JP5098033B2 - siRNA, recombinant vector expressing this siRNA, NR4A2 gene expression inhibitor, IL-17 gene expression inhibitor, and CD4 positive T cell proliferation inhibitor - Google Patents

Description

The present invention relates to siRNA, a recombinant vector expressing this siRNA, an NR4A2 gene expression inhibitor, an IL-17 gene expression inhibitor, and a CD4 positive T cell proliferation inhibitor .

  IFN-γ is produced from a detailed analysis using the mouse EAE (Experimental autoimmune encephalomyelitis) model, an animal model of MS, as recent findings on the pathogenesis of multiple sclerosis (MS). It has been reported that IL-17-producing T cells (Th17 cells) infiltrating the central nervous region are more important than Th1 cells (Current Opinion in Immunology 2006, vol.18: p.349-356). In addition, experiments using IL-17 knockout mice revealed that IL-17 is involved in the development of EAE. Furthermore, in IFN-γ knockout mice where EAE worsens, CD4-positive T It has been reported that IL-17 production is enhanced in cells (The Journal of Immunology 2006, vol. 177: p.566-573).

  On the other hand, IL-1 receptor antagonist knockout mice spontaneously develop autoimmune arthritis, but double-deficient mice that cross this mouse and IL-17 knockout mice suppress this onset of arthritis (Ernst Schering Res Found Workshop. 2006, vol.56: p.129-153).

  Furthermore, analysis using IL-17 knockout mice reveals that IL-17 is also deeply involved in the development of various inflammatory diseases such as contact hypersensitivity, delayed hypersensitivity, and airway hypersensitivity. (Immunity. 2002, vol.17: p.375-87).

  These results reveal that IL-17 is an important factor in the development of inflammatory diseases such as autoimmune encephalitis, autoimmune arthritis, and hypersensitivity, while simultaneously producing IL-17. It became clear that these inflammations can be suppressed if suppressed.

  Therefore, in order to improve the inflammation caused by IL-17, there has been a demand for new development of an IL-17 production inhibitor, a growth inhibitor of CD4-positive T cells that produce IL-17, and the like.

The present invention relates to siRNA useful for suppression of IL-17 production, suppression of proliferation of CD4-positive T cells, improvement of inflammation caused by IL-17, etc., a recombinant vector expressing this siRNA, and these as active ingredients It is intended to provide an NR4A2 gene expression inhibitor, an IL-17 gene expression inhibitor, and a CD4 positive T cell proliferation inhibitor, which are contained as follows.

  As a result of diligent research to solve the above-mentioned problems, the present inventors have identified an NR4A2 gene that is specifically upregulated in peripheral blood T cells derived from multiple sclerosis (MS) patients, and this NR4A2 gene is upregulated. It was also found in T cells infiltrating the central nervous region (CNS) of mice that induced experimental autoimmune encephalomyelitis (EAE), an animal model of MS.

  In addition, it became clear that the expression of NR4A2 gene and the production of cytokines such as IFN-γ, TNFα, IL-10, IL-4, IL-2, and IL-17 showed a positive correlation. The present inventors have found that it is directly or indirectly involved in the regulation of expression of these cytokine genes.

Furthermore, production of cytokines such as IFN-γ, TNFα, IL-10, IL-4, IL-2, and IL-17 by suppressing the expression of NR4A2 gene using siRNA that suppresses the expression of NR4A2 gene It was also found that the proliferation of CD4 positive T cells can be suppressed.
Thus, the present inventors have completed the present invention.

That is, the siRNA according to the present invention is a sense strand that is an oligonucleotide having a base sequence shown in any of SEQ ID NOs: 1 to 3 , and a sequence that is a base sequence that is complementary to the base sequences of SEQ ID NOs: 1 to 3 , respectively. It is a double-stranded RNA comprising an antisense strand, which is an oligonucleotide having the base sequences shown in Nos. 12-14 . The oligonucleotide is a sugar-modified ribonucleotide, and the 2′-OH group is substituted with a group selected from H, OR, halo, SH, SR, NH ₂ , NHR, NR ₂ or CN (provided that , R is C ₁ -C ₆ alkyl, alkenyl or alkynyl and halo is F, Cl, Br or I).

The recombinant vector according to the present invention is characterized by expressing the above-described siRNA .

The NR4A2 gene expression inhibitor, IL-17 gene expression inhibitor, and CD4 positive T cell proliferation inhibitor according to the present invention contain the above-described siRNA or the above-described recombinant vector as an active ingredient.

<Cross-reference with related literature>
This application claims the benefit of priority of Japanese Patent Application No. 2007-49769 filed on Feb. 28, 2007, and is incorporated herein by reference.

In one Example of this invention, it is a figure which shows the result of having investigated the expression level of the NR4A2 gene in MS patient and a healthy person. In one Example of this invention, it is a figure which shows the result of having investigated the change of the expression of the NR4A2 gene in the CD3 + T cell derived from SPL, dLN, or a CNS infiltrating mononuclear cell in the disease course of EAE. FIG. 2A shows the clinical score after induction of EAE, and FIG. 2B shows the NR4A2 gene in each CD3 + T cell isolated from SPL, dLN, or CNS infiltrating mononuclear cells at 0, 9, 15, and 21 days after induction of EAE. The results of examining the expression level of are shown respectively. In one Example of this invention, it is a figure which shows the result of having investigated the variation | change_quantity of IL-17 which a CNS infiltrating T cell or a SPL T cell produces after onset of EAE. In one Example of this invention, it is a figure which shows the result of having analyzed the correlation of NR4A2 gene expression and IL-17 production by a reporter assay. FIG. 4A shows the result of examining the induction of promoter activity of each cytokine gene by the expression of NR4A2 gene, and FIG. 4B shows the induction of promoter activity of IL-17 gene when the amount of plasmid introduced into the cell is changed. The results are shown respectively. In one Example of this invention, it is a figure which shows the result of having analyzed the correlation of NR4A2 gene expression and IL-17 production using the bicistronic retroviral vector which co-expresses NR4A2 protein and eGFP. FIG. 5A is a vector construction diagram of pMIG-Φ and pMIG-NR4A2, FIG. 5B is a result of examining changes in IL-17 produced in CD4 T cells after overexpression of NR4A2 gene, and FIG. 5C is an excess of NR4A2 gene. The results of examining changes in IFN-γ produced in CD4 T cells after expression are shown respectively. In one Example of this invention, it is a figure which shows the result of having screened siRNA which can suppress the expression of a human NR4A2 gene. FIG. 6A shows the result of examining the expression level of the NR4A2 gene when the amount of siRNA1 introduced is changed, and FIG. 6B shows the result of examining the expression level of the NR4A2 gene when the amount of siRNA2 and siRNA3 is changed. Each is shown. In one Example of this invention, it is a figure which shows the result of having investigated the effect | action which human NR4A2 protein exerts on inflammatory cytokine production and cell proliferation using siRNA1. In one Example of this invention, it is a figure which shows the result of having investigated the effect | action which human NR4A2 protein exerts on cytokine production, such as IFN-γ, TNFα, and IL-10, using siRNA-1. In one Example of this invention, it is a figure which shows the result of having investigated the effect | action which human NR4A2 protein exerts on cytokine production, such as IL-4, IL-2, and IL-17, using siRNA-1. In one embodiment of the present invention, siRNA-1 is used to suppress experimental autoimmune encephalomyelitis (EAE) induced in mice by suppressing the expression of the NR4A2 gene in T cells. It is a figure which shows the result.

  Hereinafter, embodiments of the present invention completed based on the above knowledge will be described in detail with reference to examples. Unless otherwise stated in the embodiments and examples, J. Sambrook, EF Fritsch & T. Maniatis (Ed.), Molecular cloning, a laboratory manual (3rd edition), Cold Spring Harbor Press, Cold Spring Harbor, New York (2001); FM Ausubel, R. Brent, RE Kingston, DD Moore, JG Seidman, JA Smith, K. Struhl (Ed.), Standard Protocols in Molecular Biology, John Wiley & Sons Ltd. The method described in the protocol collection, or a modified or modified method thereof is used. In addition, when using commercially available reagent kits and measuring devices, unless otherwise explained, protocols attached to them are used.

  The objects, features, advantages, and ideas of the present invention will be apparent to those skilled in the art from the description of the present specification, and those skilled in the art can easily reproduce the present invention from the description of the present specification. it can. The embodiments and specific examples of the invention described below show preferred embodiments of the present invention, and are shown for illustration or explanation. It is not limited. It will be apparent to those skilled in the art that various modifications and variations can be made based on the description of the present specification within the spirit and scope of the present invention disclosed herein.

== Screening method ==
As described above, the expression of NR4A2 gene and the expression of cytokine genes such as IL-17, IFN-γ, IL-2, TNFα, IL-10, and IL-4, that is, the production of these cytokines has a positive correlation. It was revealed that the NR4A2 gene may be directly or indirectly involved in the positive expression control of these cytokine genes. In addition, suppression of NR4A2 gene expression can suppress the production of cytokines such as IL-17, IFN-γ, IL-2, TNFα, IL-10, and IL-4, and proliferation of CD4-positive T cells. Became clear. Therefore, by identifying substances that can suppress the function of the NR4A2 protein in IL-17-producing T cells, substances that improve inflammation caused by IL-17, substances that suppress IL-17 production (IL- 17 substance expression-suppressing substances), and substances that suppress the proliferation of CD4-positive T cells can be screened.

  Here, to suppress the function of the NR4A2 protein in IL-17-producing T cells means to suppress the overall function level of the NR4A2 protein in the cells, and as a method therefor, for example, inhibition of the function of the NR4A2 protein Compounds and function-inhibiting antibodies may be bound to NR4A2 protein to reduce the functional level inherent to NR4A2 protein, or mRNA that encodes NR4A2 protein or NR4A2 protein is degraded, or expression of NR4A2 gene is suppressed. The method is not particularly limited, but it is preferable to suppress the expression by the RNAi method from the technical simplicity.

  Identification of a substance capable of suppressing the function performed by the NR4A2 protein is performed by, for example, causing a test substance to act on an NR4A2 gene-expressing cell, and then regarding the expression level of the NR4A2 gene, This can be done by comparing and evaluating before the test substance is allowed to act.

  Alternatively, for example, a recombinant vector in which a gene encoding a marker such as GFP, luciferase, or beta-galactosidase is inserted into an expression vector having an enhancer / promoter of the NR4A2 gene, electroporation, microinjection, lipofection Introduced into an appropriate host cell (for example, T cell) by the transfection method using calcium or calcium, and the test substance is not allowed to act on the cell for the expression of the marker. Comparison / evaluation with the same type of cells or before the test substance is allowed to act may be performed. When the recombinant vector is phage DNA or viral DNA, the gene may be introduced by infecting an appropriate host cell (for example, T cell).

  Here, the action of a test substance on a cell means that the test substance is directly or indirectly added to or administered to the cell so that the test substance acts on the cell in vitro or in vivo. This refers to demonstrating the efficacy of the test substance. The target cell is an NR4A2 gene that is endogenously introduced into the NR4A2 gene promoter, even if the NR4A2 gene or marker gene is artificially introduced by the NR4A2 gene promoter. May be a cell that expresses, and may be a cultured cell or a cell in an individual.

  When using an animal individual, for example, a test substance is administered to a vertebrate that has developed an inflammatory disease caused by IL-17, and the expression level of the NR4A2 gene in the IL-17-producing T cell of the individual is measured. This can be done by comparing and evaluating the same type of animal not administered with the test substance, or before administration of the test substance. Here, IL-17-producing T cells are not particularly limited, and examples thereof include peripheral blood T cells and CNS infiltrating T cells. In addition, administration of the test substance to the vertebrate may be performed directly in the vicinity of the affected area, or may be administered orally or parenterally such as intravenously or intraperitoneally.

  The expression level of the NR4A2 gene can be measured by measuring the amount of mRNA by, for example, quantitative RT-PCR (quantitative reverse transcription-polymerase chain reaction) method, Northern blot method, dot blot method, etc. It is preferable to use a quantitative RT-PCR method in that the amount of mRNA can be measured using RNA (isolated from blood).

  The primer used in the quantitative RT-PCR method is not particularly limited as long as it can specifically detect NR4A2 gene expression, but an oligonucleotide consisting of 12 to 26 bases is preferable. The nucleotide sequence can be determined based on the conventional sequence method of the NR4A2 gene of the target vertebrate. And the primer which has the determined arrangement | sequence can be obtained by producing using a DNA synthesizer, for example. Specifically, for example, X75918, AB017586, AB019433, BC009288, and S77154 if the animal is a human, U01146, L08595, U72345, NM_019328 if the animal is a rat, and if the animal is a mouse Primers can be prepared based on the sequence information registered in NM_013613.

== NR4A2 protein function inhibitor ==
As described above, IL-17, IFN-γ, IL-2, TNFα, IL-10, IL-4 and other cytokine production by suppressing the function of NR4A2 protein, and CD4 positive T cell proliferation Therefore, substances that suppress the function of NR4A2 protein in IL-17-producing T cells can improve inflammation caused by IL-17, suppress IL-17 production, and suppress proliferation of CD4-positive T cells. Useful.

The substance that suppresses the function performed by the NR4A2 protein means a substance that suppresses the overall function level of the NR4A2 protein in the cell, and may be, for example, a substance that suppresses the expression of the NR4A2 gene. SiRNA that suppresses the expression of the NR4A2 gene by the RNAi method (in this specification, siRNA and siDNA, or a chimeric molecule of siDNA and siRNA are collectively referred to), or a recombinant vector that expresses the siRNA , etc. Can be used. Examples of siRNAs that suppress the expression of the NR4A2 gene include 5'-GGACAGCAGUCCUCCAUUAAUU-3 '(SEQ ID NO: 1), 5'-GGUUCGCACAGACAGUUUAAUU-3' (SEQ ID NO: 2), or 5'-GGCGAACCCUGACUAUCAAAUU-3 '(SEQ ID NO: 1). 3), or an oligonucleotide having a base sequence complementary to the base sequence. A base sequence complementary to the base sequence of SEQ ID NO: 1 is 5′-UUAAUGGAGGACUGCUGUCCUU-3 ′ (SEQ ID NO: 12). The base sequence complementary to the base sequence of SEQ ID NO: 2 is 5′-UUAAACUGUCUGUGCGAACCUU-3 ′ (SEQ ID NO: 13). A base sequence complementary to the base sequence of SEQ ID NO: 3 is 5′-UUUGAUAGUCAGGGUUCGCCUU-3 ′ (SEQ ID NO: 14). In the present invention, these oligonucleotides may be sugar chains, nucleobases, or ribonucleotides (including deoxyribonucleotides) modified in the backbone.

Examples of ribonucleotides modified with sugar chains include, for example, the 2′-OH group of ribose substituted with a group selected from H, OR, halo, SH, SR, NH ₂ , NHR, NR ₂ or CN (Wherein R is C ₁ -C ₆ alkyl, alkenyl or alkynyl and halo is F, Cl, Br or I). Examples of ribonucleotides with modified nucleobases include uridine or cytidine substituted at the 5-position (eg, 5- (2-amino) -propyluridine, 5-bromouridine, etc.), adenosine substituted at the 8-position Alternatively, guanosine (for example, 8-bromoguanosine and the like), deazanucleotide (for example, 7-deazaadenosine and the like), O- or N-alkylated nucleotide (for example, N6-methyladenosine and the like) and the like can be mentioned. In addition, examples of the ribonucleotide whose backbone is modified include those in which a phosphate group is substituted with a modifying group such as a thiophosphate group.

  The above-mentioned oligonucleotide can be synthesized by a DNA / RNA synthesizer or a conventional method such as an in vitro transcription reaction, an enzyme synthesis method, a solid phase synthesis method or the like.

  Examples of the recombinant vector expressing siNA include, for example, when siNA is an unmodified RNA, an expression vector virus such as a viral vector or a plasmid incorporating a DNA encoding the siNA.

  In addition, cell lines in vitro infected with an expression vector in which the DNA is incorporated into a viral vector, viruses produced from the cell line, and the like are also useful as substances that suppress the expression of the NR4A2 gene. When a virus is used as a drug or pharmaceutical composition, it is preferable to mutate the virus so as not to proliferate by a known method such as genetic manipulation in order to prevent toxicity and side effects. For example, a retrovirus or adenovirus that is infectious but modified so as not to replicate may be used that is incorporated into an expression vector capable of expressing the above siNA. Examples of the viral vector include an adenovirus vector, a retrovirus vector, and a phage vector.

  In addition, liposomes encapsulating the above siNA are also useful as substances that suppress the expression of the NR4A2 gene. In this case, these oligonucleotides can be introduced into the target site in vivo by using Trans IT In Vivo Gene Delivery System (trade name of TaKaRa) or the like.

  Furthermore, the above-described polymeric micelle encapsulating siNA also has excellent properties as a drug carrier and is useful as a substance that suppresses the expression of the NR4A2 gene.

  In addition, a complex of the above-mentioned plasmid for expressing siNA in cells and a polymer carrier can also carry the plasmid to the target tissue where it can express siNA, thereby suppressing the expression of the NR4A2 gene. It is useful as a substance to be used. As the polymer carrier, any substance that can introduce the plasmid into cells may be used. For example, polylysine, polyethyleneimine, porotamine, chitosan, synthetic peptide, or dendrimer Etc. can be used.

  In order to suppress gene expression, the miNA method (including miRNA, miDNA, and a chimeric molecule in which some nucleotides are substituted with deoxynucleotides) may be used instead of the siNA method. The method of expressing in the same manner as described above for siNA.

== Drug / Pharmaceutical Composition of the Present Invention ==
A composition containing, as an active ingredient, a substance that suppresses the function of the NR4A2 protein may be administered as a pharmaceutical to humans or vertebrates other than humans, or may be used as a reagent for experiments. Such pharmaceuticals may be administered directly to the affected area, orally as tablets, capsules, granules, powders, syrups and the like, or as injections and intraperitoneally. Or parenteral administration by intravenous injection. In addition, pharmaceutical preparations are carried out by conventional methods using conventionally used formulation additives (for example, excipients, binders, lubricants, disintegrating agents, flavoring agents, solvents, stabilizers, etc.). be able to.

  Hereinafter, the present invention will be specifically described by way of examples.

[Experimental Example 1]
First, in comparison with healthy individuals, genes that selectively show high expression in peripheral blood T cells derived from multiple sclerosis (MS) patients were identified.

  Blood samples were collected from 57 MS patients and 19 healthy individuals, and PBMCs (peripheral blood mononuclear cells) were isolated by ficoll density gradient centrifugation. Each PBMC was reacted with magnetic microbeads coated with anti-CD3 antibody, and CD3 + T cells were separated by AutoMACS (Miltenyi Biotec, Auburn, CA). Subsequently, total RNA was isolated from each CD3 + T cell using the RNeasy Mini Kit (Qiagen, Valencia, CA), and aminoscripts were prepared using Random primer (Invitrogen) with Superscript II RNase H-Reverse Transcriptase (Invitrogen). Reverse transcription reaction was performed in the presence of dUTP (Sigma). Thereafter, the fluorescent dye Cy5 was incorporated using Cy5 mono-reactive dye (currently GE Healthcare) to label the cDNA. In this experiment, for mRNA obtained from healthy individuals, a labeled cDNA was prepared by incorporating Cy3 (Cy3 mono-reactive dye (currently GE Healthcare)) during reverse transcription reaction, and the gene expression level was standardized. Used for.

  Hybridization solution containing equal amount of Cy3- or Cy5-labeled cDNA was hybridized with cDNA microarray at 62 ° C for 10 hours, scanned using ScanArray 5000 scanner (GSI Lumonics, Boston, MA), and QuantArray software ( Data analysis was performed by GSI Lumonics (global correction). The signal intensity of Cy3 and Cy5 was corrected by global normalization, and the gene expression level was (MS patient signal intensity) / (healthy person signal intensity) as an index. Statistical analysis was performed with Cyber-T software (http://www.genomics.uci.edu). In addition, as the cDNA microarray described above, an oncogene, a transcription factor, a signal transmitter, a cell cycle-related gene, an apoptosis-related gene, and a cytokine / cell can be obtained by the method described in the literature (F. Koike et al., 2003). Growth factors and their receptors, and cDNAs of 1263 genes such as housekeeping genes were immobilized on a slide glass coated with poly-L-lysine (Hitachi Life Science, Saitama, Japan).

  As a result, a significant change was observed in 286 genes among the analyzed 1263 genes, of which 87 genes were significantly upregulated in peripheral blood T cells derived from MS patients, of which the genes with the highest upregulation were observed. As one, the NR4A2 gene encoding a transcription factor was identified. Next, quantitative RT-PCR was performed to quantify the expression level of the NR4A2 gene in MS patients and healthy individuals.

  1 μg of total RNA isolated from CD3 + T cells of 57 MS patients and 19 healthy individuals was treated with DNase and randomized as a primer, SuperScript II RNase H-reverse transcriptase (Invitrogen, Carlsbad, CA) Then, cDNA was synthesized. Using the obtained cDNA as a template, a primer specific for NR4A2 cDNA (forward primer 5'-CGACATTTCTGCCTTCTCC-3 ': SEQ ID NO: 4, reverse primer 5'-GGTAAAGTGTCCAGGAAAAG-3': SEQ ID NO: 5) and Light Cycler-FastStart DNA NR4A2 cDNA was amplified by PCR using Light Cycler ST300 (Roche Diagnostics GmbH, Mannheim, Germany) using Master SYBR Green I kit (Roche), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal standard. As quantified the expression level of NR4A2 gene. The result is shown in FIG.

  As shown in FIG. 1, it was found that the expression level of the NR4A2 gene was approximately five times higher in the CD3 + T cells of MS patients than in healthy individuals.

[Experiment 2]
In this experimental example, experimental autoimmune encephalomyelitis (EAE) was induced in mice, and the pathogenesis of EAE after induction, spleen (SPL) T cells, regional lymph node (dLN) T The correlation with the expression level of NR4A2 gene in cells and T cells infiltrating the central nervous region (CNS) was examined.

8-12 weeks old female C57BL / 6 (B6) mice (CLEA Laboratory Animal Corp, Tokyo, Japan) were subcutaneously emulsified with 100 μg of MOG _35-55 peptide and heat treated Mycobacterium tuberculosis H37RA (Difco) Immunized with 1 mg of complete Freund's adjuvant. On the day of immunization (day 0) and 2 days after immunization, pertussis toxin (200 ng in PBS; List Biological Laboratories) was administered intraperitoneally to induce EAE (day 0: d0). And scored daily (FIG. 2A).
0: no clinical symptoms, 1: reduced tail tension, 2: tail relaxation, 3: partial paralysis of the hind legs, 4: paralysis of the entire hind legs, 5: paralysis of the forefoot and hind legs

  In addition, mice were killed at 0, 9, 15, and 21 days after induction of EAE (d0, d9, d15, and d21), and mononuclear cells that had infiltrated SPL cells, dLN cells, and CNS were detected in Percoll density. Each was collected by gradient centrifugation. These cells are labeled with anti-CD3-PE antibody (2C11; BD Pharmingen), and CD3 + T cells are separated by EPICS ALTRA Cell Sorting System (Beckman Coulter, Tokyo, Japan) (CD3 + T cell purity is 93% or more) Similarly to the method described in Experimental Example 1, the expression level of the NR4A2 gene was measured by quantitative RT-PCR (FIG. 2B).

  As shown in FIG. 2, the expression of NR4A2 gene in CD3 + T cells of spleen and regional lymph nodes of EAE-induced mice did not change regardless of the pathogenesis, but NR4A2 in CD3 + T cells infiltrated into CNS. It was found that gene expression was high at the onset of the disease state and then decreased.

[Experiment 3]
In the pathogenesis of multiple sclerosis (MS), IL-17-producing T cells (Th17 cells) that have infiltrated the central nervous system have been reported to be the main body of pathogenic T cells (Current Opinion in Immunology 2006) , 18: 349-356). Therefore, it was examined whether IL-17 was produced in the spleen cells and CNS infiltrating cells described above.

  Similarly to the method described in Experimental Example 2, CD3 + T cells were separated from SPL cells and CNS infiltrating mononuclear cells on day 17 after EAE induction. Thereafter, Fc on the cell surface was blocked with an anti-CD16 / 32 antibody (2.4G2) (BD Bioscience) and stained with an anti-CD4-PerCP antibody (L3T4; BD Pharmingen). After staining, the cells were washed and fixed with Cytofix / Cytoperm solution (TC11-18H10; BD Pharmingen), permeabilized the cell membrane, and intracellular cytokines were stained with anti-IL-17-PE antibody (BD Pharmingen), and FACScaliber IL-17 production was analyzed by flow cytometer (BD bioscience). The result is shown in FIG.

  As shown in FIG. 3, IL-17 production was hardly observed in SPL CD3 + T cells in which the expression of NR4A2 gene was not changed by the induction of EAE, but the expression of NR4A2 gene was enhanced by the induction of EAE. Many of the CNS infiltrated CD3 + T cells were found to produce IL-17.

[Experimental Example 4]
Next, the correlation between NR4A2 gene expression and IL-17 production was analyzed by a reporter assay.
All open reading frames (ORF) of human NR4A2 cDNA were amplified by PCR and introduced into the BamHI / XhoI site of pcDNA4 / HisMax vector (Invitrogen). In PCR, a sequence complementary to the vector region is used, primer 5′-AGTCGGATCCCCTTGTGTTCAGGCGCAG-3 ′ (SEQ ID NO: 6) containing a BamHI recognition site is used as a forward primer, and primer 5 containing an XhoI site is used as a reverse primer. '-AGTCCTCGAGTTAGAAAGGTAAAGTGTCCAG-3' (SEQ ID NO: 7) was used respectively.

  The reporter gene plasmid includes DNA upstream of the transcription start site of IL-17, IL-2, or IFN-γ (IL-17: 1125 bp, IL-2: 568, IFN-γ: 538 bp) The fragment was generated by introducing it into pGL3-basic plasmids (Promega). The fragment containing DNA upstream of the transcription start site of IL-17 is 5′-ATGCGAGCTCGGATCTCAGGACAAACAGGTTC-3 ′ (SEQ ID NO: 8) and HindIII site as a forward primer for the IL-17 promoter region containing the SacI site. It was obtained by amplifying by PCR using 5′-ATGCAAGCTTGACTCACCACCAATGAGGTCTT-3 ′ (SEQ ID NO: 9) as a reverse primer for the IL-17 promoter region contained therein. In addition, as a fragment containing DNA upstream of the transcription start site of IL-2 and IFN-γ, HindIII fragment (−568 to +50) of IL-2 promoter and XbaI-BamHI fragment (−538) of IFN-γ promoter ~ + 64) were used respectively (J Exp Med Vol. 178 1483-1496, 1993).

Next, mice were added to RPMI 1640 medium supplemented with 10% FCS (fetal bovine serum), 2 mM L-glutamine, 100 U / ml penicillin / streptomycin, 2 mM sodium pyruvate, and 50 μM β-mercaptoethanol (complete medium). EL4 cells were suspended (1 × 10 ⁷ cells / 0.5 ml). In 0.5 ml of this cell suspension, 10 μg of a reporter gene plasmid having a DNA fragment upstream of the transcription start site of IL-17, IL-2, or IFN-γ, 0.1 μg of Renilla luciferase plasmid, and 10 μg of pcDNA4-NR4A2 Alternatively, 10 μg of pcDNA4-LacZ was added, and the plasmid was introduced into EL4 cells using GenePulser electroporator II (Bio-Rad) in the presence of DEAE (diethylaminoethyl) -dextran. Six hours later, 50 ng / ml PMA and 1 μg / ml ionomycin were added to stimulate the DNA-introduced cells, and the cells were further cultured for 18 hours. In addition, as a control, cells in which each DNA-introduced cell was cultured for 24 hours without adding PMA and ionomycin were prepared.

  After culturing, each cell is collected, lysed with 50 μl of 1XPLB, luciferase activity is measured by Dual-Luciferase Reporter Assay System (Promega) (Luminometer TD-20 / 20, Turner Designs), and the luciferase activity of Renilla is measured. Normalization was performed to obtain standard deviation (SD values) (FIG. 4A).

  Also, use a reporter gene plasmid with a DNA fragment upstream of the transcription start site of IL-17 gene, and change the amount of pcDNA4-NR4A2 or pcDNA4-LacZ added (0, 4, 8, 12, 16, or 20 μg). Except for not stimulating, luciferase activity was measured in the same manner as described above (FIG. 4B).

  As shown in FIG. 4A, in the NR4A2 gene-introduced cell, a reporter gene containing a promoter region of a gene for inflammatory cytokines such as IL-17, IFN-γ, and IL-2 regardless of the presence or absence of stimulation with PMA and ionomycin Was found to activate the transcription of. Further, as shown in FIG. 4B, it was found that the transcription activity of the reporter gene including the promoter region of IL-17 gene can be enhanced by increasing the amount of pcDNA4-NR4A2 without stimulation.

[Experimental Example 5]
In addition, the correlation between NR4A2 gene expression and IL-17 production was analyzed using a bicistronic retroviral vector that co-expresses NR4A2 protein and eGFP as an infection index.

  Using a forward primer containing a Bgl II site (5′-ATGCAGATCTATGGGGGGTTCTCATCATCA-3 ′: SEQ ID NO: 10) and a reverse primer containing the above Xho I site (SEQ ID NO: 7), the ORF of human NR4A2 cDNA was transformed into pMIG A gene was introduced into the BamHI / XhoI site of -Φ (pMIG-W bicistronic retrovirus vector; provided by L. Van Parijis (Massachusetts Institute of Technology, Cambridge, Massachusetts, USA); FIG. 5A) to prepare pMIG-NR4A2. In FIG. 5A, “LTR” means long terminal repeat, “IRES” means internal ribosome entry site, and “eGFP” means enhanced green fluorescence protein.

  Next, mouse CD4 T cells were purified from the spleen by AUTOMACS using a mouse CD4 T isolation kit (BD pharmingen). Thereafter, the cells were stimulated with anti-CD3 antibody (2C11) and anti-CD28 antibody (37.51) for 24-48 hours in complete medium supplemented with IL-2 (100 U / ml). This activated CD4 T cell was infected twice with a retrovirus prepared by co-transfection of 293T cells with pMIG retroviral vector (pMIG-NR4A2 or pMIG-Φ) and pCL-Eco Retrovirus Packaging Vector. The cells were then cultured for 3 days in complete medium containing 30 U / ml IL-2, and CD4 T cells were stimulated again with PMA (20 ng / ml) and ionomycin (1 mg / ml) for 4 hours in the presence of 2 μM monensin. Intracellular cytokines were detected using anti-CD4-PerCP antibody and anti-IL-17-PE antibody or anti-IFN-γ-PE antibody (XMG1.2; BD Pharmingen). The results are shown in FIGS. 5B and 5C, respectively. In FIGS. 5B and 5C, R1 and R3 indicate GFP-CD4 T cells, and R2 and R4 indicate GFP + CD4 T cells, respectively.

  As shown in FIGS. 5B and 5C, when the NR4A2 gene was forcibly expressed and stimulated with PMA and ionomycin, IL-17 and IFN-γ production was significantly increased in GFP-positive cells (10.3% and 31.1%, respectively). However, GFP-negative cells showed almost no increase in the production of these inflammatory cytokines. Therefore, IL-17 and IFN-γ are produced by expressing the NR4A2 gene in activated CD4 T cells.

[Example 1]
In this example, in order to analyze the effect of human NR4A2 protein on inflammatory cytokine production, siRNA capable of suppressing the expression of human NR4A2 gene was first screened.

In 4 ml of DMEM (Dulbecco's Modified Eagle Medium) supplemented with 10% FCS, 2 mM L-glutamine, 100 U / ml penicillin / streptomycin, 2 mM sodium pyruvate, and 50 μM β-mercaptoethanol, 293T cells (0.8 × 10 ⁶ cells) was suspended, and the candidate siRNA (20, 50, 100 pmol) was introduced into 293T cells together with pcDNA4-NR4A2 (0.2 μg) by lipofection using Lipofectamine 2000 (Invitrogen). In addition, as a control, those in which only pcDNA4-NR4A2 was introduced and those in which siRNA and pcDNA4-NR4A2 were not introduced were prepared. After culturing for 24 hours, the expression level of the NR4A2 gene was measured by quantitative RT-PCR according to the method described in Experimental Example 1, and siRNA useful for suppressing the expression of the NR4A2 gene was screened.

  As a result, three siRNAs [siRNA-1 sense sequence: 5'-GGACAGCAGUCCUCCAUUAAUU-3 '(SEQ ID NO: 1), siRNA-2 sense sequence: 5'-GGUUCGCACAGACAGUUUAAUU-3' ( SEQ ID NO: 2) and the sense sequence of siRNA-3: 5′-GGCGAACCCUGACUAUCAAAUU-3 ′ (SEQ ID NO: 3)] were identified (FIGS. 6A and 6B).

  Next, the most effective siRNA-1 of these siRNAs was used to suppress the expression of the NR4A2 gene, and the effects on the ability to produce cytokines from human peripheral blood T cells and CD4 positive T cell proliferation were examined. .

  Using human CD4 T cell isolation kit (Milteniyi Biotech GmbH, Bergisch Gladbach, Germany), human CD4 T cells were isolated from healthy human PBMC by the AUTOMACS cell purification system. The obtained CD4 T cells were mixed with 200 pmol siRNA-1 (siRNA) or control RNA [control RNA sense sequence: 5'-CCGAACGUUAAUCCCGGAUAUU-3 '(SEQ ID NO: 11) containing -UU-3'-overhang]. The suspension was suspended in 100 μl Nucleofector solution (Human T cell Nucleofector Kit; Amaxa Biosystems, Cologne, Germany) and nucleofected using the Amaxa Nucleofector II apparatus. Thereafter, CD4 T cells were transferred to a complete medium and cultured for 8 hours. Thereafter, CD4 T cells were further stimulated with an anti-CD3 antibody (OKT-3). As a control, cells cultured in a medium not containing anti-CD3 antibody (OKT-3) were prepared.

After incubation for 48 hours, the amount of IL-17 and IFN-γ produced in the supernatant was determined by ELISA (Human IL-17 DuoSet; R & D Systems, Minneapolis, USA) or CBA Human Th1 / 2 cytokine II kit (BD Pharmingen). Measurement and quantitative analysis of changes in cytokine production ability. In addition, after culture, CD4 T cells were further cultured for 16 hours in the presence of [ ³ H] thymidine (1 μCi / well), and the radioactivity was measured with a β-1205 counter (Pharmacia, Uppsala, Sweden). Changes in cell proliferation were quantitatively analyzed. The results are shown in FIG.

  As shown in FIG. 7, by suppressing the expression of the NR4A2 gene with siRNA specific to NR4A2 cDNA, IL-17 production, IFN-γ production, and CD4 positive T cell proliferation by stimulation with anti-CD3 antibody are significantly suppressed. I knew it was possible.

[Example 2]
Next, it was investigated whether or not the production amount of various cytokines was reduced by suppressing siRNA-1 to MS patient-derived peripheral blood T cells to suppress the expression of NR4A2 gene.

  Similar to the method described in Example 1, peripheral blood T cells were isolated from healthy PBMC and MS patient PBMC, respectively, nucleated with siRNA-1 or control RNA, cultured, and CD4 T cells were immobilized on anti-CD3 The antibody (OKT-3) was further stimulated for 48 hours, and various cytokine production amounts (IFN-γ, TNFα, IL-10, IL-4, IL-2, IL-17, etc.) were measured. The amounts of TNFα, IL-10, IL-4, and IL-2 were quantified using CBA Human Th1 / 2 cytokine II kit (BD Pharmingen). The results are shown in FIGS.

  As shown in FIG. 8 and FIG. 9, not only healthy human-derived peripheral blood T cells but also MS patient-derived peripheral blood T cells, by acting siRNA, IFN-γ, TNFα, IL-10, IL- 4. It was found that the production of cytokines such as IL-2 and IL-17 can be reduced. In particular, it was found that the production amount of IL-17 and IL-10 can be reduced to the level of peripheral blood T cells derived from healthy individuals by allowing siRNA to act on peripheral blood T cells derived from MS patients.

  From the above, it is clear that the expression of NR4A2 gene and the production of inflammatory cytokines such as IL-17, IFN-γ and IL-2 show a positive correlation, and NR4A2 protein is directly or indirectly The possibility of being involved in the regulation of cytokine gene expression was shown.

[Example 3]
10 days after immunization of Freund's complete adjuvant (CFA) emulsion containing 100 μg of PLP139-151 peptide and 1 mg of Mycobacterium tuberculosis (37RA), inguinal and popliteal lymph nodes on day 10 after immunization The lymphocytes were isolated from. Using the HVJ-E (GENOMEONE; Ishihara Sangyo, Osaka) to the resulting cells (4 × 10 ⁶ cells / ml), siRNA-1 (sense sequence is SEQ ID NO: 1) that suppresses the expression of the NR4A2 gene. Introduced. Thereafter, the cells were cultured for 3 days in a complete medium containing 35 μg / ml PLP peptide, and the collected T cell blasts (5 × 10 ⁶ cells / mouse) and pertussis were applied to SJF / J mice previously irradiated with 3 Gy radiation. EAE was induced by intraperitoneal administration of a toxin (200 ng in PBS; List Biological Laboratories), and clinical symptoms were scored as in Experimental Example 2.

  As a result, significant suppression of EAE was observed in the siRNA-treated group compared to the control RNA-treated group (FIG. 10A).

  Paraffin sections of cervical spinal cord tissue were prepared from mice sacrificed on day 31 after EAE induction, and hematoxylin / eosin staining (HE staining) and Luxol fast blue staining (LFB staining) were performed. As a result, in the siRNA-1-treated group, suppression of mononuclear cell infiltration into the tissue (HE staining) and inhibition of detachment (LFB staining) were observed as compared to the control RNA treatment group (FIG. 10B).

  Thus, by using siRNA to suppress the expression of the NR4A2 gene in T cells, EAE induced in mice can be suppressed.

  According to the present invention, IL-17 production suppression, CD4 positive T cell proliferation suppression, oligonucleotide useful for improving inflammation caused by IL-17, and the like, and NR4A2 gene expression containing it as an active ingredient Inhibitors, IL-17 gene expression inhibitors, CD4-positive T cell proliferation inhibitors, and pharmaceutical compositions can be provided.

Claims (6)

Sense strand oligonucleotide of the nucleotide sequence shown in any of SEQ ID NO: 1-3, and, the base sequence shown in SEQ ID NO: 12-14 are each a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 1-3 SiRNA that is a double-stranded RNA consisting of an antisense strand that is an oligonucleotide .
5'-GGACAGCAGUCCUCCAUUAAUU-3 '(SEQ ID NO: 1)
5'-UUAAUGGAGGACUGCUGUCCUU-3 '(SEQ ID NO: 12)
5'-GGUUCGCACAGACAGUUUAAUU-3 '(SEQ ID NO: 2)
5'-UUAAACUGUCUGUGCGAACCUU-3 '(SEQ ID NO: 13)
5'-GGCGAACCCUGACUAUCAAAUU-3 '(SEQ ID NO: 3)
5'-UUUGAUAGUCAGGGUUCGCCUU-3 '(SEQ ID NO: 14) The oligonucleotide is a sugar-modified ribonucleotide, and the 2′-OH group is substituted with a group selected from H, OR, halo, SH, SR, NH ₂ , NHR, NR ₂ or CN (provided that , R represents, C ₁ -C ₆ alkyl, alkenyl or alkynyl, halo, F, Cl, Br or I.) siRNA according to claim 1, characterized in that. A recombinant vector expressing the siRNA according to claim 1 or 2. An NR4A2 gene expression inhibitor comprising the siRNA according to claim 1 or 2 or the recombinant vector according to claim 3 as an active ingredient. An IL-17 gene expression inhibitor comprising the siRNA according to claim 1 or 2 or the recombinant vector according to claim 3 as an active ingredient. A CD4-positive T cell proliferation inhibitor containing the siRNA according to claim 1 or 2 or the recombinant vector according to claim 3 as an active ingredient.