Cpla2e inducer and use thereof

Abstract

To provide additional treatment options for treating cognitive impairments associated with brain disorders.SOLUTION: To provide a cytosolic phospholipase A2 ε (cPLA2e) inducer used as a medicine, especially used for treating cognitive disorders and / or diseases associated with cognitive disorders, for example, dementias, more specifically, age-related dementias and / or Alzheimer's diseases.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to cPLA2e inducers and to cPLA2e inducers for use as pharmaceuticals, particularly for the treatment of cognitive impairment and / or diseases associated with cognitive impairment, such as dementia, more particularly age-related dementia and / or Alzheimer's disease. [Background technology]

[0002] Mild cognitive impairment is characterized by impairment of memory, language, and / or other important cognitive functions that does not interfere with an individual's daily life. This condition often progresses to dementia, which is characterized by a global decline in cognitive ability that interferes with daily life.

[0003] Alzheimer's disease (AD) is currently the leading form of dementia in the elderly, affecting approximately 50 million people worldwide. Progressive and irreversible cognitive impairment and memory loss, combined with the presence of Aβ peptide aggregates and neurofibrillary tangles (NFTs), are key features of the disease.

[0004] To date, most therapies assayed for AD have focused on targeting one of these two histopathological hallmarks, specifically Aβ levels. However, the high failure rate of AD trials (>99%) and the high costs associated with this disease make it essential to investigate AD with the aim of finding new therapies.

[0005] The study of AD is complicated by the sometimes discrepancy between the appearance of classic AD markers and the symptoms of dementia. In several longitudinal studies, substantial AD pathology has been observed in the brains of cognitively normal elderly subjects. These findings suggest that classic AD features are not sufficient to cause dementia and open the possibility of studying these AD-resilient patients to identify new potential targets for AD treatment. do.

[0006] Cognitive impairment is a condition associated with numerous brain disorders. Brain disorders can have many causes, such as degenerative conditions, genetics, trauma, infections, and malnutrition. For example, cognitive impairment can be associated with aging and / or neurodegenerative diseases, such as Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis (ALS), psychosis, Parkinson's disease psychosis, Alzheimer's disease psychosis, dementia with Lewy bodies, prion-mediated neurodegenerative disorders, such as Creutzfeldt-Jakob disease and kuru, corticobasal degeneration, frontotemporal lobar degeneration, multiple sclerosis, normal pressure hydrocephalus, chronic organic brain syndrome, Pick's disease, progressive supranuclear palsy, or senile dementia. Cognitive impairment can also have a congenital basis, such as Prader-Willi syndrome, Down syndrome, Fragile X syndrome, Angelman syndrome, and autism spectrum disorder. Cognitive impairment may also be associated with brain trauma, such as that resulting from chronic subdural hematoma, concussion, stroke, intracerebral hemorrhage, or other brain injuries, such as those resulting from infections (e.g., encephalitis, meningitis, and sepsis), or drug addiction or abuse. Cognitive impairment may also be associated with other conditions that impair or otherwise affect the normal function of the central nervous system, including sleep deprivation, psychiatric disorders, such as anxiety disorders, dissociative disorders, mood disorders, schizophrenia, treatment with psychotropic drugs, treatment with dopamine agonists, and somatoform and factitious disorders, and may also be associated with peripheral nervous system conditions, such as chronic pain. In some cases, the cause of cognitive impairment may be unknown or unclear.

[0007] Cognitive dysfunction may include, for example, impairments in attention, information acquisition, information processing, working memory, short-term memory, long-term memory, anterograde memory, retrograde memory, memory retrieval, discrimination learning, decision-making, verbal retrieval, inhibitory response control, and attention. It may manifest in many ways, such as impaired learning and / or memory, including but not limited to, set-shifting, delayed reinforcement learning, reversal learning, temporal consolidation of spontaneous behaviors, and manifestations of concern with personal surroundings and self-care. Cognitive impairment may be characterized by a progressive loss of memory, cognition, reasoning, executive function, planning, judgment, and emotional stability.

[0008] Although much progress has been made, treatments for cognitive impairments associated with brain damage remain largely inadequate. Treatments are limited or unavailable for diseases such as Huntington's disease, Parkinson's disease, amyotrophic lateral sclerosis, Alzheimer's disease, Prader-Willi syndrome, and dementia with Lewy bodies. Additional therapeutic options for treating cognitive impairments associated with brain damage are needed. Summary of the Invention

[0009] We now surprisingly disclose that overexpression of hippocampal PLA2G4E (also called cytosolic phospholipase A2ε (cPLA2e)) mediated by treatment with AAV2 / 9-mPLA2G4E (a viral vector encoding cPLA2e) significantly rescued spatial memory impairment in aged APP / PS1 mice 2 months after treatment by stereotaxic injection, and improved memory retention in aged C57BL / 6 / SJL WT mice 3 months after treatment by stereotaxic injection.

[0010] Thus, in a first aspect, the present invention relates to a nucleic acid construct comprising a nucleotide sequence encoding cytosolic phospholipase A2ε (cPLA2e).

[0011] In certain embodiments of the above nucleic acid construct, the cPLA2e is human cPLA2e, typically human cPLA2e of SEQ ID NO: 1 or SEQ ID NO: 3, or a mutant human cPLA2e having at least 70% sequence identity to human cPLA2e of SEQ ID NO: 1 or SEQ ID NO: 3.

[0012] In more particular embodiments of the above nucleic acid construct, the nucleotide sequence encoding cPLA2e is SEQ ID NO:2 or SEQ ID NO:4.

[0013] In a specific embodiment, the nucleic acid construct further comprises a promoter operably linked to the nucleotide sequence encoding cPLA2e.

[0014] In more specific embodiments of the above nucleic acid constructs, the promoter operably linked to the nucleotide sequence encoding cPLA2e is a neuron-specific promoter, and in particular the promoter is a SYN1 promoter or a hybrid SYN1 promoter.

[0015] In a specific embodiment, the nucleic acid construct further comprises a polyadenylation signal sequence, in particular the polyadenylation signal sequence of the bovine growth hormone gene.

[0016] In a specific embodiment, the nucleic acid construct comprises 5'ITR and 3'ITR sequences, preferably 5'ITR and 3'ITR sequences of an adeno-associated virus, more preferably 5'ITR and 3'ITR sequences derived from the AAV2 serotype.

[0017] In another embodiment, the nucleic acid construct of the present invention is RNA, particularly mRNA.

[0018] In one aspect, the present invention relates to a vector comprising the nucleic acid construct of the present invention. Preferably, said vector is a viral vector, more preferably an AAV vector.

[0019] In one aspect, the present invention relates to a viral particle comprising a nucleic acid construct of the present invention.

[0020] In a specific embodiment, the viral particles are selected from AAV particles, preferably AAV particles comprising capsid proteins selected from the group consisting of AAV2, AAV5, AAV9 and AAV TT serotypes.

[0021] In one aspect, the present invention also relates to a host cell comprising the nucleic acid construct or expression vector of the present invention.

[0022] In a further aspect, the present invention provides a method for producing a composition comprising: a) culturing packaging cells containing the nucleic acid construct or vector of the present invention in a culture medium; b) harvesting viral particles from the cell culture supernatant and / or cells; The present invention relates to a method for producing a viral particle, comprising:

[0023] In another aspect, the present invention relates to a pharmaceutical composition comprising a nucleic acid construct, vector, or viral particle, or host cell of the present invention and a pharmaceutically acceptable carrier or excipient.

[0024] In another aspect, the present invention relates to the nucleic acid construct, vector, viral particle or host cell of the present invention, or a pharmaceutical composition comprising said nucleic acid construct, vector, viral particle or host cell for use as a medicament.

[0025] In yet another aspect, the present invention relates to cPLA2e inducers for use as pharmaceutical agents.

[0026] In a related aspect, the present invention relates to cPLA2e inducers for use in treating cognitive impairment and / or disorders associated with cognitive impairment in a subject in need thereof.

[0027] In a specific embodiment, the disease associated with cognitive impairment is dementia.

[0028] In a specific embodiment, the disease associated with cognitive impairment is age-related dementia or Alzheimer's disease.

[0029] In specific embodiments, the cPLA2e inducer is selected from the group consisting of a) a nucleic acid construct of the present invention, b) a vector comprising a nucleic acid construct of the present invention, c) a viral particle comprising a nucleic acid construct or vector of the present invention, d) a host cell comprising a nucleic acid construct or vector of the present invention, e) a cPLA2e polypeptide or protein, and f) a pharmaceutical composition comprising any of the above nucleic acid construct, a vector comprising a nucleic acid construct, a viral particle comprising a nucleic acid construct or vector, and a cPLA2e polypeptide or protein.

[0030] In more specific embodiments, the cPLA2e inducer is a nucleic acid construct, vector, or viral particle of the present invention, or a pharmaceutical composition comprising said nucleic acid, vector, or viral particle.

[0031] In a specific embodiment, the cPLA2e inducer is a protein having cPLA2e activity, preferably the protein of SEQ ID NO:1 or SEQ ID NO:3. [Brief explanation of the drawings]

[0032] [Figure 1A] Figure 1 shows the escape latency to the hidden platform in the MWM test for aged WT (negative control), APP / PS1 sham (sham-injected APP / PS1 mice), and APP / PS1 AAV2 / 9-mPLA2G4E (APP / PS1 mice treated with AAV2 / 9-mPLA2G4E) 2 months after stereotaxic surgery (two-way ANOVA followed by Bonferroni's post-hoc test, n = 6-9; *P ≤ 0.05 APP / PS1 sham vs. WT, **P ≤ 0.01 APP / PS1 sham vs. WT, +P ≤ 0.05 APP / PS1 AAV2 / 9-mPLA2G4E vs. WT, $P ≤ 0.05 APP / PS1 AAV2 / 9-mPLA2G4E vs. APP / PS1 sham, and $$P ≤ 0.01 APP / PS1 AAV2 / 9-mPLA2G4E vs. APP / PS1 sham). [Figure 1B]Figure 1 shows the percentage of time spent in the correct quadrant during 15-s and 60-s exploration trials on day 6 for aged WT, APP / PS1 sham, and APP / PS1 AAV2 / 9-mPLA2G4E mice 2 months after hippocampal injection (one-way ANOVA followed by Newman-Keuls post-hoc test, n = 6-9; *P ≤ 0.05 APP / PS1 sham vs. WT, **P ≤ 0.01 APP / PS1 sham vs. WT, and $P ≤ 0.05 APP / PS1 AAV2 / 9-mPLA2G4E vs. APP / PS1 sham). [Figure 2A] Representative Golgi-stained images of apical dendrites on CA1 hippocampal pyramidal neurons from aged WT (negative control), APP / PS1 sham (sham-injected APP / PS1 mice), and APP / PS1 AAV2 / 9-mPLA2G4E (AAV2 / 9-mPLA2G4E-treated APP / PS1 mice). Scale bar = 10 μm. [Figure 2B] Histoblot showing quantification of spine density in CA1 hippocampal pyramidal neurons from WT, APP / PS1 sham, and AAV2 / 9-PLA2G4E-treated mice (one-way ANOVA followed by Newman-Keuls post-hoc test, n=4, +p≦0.05 WT vs. APP / PS1 AAV2 / 9-PLA2G4E, $p≦0.05 APP / PS1 sham vs. APP / PS1 AAV2 / 9-PLA2G4E). [Figure 3A] Figure 1 shows the escape latency of the hidden platform in the MWM test for aged WT sham (sham-injected C57BL / 6 / SJL WT mice) and WT AAV2 / 9-mPLA2G4E (AAV2 / 9-mPLA2G4E-treated C57BL / 6 / SJL WT mice) 3 months after hippocampal injection by stereotaxic surgery (two-way ANOVA test followed by Bonferroni's post-hoc test, n=4 or 5). [Figure 3B] Figure 1 shows the percentage of time spent in the correct quadrant during 15-s and 60-s exploration trials on day 5 for aged WT sham and WT AAV2 / 9-mPLA2G4E mice 3 months after stereotaxic surgery (one-way ANOVA test followed by Newman-Keuls post-hoc test, n=4 or 5, *P≦0.05 WT AAV2 / 9-mPLA2G4E vs. WT sham). [Figure 4A] Figure 1 shows the experimental design of the fear conditioning paradigm used to elucidate the role of PLA2G4E in memory function.Figure 2 shows graphs showing the percentage of freezing behavior in TT mice during the training and test phases. [Figure 4B] FIG. 1 shows pCREB levels measured by immunoblotting in hippocampal extracts and normalized to β-actin (one-way ANOVA test followed by Newman-Keuls post-hoc test, n=7 or 8, **P≦0.01 naive vs. TT, ++P≦0.01 T24 vs. TT). [Figure 4C] FIG. 1 shows PLA2G4E levels measured by immunoblotting in hippocampal extracts and normalized to β-actin (one-way ANOVA test followed by Newman-Keuls post-hoc test, n=7 or 8, **P≦0.01 naive vs. TT, ++P≦0.01 T24 vs. TT). [Figure 5] pCREB, pGluA1, synapsin I, and PLA2G4E levels in primary neuronal cultures after treatment with bicuculline (Bic) and / or AAV9-shPLA2G4E (shPLA) were measured by immunoblotting and normalized to β-actin (one-way ANOVA test followed by Newman-Keuls post-hoc test, n = 3-6; **P ≤ 0.01, ***P ≤ 0.001 control vs. Bic; ++P ≤ 0.01 control vs. shPLA; $P ≤ 0.05, $$P ≤ 0.01, $$$P ≤ 0.001 Bic vs. shPLA+Bic). Data are expressed as arbitrary units (mean ± SEM) relative to control. DETAILED DESCRIPTION OF THE INVENTION

[0033] In one aspect, the present invention relates to a cytosolic phospholipase A2ε inducer for use as a medicament, more particularly for use in the treatment of cognitive impairment and / or a disease associated with cognitive impairment in a subject in need thereof.

[0034] As used herein, the terms "cytosolic phospholipase A2ε" and "phospholipase A2 group IVE," "PLA2G4E," or "cPLA2e" refer interchangeably to members of the cytosolic phospholipase A2 group IV family of calcium-dependent enzymes that selectively hydrolyze glycerophospholipids at the sn-2 position. Members of this family are involved in regulating membrane tubule-mediated transport. This enzyme interacts with clathrin PLA2G4E plays a role in transport via the clathrin-independent endocytic pathway. This enzyme regulates the recycling process through the formation of tubules that transport internalized clathrin-independent cargo proteins back to the cell surface (Capestrano M. et al. Journal of Cell Science 2014; 127: 977-993). PLA2G4E binds phosphatidylethanolamine (PE) to provide an acyl chain. It can catalyze the calcium-dependent formation of N-acylphosphatidylethanolamine (NAPE) using the donor (Ogura Y. et al. Nat Chem Biol. 2016; 12(9): 669-671). Human cPLA2e is naturally encoded by the PLA2G4E gene. Human cPLA2e is recorded, for example, in UniprotKB (https: / / www.uniprot.org / ) under entry accession number Q3MJ16. This entry describes two isoforms produced by alternative splicing: a) isoform 1 (using identifier Q3MJ16-3), which is selected as the "canonical" sequence (SEQ ID NO: 1); and b) isoform 2 (using identifier Q3MJ16-2), which differs from the canonical sequence in that amino acids 1-376 are deleted in isoform 2 (SEQ ID NO: 3). The term "cPLA2e" refers to the enzyme and any additional co- or post-translational modifications thereof.

[0035] As used herein, the term "cPLA2e inducer" refers to an agent (molecule or composition) that, when administered to a cell, directly or indirectly results in the acquisition of cPLA2e activity in the cell, particularly an agent that results in the acquisition of expression of the enzyme cPLA2e, such as a cPLA2e transgene (i.e., a nucleotide sequence encoding cPLA2e) or an expression product of the transgene.

[0036] Nucleic Acid Constructs In one embodiment, the cPLA2 inducer for use in the present invention is or comprises a nucleic acid construct comprising a nucleotide sequence encoding cytosolic phospholipase A2ε (cPLA2e).

[0037] Thus, in another aspect, the present invention relates to a nucleic acid construct comprising a nucleotide sequence encoding cytosolic phospholipase A2ε (cPLA2e).

[0038] The terms "nucleic acid" and "polynucleotide" or "nucleotide sequence" are used interchangeably herein to refer to any molecule composed of or comprising monomeric nucleotides. A nucleic acid may be an oligonucleotide or a polynucleotide. A nucleotide sequence may be DNA or RNA. The nucleotide sequences may be chemically modified or artificial. Nucleotide sequences include peptide nucleic acids (PNAs), morpholinos and locked nucleic acids (LNAs), as well as glycol nucleic acids (GNAs) and threose nucleic acids (TNAs). These sequences are distinguished from naturally occurring DNA or RNA by changes in the backbone of the molecule, respectively. Phosphorothioate nucleotides may also be used. Other deoxynucleotide analogs, including methyl phosphonates, phosphoramidates, phosphorodithioates, N3'P5'-phosphoramidates, and oligoribonucleotide phosphorothioates, as well as their 2'-O-allyl analogs, and 2'-O-methylribonucleotide methylphosphonates, can be used in the nucleotides of the present invention.

[0039] As used herein, the term "nucleic acid construct" refers to a non-naturally occurring nucleic acid obtained by the use of recombinant DNA technology. In particular, a nucleic acid construct is a nucleic acid molecule, either single-stranded or double-stranded, that has been modified to contain segments of nucleic acid sequences combined or juxtaposed in a manner that would not otherwise occur in nature.

[0040] In some embodiments, nucleic acid constructs of the invention include a nucleotide sequence encoding naturally occurring cPLA2e (wild-type cPLA2e), such as naturally occurring human cPLA2e (e.g., isoform 1 or 2), a known cPLA2e from a primate, mouse, or other mammal. In some embodiments, the cPLA2e is a variant, peptide, or polypeptide having substitutions and insertions, and / or additions, deletions, and / or covalent modifications relative to naturally occurring cPLA2e, typically human cPLA2e isoform 1 or 2. In some embodiments, the cPLA2e encoded by a nucleic acid construct of the invention is a fusion protein or polypeptide to which several amino acids (e.g., a tag) or polypeptides (e.g., a carrier polypeptide) can be added (e.g., at the N-terminus or C-terminus) to the encoded cPLA2e, for example, for localization or targeting. In some embodiments, the cPLA2e encoded by the nucleic acid construct is a fragment cPLA2e, typically human cPLA2e isoform 1 or 2, optionally lacking amino acid residues located at the carboxy-, amino-terminal, or internal regions.

[0041] In one embodiment, the nucleic acid construct of the present invention comprises a nucleotide sequence encoding human cPLA2e, preferably human cPLA2e of SEQ ID NO: 1 or SEQ ID NO: 3 corresponding to isoform 1 or 2, respectively, or a coding sequence of naturally occurring or recombinant cPLA2e, typically a mutant human cPLA2e having at least 70%, 75%, 80%, 85%, 90%, 95% or 99% sequence identity to human cPLA2e of SEQ ID NO: 1 or SEQ ID NO: 3.

[0042] As will be appreciated by those skilled in the art, human cPLA2e isoform 1 or 2 protein fragments, functional protein domains, variants, and homologous proteins (orthologs) are also considered within the scope of the cPLA2e of the nucleic acid constructs of the present invention. It is understood that different embodiments of cPLA2e have substantially the same cPLA2e activity as human cPLA2e isoform 1 or 2. Substantially the same activity can be up to ±5% of the activity, including, for example, ±4%, ±3%, ±2%, ±1% or less.

[0043] As mentioned above, cPLA2e is a calcium-dependent enzyme member of the cytosolic phospholipase A2 group IV family that selectively hydrolyzes glycerophospholipids at the sn-2 position. It has been described that it exhibits very low phospholipase (PLA) activity. Instead, cPLA2e has been shown to have calcium-dependent N-acyltransferase (Ca-NAT) activity, producing N-acylphosphatidylethanolamine (NAPE) and N-acylethanolamine (NAE) in mammalian cells. Its transacylase properties have been associated with serine hydrolase activity (Ogura et al., 2004). l., Nat Chem Biol. 2016, 12(9), 669-671).

[0044] Ca-NAT activity can be determined by measuring NAPE production in biological samples (e.g., cell lysates), e.g., N-C16:0 DOPE production in a reaction with DPPC (40 μM) and DOPE (75 μM) at 37° C. for 30 minutes with or without the addition of CaCl (3 mM) to the reaction mixture. Ca-independent activity is subtracted in calculating Ca-dependent activity. Alternatively, NAPE production (e.g., 13Targeted analysis of C16:0-containing NAPEs can be performed by incubating mammalian cells (e.g., HEK293T cells) in serum-containing culture medium with or without 2 μM ionomycin. Cells are incubated at 37°C for a period of time (e.g., 30 minutes) before lipid extraction. The extracted lipids can be separated by chromatography and analyzed by mass spectrometry (e.g., LC-MS / MS).

[0045] In a preferred embodiment of the nucleic acid construct of the present invention, the nucleotide sequence encoding cPLA2e is SEQ ID NO: 2 or SEQ ID NO: 4, or a variant nucleotide sequence having at least 75%, at least 80%, at least 85%, at least 90% or at least 95% identity to SEQ ID NO: 2 or 4.

[0046] As used herein, the term "sequence identity" or "identity" refers to the number of matches (identical nucleic acid or amino acid residues) at positions in the alignment of two polynucleotide sequences or two polypeptide sequences. Sequence identity is determined by comparing the sequences when aligned to maximize overlap and identity while minimizing sequence gaps. In particular, sequence identity can be determined using any of a number of mathematical global or local alignment algorithms, depending on the length of the two sequences. Sequences of similar length are preferably aligned using a global alignment algorithm (e.g., the Needleman and Wunsch algorithm; Needleman and Wunsch, 1970, J Mol Biol.; 48(3):443-53) that optimally aligns the sequences over their entire length, while sequences of substantially different lengths are aligned using a local alignment algorithm (e.g., the Smith and Waterman algorithm (Smith and Waterman, 1981, J Theor Biol.; 91(2):379-80) or the Altschul algorithm (Altschul SF et al., 1997, Nucleic Acids Res.; 25(17):3389-402; Altschul SF et al., 2005, Bioinformatics;21(8):1451-6). Alignment for purposes of determining percent sequence identity of nucleic acids can be achieved in a variety of ways within the skill of the art, for example, using publicly available computer software available on internet websites such as http: / / blast.ncbi.nlm.nih.gov / or http: / / www.ebi.ac.uk / Tools / emboss / . Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.For purposes herein, nucleic acid % sequence identity values ​​refer to values ​​generated using the pairwise sequence alignment program EMBOSS Needle, which uses the Needleman-Wunsch algorithm to generate an optimal global alignment of two sequences, with all search parameters set to default values: scoring matrix=BLOSUM62, gap open=10, gap extension=0.5, end gap penalty=false, end gap open=10, and end gap extension=0.5.

[0047] The nucleic acid constructs described herein can have different uses, among others, they can be used to generate viral vectors for gene therapy or to generate non-viral vectors, such as nucleic acid constructs with mRNA structures, also for gene therapy.

[0048] In one embodiment, a nucleic acid construct according to the invention comprises at least a nucleotide sequence encoding cPLA2e and suitable nucleic acid elements for its expression in a host cell.

[0049] For example, in one embodiment, a nucleic acid construct comprises a nucleotide sequence encoding cPLA2e and one or more regulatory sequences required for expression of the coding sequence in a relevant target cell type or tissue. Generally, a nucleic acid construct comprises a coding sequence and regulatory sequences preceding (5' non-coding sequences) and following (3' non-coding sequences) the coding sequence required for expression of a selected gene product. Thus, in a specific embodiment, the nucleic acid construct comprises at least (i) a nucleotide sequence encoding cPLA2e under the control of a promoter, and (iii) a 3' untranslated region, which typically includes a polyadenylation signal sequence and / or a transcription terminator. The nucleic acid construct may also comprise additional regulatory elements, such as enhancer sequences, introns, microRNA targeting sequences, polylinker sequences facilitating insertion of DNA fragments into a vector, and / or splicing signal sequences.

[0050] promoter In one embodiment, a nucleic acid construct of the invention also comprises a promoter, which initiates transgene expression after introduction into a host cell.

[0051] As used herein, the term "transgene" refers to a nucleic acid molecule, DNA, or cDNA, that encodes a gene product used as an active ingredient in gene therapy. The gene product can be RNA, a peptide, or a protein. The transgene can encode a native gene product or a recombinant non-naturally occurring gene product, such as cPLA2e.

[0052] As used herein, the term "promoter" refers to a regulatory element that directs transcription of an operably linked nucleic acid (transgene). A promoter can regulate both the rate and efficiency of transcription of an operably linked nucleic acid. A promoter may be operably linked to other regulatory elements that enhance ("enhancers") or repress ("repressors") promoter-dependent transcription of a nucleic acid. These regulatory elements include, but are not limited to, transcription factor binding sites, repressor and activator protein binding sites, and any other sequence of nucleotides known to those skilled in the art that acts directly or indirectly to regulate the amount of transcription from a promoter, including, for example, attenuators, enhancers, and silencers. A promoter is located near the transcription start site of an operably linked gene or coding sequence, on the same strand, and upstream of the DNA sequence (toward the 5' region of the sense strand). A promoter can be approximately 100 to 1000 base pairs in length. Positions in the promoter are specified relative to the transcription start site of a particular gene (ie, upstream positions are negative numbers counting backward from -1, eg, -100 is a position 100 base pairs upstream).

[0053] As used herein, the term "operably linked" refers to the linkage of polynucleotide (or polypeptide) elements in a functional relationship. A nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. For example, a promoter or transcriptional regulatory sequence is operably linked to a coding sequence if it affects the transcription of the coding sequence. Operably linked means that the polynucleotide sequences being linked are usually contiguous, and, where necessary to join two protein-coding regions, contiguous and in reading frame.

[0054] In one embodiment, the nucleic acid construct of the invention further comprises a promoter operably linked to the nucleotide sequence encoding cPLA2e.

[0055] In one embodiment, the promoter operably linked to the cPLA2e coding sequence is a heterologous promoter. As used herein, the term "heterologous" when used as an attribute of a nucleotide or peptide sequence (e.g., a heterologous promoter, a heterologous enhancer, etc.) refers to a sequence that is not naturally operably linked to another nucleotide or peptide sequence. In this particular case, "heterologous promoter" refers to a promoter sequence that is not naturally operably linked to the nucleotide sequence encoding cPLA2e.

[0056] Typically, such promoters can be tissue or cell type specific promoters, or organ specific promoters, or promoters specific to multiple organs, or systemic or ubiquitous promoters.

[0057] In certain embodiments, the nucleic acid construct of the present invention further comprises a promoter operably linked to the nucleotide sequence encoding cPLA2e, the promoter directing expression of the encoded cPLA2e in at least neurons of the hippocampus.

[0058] In certain embodiments of the nucleic acid constructs of the present invention, the promoter operably linked to the nucleotide sequence encoding cPLA2e is a neuron-specific promoter.

[0059] As used herein, the term "specific promoter" refers to a promoter whose activity is not necessarily restricted to a single cell type, but which nevertheless exhibits selectivity, being active in certain groups of cells or tissues and less active or silent in other groups. However, it may be preferable for the promoter of the nucleic acid construct of the present invention to exhibit strict cell specificity, being active at detectable levels only in neuronal cells.

[0060] Thus, as used herein, a "neuron-specific promoter" is a promoter that controls the expression of a gene that is uniquely or primarily expressed in neurons or cells derived from neurons. A neuron-specific promoter directs the expression of a gene in neurons or cells derived from neurons, but does not substantially direct the expression of the same gene in other cell types, such as glial cells, and thus has neuron-specific transcriptional activity. In some cases, some low level expression may be observed in other cell types, but such expression may be substantially lower than expression in neurons; for example, expression in neurons may be at least two-fold, at least three-fold, at least four-fold, at least five-fold, or at least ten-fold higher than the expression level in other cells. Such promoters may be strong or weak promoters and may direct constitutive expression of a gene in neurons or cells derived from neurons, or may direct expression in response to certain conditions, signals, or cellular events.

[0061] Thus, a neuron-specific promoter allows active expression of a gene linked to it in neurons and prevents its expression in other cells or tissues.

[0062] In a more particular embodiment of the nucleic acid construct of the present invention, the promoter operably linked to the nucleotide sequence encoding cPLA2e is the synapsin 1 (SYN1) gene promoter (Kuegler S et al. Gene Ther. 2003; 10(4): 337-47), a neuronal cell-specific promoter. The heterologous enolase (NSE) gene promoter (Forss-Petters S et al. Neuron. 1990;5(2):187-97, Twyman RM et al. J Mol Neurosci. 1997;8(1):63-73), the Hb9 gene promoter (Eur J Neurosci. 1997;9:452, J Neurosci Res. 2000;59:321), and prion Protein (Prnp) gene promoter (Weber P et al. Eur J Neurosci. 2001; 1 4:1777), α-calcium-calmodulin-dependent kinase II (CaMKIIα) gene promoter (Dittgen T et al. Proc Natl Acad Sci U S A. 2004; 101: 18206-18211), methyl-CpG-binding protein 2 (MECP2) gene promoter (Adachi M. et al. Hum Mol Genet. 2005; 14(23):3709-3722, Gray SJ et al. Hum Gene Ther. 2011; 22(9): 1143-1153), and tubulin α1 (Ta1) gene promoter (Gloster A. et al. J Neurosci. 1994; 14:7319).

[0063] Typically, such promoters (particularly the neuron-specific promoters from the select group above) may be a complete promoter containing core, proximal, and distal promoter elements; a promoter, e.g., a fragment of a core promoter, or any other fragment sufficient to direct gene expression in a target cell, tissue, or organ; or a chimeric or hybrid promoter, e.g., a promoter containing the core promoter of a gene and a heterologous enhancer sequence from another gene or synthetically. The term "core promoter" as used herein refers to the minimal portion of a promoter required to properly initiate transcription. It consists of a transcription initiation site and functional sequences for binding of the transcription initiation complex (TATA box) within a cell or host organism. Non-limiting examples of suitable neuron-specific hybrid promoters include, among others, hybrid promoters based on the SYN1 promoter, such as a hybrid promoter resulting from the fusion of promoter elements of the SYN1 and CMV genes (e.g., Matsuzaki Y. et al. J Neurosci Methods 2014; 223:133-143), the Hb9 promoter, such as the Hsp68 minimal promoter (Singh NR et al. Exp Neurol. 2005; 196(2):224-234) or the CMV minimal promoter (Lukashchuk V. et al. Mol Ther Methods Clin Dev. 2016; 3: 15055) is a mouse Hb9 enhancer fused to the .

[0064] In one embodiment of the nucleic acid construct of the present invention, the promoter operably linked to the nucleotide sequence encoding cPLA2e is a SYN1 promoter or a hybrid SYN1 promoter, such as a hybrid SYN1 promoter comprising the core SYN1 promoter fused to a CMV gene promoter element.

[0065] In one embodiment, the nucleic acid construct of the present invention comprises a hybrid SYN1 promoter operably linked to a nucleotide sequence encoding cPLA2e, typically cPLA2e of SEQ ID NO: 1 or 3, and preferably the encoding nucleotide sequence is SEQ ID NO: 2 or 4.

[0066] All of these promoter sequences have the property of allowing expression of the cPLA2e encoded by the nucleic acid construct in neurons of at least the hippocampus.

[0067] In a specific embodiment, the promoter used in the nucleic acid construct of the present invention may be a chemically inducible promoter. As used herein, a chemically inducible promoter is a promoter that is regulated by in vivo administration of a chemical inducer to the subject in need thereof. Examples of suitable chemically inducible promoters include, but are not limited to, the tetracycline / minocycline inducible promoter (Chtarto 2003, Neurosci Lett. 352:155-158) or the rapamycin inducible system (Sanftner 2006, Mol Ther.13:167-174).

[0068] Polyadenylation signal Embodiments of the nucleic acid construct may include a polyadenylation signal sequence, with or without any other nucleotide elements. As used herein, the term "polyadenylation signal" or "poly(A) signal" refers to a precursor The term "poly(A) signal" refers to a specific recognition sequence in the 3' untranslated region (3'UTR) of a gene that is transcribed into an mRNA molecule and directs the termination of gene transcription. The poly(A) signal acts as a signal for endonucleolytic cleavage at the 3' end of the newly formed precursor mRNA and the addition of an RNA stretch consisting only of adenine bases to the 3' end (the polyadenylation process; poly(A) tail). The poly(A) tail is important for the nuclear export, translation, and stability of mRNA. In the context of the present invention, a polyadenylation signal is a recognition sequence that can direct the polyadenylation of mammalian and / or viral genes in mammalian cells.

[0069] Poly(A) signals typically consist of a) the consensus sequence AAUAAA, which has been shown to be required for both 3'-end cleavage and polyadenylation of pre-messenger RNA (pre-mRNA) and to promote downstream transcription termination, and b) additional elements upstream and downstream of AAUAAA that control the efficiency of AAUAAA's use as a poly(A) signal. Considerable variation is observed in these motifs among mammalian genes.

[0070] In one embodiment, optionally in combination with one or more features of the various embodiments above or below, the polyadenylation signal sequence of the nucleic acid construct of the invention is a polyadenylation signal sequence of a mammalian or viral gene. Suitable polyadenylation signals include, among others, the SV40 early polyadenylation signal, the SV40 late polyadenylation signal, the HSV thymidine kinase polyadenylation signal, the protamine gene polyadenylation signal, the adenovirus 5 EIb polyadenylation signal, the growth hormone polyadenylation signal, the PBGD polyadenylation signal, computer-designed polyadenylation signals (synthetic), and the like.

[0071] In a specific embodiment, the polyadenylation signal sequence of the nucleic acid construct is a polyadenylation signal sequence based on the bovine growth hormone gene.

[0072] In a specific embodiment, a nucleic acid construct according to the invention comprises a hybrid SYN1 promoter and the polyadenylation signal sequence of the bovine growth hormone gene operably linked to a nucleotide sequence encoding cPLA2e of SEQ ID NO: 1 or 3. In a preferred embodiment, the nucleotide sequence encoding cPLA2e is SEQ ID NO: 2 or 4.

[0073] Nucleic acid constructs with mRNA structure In some embodiments, the nucleic acid construct of the present invention is RNA. In certain embodiments, the nucleic acid construct has the structure of mRNA. In certain embodiments, the mRNA can be modified. Modifications of mRNA nucleic acids are described in further detail in U.S. Patent Application Publication Nos. 20140206752, 20150086614, and 20160304552, as well as WO 2016011226, WO 2016014846, and WO 2016011306. These include, among others, chemically modified nucleobases, sugars, backbones, or any combination thereof, patterned untranslated regions (UTRs), and microRNA (miRNA) binding site(s).

[0074] Thus, in some embodiments, a nucleic acid construct comprising a nucleotide sequence encoding cPLA2e may further comprise at least one of the following features: a) a 5' cap structure, b) a 5' UTR, or c) a 3' UTR. In one aspect, the polynucleotide further comprises two of the features. In one aspect, the polynucleotide further comprises all three of these features. The UTR may be homologous or heterologous to the nucleotide sequence encoding cPLA2e.

[0075] Untranslated regions (UTRs) are nucleic acid sections of a polynucleotide that are not translated before the start codon (5'UTR) and after the stop codon (3'UTR). In some embodiments, a nucleic acid construct of the invention comprising a nucleotide sequence encoding cPLA2e further comprises a UTR (e.g., a 5'UTR or a functional fragment thereof, a 3'UTR or a functional fragment thereof, or a combination thereof).

[0076] In some embodiments, a nucleic acid construct comprises two or more 5' UTRs or functional fragments thereof, each having the same or different nucleotide sequences. In some embodiments, a nucleic acid construct comprises two or more 3' UTRs or functional fragments thereof, each having the same or different nucleotide sequences. In some embodiments, the 5' UTR or functional fragments thereof, the 3' UTR or functional fragments thereof, or any combination thereof, are sequence-optimized. In some embodiments, the 5' UTR or functional fragments thereof, the 3' UTR or functional fragments thereof, or any combination thereof, comprises at least one chemically modified nucleobase, such as 1-methylpseudouridine or 5-methoxyuracil. In some embodiments, the functional fragments of the 5' UTR or 3' UTR comprise one or more regulatory features of the full-length 5' or 3' UTR, respectively.

[0077] Typically, by manipulating the characteristics found in highly expressed genes of a particular target cell / tissue / organ, one can enhance polynucleotide stability and protein production in that particular target cell / tissue / organ.

[0078] In some embodiments, the 5'UTR and 3'UTR may be heterologous. In some embodiments, the 5'UTR may be from a different species than the 3'UTR.

[0079] WO 2014 / 164253 (incorporated herein by reference in its entirety) provides a list of exemplary UTRs that can be used in the polynucleotides of the present invention as flanking regions of the nucleotide sequence encoding cPLA2e.

[0080] Wild-type UTRs from any gene or mRNA can be incorporated into the nucleic acid constructs of the invention. In some embodiments, a UTR can be altered relative to the wild-type or native UTR, for example, by changing the orientation or position of the UTR relative to the coding nucleotide sequence, or by incorporating additional nucleotides, deleting nucleotides, exchanging nucleotides, or rearranging nucleotides, to create a mutant UTR. In some embodiments, variants of the 5' or 3' UTR can be used, such as mutants of the wild-type UTR, or variants in which one or more nucleotides are added to or removed from the end of the UTR. Additionally, one or more synthetic UTRs can be used in combination with one or more non-synthetic UTRs. See, for example, Mandal and Rossi, Nat. Protoc. 2013 8(3):568-82, and the sequences available at www.addgene.org / Derrick_Rossi / (each of which is incorporated by reference in its entirety). See, for example, the following references:

[0081] The 5' cap structure of native mRNA is involved in nuclear export, enhances mRNA stability, and binds to mRNA cap-binding protein (CBP), which is involved in mRNA stability and translational competence in cells through the association of CBP with poly(A)-binding protein, to form mature circular mRNA species. The cap further assists in the removal of 5'-proximal introns during mRNA splicing. Endogenous mRNA molecules can be 5'-end capped to generate a 5'-ppp-5'-triphosphate linkage between the terminal guanosine cap residue and the 5'-terminal transcribed sense nucleotide of the mRNA molecule. This 5'-guanylate cap can then be methylated to generate an N7-methyl-guanylate residue. The 5'-end of mRNA can be capped to generate a 5'-ppp-5'-triphosphate linkage between the terminal guanosine cap residue and the 5'-terminal transcribed sense nucleotide of the mRNA molecule. Optionally, the ribose sugars of the terminal and / or ante-terminal transcribed nucleotides are 2'- Nucleic acid molecules, such as mRNA molecules, can be targeted for degradation by 5'-decapping via hydrolysis and cleavage of the guanylate cap structure, which may be O-methylated.

[0082] In some embodiments, a nucleic acid construct of the invention incorporates a moiety or structure as a 5' cap.

[0083] In some embodiments, a nucleic acid construct of the invention (ie, a polynucleotide comprising a nucleotide sequence encoding a cPLAe) further comprises a polyA tail.

[0084] vector The nucleic acid construct of the present invention may be contained in an expression vector. Thus, in one aspect, the present invention relates to an expression vector comprising the nucleic acid construct of the present invention.

[0085] As used herein, the term "expression vector" or "vector" refers to a nucleic acid molecule used as a vehicle to transfer genetic material, particularly to deliver nucleic acids to host cells either in vitro or in vivo. Expression vectors also refer to nucleic acid molecules capable of directing the expression of a gene (transgene) in a host cell or host organism compatible with such sequences. Expression vectors typically contain at least appropriate transcriptional regulatory sequences and, optionally, a 3' transcription termination signal. Additional factors (endogenous or chimeric transcription factors) necessary or beneficial for directing expression, such as expression enhancer elements capable of responding to precise induction signals or specific to certain cells, organs, or tissues, may be present. Vectors include, but are not limited to, plasmids, phasmids, cosmids, transposable elements, viruses, and artificial chromosomes (e.g., YACs). Preferably, the vectors of the present invention are suitable for use in gene or cell therapy, particularly for targeting neural cells.

[0086] In some embodiments, the expression vector is a viral vector such as a Moloney murine leukemia virus vector (MoMLV), a vector derived from MSCV, SFFV, MPSV, or SNV, a lentiviral vector (e.g., derived from human immunodeficiency virus (HIV), simian immunodeficiency virus (SIV), feline immunodeficiency virus (FIV), bovine immunodeficiency virus (BIV), or equine infectious anemia virus (EIAV)), an adenovirus (Ad) vector, an adeno-associated virus (AAV) vector, a simian virus 40 (SV-40) vector, a bovine papillomavirus vector, an Epstein-Barr virus, a herpesvirus vector, a vaccinia virus vector, a Harvey murine sarcoma virus vector, a mouse mammary tumor virus vector, or a Rous sarcoma virus vector.

[0087] As is known in the art, depending on the particular viral vector under consideration, suitable sequences, such as AAV ITRs for AAV vectors or LTRs for lentiviral vectors, must be introduced into the vector of the invention to obtain a functional viral vector. In certain embodiments, optionally in combination with one or more features of the various embodiments described above or below, the vector is an AAV vector.

[0088] AAV has attracted great interest as a potential vector for human gene therapy. Advantageous properties of this virus include its lack of association with any human disease, its ability to infect both dividing and non-dividing cells, and its ability to infect a wide range of cell lines derived from different tissues. The AAV genome is composed of a linear single-stranded DNA molecule containing 4681 bases (Berns and Bohenzky, 1987, Advances in Virus Research (Academic Press, Inc.) 32:243-307). The genome contains inverted terminal repeats (ITRs) at both ends that function in cis as DNA replication origins and viral packaging signals. The ITRs are approximately 145 bp long. The internal non-repetitive portion of the genome contains two large open reading frames known as the AAV rep and cap genes, respectively. These genes encode viral proteins involved in virion replication and packaging. In particular, at least four viral proteins are synthesized from the AAV rep genes, Rep 78, Rep 68, Rep 52, and Rep 40, named according to their apparent molecular weights. The AAV cap gene encodes at least three proteins, VP1, VP2, and VP3. For a detailed description of the AAV genome, see, e.g., Muzyczka, N. 1992 Current Topics in Microbiol. and Immunol. 158:97-129.

[0089] Thus, in one embodiment, optionally in combination with one or more features of the various embodiments described above or below, the nucleic acid construct or expression vector of the invention (comprising a nucleotide sequence encoding cPLA2e) further comprises 5' ITR and 3' ITR sequences, preferably adeno-associated virus 5' ITR and 3' ITR sequences.

[0090] As used herein, the term "inverted terminal repeats (ITRs)" refers to the nucleotide sequences located at the 5' end (5' ITR) and the 3' end (3' ITR) of a virus, which contain palindromic sequences and can fold to form T-shaped hairpin structures that function as primers during the initiation of DNA replication. They are also required for integration of the viral genome into and rescue from the host genome, and encapsidation of viral nucleic acids into mature virions. The ITRs are required in cis for replication of the vector genome and its packaging into viral particles.

[0091] The AAV ITRs used in the viral vectors of the present invention may have a wild-type nucleotide sequence or may be altered by insertion, deletion, or substitution. The serotype of the AAV inverted terminal repeats (ITRs) can be selected from any known human or non-human AAV serotype. In a specific embodiment, a nucleic acid construct or viral expression vector can be obtained using ITRs from any AAV serotype, including AAV1, AAV2, AAV3 (including types 3A and 3B), AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, avian AAV, bovine AAV, canine AAV, equine AAV, ovine AAV, and any other AAV serotypes now known or later discovered.

[0092] In a preferred embodiment, the nucleic acid construct or expression vector further comprises the 5' and 3' ITRs of AAV of serotype AAV2.

[0093] In other embodiments, the nucleic acid constructs or expression vectors of the invention can be obtained using synthetic 5' and / or 3' ITRs, and using 5' and 3' ITRs derived from viruses of different serotypes. All other viral genes required for viral vector replication can be provided in trans in virus producer cells (packaging cells), as described below. Therefore, their incorporation into viral vectors is optional.

[0094] In one embodiment, the nucleic acid construct or viral vector of the present invention comprises a viral 5' ITR, a psi packaging signal, and a 3' ITR. The "psi packaging signal" is a cis-acting nucleotide sequence in the viral genome that is essential for the process of packaging the viral genome into viral capsids during replication in some viruses (e.g., adenovirus, lentivirus, etc.).

[0095] The construction of recombinant AAV viral particles is generally known in the art and is described, for example, in U.S. Pat. Nos. 5,173,414 and 5,139,941; WO 92 / 04094; 1070, WO 93 / 03769, Lebkowski et al. (1988) Molec. Cell. Biol. 8:3988-3996, Vincent et al. (1990) Vaccines 90 (Cold Spring Harbor Laboratory Press), Carter, BJ (1992) Current Opinion in Biotechnology 3:533-539, Muzyczka, N. (1992) Current Topics in Microbiol. and Immunol. 158:97-129, and Kotin, RM (1994) Human Gene Therapy 5:793-801.

[0096] virus particles The nucleic acid constructs or expression vectors of the present invention can be packaged into viral capsids to produce "viral particles," also called "viral vector particles."

[0097] Thus, in one aspect, the invention relates to a viral particle comprising a nucleic acid construct or an expression vector of the invention.

[0098] In one aspect, the present invention relates to viral particles comprising a nucleic acid construct or expression vector comprising a promoter operably linked to a nucleotide sequence encoding cPLA2e, and viral particles comprising a nucleic acid construct or expression vector comprising a) a promoter operably linked to a nucleotide sequence encoding cPLA2e, b) a polyadenylation signal sequence, and c) a 5'ITR and a 3'ITR, optionally in combination with one or more features of the various embodiments described above or below.

[0099] In a preferred embodiment, the viral particles of the invention are AAV particles comprising adeno-associated virus capsid proteins; i.e., the nucleic acid constructs or expression vectors of the invention are packaged in AAV-derived capsids to generate "adeno-associated viral particles" or "AAV particles." The term AAV particle encompasses any genetically engineered recombinant or mutant AAV particle. Recombinant AAV particles can be produced by encapsidating a nucleic acid construct or viral expression vector comprising ITR(s) from a particular AAV serotype into a viral particle formed by native or mutant Cap proteins corresponding to AAV of the same or a different serotype.

[0100] The viral capsid proteins of adeno-associated viruses include capsid proteins VP1, VP2, and VP3. Differences between the capsid protein sequences of various AAV serotypes result in the use of different cell surface receptors for cell entry. This, combined with alternative intracellular processing pathways, results in different tissue tropism for each AAV serotype.

[0101] In one embodiment, AAV particles according to the present invention can be produced by encapsidating a viral vector of an AAV vector / genome derived from a specific AAV serotype onto a viral particle formed by the native Cap protein corresponding to the AAV of the same specific serotype. Nevertheless, several approaches have been developed to modify and improve the structural and functional properties of naturally occurring AAV viral particles (Buenning H et al. J Gene Med, 2008; 10: 717-733; Paulk et al. Mol Ther. 2018; 26(1):289-303; Wang L et al. Mol Ther. 2015; 23(12):1877-87; Vercauteren et al. Mol Ther. 2016; 24(6):1042-1049; Zinn E et al., Cell Rep. 2015; 12(6):1056-68).

[0102] Thus, in another embodiment, the AAV viral particles according to the present invention may be, for example, a) viral particles composed of capsid proteins from the same or different AAV serotypes (e.g., AAV2 ITR and AAV9 capsid protein, AAV2 ITR and AAV TT capsid protein, etc.), b) mosaic viral particles composed of a mixture of capsid proteins from different AAV serotypes or mutants (e.g., two or AAV2 ITRs having capsids formed by proteins of multiple AAV serotypes), c) chimeric viral particles composed of capsid proteins truncated by domain swapping between different AAV serotypes or variants (e.g., AAV2 ITRs comprising AAV5 capsid proteins with AAV3 domains), or d) nucleic acid constructs comprising a nucleotide sequence encoding cPLA2e flanked by ITR(s) of a given AAV serotype to be packaged into targeted viral particles engineered to display selective binding domains that enable precise interaction with target cell-specific receptors.

[0103] In specific embodiments, examples of AAV serotypes of the capsid protein of an AAV particle according to the present invention include AAV2, AAV5, AAV9, and AAV TT. In more preferred embodiments, the AAV serotype of the capsid protein is selected from the AAV9 and AAV TT serotypes.

[0104] In certain embodiments, optionally in combination with one or more features of the various embodiments described above or below, the viral particle is an AAV particle comprising a nucleic acid construct or expression vector comprising 5' ITR and 3' ITR sequences derived from an AAV virus, preferably the AAV particle comprises capsid proteins of the AAV2, AAV5, AAV9 or AAV TT serotype, more preferably the AAV9 serotype or the AAV TT serotype, and / or the 5' ITR and 3' ITR sequences of the AAV2 serotype.

[0105] In certain embodiments, optionally in combination with one or more features of the various embodiments above or below, the viral particle comprises a nucleic acid construct or expression vector comprising a nucleotide sequence encoding human cPLA2e of amino acid sequence SEQ ID NO: 1 or SEQ ID NO: 3 under the control of a promoter, wherein the promoter enables expression of the human cPLA2e in at least hippocampal neurons, the viral particle is selected from among viral particles that target at least hippocampal neurons and is typically an AAV particle comprising a capsid protein selected from the group consisting of AAV2, AAV5, AAV9 or AAV TT serotypes, and preferably the nucleotide sequence encoding human cPLA2e is SEQ ID NO: 2 or SEQ ID NO: 4, and / or the promoter is a neuron-specific promoter, more preferably a SYN1 promoter or a hybrid SYN1 promoter.

[0106] In more specific embodiments, such recombinant AAV particles according to the invention comprise capsid proteins of AAV9 or AAV TT serotype and an AAV vector comprising (i) a nucleic acid construct comprising a hybrid SYN1 promoter operably linked to the nucleotide sequence of SEQ ID NO: 2 or 4 encoding human cPLA2e, and (ii) AAV ITRs, such as the 5' and 3' ITRs of AAV2, flanking the nucleic acid construct.

[0107] The AAV viral particles of the present invention may be derived from AAV1, AAV2, AAV3 (including types 3A and 3B), AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, avian AAV, bovine AAV, canine AAV, equine AAV, ovine AAV, synthetic AAV variants such as NP40, NP59, NP84 (Paulk et al., Mol. Ther. 2018.26(1):289-303), LK03(Wang L et al. Mol Ther. 2015. 23(12):1877-87) , AAV3-ST (Vercauteren et al. Mol Ther. 2016.24(6):1042-1049), Anc8 0 (Zinn E et al., Cell Rep. 2015;12(6):1056-68), and any currently known or future It will be understood by those skilled in the art that the capsid proteins may be derived from any AAV serotype, including any other AAV serotype that has been discovered.

[0108] Production of vectors and viral particles The production of viral particles comprising an expression viral vector as disclosed above can be achieved by This can be done using conventional methods and protocols, chosen taking into account the structural features selected by the actual embodiment of the expression vector and the viral particle of the vector.

[0109] Briefly, viral particles can be produced in host cells, particularly specific virus-producing cells (packaging cells), transfected with the nucleic acid construct or expression vector to be packaged in the presence of a helper vector or virus, or other DNA construct(s).

[0110] As used herein, the term "packaging cell" refers to a cell or cell line that may be transfected with a nucleic acid construct or expression vector of the present invention and that provides in trans all missing functions required for complete replication and packaging of a viral vector. Typically, packaging cells constitutively or inducibly express one or more of the missing viral functions. The packaging cells may be adherent or suspension cells.

[0111] Typically, a method for producing viral particles includes the steps of: a) culturing packaging cells containing the nucleic acid construct or expression vector as described above in a culture medium; and b) collecting viral particles from the cell culture supernatant and / or from within the cells.

[0112] AAV viral particles can be produced using conventional methods consisting of transient cell co-transfection with a nucleic acid construct or expression vector (e.g., a plasmid) containing a transgene of the present invention, a nucleic acid construct encoding the rep and cap genes but not the ITR sequences (e.g., an AAV helper plasmid), and a third nucleic acid construct (e.g., a plasmid) that provides the adenoviral functions required for AAV replication. Viral genes required for AAV replication are referred to herein as viral helper genes. Typically, the genes required for AAV replication are adenoviral helper genes such as E1A, E1B, E2a, E4, or VA RNA. Preferably, the adenoviral helper genes are of the Ad5 or Ad2 serotype.

[0113] Large-scale production of AAV particles according to the present disclosure can also be achieved, for example, by infection of insect cells with combinations of recombinant baculoviruses (Urabe et al. Hum. Gene Ther. 2002; 13: 1935-1943). SF9 cells are co-infected with two or three baculovirus vectors expressing AAV rep, AAV cap, and the AAV vector to be packaged, respectively. The recombinant baculovirus vectors provide viral helper gene functions required for viral replication and / or packaging. Smith et al. 2009 (Molecular Therapy, vol. 17, no. 11, pp 1888-1896) demonstrated large-scale production of AAV particles in insect cells. A dual baculovirus expression system for the production of ribosomal RNA is further described.

[0114] Suitable culture media are known to those skilled in the art. The components constituting such media may vary depending on the type of cells to be cultured. In addition to nutrient composition, osmolarity and pH are considered important parameters of culture media. Cell growth media contain numerous components known to those skilled in the art, including amino acids, vitamins, organic and inorganic salts, carbohydrate sources, lipids, trace elements (CuSO4, FeSO4, Fe(NO3)3, ZnSO4, etc.), each present in an amount that supports in vitro cell culture (i.e., cell survival and growth). The components may also contain different auxiliary substances, such as buffer substances (sodium bicarbonate, Hepes, Tris, etc.), oxidative stabilizers, stabilizers against mechanical stress, protease inhibitors, animal growth factors, plant hydrolysates, anti-caking agents, anti-foaming agents, etc. The properties and composition of cell growth media vary depending on the specific cell requirements. Examples of commercially available cell growth media include MEM (Minimum Essential Medium), BME (Basal Eagle Medium), DMEM (Dulbecco's Modified Eagle Medium), Iscove's Modified Dulbecco's Medium, GMEM, RPMI 1640, Leibovitz L-15, McCoy's, 199 medium, Ham's (Ham's medium) F10, and its derivatives Ham's F12 and DMEM / F12.

[0115] Further guidance for the construction and production of viral vectors for use in accordance with the present disclosure can be found in Viral Vectors for Gene Therapy, Methods and Protocols. Series: Methods in Molecular Biology, Vol. 737. Merten and Al-Rubeai (Eds.); 2011 Humana Press (Springer); Gene Therapy. M. Giacca. 2010 Springer-Verlag; Heilbronn R. and Weger S. Viral Vectors for Gene Transfer: Current Status of Gene Therapeutics. In: Drug Delivery, Handbook of Experimental Pharmacology 197, M. Schaefer-Korting (Ed.). 2010 Springer-Verlag; pp. 143-170; Adeno-Associated Virus: Methods and Protocols. RO Snyder and P. Moulliier (Eds). 2011 Humana Press (Springer); Buenning H. et al. Recent developments in adeno-associated virus technology. J. Gene Med. 2008; 10:717-733, Adenovirus: Methods and Protocols. M. Chillon and A. Bosch (Eds.); Third Edition. 2014 Humana Press (Springer).

[0116] In another aspect, the present invention relates to a host cell comprising the nucleic acid construct or expression vector of the present invention.

[0117] In one embodiment, the host cells according to the invention are specific virus-producing cells, also called packaging cells, which are transfected with the nucleic acid constructs or expression vectors according to the invention in the presence of a helper vector or virus or other DNA construct, and which provide in trans all missing functions required for the complete replication and packaging of viral particles. The packaging cells can be adherent or suspension cells.

[0118] For example, the packaging cells may be eukaryotic cells, such as mammalian cells, including monkey, human, canine, and rodent cells. Examples of human cells include PER.C6 cells (WO 01 / 38362), MRC-5 (ATCC CCL-171), WI-38 (ATCC CCL-75), HEK-293 cells (ATCC CRL-1573), HeLa cells (ATCC CCL2), and fetal rhesus lung cells (ATCC CL-160). Examples of non-human primate cells include Vero cells (ATCC CCL81), COS-1 cells (ATCC CRL-1650), and COS-7 cells (ATCC CRL-1651). Examples of canine cells include MDCK cells (ATCC CCL-34). Examples of rodent cells include hamster cells such as BHK21-F, HKCC, or CHO cells.

[0119] As an alternative to mammalian sources, packaging cells for production of viral particles can be derived from avian sources such as chicken, duck, goose, quail, or pheasant. Examples of avian cell lines include avian embryonic stem cells (WO 01 / 85938 and WO 03 / 076601), immortalized duck retinal cells (WO 2005 / 042728), and cells derived from avian embryonic stem cells, including chicken cells (WO 2006 / 108846) or duck cells, such as the EB66 cell line (WO 2008 / 129058 and WO 2008 / 142124).

[0120] In another embodiment, the cell can be any cell that is permissive for baculovirus infection and the replicative packaging cell. In a specific embodiment, the cell is an insect cell such as SF9 cells (ATCC CRL-1711), Sf21 cells (IPLB-Sf21), MG1 cells (BTI-TN-MG1), or High Five™ cells (BTI-TN-5B1-4).

[0121] Thus, in certain embodiments, the host cell contains a nucleic acid construct or expression vector comprising a nucleotide sequence encoding a cPLA2e according to the present invention (e.g., a cPLA2e vector according to the present invention). AAV vectors prepared by the method described above), nucleic acid constructs, such as plasmids, encoding the AAV rep and / or cap genes without ITR sequences, and / or nucleic acid constructs, such as plasmids or viruses, containing viral helper genes.

[0122] In another aspect, the invention relates to a host cell transduced with an expression vector or viral particle of the invention, the term "host cell" as used herein referring to any cell line that is susceptible to infection by the virus of interest and suitable for in vitro culture.

[0123] In other embodiments, the host cells of the invention can be used for therapeutic purposes, such as the therapeutic applications disclosed herein.

[0124] Pharmaceutical Composition Another aspect of the present invention is a pharmaceutical composition comprising a nucleic acid construct as described above, a vector as described above, a host cell as described above or a viral particle as described above in combination with one or more pharmaceutically acceptable excipients.

[0125] In another aspect, the present invention also refers to a pharmaceutical composition comprising a cPLA2e inducer in any of the embodiments disclosed above or below for use or administration in the treatment of cognitive impairment and / or diseases associated with cognitive impairment.

[0126] As used herein, the term "pharmaceutically acceptable" means approved by a regulatory agency or an official pharmacopoeia, such as the European Pharmacopoeia, for use in animals and / or humans. The term "excipient" refers to a diluent, adjuvant, carrier, or vehicle with which a therapeutic agent is administered.

[0127] Pharmaceutical compositions or medicaments of the present invention typically comprise an effective amount of a therapeutic agent (e.g., a vector or viral particle of the present invention) sufficient to produce the desired therapeutic effect, and a pharmaceutically acceptable carrier or excipient.

[0128] In a preferred embodiment, optionally in combination with one or more features of the various embodiments above or below, the present invention then relates to a pharmaceutical composition comprising a vector or viral particle as disclosed above and a pharmaceutically acceptable carrier.

[0129] Any suitable pharmaceutically acceptable carrier or excipient can be used to prepare pharmaceutical compositions (see, for example, Remington: The Science and Practice of Pharmacy, Alfonso R. Gennaro (Editor), Mack Publishing Company, April 1997). Pharmaceutical compositions are typically sterile and stable under the conditions of manufacture and storage. Pharmaceutical compositions can be formulated as a solution (e.g., saline, dextrose solution, or buffer, or other pharmaceutically acceptable sterile liquid), microemulsion, liposome, or other ordered structure (e.g., microparticles or nanoparticles) suitable for accommodating high product concentrations. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, etc.), and suitable mixtures thereof. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. In many cases, it is preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride in the composition. Preferably, the pharmaceutical composition is formulated as a solution, more preferably a saline solution that is optionally buffered.

[0130] Preferably, the pharmaceutical composition is in a solution, more preferably in saline, optionally buffered. Supplementary active compounds can also be incorporated into the pharmaceutical compositions of the present invention. Guidance regarding the co-administration of additional therapeutic agents can be found, for example, in the Canadian Pharmaceutical Association's Compendium of Pharmaceuticals and Specialties (CPS).

[0131] In one embodiment, the pharmaceutical composition is suitable for intraparenchymal, intracerebral, intravenous, or intrathecal administration. These pharmaceutical compositions are exemplary only and are not intended to be limiting of pharmaceutical compositions suitable for other parenteral and non-parenteral routes of administration. The pharmaceutical compositions described herein can be packaged in single unit dose or multi-dose form.

[0132] therapeutic use Using an animal model of Alzheimer's disease (APP / PS1 mice) and aged WT mice, the inventors surprisingly found that AAV-mediated enhancement of cPLA2e expression improved memory impairment in APP / PS1 mice and memory function in aged WT animals.

[0133] These results provide strong evidence for a possible therapeutic strategy for the treatment of cognitive impairment and / or diseases associated with cognitive impairment in subjects, more particularly for the treatment of dementia, such as age-related dementia or Alzheimer's disease.

[0134] Therefore, another aspect of the present invention relates to a method for treating cognitive impairment and / or a disease associated with cognitive impairment, such as dementia, particularly age-related dementia or Alzheimer's disease, in a subject in need of treatment, which method comprises administering a therapeutically effective amount of a cPLA2e inducer to the subject.

[0135] In a further aspect, the present invention relates to a cPLA2e inducer for use as a medicament in a subject in need of treatment, more particularly for use in the treatment of cognitive impairment and / or diseases associated with cognitive impairment, such as dementia, more particularly age-related dementia or Alzheimer's disease, in a subject in need of treatment.

[0136] In a related aspect, the present invention relates to the use of cPLA2e inducers for the manufacture of a medicament, more particularly for the treatment of cognitive impairment and / or diseases associated with cognitive impairment, such as dementia, more particularly age-related dementia or Alzheimer's disease.

[0137] The cPLA2e inducers used or administered in the treatment of cognitive impairment and diseases associated with cognitive impairment according to the present invention can be selected from the group consisting of: a) the nucleic acid construct of the present invention as described above; b) a vector comprising the nucleic acid construct of the present invention as described above; c) a viral particle comprising the nucleic acid construct or vector of the present invention as described above; d) a host cell comprising the nucleic acid construct or vector according to the present invention; e) a cPLA2e polypeptide or protein; and f) a pharmaceutical composition comprising any of the above-mentioned nucleic acid constructs, vectors comprising the nucleic acid constructs, viral particles comprising the nucleic acid constructs or vectors, and cPLA2e polypeptides or proteins.

[0138] With respect to the above cPLA2e polypeptides or proteins, any and all of the embodiments and preferred embodiments described above for cPLA2e encoded by the nucleotide sequence contained in the nucleic acid construct of the present invention are embodiments within the scope of cPLA2e polypeptides or proteins used or administered in treatment according to the present invention, particularly cPLA2e comprising or consisting solely of the amino acids of SEQ ID NO: 1 or 3, or a variant having at least 70% sequence identity thereto, optionally as a fusion protein with another polypeptide(s) such as a tag or carrier polypeptide.

[0139] In a preferred embodiment, the cPLA2e inducer for therapeutic use according to the present invention is a vector of the present invention, more preferably a viral vector or viral particle (eg, AAV particle), or a pharmaceutical composition comprising same.

[0140] As used herein, the terms "cognitive disorder" and "cognitive dysfunction" refer interchangeably to any cognitive dysfunction, such as a condition characterized by one or more of the following behaviors: impairment of at least one form of learning (e.g., associative learning), impairment of at least one form of memory function (e.g., executive function), impairment of learning, impairment of memory acquisition, impairment of memory retrieval, suppression of long-term potentiation (LTP) in the hippocampus, or a combination thereof. In humans, cognition and its potential dysfunction can be determined or assessed using any suitable method for testing hippocampal or brain function and neuroimaging. For example, cognition (e.g., memory or learning) and its potential dysfunction can be determined or assessed using, but not limited to, the "Kiel Locomotor Maze," which has features of the radial arm maze and Morris water maze optimized for assessing spatial memory and orientation in school-age children, or the "Kiel Locomotor Maze," which has features of the radial arm maze and Morris water maze optimized for assessing spatial memory span, spatial working memory, and spatial cognition. Neuropsychological function can be measured using any suitable psychological test, including the Cambridge Neuropsychological Test Automated Battery (CANTAB), a specially designed battery of computerized, non-verbal, visually presented neuropsychological tests. The results of the methods disclosed herein regarding cognitive impairment can be demonstrated by comparative studies in animals (eg, rats or mice) using the same compositions administered to humans.

[0141] The cPLA2e inducer of the present invention is particularly useful for treating cognitive impairment and diseases associated with cognitive impairment, such as dementia (e.g., age-related dementia (senile dementia), vascular dementia and / or neurodegenerative dementia diseases associated with abnormal protein aggregation, particularly Alzheimer's disease, Parkinson's disease, ALS or prion disease, Creutzfeldt-Jakob disease, or Gerstmann-Straussler-Scheinker disease), mild cognitive impairment, attention deficit disorder.Therefore, the cPLA2e inducer of the present invention is particularly used for treating cognitive impairment associated with one of these pathologies.

[0142] In a preferred embodiment, cPLA2e inducers are particularly useful for treating cognitive impairment in patients with Alzheimer's disease. As used herein, Alzheimer's disease (AD) refers to a progressive neurodegenerative disorder of the central nervous system of unknown cause. In the context of AD, the defining feature is cognitive impairment. AD is defined as a neurodegenerative disorder that constitutes the major form of dementia common in the elderly and is characterized by the accumulation in the brain of two abnormal proteins, β-amyloid peptide and hyperphosphorylated tau, in the form of amyloid plaques and neurofibrillary tangles, respectively. The criteria for diagnosing AD published in 1984 by the National Institute of Neurological and Communicative Disorders and Stroke (NINCDS) and the Alzheimer's disease and Related Disorders Association (ARRDA) include: (1) two or more These criteria include (1) the presence of progressive dementia, (2) the absence of altered consciousness, (3) the absence of altered consciousness, (4) onset between the ages of 40 and 90, and (5) the absence of other causes. Characteristic and reliable AD biomarkers are now available through structural MRI, molecular neuroimaging with PET, and cerebrospinal fluid analysis to confirm the diagnosis of AD. Furthermore, a pre-Alzheimer's disease prodromal state known as mild cognitive impairment (MCI) is defined as memory loss that is objectively abnormal for the subject's age and education level. Criteria for MCI include (1) memory complaints confirmed by a family member, (2) the presence of normal other cognitive function, (3) normal daily activities, (4) abnormal memory for age, and (5) the absence of dementia.

[0143] As used herein, the term "subject" or "patient" refers to a mammal. Mammalian species that may benefit from the disclosed therapeutic methods include, but are not limited to, humans, non-human primates such as apes, chimpanzees, monkeys and orangutans, domestic animals including dogs and cats, and livestock such as horses, cows, pigs, sheep and goats, or other mammalian species including, but not limited to, mice, rats, guinea pigs, rabbits, hamsters, etc.

[0144] As used herein, "treatment," "treating," or "treat" " refers to (i) preventing or delaying the occurrence of a disease, disorder, or condition in a subject who may be predisposed to, but has not yet been diagnosed as having, the disease, disorder, or condition; (ii) inhibiting the disease, disorder, or condition, i.e., arresting or slowing its onset or progression; and / or (iii) palliating the disease, disorder, or condition, i.e., causing regression of the disease, disorder, and / or condition. In certain embodiments, such terms refer to the amelioration or eradication of a disease or symptoms associated with a disease.

[0145] With respect to cognitive impairment, "treatment" or "treating" refers to (i) preventing or delaying the occurrence of cognitive impairment in a subject who may be susceptible to, but has not yet been diagnosed with, cognitive impairment; (ii) inhibiting cognitive impairment, i.e., arresting or slowing its onset or progression; (iii) alleviating cognitive impairment, i.e., causing its regression; and / or (iv) improving cognitive ability. The term "treating cognitive impairment," unless otherwise specified herein, refers to alleviating cognitive impairment, ameliorating at least one symptom associated with or caused by cognitive impairment (such as a symptom of a disease or disorder that may cause cognitive impairment), or both. Treatment of cognitive impairment particularly relates to the treatment of learning and memory disorders and the improvement of learning and memory ability. "Improving learning and memory ability" refers to improving or increasing the mental ability to record, retain, or recall past experiences, knowledge, ideas, sensations, thoughts, or impressions.

[0146] As used herein, a "therapeutically effective amount" refers to an amount effective, at dosages and for periods of time required, to achieve a desired therapeutic outcome, such as one or more of the following: a significant delay in the onset or progression of the disease, a significant decrease in the severity of one or more symptoms, a significant decrease in characteristics of AD, amyloid and / or tau pathology, a significant increase in synaptic plasticity, a significant decrease in mortality associated with aging and / or AD.

[0147] A therapeutically effective amount is typically also one in which any toxic or detrimental effects of the product or pharmaceutical composition are outweighed by the therapeutically beneficial effects.

[0148] In one embodiment, a nucleic acid construct, expression vector, viral particle, host cell, cPLA2e polypeptide or protein, or pharmaceutical composition for therapeutic use according to the present invention is administered to a subject or patient by a parenteral route, such as intraparenchymal, intracerebral, intracerebroventricular (icv), intrathecal, intranasal, intravenous, or subcutaneous route.

[0149] Generally, therapeutically effective amount of the above-mentioned nucleic acid construct, expression vector, viral particle, host cell, cPLA2e polypeptide or protein, or pharmaceutical composition is preferably administered by intrathecal or intraparenchymal route, and the latter is preferably administered to brain regions such as hippocampal formation or cerebral cortex.Intraparenchymal route can promote the preferred local administration to hippocampus and cortex compared with other brain regions.As used herein, "preferably local administration to hippocampus" does not mean that all cPLA2e inducer is administered to the above-mentioned brain region, but means that the majority of cPLA2e inducer, for example, at least 50%, at least 60%, at least 70% or at least 80%, is administered to the above-mentioned region.

[0150] The therapeutically effective amount of a cPLA2e inducer (e.g., a nucleic acid construct, expression vector, viral particle, host cell, or cPLA2e polypeptide or protein), or a pharmaceutical composition containing same, may vary depending on factors such as the disease state, age, sex, and weight of the individual, and the ability of the product or pharmaceutical composition to elicit a desired response in the individual. Dosage regimens can be adjusted to provide the optimal therapeutic response.

[0151] For any particular subject, specific dosage regimens can be adjusted over time according to the individual needs and the professional judgment of the person administering or supervising the administration of the compositions. Dosage ranges set forth herein are exemplary only and do not limit the dosage ranges that may be selected by a physician.

[0152] In one embodiment, the AAV viral particles according to the invention are administered to a human subject or patient for the treatment of cognitive impairment or a disease associated with cognitive impairment, such as Alzheimer's disease. 8 vg / kg~10 14 vg / kg (vg: viral genome; kg: subject or patient weight), e.g., 1 × 10 10 vg / kg~5×10 14 In a more particular embodiment, the amount or dose may be administered in the range of 1 x 10 vg / kg. 12 vg / kg~1×10 13 In an alternative embodiment, the amount is administered in the range of 1 x 10 vg / kg. 9 iu / kg~1×10 11 Amounts or doses falling within the range of iu / kg (iu: infectious unit of vector) are administered.

[0153] In another aspect, the present invention further relates to a kit comprising, in one or more containers, a nucleic acid construct, expression vector, host cell, viral particle, or pharmaceutical composition comprising said nucleic acid construct, vector, host cell, or viral particle of the invention. The kit may include instructions or packaging material describing how to administer the nucleic acid construct, expression vector, viral particle, host cell, or pharmaceutical composition contained in the kit to a patient. The containers of the kit may be made of any suitable material, such as glass, plastic, metal, etc., and may be of any suitable size, shape, or configuration. In certain embodiments, the kit may include one or more ampoules or syringes containing the product of the invention in a suitable liquid or solution form.

[0154] The following examples are offered for illustrative purposes and are not intended to limit the invention. Furthermore, the present invention includes all possible combinations of specific preferred embodiments described herein.

[0155] Methods for screening new agents useful in the treatment of cognitive impairment and / or diseases associated with cognitive impairment - Patent Application 20070122993 The inventors are also working to develop a system for screening compound candidates, such as peptides, polypeptides (e.g., antibodies), or small molecule candidates, by taking advantage of the fact that induction or increase of cPLA2e results in a highly significant increase in calcium-dependent N-acyltransferase (Ca-NAT) activity.

[0156] Therefore, the present invention provides a) contacting a compound with a mammalian assay cell; b) determining whether an effect associated with induction or increase of cPLA2e is obtained; c) if such an effect is obtained compared to a control, identifying the compound as a candidate for the treatment of cognitive impairment and / or a disease associated with cognitive impairment; Also provided are methods for identifying compounds as candidates for the treatment of cognitive impairment and / or diseases associated with cognitive impairment, comprising:

[0157] A possible embodiment of the method of the present invention is where the cells to be assayed are mammalian cells such as HEK293T cells. The in vitro method involves performing an in vitro method on mammalian cells. These cells are cultured (e.g., for 30 minutes or 1 hour) in a medium suitable for cell growth and proliferation in the presence of a candidate compound, with or without ionomycin (e.g., 2 μM), and Ca-NAT activity is tested (e.g., by targeted metabolite profiling) compared to a control cell not exposed to the candidate compound. If Ca-NAT activity is found to be increased relative to the control cell, the compound is identified as a potential candidate for the treatment of cognitive impairment and / or diseases associated with cognitive impairment.

[0158] In some embodiments, cPLA2e transfected cells (eg, with a nucleic acid construct of the invention) can be used as a positive control for induction of cPLA2e activity.

[0159] The term "induce or increase" as used herein may refer to the ability to cause an overall increase, preferably of 20% or more, more preferably of 50% or more, and most preferably of 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more. [Example]

[0160] To determine whether PLA2G4E is involved in learning and memory function, we overexpressed PLA2G4E in the brains of a) APP / PS1 mice (a model of AD) and b) aged wild-type animals, both of which suffer from cognitive impairment. To this end, an AAV vector containing Mus musculus cPLA2e as a transgene was constructed and administered to the animals. Learning and memory function was then assessed using the MWM method.

[0161] Example 1. Construction of AAV2 / 9-mPLA2G4E An AAV2 / 9-mPLA2G4E vector was constructed containing the nucleotide sequence of SEQ ID NO: 5 encoding mouse PLA2G4E fused to the flag sequence via a linker as a transgene.

[0162] First, a 3108 bp fragment containing mouse PLA2G4E fused to the FLAG sequence was cloned from the plasmid pRK5-PLA2G4E (a gift from BJ Cravatt; disclosed in Ogura Y et al. Nat. Chem. Biol. 2016; 12(9): 669-671) into CutSm The fragments were excised by digestion with XmnI and SacI in art™ buffer, separated by electrophoresis on a 1% agarose gel, extracted from the gel using the QIAquick™ Gel Extraction Kit (QIAGEN), and purified using QIAquick™. The product was purified using a QIAGEN™ PCR Purification Kit.

[0163] Next, a 4163 bp backbone fragment was obtained from the plasmid pAAV-hα-synuclein A53T (a kind gift from Dr. J. Gerez) by digestion with Xhol (in CutSmart™ buffer) followed by treatment with Klenow polymerase, dNTPs, and NEB2.1 buffer. After purification, the 4163 bp backbone fragment was then digested with SacI (in CutSmart™ buffer) and purified with shrimp alkaline phosphatase rSAP (New England Biolabs, MA, USA; Weissig, H. et al. Biochem. J. 1993; 290: 503-508) to avoid religation of the vector. The 4163 bp backbone fragment was finally isolated, extracted and purified as described above for the 3108 bp fragment.

[0164] Finally, the 3108 bp fragment was cloned into the 4163 bp backbone fragment by treatment with T4 DNA ligase (Invitrogen) to obtain the plasmid pAAV2-mPLA2G4E.

[0165] After generating pAAV2-mPLA2G4E containing the desired construct, it is then The plasmid was subjected to several amplification steps using TOP10 electrocompetent E. coli strains to generate an appropriate amount of plasmid for final virus production. The plasmid was transformed into IntCell (Invitrogen) and bacteria that had integrated the plasmid were selected by plating on LB medium containing ampicillin (50 μg / ml). The plasmid was then obtained from the bacteria and purified using a QIAprep™ Spin Miniprep Kit (QIAGEN). The presence and correct sequence of the insert were confirmed. After confirming the orientation and the presence of AAV2 ITRs, a desired amount of plasmid was isolated from the ampicillin-resistant clones using a commercially available QIAGEN™ Plasmid Maxi Kit (QIAGEN). The plasmid was obtained and purified.

[0166] After constructing and purifying the vector plasmid, AAV vector particles were generated by double transfection of HEK-293T cells with the plasmid pAAV2-mPLA2G4E and the pDP9 helper plasmid, which expresses the adenoviral molecules required for the production and packaging of AAV:AAV9 cap and AAV2 rep (Durocher, Y., S. Perret, and A. Kamen., 2002, Nucleic Acids Research, 30(2):9e-9).

[0167] Vector particles were finally purified by iodixanol gradient and titrated by quantitative PCR. Viral titration, expressed as viral particles (vp) / ml, was determined using primers for mouse PLA2G4E, Forward primer: ATGGTGACAGACTCCTTCGAG (SEQ ID NO: 6) and Reverse primer: CCTCTGCGTAAAGCTGTGG (SEQ ID NO: 7), were obtained by quantitative PCR (q-PCR) using

[0168] The resulting viral titer was 2.6 x 10 11 vp / ml.

[0169] Example 2. General Methods mouse APP / PS1 Mice. The mouse APP / PS1 model expresses human transgenes for amyloid precursor protein (APP) with the Swedish mutation (K595N / M596L) and PSEN1 with the L166P mutation, both driven by the Thy1 promoter. These mice are on an inbred C57BL / 6J genetic background. The AD mouse model APP / PS1 is a more accelerated amyloidosis model than Tg2576. In these mice, expression of the human APP transgene is approximately three-fold higher than endogenous mouse APP, resulting in preferential production of human Aβ42 over Aβ40. Furthermore, amyloid plaque deposition begins in the hippocampus at 3-4 months of age (Radde et al., 2006, EMBO reports; 7(9):940-946; Maia LF et al., 2013, Science translational medicine; 5(194):94-194), and cognitive impairment appears from 7 months of age (Serneels L et al., 2009, Science; 324(5927):639-642). For APP / PS1 and their corresponding negative littermates, both male and female mice aged 16-19 months were used.

[0170] Aged wild-type mice. Wild-type mice exhibit age-related memory deficits. Specifically, in the Morris water maze, aged wild-type mice perform significantly worse than young mice during the exploration trial and therefore do not form a robust memory for the platform location during the hidden platform phase. These mice were on an inbred C57BL / 6 / SJL genetic background.

[0171] Two-month-old male wild-type (WT) C57BL / 6 mice were also used to test the effects of PLA2G4E on synaptic activity.

[0172] Stereotactic surgery for virus administration To overexpress PLA2G4E in hippocampal neurons, mice were administered AAV2 / 9-mPLA2G4E into the CA1 region of the hippocampus by stereotaxic surgery. This procedure is based on a three-dimensional system of axial and spatial coordinates that allows localization of specific points in the mouse brain (given as three-dimensional distances in millimeters (mm)) relative to two easily identifiable points in the brain: bregma and lambda. Using a mouse atlas (G. Paxinos and K.B.J. Franklin, "The mouse brain in stereotaxic coordinates," Academic Press, 1997), the coordinates chosen for hippocampal CA1 injection were anterior-posterior -2.0 mm, hemi-lateral ±1.7 mm, and posterior-ventral -2.0 mm relative to the bregma point (formed by the intersection of the sagittal and coronal sutures). Prior to virus administration or sham procedures (no injections, only surgery), animals were anesthetized by intraperitoneal (ip) administration of 80 / 10 mg / Kg ketamine / xylazine and treated with the analgesic buprenorphine (Buprex™) at a dose of 0.1 mg / Kg. After the mice were fully anesthetized, they were placed in a stereotaxic device with their heads completely fixed. After disinfecting the area with 96-degree alcohol, a scalpel was used to cut the skin in an anterior-posterior direction, freeing the skull from the periosteum and making the bregma and lambda reference points visible. Next, a drill bill was used to drill a hole in the skull, and vector virus particles (2.6 × 10 8A 5 μl Hamilton syringe, either loaded with 1000 copies of the virus (genome copies) or unloaded (for sham procedures), was attached to the stereotaxic arm. After positioning at the correct coordinates, 1 μl of solution was injected at 0.2 μl / min, and the syringe was then maintained there for an additional 2 minutes to allow for accurate virus diffusion before slowly withdrawing it. For sham-injected mice, the syringe was left in the brain for 5 minutes before being withdrawn. The same procedure was repeated for the other hemisphere. After bilateral injections, the animals were sutured and topically administered povidone-iodine (Betadine™). The animals were then placed on an electric blanket until they woke up to avoid heat loss. Finally, to facilitate postoperative feeding, the animals were individually placed in clean cages with easy access to water-softened food. Throughout the intervention, physiological serum was continuously applied to the eyes of the mice to prevent dryness and resulting blindness.

[0173] MWM Test Spatial memory was tested using the Morris water maze test, which analyzes both spatial and working memory and is considered a consistent test for assessing hippocampal damage, one of the main features of AD in humans (D'Hooge and Deyn, 2001, Brain research reviews; 36(1):60-90).

[0174] The test was conducted in a circular pool (1.2 m diameter) filled with water at 20°C and made opaque by the addition of non-toxic white paint. The pool was divided into four virtual quadrants, one of which contained a platform whose location the mouse had to learn in order to escape the water and reach safety. Each of the four walls surrounding the pool was painted with a geometric shape that served as a guide for the mouse and was covered or uncovered depending on the stage of the test. Throughout the test, the mouse's behavior was monitored by a camera fixed to the ceiling directly above the pool and recorded by the HVS system, allowing subsequent analysis of escape latency, swimming speed, path length, and percentage of time spent in each quadrant of the pool using the software SMART-LD (Panlab).

[0175] Three different phases can be distinguished in the MWM test.

[0176] 1) Visible platform phase: In this phase, a platform, identified by a part that can be clearly recognized by the animal to facilitate location confirmation, was placed in the center of one of the quadrants, 1 cm above the water surface. Here, visual cues were kept hidden so that the mice would become accustomed to the pool and learn to go to the platform to escape from the water. For the visible platform phase, mice were trained 8 times a day for 3 consecutive days. In each test, the mice were given 60 seconds to find the platform, and if they were unable to reach it within this period, they were placed on the platform. After being placed on the platform, the animals were allowed 15 seconds to check the platform, after which they were returned to their cages. It was returned.

[0177] 2) Hidden Platform Phase: In the second phase of the test, the platform was placed in the opposite quadrant to the visible platform phase. The platform was submerged 1 cm below the water surface so that there were no parts on it. In this phase, the mice had to learn how to find the platform using cues displayed on the wall, which was not covered at this point. Therefore, the mice were trained four times a day for 7 days. As in the previous phase, the mice were given 60 seconds to reach the platform. If they did not find the platform within 60 seconds, they were guided to the platform. In both cases, they remained on the platform for 15 seconds. To avoid the development of track preference in the mice, three random starting positions were provided in each quadrant without a platform.

[0178] 3) Exploration Trial: Memory retention was assessed on days 6 and 8 of the hidden platform phase in an exploration trial conducted immediately before the start of the hidden platform test on these days. For this test, the platform was removed from the pool and the animals were allowed to swim for 60 seconds. The time the mice spent in the quadrant containing the platform during the hidden platform phase was considered an estimate of memory retention. A retention rate of more than 25% was considered to indicate learning, while a retention rate of less than 25% was considered random. Because it has been suggested that the sensitivity of the MWM test can be increased by providing shorter exploration trials, the time spent in the correct quadrant was analyzed both during the first 15 seconds and throughout the entire 60 seconds of the test (Gerlai, 2001, Behavioral Brain Research; 125(1-2):269-277).

[0179] Dendritic spine density measurement using Golgi-Cox staining To analyze the density and morphology of dendritic spines, a modified Golgi-Cox method was used (Glaser, Edmund M. and Hendrik Van der Loos, 1981, Journal of Neuroscience Methods 4(2):117-25). First, the hemibrain immediately after removal from the skull was soaked in Golgi-Cox solution (1% The brains were then incubated in a solution of 0.8% potassium chromate (1% potassium dichromate, 1% mercury chloride, 0.8% potassium chromate) at room temperature for 48 hours, protected from light. The solution was then refreshed, and the tissues were maintained there for an additional 3 weeks. The brains were then washed with distilled water and placed in 90°C ethanol for 30 minutes before being processed into 200 μm-thick coronal sections using a vibratome. The sections were then incubated in 70°C ethanol, washed with distilled water, reduced in 16% ammonia for 1 hour, and fixed in 1% sodium thiosulfate for 7 minutes. After further washing, the sections were placed on microscope slides, dehydrated in ascending alcohols, and mounted with DPX Mountant (VWR, BDH Prolabo™).

[0180] The spine density of the secondary apical dendrites of pyramidal neurons located in the CA1 region of the hippocampus was determined. Each selected neuron was photographed using a Nikon Eclipse E600 light microscope, and images were recorded with a digital camera (Nikon DXM 1200F) at a resolution of 1000 dots per inch (dpi) to 1500 dpi. Secondary dendrites photographed 100 μm to 200 μm from the cell body were taken, indicating that spine density is relatively uniform in CA1 pyramidal neurons (Megias, M., Z. Emri, T.F. Freund, and A.I. Gulyas, 2001, Neuroscience 102(3):527-40). Dendrites were used for quantification. For each mouse (n=4 per group), three dendrites from nine different neurons were used for analysis.

[0181] Fear conditioning test (FC) The effect of PLA2G4E expression on fear memory was analyzed using the FC paradigm. This behavioral test consisted of three phases: habituation, training, and testing. The test was performed using the StartFear system (Panlab). In the habituation phase, mice were habituated to the conditioning chamber without stimulation for 3 minutes. 24 hours later, in the training phase, mice were placed in the same chamber again and allowed to explore for 2 minutes. Then, two 2-second flicks were performed with a 30-second interval between each flick. A shock (0.3 mA) was administered, followed by a further 30 seconds before the mice were returned to their home cage. The following day, the mice were returned to the conditioning chamber and allowed to explore the context for 2 minutes. Freezing behavior was recorded during this time, and the freezing score was expressed as a percentage. The T24 group was sacrificed 24 hours after training, and the TT group was sacrificed 1 hour after testing. The naive group was sacrificed without any steps in the paradigm.

[0182] protein extract To obtain total protein extracts, brain samples were homogenized in lysis buffer (10 mM Tris-HCl pH=7.5, 1 mM NaF, 0.1 mM Na3VO4, 2% SDS) containing protease inhibitors, sonicated for 2 min, placed on ice for 20 min, and centrifuged at 15,700 g for 13 min at 8°C. The supernatant was stored at -80°C. Total protein was analyzed using the Pierce™ BCA Protein Assay Kit (Thermo Scientific). Protein concentrations were determined.

[0183] Immunoblotting Protein samples were mixed with 6x Laemmli sample buffer, boiled at 95°C for 5 min, resolved on an SDS-polyacrylamide gel, and transferred to a nitrocellulose membrane.

[0184] The membrane was then blocked with 5% milk in TBS and incubated with the following primary antibodies in the corresponding buffer: rabbit polyclonal anti-pGluA1-Ser831 (1:1000, Millipore ), rabbit monoclonal anti-pCREB (Ser133) (1:1000, Cell Signaling), mouse monoclonal anti-synapsin I (1:1000, Synaptic Systems), rabbit polyclonal anti-PLA2G4E (1:1000, Proteintech), and mouse monoclonal The sections were incubated overnight with a central anti-β-actin antibody (1:100,000, Synaptic Systems). After washing twice with TBS / Tween-20 and once with TBS alone, immunolabeled protein bands were detected with HRP-conjugated anti-rabbit or anti-mouse antibodies (1:5,000, Santa Cruz). Antibody binding was then visualized by an enhanced chemiluminescence system (ECL, GE Healthcare Bioscience) and autoradiography exposure to Hyperfilm™ ECL (GE Healthcare Bioscience). Quantity One™ software v.4.6.3 (Bio-Rad) was used for protein quantification.

[0185] Knockdown of PLA2G4E in primary neuronal cultures We used specific small interfering RNA (siRNA) to inhibit PLA2G4E expression in primary neuronal cultures. To identify effective targeting sequences for RNAi, we analyzed the full-length coding sequence of mouse PLA2G4E using different algorithms. After identifying sequences with high efficacy in inhibiting PLA2G4E expression, we used candidate sequences to design constructs containing the H1 promoter operably linked to an shRNA sequence (SEQ ID NO: 8; i.e., 21-nt sense and antisense sequences connected by a hairpin loop (TCAAGAGA)) followed by a poly(T) termination signal. The shRNA-containing construct was then cloned into adeno-associated virus serotype 9 (AAV9-shPLA2G4E) for stable siRNA delivery (Unitat de Produccio de Vectors, Barcelona).

[0186] To evaluate the selective inhibition of activity-dependent signaling by PLA2G4E, we used primary neuronal cultures as a model to study synaptic responses to evoked burst signals in functional neuronal networks. Primary neuronal cultures were obtained from the hippocampus and cortex of wild-type (WT) mice at embryonic day 16 (E16) (A. Ricobaraza, Neuropsychopharmacology, (2009); 34: 1721-1732), AAV9-shPLA2G4E, or AAV9-sh-scrambled control were infected on in vitro culture day (DIV) 1. These cultures were then treated with the GABA A receptor antagonist bicuculline (50 μM, 1 h) on DIV 14 to induce bursts of action potential firing (Arnold et al., 2005 J. Physiol. 564: 3-19, Rao et al., Nat. Neurosci., 2006; 9: 887-895). Proteins were then soaked in 2% SDS buffer. The cells were buffer-extracted and activation of CREB (phosphorylated at Ser133), pGluA1 and synapsin I expression was examined in the lysates by immunoblotting.

[0187] Example 3. Effect of AAV2 / 9-mPLA2G4E on memory function in APP / PS1 mice The first group (n = 9) of male and female 16- to 19-month-old APP / PS1 mice was treated with AAV2 / 9-mPLA2G4E by stereotaxic surgery as described above. Similarly, a second group (n = 6) of 16- to 19-month-old APP / PS1 mice (sham-injected) and a third group (n = 9) of age-matched non-transgenic mice (n = 9) were included as positive (memory deficit) and negative (no AD-related memory impairment) controls. Two months after stereotaxic surgery, spatial memory was tested using the MWM test as described above. Mice underwent a 3-day visible platform phase followed by a 7-day hidden platform phase. Memory retention was tested on days 6 and 8 in exploration trials conducted immediately before the start of the corresponding hidden platform phase trials.

[0188] In the final trial of the visible platform phase, no significant differences were observed between groups (data not shown), indicating that all animals were able to perform the task under the same conditions.

[0189] As expected, APP / PS1 mice performed significantly worse than WT mice during the hidden platform phase, confirming the spatial memory deficits associated with this AD mouse model (Fig. 1A).Interestingly, treatment with AAV2 / 9-mPLA2G4E rescued the spatial working memory deficits (Fig. 1A).

[0190] Furthermore, as shown in Figure 1B, AAV2 / 9-mPLA2G4E-treated mice spent longer in the correct quadrant than sham-injected mice during the exploration trial on day 6. Similar results were obtained during the exploration trial on day 8 (data not shown), indicating that PLA2G4E overexpression reversed the memory retention deficits exhibited by aged APP / PS1 mice.

[0191] Meanwhile, a 33% mortality rate was observed in sham-injected APP / PS1 mice compared with 10% and 0% in AAV2 / 9-mPLA2G4E-injected APP / PS1 and non-transgenic mice, respectively.

[0192] In conclusion, overexpression of hippocampal PLA2G4E in aged APP / PS1 mice mediated by AAV2 / 9-mPLA2G4E treatment significantly rescued spatial memory impairment 2 months after stereotaxic injection.

[0193] Using the Golgi-Cox method, we analyzed whether the behavioral recovery induced by PLA2G4E overexpression was reflected in structural changes in dendritic spine density, specifically, apical dendrites derived from pyramidal neurons in the CA1 region of the hippocampus.

[0194] As shown in Figures 2A and 2B, the 2 / 9-PLA2G4E virus significantly increased dendritic spine density in both WT and APP / PS1 sham mice, but no difference was observed between WT and APP / PS1 sham mice.

[0195] These results suggest that changes in spine density may explain the memory recovery observed in the PLA2G4E-overexpressing APP / PS1 mice.

[0196] Example 4. Effect of AAV2 / 9-mPLA2G4E on memory function in aged wild-type mice The effect of PLA2G4E overexpression on memory function in 17-month-old female C57BL / 6 / SJL WT mice was also evaluated. One group of C57BL / 6 / SJL WT mice (n = 5) was treated with AAV2 / 9-mPLA2G4E by stereotaxic surgery, and a second control group of C57BL / 6 / SJL WT mice (n = 4) received sham injections. Three months after the stereotaxic surgery procedure, spatial memory was tested using the MWM test as described above. In this case, the hidden platform phase was performed for only 6 days, and the exploration trial was performed on days 5 and 7.

[0197] No significant differences were observed between groups in the visible platform phase (data not shown), indicating that all mice were similarly able to perform the task.

[0198] Although no significant difference was observed between the two groups in the hidden platform phase (Fig. 3A), mice treated with AAV2 / 9-mPLA2G4E spent longer in the correct quadrant than sham-injected mice in the exploration trials performed on days 5 (Fig. 3B) and 7 (data not shown), indicating that hippocampal overexpression of viral PLA2G4E improves memory retention in aged WT mice.

[0199] In summary, hippocampal PLA2G4E overexpression mediated by AAV2 / 9-mPLA2G4E treatment improved memory retention in aged C57BL / 6 / SJL WT mice 3 months after injection.

[0200] Example 5. Role of PLA2G4E in memory function: Upregulation of PLA2G4E expression after fear conditioning memory retrieval To obtain more direct evidence for the functional role of PLA2G4E in learning and memory, we examined whether PLA2G4E expression is regulated in the fear conditioning (FC) test, which requires hippocampal-dependent transcription and protein synthesis and is widely used to characterize the biochemical requirements for memory formation (Huff et al., 2006 J. Neurosci., 26, pp. 1616-1623).

[0201] pCREB and PLA2G4E expression in the brain was analyzed by immunoblot after consolidation of fear memory in 2-month-old C57BL / 6J WT mice (TT group; n = 8) sacrificed 1 h after testing in the FC paradigm and compared with mice sacrificed 24 h after the training phase of FC (T24 group; n = 7) and mice not subjected to any aspect of the FC test (naive group; n = 8).

[0202] As expected, mice reintroduced into their cages (TT group) showed a significant increase (P<0.001) in freezing time (an index of memory formation) during the test phase compared with the training phase (Fig. 4A).

[0203] Because CREB-mediated transcription is required for the consolidation and reconsolidation of contextual fear memory (Kida (2002 Nat. Neurosci., 5, pp. 348-355) first analyzed pCREB as an indicator of neuroplasticity in the hippocampus of animals. Upregulation of pCREB in the hippocampus was observed in groups of mice that were re-caged.

[0204] Also, surprisingly, PLA2G4E expression was stronger in both of these regions in this group of mice compared to the others (FIG. 4C).

[0205] In summary, these data suggest that during contextual memory retention, PLA2G4E is increased in the hippocampus after retrieval of a consolidation memory.

[0206] Example 6. Role of PLA2G4E in synaptic plasticity: knockdown of PLA2G4E Un blocks the activation of synaptic proteins involved in synaptic transmission Given the plausible role of PLA2G4E in memory function, in vitro assays were performed to further characterize its role on synaptic activity.

[0207] We used a well-characterized protocol in cortical and hippocampal primary neurons (Rao et al., 2006 Nat. Neurosci., 9: 887-895) based on exposure to the GABA(A) receptor antagonist bicuculline (50 μM, 1 h), which can induce and / or increase synaptic efficacy at excitatory synapses.

[0208] To demonstrate activation of NMDA receptors, CREB activation (phosphorylation of CREB at the activation site residue Ser133) was analyzed (Ginty et al., 1993 Science 260: 238-241 As shown in Figure 5 and described by several authors (Hardingham et al., 2002 Nat. Neurosci., 5: 405-414), we demonstrated that bicuculline induces sustained CREB phosphorylation at Ser133 (through activation of NMDA receptors) and increases AMPA receptor activation as analyzed by measuring pGluA1 levels (Rao et al., 2006 Nat. Neurosci., 9: 887-895).

[0209] We also analyzed synapsin I levels because this presynaptic protein increases in the hippocampus during long-term potentiation (LTP) (Sato et al., 2000 Brain Res., 872: 219-222) and plays a fundamental role in the formation, maintenance, and rearrangement of synaptic contacts (reviewed in Cesca et al., 2010 Prog. Neurobiol., 91: 313-348). We also observed a significant increase in synapsin I in bicuculline-activated neuronal cultures.

[0210] Next, PLA2G4E expression was analyzed under the same conditions and, interestingly, it was observed to be strongly induced by bicuculline, indicating that neuronal activation indeed upregulates PLA2G4E expression.

[0211] We then analyzed the effects of chronic PLA2G4E knockdown using AAV-shPLA2G4E. In primary neuronal cultures, we demonstrated that treatment with AAV-shPLA2G4E blocked bicuculline-induced PLA2G4E expression and effectively blocked bicuculline-induced activation of CREB and GluA1 (Figure 5). Similarly, acute PLA2G4E knockdown no longer increased synapsin I expression in response to bicuculline.

[0212] In summary, these data suggest that PLA2G4E knockdown can alter synapse formation and / or stability.

[0213] Sequences of the present disclosure SEQ ID NO: 1 Human cytosolic phospholipase A2ε (isoform 1) MSLQASEGCPGLGTNVFVPQSPQTDEEGSRSGRSFSEFEDTQDLDTPGLPPFCPMAPWGSEEGLSPCHLLTVRVIRMKNVRQADMLSQTDCFVSLWLPTASQKKLRTR TISNCPNPEWNESFNFQIQSRVKNVLELSVCDEDTVTPDDHLLTVLYDLTKLCFRKKTHVKFPLNPQGMEELEVEFLLEESPSPPETLVTNGVLVSRQVSCLEVHAQSR RRRKREKMKDLLVMVNESFENTQRVRPCLEPCCPTSACFQTAACFHYPKYFQSQVHVEVPKSHWSCGLCCRSRKKGPISQPLDCLSDGQVMTLPVGESYELHMKSTPC PETLDVRLGFSLCPAELEFLQKRKVVVAKALKQVLQLEEDLQEDEVPLIAIMATGGGTRSMTSMYGHLLGLQKLNLLDCASYITGLSGATWTMATLYRDPDWSSKNLEP AIFEARRHVVKDKLPSLFPDQLRKFQEELRQRSQEGYRVTFTDFWGLLIETCLGDERNECKLSDQRAALSCGQNPLPIYLTINVKDDVSNQDFREWFEFSPYEVGLQK YGAFIPSELFGSEFFMGRLVKRIPESRICYMLGLWSSIFSLNLLDAWNLSHTSEEFFHRWTREKVQDIEDEPILPEIPKCDANILETTVVIPGSWLSNSFREILTHRSF VSEFHNFLSGLQLHTNYLQNGQFSRWKDTVLDGFPNQLTESANHLCLLDTAFFVNSSYPPLLRPERKADLIIHLNYCAGSQTKPLKQTCEYCTVQNIPFPKYELPDEN ENLKECYLMENPQEPDAPIVTFFPLINDTFRKYKAPGVERSPEELEQGQVDIYGPKTPYATKELTYTEATFDKLVKLSEYNILNNKDTLLQALRLAVEKKKRLKGQCPS SEQ ID NO: 2 Nucleotide sequence encoding human cPLA2e isoform 1 SEQ ID NO: 3 Human isoform 2 of cytosolic phospholipase A2ε MATGGGTRSMTSMYGHLLGLQKLNLLDCASYITGLSGATWTMATLYRDPDWSSKNLEPAIFEARRHVVKDKLPSLFPDQLRKFQEELRQRSQEGYRVTFTDFWGLLIETCLGDERNECKLSDQ RAALSCGQNPLPIYLTINVKDDVSNQDFREWFEFSPYEVGLQKYGAFIPSELFGSEFFMGRLVKRIPESRICYMLGLWSSIFSLNLLDAWNLSHTSEEFFHRWTREKVQDIEDEPILPEIPKC DANILETTVVIPGSWLSNSFREILTHRSFVSEFHNFLSGLQLHTNYLQNGQFSRWKDTVLDGFPNQLTESANHLCLLDTAFFVNSSYPPLLRPERKADLIIHLNYCAGSQTKPLKQTCEYCTV QNIPFPKYELPDENENLKECYLMENPQEPDAPIVTFFPLINDTFRKYKAPGVERSPEELEQGQVDIYGPKTPYATKELTYTEATFDKLVKLSEYNILNNKDTLLQALRLAVEKKKRLKGQCPS SEQ ID NO: 4 Nucleotide sequence encoding human cPLA2e isoform 2 ATGGCCACTGGGGGTGGAACAAGATCCATGACCTCCATGTATGGCCACCTGCTGGGGCTGCAGAAGCTGAACCTCCTGGACTGTGCCAGCTACATCACCGGTCTATCAGGGGCCACCTGGACCATGGCTACCTTGTACCGTGACCCTGACTGGTCCTCCAAAAACTTGGAGCCTGCTATCTTTGAGGCTCGGAGACATGTGGTAAAGGACAAGCTACCCTCCCTGTTCCCAGACCAGCTCCGCAAATTCCAGGAGGAGCTCCGGCAGCGCAGCCAGGAAGGCTACAGGGTCACCTTTACAGACTTCTGGGGCCTGCTGATAGAGACCTGCCTGGGGGACGAGAGAAATGAATGCAAACTGTCAGATCAGCGTGCTGCTTTGAGCTGCGGCCAGAACCCCCTGCCCATCTACCTCACCATCAATGTCAAGGATGATGTAAGCAACCAGGACTTCAGAGAGTGGTTCGAGTTCTCCCCCTACGAGGTGGGCCTGCAGAAGTATGGGGCCTTCATCCCCTCCGAGCTCTTCGGCTCCGAGTTCTTCATGGGGCGGCTGGTGAA GAGGATCCCGGAGTCTCGAATCTGCTACATGCTAGGCCTGTGGAGCAGCATCTTCTCCCTGAACCTGCTGGATGCCTGGAACCTGTCACACACCTCGGAGGAGTTTTTCCACAGGTGGACAAGGGAGAAAGTGCAGGACATCGAAGACGAGCCGATCCTGCCTGAAATCCCCAAATGTGATGCTAACATCCTGGAGACCACGGTAGTGATCCCAGGGTCATGGCTGTCCAATTCTTTCCGAGAAATCCTTACCCATCGGTCCTTCGTGTCTGAGTTTCACAACTTCCTGTCTGGGCTGCAGCTGCACACCAACTACCTCCAGAATGGCCAGTTCTCTAGGTGGAAAGACACAGTGCTAGATGGTTTCCCAAACCAGCTGACCGAGTCCGCGAACCACCTGTGCCTGCTGGACACTGCGTTCTTTGTCAACTCCAGCTACCCGCCCCTCCTCAGGCCAGAGCGAAAAGCCGACCTCATCATCCACCTCAACTACTGTGCTGGGTCCCAGACAAAGCCCCTGAAACAAACCTGTGAGTACTGCACTGTGCAGAACATCCCCTTCCCCAAATACGAGCTGCCAGATGAGAATGAAAATCTCAAGGAATGCTACCTGATGGAGAACCCCCAGGAACCCGATGCCCCCATCGTGACTTTCTTCCCACTCATCAATGACACTTTCCGAAAATACAAGGCACCAGGTGTAGAGCGAAGCCCTGAGGAGCTGGAGCAGGGCCAGGTGGACATTTATGGTCCCAAAACTCCCTATGCCACCAAGGAGCTGACATACACAGAGGCCACCTTTGACAAGCTGGTGAAACTCTCAGAGTATAACATCCTGAATAATAAGGACACTCTCCTCCAGGCTCTGCGGCTCGCAGTGGAGAAGAAGAAGCGCCTGAAGGGCCAGTGTCCCTCCTAG Nucleotide sequence encoding mouse PLA2G4E fused to the sequence number 5 flag sequence SEQ ID NO: 6 Forward primer fwPLA2G4E ATGGTGACAGACTCCTTCGAG SEQ ID NO: 7 Reverse primer rvPLA2G4E CCTCTGCGTAAAGCTGTGG SEQ ID NO: 8 shRNA of PLA2G4E (shPLA2G4E) GGTCTATGGTCTCCTTGTATCAAGAGTACAAGGAGACCATAGACC

Claims

1. A nucleic acid construct comprising a nucleotide sequence encoding cytosolic phospholipase A2ε (cPLA2e).

2. The nucleic acid construct of claim 1, wherein the cPLA2e is human cPLA2e, typically human cPLA2e of SEQ ID NO: 1 or SEQ ID NO: 3, or a variant human cPLA2e having at least 70% sequence identity to human cPLA2e of SEQ ID NO: 1 or SEQ ID NO:

3.

3. The nucleic acid construct of claim 2, wherein the nucleotide sequence encoding cPLA2e is SEQ ID NO: 2 or SEQ ID NO:

4.

4. The nucleic acid construct of any one of claims 1 to 3, further comprising a promoter operably linked to the nucleotide sequence encoding cPLA2e.

5. 5. The nucleic acid construct of claim 4, wherein the promoter is a neuron-specific promoter, preferably the promoter is a SYN1 promoter or a hybrid SYN1 promoter.

6. The nucleic acid construct according to any one of claims 1 to 5, further comprising a polyadenylation signal sequence, preferably the polyadenylation signal sequence of the bovine growth hormone gene.

7. 7. The nucleic acid construct according to any one of claims 1 to 6, further comprising 5'ITR and 3'ITR sequences, preferably 5'ITR and 3'ITR sequences of an adeno-associated virus, more preferably 5'ITR and 3'ITR sequences derived from the AAV2 serotype.

8. A vector comprising the nucleic acid construct of any one of claims 1 to 7.

9. The vector of claim 8 which is a viral vector.

10. The vector of claim 9, which is an AAV vector.

11. The vector of claim 10, comprising the nucleic acid construct of claim 2.

12. A viral particle comprising the nucleic acid construct according to any one of claims 1 to 7 or the vector according to any one of claims 8 to 11.

13. 13. The viral particle according to claim 12, selected from among AAV particles, preferably AAV particles comprising capsid proteins selected from the group consisting of AAV2, AAV5, AAV9 and AAV TT serotypes.

14. A host cell comprising the nucleic acid construct of any one of claims 1 to 7 or the vector of any one of claims 8 to 11.

15. a) culturing packaging cells containing the nucleic acid construct of any one of claims 1 to 7 or the vector of any one of claims 8 to 11 in a culture medium; b) harvesting viral particles from the cell culture supernatant and / or cells; A method for producing virus particles, comprising:

16. A pharmaceutical composition comprising the nucleic acid construct of any one of claims 1 to 7, the vector of any one of claims 8 to 11, the viral particle of claim 12 or 13, or the host cell of claim 14, and a pharmaceutically acceptable carrier or excipient.

17. A pharmaceutical composition comprising the nucleic acid construct according to any one of claims 1 to 7, the vector according to any one of claims 8 to 11, the viral particle according to claim 12 or 13, the host cell according to claim 14, or the pharmaceutical composition according to claim 16, for use as a drug.

18. cPLA2e inducers for use as pharmaceuticals.

19. A cPLA2e inducer for use in the treatment of cognitive impairment and / or a disease associated with cognitive impairment in a subject in need thereof.

20. The cPLA2e inducer for use according to claim 20, wherein the disease is dementia.

21. The cPLA2e inducer for use according to claim 20, wherein the disease is age-related dementia or Alzheimer's disease.

22. A cPLA2e inducer for use according to any one of claims 19 to 21, which is a nucleic acid construct according to any one of claims 1 to 7, a vector according to any one of claims 8 to 11, a viral particle according to claim 12 or 13, a host cell according to claim 14, or a pharmaceutical composition according to claim 16.

23. The cPLA2e inducer for use according to any one of claims 19 to 21, which is a protein having cPLA2e activity.

24. The cPLA2e inducer for use according to claim 23, wherein the protein having cPLA2e activity is a protein comprising or consisting solely of SEQ ID NO: 1 or SEQ ID NO: 3, or a variant thereof having at least 70% sequence identity thereto.

25. a. contacting a compound with a mammalian assay cell; b. determining whether an effect associated with induction or increase of cPLA2e is obtained; c. if such an effect is achieved, identifying said compound as a candidate for the treatment of cognitive impairment and / or a disease associated with cognitive impairment; A method for identifying a compound as a candidate for the treatment of cognitive impairment and / or a disease associated with cognitive impairment, comprising:

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