WO2016092453A1

WO2016092453A1 - Prevention or reversal of apoe4 induced brain pathology by activation of vegf receptors

Info

Publication number: WO2016092453A1
Application number: PCT/IB2015/059410
Authority: WO
Inventors: Daniel MICHAELSON; Shiran SALOMON-ZIMRI; Micaela GLAT; Daniel Offen
Original assignee: Ramot At Tel-Aviv University Ltd.
Priority date: 2014-12-07
Filing date: 2015-12-07
Publication date: 2016-06-16

Abstract

The invention relates to prevention, treatment and slowing the progression of brain pathology in a disease or condition associated with ApoE4 genotype such as Alzheimer's Disease (AD) in a subject having the ApoE4 genotype through activation of Vascular Endothelial Growth Factor Receptor (VEGF) in the hippocampus or a sub-region thereof.

Description

PREVENTION OR REVERSAL OF APOE4 INDUCED BRAIN PATHOLOGY BY ACTIVATION OF VEGF RECEPTORS

FIELD OF THE INVENTION

The invention relates to prevention and treatment of brain pathology by over-expression of Vascular Endothelial Growth Factor (VEGF) or activation of VEGF Receptors (VEGFRs) in individuals with an ApoE4 genotype.

BACKGROUND OF THE INVENTION

Alzheimer's Disease (AD) is characterized by build-up of aggregates of the peptide beta- amyloid, and is associated with neuronal and vascular dysfunction. It is still not definitively known what initiates the disease and whether amyloid beta aggregation is a cause or a result of the disease process. It is known Apolipoprotein E4 (ApoE4), a major genetic risk factor for AD, is associated with increased neurodegeneration and vascular impairments.

Apolipoprotein E (ApoE) is a class of apolipoprotein that is essential for the normal catabolism of triglyceride -rich lipoprotein constituents. In peripheral tissues, ApoE is primarily produced by the liver and macrophages, and mediates cholesterol metabolism in an isoform-dependent manner. In the central nervous system, ApoE is mainly produced by astrocytes, and transports cholesterol to neurons via ApoE receptors, which are members of the low density lipoprotein receptor gene family. ApoE is polymorphic, with three major alleles: ApoE2, ApoE3, and ApoE4. Although these allelic forms differ from each other by only one or two amino acids at positions 112 and 158, these differences alter ApoE structure and function. The ApoE4 variant is the largest known genetic risk factor for late-onset sporadic AD in a variety of ethnic groups. Caucasian and Japanese carriers of two ApoE4 alleles have between 10 and 30 times the risk of developing AD by 75 years of age, as compared to those not carrying any ApoE4 alleles. While the exact mechanism of how ApoE4 causes such dramatic effects remains to be fully determined, evidence has been presented suggesting an interaction with amyloid proteolysis. Apolipoprotein E enhances proteolytic break-down of beta-amyloid, both within and between cells. The isoform ApoE4 is not as effective as the others at promoting these reactions, resulting in increased vulnerability to AD in individuals with that gene variation. The pivotal role of ApoE in AD was first identified through linkage analysis which were followed by association analysis confirming the role of the ApoE4 allele as a strong genetic risk factor for AD (Strittmatter et al., Proc Natl Acad Sci USA, 1993; 90(5): 1977- 1981). Other forms of brain pathology may also be more prevalent in subjects with the ApoE4 genotype. McCarron and Nicoll (Ann N Y Acad Sci. 2000; 903: 176-9) showed that ApoE4 allele was found to be associated with cerebral amyloid angiopathy. Zhang et al. (Lipids Health Dis. 2014; 13: 47.) showed that the ApoE4 allele was associated with a higher risk of intracerebral hemorrhage.

Furthermore, a meta-analysis conducted by Zhou, W., et al.( J Neurotrauma. 2008 25(4):279- 90) showed that the ApoE4 genotype was significantly associated with poor outcome of traumatic brain injury (TBI) six-months after injury, but was not associated with the initial severity of brain injury following T BI.

VEGF

Vascular endothelial growth factor (VEGF) has been shown to play an important role in both the neuronal and vascular systems. VEGF-A which is the most prevalent form of VEGF, has been reported as a pivotal regulator of both normal and abnormal angiogenesis. Compared to other growth factors that contribute to the processes of vascular formation, VEGF-A is unique in its high specificity for endothelial cells within the vascular system. VEGF-A is essential for embryonic vasculogenesis and angiogenesis. Furthermore, VEGF-A is required for the cyclical blood vessel proliferation in the female reproductive tract as well as for bone growth and cartilage formation.

Substantial evidence also implicates VEGF's critical role in the development of conditions or diseases that involve pathological angiogenesis. VEGF-A mRNA is overexpressed by the majority of human tumors examined. Given its central role in promoting tumor growth, VEGF provides an attractive target for therapeutic intervention aimed at blocking VEGF or its receptor signaling system in the treatment of neoplastic diseases.

VEGFs specifically interact with one or several receptor tyrosine kinases (RTKs), VEGF receptor- 1, -2, and -3 (VEGFR-1, -2, -3), and with distinct coreceptors such as neuropilins or heparan sulfate glycosaminoglycans. VEGF receptors are classified as type V RTKs whose extracellular domains consists of seven immunoglobulin-like (Ig-like) domains. VEGF receptors are activated upon ligand-mediated dimerization. VEGF binds to VEGFR-1 (Flt-1) and VEGFR-2 (KDR/Flk-1). VEGFR-2 appears to mediate almost all of the known cellular responses to VEGF. The function of VEGFR-1 is less well defined, although it is thought to modulate VEGFR-2 signaling. Another function of VEGFR-1 is to act as a dummy/decoy receptor, sequestering VEGF from VEGFR-2 binding. In fact, an alternatively spliced form of VEGFR-1 (sFltl) is not a membrane bound protein but is secreted and functions primarily as a decoy. VEGFR-3 mediates lymphangiogenesis in response to VEGF-C and VEGF-D and does not bind VEGF-A.

Mateo et al. (Mateo et al., Acta Neurol Scand. 2007; 116(l):56-8.) showed that a decrease in serum VEGF levels could contribute to the neurodegenerative process in AD. However, no significant correlation between serum VEGF levels and age, sex, APOE alleles, AD dementia severity or VEGF gene polymorphisms was found.

European Patent No. EP1272208B discloses use of VEGF- 165 and homologues to treat neuron disorders.

Michaelson et al., (Alzheimer's & Dementia: The Journal of the Alzheimer's Association, Volume 6, Issue 4, S47, 2010) indicated the role of VEGF in mediating the synergistic pathological effects of beta-amyloid and APOE4 on the neurovascular system.

During and Cao (Current Alzheimer Research, 2006, 3, 29-33) showed that genetically increasing hippocampal VEGF in rats resulted in about 2 fold increase in neurogenesis, associated with improved cognition.

There is an unmet need for providing effective therapeutic methods for preventing and reversing the brain pathologies that may be correlated to ApoE4, including that of AD.

SUMMARY OF THE INVENTION

The present invention provides compositions and methods of treatment for Alzheimer's Disease (AD) specifically in patients identified as having the ApoE4 genotype. In particular, the compositions and methods relate to activation of VEGF receptor in hippocampus of the brain of ApoE4 patients having AD. The activation may be a direct activation by an agonist of VEGFR or indirect activation through expression of an agonist of the VEGFR in the hippocampus.

It is now disclosed for the first time that administration of a vector expressing of VEGF directly into the hippocampus or sub-regions thereof reduces or alleviates the pathology observed in a mouse model having the ApoE4 genotype. Surprisingly, the treatment is beneficial specifically for subjects having the ApoE4 genotype and it is contraindicated in other individuals.

According to the principles of the invention the elevation of VEGF in the hippocampus, may be accomplished by direct administration, by use of a targeting agent or vehicle, by specific activation of the receptors or by use of a vector which ensures expression in a specific region of the brain. In one aspect the present invention provides a pharmaceutical composition comprising an agent capable of inducing expression of a vascular endothelial growth factor receptor (VEGFR) agonist specifically in the hippocampus or in a sub-region of the hippocampus, for use in a subject having the ApoE4 genotype, in treatment or slowing the progression of a brain pathology, disease or condition associated with ApoE4 genotype.

The present invention further provides a pharmaceutical composition comprising a vascular endothelial growth factor receptor (VEGFR) agonist, for use in a subject having the ApoE4 genotype, in treatment or slowing the progression of a brain pathology, disease or condition associated with ApoE4 genotype by administering said VEGFR agonist into the hippocampus or into a sub-region of the hippocampus.

The most important example of a disease associated with the ApoE4 genotype is Alzheimer's Disease. Additional pathologies that have shown an increased association with ApoE4 are cerebral amyloid angiopathy and intracerebral hemorrhage. These compositions may also be beneficial in enhancing the recovery from traumatic brain injury in subjects having ApoE4 genotype.

In some embodiments of this aspect the present invention provides a pharmaceutical composition comprising an agent capable of inducing expression of a vascular endothelial growth factor receptor (VEGFR) agonist specifically in the hippocampus or in a sub-region of the hippocampus, for use in treatment or slowing the progression of Alzheimer's Disease (AD) in a subject having an ApoE4 genotype. In some embodiments of the invention the sub-region of the hippocampus is CA3 sub-region. The VEGFR agonist is selected from the group consisting of a vascular endothelial growth factor (VEGF), a gremlin protein and a β-hairpin peptide activating VEGFR. In one particular embodiment the agonist is VEGF. In another specific embodiment the VEGF is VEGF- A.

In some embodiments the agent capable of inducing expression of VEGFR agonist is a polynucleotide construct. Such a construct may be capable of expressing the VEGF-A specifically in the hippocampus or in a sub-region of the hippocampus. Example for such a construct is a vector, and in particularly lentiviral vector. According to some embodiments the composition of the present invention is for use by systemic administration, wherein the polynucleotide construct encoding VEGFR agonist is capable of expressing VEGFR agonist specifically in the hippocampus or in a sub-region of the hippocampus. According to some embodiments the composition of the present invention is for use by direct administration into the hippocampus or into a sub-region of the hippocampus. The present invention provides also a pharmaceutical composition comprising a vascular endothelial growth factor receptor (VEGFR) agonist, for use in treatment or slowing the progression of Alzheimer's Disease (AD) in a subject having an ApoE4 genotype by administration into the hippocampus or into a sub-region of the hippocampus. In some embodiments the composition are administered directly into the hippocampus or into a sub- region of the hippocampus.

In another aspect the present invention provides a method of treating or slowing the progression of Alzheimer's Disease in a subject having the ApoE4 genotype, said method comprising administering a pharmaceutical composition comprising a therapeutically effective amount of an agent capable of activating vascular endothelial growth factor receptor (VEGFR) specifically in the hippocampus or in a sub-region of the hippocampus. According to some embodiments activating VEGFR comprises inducing the expression of VEGFR agonist specifically in the hippocampus or in a sub-region of the hippocampus. According to other embodiments activating VEGFR comprises administration of VEGFR agonist specifically to the hippocampus or in a sub-region of the hippocampus.

In yet another aspect the present invention provides a method of treating or slowing the progression of brain pathology, disease or condition associated with ApoE4 genotype, said method comprising ascertaining the ApoE4 genotype in the subject and administering a pharmaceutical composition comprising a therapeutically effective amount of an agent capable of activating vascular endothelial growth factor receptor (VEGFR) specifically in the hippocampus or in a sub-region of the hippocampus.

BRIEF DESCRIPTION OF THE FIGURES

Fig. 1 shows the effect of ApoE4 on VEGF expression in naive CNS and periphery of young naive mice. Fig. 1A - Quantification of VEGF-A immunohistochemistry analysis of CA1 hippocampal sub-region (*p<0.05 by student t-test); Fig. IB Immunoblot analysis of hippocampal VEGF and its quantification (**p<0.01 by student t-test); Fig 1C - VEGF-A hippocampal rt-PCR measurements (*p<0.05 by student t-test); Fig ID - Immunoblot analysis of VEGF receptor-2 and its quantification (*p<0.05 by student t-test); Fig IE - VEGF receptor- 2 rt-PCR hippocampal measurements (*P<0.05 by student t-test); Fig IF - Immunoblot analysis of VEGF-A in blood serum and its quantification (***p<0.001 by student t-test); Fig 1G) HIF la (left panel) and HIF 2 (right panel) rt-PCR hippocampal measurements (*p<0.05 by student t-test). ApoE3 mice depict in white bars whereas apoE4 mice depict in black bars. Fig. 2 shows the effect of LV-VEGF treatment. Fig. 2A - representative sections of staining for NeuN in naive and GFP-injected mice; Fig. 2B - VEGF immunohistology staining of hippocampal CA3 subregion of naive, LV-GFP treated and LV-VEGF treated mice. Representative sections (left) and quantification (right) are shown (p<0.05 for group and p<0.01 for treatment effects by 2-way ANOVA; p<0.05 for treatment effect by 1-way ANOVA and *p<0.05 for the post-hoc analysis VEGF on apoE4 mice compared to the control LV-GFP group). Fig. 2C -Representative immunoblots (left) and quantification (right) of immunoblot analysis of hippocampal VEGF-A in naive, LV-GFP and LV-VEGF treated mice (p<0.05 for group and p<0.001 for treatment effects by 2-way ANOVA; p<0.05 for treatment effect by 1- way ANOVA and *p<0.05 for the post -hoc analysis of the specific effect of VEGF on apoE4 mice compared to the control LV-GFP group); Fig. 2D - Representative immunoblots (left) and quantification (right) of immunoblot analysis of VEGF receptor-2 in naive, LV-GFP treated and LV-VEGF treated mice (p<0.05 for group and p<0.001 for treatment effects by 2-way ANOVA; p<0.01 for treatment effect by 1-way ANOVA and ***p<0.001 for the post-hoc analysis of the specific effect of VEGF on apoE4 mice compared to the control LV-GFP group); Fig. 2E - HIF la mRNA levels utilized rt-PCR measurements in naive, LV-GFP treated and LV-VEGF treated mice (p<0.05 for group and p<0.0001 for treatment effects 2-way ANOVA; p<0.0001 for treatment effect by 1-way ANOVA and **p<0.01 for the post-hoc analysis of the specific effect of VEGF on apoE4 mice compared to the control LV-GFP group).

Fig. 3 shows that VEGF reverses cognitive impairments. Fig 3A - Morris water maze. The time to reach the hidden platform was measured (sec) (***p<0.001 by post hoc analysis for the specific effect of VEGF on apoE4 mice compared to the control LV-GFP group in days 3 and 4). Fig 3B - novel object recognition test. The ratio of the number of visits near the novel object from the sum of visits near both old and novel objects was measured (p<0.001 for the for the post- hoc analysis of the specific effect of VEGF on apoE4 mice compared to the control LV-GFP group). Fig. 4 shows the effect of VEGF treatment on VgluTl and Doublecortin. Fig 4A - Representative sections (left) and quantification (right) of VGluTl immunohistology staining of CA3 hippocampal sub-region (*p<0.05 for post-hoc analysis for treatment effects); Fig. 4B - Representative immunoblots (left) and quantification (right) of VGluTl immunoreactivity measurements (p< 0.05 for group and treatment by 2-way ANOVA, and p<0.001 for post-hoc analysis for the specific effect of VEGF treatment on apoE4 mice compared to the control LV- GFP group); Fig. 4C - Representative sections (left) and quantification (right) of doublecortin (DCX) immunohistology staining of CA3 hippocampal sub-region (p< 0.05 for group and treatment by 2-way ANOVA); Fig. 4D - Representative sections (left) and quantification (right) of ApoER2 immunohistology staining of CA3 hippocampal sub-regions (p< 0.05 for the effect of group by 2-way ANOVA).

Fig. 5 shows that the Alzheimer's disease's hallmarks were not affected reversed by VEGF treatment. Fig. 5A - Representative sections (left) and quantification (right) of β-amyloid immunohistology staining of CA3 hippocampal sub-regions (p<0.05 for the effect of group and p<0.01 for the effect of treatment by 2-way ANOVA, p<0.01 for the treatment effect by 1-way ANOVA); Fig. 5B Representative sections (left) and quantification (right) of AT8 immunohistology staining of CA3 hippocampal sub-regions indicating the Tau protein phosphorylation (p<0.05 for the effect of group by 2-way ANOVA); and Fig 5C - Representative blots (left) and quantification (right) of ApoE immunoreactivity measurements (p<0.05 for the effect of group and treatment by 2-way ANOVA, p<0.001 for post-hoc analysis of the effects of LV-VEGF treatment in apoE4 mice showed for the corresponding apoE3 mice in all groups).

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a pharmaceutical composition comprising an agent capable of inducing expression of a vascular endothelial growth factor receptor (VEGFR) agonist specifically in the hippocampus or in a sub-region of the hippocampus, for use in treatment or slowing the progression of Alzheimer's disease (AD) in a subject having an ApoE4 genotype. It is disclosed herein for the first time that mice with ApoE4 genotype, which is a known factor increasing the risk of developing Alzheimer's Disease, have lower levels of VEGF in the hippocampus than those expressing the ApoE3 isoform of ApoE.

Similar results were obtained for VEGF mRNA. Therefore, this is the first time that the ApoE4 genotype and the level of VEGF in the hippocampus were shown to be correlated (both in terms of the protein and RNA levels).

Without wishing to be bound by any theory or mechanism of action it is postulated that ApoE4 down-regulates the expression of VEGF. These effects are associated with cognitive impairments; decreased levels of the pre-synaptic marker VGluT and of the ApoE receptor 2 (ApoER2) in hippocampal neurons; and corresponding accumulation of hyperphosphorylated tau and beta amyloid. It is further shown herein, for the first time, that over-expression of VEGF in the hippocampus of ApoE4 mice, using a VEGF-A-expressing virus, results in reversal of cognitive brain pathological effects induced by ApoE4.

The term "pharmaceutical composition" as used herein and in the claims refers to a composition comprising at least one active agent as disclosed herein formulated together with one or more pharmaceutically acceptable carriers.

The term "pharmaceutically acceptable carrier" or "pharmaceutically acceptable excipient" as used herein refers to any and all solvents, dispersion media, preservatives, antioxidants, coatings, isotonic and absorption delaying agents, surfactants, and the like, that are compatible with pharmaceutical administration. The use of such media and agents for pharmaceutically active substances is well known in the art. The compositions may contain other active compounds providing supplemental, additional, or enhanced therapeutic functions.

The term "agent capable of inducing expression" as used herein and in the claims refers to an agent capable of increasing the transcription and/or translation of a VEGFR agonist. In one example the agent is a compound capable of increasing the transcription and/or translation of VEGF-A, and in particular of VEGVies-

The terms "vascular endothelial growth factor receptor agonist" and "VEGFR agonist" as used herein and in the claims refers to any known agonist of VEGFR. In some embodiments the VEGFR agonist is vascular endothelial growth factor (VEGF). In other embodiments it may be other agent such as a gremlin protein (accession number 060565), that is known to activate VEGFR (Claesson-Welsh L., Blood, 2010, 116(18), 3386-3387) or β-hairpin peptide that is capable of activating VEGFR. Examples of such peptides are described in Diana et al. (Journal of Biological Chemistry, 2011, 286(48), 41680-41692), which is incorporated herein in its entirety by reference. Exemplary such a peptide has a sequence set forth in SEQ ID NO: l. The term "vascular endothelial growth factor" or "VEGF" as used herein and in the claims refers to any known VEGF such as VEGF-A, VEGF-B, VEGF-C or VEGF-D. In particular VEGF is VEGF-A (accession number NM_003376). In some embodiments VEGF is an isoform of VEGF-A protein and in particular human VEGF121, VEGFmb, VEGF145, VEGF165, VEGFiesb, VEGFi₈₉ and VEGF206 (Uniport Accession Number P15692-9, P15692-12, P15692- 6, P15692-4, P15692-8, P15692-2 and P15692-14, respectively). According to one particular embodiment the VEGF is VEGF-A or an analog thereof being an agonist of VEGF receptor and having at least 90% sequence identity with VEGF-A, e.g. heaving at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% ,99% or having 100% identity with VEGF-A. The term "sub-region of the hippocampus" as used herein and in the claims refer to any sub- region of the hippocampus e.g. CAl, CA2, CA3, CA4, Dentate gyrus, or fimbria. In some embodiments the sub-region of the hippocampus is CAl sub-region. In another embodiment, the sub-region is CA2 sub-region. In still another embodiment the sub-region is CA3 sub- region. In further embodiment the sub-region refers to a sub-region comprising CAl, CA2 and CA3 sub-regions.

The term "treatment or slowing the progression" as used herein and in the claims refers to taking steps to obtain beneficial or desired results, including clinical results. Beneficial or desired clinical results include, but are not limited to, alleviation or amelioration of one or more symptoms associated Alzheimer's disease, delay or slow the processing of the disease of its symptoms , delay or slowing of that impairment, amelioration, palliation or stabilization of the symptoms, etc.

As previously discussed the composition according to the present invention is beneficial to those having ApoE4 genotype. The term "ApoE4 genotype" as used herein and in the claims refer to a subject who is homozygote to ApoE4 allele, i.e. having two ApoE4 alleles. Example 5 showed that administration of LV-VEGF to mice having ApoE3 genotype actually increased the level of β-amyloid. Thus it can be seen that the treatment according to the present invention is beneficial to subject having ApoE4 genotype only and contraindicated to others.

According to any one of the above embodiments the pharmaceutical composition comprises a polynucleotide molecule encoding the VEGFR agonist. The terms "polynucleotide" and "nucleic acid" are used herein interchangeably and refer to DNA, RNA, single stranded or double stranded and chemical modifications thereof.

According to further embodiments the pharmaceutical composition comprises a polynucleotide construct encoding the VEGFR agonist. According to some embodiments the VEGFR agonist is VEGF. According to other embodiments the VEGFR agonist is VEGF-A. In more particular embodiments the VEGF-A is VEGFi65. In some specific embodiments the VEGF is human VEGF-A. In other embodiments the VEGFR agonist is gremlin protein or β-hairpin peptide activating VEGFR.

According to some embodiments, the polynucleotide construct encoding VEGFR agonist is capable of expressing VEGFR agonist specifically in the hippocampus or in a sub-region of the hippocampus. According to one embodiment the VEGFR agonist encoding polynucleotide construct is capable of expressing VEGFR specifically in CAl, CA2, CA3 sub-region of the hippocampus or in a sub-region which is a combination of CAl, CA2 and CA3 sub-regions. According to some embodiments the VEGFR agonist is VEGF-A, and in particular VEGF₁65. According to other embodiment the VEGFR agonist is gremlin protein or β-hairpin peptide activating VEGFR.

According to some embodiments the polynucleotide construct encoding VEGFR agonist is capable of expressing said agonist constitutively. In other embodiment the expression occurs upon induction. According to some embodiments the polynucleotide encoding VEGFR agonist is operably linked to a tissue specific promoter specific to the hippocampus or to a sub-region of the hippocampus. According to some embodiments the tissue specific promoter is specific to CA1 or CA3 sub-region of the hippocampus. The term "operably linked" as used herein and in the claims refers to an arrangement of elements that allows them to be functionally related. For example, a promoter is operably linked to a coding sequence if it controls the transcription of the sequence. Any known tissue specific promoter specific to the hippocampus or to a sub- region of the hippocampus can be used. Non-limiting examples of such promoters are described in US 6,066,726 and Pan-Vazquez et al. Molecular Brain (2015) 8:40. According to some embodiments the VEGFR agonist. According to other embodiments VEGF is VEGF-A. According to some particular embodiments VEGF-A is VEGF165. In some specific embodiments the VEGF is human VEGF-A. According to other embodiment the VEGFR agonist is gremlin protein or β-hairpin peptide activating VEGFR.

According to any one of the above embodiment the polynucleotide construct encoding a VEGFR agonist is a vector. According to some embodiments the VEGFR agonist is VEGF. According to some embodiments the VEGF is VEGF-A. According to some particular embodiments VEGF-A is VEGF₁65. In some specific embodiments the VEGF is human VEGF- A. According to other embodiment the VEGFR agonist is gremlin protein or β-hairpin peptide activating VEGFR.

The term "vector" as used herein and in the claims refers to any viral or non-viral vector, as well as any plasmid, cosmid, phage or binary vector in double or single stranded linear or circular form that may or may not be self-transmissible or mobilizable, and that can transform eukaryotic host cells either by integration into the cellular genome or which can exist extrachromosomally (e.g., autonomous replicating plasmid with an origin of replication). Any vector known in the art is envisioned for use in the practice of this invention.

According to some embodiments the vector of the present invention is a plasmid. According to other embodiments the vector is a virus, a modified or engineered virus. The modification of a vector may include mutations, such as deletion or insertion mutation, gene deletion or gene inclusion. In particular, a mutation may be done in one or more regions of the viral genome. Such mutations may be introduced in a region related to internal structural proteins, replication, or reverse transcription function. Other examples of vector modification are deletion of certain genes constituting the native infectious vector such as genes related to the virus' pathogenicity and/or to its ability to replicate.

According to some embodiments the vector is a virus selected, lentivirus, adenovirus, modified adenovirus and retrovirus. In one particular embodiment the vector is lentivirus. The vector is capable of expressing VEGFR agonist. According to some embodiments the VEGF is VEGF- A. According to some embodiments the VEGF is VEGF-A. According to some particular embodiments VEGF-A is VEGF₁65. In some specific embodiments the VEGF is human VEGF- A. According to other embodiment the VEGFR agonist is gremlin protein or β-hairpin peptide activating VEGFR. According to one embodiment said virus is capable of expressing VEGFR angonist specifically in the hippocampus or in a sub-region of the hippocampus, e.g. in CA1 or CA3 sub-region. According to some embodiment the VEGF is VEGF165.

According to some embodiments the pharmaceutical composition according to the present invention comprises a vehicle capable of delivering the polynucleotide construct encoding the VEGFR agonist, as defined hereinabove, to the brain. According to some embodiments the VEGF is VEGF-A. According to other embodiments the VEGFR agonist is VEGF-A. According to some particular embodiments VEGF is VEGF165. In some specific embodiments the VEGF is human VEGF-A. According to other embodiment the VEGFR agonist is gremlin protein or β-hairpin peptide activating VEGFR. Non-limiting example for such a vehicle is a liposome. Such a polynucleotide construct, according to some embodiments, is capable of expressing VEGFR agonist, and in particular VEGF-A specifically in the hippocampus or in a sub-region of the hippocampus, e.g. in CA1 or CA3 sub-region. According to some embodiments the VEGFR agonist encoding polynucleotide is operably linked to a tissue specific promoter specific to the hippocampus or to a sub-region of the hippocampus.

According to any one of the above embodiments, the pharmaceutical composition according to the present invention is for use by direct administration into the hippocampus or into a sub- region of the hippocampus. Suitable methods for delivery of therapeutic substances into a specific region of the brain are known in the art. For example, WO 2011/123842 describes treatment of neurodegenerative diseases by targeted delivery of a nerve growth factor or a vector expressing it, into the substantia nigra or the striatum. The method of direct administration is in the competence of the person skilled in the art, and any know method may be used according to the present invention. Examples for a direct method administration are described also in Garg et al., (Curr Alzheimer Res, 2014. 11(6): p. 549-57) and include intracerebral infusion, intracerebral catheterization and so on. In one embodiment such pharmaceutical composition comprises a polynucleotide construct encoding a VEGFR agonist. According to some embodiments the VEGFR agonist is VEGF. According to other embodiments VEGFR agonist is VEGF- A. According to more particular embodiments VEGF- A is VEGFi65- According to other embodiment the VEGFR agonist is gremlin protein or β- hairpin peptide activating VEGFR. VEGF-A may be VECF121, VEGFmb, VEGF145, VEGF105, VEGFi65b, VEGF; 89 and VEGF206. According to some particular embodiments VEGF-A is VEGF₁65 or an analog thereof. In some specific embodiments the VEGF is human VEGF and more particularly human VEGF-A. In some embodiments the pharmaceutical composition is directly administered into one or more the sub-region of the hippocampus. In some embodiments said sub-region is CAl sub-region. In another embodiment, the sub-region is CA2 sub-region. In still another embodiment the sub-region is CA3 sub-region. In further the pharmaceutical composition is directly administered into more than one sub-regions, e.g. administered into CAl and CA2, into CAl and CA3 or into CAl, CA2 and CA3 sub-regions. According to some embodiments the VEGFR agnonist encoding polynucleotide construct is capable of expressing VEGF-A constitutively. In other embodiment the expression of VEGF- A occurs upon induction. According to some embodiments the VEGFR agonist encoding polynucleotide is operably linked to a tissue specific promoter specific to the hippocampus or to a sub-region of the hippocampus. According to one embodiment the polynucleotide construct is a vector capable of expressing the VEGFR agonist. According to some embodiments the VEGFR agonist is VEGF. According to some embodiments the VEGF is VEGF-A. According to some particular embodiments VEGF is VEGF₁65. In some specific embodiments the VEGF is human VEGF. According to other embodiment the VEGFR agonist is gremlin protein or β- hairpin peptide activating VEGFR. According to one further embodiment the vector is a virus such as lentivirus, adenovirus, modified adenovirus or retrovirus. According to other embodiments the pharmaceutical composition comprises a vehicle capable of delivering the polynucleotide construct as defined. Example for such a vehicle is a liposome. According to one specific embodiment the pharmaceutical composition comprises a vector such as a virus, and in particular lentivirus, encoding for VEGF-A for use by direct administration into the hippocampus or into a sub-region of the hippocampus such as CAl and CA3. The VEGF may be VEGFies. According to one more particular embodiment the pharmaceutical composition is for use by direct administration into hippocampus or sub-region of the hippocampus, e.g. into CA3 sub- region comprises a vector such as a virus encoding VEGF-A, more particularly VEGF165. According to some embodiments the pharmaceutical composition according to any one of the above embodiments is for use by systemic administration, wherein said polynucleotide construct encoding VEGFR agonist is capable of expressing the agonist specifically in the hippocampus or in a sub-region of the hippocampus. According to some embodiments the VEGFR agonist is VEGF. According to some embodiments the VEGFR agonist is VEGF-A. According to some particular embodiments VEGF is VEGF₁65. In some specific embodiments the VEGF is human VEGF-A. According to other embodiment the VEGFR agonist is gremlin protein or β-hairpin peptide activating VEGFR. According to some specific embodiments the VEGF-A is VEGF₁65. Such administration may be aimed at bypassing the blood brain barrier. Thus, according to some embodiments the systemic administration is intrathecal administration. According to the teaching of the present invention the polynucleotide construct administered intrathecally expresses VEGF-A specifically in the hippocampus or in a sub-region of the hippocampus. According to some embodiment the polynucleotide construct encoding VEGF- A is capable of expressing VEGF-A specifically in CA1, CA2, CA3 sub-region of the hippocampus or in the region which is a combination of said sub-regions. According to some embodiments the polynucleotide construct encoding VEGF-A is operably linked to a tissue specific promoter specific to the hippocampus or to a sub-region of the hippocampus. According to one embodiment the polynucleotide construct is a vector capable of expressing the VEGF-A. According to one further embodiment the vector is a virus such as lentivirus, adenovirus, modified adenovirus or retrovirus. According to other embodiment the pharmaceutical composition comprises a vehicle capable of delivering the polynucleotide construct as defined. Example for such a vehicle is a liposome.

The present invention provides a pharmaceutical composition comprising a vascular endothelial growth factor receptor (VEGFR) agonist, for use in treatment or slowing the progression of Alzheimer's Disease (AD) in a subject having an ApoE4 genotype by administration into the hippocampus or into a sub-region of the hippocampus. Such compositions are formulated for a direct administration into the hippocampus or into a sub-region of the hippocampus. According to some embodiments the VEGFR agonist is VEGF. According to other embodiments the VEGFR agonist VEGF-A and in particular VEGF₁65. According to some embodiment VEGF is human VEGF-A. According to other embodiments the VEGFR agonist is a gremlin protein or a β-hairpin peptide activating VEGFR. According to some embodiments the sub-region of the hippocampus is CA3 sub-region. In other embodiments said sub-region is CA1 sub-region. In another embodiment, the hippocampus sub-region is CA2 sub-region.

According to some embodiments the administration is a direct administration of VEGFR agonist into the hippocampus or in a sub-region of the hippocampus. The direct administration is as defined hereinabove and may be performed by any known method including direct injection, use of catheter of pump. According to some specific embodiments the pharmaceutical composition is formulated for a direct administration into hippocampus or sub-region of the hippocampus, e.g. into CA3 sub-region, and comprises VEGF-A. According to some particular embodiments VEGF-A is VEGF₁65. In some specific embodiments the VEGF-A is human VEGF-A.

According to some other embodiments the administration encompass administration of a composition that lead the VEGFR agonist to the hippocampus or to a sub-region of the hippocampus. Any method known in the art may be used. Non-limiting example of such a method is use of carrier mediated delivery, liposome etc.

According to a further aspect the present invention provides a pharmaceutical composition comprising an agent capable of inducing expression of a vascular endothelial growth factor receptor (VEGFR) agonist specifically in the hippocampus or in a sub-region of the hippocampus, for use in a subject having the ApoE4 genotype in treatment or slowing the progression of a brain pathology, disease or condition associated with ApoE4 genotype.

In one embodiment the present invention provides a pharmaceutical composition comprising a vascular endothelial growth factor receptor (VEGFR) agonist, for use in a subject having the ApoE4 genotype, in treatment or slowing the progression of a brain pathology, disease or condition associated with ApoE4 genotype by administering said VEGFR agonist into the hippocampus or into a sub-region of the hippocampus.

In one embodiment said brain pathology is selected from Alzheimer's Disease, cerebral amyloid angiopathy, and intracerebral hemorrhage. In other embodiments the pharmaceutical composition is for use in enhancing the recovery from traumatic brain injury in subjects having ApoE4 genotype.

According to some embodiments VEGFR agonist is VEGF. According to some embodiments VEGF is VEGF-A. In particular embodiments the VEGF-A is VEGF165. In specific embodiments the VEGF is human VEGF-A. In other embodiments the VEGFR agonist is gremlin protein or β-hairpin peptide activating VEGFR. According to one embodiment hippocampus sub-region is selected from CA1 , CA2, CA3 sub- region and any combination thereof.

According to some embodiments the pharmaceutical composition comprises an agent capable of inducing expression of VEGFR, wherein said agent comprises a polynucleotide construct encoding the VEGFR agonist. According to some embodiments, the polynucleotide construct encoding VEGFR agonist is capable of expressing VEGFR agonist specifically in the hippocampus or in a sub-region of the hippocampus. According to some embodiments the polynucleotide molecule encoding VEGF-A is operably linked to a tissue specific promoter specific to the hippocampus or to a sub-region of the hippocampus. The polynucleotide construct may be a vector, such as a plasmid, lentiviral vector, adenoviral vector, modified adenoviral vector and retroviral vector. According to some embodiments the pharmaceutical composition is for use by systemic administration, wherein said polynucleotide construct encoding VEGFR agonist is capable of expressing VEGFR agonist specifically in the hippocampus or in a sub-region of the hippocampus. According to other embodiments the pharmaceutical composition is for use by direct administration into the hippocampus or into a sub-region of the hippocampus. According to any of the above embodiments the VEGFR agonist is VEGF-A, and in particular VEGFi65- According to other embodiment the VEGFR agonist is a gremlin protein or a β-hairpin peptide activating VEGFR.

According to another aspect, the present invention provides a method of treating or slowing the progression of Alzheimer's disease in a subject having the ApoE4 genotype, said method comprising administering a pharmaceutical composition comprising a therapeutically effective amount of an agent capable of activating vascular endothelial growth factor receptor (VEGFR) specifically in the hippocampus or in a sub-region of the hippocampus. The terms "treating or slowing the progression", "ApoE4 genotype", "VEGFR agonist" and "sub-region of the hippocampus" are as defined hereinabove.

According to some embodiments, the agent is capable of activating the VEGFR in a sub-region of the hippocampus selected from the CA1 , CA2 and CA3 sub-region. According to other embodiments the composition is the agent is capable of activating VEGFR in more than one sub-region, e.g. it is capable of activating VEGFR in CA1 and CA3, in CA1 and CA2, or in CA1 , CA2 and CA3 subregions.

According to some embodiments, activating VEGFR refers to inducing the expression of a VEGFR agonist. In some embodiments the VEGFR agonist is vascular endothelial growth factor (VEGF). In other embodiments the VEGFR agonist may be any other agent such as a gremlin protein, that was shown to activate VEGFR (Claesson-Welsh, Blood, 2010, 116(18), 3386-3387) or β-hairpin peptide that is capable of activating VEGFR. According to some embodiments the VEGF is VEGF-A. According to other embodiments the VEGF-A is selected from a group consisting of VEGF] ??, VEGFmb, VEGF145, VEGF165, VEGFiesb, VEGF189 and VEGF206. According to some specific embodiments the VEGF is human VEGF-A.

According to some embodiments the agent capable of inducing expression of VEGFR agonist is a polynucleotide molecule encoding said agonist. According to some other embodiments the agent capable of inducing expression of VEGFR agonist is a polynucleotide construct comprising the polynucleotide molecule encoding VEGFR agonist. According to one embodiment such VEGFR agonist encoding polynucleotide construct is capable of expressing VEGFR agonist specifically in the hippocampus or in a sub-region of the hippocampus. According to any of the above embodiments the VEGFR agonist is VEGF. According to some embodiments, the VEGF is VEGF-A, and in particular VEGF₁65. According to other embodiment the VEGFR agonist is gremlin protein or β-hairpin peptide activating VEGFR. According to some embodiments the polynucleotide construct encoding VEGFR agonist is capable of expressing VEGFR agonist constitutively. In other embodiment the expression of VEGFR agonist occurs upon induction. According to some embodiments the polynucleotide encoding VEGFR agonist is operably linked to a tissue specific promoter specific to the hippocampus or to a sub-region of the hippocampus. According to one embodiment the tissue specific promoter is specific to CA1, CA2 or CA3. According to some embodiments the VEGFR agonist is VEGF. According to other embodiments, the VEGF is VEGF-A, and in particular VEGF₁65. In some embodiments, the VEGF is human VEGF-A. According to other embodiment the VEGFR agonist is gremlin protein or β-hairpin peptide activating VEGFR. According to any one of the above embodiments, the VEGFR agonist encoding polynucleotide construct is a vector. The term "vector" is as defined above. In some embodiments the vector is selected from the group consisting of a plasmid, lentiviral vector, adenoviral vector, modified adenoviral vector, and retroviral vector. In one more particular embodiment the vector is a virus. In more particular embodiments the vector is lentiviral vector, adenoviral vector, modified adenoviral vector and retroviral vector. According to any of the above embodiments the VEGFR agonist is VEGF. According to some embodiments the VEGF is VEGF-A, and in particular VEGF₁65. According to other embodiment the VEGFR agonist is gremlin protein or β-hairpin peptide activating VEGFR. According to some embodiments the composition comprises a vehicle capable of delivering the agent, and in particular polynucleotide construct, as defined herein above to the brain.

According to any one of the above embodiments the compositions according to the present invention is directly administered into the hippocampus or into a sub-region of the hippocampus. In some embodiments the composition is administered into CA1 or CA3 sub- regions of the hippocampus. According to some other embodiments the composition is administered into several sub-regions of the hippocampus. In one embodiment the composition is administered into CA1 and CA2 sub-regions or into CA1 and CA3 sub-regions or into CA1, CA2 and CA3 sub-reagions.

According to other embodiments the composition according to the present invention is administered by a systemic route, wherein the composition comprising VEGFR agonist encoding polynucleotide construct is capable of expressing VEGFR agonist specifically in the hippocampus or in a sub-region of the hippocampus. In one specific embodiment the systemic administration is intrathecal administration, i.e. such composition is administered intratechally. According to some embodiments the VEGFR agonist encoding polynucleotide is operably linked to a tissue specific promoter specific to the hippocampus or to a sub-region of the hippocampus. According to one embodiment the tissue specific promoter is specific to CA1, CA2 or CA3. According to any of the above embodiments the VEGFR agonist is VEGF. According to other embodiments the VEGF is VEGF-A, and more particularly VEGF165. According to other embodiment the VEGFR agonist is gremlin protein or β-hairpin peptide activating VEGFR.

According to some embodiments the composition according to the present invention is directly administered into the hippocampus or into a sub-region of the hippocampus. In some embodiment the composition comprises a polynucleotide construct encoding VEGF-A. In one embodiment the VEGF encoding polynucleotide construct is capable of expressing VEGF-A specifically in the hippocampus or in a sub-region of the hippocampus. According to some embodiments the polynucleotide encoding VEGF-A is operably linked to a tissue specific promoter specific to the hippocampus or to a sub-region of the hippocampus. According to one embodiment the tissue specific promoter is specific to CA1, CA2 or CA3.

The present invention provides a method of treating or slowing the progression of Alzheimer's disease in a subject having the ApoE4 genotype, said method comprising administering a vector encoding for VEGF-A to the hippocampus or to a sub-region of the hippocampus. The present invention provides a method of treating or slowing the progression of Alzheimer's Disease in a subject having the ApoE4 genotype, said method comprising administering a pharmaceutical composition comprising a vector encoding for VEGF-A, to the hippocampus or to a sub-region of the hippocampus.

According to some embodiments activating VEGFR comprises administration specifically to the hippocampus or in a sub-region of the hippocampus of a VEGFR agonist. The VEGFR agonist is as defined hereinabove. According to any of the above embodiments the VEGFR agonist is VEGF-A, and in particular VEGFi65. According to other embodiment the VEGFR agonist is a gremlin protein or a β-hairpin peptide activating VEGFR.

According to some embodiments the administration is a direct administration of VEGFR agonist into the hippocampus or into a sub-region of the hippocampus. The direct administration is as defined hereinabove and may be performed by any known method including direct injection, use of catheter of pump.

According to some other embodiments the administration encompass administration of a composition that lead the VEGFR agonist to the hippocampus or in a sub-region of the hippocampus. Any method known in the art may be used. Non-limiting example of such a method is use of carrier mediated delivery, liposome etc.

According to yet another aspect the present invention provides a method of treating or slowing the progression of brain pathology, disease or condition associated with ApoE4 genotype, said method comprising ascertaining the ApoE4 genotype in the subject and administering a pharmaceutical composition comprising a therapeutically effective amount of an agent capable of activating vascular endothelial growth factor receptor (VEGFR) specifically in the hippocampus or in a sub-region of the hippocampus. In one embodiment the brain pathology is selected from a list consisting of Alzheimer's Disease, cerebral amyloid angiopathy and intracerebral hemorrhage. In other embodiments the present invention provides a method of enhancing the recovery from traumatic brain injury in subjects having ApoE4 genotype. The terms "agent", "activating VEGFR", "sub-region of the hippocampus", as well as all other terms are as defined herein above.

According to some embodiments, activating VEGFR refers to inducing the expression of a VEGFR agonist. According to some embodiments activating VEGFR comprises administration specifically to the hippocampus or in a sub-region of the hippocampus of a VEGFR agonist. The VEGFR agonist is as defined hereinabove. According to any of the above embodiments the VEGFR agonist is VEGF. According to other embodiments the VEGF is VEGF-A. According to some embodiments the VEGF is VEGF₁65. According to one particular embodiment the VEGF is human VEGF-A. According to other embodiment the VEGFR agonist is a gremlin protein or a β-hairpin peptide activating VEGFR. Table 1. Sequence table

Having now generally described the invention, the same will be more readily understood through reference to the following examples, which are provided by way of illustration and are not intended to be limiting of the present invention.

EXAMPLES

Materials and Methods

Mice - ApoE-targeted replacement mice, in which the endogenous mouse apoE was replaced by either human apoE3 or apoE4, were created by gene targeting, and were purchased from Taconic (Germantown, NY). Mice were back-crossed to wild-type C57BL/6J mice (Harlan 2BL/610) for ten generations and were homozygous for the apoE3 (3/3) or apoE4 (4/4) alleles. These mice are referred to in the text as apoE3 and apoE4 mice, respectively. The apoE genotype of the mice was confirmed by PCR analysis, as described in Levi et al. (Neurobiol Dis, 2003. 13(3): p. 273-82) and Belinson and Michaelson (J Neural Transm, 2009. 116(11): p. 1427-34). All experiments were performed on age-matched male animals (4 months of age), and were approved by the Tel Aviv University Animal Care Committee. Every effort was made to reduce animal stress and to minimize animal usage. Following treatment, the mice were anesthetized with ketamine and xylazine and perfused transcardially with phosphate buffer saline (PBS). Their brains were then removed and halved, and each hemisphere was further processed for either biochemical or histological analysis, as outlined below. Each of the 6 groups (apoE3 or apoE4 x Naive, LV-GFPtreated or LV-VEGF treated) consisted of 8-11 mice, and the experiment was performed on 2 different cohorts of mice.

Lentivirus preparation - The human VEGF gene (SEQ ID NO: 2) was amplified from a pBluescript plasmid, purchased from Harvard Institute of Proteomics, Boston, USA, and cloned into pLenti6/R4R2/V5-DEST (Invitrogen) using the ViralPower Promoterless Lentiviral Gateway Kit (Invitrogen). For the lentiviral production, The VEGF vector, or a pLL3.7-CMV- EGFP control plasmid were co-transfected with the packaging plasmids pLPl, pLP2, and pLP/VSVG into the 293T producer cell line using Lipofectamine 2000 (Invitrogen). The supernatant was collected 48 and 72 hours post transfection and was subsequently deposited using ultracentrifugation at 25,000 RPM for 2 hours. The virus-containing pellet was aspirated using HBSS, aliquot and kept stored in -80°C until use. Lentiviral titer was determined using the Lenti-X p24 Rapid Titer Kit and the manufacturer's recommended procedure (Clontech Laboratories). The titer was estimated to be 10⁸.

Intracerebral administration of viral vectors - At the age of 4 months, when apoE4-driven pathologies are readily detected in the brain, apoE3 and apoE4 targeted replacement (TR) mice were anesthetized with a mixture of ketamine-xylazine and placed in a stereotactic apparatus (model 940; David Kopf). Subsequently, l-μΕ of the viral preparation was injected bilaterally into the CA3 region of the hippocampus by using the following coordinates: ±3.2 mm medial/lateral, -2.1 mm anterior/posterior, -2.2 mm dorsal/ventral from the bregma. The preparation was injected with a speed of 0.5 μΕ/πιίη over a period of 2 min by using a Hamilton 10-μΕ syringe and a 26 ga needle. The mice were stitched and then returned to their cages. Behavioral testing. The behavioral tests were initiated 20 days following the lentivirus injection. The mice were first subjected to the novel object recognition test for 3 days and then, following a 4-day interval, to the Morris water maze for 5 days.

(i) Novel object recognition test. This was performed as described in Salomon-Zimri et al. (Neurodegener Dis, 2014;13(2-3):86-92). In brief, the mice (9-12 per group ) were first placed in an arena (60x60 cm with 50 cm walls) in the absence of objects, after which two identical objects were added. Either 2 hours (short-term memory test, STM) or 24 hours later (long-term memory test, LTM), the mice were re-introduced to the arena in which one of the objects was replaced by a novel one. The behavior of the mice was then monitored utilizing the Etho Vision XT 11 program for 5 minutes, and the time and number of visits that the mice paid to each of the objects were measured. The results are presented as the ratio in the number of visits near the novel object relative to the total time spent near both new and old objects,

(ii) The Morris water maze. This was performed as previously described in Salomon-Zimri et al., (Neurodegener Dis, 2014;13(2-3):86-92). Accordingly, the mice were placed in a 140 cm circular pool with the water rendered opaque with milk powder and a 10 cm circular platform submerged 1 cm below the surface of the water was placed at a fixed position. The mice (9-12 per group) were subjected to 4 trials per day for 5 days, such that for each trial the mice were placed in one of equally spaced locations along the perimeter of the pool. The inter-trial interval was 30 min and the location of the platform was unchanged between days. The mice were introduced to the arena from 4 random locations, whose order was unchanged between days. The performance of the mice was monitored by measuring the time they took to reach the platform. A probe test was performed following the last trial of the 5^th day, in which the hidden platform was removed from the arena and the amount of time the mice spent in the quadrant in which the platform was located and in the other quadrants was measured. Measurements were performed using the Etho Vision XT 11 program.

Immunohistochemistry and immunofluorescence confocal microscopy. One brain hemisphere was fixed over night with 4% paraformaldehyde in 0.1M phosphate buffer, pH 7.4, and then placed in 30% sucrose for 48 h. Frozen coronal sections (30μπι) were then cut on a sliding microtome, collected serially, placed in 200μ1 of cryoprotectant (containing glycerin, ethylene glycol, and 0.1M Sodium-Phosphate buffer, pH 7.4), and stored at -20°C until use. Examination of VEGF-A ligand was conducted utilizing anti-VEGF antibody directed against the human VEGF165 which is the most abundant isoform of VEGF-A and its corresponding VEGF164 isoform in mice for both Immunohistochemistry and immunoblot assays. The free- floating sections were immunostained with the following primary antibodies (Abs): rabbit anti- VEGF (l : 1000,calbiochem); guinea-pig anti-vesicular glutamatergic transporter 1 (VGluTl ; 1 :2000; Millipore); rabbit anti-ApoER2 (aCT; J. Herz lab [28] ;1: 1000); rabbit anti- doublecortin (1 :200; santa cruz); rabbit anti-A 42 (1 :500; Chemicon, Temecula, CA); rabbit anti-202/205 phosphorylated tau (AT8; 1 :200, Innogenetics). Immunohistochemistry was performed as previously described (Belinson et al., J Neurosci, 2008. 28(18): p. 4690-701). Accordingly, sections were washed with lOmM PBS, pH 7.4, and blocked for 1 h in 20% serum diluted in PBS with 0.1% Triton X-100 (PBST), after which the primary Ab, diluted in PBST containing 2% of the appropriate serum, was applied overnight at 4°C. The sections were then rinsed in PBST, and incubated for 1 h at room temperature with the corresponding secondary Ab (Vector Laboratories, Burlingame, CA) diluted 1:200 in PBST containing 2% of the appropriate serum. After several additional rinses in PBST, the sections were incubated for 0.5 h in avidin-biotin-horseradish peroxidase complex (ABC Elite; Vector Laboratories) in PBST. After rinses in PBST, sections were placed for up to 10 min in diaminobenzidine chromagen solution (Vector Laboratories). To minimize variability, sections from all animals of the same cohort were stained simultaneously. The reaction was monitored visually and stopped by rinses in PBS. The sections were mounted on a dry gelatin-coated slide and then dehydrated and sealed with cover slips. Αβ staining was performed similarly except that the sections were pre- incubated with 70% formic acid for 7 min in order to increase antigen retrieval prior to staining. The immunostained sections were viewed using a Zeiss light microscope (Axioskop, Oberkochen, Germany) interfaced with a CCD video camera (Kodak Megaplus, Rochester, NY, USA). Pictures of stained brains were obtained at X10 magnification. Analysis and quantification of the staining (2 hippocampal images per animal at Bregma -1.7 to -2.06) were performed using the Image-Pro plus system for image analysis (v. 5.1, Media Cybernetics, Silver Spring, MD, USA). The images were analyzed by marking the area of interest and setting a threshold for all sections subjected to the same staining. The stained area above the threshold relative to the total area was then determined for each section. All the groups were stained together and the results presented correspond to the mean ± SEM of the percent area stained normalized relative to the young control apoE3 mice.

Immunofluorescence staining was performed using fluorescent chromogens. Accordingly, sections were first blocked (incubation with 20% normal donkey serum in PBST for 1 h at room temperature), and then reacted for 48 h at 4°C with the primary Abs (dissolved in 2% normal donkey serum in PBST). Next, the bound primary Abs were visualized by incubating the sections for 1 h at room temperature with Alexa-fluor 488 -conjugated goat anti-guinea-pig (1 : 1000; Invitrogen); Alexa- Alexa-fluor 488-conjugated donkey anti-rabbit (1 : 1000; Invitrogen, Eugene, OR), or Alexa-fluor 546-conjugated donkey anti -rabbit (1 : 1000; Invitrogen). The sections were then mounted on dry gelatin-coated slides. Sections stained for immunofluorescence were visualized using a confocal scanning laser microscope (Zeiss, LSM 510). Images (1024x1024 pixels, 12 bit) were acquired by averaging eight scans. Control experiments revealed no staining in sections lacking the first Ab. The intensities of immunofluorescence staining were calculated utilizing the Image-Pro Plus system (version 5.1, Media Cybernetics) as previously described [29]. All images for each immunostaining were obtained under identical conditions, and their quantitative analyses were performed with no further handling. Moderate adjustments for contrast and brightness were performed similarly on all the presented images of the different mouse groups. The images were analyzed by setting a threshold for all sections of a specific labeling. The area of staining over the threshold relative to the total area of interest was determined and averaged for each mouse and each group. Immunoblot analysis. Immunoblot analysis was performed as previously described [30, 31]. In brief, the hippocampus was rapidly removed from one freshly excised hemisphere and stored frozen at -70°C until use. The dissected hippocampus was then homogenized in 200μ1, in the following buffer [lOmM HEPES, pH 7, which contained 2mM EDTA, 2mM EGTA, 0.5mM DTT, protease inhibitor cocktail (Sigma P8340), and phosphatase inhibitor cocktail (Sigma P5726)]. The homogenates were then aliquoted and stored at -70°C. For SDS-electrophoresis, the samples were boiled for 10 min with 0.5% SDS and immunoblotted as previously described [29, 30]. The following Abs were used: Rabbit anti-VEGF (l : 1000,calbiochem); rabbit anti- VEGFR2 (1 :500, cell signaling, 55bl l); mouse anti-VGluTl (1 : 1000; Millipore); Goat anti- apoE (1 :10000, Chemicon); and Mouse anti-GAPDH (1 :1000; Abeam). Protein concentration was determined utilizing the BCA protein assay kit (Pierce 23225). The immunoblot bands were visualized utilizing the ECL chemiluminescent substrate (Pierce), after which their intensity was quantified using EZQuantGel software (EZQuant, Tel Aviv, Israel). GAPDH levels were employed as gel loading controls.

qRT-PCR analysis. RT-PCR analysis was performed as previously described [32]. In brief, the hippocampus was rapidly excised from one freshly removed hemisphere and stored frozen at -70°C until use. RNA was extracted from the tissue using the MasterPure RNA purification kit (Epicentre, USA). RNA was transformed into cDNA using the High Capacity cDNA reverse transcription kit (Applied Biosystems, USA). TaqMan qRT PCR assays were conducted according to the manufacturer's specifications (Applied Biosystems). Oligonucleotides (probes) for TaqMan qRT PCR were attached to FAM (6-carboxyfluorescin) at the 5' end and a quencher dye at the 3' end. VEGF, VEGFR2, and HIFland HIF2 gene expression levels were determined utilizing TaqMan RT-PCR specific primers (Applied Biosystems). Analysis and quantification were conducted using the 7300 system software and compared to the expression of the housekeeping HPRT-1 gene.

Statistical Analysis. The experimental design consisted of 2 genotypes (also referred as "group" : apoE3 and apoE4) and of 3 treatments (naive, LV-GFP-treated and LV-VEGF-treated mice), and the results were analyzed utilizing 2-way ANOVA testing using STATISTICA software (Version 8.0 StatSoft, Inc., Tulsa, USA). Further examination of the treatment effect conduct utilizing 1-way ANOVA followed by post-hoc Bonferroni correction was performed in order to test the specific effect of VEGF treatment on apoE4 mice group. Each of the 6 groups contained 8-11 mice and the experiment was performed on 2 different cohorts of mice (cohort for the behavioral paradigm and a different cohort for the biochemistry analysis). The naive experiment consisted of 2 genotypes (apoE3 and apoE4) in which results were normalized to apoE3 and were analyzed utilizing student t-test.

Example 1. The effects of apoE4 on VEGF levels in the hippocampus of young naive TR mice.

The extent to which apoE4 effects the levels of VEGF in the hippocampus was first examined histologically. As can be seen in Fig. 1A, apoE4 mice showed lower levels of VEGF than the corresponding apoE3 mice. These results obtained both in the CA1 and CA3 region (results presented for CA1), (p<0.05 by student t-test) there was no effect on the DG sub-region of the hippocampus. Moreover, these results were verified utilizing immunoblot assay of the total level of VEGF in the hippocampus. As can be seen in Fig. IB, the levels of VEGF were lower in the apoE4 than in the apoE3 mice (p<0.01 by student t-test). These effects were accompanied with a similar decrease in mRNA levels as presented in Fig. 1C (p<0.05 by student t-test), suggesting that the apoE4 related decrease in VEGF is driven by gene expression. Examination of VEGF receptors showed lower levels of VEGF receptor 2 both in protein and mRNA levels (Fig. ID and Fig. IE respectively, p<0.05 by student t-test). Examination of mRNA and protein levels of VEGF receptor 1 using large sample group showed that these levels were not significantly affected in ApoE4 mice (data not shown). In contrast, in the experiment exploiting larger group, the levels of VEGF receptor 1 was not significantly affected by apoE4 in both protein and mRNA levels. Interestingly, complementary measurements of VEGF levels in the serum reveled an opposite effect in which VEGF levels were higher in the apoE4 mice compared to apoE3 ( Fig IF; p<0.001 by student t-test).

The apoE4-induced decrease in VEGF levels was accompanied by decreased levels of Hypoxia- inducible factors 1 (HIF1) but not HIF2 mRNA levels in the hippocampus, suggesting that the effects of VEGF in the apoE4 mice are associated with specific stress-related conditions (Fig. 1G; ***p<0.05 by student t-test). All results of the examples are presented as mean +/-SEM; n=8-15 per group. Example 2. The effect of LV-VEGF treatment on VEGF expression in young naive mice.

We next examined whether the apoE4-related decrease in VEGF can be counteracted by lentivirus expressing VEGF construct. This was perused by intrahippocampal injection of VEGF-A or GFP construct (the latter was used as a control), to the CA3 subregion as described in materials and methods (Fig. 2A). As it can be seen in Fig. 2B, expression of VEGF as described resulted in significant specific elevation of VEGF levels in CA3 subregion compared to LV-GFP and naive control groups. Quantification revealed significant effects for group p<0.05 (i.e. significant difference between apoE3 and apoE4 mice), and for treatment p<0.01 (i.e. significant difference between LV-VEGF and naive mice or LV-GFP mice) by 2-way ANOVA; further analysis of the effects of the different treatments showed p<0.05 utilizing 1- way ANOVA and p<0.05 for the post-hoc analysis of the specific effect of VEGF on apoE4 mice compared to the control LV-GFP group.

Similar results were obtained for the CA1 subgregion. These results were invigorated by immunoblot analysis as depicted in Fig. 2C; Quantification revealed significant effects for group p<0.05, and treatment p<0.001 by 2-way ANOVA; further analysis of the effects of the different treatments showed p<0.05 utilizing 1-way ANOVA and p<0.05 for the post-hoc analysis of the specific effect of LV-VEGF on apoE4 mice compared to the control LV-GFP group.

The effects of specific elevation of VEGF was also associated by corresponding effect on VEGF receptor-2 protein level as shown in Fig 2D; Quantification of these results revealed significant effects for group p<0.05, and treatment p<0.001 by 2-way ANOVA; further analysis of the effects of the different treatments showed p<0.01 utilizing 1-way ANOVA and p<0.001 for the post -hoc analysis of the specific effect of VEGF on apoE4 mice compared to the control LV- GFP group. Corresponding mRNA analysis showed similar results as obtained in naive mice (not shown).

Examination of the Hypoxia-inducible factors showed significant decrease in the levels of HIF 1 following both control LV-GFP and LV-VEGF groups, as shown in Fig. 2E. Quantification revealed significant effects for group p<0.05, and treatment p<0.0001 by 2-way ANOVA; further analysis of the effects of the different treatments showed p<0.0001 utilizing 1-way ANOVA and p<0.01 for the post-hoc analysis of the specific effect of VEGF on apoE4 mice compared to the control LV-GFP group. Levels of the corresponding HIF 2 however, were not affected by both, genotype or treatment.

All the results are resented as mean+/-SEM; n=8-15 per group. Example 3. The effects of LV-VEGF treatment on apoE4-driven cognitive deficits.

The extent to which LV-VEGF treatment can counteract apoE4 behavioral deficits was next examined by Morris water maze test and by the novel object recognition test.

Morris water maze test. The test was performed as described in material and methods section and the results are presented in Fig. 3A. As can be seen from the figure, the mice in all the groups (e.g naive, LV-GFP and LV-VEGF treated mice) improved their performance over time and reached similar plateau level in day 5 as measured by the latency to reach the hidden platform. However both naive and LV-GFP apoE4 mice showed deficit in the learning curve. These apoE4-dependent deficits was counteracted by the VEGF treatment. None of the treatments affected the apoE3 mice (Fig 3A; ***p<0.001 by post hoc analysis for the specific effect of VEGF on apoE4 mice compared to the control LV-GFP group in days 3 and 4). Novel object recognition test. The test was performed as described in material and methods section and the results are presented in Fig. 3B. It can be clearly seen from the figure that the apoE3 mice made more visits to the novel object than to the familiar one. In contrast, the naive or LV-GFP apoE4 mice made the same amount of visits to the familiar and novel objects, indicating a deficit in the memory of the familiar object. This deficit was abolished by VEGF treatment (LV-VEGF group). As it can be seen from that figure, the treatment of ApoE4 mice with VEGF improved both the short term (upright panel) and the long term (low right panel) memory Quantification of the results revealed p<0.01 for the effect of group and p<0.05 for the effect of treatment by 2-way ANOVA. Further analysis of the effects of the different treatments showed p<0.05 utilizing 1-way ANOVA and p<0.01 for the post-hoc analysis of the specific effect of VEGF treatment on apoE4 mice compared to the control LV-GFP group.

Example 4. Synaptic and neuronal parameters Phenotype reversed by VEGF treatment.

VGluT marker. We first focused on the apoE4-induced deficit in the pre-synaptic marker VGluT Liraz et al. (Mol Neurodegener, 2013. 8: p. 16) Immunohistocemical examination in CA3 hippocampal subregion, in which the pathology of apoE4 is the most pronounced, revealed that LV-VEGF markedly increased VGluT levels in apoE4 mice, thus reversed the pathological effects of apoE4. (Fig. 4A). Quantification of these results revealed significant effects for group p<0.05, and treatment p<0.05 by 2-way ANOVA. This was confirmed by complementary immunoblot analysis (Fig 4B). Quantification revealed significant effect of p< 0.05 for both group and treatment by 2-way ANOVA and additional Post-hoc analysis revealed p<0.001 for the specific effect of VEGF treatment on apoE4 mice compared to the control LV-GFP group. All results are shown as mean+/-SEM; n=8-10 per group.

Doublecortin marker. It was previously shown that neurogenesis as measured by the marker Doublecortin (DCX) is up-regulated in apoE4 mice compared to apoE3 (Salomon-Zimri et al., Neurodegener Dis, 2014;13(2-3):86-92). We therefore examined the effect of LV-VEGF treatment on this parameter; the results are presented in Fig. 4C. This revealed that the sham control treatment (LV-GFP) by itself had a marked effect on the levels of DCX in which it inversed the level of DCX in apoE4 compared to apoE3 mice. The up-regulation of VEGF resulted in specific elevation of DCX in apoE4 mice to the levels of the corresponding apoE3 mice, thus abolishing the apoE genotype related differences. Quantification revealed significant effect of p< 0.05 for group and treatment by 2-way ANOVA. We next examined the effect of LV-VEGF treatment on the levels of apoER2. As was previously shown (Gilat-Frenkel), levels of apoE receptor 2 are lowered in apoE4 mice compared to the corresponding apoE3 mice. It can be seen from the results (Fig. 4D) that this is also correct for LV-GFP control groups, however said effect was reversed by VEGF treatment in CA3 subregion. Quantification revealed significant effect of group p< 0.05 for by 2-way ANOVA.

Example 5. AD hallmark parameters

In contrast to the effect of VEGF treatment on the cognitive, neuronal and ApoE receptor pathologies, the apoE4-induced elevated levels of neither beta amyloid (Fig. 5A) nor Tau phosphoryation (Fig 5B) were altered by the LV-VEGF treatment. These findings suggest that the pathological effects of ApoE4, i.e. neuronal and cognitive impairment, which is driven via VEGF dependent mechanism and can be reversed by up-regulation of the expression of VEGF, is not related to the standard AD hallmark parameters e.g. β-amyloid accumulation or Tau phosphorylation.

Interestingly, LV-VEGF increased the levels of beta-amyloid and AT-8 in the ApoE3 mice up to the levels of the corresponding ApoE4 mice indicating that such a treatment is contraindicated to any other genotype except for ApoE4. Quantification of beta-amyloid revealed p<0.05 for the effect of group and p<0.01 for the effect of treatment by 2-way ANOVA. Further analysis of the effects of the different treatments showed p<0.01 by 1-way ANOVA. Moreover, analysis of AT8 staining utilized 2-way ANOVA revealed p<0.05 for the effect of group. These results support the assertion that activating the VEGF receptor, in particular by inducing elevation of the expression of VEGF, is beneficial only to a person having the ApoE4 genotype and may be harmful to others.

ApoE levels are known to be down-regulated by apoE4 in both control groups (Sullivan et al., Neurobiol Aging, 2011. 32(5): p. 791-801). As it can been seen from Fig. 5C, ApoE levels were lower in apoE4 mice relative to the ApoE3 mice in all detected groups. Quantification revealed p<0.05 for the effect of group and treatment by 2-way ANOVA. Further post-hoc analysis of the effects of LV- VEGF treatment in apoE4 mice showed p<0.001 for the corresponding apoE3 mice in all groups.All results are presented as mean+/-SEM; n=8-10 per group.

While the present invention has been particularly described, persons skilled in the art will appreciate that many variations and modifications can be made. Therefore, the invention is not to be construed as restricted to the particularly described embodiments, rather the scope, spirit and concept of the invention will be more readily understood by reference to the claims which follow.

Claims

1. A pharmaceutical composition comprising an agent capable of inducing expression of a vascular endothelial growth factor receptor (VEGFR) agonist specifically in the hippocampus or in a sub-region of the hippocampus, for use in treatment or slowing the progression of Alzheimer's Disease (AD) in a subject having an ApoE4 genotype.

2. The pharmaceutical composition of claim 1 , wherein the sub-region of the hippocampus is selected from the CA1 sub-region, CA3 sub-region or both CA1 and CA3 sub-regions.

3. The pharmaceutical composition of claim 1 or claim 2, wherein said agonist is selected from a vascular endothelial growth factor (VEGF), a gremlin protein and a β-hairpin peptide activating VEGFR.

4. The pharmaceutical composition of claim 3, wherein said agonist is VEGF

5. The pharmaceutical composition of claim 4, wherein the VEGF is VEGF-A.

6. The pharmaceutical composition of claim 5, wherein the VEGF-A is selected from VEGFm, VEGFmb, VEGF₁₄₅, VEGF₁₆₅, VEGF₁₆₅b, VEGF₁₈₉ and VEGF206.

7. The pharmaceutical composition of claim 1, wherein the agent comprises a polynucleotide construct comprising a polynucleotide molecule encoding a VEGF-A.

8. The pharmaceutical composition of claim 7, wherein said polynucleotide construct is capable of expressing the VEGF-A specifically in the hippocampus or in a sub-region of the hippocampus.

9. The pharmaceutical composition of claim 8, wherein said polynucleotide molecule encoding VEGF-A is operably linked to a tissue specific promoter specific to the hippocampus or to a sub-region of the hippocampus.

10. The pharmaceutical composition of claim 8 or claim 9, wherein the construct is a vector.

11. The pharmaceutical composition of claim 10, wherein the vector is selected from a plasmid, lentiviral vector, adenoviral vector, modified adenoviral vector and retroviral vector.

12. The pharmaceutical composition of claim 11, wherein the vector is a virus.

13. The pharmaceutical composition of any one of claims 1 to 12, for use by systemic administration, wherein said polynucleotide construct encoding VEGFR agonist is capable of expressing VEGFR agonist specifically in the hippocampus or in a sub-region of the hippocampus.

14. The pharmaceutical composition of any one of claims 1 to 13, for use by direct administration into the hippocampus or into a sub-region of the hippocampus.

15. A pharmaceutical composition comprising a vascular endothelial growth factor receptor (VEGFR) agonist, for use in treatment or slowing the progression of Alzheimer's Disease (AD) in a subject having an ApoE4 genotype by administration into the hippocampus or into a sub-region of the hippocampus.

16. The pharmaceutical composition of claim 15, wherein said administration comprises a direct administration.

17. The pharmaceutical composition of claim 15 and claim 16, wherein said agonist is selected from a vascular endothelial growth factor (VEGF), a gremlin protein and a β- hairpin peptide activating VEGFR.

18. A method of treating or slowing the progression of Alzheimer's Disease in a subject having the ApoE4 genotype, said method comprising administering a pharmaceutical composition comprising a therapeutically effective amount of an agent capable of activating vascular endothelial growth factor receptor (VEGFR) specifically in the hippocampus or in a sub-region of the hippocampus.

19. The method of claim 18, wherein the sub-region of the hippocampus is selected from the group consisting of CA1 sub-region, CA3 sub-region and both CA1 and CA3 sub-regions.

20. The method of claim 18, wherein activating VEGFR comprises inducing the expression of VEGFR agonist specifically in the hippocampus or in a sub-region of the hippocampus.

21. The method of claim 20, wherein said agonist is selected from the group consisting of a vascular endothelial growth factor (VEGF), a gremlin protein and a β-hairpin peptide activating VEGFR.

22. The method of claim 21, wherein said agonist is VEGF.

23. The method of claim 22, wherein the VEGF is VEGF-A.

24. The method of claim 23, wherein VEGF-A is selected from a group consisting of VEGF121, VEGFmb, VEGF145, VEGFies, VEGFiesb, VEGF189 and VEGF206.

25. The method of claim of claim 20, wherein the agent comprises a polynucleotide construct comprising a polynucleotide molecule encoding VEGF-A.

26. The method of claim 25, wherein said polynucleotide construct is capable of expressing VEGF-A specifically in the hippocampus or in a sub-region of the hippocampus.

27. The method of claim 26, wherein said polynucleotide molecule encoding VEGF-A is operably linked to a tissue specific promoter specific to the hippocampus or to a sub- region of the hippocampus.

28. The method of claim 25, wherein said polynucleotide construct is a vector.

29. The method of claim 28, wherein the vector is selected from the group consisting of a plasmid, lentiviral vector, adenoviral vector, modified adenoviral vector, and retroviral vector.

30. The method of any one of claims 20 to 29, wherein said polynucleotide construct encoding VEGF-A is capable of expressing VEGF-A specifically in the hippocampus or in a sub-region of the hippocampus.

31. The method of claim 30, wherein said agent is systemically administered.

32. The method of any one of claims 20 to 31, wherein the agent is directly administered into the hippocampus or into a sub-region of the hippocampus.

33. The method of claim 18 , wherein activating VEGFR comprises administration of VEGFR agonist specifically to the hippocampus or in a sub-region of the hippocampus.

34. The method of claim 33, wherein the administration comprises a direct administration of an agent to the hippocampus or to a sub-region of the hippocampus.

35. The method of claim 34, wherein said agonist is selected from the group consisting of a vascular endothelial growth factor (VEGF), a gremlin protein and a β-hairpin peptide activating VEGFR.

36. A pharmaceutical composition comprising an agent capable of inducing expression of a vascular endothelial growth factor receptor (VEGFR) agonist, for use in a subject having the ApoE4 genotype in treatment or slowing the progression of a brain pathology, disease or condition associated with ApoE4 genotype.

37. A pharmaceutical composition comprising a vascular endothelial growth factor receptor (VEGFR) agonist, for use in a subject having the ApoE4 genotype in treatment or slowing the progression of a brain pathology, disease or condition associated with ApoE4 genotype by administering said VEGFR agonist into the hippocampus or into a sub-region of the hippocampus.

38. A method of treating or slowing the progression of brain pathology, disease or condition associated with ApoE4 genotype, said method comprising ascertaining the ApoE4 genotype in the subject and administering a pharmaceutical composition comprising a therapeutically effective amount of an agent capable of activating vascular endothelial growth factor receptor (VEGFR) specifically in the hippocampus or in a sub-region of the hippocampus.