cPLA2e inducers and their use

Abstract

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.

Description

[Technical Field]

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

[0002] Mild cognitive impairment is characterized by impairments in memory, language, and / or other important cognitive functions that do not significantly impair an individual's daily life. This condition often progresses to dementia, characterized by an overall decline in cognitive abilities that significantly impairs 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, coupled with the presence of Aβ peptide aggregates and neurofibrillary tangles (NFTs), are the main characteristics of AD.

[0004] To date, most therapies assayed for Alzheimer's disease (AD) have focused on targeting one of these two histopathological features, particularly Aβ levels. However, given the high failure rate of AD trials (over 99%) and the high costs associated with this disease, it is essential to investigate AD with the aim of discovering new therapies.

[0005] Research into Alzheimer's disease (AD) is complex due to the occasional discrepancies between the emergence of classic AD markers and the symptoms of dementia. Several longitudinal studies have observed substantial AD lesions in the brains of cognitively normal elderly subjects. These findings suggest that classic AD features may not be sufficient to cause dementia, opening up the possibility of studying these AD-resilient patients to identify new potential targets for AD treatment.

[0006] Cognitive impairment is a condition associated with numerous brain disorders. These brain disorders can have many causes, such as degenerative conditions, genetic factors, trauma, infections, and malnutrition. For example, cognitive impairment may 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 psychosis, Alzheimer's psychosis, Lewy body dementia, prion neurodegenerative disorders, such as Creutzfeldt-Jakob disease and Kuru disease, corticobasal degeneration, frontotemporal lobar degeneration, multiple sclerosis, normal pressure hydrocephalus, chronic organic brain syndromes, Pick's disease, progressive supranuclear palsy, or senile dementia. Cognitive impairment may also have a congenital basis, such as Prader-Willi syndrome, Down syndrome, fragile X syndrome, Angelman syndrome, and autism spectrum disorder. Cognitive impairment may be associated with traumatic brain injury such as chronic subdural hematoma, concussion, stroke, or intracerebral hemorrhage, or with infections (e.g., encephalitis, meningitis, and sepsis), or with other brain injuries resulting from drug intoxication or abuse. Cognitive impairment may also be associated with sleep deprivation, mental disorders such as anxiety disorders, dissociative disorders, mood disorders, schizophrenia, treatment with psychotropic drugs, treatment with dopamine agonists, and other conditions that impair or otherwise affect the normal functioning of the central nervous system, including 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 impairment can manifest in many forms, such as learning and / or memory impairments, including but not limited to: attention, information acquisition, information processing, working memory, short-term memory, long-term memory, anterograde memory, retrograde memory, memory retrieval, discriminative learning, decision-making, verbal retrieval, inhibitory response control, attentional set shifting, delayed reinforcement learning, reverse learning, temporary integration of spontaneous behavior, and expression of concern for personal care and self-care. Cognitive impairment may be characterized by a progressive loss of memory, cognition, reasoning, executive function, planning, judgment, and emotional stability.

[0008] Despite significant progress, treatment for cognitive impairments associated with brain damage remains largely inadequate. For conditions such as Huntington's disease, Parkinson's disease, amyotrophic lateral sclerosis (ALS), Alzheimer's disease, Prader-Willi syndrome, and Lewy body dementia, treatment options are limited or unavailable. Additional treatment options are needed to address cognitive impairments associated with brain damage. [Overview of the project]

[0009] In this study, the inventors surprisingly disclose that overexpression of hippocampal PLA2G4E (also known as cytoplasmic 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 two months after stereotactic injection treatment, and improved memory retention in aged C57BL / 6 / SJL WT mice three months after stereotactic injection treatment.

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

[0011] In a particular embodiment of the nucleic acid construct described above, 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 with human cPLA2e of SEQ ID NO: 1 or SEQ ID NO: 3.

[0012] In a more specific embodiment of the nucleic acid construct described above, 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 includes a promoter operably linked to a nucleotide sequence encoding cPLA2e.

[0014] In a more specific embodiment of the nucleic acid construct described above, 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 includes a polyadenylation signal sequence, particularly a polyadenylation signal sequence of a bovine growth hormone gene.

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

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

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

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

[0020] In specific embodiments, the above-mentioned virus particles are selected from AAV particles, preferably AAV particles containing capsid proteins selected from the group consisting of AAV2, AAV5, AAV9, and AAV TT serotypes.

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

[0022] In a further embodiment, the present invention is a) Culturing a packaging cell containing the nucleic acid construct or vector of the present invention in a culture medium; b) Collecting virus particles from the cell culture supernatant and / or intracellularly; It relates to a method for producing virus particles, comprising the above.

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

[0024] In another aspect, the present invention relates to a pharmaceutical composition comprising the nucleic acid construct, vector, virus particle or host cell of the present invention, or a medicament, which is used as a medicament and contains the nucleic acid construct, vector, virus particle or host cell.

[0025] In yet another aspect, the present invention relates to an inducer of cPLA2e used as a medicament.

[0026] In a related aspect, the present invention relates to an inducer of cPLA2e used for the treatment of cognitive impairment and / or a disease associated with cognitive impairment in a subject in need of treatment.

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

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

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

[0030] In a more specific embodiment, the cPLA2e inducer is the nucleic acid construct, vector, or virus particle of the present invention, or a pharmaceutical composition containing the nucleic acid, vector, or virus particle.

[0031] In specific embodiments, 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 drawing]

[0032] [Figure 1A] This figure 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) two months after stereotactic surgery (Bonferroni post-hoc test after two-way ANOVA 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]This figure shows the percentage of time spent in the exact quadrant during 15-second and 60-second exploratory trials on day 6 for aged WT, APP / PS1 sham, and APP / PS1 AAV2 / 9-mPLA2G4E, two months after hippocampal injection (Newman-Coils post-hoc test after one-way ANOVA 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] These are representative Golgi-stained images of the terminal 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 (APP / PS1 mice treated with AAV2 / 9-mPLA2G4E). Scale bar = 10 μm. [Figure 2B] This histoblot shows the quantification of spine density in CA1 hippocampal pyramidal neurons from mice treated with WT, APP / PS1 sham, and AAV2 / 9-PLA2G4E (Newman-Coyles post-hoc test after one-way ANOVA, 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] This figure 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 (C57BL / 6 / SJL WT mice treated with AAV2 / 9-mPLA2G4E) 3 months after hippocampal injection via stereotactic surgery (Bonferroni post-hoc test after two-way ANOVA, n=4 or 5). [Figure 3B] This figure shows the percentage of time spent in the exact quadrant during 15-second and 60-second exploration trials on day 5 of aged WT sham and WT AAV2 / 9-mPLA2G4E mice 3 months after stereotactic surgery (Newman-Coils post-hoc test after one-way ANOVA, n=4 or 5, *P≦0.05 WT AAV2 / 9-mPLA2G4E vs WT sham). [Figure 4A] This figure shows the experimental design of a fear conditioning paradigm used to elucidate the role of PLA2G4E in memory function. The graph shows the percentage of freezing behavior in TT mice during the training and testing phases. [Figure 4B] This figure shows pCREB levels normalized to β-actin, measured by immunoblotting in hippocampal extracts (Newman-Coyles post-hoc test after one-way ANOVA, n=7 or 8, **P≦0.01 naive vs. TT, ++P≦0.01 T24 vs. TT). [Figure 4C] This figure shows PLA2G4E levels normalized to β-actin, measured by immunoblotting in hippocampal extracts (Newman-Coyles post-hoc test after one-way ANOVA, n=7 or 8, **P≦0.01 naive vs. TT, ++P≦0.01 T24 vs. TT). [Figure 5] This figure shows the levels of pCREB, pGluA1, synapsin I, and PLA2G4E measured by immunoblotting in primary neuronal cultures treated with bicuculin (Bic) and / or AAV9-shPLA2G4E (shPLA), normalized to β-actin (Newman-Coyles post-hoc test after one-way ANOVA, 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 in arbitrary units relative to the control (mean ± SEM). [Modes for carrying out the invention]

[0033] In one embodiment, the present invention relates to a cytoplasmic phospholipase A2ε inducer used as a drug, more specifically for the treatment of cognitive impairment and / or cognitive impairment-related diseases in subjects requiring treatment.

[0034] As used herein, the terms “cytoplasmic phospholipase A2ε,” “phospholipase A2 group IVE,” “PLA2G4E,” and “cPLA2e” refer without distinction to members of the cytoplasmic 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. These enzymes play a role in transport via the clathrin-independent endocytosis pathway. They regulate the recirculation process through the formation of tubules that transport and return internalized clathrin-independent cargo proteins to the cell surface (Capestrano M. et al. Journal of Cell Science 2014; 127: 977-993). PLA2G4E can catalyze the calcium-dependent formation of N-acylphosphatidylethanolamine (NAPE) using phosphatidylethanolamine (PE) as the acyl chain 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 / ) with entry accession number Q3MJ16. This entry produces two isoforms by alternative splicing: a) isoform 1 selected as the "canonical" sequence (sequence number 1) (using identifier: Q3MJ16-3); and b) isoform 2 (sequence number 3), which differs from the canonical sequence in that amino acids 1-376 are deleted (using identifier: Q3MJ16-2). The term "cPLA2e" refers to the enzyme and any additional cotranslation or posttranslational modification thereof.

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

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

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

[0038] The terms “nucleic acid” and “polynucleotide” or “nucleotide sequence” are used herein without distinction to refer to any molecule composed of or containing monomeric nucleotides. Nucleic acids may be oligonucleotides or polynucleotides. Nucleic acid sequences may be DNA or RNA. Nucleic acid sequences may be chemically modified or artificial. Nucleic acid sequences include peptide nucleic acids (PNA), morpholino and roch nucleic acids (LNA), as well as glycol nucleic acids (GNA) and threose nucleic acids (TNA). Each of these sequences is distinguished from naturally occurring DNA or RNA by a change in the molecular skeleton. Phosphothioate nucleotides may also be used. Other deoxyribonucleotide analogs include methylphosphonates, phosphoramidates, phosphorodithioates, N3'P5'-phosphoamidates and oligoribonucleotide phosphorothioates, as well as their 2'-O-allyl analogs, and 2'-O-methylribonucleotide methylphosphonate, which can be used in the nucleotides of the present invention.

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

[0040] In some embodiments, the nucleic acid construct of the present invention comprises a nucleotide sequence encoding a 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 to the naturally occurring cPLA2e, typically to human cPLA2e isoform 1 or 2. In some embodiments, the cPLA2e encoded by the nucleic acid construct of the present invention is a fusion protein or polypeptide to which several amino acids (e.g., tags) or polypeptides (e.g., carrier polypeptides) 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, from which an amino acid residue located at the carboxy, amino terminus, or internal region may be optionally deleted.

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

[0042] As will be recognized 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 to be within the scope of cPLA2e of the nucleic acid construct of the present invention. Different embodiments of cPLA2e are understood to have substantially the same cPLA2e activity as human cPLA2e isoform 1 or 2. Substantially the same activity may be up to ±5% activity, including, for example, ±4%, ±3%, ±2%, ±1% or less.

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

[0044] Ca-NAT activity can be determined by measuring the production of NAPE in a biological sample (e.g., cell lysate), for example, by measuring the production of N-C16:0 DOPE in a reaction with DPPC (40 μM) and DOPE (75 μM) at 37°C for 30 minutes with or without the addition of CaCl2 (3 mM) to the reaction mixture. Ca-independent activity is subtracted in the calculation of Ca-dependent activity. Alternatively, NAPE production (e.g., 13Targeted analysis of C16:0-containing NAPE 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 set period (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 mutant nucleotide sequence having at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity with SEQ ID NO: 2 or 4.

[0046] As used herein, the terms “sequence identity” or “identity” refer to the number of positional matches (identical nucleic acid residues or amino acid residues) in the alignment of two polynucleotide sequences or two polypeptide sequences. Sequence identity is determined by comparing sequences that have been aligned to maximize overlap and identity while minimizing sequence gaps. In particular, sequence identity can be determined using one of a number of mathematical global or local alignment algorithms, depending on the lengths of the two sequences. Sequences of similar length are preferably aligned using a global alignment algorithm that optimally aligns the sequence along its entire length (e.g., Needleman and Wunsch's algorithm; Needleman and Wunsch, 1970, J Mol Biol.; 48(3):443-53), while sequences of substantially different lengths are preferably aligned using a local alignment algorithm (e.g., Smith and Waterman's algorithm (Smith and Waterman, 1981, J Theor Biol.; 91(2):379-80) or Altschul's algorithm (Altschul SF et al., 1997, Nucleic Acids Res.; 25(17):3389-402, Altschul SF et al., 2005, Alignment is preferably performed using Bioinformatics;21(8):1451-6). Alignment for the purpose of determining the sequence identity % of nucleic acids can be achieved in various ways within the scope of the skills in 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 / . A person skilled in the art can determine appropriate parameters for measuring the alignment, including any algorithm required to achieve the greatest possible alignment over the full length of the sequences being compared.For the purposes of this specification, the nucleic acid sequence identity % value refers to the value generated using the pairwise sequence alignment program EMBOSS Needle, which uses the Needleman-Wunsch algorithm to produce the optimal global alignment of two sequences, where all search parameters are set to their default values: scoring matrix = BLOSUM62, gap open = 10, gap elongation = 0.5, end gap penalty = false, end gap open = 10, and end gap elongation = 0.5.

[0047] The nucleic acid constructs described herein may have different applications. In particular, they can be used to generate viral vectors for gene therapy, or to generate non-viral vectors for gene therapy, such as nucleic acid constructs having mRNA structures.

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

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

[0050] promoter In one embodiment, the nucleic acid construct of the present invention also includes a promoter, which initiates transgene expression after being introduced 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 may be RNA, a peptide, or a protein. The transgene may encode a natural 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 the transcription of a nucleic acid (transgene) to which it is operably ligated. A promoter can regulate both the rate and efficiency of transcription of a operably ligated nucleic acid. A promoter may be operably ligated to other regulatory elements that enhance ("enhancers") or repress ("repressors") the promoter-dependent transcription of the nucleic acid. These regulatory elements include, but are not limited to, transcription factor binding sites, repressors and activating protein binding sites, and any other sequences of nucleotides known to those skilled in the art that act 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 the operably ligated gene or coding sequence, on the same strand, and upstream of the DNA sequence (towards the 5' region of the sense strand). A promoter may be approximately 100 to 1000 base pairs in length. The position in the promoter is specified relative to the transcription start site of a particular gene (i.e., upstream positions are negative numbers counting backward from -1, for example, -100 is 100 base pairs upstream).

[0053] As used herein, the term “operably linked” refers to the linking of functionally related polynucleotide (or polypeptide) elements. A nucleic acid is “operably linked” when it is placed in a functional relationship with another nucleic acid sequence. For example, a promoter or transcriptional regulatory sequence is operably linked to a coding sequence when it affects the transcription of that coding sequence. "Operatably linked" means that the linked polynucleotide sequences are typically adjacent, and if two protein coding regions need to be joined, they are adjacent and within the reading frame.

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

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

[0056] Typically, such promoters may be tissue- or cell-type-specific promoters, organ-specific promoters, 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 ligated to a nucleotide sequence encoding cPLA2e, the promoter directing the expression of encoded cPLA2e in at least hippocampal neurons.

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

[0059] As used herein, the term “specific promoter” refers to a promoter that exhibits selectivity, being active in one group of cells or tissues and having low or silent activity in another, although its activity is not necessarily limited to a single cell type. 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 a level detectable only in nerve cells.

[0060] Therefore, as used herein, “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 substantially does not 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 levels of expression may be observed in other cell types, but such expression is substantially lower than that in neurons, for example, expression in neurons may be at least 2, at least 3, at least 4, at least 5, or at least 10 times higher than the expression level in other cells. Such a promoter may be a strong promoter or a weak promoter, and may direct the 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] Therefore, neuron-specific promoters enable the active expression of the gene linked to them in neurons, while preventing its expression in other cells or tissues.

[0062] In more specific embodiments of the nucleic acid construct of the present invention, promoters operably linked to the nucleotide sequence encoding cPLA2e include the synapsin 1 (SYN1) gene promoter (Kuegler S et al. Gene Ther. 2003; 10(4): 337-47), the neuron-specific enolase (NSE) gene promoter (Forss-Petters S et al. Neuron. 1990; 5(2): 187-97, Twyman RM et a. 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 the prion protein (Prnp) gene promoter (Weber P et al. Eur J Neurosci. 2001; These are neuron-specific promoters selected from a group consisting of the α-calcium-calmodulin-dependent kinase II (CaMKIIα) gene promoter (Dittgen T et al. Proc Natl Acad Sci US A. 2004; 101: 18206-18211), the 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 the tubulin α1 (Ta1) gene promoter (Gloster A. et al. J Neurosci. 1994; 14:7319).

[0063] Typically, such promoters (in particular, the neuron-specific promoters of the above-mentioned selection group) may be complete promoters comprising core, proximal, and distal promoter elements; promoters, e.g., fragments of a core promoter, or any other fragment sufficient to direct gene expression in a target cell, tissue, or organ; or chimeric or hybrid promoters, e.g., promoters comprising a core promoter of a gene and heterologous enhancer sequences from another gene or synthesis. The term "core promoter" as used herein refers to the minimum portion of a promoter required to properly initiate transcription. This consists of a transcription initiation site and a functional sequence for binding to the transcription initiation complex (TATA box) within the cell or host organism. A non-limiting example of a suitable neuron-specific hybrid promoter is a hybrid promoter 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), or a mouse Hb9 enhancer fused to an 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).

[0064] In one embodiment of the nucleic acid construct of the present invention, the promoter operably ligated to the nucleotide sequence encoding cPLA2e is a SYN1 promoter, or a hybrid SYN1 promoter such as a hybrid SYN1 promoter including a 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 ligated to a nucleotide sequence encoding cPLA2e, typically sequence number 1 or 3, preferably sequence number 2 or 4.

[0066] All of these promoter sequences possess properties that enable the expression of cPLA2e encoded by nucleic acid constructs, at least in hippocampal neurons.

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

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

[0069] Poly(A) signaling typically consists of a) a consensus sequence AAUAAA, which is required for both 3' end cleavage and polyadenylation of premessenger RNA (premRNA) and has been shown to promote downstream transcription termination, and b) additional upstream and downstream elements of AAUAAA that control the utilization efficiency of AAUAAA as a poly(A) signal. Considerable variation is observed in these motifs in mammalian genes.

[0070] In one embodiment, the polyadenylation signal sequence of the nucleic acid construct of the present invention is a polyadenylation signal sequence of a mammalian gene or a viral gene, optionally combined with one or more features of the various embodiments described above or below. 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, and computer-designed polyadenylation signals (synthesized).

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

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

[0073] nucleic acid construct having 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, mRNA can be modified. Modifications of mRNA nucleic acids are described in more detail in U.S. Patent Publication Nos. 20140206752, 20150086614, and 20160304552, as well as International Publication Nos. 2016011226, 2016014846, and 2016011306. These modifications include chemically modified nucleic acid bases, sugars, backbone or any combination thereof, patterned untranslated regions (UTRs), and microRNA (miRNA) binding sites.

[0074] Therefore, 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 embodiment, the polynucleotide further comprises two of these features. In one embodiment, the polynucleotide may further comprise all three of these features. The UTR may be homologous or heterologous to the nucleotide sequence encoding cPLA2e.

[0075] The untranslated region (UTR) is the untranslated polynucleotide nucleic acid section before the start codon (5'UTR) and after the stop codon (3'UTR). In some embodiments, the nucleic acid construct of the present invention, which includes a nucleotide sequence encoding cPLA2e, further includes the UTR (e.g., the 5'UTR or a functional fragment thereof, the 3'UTR or a functional fragment thereof, or a combination thereof).

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

[0077] Typically, by manipulating the characteristics of genes highly expressed in specific target cells / tissues / organs, the stability of polynucleotides and protein production in those specific target cells / tissues / organs can be enhanced.

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

[0079] International Publication No. 2014 / 164253 (which in whole constitutes part of this specification) presents a list of exemplary UTRs that can be used in the polynucleotide of the present invention as adjacent regions to the nucleotide sequence encoding cPLA2e.

[0080] Wild-type UTRs derived from any gene or mRNA can be incorporated into the nucleic acid construct of the present invention. In some embodiments, mutant UTRs can be created by altering the orientation or position of the UTR relative to the coding nucleotide sequence, or by incorporating additional nucleotides, deleting nucleotides, exchanging or transposing nucleotides. In some embodiments, mutants of 5' or 3' UTRs, such as mutants of wild-type UTRs, or mutants in which one or more nucleotides are added to or removed from the terminal of the UTR can be used. Furthermore, 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 / (their entire contents constitute part of this specification by reference).

[0081] The 5' cap structure of natural mRNA is involved in nuclear export, enhances mRNA stability, and, through association with CBP and poly(A)-binding proteins, binds to mRNA cap-binding proteins (CBPs), which are involved in mRNA stability and translational ability in cells, to form mature circular mRNA species. The cap further assists in the removal of the 5' proximal intron during mRNA splicing. Endogenous mRNA molecules can be 5'-capped to create a 5'-ppp-5'-triphosphate linkage between the terminal guanosine cap residue and the 5'-terminal transcription sense nucleotide of the mRNA molecule. This 5'-guanylate cap can then be methylated to produce an N7-methyl-guanylate residue. The ribose sugar of the terminal and / or ante-terminal transcription nucleotide at the 5' end of mRNA may optionally be 2'-O-methylated. 5'-decapping by hydrolysis and cleavage of the guanylate cap structure allows nucleic acid molecules such as mRNA molecules to be targeted for degradation.

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

[0083] In some embodiments, the nucleic acid construct of the present invention (i.e., a polynucleotide comprising a nucleotide sequence encoding cPLAe) further comprises a poly-A tail.

[0084] vector The nucleic acid construct of the present invention may be contained in an expression vector. Therefore, in one embodiment, the present invention relates to an expression vector containing the nucleic acid construct of the present invention.

[0085] As used herein, the terms “expression vector” or “vector” refer to a nucleic acid molecule used as a vehicle to transport genetic material, particularly to deliver nucleic acids to host cells, either in vitro or in vivo. An expression vector also refers to a nucleic acid molecule capable of producing the expression of a gene (transgene) in a host cell or host organism that is compatible with such a sequence. An expression vector typically comprises at least a suitable transcriptional regulatory sequence and optionally a 3' transcription termination signal. Additional factors (endogenous or chimeric transcription factors) may be present that are necessary or beneficial for producing the expression of expression enhancer elements, etc., that are capable of responding to a precise induction signal or that are specific to certain cells, organs, or tissues. Examples of vectors include, but are not limited to, plasmids, fasmids, cosmids, transposition elements, viruses, and artificial chromosomes (e.g., YACs). Preferably, the vectors of the present invention are vectors suitable for use in gene or cell therapy, and are particularly suitable for targeting nerve cells.

[0086] In some embodiments, the expression vector is a viral vector such as Moloney mouse leukemia virus vector (MoMLV), MSCV, SFFV, MPSV, or SNV; lentiviral vectors (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)); adenovirus (Ad) vector; adeno-associated virus (AAV) vector; Simian virus 40 (SV-40) vector; bovine papillomavirus vector; Epstein-Barr virus; herpesvirus vector; vaccinia virus vector; Harvey mouse sarcoma virus vector; mouse mammary cancer virus vector; or Rous sarcoma virus vector.

[0087] As is known in the art, depending on the specific viral vector considered, it is necessary to introduce a suitable sequence, such as an AAV ITR for AAV vectors or an LTR for lentiviral vectors, into the vector of the present invention in order to obtain a functional viral vector. In certain embodiments, the vector is an AAV vector, optionally combined with one or more features of the various embodiments described above or below.

[0088] AAV is attracting significant interest as a potential vector for human gene therapy. Its advantageous characteristics include its lack of association with any particular 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 consists 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 reverse-end repeats (ITRs) at both ends, which function in cis as DNA replication origins and viral packaging signals. The ITRs are approximately 145 bp long. The internal non-repeat 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, which are named according to their apparent molecular weight. The AAV cap gene encodes at least three proteins, VP1, VP2, and VP3. For a detailed description of the AAV genome, see, for example, Muzyczka, N. 1992 Current Topics in Microbiol. and Immunol. 158:97-129.

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

[0090] As used herein, the term “reverse terminal repeat (ITR)” refers to the nucleotide sequences located at the 5' end (5'ITR) and the nucleotide sequences located at the 3' end (3'ITR) of a virus, which include palindromic sequences and can be folded to form a T-shaped hairpin structure that functions as a primer at the initiation of DNA replication. These are also required for the integration of the viral genome into the host genome, rescue from the host genome, and capsid formation of the viral nucleic acid into mature virions. ITRs are required in cis for the 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 modified by insertion, deletion, or substitution. The serotype of the AAV reverse terminal repeat (ITR) can be selected from any known human or non-human AAV serotype. In specific embodiments, the nucleic acid construct or viral expression vector can be obtained using ITRs of 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, sheep AAV, and any other AAV serotypes currently known or to be discovered in the future.

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

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

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

[0095] The construction of recombinant AAV virus particles is generally known in the art and is described, for example, in U.S. Patents 5,173,414 and 5,139,941, International Publication Nos. 92 / 01070 and 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 construct or expression vector of the present invention can be packaged in a viral capsid to generate "viral particles," also known as "viral vector particles."

[0097] Therefore, in one embodiment, the present invention relates to a viral particle comprising the nucleic acid construct or expression vector of the present invention.

[0098] In one embodiment, the present invention relates to a viral particle comprising a nucleic acid construct or expression vector comprising a promoter operably linked to a nucleotide sequence encoding cPLA2e, and a viral particle 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) 5'ITR and 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 present invention are AAV particles containing the capsid protein of adeno-associated virus, i.e., the nucleic acid construct or expression vector of the present invention is packaged on an AAV-derived capsid to produce “adeno-associated virus particles,” or “AAV particles.” The term AAV particles encompasses any genetically modified, recombinant AAV particles, or mutant AAV particles. Recombinant AAV particles can be produced by capsid formation on viral particles formed with native or mutant Cap proteins corresponding to the same or different serotypes of AAV, using a nucleic acid construct or viral expression vector containing ITRs (which may be more than one) derived from a specific AAV serotype.

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

[0101] In one embodiment, AAV particles according to the present invention can be produced by capsid formation on viral particles formed by a native Cap protein corresponding to the same specific serotype of AAV, using a viral vector of an AAV vector / genome derived from a particular AAV serotype. Nevertheless, several methods 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] Therefore, in another embodiment, the AAV virus particle according to the present invention comprises, for example, a) a virus particle composed of capsid proteins derived from the same or different AAV serotypes (e.g., AAV2 ITR and AAV9 capsid proteins, AAV2 ITR and AAV TT capsid proteins, etc.), b) a mosaic virus particle composed of a mixture of capsid proteins derived from different AAV serotypes or mutants (e.g., an AAV2 ITR having a capsid formed by proteins from two or more AAV serotypes), c) a chimeric virus particle composed of capsid proteins cleaved by domain exchange between different AAV serotypes or mutants (e.g., an AAV2 ITR containing an AAV5 capsid protein having an AAV3 domain), or d) a nucleic acid construct comprising a nucleotide sequence encoding a contiguous cPLA2e of a given AAV serotype, which is packaged into a targeted virus particle manipulated to exhibit a selective binding domain that enables a tight interaction with a target cell-specific receptor.

[0103] In specific embodiments, examples of AAV serotypes of the capsid protein of AAV particles according to the present invention include AAV2, AAV5, AAV9, and AAV TT. In more preferred embodiments, the above AAV serotype of the capsid protein is selected from 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 containing 5'ITR and 3'ITR sequences derived from the AAV virus, preferably the AAV particle comprising a capsid protein of an AAV2, AAV5, AAV9, or AAV TT serotype, more preferably an AAV9 serotype or AAV TT serotype, and / or 5'ITR and 3'ITR sequences of an AAV2 serotype.

[0105] In certain embodiments, optionally in combination with one or more features of the various embodiments described above or below, the viral particle comprises a nucleic acid construct or expression vector comprising a nucleotide sequence encoding the amino acid human cPLA2e of SEQ ID NO: 1 or SEQ ID NO: 3 under the control of a promoter, wherein the promoter enables the expression of the human cPLA2e in at least hippocampal neurons, the viral particle is an AAV particle comprising a capsid protein selected from among viral particles that target at least hippocampal neurons, typically from the group consisting of AAV2, AAV5, AAV9 or AAV TT serotypes, 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 a more specific embodiment, such recombinant AAV particles according to the present invention comprise a capsid protein of an 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) an AAV ITR such as the 5' and 3' ITR of AAV2 adjacent to the nucleic acid construct.

[0107] The AAV virus particles according to the present invention include synthetic AAV variants such as 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, sheep AAV, NP40, NP59, NP84, etc. (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), and Anc80 (Zinn E et al., Cell Rep. Those skilled in the art will understand that this may include capsid proteins derived from any AAV serotype, including any other AAV serotypes currently known or to be discovered in the future (2015;12(6):1056-68).

[0108] Vector and virus particle production The preparation of viral particles containing the expressing viral vectors disclosed above can be carried out using conventional methods and protocols selected in consideration of the structural features chosen by the actual embodiment of the expressing vector and the viral particles of the vector.

[0109] In short, viral particles can be produced in host cells, particularly specific virus-producing cells (packaging cells), that have been transfected with a nucleic acid construct or expression vector to be packaged in the presence of a helper vector or virus, or other DNA constructs (which may be multiple).

[0110] As used herein, the term “packaging cell” refers to a cell or cell line that may be transfected with the nucleic acid construct or expression vector of the present invention and that transfectly provide all the deficient functions required for complete replication and packaging of the viral vector. Typically, the packaging cell constitutively or inductively expresses one or more of the deficient viral functions. The packaging cell may be an adherent cell or a suspension cell.

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

[0112] AAV virus particles can be produced using a conventional method comprising transient cell co-transfection with a nucleic acid construct or expression vector (e.g., plasmid) containing the transgene of the present invention, a nucleic acid construct encoding rep and cap genes but without an ITR sequence (e.g., AAV helper plasmid), and a third nucleic acid construct (e.g., plasmid) that provides adenovirus function required for AAV replication. The viral gene required for AAV replication is referred to herein as the viral helper gene. Typically, the above-mentioned gene required for AAV replication is an adenovirus helper gene such as E1A, E1B, E2a, E4, or VA RNA. The adenovirus helper gene is preferably of the Ad5 or Ad2 serotype.

[0113] The large-scale production of AAV particles according to this disclosure can also be carried out, for example, by infection of insect cells with a combination 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 packaging AAV vector, respectively. The recombinant baculovirus vectors provide the viral helper gene functions required for viral replication and / or packaging. Smith et al 2009 (Molecular Therapy, vol.17, no.11, pp 1888-1896) further describes a dual baculovirus expression system for the large-scale production of AAV particles in insect cells.

[0114] Suitable culture media are known to those skilled in the art. The components of such media may vary depending on the type of cells to be cultured. In addition to nutritional composition, osmotic pressure and pH are considered important parameters of the culture medium. Cell growth media contain a number of components known to those skilled in the art, including amino acids, vitamins, organic and inorganic salts, sources of carbohydrates, lipids, and trace elements (CuSO4, FeSO4, Fe(NO3)3, ZnSO4, etc.), each component present in amounts that support in vitro cell culture (i.e., cell survival and growth). The components may also include different auxiliary substances such as buffers (sodium bicarbonate, Hepes, Tris, etc.), oxidative stabilizers, stabilizers to counteract mechanical stress, protease inhibitors, animal growth factors, plant hydrolysates, anticaking agents, and defoaming agents. The properties and composition of cell growth media vary depending on the specific cell requirements. Examples of commercially available cell growth media include MEM (Minimal Essential Medium), BME (Eagle's Basal Medium), DMEM (Dulbecc's Modified Eagle Medium), Iskoff DMEM (Iskoff's Modified Dulbecco's Medium), GMEM, RPMI 1640, Leibowitz L-15, McCoy's, 199 medium, HAM (HAM's Medium) F10, and its derivatives HAM F12, DMEM / F12, etc.

[0115] Further guidance for the construction and preparation of viral vectors used in accordance with this 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. Moulllier (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 embodiment, 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 cell according to the present invention is a specific virus-producing cell, also called a packaging cell, that is transfected with a nucleic acid construct or expression vector according to the present invention in the presence of a helper vector or virus or other DNA construct, and that transfects all the missing functions required for complete replication and packaging of viral particles. The packaging cell may be an adherent cell or a suspension cell.

[0118] For example, the packaging cells described above may be eukaryotic cells such as mammalian cells, including cells from monkeys, humans, dogs, and rodents. Examples of human cells include PER.C6 cells (International Publication No. 01 / 38362), MRC-5 (ATCC CCL-171), WI-38 (ATCC CCL-75), HEK-293 cells (ATCC CRL-1573), HeLa cells (ATCC CCL2), and rhesus macaque fetal lung cells (ATCC CL-160). Examples of non-human primate cells include Vero cells (ATCC CCL81), COS-1 cells (ATCC CRL-1650), or COS-7 cells (ATCC CRL-1651). An example of a canine cell is MDCK cells (ATCC CCL-34). Examples of rodent cells include hamster cells such as BHK21-F, HKCC cells, or CHO cells.

[0119] As an alternative to mammalian sources, packaging cells for the production of viral particles may be derived from avian sources such as chickens, ducks, geese, quail, or pheasants. Examples of avian cell lines include avian embryonic stem cells (International Publication Nos. 01 / 85938 and 03 / 076601), immortalized duck retinal cells (International Publication No. 2005 / 042728), and avian embryonic stem cell-derived cells, including chicken cells (International Publication No. 2006 / 108846) or duck cells, such as the EB66 cell line (International Publication Nos. 2008 / 129058 and 2008 / 142124).

[0120] In another embodiment, the cells may be any cells tolerant of baculovirus infection and replication packaging cells. In a particular embodiment, the cells may be insect cells 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] Therefore, in certain embodiments, the host cell comprises a nucleic acid construct or expression vector (e.g., an AAV vector according to the present invention) comprising a nucleotide sequence encoding cPLA2e according to the present invention, a nucleic acid construct encoding AAV rep and / or cap genes that do not contain an ITR sequence, e.g., a plasmid, and / or a nucleic acid construct comprising a viral helper gene, e.g., a plasmid or a virus.

[0122] In another embodiment, the present invention relates to host cells transduced with the expression vector or viral particles of the present invention, and the term “host cell” as used herein refers 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 present 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 embodiment, the present invention also refers to pharmaceutical compositions 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 disorders associated with cognitive impairment.

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

[0127] The pharmaceutical composition or agent of the present invention typically comprises a sufficient amount of a therapeutic agent (e.g., the vector or viral particle of the present invention) to produce a desired therapeutic effect, and a pharmaceutically acceptable carrier or excipient.

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

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

[0130] Preferably, the pharmaceutical composition is formulated as a solution, more preferably optionally as buffered saline solution. Auxiliary active compounds can also be incorporated into the pharmaceutical composition of the present invention. Guidelines regarding the simultaneous 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 illustrative and do not limit the selection to pharmaceutical compositions suitable for other parenteral and non-parenteral administration routes. The pharmaceutical compositions described herein can be packaged in single-dose or multi-dose forms.

[0132] therapeutic use Using an animal model of Alzheimer's disease (APP / PS1 mouse) and aged wild-type mice, the inventors surprisingly found enhanced cPLA2e expression mediated by AAV, improvement in memory impairment in APP / PS1 mice, and improvement in memory function in aged wild-type animals.

[0133] These results provide strong evidence for possible treatment strategies for cognitive impairment and / or cognitive impairment-related disorders in subjects, more specifically for dementia, such as age-related dementia or Alzheimer's disease.

[0134] Accordingly, 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, the method comprising administering a therapeutically effective amount of a cPLA2e inducer to the subject.

[0135] In a further embodiment, the present invention relates to a cPLA2e inducer used as a drug in a subject requiring treatment, and more specifically, in the treatment of cognitive impairment and / or cognitive impairment-related diseases, such as dementia, more specifically age-related dementia or Alzheimer's disease, in a subject requiring treatment.

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

[0137] cPLA2e inducers used or administered in the treatment of cognitive impairment and related diseases 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 containing the nucleic acid construct of the present invention as described above, c) viral particles containing the nucleic acid construct or vector of the present invention as described above, d) host cells containing the nucleic acid construct or vector of the present invention, e) cPLA2e polypeptide or protein, and f) a pharmaceutical composition containing any of the above nucleic acid construct, a vector containing the nucleic acid construct, viral particles containing the nucleic acid construct or vector, and cPLA2e polypeptide or protein.

[0138] With respect to the cPLA2e polypeptide or protein described above, any and all of the embodiments and preferred embodiments described above for cPLA2e encoded by the nucleotide sequence included in the nucleic acid construct of the present invention are embodiments within the range of cPLA2e containing or consisting solely of the amino acids of SEQ ID NO: 1 or 3, or variants having at least 70% sequence identity thereto, as a fusion protein with another polypeptide (which may be more than one) such as a tag or carrier polypeptide.

[0139] In preferred embodiments, the cPLA2e inducer for therapeutic use of the present invention is the vector of the present invention, more preferably a viral vector or viral particles (e.g., AAV particles), or a pharmaceutical composition containing the same.

[0140] As used herein, the terms “cognitive impairment” and “cognitive dysfunction” are used interchangeably and refer to any cognitive impairment 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 any combination thereof. In humans, cognitive and potential cognitive impairments can be determined or assessed using any preferred method for testing hippocampal or brain function and for neuroimaging. For example, cognitive impairments (e.g., memory or learning) and their potential dysfunctions can be measured using any suitable psychological test, including, but not limited to, the Kiel Locomotor Maze, which has features of a radial arm maze and a Morris water maze optimized for assessing spatial memory and orientation in school-aged children, or the Cambridge Neuropsychological Test Automated Battery (CANTAB), a series of computerized nonverbal visual presentation neuropsychological tests designed to test spatial memory range, spatial working memory, and spatial cognition. Furthermore, the results of the methods relating to cognitive impairment disclosed herein can be demonstrated by comparative studies in animals (e.g., 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 related diseases associated with conditions that impair or otherwise affect the normal function of the central nervous system, such as dementia (e.g., age-related dementia with abnormal protein aggregation such as Alzheimer's disease, Parkinson's disease, ALS or prion disease, Creutzfeldt-Jakob disease, or Gerstmann-Streussler-Scheinker disease, vascular dementia and / or neurodegenerative dementia), mild cognitive impairment, and attention deficit disorder. For this reason, the cPLA2e inducer of the present invention is particularly used for treating cognitive impairment associated with one of these conditions.

[0142] In preferred embodiments, 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 relation to AD, a defining characteristic is cognitive impairment. AD is defined as a neurodegenerative disorder that constitutes the major form of dementia common in older adults, and is characterized by the accumulation of two abnormal proteins in the brain: β-amyloid peptide and hyperphosphorylated tau, in the form of amyloid plaques and neurofibrillary tangles, respectively. The diagnostic criteria for Alzheimer's disease (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 lesions; (2) progressive dementia; (3) no altered consciousness; (4) onset between the ages of 40 and 90; and (5) no other cause that can explain the condition. Characteristic, highly reliable AD biomarkers are currently available through structural MRI, molecular neuroimaging using PET, and cerebrospinal fluid analysis to confirm the diagnosis of AD. Furthermore, the pre-Alzheimer's disease prodromal state known as mild cognitive impairment (MCI) is defined as objective abnormal memory loss relative to the subject's age and education level. The criteria for MCI include (1) memory complaints confirmed by a family member, (2) normal other cognitive functions, (3) normal daily activities, (4) abnormal memory for one's age, and (5) absence of dementia.

[0143] As used herein, the terms “subject” or “patient” refer to mammals. Mammalian species that may benefit from the disclosed therapeutic methods include, but are not limited to, humans, apes, chimpanzees, monkeys and orangutans and other non-human primates, domesticated animals including dogs and cats, and livestock such as horses, cattle, pigs, sheep and goats, or other mammalian species including, but not limited to, mice, rats, guinea pigs, rabbits and hamsters.

[0144] As used herein, “treating” or “treat” means (i) preventing or delaying the onset of a disease, disorder or condition in a subject who is susceptible to a disease, disorder or / or condition but has not yet been diagnosed with it; (ii) inhibiting a disease, disorder or condition, i.e., preventing or slowing its onset or progression; and / or (iii) alleviating a disease, disorder or condition, i.e., causing regression of the disease, disorder or / or condition. In certain embodiments, such terms mean improvement or elimination of a disease or symptoms associated with a disease.

[0145] With respect to cognitive impairment, “treatment” and “to treat” mean (i) preventing or delaying the onset of cognitive impairment in subjects who are susceptible to cognitive impairment but have not yet been diagnosed with it; (ii) inhibiting cognitive impairment, i.e., preventing or slowing its onset or progression; (iii) alleviating cognitive impairment, i.e., causing its regression; and / or (iv) improving cognitive abilities. Unless otherwise specified herein, the term “to treat cognitive impairment” means reducing cognitive impairment, improving at least one symptom associated with or resulting from 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 impairments, and to improving learning and memory abilities. “To improve learning and memory abilities” means improving or increasing the intellectual ability to record, retain, or recall past experiences, knowledge, thoughts, feelings, ideas, or impressions.

[0146] As used herein, “therapeutic dose” means the dose and duration effective in order to achieve one or more of the following desired therapeutic outcomes: a significant delay in the onset or progression of the disease; a significant reduction in the severity of one or more symptoms; a significant reduction in the characteristics of AD, amyloid and / or tau lesions; a significant increase in synaptic plasticity; and a significant reduction in age-related and / or AD-associated mortality.

[0147] The therapeutically effective dose is typically the amount in which the therapeutically beneficial effects of the product or pharmaceutical composition outweigh any toxic or harmful 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 via parenteral routes, such as intraparenchymal, intracerebral, intraventricular (icv), intrathecal, intranasal, intravenous, or subcutaneous routes.

[0149] Typically, a therapeutically effective amount of the above-mentioned nucleic acid construct, expression vector, viral particle, host cell, cPLA2e polypeptide or protein, or pharmaceutical composition is administered preferably by intrathecal or intraparenchymal routes, the latter preferably to a region of the brain such as the hippocampus or cerebral cortex. Intraparenchymal routes may facilitate preferred local administration to the hippocampus and cortex compared to other regions of the brain. As used herein, “preferred local administration to the hippocampus” does not mean that all cPLA2e inducers are administered to the brain region, but rather that the majority of the cPLA2e inducer, for example, at least 50%, at least 60%, at least 70%, or at least 80%, is administered to the region.

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

[0151] For any particular subject, a specific dosing regimen may be adjusted over time according to the individual's needs and the judgment of the professional administering or managing the composition. The dosing ranges described herein are illustrative and do not limit the dosing ranges that may be selected by a physician.

[0152] In one embodiment, the AAV virus particles according to the present invention are used to treat cognitive impairment such as Alzheimer's disease or diseases related to cognitive impairment by injecting 10 human subjects or patients. 8 vg / kg~10 14 vg / kg (vg: viral genome; kg: weight of the subject or patient), e.g., 1 × 10⁻¹⁴ 10 vg / kg~5×10 14 It can be administered in an amount or dose that falls within the range of vg / kg. In a more specific embodiment, 1 × 10 12 vg / kg~1×10 13Administer an amount contained within the range of vg / kg. In an alternative embodiment, 1×10 9 iu / kg to 1×10 11 iu / kg (iu: infectious unit of the vector) within the range or administer a dose.

[0153] In another aspect, the present invention further relates to a kit comprising the nucleic acid construct, expression vector, host cell, virus particle of the present invention, or a pharmaceutical composition comprising the above nucleic acid construct, vector, host cell or virus particle in one or more containers. The kit may include an instruction manual or packaging material describing a method for administering the nucleic acid construct, expression vector, virus particle, host cell or pharmaceutical composition contained in the kit to a patient. The containers of the kit may be 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 present invention in a suitable liquid or solution form.

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

[0155] Method for screening new active substances useful in the treatment of cognitive impairment and / or diseases associated with cognitive impairment The inventors have also been working on the development of a system for screening compound candidates such as peptides, polypeptides (e.g., antibodies) or small molecule candidates, taking advantage of the fact that the induction or increase of cPLA2e results in a very obvious increase in calcium-dependent N-acyltransferase (Ca-NAT) activity.

[0156] Therefore, the present invention provides a) a step of contacting a compound with mammalian assay cells, and b) a step of confirming whether an effect associated with the induction or increase of cPLA2e can be obtained, c) If such an effect is obtained compared to a control, the step of identifying the compound as a candidate for the treatment of cognitive impairment and / or related diseases, The invention also provides a method for identifying compounds as candidates for the treatment of cognitive impairment and / or related disorders.

[0157] Possible embodiments of the present invention involve performing an in vitro method in which the cells being assayed are mammalian cells such as HEK293T cells. These cells are cultured in a medium suitable for cell growth and proliferation in the presence of the candidate compound with or without ionomycin (e.g., 2 μm) (e.g., for 30 minutes or 1 hour), and Ca-NAT activity is tested compared to a control that was not exposed to the candidate compound (e.g., by targeted metabolite profiling). If an increase in Ca-NAT activity is found compared to control cells, the compound is identified as a potential candidate for the treatment of cognitive impairment and / or cognitive impairment-related diseases.

[0158] In some embodiments, cPLA2e-transfected cells (e.g., using the nucleic acid construct of the present invention) can be used as a positive control for inducing cPLA2e activity.

[0159] When used herein, the term “inducing or increasing” may refer to the ability to cause an overall increase of preferably 20% or more, more preferably 50% or more, and most preferably 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% or more. [Examples]

[0160] To determine whether PLA2G4E is involved in learning and memory functions, the inventors overexpressed PLA2G4E in the brains of a) APP / PS1 mice (a model of AD) and b) aged wild-type animals, both of which suffered from cognitive impairment. For this purpose, an AAV vector containing the mouse (Mus musculus) cPLA2e as the transgene was constructed and administered to the animals. Subsequently, learning and memory functions were assessed by the MWM method.

[0161] Example 1. Fabrication of AAV2 / 9-mPLA2G4E We constructed the AAV2 / 9-mPLA2G4E vector, which contains the nucleotide sequence of Sequence ID No. 5, encoding mouse PLA2G4E fused to the flag sequence via a linker, as the introduced gene.

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

[0163] Next, the 4163bp skeleton fragment was obtained from plasmid pAAV-hα-synuclein A53T (generated generously by Dr. J. Gerez) by digestion with Xhol (in CutSmart® buffer) followed by treatment with Klenow polymerase, dNTPs, and NEB2.1 buffer. Subsequently, after purification, the 4163 skeleton fragment was digested with SacI (in CutSmart® buffer) and dephosphorylated using 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. Finally, the 4163bp skeleton fragment was separated, extracted, and purified as described above for the 3108bp fragment.

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

[0165] After constructing pAAV2-mPLA2G4E containing the desired construct, it was then subjected to several amplification steps to generate a plasmid in an appropriate quantity for final virus production. First, chemically competent Escherichia coli (E. coli) bacteria were transformed with the plasmid using TOP10 electrocompetent cells (Invitrogen), and the plasmid-integrated bacteria were selected by plating on LB medium containing ampicillin (50 μg / ml). Next, the plasmid was obtained from the bacteria using the QIAprep® Spin Miniprep kit (QUIAGEN) and purified. After confirming the presence and correct orientation of the insertion fragment and the presence of the AAV2 ITR, the desired quantity of plasmid was obtained from the ampicillin-resistant clones using the commercially available QIAGEN® Plasmid Maxi kit (QUIAGEN) and purified.

[0166] After constructing and purifying the vector plasmid, AAV vector particles were produced by double transfection of HEK-293T cells with the plasmid pAAV2-mPLA2G4E and a pDP9 helper plasmid expressing adenovirus molecules necessary 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] The vector particles were finally purified by an iodixanol gradient and titrated by quantitative PCR. The viral titration, expressed as virus particles (vp) / ml, was performed using primers for mouse PLA2G4E. Forward primer: ATGGTGACAGACTCCTTCGAG (SEQ ID NO: 6) and, Reverse primer: CCTCTGCGTAAAGCTGTGG (SEQ ID NO: 7) It was obtained by quantitative PCR (q-PCR) using [specific method / tool].

[0168] The obtained viral titer was 2.6 × 10⁻⁶. 11 The value was vp / ml.

[0169] Example 2. General Method 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 from the inbred C57BL / 6J genetic background. The AD mouse model APP / PS1 is an accelerated amyloidosis model compared to Tg2576. In these mice, human APP transgene expression is approximately three times higher than in endogenous mouse APP, and human Aβ42 is preferentially produced over Aβ40. Furthermore, amyloid plaque deposition begins in the hippocampus at 3-4 months (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 is observed from 7 months (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] These are aged wild-type mice. Wild-type mice exhibit age-related memory impairment. Specifically, in the Morris water maze, aged wild-type mice performed significantly worse than younger mice during exploration trials, and therefore did not form a strong memory of the platform location at the hidden platform stage. These mice were from the inbred C57BL / 6 / SJL genetic background.

[0171] Additionally, 2-month-old male wild-type (WT) C57BL / 6 mice were used to test the effect of PLA2G4E on synaptic activity.

[0172] Stereotactic surgery for viral administration To overexpress PLA2G4E in hippocampal neurons, AAV2 / 9-mPLA2G4E was administered to the CA1 region of the hippocampus in mice via stereotactic surgery. This procedure is based on a three-dimensional system of axial and spatial coordinates that allows for the positional identification (given in three-dimensional distances in millimeters (mm)) of a specific point in the mouse brain, using two easily identifiable points in the brain, the bregma or lambda, as a reference. Using the mouse atlas (G. Paxinos and KBJ Franklin, The mouse brain in stereotaxic coordinates, Academic Press, 1997) as a reference, the coordinates selected for hippocampal CA1 injection were anterior-posterior-2.0 mm, hemilateral ±1.7 mm, and posterior-ventral-2.0 mm, using the bregma point (formed by the intersection of the sagittal and coronal sutures). Prior to viral administration or a sham procedure (no injection, surgery only), 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 completely anesthetizing the mice, their heads were fully fixed and placed in a stereotactic device. After disinfecting this area with 96° alcohol, the skin was cut anterior-posteriorly using a scalpel to detach the skull from the periosteum and expose the bregma and lambda reference points. Next, holes were drilled in the skull using a drill bill, and vector virus particles (2.6 × 10⁻⁶) were injected. 8A 5 μl Hamilton syringe, either filled with or unfilled (for the sham procedure) a genome copy, was mounted on a stereotactic arm. After positioning it precisely, 1 μl of the solution was injected at a rate of 0.2 μl / min, and the syringe was then held there for a further 2 minutes to allow the virus to spread precisely before the syringe was slowly withdrawn. 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 injection of the animals, the wounds were sutured and povidone-iodine (Betadine®) was administered topically. The animals were then placed on an electric blanket until they were awake to prevent heat loss. Finally, to facilitate postoperative feeding, the animals were placed individually 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 dehydration and resulting blindness.

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

[0174] This test is conducted in a circular pool (1.2m in diameter) filled with 20°C water and made opaque by adding a non-toxic white paint. The pool is divided into four virtual quadrants, one of which contains a platform that the mouse must learn to position itself to escape the water and find safety. Geometric shapes are drawn on each of the four walls surrounding the pool to guide the mouse, and these are covered or uncovered depending on the stage of the test. Throughout the test, the mouse's behavior is monitored by a camera fixed to the ceiling directly above the pool and recorded using an HVS system to allow for subsequent analysis of escape latency, swimming speed, distance, and percentage of time spent in each quadrant of the pool using SMART-LD (Panlab) software.

[0175] In the MWM test, three different stages can be distinguished.

[0176] 1) Visible Platform Stage: In this stage, a platform, identifiable by components clearly recognizable to the animals to facilitate location confirmation, was placed 1 cm above the water level in the center of one quadrant. Visual cues were kept hidden so that the mice could become accustomed to the pool and learn to go to the platform to escape the water. For the Visible Platform Stage, mice were trained eight times a day for three consecutive days. In each test, the mouse was given 60 seconds to find the platform, and if it could not reach the platform during this period, it was placed on the platform. After being placed on the platform, the animal was allowed 15 seconds to examine the platform before being returned to its cage.

[0177] 2) Hidden Platform Phase: In the second phase of the experiment, the platform was placed in the opposite quadrant to that of the visible platform phase. The platform was submerged 1 cm below the water surface, with no objects on it. In this phase, the mice needed to learn how to find the platform using cues displayed on the now uncovered wall. To this end, the mice were trained four times a day for seven days. As in the previous phase, the mice were given 60 seconds to reach the platform. If they could not find the platform within 60 seconds, they were guided to it. In both cases, they were kept on the platform for 15 seconds. To prevent the mice from developing a preference for the trajectory, three random starting positions were provided in each of the quadrants without the platform.

[0178] 3) Exploration Trials: Memory retention was assessed in exploration trials conducted immediately before the start of the hidden platform test on days 6 and 8 of the hidden platform phase. For this trial, 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 where the platform had been located during the hidden platform phase was considered an estimate of memory retention. A retention rate of over 25% was considered to indicate learning, while a retention rate of less than 25% was considered random. Since it has been suggested that the sensitivity of the MWM test may increase by providing shorter exploration trials, the time spent in the exact quadrant was analyzed for both the first 15 seconds and the entire 60 seconds of the test (Gerlai, 2001, Behavioural Brain Research; 125(1-2):269-277).

[0179] Dendrite spine density measurement by 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 half-brain immediately after removal from the skull was incubated in Golgi-Cox solution (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 tissue was maintained there for a further 3 weeks. Subsequently, the brain was washed with distilled water and maintained in 90°C ethanol for 30 minutes until it was processed into 200 μm thick coronal sections using a vibratome. The sections were then incubated in 70°C ethanol, washed with distilled water, reduced with 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 by gradually increasing the amount of alcohol, and mounted using DPX Mountant (VWR, BDH Prolabo®).

[0180] The spine density in the secondary apical dendrites of pyramidal cells located within the CA1 region of the hippocampus was determined. Each selected neuron was imaged using a Nikon Eclipse E600 optical microscope, and the images were recorded with a digital camera (Nikon DXM 1200F) at a resolution of 1000 dots per inch (dpi) to 1500 dpi. Secondary dendrites, imaged 100 μm to 200 μm away from the cell body, were used for quantification in CA1 pyramidal neurons with relatively uniform spine density (Megias, M., Z. Emri, TF Freund, and AI Gulyas, 2001, Neuroscience 102(3):527-40). 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 study consisted of three stages: habituation, training, and testing. The study was conducted using the StartFear system (Panlab). In the habituation stage, mice were habituated to the conditioning chamber for 3 minutes without stimulation. After 24 hours, in the training stage, the mice were placed back into the same chamber and allowed to explore for 2 minutes. Then, two foot shocks (0.3 mA) were administered over 2 seconds with a 30-second interval, and after another 30 seconds, they were returned to their home cage. The following day, the mice were returned to the conditioning chamber and allowed to explore the situation 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 of the paradigm being performed.

[0182] Protein extract To obtain a total protein extract, brain samples were homogenized in a lysis buffer containing a protease inhibitor (10 mM Tris-HCl pH=7.5, 1 mM NaF, 0.1 mM Na3VO4, 2% SDS), sonicated for 2 minutes, allowed to stand on ice for 20 minutes, and centrifuged at 15700 g at 8°C for 13 minutes. The supernatant was stored at -80°C. Total protein concentration was determined using the Pierce® BCA Protein Assay Kit (Thermo Scientific).

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

[0184] Next, the membranes were blocked with 5% milk in TBS and incubated overnight with the following primary antibodies in corresponding buffers: 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 anti-β-actin (1:100000, Synaptic Systems). After washing twice with TBS / Tween-20 and once with TBS alone, the immunolabeled protein bands were detected with HRP-conjugated anti-rabbit or anti-mouse antibody (1:5000, Santa Cruz). Next, antibody binding was visualized using 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 nerve cultures To inhibit PLA2G4E expression in primary neuronal cultures, specific small interfering RNA (siRNA) was used. To identify effective targeting sequences for RNAi, the full-length coding sequence of mouse PLA2G4E was analyzed using different algorithms. After exploring high efficacy in inhibiting PLA2G4E expression, a construct containing an H1 promoter was designed using candidate sequences, manipulably ligated to an shRNA sequence (SEQ ID NO: 8; i.e., a 21-nucleotide sense and antisense sequence linked 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, primary neuronal cultures were used as a model to study synaptic responses induced by evoked burst signals in functional neural 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), and AAV9-shPLA2G4E or AAV9-sh scrambled control were used to infect in vitro culture day 1 (DIV). Next, to induce a burst of action potential firing, these cultures were treated with bicuculin (50 μM, 1 hour), a GABA A receptor antagonist, in DIV 14 (Arnold et al., 2005 J. Physiol. 564: 3-19, Rao et al., Nat. Neurosci., 2006; 9: 887-895). Proteins were extracted with 2% SDS buffer, and activation of CREB (phosphorylated at Ser133), pGluA1, and synapsin I expression was tested by immunoblotting in the lysates.

[0187] Example 3. Effect of AAV2 / 9-mPLA2G4E on the memory function of APP / PS1 mouse A first group (n=9) of male and female APP / PS1 mice aged 16–19 months was treated with AAV2 / 9-mPLA2G4E by stereotactic surgery as described above. Similarly, a second group (n=6) of APP / PS1 mice aged 16–19 months (sham injection) and a third group (n=9) of non-transgenic mice of the same age were included as positive controls (with memory loss) and negative controls (without AD-related memory impairment). Two months after stereotactic surgery, spatial memory was tested by the MWM test as described above. Mice underwent a 3-day visible platform phase followed by a 7-day hidden platform phase. On days 6 and 8, memory retention was tested in exploratory trials performed immediately before the start of the corresponding hidden platform phase tests.

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

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

[0190] Furthermore, as shown in Figure 1B, mice treated with AAV2 / 9-mPLA2G4E stayed in the correct quadrant for a longer period than sham-injected mice in the exploratory trial on day 6. Similar results were obtained in the exploratory trial on day 8 (data not shown), demonstrating that PLA2G4E overexpression reverses the memory retention impairment exhibited in APP / PS1 aged mice.

[0191] On the other hand, a 33% mortality rate was observed in placebo-injected APP / PS1 mice, compared to 10% and 0% in APP / PS1 and non-transgenic mice injected with AAV2 / 9-mPLA2G4E, respectively.

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

[0193] Using the Golgi-Cox assay, we analyzed whether behavioral recovery induced by PLA2G4E overexpression is reflected in structural changes in dendritic spine density. Specifically, we studied apices of pyramidal neurons originating from 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 wild-type (WT) and APP / PS1 pseudomice. No difference was observed between WT and APP / PS1 pseudomice.

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

[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 female 17-month-old C57BL / 6 / SJL WT mice was also evaluated. A first group of C57BL / 6 / SJL WT mice (n=5) were treated with AAV2 / 9-mPLA2G4E by stereotactic surgery, while a second control group of C57BL / 6 / SJL WT mice (n=4) received a placebo injection. Three months after the stereotactic surgery procedure, spatial memory was tested using the MWM test as described above. In this case, the hidden platform phase was performed for only six days, and exploration trials were conducted on days 5 and 7.

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

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

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

[0200] Example 5. The role of PLA2G4E in memory function: Upregulation of PLA2G4E expression after recall of fear-conditioned memories. To obtain more direct evidence regarding the functional role of PLA2G4E in learning and memory, we tested whether PLA2G4E expression is regulated in fear conditioning (FC) tests. This task requires hippocampus-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 immunoblotting after fixation of fear memory in 2-month-old C57BL / 6J WT mice (TT group; n=8) sacrificed 1 hour after the FC paradigm test, and compared with mice sacrificed 24 hours after the FC training phase (T24 group; n=7) and mice not subjected to any phase of the FC test (naive group; n=8).

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

[0203] Since CREB-mediated transcription is required for the consolidation and reconsolidation of situational fear memories (Kida et al., 2002 Nat. Neurosci., 5, pp. 348-355), we initially analyzed pCREB as an indicator of neural plasticity in the hippocampus of animals. Upregulation of pCREB in the hippocampus was observed in a group of mice reintroduced into cages.

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

[0205] In summary, these data suggest that during situational memory retention, an increase in PLA2G4E is observed in the hippocampus after the retrieval of fixed memory.

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

[0207] We used a well-characterized protocol in cortical and hippocampal primary neurons based on exposure to bicuculin (50 μM, 1 hour), a GABA(A) receptor antagonist capable of inducing and / or enhancing synaptic efficacy at excitatory synapses (Rao et al., 2006 Nat. Neurosci., 9: 887-895).

[0208] To demonstrate NMDA receptor activation, we analyzed CREB activation (phosphorylation of CREB at the activation site residue Ser133) (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 bicuculin induces sustained CREB phosphorylation at Ser133 (by NMDA receptor activation) and increases AMPA receptor activation, as analyzed by measuring pGluA1 levels (Rao et al., 2006 Nat. Neurosci., 9: 887-895).

[0209] Since 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 synaptic junction formation, maintenance, and rearrangement (reviewed in Cesca et al., 2010 Prog. Neurobiol., 91: 313-348), synapsin I levels were also analyzed. Furthermore, a significant increase in synapsin I was observed in neuronal cultures activated with bicuculin.

[0210] Next, we analyzed PLA2G4E expression under the same conditions and, interestingly, observed that it was strongly induced by bicuculin, demonstrating that neuronal activation does indeed upregulate PLA2G4E expression.

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

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

[0213] The array of the present disclosure Sequence ID No. 1: Human cytoplasmic phospholipase A2ε (isoform 1) MSLQASEGCPGLGTNVFVPQSPQTDEEGSRSGRSFSEFEDTQDLDTPGLPPFCPMAPWGSEEGLSPCHLLTVRVIRMKNVRQADMLSQTDCFVSLWLPTASQKKLRTR TISNCPNPEWNESFNFQIQSRVKNVLELSVCDEDTVTPDDHLLTVLYDLTKLCFRKKTHVKFPLNPQGMEELEVEFLLEESPSPPETLVTNGVLVSRQVSCLEVHAQSR RRRKREKMKDLLVMVNESFENTQRVRPCLEPCCPTSACFQTAACFHYPKYFQSQVHVEVPKSHWSCGLCCRSRKKGPISQPLDCLSDGQVMTLPVGESYELHMKSTPC PETLDVRLGFSLCPAELEFLQKRKVVVAKALKQVLQLEEDLQEDEVPLIAIMATGGGTRSMTSMYGHLLGLQKLNLLDCASYITGLSGATWTMATLYRDPDWSSKNLEP AIFEARRHVVKDKLPSLFPQLRKFQEELRQRSQEGYRVTFTDFWGLLIETCLGDERNECKLSDQRAALSCGQNPLPIYLTINVKDDVSNQDFREWFEFSPYEVGLQK YGAFIPSELFGSEFFMGRLVKRIPESRICYMLGLWSSIFSLNLLDAWNLSHTSEEFFHRWTREKVQDIEDEPILPEIPKCDANILETTVVIPGSWLSNSFREILTHRSF VSEFHNFLSGLQLHTNYLQNGQFSRWKDTVLDGFPNQLTESANHLCLLDTAFFVNSSYPPLLRPERKADLIIHLNYCAGSQTKPLKQTCEYCTVQNIPFPKYELPDEN ENLKECYLMENPQEPDAPIVTFFPLINDTFRKYKAPGVERSPEELEQGQVDIYGPKTPYATKELTYTEATFDKLVKLSEYNILNNKDTLLQALRLAVEKKKRLKGQCPS Sequence ID 2: Nucleotide sequence encoding human cPLA2e isoform 1 Sequence ID No. 3: Human isoform 2 of cytoplasmic phospholipase A2ε MATGGGTRSMTSMYGHLLGLQKLNLLDCASYITGLSGATWTMATLYRDPDWSSKNLEPAIFEARRHVVKDKLPSLFPDQLRKFQEELRQRSQEGYRVTFTDFWGLLIETCLGDERNECKLSDQ RAALSCGQNPLPIYLTINVKDDVSNQDFREWFEFSPYEVGLQKYGAFIPSELFGSEFFMGRLVKRIPESRICYMLGLWSSIFSLNLLDAWNLSHTSEEFFHRWTREKVQDIEDEPILPEIPKC DANILETTVVIPGSWLSNSFREILTHRSFVSEFHNFLSGLQLHTNYLQNGQFSRWKDTVLDGFPNQLTESANHLCLLDTAFFVNSSYPPLLRPERKADLIIHLNYCAGSQTKPLKQTCEYCTV QNIPFPKYELPDENENLKECYLMENPQEPDAPIVTFFPLINDTFRKYKAPGVERSPEELEQGQVDIYGPKTPYATKELTYTEATFDKLVKLSEYNILNNKDTLLQALRLAVEKKKRLKGQCPS Sequence ID 4: Nucleotide sequence encoding human cPLA2e isoform 2 Nucleotide sequence encoding mouse PLA2G4E fused to the Sequence ID No. 5 flag sequence Sequence ID 6: Forward primer fwPLA2G4E ATGGTGACAGACTCCTTCGAG Sequence ID 7: Reverse primer rvPLA2G4E CCTCTGCGTAAAGCTGTGG shRNA of sequence number 8 PLA2G4E (shPLA2G4E) GGTCTATGGTCTCCTTGTATCAAGAGTACAAGGAGACCATAGACC

Claims

1. A cPLA2e inducer for the treatment of cognitive impairment and / or cognitive impairment-related diseases in subjects requiring treatment, A cPLA2e inducer comprising a viral vector comprising a nucleic acid construct comprising a nucleotide sequence encoding cPLA2e, a viral particle comprising the same, a host cell comprising the viral vector, or a pharmaceutical composition comprising any of the above and a pharmaceutically acceptable carrier or excipient.

2. The cPLA2e inducer according to claim 1, wherein the viral vector is an AAV vector.

3. - The cPLA2e is human cPLA2e; - The cPLA2e is the human cPLA2e of Sequence ID No. 1 or Sequence ID No. 3; or - The cPLA2e inducer according to claim 1 or 2, wherein the nucleotide sequence encoding cPLA2e is SEQ ID NO: 2 or SEQ ID NO:

4.

4. The cPLA2e inducer according to any one of claims 1 to 3, further comprising a neuron-specific promoter operably linked to a nucleotide sequence encoding the cPLA2e, wherein the nucleic acid construct further comprises the cPLA2e inducer according to any one of claims 1 to 3.

5. The cPLA2e inducer according to claim 4, wherein the neuron-specific promoter operably linked to the nucleotide sequence encoding the cPLA2e is a SYN1 promoter or a hybrid SYN1 promoter.

6. The cPLA2e inducer according to any one of claims 1 to 5, wherein the nucleic acid construct further comprises a polyadenylation signal sequence.

7. The nucleic acid construct is - 5' ITR and 3' ITR sequences of adeno-associated virus; or - 5'ITR and 3'ITR sequences derived from AAV2 serotype A cPLA2e inducer according to any one of claims 1 to 6, comprising:

8. The cPLA2e inducer according to any one of claims 1 to 7, wherein the viral particle comprises an AAV vector comprising a nucleic acid construct containing a nucleotide sequence encoding cPLA2e, and comprises a capsid protein selected from the group consisting of AAV2, AAV5, AAV9, and AAV TT serotypes.

9. A cPLA2e inducer according to any one of claims 1 to 8, for the treatment of Alzheimer's disease.

10. a. A step of contacting the compound with mammalian assay cells, b. A step to confirm whether an effect related to the induction or increase of cPLA2e can be obtained, c. If such effect is obtained, the step of identifying the compound as a candidate for the treatment of cognitive impairment and / or diseases associated with cognitive impairment, A method for identifying compounds, including, as candidates for the treatment of cognitive impairment and / or related diseases.

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