OF ALZHEIMER'S DISEASE

FIELD OF THE INVENTION:

The present invention relates to methods and pharmaceutical compositions for the treatment of Alzheimer's disease.

BACKGROUND OF THE INVENTION:

Synaptic dysfunction, cognitive decline, and excessive accumulation of neurotoxic β- amyloid peptides (Αβ), are hallmark features of Alzheimer's disease (AD). Αβ is generated by sequential cleavage of the amyloid precursor protein (APP) by β- and γ-secretase. In the competing and physiologically predominant non-amyloidogenic pathway a-secretase cleaves APP within the Αβ region (Lichtenthaler et al, 2011; Prox et al, 2012) thus precluding the formation of Αβ peptides. This leads to the secretion of the neuroprotective ectodomain APPsa, into the extracellular space in a process that can be stimulated by neuronal and synaptic activity (Hoe et al, 2012; Hoey et al, 2009).

Processing of APP by β-secretase within the amyloidogenic pathway leads to the generation of the large ectodomain ΑΡΡββ and membrane bound stubs termed CTFβ. CTFβ is then further cleaved by γ-secretase leading to the production of Αβ. AD is characterized by upregulation of β-secretase (BACE-1) resulting in a shift towards amyloidogenic APP processing (Ahmed et al, 2010; Holsinger et al, 2002). Increasing evidence suggests that the concomitant reduction in APPsa and the loss of its physiological functions contributes to AD pathogenesis. Reduced levels of APPsa or ADAM10 were reported in patients with amyloid deposits and AD (Dobrowolska et al, 2014; Lannfelt et al, 1995) and reviewed in (Endres & Fahrenholz, 2012). Lowered levels of CSF APPsa were also correlated with poor memory performance in both human patients and aged rats (reviewed in Endres & Fahrenholz, 2012). Moreover, a-secretase attenuating mutations have been associated with hereditary late-onset AD (Suh et al, 2013). Interestingly, APPsa has recently also been shown to reduce Αβ generation by binding to and thereby inhibiting BACE-1 activity (Obregon et al, 2012).

Substantial evidence has implicated APP and APPsa in protecting cultured neurons in vitro against various forms of stress (Kogel et al, 2012). In vivo, APP was found upregulated in response to brain injury suggesting a role in damage response (Leyssen et al, 2005; Murakami et al, 1998; Ramirez et al, 2001; Van den Heuvel et al, 1999). Importantly, in addition to neuroprotection APPsa has prominent physiological functions for neurite outgrowth, synaptogenesis, adult neurogenesis, synaptic plasticity and hippocampus- dependent behavior (Aydin et al, 2012). Previously, the inventors generated APP-KO mice that showed impaired long-term potentiation (LTP) and spatial learning that was fully rescued in APPsa knock-in mice expressing solely APPsa from the endogenous APP locus (Ring et al, 2007). Recently, to avoid functional redundancy within the APP gene family (reviewed in (Aydin et al, 2012), we generated conditional double knockout mice (cDKO) lacking APP and the close homologue APLP2 in excitatory forebrain neurons (Hick et al, 2015). These cDKO mice revealed reduced spine density and impaired synaptic plasticity that was associated with deficits in hippocampus dependent behaviors (Hick et al 2015).

Some studies showed the possible implication of APPsa in neuroprotection and neuromodulation but were limited to specific situations (acute brain injury for example) and no prove of concept on Alzheimer was demonstrated (Corrigan et al, 2012; Thornton et al, 2006).

Some other strategies to treat AD targeting a-secretase ADAM- 10 were tested but with poor specificity (ADAM- 10 has several hundred other substrates) and efficacy (Kuhn et al., 2015).

Thus; there is still a need for an effective and safe AD treatment strategy.

SUMMARY OF THE INVENTION:

The present invention relates to methods and pharmaceutical compositions for the treatment of Alzheimer's disease. In particular, the present invention is defined by the claims. DETAILED DESCRIPTION OF THE INVENTION:

Here, the inventors used direct overexpression of APPsa by AAV-mediated gene transfer into the brain to explore its potential to ameliorate or rescue structural, electrophysiological and behavioral deficits of AD model mice. Unexpectedly, they found that overexpression of APPsa in aged transgenic APP/PS1AE9 mice with well-established plaque pathology improves synaptic plasticity and partially rescues spine density deficits. Restoration of synaptic plasticity and increased spine density is also accompanied by a rescue of spatial memory. Moreover, APPsa expression leads to moderately reduced Αβ levels and significantly ameliorated plaque pathology. Interestingly, in AAV-APPsa injected mice, they observed an increased recruitment of microglia towards plaques which may have led to increased plaque clearance. Collectively, these data suggest, that even at stages with advanced plaque deposition APPsa may counteract Αβ induced synaptotoxic effects and restores cognitive functions. Unexpectedly, in contrast to AAV- APPsa, the inventors show that AAV- ΑΡΡβ injection is not able to restore memory deficits of APP/PS1AE9 and that APPsB expression in contrast to APPsa expression fails to ameliorate functional synaptic impairments of aged AD model mice. Also, in contrast to APPsa, APPsP fails to increase microglia density in the vicinity to plaques and induced no upregulation of the microglia marker Ibal. Accordingly, a first object of the present invention relates to a method of treating

Alzheimer's disease in a subject in need thereof comprising administering to the subject a therapeutically effective amount of a vector which comprises a nucleic acid molecule encoding for a polypeptide which is a soluble member of the APP (amyloid precursor protein) family.

Another object of the present invention relates to a method of treating Down syndrome in a subject in need thereof comprising administering to the subject a therapeutically effective amount of a vector which comprises a nucleic acid molecule encoding for a polypeptide which is a soluble member of the APP (amyloid precursor protein) family.

As used herein, the term "subject" denotes a mammal, such as a rodent, a feline, a canine, and a primate. Preferably a subject according to the invention is a human. In the context of the present invention, a "subject in need thereof denotes a subject, preferably a human, with Alzheimer's disease, prodromal Alzheimer's or Down syndrome (Trisomy 21).

As used herein, the term "Alzheimer's disease" has its general meaning in the art and denotes chronic neurodegenerative disease that usually starts slowly and gets worse over time. Alzheimer's disease (AD) is characterized by amyloid deposits, intracellular neurofibrillary tangles, neuronal loss and a decline in cognitive function. The most common early symptom is difficulty in remembering recent events (short-term memory loss). As the disease advances, symptoms can include: problems with language, disorientation (including easily getting lost), mood swings, loss of motivation, not managing self-care, and behavioural issues. AD is undoubtedly multifactorial, but the amyloid protein precursor (APP) is a key element in its development. The physiological functions of APP of its first cleavage product APPsa are unclear, but it has been shown to play crucial roles for spine density, morphology and plasticity. As used herein, the term "prodromal Alzheimer's" refers to the very early form of Alzheimer's when memory is deteriorating but a person remains functionally independent. As used herein, the term "Down syndrome" has its general meaning in the art and refers to a genetic disorder caused by the presence of all, or part of a third copy of chromosome 21. It is typically associated with physical growth delays, characteristic facial features, and mild to moderate intellectual disability. The Down syndrome is also called trisomy 21.

As used herein, the term "treatment" or "treat" refers to both prophylactic or preventive treatment as well as curative or disease modifying treatment, including treatment of subjects at risk of contracting the disease or suspected to have contracted the disease as well as subjects who are ill or have been diagnosed as suffering from a disease or medical condition, and includes suppression of clinical relapse. The treatment may be administered to a subject having a medical disorder or who ultimately may acquire the disorder, in order to prevent, cure, delay the onset of, reduce the severity of, or ameliorate one or more symptoms of a disorder or recurring disorder, or in order to prolong the survival of a subject beyond that expected in the absence of such treatment. By "therapeutic regimen" is meant the pattern of treatment of an illness, e.g., the pattern of dosing used during therapy. A therapeutic regimen may include an induction regimen and a maintenance regimen. The phrase "induction regimen" or "induction period" refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the initial treatment of a disease. The general goal of an induction regimen is to provide a high level of drug to a subject during the initial period of a treatment regimen. An induction regimen may employ (in part or in whole) a "loading regimen", which may include administering a greater dose of the drug than a physician would employ during a maintenance regimen, administering a drug more frequently than a physician would administer the drug during a maintenance regimen, or both. The phrase "maintenance regimen" or "maintenance period" refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the maintenance of a subject during treatment of an illness, e.g., to keep the subject in remission for long periods of time (months or years). A maintenance regimen may employ continuous therapy (e.g., administering a drug at a regular intervals, e.g., weekly, monthly, yearly, etc.) or intermittent therapy (e.g., interrupted treatment, intermittent treatment, treatment at relapse, or treatment upon achievement of a particular predetermined criteria [e.g., disease manifestation, etc.]).

In particular, the method of the present invention is particularly suitable for rescuing memory impairment, synaptic plasticity and/or spine density, ameliorating both structural and functional synaptic impairments, decreasing Αβ levels and plaque deposition, inducing microglia recruitment and activation in the vicinity of amyloid plaques, enhancing Αβ and plaque clearance and/or restoring cognitive functions. As used herein the "amyloid precursor protein (APP) family" has its general meaning in the art and represents integral membrane proteins expressed in many tissues and concentrated in the synapses of neurons. Amyloid precursor proteins include APP, APLPl (amyloid beta (A4) precursor- like protein 1) and APLP2 (amyloid beta (A4) precursor- like protein 1). Soluble members of the amyloid precursor protein (APP) family include the form cleaved by secretases. The soluble members thus include APPsa, APLPls and APLP2s.

In some embodiments, the vector of the present invention comprises a nucleic acid encoding for an APPsa polypeptide. As used herein the term "APPsa" has its general meaning in the art and refers to the protein formed by the cleavage of the amyloid precursor protein (APP) by the a-secretase. The APPsa is then secreted into the extracellular space. Exemplary amino acid sequences of APPsa include sequences a set forth in SEQ ID NO:l and SEQ ID NO:2. SEQ ID NOl : amino acid sequence of the murine APPsa protein

LEVPTDGNAGLLAEPQIAMFCGKLNMHMNVQNGKWESDPSGTKTCIGTKEGI LQYCQEVYPELQITNVVEANQPVTIQNWCK GRKQCKTHTHIVIPYRCLVGEFVSDA LLVPDKCKFLHQERMDVCETHLHWHTVAKETCSEKSTNLHDYGMLLPCGIDKFRGV EF VC CPL AEE SD S VD S AD AEEDD SD V WWGG ADTD Y AD GGEDKV VE V AEEEE V AD V EEEEADDDEDVEDGDEVEEEAEEPYEEATERTTSTATTTTTTTESVEEVVRVPTTAAS TPDAVDKYLETPGDENEHAHFQKAKERLEAKHRERMSQVMREWEEAERQAKNLPK ADKKAVIQHFQEKVESLEQEAANERQQLVETHMARVEAMLNDRRRLALENYITALQ AVPPRPHHVFNMLK YVRAEQKDRQHTLKHFEHVRMVDPKKAAQIRSQVMTHLRV IYERMNQSLSLLYNVPAVAEEIQDEVDELLQKEQNYSDDVLANMISEPRISYGNDAL MPSLTETKTTVELLPVNGEFSLDDLQPWHPFGVDSVPANTENEVEPVDARPAADRGL TTRPGSGLTNIKTEEISEVKMDAEFGHDSGFEVRHQK

SEQ ID NO:2: amino acid sequence of the human APPsa protein

LEVPTDGNAGLLAEPQIAMFCGRLNMHMNVQNGKWDSDPSGTKTCIDTKEGI LQYCQEVYPELQITNVVEANQPVTIQNWCKRGRKQCKTHPHFVIPYRCLVGEFVSDA LLVPDKCKFLHQERMDVCETHLHWHTVAKETCSEKSTNLHDYGMLLPCGIDKFRGV EFVCCPLAEESDNVDSADAEEDDSDVWWGGADTDYADGSEDKVVEVAEEEEVAEV EEEEADDDEDDEDGDEVEEEAEEPYEEATERTTSIATTTTTTTESVEEVVRVPTTAAS TPDAVDKYLETPGDENEHAHFQKAKERLEAKHRERMSQVMREWEEAERQAKNLPK ADKKAVIQHFQEKVESLEQEAANERQQLVETHMARVEAMLNDRRRLALENYITALQ AVPPRPRHVFNMLK YVRAEQKDRQHTLKHFEHVRMVDPKKAAQIRSQVMTHLRV IYERMNQSLSLLYNVPAVAEEIQDEVDELLQKEQNYSDDVLANMISEPRISYGNDAL MPSLTETKTTVELLPVNGEFSLDDLQPWHSFGADSVPANTENEVEPVDARPAADRGL TTRPGS GLTNIKTEEI SE VKMD AEFRHD S G YE VHHQK

In some embodiments, the vector of the present invention comprises a nucleic acid molecule encoding for a APPsa polypeptide comprising an amino acid sequence having at least 90% of identity with the sequence as set forth in SEQ ID NO: 1 or 2.

According to the invention a first amino acid sequence having at least 90% of identity with a second amino acid sequence means that the first sequence has 90; 91; 92; 93; 94; 95; 96; 97; 98; 99 or 100% of identity with the second amino acid sequence. Sequence identity is frequently measured in terms of percentage identity (or similarity or homology); the higher the percentage, the more similar are the two sequences. Methods of alignment of sequences for comparison are well known in the art. Various programs and alignment algorithms are described in: Smith and Waterman, Adv. Appl. Math., 2:482, 1981; Needleman and Wunsch, J. Mol. Biol, 48:443, 1970; Pearson and Lipman, Proc. Natl. Acad. Sci. U.S.A., 85:2444, 1988; Higgins and Sharp, Gene, 73:237-244, 1988; Higgins and Sharp, CABIOS, 5:151-153, 1989; Corpet et al. Nuc. Acids Res., 16:10881-10890, 1988; Huang et al, Comp. Appls Biosci., 8:155-165, 1992; and Pearson et al, Meth. Mol. Biol, 24:307-31, 1994). Altschul et al, Nat. Genet., 6:119-129, 1994, presents a detailed consideration of sequence alignment methods and homology calculations. By way of example, the alignment tools ALIGN (Myers and Miller, CABIOS 4:11-17, 1989) or LFASTA (Pearson and Lipman, 1988) may be used to perform sequence comparisons (Internet Program® 1996, W. R. Pearson and the University of Virginia, fasta20u63 version 2.0u63, release date December 1996). ALIGN compares entire sequences against one another, while LFASTA compares regions of local similarity. These alignment tools and their respective tutorials are available on the Internet at the NCSA Website, for instance. Alternatively, for comparisons of amino acid sequences of greater than about 30 amino acids, the Blast 2 sequences function can be employed using the default BLOSUM62 matrix set to default parameters, (gap existence cost of 11, and a per residue gap cost of 1). When aligning short peptides (fewer than around 30 amino acids), the alignment should be performed using the Blast 2 sequences function, employing the PAM30 matrix set to default parameters (open gap 9, extension gap 1 penalties). The BLAST sequence comparison system is available, for instance, from the NCBI web site; see also Altschul et al, J. Mol. Biol, 215:403-410, 1990; Gish. & States, Nature Genet., 3:266-272, 1993; Madden et al. Meth. EnzymoL, 266:131-141, 1996; Altschul et al, Nucleic Acids Res., 25:3389-3402, 1997; and Zhang & Madden, Genome Res., 7:649-656, 1997.

As used herein, the term "nucleic acid molecule" has its general meaning in the art and refers to a DNA or RNA molecule. However, the term captures sequences that include any of the known base analogues of DNA and RNA such as, but not limited to 4-acetylcytosine, 8- hydroxy-N6-methyladenosine, aziridinylcytosine, pseudoisocytosine, 5-

(carboxyhydroxylmethyl) uracil, 5-fiuorouracil, 5-bromouracil, 5- carboxymethylaminomethyl-2-thiouracil, 5-carboxymethyl-aminomethyluracil, dihydrouracil, inosine, N6-isopentenyladenine, 1 -methyladenine, 1 -methylpseudouracil, 1-methylguanine, 1- methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5- methylcytosine, N6-methyladenine, 7-methylguanine, 5-methylaminomethyluracil, 5- methoxyamino-methyl-2-thiouracil, beta-D-mannosylqueosine, 5'- methoxycarbonylmethyluracil, 5-methoxyuracil, 2-methylthio-N6-isopentenyladenine, uracil- 5-oxyacetic acid methylester, uracil-5-oxyacetic acid, oxybutoxosine, pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, -uracil-5- oxyacetic acid methylester, uracil-5-oxyacetic acid, pseudouracil, queosine, 2-thiocytosine, and 2,6-diaminopurine. In some embodiments, the nucleic acid molecule of the present invention comprises a sequence having at least 70% of identity with the nucleic acid sequence as set forth in SEQ ID NO:3, or SEQ ID NO:4. According to the invention a first nucleic acid sequence having at least 70% of identity with a second nucleic acid sequence means that the first sequence has 70; 71; 72; 73; 74; 75; 76; 77; 78; 79; 80; 81; 82; 83; 84; 85; 86; 87; 88; 89; 90; 91; 92; 93; 94; 95; 96; 97; 98; 99 or 100%) of identity with the second nucleic acid sequence. SEQ ID NO:3: co don-optimized nucleic acid sequence encoding for the murine form of the APPsa:

ttggaggtgcccaccgacggcaacgctggactgctggctgaaccccagatcgccatgttc tgcggcaagctgaacatgca catgaacgtgcagaacggcaagtgggagagcgaccccagcggcaccaagacctgcatcgg caccaaagagggcatcctgcagtat tgccaggaagtgtaccccgagctgcagatcaccaacgtggtggaagccaaccagcccgtg accatccagaactggtgcaagaggg gcagaaagcagtgcaagacccacacccacatcgtgatcccttacagatgcctcgtgggcg agttcgtgtccgacgctctgctggtgcc cgacaagtgcaagttcctgcatcaggaacggatggacgtgtgcgagacacatctgcactg gcacaccgtggccaaagagacatgca gcgagaagtccaccaacctgcacgactacggcatgctgctgccctgcggcatcgacaagt tcagaggcgtggaattcgtgtgctgcc ccctggccgaggaatccgactctgtggatagcgccgacgccgaagaggacgactctgacg tgtggtggggcggagccgacacaga ttacgctgatggcggcgaggacaaggtggtggaagtggctgaagaggaagaggtggccga cgtggaagaagaagaggccgacga cgacgaggatgtggaagatggcgacgaggtggaagaggaagccgaggaaccctacgagga agccaccgagagaaccaccagca ccgccaccacaaccaccaccactaccgagagcgtggaagaggtcgtgcgggtgccaacaa cagccgcctctacacctgacgccgt ggacaagtacctggaaaccccaggcgacgagaacgagcacgcccacttccagaaggctaa agagagactggaagctaagcaccg cgagagaatgagccaagtgatgagagagtgggaggaagctgagagacaggccaagaacct gcccaaggccgacaagaaagccg tgatccagcacttccaggaaaaggtggaaagcctggaacaggaagctgccaacgagagac agcagctggtggaaacccacatggc cagagtggaagctatgctgaacgacagaagaaggctggccctggaaaactacatcaccgc tctgcaggccgtgccccccagacctc accacgtgttcaacatgctgaagaaatacgtgcgggccgagcagaaggacagacagcaca ccctgaagcacttcgagcacgtgcgg atggtggaccccaagaaggccgcccagatcagatcccaagtgatgacccacctgagagtg atctacgagaggatgaaccagagcct gagcctgctgtacaacgtgcccgccgtggccgaagaaatccaggatgaggtggacgagct gctgcagaaagagcagaactacagc gacgacgtgctggccaacatgatcagcgagcccagaatcagctacggcaacgacgccctg atgcccagcctgaccgagacaaaga ccaccgtggaactgctgcccgtgaacggcgagttcagcctggatgacctgcagccctggc accctttcggcgtggactctgtgcctgc caacacagagaacgaagtggaacccgtggacgccagacctgccgctgatagaggcctgac cacaagacctggcagcggcctgac aaacatcaagaccgaagagatcagcgaagtgaagatggacgccgagttcgggcacgacag cggctttgaagtgcggcaccagaaa SEQ ID NO:4: co don-optimized nucleic acid sequence encoding for the human form of the APPsa:

ttggaggtgcccaccgacggcaacgccggactgctggccgagccccagatcgccatgttc tgcggcagactgaacatgc acatgaacgtgcagaacggcaagtgggacagcgaccccagcggcaccaagacctgcatcg acaccaaagagggcatcctgcagta ttgccaagaagtgtaccccgagctgcagatcaccaacgtggtggaagccaaccagcccgt gaccatccagaactggtgcaagcggg gcagaaagcagtgcaagacccacccccacttcgtgatcccttacagatgcctcgtgggcg agttcgtgtccgacgccctgctggtgcc cgacaagtgcaagttcctgcatcaagaacggatggacgtgtgcgagacacatctgcactg gcacaccgtggccaaagagacatgca gcgagaagtccaccaacctgcacgactacggcatgctgctgccctgcggcatcgacaagt tccggggcgtggaattcgtgtgctgcc ccctggccgaggaatccgacaacgtggacagcgccgacgccgaagaggacgacagcgacg tgtggtggggcggagccgacacc gattacgccgacggcagcgaggacaaggtggtggaagtggctgaagaggaagaggtggcc gaggtcgaggaagaggaagccga cgacgacgaggatgacgaggacggcgacgaggtggaagaagaggccgaggaaccctacga ggaagccaccgagcggaccacc tctatcgccaccaccaccacaaccactaccgagagcgtggaagaggtcgtgcgggtgcca accaccgccgcctctacccccgacgc cgtggacaagtacctggaaacccctggcgacgagaacgagcacgcccacttccagaaggc caaagagcggctggaagccaagca ccgcgagcggatgagccaggtcatgagagagtgggaagaagccgagcggcaggccaagaa cctgcccaaggccgacaagaaag ccgtgatccagcacttccaagaaaaggtcgagagcctggaacaagaagccgccaacgagc ggcagcagctggtggaaacccacat ggccagagtggaagccatgctgaacgaccggcggagactggccctggaaaactacatcac cgctctgcaggccgtgccccccaga ccccggcacgtgttcaacatgctgaagaaatacgtgcgggccgagcagaaggaccggcag cacaccctgaagcacttcgagcacgt gcggatggtggaccccaagaaggccgcccagatccgctctcaggtcatgacccacctgag agtgatctacgagagaatgaaccaga gcctgagcctgctgtacaacgtgcccgccgtggccgaagaaatccaggatgaggtggacg agctgctgcagaaagagcagaactac agcgacgacgtgctggccaacatgatcagcgagccccggatcagctacggcaacgacgcc ctgatgcccagcctgaccgagacaa agaccaccgtggaactgctgcccgtgaacggcgagttcagcctggacgacctgcagccct ggcacagcttcggcgctgatagcgtg cccgccaacaccgagaatgaggtggaacccgtggacgccagacctgccgccgatagaggc ctgaccacaagacctggcagcggc ctgaccaacatcaagaccgaagagatcagcgaagtgaagatggacgccgagttccggcac gacagcggctacgaggtgcaccacc agaaa

As used herein, the term "vector" has its general meaning in the art and refers to the vehicle by a nucleic acid molecule can be introduced into a host cell, so as to transform the host and promote expression (e.g. transcription and translation) of the introduced sequence. 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. Cells could be hematopoietic stem cells (e.g. CD34+ cell fraction) or hematopoietic progenitor cells (particularly monocytic progenitors or microglia precursors) isolated from the bone marrow or the blood of the patient (autologous) or from a donor (allogeneic) genetically modified to stably express APPsa or a fragment derived from it by transduction with a vector, particularly a lentiviral vector expressing APPsa under the control of a non-specific (e.g.: phosphoglycerate kinase, EFlalpha) or specific (monocytic- macrophage or microglia specific e.g. CD68 or CD1 lb) native or modified promoter.

In some embodiments, the vector of the present invention is a non-viral vector. Typically, the non- viral vector may be a plasmid which includes the nucleic acid molecule of the present invention.

In some embodiments, the vector of the present invention is 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. Examples of viral vector include but are not limited to adenoviral, retroviral, lentiviral, herpesvirus and adeno-associated virus (AAV) vectors.

In some embodiments, the vector of the present invention is an adeno-associated viral (AAV) vector. By an "AAV vector" is meant a vector derived from an adeno-associated virus serotype, including without limitation AAV1, AAV2, AAV3, AAV4, AA5, AAV6, AAV7, AAV8, AAV9, AAVrhlO or any other serotypes of AAV that can infect humans, monkeys or other species. 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. The ITRs need not 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 nucleic acid molecule of the present 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 "AAV ITRs" 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. See, e.g., Kotin, 1994; Berns, KI "Parvoviridae and their Replication" in Fundamental Virology, 2nd Edition, (B. N. Fields and D. M. Knipe, eds.) for the AAV-2 sequence. 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, AAV-6, 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, AAV-6, 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. In some embodiments, the AAV vector of the present invention is selected from vectors derived from AAV serotypes having tropism for and high transduction efficiencies in cells of the mammalian central and peripheral nervous system, particularly neurons, neuronal progenitors, astrocytes, oligodendrocytes and glial cells. In some embodiments, the AAV vector is an AAV4, AAV9 or an AAVrhlO that have been described to well transduce brain cells especially neurons. In some embodiments, the AAV vector of the present invention is a double-stranded, self-complementary AAV (scAAV) vector. Alternatively to the use of single- stranded AAV vector, self-complementary vectors can be used. The efficiency of AAV vector in terms of the number of genome-containing particles required for transduction, is hindered by the need to convert the single-stranded DNA (ssDNA) genome into double- stranded DNA (dsDNA) prior to expression. This step can be circumvented through the use of self-complementary vectors, which package an inverted repeat genome that can fold into dsDNA without the requirement for DNA synthesis or base-pairing between multiple vector genomes. Resulting self-complementary AAV (scAAV) vectors have increased resulting expression of the transgene. For an overview of AAV biology, ITR function, and scAAV constructs, see McCarty D M. Self-complementary AAV vectors; advances and applications. Mol. Ther. 2008 October; 16 (10): at pages 1648-51, first full paragraph, incorporated herein by reference for disclosure of AAV and scAAV constructs, ITR function, and role of ATRS ITR in scAAV constructs. A rAAV vector comprising a ATRS ITR cannot correctly be nicked during the replication cycle and, accordingly, produces a self-complementary, double- stranded AAV (scAAV) genome, which can efficiently be packaged into infectious AAV particles. Various rAAV, ssAAV, and scAAV vectors, as well as the advantages and drawbacks of each class of vector for specific applications and methods of using such vectors in gene transfer applications are well known to those of skill in the art (see, for example, Choi V W, Samulski R J, McCarty D M. Effects of adeno-associated virus DNA hairpin structure on recombination. J. Virol. 2005 June; 79(11):6801-7; McCarty D M, Young S M Jr, Samulski R J. Integration of adeno-associated virus (AAV) and recombinant AAV vectors. Annu Rev Genet. 2004; 38:819-45; McCarty D M, Monahan P E, Samulski R J. Self- complementary recombinant adeno-associated virus (scAAV) vectors promote efficient transduction independently of DNA synthesis. Gene Ther. 2001 August; 8(16): 1248-54; and McCarty D M. Self-complementary AAV vectors; advances and applications. Mol. Ther. 2008 October; 16(10): 1648-56; all references cited in this application are incorporated herein by reference for disclosure of AAV, rAAV, and scAAV vectors). The AAV vector of the present invention 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. Patents Nos. 5,173, 414 and 5,139, 941; International Publications Nos. WO 92/01070 (published 23 January 1992) and WO 93/03769 (published 4 March 1993); Lebkowski et al, 1988 ; Vincent et al, 1990; Carter, 1992 ; Muzyczka, 1992 ; Kotin,1994; Shelling and Smith, 1994 ; and Zhou et al, 1994. 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. Patent 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 and PNS cells can be used. The complete chimeric sequence is assembled from overlapping oligonucleotides prepared by standard methods. See, e. g., Edge, 1981 ; Nambair et al, 1984 ; Jay et al, 1984. 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. See, e. g. , Graham et al, 1973;, Sambrook et al. (1989) Molecular Cloning, a laboratory manual, Cold Spring Harbor Laboratories, New York, Davis etal. (1986) Basic Methods in Molecular Biology, Elsevier, and Chu et al, 1981. Particularly suitable transfection methods include calcium phosphate co -precipitation (Graham et al, 1973), direct microinjection into cultured cells (Capecchi, 1980), electroporation (Shigekawa et al, 1988), liposome mediated gene transfer (Mannino et al, 1988), lipid-mediated transduction (Feigner et al, 1987), and nucleic acid delivery using high-velocity microprojectiles (Klein et al, 1987).

Typically the vector of the present invention comprises an expression cassette. The term "expression cassette", as used herein, refers to a nucleic acid construct comprising nucleic acid elements sufficient for the expression of the nucleic acid molecule of the present invention. Typically, an expression cassette comprises the nucleic acid molecule of the present invention operatively linked to a promoter sequence. The term "operatively linked" refers to the association of two or more nucleic acid fragments on a single nucleic acid fragment so that the function of one is affected by the other. For example, a promoter is operatively linked with a coding sequence when it is capable of affecting the expression of that coding sequence (e.g., the coding sequence is under the transcriptional control of the promoter). Encoding sequences can be operatively linked to regulatory sequences in sense or antisense orientation. In some embodiments, the promoter is a heterologous promoter. The term "heterologous promoter", as used herein, refers to a promoter that is not found to be operatively linked to a given encoding sequence in nature. In some embodiments, an expression cassette may comprise additional elements, for example, an intron, an enhancer, a polyadenylation site, a woodchuck response element (WRE), and/or other elements known to affect expression levels of the encoding sequence. As used herein, the term "promoter" refers to a nucleotide sequence capable of controlling the expression of a coding sequence or functional RNA. In general, the nucleic acid molecule of the present invention is located 3' of a promoter sequence. In some embodiments, a promoter sequence consists of proximal and more distal upstream elements and can comprise an enhancer element. An "enhancer" is a nucleotide sequence that can stimulate promoter activity and may be an innate element of the promoter or a heterologous element inserted to enhance the level or tissue-specificity of a promoter. In some embodiments, the promoter is derived in its entirety from a native gene. In some embodiments, the promoter is composed of different elements derived from different naturally occurring promoters. In some embodiments, the promoter comprises a synthetic nucleotide sequence. It will be understood by those skilled in the art that different promoters will direct the expression of a gene in different tissues or cell types, or at different stages of development, or in response to different environmental conditions or to the presence or the absence of a drug or transcriptional co-factor. Ubiquitous, cell-type-specific, tissue-specific, developmental stage-specific, and conditional promoters, for example, drug-responsive promoters (e.g. tetracycline-responsive promoters) are well known to those of skill in the art. Examples of promoter include, but are not limited to, the phophoglycerate kinase (PKG) promoter, CAG (composite of the CMV enhancer the chicken beta actin promoter (CBA) and the rabbit beta globin intron.), NSE (neuronal specific enolase), synapsin or NeuN promoters, 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), SFFV promoter, rous sarcoma virus (RSV) promoter, synthetic promoters, hybrid promoters, and the like. The promoters can be of human origin or from other species, including from mice. In addition, sequences derived from non-viral genes, such as the murine metallothionein gene, will also find use herein. Such promoter sequences are commercially available from, e. g. Stratagene (San Diego, CA). In some embodiments, the expression cassette comprises an appropriate secretory signal sequence that will allow the secretion of the polypeptide encoded by the nucleic acid molecule of the present invention. As used herein, the term "secretory signal sequence" or variations thereof are intended to refer to amino acid sequences that function to enhance (as defined above) secretion of an operably linked polypeptide from the cell as compared with the level of secretion seen with the native polypeptide. As defined above, by "enhanced" secretion, it is meant that the relative proportion of the polypeptide synthesized by the cell that is secreted from the cell is increased; it is not necessary that the absolute amount of secreted protein is also increased. In some embodiments, essentially all (i.e., at least 95%, 97%, 98%o, 99%o or more) of the polypeptide is secreted. It is not necessary, however, that essentially all or even most of the polypeptide is secreted, as long as the level of secretion is enhanced as compared with the native polypeptide. Generally, secretory signal sequences are cleaved within the endoplasmic reticulum and, in some embodiments, the secretory signal sequence is cleaved prior to secretion. It is not necessary, however, that the secretory signal sequence is cleaved as long as secretion of the polypeptide from the cell is enhanced and the polypeptide is functional. Thus, in some embodiments, the secretory signal sequence is partially or entirely retained. The secretory signal sequence can be derived in whole or in part from the secretory signal of a secreted polypeptide (i.e., from the precursor) and/or can be in whole or in part synthetic. The length of the secretory signal sequence is not critical; generally, known secretory signal sequences are from about 10-15 to 50-60 amino acids in length. Further, known secretory signals from secreted polypeptides can be altered or modified (e.g., by substitution, deletion, truncation or insertion of amino acids) as long as the resulting secretory signal sequence functions to enhance secretion of an operably linked polypeptide. The secretory signal sequences of the invention can comprise, consist essentially of or consist of a naturally occurring secretory signal sequence or a modification thereof (as described above). Numerous secreted proteins and sequences that direct secretion from the cell are known in the art. The secretory signal sequence of the invention can further be in whole or in part synthetic or artificial. Synthetic or artificial secretory signal peptides are known in the art, see e.g., Barash et al, "Human secretory signal peptide description by hidden Markov model and generation of a strong artificial signal peptide for secreted protein expression," Biochem. Biophys. Res. Comm. 294:835-42 (2002); the disclosure of which is incorporated herein in its entirety.

In some embodiments, the vector of the present invention comprises the nucleic acid sequence set forth in SED ID NO: 5 or 6.

SEQ ID NO:5: complete sequence of the expression cassette of the AAV transfer vector encoding codon-optimized mouse APPsa ggggggggggggggggggttggccactccctctctgcgcgctcgctcgctcactgaggcc gggcgaccaaaggtcgcc cgacgcccgggctttgcccgggcggcctcagtgagcgagcgagcgcgcagagagggagtg gccaactccatcactaggggttcct agatctaggatcacgcgttctagaaatattaaggtacgggaggtacttggagcggccgca ataaaatatctttattttcattacatctgtgtg ttggttttttgtgtgaatcgatagtactaacatacgctctccatcaaaacaaaacgaaac aaaacaaactagcaaaataggctgtccccag tgcaagtgggttttaggaccaggatgaggcggggtgggggtgcctacctgacgaccgacc ccgacccactggacaagcacccaac ccccattccccaaattgcgcatcccctatcagagagggggaggggaaacaggatgcggcg aggcgcgtgcgcactgccagcttcag caccgcggacagtgccttcgcccccgcctggcggcgcgcgccaccgccgcctcagcactg aaggcgcgctgacgtcactcgccgg tcccccgcaaactccccttcccggccaccttggtcgcgtccgcgccgccgccggcccagc cggaccgcaccacgcgaggcgcgag ataggggggcacgggcgcgaccatctgcgctgcggcgccggcgactcagcgctgcctcag tctgcggtgggcagcggaggagtc gtgtcgtgcctgagagcgcagtcgaattgctagcggggatccaccggtcgccaccatgct gccttctctggctttgctgctgctggccg cttggacagtgcgggcctacccttacgacgtgcccgactacgcttacccctacgatgtgc ctgattatgcattggaggtgcccaccgac ggcaacgctggactgctggctgaaccccagatcgccatgttctgcggcaagctgaacatg cacatgaacgtgcagaacggcaagtg ggagagcgaccccagcggcaccaagacctgcatcggcaccaaagagggcatcctgcagta ttgccaggaagtgtaccccgagctg cagatcaccaacgtggtggaagccaaccagcccgtgaccatccagaactggtgcaagagg ggcagaaagcagtgcaagacccaca cccacatcgtgatcccttacagatgcctcgtgggcgagttcgtgtccgacgctctgctgg tgcccgacaagtgcaagttcctgcatcag gaacggatggacgtgtgcgagacacatctgcactggcacaccgtggccaaagagacatgc agcgagaagtccaccaacctgcacg actacggcatgctgctgccctgcggcatcgacaagttcagaggcgtggaattcgtgtgct gccccctggccgaggaatccgactctgt ggatagcgccgacgccgaagaggacgactctgacgtgtggtggggcggagccgacacaga ttacgctgatggcggcgaggacaa ggtggtggaagtggctgaagaggaagaggtggccgacgtggaagaagaagaggccgacga cgacgaggatgtggaagatggcg acgaggtggaagaggaagccgaggaaccctacgaggaagccaccgagagaaccaccagca ccgccaccacaaccaccaccact accgagagcgtggaagaggtcgtgcgggtgccaacaacagccgcctctacacctgacgcc gtggacaagtacctggaaaccccag gcgacgagaacgagcacgcccacttccagaaggctaaagagagactggaagctaagcacc gcgagagaatgagccaagtgatga gagagtgggaggaagctgagagacaggccaagaacctgcccaaggccgacaagaaagccg tgatccagcacttccaggaaaagg tggaaagcctggaacaggaagctgccaacgagagacagcagctggtggaaacccacatgg ccagagtggaagctatgctgaacga cagaagaaggctggccctggaaaactacatcaccgctctgcaggccgtgccccccagacc tcaccacgtgttcaacatgctgaagaa atacgtgcgggccgagcagaaggacagacagcacaccctgaagcacttcgagcacgtgcg gatggtggaccccaagaaggccgc ccagatcagatcccaagtgatgacccacctgagagtgatctacgagaggatgaaccagag cctgagcctgctgtacaacgtgcccgc cgtggccgaagaaatccaggatgaggtggacgagctgctgcagaaagagcagaactacag cgacgacgtgctggccaacatgatc agcgagcccagaatcagctacggcaacgacgccctgatgcccagcctgaccgagacaaag accaccgtggaactgctgcccgtga acggcgagttcagcctggatgacctgcagccctggcaccctttcggcgtggactctgtgc ctgccaacacagagaacgaagtggaac ccgtggacgccagacctgccgctgatagaggcctgaccacaagacctggcagcggcctga caaacatcaagaccgaagagatcag cgaagtgaagatggacgccgagttcgggcacgacagcggctttgaagtgcggcaccagaa atagaagcttatcgataatcaacctct ggattacaaaatttgtgaaagattgactggtattcttaactatgttgctccttttacgct atgtggatacgctgctttaatgcctttgtatcatgct attgcttcccgtatggctttcattttctcctccttgtataaatcctggttgctgtctctt tatgaggagttgtggcccgttgtcaggcaacgtggc gtggtgtgcactgtgtttgctgacgcaacccccactggttggggcattgccaccacctgt cagctcctttccgggactttcgctttccccct ccctattgccacggcggaactcatcgccgcctgccttgcccgctgctggacaggggctcg gctgttgggcactgacaattccgtggtgt tgtcggggaagctgacgtcctttccatggctgctcgcctgtgttgccacctggattctgc gcgggacgtccttctgctacgtcccttcggc cctcaatccagcggaccttccttcccgcggcctgctgccggctctgcggcctcttccgcg tcttcgccttcgccctcagacgagtcggat ctccctttgggccgcctccccgcctgatcgataccgtcgactagagctcgctgatcagcc tcgactgtgccttctagttgccagccatctg ttgtttgcccctcccccgtgccttccttgaccctggaaggtgccactcccactgtccttt cctaataaaatgaggaaattgcatcgcattgtc tgagtaggtgtcattctattctggggggtggggtggggcaggacagcaagggggaggatt gggaagacaatagcaggcatgctggg gagagatctgaggaacccctagtgatggagttggccactccctctctgcgcgctcgctcg ctcactgaggccgcccgggcaaagccc gggcgtcgggcgacctttggtcgcccggcctcagtgagcgagcgagcgcgcagagaggga gtggccaacccccccccccccccc c

SEQ ID N0:6: complete sequence of the expression cassette of the AAV transfer vector encoding codon-optimized human APPsa

Ggggggggggggggggggttggccactccctctctgcgcgctcgctcgctcactgaggcc gggcgaccaaaggtcgc ccgacgcccgggctttgcccgggcggcctcagtgagcgagcgagcgcgcagagagggagt ggccaactccatcactaggggttcc tagatctaggatcacgcgttctagaaatattaaggtacgggaggtacttggagcggccgc aataaaatatctttattttcattacatctgtgt gttggttttttgtgtgaatcgatagtactaacatacgctctccatcaaaacaaaacgaaa caaaacaaactagcaaaataggctgtcccca gtgcaagtgggttttaggaccaggatgaggcggggtgggggtgcctacctgacgaccgac cccgacccactggacaagcacccaa cccccattccccaaattgcgcatcccctatcagagagggggaggggaaacaggatgcggc gaggcgcgtgcgcactgccagcttca gcaccgcggacagtgccttcgcccccgcctggcggcgcgcgccaccgccgcctcagcact gaaggcgcgctgacgtcactcgccg gtcccccgcaaactccccttcccggccaccttggtcgcgtccgcgccgccgccggcccag ccggaccgcaccacgcgaggcgcga gataggggggcacgggcgcgaccatctgcgctgcggcgccggcgactcagcgctgcctca gtctgcggtgggcagcggaggagt cgtgtcgtgcctgagagcgcagtcgaattgctagcggggatccaccggtcgccacc

atgctgcctggactggctctgctgctgctggccgcctggacagccagagcctacccc tacgacgtgcccgactacgcctacccttatg atgtgcctgactatgcattggaggtgcccaccgacggcaacgccggactgctggccgagc cccagatcgccatgttctgcggcagac tgaacatgcacatgaacgtgcagaacggcaagtgggacagcgaccccagcggcaccaaga cctgcatcgacaccaaagagggcat cctgcagtattgccaagaagtgtaccccgagctgcagatcaccaacgtggtggaagccaa ccagcccgtgaccatccagaactggtg caagcggggcagaaagcagtgcaagacccacccccacttcgtgatcccttacagatgcct cgtgggcgagttcgtgtccgacgccct gctggtgcccgacaagtgcaagttcctgcatcaagaacggatggacgtgtgcgagacaca tctgcactggcacaccgtggccaaaga gacatgcagcgagaagtccaccaacctgcacgactacggcatgctgctgccctgcggcat cgacaagttccggggcgtggaattcgt gtgctgccccctggccgaggaatccgacaacgtggacagcgccgacgccgaagaggacga cagcgacgtgtggtggggcggag ccgacaccgattacgccgacggcagcgaggacaaggtggtggaagtggctgaagaggaag aggtggccgaggtcgaggaagag gaagccgacgacgacgaggatgacgaggacggcgacgaggtggaagaagaggccgaggaa ccctacgaggaagccaccgagc ggaccacctctatcgccaccaccaccacaaccactaccgagagcgtggaagaggtcgtgc gggtgccaaccaccgccgcctctacc cccgacgccgtggacaagtacctggaaacccctggcgacgagaacgagcacgcccacttc cagaaggccaaagagcggctggaa gccaagcaccgcgagcggatgagccaggtcatgagagagtgggaagaagccgagcggcag gccaagaacctgcccaaggccga caagaaagccgtgatccagcacttccaagaaaaggtcgagagcctggaacaagaagccgc caacgagcggcagcagctggtgga aacccacatggccagagtggaagccatgctgaacgaccggcggagactggccctggaaaa ctacatcaccgctctgcaggccgtgc cccccagaccccggcacgtgttcaacatgctgaagaaatacgtgcgggccgagcagaagg accggcagcacaccctgaagcactt cgagcacgtgcggatggtggaccccaagaaggccgcccagatccgctctcaggtcatgac ccacctgagagtgatctacgagagaa tgaaccagagcctgagcctgctgtacaacgtgcccgccgtggccgaagaaatccaggatg aggtggacgagctgctgcagaaaga gcagaactacagcgacgacgtgctggccaacatgatcagcgagccccggatcagctacgg caacgacgccctgatgcccagcctg accgagacaaagaccaccgtggaactgctgcccgtgaacggcgagttcagcctggacgac ctgcagccctggcacagcttcggcg ctgatagcgtgcccgccaacaccgagaatgaggtggaacccgtggacgccagacctgccg ccgatagaggcctgaccacaagacc tggcagcggcctgaccaacatcaagaccgaagagatcagcgaagtgaagatggacgccga gttccggcacgacagcggctacga ggtgcaccaccagaaatagaagcttatcgataatcaacctctggattacaaaatttgtga aagattgactggtattcttaactatgttgctcc ttttacgctatgtggatacgctgctttaatgcctttgtatcatgctattgcttcccgtat ggctttcattttctcctccttgtataaatcctggttgct gtctctttatgaggagttgtggcccgttgtcaggcaacgtggcgtggtgtgcactgtgtt tgctgacgcaacccccactggttggggcatt gccaccacctgtcagctcctttccgggactttcgctttccccctccctattgccacggcg gaactcatcgccgcctgccttgcccgctgct ggacaggggctcggctgttgggcactgacaattccgtggtgttgtcggggaagctgacgt cctttccatggctgctcgcctgtgttgcca cctggattctgcgcgggacgtccttctgctacgtcccttcggccctcaatccagcggacc ttccttcccgcggcctgctgccggctctgc ggcctcttccgcgtcttcgccttcgccctcagacgagtcggatctccctttgggccgcct ccccgcctgatcgataccgtcgactagagc tcgctgatcagcctcgactgtgccttctagttgccagccatctgttgtttgcccctcccc cgtgccttccttgaccctggaaggtgccactc ccactgtcctttcctaataaaatgaggaaattgcatcgcattgtctgagtaggtgtcatt ctattctggggggtggggtggggcaggacag caagggggaggattgggaagacaatagcaggcatgctggggagagatctgaggaacccct agtgatggagttggccactccctctct gcgcgctcgctcgctcactgaggccgcccgggcaaagcccgggcgtcgggcgacctttgg tcgcccggcctcagtgagcgagcga gcgcgcagagagggagtggccaaccccccccccccccccc By a "therapeutically effective amount" of the vector of the present invention is meant a sufficient amount of the vector for the treatment of Down syndrome and Alzheimer's disease. It will be understood, however, that the total daily usage of the vector of the present invention will be decided by the attending physician within the scope of sound medical judgment. The specific therapeutically effective dose level for any particular subject will depend upon a variety of factors including the disorder being treated and the severity of the disorder; activity of the specific compound employed; the specific composition employed, the age, body weight, general health, sex and diet of the subject; the time of administration, route of administration, and rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination or coincidental with the specific vector employed; and like factors well known in the medical arts. For example, it is well within the skill of the art to start doses of the compound at levels lower than those required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved. Typically, from 10 ⁸ to 10 ¹⁰ viral genomes (vg) are administered per dose in mice. Typically, the doses of AAV vectors to be administered in humans may range from 10 ¹⁰ to 10 ¹² vg.

Typically, the vector or the cell of the present invention are delivered directly and specifically into selected brain regions by intracerebral injections into the cerebellum, the dentate nucleus, the striatum, the cortex and particularly the entorhinal cortex, or the hippocampus. In some embodiments, the vector of the present invention or the cells transduced with the vector are delivered by intrathecal delivery. In some embodiments, the vector of the present invention of the cells are delivered into the brain by intracerebral injection and/ blood by intravenously injection, in the spinal fluid by intrathecal delivery, by or intracerebro ventricular injection or by intra-nasal injection. Particularly, any routes of administration that allow a strong expression of the vector in the spinal cord, brain, cortex, hippocampus, and dentate nucleus can be used in the invention. In some embodiments, the cells are delivered by infusion in the peripheral blood (intravenous or intra-arterial injection) or in the CSF. In some embodiments, the vector of the present invention is administrated to the subject in need thereof one time, two times, three times or more. In some embodiments, the vector of the present invention is administrated to the subject in need thereof one time and re- administered several months or years later to said subject. The 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, exosomes and liposomes.

In another aspect, the present invention relates to a method of treating Alzheimer's disease in a subject in need thereof comprising administering to the subject a therapeutically effective amount of cells transduced with a vector which comprises a nucleic acid molecule encoding for a polypeptide which is a soluble member of the APP (amyloid precursor protein) family.

In one embodiment, the cells administrated according to the invention are autologous hematopoietic stem cell or hematopoietic progenitors that could be isolated from the patient, transduced with a vector, particularly a lentiviral vector and reinfused directly or after bone marrow conditioning.

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.

FIGURES:

Figure 1: APPsa overexpression enhances Morris water maze performance in WT mice and rescues the spatial memory deficit of APP/PS1AE9 mice. Transgenic APP/PS1AE9 mice (n=8 per group) or littermate (LM) controls (n=3-4 per group) were either injected with AAV- Venus or AAV-APPsa vectors at 12 months of age and tested 2 months later at 14 months of age. (A) Escape latency and (B) swim speed of littermate controls or APP/PS1AE9 mice injected either with AAV- Venus or AAV-APPsa. Swim speed was similar between the different groups (2-way ANOVA: Group effect: F3,ioo=2.40; ns; Time effect: F ₄ , ₁₀₀ =1.41; ns; Group x Time interaction: F ₁₂ , ₁₀₀ <1; ns). Littermates injected with AAV- APPsa showed improved performance, as indicated by reduced escape latency (2-way ANOVA: Time effect: F ₄ ,ioo=7.138; p<0.0001; Group effect: p=0.0002, followed by Tukey post-hoc test: APP/PS1AE9 mice injected with AAV-APPsa versus each of the other groups, p<0.013). (C) Probe trial performance at 72h. 2-way ANOVA, Group effect: F3,i7=3.356; p<0.04; quadrant effect: F _lll7 =23.54; p<0.007; Group x quadrant interaction effect: F3,i7=3.356; p<0.04. APP/PS1AE9 mice injected with AAV-Venus were impaired in comparison to littermate mice injected with AAV-Venus (Tukey post-hoc test: p=0.023) confirmed by no preference for the trained target quadrant. Strikingly, AAV- APPsa treated APP/PS1AE9 mice spent more time in the target quadrant compared to APP/PS1AE9 mice injected with AAV-Venus (Tukey post-hoc test: p=0.017). Statistics: 2-way ANOVA (genotype and group as factors) with repeated measures followed by Tukey post-hoc test: *< 0.05. Values represent means ±SEM. Figure 2: APP/PS1AE9 mice reveal structural and functional synaptic impairments that are ameliorated by APPsa expression. (A) LTP was induced by TBS at hippocampal CA3-CA1 synapses after 20 min baseline recordings. Acute slices of AAV- Venus injected APP/PS1AE9 animals exhibited significant lower induction and maintenance of LTP compared to littermate controls (LM) showing similar expression of Venus (averaged potentiation minutes t50-80: 148.47 ± 6.04% vs. 178.01 ± 8.98%, p= 0.021). Viral expression of APPsa restored potentiation after TBS (171.48 ± 6.29%) in transgenic animals and resulted in an LTP curve progression comparable to that of LM controls. The LTP induction rate is shown as percentage % of mean baseline slope, n = number of slices, N = number of mice. (B, C) Input-Output strength of all AAV-injected mice showed no alterations between genotypes at any fiber volley (FV) amplitude or stimulus intensity tested. (D) Altered PPF at the 10 ms ISI revealed a significant impairment in the pre-synapse of APP/PS1AE9 mice injected with AAV-Venus in comparison to LM controls (*p= 0.030) that was restored after AAV-APPsa injection (#p= 0.047). (E) No differences in spine density at CA1 apical neurons between groups, but significantly less spines at basal dendrites of APP/PS1AE9 Venus injected mice (p=0.040). APPsa overexpression partially restored the spine density deficit. (F) Reduced spine density at CA3 apical (p=0.014) and basal (p=0.011) dendritic segments of APP/PS1AE9 AAV-Venus injected mice that is partially rescued at apical and completely at basal dendrites (p= 0.039) by APPsa. N = number of neurons, N = number of animals. Data represent mean ± SEM and were analyzed by one-way ANOVA followed by Bonferroni ^' s post-hoc test.

Figure 3: AAV-APPsa injection decreases Αβ and plaques load. (A) ELISA quantification of β-CTF in hippocampus (H) and cortex (Cx) of APP/PS1AE9 mice. No difference was detectable between AAV-Venus and AAV-APPsa injected animals. (B-D) Quantification (MSD immunoassay) of TBS soluble Αβ38 (Group effect: F _lll4 =3.879, p=0.07 (-36%), Αβ40 (Group effect: Fi,i ₄ =3.094, p=0.10 (-32%)) and Αβ42 (Group effect: Fi, i4=5.211, p=0.04 (-33%); region effect: F _lll4 <l, ns; Group x region interaction effect: Fi,i4<l, ns) in hippocampus and cortex of APP/PS1AE9 mice. Note that AAV-APPsa injected animals show reduced levels of Αβ in both anatomical regions analyzed (hippocampus and cortex). (E) Quantification of 4G8 immunolabeled area in hippocampus and cortex (2-way ANOVA: Treatment effect: F ₃ =5.50, p=0.04 (-24%); region effect: F ₃ =22.89, p=0.0004; Treatment x region interaction effect: Fi,i ₃ <l, ns). Note that 4G8 immunoreactive plaque area is significantly reduced in AAV-APPsa treated animals. Number of animals n=8/group. 2- way ANOVA (Genotype, treatment and region as factors) followed by Tukey post-hoc test: *p < 0.05. Values represent means ±SEM.

Figure 4: AAV-APPsa promotes microglia recruitment around plaques in APP/PS1AE9 mice. (A) Quantification signal intensity from western blot analysis showing the expression of GFAP and Ibal in the hippocampus of AAV- Venus or AAV-APPsa injected APP/PS1AE9 mice (n=8/group). Quantification signal intensities were normalized to GAPDH used as a loading control. AAV-APPsa treatment specifically increased Ibal (microglial marker) expression (t-test: ti4=3.586; p=0.003), whereas the astrocyte marker GFAP was not affected. (B) Whereas the distribution of GFAP positive astrocytes is unaltered, increased recruitment of Ibal positive microglia is observed in the vicinity of amyloid plaques (t-test: t ₁₆ =5.441; p<0.0001). (C) Western blot analysis showing the expression of IDE and TREM2 in the hippocampus of AAV- Venus or AAV-APPsa injected APP/PS1AE9 mice. Both Αβ clearance related proteins are significantly upregulated (IDE: t- test: ti4=3.984; p=0.0014; TREM2: t-test: ti ₄ =2.947; p=0.010) following AAV-APPsa treatment. Values represent means ±SEM. ***P < 0.001, **P < 0.01, *P < 0.05.

Figure 5: Unlike APPsa, APPsp overexpression does not rescue spatial memory deficit of APP/PS1AE9 mice in the Morris water maze. Littermate (LM) controls injected with Venus (n = 3) or transgenic APP/PS1AE9 mice (n = 7-8 per group) either injected with AAV- Venus, AAV-APPsa or ΑΑν-ΑΡΡββ vectors at 12 months of age were tested 2 months later at 14 months of age. Graphic showing the probe trial performance at 72 h. 2-way ANOVA, group quadrant interaction effect: F3,19 = 4.04; p = 0.02. APP/PS1AE9 mice injected with AAV- Venus were impaired in comparison to littermate mice injected with AAV- Venus (Tukey post hoc test: p = 0.04) confirmed by no preference for the trained target quadrant. Unlike AAV-APPsa treatment in APP/PS1AE9 mice which restored time spent in the target quadrant (Tukey post hoc test: p = 0.011), AAV-APPsP did not improve their performances compared to APP/PS1AE9 mice injected with AAV- Venus (Tukey post hoc test: p > 0.99). Data represent mean ± SEM and were analyzed by 2-way ANOVA (genotype and group as factors) with repeated measures followed by Tukey post hoc test. *p < 0.05.

Figure 6: AAV-APPsp injection do not restore long term potentiation in the hippocampus of aged APP/PS1AE9 mice. LTP was induced by TBS at hippocampal CA3- CA1 synapses after 20 min baseline recordings. Acute slices of AAV-Venus injected APP/PS1AE9 animals exhibited significant lower induction and maintenance of LTP compared to littermate controls (LM) indicating a significant impairment of the transgenic mice. Viral expression of APPsa restored potentiation after TBS in transgenic animals and resulted in an LTP comparable to that of LM controls. However, AAV-APPsP injection did not restore APP/PS1AE9 mice which show a similar level compared to AAV-Venus transgenic mice. The LTP induction rate is shown as percentage % of mean baseline slope, n = number of slices, N = number of mice.

Figure 7: AAV-APPsp injection does not activate microglia. A Western blot analysis showing the expression of IBA1 (microglial marker) in the hippocampus of AAV- Venus, AAV APPsa or AAV-APPsP injected APP/PS1AE9 mice (n = 7/8 per group). For quantification signal intensity was normalized to GAPDH used as a loading control (One-way ANOVA: group effect: F2,19 = 14.38, p = 0.0002). AAV-APPsa treatment specifically increased IBA1 expression (p = 0.0002) whereas AAV-APPsP did not (p = 0.82). B Quantification of IBA1 signal in the hippocampus following immunohistochemistry in APP/PS 1ΔΕ9 mice injected either with AAV-Venus, AAV-APPsa or AAV-APPsP (One-way ANOVA: group effect: F2,41 = 14.12, p < 0.0001). As seen in western blot, AAV-APPsP injection is unable to rise IBA1 levels compared to AAV-Venus treated mice. Values represent mean ± SEM. ***p < 0.001, **p < 0.01.

EXAMPLE:

Material & Methods AAV plasmid design and vector production

The mouse APPsa coding sequence (derived from Uniprot: PI 2023-2) was codon optimized (Geneart, Regensburg) and then cloned under control of the synapsin promoter into the single stranded, rAAV2-based transfer vector pAAVSynMCS-2A-Venus (Tang et al, 2009) via Nhel-Hindlll restriction sites. For easy detection, an N-terminal double HA-tag was inserted downstream of the APP signal peptide at the N-terminus of APPsa. The control vector (pAAV- Venus) encodes the yellow fluorescent protein Venus fused to a C-terminal farnesylation signal for membrane anchoring. All constructs were packaged into AAV9 by the MIRCen viral production platform as described (Berger et al, 2015).

Animals

Sixteen APPswe/PSlAE9 mice (referred as APP/PS1AE9; Jackson Laboratories) and seven age-matched littermate control mice were used for behavior, pathology and biochemistry. Eleven APP/PS1AE9 and five littermates were used for electrophysiology and spine density analysis. APP/PS1AE9 mice express the human APP gene carrying the Swedish double mutation (K595N/M596L). In addition, they express the human PS1AE9 variant lacking exon 9 (Borchelt et al, 1997; Jankowsky et al, 2004; Xiong et al, 2011). Only male mice were used throughout the study. For age at AAV injection and age at analysis/sacrifice see results section. All experiments were conducted in accordance with the ethical standards of French, German and European regulations (European Communities Council Directive of 24 November 1986).

Stereotactic injection of AAV

Mice were anesthetized by intraperitoneal injection of ketamine/xylazine (0.1/0.05 g/kg body weight) and positioned on a stereotactic frame (Stoelting, Wood Dale, USA). Vectors (either AAV- Venus or AAV- APPsa) were bilaterally injected into the hippocampus using 2 μΐ of viral preparation (10 ¹⁰ vg/site) at a rate of 0.2 μΐ/minute. Two injections sites per hippocampus were used to optimize virus spreading. Stereotactic coordinates of injection sites from bregma were: anteroposterior -2 mm; medio lateral +/-1 mm; dorsoventral -2.25 mm and anteroposterior -2mm; medio lateral +/-lmm; dorsoventral - 1.75mm.

Brain samples APP/PS1AE9 mice were sacrificed 5 months post-injection at 17 months of age. Following anesthesia, mice were transcardially perfused with 0.1 M phosphate buffered saline (PBS) before dissection. For immunohistochemistry, the left cerebral hemisphere was dissected and post-fixed in 4% paraformaldehyde (PFA) for 1 week and cryoprotected in 30% sucrose for 24 hours. 40 μιη sections were cut using a freezing microtome (Leica, Wetzlar, Germany), collected in a cryoprotective solution and stored at -20°C. The right hemisphere was dissected to segregate hippocampus and cortex for biochemical analysis. Samples were then homogenized in lysis buffer (TBS, NaCl 150mM and Triton 1%) containing phosphatase and protease inhibitors. After centrifugation (20 min, 13 000 rpm, 4°C), the supernatant was collected and the protein concentration was quantified by BCA Assay (Thermo Fisher Scientific, Waltham, USA). Lysate aliquots (3 mg of protein/ml) were stored at -80°C.

Immunostaining

Slices were washed with 0.1 M PBS and permeabilized in 0.25% PBS-Triton before blocking in PBS-Triton 0.25% containing 5% goat serum for 60 minutes. For vector encoded HA-APPsa immuno labeling, slices were incubated with an anti-FJA antibody (Covance, Princeton, USA, 1/250) overnight at 4°C. After successive washes (PBS-Triton 0.25%, PBS and PB 0.1 M), incubation with a biotinylated anti-mouse antibody was performed for one hour at room temperature. For signal amplification, samples were incubated using the ABC kit (Vector laboratories, Burlingame, USA) for one hour at room temperature. Finally, slices were incubated in Cy3-coupled streptavidine. HA-APPsa was co-immunostained overnight with the following primary antibodies: Rabbit anti-Ibal, 1/500, Wako, Richmond, USA; Mouse-GFAP Cy3 conjugate, 1/500, Sigma-Aldrich, Saint-Louis, USA. For immunofluorescent staining of plaques, slices were stained using a 30 min incubation in 1% thioflavin-S solution, rinsed twice (1 min each) in 50% EtOH and mounted in Vectashield fluorescent mounting media (Vector laboratories). Images were taken with a Nikon Eclipse Tz microscope (Nikon, Tokyo, Japan) and a Leica SP8 confocal microscope (Leica). For plaque quantification, slices were incubated in 88% formic acid solution for 15 min (antigen retrieval). To inactivate endogenous peroxidase, samples were incubated in hydrogen peroxide (30 min) before blocking and incubation with the primary antibody (4G8, Covance, 1/1000). Incubation with a horseradish coupled secondary antibody was done at RT, developed using the DAB kit (Vector laboratories) and mounted in Eukitt mounting media (Sigma-Aldrich, Saint-Louis, USA). Images were taken with a Z6 APO macroscope (Leica). Plaques, GFAP and Ibal immunoreactivity were quantified using I mage J (NIH, Bethesda, USA) or Icy (Institut Pasteur, Paris, France). Laserpower, numeric gain and magnification were kept constant between animals to avoid potential technical artefacts. Images were first converted to 8-bit gray scale and binary thresholded to highlight a positive staining. At least 2 sections per mouse (between -1.7 mm to -2.3 mm caudal to bregma) were quantified for either hippocampus or cortex. The average value per structure was calculated for each mouse. For quantification of Ibal and GFAP immunoreactivity around plaques, a region of interest (ROI) was drawn around the center of the plaque. The diameter of the circular ROI was set as 3 times the diameter of the plaque. Mean fluorescence intensity values were measured for either Ibal or GFAP immunoreactivity and were processed via Icy software (Institut Pasteur, Paris, France). Experimentators and data managers were blind with respect to treatments and genotypes.

Western blot analysis

Proteins were separated by electrophoresis using 4-12% SDS-PAGE (NuPAGE, Life Technologies, Carlsbad, USA) in MOPS buffer (NuPAGE, Life Technologies) and transferred to nitrocellulose membranes (iBlot, Life Technologies). After blocking in 5% milk-PBS 0.1M for 60 minutes, membranes were incubated with the primary antibodies overnight at 4°C (HA, 1/2000, Covance, Princeton, USA; Venus (GFP), l/1000,Vector laboratories Burlingame, USA; GAPDH, 1/4000 Abeam, Cambridge, UK; Ibal, 1/2000, Wako, Richmond, USA; GFAP, 1/4000 Dako, Glostrup, Denmark; IDE, 1/200, Santa Cruz Biotechnology, Dallas, USA; TREM2, 1/500, R&D Systems, Minneapolis, USA). Membranes were then washed with TBS-T (with 0.1% Tween), incubated with a horseradish peroxidase coupled secondary antibody and developed using enhanced chemiluminescence (ECL, GE Healthcare, Little Chalfont, UK and Super Signal, Thermo Fisher Scienfitic). Signals were detected with Fusion FX7 (Vilber Lourmat, Marne-la-Vallee, France) and analyzed and quantified using Image J.

ELISA

APPsa, β-CTF, and Αβ were quantified using the sAPPa kit (Meso Scale Discovery, Rockville, USA), Human APP β-CTF Assay Kit (IBL, Hamburg, Germany), V-PLEX Plus Αβ Peptide Panel 1 (6E10) Kit (Meso Scale Discovery). The procedures were performed according to the respective supplier instructions.

Morris Water Maze Experiments were performed in a 120-cm diameter, 50 cm deep tank filled with opacified water kept at 21°C and equipped with a 10 cm diameter platform submerged 1 cm under the water surface. Visual clues were disposed around the pool as spatial landmarks for the mouse and luminosity was kept at 430 lux. Training consisted of daily sessions (three trials per session) during 5 consecutive days. Start positions varied pseudo-randomly among the four cardinal points. Mean inter-trial interval was 15 min. Each trial ended when the animal reached the platform. A 60 second cut-off was used, after which mice were gently guided to the platform. Once on the platform, animals were given a 30-second rest before being returned to their cage. 72 hours after the last training trial (day 8), retention was assessed during probe trial in which the platform was no longer present. Animals were video tracked using Ethovision software (Noldus, Wageningen, Netherlands) and behavioral parameters (swim speed, travelled distance, latency, percentage of time spend in each quadrant) were automatically calculated. Experiments and statistical evaluation of data were performed by an experimentator blind to genotype and treatment group.

Statistics

Statistical analyses were performed as indicated for the respective experiments. Outliers were detected and rejected using maximum normed residual test (Grubbs' test). In most cases, data were analyzed using non-parametric Mann- Whitney U tests excepted for behavioral experiments. Two-way ANOVA with repeated measures were carried out when required by the experimental plan to assess statistical effects. Correlation matrices were generated using non-parametric Spearman rank correlation coefficient. For all analysis statistical significance was set to a p-value <0.05. All analyses were performed using Statistica (StatSoft Inc., Tulsa, USA) or Prism (GraphPad Software, La Jolla, USA).

Electrophysiology

In vitro extracellular recordings were performed on acute hippocampal slices of WT littermates stereotactically injected with the AAV- Venus (N= 5), APP/PS1AE9 mice injected either with AAV- Venus (N= 4) or AAV-APPsa virus (N= 6) at 8 months of age. Electrophysiological recordings were performed 4-5 months later at an age of 12-13 months. In-between animals were housed in a temperature- and humidity-controlled room with a 12h light-dark cycle and had access to food and water ad libitum.

Slice preparation Acute hippocampal transversal slices were prepared from individuals at an age of 12 to 13 months. Mice were anesthetized with isoflurane and decapitated. The brain was removed and quickly transferred into ice-cold carbogenated (95% 0 ₂ , 5% CO2) artificial cerebrospinal fluid (ACSF) containing 125 mM NaCl, 2 mM KC1, 1.25 mM NaH ₂ P0 ₄ , 2 mM MgCl ₂ , 26 mM NaHC0 ₃ , 25 mM glucose. After dissection of the two hemispheres one was used for Golgi-Cox staining and the other for electrophysiology. The hippocampus was sectioned into 400 μιη thick transversal slices with a vibrating microtome (Leica, VT1200S). Slices were maintained in carbogenated ACSF (125 mM NaCl, 2.mM KC1, 1.25 mM NaH ₂ P0 ₄ , 2 mM MgCl ₂ , 26 mM NaHCOs, 2 mM CaCl ₂ , 25 mM glucose) at room temperature for at least 1.5 h before transferred into a submerged recording chamber. Before recording, each slice of the AAV- Venus injected animals was proofed for fluorescence expression of Venus in area CA1 and CA3 (Zeiss, Axiovert 35). Slices absent of the fluorescence protein in the recording areas were excluded from further analysis. Extracellular field recordings

Slices were placed in a submerged recording chamber and perfused with carbogenated ACSF (32°C; 125 mM NaCl, 2 mM KC1, 1.25 mM NaH ₂ P0 ₄ , 1 mM MgCl ₂ , 26 mM NaHC03, 2 mM CaCl ₂ , 25 mM glucose) at a rate of 1.2 to 1.5 ml/min. Field excitatory postsynaptic potentials (fEPSPs) were recorded in stratum radiatum of CA1 region with a borosilicate glass micropipette (resistance 2-4 ΜΩ) filled with 3 M NaCl at a depth of -150- 200 μιη. Monopolar tungsten electrodes were used for stimulating the Schaffer collaterals at a frequency of 0.1 Hz. Stimulation intensity was adjusted to 40% of maximum fEPSP slope for 20 minutes baseline recording. LTP was induced by applying theta-burst stimulation (TBS: 10 trains of 4 pulses at 100 Hz in an 200 ms interval, repeated 3 times). Basal synaptic transmission properties were analyzed via input-output-(IO) measurements and short-term plasticity was examined via paired pulse facilitation (PPF). The IO- measurements were performed either by application of a defined current values (25 - 175 μΑ) or by adjusting the stimulus intensity to certain fiber volley (FV) amplitudes (0.1 - 0.7 mV). PPF was performed by applying a pair of two closely spaced stimuli in different inter-stimulus-intervals (ISI) ranging from 10 to 160 ms.

Dendrite and spine analysis

Golgi-Cox staining Golgi staining was done using the FD Rapid GolgiStain Kit according to the manufacturer's instructions. All procedures were performed under dark conditions. One hemisphere of each mouse was used for electrophysiology and the other one for Golgi-Cox staining. Hemispheres were immersed in 2 ml mixtures of equal parts of kit solutions A and B and stored at RT for 2 weeks. Afterwards brain tissues were stored in solution C at 4°C for at least 48 h and up to 7 days before sectioning. Solutions AB and C were renewed within the first 24 h. Coronal sections of 200 μιη were cut with a vibrating microtome (Leica, VT1200S) while embedded in 2% Agar in 0.1 M PBS. Each section was mounted with Solution C on an adhesive microscope slide pre-coated with 1% gelatin/0.1% chromalaun on both sides and stained according to the manufacturer ^' s protocol with the exception that AppliClear (AppliChem) was used instead of xylene. Finally slices were cover-slipped with Permount (Fisher Scientific).

Imaging and analysis of spine density in Golgi-Cox stained slices

Imaging of 2 ^nd or 3 ^rd order dendritic branches of hippocampal pyramidal neurons of area CA3 and CA1 was done with an Axioplan 2 imaging microscope (Zeiss) using a 63x oil objective and a z-stack thickness of 0.5 μιη under reflected light. The number of spines was determined per micrometer of dendritic length (in total 100 μιη) at apical and basal compartments using ImageJ (1.48v, National Instruments of Health, USA). At minimum 4 animals per genotype and 4 neurons per animal were analyzed blinded to genotype and injected virus.

Data analysis

Data of electrophysiological recordings were collected, stored and analyzed with LABVIEW software (National Instruments, Austin, TX). The initial slope of fEPSPs elicited by stimulation of the Schaffer collaterals was measured over time, normalized to baseline and plotted as average ± SEM. Analysis of the PPF data was performed by calculating the ratio of the slope of the second fEPSP divided by the slope of the first one and multiplied by 100. Data of Golgi-Cox staining were analyzed using GraphPad Prism (Version, 5.01) software. Spine density is expressed as mean ± SEM. Differences between genotypes were detected with one-way analysis of variance (ANOVA) followed by Bonferroni ^' s post hoc test using IBM SPSS Statistics 21.

Results AAV- APPsa injection mediates efficient and long lasting neuronal expression of APPsa in the hippocampus of APP/PS1AE9 mice

To assess the therapeutic potential of APPsa we used AAV -mediated overexpression of APPsa in the brain of aged (12 month-old) APP/PS1AE9 mice. APP/PS1AE9 mice show progressive plaque deposition starting at about 5-6 months of age and highly abundant plaques are observed at 12 months of age (Jankowsky et al, 2004; Xiong et al, 2011). AAV9 vectors expressing either Venus or codon optimized HA-tagged murine APPsa (HA-APPsa) under the control of the neuronal synapsin promoter (further referred to as AAV- Venus and AAV- APPsa, data not shown) were bilaterally injected into the stratum lacunosum moleculare region of the dorsal hippocampus and into the dentate gyrus (data not shown) of 12 month-old male APP/PSAE9 mice.

To monitor vector-mediated Venus and APPsa expression, mice were sacrificed 5 months after injection. Immunohistochemistry using an HA-tag specific antibody revealed widespread expression of HA-APPsa not only in the hippocampus, but also in the cortical layers above the injected hippocampus (data not shown). Analysis of serial anteroposterior coronal sections demonstrated widespread HA-APPsa immunoreactivity (over 3.5 mm) in the hippocampus from -2.6 mm posterior to +0.9 mm anterior from the injection site (data not shown) and in the adjacent cortex. More detailed analysis showed prominent expression of vector-mediated HA-APPsa in the pyramidal cells of the subiculum, in the CA1, CA2 regions and in granular neurons of dentate gyrus (data not shown). Within the CA3 sub field HA- APPsa expression was detectable but considerably lower. As APPsa expression was driven by the neuron-specific synapsin promotor, HA-APPsa expression was restricted to neuronal cells as revealed by double immunostaining against NeuN (data not shown). Consistently, no expression was detectable in microglia (Ibal, data not shown) or in astrocytes (GFAP, data not shown). The AAV -Venus expression pattern was largely similar to that of AAV -APPsa.

Western blot analysis of hippocampal extracts confirmed vector-mediated HA-APPsa protein expression in all injected animals. Comparable levels of either HA-APPsa or Venus were detected in injected APP/PS1AE9 mice or nontransgenic littermates, respectively (data not shown). Altogether we demonstrate that our AAV based approach leads to efficient and long lasting APPsa expression in the hippocampus and adjacent cortex.

AAV-APPsa treatment rescues the spatial memory impairment of APP/PS1AE9 mice To analyze the consequences of AAV-APPsa or AAV- Venus injection for spatial learning and memory, mice were tested in the Morris water maze place navigation task (Fig. 1). To this end, transgenic APP/PS1AE9 mice (n=8 per group) or nontransgenic littermate controls (n=3-4 per group) were either injected with AAV -Venus or AAV-APPsa vectors at 12 months of age and tested 2 months later at 14 months of age. Swim speed was comparable in all groups of animals (Fig. 1A) over the 5 days of training, thus excluding impairments in motor performances. While all 4 groups of mice did show learning, as evidenced by reduced latency to reach the platform over the 5 days of training, we observed a group effect resulting from an overall significantly increased performance in nontransgenic littermates that had received AAV-APPsa (Fig. IB). Injection of AAV-APPsa did not, however, improve the performance of APP/PS1AE9 mice (Fig. IB). Similar results were obtained when analyzing the path length to reach the platform (data not shown). During the probe trial that assesses spatial reference memory and was conducted 72 hours after the last trial of training APP/PS1AE9 mice injected with AAV-Venus were strongly impaired (Fig. 1C) in comparison to littermate mice injected with AAV-Venus and showed no preference for the trained target quadrant (Fig. 1C; paired t-test: ί ₇ =0.96; p=0.37). Strikingly, APP/PS1AE9 mice that had been injected with AAV-APPsa showed a clear preference for the trained target quadrant (Fig. 1C; paired t-test: t ₇ =2.516; p=0.045), that was statistically indistinguishable from the performance of littermate controls (Fig 1C; p> 0.84, 2-way ANOVA followed by Tukey's post-hoc test). Thus, vector mediated APPsa expression rescued the spatial memory impairment in aged APP/PS1AE9 mice despite established plaque deposition.

Impaired synaptic plasticity and reduced spine density of APP/PS1 ΔΕ9 mice are rescued by AAV-APPsa expression

Having established that AAV-APPsa expression restored the spatial memory deficits of APP/PS1AE9 mice we evaluated whether these improvements were also reflected at the functional neuronal network level. We analyzed synaptic plasticity which is considered to represent the basis of newly formed declarative memory, 4-5 months after AAV injection at an age of 12-13 months. To this end, we induced long term potentiation (LTP) at the Schaffer collateral to CA1 pathway by theta-burst stimulation (TBS) after baseline recording (data not shown). Consistent with our previous results in noninjected APP/PS1AE9 mice (Heneka et al, 2013) AAV-Venus injected APP/PS1AE9 mice exhibited significantly lower induction and maintenance of LTP (n= 22 slices), as compared to AAV-Venus injected littermate controls (n=22, data not shown). Nontransgenic control slices showed at the stable phase of LTP (t50- 80 min after TBS) a potentiation of 178.01 ± 8.98%, that was significantly reduced to only 148.47 ± 6.04% in AAV-Venus injected APP/PS1AE9 mice (Fig. 2A; p=0.021, 1-way ANOVA followed by Bonferroni ^' s post-hoc test). In contrast, the LTP curve recorded from AAV-APPsa injected APP/PS1AE9 slices (n=26) closely overlapped with and was statistically indistinguishable (1-Way ANOVA for t50-80, p>l) from that of nontransgenic littermate controls (data not shown). AAV mediated expression of APPsa largely ameliorated LTP deficits of APP/PS1AE9 mice as evidenced by nearly identical average potentiation at t50-80 in AAV-APPsa treated APP/PS1AE9 mice (171.48 ± 6.29%) and littermate controls (178.01 ± 8.98%o) receiving AAV-Venus control virus (Fig. 2A). While basal synaptic transmission was comparable in all groups (Fig. 2B and C), short-term synaptic plasticity evaluated by paired pulse facilitation (PPF, Fig. 2D) was significantly impaired in APP/PS1AE9 mice. Transgenic animals injected with AAV-Venus showed an overall lowered response towards the second stimulus in the PPF paradigm, reaching significance at an inter- stimulus interval (ISI) of 10 ms compared to littermate controls (p= 0.03; 1-way ANOVA followed by Bonferroni ^' s post-hoc test). Strikingly, AAV-APPsa treatment completely rescued presynaptic functionality in APP/PS1AE9 animals, as evidenced by PPF values statistically indistinguishable from littermate controls and significantly different from that of AAV-Venus injected transgenic animals 0.047; Fig. 2D).

Next we evaluated spine density as a correlate of excitatory synapses in the same set of animals as used for electrophysiology. Previous studies had indicated reduced spine density in various AD mouse models, presumably due to Αβ mediated toxic effects (reviewed in (Spires- Jones & Knafo, 2012). Spine density of basal and mid-apical dendritic segments of hippocampal CA1 and CA3 pyramidal cells was assessed using Golgi staining (data not shown). Apical dendrites of CA1 neurons showed comparable spine density between experimental groups, whereas significantly reduced spine density was observed in the basal dendrites of CA1 neurons from APP/PS1AE9 mice (n=16 neurons) as compared to littermates controls (n=24 neurons, both treated with AAV-Venus, Fig 2E). Analysis of CA3 neurons revealed significantly fewer spines in both basal (t-test, p=0.01) and apical (p=0.014) dendritic segments when comparing AAV-Venus expressing APP/PS1AE9 mice and nontransgenic littermates controls. Importantly, AAV-APPsa overexpression partially restored spine density in CA3 apical segments (n=24) and completely rescued the spine density deficit in basal dendrites of CA3 neurons from APP/PS1AE9 mice (p=0.031; Fig. 2F). Together, these data indicate that APPsa expression substantially ameliorates both structural and functional synaptic impairments of aged AD model mice. AAV-APPsa expression decreases Αβ levels and plaque deposition in aged APP/PSldE9 mice

APPsa had previously been reported to bind to BACE-1 and thereby reduce Αβ production (Obregon et al, 2012). We therefore evaluated if beneficial effects of AAV-APPsa overexpression on synaptic plasticity and cognitive function were associated with reduced amyloidogenic processing of APP. Employing a sensitive electrochemiluminescence ELISA we quantified products of amyloidogenic metabolism (Αβ and β-CTF) in the cortex (Cx) and hippocampus (H) of 17 months old APP/PS1AE9 mice (n=8/group), 5 months after viral vector injection. No significant difference in β-CTF levels were detectable in APP/PS1AE9 mice injected with AAV-APPsa vector, as compared to mice injected with AAV- Venus control vector (Fig. 3A). In contrast, we observed a significant decrease in soluble Αβ42 (reduced by about 33% vs control, Fig 3D) in both cortex and hippocampus of APP/PS1AE9 mice injected with AAV-APPsa vector, as compared to AAV- Venus control injections. Similarly, we found a trend towards decreased amounts of Αβ38 and Αβ40 that did, however, not reach statistical significance (Fig. 3B, C).

In order to assess the impact of APPsa overexpression on amyloid deposition, we used 4G8 immuno staining to quantify the area covered by plaques both in the hippocampus and cortex of 17 months old APP/PS1AE9 mice injected with viral vectors (data not shown). Interestingly, AAV-APPsa injection (n=8) resulted in a significantly reduced plaque area both in cortex and hippocampus as compared to AAV- Venus injected controls (Fig. 3E). Together, these results indicate that AAV-mediated APPsa overexpression moderately reduces both Αβ generation and amyloid plaque load in APP/PS1AE9 mice not only in the AAV injected hippocampus but also in distant cortical areas.

AAV-APPsa induces microglia recruitment and activation in the vicinity of amyloid plaques

Accumulation of amyloid plaques in APP/PS1AE9 mice has previously been shown to be accompanied by microgliosis and astrocytosis notably at advanced stages of plaque pathology (Kamphuis et al, 2012; Prokop et al, 2013). Here we evaluated the expression of GFAP (as an astrocyte-specific marker) and Ibal (as a microglial marker) by Western blot analysis (Fig. 4A) and IHC (Fig. 4B) in the hippocampus of 17 month old APP/PS1AE9 mice treated either with AAV-APPsa or control vector. While no significant difference was detectable for the astroglial marker GFAP, AAV-APPsa treatment lead to a significant increase in Ibal expression (about +44%; t-test, p=0.003; Fig. 4A), as compared to AAV- Venus control injections. Staining of brain sections further confirmed these data (data not shown) at the cellular level. We went on and quantified GFAP and Ibal immunoreactivity around amyloid plaques in the hippocampus. Consistent with Western blot analysis, GFAP immunoreactivity was not affected by AAV-APPsa injection (Fig. 4B). In contrast, the reduction of amyloid deposits observed after injection of the AAV-APPsa vector in APP/PS1AE9 mice was accompanied by a 2.3-fold increase in Ibal immunoreactivity in the vicinity of plaques (Fig. 4B). Moreover, we observed an altered morphology of microglia in AAV-APPsa treated mice characterized by increased ramifications in AAV-APPsa versus control vector injected APP/PS1AE9 mice (data not shown). Microglia contribute to Αβ clearance and are thought to play a protective role at least during early stages of AD (Prokop et al, 2013). Indeed, plaque associated microglia (from both AAV-APPsa and AAV- Venus treated mice) were also engaged in Αβ uptake as evidenced by Ibal/4G8 double staining (data not shown). Recently, genetic variants of TREM2 (Triggering Receptor Expressed on Myeloid cells) have been associated with an increased risk for AD (Guerreiro et al, 2013; Jonsson et al, 2013). Although the precise role of TREM2 for AD pathogenesis and Αβ pathology is still controversial (Jay et al, 2015; Wang et al, 2015) TREM2 expression has been consistently detected in plaque associated Ibal ⁺ cells in AD model mice (Frank et al, 2008; Jay et al, 2015). Consistent with an increase in plaque associated microglia we detected a significant increase of TREM2 expression (about 60% of control, t-test, p<0.05) by Western blot analysis in hippocampi of APP/PS1AE9 mice injected with AAV-APPsa versus controls (Fig. 4C, n=8 per group). We also determined the expression of neprilysin (NEP) and insulin- degrading enzyme (IDE) that are proteases produced by microglia that contribute to Αβ clearance (Tang 2008). Expression of NEP was identical in APP/PS1AE9 mice injected with AAV-APPsa versus control (not shown). However a significant increase (of about +20%>, t- test, p< 0.001) in IDE expression was observed after AAV-HA-APPsa vector injection (Fig. 4C). Together these data suggest that AAV mediated APPsa expression induces microglia recruitment, activation and possibly also phagocytic function which may lead to enhanced Αβ and plaque clearance.

ΑΑν-ΑΡΡββ injection induce an efficient and durable expression of hippocampal neurons in APP/PS1AE9 mice

In order to assess the consequences of ΑΡΡββ neuronal overexpression in APP/PS1AE9 mice, AAV9- Venus or ΑΑν9-ΑΡΡ8β and AAV9- APPsa both hemaglutinine tagged (thereafter referred as AAV- Venus, AAV-APPsP and AAV-APPsa) (data not shown) were bilaterally injected into the stratum lacunosum molecular e and the dentate gyrus regions of the hippocampus of aged APP/PS1AE9 mice (12 months) (data not shown). Mice were sacrificed at 17 months of age, (5 months post-injection) to evaluate the expression of both APPsa and APPsp. Efficient transduction of the hippocampus (especially in the CAl, CA2 and dentate gyrus) was evidenced. The pattern of expression in the hippocampus was similar in AAV-APPsa injected mice and diffusion of APPsP expression into the peri-hippocampal cortex was observed (data not shown). The APPsP expression showed also a nice diffusion from rostral to caudal coordinates in both cortex and hippocampus (data not shown). Further cellular analysis of the CAl layer confirmed that the synapsin promoter allowed specific and efficient neuronal transduction without any transduction of neither astrocytes nor microglia (data not shown). Levels of expression of ΑΡΡββ-ΗΑ and APPsa-HA in hippocampus analyzed by western blot were very close and consistent in every single animal injected. AAV- Venus control animals also displayed a similar level of Venus (data not shown).

AAV- APPsp does not improve spatial reference memory of aged APP/PS1AE9

We used the Morris water maze to evaluate the spatial reference of the APP/PS1AE9 mice. Transgenic mice (AAV- Venus (n=8), AAV-APPsa (n=8) or AAV-APPsP (n=7)) or littermate controls (AAV- Venus (n=3)) were injected at 12 months old and assessed 2 months later at 14 months of age. During the five days of the training phase (TQ, data not shown), every group of animals showed an efficient learning of the platform position highlighted by a decreased distance to find it over the five days (data not shown). 72 hours after the last training session, the platform was removed in order to assess spatial reference memory evidenced by the distance spent in the TQ (Figure 5). APP/PS1AE9 control mice injected with AAV- Venus showed an impaired memory as compared to AAV- Venus injected littermate mice (Tukey post-hoc test: p=0.04). AAV-APPsa injection in transgenic mice improved spatial reference memory as previously shown (p= 0.01). In contrast, however, AAV-APPsP treatment did rescue spatial reference memory as the time spent in the TQ was equivalent to AAV- Venus mice (p= 0.99) and significantly different compared to AAV- APPsa mice (p= 0.02). The lack of efficiency of AAV-APPsP to restore an efficient search strategy is highlighted by the occupancy plots (data not shown). Together, these results demonstrate that, in contrast to AAV-APPsa, AAV-ΑΡΡβ injection is not able to restore memory deficits of APP/PS1AE9. AAV-APPsp injection does not restore long-term potentiation in the hippocampus of aged APP/PS1AE9 mice.

We then evaluated whether the improvements of spatial memory deficits in APP/PS1AE9 mice were also reflected at the functional neuronal network level. Up to know the effects of APPsB on synaptic plasticity had not been studied, neither in vitro nor in vivo. To analyze synaptic plasticity we induced long-term potentiation (LTP) at the Schaffer collateral to CA1 pathway by theta-burst stimulation (TBS) after baseline recording (Fig. 6). Acute slices of AAV- Venus injected APP/PS1AE9 animals exhibited significant lower induction and maintenance of LTP compared to littermate controls indicating a significant impairment of the transgenic mice. Viral expression of APPsa restored potentiation after TBS in transgenic animals and resulted in an LTP curve progression comparable to that of LM controls, confirming previous results (Figure 2). Injection of AAV- APPsB vector in APP/PS1AE9 mice did also not correct the impairment of the presynaptic compartment as shown by altered PPF at the 10 ms ISI, This parameter was restored after AAV- APPsa injection but not with AAV-APPsP (data not shown). Together, these data indicate that APPsB expression in contrast to APPsa expression fails to ameliorate functional synaptic impairments of aged AD model mice. This in turn indicates a crucial role for the last 16 aminoacids of APPsa (that are lacking in APPsb) as a domain that mediates the rescue effect on memory and synaptic plasticity.

AAV-APPsp does not activate microglia in vivo in aged APP/PS1AE9 mice

In order to explain the discrepancy between AAV -APPsa and AAV-APPsP regarding amyloid plaques degradation, we assessed microglial activation. We first confirmed that APPsa is able activate microglia which in turn internalize Αβ and upregulate the amyloid degrading enzyme IDE and the receptor TREM2. In sharp contrast, the activation of microglia evidenced by upregulation of Ibal in both western blot and immunohistochemistry was not observed after AAV-APPsP or AAV- Venus injection (Figure 7). This result is unexpected and novel as previous in vitro studies indicated that both APPsa and APPsP may activate microglia in culture.

This results might indicate that the AAV- APPsa effects on soluble Αβ one hand and amyloid plaques the other hand are mediated by two independent mechanisms. Finally, the lack of activation following APPsP injection could explain why plaques levels are not altered. Conclusion

Despite a recent shift of research efforts towards preventive strategies, there is still an urgent lack of an effective treatment of patients with clinically established AD. So far, many therapeutic approaches targeted the secretases processing APP. However, since all secretases act on many different substrates besides APP (Prox et al, 2012; Vassar et al, 2014), these strategies have major drawbacks for clinical application, γ-secretase is physiologically essential and current clinical trials to develop γ-secretase inhibitors have been abrogated due to serious side effects, likely resulting from impaired Notch signaling (Doody et al, 2013). Also systemic upregulation of the major a-secretase ADAM 10 to boost APPsa production is problematic, as this may enhance cleavage of substrates implicated in tumorigenesis and in addition of several hundred substrates expressed in neurons (reviewed by Nhan et al, 2015; Prox et al, 2012, Kuhn et al, 2016). Thus, direct overexpression of APPsa in the brain may be more promising than pharmacological upregulation of a-secretase.

Here, the inventors explored a gene therapeutic approach and used AAV-based gene transfer to overexpress APPsa in the brain of transgenic APP/PS1AE9 mice that have been widely used in experimental studies assessing the efficacy of AD therapies. Bigenic APPSWE/PS1AE9 mice express a chimeric mouse/human APP (with Swedish double mutation) and a mutant human PSl gene (PS1AE9) both associated with familial forms of AD. They produce high amounts of ΙιιιΑβ leading to amyloid deposition starting at 5-6 months and pronounced progression of plaque pathology with age that is associated with impairments in cognitive behavior (Savonenko et al, 2005). Using bilateral injection of AAV- APPsa vector particles the inventors achieved highly efficient and widespread expression of APPsa throughout the whole hippocamus and also in adjacent cortical areas.

In this study, a single bilateral injection of AAV-APPsa particles was sufficient to mediate long-lasting APPsa expression over five months that was well tolerated without apparent adverse effects. This was a crucial prerequisite to study potential therapeutic efficacy of AAV-APPsa overexpression. To this end, they used aged (12 month old) APP/PS1AE9 mice with preexisting amyloidosis to mimic the situation in AD patients that are usually clinically diagnosed many years after the onset of pathology (Villemagne et al, 2013).

Taken together, the inventors provide evidence that APPsa as a molecule has beneficial effects on cognition, synaptic density, synaptic function and plasticity, microglia activation and reduces both soluble Αβ and insoluble Αβ deposits in the form of plaques. Moreover, they show that APPsa and not APPsP is responsible for the positive rescue effects in an Alzheimer mouse model. They show that APPsa but not APPsP rescues: a) cognition (MWM) and b) synaptic plasticity as a molecular correlate to synaptic strength. This are totally novel data not reported so far. In addition they also show that APPsa activates microglia and recruits microglia towards plaques. This is an unexpected novel in vivo finding, as previously in vitro both APPsa and APPsP could activate microglia to secrete inflammatory cytokines. The inventors show for the first time that in vivo both molecules have different properties and that APPsa could be very useful in AD and Down syndrome treatment strategy.

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