WO2013016826A1 - Modèle animal non-humain transgénique d'une maladie neurodégénérative - Google Patents

Modèle animal non-humain transgénique d'une maladie neurodégénérative Download PDF

Info

Publication number
WO2013016826A1
WO2013016826A1 PCT/CA2012/050527 CA2012050527W WO2013016826A1 WO 2013016826 A1 WO2013016826 A1 WO 2013016826A1 CA 2012050527 W CA2012050527 W CA 2012050527W WO 2013016826 A1 WO2013016826 A1 WO 2013016826A1
Authority
WO
WIPO (PCT)
Prior art keywords
human
caspase
transgenic animal
human transgenic
cre
Prior art date
Application number
PCT/CA2012/050527
Other languages
English (en)
Inventor
Andrea Leblanc
Original Assignee
The Sir Mortimer B. Davis - Jewish General Hospital
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 Sir Mortimer B. Davis - Jewish General Hospital filed Critical The Sir Mortimer B. Davis - Jewish General Hospital
Publication of WO2013016826A1 publication Critical patent/WO2013016826A1/fr

Links

Classifications

    • 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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
    • C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
    • C12N9/6472—Cysteine endopeptidases (3.4.22)
    • 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
    • A01K67/0278—Knock-in vertebrates, e.g. humanised vertebrates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
    • C12Y304/22—Cysteine endopeptidases (3.4.22)
    • C12Y304/22059—Caspase-6 (3.4.22.59)
    • 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
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5082—Supracellular entities, e.g. tissue, organisms
    • 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
    • A01K2207/00—Modified animals
    • A01K2207/15—Humanized animals
    • 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
    • A01K2217/072—Animals genetically altered by homologous recombination maintaining or altering function, i.e. knock in
    • 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
    • A01K2217/20—Animal model comprising regulated expression system
    • A01K2217/206—Animal model comprising tissue-specific expression system, e.g. tissue specific expression of transgene, of Cre recombinase
    • 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
    • A01K2267/0306—Animal model for genetic diseases
    • A01K2267/0318—Animal model for neurodegenerative disease, e.g. non- Alzheimer's
    • 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
    • C12N2517/00—Cells related to new breeds of animals
    • C12N2517/02—Cells from transgenic animals
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/28—Neurological disorders
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/28—Neurological disorders
    • G01N2800/2814—Dementia; Cognitive disorders
    • G01N2800/2821—Alzheimer

Definitions

  • AD Alzheimer Disease
  • the present invention provides a non-human transgenic animal whose genome comprises a transgene comprising a sequence encoding a human Caspase-6 polypeptide.
  • the present invention provides a cell line derived from the above- mentioned non-human transgenic animal.
  • A. The 3 day pre-training with a visible platform in the Morris water maze shows that the Casp6 Kl mice do not have any vision problems as there is no significant difference between controls and Kl. Note that the error bar is so small that it is obscured by the symbol.
  • the training with a hidden platform in the opposite quadrant and by changing the cues on the wall was done for 5 consecutive days at a rate of 3 trials per day.
  • Figure 9 shows the specificity of the anti-human Casp6 antibody used in Figure 8.
  • A shows immunopositive reactivity only in the Kl/Cre mouse hippocampus and not in the control KI/WT and WT/Cre mice.
  • B Adsorption of the LSB 477 anti-human Casp6 antibody with recombinant human Caspase-6 protein eliminates immunoreactivity.
  • C Neither Caspase-6 null nor wild type control mouse hippocampus is immunostained by the anti-human Caspase-6 LSB-477 antibody.
  • D The anti-Caspase-6 antibody LSB-477 immunostains cotton wool plaques in a familial AD case similarly than the anti-Tau Casp6 or anti-active Caspase-6 antisera.
  • Figure 10 shows that Tau is cleaved by Casp6 as demonstrated by immunostaining of Kl/Cre mouse brain using a TauACasp6 antiserum.
  • A shows hippocampus area where some fine neuritic staining is observed from the dendate gyrus projections into the CA3 (arrow) in the Kl/Cre but not in the KI/WT.
  • B and C show higher magnification of strongly stained neuropil threads in the cortex.
  • D and E show neurons with strong intracellular TauACasp6 intracellular aggregates.
  • Figure 11 shows a PHF-1 Tau immunostaining of 20 month old Kl/Cre hippocampus (panels A-E) and cortex (panels F-l).
  • Figure 12 shows ⁇ immunostaining of 20 month old Kl/Cre hippocampus.
  • Figure 18 shows the nucleotide sequence encoding human Caspase-6 (Accession NM_001226; SEQ ID NO: 1). The coding sequence corresponds to nucleotides 79-960.
  • transgenic mice represent a preferred embodiment of the invention
  • other transgenic mammals including, without limitation, transgenic rodents (for example, hamsters, guinea pigs, rabbits, and rats), and transgenic pigs, chickens, cattle, sheep, goats, non-human primates (e.g., marmosets) may be constructed by standard techniques and are included in the invention.
  • transgene means a nucleic acid sequence (encoding, e.g., human Caspase-6) that has been introduced into a cell by way of human intervention such as by way of the described methods herein.
  • a transgene could be partly or entirely heterologous, i.e., foreign, to the transgenic animal or cell into which it is introduced.
  • a transgene can include one or more transcriptional regulatory sequences and any other nucleic acid, such as introns, that may be necessary for optimal expression of a selected nucleic acid.
  • AD e.g., inhibitors of Caspase-6
  • zygote is a good target for microinjection, and methods of microinjecting zygotes are well known (see US 4,873, 191).
  • Embryonal cells at various developmental stages can also be used to introduce transgenes for the production of transgenic animals. Different methods are used depending on the stage of development of the embryonal cell.
  • Such transfected embryonic stem (ES) cells can thereafter colonize an embryo following their introduction into the blastocoele of a blastocyst-stage embryo and contribute to the germ line of the resulting chimeric animal (reviewed in Jaenisch, Science 240:1468-1474 (1988)).
  • the fragment and/or variant exhibits at least 70, 75, 80, 85, 90 or 95% identity with the full length Caspase-6 polypeptide precursor (SEQ ID NO: 2), or with a human Caspase-6 polypeptide lacking the first propeptide present in the precursor (i.e. comprising residues 24 to 293 of SEQ ID NO:2), and retains caspase-6 activity.
  • the fragment and/or variant is constitutively active or exhibits the capacity to self-activate.
  • the present invention further provides a method of determining whether a compound may be used for preventing or treating a neurodegenerative disease (e.g., AD, HD), comprising treating a non-human transgenic mammal harboring a transgene expressing human Caspase-6, as described above, with the compound and determining whether symptoms and/or pathology of the neurodegenerative disease are improved, reduced, or their onset delayed, relative to an untreated transgenic non-human mammal, wherein the improvement, reduction or delayed onset is indicative that the compound may be used for preventing or treating the neurodegenerative disease (e.g., AD, HD).
  • a neurodegenerative disease e.g., AD, HD
  • the % time and % distance swam in the target quadrant (where the platform was in the previous 5 days) was recorded as well as the number of times the mice crossed exactly where the platform was present. Mice showing difficulty to reach the visible platform in less than 15-20 seconds were removed from the analyses.
  • mice Perfusion of mice and collection of brains for histology. Mice were anaesthetized by isoflurane inhalation (2: 1) prior to cardiac perfusion through the left ventricle with a 21 G needle, with 0.9% sodium chloride solution followed by 4% paraformaldehyde solution (pH 7.4). All solutions were administered and monitored from an IV pouch and flow rate was adjusted by catheter. After complete perfusion of the animal, the brain was removed and placed in vials containing 10% neutral buffered formalin (Fisher Scientific, Kalamazoo, US) at room temperature for 24 hours to facilitate fixation. Prior to sectioning, whole brains were changed into 70% ethanol and cut with a coronal acrylic matrix to isolate brain area of interest.
  • FIGs 6 and 7 show results of Morris water maze experiments in which female and male were grouped.
  • Monitoring of the mouse learning and memory function by the Morris water maze method revealed that the Kl/Cre mice expressing human Casp6 in the CA1 of the hippocampus undergo age-dependent memory decline.
  • a 3 consecutive day pre-training trial with a visible platform indicated that Kl/Cre, KI/WT or WT/Cre mice tested did not have any visual impairment at 5, 9, or 17-18 months of age (FIG. 6).
  • the learning phase with a hidden platform did not show significant difference in performance across all three genotypes at any of the age.
  • Swim speed also were almost identical in all three groups of mice indicating that these do not suffer from motor problems.
  • the age-dependent deficit is more obvious when probe day measures are expressed relative to the age of the mice (Fig. 7B).
  • the Kl/Cre mice show a statistically significant linear regression in the % time, % distance, and number of platform crosses in the target quadrant relative to the age of the mice.
  • neither KI/WT nor WT/Cre mice display this age- dependent memory defect.
  • the results suggest that there are also anti- ⁇ immunopositive neurons in the dentate gyrus and CA1 of 20 month old Kl/Cre (FIG. 12). Only weak and diffuse immunoreactivity is observed at earlier time points.
  • the Kl/Cre also present with more GFAP-positive astrocytes (FIG. 13) and lba-1 -positive microglia (FIG. 14) in dentate gyrus and the stratum radium indicating an inflammatory reaction in areas with abundant synaptic connections.
  • the results indicate that early activation of the p20p10Casp6 in mouse brain causes age-dependent memory impairment and specific pathological lesions consistent with Alzheimer's disease brains.

Landscapes

  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical & Material Sciences (AREA)
  • Biomedical Technology (AREA)
  • General Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Zoology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Immunology (AREA)
  • Genetics & Genomics (AREA)
  • Biochemistry (AREA)
  • Wood Science & Technology (AREA)
  • Molecular Biology (AREA)
  • Biotechnology (AREA)
  • Environmental Sciences (AREA)
  • General Engineering & Computer Science (AREA)
  • Cell Biology (AREA)
  • Medicinal Chemistry (AREA)
  • Hematology (AREA)
  • Microbiology (AREA)
  • Urology & Nephrology (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Food Science & Technology (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • Toxicology (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Biodiversity & Conservation Biology (AREA)
  • Animal Husbandry (AREA)
  • Animal Behavior & Ethology (AREA)
  • Veterinary Medicine (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

L'invention concerne des mammifères non-humains transgéniques exprimant la Caspase-6 humaine, ainsi que leurs procédés de préparation de et leurs utilisations, par exemple pour l'évaluation de composés pour la thérapie et la prévention de maladies neurodégénératives, telles que la maladie d'Alzheimer.
PCT/CA2012/050527 2011-08-03 2012-08-03 Modèle animal non-humain transgénique d'une maladie neurodégénérative WO2013016826A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US201161514532P 2011-08-03 2011-08-03
US61/514,532 2011-08-03

Publications (1)

Publication Number Publication Date
WO2013016826A1 true WO2013016826A1 (fr) 2013-02-07

Family

ID=47628594

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CA2012/050527 WO2013016826A1 (fr) 2011-08-03 2012-08-03 Modèle animal non-humain transgénique d'une maladie neurodégénérative

Country Status (1)

Country Link
WO (1) WO2013016826A1 (fr)

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
BRYAN, K. ET AL.: "Chapter 1: Transgenic mouse models of Alzheimer's Disease: Behavioral Testing and Considerations.", METHODS OF BEHAVIOR ANALYSIS IN NEUROSCIENCE, 2009, Retrieved from the Internet <URL:http://www.ncbi.nlm.nih.gov/books/NBK5231/?report=printable> [retrieved on 20121005] *
FERNANDES-ALNEMRI T. ET AL.: "Mch2, a new member of the apoptotic Ced-3/Ice cysteine protease gene family.", CANCER RESEARCH, vol. 55, 1 July 1995 (1995-07-01), pages 2737 - 2742 *
GRAHAM R. ET AL.: "Caspase-6 and neurodegeneration", TRENDS IN NEUROSCIENCES, vol. 34, no. 12., December 2011 (2011-12-01), pages 646 - 656 *
LEE A; ET AL ET AL.: "Alternatively spliced caspase-6B isoform inhibits activation of Caspase-6A", JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 285, no. 42., 15 October 2010 (2010-10-15), pages 31974 - 31984 *
URIBE V. ET AL.: "Rescue from excitotoxicity and axonal degeneration accompanied by age-dependent behavioral and neuroanatomical alterations in caspase-6-deficient mice.", HUMAN MOLECULAR GENETICS, vol. 21, no. 9, 18 January 2012 (2012-01-18), pages 1954 - 1967 *

Similar Documents

Publication Publication Date Title
Von Koch et al. Generation of APLP2 KO mice and early postnatal lethality in APLP2/APP double KO mice
CA2290707C (fr) Animaux transgeniques dans lesquels l&#39;expression de l&#39;apolipoproteine e est alteree et procedes de dosage associes
AU671093B2 (en) Transgenic animal models for alzheimer&#39;s disease
US6187992B1 (en) Transgenic mouse having a disrupted amyloid precursor protein gene
Shmerling et al. Strong and ubiquitous expression of transgenes targeted into the β‐actin locus by Cre/lox cassette replacement
US7745690B2 (en) Transgenic nonhuman mammal representing the pathologic conditions of human rheumatoid arthritis
JP2001517065A (ja) トランスジェニック動物モデルを用いてアルツハイマー病治療薬を同定する方法
US6452065B2 (en) Transgenic mouse expressing non-native wild-type and familial Alzheimer&#39;s Disease mutant presenilin 1 protein on native presenilin 1 null background
JP2009178162A (ja) 神経変性性障害のトランスジェニック動物モデル
US20130133090A1 (en) Transgenic mammalls modified in bri protein expression
Bader et al. Renin gene expression and hypertension in transgenic animals
WO2013016826A1 (fr) Modèle animal non-humain transgénique d&#39;une maladie neurodégénérative
JP5083820B2 (ja) 無毛トランスジェニック動物
JP5046413B2 (ja) ヒトｆａｄプレセニリン突然変異をもつ標的遺伝子組換え非ヒト哺乳動物および生殖による子孫
EP2238252B1 (fr) Modèles murins portant une mutation du type inactivation du gène de la glutaminyl cyclase
US8993833B2 (en) Model of Alzheimer&#39;s Disease
US20060015959A1 (en) Transgenic animals expressing transglutaminase II
CA3155265A1 (fr) Modele de rongeur a densite minerale osseuse accrue
US20020157118A1 (en) Genetically engineered animals containing null mutations in the neurofilament genes
Von Koch Molecular analysis of a homologue of the mouse beta-amyloid precursor protein, APLP2: Isolation of APLP2cDNA, characterization of the APLP2 gene promoter and gene targeting of APLP2
JPWO2018012497A1 (ja) 疾患モデル動物および疾患治療剤
WO2004062627A2 (fr) Modeles de criblage in vivo pour le traitement de la maladie d&#39;alzheimer et d&#39;autres affections neurodegeneratives
TOWER Schilling et al.(43) Pub. Date: Jul. 16, 2009
Stephan5Halle et al. Demuth et al.(43) Pub. Date: Oct. 31, 2013
JP2006141283A (ja) ３−ホスホグリセレートデヒドロゲナーゼの発現が低下したノックアウト非ヒト哺乳動物

Legal Events

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

Ref document number: 12820075

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 12820075

Country of ref document: EP

Kind code of ref document: A1