EP1216297A2 - Animaux transgeniques utilises comme modeles dans les maladies neurodegeneratives - Google Patents

Animaux transgeniques utilises comme modeles dans les maladies neurodegeneratives

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Publication number
EP1216297A2
EP1216297A2 EP00949241A EP00949241A EP1216297A2 EP 1216297 A2 EP1216297 A2 EP 1216297A2 EP 00949241 A EP00949241 A EP 00949241A EP 00949241 A EP00949241 A EP 00949241A EP 1216297 A2 EP1216297 A2 EP 1216297A2
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European Patent Office
Prior art keywords
human
animal
protein
sequence
vector
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EP00949241A
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German (de)
English (en)
Inventor
Hugo Alfons Gabriel Geerts
Koenraad Frederic Florentina Spittaels
Chris Van Den Haute
Freddy Kamiel Van Leuven
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Janssen Pharmaceutica NV
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Janssen Pharmaceutica NV
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Priority claimed from GBGB9915574.9A external-priority patent/GB9915574D0/en
Application filed by Janssen Pharmaceutica NV filed Critical Janssen Pharmaceutica NV
Publication of EP1216297A2 publication Critical patent/EP1216297A2/fr
Ceased legal-status Critical Current

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    • 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
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    • 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
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    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
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    • A01K67/0276—Knock-out vertebrates
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    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
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    • C07K—PEPTIDES
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    • 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
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    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1205—Phosphotransferases with an alcohol group as acceptor (2.7.1), e.g. protein kinases
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    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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    • A01K2217/07—Animals genetically altered by homologous recombination
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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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    • A01K2227/105—Murine
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    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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    • 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
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    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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    • 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
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    • C12N2800/00—Nucleic acids vectors
    • C12N2800/30—Vector systems comprising sequences for excision in presence of a recombinase, e.g. loxP or FRT

Definitions

  • the present invention relates to cell and animal models for a disease condition and in particular to an animal model which can function as a model for neurodegenerative diseases, such as Alzheimers.
  • Alzheimers disease is a neurodegenerative disorder which is the most prevalent form of senile dementia, with approximately 5% of individuals of 65 and 20% of those over so being afflicted.
  • the disease is characterised by the appearance of two principal lesions within the brain termed neurofibrillary tangles and senile plaques.
  • Neurofibrillary tangles are intracellular inclusion bodies which comprise filamentous aggregates of paired helical filaments (PHF) .
  • PHF paired helical filaments
  • the principal component of PHF has been shown to be Tau, a microtubule associated protein involved in stabilising the cytoskeleton and in determining neuronal shape.
  • Tau is a phosphoprotein and aberrant hyper phosphorylation of Tau appears to represent one mechanism for its aggregation into PHF.
  • FTDP-17 Fronto- temporal dementia associated with Parkinson's disease
  • Cortico-basal degeneration progressive supranuclear palsy, multiple system atrophy
  • Pick' s disease Dementia Pugilistica, Dementia with tangles only, dementia with tangles and calcification, Down syndrome, Myotonic dystrophy, Niemann Pick's disease type C, Parkinsonism-dementia complex of Guam, Postencephalic Parkinsonism, Prion diseases with tangles, subacute sclerosing panencephalitis .
  • the present invention is therefore directed to providing an animal model of neurodegenerative diseases, such as Alzheimers and which model may be utilised to identify compounds useful in treating or ameliorating the symptoms of the condition.
  • the present invention provides a nucleic acid vector comprising a) a nucleic acid sequence encoding a human Tau protein; b) a sequence capable of directing expression of said Tau protein in the nervous system of said animal; and c) a sequence which facilitates integration of said vector into the genome of said animal so as to prevent functional expression of said animal Tau protein in favour of said human Tau protein.
  • This construct or vector thus permits generation of cells of non human animals which express the human Tau and which are substantially uncontaminated with endogenous Tau proteins from the animal or cell.
  • a cell or non-human animal may be particularly useful as a model to monitor the function of human Tau proteins and its potential role in the progression of neurodegenerative disorders mediated by Tau protein, such as Alzheimer's disease.
  • the sequence which facilitates integration of the vector into the genome comprises a sequence of nucleotides which exhibits a sufficient degree of homology with the Tau sequence of the animal or the flanking regions thereof, to permit homologous recombination and subsequent insertion of the vector into the genome of said animal at a location which disrupts the coding region and hence expression of the endogenous Tau in said animal in favour of the human Tau protein encoded from the sequence present on said vector.
  • the vector of the invention may be targeted to, for example, the corresponding Tau sequence of a mouse by the inclusion of a Ncol restriction fragment suitable for insertion of the vector into the unique Ncol site in exon 1 of the Tau sequence in the mouse genome, although as aforementioned a range of appropriate regions of homology to sites upstream or downstream of said Tau sequence may be used.
  • the vector according to the invention is termed a
  • such a vector further comprises a marker sequence which in one embodiment may comprise the hygromycin marker gene Pgk-hyg.
  • the sequence encoding the Tau protein is preferably a cDNA sequence, and even more preferably encodes one of the Tau 40 isoforms already known in the art (Goedert M, Trends Neuroscience 1993 Nov; 16(11): 460-465). However, although the known sequences encoding human Tau isoforms may be utilised, mutated Tau sequences may be used to investigate the role of Tau protein in the pathology of neurodegenerative disorders in an animal mediated by Tau protein.
  • a second aspect of the invention comprises a further nucleic acid vector comprising (a) a nucleic acid sequence encoding a protein capable of modulating a human Tau protein; (b) a sequence capable of directing expression of said protein in the cells of said animal; and (c) a targeting sequence capable of facilitating integration of said vector into the genome of said animal optionally at a position corresponding to a sequence in said animal equivalent to said protein capable of modulating human Tau protein, so as to prevent expression of said equivalent sequence in favour of said protein capable of modulating human Tau protein.
  • Such a vector when integrated at said equivalent sequence in the animal genome, in a similar fashion to the vector described above, permits expression of the protein capable of modulating Tau protein in favour of the related or equivalent protein in said animal.
  • the sequence capable of directing expression of said human Tau protein or the modulator thereof is preferably a transcriptional control sequence which can steer expression of the proteins to the nervous system of the non-human animal.
  • Transcriptional control sequences according to the invention comprise a suitable promoter and other regulatory regions, such as enhancer sequences, that can modulate the activity of the promoter.
  • a bacterial expression vector may include a promoter such as the lac promoter and for transcription initiation in the Shine-Dalgarno sequence and the start codon AUG.
  • a eukaryotic expression vector may include a heterologous or homologous promoter for RNA polymerase II, a downstream polyadenylation signal, the start codon AUG, and a termination codon for detachment of the ribosome.
  • Such vectors may be obtained commercially or assembled from the sequences described by methods well known in the art.
  • a promoter refers to the region of DNA that is upstream with respect to the direction of transcription of the transcription initiation site and which promoter is in a relationship permitting expression of the relevant proteins according to the invention.
  • DNA sequences that drive expression to neurons are known. They include both control systems that are neuron-specific and control systems that are more or less promiscuous but that induce high levels of expression in neurons. Depending on the nature of the construct used in the production of the transgenic animal and, in particular, the control elements, the desired proteins may be expressed in all neurons or only in restricted subsets of neurons of transgenic animals. Neuron-specific control systems, that drive expression to neuronal cell types in general, are known. They may be derived from genes encoding neuron-specific proteins. Such systems may be used to bring about expression of the desired Tau protein and/or the protein capable of it's modulation, in neurons .
  • the sequence is a promoter which directs expression of said proteins in the neurons of the brain or other such cells including astrocytes, oligodendrocytes microglia or Schwann cells.
  • the promoter is the mouse Thy-1 promoter which drives expression in mouse central neurons.
  • the vector according to this aspect of the invention may, advantageously, be used in combination with the vector incorporating the sequence encoding the human Tau protein described above to transfect a non-human animal and thus provide a transgenic animal which serves as a model permitting investigation into the interactions between the protein capable of modulating human Tau protein and said Tau protein and identification of potential therapeutic agents capable of modulating the effects of the phosphorylation of Tau.
  • transgenic animal incorporating the nucleic acid vector encoding said human Tau protein may be crossed with another transgenic animal comprising the vector encoding said protein capable of modulating human Tau protein which may result in offspring which express both of the proteins.
  • the protein capable of modulating human Tau protein is a kinase, and preferably one which is capable of phosphorylating human Tau protein , such as human GSK-3 ⁇ kinase, for example.
  • GSK-3 ⁇ glycogen synthase kinase-3 ⁇
  • GSK- 3 ⁇ As a potential protein Tau and neurofilament kinase has been obtained in transfected cells, wherein both protein Tau (Lovestone et al., 1994; Anderton et al., 1995; Lovestone et al., 1996; Lovestone and Reynolds, 1997) and NF-H were identified as substrates.
  • Cotransfection of GSK-3 ⁇ with Tau in CHO cells increased its phosphorylation concomitant with loss of prominent bundles of microtubules (Wagner et al . , 1996), while co-transfection with NF-H in COS cells caused electrophoretic mobility retardation and the appearance of phosphate-dependent antibody profiles.
  • Xenopus as part of the Wingless signaling pathway in which the kinase is a negative regulator of dorsoventral axis formation.
  • phosphorylation of ⁇ -catenin mediated by axin or conductin, controls the degradation of ⁇ -catenin by the ubiquitin-proteasome pathway (Aberle et al., 1997; Behrens et al. 1998; Ikeda et al., 1998).
  • the model is particularly useful to investigate the molecular basis for Alzheimers and other neurodegenerative disorders mediated by Tau protein and to investigate compounds which may alleviate the symptoms of the disease.
  • the vectors may be transformed into a suitable host cell which is preferably eukaryotic, which may itself be used to transform a non-human animal.
  • a suitable host cell which is preferably eukaryotic, which may itself be used to transform a non-human animal.
  • the invention provides a process for preparing human Tau protein or a protein capable of modulating Tau protein, comprising cultivating a host cell transformed or transfected with a vector according to the invention, under conditions to provide for expression by the vector of said proteins, and recovering the expressed proteins.
  • the host cell is a non-human animal cell, and even more preferably, an embryonic cell of a non-human animal .
  • incorporation of the exogenous DNA into the genome of the animal is accomplished by electroporation of the vector in embryonic stem cells.
  • the cells that have the exogenous DNA incorporated into their genome by homologous recombination may subsequently be injected into blastocysts for generation of the transgenic animals with the desired phenotype.
  • Successfully transformed cells which contain the vector according to the invention may be identified by well known techniques, such as lysing the cells and examining the DNA by, for example, Southern blotting or using the polymerase chain reaction.
  • the vectors may be, for example, plasmid, virus, cosmid or phage vectors, and may contain one or more selectable markers such as the hygromycin marker gene Pgk-hyg.
  • the present invention also advantageously provides nucleic acid sequences of at least approximately 10 contiguous nucleotides of a nucleic acid according to the invention and preferably from 10 to 50 nucleotides even more preferably, the nucleic acid sequence comprise the sequences illustrated in Table 1. These sequences may, advantageously be used as probes or primers to initiate replication, or the like. Such nucleic acid sequences may be produced according to techniques well known in the art, such as by recombinant or synthetic means. They may also be used in diagnostic kits or the like for detecting the presence of a nucleic acid according to the invention. These tests generally comprise contacting the probe with the sample under hybridising conditions and detecting for the presence of any duplex or triplex formation between the probe and any nucleic acid in the sample.
  • the probes according to this aspect of the invention may be anchored to a solid support. Preferably, they are present on an array so that multiple probes can simultaneously hybridize to a single biological sample.
  • the probes can be spotted onto the array or synthesised in si tu on the array. (See Lockhart et al . , Nature Biotechnology, vol. 14, December 1996 "Expression monitoring by hybridisation to high density oligonucleotide arrays".
  • a single array can contain more than 100, 500 or even 1,000 different probes in discrete locations.
  • the nucleic acid sequences, according to the invention may be produced using such recombinant or synthetic means, such as for example using PCR cloning mechanisms which generally involve making a pair of primers, which may be from approximately 10 to 50 nucleotides to a region of the gene which is desired to be cloned, bringing the primers into contact with mRNA, cDNA, or genomic DNA from a human cell, performing a polymerase chain reaction under conditions which bring about amplification of the desired region, isolating the amplified region or fragment and recovering the amplified DNA.
  • PCR cloning mechanisms which generally involve making a pair of primers, which may be from approximately 10 to 50 nucleotides to a region of the gene which is desired to be cloned, bringing the primers into contact with mRNA, cDNA, or genomic DNA from a human cell, performing a polymerase chain reaction under conditions which bring about amplification of the desired region, isolating the amplified region or fragment and
  • the nucleic acids or oligonucleotides according to the invention may carry a revealing label.
  • Suitable labels include radioisotopes such as 32 P or 35 S, enzyme labels or other protein labels such as biotin or fluorescent markers. Such labels may be added to the nucleic acids or oligonucleotides of the invention and may be detected using known techniques per se.
  • Antisense technology can be used to control gene expression through triple-helix formation of antisense DNA or RNA, both of which methods are based on binding of a polynucleotide to DNA or RNA.
  • the 5' coding portion or the mature protein sequence, which encodes for the protein of the present invention is used to design an antisense RNA oligonucleotide of from 10 to 50 base pairs in length.
  • a DNA oligonucleotide is designed to be complementary to a region of the gene involved in transcription (triple-helix - see Lee et al . Nucl. Acids Res.,
  • the antisense RNA oligonucleotide hybridises to the mRNA in vivo and blocks translation of an mRNA molecule (antisense - Okano, J. Neurochem. , 56:560 (1991); Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression, CRC Press, Boca Raton, FL (1988)).
  • each of the relevant proteins may be inhibited using antisense technology which may be used to selectively confirm the action of candidate compounds which may be identified as potential treatments for Alzheimers or other neurodegenerative diseases mediated by Tau protein using the transgenic non-human animal described herein, which expresses said human Tau and/or said protein capable of modulating human Tau protein.
  • antisense technology which may be used to selectively confirm the action of candidate compounds which may be identified as potential treatments for Alzheimers or other neurodegenerative diseases mediated by Tau protein using the transgenic non-human animal described herein, which expresses said human Tau and/or said protein capable of modulating human Tau protein.
  • the vectors of the invention may include a stop signal or sequence between the sequence capable of directing expression of said human Tau or the protein capable of modulating human Tau protein, which stop signal is flanked by two loxP sites.
  • the vector is used to establish the transgenic line as described above and in the examples below, expression of the relevant protein will not occur unless the Cre recombinase protein is present.
  • the Cre protein catalyses reciprocal conservative DNA recombination between the pairs of loxP sites with the resulting excision of the stop sequence located between the loxP sites.
  • the Cre protein may itself be expressed in another transgenic animal which is mated with the first, to remove the stop sequence following the reciprocal combination event between the two loxP sites to switch on expression of the appropriate sequence in the transgenic animal.
  • This technique also permits the DNA sequence encoding the proteins according to the invention to be excised by the Cre protein by including in the appropriate nucleic acid vector loxP sites flanking the sequences encoding human Tau and/or the protein capable of modulating human Tau protein.
  • Such vectors can be used to investigate the role of null mutations or knock-outs of the sequences encoding the proteins in the transgenic animal according to the invention.
  • a defined nucleic acid includes not only the identical nucleic acid but also any minor base variations including in particular, substitutions in cases which result in such as for example a synonymous codon (a different codon specifying the same amino acid residue) due to the degenerate code in conservative amino acid substitutions.
  • nucleic acid sequence also includes the complementary sequence to any single stranded sequence given regarding base variations.
  • a further aspect of the invention comprises a method of making a transgenic non-human animal which expresses a human Tau protein comprising the steps of: (a) introducing into an embryo cell of said animal a nucleic acid vector according to the invention; (b) introducing the embryo from step (a) into a female animal; (c) sustaining the female in step (b) until such time as the embryo has sufficiently developed and is borne from the female; and (d) sustaining the transgenic animal.
  • a further method of generating a transgenic non-human animal which expresses a human Tau protein comprises the steps of (a) introducing sequentially or simultaneously into an embryo cell of said animal a nucleic acid vector comprising a transgene encoding said human Tau protein; and a nucleic acid vector comprising a sequence of nucleotides which upon integration into the genome of said animal are capable of preventing expression of endogenous Tau protein from said animal; (b) introducing the embryo from step (a) into a female animal; (c) sustaining the female in step (b) until such time as the embryo has sufficiently developed and is borne from the female; and (d) sustaining the transgenic animal.
  • Another method of generating a transgenic non-human animal which is a model for diseases such as Alzheimers disease comprises crossing a first transgenic non-human animal expressing human Tau protein from a vector according to the invention with a second transgenic non-human animal expressing a protein capable of modulating human Tau protein according to the invention, selecting among the progeny those that carry both expression of said human Tau protein and said protein capable of modulating human Tau protein.
  • the Cre/lox technology can be used to manipulate expression of the proteins in each of the transgenic non-human animals described herein by incorporation of loxP sites flanking an appropriate DNA sequence.
  • the sequence may be one or both of those encoding either human Tau or the protein capable of modulating human Tau protein themselves or alternatively a stop sequence or codon which prevents expression of the above proteins unless a recombination event occurs in the presence of Cre recombinase to remove the stop sequence.
  • the vectors used according to this aspect of the invention, to generate the transgenic non-human animals, are incapable of replication in yeast.
  • a further aspect of the invention comprises a transgenic non-human animal that is a model for Alzheimers disease or for another neurodegenerative disease, which animal comprises an introduced DNA sequence encoding and capable of expressing the protein Tau in the nervous system of said animal and also comprises a DNA sequence encoding and capable of expressing a protein capable directly or indirectly of modulating the human Tau protein.
  • the human Tau and the protein capable of modulating human Tau are preferably those encoded by the sequences on the vectors according to the invention as described above.
  • a further aspect of the invention comprises a method of generating a transgenic non-human animal which is a model for Alzheimers disease or related neurodegenerative disorders, comprising the steps of crossing a first transgenic non-human animal comprising a vector having, i) a nucleic acid sequence encoding a human Tau protein, ii) a sequence capable of directing expression of said human Tau protein in the nervous system of said animal and iii) a targeting sequence which facilitates integration of said vector into the genome of said animal, with a second transgenic non-human animal comprising a vector capable of expressing a protein capable of modulating human Tau protein according to the invention, selecting among the progeny those that express both human Tau protein and said protein capable of modulating human Tau protein.
  • transgenic non-human animal is also provided by crossing a first transgenic non-human animal expressing human Tau protein with another non-human animal transgenic for the protein which modulates human Tau protein. Therefore, according to this aspect of the invention there is provided a method of generating a transgenic non-human animal which is a model for Alzheimers disease or related neurodegenerative disorders, comprising the steps of crossing a first transgenic non-human animal comprising a vector having, i) a nucleic acid sequence encoding a human Tau protein, ii) a sequence capable of directing expression of said human Tau protein in the nervous system of said animal and iii) a targeting sequence which facilitates integration of said vector into the genome of said animal, with a second transgenic non-human animal comprising a vector according to the invention, selecting among the progeny those that express both human Tau protein and said protein capable of modulating Tau protein.
  • progeny or "offspring” is intended to include the resulting product of a mating between the transgenic animals described provided it carries a vector according to the invention. Also included are germ cells from said transgenic animals which may themselves be used to produce further offspring comprising a vector according to the invention stably integrated into its genome.
  • the non-human animal used in accordance with the methods of the invention is a mammal and even more preferably a mouse.
  • the nucleic acid vectors described can be introduced into the embryonic stem cells, by for example electroporation. Microinjection of the cells is performed on the embryo when it is at the one cell stage, thus ensuring that the nucleic acid vector will be incorporated into the germ line of the animal and thus be expressed in all cells of the animals for subsequent transmission to progeny.
  • a further aspect of the invention comprises progeny of the transgenic animal according to the invention, which progeny carries any of the nucleic acid vectors according to the invention stably integrated into their genome.
  • the transgenic animal may advantageously exhibit the symptoms of Alzheimer's or other related - 1 !
  • Compounds which modulate and interfere with (either by enhancing or inhibiting) the hyperphosphorylation of human Tau protein may be identified by administering the compounds to the animal.
  • Compounds identified as enhancers may advantageously be applied to the animal to enhance development of the disease.
  • Inhibitors of the disease may be identified by monitoring the effects or the phosphorylation profile of Tau protein in the animal following application or administration of the compound to the animal.
  • the compounds may be administered by any suitable route, such as orally or intravenously.
  • the present invention provides a method of producing a compound which modulates the human kinase mediated hyperphosphorylation of human Tau protein comprising the steps of any one of the above described screening methods; and additionally:
  • the compounds isolated by the above methods also serve as lead compounds for the development of analog compounds.
  • the analogs should have a stabilized electronic configuration and molecular conformation that allows key functional groups to be presented to the Tau protein or the kinase in substantially the same way as the lead compound.
  • the analog compounds have spatial electronic properties which are comparable to the binding region, but can be smaller molecules than the lead compound, frequently having a molecular weight below about 2 kD and preferably below about 1 kD.
  • Identification of analog compounds can be performed through use of techniques such as self-consistent field (SCF) analysis, configuration interaction (CI) analysis, and normal mode dynamics analysis.
  • SCF self-consistent field
  • CI configuration interaction
  • normal mode dynamics analysis normal mode dynamics analysis.
  • transgene not every vector, which may otherwise be referred to as a transgene, will function optimally in every cell or animal type. Thus, routine experimentation may be required to identify or establish the best kinase or Tau isoform or promoter sequence for any given cell or animal type.
  • Antibodies to the protein or polypeptide of the present invention may, advantageously, be prepared by techniques which are known in the art.
  • polyclonal antibodies may be prepared by inoculating a host animal, such as a mouse, with the polypeptide according to the invention or an epitope thereof and recovering immune serum.
  • Monoclonal antibodies may be prepared according to known techniques such as described by Kohler R. and Milstein C, Nature (1975) 256, 495-497.
  • such antibodies may be included in a kit for identifying the human Tau or the kinase in a sample, together with means for contacting the antibody with the sample.
  • Figure 1 is an illustration of the recombinant
  • DNA construct used to target the mouse Tau locus The triangles represent the loxP sites.
  • the black boxes indicate a part of the exon 1 of the mouse Tau gene.
  • BSSK+ denotes the bluescript cloning vector.
  • Pgk-hyg represents the hygromycin marker gene.
  • the middle figure shows a partial structure of the wild-type mouse Tau gene.
  • Nco 1 is the unique site on exon 1 into which the entire construct is introduced.
  • the lower figure shows the construct ready for introduction into the ES cells and if homologous recombination occurs in the mouse genome, the different probes used with different enzyme digestions. Details are in the text under the section.
  • Figure 3 is an illustration of a Western Blot results indicating a 64 kDa Tau protein in three different transgenic mouse strains, and probed with antibodies HT- 7 and Tau-5.
  • Figures 4 & 5 are illustrations of the different digestions using rare cutting restriction enzymes in a restriction map of the human Tau gene.
  • Figure 6 is an illustration of the expression of human GSK-3 ⁇ in brain of transgenic mice (A) and activity of human GSK-3 ⁇ in the brain of transgenic animals using a synthetic substrate peptide
  • Figure 7 is an illustration of the results of a Western Blot of brain extracts of GSK- 3 ⁇ [S9A] /htau40 double transgenic mice,
  • Figure 8 is an illustration of the method of producing the loxP - hygromycin construct. This construct is incapable of replication and/or of expressing exogenous proteins in yeast.
  • Figure 9 is a restriction digest of the construct of Figure 8 using various restriction enzymes.
  • Figure 10 illustrates a restriction map of the construct of Figure 8.
  • Figure 11 is an illustration of the results obtained by probing a cell line to ensure the presence of the constructs.
  • FIG 12 is an illustration of Western Blotting of brain extracts of GSK-3 ⁇ [S9A] transgenic mice of 7 months old. Each panel compares brain extracts from 2 individual wild type (wt) mice and from
  • FIG. 13 is an illustration of the effect of alkaline phosphatase pretreatment on hyperhosphoryled protein tau. Brain homogenates of single and double htau- 40-5 and GSK-3 ⁇ [S9A] /htau40 transgenic mice were either applied untreated, or after incubation at 37°C for 3 hours without or with alkaline phosphatase (0.5 unites per ⁇ l) prior to Wester Blotting. For staining with antibodies
  • Tau-5 and Tau-1 the amounts of extract applied were 6 times less than for blotting with AT-8 and AT-180. Note the reduction in signals and the increase in electrophoretic mobility as described and discussed in the text.
  • Figure 17 is an illustration of results obtained from Western Blots of human and murine tau protein which remained unbound to microtubules, using antibodies Tau-1, AT-180 and AD-2. Further shown are the results of quantitative analysis of the unbound protein by densitometric scanning and normalisation to the reaction with antibody Tau-5.
  • Figure 18 is an illustration of the results obtained from a Western Blot to demonstrate that AD-2 and 12E8 epitopes are differentially present on the bound and free protein tau in the microtubule extracts .
  • Figure 21 is an illustration of the results obtained from evaluating the effect of co-expression of GSK-3 ⁇ on the motoric aspect of the phenotype in different tests in double htau 40 x GSK-3 ⁇ transgenic mice, relative to htau 40-2, GSK-3 ⁇ and Wild-type mice.
  • (a) is the result of the ⁇ uprighting reflex'
  • the human GSK-3 ⁇ protein was revealed by Western Blotting (Fig 6A) and was enzymatically active towards a GS-1 synthetic peptide. In brain homogenates of transgenic ice, GSK-3 ⁇ kinase activity was about doubled relative to the activity in wild-type mouse brain (Fig 6B) . Immunohistochemically, the human protein was localized in neuronal cell bodies and in processes in the cortex and hippocampus conform to and expected from the known expression pattern of the adapted mouse thyl gene construct used (Moechars et al., 1996 and references therein).
  • transgenic mice expressing the longest human tau- 4R isoform have been described and characterized (Spittaels et al, 1999) .
  • the present inventors have generated transgenic mice that express a mutant form of human GSK3B, denoted GSK- ⁇ [S9A] since the cDNA contained an alanine residue in position 9, instead of the wild-type serine to prevent inactivation by phosphorylation (Woodgett, 1990) .
  • the cDNA was incorporated in a recombinant DNA construct based on the mouse thy-1 gene promoter (Fig 14a) and transgenic mice were generated by micro- injection, in the FVB mouse strain (Moechars et al, 1996, 1999; Spittaels et al, 1999) .
  • the human GSK-3 ⁇ protein was demonstrated by Western blotting in brain and spinal cord (Fig 14b) .
  • the transgene was enzymatically active on a synthetic peptide substrate, resulting in a doubling of the total GSK-3 ⁇ kinase activity in GSK-3 ⁇ mouse brain homogenates, relative to wild-type mice (Fig 14c).
  • Immuno-histochemically, the human transgenic proteins were localized in neuronal cell bodies and in processes in the cortex and hippocampus (Fig 14d) , as expected for the adapted mouse thyl gene construct (Moechars et al., 1996, 1999; Spittaels et al, 1999).
  • motor neurons in the ventral horn of the spinal cord also expressed the human GSK-3 ⁇ [S9A] mutant and the human tau transgene as well (Fig 14d, g) (Spittaels et al, 1999) . Both transgenes were thus demonstrated to be expressed in the same neurons, in the same regions of brain and spinal cord.
  • Double transgenic mice were obtained by cross-breeding the single transgenic strains. Analysis of phosphorylation of protein tau
  • the epitopes of AD-2 and 12E8 encompass Ser 396 /Ser 404 (Buee-Scherrer et al., 1996) and Ser 262 /Ser 356 (Seubert et al., 1995), respectively, and have been discussed as pivotal in the tau-microtubule interaction, subject to regulation by phosphorylation (Bramblett et al., 1993; Sengupta et al., 1998). Our results suggest that phosphorylation of the AD-2 epitope, in our conditions by GSK-3 ⁇ , could indeed be essential for this interaction.
  • a pathological hallmark of the tau4R transgenic mice i.e. the presence of dilated axons (Spittaels et al., 1999) is now demonstrated to contain synapthophysin- bearing vesicles, normally rapidly transported to the synapse by the motor protein kinesin, which also accumulated in the diseased axons (Fig 19). Consequently, the excess protein tau appeared to inhibit axonal transport by binding to the microtubules in the tau4R transgenic mice, and this then caused the axonal dilatations and the axonopathy (Spittaels et al, 1999) .
  • WT denotes wild-type mice
  • n number of mice analyzed .
  • Murine protein tau extracted from the brain of young GSK- 3 ⁇ [S9A] transgenic mice was somewhat hyperphosphorylated, as manifested by the presence of isoforms with slower electrophoretic migration, with some AT-180 immunoreactivity but weak or absent AT-8 reaction on western blots.
  • endogenous protein tau isoforms with clearly retarded electrophoretic mobility and with strong AT-8 immuno-reactivity were evident in the brain. Isoforms of murine protein tau that migrated on 8% polyacrylamide gels as a broadened band, reacted with antibodies PHF-1 and Tau-5.
  • human protein tau40 containing 2 N-terminal inserts and 4 microtubule binding repeats. Using the same type of gene promoter construct assured the expression of both transgenes to coincide inside the same neurons in brain. In the single and double transgenic mice, human tau protein accounted for up to 60% of total protein tau in the brain of the highest expressing transgenic mouse line.
  • Immunodetection with HT-7 revealed a somatodendritic localisation in addition to axonal staining, similar to a previous report on human tau transgenic mice (Gotz et al. 1995), and resembling the localisation of endogenous protein tau in central neurons (Tashiro et al. 1997) .
  • the cDNA coding for human GSK-3 ⁇ [S9A] was ligated in the mouse thyl gene (Moechars et al., 1996).
  • a Pvul- Notl restriction fragment was micro-injected into 0.5 day old FVB/N prenuclear mouse embryos.
  • Transgenic founders were identified by southern blotting of Stul- restricted mouse tail-biopt DNA, hybridized with a probe of 701 bp obtained by PCR with forward primer 5'CAAGGTCCCCGTTTCTCC3' and reverse primer 5'CAGGGGATAGTGGTGTGG3' .
  • Human Tau40 was ligated in the mouse thyl gene.
  • a Pvul-Notl restriction fragment was micro-injected and transgenic founders identified by southern blotting of Stul-restricted mouse tail-biopt DNA.
  • the probe of 135 bp was obtained by PCR with forward primer 5' CCCCACCACAGAATCCA3 ' located in the mouse thyl gene and reverse primer 5' GCCCCCCTGATCTTTCC3' located in the human tau40 cDNA. Routine genotyping of transgenic offspring, bred into the FVB/N genetic background, was performed on tail-biopt DNA by PCR with a forward primer 5' CTGGGGCGGTCAATAAT3' located in the human tau40 gene and a reverse primer - 31
  • GSK-3 ⁇ [S9A] protein levels in brain extracts were estimated by Western Blotting with monoclonal antibodies TPK I/GSK-3 ⁇ (0.1 ⁇ g/ml) and htau40 protein levels with monoclonal antibodies HT-7 (0.5 ⁇ g/ml) and Tau-5 (0.5 ⁇ g/ml).
  • Kinase enzymatic activity was measured on brain homogenates after immunoprecipitation and fractionation by ion-exchange FPLC (Mono S) (Pharmacia, Uppsala, Sweden) (Van Lint et al. 1993) .
  • mice were anesthetized with nembutal and intracardially perfused with either paraformaldehyde (4% v/v) or methacarn (MC) (50% methanol, 30% chloroform, 10% acetic acid) . Brains were immersion-fixed overnight, dehydrated and embedded in paraffin (unless stated otherwise) . Microtome sections (6 ⁇ m) were dewaxed, hydrated and incubated with blocking solution, i.e. 3% BSA, 10% normal goat serum in Tris Buffered Saline (TBS) (50 mM Tris, pH7.4, 0.15 M NaCl).
  • blocking solution i.e. 3% BSA, 10% normal goat serum in Tris Buffered Saline (TBS) (50 mM Tris, pH7.4, 0.15 M NaCl).
  • brain sections were rinsed with 500 ⁇ l TBS (3x5'), incubated with biotin conjugated secondary antibody (1/1000) for one hour, washed (3x5') and pretreated with 500 ⁇ l 0.05 M Tris-HCl for 5 minutes.
  • tissue sections were submerged in 300 ⁇ l Strept-ABComplex/HRP (1 droplet of both solutions per 15 ml 0.05 ml 0.05 M Tris-HCl) for half an hour and successively washed (3x5'), pretreated with 500 ⁇ l 0.05 M Tris-HCl for 5' and stained with DAB.
  • mice brain homogenates of GSK-3 ⁇ transgenic mice were incubated with immobilized protein-G (Pierce, Illinois, USA) at 4°C for 2.5 hours and purified from mouse IgG by centrifugation (8000 rpm, 5', 4°C). The supernatant was denatured and reduced prior electrophoretical separation.
  • brain homogenates were diluted in a dephosphorylation buffer (Boehringer Mannheim) containing alkaline phosphatase (Boehringer Mannheim, 0.5 unit/ ⁇ l homogenate) and gently stirred at 37°C for 3 hours. Samples to be loaded on the gel were prepared as mentioned above.
  • the ES cell line E14 (Hooper et al., 1987) was cultured on mitomycin-treated STO fibroblasts, in Glasgow ME medium containing non-essential amino acids, 20% (w/v) fetal calf serum, 0. ImM 2- mercaptoethanol and ImM sodium pyruvate. Trypsinized ES cells (1.5-2 x 10 7 ) were resuspended in 500 ⁇ l of culture medium and electroporated with 10 to 15 ⁇ g of the linearised targeting DNA, using an electric pulser (Biorad Labs.) at settings of 200 V and 960 ⁇ F in electroporation cuvettes of 0.4 cm electrode distance.
  • an electric pulser Biorad Labs.
  • the electroporated ES cells were seeded onto mitomycin treated STO fibroblasts in 25 cm 2 flasks and 40 hours later, the medium was replaced with medium containing 100 ⁇ g/ml of Hygromycin B. Hygromycin resistant colonies were picked up 10 to 14 days later after electroporation and further expanded for genotyping.
  • DNA was isolated from the selected ES cell lines and 10 ⁇ g was digested with the desired restriction enzyme for 4 to 6 hours. The digests were separated by electrophoresis at 2 V/cm mechanism for 14 hours on 0.7% agarose gels resulting in an overnight run. The following day the gels were stained by Ethidium bromide and photographed, processed for capillary transfer to nylon membranes. After baking and pre- hybridisation, the blot was hybridised with the radiolabelled probes at a concentration of 2-5 x 10 r cpm/ml and kept overnight at 60°C. Hybridisation was carried out in 6X SSC, 5X Denhardt's solution, 1% SDS, 0.1% heparine, 10% Dextran sulphate and 0.1% Salmon Sperm DNA. Membranes were washed at 60°C for one hour in 0.3X SSC, 0.5% SDS and placed for autoradiographic exposure at 70°C.
  • DNA was transferred by capillary transfer to a nylon membrane with 10X SSC ( 1.5M sodium chloride, 150mM sodium citrate, pH 7.2). The membrane was baked for 2 hrs at 80°C, pre-hybridised for 6 hrs at 60°C in 6X SSC, 4X Denhardt's solution, 1% SDS, 100 ⁇ g salmon sperm DNA, 10% dextran sulfate and 0.05% heparin. Hybridisation was carried out overnight at 60°C in the same solution supplemented with 2-5 x 10 6 cpm/ml of the indicated [ 32 P] -labelled DNA probe.
  • 10X SSC 1.5M sodium chloride, 150mM sodium citrate, pH 7.2
  • the membrane was washed in 0.3X SSPE supplemented with 0.5% SDS for 1 hr at 60°C before autoradiographic exposure with intensifying screens at -70 C for 1-7 days .
  • the ThyI-Tau-40 probe as mentioned above, Hygromycin probe (a gift from Lieve Umans, Lutgarde Serneels and Anton Roebroek) and a 3' probe.
  • the latter was made by a BamHI-Kpn I restriction of a 13kb EcorV-Hind III fragment harbouring exon 1 and the intron between the exons 1 and 2 of the mouse Tau gene cloned in the Bluescript vector (gift by Hirokawa, 1997 ) yielding an external probe.
  • the 3' external probe thus obtained was purified from the gel and used for the first screening of the electroporated ES cells cultured on Hygromycin containing selection medium.
  • the Thyl probe used to check the 5' region of the construct is obtained by Apal digestion of the Thy I DNA (Prof .Van Der Putten) .
  • the purified PAC2 clone was characterised by the analyses of restriction fragments separated by Pulse Field Gel Electrophoresis (PFGE) and identified by Southern blotting using the probes generated by PCR (see materials and methods) .
  • PFGE Pulse Field Gel Electrophoresis
  • the results of the different digestions using rare-cutting restriction enzymes are shown on the restriction map of the human Tau gene ( Figure 4) .
  • the PAC2 clone as sized by PFGE was around 200 kb and housed the entire human Tau gene, confirmed by Southern blotting using different probes that identified the 5' , middle and 3' regions of the gene.
  • the loxP-PGK-hygromycin construct was cut out of the pGEM vector by Notl and ligated into the BamHI site of the Bluescript vector. Transformation of DH5 cells with this 4.7kb construct yielded two positive colonies out of the 20 screened. Restriction analyses with Sail and Seal enzymes gave the expected bands indicating that ligation occurred in the right orientation which was confirmed by sequencing with the T7 primer.
  • the 8 kb Thy-1 human Tau 40 construct (see materials and methods) with the loxP site was subcloned into the Smal site of the above bluescript vector. After transformation 5 out of the 20 colonies screened harboured the insert.
  • Ndel ⁇ 13.5kb, ⁇ 1.2kb, ⁇ 1.6kb
  • Figure 10 shows a restriction map of the concluded construct.
  • This construct is incapable of replicating and/or expressing the exogenous proteins in yeast.
  • This final construct was linearised with Notl and purified on a tip-100 column (Qiagen) which finally gave a concentration of 2.25 ⁇ g/ ⁇ l of which 8 ⁇ l was used for electroporation into ES cells.
  • the ES cells that survived the electroporation were grown on Hygromycin selective medium and after a fortnight well-grown 333 colonies had been picked up for culturing.
  • With the help of Southern blotting using the external 3' probe for the first screening (as mentioned in the materials and methods) , we were able to pick up 6 potential positive cell lines in the first screening.
  • This first positive cell line was used for injection into blastocysts while further screening has resulted in five more potential cell lines. Uterine transfers have so far given 20 pups from three female mice of which 6 are chimeric.
  • the switch of tau protein to an Alzheimer-like state includes the phosphorylation of two serine-proline motifs upstream of the microtubule binding region.
  • Glycogen synthase kinase-3 induces Alzheimer's disease-like phosphorylation of tau: generation of paired helical filament epitopes and neuronal localization of the kinase. Neuroscience Letters 147:58-62.
  • Glycogen synthase kinase 3 ⁇ is identical to tau protein kinase I generating several epitopes of paired helical filaments.

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Abstract

La présente invention concerne un acide nucléique vecteur comprenant : (a) une séquence nucléotidique codant une protéine Tau humaine ; (b) une séquence capable de diriger l'expression de la protéine Tau humaine dans le système nerveux d'un animal non humain ; et (c) une séquence de ciblage qui facilite l'intégration dudit vecteur dans le génome de l'animal afin d'empêcher l'expression de la protéine Tau équivalente ou d'une protéine liée ou équivalente de l'animal en faveur de la protéine Tau humaine. Dans un autre aspect, l'invention concerne un acide nucléique vecteur comprenant : (a) une séquence nucléotidique codant une protéine humaine capable de moduler la protéine Tau humaine ; (b) une séquence capable de diriger l'expression de la protéine précitée dans le système nerveux de l'animal ; et (c) une séquence de ciblage capable de faciliter l'intégration dudit vecteur dans le génome de l'animal facultativement en une position correspondant à une séquence dans l'animal codant un équivalent de la protéine humaine précitée, de façon à empêcher l'expression de la séquence équivalente en faveur de la protéine humaine capable de moduler la protéine Tau humaine. On utilise les vecteurs de l'invention pour fabriquer un animal transgénique non humain en (a) introduisant dans une cellule embryonnaire de l'animal un ou plusieurs acides nucléiques vecteurs décrits ci-dessus ; (b) en introduisant l'embryon de l'étape (a) précitée dans un animal femelle ; (c) en maintenant la femelle dans l'étape (b) jusqu'à ce que l'embryon se soit suffisamment développé et naisse ; et (d) en maintenant en vie l'animal transgénique obtenu.
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