Nucleic acid for inhibiting expression of MEX3B gene, MEX3B gene expression inhibitor, method for inhibiting MEX3B gene expression, and preventive or therapeutic agent for diseases caused by MEX3B gene expression

Abstract

Provided are a nucleic acid having less cytotoxicity as a side effect and improved ability to inhibit the expression of the MEX3B gene, a MEX3B gene expression inhibitor containing the nucleic acid, a method for inhibiting the expression of the MEX3B gene, and a preventive or therapeutic agent for a disease caused by the expression of the MEX3B gene. The nucleic acid is any one of the following (1) to (3): (1) an oligonucleotide consisting of a base sequence described in SEQ ID NO. 1 or 2 of the sequence listing; (2) an antisense oligonucleotide consisting of a base sequence obtained by deleting, substituting and / or adding one or two bases to the base sequence described in SEQ ID NO. 1 or 2 of the sequence listing and inhibiting the expression of the MEX3B gene; (3) an antisense oligonucleotide comprising a base sequence described in SEQ ID NO. 3 of the sequence listing and inhibiting the expression of the MEX3B gene.

Description

Technical Field

[0001] The present invention relates to a nucleic acid that inhibits the expression of a MEX3B gene, a MEX3B gene expression inhibitor containing the nucleic acid, a method for inhibiting the expression of a MEX3B gene, and a preventive or therapeutic agent for a disease caused by the expression of a MEX3B gene. Background Art

[0002] In recent years, it has been known that increased expression of inflammatory cytokines or chemokines such as IL-6, IL-13, TNF, G-CSF, CXCL1, CXCL2, or CXCL5 is associated with serious diseases (e.g., severe asthma, joint diseases, diabetes, inflammatory bowel disease, atopic dermatitis, systemic lupus erythematosus, cancer, mental illness, cardiovascular disease, respiratory disease, type 2 diabetes, kidney disease, etc.) (e.g., Non-Patent Document 1).

[0003] On the other hand, it is known that the MEX3B protein is a molecule that binds to various mRNAs and controls the functions (ie, translation into proteins) or stability of these mRNAs (eg, Non-Patent Document 2).

[0004] Furthermore, Patent Document 1 discloses that the MEX3B protein binds to the mRNA of the above-mentioned inflammatory cytokines or chemokines and is associated with the function (ie, translation into protein) or stability of these mRNAs, and is involved in the onset of diseases caused by the above-mentioned inflammatory cytokines or chemokines.

[0005] Therefore, inhibition of MEX3B expression may be important in the treatment or prevention of the above diseases.

[0006] Prior art literature

[0007] Patent Literature

[0008] Patent Document 1: International Publication No. 2018 / 008750A1

[0009] Patent Document 2: International Publication No. 2018 / 008749A1

[0010] Non-patent literature

[0011] Non-patent literature 1: Int Immunol. 2015 Jan; 27(1): 21-9, Cancer Discov. 2016 Jan; 6(1): 80-95.

[0012] Non-patent document 2: Nucleic Acids Res. 2007; 35(4): 1289-300. Summary of the invention

[0013] Problems to be solved by the invention

[0014] Patent Document 2 discloses nucleic acids and the like that can suppress the expression of the MEX3B gene while suppressing cytotoxicity. However, there is still room for improvement in the effect of suppressing the expression of the MEX3B gene.

[0015] The present invention is made in view of the above situation, and its purpose is to provide a nucleic acid with low cytotoxicity and improved ability to inhibit the expression of the MEX3B gene, a MEX3B gene expression inhibitor containing the above nucleic acid, a method for inhibiting the expression of the MEX3B gene, and a preventive or therapeutic agent for diseases caused by the expression of the MEX3B gene.

[0016] Means for solving problems

[0017] The inventors of the present application designed a large number of antisense oligonucleotides in a manner that covers the entire region of the human MEX3B precursor mRNA before splicing, and conducted comprehensive experiments on the inhibition of MEX3B gene expression. The results showed that specific antisense oligonucleotides having a sequence complementary to a specific sequence in the MEX3B gene can significantly inhibit the mRNA expression of the MEX3B gene, and also found that they can significantly inhibit the expression of the MEX3B protein.

[0018] The present invention has been accomplished based on the above findings.

[0019] Specifically, the present invention is as follows.

[0020] A first aspect of the present invention is a nucleic acid according to any one of the following (1) to (3).

[0021] (1) an oligonucleotide consisting of the base sequence described in SEQ ID NO: 1 or 2 in the sequence listing;

[0022] (2) an antisense oligonucleotide that is composed of a base sequence in which one or two bases are deleted, substituted, and / or added to the base sequence described in SEQ ID NO: 1 or 2 in the sequence listing and that inhibits the expression of the MEX3B gene;

[0023] (3) An antisense oligonucleotide comprising the base sequence described in SEQ ID NO: 3 in the sequence listing and inhibiting the expression of the MEX3B gene.

[0024] A second aspect of the present invention is a MEX3B gene expression inhibitor comprising the nucleic acid according to the first aspect.

[0025] The third aspect of the present invention is a method for inhibiting MEX3B gene expression, comprising the step of contacting the agent according to the second aspect with a subject (excluding human individuals).

[0026] A fourth aspect of the present invention is a preventive or therapeutic agent for a disease caused by MEX3B gene expression, comprising the agent according to the second aspect.

[0027] Effects of the Invention

[0028] According to the present invention, there can be provided a nucleic acid having low cytotoxicity and improved ability to inhibit MEX3B gene expression, a MEX3B gene expression inhibitor containing the nucleic acid, a method for inhibiting MEX3B gene expression, and a preventive or therapeutic agent for a disease caused by MEX3B gene expression. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 It is a graph showing the suppression of the human MEX3B mRNA expression level by transfection of each Gapmer-type nucleic acid.

[0030] Figure 2 It is a graph showing the suppression of the human MEX3B mRNA expression level by transfection of each Gapmer-type nucleic acid.

[0031] Figure 3 It is a graph showing the suppression of the human MEX3B mRNA expression level by transfection of each Gapmer-type nucleic acid.

[0032] Figure 4 The graphs show the suppression of the expression level of mouse MEX3B mRNA by transfection of each Gapmer-type nucleic acid.

[0033] Figure 5 This is a graph showing the results of inhibition of human MEX3B mRNA expression by free uptake of each Gapmer-type nucleic acid.

[0034] Figure 6 The graphs show the results of suppressing the expression of human and mouse MEX3B proteins by transfection of each Gapmer-type nucleic acid.

[0035] Figure 7 It is a graph showing the results of suppression of expression of human MEX3B protein by transfection of each Gapmer-type nucleic acid.

[0036] Figure 8 The graphs show the results of suppression of mouse MEX3B protein expression by transfection of each Gapmer-type nucleic acid.

[0037] Fig. 9 It is a graph showing the results of suppression of expression of human MEX3B protein by transfection of each Gapmer-type nucleic acid.

[0038] Fig.10 The graphs show the results of suppression of mouse MEX3B protein expression by transfection of each Gapmer-type nucleic acid. DETAILED DESCRIPTION

[0039] Hereinafter, embodiments of the present invention will be described in detail, but the present invention is not limited to the following embodiments at all, and can be implemented with appropriate modifications within the scope of the purpose of the present invention.

[0040] (MEX3B gene)

[0041] The MEX3B gene consists of exon 1, intron and exon 2, and this structure is highly conserved in humans, mice and other mammals.

[0042] As an untranslated region (UTR) that does not encode amino acids in an exon, a 5'UTR exists upstream of the start codon, and a 3'UTR exists downstream of the stop codon.

[0043] The human MEX3B gene encoding the mRNA of human MEX3B has the sequence represented by SEQ ID NO: 4 described below.

[0044] In SEQ ID NO: 4, the base sequence from 437 to 2146 is CDS, the base sequence from 1 to 436 is 5'UTR, and the base sequence from 2147 to 3532 is 3'UTR.

[0045] The mouse MEX3B gene encoding the mRNA of mouse MEX3B has the sequence represented by SEQ ID NO: 5 described below.

[0046] In SEQ ID NO: 5, the base sequence at positions 319 to 2049 is CDS, the base sequence at positions 1 to 318 is 5'UTR, and the base sequence at positions 2050 to 3416 is 3'UTR.

[0047] Sequence No. 6 described below shows 836 bases in the intron region of the human MEX3B gene.

[0048] Sequence number 7 represents the base sequence of the precursor mRNA encoding human MEX3B before splicing. In the sequence of the precursor mRNA encoding human MEX3B represented by sequence number 7, the base sequences at positions 437 to 692 and 1529 to 2982 are CDS, the base sequence at positions 1 to 436 is 5'UTR, the base sequence at positions 2983 to 4368 is 3'UTR, and the region at positions 693 to 1528 corresponds to the intron region of the human MEX3B gene represented by sequence number 6.

[0049] In addition, genes encoding MEX3B proteins (for example, proteins having the amino acid sequence represented by SEQ ID NO: 8 (human) or 9 (mouse)) all belong to the MEX3B gene.

[0050] (Acquisition of MEX3B gene)

[0051] The method for obtaining the MEX3B gene is not particularly limited. Suitable probes or primers can be prepared based on the information of the base sequences and amino acid sequences recorded in sequence numbers 4, 5, and 7 to 9 of the sequence listing of this specification, and used to screen the desired clones from a human cDNA library (prepared from suitable cells expressing the MEX3B gene according to conventional methods), thereby isolating the MEX3B gene.

[0052] <Nucleic Acid Inhibiting Expression of MEX3B Gene>

[0053] The nucleic acid that inhibits the expression of the MEX3B gene according to the first aspect (hereinafter, also simply referred to as "the nucleic acid according to the first aspect") is any one of the following (1) to (3).

[0054] (1) an oligonucleotide consisting of the base sequence described in SEQ ID NO: 1 or 2 in the sequence listing;

[0055] (2) an antisense oligonucleotide that is composed of a base sequence in which one or two bases are deleted, substituted, and / or added to the base sequence described in SEQ ID NO: 1 or 2 in the sequence listing and that inhibits the expression of the MEX3B gene;

[0056] (3) An antisense oligonucleotide comprising the base sequence described in SEQ ID NO: 3 in the sequence listing and inhibiting the expression of the MEX3B gene.

[0057] The suppression of MEX3B gene expression by the nucleic acid according to the first aspect is at least one of suppression of transcription level and suppression of protein expression level.

[0058] There is no particular limitation on the degree of inhibition, as long as at least one of the transcription amount and the protein expression amount is reduced compared with the negative control (or wild type). Preferably, at least one of the transcription amount and the protein expression amount is reduced by 10% or more, more preferably by 15% or more, further preferably by 20% or more, further preferably by 30% or more, particularly preferably by 40% or more, particularly preferably by 50% or more, and most preferably by 60% or more, compared with the negative control (or wild type).

[0059] Regarding (1) above, the base sequence described in SEQ ID NO: 1 (GCGGATACTA CAGCTT) and the base sequence described in SEQ ID NO: 2 (GAGCGGATACTACAG C) are the base sequences possessed by the gaper-type nucleic acids hmrGD-89-2 and hmrGD-89 described later in the Examples, respectively.

[0060] Regarding (2) above, the “one or two” in “a base sequence in which one or two bases are deleted, substituted and / or added to the base sequence” is preferably one.

[0061] Regarding the above (2), the base length of the antisense oligonucleotide is preferably 14 bases or more, and more preferably 15 bases or more.

[0062] The upper limit of the base length of the antisense oligonucleotide is preferably 18 bases or less, more preferably 17 bases or less, and even more preferably 16 bases or less.

[0063] From the viewpoint of productivity and the like, the above-mentioned “base sequence obtained by deletion, substitution and / or addition” is preferably a “base sequence obtained by deletion and / or substitution”, and more preferably a “base sequence obtained by deletion”.

[0064] As the degree of the above-mentioned DNA mutation, for example, it is preferably a degree of identity of more than 90% with the base sequence recorded in sequence number 1 or 2 in the sequence table, more preferably a degree of identity of more than 93%, further preferably a degree of identity of more than 95%, particularly preferably a degree of identity of more than 97%, and most preferably a degree of identity of more than 98%.

[0065] In the base sequence recorded in sequence number 1 or 2 of the sequence table, part of the DNA sequence may be changed by various artificial treatments, such as site-directed mutagenesis, random mutation caused by mutagen treatment, mutation, deletion, and connection of DNA fragments caused by restriction endonuclease cleavage, etc., or no artificial treatment may be performed.

[0066] Regarding (3) above, the base sequence (GCGGATACTACAGC) recorded in SEQ ID NO: 3 is a base sequence of 14 bases common to SEQ ID NOs: 1 and 2 above.

[0067] Regarding the above (3), the above-mentioned antisense oligonucleotide is preferably an antisense oligonucleotide having a sequence complementary to an oligonucleotide containing at least 12 consecutive nucleotides in the base sequence of the MEX3B gene, more preferably an antisense oligonucleotide having a sequence complementary to an oligonucleotide containing at least 13 nucleotides, and further preferably an antisense oligonucleotide having a sequence complementary to an oligonucleotide containing at least 14 nucleotides.

[0068] In addition, as the upper limit of the base length of the above-mentioned antisense oligonucleotide, it is preferably an antisense oligonucleotide having a sequence complementary to an oligonucleotide of 25 or less consecutive nucleotides in the base sequence of the MEX3B gene, more preferably an antisense oligonucleotide having a sequence complementary to an oligonucleotide of 20 or less consecutive nucleotides, further preferably an antisense oligonucleotide having a sequence complementary to an oligonucleotide of 19 or less consecutive nucleotides, still further preferably an antisense oligonucleotide having a sequence complementary to an oligonucleotide of 18 or less consecutive nucleotides, particularly preferably an antisense oligonucleotide having a sequence complementary to an oligonucleotide of 17 or less consecutive nucleotides, and particularly preferably an antisense oligonucleotide having a sequence complementary to an oligonucleotide of 16 or less consecutive nucleotides.

[0069] From the viewpoint of productivity and the like, the antisense oligonucleotide of (3) above preferably has a base sequence of 15 bases or less.

[0070] Regarding the above (3), as an example of the degree of DNA mutation, it is preferably a degree of identity of more than 90% with the base sequence recorded in sequence number 3 of the sequence table, more preferably a degree of identity of more than 93%, further preferably a degree of identity of more than 95%, particularly preferably a degree of identity of more than 97%, and most preferably a degree of identity of more than 98%.

[0071] Regarding (1) to (3) above, for the above-mentioned antisense oligonucleotide, it is preferred that, for example, a region in the MEX3B gene and the above-mentioned antisense oligonucleotide complementary thereto form a hybrid after being introduced into a cell, thereby producing a hybrid double strand, and using a nuclease (for example, ribonuclease H) specific to the hybrid double strand to degrade the mRNA of MEX3B containing the nucleotide chain.

[0072] The antisense oligonucleotide may be DNA or RNA, but is preferably DNA from the viewpoint of cleaving mRNA by the specific nuclease.

[0073] Regarding (1) to (3) above, the antisense oligonucleotide is preferably an antisense oligonucleotide comprising at least one nucleotide having at least one structure selected from the group consisting of a phosphorothioate structure, a bridge structure, and an alkoxy structure.

[0074] For example, by providing a phosphorothioate structure in the phosphodiester bond portion (which connects nucleotides to each other), nuclease resistance can be acquired, and since hydrophobicity is improved, uptake into cells or nuclei can be increased.

[0075] In addition, by making the sugar part of the nucleotide have a bridging structure such as 2',4'-BNA (2',4'-Bridged Nucleic Acid, also known as LNA (Locked Nucleic Acid)), ENA (2'-O,4'-C-ethylene-bridged Nucleic Acid (2'-O,4'-C-Ethylene-bridged Nucleic Acid)), or an alkoxy structure such as 2'-O-methylation or 2'-O-methoxyethylation (2'-MOE), nuclease resistance can be obtained and the mRNA binding ability can be improved.

[0076] In the above-mentioned antisense oligonucleotide, it is preferred that at least one phosphodiester bond portion (which connects nucleotides to each other) has a phosphorothioate structure, more preferably more than 50% of the phosphodiester bonds in the above-mentioned antisense oligonucleotide have a phosphorothioate structure, further preferably more than 70% of the phosphodiester bonds in the above-mentioned antisense oligonucleotide have a phosphorothioate structure, particularly preferably more than 90% of the phosphodiester bonds in the above-mentioned antisense oligonucleotide have a phosphorothioate structure, and most preferably all the phosphodiester bonds in the above-mentioned antisense oligonucleotide have a phosphorothioate structure.

[0077] In the above antisense oligonucleotide, it is preferred that at least one of the terminal nucleotides has a bridge structure or an alkoxy structure.

[0078] More specifically, at least at any one end, preferably any nucleotide among 4 or less bases from the end has a bridge structure or an alkoxy structure, more preferably any nucleotide among the 1st to 3rd bases from the end has a bridge structure or an alkoxy structure, and further preferably 2 or 3 bases from the end have a bridge structure or an alkoxy structure.

[0079] It is preferred that the nucleotides at both terminals of the above-mentioned antisense oligonucleotide have a bridging structure or an alkoxy structure (so-called Gapmer-type antisense oligonucleotide), and it is more preferred that at both terminals of the above-mentioned antisense oligonucleotide, any nucleotide of 4 or less bases independently from the terminal has a bridging structure or an alkoxy structure, and it is further preferred that any nucleotide of the 1st to 3rd bases from the terminal has a bridging structure or an alkoxy structure, and it is particularly preferred that 2 or 3 bases from the terminal have a bridging structure or an alkoxy structure.

[0080] The nucleic acid related to the first embodiment can be produced by a conventional method using a DNA synthesizer and known organic synthesis techniques.

[0081] In vivo or in vitro, uptake into cells can be achieved by contacting the nucleic acid of the first aspect with cells (for example, by adding the nucleic acid of the first aspect to a medium for culturing any cells).

[0082] On the other hand, by making the nucleic acid related to the first aspect have at least one structure selected from the group consisting of a phosphorothioate structure, a bridge structure, and an alkoxy structure, the uptake into cells can be further improved.

[0083] By making the nucleic acid related to the first embodiment have at least one structure selected from the group consisting of a phosphorothioate structure, a bridge structure, and an alkoxy structure, and using it in combination with a lipofection vector described later, the uptake into cells can be further improved.

[0084] On the other hand, the nucleic acid according to the first embodiment has a strong inhibitory effect on MEX3B gene expression and does not require the use of a liposome transfection vector described below. In addition, as described in Example 4, it can be taken up into cells by free uptake.

[0085] The method for introducing the nucleic acid into cells according to the first embodiment may be a method in which the nucleic acid is inserted into an appropriate vector and then introduced into an appropriate host cell.

[0086] The type of the above-mentioned suitable vector is not particularly limited, and may be, for example, an autonomously replicating vector (eg, a plasmid, etc.), preferably a vector that is integrated into the genome of a host cell when introduced into the host cell and replicated together with the integrated chromosome.

[0087] As the above-mentioned suitable vector, plasmids derived from Escherichia coli (e.g., pBR322, pUC118 and others), plasmids derived from Bacillus subtilis (e.g., pUB110, pSH19 and others), bacteriophages, retroviruses, animal viruses such as vaccinia virus, etc. can be used. When performing recombination, a translation start codon and a translation stop codon can also be added using a suitable synthetic DNA linker.

[0088] In addition, as required, the nucleic acid involved in the first embodiment can be functionally bound to an appropriate terminator, such as a human growth hormone terminator, or a TPI1 terminator or an ADH3 terminator for a fungal host. The recombinant vector may further have the following elements: a polyadenylation signal (e.g., a polyadenylation signal from the E1b region of SV40 or type 5 adenovirus); a transcription enhancer sequence (e.g., an SV40 enhancer); and a translation enhancer sequence (e.g., a sequence encoding adenovirus VARNA).

[0089] The recombinant vector may further include a DNA sequence that enables the vector to replicate in a host cell, and an example thereof is the SV40 replication origin (when the host cell is a mammalian cell).

[0090] The recombinant vector may further contain a selection marker. Examples of selection markers include genes whose complement is lacking in host cells, such as dihydrofolate reductase (DHFR) or Schizosaccharomyces pombe TPI gene, or resistance genes for drugs such as ampicillin, kanamycin, tetracycline, chloramphenicol, neomycin, or hygromycin.

[0091] Examples of host cells into which the nucleic acid according to the first embodiment or a vector containing the same may include higher eukaryotic cells, bacteria, yeast, fungi, and the like, and mammalian cells are preferred.

[0092] Examples of mammalian cells include HEK293 cells, HeLa cells, COS cells (e.g., COS-7 cells, etc.), BHK cells, CHL cells or CHO cells, BALB / c mouse cells (e.g., BALB / c mouse embryonic fibroblasts), etc. Methods for transforming mammalian cells and expressing genes introduced into the cells are also known, and for example, liposome transfection, electroporation, calcium phosphate method, etc. can be used.

[0093] The inhibition of MEX3B gene expression by the nucleic acid involved in the first embodiment can be detected in vivo or in vitro using, for example, probes or primers having a part or all of the base sequence of the gene, as long as the base sequence information of the MEX3B gene is used as the basis.

[0094] In particular, the expression level of the MEX3B gene at the mRNA level can be measured by conventional methods such as RT-PCR and Northern blotting.

[0095] When performing PCR, the primers are not particularly limited as long as they can specifically amplify only the MEX3B gene, and can be appropriately set based on the sequence information of the MEX3B gene. For example, an oligonucleotide comprising at least 10 consecutive nucleotides in the MEX3B gene, and an antisense oligonucleotide having a sequence complementary to the oligonucleotide can be used as a probe or primer. More specifically, an oligonucleotide having a base sequence of 10 to 60 consecutive residues (preferably 10 to 40 consecutive residues) in the MEX3B gene, and an antisense oligonucleotide having a sequence complementary to the oligonucleotide can be used.

[0096] In addition, the measurement of the expression level of MEX3B protein can be carried out by using common immunoassays such as Western blotting or ELISA. Specifically, it can be carried out by using conventional methods known to those skilled in the art as described in Molecular Cloning, 2nd Edition or Current Protocols in Molecular Biology, etc.

[0097] <MEX3B gene expression inhibitor>

[0098] The MEX3B gene expression inhibitor according to the second mode (hereinafter, also simply referred to as "the reagent according to the second mode") contains the nucleic acid according to the first mode.

[0099] As described above, the inhibition of MEX3B gene expression is at least one of transcriptional inhibition and protein expression inhibition.

[0100] From the viewpoint of improving the uptake into cells, the MEX3B gene expression inhibitor according to the second mode may further contain a lipofection vector. As described in Example 4, the nucleic acid according to the first mode can be taken up into cells by free uptake. From the viewpoint of achieving a strong MEX3B gene expression inhibitory effect, it may not contain a lipofection vector.

[0101] As the lipofection vector, a vector with high affinity for cell membranes (such as liposome, cholesterol, etc.) can be cited. Lipofectamine or Lipofectin is preferred, and Lipofectamine is more preferred.

[0102] By making the nucleic acid according to the first mode have at least one structure selected from the group consisting of phosphorothioate structure, bridging structure, and alkoxy structure and using it in combination with a lipofection vector, the uptake into cells can be further improved.

[0103] For example, in the presence of a lipofection vector, the nucleic acid according to the first mode having at least one structure selected from the group consisting of phosphorothioate structure, bridging structure, and alkoxy structure can be contacted with the subject described later.

[0104] The reagent according to the second mode can be in a form containing a mixture of the nucleic acid according to the first mode, the above lipofection vector, and other optional components (such as water, buffer, etc.), or in a form of a kit in which the nucleic acid according to the first mode and other optional components, and the above lipofection vector and other optional components are packaged in separate containers.

[0105] The form of the reagent involved in the second embodiment is not particularly limited, and can be used in the form of liquid, granules, tablets, capsules, patches, etc. In the case of the body, the reagent involved in the second embodiment can be directly exposed to the tissue. More preferably, the following means can be used to administer to the body: exposure (liquid, etc.) to the body; or oral administration; or injection, spraying, or coating, etc., into blood vessels such as veins or arteries, in the mouth, under the tongue, in the rectum, in the abdominal cavity, in the vagina, in the medullary cavity, in the muscle, in the skin, subcutaneous, intradermal, in the bladder, in the trachea (bronchus), eyes, nose, ears, etc.

[0106] <Method for inhibiting MEX3B gene expression>

[0107] The method for inhibiting MEX3B gene expression according to the third aspect comprises the step of contacting the agent according to the second aspect with a subject.

[0108] As described in Example 4 below, the reagent according to the second embodiment can be taken up into cells through free uptake by the above-mentioned contact, for example, without requiring calcium stimulation (calcium shock) or the like.

[0109] Examples of the target include biological individuals (for example, individuals of mammals or birds), microorganisms, protozoa, biological tissues, biological tissue sections, human cells, animal cells, and the like.

[0110] Examples of mammals or birds include cattle, sheep, goats, pigs, horses, dogs, chickens, mice, rats, guinea pigs, hamsters, rabbits, and primates. Examples of primates include monkeys, and may or may not be human.

[0111] There is no particular limitation on the contact method, and for example, the reagent according to the second embodiment may be added to a medium containing the object, or the medium may be prepared to contain the reagent according to the second embodiment. There is no particular limitation on the temperature, time, etc. during the contact, and they may be appropriately set according to the type of the object, etc.

[0112] <Preventive or therapeutic agent for a disease caused by MEX3B gene expression>

[0113] The preventive or therapeutic agent for a disease caused by MEX3B gene expression according to the fourth aspect comprises the MEX3B gene expression inhibitor according to the second aspect.

[0114] Interleukin 6 (IL-6) is an important cytokine involved in inflammation, hematopoiesis, bone metabolism, tumor progression, etc. IL-6 activity is known to mainly promote the transition from acute inflammation to acquired immune response and the onset of chronic inflammatory diseases (eg, JAsthma. 2008; 45 Suppl 1: 41-4).

[0115] Interleukin 13 (IL-13) is known to play a role as an inflammatory cytokine that further aggravates allergic inflammation in peripheral tissues. IL-13 is known to promote allergic reactions, which are the main cause of allergic asthma, and is also involved in the refractory nature of asthma (making steroid agents ineffective). In addition, IL-13 is not only involved in asthma, but also in the formation of the disease conditions in inflammatory bowel disease and atopic dermatitis (for example, J Allergy (Cairo). 2012; 2012: 316049, NEngl J Med 2011; 365: 1088-1098).

[0116] TNF-α in TNF (Tumor Necrosis Factor) is a signal factor that induces inflammatory response and is an important factor from the viewpoint of anti-infection. On the other hand, it is also known to be involved in disorders caused by hyperinflammatory reactions. That is, TNF is involved in the deterioration of various diseases, and is known to be mainly involved in joint diseases (rheumatoid arthritis, psoriatic arthritis, spondyloarthritis, ankylosing spondylitis), inflammatory bowel disease (ulcerative colitis, Crohn's disease), cancer (ovarian cancer, breast cancer), mental illness (depression, bipolar disorder, epilepsy, Alzheimer's disease, Parkinson's disease, multiple sclerosis), cardiovascular disease (heart failure, arteriosclerosis), respiratory disease (bronchial asthma, chronic bronchitis, chronic obstructive pulmonary disease, acute lung injury), type 2 diabetes, kidney disease (ischemic renal injury, post-transplant rejection, glomerulonephritis), etc. (for example, J Allergy Clin Immunol. 2008 Jan; 121(1): 5-10, J Pathol. 2008 Jan; 214(2): 149-60.).

[0117] In addition, G-CSF (Granulocyte-Colony Stimulating Factor) is known to have the effect of promoting granulocyte production and enhancing the function of neutrophils.

[0118] In addition, IL-13, TNF, and G-CSF are also known to be involved in the aggravation of asthma (for example, Curr Opin Immunol. 2013 Dec; 25(6): 755-60).

[0119] In addition, CXCL1, CXCL2, and CXCL5 belong to the CXC subfamily of inflammatory chemokines.

[0120] When CXCL1, CXCL2, and CXCL5 are secreted in the lung tissue due to excessive inflammation in the respiratory mucosa, the infiltration of neutrophils that highly express CXCR2 (which is a receptor for CXCL1, CXCL2, and CXCL5) is promoted. As a result, the infiltration of steroid-resistant neutrophils causes severe asthma, thereby inducing chronic inflammation (which induces irreversible remodeling of the respiratory tract).

[0121] The inventors of the present application have discovered that the MEX3B gene is involved in the onset of diseases caused by IL-6, IL-13, TNF, G-CSF, CXCL1, CXCL2, or CXCL5.

[0122] Therefore, the preventive or therapeutic agent involved in the fourth embodiment is considered to be effective as a preventive or therapeutic agent for diseases caused by increased expression of IL-6, IL-13, TNF, G-CSF, CXCL1, CXCL2, or CXCL5 (for example, excessive increase in expression, preferably increase in expression relative to healthy state, more preferably excessive increase in expression relative to healthy state) (for example, severe asthma, chronic obstructive pulmonary disease, rheumatoid arthritis, colitis, Crohn's disease, atopic dermatitis, systemic lupus erythematosus, cancer, etc. caused by IL-6, IL-13, TNF, G-CSF, CXCL1, CXCL2, or CXCL5 (Int Immunol. 2015 Jan; 27(1): 21-9, Cancer Discov. 2016 Jan; 6(1): 80-95.)).

[0123] In addition, the MEX3B protein is known to be a protein that induces apoptosis (for example, Japanese Patent No. 4429269).

[0124] It is known that apoptosis is involved in not only normal physiological processes but also serious diseases such as neurodegenerative diseases. For example, abnormal hyperactivation of apoptosis is considered to be the cause of neurodegenerative diseases (e.g., Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, Huntington's disease, etc.) (Wolozin, B. et al., (1996) Science, 274, 1710-1713).

[0125] Therefore, the preventive or therapeutic agent according to the fourth aspect is considered to be effective as a preventive or therapeutic agent for neurodegenerative diseases.

[0126] The prophylactic or therapeutic agent according to the fourth mode can be administered systemically or locally by oral or parenteral routes. As parenteral administration methods, intravenous injection such as drip infusion, intramuscular injection, intraperitoneal injection, subcutaneous injection, etc. can be cited. An appropriate administration method can be selected according to the age and symptoms of the patient. The dosage varies depending on the age, administration route, and number of administrations, and can be appropriately selected by those skilled in the art.

[0127] As a preparation form suitable for parenteral administration, for example, a form containing additives such as stabilizers, buffers, preservatives, and isotonic agents can be cited, and it may also contain a pharmaceutically acceptable carrier and additives. Examples of such carriers and additives include water, organic solvents, high molecular compounds (such as collagen, polyvinyl alcohol, etc.), stearic acid, human serum albumin (HSA), mannitol, sorbitol, lactose, surfactants, etc., but are not limited thereto.

[0128] Regarding the dosage of the nucleic acid according to the first mode as an active ingredient, it is usually in the range of about 0.1 μg to 100 mg per administration (relative to each 1 kg body weight).

[0129] Examples

[0130] Hereinafter, examples are shown to more specifically illustrate the present invention, but the scope of the present invention is not limited to these examples.

[0131] 《Example 1》

[0132] <Preparation of Gapmer - type nucleic acid>

[0133] A large number of antisense oligonucleotides were prepared with a 5 - base stagger to cover the entire region of the pre - mRNA of human MEX3B before splicing, and a comprehensive test on the inhibition of MEX3B gene expression was carried out.

[0134] Gapmer-type nucleic acids hmrGD-87 (SEQ ID NO: 11), hmrGD-88 (SEQ ID NO: 12), hmrGD-89 (SEQ ID NO: 2), hmrGD-90 (SEQ ID NO: 13), hmrGD-91 (SEQ ID NO: 14), hmrGD-92 (SEQ ID NO: 15), hmrGD-93 (SEQ ID NO: 16), hmrGD-94 (SEQ ID NO: 17), hmrGD-95 (SEQ ID NO: 18), hmrGD-96 (SEQ ID NO: 19), hmrGD-97 (SEQ ID NO: 20), hmrGD-98 (SEQ ID NO: 21), hmrGD-99 (SEQ ID NO: 22), hmrGD-100 (SEQ ID NO: 23), hmrGD-101 (SEQ ID NO: 24), hmrGD-102 (SEQ ID NO: 25), hmrGD-103 (SEQ ID NO: 26), hmrGD-104 (SEQ ID NO: 27), hmrGD-105 (SEQ ID NO: 28), hmrGD-106 (SEQ ID NO: 29), hmrGD-107 (SEQ ID NO: 30), hmrGD-108 (SEQ ID NO: 31), hmrGD-109 (SEQ ID NO: 32), hmrGD-110 (SEQ ID NO: 33), hmrGD-111 (SEQ ID NO: 34), hmrGD-112 (SEQ ID NO: 35), hmrGD-113 (SEQ ID NO: 36), hmrGD-114 rGD-176 (sequence number 17), hmrGD-178 (sequence number 18), hmrGD-179 (sequence number 19), hmrGD-180 (sequence number 20), hmrGD-182 (sequence number 21), hmrGD-199 (sequence number 22), hmrGD-201 (sequence number 23), hmrGD-202 (sequence number 24), hmrGD-203 (sequence number 25) and hmrGD-205 (sequence number 26) are shown in Table 1 below.

[0135] Among the above-mentioned gapmer-type nucleic acids, hmrGD-176, hmrGD-178, hmrGD-179, hmrGD-180, hmrGD-182, hmrGD-199, hmrGD-201, hmrGD-202, hmrGD-203, and hmrGD-205 are gapmer-type nucleic acids described in Patent Document 2.

[0136] In addition, a gapmer-type nucleic acid LNA-NC(2,2) (sequence number 10) was used as a negative control, and a gapmer-type nucleic acid LNA-1(2,2) which had been confirmed to exhibit a predetermined MEX3B gene expression inhibitory effect was used as a positive control. The gapmer-type nucleic acid LNA-1(2,2) is an antisense oligonucleotide complementary to a predetermined target sequence on human precursor mRNA.

[0137] It should be noted that 2 or 3 bases of LNA (2', 4'-BNA) are configured at both ends of each gapmer-type nucleic acid, and the bases used to fill the remaining fragments are ordinary DNA. The phosphodiester bonds connecting each nucleotide are phosphorothioated, and the full length of the gapmer-type nucleic acid used as a negative control is 15 bases, and the full length of the other gapmer-type nucleic acids is 16 bases.

[0138] Furthermore, the above-mentioned "(2,2)" means that the two bases at both ends of the gaper-type nucleic acid are LNAs.

[0139] [Table 1]

[0140]

[0141] <Transfection using Gapmer-type nucleic acid>

[0142] Transfection (introduction into cells) was performed using the Gapmer-type nucleic acid of the comparative example and the Gapmer-type nucleic acid of the example.

[0143] The cells used for transfection of the gapmer type nucleic acid were human lung adenocarcinoma alveolar basal epithelial cells A549 cells, and the nucleic acid was introduced into the cells at a final concentration of 20 nM using Lipofectamine RNAiMax (Invitrogen) and its recommended protocol.

[0144] <Quantitative RT-PCR assay>

[0145] After 48 hours of transfection, the state of the cells was observed under a microscope, and then the cells were recovered and total RNA was recovered using a lysis buffer TRIsure (BIOLINE). Reverse transcription was performed using Primescript (TAKARA) to obtain cDNA.

[0146] Then, quantitative RT-PCR was performed using Light Cycler 480 (manufactured by ROCHE).

[0147] The primer sequences used in the quantitative RT-PCR assay are as follows.

[0148] Human MEX3B forward primer: 5'-ACCCAGTTCTGAGCATGTCG-3' (SEQ ID NO: 27)

[0149] Human MEX3B reverse primer: 5'-CGAACTGGGGTCTTGATGTAA-3' (SEQ ID NO: 28)

[0150] Human GAPDH forward primer: 5'-GCACCGTCAAGGCTGAGAAC-3' (SEQ ID NO: 29)

[0151] Human GAPDH reverse primer: 5'-TGGTGAAGACGCCAGTGGA-3' (SEQ ID NO: 30)

[0152] GAPDH was used as the internal standard.

[0153] Depend on Figure 1 and Figure 2The results of quantitative RT-PCR shown in the figure show that the gapmer type nucleic acid of hmrGD-89 (SEQ ID NO. 2) suppressed the expression of human MEX3B mRNA to about 1 / 3 of the negative control gapmer type nucleic acid LNA-NC (2,2) (SEQ ID NO. 10), and showed an effect of suppressing the expression of human MEX3B mRNA equivalent to the positive control gapmer type nucleic acid LNA-1 (2,2). This expression suppression ability is particularly excellent compared with other gapmer type nucleic acids targeting 3'UTR.

[0154] In addition, although not shown in the figure, no side effects such as cytotoxicity were observed in cells transfected with gaper-type nucleic acids hmrGD-87, hmrGD-88, hmrGD-89, hmrGD-90, hmrGD-91, hmrGD-92, hmrGD-93, hmrGD-176, hmrGD-178, hmrGD-179, hmrGD-180, hmrGD-182, hmrGD-199, hmrGD-201, hmrGD-202, hmrGD-203 and hmrGD-205, as in the negative control.

[0155] It should be noted that slight cytotoxicity was observed in the positive control gaper-type nucleic acid LNA-1(2,2).

[0156] 《Example 2》

[0157] As described above, hmrGD-89 (sequence number 2) has a significant inhibitory effect on human MEX3B mRNA expression. Therefore, in order to further explore Gapmer-type nucleic acids with mRNA expression inhibitory effects, Gapmer-type nucleic acids with a target sequence shifted by 1 or 2 bases from hmrGD-89 were prepared and the experiment was carried out in the same manner as in Example 1.

[0158] As summarized in Table 2 below, the gapmer nucleic acid hmrGD-89-2 (SEQ ID NO: 1) is a gapmer nucleic acid in which the target sequence is shifted by two bases toward the 5′ end from the target sequence of hmrGD-89.

[0159] [Table 2]

[0160]

[0161] Depend on Figure 3The results of the quantitative RT-PCR shown show that the Gapmer-type nucleic acid of hmrGD-89 (sequence number 2) has the same inhibitory effect on the mRNA expression of human MEX3B as the positive control Gapmer-type nucleic acid LNA-1 (2, 2), and in comparison, the Gapmer-type nucleic acid hmrGD-89-2 (sequence number 1) has a superior inhibitory effect on the mRNA expression of human MEX3B.

[0162] In addition, in the cells transfected with the Gapmer-type nucleic acid hmrGD-89-2, no adverse effects such as cytotoxicity were observed, similarly to the negative control.

[0163] Example 3: Mouse MEX3B mRNA expression inhibition test

[0164] The gaper-type nucleic acids hmrGD-89-2 and hmrGD-89 were tested for their ability to inhibit the mRNA expression of mouse MEX3B.

[0165] <Transfection using Gapmer-type nucleic acid>

[0166] Transfection with Gapmer-type nucleic acids hmrGD-89-2 and hmrGD-89 was carried out in the same manner as in Example 2, except that the cells to be transfected were changed to mouse lung epithelial MLE-15 cells.

[0167] <Quantitative RT-PCR assay>

[0168] Quantitative RT-PCR was performed in the same manner as in Example 2 except that the primers used in the quantitative RT-PCR were changed to the following ones. The results are shown in Figure 4 .

[0169] Mouse MEX3B forward primer: 5'-CGTCGTCCTCTGTGGTCTTTCCCGGGGGTG-3' (SEQ ID NO: 31)

[0170] Mouse MEX3B reverse primer: 5'-TCAGGAAAAAATGCGGATGGCCTGAGTGAC-3' (SEQ ID NO: 32)

[0171] Mouse β-actin forward primer: 5'-GGATGCAGAAGGAGATTACTGC-3' (SEQ ID NO: 33)

[0172] Mouse β-actin reverse primer: 5'-CCACCGATCCACACAGAGCA-3' (SEQ ID NO: 34)

[0173] β-Actin was used as an internal standard.

[0174] Depend on Figure 4 The results of the quantitative RT-PCR shown show that both Gapmer-type nucleic acids hmrGD-89-2 and hmrGD-89 exerted the same inhibitory effect on mouse MEX3B mRNA expression as the positive control Gapmer-type nucleic acid LNA-Ex-mMex3B-4, which is known to show a specified MEX3B gene expression inhibitory effect. The Gapmer-type nucleic acid LNA-Ex-mMex3B-4 is an antisense oligonucleotide complementary to a specified target sequence on mouse precursor mRNA.

[0175] Example 4: Human MEX3B mRNA expression inhibition test using free uptake

[0176] The inhibition test of human MEX3B mRNA expression was performed by free uptake independent of the transfection method using Lipofectamine.

[0177] In addition to the LNA-type gapmer nucleic acids hmrGD-89-2 and hmrGD-89 used in Example 2, ENA-type gapmer nucleic acids hmrGD-89-2 and hmrGD-89 were prepared and tested.

[0178] <Transfection by free uptake method>

[0179] The cells were introduced in the same manner as in Example 2 except that Lipofectamine RNAiMax (manufactured by Invitrogen) was not used and the final concentration of the cells was 1 μM.

[0180] <Quantitative RT-PCR assay>

[0181] Quantitative RT-PCR was performed in the same manner as in Example 2 except that the state of the cells was observed under a microscope 72 hours after the transfection and the cells were recovered and the total RNA was recovered.

[0182] The results are shown in Figure 5 . Figure 5 The control in is the negative control.

[0183] Depend on Figure 5 The results of quantitative RT-PCR shown in the figure show that the gaper-type nucleic acid hmrGD-89-2 and the LNA type and ENA type of hmrGD-89 both showed excellent levels of suppression of human MEX3B mRNA expression even by the free uptake method, and it is expected that they can suppress the expression of the MEX3B gene when administered to a living body. Among them, the LNA type and ENA type of hmrGD-89-2 are particularly excellent.

[0184] Example 5: Protein expression inhibition test of MEX3B in humans and mice

[0185] In the same experiment as in Example 1, in which the expression inhibition of the MEX3B gene covering the entire region of the human MEX3B precursor mRNA before splicing was performed, not only the human lung adenocarcinoma alveolar basal epithelial cells A549 cells but also the mouse lung epithelial cells MLE-15 cells were transfected, and then the state of the cells was observed under a microscope 48 hours after the transfection, and then the cells were recovered, and the total protein was recovered using RIPA (Radio-Immunoprecipitation Assay) buffer (manufactured by FUJIFILM Wako Pure Chemical Corporation), and the protein expression inhibition test of human and mouse MEX3B was performed by Western blotting.

[0186] The results of using the gaper-type nucleic acids hmrGD-87 (SEQ ID NO: 11), hmrGD-88 (SEQ ID NO: 12), hmrGD-89 (SEQ ID NO: 2), hmrGD-90 (SEQ ID NO: 13), hmrGD-91 (SEQ ID NO: 14), hmrGD-92 (SEQ ID NO: 15), and hmrGD-93 (SEQ ID NO: 16) are shown in FIG. Figure 6 .

[0187] In the figure, a) shows the results of transfection of human lung adenocarcinoma alveolar basal epithelial cells A549, and b) shows the results of transfection of mouse lung epithelial cells MLE-15. α-Tubulin was used as an internal standard (the same applies hereinafter).

[0188] Lane NC shows the results using the gapmer type nucleic acid LNA-NC(2,2) (SEQ ID NO: 10) as a negative control, and lanes 1 and G4 show the results using the gapmer type nucleic acid as a positive control.

[0189] according to Figure 6 The results of Western blotting shown in a) and b) show that, for the gapmer type nucleic acid of hmrGD-89, the band of MEX3B protein became fainter in both human and mouse, similarly to the gapmer type nucleic acid of the positive control, and the expression of MEX3B protein was suppressed. This effect is particularly excellent compared with other gapmer type nucleic acids targeting 3'UTR.

[0190] In addition, among the Gapmer-type nucleic acids described in Patent Document 2, hmrGD-178, hmrGD-179, hmrGD-180, hmrGD-182, hmrGD-199, hmrGD-201, hmrGD-202, hmrGD-203, and hmrGD-205 were similarly transfected into human lung adenocarcinoma alveolar basal epithelial cells A549, and the protein expression of human MEX3B was confirmed by Western blotting. The results are shown in Figure 7 .

[0191] according to Figure 7 The results based on Western blotting shown in the figure show that the bands of MEX3B protein in hmrGD-178, hmrGD-179, hmrGD-180, hmrGD-182, hmrGD-199, hmrGD-201, hmrGD-202, hmrGD-203 and hmrGD-205 (especially hmrGD-201 and hmrGD-202) were slightly lighter than those in the negative control NC.

[0192] In addition, hmrGD-179 and hmrGD-180, among the gaper-type nucleic acids described in Patent Document 2, were transfected into mouse lung epithelial MLE-15 cells, and then the protein expression of mouse MEX3B was confirmed by Western blotting. The results are shown in Figure 8 .

[0193] Depend on Figure 8 The results of Western blotting shown above show that the bands of the MEX3B protein in hmrGD-179 and hmrGD-180 were slightly lighter than those in the negative control NC.

[0194] Example 6: Protein expression inhibition test of MEX3B in humans and mice

[0195] As described above, hmrGD-89 has a significant effect in suppressing the protein expression of MEX3B. Therefore, for hmrGD-89-2, a protein expression suppression test of human and mouse MEX3B was performed by Western blotting in the same manner as in Example 5. The results for human MEX3B are shown in Fig. 9 The results for mice are shown in Fig.10 .

[0196] according to Fig. 9 and 10The results shown show that, in the case of the gapmer nucleic acids of hmrGD-89-2 and hmrGD-89, the band of MEX3B protein became fainter in both human and mouse, similarly to the gapmer nucleic acid of the positive control, indicating that the expression of MEX3B protein was significantly suppressed.

Claims

1. A nucleic acid, which is any one of the following (1) to (3), (1) an oligonucleotide consisting of the base sequence described in SEQ ID NO: 1 or 2 in the sequence listing; (2) an antisense oligonucleotide which is obtained by adding one or two bases to the base sequence described in SEQ ID NO: 1 or 2 of the sequence listing, which consists of a base sequence complementary to the MEX3B gene in its entirety and which inhibits the expression of the MEX3B gene; (3) An antisense oligonucleotide which comprises a 16 to 18-base sequence of the base sequence described in SEQ ID NO: 3 of the sequence listing, is complementary to the MEX3B gene in full length, and inhibits the expression of the MEX3B gene.

2. The nucleic acid according to claim 1, wherein The antisense oligonucleotide of (2) is obtained by adding one base to the base sequence described in SEQ ID NO: 1 or 2 in the sequence listing.

3. The nucleic acid according to claim 2, wherein The addition in the antisense oligonucleotide of (2) is an addition to at least one of the terminals. 4 . Use of the nucleic acid according to claim 1 for producing a MEX3B gene expression inhibitor.

5. A method for inhibiting MEX3B gene expression, comprising the step of contacting the agent described in claim 4 with a subject, wherein: The object does not include individual organisms.

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