Patent Grant
10986821
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.
Alzheimer's disease (AD) is the most frequently encountered form of dementia (about 70% of dementia cases). With improved life expectancy, especially in developed countries, the incidence of dementia has dramatically increased and current forecasts speak in terms of a doubling of the number of persons affected every 20 years. In France, it is estimated that more than 850,000 people (with a majority of woman) are currently suffering from dementia, and around 225,000 new cases appear each year. AD is characterized by the accumulation of senile plaques (SP), neurofibrillary tangles (NFT), and selective synaptic and neuronal loss in plaques are composed of insoluble extracellular aggregates consisting mainly of amyloid β (Aβ) peptides derived from proteolytic cleavages of the amyloid precursor protein (APP). Genetic studies, together with the demonstration of a direct toxic effect of Aβ, led to the development of the amyloid cascade hypothesis to explain the Aβ-associated neurodegenerative process. Aβ rapidly aggregates to form amorphous and fibrillar oligomers, which then deposit to build senile plaques. A number of studies have provided evidence that βCTF and soluble Aβ oligomers are more toxic to cells than mature fibrils (Kayed et al., 2003) and these neurotoxic peptides are originally produced by the cleavage of APP.
Mutations in genes that encode APP or proteases that generate Aβ (PS1; PS2) are responsible for the familial forms (5% of cases) of AD (Selkoe et al., 2001).
Different 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.
The use of viral vectors to develop experimental models would be a valuable breakthrough in the field. AAV 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. High transduction efficiencies as well as robust and sustained transgene expression lead to the rapid appearance of functional and behavioral abnormalities and severe neurodegeneration. Targeted injections in different brain areas can be used to investigate the regional specificity of the neuropathology and eliminate potential side effects associated with a widespread overexpression of the transgene. Finally, models can be established in different mammalian species including large animals like dogs, pigs and non-human primates, thereby providing an opportunity to assess complex behavioral changes and perform longitudinal follow-up of neuropathological alterations by imaging. 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.
Thus, 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.
In one embodiment, 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.
In another embodiment, 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.
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.
In another embodiment, the invention 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.
As used herein, the term “APP” or “Amyloid Precursor Protein” denotes 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 (Aβ), 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:1 as described below:
The protein sequence of the APP protein has the sequence SEQ ID NO: 2 as described below:
In another embodiment, 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):
As used herein, the term “PS1” 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 PS1 is disclosed in Genbank under access number Gene ID: 5663 and code for the following protein sequence (SEQ ID NO:4):
In one embodiment, the PS1 protein used according to the invention may be modified (PS1 M146L) and may have the protein sequence sequence (SEQ ID NO: 5) as described below:
In another embodiment, 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.
As used herein, the term “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):
In one embodiment, 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.
In one embodiment, 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.
In another embodiment, 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.
The 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.
In one embodiment, the vector use according to the invention is a non viral vector or a viral vector.
In a particular embodiment, the non viral vector may be a plasmid comprising a nucleic acid sequence that encodes the APP protein and/or the PS1 protein.
In another particular embodiment, the vector may 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. Typically, 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.
The terms “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.
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. Detailed protocols for producing such replication-defective recombinant viruses may be found for instance in WO95/14785, WO96/22378, U.S. Pat. Nos. 5,882,877, 6,013,516, 4,861,719, 5,278,056 and WO94/19478.
In a particular embodiment, the viral vector may be an adenoviral, a retroviral, a lentiviral, an herpesvirus or an adeno-associated virus (AAV) vectors.
In a preferred embodiment, adeno-associated viral (AAV) vectors are employed.
In another preferred embodiment, the AAV vector is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10 or any other serotypes of AAV that can infect human, rodents, monkeys or other species.
In a more preferred embodiment, the AAV vector is an AAV10 or AAV9.
By 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. Thus, 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.g., by the insertion, deletion or substitution of nucleotides, so long as the sequences provide for functional rescue, replication and packaging. 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.
The 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. By “adeno-associated virus inverted terminal repeats” or “AAVITRs” is meant the art-recognized regions found at each end of the AAV genome which function together in cis as origins of DNA replication and as packaging signals for the virus. 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. As used herein, 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.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 heterologous sequence into the recipient cell genome when AAV Rep gene products are present in the cell. Additionally, 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. Furthermore, 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.
Particularly preferred are 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. In one preferred example, AAV2 based vectors have been shown to direct long-term expression of transgenes in CNS, preferably transducing neurons. In other non limiting examples, preferred vectors include vectors derived from AAV10 and AAV11 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. Such control elements can comprise control sequences normally associated with the selected gene.
Alternatively, 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. In addition, sequences derived from nonviral genes, such as the murine metallothionein gene, will also find use herein. Such promoter sequences are commercially available from, e.g., Stratagene (San Diego, Calif.). For purposes of the present invention, both heterologous promoters and other control elements, such as CNS-specific and inducible promoters, enhancers and the like, will be of particular use.
Examples of heterologous promoters include the CMV promoter. Examples of CNS specific promoters include those isolated from the genes of myelin basic protein (MBP), glial fibrillary acid protein (GFAP), and neuron specific enolase (NSE).
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. Such constructs can be designed using techniques well known in the art. See, e.g., U.S. Pat. Nos. 5,173,414 and 5,139,941; International Publications Nos. WO 92/01070 (published 23 Jan. 1992) and WO 93/03769 (published 4 Mar. 1993). Alternatively, 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. Pat. No. 5,139,941. In particular, several AAV vectors are described therein which are available from the American Type Culture Collection (“ATCC”) under Accession Numbers 53222, 53223, 53224, 53225 and 53226. Additionally, chimeric genes can be produced synthetically to include AAV ITR sequences arranged 5′ and 3′ of one or more selected nucleic acid sequences. Preferred codons for expression of the chimeric gene sequence in mammalian CNS cells can be used. The complete chimeric sequence is assembled from overlapping oligonucleotides prepared by standard methods. In order to produce AAV virions, an AAV expression vector is introduced into a suitable host cell using known techniques, such as by 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.
For instance, 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/β-actin hybrid promoter (CAG) consisting of the enhancer from the cytomegalovirus immediate gene, the promoter, splice donor and intron from the chicken β-actin gene, the splice acceptor from rabbit β-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), kanamycine, hygromycine B, geneticine, blasticidine S or puromycine.
In a particular embodiment, 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, Iranian, Austrian, German, French, Florida, Indiana or Australian mutations) and in particular APPsl (Swedish and London mutations).
In another particular embodiment, the vector of the invention contains a nucleic acid sequence that encodes the PS1 protein, PS1 M146L, or PS2.
In another particular embodiment, 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.
In another particular embodiment, 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.
In another particular embodiment, 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.
In a particular embodiment of the invention, the vector of the invention is a viral vector, for example the AAV10 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.
In a particular embodiment, the vector of the invention is a viral vector, for example the AAV10 or AAV9 vectors which contains a nucleic acid sequence that encodes the APP protein and a nucleic acid sequence that encodes the PS1 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 PS1 protein or a variant thereof.
In one embodiment, 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 PS1 protein.
In another embodiment, 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 PS1 protein or a variant thereof.
In another embodiment, 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 PS1 protein.
In another embodiment, 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 PS1 protein M146L.
In a particular embodiment, 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 PS1 protein and/or a vector containing a nucleic acid sequence that encodes the PS2 protein.
In another particular embodiment, 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.
In another particular embodiment, 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 PS1 protein and a vector containing a nucleic acid sequence that encodes the PS2 protein.
Particularly, 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. Generally, 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.
In a particular embodiment, vectors of the invention are delivered by stereotactic injections or microinjections directly in the brain and more precisely in the hippocampus.
To deliver vectors of the invention specifically to a particular region and to a particular population of cells of the CNS, vectors may be administered by stereotaxic microinjection. For example, animals have the stereotactic frame base fixed in place (screwed into the skull). The brain with stereotactic frame base (MRI compatible 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.
Preferably, the method of the invention comprises intracerebral administration through stereotaxic injections. However, other known delivery methods may also be adapted in accordance with the invention. For example, for a more widespread distribution of the vector across the CNS, it may be injected into the cerebrospinal fluid, e.g., by lumbar puncture. To direct the vector to the peripheral nervous system, it may be injected into the spinal cord or into the peripheral ganglia, or the flesh (subcutaneously or intramuscularly) of the body part of interest. In certain situations the vector can be administered via an intravascular approach. For example, the vector can be administered intra-arterially (carotid) in situations where the blood-brain barrier is disturbed or not disturbed. Moreover, for more global delivery, 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.
A colloidal dispersion system may also be used for targeted gene delivery. Colloidal dispersion systems include macromolecule complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes.
In another embodiment, the method according to the invention may further comprise injections of molecules that can help to establish the Alzheimer's disease in animals. For example, the protein ApoE can be injected or overexpressed to the animal to promote the process of establishing the disease.
Thus, 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 PS1 protein or a variant thereof and the protein ApoE.
In another embodiment, 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 PS1 protein and the protein ApoE.
In another embodiment, 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 PS1 protein and the protein ApoE.
Particularly, a vector containing a nucleic acid sequence that encodes the protein ApoE may be use in the method according to the invention.
Particularly, 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.
As used herein, the term “protein ApoE” denotes a protein which confers a risk for Alzheimer and cardiovascular disease. The ApoE gene codes for a protein which is implicated in the cholesterol regulation. There are three relatively common allelic variants of ApoE (accession number: NP_000032.1) known as ApoE2, ApoE3, and ApoE4. The most common variant overall is ApoE3 which is neutral. ApoE2 protect while ApoE4 confers a higher risk for Alzheimer and cardiovascular disease.
In another aspect, 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, Iranian, 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.
Thus, in a specific embodiment, 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).
Indeed, contrary to prior art transgenic animals, 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. Moreover, 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.
Furthermore, contrary to animal obtained by injection, animals obtained by the method product all neurotoxic metabolites derived from APP (Aβ42 and βCTF), 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.
Therefore, in a fourth object, 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 (Aβ42 and βCTF) for example. 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.
Alternatively, 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.
The invention will be further illustrated by the following figures and examples. However, these examples and figures should not be interpreted in any way as limiting the scope of the present invention.
Material & Methods
Tissue Collection
Test mice were anesthetized with ketamine/xylazine and perfused transcardially with 20 ml PBS. One hemisphere was post-fixed for 24 h in 4% PFA, cryoprotected in 30% of sucrose in PBS and cut into 40 μm 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.
ELISAs and Western Blots
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 Aβ was then measured using the MSD Human Aβ42 Kit. βCTF was measured using the IBL Human βCTF Kit and the P-Tau using the Innogenetics Phospho-Tau 181P 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 various primary antibodies: APP 6E10 (Sigma), APP Cter (Calbiochem) and Presinilin 1 (Millipore). Densitometry quantification of bands was realised with the Bio1D software.
Behavioral Analysis
Open Field:
Movement in an open field was used to assess whether APP and PS1 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.
Morris Water Maze:
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%.
Results
To evaluate the relevance of our model, we have performed a comparative study between AAV9 and AAV10 vectors encoding the codon-optimized human APP (APPsl, Swedish-London mutations, promoting the cleavage by β secretase complex) and/or PS1 M146L (M146L) transgenes in mice (
These results show that the expression of human APPsl by gene transfer leads to lowly increase the total quantity of APP. Co-express with the PS1 M146L, human APP and βCTF amount decrease due to APP metabolization by secretase complexes. Moreover, AAV10 virus seems to be better to efficiently produce human APP in mice than AAV9 virus.
AD is characterized by the amyloidogenic pathway of APP metabolism that results from the cleavage of APP by PS1 (
We confirmed by immunohistochemistry our results showed in
APP is cleaved into different metabolites like C-terminal fragment of APP (βCTF) and Aβ42 peptide with characterized neurotoxic properties. We showed that expression of PS1 M146L leads to increased metabolism of βCTF in Aβ42 peptides. Indeed decreased concentration of βCTF is observed in the hippocampus of mice co-injected with AAV10-APPsl and AAV10-PS1 M146L vectors (
The amount of βCTF showed respectively a 56- and 25-fold increase for APPsl and APPsl/PS1 M146L mice compared to PS1 M146L control mice one month after injection (
A longitudinal study was performed to analyze the kinetics of neurotoxic peptides production in mouse brain (
At 2.5 months post-injection, a behavioral study was performed in injected animals (
During the learning phase of the Morris water maze test, no learning defect was observed in APPsl/PS1 M146L compared to PS1 M146L control mice The two groups had therefore a normal learning profile. During the restitution phase of acquired information (72 hours retention time), a failure to return to platform quadrant previously acquired was observed in APPsl/PS1 M146L mice. The distance traveled by the mouse PS1 M146L in the target quadrant (TQ) was significantly greater than in other quadrants (p=0.01) confirming the presence of a spatial bias. The presence of this spatial bias was not observed for APPsl/PS1 M146L mice (p=ns). So APPsl/PS1 M146L mice traveled less distance in the quadrant previously containing the platform. These results confirm a lack of long-term memory in these mice compared to control mouse PS1 M146L (p=0.02).
In conclusion, 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.
The generation of 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).
These models have specific drawbacks:
In comparison with current transgenic models, the present AAV-APPsl/AAV-PS1 M146L model offers many advantages (see table 2):
As compared to models by injection, our model has many advantages (see table 1):
Thus, 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.
In order to confirm the relevance of this strategy compared to human physiopathology, we performed a comparative study between hippocampus homogenates from 3 months old APP/PS1 mice, human samples (age matched non dementia controls & AD Braak 6/Thal 5 patients; n=5/group) and 5 months old APP/PS1ΔE9 commonly used as gold standard.
An APP decrease was observed in both pathologic groups i.e. AAV-APP/PS1 and AD Braak VI Thal V patients (
Given the evidence that human APP is processed following the amyloidogenic pathway we examined the potential impact on the hyperphosphorylation of the murine Tau. We detected an increase in the APP/PS1 group (n=4) compared to the APP (n=4) and PS1 (n=4) groups (
It is well known that synaptic dysfunctions appear as an early event in AD (Scheff et al., 2007). Some synaptic markers like PSD-95 have been showed as reduced in AD patients (Proctor et al., 2010). We evaluated PSD-95 levels in the hippocampus of our model at 3 months post-injection. A significant decrease appeared in the APP/PS1 group compared to PS1 group (
Increasing evidences appeared these past few years about a decreased GABAergic signaling in AD patients (Gang et al., 2009; Xue et al., 2014; Tiwari et al., 2012). Using a 11.7 Tesla MRI, Magnetic Resonance Spectroscopy analysis was performed on PS1 and APP/PS1 mice at 3 months post-injection (n=6 per group). The region of interest was selected in both hippocampus of each mouse brain (data not shown). Results for the APP/PS1 were normalized to the PS1 values. APP/PS1 mice have significantly lower concentrations of Glutamine (Gln; p=0.017), GABA (p=0.018) and NAA (p=0.04) than PS1 mice indicating a decreased neuronal health and particularly a decreased GABA signaling pathway. No differences were obtained between both groups in the levels of Glu, tNAA, Ins and tChol (data not shown). Glutamine is the precursor of Glutamate which is itself the precursor of the GABA neurotransmitter. To explain why we observed a decrease of Glutamine and GABA but not of Glutamate, we looked for the Gad65 expression. Gad65 is an enzyme which catalyzes the decarboxylation of Glutamate to GABA for neurotransmission. It appeared decreased in the APP/PS1 mice at 3 months after injection compared to PS1 mice (
We generate an AAV vector coding for a fusion protein containing APP and PS1 protein spaced by a self-cleaving peptide (T2A peptide). Mice injected with CAG-APP-T2A-PS1 construction present production of neurotoxic metabolites of APP (βCTF, Aβ38/40/42) 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). Our strategy allows a stable expression of transgenes without significant APP overexpression. This leads to βAPP production and its neurotoxic catabolites such as sAPPβ, βCTF and Aβ42 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. Otherwise, they measured very close amounts of APP, βCTF and Aβ peptides compared to human homogenates and unlike what we can find in APP/PS1ΔE9 mice. Interestingly, only co-injection triggered hyperphosphorylation of the murine Tau protein resulting from an increase of GSK-3β levels. Finally, significant behavior impairments appeared from 3 months post-injection in association with an alteration of synaptic functions especially a decrease of PSD-95 associated with synaptic defects such as extrasynaptic NMDAR activity and an alteration in the GABAergic pathway.
Throughout this application, various references describe the state of the art to which this invention pertains. The disclosures of these references are hereby incorporated by reference into the present disclosure.
| Number | Date | Country | Kind |
|---|---|---|---|
| 13306518 | Nov 2013 | EP | regional |
| Number | Date | Country |
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| 2002063951 | Aug 2002 | WO |
| 2012049314 | Apr 2012 | WO |
| Entry |
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| Number | Date | Country | |
|---|---|---|---|
| 20190183102 A1 | Jun 2019 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 15034370 | US | |
| Child | 16178949 | US |
