WO2008046543A1 - Gènes protecteurs de cellules - Google Patents

Gènes protecteurs de cellules Download PDF

Info

Publication number
WO2008046543A1
WO2008046543A1 PCT/EP2007/008790 EP2007008790W WO2008046543A1 WO 2008046543 A1 WO2008046543 A1 WO 2008046543A1 EP 2007008790 W EP2007008790 W EP 2007008790W WO 2008046543 A1 WO2008046543 A1 WO 2008046543A1
Authority
WO
WIPO (PCT)
Prior art keywords
target
candidate gene
polypeptide
diseases
mammal
Prior art date
Application number
PCT/EP2007/008790
Other languages
English (en)
Inventor
Stefan Golz
Holger Summer
Andreas Geerts
Axel Methner
Original Assignee
Bayer Healthcare Ag
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Bayer Healthcare Ag filed Critical Bayer Healthcare Ag
Publication of WO2008046543A1 publication Critical patent/WO2008046543A1/fr

Links

Classifications

    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G01N2500/04—Screening involving studying the effect of compounds C directly on molecule A (e.g. C are potential ligands for a receptor A, or potential substrates for an enzyme A)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/28—Neurological disorders
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/32—Cardiovascular disorders

Definitions

  • the present invention is in the field of molecular biology, more particularly, the present invention relates to nucleic acid sequences and amino acid sequences of a set of human genes or rat genes and its regulation for the treatment, diagnostic and use as a biomarker in cell protection processes.
  • cell protection means a mechanism or process which protects a cell or tissue from apoptosis, degeneration or remodelling processes.
  • Disorders associated with a lack of cell protection are "cellular stress protection deficiency” (CSPD) diseases.
  • CSPD cellular stress protection deficiency
  • Cell stress is broadly defined to include - but not limited to - cellular responses to heat shock, oxidative stress, heavy metals, and toxic chemicals
  • neuroprotection means mechanisms within the nervous system which protect neurons from apoptosis or degeneration, for example following a braininjury or as a result of chronic neurodegenerative diseases.
  • the word derives from the words “neuron” (Greek for nerve cell) and “protection” (Latin for "saving”).
  • Gene expression technologies may be useful in several areas of drug discovery and development, such as target identification, lead optimization, and identification of mechanisms of action.
  • the TaqMan technology can be used to compare differences between expression profiles of normal tissue and diseased tissue.
  • Expression profiling has been used in identifying genes, which are up- or downregulated in a variety of diseases.
  • An interesting application of expression profiling is temporal monitoring of changes in gene expression during disease progression and drug treatment or in patients versus healthy individuals.
  • the premise in this approach is that changes in pattern of gene expression in response to physiological or environmental stimuli (e.g., drugs) may serve as indirect clues about disease-causing genes or drug targets.
  • physiological or environmental stimuli e.g., drugs
  • the effects of drugs with established efficacy on global gene expression patterns may provide a guidepost, or a genetic signature, against which a new drug candidate can be compared.
  • the nucleotide sequence of PRSS23 (Target ID NO: 37) is accessible in the databases by the accession number AF015287 (human). The sequence is given in SEQ E) NO: 125. The amino acid sequence of PRSS23 is depicted in SEQ ID NO: 37.
  • PRSS23 is described as a secreted protease with synonyms putative secreted protein ZSIG13; serine protease, umbilical endothelium; MGC5107 and SPUVE .
  • PRSS23 mRNA expression is decribed as regulated in cancer tissues [Ifon et al. (2005); Pentecost et al., (2005)].
  • PRSS23 is published (but not limited to) in patents WO02081745, EP1612281 and
  • PRSS23 The expression of PRSS23 is upregulated in cell protection processes as shown in table 1 (Fig. I)-
  • the growth hormone secretagogue receptor is a G protein-coupled receptor (GPCR) found in the pituitary gland and brain that is involved in the control of growth hormone release.
  • GPCR G protein-coupled receptor
  • McKee et al. [McKee et al., (1997)] identified 2 human genes related to GHSR, GPR38 and GPR39.
  • the predicted 453 -amino acid GPR39 protein contains the 7 transmembrane domains characteristic of GPCRs.
  • McKee et al. [McKee et al., (1997)] found that the protein sequence of GPR39 is 27% and 32% identical to that of GSHR and neurotensin receptor- 1, respectively.
  • the invention relates to the use of a candidate set of genes useful as biomarkers, targets or diagnostic tools in cell protection processes.
  • the invention also relates to novel disease associations of those polypeptides and polynucleotides.
  • the invention also relates to the use of those genes as biomarkers for cell protection processes.
  • the invention also relates to novel methods of screening for therapeutic agents for cell protection approaches in a mammal.
  • the invention also relates to pharmaceutical compositions for the treatment diseases associated with cell protection processes in a mammal comprising a polypeptide, a polynucleotide, or regulator or modulators of candidate genes activity.
  • the invention further comprises methods of diagnosing diseases associated with cell protection processes in a mammal.
  • Table 1 Table contains numerical values for genes up-regulated under cell protection conditions. Relative expression of the mentioned gene in HEK293 cells: column 6: ,,Kl" (low expression of GPR39), column 7 ,,Kl 7" (high expression of GPR39), and column 8 ,,fold change” (numerical value of fold change factor between Kl and Kl 7. positive factor : up regulation; negative factor : down regulation).
  • Table 2 Table contains numerical values for genes down-regulated under cell protection conditions. Relative expression of the mentioned gene in HEK293 cells: column 6: ,JCl" (low expression of GPR39), column 7 ,,Kl 7" (high expression of GPR39), and column 8 ,,fold change” (numerical value of fold change factor between Kl and K17. positive factor : up regulation; negative factor : down regulation).
  • Table 3 List of Target NO: l-Target NO:88 polypeptides of the candidate genes of the invention with corresponding SEQ ID NO and Genbank accession number.
  • Table 4 List of Target NO: 1-Target NO:88 polynucleotides of the candidate genes of the invention with corresponding SEQ ID NO and Genbank accession number.
  • Fig. 5 Sequence Listing
  • the first column ("Target No") of all tables assigns serial numbers to all candidate genes and corresponds in all tables.
  • the sequences in the sequence list correspond to the sequence accession numbers in tables 3 and 4 and reflect the public available sequences at time of filing. Sequences were retrieved from Genbank with the listed accession numbers.
  • oligonucleotide is a stretch of nucleotide residues which has a sufficient number of bases to be used as an oligomer, amplimer or probe in a polymerase chain reaction (PCR). Oligonucleotides are prepared from genomic or cDNA sequence and are used to amplify, reveal, or confirm the presence of a similar DNA or RNA in a particular cell or tissue. Oligonucleotides or oligomers comprise portions of a DNA sequence having at least about 10 nucleotides and as many as about 35 nucleotides, preferably about 25 nucleotides.
  • Probes may be derived from naturally occurring or recombinant single- or double-stranded nucleic acids or may be chemically synthesized. They are useful in detecting the presence of identical or similar sequences. Such probes may be labeled with reporter molecules using nick translation, Klenow fill-in reaction, PCR or other methods well known in the art. Nucleic acid probes may be used in southern, northern or in situ hybridizations to determine whether DNA or RNA encoding a certain protein is present in a cell type, tissue, or organ.
  • a “fragment of a polynucleotide” is a nucleic acid that comprises all or any part of a given nucleotide molecule, the fragment having fewer nucleotides than about 6 kb, preferably fewer than about 1 kb.
  • Reporter molecules are radionuclides, enzymes, fluorescent, chemiluminescent, or chromo- genic agents which associate with a particular nucleotide or amino acid sequence, thereby establishing the presence of a certain sequence, or allowing for the quantification of a certain sequence.
  • “Chimeric” molecules may be constructed by introducing all or part of the nucleotide sequence of this invention into a vector containing additional nucleic acid sequence which might be expected to change any one or several of the following gene characteristics: cellular location, distribution, ligand-binding affinities, interchain affinities, degradation/turnover rate, signaling, etc.
  • “Active”, with respect to a candidate polypeptide refers to those forms, fragments, or domains of a candidate polypeptide which retain the biological and/or antigenic activity of a candidate polypeptide.
  • “Naturally occurring candidate polypeptide” refers to a polypeptide produced by cells which have not been genetically engineered and specifically contemplates various polypeptides arising from post-translational modifications of the polypeptide including but not limited to acetylation, carboxylation, glycosylation, phosphorylation, lipidation and acylation.
  • Derivative refers to polypeptides which have been chemically modified by techniques such as ubiquitination, labeling (see above), pegylation (derivatization with polyethylene glycol), and chemical insertion or substitution of amino acids such as ornithine which do not normally occur in human proteins.
  • Constant amino acid substitutions result from replacing one amino acid with another having similar structural and/or chemical properties, such as the replacement of a leucine with an isoleucine or valine, an aspartate with a glutamate, or a threonine with a serine.
  • “Insertions” or “deletions” are typically in the range of about 1 to 5 amino acids. The variation allowed may be experimentally determined by producing the peptide synthetically while systematically making insertions, deletions, or substitutions of nucleotides in the sequence using recombinant DNA techniques.
  • a “signal sequence” or “leader sequence” can be used, when desired, to direct the polypeptide through a membrane of a cell.
  • Such a sequence may be naturally present on the polypeptides of the present invention or provided from heterologous sources by recombinant DNA techniques.
  • Oligopeptide is a short stretch of amino acid residues and may be expressed from an oligonucleotide. Oligopeptides comprise a stretch of amino acid residues of at least 3, 5, 10 amino acids and at most 10, 15, 25 amino acids, typically of at least 9 to 13 amino acids, and of sufficient length to display biological and/or antigenic activity.
  • inhibitor is any substance which retards or prevents a chemical or physiological reaction or response. Common inhibitors include but are not limited to antisense molecules, antibodies, and antagonists.
  • Biomarker are measurable and quantifiable biological parameters (e.g. specific enzyme concentration, specific hormone concentration, specific gene phenotype distribution in a population, presence of biological substances) which serve as indices for health - and physiology related assessments, such as disease risk, psychiatric disorders, environmental exposure and its effects, disease diagnosis, metabolic processes, substance abuse, pregnancy, cell line development, epidemiologic studies, etc.. Parameter that can be used to identify a toxic effect in an individual organism and can be used in extrapolation between species. Indicator signalling an event or condition in a biological system or sample and giving a measure of exposure, effect, or susceptibility.
  • biological parameters e.g. specific enzyme concentration, specific hormone concentration, specific gene phenotype distribution in a population, presence of biological substances
  • Parameter that can be used to identify a toxic effect in an individual organism and can be used in extrapolation between species.
  • Indicator signalling an event or condition in a biological system or sample and giving a measure of exposure, effect, or susceptibility.
  • Biomarkers can reflect a variety of disease characteristics, including the level of exposure to an environmental or genetic trigger, an element of the disease process itself, an intermediate stage between exposure and disease onset, or an independent factor associated with the disease state but not causative of pathogenesis.
  • biomarkers can be used to identify the risk of developing an illness (antecedent biomarkers), aid in identifying disease (diagnostic biomarkers), or predict future disease course, including response to therapy (prognostic biomarkers).
  • Standard expression is a quantitative or qualitative measurement for comparison. It is based on a statistically appropriate number of normal samples and is created to use as a basis of comparison when performing diagnostic assays, running clinical trials, or following patient treatment profiles.
  • Animal as used herein may be defined to include human, domestic (e.g., cats, dogs, etc.), agricultural (e.g., cows, horses, sheep, etc.) or test species (e.g., mouse, rat, rabbit, etc.).
  • domestic e.g., cats, dogs, etc.
  • agricultural e.g., cows, horses, sheep, etc.
  • test species e.g., mouse, rat, rabbit, etc.
  • nucleotide sequences encoding a candidate gene have numerous applications in techniques known to those skilled in the art of molecular biology. These techniques include use as hybridization probes, use in the construction of oligomers for PCR, use for chromosome and gene mapping, use in the recombinant production of candidate gene, and use in generation of antisense DNA or RNA, their chemical analogs and the like. Uses of nucleotides encoding a candidate gene disclosed herein are exemplary of known techniques and are not intended to limit their use in any technique known to a person of ordinary skill in the art.
  • nucleotide sequences disclosed herein may be used in molecular biology techniques that have not yet been developed, provided the new techniques rely on properties of nucleotide sequences that are currently known, e.g., the triplet genetic code, specific base pair interactions, etc.
  • nucleotide sequences which encode a candidate gene, its derivatives or its variants are preferably capable of hybridizing to the nucleotide sequence of the naturally occurring candidate gene polynucleotide under stringent conditions, it may be advantageous to produce nucleotide sequences encoding candidate gene polypeptides or its derivatives possessing a substantially different codon usage. Codons can be selected to increase the rate at which expression of the peptide occurs in a particular prokaryotic or eukaryotic expression host in accordance with the frequency with which particular codons are utilized by the host.
  • RNA transcripts having more desirable properties such as a greater half-life, than transcripts produced from the naturally occurring sequence.
  • Nucleotide sequences encoding a candidate gene polypeptide may be joined to a variety of other nucleotide sequences by means of well established recombinant DNA techniques.
  • Useful nucleotide sequences for joining to candidate gene polynucleotides include an assortment of cloning vectors such as plasmids, cosmids, lambda phage derivatives, phagemids, and the like.
  • Vectors of interest include expression vectors, replication vectors, probe generation vectors, sequencing vectors, etc.
  • vectors of interest may contain an origin of replication functional in at least one organism, convenient restriction endonuclease sensitive sites, and selectable markers for one or more host cell systems.
  • Another aspect of the subject invention is to provide for candidate gene-specific hybridization probes capable of hybridizing with- naturally occurring nucleotide sequences encoding candidate gene. Such probes may also be used for the detection of similar protease encoding sequences and should preferably show at least 40% nucleotide identity to candidate gene polynucleotides.
  • the hybridization probes of the subject invention may be derived from nucleotide sequences comprised in a group of nucleotide sequences consisting of SEQ ID NO: 88 - SEQ ID NO: 176 or from genomic sequences including promoter, enhancers or introns of the native gene. Hybridization probes may be labelled by a variety of reporter molecules using techniques well known in the art.
  • the invention relates to nucleic acid sequences that hybridize with such candidate gene encoding nucleic acid sequences under stringent conditions.
  • Nucleic acid molecules that will hybridize to candidate gene polynucleotides under stringent conditions can be identified functionally.
  • examples of the uses for hybridization probes include: histochemical uses such as identifying tissues that express candidate gene; measuring mRNA levels, for instance to identify a sample's tissue type or to identify cells that express abnormal levels of candidate gene; and detecting polymorphisms of candidate gene.
  • PCR provides additional uses for oligonucleotides based upon the nucleotide sequence which encodes candidate gene.
  • probes used in PCR may be of recombinant origin, chemically synthesized, or a mixture of both.
  • Oligomers may comprise discrete nucleotide sequences employed under optimized conditions for identification of candidate gene in specific tissues or diagnostic use. The same two oligomers, a nested set of oligomers, or even a degenerate pool of oligomers may be employed under less stringent conditions for identification of closely related DNAs or RNAs.
  • PCR primers i.e., preparations of primers that are heterogeneous at given sequence locations, can be designed to amplify nucleic acid sequences that are highly homologous to, but not identical with candidate gene.
  • Strategies are now available that allow for only one of the primers to be required to specifically hybridize with a known sequence.
  • appropriate nucleic acid primers can be ligated to the nucleic acid sought to be amplified to provide the hybridization partner for one of the primers. In this way, only one of the primers need be based on the sequence of the nucleic acid sought to be amplified.
  • PCR methods for amplifying nucleic acid will utilize at least two primers.
  • One of these primers will be capable of hybridizing to a first strand of the nucleic acid to be amplified and of priming enzyme-driven nucleic acid synthesis in a first direction.
  • the other will be capable of hybridizing the reciprocal sequence of the first strand (if the sequence to be amplified is single stranded, this sequence will initially be hypothetical, but will be synthesized in the first amplification cycle) and of priming nucleic acid synthesis from that strand in the direction opposite the first direction and towards the site of hybridization for the first primer.
  • Conditions for conducting such amplifications particularly under preferred stringent hybridization conditions, are well known.
  • RNA polymerase as T7 or SP6 RNA polymerase and the appropriate reporter molecules.
  • nucleic acid sequence can be inserted into any of the many available DNA vectors and their respective host cells using techniques which are well known in the art.
  • synthetic chemistry may be used to introduce mutations into the nucleotide sequence.
  • a portion of sequence in which a mutation is desired can be synthesized and recombined with longer portion of an existing genomic or recombinant sequence.
  • candidate gene polynucleotides may be used to produce a purified oligo-or polypeptide using well known methods of recombinant DNA technology.
  • the oligopeptide may be expressed in a variety of host cells, either prokaryotic or eukaryotic. Host cells may be from the same species from which the nucleotide sequence was derived or from a different species. Advantages of producing an oligonucleotide by recombinant DNA technology include obtaining adequate amounts of the protein for purification and the availability of simplified purification procedures.
  • Chromosome-based techniques such as comparative genomic hybridization (CGH) and fluorescent in situ hybridization (FISH) facilitate efforts to cytogenetically localize genomic regions that are altered in tumor cells. Regions of genomic alteration can be narrowed further using loss of hetero2ygosity analysis (LOH), in which disease DNA is analyzed and compared with normal DNA for the loss of a heterozygous polymorphic marker.
  • LH loss of hetero2ygosity analysis
  • the first experiments used restriction fragment length polymorphisms (RPLPs) [Johnson, (1989)], or hypervariable minisatellite DNA [Barnes, 2000].
  • Nucleic acid arrays that have been used in the present invention are those that are commercially available from Affymetrix (Santa Clara, Calif.) under the brand name GeneChip Human Genome U133 Plus 2.0 Array.® or Rat Genome U230 plus 2.0 Array respectively which represents the complete coverage of the Human Genome Ul 33 Set plus 9921 probe sets representing approximately 6,500 new genes (with a total of approximately 56 000 transcripts) or the Rat Genome respectively.
  • Affymetrix (Santa Clara, Calif.) GeneChip technology platform which consists of high-density microarrays and tools to help process and analyze those arrays, including standardized assays and reagents, instrumentation, and data management and analysis tools.
  • GeneChip microarrays consist of small DNA fragments (referred to as probes), chemically synthesized at specific locations on a coated quartz surface. By extracting and labeling nucleic acids from experimental samples, and then hybridizing those prepared samples to the array, the amount of label can be monitored enabling a measurement of gene regulation
  • the GeneChip human genome arrays include a set of human maintenance genes to facilitate the normalization and scaling of array experiments and to perform data comparison. This set of normalization genes shows consistent levels of expression over a diverse set of tissues.
  • a number of diseases are associated with changes in the copy number of a certain gene.
  • the real-time PCR method or microarray analyis can be used to determine the expression levels which are known to be linked with diseases that are associated with the symptoms the patient has.
  • Fusion proteins are useful for generating antibodies against candidate gene polypeptides and for use in various assay systems. For example, fusion proteins can be used to identify proteins which interact with portions of candidate gene polypeptides. Protein affinity chromatography or library-based assays for protein-protein interactions, such as the yeast two-hybrid or phage display systems, can be used for this purpose. Such methods are well known in the art and also can be used as drug screens.
  • a candidate gene fusion protein comprises two polypeptide segments fused together by means of a peptide bond.
  • the first polypeptide segment can comprise at least 54, 75, 100, 125, 139, 150, 175, 200, 225, 250, 275, 300, 325 or 350 contiguous amino acids of polypeptide sequences comprised in a group of polypeptide sequences consisting of SEQ ID NO:1 - SEQ ID NO:88 or of a variant, such as those described above.
  • the first polypeptide segment also can comprise full-length candidate gene.
  • the second polypeptide segment can be a full-length protein or a protein fragment.
  • Proteins commonly used in fusion protein construction include, but are not limited to ⁇ galactosidase, ⁇ - glucuronidase, green fluorescent protein (GFP), autofluorescent proteins, including blue fluorescent protein (BFP), glutathione-S-transferase (GST), luciferase, horseradish peroxidase (HRP), and chloramphenicol acetyltransferase (CAT).
  • epitope tags are used in fusion protein constructions, including histidine (His) tags, FLAG tags, influenza hemagglutinin (HA) tags, Myc tags, VSV-G tags, and thioredoxin (Trx) tags.
  • Other fusion constructions can include maltose binding protein (MBP), S-tag, Lex a DNA binding domain (DBD) fusions, GAL4 DNA binding domain fusions, and herpes simplex virus (HSV) BP 16 protein fusions.
  • a fusion protein also can be engineered to contain a cleavage site located adjacent to the candidate gene.
  • a naturally occurring candidate gene polynucleotide can be isolated free of other cellular components such as membrane components, proteins, and lipids.
  • Polynucleotides can be made by a cell and isolated using standard nucleic acid purification techniques, or synthesized using an amplification technique, such as the polymerase chain reaction (PCR), or by using an automatic synthesizer. Methods for isolating polynucleotides are routine and are known in the art. Any such technique for obtaining a polynucleotide can be used to obtain isolated candidate gene polynucleotides. For example, restriction enzymes and probes can be used to isolate polynucleotide fragments which comprise candidate gene nucleotide sequences. Isolated polynucleotides are in preparations which are free or at least 70, 80, or 90% free of other molecules.
  • candidate gene cDNA molecules can be made with standard molecular biology techniques, using candidate gene mRNA as a template, candidate gene cDNA molecules can thereafter be replicated using molecular biology techniques known in the art.
  • An amplification technique such as PCR, can be used to obtain additional copies of polynucleotides of the invention, using either human genomic DNA or cDNA as a template.
  • synthetic chemistry techniques can be used to synthesizes candidate gene polynucleotides.
  • the degeneracy of the genetic code allows alternate nucleotide sequences to be synthesized which will encode candidate gene having, for example, an amino acid sequence comprised in a group of polypeptide sequences consisting of SEQ ID NO: 1 - SEQ ID NO:88 or a biologically active variant thereof.
  • PCR-based methods can be used to extend nucleic acid sequences encoding human candidate gene, for example to detect upstream sequences of candidate gene gene such as promoters and regulatory elements.
  • restriction-site PCR uses universal primers to retrieve unknown sequence adjacent to a known locus. Genomic DNA is first amplified in the presence of a primer to a linker sequence and a primer specific to the known region. The amplified sequences are then subjected to a second round of PCR with the same linker primer and another specific primer internal to the first one. Products of each round of PCR are transcribed with an appropriate RNA polymerase and sequenced using reverse transcriptase.
  • Inverse PCR also can be used to amplify or extend sequences using divergent primers based on a known region.
  • Primers can be designed using commercially available software, such as OLIGO
  • Primer Analysis software (National Biosciences Inc., Plymouth, Minn.), to be 22-30 nucleotides in length, to have a GC content of 50% or more, and to anneal to the target sequence at temperatures about 68-72 0 C.
  • the method uses several restriction enzymes to generate a suitable fragment in the known region of a gene. The fragment is then circularized by intramolecular ligation and used as a PCR template.
  • capture PCR which involves PCR amplification of DNA fragments adjacent to a known sequence in human and yeast artificial chromosome DNA.
  • multiple restriction enzyme digestions and ligations also can be used to place an engineered double-stranded sequence into an unknown fragment of the DNA molecule before performing PCR.
  • Randomly-primed libraries are preferable, in that they will contain more sequences which contain the 5' regions of genes. Use of a randomly primed library may be especially preferable for situations in which an oligo d(T) library does not yield a full- length cDNA. Genomic libraries can be useful for extension of sequence into 5' non-transcribed regulatory regions.
  • capillary electrophoresis systems can be used to analyze the size or confirm the nucleotide sequence of PCR or sequencing products.
  • capillary sequencing can employ flowable polymers for electrophoretic separation, four different fluorescent dyes (one for each nucleotide) which are laser activated, and detection of the emitted wavelengths by a charge coupled device camera.
  • Output/light intensity can be converted to electrical signal using appropriate equipment and software (e.g., GENOTYPER and Sequence NAVIGATOR, Perkin Elmer), and the entire process from loading of samples to computer analysis and electronic data display can be computer controlled.
  • Capillary electrophoresis is especially preferable for the sequencing of small pieces of DNA which might be present in limited amounts in a particular sample.
  • candidate gene can be obtained, for example, by purification from human cells, by expression of candidate gene polynucleotides, or by direct chemical synthesis.
  • candidate gene can be purified from any human cell which expresses the enzyme, including those which have been transfected with expression constructs which express candidate gene.
  • a purified candidate gene is separated from other compounds which normally associate with candidate gene in the cell, such as certain proteins, carbohydrates, or lipids, using methods well- known in the art. Such methods include, but are not limited to, size exclusion chromatography, ammonium sulfate fractionation, ion exchange chromatography, affinity chromatography, and preparative gel electrophoresis.
  • candidate gene polynucleotides can be inserted into an expression vector which contains the necessary elements for the transcription and translation of the inserted coding sequence.
  • Methods which are well known to those skilled in the art can be used to construct expression vectors containing sequences encoding candidate gene and appropriate transcriptional and translational control elements. These methods include in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination.
  • a variety of expression vector/host systems can be utilized to contain and express sequences encoding candidate gene. These include, but are not limited to, microorganisms, such as bacteria transformed with recombinant bacteriophage, plasmid, or cosmid DNA expression vectors; yeast transformed with yeast expression vectors, insect cell systems infected with virus expression vectors ⁇ e.g., baculovirus), plant cell systems transformed with virus expression vectors ⁇ e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or with bacterial expression vectors ⁇ e.g. , Ti or pBR322 plasmids), or animal cell systems.
  • microorganisms such as bacteria transformed with recombinant bacteriophage, plasmid, or cosmid DNA expression vectors
  • yeast transformed with yeast expression vectors insect cell systems infected with virus expression vectors ⁇ e.g., baculovirus
  • plant cell systems transformed with virus expression vectors ⁇ e.g., cauliflower mosaic virus, Ca
  • control elements or regulatory sequences are those non-translated regions of the vector - enhancers, promoters, 5' and 3' untranslated regions — which interact with host cellular proteins to carry out transcription and translation. Such elements can vary in their strength and specificity.
  • any number of suitable transcription and translation elements including constitutive and inducible promoters, can be used.
  • inducible promoters such as the hybrid lacZ promoter of the BLUESCRIPT phagemid (Stratagene, LaJoIIa, Calif.) or pSPORTl plasmid (Life Technologies) and the like can be used.
  • the baculovirus polyhedrin promoter can be used in insect cells.
  • Promoters or enhancers derived from the genomes of plant cells ⁇ e.g., heat shock, RUBISCO, and storage protein genes
  • plant viruses ⁇ e.g., viral promoters or leader sequences
  • promoters from mammalian genes or from mammalian viruses are preferable. If it is necessary to generate a cell line that contains multiple copies of a nucleotide sequence encoding candidate gene, vectors based on SV40 or EBV can be used with an appropriate selectable marker.
  • a number of expression vectors can be selected. For example, when a large quantity of candidate gene is needed for the induction of antibodies, vectors which direct high level expression of fusion proteins that are readily purified can be used. Such vectors include, but are not limited to, multifunctional E. coli cloning and expression vectors such as BLUESCRIPT (Stratagene). In a BLUESCRIPT vector, a sequence encoding candidate gene can be ligated into the vector in frame with sequences for the amino-terminal Met and the subsequent 7 residues of ⁇ -galactosidase so that a hybrid protein is produced.
  • BLUESCRIPT a sequence encoding candidate gene can be ligated into the vector in frame with sequences for the amino-terminal Met and the subsequent 7 residues of ⁇ -galactosidase so that a hybrid protein is produced.
  • pIN vectors or pGEX vectors also can be used to express foreign polypeptides as fusion proteins with glutathione S-transferase (GST).
  • GST glutathione S-transferase
  • fusion proteins are soluble and can easily be purified from lysed cells by adsorption to glutathione-agarose beads followed by elution in the presence of free glutathione.
  • Proteins made in such systems can be designed to include heparin, thrombin, or factor Xa protease cleavage sites so that the cloned polypeptide of interest can be released from the GST moiety at will.
  • sequences encoding candidate gene can be driven by any of a number of promoters.
  • viral promoters such as the 35 S and 19S promoters of CaMV can be used alone or in combination with the omega leader sequence from TMV.
  • plant promoters such as the small subunit of RUBISCO or heat shock promoters can be used. These constructs can be introduced into plant cells by direct DNA transformation or by pathogen-mediated transfection.
  • An insect system also can be used to express candidate gene.
  • Autographa californica nuclear polyhidrosis virus (AcNPV) is used as a vector to express foreign genes in Spodoptera frugiperda cells or in Trichoplusia larvae.
  • Sequences encoding candidate gene can be cloned into a non-essential region of the virus, such as the polyhedrin gene, and placed under control of the polyhedrin promoter.
  • Successful insertion of candidate gene will render the polyhedrin gene inactive and produce recombinant virus lacking coat protein.
  • the recombinant viruses can then be used to infect S. frugiperda cells or Trichoplusia larvae in which candidate gene can be expressed.
  • a number of viral-based expression systems can be used to express candidate gene in mammalian host cells.
  • sequences encoding candidate gene can be ligated into an adenovirus transcription/translation complex comprising the late promoter and tripartite leader sequence. Insertion in a non-essential El or E3 region of the viral genome can be used to obtain a viable virus which is capable of expressing candidate gene in infected host cells [Engelhard, (1994)].
  • transcription enhancers such as the Rous sarcoma virus (RSV) enhancer, can be used to increase expression in mammalian host cells.
  • RSV Rous sarcoma virus
  • HACs Human artificial chromosomes
  • HACs also can be used to deliver larger fragments of DNA than can be contained and expressed in a plasmid.
  • HACs of 6M to 1OM are constructed and delivered to cells via conventional delivery methods (e.g., liposomes, polycationic amino polymers, or vesicles).
  • Specific initiation signals also can be used to achieve more efficient translation of sequences encoding candidate gene. Such signals include the ATG initiation codon and adjacent sequences. In cases where sequences encoding candidate gene, its initiation codon, and upstream sequences are inserted into the appropriate expression vector, no additional transcriptional or translational control signals may be needed.
  • exogenous translational control signals including the ATG initiation codon
  • the initiation codon should be in the correct reading frame to ensure translation of the entire insert.
  • Exogenous translational elements and initiation codons can be of various origins, both natural and synthetic.
  • a host cell strain can be chosen for its ability to modulate the expression of the inserted sequences or to process the expressed candidate gene in the desired fashion.
  • modifications of the polypeptide include, but are not limited to, acetylation, carboxylation, glycosylation, phosphorylation, lipidation, and acylation.
  • Post-translational processing which cleaves a "prepro" form of the polypeptide also can be used to facilitate correct insertion, folding and/or function.
  • Different host cells which have specific cellular machinery and characteristic mechanisms for post-translational activities (e.g., CHO, HeLa, MDCK, HEK293, and WI38), are available from the American Type Culture Collection (ATCC; 10801 University Boulevard, Manassas, VA 20110-2209) and can be chosen to ensure the correct modification and processing of the foreign protein.
  • ATCC American Type Culture Collection
  • Stable expression is preferred for long-term, high-yield production of recombinant proteins.
  • cell lines which stably express candidate gene can be transformed using expression vectors which can contain viral origins of replication and/or endogenous expression elements and a selectable marker gene on the same or on a separate vector. Following the introduction of the vector, cells can be allowed to grow for 1 -2 days in an enriched medium before they are switched to a selective medium.
  • the purpose of the selectable marker is to confer resistance to selection, and its presence allows growth and recovery of cells which successfully express the introduced candidate gene sequences.
  • Resistant clones of stably transformed cells can be proliferated using tissue culture techniques appropriate to the cell type. Any number of selection systems can be used to recover transformed cell lines.
  • herpes simplex virus thymidine kinase [Logan, (1984)] and adenine phosphoribosyltransferase [Wigler, (1977)] genes which can be employed in tk ⁇ or aprf cells, respectively.
  • antimetabolite, antibiotic, or herbicide resistance can be used as the basis for selection.
  • dhfr confers resistance to methotrexate [Lowy, (1980)]
  • npt confers resistance to the aminoglycosides, neomycin and G-418 [Wigler, (1980)]
  • als and pat confer resistance to chlorsulfuron and phosphinotricin acetyltransferase, respectively [Colbere-Garapin, 1981]. Additional selectable genes have been described.
  • trpB allows cells to utilize indole in place of tryptophan, or hisD, which allows cells to utilize histinol in place of histidine.
  • Visible markers such as anthocyanins, ⁇ -glucuronidase and its substrate GUS, and luciferase and its substrate luciferin, can be used to identify transformants and to quantify the amount of transient or stable protein expression attributable to a specific vector system
  • marker gene expression suggests that a candidate gene polynucleotide is also present, its presence and expression may need to be confirmed. For example, if a sequence encoding candidate gene is inserted within a marker gene sequence, transformed cells containing sequences which encode candidate gene can be identified by the absence of marker gene function. Alternatively, a marker gene can be placed in tandem with a sequence encoding candidate gene under the control of a single promoter. Expression of the marker gene in response to induction or selection usually indicates expression of candidate gene polynucleotide.
  • host cells which contain a candidate gene polynucleotide and which express candidate gene can be identified by a variety of procedures known to those of skill in the art. These procedures include, but are not limited to, DNA-DNA or DNA-RNA hybridizations and protein bioassay or immunoassay techniques which include membrane, solution, or chip-based technologies for the detection and/or quantification of nucleic acid or protein.
  • DNA-DNA or DNA-RNA hybridizations and protein bioassay or immunoassay techniques which include membrane, solution, or chip-based technologies for the detection and/or quantification of nucleic acid or protein.
  • the presence of a polynucleotide sequence encoding candidate gene can be detected by DNA-DNA or DNA-RNA hybridization or amplification using probes or fragments or fragments of polynucleotides encoding candidate gene.
  • Nucleic acid amplification-based assays involve the use of oligonucleotides selected from sequences encoding candidate gene to detect transformants which contain a candidate gene poly
  • a variety of protocols for detecting and measuring the expression of candidate gene, using either polyclonal or monoclonal antibodies specific for the polypeptide, are known in the art. Examples include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), and fluorescence activated cell sorting (FACS).
  • ELISA enzyme-linked immunosorbent assay
  • RIA radioimmunoassay
  • FACS fluorescence activated cell sorting
  • a two-site, monoclonal-based immunoassay using monoclonal antibodies reactive to two non-interfering epitopes on candidate gene can be used, or a competitive binding assay can be employed.
  • a wide variety of labels and conjugation techniques are known by those skilled in the art and can be used in various nucleic acid and amino acid assays.
  • Means for producing labeled hybridization or PCR probes for detecting sequences related to polynucleotides encoding candidate gene include oligolabeling, nick translation, end-labeling, or PCR amplification using a labeled nucleotide.
  • sequences encoding candidate gene can be cloned into a vector for the production of an mRNA probe.
  • Such vectors are known in the art, are commercially available, and can be used to synthesize RNA probes in vitro by addition of labeled nucleotides and an appropriate RNA polymerase such as T7, T3, or SP6. These procedures can be conducted using a variety of commercially available kits (Amersham Pharmacia Biotech, Promega, and US Biochemical).
  • Suitable reporter molecules or labels which can be used for ease of detection include radionuclides, enzymes, and fluorescent, chemiluminescent, or chromogenic agents, as well as substrates, cofactors, inhibitors, magnetic particles, and the like.
  • Host cells transformed with candidate gene polynucleotides can be cultured under conditions suitable for the expression and recovery of the protein from cell culture.
  • the polypeptide produced by a transformed cell can be secreted or contained intracellularly depending on the sequence and/or the vector used.
  • expression vectors containing candidate gene polynucleotides can be designed to contain signal sequences which direct secretion of soluble candidate gene through a prokaryotic or eukaryotic cell membrane or which direct the membrane insertion of membrane-bound candidate gene.
  • purification facilitating domains include, but are not limited to, metal chelating peptides such as histidine-tryptophan modules that allow purification on immobilized metals, protein A domains that allow purification on immobilized immunoglobulin, and the domain utilized in the FLAGS extension/affinity purification system (Immunex Corp., Seattle, Wash.).
  • cleavable linker sequences such as those specific for Factor XA or enterokinase (Invitrogen, San Diego, CA) between the purification domain and candidate gene also can be used to facilitate purification.
  • One such expression vector provides for expression of a fusion protein containing candidate gene and 6 histidine residues preceding a thioredoxin or an enterokinase cleavage site. The histidine residues facilitate purification by IMAC (immobilized metal ion affinity chromatography) Maddox, (1983)], while the enterokinase cleavage site provides a means for purifying candidate gene from the fusion protein [Porath, (1992)]. Chemical Synthesis
  • Sequences encoding candidate gene can be synthesized, in whole or in part, using chemical methods well known in the art.
  • candidate gene itself can be produced using chemical methods to synthesize its amino acid sequence, such as by direct peptide synthesis using solid-phase techniques. Protein synthesis can either be performed using manual techniques or by automation. Automated synthesis can be achieved, for example, using Applied Biosystems 43 IA Peptide Synthesizer (Perkin Elmer).
  • fragments of candidate gene can be separately synthesized and combined using chemical methods to produce a full-length molecule.
  • the newly synthesized peptide can be substantially purified by preparative high performance liquid chromatography.
  • the composition of a synthetic candidate gene can be confirmed by amino acid analysis or sequencing. Additionally, any portion of the amino acid sequence of candidate gene can be altered during direct synthesis and/or combined using chemical methods with sequences from other proteins to produce a variant polypeptide or a fusion protein.
  • codons preferred by a particular prokaryotic or eukaryotic host can be selected to increase the rate of protein expression or to produce an RNA transcript having desirable properties, such as a half-life which is longer than that of a transcript generated from the naturally occurring sequence.
  • nucleotide sequences referred to herein can be engineered using methods generally known in the art to alter candidate gene polynucleotides for a variety of reasons, including but not limited to, alterations which modify the cloning, processing, and/or expression of the polypeptide or mRNA product.
  • DNA shuffling by random fragmentation and PCR reassembly of gene fragments and synthetic oligonucleotides can be used to engineer the nucleotide sequences.
  • site-directed mutagenesis can be used to insert new restriction sites, alter glycosylation patterns, change codon preference, produce splice variants, introduce mutations, and so forth.
  • candidate gene analogs are variants having an amino acid sequence that is a mutation of the amino acid sequence disclosed herein.
  • Another general class of candidate gene analogs is provided by anti-idiotype antibodies, and fragments thereof, as described below.
  • variable domains of antiidiotype candidate gene antibodies mimic candidate gene, these domains can provide candidate gene enzymatic activity.
  • Methods of producing anti-idiotypic catalytic antibodies are known to those of skill in the art [Joron et al., (1992), Friboulet et al. (1994), Avalle et al., (1998)].
  • candidate gene and its analogs are produced of labeled peptides from a labeled protein substrate.
  • Proteases can also be used in detergents and cleaning solutions.
  • serine proteases are used in solutions to clean and to disinfect contact lenses (see, for example, [U.S. 5,985,629]).
  • Another use for a serine protease is in the formulation of vaccines (see, for example, [U.S. 5,885,814]).
  • Those of skill in the art can devise other uses for molecules having candidate gene activity.
  • Any type of antibody known in the art can be generated to bind specifically to an epitope of candidate gene.
  • Antibody as used herein includes intact immunoglobulin molecules, as well as fragments thereof, such as Fab, F(ab') 2 , and Fv, which are capable of binding an epitope of candidate gene. Typically, at least 6, 8, 10, or 12 contiguous amino acids are required to form an epitope. However, epitopes which involve non-contiguous amino acids may require more, e.g., at least 15, 25, or 50 amino acid.
  • An antibody which specifically binds to an epitope of candidate gene can be used therapeutically, as well as in immunochemical assays, such as Western blots, ELISAs, radioimmunoassays, immunohistochemical assays, immunoprecipitations, or other immunochemical assays known in the art.
  • immunoassays can be used to identify antibodies having the desired specificity. Numerous protocols for competitive binding or immunoradiometric assays are well known in the art. Such immunoassays typically involve the measurement of complex formation between an immunogen and an antibody which specifically binds to the candidate gene immunogen.
  • an antibody which specifically binds to candidate gene provides a detection signal at least 5-, 10-, or 20-fold higher than a detection signal provided with other proteins when used in an immunochemical assay.
  • antibodies which specifically bind to candidate gene do not detect other proteins in immunochemical assays and can immunoprecipitate candidate gene from solution.
  • candidate gene can be used to immunize a mammal, such as a mouse, rat, rabbit, guinea pig, monkey, or human, to produce polyclonal antibodies.
  • candidate gene can be conjugated to a carrier protein, such as bovine serum albumin, thyroglobulin, and keyhole limpet hemocyanin.
  • carrier protein such as bovine serum albumin, thyroglobulin, and keyhole limpet hemocyanin.
  • various adjuvants can be used to increase the immunological response.
  • adjuvants include, but are not limited to, Freund's adjuvant, mineral gels (e.g., aluminum hydroxide), and surface active substances (e.g., lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, and dinitrophenol).
  • mineral gels e.g., aluminum hydroxide
  • surface active substances e.g., lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, and dinitrophenol.
  • BCG Bacilli Calmette-Gueri ⁇
  • Corynebacterium parvum are especially useful.
  • Monoclonal antibodies which specifically bind to candidate gene can be prepared using any technique which provides for the production of antibody molecules by continuous cell lines in culture. These techniques include, but are not limited to, the hybridoma technique, the human B- cell hybridoma technique, and the EBV-hybridoma technique [Roberge, (1995)].
  • chimeric antibodies the splicing of mouse antibody genes to human antibody genes to obtain a molecule with appropriate antigen specificity and biological activity
  • Monoclonal and other antibodies also can be "humanized” to prevent a patient from mounting an immune response against the antibody when it is used therapeutically.
  • Such antibodies may be sufficiently similar in sequence to human antibodies to be used directly in therapy or may require alteration of a few key residues. Sequence differences between rodent antibodies and human sequences can be minimized by replacing residues which differ from those in the human sequences by site directed mutagenesis of individual residues or by grating of entire complementarity determining regions.
  • Antibodies which specifically bind to candidate gene can contain antigen binding sites which are either partially or fully humanized, as disclosed in U.S. 5,565,332.
  • single chain antibodies can be adapted using methods known in the art to produce single chain antibodies which specifically bind to candidate gene.
  • Antibodies with related specificity, but of distinct idiotypic composition can be generated by chain shuffling from random combinatorial immunoglobin libraries.
  • Single-chain antibodies also can be constructed using a DNA amplification method, such as PCR, using hybridoma cDNA as a template.
  • Single-chain antibodies can be mono- or bispeciflc, and can be bivalent or tetravalent. Construction of tetravalent, bispecific single-chain antibodies is taught.
  • a nucleotide sequence encoding a single-chain antibody can be constructed using manual or automated nucleotide synthesis, cloned into an expression construct using standard recombinant DNA methods, and introduced into a cell to express the coding sequence, as described below.
  • single-chain antibodies can be produced directly using, for example, filamentous phage technology.
  • Antibodies which specifically bind to candidate gene also can be produced by inducing in vivo production in the lymphocyte population or by screening immunoglobulin libraries or panels of highly specific binding reagents.
  • Other types of antibodies can be constructed and used therapeutically in methods of the invention.
  • chimeric antibodies can be constructed as disclosed in WO 93/03151.
  • Antibodies according to the invention can be purified by methods well known in the art. For example, antibodies can be affinity purified by passage over a column to which candidate gene is bound. The bound antibodies can then be eluted from the column using a buffer with a high salt concentration.
  • Antisense oligonucleotides are nucleotide sequences which are complementary to a specific DNA or RNA sequence. Once introduced into a cell, the complementary nucleotides combine with natural sequences produced by the cell to form complexes and block either transcription or translation. Preferably, an antisense oligonucleotide is at least 11 nucleotides in length, but can be at least 12, 15, 20, 25, 30, 35, 40, 45, or 50 or more nucleotides long. Longer sequences also can be used. Antisense oligonucleotide molecules can be provided in a DNA construct and introduced into a cell as described above to decrease the level of candidate gene gene products in the cell.
  • Antisense oligonucleotides can be deoxyribonucleotides, ribonucleotides, or a combination of both. Oligonucleotides can be synthesized manually or by an automated synthesizer, by covalently linking the 5' end of one nucleotide with the 3' end of another nucleotide with non- phosphodiester internucleotide linkages such alkylphosphonates, phosphorothioates, phos- phorodithioates, alkylphosphonothioates, alkylphosphonates, phosphoramidates, phosphate esters, carbamates, acetamidate, carboxymethyl esters, carbonates, and phosphate triesters.
  • Modifications of candidate gene gene expression can be obtained by designing antisense oligonucleotides which will form duplexes to the control, 5', or regulatory regions of the candidate gene gene. Oligonucleotides derived from the transcription initiation site, e.g., between positions -10 and +10 from the start site, are preferred. Similarly, inhibition can be achieved using "triple helix" base-pairing methodology. Triple helix pairing is useful because it causes inhibition of the ability of the double helix to open sufficiently for the binding of polymerases, transcription factors, or chaperons. Therapeutic advances using triplex DNA have been described in the literature [Nicholls, (1993)]. An antisense oligonucleotide also can be designed to block translation of mRNA by preventing the transcript from binding to ribosomes.
  • Antisense oligonucleotides which comprise, for example, 2, 3, 4, or 5 or more stretches of contiguous nucleotides which are precisely complementary to a candidate gene polynucleotide, each separated by a stretch of contiguous nucleotides which are not complementary to adjacent candidate gene nucleotides, can provide sufficient targeting specificity for candidate gene mRNA.
  • each stretch of complementary contiguous nucleotides is at least 4, 5, 6, 7, or 8 or more nucleotides in length.
  • Non-complementary intervening sequences are preferably 1, 2, 3, or 4 nucleotides in length.
  • One skilled in the art can easily use the calculated melting point of an antisense-sense pair to determine the degree of mismatching which will be tolerated between a particular antisense oligonucleotide and a particular candidate gene polynucleotide sequence.
  • Antisense oligonucleotides can be modified without affecting their ability to hybridize to a candidate gene polynucleotide. These modifications can be internal or at one or both ends of the antisense molecule.
  • internucleoside phosphate linkages can be modified by adding cholesteryl or diamine moieties with varying numbers of carbon residues between the amino groups and terminal ribose.
  • Modified bases and/or sugars such as arabinose instead of ribose, or a 3', 5'-substituted oligonucleotide in which the 3' hydroxyl group or the 5' phosphate group are substituted, also can be employed in a modified antisense oligonucleotide.
  • modified oligonucleotides can be prepared by methods well known in the art.
  • Ribozymes are RNA molecules with catalytic activity [Uhlmann, (1987)]. Ribo2ymes can be used to inhibit gene function by cleaving an RNA sequence, as is known in the art. The mechanism of ribozyme action involves sequence-specific hybridization of the ribozyme molecule to complementary target RNA, followed by endonucleolytic cleavage. Examples include engineered hammerhead motif ribozyme molecules that can specifically and efficiently catalyze endonucleolytic cleavage of specific nucleotide sequences. The coding sequence of a candidate gene polynucleotide can be used to generate ribozymes which will specifically bind to mRNA transcribed from a candidate gene polynucleotide.
  • ribozymes which can cleave other RNA molecules in trans in a highly sequence specific manner have been developed and described in the art.
  • the cleavage activity of ribozymes can be targeted to specific RNAs by engineering a discrete "hybridization" region into the ribozyme.
  • the hybridization region contains a sequence complementary to the target RNA and thus specifically hybridizes with the target RNA.
  • Specific ribozyme cleavage sites within a candidate gene RNA target can be identified by scanning the target molecule for ribozyme cleavage sites which include the following sequences: GUA, GUU, and GUC. Once identified, short RNA sequences of between 15 and 20 ribonucleotides corresponding to the region of the target RNA containing the cleavage site can be evaluated for secondary structural features which may render the target inoperable. Suitability of candidate candidate gene RNA targets also can be evaluated by testing accessibility to hybridization with complementary oligonucleotides using ribonuclease protection assays.
  • nucleotide sequences comprised in a group of nucleotide sequences consisting of SEQ ID NO: 88 - SEQ ID NO: 176 and its complement provide sources of suitable hybridization region sequences. Longer complementary sequences can be used to increase the affinity of the hybridization sequence for the target.
  • the hybridizing and cleavage regions of the ribozyme can be integrally related such that upon hybridizing to the target RNA through the complementary regions, the catalytic region of the ribozyme can cleave the target.
  • Ribozymes can be introduced into cells as part of a DNA construct. Mechanical methods, such as microinjection, liposome-mediated transfection, electroporation, or calcium phosphate precipitation, can be used to introduce a ribozyme-containing DNA construct into cells in which it is desired to decrease candidate gene expression. Alternatively, if it is desired that the cells stably retain the DNA construct, the construct can be supplied on a plasmid and maintained as a separate element or integrated into the genome of the cells, as is known in the art.
  • a ribozyme- encoding DNA construct can include transcriptional regulatory elements, such as a promoter element, an enhancer or UAS element, and a transcriptional terminator signal, for controlling transcription of ribozymes in the cells. Ribozymes also can be engineered to provide an additional level of regulation, so that destruction of mRNA occurs only when both a ribozyme and a target gene are induced in the cells. Screening / Screening Assays
  • Regulators as used herein refer to compounds that affect the activity of candidate gene in vivo and/or in vitro. Regulators can be agonists and antagonists of candidate gene polypeptide and can be compounds that exert their effect on the candidate gene activity via the enzymatic activity, expression, post-translational modifications or by other means.
  • Agonists of candidate gene are molecules which, when bound to candidate gene, increase or prolong the activity of candidate gene.
  • Agonists of candidate gene include proteins, nucleic acids, carbohydrates, small molecules, or any other molecule which activate candidate gene.
  • Antagonists of candidate gene are molecules which, when bound to candidate gene, decrease the amount or the duration of the activity of candidate gene. Antagonists include proteins, nucleic acids, carbohydrates, antibodies, small molecules, or any other molecule which decrease the activity of candidate gene.
  • modulate refers to a change in the activity of candidate gene polypeptide. For example, modulation may cause an increase or a decrease in enzymatic activity, binding characteristics, or any other biological, functional, or immunological properties of candidate gene.
  • the terms “specific binding” or “specifically binding” refer to that interaction between a protein or peptide and an agonist, an antibody, or an antagonist. The interaction is dependent upon the presence of a particular structure of the protein recognized by the binding molecule (i.e., the antigenic determinant or epitope). For example, if an antibody is specific for epitope "A" the presence of a polypeptide containing the epitope A, or the presence of free unlabeled A, in a reaction containing free labeled A and the antibody will reduce the amount of labeled A that binds to the antibody.
  • the invention provides methods (also referred to herein as "screening assays") for identifying compounds which can be used for the treatment of diseases related to candidate gene.
  • the methods entail the identification of candidate or test compounds or agents (e.g., peptides, peptidomimetics, small molecules or other molecules) which bind to candidate gene and/or have a stimulatory or inhibitory effect on the biological activity of candidate gene or its expression and then determining which of these compounds have an effect on symptoms or diseases related to candidate gene in an in vivo assay.
  • candidate or test compounds or agents e.g., peptides, peptidomimetics, small molecules or other molecules
  • Candidate or test compounds or agents which bind to candidate gene and/or have a stimulatory or inhibitory effect on the activity or the expression of candidate gene are identified either in assays that employ cells which express candidate gene (cell-based assays) or in assays with isolated candidate gene (cell-free assays).
  • the various assays can employ a variety of variants of candidate gene (e.g., full-length candidate gene, a biologically active fragment of candidate gene, or a fusion protein which includes all or a portion of candidate gene).
  • candidate gene can be derived from any suitable mammalian species (e.g., human candidate gene, rat candidate gene or murine candidate gene).
  • the assay can be a binding assay entailing direct or indirect measurement of the binding of a test compound or a known candidate gene ligand to candidate gene.
  • the assay can also be an activity assay entailing direct or indirect measurement of the activity of candidate gene.
  • the assay can also be an expression assay entailing direct or indirect measurement of the expression of candidate gene mRNA or candidate gene protein.
  • the various screening assays are combined with an in vivo assay entailing measuring the effect of the test compound on the symptoms of diseases related to candidate gene.
  • the present invention includes biochemical, cell free assays that allow the identification of inhibitors and agonists of proteases suitable as lead structures for pharmacological drug development.
  • assays involve contacting a form of candidate gene (e.g., full-length candidate gene, a biologically active fragment of candidate gene, or a fusion protein comprising all or a portion of candidate gene) with a test compound and determining the ability of the test compound to act as an antagonist (preferably) or an agonist of the enzymatic activity of candidate gene.
  • candidate gene molecules of the present invention can be measured using a variety of assays that measure candidate gene activity.
  • candidate gene enzyme activity can be assessed by a standard in vitro serine/metallo/... protease assay (see, for example, [U.S. 5,057,414]).
  • substrates suitable for in vitro assays such as SucAla-Ala-Pro-Phe-pNA, fluorescein mono-p-guanidinobenzoate hydrochloride, benzyloxycarbonyl-L-Arginyl-S-benzylester, Nalpha-Benzoyl-L-arginine ethyl ester hydrochloride, and the like.
  • protease assay kits available from commercial sources, such as CalbiochemTM (San Diego, Calif.). For general references, see Barrett (Ed.), Methods in Enzymology, Proteolytic Enzymes: Serine and Cysteine Peptidases (Academic Press Inc. 1994), and Barrett et al., (Eds.), Handbook of Proteolytic Enzymes (Academic Press Inc. 1998).
  • Solution in vitro assays can be used to identify a candidate gene substrate or inhibitor.
  • Solid phase systems can also be used to identify a substrate or inhibitor of a candidate gene polypeptide.
  • a candidate gene polypeptide or candidate gene fusion protein can be immobilized onto the surface of a receptor chip of a commercially available biosensor instrument (BIACORE, Biacore AB; Uppsala, Sweden). The use of this instrument is disclosed, for example, by [Karlsson, (1991), and Cunningham and Wells, (1993)].
  • a candidate gene polypeptide or fusion protein is covalently attached, using amine or sulfhydryl chemistry, to dextran fibers that are attached to gold film within a flow cell.
  • a test sample is then passed through the cell. If a candidate gene substrate or inhibitor is present in the sample, it will bind to the immobilized polypeptide or fusion protein, causing a change in the refractive index of the medium, which is detected as a change in surface plasmon resonance of the gold film.
  • This system allows the determination on- and off-rates, from which binding affinity can be calculated, and assessment of the stoichiometry of binding, as well as the kinetic effects of candidate gene mutation.
  • This system can also be used to examine antibody-antigen interactions, and the interactions of other complement/anti-complement pairs.
  • the invention provides assays for screening candidate or test compounds which bind to or modulate the activity of candidate gene.
  • assays can employ full-length candidate gene, a biologically active fragment of candidate gene, or a fusion protein which includes all or a portion of candidate gene.
  • the test compound can be obtained by any suitable means, e.g., from conventional compound libraries.
  • Determining the ability of the test compound to modulate the activity of candidate gene can be accomplished, for example, by determining the ability of candidate gene to bind to or interact with a target molecule.
  • the target molecule can be a molecule with which candidate gene binds or interacts with in nature.
  • the target molecule can be a component of a signal transduction pathway which facilitates transduction of an extracellular signal.
  • the target candidate gene molecule can be, for example, a second intracellular protein which has catalytic activity or a protein which facilitates the association of downstream signaling molecules with candidate gene.
  • Determining the ability of candidate gene to bind to or interact with a target molecule can be accomplished by one of the methods described above for determining direct binding. In one embodiment, determining the ability of a polypeptide of the invention to bind to or interact with a target molecule can be accomplished by determining the activity of the target molecule.
  • the activity of the target molecule can be determined by detecting induction of a cellular second messenger of the target (e.g., intracellular Ca 2+ , diacylglycerol, IP 3 , etc.), detecting catalytic/enzymatic activity of the target on an appropriate substrate, detecting the induction of a reporter gene (e.g., a regulatory element that is responsive to a polypeptide of the invention operably linked to a nucleic acid encoding a detectable marker, e.g., luciferase), or detecting a cellular response.
  • a reporter gene e.g., a regulatory element that is responsive to a polypeptide of the invention operably linked to a nucleic acid encoding a detectable marker, e.g., luciferase
  • binding of a test compound to candidate gene, or interaction of candidate gene with a target molecule in the presence and absence of a candidate compound can be accomplished in any vessel suitable for containing the reactants. Examples of such vessels include microtitre plates, test tubes, and micro-centrifuge tubes.
  • a fusion protein can be provided which adds a domain that allows one or both of the proteins to be bound to a matrix.
  • glutathione-S-transferase (GST) fusion proteins or glutathione-S- transferase fusion proteins can be adsorbed onto glutathione sepharose beads (Sigma Chemical; St. Louis, Mo.) or glutathione derivatized microtitre plates, which are then combined with the test compound or the test compound and either the non-adsorbed target protein or candidate gene, and the mixture incubated under conditions conducive to complex formation (e.g., at physiological conditions for salt and pH). Following incubation, the beads or microtitre plate wells are washed to remove any unbound components and complex formation is measured either directly or indirectly, for example, as described above. Alternatively, the complexes can be dissociated from the matrix, and the level of binding or activity of candidate gene can be determined using standard techniques.
  • candidate gene or its target molecule can be immobilized utilizing conjugation of biotin and streptavidin.
  • Biotinylated polypeptide of the invention or target molecules 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 plates (Pierce Chemical).
  • antibodies reactive with candidate gene or target molecules but which do not interfere with binding of the polypeptide of the invention to its target molecule can be derivatized to the wells of the plate, and unbound target or polypeptide of the invention trapped in the wells by antibody conjugation.
  • Methods for detecting such complexes include immunodetection of complexes using antibodies reactive with candidate gene or target molecule, as well as enzyme-linked assays which rely on detecting an enzymatic activity associated with candidate gene or target molecule.
  • Another technique for drug screening which may be used provides for high throughput screening of compounds having suitable binding affinity to the protein of interest as described in published
  • test compounds are synthesized on a solid substrate, such as plastic pins or some other surface.
  • the test compounds are reacted with candidate gene, or fragments thereof, and washed.
  • Bound candidate gene is then detected by methods well known in the art.
  • Purified candidate gene can also be coated directly onto plates for use in the aforementioned drug screening techniques.
  • non-neutralizing antibodies can be used to capture the peptide and immobilize it on a solid support.
  • the screening assay can also involve monitoring the expression of candidate gene.
  • regulators of expression of candidate gene can be identified in a method in which a cell is contacted with a candidate compound and the expression of candidate gene protein or mRNA in the cell is determined. The level of expression of candidate gene protein or mRNA the presence of the candidate compound is compared to the level of expression of candidate gene protein or mRNA in the absence of the candidate compound. The candidate compound can then be identified as a regulator of expression of candidate gene based on this comparison. For example, when expression of candidate gene protein or mRNA protein is greater (statistically significantly greater) in the presence of the candidate compound than in its absence, the candidate compound is identified as a stimulator of candidate gene protein or mRNA expression.
  • candidate gene protein or mRNA when expression of candidate gene protein or mRNA is less (statistically significantly less) in the presence of the candidate compound than in its absence, the candidate compound is identified as an inhibitor of candidate gene protein or mRNA expression.
  • the level of candidate gene protein or mRNA expression in the cells can be determined by methods described below.
  • the test compound is preferably a small molecule which binds to and occupies the active site of candidate gene polypeptide, thereby making the ligand binding site inaccessible to substrate such that normal biological activity is prevented.
  • small molecules include, but are not limited to, small peptides or peptide-like. molecules.
  • Potential ligands which bind to a polypeptide of the invention include, but are not limited to, the natural ligands of known candidate gene proteases and analogues or derivatives thereof.
  • either the test compound or the candidate gene polypeptide can comprise a detectable label, such as a fluorescent, radioisotopic, chemiluminescent, or enzymatic label, such as horseradish peroxidase, alkaline phosphatase, or luciferase.
  • a detectable label such as a fluorescent, radioisotopic, chemiluminescent, or enzymatic label, such as horseradish peroxidase, alkaline phosphatase, or luciferase.
  • Detection of a test compound which is bound to candidate gene polypeptide can then be accomplished, for example, by direct counting of radioemmission, by scintillation counting, or by determining conversion of an appropriate substrate to a detectable product.
  • binding of a test compound to a candidate gene polypeptide can be determined without labeling either of the interactants.
  • a microphysiometer can be used to detect binding of a test compound with a candidate gene polypeptide.
  • a microphysiometer e.g., CytosensorTM
  • LAPS light-addressable potentiometric sensor
  • BIA Bimolecular Interaction Analysis
  • a candidate gene-like polypeptide can be used as a "bait protein" in a two-hybrid assay or three-hybrid assay [Szabo, (1995); U.S. 5,283,317), to identify other proteins which bind to or interact with candidate gene and modulate its activity.
  • the two-hybrid system is based on the modular nature of most transcription factors, which consist of separable DNA-binding and activation domains.
  • the assay utilizes two different DNA constructs.
  • polynucleotide encoding candidate gene can be fused to a polynucleotide encoding the DNA binding domain of a known transcription factor (e.g., GAL-4).
  • a DNA sequence that encodes an unidentified protein (“prey" or "sample” can be fused to a polynucleotide that codes for the activation domain of the known transcription factor.
  • the DNA-binding and activation domains of the transcription factor are brought into close proximity. This proximity allows tran- scription of a reporter gene (e.g., LacZ), which is operably linked to a transcriptional regulatory site responsive to the transcription factor. Expression of the reporter gene can be detected, and cell colonies containing the functional transcription factor can be isolated and used to obtain the DNA sequence encoding the protein which interacts with candidate gene.
  • a reporter gene e.g., LacZ
  • either the candidate gene- like polypeptide (or polynucleotide) or the test compound can be bound to a solid support.
  • suitable solid supports include, but are not limited to, glass or plastic slides, tissue culture plates, microtiter wells, tubes, silicon chips, or particles such as beads (including, but not limited to, latex, polystyrene, or glass beads).
  • test compounds can be used to attach candidate gene-like polypeptide (or polynucleotide) or test compound to a solid support, including use of covalent and non-covalent linkages, passive absorption, or pairs of binding moieties attached respectively to the polypeptide (or polynucleotide) or test compound and the solid support.
  • Test compounds are preferably bound to the solid support in an array, so that the location of individual test compounds can be tracked. Binding of a test compound to candidate gene (or a polynucleotide encoding for candidate gene) can be accomplished in any vessel suitable for containing the reactants. Examples of such vessels include microtiter plates, test tubes, and microcentrifuge tubes.
  • candidate gene is a fusion protein comprising a domain that allows binding of candidate gene to a solid support.
  • glutathione-S-transferase fusion proteins can be adsorbed onto glutathione sepharose beads (Sigma Chemical, St. Louis, Mo.) or glutathione derivatized microtiter plates, which are then combined with the test compound or the test compound and the non-adsorbed candidate gene; the mixture is then incubated under conditions conducive to complex formation (e.g., at physiological conditions for salt and pH). Following incubation, the beads or microtiter plate wells are washed to remove any unbound components. Binding of the interactants can be determined either directly or indirectly, as described above. Alternatively, the complexes can be dissociated from the solid support before binding is determined.
  • candidate gene or a polynucleotide encoding candidate gene
  • a test compound can be immobilized utilizing conjugation of biotin and streptavidin.
  • Biotinylated candidate gene or a polynucleotide encoding biotinylated candidate gene
  • test compounds can be prepared from biotin-NHS (N- hydroxysuccinimide) using techniques well known in the art (e.g., biotinylation kit, Pierce Chemicals, Rockford, 111.) and immobilized in the wells of streptavidin-coated plates (Pierce Chemical).
  • antibodies which specifically bind to candidate gene, polynucleotide, or a test compound, but which do not interfere with a desired binding site, such as the active site of candidate gene can be derivatized to the wells of the plate. Unbound target or protein can be trapped in the wells by antibody conjugation.
  • Methods for detecting such complexes include immunodetection of complexes using antibodies which specifically bind to candidate gene polypeptide or test compound, enzyme-linked assays which rely on detecting an activity of candidate gene polypeptide, and SDS gel electrophoresis under non-reducing conditions.
  • Screening for test compounds which bind to a candidate gene polypeptide or polynucleotide also can be carried out in an intact cell. Any cell which comprises a candidate gene polypeptide or polynucleotide can be used in a cell-based assay system. A candidate gene polynucleotide can be naturally occurring in the cell or can be introduced using techniques such as those described above. Binding of the test compound to candidate gene or a polynucleotide encoding candidate gene is determined as described above.
  • Test compounds can be tested for the ability to increase or decrease candidate gene activity of a candidate gene polypeptide.
  • the candidate gene activity can be measured, for example, using methods described in the specific examples, below, candidate gene activity can be measured after contacting either a purified candidate gene or an intact cell with a test compound.
  • a test compound which decreases candidate gene activity by at least about 10, preferably about 50, more preferably about 75, 90, or 100% is identified as a potential agent for decreasing candidate gene activity.
  • a test compound which increases candidate gene activity by at least about 10, preferably about 50, more preferably about 75, 90, or 100% is identified as a potential agent for increasing candidate gene activity.
  • test compounds which increase or decrease candidate gene gene expression are identified.
  • the term "correlates with expression of a polynucleotide” indicates that the detection of the presence of nucleic acids, the same or related to a nucleic acid sequence encoding candidate gene, by northern analysis or realtime PCR is indicative of the presence of nucleic acids encoding candidate gene in a sample, and thereby correlates with expression of the transcript from the polynucleotide encoding candidate gene.
  • microarray refers to an array of distinct polynucleotides or oligonucleotides arrayed on a substrate, such as paper, nylon or any other type of membrane, filter, chip, glass slide, or any other suitable solid support.
  • a candidate gene polynucleotide is contacted with a test compound, and the expression of an RNA or polypeptide product of candidate gene polynucleotide is determined.
  • the level of expression of appropriate mRNA or polypeptide in the presence of the test compound is compared to the level of expression of mRNA or polypeptide in the absence of the test compound.
  • the test compound can then be identified as a regulator of expression based on this comparison.
  • test compound when expression of mRNA or polypeptide is greater in the presence of the test compound than in its absence, the test compound is identified as a stimulator or enhancer of the mRNA or polypeptide expression.
  • test compound when expression of the mRNA or polypeptide is less in the presence of the test compound than in its absence, the test compound is identified as an inhibitor of the mRNA or polypeptide expression.
  • the level of candidate gene mRNA or polypeptide expression in the cells can be determined by methods well known in the art for detecting mRNA or polypeptide. Either qualitative or quantitative methods can be used.
  • the presence of polypeptide products of candidate gene polynucleotide can be determined, for example, using a variety of techniques known in the art, including immunochemical methods such as radioimmunoassay, Western blotting, and immunohistochemistry.
  • polypeptide synthesis can be determined in vivo, in a cell culture, or in an in vitro translation system by detecting incorporation of labelled amino acids into candidate gene.
  • Such screening can be carried out either in a cell-free assay system or in an intact cell.
  • Any cell which expresses candidate gene polynucleotide can be used in a cell-based assay system.
  • the candidate gene polynucleotide can be naturally occurring in the cell or can be introduced using techniques such as those described above. Either a primary culture or an established cell line can be used.
  • test compounds for use in the screening assays of the invention can be obtained from any suitable source, e.g., conventional compound libraries.
  • the test compounds can also be obtained using any of the numerous approaches in combinatorial library methods known in the art, including: biological libraries; spatially addressable parallel solid phase or solution phase libraries; synthetic library methods requiring deconvolution; the "one-bead one-compound” library method; and synthetic library methods using affinity chromatography selection.
  • the biological library approach is limited to peptide libraries, while the other four approaches are applicable to peptide, non-peptide oligomer or small molecule libraries of compounds [Lam, (1997)]. Examples of methods for the synthesis of molecular libraries can be found in the art. Libraries of compounds may be presented in solution or on beads, bacteria, spores, plasmids or phage. Modeling of Regulators
  • Computer modeling and searching technologies permit identification of compounds, or the improvement of already identified compounds, that can modulate candidate gene expression or activity. Having identified such a compound or composition, the active sites or regions are identified. Such sites might typically be the enzymatic active site, regulator binding sites, or ligand binding sites.
  • the active site can be identified using methods known in the art including, for example, from the amino acid sequences of peptides, from the nucleotide sequences of nucleic acids, or from study of complexes of the relevant compound or composition with its natural ligand. In the latter case, chemical or X-ray crystallographic methods can be used to find the active site by finding where on the factor the complexed ligand is found.
  • the three dimensional geometric structure of the active site is determined. This can be done by known methods, including X-ray crystallography, which can determine a complete molecular structure. On the other hand, solid or liquid phase NMR can be used to determine certain intramolecular distances. Any other experimental method of structure determination can be used to obtain partial or complete geometric structures.
  • the geometric structures may be measured with a complexed ligand, natural or artificial, which may increase the accuracy of the active site structure determined.
  • the methods of computer based numerical modeling can be used to complete the structure or improve its accuracy.
  • Any recognized modeling method may be used, including parameterized models specific to particular biopolymers such as proteins or nucleic acids, molecular dynamics models based on computing molecular motions, statistical mechanics models based on thermal ensembles, or combined models.
  • standard molecular force fields representing the forces between constituent atoms and groups, are necessary, and can be selected from force fields known in physical chemistry.
  • the incomplete or less accurate experimental structures can serve as constraints on the complete and more accurate structures computed by these modeling methods.
  • candidate modulating compounds can be identified by searching databases containing compounds along with information on their molecular structure. Such a search seeks compounds having structures that match the determined active site structure and that interact with the groups defining the active site. Such a search can be manual, but is preferably computer assisted. These compounds found from this search are potential candidate gene modulating compounds. Alternatively, these methods can be used to identify improved modulating compounds from an already known modulating compound or ligand. The composition of the known compound can be modified and the structural effects of modification can be determined using the experimental and computer modeling methods described above applied to the new composition. The altered structure is then compared to the active site structure of the compound to determine if an improved fit or interaction results. In this manner systematic variations in composition, such as by varying side groups, can be quickly evaluated to obtain modified modulating compounds or ligands of improved specificity or activity.
  • the present invention provides candidate genes for prophylactic, therapeutic and diagnostic methods for "cellular stress protection deficiency" (CSPD) disorders.
  • CSPD cellular stress protection deficiency
  • the term cell stress protection means a mechanism or process, which protects a cell or tissue from necrosis, apoptosis, degeneration or remodeling processes. Disorders associated with a lack of cell protection are CSPD diseases.
  • organ failure remains the major cause of death in critically ill patients.
  • current research activities focus on the cellular response to different kinds of cellular stress (hypoxia, oxidative damage and mechanical distress, others) in the pathogenic squeal of organ failure.
  • the cellular stress reactions are characterized by induction of adaptive programs of gene expression (e.g. acute phase proteins, heat shock proteins, hypoxia-associated proteins) to protect the cells from energy depletion and cell death.
  • adaptive programs of gene expression e.g. acute phase proteins, heat shock proteins, hypoxia-associated proteins
  • the cell dies due to necrosis, apoptosis or other processes. Deregulation of the balance of apoptosis and necrosis in different organs seems to be an important mechanism in the development of organ failure.
  • New insights into the cellular mechanisms during organ dysfunction promote the development of new diagnostic (e. g. optical, spectroscopic, real time PCR, etc.) and pharmacological tools leading to a better prevention and therapy of organ failure.
  • Those processes could be activated or affected by - but not limited to - radical formation, ischemia, hypoxia or other cell stress mediators.
  • Cell stress is broadly defined to include - but not limited to - cellular responses to heat shock, oxidative stress, heavy metals, and toxic chemicals.
  • the term neuroprotection means mechanisms within the nervous system which protect neurons from apoptosis or degeneration, for example following a brain injury or as a result of chronic neurodegenerative diseases. Oxidative stress and neurological disease.
  • Oxidative stress plays a predominant role in the development of several neurological diseases. This is caused by an increased vulnerability of the brain to oxidative processes due to its high energy consumption and almost entire dependency on oxidative phosphorylation reaction for the generation of ATP, which leads to a high production of reactive oxygen species (ROS) in the mitochondria.
  • ROS reactive oxygen species
  • the brain also contains many polyunsaturated fatty acids that are potential substrates for peroxidation and relatively high amounts of iron and copper that can catalyze the formation of radicals by the Fenton reaction.
  • AD Alzheimer's dementia
  • the chronic neurodegenerative disease is neuropathologically characterized by the degeneration of neurons and the presence of intracellular neurofibrillary tangles and extracellular senile plaques loaded with the amyloid ⁇ protein A ⁇ (Braak and Braak, 1991; Glenner and Wong, 1984).
  • a ⁇ peptides are toxic to nerve cells in vitro and in vivo (Kowall et al., 1992; Yankner et al., 1989) and antioxidants can inhibit A ⁇ -induced cell death in vitro (Behl et al., 1992; Behl et al., 1994).
  • PD Parkinson's disease
  • PD Parkinson's disease
  • rigidity and bradykinesia caused by the loss of dopaminergic neurons in the substantia nigra pars compacta.
  • Its neuropathological hallmark is the intraneuronal accumulation of ⁇ -synuclein in Lewy bodies (Olanow and Tatton, 1999; Spillantini et al., 1997).
  • Oxidative stress seems to be involved in the pathophysiology of this disease because the metabolism of dopamine involves the generation of ROS like H2O2 by monoamino oxidases (Maker et al., 1981). Oxidation of the dopamine catechol ring generates dopamine quinones and semiquinones (Graham, 1978; Hastings, 1995), which can react with glutathione, the main antioxidant in the cell, and cause its depletion and formation of glutathionyl conjugates (Spencer et al., 1998; Spencer et al., 1995; Spencer et al., 2002).
  • ROS reactive oxygen species
  • en2ymes Members of the antioxidant 5 defence system like the 02— detoxifying SODs, and the group of peroxide-detoxifying en2ymes, such as glutathione peroxidase and catalase have been used to suppress cell proliferation of transformed cells. These en2ymes are also expressed with great regularity in brain tumors (Pu et al., 1996).
  • Antioxidant enzymes such as superoxide dismutase (SOD), catalase, glutathione peroxidase (GPX), glutathione reductase (GR), were found to be upregulated in radio-resistant 10 glioma cells also confering cross-resistance to the antitumor agent cisplatin (Lee et al., 2004).
  • ALS is a late-onset neurodegenerative disorder characterized by the loss of motor neurons in the motor cortex, brainstem, and spinal cord resulting in progressive muscle weakness, wasting and death within 2-5 years.
  • cardiovascular diseases represent the most important health risk factors because they are responsible for more than 50% of total mortality.
  • acute coronary occlusion is the leading cause of morbidity and mortality, and according to the World Health Organization, it will be the major global cause of death by the year 2020.
  • the history of ischemic heart disease is relatively brief and represents a very convincing example of the rapid development of cardiology
  • Endothelial dysfunction is characterized by oxidative stress on the endothelial cells of the cascular system.
  • the degree of ischemic injury depends not only on the intensity and duration of the ischemic stimulus but also on the level of cardiac tolerance to O2 deprivation and other components of ischemia.
  • the first observations appeared showing that the incidence of myocardial infarction was lower in people living at high altitude.
  • These epidemiological observations were later repeatedly confirmed in experimental studies using simulated hypoxia (Ostadal B et al. 1998).
  • interest concentrated on the possibilities of limiting infarct size pharmacologically; however, this effort was not successful because it became increasingly obvious that the clinical observations did not correspond with the optimism of experimental results.
  • CH cardiovascular disease
  • cardiopulmonary diseases such as chronic ischemic heart disease and chronic obstructive lung disease.
  • Clinical profit from this phenomenon depends on the balance between the benefit (cardiac protection) and potential risks (pulmonary hypertension, right ventricular hypertrophy).
  • An interesting aspect that should be analyzed to understand human susceptibility to myocardial infarction may be the sex and age differences in cardioprotection.
  • a particularly rewarding aspect of basic research in the field of cardioprotection should be the possibility of the immediate translation of new discoveries obtained from in vitro experiments into the clinical setting, particularly those of coronary bypass surgery and percutaneous coronary intervention.
  • Myocardial regeneration consists of the repopulation of irreversibly damaged muscle with new contractile cells to restore function in the necrotic areas, thereby improving global heart function (Menasche P et al., 2003).
  • cardiomyocytes were thought to be terminally differentiated cells, implying that hypertrophy was the only available form of growth.
  • This concept was recently revised on the basis of both experimental and clinical studies which showed that some adult cells can re-enter a mitotic cycle.
  • the magnitude of this self-repair mechanism is far too limited to compensate for the massive loss of cardiomyocytes resulting from a large infarction. Therefore, the only practicable perspective is an exogenous supply of cells for effective remodeling of injured areas.
  • the present invention provides targets for prophylactic, therapeutic and diagnostic methods for "cellular stress protection deficiency” (CSPD) disorders.
  • CSPD cellular stress protection deficiency
  • CSPD diseases which are characterized by a lack of protection to cell stress leading to cell and tissue necrosis, apoptosis, degeneration and remodelling.
  • CSPD diseases which are diseases characterized by a lack of induction of protective programs of gene expression (e.g. acute phase proteins, heat shock proteins, hypoxia-associated proteins) to protect cells from energy depletion and cell death.
  • CSPD diseases that are diseases comprised in a group of diseases consisting of neurological and cardiovascular diseases due to stress.
  • CSPD diseases that are diseases comprised in a group of diseases consisting of neurological and cardiovascular diseases due to oxidative stress.
  • cardiovascluar CSPD diseases that are diseases comprised in a group of diseases consisting of acute coronary occlusion, ischemic heart disease, myocardial infarction, chronic ischemic heart diseas, chronic obstructive lung disease, pulmonary hypertension, right ventricular hypertrophy, reperfusion injury, arrhythmias, postischemic contractile dysfunction endothelial dysfunction, and cardiac hypoxia
  • the most preferred embodiment of the invention are neurological CSPD diseases that are diseases comprised in a group of diseases consisting of chronic neurodegenerative diseases, Alzheimer's dementia, Parkinson's disease, ischemic stroke, stroke, ALS (Amyotrophic lateral sclerosis), MS (multiple sclerosis), progressive muscle weekness and brain trauma.
  • diseases comprised in a group of diseases consisting of chronic neurodegenerative diseases, Alzheimer's dementia, Parkinson's disease, ischemic stroke, stroke, ALS (Amyotrophic lateral sclerosis), MS (multiple sclerosis), progressive muscle weekness and brain trauma.
  • the present invention provides candidate gene for prophylactic, therapeutic and diagnostic methods for CSPD diseases.
  • the regulatory method of the invention involves contacting a cell with an agent that modulates one or more of the activities of candidate gene.
  • An agent that modulates activity can be an agent as described herein, such as a nucleic acid or a protein, a naturally-occurring cognate ligand of the polypeptide, a peptide, a peptidomimetic, or any small molecule, hi one embodiment, the agent stimulates one or more of the biological activities of candidate gene.
  • stimulatory agents include the active candidate gene and nucleic acid molecules encoding a portion of candidate gene, hi another embodiment, the agent inhibits one or more of the biological activities of candidate gene.
  • inhibitory agents include antisense nucleic acid molecules and antibodies.
  • the present invention provides methods of treating an individual afflicted with a disease or disorder characterized by unwanted expression or activity of candidate gene or a protein in the candidate gene signaling pathway.
  • the method involves administering an agent like any agent identified or being identifiable by a screening assay as described herein, or combination of such agents that modulate say upregulate or downregulate the expression or activity of candidate gene or of any protein in the candidate gene signaling pathway.
  • the method involves administering a regulator of candidate gene as therapy to compensate for reduced or undesirably low expression or activity of candidate gene or a protein in the candidate gene signaling pathway.
  • Stimulation of activity or expression of candidate gene is desirable in situations in which enzymatic activity or expression is abnormally low and in which increased activity is likely to have a beneficial effect. Conversely, inhibition of enzymatic activity or expression of candidate gene is desirable in situations in which activity or expression of candidate gene is abnormally high and in which decreasing its activity is likely to have a beneficial effect.
  • the present invention provides for the use of candidate gene or fragments of candidate gene as a biomarker for CSPD diseases.
  • This invention further pertains to novel agents identified by the above-described screening assays and uses thereof for treatments as described herein.
  • compositions suitable for administration typically comprise the nucleic acid molecule, protein, or antibody and a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable carrier is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration.
  • the use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the compositions is contemplated. Supplementary active compounds can also be incorporated into the compositions.
  • the invention includes pharmaceutical compositions comprising a regulator of candidate gene expression or activity (and/or a regulator of the activity or expression of a protein in the candidate gene signaling pathway) as well as methods for preparing such compositions by combining one or more such regulators and a pharmaceutically acceptable carrier. Also within the invention are pharmaceutical compositions comprising a regulator identified using the screening assays of the invention packaged with instructions for use. For regulators that are antagonists of candidate gene activity or which reduce candidate gene expression, the instructions would specify use of the pharmaceutical composition for treatment of CSPD diseases. For regulators that are agonists of candidate gene activity or increase candidate gene expression, the instructions would specify use of the pharmaceutical composition for treatment of CSPD diseases.
  • An inhibitor of candidate gene may be produced using methods which are generally known in the art.
  • purified candidate gene may be used to produce antibodies or to screen libraries of pharmaceutical agents to identify those which specifically bind candidate gene.
  • Antibodies to candidate gene may also be generated using methods that are well known in the art.
  • Such antibodies may include, but are not limited to, polyclonal, monoclonal, chimeric, single chain antibodies, Fab fragments, and fragments produced by a Fab expression library. Neutralizing antibodies like those which inhibit dimer formation are especially preferred for therapeutic use.
  • the polynucleotides encoding candidate gene, or any fragment or complement thereof may be used for therapeutic purposes.
  • the complement of the polynucleotide encoding candidate gene may be used in situations in which it would be desirable to block the transcription of the mRNA.
  • cells may be transformed with sequences complementary to polynucleotides encoding candidate gene.
  • complementary molecules or fragments may be used to modulate candidate gene activity, or to achieve regulation of gene function.
  • sense or antisense oligonucleotides or larger fragments can be designed from various locations along the coding or control regions of sequences encoding candidate gene.
  • Expression vectors derived from retroviruses, adenoviruses, or herpes or vaccinia viruses, or from various bacterial plasmids may be used for delivery of nucleotide sequences to the targeted organ, tissue, or cell population. Methods which are well known to those skilled in the art can be used to construct vectors which will express nucleic acid sequence complementary to the polynucleotides of the gene encoding candidate gene. These techniques are described, for example, in [Scott and Smith (1990)].
  • compositions containing candidate gene in conjunction with a pharmaceutically acceptable carrier, for any of the therapeutic effects discussed above.
  • Such pharmaceutical compositions may consist of candidate gene, antibodies to candidate gene, and mimetics, agonists, antagonists, 5 or inhibitors of candidate gene.
  • the compositions may be administered alone or in combination with at least one other agent, such as a stabilizing compound, which may be administered in any sterile, biocompatible pharmaceutical carrier including, but not limited to, saline, buffered saline, dextrose, and water.
  • the compositions may be administered to a patient alone, or in combination with other agents, drugs or hormones.
  • a pharmaceutical composition of the invention is formulated to be compatible with its intended route of administration.
  • routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (topical), transmucosal, and rectal administration.
  • Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent
  • bases such as hydrochloric acid or sodium hydroxide.
  • the parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.
  • compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions.
  • suitable carriers include
  • composition 25 physiological saline, bacteriostatic water, Cremophor EMTM (BASF, Parsippany, NJ.) or phosphate buffered saline (PBS). hi all cases, the composition must be sterile and should be
  • the carrier can be a solvent or dispersion medium
  • ⁇ 30 containing, for example, water, ethanol, a pharmaceutically acceptable polyol like glycerol, propylene glycol, liquid polyetheylene glycol, and suitable mixtures thereof.
  • a pharmaceutically acceptable polyol like glycerol, propylene glycol, liquid polyetheylene glycol, and suitable mixtures thereof.
  • the proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
  • Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, water, ethanol, a pharmaceutically acceptable polyol like glycerol, propylene glycol, liquid polyetheylene glycol, and suitable mixtures thereof.
  • the proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfact
  • compositions 35 example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like.
  • isotonic agents for example, sugars, polyalcohols such as mannitol, sorbitol, sodium chloride in the composition.
  • Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.
  • Sterile injectable solutions can be prepared by incorporating the active compound (e.g., a polypeptide or antibody) in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization.
  • dispersions are prepared by incorporating the active compound into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated above.
  • a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated above.
  • the preferred methods of preparation are vacuum drying and freeze-drying which yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
  • Oral compositions generally include an inert diluent or an edible carrier. They can be enclosed in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, the active compound can be incorporated with excipients and used in the form of tablets, troches, or capsules. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash, wherein the compound in the fluid carrier is applied orally and swished and expectorated or swallowed.
  • compositions can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.
  • a binder such as microcrystalline cellulose, gum tragacanth or gelatin
  • an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch
  • a lubricant such as magnesium stearate or sterotes
  • a glidant such as colloidal silicon dioxide
  • a sweetening agent such as sucrose or saccharin
  • the compounds are delivered in the form of an aerosol spray from a pressurized container or dispenser which contains a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer.
  • a suitable propellant e.g., a gas such as carbon dioxide, or a nebulizer.
  • Systemic administration can also be by transmucosal or transdermal means.
  • penetrants appropriate to the barrier to be permeated are used in the formulation.
  • penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives.
  • Transmucosal administration can be accomplished through the use of nasal sprays or suppositories.
  • the active compounds are formulated into ointments, salves, gels, or creams as generally known in the art.
  • the compounds can also be prepared in the form of suppositories (e.g., with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.
  • suppositories e.g., with conventional suppository bases such as cocoa butter and other glycerides
  • retention enemas for rectal delivery.
  • the active compounds are prepared with carriers that will protect the compound against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems.
  • a controlled release formulation including implants and microencapsulated delivery systems.
  • Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art.
  • the materials can also be obtained commercially from Alza Corporation and Nova Pharmaceuticals, Inc.
  • Liposomal suspensions (including liposomes targeted to infected cells with monoclonal antibodies to viral antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. 4,522,811.
  • Dosage unit form refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.
  • the specification for the dosage unit forms of the invention are dictated by and directly dependent on the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and the limitations inherent in the art of compounding such an active compound for the treatment of individuals.
  • compositions can be included in a container, pack, or dispenser together with instructions for administration.
  • instructions for administration will specify use of the composition for CSPD diseases.
  • the instructions for administration will specify use of the composition for CSPD diseases.
  • One embodiment of the invention describes candidate gene as a biomarker for diagnostic use.
  • candidate gene as a biomarker in diagnostics is based by the comparison of candidate gene level in a biological sample from a diseased mammal with the candidate gene level in a control sample from a healthy or normal mammal. Does the candidate gene level in the diseased mammal differs from the candidate gene level in a normal or healthy mammal then the diseased mammal is diagnosed with a disease associated with an altered candidate gene level. Furthermore, comparing candidate gene levels of a biological sample from a diseased mammal with candidate gene levels of control samples from mammals with a candidate gene-associated disease already diagnosed with different stages or severity of said disease, allows the diagnose of a candidate gene-associated disease of said first diseased mammal and specifying the severity of the candidate gene-associated disease.
  • the biological sample is taken from the analogue tissue or body fluid than the control sample.
  • Normal or standard values for candidate gene expression are established by using control samples from healthy or diseased mammalian subjects.
  • a control sample can be obtained by collecting separate or combined body fluids or cell extracts taken from normal mammalian subjects, preferably human, achieving statistical relevant numbers.
  • the samples were subjected to suitable detection methods to detect candidate gene polypeptide, polynucleotide or activity.
  • the determination of candidate gene level in a mammal subjected to diagnosis is performed analogously by collecting a biological sample from said mammal. Quantities of candidate gene levels in biological samples from a mammal subjected to diagnosis are compared with the standard or normal values measured from a control sample.
  • Deviation between standard value (determined from control sample) and subject value (determined from biological sample) establishes the parameters for diagnosing disease.
  • Absolute quantification of candidate gene levels measured from biological or. control samples may be achieved by comparing those values with values obtained from an experiment in which a known amount of a substantially purified polypeptide is used.
  • Antibodies which specifically bind candidate gene may be used for the diagnosis of disorders characterized by the expression of the biomarker candidate gene, or in diagnostic assays to monitor patients being treated achieving guidance for therapy for such a disease. Such a treatment includes medication suitable to treat such a disease, and treatment with candidate gene polypeptides or polynucleotides, or agonists, antagonists, and inhibitors of candidate gene.
  • Antibodies useful for diagnostic purposes may be prepared in the same manner as those described above for therapeutics. Diagnostic assays for candidate gene include methods which utilize the antibody and a label to detect candidate gene in human body fluids or in extracts of cells or tissues.
  • the antibodies may be used with or without modification, and may be labeled by covalent or non-covalent joining with a reporter molecule. A wide variety of reporter molecules, several of which are described above, are known in the art and may be used.
  • Planar Waveguide Technology bioassays are designed to perform multiplexed nucleic acid hybridization assays, irnmunoaffinity reactions and membrane receptor based assays with high sensitivity and selectivity.
  • the recognition elements specific for the analytes of interest are bound onto the surface in small discrete spots; the transfer of the recognition elements onto the surface is performed using an adequate spotting technology, which requires only minute amounts of recognition elements.
  • Such an arrangement of different recognition elements in an array format allows the simultaneous detection and quantification of hundreds to thousands of different analytes per sample including replicates.
  • Recognition elements e.g. oligonucleotides, cDNAs, or antibodies
  • Recognition elements are spotted onto the chemically modified planar waveguide surface with typical spot diameters of 100 - 200 ⁇ m. The remaining free binding sites on the surface subsequently are being blocked to reduce or eliminate nonspecific binding.
  • the sample e.g. fluorescently labeled cDNA or pre-incubated analyte / fluorescently labeled antibody complex
  • the incubation time where a selective recognition and binding between recognition elements and corresponding target molecules e.g. DNA - DNA hybridization or antigen - antibody interaction
  • the resulting fluorescing spots can then be detected during readout.
  • Normal or standard values for candidate gene expression are established by using control samples from healthy or diseased mammalian subjects.
  • a control sample can be obtained by collecting separate or combined body fluids or cell extracts taken from normal mammalian subjects, preferably human, achieving statistical relevant numbers.
  • the control samples are combined with an antibody to candidate gene under conditions suitable for complex formation.
  • the amount of standard complex formation may be quantified by various methods, preferably by photometric means.
  • the determination of candidate gene level in a mammal subjected to diagnosis is performed analogously by collecting a biological sample from said mammal, combining said sample with an antibody to candidate gene and determination of complex formation. Quantities of candidate gene expressed in biological samples from a mammal subjected to diagnosis are compared with the standard or normal values measured from a control sample.
  • Deviation between standard value (determined from control sample) and subject value (determined from biological sample) establishes the parameters for diagnosing disease.
  • Absolute quantification of candidate gene levels measured from biological or control samples may be achieved by comparing those values with values obtained from an experiment in which a known amount of a substantially purified polypeptide is used.
  • the polynucleotides encoding candidate gene may be used for diagnostic purposes.
  • the polynucleotides which may be used include oligonucleotide sequences, complementary RNA and DNA molecules, and PNAs.
  • the polynucleotides may be used to detect and quantified gene expression in control and biological samples in which expression of the biomarker candidate gene may be correlated with disease.
  • the diagnostic assay may be used to distinguish between absence, presence, and excess expression of candidate gene, and to monitor regulation of candidate gene levels during therapeutic intervention.
  • Polynucleotide sequences encoding candidate gene may be used for the diagnosis of CSPD diseases associated with expression of candidate gene.
  • the polynucleotide sequences encoding candidate gene may be used in Southern, Northern, or dot-blot analysis, or other membrane-based technologies; in PCR technologies; in dipstick, pin, and ELISA assays; bDNA (branched DNA technology) and Planar Waveguide Technology; and in microarrays utilizing a biological sample from diseased mammals to detect altered candidate gene expression.
  • Such qualitative or quantitative methods are well known in the art.
  • the nucleotide sequences encoding candidate gene may be useful in assays that detect the presence of associated disorders, particularly those mentioned above.
  • the nucleotide sequences encoding candidate gene may be labeled by standard methods and added to a biological sample from diseased mammals under conditions suitable for the formation of hybridization complexes. After a suitable incubation period, the sample is washed and the signal is quantified and compared with a standard value. If the amount of signal in the patient sample is altered from that of a comparable control sample, the nucleotide sequences have hybridized with nucleotide sequences in the sample, and the presence of altered levels of nucleotide sequences encoding candidate gene in the sample indicates the presence of the associated disorder.
  • Such assays may also be used to evaluate the efficacy of a particular therapeutic treatment regimen in animal studies, in clinical trials, or in monitoring the treatment of an individual patient.
  • candidate gene activity can be used for diagnostic purposes.
  • the activity of candidate gene molecules of the present invention can be measured using a variety of assays that measure candidate gene activity.
  • candidate gene enzyme activity can be assessed by a standard protease assay (see, for example, [U.S. 5,057,414]).
  • a normal or standard profile for expression is established. This may be accomplished by combining body fluids or cell extracts taken from normal subjects, either animal or human, with a sequence, or a fragment thereof, encoding candidate gene, under conditions suitable for hybridization or amplification. Quantification of candidate gene levels measured from biological or control samples may be achieved by comparing those values with values obtained from an experiment in which a known amount of a substantially purified polynucleotide is used. Standard values obtained from normal samples may be compared with values obtained from samples from patients who are symptomatic for a disorder. Deviation from standard values is used to establish the presence of a disorder.
  • immunoassay devices and methods are often used. These devices and methods can utilize labelled molecules in various sandwich, competitive, or non-competitive assay formats, to generate a signal that is related to the presence or amount of an analyte of interest. Additionally, certain methods and devices, such as biosensors and optical immunoassays, may be employed to determine the presence or amount of analytes without the need for a labelled molecule.
  • the markers are analyzed using an immunoassay, although other methods are well known to those skilled in the art (for example, the measurement of marker RNA levels).
  • the presence or amount of a marker is generally determined using antibodies specific for each marker and detecting specific binding.
  • Any suitable immunoassay may be utilized, for example, enzyme- linked immunoassays (ELISA), radioimmunoassay (RIAs), competitive binding assays, planar waveguide technology, and the like.
  • ELISA enzyme- linked immunoassays
  • RIAs radioimmunoassay
  • Specific immunological binding of the antibody to the marker can be detected directly or indirectly.
  • Direct labels include fluorescent or luminescent tags, metals, dyes, radionuclides, and the like, attached to the antibody.
  • Indirect labels include various enzymes well known in the art, such as alkaline phosphatase, horseradish peroxidase and the like.
  • RAMP Biomedical device called the Clinical Reader supTM, which uses the fluorescent tag method, though the skilled artisan will know of many different machines and manual protocols to perform the same assay.
  • Diluted whole blood is applied to the sample well.
  • the red blood cells are retained in the sample pad, and the separated plasma migrates along the strip.
  • Fluorescent dyed latex particles bind to the analyte and are immobilized at the detection zone. Additional particles are immobilized at the internal control zone.
  • the fluorescence of the detection and internal control zones are measured on the RAMP Clinical Reader supTM, and the ratio between these values is calculated. This ratio is used to determine the analyte concentration by interpolation from a lot-specific standard curve supplied by the manufacturer in each test kit for each assay.
  • immobilized antibodies specific for the markers is also contemplated by the present invention and is well known by one of ordinary skill in the art.
  • the antibodies could be immobilized onto a variety of solid supports, such as magnetic or chromatographic matrix particles, the surface of an assay place (such as microtiter wells) , pieces of a solid substrate material (such as plastic, nylon, paper), and the like.
  • An assay strip could be prepared by coating the antibody or a plurality of antibodies in an array on solid support. This strip could then be dipped into the test sample and then processed quickly through washes and detection steps to generate a measurable signal, such as a coloured spot.
  • the analysis of a plurality of markers may be carried out separately or simultaneously with one test sample. Several markers may be combined into one test for efficient processing of a multiple of samples. In addition, one skilled in the art would recognize the value of testing multiple samples (for example, at successive time points) from the same individual. Such testing of serial samples will allow the identification of changes in marker levels over time. Increases or decreases in marker levels, as well as the absence of change in marker levels, would provide useful information about the disease status that includes, but is not limited to identifying the approximate time from onset of the event, the presence and amount of salvagable tissue, the appropriateness of drug therapies, the effectiveness of various therapies, identification of the severity of the event, identification of the disease severity, and identification of the patient's outcome, including risk of future events.
  • An assay consisting of a combination of the markers referenced in the instant invention may be constructed to provide relevant information related to differential diagnosis.
  • a panel may be constucted using 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or more or individual markers.
  • the analysis of a single marker or subsets of markers comprising a larger panel of markers could be carried out methods described within the instant invention to optimize clinical sensitivity or specificity in various clinical settings.
  • markers could be carried out in a variety of physical formats as well.
  • the use of microtiter plates or automation could be used to facilitate the processing of large numbers of test samples.
  • single sample formats could be developed to facilitate immediate treatment and diagnosis in a timely fashion, for example, in ambulatory transport or emergency room settings.
  • Particularly useful physical formats comprise surfaces having a plurality of discrete, addressable locations for the detection of a plurality of different analytes.
  • Such formats include protein microarrays, or "protein chips" and capillary devices.
  • Cardiac markers serve an important role in the early detection and monitoring of cardiovascular disease. Markers of disease are typically substances found in a bodily sample that can be easily measured. The measured amount can correlate to underlying disease pathophysiology, presence or absence of a current or imminent cardiac event, probability of a cardiac event in the future. In patients receiving treatment for their condition -the measured amount will also correlate with responsiveness to therapy. Markers can include elevated levels of blood pressure, cholesterol, blood sugar, homocysteine and C- reactive protein (CRP).
  • CRP C- reactive protein
  • current markers even in combination with other measurements or risk factors, do not adequately identify patients at risk, accurately detect events (i.e., heart attacks), or correlate with therapy. For example, half of patients do not have elevated serum cholesterol or other traditional risk factors.
  • BNP B-type natriuretic peptide
  • brain- type natriuretic peptide is a 32 amino acid, 4 kDa peptide that is involved in the natriuresis system to regulate blood pressure and fluid balance.
  • the precursor to BNP is synthesized as a 108-amino acid molecule, referred to as "pre pro BNP,” that is proteolytically processed into a 76-amino acid N-terminal peptide (amino acids 1-76), referred to as "NT pro BNP” and the 32-amino acid mature hormone, referred to as BNP or BNP 32 (amino acids 77-108).
  • NT pro- BNP, BNP-32, and the pre pro BNP ⁇ can circulate in human plasma.
  • the 2 forms, pre pro BNP and NT pro BNP, and peptides which are derived from BNP, pre pro BNP and NT pro BNP and which are present in the blood as a result of proteolyses of BNP, NT pro BNP and pre pro BNP, are collectively described as markers related to or associated with BNP.
  • Proteolytic degradation of BNP and of peptides related to BNP have also been described in the literature and these proteolytic fragments are also encompassed it the term "BNP related peptides”.
  • BNP and BNP-related peptides are predominantly found in the secretory granules of the cardiac ventricles, and are released from the heart in response to both ventricular volume expansion and pressure overload. Elevations of BNP are associated with raised atrial and pulmonary wedge pressures, reduced ventricular systolic and diastolic function, left ventricular hypertrophy, and myocardial infarction [Sagnella, (1998)]. Furthermore, there are numerous reports of elevated BNP concentration associated with congestive heart failure and renal failure.
  • BNP and BNP-related peptides are likely not specific for ACS, they may be sensitive markers of ACS because they may indicate not only cellular damage due to ischemia, but also a perturbation of the natriuretic system associated with ACS.
  • the term "BNP" as used herein refers to the mature 32- amino acid BNP molecule itself.
  • markers related to BNP may also serve as diagnostic or prognostic indicators in patients with ACS.
  • BNP is synthesized as a 108-amino acid pre pro-BNP molecule that is proteolytically processed into a 76-amino acid "NT pro BNP" and the 32- amino acid BNP molecule.
  • NT pro-BNP Because of its relationship to BNP, the concentration of NT pro-BNP molecule can also provide diagnostic or prognostic information in patients.
  • the phrase "marker related to BNP or BNP related peptide" refers to any polypeptide that originates from the pre pro-BNP molecule, other than the 32-amino acid BNP molecule itself.
  • a marker related to or associated with BNP includes the NT pro-BNP molecule, the pro domain, a fragment of BNP that is smaller than the entire 32- amino acid sequence, a fragment of pre pro-BNP other than BNP, and a fragment of the pro domain.
  • candidate gene could be used as a biomarker for CSPD diseases in different classes :
  • Biomarker a biomarker that relates to a clinical outcome or measure of disease.
  • Efficacy Biomarker a biomarker that reflects beneficial effect of a given treatment.
  • Staging Biomarker a biomarker that distinguishes between different stages of a chronic disorder.
  • Surrogate Biomarker a biomarker that is regarded as a valid substitute for a clinical outcomes measure.
  • Toxicity Biomarker a biomarker that reports a toxicological effect of a drug on an in vitro or in vivo system.
  • Biomarker a biomarker that reports a downstream effect of a drug.
  • Target Biomarker a biomarker that reports interaction of the drug with its target.
  • One embodiment of the invention is a method of use of a target comprised in a group of targets consisting of Target NO:1 - Target NO:88 as a biomarker for a disease comprising :
  • the biological sample in step (a) of the methods is in a preferred embodiment a biological sample comprised in a group of samples consisting of a blood sample, a plasma sample, a serum sample, a tissue sample, a oral mucosa sample, a saliva sample, an interstitial fluid sample or an urine sample.
  • the blood sample is for example a whole blood sample, a fractionated blood sample, a platelet sample, a neutrophil sample, a leukocyte sample, a white blood cell sample, a monocyte sample, a red blood cell sample, a granulocyte sample, and a erythrocyte sample.
  • a tissue sample is for example a sample collected from muscle, adipose, heart or skin, (sollten hier thoughts die Gewebe aus TaqMan auftim warden?)
  • candidate gene is used as a biomarker diagnosing a disease which is associated with altered candidate gene levels.
  • Another preferred embodiment candidate gene is used as a biomarker for identifying an individual risk for developing a disease, or for predicting an adverse outcome in a patient diagnosed with a disease,
  • candidate gene as a disease biomarker in diagnostics is based by the comparison of candidate gene level in a biological sample from a diseased mammal with the candidate gene level in a control sample from a healthy or normal mammal or a group of healthy or normal mammals. Does the candidate gene level in the diseased mammal differs from the candidate gene level in a normal or healthy mammal then the diseased mammal is diagnosed with a disease associated with altered candidate gene level.
  • candidate gene as a staging biomarker, the candidate gene levels of a diseased mammal are compared with candidate gene levels of a mammal with a candidate gene-associated disease already diagnosed with different stages or severity of said disease, allows the diagnose of said first diseased mammal specifying the severity of the candidate gene-associated disease.
  • a control sample can be a sample taken from a mammal.
  • a control sample can be a previously taken sample from a mammal, as a candidate gene level in a control sample can be a predetermined level of candidate gene measured in a previously taken sample.
  • the level of candidate gene in a control sample or in a biological sample can be determined for example as a relative value and as an absolute value.
  • a previously measured candidate gene level from a control sample can be for example stored in a database, in an internet publication, in an electronically accessible form, in a publication. Comparing the level of candidate gene of a biological sample to a control sample may be comparing relative values or absolute quantified values.
  • Another embodiment is a method of use of a target comprised in a group of targets consisting of Target NO: 1 - Target NO: 88 as a biomarker for guiding a therapy of a disease comprising:
  • candidate gene is used as a biomarker for guiding a therapy in a disease which is associated with altered candidate gene levels.
  • candidate gene as a disease, efficacy or surrogate endpoint biomarker in diagnostics is based by the comparison of candidate gene level in a biological sample from a diseased mammal before treatment (the baseline sample level) with the candidate gene level in subsequent samples from said mammal receiving a treatment for the disease. Does the candidate gene level in the baseline sample differs from the candidate gene level in the subsequent samples then the therapy can be considered as successful. Does the candidate gene level in the baseline sample does not differ or differs only slightly from the candidate gene level in the subsequent samples then the therapy can be considered as not successful. If the therapy is considered not successful increased dosages of the same therapy, repeat of the same therapy or an alternative treatment which is different from the first therapy can be considered.
  • the biological sample in step (a) of the methods is in a preferred embodiment a biological sample comprised in a group of samples consisting of a blood sample, a plasma sample, a serum sample, a tissue sample, a oral mucosa sample, a saliva sample, an interstitial fluid sample or an urine sample.
  • the blood sample is for example a whole blood sample, a fractionated blood sample, a platelet sample, a neutrophil sample, a leukocyte sample, a white blood cell sample, a monocyte sample, a red blood cell sample, a granulocyte sample, and a erythrocyte sample.
  • a tissue sample is for example a sample collected from muscle, adipose, heart, brain, CSPD diseased tissues, skin or a biopsy.
  • the level of candidate gene is determined by determining the level of candidate gene polynucleotide.
  • the level of candidate gene is determined by determining the level of candidate gene polypeptide. %
  • the level of candidate gene is determined by determining the level of candidate gene activity.
  • the disease associated with a target comprised in a group of targets consisting of Target NO:1 - Target NO:88 is a CSPD disease.
  • the mammal is a human.
  • the level of a target comprised in a group of targets consisting of Target NO:1 - Target NO: 88 of the biological sample is elevated compared to the control sample.
  • Another embodiment of the present invention prefers the use of a target comprised in a group of targets consisting of Target NO: 1 - Target NO:88 in combination with the use of one or more biomarkers, more preferably with biomarkers used in diagnosing candidate gene-associated diseases.
  • a target comprised in a group of targets consisting of Target NO: 1 - Target NO:88 is combined with the use of one or more clinical biomarkers which are comprised in a group of biomarkers consisting of CRTAC, FNl, NPR3, LTBP2, TGFB2, PRS S23, CTGF, BNP, ANP, Troponin, CRP, Myoglobin, CK-MB or metabolites.
  • a target comprised in a group of targets consisting of Target NO: 1 - Target NO: 88 is combined with the use of one or more clinical biomarkers which are comprised in a group of biomarkers consisting of blood pressure, heart rate, pulmonary artery pressure, or systemic vascular resistance.
  • the use of candidate gene is combined with the use of one or more diagnostic imaging methods which are comprised in a group of methods consisting of PET (Positron Emission Tomography), CT (Computed Tomography), ultrasonic, SPECT (Single Photon Emission Computed Tomography), Echocardiography, or Impedance Cardiography.
  • kits for identifying an individual risk for developing a disease, for predicting a disease or an adverse outcome in a patient diagnosed with a disease, or for guiding a therapy in a patient with a disease comprising one ore more antibodies which specifically binds candidate gene, detection means, one or more containers for collecting and or holding the biological sample, and an instruction for its use.
  • kits for identifying an individual risk for developing a disease, for predicting a disease or an adverse outcome in a patient diagnosed with a disease, or for guiding a therapy in a patient with a disease comprising one or more probes or primers for detecting candidate gene mRNA, detection means, one or more containers for collecting and or holding the biological sample, and an instruction for its use.
  • kits for identifying an individual risk for developing a disease, for predicting a disease or an adverse outcome in a patient diagnosed with a disease, or for guiding a therapy in a patient with a disease comprising one or more substrates for detecting candidate gene activity, detection means, one or more containers for collecting and or holding the biological sample, and an instruction for its use.
  • a therapeutically effective dose refers to that amount of active ingredient which increases or decreases candidate gene activity relative to candidate gene activity which occurs in the absence of the therapeutically effective dose.
  • 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 also can be used to determine the appropriate concentration range and route of administration. Such information can then be used to determine useful doses and routes for administration in humans.
  • Therapeutic efficacy and toxicity e.g., ED 50 (the dose therapeutically effective in 50% of the population) and LD 50 (the dose lethal to 50% of the population), can be determined by standard pharmaceutical procedures in cell cultures or experimental animals.
  • the dose ratio of toxic to therapeutic effects is the therapeutic index, and it can be expressed as the ratio, LD 50 ZED 5O .
  • Pharmaceutical compositions which 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 contained in such compositions is preferably within a range of circulating concentrations that include the ED 50 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 exact dosage will be determined by the practitioner, in light of factors related to the subject that requires treatment. Dosage and administration are adjusted to provide sufficient levels of the active ingredient or to maintain the desired effect. Factors which can be taken into account include the severity of the disease state, general health of the subject, age, weight, and gender of the subject, diet, time and frequency of administration, drug combination(s), reaction sensitivities, and tolerance/response to therapy. Long-acting pharmaceutical compositions can be administered every 3 to 4 days, every week, or once every two weeks depending on the half-life and clearance rate of the particular formulation.
  • Normal dosage amounts can vary from 0.1 micrograms to 100,000 micrograms, up to a total dose of about 1 g, depending upon the route of administration.
  • Guidance as to particular dosages and methods of delivery is provided in the literature and generally available to practitioners in the art. Those skilled in the art will employ different formulations for nucleotides than for proteins or their inhibitors. Similarly, " delivery of polynucleotides or polypeptides will be specific to particular cells, conditions, locations, etc.
  • the reagent is a single-chain antibody
  • polynucleotides encoding the antibody can be constructed and introduced into a cell either ex vivo or in vivo using well-established techniques including, but not limited to, transferrin-polycation- mediated DNA transfer, transfection with naked or encapsulated nucleic acids, liposome- mediated cellular fusion, intracellular transportation of DNA-coated latex beads, protoplast fusion, viral infection, electroporation, "gene gun", and DEAE- or calcium phosphate-mediated transfection.
  • the expression product is mRNA
  • the reagent is preferably an antisense oligonucleotide or a ribozyme.
  • Polynucleotides which express antisense oligonucleotides or ribozymes can be introduced into cells by a variety of methods, as described above.
  • a reagent reduces expression of candidate gene gene or the activity of candidate gene by at least about 10, preferably about 50, more preferably about 75, 90, or 100% relative to the absence of the reagent.
  • the effectiveness of the mechanism chosen to decrease the level of expression of candidate gene gene or the activity of candidate gene can be assessed using methods well known in the art, such as hybridization of nucleotide probes to candidate gene-specific mRNA, quantitative RT-PCR, immunologic detection of candidate gene, or measurement of candidate gene activity.
  • any of the pharmaceutical compositions of the invention can be administered in combination with other appropriate therapeutic agents. Selection of the appropriate agents for use in combination therapy can be made by one of ordinary skill in the art, according to conventional pharmaceutical principles.
  • the combination of therapeutic agents can act synergistically to effect the treatment or prevention of the various disorders described above. Using this approach, one may be able to achieve therapeutic efficacy with lower dosages of each agent, thus reducing the potential for adverse side effects.
  • Any of the therapeutic methods described above can be applied to any subject in need of such therapy, including, for example, mammals such as dogs, cats, cows, horses, rabbits, monkeys, and most preferably, humans.
  • Nucleic acid molecules of the invention are those nucleic acid molecules which are contained in a group of nucleic acid molecules consisting of (i) nucleic acid molecules encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 1- SEQ ID NO: 88, (ii) nucleic acid molecules comprising the sequence of SEQ ID NO: 89 - SEQ ID NO: 176, (iii) nucleic acid molecules having the sequence of SEQ ID NO: 89 - SEQ ID NO: 176, (iv) nucleic acid molecules the complementary strand of which hybridizes under stringent conditions to a nucleic acid molecule of (i), (ii), or (iii), (v) nucleic acid molecules the sequence of which differs from the
  • nucleic acid molecules which have a sequence identity of at least 80%, 85%, 90%, 95%, 98% or
  • Polypeptides of the invention are those polypeptides which are contained in a group of polypeptides consisting of (i) polypeptides having the sequence of SEQ ED NO: 1- SEQ ID NO:88, (ii) polypeptides comprising the sequence of SEQ ID NO: 1- SEQ ID NO:88, (iii) polypeptides encoded by nucleic acid molecules of the invention and (iv) polypeptides which show at least 99%, 98%, 95%, 90%, or 80% identity with a polypeptide of (i), (ii), or (iii).
  • An object of the invention is a method of screening for therapeutic agents useful in the treatment of a disease comprised in a group of diseases consisting of CSPD diseases in a mammal comprising the steps of (i) contacting a test compound with a candidate gene polypeptide, (ii) detect binding of said test compound to said candidate gene polypeptide.
  • a test compound with a candidate gene polypeptide
  • detect binding of said test compound to said candidate gene polypeptide e.g., compounds that bind to the candidate gene polypeptide are identified potential therapeutic agents for such a disease.
  • Another object of the invention is a method of screening for therapeutic agents useful in the treatment of a disease comprised in a group of diseases consisting of CSPD diseases in a mammal comprising the steps of (i) determining the activity of a candidate gene polypeptide at a certain concentration of a test compound or in the absence of said test compound, (ii) determining the activity of said polypeptide at a different concentration of said test compound.
  • Another object of the invention is a method of screening for therapeutic agents useful in the treatment of a disease comprised in a group of diseases consisting of CSPD diseases in a mammal comprising the steps of (i) determining the activity of a candidate gene polypeptide at a certain concentration of a test compound, (ii) determining the activity of a candidate gene polypeptide at the presence of a compound known to be a regulator of a candidate gene polypeptide.
  • compounds that show similar effects on the activity of the candidate gene polypeptide in (i) as compared to compounds used in (ii) are identified potential therapeutic agents for such a disease.
  • test compound displaces a ligand which is first bound to the polypeptide.
  • Another object of the invention is a method of screening for therapeutic agents useful in the treatment of a disease comprised in a group of diseases consisting of CSPD diseases in a mammal comprising the steps of (i) contacting a test compound with a candidate gene polynucleotide, (ii) detect binding of said test compound to said candidate gene polynucleotide.
  • Compounds that, e.g., bind to the candidate gene polynucleotide are potential therapeutic agents for the treatment of such diseases.
  • Another object of the invention is the method of the above, wherein the nucleic acid molecule is RNA.
  • Another object of the invention is a method of the above, wherein the contacting step is in or at the surface of a cell.
  • Another object of the invention is a method of the above, wherein the contacting step is in a cell- free system.
  • Another object of the invention is a method of the above, wherein the polynucleotide is coupled to a detectable label.
  • Another object of the invention is a method of the above, wherein the test compound is coupled to a detectable label.
  • Another object of the invention is a method of diagnosing a disease comprised in a group of diseases consisting of CSPD diseases in a mammal comprising the steps of (i) determining the amount of a candidate gene polynucleotide in a sample taken from said mammal, (ii) determining the amount of candidate gene polynucleotide in healthy and/or diseased mammal.
  • a disease is diagnosed, e.g., if there is a substantial similarity in the amount of candidate gene polynucleotide in said test mammal as compared to a diseased mammal.
  • Another object of the invention is a pharmaceutical composition for the treatment of a disease comprised in a group of diseases consisting of CSPD diseases in a mammal comprising a therapeutic agent which binds to a candidate gene polypeptide.
  • Another object of the invention is a pharmaceutical composition for the treatment of a disease comprised in a group of diseases consisting of CSPD diseases in a mammal comprising a therapeutic agent which regulates the activity of a candidate gene polypeptide.
  • Another object of the invention is a pharmaceutical composition for the treatment of a disease comprised in a group of diseases consisting of CSPD diseases in a mammal comprising a therapeutic agent which regulates the activity of a candidate gene polypeptide, wherein said therapeutic agent is (i) a small molecule, (ii) an RNA molecule, (iii) an antisense oligonucleotide, (iv) a polypeptide, (v) an antibody, or (vi) a ribozyme.
  • Another object of the invention is a pharmaceutical composition for the treatment of a disease comprised in a group of diseases consisting of CSPD diseases in a mammal comprising a candidate gene polynucleotide.
  • Another object of the invention is a pharmaceutical composition for the treatment of a disease comprised in a group of diseases consisting of CSPD diseases in a mammal comprising a candidate gene polypeptide.
  • Another object of the invention is the use of regulators of a candidate gene for the preparation of a pharmaceutical composition for the treatment of a disease comprised in a group of diseases consisting of CSPD diseases in a mammal.
  • Another object of the invention is a method for the preparation of a pharmaceutical composition useful for the treatment of a disease comprised in a group of diseases consisting of CSPD diseases in a mammal comprising the steps of (i) identifying a regulator of candidate gene, (ii) determining whether said regulator ameliorates the symptoms of a- disease comprised in a group of diseases consisting of CSPD diseases in a mammal; and (iii) combining of said regulator with an acceptable pharmaceutical carrier.
  • Another object of the invention is the use of a regulator of candidate gene for the regulation of candidate gene activity in a mammal having a disease comprised in a group of diseases consisting of CSPD diseases.
  • cDNA sequence coding for candidate gene enables its use as a tool for antisense technology in the investigation of gene function.
  • Oligonucleotides, cDNA or genomic fragments comprising the antisense strand of a polynucleotide coding for candidate gene are used either in vitro or in vivo to inhibit translation of the mRNA.
  • antisense molecules can be designed at various locations along the nucleotide sequences.
  • modifications of gene expression is obtained by designing antisense sequences to intron regions, promoter/enhancer elements, or even to trans-acting regulatory genes.
  • Expression of candidate gene is accomplished by subcloning the cDNAs into appropriate expression vectors and transfecting the vectors into expression hosts such as, e.g., E. coli.
  • the vector is engineered such that it contains a promoter for ⁇ -galactosidase, upstream of the cloning site, followed by sequence containing the amino-terminal Methionine and the subsequent seven residues of ⁇ -galactosidase.
  • an engineered bacteriophage promoter useful for artificial priming and transcription and for providing a number of unique endonuclease restriction sites for cloning.
  • IPTG Isopropyl- ⁇ -D-thiogalactopyranoside
  • the cDNA is not in the proper reading frame, it is obtained by deletion or insertion of the appropriate number of bases using well known methods including in vitro mutagenesis, digestion with exonuclease IH or mung bean nuclease, or the inclusion of an oligonucleotide linker of appropriate length.
  • the candidate gene cDNA is shuttled into other vectors known to be useful for expression of proteins in specific hosts.
  • Oligonucleotide primers containing cloning sites as well as a segment of DNA (about 25 bases) sufficient to hybridize to stretches at both ends of the target cDNA is synthesized chemically by standard methods. These primers are then used to amplify the desired gene segment by PCR.
  • the resulting gene segment is digested with appropriate restriction enzymes under standard conditions and isolated by gel electrophoresis. Alternately, similar gene segments are produced by digestion of the cDNA with appropriate restriction enzymes. Using appropriate primers, segments of coding sequence from more than one gene are ligated together and cloned in appropriate vectors. It is possible to optimize expression by construction of such chimeric sequences.
  • Suitable expression hosts for such chimeric molecules include, but are not limited to, mammalian cells such as Chinese Hamster Ovary (CHO) and human 293 cells., insect cells such as Sf9 cells, yeast cells such as Saccharomyces cerevisiae and bacterial cells such as E. coli.
  • a useful expression vector also includes an origin of replication to allow propagation in bacteria, and a selectable marker such as the ⁇ -lactamase antibiotic resistance gene to allow plasmid selection in bacteria.
  • the vector may include a second selectable marker such as the neomycin phosphotransferase gene to allow selection in transfected eukaryotic host cells.
  • Vectors for use in eukaryotic expression hosts require RNA processing elements such as 3' polyadenylation sequences if such are not part of the cDNA of interest.
  • the vector contains promoters or enhancers which increase gene expression.
  • promoters are host specific and include MMTV, SV40, and metallothionine promoters for CHO cells; trp, lac, tac and T7 promoters for bacterial hosts; and alpha factor, alcohol oxidase and PGH promoters for yeast.
  • Transcription enhancers such as the rous sarcoma virus enhancer, are used in mammalian host cells. Once homogeneous cultures of recombinant cells are obtained through standard culture methods, large quantities of recombinantly produced candidate gene are recovered from the conditioned medium and analyzed using chromatographic methods known in the art.
  • candidate gene can be cloned into the expression vector pcDNA3, as exemplified herein.
  • This product can be used to transform, for example, HEK293 or COS by methodology standard in the art. Specifically, for example, using Lipofectamine (Gibco BRL catolog no. 18324-020) mediated gene transfer.
  • candidate gene is expressed as a chimeric protein with one or more additional polypeptide domains added to facilitate protein purification.
  • purification facilitating domains include, but are not limited to, metal chelating peptides such as histidine-tryptophan modules that allow purif ⁇ cation on immobilized metals [Appa Rao, 1997] and the domain utilized in the FLAGS extension/affinity purification system (Immunex Corp., Seattle, Washington).
  • the inclusion of a cleavable linker sequence such as Factor Xa or enterokinase (Invitrogen, Groningen, The Netherlands) between the purification domain and the candidate gene sequence is useful to facilitate expression of candidate gene.
  • the following example provides a method for purifying candidate gene.
  • candidate gene is generated using the baculovirus expression system BAC-TO-BAC (GIBCO BRL) based on Autographa californica nuclear polyhedrosis virus (AcNPV) infection of Spodoptera frugiperda insect cells (Sf9 cells).
  • BAC-TO-BAC baculovirus expression system
  • AcNPV Autographa californica nuclear polyhedrosis virus
  • the recombinant plasmid is transformed into DHlOBAC competent cells which contain the parent bacmid bMON14272 (AcNPV infectious DNA) and a helper plasmid.
  • the mini-Tn7 element on the pFASTBAC donor can transpose to the attTn7 attachment site on the bacmid thus introducing the protease gene into the viral genome. Colonies containing recombinant bacmids are identified by disruption of the lacZ gene.
  • protease/bacmid construct can then be isolated and infected into insect cells (Sf9 cells) resulting in the production of infectious recombinant baculovirus particles and expression of either unfiised recombinant enzyme (pFastbacl) or candidate gene-His fusion protein (pFastbacHT).
  • pFastbacl unfiised recombinant enzyme
  • pFastbacHT candidate gene-His fusion protein
  • Cells are harvested and extracts prepared 24, 48 and 72 hours after transfection. Expression of candidate gene is confirmed by coomassie staining after sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and western blotting onto a PVDF membrane of an unstained SDS-PAGE.
  • SDS-PAGE sodium dodecyl sulphate-polyacrylamide gel electrophoresis
  • the protease-His fusion protein is detected due to the interaction between the Ni- NTA HRP conjugate and the His-tag which is fused to candidate gene.
  • denatured protein from reverse phase HPLC separation is obtained in quantities up to 75 mg. This denatured protein is used to immunize mice or rabbits using standard protocols; about 100 ⁇ g are adequate for immunization of a mouse, while up to 1 mg might be used to immunize a rabbit.
  • the denatured protein is radioiodinated and used to screen potential murine B-cell hybridomas for those which produce antibody. This procedure requires only small quantities of protein, such that 20 mg is sufficient for labeling and screening of several thousand clones.
  • the amino acid sequence of an appropriate candidate gene domain is analyzed to determine regions of high antigenicity.
  • Oligopeptides comprising appropriate hydrophilic regions are synthesized and used in suitable immunization protocols to raise antibodies.
  • the optimal amino acid sequences for immunization are usually at the C-terminus, the N-terminus and those intervening, hydrophilic regions of the polypeptide which are likely to be exposed to the external environment when the protein is in its natural conformation.
  • selected peptides typically, about 15 residues in length, are synthesized using an Applied Biosystems Peptide Synthesizer Model 43 IA using fmoc-chemistry and coupled to keyhole limpet hemocyanin (KLH; Sigma, St. Louis, MO) by reaction with M-maleimidobenzoyl-N- hydroxysuccinimide ester, MBS. If necessary, a cysteine is introduced at the N-terminus of the peptide to permit coupling to KLH. Rabbits are immunized with the peptide-KLH complex in complete Freund's adjuvant.
  • KLH keyhole limpet hemocyanin
  • the resulting antisera are tested for antipeptide activity by binding the peptide to plastic, blocking with 1% bovine serum albumin, reacting with antisera, washing and reacting with labeled (radioactive or fluorescent), affinity purified, specific goat anti-rabbit IgG.
  • Hybridomas are prepared and screened using standard techniques. Hybridomas of interest are detected by screening with labeled candidate gene to identify those fusions producing the monoclonal antibody with the desired specificity. In a typical protocol, wells of plates (FAST;
  • Becton-Dickinson, Palo Alto, CA) are coated during incubation with affinity purified, specific rabbit anti-mouse (or suitable antispecies 1 g) antibodies at 10 mg/ml.
  • the coated wells are blocked with 1% bovine serum albumin, (BSA), washed and incubated with supernatants from hybridomas. After washing the wells are incubated with labeled candidate gene at 1 mg/ml.
  • BSA bovine serum albumin
  • Particular candidate gene antibodies are useful for investigating signal transduction and the diagnosis of infectious or hereditary conditions which are characterized by differences in the amount or distribution of candidate gene or downstream products of an active signaling cascade.
  • Diagnostic tests for candidate gene include methods utilizing antibody and a label to detect candidate gene in human body fluids, membranes, cells, tissues or extracts of such.
  • the polypeptides and antibodies of the present invention are used with or without modification. Frequently, the polypeptides and antibodies are labeled by joining them, either covalently or noncovalently, with a substance which provides for a detectable signal.
  • labels and conjugation techniques are known and have been reported extensively in both the scientific and patent literature. Suitable labels include radionuclides, enzymes, substrates, cofactors, inhibitors, fluorescent agents, chemiluminescent agents, chromogenic agents, magnetic particles and the like.
  • a variety of protocols for measuring soluble or membrane-bound candidate gene, using either polyclonal or monoclonal antibodies specific for the protein, are known in the art. Examples include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA) and fluorescent activated cell sorting (FACS).
  • ELISA enzyme-linked immunosorbent assay
  • RIA radioimmunoassay
  • FACS fluorescent activated cell sorting
  • a two-site monoclonal-based immunoassay utilizing monoclonal antibodies reactive to two non-interfering epitopes on candidate gene is preferred, but a competitive binding assay may be employed.
  • Native or recombinant candidate gene is purified by immunoaffmity chromatography using antibodies specific for candidate gene.
  • an immunoaffinity column is constructed by covalently coupling the anti-TRH antibody to an activated chromatographic resin.
  • Polyclonal immunoglobulins are prepared from immune sera either by precipitation " with ammonium sulfate or by purification on immobilized Protein A (Pharmacia LKB Biotechnology,
  • monoclonal antibodies are prepared from mouse ascites fluid by ammonium sulfate precipitation or chromatography on immobilized Protein A. Partially purified immunoglobulin is covalently attached to a chromatographic resin such as CnBr-activated
  • Sepharose (Pharmacia LKB Biotechnology). The antibody is coupled to the resin, the resin is blocked, and the derivative resin is washed according to the manufacturer's instructions.
  • Such immunoaffinity columns are utilized in the purification of candidate gene by preparing a fraction from cells containing candidate gene in a soluble form. This preparation is derived by solubilization of whole cells or of a subcellular fraction obtained via differential centrifugation (with or without addition of detergent) or by other methods well known in the art. Alternatively, soluble candidate gene containing a signal sequence is secreted in useful quantity into the medium in which the cells are grown.
  • a soluble candidate gene-containing preparation is passed over the immunoaff ⁇ nity column, and the column is washed under conditions that allow the preferential absorbance of candidate gene (e.g., high ionic strength buffers in the presence of detergent). Then, the column is eluted under conditions that disrupt antibody/protein binding (e.g., a buffer of pH 2-3 or a high concentration of a chaotrope such as urea or thiocyanate ion), and candidate gene is collected.
  • a buffer of pH 2-3 or a high concentration of a chaotrope such as urea or thiocyanate ion
  • This invention is particularly useful for screening therapeutic compounds by using candidate gene or fragments thereof in any of a variety of drug screening techniques.
  • the following example provides a system for drug screening measuring the protease activity.
  • the recombinant protease-His fusion protein can be purified from the crude lysate by metal- affinity chromatography using Ni-NTA agarose. This allows the specific retention of the recombinant material (since this is fused to the His-tag) whilst the endogenous insect proteins are washed off. The recombinant material is then eluted by competition with imidazol.
  • candidate gene molecules of the present invention can be measured using a variety of assays that measure candidate gene activity.
  • candidate gene enzyme activity can be assessed by a standard in vitro serine/metallo/... protease assay (see, for example, [U.S. 5,057,414]).
  • substrates suitable for in vitro assays such as SucAla-Ala-Pro-Phe-pNA, fluorescein mono-p-guanidinobenzoate hydrochloride, benzyloxycarbonyl-L-Arginyl-S-benzylester, Nalpha-Benzoyl-L-arginine ethyl ester hydrochloride, and the like.
  • protease assay kits available from commercial sources, such as CalbiochemTM (San Diego, Calif.). For general references, see Barrett (Ed.), Methods in Enzymology, Proteolytic Enzymes: Serine and Cysteine Peptidases (Academic Press Inc. 1994), and Barrett et al., (Eds.), Handbook of Proteolytic Enzymes (Academic Press Inc. 1998).
  • the goal of rational drug design is to produce structural analogs of biologically active polypeptides of interest or of small molecules with which they interact, agonists, antagonists, or inhibitors. Any of these examples are used to fashion drugs which are more active or stable forms of the polypeptide or which enhance or interfere with the function of a polypeptide in vivo.
  • the three-dimensional structure of a protein of interest, or of a protein-inhibitor complex is determined by x-ray crystallography, by computer modeling or, most typically, by a combination of the two approaches. Both the shape and charges of the polypeptide must be ascertained to elucidate the structure and to determine active site(s) of the molecule. Less often, useful information regarding the structure of a polypeptide is gained by modeling based on the structure of homologous proteins. In both cases, relevant structural information is used to design efficient inhibitors. Useful examples of rational drug design include molecules which have improved activity or stability or which act as inhibitors, agonists, or antagonists of native peptides.
  • a target-specific antibody selected by functional assay, as described above, and then to solve its crystal structure.
  • This approach in principle, yields a pharmacore upon which subsequent drug design is based. It is possible to bypass protein crystallography altogether by generating anti-idiotypic antibodies (anti-ids) to a functional, pharmacologically active antibody. As a mirror image of a mirror image, the binding site of the anti-ids is expected to be an analog of the original receptor. The anti-id is then used to identify and isolate peptides from banks of chemically or biologically produced peptides. The isolated peptides then act as the pharmacore.
  • anti-ids anti-idiotypic antibodies
  • Labeled candidate gene is useful as a reagent " for the purification of molecules with which it interacts.
  • candidate gene is covalently coupled to a chromatography column.
  • Cell-free extract derived from synovial cells or putative target cells is passed over the column, and molecules with appropriate affinity bind to candidate gene, candidate gene-complex is recovered from the column, and the candidate gene-binding ligand disassociated and subjected to N-terminal protein sequencing.
  • the amino acid sequence information is then used to identify the captured molecule or to design degenerate oligonucleotide probes for cloning the relevant gene from an appropriate cDNA library.
  • antibodies are raised against candidate gene, specifically monoclonal antibodies. The monoclonal antibodies are screened to identify those which inhibit the binding of labeled candidate gene. These monoclonal antibodies are then used therapeutically.
  • Example 10 Use and Administration of Antibodies, Inhibitors, or Antagonists
  • LSTs are formulated in a nontoxic, inert, pharmaceutically acceptable aqueous carrier medium preferably at a pH of about 5 to 8, more preferably 6 to 8, although pH may vary according to the characteristics of the antibody, inhibitor, or antagonist being formulated and the condition to be treated. Characteristics of LSTs include solubility of the molecule, its half-life and antigenicity/immunogenicity. These and other characteristics aid in defining an effective carrier. Native human proteins are preferred as LSTs, but organic or synthetic molecules resulting from drug screens are equally effective in particular situations.
  • LSTs are delivered by known routes of administration including but not limited to topical creams and gels; transmucosal spray and aerosol; transdermal patch and bandage; injectable, intravenous and lavage formulations; and orally administered liquids and pills particularly formulated to resist stomach acid and enzymes.
  • routes of administration including but not limited to topical creams and gels; transmucosal spray and aerosol; transdermal patch and bandage; injectable, intravenous and lavage formulations; and orally administered liquids and pills particularly formulated to resist stomach acid and enzymes.
  • the particular formulation, exact dosage, and route of administration is determined by the attending physician and varies according to each specific situation.
  • Such determinations are made by considering multiple variables such as the condition to be treated, the LST to be administered, and the pharmacokinetic profile of a particular LST. Additional factors which are taken into account include severity of the disease state, patient's age, weight, gender and diet, time and frequency of LST administration, possible combination with other drugs, reaction sensitivities, and tolerance/response to therapy. Long acting LST formulations 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 LST.
  • Normal dosage amounts vary from 0.1 to 10 5 ⁇ g, up to a total dose of about 1 g, depending upon the route of administration.
  • Guidance as to particular dosages and methods of delivery is provided in the literature; see U.S. Pat. Nos. 4,657,760; 5,206,344; or 5,225,212.
  • Those skilled in the art employ different formulations for different LSTs.
  • Administration to cells such as nerve cells necessitates delivery in a manner different from that to other cells such as vascular endothelial cells. It is contemplated that abnormal signal transduction, trauma, or diseases which trigger candidate gene activity are treatable with LSTs.
  • Animal model systems which elucidate the physiological and behavioral roles of the candidate gene are produced by creating nonhuman transgenic animals in which the activity of the candidate gene is either increased or decreased, or the amino acid sequence of the expressed candidate gene is altered, by a variety of techniques.
  • these techniques include, but are not limited to: 1) Insertion of normal or mutant versions of DNA encoding a candidate gene, by microinjection, electroporation, retroviral transfection or other means well known to those skilled in the art, into appropriately fertilized embryos in order to produce a transgenic animal or 2) homologous recombination of mutant or normal, human or animal versions of these genes with the native gene locus in transgenic animals to alter the regulation of expression or the structure of these candidate gene sequences.
  • the technique of homologous recombination is well known in the art. It replaces the native gene with the inserted gene and hence is useful for producing an animal that cannot express native candidate genes but does express, for example, an inserted mutant candidate gene, which has replaced the native candidate gene in the animal's genome by recombination, resulting in underexpression of the transporter. Microinjection adds genes to the genome, but does not remove them, and the technique is useful for producing an animal which expresses its own and added candidate gene, resulting in overexpression of the candidate gene.
  • transgenic animal One means available for producing a transgenic animal, with a mouse as an example, is as follows: Female mice are mated, and the resulting fertilized eggs are dissected out of their oviducts. The eggs are stored in an appropriate medium such as cesiumchloride M2 medium. DNA or cDNA encoding candidate gene is purified from a vector by methods well known to the one skilled in the art. Inducible promoters may be fused with the coding region of the DNA to provide an experimental means to regulate expression of the transgene. Alternatively or in addition, tissue specific regulatory elements may be fused with the coding region to permit tissue-specific expression of the transgene.
  • microinjection needle which may be made from capillary tubing using a piper puller
  • the egg to be injected is put in a depression slide.
  • the needle is inserted into the pronucleus of the egg, and the DNA solution is injected.
  • the injected egg is then transferred into the oviduct of a pseudopregnant mouse which is a mouse stimulated by the appropriate hormones in order to maintain false pregnancy, where it proceeds to the uterus, implants, and develops to term.
  • microinjection is not the only method for inserting DNA into the egg but is used here only for exemplary purposes.
  • HEK293 cells and HEK293R cells were cultured as previously described [Sahin et al., (2006), Lewerenz et al. (2006)].
  • High purity plasmids were obtained using the Nucleobond AX 500 columns (Machery & Nagel, D ⁇ ren, Germany) according to the manufacturer.
  • confluent HEK293 cells were harvested by trypsination. 15x10 6 HEK293 cells resuspended in 500 ⁇ l of PBS and incubated on ice for 10 minutes in the presence of 10 ⁇ g plasmid DNA. Empty pCI-neo vector served as control.
  • Electroporation was carried our with a Genepulser (Biorad, Kunststoff, Germany) at 960 F and 250 mA with a time constant of 17-20 sec. Immediately after transfection, cells of each electroporation were plated in three 92 mm culture-dishes. 24 hours after plating, cells were trypsinized and 5x103 cells were seeded onto 96 well-cell culture plates for toxicity assays or GSH measurement.
  • GPR39 cDNA Full length GPR39 cDNA were cloned by high-fidelity PCR from HEK293 cDNA using EST data into pcDNA3.
  • 5000 HEK293 cells were seeded in 100 ⁇ l into 96-well plates and glutamate (250 mM stock solution, pH 7.4) added 24 hours later and left for 8 hours.
  • the clones are defined as Kl : low expression of GPR39; Kl 7 : high expression of GPR39.
  • Cell viability was measured by the amount of blue formazan produced by viable cells from the tetrazolium salt 3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide (MTT, Sigma), which is proportional to the number of viable cells as previously described [Sahin et al., (2006), Lewerenz et al. (2006)].
  • RNAs from cultured cells were prepared with a RNeasy miniprep kit according to the manufacturer's (Qiagen, Hilden, Germany) protocol.
  • the RNA was quantified by a NanoDrop ND- 1000 spectrophotometer (NanoDrop Technologies, Wilmington, DE), and quality was assessed by a 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA).
  • Double stranded cDNA synthesis and in vitro transcription (IVT) were carried out according to Affymetrix protocols.
  • RNA from cultured cells were reverse transcribed with SuperScript ⁇ and T7- Oligo(dT) Promotor Primer for 1 h at 42 0 C, followed by 2 h incubation at 16 ° for second-strand cDNA synthesis.
  • the cDNA served as a template for subsequent in vitro transcription (IVT).
  • the IVT reaction was carried out in the presence of T7 RNA polymerase and a biotinylated nucleotide analog/ribonucleotide mix for complementary (cRNA) amplification and biotin labeling for 16 h at 37 0 C.
  • the quality of cRNA was assessed by a 2100 Bioanalyzer.
  • Biotinylated cRNA was cleaned up and 15 ⁇ g were fragmented by metal/heat induced fragmentation. The fragmented cRNA was hybridized against GeneChip Human genome Ul 33 Plus 2.0 in a Affymetrix GeneChip Hybridization Oven for 16 h at 45 0 C and 60 rpm. Each microarray was washed and stained with streptavidin-phycoerythrin using Affymetrix GeneChip Fluidics Station 450 and then scanned with Affymetrix Scanner 3000. Raw data was analyzed with Affymetrix MAS5 algorithm and probesets with a detection p-value of p ⁇ 0.01 were used for further analysis.
  • Ostadal B Ostadalova I, Kolar F, Pelouch V, Dhalla NS. Cardiac adaptation to chronic hypoxia.
  • Becker LB New concepts in reactive oxygen species and cardiovascular reperfusion physiology.

Landscapes

  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Molecular Biology (AREA)
  • Chemical & Material Sciences (AREA)
  • Biomedical Technology (AREA)
  • Urology & Nephrology (AREA)
  • Hematology (AREA)
  • Immunology (AREA)
  • Biotechnology (AREA)
  • Analytical Chemistry (AREA)
  • Cell Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Food Science & Technology (AREA)
  • Medicinal Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Microbiology (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

L'invention porte sur des gènes candidats associés à des procédés protecteurs de cellules. L'invention concerne également des dosages permettant d'identifier des composés utiles dans le traitement, le diagnostic ou la prévention de maladies CSPD. L'invention porte également sur des composés qui se lient et/ou activent ou inhibent l'activité de gènes candidats ainsi que sur des compositions pharmaceutiques comprenant de tels composés. L'invention porte aussi sur des gènes candidats en tant que biomarqueurs de maladies associées à CSPD.
PCT/EP2007/008790 2006-10-16 2007-10-10 Gènes protecteurs de cellules WO2008046543A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
EP06021591 2006-10-16
EP06021591.0 2006-10-16

Publications (1)

Publication Number Publication Date
WO2008046543A1 true WO2008046543A1 (fr) 2008-04-24

Family

ID=38922429

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/EP2007/008790 WO2008046543A1 (fr) 2006-10-16 2007-10-10 Gènes protecteurs de cellules

Country Status (1)

Country Link
WO (1) WO2008046543A1 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20150175969A1 (en) * 2012-09-26 2015-06-25 Fujifilm Corporation Polypeptide, scaffold composition, composition for cartilage tissue restoration, composition for cartilage cell culture, and composition for promoting glycosaminoglycan production

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003065984A2 (fr) * 2002-02-01 2003-08-14 Millennium Pharmaceuticals, Inc. Methodes et compositions permettant de traiter les maladies cardio-vasculaires avec les molecules 1682, 6169, 6193, 7771, 14395, 29002, 33216, 43726, 69292, 21656, 32427, 2402, 7747,1720, 9151, 60491, 1371, 7077, 33207, 1419, 18036, 16105, 38650, 14245, 58848, 1870, 25856, 32394, 3484, 345, 9252, 9135, 10532, 18610, 8165, 2

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003065984A2 (fr) * 2002-02-01 2003-08-14 Millennium Pharmaceuticals, Inc. Methodes et compositions permettant de traiter les maladies cardio-vasculaires avec les molecules 1682, 6169, 6193, 7771, 14395, 29002, 33216, 43726, 69292, 21656, 32427, 2402, 7747,1720, 9151, 60491, 1371, 7077, 33207, 1419, 18036, 16105, 38650, 14245, 58848, 1870, 25856, 32394, 3484, 345, 9252, 9135, 10532, 18610, 8165, 2

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20150175969A1 (en) * 2012-09-26 2015-06-25 Fujifilm Corporation Polypeptide, scaffold composition, composition for cartilage tissue restoration, composition for cartilage cell culture, and composition for promoting glycosaminoglycan production
US9932557B2 (en) * 2012-09-26 2018-04-03 Fujifilm Corporation Polypeptide, scaffold composition, composition for cartilage tissue restoration, composition for cartilage cell culture, and composition for promoting glycosaminoglycan production

Similar Documents

Publication Publication Date Title
US8088584B2 (en) LTBP2 as a biomarker and diagnostic target
US20100081136A1 (en) Crtac as a biomarker, therapeutic and diagnostic target
US20100104501A1 (en) Prss23 as a biomarker, therapeutic and diagnostic target
WO2008046510A1 (fr) Fn1 utilisé comme biomarqueur, cible thérapeutique et diagnostique
US20110033844A1 (en) Use of lysosomal carboxypeptidase c (prcp) as a therapeutic or diagnostic target
US20110111394A1 (en) Ctgf as a biomarker, therapeutic and diagnostic target
WO2007147497A2 (fr) Utilisation d'un domaine du type désintégrine et métalloprotéinase à motif de thrombospondine de type 1 (adamts1) comme cible thérapeutique et diagnostique
WO2008046528A1 (fr) Npr3 utilisé comme biomarqueur, cible thérapeutique et diagnostique
WO2007141004A1 (fr) Utilisation de l'adipsine (adn) comme cible thérapeutique ou diagnostique
WO2008046508A1 (fr) Facteur tgfb2 utilisé en tant que biomarqueur, cible thérapeutique et diagnostique
WO2008046543A1 (fr) Gènes protecteurs de cellules
US20110130347A1 (en) Identification of new splice-variants of g-protein coupled receptor ep3 and uses thereof
US7338781B2 (en) Organic anion transporting (oat)-like protein UST3-LIKE1 and uses thereof
WO2007140907A1 (fr) Utilisation de sérine endopeptidase 22 (prss22) en tant que cible thérapeutique ou diagnostique
EP1633882B1 (fr) Diagnostic et therapeutique de maladies associees a une aminopeptidase specifique a leucyle insensible a la puromycine (pils)
WO2005075510A1 (fr) Agents diagnostiques et therapeutiques pour des maladies associees au canal potassique, agents membres 9 de la sous-famille k (kcnk9)
WO2007140895A1 (fr) Utilisation de la leucyl aminopeptidase 3 (lap3) en tant que cible thérapeutique ou diagnostique
EP1606633B1 (fr) Diagnostics et traitements contre des maladies associees au recepteur d3 de la dopamine (drd3)
WO2005054848A2 (fr) Diagnostics et therapeutique pour maladies associees au canal potassique a rectification entrante couple a une proteine (girk2)
WO2007140906A1 (fr) Utilisation de la leucyl/cystinyl aminopeptidase (lnpep) en tant que cible thérapeutique ou diagnostique
US20050244896A1 (en) Diagnostics and therapeutics for diseases associated with neuropeptide ff receptor 2 (npff2)
WO2007141005A1 (fr) Utilisation de glutamyl aminopeptidase (enpep) comme cible thérapeutique ou diagnostique
WO2005054866A2 (fr) Agents diagnostiques et therapeutiques utilises dans le traitement des maladies associees au canal potassique de domaine a deux pores (kcnk2)
US20070059694A1 (en) Diagnostics and therapeutics for diseases associated with g-protein coupled receptor prostaglandin e2 ep1 (prostaglandin e2 ep1)
WO2005075989A1 (fr) Diagnostic et traitements des maladies associees au recepteur metabotropique du glutamate 5 (mglur5)

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 07818863

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 07818863

Country of ref document: EP

Kind code of ref document: A1