saturate exactly half of the binding-sites on the polypeptide. Compounds with a high affinity binding have low Kd, IC50 and EC50 values, i.e. in the range of 100 nM to 1 pM; a moderate to low affinity binding relates to a high Kd, IC50 and EC50 values, i.e. in the micromolar range. The present assay method may also be practiced in a cellular assay, A host cell expressing TARGET can be a cell with endogenous expression or a cell over-expressing the TARGET e.g. by transduction. When the endogenous expression of the polypeptide is not sufficient to determine a baseline that can easily be measured, one may use using host cells that over-express TARGET. Over-expression has the advantage that the level of the TARGET substrate end products is higher than the activity level by endogenous expression. Accordingly, measuring such levels using presently available techniques is easier. In such cellular assay, the biological activity of TARGET may be measured by following the production of cartilage component synthesis. The present invention further relates to a method for identifying a compound that induces anabolic stimulation of chondrocytes, comprising: (a) contacting a compound with a.polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 101-128 and 401-594; (b) determining the binding affinity of the compound to the polypeptide; (c) contacting a population of mammalian cells expressing said polypeptide with the compound that exhibits a binding affinity of at least 10 micromolar; and (d) identifying the compound that induces the synthesis of proteins that are a constituent of normal cartilage and/or that are required for the formation of cartilage. For high-throughput purposes, libraries of compounds may be used such as antibody fragment libraries, peptide phage display libraries, peptide libraries (e.g. LOPAP™, Sigma Aldrich), lipid libraries (BioMol), synthetic compound libraries (e.g. LOPAC™, Sigma
Aldrich) or natural compound libraries (Specs, TimTec). Preferred drug candidate compounds are low molecular weight compounds. Low molecular weight compounds, i.e. with a molecular weight of 500 Dalton or less, are likely to have good absorption and permeation in biological systems and are consequently more likely to be successful drug candidates than compounds with a molecular weight above 500 Dalton (Lipinski et al. (1997)). Peptides comprise another preferred class of drug candidate compounds. Peptides may be excellent drug candidates and there are multiple examples of commercially valuable peptides such as fertility hormones and platelet aggregation inhibitors. Natural compounds are another preferred class of drug candidate compound. Such compounds are found in and extracted from natural sources, and which may thereafter be synthesized. The lipids are another preferred class of drug candidate compound. Another preferred class of drug candidate compounds is an antibody. The present invention also provides antibodies directed against a TARGET. These antibodies may be endogenously produced to bind to the TARGET within the cell, or added to the tissue to bind to TARGET polypeptide present outside the cell. These antibodies may be monoclonal antibodies or polyclonal antibodies. The present invention includes chimeric, single chain, and humanized antibodies, as well as FAb fragments and the products of a FAb expression library, and Fv fragments and the products of an Fv expression library. In certain embodiments, polyclonal antibodies may be used in the practice of the invention. The skilled artisan knows methods of preparing polyclonal antibodies. Polyclonal antibodies can be raised in a mammal, for example, by one or more injections of an immunizing agent and, if desired, an adjuvant. Typically, the immunizing agent and/or adjuvant will be injected in the mammal by multiple subcutaneous or intraperitoneal injections,,^ Antibodies may also be generated against the intact TARGET protein or polypeptide, or against a fragment, derivatives including conjugates, or other epitope of the TARGET protein or polypeptide, such as the TARGET embedded in a cellular membrane, or a library of antibody variable regions, such as a phage display library. It may be useful to conjugate the immunizing agent to a protein known to be immunogenic in the mammal being immunized. Examples of such immunogenic proteins include but are not limited to keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, and soybean trypsin inhibitor. Examples of adjuvants that may be employed include Freund's complete adjuvant and MPL-TDM adjuvant (monophosphoryl Lipid A, synthetic trehalose dicorynomycolate). One skilled in the art without undue experimentation may select the immunization protocol. In some embodiments, the antibodies may be monoclonal antibodies. Monoclonal antibodies may be prepared using methods known in the art. The monoclonal antibodies of the present invention may be "humanized" to prevent the host from mounting an immune response to the antibodies. A "humanized antibody" is one in which the complementarity deterrnining regions (CDRs) and/or other portions of the light and/or heavy variable domain framework are derived from a non-human immunoglobulin, but the remaining portions of the molecule are derived from one or more human immunoglobulins. Humanized antibodies also include antibodies characterized by a humanized heavy chain associated with a donor or acceptor unmodified light chain or a chimeric light chain, or vice versa. The humanization of antibodies may be accomplished by methods known in the art (see, e.g. Mark and Padlan, (1994) "Chapter 4. Humanization of Monoclonal Antibodies", The Handbook of Experimental Pharmacology Vol. 113, Springer- Verlag, New York). Transgenic animals may be used to express humanized antibodies. Human antibodies can also be produced using various techniques known in the art, including phage display libraries (Hoogenboom and Winter, (1991) J. Mol. Biol. 227:381- 8; Marks et al. (1991). J. Mol. Biol. 222:581-97). The techniques of Cole, et al. and Boerner, et al. are also available for the preparation of human monoclonal antibodies (Cole, et al. (1985) Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77; Boerner, et al (1991). J. Immunol., 147(l):86-95). Techniques known in the art for the production of single chain antibodies can be adapted to produce single chain antibodies to the TARGET polypeptides and proteins of the present invention. The antibodies may be monovalent antibodies. Methods for preparing monovalent antibodies are well known in the art. For example, one method involves recombinant expression of immunoglobulin light chain and modified heavy chain. The heavy chain is truncated generally at any point in the Fc region so as to prevent heavy chain cross-linking. Alternatively; the relevant cysteine residues are substituted with another amino acid residue or are deleted so as to prevent cross-linking. Bispecific antibodies are monoclonal, preferably human or humanized, antibodies that have binding specificities for at least two different antigens and preferably for a cell-surface protein or receptor or receptor subunit. In the present case, one of the binding specificities is for one domain of the TARGET; the other one is for another domain of the same or different TARGET. Methods for making bispecific antibodies are known in the art. Traditionally, the recombinant production of bispecific antibodies is based on the co-expression of two immunoglobulin heavy-chain/light-chain pairs, where the two heavy chains have different specificities (Milstein and Cuello, (1983) Nature 305:537-9). Because of the random assortment of immunoglobulin heavy and light chains, these hybridomas (quadromas) produce a potential mixture of ten different antibody molecules, of which only one has the correct bispecific structure. Affinity chromatography steps usually accomplish the purification of the correct molecule. Similar procedures are disclosed in Trauneeker, et al. (1991) EMBO J. 10:3655-9. According to another preferred embodiment, the assay method uses a drug candidate compound identified as having a binding affinity for a TARGET, and/or has already been identified as having down-regulating activity such as antagonist activity vis-a-vis one or more TARGET. The present invention further relates to a method for inducing anabolic stimulation of chondrocytes comprising contacting said cells with an expression inhibitory agent comprising a polynucleotide sequence that complements at least about 17 nucleotides of the polyribonucleotide comprising a nucleotide sequence selected from the group consisting of SEQ D NO: 1-28. In a preferred embodiment the expression-inhibitory agent comprises a polynucleotide sequence that complements a nucleotide sequence selected from the group consisting of SEQ TD NO: 202-315. Another aspect of the present invention relates to a method for inducing the anabolic stimulation of chondrocytes, comprising by contacting said cell with an expression-inhibiting agent that inhibits the translation in the cell of a polyribonucleotide encoding a TARGET polypeptide. A particular embodiment relates to a composition comprising a polynucleotide including at least one antisense strand that functions to pair the agent with the TARGET TARGET mRNA, and thereby down-regulate or block the expression of TARGET polypeptide. The inhibitory agent preferably comprises antisense polynucleotide, a ribozyme, and a small interfering RNA (siRNA), wherein said agent comprises a nucleic acid sequence complementary to, or engineered from, a natøally-occurring polynucleotide sequence encoding a portion of a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ TD NO: 101-128. In a preferred embodiment the expression-inhibiting agent is complementary to a polynucleotide sequence selected from the group consisting of SEQ ID NO: 1-28. In an especially preferred embodiment the expression-inhibiting agent is complementary to a polynucleotide sequence selected from the group consisting of SEQ ED NO: 202-315. An embodiment of the present invention relates to a method wherein the expression- inhibiting agent is selected from the group consisting of antisense RNA, antisense oligodeoxynucleotide (ODN), a ribozyme that cleaves the polyribonucleotide coding for SEQ ID NO: 101-128, a small interfering RNA (siRNA, preferably shRNA,) that is sufficiently complementary to a portion of the polyribonucleotide coding for SEQ ID NO: 101-128, such that the siRNA, preferably sbRNA, interferes with the translation of the TARGET polyribonucleotide to the TARGET polypeptide. Preferably the expression-inhibiting agent is an antisense RNA, ribozyme, antisense oligodeoxynucleotide, or siRNA, preferably shRNA, complementary to a nucleotide sequence selected from the group consisting of SEQ ID NO: 1-28. In an especially preferred embodiment the expression-inhibiting agent is complementary to a polynucleotide sequence selected from the group consisting of SEQ ID NO: 202-315. A special embodiment of the present invention relates to a method wherein the expression-inhibiting agent is a nucleic acid expressing the antisense RNA, antisense oligodeoxynucleotide (ODN), a ribozyme that cleaves the polyribonucleotide coding for SEQ ED NO: 101-128, a small interfering RNA (siRNA, preferably shRNA,) that is sufficiently complementary to a portion of the polyribonucleotide coding for SEQ ID NO: 101-128, such that the siRNA, preferably shRNA, interferes with the translation of the TARGET polyribonucleotide to the TARGET polypeptide. Preferably the nucleotide sequence is complementary to a polynucleotide selected from the group consisting of SEQ ID NO: 1-28. In an especially preferred embodiment nucleotide sequence is complementary to a polynucleotide selected from the group consisting of SEQ ID NO: 202-315. The down regulation of gene expression using antisense nucleic acids can be achieved at the translational or transcriptional level. Antisense nucleic acids of the invention are preferably nucleic acid fragments capable of specifically hybridizing with all or part of a nucleic acid encoding a TARGET polypeptide or the corresponding messenger RNA. In addition, antisense nucleic acids may be designed which decrease expression of the nucleic acid sequence capable of encoding a TARGET polypeptide by inhibiting splicing of its primary transcript. Any length of antisense sequence is suitable for practice of the invention so long as it is capable of down-regulating or blocking expression of a nucleic acid coding for a TARGET. Preferably, the antisense sequence is at least about 17 nucleotides in length. The preparation and use of antisense nucleic acids, DNA encoding antisense RNAs and the use of oligo and genetic antisense is known in the art. One embodiment of expression-inhibitory agent is a nucleic acid that is antisense to a nucleic acid comprising SEQ ID NO: 1-28. For example, an antisense nucleic acid (e.g. DNA) may be introduced into cells in vitro, or administered to a subject in vivo, as gene therapy to inhibit cellular expression of nucleic acids comprising SEQ ID NO: 1-28. Antisense oligonucleotides preferably comprise a sequence containing from about 17 to about 100 nucleotides and more preferably the antisense oligonucleotides comprise from about 18 to about 30 nucleotides. Antisense nucleic acids may be prepared from about 10 to about 30 contiguous nucleotides complementary to a nucleic acid sequence selected from the sequences of SEQ ID NO: 1-28. The antisense nucleic acids are preferably oligonucleotides and may consist entirely of deoxyribo-nucleotides, modified deoxyribonucleotides, or some combination of both. The antisense nucleic acids can be synthetic oligonucleotides. The oligonucleotides may be chemically modified, if desired, to improve stability and/or selectivity. Since oligonucleotides are susceptible to degradation by intracellular nucleases, the modifications can include, for example, the use of a sulfur group to replace the free oxygen of the phosphodiester bond. This modification is called a phosphorothioate linkage. Phosphorothioate antisense oligonucleotides are water soluble, polyanionic, and resistant to endogenous nucleases. In addition, when a phosphorothioate antisense oligonucleotide hybridizes to its TARGET site, the RN202-315NA duplex activates the endogenous enzyme ribonuclease (RNase) H, which cleaves the mRNA component of the hybrid molecule. In addition, antisense oligonucleotides with phosphoramidite and polyamide (peptide) linkages can be synthesized. These molecules should be very resistant to nuclease degradation. Furthermore, chemical groups can be added to the 21 carbon of the sugar moiety and the 5 carbon (C-5) of pyrimidines to enhance stability and facilitate the binding of the antisense oligonucleotide to its TARGET site. Modifications may include 2'-deoxy, O- pentoxy, O-propoxy, O-methoxy, fluoro, methoxyethoxy phosphorothioates, modified bases, as well as other modifications known to those of skill in the art. Another type of expression-inhibitory agent that reduces the levels of TARGETS is the ribozyme. Ribozymes are catalytic RNA molecules (RNA enzymes) that have separate catalytic and substrate binding domains. The substrate binding sequence combines by nucleotide complementarity and, possibly, non-hydrogen bond interactions with its TARGET sequence. The catalytic portion cleaves the TARGET RNA at a specific site. The substrate domain of a ribozyme can be engineered to direct it to a specified mRNA sequence. The ribozyme recognizes and then binds a TARGET mRNA through complementary base pairing. Once it is bound to the correct TARGET site, the ribozyme acts enzymatically to cut the TARGET mRNA. Cleavage of the mRNA by a ribozyme destroys its ability to direct synthesis of the corresponding polypeptide. Once the ribozyme has cleaved its TARGET sequence, it is released and can repeatedly bind and cleave at other mRNAs. Ribozyme forms include a hammerhead motif, a hairpin motif, a hepatitis delta virus, group I intron or RNaseP RNA (in association with an RNA guide sequence) motif or Neurospora VS RNA motif. Ribozymes possessing a hammerhead or hairpin structure are readily prepared since these catalytic RNA molecules can be expressed within cells from eukaryotic promoters (Chen, et al. (1992) Nucleic Acids Res. 20:4581-9). A ribozyme of the present invention can be expressed in eukaryotic cells from the appropriate DNA vector. If desired, the activity of the ribozyme may be augmented by its release from the primary transcript by a second ribozyme (Ventura, et al. (1993) Nucleic Acids Res. 21 :3249-55). Ribozymes may be chemically synthesized by combining an oligodeoxyribonucleotide with a ribozyme catalytic domain (20 nucleotides) flanked by sequences that hybridize to the TARGET mRNA after transcription. The oligodeoxyribonucleotide is amplified by using the substrate binding sequences as primers. The amplification product is cloned into a eukaryotic expression vector. Ribozymes are expressed from transcription units inserted into DNA, RNA, or viral vectors. Transcription of the ribozyme sequences are driven from a promoter for eukaryotic RNA polymerase I (pol (I), RNA polymerase II (pol IT), or RNA polymerase IH (pol IH). Transcripts from pol II or pol DI promoters will be expressed at high levels in all cells; the levels of a given pol II promoter in a given cell type will depend on nearby gene regulatory sequences. Prokaryotic RNA polymerase promoters are also used, providing that the prokaryotic RNA polymerase enzyme is expressed in the appropriate cells (Gao and Huang, (1993) Nucleic Acids Res. 21:2867-72). It has been demonstrated that ribozymes expressed from these promoters can function in mammalian cells (Kashani-Sabet, et al. (1992) Antisense Res. Dev. 2:3-15). A particularly preferred inhibitory agent is a small interfering RNA (siRNA, preferably shRNA). siRNA, preferably shRNA, mediate the post-transcriptional process of gene silencing by double stranded RNA (dsRNA) that is homologous in sequence to the silenced RNA. siRNA according to the present invention comprises a sense strand of 17-25 nucleotides complementary or homologous to a contiguous 17-25 nucleotide sequence selected from the group of sequences described in SEQ ID NO: 1-28, preferably from the group of sequences described in SEQ ID No: 202-315, and an antisense strand of 17-23 nucleotides complementary to the sense strand. Exemplary sequences are described as sequences complementary to SEQ ID NO: 202-315. The most preferred siRNA comprises sense and anti-sense strands that are 100 per cent complementary to each other and the TARGET polynucleotide sequence. Preferably the siRNA further comprises a loop region linking the sense and the antisense strand. A self-complementing single stranded siRNA molecule polynucleotide according to the present invention comprises a sense portion and an antisense portion connected by a loop region linker. Preferably, the loop region sequence is 4-30 nucleotides long, more preferably 5-15 nucleotides long and most preferably 8 nucleotides long. In a most preferred embodiment the linker sequence is UUGCUAUA (SEQ ID NO: 201). Self-complementary single stranded siRNAs form hairpin loops and are more stable than ordinary dsRNA. In addition, they are more easily produced from vectors. Analogous to antisense RNA, the siRNA can be modified to confirm resistance to nucleolytic degradation, or to enhance activity, or to enhance cellular distribution, or to enhance cellular uptake, such modifications may consist of modified intemucleoside linkages, modified nucleic acid bases, modified sugars and or chemical linkage the siRNA to one or more moieties or conjugates. The nucleotide sequences are selected according to siRNA designing rules that give an improved reduction of the TARGET sequences compared to nucleotide sequences that do not comply with these siRNA designing rules (For a discussion of these rules and examples of the preparation of siRNA, WO2004094636, published November 4, 2004, and UA20030198627, are hereby incorporated by reference). The present invention also relates to compositions, and methods using said compositions, comprising a DNA expression vector capable of expressing a polynucleotide capable of inducing anabolic stimulation of chondrocytes and described hereinabove as an expression inhibition agent. A special aspect of these compositions and methods relates to the down-regulation or blocking of the expression of a TARGET polypeptide by the induced expression of a polynucleotide encoding an intracellular binding protein that is capable of selectively interacting with the TARGET polypeptide. An intracellular binding protein includes any protein capable of selectively interacting, or binding, with the polypeptide in the cell in which it is expressed and neutralizing the function of the polypeptide. Preferably, the intracellular binding protein is a neutralizing antibody or a fragment of a neutralizing antibody having binding affinity to an epitope of the TARGET polypeptide of SEQ ID NO: 101-128 and 401- 594. More preferably, the intracellular binding protein is a single chain antibody, A special embodiment of this composition comprises the expression-inhibiting agent selected from the group consisting of antisense RNA, antisense oligodeoxynucleotide (ODN), a ribozyme that cleaves the polyribonucleotide coding for SEQ ID NO: 101-128, and a small interfering RNA (siRNA) that is sufficiently homologous to a portion of the polyribonucleotide coding for SEQ ID NO: 101-128, such that the siRNA interferes with the translation of the TARGET polyribonucleotide to the TARGET polypeptide, The polynucleotide expressing the expression-inhibiting agent is preferably included within a vector. The polynucleic acid is operably linked to signals enabling expression of the nucleic acid sequence and is introduced into a cell utilizing, preferably, recombinant vector constructs, which will express the antisense nucleic acid once the vector is introduced into the cell. A variety of viral-based systems are available, including adenoviral, retroviral, adeno-associated viral, lentiviral, herpes simplex viral or a sendaviral vector systems, and all may be used to introduce and express polynucleotide sequence for the expression-inhibiting agents in TARGET cells. Preferably, the viral vectors used in the methods of the present invention are replication defective. Such replication defective vectors will usually pack at least one region that is necessary for the replication of the virus in the infected cell. These regions can either be eliminated .(in whole or in part), or be rendered non-functional by any technique known to a person skilled in the art. These techniques include the total removal, substitution, partial deletion or addition of one or more bases to an essential (for replication) region. Such techniques may be performed in vitro (on the isolated DNA) or in situ, using the techniques of genetic manipulation or by treatment with mutagenic agents. Preferably, the replication defective virus retains the sequences of its genome, which are necessary for encapsidating, the viral particles. In a preferred embodiment, the viral element is derived from an adenovirus. Preferably, the vehicle includes an adenoviral vector packaged into an adenoviral capsid, or a functional part, derivative, and/or analogue thereof. Adenovirus biology is also comparatively well known on the molecular level. Many tools for adenoviral vectors have been and continue to be developed, thus making an adenoviral capsid a preferred vehicle for incorporating in a library of the invention. An adenovirus is capable of infecting a wide variety of cells. However, different adenoviral serotypes have different preferences for cells. To combine and widen the TARGET cell population that an adenoviral capsid of the invention can enter in a preferred embodiment, the vehicle includes adenoviral fiber proteins from at least two adenoviruses. Preferred adenoviral fiber protein sequences are serotype 17, 45 and 51. Techniques or construction and expression of these chimeric vectors are disclosed in US Published Patent Applications 20030180258 and 20040071660, hereby incorporated by reference. In a preferred embodiment, the nucleic acid derived from an adenovirus includes the nucleic acid encoding an adenoviral late protein or a functional part, derivative, and/or analogue thereof. An adenoviral late protein, for instance an adenoviral fiber protein, may be favorably used to TARGET the vehicle to a certain cell or to induce enhanced delivery of the vehicle to the cell. Preferably, the nucleic acid derived from an adenovirus encodes for essentially all adenoviral late proteins, enabling the formation of entire adenoviral capsids or functional parts, analogues, and/or derivatives thereof. Preferably, the nucleic acid derived from an adenovirus includes the nucleic acid encoding adenovirus E2A or a functional part, derivative, and or analogue thereof. Preferably, the nucleic acid derived from an adenovirus includes the nucleic acid encoding at least one E4-region protein or a functional part, derivative, and or analogue thereof, which facilitates, at least in part, replication of an adenoviral derived nucleic acid in a cell. The adenoviral vectors used in the examples of this application are exemplary of the vectors useful in the present method of treatment invention. Certain embodiments of the present invention use retroviral vector systems. Retroviruses are integrating viruses that infect dividing cells, and their construction is known in the art. Retroviral vectors can be constructed from different types of retrovirus, such as, MoMuLV ("murine Moloney leukemia virus" MSV ("murine Moloney sarcoma virus"), HaSV ("Harvey sarcoma virus"); SNV ("spleen necrosis virus"); RSV ("Rous sarcoma virus") and Friend virus. Lentiviral vector systems may also be used in the practice of the present invention. In other embodiments of the present invention, adeno-associated viruses ("AAV") are utilized. The AAV viruses are DNA viruses of relatively small size that integrate, in a stable and site-specific manner, into the genome of the infected cells. They are able to infect a wide spectrum of cells without inducing any effects on cellular growth, morphology or differentiation, and they do not appear to be involved in human pathologies. In the vector construction, the polynucleotide agents of the present invention may be linked to one or more regulatory regions. Selection of the appropriate regulatory region or regions is a routine matter, within the level of ordinary skill in the art. Regulatory regions include promoters, and may include enhancers, suppressors, etc. Promoters that may be used in the expression vectors of the present invention include both constitutive promoters and regulated (inducible) promoters. The promoters may be prokaryotic or eukaryotic depending on the host. Among the prokaryotic (mcluding bacteriophage) promoters useful for practice of this invention are lac, lacZ, T3, 17, lambda Pr, Pi, and tip promoters. Among the eukaryotic (including viral) promoters useful for practice of this invention are ubiquitous promoters (e.g. HPRT, vhnentin, actin, tubulin), intermediate filament promoters (e.g. desmin, neurofilaments, keratin, GFAP), therapeutic gene promoters (e.g. MDR type, CFTR, factor VTfi), tissue-specific promoters (e.g. actin promoter in smooth muscle cells, or Fit and Flk promoters active in endothelial cells), including animal transcriptional control regions, which exhibit tissue specificity and have been utilized in transgenic animals: elastase I gene control region which is active in pancreatic acinar cells (Swift, et al. (1984) Cell 38:639-46; Ornitz, et al. (1986) Cold Spring Harbor Symp. Quant. Biol. 50:399-409; MacDonald, (1987) Hepatology 7:425-515); insulin gene control region which is active in pancreatic beta cells (Hanahan, (1985) Nature 315:115-22), immunoglobulin gene control region which is active in lymphoid cells (Grosschedl, et al. (1984) Cell 38:647-58; Adames, et al. (1985) Nature 318:533-8; Alexander, et al. (1987) Mol. Cell. Biol. 7:1436-44), mouse mammary tumor virus control region which is active in testicular, breast, lymphoid and mast cells (Leder, et al. (1986) Cell 45:485-95), dbumin gene control region which is active in liver (Pinkert, et al. (1987) Genes and Devel. 1:268-76), alpha-fetoprotein gene control region which is active in liver (Xrumlauf, et al. (1985) Mol. Cell. Biol., 5:1639-48; Hammer, et al. (1987) Science 235:53-8), alpha 1-antitrypsin gene control region which is active in the liver (Kelsey, et al. (1987) Genes and Devel., 1: 161-71), beta-globin gene control region which is active in myeloid cells (Mogram, et al. (1985) Nature 315:338-40; Kollias, et al. (1986) Cell 46:89- 94), myelin basic protein gene control region which is active in oligodendrocyte cells in the brain (Readhead, et al. (1987) Cell 48:703-12), myosin light chain-2 gene control region which is active in skeletal muscle (Sani, (1985) Nature 314.283-6), and gonadotropic releasing hormone gene control region which is active in the hypothalamus (Mason, et al. (1986) Science 234:1372-8). Other promoters which may be used in the practice of the invention include promoters which are preferentially activated in dividing cells, promoters which respond to a stimulus (e.g. steroid hormone receptor, retinoic acid receptor), tetracycline-regulated transcriptional modulators, cytomegalovirus immediate-early, retroviral LTR, metallothionein, SV-40, El a, and MLP promoters. Additional vector systems include the non-viral systems that facilitate introduction of polynucleotide agents into a patient. For example, a DNA vector encoding a desired sequence can be introduced in vivo by lipofection. Synthetic cationic lipids designed to limit the difficulties encountered with liposome-mediated transfection can be used to prepare liposomes for in vivo transfection of a gene encoding a marker (Feigner, et. al. (1987) Proc. Natl. Acad Sci. USA 84:7413-7); see Mackey, et al. (1988) Proc. Natl. Acad. Sci. USA 85:8027-31; Uhner, et al. (1993) Science 259:1745-8). The use of cationic lipids may promote encapsulation of negatively charged nucleic acids, and also promote fusion with negatively charged cell membranes (Feigner and Ringold, (1989) Nature 337:387-8). Particularly useful lipid compounds and compositions for transfer of nucleic acids are described in International Patent Publications WO 95/18863 and WO 96/17823, and in U.S. Pat. No. 5,459,127. The use of lipofection to introduce exogenous genes into the specific organs in vivo has certain practical advantages and directing transfection to particular cell types would be particularly advantageous in a tissue with cellular heterogeneity, for example, pancreas, liver, kidney, and the brain. Lipids may be chemically coupled to other molecules for the purpose of targeting. Targeted peptides, e.g., hormones or neurotransmitters, and proteins for example, antibodies, or non-peptide molecules could be coupled to liposomes chemically. Other molecules are also useful for facilitating transfection of a nucleic acid in vivo, for example, a cationic oligopepti.de (e.g., International Patent Publication WO 95/21931), peptides derived from DNA binding proteins (e.g., International Patent Publication WO 96/25508), or a cationic polymer (e.g., International Patent Publication WO 95/21931). It is also possible to introduce a DNA vector in vivo as a naked DNA plasmid (see U.S. Pat. Nos. 5,693,622, 5,589,466 and 5,580,859). Naked DNA vectors for therapeutic purposes can be introduced into the desired host cells by methods known in the art, e.g., transfection, electroporation, microinjection, transduction, cell fusion, DEAE dextran, calcium phosphate precipitation, use of a gene gun, or use of a DNA vector transporter (see, e.g., Wilson, et al. (1992) J. Biol. Chem 267:963-7; Wu and Wu, (1988) J. Biol. Chem. 263:14621-4; Hartmut, et al. Canadian Patent Application No. 2,012,311, filed Mar. 15, 1990; Williams, et al (1991). Proc. Natl. Acad. Sci. USA 88:2726-30). Receptor-mediated DNA delivery approaches can also be used (Curiel, et al. (1992) Hum. Gene Ther. 3:147- 54; Wu andWu, (1987) J. Biol. Chem. 262:4429-32). The present invention also provides biologically compatible, cartilage formation- enhancing compositions comprising an effective amount of one or more compounds identified as TARGET inhibitors, and/or the expression-inhibiting agents as described hereinabove. A biologically compatible composition is a composition, that may be solid, liquid, gel, or other form, in which the compound, polynucleotide, vector, and antibody of the invention is maintained in an active form, e.g., in a form able to effect a biological activity. For example, a compound of the invention would have inverse agonist or antagonist activity on the TARGET; a nucleic acid would be able to replicate, translate a message, or hybridize to a complementary mRNA of a TARGET; a vector would be able to transfect a TARGET cell and expression the antisense, antibody, ribozyme or siRNA as described hereinabove; an antibody would bind a TARGET polypeptide domain. A preferred biologically compatible composition is an aqueous solution that is buffered using, e.g., Tris, phosphate, or HEPES buffer, containing salt ions. Usually the concentration of salt ions will be similar to physiological levels. Biologically compatible solutions may include stabilizing agents and preservatives. In a more preferred embodiment, the biocompatible composition is a pharmaceutically acceptable composition. Such compositions can be formulated for administration by topical, oral, parenteral, intranasal, subcutaneous, and intraocular, routes. Parenteral administration is meant to include intravenous injection, intramuscular injection, intraarterial injection or infusion techniques. The composition may be administered parenterally in dosage unit formulations containing standard, well-known non-toxic physiologically acceptable carriers, adjuvants and vehicles as desired. A particularly preferred embodiment of the present composition invention is a cartilage formation-enhancing pharmaceutical composition comprising a therapeutically effective amount of an expression-inhibiting agent as described hereinabove, in admixture with a pharmaceutically acceptable carrier. Another preferred embodiment is a pharmaceutical composition for the treatment or prevention of a condition a systemic or local decrease in mean cartilage thickness, or a susceptibility to the condition, comprising an effective cartilage formation-enhancing amount of a TARGET antagonist or inverse agonist, its pharmaceutically acceptable salts, hydrates, solvates, or prodrugs thereof in admixture with a pharmaceutically acceptable carrier. Pharmaceutical compositions for oral administration can be formulated using pharmaceutically acceptable carriers well known in the art in dosages suitable for oral administration. Such carriers enable the pharmaceutical compositions to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, and the like, for ingestion by the patient. Pharmaceutical compositions for oral use can be prepared by combining active compounds with solid excipient, optionally grinding a resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries, if desired, to obtain tablets or dragee cores. Suitable excipients are carbohydrate or protein fillers, such as sugars, including lactose, sucrose, mannitol, or sorbitol; starch from corn, wheat, rice, potato, or other plants; cellulose, such as methyl cellulose, hydroxypropylmethyl-cellulose, or sodium carboxymethyl-cellulose; gums including arabic and tragacanth; and proteins such as gelatin and collagen. If desired, disintegrating or solubilizing agents may be added, such as the cross-linked polyvinyl pyrrolidone, agar, alginic acid, or a salt thereof, such as sodium alginate. Dragee cores may be used in conjunction with suitable coatings, such as concentrated sugar solutions, which may also contain gum arabic, talc, polyvinylpyrrolidone, carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures. Dyestuffs or pigments may be added to the tablets or dragee coatings for product identification or to characterize the quantity of active compound, i.e., dosage. Pharmaceutical preparations that can be used orally include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a coating, such as glycerol or sorbitol. Push-fit capsules can contain active ingredients mixed with filler or binders, such as lactose or starches, lubricants, such as talc or magnesium stearate, and, optionally, stabilizers. In soft capsules, the active compounds may be dissolved or suspended in suitable liquids, such as fatty oils, liquid, or liquid polyethylene glycol with or without stabilizers. Preferred sterile injectable preparations can be a solution or suspension in a non-toxic parenterally acceptable solvent or diluent. Examples of pharmaceutically acceptable carriers are saline, buffered saline, isotonic saline (e.g. monosodium or disodium phosphate, sodium, potassium; calcium or magnesium chloride, or mixtures of such salts), Ringer's solution, dextrose, water, sterile water, glycerol ethanol, and combinations thereof 1,3-butanediol and sterile fixed oils are conveniently employed as solvents or suspending media. Any bland fixed oil can be employed including synthetic mono- or di-glycerides. Fatty acids such as oleic acid also find use in the preparation of injectables. The composition medium can also be a hydrogel, which is prepared from any biocompatible or non-cytotoxic homo- or hetero-polymer, such as a hydrophilic polyacrylic acid polymer that can act as a drug absorbing sponge. Certain of them, such as, in particular, those obtained from ethylene and/or propylene oxide are commercially available. A hydrogel can be deposited directly onto the surface of the tissue to be treated, for example during surgical intervention. Embodiments of pharmaceutical compositions of the present invention comprise a replication defective recombinant viral vector encoding the polynucleotide inhibitory agent of the present invention and a transfection enhancer, such as poloxamer. An example of a poloxamer is Poloxamer 407, which is commercially available (BASF, Parsippany, N. J.) and is a non-toxic, biocompatible polyol. A poloxamer impregnated with recombinant viruses may be deposited directly on the surface of the tissue to be treated, for example during a surgical intervention. Poloxamer possesses essentially the same advantages as hydrogel while having a lower viscosity. The active expression-inhibiting agents may also be entrapped in microcapsules prepared, for example, by interfacial polymerization, for example, hydroxymethylcellulose or gelatin-microcapsules and poly-(methyhnethacylate) microcapsules, respectively, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules) or in macroemulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences (1980) 16th edition, Osol, A. Ed. Sustained-release preparations may be prepared. Suitable examples of sustained- release preparations include semi-permeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e.g. films, or microcapsules. Examples of sustained-release matrices include polyesters, hydrogels (for example, poly(2-hydroxyethyl-methacrylate), or ρoly(vinylalcohol)), polylactides (U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and gamma-ethyl-L-glutamate, non- degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers such as the LUPRON DEPOT™, (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), and poly-D-(-)-3-hydroxybutyric acid. While polymers such as ethylene-vinyl acetate and lactic acid-glycolic acid enable release of molecules for over 100 days, certain hydrogels release proteins for shorter time periods. When encapsulated antibodies remain in the body for a long time, they may denature or aggregate as a result of exposure to moisture at 37°C, resulting in a loss of biological activity and possible changes in immunogenicity. Rational strategies can be devised for stabilization depending on the mechanism involved. For example, if the aggregation mechamsm is discovered to be intermolecular S-S bond formation through thio-disulfide interchange, stabilization may be achieved by modifying sulfhydryl residues, lyophilizing from acidic solutions, controlling moisture content, using appropriate additives, and developing specific polymer matrix compositions. As defined above, therapeutically effective dose means that amount of protein, polynucleotide, peptide, or its antibodies, agonists or antagonists, which ameliorate the symptoms or condition. Therapeutic efficacy and toxicity of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., ED50 (the dose therapeutically effective in 50% of the population) and LD50 (the dose lethal to 50% of the population). The dose ratio of toxic to therapeutic effects is the therapeutic index, and it can be expressed as the ratio, LD50/ED50. Pharmaceutical compositions that exhibit large therapeutic indices are preferred. The data obtained from cell culture assays and animal studies is used in formulating a range of dosage for human use. The dosage of such compounds lies preferably within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage varies within this range depending upon the dosage form employed, sensitivity of the patient, and the route of administration. For any compound, the therapeutically effective dose can be estimated initially either in cell culture assays or in animal models, usually mice, rabbits, dogs, or pigs. The animal model is also used to achieve a desirable concentration range and route of administration. Such information can then be used to determine useful doses and routes for administration in humans. The exact dosage is chosen by the individual physician in view of the patient to be treated. Dosage and administration are adjusted to provide sufficient levels of the active moiety or to maintain the desired effect. Additional factors which may be taken into account include the severity of the disease state, age, weight and gender of the patient; diet, desired duration of treatment, method of aciministration, time and frequency of administration, drug combination(s), reaction sensitivities, and tolerance/response to therapy. Long acting pharmaceutical compositions might be administered every 3 to 4 days, every week, or once every two weeks depending on half-life and clearance rate of the particular formulation. The pharmaceutical compositions according to this invention may be administered to a subject by a variety of methods. They may be added directly to TARGET tissues, complexed with cationic lipids, packaged within liposomes, or delivered to TARGET cells by other methods known in the art. Localized administration to the desired tissues may be done by direct injection, transdermal absoφtion, catheter, infusion pump or stent. The DNA, DNA/vehicle complexes, or the recombinant virus particles are locally administered to the site of treatment. Alternative routes of delivery include, but are not limited to, intravenous injection, intramuscular injection, subcutaneous injection, aerosol inhalation, oral (tablet or pill form), topical, systemic, ocular, intraperitoneal and/or intrathecal delivery. Examples of ribozyme delivery and aciministration are provided in Sullivan et al. WO 94/02595. Antibodies according to the invention may be delivered as a bolus only, infused over time or both administered as a bolus and infused over time. Those skilled in the art may employ different formulations for polynucleotides than for proteins. Similarly, delivery of polynucleotides or polypeptides will be specific to particular cells, conditions, locations, etc. As discussed hereinabove, recombinant viruses may be used to introduce DNA encoding polynucleotide agents useful in the present invention. Recombinant viruses according to the invention are generally formulated and administered in the form of doses of between about 104 and about 1014 pfu. In the case of AAVs and adenoviruses, doses of from about 106 to about 1011 pfu are preferably used. The term pfu ("plaque-forming unit") corresponds to the infective power of a suspension of virions and is determined by infecting an appropriate cell culture and measuring the number of plaques formed. The techniques for determining the pfu titre of a viral solution are well documented in the prior art. The present invention also provides methods of enhancing cartilage formation, which comprise the administration to said subject a therapeutically effective amount of an expression-inhibiting agent of the invention. A further aspect of the invention relates to a method of treating or preventing a disease involving chondrocyte anabolic stimulation, comprising aάniinistering to said subject a cartilage formation-enhancing pharmaceutical composition as described herein. Examples of diseases involving anabolic stimulation of chondrocytes that are treatable using the means and methods of the present invention include, but are not limited to osteoarthritis, rheumatoid arthritis, psoriatic arthritis, juvenile rheumatoid arthritis, gouty arthritis, septic or infectious arthritis, reactive arthritis, reflex sympathetic dystrophy, algodystrophy, Tietze syndrome or costal chondritis, fibromyalgia, osteochondritis, neurogenic or neuropathic arthritis, arthropathy, endemic forms of arthritis like osteoarthritis deformans endemica, Mseleni disease, and Handigodu disease; degeneration resulting from fibromyalgia, systemic lupus erythematosus, scleroderma, and ankylosing spondylitis. Furthermore, people suffering from congenital cartilage malformations, including hereditary chondrolysis, chondrodysplasias and pseudoachondrodysplasias, are likely to benefit from programs that result in anabolic stimulation of chondrocytes, and these diseases therefore may also be treated by using the methods and means of the present invention. Non-limiting examples of congenital cartilage malformation related diseases are microtia, anotia, and metaphyseal chondrodysplasia. The polypeptides or the polynucleotides employed in the methods of the present invention may be free in solution, affixed to a solid support, borne on a cell surface, or located intracellularly. To perform the methods it is feasible to immobilize either the polypeptide of the present invention or the compound to facilitate separation of complexes from uncomplexed forms of the polypeptide, as well as to accommodate automation of the assay. Interaction (e.g., binding of) of the polypeptide of the present invention with a compound can be accomplished in any vessel suitable for containing the reactants. Examples of such vessels include microtitre plates, test tubes, and microcentrifuge tubes. In one embodiment, a fusion protein can be provided which adds a domain that allows the polypeptide to be bound to a matrix. For example, the polypeptide of the present invention can be "His" tagged, and subsequently adsorbed onto Ni-NTA microtitre plates, or ProtA fusions with the polypeptides of the present invention can be adsorbed to IgG, which are then combined with the cell lysates (e.g., (35S-labelled) and the candidate compound, and the mixture incubated under conditions favorable for complex formation (e.g., at physiological conditions for salt and pH). Following incubation, the plates are washed to remove any unbound label, and the matrix is immobilized. The amount of radioactivity can be determined directly, or in the supernatant after dissociation of the complexes. Alternatively, the complexes can be dissociated from the matrix, separated by SDS-PAGE, and the level of the protein binding to the protein of the present invention quantitated from the gel using standard electrophoretic techniques. Other techniques for immobilizing protein on matrices can also be used in the method of identifying compounds. For example, either the polypeptide of the present invention or the compound can be immobilized utilizing conjugation of biotin and streptavidin. Biotinylated protein molecules of the present invention can be prepared from biotin-NHS (N-hydroxy- succinimide) using techniques well known in the art (e.g., biotinylation kit, Pierce Chemicals, Rockford, 111.), and immobilized in the wells of streptavidin-coated 96 well plates (Pierce Chemical). Alternatively, antibodies reactive with the polypeptides of the present invention but which do not interfere with binding of the polypeptide to the compound can be derivatized to the wells of the plate, and the polypeptide of the present invention can be trapped in the wells by antibody conjugation. As described above, preparations of a labeled candidate compound are incubated in the wells of the plate presenting the polypeptide of the present invention, and the amount of complex trapped in the well can be quantitated. Another embodiment of the present invention relates to a method for in vitro production of cartilage tissue, comprising the steps of contacting chondrocyte cells with a polynucleotide sequence comprising a sequence selected from the group consisting of sequences complementary to SEQ ID No: 1-28, preferably selected from the group consisting of sequences complementary to SEQ ID NO: 202-315 for a time sufficient to re-differentiate the chondrocytes thereby producing a cartilaginous matrix. In a preferred embodiment, the method comprises the steps of: (a) applying chondrocyte cells on a substrate to form a cellular substrate, (b) introducing a polynucleotide comprising a nucleotide sequence selected from the group consisting of sequences complementary to SEQ ID No: 1-28, preferably selected from the group consisting of sequences complementary to SEQ TD NO: 202-315, for a time sufficient to re-differentiate the chondrocyte cells, thereby producing a cartilaginous matrix. The invention thus provides a method for producing a substrate with a matrix grown thereon, which matrix may be used for the provision of load-bearing implants, including joint prostheses, such as artificial knee joints and finger joints, and maxillofacial implants. It can also be used for special surgery devices, such as spacers, or cartilage fillers, and for use in augmentation, obliteration or reconstitution of cartilage defects and damaged or lost cartilage. The present invention also relates to a combination of a load-bearing implant (preferably coated with a matrix as described above) with a cartilage filler comprising a matrix as described. The method of the invention is also very suitable in relation to revision surgery, i.e., when previous surgical devices require replacement. Suitable cells are stem cells cells, including mesenchymal stem cells cells and in particular chondrosyte precursor cells. The mesenchymal stem cells, and especially the chondrosyte precursor cells are found to be very effective in the cartilage producing process when taken from their original environment. In addition, cells derived from cartilage biopsies of a subject may be cultured and utilized with the present invention. The mesenchymal stem cells can be directly applied on the substrate or they can advantageously be multiplied in the absence of the substrate before being applied on the substrate. In the latter mode, the cells are still largely multipotent after multiplication and, for the puφose of the invention, they are still referred to as undifferentiated. Subsequently, the cells are allowed to differentiate. Differentiation can be induced or enhanced by the presence of suitable inductors, - such as bone morphogenic proteins (BMP2; BM4; BMP7), tiansforming growth factor beta (TGFbeta), CDMP1 and CDMP2. Especially suitable inductors of differentiation are the expression inhibitory agents of the present invention. The use of mesenchymal stem cells provides several advantages. Firstly, their lower differentiation implies a higher proUferation rate and allows the eventual functionality to be better directed and controlled. Moreover, culturing these cells not only produces the required cartilage matrix containing organic and inorganic components, but also results in the presence, in the culture medium and in the matrix, of several factors which are essential for growth of the tissue and for adaptation to existing living tissue. Also, the culture medium can be a source of active factors such as growth factors, to be used in connection with the implanting process. Furthermore, such undifferentiated cells are often available in large quantities and more conveniently than e.g., mature cartilage cells, and exhibit a lower morbidity during recovery. Moreover, the undifferentiated cells can be obtained from the patient for whom the implant is intended. The cartilage resulting from these cells is autologous to the patient and thus no immune response will be induced. Matrices as thick as 100 μm can be produced as a result of the use of undifferentiated cells. The substrate on which the undifferentiated cells can be applied and cultured can be a metal, such as titanium, cobalt chromium alloy or stainless steel, a bioactive surface such as a calcium phosphate, polymer surfaces such as polyethylene, and the like. Although less preferred, siliceous material such as glass ceramics, can also be used as a substrate. Most preferred are metals, such as titanium, and calcium phosphates, even though calcium phosphate is not an indispensable component of the substrate. The substrate may be porous or non-porous. The cells can be applied at a rate of e.g., 103 -106 per cm2, in particular 104 - 2 X 105 cells per cm2. The culture medium to be used in the method according to the invention can be a commonly known culture medium such as MEM (minimum essential medium). Advantageously, the medium can be a conditioned medium. In this context, a conditioned medium is understood to be a medium wherein similar cells have previously been incubated, causing the medium to contain factors such as polypeptides, secreted by the cells which are important for cell growth and cell differentiation. The cells are cultured for a time sufficient to produce a matrix layer, e.g, a matrix layer having a thickness of at least 0.5 μm, in particular from 1 up to 100 μm, more in particular of 10-50 μm. The cells may be contacted with the culture medium for e.g. 2-15 weeks, in particular 4-10 weeks. The production of the matrix, when applied on a substrate, results in a continuous or quasi-continuous coating covering the substrate for at least 50%, in particular at least 80% of its surface area. In yet another aspect of the invention, the invention provides a method for diagnosing a pathological condition involving chondrocyte de-differentiation, said method comprising the steps of: determining the nucleic acid sequence of any one of the genes encoding the polypeptides listed in Table 1A in a genomic DNA sample; comparing the sequence from step (a) with the nucleic acid sequence of a healthy subject; and identifying any difference(s) related to the pathological condition. Such differences may be further checked in in vitro assays applying similar marker genes as disclosed herein. Such assays will reveal the role of the gene or its encoded polypeptide in anabolic stimulation processes of chondrocytes. If such mutations are identified this knowledge can be further exploited in test-kits for diagnosis of similar diseases. Still another aspect or the invention relates to a method for diagnosing a pathological condition involving chondrocyte anabolic stimulation or a susceptibility to the condition in a subject, comprising deterrnining the amount of polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 101-128 and 401-594 in a biological sample, and comparing the amount with the amount of the polypeptide in a healthy subject, wherein an increase of the amount of polypeptide compared to the healthy subject is indicative of the presence of the pathological condition. Clearly, the activity and/or expression levels of the target genes as disclosed herein may have an effect on anabolic stimulation of chondrocytes. It remains to be determined to what level the activity should be elevated to diagnose for the disease. However, by comparing levels found in patients, individuals without symptoms and clearly healthy individuals the skilled person may easily determine these relevant levels. Since the skilled person is now aware which polypeptides should be monitored, the present invention provides novel tools for test assays for such diagnostics. A prominent disease that may be controlled, checked and diagnosed by using the knowledge provided by the present invention is osteoarthritis. The rate of chondrocyte anabolic stimulation can typically be measured by determining the deposition of cartilage, or cartilage components, or cartilage-containing extra-cellular matrix produced by the chondrocytes, in the medium. A cell-based ELISA, enzymatic assays, or other general techniques known in the art can be used to measure cartilage components, like the ones described in Walsh G., Proteins: Biotechnology and Biochemistry. John Wiley and Sons, 2001. The invention is further illustrated in the following figures and examples.
EXAMPLES
Example 1. Development of a high-throughput screening method for the detection of endogenous collagen type IL alpha- 1 (col2αl) Principle of the assay: Normal human articular chondrocytes (NHAC's) that are grown in two-dimensional cultures become dedifferentiated and gradually cease to synthesize cartilage. They can be re- differentiated into anabolic, active chondrocytes in the presence of appropriate factors (e.g. BMP2). An assay to screen for such factors was developed by monitoring the levels of collagen type π, alpha-1 (col2αl), a major constituent of normal cartilage. NHAC's are seeded in 384 well plates and 1 day after plating infected with individual siRNA adenoviruses (Ad-siRNA) from the SilenceSelect collection (see WO03/020931). Col2αl deposition is determined at 14 days after the start of the infection (14 dpi).
Control viruses
Ad-BMP2; described in WO 03/018799
BMP4; Ad5 dEl/E2A adenoviruses that mediate the expression of full length bone moφhogenetic protein 4 pre-protein (see NP_570912).
Ad-LacZ; referred to as pIPspAdApt6-lacZ in WO 02/070744 Ad-eGFP; referred to as ρIPspAdApt6-eGFP in WO 02/070744
Ad-Empty; described in WO 02/070744
Development of the assay NHAC's were isolated from donors who died from unrelated causes, and were obtained after informed consent (Cambrex, Verviers, Belgium). In a series of experiments, carried out in 384- well plates, several parameters are optimized: cell seeding density, multiplicities of infection (MOD of control viruses (Ad-
BMP2 or Ad-eGFP), duration of infection, toxicity, infection efficiency (using Ad-eGFP) and the day of readout. Using Ad-BMP2 (BMP2 over-expression) as a positive control for assay development, the following protocol resulted in the highest dynamic range for the assay with the lowest standard deviation on the background signal: NHAC's are seeded on day 0 at 1500 cells/well of a 384- well plate in 60 μl of
DMEM/F12 (InVitrogen), containing 10% heat-inactivated fetal calf serum (FBS-HI) and Pen/Strep) and infected the next day with 2.5 μl of Ad-control- virus (Ad-BMP2 or Ad-eGFP; this corresponds to an assumed MOI of 2000). After 7 days, 10 μl of a 50 μg/ml 2-Phospho-
L-ascorbic acid in assay culture medium is added to each well. Up-regulation of Col2al is read at 10 dpi: The medium is removed with a VacuSafe; 50 μl ice-cold MeOH is added with a multidrop and removed immediately with a VacuSafe; 80 μl of ice-cold MeOH is added with a multidrop to fixate the cells, and plates are incubated for 20 min at -20°C; MeOH is removed with a VacuSafe; plates are air-dried for 20 min, followed by 2x washing with 80 μl of phosphate buffered saline (PBS); 75 μl of blocking buffer (0.1% casein in PBS) is added and plates are incubated for at least 2 h at room temperature (RT); blocking buffer is removed; cells are washed with 25 μl of EC buffer (20 mM sodium phosphate, 2mM EDTA, 400 mM NaCl, 0.2% BSA, 0.05% CHAPS, 0.4% casein, 0.05% NaN3, pH 7) and 35 μl of the primary antibody (Collagen IT Ab-2 Neomarkers. Catalogue number MS-235-P) diluted 1/450, 1/225 in buffer C (20 mM sodium phosphate, 2 mM EDTA, 400 mM NaCl, 1% BSA, pH 7)) is added with a multichannel pipette; plates are incubated overnight at 4°C; primary antibody is removed; cells are washed twice with 80 μl of PBST (0.5% Tween 20 in PBS) and once with 80 μl PBS; 35 μl of the secondary antibody (Goat-anti-mouse Immunoglobulins/HRP. DAKO. Catalogue number P0477; diluted 1/2000 in buffer C) is added with multichannel pipette; plates are incubated at RT for 1 h; secondary antibody is removed and cells are washed twice with 80 μl PBST and once with 80 μl PBS; 50 μl of luminol substrate is added and after 5 minutes read-out is determined on a luminometer. After optimization of the assay (see Figure 1), a 384 well control plate is prepared that contains positive control viruses (BMP2 and BMP4) and neutral viruses (eGFP; lacZ and empty, see figure 1). Aliquots of the control plate are prepared and frozen at -20°C. A control plate is and taken along in every screening batch.
Example 2. Screening of 9216 adenoviral siRNA vectors in the chondrogenesis assay The optimized protocol for screening the SilenceSelect library runs as follows: on day 0, propagated human primary chondrocytes are seeded in Greiner white, flat bottom, TC- treated 384 well plates with clear bottom (Catalogue number 781080) in 60 μl medium at a density of 1500 cells per well. One day later, 2.5 μl Ad-siRNA virus from each well of the SilenceSelect™ collection (WO 03/020931), stored in 384 well plates (estimated titer of 1 x 109 viral particles per ml) is transferred with the aid of a 96/384 channel dispenser (Tecan Freedom 200 equipped with TeMO96, TeMO384 and RoMa, Tecan AG, Switzerland) to individual wells of the 384 well plates containing chondrocytes. The control plate is run under the same conditions as the aliquot plates from the SilenceSelect collection. All Ad- siRNA viruses are screened in duplicate on independent assay plates. After infection, plates are incubated at 37°C. Seven days post infection the medium containing the adenoviruses is replaced by fresh medium. Thirteen days post infection, the amounts of col2αl depositions per well is determined with the cELISA method. A typical result of a 384 well screening plate is depicted in figure 2. The duplicate screen is repeated once. Ad-siRNA viruses are nominated as hits if at least 2 data points of the four tested (two times screened in duplicate) score above threshold. Threshold is set at average plus 2.5 times standard deviation of all data points per plate. A total of 282 hits are isolated that scored above the threshold, representing 274 independent genes. A representative example is provided in Figure 2, in which the "times standard deviation" of duplicate data points are indicated on the X-axis and Y-axis. The threshold (2.5 times standard deviation) is indicated by dotted lines. Negative values indicate data points that scored below average. The results for some of the genes are shown in figure 3. A clear induction of the collagen II levels is observed upon infection of the Ad-siRNA targeting the indicated gene. The data are represented as relative light units (rlu) correlating to collagen II levels.
Example 3. Propagation of hits The 282 Ad-siRNA hits are subjected to further analysis to establish their therapeutic potential to induce chondrocyte anabolic stimulation. A first step entails a quality control on the Ad-siRNA selected for further analysis (this example). Next steps are the screening of the targets in other assays to validate their role in chondrocyte anabolic stimulation such as the induction of aggrecan, another main constituent of cartilage besides collagen IT (Example 4), the ability to induce chondrocyte anabolic stimulation in chondrocytes from other donors (Example 5), the induction of a correct marker profile in tluee-dimensional chondrocyte cultures (example 11), the presence of posttranslational modifications on aggrecan (example 9) and collagen II (example 10) in three-dimensional chondrocyte cultures, the development of additional Ad-siRNAs targeting the identified transcripts (example 7), and confirmation that the corresponding genes are indeed expressed in residing chondrocytes (example 12). To propagate the 282 hits of the chondrogenesis assay, 2.25 x 104 PerC6.E2A cells are seeded in 200 μl of DMEM containing 10% non-heat inactivated FCS into each well of a 96 well plate and incubated overnight at 39°C in a humidified incubator at 10% CO2. Subsequently, 1 μl of crude lysate from the siRNA adenovirus stocks in matrix tubes is added and incubation proceeds at 34°C in a humidified incubator at 10% CO2 for 7 days. All hits are propagated in duplicate on two independent plates. The two lysates are pooled and aliquots are frozen at -20°C. The propagated Ad-siRNAs are re-screened at three MOFs in the chondrogenesis assay in duplicate (see Example 1). The Ad-siRNAs have to score at least once above threshold (average + 2.5 times standard deviation) to pass this quality control step.
Example 4. Aggrecan induction A second assay to screen for chondrocyte anabolic factors is developed by monitoring the levels of aggrecan, another major constituent of cartilage. In this assay, glycosaminoglycans on aggrecan are stained by Alcian blue. NHACs are seeded in 384 well plates and 1 day after plating infected with individual Ad-siRNA from the SilenceSelect™ collection. Aggrecan deposition is determined at 14 days post infection. Using Ad-BMP2 as a positive control, we confirm in a series of experiments that several parameters optimized for the Coin cELISA assay are also applicable for the Alcian blue stain assay for aggrecan. These parameters include cell seeding density, MOIs of control viruses, duration of infection, and the day of readout. NHAC's are seeded on day 0 at 1500 cells/well of a 384 well black-plate with clear bottom in 60 μl of DMEM/F12, containing 10% FBS-HI and Pen/Strep and infected the next day with 2.5 μl of Ad-BMP2 or Ad-eGFP; at an MOI of 2000. After 7 days, 10 μl of a 50 μg/ml 2-Phospho-L-ascorbic acid in assay culture medium is added to each well. Up- regulation of aggrecan is read at 10 dpi with Alcian blue staining: The medium is removed with a VacuSafe; 50 μl ice-cold MeOH is added with a multidrop and removed immediately with a VacuSafe; 80 μl of ice-cold MeOH is added with a multidrop to fixate the cells, and plates are incubated for 20 min at -20°C; MeOH is removed with a VacuSafe; plates are air dried for 20 min. After washing once with 80 μl of PBS; 80 μl of 0.05% Alcian blue stain buffer (0.05% Alcian blue, Sigma, catalog number S-2889; 0.4 M MgCl2/25 mM sodium acetate, pH5.5) is added and plates are incubated overnight at RT. The next day cells are washed subsequently in 80 μl of 3% acetic acid, 25% ethanol/3% acetic acid, and 50% ethanol/3% acetic acid. Solutions are added with a multidrop and removed with a Vacusafe. After replacing 50% ethanol/3% acetic acid with 70% ethanol/3% acetic acid, each individual well is photographed with a SONY CCD camera, images are analyzed using a Galapagos Alcian blue quantification algorithm based on the separation of the blue signal through a color threshold procedure after a noise reduction filter. The amount of blue staining, which is proportional to aggrecan content, is expressed in pixel unit (see Figure 4). The propagated hits from Example 3 are used to transduce NHAc cells at three MOFs in duplicate in the chondrogenesis assay (see Example 1). The Ad-siRNAs have to score in duplicate in at least one MOI above threshold (average + 2.5 x standard deviation) to pass this secondary assay. In total, 101 out of 282 hits passed the Alcian blue assay for aggrecan (see Table 1). The results for some of the genes are shown in figure 5. Values represent the numerical output of the described algorithm and are correlated to Alcian blue staining levels. A clear induction of the aggrecan levels is observed upon infection of the Ad-siRNA targeting the indicated gene. The Ad-siRNA targeting FZDl does not seem to induce Alcian blue staining.
Table 1. Overview of the 101 target sequences and their respective SEQ ID NOS (316-416) corresponding to the genes encoding the different polypeptides involved in chondrogenic differentiation. The GenBank numbers and target gene symbols (general names) are also given.
Figure imgf000049_0001
Figure imgf000050_0001
Figure imgf000051_0001
Figure imgf000052_0001
Figure imgf000053_0001
Figure imgf000054_0001
Figure imgf000055_0001
Figure imgf000056_0001
Example 5. Donor dependency The 282 hits identified by the ColII cELISA assay are further subject to a donor dependency test to demonstrate that the induction of Coin production by a given hit is not restricted to a single donor. In addition to the 11 -year-old donor (donor I) previously used, NHAC's from multiple donors with ages of 24 (donor II), 41 (donor DI), and 50 (donor TV), are obtained after informed consent (Cambrex, Venders, Belgium). Cells are seeded as described in Examples 1 and 3. The propagated Ad-siRNAs are used to transduce NHAc cells from these different donors at three MOI's in duplicate in the chondrogenesis assay (see Examples 1 and 3). The Ad-siRNAs have to score at least once above threshold (average + 2.5 times standard deviation) to pass this donor dependency test. Out of 101 hits that passed the Alcian blue assay for aggrecan, 97 score positive in Donor TV. Of the remaining 4, 1 score positive in Donor II. The other 3 did not score in the other donors tested. In addition, 40 of the 101 hits score positive in all three additional donors. These results demonstrate that 98 out of these 101 hits function in a non-donor- dependent manner. Exceptions are H33-145; H33-182; and H33-263 (see Table 1). Indicated in this Table 1 are the Target Gene Symbol, Gene Bank Accession Number, and drugability class of the genes that correspond to the target sequences. The results for some of the genes are shown in Table 2. These data show that knocking down the RNA levels of the indicated genes induces collagen II levels in at least 2 donors. The values represent times standard deviation of the background. Table 2: overview donor dependency data. N=2 for every condition. All data points are represented as fold standard deviation of the background. All values above 2.5 are considered to be positive and are shaded grey.
Figure imgf000057_0001
Figure imgf000058_0001
57
Figure imgf000059_0001
Figure imgf000059_0002
Figure imgf000060_0001
Example 6. Quality control of target Ad-siRNAs: Target Ad-siRNAs are propagated using derivatives of PER.C6® cells (Crucell, Leiden, The Netherlands) at a 96-well plate level, followed by re-screening these viruses at several MOFs in the primary assay (see Example 1) and by sequencing the siRNAs encoded by the target Ad-siRNA viruses. PER.E2A cells are seeded in 96 well plates at a density of 40,000 cells per well in 180 μl PER.E2A medium. Cells are then incubated overnight at 39°C in a 10% C02 humidified incubator. One day later, cells are infected with 1 μl of crude cell lysate from SilenceSelect stocks containing target Ad-siRNAs. Cells are incubated further at 34°C, 10% CO2 until appearance of cytopathic effect (as revealed by the swelling and rounding up of the cells, typically 7 days post infection). The supernatant is collected and the virus crude lysate is treated with proteinase K: 12 μl crude lysate is added to 4 μl Lysis buffer (lx Expand High Fidelity buffer with MgCl2 (Roche Molecular Biochemicals, Cat. No 1332465) supplemented with 1 mg/ml proteinase K (Roche Molecular Biochemicals, Cat No 745 723) and 0.45% Tween-20 (Roche Molecular Biochemicals, Cat No 1335465) in sterile PCR tubes. These are incubated at 55°C for 2 h followed by a 15 min inactivation step at 95°C. For the PCR reaction, 1 μl lysate is added to a PCR master mix composed of 5 μl lOx Expand High Fidelity buffer with MgCl2, 0.5 μl of dNTP mix (10 mM for each dNTP), 1 μl of 'Forward primer' (10 mM stock, sequence: 5' CCG TTT ACG TGG AGA CTC GCC, SEQ ID NO: 29), 1 μl of 'Reverse Primer' (10 mM stock, sequence: 5' CCC CCA CCT TAT ATA TAT TCT TTC C, SEQ ID NO: 30), 0.2 μl of Expand High Fidelity DNA polymerase (3.5 U/μl, Roche Molecular Biochemicals) and 41.3 μl of H2O. PCR is performed in a PE Biosystems GeneAmp PCR system 9700 as follows: the PCR mixture (50 μl in total) is incubated at 95°C for 5 min; each cycle runs at 95°C for 15 sec, 55°C for 30 sec, 68°C for 4 min, and is repeated for 35 cycles. A final incubation at 68°C is performed for 7 min. 5 μl of the PCR mixture is mixed with 2 μl of 6 x gel loading buffer, loaded on a 0.8% agarose gel containing 0.5 μg/μl ethidium bromide to resolve the amplification products. The size of the amplified fragments is estimated from a standard DNA ladder loaded on the same gel. The expected size is ~500 bp. For sequencing analysis, the siRNA constructs expressed by the target adenoviruses are amplified by PCR using primers complementary to vector sequences flanking the Sapl site of the pIPspAdapt6-U6 plasmid. The sequence of the PCR fragments is determined and compared with the expected sequence. All sequences are, found to be identical to the expected sequence.
Example 7: Evaluation of the on target effect of the identified siRNA sequences. To evaluate whether the identified siRNA sequences really increase collagen π and aggrecan levels through the knock down of the target mRNA, a second siRNA sequence is identified that exerts the same effect. A number of additional siRNA sequences targeting the HIT sequences are designed and incorporated in adenoviruses according to WO 03/020931. After production of these adenoviruses, the Ad-siRNAs are infected at different volumes (1.5 μL 5 μl and 15 μl) in the chondrocytes and their effect on collagen π is assessed as described in example 1. The threshold (average 4- 2.5 standard deviation) is calculated for every volume. If a virus scores above the threshold for one or more of the different infection volumes, it is considered to be positive. The on target results are shown in figure 6. These results indicate that at least one additional siRNA, which targets the HIT sequence, can be identified. This underscores the on target effect of the siRNA sequences identified during the screening of the Silence Select library.
Example 8. Development of a tlnee-dimensional-alginate culture system for a quantitative marker analysis for stable cartilage and the assessment of glycosaminoglycans (GAG) and hvdroxyprolines (Hyp) synthesis. Principle of the assay: Normal human articular chondrocytes (NHAC's) grown in three-dimensional cultures are able to maintain a "differentiated state" as measured by the expression of Collagen type II and aggrecan. De-differentiated chondrocytes cultured in a two-dimensional system for a limited amount of passages can revert to a differentiated state when transferred into a three- dimensional culture system. This system was established to test the capability of siRNA adenoviruses (Ad-siRNA) 1) to induce a mRNA expression pattern that correlates with anabolic active chondrocytes and 2) to induce protein modification of collagen type π (hydroxyprolines) and aggrecan (glycosaminoglycans) involved in the stability of cartilage. Normal cartilage NHAC's are cultured in a two-dimensional culture system for two or three passages for cell expansion purposes. Cells are transduced with individual siRNA adenoviruses (Ad-siRNA) in the two-dimensional culture system and three days later are transferred into the three-dimensional alginate culture system. After 10 days in the alginate culture, various parameters can be assessed (e.g. mRNA marker analysis and protein modifications).
Assay procedure Using Ad-BMP2 (BMP2: "strong" collagen π inducer) and Ad-BMP7 (BMP7:
"weak" collagen π inducer) as positive controls and Ad-ALPL as negative control the following protocol is set up (for both mRNA marker analysis and protein modification assessment): After two or three passages in monolayer culture conditions NHAC's are seeded at 2.10E+06 cells/T175 flask in 30 ml of chondrocyte growing medium (Cambrex) and transduced the following day with control-viruses (Ad-BMP2, Ad-BMP4, Ad-BMP7, Ad- ALPL) using an MOI of 2000. After three days, cells are trypsinised using the chondrocyte reagent pack (Cambrex) and washed once with 155 mM sodium chloride/20 mM Hepes ph 7.4 (Cambrex). Cells are re-suspended at a density of 2x106 cells/ml in 1.2% sodium alginate (Cambrex). The cell suspension is transferred into a syringe attached to a 21-22 gauge needle and expelled in a drop-wise fashion into 102 mM calcium chloride/5 mM Hepes pH 7.4 (1 ml and 5 ml respectively in 24-well and 6-well plate) Five and 50 beads per well are produced respectively in the 24-well and 6-well plates. Plates containing the alginate beads are incubated for 10 minutes with gentle shaking every 2 minutes. The calcium chloride solution is aspirated with a Vacusafe system and beads are washed three times with the sodium chloride solution using the Vacusafe and once with DMEMF/12 supplemented with 10% heat inactivated fetal bovine serum (FBS-HI) and 1% penicillin/streptomycin. Alginate beads are finally re-suspended into 0.5 ml and 3 ml (respectively in 24-well and 6-well plate) of DMEMF/12 supplemented with 10% heat inactivated fetal calf serum (FBS-HI), 1% penicillin/streptomycin and 25 μg/ml of ascorbic acid (Fluka, SigmlOl-128 and 401- 5941drich). The alginate cultures are incubated in a humidified incubator at 37°C and 5% CO2 during 10 days with a medium refreshment every 48/72h. For each adenoviral transduction, 60 alginate beads are generated: 2x5 beads were cultured in 24-well plates for the GAGs/Hyps assessments and 50 beads are cultured in a single well of a 6-well plate for the mRNA expression pattern determination. Control viruses
Ad-BMP2; described in WO 03/018799
Ad-BMP7; Ad5 dEl/E2A adenoviruses that mediate the expression of full length bone morphogenetic protein 7 pre-protein (NP_001710). Ad-BMP4; Ad5 dEl E2A adenoviruses that mediate the expression of full length bone morphogenetic protein 4 pre-protein (see NP_570912).
Ad-ALPL; Ad5 dEl/E2A adenoviruses that mediate the expression of full length liver/bone/kidney alkaline phosphatase (NP_000469).
Example 9: Effect of knock down of target genes in chondrocytes embedded in alginate beads, on the glycosaminoglycans fGAGs levels. Chondrocytes are infected and embedded in alginate beads according to example 8.
After 10 days in culture the alginate beads are treated with papain in order to solubilise the glycosaminoglycans prior to quantification: Beads cultured in the 24-well plates are washed once with a 50 mM Phosphate buffer pH 6.5 and incubated for 3 to 4 h at 65°C with 250 μl/well of the same buffer containing 2 mM EDTA, 2 mM L-cystein and 126 μg/ml papain
(Sigma). Complete digestion of the beads is assessed by microscopic observation. Papain digests are frozen at -20°C until glycosaminoglycans quantification is performed. The GAGs produced by the primary chondrocytes in the alginate culture system are measured using the Blyscan™ assay (Biocolor Ltd, Newtownabbey, Northern Ireland).
Principle of the Blvscan assay The Blyscan Assay is a quantitative dye-binding method for the analysis of sulfated
GAGs. The dye label used in the assay is 1,9-dimethylmethylene blue employed under conditions that produce a specific label for the sulfated polysaccharides component of proteoglycans and/or the protein-free sulfated glycosaminoglycan chains. Aggrecan is the predomninant proteoglycan in articular cartilage, representing ± 90% of the cartilage proteoglycans. It is composed of a central core protein attached to ± 50 keratan sulfate and ± 100 chondroitin sulfate chains known as GAGs required for the biological function and the stability of aggrecan.
Assay description The papain digests generated after culturing the chondrocytes for 10 days in alginate beads (Example 7) are diluted in water 1:100, 1:200 if cells were originally transduced with the positives controls and 1:50, 1:100 if transduced with the negative controls. This dilution step allows readout values within the standard range of the assay. The GAG standard provided by the manufacturer contains 100 μg/ml of chondroitin 4-sulfate purified from bovine trachea. This standard is run in duplicate at four concentrations corresponding to 1, 2, 3 and 5 μg of GAGs. Standard and controls are individually diluted in lOOμl final volume in eppendorf tubes. One millihter of the Blyscan Dye Reagent is added to each tube and incubated for 30 minutes at room temperature with continuous shaking. When formed, the GAG-dye complex becomes insoluble and is then separated from the remaining excess soluble unbound dye by centrifugation (10000 x g for 10 minutes). Supernatant are discarded by inverting and careful draining of the tube contents. One milliliter of Blyscan Dissociation Reagent is added to each tube and incubated for one hour to one and a half hour with continuous shaking. This reagent brings the GAG-bound dye back into solution. The GAG content of the assayed samples is spectrophotometrically determined by the amount of dye recovered from the GAGs in the test sample. Two hundred microhters of the dissociation dye solutions are added to the wells of a 96-well plate and reading is performed on an automatic plate reader set at a dual wavelength (656 and 450 nm). The GAGs concentrations measured in the Blyscan assay are normalized to DNA content by performing a fluorimetric Hoechst assay on the same papain digests. Hoechst 33342 dye reagent (Molecular Probes), a bisbenzimidasole dye that binds to adenine/thymine rich regions on DNA. Papain digests were diluted 1:1.7 and 1:3.3 in TE buffer, this dilution step allows readout values within the standard range of the assay. Purified calf thymus DNA (SigmlOl-128 and 401-5941drich) is used as standard DNA: the initial stock solution (2 μg/ml) is sequentially diluted in TE buffer in order to obtain the following concentration range: 1.5, 1.0, 0.75, 0.50, 0.2 μg/ml. Standard and test samples are diluted in TE to a final volume of 100 μl and added to a 96-well plate. One hundred microliters of Hoechst 33342 dye reagent are added to the wells, reading is performed on a multifunctional microplate reader (Fluostar Galaxy, BMG Labtechnologies GmbH) with an excitation wavelength set at 360nm and an emission wavelength set at 440 nm. When GAG concentrations are normalized to DNA content the resulting values expressed as the ratio GAG concentration (μg/ml)/DNA concentration (μg/ml) are used to calculate the assay window. This window is calculated as the ratio normalized GAG Ad- BMP2 (or Ad-BMP4)/normalized GAG Ad-ALPL. The effect of the knock down of the 14 target genes on the GAG levels is assessed as described. The results are shown in figure 7. The results are expressed as fold induction of the GAG levels compared to the average of the normalized GAG values obtained for two negative control knock-down adenoviruses (Ad-PTGER4 and Ad-GRM7). Knock down of the mRNA of the respective genes results in an increase of the GAG levels.
Control viruses
Ad-BMP2: described in WO 03/018799
Ad-BMP4: Ad5 dEl E2A adenoviruses that mediate the expression of full length bone morphogenetic protein 4 pre-protein (see NP_570912). Ad-ALPL: Ad5 dEl/E2A adenoviruses that mediate the expression of full length liver/bone/kidney alkaline phosphatase (NP_000469). Ad-PTGER4: Ad5 dEl/E2A adenoviruses that comprise the siRNA sequence CCATGCCTATTTCTACAGC (SEQ ID NO: 31) to knock down the prostaglandin E receptor 4 mRNA. Ad-GRM7: Ad5 dEl/E2A adenoviruses that comprise the siRNA sequence TCAGTAACAGCTCCCAGAC (SEQ ID NO: 32) to knock down the metabotropic glutamate receptor 7 mRNA.
Example 10: Quantitative analysis of Hvdroxyprolines (Hyps). In articular cartilage, approximately 95% of the collagen is type II collagen. Its polymers are the fibrils that form the basic cohesive framework of the tissue. The collagen biosynthesis involves several unique posttranslational modifications including hydroxylation of proline and lysine residues. These modifications are crucial for collagen stability and resistance to proteolytic enzymes. Chondrocytes are infected and embedded in alginate beads according to example 8. After 10 days in culture the alginate beads are treated with papain : Beads cultured in the 24- well plates are washed once with a 50 mM Phosphate buffer pH 6.5 and incubated for 3 to 4 h at 65°C with 250 μl/well of the same buffer containing 2 mM EDTA, 2 mM L-cystein and 126 μg/ml papain (Sigma). Complete digestion of the beads is assessed by microscopic observation. Papain digests are frozen at -20°C until hydroxyproline quantification is performed. Hydroxyproline asessment in the papain digests is performed by HPLC after acid hydrolysis and FMOC (9-fluorenyhnethyl chloroformate) derivatisation of the samples. This method is described in "Bank RA, Jansen EJ, Beekman B and Te Koppele JM. (1996) Amino acid analysis by reverse-phase high performance liquid chromatography: Improved derivatisation and detection conditions with 9-fluorenyhnethyl chloroformate. Anal Biochem. 240 (2): 167-176. The effect of the knock down of the 14 target genes on the hydroxyproline levels is assessed as described. The results are shown in figure 8. The results are expressed as fold induction compared to the average of Hyp concentrations measured for the two KD controls (Ad-PTGER4 and AD-GRM7). Knock down of the mRNA of the respective genes results in an increase of the hydroxyproline levels. Control viruses Ad-BMP2; described in WO 03/018799
Ad-BMP4; Ad5 ; dEl E2A adenoviruses that mediate the expression of full length bone morphogenetic protein 4 pre-protein (see NP_570912).
Ad-ALPL: Ad5 dEl/E2A adenoviruses that mediate the expression of full length liver/bone/kidney alkaline phosphatase (NP 000469). Ad-PTGER4: Ad5 dEl/E2A adenoviruses that comprise the siRNA sequence CCATGCCTATTTCTACAGC (SEQ ID NO: 33) to knock down the prostaglandin E receptor 4 mRNA. Ad-GRM7: Ad5 dEl/E2A adenoviruses that comprise the siRNA sequence TCAGTAACAGCTCCCAGAC (SEQ ID NO: 34) to knock down the metabotropic glutamate receptor 7 rnRNA.
Example 11: Quantitative analysis of markers for stable cartilage- Assay general principle: Chondrocyte phenotypes can be categorized by characteristic patterns of gene expression. Quantitative RT-PCR techniques are used to monitor the expression pattern of a set of key marker molecules to define which phenotype is induced on the chondrocytes. Positive markers included collagen type II and FGFR3, typically expressed by cartilage chondrocytes. Negative makers included (1) collagens types I and III for dedifferentiated or fibroblast-like chondrocytes, a phenotype that can also be induced by retinoic acid or interleukin-1; (2) collagen X, PTHLH and ALK-1 for hypertrophic chondrocytes that are found in the calcified zone of adult cartilage and the lower hypertrophic zone of the fetal growth-plate cartilage; and (3) MMP13 as proteolytic enzyme involved in cartilage degradation. Functional cartilage chondrocytes should express high levels of positive markers but low or not detectable levels of negative markers. Assay description Chondrocytes are infected and embedded in alginate beads according to example 8. After 10 days in culture the alginate beads are treated with 55 mM sodium citrate (Cambrex) in order to recover the chondrocytes from the alginate beads and harvest RNA: Incubation medium was removed from the 6-well plates with the Vacusafe and 5 ml of 55 mM sodium citrate are added to each well and incubated for 15 rninutes at room temperature. The partially solubilised beads are gently mixed and transferred to a FALCON tube, wells are rinsed once with 2 ml of sodium citrate solution to collect the remaining beads and released cells. Tubes are laid on their side and gently mixed every 2-3 minutes until beads have completely solubilised (+/- 15 minutes). Tubes are centrifuged at 1000 rpm for 10 minutes. Supernatant is discarded and the cell pellet is re-suspended in 2 ml of sodium citrate solution and left for 5 minutes. Six mϋliliters of 155 mM sodium chloride/20 mM Hepes ph 7.4 (Cambrex) are added to each tube prior to a 5 minutes centrifugation at 210 g. Cell pellet is lysed in 180 μl SV40 lysing buffer (Promega SV40 total RNA extraction kit) and frozen at - 20°C until RNA isolation is performed with the Promega SV40 total RNA extraction kit according to the manufacturer's instructions. Purified RNA is quantified using ribogreen reagent (Molecular Probes) and yeast RNA (Ambion) as standard RNA. Purified RNA from chondrocytes transduced with control adenoviruses and cultured for 10 days in the alginate 3-dimensional culture system is used in a reverse transcription (RT) reaction. RNA is first diluted in water (Life Technologies-Invitrogen, Breda, The Netherlands) depending on the initial concentration of the sample: For RNA concentration below 25 ng/ml, samples are used undiluted, for RNA concentration between 25 and 50 ng/ml samples are diluted 1:2.5, For RNA concentration between 50 and 100 ng/ml samples are diluted 1:5 and for RNA concentration between 100 and 160 ng/ml samples are diluted 1:10. Two microliters of diluted/undiluted RNA are added to 5 μl of a reaction mix consisting of: lx Taqman RT buffer, 5 mM MgC12, 500 μM dNTPs (2.5 mM each), 2.5 μM Random hexamers, 0.4 U/μl of RNAse inhibitor and 1.25 U/μl of MultiScribe Reverse Transcriptase (all reagents purchased from Applied Biosystems). The PCR reaction is performed in a Peltier Thermal Cycler-200 (BlOzym, Landgraaf, The Netherlands) as followed: the PCR total mixture (60 μl) is incubated 10 minutes at 25°C followed by 30 minutes at 48°C, followed by 5 minutes at 95°C. Each reaction is run in parallel with a control that does not contain any RNAse inhibitor or Reverse Transcriptase. Reverse transcription is followed by a quantitative PCR for specific amplification of the selected positive and negative marker genes, GAPDH is included as the endogenous control. Five microliters of cDNA (from RT reaction) are added to 20 μl of a PCR reaction mix consisting of: IX Brilliant® SYBR® Green QPCR Master Mix (Sratagene Europe, Amsterdam, The Netherlands), 300 μM of each forward and reverse primers (table 3) (except for GAPDH and ALK-1, used at 100 μM)(Life Technologies-rnvitrogen, Breda, The Netherlands) and 300 nM of reference dye (Stratagene) (diluted 1:100 in H2O). The PCR mixture (25 μl) is incubated for 10 minutes at 95°C followed by 40 cycles: 15 seconds at 95°C followed by 1 minute at 60°C in the ABI PRISM® 7000 Sequence Detection System (Applied Biosystems, Nieuwerkerk AID Ijssel, The Netherlands). Table 3: primer sequences for the positive and negative markers:
Figure imgf000068_0001
Figure imgf000069_0001
Results are expressed for each tested marker gene as the relative expression in the sample (transduction with tested adenovirus) versus control (knock-down control adenoviruses) (Relative expression = 2ddCt, where ddCt = dCtκo sam le - dCtκo control and dCt = Ctsampie - CtGAPDH). The results for the 14 genes are shown in figure 9. The knock down of the mRNA of the respective genes results in expression of the positive markers, while the levels of the negative markers are either low or not detectable.
Control viruses Ad-BMP2; described in WO 03/018799
Ad-ALPL: Ad5 dEl/E2A adenoviruses that mediate the expression of full length liver/bone/kidney alkaline phosphatase (NP_000469). Example 12: Expression of cDNA's in human cartilage Upon identification of a modulator of cartilage synthesis, it is of the highest importance to evaluate whether the modulator is expressed in the tissue and the cells of interest. This can be achieved by measuring the RNA and/or protein levels. In recent years, RNA levels are being quantified through real time PCR technologies, whereby the RNA is first transcribed to cDNA and then the amplification of the cDNA of interest is monitored during a quantitative PCR reaction. The amplification plot and the resulting Ct value are indicators for the amount of a specific RNA transcript present in the sample. Ct values are determined in the presence or absence of the reverse transcriptase step (+RT versus -RT). An amplification signal in the -RT condition indicates the occurrence of non-specific PCR products originating from the genomic DNA. If the +RT Ct value is 3 Ct values higher than the -RT Ct value, then the investigated RNA is present in the sample. To assess whether the polypeptides of the genes identified in the above assays are expressed in human cartilage, real time PCR with specific primers for the polynucleotides ("Assay on Demand" Applied Biosystems) is performed on human cartilage total RNA (Clinomics Biosciences). 2 samples of non-osteoarthritis and 2 of osteoarthritis patients are analyzed. In short, 40 ng of RNA is transcribed to DNA using the MultiScribe Reverse Transcriptase (50 U/μl) enzyme (Applied BioSystems). The resulting cDNA is amplified with AmphTaq Gold DNA polymerase (Applied BioSystems) during 40 cycles using an ABI PRISM® 7000 Sequence Detection System. Amplification of the transcript is detected via SybrGreen which results in a fluorescent signal upon intercalation in double stranded DNA. Total RNA isolated from human cartilage is analyzed for the presence of transcripts listed in Table 4 via quantitative real time PCR. For the genes listed in Table 5 the obtained Ct values indicate that they are detected in all RNA samples. ELA1 RNA is not detected in the real time PCR analysis, underscoring the need for additional patient analysis.
Table 4: Ct values
Figure imgf000071_0001
Example 13: Identification Of Small Molecules That Inhibit TARGET Kinase Activity Compounds are screened for inhibition of the activity of the TARGETS that are kinase polypeptides. The affinity of the compounds to the polypeptides is determined in an experiment detecting changed reaction conditions after phosphorylation. The TARGET kinase polypeptides are incubated with its substrate and ATP in an appropriate buffer. The combination of these components results in the in vitro phosphorylation of the substrate. Sources of compounds include commercially available screening library, peptides in a phage display library or an antibody fragment library, and compounds that have been demonstrated to have binding affinity for a TARGET kinase. The TARGET kinase polypeptides can be prepared in a number of ways depending on whether the assay will be run using cells, cell fractions or biochemically, on purified proteins. The polypeptides can be applied as complete polypeptides or as polypeptide fragments, which still comprise TARGET kinase catalytic activity. Identification of small molecules inhibiting the activity of the TARGET kinase polypeptides is performed by measuring changes in levels of phosphorylated substrate or ATP. Since ATP is consumed during the phosphorylation of the substrate, its levels correlate with the kinase activity. Measuring ATP levels via chemiluminescent reactions therefore represents a method to measure kinase activity in vitro (Perkin Elmer). In a second type of assay, changes in the levels of phosphorylated substrate are detected with phosphospecific agents and are correlated to kinase activity. These levels are detected in solution or after immobilization of the substrate on a microtiter plate or other carrier. In solution, the phosphorylated substrate is detected via fluorescence resonance energy transfer (FRET) between the Eu labeled substrate and an APC labeled phosphospecific antibody (Perkin Elmer), via fluorescence polarization (FP) after binding of a phosphospecific antibody to the fluorescently labeled phosphorylated substrate (Panvera), via an Amplified Luminescent Proximity Homogeneous Assay (ALPHA) using the phosphorylated substrate and phosphospecific antibody, both coupled to ALPHA beads (Perkin Elmer) or using the IMAP binding reagent that specifically detects phosphate groups and thus alleviates the use of the phosphospecific antibody (Molecular Devices). Alternatively, the substrate is immobilized directly or by using biotm-stieptavidin on a microtiter plate. After immobihzation, the level of phosphorylated substrate is detected using a classical ELISA where binding of the phosphospecific antibody is either monitored via an enzyme such as horseradish peroxidase (HRP) or alkaline phospahtase (AP) which are either directly coupled to the phosphospecific antibody or are coupled to a secondary antibody. Enzymatic activity correlates to phosphorylated substrate levels. Alternatively, binding of the Eu-labeled phosphospecific antibody to the immobilized phosphorylated substrate is determined via time resolved fluorescence energy (TRF) (Perkin Elmer). In addition, the substrate can be coated on FLASH plates (Perkin Elmer) and phosphorylation of the substrate is detected using 33P labeled ATP or 5l labeled phosphospecific antibody. Small molecules are randomly screened or are preselected based upon drug class, (i.e. known kinase inhibitors), or upon virtual ligand screening (VLS) results. VLS uses virtual docking technology to test large numbers of small molecules in silico for their binding to the polypeptide of the invention. Small molecules are added to the kinase reaction and their effect on levels of phosphorylated substrate is measured with one or more of the above- described technologies. Small molecules that inhibit the kinase activity are identified and are subsequently tested at different concentrations. IC50 values are calculated from these dose response curves. Strong binders have an IC50 in the nanomolar and even picomolar range. Compounds that have an IC50 of at least 10 micromol or better (nmol to pmol) are applied in collagen II assay to check for their effect on the induction of chondrocyte anabolic stimulation.
Example 14: Ligand Screens For TARGET GPCRs. Example 14 A: Reporter Gene Screen. Mammalian cells such as Hek293 or CHO-K1 cells are either stably transfected with a plasmid harboring the luciferase gene under the control of a cAMP dependent promoter (CRE elements) or transduced with an adenovirus harboring a luciferase gene under the control of a cAMP dependent promoter. In addition reporter constructs can be used with the luciferase gene under the control of a Ca2+ dependent promoter (NF-AT elements) or a promoter that is controlled by activated NF-κB. These cells, expressing the reporter construct, are then transduced with an adenovirus harboring the cDNA of a TARGET GPCR. Forty (40) hours after transduction the cells are treated with the following: a) an agonist for the receptor and screened against a large collection of reference compounds comprising peptides (LOPAP, Sigma Aldrich), hpids (Biomol, TimTech), carbohydrates (Specs), natural compounds (Specs, TimTech), small chemical compounds (Tocris), commercially available screening libraries, and compounds that have been demonstrated to have binding affinity for a polypeptide comprising an amino acid sequence selected from the group consisting of the SEQ ID NOs of the TARGET GPCRs; or b) a large collection of reference compounds comprising peptides (LOPAP, Sigma
Aldrich), lipids (BiomoL TimTech), carbohydrates (Specs), natural compounds (Specs, TimTech), small chemical compounds (Tocris), commercially available screening libraries, and compounds that have been demonstrated to have binding affinity for a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs of the TARGET GPCRs. Compounds, which decrease the agonist induced increase in luciferase activity or the constitutive activity, are considered to be antagonists or inverse agonists for a TARGET GPCR. These compounds are screened again for verification and screened against their effect on anabolic stimulation of chondrocytes. The compounds are also screened to verify binding to the GPCR. The binding and reporter activity assays can be performed in essentially any order to screen compounds. In addition, cells expressing the NF-AT reporter gene can be transduced with an adenovirus harboring the cDNA encoding the α-subunit of G15 or chimerical Got subunits. Gi5 is a promiscuous G protein of the Gq class that couples to many different GPCRs and as such re-directs their signaling towards the release of intracellular Ca2+ stores. The chimerical G alpha subunits are members of the Gs and Gj/0 family by which the last 5 C-terminal residues are replaced by those of Gα , these chimerical G-proteins also redirect cAMP signaling to Ca2+ signaling. Example 14 B: FLIPR screen. Mammalian cells such as Hek293 or CHO-K1 cells are stably transfected with an expression plasmid construct harboring the cDNA of a TARGET GPCR. Cells are seeded, grown, and selected until sufficient stable cells can be obtained. Cells are loaded with a Ca2+ dependent fluorophore such as Fura3 or Fura4. After washing away the excess of fluorophore the cells are screened against a large collection of reference compounds comprising peptides (LOPAP, Sigma Aldrich), lipids (BiomoL TimTech), carbohydrates (Specs), natural compounds (Specs, TimTech), small chemical compounds (Tocris), commercially available screening libraries, and compounds that have been demonstrated to have binding affinity for a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ TD NOs of the TARGET GPCRs, by simultaneously adding an agonist (alternatively no agonist need be added if the constitutive activity of the receptor is used) and a compound to the cells. Activation of the receptor is measured as an almost instantaneously increase in fluorescence due to the interaction of the fluorophore and the Ca2+ that is released. Compounds that reduce or inhibit the agonist induced increase in fluorescence (or constitutive fluorescence) are considered to be antagonists or inverse agonists for the receptor they are screened against. These compounds are screened again to measure the amount of anabolic stimulation of chondrocytes as well as binding to a TARGET GPCR. Example 14 C: AequoScreen. CHO cells, stably expressing Apoaequorin are stably transfected with a plasmid construct harboring the cDNA of a TARGET GPCR. Cells are seeded, grown, and selected until sufficient stable cells can be obtained. The cells are loaded with coelenterazine, a cofactor for apoaequorin. Upon receptor activation intracellular Ca2+ stores are emptied and the aequorin will react with the coelenterazine in a light emitting process. The emitted light is a measure for receptor activation. The CHO, stable expressing both the apoaequorin and the receptor are screened against a large collection of reference compounds comprising peptides (LOPAP, Sigma Aldrich), lipids (Biomol, TimTech), carbohydrates (Specs), natural compounds (Specs, TimTech), small chemical compounds (Tocris), commercially available screening libraries, and compounds that have been demonstrated to have binding affinity for a polypeptide comprising an an ino acid sequence selected from the group consisting of SEQ ID NOS of the TARGET GPCRs, by simultaneously adding an agonist (alternatively no agonist need be added if the constitutive activity of the receptor is used) and a compound to the cells. Activation of the receptor is measured as an almost instantaneously light flash due to the interaction of the apoaequorin, coelenterazine, and the Ca2+ that is released. Compounds that reduce or inhibit the agonist induced increase in light or the constitutive activity are considered to be antagonists or inverse agonists for the receptor they are screened against. These compounds are screened again to measure the amount of anabolic stimulation of chondrocytes as well as binding to a TARGET GPCR. In addition, CHO cells stable expressing the apoaequorin gene are stably transfected with a plasmid construct harboring the cDNA encoding the α-subunit of G15 or chimerical Gα subunits. G15 is a promiscuous G protein of the Gq class that couples to many different GPCRs and as such redirects their signaling towards the release of intracellular Ca2+ stores. The chimerical G alpha subunits are members of the Gs and Gy0 family by which the last 5 C- terminal residues are replaced by those of Gαq, these chimerical G-proteins also redirect cAMP signaling to Ca2+ signaling.
Example 14 D: Screening for compounds that bind to the GPCR polypeptides (displacement experiment) Compounds are screened for binding to the TARGET, GPCR polypeptides. The affinity of the compounds to the polypeptides is determined in a displacement experiment. In brief, the GPCR polypeptides are incubated with a labeled (radiolabeled, fluorescent labeled) hgand that is known to bind to the polypeptide and with an unlabeled compound. The displacement of the labeled ligand from the polypeptide is determined by measuring the amount of labeled hgand that is still associated with the polypeptide. The amount associated with the polypeptide is plotted against the concentration of the compound to calculate IC50 values. This value reflects the binding affinity of the compound to its TARGET, i.e. the TARGET GPCR polypeptides. Strong binders have an IC50 in the nanomolar and even picomolar range. Compounds that have an IC50 of at least 10 micromol or better (nmol to pmol) are applied an anabohc stimulation of chondrocytes assay to check for their effect on osteogenesis. The TARGET GPCR polypeptides can be prepared in a number of ways depending on whether the assay are run on cells, cell fractions or biochemically, on purified proteins. Example 14 E: Screening for compounds that bind to a TARGET GPCR (generic GPCR screening assay) When a G protein receptor becomes constitutively active, it binds to a G protein (Gq, Gs, Gi, G0) and stimulates the binding of GTP to the G protein. The G protein then acts as a GTPase and slowly hydrolyses the GTP to GDP, whereby the receptor, under normal conditions, becomes deactivated. However, constitutively activated receptors continue to exchange GDP to GTP. A non-hydrolyzable analog of GTP, [35S]GTPγS, can be used to monitor enhanced binding to membranes which express constitutively activated receptors. It is reported that [35S]GTPγS can be used to monitor G protein coupling to membranes in the absence and presence of ligand. Moreover, a preferred approach is the use of a GPCR-G protein fusion protein. The strategy to generate a TARGET GPCR-G protein fusion protein is well known for those known in the art. Membranes expressing TARGET GPCR-G protein fusion protein are prepared for use in the direct identification of candidate compounds such as inverse agonist. Homogenized membranes with TARGET GPCR-G protein fusion protein are transferred in a 96-well plate. A pin-tool is used to transfer a candidate compound in each well plus [35S]GTPγS, followed by incubation on a shaker for 60 minutes at room temperature. The assay is stopped by spinning of the plates at 4000 RPM for 15 minutes at 22°C. The plates are then aspirated and radioactivity is then read. Example 14 F: Receptor Ligand Binding Study On Cell Surface The receptor is expressed in mammalian cells (Hek293, CHO, COS7) by adenoviral transducing the cells (see US 6,340,595). The cells are incubated with both labeled ligand (iodinated, tritiated, or fluorescent) and the unlabeled compound at various concentrations, ranging from 10 pM to 10 μM (3 hours at 4°C: 25 mM HEPES, 140 mM NaCl, 1 mM CaCb, 5 mM MgCl2 and 0.2% BSA, adjusted to pH 7.4). Reactions mixtures are aspirated onto PEI-treated GF/B glass filters using a cell harvester (Packard). The filters are washed twice with ice cold wash buffer (25 mM HEPES, 500 mM NaCl, 1 mM CaCl2, 5 mM MgCl2, adjusted to pH 7.4). Scintillant (MicroScint-10; 35 μl) is added to dried filters and the filters counted in a (Packard Topcount) scintillation counter. Data are analyzed and plotted using Prism software (GraphPad Software, San Diego, Calif.). Competition curves are analyzed and IC50 values calculated. If one or more data points do not fall within the sigmoidal range of the competition curve or close to the sigmoidal range the assay is repeated and concentrations of labeled hgand and unlabeled compound adapted to have more data points close to or in the sigmoidal range of the curve. Example 14 G: Receptor Ligand Binding Studies On Membrane Preparations Membranes preparations are isolated from mammalian cells (Hek293, CHO, COS7) cells over expressing the receptor is done as follows: Medium is aspirated from the transduced cells and cells are harvested in 1 x PBS by gentle scraping. Cells are pelleted (2500 rpm 5 min) and resuspended in 50 mM Tris pH 7.4 (10 x 106 cells/ml). The cell pellet is homogenized by sonicating 3 x 5 sec (UP50H; sonotrode MSI; max amplitude: 140 μm; max Sonic Power Thickness: 125W/cm2). Membrane fractions are prepared by centrifuging 20 min at maximal speed (13,000 rpm ~15,000 to 20,000g or rcf). The resulting pellet is resuspended in 500 μl 50 mM Tris pH 7.4 and sonicated again for 3 x 5 sec. The membrane fraction is isolated by centrifugation and finally resuspended in PBS. Binding competition and derivation of IC50 values are determined as described above. Example 14 H: Internalization screen (1) Activation of a GPCR-associated signal transduction pathway commonly leads to translocation of specific signal transduction molecules from the cytoplasm to the plasma membrane or from the cytoplasm to the nucleus. Norak has developed their transfluor assay based on agonist-induced translocation of receptor-β-arrestin-GFP complex from the cytosol to the plasma membrane and subsequent internalization of this complex, which occurs during receptor desensitization. A similar assay uses GFP tagged receptor instead of β-arrestin. Hek293 cells are transduced with a TARGET GPCR vector that translates for a TARGET GPCR-eGFP fusion protein. 48 hours after transduction, the cells are set to fresh serum-free medium for 60 minutes and treated with a hgand for 15, 30, 60 or 120 minutes at 37°C and 5% CO2. After indicated exposure times, cells are washed with PBS and fixed with 5% paraformaldehyde for 20 minutes at RT. GFP fluorescence is visualized with a Zeiss microscope with a digital camera. This method aims for the identification of compounds that inhibit a ligand-mediated (constitutive activity-mediated) translocation of the fusion protein to intracellular compartments.
Example 141: Internalization screen 2) Various variations on translocation assays exists using β-arrestin and β-galactosidase enzyme complementation and BRET based assays with receptor as energy donor and β- arrestin as energy acceptor. Also the use of specific receptor antibodies labeled with pH sensitive dyes are used to detect agonist induced receptor translocation to acidic lysosomes. All of the translocation assays are used for screening for both agonistic and antagonistic acting ligands. Example 14 J: Melanophore assay f Arena Pharmaceutical) The melanophore assay is based on the ability of GPCRs to alter the distribution of melanin containing melanosomes in Xenopus melanophores. The distribution of the melanosomes depends on the exogenous receptor that is either Gi/0 or Gs/q coupled. The distribution of the melanosomes (dispersed or aggregated) is easily detected by measuring light absorption. This type of assay is used for both agonist as well as antagonist compound screens.
Example 15: Identification of small molecules that inhibit protease activity. Compounds are screened for inhibition of the activity of the polypeptides of the present invention. The affinity of the compounds to the polypeptides is determined in an experiment detecting changes in levels of cleaved substrate. In brief, the polypeptides of the present invention are incubated with its substrate in an appropriate buffer. The combination of these components results in the cleavage of the substrate. The polypeptides can be applied as complete polypeptides or as polypeptide fragments, which still comprise the catalytic activity of the polypeptide of the invention. Cleavage of the substrate can be followed in several ways. In a first method, the substrate protein is heavily labeled with a fluorescent dye, like fluorescent, resulting in a complete quenching of the fluorescent signal. Cleavage of the substrate however, releases individual fragments, which contain less fluorescent labels. This results in the loss of quenching and the generation of a fluorescent signal, which correlates to the levels of cleaved substrate. Cleavage of the protein, which results in smaller peptide fragments, can also be measured using fluorescent polarization (FP). Alternatively, cleavage of the substrate can also be detected using fluorescence resonance energy transfer (FRET): a peptide substrate is labeled on both sides with either a quencher and fluorescent molecule, like DABCYL and ED ANS. Upon cleavage of the substrate both molecules are separated resulting in fluorescent signal correlating to. the levels of cleaved substrate. In addition, cleavage of a peptide substrate can also generate a new substrate for another enzymatic reaction, which is then detected via a fluorescent, chermuminescent or colorimetric method. Small molecules are randomly screened or are preselected based upon drug class, i.e. protease, or upon virtual ligand screening (VLS) results. VLS uses virtual docking technology to test large numbers of small molecules in silico for their binding to the polypeptide of the invention. Small molecules are added to the proteolytic reaction and their effect on levels of cleaved substrate is measured with the described technologies. Small molecules that inhibit the protease activity are identified and are subsequently tested at different concentrations. IC50 values are calculated from these dose response curves. Strong binders have an IC50 in the nanomolar and even picomolar range. Compounds that have an IC50 of at least 10 micromol or better (nmol to pmol) are applied in amyloid beta secretion assay to check for their effect on the beta amyloid secretion and processing.
EXAMPLE 16: Identification of small molecules that inhibit phosphodiesterase activity. Compounds are screened for inhibition of the activity of the polypeptides of the present invention. The affinity of the compounds to the polypeptides is determined in an experiment detecting changes in levels of substrate or product. In brief, the polypeptides of the present invention are incubated with its substrate in an appropriate buffer. The combination of these components results in the conversion of the substrate into its product. The polypeptides can be applied as complete polypeptides or as polypeptide fragments, which still comprise the catalytic activity of the polypeptide of the invention. Conversion of cAMP or cGMP in AMP or GMP can be followed 1) by determining the cAMP or cGMP levels using e.g. ELISA. Alpha screen technology, Time resolved fluorescent technology, IMAP 2) by deterrnining the levels of the products AMP and GMP using a colorimetric assay. The basis for the latter assay is the cleavage of cAMP or cGMP by a cyclic nucleotide phosphodiesterase. The 5 '-nucleotide released is further cleaved into the nucleoside and phosphate by the enzyme 5'-nucleotidase. The phosphate released due to enzymatic cleavage is quantified using BIOMOL GREEN™ reagent in a modified Malachite Green assay. Small molecules are randomly screened or are preselected based upon drug class, i.e.
PDE, or upon virtual ligand screening (VLS) results. VLS uses virtual docking technology to test large numbers of small molecules in silico for their binding to the poljφeptide of the invention. Small molecules are added to the PDE reaction and their effect on cyclic nucleotide levels is measured with the described technologies. Small molecules that inhibit the PDE activity are identified and are subsequently tested at different concentrations. IC50 values are calculated from these dose response curves. Strong binders have an IC50 in the nanomolar and even picomolar range. Compounds that have an IC50 of at least 10 micromol or better (nmol to pmol) are applied in assays evaluating the anabolic activity of chondrocytes. This can be achieved by determining col2αl and aggrecan levels produced by the chondrocytes. REFERENCES
Arts et al. (2003) Genome Res 10:2325-32 Chubinskaya and Kuettner (2003). Int J Biochem Cell Biol 35; 1323-40 Cortez-Retamozo et al. (2004). Cancer Res 64: 2853-7 Dorsett Y, Tuschl T. (2004). Nature Reviews Drug Discovery 3: 318-329 Harborth J, et al. (2003) Antisense Nucleic Acid Drug Dev 13(2): 83-105 Khvorova A, et al. (2003) Cell. 115(2): 209-16 Lipinsky, CA, et al. (2001). Adv Drug Deliv Rev 46: 3-26 Reynolds A, et al. (2004) Nat Biotechnol 22: 326-30 Tuschl, (2002). Nature Biotechnol. 20:446-8 Ui-Tei K, et al. (2004) Nucleic Acids Res. 32(3): 936-48 Wang L and Mu FY. (2004) Bioinformatics (In press)
Sequence Listing
<SEQ ID NO: 1; DNA; Homo sapiens>
ATGGGGACGACGGCCCCAGGGCCCATTCACCTGCTGGAGCTATGTGACCAGAAGCTCATG GAGTTTCTCTGCAACATGGATAATAAGGACTTGGTGTGGCTTGAGGAGATCCAAGAGGAG GCCGAGCGCATGTTCACCAGAGAATTCAGCAAAGAGCCAGAGCTGATGCCCAAAACACCT TCTCAGAAGAACCGACGGAAGAAGAGACGGATTTCTTATGTTCAGGATGAAAACAGAGAT CCCATCAGGAGAAGGTTATCCCGCAGAAAGTCTCGGAGCAGCCAGCTGAGCTCCCGACGC CTCCGCAGCAAGGACAGTGTAGAGAAGCTGGCTACAGTGGTCGGGGAGAACGGCTCCGTT CTGCGGCGTGTGACCCGTGCTGCGGCTGCAGCTGCCGCGGCTACCATGGCATTGGCTGCA CCTTCTTCACCCACCCCTGAGTCTCCCACGATGCTGACTAAGAAGCCCGAGGATAACCAC ACCCAGTGCCAGCTGGTGCCTGTGGTGGAGATCGGCATCAGTGAGCGCCAGAATGCTGAG CAGCATGTCACCCAGCTCATGTCCACCGAGCCTCTGCCCCGCACTCTGTCCCCGACTCCA GCTTCAGCCACAGCTCCAACCTCCCAGGGCATCCCGACATCAGATGAGGAATCAACACCT AAGAAGTCGAAGGCCAGGATACTGGAGTCCATCACAGTGAGCTCCCTGATGGCTACACCC CAGGACCCCAAGGGTCAAGGGGTCGGGACGGGGCGGTCTGCGTCTAAGCTCAGGATTGCG CAGGTCTCCCCTGGCCCACGGGACTCGCCAGCCTTTCCAGATTCTCCATGGCGGGAGCGG GTGCTGGCTCCCATCCTGCCGGATAACTTCTCCACGCCCACGGGCTCTCGCACGGACTCT CAATCGGTGCGGCACAGCCCGATCGCCCCGTCTTCCCCGAGTCCCCAAGTCTTAGCCCAG AAGTACTCTCTGGTGGCCAAACAGGAAAGTGTTGTCCGCAGGGCGAGCAGAAGGCTTGCC AAGAAGACTGCCGAAGAGCCAGCTGCCTCTGGCCGCATCATCTGTCACAGTTACCTGGAG AGGCTCCTGAATGTTGAGGTGCCCCAGAAAGTTGGTTCTGAGCAGAAGGAACCCCCCGAG GAGGCTGAGCCTGTGGCGGCAGCTGAGCCAGAGGTCCCTGAGAACAACGGAAATAACTCG TGGCCCCACAATGACACGGAGATTGCCAACAGCACACCCAACCCGAAGCCTGCAGCCAGC AGCCCGGAAACACCCTCTGCAGGGCAGCAAGAGGCCAAGACGGACCAAGCAGATGGACCC AGAGAGCCACCGCAGAGTGCCAGGAGGAAGCGCAGCTACAAGCAGGCCGTGAGTGAGCTG GACGAGGAGCAGCACCTGGAGGATGAGGAGCTGCAGCCCCCCAGGAGCAAGACCCCTTCC TCACCCTGCCCAGCCAGCAAGGTGGTACGGCCCCTCCGGACCTTTCTGCACACAGTGCAG AGGAACCAGATGCTCATGACCCCTACCTCAGCCCCACGCAGCGTCATGAAGTCCTTTATT AAGCGCAACACTCCCCTGCGCATGGACCCCAAGGAGAAGGAGCGGCAGCGCCTGGAGAAT CTGCGGCGGAAGGAGGAGGCCGAGCAGCTGCGCAGGCAGAAGGTGGAGGAGGACAAGCGG CGGCGGCTGGAGGAGGTGAAGCTGAAGCGTGAGGAACGCCTCCGCAAGGTGCTGCAGGCC CGCGAGCGGGTGGAGCAGATGAAGGAGGAGAAGAAGAAGCAGATTGAGCAGAAGTTTGCT CAGATCGACGAGAAGACTGAGAAGGCCAAGGAGGAGCGGCTGGCAGAGGAGAAGGCCAAG AAAAAGGCGGCGGCCAAGAAGATGGAGGAGGTGGAAGCACGCAGGAAGCAGGAAGAGGAT GCACGTAGGCTCAGGTGGCTGCAGCAGGAGGAGGAAGAGCGGCGGCACCAAGAGCTGCTG CAGAAGAAGAAGGAAGAGGAGCAGGAGCGGCTGCGGAAGGCGGCCGAGGCTAAGCGGCTG GCAGAGCAGCGGGAGCAGGAGCGGCGGGAGCAGGAGCGGCGGGAGCAGGAGCGGCGCGAG CAGGAGCGGCGCGAGCAGGAGCGGCGGGAGCAGGAGCGGCGCGAGCAGGAGCGACAGCTG GCAGAGCAGGAGCGTCGGCGGGAGCAGGAGCGGCTCCAAGCCGAGAGGGAGCTGCAGGAG CGGGAGAAGGCCCTGCGGCTGCAGAAGGAGCAGCTGCAGAGGGAACTGGAGGAGAAGAAG AAGAAGGAAGAGCAGCAGCGTCTGGCTGAGCGGCAGCTGCAGGAGGAGCAAGAGAAGAAA GCCAAGGAGGCAGCAGGGGCCAGCAAGGCCCTGAATGTGACTGTGGACGTGCAGTCTCCA GCTTGTACCTCATCTCCCATCACTCCGCAAGGGCACAAGGCCCCTCCCCAGATCAACCCC CACAACTACGGGATGGATCTGAATAGCGACGACTCCACCGATGATGAGGCCCATCCCCGG AAGCCCATCCCCACCTGGGCCCGAGGCACCCCGCTCAGCCAGGCTATCATTCACCAGTAC TACCAGCCACCGAACCTTCTGGAGCTCTTTGGAACCATTCTCCCACTGGACTTGGAGGAT ATCTTCAAGAAGAGCAAGCCCCGCTATCACAAGCGCACCAGCTCTGCTGTCTGGAACTCA CCGCCCCTGCAGGGCGCCAGGGTCCCCAGCAGCCTGGCCTACAGCCTGAAGAAGCACTGA GGCTGGCCTGCGGCCTTCTTGGCAGCCTCGCCTCCTGTCCATGTCTATCTGTCTGTCTGT CGGTCTGTGTCTTGGTCTGTTGCCCTCCTTCTTGGCATGCCATTGTGGAGGGCTTGGCCA GGTGTATATAAACGTCCTCTGTGCTGGGTGTTTCTGCTGCAGGTGGCAGGTGGCCCCAGG CCTGTTTGGAGGATGGGCTGGGTGGGTGGGTGGGGAAGAAATGGGCCCAGCCCCACATGG CCTGCAGACAGTGCTCTGTAAATAGTTGTTTTAATTTAGCTGAATGTTAGCATTTTAGTC TTTGGCATTTTAGCGTTTGGGAGGTAGATTAATAAAGTATATTCCTTCAAGCCTGCTGTT GATACCATGAAGACTGGGCGCCTCAGTCCCAGCCCTGTAGCTGTGTGTCTTGGGCCACCA GTGGCCTGCAGGACGAAGGTACTGTTCCATCACCTGCGGTGTGCCTCAGGATCACCAGGT GCAGGCCCCCACCCTCGGAGATGCTGCTGCAGTGAGTGGTTCCACTGCCTGGATAACCCT TGAGGAACACGTCAGTTACTGTCACGATGGGGCAGGTGGAGCTCCTTCCTATTTTTTGGG GTGCTCCCTGTTTGTAAAGGGGAGTTTGTTCATTGGGAAAGACCTGGGTCTTGACACGGC CCTGCCACTTAGTCCCCTACCCTCTCCATTCCCCAGGCTCCACCCGTGCTGCTCAAGTGC AAATGGACTTGAGAGTATTTATGTGCTGGTGAAGTATGAGGTCTGAGTAGAAAAGG
<SEQ ID NO: 2; DNA; Homo sapiens> ATGACAAACCAGGAAAAATGGGCCCACCTCAGCCCTTCGGAATTTTCCCAACTTCAGAAA TATGCTGAGTATTCTACAAAGAAATTAAAGGATGTTCTTGAAGAATTCCATGGTAATGGT GTGCTTGCAAAGTATAATCCTGAAGGGAAACAAGACATTCTTAACCAAACAATAGATTTT GAAGGTTTCAAACTATTCATGAAGACATTCCTGGAAGCCGAGCTTCCTGATGATTTCACT GCACACCTTTTCATGTCATTTAGCAACAAGTTTCCTCATTCTAGTCCAATGGTAAAAAGT AAGCCTGCTCTCCTATCAGGCGGTCTGAGAATGAATAAAGGTGCCATCACCCCTCCCCGA ACTACTTCTCCTGCAAATACGTGTTCCCCAGAAGTAATCCATCTGAAGGACATTGTCTGT TACCTGTCTCTGCTTGAAAGAGGAAGACCTGAGGATAAGCTTGAGTTTATGTTTCGCCTT TATGACACGGATGGGAATGGCTTCCTGGACAGCTCGGAGCTAGAAAATATCATCAGTCAG ATGATGCATGTTGCAGAATACCTTGAGTGGGATGTCACTGAACTTAATCCAATCCTCCAT GAAATGATGGAAGAAATTGACTATGATCATGATGGAACCGTGTCTCTGGAGGAATGGATT CAAGGAGGAATGACAACGATTCCACTTCTTGTGCTCCTGGGCTTAGAAAATAACGTGAAG GATGATGGACAGCACGTGTGGCGACTGAAGCACTTTAACAAACCTGCCTATTGCAACCTT TGCCTGAACATGCTGATTGGCGTGGGGAAGCAGGGCCTCTGCTGTTCCTTCTGCAAGTAC ACAGTCCATGAGCGCTGTGTGGCTCGAGCACCTCCCTCTTGCATCAAGACCTATGTGAAG TCCAAAAGGAACACTGATGTCATGCACCATTACTGGGTTGAAGGTAACTGCCCAACCAAG TGTGATAAGTGCCACAAAACTGTTAAATGTTACCAGGGCCTGACAGGACTGCATTGTGTT TGGTGTCAGATCACACTGCATAATAAATGTGCTTCTCATCTAAAACCTGAATGTGACTGT GGACCTTTGAAGGACCATATTTTACCACCCACAACAATCTGTCCAGTGGTACTGCAGACT CTGCCCACTTCAGGAGTTTCAGTTCCTGAGGAAAGACAATCAACAGTGAAAAAGGAAAAG AGTGGTTCCCAGCAGCCAAACAAAGTGATTGACAAGAATAAAATGCAAAGAGCCAACTCT
GTTACTGTAGATGGACAAGGCCTGCAGGTCACTCCTGTGCCTGGTACTCACCCACTTTTA GTTTTTGTGAACCCCAAAAGTGGTGGAAAACAAGGAGAACGAATTTACAGAAAATTCCAG
TATCTATTAAATCCTCGTCAGGTTTACAGTCTTTCTGGAAATGGACCAATGCCAGGGTTA AACTTTTTCCGTGATGTTCCTGACTTCAGAGTGTTAGCCTGTGGTGGAGATGGAACCGTG GGCTGGGTTTTGGATTGCATAGAAAAGGCCAATGTAGGCAAGCATCCTCCAGTTGCGATT CTGCCTCTTGGGACTGGCAATGATCTAGCAAGATGCCTGCGATGGGGAGGAGGTTACGAA GGTGAGAATCTGATGAAAATTCTAAAAGACATTGAAAACAGCACAGAAATCATGTTGGAC AGGTGGAAGTTTGAAGTCATACCTAATGACAAAGATGAGAAAGGAGACCCAGTGCCTTAC AGTATCATCAATAATTACTTTTCCATTGGCGTGGATGCCTCCATTGCACACAGATTCCAC ATCATGAGAGAAAAACACCCAGAGAAATTCAACAGTAGAATGAAGAACAAATTTTGGTAT TTTGAGTTTGGCACATCTGAAACTTTCTCAGCCACCTGCAAGAAGCTACATGAATCTGTA GAAATAGAATGTGATGGAGTACAGATAGATTTAATAAACATCTCTCTGGAAGGAATTGCT ATTTTGAATATACCAAGCATGCATGGAGGATCCAATCTTTGGGGAGAGTCTAAGAAAAGA CGAAGCCATCGACGAATAGAGAAAAAAGGGTCTGACAAAAGGACCACCGTCACAGATGCC AAAGAGTTGAAGTTTGCAAGTCAAGATCTCAGTGACCAGCTGCTGGAGGTGGTCGGCTTG GAAGGAGCCATGGAGATGGGGCAAATATACACAGGCCTGAAAAGTGCTGGCCGGCGGCTG GCTCAGTGCTCCTGCGTGGTCATCAGGACGAGCAAGTCTCTGCCAATGCAAATTGATGGG GAGCCATGGATGCAGACCCCATGCACAATAAAAATTACACACAAGAACCAAGCCCCAATG CTGATGGGCCCGCCTCCAAAAACCGGTTTATTCTGCTCCCTCGTCAAAAGGACAAGAAAC CGAAGCAAGGAATAATCCTGTGTTGTTTCACTCTTAGAAATTGAATTAGCATAATTGGGC CATGGAACACATATGCTGGAAATCTTTGAACCATTTCAAGTCTCCTGCTCATGCAAAATC ATGGAAGTGGTTTAACAGTTTTTGTTACTAAGCTAATGTAAAATTCAGCTATTAGAAAAT TTATTGTCTCAGTTTTTATAGGCATCTTTGCATGAAGAAAGCAGAAGTTTACCTGAAGTG ATACTGCATATTTTTGGTGCATGCATTCCCATAGATTTTTACATCTCCCACCCAACTCTT CCCCAATTTCCTTTTACTAACCTGTGAGAAAAACCCGTGAAACATGAAAAAGGAAATACC ATGGGAAACGTGATTCTCAGTGTGATTCCAATTATTACGAAGCACTAATCAGTAACGCTA CAATGATCATAATTGCAGATTGCTATACGTTTCCCTTTTAGAATCAGTGTATCAGTGACC TATGACTTGAGGAGAAACTTTTAATTCGAAGATTTTATTAAATAGTTGACTACAATACCT TGCTATATATACATAGTTTTTCTTCAACATCTTAACTCTTCTGAGTGGAAATAAAAATAT CAGGCATAAGGTTTTCTCATGCTGAAAAATAGAACGCGGTTTTTATTTTGCTTAGTTTTC TTTTTAATTCCAGAAATAAGTGAAAACATGTTACTTGACAGTCAAGTGTGGTAATATGGC AAGCCTTGTTCCTTTCTGCATGAGAATCTAGGAGAGAATTCATAACCACACCAATAACGA AATAGAAGTTTTAAACTATGTGCCTAATCAATGTGTTTCCCACCAAAGATTCAGAAAACA ATGCTTGAGAGAAATGGGTTAATGCATAATTAATTAAGCATTGTGGAGCAAATTTAGGGT TCCTGTGATTAATTTTGTGATGACTAAAATGCTGGAAAGCAAGTGAGTTGCCCATTAATT ATGATTAAAATTCTCACCTTTCACAGACAGACAATAAGCCAGACAACACAATCAAAGCTC AATAGATGATTTCTTGCTTTTTTCAGTCATTTATAAATATAGGTGTAATTTTTCATGGAT CAGTTAAGTACACTTGAAGGAAGTAAATGATTGTATCAGTTTATTTCTAGTATAAATGGG TACCTGTAATAATACTGAGCTCTTGGAAGCGAATCATGCATGCAATTAGCTCCCTCCTCC TCACCTACTCCACTCCCATCTTTATGACATTTCAAATGTTTATTTGGAAACAACAGCCTA GATCACTGTTGAAGGTGTTCATGGCATAGTTGGAGTCTCTGACTGTTTAAAGAAATCACA GAACAGTACTTTTCTTTTAGTGTTTCATTAAGCCTATGATGTAAAATGAAATGCTTCTGA GCAGTCTTGTAATATTGTTCATTCATATTGACCTGCATCTCATCATTGCATGTTTTATGT TTTCAAACATGCCATAAGGAAAACGAGTGCCTGAACTGCATGATTTATTAGTTTCTCTCC ACTCTGCATTAAAGTGCTAATGATTT
<SEQ ID NO: 3; DNA; Homo sapiens>
ATGACAAACCAGGAAAAATGGGCCCACCTCAGCCCTTCGGAATTTTCCCAACTTCAGAAA
TATGCTGAGTATTCTACAAAGAAATTAAAGGATGTTCTTGAAGAATTCCATGGTAATGGT GTGCTTGCAAAGTATAATCCTGAAGGGAAACAAGACATTCTTAACCAAACAATAGATTTT GAAGGTTTCAAACTATTCATGAAGACATTCCTGGAAGCCGAGCTTCCTGATGATTTCACT GCACACCTTTTCATGTCATTTAGCAACAAGTTTCCTCATTCTAGTCCAATGGTAAAAAGT AAGCCTGCTCTCCTATCAGGCGGTCTGAGAATGAATAAAGGTGCCATCACCCCTCCCCGA ACTACTTCTCCTGCAAATACGTGTTCCCCAGAAGTAATCCATCTGAAGGACATTGTCTGT TACCTGTCTCTGCTTGAAAGAGGAAGACCTGAGGATAAGCTTGAGTTTATGTTTCGCCTT TATGACACGGATGGGAATGGCTTCCTGGACAGCTCGGAGCTAGAAAATATCATCAGTCAG ATGATGCATGTTGCAGAATACCTTGAGTGGGATGTCACTGAACTTAATCCAATCCTCCAT GAAATGATGGAAGAAATTGACTATGATCATGATGGAACCGTG,a?CTCTGGAGGAATGGATT CAAGGAGGAATGACAACGATTCCACTTCTTGTGCTCCTGGGCTTAGAAAATAACGTGAAG GATGATGGACAGCACGTGTGGCGACTGAAGCACTTTAACAAACCTGCCTATTGCAACCTT TGCCTGAACATGCTGATTGGCGTGGGGAAGCAGGGCCTCTGCTGTTCCTTCTGCAAGTAC ACAGTCCATGAGCGCTGTGTGGCTCGAGCACCTCCCTCTTGCATCAAGACCTATGTGAAG TCCAAAAGGAACACTGATGTCATGCACCATTACTGGGTTGAAGGTAACTGCCCAACCAAG TGTGATAAGTGCCACAAAACTGTTAAATGTTACCAGGGCCTGACAGGACTGCATTGTGTT TGGTGTCAGATCACACTGCATAATAAATGTGCTTCTCATCTAAAACCTGAATGTGACTGT GGACCTTTGAAGGACCATATTTTACCACCCACAACAATCTGTCCAGTGGTACTGCAGACT CTGCCCACTTCAGGAGTTTCAGTTCCTGAGGAAAGACAATCAACAGTGAAAAAGGAAAAG AGTGGTTCCCAGCAGCCAAACAAAGTGATTGACAAGAATAAAATGCAAAGAGCCAACTCT GTTACTGTAGATGGACAAGGCCTGCAGGTCACTCCTGTGCCTGGTACTCACCCACTTTTA GTTTTTGTGAACCCCAAAAGTGGTGGAAAACAAGGAGAACGAATTTACAGAAAATTCCAG TATCTATTAAATCCTCGTCAGGTTTACAGTCTTTCTGGAAATGGACCAATGCCAGGGTTA AACTTTTTCCGTGATGTTCCTGACTTCAGAGTGTTAGCCTGTGGTGGAGATGGAACCGTG GGCTGGGTTTTGGATTGCATAGAAAAGGCCAATGTAGGCAAGCATCCTCCAGTTGCGATT CTGCCTCTTGGGACTGGCAATGATCTAGCAAGATGCCTGCGATGGGGAGGAGGTTACGAA GGTGAGAATCTGATGAAAATTCTAAAAGACATTGAAAACAGCACAGAAATCATGTTGGAC AGGTGGAAGTTTGAAGTCATACCTAATGACAAAGATGAGAAAGGAGACCCAGTGCCTTAC AGTATCATCAATAATTACTTTTCCATTGGCGTGGATGCCTCCATTGCACACAGATTCCAC ATCATGAGAGAAAAACACCCAGAGAAATTCAACAGTAGAATGAAGAACAAATTTTGGTAT TTTGAGTTTGGCACATCTGAAACTTTCTCAGCCACCTGCAAGAAGCTACATGAATCTGTA GAAATAGAATGTGATGGAGTACAGATAGATTTAATAAACATCTCTCTGGAAGGAATTGCT ATTTTGAATATACCAAGCATGCATGGAGGATCCAATCTTTGGGGAGAGTCTAAGAAAAGA CGAAGCCATCGACGAATAGAGAAAAAAGGGTCTGACAAAAGGACCACCGTCACAGATGCC AAAGAGTTGAAGTTTGCAAGTCAAGATCTCAGTGACCAGCTGCTGGAGGTGGTCGGCTTG GAAGGAGCCATGGAGATGGGGCAAATATACACAGGCCTGAAAAGTGCTGGCCGGCGGCTG GCTCAGTGCTCCTGCGTGGTCATCAGGACGAGCAAGTCTCTGCCAATGCAAATTGATGGG GAGCCATGGATGCAGACCCCATGCACAGTGAGTACAGAGTAGTTGATATGCTATGTCAAT CTCAGTTTTGCTTTCCTCTTTGACTAAATAACCACAATAACTGATTTTTTTCTTTATTTC TTTTCAACCTATCAGCAAATAGTCTTTTTGTTGTTGTTGTTATGTGTGTGTCAGAGCCAC TACATTTAGGCTGTAGACATTATATACCCTTGGCAATGATTTAGCTCTTGAATGTTTGTG CTAGCCTAAGTATAAATAGATCTTTTAAATAGATCAATTATAAACCATAGATCAATTATA AACTATGGAGCTAAACAAAATATTAATAAAAGTTTATCTGAAACTTTTTTGTTTATTTCA GAGCACATTATTAGAATATTATTTGCGAGAAATGCAGACCTAAGCTTATATGTGAACTTA TTTCTCAGCTTTTCTATGCCTCCATTTGGGGATTTGAGGGCTTTCTTCTCCATAAGAAAA AAATTTCTCTCCAGTTTCTACCATAATTAATTGTGTTTTCCAGAATGAGGTATTATTTAA GGCAGACACTGCCCCTCTCAAAAAAAATCAGTTTTCATTTGCATAGTGAATATTTTATTG CATTTCAAAAACATGCTAGGAACTGCTTTTGGCACTGGGAGTAGACACATGAACAAGACC AACAGTGTAATTTCCTTCAAGTTACTTACATTCCTATAATAGAGGACCGAATAAATAAAC AACTACATGATAAATATAACTTCAGACTGTGAGAGTTATTAAAAAATAAGGTGAAATGAT GATAAGAAGCTGGATTAGGTGTGGAGAATAAATACTACTTGAGATAAGGGAGACCTCTTT GAAAGGACATAGCCAAAAGCTTAGTATAAAATTAAAAAAAATAAAAAAAAAA
<SEQ ID NO: 4; DNA; Homo sapiens>
CGGGTTCCTGCTGCACTACCATGCGCCGTGCGGCCCGTGCGACTCGCCGGACCTCGCGGG CGTCCCTGTACGGAGCCCTCGGCCGGTCCTAGCAGGGATTGTCCCCATTTCCAGCTCCGG AGCGGGCGGCTGCGCCCCGCTCGTCGAGGAGCTGCGCTCACCTCAGGGGCGGGCCCCCGC CTGCGTTCGCGGCGCCAAGAAGAGTATTCCTTTCCACTTACAACTAAATAAGACCTGCTT TTGCTGGAGCTGTGCTAGGCTGAGGGAATTCCACACTGAATTTTACAAGCGGGATGGATT TCTCTAAGGCAGAAGACTGATTTTTGGAAATATGTATTTGGGAGACAGTCACGTCCTATT GAATACCTTGTGCTGGTGCTGCCATCGAAAAATCTGGTTACACTCTGGGGAGGACTGCTA CCACTGCAGAACTGAACCACTTCGGCCGTGAGATGAGTGTCCGGCCTGAGCAGGCACACC ATGAATAGATACACAACAATCAGGCAGCTCGGGGATGGAACCTACGGTTCCGTCCTGCTG GGAAGAAGCATTGAGTCTGGGGAGCTGATCGCTATTAAAAAAATGAAAAGAAAATTTTAT TCCTGGGAGGAATGCAΈGAACCTTCGGGAGGTTAAGTCTTTAAAGAAGCTCAACCATGCC AATGTAGTCAAATTAAAAGAAGTTATCAGGGAAAATGATCATCTTTATTTTATCTTCGAG TACATGAAGGAAAATCTTTACCAGCTCATTAAAGAGAGAAATAAGTTGTTTCCTGAGTCT GCTATAAGGAATATCATGTATCAGATATTACAAGGACTCGCATTTATTCACAAACACGGC TTCTTTCATCGAGACTTAAAGCCTGAGAACCTCCTCTGCATGGGACCAGAACTTGTGAAA ATTGCAGACTTTGGTTTGGCCCGAGAAATACGATCAAAACCTCCATATACAGATTATGTA TCTACCAGATGGTACAGGGCTCCAGAAGTACTCCTGAGGTCTACCAACTACAGCTCCCCC ATTGACGTCTGGGCGGTGGGCTGCATCATGGCAGAAGTTTACACCCTCAGGCCACTCTTC CCTGGAGCCAGTGAAATTGACACAATATTCAAAATTTGCCAAGTGCTGGGGACACCAAAA AAGACTGACTGGCCTGAAGGCTATCAACTTTCAAGTGCAATGAACTTCCGTTGGCCACAG TGTGTACCCAATAACTTAAAGACCTTGATTCCCAATGCTAGCAGTGAAGCAGTCCAGCTC CTGAGAGACATGCTTCAGTGGGATCCCAAGAAACGACCAACAGCTAGTCAGGCACTTCGA TATCCTTACTTCCAAGTTGGACACCCACTAGGCAGCACCACACAAAACCTTCAGGATTCA GAAAAACCACAGAAAGGCATCCTGGAAAAGGCAGGCCCACCTCCTTATATTAAGCCAGTC CCACCTGCCCAGCCACCAGCCAAGCCACACACACGAATTTCTTCACGACAGCATCAAGCC AGCCAGCCCCCTCTGCATCTCACGTACCCCTACAAAGCAGAGGTCTCCAGGACAGATCAC CCAAGCCATCTCCAGGAGGACAAGCCAAGCCCGTTGCTTTTCCCATCCCTCCACAACAAG CATCCACAGTCGAAAATCACAGCTGGCCTGGAGCACAAAAATGGTGAGATAAAGCCAAAG AGTAGGAGAAGGTGGGGTCTTATTTCCAGGTCAACAAAGGATTCAGATGATTGGGCTGAC TTGGATGACTTGGATTTCAGTCCATCCCTCAGCAGGATTGACCTGAAAAACAAGAAAAGA CAGAGTGATGACACTCTCTGCAGGTTTGAGAGTGTTTTGGACCTGAAGCCCTCTGAGCCT GTGGGCACAGGAAACAGTGCCCCCACCCAGACGTCATATCAGCGGCGAGACACGCCCACC CTGAGATCTGCAGCCAAGCAGCACTATTTGAAGCACTCTCGATACTTGCCTGGGATCAGT ATAAGAAATGGCATACTCTCGAATCCAGGCAAGGAATTTATTCCACCTAATCCATGGTCT AGTTCTGGCTTGTCTGGAAAATCTTCAGGGACAATGTCAGTAATCAGCAAAGTAAATTCA GTTGGTTCCAGCTCTACAAGTTCTAGTGGACTGACTGGAAACTATGTCCCTTCCTTTCTG AAAAAAGAAATCGGTTCTGCTATGCAGAGGGTACACCTAGCACCTATTCCAGACCCTTCC CCTGGTTATTCCTCCCTGAAGGCCATGAGACCTCATCCTGGGCGACCATTCTTCCACACC CAGCCTAGAAGCACTCCTGGGTTGATACCACGGCCTCCAGCCGCCCAGCCAGTGCATGGC CGGACAGACTGGGCTTCCAAGTACGCATCTCGGCGATGACTGTCTGCCTTGGTGATGAAT CTCTTCCTAGGGAGAAGCAGGATACTTTCCCTCAGCTGACTGGTGTTCTACCTGCAAGAT GTGCAGAGGGCATAAAAGCAAATCAACACTTTATAGTTATTCTTCTGAACTAAGACATGT CAATATTCTTTTTTAAAGTTTTTTTTTAAAATATTGATTTGAATGCAGTAGGCTTTTTTG TATAAAATTATTTTATTCTAAAACTGGGTCCCATTATTTTCTTAAACAACAGCATTTTGT ATATATGGATTATGTTTTAGCATTTTATACAGTCAACTTTGTAATGAACTTTTTAAAAAT TAATTGATTTTCCTTTGGGGTTCCAGATAATATTTTCTACAGATTTTGAAAAATGTAATA ATATTAATGCAGTATTGCAACAGGGGTGCAATTTAAGGCTATGTGATAGAGGGTTATTTA CTCAGTGTGTGCAGATATTTATGAAGTGGTGAAATTTCAAGTGTGGCTCACTAGGTACTT CAGGCCTTCTTGGACTGTTGTTAGAAAAGTGATCCTCTGCTTTTCTTAGTAGGTCATTGG TTTGATTTTTGGATACCACTCTGCTGTTCTAAAAGGACTATTATATTATATAATTCACTT TGTTTTACTTTTGTTCCCCAGATGAAAGAACTCTAAGTAAATACATTTTAAAAAATTTTT CTGACACCCTTTAATGTGGTTGCAGATCTCAGATGAAACCAAGCTTAATTATACTATGCC ATTATATTCTAATTTATTCCATTTTTGAAATCAAGTTGTATGTGTACCAATAAAAGAGAT TTCTGCTTCAAAAGGCTCTCAACATGAAGGTTAACACAGTCAATCAAACTTACATTCCTG CCAAGATGCATGGCCAAAAAACTAAGTATCAAAGCAGCAGAAGGTTTTTGATTATAGTAA CTGAGATGGAATTTTGTGCCTAGCTCAGTTCTCCAGATCTGGCTAGGAGCAGTCAATGAC TAATGTTCTGTCCTAGCCAAATTCTCAGGACAATTTGGGGAGCAGAAAGAGTTATGGCAG AGGTTCCACTCATCTACAAAGTCACAGTCACATGCCACATTTGATCTCCTAACCCTGGTG TAGTTTCTTTCAAGAGTGAGAACTTTATTTGTTGGGCAGAGGCTGTTCCATTGAGAGGAA TGTTTACAGCAGTTTCAAAAATGACAAAGTCAGTTTGGAGACAGAAAAAGACAAAAGGTC CAGTCTCATCCATCTCTATATGGTACATTTGCCTCACTTATGGTTGCCTTAAAGGCAAGA GGGAAGGTCACCATCAGTGAACGCAATGCAATCTCAACAGTGTATTGATTCATATTCTCC TAGGGCTCAAACTACTCTCTATTGGTTCCAGGATAATGACAAATTGAACCATATGTAAGT AATCTTTTATTTTTTATTTTTTTTTTGAGACAGAGTCTCACTCTGTCACCCAGGCTGGAG TGCAGTGGCGCGATCTTAGCTCTCTGCAACCTCTGCCTCCCAGGTTCAAGCCTCCTGAGT AACTGGGACTACAGGCGCCCGCCACCACGCCCAGCTAAT-ITTTTGTATTTTTAGTAGAGA CGGGGTTTCACTGTGTTAGCCAGGACGGCCTCGATCTCCTGACCTCGTGATCCACCCTCC TCCACCTCCCAAAGTACTGGGATTACAGGCATGAGCCACTGCACCCAGCCAAGTGATCAT TTTTATAGGTTAAAATGATAGGTGAAATGAATATAGACACTTTCATATGGTTCAACCTAA TGACTTGGTAAATTATTGCCTTGGTGTATTAATAATATGTTGCATTCTGAACAAATAACC ATGGCTTCCAAAGGGCCCTAACCTAAAATCGGAGAGTAATTTATGCTTTGGAGAATTTGA CTCAAATATATACTTGACCAAGCACCATGATCCCTAGGGGCATGAGAAAAGCACATAATG GATGTGGATGTGATAGGTGGTCTTTTCCTGTTAACAAGCTGGCAGCAAAGCTTCAGAAAA TATATATGCAAGCACAACTTGAAGCTGAATTCATTTCTGTATTATATTCTCAACTCGTTA TCTAAAGCATCAGAACATGTGTTTTCAGAGATGAGTCCTTTACTATAAGGTTAATATTTA TTTTCATTTTCTGTATTATATATGAAAAGTAAATTAATGTGAAACCTGGCCCAGCTTGCT GGAAAGCAGGTTTTAAATTGTAAATATTCCTTAGAGGAGCAAATGGATTGTTTAATACCA TAGTCTCAGTAATCTAGCTTATATAAGGTCATTACATTTTTTAACTGAAAAACCTAGTTA CCTGATTATTGCACATTATAAAATTGTTTTTCTAATACTTTATAGGGCCCAACTTCAGAA AATACTTCGCTTTTTTCTTTTTATGCTTTCGTTTGTTTACCAGCAAGCAACTTCCCTGGG GAAGCCAAACACATATTCATAAAAAAAATCAAGTAGCTGATGTGCAGTTGAGAAAACTAG AGGACTGAAAAAACAAATTTTAACTAGCAAATGCTGTGAATTACTCTTCCTCCCCTTCTC TGAAATGGGTAAAGGACAAATTGTGTAAAAAAACCTATGCACTATAGAAGGGAATAGTAA CCATTTCTTTTGTCTCTCTGTTTCTGTTCTGACTGAGAACCTGCAGCCATTTCTTGTTAC ATGAAAACAAAATGCTACTTGTTACCTCTATTTTTTGTTACTATACAATTATGAAATGTA ATGTAAGACACCAACAGAAATGATATACCTGTAACTGTACCTATCAGGACTATACCTCAT TTACAGTCAGAAAGCTTACTGGGATGTCAGGAAATGATACAGGGTTGGTTCTCATTTCGT GCCGAAATGAGACAGAAATTCAGTGACGAAGGTGCGTTGTAGGGGTATTGATGTGCCCCA GGTAGTGCCAGCAGAGTAGGGAAAACTGCATTTGCATAAAAACTACTCTTGACATGATTG TTCATTTTACAAAAAAATTCCATTAATTACCAAGCCCTCACCCAGCCCATGTGTGATAGG ATTTATGTAGGAAGAAACTTGATTTTCAAATAATTTTTTAAATGTATCTCTTGCCTAAAG GACTATATACATCTAATAAAGTAACACTGTGTCATCTTCTGGAGTTATCAAAAATTGTAT ACAATCAAGACAACACAAGAATTATTTTATTTTTGAGTGCAAATACAGGTACTGTTGGAG TTGATGGGCACCATGCTTTCTCATGAAGTAGCATTTCCCTACCATCAAGCCATTGTTTTG TGCCATTCAGGAGAGGAAAAAAAGGAATTTATGCTGTACATTTCAGTTCAGTGTATGACC AAAAGCAATATGTTTATAAGAAGATGTTTGACATACTAATTATTTTATATCATTTAAACC ATACTGTAGCAACATAATATATGGAGCTAATTTGTAGAATTATTTTTACGATTTCCAAAC AAATGTACTGTACTGTTATATAATTTATTGTGAGGACCTTCTCATGGAAGCCATTAGGAA AACAAACTAGAGGTAAATATCACATTAATCTGTATTATCAATTTCTCATAGACACTGTGC TAATGTGAATTTTAAATGACCTGCATCAAGTCTTCTGATCTCAGATAACTCAGTACAGAT AGCAATTAGTCAGCTGATTTGATTACAATGGAGTAACCGACAATATATTTATTTATAAAG CACATATTCATAATAACGAGAAGAATTCAGAAAACCACTTAAGCAAGACCCTTCTGAAAT AAAAAATGTTGCTTTTTAAATAGTTTGTCCTAAGGTGTTTAAAACATGTCAACCTTATGT AAGGAAAAATTTCCTGGTCCAAATAAAGTTGAAGTTTAAGAAAAATTG
<SEQ ID NO: 5; DNA; Homo sapiens> GCGCCGTGCGGCCCGTGCGACTCGCCGGACCTCGCGGGCGTCCCTGTACGGAGCCCTCGG CCGGTCCTAGCAGGGATTGTCCCCATTTCCAGCTCCGGAGCGGGCGGCTGCGCCCCGCTC GTCGAGGAGCTGCGCTCACCTCAGGGGCGGGCCCCCGCCTGCGTTCGCGGCGCCAGCAGA AGACTGATTTTTGGAAATATGTATTTGGGAGACAGTCACGTCCTATTGAATACCTTGTGC TGGTGCTGCCATCGAAAAATCTGGTTACACTCTGGGGAGGACTGCTACCACTGCAGAACT GAACCACTTCGGCCGTGAGATGAGTGTCCGGCCTGAGCAGGCACACCATGAATAGATACA CAACAATCAGGCAGCTCGGGGATGGAACCTACGGTTCCGTCCTGCTGGGAAGAAGCATTG AGTCTGGGGAGCTGATCGCTATTAAAAAAATGAAAAGAAAATTTTATTCCTGGGAGGAAT GCATGAACCTTCGGGAGGTTAAGTCTTTAAAGAAGCTCAACCATGCCAATGTAGTCAAAT TAAAAGAAGTTATCAGGGAAAATGATCATCTTTATTTTATCTTCGAGTACATGAAGGAAA ATCTTTACCAGCTCATTAAAGAGAGAAATAAGTTGTTTCCTGAGTCTGCTATAAGGAATA TCATGTATCAGATATTACAAGGACTCGCATTTATTCACAAACACGGCTTCTTTCATCGAG ACTTAAAGCCTGAGAACCTCCTCTGCATGGGACCAGAACTTGTGAAAATTGCAGACTTTG GTTTGGCCCGAGAAATACGATCAAAACCTCCATATACAGATTATGTATCTACCAGATGGT ACAGGGCTCCAGAAGTACTCCTGAGGTCTACCAACTACAGCTCCCCCATTGACGTCTGGG. CGGTGGGCTGCATCATGGCAGAAGTTTACACCCTCAGGCCACTCTTCCCTGGAGCCAGTG AAATTGACACAATATTCAAAATTTGCCAAGTGCTGGGGACACCAAAAAAGACTGACTGGC CTGAAGGCTATCAACTTTCAAGTGCAATGAACTTCCGTTGGCCACAGTGTGTACCCAATA ACTTAAAGACCTTGATTCCCAATGCTAGCAGTGAAGCAGTCCAGCTCCTGAGAGACATGC TTCAGTGGGATCCCAAGAAACGACCAACAGCTAGTCAGGCACTTCGATATCCTTACTTCC AAGTTGGACACCCACTAGGCAGCACCACACAAAACCTTCAGGATTCAGAAAAACCACAGA AAGGCATCCTGGAAAAGGCAGGCCCACCTCCTTATATTAAGCCAGTCCCACCTGCCCAGC CACCAGCCAAGCCACACACACGAATTTCTTCACGACAGCATCAAGCCAGCCAGCCCCCTC TGCATCTCACGTACCCCTACAAAGCAGAGGTCTCCAGGACAGATCACCCAAGCCATCTCC AGGAGGACAAGCCAAGCCCGTTGCTTTTCCCATCCCTCCACAACAAGCATCCACAGTCGA AAATCACAGCTGGCCTGGAGCACAAAAATGGTGAGATAAAGCCAAAGAGTAGGAGAAGGT GGGGTCTTATTTCCAGGTCAACAAAGGATTCAGATGATTGGGCTGACTTGGATGACTTGG ATTTCAGTCCATCCCTCAGCAGGATTGACCTGAAAAACAAGAAAAGACAGAGTGATGACA CTCTCTGCAGGTTTGAGAGTGTTTTGGACCTGAAGCCCTCTGAGCCTGTGGGCACAGGAA ACAGTGCCCCCACCCAGACGTCATATCAGCGGCGAGACACGCCCACCCTGAGATCTGCAG CCAAGCAGCACTATTTGAAGCACTCTCGATACTTGCCTGGGATCAGTATAAGAAATGGCA TACTCTCGAATCCAGGCAAGGAATTTATTCCACCTAATCCATGGTCTAGTTCTGGCTTGT CTGGAAAATCTTCAGGGACAATGTCAGTAATCAGCAAAGTAAATTCAGTTGGTTCCAGCT CTACAAGTTCTAGTGGACTGACTGGAAACTATGTCCCTTCCTTTCTGAAAAAAGAAATCG GTTCTGCTATGCAGAGGGTACACCTAGCACCTATTCCAGACCCTTCCCCTGGTTATTCCT CCCTGAAGGCCATGAGACCTCATCCTGGGCGACCATTCTTCCACACCCAGCCTAGAAGCA CTCCTGGGTTGATACCACGGCCTCCAGCCGCCCAGCCAGTGCATGGCCGGACAGACTGGG CTTCCAAGTACGCATCTCGGCGATGACTGTCTGCCTTGGTGATGAATCTCTTCCTAGGGA GAAGCAGGATACTTTCCCTCAGCTGACTGGTGTTCTACCTGCAAGATGTGCAGAGGGCAT AAAAGCAAATCAACACTTTATAGTTATTCTTCTGAACTAAGACATGTCAATATTCTTTTT TAAAGTTTTTTTTTAAAATATTGATTTGAATGCAGTAGGCTTTTTTGTATAAAATTATTT TATTCTAAAACTGGGTCCCATTATTTTCTTAAACAACAGCATTTTGTATATATGGATTAT GTTTTAGCATTTTATACAGTCAACTTTGTAATGAACTTTTTAAAAATTAATTGATTTTCC TTTGGGGTTCCAGATAATATTTTCTACAGATTTTGAAAAATGTAATAATATTAATGCAGT ATTGCAACAGGGGTGCAATTTAAGGCTATGTGATAGAGGGTTATTTACTCAGTGTGTGCA GATATTTATGAAGTGGTGAAATTTCAAGTGTGGCTCACTAGGTACTTCAGGCCTTCTTGG ACTGTTGTTAGAAAAGTGATCCTCTGCTTTTCTTAGTAGGTCATTGGTTTGATTTTTGGA TACCACTCTGCTGTTCTAAAAGGACTATTATATTATATAATTCACTTTGTTTTACTTTTG TTCCCCAGATGAAAGAACTCTAAGTAAATACATTTTAAAAAATTTTTCTGACACCCTTTA ATGTGGTTGCAGATCTCAGATGAAACCAAGCTTAATTATACTATGCCATTATATTCTAAT TTATTCCATTTTTGAAATCAAGTTGTATGTGTACCAATAAAAGAGATTTCTGCTTCAAAA GGCTCTCAACATGAAGGTTAACACAGTCAATCAAACTTACATTCCTGCCAAGATGCATGG CCAAAAAACTAAGTATCAAAGCAGCAGAAGGTTTTTGATTATAGTAACTGAGATGGAATT TTGTGCCTAGCTCAGTTCTCCAGATCTGGCTAGGAGCAGTCAATGACTAATGTTCTGTCC TAGCCAAATTCTCAGGACAATTTGGGGAGCAGAAAGAGTTATGGCAGAGGTTCCACTCAT CTACAAAGTCACAGTCACATGCCACATTTGATCTCCTAACCCTGGTGTAGTTTCTTTCAA GAGTGAGAACTTTATTTGTTGGGCAGAGGCTGTTCCATTGAGAGGAATGTTTACAGCAGT TTCAAAAATGACAAAGTCAGTTTGGAGACAGAAAAAGACAAAAGGTCCAGTCTCATCCAT CTCTATATGGTACATTTGCCTCACTTATGGTTGCCTTAAAGGCAAGAGGGAAGGTCACCA TCAGTGAACGCAATGCAATCTCAACAGTGTATTGATTCATATTCTCCTAGGGCTCAAACT ACTCTCTATTGGTTCCAGGATAATGACAAATTGAACCATATGTAAGTAATCTTTTATTTT TTATTTTTTTTTTGAGACAGAGTCTCACTCTGTCACCCAGGCTGGAGTGCAGTGGCGCGA TCTTAGCTCTCTGCAACCTCTGCCTCCCAGGTTCAAGCCTCCTGAGTAACTGGGACTACA GGCGCCCGCCACCACGCCCAGCTAATTTTTTGTATTTTTAGTAGAGACGGGGTTTCACTG TGTTAGCCAGGACGGCCTCGATCTCCTGACCTCGTGATCCACCCTCCTCCACCTCCCAAA GTACTGGGATTACAGGCATGAGCCACTGCACCCAGCCAAGTGATCATTTTTATAGGTTAA AATGATAGGTGAAATGAATATAGACACTTTCATATGGTTCAACCTAATGACTTGGTAAAT ΓATTGCCTTGGTGTATTAATAATATGTTGCATTCTGAACAAATAACCATGGCTTCCAAAG GGCCCTAACCTAAAATCGGAGAGTAATTTATGCTTTGGAGAATTTGACTCAAATATATAC TTGACCAAGCACCATGATCCCTAGGGGCATGAGAAAAGCACATAATGGATGTGGATGTGA TAGGTGGTCTTTTCCTGTTAACAAGCTGGCAGCAAAGCTTCAGAAAATATATATGCAAGC ACAACTTGAAGCTGAATTCATTTCTGTATTATATTCTCAACTCGTTATCTAAAGCATCAG AACATGTGTTTTCAGAGATGAGTCCTTTACTATAAGGTTAATATTTATTTTCATTTTCTG TATTATATATGAAAAGTAAATTAATGTGAAACCTGGCCCAGCTTGCTGGAAAGCAGGTTT TAAATTGTAAATATTCCTTAGAGGAGCAAATGGATTGTTTAATACCATAGTCTCAGTAAT CTAGCTTATATAAGGTCATTACATTTTTTAACTGAAAAACCTAGTTACCTGATTATTGCA CATTATAAAATTGTTTTTCTAATACTTTATAGGGCCCAACTTCAGAAAATACTTCGCTTT TTTCTTTTTATGCTTTCGTTTGTTTACCAGCAAGCAACTTCCCTGGGGAAGCCAAACACA TATTCATAAAAAAAATCAAGTAGCTGATGTGCAGTTGAGAAAACTAGAGGACTGAAAAAA CAAATTTTAACTAGCAAATGCTGTGAATTACTCTTCCTCCCCTTCTCTGAAATGGGTAAA GGACAAATTGTGTAAAAAAACCTATGCACTATAGAAGGGAATAGTAACCATTTCTTTTGT CTCTCTGTTTCTGTTCTGACTGAGAACCTGCAGCCATTTCTTGTTACATGAAAACAAAAT GCTACTTGTTACCTCTATTTTTTGTTACTATACAATTATGAAATGTAATGTAAGACACCA ACAGAAATGATATACCTGTAACTGTACCTATCAGGACTATACCTCATTTACAGTCAGAAA GCTTACTGGGATGTCAGGAAATGATACAGGGTTGGTTCTCATTTCGTGCCGAAATGAGAC AGAAATTCAGTGACGAAGGTGCGTTGTAGGGGTATTGATGTGCCCCAGGTAGTGCCAGCA GAGTAGGGAAAACTGCATTTGCATAAAAACTACTCTTGACATGATTGTTCATTTTACAAA AAAATTCCATTAATTACCAAGCCCTCACCCAGCCCATGTGTGATAGGATTTATGTAGGAA GAAACTTGATTTTCAAATAATTTTTTAAATGTATCTCTTGCCTAAAGGACTATATACATC TAATAAAGTAACACTGTGTCATCTTCTGGAGTTATCAAAAATTGTATACAATCAAGACAA CACAAGAATTATTTTATTTTTGAGTGCAAATACAGGTACTGTTGGAGTTGATGGGCACCA TGCTTTCTCATGAAGTAGCATTTCCCTACCATCAAGCCATTGTTTTGTGCCATTCAGGAG AGGAAAAAAAGGAATTTATGCTGTACATTTCAGTTCAGTGTATGACCAAAAGCAATATGT TTATAAGAAGATGTTTGACATACTAATTATTTTATATCATTTAAACCATACTGTAGCAAC ATAATATATGGAGCTAATTTGTAGAATTATTTTTACGATTTCCAAACAAATGTACTGTAC TGTTATATAATTTATTGTGAGGACCTTCTCATGGAAGCCATTAGGAAAACAAACTAGAGG TAAATATCACATTAATCTGTATTATCAATTTCTCATAGACACTGTGCTAATGTGAATTTT AAATGACCTGCATCAAGTCTTCTGATCTCAGATAACTCAGTACAGATAGCAATTAGTCAG CTGATTTGATTACAATGGAGTAACCGACAATATATTTATTTATAAAGCACATATTCATAA TAACGAGAAGAATTCAGAAAACCACTTAAGCAAGACCCTTCTGAAATAAAAAATGTTGCT TTTTAAATAGTTTGTCCTAAGGTGTTTAAAACATGTCAACCTTATGTAAGGAAAAATTTC CTGGTCCAAATAAAGTTGAAGTTTAAGAAAAATTG
<SEQ ID NO: 6; DNA; Homo sapiens>
ATGATCTGCTGCAGTGCTCTGAGCCCTAGGATTCATCTTTCTTTTCACCGTAGCCTGACT
GGCATTGTATTAGCAAACTCATCACTAGACATCGTACTACACGACACGTACTACGTTGTA GCCCACTGCGGGGGAAATGTTAGGCGCCTGCATTGCGGTGGCCCCGCGTCCCGGGAGCGC ACAGCAATGCAGGCGCTTAACATTACCCCGGAGCAGTTCTCTCGGCTGCTGCGGGACCAC AACCTGACGCGGGAGCAGTTCATCGCTCTGTACCGGCTGCGACCGCTCGTCTACACCCCA GAGCTGCCGGGACGCGCCAAGCTGGCCCTCGTGCTCACCGGCGTGCTCATCTTCGCCCTG GCACTCTTTGGCAATGCTCTGGTGTTCTACGTGGTGACCCGCAGCAAGGCCATGCGCACC GTCACCAACATCTTTATCTGCTCCTTGGCGCTCAGTGACCTGCTCATCACCTTCTTCTGC ATTCCCGTCACCATGCTCCAGAACATTTCCGACAACTGGCTGGGGGGTGCTTTCATTTGC AAGATGGTGCCATTTGTCCAGTCTACCGCTGTTGTGACAGAAATCCTCACTATGACCTGC ATTGCTGTGGAAAGGCACCAGGGACTTGTGCATCCTTTTAAAATGAAGTGGCAATACACC AACCGAAGGGCTTTCACAATGCTAGGTGTGGTCTGGCTGGTGGCAGTCATCGTAGGATCA CCCATGTGGCACGTGCAACAACTTGAGATCAAATATGACTTCCTATATGAAAAGGAACAC ATCTGCTGCTTAGAAGAGTGGACCAGCCCTGTGCACCAGAAGATCTACACCACCTTCATC CTGTCATCCTCTTCCTCCTGCCTCTTATGGAAGAAGAAACGAGCTGTCATTATGATGGTG ACAGTGGTGGCTCTCTTTGCTGTGTGCTGGGCACCATTCCATGTTGTCCATATGATGATT GAATACAGTAATTTTGAAAAGGAATATGATGATGTCACAATCAAGATGATTTTTGCTATC GTGCAAATTATTGGATTTTCCAACTCCATCTGTAATCCCATTGTCTATGCATTTATGAAT GAAAACTTCAAAAAAAATGTTTTGTCTGCAGTTTGTTATTGCATAGTAAATAAAACCTTC TCTCCAGCACAAAGGCATGGAAATTCAGGAATTACAATGATGCGGAAGAAAGCAAAGTTT T£CCTCAGAGAGAATCCAGTGGAGGAAACCAAAGGAGAAGCATTCAGTGAΕGGCAACATT GAAGTCAAATTGTGTGAACAGACAGAGGAGAAGAAAAAGCTCAAACGACATCTTGCTCTC TTTAGGTCTGAACTGGCTGAGAATTCTCCTTTAGACAGTGGGCATTAA
<SEQ ID NO: 7; DNA; Homo sapiens>
ATGCAGGCGCTTAACATTACCCCGGAGCAGTTCTCTCGGCTGCTGCGGGACCACAACCTG
ACGCGGGAGCAGTTCATCGCAGTTCATCGCCTGCGACCGCTCGTCTACACCCCAGAGCTG CCGGGACGCGCCAAGCTGGCCCTCGTGCTCACCGGCGTGCTCATCTTCGCCCTGGCGCTC TTTGGCAATGCTCTGGTGTTCTACGTGGTGACCCGCAGCAAGGCCATGCGCACCGTCACC AACATCTTTATCTGCTCCTTGGCGCTCAGTGACCTGCTCATCACCTTCTTCTGCATTCCC GTCACCATGCTCCAGAACATTTCCGACAACTGGCTGGGGGGTGCTTTCATTTGCAAGATG GTGCCATTTGTCCAGTCTACCGCTGTTGTGACAGAAATCCTCACTATGACCTGCATTGCT GTGGAAAGGCACCAGGGACTTGTGCATCCTTTTAAAATGAAGTGGCAATACACCAACCGA AGGGCTTTCACAATGCTAGGTGTGGTCTGGCTGGTGGCAGTCATCGTAGGATCACCCATG TGGCACGTGCAACAACTTGAGATCAAATATGACTTCCTATATGAAAAGGAACACATCTGC TGCTTAGAAGAGTGGACCAGCCCTGTGCACCAGAAGATCTACACCACCTTCATCCTTGTC ATCCTCTTCCTCCTGCCTCTTATGGTGATGCTTATTCTGTACAGTAAAATTGGTTATGAA CTTTGGATAAAGAAAAGAGTTGGGGATGGTTCAGTGCTTCGAACTATTCATGGAAAAGAA ATGTCCAAAATAGCCAGGAAGAAGAAACGAGCTGTCATTATGATGGTGACAGTGGTGGCT CTCTTTGCTGTGTGCTGGGCACCATTCCATGTTGTCCATATGATGATTGAATACAGTAAT TTTGAAAAGGAATATGATGATGTCACAATCAAGATGATTTTTGCTATCGTGCAAATTATT GGATTTTCCAACTCCATCTGTAATCCCATTGTCTATGCATTTATGAATGAAAACTTCAAA AAAAATGTTTTGTCTGCAGTTTGTTATTGCATAGTAAATAAAACCTTCTCTCCAGCACAA AGGCATGGAAATTCAGGAATTACAATGATGCGGAAGAAAGCAAAGTTTTCCCTCAGAGAG AATCCAGTGGAGGAAACCAAAGGAGAAGCATTCAGTGATGGCAACATTGAAGTCAAATTG TGTGAACAGACAGAGGAGAAGAAAAAGCTCAAACGACATCTTGCTCTCTTTAGGTCTGAA CTGGCTGAGAATTCTCCTTTAGACAGTGGGCATTAA
<SEQ ID NO: 8; DNA; Homo sapiens>
AGTTGAGGGATTGACACAAATGGTCAGGCGGCGGCGGCGGAGAAGGAGGCGGAGGCGCAG GGGGGAGCCGAGCCCGCTGGGCTGCGGAGAGTTGCGCTCTCTACGGGGCCGCGGCCACTA GCGCGGCGCCGCCAGCCGGGAGCCAGCGAGCCGAGGGCCAGGAAGGCGGGACACGACCCC GGCGCGCCCTAGCCACCCGGGTTCTCCCCGCCGCCCGCGCTTCATGAATCGCAAGTTTCC GCGGCGGCGGCGGCTGCGGTACGCAGAACAGGAGCCGGGGGAGCGGGCCGAAAGCGGCTT GGGCTCGACGGAGGGCACCCGCGCAGAGGTCTCCCTGGCCGCAGGGGGAGCCGCCGCCGG CCGTGCCCCTGGCAGCCCCAGCGGAGCGGCGCCAAGAGAGGAGCCGAGAAAGTATGGCTG AGGAGGAGGCGCCTAAGAAGTCCCGGGCCGCCGGCGGTGGCGCGAGCTGGGAACTTTGTG CCGGGGCGCTCTCGGCCCGGCTGGCGGAGGAGGGCAGCGGGGACGCCGGTGGCCGCCGCC GCCCGCCAGTTGACCCCCGGCGATTGGCGCGCCAGCTGCTGCTGCTGCTTTGGCTGCTGG AGGCTCCGCTGCTGCTGGGGGTCCGGGCCCAGGCGGCGGGCCAGGGGCCAGGCCAGGGGC CCGGGCCGGGGCAGCAACCGCCGCCGCCGCCTCAGCAGCAACAGAGCGGGCAGCAGTACA ACGGCGAGCGGGGCATCTCCGTCCCGGACCACGGCTATTGCCAGCCCATCTCCATCCCGC TGTGCACGGACATCGCGTACAACCAGACCATCATGCCCAACCTGCTGGGCCACACGAACC AGGAGGACGCGGGCCTGGAGGTGCACCAGTTCTACCCTCTAGTGAAAGTGCAGTGTTCCG CTGAGCTCAAGTTCTTCCTGTGCTCCATGTACGCGCCCGTGTGCACCGTGCTAGAGCAGG CGCTGCCGCCCTGCCGCTCCCTGTGCGAGCGCGCGCGCCAGGGCTGCGAGGCGCTCATGA ACAAGTTCGGCTTCCAGTGGCCAGACACGCTCAAGTGTGAGAAGTTCCCGGTGCACGGCG CCGGCGAGCTGTGCGTGGGCCAGAACACGTCCGACAAGGGCACCCCGACGCCCTCGCTGC TTCCAGAGTTCTGGACCAGCAACCCTCAGCACGGCGGCGGAGGGCACCGTGGCGGCTTCC CGGGGGGCGCCGGCGCGTCGGAGCGAGGCAAGTTCTCCTGCCCGCGCGCCCTCAAGGTGC CCTCCTACCTCAACTACCACTTCCTGGGGGAGAAGGACTGCGGCGCACCTTGTGAGCCGA CCAAGGTGTATGGGCTCATGTACTTCGGGCCCGAGGAGCTGCGCTTCTCGCGCACCTGGA TTGGCATTTGGTCAGTGCTGTGCTGCGCCTCCACGCTCTTCACGGTGCTTACGTACCTGG TGGACATGCGGCGCTTCAGCTACCCGGAGCGGCCCATCATCTTCTTGTCCGGCTGTTACA CGGCCGTGGCCGTGGCCTACATCGCCGGCTTCCTCCTGGAAGACCGAGTGGTGTGTAATG ACAAGTTCGCCGAGGACGGGGCACGCACTGTGGCGCAGGGCACCAAGAAGGAGGGCTGCA CCATCCTCTTCATGATGCTCTACTTCTTCAGCATGGCCAGCTCCATCTGGTGGGTGATCC TGTCGCTCACCTGGTTCCTGGCGGCTGGCATGAAGTGGGGCCACGAGGCCATCGAAGCCA ACTCACAGTATTTTCACCTGGCCGCCTGGGCTGTGCCGGCCATCAAGACCATCACCATCC TGGCGCTGGGCCAGGTGGACGGCGATGTGCTGAGCGGAGTGTGCTTCGTGGGGCTTAACA ACGTGGACGCGCTGCGTGGCTTCGTGCTGGCGCCCCTCTTCGTGTACCTGTTTATCGGCA CGTCCTTTCTGCTGGCCGGCTTTGTGTCGCTCTTCCGCATCCGCACCATCATGAAGCACG ATGGCACCAAGACCGAGAAGCTGGAGAAGCTCATGGTGCGCATTGGCGTCTTCAGCGTGC TGTACACTGTGCCAGCCACCATCGTCATCGCCTGCTACTTCTACGAGCAGGCCTTCCGGG ACCAGTGGGAACGCAGCTGGGTGGCCCAGAGCTGCAAGAGCTACGCTATCCCCTGCCCTC ACCTCCAGGCGGGCGGAGGCGCCCCGCCGCACCCGCCCATGAGCCCGGACTTCACGGTCT TCATGATTAAGTACCTTATGACGCTGATCGTGGGCATCACGTCGGGCTTCTGGATCTGGT CCGGCAAGACCCTCAACTCCTGGAGGAAGTTCTACACGAGGCTCACCAACAGCAAACAAG GGGAGACTACAGTCTGAGACCCGGGGCTCAGCCCATGCCCAGGCCTCGGCCGGGGCGCAG CGATCCCCCAAAGCCAGCGCCGTGGAGTTCGTGCCAATCCTGACATCTCGAGGTTTCCTC ACTAGACAACTCTCTTTCGCAGGCTCCTTTGAACAACTCAGCTCCTGCAAAAGCTTCCGT CCCTGAGGCAAAAGGACACGAGGGCCCGACTGCCAGAGGGAGGATGGACAGACCTCTTGC CCTCACACTCTGGTACCAGGACTGTTCGCTTTTATGATTGTAAATAGCCTGTGTAAGATT TTTGTAAGTATATTTGTATTTAAATGACGACCGATCACGCGTTTTTCTTTTTCAAAAGTT TTTAATTATTTAGGGCGGTTTAACCATTTGAGGCTTTTCCTTCTTGCCCTTTTCGGAGTA TTGCAAAGGAGCTAAAACTGGTGTGCAACCGCACAGCGCTCCTGGTCGTCCTCGCGCGCC TCTCCCTACCACGGGTGCTCGGGACGGCTGGGCGCCAGCTCCGGGGCGAGTTCAGCACTG CGGGGTGCGACTAGGGCTGCGCTGCCAGGGTCACTTCCCGCCTCCTCCTTTTGCCCCCTC CCCCTCCTTCTGTCCCCTCCCTTTCTTTCCTGGCTTGAGGTAGGGGCTCTTAAGGTACAG AACTCCACAAACCTTCCAAATCTGGAGGAGGGCCCCCATACATTACAATTCCTCCCTTGC TCGGCGGTGGATTGCGAAGGCCCGTCCCTTCGACTTCCTGAAGCTGGATTTTTAACTGTC CAGAACTTTCCTCCAACTTCATGGGGGCCCACGGGTGTGGGCGCTGGCAGTCTCAGCCTC CCTCCACGGTCACCTTCAACGCCCAGACACTCCCTTCTCCCACCTTAGTTGGTTACAGGG TGAGTGAGATAACCAATGCCAAACTTTTTGAAGTCTAATTTTTGAGGGGTGAGCTCATTT CATTCTCTAGTGTCTAAAACCTGGTATGGGTTTGGCCAGCGTCATGGAAAGATGTGGTTA CTGAGATTTGGGAAGAAGCATGAAGCTTTGTGTGGGTTGGAAGAGACTGAAGATATGGGT TATAAAATGTTAATTCTAATTGCATACGGATGCCTGGCAACCTTGCCTTTGAGAATGAGA CAGCCTGCGCTTAGATTTTACCGGTCTGTAAAATGGAAATGTTGAGGTCACCTGGAAAGC TTTGTTAAGGAGTTGATGTTTGCTTTCCTTAACAAGACAGCAAAACGTAAACAGAAATTG AAAACTTGAAGGATATTTCAGTGTCATGGACTTCCTCAAAATGAAGTGCTATTTTCTTAT TTTTAATCAAATAACTAGACATATATCAGAAACTTTAAAATGTAAAAGTTGTACACTTTC AACATTTTATTACGATTATTATTCAGCAGCACATTCTGAGGGGGGAACAATTCACACCAC CAATAATAACCTGGTAAGATTTCAGGAGGTAAAGAAGGTGGAATAATTGACGGGGAGATA GCGCCTGAAATAAACAAAATATGGGCATGCATGCTAAAGGGAAAATGTGTGCAGGTCTAC TGCATTAAATCCTGTGTGCTCCTCTTTTGGATTTACAGAAATGTGTCAAATGTAAATCTT TCAAAGCCATTTAAAAATATTCACTTTAGTTCTCTGTGAAGAAGAGGAGAAAAGCAATCC TCCTGATTGTATTGTTTTAAACTTTAAGAATTTATCAAAATGCCGGTACTTAGGACCTAA ATTTATCTATGTCTGTCATACGCTAAAATGATATTGGTCTTTGAATTTGGTATACATTTA TTCTGTTCACTATCACAAAATCATCTATATTTATAGAGGAATAGAAGTTTATATATATAT AATACCATATTTTTAATTTCACAAATAAAAAATTCAAAGTTTTGTACAAAATTATATGGA TTTTGTGCCTGAAAATAATAGAGCTTGAGCTGTCTGAACTATTTTACATTTTATGGTGTC TCATAGCCAATCCCACAGTGTAAAAATTCA
<SEQ ID NO: 9; DNA; Homo sapiens>
TTGGTCCAAGCAAGAAGGCAGTGGTCTACTCCATCGGCAACATGCTGGTCCTTTATGGAC ACAGCACCCAGGACCTTCCGGAAACCAATGCCCGCGTAGTCGGAGGGACTGAGGCCGGGA GGAATTCCTGGCCCTCTCAGATTTCCCTCCAGTACCGGTCTGGAGGTTCCCGGTATCACA CCTGTGGAGGGACCCTTATCAGACAGAACTGGGTGATGACAGCTGCTCACTGCGTGGATT ACCAGAAGACTTTCCGCGTGGTGGCTGGAGACCATAACCTGAGCCAGAATGATGGCACTG AGCAGTACGTGAGTGTGCAGAAGATCGTGGTGCATCCATACTGGAACAGCGATAACGTGG CTGCCGGCTATGACATCGCCCTGCTGCGCCTGGCCCAGAGCGTTACCCTCAATAGCTATG TCCAGCTGGGTGTTCTGCCCCAGGAGGGAGCCATCCTGGCTAACAACAGTCCCTGCTACA TCACAGGCTGGGGCAAGACCAAGACCAATGGGCAGCTGGCCCAGACCCTGCAGCAGGCTT ACCTGCCCTCTGTGGACTACGCCATCTGCTCCAGCTCCTCCTACTGGGGCTCCACTGTGA AGAACACCATGGTGTGTGCTGGTGGAGATGGAGTTCGCTCTGGATGCCAGGGTGACTCTG GGGGCCCCCTCCATTGCTTGGTGAATGGCAAGTATTCTGTCCATGGAGTGACCAGCTTTG TGTCCAGCCGGGGCTGTAATGTCTCCAGGAAGCCTACAGTCTTCACCCAGGTCTCTGCTT ACATCTCCTGGATAAATAATGTCATCGCCTCCAACTGAACATTTTCCTGAGTCCAACGAC CTTCCCAAAATGGTTCTTAGATCTGCAATAGGACTTGCGATCAAAAAGTAAAACACATTC TGAAAGACTATTGAGCCATTGATAGAAAAGCAAATAAAACTAGATATACATT
<SEQ ID NO: 10; DNA; Homo sapiens> TGAATAATTGAACTTTGTTTATTTCTCCATATTTTTGCAGTGGTAATTCCATTATAAAAC CTAATGAAACAATGTTTTTATAGATGGTGTGGAAAGACTTTTCTGGGCTCAGAGGTGAAA CTGACCCTTGTGTATCAGCAGCATTTCTGACTGACTGAGAGAGTGTAGTGATTAACAGAG TTGTGATGTTAGTTAAGAAACTTAGATTTGCCATTGTAGCTTTTCTACCAATTAGCAGAT TGTTTAACTCACTGAAATTGTAAAGTGGTAGACGTGGACTTAGTCATTACTGGGCAGCTT ATGAATTGTATTCATTTACTCATGATGTAAAAATGGTTAGTCTCCACTTTTAAGGCTCTA GTTCTAGTGGCTAAATAGGTACTTATTTATACAGTATGATAACTGCTGTATTAAAATACA TGTCTCAAATGTGGAATAGTAGAAGAGGTGAAGAAAATCATAGTTTGAGGTAGAATACTG TTTGCTGGTCTTAAAAACTGTGGTATTTTGGTGATTCCATAAATTAGGTCAGATACTTCC ACTGGAGGGAAACAGTTTAAAGGATATATGTGATACTATTAATAGAATGAGGAAGACACA CCAGATATTTAGGAGGGAATTAGCGAGCTTGAAACTAAGAGCTGGTTTGAATGAGACTGG GTCATAAGTGATTTCAAGTACCAGATTAAGGCACTGAGATTTTATTTTTAAGCACTGAAG TCAGATTTTTTCCTTTTAAAAGAAAGGATTCATGATGAAATCTGCTTTTTGTTTTGCAGA GAGCTTGGAGATAATTCTGGTGGCTGTGTGGAGTATGTGTTGGAGGTATTAAATTTTCAC AGTATATATAAGGCAGCAATTGATAGGCCTTTCACAGATTCTTCTGATAACTACATAAAG AGACAAAAAAAAGAAAAAAGAGCAAAGATCTGTGCTGTGTCAAGTATGACAGCCATCACT CATGGCTCTCCAGTAGGAGGGAACGACAGCCAGGGCCAGGTTCTTGATGGCCAGTCTCAG CATCTCTTCCAACAGAACCAGACTTCATCACCTGATTCTTCCAATGAGAATTCCGTAGCA ACTCCTCCTCCAGAGGAACAAGGGCAAGGTGATGCCCCACCACAGCATGAAGATGAAGAG CCTGCATTTCCACATACTGAGCTGGCAAACCTGGATGACATGATCAACAGGCCTCGATGG GTGGTTCCTGTTTTGCCAAAAGGGGAATTAGAAGTGCTTTTAGAAGCTGCTATTGATCTT AGTGTAAAAGGCCTTGATGTTAAAAGTGAAGCATGCCAACGTTTTTTTCGAGATGGACTA ACAATATCTTTCACTAAAATTCTTATGGATGAGGCTGTGAGTGGCTGGAAGTTTGAAATT CATAGATGTATTATTAACAATACTCATCGCCTAGTGGAGCTTTGTGTGGCCAAGTTGTCC CAAGATTGGTTTCCACTTCTAGAACTTCTCGCCATGGCCTTAAATCCTCACTGCAAGTTT CATATCTACAATGGTACACGTCCGTGTGAATTAATTTCCTCAAATGCTCAGTTGCCTGAA GAAGAATTATTTGCTCGTTCTTCAGATCCTCGATCACCAAAAGGTTGGCTAGTGGATCTC ATCAATAAATTTGGCACATTAAATGGGTTCCAGATTTTGCATGATCGTTTTTTTAATGGA TCAGCATTAAATATTCAAATAATTGCAGCTCTTATTAAACCATTTGGACAATGCTATGAG TTTCTCAGTCAACATACACTGAAAAAGTACTTCATTCCAGTTATAGAAATAGTTCCACAT TTATTGGAAAACTTAACTGATGAAGAACTGAAAAAGGAGGCAAAGAATGAAGCCAAAAAT
GATGCCCTTTCAATGATTATTAAATCTTTGAAGAACTTAGCTTCAAGAATTTCAGGACAA GATGAGACTATAAAAAATTTGGAAATTTTTAGGTTAAAGATGATACTCAGATTGTTGCAA ATTTCCTCTTTTAATGGAAAGATGAATGCACTGAATGAAATAAATAAGGTTATATCTAGT GTATCATATTATACTCATCGGCATAGTAATCCTGAGGAGGAAGAATGGCTGACAGCTGAG CGAATGGCAGAATGGATACAGCAAAATAATATCTTATCCATAGTCTTGCAAGACAGTCTT CATCAACCACAATATGTAGAAAAGCTAGAGAAAATTCTTCGTTTTGTGATTAAAGAAAAG GCTCTTACATTACAGGACCTTGATAATATCTGGGCAGCACAGGCAGGAAAACATGAAGCC ATTGTGAAGAATGTACATGATCTGCTAGCAAAGTTGGCTTGGGATTTTTCTCCTGGACAA CTTGATCATCTTTTTGATTGCTTTAAGGCAAGTTGGACAAATGCAAGTAAAAAGCAACGT GAAAAGCTCCTTGAGTTGATACGCCGTCTTGCAGAAGATGATAAAGATGGTGTGATGGCA CACAAAGTGTTGAACCTTCTTTGGAACCTGGCTCAGAGTGATGATGTGCCTGTAGACATC ATGGACCTTGCTCTTAGTGCCCACATAAAAATACTAGATTATAGTTGTTCCCAGGATCGA GATGCACAGAAGATCCAGTGGATAGATCACTTTATAGAAGAACTTCGCACAAATGAGAAG TGGGTAATTCCTGCTCTGAAACAAATAAGAGAAATTTGTAGTTTGTTTGGTGAAGCATCT CAAAATTTGAGTCAAACTCAGCGAAGTCCCCACATATTTTATCGCCATGATTTAATCAAC CAGCTTCAACAAAATCATGCTTTAGTTACTTTGGTAGCAGAAAACCTTGCAACCTACATG AATAGCATCAGATTGTATGCTGGAGATCATGAAGACTATGATCCACAAACAGTGAGGCTT GGAAGTCGATACAGTCATGTTCAAGAAGTTCAAGAACGACTAAACTTCCTTAGATTTTTA CTGAAGGATGGCCAACTGTGGCTCTGTGCTCCTCAGGCAAAACAAATATGGAAGTGCTTA GCAGAAAATGCAGTTTATCTTTGTGATCGTGAAGCCTGTTTTAAGTGGTATTCCAAGTTA ATGGGGGATGAACCAGACTTGGATCCTGATATTAATAAGGACTTCTTTGAAAGTAATGTA CTTCAGCTTGATCCTTCCCΓTTTAACTGAAAATGGAATGAAATGCTTTGAAAGATTTTTC AAAGCTGTCAATTGTCGAGAAAGGAAACTAATAGCAAAAAGAAGATCCTATATGATGGAT GATTTGGAATTAATTGGACTAGACTACCTTTGGAGGGTTGTGATTCAGAGTAGTGACGAG ATTGCTAACAGAGCTATAGATCTTCTTAAAGAGATATACACAAACCTTGGCCCAAGATTA AAAGCCAATCAGGTGGTTATCCATGAAGACTTCATTCAGTCTTGCTTTGATCGTTTAAAA GCATCATATGATACACTGTGTGTTTTTGATGGTGACAAAAACAGCATTAATTGTGCAAGA CAAGAAGCCATTCGAATGGTTAGAGTATTAACTGTTATAAAAGAGTACATTAATGAATGT GACAGTGATTATCACAAGGAAAGAATGATTTTACCTATGTCGAGAGCATTTTGTGGCAAA CACCTCTCTCTTATAGTTCGGTTTCCAAACCAGGGCAGACAGGTTGATGAGTTGGATATA TGGTTTCATACGAATGACACAATTGGTTCAGTACGGCGATGTATTGTTAATCGTATTAAA GCCAATGTAGCCCACAAAAAAATTGAACTTTTTGTGGGTGGTGAGCTGATAGATTCTGAA AATGACAGAAAGCTAATTGGACAATTAAACTTAAAAGATAAATCTCTAATTACAGCCAAA CTTACACAAATAAATTTCAATATGCCATCAAGTCCTGATAGCTCTTCCGATTCCTCAACT GCATCTCCTGGAAACCACCGTAATCATTACAATGATGGTCCCAATCTAAAGGTGGAAAGT TGTTTGCCTGGGGTGATAATGTCAGTGCATCCCAAATACATCTCTTTCCTTTGGCAATTT GCAAACTTAGGTAGCAACCTGAATATGCCACCTCTΓAAAAATGGAGCAAGAGTACTTATG AAACTTATGCCACCAGATAGAACAGCTGTAGAAAAATTACGAACTGTTTGTTTGGACCAT GCAAACCTTGGAGAAGGCAAACTTAGTCCACCCCTTGACTCCCTTTTCTTTGGTCCTTCT GCCTCCCAAGTTCTATACCTAACAGAGGTAGTTTATGCCTTGTTAATGCCTGCTGGTGTG CCTCTAACTGATGGGTCCTCTGACTTTCAAGTTCACTTCTTGAAAAGTGGTGGCTTACCT CTTGTACTGAGTATGCTAATAAGAAATAACTTCTTGCCAAATACAGATATGGAAACTCGA AGGGGTGCTTATTTAAATGCTCTTAAAATAGCCAAACTGTTGTTAACTGCGATTGGCTAT GGCCATGTTCGAGCTGTAGCAGAAGCTTGTCAGCCAGTTGTAGATGGTACAGACCCCATA ACACAGATTAACCAAGTTACTCATGATCAAGCAGTGGTGCTACAAAGTGCCCTTCAGAGC ATTCCTAATCCCTCATCCGAGTGCGTACTTAGAAATGAGTCCATACTTCTTGCTCAGGAA ATATCTAATGAGGCTTCAAGATATATGCCTGATATTTGTGTAATTAGGGCTATACAGAAA ATTATCTGGGCATCAGCATGTGGGGCATTAGGACTATTTTTTAGCCCAAATGAAGAAATA ACTAAAATTTATCAGATGACCACCAATGGAAGCAATAAGCTGGAGGTGGAAGATGAACAA GTTTGTTGTGAAGCACTGGAAGTGATGACCTTATGTTTTGCTTTACTTCCAACAGCGTTG GATGCACTTAGTAAAGAAAAAGCCTGGCAGACCTTCATCATTGACTTATTATTGCACTGT CCAAGCAAAACTGTTCGTCAGTTGGCACAGGAGCAGTTCTTTTTAATGTGCACCAGATGT TGCATGGGACACAGGCCTCTGCTTTTCTTCATTACTTTACTCTTTACCATACTGGGGAGC ACAGCAAGAGAGAAGGGTAAATATTCAGGTGATTATTTCACACTTTTACGGCACCTTCTC AATTATGCTTACAATGGCAATATTAACATACCCAATGCTGAAGTTCTTCTTGTCAGTGAA ATTGATTGGCTCAAAAGGATTAGGGATAATGTTAAAAACACAGGTGAAACAGGTGTCGAA GAGCCAATACTGGAAGGCCACCTTGGGGTAACAAAAGAGTTATTGGCCTTTCAAACTTCT GAGAAAAAGTATCACTTTGGTTGTGAAAAAGGAGGTGCTAATCTCATTAAAGAATTAATT GATGATTTCATCTTTCCCGCATCCAAAGTTTACCTGCAGTATTTAAGAAGTGGAGAACTA CCAGCTGAGCAGGCTATTCCAGTCTGTAGTTCACCCGTTACCATCAATGCCGGTTTTGAG CTACTTGTAGCATTAGCTATTGGCTGTGTGAGGAATCTCAAACAGATAGTAGACTGTTTG ACTGAAATGTATTACATGGGCACAGCAATTACTACTTGTGAAGCACTTACTGAGTGGGAA TATCTGCCCCCTGTTGGACCCCGCCCACCAAAAGGATTTGTGGGACTCAAAAATGCTGGT GCTACGTGTTACATGAACTCTGTGATCCAGCAGCTATACATGATTCCTTCTATCAGGAAC AGTATTCTTGCAATTGAAGGCACAGGTAGTGATTTACACGATGATATGTTCGGGGATGAG AAGCAGGACAGTGAGAGTAATGTTGATCCCCGAGATGATGTATTTGGATATCCTCATCAA TTTGAAGACAAGCCAGCATTAAGTAAGACAGAAGATAGGAAAGAGTATAATATTGGTGTC CTAAGACACCTTCAGGTCATCTTTGGTCAΦTTAGCTGCTTCCCAACTACAATACTATGTA CCCAGAGGATTTTGGAAACAGTTCAGGCTTTGGGGTGAACCTGTTAATCTCCGTGAACAA CATGATGCCTTAGAGTTTTTTAATTCTTTGGTGGATAGTTTAGATGAAGCTTTAAAAGCT TTAGGACACCCGGCTATACTAAGTAAAGTCCTAGGAGGCTCCTTTGCTGATCAGAAGATC TGCCAGGGCTGCCCACATAGGTTTGAATGTGAAGAATCTTTTACAACTTTGAATGTGGAT ATTAGAAATCATCAAAATCTTCTTGACTCTTTGGAACAGTATATCAAAGGAGATTTATTG GAAGGTGCAAATGCATATCATTGTGAAAAATGTGATAAAAAGGTTGACACAGTAAAGCGC CTGCTAATTAAAAAATTGCCTCGGGTTCTTGCTATCCAACTCAAACGATTTGACTATGAC TGGGAAAGAGAATGTGCAATTAAATTCAATGATTATTTTGAATTTCCTCGAGAGCTGGAT ATGGGACCTTACACAGTAGCAGGTGTTGCAAACCTGGAAAGGGATAATGTAAACTCAGAA AATGAGTTGATTGAACAGAAAGAGCAGTCTGACAATGAAACTGCAGGAGGCACAAAGTAC AGACTTGTAGGAGTGCTTGTACACAGTGGTCAAGCAAGCGGTGGGCATTATTATTCTTAC ATCATTCAAAGGAATGGTAAAGATGATCAGACAGATCACTGGTATAAATTTGATGATGGA GATGTAACAGAATGCAAAATGGATGATGATGAAGAAATGAAAAATCAGTGTTTTGGTGGA GAGTACATGGGAGAAGTATTTGATCACATGATGAAGCGCATGTCATATAGGCGACAGAAG AGGTGGTGGAATGCTTACATACCTTTTTATGAACAAATGGATATGATAGATGAAGATGAT GAGATGATAAGATACATATCAGAGCTAACTATTGCAAGACCCCATCAGATCATTATGTCA CCAGCCATTGAGAGAAGTGTACGGAAACAAAATGTGAAATTTATGCATAACCGATTGCAA TATAGTTTAGAGTATTTTCAGTTTGTGAAAAAACTGCTTACATGTAATGGTGTTTATTTA AACCCTGCTCCAGGGCAGGATTATTTGTTGCCTGAAGCAGAAGAAATTACTATGATTAGT ATTCAGCTTGCTGCTAGATTCCTCTTTACCACTGGATTTCACACCAAGAAAATAGTTCGT GGTCCTGCCAGTGACTGGTATGATGCACTGTGCGTTCTTCTCCGTCACAGCAAAAATGTA CGTTTTTGGTTTACTCATAATGTCCTTTTTAATGTATCAAATCGCTTCTCTGAATACCTC CTGGAGTGCCCTAGTGCAGAAGTGAGGGGTGCATTTGCAAAACTTATAGTGTTTATTGCA CACTTTTCCTTGCAAGATGGGTCTTGTCCTTCTCCTTTTGCATCTCCAGGACCTTCTAGT CAGGCATGTGATAACTTGAGCTTGAGTGACCACTTACTAAGAGCCACACTAAATCTCTTG AGAAGGGAAGTTTCAGAGCATGGACATCATTTACAGCAATATTTTAATTTGTTTGTAATG TATGCCAATTTAGGTGTGGCAGAAAAAACACAGCTTCTGAAATTGAATGTACCTGCTACC TTTATGCTTGTGTCTTTAGACGAGGGACCAGGTCCTCCAATCAAATATCAGTATGCTGAA TTAGGCAAGTTATATTCAGTAGTGTCTCAGCTGATTCGTTGTTGCAATGTGTCATCAACA ATGCAGTCTTCAATCAATGGTAATCCCCCTCTCCCCAATCCTTTCGGTGACCTTAATTTA TCACAGCCTATAATGCCAATTCAGCAGAATGTGTTAGACATTTTATTTGTGAGAACAAGT TATGTGAAGAAAATTATTGAAGACTGCAGTAACTCAGAGGATACCATCAAATTACTTCGC TTTTGCTCTTGGGAGAATCCTCAGTTCTCATCTACTGTCCTCAGCGAACTTCTCTGGCAG GTTGCATATTCATATACCTATGAACTTCGGCCATATTTAGATCTACTTTTCCAAATTTTA CTGATTGAGGACTCCTGGCAGACTCACAGAATTCATAATGCACTTAAAGGAATTCCAGAT GACAGAGATGGGCTGTTCGATACAATACAGCGCTCGAAGAATCACTATCAAAAACGAGCA TATCAGTGCATAAAATGTATGGTAGCTCTATTTAGCAGTTGTCCTGTTGCTTACCAGATC TTACAGGGTAACGGAGATCTTAAAAGAAAATGGACCTGGGCAGTGGAATGGCTAGGAGAT GAACTTGAAAGAAGACCATATACTGGCAATCCTCAGTATAGTTACAACAATTGGTCTCCT CCAGTACAAAGCAATGAAACAGCAAATGGTTATTTCTTAGAAAGATCACATAGTGCTAGG ATGACACTTGCAAAAGCTTGTGAACTCTGTCCAGAAGAGGAGCCAGATGACCAGGATGCC CCAGATGAGCATGAGCCCTCTCCATCAGAAGATGCCCCATTATATCCTCATTCACCTGCC TCTCAGTATCAACAGAATAATCATGTACATGGACAGCCATATACAGGACCAGCAGCACAT CACTTGAACAACCCTCAGAAAACAGGCCAACGAACACAAGAAAATTATGAAGGCAATGAA GAAGTATCCTCACCTCAGATGAAGGATCAGTGAAAAGCAATAATTAACTGCTTCCTTTAT GACTATGCACTAAGGTCTTATAGTCCAAACTTTCTCTGTGTCTGGCTAGTATTGAAAACT AGATAAACTGCTCCAAACCAACATGGAGTAAAGAGCATATTCACTGGTTTATTTGCAGTA ATTTGCAATTTGTCAGTGTATAAGACACATGCAGGGTGAAGTGTACAGAGTTTTGTAACA AATGACTGGTCCTAATCTGTAAATGAGAAAGGTATATATACTATGTTAATGTCTGACTGT TAATTCTTAAGCAAGAAACTTTTTTTGATGAAAACAAGTCAGATCTACACAGTCACACAA TTATTTTTTGTTGTGTTCACTACATTGTGCAATTGATATTGCCTGCTTTGAGCAGTTTGG TCAACTTACCAACTTCCCCCCAAAAAAGGGAACATAAAAGAGCCCATCTTTGTCAGTTTA CACCAATAGTTTCTTGTTAATCCTTCTTTCCTGGATATATAAGGCTGGTGGTAACTTTTG AATTATATGGTTGATGTGGAAAATTGGCAGTGTAACATTTCTAGATACTTTTCATTACCT TTTTATTCTGGTATATAGGCTAACCACTTTAAAGCTATTCTTAtCGCTGTAACAGTTAGCA TGGCTTCACACTGTTTGTGTAGCCAAGAGGACAGAATTACATGAATGACAGTGCCCAGAG TGACAGCTGTATATTGCTCAGAGCTTTTATTTCTTATACCTAGAATAAATATAAAATGGG GGAAAAAAAAAA
<SEQ ID NO: 11; DNA; Homo sapiens> CTGGGAGCGCGGCGTAGGTGGCTGCCGAGTCTTTTCCTGTTTAGGGTCTTATCCTGGCAT TGAGGGCGCCGGACTGGCGCTTTTGGCCGGCTTGGCATTGGGTGGGCGGCTTCTTGGGAC CCACATGAGCCAGTGGCATCATCCCCGCAGTGGCTGGGGCCGGAGACGCGACTTTTCAGG ACGCTCCTCAGCCAAGAAGAAGGGCGGAAACCACATCCCCGAAAGGTGGAAAGACTATCT CCCAGTTGGACAGCGGATGCCTGGGACTCGTTTCATTGCTTTCAAAGTTCCTTTGCAAAA GAGTTTTGAAAAGAAACTTGCTCCAGAAGAATGCTTTTCCCCTTTGGATCTTTTTAACAA AATCCGAGAACAAAATGAAGAACTTGGACTGATTATTGATTTAACATATACTCAACGCTA TTATAAACCAGAGGATTTGCCAGAAACTGTTCCTTACTTAAAAATTTTTACAGTTGGACA TCAAGTGCCTGATGATGAGACTATTTTTAAATTCAAACACGCTGTTAATGGGTTTTTGAA AGAAAATAAAGATAATGATAAACTTATTGGTGTCCACTGTACCCATGGTTTAAACAGGAC TGGCTACCTCATTTGCAGATATTTGATTGATGTAGAAGGCGTGAGGCCAGATGATGCAAT TGAATTATTCAATAGGTGCCGGGGACATTGCTTAGAAAGACAAAACTACATTGAAGACCT TCAGAATGGTCCTATCAGAAAGAATTGGAATTCCAGTGTACCCAGGTCAAGTGATTTTGA AGACTCAGCACATCTCATGCAACCAGTCCACAATAAGCCTGTTAAACAAGGACCTAGGTA TAATCTACATCAGATCCAGGGTCACTCAGCTCCTCGACATTTCCACACCCAGACCCAAAG TTTGCAACAATCAGTCAGAAAATTTTCAGAGAATCCACATGTTTACCAGAGACACCATCT CCCTCCTCCTGGTCCCCCTGGAGAGGACTATTCACACAGGAGGTATTCTTGGAATGTGAA GCCCAATGCCAGTCGGGCAGCCCAGGATAGAAGAAGGTGGTATCCTTATAATTACTCCAG ACTCTCCTATCCAGCCTGTTGGGAATGGACCCAGTGATACAAACCTGTCCTGGAATTCTA CCTGGAGACCAGAGCTGGCCTGAAAATTACTGGTGTGACTTTTAATTAGTTCAGGTCTAA TCAGGTTTCTTTATTGTTCCCTTATGTATTCAAGCTTAAGGAAAAATTGCATTGCTGTTT ACCTCTTTGCTGATAAATTTGCAGTAATTACAGCATTGCAGGAAAAACAATCTGTTATTC CAGTCTTAAATTTTTCTAAAAGAAGACAATATTTTAGAACTGAAGCATTGAGAACTTCCC TTGCAAATTATTTTTAAAATTCTATCTTGTTTTTCTATGTATTTCTTTCTGACTAGACTT GTGATATGCGTGTGTTTATGTACAGAAATTTTTAGTGTTTTTGTTATGTTCTGTTATTGA CCCAAAGGCCATCTTTATTTTCTATAACTGTTCAAAATTTATATTAAAATCTACTTAGGA GATAATTTCTTTAGAAAAAAAAAAAAAAAAAAA
<SEQ ID NO: 12; DNA; Homo sapiens>
CGCCGCTCTCCGCCTCGCTTGCTCCTGCCGGGCGTGCAGGGCCCCGCCGCCGCCATGTCG
GGCTCGTTCGAGCTCTCGGTGCAGGATCTCAACGACCTGCTCTCGGACGGCAGCGGCTGC TACAGCCTCCCGAGCCAGCCCTGCAACGAGGTCACCCCGCGGATCTACGTGGGCAACGCG TCTGTGGCTCAGGACATCCCCAAGCTGCAGAAACTAGGCATCACCCATGTGCTGAACGCG GCTGAGGGCAGGTCCTTCATGCACGTCAACACCAATGCCAACTTCTACAAGGACTCCGGC ATCACATACCTGGGCATCAAGGCCAACGACACACAGGAGTTCAACCTCAGCGCTTACTTT GAAAGGGCTGCCGACTTCATTGACCAGGCTTTGGCTCAAAAGAATGGCCGGGTGCTCGTC CACTGCCGGGAAGGTTATAGCCGCTCCCCAACGCTAGTTATCGCCTACCTCATGATGCGG CAGAAGATGGACGTCAAGTCTGCCCTGAGCATCGTGAGGCAGAACCGTGAGATCGGCCCC AACGATGGCTTCCTGGCCCAGCTCTGCCAGCTCAATGACAGACTAGCCAAGGAGGGGAAG TTGAAACCCTAGGGCACCCCCACCGCCTCTGCTCGAGAGGTCCGTGGGGGAGGCCGTGGG CAAAGGTGTCCCGAGCTGCCATGTTTAGGAAACACACTGTACCCTGCTCCCAGCATCACA AGGCACTTGTCTACAAGTGTGTCCCAACACAGTCCTGGGCCACTTTCCCCACCCTGGGGA GCACATAAAGAAGCTTGCCAAGGGGGGCGTCCTTGCTCCCCAGTTGTCCTGTTTCTGTAA CTTATGATGTCTTTTCCCTGAGATGGGGGCTCAGAGGGGGAAGGCCTGTGGCCTGCATGC TTCCCGATGGCCCACGGCAGGAGGTGTGTGGAAGTGTAAGGCCTAAGATGCTCACAGAGG TCCCTCATGACCTCCCTTCCCCAACTCCCGAATCCTCTCTTGAGTGTGGACCTCAACACC TTGAGCCCTAGTAAAGGAACTATGCAAATGCAGGCCACTCTCCCCACCACGTCTGTGCCC CGCACTGTCCCCACAGCCTTCCACACCCTGTGCATAGGCAGCCCTCTCACGTCTTGAGGT CCGAAGCTGGGGTGGGGGTGTCCGTCAGTTATTAGTGGATGGAGATTCCCACAGCAAGGC TGCATTTGAATGATTTGCTTAGGATGAATGGTCCCTACACAAAGAGGCCTTGTGGGCAAA CCTGGAGAACCCTCCTAAATCCATAGAGTTTTCAAAATGTGAATCTTTGGAAGCCTTGAG TTCAGAATCTGCTGCTCTGGAATATTTCCCTTCGATCTTATCTCAGTCACTTCGTTTTTG AGAAGAGTGATGCCTTGGGCATGCTTTTTTTTTTTTCTTTTTTAGAAAACAGGGAGTTGA AGTCCAACCTATTTAAAAACCCCACCATTTGGAGAATTACAAGGGTTTTGTCCTGAATTG TAGTGTTGGCAAGCCCAAGCCACTCGTGCTAACTGCTTTTTGTCTCGGTTGCTATTCCAA GAACAGAAGGAGGAAGTTGGCCAATTACAGCGTGTGTGCATGGATGTGTGTGGGGGGCGT GCCTCTCAGAAACGCGGCCAGAAGACAAGCAGGGAAGTGAAAGGTCCCAGGCACACACCC TGCCCATTGCAGGTGGCTCTTACAGCTCTCTGGTGCCAGCACGGGATCCCTGAAGTGACT CAGCCAGGCAGACATGAGACATGGCGGAGTGTCCAAATGGATCCTTTATTGGTGGTAGAG CAAAAAAACCCAAACACGATAAACCTTTCAAAAGACTTTCTAAGGATGATATTGGAATGC ACCAGCCCTCACATGTGTATGCACATTTGCCAGAATATAAGAGTTTTGTTTTAAATACAG TCTTGTTAGGATTTTACGTTATTGTTATTATGGAAAGTGATTGTGATGCTATTTATCTTC AGGGTCACTCTGGGCAAAGAGAAGGTCCTCAGCCATGCCCCCAGCACCTTGCACATAGGT GTCTGATAAAAGTTTAAGAAATTAAACACTTTTTGAGCACCAAATATATATAGGGCATTG TTCTGGTGGGTGTGTCACGCTCCCAGAAGACTGAATTTATGGTAGGATCACTCGCAAGGC CTTGTGAAGGAGTCTTACCTAAAACAAAAGAAATATCAGGGACTTTTGTTGACTATTTAC AACTCAGTTTTACATTTAAATTCAGGCAGTGTTAATATGCCAAGGTAGGGAATGTGCCTT TTTCAGAGTTGGCCAGGAGCTCCTGGCTGGGACACGGAGAGGCAGGTGTGGCGTAAGGCC TCACTCCCGGCTGTGAAGGTCTCTGATCACACAGAAGCAGCCCTGCCCAGCCTGGTCATT TGCTGTCCGCTTTTCTCTGTGACCACAGCAGCCCTGAACAACCAGTATGTGTCTTCTTCT CCAGATAGTGAAAAAGGTGTCCAGATAAACCCACCTAAGTGAAATGGCCATCCTCTAAAC TGGGTACCTCACTGCACAGCTTCTAGGTAGCCTTCCAACTTAATCTAACTTGAGCCTCAC GTAACCCTGTAAAGTTAGTAGAGCTTGTTCTTGTATTGTGACCTTTTTTAAAAAAAAGG AACTGAGGTTCAGAATGATTAAGGGCCTGGCCCCCAGGGTTGTCCAGCTCCATAAGGTGG AGCTGGGCAAGATTTTGGGTTTGCTGCTCCCTGAAGCTGGATTCTTTCATACGATACTCT TTCTCAAGAAGGGGGCTCCCTGGGATCTCCAGGTGTACTGCACTTACCCTCAATCCAGCC CCGGAGAAGCAAGTGAAAAGGGTGGGTCCCTCATAGGCTAGAATGTGCAGCTCTTTCTCC AGGTGGGATGTAGCACCCCAAAGTAGAGCTTTCTGCTCTGCTCCTGGAAAAGGCTAGGGA GCTGGGGCTGGGGCTCCCCTCCCATGACCAGGCAGTGGTCACCCCATGGGACAGGCACAG CTACTTACGCGAACACAGCAGGTTGGTGTGGCTGGCTAACTAGGACCTCTCGAAAGTCTC TGTGGGGGCATGAGGGAGAAAAGGCCATTGGGAGAATTACTGCCTTTACTTTGGGACTAC TTTTATGCTGATAACTTGGGATTTCTTGATAGTCCTTCACCCCTGAAACCCCGTATTTAC TTAACAAGATTTAGCTCTTAGTTCTTCAAGTAAAATTAAAGTCTCTTGTGTAAGAGCCAA CACATGCCCAGCTGCGGATGGGAGCTGTTCCTGGACAGCCTTCTACTGCCTGGGAAGTGA TGGAACAGGAACTCAGGGTGCCCTTACCCCCTCCCCAGACCTGTTCCCTTTCTTTGACTG ACAGAGCACCATCCAGGCAAAATTAGAGCGCCAAATGGTTTΓCTTCTCAATCTTAAAGCA GTATACCTTTCCACAGGCTCGTCTGTGTCCCTGCCACTCTGAGTTATCCAGAAACCACCA CCTACAAATGAGGGGACTCATCTAGAAGACCTCTAAGGTCCCCTTTTGGCTCTGAGGGGT CTCTAATAATCCCCACTTGGAATTCAGCACCGCAAGGAAATTATGGGTATGTGAGCCATA ATATGATGGCCAGCAGGTGGCGCTGCCTTCCACCCATGGTGATGGATGGTTTGGAAAGGG AATGTTGGTGCCTTTTGTGCCACAAGTTAAGATGCTACTGTTTTAAAGGAAAAAAAAAAA AAAAAGTACTGATCTTCAATATGAAGACATGAGCTTTTCTCGCAGGAAATTTTCTTTTTC ACAGAACTGGTGTCAGGAATCACTGAAGGGCTAACCGTGATAGTCCTTGCAAGTAAGTCA AGGTTTTATCCTGATTGGAAATAGAAGACATTTCCGGTTGAGAGAACAGATTCGTTGGAA GCTTAACTTTTGTTGCCTCTTAACGCCACCAAATTTTAGGGTAATTTGATTATGAAAGAG TGAATTTTTCTGGACAGAAAAGGGAGAGCTACCAAATTGTTTTTTTCTTTTTAAAAGGAA GTTTAATGTCCGTTGTATCACAAATCAGTGTTAAAACACCAGAACTTTAGCCAAAATAAA TGTCTTACATTACAAAGGTAAAAAAAAAAAAAAAAA
<SEQ ID NO: 13; DNA; Homo sapiens> CCAGTTTGCTTCTTGGAGAACACTGGACAGCTGAATAAATGCAGTATCTAAATATAAAAG AGGACTGCAATGCCATGGCTTTCTGTGCTAAAATGAGGAGCTCCAAGAAGACTGAGGTGA ACCTGGAGGCCCCTGAGCCAGGGGTGGAAGTGATCTTCTATCTGTCGGACAGGGAGCCCC TCCGGCTGGGCAGTGGAGAGTACACAGCAGAGGAACTGTGCATCAGGGCTGCACAGGCAT GCCGTATCTCTCCTCTTTGTCACAACCTCTαLTGCCCTGTATGACGAGAACACCAAGCTCT GGTATGCTCCAAATCGCACCATCACCGTTGATGACAAGATGTCCCTCCGGCTCCACTACC GGATGAGGTTCTATTTCACCAATTGGCATGGAACCAACGACAATGAGCAGTCAGTGTGGC GTCATTCTCCAAAGAAGCAGAAAAATGGCTACGAGAAAAAAAAGATTCCAGATGCAACCC CTCTCCTTGATGCCAGCTCACTGGAGTATCTGTTTGCTCAGGGACAGTATGATTTGGTGA AATGCCTGGCTCCTATTCGAGACCCCAAGACCGAGCAGGATGGACATGATATTGAGAACG AGTGTCTAGGGATGGCTGTCCTGGCCATCTCACACTATGCCATGATGAAGAAGATGCAGT TGCCAGAACTGCCCAAGGACATCAGCTACAAGCGATATATTCCAGAAACATTGAATAAGT CCATCAGACAGAGGAACCTTCTCACCAGGATGCGGATAAATAATGTTTTCAAGGATTTCC TAAAGGAATTTAACAACAAGACCATTTGTGACAGCAGCGTGTCCACGCATGACCTGAAGG TGAAATACTTGGCTACCTTGGAAACTTTGACAAAACATTACGGTGCTGAAATATTTGAGA CTTCCATGTTACTGATTTCATCAGAAAATGAGATGAATTGGTTTCATTCGAATGACGGTG GAAACGTTCTCTACTACGAAGTGATGGTGACTGGGAATCTTGGAATCCAGTGGAGGCATA AACCAAATGTTGTTTCTGTTGAAAAGGAAAAAAATAAACTGAAGCGGAAAAAACTGGAAA ATAAAGACAAGAAGGATGAGGAGAAAAACAAGATCCGGGAAGAGTGGAACAATTTTTCAT TCTTCCCTGAAATCACTCACATTGTAATAAAGGAGTCTGTGGTCAGCATTAACAAGCAGG ACAACAAGAAAATGGAACTGAAGCTCTCTTCCCACGAGGAGGCCTTGTCCTTTGTGTCCC TGGTAGATGGCTACTTCCGGCTCACAGCAGATGCCCATCATTACCTCTGCACCGACGTGG CCCCCCCGTTGATCGTCCACAACATACAGAATGGCTGTCATGGTCCAATCTGTACAGAAT ACGCCATCAATAAATTGCGGCAAGAAGGAAGCGAGGAGGGGATGTACGTGCTGAGGTGGA GCTGCACCGACTTTGACAACATCCTCATGACCGTCACCTGCTTTGAGAAGTCTGAGCAGG TGCAGGGTGCCCAGAAGCAGTTCAAGAACTTTCAGATCGAGGTGCAGAAGGGCCGCTACA GTCTGCACGGTTCGGACCGCAGCTTCCCCAGCTTGGGAGACCTCATGAGCCACCTCAAGA AGCAGATCCTGCGCACGGATAACATCAGCTTCATGCTAAAACGCTGCTGCCAGCCCAAGC CCCGAGAAATCTCCAACCTGCTGGTGGCTACTAAGAAAGCCCAGGAGTGGCAGCCCGTCT ACCCCATGAGCCAGCTGAGTTTCGATCGGATCCTCAAGAAGGATCTGGTGCAGGGCGAGC ACCTTGGGAGAGGCACGAGAACACACATCTATTCTGGGACCCTGATGGATTACAAGGATG ACGAAGGAACTTCTGAAGAGAAGAAGATAAAAGTGATCCTCAAAGTCTTAGACCCCAGCC ACAGGGATATTTCCCTGGCCTTCTTCGAGGCAGCCAGCATGATGAGACAGGTCTCCCACA AACACATCGTGTACCTCTATGGCGTCTGTGTCCGCGACGTGGAGAATATCATGGTGGAAG AGTTTGTGGAAGGGGGTCCTCTGGATCTCTTCATGCACCGGAAAAGTGATGTCCTTACCA CACCATGGAAATTCAAAGTTGCCAAACAGCTGGCCAGTGCCCTGAGCTACTTGGAGGATA AAGACCTGGTCCATGGAAATGTGTGTACTAAAAACCTCCTCCTGGCCCGTGAGGGAATCG ACAGTGAGTGTGGCCCATTCATCAAGCTCAGTGACCCCGGCATCCCCATTACGGTGCTGT CTAGGCAAGAATGCATTGAACGAATCCCATGGATTGCTCCTGAGTGTGTTGAGGACTCCA AGAACCTGAGTGTGGCTGCTGACAAGTGGAGCTTTGGAACCACGCTCTGGGAAATCTGCT ACAATGGCGAGATCCCCTTGAAAGACAAGACGCTGATTGAGAAAGAGAGATTCTATGAAA GCCGGTGCAGGCCAGTGACACCATCATGTAAGGAGCTGGCTGACCTCATGACCCGCTGCA TGAACTATGACCCCAATCAGAGGCCTTTCTTCCGAGCCATCATGAGAGACATTAATAAGC TTGAAGAGCAGAATCCAGATATTGTTTCCAGAAAAAAAAACCAGCCAACTGAAGTGGACC CCACACATTTTGAGAAGCGCTTCCTAAAGAGGATCCGTGACTTGGGAGAGGGCCACTTTG GGAAGGTTGAGCTCTGCAGGTATGACCCCGAAGACAATACAGGGGAGCAGGTGGCTGTTA AATCTCTGAAGCCTGAGAGTGGAGGTAACCACATAGCTGATCTGAAAAAGGAAATCGAGA TCTTAAGGAACCTCTATpATGAGAACATTGTGAAGTACAAAGGAATCTGCACAGAAGACG GAGGAAATGGTATTAAGCTCATCATGGAATTTCTGCCTTCGGGAAGCCTTAAGGAATATC TTCCAAAGAATAAGAACAAAATAAACCTCAAACAGCAGCTAAAATATGCCGTTCAGATTT GTAAGGGGATGGACTATTTGGGTTCTCGGCAATACGTTCACCGGGACTTGGCAGCAAGAA ATGTCCTTGTTGAGAGTGAACACCAAGTGAAAATTGGAGACTTCGGTTTAACCAAAGCAA TTGAAACCGATAAGGAGTATTACACCGTCAAGGATGACCGGGACAGCCCTGTGTTTTGGT ATGCTCCAGAATGTTTAATGCAATCTAAATTTTATATTGCCTCTGACGTCTGGTCTTTTG GAGTCACTCTGCATGAGCTGCTGACTTACTGTGATTCAGATTCTAGTCCCATGGCTTTGT TCCTGAAAATGATAGGCCCAACCCATGGCCAGATGACAGTCACAAGACTTGTGAATACGT TAAAAGAAGGAAAACGCCTGCCGTGCCCACCTAACTGTCCAGATGAGGTTTATCAGCTTA TGAGAAAATGCTGGGAATTCCAACCATCCAATCGGACAAGCTTTCAGAACCTTATTGAAG GATTTGAAGCACTTTTAAAATAAGAAGCATGAATAACATTTAAATTCCACAGATTATCAA
<SEQ ID NO: 14; DNA; Homo sapiens> TCCAGTTTGCTTCTTGGAGAACACTGGACAGCTGAATAAATGCAGTATCTAAATATAAAA GAGGACTGCAATGCCATGGCTTTCTGTGCTAAAATGAGGAGCTCCAAGAAGACTGAGGTG AACCTGGAGGCCCCTGAGCCAGGGGTGGAAGTGATCTTCTATCTGTCGGACAGGGAGCCC CTCCGGCTGGGCAGTGGAGAGTACACAGCAGAGGAACTGTGCATCAGGGCTGCACAGGCA TGCCGTATCTCTCCTCTTTGTCACAACCTCTTTGCCCTGTATGACGAGAACACCAAGCTC TGGTATGCTCCAAATCGCACCATCACCGTTGATGACAAGATGTCCCTCCGGCTCCACTAC CGGATGAGGTTCTATTTCACCAATTGGCATGGAACCAACGACAATGAGCAGTCAGTGTGG CGTCATTCTCCAAAGAAGCAGAAAAATGGCTACGAGAAAAAAAAGATTCCAGATGCAACC CCTCTCCTTGATGCCAGCTCACTGGAGTATCTGTTTGCTCAGGGACAGTATGATTTGGTG AAATGCCTGGCTCCTATTCGAGACCCCAAGACCGAGCAGGATGGACATGATATTGAGAAC GAGTGTCTAGGGATGGCTGTCCTGGCCATCTCACACTATGCCATGATGAAGAAGATGCAG TTGCCAGAACTGCCCAAGGACATCAGCTACAAGCGATATATTCCAGAAACATTGAATAAG TCCATCAGACAGAGGAACCTTCTCACCAGGATGCGGATAAATAATGTTTTCAAGGATTTC CTAAAGGAATTTAACAACAAGACCATTTGTGACAGCAGCGTGTCCACGCATGACCTGAAG GTGAAATACTTGGCTACCTTGGAAACTTTGACAAAACATTACGGTGCTGAAATATTTGAG ACTTCCATGTTACTGATTTCATCAGAAAATGAGATGAATTGGTTTCATTCGAATGACGGT GGAAACGTTCTCTACTACGAAGTGATGGTGACTGGGAATCTTGGAATCCAGTGGAGGCAT AAACCAAATGTTGTTTCTGTTGAAAAGGAAAAAAATAAACTGAAGCGGAAAAAACTGGAA AATAAAGACAAGAAGGATGAGGAGAAAAACAAGATCCGGGAAGAGTGGAACAATTTTTCA TTCTTCCCTGAAATCACTCACATTGTAATAAAGGAGTCTGTGGTCAGCATTAACAAGCAG GACAACAAGAAAATGGAACTGAAGCTCTCTTCCCACGAGGAGGCCTTGTCCTTTGTGTCC CTGGTAGATGGCTACTTCCGGCTCACAGCAGATGCCCATCATTACCTCTGCACCGACGTG GCCCCCCCGTTGATCGTCCACAACATACAGAATGGCTGTCATGGTCCAATCTGTACAGAA TACGCCATCAATAAATTGCGGCAAGAAGGAAGCGAGGAGGGGATGTACGTGCTGAGGTGG AGCTGCACCGACTTTGACAACATCCTCATGACCGTCACCTGCTTTGAGAAGTCTGAGCAG GTGCAGGGTGCCCAGAAGCAGTTCAAGAACTTTCAGATCGAGGTGCAGAAGGGCCGCTAC AGTCTGCACGGTTCGGACCGCAGCTTCCCCAGCTTGGGAGACCTCATGAGCCACCTCAAG AAGCAGATCCTGCGCACGGATAACATCAGCTTCATGCTAAAACGCTGCTGCCAGCCCAAG CCCCGAGAAATCTCCAACCTGCTGGTGGCTACTAAGAAAGCCCAGGAGTGGCAGCCCGTC TACCCCATGAGCCAGCTGAGTTTCGATCGGATCCTCAAGAAGGATCTGGTGCAGGGCGAG CACCTTGGGAGAGGCACGAGAACACACATCTATTCTGGGACCCTGATGGATTACAAGGAT GACGAAGGAACTTCTGAAGAGAAGAAGATAAAAGTGATCCTCAAAGTCTTAGACCCCAGC CACAGGGATATTTCCCTGGCCTTCTTCGAGGCAGCCAGCATGATGAGACAGGTCTCCCAC AAACACATCGTGTACCTCTATGGCGTCTGTGTCCGCGACGTGGAGAATATCATGGTGGAA GAGTTTGTGGAAGGGGGTCCTCTGGATCTCTTCATGCACCGGAAAAGTGATGTCCTTACC ACACCATGGAAATTCAAAGTTGCCAAACAGCTGGCCAGTGCCCTGAGCTACTTGGAGGAT AAAGACCTGGTCCATGGAAATGTGTGTACTAAAAACCTCCTCCTGGCCCGTGAGGGAATC GACAGTGAGTGTGGCCCATTCATCAAGCTCAGTGACCCCGGCATCCCCATTACGGTGCTG TCTAGGCAAGAATGCATTGAACGAATCCCATGGATTGCTCCTGAGTGTGTTGAGGACTCC AAGAACCTGAGTGTGGCTGCTGACAAGTGGAGCTTTGGAACCACGCTCTGGGAAATCTGC TACAATGGCGAGATCCCCTTGAAAGACAAGACGCTGATTGAGAAAGAGAGATTCTATGAA AGCCGGTGCAGGCCAGTGACACCATCATGTAAGGAGCTGGCTGACCTCATGACCCGCTGC ATGAACTATGACCCCAATCAGAGGCCTTTCTTCCGAGCCATCATGAGAGACATTAATAAG CTTGAAGAGCAGAATCCAGATATTGTTTCCAGAAAAAAAAACCAGCCAACTGAAGTGGAC CCCACACATTTTGAGAAGCGCTTCCTAAAGAGGATCCGTGACTTGGGAGAGGGCCACTTT GGGAAGGTTGAGCTCTGCAGGTATGACCCCGAAGACAATACAGGGGAGCAGGTGGCTGTT AAATCTCTGAAGCCTGAGAGTGGAGGTAACCACATAGCTGATCTGAAAAAGGAAATCGAG ATCTTAAGGAACCTCTATCATGAGAACATTGTGAAGTACAAAGGAATCTGCACAGAAGAC GGAGGAAATGGTATTAAGCTCATCATGGAATTTCTGCCTTCGGGAAGCCTTAAGGAATAT CTTCCAAAGAATAAGAACAAAATAAACCTCAAACAGCAGCTAAAATATGCCGTTCAGATT TGTAAGGGGATGGACTATTTGGGTTCTCGGCAATACGTTCACCGGGACTTGGCAGCAAGA AATGTCCTTGTTGAGAGTGAACACCAAGTGAAAATTGGAGACTTCGGTTTAACCAAAGCA ATTGAAACCGATAAGGAGTATTACACCGTCAAGGATGACCGGGACAGCCCTGTGTTTTGG TATGCTCCAGAATGTTTAATGCAATCTAAATTTTATATTGCCTCTGACGTCTGGTCTTTT GGAGTCACTCTGCATGAGCTGCTGACTTACTGTGATTCAGATTCTAGTCCCATGGCTTTG TTCCTGAAAATGATAGGCCCAACCCATGGCCAGATGACAGTCACAAGACTTGTGAATACG TTAAAAGAAGGAAAACGCCTGCCGTGCCCACCTAACTGTCCAGATGAGGTTTATCAGCTT ATGAGAAAATGCTGGGAATTCCAACCATCCAATCGGACAAGCTTTCAGAACCTTATTGAA GGATTTGAAGCACTTTTAAAATAAGAAGCATGAATAACATTTAAATTCCACAGATTATCA A
<SEQ ID NO: 15; DNA; Homo sapiens>
CGCACCCCGCGCAGCGGCTGAGCCGGGAGCCAGCGCAGCCTCGGCCCCGCAGCTCAAGCC TCGTCCCCGCCGCCGCCGCCGCCGCCGCCGCCGCCGCCCCCGGGGCATGGCCTGTCTGAT GGCCGCTTTCTCGGTCGGCACCGCCATGAATGCCAGCAGTTACTCTGCAGAGATGACGGA GCCCAAGTCGGTGTGTGTCTCGGTGGATGAGGTGGTGTCCAGCAACATGGAGGCCACTGA GACGGACCTGCTGAATGGACATCTGAAAAAAGTAGATAATAACCTCACGGAAGCCCAGCG CTTCTCCTCCTTGCCTCGGAGGGCAGCTGTGAACATTGAATTCAGGGACCTTTCCTATTC GGTTCCTGAAGGACCCTGGTGGAGGAAGAAAGGATACAAGACCCTCCTGAAAGGAATTTC CGGGAAGTTCAATAGTGGTGAGTTGGTGGCCATTATGGGTCCTTCCGGGGCCGGGAAGTC CACGCTGATGAACATCCTGGCTGGATACAGGGAGACGGGCATGAAGGGGGCCGTCCTCAT CAACGGCCTGCCCCGGGACCTGCGCTGCTTCCGGAAGGTGTCCTGCTACATCATGCAGGA TGACATGCTGCTGCCGCATCTCACTGTGCAGGAGGCCATGATGGTGTCGGCACATCTGAA GCTTCAGGAGAAGGATGAAGGCAGAAGGGAAATGGTCAAGGAGATACTGACAGCGCTGGG CTTGCTGTCTTGCGCCAACACGCGGACCGGGAGCCTGTCAGGTGGTCAGCGCAAGCGCCT GGCCATCGCGCTGGAGCTGGTGAACAACCCTCCAGTCATGTTCTTCGATGAGCCCACCAG CGGCCTGGACAGCGCCTCCTGCTTCCAGGTGGTCTCGCTGATGAAAGGGCTCGCTCAAGG GGGTCGCTCCATCATTTGCACCATCCACCAGCCCAGCGCCAAACTCTTCGAGCTGTTCGA CCAGCTTTACGTCCTGAGTCAAGGACAATGTGTGTACCGGGGAAAAGTCTGCAATCTTGT GCCATATTTGAGGGATTTGGGTCTGAACTGCCCAACCTACCACAACCCAGCAGATTTTGT CATGGAGGTTGCATCCGGCGAGTACGGTGATCAGAACAGTCGGCTGGTGAGAGCGGTTCG GGAGGGCATGTGTGACTCAGACCACAAGAGAGACCTCGGGGGTGATGCCGAGGTGAACCC TTTTCTTTGGCACCGGCCCTCTGAAGAGGACTCCTCGTCCATGGAAGGCTGCCACAGCTT CTCTGCCAGCTGCCTCACGCAGTTCTGCATCCTCTTCAAGAGGACCTTCCTCAGCATCAT GAGGGACTCGGTCCTGACACACCTGCGCATCACCTCGCACATTGGGATCGGCCTCCTCAT TGGCCTGCTGTACTTGGGGATCGGGAACGAAGCCAAGAAGGTCTTGAGCAACTCCGGCTT CCTCTTCTTCTCCATGCTGTTCCTCATGTTCGCGGCCCTCATGCCTACTGTTCTGACATT TCCCCTGGAGATGGGAGTCTTTCTTCGGGAACACCTGAACTACTGGTACAGCCTGAAGGC CTACTACCTGGCCAAGACCATGGCAGACGTGCCCTTTCAGATCATGTTCCCAGTGGCCTA CTGCAGCATCGTGTACTGGATGACGTCGCAGCCGTCCGACGCCGTGCGCTTTGTGCTGTT TGCCGCGCTGGGCACCATGACCTCCCTGGTGGCACAGTCCCTGGGCCTGCTGATCGGAGC CGCCTCCACGTCCCTGCAGGTGGCCACTTTCGTGGGCCCAGTGACAGCCATCCCGGTGCT CCTGTTCTCGGGGTTCTTCGTCAGCTTCGACACCATCCCCACGTACCTACAGTGGATGTC CTACATCTCCTATGTCAGGTATGGGTTCGAAGGGGTCATCCTCTCCATCTATGGCTTAGA CCGGGAAGATCTGCACTGTGACATCGACGAGACGTGCCACTTCCAGAAGTCGGAGGCCAT CCTGCGGGAGCTGGACGTGGAAAATGCCAAGCTGTACCTGGACTTCATCGTACTCGGGAT TTTCTTCATCTCCCTCCGCCTCATTGCCTATTTTGTCCTCAGGTACAAAATCCGGGCAGA GAGGTAAAACACCTGAATGCCAGGAAACAGGAAGATTAGACACTGTGGCCGAGGGCACGT CTAGAATCGAGGAGGCAAGCCTGTGCCCGACCGACGACACAGAGACTCTTCTGATCCAAC CCCTAGAACCGCGTTGGGTTTGTGGGTGTCTCGTGCTCAGCCACTCTGCCCAGCTGGGTT GGATCTTCTCTCCATTCCCCTTTCTAGCTTTAACTAGGAAGATGTAGGCAGATTGGTGGT TTTTTTTTTTTTAACATACAGAATTTTAAATACCACAACTGGGGCAGAATTTAAAGCTC^ AACACAGCTGGTGATGAGAGGCTTCCTCAGTCCAGTCGCTCCTTAGCACCAGGCACCGTG GGTCCTGGATGGGGAACTGCAAGCAGCCTCTCAGCTGATGGCTGCACAGTCAGATGTCTG GTGGCAGAGAGTCCGAGCATGGAGCGATTCCATTTTATGACTGTTGTTTTTCACATTTTC ATCTTTCTAAGGTGTGTCTCTTTTCCAATGAGAAGTCATTTTTGCAAGCCAAAAGTCGAT CAATCGCATTCATTTTAAGAAATTATACCTTTTTAGTACTTGCTGAAGAATGATTCAGGG TAAATCACATACTTTGTTTAGAGAGGCGAGGGGTTTAACCGAGTCACCCAGCTGGTCTCA TACATAGACAGCACTTGTGAAGGATTGAATGCAGGTTCCAGGTGGAGGGAAGACGTGGAC ACCATCTCCACTGAGCCATGCAGACATTTTTAAAAGCTATACAAAAAATTGTGAGAAGAC ATTGGCCAACTCTTTCAAAGTCTTTCTTTTTCCACGTGCTTCTTATTTTAAGCGAAATAT ATTGTTTGTTTCTTCCTAAAAAAAAAAAAAAAAAAAAAAAAA
<SEQ ID NO: 16; DNA; Homo sapiens> CGCACCCCGCGCAGCGGCTGAGCCGGGAGCCAGCGCAGCCTCGGCCCCGCAGCTCAAGCC TCGTCCCCGCCGCCGCCGCCGCCGCCGCCGCCGCCGCCCCCGGGGCATGGCCTGTCTGAT GGCCGCTTTCTCGGTCGGCACCGCCATGAATGCCAGCAGTTACTCTGCAGAGATGACGGA GCCCAAGTCGGTGTGTGTCTCGGTGGATGAGGTGGTGTCCAGCAACATGGAGGCCACTGA GACGGACCTGCTGAATGGACATCTGAAAAAAGTAGATAATAACCTCACGGAAGCCCAGCG CTTCTCCTCCTTGCCTCGGAGGGCAGCTGTGAACATTGAATTCAGGGACCTTTCCTATTC GGTTCCTGAAGGACCCTGGTGGAGGAAGAAAGGATACAAGACCCTCCTGAAAGGAATTTC CGGGAAGTTCAATAGTGGTGAGTTGGTGGCCATTATGGGTCCTTCCGGGGCCGGGAAGTC CACGCTGATGAACATCCTGGCTGGATACAGGGAGACGGGCATGAAGGGGGCCGTCCTCAT CAACGGCCTGCCCCGGGACCTGCGCTGCTTCCGGAAGGTGTCCTGCTACATCATGCAGGA TGACATGCTGCTGCCGCATCTCACTGTGCAGGAGGCCATGATGGTGTCGGCACATCTGAA GCTTCAGGAGAAGGATGAAGGCAGAAGGGAAATGGTCAAGGAGATACTGACAGCGCTGGG CTTGCTGTCTTGCGCCAACACGCGGACCGGGAGCCTGTCAGGTGGTCAGCGCAAGCGCCT GGCCATCGCGCTGGAGCTGGTGAACAACCCTCCAGTCATGTTCTTCGATGAGCCCACCAG CGGCCTGGACAGCGCCTCCTGCTTCCAGGTGGTCTCGCTGATGAAAGGGCTCGCTCAAGG GGGTCGCTCCATCATTTGCACCATCCACCAGCCCAGCGCCAAACTCTTCGAGCTGTTCGA CCAGCTTTACGTCCTGAGTCAAGGACAATGTGTGTACCGGGGAAAAGTCTGCAATCTTGT GCCATATTTGAGGGATTTGGGTCTGAACTGCCCAACCTACCACAACCCAGCAGATTTTGT
CATGGAGGTTGCATCCGGCGAGTACGGTGATCAGAACAGTCGGCTGGTGAGAGCGGTTCG
GGAGGGCATGTGTGACTCAGACCACAAGAGAGACCTCGGGGGTGATGCCGAGGTGAACCC TTTTCTTTGGCACCGGCCCTCTGAAGAGGTAAAGCAGACAAAACGATTAAAGGGGTTGAG AAAGGACTCCTCGTCCATGGAAGGCTGCCACAGCTTCTCTGCCAGCTGCCTCACGCAGTT CTGCATCCTCTTCAAGAGGACCTTCCTCAGCATCATGAGGGACTCGGTCCTGACACACCT GCGCATCACCTCGCACATTGGGATCGGCCTCCTCATTGGCCTGCTGTACTTGGGGATCGG GAACGAAGCCAAGAAGGTCTTGAGCAACTCCGGCTTCCTCTTCTTCTCCATGCTGTTCCT CATGTTCGCGGCCCTCATGCCTACTGTTCTGACATTTCCCCTGGAGATGGGAGTCTTTCT TCGGGAACACCTGAACTACTGGTACAGCCTGAAGGCCTACTACCTGGCCAAGACCATGGC AGACGTGCCCTTTCAGATCATGTTCCCAGTGGCCTACTGCAGCATCGTGTACTGGATGAC GTCGCAGCCGTCCGACGCCGTGCGCTTTGTGCTGTTTGCCGCGCTGGGCACCATGACCTC CCTGGTGGCACAGTCCCTGGGCCTGCTGATCGGAGCCGCCTCCACGTCCCTGCAGGTGGC CACTTTCGTGGGCCCAGTGACAGCCATCCCGGTGCTCCTGTTCTCGGGGTTCTTCGTCAG CTTCGACACCATCCCCACGTACCTACAGTGGATGTCCTACATCTCCTATGTCAGGTATGG GTTCGAAGGGGTCATCCTCTCCATCTATGGCTTAGACCGGGAAGATCTGCACTGTGACAT CGACGAGACGTGCCACTTCCAGAAGTCGGAGGCCATCCTGCGGGAGCTGGACGTGGAAAA TGCCAAGCTGTACCTGGACTTCATCGTACTCGGGATTTTCTTCATCTCCCTCCGCCTCAT TGCCTATTTTGTCCTCAGGTACAAAATCCGGGCAGAGAGGTAAAACACCTGAATGCCAGG AAACAGGAAGATTAGACACTGTGGCCGAGGGCACGTCTAGAATCGAGGAGGCAAGCCTGT GCCCGACCGACGACACAGAGACTCTTCTGATCCAACCCCTAGAACCGCGTTGGGTTTGTG GGTGTCTCGTGCTCAGCCACTCTGCCCAGCTGGGTTGGATCTTCTCTCCATTCCCCTTTC TAGCTTTAACTAGGAAGATGTAGGCAGATTGGTGGTTTTTTTTTTTTTAACATACAGAAT TTTAAATACCACAACTGGGGCAGAATTTAAAGCTGCAACACAGCTGGTGATGAGAGGCTT CCTCAGTCCAGTCGCTCCTTAGCACCAGGCACCGTGGGTCCTGGATGGGGAACTGCAAGC AGCCTCTCAGCTGATGGCTGCACAGTCAGATGTCTGGTGGCAGAGAGTCCGAGCATGGAG CGATTCCATTTTATGACTGTTGTTTTTCACATTTTCATCTTTCTAAGGTGTGTCTCTTTT CCAATGAGAAGTCATTTTTGCAAGCCAAAAGTCGATCAATCGCATTCATTTTAAGAAATT ATACCTTTTTAGTACTTGCTGAAGAATGATTCAGGGTAAATCACATACTTTGTTTAGAGA GGCGAGGGGTTTAACCGAGTCACCCAGCTGGTCTCATACATAGACAGCACTTGTGAAGGA TTGAATGCAGGTTCCAGGTGGAGGGAAGACGTGGACACCATCTCCACTGAGCCATGCAGA CATTTTTAAAAGCTATACAAAAAATTGTGAGAAGACATTGGCCAACTCTTTCAAAGTCTT TCTTTTTCCACGTGCTTCTTATTTTAAGCGAAATATATTGTTTGTTTCTTCCTAAAAAAA AAAAAAAAAAAAAAAAAA
<SEQ ID NO: 17; DNA; Homo sapiens>
GCGAGGGGCAAGCCCGGATTCCTGCCGGCCGCCTTTCTGCGCGCGCCGGAGAGAGAGACG CGGTGGGGACAGGGATGCGCATTTCACTTCCCCGAGCTCCGGAGAGGGATGGCGGGGTGT CGGCGAGTTCACTGCTGGACACAGTTACTAATGCCAGCAGTTACTCTGCAGAGATGACGG AGCCCAAGTCGGTGTGTGTCTCGGTGGATGAGGTGGTGTCCAGCAACATGGAGGCCACTG AGACGGACCTGCTGAATGGACATCTGAAAAAAGTAGATAATAACCTCACGGAAGCCCAGC GCTTCTCCTCCTTGCCTCGGAGGGCAGCTGTGAACATTGAATTCAGGGACCTTTCCTATT CGGTTCCTGAAGGACCCTGGTGGAGGAAGAAAGGATACAAGACCCTCCTGAAAGGAATTT CCGGGAAGTTCAATAGTGGTGAGTTGGTGGCCATTATGGGTCCTTCCGGGGCCGGGAAGT CCACGCTGATGAACATCCTGGCTGGATACAGGGAGACGGGCATGAAGGGGGCCGTCCTCA TCAACGGCCTGCCCCGGGACCTGCGCTGCTTCCGGAAGGTGTCCTGCTACATCATGCAGG ATGACATGCTGCTGCCGCATCTCACTGTGCAGGAGGCCATGATGGTGTCGGCACATCTGA AGCTTCAGGAGAAGGATGAAGGCAGAAGGGAAATGGTCAAGGAGATACTGACAGCGCTGG GCTTGCTGTCTTGCGCCAACACGCGGACCGGGAGCCTGTCAGGTGGTCAGCGCAAGCGCC TGGCCATCGCGCTGGAGCTGGTGAACAACCCTCCAGTCATGTTCTTCGATGAGCCCACCA GCGGCCTGGACAGCGCCTCCTGCTTCCAGGTGGTCTCGCTGATGAAAGGGCTCGCTCAAG GGGGTCGCTCCATCATTTGCACCATCCACCAGCCCAGCGCCAAACTCTTCGAGCTGTTCG ACCAGCTTTACGTCCTGAGTCAAGGACAATGTGTGTACCGGGGAAAAGTCTGCAATCTTG TGCCATATTTGAGGGATTTGGGTCTGAACTGCCCAACCTACCACAACCCAGCAGATTTTG TCATGGAGGTTGCATCCGGCGAGTACGGTGATCAGAACAGTCGGCTGGTGAGAGCGGTTC GGGAGGGCATGTGTGACTCAGACCACAAGAGAGACCTCGGGGGTGATGCCGAGGTGAACC CTTTTCTTTGGCACCGGCCCTCTGAAGAGGACTCCTCGTCCATGGAAGGCTGCCACAGCT TCTCTGCCAGCTGCCTCACGCAGTTCTGCATCCTCTTCAAGAGGACCTTCCTCAGCATCA TGAGGGACTCGGTCCTGACACACCTGCGCATCACCTCGCACATTGGGATCGGCCTCCTCA TTGGCCTGCTGTACTTGGGGATCGGGAACGAAGCCAAGAAGGTCTTGAGCAACTCCGGCT TCCTCTTCTTCTCCATGCTGTTCCTCATGTTCGCGGCCCTCATGCCTACTGTTCTGACAT TTCCCCTGGAGATGGGAGTCTTTCTTCGGGAACACCTGAACTACTGGTACAGCCTGAAGG CCTACTACCTGGCCAAGACCATGGCAGACGTGCCCTTTCAGATCATGTTCCCAGTGGCCT ACTGCAGCATCGTGTACTGGATGACGTCGCAGCCGTCCGACGCCGTGCGCTTTGTGCTGT TTGCCGCGCTGGGCACCATGACCTCCCTGGTGGCACAGTCCCTGGGCCTGCTGATCGGAG CCGCCTCCACGTCCCTGCAGGTGGCCACTTTCGTGGGCCCAGTGACAGCCATCCCGGTGC TCCTGTTCTCGGGGTTCTTCGTCAGCTTCGACACCATCCCCACGTACCTACAGTGGATGT CCTACATCTCCTATGTCAGGTATGGGTTCGAAGGGGTCATCCTCTCCATCTATGGCTTAG ACCGGGAAGATCTGCACTGTGACATCGACGAGACGTGCCACTTCCAGAAGTCGGAGGCCA TCCTGCGGGAGCTGGACGTGGAAAATGCCAAGCTGTACCTGGACTTCATCGTACTCGGGA TTTTCTTCATCTCCCTCCGCCTCATTGCCTATTTTGTCCTCAGGTACAAAATCCGGGCAG AGAGGTAAAACACCTGAATGCCAGGAAACAGGAAGATTAGACACTGTGGCCGAGGGCACG TCTAGAATCGAGGAGGCAAGCCTGTGCCCGACCGACGACACAGAGACTCTTCTGATCCAA CCCCTAGAACCGCGTTGGGTTTGTGGGTGTCTCGTGCTCAGCCACTCTGCCCAGCTGGGT TGGATCTTCTCTCCATTCCCCTTTCTAGCTTTAACTAGGAAGATGTAGGCAGATTGGTGG TTTTTTTTTTTTTAACATACAGAATTTTAAATACCACAACTGGGGCAGAATTTAAAGCTG CAACACAGCTGGTGATGAGAGGCTTCCTCAGTCCAGTCGCTCCTTAGCACCAGGCACCGT GGGTCCTGGATGGGGAACTGCAAGCAGCCTCTCAGCTGATGGCTGCACAGTCAGATGTCT GGTGGCAGAGAGTCCGAGCATGGAGCGATTCCATTTTATGACTGTTGTTTTTCACATTTT CATCTTTCTAAGGTGTGTCTCTTTTCCAATGAGAAGTCATTTTTGCAAGCCAAAAGTCGA TCAATCGCATTCATTTTAAGAAATTATACCΓTTTTAGTACTTGCTGAAGAATGATTCAGG GTAAATCACATACTTTGTTTAGAGAGGCGAGGGGTTTAACCGAGTCACCCAGCTGGTCTC ATACATAGACAGCACTTGTGAAGGATTGAATGCAGGTTCCAGGTGGAGGGAAGACGTGGA CACCATCTCCACTGAGCCATGCAGACATTTTTAAAAGCTATACAAAAAATTGTGAGAAGA CATTGGCCAACTCTTTCAAAGTCTTTCTTTTTCCACGTGCTTCTTATTTTAAGCGAAATA TATTGTTTGTTTCTTCCTAAAAAAAAAAAAAAAAAAAAAAAAA
<SEQ ID NO: 18; DNA; Homo sapiens> Λ AGACACTGAATCATCATTTGTAGTTTGGGGGGCTTTACATGCCTGCAGTGGTGAAAACTG AAATTTTGTCCCACTTAAGGGAGTTTCTTCTTCCCTTTATTAATTGCAAAATAAATATAT GTCACTTCAGAGGGCAGCAGCTGGACTACCTATGTTTGTGGCGCTACACCAACCTGAACT TCGCTTCCTGAGACCTAAGATTCAGCCCCGTGCTCAGCAGACATCAGGGATCACCGACTC TGTGCCAGGAGCTGTTCTTGATGCTGGGAACGCAGGGGTGGACAAAACAGAGAAAGCCCT GCCCTCAGAATGCCAGCAGTTACTCTGCAGAGATGACGGAGCCCAAGTCGGTGTGTGTCT CGGTGGATGAGGTGGTGTCCAGCAACATGGAGGCCACTGAGACGGACCTGCTGAATGGAC ATCTGAAAAAAGTAGATAATAACCTCACGGAAGCCCAGCGCTTCTCCTCCTTGCCTCGGA GGGCAGCTGTGAACATTGAATTCAGGGACCTTTCCTATTCGGTTCCTGAAGGACCCTGGT GGAGGAAGAAAGGATACAAGACCCTCCTGAAAGGAATTTCCGGGAAGTTCAATAGTGGTG AGTTGGTGGCCATTATGGGTCCTTCCGGGGCCGGGAAGTCCACGCTGATGAACATCCTGG CTGGATACAGGGAGACGGGCATGAAGGGGGCCGTCCTCATCAACGGCCTGCCCCGGGACC TGCGCTGCTTCCGGAAGGTGTCCTGCTACATCATGCAGGATGACATGCTGCTGCCGCATC TCACTGTGCAGGAGGCCATGATGGTGTCGGCACATCTGAAGCTTCAGGAGAAGGATGAAG GCAGAAGGGAAATGGTCAAGGAGATACTGACAGCGCTGGGCTTGCTGTCTTGCGCCAACA CGCGGACCGGGAGCCTGTCAGGTGGTCAGCGCAAGCGCCTGGCCATCGCGCTGGAGCTGG TGAACAACCCTCCAGTCATGTTCTTCGATGAGCCCACCAGCGGCCTGGACAGCGCCTCCT GCTTCCAGGTGGTCTCGCTGATGAAAGGGCTCGCTCAAGGGGGTCGCTCCATCATTTGCA CCATCCACCAGCCCAGCGCCAAACTCTTCGAGCTGTTCGACCAGCTTTACGTCCTGAGTC AAGGACAATGTGTGTACCGGGGAAAAGTCTGCAATCTTGTGCCATATTTGAGGGATTTGG GTCTGAACTGCCCAACCTACCACAACCCAGCAGATTTTGTCATGGAGGTTGCATCCGGCG AGTACGGTGATCAGAACAGTCGGCTGGTGAGAGCGGTTCGGGAGGGCATGTGTGACTCAG ACCACAAGAGAGACCTCGGGGGTGATGCCGAGGTGAACCCTTTTCTTTGGCACCGGCCCT CTGAAGAGGACTCCTCGTCCATGGAAGGCTGCCACAGCTTCTCTGCCAGCTGCCTCACGC AGTTCTGCATCCTCTTCAAGAGGACCTTCCTCAGCATCATGAGGGACTCGGTCCTGACAC ACCTGCGCATCACCTCGCACATTGGGATCGGCCTCCTCATTGGCCTGCTGTACTTGGGGA TCGGGAACGAAGCCAAGAAGGTCTTGAGCAACTCCGGCTTCCTCTTCTTCTCCATGCTGT TCCTCATGTTCGCGGCCCTCATGCCTACTGTTCTGACATTTCCCCTGGAGATGGGAGTCT TTCTTCGGGAACACCTGAACTACTGGTACAGCCTGAAGGCCTACTACCTGGCCAAGACCA TGGCAGACGTGCCCTTTCAGATCATGTTCCCAGTGGCCTACTGCAGCATCGTGTACTGGA TGACGTCGCAGCCGTCCGACGCCGTGCGCTTTGTGCTGTTTGCCGCGCTGGGCACCATGA CCTCCCTGGTGGCACAGTCCCTGGGCCTGCTGATCGGAGCCGCCTCCACGTCCCTGCAGG TGGCCACTTTCGTGGGCCCAGTGACAGCCATCCCGGTGCTCCTGTTCTCGGGGTTCTTCG TCAGCTTCGACACCATCCCCACGTACCTACAGTGGATGTCCTACATCTCCTATGTCAGGT ATGGGTTCGAAGGGGTCATCCTCTCCATCTATGGCTTAGACCGGGAAGATCTGCACTGTG ACATCGACGAGACGTGCCACTTCCAGAAGTCGGAGGCCATCCTGCGGGAGCTGGACGTGG AAAATGCCAAGCTGTACCTGGACTTCATCGTACTCGGGATTTTCTTCATCTCCCTCCGCC TCATTGCCTATTTTGTCCTCAGGTACAAAATCCGGGCAGAGAGGTAAAACACCTGAATGC CAGGAAACAGGAAGATTAGACACTGTGGCCGAGGGCACGTCTAGAATCGAGGAGGCAAGC CTGTGCCCGACCGACGACACAGAGACTCTTCTGATCCAACCCCTAGAACCGCGTTGGGTT TGTGGGTGTCTCGTGCTCAGCCACTCTGCCCAGCTGGGTTGGATCTTCTCTCCATTCCCC TTTCTAGCTTTAACTAGGAAGATGTAGGCAGATTGGTGGTTTTTTTTTTTTTAACATACA GAATTTTAAATACCACAACTGGGGCAGAATTTAAAGCTGCAACACAGCTGGTGATGAGAG
GCTTCCTCAGTCCAGTCGCTCCTTAGCACCAGGCACCGTGGGTCCTGGATGGGGAACTGC AAGCAGCCTCTCAGCTGATGGCTGCACAGTCAGATGTCTGGTGGCAGAGAGTCCGAGCAT GGAGCGATTCCATTTTATGACTGTTGTTTTTCACATTTTCATCTTTCTAAGGTGTGTCTC TTTTCCAATGAGAAGTCATTTTTGCAAGCCAAAAGTCGATCAATCGCATTCATTTTAAGA AATTATACCTTTTTAGTACTTGCTGAAGAATGATTCAGGGTAAATCACATACTTTGTTTA GAGAGGCGAGGGGTTTAACCGAGTCACCCAGCTGGTCTCATACATAGACAGCACTTGTGA AGGATTGAATGCAGGTTCCAGGTGGAGGGAAGACGTGGACACCATCTCCACTGAGCCATG CAGACATTTTTAAAAGCTATACAAAAAATTGTGAGAAGACATTGGCCAACTCTTTCAAAG TCTTTCTTTTTCCACGTGCTTCTTATTTTAAGCGAAATATATTGTTTGTTTCTTCCTAAA AAAAAAAAAAAAAAAAAAAAAA SEQ ID NO: 19; DNA; Homo sapiens> ACTTCAGAGGGCAGCAGCTGGACTACCTATGTTTGTGGCGCTACACCAACCTGAACTTCG CTTCCTGAGACCTAAGATTCAGCCCCGTGCTCAGCAGACATCAGGGATCACCGACTCTGT GCCAGGAGCTGTTCTTGATGCTGGGAACGCAGGGGTGGACAAAACAGAGAAAGCCCTGCC CTCAGTGAGAAATATGCTGTCATGTAAATTGCTTTTTCCCCTATAGAATGCCAGCAGTTA CTCTGCAGAGATGACGGAGCCCAAGTCGGTGTGTGTCTCGGTGGATGAGGTGGTGTCCAG CAACATGGAGGCCACTGAGACGGACCTGCTGAATGGACATCTGAAAAAAGTAGATAATAA CCTCACGGAAGCCCAGCGCTTCTCCTCCTTGCCTCGGAGGGCAGCTGTGAACATTGAATT CAGGGACCTTTCCTATTCGGTTCCTGAAGGACCCTGGTGGAGGAAGAAAGGATACAAGAC CCTCCTGAAAGGAATTTCCGGGAAGTTCAATAGTGGTGAGTTGGTGGCCATTATGGGTCC TTCCGGGGCCGGGAAGTCCACGCTGATGAACATCCTGGCTGGATACAGGGAGACGGGCAT GAAGGGGGCCGTCCTCATCAACGGCCTGCCCCGGGACCTGCGCTGCTTCCGGAAGGTGTC CTGCTACATCATGCAGGATGACATGCTGCTGCCGCATCTCACTGTGCAGGAGGCCATGAT GGTGTCGGCACATCTGAAGCTTCAGGAGAAGGATGAAGGCAGAAGGGAAATGGTCAAGGA GATACTGACAGCGCTGGGCTTGCTGTCTTGCGCCAACACGCGGACCGGGAGCCTGTCAGG TGGTCAGCGCAAGCGCCTGGCCATCGCGCTGGAGCTGGTGAACAACCCTCCAGTCATGTT CTTCGATGAGCCCACCAGCGGCCTGGACAGCGCCTCCTGCTTCCAGGTGGTCTCGCTGAT GAAAGGGCTCGCTCAAGGGGGTCGCTCCATCATTTGCACCATCCACCAGCCCAGCGCCAA ACTCTTCGAGCTGTTCGACCAGCTTTACGTCCTGAGTCAAGGACAATGTGTGTACCGGGG AAAAGTCTGCAATCTTGTGCCATATTTGAGGGATTTGGGTCTGAACTGCCCAACCTACCA CAACCCAGCAGATTTTGTCATGGAGGTTGCATCCGGCGAGTACGGTGATCAGAACAGTCG GCTGGTGAGAGCGGTTCGGGAGGGCATGTGTGACTCAGACCACAAGAGAGACCTCGGGGG TGATGCCGAGGTGAACCCTTTTCTTTGGCACCGGCCCTCTGAAGAGGACTCCTCGTCCAT GGAAGGCTGCCACAGCTTCTCTGCCAGCTGCCTCACGCAGTTCTGCATCCTCTTCAAGAG GACCTTCCTCAGCATCATGAGGGACTCGGTCCTGACACACCTGCGCATCACCTCGCACAT TGGGATCGGCCTCCTCATTGGCCTGCTGTACTTGGGGATCGGGAACGAAGCCAAGAAGGT CTTGAGCAACTCCGGCTTCCTCTTCTTCTCCATGCTGTTCCTCATGTTCGCGGCCCTCAT GCCTACTGTTCTGACATTTCCCCTGGAGATGGGAGTCTTTCTTCGGGAACACCTGAACTA CTGGTACAGCCTGAAGGCCTACTACCTGGCCAAGACCATGGCAGACGTGCCCTTTCAGAT CATGTTCCCAGTGGCCTACTGCAGCATCGTGTACTGGATGACGTCGCAGCCGTCCGACGC CGTGCGCTTTGTGCTGTTTGCCGCGCTGGGCACCATGACCTCCCTGGTGGCACAGTCCCT GGGCCTGCTGATCGGAGCCGCCTCCACGTCCCTGCAGGTGGCCACTTTCGTGGGCCCAGT GACAGCCATCCCGGTGCTCCTGTTCTCGGGGTTCTTCGTCAGCTTCGACACCATCCCCAC GTACCTACAGTGGATGTCCTACATCTCCTATGTCAGGTATGGGTTCGAAGGGGTCATCCT CTCCATCTATGGCTTAGACCGGGAAGATCTGCACTGTGACATCGACGAGACGTGCCACTT CCAGAAGTCGGAGGCCATCCTGCGGGAGCTGGACGTGGAAAATGCCAAGCTGTACCTGGA CTTCATCGTACTCGGGATTTTCTTCATCTCCCTCCGCCTCATTGCCTATTTTGTCCTCAG GTACAAAATCCGGGCAGAGAGGTAAAACACCTGAATGCCAGGAAACAGGAAGATTAGACA CTGTGGCCGAGGGCACGTCTAGAATCGAGGAGGCAAGCCTGTGCCCGACCGACGACACAG AGACTCTTCTGATCCAACCCCTAGAACCGCGTTGGGTTTGTGGGTGTCTCGTGCTCAGCC ACTCTGCCCAGCTGGGTTGGATCTTCTCTCCATTCCCCTTTCTAGCTTTAACTAGGAAGA TGTAGGCAGATTGGTGGTTTTTTTTTTTTTAACATACAGAATTTTAAATACCACAACTGG GGCAGAATTTAAAGCTGCAACACAGCTGGTGATGAGAGGCTTCCTCAGTCCAGTCGCTCC TTAGCACCAGGCACCGTGGGTCCTGGATGGGGAACTGCAAGCAGCCTCTCAGCTGATGGC TGCACAGTCAGATGTCTGGTGGCAGAGAGTCCGAGCATGGAGCGATTCCATTTTATGACT GTTGTTTTTCACATTTTCATCTTTCTAAGGTGTGTCTCTTTTCCAATGAGAAGTCATTTT TGCAAGCCAAAAGTCGATCAATCGCATTCATTTTAAGAAATTATACCTTTTTAGTACTTG CTGAAGAATGATTCAGGGTAAATCACATACTTTGTTTAGAGAGGCGAGGGGTTTAACCGA GTCACCCAGCTGGTCTCATACATAGACAGCACTTGTGAAGGATTGAATGCAGGTTCCAGG TGGAGGGAAGACGTGGACACCATCTCCACTGAGCCATGCAGACATTTTTAAAAGCTATAC AAAAAATTGTGAGAAGACATTGGCCAACTCTTTCAAAGTCTTTCTTTTTCCACGTGCTTC TTATTTTAAGCGAAATATATTGTTTGTTTCTTCCTAAAAAAAAAAAAAAAAAAAAAAAAA
<SEQ ID NO: 20; DNA; Homo sapiens>
GCTTTATAAAGGGGAGTTTCCCTGCACAAGCTCTCTCTCTTGTCTGCCGCCATGTGAGAC ATGCCTTTGACCTTCCGCCATGATCATGAGGCTTCCCCAGCCACATGGAACTAATGCCAG CAGTTACTCTGCAGAGATGACGGAGCCCAAGTCGGTGTGTGTCTCGGTGGATGAGGTGGT GTCCAGCAACATGGAGGCCACTGAGACGGACCTGCTGAATGGACATCTGAAAAAAGTAGA TAATAACCTCACGGAAGCCCAGCGCTTCTCCTCCTTGCCTCGGAGGGCAGCTGTGAACAT TGAATTCAGGGACCTTTCCTATTCGGTTCCTGAAGGACCCTGGTGGAGGAAGAAAGGATA CAAGACCCTCCTGAAAGGAATTTCCGGGAAGTTCAATAGTGGTGAGTTGGTGGCCATTAT GGGTCCTTCCGGGGCCGGGAAGTCCACGCTGATGAACATCCTGGCTGGATACAGGGAGAC GGGCATGAAGGGGGCCGTCCTCATCAACGGCCTGCCCCGGGACCTGCGCTGCTTCCGGAA GGTGTCCTGCTACATCATGCAGGATGACATGCTGCTGCCGCATCTCACTGTGCAGGAGGC CATGATGGTGTCGGCACATCTGAAGCTTCAGGAGAAGGATGAAGGCAGAAGGGAAATGGT CAAGGAGATACTGACAGCGCTGGGCTTGCTGTCTTGCGCCAACACGCGGACCGGGAGCCT GTCAGGTGGTCAGCGCAAGCGCCTGGCCATCGCGCTGGAGCTGGTGAACAACCCTCCAGT CATGTTCTTCGATGAGCCCACCAGCGGCCTGGACAGCGCCTCCTGCTTCCAGGTGGTCTC GCTGATGAAAGGGCTCGCTCAAGGGGGTCGCTCCATCATTTGCACCATCCACCAGCCCAG CGCCAAACTCTTCGAGCTGTTCGACCAGCTTTACGTCCTGAGTCAAGGACAATGTGTGTA CCGGGGAAAAGTCTGCAATCTTGTGCCATATTTGAGGGATTTGGGTCTGAACTGCCCAAC CTACCACAACCCAGCAGATTTTGTCATGGAGGTTGCATCCGGCGAGTACGGTGATCAGAA CAGTCGGCTGGTGAGAGCGGTTCGGGAGGGCATGTGTGACTCAGACCACAAGAGAGACCT CGGGGGTGATGCCGAGGTGAACCCTTTTCTTTGGCACCGGCCCTCTGAAGAGGACTCCTC GTCCATGGAAGGCTGCCACAGCTTCTCTGCCAGCTGCCTCACGCAGTTCTGCATCCTCTT CAAGAGGACCTTCCTCAGCATCATGAGGGACTCGGTCCTGACACACCTGCGCATCACCTC GCACATTGGGATCGGCCTCCTCATTGGCCTGCTGTACTTGGGGATCGGGAACGAAGCCAA GAAGGTCTTGAGCAACTCCGGCTTCCTCTTCTTCTCCATGCTGTTCCTCATGTTCGCGGC CCTCATGCCTACTGTTCTGACATTTCCCCTGGAGATGGGAGTCTTTCTTCGGGAACACCT GAACTACTGGTACAGCCTGAAGGCCTACTACCTGGCCAAGACCATGGCAGACGTGCCCTT TCAGATCATGTTCCCAGTGGCCTACTGCAGCATCGTGTACTGGATGACGTCGCAGCCGTC CGACGCCGTGCGCTTTGTGCTGTTTGCCGCGCTGGGCACCATGACCTCCCTGGTGGCACA GTCCCTGGGCCTGCTGATCGGAGCCGCCTCCACGTCCCTGCAGGTGGCCACTTTCGTGGG CCCAGTGACAGCCATCCCGGTGCTCCTGTTCTCGGGGTTCTTCGTCAGCTTCGACACCAT CCCCACGTACCTACAGTGGATGTCCTACATCTCCTATGTCAGGTATGGGTTCGAAGGGGT CATCCTCTCCATCTATGGCTTAGACCGGGAAGATCTGCACTGTGACATCGACGAGACGTG CCACTTCCAGAAGTCGGAGGCCATCCTGCGGGAGCTGGACGTGGAAAATGCCAAGCTGTA CCTGGACTTCATCGTACTCGGGATTTTCTTCATCTCCCTCCGCCTCATTGCCTATTTTGT CCTCAGGTACAAAATCCGGGCAGAGAGGTAAAACACCTGAATGCCAGGAAACAGGAAGAT TAGACACTGTGGCCGAGGGCACGTCTAGAATCGAGGAGGCAAGCCTGTGCCCGACCGACG ACACAGAGACTCTTCTGATCCAACCCCTAGAACCGCGTTGGGTTTGTGGGTGTCTCGTGC TCAGCCACTCTGCCCAGCTGGGTTGGATCTTCTCTCCATTCCCCTTTCTAGCTTTAACTA GGAAGATGTAGGCAGATTGGTGGTTTTTTTTTTTTTAACATACAGAATTTTAAATACCAC AACTGGGGCAGAATTTAAAGCTGCAACACAGCTGGTGATGAGAGGCTTCCTCAGTCCAGT CGCTCCTTAGCACCAGGCACCGTGGGTCCTGGATGGGGAACTGCAAGCAGCCTCTCAGCT GATGGCTGCACAGTCAGATGTCTGGTGGCAGAGAGTCCGAGCATGGAGCGATTCCATTTT ATGACTGTTGTTTTTCACATTTTCATCTTTCTAAGGTGTGTCTCTTTTCCAATGAGAAGT CATTTTTGCAAGCCAAAAGTCGATCAATCGCATTCATTTTAAGAAATTATACCTTTTTAG TACTTGCTGAAGAATGATTCAGGGTAAATCACATACTTTGTTTAGAGAGGCGAGGGGTTT AACCGAGTCACCCAGCTGGTCTCATACATAGACAGCACTTGTGAAGGATTGAATGCAGGT TCCAGGTGGAGGGAAGACGTGGACACCATCTCCACTGAGCCATGCAGACATTTTTAAAAG CTATACAAAAAATTGTGAGAAGACATTGGCCAACTCTTTCAAAGTCTTTCTTTTTCCACG TGCTTCTTATTTTAAGCGAAATATATTGTTTGTTTCTTCCTAAAAAAAAAAAAAAAAAAA AAAAAA
<SEQ ID NO: 21; DNA; Homo sapiens> TTCTTTCCAAGGGTCTCTGGGTGAGGCCCGTGACCTTCCCAAGCCTCTCCCTGTCTTGTG AAACCTGGGCGTGATATACCTCCCTTTTAGGGCTGCTGCGATCATTTAGGCAGATTAAAC CTCATAAGTGGTTTCCCATACAAGAAAGATGCTAGCAGTGCAACAGACAGAACACTTACC TGCCTGCCCTCCCGCCAGGAGGTGGTCTTCCAACTTTTGCCCGGAGTCTACAGAGGGTGG GCCCTCTCTGCTGGGGCTCCGGGACAHGGTCAGGAGAGGTTGGTCTGTCTGTACCGCCAT ICTCTTGGCCAGACTGTGGTGTCTGGTCCCTACTCACACCTTCCTGTCAGAGTATCCAGA GGCCGCAGAGTATCCACACCCTGGCTGGGTGTACTGGCTACAGATGGCTGTGGCTCCAGG TCACCTGCGTGCCTGGGTGATGAGAAATAATGTCACAACAAATATCCCATCTGCATTCTC TGGGACACTGACCCATGAAGAGAAAGCAGTTCTCACAGTTTTTACAGGCACAGCCACAGC CGTGCATGTACAGGTGGCAGCTTTAGCTTCTGCTAAACTGGAGAGCTCAGTGTTTGTGAC AGACTGCGTGTCCTGCAAAATCGAAAATGTCTGTGATTCAGCTCTTCAGGGAAAAAGGGT GCCGATGTCTGGCCTACAGGGCTCAAGCATTGTCATCATGCCCCCATCCAACCGTCCACT CGCCAGTGCGGCATCCTGCACGTGGTCAGTCCAAGTCCAGGGAGGGCCCCATCACCTGGG GGTGGTCGCTATCAGTGGCAAAGTCTTGTCAGCAGCTCATGGGGCAGGAAGGGCCTATGG TTGGGGGTTTCCTGGCGATCCCATGGAGGAAGGATACAAGACCCTCCTGAAAGGAATTTC CGGGAAGTTCAATAGTGGTGAGTTGGTGGCCATTATGGGTCCTTCCGGGGCCGGGAAGTC CACGCTGATGAACATCCTGGCTGGATACAGGGAGACGGGCATGAAGGGGGCCGTCCTCAT CAACGGCCTGCCCCGGGACCTGCGCTGCTTCCGGAAGGTGTCCTGCTACATCATGCAGGA TGACATGCTGCTGCCGCATCTCACTGTGCAGGAGGCCATGATGGTGTCGGCACATCTGAA GCTTCAGGAGAAGGATGAAGGCAGAAGGGAAATGGTCAAGGAGATACTGACAGCGCTGGG CTTGCTGTCTTGCGCCAACACGCGGACCGGGAGCCTGTCAGGTGGTCAGCGCAAGCGCCT GGCCATCGCGCTGGAGCTGGTGAACAACCCTCCAGTCATGTTCTTCGATGAGCCCACCAG CGGCCTGGACAGCGCCTCCTGCTTCCAGGTGGTCTCGCTGATGAAAGGGCTCGCTCAAGG GGGTCGCTCCATCATTTGCACCATCCACCAGCCCAGCGCCAAACTCTTCGAGCTGTTCGA CCAGCTTTACGTCCTGAGTCAAGGACAATGTGTGTACCGGGGAAAAGTCTGCAATCTTGT GCCATATTTGAGGGATTTGGGTCTGAACTGCCCAACCTACCACAACCCAGCAGATTTTGT CATGGAGGTTGCATCCGGCGAGTACGGTGATCAGAACAGTCGGCTGGTGAGAGCGGTTCG GGAGGGCATGTGTGACTCAGACCACAAGAGAGACCTCGGGGGTGATGCCGAGGTGAACCC TTTTCTTTGGCACCGGCCCTCTGAAGAGGTAAAGCAGACAAAACGATTAAAGGGGTTGAG AAAGGACTCCTCGTCCATGGAAGGCTGCCACAGCTTCTCTGCCAGCTGCCTCACGCAGTT CTGCATCCTCTTCAAGAGGACCTTCCTCAGCATCATGAGGGACTCGGTCCTGACACACCT GCGCATCACCTCGCACATTGGGATCGGCCTCCTCATTGGCCTGCTGTACTTGGGGATCGG GAACGAAGCCAAGAAGGTCTTGAGCAACTCCGGCTTCCTCTTCTTCTCCATGCTGTTCCT CATGTTCGCGGCCCTCATGCCTACTGTTCTGACATTTCCCCTGGAGATGGGAGTCTTTCT TCGGGAACACCTGAACTACTGGTACAGCCTGAAGGCCTACTACCTGGCCAAGACCATGGC AGACGTGCCCTTTCAGATCATGTTCCCAGTGGCCTACTGCAGCATCGTGTACTGGATGAC GTCGCAGCCGTCCGACGCCGTGCGCTTTGTGCTGTTTGCCGCGCTGGGCACCATGACCTC CCTGGTGGCACAGTCCCTGGGCCTGCTGATCGGAGCCGCCTCCACGTCCCTGCAGGTGGC CACTTTCGTGGGCCCAGTGACAGCCATCCCGGTGCTCCTGTTCTCGGGGTTCTTCGTCAG CTTCGACACCATCCCCACGTACCTACAGTGGATGTCCTACATCTCCTATGTCAGGTATGG GTTCGAAGGGGTCATCCTCTCCATCTATGGCTTAGACCGGGAAGATCTGCACTGTGACAT CGACGAGACGTGCCACTTCCAGAAGTCGGAGGCCATCCTGCGGGAGCTGGACGTGGAAAA TGCCAAGCTGTACCTGGACTTCATCGTACTCGGGATTTTCTTCATCTCCCTCCGCCTCAT TGCCTATTTTGTCCTCAGGTACAAAATCCGGGCAGAGAGGTAAAACACCTGAATGCCAGG AAACAGGAAGATTAGACACTGTGGCCGAGGGCACGTCTAGAATCGAGGAGGCAAGCCTGT GCCCGACCGACGACACAGAGACTCTTCTGATCCAACCCCTAGAACCGCGTTGGGTTTGTG GGTGTCTCGTGCTCAGCCACTCTGCCCAGCTGGGTTGGATCTTCTCTCCATTCCCCTTTC TAGCTTTAACTAGGAAGATGTAGGCAGATTGGTGGTTTTTTTTTTTTTAACATACAGAAT TTTAAATACCACAACTGGGGCAGAATTTAAAGCTGCAACACAGCTGGTGATGAGAGGCTT CCTCAGTCCAGTCGCTCCTTAGCACCAGGCACCGTGGGTCCTGGATGGGGAACTGCAAGC AGCCTCTCAGCTGATGGCTGCACAGTCAGATGTCTGGTGGCAGAGAGTCCGAGCATGGAG CGATTCCATTTTATGACTGTTGTTTTTCACATTTTCATCTTTCTAAGGTGTGTCTCTTTT CCAATGAGAAGTCATTTTTGCAAGCCAAAAGTCGATCAATCGCATTCATTTTAAGAAATT ATACCTTTTTAGTACTTGCTGAAGAATGATTCAGGGTAAATCACATACTTTGTTTAGAGA GGCGAGGGGTTTAACCGAGTCACCCAGCTGGTCTCATACATAGACAGCACTTGTGAAGGA TTGAATGCAGGTTCCAGGTGGAGGGAAGACGTGGACACCATCTCCACTGAGCCATGCAGA CATTTTTAAAAGCTATACAAAAAATTGTGAGAAGACATTGGCCAACTCTTTCAAAGTCTT TCTTTTTCCACGTGCTTCTTATTTTAAGCGAAATATATTGTTTGTTTCTTCCTAAAAAAA AAAAAAAAAAAAAAAAAA
<SEQ ID NO: 22; DNA; Homo sapiens> GGAACCGCCGCCGGTATCCGCGTCCGCAGCGCCGCCAGCCAGGCGAGAGCCGTGTGGGAT CCCAGCGCCCGCACTCCCGCCCCCGCCAAGGAGCCAGGAATGGCACAACTAGAGAGGAGC GCCATCTCTGGCTTCAGCTCTAAGTCCAGGCGAAACTCATTCGCATATGATGTTAAGCGT GAAGTATACAATGAGGAGACCTTTCAACAGGAACACAAAAGGAAGGCCTCCTCTTCTGGG AACATGAACATCAACATCACCACCTTCAGACACCACGTCCAGTGCCGCTGCTCATGGCAC AGGTTCCTACGATGCGTGCTTACAATCTTTCCCTTCCTAGAATGGATGTGTATGTATCGA TTAAAGGATTGGCTTCTGGGAGACTTACTTGCTGGTATAAGTGTTGGCCTTGTGCAAGTT CCCCAAGGCCTGACACTTAGTTTGCTGGCAAGGCAACTGATTCCTCCTCTCAACATCGCT TATGCAGCTTTCTGTTCTTCGGTAATCTATGTAATTTTTGGATCGTGTCATCAAATGTCC ATTGGTTCCTTCTTCCTGGTGAGTGCTCTGCTGATCAACGTTCTGAAAGTGAGCCCATTC AACAACGGTCAACTGGTCATGGGATCTTTCGTCAAGAATGAGTTTTCGGCCCCCTCCTAC CTTATGGGCTATAATAAATCCTTGAGTGTGGTGGCAACCACAACTTTTCTGACTGGGATT ATTCAGCTAATAATGGGCGTATTGGGTTTGGGCTTCATTGCCACTTACCTTCCGGAGTCT GCAATGAGTGCTTACCTGGCTGCTGTGGCACTTCATATCATGCTGTCCCAGCTGACTTTC ATCTTTGGGATTATGATTAGTTTCCATGCCGGTCCCATCTCCTTCTTCTATGACATAATT AATTACTGTGTAGCTCTCCCAAAAGCGAATTCCACCAGCATTCTAGTATTTCTAACTGTT GTTGTTGCTCTGCGAATCAACAAATGTATCAGAATTTCTTTCAATCAGTATCCCATTGAG TTTCCCATGGAATTATTTCTGATTATTGGCTTCACTGTGATTGCAAACAAGATAAGCATG GCCACAGAAACCAGCCAGACGCTTATTGACATGATTCCTTATAGCTTTCTGCTTCCTGTA ACACCAGATTTCAGCCTTCTTCCCAAGATAATTTTACAAGCCTTCTCCTTATCTTTGGTG AGCTCCTTTCTGCTCATATTTCTGGGCAAGAAGATTGCCAGTCTTCACAATTACAGTGTC AATTCCAACCAGGATTTAATAGCCATCGGCCTTTGCAATGTCGTCAGTTCATTTTTCAGA TCTTGTGTGTTTACTGGTGCTATTGCTAGGACTATTATCCAGGATAAATCTGGAGGAAGA CAACAGTTTGCATCTCTGGTAGGCGCAGGTGTGATGCTGCTCCTGATGGTGAAGATGGGA CACTTTTTCTACACACTGCCAAATGCTGTGCTGGCTGGTATTATTCTGAGCAACGTCATT CCCTACCTTGAAACCATTTCTAACCTACCCAGCCTGTGGAGGCAGGACCAATATGACTGT GCTCTTTGGATGATGACATTCTCATCTTCAATTTTCCTGGGACTGGACATTGGACTAATT ATCTCAGTAGTTTCTGCTTTCTTCATCACCACTGTTCGTTCACACAGAGCTAAGATTCTT CTCCTGGGTCAAATCCCTAACACCAACATTTATAGAAGCATCAATGATTATCGGGAGATC ATCACCATTCCTGGGGTGAAAATCTTCCAGTGCTGCAGCTCAATTACATTTGTAAATGTT TACTACCTAAAGCATAAGCTGTTAAAAGAGGTTGATATGGTAAAGGTGCCTCTTAAAGAA GAAGAAATTTTCAGCTTGTTTAATTCAAGTGACACCAATCTACAAGGAGGAAAGATTTGC AGGTGTTTCTGCAACTGTGATGATCTGGAGCCGCTGCCCAGGATTCTTTACACAGAGCGA TTTGAAAATAAACTGGATCCCGAAGCATCCTCCATTAACCTGATTCACTGCTCACATTTT GAGAGCATGAACACAAGCCAAACTGCATCCGAAGACCAAGTGCCATACACAGTATCGTCC GTGTCTCAGAAAAATCAAGGGCAACAGTATGAGGAGGTGGAGGAAGTTTGGCTTCCTAAT AACTCATCAAGAAACAGCTCACCAGGACTGCCTGATGTGGCGGAAAGCCAGGGGAGGAGA TCACTCATCCCTTACTCAGATGCGTCTCTACTGCCCAGTGTCCACACCATCATCCTGGAT TTCTCCATGGTACACTACGTGGATTCACGGGGGTTAGTCGTATTAAGACAGATATGCAAT GCCTTTCAAAACGCCAACATTTTGATACTCATTGCAGGGTGTCACTCTTCCATAGTCAGG GCATTTGAGAGGAATGATTTCTTTGACGCTGGCATCACCAAGACCCAGCTGTTCCTCAGC GTTCACGACGCCGTGCTGTTTGCCTTGTCAAGGAAGGTCATAGGCTCCTCTGAGTTAAGC ATCGATGAATCCGAGACAGTGATACGGGAAACCTACTCAGAAACAGACAAGAATGACAAT TCAAGATATAAAATGAGCAGCAGTTTTCTAGGAAGCCAAAAAAATGTAAGTCCAGGCTTC ATCAAGATCCAACAGCCTGTAGAAGAGGAGTCGGAGTTGGATTTGGAGCTGGAATCAGAA CAAGAGGCTGGGCTGGGTCTGGACCTAGACCTGGATCGGGAGCTGGAGCCTGAAATGGAG CCCAAGGCTGAGACCGAGACCAAGACCCAGACCGAGATGGAGCCCCAGCCTGAGACTGAG CCTGAGATGGAGCCCAACCCCAAATCTAGGCCAAGAGCTCACACTTTTCCTCAGCAGCGT TACTGGCCTATGTATCATCCGTCTATGGCTTCCACCCAGTCTCAGACTCAGACTCGGACA TGGTCAGTGGAGAGGAGACGCCATCCTATGGATTCATACTCACCAGAGGGCAACAGCAAT GAAGATGTCTAGGAGATGAACTAGAAATAAGGGGTCAGATAATGCTGGCAAATCCTCCTA CCCAAAAAGGGGTCAATTGTCCAGAGACCTAGACTGGATACGAACTAGCAGTACTTCCTT CCTGACTGTGACTCCTACTACCTGCCAGCCTTCTTCCTTGCTCTGCGCTGGGATCATACT CCGAAATCACATTACTAAATGCCAACAATTATCTCTGAATTCCCTATCCAGG.CTCCCCTC ATTTCACCTTCAGCATATATTCTAGTCATGAATTTCCTTCTTCACACACCCCACATCTCT GGGCTTTGTGCCAGACCATCTCTAACTTAATCCTCTCATCCCTGTTCCCCTTTCTCCAAA GAGATGAAGCTCAAATAAAATGTATAACTCTAGTAAAAAA
<SEQ ID NO: 23; DNA; Homo sapiens> ATTGAATGCAGCAAGGGTCTGGAGGCTGAGGACCAGGCAGACAAACATTCAGAGTTGCTG GAATGCGACAGAGACAGGGAGTCAGACTGGTCATGCAAGGCCAGCTCTGGGGTCTCGGCA GGTGGTCCGCGACATGACCTCCGAGTTCTTCTCTGCCCAGCTCCGGGCCCAGATCTCTGA CGACACCACTCACCCGATCTCCTACTACAAGCCCGAGTTCTACATGCCGGATGACGGGGG CACTGCTCACCTGTCTGTGGTCGCAGAGGACGGCAGTGCTGTGTCCGCCACCAGCACCAT CAACCTCTACTTTGGCTCCAAGGTGCGCTCCCCAGTCAGCGGGATCCTGCTCAATAATGA AATGGATGACTTCAGCTCTACCAGCATCACCAACGAGTTTGGGGTACCCCCCTCACCTGC CAATTTCATCCAGCCAGGGAAGCAGCCGCTCTCGTCCATGTGCCCGACGATCATGGTGGG CCAGGACGGCCAGGTCCGGATGGTGGTGGGAGCTGCCGGGGGCACGCAGATCACCATGGC CACTGCACTGGCCATCATCTACAACCTCTGGTTCGGCTATGACGTGAAGTGGGCCGTGGA GGAGCCCCGGCTGCACAACCAGCTTCTGCCCAACGTCACGACAGTGGAGAGAAACATTGA CCAGGAAGTGACTGCAGCCCTGGAGACCCGGCACCATCACACCCAGATCACGTCCACCTT CATTGCTGTGGTGCAAGCCATCGTCCGCATGGCTGGTGGCTGGGCAGCTGCCTCGGACTC CAGGAAAGGTGGGGAACCTGCTGGCTACTGATTGCTCCAGGCGGACAAGGCTGACAAGCA ATCCAGGAACAAAATACTCACCAGGACGAGGAAGAGGACTTTGGGGGACAGGCTTCTCCT GTGAGCAGCAGAGCAGCACAATAAATGAGGCCACTGTGCCAGGCTCCAGGTGGCCTCCCT GGCCTGTCTCCCCACTC
<SEQ ID NO: 24; DNA; Homo sapiens>
CTCGAGAGCTGGGCTCTGCGTCCTCGTCCAGCCGCCAACTCGGCCAAAGGCGAAGCCAGC AGTTTCTTCTGCTGCCGGGCAACGCGCCTTTTAAACCTGAGGGAGTGGGCGCGTGAGCAC TTAATGGCGCCGGTGACAGAGTGAGCTTAACGGATTAATAAGCGCAGCCAGGCCAGCTCT GGGGTCTCGGCAGGTGGTCCGCGACATGACCTCCGAGTTCTTCTCTGCCCAGCTCCGGGC CCAGATCTCTGACGACACCACTCACCCGATCTCCTACTACAAGCCCGAGTTCTACATGCC GGATGACGGGGGCACTGCTCACCTGTCTGTGGTCGCAGAGGACGGCAGTGCTGTGTCCGC CACCAGCACCATCAACCTCTACTTTGGCTCCAAGGTGCGCTCCCCAGTCAGCGGGATCCT GCTCAATAATGAAATGGATGACTTCAGCTCTACCAGCATCACCAACGAGTTTGGGGTACC CCCCTCACCTGCCAATTTCATCCAGCCAGGGAAGCAGCCGCTCTCGTCCATGTGCCCGAC GATCATGGTGGGCCAGGACGGCCAGGTCCGGATGGTGGTGGGAGCTGCCGGGGGCACGCA GATCACCATGGCCACTGCACTGGCCATCATCTACAACCTCTGGTTCGGCTATGACGTGAA GTGGGCCGTGGAGGAGCCCCGGCTGCACAACCAGCTTCTGCCCAACGTCACGACAGTGGA GAGAAACATTGACCAGGAAGTGACTGCAGCCCTGGAGACCCGGCACCATCACACCCAGAT CACGTCCACCTTCATTGCTGTGGTGCAAGCCATCGTCCGCATGGCTGGTGGCTGGGCAGC TGCCTCGGACTCCAGGAAAGGTGGGGAACCTGCTGGCTACTGATTGCTCCAGGCGGACAA GGCTGACAAGCAATCCAGGAACAAAATACTCACCAGGACGAGGAAGAGGACTTTGGGGGA CAGGCTTCTCCTGTGAGCAGCAGAGCAGCACAATAAATGAGGCCACTGTGCCAGGCTCCA GGTGGCCTCCCTGGCCTGTCTCCCCACTC
<SEQ ID NO: 25; DNA; Homo sapiens> ATTGAATGCAGCAAGGGTCTGGAGGCTGAGGACCAGGCAGACAAACATTCAGAGTTGCTG GAATGCGACAGAGACAGGGAGTCAGACTGGTCATGCAAGGTCCTGGGCCTGCCCTTGGGT CCTGGGGAGCCACGGAAGGTTGTGGGTGCCAGAGGGTTGTGGTCAGAGCCACAGTCAGGG GCCTTCTGAGACCTGTGCCCCCTCCCCACCCTCCCTCCCCACCTCCCTAGGCCAGCTCTG GGGTCTCGGCAGGTGGTCCGCGACATGACCTCCGAGTTCTTCTCTGCCCAGCTCCGGGCC CAGATCTCTGACGACACCACTCACCCGATCTCCTACTACAAGCCCGAGTTCTACATGCCG GATGACGGGGGCACTGCTCACCTGTCTGTGGTCGCAGAGGACGGCAGTGCTGTGTCCGCC ACCAGCACCATCAACCTCTACTTTGGCTCCAAGGTGCGCTCCCCAGTCAGCGGGATCCTG CTCAATAATGAAATGGATGACTTCAGCTCTACCAGCATCACCAACGAGTTTGGGGTACCC CCCTCACCTGCCAATTTCATCCAGCCAGGGAAGCAGCCGCTCTCGTCCATGTGCCCGACG ATCATGGTGGGCCAGGACGGCCAGGTCCGGATGGTGGTGGGAGCTGCCGGGGGCACGCAG ATCACCATGGCCACTGCACTGGCCATCATCTACAACCTCTGGTTCGGCTATGACGTGAAG TGGGCCGTGGAGGAGCCCCGGCTGCACAACCAGCTTCTGCCCAACGTCACGACAGTGGAG AGAAACATTGACCAGGAAGTGACTGCAGCCCTGGAGACCCGGCACCATCACACCCAGATC ACGTCCACCTTCATTGCTGTGGTGCAAGCCATCGTCCGCATGGCTGGTGGCTGGGCAGCT GCCTCGGACTCCAGGAAAGGTGGGGAACCTGCTGGCTACTGATTGCTCCAGGCGGACAAG GCTGACAAGCAATCCAGGAACAAAATACTCACCAGGACGAGGAAGAGGACTTTGGGGGAC AGGCTTCTCCTGTGAGCAGCAGAGCAGCACAATAAATGAGGCCACTGTGCCAGGCTCCAG GTGGCCTCCCTGGCCTGTCTCCCCACTC
<SEQ ID NO: 26; DNA; Homo sapiens> GTCAGAGTCTTCCCTCAGCTTTGTACATCAGCACTGTTTTGATAGATAAGAGAGGCTACT GTATAAGTGTGTAAGATTTGTAATGGACTGAGGAATGCTTGTTCTAGCTGCTGAAACTGA CTGTAACTGTATTGTCTTAGGAGCATCATCATGGGGTCTAGTGCCACAGAGATTGAAGAA TTGGAAAACACCACTTTTAAGTATCTTACAGGAGAACAGACTGAAAAAATGTGGCAGCGC CTGAAAGGAATACTAAGATGCTTGGTGAAGCAGCTGGAAAGAGGTGATGTTAACGTCGTC GACTTAAAGAAGAATATTGAATATGCGGCATCTGTGCTGGAAGCAGTTTATATCGATGAA ACAAGAAGACTTCTGGATACTGAAGATGAGCTCAGTGACATTCAGACTGACTCAGTCCCA TCTGAAGTCCGGGACTGGTTGGCTTCTACCTTTACACGGAAAATGGGGATGACAAAAAAG AAACCTGAGGAAAAACCAAAATTTCGGAGCATTGTGCATGCTGTTCAAGCTGGAATTTTT GTGGAAAGAATGTACCGAAAAACATATCATATGGTTGGTTTGGCATATCCAGCAGCTGTC ATCGTAACATTAAAGGATGTTGATAAATGGTCTTTCGATGTATTTGCCCTAAATGAAGCA AGTGGAGAGCATAGTCTGAAGTTTATGATTTATGAACTGTTTACCAGATATGATCTTATC AACCGTTTCAAGATTCCTGTTTCTTGCCTAATCACCTTTGCAGAAGCTTTAGAAGTTGGT TACAGCAAGTACAAAAATCCATATCACAATTTGATTCATGCAGCTGATGTCACTCAAACT GTGCATTACATAATGCTTCATACAGGTATCATGCACTGGCTCACTGAACTGGAAATTTTA GCAATGGTCTTTGCTGCTGCCATTCATGATTATGAGCATACAGGGACAACAAACAACTTT CACATTCAGACAAGGTCAGATGTTGCCATTTTGTATAATGATCGCTCTGTCCTTGAGAAT CACCACGTGAGTGCAGCTTATCGACTTATGCAAGAAGAAGAAATGAATATCTTGATAAAT TTATCCAAAGATGACTGGAGGGATCTTCGGAACCTAGTGATTGAAATGGTTTTATCTACA GACATGTCAGGTCACTTCCAGCAAATTAAAAATATAAGAAACAGTTTGCAGCAGCCTGAA GGGATTGACAGAGCCAAAACCATGTCCCTGATTCTCCACGCAGCAGACATCAGCCACCCA GCCAAATCCTGGAAGCTGCATTATCGGTGGACCATGGCCCTAATGGAGGAGTTTTTCCTG CAGGGAGATAAAGAAGCTGAATTAGGGCTTCCATTTTCCCCACTTTGTGATCGGAAGTCA ACCATGGTGGCCCAGTCACAAATAGGTTTCATCGATTTCATAGTAGAGCCAACATTTTCT CTTCTGACAGACTCAACAGAGAAAATTGTTATTCCTCTTATAGAGGAAGCCTCAAAAGCC GAAACTTCTTCCTATGTGGCAAGCAGCTCAACCACCATTGTGGGGTTACACATTGCTGAT GCACTAAGACGATCAAATACAAAAGGCTCCATGAGTGATGGGTCCTATTCCCCAGACTAC TCCCTTGCAGCAGTGGACCTGAAGAGTTTCAAGAACAACCTGGTGGACATCATTCAGCAG AACAAAGAGAGGTGGAAAGAGTTAGCTGCACAAGGTGAATCTGATCTTCATAAGAACTCA GAAGACTTAGTAAATGCTGAAGAAAAACATGATGAGACACATTCATAGGCCCGAAACACC TTAAAGACTTCTGTCATTTTAAACATGAGAGGACAATGAAATCAGCATGAAAACATCCTA AATTCTCAACTTTCCACAAAGCTATGGCTCTTCTTTCAACATAGAATTGGATTGGGCCAT TTTAATTGACTCCTATACAAGGAATTAAGAAGAACATAAATTTTGAGCTAGTAACTCTGG CCAAATAAATACACTCAAGTTTTTATCAGAGTTTTTGGCCAGTGCTTCTGCCATTTTTTT CCCTCCACAATTTGGCCTTCTTCAATCAAGCCAGATAAATTTTTGAGACAAAAGTCAGAC AGTTTTTAATTTTTCTTGCTTTGAACCCTGTCATAATGACTGTGCAATACATGTGCAGAA GATGAGGTATTTTAAAATTTACTTCCTTGCACTGTCTTACACAGAGTGCTATAACTATAA ATTTTTCAAGGTCTTAAATAAAAGGAAGCAAAAACAAAATTATTGAAAAATTTTTTTTGT TGTGCTGGGGAATATACTATTTAGATTGTCCTTCTTATTTTAAATGCATGGGAACAGAAT GACAGGGGGGATGCTGAGGAGCTGGTTGAAGCATCAGAGCAATGCTACAGTCCAACAATG GAGCATTAGATCCC
<SEQ ID NO: 27; DNA; Homo sapiens>
GAATTCTGATGTGCTTCAGTGCACAGAACAGTAACAGATGAGCTGGTTTTGGGGAGAGCT
TGAGTACTCAGTCGGAGCATCATCATGGGGTCTAGTGCCACAGAGATTGAAGAATTGGAA AACACCACTTTTAAGTATCTTACAGGAGAACAGACTGAAAAAATGTGGCAGCGCCTGAAA GGAATACTAAGATGCTTGGTGAAGCAGCTGGAAAGAGGTGATGTTAACGTCGTCGACTTA AAGAAGAATATTGAATATGCGGCATCTGTGCTGGAAGCAGTTTATATCGATGAAACAAGA AGACTTCTGGATACTGAAGATGAGCTCAGTGACATTCAGACTGACTCAGTCCCATCTGAA GTCCGGGACTGGTTGGCTTCTACCTTTACACGGAAAATGGGGATGACAAAAAAGAAACCT GAGGAAAAACCAAAATTTCGGAGCATTGTGCATGCTGTTCAAGCTGGAATTTTTGTGGAA AGAATGTACCGAAAAACATATCATATGGTTGGTTTGGCATATCCAGCAGCTGTCATCGTA ACATTAAAGGATGTTGATAAATGGTCTTTCGATGTATTTGCCCTAAATGAAGCAAGTGGA GAGCATAGTCTGAAGTTTATGATTTATGAACTGTTTACCAGATATGATCTTATCAACCGT TTCAAGATTCCTGTTTCTTGCCTAATCACCTTTGCAGAAGCTTTAGAAGTTGGTTACAGC AAGTACAAAAATCCATATCACAATTTGATTCATGCAGCTGATGTCACTCAAACTGTGCAT TACATAATGCTTCATACAGGTATCATGCACTGGCTCACTGAACTGGAAATTTTAGCAATG GTCTTTGCTGCTGCCATTCATGATTATGAGCATACAGGGACAACAAACAACTTTCACATT CAGACAAGGTCAGATGTTGCCATTTTGTATAATGATCGCTCTGTCCTTGAGAATCACCAC GTGAGTGCAGCTTATCGACTTATGCAAGAAGAAGAAATGAATATCTTGATAAATTTATCC AAAGATGACTGGAGGGATCTTCGGAACCTAGTGATTGAAATGGTTTTATCTACAGACATG TCAGGTCACTTCCAGCAAATTAAAAATATAAGAAACAGTTTGCAGCAGCCTGAAGGGATT GACAGAGCCAAAACCATGTCCCTGATTCTCCACGCAGCAGACATCAGCCACCCAGCCAAA TCCTGGAAGCTGCATTATCGGTGGACCATGGCCCTAATGGAGGAGTTTTTCCTGCAGGGA GATAAAGAAGCTGAATTAGGGCTTCCATTTTCCCCACTTTGTGATCGGAAGTCAACCATG GTGGCCCAGTCACAAATAGGTTTCATCGATTTCATAGTAGAGCCAACATTTTCTCTTCTG ACAGACTCAACAGAGAAAATTGTTATTCCTCTTATAGAGGAAGCCTCAAAAGCCGAAACT TCTTCCTATGTGGCAAGCAGCTCAACCACCATTGTGGGGTTACACATTGCTGATGCACTA AGACGATCAAATACAAAAGGCTCCATGAGTGATGGGTCCTATTCCCCAGACTACTCCCTT GCAGCAGTGGACCTGAAGAGTTTCAAGAACAACCTGGTGGACATCATTCAGCAGAACAAA GAGAGGTGGAAAGAGTTAGCTGCACAAGAAGCAAGAACCAGTTCACAGAAGTGTGAGTTT ATTCATCAGTAAACACCTTTAAGTAAAACCTCGTGCATGGTGGCAGCTCTAATTTGACCA AAAGACTTGGAGATTTTGATTATGCTTGCTGGAAATCTACCCTGTCCTGTGTGAGACAGG AAATCTATTTTTGCAGATTGCTCAATAAGCATCATGAGCCACATAAATAACAGCTGTAAA CTCCTTAATTCACCGGGCTCAACTGCTACCGAACAGATTCATCTAGTGGCTACATCAGCA CCTTGTGCTTTCAGATATCTGTTTCAATGGCATTTTGTGGCATTTGTCTTTACCGAGTGC CAATAAATTTTCTTTGAGCAGCTAAAAAA
<SE ID NO: 28; DNA; Homo sapiens> AAATGCTTGAGGAGAGAGAGAGAGTAAGGAGCCAGCCATGAATCCTTTCCAGAAAAATGA GTCCAAGGAAACTCTTTTTTCACCTGTCTCCATTGAAGAGGTACCACCTCGACCACCTAG CCCTCCAAAGAAGCCATCTCCGACAATCTGTGGCTCCAACTATCCACTGAGCATTGCCTT CATTGTGGTGAATGAATTCTGCGAGCGCTTTTCCTATTATGGAATGAAAGCTGTGCTGAT CCTGTATTTCCTGTATTTCCTGCACTGGAATGAAGATACCTCCACATCTATATACCATGC CTTCAGCAGCCTCTGTTATTTTACTCCCATCCTGGGAGCAGCCATTGCTGACTCGTGGTT GGGAAAATTCAAGACAATCATCTATCTCTCCTTGGTGTATGTGCTTGGCCATGTGATCAA GTCCTTGGGTGCCTTACCAATACTGGGAGGACAAGTGGTACACACAGTCCTATCATTGAT CGGCCTGAGTCTAATAGCTTTGGGGACAGGAGGCATCAAACCCTGTGTGGCAGCTTTTGG TGGAGACCAGTTTGAAGAAAAACATGCAGAGGAACGGACTAGATACTTCTCAGTCTTCTA CCTGTCCATCAATGCAGGGAGCTTGATTTCTACATTTATCACACCCATGCTGAGAGGAGA TGTGCAATGTTTTGGAGAAGACTGCTATGCATTGGCTTTTGGAGTTCCAGGACTGCTCAT GGTAATTGCACTTGTTGTGTTTGCAATGGGAAGCAAAATATACAATAAACCACCCCCTGA AGGAAACATAGTGGCTCAAGTTTTCAAATGTATCTGGTTTGCTATTTCCAATCGTTTCAA GAACCGTTCTGGAGACATTCCAAAGCGACAGCACTGGCTAGACTGGGCGGCTGAGAAATA TCCAAAGCAGCTCATTATGGATGTAAAGGCACTGACCAGGGTACTATTCCTTTATATCCC ATTGCCCATGTTCTGGGCTCTTTTGGATCAGCAGGGTTCACGATGGACTTTGCAAGCCAT CAGGATGAATAGGAATTTGGGGTTTTTTGTGCTTCAGCCGGACCAGATGCAGGTTCTAAA TCCCCTTCTGGTTCTTATCTTCATCCCGTTGTTTGACTTTGTCATTTATCGTCTGGTCTC CAAGTGTGGAATTAACTTCTCATCACTTAGGAAAATGGCTGTTGGTATGATGCTAGCATG CCTGGCATTTGCAGTTGCGGCAGCTGTAGAGATAAAAATAAATGAAATGGCCCCAGCCCA GCCAGGTCCCCAGGAGGTTTTCCTACAAGTCTTGAATCTGGCAGATGATGAGGTGAAGGT GACAGTGGTGGGAAATGAAAACAATTCTCTGTTGATAGAGTCCATCAAATCCTTTCAGAA AACACCACACTATTCCAAACTGCACCTGAAAACAAAAAGCCAGGATTTTCACTTCCACCT GAAATATCACAATTTGTCTCTCTACACTGAGCATTCTGTGCAGGAGAAGAACTGGTACAG TCTTGTCATTCGTGAAGATGGGAAGAGTATCTCCAGCATGATGGTAAAGGATACAGAAAG CAGAACAACCAATGGGATGACAACCGTGAGGTTTGTTAACACTTTGCATAAAGATGTCAA CATCTCCCTGAGTACAGATACCTCTCTCAATGTTGGTGAAGACTATGGTGTGTCTGCTTA TAGAACTGTGCAAAGAGGAGAATACCCTGCAGTGCACTGTAGAACAGAAGATAAGAACTT TTCTCTGAATTTGGGTCTTCTAGACTTTGGTGCAGCATATCTGTTTGTTATTACTAATAA CACCAATCAGGGTCTTCAGGCCTGGAAGATTGAAGACATTCCAGCCAACAAAATGTCCAT TGCGTGGCAGCTACCACAATATGCCCTGGTTACAGCTGGGGAGGTCATGTTCTCTGTCAC AGGTCTTGAGTTTTCTTATTCTCAGGCTCCCTCTGGCATGAAATCTGTGCTCCAGGCAGC TTGGCTATTGACAATTGCAGTTGGGAATATCATCGTGCTTGTTGTGGCACAGTTCAGTGG CCTGGTACAGTGGGCCGAATTCATTTTGTTTTCCTGCCTCCTGCTGGTGATCTGCCTGAT CTTCTCCATCATGGGCTACTACTATGTTCCTGTAAAGACAGAGGATATGCGGGGTCCAGC AGATAAGCACATTCCTCACATCCAGGGGAACATGATCAAACTAGAGACCAAGAAGACAAA ACTCTGATGACTCCCTAGATTCTGTCCTGACCCCAATTCCTGGCCCTGTCTTGAAGCATT TTTTTTCTTCTACTGGATTAGACAAGAGAGATAGCAGCATATCAGAGCTGATCTCCTCCA CCTTTCTCCAATGACAGAAGTTCCAGGACTGGTTTTCCAGTACATCTTTAAACAAGGCCC CAGAGACTCTATGTCTGCCCGTCCATCAGTGAACTCATTAAAACTTGTGCAGTGTTGCTG GAGCTGGCCTGGTGTCTCCAAATGACCATGAAAATACACACGTATAATGGAGATCATTCT CTGTGGGTATGCAAAGTTATGGGAATTCCTTTATAGGTAACTGCCATTTAGGACTGATGG CCCTAATTTTTGAGGTGCTGATTTAGAGGCAAAATTGCAGAATAACAAAGAAATGGTATT TCAAGTTTTTTTTTTTATAAGCAATGTAATTATGCTATTCACAGGGGCCTCAAGAATTGG TATGTATGATGTGATCTGGTCCAGCCAGGGCCTGGCTTGTCAGCTCTCTAGGTTTGATAT GACTTTAGTAAATTTGTCAATATAGATGGTAGGAAGCAGAATGCCATTTTATTAAAACAC AGGAGAAGTTAAAAAAAAAAAAAAAA
<SEQ ID NO: 101; PRT; Homo sapiens> GTTAPGPIHLLELCDQKLMEFLCNMDNKDLVWLEEIQEEAERMFTREFSKEPELMPKTP ΞQKNRRKKRRISYVQDENRDPIRRRLSRRKSRSSQLSSRRLRSKDSVEKLATVVGENGSV LRRVTRAAAAAAAATMALAAPSSPTPESPTMLTKKPEDNHTQCQLVPWEIGISERQNAE QHVTQLMSTEPLPRTLSPTPASATAPTSQGIPTSDEESTPKKSKARILESITVSSLMATP QDPKGQGVGTGRSASKLRIAQVSPGPRDSPAFPDSPWRERVLAPI PDNFSTPTGSRTDS QSVRHSPIAPSSPSPQVAQKYSLVAKQESWRRASRRLAKKTAEEPAASGRIICHSYLE RLLNVEVPQKVGSEQKEPPEEAEPVAAAEPEVPENNGNNSWPHNDTEIANSTPNPKPAAS SPETPSAGQQEAKTDQADGPREPPQSARRKRSYKQAVSELDEEQHLEDEELQPPRSKTPS SPCPASKWRPLRTFLHTVQRNQMLMTPTSAPRSVMKSFIKRNTPLRMDPKEKERQRLEN LRRKEEAEQLRRQKVEEDI^RRRLEEVKLKREERLRKVLQARERVEQMKEEKKKQIEQKFA QIDEKTEKAKEERLAEEKAKKKAAAKKMEEVEARRKQEEDARRLRWLQQEEEERRHQELL QKKKEEEQERLRKAAEAKR AEQREQERREQERREQERREQERREQERREQERREQERQ AEQERRREQERLQAERELQEREKALRLQKEQLQRELEEKKKKEEQQRLAERQLQEEQEKK AKEAAGASKALNVTVDVQSPACTSSPITPQGHKAPPQINPHNYGMDLNSDDSTDDEAHPR KPIPTWARGTPLSQAIIHQYYQPPNLLE FGTILPLDLEDIFKKSKPRYHKRTSSAVWNS PPLQGARVPSSLAYSLKKH
<SEQ ID NO: 102; PRT; Homo sapiens>
MTNQEKAHLSPSEFSQLQKYAEYSTKKLKDVLEEFHGNGVLAKYNPEGKQDILNQTIDF EGFKLFMKTFLEAELPDDFTAHLFMSFSNKFPHSSPMVKSKPALLSGG RMNKGAITPPR TTSPANTCSPEVIHLKDIVCYLSLLERGRPEDKLEFMFRLYDTDGNGFLDSSELENIISQ MHVAEYLEWDVTELNPILHE MEEIDYDHDGTVSLEE IQGG TTIPLLVLLG ENNVK DDGQHV RLKHFNKPAYCNLCLNMLIGVGKQGLCCSFCKYTVHERCVARAPPSCIKTYVK SKRNTDVMHHYWVEGNCPTKCDKCHKTVKCYQGLTGLHCVWCQITLHNKCASHLKPECDC GPLKDHILPPTTICPWLQTLPTSGVSVPEERQSTVKKEKSGSQQPNKVIDKNKMQRANS VTVDGQG QVTPVPGTHPLLVFVNPKSGGKQGERIYRKFQYLLNPRQVYSLSGNGPMPGL NFFRDVPDFRVLACGGDGTVG VLDCIEKANVGKHPPVAILPLGTGNDLARCLRWGGGYE GENLMKILKDIENSTEIMLDR KFEVIPNDKDEKGDPVPYSIINNYFSIGVDASIAHRFH IMREKHPEKFNSRMKNKFWYFEFGTSETFSATCKKLHESVEIECDGVQID INISLEGIA ILNIPSMHGGSNL GESKKRRSHRRIEKKGSDKRTTVTDAKELKFASQDLSDQL EWGL EGAMEMGQIYTGLKSAGRRLAQCSCVVIRTSKSLPMQIDGEPWMQTPCTIKITHKNQAPM LMGPPPKTGLFCSLVKRTRNRSKE
<SEQ ID NO: 103; PRT; Homo sapiens>
MTNQEKAHLSPSEFSQLQKYAEYSTKKLKDVLEEFHGNGVLAKYNPEGKQDILNQTIDF
EGFKLFMKTFLEAELPDDFTAHLFMSFSNKFPHSSPMVKSKPAL SGGLRMNKGAITPPR TTSPANTCSPEVIHLKDIVCYLSLLERGRPEDKLEFMFRLYDTDGNGFLDSSELENIISQ MMHVAEYLE DVTELNPILHEMMEEIDYDHDGTVSLEE IQGGMTTIPLLVLLGLENNVK DDGQHVWRLKHFNKPAYCNLCLN LIGVGKQGLCCSFCKYTVHERCVARAPPSCIKTYVK SKRNTDVMHHY VEGNCPTKCDKCHKTVKCYQGLTGLHCV CQITLHNKCASHLKPECDC GPLKDHILPPTTICPVVLQTLPTSGVSVPEERQSTVKΪEKSGSQQPNKVIDKNKMQRANS VTVDGQGLQVTPVPGTHPLLVFVNPKSGGKQGERIYRKFQYLLNPRQVYSLSGNGPMPGL NFFRDVPDFRVLACGGDGTVGWVLDCIEKANVGKHPPVAILPLGTGNDLARCLRWGGGYE GENLMKILKDIENSTEIMLDR KFEVIPNDKDEKGDPVPYSIINNYFSIGVDASIAHRFH IMREKHPEKFNSRMKN FWYFEFGTSETFSATCKKLHESVEIECDGVQIDLINISLEGIA ILNIPSMHGGSNL GESKKRRSHRRIEKKGSDΪORTTVTDAELKFASQDLSDQLLEWGL EGAMEMGQIYTGLKSAGRRLAQCSCWIRTSKSLPMQIDGEP MQTPCTVSTE
<SEQ ID NO: 104; PRT; Homo sapiens>
MNRYTTIRQLGDGTYGSVLLGRSIESGELIAIKKMKRKFYS EECMN REVKSLKKLNHA NWKLKEVIRENDHLYFIFEYMKENLYQLIKERNKLFPESAIRNIMYQILQGLAFIHKHG FFHRDLKPEN LCMGPELVKIADFGLAREIRSKPPYTDYVSTRWYRAPEVLLRSTNYSSP IDVWAVGCIMAEVYT RPLFPGASEIDTIFKICQVLGTPKKTDWPEGYQ SSAMNFRWPQ CVPNNLKTLIPNASSEAVQLLRDMLQWDPKKRPTASQALRYPYFQVGHPLGSTTQNLQDS EKPQKGI EKAGPPPYIKPVPPAQPPAKPHTRISSRQHQASQPPLHLTYPYKAEVSRTDH PSHLQEDKPSPLLFPSLHNKHPQSKITAGLEHKNGEIKPKSRRR GLISRSTKDSDD AD LDDLDFSPSLSRID KNKKRQSDDTLCRFESVLDLKPSEPVGTGNSAPTQTSYQRRDTPT LRSAAKQHYLKHSRYLPGISIRNGI SNPGKEFIPPNPWSSSGLSGKSSGTMSVISKVNS VGSSSTSSSGLTGNYVPSFLKKEIGSAMQRVH APIPDPSPGYSSLKA RPHPGRPFFHT QPRSTPGLIPRPPAAQPVHGRTD ASKYASRR
<SEQ ID NO: 105; PRT; Homo sapiens>
MNRYTTIRQLGDGTYGSVLLGRSIESGELIAIKIOMKRKFYSWEECMNLREVKSLKKLNHA NWKLKEVIRENDHLYFIFEYMKENLYQLIKERNKLFPESAIRNIMYQILQGLAFIHKHG FFHRDLKPENLLCMGPELVKIADFGLAREIRSKPPYTDYVSTRWYRAPEVLLRSTNYSSP IDVAVGCIMAEVYTLRPLFPGASEIDTIFKICQVLGTPKKTDWPEGYQLSSAMNFRWPQ CVPNNLKTLIPNASSEAVQLLRDMLQ DPKECRPTASQALRYPYFQVGHPLGSTTQNLQDS EKPQKGI EKAGPPPYIKPVPPAQPPAKPHTRISSRQHQASQPPLHLTYPYKAEVSRTDH PSHLQEDKPSP LFPSLHNKHPQSKITAGLEHKNGEIKPKSRRRWGLISRSTKDSDDWAD LDDLDFSPSLSRIDLKNKKRQSDDTLCRFESVLDLKPSEPVGTGNSAPTQTSYQRRDTPT LRSAAKQHYLKHSRYLPGISIRNGI SNPGKEFIPPNPWSSSGLSGKSSGTMSVISKVNS VGSSSTSSSGLTGNYVPSFLKKEIGSAMQRVHLAPIPDPSPGYSSLKAMRPHPGRPFFHT QPRSTPGLIPRPPAAQPVHGRTD ASKYASRR
<SEQ ID NO: 106; PRT; Homo sapiens> MICCSALSPRIHLSFHRSLTGIVLANSSLDIVLHDTYYWAHCGGNVRR HCGGPASRER TAMQALNITPEQFSRLLRDHNLTREQFIALYRLRPLVYTPELPGRAKLALVLTGVLIFAL ALFGNALVFYWTRSKAMRTVTNIFICSLA SDLLITFFCIPVTMLQNISDN LGGAFIC EMVPFVQSTAVVTEILTMTCIAVERHQGLVHPFK KWQYTNRRAFTMLGVV LVAVIVGS PM HVQQ EIKYDFLYEKEHICC EE TSPVHQKIYTTFILSSSSSCLLWKKKRAVIMMV TWALFAVCWAPFHVVHMMIEYSNFEKEYDDVTIK IFAIVQIIGFSNSICNPIVYAFMN ENFKKNVLSAVCYCIVNKTFSPAQRHGNSGIT MRKKAKFSLRENPVEETKGEAFSDGNI EVK CEQTEEKKK KRHLALFRSELAENSP DSGH
<SEQ ID NO: 107; PRT; Homo sapiens> MQALNITPEQFSRLLRDHNLTREQFIAVHRLRPLVYTPELPGRAKLALVLTGVLIFALAL FGNALVFYWTRSKAMRTVTNIFICSLA SDLLITFFCIPVTM QNISDN LGGAFICKM VPFVQSTAVVTEILTMTCIAVERHQGVHPFKMK QYTNRRAFT LGVV LVAVIVGSPM WHVQQLEIKYDFLYEKEHICCLEE TSPVHQKIYTTFILVILF LPLMVMLILYSKIGYE LWIKKRVGDGSVLRTIHGKEMSKIARKKIRAVIMMVTVVALFAVC APFHVVHMMIEYSN FEKEYDDVTIKMIFAIVQIIGFSNSICNPIVYAF NENFKKNVLSAVCYCIVNKTFSPAQ RHGNSGITMMRKKAKFSLRENPVEETKGEAFSDGNIEVKLCEQTEEKKKLKRHLALFRSE LAENSPLDSGH
<SEQ ID NO: 108; PRT; Homo sapiens> MAEEEAPKKSRAAGGGASWE CAGALSARAEEGSGDAGGRRRPPVDPRRLARQ LLLL LLEAPLL GVRAQAAGQGPGQGPGPGQQPPPPPQQQQSGQQYNGERGISVPDHGYCQPIS IPLCTDIAYNQTIMPNLLGHTNQEDAGLEVHQFYPLVKVQCSAELKFFLCSMYAPVCTVL EQA PPCRSLCERARQGCEAL NKFGFQ PDTLKCEKFPVHGAGELCVGQNTSDKGTPTP SLLPEFWTSNPQHGGGGHRGGFPGGAGASERGKFSCPRALKVPSY NYHFLGEKDCGAPC EPTKVYGLMYFGPEELRFSRTWIGI SVLCCASTLFTVLTYLVDMRRFSYPERPIIF SG CYTAVAVAYIAGFLLEDRVVCNDKFAEDGARTVAQGTKKEGCTILFM LYFFSMASSI VILSLT F AAG K GHEAIEANSQYFHLAAWAVPAIKTITILALGQVDGDVLSGVCFVG LNNVDALRGFVLAPLFVYLFIGTSFLLAGFVSLFRIRTI KHDGTKTEKLEKLMVRIGVF SVLYTVPATIVIACYFYEQAFRDQ ERS VAQSCKSYAIPCPHLQAGGGAPPHPPMSPDF TVFMIKYLMTLIVGITSGF I SGKTLNS RKFYTRLTNSKQGETTV <SEQ ID NO: 109; PRT; Homo sapiens> MLVLYGHSTQD PETNARVVGGTEAGRNS PSQISLQYRSGGSRYHTCGGTLIRQNV T AAHCVDYQKTFRWAGDHNLSQNDGTEQYVSVQKIWHPYWNSDNVAAGYDIAL RLAQS VTLNSYVQLGVLPQEGAILANNSPCYITG GKTKTNGQ AQTLQQAYLPSVDYAICSSSS Y GSTVKNTMVCAGGDGVRSGCQGDSGGPLHCLVNGKYSVHGVTSFVSSRGCNVSRKPTV FTQVSAYIS INNVIASN <SEQ ID NO: 110; PRT; Homo sapiens> MTAITHGSPVGGNDSQGQVLDGQSQHLFQQNQTSSPDSSNENSVATPPPEEQGQGDAPPQ HEDEEPAFPHTELANLDDMINRPRWPVLPKGELEVLLEAAIDLSVKG DVKSEACQRF FRDGLTISFTKILMDEAVSG KFEIHRCIINNTHRLVE CVAKLSQDWFPLLELLAMALN PHCKFHIYNGTRPCELISSNAQLPEEELFARSSDPRSPKGWLVDLINKFGTLNGFQILHD RFFNGSALNIQIIAALIKPFGQCYEFLSQHTLKKYFIPVIEIVPHLLENLTDEELKKEAK NEAKNDALSMIIKSLKNLASRISGQDETIKNLEIFRLΪ ILRLLQISSFNGK NALNEIN KVISSVSYYTHRHSNPEEEE LTAERMAEWIQQNNILSIVLQDSLHQPQYVEKLEKILRF VIKEKALTLQDLDNIWAAQAGKHEAIVKNVHDLLAKLAWDFSPGQ DHLFDCFKASWTNA SKKQREKLLELIRRLAEDDKDGV AHECVLNLLWNLAQSDDVPVDIMDLALSAHIKILDYS CSQDRDAQKIQ IDHFIEELRTNDKVIPALKQIREICSLFGEASQNLSQTQRSPHIFYR HDLINQLQQNHALVTLVAENLATYMNSIRYAGDHEDYDPQTVRLGSRYSHVQEVQERLN FLRFLLKDGQLWLCAPQAKQIWKCLAENAVYLCDREACFKWYSKLMGDEPDLDPDINKDF FESNVLQLDPSLLTENGMKCFERFFKAVNCRERKLIAKRRSYMMDD ELIGLDYL RWI QSSDEIANRAIDLLKEIYTNLGPRLKANQVVIHEDFIQSCFDRLKASYDTLCVFDGDKNS INCARQEAIRMVRVLTVIKEYINECDSDYHKERMILPMSRAFCGKHLSLIVRFPNQGRQV DELDIWFHTNDTIGSVRRCIVNRIKAWAHKKIELFVGGELIDSENDRKLIGQ NLKDKS LITAKLTQINFNMPSSPDSSSDSSTASPGNHRNHYNDGPNLKVESCLPGVIMSVHPKYIS FLWQFANLGSNLNMPPLKNGARVLMKL PPDRTAVEELRTVCLDHANLGEGKLSPPLDSL
^iFFGPSASQVLYLTEWYALLMPAGVPLTDGSSDFQVHFLKSGGLPLVL§MLIRNNFLPNT DMETRRGAYLNALKIAKLLLTAIGyGHVRAVAEACQPWDGTDPITQINQVTHDQAWLQ SALQSIPNPSSECVLRNESILLAQEISNEASRYMPDICVIRAIQKII ASACGALGLFFS PNEEITKIYQ TTNGSNKLEVEDEQVCCEALEV TLCFALLPTALDALSKEKAQTFIID LLLHCPSKTVRQLAQEQFFLMCTRCCMGHRPLLFFITLLFTILGSTAREKGKYSGDYFT LRHLLNYAYNGNINIPNAEVLLVSEID LKRIRDNVKNTGETGVEEPILEGHLGVTKELL AFQTSEKKYHFGCEKGGANLIKELIDDFIFPASKVYLQYLRSGELPAEQAIPVCSSPVTI NAGFELLVALAIGCVRNLKQIVDCLTEMYYMGTAITTCEALTEWEY PPVGPRPPKGFVG LKNAGATCYMNSVIQQ YMIPSIRNSILAIEGTGSDLHDDMFGDEKQDSESNVDPRDDVF GYPHQFEDKPALSKTEDRKEYNIGVLRHLQVIFGHLAASQLQYYVPRGF KQFRLWGEPV NLREQHDALEFFNSLVDSLDEALKALGHPAILSKVLGGSFADQKICQGCPHRFECEESFT TLNVDIRNHQNLLDSLEQYIKGDLLEGANAYHCEKCDKKVDTVKRLLIKK PRVLAIQLK RFDYDWERECAIKFNDYFEFPRELDMGPYTVAGVANLERDNVNSENE IEQKEQSDNETA GGTKYRLVGVLVHSGQASGGHYYSYIIQRNGKDDQTDHWYKFDDGDVTECKMDDDEEMKN QCFGGEYMGEVFDHM KRMSYRRQKRWWNAYIPFYEQMDMIDEDDEMIRYISELTIARPH QIIMSPAIERSVRKQNVKFMHNRLQYSLEYFQFVK LLTCNGVYLNPAPGQDYLLPEAEE ITMISIQLAARFLFTTGFHTKKIVRGPASDWYDALCVLLRHSKNVRFWFTHNVLFNVSNR FSEYLLECPSAEVRGAFAKLIVFIAHFS QDGSCPSPFASPGPSSQACDNLSLSDHLLRA TLNLLRREVSEHGHHLQQYFNLFVMYANLGVAEKTQLLKLNVPATFMLVSLDEGPGPPIK YQYAELGKLYSWSQ IRCCNVSSTMQSSINGNPPLPNPFGDLNLSQPI PIQQNVLDI FVRTSYVKKIIEDCSNSEDTIKLLRFCSWENPQFSSTVLSELL QVAYSYTYELRPYLDL LFQILLIEDS QTHRIHNALKGIPDDRDGLFDTIQRSKNHYQKRAYQCIKC VALFSSCP VAYQILQGNGDLKRKWTWAVEWLGDELERRPYTGNPQYSYISINWSPPVQSNETANGYFLER SHSARMTLAKACELCPEEEPDDQDAPDEHEPSPSEDAPLYPHSPASQYQQNNHVHGQPYT GPAAHHLNNPQKTGQRTQENYEGNEEVSSPQMKDQ
<SEQ ID NO: 111; PRT; Homo sapiens> MSQ HHPRSG GRRRDFSGRSSAKKKGGNHIPER KDYLPVGQRMPGTRFIAFKVPLQKS FEKKLAPEECFSP DLFNKIREQNEELGLIID TYTQRYYKPEDLPETVPYLKIFTVGHQ VPDDETIFKFKHAVNGFLKENKDNDKLIGVHCTHGLNRTGYLICRYLIDVEGVRPDDAIE LFNRCRGHCLERQNYIEDLQNGPIRKN NSSVPRSSDFEDSAHLMQPVHNKPVKQGPRYN LHQIQGHSAPRHFHTQTQSLQQSVRKFSENPHVYQRHHLPPPGPPGEDYSHRRYSWNVKP NASRAAQDRRR YPYNYSRLSYPACWEWTQ
<SEQ ID NO: 112; PRT; Homo sapiens>
MSGSFELSVQDLNDLLSDGSGCYSLPSQPCNEVTPRIYVGNASVAQDIPKLQKLGITHVL NAAEGRSFMHVNTNANFYKDSGITYLGIKANDTQEFNLSAYFERAADFIDQALAQKNGRV LVHCREGYSRSPT VIAYLMMRQKMDVKSALSIVRQNREIGPNDGFLAQLCQLNDRLAKE GKLKP
<SEQ ID NO: 113; PRT; Homo sapiens> MQYLNIKEDCNAMAFCAKMRSSKKTEVNLEAPEPGVEVIFYLSDREPLRLGSGEYTAEEL CIRAAQACRISP CHNLFALYDENTK WYAPNRTITVDDKMSLRLHYRMRFYFTN HGTN DNEQSVWRHSPKKQKNGYEKKKIPDATPLLDASSLEYLFAQGQYDLVKCLAPIRDPKTEQ DGHDIENECLG AVLAISHYAMMKKMQLPELPKDISYKRYIPETLNKSIRQRNLLTRMRI NNVFKDFLKEFNNKTICDSSVSTHDLKVKYLATLET TKHYGAEIFETSM LISSENEMN FHSNDGGNVLYYEVMVTGNLGIQ RHKPNWSVEKEKNKLKRKKLENKDKKDEEKNKIR EΞ NNFSFFPEITHIVIKESWSINKQDNKKMELKLSSHEEA SFVSLVDGYFRLTADAH HYLCTDVAPPLIVHNIQNGCHGPICTEYAINKLRQEGSEEGMYVLRWSCTDFDNI MTVT CFEKSEQVQGAQKQF NFQIEVQKGRYSLHGSDRSFPSLGDLMSHLKKQILRTDNISFML KRCCQPKPREISNLLVATKKAQEWQPVYPMSQLSFDRI KKDLVQGEHLGRGTRTHIYSG TLMDYKDDEGTSEEKKIKVI KVLDPSHRDISLAFFEAASMMRQVSHKHIVYLYGVCVRD VENIMVEEFVEGGPLDLFMHRKSDVLTTPWKFKVAKQLASALSYLEDKDLVHGNVCTKNL AREGIDSECGPFIK SDPGIPITVLSRQECIERIPWIAPECVEDSKNLSVAADKWSFG TTL EICYNGEIPLKDKTLIEKERFYESRCRPVTPSCKELADLMTRCMNYDPNQRPFFRA IMRDINKLEEQNPDIVSRKKNQPTEVDPTHFEKRFLECRIRDLGEGHFGKVELCRYDPEDN TGEQVAVKSLKPESGGNHIAD KKEIEILRNLYHENIVKYKGICTEDGGNGIKLIMEFLP SGSLKEYLPϊaiKNKINLKQQLKYAVQICKGMDYLGSRQYVHRDLAARNVLVESEHQVKIG DFGLTKAIETDKEYYTVKDDRDSPVFWYAPECLMQSKFYIASDVWSFGVTLHELLTYCDS DSSPMALFLKMIGPTHGQMTVTRLVNTLKEGKRLPCPPNCPDEVYQLMRKC EFQPSNRT SFQNLIEGFEALLK <SEQ ID NO: 114; PRT; Homo sapiens>
MAFCAKMRSSKKTEVNLEAPEPGVEVIFYLSDREPLRLGSGEYTAEELCIRAAQACRISP LCHNLFALYDENTKL YAPNRTITVDDKMSLR HYRMRFYFTN HGTNDNEQSVRHSPK KQKNGYEKKKIPDATP LDASSLEYLFAQGQYD VKCLAPIRDPKTEQDGHDIENECLGM AVLAISHYAMMKK Q PELPKDISYKRYIPETLNKSIRQRNL TRMRINNVFKDFLKEFN NKTICDSSVSTHD KVKYLATLETLTKHYGAEIFETSMLLISSENEMNWFHSNDGGNVLY YEVMVTGNLGIQWRHKPNWSVEKEKNKLKRKKLENKDKKDEEKNKIREE NNFSFFPEI THIVIKESWSINKQDNKKMELKLSSHEEALSFVSLVDGYFRLTADAHHYLCTDVAPPLI VHNIQNGCHGPICTEYAINΪ RQEGSEEGMYV R SCTDFDNIL TVTCFEKSEQVQGAQ KQFKWFQIEVQKGRYSLHGSDRSFPSLGDLMSHLKKQILRTDNISFMLKRCCQPKPREIS NLLVATKΪCAQE QPVYP SQLSFDRILKKDLVQGEHLGRGTRTHIYSGTLMDYKDDEGTS EEKKIKVILKVLDPSHRDISLAFFEAASMMRQVSHKHIVYLYGVCVRDVENIMVEEFVEG GPLD FMHRKSDVLTTP KFKVAKQLASALSY EDKDLVHGNVCTKNLLLAREGIDSECG PFIKLSDPGIPITVLSRQECIERIP IAPECVEDSKNLSVAADKWSFGTTLWEICYNGEI PLKDKTLIEECERFYESRCRPVTPSCKELADLMTRCMNYDPNQRPFFRAIMRDINKLEEQN PDIVSRKKNQPTEVDPTHFEKRFLKRIRDLGEGHFGKVELCRYDPEDNTGEQVAVKSLKP ESGGNHIADLKΪEIEILRNLYHENIVKYKGICTEDGGNGIKLIMEFLPSGSLKEYLPKNK NKINLKQQLKYAVQICKGMDYLGSRQYVHRDLAARNVLVESEHQVKIGDFGLTKAIETDK EYYTVKDDRDSPVFWYAPECLMQSKFYIASDV SFGVTLHELLTYCDSDSSP ALFLKMI GPTHGQMTVTRLVNTLKEGKRLPCPPNCPDEVYQLMRKCWEFQPSNRTSFQNLIEGFEAL LK
<SEQ ID NO: 115; PRT; Homo sapiens>
MACLMAAFSVGTAMNASSYSAEMTEPKSVCVSVDEWSSNMEATETDLLNGHLKKVDNNL
TEAQRFSSLPRRAAVNIEFRDLSYSVPEGPWWRKKGYKTLLKGISGKFNSGELVAIMGPS GAGKSTLIΦJILAGYRETGMKGAVLINGLPRDLRCFRKVSCYIMQDDMLLPHLTVQEAMMV SAHLKLQEKDEGRREMVKEILTALGLLSCANTRTGSLSGGQRKRLAIALELVNNPPVMFF DEPTSGLDSASCFQVVSLMKGLAQGGRSIICTIHQPSAKLFELFDQLYVLSQGQCVYRGK VCNLVPYLRDLGLNCPTYHNPADFVMEVASGEYGDQNSRLVRAVREGMCDSDHKRDLGGD AEVNPFL HRPSEEDSSSMEGCHSFSASCLTQFCILFKRTFLSIMRDSVLTHLRITSHIG IGLLIGLLYLGIGNEAKKVLSNSGFLFFSMLFMFAALMPTVLTFPLEMGVFLREHLNY YSLKAYYLAKTMADVPFQIMFPVAYCSIVYWMTSQPSDAVRFVLFAALGTMTSLVAQSLG LLIGAASTSLQVATFVGPVTAIPVLLFSGFFVSFDTIPTYLQ MSYISYVRYGFEGVILS IYGLDREDLHCDIDETCHFQKSEAILRELDVENAKLYLDFIVLGIFFISLRLIAYFVLRY KIRAER
<SEQ ID NO: 116; PRT; Homo sapiens> ACLMAAFSVGTAMNASSYSAEMTEPKSVCVSVDEVVSSNMEATETDLLNGHLKKVDNNL TEAQRFSSLPRRAAVNIEFRDLSYSVPEGP RKKGYKTLLKGISGKFNSGELVAIMGPS GAGKSTLI^ILAGYRETGMKGAVLINGLPRDLRCFRKVSCYIMQDDMLLPHLTVQEAMMV SAHLKLQEKDEGRREMVKEILTALGLLSCANTRTGSLSGGQRKRLAIALELVNNPPVMFF DEPTSGLDSASCFQWSLMKGLAQGGRSIICTIHQPSAKLFELFDQLYVLSQGQCVYRGK VCNLVPYLRDLGLNCPTYHNPADFVMEVASGEYGDQNSRLVRAVREGMCDSDHKRDLGGD AEVNPFL HRPSEEVKQTKRLKGLRKDSSSMEGCHSFSASCLTQFCILFKRTFLSIMRDS VLTHLRITSHIGIGLLIGLLYLGIGNEAKKVLSNSGFLFFS LFLMFAALMPTVLTFPLE MGVFLREHLNY YSLKAYYLAKTMADVPFQIMFPVAYCSIVYWMTSQPSDAVRFVLFAAL GTMTSLVAQSLGLLIGAASTSLQVATFVGPVTAIPVLLFSGFFVSFDTIPTYLQ MSYIS YVRYGFEGVILSIYGLDREDLHCDIDETCHFQKSEAILRELDVENAKLYLDFIVLGIFFI SLRLIAYFVLRYKIRAER <SEQ ID NO: 117; PRT; Homo sapiens>
MRISLPRAPERDGGVSASSLLDTVTNASSYSAEMTEPKSVCVSVDEVVSSNMEATETDLL NGHLKKVDNNLTEAQRFSSLPRRAAVNIEFRDLSYSVPEGP WRKKGYKTLLKGISGKFN SGELVAIMGPSGAGKSTLMNILAGYRETGMKGAVLINGLPRDLRCFRKVSCYIMQDDMLL PHLTVQEAMMVSAHLKLQEKDEGRREMVKEILTALGLLSCANTRTGSLSGGQRKRLAIAL ELVNNPPVMFFDEPTSGLDSASCFQVVSL KGLAQGGRSIICTIHQPSAKLFELFDQLYV LSQGQCVYRGKVCNLVPYLRDLGLNCPTYHNPADFVMEVASGEYGDQNSRLVRAVREG C DSDHKRDLGGDAEVNPFL HRPSEEDSSSMEGCHSFSASCLTQFCILFKRTFLSIMRDSV LTHLRITSHIGIGLLIGLLYLGIGNEAKKVLSNSGFLFFSMLFL FAALMPTVLTFPLEM GVFLREHLNYWYSLKA.YYLAKTMADVPFQIMFPVAYCSIVYWMTSQPSDAVRFVLFAALG TMTSLVAQSLGLLIGAASTSLQVATFVGPVTAIPVLLFSGFFVSFDTIPTYLQWMSYISY VRYGFEGVILSIYGLDREDLHCDIDETCHFQKSEAILRELDVENAKLYLDFIVLGIFFIS LRLIAYFVLRYKIRAER
<SEQ ID NO: 118; PRT; Homo sapiens> MLGTQGWTKQRKPCPQNASSYSAEMTEPKSVCVSVDEWSSNMEATETDLLNGHLKKVDN NLTEAQRFSSLPRRAAVNIEFRDLSYSVPEGPWWRKKGYKTLLKGISGKFNSGELVAIMG PSGAGKSTL NILAGYRETGMKGAVLINGLPRDLRCFRKVSCYIMQDDM LPHLTVQEAM MVSAHLECLQEKDEGRRE VKEILTALGLLSCANTRTGSLSGGQRKRLAIALELVNNPPVM FFDEPTSGLDSASCFQWSLMKGLAQGGRSIICTIHQPSAKLFELFDQLYVLSQGQCVYR GKVCNLVPYLRDLGLNCPTYHNPADFVMEVASGEYGDQNSRLVRAVREGMCDSDHKRDLG GDAEVNPFLWHRPSEEDSSSMEGCHSFSASCLTQFCILFKRTFLSIMRDSVLTHLRITSH IGIGLLIGLLYLGIGNEAKKVLSNSGFLFFSMLFLMFAALMPTVLTFPLEMGVFLREHLN YWYSLKAYYLAKTMADVPFQIMFPVAYCSIVYWMTSQPSDAVRFVLFAALGTMTSLVAQS LGLLIGAASTSLQVATFVGPVTAIPVLLFSGFFVSFDTIPTYLQWMSYISYVRYGFEGVI LSIYGLDREDLHCDIDETCHFQKSEAILRELDVENAKLYLDFIVLGIFFISLRLIAYFVL RYKIRAER
<SEQ ID NO: 119; PRT; Homo sapiens> MTEPKSVCVSVDEVVSSNMEATETDLLNGHLKKVDNNLTEAQRFSSLPRRAAVNIEFRDL SYSVPEGPWWRKKGYKTLLKGISGKFNSGELVAIMGPSGAGKSTLMNILAGYRETGMKGA VLINGLPRDLRCFR VSCYIMQDDMLLPHLTVQEAMMVSAHLKLQEKDEGRRE VKEILT ALGLLSCANTRTGSLSGGQRKRLAIALELVNNPPVMFFDEPTSGLDSASCFQVVSLMKGL AQGGRSIICTIHQPSAKLFELFDQLYVLSQGQCVYRGKVCNLVPYLRDLGLNCPTYHNPA DFVMEVASGEYGDQNSRLVRAVREGMCDSDHKRDLGGDAEVNPFL HRPSEEDSSSMEGC HSFSASCLTQFCILFKRTFLSIMRDSVLTHLRITSHIGIGLLIGLLYLGIGNEAKKVLSN SGFLFFSMLFLMFAALMPTVLTFPLEMGVFLREHLNY YSLKAYYLAKTMADVPFQIMFP VAYCSIVYWMTSQPSDAVRFVLFAALGTMTSLVAQSLGLLIGAASTSLQVATFVGPVTAI PVLLFSGFFVSFDTIPTYLQ MSYISYVRYGFEGVILSIYGLDREDLHCDIDETCHFQKS EAILRELDVENAKLYLDFIVLGIFFISLRLIAYFVLRYKIRAER
<SEQ ID NO: 120; PRT; Homo sapiens>
MIMRLPQPHGTNASSYSAEMTEPKSVCVSVDEVVSSNMEATETDLLNGHLKKVDNNLTEA QRFSSLPRRAAVNIEFRDLSYSVPEGP WRKKGYKTLLKGISGKFNSGELVAIMGPSGAG KSTLMNILAGYRETGMKGAVLINGLPRDLRCFRKVSCYIMQDDMLLPHLTVQEAMMVSAH LKLQEKDEGRRE VKEILTALGLLSCANTRTGSLSGGQRKRLAIALELVNNPPVMFFDEP TSGLDSASCFQWSLMKGLAQGGRSIICTIHQPSAKLFELFDQLYVLSQGQCVYRGKVCN LVPYLRDLGLNCPTYHNPADFVMEVASGEYGDQNSRLVRAVREGMCDSDHKRDLGGDAEV NPFL HRPSEEDSSS EGCHSFSASCLTQFCILFKRTFLSIMRDSVLTHLRITSHIGIGL LIGLLYLGIGNEAKΪVLSNSGFLFFSMLFLMFAALMPTVLTFPLEMGVFLREHLNY YSL KAYYLAKTMADVPFQIMFPVAYCSIVYWMTSQPSDAVRFVLFAALGTMTSLVAQSLGLLI GAASTSLQVATFVGPVTAIPVLLFSGFFVSFDTIPTYLQ SYISYVRYGFEGVILSIYG LDREDLHCDIDETCHFQKSEAILRELDVENAKLYLDFIVLGIFFISLRLIAYFVLRYKIR AER <SEQ ID NO: 121; PRT; Homo sapiens>
MLAVQQTEHLPACPPARRWSSNFCPESTEGGPSLLGLRDMVRRG SVCTAILLARL CLV PTHTFLSEYPEAAEYPHPG VYWLQMAVAPGHLRAWVMRNNVTTNIPSAFSGTLTHEEiA VLTVFTGTATAVHVQVAALASAKLESSVFVTDCVSCKIENVCDSALQGKRVPMSGLQGSS IVIMPPSNRPLASAASCTWSVQVQGGPHHLGWAISGKVLSAAHGAGRAYGWGFPGDPME EGYKTLLKGISGKFNSGELVAIMGPSGAGKSTLMNILAGYRETGMKGAVLINGLPRDLRC FRKVSCYIMQDDMLLPHLTVQEAM SAHLECLQEKDEGRREMVKEILTALGLLSCANTRT GSLSGGQRKRLAIALELVNNPPVMFFDEPTSGLDSASCFQVVSLMKGLAQGGRSIICTIH QPSAKLFELFDQLYVLSQGQCVYRGKVCNLVPYLRDLGLNCPTYHNPADFVMEVASGEYG DQNSRLVRAVREGMCDSDHKRDLGGDAEVNPFLWHRPSEEVKQTKRLKGLRKDSSSMEGC HSFSASCLTQFCILFKRTFLSI RDSVLTHLRITSHIGIGLLIGLLYLGIGNEAKKVLSN SGFLFFSI^FLMFAALMPTVLTFPLEMGVFLREHLNYWYSLKAYYLAKTMADVPFQIMFP VAYCSIVYMTSQPSDAVRFVLFAALGT TSLVAQSLGLLIGAASTSLQVATFVGPVTAI PVLLFSGFFVSFDTIPTYLQ MSYISYVRYGFEGVILSIYGLDREDLHCDIDETCHFQKS EAILRELDVENAKLYLDFIVLGIFFISLRLIAYFVLRYKIRAER
<SEQ ID NO: 122; PRT; Homo sapiens>
MAQLERSAISGFSSKSRRNSFAYDVKREVYNEETFQQEHKRKASSSGNMNINITTFRHHV QCRCSWHRFLRCVLTIFPFLE MCMYRLKD LLGDLLAGISVGLVQVPQGLTLSLLARQL IPPLNIAYAAFCSSVIYVIFGSCHQMSIGSFFLVSALLINVLKVSPFNNGQLVMGSFVKN EFSAPSYLMGYNKSLSWATTTFLTGIIQLIMGVLGLGFIATYLPESAMSAYLAAVALHI MLSQLTFIFGIMISFHAGPISFFYDIINYCVALPKANSTSILVFLTVWALRINKCIRIS FNQYPIEFPMELFLIIGFTVIANKISMATETSQTLIDMIPYSFLLPVTPDFSLLPKIILQ AFSLSLVSSFLLIFLGKKIASLHNYSVNSNQDLIAIGLCNWSSFFRSCVFTGAIARTII QDKSGGRQQFASLVGAGV LLL VKMGHFFYTLPNAVLAGIILSNVIPYLETISNLPSL RQDQYDCALWMMTFSSSIFLGLDIGLIISVVSAFFITTVRSHRAKILLLGQIPNTNIYRS INDYREIITIPGVKIFQCCSSITFVNVYYLKHKLLKEVDMVKVPLKEEEIFSLFNSSDTN LQGGKICRCFCNCDDLEPLPRILYTERFENKLDPEASSINLIHCSHFESMNTSQTASEDQ VPYTVSSVSQKNQGQQYEEVEEVWLPNNSSRNSSPGLPDVAESQGRRSLIPYSDASLLPS VHTIILDFSMVHYVDSRGLWLRQICNAFQNANILILIAGCHSSIVRAFERNDFFDAGIT KTQLFLSVHDAVLFALSRKVIGSSELSIDESETVIRETYSETDKNDNSRYKMSSSFLGSQ KNVSPGFIKIQQPVEEESELDLELESEQEAGLGLDLDLDRELEPEMEPKAETETKTQTEM EPQPETEPEMEPNPKSRPRAHTFPQQRY PMYHPSMASTQSQTQTRT SVERRRHPMDSY SPEGNSNEDV <SEQ ID NO: 123; PRT; Homo sapiens>
MTSEFFSAQLRAQISDDTTHPISYYKPEFYMPDDGGTAHLSWAEDGSAVSATSTINLYF GSKVRSPVSGILLNNEMDDFSSTSITNEFGVPPSPANFIQPGKQPLSSMCPTIMVGQDGQ VRMVVGAAGGTQITMATALAIIYNLWFGYDVKWAVEEPRLHNQLLPNVTTVERNIDQEVT AALETRHHHTQITSTFIAWQAIVRMAGGWAAASDSRKGGEPAGY
<SEQ ID NO: 124; PRT; Homo sapiens>
MTSEFFSAQLRAQISDDTTHPISYYKPEFYMPDDGGTAHLSWAEDGSAVSATSTINLYF GSKVRSPVSGILLNNEMDDFSSTSITNEFGVPPSPANFIQPGKQPLSSMCPTIMVGQDGQ VRMWGAAGGTQITMATALAIIYNLWFGYDVKWAVEEPRLHNQLLPNVTTVERNIDQEVT AALETRHHHTQITSTFIAVVQAIVRAGGAAASDSRKGGEPAGY
<SEQ ID NO: 125; PRT; Homo sapiens>
MTSEFFSAQLRAQISDDTTHPISYYKPEFYMPDDGGTAHLSWAEDGSAVSATSTINLYF GSKVRSPVSGILLNNEMDDFSSTSITNEFGVPPSPANFIQPGKQPLSSMCPTIMVGQDGQ VRMWGAAGGTQITMATALAIIYNL FGYDVK AVEEPRLHNQLLPNVTTVERNIDQEVT AALETRHHHTQITSTFIAVVQAIVRMAGG AAASDSRKGGEPAGY <SEQ ID NO: 126; PRT; Homo sapiens>
MGSSATEIEELENTTFKYLTGEQTEKM QRLKGILRCLVKQLERGDVNWDLKKNIEYAA SVLEAVYIDETRRLLDTEDELSDIQTDSVPSEVRD LASTFTRKMGMTKKKPEEKPKFRS IVHAVQAGIFVERMYRKTYHMVGLAYPAAVIVTLKDVDKWSFDVFALNEASGEHSLKFMI YELFTRYDLINRFKIPVSCLITFAEALEVGYSKYKNPYHNLIHAADVTQTVHYIMLHTGI MH LTELEILAMVFAAAIHDYEHTGTTNNFHIQTRSDVAILYNDRSVLENHHVSAAYRLM QEEEMNILINLSKDDWRDLRNLVIEMVLSTDMSGHFQQIKNIRNSLQQPEGIDRAKTMSL ILHAADISHPAKSWKLHYR TMALMEEFFLQGDKEAELGLPFSPLCDRKSTMVAQSQIGF IDFIVEPTFSLLTDSTEKIVIPLIEEASKAETSSYVASSSTTIVGLHIADALRRSNTKGS MSDGSYSPDYSLAAVDLKSFKNNLVDIIQQNKER KELAAQGESDLHKNSEDLVNAEEKH DETHS <SEQ ID NO: 127; PRT; Homo sapiens>
MGSSATEIEELENTTFKYLTGEQTEKM QRLKGILRCLVKQLERGDVNVVDLKKNIEYAA SVLEAVYIDETRRLLDTEDELSDIQTDSVPSEVRDWLASTFTRKMGMTKECKPEEKPKFRS IVHAVQAGIFVERMYRKTYHMVGLAYPAAVIVTLKDVDKWSFDVFALNEASGEHSLKFMI YELFTRYDLINRFKIPVSCLITFAEALEVGYSKYKNPYHNLIHAADVTQTVHYIMLHTGI MH LTELEILAMVFAAAIHDYEHTGTTNNFHIQTRSDVAILYNDRSVLENHHVSAAYRLM QEEEMNILINLSKDD RDLRNLVIEMVLSTDMSGHFQQIKNIRNSLQQPEGIDRAKTMSL ILHAADISHPAKS KLHYRWTMALMEEFFLQGDKEAELGLPFSPLCDRKSTMVAQSQIGF IDFIVEPTFSLLTDSTEKIVIPLIEEASKAETSSYVASSSTTIVGLHIADALRRSNTKGS MSDGSYSPDYSLAAVDLKSFKNNLVDIIQQNKER KELAAQEARTSSQKCEFIHQ <SEQ ID NO: 128; PRT; Homo sapiens>
MNPFQKNESKETLFSPVSIEEVPPRPPSPPKKPSPTICGSNYPLSIAFIWNEFCERFSY
YGMKAVLILYFLYFLH NEDTSTSIYHAFSSLCYFTPILGAAIADS LGKFKTIIYLSLV
YVLGHVIKSLGALPILGGQWHTVLSLIGLSLIALGTGGIKPCVAAFGGDQFEEKHAEER
TRYFSVFYLSINAGSLISTFITPMLRGDVQCFGEDCYALAFGVPGLLMVIALWFAMGSK
IYNKPPPEGNIVAQVFKCIWFAISNRFKNRSGDIPKRQHWLDWAAEKYPKQLIMDVKALT
RVLFLYIPLPMFWALLDQQGSRWTLQAIRMNRNLGFFVLQPDQMQVLNPLLVLIFIPLFD
FVIYRLVSKCGINFSSLRKMAVGMILACLAFAVAAAVEIKINEMAPAQPGPQEVFLQVLN
LADDEVKVTWGNENNSLLIESIKSFQKTPHYSKLHLKTKSQDFHFHLKYHNLSLYTEHS
VQEKN YSLVIREDGNSISSMMVKDTESRTTNGMTTVRFVNTLHKDVNISLSTDTSLNVG
EDYGVSAYRTVQRGEYPAVHCRTEDKNFSLNLGLLDFGAAYLFVITNNTNQGLQA KIED
IPANKMSIA QLPQYALVTAGEVMFSVTGLEFSYSQAPSGMKSVLQAA LLTIAVGNIIV
LWAQFSGLVQ AEFILFSCLLLVICLIFSIMGYYYVPVKTEDMRGPADKHIPHIQGNMI
KLETKKTKL
<SEQ ID NO: 202; DNA; Artificial Sequence> TTCCATGGTAATGGTGTGC
<SEQ ID NO: 203; DNA; Artificial Sequence> CCGAAGCAAGGAATAATCC
<SEQ ID NO: 204; DNA; Artificial Sequence> TATGTTTCGCCTTTATGAC <SEQ ID NO: 205; DNA; Artificial Sequence> GGATTCAAGGAGGAATGAC
<SEQ ID NO: 206; DNA; Artificial Sequence> CTCCCTCTTGCATCAAGAC
<SEQ ID NO: 207; DNA; Artificial Sequence> AAATCCTCGTCAGGTTTAC
<SEQ ID NO: 208; DNA; Artificial Sequence> AGTGCCTTACAGTATCATC
<SEQ ID NO: 209; DNA; Artificial Sequence> CCTGAATGTGACTGTGGAC <SEQ ID NO: 210; DNA; Artificial Sequence> GACTGACTGGCCTGAAGGC
<SEQ ID NO: 211; DNA; Artificial Sequence> GCAGCACTATTTGAAGCAC
<SEQ ID NO: 212; DNA; Artificial Sequence> GCCTGAGAACCTCCTCTGC
<SEQ ID NO: 213; DNA; Artificial Sequence> ACAGCTAGTCAGGCACTTC
<SEQ ID NO: 214; DNA; Artificial Sequence> TATAAGAAATGGCATACTC <SEQ ID NO: 215; DNA; Artificial Sequence> CTAATCCATGGTCTAGTTC
<SEQ ID NO: 216; DNA; Artificial Sequence> GTCTGCTATAAGGAATATC
<SEQ ID NO: 217; DNA; Artificial Sequence> AAGTACTCCTGAGGTCTAC <SEQ ID NO: 218; DNA; Artificial Sequence> AGGCACCAGGGACTTGTGC
<SEQ ID NO: 219; DNA; Artificial Sequence> GATCTACACCACCTTCATC
<SEQ ID NO: 220; DNA; Artificial Sequence> TGGTGTTCTACGTGGTGAC
<SEQ ID NO: 221; DNA; Artificial Sequence> TGTTAGGCGCCTGCATTGC
<SEQ ID NO: 222; DNA; Artificial Sequence> CAACATCTTTATCTGCTCC <SEQ ID NO: 223; DNA; Artificial Sequence> CGAAGGGCTTTCACAATGC
<SEQ ID NO: 224; DNA; Artificial Sequence> GTACTACGTTGTAGCCCAC
<SEQ ID NO: 225; DNA; Artificial Sequence> TGCAGGCGCTTAACATTAC
<SEQ ID NO: 226; DNA; Artificial Sequence> GGTGTATGGGCTCATGTAC
<SEQ ID NO: 227; DNA; Artificial Sequence> CATCGTCATCGCCTGCTAC <SEQ ID NO: 228; DNA; Artificial Sequence> GCTCATGGTGCGCATTGGC
<SEQ ID NO: 229; DNA; Artificial Sequence> GTACCTTATGACGCTGATC
<SEQ ID NO: 230; DNA; Artificial Sequence> ACCTGGTATGGGTTTGGCC
<SEQ ID NO: 231; DNA; Artificial Sequence> ATGTGTGCAGGTCTACTGC
<SEQ ID NO: 232; DNA; Artificial Sequence> TTATTTAGGGCGGTTTAAC <SEQ ID NO: 233; DNA; Artificial Sequence> TAATGTCATCGCCTCCAAC
<SEQ ID NO: 234; DNA; Artificial Sequence> AGAACTGGGTGATGACAGC
<SEQ ID NO: 235; DNA; Artificial Sequence> CAACAGTCCCTGCTACATC <SEQ ID NO: 236; DNA; Artificial Sequence> GATCGTGGTGCATCCATAC
<SEQ ID NO: 237; DNA; Artificial Sequence> GCGTGGATTACCAGAAGAC
<SEQ ID NO: 238; DNA; Artificial Sequence> TAACAACAGTCCCTGCTAC
<SEQ ID NO: 239; DNA; Artificial Sequence> CCATTGCTTGGTGAATGGC
<SEQ ID NO: 240; DNA; Artificial Sequence> ATTCTCTCCATGGAGTGAC <SEQ ID NO: 241; DNA; Artificial Sequence> ATGAACTCTGTGATCCAGC
<SEQ ID NO: 242; DNA; Artificial Sequence> AGGTTGGCTAGTGGATCTC
<SEQ ID NO: 243; DNA; Artificial Sequence> AAGTGGGTAATTCCTGCTC
<SEQ ID NO: 244; DNA; Artificial Sequence> CGAATGGCAGAATGGATAC
<SEQ ID NO: 245; DNA; Artificial Sequence> TCTGGCAGGTTGCATATTC <SEQ ID NO: 246; DNA; Artificial Sequence> CTGCAAGTTTCATATCTAC
<SEQ ID NO: 247; DNA; Artificial Sequence> ATAGCATCAGATTGTATGC
<SEQ ID NO: 248; DNA; Artificial Sequence> TTTACACGATGATATGTTC
<SEQ ID NO: 249; DNA; Artificial Sequence> CAGAGGATTTGCCAGAAAC
<SEQ ID NO: 250; DNA; Artificial Sequence> TTGGAATTCCAGTGTACCC <SEQ ID NO: 251; DNA; Artificial Sequence> CAGAGACACCATCTCCCTC
<SEQ ID NO: 252; DNA; Artificial Sequence> ACCCAGACCCAAAGTTTGC
<SEQ ID NO: 253; DNA; Artificial Sequence> AAGGTGGAAAGACTATCTC <SEQ ID NO: 254; DNA; Artificial Sequence> TATAAACCAGAGGATTTGC
<SEQ ID NO: 255; DNA; Artificial Sequence> GTATAATCTACATCAGATC
<SEQ ID NO: 256; DNA; Artificial Sequence> CCACATGTTTACCAGAGAC
<SEQ ID NO: 257; DNA; Artificial Sequence> GCTAGTTATCGCCTACCTC
<SEQ ID NO: 258; DNA; Artificial Sequence> GACACACAGGAGTTCAACC <SEQ ID NO: 259; DNA; Artificial Sequence> GCTGCAGAAACTAGGCATC
<SEQ ID NO: 260; DNA; Artificial Sequence> TGCCAACTTCTACAAGGAC
<SEQ ID NO: 261; DNA; Artificial Sequence> CGACACACAGGAGTTCAAC
<SEQ ID NO: 262; DNA; Artificial Sequence> GATGGACGTCAAGTCTGCC
<SEQ ID NO: 263; DNA; Artificial Sequence> ACAGGAGTTCAACCTCAGC <SEQ ID NO: 264; DNA; Artificial Sequence> TTGGCATGGAACCAACGAC
<SEQ ID NO: 265; DNA; Artificial Sequence> CCTCTTTGCCCTGTATGAC
<SEQ ID NO: 266; DNA; Artificial Sequence> AGATTCCAGATGCAACCCC
<SEQ ID NO: 267; DNA; Artificial Sequence> CATGAGCCAGCTGAGTTTC
<SEQ ID NO: 268; DNA; Artificial Sequence> GTGGAAGTGATCTTCTATC <SEQ ID NO: 269; DNA; Artificial Sequence> TGGCTGTCATGGTCCAATC
<SEQ ID NO: 270; DNA; Artificial Sequence> CCGCTGCATGAACTATGAC
<SEQ ID NO: 271; DNA; Artificial Sequence> TTGGAGACTTCGGTTTAAC <SEQ ID NO: 272; DNA; Artificial Sequence> TGTGATTCAGATTCTAGTC
<SEQ ID NO: 273; DNA; Artificial Sequence> AGTGGATGTCCTACATCTC
<SEQ ID NO: 274; DNA; Artificial Sequence> ATCATGCAGGATGACATGC
<SEQ ID NO: 275; DNA; Artificial Sequence> CTGAACTACTGGTACAGCC
<SEQ ID NO: 276; DNA; Artificial Sequence> CAGCTTTACGTCCTGAGTC <SEQ ID NO: 277; DNA; Artificial Sequence> TCAGACCACAAGAGAGACC
<SEQ ID NO: 278; DNA; Artificial Sequence> GTACCTACAGTGGATGTCC
<SEQ ID NO: 279; DNA; Artificial Sequence> TGGTCAAGGAGATACTGAC
<SEQ ID NO: 280; DNA; Artificial Sequence> CCCTCCAGTCATGTTCTTC
<SEQ ID NO: 281; DNA; Artificial Sequence> TTCTGCAACTGTGATGATC <SEQ ID NO: 282; DNA; Artificial Sequence> GTACACTACGTGGATTCAC
<SEQ ID NO: 283; DNA; Artificial Sequence> GCGAATTCCACCAGCATTC
<SEQ ID NO: 284; DNA; Artificial Sequence> TCTTCCAGTGCTGCAGCTC
<SEQ ID NO: 285; DNA; Artificial Sequence> TCAGAACAAGAGGCTGGGC
<SEQ ID NO: 286; DNA; Artificial Sequence> GAAGATTGCCAGTCTTCAC <SEQ ID NO: 287; DNA; Artificial Sequence> GATTCCTCCTCTCAACATC
<SEQ ID NO: 288; DNA; Artificial Sequence> GCATTCTAGTATTTCTAAC
<SEQ ID NO: 289; DNA; Artificial Sequence> TTACAGTGTCAATTCCAAC <SEQ ID NO: 290; DNA; Artificial Sequence> TGATTATCGGGAGATCATC
<SEQ ID NO: 291; DNA; Artificial Sequence> GAATGGATGTGTATGTATC
<SEQ ID NO: 292; DNA; Artificial Sequence> TGACATGATTCCTTATAGC
<SEQ ID NO: 293; DNA; Artificial Sequence> TCTACACACTGCCAAATGC
<SEQ ID NO: 294; DNA; Artificial Sequence> ACTGGCCATCATCTACAAC <SEQ ID NO: 295; DNA; Artificial Sequence> TGCTCACCTGTCTGTGGTC
<SEQ ID NO: 296; DNA; Artificial Sequence> ACATTGACCAGGAAGTGAC
<SEQ ID NO: 297; DNA; Artificial Sequence> TGGATGACTTCAGCTCTAC
<SEQ ID NO: 298; DNA; Artificial Sequence> CTACAACCTCTGGTTCGGC
<SEQ ID NO: 299; DNA; Artificial Sequence> CACGACAGTGGAGAGAAAC <SEQ ID NO: 300; DNA; Artificial Sequence> GTTCTACATGCCGGATGAC
<SEQ ID NO: 301; DNA; Artificial Sequence> AGGTATCATGCACTGGCTC
<SEQ ID NO: 302; DNA; Artificial Sequence> GAAGCTGAATTAGGGCTTC
<SEQ ID NO: 303; DNA; Artificial Seq ence> CTGGTGGACATCATTCAGC
<SEQ ID NO: 304; DNA; Artificial Sequence> TTTGTGATCGGAAGTCAAC <SEQ ID NO: 305; DNA; Artificial Sequence> ATTGCTGATGCACTAAGAC
<SEQ ID NO: 306; DNA; Artificial Sequence> CAGATATGATCTTATCAAC
<SEQ ID NO: 307; DNA; Artificial Sequence> ACTGTGCATTACATAATGC <SEQ ID NO: 308; DNA; Artificial Sequence> CACGTGAGTGCAGCTTATC
<SEQ ID NO: 309; DNA; Artificial Sequence> AGTCCTATCATTGATCGGC
<SEQ ID NO: 310; DNA; Artificial Sequence> GAAGCCATCTCCGACAATC
<SEQ ID NO: 311; DNA; Artificial Sequence> ATGGCTGTTGGTATGATCC
<SEQ ID NO: 312; DNA; Artificial Sequence> CCGTGAGGTTTGTTAACAC <SEQ ID NO: 313; DNA; Artificial Sequence> TTGGGTGCCTTACCAATAC
<SEQ ID NO: 314; DNA; Artificial Sequence> CTCCAAGTGTGGAATTAAC
<SEQ ID NO: 315; DNA; Artificial Sequence> GCATGATGGTAAAGGATAC
<SEQ ID NO: 401; PRT; Homo sapiens> MICCSALSPRIHLSFHRSLTGIVLANSSLDIVLHDTYYWAHCGGNVRRLHCGGPASRER TAMQALNITPEQFSRLLRDHNLTREQFIALYRLRPLVYTPELPGRAKLA
<SEQ ID NO: 402; PRT; Homo sapiens> LVLTGVLIFALALFGNALVFYW
<SEQ ID NO: 403; PRT; Homo sapiens> TRSKAMRTVTN
<SEQ ID NO: 404; PRT; Homo sapiens> IFICSLALSDLLITFFCIPVTML
<SEQ ID NO: 405; PRT; Homo sapiens> QNISDNWLGGAFIC <SEQ ID NO: 406; PRT; Homo sapiens> KMVPFVQSTAWTEILTMTCIAV
<SEQ ID NO: 407; PRT; Homo sapiens> ERHQGLVHPFKMK QYTNRR <SEQ ID NO: 408; PRT; Homo sapiens> AFTMLGW LVAVIVGSPMWHVQ
<SEQ ID NO: 409; PRT; Homo sapiens> QLEIKYDFLYEKEHICCLEEWTSPVHQKIYTTFILSSSSSCLLWKKKR
<SEQ ID NO: 410; PRT; Homo sapiens> AVIMMVTWALFAVC APFHWH <SEQ ID NO: 411; PRT; Homo sapiens> MMIEYSNFEKEYDDVTIKM
<SEQ ID NO: 412; PRT; Homo sapiens> IFAIVQIIGFSNSICNPIVYAFM
<SEQ ID NO: 413; PRT; Homo sapiens>
NENFKKNVLSAVCYCIVNKTFSPAQRHGNSGITMMRKKAKFSLRENPVEETKGEAFSDGN
IEVKLCEQTEEKKKLKRHLALFRSELAENSPLDSGH
<SEQ ID NO: 414; PRT; Homo sapiens> MQALNITPEQFSRLLRDHNLTREQFIAVHRLRPLVYTPELPGRAKLA
<SEQ ID NO: 415; PRT; Homo sapiens> LVLTGVLIFALALFGNALVFYW
<SEQ ID NO: 416; PRT; Homo sapiens> TRSKAMRTVTN
<SEQ ID NO: 417; PRT; Homo sapiens> IFICSLALSDLLITFFCIPVTML
<SEQ ID NO: 418; PRT; Homo sapiens> QNISDN LGGAFIC <SEQ ID NO: 419; PRT; Homo sapiens> KMVPFVQSTAWTEILTMTCIAV
<SEQ ID NO: 420; PRT; Homo sapiens> ERHQGLVHPFKMKQYTNRR
<SEQ ID NO: 421; PRT; Homo sapiens> AFTMLGW LVAVIVGSPM HVQ
<SEQ ID NO: 422; PRT; Homo sapiens> QLEIKYDFLYEKEHICCLEEWTSPVHQK
<SEQ ID NO: 423; PRT; Homo sapiens> IYTTFILVILFLLPLMVMLILYS <SEQ ID NO: 424; PRT; Homo sapiens> KIGYELWIKKRVGDGSVLRTIHGKEMSKIARKKK
<SEQ ID NO: 425; PRT; Homo sapiens> RAVIMMVTWALFAVC APFHW <SEQ ID NO: 426; PRT; Homo sapiens> HMMIEYSNFEKEYDDVTIK
<SEQ ID NO: 427; PRT; Homo sapiens> MIFAIVQIIGFSNSICNPIVYAF
<SEQ ID NO: 428; PRT; Homo sapiens>
MNENFKKNVLSAVCYCIVNKTFSPAQRHGNSGITMMRKKAKFSLRENPVEETKGEAFSDGNI
EVKLCEQTEEKKKLKRHLALFRSELAENSPLDSGH
<SEQ ID NO: 429; PRT; Homo sapiens>
MAEEEAPKKSRAAGGGASWELCAGALSARLAEEGSGDAGGRRRPPVDPRRLARQLLLLL
LLEAPLLLGVRAQAAGQGPGQGPGPGQQPPPPPQQQQSGQQYNGERGISVPDHGYCQPIS
IPLCTDIAYNQTIMPNLLGHTNQEDAGLEVHQFYPLVKVQCSAELKFFLCSMYAPVCTVL
EQALPPCRSLCERARQGCEALMNKFGFQ PDTLKCEKFPVHGAGELCVGQNTSDKGTPTP
SLLPEF TSNPQHGGGGHRGGFPGGAGASERGKFSCPRALKVPSYLNYHFLGEKDCGAPC
EPTKVYGLMYFGPEELRFSR
<SEQ ID NO: 430; PRT; Homo sapiens> TWIGIWSVLCCASTLFTVLTYLV
<SEQ ID NO: 431; PRT; Homo sapiens> DMRRFSYPERPI <SEQ ID NO: 432; PRT; Homo sapiens> IFLSGCYTAVAVAYIAGFLL
<SEQ ID NO: 433; PRT; Homo sapiens> EDRWCNDKFAEDGARTVAQGTKKEGC
<SEQ ID NO: 434; PRT; Homo sapiens> TILFMMLYFFSMASSIWVILSL
<SEQ ID NO: 435; PRT; Homo sapiens> TWFLAAGMKWGHEAIEANSQ
<SEQ ID NO: 436; PRT; Homo sapiens> YFHLAAAVPAIKTITILALGQV <SEQ ID NO: 437; PRT; Homo sapiens> DGDVLSGVCFVGLNNVDALRGFV
<SEQ ID NO: 438; PRT; Homo sapiens> LAPLFVYLFIGTSFLLAGFVSLF
<SEQ ID NO: 439; PRT; Homo sapiens> RIRTIMKHDGTKTEKLEKLM
<SEQ ID NO: 440; PRT; Homo sapiens> VRIGVFSVLYTVPATIVIACYFY
<SEQ ID NO: 441; PRT; Homo sapiens> EQAFRDQWERSWVAQSCKSYAIPCPHLQAGGGAPPHPPMSPD <SEQ ID NO: 442; PRT; Homo sapiens> FTVFMIKYLMTLIVGITSGFWIW
<SEQ ID NO: 443; PRT; Homo saρiens> SGKTLNSWRKFYTRLTNSKQGETTV
<SEQ ID NO: 444; PRT; Homo sapiens>
MACLMAAFSVGTAMNASSYSAEMTEPKSVCVSVDEWSSNMEATETDLLNGHLKKVDNNL TEAQRFSSLPRRAAVNIEFRDLSYSVPEGPWWRKKGYKTLLKGISGKFNSGELVAIMGPS GAGKSTLMNILAGYRETGMKGAVLINGLPRDLRCFRKVSCYIMQDDMLLPHLTVQEAMMV SAHLKLQEKDEGRREMVKEILTALGLLSCANTRTGSLSGGQRKRLAIALELVNNPPVMFF DEPTSGLDSASCFQWSLMKGLAQGGRSIICTIHQPSAKLFELFDQLYVLSQGQCVYRGK VCNLVPYLRDLGLNCPTYHNPADFVMEVASGEYGDQNSRLVRAVREGMCDSDHKRDLGGD AEVNPFL HRPSEEDSSSMEGCHSFSASCLTQFCILFKRTFLSIMRDSV
<SEQ ID NO: 445; PRT; Homo sapiens> LTHLRITSHIGIGLLIGLLYLGI
<SEQ ID NO: 446; PRT; Homo sapiens> GNEAKKVLSNSG
<SEQ ID NO: 447; PRT; Homo sapiens> FLFFSMLFLMFAALMPTVLTFPL <SEQ ID NO: 448; PRT; Homo sapiens> EMGVFLREHLNY YSLKAYYLAK
<SEQ ID NO: 449; PRT; Homo sapiens> TMADVPFQIMFPVAYCSIVYWMT
<SEQ ID NO: 450; PRT; Homo sapiens> SQPSDA
<SEQ ID NO: 451; PRT; Homo sapiens> VRFVLFAALGTMTSLVAQSLGLL
<SEQ ID NO: 452; PRT; Homo sapiens> IGAASTSLQ <SEQ ID NO: 453; PRT; Homo sapiens> VATFVGPVTAIPVLLFSGFFVSF
<SEQ ID NO: 454; PRT; Homo sapiens> DTIPTYLQ MSY
<SEQ ID NO: 455; PRT; Homo sapiens> ISYVRYGFEGVILSIYGL
<SEQ ID NO: 456; PRT; Homo sapiens> DREDLHCDIDETCHFQKSEAILRELDVENAK
<SEQ ID NO: 457; PRT; Homo sapiens> LYLDFIVLGIFFISLRLIAYFVL <SEQ ID NO: 458; PRT; Homo sapiens> RYKIRAER
<SEQ ID NO: 459; PRT; Homo sapiens> MACLMAAFSVGTAMNASSYSAEMTEPKSVCVSVDEWSSNMEATETDLLNGHLKKVDNNL TEAQRFSSLPRRAAVNIEFRDLSYSVPEGPWWRKKGYKTLLKGISGKFNSGELVAIMGPS GAGKSTLMNILAGYRETGMKGAVLINGLPRDLRCFRKVSCYIMQDDMLLPHLTVQEAMMV SAHLKLQEKDEGRREMVKEILTALGLLSCANTRTGSLSGGQRKRLAIALELVNNPPVMFF DEPTSGLDSASCFQWSLMKGLAQGGRSIICTIHQPSAKLFELFDQLYVLSQGQCVYRGK VCNLVPYLRDLGLNCPTYHNPADFVMEVASGEYGDQNSRLVRAVREGMCDSDHKRDLGGD AEVNPFL HRPSEEVKQTKRLKGLRKDSSSMEGCHSFSASCLTQFCILFKRTFLSIMRDS V
<SEQ ID NO: 460; PRT; Homo sapiens> LTHLRITSHIGIGLLIGLLYLGI
<SEQ ID NO: 461; PRT; Homo sapiens> GNEAKKVLSNSG <SEQ ID NO: 462; PRT; Homo sapiens> FLFFSMLFLMFAALMPTVLTFPL
<SEQ ID NO: 463; PRT; Homo sapiens> EMGVFLREHLNYWYSLKAYYLAK
<SEQ ID NO: 464; PRT; Homo sapiens> TMADVPFQIMFPVAYCSIVYWMT
<SEQ ID NO: 465; PRT; Homo sapiens> SQPSDA
<SEQ ID NO: 466; PRT; Homo sapiens> VRFVLFAALGTMTSLVAQSLGLL <SEQ ID NO: 467; PRT; Homo sapiens> IGAASTSLQ
<SEQ ID NO: 468; PRT; Homo sapiens> VATFVGPVTAIPVLLFSGFFVSF
<SEQ ID NO: 469; PRT; Homo sapiens> DTIPTYLQWMSY
<SEQ ID NO: 470; PRT; Homo sapiens> ISYVRYGFEGVILSIYGL
<SEQ ID NO: 471; PRT; Homo sapiens> DREDLHCDIDETCHFQKSEAILRELDVENAK <SEQ ID NO: 472; PRT; Homo sapiens> LYLDFIVLGIFFISLRLIAYFVL
<SEQ ID NO: 473; PRT; Homo sapiens> RYKIRAER <SEQ ID NO: 474; PRT; Homo sapiens>
MRISLPRAPERDGGVSASSLLDTVTNASSYSAEMTEPKSVCVSVDEWSSNMEATETDLL
NGHLKKVDNNLTEAQRFSSLPRRAAVNIEFRDLSYSVPEGPWWRKKGYKTLLKGISGKFN
SGELVAIMGPSGAGKSTLMNILAGYRETGMKGAVLINGLPRDLRCFRKVSCYIMQDDMLL
PHLTVQEAMMVSAHLKLQEKDEGRREMVKEILTALGLLSCANTRTGSLSGGQRKRLAIAL
ELVNNPPVMFFDEPTSGLDSASCFQWSLMKGLAQGGRSIICTIHQPSAKLFELFDQLYV
LSQGQCVYRGKVCNLVPYLRDLGLNCPTYHNPADFVMEVASGEYGDQNSRLVRAVREGMC
DSDHKRDLGGDAEVNPFL HRPSEEDSSSMEGCHSFSASCLTQFCILFKRTFLSIMRDSV <SEQ ID NO: 475; PRT; Homo sapiens> LTHLRITSHIGIGLLIGLLYLGI
<SEQ ID NO: 476; PRT; Homo sapiens> GNEAKKVLSNSG
<SEQ ID NO: 477; PRT; Homo sapiens> FLFFSMLFLMFAALMPTVLTFPL
<SEQ ID NO: 478; PRT; Homo sapiens> EMGVFLREHLNYWYSLKAYYLAK
<SEQ ID NO: 479; PRT; Homo sapiens> TMADVPFQIMFPVAYCSIVYWMT <SEQ ID NO: 480; PRT; Homo sapiens> SQPSDA
<SEQ ID NO: 481; PRT; Homo sapiens> VRFVLFAALGTMTSLVAQSLGLL
<SEQ ID NO: 482; PRT; Homo sapiens> IGAASTSLQ
<SEQ ID NO: 483; PRT; Homo sapiens> VATFVGPVTAIPVLLFSGFFVSF
<SEQ ID NO: 484; PRT; Homo sapiens> DTIPTYLQWMSY <SEQ ID NO: 485; PRT; Homo sapiens> ISYVRYGFEGVILSIYGL
<SEQ ID NO: 486; PRT; Homo sapiens> DREDLHCDIDETCHFQKSEAILRELDVENAK
<SEQ ID NO: 487; PRT; Homo sapiens> LYLDFIVLGIFFISLRLIAYFVL
<SEQ ID NO: 488; PRT; Homo sapiens> RYKIRAER
<SEQ ID NO: 489; PRT; Homo sapiens>
MLGTQG TKQRKPCPQNASSYSAEMTEPKSVCVSVDEWSSNMEATETDLLNGHLKKVDN
NLTEAQRFSSLPRRAAVNIEFRDLSYSVPEGPWWRKKGYKTLLKGISGKFNSGELVAIMG PSGAGKSTLMNILAGYRETGMKGAVLINGLPRDLRCFRKVSCYIMQDDMLLPHLTVQEAM MVSAHLKLQEKDEGRREMVKEILTALGLLSCANTRTGSLSGGQRKRLAIALELVNNPPVM FFDEPTSGLDSASCFQWSLMKGLAQGGRSIICTIHQPSAKLFELFDQLYVLSQGQCVYR GKVCNLVPYLRDLGLNCPTYHNPADFVMEVASGEYGDQNSRLVRAVREGMCDSDHKRDLG GDAEVNPFL HRPSEEDSSSMEGCHSFSASCLTQFCILFKRTFLSIMRDSV
<SEQ ID NO: 490; PRT; Homo sapiens> LTHLRITSHIGIGLLIGLLYLGI
<SEQ ID NO: 491; PRT; Homo sapiens> GNEAKKVLSNSG
<SEQ ID NO: 492; PRT; Homo sapiens> FLFFSMLFLMFAALMPTVLTFPL <SEQ ID NO: 493; PRT; Homo sapiens> EMGVFLREHLNYWYSLKAYYLAK
<SEQ ID NO: 494; PRT; Homo sapiens> TMADVPFQIMFPVAYCSIVYWMT
<SEQ ID NO: 495; PRT; Homo sapiens> SQPSDA
<SEQ ID NO: 496; PRT; Homo sapiens> VRFVLFAALGTMTSLVAQSLGLL
<SEQ ID NO: 497; PRT; Homo sapiens> IGAASTSLQ <SEQ ID NO: 498; PRT; Homo sapiens> VATFVGPVTAIPVLLFSGFFVSF
<SEQ ID NO: 499; PRT; Homo sapiens> DTIPTYLQWMSY
<SEQ ID NO: 500; PRT; Homo sapiens> ISYVRYGFEGVILSIYGL
<SEQ ID NO: 501; PRT; Homo sapiens> DREDLHCDIDETCHFQKSEAILRELDVENAK
<SEQ ID NO: 502; PRT; Homo sapiens> LYLDFIVLGIFFISLRLIAYFVL <SEQ ID NO: 503; PRT; Homo sapiens> RYKIRAER
<SEQ ID NO: 504; PRT; Homo sapiens>
MTEPKSVCVSVDEWSSNMEATETDLLNGHLKKVDNNLTEAQRFSSLPRRAAVNIEFRDL SYSVPEGPWWRKKGYKTLLKGISGKFNSGELVAIMGPSGAGKSTLMNILAGYRETGMKGA VLINGLPRDLRCFRKVSCYIMQDDMLLPHLTVQEAMMVSAHLKLQEKDEGRREMVKEILT ALGLLSCANTRTGSLSGGQRKRLAIALELVNNPPVMFFDEPTSGLDSASCFQWSLMKGL AQGGRSIICTIHQPSAKLFELFDQLYVLSQGQCVYRGKVCNLVPYLRDLGLNCPTYHNPA DFVMEVASGEYGDQNSRLVRAVREGMCDSDHKRDLGGDAEVNPFLWHRPSEEDSSSMEGC HSFSASCLTQFCILFKRTFLSIMRDSV
<SEQ ID NO: 505; PRT; Homo sapiens> LTHLRITSHIGIGLLIGLLYLGI
<SEQ ID NO: 506; PRT; Homo sapiens> GNEAKKVLSNSG
<SEQ ID NO: 507; PRT; Homo sapiens> FLFFSMLFLMFAALMPTVLTFPL
<SEQ ID NO: 508; PRT; Homo sapiens> EMGVFLREHLNYWYSLKAYYLAK <SEQ ID NO: 509; PRT; Homo sapiens> TMADVPFQIMFPVAYCSIVYWMT
<SEQ ID NO: 510; PRT; Homo sapiens> SQPSDA
<SEQ ID NO: 511; PRT; Homo sapiens> VRFVLFAALGTMTSLVAQSLGLL
<SEQ ID NO: 512; PRT; Homo sapiens> IGAASTSLQ
<SEQ ID NO: 513; PRT; Homo sapiens> VATFVGPVTAIPVLLFSGFFVSF <SEQ ID NO: 514; PRT; Homo sapiens> DTIPTYLQWMSY
<SEQ ID NO: 515; PRT; Homo sapiens> ISYVRYGFEGVILSIYGL
<SEQ ID NO: 516; PRT; Homo sapiens> DREDLHCDIDETCHFQKSEAILRELDVENAK
<SEQ ID NO: 517; PRT; Homo sapiens> LYLDFIVLGIFFISLRLIAYFVL
<SEQ ID NO: 518; PRT; Homo sapiens> RYKIRAER <SEQ ID NO: 519; PRT; Homo sapiens>
MIMRLPQPHGTNASSYSAEMTEPKSVCVSVDEWSSNMEATETDLLNGHLKKVDNNLTEA QRFSSLPRRAAVNIEFRDLSYSVPEGPWWRKKGYKTLLKGISGKFNSGELVAIMGPSGAG KSTLMNILAGYRETGMKGAVLINGLPRDLRCFRKVSCYIMQDDMLLPHLTVQEAMMVSAH LKLQEKDEGRREMVKEILTALGLLSCANTRTGSLSGGQRKRLAIALELVNNPPVMFFDEP TSGLDSASCFQWSLMKGLAQGGRSIICTIHQPSAKLFELFDQLYVLSQGQCVYRGKVCN LVPYLRDLGLNCPTYHNPADFVMEVASGEYGDQNSRLVRAVREGMCDSDHKRDLGGDAEV NPFLWHRPSEEDSSSMEGCHSFSASCLTQFCILFKRTFLSIMRDSV <SEQ ID NO: 520; PRT; Homo sapiens> LTHLRITSHIGIGLLIGLLYLGI
<SEQ ID NO: 521; PRT; Homo sapiens> GNEAKKVLSNSG
<SEQ ID NO: 522; PRT; Homo sapiens> FLFFSMLFLMFAALMPTVLTFPL <SEQ ID NO: 523; PRT; Homo sapiens> EMGVFLREHLNYWYSLKAYYLAK
<SEQ ID NO: 524; PRT; Homo sapiens> TMADVPFQIMFPVAYCSIVYWMT
<SEQ ID NO: 525; PRT; Homo sapiens> SQPSDA
<SEQ ID NO: 526; PRT; Homo sapiens> VRFVLFAALGTMTSLVAQSLGLL
<SEQ ID NO: 527; PRT; Homo sapiens> IGAASTSLQ <SEQ ID NO: 528; PRT; Homo sapiens> VATFVGPVTAIPVLLFSGFFVSF
<SEQ ID NO: 529; PRT; Homo sapiens> DTIPTYLQWMSY
<SEQ ID NO: 530; PRT; Homo sapiens> ISYVRYGFEGVILSIYGL
<SEQ ID NO: 531; PRT; Homo sapiens> DREDLHCDIDETCHFQKSEAILRELDVENAK
<SEQ ID NO: 532; PRT; Homo sapiens> LYLDFIVLGIFFISLRLIAYFVL <SEQ ID NO: 533; PRT; Homo sapiens> RYKIRAER
<SEQ ID NO: 534; PRT; Homo sapiens> MLAVQQTEHLPACPPARRWSSNFCPESTEGGPSLLGLRDMVRRGWSVCTAILLARLWCLV PTHTFLSEYPEAAEYPHPGWVYWLQMAVAPGHLRAWVMRNNVTTNIPSAFSGTLTHEEKA VLTVFTGTATAVHVQVAALASAKLESSVFVTDCVSCKIENVCDSALQGKRVPMSGLQGSS IVIMPPSNRPLASAASCTWSVQVQGGPHHLGWAISGKVLSAAHGAGRAYGWGFPGDPME EGYKTLLKGISGKFNSGELVAIMGPSGAGKSTLMNILAGYRETGMKGAVLINGLPRDLRC FRKVSCYIMQDDMLLPHLTVQEAMMVSAHLKLQEKDEGRREMVKEILTALGLLSCANTRT GSLSGGQRKRLAIALELVNNPPVMFFDEPTSGLDSASCFQWSLMKGLAQGGRSIICTIH QPSAKLFELFDQLYVLSQGQCVYRGKVCNLVPYLRDLGLNCPTYHNPADFVMEVASGEYG DQNSRLVRAVREGMCDSDHKRDLGGDAEVNPFLWHRPSEEVKQTKRLKGLRKDSSSMEGC HSFSASCLTQFCILFKRTFLSIMRDSV <SEQ ID NO: 535; PRT; Homo sapiens> LTHLRITSHIGIGLLIGLLYLGI
<SEQ ID NO: 536; PRT; Homo sapiens> GNEAKKVLSNSG
<SEQ ID NO: 537; PRT; Homo sapiens> FLFFSMLFLMFAALMPTVLTFPL <SEQ ID NO: 538; PRT; Homo sapiens> EMGVFLREHLNYWYSLKAYYLAK
<SEQ ID NO: 539; PRT; Homo sapiens> TMADVPFQIMFPVAYCSIVYWMT
<SEQ ID NO: 540; PRT; Homo sapiens> SQPSDA
<SEQ ID NO: 541; PRT; Homo sapiens> VRFVLFAALGTMTSLVAQSLGLL
<SEQ ID NO: 542; PRT; Homo sapiens> IGAASTSLQ <SEQ ID NO: 543; PRT; Homo sapiens> VATFVGPVTAIPVLLFSGFFVSF
<SEQ ID NO: 544; PRT; Homo sapiens> DTIPTYLQWMSY
<SEQ ID NO: 545; PRT; Homo sapiens> ISYVRYGFEGVILSIYGL
<SEQ ID NO: 546; PRT; Homo sapiens> DREDLHCDIDETCHFQKSEAILRELDVENAK
<SEQ ID NO: 547; PRT; Homo sapiens> LYLDFIVLGIFFISLRLIAYFVL <SEQ ID NO: 548; PRT; Homo sapiens> RYKIRAER
<SEQ ID NO: 549; PRT; Homo sapiens>
MAQLERSAISGFSSKSRRNSFAYDVKREVYNEETFQQEHKRKASSSGNMNINITTFRHHV QCRCSWHRFLRCVLTIFPFLEWMCMYRLKD
<SEQ ID NO: 550; PRT; Homo sapiens> WLLGDLLAGISVGLVQVPQGLTL <SEQ ID NO: 551; PRT; Homo sapiens> SLLAR
<SEQ ID NO: 552; PRT; Homo sapiens> QLIPPLNIAYAAFCSSVIYVIFG <SEQ ID NO: 553; PRT; Homo sapiens> SCHQMS
<SEQ ID NO: 554; PRT; Homo sapiens> IGSFFLVSALLINVLKVSPF
<SEQ ID NO: 555; PRT; Homo sapiens> NNGQLVMGSFVKNEFSAPSYLMGYNKSLS <SEQ ID NO: 556; PRT; Homo sapiens> WATTTFLTGIIQLIMGVLGLGF
<SEQ ID NO: 557; PRT; Homo sapiens> IATYLPESAMSA
<SEQ ID NO: 558; PRT; Homo sapiens> YLAAVALHIMLSQLTFIFGIMIS
<SEQ ID NO: 559; PRT; Homo sapiens> FHAGPISFFYDIIN
<SEQ ID NO: 560; PRT; Homo sapiens> YCVALPKANSTSILVFLTVWAL <SEQ ID NO: 561; PRT; Homo sapiens> RINKCIRISFNQYPIEFPME
<SEQ ID NO: 562; PRT; Homo sapiens> LFLIIGFTVIANKISMAT
<SEQ ID NO: 563; PRT; Homo sapiens> ETSQTLIDMIPYSFLLPVTPDFSLLPK
<SEQ ID NO: 564; PRT; Homo sapiens> IILQAFSLSLVSSFLLIFLG
<SEQ ID NO: 565; PRT; Homo sapiens> KKIASLHNYSVNSNQDLIAIGLCNWSSFFRSCVFTGAIARTIIQDKSGGRQQ <SEQ ID NO: 566; PRT; Homo sapiens> FASLVGAGVMLLLMVKMGHFFYT
<SEQ ID NO: 567; PRT; Homo sapiens> LPNA
<SEQ ID NO: 568; PRT; Homo sapiens> VLAGIILSNVIPYLETISNL
<SEQ ID NO: 569; PRT; Homo sapiens> PSLWRQDQYDCALWMMTFSS
<SEQ ID NO: 570; PRT; Homo sapiens> SIFLGLDIGLIISWSAFFITTV <SEQ ID NO: 571; PRT; Homo sapiens>
RSHRAKILLLGQIPNTNIYRSINDYREIITIPGVKIFQCCSSITFVNVYYLKHKLLKEVD
MVKVPLKEEEIFSLFNSSDTNLQGGKICRCFCNCDDLEPLPRILYTERFENKLDPEASSI
NLIHCSHFESMNTSQTASEDQVPYTVSSVSQKNQGQQYEEVEEVWLPNNSSRNSSPGLPD
VAESQGRRSLIPYSDASLLPSVHTIILDFSMVHYVDSRGLWLRQICNAFQNANILILIA
GCHSSIVRAFERNDFFDAGITKTQLFLSVHDAVLFALSRKVIGSSELSIDESETVIRETY
SETDKNDNSRYKMSSSFLGSQKNVSPGFIKIQQPVEEESELDLELESEQEAGLGLDLDLD
RELEPEMEPKAETETKTQTEMEPQPETEPEMEPNPKSRPRAHTFPQQRYWPMYHPSMAST
QSQTQTRTWSVERRRHPMDSYSPEGNSNEDV
<SEQ ID NO: 572; PRT; Homo sapiens> MNPFQKNESKETLFSPVSIEEVPPRPPSPPKKPSPTICGSNYPLSIAFIWNEFCER
<SEQ ID NO: 573; PRT; Homo sapiens> FSYYGMKAVLILYFLYFL
<SEQ ID NO: 574; PRT; Homo sapiens> HWNEDTSTS <SEQ ID NO: 575; PRT; Homo sapiens> lYHAFSSLCYFTPILGAAIADSW
<SEQ ID NO: 576; PRT; Homo sapiens> LGKFKT
<SEQ ID NO: 577; PRT; Homo sapiens> IIYLSLVYVLGHVIKSLGALPIL
<SEQ ID NO: 578; PRT; Homo sapiens> GGQ
<SEQ ID NO: 579; PRT; Homo sapiens> WHTVLSLIGLSLIALGTGGIKP <SEQ ID NO: 580; PRT; Homo sapiens> CVAAFGGDQFEEKHAEERTR
<SEQ ID NO: 581; PRT; Homo sapiens> YFSVFYLSINAGSLISTFIT
<SEQ ID NO: 582; PRT; Homo sapiens> PMLRGDVQCFGEDC
<SEQ ID NO: 583; PRT; Homo sapiens> YALAFGVPGLLMVIALWFA
<SEQ ID NO: 584; PRT; Homo sapiens>
MGSKIYNKPPPEGNIVAQVFKCIWFAISNRFKNRSGDIPKRQHWLDWAAEKYPKQLIMDV
K
<SEQ ID NO: 585; PRT; Homo sapiens> ALTRVLFLYIPLPMFWALL <SEQ ID NO: 586; PRT; Homo saρiens> DQQGSRWTLQAIRMNRNLGFFVLQPDQMQ
<SEQ ID NO: 587; PRT; Homo sapiens> VLNPLLVLIFIPLFDFVIYRLVS
<SEQ ID NO: 588; PRT; Homo saρiens> KCGINFSSLRK <SEQ ID NO: 589; PRT; Homo saρiens> MAVGMILACLAFAVAAAV
<SEQ ID NO: 590; PRT; Homo sapiens>
EIKINEMAPAQPGPQEVFLQVLNLADDEVKVTWGNENNSLLIESIKSFQKTPHYSKLHL KTKSQDFHFHLKYHNLSLYTEHSVQEKNWYSLVIREDGNSISSMMVKDTESRTTNGMTTV RFVNTLHKDVNISLSTDTSLNVGEDYGVSAYRTVQRGEYPAVHCRTEDKNFSLNLGLLDF GAAYLFVITNNTNQGLQAWKIEDIPANKMSIAWQLPQYALVTAGEVMFSVTGLEFSYSQA PSGMKSVLQA <SEQ ID NO: 591; PRT; Homo sapiens> AWLLTIAVGNIIVLWAQFSGLV
<SEQ ID NO: 592; PRT; Homo sapiens> QWAE
<SEQ ID NO: 593; PRT; Homo sapiens> FILFSCLLLVICLIFSIMGYYYV
<SEQ ID NO: 594; PRT; Homo sapiens> PVKTEDMRGPADKHIPHIQGNMIKLETKKTKL

Claims

We claim:
1. Method for identifying a compound that induces chondrocyte anabolic stimulation, comprising contacting a compound with a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 101-128 and 401-594; and measuring a compound-polypeptide property related to the anabolic stimulation of chondrocytes.
2. The method according to claim 1, wherein said polypeptide is in an in vitro cell- free preparation.
3. The method according to claim 2, wherein said polypeptide is present in a mammalian cell.
4. The method of claim 1, wherein said property is a bmding affinity of said compound to said polypeptide.
5. The method of claim 3, wherein said property is activation of a biological pathway producing a biochemical marker indicative of the anabolic stimulation of chondrocytes.
6. The method of claim 5 wherein said indicator is selected from the group consisting of collagen type II, alpha-1 (co!2αl) and aggrecan.
7. The method of claim 6 wherein said polypeptide comprises an amino acid sequence selected from the group consisting of SEQ TD NO: 101-128.
8. The method according to claim 1, wherein said compound is selected from the group consisting of compounds of a commercially available screening library and compounds having binding affinity for a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 101-128 and 401- 594.
9. The method according to claim 2, wherein said compound is a peptide in a phage display library or an antibody fragment library.
10. An agent for inducing the anabolic stimulation of chondrocytes, selected from the group consisting of an antisense polynucleotide, a ribozyme, and a small interfering RNA (siRNA), wherein said agent comprises a nucleic acid sequence complementary to, or engineered from, a natmally-occurring polynucleotide sequence encoding a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ TD NO: 101-128 and 401-594.
11. The agent according to claim 10, wherein polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 101-128.
12. The agent according to claim 11, wherein a vector in a mammahan cell expresses said agent.
13. The agent according to claim 12, wherein said vector is an adenoviral, retroviral, adeno-associated viral, lentiviral, a herpes simplex viral or a sendaiviral vector.
14. The agent according to claim 10, wherein said antisense polynucleotide and said siRNA comprise an antisense strand of 17-25 nucleotides complementary to a sense strand, wherein said sense strand is selected from 17-25 continuous nucleotides of a nucleic acid sequence encoding a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 101-128.
15. The agent according to claims 14, wherein said siRNA further comprises said sense strand.
16. The agent according to claim 15, wherein said sense strand is selected from 17-25 continuous nucleotides of a nucleic acid sequence selected from the group consisting of SEQ ID NO: 1-28.
17. The agent according to claim 16, wherein said siRNA further comprises a loop region connecting said sense .and said antisense strand.
18. The agent according to claim 17, wherein said loop region comprises a nucleic acid sequence defined of SEQ ID NO: 201.
19. The agent according to claim 18, wherein said agent is an antisense polynucleotide, ribozyme, or siRNA comprising a nucleic acid sequence complementary to a polynucleotide selected from the group consisting of SEQ ID NO: 202-315.
20. A chondrocyte anabohc stimulation enhancing pharmaceutical composition comprising a therapeutically effective amount of an agent of claim 10 in admixture with a pharmaceutically acceptable carrier.
21. A method of treating and/or preventing a disease involving a systemic or local decrease in cartilege in a subject suffering from or susceptible to the disease, comprising a<lnrinistering to said subject a pharmaceutical composition according to claim 20.
22. The method according to claim 21 wherein the disease is selected from the group consisting of osteoarthritis, rheumatoid arthritis, psoriatic arthritis, juvenile rheumatoid arthritis, gouty arthritis, septic or infectious arthritis, reactive arthritis, reflex sympathetic dystrophy, algodystrophy, Tietze syndrome or costal chondritis, fibromyalgia, osteochondritis, neurogenic or neuropathic arthritis, arthropathy, osteoarthritis deformans endemica, Mseleni disease, Handigodu disease, degeneration resulting from fibromyalgia, systemic lupus erythematosus, scleroderma, ankylosing spondylitis, hereditary chondrolysis, chondrodysplasias, pseudoachondrodysplasias, microtia, anotia, and metaphyseal chondrodysplasia.
23. The method according to claim 22, wherein the disease is osteoarthritis.
24. Use of an agent according to claims 10-19 in the manufacture of a medicament for the treatment and/or prevention of a disease involving a decrease in mean cartilage thickness.
25. Use according to claim 24, wherein the disease is selected from the group consisting of osteoartnritis, hypercalcemia of malignancy, multiple myelomatosis, hyperparathyroidism, and hyperthyroidism.
26. Use according to claim 25, wherein the disease is osteoarthritis.
27. A method for in vitro production of cartilage tissue, comprising contacting de- differentiated chondrocyte cells with a polynucleotide sequence comprising a nucleic acid sequence complementary to a polynucleotide selected from the' group consisting of SEQ ID NO: 1-28 for a time sufficient to re-differentiate the chondrocytes, thereby producing a cartilaginous matrix.
28. A method according to claim 27, comprising: applying a mixture of chondrocytes and de-differentiated chondrocytes on a substrate to form a cellular substrate, and contacting said cells with a polynucleotide sequence comprising a nucleic acid sequence complementary to a polynucleotide selected from the group consisting of SEQ ID NO: 202-315, thereby producing a continuous cartilaginous matrix.
29. A method for diagnosing a pathological condition involving a systemic or local decrease in mean cartilage thickness or a susceptibility to the condition in a subject, comprising determining a first amount of polypeptide comprising an a ino acid sequence selected from the group consisting of SEQ TD NO: 101-128 and 401-594 present in a biological sample obtained from said subject, and comparing said first amount with the ranges of amounts of the polypeptide determined in a population of healthy subjects, wherein an increase of the amount of polypeptide in said biological sample compared to the range of amounts determined for healthy subjects is indicative of the presence of the pathological condition.
30. A method for diagnosing a pathological condition involving chondrocyte de- differentiation, said method comprising the steps of: deterrnining the nucleic acid sequence of any one of the genes of SEQ. ID NOS. 1-28 in a genomic DNA sample; comparing the sequence from step (a) with the nucleic acid sequence of a healthy subject; and identifying any difference(s) related to the pathological condition.
31. A method for diagnosing a pathological condition involving chondrocyte de- differentiation, said method comprising the steps of: deterrnining the amount of any one of the polypeptides of SEQ ID NOS. 101-128 in a sample; and comparing the amount determined in step (a) with the amount of said polypeptide in a sample from a healthy individual; wherein an increase as compared to the sample of the healthy individual is indicative for the onset or presence of said pathological condition.
32. Method according to claim 31, wherein the pathological.eondition is osteoarthritis.
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