Title of the invention: Nucleic acid that suppresses MEX3B gene expression, MEX3B gene expression suppressant, method that suppresses MEX3B gene expression, and preventive or therapeutic agent for diseases caused by MEX3B gene expression.
Technical field
[0001]
The present invention relates to a nucleic acid that suppresses the expression of the MEX3B gene, a MEX3B gene expression inhibitor containing the above nucleic acid, a method of suppressing the MEX3B gene expression, and a preventive or therapeutic agent for a disease caused by the MEX3B gene expression.
Background technology
[0002]
In recent years, diseases with serious increased expression of inflammatory cytokines or chemocaines such as IL-6, IL-13, TNF, G-CSF, CXCL1, CXCL2, or CXCL5 (eg, severe asthma, joint disease, diabetes, inflammatory) It has been clarified to be involved in intestinal diseases, atopic dermatitis, systemic erythematosus, cancer, mental diseases, cardiovascular diseases, respiratory diseases, type 2 diabetes, renal diseases, etc. (for example, non-patent documents). 1).
On the other hand, the MEX3B protein is known to be a molecule that binds to various mRNAs and controls the function (that is, translation into protein) or stability of those mRNAs (for example, Non-Patent Document 2).
Then, in Patent Document 1, MEX3B protein binds to the mRNA of the inflammatory cytokine or chemokine and is involved in the function (that is, translation into protein) or stability of the mRNA, and the disease caused by the inflammatory cytokine or chemokine. It is disclosed that it is involved in the onset of.
Therefore, suppression of MEX3B expression may be important in the treatment or prevention of the above-mentioned diseases.
prior art documents
patent literature
[0003]
Patent Document 1: International Publication No. 2018/008750A1
Patent Document 2: International Publication No. 2018/008749A1
Non-patent literature
[0004]
Non-Patent Document 1: Int Immunol. 2015 Jan; 27 (1): 21-9, Cancer Discov. 2016 Jan; 6 (1): 80-95.
Non-Patent Document 2: Nucleic Acids Res. 2007; 35 (4): 1289-300.
SUMMARY OF THE INVENTION
Problems to be Solved by the Invention
[0005]
Patent Document 2 discloses a nucleic acid or the like that can suppress the expression of the MEX3B gene while suppressing the cytotoxicity. However, there was room for improvement in the effect of suppressing the expression of the MEX3B gene.
[0006]
The present invention has been made in view of the above circumstances, and is a nucleic acid having less cytotoxicity and improved ability to suppress MEX3B gene expression, a MEX3B gene expression inhibitor containing the above nucleic acid, a method for suppressing MEX3B gene expression, and MEX3B. The purpose is to provide a preventive or therapeutic agent for diseases caused by gene expression.
Means to solve problems
[0007]
The present inventors designed a large number of antisense oligonucleotides to cover the entire pre-mRNA region of human MEX3B before splicing, and comprehensively tested the suppression of MEX3B gene expression. As a result, a specific sequence in the MEX3B gene was obtained. It has been found that a specific antisense oligonucleotide having a sequence complementary to the above can significantly suppress the mRNA expression of the MEX3B gene, and also can significantly suppress the expression of the MEX3B protein.
The present invention has been completed based on the above findings.
Specifically, the present invention is as follows.
[0008]
The first aspect of the present invention is any of the following nucleic acids (1) to (3).
(1) Oligonucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 1 or 2 in the sequence listing
(2) An antisense oligonucleotide that consists of a base sequence in which one or two bases are deleted, substituted, and / or added in the base sequence shown in SEQ ID NO: 1 or 2 in the sequence listing, and suppresses the expression of the MEX3B gene.
(3) An antisense oligonucleotide that contains the nucleotide sequence shown in SEQ ID NO: 3 in the sequence listing and suppresses the expression of the MEX3B gene.
[0009]
The second aspect of the present invention is
A MEX3B gene expression inhibitor containing the nucleic acid according to the first aspect.
The third aspect of the present invention is
A method for suppressing MEX3B gene expression, which comprises contacting a subject (excluding a human individual) with an agent according to the second aspect.
The fourth aspect of the present invention is
A preventive or therapeutic agent for a disease caused by MEX3B gene expression, which comprises an agent according to the second aspect.
Effect of the invention
[0010]
According to the present invention, a nucleic acid having less cytotoxicity and improved ability to suppress MEX3B gene expression, a MEX3B gene expression inhibitor containing the above nucleic acid, a method for suppressing MEX3B gene expression, and prevention of diseases caused by MEX3B gene expression or A therapeutic agent can be provided.
A brief description of the drawing
[0011]
FIG. 1 is a diagram showing suppression of human MEX3B mRNA expression level by transfection of each gapmer type nucleic acid.
FIG. 2 is a diagram showing suppression of human MEX3B mRNA expression level by transfection of each gapmer type nucleic acid.
FIG. 3 is a diagram showing suppression of human MEX3B mRNA expression level by transfection of each gapmer type nucleic acid.
FIG. 4 is a diagram showing suppression of mouse MEX3B mRNA expression level by transfection of each gapmer type nucleic acid.
FIG. 5 is a diagram showing the results of suppressing mRNA expression of human MEX3B by free uptake of each gapmer-type nucleic acid.
FIG. 6 is a diagram showing the results of suppressing the expression of human and mouse MEX3B proteins by transfection of each gapmer-type nucleic acid.
FIG. 7 is a diagram showing the results of suppressing the expression of human MEX3B protein by transfection of each gapmer-type nucleic acid.
FIG. 8 is a diagram showing the results of suppressing the expression of mouse MEX3B protein by transfection of each gapmer type nucleic acid.
FIG. 9 is a diagram showing the results of suppressing the expression of human MEX3B protein by transfection of each gapmer-type nucleic acid.
FIG. 10 is a diagram showing the results of suppressing the expression of mouse MEX3B protein by transfection of each gapmer type nucleic acid.
Mode for carrying out the invention
[0012]
Hereinafter, embodiments of the present invention will be described in detail, but the present invention is not limited to the following embodiments, and can be carried out with appropriate modifications within the scope of the object of the present invention. ..
[0013]
(MEX3B gene)
The MEX3B gene contains exon 1, intron and exon 2, and this composition is highly conserved in humans, mice and other mammals.
As the untranslated region (UTR) that does not encode amino acids in exons, there is a 5'UTR upstream of the start codon and a 3'UTR downstream of the start codon.
The human MEX3B gene encoding human MEX3B mRNA has the sequence represented by SEQ ID NO: 4 below.
In SEQ ID NO: 4, the 437th to 2146th base sequence is CDS, the 1st to 436th base sequence is 5'UTR, and the 2147 to 3532th base sequence is 3'UTR.
The mouse MEX3B gene encoding the mouse MEX3B mRNA has the sequence represented by SEQ ID NO: 5 below.
In SEQ ID NO: 5, the 319th to 2049th base sequence is CDS, the 1st to 318th base sequence is 5'UTR, and the 2050 to 3416th base sequence is 3'UTR.
SEQ ID NO: 6 below indicates 836 bases in the intron region of the human MEX3B gene.
SEQ ID NO: 7 shows the base sequence encoding the pre-mRNA of human MEX3B before splicing. In the sequence encoding the pre-mRNA of human MEX3B represented by SEQ ID NO: 7, the base sequences 437 to 692 and 1529 to 2892 are CDS, and the base sequences 1 to 436 are 5'UTR, 2983 to. The 4368th base sequence is 3'UTR, and the 693th to 1528th regions correspond to the intron region of the human MEX3B gene represented by SEQ ID NO: 6.
Further, all the genes encoding the MEX3B protein (for example, the protein having the amino acid sequence represented by SEQ ID NO: 8 (human) or 9 (mouse)) belong to the MEX3B gene.
[0014]
(Acquisition of MEX3B gene)
The method for obtaining the MEX3B gene is not particularly limited. Based on the nucleotide sequence and amino acid sequence information set forth in SEQ ID NOs: 4, 5, and 7-9 of the sequence listing of the present specification, appropriate probes or primers are prepared, and they are used in a human cDNA library (MEX3B). The MEX3B gene can be isolated by selecting the desired clone from suitable cells in which the gene is expressed (prepared according to a conventional method).
[0015]

The nucleic acid that suppresses the expression of the MEX3B gene according to the first aspect (hereinafter, also simply referred to as “nucleic acid according to the first aspect”) is any of the following (1) to (3).
(1) Oligonucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 1 or 2 in the sequence listing
(2) An antisense oligonucleotide that consists of a base sequence in which one or two bases are deleted, substituted, and / or added in the base sequence shown in SEQ ID NO: 1 or 2 in the sequence listing, and suppresses the expression of the MEX3B gene.
(3) An antisense oligonucleotide that contains the nucleotide sequence shown in SEQ ID NO: 3 in the sequence listing and suppresses the expression of the MEX3B gene.
[0016]
Suppression of MEX3B gene expression by nucleic acid according to the first aspect is at least one of transcription amount suppression and protein expression amount suppression.
The degree of suppression is not particularly limited as long as at least one of the transcription amount and the protein expression level is lower than that of the negative control (or wild type), but the transcription amount is higher than that of the negative control (or wild type). And at least one of the protein expression levels is preferably reduced by 10% or more, more preferably 15% or more, further preferably 20% or more, and 30% or more. It is even more preferable, it is particularly preferable that it is reduced by 40% or more, it is particularly preferable that it is reduced by 50% or more, and it is most preferable that it is reduced by 60% or more.
Regarding the above (1), the nucleotide sequence shown in SEQ ID NO: 1 (GCGGACTACAGCTT) and the nucleotide sequence shown in SEQ ID NO: 2 (GAGGGATACTACAGC) are the gapmer-type nucleic acids hmrGD-89-2 and hmrGD-, which will be described later in Examples, respectively. It is a base sequence possessed by 89.
[0017]
Regarding (2) above, the number of "1 or 2" in the "base sequence in which 1 or 2 bases are deleted, substituted and / or added in the base sequence" is preferably 1.
Regarding (2) above, the base length of the antisense oligonucleotide is preferably 14 bases or more, and more preferably 15 bases or more.
The upper limit of the base length of the antisense oligonucleotide is preferably 18 bases or less, more preferably 17 bases or less, and further preferably 16 bases or less.
The above-mentioned "deleted, substituted and / or added base sequence" is preferably a "deleted and / or substituted base sequence" from the viewpoint of manufacturability and the like, and is preferably a "deleted base sequence". Is more preferable.
The degree of the above DNA mutation is, for example, preferably 90% or more, more preferably 93% or more, still more preferably 93% or more, with the base sequence shown in SEQ ID NO: 1 or 2 in the sequence listing. Those having an identity of 95% or more, particularly preferably 97% or more, and most preferably 98% or more can be mentioned.
In the nucleotide sequence shown in SEQ ID NO: 1 or 2 in the sequence listing, by various artificial treatments such as site-specific mutagenesis, random mutation by mutagenesis treatment, mutation / deletion / ligation of DNA fragment by restriction enzyme cleavage, etc. , The DNA sequence may or may not be partially altered.
[0018]
Regarding (3) above, the nucleotide sequence (GCGGATACTACAGC) shown in SEQ ID NO: 3 is the above-mentioned SEQ ID NOs: 1 and 2.It is a 14-base base sequence common to .
Regarding (3) above, the antisense oligonucleotide is preferably an antisense oligonucleotide having a sequence complementary to an oligonucleotide containing at least 12 consecutive nucleotides in the nucleotide sequence of the MEX3B gene, and at least 13 nucleotides. More preferred are antisense oligonucleotides having a sequence complementary to an oligonucleotide comprising: and even more preferred are antisense oligonucleotides having a sequence complementary to an oligonucleotide comprising at least 14 nucleotides.
In addition, the upper limit of the base length of the antisense oligonucleotide is preferably an antisense oligonucleotide having a sequence complementary to a continuous oligonucleotide of 25 nucleotides or less in the base sequence of the MEX3B gene. More preferably, it is an antisense oligonucleotide having a sequence complementary to an oligonucleotide of 20 nucleotides or less, more preferably an antisense oligonucleotide having a sequence complementary to a continuous oligonucleotide of 19 nucleotides or less, It is even more preferred that the antisense oligonucleotide has a sequence complementary to a continuous oligonucleotide of 18 nucleotides or less, and an antisense oligonucleotide having a sequence complementary to a continuous oligonucleotide of 17 nucleotides or less. Particularly preferred are antisense oligonucleotides having a sequence complementary to an oligonucleotide of 16 consecutive nucleotides or less.
[0019]
From the standpoint of manufacturability, etc., the antisense oligonucleotide of (3) above preferably has a base sequence of 15 bases or less.
Regarding (3) above, the degree of DNA mutation, for example, preferably has 90% or more identity with the nucleotide sequence set forth in SEQ ID NO: 3 in the sequence listing, more preferably 93% or more, and furthermore Preferably, those having 95% or more, particularly preferably 97% or more, and most preferably 98% or more are included.
[0020]
With respect to (1) to (3) above, the antisense oligonucleotide is, for example, an oligonucleotide in the MEX3B gene and a complementary antisense oligonucleotide, which are hybridized after introduction into a cell. Preferably, the MEX3B mRNA containing nucleotide strand is degraded by a nuclease (eg, RNase H) specific for the resulting hybrid duplex.
The antisense oligonucleotide may be either DNA or RNA, but is preferably DNA from the viewpoint that mRNA is cleaved by the specific nuclease.
[0021]
Regarding (1) to (3) above, the antisense oligonucleotide is an antisense oligonucleotide containing at least one nucleotide having at least one structure selected from the group consisting of a phosphorothioate structure, a crosslinked structure and an alkoxy structure. Preferably.
For example, nuclease resistance can be acquired by having a phosphorothioate structure in the phosphodiester bond that connects nucleotides, and uptake into cells or nuclei can also be improved due to improved hydrophobicity. can.
In addition, the sugar moiety of the nucleotide is 2',4'-BNA (2',4'-Bridged Nucleic Acid; also known as LNA (Locked Nucleic Acid)), ENA (2'-O,4'-C-Ethylene-bridged Acquires nuclease resistance and improves mRNA binding ability by having a crosslinked structure such as Nucleic Acid) and an alkoxy structure such as 2'-O-methylation and 2'-O-methoxyethylation (2'-MOE) be able to.
[0022]
In the antisense oligonucleotide, at least one phosphodiester bond connecting nucleotides preferably has a phosphorothioate structure, and 50% or more of the phosphodiester bonds in the antisense oligonucleotide have a phosphorothioate structure. More preferably, 70% or more of the phosphodiester bonds in the antisense oligonucleotide have a phosphorothioate structure, and 90% or more of the phosphodiester bonds in the antisense oligonucleotide are Having a phosphorothioate structure is particularly preferred, and most preferably all phosphodiester bonds in the antisense oligonucleotide have a phosphorothioate structure.
In the above antisense oligonucleotide, at least one terminal nucleotide preferably has a crosslinked structure or an alkoxy structure.
More specifically, on at least one end, any nucleotide from the end to 4 bases preferably has a crosslinked structure or an alkoxy structure, and any nucleotide from the 1st to 3rd bases from the end more preferably has a crosslinked structure or an alkoxy structure, and more preferably 2 or 3 bases from the terminal have a crosslinked structure or an alkoxy structure.
It is preferable that the nucleotides at both ends of the antisense oligonucleotide have a crosslinked structure or an alkoxy structure (so-called Gapmer-type antisense oligonucleotides). More preferably, any nucleotide from to 4 bases has a crosslinked structure or an alkoxy structure, more preferably any nucleotide from the 1st to 3rd bases from the end has a crosslinked structure or an alkoxy structure, the terminal It is particularly preferred that 2 or 3 bases from have a crosslinked structure or an alkoxy structure.
The nucleic acid according to the first aspect can be produced by a conventional method using a DNA synthesizer and known organic synthesis technology.
[0023]
Uptake into cells, in vivo or in vitro, involves contacting the nucleic acid of the first aspect with a cell, e.g. can be achieved by adding a nucleic acid according to
On the other hand, when the nucleic acid according to the first aspect has at least one structure selected from the group consisting of a phosphorothioate structure, a crosslinked structure and an alkoxy structure, it is possible to further improve uptake into cells.
The nucleic acid according to the first aspect has at least one structure selected from the group consisting of a phosphorothioate structure, a crosslinked structure and an alkoxy structure, and is used in combination with a carrier for lipofection described later to be incorporated into cells. can be further improved.
On the other hand, the nucleic acid according to the first aspect has such a strong MEX3B gene expression-suppressing effect that the lipofection carrier described later is not used. In addition, as described later in Example 4, it can be taken up into cells by free uptake.
[0024]
The method of introducing the nucleic acid according to the first aspect into a cell may be a method of inserting it into an appropriate vector and further introducing it into an appropriate host cell.
The type of suitable vector is not particularly limited, and may be, for example, an autonomously replicating vector (e.g., a plasmid). It is preferably one that is replicated with.
Suitable vectors include E. coli-derived plasmids (eg, pBR322, pUC118, etc.), Bacillus subtilis-derived plasmids (eg, pUB110, pSH19, etc.), and animal viruses such as bacteriophages, retroviruses, and vaccinia viruses. can. Upon recombination, it is also possible to add a translation initiation codon and a translation termination codon using an appropriate synthetic DNA adapter.
[0025]
Alternatively, the nucleic acid according to the first aspect may optionally be functionally linked to a suitable terminator such as the human growth hormone terminator or, for fungal hosts, the TPI1 terminator or the ADH3 terminator. Recombinant vectors may further include elements such as polyadenylation signals (eg, from the SV40 or adenovirus 5E1b regions), transcriptional enhancer sequences (eg, the SV40 enhancer) and translational enhancer sequences (eg, those encoding adenovirus VA RNA). You may have
The recombinant vector may further comprise a DNA sequence that enables the vector to replicate in the host cell, an example of which is the SV40 origin of replication (when the host cell is a mammalian cell).
The recombinant vector may further contain a selectable marker. Selectable markers include, for example, dihydrofolate reductase (DHFR) or genes whose complement is lacking in the host cell, such as the Schizosaccharomyces pombe TPI gene, or, for example, ampicillin, kanamycin, tetracycline, chloramphenicol, Drug resistance genes such as neomycin or hygromycin can be mentioned.
[0026]
Host cells into which the nucleic acid according to the first aspect or a vector containing it is introduced include higher eukaryotic cells, bacteria, yeast, fungi, etc., but are preferably mammalian cells.
Examples of mammalian cells include HEK293 cells, HeLa cells, COS cells (such as COS-7 cells), BHK cells, CHL cells or CHO cells, BALB/c mouse cells (such as BALB/c mouse embryonic fibroblasts). ) and the like. Methods for transforming mammalian cells and expressing genes introduced into the cells are also known, and for example, the lipofection method, electroporation method, calcium phosphate method and the like can be used.
[0027]
Suppression of MEX3B gene expression by the nucleic acid according to the first aspect can be performed in vivo or in vitro based on the nucleotide sequence information of the MEX3B gene, for example, using a probe or primer having the nucleotide sequence of part or all of the gene. can be detected by
In particular, measurement of the expression level of the MEX3B gene at the mRNA level can be performed by conventional methods such as RT-PCR and Northern blotting.
[0028]
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 containing at least 10 contiguous nucleotides in the MEX3B gene, as well as an antisense oligonucleotide having a sequence complementary to the oligonucleotide can be used as probes or primers. More specifically, an oligonucleotide having a nucleotide sequence of 10 to 60 consecutive residues, preferably 10 to 40 residues in the MEX3B gene, and an antisense oligonucleotide having a sequence complementary to the oligonucleotide are used. can do.
[0029]
In addition, measurement of the expression level at the MEX3B protein level can be performed by a normal immunoassay such as Western blot or ELISA. Specifically, it can be performed by a conventional method known to those skilled in the art described in Molecular Cloning, Second Edition, Current Protocols in Molecular Biology, or the like.
[0030]

The MEX3B gene expression inhibitor according to the second aspect (hereinafter also simply referred to as "the agent according to the second aspect") contains the nucleic acid according to the first aspect.
As described above, suppression of MEX3B gene expression is at least one of transcription level suppression and protein expression level suppression.
The MEX3B gene expression inhibitor according to the second aspect is a liposome from the viewpoint of improving uptake into cells.The nucleic acid according to the first aspect may be further contained as a carrier for infection, but the nucleic acid according to the first aspect can be taken up into cells by free uptake as described later in Example 4, and strong MEX3B gene expression is expressed. From the viewpoint of achieving the inhibitory effect, the carrier for lipofection may not be included.
Examples of the lipofection carrier include carriers having a high affinity for cell membranes (for example, liposomes, cholesterol, etc.), and lipofectamine or lipofectin is preferable, and lipofectamine is more preferable.
The nucleic acid according to the first aspect has at least one structure selected from the group consisting of a phosphorothioate structure, a crosslinked structure and an alkoxy structure, and is used in combination with a carrier for lipofection to further improve intracellular uptake. Can be made to.
For example, the nucleic acid according to the first aspect having at least one structure selected from the group consisting of a phosphorothioate structure, a crosslinked structure and an alkoxy structure may be brought into contact with a target described later in the presence of a carrier for lipofection.
[0031]
The agent according to the second aspect may be in a form in which the nucleic acid according to the first aspect, the carrier for lipofection and other optional components (for example, water, buffer solution, etc.) are mixed and contained, but the first aspect may be used. The nucleic acid and other optional components according to the embodiment may be in the form of a kit in which the carrier for lipofection and other optional components are packaged in separate containers.
The form of the agent according to the second aspect is not particularly limited, but can be used in the form of liquid, granules, tablets, capsules, patches and the like. In the case of in vivo, the agent according to the second aspect may be directly exposed to the tissue. More preferably, it is exposed to a living body (liquid, etc.) or orally administered, or intravascular, oral, sublingual, rectal, intraperitoneal, vaginal, intrathecal, intramuscular, skin, subcutaneous, etc. It may be administered in vivo by means such as injection, spraying, or application into the skin, bladder, trachea (bronchi), eyes, nose, ears, and the like.
[0032]

The method for suppressing MEX3B gene expression according to the third aspect includes contacting the subject with the agent according to the second aspect.
As will be described later in Example 4, the agent according to the second aspect can be taken up into cells by free uptake by the above contact without requiring, for example, calcium shock or the like.
Targets include individual organisms (for example, individuals of mammals or birds), microorganisms, protozoa, biological tissues, biological tissue sections, human cells, animal cells, and the like.
Examples of mammals or birds include cows, sheep, goats, pigs, horses, dogs, chickens, mice, rats, guinea pigs, hamsters, rabbits, and primates. Examples of primate individuals include monkey individuals and the like, which may or may not be human individuals.
The form of contact is not particularly limited, but for example, the agent according to the second aspect may be added to the medium containing the target, or the medium containing the agent according to the second aspect in advance may be prepared. .. The temperature, time, etc. at the time of contact are not particularly limited, but are appropriately set according to the type of the object and the like.
[0033]

The preventive or therapeutic agent for a disease caused by MEX3B gene expression according to the fourth aspect includes the MEX3B gene expression inhibitor according to the second aspect.
Interleukin 6 (IL-6) is an important cytokine involved in inflammation, hematopoiesis, bone metabolism, tumor exacerbation, etc. It is known that the activity of IL-6 mainly contributes to the transition from acute inflammation to acquired immune response, the onset of chronic inflammatory diseases, etc. (for example, J Asthma. 2008; 45 Suppl 1: 41-4. ).
Interleukin 13 (IL-13) is known to play a role as an inflammatory cytokine in enhancing allergic inflammation in peripheral tissues. IL-13 is known to be involved not only in promoting the allergic reaction, which is a major cause of allergic asthma, but also in intracting asthma, which is the ineffectiveness of steroids. In addition, IL-13 is involved in the pathogenesis of inflammatory bowel disease and atopic dermatitis as well as asthma (for example, J Allergy (Cairo) 2012; 2012: 316049, N Engl J Med 2011). 365: 1088-1098).
TNF (tumor necrosis factor: Tumor Necrosis Factor), especially TNF-α, is a signal factor that induces an inflammatory reaction and is an important factor from the viewpoint of infection protection. On the other hand, it is known that it is also involved in disorders caused by increased inflammation. That is, TNF is involved in exacerbation of pathological conditions in various diseases, mainly joint diseases (rheumatoid arthritis, psoriatic arthritis, spondyloarthropathies, tonic spondylitis), inflammatory bowel diseases (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) It is known to be involved in asthma, chronic bronchitis, chronic obstructive pulmonary disease, acute pulmonary disorder), type 2 diabetes, renal disease (ischemic nephropathy, post-transplant rejection, glomerular nephritis), etc. , J Allergy Clin Immunol. 2008 Jan; 121 (1): 5-10, J Pathol. 2008 Jan; 214 (2): 149-60.).
In addition, G-CSF (granulocyte colony stimulating factor: Granulocyte Colony Stimulating Factor) is known to have an effect of promoting granulocyte production and enhancing neutrophil function.
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.).
[0034]
In addition, CXCL1, CXCL2, and CXCL5 belong to the inflammatory chemokine CXC subfamily.
When CXCL1, CXCL2, and CXCL5 are secreted in lung tissue due to excessive inflammation in the airway mucosa, infiltration of neutrophils that highly express CXCR2, which is a receptor for CXCL1, CXCL2, and CXCL5, is promoted. As a result, steroid-resistant neutrophil infiltration induces chronic inflammation that induces irreversible remodeling of the airways by causing severe asthma.
The present inventors have found that the MEX3B gene is involved in the development of diseases caused by IL-6, IL-13, TNF, G-CSF, CXCL1, CXCL2, or CXCL5.
Therefore, the prophylactic or therapeutic agent according to the fourth aspect has an increased expression of IL-6, IL-13, TNF, G-CSF, CXCL1, CXCL2, or CXCL5 (eg, for an excessive increase in expression, preferably for normal conditions). (Eg, severe asthma, chronic obstructive pulmonary disease, rheumatoid arthritis, colitis, Crohn's disease, atopy dermatitis, systemic erythematosus) , Severe asthma caused by IL-6, IL-13, TNF, G-CSF, CXCL1, CXCL2, or CXCL5, chronic obstructive pulmonary disease, rheumatoid arthritis, colitis, Crohn's disease, atopy dermatitis , Systemic erythematosus, cancer, etc. (Int Immunol. 2015 Jan; 27 (1): 21-9, Cancer Discov. 2016 Jan; 6 (1): 80-95.)) Is effective as a preventive or therapeutic agent. it is conceivable that.
[0035]
Further, the MEX3B protein is known to be a protein that induces apoptosis (for example, Japanese Patent No. 4429269).
Apoptosis is known to be involved in the development of serious diseases such as neurodegenerative diseases in addition to normal physiological processes. For example, neurodegenerative diseases (eg, Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, Huntington's chorea, etc.) are thought to be caused by an abnormal increase in apoptosis (Wolozin, B., et al. , (1996) Science, 274, 1710-1713).
Therefore, the prophylactic or therapeutic agent according to the fourth aspect is considered to be effective as a prophylactic or therapeutic agent for neurodegenerative diseases.
[0036]
The prophylactic or therapeutic agent according to the fourth aspect can be orally or parenterally administered systemically or topically. Examples of the parenteral administration method include intravenous injection such as infusion, intramuscular injection, intraperitoneal injection, and subcutaneous injection. The administration method can be appropriately selected according to the patient's age and symptoms. The dose varies depending on the age, the route of administration, and the number of administrations, and can be appropriately selected by those skilled in the art.
Examples of the formulation form suitable for parenteral administration include those containing additives such as stabilizers, buffers, preservatives, and tonicity agents, and those containing pharmaceutically acceptable carriers and additives. It may be. Examples of such carriers and additives include water, organic solvents, polymeric compounds (collagen, polyvinyl alcohol, etc.), stearic acid, human serum albumin (HSA), mannitol, turbitol, lactose, surfactants and the like. However, it is not limited to these.
[0037]
The dose of the nucleic acid according to the first aspect, which is the active ingredient, is generally in the range of about 0.1 μg to 100 mg per 1 kg of body weight at a time.
Example
[0038]
Hereinafter, the present invention will be described in more detail with reference to Examples, but the scope of the present invention is not limited to these Examples.
[0039]
<< Example 1 >>

A large number of antisense oligonucleotides shifted by 5 bases were prepared so as to cover the entire premRNA region of human MEX3B before splicing, and suppression of MEX3B gene expression was comprehensively tested.
Among a large number of gapmer-type nucleic acids shifted by 5 bases prepared as antisense oligonucleotides complementary to human MEX3B premRNA, gapmer-type nucleic acids hmrGD-87 (SEQ ID NO: 11) and 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-176. (SEQ ID NO: 17), hmrGD-178 (SEQ ID NO: 18), hmrGD-179 (SEQ ID NO: 19), hmrGD-180 (SEQ ID NO: 20), hmrGD-182 (SEQ ID NO: 21), hmrGD-199 (SEQ ID NO: 22). , HmrGD-201 (SEQ ID NO: 23), hmrGD-202 (SEQ ID NO: 24), hmrGD-203 (SEQ ID NO: 25) and hmrGD-205 (SEQ ID NO: 26) are shown in Table 1 below.
Among the 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 patented. It is a gapmer type nucleic acid described in Document 2.
In addition, a gapmer-type nucleic acid LNA-NC (2,2) (SEQ ID NO: 10) as a negative control and an antisense oligonucleotide complementary to a predetermined target sequence on human pre-mRNA as a positive control, which are predetermined MEX3B. The gapmer-type nucleic acid LNA-1 (2,2), which has been confirmed to have a gene expression inhibitory effect, was used.
Two or three bases of LNA (2', 4'-BNA) are placed at both ends of each gapmer-type nucleic acid, and the bases that fill the other spaces are normal DNA, and the phosphoric acid that connects each nucleotide. The diester bond was phosphorothioated, and the total length of the negative control gapmer-type nucleic acid was 15 bases, and the total length of the other gapmer-type nucleic acids was 16 bases.
Further, the above "(2,2)" means that each of the two bases at both ends of the gapmer-type nucleic acid is LNA.
[0040]
[table 1]

[0041]

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.
Human alveolar basal epithelial adenocarcinoma A549 cells were used for the transfection of gapmer-type nucleic acids, and Lipofectamine RNAiMax (manufactured by Invitrogen) and its recommended protocol were used to introduce cells at a final concentration of 20 nM. did.
[0042]

After 48 hours after transfection, the state of the cells was observed under a microscope, the cells were collected, and total RNA was collected using the lysis buffer TRIsure (manufactured by BIOLINE). A reverse transcription reaction was performed using Primescript (manufactured by TAKARA) to obtain cDNA.
After that, quantitative RT-PCR was performed using Light Cycler 480 (manufactured by ROCHE).
The primer sequences used for the quantitative RT-PCR test are as follows.
Human MEX3B Primer Forward: 5'-ACCCAGTTCTGAGCATGTCG-3' (SEQ ID NO: 27)
Human MEX3B primer Reverse: 5'-CGAACTGGGGTCTTGATGTAA-3' (SEQ ID NO: 28)
Human GAPDH primer Forward: 5'-GCACCGTCAAGGCTGAGAAC-3' (SEQ ID NO: 29)
Human GAPDH primer Reverse: 5'-TGGTGAAGACGCCAGTGGA-3' (SEQ ID NO: 30)
GAPDH is an internal standard.
[0043]
As is clear from the quantitative RT-PCR results shown in FIGS. 1 and 2, the gapmer-type nucleic acid of hmrGD-89 (SEQ ID NO: 2) is the negative control gapmer-type nucleic acid LNA-NC(2,2). (SEQ ID NO: 10), it inhibited human MEX3B mRNA expression by about 1/3 and exhibited an effect of suppressing human MEX3B mRNA expression equivalent to that of the positive control gapmer-type nucleic acid LNA-1(2,2). This expression-suppressing ability was remarkably superior to other gapmer-type nucleic acids targeting 3'UTR.
In addition, although not shown, gapmer-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, all cells transfected with no side effects were observed.
In addition, some cytotoxicity was observed in the positive control gapmer-type nucleic acid LNA-1 (2, 2).
[0044]
<>
As described above, hmrGD-89 (SEQ ID NO: 2) had a remarkable effect of suppressing the expression of human MEX3B mRNA. A gapmer-type nucleic acid shifted by 1 or 2 bases from the target sequence of was prepared and tested in the same manner as in Example 1.
Gapmer-type nucleic acid hmrGD-89-2 (SEQ ID NO: 1) is a gapmer-type nucleic acid in which the target sequence is shifted from the target sequence of hmrGD-89 to the 5′ end by two bases, as summarized in Table 2 below. be.
[0045]
[Table 2]

As is clear from the quantitative RT-PCR results shown in FIG. 3, the gapmer-type nucleic acid of hmrGD-89 (SEQ ID NO: 2) is equivalent to the positive control gapmer-type nucleic acid LNA-1(2,2). , whereas the gapmer-type nucleic acid hmrGD-89-2 (SEQ ID NO: 1) has a superior effect of suppressing the expression of human MEX3B mRNA.
In addition, no side effects such as cytotoxicity were observed in cells transfected with the gapmer-type nucleic acid hmrGD-89-2, as in the negative control.
[0046]
<> Mouse MEX3B mRNA expression suppression test
A mouse MEX3B mRNA expression suppression test was performed on gapmer-type nucleic acids hmrGD-89-2 and hmrGD-89.

Transfection with gapmer-type nucleic acids hmrGD-89-2 and hmrGD-89 was performed in the same manner as in Example 2, except that the cells to be transfected were mouse lung epithelial cell MLE-15 cells.

A quantitative RT-PCR test was performed in the same manner as in Example 2, except that the primers used in the quantitative RT-PCR test were changed as follows. The results are shown in FIG.

Mouse MEX3B Primer Forward: 5'-CGTCGTCCTCTGTGGTCTTTCCCGGGGGTG-3' (SEQ ID NO: 31)
Mouse MEX3B primer Reverse: 5'-TCAGGAAAAATGCGGATGGCCTGAGTGAC-3' (SEQ ID NO: 32)
Mouse β-actin primer Forward: 5′-GGATGCAGAAGGAGATTACTGC-3′ (SEQ ID NO: 33)
Mouse β-actin primer Reverse: 5′-CCACCGATCCACACAGAGCA-3′ (SEQ ID NO: 34)
β-actin is an internal standard.
[0047]
As is clear from the results of quantitative RT-PCR shown in FIG. 4, both gapmer-type nucleic acids hmrGD-89-2 and hmrGD-89 are antisense oligos complementary to predetermined target sequences on mouse pre-mRNA. It can be seen that the positive control gapmer-type nucleic acid LNA-Ex-mMex3B-4, which is a nucleotide and is known to exhibit a predetermined MEX3B gene expression-suppressing effect, exhibits the same mouse MEX3B mRNA expression-suppressing effect.
[0048]
<> Human MEX3B mRNA expression suppression test by free uptake
A human MEX3B mRNA expression suppression test was performed by free uptake without the transfection method using lipofectamine.
In addition to the LNA-type gapmer-type nucleic acids hmrGD-89-2 and hmrGD-89 used in Example 2, ENA-type gapmer-type nucleic acids hmrGD-89-2 and hmrGD-89 were also prepared and tested. .

Cells were introduced in the same manner as in Example 2, except that Lipofectamine RNAiMax (manufactured by Invitrogen) was not used and the cells were introduced at a final concentration of 1 μM.

Quantitative RT-PCR was performed in the same manner as in Example 2, except that 72 hours after transfection, the state of the cells was observed under a microscope, and then the cells were collected and total RNA was collected.
The results are shown in Figure 5. The control in Figure 5 is a negative control.
[0049]
As is clear from the results of quantitative RT-PCR shown in FIG. 5, both the LNA and ENA forms of the gapmer nucleic acids hmrGD-89-2 and hmrGD-89 are human MEX3B even in the free uptake method. It is expected that the expression of the MEX3B gene can be suppressed even when administered to a living body. Among them, the LNA type and ENA type of hmrGD-89-2 were particularly excellent.
[0050]
<> Human and mouse MEX3B protein expression suppression test
Similar to Example 1, in a comprehensive MEX3B gene expression suppression test for the entire pre-mRNA region of human MEX3B before splicing, not only human alveolar basal epithelial adenocarcinoma cell A549 cells, but also mouse lung epithelial cell MLE-15 Cells were also transfected, and 48 hours after transfection, the state of the cells was observed under a microscope, and then the cells were collected and treated with RIPA (Radio-Immunoprecipitation Assay) buffer (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.). was used to recover the total protein, and a protein expression suppression test for human and mouse MEX3B was performed by Western blotting.
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) and hmrGD-93 (SEQ ID NO: 16) are shown in FIG.
In the figure, a) is the result of transfection with human alveolar basal epithelial adenocarcinoma cell A549 cells, and b) is the result of transfection with mouse lung epithelial cell MLE-15 cells. α-Tubulin is the internal standard (and so on).
Lane NC is the result of using a gapmer-type nucleic acid LNA-NC(2,2) (SEQ ID NO: 10) as a negative control, and lanes 1 and G4 both use a gapmer-type nucleic acid as a positive control. This is the result of
[0051]
From the results of Western blotting shown in FIGS. 6a) and b), the hmrGD-89 gapmer-type nucleic acid makes the MEX3B protein band as faint as the positive control gapmer-type nucleic acid in both humans and mice. and the expression of MEX3B protein was suppressed. This effect was significantly superior to other 3'UTR-targeted gapmer-type nucleic acids.
[0052]
Further, 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 Similarly, human MEX3B protein expression suppression was confirmed by Western blotting after human alveolar basal epithelial adenocarcinoma A549 cells were transfected. The results are shown in FIG.
From the Western blot results shown in FIG. 7, hmrGD-178, hmrGD-179, hmrGD-180, hmrGD-182, hmrGD-199, hmrGD-201, hmrGD-202, hmrGD-203 and hmrGD-203 compared to the negative control NC. The MEX3B protein band for hmrGD-205 (particularly hmrGD-201 and hmrGD-202) was only slightly faint.
[0053]
In addition, among the gapmer-type nucleic acids described in Patent Document 2, for hmrGD-179 and hmrGD-180, mouse lung epithelial cell MLE-15 cells were transfected, and Western blotting confirmed suppression of mouse MEX3B protein expression. did. The results are shown in FIG.
As can be seen from the results of Western blotting shown in Figure 8, the MEX3B protein band for hmrGD-179 and hmrGD-180 was only slightly faint compared to the negative control NC.
[0054]
<> Human and mouse MEX3B protein expression suppression test
As described above, since the effect of hmrGD-89 on suppressing MEX3B protein expression was remarkable, hmrGD-89-2 was also subjected to Western blotting in the same manner as in Example 5 to suppress human and mouse MEX3B protein expression. did the test. Human MEX3B results are shown in FIG. 9 and mouse results are shown in FIG.
[0055]
From the results shown in FIGS. 9 and 10, the gapmer-type nucleic acids of hmrGD-89-2 and hmrGD-89 have the same faint MEX3B protein band as the positive control gapmer-type nucleic acids in both humans and mice. It can be said that the expression of the MEX3B protein is remarkably suppressed.
The scope of the claims
[Claim 1]
A nucleic acid that is any of the following (1) to (3).
(1) sequence listing Oligonucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 1 or 2.
(2) An antisense oligonucleotide that consists of a base sequence in which one or two bases are deleted, substituted, and / or added in the base sequence shown in SEQ ID NO: 1 or 2 in the sequence listing, and suppresses the expression of the MEX3B gene.
(3) An antisense oligonucleotide that contains the nucleotide sequence shown in SEQ ID NO: 3 in the sequence listing and suppresses the expression of the MEX3B gene.
[Claim 2]
The nucleic acid according to claim 1, wherein the deleted, substituted and / or added base sequence in the above (2) is the deleted base sequence.
[Claim 3]
The nucleic acid according to claim 1, wherein the antisense oligonucleotide of (3) has a base sequence of 15 bases or less.
[Claim 4]
A MEX3B gene expression inhibitor containing the nucleic acid according to any one of claims 1 to 3.
[Claim 5]
A method for suppressing MEX3B gene expression, which comprises contacting the agent according to claim 4 with a target (excluding a human individual).
[Claim 6]
A prophylactic or therapeutic agent for a disease caused by MEX3B gene expression, which comprises the agent according to claim 4.