Prophylactic or therapeutic agent for crohn's disease and method for examining crohn's disease

Abstract

Provided are a prophylactic or therapeutic agent for Crohn's disease, and a method for examining Crohn's disease. The prophylactic or therapeutic agent for Crohn's disease contains at least one selected from the group consisting of a RUNX2 inhibitor and a BHLHE40 inhibitor. The method for examining Crohn's disease includes (1) a step for detecting a protein and / or mRNA of at least one gene selected from the group consisting of RUNX2 and BHLHE40 in digestive tract-derived immune cells collected from a subject.

Description

Preventive or therapeutic agent for Crohn's disease and method for testing for Crohn's disease

[0001] The present invention relates to a preventive or therapeutic agent for Crohn's disease, a method for testing for Crohn's disease, and the like.

[0002] Crohn's disease is a designated intractable disease that causes chronic inflammation in the gastrointestinal tract, including the intestines, and Th1 / Th17 cells are known to be involved in the pathogenesis of chronic intestinal inflammation. However, it was not clear what kind of diversity T cells exhibit under the pathology of Crohn's disease, and the specific T cell subsets that contribute to the pathology of Crohn's disease and the mechanism by which they are induced were also not clarified.

[0003] In this context, Non-Patent Document 1 reports that Crohn's disease is characterized by a disease-specific CD4 + Trm cells are present, and Crohn's disease-specific CD4 + Trm cells secrete IFN-γ upon cytokine stimulation and have epithelial damage effects. + It has been reported that the Trm cell content correlates with indicators of clinical severity, and part of the pathogenesis of Crohn's disease has been elucidated.

[0004] Proc Natl Acad Sci US A. 2023 Jan 3; 120(1): e2204269120.

[0005] An objective of the present invention is to provide a preventive or therapeutic agent for Crohn's disease and a method for testing for Crohn's disease.

[0006] Based on the above findings in Non-Patent Document 1, the present inventors conceived the possibility that a factor may be responsible for Trm induction and IFN-γ expression induction in Crohn's disease. Based on this concept, the present inventors conducted extensive research and found that RUNX2 / BHLHE40 has the ability to induce Trm and IFN-γ expression. Based on this finding, the present inventors conducted further research and completed the present invention. That is, the present invention encompasses the following aspects.

[0007] Item 1. A preventive or therapeutic agent for Crohn's disease, comprising at least one agent selected from the group consisting of a RUNX2 inhibitor and a BHLHE40 inhibitor.

[0008] Item 2. The preventive or therapeutic agent according to Item 1, wherein the RUNX2 inhibitor is at least one selected from the group consisting of an inhibitor of RUNX2 function and an inhibitor of RUNX2 expression, and / or the BHLHE40 inhibitor is at least one selected from the group consisting of an inhibitor of BHLHE40 function and an inhibitor of BHLHE40 expression.

[0009] Item 3. The preventive or therapeutic agent according to Item 1 or 2, wherein the RUNX2 inhibitor is at least one selected from the group consisting of a polynucleotide targeting RUNX2, an expression cassette for the polynucleotide, a low-molecular-weight compound, a peptide, a protein, and an antibody, and / or the BHLHE40 inhibitor is at least one selected from the group consisting of a polynucleotide targeting BHLHE40, an expression cassette for the polynucleotide, a low-molecular-weight compound, a peptide, a protein, and an antibody.

[0010] Item 4. The preventive or therapeutic agent according to any one of Items 1 to 3, wherein the RUNX2 inhibitor is an inhibitor of RUNX2 in immune cells and / or the BHLHE40 inhibitor is an inhibitor of BHLHE40 in immune cells.

[0011] Item 5. The preventive or therapeutic agent according to Item 4, wherein the immune cells are T cells.

[0012] Item 6. (1) A method for testing for Crohn's disease, comprising the step of detecting a protein and / or mRNA of at least one gene selected from the group consisting of RUNX2 and BHLHE40 in immune cells derived from the gastrointestinal tract collected from a subject.

[0013] Item 7. The method according to Item 6, further comprising: (2) determining the presence or absence of Crohn's disease or recurrence of Crohn's disease in the subject, or the prognosis of Crohn's disease, based on the amount or concentration of the protein and / or the mRNA detected in step (1).

[0014] Item 8. The method according to Item 7, wherein the step (2) comprises: (2A) determining that the subject has Crohn's disease or has relapsed, or that the prognosis for Crohn's disease is poor, if the amount or concentration of the protein and / or the mRNA detected in the step (1) is equal to or greater than a cutoff value; and / or (2B) determining that the subject does not have Crohn's disease or has not relapsed, or that the prognosis for Crohn's disease is good, if the amount or concentration of the protein and / or the mRNA detected in the step (1) is equal to or less than a cutoff value.

[0015] Item 9. A diagnostic agent for Crohn's disease, comprising a molecule that binds to the protein and / or mRNA of at least one gene selected from the group consisting of RUNX2 and BHLHE40.

[0016] Item 10. The test agent according to Item 9, for use in the method according to any one of Items 6 to 8.

[0017] According to the present invention, it is possible to provide a preventive or therapeutic agent for Crohn's disease and a method for testing for Crohn's disease.

[0018] The following shows an outline of the method for searching for transcription factors highly expressed in Crohn's disease-specific CD4+ Trm cells (Test Example 1). The following shows an outline of the method for evaluating the Trm-inducing ability and IFN-γ expression-inducing ability of RUNX2 and BHLHE40 (Test Example 2). + The graph shows the results of measuring the Trm induction rate in T cells (Test Example 2). The vertical axis shows human CD4 + The percentage of Trm cells among T cells is shown. On the horizontal axis, "Control" indicates the case where an empty vector was introduced, "Runx2 / Bhlhe40" indicates the case where one of the expression vectors was introduced, and "both" indicates the case where both the expression vectors for Runx2 / Bhlhe40 were introduced. + The figure shows the results of measuring the induction rate of IFN-γ expression in T cells (Test Example 2). +The figures show the percentage of IFN-γ-expressing cells among T cells. On the horizontal axis, "Control" indicates the case where an empty vector was introduced, "Runx2 / Bhlhe40" indicates the case where one expression vector was introduced, and "both" indicates the case where both expression vectors were introduced. An outline of the method for RUNX2 and BHLHE40 knockdown analysis (Test Example 3) is shown. The figures show the results of measuring the expression levels of each gene after RUNX2 and BHLHE40 knockdown. On the horizontal axis, "NC" indicates the negative control, "RUNX2 i" indicates RUNX2 knockdown, and "BHLHE40 i" indicates BHLHE40 knockdown.

[0019] In this specification, the expressions "contain" and "comprise" include the concepts of "contain", "include", "consist essentially of" and "consist only of".

[0020] 1. Preventive or Therapeutic Agent In one aspect, the present invention relates to a preventive or therapeutic agent for Crohn's disease (sometimes referred to herein as the "agent of the present invention"), which contains at least one agent selected from the group consisting of a RUNX2 inhibitor and a BHLHE40 inhibitor. This will be explained below.

[0021] 1-1. Active ingredient 1-1-1. Target of inhibition The RUNX2 gene is a gene encoding the RUNX2 (Runt-related transcription factor 2) protein. The BHLHE40 gene is a gene encoding the BHLHE40 (Basic Helix-Loop-Helix Family Member E40) protein. The target of inhibition, RUNX2 (RUNX2 protein, RUNX2 mRNA), is an expression product of the RUNX2 gene, and is expressed in an organism or its cells (particularly, immune cells (preferably T cells, more preferably CD4 +The target of inhibition, BHLHE40 (BHLHE40 protein, BHLHE40 mRNA), is an expression product of the BHLHE40 gene, and is expressed by an organism to which the agent of the present invention is applied or its cells (particularly, immune cells (preferably T cells, more preferably CD4 T cells)), preferably gastrointestinal (more preferably intestinal) immune cells, more preferably gastrointestinal mucosal lamina propria immune cells). + The target RUNX2 protein / mRNA and BHLHE40 protein / mRNA are expressed by immune cells in the gastrointestinal tract (more preferably intestinal T cells), preferably gastrointestinal (more preferably intestinal) immune cells, more preferably gastrointestinal lamina propria immune cells. Therefore, depending on the target species, the target RUNX2 protein / mRNA and BHLHE40 protein / mRNA can be varied as necessary. The target species is not particularly limited, and examples include various mammals such as humans, monkeys, mice, rats, dogs, cats, rabbits, pigs, horses, cattle, sheep, goats, and deer.

[0022] The amino acid and nucleotide sequences of RUNX2 protein / mRNA and BHLHE40 protein / mRNA derived from various biological species are known. Specifically, for example, the human RUNX2 gene is identified by NCBI gene ID: 860, and the amino acid sequence of the human RUNX2 protein is, for example, the amino acid sequence shown in SEQ ID NO: 1, and the nucleotide sequence of the human RUNX2 coding sequence is, for example, the nucleotide sequence shown in SEQ ID NO: 2. Furthermore, for example, the human BHLHE40 gene is identified by NCBI gene ID: 8553, and the amino acid sequence of the human BHLHE40 protein is, for example, the amino acid sequence shown in SEQ ID NO: 3, and the nucleotide sequence of the human BHLHE40 coding sequence is, for example, the nucleotide sequence shown in SEQ ID NO: 4.

[0023] The amino acid sequences and nucleotide sequences of various species can be obtained or estimated from the above information. The RUNX2 protein / mRNA and BHLHE40 protein / mRNA may also include the above splicing variants.

[0024] The RUNX2 protein to be inhibited may have amino acid mutations such as substitutions, deletions, additions, and insertions, as long as it retains its original properties (transcription factor activity).The RUNX2 mRNA to be inhibited may also have base mutations such as substitutions, deletions, additions, and insertions, as long as the protein translated from the mRNA retains its original properties (transcription factor activity).

[0025] The BHLHE40 protein to be inhibited may have amino acid mutations such as substitutions, deletions, additions, and insertions, as long as it retains its original properties (transcription factor activity).The BHLHE40 mRNA to be inhibited may also have base mutations such as substitutions, deletions, additions, and insertions, as long as the protein translated from the mRNA retains its original properties (transcription factor activity).

[0026] The amino acid sequence mutation is preferably a substitution, more preferably a conservative substitution, from the viewpoint of less loss of activity. The nucleotide sequence mutation is preferably a mutation that does not result in an amino acid substitution in the protein translated from the mRNA, or a mutation that results in a conservative amino acid substitution.

[0027] A preferred example of the RUNX2 protein to be inhibited is a protein consisting of an amino acid sequence that is 85 to 100% identical to the amino acid sequence of the wild-type RUNX2 protein (e.g., SEQ ID NO: 1) and that retains its original properties (transcription factor activity).

[0028] A preferred example of the RUNX2 mRNA to be inhibited is a base sequence that has 85 to 100% identity to an mRNA containing the base sequence of the wild-type RUNX2 coding sequence (e.g., SEQ ID NO: 2) and encodes a protein with its original properties (transcription factor activity).

[0029] Preferred examples of the BHLHE40 protein to be inhibited include proteins consisting of an amino acid sequence that is 85 to 100% identical to the amino acid sequence of the wild-type BHLHE40 protein (e.g., SEQ ID NO: 3) and that retain its original properties (transcription factor activity).

[0030] A preferred example of the BHLHE40 mRNA to be inhibited is a base sequence that is 85 to 100% identical to an mRNA containing the base sequence of the wild-type BHLHE40 coding sequence (e.g., SEQ ID NO: 4) and encodes a protein that has its original properties (transcription factor activity).

[0031] The identity is more preferably 90% or more, even more preferably 95% or more, and even more preferably 98% or more.

[0032] As used herein, the term "identity" of an amino acid sequence refers to the degree of correspondence between two or more comparable amino acid sequences. Therefore, the greater the correspondence between two amino acid sequences, the greater the identity or similarity between those sequences. The level of identity between amino acid sequences can be determined, for example, using the sequence analysis tool FASTA with default parameters. Alternatively, it can be determined using the BLAST algorithm by Karlin and Altschul (Karlin S, Altschul SF, "Methods for assessing the statistical significance of molecular sequence features by using general scoring schemes," Proc Natl Acad Sci USA. 87:2264-2268 (1990); Karlin S, Altschul SF, "Applications and statistics for multiple high-scoring segments in molecular sequences," Proc Natl Acad Sci USA. 90:5873-7 (1993)). A program called BLASTX, based on the BLAST algorithm, has been developed. Specific techniques for these analysis methods are known and can be found on the National Center of Biotechnology Information (NCBI) website (http: / / www.ncbi.nlm.nih.gov / ). The "identity" of nucleotide sequences is also defined in the same manner as above.

[0033] As used herein, the term "conservative substitution" refers to the substitution of an amino acid residue with an amino acid residue having a similar side chain. For example, substitution between amino acid residues having basic side chains such as lysine, arginine, and histidine constitutes a conservative substitution. Other examples of conservative substitutions include substitution between amino acid residues having acidic side chains such as aspartic acid and glutamic acid; amino acid residues having uncharged polar side chains such as glycine, asparagine, glutamine, serine, threonine, tyrosine, and cysteine; amino acid residues having nonpolar side chains such as alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, and tryptophan; amino acid residues having β-branched side chains such as threonine, valine, and isoleucine; and amino acid residues having aromatic side chains such as tyrosine, phenylalanine, tryptophan, and histidine.

[0034] 1-1-2. Inhibitors RUNX2 / BHLHE40 inhibitors are not particularly limited, as long as they are components capable of inhibiting the function and / or expression of RUNX2 / BHLHE40. Preferable examples of RUNX2 / BHLHE40 inhibitors include polynucleotides targeting RUNX2 / BHLHE40, expression cassettes for such polynucleotides, low-molecular-weight compounds, peptides, proteins, antibodies, and the like. RUNX2 / BHLHE40 inhibitors can be used alone or in combination of two or more.

[0035] In a preferred embodiment of the present invention, the RUNX2 inhibitor is administered to immune cells (preferably T cells, more preferably CD4 + and / or a BHLHE40 inhibitor is an inhibitor of RUNX2 in immune cells (preferably T cells, more preferably CD4 T cells), preferably in gastrointestinal (more preferably intestinal) immune cells, more preferably in the gastrointestinal mucosal lamina propria immune cells (e.g., an immune cell-targeting RUNX2 inhibitor, an immune cell-specific RUNX2 inhibitor). +T cells), preferably gastrointestinal (more preferably intestinal) immune cells, more preferably gastrointestinal mucosal lamina propria immune cells (e.g., a BHLHE40 inhibitor targeting immune cells, an immune cell-specific BHLHE40 inhibitor).

[0036] 1-1-2-1. Function Inhibitors The RUNX2 / BHLHE40 function inhibitor is not particularly limited, as long as it is capable of inhibiting the function of the RUNX2 / BHLHE40 protein expressed in an organism or cells thereof to which the agent of the present invention is applied. RUNX2 / BHLHE40 function inhibitors can be used alone or in combination of two or more.

[0037] Examples of RUNX2 function inhibitors include substances that can inhibit the DNA binding of RUNX2, more specifically, substances that have binding affinity to the DNA-binding domain of RUNX2. Examples of BHLHE40 function inhibitors include substances that can inhibit the DNA binding of BHLHE40, more specifically, substances that have binding affinity to the DNA-binding domain of BHLHE40. Examples of these function inhibitors include low-molecular-weight compounds (e.g., molecular weights of 1000 or less, 800 or less, 700 or less, or 600 or less; e.g., molecular weights of 100 or more, 150 or more, or 200 or more), antibodies, etc.

[0038] The binding region can be determined based on known information and / or can be predicted based on known information (for example, by building a docking model).

[0039] The antibody includes polyclonal antibodies, monoclonal antibodies, chimeric antibodies, single-chain antibodies, and portions of the above antibodies that have antigen-binding ability, such as Fab fragments and fragments produced by an Fab expression library. The antibody of the present invention also includes an antibody that has antigen-binding ability to a polypeptide consisting of at least 8 consecutive amino acids, preferably 15 amino acids, and more preferably 20 consecutive amino acids, of the amino acid sequence of RUNX2 / BHLHE40.

[0040] In addition to the above, the RUNX2 / BHLHE40 function inhibitor can be any molecule (e.g., peptide, protein, artificial antibody, aptamer, etc.) that has binding ability (preferably specific binding ability) to RUNX2 / BHLHE40. When a protein or peptide such as an antibody is used as the RUNX2 / BHLHE40 function inhibitor, its expression cassette can also be used instead.

[0041] 1-1-2-2. Expression Inhibitors The RUNX2 / BHLHE40 expression inhibitor is not particularly limited, as long as it can suppress the expression level of RUNX2 / BHLHE40 protein and / or RUNX2 / BHLHE40 mRNA expressed in an organism or cells thereof to which the agent of the present invention is applied. RUNX2 / BHLHE40 expression inhibitors can be used alone or in combination of two or more.

[0042] Examples of RUNX2 / BHLHE40 expression inhibitors include RUNX2 / BHLHE40-specific small interfering RNA (siRNA), RUNX2 / BHLHE40-specific microRNA (miRNA), RUNX2 / BHLHE40-specific antisense nucleic acids, and expression cassettes thereof; RUNX2 / BHLHE40-specific ribozymes; and RUNX2 / BHLHE40 expression inhibitors using target-specific nuclease systems (e.g., CRISPR / Cas systems).

[0043] In addition, suppression of expression means suppressing the expression level of RUNX2 / BHLHE40 protein, RUNX2 / BHLHE40 mRNA, etc. to, for example, 1 / 2, 1 / 3, 1 / 5, 1 / 10, 1 / 20, 1 / 30, 1 / 50, 1 / 100, 1 / 200, 1 / 300, 1 / 500, 1 / 1000, or 1 / 10,000 or less, and also includes reducing these expression levels to zero.

[0044] 1-1-2-2-1. siRNA, miRNA, Antisense Nucleic Acids RUNX2 / BHLHE40-specific siRNA is not particularly limited as long as it is a double-stranded RNA molecule that specifically suppresses the expression of the gene encoding RUNX2 / BHLHE40. In one embodiment, the siRNA preferably has a length of, for example, 18 or more bases, 19 or more bases, 20 or more bases, or 21 or more bases. Furthermore, the siRNA preferably has a length of, for example, 25 or less bases, 24 or less bases, 23 or less bases, or 22 or less bases. It is contemplated that the upper and lower limits of the siRNA length described herein may be arbitrarily combined.

[0045] The structure of the siRNA is not particularly limited. The siRNA may be a small hairpin RNA (shRNA). The siRNA may have additional bases at the 5' or 3' end. The siRNA may have a protruding sequence (overhang) at the 3' end, specifically, for example, dTdT (dT represents deoxythymidine) added thereto.

[0046] siRNA and / or shRNA sequences can be searched for using search software provided free of charge on various websites, including, for example, the siRNA Target Finder provided by Ambion (http: / / www.ambion.com / jp / techlib / misc / siRNA_finder.html), the insert design tool for pSilencer® Expression Vector (http: / / www.ambion.com / jp / techlib / misc / psilencer_converter.html), and GeneSeer provided by RNAi Codex (http: / / codex.cshl.edu / scripts / newsearchhairpin.cgi).

[0047] The RUNX2 / BHLHE40-specific miRNA may be any miRNA as long as it inhibits the translation of the gene encoding RUNX2 / BHLHE40. For example, instead of cleaving the target mRNA like siRNA, the miRNA may inhibit its translation by pairing with the target's 3' untranslated region (UTR). The miRNA may be a pri-miRNA (primary miRNA), a pre-miRNA (precursor miRNA), or a mature miRNA. The length of the miRNA is not particularly limited; the length of the pri-miRNA is typically several hundred to several thousand bases, the length of the pre-miRNA is typically 50 to 80 bases, and the length of the mature miRNA is typically 18 to 30 bases. In one embodiment, the RUNX2 / BHLHE40-specific miRNA is preferably a pre-miRNA or a mature miRNA, and more preferably a mature miRNA. Such RUNX2 / BHLHE40-specific miRNA may be synthesized by known techniques or purchased from a company that provides synthetic RNAs.

[0048] RUNX2 / BHLHE40-specific antisense nucleic acids are nucleic acids containing a base sequence complementary or substantially complementary to the base sequence of the mRNA of the gene encoding RUNX2 / BHLHE40, or a portion thereof. These nucleic acids inhibit RUNX2 / BHLHE40 protein synthesis by binding to the mRNA to form a specific and stable duplex. Antisense nucleic acids may be DNA, RNA, or DNA / RNA chimeras. When the antisense nucleic acid is DNA, the RNA:DNA hybrid formed by the target RNA and the antisense DNA is recognized by endogenous ribonuclease H (RNase H), causing selective degradation of the target RNA. Therefore, in the case of antisense DNA directed against degradation by RNase H, the target sequence may be not only a sequence in the mRNA but also a sequence in an intron region in the initial translation product of the RUNX2 / BHLHE40 gene. Intron sequences can be determined by comparing the genomic sequence with the cDNA base sequence of the RUNX2 / BHLHE40 gene using homology search programs such as BLAST and FASTA. The target region of a RUNX2 / BHLHE40-specific antisense nucleic acid may be any length, as long as hybridization of the antisense nucleic acid results in inhibition of translation into RUNX2 / BHLHE40 protein. The RUNX2 / BHLHE40-specific antisense nucleic acid may be the entire sequence or a partial sequence of the mRNA encoding RUNX2 / BHLHE40. Considering ease of synthesis, antigenicity, and intracellular internalization, oligonucleotides consisting of approximately 10 to approximately 40 bases, particularly approximately 15 to approximately 30 bases, are preferred, but are not limited to these. More specifically, preferred target regions of the RUNX2 / BHLHE40 gene include, but are not limited to, the 5'-end hairpin loop, 5'-end untranslated region, translation initiation codon, protein-coding region, ORF translation termination codon, 3'-end untranslated region, 3'-end palindrome region, and 3'-end hairpin loop.

[0049] As used herein, "complementary" refers not only to binding based on a perfect complementary relationship (A and T, and G and C), but also to binding based on a complementary relationship sufficient to allow hybridization under stringent conditions. Stringent conditions can be determined based on the melting temperature (Tm) of the nucleic acid to which the complex or probe binds, as taught by Berger and Kimmel (1987, Guide to Molecular Cloning Techniques Methods in Enzymology, Vol. 152, Academic Press, San Diego, CA). For example, typical post-hybridization washing conditions include 1×SSC, 0.1% SDS, and 37°C. It is preferable that the hybridized state is maintained even after washing under such conditions. Although not particularly limited, examples of more stringent hybridization conditions include approximately "0.5xSSC, 0.1% SDS, 42°C," and examples of even more stringent hybridization conditions include washing conditions of approximately "0.1xSSC, 0.1% SDS, 65°C."

[0050] RUNX2 / BHLHE40-specific siRNA, RUNX2 / BHLHE40-specific miRNA, and RUNX2 / BHLHE40-specific antisense nucleic acids can be prepared by determining the target sequence of mRNA or an initial transcription product based on the cDNA sequence or genomic DNA sequence of the RUNX2 / BHLHE40 gene and synthesizing a complementary sequence using a commercially available DNA / RNA automatic synthesizer. Antisense nucleic acids containing various modifications can also be chemically synthesized by known techniques.

[0051] The expression cassette for RUNX2 / BHLHE40-specific siRNA, RUNX2 / BHLHE40-specific miRNA, or RUNX2 / BHLHE40-specific antisense nucleic acid is not particularly limited, so long as it is a polynucleotide into which RUNX2 / BHLHE40-specific siRNA, RUNX2 / BHLHE40-specific miRNA, or RUNX2 / BHLHE40-specific antisense nucleic acid has been incorporated in an expressible state. Typically, the expression cassette includes a polynucleotide including a promoter sequence and a coding sequence for the RUNX2 / BHLHE40-specific siRNA, RUNX2 / BHLHE40-specific miRNA, or RUNX2 / BHLHE40-specific antisense nucleic acid (and, if necessary, a transcription termination signal sequence), as well as other sequences as necessary.

[0052] As used herein, the terms "nucleic acid" and "polynucleotide" are not particularly limited and encompass both natural and artificial nucleic acids. Specifically, in addition to DNA, RNA, and the like, known chemical modifications may be used, as exemplified below. To prevent degradation by hydrolases such as nucleases, the phosphate residue of each nucleotide may be substituted with a chemically modified phosphate residue, such as phosphorothioate (PS), methylphosphonate, or phosphorodithioate. Furthermore, the hydroxyl group at the 2-position of the sugar (ribose) of each ribonucleotide may be substituted with -OR (where R represents, for example, CH3(2'-O-Me), CH2CHOCH3(2'-O-MOE), CH2CH2NHC(NH)NH2, CH2CONHCH3, or CH2CH2CN). Furthermore, the base moiety (pyrimidine or purine) may be chemically modified, for example, by introducing a methyl group or a cationic functional group into the 5-position of the pyrimidine base, or by substituting a thiocarbonyl group for the carbonyl group at the 2-position. Further examples include, but are not limited to, those in which the phosphate moiety or hydroxyl moiety is modified with, for example, biotin, an amino group, a lower alkylamine group, an acetyl group, etc. Also usable are BNA (LNA), in which the conformation of the sugar moiety of the nucleotide is fixed to N-type by bridging the 2' oxygen and 4' carbon of the sugar moiety.

[0053] 1-1-2-2-2. Expression Inhibitors Using a Target-Specific Nuclease System The expression inhibitors using a target-specific nuclease system are not particularly limited, as long as they are capable of suppressing the expression of the RUNX2 / BHLHE40 gene using a target-specific nuclease system (e.g., a CRISPR / Cas system). The expression of the RUNX2 / BHLHE40 gene can be suppressed, for example, by disrupting the RUNX2 / BHLHE40 gene, modifying the RUNX2 / BHLHE40 gene promoter to suppress promoter activity, or by transcriptional suppression (CRISPRi) through binding of an expression inhibitor to the RUNX2 / BHLHE40 gene region.

[0054] When the CRISPR / Cas system is employed, for example, a vector containing a guide RNA expression cassette targeting the RUNX2 / BHLHE40 gene or its promoter and a Cas protein expression cassette can typically be used as the expression inhibitor, but is not limited to this. In addition to this typical example, a combination of a vector containing a guide RNA and / or its expression cassette targeting the RUNX2 / BHLHE40 gene or its promoter, and a vector containing a Cas protein and / or its expression cassette can also be used as the expression inhibitor.

[0055] There are no particular limitations on the guide RNA as long as it is used in the CRISPR / Cas system. For example, various guide RNAs can be used that can bind to a target site in genomic DNA (e.g., the RUNX2 / BHLHE40 gene, its promoter, etc.) and bind to a Cas protein, thereby guiding the Cas protein to the target site in genomic DNA.

[0056] As used herein, the term "target site" refers to a site on genomic DNA that consists of a DNA strand (target strand) and its complementary DNA strand (non-target strand), which consists of a PAM (Proto-spacer Adjacent Motif) sequence and a sequence adjacent to the 5' side of the PAM sequence that is approximately 17 to 30 bases long (preferably 18 to 25 bases long, more preferably 19 to 22 bases long, and particularly preferably 20 bases long).

[0057] The guide RNA has a sequence involved in binding to a target site in genomic DNA (sometimes referred to as a crRNA (CRISPR RNA) sequence), and this crRNA sequence binds complementary (preferably complementary and specific) to a sequence excluding the PAM sequence complementary sequence of the non-target strand, thereby enabling the guide RNA to bind to the target site in genomic DNA. Furthermore, the guide RNA has a sequence involved in binding to a Cas protein (sometimes referred to as a tracrRNA (trans-activating crRNA) sequence), and this tracrRNA sequence binds to the Cas protein, thereby guiding the Cas protein to the target site in genomic DNA.

[0058] The tracrRNA sequence is not particularly limited. The tracrRNA sequence is typically an RNA sequence of approximately 50 to 100 bases long that can form multiple (usually three) stem-loops, and the sequence varies depending on the type of Cas protein used. Various known sequences can be used as the tracrRNA sequence depending on the type of Cas protein used.

[0059] The guide RNA typically contains the above-mentioned crRNA sequence and tracrRNA sequence. The guide RNA may be a single-stranded RNA (sgRNA) containing the crRNA sequence and the tracrRNA sequence, or an RNA complex formed by complementary binding of an RNA containing the crRNA sequence and an RNA containing the tracrRNA sequence.

[0060] The Cas protein is not particularly limited as long as it is used in the CRISPR / Cas system; for example, various proteins that bind to target sites in genomic DNA in a complex with a guide RNA can be used. Cas proteins with impaired DNA cleavage activity can also be used. In this case, transcriptional repressors (e.g., SALL1, SDS3, etc.) can be induced as needed to exert transcriptional repression. Cas proteins derived from various organisms are known, including the Cas9 protein, and more preferably the Cas9 protein endogenously contained in bacteria belonging to the genus Streptococcus. Information on the amino acid sequences and coding sequences of various Cas proteins can be easily obtained from various databases, such as NCBI.

[0061] The expression inhibitor can be easily prepared using known genetic engineering techniques, such as PCR, restriction enzyme digestion, DNA ligation, in vitro transcription / translation, and recombinant protein production techniques.

[0062] 1-2. Uses At least one selected from the group consisting of RUNX2 inhibitors and BHLHE40 inhibitors can be used as an active ingredient of an agent for preventing or treating Crohn's disease.

[0063] In one embodiment, the agent of the present invention can prevent or treat Crohn's disease by suppressing Th1 effector activity and promoting migration of T cells from intestinal tissue. More specifically, for example, Crohn's disease-specific CD4 + The induction of Trm cells and the induction of IFN-γ expression are suppressed, thereby making it possible to prevent or treat Crohn's disease.

[0064] As used herein, "treatment" can include concepts such as cure, remission, alleviation, mitigation, and suppression of progression of symptoms. Furthermore, "prevention" can include concepts such as not only preventing the onset of a disease, but also delaying the onset of the disease, suppressing symptoms once the disease has occurred, preventing recurrence after cure or remission of symptoms, and suppressing exacerbation after alleviation or alleviation of symptoms.

[0065] The content of the active ingredient in the agent of the present invention can be appropriately determined taking into consideration the type of target disease, the desired therapeutic effect, the administration method, the treatment period, the age and body weight of the patient, etc. For example, the content of the active ingredient in the agent of the present invention can be about 0.0001 to 100 parts by weight, assuming that the total amount of the agent of the present invention is 100 parts by weight.

[0066] The administration form of the agent of the present invention is not particularly limited as long as the desired effect is obtained, and it can be administered to mammals, including humans, by either oral administration or parenteral administration (e.g., intravenous injection, intramuscular injection, subcutaneous administration, rectal administration, transdermal administration, or topical administration). Parenteral administration is preferred. Dosage forms for oral and parenteral administration and their preparation methods are well known to those skilled in the art, and can be prepared according to conventional methods by mixing the active ingredient with a pharmaceutically acceptable carrier, etc.

[0067] Dosage forms for parenteral administration include injectable preparations (e.g., drip infusions, intravenous injections, intramuscular injections, subcutaneous injections, and intradermal injections), topical preparations (e.g., ointments, poultices, lotions, creams, and gels), suppositories, inhalants, eye preparations, eye ointments, nasal drops, ear drops, liposomes, and LNP (Lipid Nano Particle) preparations. For example, injectable preparations are prepared by dissolving the active ingredient in distilled water for injection, and solubilizers, buffers, pH adjusters, isotonicity agents, soothing agents, preservatives, stabilizers, and the like can be added as needed. The agent of the present invention can also be prepared as a lyophilized preparation for immediate use.

[0068] The agent of the present invention may further contain other drugs that are effective in treating or preventing diseases.

[0069] The agent of the present invention can contain any carrier or additive, for example, a pharmaceutically acceptable carrier or additive.

[0070] Pharmaceutically acceptable carriers and additives include, but are not limited to, excipients such as sucrose and starch; binders such as cellulose and methylcellulose; disintegrants such as starch and carboxymethylcellulose; lubricants such as magnesium stearate and aerosil; flavorings such as citric acid and menthol; preservatives such as sodium benzoate and sodium bisulfite; stabilizers such as citric acid and sodium citrate; suspending agents such as methylcellulose and polyvinylpyrrolide; dispersing agents such as surfactants; diluents such as water and physiological saline; base waxes, etc.

[0071] The dosage of the agent of the present invention can be determined based on various factors, such as the route of administration, the type of disease, the severity of symptoms, the patient's age, sex, and body weight, the severity of the disease, pharmacological findings such as pharmacokinetic and toxicological characteristics, whether a drug delivery system is used, and whether the agent is administered as part of a combination of other drugs. The dosage of the agent of the present invention can be, for example, approximately 1 μg / kg (body weight) to 10 g / kg (body weight) per day. The administration schedule of the agent of the present invention can also be determined taking into account factors similar to those for the dosage. For example, the above daily dosage can be administered 1 to 5 times per day to 1 month.

[0072] 2. Crohn's Disease Testing Method In one aspect, the present invention relates to (1) a method for testing for Crohn's disease (also referred to herein as the "testing method of the present invention"), which comprises the step of detecting protein and / or mRNA of at least one gene selected from the group consisting of RUNX2 and BHLHE40 in gastrointestinal-derived immune cells collected from a subject (also referred to herein as the "target molecule of the present invention"). This method is described below.

[0073] 2-1. Step (1) The subject may be any mammal, but in one embodiment, the subject may be a subject whose Crohn's disease status is unknown, a subject suspected of having Crohn's disease, a subject whose symptoms of Crohn's disease have been cured, in remission, alleviated, or alleviated, a subject suffering from Crohn's disease, a subject with recurrent Crohn's disease, etc. Examples of mammals include laboratory animals such as rodents (e.g., mice, rats, hamsters, guinea pigs) and rabbits, pets (e.g., dogs and cats), livestock (e.g., cows, pigs, goats, horses, sheep), primates (e.g., monkeys, orangutans, chimpanzees), and humans, with humans being particularly preferred.

[0074] The gastrointestinal tract-derived immune cells are immune cells (preferably T cells, more preferably CD4 cells) contained in the gastrointestinal tract (more preferably intestinal tract). + T cells), preferably gastrointestinal lamina propria immune cells. Gastrointestinal-derived immune cells can be collected, isolated, and separated from a subject by methods known to those skilled in the art.

[0075] The RUNX2 gene and BHLHE40 gene, as well as their proteins and mRNAs, are as explained above in "1. Preventive or therapeutic agents."

[0076] The target molecules of the present invention that are the detection targets in step (1) also include isoforms and those containing mutations that occur between individuals.

[0077] When detecting a target molecule of the present invention that is a protein, the method for detecting the target molecule of the present invention (preferably a method for measuring the amount or concentration of the target molecule of the present invention) is not particularly limited, as long as it is a method that can detect the target molecule of the present invention. Examples of such methods include immunoassays and flow cytometry. Immunoassays can be widely used, regardless of whether they are direct, indirect, homogeneous, heterogeneous, competitive, or non-competitive. More specific examples of immunoassays include ELISA (e.g., direct, indirect, sandwich, or competitive), radioimmunoassay (RIA), immunoradiometric assay (IRMA), enzyme immunoassay (EIA), sandwich EIA, immunochromatography, Western blot, immunoprecipitation, slot or dot blot assay, immunohistochemical staining, fluorescent immunoassay, immunoassay using an avidin-biotin or streptavidin-biotin system, and immunoassay using surface plasmon resonance (SPR). Specifically, the target molecule of the present invention can be detected by immunoassay, for example, by contacting a labeled antibody directly or indirectly with a molecule binding to the target molecule of the present invention that has bound to the target molecule of the present invention, and quantifying the signal derived from the label of the bound labeled antibody. The labeled antibody used in this case and the antibody that mediates between the labeled antibody and the molecule binding to the target molecule of the present invention or the target molecule of the present invention are not particularly limited, and examples that can be used include antibodies against antibody constant regions and anti-idiotype antibodies.

[0078] When detecting a protein target molecule of the present invention, binding molecules to the target molecule of the present invention include, for example, polyclonal antibodies, monoclonal antibodies, chimeric antibodies, single-chain antibodies, or molecules containing portions of the above antibodies that have antigen-binding ability, such as Fab fragments and fragments produced by an Fab expression library. Binding molecules to the target molecule of the present invention that have antigen-binding ability to a polypeptide consisting of at least 8 consecutive amino acids, preferably 15 amino acids, and more preferably 20 amino acids, from the amino acid sequence of the target molecule of the present invention are also included in the binding molecules to the target molecule of the present invention.

[0079] The type of label used in the label (e.g., labeled antibody) for detecting the target molecule of the present invention, which is a protein, is not particularly limited. Examples of labels include fluorescent substances, luminescent substances, dyes, enzymes, gold colloids, and radioisotopes. Among these, enzyme labels such as peroxidase and alkaline phosphatase are preferred from the viewpoints of safety, economy, detection sensitivity, and the like.

[0080] When detecting a target molecule of the present invention that is mRNA, the method for detecting the target molecule of the present invention (preferably a method for measuring the amount or concentration of the target molecule of the present invention) is not particularly limited, as long as it is capable of detecting the target molecule of the present invention. Examples of such methods include RNA-Seq, Northern blotting, RNase protection assay, reverse transcription polymerase chain reaction (RT-PCR) (Weis JH et al., Trends in Genetics 1992;8:263-264), and quantitative real-time RT-PCR (Held CA et al., Genome Research 1996;6:986-994). In one embodiment, the above methods can use molecules (e.g., primers, probes, etc.) that bind to the target molecule of the present invention. The primer pair and probe contain sequences that can complementarily bind to the nucleotide sequence of the target molecule mRNA of the present invention or its complementary sequence, and can be synthesized based on known base sequences. The base lengths of the primers and probes are not particularly limited. The primers can each be 10 to 50 nucleotides in length, preferably 15 to 30. The probes can range in length from 10 nucleotides to the full length of the nucleotide sequence complementary to the nucleotide sequence of the mRNA of the molecule of interest of the present invention, preferably 20 to 150 nucleotides.

[0081] The primer pair and probe may be made of natural nucleic acids such as RNA and DNA, or may be made of a combination of natural nucleic acids with chemically modified nucleic acids or pseudo nucleic acids, if necessary. Examples of chemically modified nucleic acids and pseudo nucleic acids include PNA (Peptide Nucleic Acid), LNA (Locked Nucleic Acid; registered trademark), methylphosphonate DNA, phosphorothioate DNA, and 2'-O-methyl RNA. Furthermore, the primer and probe may contain a fluorescent substance and / or a quencher substance, or a radioisotope (e.g., 32 P, 33 P, 35 Labeling or modification may be performed using a labeling substance such as Fluorescent Protein I (FITC), Texas Amino Acids (TFA), or a modifying substance such as biotin, (streptavidin), or magnetic beads. The labeling substance is not limited, and commercially available substances can be used. For example, fluorescent substances such as FITC, Texas Amino Acids (TFA), Cy3, Cy5, Cy7, Cyanine 3, Cyanine 5, Cyanine 7, FAM, HEX, VIC, fluorescamine and its derivatives, and rhodamine and its derivatives can be used. Quencher substances such as AMRA, DABCYL, BHQ-1, BHQ-2, or BHQ-3 can be used. The labeling position of the labeling substance in the primer and probe can be determined appropriately depending on the properties of the modifying substance and the intended use. Generally, modification is performed at the 5' or 3' end. Furthermore, a single primer and probe molecule may be labeled with one or more types of labeling substances. The design of the nucleotide sequences of primers and probes and the selection of labeling substances are well known and are disclosed in molecular biology experimental protocol books such as Molecular Cloning: A Laboratory Manual by Sambrook, J and Russell, DW (3rd ed., Cold Spring Harbor Laboratory Press, 2001).

[0082] The detection method may be adopted singly or in combination of two or more.

[0083] According to the testing method of the present invention including step (1), it is possible to provide the amount and / or concentration of the target molecule of the present invention, which is an indicator for testing for Crohn's disease, thereby assisting in testing for Crohn's disease, etc.

[0084] 2-2. Step (2) In one embodiment, the testing method of the present invention preferably further comprises: (2) a step of determining the presence or absence of Crohn's disease or recurrence or the prognosis of Crohn's disease in the subject based on the amount or concentration of the protein and / or the mRNA detected in step (1).

[0085] More specifically, step (2) can include: (2A) determining that the subject has Crohn's disease or has relapsed, or that the prognosis for Crohn's disease is poor, when the amount or concentration of the protein and / or the mRNA detected in step (1) is equal to or greater than a cutoff value; and / or (2B) determining that the subject does not have Crohn's disease or has not relapsed, or that the prognosis for Crohn's disease is good, when the amount or concentration of the protein and / or the mRNA detected in step (1) is equal to or less than a cutoff value.

[0086] The cutoff value is set in advance based on a statistical analysis or ROC analysis of the amount or concentration of each target molecule of the present invention, and the presence or absence of Crohn's disease, the presence or absence of recurrence, or the degree of prognosis, of the evaluation population, prepared as a database. Alternatively, the cutoff value can be set on a case-by-case basis. When the cutoff value is determined by statistical analysis, for example, the median, arithmetic mean, or other average value of the amount or concentration data of the target molecule of the present invention in the evaluation population can be used. When the cutoff value is determined by ROC analysis, for example, the cutoff value based on ROC analysis can be the amount or concentration of the target molecule of the present invention at the point on the ROC curve where the distance between the point on the vertical axis (sensitivity or true positive) of the ROC curve graph that is 1.0 and the point on the horizontal axis (1 - specificity) that is 0.0 is the shortest. Alternatively, the cutoff value can be derived from the Youden index of the ROC curve (Cancer 1950;3:32-35.). Once established, the database of the population for evaluation may be used to set a cutoff value in the method of testing a subject for Crohn's disease of the present invention without any changes. Alternatively, new subjects, including the subjects of the present invention, may be incorporated into the population for evaluation, and the database of the population for evaluation for Crohn's disease may be updated as appropriate and used to set a cutoff value in the method of testing a subject for Crohn's disease of the present invention. The cutoff value may be, for example, a percentile value of the amount or concentration of the target molecule of the present invention in a biological sample from a reference group of subjects, such as any of the 10th to 90th percentile values, any of the 30th to 70th percentile values, or any of the 40th to 60th percentile values.

[0087] A subject determined to have Crohn's disease, to be undergoing recurrence, or to have a poor prognosis for Crohn's disease in step (2) can be determined to be a target for administration of the agent of the present invention. That is, the testing method of the present invention can be used as a companion diagnosis for the agent of the present invention.

[0088] 3. Test Agent for Crohn's Disease In one aspect, the present invention relates to a test agent for Crohn's disease (the test agent of the present invention) that contains a binding molecule for the target molecule of the present invention. This will be explained below.

[0089] The diagnostic agent of the present invention is a drug for testing for Crohn's disease (specifically, for testing for the presence or absence of Crohn's disease or recurrence, or the prognosis of Crohn's disease). The diagnostic agent of the present invention can be used in the testing method of the present invention. Furthermore, the diagnostic agent of the present invention can be used as a companion diagnostic agent for the agent of the present invention.

[0090] The test agent of the present invention may be in the form of a composition containing a molecule capable of binding to a target molecule of the present invention. The composition may contain other components as necessary. Examples of other components include bases, carriers, solvents, dispersants, emulsifiers, buffers, stabilizers, excipients, binders, disintegrants, lubricants, thickeners, moisturizers, colorants, fragrances, chelating agents, etc.

[0091] The test agent of the present invention may be in the form of a kit containing a molecule capable of binding to a target molecule of the present invention. The kit may also contain instruments, reagents, and the like that can be used to carry out the test method of the present invention.

[0092] The binding molecule for the target molecule of the present invention can be immobilized on any solid phase, and therefore the test agent of the present invention can be provided in the form of a substrate on which the binding molecule for the target molecule of the present invention is immobilized (for example, a microarray chip on which a probe is immobilized, or another example, an ELISA plate on which an antibody is immobilized).

[0093] The solid phase used for immobilization is not particularly limited as long as it can immobilize antibodies, etc., and examples thereof include glass plates, nylon membranes, microbeads, silicon chips, capillaries, and other substrates. There are no particular limitations on the immobilization of the detection agent to the solid phase.

[0094] Examples of the apparatus include test tubes, microtiter plates, agarose particles, latex particles, purification columns, epoxy-coated slide glasses, and gold colloid-coated slide glasses.

[0095] Examples of the reagent include labeled antibodies and standard samples (positive control, negative control).

[0096] As the labeled antibody, various commercially available antibodies can be used depending on the type (for example, isotype) of the molecule that binds to the target molecule of the present invention.

[0097] The target molecule of the present invention can be used as a standard sample. The target molecule of the present invention can be obtained, for example, by culturing cells into which an expression vector for the target molecule of the present invention has been introduced and purifying the target molecule from the cells or the culture supernatant.

[0098] The present invention will be described in detail below based on examples, but the present invention is not limited to these examples.

[0099] Test Example 1. Crohn's disease-specific CD4 + Search for transcription factors highly expressed in Trm cells: Crohn's disease-specific CD4 + The existence of Trm cells has been reported (Non-Patent Document 1). In this test example, we searched for transcription factors that are highly expressed in these cells. An overview of the search method is shown in Figure 1. The detailed method is described below.

[0100] Test Example 1-1. Isolation of Lamina Propria Mononuclear Cells (LPMCs) Control, unaffected intestinal mucosa was obtained from macroscopically intact sites of colorectal cancer patients. Inflamed intestinal mucosa was obtained from surgically resected specimens of Crohn's disease patients. Specifically, the procedure was as follows: Intestinal epithelial cells were dissociated by shaking in 5 mM ethylenediaminetetraacetic acid (EDTA) in Hank's balanced salt solution, and the muscularis layer was then removed. The mucosal layer was cut into fragments and digested in RPMI 1640 with 1 mg / mL collagenase type 1 (Sigma-Aldrich) mixed with 0.07 IU / mL DNase for 30 minutes at 37°C. The cells were centrifuged at 800 g, dispersed in EDTA solution, and washed with phosphate-buffered saline (PBS). LPMCs were frozen in liquid nitrogen until analysis.

[0101] Test Example 1-2. CD4 + Isolation of CD4 T cells + Isolation of T cells is performed using CD4 + The T cell isolation kit (Miltenyi Biotec) was used according to the manufacturer's protocol, followed by FACS.7 Resuspended in 40 μL of FACS Buffer per CD4 + The T cell biotin-antibody cocktail was added and incubated at 4°C for 5 minutes. FACS Buffer and CD4+ T cell MicroBeads Cocktail were added and incubated at 4°C for 10 minutes. The LS column was washed with 3 mL of FACS Buffer, and the mononuclear cells were applied to the column. The flow-through was collected. CD4 T cells were isolated from LPMCs. + To isolate T cells, CD4 + The T cell enriched cell suspension was stained with CD3-APC / Cy7, CD4-PE / Cy7 for 30 min at 4°C, washed twice with FACS Buffer, stained with 7AAD, and transferred to BD FACSMelody for further purification. TM was incorporated.

[0102] Test Example 1-3. Nuclear extract CD4 + T cells were centrifuged at 500 x g for 5 min at 4°C, the cell pellet was resuspended, and lysed in 300 μL of chilled 0.1× lysis buffer [10 mM Tris-HCl pH 7.4, 10 mM NaCl, 3 mM MgCl2, 1% BSA, 1 mM DTT, 1 U / μL RNase inhibitor, 0.01% Tween-20, 0.01% Nonidet P40 Substitute, 0.001% Digitonin] for 3 min on ice. Lysis was quenched with 1 mL of chilled wash buffer [10 mM Tris-HCl pH 7.4, 10 mM NaCl, 3 mM MgCl2, 1% BSA, 1 mM DTT, 1 U / μL RNase inhibitor, 0.1% Tween-20]. Cells were then harvested by centrifugation at 500 x g for 5 min at 4°C and washed twice with 1 mL of wash buffer. During the second wash, cells were filtered through a 40 μL FLOWMI Cell Strainer. Nuclei were resuspended in diluted Nuclei Buffer [1× Nuclei Resuspension Buffer, 1 mM DTT, 1 U / μL RNase inhibitor] at a concentration of 3200 Nuclei / μL.

[0103] Experimental Example 1-4: Single-Cell and Multiome Data Generation. Single-cell libraries were constructed according to the 10× Genomics Chromium Next GEM Single Cell Multiome ATAC + Gene Expression protocol (CG000338). Specifically, the nuclei suspension was incubated with transposase to fragment DNA in open chromatin regions and add adapter sequences to the ends of the DNA fragments. The transposed nuclei were loaded onto a Chromium Next GEMChip J (PN-1000234, 10× Genomics, Pleasanton, CA) along with partitioning oil and barcoded single-cell gel beads, followed by PCR amplification. ATAC and gene expression libraries were then prepared separately. Library quality was assessed using the Agilent High Sensitive DNA Kit (#5067-4626) on an Agilent Bioanalyzer 2100 (Agilent Technologies, Santa Clara, USA). Library concentrations were determined by qPCR using the Collibri Library Quantification kit (#A38524500, ThermoFisher Scientific) in a QuantStudio3 (ThermoFisher Scientific). Libraries were then pooled equally and subjected to paired-end sequencing on an Illumina NextSeq 550 System (Illumina, Inc., USA) using the High Output Kit v2.5 (#20024907, Illumina, Inc., USA).

[0104] Test Examples 1-5. Single-Cell Multi-ome Data Processing For the processing of multi-ome data, the 10×Genomics Cell Ranger ARC (v2.0.0) pipeline was used. Raw sequence data was first converted to fastq format using "cellranger-arc mkfastq". The raw files of RNA-seq and ATAC-seq libraries of the same sample were aligned to the UCSC human genome (hg38) and quantified using "cellranger-arc count". Samples were aggregated using "cellranger-arc aggr" to normalize the sequencing depth. The raw data of the RNA count matrix and ATAC fragments were further processed using the Seurat (v5.1.0) 55 and Signac (v1.13.0) of the R package, respectively. Based on the filtering results of RNA-assay metrics (500 < nCount_RNA < 30,000, < 7500, percnt.mt < 25) and ATAC-assay metrics (500 < nCount_ATAC < 100,000, nucleosome_signal < 2, TSS.enrichment > 1), 42,553 cells were obtained. The gene expression count matrix was normalized using the NormalizeData function. Principal component (PC) analysis was performed based on the top 2000 highly variable features. For ATAC data, peak calling was performed using the MACS2 package with the CallPeaks function of Signac. Peaks overlapping with the genomic blacklist regions of the Hg38 genome were removed. Each peak represents one potential regulatory DNA element. The peak count matrix was normalized using latent semantic indexing (LSI) including term frequency (TF) inverse document frequency (IDF) and singular value decomposition (SVD). The first LSI component was removed from downstream analysis as follows. This was highly correlated with the sequencing depth.

[0105] Test Example 1-6: Single-cell multi-ome data analysis. Data integration was performed using the R package Harmony (v1.2.0). The FindMarkers function was used to calculate differentially expressed genes and differentially accessible open chromatin regions in each cluster. Transcription factor enrichment analysis was performed using CHIP-Atlas. Motif analysis was performed using the FindMotifs function in Signac. Knockdown simulation was performed using the CellOracle Package.

[0106] Results: 73 transcription factors with logP-Val<-50 were identified by scATAC-seq, and 13 transcription factors with logP-Val<-50 and FC>2 were identified by scRNA-seq. Two transcription factors (RUNX2 and BHLHE40) overlapped between the two groups and were associated with Crohn's disease-specific CD4 + It was found to be a transcription factor highly expressed in Trm cells.

[0107] Test Example 2: Evaluation of the Trm-inducing ability and IFN-γ expression-inducing ability of RUNX2 and BHLHE40 Crohn's disease-specific CD4 + It has been reported that Trm cells secrete IFN-γ, which activates immune cells and damages epithelial cells, and are involved in the pathogenesis of Crohn's disease (Non-Patent Document 1). + Two factors (RUNX2 and BHLHE40) found in Test Example 1 to be transcription factors highly expressed in Trm cells were evaluated for their ability to induce Trm and IFN-γ expression. An overview of the evaluation method is shown in Figure 2. The details of the method are described below.

[0108] Test Example 2-1. Peripheral blood mononuclear cell (PBMC) isolation and CD4 +T Cell Isolation PBMCs were isolated using Ficoll-Paque PREMIUM 1.084 (Cytiva, Marlborough, MA, USA) according to the manufacturer's protocol. Briefly, 10 ml of human peripheral blood was diluted with an equal volume of PBS. The diluted blood sample was layered on Ficoll-Paque medium solution and then centrifuged at 400 g at room temperature (RT) for 30 minutes. After removing the upper layer, the mononuclear cell layer was collected in a new tube and washed twice with PBS. The isolated PBMCs were stored in liquid nitrogen until further analysis. CD4 T cells were isolated from the isolated PBMCs as described in Test Example 1-2. + T cells were isolated.

[0109] Test Example 2-2. Vector Construction: RNA was extracted from human T cells using the FAST gene RNA Premium Kit (Nippon Genetics), and cDNA was synthesized using ReverTra Ace qPCR RT Master Mix (Toyobo). RUNX2 was amplified with the oligo primers 5'-GGTGTCGTGACGTACGGCCACCATGCGTATTCCCGTAGATCCG-3' (SEQ ID NO: 5) and 5'-CTCCACTGCCGCTAGCATATGGTCGCCAAACAGATTCATC-3' (SEQ ID NO: 6). BHLHE40 was amplified with the oligo primers 5'-GGTGTCGTGACGTACGGCCACCATGGAGCGGATCCCCAGC-3' (SEQ ID NO: 7) and 5'-CTCCACTGCCGCTAGCGTCTTTGGTTTCTAAGTTTAAAGGGGGGATT-3' (SEQ ID NO: 8). The cDNAs were then cloned into a lentiviral vector using a DNA ligation kit (Takara).

[0110] Test Example 2-3. Lentivirus Production On day 1, LentiX293T cells were cultured in a virus harvest medium (Opti-MEM I reduced serum medium, 5% FBS, 100 mM sodium pyruvate) at 7 × 10 cells / well in a collagen-treated 10 cm dish. 6On day 2, cells were transfected with 3.8 μg of VSVg, 5.85 μg of psPAX2, and 7.6 μg of the transgene lentiviral vector using Opti-MEM I Reduced Serum Medium (Gibco) and Lipofectamine 3000 (Thermo Fisher Scientific). After 6 hours, the culture medium was transferred to Viral Harvest Medium containing Viral Boost Reagent (ALSTEM). Viral supernatants were collected on days 3 and 4 and stored at 4°C. To concentrate the viral supernatant, the supernatant was centrifuged at 500g for 10 minutes at 4°C, mixed with Lentiviral Precipitation Solution (ALSTEM), and incubated overnight at 4°C. The next day, the combined supernatants were centrifuged at 1500g for 40 minutes at 4°C, and the supernatant was removed. The virus-containing pellet was resuspended in cold PMS and stored at -80°C until use.

[0111] Test Example 2-4. Overexpression of lentiviral RUNX2 and BHLHE40 in naive T cells On day 0, a 96-well plate was incubated with 10 μg / mL of anti-hCD3 antibody and 5 μg / mL of anti-hCD28 antibody for 3 hours. After washing twice with 100 μL of 2% FBS RPMI, 1.0 × 10 6 CD4 + T cells were seeded and cultured in RPMI-1640 medium containing D-glucose and glutamine, supplemented with 10% heat-inactivated FBS, 100 U / mL penicillin, 100 μg / mL streptomycin sulfate, and 100 U / mL hIL-2. On day 1, control, RUNX2-GFP, or BHLHE40-GFP lentivirus was added to the culture medium and incubated for 10 minutes at 37°C in a 5% CO2 atmosphere. After incubation, the culture plates were centrifuged at 1200 g for 90 minutes at 32°C and returned to the incubator. On day 3, the cells were passaged into 24-well plates. On day 4, T cells were harvested and analyzed by flow cytometry.

[0112] Test Example 2-5. Flow Cytometry. Cells were activated with PMA (10 ng / ml) and ionomycin (250 ng / ml), then incubated with monensin (GolgiStop; BD Pharmingen, Franklin Lakes, NJ, USA) at 37°C for 2 hours. After stimulation, cells were harvested, washed twice with 2% FBS-supplemented PBS, and stained for surface markers (anti-human CD4 antibody and anti-human CD103 antibody) at 4°C for 30 minutes. Cells were then washed with PBS, treated with Cytofix / Cytoperm (BD Biosciences) for 20 minutes at 4°C, washed with perm / wash buffer, and centrifuged at 800 g. IFN-γ-FITC (Biolegend) dissolved in perm / wash buffer was added to the pellet, incubated for 30 minutes at 4°C, and washed with perm / wash buffer. Cells were analyzed using a BD FACSCanto TM Flow Cytometry Standard (FCS) files were acquired using FlowJo. TM The data was uploaded to the software and analyzed.

[0113] Results Human CD4 + The results of measuring the Trm induction rate in T cells are shown in Figure 3. + The results of measuring the induction rate of IFN-γ expression in T cells are shown in Figure 4. It was found that RUNX2 and BHLHE40 have the ability to induce Trm and IFN-γ expression.

[0114] Combining the findings of Non-Patent Document 1 with the results of Test Examples 1 and 2, it is clear that inhibition of RUNX2 and / or BHLHE40 increases Crohn's disease-specific CD4 + It was found that the induction of Trm cells and the induction of IFN-γ expression can be suppressed, making it possible to prevent or treat Crohn's disease. Furthermore, it was found that detecting RUNX2 / BHLHE40 in immune cells derived from the gastrointestinal tract collected from a subject can be used to test for Crohn's disease, more specifically, to determine the presence or recurrence of Crohn's disease or the prognosis of Crohn's disease.

[0115] Test Example 3. Analysis of knockdown of RUNX2 and BHLHE40 RUNX2 and BHLHE40 were knocked down and the effects were analyzed. An overview of the analysis method is shown in Figure 5. The detailed method is described below.

[0116] Test Example 3-1. Construction of CRISPR Knockdown Lentiviral Vector: The EF1α core promoter was removed from lentiCRISPR v2-dCas9 (#112233, Addgene, Cambridge, MA, USA) and replaced with an MSCV promoter fragment (IDT). The Cas9-P2A-Puromycin cassette was replaced with a gene fragment encoding dCas9-ZIM3-T2A-mScarlet (IDT, Coralville, IA, USA) and integrated using the Gibson Assembly method. Oligonucleotides for the gRNA sequence were purchased from Thermo Fisher Scientific. Oligonucleotide annealing was performed by mixing 1 μL each of forward and reverse strand oligos, 7 μL of nuclease-free water, and 1 μL of 10x TNE buffer (100 mM Tris HCl pH 8.0, 10 mM EDTA, 500 mM NaCl), heating at 95°C for 5 minutes, and then allowing to cool to room temperature. DNA ligation was performed using a Takara Bio DNA ligation kit according to the manufacturer's protocol. The sequences used were: Control 5'-CACCGTGTCTTTAAACACGCCATCG-3' (SEQ ID NO: 9) 5'-AAACCGATGGCGTGTTTAAAGACAC-3' (SEQ ID NO: 10) RUNX2 KD: 5'-CACCGGGCGGGTAGGGAGACCCGGG-3' (SEQ ID NO: 11) 5'-AAACCCCGGGTCTCCCTACCCGCCC-3' (SEQ ID NO: 12) BHLHE40 KD: 5'-CACCGACGGCGCAGACAGACCGCGC-3' (SEQ ID NO: 13) 5'-AAACGCGCGGTCTGTCTGCGCCGTC-3' (SEQ ID NO: 14).

[0117] Test Example 3-2. Lentiviral knockdown: Lamina propria mononuclear cells (LPMCs) were isolated from the intestines of patients with Crohn's disease. The isolated LPMCs were stained with CD3-APC / Cy7 (#300426, BioLegend) and CD4-PE / Cy7 (#557852, BD Biosciences) at 4°C for 30 minutes and washed twice with FACS buffer. Dead cell staining was performed using 7AAD (#420404, BioLegend) and BD FACSMelody. TM Similar to the overexpression experiments, CD3 expression was measured using lentiviral vectors incorporating RUNX2 knockdown and BHLHE40 knockdown sequences. + CD4 + 7AAD - T cells were infected with the virus. On day 3, the medium was replaced with fresh medium. On day 5, T cells were harvested, mScarlet3-positive cells were isolated by flow cytometry, and knockdown cells were selected.

[0118] Test Example 3-2. Quantitative PCR (qPCR) Total RNA was extracted using the Fast Gene RNA Premium Kit (NIPPON Genetics, Tokyo) and reverse transcribed using ReverTra Ace qPCR RT Master Mix with gDNA Remover (TOYOBO). Lamnia propria CD4 + For knockdown experiments using T cells, RNA was extracted from 500 cells and cDNA was synthesized using the RT-RamDA cDNA Synthesis Kit (TOYOBO). Real-time PCR was performed using StepOnePlus TMPCR was performed using a Real-Time PCR System (Applied Biosystems) and Power SYBR Green PCR Master Mix (Applied Biosystems). All expression levels were normalized to GAPDH. PCR conditions: 50°C (2 min), 95°C (10 min), 40 cycles of 95°C (15 sec), 64°C (60 sec). The sequences of the primer sets used are as follows: GAPDH: 5'-GTCGGAGTCAACGGATT-3' (SEQ ID NO: 15), 5'-AAGCTTCCCGTTCTCAG-3' (SEQ ID NO: 16) RUNX2 variant 1: 5'-ATGCGTATTCCCGTAGATCC-3' (SEQ ID NO: 17), 5'-GGGCTCACGTCGCTCATTT-3' (SEQ ID NO: 18) BHLHE40: 5'-TAAAGCGGAGCGAGGACAGCAA-3' (SEQ ID NO: 19), 5'-GATGTTCGGGTAGGAGATCCTTC-3' (SEQ ID NO: 20) IFNG: 5'-TCCCATGGGTTGTGTGTTTA-3' (SEQ ID NO: 21), 5'-AAGCACCAGGCATGAAATCT-3' (SEQ ID NO: 22) GZMB: 5'-CGACAGTACCATTGAGTTGTGCG-3' (SEQ ID NO: 23), 5'-TTCGTCCATAGGAGACAATGCCC-3' (SEQ ID NO: 24) PRF1: 5'-ACTCACAGGCAGCCAACTTTGC-3' (SEQ ID NO: 25), 5'-CTCTTGAAGTCAGGGTGCAGCG-3' (SEQ ID NO: 26) S1PR1: 5'-CCTGTGACATCCTCTTCAGAGC-3' (SEQ ID NO: 27), 5'-CACTTGCAGCAGGACATGATCC-3' (SEQ ID NO: 28).

[0119] The results are shown in Figure 6. Suppression of RUNX2 and BHLHE40 in diseased CD4+ T cells suppressed Th1-type effector activity and cytotoxicity, and promoted T cell migration from the tissue.

Claims

1. A preventive or therapeutic agent for Crohn's disease, comprising at least one selected from the group consisting of a RUNX2 inhibitor and a BHLHE40 inhibitor.

2. The preventive or therapeutic agent according to claim 1, wherein the RUNX2 inhibitor is at least one selected from the group consisting of RUNX2 function inhibitors and RUNX2 expression inhibitors, and / or the BHLHE40 inhibitor is at least one selected from the group consisting of BHLHE40 function inhibitors and BHLHE40 expression inhibitors.

3. The preventive or therapeutic agent according to claim 1, wherein the RUNX2 inhibitor is at least one selected from the group consisting of a polynucleotide targeting RUNX2, an expression cassette for the polynucleotide, a low molecular weight compound, a peptide, a protein, and an antibody, and / or the BHLHE40 inhibitor is at least one selected from the group consisting of a polynucleotide targeting BHLHE40, an expression cassette for the polynucleotide, a low molecular weight compound, a peptide, a protein, and an antibody.

4. The preventive or therapeutic agent according to any one of claims 1 to 3, wherein the RUNX2 inhibitor is an inhibitor of RUNX2 in immune cells, and / or the BHLHE40 inhibitor is an inhibitor of BHLHE40 in immune cells.

5. The preventive or therapeutic agent according to claim 4, wherein the immune cells are T cells.

6. (1) A method for testing for Crohn's disease, comprising the step of detecting the protein and / or mRNA of at least one gene selected from the group consisting of RUNX2 and BHLHE40 in gastrointestinal-derived immune cells collected from a subject.

7. The method according to claim 6, further comprising: (2) determining the presence or absence of Crohn's disease or recurrence or the prognosis of Crohn's disease in the subject based on the amount or concentration of the protein and / or the mRNA detected in step (1).

8. The method according to claim 7, wherein the step (2) comprises: (2A) determining that the subject has Crohn's disease or has relapsed, or that the prognosis for Crohn's disease is poor, if the amount or concentration of the protein and / or the mRNA detected in the step (1) is equal to or greater than a cutoff value; and / or (2B) determining that the subject does not have Crohn's disease or has not relapsed, or that the prognosis for Crohn's disease is good, if the amount or concentration of the protein and / or the mRNA detected in the step (1) is equal to or less than a cutoff value.

9. A diagnostic agent for Crohn's disease, comprising a binding molecule for the protein and / or mRNA of at least one gene selected from the group consisting of RUNX2 and BHLHE40.

10. The test agent according to claim 9, for use in the method according to any one of claims 6 to 8.

Images