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WO1998003643A2 - Animaux transgenic avec sequences app ou a4ct mutantes humaines - Google Patents

Animaux transgenic avec sequences app ou a4ct mutantes humaines Download PDF

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Publication number
WO1998003643A2
WO1998003643A2 PCT/EP1997/003960 EP9703960W WO9803643A2 WO 1998003643 A2 WO1998003643 A2 WO 1998003643A2 EP 9703960 W EP9703960 W EP 9703960W WO 9803643 A2 WO9803643 A2 WO 9803643A2
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WO
WIPO (PCT)
Prior art keywords
construct
app
a4ct
construct according
seq
Prior art date
Application number
PCT/EP1997/003960
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English (en)
Other versions
WO1998003643A3 (fr
Inventor
Stefan Lichtenthaler
Peter Prior
Colin Louis Masters
Konrad Beyreuther
Original Assignee
Smithkline Beecham Pharma Gmbh
Smithkline Beecham Australia Pty. Ltd.
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
Priority claimed from GBGB9615351.5A external-priority patent/GB9615351D0/en
Priority claimed from GBGB9618804.0A external-priority patent/GB9618804D0/en
Priority claimed from GBGB9709239.9A external-priority patent/GB9709239D0/en
Application filed by Smithkline Beecham Pharma Gmbh, Smithkline Beecham Australia Pty. Ltd. filed Critical Smithkline Beecham Pharma Gmbh
Publication of WO1998003643A2 publication Critical patent/WO1998003643A2/fr
Publication of WO1998003643A3 publication Critical patent/WO1998003643A3/fr

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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
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4711—Alzheimer's disease; Amyloid plaque core protein
    • 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
    • A01K2217/20—Animal model comprising regulated expression system
    • 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
    • A01K2267/0312—Animal model for Alzheimer's disease
    • 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
    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/008—Vector systems having a special element relevant for transcription cell type or tissue specific enhancer/promoter combination

Definitions

  • This invention relates to modified amyloid precursor proteins and their use in the production of transgenic animals.
  • the main protein component of the amyloid plaques found in the brain of Alzheimer's disease (AD) patients is ⁇ A4, a 4 kDa peptide consisting of mainly forty and forty-two residues ( ⁇ A4 0 , ⁇ A4 2 ) being derived from the amyloid precursor protein (APP).
  • APP amyloid precursor protein
  • ⁇ -secretase APP can be cleaved at the N-terminus of ⁇ A4 generating a soluble APP and the C-terminal fragment A4CT (C99).
  • This 99 residue long membrane protein A4CT (ref. 1) which is the direct precursor for ⁇ A4 contains the entire ⁇ A4 domain, the membrane domain and the cytoplasmic tail of APP.
  • ⁇ -secretase Alternative processing of APP in a post-Golgi-compartment by a protease termed ⁇ -secretase leads to the cleavage of APP within the ⁇ A4 domain yielding secretory APP and the transmembrane fragment p3CT which is the direct precursor for p3.
  • Both C-terminal fragments of APP, A4CT and p3CT are cleaved within the membrane domain by a ⁇ -cleavage activity, thereby releasing ⁇ A4 and p3 into the medium (refs. 2, 3).
  • the site of ⁇ -cleavage is mainly the peptide bond Val(40)-Ile(41 )of A4CT and to a minor extent the bond Ala(42)- Thr(43).
  • APP with the Familial AD linked mutations at Val 717 (based on APP770, Val 46 of A4CT) an increased ⁇ -cleavage occurs behind Val(42), thus producing larger amounts of ⁇ A4 1-42 (ref. 4).
  • Transgenic mice expressing A4CT have been produced using the human APP promoter (ref. 5), the human thy- 1 promoter (ref. 6) and the JC viral early region promoter (ref. 7). Numerous promoters have been used in conjunction with the full length APP cDNA (ref. 12).
  • transgenic mammals bearing APP derived DNA sequences are also described in WO93/14200 (TSI Corporation), WO91/19810 (California Biotechnology Inc), WO93/02189 (University of Calfornia), WO89/00689, WO92/06187 (The Upjohn Company), EP0451700 (Miles Inc.), WO92/13069 (Imperial College of Science Technology and Medicine) and WO89/06689 (McClean Hospital Corporation). Results obtained depend upon the source of the promoter and the protein coding sequence used.
  • ⁇ A4,_, j peptide is the major subunit of amorphous and neuritic plaques in
  • references herein to A4CT, ⁇ A4, J2 and ⁇ A4 1J0 include all N-terminal va ⁇ ants produced by alternative cleavage du ⁇ ng processing.
  • a non-human transgenic mammal whose cells contain a construct comp ⁇ sing a human APP or A4CT DNA sequence encoding a mutation selected from: (i) T43A, T43S, T43G, T43V, T43L, T43I or T43F; and
  • Mutation (I) is preferably selected from T43A and T43S
  • Insertion (n) is preferably located between residues 42 and 53, more preferably between 46 and 53. In a preferred embodiment the insertion is located between T48 and L49.
  • the residues for insertion (n) are preferably selected from F, I, G, Y, L, A, P, W, M, S, T, N and Q.
  • the insertion (n) is preferably 2 to 6 residues long. In a preferred embodiment the insertion (n) is LV
  • construct additionally encodes a mutation selected from-
  • V46F V46I, V46G, V46Y, V46L, V46A.
  • V46P V46W, V46M, V46S, V46T, V46N or V46Q.
  • the additional mutation is V46F
  • the construct further encodes the APP signal sequence
  • APP residues 1 to 17 immediately upstream of the APP or A4CT DNA sequence.
  • Hydrophobic residue inserts such as LeuGlu or Met are necessary for processing of A4CT to ⁇ A4 and should preferably be included between the signal peptide and A4CT coding regions and will remain attached to the processed A4CT.
  • the invention also relates to mammalian cells expressing the construct and to the
  • transgenic mammals of the invention may be carried out conventionally, for example as described in WO93/14200, WO91/19810, WO93/02189, WO89/00689, WO92/061 7, EP0451700, WO92/13069 and WO89/06689.
  • the APP or A4CT coding DNA is obtained by probing a human cDNA library.
  • Suitable promoters for use in the present invention include: Human APP (ref. 5); rat neuron specific enolase (neurons) (ref. 18); human ⁇ actin (ref. 19); human PDGF ⁇ (ref. 20); mouse Thy 1 (ref. 21); mouse P ⁇ on protein promoter (PrP) (ref. 14); rat synapsin 1 (brain) (ref. 22), human FMR1 (brain) (ref. 23); human neurofilament low (ref. 24), middle (brain) (ref. 25), NEX-1 (brain) (ref.
  • mouseAPLP2 (brain) (ref. 27); rat alpha tubu n (ref. 28), mouse transfer ⁇ n (ref. 29), mouse HMGCR (3-hydroxy- 3-methyiglutaryl coenzyme A reductase, oligodendrocytes) (ref. 30) and mouse myelin basic protein (ref. 31).
  • a tetracycline-inducible system may also be used, which has the advantage of regulating the gene expression (induction/repression) (refs. 33, 32).
  • This system uses two constructs, a minimal promoter (PhCMV*-l ) fused to seven tetracychc operator sequences and the cDNA in question, and a trangene containing the tetracycline- controlled tran-activator protein (tTA) coding sequence under the control of a promoter, for example taken from the above list.
  • tTA tetracycline- controlled tran-activator protein
  • a preferred promoter is the mouse P ⁇ on protein promoter (ref. 14)
  • the construct is prepared by conventional recombinant DNA techniques (ref. 10).
  • the transgenic mammal is produced by conventional techniques (refs. 8, 15, 16, 17).
  • the transgenic mammal is produced by introduction of the construct into an embryo, insertion of the embryo into a surrogate mother and allowing the embryo to develop to term.
  • the construct is prepared for transfer to the host animal by cleavage of vector containing the construct and purification of the DNA (ref. 8)
  • the transfer is carried out conventionally preferably using microinjection as described in detail in reference 8.
  • the transgenic mammal is produced by introduction of the construct into embryonic stem cells by conventional methods such as calcium phosphate/DNA precipitation, direct inaction or electroporation (ref. 9) followed by injection of the transformed cells into blastocytes and insertion of the resulting embryo into a surrogate mother as described above
  • Transgenic animals are identified by DNA analyis using Southern blot and PCR to detect founder animals
  • the transgenic mammal is preferably a rodent such as rat or mouse, more preferably a mouse.
  • Mammalian cells expressing the construct may be prepared by conventional methods.
  • Host cells are genetically engineered (transduced or transformed or transfected) with the vectors of this invention which may be, for example, a cloning vector or an expression vector.
  • the vector may be, for example, in the form of a plasmid, a viral particle, a phage, etc.
  • the engineered host cells can be cultured in conventional nutrient media modified as appropriate for activating promoters, selecting transformants or amplifying the human genes.
  • the culture conditions such as temperature, pH and the like will be apparent to the ordinarily skilled artisan.
  • mammalian cell culture systems can be employed to express recombinant protein.
  • mammalian expression systems include the COS-7 lines of monkey kidney fibroblasts, described by Gluzman, Cell, 23.175 ( 1981 ), and other cell lines capable of expressing a compatible vector, for example, the SH-S Y5Y, CHO and HeLa cell lines.
  • Mammalian expression vectors will comprise an origin of replication, a suitable promoter and enhancer, and also any necessary ribosome binding sites, polyadenylation site, splice donor and acceptor sites, transcriptional termination sequences, and 5' flanking nontranscribed sequences. DNA sequences derived from the SV40 splice, and polyadenylation sites may be used to provide the required nontranscribed genetic elements.
  • the expression vectors preferably contain one or more selectable marker genes to provide a phenotypic trait for selection of transformed host cells such as hygromycin or neomycin resistance for eukaryotic cell culture.
  • the approp ⁇ ate DNA sequence may be inserted into the vector by a variety of procedures.
  • the DNA sequence is inserted into an approp ⁇ ate restriction endonuciease s ⁇ te(s) by procedures known in the art. Such procedures and others are deemed to be within the scope of those skilled in the art.
  • the DNA sequence in the expression vector is operatively linked to an appropriate expression control sequence(s) (promoter) to direct mRNA synthesis.
  • promoters include the CMV promoter, pCEP4 (Invitrogen) and other promoters known to control expression of genes in eukaryotic cells or their viruses and replicable and viable in the host
  • Introduction of the construct into the host cell can be effected by calcium phosphate transfection, lipofectin-mediated transfection, or electroporation (Davis, L., Dibner, M., Battey, I., Basic Methods in Molecular Biology, (1986))
  • the transgenic mammal or cells of the invention may be used to screen for drugs which inhibit deposit of ⁇ A4 by administe ⁇ ng test drug to the mammal or cell culture medium and observing changes in APP expression and processing, histopathology and/or behavioural changes.
  • the invention extends to such method of screening
  • SPA4CT C-terminal insertion ('CTI') ⁇ n the transmembrane domain (LV between T48 and L49) (SEQ ID NOs: 1 1 and 12) as well as a construct carrying the FAD mutation SPA4CT V46F wtSPA4CT consists of the 17 amino acid long signal peptide of APP followed by two additional amino acids of APP 695 (Leu and Glu) and then continuing with the ⁇ A4 sequence and the whole C-terminal domain and the mutagenesis was earned out in vector pSP65/SPA4CT (ref.
  • SPA4CT T43F was carried out in pBS/SPA4CT rev
  • pBS/SPA4CT rev was obtained by cloning the Kpnl/Nhe fragment of pCEP/SPA4CT (ref. 2) in the pBS/SPC99 vector (ref.
  • constructs were inserted into pCEP vector (ref. 2) and were stably transfected into COS7 cells.
  • G2-1 1 (monoclonal) against synthetic peptide ⁇ A4 35-42 for the immunoprecipitation of ⁇ A4 ⁇ -42
  • 692 polyclonal rabbit serum against synthetic peptide ⁇ A4 1-40 for the immunoprecipitation of ⁇ A4.
  • n-40 and n-42 stand for peptides with a defined C-termmus (ie residue 40 or 42 respectively of ⁇ A4) but allowing for possible N-terminal homogeneity.
  • the full length ⁇ A4 forms produced by the particular constructs desc ⁇ bed herein contain Leu, Glu at positions -2, - 1.
  • the stably transfected COS7 cells were metabohcally labeled for 10 min in methionine free MEM-medium containing 133 ⁇ G/ml 3S S-methion ⁇ ne.
  • A4CT was immunoprecipitated with polyclonal antibody against A4CT (ref. 2), separated on a 10% T ⁇ s-T ⁇ cine gel and quantified by phospho ⁇ maging.
  • the mutations near the C-terminus of ⁇ A4 are able to influence the ⁇ -cleavage site, whereas the overall amount of generated ⁇ A4 as well as the ratio of ⁇ A4/p3 remain unchanged.
  • the single mutants ( I ), (2) and (3) have an increased ratio ⁇ A4 2 / ⁇ A4 1 40 relative to wt.
  • the double mutants (4), (5) and (6) lead, respectively, to a 5.6-, 5.8 and 4.1 -fold increase in ⁇ A4 2 / ⁇ A4, 4n relative to wt.
  • constructs which produce enhanced production of ⁇ A4 2 .relative to ⁇ A4, 4fl are useful for the generation of transgenic mice developing amyloid plaques.
  • construct SPA4CT T43A+V46F driven by the mouse Prion protein promoter (ref. 14) is used to transform a mouse by the following procedures:
  • the construct is prepared and purified. Female mice are induced to superovulate and embryos are recovered. DNA is microinjected into the pronucleus of embryos.
  • Embryos are transferred into pseudopregnant mice (female mice previously paired with vasectomised males).
  • mice Embryos are developed and mice are born. Founder mice are identified by Southern blot and PCR and bred on.
  • mice are as follows: Donor mice (embryos for pronucleus injection): B6D2F2 Acceptor mice: NMRI Mice for further breeding: C57BL6
  • transgenic mice described above may be used to screen for potential activity of test drugs in the treatment of Alzheimer's disease.
  • APP expression and processing may be examined using detection of mRNA by
  • Histopathological observations may be made using immunohistological techniques to permit identification of amyloid plaques and in situ hybridisation using labelled probes to target mRNA.
  • Observation of behavioural changes may employ conventional tests used to assess learning and memory deficits.

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Abstract

Produits de recombinaison qui comportent une séquence d'ADN APP ou A4CT codant des mutations qui entraînent un taux plus élevé de βA41-42/βA41-40 que le type sauvage et utilisation desdits produits dans la production d'animaux transgéniques développant des plaques amyloïdes, destinés à servir de modèles pour la maladie d'Alzheimer.
PCT/EP1997/003960 1996-07-22 1997-07-17 Animaux transgenic avec sequences app ou a4ct mutantes humaines WO1998003643A2 (fr)

Applications Claiming Priority (6)

Application Number Priority Date Filing Date Title
GB9615351.5 1996-07-22
GBGB9615351.5A GB9615351D0 (en) 1996-07-22 1996-07-22 Compounds
GBGB9618804.0A GB9618804D0 (en) 1996-09-09 1996-09-09 Compounds
GB9618804.0 1996-09-09
GBGB9709239.9A GB9709239D0 (en) 1997-05-08 1997-05-08 APP transgenic model
GB9709239.9 1997-05-08

Publications (2)

Publication Number Publication Date
WO1998003643A2 true WO1998003643A2 (fr) 1998-01-29
WO1998003643A3 WO1998003643A3 (fr) 1998-04-30

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998058060A1 (fr) * 1997-06-18 1998-12-23 Smithkline Beecham Pharma Gmbh Sequences app ou a4ct humaines codant la mutation 145f
GB2380196A (en) * 2001-08-21 2003-04-02 Smithkline Beecham Plc Transgenic animals with mutant human amyloid precursor protein sequences
WO2004026331A1 (fr) * 2002-09-17 2004-04-01 Vlaams Interuniversitair Instituut Voor Biotechnologie Vzw Peptides inhibant les activites de clivage specifiques des presenilines
US6815175B2 (en) 2001-03-16 2004-11-09 Cornell Research Foundation, Inc. Anti-amyloid peptide antibody based diagnosis and treatment of a neurological disease or disorder
WO2006134342A3 (fr) * 2005-06-13 2007-05-18 Merck Sharp & Dohme Proteines

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE69233109T2 (de) * 1992-01-07 2004-05-19 Elan Pharmaceuticals, Inc., San Francisco Transgene tiermodelle fur alzheimer-krankheit

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998058060A1 (fr) * 1997-06-18 1998-12-23 Smithkline Beecham Pharma Gmbh Sequences app ou a4ct humaines codant la mutation 145f
US6815175B2 (en) 2001-03-16 2004-11-09 Cornell Research Foundation, Inc. Anti-amyloid peptide antibody based diagnosis and treatment of a neurological disease or disorder
GB2380196A (en) * 2001-08-21 2003-04-02 Smithkline Beecham Plc Transgenic animals with mutant human amyloid precursor protein sequences
WO2004026331A1 (fr) * 2002-09-17 2004-04-01 Vlaams Interuniversitair Instituut Voor Biotechnologie Vzw Peptides inhibant les activites de clivage specifiques des presenilines
WO2006134342A3 (fr) * 2005-06-13 2007-05-18 Merck Sharp & Dohme Proteines

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