WO2012153854A1 - Cytokine-chemokine modulator - Google Patents

Abstract

The invention provides novel means for modulating the expression of cytokine-chemokine genes associated with inflammation and infection. Specifically, the invention provides an antisense nucleotide that comprises the sequence complementary to the 3'UTR of the CCL2, CCL20, CX3CL1, IL-23A, CD69, NF-κB p65, TNF-α, Fam89a, Grk5, phosopholipid scramblase 1, Runx1, semaphorin 4A, Steap4, lymphotoxin β Psmb10 or TLR2 gene induced by IL-1β and that is capable of modulating the expression of the gene; sense oligonucleotide that contains the sequence complementary to the endogenous antisense transcription product containing the sequence complementary to the 3'UTR of the gene and that is capable of modulating the expression of the gene; and an agent that prevents and/or treats inflammatory disease or infection and contains the sense or antisense nucleotide.

Description

Cytokine chemokine modulator

The present invention relates to a nucleic acid (antisense nucleotide) that is complementary to a natural (endogenous) antisense transcript that is complementary to the mRNA of a cytokine chemokine involved in inflammation or infection, and changes the stability of the mRNA, and Utilization of its cytokine / chemokine mRNA stability regulating action, and a natural (endogenous) antisense transcript that is complementary to the cytokine / chemokine mRNA involved in inflammation and infection and changes the stability of the mRNA The present invention relates to a complementary nucleic acid (sense nucleotide) and use of its cytokine / chemokine mRNA stability regulating action.

Natural (endogenous) antisense transcript (N atural A ntisense T ranscript; hereinafter also referred to as "NAT") and, inherent in the organism, a RNA having a nucleotide sequence complementary to mRNA, specifically Specifically, it is RNA synthesized using a DNA strand encoding a sense gene (that is, a non-template strand of mRNA) as a template. Sense-antisense RNA has the ability to form double strands. For example, double-stranded RNA is necessary for RNA interference, and is used to control protein translation by small RNAs called microRNAs (miRNAs). It is known that double-stranded RNA is involved. Recent comprehensive cDNA analysis revealed that a considerable amount of NAT was transcribed (Non-patent Documents 1 and 2). For example, it has been suggested that about 2,500 pairs (Non-patent Document 3) exist in mice and about 2,600 pairs (Non-patent Document 4) exist in humans. Does not contain a lot of untranslatable NAT.

Although the physiological function of NAT has not been fully elucidated, NAT has shown that various regulatory RNAs with different physiological functions such as stabilization or destabilization of mRNA, translational suppression from mRNA, etc. It has been found that it belongs to a group (Non-Patent Document 5).
For example, it has been reported that NAT complementary to endothelial nitric oxide synthase (eNOS) mRNA increases in the presence of a histone deacetylase inhibitor and decreases the amount of eNOS mRNA (non-patent literature). 6). Also in yeast, it is known that NAT of the PHO84 gene suppresses mRNA expression through histone deacetylation (Non-patent Document 7).

On the other hand, the present inventors have a NAT complementary to the 3′-untranslated region (UTR) of inducible nitric oxide synthase (iNOS) mRNA, and that this antisense RNA hybridizes with iNOS RNA mRNA. The mRNA is stabilized and the production amount of iNOS and the synthesis amount of NO increase, and the sense oligodeoxynucleotide (ODN) complementary to the NAT inhibits the binding of NAT to the iNOS mRNA. It was reported that iNOS production and NO synthesis by iNOS can be suppressed (Patent Documents 1 and 2, Non-Patent Document 8). In addition, Nishizawa and its collaborators have a complementary NAT in the region that forms a secondary structure with two stem-loops, which is important for the nuclear export of interferon (IFN) -α mRNA. Antisense RNA contributes to the stabilization of IFN-α mRNA, and NAT knockdown by sense ODN leads to mRNA instability, whereas overexpression of antisense RNA significantly stabilizes the mRNA (Patent Document 3). The present inventors named the technique of controlling NAT by this sense ODN as “NAT-targeted REgulation (NATRE) technology”.

By the way, many genes related to inflammation and infection such as cytokines and chemokines are expressed in hepatocytes in response to the pro-inflammatory cytokine interleukin-1β (IL-1β), which is induced by IL-1β. It has not been reported so far whether or not NAT is transcribed from the early response gene group, and whether or not expression control using sense ODN is possible in these inflammation / infection-related genes remains unclear. .

WO 2007/142303 pamphlet WO 2007/142304 pamphlet WO 2011/046221 pamphlet

Cheng et al., Science, 308: 1149-1154 (2005) Katayama et al., Science, 309: 1564-1566 (2005) Kiyosawa et al., Genome Res., 13: 1324-1334 (2003) Yelin et al., Nat. Biotechnol., 21: 379-386 (2003) Faghihi and Wahlestedt, Nat. Rev. Mol. Cell Biol., 10: 637-643 (2009) Robb et al., J. Biol. Chem., 279: 37982-37996 (2004) Camblong et al., Cell, 131: 706-717 (2007) Matsui et al., Hepatology, 47: 686-697 (2008)

The object of the present invention was to search for NAT capable of regulating the expression of cytokines and chemokine genes related to inflammation and infection, and to use a sense oligonucleotide complementary to the NAT and an antisense oligonucleotide homologous to the NAT, It is to provide a novel means for preventing and treating diseases such as inflammatory diseases and viral infections.

The present inventors selected 21 genes (including cytokines and chemokine genes) induced by IL-1β and attempted to detect NAT complementary to the 3 ′ UTR of mRNA. As a result of strand-specific RT-PCR, it was found that NAT was transcribed from 16 out of 21 genes (76%). Next, we have 7 genes (CCL2, CCL20, CX3CL1, IL-23 p19 subunit, CD69, NF-κB p65 subunit, TNF-α) in which both mRNA and NAT are abundantly transcribed. Predict the secondary structure of the 3'UTR of these mRNAs, design and synthesize a sense ODN that is homologous to an RNA sequence that contains at least one loop within a region conserved between human and rat, Introduced into rat hepatocytes, the mRNA level of each gene in the cells after stimulation with IL-1β was compared with that in cells not transfected with sense ODN.
As a result, most sense ODNs designed for RNA sequences containing loop portions within the conserved region of the predicted secondary structure have altered (increased or decreased) the expression level of the target mRNA. In contrast, a sense ODN designed outside the conserved region of the secondary structure did not affect the expression level of the target mRNA.

Based on these findings, the present inventors have a NAT containing a region corresponding to 3′UTR in many cytokine-chemokine genes induced by IL-1β, and the expression of the gene is regulated by NAT. Furthermore, using a sense oligonucleotide having a sequence complementary to the region containing the loop portion of the secondary structure that can be taken by the NAT (hence, homologous to mRNA), expression control (up-regulation and down-regulation) of the gene is performed. It was concluded that (regulation) is possible, and the present invention was completed.

That is, the present invention is as follows.
[1] Induced by IL-1β, CCL2, CCL20, CX3CL1, IL-23A, CD69, NF-κB p65, TNF-α, Fam89a, Grk5, phosopholipid scramblase 1, Runx1, semaphorin 4A, Steap4, lymphphotoxin β Psmb10 Alternatively, an antisense nucleotide comprising a sequence complementary to the 3′UTR of the TLR2 gene and capable of regulating the expression of the gene.
[2] The antisense nucleotide according to [1] above, wherein the gene induced by IL-1β is CCL2, CCL20, CX3CL1, IL-23A, CD69, NF-κB p65 or TNF-α.
[3] CCL2, CCL20, CX3CL1, IL-23A, CD69, NF-κB p65, TNF-α, Fam89a, Grk5, phosopholipid scramblase 1, Runx1, semaphorin, comprising the antisense nucleotide described in [1] above 4A, Steap4, lymphphotoxin β Psmb10 or TLR2 expression regulator.
[4] The agent according to [3] above, which is used for prevention or treatment of inflammatory diseases or infectious diseases.
[5] Induced by IL-1β, CCL2, CCL20, CX3CL1, IL-23A, CD69, NF-κB p65, TNF-α, Fam89a, Grk5, phosopholipid scramblase 1, Runx1, semaphorin 4A, Steap4, lymphphotoxin β Psmb10 Alternatively, a sense oligonucleotide comprising a sequence complementary to an endogenous antisense transcript comprising a sequence complementary to the 3 ′ UTR of the TLR2 gene and capable of regulating expression of the gene.
[6] The sense oligonucleotide according to [5] above, wherein the gene induced by IL-1β is CCL2, CCL20, CX3CL1, IL-23A, CD69, NF-κB p65 or TNF-α.
[7] The sense oligonucleotide according to [6] above, which is any of the following (a) to (g):
(a) In the 3′UTR sequence of CCL2 mRNA represented by SEQ ID NO: 1, domain 1 represented by nucleotide numbers 25-51 or domain 2 represented by nucleotide numbers 103-175, or those of orthologs in other mammals Sense oligonucleotide capable of regulating the expression of the CCL2 gene, comprising a nucleotide sequence having 90% or more identity with a sequence comprising at least part of one or more loop structures in the corresponding domain
(b) In the 3'UTR sequence of CCL20 mRNA represented by SEQ ID NO: 2, domain 1 represented by nucleotide numbers 65-107, domain 2 represented by nucleotide numbers 124-155, or domain 3 represented by nucleotide numbers 214-282 Or expression of the CCL20 gene comprising a nucleotide sequence having at least 90% identity with a sequence comprising at least a portion of one or more loop structures within a domain corresponding to those of orthologs in other mammals Regulatable sense oligonucleotide
(c) In the 3′UTR sequence of CX3CL1 mRNA represented by SEQ ID NO: 3, domain 1 represented by nucleotide numbers 1103-1202, domain 2 represented by nucleotide numbers 1305-1369, and domain 3 represented by nucleotide numbers 1416-1482 In domain 4, represented by nucleotide number 1633-1681, domain 5 represented by nucleotide number 1688-1745, or domain 6 represented by nucleotide number 1784-1810, or a domain corresponding to those of orthologs in other mammals A sense oligonucleotide capable of regulating the expression of the CX3CL1 gene, comprising a nucleotide sequence having at least 90% identity with a sequence comprising at least a part of one or more loop structures
(d) In the 3'UTR sequence of IL-23A mRNA shown in SEQ ID NO: 4, domain 1 shown by nucleotide numbers 37-55, domain 2 shown by nucleotide numbers 193-303, and nucleotide numbers 380-448 90% or more identity with a sequence comprising at least part of one or more loop structures within domain 3, or domain 4 represented by nucleotide numbers 555-600, or domains corresponding to those of orthologs in other mammals Sense oligonucleotide capable of regulating IL-23A gene expression comprising a nucleotide sequence having
(e) In the 3'UTR sequence of CD69 mRNA represented by SEQ ID NO: 5, domain 1 represented by nucleotide numbers 15-32, domain 2 represented by nucleotide numbers 120-186, domain 3 represented by nucleotide numbers 218-252 In domain 4, indicated by nucleotide numbers 344-370, domain 5 indicated by nucleotide numbers 389-633, or domain 6 indicated by nucleotide numbers 656-786, or domains corresponding to those of orthologs in other mammals A sense oligonucleotide capable of regulating the expression of the CD69 gene, comprising a nucleotide sequence having 90% or more identity with a sequence comprising at least a part of one or more loop structures
(f) In the 3′UTR sequence of NF-κB p65 mRNA represented by SEQ ID NO: 6, domain 1 represented by nucleotide numbers 161-301, domain 2 represented by nucleotide numbers 343-380, represented by nucleotide numbers 401-412 90% or more identical to a sequence comprising at least a part of one or more loop structures in the domain 3, or the domain 4 indicated by nucleotide numbers 473-523, or the domain corresponding to those of orthologs in other mammals Sense oligonucleotide capable of regulating expression of NF-κB p65 gene, comprising a nucleotide sequence having sex
(g) In the 3′UTR sequence of TNF-α mRNA represented by SEQ ID NO: 7, domain 1 represented by nucleotide numbers 304-405, domain 2 represented by nucleotide numbers 430-546, and nucleotide numbers 687-713 90% or more identity with a sequence comprising at least part of one or more loop structures within domain 3, or domain 4 represented by nucleotide numbers 752-776, or domains corresponding to those of orthologs in other mammals A sense oligonucleotide capable of regulating the expression of a TNF-α gene comprising a nucleotide sequence having the following sequence: [8] CCL2, CCL20, CX3CL1, IL-23A comprising the sense oligonucleotide according to [5] above, Expression regulator of CD69, NF-κB p65, TNF-α, Fam89a, Grk5, phosopholipid scramblase 1, Runx1, semaphorin 4A, Steap4, lymphphotoxin β Psmb10 or TLR2.
[9] The agent according to [8] above, which is used for prevention or treatment of inflammatory diseases or infectious diseases.
[10] In the presence and absence of the test substance, CCL2, CCL20, CX3CL1, IL-23A, CD69, NF-κB p65, TNF-α, Fam89a, Grk5, phosopholipid scramblase 1, Runx1, semaphorin 4A, Steap4, A screening method for an expression regulator of the gene, comprising detecting and comparing the hybridization between the mRNA of the lymphotoxin β Psmb10 or TLR2 gene and the endogenous antisense transcript thereto.
[11] contacting a test substance with a cell expressing mRNA of the gene and an endogenous antisense transcript thereto, and measuring a change in the amount of the mRNA and / or protein encoded in the cell. The method according to [10] above, characterized in that it is characterized.

The antisense nucleotide of the present invention can regulate the expression of the gene by interacting with the 3 'UTR of the mRNA in the same manner as NAT for mRNA of various genes induced by IL-1β. On the other hand, the sense oligonucleotide of the present invention can regulate the expression of a target gene to a desired level by interacting with NAT to regulate its gene expression regulation action positively or negatively.

It is a figure which shows the predicted secondary structure of 3'UTR of CCL2 mRNA. FIG. 5 shows the position of a conserved region (indicated by domain “+”) containing one or more loop structures of 3′UTR of CCL2 mRNA and designed sense ODN (indicated by underline). It is a figure which shows the predicted secondary structure of 3'UTR of CCL20 * mRNA. FIG. 4 shows the position of a conserved region (indicated by domain “+”) containing one or more loop structures of 3′UTR of CCL20 mRNA and the designed sense ODN (indicated by underline). It is a figure which shows the predicted secondary structure of 3'UTR of CX3CL1 mRNA. FIG. 3 shows the position of a conserved region (indicated by domain “+”) containing one or more loop structures of 3′UTR of CX3CL13 mRNA and the designed sense ODN (indicated by underline). FIG. 4 shows the predicted secondary structure of 3′UTR of IL-23A mRNA. FIG. 3 shows the position of a conserved region (indicated by domain “+”) containing one or more loop structures of 3′UTR of IL-23A mRNA and the designed sense ODN (indicated by underline). FIG. 3 shows the predicted secondary structure of 3 ′ UTR of CD69 mRNA. FIG. 3 shows the position of a conserved region (indicated by domain “+”) containing one or more loop structures of 3′UTR of CD69 mRNA and the designed sense ODN (indicated by underline). It is a figure which shows the predicted secondary structure of 3'UTR of NF-κB p65 mRNA. FIG. 3 shows the position of a conserved region (indicated by domain “+”) containing one or more loop structures of 3 ′ UTR of NF-κB p65 mRNA and designed sense ODN (indicated by underline). It is a figure which shows the predicted secondary structure of 3'UTR of TNF- (alpha) mRNA. FIG. 3 shows the position of a conserved region (indicated by domain “+”) containing one or more loop structures of 3′UTR of TNF-α mRNA and the designed sense ODN (indicated by underline). It is a figure which shows the expression fluctuation | variation of CCL2, CCL20, CX3CL1, IL-23A, CD69, NF- (kappa) B65 and TNF- (alpha) in the hepatocytes which introduce | transduced various sense ODN.

The present invention provides antisense nucleotides of these genes, which have an effect of regulating the expression of genes related to various inflammations / infections including cytokines and chemokines induced by IL-1β.
The target gene (mRNA) in the present invention is an endogenous antisense transcript containing a sequence complementary to its 3 ′ untranslated region (3′UTR) in the gene group induced by IL-1β. There is no particular limitation as long as it is a gene present, but specifically, for example, chemokine (CC motif) ligand 2 (CCL2); chemokine (CC motif) ligand 20 (CCL20); chemokine (C-X3-C motif) ligand 1 (CX3CL1); interleukin-23 α-subunit p19 (IL-23A); cluster of differentiation 69 (CD69); nuclear factor-κB p65 subunit (NF-κB p65); tumor necrosis factor-α (TNF-α) ; family with sequence similarity 89 member A (Fam89a); G protein-coupled receptor kinase 5 (Grk5); phosopholipid scramblase 1; runt-related transcription factor 1 (Runx1); sema domain, immunoglobulin domain, transmembrane domain and short cytoplasmic domain 4A (semaphorin 4A); six-transmembrane epithelial antigen of prostate 4 (Steap4); lymphotoxin β; proteasome subunit, beta type 10 (Psmb10); Toll-like recep and tor 2 (TLR2). CCL2, CCL20, CX3CL1, IL-23A, CD69, NF-κB p65 or TNF-α is preferable.

The antisense nucleotide of the present invention may be any nucleotide as long as it contains a sequence complementary to the 3 'UTR of the target mRNA and can regulate the expression (protein production) of the target gene. Here, the “complementary” sequence is not only a sequence that is completely complementary to mRNA, but also 1 to a number (as long as it can hybridize with and interact with mRNA under physiological conditions of cells). 2, 3, 4 or 5) may contain base mismatches. Preferably, the sequence complementary to the 3′UTR of the target mRNA is a stringent condition such as those described in Current Protocols in Molecular Biology, John Wiley & Sons, 6.3.1-6.3.6, 1999 For example, hybridization at 6 × SSC (sodium chloride / sodium citrate) / 45 ° C., followed by one or more washings at 0.2 × SSC / 0.1% SDS / 50-65 ° C.) It is a sequence that can hybridize with.

Specifically, since the endogenous antisense transcript (NAT) for a target mRNA is believed to interact with a thermodynamically unstable portion of the mRNA, the antisense nucleotides of the present invention may contain 3′UTR of the target mRNA. It is preferred to include a sequence complementary to a portion that is not thermodynamically stable therein. The portion that is not thermodynamically stable includes a region that is in a single-stranded state when the mRNA has a secondary structure (for example, a region corresponding to the loop portion of the stem-loop structure). The secondary structure of the 3'UTR of the target mRNA is represented by mfold (see GCG Software; Proc. Natl. Acad. Sci. USA, 86: 7706-10 (1989)) based on the nucleotide sequence information of the region. Can be predicted using an existing RNA secondary structure prediction program. Any of these nucleotide sequence information is readily available. For example, CCL2, CCL20, CX3CL1, IL-23A, CD69, NF-κB p65 and TNF-α mRNA 3′UTR sequences (provided U is replaced with T) are shown in SEQ ID NOs: 1-7, respectively. Show.

Preferably, the antisense nucleotide against CCL2 of the present invention is the domain 1 and / or nucleotide number 103-175 represented by nucleotide number 25-51 in the rat CCL2 mRNA 3'UTR sequence represented by SEQ ID NO: 1. In the domain 2 shown, in the CCL2 ortholog in other mammals, a sequence containing at least a part of one or more loop structures (for example, 3 bases or more) in the domain corresponding to each domain in the rat CCL2 Containing a complementary nucleotide sequence.

Preferably, the antisense nucleotide against CCL20 of the present invention, in the rat CCL20 ヌ ク レ オ チ ド mRNA 3'UTR sequence shown in SEQ ID NO: 2, is in domain 1 and / or nucleotide numbers 124-155 shown in nucleotide numbers 65-107 In the domain 2 shown and / or in the domain 3 shown by nucleotide numbers 214-282, in the CCL20 ortholog in other mammals, one or more loop structures in the domain corresponding to each domain in the rat CCL20 A nucleotide sequence complementary to a sequence containing at least a part (for example, 3 bases or more) is included.

Preferably, the antisense nucleotide against CX3CL1 of the present invention is the domain 1 and / or nucleotide number 1305-1369 represented by nucleotide number 1103-1202 in the 3′UTR sequence of rat CX3CL1 mRNA represented by SEQ ID NO: 3. Domain 2 and / or domain 3 shown by nucleotide number 1416-1482 and / or domain 4 shown by nucleotide number 1633-1681 and / or domain 5 shown by nucleotide number 1688-1745 and / or nucleotide number 1784 In domain 6 indicated by -1810, in the CX3CL1 ortholog in other mammals, the domain corresponding to each domain in the rat CX3CL1 is complementary to a sequence containing at least a part of one or more loop structures. Contains nucleotide sequence.

Preferably, the antisense nucleotide against IL-23A of the present invention is the domain 1 and / or nucleotide represented by nucleotide number 37-55 in the 3′UTR sequence of rat IL-23A mRNA represented by SEQ ID NO: 4 In the domain 2 shown by the number 193-303 and / or the domain 3 shown by the nucleotide number 380-448 and / or the domain 4 shown by the nucleotide number 555-600, in the IL-23A ortholog in other mammals A nucleotide sequence complementary to a sequence containing at least a part of one or more loop structures in a domain corresponding to each domain of the rat IL-23A.

Preferably, the antisense nucleotide against CD69 of the present invention is the domain 1 and / or nucleotide number 120-186 represented by nucleotide number 15-32 in the 3′UTR sequence of rat CD69 mRNA represented by SEQ ID NO: 5. 2 and / or domain 3 shown by nucleotide number 218-252 and / or domain 4 shown by nucleotide number 344-370 and / or domain 5 shown by nucleotide number 389-633 and / or nucleotide number 656 In domain 6 shown by -786, in the CD69 ortholog in other mammals, the domain corresponding to each domain in rat CD69 is complementary to a sequence containing at least a part of one or more loop structures. Contains nucleotide sequence.

Preferably, the antisense nucleotide for NF-κB p65 of the present invention is the domain 1 and / or the nucleotides 161-301 in the 3′UTR sequence of rat NF-κB p65 mRNA shown in SEQ ID NO: 6. Or in domain 2 indicated by nucleotide number 343-380 and / or domain 3 indicated by nucleotide number 401-412 and / or domain 4 indicated by nucleotide number 473-523, to NF-κB p65 ortholog in other mammals In that case, it comprises a nucleotide sequence complementary to a sequence containing at least a part of one or more loop structures in the domain corresponding to each domain in the rat NF-κB p65.

Preferably, the antisense nucleotide against TNF-α of the present invention is the domain 1 and / or nucleotide represented by nucleotide number 304-405 in the 3′UTR sequence of rat TNF-α mRNA represented by SEQ ID NO: 7 In the domain 2 shown by the number 430-546 and / or the domain 3 shown by the nucleotide number 687-713 and / or the domain 4 shown by the nucleotide number 752-776, in the TNF-α ortholog in other mammals A nucleotide sequence complementary to a sequence containing at least a part of one or more loop structures in the domain corresponding to each domain in the rat TNF-α.

As for the corresponding domain of orthologs in mammals other than rats, in the case of a gene having an ARE motif in 3'UTR, the nucleotide sequence of the peripheral region of the ARE motif common to multiple species is well conserved among mammalian species. Since the secondary structure is similar, the region can be preferably selected. For example, Table 1 shows the number of ARE motifs present in the 3'UTR of the above-mentioned 7 genes of rat, mouse and human and the common ARE motifs between human-rat and among three species.

The length of the antisense nucleotide of the present invention is not particularly limited, and a nucleotide sequence complementary to a sequence comprising at least a part of one or more loop structures in the domain in the full length of NAT or 3′UTR of the target mRNA. It may contain a partial sequence of NAT, but from the viewpoint of sequence specificity, it contains at least 10 bases, preferably about 12 bases or more, more preferably about 15 bases or more, a portion complementary to the target sequence. . In addition, from the viewpoint of ease of administration when used as a pharmaceutical, those having a base length of 500 bases or less, preferably 300 bases or less, more preferably 150 bases or less are mentioned.

Furthermore, when the antisense nucleotide of the present invention is an oligonucleotide having about 10 to 50 bases, the nucleotide is a sequence that causes a sequence-nonspecific reaction (for example, 5′-CG-3 ′, 5′-GGGG-3 ', 5'-GGGGG-3' and the like are preferably selected, and are preferably selected from those having no similar complementary strand sequence in RNA other than the target mRNA. The absence of a similar complementary strand sequence in other RNAs can be confirmed by performing a homology search against the target mammalian genome sequence using the antisense oligonucleotide candidate sequence as a query. . Here, as homology search means, known nucleic acid homology search software (for example, NCBI BLAST (National Center Biotechnology Information Basic Basic Local Alignment Search Tool) NBLAST and XBLAST programs (version 2.0), FASTA program in GCG software package Etc.) can be used. Moreover, as a genomic DNA data set, for example, all human genome data provided by Celera can be used.

The antisense nucleotide of the present invention is used in various forms depending on the method of introduction into cells. For example, when the antisense nucleotide is an oligonucleotide having about 10 to 50 bases, it may be any one of single-stranded DNA, single-stranded RNA, and DNA / RNA chimera, and it is further added with known modifications. There may be. Here, the “nucleotide” may include not only purine and pyrimidine bases but also those having other modified heterocyclic bases.

The nucleotide molecule constituting the antisense nucleotide may be natural DNA or RNA, but various chemicals may be used to improve stability (chemical and / or enzyme) and specific activity (affinity with RNA). Modifications can be included. For example, in order to prevent degradation by nuclease, etc., phosphate residues (phosphates) of each nucleotide constituting the antisense oligonucleotide may be changed to chemically modified phosphate residues such as phosphorothioate (PS), methylphosphonate, phosphorodithionate, etc. Substituents can be substituted. In addition, the 2′-position hydroxyl group of the sugar (ribose) of each nucleotide is represented by —OR (R is, for example, CH ₃ (2′-O-Me), CH ₂ CH ₂ OCH ₃ (2′-O-MOE), CH ₂ CH ₂ NHC (NH) NH ₂ , CH ₂ CONHCH ₃ , CH ₂ CH ₂ CN and the like may be substituted). Furthermore, the base moiety (pyrimidine, purine) may be chemically modified, for example, introduction of a methyl group or a cationic functional group at the 5-position of the pyrimidine base, or substitution of the carbonyl group at the 2-position with thiocarbonyl. Is mentioned.
The conformation of the sugar part of RNA is dominated by C2'-endo (S type) and C3'-endo (N type). In single-stranded RNA, it exists as an equilibrium between the two, but double-stranded Is fixed to the N type. Therefore, BNA (LNA) (Imanishi) is an RNA derivative in which the conformation of the sugar moiety is fixed to the N-type by bridging the 2 'oxygen and 4' carbon to give strong binding ability to the target mRNA. , T. et al., Chem. Commun., 1653-9, 2002; Jepsen, JS et al., Oligonucleotides, 14, 130-46, 2004) and ENA (Morita, K. et al., Nucleosides Nucleotides Nucleicides Nucleic Acids , 22, 1619-21, 2003) can also be preferably used.

The antisense oligonucleotide of the present invention synthesizes a complementary sequence based on the target mRNA (cDNA) sequence using a commercially available DNA / RNA automatic synthesizer (Applied Biosystems, Beckman, etc.). Can be prepared.

The antisense oligonucleotide of the present invention is provided in a special form such as a liposome or a microsphere, or a hydrophobic substance such as a polycationic substance such as polylysine or a lipid (eg, phospholipid, cholesterol, etc.) is added thereto. It can be provided in the form. Alternatively, the antisense oligonucleotide of the present invention is converted to a peptide having a membrane permeation function (for example, Drosophila-derived Antennapedia homeodomain (AntP), human immunodeficiency virus (HIV) -derived TAT, herpes simplex virus (HSV) -derived Modification with a cell-passing domain such as VP22) can promote the uptake of the oligonucleotide into cells.

On the other hand, when the antisense nucleotide of the present invention is a polynucleotide having a longer base length, introduction of the nucleotide into a cell can be carried out using a gene transfer method known per se. In this case, double-stranded DNA is preferably used as the nucleotide. The antisense nucleotide of the present invention extracts, for example, total RNA from a cell that expresses the target gene NAT (for example, a cell that highly expresses target mRNA and NAT by IL-1β stimulation). For example, SEQ ID NOs: 1-7 Based on the sequence information of the target mRNA shown in the above, a primer that can amplify an appropriate region of the complementary strand sequence is designed, and RT-PCR is performed (see Examples below). The obtained cDNA is inserted into an appropriate expression vector containing a promoter that can function in host animal cells. Examples of expression vectors include retroviruses, lentiviruses, adenoviruses, adeno-associated viruses, herpes viruses, Sendai virus and other viral vectors, animal cell expression plasmids (eg, pA1-11, pXT1, pRc / CMV, pRc / RSV). , PcDNAI / Neo) or the like.
Examples of the promoter used in the expression vector include EF1α promoter, CAG promoter, SRα promoter, SV40 promoter, LTR promoter, CMV (cytomegalovirus) promoter, RSV (rous sarcoma virus) promoter, MoMuLV (Molone murine leukemia virus) LTR. HSV-TK (herpes simplex virus thymidine kinase) promoter and the like are used. Of these, EF1α promoter, CAG promoter, MoMuLV LTR, CMV promoter, SRα promoter and the like are preferable.
In addition to the promoter, the expression vector may optionally contain an enhancer, a poly A addition signal, a selection marker gene, an SV40 replication origin, and the like. Examples of the selection marker gene include a dihydrofolate reductase gene, a neomycin resistance gene, a puromycin resistance gene, and the like.

The antisense nucleotide of the present invention can regulate target gene expression (protein production) by interacting with the target mRNA. Various genes including cytokine / chemokine gene induced by IL-1β have various physiological activities such as inflammation-inducing or anti-inflammatory action, infection defense, regulation of immune response, etc. The contained drug can be used as a target gene expression regulator for the prevention and / or treatment of various diseases including inflammatory diseases and infectious diseases.
For example, antisense nucleotides that can enhance the expression of CCL2 can be used for angiogenesis and wound healing, while CCL2 migrates to monocytes and eosinophils, so that it can suppress the expression of CCL2. Nucleotides are useful for the prevention and treatment of chronic inflammatory diseases including arteriosclerosis and allergic inflammation.
Since CCL20 exhibits antibacterial activity against a wide range of microorganisms, antisense nucleotides that can enhance the expression of CCL20 are effective in preventing and treating various microbial infections. On the other hand, since CCL20 plays an important role in the initiation and maintenance of adaptive immunity, antisense nucleotides that can suppress the expression of CCL20 are bronchial asthma, multiple sclerosis, rheumatoid arthritis, various dermatitis, inflammatory It is effective for the prevention and treatment of autoimmune diseases such as bowel disease.
Antisense nucleotides that can enhance CX3CL1 expression can be used for angiogenesis, while antisense nucleotides that can suppress CX3CL1 expression prevent or treat inflammatory diseases including arteriosclerosis and glomerulonephritis It is useful for the prevention and treatment of anti-rejection and rheumatoid arthritis.
IL-23A is important for the maintenance of cellular immunity, and its overexpression causes multi-organ inflammation. Therefore, antisense nucleotides that can enhance IL-23A expression are effective in the prevention and treatment of infectious diseases. On the other hand, antisense nucleotides that can suppress the expression of IL-23A are useful for the prevention and treatment of autoimmune diseases and inflammatory diseases.
Since CD69 plays an important role in the differentiation and activation of lymphocytes, antisense nucleotides that can enhance the expression of CD69 are effective in the prevention and treatment of infectious diseases. On the other hand, CD69 is expressed in most inflammatory cells that are expressed locally, and an anti-antibody that can suppress the expression of CD69 due to detection of autoantibodies against CD69 from patients with rheumatoid arthritis and systemic lupus erythematosus (SLE). Nucleotides are useful for the prevention and treatment of autoimmune diseases and inflammatory diseases.
Since NF-κB is a transcription factor having an important role in the immune system, antisense nucleotides that can enhance the expression of NF-κB p65 are effective in preventing and treating infectious diseases. On the other hand, NF-κB is constantly activated in cancer cells and inhibits apoptosis, and is also involved in pathogenesis such as bone metabolism, bronchial asthma, arthritis, inflammatory bowel disease, sepsis, Antisense nucleotides that can suppress the expression of NF-κB p65 are useful for the prevention and treatment of cancer, osteoporosis, autoimmune diseases and inflammatory diseases.
Since TNF-α shows protection against infection and anti-tumor action by inducing apoptosis and enhancing antibody production by inflammatory mediators and plasma cells, antisense nucleotides that can enhance the expression of TNF-α prevent infection and cancer. It is effective for treatment. On the other hand, since TNF-α is involved in the pathogenesis of rheumatoid arthritis, psoriasis, diabetes, hyperlipidemia, sepsis, and osteoporosis, antisense nucleotides that can suppress the expression of TNF-α are used to prevent and treat these diseases. Useful for.

The medicament containing the antisense nucleotide of the present invention has low toxicity, and as such a solution or a pharmaceutical composition of an appropriate dosage form, can be used as a human or non-human mammal (eg, mouse, rat, guinea pig, rabbit, sheep, Pigs, cows, cats, dogs, monkeys, etc.) can be administered orally or parenterally (eg, inhalation administration, intravascular administration, subcutaneous administration, transmucosal administration, etc.).
When these nucleic acids are used as a prophylactic / therapeutic agent for the above-mentioned various diseases, they can be formulated and administered according to a method known per se. That is, the antisense nucleotide of the present invention is inserted alone or in a functional manner into the appropriate expression vector for mammalian cells such as a retroviral vector, lentiviral vector, adenoviral vector, adeno-associated viral vector and the like. Then, it can be formulated according to conventional means. The nucleotide can be administered as it is or together with an auxiliary agent for promoting intake by a gene gun or a catheter such as a hydrogel catheter. Alternatively, it can be aerosolized and locally administered into the trachea as an inhalant.
Furthermore, for the purpose of improving pharmacokinetics, extending the half-life, and improving cellular uptake efficiency, the nucleotide may be formulated (injection) alone or with a carrier such as a liposome and administered intravenously, subcutaneously, etc. .

The antisense nucleotide of the present invention may be administered per se or as an appropriate pharmaceutical composition. The pharmaceutical composition used for administration may contain the antisense nucleotide of the present invention and a pharmacologically acceptable carrier, diluent or excipient. Such a pharmaceutical composition is provided as a dosage form suitable for oral or parenteral administration.

As a composition for parenteral administration, for example, injections, aerosols, suppositories and the like are used, and injections are intravenous injections, subcutaneous injections, intradermal injections, intramuscular injections, intravenous injections, etc. May be included. Such an injection can be prepared according to a known method. Aerosol formulations can be placed in compressed acceptable propellants such as dichlorodifluoromethane, propane, nitrogen and the like. Alternatively, it may be formulated as an incompressible pharmaceutical product such as a nebulizer or an atomizer. Suppositories used for rectal administration may be prepared by mixing the nucleic acid with a normal suppository base.

Compositions for oral administration include solid or liquid dosage forms, specifically tablets (including sugar-coated tablets and film-coated tablets), pills, granules, powders, capsules (including soft capsules), and syrups. Agents, emulsions, suspensions and the like. Such a composition is produced by a known method and may contain a carrier, a diluent or an excipient usually used in the pharmaceutical field. As the carrier and excipient for tablets, for example, lactose, starch, sucrose, and magnesium stearate are used.

The above parenteral or oral pharmaceutical composition is conveniently prepared in a dosage unit form suitable for the dosage of the active ingredient. Examples of such dosage forms include tablets, pills, capsules, injections (ampoules), aerosols, and suppositories. The antisense nucleotide of the present invention is preferably contained, for example, usually 5 to 500 mg per dosage unit dosage form, particularly 5 to 100 mg for injections, and 10 to 250 mg for other dosage forms.

The dosage of the above-mentioned medicament containing the antisense nucleotide of the present invention varies depending on the administration subject, target disease, symptom, administration route, etc., but for example, the dosage of the antisense nucleotide of the present invention is usually 0.01 to 20 mg. It is convenient to administer about 0.1 kg / kg body weight, preferably about 0.1-10 mg / kg body weight, more preferably about 0.1-5 mg / kg body weight by intravenous injection or inhalation once to several times a day. In the case of other parenteral administration and oral administration, an equivalent amount can be administered. If symptoms are particularly severe, the dose may be increased according to the symptoms.

Note that the above-described pharmaceutical composition may contain other drugs as long as an undesirable interaction is not caused by the combination with the antisense nucleotide of the present invention. Other drugs include, for example, antiviral drugs, antitumor drugs, antibacterial drugs, antifungal drugs, antiprotozoal drugs, antibiotics, antiseptic drugs, antiseptic shock drugs, endotoxin antagonists, immunomodulators, non-steroidal drugs Examples include anti-inflammatory drugs, steroid drugs, inflammatory mediator action inhibitors, inflammatory mediator production inhibitors, anti-inflammatory mediator action inhibitors, anti-inflammatory mediator production inhibitors.

Oligonucleotides that contain a nucleotide sequence that is complementary to the nucleotide sequence of the endogenous antisense transcript (NAT) (ie, the sense oligonucleotide of the NAT transcribed gene) inhibits NAT's regulation of expression on the target mRNA. It is possible to regulate the stability of the target mRNA and regulate the expression of the target gene (protein production) positively or negatively. Therefore, the present invention also provides a sense oligonucleotide homologous to the 3 'UTR of a gene having an expression-regulating activity of a gene in which NAT is induced, induced by IL-1β. Here, the target gene (mRNA) is the same as described above for the antisense nucleotide of the present invention.

The sense oligonucleotide of the present invention includes a sequence complementary to NAT (and thus a sequence homologous to the 3 ′ UTR), which contains a sequence complementary to the 3 ′ UTR of the target mRNA, and changes the stability of the target mRNA. Any oligonucleotide can be used as long as it can be used. Here, the “complementary” sequence is not only a sequence that is completely complementary to NAT, but as long as it can hybridize with NAT under physiological conditions of cells and inhibit the action of NAT on mRNA, It may contain one to several (2, 3, 4 or 5) base mismatches. Preferably, the sequence complementary to the target NAT refers to stringent conditions such as those described in Current Protocols in Molecular Biology, John Wiley & Sons, 6.3.1-6.3.6, 1999 (eg 6 × Under the condition of SSC (sodium chloride / sodium citrate) / 45 ° C hybridization, followed by 0.2 × SSC / 0.1% SDS / 50-65 ° C one or more washings). The sequence to get.
As described above, since the antisense nucleotide against the target mRNA interacts with a thermodynamically unstable portion of the mRNA to regulate the stability of the mRNA, the sense oligonucleotide of the present invention contains the target oligonucleotide in the target mRNA. It preferably contains a sequence homologous to a portion that is not thermodynamically stable. Examples of the portion that is not thermodynamically stable include a region that is in a single-stranded state when the mRNA has a secondary structure (for example, a region corresponding to the loop portion of the stem-loop structure).
The “homologous” sequence is not only a sequence that is completely identical to the specific partial nucleotide sequence of the target mRNA, but also the action of NAT on the mRNA by hybridizing with NAT on the mRNA under physiological conditions of the cell. 1 to several (2, 3, 4 or 5) bases may be different as long as they can be inhibited. More preferably, the “homologous” sequence is 90% or more identity to the target mRNA in the overlapping region when the sequence of the target site of the target mRNA and the sequence of the sense oligonucleotide are aligned. Means a nucleotide sequence having

Furthermore, the sense oligonucleotide of the present invention does not contain a sequence that causes a non-sequence-specific reaction (for example, 5′-CG-3 ′, 5′-GGGG-3 ′, 5′-GGGGG-3 ′, etc.). It is preferable to select from those having no similar sequence in RNA other than the target mRNA. The absence of similar sequences in other RNAs can be confirmed by methods similar to those described above for antisense oligonucleotides.

Preferably, the sense oligonucleotide for CCL2 NAT of the present invention, in the rat CCL2 mRNA 3'UTR sequence shown in SEQ ID NO: 1, is represented by domain 1 shown by nucleotide numbers 25-51 or nucleotide numbers 103-175 In the CCL2 ortholog in other mammals in the domain 2, the sequence corresponding to each domain in the rat CCL2 includes at least a part of one or more loop structures (for example, 3 bases or more). Contains a homologous nucleotide sequence. For example, TTAAGTAATGTTAAACTTAT (CCL2-Se1; SEQ ID NO: 8) that can enhance the expression of CCL2 mRNA as a sense oligonucleotide homologous to domain 1, and CCL2 mRNA as a sense oligonucleotide that is homologous to domain 2 Examples include, but are not limited to, TCCATTTTTTTATTTCTCTG (CCL2-Se2; SEQ ID NO: 9) that can be enhanced.

Preferably, the sense oligonucleotide for CCL20 NAT of the present invention is represented by the domain 1 shown by nucleotide numbers 65-107 and nucleotide numbers 124-155 in the rat CCL20 mRNA 3'UTR sequence shown by SEQ ID NO: 2. In domain 2 indicated by nucleotide 2 or nucleotide number 214-282, or in the CCL20 ortholog in other mammals, at least a part of one or more loop structures in the domain corresponding to each domain in the rat CCL20 It includes a nucleotide sequence that is homologous to a sequence that includes (eg, 3 bases or more). For example, as a sense oligonucleotide homologous to domain 1, GGTTTCACCTGCACATCACT (CCL20-Se1; SEQ ID NO: 10) can suppress the expression of CCL20 mRNA, and as a sense oligonucleotide homologous to domain 3, expression of CCL20 mRNA Examples include, but are not limited to, GTTTAGCTATTTAATGTTAA (CCL2-Se2; SEQ ID NO: 11) that can be enhanced.

Preferably, the sense oligonucleotide for CX3CL1 NAT of the present invention, in the 3′UTR sequence of rat CX3CL1 mRNA shown in SEQ ID NO: 3, is in domain 1, nucleotide numbers 1305-1369 shown as nucleotide numbers 1103-1202. Domain 2 shown, Domain 3 shown by nucleotide number 1416-1482, Domain 4 shown by nucleotide number 1633-1681, Domain 5 shown by nucleotide number 1688-1745 or Domain 6 shown by nucleotide number 1784-1810, The CX3CL1 ortholog in other mammals contains a nucleotide sequence homologous to a sequence containing at least a part of one or more loop structures in the domain corresponding to each domain in the rat CX3CL1. For example, ACTTGTGCATGTGTGTACTT (CX3CL1-Se1; SEQ ID NO: 13) can enhance the expression of CX3CL1 mRNA as a sense oligonucleotide homologous to domain 1, and CX3CL1 mRNA as a sense oligonucleotide homologous to domain 2 Examples include, but are not limited to, ACAAAGTGTCTACTGAAGCA (CX3CL1-Se2; SEQ ID NO: 14) and CTACTGAAGCAGAGAGCAGC (CX3CL1-Se3; SEQ ID NO: 15) that can suppress the expression of CX3CL1 mRNA.

Preferably, the sense oligonucleotide for IL-23A NAT of the present invention, in the 3′UTR sequence of rat IL-23A mRNA shown in SEQ ID NO: 4, is the domain 1, nucleotide number shown by nucleotide numbers 37-55 In the domain 2 represented by 193-303, the domain 3 represented by nucleotide numbers 380-448 or the domain 4 represented by nucleotide numbers 555-600, and the IL-23A ortholog in other mammals, the rat IL- A nucleotide sequence homologous to a sequence comprising at least part of one or more loop structures within the domain corresponding to each domain of 23A. For example, as a sense oligonucleotide homologous to domain 1, AATCCATCAATGCAGACATC (IL23-Se1; SEQ ID NO: 16) capable of suppressing the expression of IL-23A mRNA, and as a sense oligonucleotide homologous to domain 2, IL-23A GAAGCTGGCAGACAGCTGCA (IL23-Se2; SEQ ID NO: 17) capable of enhancing mRNA expression, TCCTTCAGTTCTAACAGAAC (IL23-Se3; SEQ ID NO: 18) capable of suppressing IL-23A 発 現 mRNA expression as a sense oligonucleotide homologous to domain 3 Examples of the sense oligonucleotide homologous to domain 4 include, but are not limited to, AACAGTTTAGAGGATTGTTA (IL23-Se4; SEQ ID NO: 19) that can suppress the expression of IL-23A mRNA.

Preferably, the sense oligonucleotide for CD69 NAT of the present invention is the domain 1 represented by nucleotide numbers 15-32 and nucleotide numbers 120-186 in the 3′UTR sequence of rat CD69 mRNA represented by SEQ ID NO: 5. In domain 2, indicated in domain 3 indicated by nucleotide numbers 218-252, domain 4 indicated by nucleotide numbers 344-370, domain 5 indicated by nucleotide numbers 389-633 or domain 6 indicated by nucleotide numbers 656-786, The CD69 ortholog in other mammals contains a nucleotide sequence homologous to a sequence containing at least a part of one or more loop structures in the domain corresponding to each domain in the rat CD69. For example, as a sense oligonucleotide homologous to domain 5, GCCAATGCTTATGAAAACA (CD69-Se2; SEQ ID NO: 21) can enhance the expression of CD69 mRNA, and as a sense oligonucleotide homologous to domain 6, IL-23A の mRNA Examples include, but are not limited to, GTGGCAGATCTCTGTCAGGA (CD69-Se3; SEQ ID NO: 22) that can enhance expression.

Preferably, the sense oligonucleotide for NF-κB p65 NAT of the present invention is the domain 1 represented by nucleotide numbers 161-301 in the 3 ′ UTR sequence of rat NF-κB p65 mRNA represented by SEQ ID NO: 6, In the domain 2 represented by nucleotide number 343-380, domain 3 represented by nucleotide number 401-412 or domain 4 represented by nucleotide number 473-523, and the NF-κB p65 ortholog in other mammals, A nucleotide sequence homologous to a sequence containing at least a part of one or more loop structures in the domain corresponding to each domain in rat NF-κB p65. For example, as a sense oligonucleotide homologous to domain 1, GAACTCTTGAGACCCTGCTT (p65-Se1; SEQ ID NO: 23) capable of enhancing the expression of NF-κB 相同 p65 mRNA, and as a sense oligonucleotide homologous to domain 3, NF- GCAACGCTCCTAGGAGCAGC (p65-Se3; SEQ ID NO: 25) that can suppress the expression of κB p65 mRNA, AACTCTCCATGCTGAGCAGT (p65-Se4; sequence that can suppress the expression of NF-κB p65 mRNA as a homologous sense oligonucleotide to domain 4 No. 26), respectively, but is not limited thereto.

Preferably, the sense oligonucleotide for TNF-αNAT of the present invention is the domain 1, nucleotide number 430 represented by nucleotide number 304-405 in the 3′UTR sequence of rat TNF-α mRNA represented by SEQ ID NO: 7. In the domain 2 represented by -546, domain 3 represented by nucleotide numbers 687-713 or domain 4 represented by nucleotide numbers 752-776, and the TNF-α ortholog in other mammals, the rat TNF-α A nucleotide sequence homologous to a sequence comprising at least part of one or more loop structures within the domain corresponding to each domain in For example, AGATGTCTCAGGCCTCCCTT (TNF-Se1; SEQ ID NO: 27) capable of enhancing the expression of TNF-α mRNA as a sense oligonucleotide homologous to domain 1, and TNF-α as a sense oligonucleotide homologous to domain 2 GGAACCCCCTATATTTATAA (TNF-Se2; SEQ ID NO: 28) that can enhance the expression of mRNA and TAATTGCACCTGTGACTATT (TNF-Se3; SEQ ID NO: 29) that can suppress the expression of TNF-α mRNA, as a sense oligonucleotide homologous to domain 3 , AACAAGATATTTATCTAACC (TNF-Se5; SEQ ID NO: 31) that can suppress the expression of TNF-α mRNA, ACTGAACCTCTGCTCCCCAC (TNF-Se7; TNF-α mRNA that can suppress the expression of TNF-α mRNA as a sense oligonucleotide homologous to domain 4 SEQ ID NO: 33), respectively, but is not limited thereto.

The length of the sense oligonucleotide of the present invention is not particularly limited, but from the viewpoint of sequence specificity, the portion complementary to the target sequence in the target NAT is at least 10 bases or more, preferably about 12 bases or more, more preferably Contains about 15 bases or more. In addition, in view of ease of synthesis, production cost, ease of administration, and the like, those having a base length of 50 bases or less, preferably 40 bases or less, more preferably 30 bases or less are mentioned.

The sense oligonucleotide of the present invention may be any of single-stranded DNA, single-stranded RNA, and DNA / RNA chimera, and may be those with known modifications. Here, the “nucleotide” may include not only purine and pyrimidine bases but also those having other modified heterocyclic bases. When the sense oligonucleotide is DNA (ODN), the RNA: DNA hybrid formed by the target NAT and the sense ODN can be recognized by endogenous RNase H and cause selective degradation of the target NAT.

The nucleotide molecule constituting the sense oligonucleotide may be natural DNA or RNA, but in order to improve stability (chemical and / or enzyme) and specific activity (affinity with RNA), the above antisense As with nucleotides, various chemical modifications can be included.

The sense oligonucleotide of the present invention synthesizes a sequence homologous to the target mRNA (cDNA) sequence using a commercially available DNA / RNA automatic synthesizer (Applied Biosystems, Beckman, etc.). Can be prepared.

The sense oligonucleotide of the present invention is provided in a special form such as a liposome or a microsphere, or a form to which a hydrophobic substance such as a polycationic substance such as polylysine or a lipid (eg, phospholipid, cholesterol, etc.) is added. Can be provided at. Alternatively, the sense oligonucleotide of the present invention is converted into a peptide having a membrane permeation function (for example, Drosophila-derived Antennapedia homeodomain (AntP), human immunodeficiency virus (HIV) -derived TAT, herpes simplex virus (HSV) -derived VP22. And the like can be promoted by incorporating the oligonucleotide into a cell.

The sense oligonucleotide of the present invention can change the target gene expression (protein production) regulating action by the NAT by interacting with the target NAT. Since various genes including cytokine / chemokine gene induced by IL-1β have various physiological activities such as inflammation-inducing or anti-inflammatory action, infection defense, and regulation of immune response, the sense oligonucleotide of the present invention is used. The contained drug can be used as a target gene expression regulator for the prevention and / or treatment of various diseases including inflammatory diseases and infectious diseases.
For example, sense oligonucleotides that can enhance the expression of CCL2 can be used for angiogenesis and wound healing, while CCL2 migrates to monocytes and eosinophils, so that it can suppress the expression of CCL2. Nucleotides are useful for the prevention and treatment of chronic inflammatory diseases including arteriosclerosis and allergic inflammation.
Since CCL20 exhibits antibacterial activity against a wide range of microorganisms, sense oligonucleotides that can enhance the expression of CCL20 are effective in the prevention and treatment of various microbial infections. On the other hand, since CCL20 plays an important role in the initiation and maintenance of adaptive immunity, sense oligonucleotides that can suppress the expression of CCL20 are bronchial asthma, multiple sclerosis, rheumatoid arthritis, various dermatitis, inflammatory It is effective for the prevention and treatment of autoimmune diseases such as bowel disease.
Sense oligonucleotides that can enhance CX3CL1 expression can be used for angiogenesis, while sense oligonucleotides that can suppress CX3CL1 expression prevent or treat inflammatory diseases including arteriosclerosis and glomerulonephritis It is useful for the prevention and treatment of anti-rejection and rheumatoid arthritis.
IL-23A is important for the maintenance of cellular immunity, and its overexpression causes multi-organ inflammation. Therefore, sense oligonucleotides that can enhance IL-23A expression are effective in the prevention and treatment of infectious diseases. On the other hand, sense oligonucleotides that can suppress the expression of IL-23A are useful for the prevention and treatment of autoimmune diseases and inflammatory diseases.
Since CD69 plays an important role in the differentiation and activation of lymphocytes, a sense oligonucleotide that can enhance the expression of CD69 is effective in the prevention and treatment of infectious diseases. On the other hand, CD69 is expressed in most inflammatory cells that are expressed locally, and autoantibodies against CD69 are detected in patients with rheumatoid arthritis and systemic lupus erythematosus (SLE). Nucleotides are useful for the prevention and treatment of autoimmune diseases and inflammatory diseases.
Since NF-κB is a transcription factor having an important role in the immune system, a sense oligonucleotide that can enhance the expression of NF-κB p65 is effective in the prevention and treatment of infectious diseases. On the other hand, NF-κB is constantly activated in cancer cells and inhibits apoptosis, and is also involved in pathogenesis such as bone metabolism, bronchial asthma, arthritis, inflammatory bowel disease, sepsis, A sense oligonucleotide capable of suppressing the expression of NF-κB p65 is useful for the prevention and treatment of cancer, osteoporosis, autoimmune diseases and inflammatory diseases.
TNF-α shows protection against infection and anti-tumor action by inducing apoptosis and enhancing antibody production by inflammatory mediators and plasma cells. Therefore, sense oligonucleotides that can enhance the expression of TNF-α prevent infection and cancer. It is effective for treatment. On the other hand, since TNF-α is involved in the pathogenesis of rheumatoid arthritis, psoriasis, diabetes, hyperlipidemia, sepsis, and osteoporosis, sense oligonucleotides that can suppress the expression of TNF-α are used to prevent and treat these diseases. Useful for.

The medicament containing the sense oligonucleotide of the present invention has low toxicity and can be used as a liquid or as a pharmaceutical composition of an appropriate dosage form as a human or non-human mammal (eg, mouse, rat, guinea pig, rabbit, sheep, Pigs, cows, cats, dogs, monkeys, etc.) can be administered orally or parenterally (eg, inhalation administration, intravascular administration, subcutaneous administration, transmucosal administration etc.).
When these nucleic acids are used as a prophylactic / therapeutic agent for the above-mentioned various diseases, they can be formulated and administered in the same manner as the antisense nucleotide of the present invention.

The dose of the above-mentioned medicament containing the sense oligonucleotide of the present invention varies depending on the administration subject, target disease, symptom, administration route, etc. For example, the dose of the sense oligonucleotide of the present invention is usually 0.01 to 20 mg. It is convenient to administer about 0.1 kg / kg body weight, preferably about 0.1-10 mg / kg body weight, more preferably about 0.1-5 mg / kg body weight by intravenous injection or inhalation once to several times a day. In the case of other parenteral administration and oral administration, an equivalent amount can be administered. If symptoms are particularly severe, the dose may be increased according to the symptoms.

The above-described pharmaceutical composition may contain other drugs as long as no undesirable interaction is caused by the combination with the sense oligonucleotide of the present invention. Other drugs include, for example, antiviral drugs, antitumor drugs, antibacterial drugs, antifungal drugs, antiprotozoal drugs, antibiotics, antiseptic drugs, antiseptic shock drugs, endotoxin antagonists, immunomodulators, non-steroidal drugs Examples include anti-inflammatory drugs, steroid drugs, inflammatory mediator action inhibitors, inflammatory mediator production inhibitors, anti-inflammatory mediator action inhibitors, anti-inflammatory mediator production inhibitors.

The present invention also provides a method of screening for a substance that regulates target gene expression (protein production) by regulating the action of NAT on the target mRNA. The screening method of the present invention is characterized by detecting the hybridization between a target mRNA and NAT for the target mRNA in the presence and absence of a test substance and comparing the degree thereof.
For example, target mRNA and NAT are isolated by a conventional method, one of them is solid-phased and the other is labeled with an appropriate labeling agent, and the test is performed under conditions where RNA can form a physiological secondary structure. Examples include a method of hybridizing both in the presence and absence of a substance and comparing the amount of label bound to the solid phase under both conditions. Here, as mRNA and NAT, the full length thereof may be used, respectively, or the 3 ′ UTR of mRNA and a fragment containing a NAT sequence complementary to the region may be used.

Examples of the solid phase material include semiconductors such as silicon, inorganic materials such as glass and diamond, films mainly composed of high molecular substances such as polyethylene terephthalate and polypropylene, and the shape of the solid phase includes a slide glass, Examples include, but are not limited to, microwell plates, microbeads, and fiber types. As a method for immobilizing mRNA or NAT on a solid phase, functional groups such as amino group, aldehyde group, SH group, and biotin are introduced into the RNA in advance, while reacting with the RNA on the solid phase. Functional groups (eg, aldehyde group, amino group, SH group, streptavidin, etc.) are introduced, and the solid phase and RNA are cross-linked by covalent bond between the two functional groups, or the polyanionic RNA is immobilized. Examples of the method include, but are not limited to, a method of immobilizing RNA using polycation coating of a phase and electrostatic bonding. The solid-phase RNA preparation methods include the Affymetrix method, which synthesizes RNA one nucleotide at a time on a substrate (glass, silicon, etc.) using a photolithography method, the micro spotting method, the inkjet method, and the bubble jet (registered trademark) method The Stanford method in which RNA prepared in advance is spotted on the substrate using the above method, but considering the base length of the RNA to be used, it is preferable to use the Stanford method or a method combining both.

As the labeling agent, for example, a radioisotope, an enzyme, a fluorescent substance, a luminescent substance, or the like is used. As the radioisotope, for example, [ ³² P], [ ³ H], [ ¹⁴ C] and the like are used. As the enzyme, a stable enzyme having a large specific activity is preferable. For example, β-galactosidase, β-glucosidase, alkaline phosphatase, peroxidase, malate dehydrogenase and the like are used. As the fluorescent material, for example, fluorescamine, fluorescein isothiocyanate, Cy3, Cy5 and the like are used. As the luminescent substance, for example, luminol, luminol derivatives, luciferin, lucigenin and the like are used. Furthermore, biotin- (strept) avidin can also be used for binding between the probe and the labeling agent.

The test substance may be any known substance or new substance, such as a nucleic acid, carbohydrate, lipid, protein, peptide, organic low molecular weight compound, compound library prepared using combinatorial chemistry techniques, solid phase Examples include random peptide libraries prepared by synthesis or phage display methods, or natural components derived from microorganisms, animals and plants, marine organisms, and the like. The concentration of the test substance to be added varies depending on the type of compound (solubility, toxicity, etc.), but is appropriately selected within the range of, for example, about 0.1 nM to about 100 nM. Examples of the incubation time include about 1 to about 24 hours.

After bringing the RNA on the solid phase into contact with the labeled RNA (and the test substance) and incubating, the RNA that did not bind to the solid phase is washed away, and the amount of RNA bound to the solid phase is detected. When the amount of the label bound to the solid phase is significantly increased in the presence of the test substance compared to the absence, the test substance can be selected as a candidate for a substance that enhances the action of NAT on the target mRNA. On the other hand, if the amount of label bound to the solid phase is significantly reduced in the presence of the test substance compared to the absence, the test substance can be selected as a candidate for a substance that suppresses the action of NAT on the target mRNA. it can.

In a preferred embodiment, a target substance is contacted more directly by contacting a test substance with a cell that expresses the target mRNA and NAT, and measuring a change in the amount of the mRNA and / or protein encoded in the cell. Substances that enhance or suppress gene expression can be selected. The cell that expresses the target mRNA and NAT may be a cell that can naturally express both RNAs (for example, IL-1β-stimulated hepatocytes), or a DNA that expresses either or both of them. The introduced recombinant cell may also be used. In the case of recombinant cells, examples of host cells include animal cells such as H4IIE-C3 cells, HepG2 cells, 293T cells, HEK293 cells, COS7 cells, 2B4T cells, CHO cells, MCF-7 cells, and H295R cells. it can. The target mRNA and DNA encoding NAT are both isolated by conventional methods, converted into double-stranded DNA by reverse transcription, etc., and then inserted into an expression vector having a promoter that can function in the host cell. For example, it can be prepared by introducing this vector into a host cell by the calcium phosphate coprecipitation method, PEG method, electroporation method, microinjection method, lipofection method or the like.

As the test substance, those described above are used. The contact between the test substance and the cell is, for example, a medium suitable for culturing the cell (for example, a minimum essential medium (MEM) containing about 5 to 20% fetal calf serum, Dulbecco's modified Eagle medium (DMEM), RPMI1640 Medium, 199 medium, F12 medium, etc.) and various buffers (eg, HEPES buffer, phosphate buffer, phosphate buffered saline, Tris-HCl buffer, borate buffer, acetate buffer, etc.) It can be carried out by adding a test substance and incubating the cells for a certain period of time. The concentration of the test substance to be added varies depending on the type of compound (solubility, toxicity, etc.), but is appropriately selected within the range of, for example, about 0.1 nM to about 100 nM. Examples of the incubation time include about 1 to about 48 hours. If necessary, the cells may be infected with the virus during the incubation.

After incubation (or over time), RNA is extracted from the cells and the target mRNA level is measured by RT-PCR, real-time PCR or Northern blot analysis, or the culture supernatant is collected and various immunoassays known per se Or the amount of protein encoded by the target gene is measured by Western blotting or the like. When the amount of target mRNA and / or protein in a cell is significantly increased by adding a test substance, the test substance can be selected as a candidate for a target gene expression enhancing substance. On the other hand, when the amount of mRNA and / or protein is significantly decreased by the addition of a test substance, the test substance can be selected as a candidate for a target gene expression inhibitor.

Substances capable of enhancing or suppressing the expression of the target gene selected by the screening method described above are used for the prevention and / or prevention of the above-mentioned various diseases in the same manner as antisense nucleotides and sense oligonucleotides that can enhance or suppress the expression of the gene. Alternatively, it can be used as a therapeutic drug.

A medicine containing a substance selected by the above screening method has low toxicity, and can be used as a liquid or as a pharmaceutical composition of an appropriate dosage form as a human or non-human mammal (eg, mouse, rat, guinea pig, rabbit). , Sheep, pigs, cows, cats, dogs, monkeys, etc.) or orally (eg, inhalation administration, intravascular administration, subcutaneous administration, etc.). The pharmaceutical composition used for administration may comprise a selected substance and a pharmacologically acceptable carrier, diluent or excipient.

As a composition for parenteral administration, for example, injections, aerosols, suppositories and the like are used, and injections are intravenous injections, subcutaneous injections, intradermal injections, intramuscular injections, intravenous injections, etc. May be included. Such an injection can be prepared according to a known method. As a method for preparing an injection, it can be prepared, for example, by dissolving, suspending or emulsifying a substance for enhancing or suppressing the expression of a selected target gene in a sterile aqueous liquid or oily liquid that is usually used for injection. As an aqueous solution for injection, for example, an isotonic solution containing physiological saline, glucose and other adjuvants, and the like are used, and suitable solubilizers such as alcohol (eg, ethanol), polyalcohol (eg, Propylene glycol, polyethylene glycol), nonionic surfactants (eg, polysorbate 80, HCO-50 (polyoxyethylene (50 mol) adduct-of-hydrogenated-castor-oil)) and the like may be used in combination. As the oily liquid, for example, sesame oil, soybean oil and the like are used, and benzyl benzoate, benzyl alcohol and the like may be used in combination as a solubilizing agent. The prepared injection solution is preferably filled in a suitable ampoule. Aerosol formulations can be placed in compressed acceptable propellants such as dichlorodifluoromethane, propane, nitrogen and the like. Alternatively, it may be formulated as a non-compressible pharmaceutical product such as a nebulizer or an atomizer. A suppository used for rectal administration may be prepared by mixing a substance for enhancing or suppressing the expression of the target gene with a normal suppository base.

Compositions for oral administration include solid or liquid dosage forms, specifically tablets (including sugar-coated tablets and film-coated tablets), pills, granules, powders, capsules (including soft capsules), and syrups. Agents, emulsions, suspensions and the like. Such a composition is produced by a known method and may contain a carrier, a diluent or an excipient usually used in the pharmaceutical field. As the carrier and excipient for tablets, for example, lactose, starch, sucrose, and magnesium stearate are used.

The above parenteral or oral pharmaceutical composition is conveniently prepared in a dosage unit form suitable for the dosage of the active ingredient. Examples of such dosage forms include tablets, pills, capsules, injections (ampoules), aerosols, and suppositories. It is preferable that the substance for enhancing or suppressing the expression of the target gene is usually contained in an amount of 5 to 500 mg per dosage unit dosage form, particularly 5 to 100 mg for injections and 10 to 250 mg for other dosage forms.

The dose of the above-mentioned pharmaceutical containing a substance that enhances or suppresses the expression of the target gene varies depending on the administration subject, target disease, symptom, administration route, etc., but for example, the substance is usually 0.01 to 20 mg per dose It is convenient to administer about 0.1 kg / kg body weight, preferably about 0.1 to 10 mg / kg body weight, more preferably about 0.1 to 5 mg / kg body weight by intravenous injection once to several times a day. In the case of other parenteral administration and oral administration, an equivalent amount can be administered. If symptoms are particularly severe, the dose may be increased according to the symptoms.

Hereinafter, the present invention will be described more specifically with reference to examples. However, these are merely examples and do not limit the scope of the present invention.

(Materials and methods)
Transfection of hepatocytes Hepatocytes were isolated from male Wistar rats (SPF / VAF Crlj: WI; Charles River Japan) as described in Prostaglandins 1993; 45: 459-474 and seeded at 37 ° C. Incubated overnight. Hepatocytes (3.0 × 10 ⁵ cells / dish) were transfected with sense ODN. Each ODN (1.5 μg) was mixed with 1.5 μL of magnet-assisted transfection A reagent (IBA), added to each well, incubated on the magnetic plate for 15 minutes, and then the medium was treated with fresh William's E medium (Sigma-Aldrich). ). The cells were cultured overnight and treated with 1 nM human IL-1β (Otsuka Pharmaceutical Co., Ltd.) for 4 hours (RNA preparation) or 8 hours (cell extraction). Animal experiments were conducted with the approval of the Animal Experiment Committee of Ritsumeikan University Biwako Kusatsu Campus.

Secondary structure prediction of mRNA The secondary structure of 3'UTR of mRNA was predicted by the mfold program (Zuker). A common conserved region was selected from the predicted structure of the 3'UTR of mRNA. Each region contained at least one stem-loop structure.

Design of Sense ODN Sense ODN protected with a phosphothioate bond was designed according to the method described in J. Neurochem. 2003; 86: 374-382 (Gene Design). The designed sense ODN sequence corresponds to an mRNA sequence containing at least one loop of a common region conserved between human and rat, and CpG motifs and G-quartets were avoided.

Strand-specific reverse transcription-polymerase chain reaction (RT-PCR)
Total RNA was prepared using Sepasol I Super (Nacalai Tesque) and treated with TURBO DNA-free kit (Ambion-Applied Biosystems). A complementary DNA (cDNA) was synthesized using an oligo dT primer for mRNA and a strand-specific primer for NAT, and a pair of primers (operon and operon) were synthesized according to the method described in Genes Cells. 2000; 5: 111-125. Biotechnologies) was used to perform step-down PCR.
Tables 2 and 3 summarize the PCR primers, NAT reverse transcription primers and PCR primers used for mRNA amplification for the seven genes CCL2, CCL20, CX3CL1, IL-23A, CD69, NF-κB p65 and TNF-α. It was.

(result)
In order to search for genes that transcribe both mRNA and NAT, we selected 21 genes (including cytokines and chemokine genes) induced by IL-1β and complementary to their 3 'UTRs. Attempted to detect NAT. That is, whether or not NAT was transcribed from these 21 genes was examined by chain-specific RT-PCR using a gene-specific sense primer for RT (Table 3). As shown in Table 4, as a result of the strand-specific RT-PCR analysis, it was found that NAT was transcribed from 16 out of 21 genes (76%). Interestingly, the ARE motif in 3'UTRR was found in 13 NAT-transcribed genes (81%). These data suggest that NAT corresponding to the 3'UTR of mRNA is frequently transcribed from IL-1β-inducible genes, supporting the idea that NAT is widely expressed. It was.

The present inventors selected 7 genes (CCL2, CCL20, CX3CL1, IL-23A, CD69, NF-κB p65 and TNF-α) in which both mRNA and NAT are abundantly transcribed, and applied NATRE technology. . The present inventors predicted the secondary structure of the 3′UTR of these mRNAs (FIGS. 1A-7A), and each sense ODN competes with the mRNA that leads to inhibition of the mRNA-NAT interaction. Sense ODNs were designed according to the rule of containing at least one loop of the storage region (FIGS. 1B-7B) (Table 5 and FIGS. 1B-7B).

After transfecting rat hepatocytes with each sense ODN and stimulating the cells with IL-1β, the expression level of the target mRNA was determined using SYBR Green I as described in Hepatology,; 2008; 47: 686-697. Measured by real-time PCR. The results are shown in FIG. Elongation factor 1alpha (EF) mRNA was used as an internal standard, and the values in the graph indicate each mRNA / EF mRNA (%), and were expressed as a relative amount with respect to the expression level in non-sense ODN-introduced cells. Most sense ODNs designed for RNA sequences that contain loop portions within conserved regions of predicted secondary structure have altered (increased or decreased) the expression level of the target mRNA, whereas Sense ODNs (TNF-Se4 and TNF-Se6) designed outside the conserved region of secondary structure did not affect the expression level of the target mRNA.

The antisense nucleotide and sense oligonucleotide of the present invention are used as a regulator of the expression of an inflammation / infection-related gene in which an endogenous antisense transcript is present, which is induced by IL-1β. Useful for treatment.

This application is based on Japanese Patent Application No. 2011-107707, filed in Japan on May 12, 2011, the contents of which are hereby incorporated by reference.

Claims (11)

IL-1β induced CCL2, CCL20, CX3CL1, IL-23A, CD69, NF-κB p65, TNF-α, Fam89a, Grk5, phosopholipid scramblase 1, Runx1, semaphorin 4A, Steap4, lymphphotoxin β10Psmb10 or TLR2 gene An antisense nucleotide comprising a sequence complementary to the 3 ′ UTR of the gene and capable of regulating expression of the gene. The antisense nucleotide according to claim 1, wherein the gene induced by IL-1β is CCL2, CCL20, CX3CL1, IL-23A, CD69, NF-κBp65 or TNF-α. CCL2, CCL20, CX3CL1, IL-23A, CD69, NF-κB p65, TNF-α, Fam89a, Grk5, phosopholipid scramblase 1, Runx1, semaphorin 4A, Steap4, comprising the antisense nucleotide of claim 1 Lymphotoxin β Psmb10 or TLR2 expression regulator. The agent according to claim 3, which is used for prevention or treatment of inflammatory diseases or infectious diseases. IL-1β induced CCL2, CCL20, CX3CL1, IL-23A, CD69, NF-κB p65, TNF-α, Fam89a, Grk5, phosopholipid scramblase 1, Runx1, semaphorin 4A, Steap4, lymphphotoxin β10Psmb10 or TLR2 gene A sense oligonucleotide comprising a sequence complementary to an endogenous antisense transcript comprising a sequence complementary to the 3 ′ UTR of the gene and capable of regulating expression of the gene. The sense oligonucleotide according to claim 5, wherein the gene induced by IL-1β is CCL2, CCL20, CX3CL1, IL-23A, CD69, NF-κBp65 or TNF-α. The sense oligonucleotide according to claim 6, which is any one of the following (a) to (g).
(a) In the 3′UTR sequence of CCL2 mRNA represented by SEQ ID NO: 1, domain 1 represented by nucleotide numbers 25-51 or domain 2 represented by nucleotide numbers 103-175, or those of orthologs in other mammals Sense oligonucleotide capable of regulating the expression of the CCL2 gene, comprising a nucleotide sequence having 90% or more identity with a sequence comprising at least part of one or more loop structures in the corresponding domain
(b) In the 3'UTR sequence of CCL20 mRNA represented by SEQ ID NO: 2, domain 1 represented by nucleotide numbers 65-107, domain 2 represented by nucleotide numbers 124-155, or domain 3 represented by nucleotide numbers 214-282 Or expression of the CCL20 gene comprising a nucleotide sequence having at least 90% identity with a sequence comprising at least a portion of one or more loop structures within a domain corresponding to those of orthologs in other mammals Regulatable sense oligonucleotide
(c) In the 3′UTR sequence of CX3CL1 mRNA represented by SEQ ID NO: 3, domain 1 represented by nucleotide numbers 1103-1202, domain 2 represented by nucleotide numbers 1305-1369, and domain 3 represented by nucleotide numbers 1416-1482 In domain 4, represented by nucleotide number 1633-1681, domain 5 represented by nucleotide number 1688-1745, or domain 6 represented by nucleotide number 1784-1810, or a domain corresponding to those of orthologs in other mammals A sense oligonucleotide capable of regulating the expression of the CX3CL1 gene, comprising a nucleotide sequence having at least 90% identity with a sequence comprising at least a part of one or more loop structures
(d) In the 3'UTR sequence of IL-23A mRNA shown in SEQ ID NO: 4, domain 1 shown by nucleotide numbers 37-55, domain 2 shown by nucleotide numbers 193-303, and nucleotide numbers 380-448 90% or more identity with a sequence comprising at least part of one or more loop structures within domain 3, or domain 4 represented by nucleotide numbers 555-600, or domains corresponding to those of orthologs in other mammals Sense oligonucleotide capable of regulating IL-23A gene expression comprising a nucleotide sequence having
(e) In the 3'UTR sequence of CD69 mRNA represented by SEQ ID NO: 5, domain 1 represented by nucleotide numbers 15-32, domain 2 represented by nucleotide numbers 120-186, domain 3 represented by nucleotide numbers 218-252 In domain 4, indicated by nucleotide numbers 344-370, domain 5 indicated by nucleotide numbers 389-633, or domain 6 indicated by nucleotide numbers 656-786, or domains corresponding to those of orthologs in other mammals A sense oligonucleotide capable of regulating the expression of the CD69 gene, comprising a nucleotide sequence having 90% or more identity with a sequence comprising at least a part of one or more loop structures
(f) In the 3′UTR sequence of NF-κB p65 mRNA represented by SEQ ID NO: 6, domain 1 represented by nucleotide numbers 161-301, domain 2 represented by nucleotide numbers 343-380, represented by nucleotide numbers 401-412 90% or more identical to a sequence comprising at least a part of one or more loop structures in the domain 3, or the domain 4 indicated by nucleotide numbers 473-523, or the domain corresponding to those of orthologs in other mammals Sense oligonucleotide capable of regulating expression of NF-κB p65 gene, comprising a nucleotide sequence having sex
(g) In the 3′UTR sequence of TNF-α mRNA represented by SEQ ID NO: 7, domain 1 represented by nucleotide numbers 304-405, domain 2 represented by nucleotide numbers 430-546, and nucleotide numbers 687-713 90% or more identity with a sequence comprising at least part of one or more loop structures within domain 3, or domain 4 represented by nucleotide numbers 752-776, or domains corresponding to those of orthologs in other mammals A sense oligonucleotide capable of regulating the expression of the TNF-α gene, comprising a nucleotide sequence having CCL2, CCL20, CX3CL1, IL-23A, CD69, NF-κB p65, TNF-α, Fam89a, Grk5, phosopholipid scramblase 1, Runx1, semaphorin 4A, Steap4, comprising the sense oligonucleotide according to claim 5 Lymphotoxin β Psmb10 or TLR2 expression regulator. The agent according to claim 8, which is used for prevention or treatment of inflammatory diseases or infectious diseases. CCL2, CCL20, CX3CL1, IL-23A, CD69, NF-κB p65, TNF-α, Fam89a, Grk5, phosopholipid scramblase 1, Runx1, semaphorin 4A, Steap4, lymphphotoxin β Psmb10 in the presence and absence of the test substance Alternatively, a method for screening an expression regulator of the gene, comprising detecting and comparing the hybridization between the mRNA of the TLR2 gene and the endogenous antisense transcript thereto. A test substance is brought into contact with a cell expressing mRNA of the gene and an endogenous antisense transcript thereto, and a change in the amount of mRNA and / or protein encoded in the cell is measured. The method according to claim 10.

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