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  • A01K2267/0312—Animal model for Alzheimer's disease
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  • A01K2267/03—Animal model, e.g. for test or diseases
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  • 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
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  • 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
  • C12N2015/8527—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells for producing genetically modified animals, e.g. transgenic for producing animal models, e.g. for tests or diseases
  • C12N2015/8536—Animal models for genetic diseases
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  • C12N2750/14011—Parvoviridae
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  • C12N2750/14141—Use of virus, viral particle or viral elements as a vector
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• • G—PHYSICS
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  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
  • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
  • G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
  • G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups

Definitions

• the present invention relates to a vector comprising a nucleic acid sequence that encodes the APP protein and/or the PS1 protein or variants thereof.
• the invention also relates to a method for inducing Alzheimer's disease in an animal using the vector of the invention and to animal model having Alzheimer's disease obtained by said method.
• AD Alzheimer's disease
• SP senile plaques
• NFT neurofibrillary tangles
• APP amyloid precursor protein
• AD animal models have been developed, most of them being transgenic mouse models obtained by transferring genes carrying mutations identified in familial AD including APP, PS1 and PS2 (Lee and Han, 2013). Although not perfect, these models offer a mean to gain knowledge on the physiopathology of AD but they also suffer from various limitations (expression of neurotoxic peptides from in utero development, associated compensatory effect, genetic drift in particular) which impair their use in research.
• viral vectors are attractive tools for gene transfer in the central nervous system (CNS) due to their lack of toxicity, their strong capacity to transduce neurons and to stably express recombinant proteins (for several years in rodents, dogs and primates).
• Viral vectors have already and are currently being used in several clinical trials in human patients worldwide (Cartier et al, 2009).
• the use of viral vectors to develop new animal models of neurodegenerative disorders is currently under investigation (Deglon and Hantraye, 2005). This strategy holds various advantages compared to classical transgenic approaches: viral vectors are versatile, highly flexible tools to perform in vivo studies and multiple genetic models can be created in a short period of time.
• Lentiviral or AAV vectors were successfully injected in the brain of adult mice, rats or primates to create models of various neurodegenerative diseases such as Huntington's, Parkinson's, Machado-Joseph diseases (Kirik et al, 2002; Lo Bianco et al, 2002).
• the inventors have now developed an efficient and powerful animal model of Alzheimer's disease by using optimized APPsl and PS1 genes and Adeno-associated virus (AAV) vectors.
• AAV Adeno-associated virus
• the invention relates to a vector comprising a nucleic acid sequence that encodes the APP protein and/or the PS1 protein or variants thereof.
• the invention also relates to a method for inducing the Alzheimer's disease in an animal using the vector of the invention and to animal model having the Alzheimer's disease obtained by said method.
• a first object of the invention relates to a vector comprising a nucleic acid sequence that encodes the APP protein and/or the PS1 protein or variants thereof.
• the vector of the invention comprises a nucleic acid sequence that encodes the APP protein and a nucleic acid sequence that encodes the PS1 protein.
• the vector of the invention may comprises any variant of the nucleic acid sequence which encodes for the APP protein and/or any variant of the nucleic acid sequence which encodes for the PS1 protein.
• the vector of the invention may comprises any variant of the nucleic acid sequence which encodes for any variant of the APP protein and/or any variant of the nucleic acid sequence which encodes for any variant of the PS1 protein.
• the invention in another embodiment, relates to a vector comprising a nucleic acid sequence that encodes the APP protein or variants thereof and a vector comprising a nucleic acid sequence that encodes the PS1 protein or variants thereof.
• APP Amyloid Precursor Protein
• APP is an integral membrane protein expressed in many tissues and concentrated in the synapses of neurons. Its primary function is not known, though it has been implicated as a regulator of synapse formation, neural plasticity and iron export.
• APP is best known as the precursor molecule whose proteolysis generates beta amyloid ( ⁇ ), a 37 to 49 amino acid peptide whose amyloid fibrillar form is the primary component of amyloid plaques found in the brains of Alzheimer's disease patients.
• the cDNA sequence for APP is disclosed in Genbank under access number Gene ID: 351 and has the sequence SEQ ID NO: l as described below: ATGCTGCCCGGACTGGCTCTGCTGCTGCTGGCCGCTTGGACCGCCAGAGCC CTGGAAGTGCCCACCGATGGCAATGCTGGCCTGCTGGCCGAGCCCCAGATCGCCA TGTTCTGCGGCAGACTGAACATGCACATGAACGTGCAGAACGGCAAGTGGGACA GCGACCCCAGCGGCACCAAGACCTGCATCGACACCAAAGAGGGCATCCTGCAGT ATTGCCAGGAAGTGTACCCCGAGCTGCAGATCACCAACGTGGTGGAAGCCAACC AGCCCGTGACCATCCAGAACTGGTGCAAGCGGGGCAGAAAGCAGTGCAAGACCC ACCCCCACTTCGTGATCCCTTACCGGTGCCTGGTCGGAGAGTTCGTGTCCGACGC CCTGCTGGTGCCCGACAAGTGCAAGTTCCTGCATCAGGAACGGATGGACGTCTGC GAGACACATCTGC
• the APP used according to the invention is the APP (SEQ ID NO: 2) with the Swedish and London mutations (APPsl) which has the following protein sequence (SEQ ID NO: 3):
• PSl or "Presenilin 1" denotes a protein encoded by the PSEN1 gene.
• Presenilin 1 is one of the four core proteins in presenilin complex, which mediate the regulated proteolytic events of several proteins in the cell, including gamma secretase.
• Gamma-secretase is considered to play a strong role in generation of beta amyloid, accumulation of which is related to the onset of Alzheimer 's disease, from the beta-amyloid precursor protein.
• the cDNA sequence for PSl is disclosed in Genbank under access number Gene ID: 5663 and code for the following protein sequence (SEQ ID NO:4):
• the PSl protein used according to the invention may be modified (PSl M146L) and may have the protein sequence sequence (SEQ ID NO: 5) as described below: MTELPAPLSYFQNAQMSEDNHLSNTVRSQNDNRERQEHNDRRSLGHPEPLSN GRPQGNSRQVVEQDEEEDEELTLKYGAKHVIMLFVPVTLCMVVVVATIKSVSFYTRK DGQLIYTPFTEDTETVGQRALHSILNAAIMISVIVVMTILLVVLYKYRCYKVIHAWLII SSLLLLFFFSFIYLGEVFKTYNVAVDYITVALLIWNFGVVGMISIHWKGPLRLQQAYLI MISALMAL VFIKYLPEWT A WLILAVISVYDLV AVLCPKGPLRML VET AQERNETLFP ALIYSSTMVWLVNMAEGDPEAQR VSKNSKYNAESTERESQDTVAENDDGGFSEEW EAQRDSHLGPHRSTPES
• the vector of the invention comprising a nucleic acid sequence that encodes the APP protein and/or the PS1 or PS2 proteins or variants thereof.
• PS2 or "Presenilin 2” denotes a protein encoded by the PSEN2 gene.
• Presenilin 2 is one of the four core proteins in presenilin complex, which mediate the regulated proteolytic events of several proteins in the cell, including gamma secretase.
• Gamma-secretase is considered to play a strong role in generation of beta amyloid, accumulation of which is related to the onset of Alzheimer's Disease, from the beta-amyloid precursor protein.
• the cDNA sequence for PS2 is disclosed in Genbank under access number Gene ID: 5664 and code for the following protein (SEQ ID NO: 6):
• the vector of the invention comprises a nucleic acid sequence that encodes the APP protein and a nucleic acid sequence that encodes the PS2 protein.
• the vector of the invention comprises a nucleic acid sequence that encodes the APP protein, a nucleic acid sequence that encodes the PS1 protein and a nucleic acid sequence that encodes the PS2 protein.
• the vector of the invention may comprises any variant of the nucleic acid sequence which encodes for the APP protein and/or any variant of the nucleic acid sequence which encodes for the PS1 protein and/or variant of the nucleic acid sequence which encodes for the PS2 protein. In another embodiment, the vector of the invention may comprises any variant of the nucleic acid sequence which encodes for any variant of the APP protein and/or any variant of the nucleic acid sequence which encodes for any variant of the PS1 protein and/or any variant of the nucleic acid sequence which encodes for any variant of the PS2 protein.
• variants include, for instance, naturally-occurring variants due to allelic variations between individuals (e.g., polymorphisms), alternative splicing forms, etc.
• the term variant also includes genes sequences of the invention from other sources or organisms. Variants are preferably substantially homologous to sequences according to the invention, i.e., exhibit a nucleotide sequence identity of typically at least about 75%, preferably at least about 85%, more preferably at least about 90%, more preferably at least about 95% with sequences of the invention. Variants of the genes of the invention also include nucleic acid sequences, which hybridize to a sequence as defined above (or a complementary strand thereof) under stringent hybridization conditions.
• Typical stringent hybridisation conditions include temperatures above 30° C, preferably above 35°C, more preferably in excess of 42°C, and/or salinity of less than about 500 mM, preferably less than 200 mM.
• Hybridization conditions may be adjusted by the skilled person by modifying the temperature, salinity and/or the concentration of other reagents such as SDS, SSC, etc.
• the vector use according to the invention is a non viral vector or a viral vector.
• the non viral vector may be a plasmid comprising a nucleic acid sequence that encodes the APP protein and/or the PS1 protein.
• the vector may be a viral vector.
• Gene delivery viral vectors useful in the practice of the present invention can be constructed utilizing methodologies well known in the art of molecular biology.
• viral vectors carrying transgenes are assembled from polynucleotides encoding the transgene, suitable regulatory elements and elements necessary for production of viral proteins which mediate cell transduction.
• gene transfer or “gene delivery” refer to methods or systems for reliably inserting foreign DNA into host cells. Such methods can result in transient expression of non- integrated transferred DNA, extrachromosomal replication and expression of transferred replicons (e. g. episomes), or integration of transferred genetic material into the genomic DNA of host cells.
• transferred replicons e. g. episomes
• Such recombinant viruses may be produced by techniques known in the art, such as by transfecting packaging cells or by transient transfection with helper plasmids or viruses. Typical examples of virus packaging cells include PA317 cells, PsiCRIP cells, GPenv+ cells, 293 cells, etc.
• the viral vector may be an adenoviral, a retroviral, a lentiviral, an herpesvirus or an adeno-associated virus (AAV) vectors.
• AAV adeno-associated virus
• adeno-associated viral (AAV) vectors are employed.
• the AAV vector is AAV1, AAV2, AAV3, AAV4, AA5, AAV6, AAV7, AAV8, AAV9, AAV10 or any other serotypes of AAV that can infect human, rodents, monkeys or other species.
• the AAV vector is an AAV 10 or AAV9.
• an “AAV vector” is meant a vector derived from an adeno-associated virus serotype, including without limitation, AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV6, etc.
• AAV vectors can have one or more of the AAV wild-type genes deleted in whole or part, preferably the rep and/or cap genes, but retain functional flanking ITR sequences. Functional ITR sequences are necessary for the rescue, replication and packaging of the AAV virion.
• an AAV vector is defined herein to include at least those sequences required in cis for replication and packaging (e. g., functional ITRs) of the virus. ITRs don't need to be the wild- type nucleotide sequences, and may be altered, e.
• AAV expression vectors are constructed using known techniques to at least provide as operatively linked components in the direction of transcription, control elements including a transcriptional initiation region, the DNA of interest (i.e. the nucleic acid sequences of the invention) and a transcriptional termination region.
• control elements are selected to be functional in a mammalian cell.
• the resulting construct which contains the operatively linked components is bounded (5' and 3 ') with functional AAV ITR sequences.
• AAV ITRs adeno-associated virus inverted terminal repeats
• AAV ITRs together with the AAV rep coding region, provide for the efficient excision and rescue from, and integration of a nucleotide sequence interposed between two flanking ITRs into a mammalian cell genome.
• the nucleotide sequences of AAV ITR regions are known.
• an "AAV ITR” does not necessarily comprise the wild-type nucleotide sequence, but may be altered, e. g., by the insertion, deletion or substitution of nucleotides. Additionally, the AAV ITR may be derived from any of several AAV serotypes, including without limitation, AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV6, etc. Furthermore, 5' and 3' ITRs which flank a selected nucleotide sequence in an AAV vector need not necessarily be identical or derived from the same AAV serotype or isolate, so long as they function as intended, i.
• AAV ITRs may be derived from any of several AAV serotypes, including without limitation, AAV-1, AAV-2, AAV-3, AAV-4, AAV 5, AAV6, etc.
• 5' and 3' ITRs which flank a selected nucleotide sequence in an AAV expression vector need not necessarily be identical or derived from the same AAV serotype or isolate, so long as they function as intended, i. e., to allow for excision and rescue of the sequence of interest from a host cell genome or vector, and to allow integration of the DNA molecule into the recipient cell genome when AAV Rep gene products are present in the cell.
• vectors derived from AAV serotypes having tropism for and high transduction efficiencies in cells of the mammalian CNS, particularly neurons.
• a review and comparison of transduction efficiencies of different serotypes is provided in this patent application.
• AAV2 based vectors have been shown to direct long- term expression of transgenes in CNS, preferably transducing neurons.
• preferred vectors include vectors derived from AAV 10 and AAV 11 serotypes, which have also been shown to transduce cells of the CNS.
• the selected nucleotide sequence is operably linked to control elements that direct the transcription or expression thereof in the subject in vivo.
• control elements can comprise control sequences normally associated with the selected gene.
• heterologous control sequences can be employed.
• Useful heterologous control sequences generally include those derived from sequences encoding mammalian or viral genes. Examples include, but are not limited to, the phophoglycerate kinase (PKG) promoter, CAG, neuronal promoters, promoter of Dopamine- 1 receptor and Dopamine-2 receptor, the SV40 early promoter, mouse mammary tumor virus LTR promoter; adenovirus major late promoter (Ad MLP); a herpes simplex virus (HSV) promoter, a cytomegalovirus (CMV) promoter such as the CMV immediate early promoter region (CMVIE), rous sarcoma virus (RSV) promoter, synthetic promoters, hybrid promoters, and the like.
• PKG phophoglycerate kinase
• CAG phophoglycerate kinase
• CAG phophoglycerate kinase
• sequences derived from nonviral genes will also find use herein.
• Such promoter sequences are commercially available from, e. g. , Stratagene (San Diego, CA).
• heterologous promoters and other control elements such as CNS-specific and inducible promoters, enhancers and the like, will be of particular use.
• heterologous promoters include the CMV promoter.
• CNS specific promoters include those isolated from the genes of myelin basic protein (MBP), glial fibrillary acid protein (GFAP), and neuron specific enolase (NSE).
• MBP myelin basic protein
• GFAP glial fibrillary acid protein
• NSE neuron specific enolase
• the AAV expression vector which harbors the DNA molecule of interest bounded by AAV ITRs can be constructed by directly inserting the selected sequence (s) into an AAV genome which has had the major AAV open reading frames ("ORFs") excised therefrom. Other portions of the AAV genome can also be deleted, so long as a sufficient portion of the ITRs remain to allow for replication and packaging functions.
• ORFs major AAV open reading frames
• Such constructs can be designed using techniques well known in the art. See, e. g. , U. S. Patents Nos. 5,173, 414 and 5,139, 941; International Publications Nos. WO 92/01070 (published 23 January 1992) and WO 93/03769 (published 4 March 1993).
• AAV ITRs can be excised from the viral genome or from an AAV vector containing the same and fused 5' and 3 Of a selected nucleic acid construct that is present in another vector using standard ligation techniques.
• AAV vectors which contain ITRs have been described in, e. g. , U. S. Patent no. 5, 139, 941.
• AAV vectors are described therein which are available from the American Type Culture Collection ("ATCC”) under Accession Numbers 53222,53223, 53224,53225 and 53226.
• chimeric genes can be produced synthetically to include AAV ITR sequences arranged 5'and 3'of one or more selected nucleic acid sequences.
• AAV expression vector is introduced into a suitable host cell using known techniques, such as by transfection.
• transfection A number of transfection techniques are generally known in the art. Particularly suitable transfection methods include calcium phosphate co -precipitation, direct microinjection into cultured cells, electroporation, liposome mediated gene transfer, lipid-mediated transduction, and nucleic acid delivery using high- velocity microprojectiles.
• a particular viral vector such as the AAV10 or AAV9, comprises, in addition to a nucleic acid sequences of the invention, the backbone of AAV vector with ITR derived from AAV-2, the promoter, such as the mouse PGK (phosphoglycerate kinase) gene or the cytomegalovirus/p-actin hybrid promoter (CAG) consisting of the enhancer from the cytomegalovirus immediate gene, the promoter, splice donor and intron from the chicken ⁇ - actin gene, the splice acceptor from rabbit ⁇ -globin, or any neuronal promoter such as the promoter of Dopamine- 1 receptor or Dopamine-2 receptor, or the synapsin promoter, with or without the wild-type or mutant form of woodchuck hepatitis virus post-transcriptional regulatory element (WPRE).
• the viral vector may comprise in addition, a nucleic acid sequence encoding an antibiotic resistance gene such as the genes of resistance ampicilline (AmpR),
• the vector of the invention contains a nucleic acid sequence that encodes the APP protein or APP mutated familiar forms (for example Tottori, Flemish, Arctic, Dutch, Iowa, Egyptian, Austrian, German, French, Florida, Indiana or Australian mutations) and in particular APPsl (Swedish and London mutations).
• the vector of the invention contains a nucleic acid sequence that encodes the PS1 protein, PS1 M146L, or PS2.
• the vector of the invention contains a nucleic acid sequence that encodes the APPsl protein and a nucleic acid sequence that encodes the PS1 protein.
• the vector of the invention contains a nucleic acid sequence that encodes the APPsl protein and a nucleic acid sequence that encodes the PS1 protein or a nucleic sequence that encodes the PS2 protein.
• the vector of the invention contains a nucleic acid sequence that encodes the APPsl protein, a nucleic acid sequence that encodes the PS1 protein and a nucleic sequence that encodes the PS2 protein.
• the vector of the invention is a viral vector, for example the AAV 10 or AAV9 vectors which contains a nucleic acid sequence that encodes the APPsl protein and a nucleic acid sequence that encodes the PS1 protein M146L and/or a nucleic sequence that encodes the PS2 protein, the gene AmpR, sequences ITR and the promoter CAG.
• the vector of the invention is a viral vector, for example the AAV 10 or AAV9 vectors which contains a nucleic acid sequence that encodes the APP protein and a nucleic acid sequence that encodes the PSl protein spaced by a nucleic acid sequence that encodes a self-cleaving peptide (especially T2A peptide) and the promoter CAG.
• a viral vector for example the AAV 10 or AAV9 vectors which contains a nucleic acid sequence that encodes the APP protein and a nucleic acid sequence that encodes the PSl protein spaced by a nucleic acid sequence that encodes a self-cleaving peptide (especially T2A peptide) and the promoter CAG.
• a second object of the invention relates to a method for inducing the Alzheimer's disease in an animal, said method comprising the administration of at least one vector containing a nucleic acid sequence that encodes the APP protein and/ or the PSl protein or a variant thereof.
• the vector used for inducing the Alzheimer's disease comprises the nucleic acid sequence that encodes the APP protein and the nucleic acid sequence that encodes the PSl protein.
• the method for inducing the Alzheimer's disease in an animal comprises the administration of a vector containing a nucleic acid sequence that encodes the APP protein or a variant thereof and a vector containing a nucleic acid sequence that encodes the PSl protein or a variant thereof.
• the method for inducing the Alzheimer's disease in an animal comprises the administration of a vector containing a nucleic acid sequence that encodes the APP protein and a vector containing a nucleic acid sequence that encodes the PSl protein.
• the method for inducing the Alzheimer's disease in an animal comprises the administration of a vector containing a nucleic acid sequence that encodes the APPsl protein and a vector containing a nucleic acid sequence that encodes the PSl protein M146L.
• the method for inducing the Alzheimer's disease in an animal comprises the administration of a vector containing a nucleic acid sequence that encodes the APP protein and/or a vector containing a nucleic acid sequence that encodes the PSl protein and/or a vector containing a nucleic acid sequence that encodes the PS2 protein.
• the method for inducing the Alzheimer's disease in an animal comprises the administration of a vector containing a nucleic acid sequence that encodes the APP protein and a vector containing a nucleic acid sequence that encodes the PS2 protein.
• the method for inducing the Alzheimer's disease in an animal comprises the administration of a vector containing a nucleic acid sequence that encodes the APP protein and a vector containing a nucleic acid sequence that encodes the PS 1 protein and a vector containing a nucleic acid sequence that encodes the PS2 protein.
• the method according to the invention is not a method of treatment, in particular a method of treatment of the human or animal body by surgery or therapy.
• Methods of delivery of vectors to neurons and/or astrocytes of the animal model includes generally any method suitable for delivery vectors to the neurons and/or astrocytes such that at least a portion of cells of a selected synaptically connected cell population is transduced.
• Vectors may be delivered to any cells of the central nervous system, or both.
• the vector is delivered to the cells of the central nervous system, including for example cells of the spinal cord, brainstem (medulla, pons, and midbrain), cerebellum, diencephalon (thalamus, hypothalamus), telencephalon (corpus striatum, cerebral cortex, or, within the cortex, the occipital, temporal, parietal or frontal lobes), or combinations thereof, or preferably any suitable subpopulation thereof.
• Further preferred sites for delivery include the ruber nucleus, corpus amygdaloideum, entorhinal cortex and neurons in ventralis lateralis, or to the anterior nuclei of the thalamus.
• vectors of the invention are delivered by stereotactic injections or microinjections directly in the brain and more precisely in the hippocampus.
• vectors may be administered by stereotaxic microinjection.
• animals have the stereotactic frame base fixed in place (screwed into the skull).
• the brain with stereotactic frame base (MRIcompatible with fiducial markings) is imaged using high resolution MRI.
• the MRI images are then transferred to a computer which runs stereotactic software.
• a series of coronal, sagittal and axial images are used to determine the target (site of AAV vector injection) and trajectory.
• the software directly translates the trajectory into 3 dimensional coordinates appropriate for the stereotactic frame. Holes are drilled above the entry site and the stereotactic apparatus positioned with the needle implanted at the given depth.
• the AAV vector is then injected at the target sites. Since the AAV vector integrates into the target cells, rather than producing viral particles, the subsequent spread of the vector is minor, and mainly a function of passive diffusion from the site of injection and of course the desired transsynaptic transport, prior to integration.
• the degree of diffusion may be controlled by adjusting the ratio of vector to fluid carrier.
• Additional routes of administration may also comprise local application of the vector under direct visualization, e. g., superficial cortical application, or other nonstereotactic application.
• the vector may be delivered intrathecally, in the ventricules or by intravenous injection.
• the method of the invention comprises intracerebral administration through stereotaxic injections.
• other known delivery methods may also be adapted in accordance with the invention.
• the vector may be injected into the cerebrospinal fluid, e. g. , by lumbar puncture.
• the vector may be injected into the spinal cord or into the peripheral ganglia, or the flesh (subcutaneously or intramuscularly) of the body part of interest.
• the vector can be administered via an intravascular approach.
• the vector can be administered intra-arterially (carotid) in situations where the blood-brain barrier is disturbed or not disturbed.
• the vector can be administered during the "opening" of the blood-brain barrier achieved by infusion of hypertonic solutions including mannitol.
• Vectors used herein may be formulated in any suitable vehicle for delivery. For instance they may be placed into a pharmaceutically acceptable suspension, solution or emulsion. Suitable mediums include saline and liposomal preparations. More specifically, pharmaceutically acceptable carriers may include sterile aqueous of non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose), and the like.
• Preservatives and other additives may also be present such as, for example, antimicrobials, antioxidants, chelating agents, and inert gases and the like.
• colloidal dispersion systems include macromolecule complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes.
• the method according to the invention may further comprise injections of molecules that can help to establish the Alzheimer's disease in animals.
• the protein ApoE can be injected or overexpressed to the animal to promote the process of establishing the disease.
• the invention may relates to a method for inducing the Alzheimer's disease in an animal, said method comprising the administration of at least one vector containing a nucleic acid sequence that encodes the APP protein and/or the PSl protein or a variant thereof and the protein ApoE.
• the method for inducing the Alzheimer's disease in an animal comprises the administration of a vector comprising the nucleic acid sequence that encodes the APPsl protein and the nucleic acid sequence that encodes the PSl protein and the protein ApoE.
• the method for inducing the Alzheimer's disease in an animal comprises the administration of a vector containing a nucleic acid sequence that encodes the APP protein and a vector containing a nucleic acid sequence that encodes the PSl protein and the protein ApoE.
• ApoE may be use in the method according to the invention.
• a vector containing a nucleic acid sequence that encodes the protein ApoE2 or ApoE3 or ApoE4 may be use in the method according to the invention
• protein ApoE denotes a protein which confers a risk for
• the ApoE gene codes for a protein which is implicated in the cholesterol regulation.
• ApoE2 allelic variants of ApoE
• ApoE3 allelic variants of ApoE
• ApoE4 confers a higher risk for Alzheimer and cardiovascular disease.
• the invention relates to a vector containing a nucleic acid sequence that encodes the APP mutated familiar forms (for example Tottori, Flemish, Arctic, Dutch, Iowa, Egyptian, Austrian, German, French, Florida, Indiana or Australian mutations) and in particular APPsl and/or the PS1 protein for use in a method for inducing the Alzheimer's disease in an animal.
• a nucleic acid sequence that encodes the APP mutated familiar forms (for example Tottori, Flemish, Arctic, Dutch, Iowa, Egyptian, Austrian, German, French, Florida, Indiana or Australian mutations) and in particular APPsl and/or the PS1 protein for use in a method for inducing the Alzheimer's disease in an animal.
• a third object of the invention relates to an animal having the Alzheimer's disease, said animal being obtained by the method according to the invention.
• An animal obtained by the method of the invention will preferably display increased production of amyloid peptides, hyperphosphorylation of endogenous Tau protein and cognitive deficits, parameters which are characteristics of Alzheimer's disease.
• said animal is for use as a model of Alzheimer's disease.
• the invention further relates to the use of an animal having increased production of amyloid peptides, hyperphosphorylation of endogenous Tau protein and cognitive deficits as a model of Alzheimer's disease, said animal being obtained by the method of the invention.
• the animal obtained by the method of the invention may be of any species. It may for instance be a rodent or a non-human primate. Particularly, the animal obtained by the method of the invention is not a human. Typically, the animal obtained by the method of the invention may be a rat, a mouse or a macacus microceb. The animal may be a genetically modified animal, such as a 'knockout' animal in which the function or expression of a gene has been reduced or eliminated.
• Animals obtained by the method of the invention can be easily distinguished from prior art Alzheimer models and offer many advantages (see examples).
• animals obtained by the method of the invention can be obtained rapidly e.g. in one month and can be obtained in several animal lines for example in most of the mouse lines.
• the animal obtained by the method of the invention overcomes two major drawbacks of transgenic models: 1) continuous transgenes expression from in utero, 2) limitation of the transgenesis to mice.
• animals obtained by the method product all neurotoxic metabolites derived from APP ( ⁇ 42 and PCTF), in a continuous manner and in a pathophysiologic level.
• Such animal model may for instance be of major interest for industrial validation of current and future treatments against this disease.
• the invention relates to a method of screening a compound for therapeutic use in the treatment of Alzheimer's disease, using the animal of the invention.
• the invention also concerns the use of said animal for assessing potential side-effects of treatment of Alzheimer's disease.
• Said treatment may include, for example, administration of therapeutic compounds that act on APP accumulation, as described below.
• the compound to be screened for therapeutic use against Alzheimer's disease may be used for preventing or treating Alzheimer's disease.
• Such compound may be any kind of compound that may act Alzheimer's disease. It may for instance decrease accumulation of APP and/or decrease accumulation of neurotoxic metabolites derived from APP ( ⁇ 42 and
• the compound to be screened for therapeutic use against Alzheimer's disease should preferably display a low toxicity.
• the screening may for instance include the steps of administering a compound to be screened to the animal of the invention, waiting for a certain period of time, optionally repeating the administration, measuring the accumulation of APP and/or neurotoxic metabolites, and selecting the compound according to its effect on the accumulation of APP and/or neurotoxic metabolites. For example, if the compound tested allows a decrease of the accumulation of APP and/or neurotoxic metabolites, it could be select as potential therapeutic drug against Alzheimer's disease.
• the animal of the invention may also be for use for studying the mechanism of Alzheimer's disease.
• Another embodiment concerns the use of an animal having Alzheimer's disease for studying the mechanism of the disease, said animal being obtained by the method of the invention. For instance, such an animal can be useful for understanding the physio-pathology or the molecular mechanism involved in Alzheimer's disease.
• FIG. 1 Western blot analysis of transgene expression.
• A Representative Western blots of PSl M146L, human APP (hAPP) and human + murine APP (total APP: tAPP) in hippocampus homogenates transduced by AAV9 or AAV 10 vectors carrying the PSl M146L (PSl *), APPsl (APP) and/or APPsl + PSl M146L (APP/PS1 *) transgenes.
• PSl * PSl M146L
• APP APPsl
• APP/PS1 * total APP
• FIG. 1 Amyloidogenic processing of APP.
• APP protein is cleaved by the ⁇ secretase which leads to the production of soluble fragment sAPPp and PCTF fragment that remains anchored in the membrane.
• the PCTF is then cleaved by the PSl (belonging to the ⁇ secretase complex) allowing production of ⁇ 42 and ⁇ 40 peptides.
• FIG. 3 Simultaneous intracerebral injection of AAVlO-APPsl and AAVIO-PSI M146L in 8 weeks old C57BL/6J mice induce amyloid cascade one month after injection.
• APP is metabolized to (A) PCTF and then (B) ⁇ 42 peptide.
• C In contrast to APP and PCTF that decreased with PSl M146L overexpression, ⁇ 42 production is increased confirming the interest of the simultaneous intracerebral injection of both vectors.
• D In accordance, abnormal phosphorylation on Threonine residue 181 of murine Tau was stimulated by the double injection of AAV coding for APP and PSl M146L genes. In all cases, AAV 10 induced greater production of neurotoxic metabolites.
• FIG. 4 Simultaneous intracerebral injection of AAVlO-APPsl and AAVIO-PS I M146L in 8 weeks old C57BL/6J mice induce amyloid cascade at least up to five months.
• A The amyloidogenic pathway of APP involved in AD leads to the production of two metabolites of the neurotoxic APP peptides, ⁇ 42 and PCTF into hippocampus.
• B Hippocampal injection of AAVIO vectors encoding the APPsl and PSl M146L proteins allowed, within the first month, the induction of a significant production of ⁇ 42 as well as PCTF. This production was stable with time (analyzed up to 5 months).
• FIG. 6 AAV- APP and AAV-PSl co-injection leads to AD-like production levels of amyloid derivatives.
• APP levels were normalized to GAPDH. Data are means ⁇ s.e.m. One Way Anova: ***p ⁇ 0.0001.
• FIG. 7 AAV- APP and AAV-PSl co-injection allows a hyperphosphorylation of the murine Tau protein from 1 month post-injection.
• FIG. 8 AAV-APP and AAV-PSl co-injection causes a neuronal network failure 3 months post-injection.
• B Tonic Glutamatergic Current recorded at a holding potential of +40 mV by whole-cell patch-clamp of CA1 pyramidal neurons.
• FIG. 9 AAV-APP and AAV-PSl co-injection leads to a reduction of gabaergic synaptic marker Gad65.
• Table 1 Comparative table of some AAV models of AD.
• Table 2 Comparative table of some transgenic models of AD.
• Test mice were anesthetized with ketamine/xylazine and perfused transcardially with 20 ml PBS. One hemisphere was post-fixed for 24h in 4% PFA, cryoprotected in 30% of sucrose in PBS and cut into 40 ⁇ sections using a freezing microtome for immunohistochemical and histological analyses (data not shown). The other half was frozen immediately on dry ice and used for Western blots and ELISAs.
• mice hippocampal tissue was homogenized in a lysis buffer (TBS, NaCl 150 mM, Triton 1%, Phosphatase and Protease inhibitors) and centrifugated 20' at 13000 rpm. Protein levels were normalized by BCA protein assay (Pierce Biotechnology). Extracted ⁇ was then measured using the MSD Human ⁇ 42 Kit. PCTF was measured using the IBL Human PCTF Kit and the P-Tau using the Innogenetics Phospho-Tau 18 IP Kit. Aliquots of protein were electrophoretically separated using NuPAGE Bis-Tris Gels (Life Technologies).
• Electrophoresed proteins were then transferred to nitrocellulose membranes using the iBlot 7- Minute Blotting System, blocked in Tris-buffered saline containing 5% non-fat dry milk and subsequently hybridized with vaarious primary antibodies: APP 6E10 (Sigma), APP Cter (Calbiochem) and Presinilin 1 (Millipore). Densitometry quantification of bands was realised with the Bio ID software. Behavioral analysis
• Open field Movement in an open field was used to assess whether APP and PS 1 injection had an effect on anxiety which may affect memory and learning behaviors. Mice were placed in the center of a square field. The amount of time spent at the periphery along the walls was recorded as measures of anxiety.
• the Morris water maze (MWM) task quantifies mice memory abilities (Morris, 1984). This test was used as a measure of spatial learning, the mouse must learn the location of a hidden platform by referring to visual cues placed around the room. The platform location was kept constant throughout training but the starting point varied between trials. MWM consists of five consecutive learning days (3 trials per day). Seventy- two hours after the last trial of the fifth day a probe trial is realized to quantify long-term memory. In both testing phases, distance traveled in the quadrant containing the platform or target quadrant is quantified. An effective memory storage must therefore be accompanied by the establishment of a spatial bias characterized by a distance travelled in the target quadrant over than 25%.
• AD is characterized by the amyloidogenic pathway of APP metabolism that results from the cleavage of APP by PSl ( Figure 2).
• Animals injected with AAV vector encoding human PSl M146L protein only (control animal) or with AAV vectors encoding the APPsl and PSl M146L were sacrificed at 1 month post-injection for histopathological (data not shown), and biochemical and molecular studies ( Figures 3).
• Example 2 Production of metabolites in the animal model
• APP is cleaved into different metabolites like C-terminal fragment of APP (PCTF) and ⁇ 42 peptide with characterized neurotoxic properties.
• PCTF C-terminal fragment of APP
• ⁇ 42 peptide with characterized neurotoxic properties.
• PS l M146L leads to increased metabolism of PCTF in ⁇ 42 peptides.
• concentration of PCTF is observed in the hippocampus of mice co-injected with AAVIO- APPsl and AAV10-PS1 M146L vectors (Figure 3).
• PCTF The amount of PCTF showed respectively a 56- and 25-fold increase for APPsl and APPsl/PSl M146L mice compared to PSl M146L control mice one month after injection (Figure 3A).
• Overexpression of human APPsl thus significantly promotes the production of PCTF.
• PCTF concentration is decreased in APPsl/PSl M146L mice compared to APPsl mice, demonstrating increased metabolism of PCTF with overexpression of PSl M146L.
• PCTF was also detected in cortical structures in the absence of cortical production sites which argues for diffusion of PCTF produced into the hippocampus towards the cortical structures (data not shown).
• ⁇ 42 the main neurotoxic peptide in AD production is, on the other side, strongly increased in APPsl/PSl M146L mice.
• AAV-APPsl and AAV-PS1 M146L injection in wild type mouse leads to rapid (1 month) and stable (evaluated up to 5 months) increased production of amyloid peptides, hyperphosphorylation of endogenous Tau protein and cognitive deficits in mice, parameters which are characteristics of Alzheimer's disease.
• Such models could be useful to analyze deleterious mechanisms induced by amyloid pathway, as well as to evaluate biomarkers or screen therapeutic approaches.
• Example 4 Advantages of animal model of the invention from other models
• AD animal models aims to reproduce symptoms, injuries or causes similar to those observed in the human disease. Many strains of transgenic mice are successful to reproduce these lesions: extracellular deposits of AB peptide and intracellular accumulation of Tau protein. However the existing models are imperfect. To identify new therapeutic targets and the effectiveness of treatments in AD, various pharmaceutical companies have developed their own mouse models. Some companies also developed / used different models for provision of services as Contract Research Organizations (CROs).
• CROs Contract Research Organizations
• Transgenic models have an important expression of transgenes from the embryonic stages of development which will ultimately lead to the establishment of adaptive mechanisms. In addition, the cost of production is very high. They often imperfectly reproduce the AD phenotype and are difficult to transpose to larger species. Obtain models of AD in large species (rats and primates in particular) would be crucial to develop biomarkers and validate therapeutic approaches in a context as close as possible to the human pathophysiology.
• Models by intracerebral injection of amyloid peptides, truncated or not, are very easy to develop, relatively inexpensive and do not induce adaptive mechanisms. However, they suffer from several drawbacks:in addition to providing a partial model of AD, they do not have all the neurotoxic products generated in AD and in particular PCTF, products described as highly neurotoxic even at low doses. The administered concentrations of ⁇ 42 or 25- 35 are much higher than those observed in human pathological conditions. These models are therefore particularly suitable for measuring the neuroprotective ability of drugs but have a reduced interest to characterize compounds that modulate thepathological APP metabolism or intracellular changes resulting from the production of neurotoxic metabolites derived from APP.
• a mouse model (and/or rat) of Alzheimer's disease by gene transfer would be a powerful tool that would combine the advantages of transgenic animals (complete and stable modeling of the amyloid cascade) without the inconvenience of adaptive mechanisms, and with reduced production costs.
• Such model could be a major alternative for companies like CROs.
• Example 5 Gene transfer leads to APP and cleavage products levels close to humans
• Example 6 APP/PSl co-injection triggers a hyperphosphorylation of the endogenous Tau protein
• Glutamine is the precursor of Glutamate which is itself the precursor of the GABA neurotransmitter.
• Gad65 is an enzyme which catalyzes the decarboxylation of Glutamate to GABA for neurotransmission.
• mice injected with CAG-APP-T2A- PS1 construction present production of neurotoxic metabolites of APP (PCTF, ⁇ 38/40/42) close to human amounts. Hyperphosphorylation of murine TAU protein is also observable. These cerebral changes lead to behavioral defects in Morris water maze.
• the inventors describe here the development of an alternative AAV-based mouse model with two major objectives: create a relevant model closer to human physiopathology and mimic the early stages of AD.
• This model was obtained by co-injection, in the hippocampus of wild-type mice, of two AAV vectors coding the human Amyloid Protein Precursor (APPsl) and the human Presinilin 1 (PS1M146L).
• APPsl human Amyloid Protein Precursor
• PS1M146L human Presinilin 1
• Our strategy allows a stable expression of transgenes without significant APP overexpression. This leads to ⁇ production and its neurotoxic catabolites such as sAPPp, PCTF and ⁇ 42 as soon as one month post-injection and stable during at least 12 months without classical late symptoms appearance such as senile plaque, inflammation or atrophy.
• Devi, L. & Ohno, M. Phospho-eIF2alpha level is important for determining abilities of
• PDAPP V717F mice Neurobiology of disease 11 , 425-433 (2002).

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