WO2007085706A1 - Procédé de criblage de substances pour le traitement de maladies neuropsychiatriques et matériels pour ce procédé - Google Patents

Procédé de criblage de substances pour le traitement de maladies neuropsychiatriques et matériels pour ce procédé Download PDF

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WO2007085706A1
WO2007085706A1 PCT/FI2007/050051 FI2007050051W WO2007085706A1 WO 2007085706 A1 WO2007085706 A1 WO 2007085706A1 FI 2007050051 W FI2007050051 W FI 2007050051W WO 2007085706 A1 WO2007085706 A1 WO 2007085706A1
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amigo
knockout
substance
mouse
animal
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PCT/FI2007/050051
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WO2007085706A8 (fr
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Heikki Rauvala
Juha Kuja-Panula
Vootele Voikar
Natalia Kulesskaya
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Licentia Ltd.
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    • 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
    • G01N33/94—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving narcotics or drugs or pharmaceuticals, neurotransmitters or associated receptors
    • G01N33/9466—Antidepressants
    • 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
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
    • A01K67/027—New or modified breeds of vertebrates
    • A01K67/0275—Genetically modified vertebrates, e.g. transgenic
    • A01K67/0276—Knock-out vertebrates
    • 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
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/8509—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells for producing genetically modified animals, e.g. 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
    • 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
    • A01K2227/00—Animals characterised by species
    • A01K2227/10—Mammal
    • A01K2227/105—Murine
    • 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
    • A01K2267/00—Animals characterised by purpose
    • A01K2267/03—Animal model, e.g. for test or diseases
    • A01K2267/035—Animal model for multifactorial diseases
    • A01K2267/0356—Animal model for processes and diseases of the central nervous system, e.g. stress, learning, schizophrenia, pain, epilepsy

Definitions

  • the present invention relates to the field of non-human knockout animals and provides a mouse having a defect in AMIGO (amphoterin-induced gene and ORF).
  • AMIGO amphoterin-induced gene and ORF
  • the AMIGO knockout mouse which exhibit deficit in prepulse inhibition and enhanced locomotor activity, provides a test system used in the development of therapeutic drugs for related disorders.
  • the present invention is directed to a method for screening substances for treatment of neuropsychiatric diseases by the use of a mouse having a defective AMIGO, a tissue, or a cell of said mouse.
  • Amphoterin/RAGE receptor for advanced glycation end products
  • AMIGO is specifically expressed in the nervous system whereas AMIGO2 and AMIGO3 display a wider tissue distribution.
  • AMIGO mediates axon-axon interaction during construction of neuronal connections of the brain (Kuja-Panula et al., 2003). Further, a homophilic and heterophilic binding mechanism was found between the members of the AMIGO family.
  • FIG. 1 Domain structure of the three AMIGOs.
  • the proteins consist of 6 leucine-rich repeats (LRRs) and one immunoglobulin domain (IG-domain) in their extracellular moieties, followed by a transmembrane domain (TM-domain) and the cytosolic tail. Similarity of the three AMIGOs at the amino acid level is about 50 %.
  • FIGS 2a, 2b and 2c Targeted disruption of the AMIGO gene, (a) Structures of the targeting vector, genomic AMIGO allele and mutated knockout allele. Exons for AMIGO are marked with hatched boxes and inside the exon 2 the couding sequence is marked as CDS. The location of the probe used for Southern blotting is shown (3 'probe).
  • the start codon of LacZ gene was inserted into same location as the start codon of AMIGO gene; Neo, neomycin resistance cassette; TK, herpes simplex virus thymidine kinase.
  • This invention is based on the production and use of a knockout mouse having a defect in murine gene/protein termed AMIGO (SEQ ID NO:1) and its homologous counterparts designated as AMIGO2 (SEQ ID NO:3) and AMIGO3 (SEQ ID NO:5). Together these three proteins form a iamily of transmembrane proteins described previously in WO 2004/055055 (for simplicity, all of these proteins are hereinafter referred as AMIGO or AMIGOs). The use of terms and abbreviations herein is identical to the use of the same in WO 2004/055055.
  • non-human homo logs of AMIGO than murine AMIGOs can be used to construct an AMIGO "knock out" animal, i.e., having a defective or altered gene encoding AMIGO, as a result of homologous recombination between the endogenous AMIGO gene and an altered genomic AMIGO DNA introduced into an embryonic cell of the animal.
  • murine AMIGO cDNA can be used to clone genomic AMIGO DNA in accordance with established techniques.
  • a portion of the genomic AMIGO DNA can be deleted or replaced with another gene, such as a gene encoding a selectable marker which can be used to monitor integration.
  • flanking DNA typically, several kilobases of unaltered flanking DNA (both at the 5' and 3' ends) are included in the vector (see e.g., Thomas and Capecchi, Cell 51:503 (1987) for a description of homologous recombination vectors).
  • the vector is introduced into an embryonic stem cell line (e.g., by electroporation) and cells in which the introduced DNA has homologously recombined with the endogenous DNA are selected (see e.g., Li et al., Cell 69:915 (1992)).
  • the selected cells are then injected into a blastocyst of an animal (e.g., a mouse) to form aggregation chimeras (see e.g., Bradley, in Teratocarcinomas and Embryonic Stem Cells: A Practical Approach, E. J. Robertson, ed. (IRL, Oxford, 1987), pp. 113-152).
  • a chimeric embryo can then be implanted into a suitable pseudopregnant female foster animal and the embryo brought to term to create a "knock out" animal.
  • Progeny harbouring the homologously recombined DNA in their germ cells can be identified by standard techniques and used to breed animals in which all cells of the animal contain the homologously recombined DNA. Knockout animals can be characterized for their ability to mimic human neurological disorders and defects.
  • One of the embodiments of the present invention is a method for screening candidate substances for antipsychotic drugs, which are preferably for the treatment of neuropsychiatric condition such as schizophrenia, the method comprising the steps of
  • said non-human AMIGO knockout animal is a mouse.
  • the production of such a mouse is disclosed in the experimental section below and the knockout construction of said mouse is preferably as shown in Figure 2A.
  • the present invention is also directed to the knockout mouse as such and the method of preparing a non-human AMIGO knockout mouse. It is also clear that a person skilled in the art can use the teaching of this application in order to prepare other non-human AMIGO knockout animals, such as a knockout rat. Thus, the invention is also directed to a method for preparing a non-human AMIGO knockout animal.
  • Another embodiment of the invention is an isolated cell or tissue of the AMIGO knockout mouse as defined above.
  • Cells or tissues of the AMIGO knockout mouse can be used in a method for screening candidate substances for antipsychotic drugs. This method may comprise the steps of
  • the present invention is also directed to the use of a substance obtained by the screening method of the invention for manufacture of a medicament for the treatment of a neuropsychiatric condition, such as schizophrenia.
  • a neuropsychiatric condition such as schizophrenia.
  • FIG. 2 For AMIGO gene targeting (Figure 2), we constructed a replacement vector by using genomic DNA fracments from mouse phage library (strain 129SV). The whole coding region of AMIGO gene was replaced by inserting LacZ gene under the promoter of AMIGO gene using tailored PCR primers: 5 'primer
  • the primers were a 5' primer (5 1 - AGCAACATCCTCAGCTGCTC-3') and a 3' primer (5 1 -
  • primers were a 5' primer (5 1 - CAACGACCCCTTCATTGACC-3') and a 3' primer (5 1 - AGTGATGGCATGGACTGTGG-3').
  • the subsequent PCR reaction was performed in a PCR mix (2.5 ⁇ M dNTP, 10 mM Tris-HCL, pH 8.8, 150 mM KCL, 1.5 mM MgCl 2 , and 0.1% Triton X-100) containing 0.2 ⁇ M 5' primer and 3' primer and 1 U DyNAzymeTM II DNA Polymerase (Finnzymes).
  • the amplification products were separated on 1.5% agarose gel and stained with EtBr.
  • mice All mice were housed in a specific-pathogen- free barries animal facility. AMIGO knockout mice were backcrossed to the C57BL/6J strain at least 8 times before being used for behavioral experiments.
  • the AMIGO knockout mice have been analyzed using our test battery (V ⁇ ikar et al., 2001, 2002 and 2004) to screen for alterations in behaviour.
  • the knockout mice displayed significantly enhanced locomotor activity in the plus maze and open field tests (Figure 3). Novelty- induced hyperactivity is a common feature in genetic animal models of schizophrenia.
  • Deficient sensorimotor gating measured by prepulse inhibition, is a robust symptom in schizophrenia (although reduced PPI has been found also in patients with obsessive- compulsive disorder, Huntington's disease and attention deficit disorder).
  • PPI is a phenomenon in which a weak prepulse stimulus attenuates the response to a subsequent startling stimulus like voice. PPI can be measured for example in humans and rodents. Measurement of PPI is an essential component in rodent models of schizophrenia. Here we found that the knockout mice displayed significantly reduced PPI as compared to control mice ( Figure 4).
  • the question whether the AMIGO -/- mice could be used as a model of psychiatric disorders charagterized by delicts in prepulse inhibition and/or enhanced locomotor activity such as schizophrenia is addressed by testing several antipsychotic drugs for the ability to reverse the PPI phenotype in the AMIGO -/- mice.
  • the data obtained with antipsychotics clozapine (1 mg/kg) and haloperidol (1 mg/kg) indicate that both drugs alleviate the deficit in the knockout mice and the selected doses without significantly affecting the level of PPI in the wild type animals (Figure 5).
  • the knockout mice displayed good spatial learning and memory in the Morris water maze test.
  • Another memory test Pavlovian fear conditioning, revealed reduced context-dependent memory in knockout mice ( Figure 6). This finding can be explained by hyperactivity of the knockout mice.
  • mice were placed in the square box (30 x 30 cm) equipped with infrared emitter-detector frames and left there for 30 minutes. During this time, the horizontal (distance moved) and vertical (rearings) activity was measured based on the IR beam brakes.
  • mice Circadian activity.
  • the mice were housed singly in metabolic cages (Comprehensive Laboratory Animal Monitorin System) for 72 hours and activity, food and water intake and respiratory gases (O2 consumption, CO2 production) were monitored continuously over whole period.
  • metabolic cages Comprehensive Laboratory Animal Monitorin System
  • O2 consumption, CO2 production food and water intake and respiratory gases
  • Prepulse inhibition The startle responses were measured in the commercially available startle system. Briefly, the mouse was restrained in the Plexiglas cylinder and after short (5 min) acclimation period the acoustic stimuli were applied with varying interval (10-20 seconds).
  • the startle stimulus (SS) applied was 40 ms 120 dB burst of white noise, the onset of prepulse was 100 ms before the onset of the startle stimulus.
  • the 20 ms prepulse stimuli (PPS) were noise bursts of 68, 72, 76 and 80 dB.
  • the five trial types (SS alone or PPS+SS) were presented in pseudorandom order such that each trial type was presented once within a block of 5 trials (altogether 10 blocks). The PPI was calculated as a percentage of inhibition in PPS+SS trials compared with SS trials.
  • the drugs (clozapine and haloperidol) were administered in a dose of 1 mg/kg 30 min before the test.
  • Fear conditioning This is a form of classical conditioning where two memory components can be analysed - association of unconditioned stimulus (foot-shock, US) with a particular compartment (contextual memory) and simple association of conditioned stimulus (tone, CS) with shock.
  • the experiments were carried out employing a computer- controlled fear conditioning system. Training was performed in a clear acrylic cage (35x20x20 cm) within a constantly illuminated ( ⁇ 550 Ix) fear conditioning box.
  • a loudspeaker provided a constant, white background noise (68 dB) for 120 s followed by 10 kHz tone (CS, 75 dB, pulsed 5 Hz) for 30 s.
  • the tone was terminated by a footshock (US, 0.7 mA, 2 s, constant current) delivered through a stainless steel floor grid.
  • a footshock US, 0.7 mA, 2 s, constant current
  • Two CS-US pairings were separated by a 30 s pause.
  • Contextual memory was tested 24 h after the training.
  • the animals were returned to the conditioning box and total time of freezing (defined as an absence of any movements for more than 3 s) was measured by infrared light barriers scanned continuously with a frequency of 10 Hz.
  • the CS was not used during this time.
  • Memory to the CS (tone) was tested 2 h later in a novel context.
  • the new context was a similarly sized acrylic box.
  • the light intensity was reduced to 100 Ix, the floor was flat (without shock grid) and the background colour was black (as opposed to white colour in training context).
  • the CS was applied for additional 120 s and freezing was measured as above.
  • the activity was registered in all phases of training and testing.
  • AMIGO a transmembrane protein implicated in axon tract development, defines a novel protein family with leucine-rich repeats. J. Cell. Biol. 160(6): 963-973. Kaksonen, M., Pavlov, L, V ⁇ ikar, V., Lauri, S.E., Hienola, A., Riekki, R., Lakso, M., Taira, T., and Rauvala, H. (2002). Syndecan-3 deficient mice exhibit enhanced LTP and impaired hippocampus-dependent memory. MoI. Cell. Neurosci. 21: 158-172.
  • HB-GAM heparin-binding growth-associated molecule
  • HB-GAM heparin-binding growth-associated molecule

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Abstract

La présente invention concerne un animal knock-out pour AMIGO (amphoterin-induced gene and ORF = gène induit par l'amphotérine et cadre de lecture ouvert), par exemple une souris, qui présente une activité locomotrice augmentée. La présente invention concerne un procédé de criblage de substances destinées au traitement de maladies neuropsychiatriques en utilisant une souris ayant un AMIGO défecteux, un tissu ou une cellule de cette souris.
PCT/FI2007/050051 2006-01-30 2007-01-30 Procédé de criblage de substances pour le traitement de maladies neuropsychiatriques et matériels pour ce procédé WO2007085706A1 (fr)

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FI20060089A FI20060089A0 (fi) 2006-01-30 2006-01-30 Käyttäytymishäiriöiden hoito

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004055055A1 (fr) * 2002-12-13 2004-07-01 Licentia Ltd Proteine transmembranaire amigo et utilisations de celle-ci
WO2005104834A2 (fr) * 2004-04-14 2005-11-10 Euro-Celtique S.A. Gene pnpg5 associe a la douleur

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004055055A1 (fr) * 2002-12-13 2004-07-01 Licentia Ltd Proteine transmembranaire amigo et utilisations de celle-ci
WO2005104834A2 (fr) * 2004-04-14 2005-11-10 Euro-Celtique S.A. Gene pnpg5 associe a la douleur

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
KAKSONEN M. ET AL.: "Syndecan-3-Deficient Mice Exhibit Enhanced LTP and Impaired Hippocampus-Dependent Memory", MOLECULAR AND CELLULAR NEUROSCIENCE, vol. 21, 2002, pages 158 - 172, XP003011305 *
KUJA-PANULA J. ET AL.: "AMIGO, a transmembrane protein implicated in axon tract development, defines a novel protein family with leucine-rich repeats", THE JOURNAL OF CELL BIOLOGY, vol. 160, no. 6, 2003, pages 963 - 973, XP003011304 *

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