EP0734447A1 - Animal model of the alzheimer disease, preparation and utilisations - Google Patents
Animal model of the alzheimer disease, preparation and utilisationsInfo
- Publication number
- EP0734447A1 EP0734447A1 EP95909839A EP95909839A EP0734447A1 EP 0734447 A1 EP0734447 A1 EP 0734447A1 EP 95909839 A EP95909839 A EP 95909839A EP 95909839 A EP95909839 A EP 95909839A EP 0734447 A1 EP0734447 A1 EP 0734447A1
- Authority
- EP
- European Patent Office
- Prior art keywords
- adenovirus
- animal model
- disease
- alzheimer
- administered
- Prior art date
- Legal status (The legal status 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 status listed.)
- Withdrawn
Links
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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
-
- 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
-
- 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
-
- 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
- C12N15/86—Viral vectors
-
- 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
- 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
- C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
- C12N2710/00011—Details
- C12N2710/10011—Adenoviridae
- C12N2710/10311—Mastadenovirus, e.g. human or simian adenoviruses
- C12N2710/10341—Use of virus, viral particle or viral elements as a vector
- C12N2710/10343—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
Definitions
- the present invention relates to an animal model of Alzheimer's disease, its preparation by means of recombinant viruses, and its use, in particular for the detection of active compounds. More particularly, it relates to recombinant adenoviruses comprising a DNA sequence coding for a form of the precursor of the amyloid peptide, their preparation and their use for the preparation of animal models of Alzheimer's disease. It also relates to the use of these animal models, in particular for the detection of compounds active on Alzheimer's disease, as well as the compounds identified.
- Alzheimer's disease currently affects 10% of people over the age of 65, and 20% over the age of 80. Estimates predict for the year 2000, 5 to 8 million cases in the United States and 23 million worldwide.
- This senile dementia is characterized by the post-mortem presence of senile plaques (comprising a central amyloid deposit) and neurofibrillary degeneration. Although the etiology of the disease is not yet fully known, the formation of amyloid deposits seems to correspond to an early and decisive event.
- the major component of these deposits is the ⁇ A4 peptide from 39 to 43 amino acids whose precursor, designated precursor of the amyloid peptide (APP for "Amyloid Precursor Protein"), has been cloned and sequenced (Kang et al., Nature 325 (1987 ) 733). Since then, various isoforms of the precursor of the amyloid peptide have been identified, corresponding to an alternative splicing of the premeaser RNA (Ponte et al., Nature 331 (1988) 525). In addition, the recent identification in certain familial forms of Alzheimer's disease of mutations in APP has reinforced the involvement of this protein in the etiology of the disease.
- APP amyloid Precursor Protein
- the models according to the invention were prepared by means of vectors viral, more particularly from vectors derived from adenoviruses, containing a DNA sequence coding for a form of the precursor of the amyloid peptide.
- the Applicant has indeed shown that it is possible to construct recombinant adenoviruses containing such a sequence coding for a form of the precursor of the amyloid peptide, to administer these recombinant adenoviruses to an animal, that this administration allows expression of this sequence in said animal and in particular in the nervous system of said animal, and that the expression obtained makes it possible to induce on said animal certain anatomohistological and / or behavioral characteristics of Alzheimer's disease.
- a first object of the present invention therefore resides in a defective recombinant adenovirus comprising a DNA sequence coding for a form of the precursor of the amyloid peptide.
- adenoviruses could be used for the transfer of genes in vivo in the nervous system.
- the recombinant adenoviruses of the present invention are particularly advantageous since they are capable of inducing the appearance of symptoms of Alzheimer's disease in animals.
- the adenoviruses of the invention are capable of inducing the appearance of anatomohistological and / or behavioral characteristics of Alzheimer's disease, which allows the production of an animal physiopathological model which is particularly advantageous for the study and development of a treatment for this disease.
- the particularly advantageous properties of the models of the invention essentially result from the virus used (defective adenovirus, deleted from certain viral regions), from the promoter used for the expression of the sequence coding for APP (preferably viral promoter), and from the methods of administration of said vector, allowing efficient expression and in appropriate tissues of the APP sequence.
- an adenovirus is used which lacks the regions of its genome which are necessary for its replication in the target cell.
- the defective adenoviruses according to the invention are therefore incapable of replicating autonomously in the target cell.
- the genome of the defective adenoviruses used in the context of the present invention lacks at least the sequences necessary for the replication of said virus in the infected cell. These regions can either be eliminated (altogether or in part), either made non-functional, or substituted by other sequences and in particular by the sequence coding for a form of the precursor.
- the defective virus nevertheless retains the sequences of its genome which are necessary for the packaging of the viral particles.
- serotypes of adenoviruses the structure and properties of which vary somewhat.
- human adenoviruses of type 2 or 5 Ad 2 or Ad 5
- Ad 2 or Ad 5 adenoviruses of animal origin.
- the adenovirus of animal origin is a canine adenovirus, more preferably wine adenovirus obtained from a CAV2 strain [manhattan or A26 / 61 strain (ATCC VR-800) for example]. It can also be an adenovirus of mixed origin (containing human and animal sequences).
- the adenovirus of the invention is a human adenovirus of the Ad2 or Ad5 type.
- the adenovirus of the invention is a canine adenovirus of the CAV-2 type.
- the adenoviruses of the invention carry a DNA sequence coding for a form of the precursor of the amyloid peptide.
- the term precursor of the amyloid peptide designates any peptide capable of maturing into an amyloid peptide.
- the precursor of the amyloid peptide according to the invention is capable of maturing into a form of the amyloid peptide ⁇ A4 or of a variant thereof. In a particularly advantageous manner, it must be able to mature into one of the forms comprising 39 to 43 amino acids of the ⁇ A4 peptide.
- the precursor of the amyloid peptide within the meaning of the invention is represented by any natural or artificial variant of the APP.
- different natural variants of APP exist, as well as certain mutated forms.
- artificial variants of the APP can be used, in particular variants containing a truncated N-terminal region.
- the mature amyloid peptide is located in the center of the molecule, at residues 597 to 638 (Kang et al., Nature 325 (1987) 733).
- a DNA sequence coding for the precursor of the truncated amyloid peptide in its N-terminal region is used. More preferably, a DNA sequence coding for the precursor of the truncated amyloid peptide of the N-terminal region preceding the amyloid peptide [variant A4CT or SpA4CT (preceded by a signal sequence)] is used.
- the adenovirus of the invention comprises a DNA sequence coding for any natural or synthetic variant of the APP. Even more preferably, the adenovirus of the invention comprises a DNA sequence coding for 1 ⁇ PP695, 1 ⁇ PP751 or 1 ⁇ PP770.
- the adenovirus of the invention comprises a DNA sequence coding for the precursor of the truncated amyloid peptide in its N-terminal region, such as in particular the A4CT or SpA4CT variants.
- the DNA sequence may also be any sequence homologous to those described above, coding for a form of the precursor of the amyloid peptide as defined above.
- Such homologous sequences can in particular be obtained by techniques known to those skilled in the art, such as mutation, deletion, addition and / or hybridization with the sequences described above.
- mutations observed in familial forms of Alzheimer's disease.
- the sequence coding for the precursor of the amyloid peptide is placed under the control of heterologous expression signals allowing its expression in the infected cells.
- heterologous expression signals signals different from those naturally responsible for the expression of the precursor of the amyloid peptide. They may in particular be sequences responsible for the expression of other proteins, or synthetic sequences.
- they may be promoter sequences of eukaryotic or viral genes.
- they may be promoter sequences originating from the genome of the cell which it is desired to infect.
- they may be promoter sequences originating from the genome of a virus, including the adenovirus used.
- the DNA sequence is placed under the control of expression signals allowing its expression in the nervous system of an animal, preferably chosen from promoters of viral origin.
- the expression sequences can be modified by the addition of regulatory activation sequences, or allowing tissue-specific expression. It may in fact be particularly advantageous to use expression signals which are active specifically or mainly in nerve cells, so that the DNA sequence is only expressed and produces its effect when the virus has actually infected a nerve cell.
- expression signals which are active specifically or mainly in nerve cells, so that the DNA sequence is only expressed and produces its effect when the virus has actually infected a nerve cell.
- the promoter of GFAP may be mentioned, for example, the promoter of GFAP.
- the adenovirus of the invention comprises a DNA sequence coding for a form of the precursor of the amyloid peptide under the control of LTR-RSV.
- this construction indeed makes it possible to express in a very efficient and lasting manner said DNA sequence in the nervous system of animals, and to induce the appearance of easily detectable symptoms of Alzheimer's disease. .
- the defective recombinant adenoviruses according to the invention can be prepared by any technique known to a person skilled in the art (Levrero et al., Gene 101 (1991)
- they can be prepared by homologous recombination between an adenovirus and a plasmid carrying, inter alia, the DNA sequence. Homologous recombination occurs after co-transfection of said adenovirus and plasmid into an appropriate cell line.
- the cell line used must preferably (i) be transformable by said elements, and (ii), contain the sequences capable of complementing the part of the genome of the defective adenovirus, preferably in integrated form to avoid the risks of recombination.
- a line mention may be made of the human embryonic kidney line 293 (Graham et al., J. Gen. Virol. 36 (1977) 59) which contains in particular, integrated into its genome, the left part of the genome of an adenovirus
- Ad5 (12%). Strategies for the construction of vectors derived from adenoviruses have also been described in applications Nos. FR 93 05954 and FR 93 08596. Then, the adenoviruses which have multiplied are recovered and purified according to conventional techniques of molecular biology, as illustrated in the examples.
- Another subject of the invention relates to the use of an adenovirus as defined above for the preparation of an animal model of Alzheimer's disease.
- the invention also relates to any animal model of Alzheimer's disease comprising a non-human mammal containing a recombinant adenovirus as defined above.
- the use of the vectors described above makes it entirely advantageous to prepare animal models of Alzheimer's disease which are reliable and particularly simple to use. More particularly, in these models, the recombinant adenovirus is administered by injection and, even more preferably, by direct injection into the central nervous system. This mode of administration is particularly advantageous since it allows the vector to quickly and effectively infect nerve cells, practically without being disseminated throughout the rest of the body.
- Direct injection into the central nervous system is preferably carried out using a stereotaxic injection device.
- a stereotaxic injection device makes it possible to target with great precision the injection site, as the examples show.
- the recombinant adenovirus is administered in the form of solutions, and in particular of sterile, isotonic solutions, or of dry compositions, in particular lyophilized, which, by addition according to the case of sterilized water or physiological serum, allow the constitution of injectable solutions.
- the doses of defective recombinant adenovirus used for the injection and the number of injections can be adapted according to different parameters, and in particular according to the mode of administration used, the animal used, etc.
- the recombinant adenoviruses according to the invention are formulated and administered in the form of doses of between 10 4 and 10 14 pfu / ml, and preferably 10 6 to 10 10 pfu / ml.
- the term pfu (“plaque forming unit”) corresponds to the infectious power of a virus solution, and is determined by infection of an appropriate cell culture, and measures, generally after 48 hours, the number of cell areas infected. The techniques for determining the pfu titer of a viral solution are well documented in the literature.
- the number of injections can vary depending on the case, between 1 and 5.
- the recombinant adenoviruses are administered to an animal aged 1 to 25 weeks, preferably 4 to 20 weeks. Even more preferably, the injection is carried out on animals 8 to 10 weeks old.
- the non-human mammal used for the preparation of the models according to the invention is advantageously chosen from rodents. More preferably, it is a mouse, a rat, a guinea pig or a rabbit. However, other animals, such as the lemur, can also be used.
- the present invention also describes a method for preparing an animal model of Alzheimer's disease according to which a non-human mammal is administered a recombinant adenovirus comprising a DNA sequence coding for a form of the precursor of the amyloid peptide. .
- the subject of the present invention is also a process for detecting compounds or compositions active with respect to Alzheimer's disease according to which said compound or said composition is administered to an animal as described above, and determines its activity on the effects induced by the recombinant adenovirus.
- the compound or composition is administered by the oral, topical, parenteral, intranasal, intravenous, intramuscular, subcutaneous, intraocular, transdermal, etc. route.
- the invention also includes any compound or composition active with respect to Alzheimer's disease, obtained by the method described above.
- Figure 1 Representation of the vector pXL2244
- Figure 2 Representation of the vector ⁇ Sh-Ad-APP695
- the pBR322, pUC and phage plasmids of the M13 series are of commercial origin (Bethesda Research Laboratories).
- the DNA fragments can be separated according to their size by electrophoresis in agarose or acrylamide gels, extracted with phenol or with a phenol / chloroform mixture, precipitated with ethanol and then incubated in the presence of the DNA ligase from phage T4 (Biolabs) according to the supplier's recommendations.
- the filling of the protruding 5 ′ ends can be carried out by the Klenow fragment of DNA Polymerase I of E. coli (Biolabs) according to the supplier's specifications.
- the destruction of the protruding 3 ′ ends is carried out in the presence of the DNA polymerase of phage T4 (Biolabs) used according to the manufacturer's recommendations.
- the destruction of the protruding 5 ′ ends is carried out by gentle treatment with nuclease SI.
- Mutagenesis directed in vitro by synthetic oligodeoxynucleotides can be carried out according to the method developed by Taylor et al. [Nucleic Acids Res. 23 (1985) 8749-8764] using the kit distributed by Amersham.
- Verification of the nucleotide sequences can be carried out by the method developed by Sanger et al. [Proc. Natl. Acad. Sci. USA, 74 (1977) 5463-5467]. Examples
- Example 1 Construction of the vector pSh-Ad-APP695.
- This example describes the construction of a vector comprising a DNA sequence coding for the precursor of the amyloid peptide APP695 under the control of a promoter constituted by the LTR of the russet sarcoma virus (LTR-RSV).
- the plasmid pXL2244 contains the ApoAI cDNA under the control of the LTR promoter of the RSV virus, as well as sequences of the Ad5 adenovirus (FIG. 1). It was constructed by insertion of a Clal-EcoRV fragment containing the cDNA coding for preproApoAI into the vector pLTR RSV- ⁇ gal (Stratford-Perricaudet et al., J. Clin. Invest. 90 (1992) 626) by the same enzymes.
- This vector contains a sequence coding for the isoform 695 of the precursor of the amyloid peptide (supplied by Dr. J.N. Octave) (Kang et al. Cited above).
- This example describes the construction of the vector pSh-Ad-APP695 containing the sequence coding for the isoform 695 of the precursor of the amyloid peptide under control of the LTR of the RSV virus, as well as sequences of the adenovirus Ad5 allowing the recombination in vivo.
- the vector pSVK3 APP695 was digested with the enzymes EcoRV and Sali.
- the 2 kb EcoRV-SalI fragment containing the sequence coding for isoform 695 of the precursor of the amyloid peptide was then isolated and purified by electrophoresis on an agarose gel LMP ("Low Melting Point").
- the vector pXL2244 was treated with the restriction enzyme Clal (site at position 1089 in FIG. 1). The ends were then made blunt by treatment in the presence of the klenow fragment of the DNA polymerase.
- the vector was then digested with the enzyme Sali (site in position 1930 in FIG. 1).
- the capacity of the vector pSh-Ad-APP695 to express on cell culture the isoform 695 of APP has been demonstrated by transient transfection of COS1 cells.
- the cells (10 ⁇ cells per 10 cm diameter dish) were transfected (8 ⁇ g of vector) in the presence of Transfectam.
- the total and secreted proteins were analyzed in western blot.
- the proteins are separated according to their mass by electrophoresis on a polyacrylamide gel and then transferred to a nitrocellulose membrane.
- the presence of APP is then confirmed by immunological reaction according to the ECL method (Amersham) using the specific antibody 22C11 (Boehringer).
- the vector pSh-Ad-APP695 was linearized and cotransfected with a deficient adenoviral vector, in helper cells (line 293) providing in trans the functions coded by the E1 (E1A and E1B) regions of adenovirus.
- the adenovirus Ad-APP695 was obtained by homologous in vivo recombination between the mutant adenovirus Ad-dll324 (Thimmappaya et al., Cell 31 (1982) 543) and the vector pSh-Ad-APP695, according to the protocol following: the plasmid pSh-Ad-APP695 and the adenovirus Ad-dll324, linearized by the enzyme Clal, were co-transfected in line 293 in the presence of calcium phosphate, to allow homologous recombination. The recombinant adenoviruses thus generated were selected by plaque purification.
- the DNA of the recombinant adenovirus was amplified in the cell line 293, which leads to a culture supernatant containing the unpurified recombinant defective adenovirus having a titre of approximately 10 10 pfu / ml.
- the viral particles are then purified by centrifugation on a cesium chloride gradient according to known techniques (see in particular Graham et al., Virology 52 (1973) 456).
- the Ad-APP695 adenovirus can be stored at -80 ° C in 20% glycerol.
- Ad-APP695 The capacity of the adenovirus Ad-APP695 to express APP isoform 695 on cell culture has been demonstrated by infection of cells of line 293. The presence of APP in the cells was then determined under the same conditions as in Example 2.
- This example describes the construction of an ammal model of Alzheimer's disease containing an adenovirus capable of expressing variant 695 of APP.
- the adenovirus prepared in Example 3 is used in purified form (3.5 10 ⁇ pfu / ⁇ l), in a saline phosphate solution (PBS).
- the animals previously anesthetized, are prepared by direct injections into the central nervous system and, more specifically, in the hippocampus and the entorhinal cortex. The injections are carried out using a cannula (outside diameter 280 ⁇ m) connected to a pump. The injection speed is fixed at 0.5 ⁇ l / min, after which the cannula remains in place for an additional 4 minutes before being reassembled.
- the injection volume in the hippocampus and the entorhinal cortex is 1 ⁇ l.
- the dose of adenovirus injected is 3.5. 10 ° pfu / ⁇ l.
- 1 ⁇ PP695 at the intracerebral injection site is analyzed by immunohistology.
- the animals are sacrificed, under anesthesia, by intracardiac perfusion of 4% paraformaldehyde. After removal and post-fixation (2 hours), the brain is cut in a cryomat: sections 40 ⁇ m thick are collected in buffer. These floating sections are then treated for the immunohistological revelation of 1 ⁇ PP695 with a specific antibody 22C11 (Boehringer) (streptavidin-biotin peroxidase method).
- the animals of the invention make it possible to highlight the impact of this overexpression on the development of brain pathologies linked to Alzheimer's disease, such as the presence of ⁇ protein. -amyloid in diffuse deposits or in plaques, the presence of neurofibrillary degeneration and neuritic plaques, or even neuronal and synaptic losses.
- ⁇ protein. -amyloid in diffuse deposits or in plaques, the presence of neurofibrillary degeneration and neuritic plaques, or even neuronal and synaptic losses.
- the presence of beta-A4, PHF, Ubiquitin and GFAP is determined by immunohistology. In this case, immunohistology is performed on paraffin sections, the brain having been previously dehydrated and included in paraffin.
- the animals of the invention also make it possible to demonstrate the impact of the overexpression of APP695 on behavior, and in particular, on memory processes.
- the pool test (R. Morris, J. Neurosc. Meth. 11 (1984) 47) makes it possible to measure the effect of the injection of adeno-APP, and possibly a compound or of a composition to be tested, on some of these processes.
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Abstract
The present invention relates to an animal model of the Alzheimer disease, its preparation by means of recombinant viruses, and its utilisation particularly for eliciting active compounds.
Description
MODELE ANIMAL DE LA MALADIE D'ALZHEIMER. PREPARATION ET UTILISATIONS ANIMAL MODEL OF ALZHEIMER'S DISEASE. PREPARATION AND USES
La présente invention concerne un modèle animal de la maladie d'Alzheimer, sa préparation au moyen de virus recombinants, et son utilisation, notamment pour la mise en évidence de composés actifs. Plus particulièrement, elle concerne des adénovirus recombinants comportant une séquence d'ADN codant pour une forme du précurseur du peptide amyloïde, leur préparation et leur utilisation pour la préparation de modèles animaux de la maladie d'Alzheimer. Elle concerne aussi l'utilisation de ces modèles animaux, notamment pour la mise en évidence de composés actifs sur la maladie d'Alzheimer, ainsi que les composés identifiés.The present invention relates to an animal model of Alzheimer's disease, its preparation by means of recombinant viruses, and its use, in particular for the detection of active compounds. More particularly, it relates to recombinant adenoviruses comprising a DNA sequence coding for a form of the precursor of the amyloid peptide, their preparation and their use for the preparation of animal models of Alzheimer's disease. It also relates to the use of these animal models, in particular for the detection of compounds active on Alzheimer's disease, as well as the compounds identified.
La maladie d'Alzheimer atteint actuellement 10 % des personnes âgées de plus de 65 ans, et 20 % au delà de 80 ans. Les estimations prévoient pour l'an 2000, 5 à 8 millions de cas aux Etats-Unis et 23 millions dans le monde. Cette démence sénile est caractérisée par la présence post-mortem de plaques séniles (comportant un dépôt amyloïde central) et de dégénérescences neurofibrillaires. Bien que l'étiologie de la maladie ne soit pas encore entièrement connue, la formation des dépôts amyloïdes semble correspondre à un événement précoce et déterminant.Alzheimer's disease currently affects 10% of people over the age of 65, and 20% over the age of 80. Estimates predict for the year 2000, 5 to 8 million cases in the United States and 23 million worldwide. This senile dementia is characterized by the post-mortem presence of senile plaques (comprising a central amyloid deposit) and neurofibrillary degeneration. Although the etiology of the disease is not yet fully known, the formation of amyloid deposits seems to correspond to an early and decisive event.
Le composant majoritaire de ces dépôts est le peptide βA4 de 39 à 43 acides aminés dont le précurseur, désigné précurseur du peptide amyloïde (APP pour "Amyloid Precursor Protein"), a été clone et séquence (Kang et al., Nature 325 (1987) 733). Depuis, différentes isoformes du précurseur du peptide amyloïde ont été identifiées, correspondant à un épissage alternatif de l'ARN préméssager (Ponte et al., Nature 331 (1988) 525). De plus, l'identification récente dans certaines formes familiales de la maladie d'Alzheimer de mutations dans l'APP a renforcé l'implication de cette protéine dans l'étiologie de la maladie.The major component of these deposits is the βA4 peptide from 39 to 43 amino acids whose precursor, designated precursor of the amyloid peptide (APP for "Amyloid Precursor Protein"), has been cloned and sequenced (Kang et al., Nature 325 (1987 ) 733). Since then, various isoforms of the precursor of the amyloid peptide have been identified, corresponding to an alternative splicing of the premeaser RNA (Ponte et al., Nature 331 (1988) 525). In addition, the recent identification in certain familial forms of Alzheimer's disease of mutations in APP has reinforced the involvement of this protein in the etiology of the disease.
Actuellement, l'étude de la maladie d'Alzheimer et la mise au point de composés actifs sur cette maladie sont extrêmement difficiles, notamment en raison de l'absence de modèles animaux de cette pathologie. Les seuls modèles disponibles actuellement reposent sur l'étude in vitro de l'agrégation du peptide β amyloïde ou de la toxicité de ce peptide sur cellules en culture. Mais ce type de modèle ne peut être prédictif d'une activité in vivo, en particulier pour une pathologie aussi complexe que la maladie d'Alzheimer. La présente invention apporte une solution à ce problème. Elle décrit en effet la préparation de modèles animaux de la maladie d'Alzheimer, ainsi que la possibilité d'utiliser ces modèles pour la mise en évidence de composés actifs sur cette pathologie. Les modèles selon l'invention ont été préparés au moyen de vecteurs
viraux, plus particulièrement de vecteurs dérivés des adénovirus, contenant une séquence d'ADN codant pour une forme du précurseur du peptide amyloïde. La demanderesse a en effet montré qu'il est possible de construire des adénovirus recombinants contenant une telle séquence codant pour une forme du précurseur du peptide amyloïde, d'administrer ces adénovirus recombinants à un animal, que cette administration permet une expression de cette séquence dans ledit animal et en particulier dans le système nerveux dudit animal, et que l'expression obtenue permet d'induire sur ledit animal certaines caractéristiques anatomohistologiques et/ou comportementales de la maladie d'Alzheimer. Un premier objet de la présente invention réside donc dans un adénovirus recombinant défectif comprenant une séquence d'ADN codant pour une forme du précurseur du peptide amyloïde.Currently, the study of Alzheimer's disease and the development of compounds active on this disease are extremely difficult, in particular because of the absence of animal models of this pathology. The only models currently available are based on the in vitro study of the aggregation of the amyloid β peptide or of the toxicity of this peptide on cells in culture. But this type of model cannot be predictive of activity in vivo, in particular for a pathology as complex as Alzheimer's disease. The present invention provides a solution to this problem. It indeed describes the preparation of animal models of Alzheimer's disease, as well as the possibility of using these models for the detection of active compounds on this pathology. The models according to the invention were prepared by means of vectors viral, more particularly from vectors derived from adenoviruses, containing a DNA sequence coding for a form of the precursor of the amyloid peptide. The Applicant has indeed shown that it is possible to construct recombinant adenoviruses containing such a sequence coding for a form of the precursor of the amyloid peptide, to administer these recombinant adenoviruses to an animal, that this administration allows expression of this sequence in said animal and in particular in the nervous system of said animal, and that the expression obtained makes it possible to induce on said animal certain anatomohistological and / or behavioral characteristics of Alzheimer's disease. A first object of the present invention therefore resides in a defective recombinant adenovirus comprising a DNA sequence coding for a form of the precursor of the amyloid peptide.
Il a été montré dans la demande copendante n° PCT/EP93/02519 que les adénovirus pouvaient être utilisés pour le transfert de gènes in vivo dans le système nerveux. Les adénovirus recombinants de la présente invention sont particulièrement avantageux puisqu'ils sont capables d'induire l'apparition de symptômes de la maladie d'Alzheimer chez l'animal. En particulier, de manière surprenante, les adénovirus de l'invention sont capables d'induire l'apparition de caractéristiques anatomohistologiques et/ou comportementales de la maladie d'Alzheimer, ce qui permet la réalisation d'un modèle physiopathologique animal particulièrement avantageux pour l'étude et la mise au point d'un traitement de cette maladie.It was shown in copending application No. PCT / EP93 / 02519 that adenoviruses could be used for the transfer of genes in vivo in the nervous system. The recombinant adenoviruses of the present invention are particularly advantageous since they are capable of inducing the appearance of symptoms of Alzheimer's disease in animals. In particular, surprisingly, the adenoviruses of the invention are capable of inducing the appearance of anatomohistological and / or behavioral characteristics of Alzheimer's disease, which allows the production of an animal physiopathological model which is particularly advantageous for the study and development of a treatment for this disease.
Les propriétés particulièrement avantageuses des modèles de l'invention découlent essentiellement du virus utilisé (adénovirus défectif, délété de certaines régions virales), du promoteur utilisé pour l'expression de la séquence codant pour l'APP (promoteur viral de préférence), et des méthodes d'administration dudit vecteur, permettant l'expression efficace et dans les tissus appropriés de la séquence APP.The particularly advantageous properties of the models of the invention essentially result from the virus used (defective adenovirus, deleted from certain viral regions), from the promoter used for the expression of the sequence coding for APP (preferably viral promoter), and from the methods of administration of said vector, allowing efficient expression and in appropriate tissues of the APP sequence.
Préférentiel lement, on utilise dans le cadre de la présente invention un adénovirus dépourvu des régions de son génome qui sont nécessaires à sa réplication dans la cellule cible. Les adénovirus défectifs selon l'invention sont donc incapables de se répliquer de façon autonome dans la cellule cible.Preferably, in the context of the present invention, an adenovirus is used which lacks the regions of its genome which are necessary for its replication in the target cell. The defective adenoviruses according to the invention are therefore incapable of replicating autonomously in the target cell.
Généralement, le génome des adénovirus défectifs utilisés dans le cadre de la présente invention est dépourvu au moins des séquences nécessaires à la réplication dudit virus dans la cellule infectée. Ces régions peuvent être soit éliminées (en tout ou
en partie), soit rendues non-fonctionnelles, soit substituées par d'autres séquences et notamment par la séquence codant pour une forme du précurseur.Generally, the genome of the defective adenoviruses used in the context of the present invention lacks at least the sequences necessary for the replication of said virus in the infected cell. These regions can either be eliminated (altogether or in part), either made non-functional, or substituted by other sequences and in particular by the sequence coding for a form of the precursor.
Préférentiellement, le virus défectif conserve néanmoins les séquences de son génome qui sont nécessaires à l'encapsidation des particules virales. II existe différents sérotypes d'adénovirus, dont la structure et les propriétés varient quelque peu. Parmi ces sérotypes, on préfère utiliser dans le cadre de la présente invention les adénovirus humains de type 2 ou 5 (Ad 2 ou Ad 5) ou les adénovirus d'origine animale. Parmi les adénovirus d'origine animale utilisables dans le cadre de la présente invention on peut citer les adénovirus d'origine canine, bovine, murine, (exemple : Mavl, Beard et al., Virology 75 (1990) 81), ovine, porcine, aviaire ou encore simienne (exemple : SAV) (voir demande FR 93 05954). De préférence, l'adénovirus d'origine animale est un adénovirus canin, plus préférentiellement vin adénovirus obtenu à partir d'une souche CAV2 [souche manhattan ou A26/61 (ATCC VR-800) par exemple]. Il peut également s'agir d'un adénovirus d'origine mixte (contenant des séquences humaines et des séquence animales).Preferably, the defective virus nevertheless retains the sequences of its genome which are necessary for the packaging of the viral particles. There are different serotypes of adenoviruses, the structure and properties of which vary somewhat. Among these serotypes, it is preferred to use, within the framework of the present invention, human adenoviruses of type 2 or 5 (Ad 2 or Ad 5) or adenoviruses of animal origin. Among the adenoviruses of animal origin which can be used in the context of the present invention, mention may be made of adenoviruses of canine, bovine, murine origin (example: Mavl, Beard et al., Virology 75 (1990) 81), ovine, porcine , avian or even simian (example: after-sales service) (see request FR 93 05954). Preferably, the adenovirus of animal origin is a canine adenovirus, more preferably wine adenovirus obtained from a CAV2 strain [manhattan or A26 / 61 strain (ATCC VR-800) for example]. It can also be an adenovirus of mixed origin (containing human and animal sequences).
Dans un mode préféré, l'adénovirus de l'invention est un adénovirus humain de type Ad2 ou Ad5.In a preferred embodiment, the adenovirus of the invention is a human adenovirus of the Ad2 or Ad5 type.
Dans un autre mode préféré, l'adénovirus de l'invention est un adénovirus canin de type CAV-2.In another preferred embodiment, the adenovirus of the invention is a canine adenovirus of the CAV-2 type.
Comme indiqué ci-dessus, les adénovirus de l'invention portent une séquence d'ADN codant pour une forme du précurseur du peptide amyloïde. Le terme précurseur du peptide amyloïde désigne au sens de la présente invention tout peptide capable de maturer en peptide amyloïde. De préférence, le précurseur du peptide amyloïde selon l'invention est capable de maturer en une forme du peptide amyloïde βA4 ou d'un variant de celle-ci. De manière particulièrement avantageuse, il doit pouvoir maturer en une des formes comprenant 39 à 43 acides aminés du peptide βA4.As indicated above, the adenoviruses of the invention carry a DNA sequence coding for a form of the precursor of the amyloid peptide. Within the meaning of the present invention, the term precursor of the amyloid peptide designates any peptide capable of maturing into an amyloid peptide. Preferably, the precursor of the amyloid peptide according to the invention is capable of maturing into a form of the amyloid peptide βA4 or of a variant thereof. In a particularly advantageous manner, it must be able to mature into one of the forms comprising 39 to 43 amino acids of the βA4 peptide.
De préférence, le précurseur du peptide amyloïde au sens de l'invention est représenté par tout variant naturel ou artificiel de l'APP. Comme indiqué ci-avant, différents variants naturels de l'APP existent, ainsi que certaines formes mutées. Parmi les formes naturelles du précurseur, on peut citer plus particulièrement les isoformes APP695, APP751 et APP770. Par ailleurs, des variants artificiels de l'APP peuvent être utilisés, notamment des variants contenant une région N-terminale tronquée. En effet, dans les variants naturels de l'APP, et en particulier pour l'isoforme de l'APP à
695 acides aminés, le peptide amyloïde mature est situé au centre de la molécule, au niveau des résidus 597 à 638 (Kang et al., Nature 325 (1987) 733). Dans un mode de réalisation avantageux de l'invention, on utilise une séquence d'ADN codant pour le précurseur du peptide amyloïde tronqué dans sa région N-terminale. Plus préférentiellement, on utilise une séquence d'ADN codant pour le précurseur du peptide amyloïde tronqué de la région N-terminale précédent le peptide amyloïde [variant A4CT ou SpA4CT (précédé d'une séquence signal)].Preferably, the precursor of the amyloid peptide within the meaning of the invention is represented by any natural or artificial variant of the APP. As indicated above, different natural variants of APP exist, as well as certain mutated forms. Among the natural forms of the precursor, mention may be made more particularly of the isoforms APP695, APP751 and APP770. Furthermore, artificial variants of the APP can be used, in particular variants containing a truncated N-terminal region. Indeed, in the natural variants of APP, and in particular for the isoform of APP at 695 amino acids, the mature amyloid peptide is located in the center of the molecule, at residues 597 to 638 (Kang et al., Nature 325 (1987) 733). In an advantageous embodiment of the invention, a DNA sequence coding for the precursor of the truncated amyloid peptide in its N-terminal region is used. More preferably, a DNA sequence coding for the precursor of the truncated amyloid peptide of the N-terminal region preceding the amyloid peptide [variant A4CT or SpA4CT (preceded by a signal sequence)] is used.
Préférentiellement, l'adénovirus de l'invention comprend une séquence d'ADN codant pour tout variant naturel ou synthétique de l'APP. Encore plus préférentiellement, l'adénovirus de l'invention comprend une séquence d'ADN codant pour 1ΑPP695, 1ΑPP751 ou 1ΑPP770.Preferably, the adenovirus of the invention comprises a DNA sequence coding for any natural or synthetic variant of the APP. Even more preferably, the adenovirus of the invention comprises a DNA sequence coding for 1ΑPP695, 1ΑPP751 or 1ΑPP770.
Dans un mode particulier, l'adénovirus de l'invention comprend une séquence d'ADN codant pour le précurseur du peptide amyloïde tronqué dans sa région N- terminale, tel que notamment les variants A4CT ou SpA4CT.In a particular embodiment, the adenovirus of the invention comprises a DNA sequence coding for the precursor of the truncated amyloid peptide in its N-terminal region, such as in particular the A4CT or SpA4CT variants.
Par ailleurs, il est entendu que la séquence d'ADN peut être également toute séquence homologue à celles décrites ci-dessus, codant pour une forme du précurseur du peptide amyloïde tel que défini ci-avant. De telles séquences homologues peuvent en particulier être obtenues par des techniques connues de l'homme du métier, telles que la mutation, délétion, addition et/ou hybridation avec les séquences décrites ci- dessus. Parmi les formes mutées, on peut citer à titre d'exemple les mutations observées dans les formes familiales de la maladie d'Alzheimer.Furthermore, it is understood that the DNA sequence may also be any sequence homologous to those described above, coding for a form of the precursor of the amyloid peptide as defined above. Such homologous sequences can in particular be obtained by techniques known to those skilled in the art, such as mutation, deletion, addition and / or hybridization with the sequences described above. Among the mutated forms, mention may be made, by way of example, of the mutations observed in familial forms of Alzheimer's disease.
Avantageusement, la séquence codant pour le précurseur du peptide amyloïde est placée sous le contrôle de signaux d'expression hétérologues permettant son expression dans les cellules infectées. Au sens de la présente invention, on entend par signaux d'expression hétérologues des signaux différents de ceux naturellement responsables de l'expression du précurseur du peptide amyloïde. Il peut s'agir en particulier de séquences responsables de l'expression d'autres protéines, ou de séquences synthétiques. Notamment, il peut s'agir de séquences promotrices de gènes eucaryotes ou viraux. Par exemple, il peut s'agir de séquences promotrices issues du génome de la cellule que l'on désire infecter. De même, il peut s'agir de séquences promotrices issues du génome d'un virus, y compris l'adénovirus utilisé.
Préférentiellement, la séquence d'ADN est placée sous le contrôle de signaux d'expression permettant son expression dans le système nerveux d'un animal, choisis de préférence parmi les promoteurs d'origine virale.Advantageously, the sequence coding for the precursor of the amyloid peptide is placed under the control of heterologous expression signals allowing its expression in the infected cells. Within the meaning of the present invention, by heterologous expression signals is meant signals different from those naturally responsible for the expression of the precursor of the amyloid peptide. They may in particular be sequences responsible for the expression of other proteins, or synthetic sequences. In particular, they may be promoter sequences of eukaryotic or viral genes. For example, they may be promoter sequences originating from the genome of the cell which it is desired to infect. Likewise, they may be promoter sequences originating from the genome of a virus, including the adenovirus used. Preferably, the DNA sequence is placed under the control of expression signals allowing its expression in the nervous system of an animal, preferably chosen from promoters of viral origin.
Plus particulièrement, on peut citer par exemple les promoteurs E1A, MLP, CMV, LTR-RSV, etc.More particularly, mention may be made, for example, of the promoters E1A, MLP, CMV, LTR-RSV, etc.
En outre, les séquences d'expression peuvent être modifiées par addition de séquences d'activation de régulation, ou permettant une expression tissu-spécifique. Il peut en effet être particulièrement intéressant d'utiliser des signaux d'expression actifs spécifiquement ou majoritairement dans les cellules nerveuses, de manière à ce que la séquence d'ADN ne soit exprimée et ne produise son effet que lorsque le virus a effectivement infecté une cellule nerveuse. A cet égard, on peut citer par exemple le promoteur de la GFAP.In addition, the expression sequences can be modified by the addition of regulatory activation sequences, or allowing tissue-specific expression. It may in fact be particularly advantageous to use expression signals which are active specifically or mainly in nerve cells, so that the DNA sequence is only expressed and produces its effect when the virus has actually infected a nerve cell. In this regard, there may be mentioned, for example, the promoter of GFAP.
Dans un mode particulièrement avantageux, l'adénovirus de l'invention comprend une séquence d'ADN codant pour une forme du précurseur du peptide amyloïde sous contrôle du LTR-RSV. Comme le montrent les exemples, cette construction permet en effet d'exprimer de manière très efficace et durable ladite séquence d'ADN dans le système nerveux des animaux, et d'induire l'apparition de symptômes facilement détectables de la maladie d'-Alzheimer.In a particularly advantageous embodiment, the adenovirus of the invention comprises a DNA sequence coding for a form of the precursor of the amyloid peptide under the control of LTR-RSV. As the examples show, this construction indeed makes it possible to express in a very efficient and lasting manner said DNA sequence in the nervous system of animals, and to induce the appearance of easily detectable symptoms of Alzheimer's disease. .
Les adénovirus recombinants défectifs selon l'invention peuvent être préparés par toute technique connue de l'homme du métier (Levrero et al., Gène 101 (1991)The defective recombinant adenoviruses according to the invention can be prepared by any technique known to a person skilled in the art (Levrero et al., Gene 101 (1991)
195, EP 185 573; Graham, EMBO J. 3 (1984) 2917). En particulier, ils peuvent être préparés par recombinaison homologue entre un adénovirus et un plasmide portant entre autre la séquence d'ADN. La recombinaison homologue se produit après co- transfection desdits adénovirus et plasmide dans une lignée cellulaire appropriée. La lignée cellulaire utilisée doit de préférence (i) être transformable par lesdits éléments, et (ii), comporter les séquences capables de complémenter la partie du génome de l'adénovirus défectif, de préférence sous forme intégrée pour éviter les risques de recombinaison. A titre d'exemple de lignée, on peut mentionner la lignée de rein embryonnaire humain 293 (Graham et al., J. Gen. Virol. 36 (1977) 59) qui contient notamment, intégrée dans son génome, la partie gauche du génome d'un adénovirus195, EP 185,573; Graham, EMBO J. 3 (1984) 2917). In particular, they can be prepared by homologous recombination between an adenovirus and a plasmid carrying, inter alia, the DNA sequence. Homologous recombination occurs after co-transfection of said adenovirus and plasmid into an appropriate cell line. The cell line used must preferably (i) be transformable by said elements, and (ii), contain the sequences capable of complementing the part of the genome of the defective adenovirus, preferably in integrated form to avoid the risks of recombination. As an example of a line, mention may be made of the human embryonic kidney line 293 (Graham et al., J. Gen. Virol. 36 (1977) 59) which contains in particular, integrated into its genome, the left part of the genome of an adenovirus
Ad5 (12 %). Des stratégies de construction de vecteurs dérivés des adénovirus ont également été décrites dans les demandes n° FR 93 05954 et FR 93 08596.
Ensuite, les adénovirus qui se sont multipliés sont récupérés et purifiés selon les techniques classiques de biologie moléculaire, comme illustré dans les exemples.Ad5 (12%). Strategies for the construction of vectors derived from adenoviruses have also been described in applications Nos. FR 93 05954 and FR 93 08596. Then, the adenoviruses which have multiplied are recovered and purified according to conventional techniques of molecular biology, as illustrated in the examples.
Un autre objet de l'invention concerne l'utilisation d'un adénovirus tel que défini ci-avant pour la préparation d'un modèle animal de la maladie d'Alzheimer.Another subject of the invention relates to the use of an adenovirus as defined above for the preparation of an animal model of Alzheimer's disease.
L'invention concerne également tout modèle animal de la maladie d'Alzheimer comprenant un mammifère non-humain contenant un adénovirus recombinant tel que défini ci-avant.The invention also relates to any animal model of Alzheimer's disease comprising a non-human mammal containing a recombinant adenovirus as defined above.
Comme indiqué ci-avant, l'utilisation des vecteurs décrits ci-avant permet de manière tout à fait avantageuse de préparer des modèles animaux de la maladie d'Alzheimer fiables et particulièrement simples à utiliser. Plus particulièrement, dans ces modèles, l'adénovirus recombinant est administré par injection et, encore plus préférentiellement, par injection directe dans le système nerveux central. Ce mode d'administration est particulièrement avantageux puisqu'il permet au vecteur d'infecter rapidement et efficacement les cellules nerveuses, pratiquement sans être disséminé dans le reste de l'organisme.As indicated above, the use of the vectors described above makes it entirely advantageous to prepare animal models of Alzheimer's disease which are reliable and particularly simple to use. More particularly, in these models, the recombinant adenovirus is administered by injection and, even more preferably, by direct injection into the central nervous system. This mode of administration is particularly advantageous since it allows the vector to quickly and effectively infect nerve cells, practically without being disseminated throughout the rest of the body.
L'injection directe dans le système nerveux central est réalisée de préférence au moyen d'un appareil d'injection stéréotaxique. L'emploi de ce type d'appareil permet en effet de cibler avec une grande précision le site d'injection, comme le montrent les exemples.Direct injection into the central nervous system is preferably carried out using a stereotaxic injection device. The use of this type of device makes it possible to target with great precision the injection site, as the examples show.
Avantageusement, l'adénovirus recombinant est administré sous forme de solutions, et en particulier de solutions stériles, isotoniques, ou de compositions sèches, notamment lyophilisées, qui, par addition selon le cas d'eau stérilisée ou de sérum physiologique, permettent la constitution de solutés injectables.Advantageously, the recombinant adenovirus is administered in the form of solutions, and in particular of sterile, isotonic solutions, or of dry compositions, in particular lyophilized, which, by addition according to the case of sterilized water or physiological serum, allow the constitution of injectable solutions.
Les doses d'adénovirus recombinant défectif utilisées pour l'injection et le nombre d'injections peuvent être adaptées en fonction de différents paramètres, et notamment en fonction du mode d'administration utilisé, de l'animal utilisé, etc. D'une manière générale, les adénovirus recombinants selon l'invention sont formulés et administrés sous forme de doses comprises entre 104 et 1014 pfu/ml, et de préférence 106 à 1010 pfu/ml. Le terme pfu ("plaque forming unit") correspond au pouvoir infectieux d'une solution de virus, et est déterminé par infection d'une culture cellulaire appropriée, et mesure, généralement après 48 heures, du nombre de plages de cellules
infectées. Les techniques de détermination du titre pfu d'une solution virale sont bien documentées dans la littérature.The doses of defective recombinant adenovirus used for the injection and the number of injections can be adapted according to different parameters, and in particular according to the mode of administration used, the animal used, etc. In general, the recombinant adenoviruses according to the invention are formulated and administered in the form of doses of between 10 4 and 10 14 pfu / ml, and preferably 10 6 to 10 10 pfu / ml. The term pfu ("plaque forming unit") corresponds to the infectious power of a virus solution, and is determined by infection of an appropriate cell culture, and measures, generally after 48 hours, the number of cell areas infected. The techniques for determining the pfu titer of a viral solution are well documented in the literature.
Le nombre d'injections peut varier selon les cas, entre 1 et 5.The number of injections can vary depending on the case, between 1 and 5.
De préférence, les adénovirus recombinants sont administrés à un animal âgé de 1 à 25 semaines, de préférence de 4 à 20 semaines. Plus préférentiellement encore, l'injection est réalisée sur des animaux de 8 à 10 semaines.Preferably, the recombinant adenoviruses are administered to an animal aged 1 to 25 weeks, preferably 4 to 20 weeks. Even more preferably, the injection is carried out on animals 8 to 10 weeks old.
Le mammifère non-humain utilisé pour la préparation des modèles selon l'invention est avantageusement choisi parmi les rongeurs. Plus préférentiellement, il s'agit d'une souris, d'un rat, d'un cobaye ou d'un lapin. Toutefois, d'autres animaux, comme le lémurien, peuvent également être utilisés.The non-human mammal used for the preparation of the models according to the invention is advantageously chosen from rodents. More preferably, it is a mouse, a rat, a guinea pig or a rabbit. However, other animals, such as the lemur, can also be used.
La présente invention décrit également un procédé de préparation d'un modèle animal de la maladie d'Alzheimer selon lequel on administre, à un mammifère non-humain, un adénovirus recombinant comprenant une séquence d'ADN codant pour une forme du précurseur du peptide amyloïde.The present invention also describes a method for preparing an animal model of Alzheimer's disease according to which a non-human mammal is administered a recombinant adenovirus comprising a DNA sequence coding for a form of the precursor of the amyloid peptide. .
La présente invention a également pour objet un procédé de mise en évidence de composés ou compositions actifs vis-à-vis de la maladie d'Alzheimer selon lequel on administre ledit composé ou ladite composition à un animal tel que décrit ci-dessus, et on détermine son activité sur les effets induits par l'adénovirus recombinant.The subject of the present invention is also a process for detecting compounds or compositions active with respect to Alzheimer's disease according to which said compound or said composition is administered to an animal as described above, and determines its activity on the effects induced by the recombinant adenovirus.
Avantageusement, le composé ou la composition est administré par voie orale, topique, parentérale, intranasale, intraveineuse, intramusculaire, sous-cutanée, intraoculaire, transdermique, etc.Advantageously, the compound or composition is administered by the oral, topical, parenteral, intranasal, intravenous, intramuscular, subcutaneous, intraocular, transdermal, etc. route.
L'invention comprend aussi tout composé ou composition actif vis-à-vis de la maladie d'Alzheimer, obtenu par le procédé décrit ci-avant.The invention also includes any compound or composition active with respect to Alzheimer's disease, obtained by the method described above.
La présente invention sera plus complètement décrite à l'aide des exemples qui suivent, qui doivent être considérés comme illustratifs et non limitatifs.The present invention will be more fully described with the aid of the following examples, which should be considered as illustrative and not limiting.
Légende des figuresLegend of figures
Figure 1 : Représentation du vecteur pXL2244 Figure 2 : Représentation du vecteur ρSh-Ad-APP695
Techniques générales de biologie moléculaireFigure 1: Representation of the vector pXL2244 Figure 2: Representation of the vector ρSh-Ad-APP695 General molecular biology techniques
Les méthodes classiquement utilisées en biologie moléculaire telles que les extractions préparatives d'ADN plasmidique, la centrifugation d'ADN plasmidique en gradient de chlorure de césium, l'électrophorèse sur gels d'agarose ou d'acrylamide, la purification de fragments d'ADN par électroélution, les extraction de protéines au phénol ou au phénol-chloroforme, la précipitation d'ADN en milieu salin par de l'éthanol ou de l'isopropanol, la transformation dans Escherichia coli, etc ... sont bien connues de l'homme de métier et sont abondament décrites dans la littérature [Maniatis- T. et al., "Molecular Cloning, a Laboratory Manual", Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1982; Ausubel F.M. et al. (eds), "Current Protocols in Molecular Biology", John Wiley & Sons, New York, 1987].Classically used methods in molecular biology such as preparative extractions of plasmid DNA, centrifugation of plasmid DNA in cesium chloride gradient, electrophoresis on agarose or acrylamide gels, purification of DNA fragments by electroelution, the extraction of proteins with phenol or phenol-chloroform, the precipitation of DNA in a saline medium with ethanol or isopropanol, the transformation in Escherichia coli, etc. are well known in the art. skilled in the art and are abundantly described in the literature [Maniatis- T. et al., "Molecular Cloning, a Laboratory Manual", Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1982; Ausubel F.M. et al. (eds), "Current Protocols in Molecular Biology", John Wiley & Sons, New York, 1987].
Les plasmides de type pBR322, pUC et les phages de la série M13 sont d'origine commerciale (Bethesda Research Laboratories).The pBR322, pUC and phage plasmids of the M13 series are of commercial origin (Bethesda Research Laboratories).
Pour les ligatures, les fragments d'ADN peuvent être séparés selon leur taille par électrophorèse en gels d'agarose ou d'acrylamide, extraits au phénol ou par un mélange phénol/chloroforme, précipités à l'éthanol puis incubés en présence de l'ADN ligase du phage T4 (Biolabs) selon les recommandations du fournisseur.For the ligations, the DNA fragments can be separated according to their size by electrophoresis in agarose or acrylamide gels, extracted with phenol or with a phenol / chloroform mixture, precipitated with ethanol and then incubated in the presence of the DNA ligase from phage T4 (Biolabs) according to the supplier's recommendations.
Le remplissage des extrémités 5' proéminentes peut être effectué par le fragment de Klenow de l'ADN Polymérase I d'E. coli (Biolabs) selon les spécifications du fournisseur. La destruction des extrémités 3' proéminentes est effectuée en présence de l'ADN Polymérase du phage T4 (Biolabs) utilisée selon les recommandations du fabricant. La destruction des extrémités 5' proéminentes est effectuée par un traitement ménagé par la nucléase SI.The filling of the protruding 5 ′ ends can be carried out by the Klenow fragment of DNA Polymerase I of E. coli (Biolabs) according to the supplier's specifications. The destruction of the protruding 3 ′ ends is carried out in the presence of the DNA polymerase of phage T4 (Biolabs) used according to the manufacturer's recommendations. The destruction of the protruding 5 ′ ends is carried out by gentle treatment with nuclease SI.
La mutagénèse dirigée in vitro par oligodéoxynucléotides synthétiques peut être effectuée selon la méthode développée par Taylor et al. [Nucleic Acids Res. 23 (1985) 8749-8764] en utilisant le kit distribué par Amersham.Mutagenesis directed in vitro by synthetic oligodeoxynucleotides can be carried out according to the method developed by Taylor et al. [Nucleic Acids Res. 23 (1985) 8749-8764] using the kit distributed by Amersham.
L'amplification enzymatique de fragments d'ADN par la technique dite de PCREnzymatic amplification of DNA fragments by the so-called PCR technique
[Polymérase-catalyzed Chain Reaction, Saiki R.K. et al., Science 22Q (1985) 1350-[Polymerase-catalyzed Chain Reaction, Saiki R.K. et al., Science 22Q (1985) 1350-
1354; Mullis K.B. et Faloona F.A., Meth. Enzym. 155 (1987) 335-350] peut être effectuée en utilisant un "DNA thermal cycler" (Perkin Elmer Cetus) selon les spécifications du fabricant.1354; Mullis K.B. and Faloona F.A., Meth. Enzym. 155 (1987) 335-350] can be performed using a "DNA thermal cycler" (Perkin Elmer Cetus) according to the manufacturer's specifications.
La vérification des séquences nucléotidiques peut être effectuée par la méthode développée par Sanger et al. [Proc. Natl. Acad. Sci. USA, 74 (1977) 5463-5467].
ExemplesVerification of the nucleotide sequences can be carried out by the method developed by Sanger et al. [Proc. Natl. Acad. Sci. USA, 74 (1977) 5463-5467]. Examples
Exemple 1. Construction du vecteur pSh-Ad-APP695.Example 1. Construction of the vector pSh-Ad-APP695.
Cet exemple décrit la construction d'un vecteur comprenant une séquence d'ADN codant pour le précurseur du peptide amyloïde APP695 sous le contrôle d'un promoteur constitué par le LTR du virus du sarcome de rous (LTR-RSV).This example describes the construction of a vector comprising a DNA sequence coding for the precursor of the amyloid peptide APP695 under the control of a promoter constituted by the LTR of the russet sarcoma virus (LTR-RSV).
1.1. Vecteurs de départ :1.1. Starting vectors:
- Vecteur pXL2244 : Le plasmide pXL2244 contient l'ADNc de l'ApoAI sous le contrôle du promoteur LTR du virus RSV, ainsi que des séquences de l'adénovirus Ad5 (figure 1). Il a été construit par insertion d'un fragment Clal-EcoRV contenant l'ADNc codant pour la préproApoAI dans le vecteur pLTR RSV-βgal (Stratford- Perricaudet et al., J. Clin. Invest. 90 (1992) 626), digéré par les même enzymes.- Vector pXL2244: The plasmid pXL2244 contains the ApoAI cDNA under the control of the LTR promoter of the RSV virus, as well as sequences of the Ad5 adenovirus (FIG. 1). It was constructed by insertion of a Clal-EcoRV fragment containing the cDNA coding for preproApoAI into the vector pLTR RSV-βgal (Stratford-Perricaudet et al., J. Clin. Invest. 90 (1992) 626) by the same enzymes.
- Vecteur pSVK3 APP695 : Ce vecteur contient une séquence codant pour l'isoforme 695 du précurseur du peptide amyloïde (fourni par le Dr. J.N. Octave) (Kang et al. précitée).- Vector pSVK3 APP695: This vector contains a sequence coding for the isoform 695 of the precursor of the amyloid peptide (supplied by Dr. J.N. Octave) (Kang et al. Cited above).
1.2. Construction du vecteur pSh-Ad-APP6951.2. Construction of the vector pSh-Ad-APP695
Cet exemple décrit la construction du vecteur pSh-Ad-APP695 contenant la séquence codant pour l'isoforme 695 du précurseur du peptide amyloïde sous contrôle du LTR du virus RSV, ainsi que des séquences de l'adénovirus Ad5 permettant la recombinaison in vivo.This example describes the construction of the vector pSh-Ad-APP695 containing the sequence coding for the isoform 695 of the precursor of the amyloid peptide under control of the LTR of the RSV virus, as well as sequences of the adenovirus Ad5 allowing the recombination in vivo.
Le vecteur pSVK3 APP695 a été digéré par les enzymes EcoRV et Sali. Le fragment EcoRV-SalI de 2 kb contenant la séquence codant pour l'isoforme 695 du précurseur du peptide amyloïde a ensuite été isolé et purifié par électrophorèse sur un gel d'agarose LMP ("Low Melting Point"). Parallèlement, le vecteur pXL2244 a été traité par l'enzyme de restriction Clal (site en position 1089 sur la figure 1). Les extrémités ont ensuite été rendues franches par traitement en présence du fragment klenow de l'ADN polymérase. Le vecteur a ensuite été digéré par l'enzyme Sali (site en position 1930 sur la figure 1). Le vecteur linéaire de 7 kb résultant a ensuite été isolé et purifié par électrophorèse sur un gel d'agarose LMP. Les fragments de 2 et 7 kb ainsi obtenus ont ensuite été ligaturés pour générer le vecteur pSh-Ad-APP695 (figure 2).
Exemple 2. Fonctionalité du vecteur pSh-Ad-APP695The vector pSVK3 APP695 was digested with the enzymes EcoRV and Sali. The 2 kb EcoRV-SalI fragment containing the sequence coding for isoform 695 of the precursor of the amyloid peptide was then isolated and purified by electrophoresis on an agarose gel LMP ("Low Melting Point"). In parallel, the vector pXL2244 was treated with the restriction enzyme Clal (site at position 1089 in FIG. 1). The ends were then made blunt by treatment in the presence of the klenow fragment of the DNA polymerase. The vector was then digested with the enzyme Sali (site in position 1930 in FIG. 1). The resulting 7 kb linear vector was then isolated and purified by electrophoresis on an LMP agarose gel. The 2 and 7 kb fragments thus obtained were then ligated to generate the vector pSh-Ad-APP695 (FIG. 2). Example 2. Functionality of the vector pSh-Ad-APP695
La capacité du vecteur pSh-Ad-APP695 à exprimer sur culture cellulaire l'isoforme 695 de l'APP a été démontrée par transfection transitoire de cellules COS1. Pour cela, les cellules (10^ cellules par boite de 10 cm de diamètre) ont été transfectées (8 μg de vecteur) en présence de Transfectam. Après 72 heures, les protéines totales et sécrétées ont été analysées en western blot. A cet effet, les protéines sont séparées selon leur masse par électrophorèse sur un gel de polyacrylamide puis transférées sur une membrane de nitrocellulose. La présence d'APP est ensuite confirmée par réaction immunologique selon la méthode ECL (Amersham) en utilisant l'anticorps spécifique 22C11 (Boehringer).The capacity of the vector pSh-Ad-APP695 to express on cell culture the isoform 695 of APP has been demonstrated by transient transfection of COS1 cells. For this, the cells (10 ^ cells per 10 cm diameter dish) were transfected (8 μg of vector) in the presence of Transfectam. After 72 hours, the total and secreted proteins were analyzed in western blot. To this end, the proteins are separated according to their mass by electrophoresis on a polyacrylamide gel and then transferred to a nitrocellulose membrane. The presence of APP is then confirmed by immunological reaction according to the ECL method (Amersham) using the specific antibody 22C11 (Boehringer).
Exemple 3. Construction de l'adénovirus Ad-APP695Example 3. Construction of the Ad-APP695 Adenovirus
Le vecteur pSh-Ad-APP695 a été linéarisé et cotransfecté avec un vecteur adénoviral déficient, dans les cellules helper (lignée 293) apportant en trans les fonctions codées par les régions El (E1A et E1B) d'adénovirus. Plus précisément, l'adénovirus Ad-APP695 a été obtenu par recombinaison homologue in vivo entre l'adénovirus mutant Ad-dll324 (Thimmappaya et al., Cell 31 (1982) 543) et le vecteur pSh-Ad-APP695, selon le protocole suivant : le plasmide pSh-Ad-APP695 et l'adénovirus Ad-dll324, linéarisé par l'enzyme Clal, ont été co- transfectés dans la lignée 293 en présence de phosphate de calcium, pour permettre la recombinaison homologue. Les adénovirus recombinants ainsi générés ont été sélectionnés par purification sur plaque. Après isolement, l'ADN de l'adénovirus recombinant a été amplifié dans la lignée cellulaire 293, ce qui conduit à un surnageant de culture contenant l'adénovirus défectif recombinant non purifié ayant un titre d'environ 1010 pfu/ml. Les particules virales sont ensuite purifiées par centrifugation sur gradient de chlorure de césium selon les techniques connues (voir notamment Graham et al., Virology 52 (1973) 456). L'adénovirus Ad-APP695 peut être conservé à -80°C dans 20 % de glycérol.The vector pSh-Ad-APP695 was linearized and cotransfected with a deficient adenoviral vector, in helper cells (line 293) providing in trans the functions coded by the E1 (E1A and E1B) regions of adenovirus. More specifically, the adenovirus Ad-APP695 was obtained by homologous in vivo recombination between the mutant adenovirus Ad-dll324 (Thimmappaya et al., Cell 31 (1982) 543) and the vector pSh-Ad-APP695, according to the protocol following: the plasmid pSh-Ad-APP695 and the adenovirus Ad-dll324, linearized by the enzyme Clal, were co-transfected in line 293 in the presence of calcium phosphate, to allow homologous recombination. The recombinant adenoviruses thus generated were selected by plaque purification. After isolation, the DNA of the recombinant adenovirus was amplified in the cell line 293, which leads to a culture supernatant containing the unpurified recombinant defective adenovirus having a titre of approximately 10 10 pfu / ml. The viral particles are then purified by centrifugation on a cesium chloride gradient according to known techniques (see in particular Graham et al., Virology 52 (1973) 456). The Ad-APP695 adenovirus can be stored at -80 ° C in 20% glycerol.
Exemple 4. Fonctionalité de l'adénovirus Ad-APP695Example 4. Functionality of the Ad-APP695 Adenovirus
La capacité de l'adénovirus Ad-APP695 à exprimer sur culture cellulaire l'isoforme 695 de l'APP a été démontrée par infection des cellules de la lignée 293. La
présence d'APP dans les cellules a ensuite été déterminée dans les mêmes conditions que dans l'exemple 2.The capacity of the adenovirus Ad-APP695 to express APP isoform 695 on cell culture has been demonstrated by infection of cells of line 293. The presence of APP in the cells was then determined under the same conditions as in Example 2.
Ces études permettent de montrer que l'adénovirus exprime bien le variant 695 de l'APP en culture cellulaire.These studies make it possible to show that the adenovirus expresses the 695 variant of APP well in cell culture.
Exemple 5. Construction d'un modèle animalExample 5. Building an animal model
Cet exemple décrit la construction d'un modèle ammal de la maladie d'Alzheimer contenant un adénovirus capable d'exprimer le variant 695 de l'APP.This example describes the construction of an ammal model of Alzheimer's disease containing an adenovirus capable of expressing variant 695 of APP.
L'adénovirus préparé dans l'exemple 3 est utilisé sous forme purifiée (3,5 10^ pfu/μl), dans une solution saline phosphate (PBS). Les animaux, préalablement anesthésiés, sont préparés par injections directes dans le système nerveux central et, plus précisément, au niveau de l'hippocampe et du cortex entorhinal. Les injections sont réalisées à l'aide d'une canule (diamètre extérieur 280 μm) connectée à une pompe. La vitesse d'injection est fixée à 0,5 μl/min, après quoi, la canule reste en place pendant 4 minutes supplémentaires avant d'être remontée. Le volume d'injection dans l'hippocampe et le cortex entorhinal est de 1 μl. La dose d'adénovirus injectée est de 3,5. 10° pfu/μl.The adenovirus prepared in Example 3 is used in purified form (3.5 10 ^ pfu / μl), in a saline phosphate solution (PBS). The animals, previously anesthetized, are prepared by direct injections into the central nervous system and, more specifically, in the hippocampus and the entorhinal cortex. The injections are carried out using a cannula (outside diameter 280 μm) connected to a pump. The injection speed is fixed at 0.5 μl / min, after which the cannula remains in place for an additional 4 minutes before being reassembled. The injection volume in the hippocampus and the entorhinal cortex is 1 μl. The dose of adenovirus injected is 3.5. 10 ° pfu / μl.
Pour l'injection dans l'hippocampe, les coordonnées stéréotaxiques sont les suivantes : AP=-2.0; ML=1,5; V=-2,5 (les coordonnées AP et ML sont déterminées par rapport au bregma, la coordonnée V par rapport à la surface de l'os crânien au niveau du bregma.For injection into the hippocampus, the stereotaxic coordinates are as follows: AP = -2.0; ML = 1.5; V = -2.5 (the AP and ML coordinates are determined relative to the bregma, the V coordinate relative to the surface of the cranial bone at the bregma level.
Pour l'injection dans le cortex entorhinal, les coordonnées stéréotaxiques sont les suivantes : AP=-3,5; ML=3,5; V=-3,7.For injection into the entorhinal cortex, the stereotaxic coordinates are as follows: AP = -3.5; ML = 3.5; V = -3.7.
Les injections suivantes sont réalisées :The following injections are carried out:
- 11 souris C57B1/6 mâles, âgées de 2 mois, sont injectées avec 3,5. 10^ pfu/μl d'adénovirus ad-APP et 4 avec 3,5. 10^ pfu/μl d'adénovirus ad-APP + 3,5. 10*-* pfu/μl d'adénovirus ad-BGal (Le Gai La Salle et al., Science 259 (1993) 988).- 11 male C57B1 / 6 mice, 2 months old, are injected with 3.5. 10 ^ pfu / μl of ad-APP adenovirus and 4 with 3.5. 10 ^ pfu / μl of ad-APP adenovirus + 3.5. 10 * - * pfu / μl of ad-BGal adenovirus (Le Gai La Salle et al., Science 259 (1993) 988).
- 5 souris C57B1/6 mâles, âgées de 2 mois, sont injectées avec 3,5. 10^ pfu/μl d'adénovirus ad-BGal.- 5 male C57B1 / 6 mice, 2 months old, are injected with 3.5. 10 ^ pfu / μl of ad-BGal adenovirus.
Analyse histologiqueHistological analysis
Au cours du temps (7, 15, 30 et 60 jours après l'injection) la surexpression deOver time (7, 15, 30 and 60 days after the injection) the overexpression of
1ΑPP695 au niveau du site d'injection intracérébral est analysée par immunohistologie.
Les animaux sont sacrifiés, sous anesthésie, par perfusion intracardiaque de 4% paraformaldéhyde. Après prélèvement et post-fixation (2 heures), le cerveau est coupé au cryomat : des coupes de 40 μm d'épaisseur sont recueillies dans du tampon. Ces coupes flottantes sont ensuite traitées pour la révélation immunohistologique de 1ΑPP695 par un anticorps spécifique 22C11 (Boehringer) (méthode de streptavidine- biotine peroxydase).1ΑPP695 at the intracerebral injection site is analyzed by immunohistology. The animals are sacrificed, under anesthesia, by intracardiac perfusion of 4% paraformaldehyde. After removal and post-fixation (2 hours), the brain is cut in a cryomat: sections 40 μm thick are collected in buffer. These floating sections are then treated for the immunohistological revelation of 1ΑPP695 with a specific antibody 22C11 (Boehringer) (streptavidin-biotin peroxidase method).
En plus de l'analyse histologique démontrant la surexpression de 1ΑPP695, les animaux de l'invention permettent de mettre en évidence l'incidence de cette surexpression sur le développement de pathologies cérébrales liées à la maladie d'Alzheimer, comme la présence de protéine β-amyloïde en dépots diffus ou en plaques, la présence de dégénérescence neurofibrillaires et de plaques neuritiques, ou encore des pertes neuronales et synaptiques. Comme pour 1ΑPP695, la présence de béta-A4, PHF, Ubiquitine et GFAP est déterminée par immunohistologie. Dans ce cas, l'immunohistologie est réalisée sur coupes en paraffine, le cerveau ayant été préalablement deshydraté et inclu en paraffine.In addition to the histological analysis demonstrating the overexpression of 1ΑPP695, the animals of the invention make it possible to highlight the impact of this overexpression on the development of brain pathologies linked to Alzheimer's disease, such as the presence of β protein. -amyloid in diffuse deposits or in plaques, the presence of neurofibrillary degeneration and neuritic plaques, or even neuronal and synaptic losses. As for 1ΑPP695, the presence of beta-A4, PHF, Ubiquitin and GFAP is determined by immunohistology. In this case, immunohistology is performed on paraffin sections, the brain having been previously dehydrated and included in paraffin.
Analyse comportementaleBehavioral analysis
Les animaux de l'invention permettent également de mettre en évidence l'incidence de la surexpression d'APP695 sur le comportement, et en particulier, sur les processus mnésiques. A cet égard, le test de la piscine (R. Morris, J. Neurosc. Meth. 11 (1984) 47) permet de mesurer l'effet de l'injection de l'adéno-APP, et éventuellemet d'un composé ou d'une composition à tester, sur certains de ces processus.
The animals of the invention also make it possible to demonstrate the impact of the overexpression of APP695 on behavior, and in particular, on memory processes. In this regard, the pool test (R. Morris, J. Neurosc. Meth. 11 (1984) 47) makes it possible to measure the effect of the injection of adeno-APP, and possibly a compound or of a composition to be tested, on some of these processes.
Claims
1. Adénovirus recombinant défectif comprenant une séquence d'ADN codant pour une forme du précurseur du peptide amyloïde.1. Defective recombinant adenovirus comprising a DNA sequence coding for a form of the precursor of the amyloid peptide.
2. Adénovirus selon la revendication 1 caractérisé en ce qu'il est dépourvu des régions de son génome qui sont nécessaires à sa réplication dans la cellule cible.2. Adenovirus according to claim 1 characterized in that it lacks the regions of its genome which are necessary for its replication in the target cell.
3. Adénovirus selon la revendication 2 caractérisé en ce qu'il s'agit d'un adénovirus humain de type Ad2 ou Ad5 ou canin de type CAV-2.3. Adenovirus according to claim 2 characterized in that it is a human adenovirus of type Ad2 or Ad5 or canine of type CAV-2.
4. Adénovirus selon l'une des revendications 1 à 3 caractérisé en ce que la séquence d'ADN hétérologue code pour tout variant naturel ou synthétique de l'APP.4. Adenovirus according to one of claims 1 to 3 characterized in that the heterologous DNA sequence codes for any natural or synthetic variant of APP.
5. Adénovirus selon la revendication 4 caractérisé en ce que la séquence d'ADN hétérologue code pour 1ΑPP695, 1ΑPP751 ou 1ΑPP770.5. Adenovirus according to claim 4 characterized in that the heterologous DNA sequence codes for 1ΑPP695, 1ΑPP751 or 1ΑPP770.
6. Adénovirus selon la revendication 4 caractérisé en ce que la séquence d'ADN hétérologue code pour le précurseur du peptide amyloïde tronqué dans sa région N-terminale.6. Adenovirus according to claim 4 characterized in that the heterologous DNA sequence codes for the precursor of the truncated amyloid peptide in its N-terminal region.
7. Adénovirus selon l'une des revendications précédentes caractérisé en ce que la séquence d'ADN est placée sous le contrôle de signaux d'expression permettant son expression dans le système nerveux d'un animal, choisis de préférence parmi les promoteurs d'origine virale.7. Adenovirus according to one of the preceding claims, characterized in that the DNA sequence is placed under the control of expression signals allowing its expression in the nervous system of an animal, preferably chosen from the promoters of origin viral.
8. Adénovirus selon la revendication 7 caractérisé en ce que les signaux d'expression sont choisis parmi les promoteurs E1A, MLP, CMV et LTR-RSV.8. Adenovirus according to claim 7 characterized in that the expression signals are chosen from the promoters E1A, MLP, CMV and LTR-RSV.
9. Adénovirus recombinant défectif caractérisé en ce qu'il comprend une séquence d'ADN codant pour une forme du précurseur du peptide amyloïde sous contrôle du LTR-RSV.9. Defective recombinant adenovirus characterized in that it comprises a DNA sequence coding for a form of the precursor of the amyloid peptide under the control of LTR-RSV.
10. Utilisation d'un adénovirus selon l'une des revendications 1 à 9 pour la préparation d'un modèle animal de la maladie d'Alzheimer.10. Use of an adenovirus according to one of claims 1 to 9 for the preparation of an animal model of Alzheimer's disease.
11. Modèle animal de la maladie d'Alzheimer caractérisé en ce qu'il s'agit d'un mammifère non-humain contenant un adénovirus recombinant selon l'une des revendications 1 à 9. 11. Animal model of Alzheimer's disease characterized in that it is a non-human mammal containing a recombinant adenovirus according to one of claims 1 to 9.
12. Modèle animal selon la revendication 11 caractérisé en ce que l'adénovirus recombinant est administré par injection.12. Animal model according to claim 11 characterized in that the recombinant adenovirus is administered by injection.
13. Modèle animal selon la revendication 12 caractérisé en ce que l'adénovirus recombinant est administré par injection dans le système nerveux central.13. Animal model according to claim 12 characterized in that the recombinant adenovirus is administered by injection into the central nervous system.
14. Modèle animal selon la revendication 13 caractérisé en ce que l'adénovirus recombinant est administré par injection dans le système nerveux central au moyen d'un appareil d'injection stéréotaxique.14. Animal model according to claim 13 characterized in that the recombinant adenovirus is administered by injection into the central nervous system by means of a stereotaxic injection device.
15. Modèle animal selon l'une des revendications 11 à 14 caractérisé en ce que l'adénovirus recombinant est administré à un animal âgé de 1 à 25 semaines, et de préférence, de 4 à 20 semaines.15. Animal model according to one of claims 11 to 14 characterized in that the recombinant adenovirus is administered to an animal aged from 1 to 25 weeks, and preferably from 4 to 20 weeks.
16. Modèle animal selon l'une des revendications 11 à 15 caractérisé en ce que l'adénovirus recombinant est administré sous forme d'une solution stérile partiellement purifiée.16. Animal model according to one of claims 11 to 15 characterized in that the recombinant adenovirus is administered in the form of a partially purified sterile solution.
17. Modèle animal selon l'une des revendications 11 à 16 caractérisé en ce que le mammifère non-humain est choisi parmi les rongeurs.17. Animal model according to one of claims 11 to 16 characterized in that the non-human mammal is chosen from rodents.
18. Modèle animal selon la revendication 17 caractérisé en ce qu'il s'agit d'une souris, d'un rat, d'un cobaye, d'un lémurien ou d'un lapin.18. Animal model according to claim 17 characterized in that it is a mouse, a rat, a guinea pig, a lemur or a rabbit.
19. Procédé de préparation d'un modèle animal de la maladie d'Alzheimer caractérisé en ce que l'on administre, à un mammifère non-humain, un adénovirus recombinant comprenant une séquence d'ADN codant pour une forme du précurseur du peptide amyloïde.19. Method for preparing an animal model of Alzheimer's disease, characterized in that a non-human mammal is administered a recombinant adenovirus comprising a DNA sequence coding for a form of the precursor of the amyloid peptide .
20. Procédé de mise en évidence de composés ou compositions actifs vis-à- vis de la maladie d'Alzheimer caractérisé en ce que l'on administre ledit composé ou ladite composition à un animal selon l'une des revendications 11 à 18, et on détermine son activité sur l'effet induit par l'adénovirus recombinant.20. Method for demonstrating compounds or compositions active with respect to Alzheimer's disease, characterized in that said compound or said composition is administered to an animal according to one of claims 11 to 18, and its activity is determined on the effect induced by the recombinant adenovirus.
21. Procédé selon la revendication 20 caractérisé en ce que le composé ou la composition sont administrés par voie orale, topique, parentérale, intranasale, intraveineuse, intramusculaire, sous-cutanée, intraoculaire ou transdermique. 21. The method of claim 20 characterized in that the compound or composition are administered orally, topically, parenterally, intranasally, intravenously, intramuscularly, subcutaneously, intraocularly or transdermally.
22. Composé actif vis-à-vis de la maladie d'Alzheimer, obtenu par le procédé selon les revendications 20 à 21.22. Active compound vis-à-vis Alzheimer's disease, obtained by the method according to claims 20 to 21.
23. Composition active vis-à-vis de la maladie d'Alzheimer, obtenue par le procédé selon les revendications 20 à 21. 23. Active composition vis-à-vis Alzheimer's disease, obtained by the method according to claims 20 to 21.
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| FR9401922 | 1994-02-21 | ||
| FR9401922A FR2716460B1 (en) | 1994-02-21 | 1994-02-21 | Animal model of Alzheimer's disease, preparation and uses. |
| PCT/FR1995/000187 WO1995022616A1 (en) | 1994-02-21 | 1995-02-17 | Animal model of the alzheimer disease, preparation and utilisations |
Publications (1)
| Publication Number | Publication Date |
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| EP0734447A1 true EP0734447A1 (en) | 1996-10-02 |
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| EP95909839A Withdrawn EP0734447A1 (en) | 1994-02-21 | 1995-02-17 | Animal model of the alzheimer disease, preparation and utilisations |
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| EP (1) | EP0734447A1 (en) |
| JP (1) | JPH09509059A (en) |
| AU (1) | AU1815695A (en) |
| CA (1) | CA2182724A1 (en) |
| FR (1) | FR2716460B1 (en) |
| WO (1) | WO1995022616A1 (en) |
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| GB9706463D0 (en) * | 1997-03-27 | 1997-05-14 | Medical Res Council | A model of inflamation in the central nervous system for use in the study of disease |
| JP2003516147A (en) * | 1999-12-10 | 2003-05-13 | アリアド ジーン セラピューティクス インコーポレイテッド | High levels of gene expression in primates |
| US20040093623A1 (en) * | 2000-12-20 | 2004-05-13 | Zeger Debyser | Non-human animal disease models |
| KR101890978B1 (en) | 2014-06-17 | 2018-08-24 | 서울대학교산학협력단 | Transgenic cloned porcine Models for alzheimer's disease and the Use thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| GB8927908D0 (en) * | 1989-12-09 | 1990-02-14 | Medical Res Council | Improvements in or relating to alzheimer's disease |
| EP0451700A1 (en) * | 1990-04-10 | 1991-10-16 | Miles Inc. | Recombinant APP minigenes for expression in transgenic mice as models for Alzheimers's disease |
| WO1993014200A1 (en) * | 1992-01-07 | 1993-07-22 | Tsi Corporation | Transgenic animal models for alzheimer's disease |
| WO1994008026A1 (en) * | 1992-09-25 | 1994-04-14 | Rhone-Poulenc Rorer S.A. | Adenovirus vectors for the transfer of foreign genes into cells of the central nervous system, particularly in brain |
| GB9220777D0 (en) * | 1992-10-02 | 1992-11-18 | Univ Leicester | Oligonucleotide probes |
-
1994
- 1994-02-21 FR FR9401922A patent/FR2716460B1/en not_active Expired - Fee Related
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1995
- 1995-02-17 AU AU18156/95A patent/AU1815695A/en not_active Abandoned
- 1995-02-17 EP EP95909839A patent/EP0734447A1/en not_active Withdrawn
- 1995-02-17 CA CA 2182724 patent/CA2182724A1/en not_active Abandoned
- 1995-02-17 WO PCT/FR1995/000187 patent/WO1995022616A1/en not_active Application Discontinuation
- 1995-02-17 JP JP7521633A patent/JPH09509059A/en not_active Ceased
Non-Patent Citations (1)
| Title |
|---|
| JANUS A.C. et al: 'Transgenic mouse models of Alzheimer's disease', BIOCHIMICA ET BIOPHYSICA ACTA, 2000-07-26, vol: 1502, pages 63-75 * |
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| AU1815695A (en) | 1995-09-04 |
| JPH09509059A (en) | 1997-09-16 |
| FR2716460B1 (en) | 2002-08-09 |
| FR2716460A1 (en) | 1995-08-25 |
| WO1995022616A1 (en) | 1995-08-24 |
| CA2182724A1 (en) | 1995-08-24 |
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