+

WO1998011222A1 - Methodes de selection de composes utilisables pour traiter la schizophrenie - Google Patents

Methodes de selection de composes utilisables pour traiter la schizophrenie Download PDF

Info

Publication number
WO1998011222A1
WO1998011222A1 PCT/US1997/016219 US9716219W WO9811222A1 WO 1998011222 A1 WO1998011222 A1 WO 1998011222A1 US 9716219 W US9716219 W US 9716219W WO 9811222 A1 WO9811222 A1 WO 9811222A1
Authority
WO
WIPO (PCT)
Prior art keywords
gtiii
cell
gene
compounds
expression
Prior art date
Application number
PCT/US1997/016219
Other languages
English (en)
Inventor
Pullanipally Shashidharan
Original Assignee
The Mount Sinai School Of Medicine Of The City University Of New York
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by The Mount Sinai School Of Medicine Of The City University Of New York filed Critical The Mount Sinai School Of Medicine Of The City University Of New York
Priority to AU44809/97A priority Critical patent/AU4480997A/en
Publication of WO1998011222A1 publication Critical patent/WO1998011222A1/fr

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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5091—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing the pathological state of an organism
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/07—Animals genetically altered by homologous recombination
    • A01K2217/075—Animals genetically altered by homologous recombination inducing loss of function, i.e. knock out
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/30—Psychoses; Psychiatry
    • G01N2800/302—Schizophrenia

Definitions

  • the present invention relates to screening assays for compounds that modulate the activity of the neuron-specific glutamate transporter (glutamate transporter III) , and their use in the treatment of schizophrenia and other neurological disorders mediated by abnormal activity of glutamate transporter III.
  • glutamate transporter III neuron-specific glutamate transporter
  • glutamate is released from nerve terminals during neurotransmission serving as an excitatory neurotransmitter .
  • Synaptic function of glutamate is thought to be terminated by rapid uptake into the surrounding glial cells or into the pre-synaptic nerve terminals (Nicholls, D.G., 1993, Eur. J. Biochem. 212:613- 631) .
  • This high affinity uptake is accomplished by carrier proteins, located in the plasma membrane of glutamatergic nerve terminals and of glial cells (Nicholls, D.G. , 1993, Eur. J. Biochem. 212:613-631).
  • the uptake does not discriminate between aspartate or glutamate and depends on the co-transport of Na + , and counter-transport of K + .
  • these proteins are known as Na + /K * dependent aspartate/glutamate transporters .
  • the human aspartate/glutamate transporter III (Shashidharan et al., 1994, Brain Research 662:245-250 which is incorporated by reference herein in its entirety) , is homologous to a rabbit glutamate carrier (Kanai, Y., et al., 1992, Nature, 360:467-471) but it shows low homology to two human glutamate transporters previously described (Shashidharan, P., et al., 1993, Biochim. Biophys. Acta. 1216:161-164); (Shashidharan, P., et al., 1994, Biochim. Biophys. Acta. 1191:393-396).
  • human aspartate/glutamate transporter III is expressed in both neural and non-neural tissues.
  • this transporter mRNA is present in neurons and shows an extensive distribution pattern in cerebral cortical layers, substantia nigra, rad nucleus and hippocampus.
  • the glutamate transporter III is the only glutamate transporter known to be expressed outside the central nervous system.
  • glutamatergic pre- synaptic mechanisms are altered in neurodegenerative disorders, such as amyotrophic lateral sclerosis, and in metabolic insults (ischemia, hypoglycemia, and thiamine deficiency) that lead to neuronal degeneration through excitotoxic mechanisms.
  • metabolic insults ischemia, hypoglycemia, and thiamine deficiency
  • glutamate transporters in various neurologic disorders is unknown.
  • the present invention relates to screening assays to identify compounds that modulate the activity of the glutamate transporter III (hereinafter "GTIII") and their use for the treatment of schizophrenia, and other neurological disorders mediated by GTIII.
  • GTIII glutamate transporter III
  • the invention also relates to the compounds identified in the assays, diagnostic methods and treatment.
  • the invention is based, in part, on the discovery of a selective and significant reduction of the mRNA that encodes the neuron-specific GTIII in the brains of schizophrenics, implicating a role of the transporter in the pathophysiology of schizophrenia.
  • the compounds which modulate the activity of GTIII may be useful as antipsychotics for treating schizophrenia or other neurological disorders mediated by GTIII, including but not limited to Parkinsons or Alzheimer's disease.
  • the invention relates to assays designed to screen for compounds or compositions that modulate GTIII activity.
  • cell-based assays or non-cell based assays can be used to detect compounds that interact with, e.g.. bind to the GTIII.
  • the cell-based assays have the advantage in that they can be used to identify compounds that affect GTIII biological activity, i.e. , glutamate transport.
  • the invention also relates to assays designed to screen for compounds or compositions that modulate GTIII gene expression.
  • cell-based assays, or cell-lysate assays e.g. , in vitro transcription or translation assays
  • can be used to screen for compounds or compositions that modulate GTIII transcription e.g. , compounds that modulate expression, production or activity of transcription factors involved in GTIII gene expression; polynucleotides that form triple helical structures with an GTIII regulatory region and inhibit transcription of the GTIII gene, etc.
  • cell-based assays or cell-lysate assays can be used to screen for compounds or compositions that modulate translation of GTIII transcripts (e.g. , antisense and ribozyme molecules) .
  • the cell-based assays or cell-lysate assays can be used to test polynucleotide constructs designed to modify the expression of the GTIII gene jLn vivo.
  • constructs include polynucleotide constructs designed for gene therapy; e.g. , expression constructs or gene replacement constructs that place the GTIII gene under the control of a strong promoter system, an inducible promoter system or a constitutive promoter system.
  • the invention also encompasses agonists and antagonists of GTIII, including small molecules, large molecules, and antibodies, as well as nucleotide sequences that can be used to inhibit GTIII gene expression (e.g.
  • Such compounds may be used to treat schizophrenia, or other GTIII- mediated neuropathologies.
  • the invention also encompasses the use of such compounds and compositions, including gene therapy approaches, that modulate GTIII activity or GTIII gene expression to treat schizophrenia, or other GTIII-mediated neuropathologies.
  • GTIII nucleotides or coding sequences means DNA sequences encoding GTIII mRNA transcripts, GTIII protein, polypeptide or peptide fragments of GTIII protein, or GTIII fusion proteins.
  • GTIII nucleotide sequences encompass DNA, including genomic DNA (e.g. the GTIII gene) or cDNA.
  • GTIII means GTIII gene products, e.g.. transcripts and the GTIII protein. Polypeptides or peptide fragments of the GTIII protein are referred to as GTIII polypeptides or GTIII peptides. Fusions of GTIII, or GTIII polypeptides, or peptide fragments to an unrelated protein are referred to herein as GTIII fusion proteins.
  • a functional GTIII refers to a protein which transports glutamate into a cell.
  • ECD means "extracellular domain”.
  • TM means “transmembrane domain”.
  • CD means "cytoplasmic domain”.
  • FIG. 1 Nucleotide and deduced amino acid sequence of the human glutamate transporter III cDNA. Nucleotides corresponding to the homologous rabbit glutamate transporter (Kanai, Y. , et al., 1992, Nature, 360:467-471) are shown in italics directly above the human nucleotide sequence only where they are different. Also, predicted amino acids corresponding to the rabbit cDNA (Kanai, Y., et al., 1992, Nature, 360:467-471) are also shown in italics directly below the human amino acid sequence only where they are different. Insertions are marked by relevant bases and deletions by dashes. The nucleotide number is indicated on the left and the stop codon is indicated with an asterisk. The ten putative transmembrane regions are underlined; the potential W-glycosylation sites are indicated by double underlines.
  • the invention encompasses screening methods (e.g. , assays) for the identification of compounds which modulate the activity of GTIII.
  • the invention also encompasses agonists and antagonists of GTIII, including small molecules, large molecules, and antibodies, as well as nucleotide sequences that can be used to inhibit GTIII gene expression (e.g. , antisense and ribozyme molecules) , and gene or regulatory sequence replacement constructs designed to enhance GTIII gene expression (e.g. , expression constructs that place the GTIII gene under the control of a strong promoter system) .
  • Such compounds may be used to treat schizophrenics, or patients suffering from other neurological disorders mediated by GTIII.
  • cellular and non-cellular assays are described that can be used to identify compounds that interact with the GTIII, e.g. , modulate the activity of the GTIII and/or bind to the GTIII.
  • Such cell-based assays of the invention utilize cells, cell lines, or engineered cells that express the GTIII; including genetically engineered host cells that express GTIII, or GABAergic cells or cell lines.
  • the invention also encompasses the use of cell-based assays or cell-lysate assays (e.g. , in vitro transcription or translation assays) to screen for compounds or compositions that modulate GTIII gene expression.
  • constructs containing a reporter sequence linked to a regulatory element of the GTIII gene can be used in engineered cells, or in cell lysate extracts, to screen for compounds that modulate the expression of the reporter gene product at the level of transcription.
  • assays could be used to identify compounds that modulate the expression or activity of transcription factors involved in GTIII gene expression, or to test the activity of triple helix polynucleotides.
  • engineered cells or translation extracts can be used to screen for compounds (including antisense and ribozyme constructs) that modulate the translation of GTIII mRNA transcripts, and therefore, affect expression of the GTIII.
  • the invention also encompasses GTIII proteins, polypeptides (including soluble GTIII polypeptides or peptides) , GTIII fusion proteins, and peptides (including synthetic peptides) derived from the GTIII amino acid sequence for use in non-cell based screening assays, for use in generating antibodies, for diagnostics and therapeutics.
  • the GTIII is predicted to traverse the cell membrane ten times (see FIG. 1) .
  • the domains located between the transmembrane domain contribute to the extracellular domain and cytoplasraic domain of GTIII.
  • Peptides corresponding to each domain or polypeptides composed of two or more of the domains linked together can be engineered as described in Section 5.4.1, infra.
  • Such peptides or polypeptides can be fused to a heterologous protein, e.g.. a reporter, an Ig Fc region, etc., to yield a fusion protein.
  • a heterologous protein e.g.. a reporter, an Ig Fc region, etc.
  • Such peptides, polypeptides and fusion proteins may be used in the non-cell based assays for screening compounds that interact with or bind to the GTIII.
  • the invention also encompasses diagnostic assays for schizophrenia, or other disorders mediated by GTIII, or other disorders mediated by GTIII, using hybridization analyses to detect mutations in a patient's GTIII gene, or the level of transcription of GTIII protein in patient samples.
  • Analysis of genomic DNA for mutations in the patient's GTIII gene can be performed on any cell sample taken from the patient, e.g. , blood samples, skin, bone marrow, etc.
  • the patient material used to analyze transcription levels of GTIII should be obtained from tissues that express the GTIII gene, e.g.. hippocampus, brain, neuronal tissue samples.
  • antibodies to GTIII can be used to detect levels of expression of GTIII protein in patient samples, or in situ using imaging techniques; e.g..
  • GTIII expression is also within the scope of the invention.
  • nucleotide constructs encoding functional GTIIIs, mutant GTIIIs, as well as antisense and ribozyme molecules can be used to modulate GTIII expression.
  • the invention also encompasses pharmaceutical formulations and methods for treatment.
  • GTIII The specific role of GTIII in schizophrenia was investigated by studying the expression of GTIII in the brains of schizophrenics vs. normal controls.
  • a Taq 1 fragment of the GTIII cDNA i.e.. nucleotide 1065-1319 of FIG. 1 was used as a probe.
  • a two-way ANOVA group by region indicated that there was a reduction in GTIII mRNA in schizophrenics (p ⁇ .0001) when compared to normal controls. Comparisons for each region indicated that the difference was statistically significant (p ⁇ 105) in dentate, CA1 and subiculum where levels were 69, 67 and 61% respectively, of normal levels. Decreased levels were also observed (.08> p>.05) in CA4 , presubiculum and parahippocampal layers ii/iii.
  • RNA protection studies and the in situ hybridization studies indicate that aberrant hippocampal expression of GTIII plays a role in the neuropathology of schizophrenia.
  • At least three different assay systems can be designed and used to identify compounds or compositions that modulate GTIII activity or
  • kits The systems described below may be formulated into kits.
  • GTIII or cells expressing GTIII can be packaged in a variety of containers, e.g. , vials, tubes, microtitre well plates, bottles, and the like.
  • Other reagents can be included in separate containers and provided with the kit; e.g. , positive controls samples, negative control samples, buffers, cell culture media, etc.
  • a cell-based assay system can be used to screen for compounds that modulate the activity of the GTIII, and, therefore, may be useful to treat schizophrenia and other neurological disorders mediated by GTIII.
  • Such disorders may include Parkinsons and Alzheimer's.
  • cells that endogenously express GTIII can be used in the assay, e.g. , GABAergic cells such as Purkinje cells of the cerebellum.
  • GABAergic cells such as Purkinje cells of the cerebellum.
  • cell lines such as 293 cells, COS cells, CHO cells, fibroblasts, and the like, genetically engineered to express GTIII can be used for screening purposes.
  • host cells genetically engineered to express a functional GTIII that transports glutamate should be used.
  • Glutamate transport can be measured using standard methods known to those skilled in the art to assay for glutamate uptake by the host cell. For example, cellular uptake of labeled glutamate from the culture media can be measured. Alternatively, when GABAergic cells are used, host cell production of GABA can be measured (glutamate taken up by such cells is a precursor for the synthesis of GABA) .
  • the cells expressing GTIII are exposed to a test compound or to vehicle controls (e.g. , placebos) . After exposure, the cells can be assayed to measure glutamate transport. The ability of a test compound to increase levels of transport, above those levels seen with cells treated with a vehicle control, indicates that the test compound enhances the activity of GTIII expressed by the host cell.
  • vehicle controls e.g. , placebos
  • non-cell based assay systems may be used to identify compounds that interact with, e.g. , bind to GTIII. Such compounds may act as antagonists or agonists of GTIII activity.
  • Isolated membranes may be used to identify compounds that interact with GTIII. For example, in a typical experiment using isolated membranes, 293 cells may be genetically engineered to express the GTIII. Membranes can be harvested by standard techniques and used in an . in vitro binding assay, e.g. , labeled test compound.
  • soluble GTIII may be recombinantly expressed and utilized in non-cell based assays to identify compounds that bind to GTIII.
  • Recombinantly expressed GTIII polypeptides, peptides, or fusion proteins containing one or more of the domains that contribute to the ECD prepared as described in Section 5.4.1, infra can be used in the non- cell based screening assays.
  • peptides corresponding to the domains that contribute to the CD of GTIII, or fusion proteins containing one or more of these peptides can be used in non-cell based assay systems to identify compounds that bind to the cytoplasmic portion of the GTIII; such compounds may be useful to modulate the activity of GTIII.
  • the recombinantly expressed GTIII can be conveniently attached to a solid substrate such as a test tube, microtitre well or a column, by means well known to those in the art (see Ausubel et al., supra) .
  • the test compounds are then assayed for their ability to bind to the GTIII.
  • In vitro cell based assays may be designed to screen for compounds that regulate GTIII expression at either the transcriptional or translational level.
  • DNA encoding a reporter molecule can be linked to a regulatory element of the GTIII gene and used in appropriate intact cells, cell extracts or lysates to identify compounds that modulate GTIII gene expression.
  • Appropriate cells or cell extracts are prepared from any cell type that normally expresses the GTIII gene, thereby ensuring that the cell extracts contain the transcription factors required for .in vitro or in vivo transcription.
  • the screen can be used to identify compounds that modulate the expression of the reporter construct. In such screens, the level of reporter gene expression is determined in the presence of the test compound and compared to the level of expression in the absence of the test compound.
  • cells or in vitro cell lysates containing GTIII transcripts may be tested for modulation of GTIII mRNA translation.
  • test compounds are assayed for their ability to modulate the translation of GTIII mRNA in in vitro translation extracts.
  • Compounds that increase the level of GTIII expression, either at the transcriptional or translational level, may be useful for treatment of schizophrenia.
  • the compounds which may be screened in accordance with the invention include, but are not limited to peptides, antibodies and fragments thereof, and other organic compounds (e.g. , peptidomimetics) that bind to GTIII and either enhance the activity of GTIII as well as peptides, antibodies or fragments thereof, and other organic compounds.
  • organic compounds e.g. , peptidomimetics
  • Compounds may include, but are not limited to, peptides such as, for example, soluble peptides, including but not limited to members of random peptide libraries; (see, e.g. , Lam, K.S. et al. , 1991, Nature 354:82-84 ; Houghten, R. 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, Z.
  • peptides such as, for example, soluble peptides, including but not limited to members of random peptide libraries; (see, e.g. , Lam, K.S. et al. , 1991, Nature 354:82-84 ; Houghten, R. et al., 1991, Nature 3
  • 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.
  • Other compounds which can be screened in accordance with the invention include but are not limited to small organic molecules that are able to cross the blood-brain barrier, gain entry into an appropriate cell and affect the expression of the GTIII gene; or such compounds that affect the activity of the GTIII or the activity.
  • Computer modelling and searching technologies permit identification of compounds, or the improvement of already identified compounds, that can modulate GTIII expression or activity. Having identified such a compound or composition, the active sites or regions are identified. Such active sites might typically be ligand binding sites.
  • the active site can be identified using methods known in the art including, for example, from the amino acid sequences of peptides, from the nucleotide sequences of nucleic acids, or from study of complexes of the relevant compound or composition with its natural ligand. In the latter case, chemical or X-ray crystallographic methods can be used to find the active site by finding where on the factor the complexed ligand is found.
  • the three dimensional geometric structure of the active site is determined. This can be done by known methods, including X-ray crystallography, which can determine a complete molecular structure. On the other hand, solid or liquid phase NMR can be used to determine certain intramolecular distances. Any other experimental method of structure determination can be used to obtain partial or complete geometric structures.
  • the geometric structures may be measured with a complexed ligand, natural or artificial, which may increase the accuracy of the active site structure determined.
  • the methods of computer based numerical modelling can be used to complete the structure or improve its accuracy.
  • Any recognized modelling method may be used, including parameterized models specific to particular biopolymers such as proteins or nucleic acids, molecular dynamics models based on computing molecular motions, statistical mechanics models based on thermal ensembles, or combined models.
  • standard molecular force fields representing the forces between constituent atoms and groups, are necessary, and can be selected from force fields known in physical chemistry.
  • the incomplete or less accurate experimental structures can serve as constraints on the complete and more accurate structures computed by these modeling methods.
  • candidate modulating compounds can be identified by searching databases containing compounds along with information on their molecular structure. Such a search seeks compounds having structures that match the determined active site structure and that interact with the groups defining the active site. Such a search can be manual, but is preferably computer assisted. These compounds found from this search are potential GTIII modulating compounds.
  • these methods can be used to identify improved modulating compounds from an already known modulating compound or ligand.
  • the composition of the known compound can be modified and the structural effects of modification can be determined using the experimental and computer modelling methods described above applied to the new composition.
  • the altered structure is then compared to the active site structure of the compound to determine if an improved fit or interaction results. In this manner systematic variations in composition, such as by varying side groups, can be quickly evaluated to obtain modified modulating compounds or ligands of improved specificity or activity.
  • CHARMm performs the energy minimization and molecular dynamics functions.
  • QUANTA performs the construction, graphic modelling and analysis of molecular structure.
  • QUANTA allows interactive construction, modification, visualization, and analysis of the behavior of molecules with each other.
  • a number of articles review computer modelling of drugs interactive with specific proteins, such as Rotivinen, et al.) 1988, Acta Pharmaceutical Fennica 97:159-166); Ripka (1988 New Scientist 54-57); McKinaly and Rossmann (1989, Annu. Rev. Pharmacol. Toxiciol. 29:111-122); Perry and
  • Compounds identified via assays such as those described herein may be useful, for example, in elaborating the biological function of the GTIII gene product, and for ameliorating schizophrenia, or other GTIII-mediated disorders.
  • Assays for testing the efficacy of compounds identified in the cellular screen can be tested in animal model systems.
  • animal models may be used as test substrates for the identification of drugs, pharmaceuticals, therapies and interventions which may be effective in treating schizophrenia and other neurological disorders.
  • animal models may be exposed to a compound. The response of the animals to the exposure may be monitored by assessing the level of glutamate in the nervous tissue of the animals, or the plasma concentration of glutamate.
  • Compounds that stimulate glutamate transport (and reduce plasma concentration and glutamate accumulation in the nervous tissue) should be useful for treating schizophrenia. Dosages of test agents may be determined by deriving dose-response curves, as discussed in Section 5.6, below.
  • Transgenic animals that express the human GTIII gene products, or defective GTIII can be used as model systems. Animals of any species, including, but not limited to, mice, rats, rabbits, guinea pigs, pigs, micro-pigs, goats, and non- human primates, e.g. , baboons, monkeys, and chimpanzees may be used to generate GTIII transgenic animals.
  • Any technique known in the art may be used to introduce the human GTIII transgene into animals to produce the founder lines of transgenic animals.
  • Such techniques include, but are not limited to pronuclear microinjection (Hoppe, P.C. 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 .
  • the present invention provides for transgenic animals that carry the GTIII transgene in all their cells, as well as animals which 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, M. et al., 1992, Proc. Natl. Acad. Sci. USA 89: 6232-6236) .
  • the regulatory sequences required for such a cell-type specific activation will depend upon the particular cell type of interest, and will be apparent to those of skill in the art.
  • gene targeting is preferred. Briefly, when such a technique is to be utilized, vectors containing nucleotide sequences homologous to the endogenous GTIII gene and/or sequences flanking the gene are designed for the purpose of integrating, via homologous recombination with chromosomal sequences, into and disrupting the function of the endogenous GTIII gene.
  • the transgene may also be selectively expressed in a particular cell type with concomitant inactivation of the endogenous GTIII 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 recombination will depend upon the particular cell type of interest, and will be apparent to those of skill in the art.
  • founder animals have been generated, standard techniques such as Southern blot analysis or PCR techniques are used to analyze animal tissues to determine whether integration of the transgene has taken place.
  • the level of mRNA expression of the transgene in the tissues of the founder animals may also be assessed using techniques which include but are not limited to Northern blot analysis of tissue samples obtained from the animal, in situ hybridization analysis, and RT-PCR. Samples of the GTIII gene-expressing tissue, may also be evaluated i munocytochemically using antibodies specific for the GTIII transgene product. 5.3.2. INHIBITION OF GTIII EXPRESSION AND GENERATION OF ANIMAL MODELS
  • animal models for schizophrenia, or other GTIII-mediated neurological disorders can be designed by reducing the level of endogenous GTIII expression, e.g. , using antisense or ribozyme approaches to inhibit or prevent translation of GTIII mRNA transcripts; triple helix approaches to inhibit transcription of the GTIII gene; or targeted homologous recombination to inactivate or "knock out" the GTIII gene or its endogenous promoter.
  • delivery techniques should be preferably designed to cross the blood-brain barrier (see PCT WO89/10134, which is incorporated by reference herein in its entirety) .
  • the antisense, ribozyme or DNA constructs described herein could be administered directly to the site containing the target cells.
  • Antisense approaches involve the design of oligonucleotides (either DNA or RNA) that are complementary to mRNA.
  • the antisense oligonucleotides will bind to the complementary mRNA transcripts and prevent translation. Absolute complementarily, although preferred, is not required.
  • a sequence "complementary" to a portion of an RNA means a sequence having sufficient complementarily to be able to hybridize with the RNA, forming a stable duplex; in the case of double-stranded antisense nucleic acids, a single strand of the duplex DNA may thus be tested, or triplex formation may be assayed.
  • the ability to hybridize will depend on both the degree of complementarily 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. While antisense nucleotides complementary to the coding region sequence could be used, those complementary to the transcribed untranslated region are most preferred. Oligonucleotides that are complementary to the 5' end of the message, e.g.
  • oligonucleotides complementary to either the 5'- or 3'- non- translated, non-coding regions of GTIII could be used in an antisense approach to inhibit translation of endogenous mRNA.
  • Oligonucleotides complementary to the 5' untranslated region of the mRNA should include the complement of the AUG start codon.
  • Antisense oligonucleotides complementary to mRNA coding regions are less efficient inhibitors of translation but could be used in accordance with the invention.
  • antisense nucleic acids should be at least six nucleotides in length, and are preferably oligonucleotides ranging from 6 to about 50 nucleotides in length.
  • 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, xantine, 4-acetylcytosine, 5- (carboxyhydroxylmethyl) uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D- galactosylqueosine, inosine, N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2 , 2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7-methylguanine, 5-methylaminomethyluracil , 5-methoxyaminomethyl-2-thiouracil , beta-D-mannos
  • the antisense oligonucleotide comprises at least one modified phosphate backbone selected from the group consisting of a phosphorothioate, a phosphorodithioate, a phosphoramidothioate, a phosphoramidate, a phosphordiamidate, a methylphosphonate, an alkyI phosphotriester, and a formacetal or analog thereof.
  • the antisense oligonucleotide is an ⁇ -anomeric oligonucleotide.
  • oligonucleotide forms specific double-stranded hybrids with complementary RNA in which, contrary to the usual ⁇ -units, the strands run parallel to each other (Gautier et al., 1987, Nucl. Acids Res. 15:6625-6641).
  • the oligonucleotide is a 2'- O-methylribonucleotide (Inoue et al., 1987, Nucl. Acids Res. 15:6131-6148), or a chimeric RNA-DNA analogue (Inoue et al., 1987, FEBS Lett. 215:327-330).
  • Oligonucleotides of the invention may be synthesized by standard methods known in the art, e.g. by use of an automated DNA synthesizer (such as are commercially available from Biosearch, Applied Biosystems, etc.).
  • an automated DNA synthesizer such as are commercially available from Biosearch, Applied Biosystems, etc.
  • phosphorothioate oligonucleotides may be synthesized by the method of Stein et 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.
  • the antisense molecules should be delivered to cells which express GTIII in vivo, e.g. , neural tissue.
  • 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 GTIII transcripts and thereby prevent translation of the GTIII mRNA.
  • a vector can be introduced in vivo 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 chromoso ally integrated, as long as it can be transcribed to produce the desired antisense RNA.
  • 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. Any type of plasmid, cosmid, YAC or viral vector can be used to prepare the recombinant DNA construct which can be introduced directly into the tissue site; e.g. , the hippocampus.
  • viral vectors can be used which selectively infect the desired tissue; (e.g.. for brain, herpesvirus vectors may be used) , in which case administration may be accomplished by another route (e.g. , systemically) .
  • Ribozyme molecules designed to catalytically cleave GTIII mRNA transcripts can also be used to prevent translation of GTIII mRNA and expression of GTIII. (See, e.g. , PCT International Publication WO90/11364, published October 4, 1990; Sarver et al., 1990, Science 247:1222-1225).
  • ribozymes that cleave mRNA at site specific recognition sequences can be used to destroy GTIII 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 construction and production of hammerhead ribozymes is well known in the art and is described more fully in Haseloff and Gerlach, 1988, Nature, 334:585-591.
  • the ribozyme is engineered so that the cleavage recognition site is located near the 5' end of the GTIII mRNA; i.e. , to increase efficiency and minimize the intracellular accumulation of non-functional mRNA transcripts.
  • the ribozymes of the present invention also include RNA endoribonucleases (hereinafter "Cech-type ribozymes”) such as the one which occurs naturally in Tetrahymena Thermophila (known as the IVS, or L-19 IVS RNA) and which has been extensively described by Thomas Cech and collaborators (Zaug, et al., 1984, Science, 224:574-578; Zaug and Cech, 1986, Science, 231:470-475; Zaug, et al., 1986, Nature, 324:429- 433; published International patent application No. WO 88/04300 by University Patents Inc.; Been and Cech, 1986, Cell, 47:207-216).
  • Cech-type ribozymes such as the one which occurs naturally in Tetrahymena Thermophila (known as the IVS, or L-19 IVS RNA) and which has been extensively described by Thomas Cech and collaborators (Zaug, et al., 1984, Science,
  • the Cech-type ribozymes have an eiqht base pair active site which hybridizes to a target RNA sequence whereafter cleavage of the target RNA takes place.
  • the invention encompasses those Cech-type ribozymes which target eight base-pair active site sequences that are present in GTIII mRNA.
  • the ribozymes can be composed of modified oligonucleotides (e.g. for improved stability, targeting, etc.) and should be delivered to cells which express the GTIII in vivo, e.g. , hippocampus.
  • a preferred method of delivery involves using a DNA construct "encoding" the ribozyme under the control of a strong constitutive pol III or pol II promoter, so that transfected cells will produce sufficient quantities of the ribozyme to destroy endogenous GTIII messages and inhibit translation. Because ribozymes unlike antisense molecules, are catalytic, a lower intracellular concentration is required for efficiency.
  • Endogenous GTIII gene expression can also be reduced by inactivating or "knocking out" the GTIII gene or its promoter using targeted homologous recombination (e.g. , see Smithies et al., 1985, Nature 317:230-234; Thomas & 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 GTIII (or a completely unrelated DNA sequence) flanked by DNA homologous to the endogenous GTIII gene can be used, with or without a selectable marker and/or a negative selectable marker, to transfect cells that express GTIII in vivo.
  • Such approaches are particularly suited in the agricultural field where modifications to ES (embryonic stem) cells can be used to generate animal offspring with an inactive GTIII (e.g. , see Thomas & Capecchi 1987 and Thompson 1989, supra) .
  • endogenous GTIII gene expression can be reduced by targeting deoxyribonucleotide sequences complementary to the regulatory region of the GTIII gene (i.e. , the GTIII promoter and/or enhancers) to form triple helical structures that prevent transcription of the GTIII gene in target cells in the body.
  • deoxyribonucleotide sequences complementary to the regulatory region of the GTIII gene i.e. , the GTIII promoter and/or enhancers
  • GTIII PROTEINS POLYPEPTIDES, AND ANTIBODIES
  • GTIII protein polypeptides and peptide fragments, mutated, truncated or deleted forms of the GTIII and/or GTIII fusion proteins can be prepared for a variety of uses, including but not limited to the generation of antibodies, as reagents in diagnostic assays, as reagents in assays for screening for compounds that can be used in the treatment of schizophrenia and other disorders mediated by GTIII, and as pharmaceuticals useful in the treatment of schizophrenia or other disorders mediated oy GTIII.
  • the deduced amino acid sequences of the human GTIII is shown in FIG. 1, where the predicted ten transmembrane domains are underlined.
  • the hydrophilic domains located between the TM domains contribute to the structure of ECD and the CD of the transporter.
  • Peptides corresponding to one or more domains of the GTIII e.g. , ECD, TM or CD
  • truncated or deleted GTIII e.g. , GTIII in which one or more of the domains of the ECD, TM and/or CD is deleted
  • fusion proteins in which the full length GTIII, a GTIII peptide or truncated GTIII is fused to an unrelated protein are also within the scope of the invention.
  • peptides corresponding to individual domains located between the ten transmembrane domains, or a polypeptide consisting of each of these domains linked together can be fused to another polypeptide (e.g. , an IgFc polypeptide) .
  • another polypeptide e.g. , an IgFc polypeptide
  • mutant GTIII proteins or polypeptides can be prepared; such mutants include but are not limited to GTIII in which the glycosylation sites are eliminated to improve production and yield.
  • Such peptides, polypeptides, and fusion proteins can be prepared by recombinant DNA techniques. For example, nucleotide sequences encoding one or more of the domains that occur between the transmembrane domains cloned and ligated together to encode a soluble molecule. The DNA sequences encoding one or more of these domains can be ligated together directly or via a linker oligonucleotide that encodes a peptide spacer. Such linkers may encode flexible, glycine- rich amino acid sequences thereby allowing the domains that are strung together to assume a conformation that corresponds to their native conformation. Alternatively, nucleotide sequences encoding individual domains within the ECD can be used to express GTIII peptides.
  • a variety of host-expression vector systems may be utilized to express nucleotide sequences encoding the appropriate regions of the GTIII to produce such polypeptides.
  • the resulting peptide or polypeptide is a soluble derivative (e.g. , peptides corresponding to the domains located between the TMs, or truncated or deleted GTIII in which the TMs and/or CDs are deleted) the peptide or polypeptide can be recovered from the culture media.
  • the GTIII product can be recovered from the host cell itself.
  • the host-expression vector systems also encompass engineered host cells that express the GTIII or functional equivalents in situ, i.e. , anchored in the cell membrane. Purification or enrichment of the GTIII from such expression systems can be accomplished using appropriate detergents and lipid micelles and methods well known to those skilled in the art. However, such engineered host cells themselves may be used in situations where it is important not only to retain the structural and functional characteristics of the GTIII, but to assess biological activity, e.g. , in drug screening assays.
  • the host-expression vector systems that may be used for purposes of the invention include but are not limited to microorganisms such as bacteria (e.g. , E. coli. B.
  • subtilis transformed with recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vectors containing GTIII nucleotide sequences; yeast (e.g. , Saccharomyces , Pichia. transformed with recombinant yeast expression vectors containing the GTIII nucleotide sequences; insect cell systems infected with recombinant virus expression vectors (e.g. , baculovirus) containing the GTIII sequences; plant cell systems 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 GTIII nucleotide sequences
  • mammalian cell systems e.g. , COS, CHO, BHK, 293, 3T3 harboring recombinant expression constructs containing promoters derived from the genome of mammalian cells (e.g. , metallothionein promoter) or from mammalian viruses (e.g. , the adenovirus late promoter; the vaccinia virus 7.5K promoter) .
  • a number of expression vectors may be advantageously selected depending upon the use intended for the GTIII gene product being expressed. For example, when a large quantity of such a protein is to be produced, for the generation of pharmaceutical compositions of GTIII protein or for raising antibodies to the GTIII protein, for example, vectors which direct the expression of high levels of fusion protein products that are readily purified may be desirable.
  • vectors include, but are not limited, to the E. coli expression vector pUR278 (Ruther et al. , 1983, EMBO J.
  • GTIII coding sequence may be ligated individually into the vector in frame with the lacZ coding region so that a fusion protein is produced;
  • pIN vectors Inouye & Inouye, 1985, Nucleic Acids Res. 13:3101-3109; Van Heeke & Schuster, 1989, J. Biol. Chem. 264:5503-5509; and the like.
  • pGEX vectors may also be used to express foreign polypeptides as fusion proteins with glutathione S-transferase (GST) .
  • fusion proteins are soluble and can easily be purified from lysed cells by adsorption to glutathione-agarose beads followed by elution in the presence of free glutathione.
  • the PGEX vectors are designed to include thrombin or factor Xa protease cleavage sites so that the cloned target gene product can be released from the GST moiety.
  • 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.
  • Autographa californica nuclear polyhidrosis virus (AcNPV) is used as a vector to express foreign genes.
  • the virus grows in Spodoptera frugiperda cells.
  • the GTIII 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 GTIII 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) .
  • the recombinant viruses are then used to infect cells in which the inserted gene is expressed. (E.g.. see Smith et al., 1983, J. Virol. 46: 584; Smith, U.S. Patent No. 4,215,051).
  • the GTIII nucleotide 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 the GTIII gene product in infected hosts.
  • a non- essential region of the viral genome e.g. , region El or E3
  • Specific initiation signals may also be required for efficient translation of inserted GTIII nucleotide sequences. These signals include the ATG initiation codon and adjacent sequences. In cases where an entire GTIII gene or cDNA, 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 GTIII coding sequence is inserted, exogenous translational control signals, including, perhaps, the ATG initiation codon, must be provided. Furthermore, the initiation codon must be in frame with the reading frame of the desired coding sequence to ensure translation of the entire insert.
  • exogenous translational control signals and initiation codons can be of a variety of origins, both natural and synthetic.
  • the efficiency of expression may be enhanced by the inclusion of appropriate transcription enhancer elements, transcription terminators, etc. (See Bittner et al., 1987, Methods in Enzymol. 153:516-544).
  • a host cell strain may be chosen which 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. Accordingly, eukaryotic host cells which possess the cellular machinery for proper processing of the primary transcript, glycosylation, and phosphorylation of the gene product may be used. Such mammalian host cells include, but are not limited to, CHO, VERO, BHK, HeLa, COS, MDCK, 293, 3T3 and WI38 cell lines. For long-term, high-yield production of recombinant proteins, stable expression is preferred. For example, cell lines which stably express the GTIII sequences described above may be engineered. Rather than using expression vectors which contain viral origins of replication, host cells can be transformed with DNA controlled by appropriate expression control elements (e.g.
  • 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 which in turn can be cloned and expanded into cell lines.
  • This method may advantageously be used to engineer cell lines which express the GTIII gene product.
  • Such engineered cell lines may be particularly useful in screening and evaluation of compounds that affect the endogenous activity of the GTIII gene product.
  • a number of selection systems may be used, including but not limited to the herpes simplex virus thymidine kinase (Wigler, et al., 1977, Cell 11:223), hypoxanthine-guanine phosphoribosyltransferase (Szybalska & 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 & Berg, 1981, Proc. Natl.
  • Antibodies that specifically recognize one or more epitopes of GTIII, or peptide fragments of the GTIII are also encompassed by the invention.
  • Such antibodies include but are not limited to polyclonal antibodies, monoclonal antibodies ( Abs) , 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.
  • the antibodies of the invention may be used, for example, in the detection of the GTIII in a biological sample and may, therefore, be utilized as part of a diagnostic or prognostic technique whereby patients may be tested for abnormal amounts of GTIII.
  • Such antibodies may also be utilized in conjunction with, for example, compound screening schemes, as described, above, for the evaluation of the effect of test compounds on expression and/or activity of the GTIII gene product.
  • such antibodies can be used in conjunction with the gene therapy techniques described, below, e.g., to evaluate the normal and/or engineered GTIII-expressing cells prior to their introduction into the patient.
  • Such antibodies may additionally be used as a method for the inhibition of abnormal GTIII activity.
  • various host animals may be immunized by injection with the GTIII, or a GTIII polypeptide or peptide.
  • a synthetic peptide corresponding to amino acid residues 161-177 of FIG. 1 (QYKTKREEVKPPSDPEM) was used to generate monoclonal antibodies to GTIII.
  • Such host animals may include but are not limited to rabbits, mice, hamsters 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 as BCG (bacille Calmette-Guerin) and Corynebacteriu parvum.
  • BCG Bacille Calmette-Guerin
  • Monoclonal antibodies which are homogeneous populations of antibodies to a particular antigen, may be obtained by any technique which 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 i munoglobulin class including IgG, IgM, IgE, IgA, IgD and any subclass thereof.
  • the hybridoma producing the Ab of this invention may be cultivated n 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.
  • single chain antibodies can be adapted to produce single chain antibodies against GTIII gene products.
  • Single chain antibodies are formed by linking the heavy and light chain fragments of the Fv region via an amino acid bridge, resulting in a single chain polypeptide.
  • Antibody fragments which 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.
  • Antibodies to the GTIII can, in turn, be utilized to generate anti-idiotype antibodies that "mimic" epitopes of the GTIII, using techniques well known to those skilled in the art. (See, e.g. , Greenspan & Bona, 1993, FASEB J 7 (5) :437-444; and Nissinoff, 1991, J. Immunol. 147(8) :2429- 2438) .
  • GTIII can be controlled in vivo (e.g. at the transcriptional or translational level) using gene therapy approaches to regulate GTIII activity. Such an approach may be useful to treat schizophrenia and other GTIII-mediated disorders. Certain approaches are described below.
  • GTIII nucleic acid sequences can be utilized. Where a defective GTIII gene is diagnosed, treatment can be administered, for example, in the form of gene replacement therapy.
  • one or more copies of a normal GTIII gene or a portion of the GTIII gene that directs the production of a GTIII gene product exhibiting normal function may be inserted into the appropriate cells within a patient or animal subject, using vectors which include, but are not limited to adenovirus, adeno-associated virus, retrovirus and herpes virus vectors, in addition to other particles that introduce DNA into cells, such as liposomes. Because the GTIII gene is expressed in the brain, e.g.
  • the hippocampus such gene replacement therapy techniques should be capable of delivering GTIII gene sequences to these cell types within patients.
  • the techniques for delivery of the GTIII gene sequences should be designed to readily cross the blood-brain barrier, which are well known to those of skill in the art (see, e.g. , PCT application, publication No. WO89/10134, which is incorporated herein by reference in its entirety) , or, alternatively, should involve direct administration of such GTIII gene sequences to the site of the cells in which the GTIII gene sequences are to be expressed.
  • targeted homologous recombination can be utilized to correct the defective endogenous GTIII gene in the appropriate tissue; e.g. , brain tissue.
  • targeted homologous recombination can be used to correct the defect in ES cells in order to generate offspring with a corrected trait.
  • Additional methods which may be utilized to increase the overall level of GTIII gene expression and/or GTIII activity include the introduction of appropriate GTIII-expressing cells, preferably autologous cells, into a patient at positions and in numbers which are sufficient to ameliorate the symptoms of schizophrenia.
  • Such cells may be either recombinant or non-recombinant.
  • the cells which can be administered to increase the overall level of GTIII gene expression in a patient are normal cells, or hippocampal cells which express the GTIII gene.
  • the cells can be administered at the anatomical site in the brain, or as part of a tissue graft located at a different site in the body.
  • Such cell-based gene therapy techniques are well known to those skilled in the art, see, e.g. , Anderson, et al., U.S. Patent No. 5,399,349; Mulligan & Wilson, U.S. Patent No. 5,460,959.
  • the invention encompasses methods and compositions for modulating the activity of GTIII and treating schizophrenia. Because a loss of normal GTIII gene product function correlates with schizophrenia, an increase in GTIII activity would facilitate progress towards treatment.
  • Toxicity and therapeutic efficacy of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g. , for determining the LD 50 (the dose lethal to 50% of the population) and the ED 50 (the dose therapeutically effective in 50% of the population) .
  • the dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD 50 /ED 50 .
  • Compounds which 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 concent ' rations that include the ED 50 with little or no toxicity.
  • the dosage may vary within this range depending upon the dosage form employed and the route of administration utilized.
  • the therapeutically effective dose can be estimated initially from cell culture assays.
  • a dose may be formulated in animal models to achieve a circulating plasma concentration range that includes the IC b0 (i.e. , the concentration of the test compound which achieves a half-maximal inhibition of symptoms) as determined in cell culture.
  • IC b0 i.e. , the concentration of the test compound which achieves a half-maximal inhibition of symptoms
  • levels in plasma may be measured, for example, by high performance liquid chromatography.
  • compositions for use in accordance with the present invention may be formulated in conventional manner using one or more physiologically acceptable carriers or excipients.
  • the compounds and their physiologically acceptable salts and solvates may be formulated for administration by inhalation or insufflation (either through the mouth or the nose) or oral, buccal, parenteral or rectal administration.
  • the pharmaceutical compositions may take the form of, for example, tablets or capsules prepared by conventional means with pharmaceutically acceptable excipients such as binding agents (e.g. , pregelatinised maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose) ; fillers (e.g.. lactose, macrocrystalline 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 methylcellulose
  • fillers e.g.. lactose, macrocrystalline cellulose or calcium hydrogen phosphate
  • lubricants e.g. , magnesium stearate, talc or si
  • the tablets may be coated by methods well known in the art.
  • 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.
  • compositions for oral administration may be suitably formulated to give controlled release of the active compound.
  • compositions for buccal administration may take the form of tablets or lozenges formulated in conventional manner .
  • the compounds for use according to the present invention are conveniently delivered in the form of an aerosol spray presentation from pressurized packs or a nebulizer, with the use of a suitable propellant, e.g. , dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas.
  • a suitable propellant e.g. , dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas.
  • a suitable propellant e.g. , dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas.
  • a suitable propellant e.g. , dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane
  • 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 which may contain one or more unit dosage forms containing the active ingredient.
  • the pack may for example comprise metal or plastic foil, such as a blister pack.
  • the pack or dispenser device may be accompanied by instructions for administration.
  • Glutamate is a major excitatory amino acid neurotransmitter in the mammalian central nervous system.
  • the synaptic action of glutamate is thought to be terminated by a high-affinity uptake system, which is dependent on Na+ and K+.
  • This example describes the production and characterization of a monoclonal antibody for human neuron- specific glutamate transporter.
  • a monoclonal antibody was raised against a synthetic peptide (QYKTKREEVKPPSDPEM) that corresponds to amino acid residues 161-177 of the human neuron-specific glutamate transporter cDNA.
  • the hybridomas were screened against the free peptide by ELISA initially and the clones were selected on the basis of their immunoreactivity.
  • the large pyramidal neurons showed intense immunoreactivity, although there were other smaller neurons through the cortex showing less immunoreactivity.
  • neuronal somata and dendrites of the sub-fields CA1 to CA3 and the dentate gyrus were intensely immunoreactive.
  • substantia nigra in addition to neuronal somata with proximal dendritic labeling, the antibody stained fine caliber fibers and numerous puncta.
  • Glutamate abnormalities have been implicated in the pathophysiology of schizophrenia.
  • HIPPOCAMPUS OF SCHIZOPHRENICS A neural circuit involving striatu /nucleus accumbens, hippocampus/parahippocampal cortex and prefrontal cortex may be involved in the pathophysiology of schizophrenia. As these regions are connected by glutamatergic neurons, a study of these neurons in the hippocampus/parahippocampal cortex seemed indicated.
  • a two-way ANOVA (group by region) indicated that there was a reduction in GTIII mRNA in schizophrenics (p(.0001). Post-hoc comparisons for each region indicted that this difference attained statistical significance (p ⁇ 05) in dentate, CA1, and subiculum, where levels were 69, 67 and 61%, respectively, of normal levels. Levels were also decreased (.08>p).05) in CA , presubiculum and parahippocampai layers ii/iii.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Immunology (AREA)
  • Molecular Biology (AREA)
  • Urology & Nephrology (AREA)
  • Biomedical Technology (AREA)
  • Hematology (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Medicinal Chemistry (AREA)
  • Food Science & Technology (AREA)
  • Pathology (AREA)
  • Cell Biology (AREA)
  • Biotechnology (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • Organic Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • General Physics & Mathematics (AREA)
  • Microbiology (AREA)
  • Physiology (AREA)
  • Toxicology (AREA)
  • Zoology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Biophysics (AREA)
  • Genetics & Genomics (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Abstract

Cette invention porte sur des méthodes de sélection de médicaments, ainsi que sur des méthodes diagnostiques et thérapeutiques aux fins du traitement de la schizophrénie à l'aide du transporteur de glutamate spécifique des neurones humains, dénommé transporteur III du glutamate, comme cible pour intervention. Elle porte également sur des composés modulant l'activité ou l'expression de ce transporteur ainsi que sur l'utilisation de ces composés pour traiter la schizophrénie et d'autres troubles induits par une activité anormale dudit transporteur.
PCT/US1997/016219 1996-09-13 1997-09-15 Methodes de selection de composes utilisables pour traiter la schizophrenie WO1998011222A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU44809/97A AU4480997A (en) 1996-09-13 1997-09-15 Screening methods for compounds useful in the treatment of schizophrenia

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
US2525596P 1996-09-13 1996-09-13
US60/025,255 1996-09-13
US92831697A 1997-09-12 1997-09-12
US08/928,316 1997-09-12

Publications (1)

Publication Number Publication Date
WO1998011222A1 true WO1998011222A1 (fr) 1998-03-19

Family

ID=26699504

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US1997/016219 WO1998011222A1 (fr) 1996-09-13 1997-09-15 Methodes de selection de composes utilisables pour traiter la schizophrenie

Country Status (2)

Country Link
AU (1) AU4480997A (fr)
WO (1) WO1998011222A1 (fr)

Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000022122A3 (fr) * 1998-10-13 2000-10-05 Genset Sa Genes, proteines et marqueurs bialleliques lies a une maladie du systeme nerveux central
WO2001063293A3 (fr) * 2000-02-24 2002-04-11 Oxford Glycosciences Uk Ltd Proteines et genes et leur utilisation dans le diagnostic et le traitement de la schizophrenie
WO2002008384A3 (fr) * 2000-07-25 2002-06-06 Univ California Nouveaux transporteurs de glutamate
WO2001040493A3 (fr) * 1999-11-30 2002-07-18 Genset Sa Genes associes a la schizophrenie et proteines et marqueurs bialleliques correspondants
US6476208B1 (en) 1998-10-13 2002-11-05 Genset Schizophrenia associated genes, proteins and biallelic markers
US6555316B1 (en) 1999-10-12 2003-04-29 Genset S.A. Schizophrenia associated gene, proteins and biallelic markers
US7371811B2 (en) 1998-10-13 2008-05-13 Serono Genetics Institute S.A. Schizophrenia associated genes, proteins and biallelic markers
WO2008139170A2 (fr) * 2007-05-09 2008-11-20 Sinclair Pharmaceuticals Limited Agents améliorant l'administration transdermique et transmucosale de médicament

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
BRAIN RESEARCH, 1994, Vol. 662, No. 1-2, SHASHIDHARAN et al., "Neuron-Specific Human Glutamate Transporter: Molecular Cloning, Characterization and Expression in Human Brain", pages 245-250. *
NATURE, 03 December 1992, Vol. 360, No. 6403, KANAI et al., "Primary Structure and Functional Characterization of a High-Affinity Glutamate Transporter", pages 467-471. *
NEUROCHEMICAL RESEARCH, October 1991, Vol. 16, No. 10, SQUIRES et al., "A Review of Evidence for GABergic Predominance/Glutamatergic Deficit as a Common Etiological Factor in Both Schizophrenia and Affective Psychoses: More Support for a Continuum Hypothesis of 'Functional' Psychosis", pages 1099-1111. *

Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000022122A3 (fr) * 1998-10-13 2000-10-05 Genset Sa Genes, proteines et marqueurs bialleliques lies a une maladie du systeme nerveux central
US6476208B1 (en) 1998-10-13 2002-11-05 Genset Schizophrenia associated genes, proteins and biallelic markers
US7371811B2 (en) 1998-10-13 2008-05-13 Serono Genetics Institute S.A. Schizophrenia associated genes, proteins and biallelic markers
US6555316B1 (en) 1999-10-12 2003-04-29 Genset S.A. Schizophrenia associated gene, proteins and biallelic markers
WO2001040493A3 (fr) * 1999-11-30 2002-07-18 Genset Sa Genes associes a la schizophrenie et proteines et marqueurs bialleliques correspondants
WO2001063293A3 (fr) * 2000-02-24 2002-04-11 Oxford Glycosciences Uk Ltd Proteines et genes et leur utilisation dans le diagnostic et le traitement de la schizophrenie
WO2002008384A3 (fr) * 2000-07-25 2002-06-06 Univ California Nouveaux transporteurs de glutamate
US6818391B2 (en) 2000-07-25 2004-11-16 The Regents Of The University Of California Glutamate transporters
WO2008139170A2 (fr) * 2007-05-09 2008-11-20 Sinclair Pharmaceuticals Limited Agents améliorant l'administration transdermique et transmucosale de médicament
WO2008139170A3 (fr) * 2007-05-09 2009-07-23 Sinclair Pharmaceuticals Ltd Agents améliorant l'administration transdermique et transmucosale de médicament

Also Published As

Publication number Publication date
AU4480997A (en) 1998-04-02

Similar Documents

Publication Publication Date Title
US5891720A (en) Isolated DNA encoding a novel human G-protein coupled receptor
US5932779A (en) Screening methods for compounds useful in the regulation of body weight
US5908609A (en) Screening methods for compounds useful in the regulation of body weight
WO1998046620A9 (fr) Nouveau recepteur couple a la proteine g humaine
US6248528B1 (en) Methods and compositions for the diagnosis and treatment of neuropsychiatric disorders
WO1998011222A1 (fr) Methodes de selection de composes utilisables pour traiter la schizophrenie
US5866412A (en) Chromosome 18 marker
US6987178B2 (en) Human uncoupling proteins and polynucleotides encoding the same
EP0981357A1 (fr) Procedes et compositions s'appliquant au diagnostique et au traitement des troubles neuropsychiatriques
US20040038345A1 (en) Novel human seven-transmembrane receptors
AU780591B2 (en) Novel human ion channel proteins
WO1998042362A9 (fr) Procedes et compositions s'appliquant au diagnostique et au traitement des troubles neuropsychiatriques
US5914394A (en) Methods and compositions for the diagnosis and treatment of neuropsychiatric disorders
US20090081212A1 (en) Use and targeting of cd98 light-chain proteins in therapies for thyroid hormone disorders
EP1226170B1 (fr) Chordin humaine et polynucleotides codant pour ce meme
US20050130890A1 (en) Amyloid beta is a ligand for FPR class receptors
US20020142298A1 (en) Methods and compositions for the diagnosis and treatment of neuropsychiatric disorders
US20040127440A1 (en) Methods and compositions for control of bone formation via modulation of neuropeptide Y activity
JP2003528804A (ja) レプチン活性の調節を介して骨形成を制御するための方法および組成物
WO1998042723A9 (fr) Procedes et compositions s'appliquant au diagnostique et au traitement des troubles neuropsychiatriques
US6806083B2 (en) Human LDL receptor encoding polynucleotides
WO2002064021A2 (fr) Procedes et compositions pour reguler la formation osseuse par modulation de l'activite ciliaire du facteur neurotrophique
US20040229316A1 (en) Novel human G-protein coupled receptor
AU7744900A (en) Novel human membrane proteins
US20040048285A1 (en) Novel seven transmembrane proteins and polynucleotides encoding the same

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AU CA IL 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
NENP Non-entry into the national phase

Ref country code: JP

Ref document number: 1998513911

Format of ref document f/p: F

122 Ep: pct application non-entry in european phase
NENP Non-entry into the national phase

Ref country code: CA

点击 这是indexloc提供的php浏览器服务，不要输入任何密码和下载