WO2016031996A1

WO2016031996A1 - Prophylactic/therapeutic agent for arthritis, test kit for arthritis, and method for screening for prophylactic/therapeutic agent for arthritis

Info

Publication number: WO2016031996A1
Application number: PCT/JP2015/074556
Authority: WO
Inventors: 和久中野
Original assignee: 学校法人産業医科大学
Priority date: 2014-08-28
Filing date: 2015-08-24
Publication date: 2016-03-03
Also published as: JPWO2016031996A1

Abstract

The present invention provides: a prophylactic/therapeutic agent for arthritis including rheumatoid arthritis, which comprises a Tet3 expression inhibitor; a kit for diagnosing arthritis including rheumatoid arthritis; and a means for searching for a novel substance having a prophylactic and/or therapeutic activity on arthritis including rheumatoid arthritis.

Description

Arthritis prevention / treatment agent, test kit, and screening method for arthritis prevention / treatment agent

The present invention relates to a prophylactic / therapeutic agent for arthritis, particularly rheumatoid arthritis (RA), and a screening method for a prophylactic / therapeutic agent for arthritis, particularly rheumatoid arthritis. More specifically, arthritis, particularly a prophylactic and / or therapeutic agent for rheumatoid arthritis, which contains a substance that inhibits the function of Tet 3 (Ten-Eleven tranlocation 3), and arthritis using Tet 3 function inhibition as an index, In particular, the present invention relates to a method for screening a candidate substance for a prophylactic / therapeutic agent for rheumatoid arthritis.

Rheumatoid arthritis is an intractable autoimmune disease that causes chronic inflammation in joints throughout the body. The synovial tissue in the joint grows and progressively destroys cartilage and bone. Rheumatoid arthritis synovial fibroblasts (FLS) are activated by stimulation (inflammatory cytokines such as TNFα and IL-1β) by macrophages and lymphocytes, and activated synovial fibroblasts While chemokine production and substrate-degrading enzyme secretion cause joint destruction, it also has properties similar to cancer cells, such as increased proliferation and invasion and decreased apoptosis sensitivity. In recent years, from the results of GWAS (Genome-Wide Association Study), it has become known that about 30 gene polymorphisms are related to the onset and severity of rheumatoid arthritis. On the other hand, it has been suggested that abnormalities in epigenetics are deeply related to the onset and severity of rheumatoid arthritis. DNA methylation is one of the typical mechanisms of epigenetics, and it has been reported that rheumatoid arthritis also has abnormal DNA methylation in the entire genome or the promoter region of a specific gene. The inventors have recently conducted genome-wide DNA methylation analyses, and there are DNA methylation patterns peculiar to diseases in synovial fibroblasts derived from rheumatoid arthritis patients, and many of the genes exhibiting abnormal methylation have been found in rheumatoid arthritis. It was shown to be deeply involved in the pathological condition (Non-patent Document 1).
Rheumatoid arthritis treatment targeting inflammatory cytokines with biologics has made remission induction familiar, but there is a so-called “Window of Opportunity” in rheumatoid arthritis treatment, and the delay in treatment start has a therapeutic effect. Limited. This suggests that the persistence of the inflammatory environment itself may transform the cells present at the site of synovial inflammation into a more aggressive and treatment-resistant phenotype, but this detailed mechanism is unknown. there were. The inventor found that TNFα and IL-1β, which play a central role in synovial inflammation and bone joint destruction in rheumatoid arthritis, decrease the expression of DNA methylase (DNMT) in FLS and promote passive demethylation This indicates that the molecular mechanism of disease-specific DNA methylation pattern formation was clarified (Non-patent Document 2).
However, since the mechanism of active DNA demethylation was unknown until now, the whole picture of the dynamism of DNA methylation / demethylation was unknown. Recently, a Tet (Ten-Eleven transcription) protein family that functions as a DNA demethylase has been identified, and the Tet protein hydroxylates 5-methylcytosine (5mC) to synthesize 5-hydroxymethylcytosine (5hmC). The research on DNA methylation dynamism in ES cells and the like is making rapid progress.
However, the involvement of Tet protein in rheumatoid arthritis has not yet been clarified, and it remains unclear whether Tet protein can be a therapeutic target for rheumatoid arthritis.

Accordingly, an object of the present invention is to clarify the function of Tet protein, particularly Tet3 protein, in arthritis, particularly rheumatoid arthritis, and based on this function, a prophylactic / therapeutic agent and test kit for arthritis targeting Tet3 And a means for searching for a novel substance having arthritis preventive and / or therapeutic activity using Tet 3 function regulation as an index.

In order to achieve the above object, the present inventor has expressed the expression level of Tet protein family and DNA in synovial fibroblasts (FLS) derived from rheumatoid arthritis patients stimulated with synovium or inflammatory cytokines derived from rheumatoid arthritis patients. Methylation levels were measured. As a result, compared with healthy subjects and osteoarthritis (OA) patients, the synovial fibroblasts (FLS) derived from rheumatoid arthritis patients stimulated with rheumatoid arthritis patient-derived synovial surface cell layers and inflammatory cytokines We found that the expression level of Tet 3 was high and the DNA methylation level was low. In addition, when Tet3 of FLS derived from patients with rheumatoid arthritis is knocked down, expression of chemokine CCL2 and adhesion molecule ICAM1 involved in FLS's own tissue (cartilage, bone) infiltration is inhibited, and the invasion ability of FLS is also suppressed. It was done. These results indicate that selective inhibition of Tet 3 inhibits the expression of CCL2 and ICAM1 through the maintenance of DNA methylation levels, and accordingly inhibits the activation of synovial fibroblasts represented by invasiveness. This suggests that this may lead to a novel treatment for arthritis, particularly rheumatoid arthritis.
As a result of further studies based on these findings, the present inventors have completed the present invention.
That is, the present invention is as follows.
[1] A prophylactic and / or therapeutic agent for arthritis, which contains an inhibitor of expression of Tet 3 (Ten-Eleven transcription 3).
[2] An expression inhibitor of Tet 3 is
(A) an antisense nucleic acid against a transcript of the Tet 3 gene,
The agent according to [1], which is (b) a ribozyme nucleic acid for a transcription product of the Tet 3 gene, or (c) a nucleic acid having RNAi activity for a transcription product of the Tet 3 gene or a precursor thereof.
[3] The agent according to [1] or [2], wherein the arthritis is rheumatoid arthritis, psoriatic arthritis or spondyloarthritis.
[4] A screening method for a prophylactic and / or therapeutic drug for arthritis, comprising the following steps (1) to (3):
(1) contacting a cell containing a nucleic acid encoding a Tet 3 gene or a reporter protein under the control of a transcriptional regulatory region of the gene with a test substance;
(2) measuring the expression level of the Tet 3 gene, Tet 3 protein or reporter protein in the cell,
(3) A test substance in which the expression level of the Tet 3 gene, Tet 3 protein, or reporter protein is reduced as compared with the case where it is measured in the absence of the test substance is used as a prophylactic and / or therapeutic drug for arthritis. Selecting as a candidate.
[5] Contact with Tet 3 and a test substance, and select a test substance capable of binding to Tet 3 as a prophylactic and / or therapeutic drug for arthritis, and / or prevention of arthritis A screening method for therapeutic drugs.
[6] The method further includes applying a test substance selected as a candidate for a prophylactic and / or therapeutic agent for arthritis to an arthritis model and testing whether or not to suppress an inflammatory reaction in the model. The method described.
[7] A screening method for a prophylactic and / or therapeutic agent for arthritis, comprising the following steps (1) to (3):
(1) a step of bringing synovial fibroblasts into contact with a test substance;
(2) measuring the degree of demethylation or invasiveness of 5-methylcytosine (5 mC) in the genome of the cell,
(3) Compared to the measurement in the absence of the test substance, the test substance that suppresses the demethylation or invasiveness of 5-methylcytosine (5mC) in the genome of the cell is used to prevent arthritis and And / or selecting as a candidate for a therapeutic agent.
[8] The method according to any one of [4] to [7], wherein the arthritis is rheumatoid arthritis, psoriatic arthritis or spondyloarthritis.
[9] A method for examining arthritis, comprising detecting or quantifying a transcription product or translation product of the Tet 3 gene from a sample derived from a subject using the following (a) or (b):
(A) a nucleic acid probe or nucleic acid primer capable of specifically detecting a transcription product of the Tet 3 gene; and (b) an antibody that specifically recognizes a translation product of the Tet 3 gene.
[10] The method according to [9], wherein the arthritis is rheumatoid arthritis, psoriatic arthritis or spondyloarthritis.
[11] The following (a) and / or (b):
(A) a nucleic acid probe or nucleic acid primer capable of specifically detecting a Tet 3 gene transcription product; and (b) an arthritis test kit comprising an antibody that specifically recognizes a translation product of the Tet 3 gene.
[12] The kit according to [11], wherein the arthritis is rheumatoid arthritis, psoriatic arthritis or spondyloarthritis.
[13] A method for preventing and / or treating arthritis, comprising administering to a subject an effective amount of an expression inhibitor of Tet 3 (Ten-Eleven transcription 3).
[14] An expression inhibitor of Tet 3 (Ten-Eleven transcription 3) for use in the prevention and / or treatment of arthritis.
[15] Use of a Tet 3 (Ten-Eleven tranlation 3) expression inhibitor for the manufacture of an agent for preventing and / or treating arthritis.

Since the present invention has revealed that Tet 3 is involved in the pathological deterioration of rheumatoid arthritis, arthritis can be treated or prevented by inhibiting the expression or function of Tet 3. In addition, therapeutic or prophylactic agents for arthritis can be screened using Tet 3 expression or function inhibition as an index. Furthermore, arthritis can be examined using the expression of Tet 3 as an index.

FIG. 1 shows expression of Tet protein family (Tet1,2,3), DNA methylation (5-methylcytosine (5 mC), 5-hydroxymethylcytosine (5 hmC)) in synovial tissue derived from patients with rheumatoid arthritis (RA). It is an immunohistochemical dyeing | staining image which shows.
FIG. 2 shows the expression of Tet protein family (Tet1,2,3), DNA methylation (5-methylcytosine (5 mC), 5-hydroxymethylcytosine (5 hmC) in synovial tissue from osteoarthritis (OA) patients. It is an immunohistochemical staining image showing)).
FIG. 3 shows immunity showing double staining of Tet3 protein (blue) and CD55 protein (brown) and double staining of Tet3 protein (blue) and CD68 protein (brown) in synovial tissue from patients with rheumatoid arthritis (RA). It is a histochemical staining image.
FIG. 4 is a diagram showing the relative mRNA expression levels of the Tet family (Tet1, Tet 2, Tet 3) in cytokine-unstimulated FLS (normal subjects, OA, RA).
FIG. 5 is an immunohistochemically stained image showing the expression of the Tet family (green) (Tet1, Tet 2, Tet 3) and the nucleus (blue) in cytokine-unstimulated FLS (OA, RA).
FIG. 6 is a graph showing temporal changes in the mRNA expression level of the Tet family (Tet 1, Tet 2, Tet 3) in FLS (n = 4 (RA 2, OA 2)) after stimulation with TNFα or IL-1β. is there.
FIG. 7 is a graph showing the change over time in the N / C ratio of Tet3 protein in FLS after stimulation with TNFα.
FIG. 8A is a diagram showing the protein expression level of Tet3 in OA-derived or healthy subject-derived FLS (OA, Nr) after stimulation with TNFα. FIG. 8B shows the protein expression level of Tet 3 in AR-derived FLS after stimulation with TNFα.
FIG. 9A is a diagram showing a test process of Example 4. FIG. 9B is a dot blot image showing 5 hmC levels in AR-derived FLS after stimulation with TNFα. FIG. 9C is a graph showing the 5hmC level in AR-derived FLS.
FIG. 10A is a diagram showing a test process of Example 5. FIG. 10B is a graph showing the secretion level of inflammatory cytokines in AR-derived FLS after knocking down Tet3 and stimulation with TNFα.
FIG. 11A is a diagram showing a test process of Example 6. FIG. 11B is a micrograph showing Scratch assay of AR-derived FLS after knocking down Tet3 and stimulation with TNFα. FIG. 11C is a graph showing the number of infiltrating cells per unit area of AR-derived FLS after knocking down Tet 3, stimulating with TNFα, and 24 hours after scratch.

The present invention is based, at least in part, on the discovery that Tet 3 contributes to exacerbation of rheumatoid arthritis. This finding indicates that Tet 3 can be used not only as a marker for arthritis, particularly rheumatoid arthritis, but also as a drug discovery target for arthritis, particularly rheumatoid arthritis. That is, a known inhibitor of Tet 3 is useful for prevention and / or treatment of arthritis, and a novel Tet 3 inhibitor, and thus prevention / treatment of arthritis, using Tet3 protein and cells / animals expressing it. You can also search for substances that will be drugs.
I. Tet 3 or nucleic acid encoding it
In this specification, Tet 3 is a known protein, and Genbank Accession No. : A protein containing the amino acid sequence of human Tet 3 represented by SEQ ID NO: 2, or the amino acid sequence substantially identical thereto, known as 043151. In the present specification, proteins and peptides are described with the N-terminus (amino terminus) at the left end and the C-terminus (carboxyl terminus) at the right end according to the convention of peptide notation.
As used herein, Tet 3 refers to cells of humans and other warm-blooded animals (eg, mice, rats, cows, monkeys, dogs, pigs, sheep, rabbits, guinea pigs, hamsters, chickens, etc.) [eg, synovial fibroblasts. Cells, synovial surface cells, etc.] or tissues [e.g., synovial membranes (especially, synovial surface cell layers), etc.] and the like may be isolated and purified by known protein separation and purification techniques.
Examples of the “amino acid sequence represented by SEQ ID NO: 2 or an amino acid sequence substantially identical thereto” include the following (a) to (e):
(A) the amino acid sequence represented by SEQ ID NO: 2;
(B) In the amino acid sequence represented by SEQ ID NO: 2, one or more amino acids are deleted, added, inserted or substituted, and demethylating activity of 5-methylcytosine (5 mC) or synovial fibroblasts An amino acid sequence having invasion promoting activity;
(C) an amino acid having 90% or more homology with the amino acid sequence represented by SEQ ID NO: 2 and having a demethylating activity of 5-methylcytosine (5 mC) or an activity of promoting invasion of synovial fibroblasts Sequence;
(D) an amino acid sequence encoded by DNA having the base sequence represented by SEQ ID NO: 1;
(E) Demethylation activity of 5-methylcytosine (5 mC) or synovial fiber encoded by DNA that hybridizes under stringent conditions with DNA having a complementary strand sequence of the base sequence represented by SEQ ID NO: 1 An amino acid sequence having an activity of promoting blast infiltration.
Specifically, the human Tet 3 protein consisting of the amino acid sequence represented by SEQ ID NO: 2 or the amino acid sequence of an ortholog in mammals or the human Tet 3 protein consisting of the amino acid sequence represented by SEQ ID NO: 2 or The amino acid sequence in the splice variant, allelic variant or polymorphic variant of the ortholog may be mentioned.
As used herein, “homology” refers to an optimal alignment when two amino acid sequences are aligned using a mathematical algorithm known in the art (preferably the algorithm uses a sequence of sequences for optimal alignment). The percentage of identical and similar amino acid residues relative to all overlapping amino acid residues in which one or both of the gaps can be considered). “Similar amino acids” mean amino acids that are similar in physicochemical properties, such as aromatic amino acids (Phe, Trp, Tyr), aliphatic amino acids (Ala, Leu, Ile, Val), polar amino acids (Gln, Asn). ), Basic amino acids (Lys, Arg, His), acidic amino acids (Glu, Asp), amino acids having hydroxyl groups (Ser, Thr), amino acids with small side chains (Gly, Ala, Ser, Thr, Met), etc. Examples include amino acids classified into groups. It is expected that substitution with such similar amino acids will not change the phenotype of the protein (ie, is a conservative amino acid substitution). Specific examples of conservative amino acid substitutions are well known in the art and are described in various literature (see, for example, Bowie et al., Science, 247: 1306-1310 (1990)).
The homology of the amino acid sequences in this specification is determined using the homology calculation algorithm NCBI BLAST (National Center for Biotechnology Information Local Alignment Search Tool), and the following conditions (expectation value = 10; allow gap; matrix = BLOSUM62; filtering) = OFF). Other algorithms for determining amino acid sequence homology include, for example, Karlin et al., Proc. Natl. Acad. Sci. USA, 90: 5873-5877 (1993) [the algorithm is incorporated into the NBLAST and XBLAST programs (version 2.0) (Altschul et al., Nucleic Acids Res., 25: 3389-3402 (1997)]. )], Needleman et al., J. MoI. Mol. Biol. 48: 444-453 (1970) [the algorithm is incorporated into the GAP program in the GCG software package], Myers and Miller, CABIOS, 4: 11-17 (1988) [ The algorithm is incorporated into the ALIGN program (version 2.0) which is part of the CGC sequence alignment software package], Pearson et al., Proc. Natl. Acad. Sci. USA, 85: 2444-2448 (1988) [the algorithm is incorporated in the FASTA program in the GCG software package] and the like, and they can be preferably used as well.
The stringent conditions in (e) above are, for example, the conditions described in Current Protocols in Molecular Biology, John Wiley & Sons, 6.3.1-6.3.6, 1999, for example, 6 × SSC ( sodium chloride / sodium citrate) / hybridization at 45 ° C., followed by one or more washings at 0.2 × SSC / 0.1% SDS / 50 to 65 ° C. Hybridization conditions that give the same stringency can be selected as appropriate.
More preferably, as “an amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 2”, the amino acid sequence represented by SEQ ID NO: 2 is about 90% or more, preferably about 95% or more, More preferred is an amino acid sequence having an identity of about 96% or more, more preferably about 97% or more, particularly preferably about 98% or more, and most preferably about 99% or more.
“A protein comprising an amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 2” comprises an amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 2, and SEQ ID NO: 2 is a protein having substantially the same function as the protein consisting of the amino acid sequence represented by 2.
Here, “substantially the same function” means that the properties are qualitatively the same, for example, physiologically or pharmacologically, and the degree of function (eg, about 0.1 to about Quantities such as about 10 times, preferably 0.5 to 2 times) and the molecular weight of the protein may be different. Tet3 has a function of converting 5-methylcytosine (5mC) into 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) or 5-carboxyl cytosine (5CaC) (hereinafter referred to as “5 -Demethylation activity of methylcytosine (5 mC) ”), promotion activity of synovial fibroblast invasion, etc., and a protein having the above activity is regarded as“ a protein having substantially the same function ” be able to.
Here, the demethylation activity of 5-methylcytosine (5 mC) and the promotion activity of infiltration of synovial fibroblasts can be measured, for example, as described in Examples described later.
Examples of the Tet3 protein in the present invention include (i) 1 to 30, preferably 1 to 10, more preferably 1 to the number (5, 4, 3, or 2) in the amino acid sequence represented by SEQ ID NO: 2. ) An amino acid sequence in which one amino acid is deleted, (ii) 1 to 30, preferably 1 to 10, more preferably 1 to a number (5, 4, 3, or 3) in the amino acid sequence represented by SEQ ID NO: 2. 2) an amino acid sequence to which one amino acid is added, (iii) 1 to 30, preferably 1 to 10, more preferably 1 to a number (5, 4, 3, or 3) in the amino acid sequence represented by SEQ ID NO: 2. 2) an amino acid sequence in which one amino acid is inserted, (iv) 1 to 30, preferably 1 to 10, more preferably 1 to a number (5, 4, or 4) in the amino acid sequence represented by SEQ ID NO: 2. 3 or 2) substitution of another amino acid with another amino acid The amino acid sequence was, or (v) is also included, such as protein comprising the amino acid sequence comprising a combination thereof.
When the amino acid sequence is inserted, deleted, added or substituted as described above, the position of the insertion, deletion, addition or substitution is determined by demethylation of 5-methylcytosine (5 mC) or synovial membrane. There is no particular limitation as long as fibroblast infiltration can be promoted.
Here, as a technique for artificially performing amino acid deletion, addition, insertion or substitution, for example, conventional site-directed mutagenesis is applied to DNA encoding the amino acid sequence represented by SEQ ID NO: 2. Thereafter, a technique for expressing this DNA by a conventional method can be mentioned. Here, as a site-specific mutagenesis method, for example, a method utilizing amber mutation (gapped duplex method, Nucleic Acids Res., 12, 9441-9456 (1984)), a PCR method using a mutagenesis primer. Etc.
Preferred examples of Tet 3 include, for example, a human protein consisting of the amino acid sequence represented by SEQ ID NO: 2 (Genbank Accession No. 0431151), or an ortholog, allelic variant, and polymorphic variant thereof in other mammals [eg, Single nucleotide polymorphisms (SNPs)] and the like.
“Nucleic acid encoding Tet 3” is a nucleic acid comprising the amino acid sequence represented by SEQ ID NO: 2 shown in the above (a) to (e) or a base sequence encoding an amino acid sequence substantially identical thereto. Represents. Specifically, the following (f) to (j):
(F) a base sequence encoding the amino acid sequence represented by SEQ ID NO: 2,
(G) In the amino acid sequence represented by SEQ ID NO: 2, one or more amino acids are deleted, added, inserted or substituted, and demethylating activity of 5-methylcytosine (5 mC) or synovial fibroblasts A base sequence encoding an amino acid sequence having an invasion promoting activity,
(H) an amino acid having 90% or more homology with the amino acid sequence represented by SEQ ID NO: 2 and having a demethylating activity of 5-methylcytosine (5 mC) or an activity of promoting invasion of synovial fibroblasts A base sequence encoding the sequence,
(I) the base sequence represented by SEQ ID NO: 1,
(J) a base sequence that hybridizes with a DNA having a complementary strand sequence of the base sequence represented by SEQ ID NO: 1 under stringent conditions, and the demethylation activity or synovium of 5-methylcytosine (5 mC) A base sequence encoding an amino acid sequence having an activity of promoting fibroblast infiltration,
The nucleic acid which has is mentioned.
Here, the gene may be DNA such as cDNA or genomic DNA, or RNA such as mRNA, and is a concept including both a single-stranded nucleic acid sequence and a double-stranded nucleic acid sequence. In this specification, the nucleic acid sequence shown in SEQ ID NO: 1 or the like is a DNA sequence for convenience, but when an RNA sequence such as mRNA is shown, thymine (T) is interpreted as uracil (U). .
Preferred examples of the nucleic acid encoding Tet 3 include, for example, human Tet 3 cDNA (Genbank Accession No. NM_001287491) consisting of the base sequence represented by SEQ ID NO: 1, or its orthologs and allelic variants in other mammals And polymorphic variants [for example, single nucleotide polymorphisms (SNPs)] and the like.
The present invention provides a prophylactic and / or therapeutic agent for arthritis, comprising a substance that inhibits the expression of Tet 3.
II. Substances that inhibit the expression of Tet 3
In the present invention, the “substance that inhibits the expression of Tet3” refers to any transcription level of nucleic acid encoding Tet3 (Tet3 gene), level of post-transcriptional regulation, level of translation into Tet 3 protein, level of post-translational modification, etc. It may act in stages. Therefore, substances that inhibit Tet3 expression include, for example, substances that inhibit Tet3 gene transcription (eg, antigenes), substances that inhibit the processing of early transcription products into mRNA, and mRNA transport to the cytoplasm. Substances that inhibit translation of Tet 3 from mRNA (eg, antisense nucleic acid, miRNA), substances that degrade mRNA (eg, siRNA, ribozyme), and substances that inhibit post-translational modification of the initial translation product It is. Any substance acting at any stage can be preferably used, but more preferably, a substance selected from the group consisting of the following (1) to (3) is exemplified.
(1) an antisense nucleic acid against a transcript of the Tet 3 gene,
(2) a ribozyme nucleic acid for the transcription product of the Tet 3 gene,
(3) A nucleic acid having RNAi activity for a transcription product of the Tet3 gene or a precursor thereof.
Here, a preferable example of the transcription product is mRNA.
As a substance that specifically inhibits the translation of Tet 3 gene from mRNA to Tet 3 (or degrades mRNA), it is preferable that the base sequence is complementary or substantially complementary to the base sequence of these mRNAs or its A nucleic acid containing a part is mentioned.
The base sequence substantially complementary to the base sequence of mRNA of Tet 3 gene is the physiological condition of Tet 3 producing cells (eg, synovial fibroblasts, synovial surface cells) in the mammal to be administered. The base sequence having a degree of complementarity capable of binding to the target sequence of the mRNA and inhibiting its translation (or cleaving the target sequence), specifically, for example, the base sequence of the mRNA About 90% or more, preferably about 95% or more, more preferably about 97% or more of the homologous base sequence (that is, the base sequence of the complementary strand of mRNA) with the overlapping region. It is a base sequence.
The “base sequence homology” in the present invention uses the homology calculation algorithm NCBI BLAST (National Center for Biotechnology Information Basic Alignment Search Tool) and the following conditions (expectation value = 10; allow gap; filtering = ON; Match score = 1; mismatch score = -3).
More specifically, the base sequence complementary or substantially complementary to the base sequence of mRNA of the Tet 3 gene includes the following (k) or (l):
(K) a base sequence complementary or substantially complementary to the base sequence represented by SEQ ID NO: 1;
(L) a base sequence that hybridizes with a complementary strand sequence of the base sequence represented by SEQ ID NO: 1 under stringent conditions, and has a demethylation activity of 5-methylcytosine (5mC) or synovial fiber A base sequence complementary to or substantially complementary to a sequence encoding a protein having an activity of promoting blast infiltration;
Is mentioned.
The stringent conditions are as described above.
Preferred examples of Tet 3 gene mRNA include human Tet 3 mRNA comprising the nucleotide sequence represented by SEQ ID NO: 1 (Genbank Accession No. NM_001287491), or their orthologs in other mammals, Examples include splice variants, allelic variants, and polymorphic variants.
The nucleotide sequence of the Tet 3 gene mRNA and the “part of the complementary or substantially complementary nucleotide sequence” are capable of specifically binding to the Tet 3 gene mRNA and of the protein from the mRNA. The length and position are not particularly limited as long as they can inhibit translation (or degrade the mRNA). However, from the viewpoint of sequence specificity, at least a portion complementary or substantially complementary to the target sequence is required. It contains 10 bases or more, preferably about 15 bases or more.
Specifically, the nucleic acid containing any one of the following (1) to (3) is preferable as a nucleic acid containing a base sequence complementary to or substantially complementary to the base sequence of mRNA of the Tet 3 gene or a part thereof. Illustrated:
(1) an antisense nucleic acid against mRNA of the Tet 3 gene,
(2) a ribozyme nucleic acid against mRNA of the Tet 3 gene,
(3) A nucleic acid having RNAi activity against Tet 3 gene mRNA or a precursor thereof.
(1) Antisense nucleic acid against Tet 3 gene mRNA
The “antisense nucleic acid against Tet3 gene mRNA” in the present invention is a nucleic acid comprising a base sequence complementary to or substantially complementary to the base sequence of the mRNA or a part thereof, which is specific to the target mRNA and It has a function of suppressing protein synthesis by forming a stable double chain and binding.
Antisense nucleic acids are polydeoxyribonucleotides containing 2-deoxy-D-ribose, polyribonucleotides containing D-ribose, other types of polynucleotides that are N-glycosides of purine or pyrimidine bases, Other polymers with non-nucleotide backbones (eg, commercially available protein nucleic acids and synthetic sequence specific nucleic acid polymers) or other polymers containing special linkages, provided that the polymer is a base as found in DNA or RNA And a nucleotide having a configuration that allows attachment of a base). They may be double-stranded DNA, single-stranded DNA, double-stranded RNA, single-stranded RNA, DNA: RNA hybrids, unmodified polynucleotides (or unmodified oligonucleotides), known modifications Additions, such as those with labels known in the art, capped, methylated, one or more natural nucleotides replaced with analogs, intramolecular nucleotide modifications Such as those having uncharged bonds (eg methylphosphonates, phosphotriesters, phosphoramidates, carbamates, etc.), charged bonds or sulfur-containing bonds (eg phosphorothioates, phosphorodithioates, etc.) Things such as proteins (eg, nucleases, nuclease inhibitors, toxins, antibodies, Null peptide, poly-L-lysine, etc.) and sugars (eg, monosaccharides), etc., side chain groups, intercurrent compounds (eg, acridine, psoralen, etc.), chelate compounds (eg, , Metals, radioactive metals, boron, oxidizing metals, etc.), alkylating agents, and modified bonds (eg, alpha anomeric nucleic acids) Also good. Here, the “nucleoside”, “nucleotide” and “nucleic acid” may include not only purine and pyrimidine bases but also those having other modified heterocyclic bases. Such modifications may include methylated purines and pyrimidines, acylated purines and pyrimidines, or other heterocycles. Modified nucleosides and modified nucleotides may also be modified at the sugar moiety, for example, one or more hydroxyl groups are replaced by halogens, aliphatic groups, etc., or functional groups such as ethers, amines, etc. It may be converted.
As described above, the antisense nucleic acid may be DNA or RNA, or may be a DNA / RNA chimera. When the antisense nucleic acid is DNA, the RNA: DNA hybrid formed by the target RNA and the antisense DNA can be recognized by endogenous RNase H and cause selective degradation of the target RNA. Therefore, in the case of antisense DNA directed to degradation by RNase H, the target sequence may be not only the sequence in mRNA but also the sequence of the intron region in the initial translation product of the Tet3 gene. The intron sequence can be determined by comparing the genomic sequence and the cDNA base sequence of the Tet3 gene using a homology search program such as BLAST or FASTA.
The target region of the antisense nucleic acid of the present invention is not particularly limited in length as long as the antisense nucleic acid hybridizes, and as a result, the translation into the protein: Tet 3 is inhibited. May be the entire sequence or a partial sequence of mRNA that encodes, and may include a short sequence of about 10 bases and a long sequence of mRNA or the initial transcript. In view of easiness of synthesis, antigenicity, intracellular migration, etc., an oligonucleotide consisting of about 10 to about 40 bases, particularly about 15 to about 30 bases is preferred, but is not limited thereto. Specifically, 5 ′ end hairpin loop, 5 ′ end 6-base pair repeat, 5 ′ end untranslated region, translation start codon, protein coding region, ORF translation stop codon, 3 ′ end untranslated region of Tet3 gene A 3 ′ end palindromic region or a 3 ′ end hairpin loop or the like may be selected as a preferred target region of an antisense nucleic acid, but is not limited thereto.
Furthermore, the antisense nucleic acid of the present invention not only hybridizes with Tet 3 gene mRNA and initial transcription products to inhibit translation into proteins, but also binds to these genes that are double-stranded DNA to form triplex. It may be a (triplex) -forming (antigene) that can inhibit transcription to RNA.
The nucleotide molecules constituting the antisense nucleic acid 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 a hydrolase such as nuclease, the phosphate residue (phosphate) of each nucleotide constituting the antisense nucleic acid is chemically modified, for example, phosphorothioate (PS), methylphosphonate, phosphorodithionate, etc. It can be substituted with a phosphate residue. In addition, the 2′-position hydroxyl group of the sugar (ribose) of each nucleotide is changed to —OR (R═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, etc.) 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, etc. 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, in order to give strong binding ability to the target RNA, BNA (LNA) (Imanishi), which is an RNA derivative in which the conformation of the sugar moiety is fixed to the N-type by cross-linking 2 ′ oxygen and 4 ′ carbon , 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 Nucleic Acids, 22, 1619-21, 2003) can also be preferably used.
The antisense oligonucleotide of the present invention determines the target sequence of mRNA or initial transcript based on the cDNA sequence or genomic DNA sequence of the Tet 3 gene, and is a commercially available DNA / RNA automatic synthesizer (Applied Biosystems, Beckman). Etc.) can be prepared by synthesizing a complementary sequence thereto. In addition, any of the above-described antisense nucleic acids containing various modifications can be chemically synthesized by a method known per se.
(2) Ribozyme nucleic acid against Tet 3 gene mRNA
As another preferred example of a nucleic acid comprising a base sequence complementary to or substantially complementary to the base sequence of mRNA of the Tet 3 gene or a part thereof, a ribozyme capable of specifically cleaving the mRNA within the coding region A nucleic acid is mentioned. “Ribozyme” refers to RNA having an enzyme activity that cleaves nucleic acids in a narrow sense, but in this specification, it is used as a concept including DNA as long as it has sequence-specific nucleic acid cleaving activity. The most versatile ribozyme nucleic acid is self-splicing RNA found in infectious RNA such as viroid and virusoid, and hammerhead type and hairpin type are known. The hammerhead type exhibits enzyme activity at about 40 bases, and a few bases at both ends adjacent to the part having the hammerhead structure (about 10 bases in total) are made into a sequence complementary to the desired cleavage site of mRNA. By doing so, it is possible to specifically cleave only the target mRNA. This type of ribozyme nucleic acid has the additional advantage of not attacking genomic DNA because it uses only RNA as a substrate. When the Tet 3 gene mRNA has a double-stranded structure by itself, the target sequence is made single-stranded by using a hybrid ribozyme linked with an RNA motif derived from a viral nucleic acid that can specifically bind to an RNA helicase. [Proc. Natl. Acad. Sci. USA, 98 (10): 5572-5577 (2001)]. Furthermore, when the ribozyme is used in the form of an expression vector containing the DNA encoding the ribozyme, in order to promote the transfer of the transcription product to the cytoplasm, the ribozyme should be a hybrid ribozyme further linked with a tRNA-modified sequence. [Nucleic Acids Res. 29 (13): 2780-2788 (2001)].
(3) siRNA against Tet 3 gene mRNA
In the present specification, a double-stranded RNA composed of an oligo RNA complementary to Tet 3 gene mRNA and its complementary strand, so-called siRNA, is also complementary or substantially complementary to the base sequence of Tet 3 gene mRNA. Defined as being encompassed by a nucleic acid comprising a basic nucleotide sequence or a portion thereof. When a short double-stranded RNA is introduced into a cell, a phenomenon called RNA interference (RNAi), in which mRNA complementary to the RNA is degraded, has been known in nematodes, insects, plants, etc. Since it has been confirmed that this phenomenon occurs widely in animal cells [Nature, 411 (6836): 494-498 (2001)], it has been widely used as an alternative technique to the above-described antisense nucleic acids and ribozymes.
Based on the cDNA sequence information of the target gene, siRNA can be obtained, for example, from Elbashir et al. (Genes Dev., 15, 188-200 (2001)), Teramoto et al. (FEBS Lett. 579 (13): p2878-82 (2005)). Can be designed according to the rules proposed by The target sequence of siRNA has a length of 15 to 50 bases, preferably 19 to 49 bases, more preferably 19 to 27 bases in principle. For example, AA + (N) 19 (following AA, 19 Base sequence), AA + (N) 21 (base sequence of 21 bases following AA) or A + (N) 21 (base sequence of 21 bases following A).
The nucleic acid of the present invention may have an additional base at the 5 ′ or 3 ′ end. The length of the additional base is usually about 2 to 4 bases, and the total length of siRNA is 19 bases or more. The additional base may be DNA or RNA, but the use of DNA may improve the stability of the nucleic acid. Examples of such an additional base sequence include ug-3 ′, uu-3 ′, tg-3 ′, tt-3 ′, ggg-3 ′, guuu-3 ′, gttt-3 ′, and tttt-3. Examples of such sequences include, but are not limited to, ', uuuu-3'.
Moreover, siRNA may have an overhang | projection part sequence (overhang) in 3 'terminal, and specifically, what added dTdT (dT represents the deoxythymidine residue of deoxyribonucleic acid) is mentioned. . Further, it may be a blunt end (blunt end) without end addition.
The siRNA may have a different number of bases in the sense strand and the antisense strand, for example, “aiRNA” in which the antisense strand has a protruding portion sequence (overhang) at the 3 ′ end and the 5 ′ end. Can be mentioned. A typical aiRNA has an antisense strand consisting of 21 bases, a sense strand consisting of 15 bases, and has an overhang structure of 3 bases at each end of the antisense strand.
(Sun, X. et al., Nature Biotechnology Vol. 26 No. 12 p1379, International Publication No. WO2009 / 029688 pamphlet).
The position of the target sequence is not particularly limited, but it is desirable to select the target sequence from the 5′-UTR and the start codon to about 50 bases and from a region other than the 3′-UTR. For a candidate group of target sequences selected based on the above-mentioned rules and others, whether or not there is a homology in a 16-17 base sequence in mRNA other than the target is determined by BLAST (http: //www.ncbi.nlm Investigate using homology search software such as .nih.gov / BLAST /) to confirm the specificity of the selected target sequence. For the target sequence confirmed to be specific, a sense strand having a 3 'end overhang of TT or UU at 19-21 bases after AA (or NA), a sequence complementary to the 19-21 bases and TT or A double-stranded RNA consisting of an antisense strand having a 3 ′ terminal overhang of UU may be designed as siRNA. In addition, for the short hairpin RNA (shRNA) that is a precursor of siRNA, an arbitrary linker sequence (for example, about 5-25 bases) capable of forming a loop structure is appropriately selected, and the sense strand and the antisense strand are combined with each other. It can be designed by linking via a linker sequence.
The sequence of siRNA and / or shRNA can be searched using search software provided free of charge on various web sites. Such sites include, for example, siRNA Target Finders provided by Ambion
(Http://www.ambion.com/jp/techlib/misc/siRNA_finder.html) and pSilencer® Expression Vector insert design tool
(Http://www.ambion.com/jp/techlib/misc/psilencer_converter.html), GeneSee provided by RNAi Codex
(Http://codex.cshl.edu/scripts/newsearchhairpin.cgi), but is not limited thereto.
The ribonucleoside molecule constituting the siRNA may also be modified in the same manner as in the above-described antisense nucleic acid in order to improve stability, specific activity and the like. However, in the case of siRNA, if all ribonucleoside molecules in natural RNA are replaced with a modified form, RNAi activity may be lost, and therefore it is necessary to introduce a minimum modified nucleoside that allows the RISC complex to function. .
Specifically, in order to improve a part of the nucleotide molecule constituting siRNA, natural DNA, stability (chemical and / or enzyme) and specific activity (affinity with RNA) as the modification. Can be substituted with various chemically modified RNAs (see Usman and Cedergren, 1992, TIBS 17, 34; Usman et al., 1994, Nucleic Acids Sym. Ser. 31, 163). For example, in order to prevent degradation by a hydrolase such as nuclease, a phosphate residue (phosphate) of each nucleotide constituting siRNA is changed to a chemically modified phosphate such as phosphorothioate (PS), methylphosphonate, phosphorodithionate, etc. It can be substituted with a residue. In addition, the 2′-position hydroxyl group of the sugar (ribose) of each nucleotide is changed to —OR (R═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, etc.) and a fluorine atom (-F) 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, etc. Is mentioned. In addition, the method for modifying an antisense nucleic acid described in (1) above can be used. Or you may give the chemical modification (2'-deoxylation, 2'-H) which substitutes a part of RNA in siRNA with DNA. In addition, the 2′-position and 4′-position of sugar (ribose) are —O—CH ₂ An artificial nucleic acid (LNA: Locked Nucleic Acid) in which the conformation is fixed to N-type by crosslinking with − may be used.
In addition, the sense strand and antisense strand constituting siRNA are linked via a linker to a ligand, peptide, sugar chain, antibody, lipid, positive charge, or molecular structure that specifically recognizes a receptor present on the cell surface. It may be chemically bonded to oligoarginine, Tat peptide, Rev peptide, Ant peptide or the like that adsorbs and penetrates the surface layer.
The siRNA is synthesized by synthesizing the sense strand and antisense strand of the target sequence on the mRNA with a DNA / RNA automatic synthesizer and denatured in an appropriate annealing buffer at about 90 to about 95 ° C. for about 1 minute, It can be prepared by annealing at about 30 to about 70 ° C. for about 1 to about 8 hours. It can also be prepared by synthesizing a short hairpin RNA (shRNA) serving as a precursor of siRNA and cleaving it with a dicer.
In the present specification, a nucleic acid designed to generate siRNA against Tet3 gene mRNA in vivo also includes a nucleotide sequence complementary or substantially complementary to the nucleotide sequence of Tet3 gene mRNA or a nucleotide sequence thereof. Defined as encompassed by a portion of nucleic acid. Examples of such a nucleic acid include an expression vector constructed so as to express the above-mentioned shRNA and siRNA. The shRNA is an oligo comprising a base sequence in which a sense sequence and an antisense strand of a target sequence on mRNA are inserted by interposing a spacer sequence having a length (for example, about 5 to 25 bases) capable of forming an appropriate loop structure. It can be prepared by designing RNA and synthesizing it with an automatic DNA / RNA synthesizer. There are tandem type and stem loop (hairpin) type in vectors expressing shRNA. In the former, siRNA sense strand expression cassette and antisense strand expression cassette are connected in tandem, and each strand is expressed and annealed in the cell to form double stranded siRNA (dsRNA). It is. On the other hand, the latter is one in which an shRNA expression cassette is inserted into a vector, in which shRNA is expressed in cells and processed by dicer to form dsRNA. As a promoter, a pol II promoter (for example, a CMV immediate early promoter) can be used, but a pol III promoter is generally used in order to cause transcription of a short RNA accurately. Examples of the polIII promoter include mouse and human U6-snRNA promoter, human H1-RNase P RNA promoter, human valine-tRNA promoter, and the like. Further, a sequence in which 4 or more Ts are continuous is used as a transcription termination signal.
The siRNA or shRNA expression cassette thus constructed is then inserted into a plasmid vector or viral vector. As such vectors, virus vectors such as retrovirus, lentivirus, adenovirus, adeno-associated virus, herpes virus, Sendai virus, animal cell expression plasmids and the like are used.
The siRNA is based on nucleotide sequence information, for example, 394 Applied Biosystems, Inc. It can be chemically synthesized according to a conventional method using an automatic DNA / RNA synthesizer such as a synthesizer. For example, Caruthers et al. , 1992, Methods in Enzymology 211, 3-19, Thompson et al. , International Publication No. 99/54459, Wincott et al. , 1995, Nucleic Acids Res. 23, 2677-2684, Wincott et al. , 1997, Methods Mol. Bio. 74, 59, Brennan et al. , 1998, Biotechnol Bioeng. 61, 33-45, Usman et al. , 1987
J. et al. Am. Chem. Soc. 109, 7845; Scaringe et al. 1990 Nucleic Acids Res. , 18, 5433, and the method described in US Pat. No. 6,00131111. Specifically, it can be synthesized using a nucleic acid protecting group (for example, dimethoxytrityl group at the 5 ′ end) and a coupling group (for example, phosphoramidite at the 3 ′ end) known to those skilled in the art. That is, the protecting group at the 5 ′ end is deprotected with an acid such as TCA (trichloroacetic acid) and a coupling reaction is performed. Then, after capping with an acetyl group, the next nucleic acid condensation reaction is performed. In the case of siRNA containing modified RNA or DNA, a modified RNA (for example, 2′-O-methyl nucleotide, 2′-deoxy-2′-fluoronucleotide) may be used as a raw material, and the coupling reaction These conditions can be adjusted as appropriate. When introducing a phosphorothioate bond in which the phosphate binding moiety is modified, a borage reagent (3H-1,2-benzodithiol-3-one 1,1-dioxide) can be used.
Alternatively, oligonucleotides may be synthesized separately and joined together after synthesis, for example by ligation (Moore et al., 1992, Science 256, 9923; Draper et al. International Publication WO 93/23569; Shabarova et al. , 1991, Nucleic Acids Research 19, 4247; Bellon et al., 1997, Nucleosides & Nucleotides, 16, 951: Bellon et al., 1997, Nucleosides & Nucleotides, j. Et al. And / or connected together by hybridization after deprotection It may be. siRNA molecules can also be synthesized by tandem synthesis. That is, both siRNA strands are synthesized as a single continuous oligonucleotide separated by a cleavable linker, which is then cleaved to generate separate siRNA fragments that are hybridized and purified. . The linker may be a polynucleotide linker or a non-nucleotide linker.
The synthesized siRNA molecules can be purified using methods known to those skilled in the art. For example, a method of purification by gel electrophoresis or a method of purification using high performance liquid chromatography (HPLC) can be mentioned.
In the present invention, the oligo RNA complementary to the Tet 3 gene mRNA that constitutes the siRNA against the Tet 3 gene and its complementary strand include a sequence comprising SEQ ID NO: 3 and a sequence comprising SEQ ID NO: 4, respectively. Preferably, the sequence consisting of SEQ ID NO: 3 and the sequence consisting of SEQ ID NO: 4 are exemplified.
Another preferred example of a nucleic acid containing a base sequence complementary to or substantially complementary to the base sequence of mRNA of the Tet 3 gene or a part thereof is micro RNA (miRNA) targeting the mRNA. Examples of human miRNA targeting human Tet 3 mRNA include hsa-miR-22, hsa-miR-26a, hsa-miR-301a, hsa-miR-301b, hsa-miR-372, hsa-miR-29c. Hsa-miR-29a, hsa-miR-29b, hsa-miR-374b, hsa-miR-374a, and the like. miRNA can also be prepared according to the method described for siRNA.
A nucleic acid containing a nucleotide sequence complementary to or substantially complementary to the nucleotide sequence of Tet 3 gene mRNA or a part thereof is provided in a special form such as a liposome or a microsphere, or other molecules are added. Can be provided in different forms. Examples of additional forms that can be used include polycationic substances such as polylysine that act to neutralize the charge of the phosphate group skeleton, and lipids that enhance interaction with cell membranes and increase nucleic acid uptake. Hydrophobic substances such as (eg, phospholipid, cholesterol, etc.) can be mentioned. Preferred lipids for addition include cholesterol and derivatives thereof (eg, cholesteryl chloroformate, cholic acid, etc.). Such can be attached to the 3 'or 5' end of the nucleic acid and can be attached via a base, sugar, intramolecular nucleoside bond. Examples of the other group include a cap group specifically arranged at the 3 ′ end or 5 ′ end of a nucleic acid, which prevents degradation by a nuclease such as exonuclease or RNase. Such capping groups include, but are not limited to, hydroxyl protecting groups known in the art, including glycols such as polyethylene glycol and tetraethylene glycol.
Tet 3 expression inhibitory activity of these nucleic acids can be determined by transforming a nucleic acid encoding Tet 3 into a Tet 3 gene expression system in vivo or in vitro, or a translation system for Tet 3 protein in vivo or in vitro. Can be used to investigate.
The substance that inhibits the expression of Tet3 in the present invention is not limited to a nucleic acid containing a base sequence complementary to or substantially complementary to the base sequence of mRNA of Tet3 gene as described above, or a part thereof. Other substances such as small molecule compounds may be used as long as production is directly or indirectly inhibited. Such a substance can be obtained, for example, by the screening method of the present invention described later.
A substance that inhibits the expression of Tet 3 exhibits the activity of suppressing the demethylation activity of 5-methylcytosine (5mC) possessed by Tet 3 or the activity of promoting the invasion of synovial fibroblasts. Useful for treatment.
Therefore, a medicament containing a substance that inhibits the expression of Tet 3 can be used as an agent for preventing and / or treating arthritis.
III. Medicament containing antisense nucleic acid, ribozyme nucleic acid, siRNA and precursor thereof
An antisense nucleic acid of the present invention that can complementarily bind to a transcription product of the Tet 3 gene and suppress protein translation from the transcription product, or a homology (or complementary) to the transcription product (mRNA) of the Tet 3 gene. SiRNA (or ribozyme) having a nucleotide sequence and capable of cleaving the transcription product as a target, and shRNA that is a precursor of the siRNA (hereinafter, comprehensively referred to as “the nucleic acid of the present invention”) May suppress the expression of Tet3 in vivo and suppress the activity of promoting the demethylation of 5-methylcytosine (5mC) or the infiltration of synovial fibroblasts, and thus preventing arthritis and / or It can be used as a therapeutic agent.
The medicament containing the nucleic acid 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, rat, rabbit, sheep, pig, cow, cat, It can be administered orally or parenterally (eg, intravascular administration, subcutaneous administration, etc.) to dogs, monkeys, etc.
When the nucleic acid of the present invention is used as a prophylactic and / or therapeutic agent for the arthritis, it can be formulated and administered according to a method known per se. That is, the nucleic acid of the present invention is inserted alone or in a functional manner into an appropriate expression vector for mammalian cells such as a retrovirus vector, adenovirus vector, adenovirus associated virus vector, etc., and then formulated according to conventional means. can do. The nucleic acid can be administered as it is or together with an auxiliary agent for promoting intake by a catheter such as a gene gun or a hydrogel catheter. Alternatively, it can be aerosolized and locally administered into the trachea as an inhalant.
Furthermore, for the purpose of improving the pharmacokinetics, prolonging the half-life, and improving the efficiency of cellular uptake, the nucleic acid may be formulated (injection) alone or with a carrier such as liposome and administered intravenously, subcutaneously, etc. .
The nucleic acids of the invention may be administered per se or as a suitable pharmaceutical composition. The pharmaceutical composition used for administration may contain the nucleic acid of the present invention and a pharmacologically acceptable carrier, diluent or excipient. Such pharmaceutical compositions are provided as dosage forms suitable for oral or parenteral administration.
As a composition for parenteral administration, for example, injections, suppositories and the like are used. Injections are dosage forms such as intravenous injections, subcutaneous injections, intradermal injections, intramuscular injections, infusions, and the like. 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 the nucleic acid of the present invention in a sterile aqueous liquid or oily liquid 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) additive of hydrogenated castor oil)), and the like. 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. 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 dragees and film-coated tablets), pills, granules, powders, capsules (including soft capsules), 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 compositions are conveniently prepared in dosage unit form to suit the dosage of the active ingredient. Examples of the dosage form of such a dosage unit include tablets, pills, capsules, injections (ampoules), and suppositories. The nucleic acid of the present invention is preferably contained, for example, usually 5 to 500 mg per dosage unit form, especially 5 to 100 mg for injections and 10 to 250 mg for other dosage forms.
The dose of the above-mentioned medicament containing the nucleic acid of the present invention varies depending on the administration subject, target disease, symptom, administration route, etc., but for example, when used for the treatment / prevention of rheumatoid arthritis, The amount of nucleic acid is usually about 0.01 to 20 mg / kg body weight, preferably about 0.1 to 10 mg / kg body weight, more preferably about 0.1 to 5 mg / kg body weight 1 to 5 times a day. It is convenient to administer by intravenous injection to a degree, preferably about 1 to 3 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.
Each of the above-described compositions may appropriately contain other active ingredients as long as an undesirable interaction is not caused by blending with the nucleic acid of the present invention.
The pharmaceutical composition containing the above-mentioned antisense nucleic acid, ribozyme nucleic acid, siRNA and its precursor to Tet 3, or a low-molecular compound that suppresses the expression of Tet 3, etc. is used for the treatment, prevention, or progression of arthritis. Can be used for prevention. Specific examples of arthritis include rheumatoid arthritis, psoriatic arthritis, spondyloarthritis (eg, ankylosing spondylitis), and preferably rheumatoid arthritis. In addition, any disease in which Tet 3 is involved in the deterioration of the disease state is included in the target disease of the present invention.
When the pharmaceutical composition containing the above-mentioned antisense nucleic acid, ribozyme nucleic acid, siRNA and its precursor against Tet 3 or a low molecular weight compound that suppresses the expression of Tet 3 is used for the treatment or prevention of arthritis May be used alone or in combination with one or more drugs having anti-inflammatory activity.
The drug used in combination is not particularly limited. For example, mesalazine, corticosteroids (eg, betamethasone, prednisolone, hydrocortisone, dexamethasone, etc.), nonsteroidal anti-inflammatory drugs (NSAIDs; eg, salicylic acid, anthranilic acid) , Aryl acids, propionates, oxicams, pilins), anti-TNFα antibodies (influximab, adalimumab), anti-rheumatic drugs (eg, immunomodulators such as actarit, immunosuppressants such as methotrexate, anti-TNFα antibodies And biological preparations such as etanercept).
IV. Screening drug candidate compounds for disease
As mentioned above, inhibition of Tet 3 expression and / or function suppresses the demethylation of 5-methylcytosine (5mC), which is the most important aggressive phenotype of activated synovial fibroblasts One invasive ability is suppressed. This indicates that selective inhibition of Tet3 inhibits the expression of CCL2 and ICAM1 through the maintenance of DNA methylation levels, and accordingly, inhibits the activation of synovial fibroblasts represented by invasiveness. Means that it leads to treatment of arthritis. Therefore, a compound that inhibits the expression and / or function of Tet 3 can be used as a prophylactic and / or therapeutic agent for arthritis.
Therefore, cells that produce Tet 3 can be used as a tool for screening for preventive and / or therapeutic drugs for arthritis by using the expression level and / or function of Tet 3 (or Tet 3 gene) as an index. it can.
When screening for a compound that inhibits the expression or function of Tet 3, the screening method involves culturing cells capable of producing Tet 3 in the presence and absence of a test substance, 3 comparing the expression level or degree of function. A compound that inhibits the function of Tet 3 can also be screened by testing its ability to bind to purified Tet 3 protein.
Cells having the ability to produce Tet 3 used in the above screening methods include human or other mammalian cells (eg, synovial fibroblasts, synovial surface cells, etc.) that naturally express them, or those If it is a biological sample containing (for example: synovium (especially synovial surface cell layer) etc.) etc., there will be no restriction | limiting. In the case of synovial membranes derived from non-human animals, they may be isolated from the living body and cultured, or the test substance may be administered to the living body itself, and the biological sample may be isolated after a certain period of time. .
Moreover, various transformants prepared by known and commonly used genetic engineering techniques can also be used as the cells having the ability to produce Tet 3. As the host, for example, animal cells such as H4IIE-C3 cells, HepG2 cells, HEK293 cells, COS7 cells and CHO cells are preferably used.
Specifically, it hybridizes under stringent conditions with a DNA encoding Tet3 (that is, the base sequence represented by SEQ ID NO: 1 or a base sequence complementary to the base sequence, and SEQ ID NO: A DNA comprising a base sequence encoding a polypeptide having the same function as that of the protein consisting of the amino acid sequence represented by 2) is introduced downstream into a promoter in an appropriate expression vector and introduced into a host animal cell. Can be prepared.
A method for preparing a gene encoding Tet 3 will be described below.
The gene encoding Tet 3 can be obtained by a conventional genetic engineering method (for example, Sambrook J., Frisch EF, Maniatis T., Molecular Cloning 2nd edition), published by Cold Spring Harbor Laboratory ( The method can be obtained according to the method described in Cold Spring Harbor Laboratory Press). That is, DNA encoding Tet 3 is derived from, for example, a cell or tissue that produces Tet 3 described above by synthesizing an appropriate oligonucleotide as a probe or primer based on the nucleotide sequence represented by SEQ ID NO: 1. Cloning can be performed from cDNA or cDNA library using hybridization method or PCR method. Hybridization can be performed, for example, according to the method described in Molecular Cloning 2nd edition (above). When a commercially available library is used, hybridization can be performed according to the method described in the instruction manual attached to the library.
The nucleotide sequence of DNA can be determined using a known kit such as Mutan. ^TM -Super Express Km (Takara Shuzo Co., Ltd.), Mutan ^TM -K (Takara Shuzo Co., Ltd.) and the like can be converted according to a method known per se such as the ODA-LA PCR method, the Gapped duplex method, the Kunkel method, or the like.
The cloned DNA can be used as it is or after digestion with a restriction enzyme or addition of a linker, if desired. The DNA may have ATG as a translation initiation codon on the 5 ′ end side, and may have TAA, TGA, or TAG as a translation termination codon on the 3 ′ end side. These translation initiation codon and translation termination codon can be added using an appropriate synthetic DNA adapter.
Next, using the obtained Tet3 gene, cells expressing the Tet3 protein can be produced and obtained according to a normal genetic engineering method.
For example, a plasmid is prepared so that the Tet 3 gene can be expressed in the host cell, this is introduced into the host cell, transformed, and the transformed host cell (transformant) is cultured. What is necessary is just to acquire the cell which expresses protein. Examples of the plasmid include a promoter that can replicate autonomously in a host cell, can replicate autonomously, can be easily isolated and purified from the host cell, and can function in the host cell. Preferred examples include those in which a gene encoding Tet 3 is introduced into an expression vector having a detectable marker. Various types of expression vectors are commercially available.
For example, an expression vector used for expression in E. coli is an expression vector containing a promoter such as lac, trp, tac, etc., and these are commercially available from Pharmacia, Takara Bio and the like. Restriction enzymes used for introducing a gene encoding Tet 3 into the expression vector are also commercially available from Takara Bio and others. If it is necessary to direct further expression, a ribosome binding region may be linked upstream of the DNA encoding Tet3. Examples of the ribosome binding region used include Guarente L. et al. (Cell 20, p543) and Taniguchi et al. (Genetics of Industrial Microorganisms, p202, Kodansha).
In addition, animal cell expression plasmids (eg, pA1-11, pXT1, pRc / CMV, pRc / RSV, pcDNAI / Neo); bacteriophages such as λ phage; animal virus vectors such as retrovirus, vaccinia virus, adenovirus, etc. It can also be used. The promoter may be any promoter as long as it is appropriate for the host used for gene expression. For example, SRα promoter, SV40 promoter, LTR promoter, CMV (cytomegalovirus) promoter, RSV (rous sarcoma virus) promoter, MoMuLV (Moloney murine leukemia virus) LTR, HSV-TK (herpes simplex virus thymidine kinase) promoter, β actin A gene promoter, aP2 gene promoter, etc. are used. Of these, EF-α promoter, CAG promoter, CMV promoter, SRα promoter and the like are preferable.
In addition to the above, an expression vector containing an enhancer, a splicing signal, a poly A addition signal, a selection marker, an SV40 origin of replication (hereinafter sometimes abbreviated as SV40ori) and the like is used as desired. it can.
Examples of the selectable marker include a dihydrofolate reductase gene (hereinafter abbreviated as dhfr, methotrexate (MTX) resistance), an ampicillin resistance gene (hereinafter amp). ^r A neomycin resistance gene (hereinafter, neo) ^r G418 resistance) and the like. In particular, when dhfr gene-deficient Chinese hamster cells are used and the dhfr gene is used as a selection marker, the target gene can be selected using a medium not containing thymidine.
A Tet 3 expressing cell can be produced by transforming a host with an expression vector containing the DNA encoding Tet 3 described above.
Examples of host cells include prokaryotic or eukaryotic microbial cells, insect cells, or mammalian cells. Examples of mammalian cells include HepG2 cells, HEK293 cells, HeLa cells, human FL cells, monkey COS-7 cells, monkey Vero cells, Chinese hamster ovary cells (hereinafter abbreviated as CHO cells), dhfr gene-deficient CHO cells ( Hereinafter, CHO (dhfr ⁻ Abbreviated as cells), mouse L cells, mouse AtT-20 cells, mouse myeloma cells, rat H4IIE-C3 cells, rat GH3 cells, and the like.
The plasmid obtained as described above can be introduced into the host cell by an ordinary genetic engineering method. The transformant can be cultured by a conventional method used for culturing microorganisms, insect cells or mammalian cells. For example, in the case of Escherichia coli, culturing is performed in a medium appropriately containing an appropriate carbon source, nitrogen source, and micronutrients such as vitamins. The culture method may be any of solid culture and liquid culture, and preferred examples include liquid culture such as aeration and agitation culture.
Transformation can be performed by calcium phosphate coprecipitation method, PEG method, electroporation method, microinjection method, lipofection method and the like. For example, the method described in Cell Engineering Supplement 8 New Cell Engineering Experiment Protocol, 263-267 (1995) (published by Shujunsha), Virology, Volume 52, 456 (1973) can be used.
A transformed cell obtained as described above, a mammalian cell having an ability to produce Tet 3 or a tissue / organ containing the cell is, for example, a minimum essential medium containing about 5 to 20% fetal calf serum ( (MEM) [Science, 122, 501 (1952)], Dulbecco's modified Eagle medium (DMEM) [Virology, 8, 396 (1959)], RPMI 1640 medium [The Journal of the American Medical Association, 199 (199) 1967)] and 199 medium [Proceeding of the Society for the Biological Medicine, Vol. 73, 1 (1950)]. The pH of the medium is preferably about 6-8. Culture is usually performed at about 30 to 40 ° C., and aeration and agitation are added as necessary.
The Tet 3 protein may be obtained by combining methods commonly used for general protein isolation / purification. For example, the transformant obtained by the above culture may be removed by centrifugation or the like, and Tet3 may be purified from the culture supernatant in the same manner as described above. Further, when the Tet 3 protein accumulates in the cells of the transformant obtained by the above culture, for example, the transformant is collected by centrifugation or the like, and then the cells are disrupted or lysed. If there is, the protein may be solubilized and purified by singly or in combination with various chromatographic processes such as ion exchange, hydrophobicity, and gel filtration. An operation of restoring the higher order structure of the purified protein may be further performed.
In conducting the screening of the present invention, examples of the test substance include proteins, peptides, non-peptide compounds, synthetic compounds, fermentation products, cell extracts, plant extracts, animal tissue extracts, and the like. These substances may be novel or may be known ones.
In addition, when selecting a substance that decreases the expression level of Tet 3 or the Tet 3 gene or a substance that decreases the function of Tet 3, a control cell that is not brought into contact with the test substance can also be used as a comparative control. Here, “do not contact the test substance” means that the same amount of solvent (blank) as the test substance is added instead of the test substance, the expression level of Tet 3 or Tet 3 gene, or the function of Tet 3 The case where a negative control substance that does not affect the above is added is also included.
The contact of the test substance with the cells is, for example, the above-mentioned medium or various buffers (for example, HEPES buffer, phosphate buffer, phosphate buffered saline, Tris-HCl buffer, borate buffer, acetic acid). The test substance can be added to a buffer solution or the like, and the cells can be incubated for a certain 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 about 0.1 nM to about 100 μM, for example. Examples of the incubation time include about 10 minutes to about 24 hours.
When cells producing Tet 3 are provided in the form of a non-human mammal individual, the state of the animal individual is not particularly limited. For example, an arthritis model animal in which arthritis is induced by a drug or genetic modification (for example, collagen RA model animals such as induced arthritis (CIA) mice, SKG mice, PD-1 knockout mice, K / BxN mice, Synoviolin Tg mice, etc.). There are no particular restrictions on the breeding conditions of the animals used, but it is preferable that the animals are raised in an environment of SPF grade or higher. Contact of the test substance with the cells is carried out by administering the test substance to the animal individual. The administration route is not particularly limited, and examples thereof include intravenous administration, intraarterial administration, subcutaneous administration, intradermal administration, intraperitoneal administration, oral administration, intratracheal administration, and rectal administration. The dose is not particularly limited. For example, a dose of about 0.5 to 20 mg / kg can be administered 1 to 5 times a day, preferably 1 to 3 times a day for 1 to 14 days. .
Alternatively, the above screening method may be performed by contacting a test substance with an extract of the cells or Tet 3 isolated and purified from the cells, instead of the cells having the ability to produce Tet 3. it can.
(Measurement of expression level of Tet 3 gene or Tet 3)
The present invention relates to a prophylactic and / or therapeutic agent for arthritis, characterized by comparing the expression of the protein (gene) in cells having the ability to produce Tet 3 in the presence and absence of a test substance. A screening method is provided. The cells used in this method, the type of test substance, the mode of contact between the test substance and cells, etc. are the same as described above.
The expression level of Tet 3 is a nucleic acid that can hybridize with the above-described DNA encoding Tet 3 under stringent conditions, that is, the base sequence represented by SEQ ID NO: 1 or a complementary base sequence and stringent. Can be measured at the RNA level by detecting mRNA of the Tet 3 gene using a nucleic acid (DNA) that can hybridize under various conditions (hereinafter sometimes referred to as “the nucleic acid for detection of the present invention”). it can. Alternatively, the expression level can also be measured at the protein level by detecting these proteins using the above-described antibody against Tet 3 (hereinafter sometimes referred to as “the detection antibody of the present invention”). .
Therefore, more specifically, the present invention
(A) Cells having the ability to produce Tet 3 are cultured in the presence and absence of a test substance, and the amount of mRNA encoding the protein under both conditions is determined using the nucleic acid for detection of the present invention. A method for screening a prophylactic and / or therapeutic agent for arthritis, characterized by measuring and comparing; and
(B) Cells having the ability to produce Tet 3 are cultured in the presence and absence of a test substance, and the amount of the protein under both conditions is measured and compared using the detection antibody of the present invention. A screening method for a prophylactic and / or therapeutic agent for arthritis is provided.
That is, screening for a substance that changes the expression level of Tet 3 can be performed as follows.
(I) administering a test substance to a normal or disease model (eg, RA model animal) non-human mammal (eg, mouse, rat, rabbit, sheep, pig, cow, cat, dog, monkey, etc.) After a certain period of time (after 30 minutes to 3 days, preferably 1 hour to 2 days, more preferably 1 hour to 24 hours), blood or a specific organ (for example, synovial membrane), Alternatively, tissue or cells isolated from the organ are obtained.
Tet 3 mRNA can be quantified by extracting mRNA from cells or the like by a conventional method, or can be quantified by Northern blot analysis known per se. On the other hand, the amount of Tet 3 protein can be quantified using Western blot analysis or various immunoassay methods described in detail below.
(Ii) A test substance when a cell expressing Tet 3 gene (for example, a synovial fibroblast, a synovial surface cell, or a transformant introduced with Tet3) is prepared according to the above method and cultured according to a conventional method Is added to the medium or buffer, and after incubation for a certain period of time (from 1 day to 7 days, preferably from 1 day to 3 days, more preferably from 2 days to 3 days), Tet3 contained in the cells or the code thereof is encoded. MRNA can be quantified and analyzed in the same manner as (i) above.
Detection and quantification of the expression level of the Tet3 gene (mRNA) can be performed by a known method such as Northern blotting or RT-PCR using RNA prepared from the cells or a complementary polynucleotide transcribed therefrom. Specifically, by using a polynucleotide having at least 15 bases continuous in the base sequence of the Tet 3 gene and / or a complementary polynucleotide thereof as a primer or a probe, the presence or absence of expression of the Tet 3 gene in RNA, Its expression level can be detected and measured. Such a probe or primer can be designed based on the base sequence of the Tet 3 gene, for example, using primer 3 (http://primer3.sourceforge.net/) or vector NTI (manufactured by Infomax). it can.
When using Northern blotting, the primer or probe is a radioisotope ( ³² P, ³³ P or the like: labeled with RI) or a fluorescent substance, and hybridized with cell-derived RNA transferred to a nylon membrane or the like according to a conventional method, and then the primer or probe (DNA or RNA) and RNA formed And a method of detecting and measuring a double strand with a radiation detector (BAS-1800II, manufactured by Fuji Film Co., Ltd.) or a fluorescence detector as a signal derived from the primer or probe label (RI or fluorescent substance) can do. In addition, using AlkPhos Direct Labeling and Detection System (manufactured by Amersham Pharmacia Biotech), the probe is labeled according to the protocol, hybridized with cell-derived RNA, and then the signal derived from the probe label is multibiotic. A method of detecting and measuring with a major STORM860 (manufactured by Amersham Pharmacia Biotech) can also be used.
When using the RT-PCR method, cDNA was prepared from cell-derived RNA according to a conventional method, and was prepared based on the Tet 3 gene sequence so that the target Tet 3 gene region could be amplified using this as a template. A pair of primers (a normal strand that binds to the cDNA (− strand) and a reverse strand that binds to the + strand) are hybridized with this, and PCR is performed according to a conventional method, and the resulting amplified double-stranded DNA is detected. The method of doing can be illustrated. In addition, the detection of the amplified double-stranded DNA was performed by a method for detecting the labeled double-stranded DNA produced by performing the PCR using a primer previously labeled with RI or a fluorescent substance. For example, a method may be used in which double-stranded DNA is transferred to a nylon membrane or the like according to a conventional method, and the labeled primer is used as a probe to hybridize with this to detect it. The produced labeled double-stranded DNA product can be measured with an Agilent 2100 Bioanalyzer (manufactured by Yokogawa Analytical Systems). Also, an RT-PCR reaction solution is prepared according to the protocol using SYBR Green RT-PCR Reagents (manufactured by Applied Biosystems), and reacted with ABI PRIME 7900 Sequence Detection System (manufactured by Applied Biosystems). You can also
Screening for a substance that changes the expression level of Tet 3 can also be performed by a reporter gene assay using the transcriptional regulatory region of Tet 3 gene. Here, the “transcriptional regulatory region” usually refers to a range of several kb to several tens of kb upstream of the chromosomal gene. For example, (i) 5′-race method (5′-RACE method) (for example, 5 (Ii) a step of determining the 5 ′ end by a conventional method such as oligocap method, S1 primer mapping, etc. (ii) Genome Walker Kit (which can be carried out using “-full Race Core Kit (manufactured by Takara Bio Inc.), etc.); The 5′-upstream region can be obtained using Clontech, etc., and the obtained upstream region can be identified by a technique including a step of measuring promoter activity.
A reporter protein expression vector is constructed by linking a nucleic acid encoding a reporter protein (hereinafter referred to as “reporter gene”) in a functional form downstream of the transcriptional regulatory region of the Tet 3 gene. The vector may be prepared by a method known to those skilled in the art. That is, “Molecular Cloning: A Laboratory Manual 2nd edition” (1989), Cold Spring Harbor Laboratory Press, “Current Protocols in Molecular Biology” (1987), John Ins. The transcriptional regulatory region of the Tet 3 gene excised according to the usual genetic engineering technique described in the above can be incorporated on a plasmid containing a reporter gene.
Reporter proteins include β-glucuronidase (GUS), luciferase, chloramphenicol transacetylase (CAT), β-galactosidase (GAS), green fluorescent protein (GFP), yellow fluorescent protein (YFP), blue fluorescent protein ( CFP), red fluorescent protein (RFP) and the like.
A reporter gene in which the transcriptional regulatory region of the prepared Tet 3 gene is operably linked is inserted into a vector that can be used in a cell into which the reporter gene is introduced, using a normal genetic engineering technique, and a plasmid Can be prepared and introduced into a suitable host cell. Stable transformed cells can be obtained by culturing in a medium with selection conditions according to the selection marker gene mounted on the vector. Alternatively, a reporter gene in which the transcriptional regulatory region of the Tet 3 gene is operably linked may be transiently expressed in the host cell.
In addition, as a method for measuring the expression level of the reporter gene, a method corresponding to each reporter gene may be used. For example, when a luciferase gene is used as a reporter gene, the transformed cell is cultured for several days, an extract of the cell is obtained, and then the extract is reacted with luciferin and ATP to cause chemiluminescence, and the luminescence intensity Promoter activity can be detected by measuring. In this case, a commercially available luciferase reaction detection kit such as Picker Gene Dual Kit (registered trademark; manufactured by Toyo Ink) can be used.
As a method for measuring the protein amount of Tet 3, specifically, for example,
(I) The detection antibody of the present invention is reacted competitively with the sample solution and labeled Tet3, and the labeled protein bound to the antibody is detected to quantify Tet3 in the sample solution. How,
(Ii) The labeling agent on the insolubilized carrier after reacting the sample solution with the detection antibody of the present invention insolubilized on the carrier and another labeled detection antibody of the present invention simultaneously or continuously For example, a method of quantifying Tet 3 in a sample solution by measuring the amount (activity) of.
Detection and quantification of the protein expression level of Tet 3 can be quantified according to a known method such as Western blotting using an antibody recognizing Tet3. Western blotting uses an antibody that recognizes Tet 3 as a primary antibody, and then uses a secondary antibody as a secondary antibody. ¹²⁵ Labeled with an antibody that binds to a primary antibody labeled with a radioisotope such as I, a fluorescent substance, an enzyme such as horseradish peroxidase (HRP), etc., and signals derived from these labeled substances are measured with a radiation measuring instrument (BAI-1800II: It can be carried out by measuring with a fluorescence detector or the like. In addition, after using an antibody that recognizes Tet 3 as a primary antibody, detection is performed according to the protocol using an ECL Plus Western Blotting Detection System (Amersham Pharmacia Biotech), and a multi-biometric STORM860 (Amersham Pharmacia Biotech) Can also be measured.
The form of the antibody is not particularly limited, and may be a polyclonal antibody having Tet 3 as an immunogen or a monoclonal antibody, and further, at least a sequence of amino acid sequences constituting Tet 3 is continuous. In general, an antibody having antigen-binding property against a polypeptide consisting of 8 amino acids, preferably 15 amino acids, more preferably 20 amino acids can also be used.
Methods for producing these antibodies are already well known, and the antibodies of the present invention can also be produced according to these conventional methods (Current protocol in Molecular Biology edit. Ausubel et al. (1987) Publ. John Wiley and Sons. Section 11.12-11.13).
In the above quantification method (ii), it is desirable that the two types of antibodies recognize different portions of Tet 3. For example, if one antibody recognizes the N-terminal part of Tet 3, one that reacts with the C-terminal part of the protein can be used as the other antibody.
As a labeling agent used in a measurement method using a labeling substance, for example, a radioisotope, an enzyme, a fluorescent substance, a luminescent substance, or the like is used. Examples of radioisotopes include [ ¹²⁵ I], [ ¹³¹ I], [ 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 substance, for example, fluorescamine, fluorescein isothiocyanate and the like are used. As the luminescent substance, for example, luminol, luminol derivatives, luciferin, lucigenin and the like are used. Furthermore, a biotin- (strept) avidin system can also be used for binding of an antibody or antigen and a labeling agent.
The Tet 3 quantification method using the detection antibody of the present invention is not particularly limited, and the amount of antibody, antigen or antibody-antigen complex corresponding to the amount of antigen in the sample solution is chemically or physically determined. Any measurement method may be used as long as it is a measurement method that is detected by a standard means and calculated from a standard curve prepared using a standard solution containing a known amount of antigen. For example, nephrometry, competition method, immunometric method and sandwich method are preferably used. In view of sensitivity and specificity, for example, the sandwich method described later is preferably used.
In the insolubilization of the antigen or antibody, physical adsorption may be used, or a chemical bond usually used for insolubilizing and immobilizing proteins or enzymes may be used. Examples of the carrier include insoluble polysaccharides such as agarose, dextran, and cellulose, synthetic resins such as polystyrene, polyacrylamide, and silicon, or glass.
In the sandwich method, the sample solution is reacted with the insolubilized detection antibody of the present invention (primary reaction), and further labeled with another detection antibody of the present invention (secondary reaction), and then on the insolubilized carrier. By measuring the amount or activity of the labeling agent, Tet 3 in the sample solution can be quantified. The primary reaction and the secondary reaction may be performed in the reverse order, may be performed simultaneously, or may be performed at different times. The labeling agent and the insolubilization method can be the same as those described above. Further, in the immunoassay by the sandwich method, the antibody used for the immobilized antibody or the labeled antibody is not necessarily one type, and a mixture of two or more types of antibodies is used for the purpose of improving measurement sensitivity. May be.
The detection antibody of the present invention can also be used in measurement systems other than the sandwich method, such as a competitive method, an immunometric method, or nephrometry.
In the competition method, Tet 3 in a sample solution and labeled Tet 3 are reacted competitively with an antibody, and then an unreacted labeled antigen (F) and a labeled antigen (B) bound to the antibody are combined. Separation (B / F separation) and the amount of labeling of either B or F is measured to quantify Tet 3 in the sample solution. In this reaction method, a soluble antibody is used as an antibody, B / F separation is performed using polyethylene glycol or a secondary antibody against the antibody (primary antibody), and a solid phase is used as the primary antibody. Either an antibody is used (direct method), or a primary antibody is soluble, and a solid phase antibody is used as a secondary antibody (indirect method).
In the immunometric method, Tet 3 in a sample solution and Tet 3 immobilized on a solid phase are competitively reacted with a certain amount of labeled antibody, and then the solid phase and the liquid phase are separated, or the Tet 3 in the sample solution is separated. Tet 3 is reacted with an excess amount of labeled antibody, and then solid-phased Tet 3 is added to bind unreacted labeled antibody to the solid phase, and then the solid phase and the liquid phase are separated. Next, the amount of label in any phase is measured to quantify the amount of antigen in the sample solution.
In nephrometry, the amount of insoluble precipitate produced as a result of the antigen-antibody reaction in a gel or solution is measured. Even when the amount of Tet 3 in the sample solution is very small and only a small amount of precipitate can be obtained, laser nephrometry using laser scattering is preferably used.
In applying these individual immunological measurement methods to the quantification method of the present invention, special conditions, operations and the like are not required to be set. What is necessary is just to construct | assemble the measurement system of Tet3, adding the usual technical consideration of those skilled in the art to the usual conditions and operation method in each method. For details of these general technical means, it is possible to refer to reviews, books and the like.
For example, Hiroshi Irie “Radioimmunoassay” (Kodansha, published in 1974), Hiroshi Irie “Continue Radioimmunoassay” (published in Kodansha, 1979), “Enzyme Immunoassay” edited by Eiji Ishikawa et al. 53)), edited by Eiji Ishikawa et al. "Enzyme Immunoassay" (2nd edition) (Medical Shoin, published in 1982), edited by Eiji Ishikawa et al. "Enzyme Immunoassay" (3rd edition) (Medical Shoin, Showa 62)), “Methods in ENZYMOLOGY” Vol. 70 (Immunochemical Technologies (Part A)), ibid., Vol. 73 (Immunochemical Technologies (Part B)), ibid., Vol. 74 (Immunochemical Technologies (Part C)), ibid., Vol. 84 (Immunochemical Technologies (Part D: Selected Immunoassays)), ibid., Vol. 92 (Immunochemical Technologies (Part E: Monoclonal Antibodies and General Immunoassay Methods)), Vol. 121 (Immunochemical Technologies (Part I: Hybridoma Technology and Monoclonal Antibodies)) (published by Academic Press).
As described above, the amount of Tet3 in a cell can be quantified with high sensitivity by using the detection antibody of the present invention.
For example, in the above screening method, the expression level of Tet 3 (mRNA level or protein level) in the presence of the test substance is about 20% or more, preferably about 30%, compared to the case in the absence of the test substance. %, More preferably about 50% or more, the test substance can be selected as a Tet 3 expression inhibitor, and thus as a candidate for a prophylactic and / or therapeutic drug for arthritis.
Alternatively, in the screening method described above, a cell containing a reporter gene under the control of the transcriptional regulatory region in the Tet3 gene can be used instead of the cell expressing the Tet3 gene. Such cells may be cells, tissues, organs or individuals of transgenic animals into which a reporter gene (eg, luciferase, GFP, etc.) under the control of the transcriptional regulatory region of the Tet 3 gene has been introduced. When such cells are used, the expression level of Tet 3 can be evaluated by measuring the expression level of the reporter gene using a conventional method.
(Measurement of Tet 3 function)
The screening method of the present invention can also be performed using as an index whether or not the test substance inhibits the function of Tet3.
Since Tet 3 is a demethylated protein, it is considered that a substance capable of binding to Tet 3 protein can inhibit the function of Tet 3 by suppressing the demethylation activity. Therefore, a candidate for a Tet 3 function inhibitor can be screened using the binding ability to Tet 3 as an index.
For example, a test substance is adsorbed to each well of a well plate, a Tet 3 solution labeled with an appropriate labeling agent is added to each well and incubated, the liquid phase is removed, and the amount of label bound to the solid phase after washing is determined. By measuring, a test substance having the ability to bind to Tet 3 can be detected. Instead of directly labeling Tet 3, it is also possible to detect Tet 3 bound to the solid phase using a labeled anti-Tet 3 antibody. Alternatively, a test substance solution is passed through a carrier on which Tet 3 is immobilized (for example, an affinity column), and the test substance retained on the carrier is used as a substance capable of binding to Tet 3, ie, prevention of arthritis and It can also be selected as a candidate for a therapeutic agent.
Whether or not the candidate substance thus obtained actually has an anti-inflammatory action is confirmed by applying the candidate substance to an arthritis model and testing whether or not the inflammatory reaction in the model is suppressed. be able to. In vivo and in vitro models can be used as such arthritis models. Examples of in vivo models include CIA models (can be prepared by immunizing non-human animals with type II collagen emulsified with complete Freund's adjuvant), CAIA models (recognizing epitopes in CB11 of type II collagen) RA models such as those that can be prepared by injecting a monoclonal antibody cocktail into a non-human animal) can be used, but are not limited thereto. On the other hand, examples of in vitro models include culture systems of target cells in arthritis (for example, synovial cells in RA) (for example, culture systems of synovial fibroblasts derived from synovial tissue of RA patients). However, it is not limited to these. These in vitro models can be stimulated by inflammatory cytokines such as TNFα as necessary, or complex cultures with inflammatory cytokine producing cells such as monocytes, macrophages, neutrophils (eg, transwells). ^TM Culture using target culture cells (eg, synovial fibroblasts) in the upper compartment and inflammatory cytokine-producing cells (macrophage-like THP-1 cells, RAW264.7 cells) in the lower compartment using a culture system, etc. System) and the like can induce an inflammatory reaction.
Whether or not the candidate substance has an anti-inflammatory action can be determined by whether or not the inflammatory reaction in the arthritis model is suppressed by the addition of the candidate substance. For example, in the case of the above RA model animal, the presence or absence and / or degree of arthritis treatment effect can be determined by using synovitis, the degree of inflammatory cell infiltration, the presence or absence of rheumatoid factor, and the like as indices. On the other hand, when a synovial fibroblast monolayer culture system, which is an in vitro arthritis model, was used, the degree of demethylation of genomic 5-methylcytosine (5 mC) or the result of scratch assay, as described in the examples below. The degree of inflammatory reaction can be evaluated using as an index.
In yet another embodiment of the present invention, the in vitro arthritis model can be used to screen in one step a substance that inhibits the function of Tet 3 and exhibits an effect of preventing and / or treating arthritis. The method includes the following steps (1) to (3).
(1) a step of bringing synovial fibroblasts into contact with a test substance;
(2) measuring the degree of demethylation or invasiveness of 5-methylcytosine (5 mC) in the genome of the cell,
(3) Compared to the measurement in the absence of the test substance, the test substance that suppresses the demethylation or invasiveness of 5-methylcytosine (5mC) in the genome of the cell is used to prevent arthritis and And / or selecting as a candidate for a therapeutic agent.
The method may further include a step of inducing inflammation at the same time as or before or after the step (1), if necessary. Examples of methods for inducing inflammation include stimulation with inflammatory cytokines such as TNFα, and complex culture with inflammatory cytokine-producing cells such as monocytes, macrophages and neutrophils. In a preferred embodiment, for example, transwell ^TM A method of culturing target cells (eg, synovial fibroblasts) in the upper compartment and inflammatory cytokine-producing cells (macrophage-like THP-1 cells, RAW264.7 cells) in the lower compartment using a culture system or the like, respectively. Is mentioned. The test substance is usually added to the medium in the lower compartment. For example, screening for ingredients contained in foods that can be absorbed into the intestine and inhibit the function of Tet 3, and orally administrable Tet 3 function inhibitors In the case where it is intended to do so, a test substance can be added to the medium in the upper compartment.
The degree of demethylation of 5-methylcytosine (5mC) in the cell genome is measured according to a known method such as Western blotting using an antibody that recognizes 5hmC or 5mC using the genomic DNA prepared from the cell. Can be quantified. The Western blot method used in this screening method may be the same as the Western blot method described in the above-described method for measuring Tet 3 protein. The form of the antibody recognizing 5hmC or 5mC is not particularly limited, and may be a polyclonal antibody having 5hmC or 5mC as an immunogen or a monoclonal antibody.
Moreover, the measurement of the invasive degree of a cell can be measured according to the method as described in the Example mentioned later, for example. Specifically, cells that are brought into contact with or not in contact with the test substance are cultured in a medium (10% FCS-containing DMEM (which may contain inflammatory cytokine (eg, TNFα) if necessary)) ( For example, after 96 hours), scraping the surface of the culture dish to which the cells adhere, and measuring the number of cells that infiltrate the scratched surface while culturing again, thereby measuring and comparing the degree of invasiveness of the cells. Can do.
A substance that inhibits the expression or function of Tet 3 obtained by using any one of the screening methods of the present invention is useful as a medicament for the prevention and / or treatment of inflammatory diseases.
When the compound obtained by using the screening method of the present invention is used as the above-mentioned prophylactic / therapeutic agent, it can be formulated in the same manner as the low molecular weight compound that inhibits the expression or function of Tet 3, and the same administration route And administered orally or parenterally to humans or mammals (eg, mice, rats, rabbits, sheep, pigs, cows, horses, cats, dogs, monkeys, chimpanzees, etc.) Can do.
V. Testing method for disease
As described above, it was found that the expression level of Tet 3 was high in synovial fibroblasts derived from rheumatoid arthritis patients stimulated with a synovial surface cell layer derived from rheumatoid arthritis patients and inflammatory cytokines.
Therefore, arthritis can be examined by using the expression level of Tet 3 (or Tet 3 gene) in a sample derived from a subject as an index.
The present invention provides a method for examining arthritis, which comprises detecting or quantifying a Tet 3 gene transcription product or translation product from a subject-derived sample using a Tet 3 detection substance. Specific examples of the Tet3 detection substance include the following (a) or (b):
(A) a nucleic acid probe or a nucleic acid primer capable of specifically detecting a transcript of the Tet 3 gene
(B) An antibody that specifically recognizes the translation product of the Tet 3 gene.
Specific examples of arthritis include rheumatoid arthritis, psoriatic arthritis, spondyloarthritis (eg, ankylosing spondylitis), and preferably rheumatoid arthritis.
The sample used in the test method of the present invention contains a Tet3 gene product (eg, RNA, protein, its degradation product, etc.) that is to be detected or quantified and that is separated from the test subject. Any tissue or cell that can be used is not particularly limited. Examples thereof include synovium, synovial fibroblasts, and synovial surface cells.
Detection or quantification of Tet 3 in a sample isolated from a subject is performed by preparing a RNA (eg, total RNA, mRNA) fraction from the subject and detecting or quantifying a transcript of the Tet 3 gene contained in the fraction. Can be examined.
Therefore, in one embodiment, the test method of the present invention is characterized by measuring using a nucleic acid probe or a nucleic acid primer capable of specifically detecting a transcript of the Tet 3 gene.
The RNA fraction can be prepared using a known method such as guanidine-CsCl ultracentrifugation, AGPC, etc., but using a commercially available RNA extraction kit (eg, RNeasy Mini Kit; manufactured by QIAGEN, etc.) High-purity total RNA can be prepared quickly and easily from a small amount of sample. As a means for detecting the transcript of the Tet 3 gene in the RNA fraction, for example, a method using hybridization (Northern blot, dot blot, DNA chip analysis, etc.) or PCR (RT-PCR, competitive PCR, real-time PCR) Etc.). Quantitative PCR methods such as competitive PCR and real-time PCR are preferable because they can detect changes in the expression of the Tet 3 gene quickly and easily from a small amount of sample with high quantitativeness.
The nucleic acid probe or nucleic acid primer capable of specifically detecting the transcript of the Tet 3 gene and the hybridization method using the nucleic acid probe or nucleic acid primer are the above-described screening methods for preventive and / or therapeutic agents for arthritis of the present invention. It may be the same as the nucleic acid for detection and the hybridization method of the present invention described.
Alternatively, the detection or quantification of Tet 3 in a sample isolated from a subject comprises preparing a protein fraction from the specimen and detecting or quantifying the translation product of the gene (ie, Tet3 protein) contained in the fraction. Can be examined. Detection or quantification of Tet3 can also be performed by an immunoassay (eg, ELISA, FIA, RIA, Western blot, etc.) using an antibody that specifically recognizes Tet3 protein.
Therefore, in one embodiment, the test method of the present invention is characterized by measuring using an antibody capable of specifically detecting the translation product of Tet 3.
The antibody specifically recognizing the translation product of Tet 3 and the immunological measurement method using the antibody are the detection antibody of the present invention described in the screening method for the prevention and / or treatment of arthritis of the present invention. And it may be the same as the immunological measurement method.
In the arthritis inspection method of the present invention, arthritis can be examined by detecting or quantifying Tet3. Specifically, it may be a method including the following steps.
(1) detecting or quantifying Tet 3 for a sample separated from a control group and a subject;
(2) A step of comparing Tet3 detected or quantified in the control group with Tet3 detected or quantified in the subject.
As shown in Examples described later, the Tet3 concentration in the synovium is higher in patients with rheumatoid arthritis compared to osteoarthritis patients (control group). Arthritis testing is based on such a positive correlation between the concentration of Tet3 and the prevalence of arthritis.
For example, the concentration of Tet 3 in a sample from a control group and subject that does not develop arthritis is quantified, and the concentration of Tet 3 in a sample from the subject is compared to the concentration of Tet 3 in a sample from the control group. Alternatively, a correlation diagram between the concentration of Tet 3 and the presence or absence of the onset of arthritis may be prepared in advance, and the Tet 3 concentration in the subject may be compared with the correlation diagram. The concentration comparison is preferably performed based on the presence or absence of a significant difference.
When Tet 3 is detected or quantified at a higher value in the subject than in the control group, it can be determined that the possibility of developing arthritis as described above is high. Therefore, in addition to the steps (1) and (2) above, the test method of the present invention (3) develops arthritis when Tet3 is detected or quantified in subjects at a higher value than the control group. It may also include a step of determining that it is.
Furthermore, the present invention extends to a kit (diagnostic agent) for arthritis examination. The arthritis test kit of the present invention is not particularly limited as long as it is a kit for simply carrying out the above-described test method of the present invention. The test kit is
(A) a nucleic acid probe or nucleic acid primer capable of specifically detecting a transcript of the Tet 3 gene, and / or
(B) an antibody that specifically recognizes the translation product of the Tet 3 gene
It contains. When the determination kit includes two or more of the above-described nucleic acids and / or antibodies, each nucleic acid or antibody specifically recognizes a different part of the base sequence of the Tet 3 gene from each other, or is a translation product of the Tet 3 gene. Different epitopes can be specifically recognized.
When the kit of the present invention includes the reagent containing the nucleic acid (a) as a component, examples of the nucleic acid include the probe nucleic acid or the primer oligonucleotide described above in the test method of the present invention.
The nucleic acid capable of detecting the expression of the Tet 3 gene can be provided as a solid in a dry state or in an alcohol precipitate state, or dissolved in water or an appropriate buffer (eg, TE buffer). Can also be provided. When used as a labeled probe, the nucleic acid can be provided in a state of being previously labeled with any of the above-mentioned labeling substances, or can be provided separately from the labeling substance and can be used after labeling.
Alternatively, the nucleic acid can be provided in a state immobilized on an appropriate solid phase. Examples of the solid phase include, but are not limited to, glass, silicon, plastic, nitrocellulose, nylon, polyvinylidene difluoride, and the like. As immobilization means, a functional group such as an amino group, an aldehyde group, an SH group, or biotin is introduced into a nucleic acid in advance, and a functional group capable of reacting with the nucleic acid on a solid phase (eg, aldehyde) Group, amino group, SH group, streptavidin, etc.), and the solid phase and the nucleic acid are cross-linked by covalent bond between the two functional groups, or the polyanionic nucleic acid is coated with the polycation and the static phase is coated. Examples of the method include immobilization of nucleic acid using electric coupling, but are not limited thereto.
The nucleic acid contained in the test kit is constructed so that the expression of the Tet 3 gene can be detected by the same method (eg, Northern blot, dot blot, DNA array technology, quantitative RT-PCR, etc.). It is particularly preferable.
When the kit of the present invention contains the reagent containing the antibody (b) as a component, examples of the antibody include the antibodies described above in the test method of the present invention.
The reagent constituting the kit of the present invention is a nucleic acid or antibody that can detect the expression of the Tet 3 gene and other substances necessary for the reaction for detecting the expression of the gene, and is stored in a coexisting state. Thus, a substance that does not adversely influence the reaction can be further contained. Alternatively, the reagent may be provided with a separate reagent containing other substances necessary in the reaction to detect the expression of the Tet 3 gene. For example, when the reaction for detecting the expression of the Tet3 gene is PCR, examples of the other substance include a reaction buffer, dNTPs, and a heat-resistant DNA polymerase. When competing PCR or real-time PCR is used, it can further include a competitor nucleic acid, a fluorescent reagent (such as the above intercalator and fluorescent probe), and the like. In addition, when the reaction for detecting the expression of the Tet 3 gene is an antigen-antibody reaction, examples of the other substance include a reaction buffer, a competitor antibody, and a labeled secondary antibody (for example, the primary antibody is a rabbit anti-antibody reaction). In the case of human Tet 3 antibody, mouse anti-rabbit IgG labeled with peroxidase, alkaline phosphatase, etc.), blocking solution, etc. may be mentioned.
The following examples are merely to illustrate the present invention more specifically, and do not limit the scope of the present invention.

(Example 1) Expression level of Tet protein family in synovium Tet protein family has three subtypes, Tet 1, Tet 2 and Tet 3, and the expression level varies from cell to cell. It is known that the genetic locus to be different is different. Therefore, the expression level of the Tet protein family was first examined by immunohistochemical staining in synovium from rheumatoid arthritis (RA) patients and osteoarthritis (OA) patients as controls. As for the synovium, a part of synovial tissue obtained at the time of joint surgery was cryopreserved and a tissue section was prepared later. The antibodies are Goat-anti-human TET1 Ab (Santa Cruz biotech, sc-1634443), mouse anti-human TET2 Ab (sc-136926), rabbit anti-human TET3 Ab (sc-139itx) 5-hmC) polyclonal Ab (ActiveMotif), rabbit anti-5-methylcytosine (5-mC) polyclonal Ab (ActiveMotif), mouse monoclonal anti-CD89A (Ab7470 ), The secondary antibody was used MACH 2 double stain 2 (mouse-HRP + rabbit-AP). For observation, BIOREVO (keyence, Japan) was used.
As a result, there was no difference in the expression levels of Tet 1 and Tet 2 in both OA and RA, and Tet 3 was more strongly expressed in RA than OA in the synovial surface cell layer (FIGS. 1 to 3). .
In addition, in order to examine the DNA methylation level in the RA, OA-derived synovium and the demethylation level by the Tet protein family, 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) were immunohistochemically stained. .
As a result, the level of 5 mC was low for both RA and OA, and the level of 5 hmC was moderate, giving a stronger impression on the synovial surface (FIGS. 1 and 2).
(Example 2) Expression level of Tet protein family in synovial fibroblasts (FLS) In subcultured FLS, there is no direct effect of inflammatory cytokines, but RA-specific DNA methylation profile is maintained. (Nakano K et al. Ann Rheum Dis 2013). Therefore, the expression levels (mRNA and protein) of the Tet family when cultured in the absence of inflammatory cytokines were compared between RA, OA, and healthy human FLS. As FLS, synovial cells extracted by adding collagenase treatment to the synovial tissue of Example 1 were used for passage 4-6, and FLS in which purity of 99% or more was confirmed was used. mRNA expression was analyzed by real-time PCR using StepOnePlus ^™ . For primer / probe, TET1 (Hs00286756_m1), TET2 (Hs00325999_m1), and TET3 (00379125_m1) from TaqMan Gene Expression Assays were used, and the expression level was corrected with GAPDH (Hs9999999_m1). For fluorescent staining, as primary antibodies, Goat-anti-human TET1 Ab (Santa Cruz biotech, sc-1634443), mouse anti-human TET2 Ab (sc-136926), rabbit anti-human TET3 Ab, sc-19616 FITC-conjugated anti-goat IgG, FITC-conjugated anti-mouse IgG, and FITC-conjugated anti-rabbit IgG were used as antibodies. BIOREVO (keyence, Japan) was used for observation, and BZ-II Viewer software (keyence) was used for image analysis.
As a result, there was no difference in the mRNA expression level of the Tet protein family in FLS without stimulation by inflammatory cytokine among RA, OA, and healthy individuals (FIG. 4). On the other hand, at the protein expression level, the expression levels of Tet1 and Tet2 were very low, and Tet3 was observed to be stronger in OA and RA-derived FLS than Tet1 and Tet2 (FIG. 5).
(Example 3) Expression level of Tet protein family in FLS after stimulation with inflammatory cytokines In the case of culturing with addition of inflammatory cytokines TNFα and IL-1β which are considered to be mainly involved in the pathological condition of RA in FLS of Example 2 Changes in expression level (mRNA, protein) of the Tet protein family were examined. TNFα was stimulated with recombinant human TNF-alpha (R & D), and IL-1β was stimulated with 50 ng / ml and 1 ng / ml, respectively, using recombinant human IL-1β (RELIATech GmbH). In addition, fluorescence staining was performed, and the fluorescence intensity of the nucleus and cytoplasm of 20 cells was randomly analyzed, and the ratio of the fluorescence intensity was defined as the N / C ratio. Nucleo Extraction Kit (Affymetrix, Fremont, Calif.) Was used for the extraction of nucleoprotein, and protein expression was analyzed by Western blot method.
As a result, when FLS was stimulated with TNFα or IL-1β, Tet 1 significantly decreased 2 hours after stimulation, whereas Tet 3 2 hours after stimulation, regardless of OA and RA. The expression increased significantly. There was no change in the expression of Tet2 (FIG. 6). Regarding the protein expression level, regardless of healthy subjects, OA, and RA, the Tet 3 expression level in the FLS nucleus increased significantly from 24 hours after TNFα stimulation and became prominent at 96 hours after stimulation (FIG. 7, 8).
(Example 4) DNA methylation level in FLS after stimulation with inflammatory cytokines In order to examine the DNA demethylation level, stimulation with TNFα was performed on RA-derived FLS, and the change in the level of 5hmC (genomic DNA) was examined. For the extraction of gDNA, the Spingen genomic DNA purification kit (m. biotech) was used, and the expression level of 5hmC in gDNA was analyzed by the Dot Blot method. Rabbit anti-5-methylcytosine (5-mC) polyclonal Ab (ActiveMotif) for primary antibodies, ECL ^TM Anti-Rabbit IgG for secondary antibodies, HRP linked whole antibody, GE health EC Blotting detection reagent (GE healthcare, UK) was used. As controls, 5-Methylcytosine & 5-Hydroxymethylcytosine DNA Standard Set was used. Quantification was performed using Light-capture (AE-6972, Atto, Tokyo, Japan) for image capturing, and CS Analyzer, version 3.0 (Atto) for image analysis.
As a result, FLS stimulated with TNFα significantly increased the level of 5 hmC at 96 hours after stimulation (FIG. 9).
(Example 5) Secretion level of inflammatory cytokine in FLS after suppression of Tet 3 expression Inhibition of Tet 3 expression using Tet 3 siRNA, followed by stimulation with TNFα and secretion of RA-derived FLS Evaluated. Specifically, FLS obtained by knocking down Tet 3 with siRNAs consisting of the nucleotide sequences of SEQ ID NO: 3 and SEQ ID NO: 4 was cultured in a medium containing TNFα (1 ng / ml) (10% FCS-containing DMEM) for 96 hours. After washing, the cells were further cultured for 48 hours in DMEM containing 0.1% FCS without TNFα. Tet 1 siRNA was used as a control. Two types of siRNA (Primer Set Human from Life technologies) used in the preliminary study were used for each gene, that is, siRNA IDs: HSS129586, HSS123253 for TET1, HSS123253, HSS1233Hor TET2, HSS15364T used. As a control, Low GC Duplex (Invitrogen) from Stealth ^™ RNAi Negative Control Duplexes was used. The siRNA gene was introduced by lipofection using Lipofectamine RNAiMAX Transfection Reagent (Life technologies). As a result of preliminary examination of gene transfer efficiency, it was decided to use HSS1533381 as TET3 siRNA and HSS129578 as TET1 siRNA. Regarding the amount of secreted protein such as IL-6, IL-8, VEGF, CCL2, etc., the BD ^™ Cytometric Bead Array (CBA, BD, Japan) asked the SRL to measure the secreted amount of MMP3 (the test method was the LTIA method) . The expression of adhesion molecules ICAM-1 and VCAM-1 was measured by flow-Cytometry method (FACSVerse ^TM, BD), and analyzed with FlowJo (Miltenyi Biotec). TaqMan (R) GeneExpress Assays CCL2 (Hs00234140_m1) was used for expression analysis of CCL2 mRNA.
As a result, among many cytokines, chemokines, and MMPs that have been suggested to be related to the pathological condition of RA, IL-6, IL-8, VEGF, CCL2, MMP3, etc. were found to induce TNFα-dependent expression. However, among these, only CCL2 inhibited TNFα-dependent expression induction by Tet 3 knockdown (FIG. 10). This phenomenon was also confirmed at the mRNA and secretory protein levels. In addition, among the adhesion molecules, TAMα-dependent expression induction of ICAM1 was similarly inhibited by Tet 3 knockdown. It is known that CCL2 and ICAM-1 are involved in the invasion of FLS into bone and cartilage tissues and deeply involved in the pathogenesis of RA. Therefore, it was suggested that Tet 3 knockdown is effective in the prevention and treatment of RA by suppressing the expression of CCL2 and ICAM-1 through maintenance of DNA methylation level. Although detection was attempted for IL-1β, TNFα, and IL-17α, they could not be detected because the values were below detection sensitivity.
(Example 6) Invasion ability of FLS after suppression of Tet 3 expression After inhibiting Tet 3 expression using Tet 3 siRNA, stimulation with TNFα was performed, and the invasion ability of RA-derived FLS was evaluated by Scratch assay. Specifically, FLS obtained by knocking down Tet 3 with siRNA was cultured for 96 hours in a medium (10% FCS-containing DMEM) containing TNFα (1 ng / ml), washed, and scratch was applied to the dish surface to which FLS was adhered. The observation was performed for a maximum of 48 hours while culturing in 0.1% FCS-containing DMEM not containing TNFα. Tet 1 siRNA was used as a control.
As a result, TNF3 knockdown completely inhibited TNFα-dependent induction of FLS infiltration (FIG. 11). Suggested that selective inhibition of Tet 3 leads to the prevention and treatment of RA by inhibiting invasiveness, one of the most important aggressive phenotypes found in synovial fibroblasts of rheumatoid arthritis patients It was done.

According to the present invention, an agent for preventing and / or treating arthritis or a diagnostic kit targeting Tet 3, which is a factor different from the target of conventional therapeutic agents, is provided. By providing therapeutic drugs with new mechanisms of action, patients with arthritis who do not have a pathological recovery effect with existing therapies or who do not have sufficient pathological recovery effects, and continue to use existing drugs Better treatment can also be provided for arthritic patients who have acquired resistance to the therapeutic agent. Furthermore, the present invention can also be used for the purpose of preventing arthritis in advance, and for the purpose of preventing recurrence of disease in patients whose arthritis has been temporarily ameliorated. In addition, according to the present invention, it is possible to screen for a candidate substance for a novel prophylactic / therapeutic agent for arthritis that exhibits an effect of preventing and / or treating arthritis by inhibiting the expression or function of Tet3.
This application is based on Japanese Patent Application No. 2014-174638 (filing date: August 28, 2014) filed in Japan, the contents of which are incorporated in full herein.

Claims

A preventive and / or therapeutic agent for arthritis, which contains an expression inhibitor of Tet 3 (Ten-Eleven tranlocation 3).
Tet 3 expression inhibitor is
(A) an antisense nucleic acid against a transcript of the Tet 3 gene,
The agent according to claim 1, which is (b) a ribozyme nucleic acid for a transcription product of Tet 3 gene, or (c) a nucleic acid having RNAi activity for a transcription product of Tet 3 gene or a precursor thereof.
The agent according to claim 1 or 2, wherein the arthritis is rheumatoid arthritis, psoriatic arthritis or spondyloarthritis.
A method for screening a prophylactic and / or therapeutic agent for arthritis, comprising the following steps (1) to (3):
(1) contacting a cell containing a nucleic acid encoding a Tet 3 gene or a reporter protein under the control of a transcriptional regulatory region of the gene with a test substance;
(2) measuring the expression level of the Tet 3 gene, Tet 3 protein or reporter protein in the cell,
(3) A test substance in which the expression level of the Tet 3 gene, Tet 3 protein, or reporter protein is reduced as compared with the case where it is measured in the absence of the test substance is used as a prophylactic and / or therapeutic drug for arthritis. Selecting as a candidate.
A prophylactic and / or therapeutic drug for arthritis, characterized by contacting Tet 3 with a test substance and selecting a test substance capable of binding to Tet 3 as a prophylactic and / or therapeutic drug for arthritis. Screening method.
The method according to claim 5, further comprising applying a test substance selected as a candidate for a prophylactic and / or therapeutic agent for arthritis to an arthritis model and testing whether to suppress an inflammatory response in the model. .
A method for screening a prophylactic and / or therapeutic agent for arthritis, comprising the following steps (1) to (3):
(1) a step of bringing synovial fibroblasts into contact with a test substance;
(2) measuring the degree of demethylation or invasiveness of 5-methylcytosine (5 mC) in the genome of the cell,
(3) Compared to the measurement in the absence of the test substance, the test substance that suppresses the demethylation or invasiveness of 5-methylcytosine (5mC) in the genome of the cell is used to prevent arthritis and And / or selecting as a candidate for a therapeutic agent.
The method according to any one of claims 4 to 7, wherein the arthritis is rheumatoid arthritis, psoriatic arthritis or spondyloarthritis.
A method for examining arthritis, comprising detecting or quantifying a transcription product or translation product of the Tet 3 gene from a sample derived from a subject using the following (a) or (b):
(A) a nucleic acid probe or nucleic acid primer capable of specifically detecting a transcription product of the Tet 3 gene; and (b) an antibody that specifically recognizes a translation product of the Tet 3 gene.
The method according to claim 9, wherein the arthritis is rheumatoid arthritis, psoriatic arthritis or spondyloarthritis.
(A) and / or (b) below:
(A) a nucleic acid probe or nucleic acid primer capable of specifically detecting a Tet 3 gene transcription product; and (b) an arthritis test kit comprising an antibody that specifically recognizes a translation product of the Tet 3 gene.
The kit according to claim 11, wherein the arthritis is rheumatoid arthritis, psoriatic arthritis or spondyloarthritis.
A method for preventing and / or treating arthritis, comprising administering to a subject an effective amount of an expression-inhibiting substance of Tet 3 (Ten-Eleven tranlocation 3).
Expression inhibitor of Tet 3 (Ten-Eleven tranlation 3) for use in the prevention and / or treatment of arthritis.
Use of an expression-inhibiting substance of Tet 3 (Ten-Eleven tranlocation 3) to produce an agent for preventing and / or treating arthritis.