WO1999035290A1 - Procedes et compositions permettant de diagnostiquer et de traiter des troubles neuropsychiatriques - Google Patents

Procedes et compositions permettant de diagnostiquer et de traiter des troubles neuropsychiatriques Download PDF

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WO1999035290A1
WO1999035290A1 PCT/US1999/000297 US9900297W WO9935290A1 WO 1999035290 A1 WO1999035290 A1 WO 1999035290A1 US 9900297 W US9900297 W US 9900297W WO 9935290 A1 WO9935290 A1 WO 9935290A1
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yesl
gene
protein
nucleic acid
gene product
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PCT/US1999/000297
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WO1999035290A9 (fr
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Hong Chen
Nelson B. Freimer
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Millennium Pharmaceuticals, Inc.
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Priority to AU22150/99A priority Critical patent/AU2215099A/en
Publication of WO1999035290A1 publication Critical patent/WO1999035290A1/fr
Publication of WO1999035290A9 publication Critical patent/WO1999035290A9/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers

Definitions

  • the present invention relates to drug screening assays, and diagnostic and therapeutic methods for the treatment of neuropsychiatric disorders, mediated by the expression of a mutant form of the yesl gene or by aberrant levels of yesl expression.
  • the invention is based on Applicant's discovery that the yes2 gene is linked to the short arm of chromosome 18 in a region of the chromosome involved in mediating neuropsychiatric disorders such as bipolar-affected disorder (BAD) .
  • BAD bipolar-affected disorder
  • the invention relates to methods for the identification of compounds that modulate the expression, synthesis and activity of the yesl protein/gene and to using compounds such as those identified as therapeutic agents in the treatment of an yesl mediated disorder; a neuropsychiatric disorder, including, by way of example and not of limitation, bipolar affective disorder.
  • the _ invention also relates to methods for the diagnostic evaluation, genetic testing and prognosis of yesl mediated disorders.
  • Huntington's disease which can be traced to a mutation in a single gene and in which neurons in the striatum degenerate
  • Parkinson's disease in which dopaminergic neurons in the nigro-striatal pathway degenerate.
  • Neuropsychiatric disorders such as schizophrenia, attention deficit disorders, schizoaffective disorder, bipolar affective disorders, or unipolar affective disorder, from neurological disorders, in which anatomical or biochemical pathologies are manifest.
  • identification of the causative defects and the neuropathologies of neuropsychiatric disorders are needed in order to enable clinicians to evaluate and prescribe appropriate courses of treatment to cure or ameliorate the symptoms of these disorders.
  • bipolar affective disorder also known as bipolar mood disorder (BP) or manic- depressive illness, which is characterized by episodes of elevated mood (mania) and depression (Goodwin, et al . , 1990, Manic Depressive Illness , Oxford University Press, New York) .
  • BP-I severe bipolar affective (mood) disorder
  • SAD-M schizoaffective disorder manic type
  • BP-I often co-segregates in families with more etiologically heterogeneous syndromes, such as with a unipolar affective disorder such as unipolar major depressive disorder (MDD) , which is a more broadly defined phenotype (Freimer and Reus, 1992, in The Molecular and Genetic Basis of Neurological Disease , Rosenberg, et al . , eds., Butterworths, New York, pp. 951-965; Mclnnes and Freimer, 1995, Curr. Opin. Genet.
  • MDD unipolar major depressive disorder
  • BP-I and SAD-M are severe mood disorders that are frequently difficult to distinguish from one another on a cross-sectional basis, follow similar clinical courses, and segregate together in family studies (Rosenthal, et al . , 1980, Arch. General Psychiat. 37, 804- 810; Levinson and Levitt, 1987, Am. J. Psychiat. 144, 415- 426; Goodwin, et al . , 1990, Manic Depressive Illness , Oxford University Press, New York) .
  • methods for distinguishing neuropsychiatric disorders such as these are needed in order to effectively diagnose and treat afflicted individuals.
  • Mapping genes for common diseases believed to be caused by multiple genes, such as BAD may be complicated by the typically imprecise definition of phenotypes, by etiologic heterogeneity, and by uncertainty about the mode of genetic transmission of the disease trait. With neuropsychiatric disorders there is even greater ambiguity in distinguishing individuals who likely carry an affected genotype from those who are genetically unaffected. For example, one can define an affected phenotype for BAD by including one or more of the broad grouping of diagnostic classifications that constitute the mood disorders: BP-I, SAD-M, MDD, and bipolar affective (mood) disorder with hypomania and major depression (BP-II) .
  • neuropsychiatric disorder phenotypes do not exhibit classic Mendelian recessive or dominant inheritance patterns attributable to a single genetic locus, (2) there may be incomplete penetrance, i.e., individuals who inherit a predisposing allele may not manifest disease; (3) a phenocopy phenomenon may occur, i .e . , individuals who do not inherit a predisposing allele may nevertheless develop disease due to environmental or random causes; (4) genetic heterogeneity may exist, in which case mutations in any one of several genes may result in identical phenotypes.
  • the human cellular yes gene, yesl was initially identified as a homology of r-yes , the oncogene of avian sarcoma virus Y73.
  • the product of the r-yes gene is associated with tyrosine-specific protein kinase activity and its amino acid sequence shows a high degree of homology with that of that src gene product of Rous sarcoma virus.
  • the src family of protein tyrosine kinases functionally interacts with several receptor and non-receptor protein tyrosine kinases and plays an important role in signal transduction pathways involved in all development.
  • the unique N-terminal regions of the src-family tyrosine kinases may be involved in their interacting with distinct cellular proteins, such as membrane receptors, unique effectors or specific substrates.
  • yeast artificial chromosomes containing the yesl gene as probes and flourescence in situ hybridization
  • Silverman et al. (1993) detected a strong signal in the region corresponding to the chromosomal 18pll.3 location.
  • the present inventors have found that abnormal levels of expression, or expression of mutant forms, of the yesl gene is associated with neuropsychiatric disorders. This association identifies an activity for yesl different than that previously suggested in the art.
  • the present invention relates, first, to the identification of the association of the mammalian yesl gene with neuropsychiatric disorders in humans, e.g. , bipolar affective disorder.
  • the invention further relates to methods for the treatment of yesl mediated neuropsychiatric disorders, wherein such methods comprise administering a compound which modulates the expression of a mammalian yesl gene and/or the synthesis or activity of a mammalian yesl gene product so symptoms of the disorder are ameliorated.
  • the invention further relates to methods for the treatment of mammalian yesl mediated neuropsychiatric disorders resulting from yesl gene mutations, wherein such methods comprise supplying the mammal with a nucleic acid molecule encoding an unimpaired yesl gene product such that an unimpaired yesl gene product is expressed and symptoms of the disorder are ameliorated.
  • the invention further relates to methods for the treatment of mammalian yesl mediated neuropsychiatric disorders resulting from yesl gene mutations, wherein such methods comprise supplying the mammal with a cell comprising a nucleic acid molecule that encodes an unimpaired yesl gene product such that the cell expresses the unimpaired yesl gene product and symptoms of the disorder are ameliorated.
  • the present invention is directed to methods that utilize the yesl gene and/or gene product sequences for the diagnostic evaluation, genetic testing and prognosis of an yesl mediated neuropsychiatric disorder.
  • the invention relates to methods for diagnosing yesl mediated neuropsychiatric disorders, wherein such methods comprise measuring yesl gene expression in a patient sample, or detecting an yesl mutation in the genome of the mammal suspected of exhibiting such a disorder.
  • the invention still further relates to methods for identifying compounds capable of modulating the expression of the mammalian yesl gene and/or the synthesis or activity of the mammalian yesl gene products, wherein such methods comprise contacting a compound to a cell that expresses an yesl gene, measuring the level of yesl gene expression, gene product expression or gene product activity, and comparing this level to the level of yesl gene expression, gene product expression or gene product activity produced by the cell in the absence of the compound, such that if the level obtained in the presence of the compound differs from that obtained in its absence, a compound capable of modulating the expression of the mammalian yesl gene and/or the synthesis or activity of the mammalian yesl gene products has been identified.
  • yesl gene and/or gene products can also be utilized as markers for fine structure mapping of the region of the short arm of human chromosome 18 between the telomere and D185481. Further, several polymorphic markers on the yesl gene can be used for genotyping analysis.
  • the yesl mediated neuropsychiatric disorders referred to herein include, but are not limited to, bipolar affective disorder, e .g. , severe bipolar affective (mood) disorder (BP- I) , bipolar affective (mood) disorder with hypomania and major depression (BP-II) .
  • yesl mediated neuropsychiatric disorder refers to a disorder involving an aberrant level of yesl gene expression, gene product synthesis and/or gene product activity relative to levels found in normal, unaffected, unimpaired individuals, levels found in clinically normal individuals, and/or levels found in a population whose level represents a baseline, average yesl level.
  • BP-II bipolar affective (mood) disorder with 10 hypomania and major depression bp, base pair(s)
  • STS short tag sequence 20.
  • YAC yeast artificial chromosome
  • FIG. 1 Sequence of the human yesl cDNA. Amino acid sequences are indicated. 25 FIG. 2. Genomic Sequence of the human yesl gene. Exons are in bold and the 3' UTR is underlined and bold.
  • ABD human bipolar affective disorder
  • the invention described in the subsections below encompasses screening methods (e.g.. assays) for the identification of compounds which can be used to treat individuals suffering from an yesl mediated neuropsychiatric disorder.
  • the invention also encompasses agonists and antagonists of the yesl gene product, including small molecules, large molecules, and antibodies, as well as nucleotide sequences that can be used to inhibit yesl gene expression (e.g. , antisense and ribozyme molecules) , and gene or regulatory sequence replacement constructs designed to enhance yesl gene expression (e.g. , expression constructs that place the yesl gene under the control of a strong promoter system) .
  • cellular and non-cellular assays done in combination with animal and human clinical studies, are described that can be used to identify compounds that interact with the yesl gene product, e.g. , modulate the activity of the yesl and/or bind to the yesl gene product for use is treating neuropsychiatric disorders associated with abnormal yesl activity.
  • Such assays of the invention preferably utilize cells, cell lines, or engineered cells or cell lines that express the yesl gene product.
  • the invention also encompasses the use of cell-based assays or cell-lysate assays (e.g.. in vitro transcription or translation assays) to screen for compounds or compositions that modulate yesl gene expression.
  • constructs containing a reporter sequence linked to a regulatory element of the yesl gene sequences found 5' to exon-1 shown in
  • Figure 2 can be used in engineered cells, or in cell lysate extracts, to screen for compounds that modulate the expression of the reporter gene product at the level of transcription.
  • assays could be used to identify compounds that modulate the expression or activity of transcription factors involved in yesl gene expression, or to test the activity of triple helix polynucleotides.
  • engineered cells or translation extracts can be used to screen for compounds (including antisense and ribozyme constructs) that modulate the translation of yesl mRNA transcripts, and therefore, affect expression of the yesl gene product.
  • the invention also encompasses yesl gene products, polypeptides (including soluble yesl polypeptides or peptides) and yesl fusion proteins for use in non-cell based screening assays, for use in generating antibodies, for diagnostics and therapeutics.
  • Such peptides or polypeptides can be fused to a heterologous protein, e.g.. reporter, an Ig Fc region, etc., to yield a fusion protein.
  • peptides, polypeptides and fusion proteins can be used in the non-cell based assays for screening compounds that interact with, e.g. , modulate the activity of the yesl gene product and/or bind to the yesl gene product.
  • yesl proteins can be used to treat neuropsychiatric disorders.
  • Such yesl gene products include but are not limited to soluble derivatives such as peptides or polypeptides corresponding to one or more domains of the yesl gene product.
  • antibodies to the yesl protein or anti-idiotypic antibodies that mimic the yesl gene product (including Fab fragments) , antagonists or agonists can be used to treat neuropsychiatric disorders involving yesl.
  • nucleotide constructs encoding such yesl gene products can be used to genetically engineer host cells to express such yesl gene products in vivo; these genetically engineered cells can function as "biorea ⁇ tors" in the body delivering a continuous supply of yesl gene product, yesl peptides, soluble yesl polypeptides.
  • this invention presents methods for the diagnostic evaluation and prognosis of neuropsychiatric disorders, including BAD.
  • nucleic acid molecules encoding yesl can be used as diagnostic hybridization probes or as primers for diagnostic PCR analysis for the identification of yesl gene mutations, allelic variations and regulatory defects in the yesl gene.
  • polymorphisms found in coding and non-coding regions of yesl can be used as genotypic markers, particularly in assessing a risk for BAD.
  • Gene therapy approaches for the modulation of yesl gene expression and/or activity in the treatment of neuropsychiatric disorders are within the scope of the invention.
  • nucleotide constructs encoding functional yesl gene, mutant yesl gene, as well as antisense and ribozyme molecules can be used to modulate yesl gene expression.
  • the invention also encompasses pharmaceutical formulations and methods for treating neuropsychiatric disorders involving the yesl gene.
  • the present invention presents methods for selecting an effective drug to administer to an individual having an yesl medicated disorder. Such methods are based on the detection of genetic polymorphisms in the yesl gene or variations in yesl gene expression due to altered methylation, differential spinning, or post-transductional modification of the yesl gene product which can affect the safety and efficacy of a therapeutic agent.
  • BAC bacterial artificial chromosome
  • sheared libraries can be made from BAC54. Fragments of a convenient size, e .g. , in the size range of approximately 1 kb, are cloned into a standard plasmid, and sequenced. Further yesl sequences can then readily be identified by alignment of the BAC sequences with the yesl sequences depicted in Figure 1. Alternatively, BAC subclones containing additional yesl sequences can be identified by identifying those subclones which hybridize to probes derived from the yesl sequences depicted in Figure 1.
  • the isolated yesl gene sequences disclosed herein may be labeled and used to screen a cDNA library constructed from mRNA obtained from appropriate cells or tissues (e.g., brain tissues) derived from the organism (e.g., mouse) of interest.
  • the hybridization conditions used should be of a lower stringency when the cDNA library is derived from an organism different from the type of organism from which the labeled sequence was derived.
  • the labeled fragment may be used to screen a genomic library derived from the organism of interest, again, using appropriately stringent conditions.
  • Low stringency conditions are well known to those of skill in the art, and will vary predictably depending on the specific organisms from which the library and the labeled sequences are derived. For guidance regarding such conditions see, for example, Sambrook, et al . , 1989, Molecular Cloning, A Laboratory Manual, Second Edition, Cold Spring Harbor Press, N.Y.; and Ausubel, et al . , 1989, Current Protocols in Molecular Biology, Green Publishing Associates and Wiley Interscience, N.Y.
  • an ye ⁇ rl gene allelic variant may be isolated from, for example, human nucleic acid, by performing PCR using two degenerate oligonucleotide primer pools designed on the basis of amino acid sequences within the yesl gene product disclosed herein.
  • the template for the reaction may be cDNA obtained by reverse transcription of mRNA prepared from, for example, human or non-human cell lines or tissue known or suspected to express an yesl gene allele (such as human leukemia cell lines e.g., K562 B1A, H630 and H630-1, e.g.
  • allelic variant will be isolated from an individual who has an yesl mediated neuropsychiatric disorder.
  • One such variant is described in the examples below.
  • the PCR product may be subcloned and sequenced to ensure that the amplified sequences represent the sequences of an yesl gene nucleic acid sequence.
  • the PCR fragment may then be used to isolate a full length cDNA clone by a variety of methods.
  • the amplified fragment may be labeled and used to screen a bacteriophage cDNA library.
  • the labeled fragment may be used to isolate genomic clones via the screening of a genomic library.
  • PCR technology may also be utilized to isolate full length cDNA sequences.
  • RNA may be isolated, following standard procedures, from an appropriate cellular or tissue source (i.e., one known, or suspected, to express the yesl gene, such as, for example, brain tissue samples obtained through biopsy or post-mortem) .
  • a reverse transcription reaction may be performed on the RNA using an oligonucleotide primer specific for the most 5' end of the amplified fragment for the priming of first strand synthesis.
  • the resulting RNA/DNA hybrid may then be "tailed" with guanines using a standard terminal transferase reaction, the hybrid may be digested with RNAase H, and second strand synthesis may then be primed with a poly-C primer.
  • cDNA sequences upstream of the amplified fragment may easily be isolated.
  • the yesl gene sequences may be used to isolate mutant yesl gene alleles, preferably from a human subject. Such mutant alleles may be isolated from individuals either known or proposed to have a genotype that contributes to the symptoms of an yesl mediated neuropsychiatric disorder, such as BAD. Mutant alleles and mutant allele products may then be utilized in the therapeutic and diagnostic systems described below. Additionally, such yesl gene sequences can be used to detect yesl gene regulatory (e.g., promoter) defects which can be associated with an yesl mediated neuropsychiatric disorder, a neuropsychiatric disorder such as BAD.
  • yesl gene regulatory e.g., promoter
  • a cDNA of a mutant allelic variant of the yesl gene may be isolated, for example, by using PCR, a technique that is well known to those of skill in the art.
  • the first cDNA strand may be synthesized by hybridizing an oligo- dT oligonucleotide to mRNA isolated from tissue known or suspected to be expressed in an individual putatively carrying the mutant yesl allele, and by extending the new strand with reverse transcriptase.
  • the second strand of the cDNA is then synthesized using an oligonucleotide that hybridizes specifically to the 5' end of the normal gene.
  • the product is then amplified via PCR, cloned into a suitable vector, and subjected to DNA sequence analysis through methods well known to those of skill in the art.
  • DNA sequence analysis By comparing the DNA sequence of the mutant yesl allele to that of the normal yesl allele, the mutation (s) responsible for the loss or alteration of function of the mutant yesl gene product can be ascertained.
  • a genomic library can be constructed using DNA obtained from an individual suspected of or known to carry a mutant yesl allele, or a cDNA library can be constructed using RNA from, a tissue known, or suspected, to express a mutant yesl allele.
  • An unimpaired yesl gene or any suitable fragment thereof may then be labeled and used as a probe to identify the corresponding mutant yesl allele in such libraries.
  • Clones containing the mutant yesl gene sequences may then be purified and subjected to sequence analysis according to methods well known to those of skill in the art.
  • an expression library can be constructed utilizing cDNA synthesized from, for example, RNA isolated from a tissue known, or suspected, to express a mutant yesl allele in an individual suspected of or known--to carry such a mutant allele.
  • gene products made by the putatively mutant tissue may be expressed and screened using standard antibody screening techniques in conjunction with antibodies raised against the normal yesl gene product, as described, below, in Section 5.3.
  • For screening techniques see, for example, Harlow and Lane, eds., 1988, "Antibodies: A Laboratory Manual", Cold Spring Harbor Press, Cold Spring Harbor. )
  • a polyclonal set of anti- yesl gene product antibodies are likely to cross-react with the mutant yesl gene product.
  • Library clones detected via their reaction with such labeled antibodies can be purified and subjected to sequence analysis according to methods well known to those of skill in the art. yesl mutations can further be detected using PCR amplification techniques.
  • Primers can routinely be designed to amplify overlapping regions of the whole yesl sequence including the promoter region. In one embodiment, primers are designed to cover the exon-intron boundaries such that, coding regions can be scanned for mutations (see. Figure 2) .
  • Genomic DNA isolated from lymphocytes of normal and affected individuals is used as PCR template.
  • PCR products from normal and affected individuals are compared, either by single strand conformational polymorphism (SSCP) mutation detection techniques and/or by sequencing.
  • SSCP single strand conformational polymorphism
  • PROTEIN PRODUCTS OF THE yesl GENE yesl gene products, or peptide fragments thereof can be prepared for a variety of uses.
  • gene products, or peptide fragments thereof can be used for the generation of antibodies, in diagnostic assays, or for the identification of other cellular or extracellular gene products involved in the regulation of yesl mediated neuropsychiatric disorders, such as BAD.
  • the amino acid sequence depicted in Figure l represents an yesl gene product.
  • the yesl gene product sometimes referred to herein as an "yesl protein" includes those gene products encoded by the yesl gene sequences depicted in Figure 1, as well as other human allelic variants of yesl that can be identified by the methods herein described.
  • allelic variants of yesl There are two classes of allelic variants of yesl, functional variants and altered functional variants.
  • a functional variant is one which has all the activities of wild-type yesl at levels that do not mediate a pathological condition. Functional variants will typically contain only minor variation in amino acid sequence.
  • An altered- functional variant includes variants having an alteration in one or more yesl activities, for example, a loss or increase in yesl activity.
  • An altered functional variant of yesl can either have a minor or significant change in amino acid sequence when compared to wild-type yesl.
  • yesl gene products may include proteins that represent functionally equivalent gene products.
  • Such an equivalent yesl gene product may contain deletions, including internal deletions, additions, including additions yielding fusion proteins, or substitutions of amino acid residues within and/or adjacent to the amino acid sequence encoded by the yesl gene sequences described, above, in Section 5.1, but that result in a "silent" change, in that the change produces a functionally equivalent yesl gene product.
  • Amino acid substitutions may be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues involved.
  • nonpolar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, and methionine; polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine; positively charged (basic) amino acids include arginine, lysine, and histidine; and negatively charged (acidic) amino acids include aspartic acid and glutamic acid.
  • deletion or non-conservative alterations can be engineered to produce altered, including reduced yesl gene products.
  • Such alterations can, for example, alter one or more of the biological functions of the yesl gene product. Further, such alterations can be selected so as to generate yesl gene products that are better suited for expression, scale up, etc. in the host cells chosen. For example, cysteine residues can be deleted or substituted with another amino acid residue in order to eliminate disulfide bridges.
  • the yesl gene products, peptide fragments thereof and fusion proteins thereof may be produced by recombinant DNA technology using techniques well known in the art.
  • methods for preparing the yesl gene products, polypeptides, peptides, fusion peptide and fusion polypeptides of the invention by expressing nucleic acid containing yesl gene sequences are described herein.
  • Methods that are well known to those skilled in the art can be used to construct expression vectors containing yesl gene product coding sequences and appropriate transcriptional and translational control signals. These methods include, for example, in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. See, for example, the techniques described in Sambrook, et al .
  • RNA capable of encoding yesl gene product sequences may be chemically synthesized using, for example, synthesizers. See, for example, the techniques described in "Oligonucleotide Synthesis", 1984, Gait, ed., IRL Press, Oxford.
  • a variety of host-expression vector systems may be utilized to express the yesl gene product coding sequences of the invention. Such host-expression systems represent vehicles by which the coding sequences of interest may be produced and subsequently purified, but also represent cells that may, when transformed or transfected with the appropriate nucleotide coding sequences, exhibit the yesl gene product of the invention in situ .
  • microorganisms such as bacteria (e.g., E. coli , B . subtilis) transformed with recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vectors containing yesl gene product coding sequences; yeast (e .g. , 5 Saccharomyces , Pichia) transformed with recombinant yeast expression vectors containing the yesl gene product coding sequences; insect cell systems infected with recombinant virus expression vectors (e.g., baculovirus) containing the yesl gene product coding sequences; plant cell systems
  • bacteria e.g., E. coli , B . subtilis
  • yeast e.g. , 5 Saccharomyces , Pichia
  • insect cell systems infected with recombinant virus expression vectors e.g., baculovirus
  • virus expression vectors e.g. , cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV
  • plasmid expression vectors e.g. , Ti plasmid
  • mammalian cell systems e.g., COS, CHO, BHK
  • promoters derived from the genome of mammalian cells (e.g., metallothionein promoter) or from mammalian viruses (e.g., the adenovirus late promoter; the vaccinia virus 7.5K promoter).
  • mammalian cells e.g., metallothionein promoter
  • mammalian viruses e.g., the adenovirus late promoter; the vaccinia virus 7.5K promoter.
  • a number of expression vectors may be advantageously selected depending upon the use intended for the yesl gene product being expressed. For example, when a large quantity of such a protein is to be produced, for the generation of pharmaceutical compositions of yesl gene
  • vectors that direct the expression of high levels of fusion protein products that are readily purified may be desirable.
  • vectors include, but are not limited, to the E. coli expression vector.
  • pUR278 (Ruther et al . , 1983, EMBO
  • pGEX vectors may also be used to express foreign polypeptides as fusion proteins with gluta- thione S-transferase (GST) .
  • GST gluta- thione 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.
  • the pGEX vectors are designed to include thrombin or factor Xa protease cleavage sites so that the cloned target gene product can be released from the GST moiety.
  • AcNPV nuclear polyhedrosis virus
  • the virus grows in Spodoptera frugiperda cells.
  • the yesl gene product coding sequence may be cloned individually into non-essential regions (for example the polyhedrin gene) of the virus and placed under control of an AcNPV promoter (for example the polyhedrin promoter) .
  • Successful insertion of yesl gene product coding sequence will result in inactivation of the polyhedrin gene and production of non-occluded recombinant virus (i.e., virus lacking the proteinaceous coat coded for by the polyhedrin gene) .
  • a number of viral-based expression systems may be utilized.
  • the yesl gene product coding sequence of interest may be ligated to an adenovirus transcription/translation control complex, e.g., the late promoter and tripartite leader sequence.
  • This chimeric gene may then be inserted in the adenovirus genome by in vitro or in vivo recombination. Insertion in a non- essential region of the viral genome (e.g., region El or E3) will result in a recombinant virus that is viable and capable of expressing yesl gene product in infected hosts. (e.g.. See Logan and Shenk, 1984, Proc. Natl.
  • Specific initiation signals may also be required for efficient translation of inserted yesl gene product coding sequences. These signals include the ATG initiation codon and adjacent sequences. In cases where an entire yesl gene, including its own initiation codon and adjacent sequences, is inserted into the appropriate expression vector, no additional translational control signals may be needed. However, in cases where only a portion of the yesl gene coding sequence is inserted, exogenous translational control signals, including, perhaps, the ATG initiation codon, must be provided. Furthermore, the initiation codon must be in phase with the reading frame of the desired coding sequence to ensure translation of the entire insert.
  • exogenous translational control signals and initiation codons can be of a variety of origins, both natural and synthetic.
  • the efficiency of expression may be enhanced by the inclusion of appropriate transcription enhancer elements, transcription terminators, etc. (see Bittner, et al . , 1987, Methods in Enzymol. 153, 516-544).
  • a host cell strain may be chosen that modulates the expression of the inserted sequences, or modifies and processes the gene product in the specific fashion desired. Such modifications (e.g., gly ⁇ osylation) and processing (e.g., cleavage) of protein products may be important for the function of the protein.
  • Different host cells have characteristic and specific mechanisms for the post-translational processing and modification of proteins and gene products. Appropriate cell lines or host systems can be chosen to ensure the correct modification and processing of the foreign protein expressed.
  • eukaryotic host cells that possess the cellular machinery for proper processing of the primary transcript, glycosylation, and phosphorylation of the gene product may be used.
  • mammalian host cells include but are not limited to CHO, VERO, BHK, HeLa, COS, MDCK, 293, 3T3 , and WI38.
  • cell lines that stably express the yesl gene product may be engineered.
  • host cells can be transformed with DNA controlled by appropriate expression control elements (e.g., promoter, enhancer, sequences, transcription terminators, polyadenylation sites, etc.), and a selectable marker.
  • appropriate expression control elements e.g., promoter, enhancer, sequences, transcription terminators, polyadenylation sites, etc.
  • engineered cells may be allowed to grow for 1-2 days in an enriched media, and then are switched to a selective media.
  • the selectable marker in the recombinant plasmid confers resistance to the selection and allows cells to stably integrate the plasmid into their chromosomes and grow to form foci that in turn can be cloned and expanded into cell lines.
  • This method may advantageously be used to engineer cell lines that express the yesl gene product.
  • Such engineered cell lines may be particularly useful in screening and evaluation of compounds that affect the endogenous activity of the yesl gene product.
  • a number of selection systems may be used, including but not limited to the herpes simplex virus thymidine kinase (Wigler, et al .
  • genes can be employed in tk " , hgprt” or aprt " cells, respectively.
  • antimetabolite resistance can be used as the basis of selection for the following genes: dhfr, which confers resistance to methotrexate (Wigler, et al . , 1980, Natl. Acad. Sci.
  • any fusion protein may be readily purified by utilizing an antibody specific for the fusion protein being expressed.
  • a system described by Janknecht, et al allows for the ready purification of non- denatured fusion proteins expressed in human cell lines (Janknecht, et ai., 1991, Proc. Natl. Acad. Sci. USA 88, 8972-8976) .
  • the gene of interest is subcloned into a vaccinia recombination plasmid such that the gene's open reading frame is translationally fused to an amino-terminal tag consisting of six histidine residues.
  • Extracts from cells infected with recombinant vaccinia virus are loaded onto Ni 2+ -nitriloacetic acid-agarose columns and histidine-tagged proteins are selectively eluted with imidazole-containing buffers.
  • the yesl gene products can also be expressed in
  • transgenic animals Animals of any species, including, but not limited to, mice, rats, rabbits, guinea pigs, pigs, micro-pigs, goats, sheep, and non-human primates, e.g., baboons, monkeys, and chimpanzees may be used to generate yesl transgenic animals.
  • 15 herein refers to animals expressing yesl gene sequences from a different species (e .g . , mice expressing human yesl gene sequences) , as well as animals that have been genetically engineered to overexpress endogenous (i .e . , same species) yesl sequences or animals that have been genetically
  • Any technique known in the art may be used to introduce an yesl gene transgene into animals to produce the founder lines of transgenic animals. Such techniques include, but
  • transgenic animal clones containing an yesl transgene for example, nuclear transfer into enucleated oocytes of nuclei from cultured embryonic, fetal or adult cells induced to quiescence (Campbell, et al . , 1996, Nature 380, 64-66; Wilmut, et al . , Nature 385, 810-813).
  • the present invention provides for transgenic animals that carry an yesl transgene in all their cells, as well as animals that carry the transgene in some, but not all their cells, i.e., mosaic animals.
  • the transgene may be integrated as a single transgene or in concatamers, e .g. , head-to-head tandems or head-to-tail tandems.
  • the transgene may also be selectively introduced into and activated in a particular cell type by following, for example, the teaching of Lasko et al . (Lasko, et al . , 1992, Proc. Natl. Acad. Sci. USA 89, 6232-6236) .
  • the regulatory sequences required for such a cell-type specific activation will, depend upon the particular cell type of interest, and will be apparent to those of skill in the art.
  • gene targeting is preferred.
  • vectors containing some nucleotide sequences homologous to the endogenous yesl gene are designed for the purpose of integrating, via homologous recombination with chromosomal sequences, into and disrupting the function of the nucleotide sequence of the endogenous yesl gene.
  • the transgene may also be selectively introduced into a particular cell type, thus inactivating the endogenous yesl gene in only that cell type, by following, for example, the teaching of Gu, et al . (Gu, et al . , 1994, Science 265, 103-106) .
  • the regulatory sequences required for such a cell- type specific inactivation will depend upon the particular cell type of interest, and will be apparent to those of skill in the art.
  • the expression of the recombinant yesl gene may be assayed utilizing standard techniques. Initial screening may be accomplished by Southern blot analysis or PCR techniques to analyze animal tissues to assay whether integration of the transgene has taken place. The level of mRNA expression of the transgene in the tissues of the transgenic animals may also be assessed using techniques that include but are not limited to Northern blot analysis of tissue samples obtained from the animal, in situ hybridization analysis, and RT-PCR (reverse transcriptase PCR) . Samples of yesl gene-expressing tissue, may also be evaluated immunocytochemically using antibodies specific for the yesl transgene product.
  • ANTIBODIES TO vesl GENE PRODUCTS Described herein are methods for the production of antibodies capable of specifically recognizing one or more yesl gene product epitopes, or epitopes of functional or altered functional variants, or peptide fragments of the yesl gene products. Further, antibodies that specifically recognize mutant forms of yesl, are encompassed by the invention.
  • Such antibodies may include, but are not limited to, polyclonal antibodies, monoclonal antibodies (mAbs) , humanized or chimeric antibodies, single chain antibodies, Fab fragments, F(ab') 2 fragments, fragments produced by a Fab expression library, anti-idiotypic (anti-Id) antibodies, and epitope-binding fragments of any of the above.
  • mAbs monoclonal antibodies
  • Such antibodies may be used, for example, in the detection of an yesl gene product in an biological sample and may, therefore, be utilized as part of a diagnostic or prognostic technique whereby patients may be tested for abnormal levels of yesl gene products, and/or for the presence of abnormal forms of such gene products.
  • Such antibodies may also be utilized in conjunction with, for example, compound screening schemes, as described, below, in Section 5.8, for the evaluation of the effect of test compounds on yesl gene product levels and/or activity. Additionally, such antibodies can be used in conjunction with the gene therapy techniques described, below, in Section 5.9.2 to, for example, evaluate the normal and/or engineered yesl-expressing cells prior to their introduction into the patient. Anti-yesl gene product antibodies may additionally be used in methods for inhibiting abnormal yesl gene product activity. Thus, such antibodies may, therefore, be utilized as part of treatment methods for an yesl ⁇ mediated neuropsychiatric disorder, such as BAD.
  • an yesl ⁇ mediated neuropsychiatric disorder such as BAD.
  • various host animals may be immunized by injection with an yesl gene product, or a portion thereof.
  • Such host animals may include, but are not limited to rabbits, mice, and rats, to name but a few.
  • Various adjuvants may be used to increase the immunological response, depending on the host species, including but not limited to Freund's (complete and incomplete) , mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, dinitrophenol, and potentially useful human adjuvants such as BCG (bacille Calmette-Guerin) and Corynebacterium parvum.
  • BCG Bacille Calmette-Guerin
  • Polyclonal antibodies are heterogeneous populations of antibody molecules derived from the sera of animals immunized with an antigen, such as an yesl gene product, or an antigenic functional derivative thereof.
  • an antigen such as an yesl gene product, or an antigenic functional derivative thereof.
  • host animals such as those described above, may be immunized by injection with yesl gene product supplemented with adjuvants as also described above.
  • Monoclonal antibodies, which are homogeneous populations of antibodies to a particular antigen may be obtained by any technique that provides for the production of antibody molecules by continuous cell lines in culture. These include, but are not limited to, the hybridoma technique of Kohler and Milstein, (1975, Nature 256, 495-497; and U.S. Patent No.
  • Such antibodies may be of any immunoglobulin class including IgG, IgM, IgE, IgA, IgD and any subclass thereof.
  • the hybridoma producing the mAb of this invention may be cultivated in vitro or in vivo.
  • a chimeric antibody is a molecule in which different portions are derived from different animal species, such as those having a variable region derived from a murine mAb and a human immunoglobulin constant region. (See, e.g., Cabilly et al., U.S. Patent No. 4,816,567; and Boss et al. , U.S. Patent No. 4,816397, which are incorporated herein by reference in their entirety.)
  • An immunoglobulin light or heavy chain variable region consists of a "framework" region interrupted by three hypervariable regions, referred to as comple entarily determining regions (CDRs) .
  • CDRs comple entarily determining regions
  • the extent of the framework region and CDRs have been precisely defined (see, "Sequences of Proteins of Immunological Interest", Kabat, E. et al., U.S.Department of Health and Human Services (1983) .
  • humanized antibodies are antibody molecules from non-human species having one or more CDRs from the non- human species and a framework region from a human immunoglobulin molecule.
  • Single chain antibodies are formed by linking the heavy and light chain fragments of the Fv region via an amino acid bridge, resulting in a single chain polypeptide.
  • Antibody fragments that recognize specific epitopes may be generated by known techniques. For example, such fragments include but are not limited to: the F(ab') 2 fragments, which can be produced by pepsin digestion of the antibody molecule and the Fab fragments, which can be
  • Fab expression libraries may be constructed (Huse, et al . , 1989, Science, 246, 1275-1281) to allow rapid and easy identification of monoclonal Fab fragments with the desired specificity.
  • Such applications include, for example, prognostic and diagnostic evaluation of an yesl mediated neuropsychiatric disorder, such as BAD, and the identification of subjects
  • Such applications include methods for the treatment of an yesl mediated neuropsychiatric disorder, such as BAD as described, below, in Section 5.9, and for the identification of compounds that
  • Such compounds can include, for example, other cellular products that are involved in mood regulation and in yesl mediated neuropsychiatric disorders, such as BAD.
  • 3 compounds can be used, for example, in the amelioration of yesl mediated neuropsychiatric disorders, such as BAD. 5.5. DIAGNOSIS OF ABNORMALITIES OF AN yesl, MEDIATED NEUROPSYCHIATRIC DISORDER
  • a variety of methods can be employed for the diagnostic and prognostic evaluation of yesl mediated neuropsychiatric _ disorders and for the identification of subjects having a predisposition to such disorders.
  • Such methods may, for example, utilize reagents such as the yesl gene nucleotide sequences described in Sections 5.1, or antibodies directed against yesl gene products, including peptide fragments thereof, as described, above, in Section 5.3.
  • reagents such as the yesl gene nucleotide sequences described in Sections 5.1, or antibodies directed against yesl gene products, including peptide fragments thereof, as described, above, in Section 5.3.
  • reagents may be used, for example, for:
  • yesl gene nucleotide sequences can, for example, be used to diagnose an yesl mediated neuropsychiatric disorder using, for example, the techniques for yesl mutation detection described above in Section 5.1.
  • Mutations at a number of different genetic loci may lead to phenotypes related to neuropsychiatric disorders.
  • the treatment of patients suffering from such neuropsychiatric disorder will be designed to target the particular genetic loci containing the mutation mediating the disorder.
  • Genetic polymorphisms have been linked to differences in drug effectiveness. Thus, identification of alterations in the yesl gene or protein can be utilized to optimize therapeutic drug treatments.
  • polymorphisms in the yesl gene sequence may be utilized to identify an individual having, or at risk in developing, a disease or condition resulting from an yesl mediated disorder and thus define the most effective and safest drug treatment.
  • Assays such as those described herein may be used to identify such polymorphisms or variations in yesl gene expression activity.
  • the methods described herein may be performed, for example, by utilizing pre-packaged diagnostic kits comprising at least one specific yesl gene nucleic acid or anti-yesl gene product antibody reagent described herein, which may be conveniently used, e .g. , in clinical settings, to diagnose patients exhibiting abnormalities of an yesl mediated neuropsychiatric disorder, such as BAD.
  • any nucleated cell can be used as a starting source for genomic nucleic acid.
  • any cell type or tissue in which the yesl gene is expressed may be utilized.
  • Nucleic acid-based detection techniques are described, below, in Section 5.6.
  • Peptide detection techniques are described, below, in Section 5.7.
  • a variety of methods can be employed to screen for the presence of yesl gene-specific mutations and to detect and/or assay levels of yesl nucleic acid sequences. Mutations within the yesl gene can be detected by utilizing a number of techniques. Nucleic acid from any nucleated cell can be used as the starting point for such assay techniques, and may be isolated according to standard nucleic acid preparation procedures that are well known to those of skill in the art. yesl nucleic acid sequences may be used in hybridization or amplification assays of biological samples to detect abnormalities involving yesl gene structure, including point mutations, insertions, deletions, inversions, translocations and chromosomal rearrangements. Such assays may include, but are not limited to, Southern analyses, single-stranded conformational polymorphism analyses (SSCP) , and PCR analyses.
  • SSCP single-stranded conformational polymorphism analyses
  • Diagnostic methods for the detection of yesl gene- specific mutations can involve for example, contacting and incubating nucleic acids including recombinant DNA molecules, cloned genes or degenerate variants thereof, obtained from a sample, e.g., derived from a patient sample or other appropriate cellular source, with one or more labeled nucleic acid reagents including recombinant DNA molecules, cloned genes or degenerate variants thereof, as described in Section 5.1, under conditions favorable for the specific annealing of these reagents to their complementary sequences within the yesl gene.
  • the diagnostic methods of the present invention further encompass contacting and incubating nucleic acids for the detection of single nucleotide mutations or polymorphisms of the yesl gene.
  • the lengths of these nucleic acid reagents are at least 15 to 30 nucleotides.
  • all non-annealed nucleic acids are removed from the nucleic acid: yesl molecule hybrid.
  • the presence of nucleic acids that have hybridized, if any such molecules exist, is then detected.
  • the nucleic acid from the cell type or tissue of interest can be immobilized, for example, to a solid support such as a membrane, or a plastic surface such as that on a microtiter plate or polystyrene beads.
  • nucleic acid reagents of the type described in Section 5.1 are easily removed. Detection of the remaining, annealed, labeled yesl nucleic acid reagents is accomplished using standard techniques well-known to those in the art.
  • the yesl gene sequences to which the nucleic acid reagents have annealed can be compared to the annealing pattern expected from a normal yesl gene sequence in order to determine whether an yesl gene mutation is present.
  • yesl mutations or polymorphisms can be detected by using a microassay of yesl nucleic acid sequences immobilized to a substrate or "gene chip" (see, e.g. Cronin, et al., 1996, Human Mutation 7:244- 255) .
  • Alternative diagnostic methods for the detection of yesl gene specific nucleic acid molecules, in patient samples or other appropriate cell sources may involve their amplification, e.g., by PCR (the experimental embodiment set forth in Mullis, 1987, U.S. Patent No. 4,683,202), followed by the detection of the amplified molecules using techniques well known to those of skill in the art.
  • the resulting amplified sequences can be compared to those that would be expected if the nucleic acid being amplified contained only normal copies of the yesl gene in order to determine whether an yesl gene mutation exists.
  • genotyping techniques can be performed to identify individuals carrying yesl gene mutations. Such techniques include, for example, the use of restriction fragment length polymorphisms (RFLPs) , which involve sequence variations in one of the recognition sites for the specific restriction enzyme used.
  • RFLPs restriction fragment length polymorphisms
  • Caskey et al . U.S. Pat.No. 5,364,759 describe a DNA profiling assay for detecting short tri and tetra nucleotide repeat sequences. The process includes extracting the DNA of interest, such as the yesl gene, amplifying the extracted DNA, and labelling the repeat sequences to form a genotypic map of the individual's DNA.
  • the level of yesl gene expression can also be assayed.
  • RNA from a cell type or tissue known, or suspected, to express the yesl gene, such as brain may be isolated and tested utilizing hybridization or PCR techniques such as are described, above.
  • the isolated cells can be derived from cell culture or from a patient.
  • the analysis of cells taken from culture may be a necessary step in the assessment of cells to be used as part of a cell-based gene therapy technique or, alternatively, to test the effect of compounds on the expression of the yesl gene.
  • Such analyses may reveal both quantitative and qualitative aspects of the expression pattern of the yesl gene, including activation or inactivation of yesl gene expression.
  • a cDNA molecule is synthesized from an RNA molecule of interest (e .g. , by reverse transcription of the RNA molecule into cDNA) .
  • a sequence within the cDNA is then used as the template for a nucleic acid amplification reaction, such as a PCR amplification reaction, or the like.
  • the nucleic acid reagents used as synthesis initiation reagents (e .g. , primers) in the reverse transcription and nucleic acid amplification steps of this method are chosen from among the yesl gene nucleic acid reagents described in Section 5.1.
  • the preferred lengths of such nucleic acid reagents are at least 9-30 nucleotides.
  • the nucleic acid amplification may be performed using radioactively or non-radioactively labeled nucleotides.
  • enough amplified product may be made such that the product may be visualized by standard ethidium bromide staining or by utilizing any other suitable nucleic acid staining method.
  • Nucleic acid reagents such as those described in Section 5.1 may be used as probes and/or primers for such in situ procedures (see, for example, Nuovo, G.J., 1992, “PCR In Situ Hybridization: Protocols And Applications", Raven Press, NY) .
  • standard Northern analysis can be performed to determine the level of mRNA expression of the yesl gene.
  • Antibodies directed against wild-type or mutant yesl gene products or conserved variants or peptide fragments thereof, which are discussed, above, in Section 5.3, may also be used as diagnostics and prognostics for an yesl mediated neuropsychiatric disorder, such as BAD. Such methods may be used to detect abnormalities in the level of yesl gene product synthesis or expression, or abnormalities in the structure, temporal expression, and/or physical location of yesl gene product.
  • the antibodies and immunoassay methods described herein have, for example, important in vitro applications in assessing the efficacy of treatments for yesl mediated neuropsychiatric disorders, such as BAD.
  • Antibodies, or fragments of antibodies, such as those described below, may be used to screen potentially therapeutic compounds in vitro to determine their effects on yesl gene expression and yesl gene product production, particularly to identify compounds that have beneficial effects on an yesl mediated neuropsychiatric disorder, such as BAD.
  • In vitro immunoassays may also be used, for example, to assess the efficacy of cell-based gene therapy for an yesl mediated neuropsychiatric disorder, such as BAD.
  • Antibodies directed against yesl gene products may be used in vitro to determine, for example, the level of yesl gene expression achieved in cells genetically engineered to produce yesl gene product. In the case of intracellular yesl gene products, such an assessment is done, preferably, using cell lysates or extracts.
  • tissue or cell type to be analyzed will generally include those that are known, or suspected, to express the yesl gene.
  • the protein isolation methods employed herein may, for example, be such as those described in Harlow and Lane (1988, "Antibodies: A Laboratory Manual", Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York) .
  • the isolated cells can be derived from cell culture or from a patient.
  • the analysis of cells taken from culture may be a necessary step in the assessment of cells to be used as part of a cell-based gene therapy technique or, alternatively, to test the effect of compounds on the expression of the yesl gene.
  • Preferred diagnostic methods for the detection of yesl gene products, functional and altered functional variants, or peptide fragments thereof may involve, for example, immunoassays wherein the yesl gene products or variants or peptide fragments are detected by their interaction with an anti-yesl gene product-specific antibody.
  • antibodies, or fragments of antibodies may be used to quantitatively or qualitatively detect the presence of yesl gene products or variants or peptide fragments thereof.
  • This can be accomplished, for example, by immunofluorescence techniques employing a fluores ⁇ ently labeled antibody (see below, this Section) coupled with light microscopic, flow cytometric, or fluorimetric detection. Such techniques are especially preferred for yesl gene products that are expressed on the cell surface.
  • the antibodies (or fragments thereof) useful in the present invention may, additionally, be employed histologically, as in imunofluorescence or immunoelectron microscopy, for in situ detection of yesl gene products, variants, or peptide fragments thereof.
  • In situ detection may be accomplished by removing a histological specimen from a patient, and applying thereto a labeled antibody that binds to an a yesl polypeptide.
  • the antibody (or fragment) is preferably applied by overlaying the labeled antibody (or fragment) onto a biological sample.
  • Immunoassays for yesl gene products, variants, or peptide fragments thereof will typically comprise incubating a sample, such as a biological fluid, a tissue extract, freshly harvested cells, or lysates of cells in the presence of a detectably labeled antibody capable of identifying yesl gene product, variants, or peptide fragments thereof, and detecting the bound antibody by any of a number of techniques well-known in the art.
  • the biological sample may be brought in contact with and immobilized onto a solid phase support or carrier, such as nitrocellulose, that is capable of immobilizing cells, cell particles or soluble proteins.
  • a solid phase support or carrier such as nitrocellulose, that is capable of immobilizing cells, cell particles or soluble proteins.
  • the support may then be washed with suitable buffers followed by treatment with the detectably labeled yesl gene product specific antibody.
  • the solid phase support may then be washed with the buffer a second time to remove unbound antibody.
  • the amount of bound label on the solid support may then be detected by conventional means.
  • solid phase support or carrier any support capable of binding an antigen or an antibody.
  • supports or carriers include glass, polystyrene, polypropylene, polyethylene, dextran, nylon, amylases, natural and modified celluloses, polyacrylamides, gabbros, and magnetite.
  • the nature of the carrier can be either soluble to some extent or insoluble for the purposes of the present invention.
  • the support material may have virtually any possible structural configuration so long as the coupled molecule is capable of binding to an antigen or antibody.
  • the support configuration may be spherical, as in a bead, or cylindrical, as in the inside surface of a test tube, or the external surface of a rod.
  • the surface may be flat such as a sheet, test strip, etc.
  • Preferred supports include polystyrene beads. Those skilled in the art will know many other suitable carriers for binding antibody or antigen, or will be able to ascertain the same by use of routine experimentation.
  • EIA enzyme immunoassay
  • ELISA Enzyme Linked Immunosorbent Assay
  • the enzyme which is bound to the antibody will react with an appropriate substrate, preferably a chromogenic substrate, in such a manner as to produce a chemical moiety that can be detected, for example, by spectrophotometric, fluorimetric or by visual means.
  • Enzymes that can be used to detectably label the antibody include, but are not limited to, malate dehydrogenase, staphylococcal nuclease, delta-5-steroid isomerase, yeast alcohol dehydrogenase, ⁇ -glycerophosphate, dehydrogenase, triose phosphate isomerase, horseradish peroxidase, alkaline phosphatase, asparaginase , glucose oxidase, 9-galactosidase, ribonuclease, urease, catalase, glucose-6-phosphate dehydrogenase, glucoamylase and acetylcholinesterase.
  • the detection can be accomplished by calorimetric methods that employ a chromogenic substrate for the enzyme. Detection may also be accomplished by visual comparison of the extent of enzymatic reaction of a substrate in comparison with similarly prepared standards.
  • Detection may also be accomplished using any of a variety of other immunoassays.
  • a radioimmunoassay RIA
  • the radioactive isotope can be detected by such means as the use of a gamma counter or a scintillation counter or by autoradiography.
  • the antibody can also be labeled with a fluorescent compound.
  • fluorescent labeling compounds are fluorescein isothiocyanate, rhodamine, phycoerythrin , phycocyanin, allophycocyanin, o-phthaldehyde and fluorescamine.
  • the antibody can also be detectably labeled using fluorescence emitting metals such as 152 Eu, or others of the lanthanide series. These metals can be attached to the antibody using such metal chelating groups as diethylenetriaminepentacetic acid (DTPA) or ethylenediaminetetraacetic acid (EDTA) .
  • DTPA diethylenetriaminepentacetic acid
  • EDTA ethylenediaminetetraacetic acid
  • the antibody also can be detectably labeled by coupling it to a chemiluminescent compound.
  • the presence of the chemiluminescent-tagged antibody is then determined by detecting the presence of luminescence that arises during the course of a chemical reaction.
  • chemiluminescent labeling compounds are luminol, isoluminol, theromatic acridinium ester, imidazole, acridinium salt and oxalate ester.
  • Bioluminescence is a 5 type of chemiluminescence found in biological systems in which a catalytic protein increases the efficiency of the chemiluminescent reaction. The presence of a bioluminescent protein is determined by detecting the presence of luminescence. Important bioluminescent compounds for 10 purposes of labeling are luciferin, luciferase and aequorin.
  • the following assays are designed to identify compounds ⁇ that bind to an yesl gene product, compounds that bind to intracellular proteins, or portions of proteins that interact with an yesl gene product, compounds that interfere with the interaction of an yesl gene product with intracellular proteins and compounds that, modulate the activity of the yesl
  • Assays may additionally be utilized that identify compounds that bind to yesl gene regulatory sequences (e.g., promoter sequences; see e.g., Platt, 1994, J. Biol. Chem. 269,
  • Such compounds may include, but are not limited to, small organic molecules, such as ones that are able to cross the blood-brain barrier, gain entry into an appropriate cell and affect expression of the yesl gene or some other
  • intracellular proteins may be involved in the control and/or regulation of mood.
  • compounds that affect the level of yesl gene expression and/or yesl gene product activity and that can be used in the therapeutic treatment of yesl mediated neuropsychiatric disorders such as BAD as described, below, in Section 5.9.
  • Compounds may include, but are not limited to, peptides such as, for example, soluble peptides, including but not limited to, Ig-tailed fusion peptides, and members of random peptide libraries; (see, e.g.. Lam, et al . , 1991, Nature 354, 82-84; Houghten, et al . , 1991, Nature 354, 84-86), and combinatorial chemistry-derived molecular library made of D- and/or L- configuration amino acids, phosphopeptides (including, but not limited to members of random or partially degenerate, directed phosphopeptide libraries; see, e.g., Songyang, et al .
  • peptides such as, for example, soluble peptides, including but not limited to, Ig-tailed fusion peptides, and members of random peptide libraries; (see, e.g.. Lam, et al . , 1991, Nature 354, 82-84;
  • antibodies including, but not limited to, polyclonal, monoclonal, humanized, anti-idiotypic, chimeric or single chain antibodies, and FAb, F(ab') 2 and FAb expression library fragments, and epitope-binding fragments thereof) , and small organic or inorganic molecules.
  • Such compounds may further comprise compounds, in particular drugs or members of classes or families of drugs, known to ameliorate or exacerbate the symptoms of a neuropsychiatric disorder such as BAD.
  • antidepressants such as lithium salts, carbamazepine, valproic acid, lysergic acid diethylamide (LSD) , p- chlorophenylalanine, p-propyldopacetamide dithiocarbamate derivatives e.g., FLA 63; anti-anxiety " drugs, e.g., diazepam; monoamine oxidase (MAO) inhibitors, e.g., iproniazid, clorgyline, phenelzine and isocarboxazid; biogenic amine uptake blockers, e.g., tri ⁇ yclic antidepressants such as desipramine, imipramine and amitriptyline; serotonin reuptake inhibitors e.g.,
  • chlorpromazine thorazine and trifluopromazine
  • butyrophenones e.g., haloperidol (Haldol)
  • thioxanthene derivatives e.g., chlorprothixene
  • dibenzodiazepines e.g.
  • clozapine a compound that has a therapeutically active property
  • benzodiazepines dopaminergic agonists and antagonists e.g., L-DOPA, cocaine, amphetamine, o-methyl-tyrosine, reserpine, tetrabenazine, benzotropine, pargyline
  • noradrenergic agonists and antagonists e.g., clonidine, phenoxybenzamine , phentolamine , tropolone.
  • Compounds identified via assays such as those described herein may be useful, for example, in elaborating the 5 biological function of the yesl gene product and for ameliorating yesl mediated neuropsychiatric disorders, such as BAD.
  • Assays for testing the effectiveness of compounds identified by, for example, techniques such as those described in Sections 5.8.1 - 5.8.3, are discussed, below, in 0 Section 5.8.4.
  • In vitro systems may be designed to identify compounds capable of binding the yesl gene products of the invention.
  • Such compounds can then be used in animal and human assays to test for the compounds ability to modulate the activity of the yesl gene product in vivo .
  • Compounds thus identified may be useful, for example, in modulating the activity of
  • 0 unimpaired and/or mutant yesl gene products may be useful in elaborating the biological function of the yesl gene, product, may be utilized in screens for identifying compounds that disrupt normal yesl gene product interactions, or may in themselves disrupt such interactions.
  • the principle of the assays used to identify compounds that bind to the yesl gene product involves preparing a reaction mixture of the yesl gene product and the test compound under conditions and for a time sufficient to allow the two components to interact and bind, thus forming a 0 complex that can be removed and/or detected in the reaction mixture. These assays can be conducted in a variety of ways.
  • one method to conduct such an assay involves anchoring an yesl gene product or a test substance onto a solid support and detecting yesl gene product/test compound S complexes formed on the solid support at the end of the reaction.
  • the yesl gene product may be anchored onto a solid support, and the test compound, which is not anchored, may be labeled, either directly or indirectly.
  • microtiter plates are conveniently utilized as the solid support.
  • the anchored component may be immobilized by non-covalent or covalent attachments.
  • Non- covalent attachment may be accomplished by simply coating the solid surface with a solution of the protein and drying.
  • an immobilized antibody preferably a monoclonal antibody, specific for the protein to be immobilized may be used to anchor the protein to the solid surface.
  • the surfaces may be prepared in advance and stored.
  • the non-immobilized component is added to the coated surface containing the anchored component. After the reaction is complete, unreacted components are removed (e.g., by washing) under conditions such that any complexes formed will remain immobilized on the solid surface.
  • the detection of complexes anchored on the solid surface can be accomplished in a number of ways. Where the previously non-immobilized component is pre-labeled, the detection of label immobilized on the surface indicates that complexes were formed.
  • an indirect label can be used to detect complexes anchored on the surface; e.g., using a labeled antibody specific for the previously non-immobilized component (the antibody, in turn, may be directly labeled or indirectly labeled with a labeled anti-Ig antibody) .
  • a reaction can be conducted in a liquid phase, the reaction products separated from unreacted components, and complexes detected; e.g., using an immobilized antibody specific for yesl gene product or the test compound to anchor any complexes formed in solution, and a labeled antibody specific for the other component of the possible complex to detect anchored complexes. 5.8.2. ASSAYS FOR INTRACELLULAR PROTEINS THAT INTERACT WITH vesl GENE PRODUCTS
  • Any method suitable for detecting protein-protein interactions may be employed for identifying yesl gene
  • proteins including intracellular proteins, that interact with yesl gene products. Once isolated, such a protein can be identified and can be used in conjunction with standard techniques, to identify proteins it interacts with. For example, at least a portion of the amino acid sequence of a
  • oligonucleotide mixtures may be used as a guide for the generation of oligonucleotide mixtures that can be used to screen for gene sequences encoding such proteins. Screening made be accomplished, for example, by standard hybridization or PCR techniques. Techniques for the generation of
  • plasmids are constructed that encode two hybrid proteins: one consists of the DNA-binding domain of a transcription activator protein fused to the yesl gene product and the other consists of the transcription activator protein's activation domain fused to an unknown protein that is encoded by a cDNA that has been recombined into this plasmid as part of a cDNA library.
  • the DNA-binding domain fusion plasmid and the cDNA library are transformed into a strain of the yeast Saccharomyces cerevi ⁇ iae that contains a reporter gene (e.g., HBS or lacZ) whose regulatory region contains the transcription activator's binding site.
  • a reporter gene e.g., HBS or lacZ
  • the two-hybrid system or related methodologies may be used to screen activation domain libraries for proteins that interact with the "bait" gene product.
  • yesl gene products may be used as the bait gene product.
  • Total genomic, cDNA sequences, or fragments thereof, are fused to the DNA encoding an activation domain.
  • This library and a plasmid encoding a hybrid of a bait yesl gene product fused to the DNA-binding domain are co-transformed into a yeast reporter strain, and the resulting transformants are screened for those that express the reporter gene.
  • a bait yesl gene sequence such as the open reading frame of the yesl gene
  • a vector such that it is translationally fused to the DNA encoding the DNA-binding domain of the GAL4 protein.
  • a cDNA library of the cell line from which proteins that interact with bait yesl gene product are to be detected can be made using methods routinely practiced in the art. According to the particular system described herein, for example, the cDNA fragments can be inserted into a vector such that they are translationally fused to the transcriptional activation domain of GAL4. Such a library can be co-transformed along with the bait yesl gene-GAL4 fusion plasmid into a yeast strain that contains a lacZ gene driven by a promoter that contains GAL4 activation sequence. A cDNA encoded protein, fused to a ⁇ GAL4 transcriptional activation domain that interacts with bait yesl gene product will reconstitute an active GAL4 protein...and thereby drive expression of the HIS3 gene.
  • Colonies that express HIS3 can be detected by their growth on petri dishes containing semi- solid agar based media lacking histidine. The cDNA can then be purified from these strains, and used to produce and isolate the bait yesl gene product-interacting protein using techniques routinely practiced in the art.
  • the yesl gene products may, in vivo, interact with one or more macromolecules, including intracellular macromolecules, such as proteins.
  • macromolecules may include, but are not limited to, nucleic acid molecules and those proteins identified via methods such as those described, above, in Sections 5.8.1 - 5.8.2.
  • the macromolecules are referred to herein as
  • binding partners Compounds that disrupt yesl gene product binding to a binding partner, by binding to yesl or the binding partner, may be useful in regulating the activity of the yesl gene product, especially mutant yesl gene products.
  • Such compounds may include, but are not limited to molecules such as peptides, and the like, as described, for example, in Section 5.8.2 above.
  • the basic principle of an assay system used to identify compounds that interfere with the interaction between the yesl gene product and a binding partner, or the binding partner involves preparing a reaction mixture containing the yesl gene product and the binding partner under conditions and for a time sufficient to allow the two to interact and bind, thus forming a complex. In order to test a compound for inhibitory activity, the reaction mixture is prepared in the presence and absence of the test compound.
  • the test compound may be initially included in the reaction mixture, or may be added at a time subsequent to the addition of yesl gene product and its binding partner.
  • Control reaction mixtures are incubated without the test compound or with a compound which is known not to block complex formation.
  • the formation of any complexes between the yesl gene product and the binding partner is then detected.
  • the formation of a complex in the control reaction, but not in the reaction mixture containing the test compound, indicates that the compound interferes with the interaction of the yesl gene product and the binding partner.
  • complex formation within reaction mixtures containing the test compound and normal yesl gene product may also be compared to complex formation within reaction mixtures containing the test compound and a mutant yesl gene product. This comparison may be important in those cases wherein it is desirable to identify compounds that disrupt interactions of mutant but not normal yesl gene product.
  • the assay for compounds that interfere with the interaction of the yesl gene products and binding partners can be conducted in a heterogeneous or homogeneous format.
  • Heterogeneous assays involve anchoring either the yesl gene product or the binding partner onto a solid support and detecting complexes formed on the solid support at the end of the reaction.
  • homogeneous assays the entire reaction is carried out in a liquid phase.
  • the order of addition of reactants can be varied to obtain different information about the compounds being tested. For example, test compounds that interfere with the interaction between the yesl gene products and the binding partners, e .g.
  • test compounds that disrupt preformed complexes e.g., compounds with higher binding constants that displace one of the components from the complex, can be tested by adding the test compound to the reaction mixture after complexes have been formed.
  • test compounds that disrupt preformed complexes e.g., compounds with higher binding constants that displace one of the components from the complex.
  • either the yesl gene product or the interactive binding partner is anchored onto a solid surface, while the non-anchored species is labeled, either directly or indirectly.
  • the anchored species may be immobilized by non-covalent or covalent attachments. Non- covalent attachment may be accomplished simply by coating the solid surface with a solution of the yesl gene product or binding partner and drying. Alternatively, an immobilized antibody specific for the species to be anchored may be used to anchor the species to the solid surface. The surfaces may be prepared in advance and stored.
  • the partner of the immobilized species is exposed to the coated surface with or without the test compound. After the reaction is complete, unreacted components are removed (e.g., by washing) and any complexes formed will remain immobilized on the solid surface.
  • the detection of complexes anchored on the solid surface can be accomplished in a number of ways. Where the non-immobilized species is pre-labeled, the detection of label immobilized on the surface indicates that complexes were formed. Where the non-immobilized species is not pre- labeled, an indirect label can be used to detect complexes anchored on the surface; e .g.
  • the antibody in turn, may be directly labeled or indirectly labeled with a labeled anti-Ig antibody
  • test compounds that inhibit complex formation or that disrupt preformed complexes can be detected.
  • the reaction can be conducted in a liquid phase in the presence or absence of the test compound, the reaction products separated from unreacted components, and complexes detected; e.g., using an immobilized antibody specific for one of the binding components to anchor any complexes formed in solution, and a labeled antibody specific for the other partner to detect anchored complexes.
  • test compounds that inhibit complex formation or that disrupt preformed complexes can be identified.
  • a homogeneous assay can be used.
  • a preformed complex of the yesl gene product and the interactive binding partner is prepared in which either the yesl gene product or its binding partners is labeled, but the signal generated by the label is quenched due to complex formation (see, e.g., U.S. Patent No. 4,109,496 by Rubenstein which utilizes this approach for immunoassays) .
  • the addition of a test substance that competes with and displaces one of the species from the preformed complex will result in the generation of a signal above background. In this way, test substances that disrupt yesl gene product/binding partner interaction can be identified.
  • these same techniques can be employed using peptide fragments that correspond to the binding domains of the yesl product and/or the binding partner (in cases where the binding partner is a protein) , in place of one or both of the full length proteins.
  • Any number of methods routinely practiced in the art can be used to identify and isolate the binding sites. These methods include, but are not limited to, mutagenesis of the gene encoding one of the proteins and screening for disruption of binding in a co-immunoprecipitation assay. Compensating mutations in the gene encoding the second species in the complex can then be selected. Sequence analysis of the genes encoding the respective proteins will reveal the mutations that correspond to the region of the protein involved in interactive binding.
  • one protein can be anchored to a solid surface using methods described in this Section above, and allowed to interact with and bind to its labeled binding partner, which has been treated with a proteolytic enzyme, such as trypsin. After washing, a short, labeled peptide comprising the binding domain may remain associated with the solid material, which can be isolated and identified by amino acid sequencing. Also, once the gene coding for the segments is engineered to express peptide fragments of the protein, it can then be tested for binding activity and purified or synthesized.
  • a proteolytic enzyme such as trypsin
  • an yesl gene product can be anchored to a solid material as described, above, in this Section by making a GST-yesl fusion protein and allowing it to bind to glutathione agarose beads.
  • the binding partner can be labeled with a radioactive isotope, such as 35 S, and cleaved with a proteolytic enzyme such as trypsin. Cleavage products can then be added to the anchored GST-yesl fusion protein and allowed to bind. After washing away unbound peptides, labeled bound material, representing the binding partner binding domain, can be eluted, purified, and analyzed for amino acid sequence by well-known methods.
  • Peptides so identified can be produced synthetically or produced using recombinant DNA technology. 5.8.4. ASSAYS FOR IDENTIFICATION OF COMPOUNDS THAT AMELIORATE AN yesl MEDIATED NEUROPSYCHIATRIC DISORDER
  • Compounds including but not limited to binding compounds identified via assay techniques such as those ' described, above, in Sections 5.8.1 - 5.8.4, can be tested for the ability to ameliorate symptoms of an yesl mediated neuropsychiatric disorder, bipolar affective (mood) disorders, such as severe bipolar affective (mood) disorder (BP-I) , bipolar affective (mood) disorder with hypomania and major depression (BP-II) , in cellular, animal and human based tests.
  • bipolar affective (mood) disorders such as severe bipolar affective (mood) disorder (BP-I)
  • BP-II bipolar affective disorder with hypomania and major depression
  • the assays described herein can identify compounds that affect yesl activity by either affecting yesl gene expression or by affecting the level of yesl gene product activity.
  • compounds may be identified that are involved in another step in the pathway in which the yesl gene and/or yesl gene product is involved and, by affecting this same pathway may modulate the effect of yesl on the development of an yesl mediated neuropsychiatric disorder, such as BAD.
  • Such compounds can be used as part of a therapeutic method for the treatment of the disorder.
  • Described below are cell-based and animal model-based assays for the identification of compounds exhibiting such an ability to ameliorate symptoms of an yesl mediated neuropsychiatric disorder, such as BAD.
  • Such assays can be used alone or in combination with the cellular and non- cellular assays described above.
  • cell-based systems can be used to identify compounds that may act to ameliorate symptoms of an yesl mediated neuropsychiatric disorder, such as BAD.
  • Such cell systems can include, for example, recombinant or non- recombinant cell, such as cell lines, that express the yesl gene.
  • cells that express yesl may be exposed to a compound suspected of exhibiting an ability to ameliorate symptoms of an yesl mediated neuropsychiatric disorder, such as BAD, at a sufficient concentration and for a sufficient time to elicit such an amelioration of such symptoms in the exposed cells.
  • the cells can be assayed to measure alterations in the expression of the yesl gene, e . g. , by assaying cell lysates for yesl mRNA transcripts (e.g., by Northern analysis) or for yesl gene products expressed by the cell; compounds that modulate expression of the yesl gene are good candidates as therapeutics.
  • animal-based systems or models for an yesl mediated neuropsychiatric disorder may be used to identify compounds capable of ameliorating symptoms of the disorder.
  • Such animal models may be used as test substrates for the identification of drugs, pharmaceuticals, therapies and interventions.
  • animal models may be exposed to a compound suspected of exhibiting an ability to ameliorate symptoms, at a sufficient concentration and for a sufficient time to elicit such an amelioration of symptoms of an yesl neuropsychiatric disorder. The response of the animals to the exposure may be monitored by assessing the reversal of the symptoms of the disorder.
  • any treatments that reverse any aspect of symptoms of an yesl mediated neuropsychiatric disorder should be considered as candidates for human therapeutic intervention in such a disorder.
  • Dosages of test agents may be determined by deriving dose-response curves, as discussed in Section 5.10.1, below.
  • such methods can comprise administering compounds which modulate the expression of a mammalian yesl gene and/or the synthesis or activity of a mammalian yesl gene product so symptoms of the disorder are ameliorated.
  • such methods can comprise supplying the mammal with a nucleic acid molecule encoding an unimpaired yesl gene product such that an unimpaired yesl gene product is expressed and symptoms of the disorder are ameliorated.
  • methods for the treatment of mammalian yesl mediated neuropsychiatric disorders resulting from yesl gene mutations can comprise supplying the mammal with a cell comprising a nucleic acid molecule that encodes an unimpaired yesl gene product such that the cell expresses the unimpaired yesl gene product and symptoms of the disorder are ameliorated.
  • yesl gene product activity In cases in which a loss of normal yesl gene product function results in the development of a yesl mediated neuropsychiatric disorder an increase in yesl gene product activity would facilitate progress towards an asymptomatic state in individuals exhibiting a deficient level of yesl gene expression and/or yesl gene product activity.
  • Methods for enhancing the expression or synthesis of yesl can include, for example, methods such as those described below, in Section 5.9.2.
  • symptoms of yesl mediated neuropsychiatric disorders may be ameliorated by administering a compound that decreases the level of yesl gene expression and/or yesl gene product activity.
  • Methods for inhibiting or reducing the level of yesl gene product synthesis or expression can include, for example, methods such as those described in Section 5.9.1.
  • the compounds administered comprise compounds, in particular drugs, reported to ameliorate or exacerbate the symptoms of a neuropsychiatric disorder, such as BAD.
  • Such compounds include antidepressants such as lithium salts, carbamazepine, valproic acid, lysergic acid diethylamide (LSD) , p- chlorophenylalanine, p-propyldopacetamide dithiocarbamate derivatives e .g. , FLA 63; anti-anxiety drugs, e .g. , diazepam; monoamine oxidase (MAO) inhibitors, e .g .
  • antidepressants such as lithium salts, carbamazepine, valproic acid, lysergic acid diethylamide (LSD) , p- chlorophenylalanine, p-propyldopacetamide dithiocarbamate derivatives e .g. , FLA 63
  • anti-anxiety drugs e .g. , diazepam
  • MAO monoamine oxidase
  • biogenic amine uptake blockers e.g., tricyclic antidepressants such as desipramine, imipramine and amitriptyline
  • serotonin reuptake inhibitors e.g., fluoxetine
  • antipsychotic drugs such as phenothiazine derivatives (e.g., chlorpromazine (thorazine) and trifluopromazine) ) , butyrophenones (e.g., haloperidol (Haldol) ) , thioxanthene derivatives (e.g., chlorprothixene) , and dibenzodiazepines (e.g., clozapine) ; benzodiazepines; dopaminergic agonists and antagonists e.g., L-DOPA, cocaine, amphetamine, ⁇ -methyl-tyrosine, res
  • dopaminergic agonists and antagonists e.g., L-DOPA, cocaine, amphetamine,
  • symptoms of yesl mediated neuropsychiatric disorders may be ameliorated by decreasing the level of yesl gene expression and/or yesl gene product activity by using yesl gene sequences in conjunction with well-known antisense, gene "knock-out,” ribozyme and/or triple helix methods to decrease the level of yesl gene expression.
  • the compounds that may exhibit the ability to modulate the activity, expression or synthesis of the yesl gene including the ability to ameliorate the symptoms of a yesl mediated neuropsychiatric disorder, such as BAD, are antisense, ribozyme, and triple helix molecules. Such molecules may be designed to reduce or inhibit either unimpaired, or if appropriate, mutant target gene activity.
  • Antisense RNA and DNA molecules act to directly block the translation of mRNA by hybridizing to targeted mRNA and preventing protein translation.
  • Antisense approaches involve the design of oligonucleotides that are complementary to a target gene mRNA. The antisense oligonucleotides will bind to the complementary target gene mRNA transcripts and prevent translation. Absolute complementarity, although preferred, is not required.
  • a sequence "complementary" to a portion of an RNA means a sequence having sufficient complementarity to be able to hybridize with the RNA, forming a stable duplex; in the case of double-stranded antisense nucleic acids, a single strand of the duplex DNA may thus be tested, or triplex formation may be assayed.
  • the ability to hybridize will depend on both the degree of complementarity and the length of the antisense nucleic acid. Generally, the longer the hybridizing nucleic acid, the more base mismatches with an RNA it may contain and still form a stable duplex (or triplex, as the case may be) .
  • One skilled in the art can ascertain a tolerable degree of mismatch by use of standard procedures to determine the melting point of the hybridized complex.
  • oligonucleotides complementary to non-coding regions of the yesl gene could be used in an antisense approach to inhibit translation of endogenous yesl mRNA.
  • Antisense nucleic acids should be at least six nucleotides in length, and are preferably oligonucleotides ranging from 6 to about 50 nucleotides in length. In specific aspects the oligonucleotide is at least 10 nucleotides, at least 17 nucleotides, at least 25 nucleotides or at least 50 nucleotides.
  • in vitro studies are first performed to quantitate the ability of the antisense oligonucleotide to inhibit gene expression. It is preferred that these studies utilize controls that distinguish between antisense gene inhibition and nonspecific biological effects of oligonucleotides. It is also preferred that these studies compare levels of the target RNA or protein with that of an internal control RNA or protein. Additionally, it is envisioned that results obtained using the antisense oligonucleotide are compared with those obtained using a control oligonucleotide.
  • control oligonucleotide is of approximately the same length as the test oligonucleotide and that the nucleotide sequence of the oligonucleotide differs from the antisense. sequence no more than is necessary to prevent specific hybridization to the target sequence.
  • the oligonucleotides can be DNA or RNA or chimeric mixtures or derivatives or modified versions thereof, single- stranded or double-stranded.
  • the oligonucleotide can be modified at the base moiety, sugar moiety, or phosphate backbone, for example, to improve stability of the molecule, hybridization, etc.
  • the oligonucleotide may include other appended groups such as peptides (e .g. , for targeting host cell receptors in vivo) , or agents facilitating transport across the cell membrane (see, e.g., Letsinger, et al . , 1989, Proc. Natl. Acad. Sci. U.S.A.
  • the oligonucleotide may be conjugated to another molecule, e.g., a peptide, hybridization triggered cross-linking agent, transport agent, hybridization-triggered cleavage agent, etc.
  • the antisense oligonucleotide may comprise at least one modified base moiety which is selected from the group including but not limited to 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl) uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D- galactosylqueosine, inosine, N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcyto
  • the antisense oligonucleotide may also comprise at least one modified sugar moiety selected from the group including but
  • the antisense oligonucleotide comprises at least one modified phosphate backbone selected from the group consisting of a phosphorothioate, a phosphorodithioate, a phosphoramidothioate, a phosphoramidate, a phosphordiamidate, a methylphosphonate, an alkyl phosphotriester, and a formacetal or analog thereof.
  • the antisense oligonucleotide is an ⁇ -anomeric oligonucleotide.
  • oligonucleotide forms specific double-stranded hybrids with complementary RNA in which, contrary to the usual .-units, the strands run parallel to each other (Gautier, et al . , 1987, Nucl. Acids Res. 15, 6625-6641).
  • the oligonucleotide is a 2 ' -O-methylribonucleotide (Inoue, et al . , 1987, Nucl. Acids Res. 15, 6131-6148) , or a chimeric RNA-DNA analogue (Inoue, et ai., 1987, FEBS Lett. 215, 327-330).
  • Oligonucleotides of the invention may be synthesized by standard methods known in the art, e .g. , by use of an automated DNA synthesizer (such as are commercially available from Biosearch, Applied Biosystems, etc.).
  • an automated DNA synthesizer such as are commercially available from Biosearch, Applied Biosystems, etc.
  • phosphorothioate oligonucleotides may be synthesized by the method of Stein, et ai . (1988, Nucl. Acids Res. 16, 3209)
  • methylphosphonate oligonucleotides can be prepared by use of controlled pore glass polymer supports (Sarin, et al . , 1988, Proc. Natl. Acad. Sci. U.S.A. 85, 7448-7451), etc.
  • Antisense molecules should be delivered to cells that express the target gene in vivo.
  • a number of methods have been developed for delivering antisense DNA or RNA to cells; e.g., antisense molecules can be injected directly into the tissue site, or modified antisense molecules, designed to target the desired cells (e.g., antisense linked to peptides or antibodies that specifically bind receptors or antigens expressed on the target cell surface) can be administered systemically.
  • a preferred approach utilizes a recombinant DNA construct in which the antisense oligonucleotide is placed under the control of a strong pol III or pol II promoter.
  • the use of such a construct to transfect target cells in the patient will result in the transcription of sufficient amounts of single stranded RNAs that will form complementary base pairs with the endogenous target gene transcripts and thereby prevent translation of the target gene mRNA.
  • a vector can be introduced e.g., such that it is taken up by a cell and directs the transcription of an antisense RNA.
  • Such a vector can remain episomal or become chromosomally integrated, as long as it can be transcribed to produce the desired antisense RNA.
  • Such vectors can be constructed by recombinant DNA technology methods standard in the art.
  • Vectors can be plasmid, viral, or others known in the art, used for replication and expression in mammalian cells.
  • Expression of the sequence encoding the antisense RNA can be by any promoter known in the art to act in mammalian, preferably human cells. Such promoters can be inducible or constitutive.
  • Such promoters include but are not limited to: 5 the SV40 early promoter region (Bernoist and Chambon, 1981, Nature 290, 304-310), the promoter contained in the 3' long terminal repeat of Rous sarcoma virus (Yamamoto, et ai., 1980, Cell 22, 787-797), the herpes thy idine kinase promoter (Wagner, et al . , 1981, Proc. Natl. Acad. Sci. U.S.A. 78,
  • 15 can be used that selectively infect the desired tissue, in which case administration may be accomplished by another route (e.g., systemically) .
  • Ribozyme molecules designed to catalytically cleave target gene mRNA transcripts can also be used to prevent 0 translation of target gene mRNA and, therefore, expression of target gene product.
  • PCT International Publication WO90/11364 published October 4, 1990; Sarver, et ai., 1990, Science 247, 1222-1225.
  • Ribozymes are enzymatic RNA molecules capable of 5 catalyzing the specific cleavage of RNA. (For a review, see Rossi, 1994, Current Biology 4, 469-471). The mechanism of ribozyme action involves sequence specific hybridization of the ribozyme molecule to complementary target RNA, followed by an endonucleolytic cleavage event.
  • the composition of 0 ribozyme molecules must include one or more sequences complementary to the target gene mRNA, and must include the well known catalytic sequence responsible for mRNA cleavage. For this sequence, see, e.g., U.S. Patent No. 5,093,246, which is incorporated herein by reference in its entirety.
  • ribozymes that cleave mRNA at site specific recognition sequences can be used to destroy target gene RNAs, the use of hammerhead ribozymes is preferred.
  • Hammerhead ribozymes cleave mRNAs at locations dictated by flanking regions that form complementary base pairs with the target mRNA. The sole requirement is that the target mRNA have the following sequence of two bases: 5'-UG-3'.
  • the ribozyme is engineered so that the cleavage recognition site is located near the 5' end of the target gene mRNA, i .e . , to increase efficiency and minimize the intracellular accumulation of non-functional mRNA transcripts.
  • the ribozymes of the present invention also include RNA endoribonucleases (hereinafter "Cech-type ribozymes”) such as the one that occurs naturally in Tetrahymena thermophila (known as the IVS, or L-19 IVS RNA) and that has been extensively described by Thomas Cech and collaborators (Zaug, et al . , 1984, Science, 224, 574-578; Zaug and Cech, 1986, Science, 231, 470-475; Zaug, et al . , 1986, Nature, 324, 429- 433; published International patent application No. WO 88/04300 by University Patents Inc.; Been and Cech, 1986, Cell, 47, 207-216) .
  • Cech-type ribozymes such as the one that occurs naturally in Tetrahymena thermophila (known as the IVS, or L-19 IVS RNA) and that has been extensively described by Thomas Cech and collaborators (Zaug, et al .
  • the Cech-type ribozymes have an eight base pair active site which hybridizes to a target RNA sequence whereafter cleavage of the target RNA takes place.
  • the invention encompasses those Cech-type ribozymes which target eight base-pair active site sequences that are present in the target gene.
  • the ribozymes can be composed of modified oligonucleotides (e.g., for improved stability, targeting, etc.) and should be delivered to cells that express the target gene in vivo.
  • a preferred method of delivery involves using a DNA construct "encoding" the ribozyme under the control of a strong constitutive pol III or pol II promoter, so that transfected cells will produce sufficient quantities of the ribozyme to destroy endogenous target gene messages and inhibit translation. Because ribozymes unlike antisense molecules, are catalytic, a lower intracellular concentration is required for efficiency.
  • Endogenous target gene expression can also be reduced by inactivating or "knocking out” the target gene or its promoter using targeted homologous recombination (e.g., see Smithies, et al . , 1985, Nature 317, 230-234; Thomas and Capecchi, 1987, Cell 51, 503-512; Thompson, et al . , 1989, Cell 5, 313-321; each of which is incorporated by reference herein in its entirety) .
  • targeted homologous recombination e.g., see Smithies, et al . , 1985, Nature 317, 230-234; Thomas and Capecchi, 1987, Cell 51, 503-512; Thompson, et al . , 1989, Cell 5, 313-321; each of which is incorporated by reference herein in its entirety
  • a mutant, nonfunctional target gene flanked by DNA homologous to the endogenous target gene (either the coding regions or regulatory regions of the target gene) can be used, with or without a selectable marker and/or a negative selectable marker, to transfect cells that express the target gene in vivo. Insertion of the DNA construct, via targeted homologous recombination, results in inactivation of the target gene.
  • ES embryonic stem
  • Such approaches are particularly suited in the agricultural field where modifications to ES (embryonic stem) cells can be used to generate animal offspring with an inactive target gene (e .g. , see Thomas and Capecchi, 1987 and Thompson, 1989, supra) .
  • this approach can be adapted for use in humans provided the recombinant DNA constructs are directly administered or targeted to the required site in vivo using appropriate viral vectors.
  • endogenous target gene expression can be reduced by targeting deoxyribonucleotide sequences complementary to the regulatory region of the target gene (i.e., the target gene promoter and/or enhancers) to form triple helical structures that prevent transcription of the target gene in target cells in the body.
  • deoxyribonucleotide sequences complementary to the regulatory region of the target gene i.e., the target gene promoter and/or enhancers
  • triple helical structures that prevent transcription of the target gene in target cells in the body.
  • Nucleic acid molecules to be used in triplex helix formation for the inhibition of transcription should be single stranded and composed of deoxynucleotides.
  • the base composition of these oligonucleotides must be designed to promote triple helix formation via Hoogsteen base pairing rules, which generally require sizeable stretches of either purines or pyrimidines to be present on one strand of a duplex.
  • Nucleotide sequences may be pyrimidine-based, which will result in TAT and CGC + triplets across the three associated strands of the resulting triple helix.
  • the pyrimidine-rich molecules provide base complementarity to a purine-rich region of a single strand of the duplex in a parallel orientation to that strand.
  • nucleic acid molecules may be chosen that are purine-rich, for example, contain a stretch of G residues.
  • Switchback molecules will form a triple helix with a DNA duplex that is rich in GC pairs, in which the majority of the purine residues are located on a single strand of the targeted duplex, resulting in GGC triplets across the three strands in the triplex.
  • the potential sequences that can be targeted for triple helix formation may be increased by creating a so called “switchback" nucleic acid molecule.
  • Switchback molecules are synthesized in an alternating 5'-3', 3 '-5' manner, such that they base pair with first one strand of a duplex and then the other, eliminating the necessity for a sizeable stretch of either purines or pyrimidines to be present on one strand of a duplex.
  • the technique may so efficiently reduce or inhibit the transcription (triple helix) and/or translation (antisense, ribozyme) of mRNA produced by normal target gene alleles that the possibility may arise wherein the concentration of normal target gene product present may be lower than is necessary for a normal phenotype.
  • nucleic acid molecules that encode and express target gene polypeptides exhibiting normal target gene activity may, be introduced into cells via gene therapy methods such as those described, below, in Section 5.9.2 that do not contain sequences susceptible to whatever antisense, ribozyme, or triple helix treatments are being utilized.
  • the target gene encodes an extracellular protein, it may be preferable to co- administer normal target gene protein in order to maintain the requisite level of target gene activity.
  • Anti-sense RNA and DNA, ribozyme, and triple helix molecules of the invention may be prepared by any method known in the art for the synthesis of DNA and RNA molecules, as discussed above. These include techniques for chemically synthesizing oligodeoxyribonucleotides and oligoribonucleotides Well known in the art such as for example solid phase phosphoramidite chemical synthesis.
  • RNA molecules may be generated by in vitro and in vivo transcription of DNA sequences encoding the antisense RNA molecule. Such DNA sequences may be incorporated into a wide variety of vectors, that incorporate suitable RNA polymerase promoters such as the T7 or SP6 polymerase promoters.
  • antisense cDNA constructs that synthesize antisense RNA constitutively or inducibly, depending on the promoter used, can be introduced stably into cell lines.
  • GENE REPLACEMENT THERAPY yesl gene nucleic acid sequences can be utilized for the treatment of an yesl mediated neuropsychiatric disorder. Such treatment can be in the form of gene replacement therapy. Specifically, one or more copies of a normal yesl gene or a portion of the yesl gene that directs the production of an yesl gene product exhibiting normal yesl gene function, may be inserted into the appropriate cells within a patient, using vectors that include, but are not limited to adenovirus, adeno-associated virus, and retrovirus vectors, in addition to other particles that introduce DNA into cells, such as liposomes.
  • yesl gene is expressed in the brain
  • gene replacement therapy techniques should be capable delivering yesl gene sequences to these cell types within patients.
  • techniques that are well known to those of skill in the art can be used to enable yesl gene sequences to cross the blood-brain barrier readily and to deliver the sequences to cells in the brain.
  • viral vectors such as, for example, those described above, are preferable.
  • techniques for delivery involve direct administration of such yesl gene sequences to the site of the cells in which the yesl gene sequences are to be expressed.
  • Additional methods that may be utilized to increase the overall level of yesl gene expression and/or yesl gene product activity include the introduction of appropriate yesl-expressing cells, preferably autologous cells, into a patient at positions and in numbers that are sufficient to ameliorate the symptoms of an yesl mediated neuropsychiatric disorder.
  • Such cells may be either recombinant or non- recombinant.
  • cells that can be administered to increase the overall level of yesl gene expression in a patient are normal cells, preferably brain cells, that express the yesl gene.
  • cells preferably autologous cells, can be engineered to express yesl gene sequences, and may then be introduced into a patient in positions appropriate for the amelioration of the symptoms of an yesl mediated neuropsychiatric disorder.
  • cells that express an unimpaired yesl gene and that are from a MHC matched individual can be utilized, and may include, for example, brain cells.
  • the expression of the yesl gene sequences is controlled by the appropriate gene regulatory sequences to allow such expression in the necessary cell types.
  • gene regulatory sequences are well known to the skilled artisan.
  • Such cell-based gene therapy techniques are well known to those skilled in the art, see, e.g., Anderson, U.S. Patent No. 5,399,349.
  • the cells to be administered are non-autologous cells, they can be administered using well known techniques that prevent a host immune response against the introduced cells from developing.
  • the cells may be introduced in an encapsulated form which, while allowing for an exchange of components with the immediate extracellular environment, does not allow the introduced cells to be recognized by the host immune system.
  • compounds such as those identified via techniques such as those described, above, in Section 5.8, that are capable of modulating yesl gene product activity can be administered using standard techniques that are well known to those of skill in the art.
  • the administration techniques should include well known ones that allow for a crossing of the blood-brain barrier.
  • the compounds that are determined to affect yesl gene expression or gene product activity can be administered to a patient at therapeutically effective doses to treat or ameliorate an yesl mediated neuropsychiatric disorder.
  • a therapeutically effective dose refers to that amount of the compound sufficient to result in amelioration of symptoms of such a disorder.
  • EFFECTIVE DOSE Toxicity and therapeutic efficacy of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD 50 (the dose lethal to 50% of the population) and the ED 50 (the dose therapeutically effective in 50% of the population) .
  • the dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD 50 /ED S0 .
  • Compounds that exhibit large therapeutic indices are preferred. While compounds that exhibit toxic side effects may be used, care should be taken to design a delivery system that targets such compounds to the site of affected tissue in order to minimize potential damage to uninfected cells and, thereby, reduce side effects.
  • the data obtained from the cell culture assays and animal studies can be used in formulating a range of dosage for use in humans.
  • the dosage of such compounds lies preferably within a range of circulating concentrations that include the ED 50 with little or no toxicity.
  • the dosage may vary within this range depending upon the dosage form employed and the route of administration utilized.
  • the therapeutically effective dose can be estimated initially from cell culture assays.
  • a dose may be formulated in animal models to achieve a circulating plasma concentration range that includes the IC 50 (i.e., the concentration of the test compound that achieves a half-maximal inhibition of symptoms) as determined in cell culture.
  • IC 50 i.e., the concentration of the test compound that achieves a half-maximal inhibition of symptoms
  • levels in plasma may be measured, for example, by high performance liquid chromatography.
  • compositions for use in accordance with the present invention may be formulated in conventional manner using one or more physiologically acceptable carriers or excipients.
  • the compounds and their physiologically acceptable salts and solvates may be formulated for administration by inhalation or insufflation (either through the mouth or the nose) or oral, buccal, parenteral or rectal administration.
  • the pharmaceutical compositions may take the form of, for example, tablets or capsules prepared by conventional means with pharmaceutically acceptable excipients such as binding agents (e.g., pregelatinised maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose) ; fillers (e.g., lactose, microcrystalline cellulose or calcium hydrogen phosphate) ; lubricants (e .g.
  • Liquid preparations for oral administration may take the form of, for example, solutions, syrups or suspensions, or they may be presented as a dry product for constitution with water or other suitable vehicle before use. Such liquid preparations may be prepared by conventional means with pharmaceutically acceptable additives such as suspending agents (e .g. , sorbitol syrup, cellulose derivatives or hydrogenated edible fats); emulsifying agents (e .g.
  • non-aqueous vehicles e.g., almond oil, oily esters, ethyl alcohol or fractionated vegetable oils
  • preservatives e.g., methyl or propyl-p-hydroxybenzoates or sorbic acid
  • the preparations may also contain buffer salts, flavoring, coloring and sweetening agents as appropriate.
  • compositions for oral administration may be suitably formulated to give controlled release of the active compound.
  • compositions for buccal administration may take the form of tablets or lozenges formulated in conventional manner.
  • the compounds for use according to the present invention are conveniently delivered in the form of an aerosol spray presentation from pressurized packs or a nebuliser, with the use of a suitable propellant, e .g. , dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas.
  • a suitable propellant e .g. , dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas.
  • a suitable propellant e .g. , dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas.
  • the dosage unit may be determined by providing a valve to deliver a metered amount.
  • the compounds may be formulated for parenteral administration by injection, e.g., by bolus injection or continuous infusion.
  • Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multi- dose containers, with an added preservative.
  • the compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.
  • the active ingredient may be in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.
  • the compounds may also be formulated in rectal compositions such, as suppositories or retention enemas, e.g., containing conventional suppository bases such as cocoa butter or other glycerides.
  • the compounds may also be formulated as a depot preparation. Such long acting formulations may be administered by implantation (for example subcutaneously or intramuscularly) or by intramuscular injection.
  • the compounds may be formulated with suitable polymeric or hydrophobic materials (for example as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt.
  • compositions may, if desired, be presented in a pack or dispenser device that may contain one or more unit dosage forms containing the active ingredient.
  • the pack may for example comprise metal or plastic foil, such as a blister pack.
  • the pack or dispenser device may be accompanied by instructions for administration. 6. EXAMPLE: LOCALIZATION OF THE yesl GENE TO CHROMOSOME 18
  • LD Linkage disequilibrium
  • YEAST ARTIFICIAL CHROMOSOME YAC MAPPING
  • yeast artificial chromosomes YACs
  • the YACs were then ordered and contig reconstructed by performing standard short tag sequence (STS) -content mapping with microsatellite markers and non-polymorphic STSs available from databases that surround the genetically defined candidate region.
  • STS standard short tag sequence
  • BACTERIAL ARTIFICIAL CHROMOSOME .BAC MAPPING STSs from the short arm of human chromosome 18 were used to screen a human BAC library (Research Genetics, Huntsville, AL) . The ends of the BACs were cloned or directly sequenced. The end sequences were used to amplify the next overlapping BACs. From each BAC, additional icrosatellites were identified. Specifically, random sheared libraries were prepared from overlapping BACs within the defined genetic interval. BAC DNA was sheared with a nebulizer (CIS-US Inc., Bedford, MA). Fragments in the size range of 600 to 1,000 bp were utilized for the sublibrary production. Microsatellite sequences from the sublibraries were identified by corresponding microsatellite probes. Sequences around such repeats were obtained to enable development of PCR primers for genomic DNA.
  • Standard RH mapping techniques were applied to a Stanford G3 RH mapping panel (Research Genetics, Huntsville, AL) to order all microsatellite markers and non-polymorphic STSs in the region being analyzed.
  • BAD bipolar affective disorder
  • LD linkage disequilibrium
  • the radiation hybrid (RH) mapping technique was independently applied to the region being analyzed.
  • RH was used to order all microsatellite markers and non-polymorphic STSs in the region.
  • the high resolution physical map ultimately constructed was obtained using data from RH mapping and STS- content mapping.
  • BAC clones within the newly identified 310 kb neuropsychiatric disorder region were analyzed to identify specific genes within the region.
  • a combination of sample sequencing, cDNA selection and transcription mapping analyses were combined to arrange sequences into tentative transcription units, that is, tentatively delineating the coding sequences of genes within this genomic region of interest.
  • yesl One of the transcription units identified was termed yesl.
  • the position of the yesl gene within the disease associate chromosomal interval identifies the yesl gene as mediating BAD.
  • Biallelic polymorphisms were identified in exon 1 of yesl using the primers described above. Polymorphisms were also identified in exon 7 and 8 which co-segregate with BAD; 20 base pairs 5' to exon 7 in Figure 2, a A to G substitution was found which co-segregates with BAD; 34 base pairs 5' to exon 7 in Figure 2, a T to C substitution was found that co- segregates with BAD; and 59 base pairs 5' to exon 8 in Figure 2, a G to A substitution was found that co-segregates with BAD. Each of these can be used as genetic markers of BAD.

Abstract

Cette invention se rapporte au gène yes1 chez les mammifères, un gène qui est associé aux troubles affectifs bipolaires chez les êtres humains. Cette invention concerne des procédés d'identification de composés qui modulent l'expression de yes1, ainsi que l'utilisation de ces composés en qualité d'agents thérapeutiques dans le traitement de troubles liés au yes1 et de troubles neuropsychiatriques. Cette invention concerne également des procédés d'évaluation diagnostique, de test génétique et de pronostic de troubles neuropsychiatriques liés au yes1 comme, par exemple, la schizophrénie, le trouble déficitaire de l'attention, un trouble schizo-affectif, un trouble affectif bipolaire ou un trouble affectif unipolaire. Cette invention concerne également des procédés et des compositions permettant de traiter ces troubles.
PCT/US1999/000297 1998-01-08 1999-01-07 Procedes et compositions permettant de diagnostiquer et de traiter des troubles neuropsychiatriques WO1999035290A1 (fr)

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001055440A1 (fr) * 2000-01-31 2001-08-02 Human Genome Sciences, Inc. Acides nucleiques, proteines et anticorps
WO2001063294A2 (fr) * 2000-02-24 2001-08-30 Oxford Glycosciences (Uk) Limited Proteines, genes et leur utilisation dans le diagnostic et le traitement de maladies affectives bipolaires (bad) et de depressions unipolaires
WO2004056386A2 (fr) * 2002-12-19 2004-07-08 To-Bbb Holding B.V. Acides nucleiques intervenant dans la regulation de la barriere hemato-encephalique

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
FREIMER N. B., ET AL.: "GENETIC MAPPING USING HAPLOTYPE, ASSOCIATION AND LINKAGE METHODS SUGGESTS A LOCUS FOR SEVERE BIPOLAR DISORDER (BPI) AT 18Q22-Q23.", NATURE GENETICS., NATURE PUBLISHING GROUP, NEW YORK, US, vol. 12., 1 April 1996 (1996-04-01), NEW YORK, US, pages 436 - 441., XP002920383, ISSN: 1061-4036, DOI: 10.1038/ng0496-436 *

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001055440A1 (fr) * 2000-01-31 2001-08-02 Human Genome Sciences, Inc. Acides nucleiques, proteines et anticorps
WO2001063294A2 (fr) * 2000-02-24 2001-08-30 Oxford Glycosciences (Uk) Limited Proteines, genes et leur utilisation dans le diagnostic et le traitement de maladies affectives bipolaires (bad) et de depressions unipolaires
WO2001063294A3 (fr) * 2000-02-24 2002-04-11 Oxford Glycosciences Uk Ltd Proteines, genes et leur utilisation dans le diagnostic et le traitement de maladies affectives bipolaires (bad) et de depressions unipolaires
WO2004056386A2 (fr) * 2002-12-19 2004-07-08 To-Bbb Holding B.V. Acides nucleiques intervenant dans la regulation de la barriere hemato-encephalique
WO2004056386A3 (fr) * 2002-12-19 2005-02-24 To Bbb Holding B V Acides nucleiques intervenant dans la regulation de la barriere hemato-encephalique

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WO1999035290A9 (fr) 1999-10-07

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