WO1998042724A1 - Methods and compositions for the diagnosis and treatment of neuropsychiatric disorders - Google Patents
Methods and compositions for the diagnosis and treatment of neuropsychiatric disorders Download PDFInfo
- Publication number
- WO1998042724A1 WO1998042724A1 PCT/US1998/006211 US9806211W WO9842724A1 WO 1998042724 A1 WO1998042724 A1 WO 1998042724A1 US 9806211 W US9806211 W US 9806211W WO 9842724 A1 WO9842724 A1 WO 9842724A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- disorder
- gene
- nucleic acid
- expression
- compound
- Prior art date
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
- C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
- C07K14/4702—Regulators; Modulating activity
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
Definitions
- the present invention relates to the mammalian fshl5w6 gene, a novel gene associated with neuropsychiatric disorders in humans.
- the invention encompasses f ⁇ hl5w6 nucleic acids, recombinant DNA molecules, cloned genes or degenerate variants thereof, fshl5w6 gene products and antibodies directed against such gene products, cloning vectors containing mammalian fshl5w6 gene molecules, and hosts that have been genetically engineered to express such molecules.
- the invention further relates to methods for the identification of compounds that modulate the expression, synthesis and activity of f ⁇ hl5w6 and to using compounds such as those identified as therapeutic agents in the treatment of a fshl5w6 disorder or a neuropsychiatric disorder, including, by way of example and not of limitation, schizophrenia, attention deficit disorder, a schizoaffective disorder, a bipolar affective disorder or a unipolar affective disorder.
- the invention also relates to methods for the diagnostic evaluation, genetic testing and prognosis of a fshl5w6 disorder or of a neuropsychiatric disorder, including, by way of example and not of limitation, schizophrenia, attention deficit disorder, a schizoaffective disorder, a bipolar affective disorder or a unipolar affective disorder.
- psychiatric disorders there are only a. few psychiatric disorders in which clinical manifestations of the disorder can be correlated with demonstrable defects in the structure and/or function of the nervous system.
- Well-known examples of such disorders include Huntington's disease, which can be traced to a mutation in a single gene and in which neurons in the striatum degenerate, and Parkinson's disease, in which dopaminergic neurons in the nigro-striatal pathway degenerate.
- the vast majority of psychiatric disorders however, presumably involve subtle and/or undetectable changes, at the cellular and/or molecular levels, in nervous system structure and function.
- Neuropsychiatric disorders such as schizophrenia, attention deficit disorders, schizoaffective disorder, bipolar affective disorders, or unipolar affective disorder., from neurological disorders, in which anatomical or biochemi-cal 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 aid 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 wi ⁇ _n 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 present invention relates, first, to the mammalian fsh!5w6 gene, a novel gene associated with neuropsychiatric disorders in humans, e.g., schizophrenia, attention deficit disorders, schizoaffective disorders, bipolar affective disorders, and/or unipolar affective disorders, including fshl5w6 nucleic acids, recombinant DNA molecules, cloned genes or degenerate variants thereof.
- the invention further relates to novel mammalian fshl5w6 gene products and to antibodies directed against such mammalian fshl5w6 gene products, or conserved variants or fragments thereof.
- fshl5w6 nucleic acid and amino acid sequences are provided herein.
- the invention also relates to vectors, including expression vectors, containing mammalian fshl5w6 gene molecules, and hosts that have been genetically engineered to express such fshl5w6 gene products.
- the invention further relates to methods for the treatment of fshl5w6 or neuropsychiatric disorders, wherein such methods comprise administering compounds which modulate the expression of a mammalian fshl5w6 gene and/or the synthesis or activity of a mammalian fshl ⁇ w ⁇ gene product so symptoms of the disorder are ameliorated.
- the invention further relates to methods for the treatment of mammalian fshl5w6 or neuropsychiatric disorders resulting from fshl5w6 gene mutations, wherein such methods comprise supplying the mammal with a nucleic acid molecule encoding an unimpaired fshl5w6 gene product such that an unimpaired fshl ⁇ w ⁇ gene product is expressed and symptoms of the disorder are ameliorated.
- the invention further relates to methods for the treatment of mammalian fshl ⁇ w ⁇ or neuropsychiatric disorders resulting from f ⁇ hl5w6 gene mutations, wherein such methods comprise supplying the mammal with a cell comprising a nucleic acid molecule that encodes an unimpaired fshlSw ⁇ gene product such that the cell expresses the unimpaired fshl5w6 gene product and symptoms of the disorder are ameliorated.
- the present invention is directed to methods that utilize the fshl5w6 gene and/or gene product sequences for the diagnostic evaluation, genetic testing and prognosis of a fshl ⁇ w ⁇ disorder or a neuropsychiatric disorder.
- the invention relates to methods for diagnosing fshl ⁇ w ⁇ or neuropsychiatric disorders, wherein such methods comprise measuring fshl5w6 gene expression in a patient sample, or detecting a fshl ⁇ w ⁇ 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 modulate the expression of the mammalian fshlSw ⁇ gene and/or the synthesis or activity of the mammalian fshlSw ⁇ gene products, wherein such methods comprise contacting a compound to a cell that expresses a fshlSw ⁇ gene, measuring the level of fshl5w ⁇ gene expression, gene product expression or gene product activity, and comparing this level to the level of fshl ⁇ w ⁇ 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 fshlSw ⁇ gene and/or the synthesis or activity of the mammalian fshl ⁇ w ⁇ gene products has been identified.
- f ⁇ hl5w ⁇ gene and/or gene produces can also be utilized as markers for mapping of the region of the long arm
- the neuropsychiatric disorders referred to herein can include, but are not limited to, schizophrenia; attention deficit disorder; a schizoaffective disorder; a bipolar affective disorder, e.g., severe bipolar affective (mood) disorder (BP-I) , bipolar affective (mood) disorder with hypomania and major depression (BP-II) ; schizoaffective disorder manic type (SAD-M) ; or a unipolar affective disorder e.g., unipolar major depressive disorder (MDD) .
- BP-I severe bipolar affective
- BP-II bipolar affective disorder with hypomania and major depression
- SAD-M schizoaffective disorder manic type
- MDD unipolar major depressive disorder
- fshlSw ⁇ disorder refers to a disorder involving an aberrant level of fshlSw ⁇ 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 fshl ⁇ w ⁇ level.
- BAD bipolar affective disorder (s) BP
- bipolar mood disorder bipolar affective disorder (s) BP
- MDD unipolar major depressive disorder RT-PCR, reverse transcriptase PCR SSCP, single-stranded conformational polymorphism
- Figure 1 depicts fshl5w6 nucleotide (SEQ ID NO:l) and amino acid sequences (SEQ ID NO: 2).
- f ⁇ hlSw ⁇ gene is associated with neuropsychiatric disorders such as human bipolar affective disorder (BAD) .
- BAD bipolar affective disorder
- f ⁇ hlSw ⁇ gene and gene product sequences are described in the example presented below in Section 6. This invention is based, in part, on the genetic and physical mapping of the f ⁇ hlSw ⁇ gene to a specific, narrow portion of chromosome 18 , also described in the Example presented below in Section 6.
- f ⁇ hlSw ⁇ gene is a novel gene associated with neuropsychiatric disorders, including BAD. Nucleic acid sequences of the identified fshl5w ⁇ gene are described herein. As used herein, "f ⁇ hl5w ⁇ gene” refers to: (a) a nucleic acid molecule containing the DNA sequence shown in Figure 1 (SEQ ID NO:l) or contained in the cDNA clone Epfshl5w6 (ATCC accession No. 98351) , as deposited with the American Type Culture Collection (ATCC) ;
- fshlSw ⁇ gene product refers to a gene product that exhibits at least one of the biological activities of an endogenous, unimpaired fshlSw ⁇ gene.
- a functionally equivalent fshlSw ⁇ gene product is one that, when present in an appropriate cell type, is capable of ameliorating, preventing or delaying the onset of one or more symptoms of a fshlSw ⁇ disorder.
- a functionally equivalent f ⁇ hl5w ⁇ gene product is one that, when present in an appropriate cell type, is capable of ameliorating, preventing or delaying the onset of one or more symptoms of a neuropsychiatric disorder.
- a functionally equivalent f ⁇ hlSw ⁇ gene product is one that, when present in an appropriate cell type, is capable of ameliorating, preventing or delaying the onset of one or more symptoms of a BAD, such as, for example, severe bipolar affective (mood) disorder, bipolar affective (mood) disorder with hypomania and major depression, or schizoaffective disorder manic type.
- fshlSw ⁇ sequences can include, for example either genomic DNA (gDNA) or cDNA sequences.
- nucleic acid which encodes a given amino acid sequence
- the nucleic acid need not only be a cDNA molecule, but can also,, for example, refer to a gDNA sequence from which an mRNA species is transcribed that is processed to encode the given amino acid sequence.
- f ⁇ hlSw ⁇ gene may also refer to degenerate variants of DNA sequences (a) through (d) .
- the invention also includes nucleic acid molecules, preferably DNA molecules, that hybridize to, and are therefore the complements of, the DNA sequences (a) through (d) , in the preceding paragraph.
- Such hybridization conditions may be highly stringent or less highly stringent, as described above.
- highly stringent conditions may refer, e.g., to washing in 6xSSC/0.05% sodium pyrophosphate at 37°C (for 14-base oligos), 48°C (for 17-base oligos), 55°C (for 20-base oligos) , and 60°C (for 23-base oligos) .
- nucleic acid molecules may encode or act as fshlSw ⁇ gene antisense molecules, useful, for example, in fshlSw ⁇ gene regulation (for and/or as antisense primers in amplification reactions of fshlSw ⁇ gene nucleic acid sequences) .
- fshlSw ⁇ gene regulation such techniques can be used to regulate, for example, a fshlSw ⁇ disorder or a neuropsychiatric disorder, such as BAD.
- sequences may be used as part of ribozyme and/or triple helix sequences, also useful for fshlSw ⁇ gene regulation.
- such molecules may be used as components of diagnostic methods whereby, for example, the presence of a particular fshlSw ⁇ allele responsible for causing a fshlSw ⁇ disorder or neuropsychiatric disorder such as BAD, e .g. , manic-depression, may be detected.
- a particular fshlSw ⁇ allele responsible for causing a fshlSw ⁇ disorder or neuropsychiatric disorder such as BAD, e .g. , manic-depression
- the invention also encompasses:
- DNA vectors that contain any of the foregoing fshlSw ⁇ coding sequences and/or their complements (i.e., antisense) (b) DNA expression vectors that contain any of the foregoing fshlSw ⁇ coding sequences operatively associated with a regulatory element that directs the expression of the coding sequences;
- regulatory elements include but are not limited to inducible and non-inducible promoters, enhancers, operators and other elements known to those skilled in the art that drive and regulate expression.
- Such regulatory elements include but are not limited to the cytomegalovirus hCMV immediate early gene, the early or late promoters of SV40 adenovirus, the lac system, the trp system, the TAC system, the TRC system, the major operator and promoter regions of phage A, the control regions of fd coat protein, the promoter for 3-phosphoglycerate kinase, the promoters of acid phosphatase, and the promoters of the yeast ⁇ -mating factors.
- the invention further includes fragments of any of the DNA sequences disclosed herein.
- the f ⁇ hl5w ⁇ gene sequences of the invention are mammalian gene sequences, with human sequences being preferred.
- the f ⁇ hlSw ⁇ gene sequences of the invention are gene sequences encoding f ⁇ hlSw ⁇ gene products containing polypeptide portions corresponding to (that is, polypeptide portions exhibiting amino acid sequence similiarity to) the amino acid sequence depicted in Figure 1, wherein the corresponding portion exhibits greater than about 50% amino acid identity with the Figure 1 sequence.
- the fshlSw ⁇ gene sequences of the invention are gene sequences encoding f ⁇ hlSw ⁇ gene products containing polypeptide portions corresponding to (that is, polypeptide portions exhibiting amino acid sequence similiarity to) the amino acid sequence depicted in Figure 1, wherein the corresponding portion exhibits greater than about 50% amino acid identity with the Figure 1 sequence, averaged across the f ⁇ hlSw ⁇ gene product's entire ,length.
- additional f ⁇ hl5w ⁇ gene sequences can be identified and readily isolated, without undue experimentation, by molecular biological techniques well known in the art, used in conjunction with the fshlSw ⁇ sequences disclosed herein.
- additional human fshl5w ⁇ gene sequences at the same or at different genetic loci as those disclosed in Figure 1 can be isolated readily.
- genes at other genetic or physical loci within the human genome that encode proteins that have extensive homology to one or more domains of the fshl5w ⁇ gene product and that encode gene products functionally equivalent to a f ⁇ hlSw ⁇ gene product.
- homologous f ⁇ hlSw ⁇ gene sequences present in other species can be identified and isolated readily.
- fshlSw ⁇ gene sequences present at the same genetic or physical locus as those sequences disclosed in Figure 1 can, for example, be obtained readily by utilizing standard « - jquencing and bacterial artificial chromosome (BAC) technologies in connection with BAC54 (Identification Reference EpHS996, ATCC Accession No. 98363).
- BAC54 Identification Reference EpHS996, ATCC Accession No. 98363
- 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 f ⁇ hlSw ⁇ sequences can then readily be identified by alignment of the BAC sequences with the f ⁇ hlSw ⁇ sequences depicted in Figure 1. Alternatively, BAC subclones containing additional f ⁇ hlSw ⁇ sequences can be identified by identifying those subclones which hybridize to probes derived from the fshlSw ⁇ sequences depicted in Figure 1.
- the isolated fshlSw ⁇ gene sequences disclosed herein may be labeled and used to screen a cDNA library constructed from mRNA obtained from appropriate cells or ⁇ issues (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
- fshlSw ⁇ gene homologue 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 fshlSw ⁇ 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 a f ⁇ hlSw ⁇ gene allele (such as human brain cell lines e.g., ATCC CRL-7605, ATCC CRL-7948, ATCC CRL-2060 PFSK-1, ATCC CRL-2176 SW 598, American Type Culture Collection, Rockville, MD; cortical neuronal cell lines, e.g., Ronnett, et al . , 1990, Science 248, 603-605; Ronnett, et al . , 1994, Neuroscience 63, 1081-1099; and Dunn, et al . , 1996, Int. J. Dev. Neurosci. 14, 61-68; neuronal line HCN-1A, Westlund et al . , 1992, Int. J. Dev. Neurosci. 10, 361-373) .
- the PCR product may be subcloned and sequenced to ensure that the amplified sequences represent the sequences of a f ⁇ hlSw ⁇ gene nucleic acid sequence.
- the PCR fragment may then be used to isolate a full length cDNA clone by a variety of methods.
- the ampl-ified 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.
- RNA may be isolated, following standard procedures, from an appropriate cellular or tissue source (i.e., one known, or suspected, to express the f ⁇ hl5w ⁇ gene, such as, for example, blood samples or 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.
- 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.
- fshlSw ⁇ gene sequences may additionally be used to isolate mutant fshlSw ⁇ gene alleles.
- mutant alleles may be isolated from individuals either known or proposed to have a genotype that contributes to the symptoms of a fshlSw ⁇ disorder, or a neuropsychiatric disorder such as BAD, for example, manic-depression. Mutant alleles and mutant allele products may then be utilized in the therapeutic and diagnostic systems described below. Additionally, such fshlSw ⁇ gene sequences can be used to detect fshl5w ⁇ gene regulatory (e.g., promoter) defects which can be associated with a f ⁇ hl5w ⁇ disorder, or a neuropsychiatric disorder such as BAD.
- fshlSw ⁇ gene regulatory e.g., promoter
- a cDNA of a mutant f ⁇ hl5w ⁇ 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 f ⁇ hlSw ⁇ 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 f ⁇ hl5w ⁇ allele to that of the normal f ⁇ hlSw ⁇ allele, the mutation (s) responsible for the loss or alteration of function of the mutant f ⁇ hl5w ⁇ 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 f ⁇ hlSw ⁇ allele, or a cDNA library can be constructed using RNA from a tissue known, or suspected, to express a mutant fshlSw ⁇ allele.
- An unimpaired f ⁇ hlSw ⁇ gene or any suitable fragment thereof may then be labeled and used as a probe to identify the corresponding mutant fshlSw ⁇ allele in such libraries.
- Clones containing the mutant f ⁇ hlSw ⁇ 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 fshlSw ⁇ 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 fshl5w ⁇ 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.)
- f ⁇ hl5w ⁇ mutation results in an expressed gene product with altered function (e.g., as a result of a missense or a frameshift mutation)
- a polyclonal set of ant ⁇ -f ⁇ hl5w ⁇ gene product antibodies are likely to cross-react with the mutant f ⁇ hlSw ⁇ 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.
- f ⁇ hlSw ⁇ mutations can further be detected using PCR amplification techniques. Primers can routinely be designed to amplify overlapping regions of the whole f ⁇ hlSw ⁇ sequence including the promoter region.
- primers are designed to cover the exon-intron boundaries such that, first, coding regions can be scanned for mutations.
- 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
- f ⁇ hlSw ⁇ 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 a f ⁇ hl5w ⁇ disorder, or a neuropsychiatric disorder such as BAD.
- the amino acid sequence depicted in Figure 1 represents a fshlSw ⁇ gene product.
- the f ⁇ hlSw ⁇ gene product sometimes referred to herein as a "fshl5w ⁇ protein", includes those gene products encoded by the fshl5w ⁇ gene sequences described in Section 5.1, above.
- fshl5w ⁇ gene products may include proteins that represent functionally equivalent (see Section 5.1 for a definition) gene pr ⁇ ducts.
- Such- an equivalent f ⁇ hl ⁇ w ⁇ 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 the amino acid sequence encoded by the f ⁇ hlSw ⁇ gene sequences described, above, in Section 5.1, but that result in a "silent" change, in that the change produces a functionally equivalent fshlSw ⁇ gene product.
- 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
- negatively charged (acidic) amino acids include aspartic acid and glutamic acid.
- deletion or non-conservative alterations can be engineered to produce altered f ⁇ hlSw ⁇ gene products.
- Such alterations can, for example, alter one or more of the biological functions of the f ⁇ hlSw ⁇ gene product. Further, such alterations can be selected so as to generate f ⁇ hlSw ⁇ gene products that are better suited for expression, scale up, etc. in the host cells chosen.
- cysteine residues can be deleted or substituted with another amino acid residue in order to eliminate disulfide bridges.
- the fshlSw ⁇ gene products, peptide fragments thereof and fusion proteins thereof may be produced by recombinant DNA technology using techniques well known in the art.
- fshl5w ⁇ gene polypeptides, peptides, fusion peptide and fusion polypeptides of the invention by expressing nucleic acid containing f ⁇ hlSw ⁇ gene sequences are described herein.
- Methods that are well known to those skilled in the art can be used to construct expression vectors containing fshlSw ⁇ 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 . , 1989, supra , and Ausubel, et al . , 1989, supra .
- RNA capable of encoding f ⁇ hlSw ⁇ 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 f ⁇ hlSw ⁇ gene 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 f ⁇ hlSw ⁇ gene product of the invention in ⁇ itu .
- microorganisms such as bacteria (e.g., E. coli , B . ⁇ ubtili ⁇ ) transformed with recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vectors containing f ⁇ hl5w ⁇ gene product coding sequences; yeast (e.g., Saccharomyce ⁇ , Pichia) transformed with recombinant yeast expression vectors containing the f ⁇ hl ⁇ w ⁇ gene product coding sequences; insect cell systems infected with recombinant virus expression vectors (e.g., baculovirus) containing the fshlSw ⁇ gene product coding sequences; plant cell systems 5 infected with recombinant virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with recombinant plasmid expression vectors (e.g., Ti plasmid) containing f ⁇ hlSw ⁇ gene product coding
- a number of expression vectors may be advantageously selected depending upon the use intended for the f ⁇ hlSw ⁇ gene product being expressed. For example, when a large quantity of such a protein is to be produced, for the generation of pharmaceutical compositions
- 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
- pGEX vectors may also be used to express foreign polypeptides as fusion proteins with glutathione S-transferase (GST) .
- GST glutathione S-transferase
- fusion proteins are soluble and can easily be purified from lysed cells by adsorption to glutathione-
- 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.
- Autographa califomica nuclear polyhedrosis virus (AcNPV) is used as a vector to express foreign genes.
- the virus grows in Spodoptera frugiperda cells.
- the f ⁇ hl5w ⁇ gene 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 f ⁇ hl5w ⁇ gene 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 fshlSw ⁇ gene 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 fshl5w ⁇ gene product in infected hosts.
- Specific initiation signals may also be required for efficient translation of inserted fshl5w ⁇ gene product coding sequences. These signals include the ATG initiation codon and adjacent sequences. In cases where an entire f ⁇ hlSw ⁇ 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 f ⁇ hl5w ⁇ gene coding sequence is inserted, exogenous translational control signals, including, perhaps, the ATG initiation codon, must be provided.
- 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 strairi 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., glycosylation) 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 f ⁇ hl5w ⁇ 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 f ⁇ hl5w ⁇ gene product.
- Such engineered cell lines may be particularly useful in screening and evaluation of compounds that affect the endogenous activity of the fshlSw ⁇ gene product.
- a number of selection systems may be used, including but not limited to the herpes s,implex virus thymidine kinase (Wigler, et al . , 1977, Ce-11 11, 223), hypoxanthine-guanine phosphoribosyltransferase (Szybalska and Szybalski, 1962, Proc. Natl. Acad. Sci. USA 48, 2026), and adenine phosphoribosyltransferase (Lowy, et al . , 1980, Cell 22, 817) 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. USA 77, 3567; O'Hare, et al . , 1981, Proc. Natl. Acad. Sci. USA 78, 1527); gpt, which confers resistance to mycophenolic acid (Mulligan and Berg, 1981, Proc. Natl. Acad. Sci. USA 78, 2072); neo, which confers resistance to the aminoglycoside G-418 (Colberre-Garapin, et al . , 1981, J. Mol. Biol. 150, 1) ; and hygro, which confers resistance to hygromycin (Santerre, et al . , 1984, Gene 30, 147).
- 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 al . , 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 fshlSw ⁇ 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 fshl5w ⁇ transgenic animals.
- transgenic refers to animals expressing fshlSw ⁇ gene sequences from a different species (e.g., mice expressing human f ⁇ hlSw ⁇ sequences) , as well as animals that have been genetically engineered to overexpress endogenous (i.e., same species) fshlSw ⁇ sequences or animals that have been genetically engineered to no longer express endogenous fshlSw ⁇ gene sequences (i.e., "knock-out" animals), and their progeny.
- Any technique known in the art may be used to introduce an fshl5w ⁇ gene transgene into animals to produce the founder lines of transgenic animals.
- Such techniques include, but are not limited to pronuclear microinjection (Hoppe and Wagner, 1989, U.S. Pat. No. 4,873,191); retrovirus mediated gene transfer into germ lines (Van der Putten, et al . , 1985, Proc. Natl. Acad. Sci., USA 82, 6148-6152); gene targeting in embryonic stem cells (Thompson, et al . , 1989, Cell 56, 313-321); electroporation of embryos (Lo, 1983, Mol. Cell. Biol.
- transgenic animal clones containing an fsh!5w ⁇ 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 f ⁇ hlSw ⁇ 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.
- vectors containing some nucleotide sequences homologous to the endogenous f ⁇ hlSw ⁇ 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 f ⁇ hlSw ⁇ gene.
- the transgene may also be selectively introduced into a particular cell type, thus inactivating the endogenous fshlSw ⁇ 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 f ⁇ hlSw ⁇ 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 fshl5w ⁇ gene- expressing tissue, may also be evaluated immunocytochemically using antibodies specific for the fshl ⁇ w ⁇ transgene product.
- ANTIBODIES TO fsh!5w6 GENE PRODUCTS Described herein are methods for the production of antibodies capable of specifically recognizing one or more fshlSw ⁇ gene product epitopes or epitopes of conserved variants or peptide fragments of the fshlSw ⁇ gene products.
- 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.
- Such antibodies may be used, for example, in the detection of a fshlSw ⁇ 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 f ⁇ hl5w ⁇ 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 f ⁇ hl5w ⁇ gene product levels and/or activity. Additionally, such antibodies can be used in conjunction with the gene therapy techniques described, below, in Section 5.9.0.2 to, for example, evaluate the normal and/or engineered f ⁇ hlSw ⁇ - expressing cells prior to their introduction into the patient.
- An ⁇ j.-f ⁇ hl5w ⁇ gene product antibodies may additionally be used as a method for the inhibition of abnormal f ⁇ hl5w ⁇ gene product activity.
- Such antibodies may, therefore, be utilized as part of treatment methods for an f ⁇ hlSw ⁇ disorder or a neuropsychiatric disorder, such as BAD.
- various host animals may be immunized by injection with a fshlSw ⁇ gene product, or a portion thereof.
- host animals may include, but are not limited to rabbits, mice, and rats, to name but a few.
- 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 a,s BCG (bacille Calmette-Guerin) and CorynebaQterium parvum .
- Polyclonal antibodies are heterogeneous populations of antibody molecules derived from the sera of animals immunized with an antigen, such as a fshl5w ⁇ gene product, or an antigenic functional derivative thereof.
- an antigen such as a fshl5w ⁇ gene product, or an antigenic functional derivative thereof.
- host animals such as those described above, may be immunized by injection with f ⁇ hlSw ⁇ 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. 4,376,110), the human B-cell hybridoma technique (Kosbor et al . , 1983, Immunology Today 4, 72; Cole et al . , 1983, Proc. Natl. Acad. Sci. USA 80, 2026-2030), and the EBV-hybridoma technique (Cole et al . , 1985, Monoclonal Antibodies And Cancer Therapy, Alan R.
- 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 . Production of high titers of mAbs in vivo makes this the presently preferred method of production.
- chimeric antibodies In addition, techniques developed for the production of "chimeric antibodies" (Morrison, et al . , 1984, Proc. Natl. Acad. Sci., 81, 6851-6855; Neuberger, et al . , 1984, Nature 312, 604-608; Takeda, et al . , 1985, Nature, 314, 452-454) by splicing the genes from a mouse antibody molecule of appropriate antigen specificity together with genes from a human antibody molecule of appropriate biological activity can be used.
- 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
- An immunoglobuin light or heavy chain variable region consists of a "framework" region interrupted by three hypervariable regions, referred to as complementarity determining regions (CDRs) .
- CDRs complementarity determining regions
- 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.
- Antibody fragments that recognize specific epitopes may be generated by known techniques.
- 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 generated by reducing the disulfide bridges of the F(ab') 2 fragments.
- 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.
- fshlSw ⁇ gene sequences fshl ⁇ w ⁇ gene products, including peptide fragments and fusion proteins thereof, and of antibodies directed against f ⁇ hl5w ⁇ gene products and peptide fragments thereof.
- Such applications include, for example, prognostic and diagnostic evaluation of a f ⁇ hlSw ⁇ disorder or a neuropsychiatric disorder, such as BAD, and the identification of subjects with a predisposition to such disorders, as described, below, in Section 5.5.
- Such applications include methods for the treatment of a f ⁇ hlSw ⁇ disorder or a neuropsychiatric disorder, such as BAD, as described, below, in Section 5.9, and for the identification of compounds that modulate the expression of the f ⁇ hlSw ⁇ gene and/or the synthesis or activity of the fshl5w ⁇ gene product, as described below, in Section 5.8.
- Such compounds can include, for example, other cellular products that are involved in mood regulation and in fshlSw ⁇ disorders and neuropsychiatric disorders, such as BAD. These compounds can be used, for example, in the amelioration of fshlSw ⁇ disorders and neuropsychiatric disorders, such as BAD. 5.5. DIAGNOSIS OF ABNORMALITIES OF A fshl5w ⁇ OR A NEUROPSYCHIATRIC DISORDER
- a variety of methods can be employed for the diagnostic and prognostic evaluation of fshlSw ⁇ disorders and neuropsychiatric disorders, such as BAD, and for the identification of subjects having a predisposition to such disorders.
- Such methods may, for example, utilize reagents such as the fshlSw ⁇ gene nucleotide sequences described in Sections 5.1, and antibodies directed against fshlSw ⁇ gene products, including peptide fragments thereof, as described, above, in Section 5.3.
- reagents such as the fshlSw ⁇ gene nucleotide sequences described in Sections 5.1, and antibodies directed against fshlSw ⁇ gene products, including peptide fragments thereof, as described, above, in Section 5.3.
- reagents may be used, for example, for:
- f ⁇ hlSw ⁇ gene nucleotide sequences can, for example, be used to diagnose an f ⁇ hlSw ⁇ or neuropsychiatric disorder using, for example, the techniques for f ⁇ hlSw ⁇ mutation detection described above in Section 5.1.
- the methods described herein may be performed, for example, by utilizing pre-packaged diagnostic kits comprising at least one specific f ⁇ hlSw ⁇ gene nucleic acid or anti- f ⁇ hlSw ⁇ gene antibody reagent described herein, which may be conveniently used, e.g., in clinical settings, to diagnose patients exhibiting abnormalities of a fshlSw ⁇ d. ⁇ sorder or a 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 f ⁇ hlSw ⁇ 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 f ⁇ hl5w ⁇ mutations and to detect and/or assay levels of f ⁇ h!5w ⁇ nucleic acid sequences.
- Mutations within the f ⁇ hl5w ⁇ 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. fshlSw ⁇ nucleic acid sequences may be used in hybridization or amplification assays of biological samples to detect abnormalities involving fshlSw ⁇ 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 f ⁇ hlSw ⁇ 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 f ⁇ hlSw ⁇ gene.
- the lengths of these nucleic acid reagents are at least 15 to 30 nucleotides.
- nucleic acid reagents After incubation, all non-annealed nucleic acids are removed from the nucleic acid '. f ⁇ hlSw ⁇ molecule hybrid. The presence of nucleic acids that have hybridized, if any such molecules exist, is then detected. Using such a detection scheme, 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. In this case, after incubation, non-annealed, labeled nucleic acid reagents of the type described in Section 5.1 are easily removed.
- Detection of the remaining, annealed, labeled f ⁇ hl5w ⁇ nucleic acid reagents is accomplished using standard techniques well-known to those in the art.-
- the fshl5w6 gene sequences to which the nucleic acid reagents have annealed can be compared to the annealing pattern expected from a normal f ⁇ hlSw ⁇ gene sequence in order to determine whether a fshl5w ⁇ gene mutation is present.
- Alternative diagnostic methods for the detection of f ⁇ hlSw ⁇ 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 f ⁇ hl5w ⁇ gene in order to determine whether a fshlSw ⁇ gene mutation exists.
- genotyping techniques can be performed to identify individuals carrying f ⁇ hl ⁇ w ⁇ 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
- improved methods for analyzing DNA polymorphisms which can be utilized for the identification of f ⁇ hlSw ⁇ gene mutations, have been described that capitalize on the presence of variable numbers of short, tandemly repeated DNA sequences between the restriction enzyme sites.
- Weber U.S. Pat. No. 5,075,21-7 describes a DNA marker based on length polymorphisms in blocks of (dC-dA) n- (dG-dT) n short tandem repeats.
- the average separation of (dC-dA)n- (dG-dT) n blocks is estimated to be 30,000-60,000 bp. Markers that are so closely spaced exhibit a high frequency co-inheritance, and are extremely useful in the identification of genetic mutations, such as, for example, mutations within the f ⁇ hl5w ⁇ gene, and the diagnosis of diseases and disorders related to f ⁇ hlSw ⁇ mutations.
- 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 fshlSw ⁇ gene, amplifying the extracted DNA, and labelling the repeat sequences to form a genotypic map of the individual's DNA.
- the level of f ⁇ hl5w ⁇ gene expression can also be assayed.
- RNA from a cell type or tissue known, or suspected, to express the fshlSw ⁇ 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 fshlSw ⁇ gene.
- Such analyses may reveal both quantitative and qualitative aspects of the expression pattern of the fshl5w ⁇ gene, including activation or inactivation of fshl ⁇ w ⁇ 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 f ⁇ hlSw ⁇ 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 fshlSw ⁇ gene.
- the antibodies and immunoassay methods described below have, for example, important in vitro applications in assessing the efficacy of treatments for fshl5w ⁇ disorders or 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 fshl5w ⁇ gene expression and f ⁇ hlSw ⁇ peptide production.
- the compounds that have beneficial effects on an f ⁇ hlSw ⁇ disorder or a neuropsychiatric disorder, such as BAD can be identified, and a therapeutically effective dose determined.
- In vitro immunoassays may also be used, for example, to assess the efficacy of cell-b,ased gene therapy for an f ⁇ hlSw ⁇ disorder or a neuropsychiatric disorder, such as BAD.
- Antibodies directed against f ⁇ hlSw ⁇ peptides may be used in vitro to determine, for example, the level of f ⁇ hl5w ⁇ gene expression achieved in cells genetically engineered to produce f ⁇ hl5w ⁇ peptides.
- intracel lular f ⁇ hlSw ⁇ 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 fshlSw ⁇ 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 fshl5w ⁇ gene.
- Preferred diagnostic methods for the detection of fshlSw ⁇ gene products or conserved variants or peptide fragments thereof may involve, for example, immunoassays wherein the fsh!5w ⁇ gene products or conserved variants or peptide fragments are detected by their interaction with an anti-f ⁇ hl5w ⁇ gene product-specific antibody.
- antibodies, or fragments of antibodies, such as those described, above, in Section 5.3, useful in the present invention may be used to quantitatively or qualitatively detect the presence of fshlSw ⁇ gene products or conserved variants or peptide fragments thereof.
- This can be accomplished, for example, by immunofluorescence techniques employing a fluorescently labeled antibody (see below, this Section) coupled with light microscopic, flow cytometric, or fluorimetric detection.
- fluorescently labeled antibody see below, this Section
- the antibodies (or fragments thereof) useful in the present invention may, additionally, be employed histologically, as in immunofluorescence or immunoelectron microscopy, for in situ detection of f ⁇ hlSw ⁇ gene products or conserved variants or peptide fragments thereof.
- ⁇ it ⁇ detection may be accomplished by removing a histological specimen from a patient, and applying thereto a labeled antibody of the present invention.
- the antibody (or fragment) is preferably applied by overlaying the labeled antibody (or fragment) onto a biological sample.
- Immunoassays for f ⁇ hlSw ⁇ gene products or conserved 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, that have been incubated in cell culture, in the presence of a detectably labeled antibody capable of identifying f ⁇ hl5w6 gene products or conserved 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, or other solid support that is capable of immobilizing cells, cell particles or soluble proteins.
- a solid phase support or carrier such as nitrocellulose, or other solid support 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 fshlSw ⁇ gene 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 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, polyacryla ides, 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.
- the binding activity of a given lot of anti-fshl5w6 gene product antibody may be determined according to well known methods. Those skilled in the art will be able to determine operative and optimal assay conditions for each determination by employing routine experimentation.
- One of the ways in which the fshl5w ⁇ gene peptide- specific antibody can be detectably labeled is by linking the same to an enzyme and use in an enzyme immunoassay (EIA) (Voller, A., "The Enzyme Linked Immunosorbent Assay (ELISA)", 5 1978, Diagnostic Horizons 2, 1-7, Microbiological Associates Quarterly Publication, Walkersville, MD) ; Voller, A. et al . , 1978, J. Clin. Pathol.
- EIA enzyme immunoassay
- 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
- 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, ⁇ g.lycerophosphate, dehydrogenase, triose phosphate isomerase, horseradish
- peroxidase alkaline phosphatase, asparaginase, glucose oxidase, ⁇ -galactosidase, ribonuclease, urease, catalase, glucose-6-phosphate dehydrogenase, glucoamylase and acetylcholinesterase.
- the detection can be accomplished by colorimetric methods that employ a chromogenic substrate for
- 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. For example, by radioactively
- RIA radioj-uimunoassay
- the radioactive isotope can be detected by such means as the use of a gamma counter or a scintillation counter or by autoradiography. It is also possible to label the antibody with a fluorescent compound. When the fluorescently labeled antibody is exposed to light of the proper wave length, its presence can then be detected due to fluorescence.
- 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. Examples cf particularly useful chemiluminescent labeling compounds are luminol , isoluminol, theromatic acridinium ester, imidazole, acridinium salt and oxalate ester.
- Bioluminescence is a 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 purposes of labeling are luciferin, luciferase and aequorin. 5.8. SCREENING ASSAYS FOR COMPOUNDS
- the following assays are designed to identify compounds that bind to a f ⁇ h!5w ⁇ gene product, intracellular proteins or portions of proteins that interact with a f ⁇ hlSw ⁇ gene product, compounds that interfere with the interaction of a f ⁇ hl5w ⁇ gene product with intracellular proteins and compounds that modulate the activity of f ⁇ hl ⁇ w ⁇ gene (i.e., modulate the level of f ⁇ hl ⁇ w ⁇ gene expression and/or modulate the level of f ⁇ hlSw ⁇ gene product activity) .
- Assays may additionally be utilized that identify compounds that bind to f ⁇ hlSw ⁇ gene regulatory sequences (e.g. , ⁇ promoter sequences; see e.g., Platt, 1994, J. Biol.
- 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 f ⁇ hlSw ⁇ gene or some other gene involved in a f ⁇ hlSw ⁇ regulatory pathway, or intracellular proteins. Methods for the identification of such intracellular proteins are described, below, in Section 5.8.2. Such intracellular proteins may be involved in the control and/or regulation of mood.
- f ⁇ hlSw ⁇ gene expression and/or f ⁇ hlSw ⁇ gene product activity are compounds that affect the level of f ⁇ hlSw ⁇ gene expression and/or f ⁇ hlSw ⁇ gene product activity and that can be used in the therapeutic treatment of f ⁇ hlSw ⁇ disorders or 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 librarie-- ; (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 librarie-- ; (see, e.g., Lam, et
- 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 v (LSD) , p- chlorophenylalanine, p-propyldopacetamide dithiocarbamate derivatives e.g., FLA 63; anti-anxiety drugs, e . g .
- diazepam diazepam
- monoamine oxidase (MAO) inhibitors e.g., iproniazid, clorgyline, phenelzine and isocarboxazid
- 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
- Compounds identified via assays such as those described herein may be useful, for example, in elaborating the biological function of the gene product, and for ameliorating f ⁇ hl5w ⁇ disorders or 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 Section 5.8.4. 5.8.1. IN VITRO SCREENING ASSAYS FOR COMPOUNDS THAT BIND TO THE fsh!5w6 GENE PRODUCT
- In vitro systems may be designed to identify compounds capable of binding the f ⁇ h!5w ⁇ gene products of the invention.
- Compounds identified may be useful, for example, in modulating the activity of unimpaired and/or mutant fshl5w ⁇ gene products, may be useful in elaborating the biological function of the f ⁇ hlSw ⁇ gene product, may be utilized in screens for identifying compounds that disrupt normal f ⁇ hl5w ⁇ gene product interactions, or may in themselves disrupt such interactions.
- the principle of the assays used to identify compounds that bind to the fshl5w ⁇ gene product involves preparing a reaction mixture of the f ⁇ hlSw ⁇ 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 complex that can be removed and/or detected in the reaction mixture.
- These assays can be conducted in a variety of ways. For example, one method to conduct such an assay would involve anchoring f ⁇ hlSw ⁇ gene product or the test substance onto a solid phase and detecting f ⁇ hlSw ⁇ gene product/test compound complexes anchored on the solid phase at the end of the reaction.
- the f ⁇ hlSw ⁇ gene product may be anchored onto a solid surface, and the test compound, which is not anchored, may be labeled, either directly or indirectly.
- microtiter plates may conveniently be utilized as the solid phase.
- 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 fshlSw ⁇ 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.
- the amino acid sequence obtained 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 oligonucleotide mixtures and the screening are well-known. (See, e.g., Ausubel, supra , and 1990, "PCR Protocols: A Guide to Methods and Applications," Innis,, et al . , eds. Academic Press, Inc. , New York) .
- methods may be employed that result in the simultaneous identification of genes that encode the a protein which interacts with an f ⁇ hlSw ⁇ protein. These methods include, for example, probing expression libraries with labeled f ⁇ hlSw ⁇ protein, using fshlSw ⁇ protein in a manner similar to the well known technique of antibody probing of ⁇ gtll libraries.
- plasmids are constructed that encode two hybrid proteins: one consists of the DNA-binding domain of a transcription activator protein fused to the fshlSw ⁇ 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 b ⁇ -.n 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 proteins reconstitutes the functional activator protein and results in expression of the reporter gene, which is detected by an assay for the reporter gene product.
- the two-hybrid system or related methodology may be used to screen activation domain libraries for proteins that interact with the "bait" gene product.
- _ f ⁇ hlSw ⁇ gene products may be used as the bait gene product.
- Total genomic or cDNA sequences are fused to the DNA encoding an activation domain.
- This library and a plasmid encoding a hybrid of a bait f ⁇ hlSw ⁇ 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 f ⁇ hlSw ⁇ gene sequence such as the open reading frame of the f ⁇ hlSw ⁇ gene, can be cloned into 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 f ⁇ hlSw ⁇ 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. This library can be co-transformed along with the bait f ⁇ hlSw ⁇ 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 GAL4 transcriptional activation domain, that interacts with bait f ⁇ hlSw ⁇ 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 f ⁇ hlSw ⁇ gene-interacting protein using techniques routinely practiced in the art.
- ASSAYS FOR COMPOUNDS THAT INTERFERE WITH fshlSw ⁇ GENE PRODUCT MACROMOLECULE INTERACTION fshlSw ⁇ gene products of the invention 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 fsh!5w ⁇ binding in this way may be useful in regulating the activity of the fshlSw ⁇ gene product, especially mutant f ⁇ hl5w ⁇ 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, which would be capable of gaining access to an f ⁇ hl5w ⁇ gene product.
- the basic principle of the assay systems used to identify compounds that interfere with the interaction between the f ⁇ hlSw ⁇ gene product and its binding partner or partners involves preparing a reaction mixture containing the f ⁇ hlSw ⁇ 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.
- 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 fshlSw ⁇ gene product and its binding partner. Control reaction mixtures are incubated without the test compound or with a placebo.
- any complexes between the f ⁇ hlSw ⁇ gene protein 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 fshlSw ⁇ gene protein and the interactive binding partner.
- complex formation within reaction mixtures containing the test compound and normal f ⁇ hlSw ⁇ gene protein may also be compared to complex formation within reaction mixtures containing the test compound and a mutant f ⁇ hl5w ⁇ gene protein. This comparison may be important in those cases wherein it is desirable to identify compounds that disrupt interactions of mutant but not normal f ⁇ hlSw ⁇ gene proteins.
- the assay for compounds that interfere with the interaction of the f hlSw ⁇ gene products and binding partners can be conducted in a heterogeneous or homogeneous format.
- Heterogeneous assays involve anchoring either the f ⁇ hlSw ⁇ gene product or the binding partner onto a solid phase and detecting complexes anchored on the solid phase at the end of the reaction.
- homogeneous assays the entire reaction is carried out in a liquid phase. In either approach, the order of addition of reactants can be varied to obtain different information about the compounds being tested.
- test compounds that interfere with the interaction between the f ⁇ hl5w ⁇ gene products and the binding partners can be identified by conducting the reaction in the presence of the test substance; i.e., by adding the test substance to the reaction mixture prior to or simultaneously with the f ⁇ hlSw ⁇ gene protein and interactive intracellular binding partner.
- 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.
- the various formats are described briefly below.
- either the f ⁇ hlSw ⁇ 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 f ⁇ hlSw ⁇ 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.
- an indirect label can be used to detect complexes anchored on the surface; e.g., using a labeled antibody specific for the initially non-immobilized species (the antibody, in turn, may be directly labeled or indirectly labeled with a labeled anti-Ig antibody) .
- 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 or that disrupt preformed complexes can be identified.
- a homogeneous assay can be used.
- a preformed complex of the fshlSw ⁇ gene protein and the interactive binding partner is prepared in which either the f ⁇ h!5w ⁇ 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 f ⁇ h!5w ⁇ gene protein/binding partner interaction can be identified.
- the fshlSw ⁇ gene product can be prepared for immobilization using recombinant DNA techniques described in Section 5.2. above.
- the fshl5w ⁇ coding region can be fused to a glutathione-S- transferase (GST) gene using a fusion vector, such as pGEX- 5X-1, in such a manner that its binding activity is maintained in the resulting fusion protein.
- GST glutathione-S- transferase
- the interactive binding partner can be purified and used to raise a monoclonal antibody, using methods routinely practiced in the art and described above, in Section 5.3. This antibody can be labeled with the radioactive isotope 125 I, for example, by methods routinely practiced in the art.
- the GST-f " sh25w6 fusion protein can be anchored to glutathione-agarose beads.
- the interactive binding partner can then be added in the presence or absence of the test compound in a manner that allows interaction and binding to occur.
- unbound material can be washed away, and the labeled monoclonal antibody can be added to the system and allowed to bind to the complexed components.
- the interaction between the fshl5w ⁇ gene protein and the interactive binding partner can be detected by measuring the amount of radioactivity that remains associated with the glutathione-agarose beads. A successful inhibition of the interaction by the test compound will result in a decrease in measured radioactivity.
- the GST-fshl5w6 gene fusion protein and the interactive binding partner can be mixed together in liquid in the absence of the solid glutathione-agarose beads.
- the test compound can be added either during or after the species are allowed to interact. This mixture can then be added to the glutathione-agarose beads and. unbound material is washed away. Again the extent of inhibition of the fshlSw ⁇ gene product/binding partner interaction can be detected by adding the labeled antibody and measuring the radioactivity associated with the beads.
- these same techniques can be employed using peptide fragments that correspond to the binding domains of the f ⁇ hlSw ⁇ protein and/or the interactive or 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 ' o 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 can be engineered to express peptide fragments of the protein, which can then be tested for binding activity and purified or synthesized.
- a proteolytic enzyme such as trypsin
- a fshlSw ⁇ gene product can be anchored to a solid material as described, above, in this Section by making a GST-fshl5w ⁇ fusion protein and allowing it to bind to glutathione agarose beads.
- the interactive binding partner obtained 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-f ⁇ hl5w ⁇ fusion protein and allowed to bind.
- labeled bound material representing the binding partner binding domain
- labeled bound material representing the binding partner binding domain
- Peptides so identified can be produced synthetically or fused to appropriate facilitative proteins using recombinant DNA technology .
- 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 a f ⁇ hlSw ⁇ disorder or a disorder of thought and/or mood, including thought disorders such as schizophrenia, schizotypal personality disorder; psychosis; mood disorders, such as schizoaffective disorders (e.g., schizoaffective disorder manic type (SAD-M); bipolar affective (mood) disorders, such as severe bipolar affective (mood) disorder (BP-I) , bipolar affective (mood) disorder with hypomania and major depression (BP-II) ; unipolar affective disorders, such as unipolar major depressive disorder (MDD) , dysthymic disorder; obsessive- compulsive disorders; phobias, e.g., agoraphobia; panic disorders; generalized anxiety disorders; somatization disorders and hypochondriasis;
- the assays described herein can identify compounds that affect fshlSw ⁇ gene activity by either affecting fshl5w ⁇ gene expression or by affecting the level of f ⁇ h!5w ⁇ gene product activity.
- compounds may be identified that are involved in another step in the pathway in which the f ⁇ hl5w ⁇ gene and/or f ⁇ hlSw ⁇ gene product is involved and, by affecting this same pathway may modulate the effect of f ⁇ hl5w ⁇ on the development of a neuropsychiatric disorder such as BAD..
- Such compounds can be used as part of a therapeutic method for the treatment of the disorder.
- cell-based and animal model- based assays for the identification of compounds exhibiting such an ability to ameliorate symptoms of a f ⁇ hlSw ⁇ disorder or a neuropsychiatric disorder, such as BAD.
- cell-based systems can be used to identify compounds that may act to ameliorate symptoms of a f ⁇ hlSw ⁇ disorder or a neuropsychiatric disorder, such as BAD.
- Such cell systems can include, for example, recombinant or non- recombinant cell, such as cell lines, that express the f ⁇ hlSw ⁇ gene.
- cells that express f ⁇ hlSw ⁇ may be exposed to a compound suspected of exhibiting an ability to ameliorate symptoms of a fshlSw ⁇ disorder or a 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.
- a compound suspected of exhibiting an ability to ameliorate symptoms of a fshlSw ⁇ disorder or a 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 eel "1 _> can be assayed to measure alterations in the expression of the f ⁇ hlSw ⁇ gene, e.g., by assaying cell lysates for fshlSw ⁇ mRNA transcripts (e.g., by Northern analysis) or for fshlSw ⁇ gene products expressed by the cell; compounds that modulate expression of the fshlSw ⁇ gene are good candidates as therapeutics.
- the cells are examined to determine whether one or more cellular phenotypes associated with an f ⁇ hlSw ⁇ disorder or a neuropsychiatric disorder, such as BAD, has been altered to resemble a more normal or unimpaired, unaffected phenotype, or a phenotype more likely to produce a lower incidence or severity of disorder symptoms.
- a neuropsychiatric disorder such as BAD
- animal-based systems or models for a f ⁇ hlSw ⁇ disorder or a neuropsychiatric disorder, such as BAD 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 that may be effective in treating such disorders.
- 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 a f ⁇ hlSw ⁇ disorder or a neuropsychiatric disorder, such as BAD, in the exposed animals.
- the response of the animals to the exposure may be monitored by assessing the reversal of such symptoms.
- any treatments that reverse any aspect of symptoms of a fshlSw ⁇ disorder or a neuropsychiatric disorder, such as BAD 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.
- a fshlSw ⁇ disorder ov a disorder of thought and/or mood, such as BAD, may be treated.
- such methods can comprise administering compounds which modulate the expression of a mammalian f ⁇ hl5w ⁇ gene and/or the synthesis or activity of a mammalian fshlSw ⁇ gene product so symptoms of the disorder are ameliorated.
- such methods can comprise supplying the mammal with a nucleic acid molecule encoding an unimpaired f ⁇ hl5w ⁇ gene product such that an unimpaired f ⁇ hlSw ⁇ gene product is expressed and symptoms of the disorder are ameliorated.
- such methods can comprise supplying the mammal with a cell comprising a nucleic acid molecule that encodes an unimpaired f ⁇ h!5w ⁇ gene product such that the cell expresses the unimpaired f ⁇ hlSw ⁇ gene product and symptoms of the disorder are ameliorated.
- fshlSw ⁇ gene product activity would facilitate progress towards an asymptomatic state in individuals exhibiting a deficient level of f ⁇ hlSw ⁇ gene expression and/or f ⁇ hlSw ⁇ gene product activity.
- Methods for enhancing the expression or synthesis of f ⁇ h!5w ⁇ can include, for example, methods such as those described below, in Section 5.9.2.
- symptoms of fshoOS disorders or neuropsychiatric disorders may be ameliorated by administering a compound that decreases the level of fshlSw ⁇ gene expression and/or f ⁇ hlSw ⁇ gene product activity.
- Methods for inhibiting or reducing the level of fshlSw ⁇ synthesis or expression can include, for example, methods such as those described in Section T.9.1.
- the compounds administered do not comprise compounds, in particular drugs, reported to ameliorate or exacerbate the symptoms of a neuropsychiatric disorder, such as BAD.
- 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 .
- anti-anxiety drugs e.g., diazepam
- monoamine oxidase (MAO) inhibitors e.g., iproniazid, clorgyline, phenelzine and isocarboxazid
- 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 (e
- symptoms of certain fsho05 disorders or neuropsychiatric disorders may be ameliorated by decreasing the level of fshl5w ⁇ gene expression and/or fshlSw ⁇ gene product activity by using f ⁇ hlSw ⁇ gene sequences in conjunction with well-known antisense, gene "knock-out,” ribozyme and/or triple helix methods to decrease the level of f ⁇ hl5w ⁇ gene expression.
- the compounds that may exhibit the ability to modulate the activity, expression or synthesis of the f ⁇ hl5w ⁇ gene including the ability to ameliorate the symptoms of a fshlSw ⁇ disorder or a 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-st v randed 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 f ⁇ hlSw ⁇ gene could be used in an antisense approach to inhibit translation of endogenous f ⁇ hlSw ⁇ 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, l-methylguanine, 1-methylinosine, 2 ,2-dimethylguanine, 2-methyladenine, 2-methylguanine
- 5-methylaminomethyluracil 5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine, 5 ' -methoxycarboxymethyluracil , 5-methoxyuracil, 2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v) , wybutoxosine, pseudouracil,
- the antisense oligonucleotide may also comprise at least one modified sugar moiety selected from the group including but not limited to arabinose, 2-fluoroarabinose, xylulose, and hexose.
- the antisense 0 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 5 formacetal or analog thereof.
- the antisense oligonucleotide is an ⁇ -anomeric oligonucleotide.
- An ⁇ - anomeric oligonucleotide forms specific double-stranded hybrids with complementary RNA in which, contrary to the
- 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 al . , 1987, FEBS Lett. 215, 327-
- 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 al . (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 nucleotides complementary to the target gene coding region sequence could be used, those complementary to the transcribed, untranslated region are most preferred.
- antisense oligonucleotides having the following sequences can be utilized in accordance with the invention: 1. 5'-GTAAAAGACA-3'
- 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: 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 al . , 1980, Cell 22, 787-797), the herpes thymidine kinase promoter (Wagner, et al . , 1981, Proc. Natl. Acad. Sci. U.S.A. 78, 1441-1445) , the regulatory sequences of the metallothionein gene (Brinster, et al . , 1982, Nature 296, 39-42), etc.
- plasmid, cosmid, YAC or viral vector can be used to prepare the recombinant DNA construct which can be introduced directly into the tissue site.
- viral vectors 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 translation of target gene mRNA and, therefore, expression of target gene product. (See, e.g., PCT International Publication WO90/11364, published October 4, 1990; Sarver, et al . , 1990, Science 247, 1222-1225).
- Ribozymes are enzymatic RNA molecules capable of catalyzing the specific cleavage of RNA.
- 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 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 mRNAs
- 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.
- hammerhead ribozymes having the following sequences can be utilized in accordance with the invention: 1. 5 ' - AUUCUAUUGUGCAAAUAUCAAAGCAGGNNNNCCUGAGNAGUCUUAUACUA UUCACAGAAA-3 ' which will cleave between nucleotides 21 and 22 in Figure 1.
- the ribozymes of the present invention also include
- Ciech-type ribozymes such as the one that occurs naturally in Tetrahymena thermophila (known as the IVS, or L-19 IV,S RNA) and that has been extensively described by-Thomas Cech and collaborators (Zaug, et al . , 1984, Science, 224, 574-578; Zaug and Cech,
- Cech-type ribozymes have an eight base pair active site which hybridizes to a target RNA
- 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 any suitable ribozymes. As in the antisense approach, the ribozymes can be any suitable ribozymes.
- a preferred method of delivery involves using a DNA construct "encoding" the ribozyme under the control of a strong constitutive pol III
- ribozymes unlike antisense molecules, are catalytic, a lower intracellular concentration is required for efficiency.
- 35 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, non- functional 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
- 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.
- 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 activi./ 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.
- f ⁇ hlSw ⁇ gene nucleic acid sequences can, for example, be utilized for the treatment of a fshlSw ⁇ disorder or a neuropsychiatric disorder, such as BAD. Such treatment can be administered, for example, in the form of gene replacement therapy.
- one or more copies of a normal fshl5w ⁇ gene or a portion of the fshl5w ⁇ gene that directs the production of a fshlSw ⁇ gene product exhibiting normal fshl5w ⁇ 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. Because the f ⁇ hlSw ⁇ gene is expressed in the brain, such gene replacement therapy techniques should be capable delivering f ⁇ hlSw ⁇ gene sequences to these cell types within patients.
- techniques that are well known to those of skill in the art (see, e.g., PCT Publication No. WO89/10134, published April 25, 1988) can be used to enable f ⁇ hlSw ⁇ 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 f ⁇ hlSw ⁇ gene sequences to the site of the cells in which the f ⁇ hlSw ⁇ gene sequences are to be expressed.
- Additional methods that may be utilized to increase the overall level of f ⁇ hlSw ⁇ gene expression and/or f ⁇ hlSw ⁇ gene product activity include the introduction of appropriate f ⁇ hlSw ⁇ -expressing cells, preferably autologous cells, into a patient at positions and in numbers that are sufficient to ameliorate the symptoms of a f ⁇ hl5w ⁇ disorder or a neuropsychiatric disorder, such as BAD.
- Such cells may be either recombinant or non-recombinant.
- cells that can be administered to increase the overall level of fshlSw ⁇ gene expression in a patient are normal cells, preferably brain cells, that. express the fshlSw ⁇ gene.
- cells preferably autologous cells, can be engineered to express fshlSw ⁇ gene sequences, and may then be introduced into a patient in positions appropriate for the amelioration of the symptoms of a f ⁇ hlSw ⁇ disorder or a neuropsychiatric disorder, such as BAD.
- cells that express an unimpaired fshlSw ⁇ gene and that are from a MHC matched individual can be utilized, and may include, for example, brain cells.
- the expression of the fshl5w ⁇ 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.
- 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 fshlSw ⁇ 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 f ⁇ hl5w ⁇ gene expression or gene product activity can be administered to a patient at therapeutically effective doses to treat or ameliorate a fshl5w ⁇ disorder or a neuropsychiatric disorder, such as BAD.
- 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 S0 (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 50 .
- 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 concentrat on 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 ethylcellulose) ; fillers (e.g., lactose, microcrystalline cellulose or calcium hydrogen phosphate) ; lubricants (e.g., magnesium stearate, talc or silica); disintegrants (e.g., potato starch or sodium starch glycolate); or wetting agents (e.g., sodium lauryl sulphate).
- binding agents e.g., pregelatinised maize starch, polyvinylpyrrolidone or hydroxypropyl ethylcellulose
- fillers e.g., lactose, microcrystalline cellulose or calcium hydrogen phosphate
- lubricants e.g., magnesium stearate, talc or silica
- 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., lecithin or acacia); non-aqueous vehicles (e.g., almond oil, oily esters, ethyl alcohol or fractionated vegetable oils); and 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.
- Preparations for oral administration may be suitably formulated to give controlled release of the active compound.
- compositions 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.
- the dosage unit may be determined by providing a valve to deliver a metered amount.
- Capsules and cartridges of e.g., gelatin for use in an inhaler or insufflator may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.
- 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 tne 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 IDENTIFICATION AND CLONING OF THE fsh!5w6 GENE
- studies are described that, first, define an interval approximately 500 kb on the long arm of human chromosome 18 within which a region associated with a neuropsychiatric disorder is located and, second, identify and clone a novel gene, referred to herein as f ⁇ hl5w ⁇ , which lies within this region and which can be involved in neuropsychiatric disorders.
- Linkage Diseguilibrium Linkage disequilibrium (LD) studies were performed using DNA from a population sample of neuropsychiatric disorder (BP-I) - patients. The population sample and LD techniques were as described in Friemer et al., 1996, Nature Genetics .12:436-441. The present LD study took advantage of the additional physical markers identified via the physical mapping techniques described below.
- Yeast artificial chromosome mapping.
- yeast artificial chromosomes containing human sequences were mapped to the region being analyzed based on publicly available maps (Cohen et al . , 1993, CR. Acad. Sci. 316, 1484-1488).
- 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
- BAC Bacterial artificial chromosome mapping.
- the STSs from the region 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 microsatellites 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.
- cDNA selection was used as an additional method for gene identification of transcribed sequences over 1 arge regions of the genome. Through a combination of characterizations including physical mapping
- RNA hybridization 25 and RNA hybridization, the selected cDNAs were arranged into transcription units.
- the cDNA selection technique was carried out as described by Rommens, et al . (1994, in Identification of Tran ⁇ cribed Sequences , Hochgeschwender and Gardiner, eds., Plenum Press, New York, pp. 65-79).
- 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.
- the new markers identified via physical mapping were typed in an LD analysis of samples collected from families affected with bipolar affective disorder.
- One interpretation of the results of this LD analysis narrows down the chromosome 18 long arm region within which a gene involved in neuropsychiatric disorders lies to an interval of about 500 kb between the publicly available markers D18S1121 and D18S380.
- the BAC clones within the newly identified 500 kb neuropsychiatric disorder region were further 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 .
- f ⁇ hl5w ⁇ The corresponding f ⁇ hlSw ⁇ gene can, therefore, can be involved in neuropsychiatric disorders.
- f ⁇ hlSw ⁇ cDNA clones were isolated through screening and random sequencing of a human fetal brain cDNA library. Among the cDNA clones identified was Epfshl5w6 (ATCC accession No. 98351) . Upon further analysis of genomic sequences, it was determined that the full length fshlSw ⁇ gene sequence is contained within BAC54 (Identification Reference EpHS996, ATCC Accession No. 98363) . f ⁇ hl ⁇ w ⁇ nucleotide and amino acid sequences are shown in Figure l.
- the fshl ⁇ w ⁇ gene product sequence depicted in Figure l exhibits some amino acid sequence similarity with two known genes identified from other 5 distantly related species.
- the depicted portion of the fshl5w ⁇ gene product exhibits approximately 45% amino acid sequence similarity with a 295 amino acid residue portion of P36, a possible Lei ⁇ hmania amazonensi ⁇ quinone oxidoreductase (Liu and Chang, 1994, Mol. Biochem. Parasitol.
- the depicted portion of the f ⁇ hl5w ⁇ gene product also exhibits approximately 45% amino acid sequence similarity with a 292 amino acid portion-,of ARP, an Arabidopsis thaliana NADPH oxidoreductase homolog (Babiychuk, et al . , 1995, J. Biol. Chem. 270, 26224-26231).
Abstract
Description
Claims
Priority Applications (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CA002285013A CA2285013A1 (en) | 1997-03-27 | 1998-03-27 | Methods and compositions for the diagnosis and treatment of neuropsychiatric disorders |
AU67868/98A AU6786898A (en) | 1997-03-27 | 1998-03-27 | Methods and compositions for the diagnosis and treatment of neuropsychiatric di sorders |
EP98913275A EP0977768A1 (en) | 1997-03-27 | 1998-03-27 | Methods and compositions for the diagnosis and treatment of neuropsychiatric disorders |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US08/828,007 | 1997-03-27 | ||
US08/828,007 US5866412A (en) | 1997-03-27 | 1997-03-27 | Chromosome 18 marker |
Publications (1)
Publication Number | Publication Date |
---|---|
WO1998042724A1 true WO1998042724A1 (en) | 1998-10-01 |
Family
ID=25250694
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/US1998/006211 WO1998042724A1 (en) | 1997-03-27 | 1998-03-27 | Methods and compositions for the diagnosis and treatment of neuropsychiatric disorders |
Country Status (5)
Country | Link |
---|---|
US (1) | US5866412A (en) |
EP (1) | EP0977768A1 (en) |
AU (1) | AU6786898A (en) |
CA (1) | CA2285013A1 (en) |
WO (1) | WO1998042724A1 (en) |
Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2001034173A1 (en) * | 1999-11-08 | 2001-05-17 | Millennium Pharmaceuticals, Inc. | METHODS AND COMPOSITIONS FOR DIAGNOSING AND TREATING CHROMOSOME 18q RELATED DISORDERS |
WO2001063294A2 (en) * | 2000-02-24 | 2001-08-30 | Oxford Glycosciences (Uk) Limited | Diagnosis of bipolar affective disorder (bad) and unipolar depression |
US6399762B1 (en) | 1998-03-16 | 2002-06-04 | The Regents Of The University Of California | Methods and compositions for diagnosing and treating chromosome -18p related disorders |
Families Citing this family (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2000024875A1 (en) * | 1998-10-23 | 2000-05-04 | Northwestern University | Compositions and methods useful for treatment of depressive disorder based on an animal model |
EP1232245A1 (en) | 1999-10-14 | 2002-08-21 | The Trustees Of The University Of Pennsylvania | Gene and methods for diagnosing neuropsychiatric disorders and treating such disorders |
AU3008601A (en) * | 1999-12-08 | 2001-06-18 | International Centre For Genetic Engineering And Biotechnology | Plastid transformation |
JP3507884B2 (en) * | 2000-03-07 | 2004-03-15 | 新潟大学長 | Objective diagnosis of schizophrenia using gene expression as an index |
US7101666B2 (en) * | 2001-08-31 | 2006-09-05 | Centre For Addiction And Mental Health | Involvement of the BDNF gene in mood disorders |
EP1594985A1 (en) * | 2003-02-19 | 2005-11-16 | Universite Laval | Method for determining susceptibility to schizophrenia |
Family Cites Families (14)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US4109496A (en) * | 1977-12-20 | 1978-08-29 | Norris Industries | Trapped key mechanism |
US4215051A (en) * | 1979-08-29 | 1980-07-29 | Standard Oil Company (Indiana) | Formation, purification and recovery of phthalic anhydride |
US4376110A (en) * | 1980-08-04 | 1983-03-08 | Hybritech, Incorporated | Immunometric assays using monoclonal antibodies |
US4873191A (en) * | 1981-06-12 | 1989-10-10 | Ohio University | Genetic transformation of zygotes |
GB8308235D0 (en) * | 1983-03-25 | 1983-05-05 | Celltech Ltd | Polypeptides |
US4902505A (en) * | 1986-07-30 | 1990-02-20 | Alkermes | Chimeric peptides for neuropeptide delivery through the blood-brain barrier |
US4946778A (en) * | 1987-09-21 | 1990-08-07 | Genex Corporation | Single polypeptide chain binding molecules |
US5116742A (en) * | 1986-12-03 | 1992-05-26 | University Patents, Inc. | RNA ribozyme restriction endoribonucleases and methods |
US4987071A (en) * | 1986-12-03 | 1991-01-22 | University Patents, Inc. | RNA ribozyme polymerases, dephosphorylases, restriction endoribonucleases and methods |
US4904582A (en) * | 1987-06-11 | 1990-02-27 | Synthetic Genetics | Novel amphiphilic nucleic acid conjugates |
US5530101A (en) * | 1988-12-28 | 1996-06-25 | Protein Design Labs, Inc. | Humanized immunoglobulins |
US5075217A (en) * | 1989-04-21 | 1991-12-24 | Marshfield Clinic | Length polymorphisms in (dC-dA)n ·(dG-dT)n sequences |
US5399346A (en) * | 1989-06-14 | 1995-03-21 | The United States Of America As Represented By The Department Of Health And Human Services | Gene therapy |
US5364759B2 (en) * | 1991-01-31 | 1999-07-20 | Baylor College Medicine | Dna typing with short tandem repeat polymorphisms and identification of polymorphic short tandem repeats |
-
1997
- 1997-03-27 US US08/828,007 patent/US5866412A/en not_active Expired - Fee Related
-
1998
- 1998-03-27 CA CA002285013A patent/CA2285013A1/en not_active Abandoned
- 1998-03-27 EP EP98913275A patent/EP0977768A1/en not_active Withdrawn
- 1998-03-27 WO PCT/US1998/006211 patent/WO1998042724A1/en not_active Application Discontinuation
- 1998-03-27 AU AU67868/98A patent/AU6786898A/en not_active Abandoned
Non-Patent Citations (4)
Title |
---|
BARON M.: "GENETIC LINKAGE AND BIPOLAR AFFECTIVE DISORDER: PROGRESS AND PITFALLS.", MOLECULAR PSYCHIATRY, BASINGSTOKE, GB, vol. 02., 1 January 1997 (1997-01-01), GB, pages 200 - 210., XP002911545, ISSN: 1359-4184, DOI: 10.1038/sj.mp.4000211 * |
EWALD H., ET AL.: "SUSCEPTIBILITY LOCI FOR BIPOLAR AFFECTIVE DISORDER ON CHROMOSOME 18? A REVIEW AND A STUDY OF DANISH FAMILIES.", PSYCHIATRIC GENETICS., RAPID COMMUNICATIONS OF OXFORD LTD., XX, vol. 07., 1 January 1997 (1997-01-01), XX, pages 01 - 12., XP002911544, ISSN: 0955-8829 * |
LEWIN B.: "GENES 4.", 1 January 1990, OXFORD, OUP., GB, article LEWIN B.: "GENESIS IV GLOSSARY, PASSAGE.", pages: 805., XP002911547, 017981 * |
MACKINNON D. F., ET AL.: "GENETICS OF MANIC DEPRESSIVE ILLNESS.", ANNUAL REVIEW OF NEUROSCIENCE., ANNUAL REVIEWS INC., PALO ALTO, CA, US, vol. 02., 1 January 1997 (1997-01-01), US, pages 355 - 373., XP002911546, ISSN: 0147-006X, DOI: 10.1146/annurev.neuro.20.1.355 * |
Cited By (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US6399762B1 (en) | 1998-03-16 | 2002-06-04 | The Regents Of The University Of California | Methods and compositions for diagnosing and treating chromosome -18p related disorders |
WO2001034173A1 (en) * | 1999-11-08 | 2001-05-17 | Millennium Pharmaceuticals, Inc. | METHODS AND COMPOSITIONS FOR DIAGNOSING AND TREATING CHROMOSOME 18q RELATED DISORDERS |
WO2001063294A2 (en) * | 2000-02-24 | 2001-08-30 | Oxford Glycosciences (Uk) Limited | Diagnosis of bipolar affective disorder (bad) and unipolar depression |
WO2001063294A3 (en) * | 2000-02-24 | 2002-04-11 | Oxford Glycosciences Uk Ltd | Diagnosis of bipolar affective disorder (bad) and unipolar depression |
Also Published As
Publication number | Publication date |
---|---|
CA2285013A1 (en) | 1998-10-01 |
EP0977768A1 (en) | 2000-02-09 |
AU6786898A (en) | 1998-10-20 |
US5866412A (en) | 1999-02-02 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US6248528B1 (en) | Methods and compositions for the diagnosis and treatment of neuropsychiatric disorders | |
US5866412A (en) | Chromosome 18 marker | |
US5955355A (en) | Chromosome 18 marker | |
WO2004040016A2 (en) | Genetic markers on chromosome 12 associated to bipolar or unipolar disorders | |
WO1998042362A9 (en) | Methods and compositions for the diagnosis and treatment of neuropsychiatric disorders | |
CA2335378A1 (en) | Methods and compositions for the diagnosis and treatment of body weight disorders, including obesity | |
US5914394A (en) | Methods and compositions for the diagnosis and treatment of neuropsychiatric disorders | |
AU3354999A (en) | Methods and compositions for diagnosing and treating chromosome-18p related disorders | |
US5939316A (en) | Chromosome 18 marker | |
EP1068347A1 (en) | Methods and compositions for the diagnosis and treatment of neuropsychiatric disorders | |
CA2298474A1 (en) | Methods and compositions for the diagnosis and treatment of neuropsychiatric disorders | |
WO1998042723A9 (en) | Methods and compositions for the diagnosis and treatment of neuropsychiatric disorders | |
WO1999035290A1 (en) | Methods and compositions for the diagnosis and treatment of neuropsychiatric disorders | |
WO2001034173A1 (en) | METHODS AND COMPOSITIONS FOR DIAGNOSING AND TREATING CHROMOSOME 18q RELATED DISORDERS | |
WO1998005969A1 (en) | Mammalian and human fxi-t1 | |
MXPA00009702A (en) | Methods and compositions for the diagnosis and treatment of neuropsychiatric disorders | |
WO2001034630A1 (en) | METHODS AND COMPOSITIONS FOR DIAGNOSING AND TREATING CHROMOSOME 18q RELATED DISORDERS | |
MXPA00009801A (en) | Methods and compositions for the diagnosis and treatment of neuropsychiatric disorders | |
WO2001034771A2 (en) | METHODS AND COMPOSITIONS FOR DIAGNOSING AND TREATING CHROMOSOME 18q RELATED DISORDERS | |
WO2001034841A1 (en) | METHODS AND COMPOSITIONS FOR DIAGNOSING AND TREATING CHROMOSOME 18q RELATED DISORDERS | |
WO2001033964A1 (en) | METHODS AND COMPOSITIONS FOR DIAGNOSING AND TREATING CHROMOSOME 18q RELATED DISORDERS | |
AU5118399A (en) | Methods and compositions for the diagnosis and treatment of body weight disorders, including obesity |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
AK | Designated states |
Kind code of ref document: A1 Designated state(s): AU CA JP |
|
AL | Designated countries for regional patents |
Kind code of ref document: A1 Designated state(s): AT BE CH DE DK ES FI FR GB GR IE IT LU MC NL PT SE |
|
DFPE | Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101) | ||
121 | Ep: the epo has been informed by wipo that ep was designated in this application | ||
ENP | Entry into the national phase |
Ref document number: 2285013 Country of ref document: CA Ref country code: CA Ref document number: 2285013 Kind code of ref document: A Format of ref document f/p: F |
|
WWE | Wipo information: entry into national phase |
Ref document number: 1998913275 Country of ref document: EP |
|
WWP | Wipo information: published in national office |
Ref document number: 1998913275 Country of ref document: EP |
|
NENP | Non-entry into the national phase |
Ref country code: JP Ref document number: 1998546043 Format of ref document f/p: F |
|
WWW | Wipo information: withdrawn in national office |
Ref document number: 1998913275 Country of ref document: EP |