WO2010004816A1

WO2010004816A1 - Inhibitor of degranulation of mast cell

Info

Publication number: WO2010004816A1
Application number: PCT/JP2009/060220
Authority: WO
Inventors: 陽史山田; 哲郎吉田
Original assignee: 協和発酵キリン株式会社
Priority date: 2008-07-07
Filing date: 2009-06-04
Publication date: 2010-01-14
Also published as: JP2011190176A

Abstract

Disclosed are: an inhibitor of the degranulation of a mast cell, which comprises, as an active ingredient, a substance capable of inhibiting the process of biosynthesis of microRNA from a microRNA precursor, such as a substance capable of inhibiting the expression of Dicer1 gene; a method for inhibiting the degranulation of a mast cell; and a therapeutic agent for a disease induced by the abnormality in a mast cell.

Description

Mast cell degranulation inhibitor

The present invention relates to a degranulation inhibitor for mast cells, a method for inhibiting degranulation of mast cells, and a therapeutic agent for diseases caused by abnormal mast cells.

It is known that mast cells are activated by various stimuli, cause degranulation, and release and produce many inflammatory mediators (Non-patent Documents 1 and 2). The main ones are a wide variety of amines, arachidonic acid metabolites, proteases, cytokines, chemokines and the like. For example, when an antigen is recognized by mast cells, histamine and tryptase are rapidly released by degranulation, and chemical mediators such as prostaglandin D2 (PGD2), leukotriene (LT), and platelet activating factor (PAF) It is known that various chemokines such as macrophage inflammatory protein (MIP) -1α and various cytokines such as granulocyte macrophage colony stimulating factor (GM-CSF) are newly synthesized and released. In addition, it has recently been clarified that the major basic protein (major basic protein), which is a cytotoxic protein previously thought to be produced by eosinophils, is produced in large amounts by mast cells in humans. (Non-Patent Document 3).

Thus, since mast cells are thought to play a major role in the pathogenesis of various allergic diseases, it is considered that allergic diseases can be treated by controlling the function of mast cells.

Information on genes expressed in mast cells is important in that they can be targets for therapeutic agents for various allergic diseases. For example, drugs that act on GPCRs expressed in mast cells are affected by inflammation from cultured human mast cells. It is known to significantly suppress the production of sex mediators. Specifically, the β2-adrenergic receptor agonist isoproterenol has a low concentration of 10 nmol / l and suppresses the release of histamine, LT, PGD2, GM-CSF and MIP-1α by 80% or more from cultured human mast cells. (Non-Patent Document 4).

Micro RNA (miRNA) is a small non-coding single-stranded RNA consisting of about 22 nucleotides that is not translated into protein, and is known to exist in many organisms including humans (Non-patent Documents 5 and 6). MicroRNAs are biosynthesized from genes that are transcribed into single or clustered microRNA precursors. In other words, the primary transcript, primary-microRNA (pri-miRNA), is first transcribed from the gene, and then in a stepwise process from pri-miRNA to mature microRNA, a precursor of about 70 bases with a characteristic hairpin structure. -microRNA (pre-miRNA) is biosynthesized from pri-miRNA. It is known that Drosha and DGCR8 are involved in this processing. Thereafter, pre-miRNA is transported from the nucleus to the cytoplasm via Exportin-5, and then processed by Dicer and TRBP, and mature microRNA is biosynthesized from pre-miRNA (Non-patent Document 7).

Mature microRNAs are thought to be involved in post-transcriptional regulation of gene expression by binding complementarily to the target mRNA and suppressing mRNA translation or by degrading mRNA.
It is known that microRNAs expressed in mammals including humans have an effect on mast cell degranulation (Patent Document 1). It is not known how to react like that.

International Publication No. 2008/029790 Pamphlet

An object of the present invention is to provide a degranulation inhibitor for mast cells, a method for inhibiting degranulation of mast cells, and a therapeutic agent for diseases caused by abnormal degranulation control of mast cells.

The present invention relates to the following.
[1] A mast cell degranulation inhibitor comprising as an active ingredient a substance that inhibits the process of biosynthesis of microRNA from a microRNA precursor.
[2] The degranulation inhibitor according to [1], wherein the substance that inhibits the process of biosynthesis of microRNA from a microRNA precursor is a substance that suppresses the expression of Dicer1 gene.
[3] The degranulation inhibitor according to [2], wherein the substance that suppresses the expression of the Dicer1 gene is a nucleic acid.
[4] The degranulation inhibitor according to [3], wherein the nucleic acid is siRNA.
[5] A mast cell degranulation inhibitor comprising, as an active ingredient, a vector that expresses siRNA that suppresses the expression of Dicer1 gene.
[6] The degranulation inhibitor according to [4] or [5], wherein the siRNA is a siRNA having a base sequence represented by any one of SEQ ID NOs: 1 to 5 as a target sequence.
[7] A method for inhibiting degranulation of mast cells, comprising using a substance that inhibits the process of biosynthesis of microRNA from a microRNA precursor.
[8] The method for inhibiting degranulation according to [7], wherein the substance that inhibits the process of biosynthesis of microRNA from a microRNA precursor is a substance that suppresses the expression of Dicer1 gene.
[9] The method for inhibiting degranulation according to [8], wherein the substance that suppresses the expression of the Dicer1 gene is a nucleic acid.
[10] The method for inhibiting degranulation according to [9], wherein the nucleic acid is siRNA.
[11] A method for inhibiting degranulation of mast cells, comprising using a vector that expresses siRNA that suppresses expression of Dicer1 gene.
[12] The method for inhibiting degranulation according to [10] or [11], wherein the siRNA is an siRNA having a base sequence represented by any one of SEQ ID NOs: 1 to 5 as a target sequence.
[13] A therapeutic agent for a disease caused by abnormal mast cells, comprising the degranulation inhibitor according to any one of [1] to [6] as an active ingredient.
[14] The therapeutic agent according to [13], wherein the disease caused by abnormality of mast cells is a disease selected from the group consisting of atopic dermatitis, asthma, chronic obstructive pulmonary disease and allergic disease.
[15] A method for treating a disease caused by abnormal mast cells, comprising administering an effective amount of the degranulation inhibitor according to any one of [1] to [6] to a subject in need thereof.
[16] The treatment method according to [15], wherein the disease caused by abnormality of mast cells is a disease selected from the group consisting of atopic dermatitis, asthma, chronic obstructive pulmonary disease, and allergic disease.
[17] Use of the degranulation inhibitor according to any one of [1] to [6] for the manufacture of a therapeutic agent for a disease caused by abnormality of mast cells.
[18] The use according to [17], wherein the disease caused by abnormality of mast cells is a disease selected from the group consisting of atopic dermatitis, asthma, chronic obstructive pulmonary disease, and allergic disease.

According to the present invention, a degranulation inhibitor for mast cells, a method for inhibiting degranulation of mast cells, and a therapeutic drug for diseases caused by abnormal mast cells can be provided.

The present invention provides a mast cell degranulation inhibitor comprising as an active ingredient a substance that inhibits the process of biosynthesis of microRNA from a microRNA precursor.

In the present invention, microRNA refers to single-stranded RNA having a length of 17 to 28 bases. The surrounding genomic sequence including the sequence of the microRNA has a sequence capable of forming a hairpin structure, and the microRNA can be cut out from any one strand of the hairpin. MicroRNAs complementarily bind to their target mRNA and suppress mRNA translation, or promote post-transcriptional control of gene expression by promoting mRNA degradation. The microRNA of the present invention includes mature microRNA.

In the present invention, the microRNA precursor is a nucleic acid having a length of about 50 to about 200 bases, more preferably about 70 to about 100 bases, and a hairpin structure is formed and microRNA is contained in one strand. Nucleic acid. The microRNA precursors of the present invention include primary-microRNA (pri-miRNA) and precursor-microRNA (pre-miRNA).

As a substance that inhibits the process of biosynthesis of microRNA from a microRNA precursor, a factor essential for biosynthesis of microRNA from the microRNA precursor (hereinafter also referred to as a factor essential for biosynthesis of microRNA). Any substance may be used as long as it inhibits the function of.

Factors essential for microRNA biosynthesis include, for example, Drosha and DGCR8, which are involved in the process of pre-miRNA biosynthesis from pri-miRNA, and the process of transport of pre-miRNA from the nucleus to the cytoplasm Examples include Dicer and TRBP involved in the process of biosynthesis of mature microRNA from Exportin-5 and pre-miRNA.

As a substance that inhibits the function of a factor essential for microRNA biosynthesis, for example, a substance that inhibits the expression of a gene encoding the factor, a substance that inhibits the function of the protein of the factor, a nucleic acid, an antibody, Any of low-molecular substances and the like may be used, but preferred is nucleic acid.

Substances that inhibit the function of factors essential for microRNA biosynthesis, preferably substances that inhibit the expression of the gene encoding Dicer (hereinafter also referred to as Dicer gene), and substances that inhibit the function of Dicer protein Can be given.

Examples of the Dicer gene include a Dicer1 gene, such as a human Dicer1 gene, and an ortholog that is a homologous gene of another species corresponding thereto, and preferably a human Dicer1 gene contained in the nucleotide sequence represented by SEQ ID NO: 6 or 7 can give. Moreover, Dicer1 is mentioned as a protein of Dicer, Preferably human Dicer1 is mention | raise | lifted.

Examples of substances that inhibit gene expression include substances that suppress the biosynthesis and translation of mRNA, and substances that reduce the amount of protein translated from mRNA by cleaving or decomposing mRNA.

Examples of nucleic acids that inhibit the function of factors essential for microRNA biosynthesis include the nucleic acids described in the following (a) to (c), and preferably the nucleic acids described in (a).
(A) siRNA including a base sequence of a target nucleic acid of a gene encoding the factor (short interferance RNA)
(B) an antisense nucleic acid for the transcription product of the gene encoding the factor or a part thereof (c) a nucleic acid having a ribozyme activity that specifically cleaves the transcription product of the gene encoding the factor

The nucleic acid may be any molecule as long as it is a molecule obtained by polymerizing nucleotides or molecules having functions equivalent to those of the nucleotides. Examples of nucleotides include RNAs that are polymers of ribonucleotides, DNAs that are polymers of deoxyribonucleotides, polymers in which RNA and DNA are mixed, and molecules in which molecules having functions equivalent to the nucleotides are polymerized. Examples thereof include a nucleotide polymer containing a nucleotide analog and a nucleotide polymer containing a nucleic acid derivative.

Nucleotide analogs include, for example, ribonucleic acid, to improve or stabilize nuclease resistance, increase affinity with complementary strand nucleic acids, increase cell permeability, or visualize, compared to RNA or DNA. Any molecule may be used as long as it is a modification of nucleotide, deoxyribonucleotide, RNA or DNA. Examples thereof include sugar moiety-modified nucleotide analogs and phosphodiester bond-modified nucleotide analogs.

The sugar moiety-modified nucleotide analog may be any one obtained by adding or substituting any chemical structural substance to a part or all of the chemical structure of the sugar of the nucleotide. For example, 2'-O-methyl Nucleotide analogues substituted with ribose, nucleotide analogues substituted with 2'-O-propylribose, nucleotide analogues substituted with 2'-methoxyethoxyribose, substituted with 2'-O-methoxyethylribose Nucleotide analogues, nucleotide analogues substituted with 2'-O- [2- (guanidinium) ethyl] ribose, nucleotide analogues substituted with 2'-O-fluororibose, introducing a bridging structure into the sugar moiety Bridged Nucleic Acid (BNA), more specifically, 2′-position oxygen atom and 4′-position carbon Locked Nucleic Acid (LNA) and ethylene-bridged nucleic acid (ENA) [Nucleic Acid search Research, 32, e175 (2004)] Furthermore, peptide nucleic acids (PNA) [Acc. Chem. Res., 32, 624 (1999)], oxypeptide nucleic acids (OPNA) [J. Am. Chem. Soc., 123, 4653 (2001)], and peptides Ribonucleic acid (PRNA) [J. Am. Chem. Soc., 122, 6900 (2000)] and the like.

The phosphodiester bond-modified nucleotide analog may be any one obtained by adding or substituting an arbitrary chemical substance to a part or all of the chemical structure of a phosphodiester bond of a nucleotide. Examples include nucleotide analogues substituted with thioate linkages, nucleotide analogues substituted with N3'-P5 'phosphoramidate linkages [Cell engineering, 16, 1463-1473 (1997)] [RNAi method And Antisense, Kodansha (2005)].

Examples of nucleic acid derivatives include atoms (for example, hydrogen atom, oxygen atom) or functional groups (for example, hydroxyl group, amino group) such as base portion, ribose portion, and phosphodiester bond portion of nucleic acid as other atoms (for example, hydrogen atom). , Sulfur atom), functional group (for example, amino group), or substituted with an alkyl group having 1 to 6 carbon atoms or protected with a protecting group (for example, methyl group or acyl group), nucleic acid, for example, lipid , Phospholipid, phenazine, folate, phenanthridine, anthraquinone, acridine, fluorescein, rhodamine, coumarin, and a molecule to which another chemical substance is added. In addition, examples of molecules obtained by adding another chemical substance to nucleic acid include 5′-polyamine addition derivatives, cholesterol addition derivatives, steroid addition derivatives, bile acid addition derivatives, vitamin addition derivatives, Cy5 addition derivatives, Cy3 addition derivatives, 6 -FAM addition derivatives, biotin addition derivatives, and the like are also included.

The siRNA containing the base sequence of the target nucleic acid of the gene encoding the factor essential for microRNA biosynthesis is a short double-stranded RNA containing the base sequence of a certain target nucleic acid, which is caused by RNA interference (RNAi). Any one can be used as long as it can suppress the expression of the target nucleic acid. The number of bases constituting one strand of siRNA is preferably 17 to 30 bases, more preferably 18 to 25 bases, and still more preferably 19 to 23 bases.

siRNA may be composed only of RNA, but as long as the expression of the target nucleic acid can be suppressed, DNA, RNA and DNA mixed polymers, nucleotide polymers including nucleotide analogs, nucleotide polymers including nucleic acid derivatives Any nucleic acid may be used, such as a substituted one.

The siRNA may also contain mismatches, bumps, loops, or wobble base pairs as necessary in the double-stranded molecule in order to regulate the ability to mediate inhibition of target gene expression. Such mismatches, bumps, loops or wobble base pairs are preferably placed in the terminal or internal region of the siRNA so that the siRNA does not significantly impair the ability to mediate inhibition of target gene expression.

Specifically, it is known that a double-stranded RNA having an overhang of several bases at the end has a high RNAi effect. Therefore, the double-stranded RNA used in the present invention may have an overhang of several bases at the end. The length and sequence of the base that forms this overhang are not particularly limited. The overhang may be either DNA or RNA. For example, two base overhangs such as TT (two thymines), UU (two uracils), and other base overhangs. For example, a molecule having a 19-base double-stranded RNA and a 2-base (TT) overhang can be preferably used. Double-stranded RNA includes molecules in which the base that forms the overhang is DNA.

The siRNA that suppresses the expression of the target nucleic acid is, for example, an algorithm for designing an siRNA for the gene based on the sequence [Nucleic Acids Res., 32, 936 (2004); Nature Biotechnology, 22, 326 (2004); Nature Biotechnology , 23, 995 (2005)]. SiRNAs designed using the above algorithm are commercially available from Qiagen, Applied Biosystems, Invitrogen, etc., and these may be used.

The siRNA preferably includes siRNA containing the base sequence of the target nucleic acid of human Dicer1 gene. The siRNA may be any siRNA that suppresses the expression of the endogenous human Dicer1 gene by introducing the siRNA into the cell.

SiRNA against human Dicer1 gene and the like can also be designed using the above algorithm. The siRNA in which the expression level of the human Dicer1 gene is reduced or suppressed is preferably SEQ ID NO: 1, 2, 3 [Molecular Cell 24, 157 (2006)], 4 [Nature, 436, 740 (2005)] or 5 Examples include siRNAs having a base sequence represented by [Nature, 436, 740 (2005)] as a target sequence.

For example, examples of the siRNA having the base sequence represented by SEQ ID NO: 1 as a target sequence include the base sequences shown below.
Antisense strand: 5'-CUAGGAUCCAGAUAGCACAdTdT-3 '(SEQ ID NO: 8)
Sense strand: 5'-UGUGCUAUCUGGAUCCUAGdTdT-3 '(SEQ ID NO: 9)

Moreover, as siRNA which makes the base sequence represented by the said sequence number 2 a target sequence, the base sequence shown below can be mention | raise | lifted, for example.
Antisense strand: 5'-UCCAGAGCUGCUUCAAGCAdTdT-3 '(SEQ ID NO: 10)
Sense strand: 5'-UGCUUGAAGCAGCUCUGGAdTdT-3 '(SEQ ID NO: 11)

In the above SEQ ID NOs: 8 to 11, “dT” is T (overhang) of DNA, and the other is RNA.

Furthermore, the present invention provides a degranulation inhibitor for mast cells, containing as an active ingredient a vector that expresses siRNA that suppresses the expression of Dicer1 gene. Those skilled in the art can easily prepare a vector capable of expressing the siRNA by a general genetic engineering technique. For example, the expression vector described in 3 below can be used.

The antisense nucleic acid for the transcription product of a gene encoding a factor essential for microRNA biosynthesis or a part thereof preferably has a sequence complementary to the gene encoding the factor or a part thereof. . The antisense nucleic acid may not be completely complementary as long as it can effectively suppress the expression of the gene encoding the factor, but is preferably 90% or more with respect to the transcription product of the gene encoding the factor. Preferably it has a complementarity of 95% or more. In order to effectively suppress the expression of a target gene using an antisense nucleic acid, the length of the antisense nucleic acid is at least 15 bases, preferably 100 bases or more, more preferably 500 bases or more.

As an antisense nucleic acid, if an antisense sequence complementary to the untranslated region near the 5 ′ end of the mRNA of the gene encoding the factor is designed, it is considered effective for inhibiting the translation of the gene. In addition, a sequence complementary to the coding region or the 3 ′ untranslated region can also be used.

The antisense nucleic acid can be transformed into a desired animal using a known method after ligating, for example, downstream of an appropriate promoter, and preferably ligating a sequence containing a transcription termination signal on the 3 ′ side.

As a nucleic acid having ribozyme activity that specifically cleaves a transcript of a gene encoding a factor essential for microRNA biosynthesis, any RNA molecule having catalytic activity may be used, such as group I intron type or RNase P. Such as those having a size of 400 bases or more, such as the M1 RNA contained in, and those having an active domain of about 40 bases called hammerhead type or hairpin type.

The self-cleaving domain of the hammerhead ribozyme cleaves 3 'of C15 in the sequence G13U14C15, but base pairing between U14 and A9 is important for its activity. Instead of C15, A15 or U15 Can be cut [FEBS Lett., 228, 228 (1988)]. By designing a ribozyme whose substrate binding site is complementary to the RNA sequence in the vicinity of the target site, it is possible to create a restriction enzyme-like RNA-cleaving ribozyme that recognizes the sequence UC, UU or UA in the target RNA [FEBSettLett , 239, 285 (1988); Protein Nucleic Acid Enzyme, 35, 2191 (1990); Nucl. Acids, Res 17, 7059 (1989)].

Hairpin ribozymes are found, for example, in the minus strand of satellite RNA of tobacco ring spot virus [Nature, 323, 349 (1986)]. By using a known method [Nucl. Acids Res, 19, 6751 (1991); Chemistry and Biology, 30, 112 (1992)] from a hairpin ribozyme, an RNA-cleaving ribozyme specific for the base sequence of the target nucleic acid May be produced. Examples of the substance that inhibits the function of the protein of the factor essential for microRNA biosynthesis include the antibody described in (a) below or the low molecular compound described in (b).
(A) an antibody that binds to the factor (b) a low molecular compound that binds to the factor

Antibodies that bind to factors essential for biosynthesis of microRNAs are prepared by methods known to those skilled in the art using recombinant proteins prepared using genetic recombination techniques in addition to the natural proteins of the factors. be able to.

For example, a polyclonal antibody can be prepared as follows. A small protein such as a rabbit is immunized with a recombinant protein expressed in a microorganism such as Escherichia coli or a partial peptide thereof as a protein of the factor or a fusion protein with GST to obtain serum. This can be prepared, for example, by purification using ammonium sulfate precipitation, protein A, protein G column, DEAE ion exchange chromatography, an affinity column coupled with the protein of the factor or a synthetic peptide, or the like.

In the case of a monoclonal antibody, for example, the protein of the factor or a partial peptide thereof is immunized to a small animal such as a mouse, the spleen is removed from the mouse, and this is ground to separate the cells, and the cells and mouse myeloma cells Are fused using a reagent such as polyethylene glycol, and a clone that produces an antibody that binds to the factor is selected from the fused cells (hybridoma). Next, the obtained hybridoma was transplanted into the abdominal cavity of the mouse, and ascites was collected from the mouse, and the obtained monoclonal antibody was obtained by, for example, ammonium sulfate precipitation, protein A, protein G column, DEAE ion exchange chromatography, It can be prepared by purification using an affinity column coupled with protein or synthetic peptide.

The form of the antibody is not particularly limited, and as long as it binds to the factor, in addition to the polyclonal antibody and the monoclonal antibody, a human antibody, a humanized antibody by genetic recombination, an antibody fragment thereof, and an antibody modification product are also included. It is.

The protein of the factor used as a sensitizing antigen for antibody acquisition is not limited to the animal species from which it is derived, but is preferably a protein derived from a mammal such as a mouse or a human, and particularly preferably a protein derived from a human. Human-derived proteins can be obtained using the gene sequences disclosed in the present specification.

The protein used as the sensitizing antigen may be a complete protein or a partial peptide of the protein. Examples of the partial peptide of the protein include an amino group (N) terminal fragment and a carboxy (C) terminal fragment of the protein.

In addition to immunizing non-human animals with antigens to obtain the above hybridomas, human lymphocytes such as human lymphocytes infected with EB virus are sensitized with proteins, protein-expressing cells or lysates thereof in vitro. Lymphocytes can be fused with human-derived myeloma cells having permanent mitotic activity, such as U266, to obtain hybridomas that produce desired human antibodies having protein-binding activity.

When an antibody that binds to the protein of the factor is used for the purpose of administering it to the human body (antibody treatment), a human antibody or a human type antibody is preferable in order to reduce immunogenicity.

Examples of the substance that can inhibit the function of the protein of the factor include low molecular weight substances that bind to the protein of the factor. The low molecular weight substance that binds to the protein of the factor may be a natural or artificial compound. Usually, it is a compound that can be produced or obtained by using methods known to those skilled in the art.
As a factor to which the above antibody or low molecular weight compound binds, Dicer is preferable, Dicer1 is more preferable, and human Dicer1 is more preferable.

The present invention also provides a method for suppressing degranulation of mast cells, characterized by using a substance that inhibits the process of biosynthesis of microRNA from a microRNA precursor.

In the degranulation suppression method of the present invention, the substance that inhibits the process of biosynthesis of microRNA from a microRNA precursor inhibits the process of biosynthesis of microRNA from the microRNA precursor in the degranulation inhibitor described above. It is the same as the substance to do.

The present invention also provides a method for inhibiting degranulation of mast cells, characterized by using a vector that expresses siRNA that suppresses the expression of Dicer1 gene.
The siRNA used in the degranulation suppressing method of the present invention is preferably an siRNA having a base sequence represented by any one of SEQ ID NOs: 1 to 5 as a target sequence.

Also, the mast cell degranulation inhibitor provided by the present invention suppresses mast cell degranulation, and is therefore expected to have a therapeutic effect on diseases caused by mast cell abnormalities. Therefore, this invention relates to the therapeutic agent (pharmaceutical composition) of the disease resulting from the abnormality of a mast cell which contains the degranulation inhibitor of the mast cell of this invention as an active ingredient. The degranulation inhibitor of the present invention can be used not only for treatment of diseases caused by abnormalities of mast cells but also for diagnosis.

Moreover, since the degranulation inhibitor provided by the present invention is expected to have a therapeutic effect on diseases caused by abnormalities of mast cells as described above, an effective amount of the degranulation inhibitor is required. By administering to a subject (for example, a patient having a disease caused by abnormal mast cells), a disease caused by abnormal mast cells can be treated. That is, the present invention relates to a method for treating a disease caused by abnormal mast cells, comprising administering an effective amount of the degranulation inhibitor of the present invention to a subject in need.

The present invention also relates to the use of the degranulation inhibitor of the present invention for producing a therapeutic agent for diseases caused by abnormalities of mast cells. The present invention also relates to the use of the degranulation inhibitor of the present invention in the manufacture of a therapeutic agent for diseases caused by abnormal mast cells.

Examples of abnormalities in mast cells include abnormalities such as mast cell differentiation and degranulation, inflammatory mediator production, cytokine production, and chemokine production. Specific examples of diseases caused by abnormal mast cells include atopic dermatitis, asthma, chronic obstructive pulmonary disease, and allergic diseases.

Hereinafter, the present invention will be described in detail.
1. Method for measuring the degree of mast cell activation (1-1) Acquisition and culture of mast cells The mast cells used in the present invention are preferably human mast cells.
Human mast cells are not particularly limited as long as they are safe and efficient, and for example, known methods from human lung, skin, fetal liver and the like [J. Immunol. Methods, 169, 153 (1994) J. Immunol., 138, 861 (1987); J. Allergy Clin. Immunol., 107, 322 (2001); J. Immunol. Methods., 240, 101 (2000)]. Further, known methods [J. Immunol., 157, 343, (1996); Blood, 91, 187 (1998); J. Allergy Clin. Immunol., 106, 141 (2000); Blood, 97, 1016 (2001) ); Blood, 98, 1127 (2001); Blood, 100, 3861 (2002); Blood, 97, 2045 (2001)], mononuclear cells prepared from human umbilical cord blood, peripheral blood, bone marrow, lung or skin Can be prepared by culturing in the presence of stem cell factor (hereinafter abbreviated as SCF) and differentiating into mast cells.

In addition, a cell line established from human mast cells can also be used. As human mast cell lines, LAD2 [Leuk. Res., 27, 671, which is known to retain the properties of human mast cells, is known. (2003); Leuk. Res., 27, 677 (2003)].

(1-2) Measurement of the degree of activation of mast cells A substance has at least one of the following actions on mast cells: inhibition of activation, inhibition of degranulation, production of inflammatory mediators, inhibition of cytokine production and inhibition of chemokine production. Having, for example, after introducing a substance that inhibits the process of biosynthesis of microRNA from the microRNA precursor of the present invention into mast cells, the mast cells were stimulated and released (i) Histamine and β-hexosaminidase as indicators of degranulation, (ii) Inflammatory mediators such as LTC4, LTD4, LTE4, PGD2, (iii) Cytokines such as TNF-α and GM-CSF, (iv) IL- 8, It can be confirmed by measuring chemokines such as I-309, MIP-1α, etc., and comparing with a case where a substance that inhibits the process of biosynthesis of microRNA from the microRNA precursor of the present invention was not introduced. .

Methods for stimulating mast cells include, for example, a method of adding anti-IgE antibody after incubation with IgE, a method of adding compound 48/80, a method of adding polymyxin B, a method of adding dextran, calcium Method of adding ionophore, method of adding acetylcholine, method of adding carbachol, method of adding thrombin, method of adding concanavalin A, method of adding calcium ionophore, method of adding ATP, method of adding doxorubicin, Etc.

Instead of degranulation, the activation of mast cells involves cytokine production such as TNF-α and GM-CSF, chemokine production such as IL-8, I-309, MIP-1α, LTC4, LTD4, LTE4, PGD2, etc. It can also be examined by measuring inflammatory mediator production etc. [Blood, 100, 3861 (2002)].

2. Methods for detecting the expression of nucleic acids such as microRNAs and microRNA precursors expressed in mast cells (2-1) Acquisition of RNA Methods for extracting total RNA from mast cells include small RNAs such as microRNAs Although it will not specifically limit if it is a method, For example, it can carry out by the method as described in Molecular Cloning 3rd edition. Alternatively, extraction can be performed using Trizol (Invitrogen), ISOGEN (Nippon Gene), mirVana ^™ miRNA Isolation Kit (Ambion), miRNeasy Mini Kit (Qiagen), and the like.

Also, low molecular weight RNA can be cloned from total RNA including low molecular weight RNA. As a method for cloning small RNA, specifically, according to the method described in Genes & Development, 15, 188-200 (2000), separation and excision of low molecular RNA by 15% polyacrylamide gel electrophoresis, 5'-terminal dephosphorylation, 3'-adapter ligation, phosphorylation, 5'-adapter ligation, reverse transcription, PCR amplification, concatamerization, ligation to vector, and then cloning of small RNA, the base of the clone Examples include a method for determining the sequence. Alternatively, for example, according to the method described in Science, 294, 858-862 (2001), separation and excision of small RNA by 15% polyacrylamide gel electrophoresis, 5′-adenylation 3′-adapter ligation, 5 '-Adapter ligation, reverse transcription, PCR amplification, concatamerization, ligation to a vector, cloning of low molecular RNA, and determination of the base sequence of the clone, etc. can be mentioned.

Alternatively, separation and excision of small RNA by 15% polyacrylamide gel electrophoresis, 5′-end dephosphorylation, 3′-adapter ligation, phosphorylation by the method described in Nucleic® Acids® Research, 34, 1765-1771 (2006) Low molecular weight is determined by cloning low molecular RNA through sequential oxidation, 5'-adapter ligation, reverse transcription, PCR amplification, and ligation to a microbead vector, and determining the base sequence by reading the base sequence of the microbead. RNA can also be obtained.

Also, small RNAs can be cloned using small RNA Cloning Kit (Takara Bio Inc.).

(2-2) Identification of microRNA Whether a small RNA sequence is a microRNA can be determined by following the criteria described in RNA, 9, 277-279 (2003). For example, in the case of a low molecular weight RNA that has been newly acquired and whose base sequence has been determined, it can be performed as follows.

Obtain a peripheral genomic sequence obtained by extending the DNA sequence corresponding to the obtained low molecular RNA base sequence by about 50 bases to the 5 'end and 3' end, respectively, and RNA that is predicted to be transcribed from the genomic sequence Predict secondary structure. As a result, when it has a hairpin structure and the base sequence of the low molecular weight RNA is located on one strand of the hairpin, it can be determined that the low molecular weight RNA is a microRNA. Genome sequences are publicly available and can be obtained from, for example, UCSC Genome Bioinformatics (http://genome.ucsc.edu/). Also, various programs for secondary structure prediction have been published, such as RNAfold [Nucleic Acids Research, 31, 3429-3431 (2003)], Mfold [Nucleic Acids Research, 31, 3406-3415 (2003)], etc. Can be used. In addition, existing microRNA sequences are registered in the database miRBase (http://microrna.sanger.ac.uk/) 、. It can be determined whether or not.

(2-3) Method for Detecting Expression Level of MicroRNA or its Precursor Examples Methods for detecting the expression level of microRNA or its precursor include, for example, (1) Northern hybridization, (2) Dot blot high Hybridization, (3) in situ hybridization, (4) quantitative PCR, (5) differential hybridization, (6) microarray, (7) ribonuclease protection assay and the like.

Northern hybridization involves separating the sample-derived RNA by gel electrophoresis and then transferring it to a support such as a nylon filter to prepare a probe appropriately labeled based on the base sequence of the microRNA or its precursor. It is a method for detecting a band specifically bound to the microRNA or its precursor by performing hybridization and washing, and specifically, for example, the method described in Science, 294, 853-858 (2001) Etc. can be performed according to the above.

The labeled probe can be prepared by, for example, radioisotope, biotin, digoxigenin, fluorescent group, chemiluminescent group, etc. by the method such as nick translation, random priming, or phosphorylation at the 5 ′ end, and the micro or its RNA precursor. It can be prepared by incorporating it into DNA, RNA or LNA having a sequence complementary to the base sequence. Since the binding amount of the labeled probe reflects the expression level of the microRNA or its precursor, the expression level of the microRNA or its precursor can be determined by quantifying the amount of the labeled probe bound. . Electrophoresis, membrane transfer, probe preparation, hybridization, and nucleic acid detection can be performed by the methods described in Molecular Cloning 3rd edition.

In dot blot hybridization, RNA extracted from tissues or cells is spot-fixed on a membrane in a dotted manner, and then hybridized with a labeled polynucleotide that serves as a probe to detect RNA that specifically hybridizes with the probe. It is a method to do. As the probe, the same probe as in Northern hybridization can be used. Preparation of RNA, RNA spot, hybridization, and detection of RNA can be performed by the methods described in Molecular Cloning 3rd edition.

In situ hybridization uses paraffin or cryostat sections of tissue obtained from a living body, or immobilized cells as specimens, performs hybridization and washing steps with a labeled probe, and performs microscopic observation. This is a method for investigating the distribution and localization of RNA or its precursor in tissues and cells [Methods in Enzymology, 254, 419 (1995)]. As the probe, the same probe as in Northern hybridization can be used. Specifically, microRNA can be detected according to the method described in Nature Method, 3, 27 (2006).

In quantitative PCR, cDNA synthesized from a sample-derived RNA using a reverse transcription primer and a reverse transcriptase (hereinafter, the cDNA is also referred to as a sample-derived cDNA) is used for measurement. A random primer or a specific RT primer can be used as a reverse transcription primer for cDNA synthesis. The specific RT primer refers to a primer having a sequence complementary to a base sequence corresponding to the microRNA or a precursor thereof and a surrounding genomic sequence.

For example, after synthesizing a specimen-derived cDNA, using this as a template, a template-specific primer designed from the base sequence corresponding to the microRNA or its precursor and its surrounding genomic sequence, or the base sequence corresponding to the reverse transcription primer PCR is used to amplify the cDNA fragment containing the micro or its RNA precursor, and the amount of the micro RNA or its precursor contained in the sample-derived RNA is detected from the number of cycles to reach a certain amount To do. As a template-specific primer, an appropriate region corresponding to the microRNA or its precursor and its surrounding genomic sequence is selected, and DNA or LNA comprising a sequence of 20 to 40 bases at the 5 ′ end of the base sequence of the region And a DNA or LNA pair consisting of a sequence complementary to 20 to 40 bases at the 3 ′ end can be used. Specifically, it can be carried out according to the method described in Nucleic Acids Research, 32, e43 (2004).

Alternatively, a specific RT primer having a stem / loop structure can also be used as a reverse transcription primer for cDNA synthesis. Specifically, it can be carried out using the method described in Nucleic® Acid Research, 33, 179 (2005) or TaqMan® MicroRNA Assays (Applied Biosystems).

Furthermore, sample-derived cDNA is hybridized to a filter or slide glass, silicon, or other substrate on which DNA corresponding to a base sequence containing at least one of the microRNAs or a precursor thereof, or LNA is immobilized, and washed. By performing the above, it is possible to detect fluctuations in the amount of the microRNA or its precursor. Examples of methods based on such hybridization include methods using differential hybridization [Trends Genet., 7, 314 (1991)] and microarrays [Genome Res., 6, 639 (1996)]. Each method immobilizes an internal control such as a nucleotide sequence corresponding to U6RNA on a filter or substrate to accurately detect the difference in the amount of the microRNA or its precursor between the control sample and the target sample. can do. In addition, labeled cDNA synthesis using differently labeled dNTPs (mixtures of dATP, dGTP, dCTP, and dTTP) based on RNA derived from the control sample and the target sample, and two filters on one filter or one substrate. By simultaneously hybridizing the labeled cDNA, the microRNA or a precursor thereof can be quantified. For example, microRNAs can be detected using a microarray described in Proc. Natl. Acad. Sci. USA, 101, 9740-9744 (2004), Nucleic Acid Research, 32, e188 (2004). Specifically, it can be detected or quantified in the same manner as mirVana miRNA Bioarray (Ambion).

In the ribonuclease protection assay, first, a promoter sequence such as T7 promoter or SP6 promoter is bound to the 3 ′ end of the base sequence corresponding to the microRNA or a precursor thereof or the surrounding genomic sequence, and labeled NTP (ATP, GTP, CTP). , UTP mixture) and an in vitro transcription system using RNA polymerase to synthesize labeled antisense RNA. The labeled antisense RNA is bound to the sample-derived RNA to form an RNA-RNA hybrid, and then digested with ribonuclease A that degrades only single-stranded RNA. The digested product is subjected to gel electrophoresis, and an RNA fragment protected from digestion by forming an RNA-RNA hybrid is detected or quantified as the nucleic acid or microRNA precursor of the present invention. Specifically, it can be detected or quantified using mirVana miRNA Detection Kit (Ambion).

3. How to inhibit the process of biosynthesis of microRNAs MicroRNAs are transcribed from a gene into a primary transcript, primary-microRNA (pri-miRNA), followed by step-wise processing from pri-miRNA to mature microRNA. About 70 bases of precursor-microRNA (pre-miRNA) with a characteristic hairpin structure was biosynthesized from pri-miRNA, then transported from the nucleus to the cytoplasm via Exportin-5, and finally Mature microRNA is biosynthesized from pre-miRNA. As a method for inhibiting the biosynthesis of microRNA, any method may be used as long as one of the above processes is inhibited and the amount of mature microRNA decreases.

Specifically, a substance that inhibits the function of the factor essential for the above-described microRNA biosynthesis can be used in the method.

Also, a vector that expresses siRNA can be prepared by inserting a DNA corresponding to a base sequence selected from a target base sequence into an expression vector. As the expression vector, a vector that can replicate autonomously in a host cell, can be integrated into a chromosome, and contains a promoter at a position where a genomic gene containing a nucleic acid base sequence can be transcribed is used. As the promoter, any promoter can be used so long as it can be expressed in the host cell. For example, RNA polymerase II (pol II) type promoter or U RNA or H1RNA transcription system such as RNA polymerase III (pol III) type promoter. Can give. Examples of the pol II promoter include cytomegalovirus (human CMV) IE (immediate early) gene promoter, SV40 early promoter, and the like. Examples of expression vectors using them include pCDNA6.2-GW / miR (Invitrogen), pSilencer® 4.1-CMV (Ambion), and the like. Examples of pol III promoters include U6 RNA, H1 RNA, and tRNA promoters. Examples of expression vectors using them include pSINsi-hH1 DNA (Takara Bio), pSINsi-hU6 DNA (Takara Bio), pENTR / U6 (Invitrogen) and the like.

Further, a DNA corresponding to the selected base sequence is inserted downstream of the promoter in the viral vector to construct a recombinant viral vector, and the vector is introduced into a packaging cell to produce a recombinant virus. The siRNA can also be expressed. The packaging cell may be any cell as long as it can replenish the deficient protein of the recombinant viral vector deficient in any of the genes encoding the proteins required for viral packaging, such as human kidney. HEK293 cells derived from mouse, mouse fibroblast NIH3T3, and the like can be used. Proteins supplemented by packaging cells include mouse retrovirus-derived gag, pol, env, etc. for retrovirus vectors, and HIV virus-derived gag, pol, env, vpr, vpu for lentiviral vectors. , Vif, tat, rev, nef, etc., adenovirus vectors, E1A, E1B, etc. Can be used.

Whether or not the biosynthesis of microRNA is actually inhibited can be confirmed by measuring the expression level for any microRNA according to the method described in 2 above. In addition, the amount of mRNA and protein of factors essential for biosynthesis of microRNA can be measured by a real-time RT-PCR method or western blotting method, respectively, and it can be confirmed whether or not these amounts have decreased.

4). Mast cell degranulation inhibitor using a substance that inhibits microRNA biosynthesis The substance that inhibits the process of biosynthesis of microRNA from a microRNA precursor according to the present invention is used as a mast cell degranulation inhibitor. Can be used. As described in 3 above, the substance that inhibits the process of biosynthesis of microRNA from a microRNA precursor may be any substance as long as it inhibits the function of factors essential for biosynthesis. However, for example, siRNA for human Dicer1 gene can be mentioned.

Regarding the degranulation inhibitory effect of mast cells, which is a substance that inhibits the process of biosynthesis of microRNA from the microRNA precursor of the present invention, as described in 1 above, microRNA from the microRNA precursor of the present invention After introducing into the mast cells a substance that inhibits the process of biosynthesis, the mast cells are stimulated and released (i) histamine and β-hexosaminidase, which are indicators of degranulation, (ii) LTC4, Measure inflammatory mediators such as LTD4, LTE4, PGD2, etc., (iii) cytokines such as TNF-α and GM-CSF, (iv) chemokines such as IL-8, I-309, MIP-1α, etc. This can be confirmed by comparing with a case where a substance that inhibits the process of biosynthesis of microRNA from a microRNA precursor was not introduced.

The formulation form and administration method of the mast cell degranulation inhibitor of the present invention are the same as those of the therapeutic agent containing a substance that inhibits biosynthesis of microRNA, which will be described later in 5.

5). Therapeutic drugs containing substances that inhibit the biosynthesis of microRNA Substances that inhibit the process of biosynthesis of microRNAs from microRNA precursors can suppress mast cell degranulation, thereby preventing abnormalities in mast cells. It can be used as a therapeutic agent for diseases caused by it. Abnormalities in mast cells include abnormalities such as mast cell differentiation and degranulation, inflammatory mediator production, cytokine production, chemokine production, etc. Diseases resulting from them include atopic dermatitis, asthma, chronic obstructive pulmonary disease And allergic diseases.

A therapeutic agent containing as an active ingredient a substance that inhibits the process of biosynthesis of microRNA from a microRNA precursor can be administered alone, but usually one or more pharmacologically acceptable It is desirable to administer it as a pharmaceutical preparation prepared by any method well known in the pharmaceutical arts.

It is desirable to use the most effective route for treatment, and oral administration or parenteral administration such as buccal, respiratory tract, rectal, subcutaneous, intramuscular and intravenous is desirable. Can be given intravenously.

Administration forms include sprays, capsules, tablets, granules, syrups, emulsions, suppositories, injections, ointments, tapes and the like.

Preparations suitable for oral administration include emulsions, syrups, capsules, tablets, powders, granules and the like.

Liquid preparations such as emulsions and syrups include sugars such as water, sucrose, sorbitol and fructose, glycols such as polyethylene glycol and propylene glycol, oils such as sesame oil, olive oil and soybean oil, p-hydroxybenzoic acid Preservatives such as esters, and flavors such as strawberry flavor and peppermint can be used as additives.

For capsules, tablets, powders, granules, etc., excipients such as lactose, glucose, sucrose, mannitol, disintegrants such as starch and sodium alginate, lubricants such as magnesium stearate and talc, polyvinyl alcohol, hydroxy A binder such as propylcellulose and gelatin, a surfactant such as fatty acid ester, and a plasticizer such as glycerin can be used as additives.

Preparations suitable for parenteral administration include injections, suppositories, sprays and the like.

Injection is prepared using a carrier made of a salt solution, a glucose solution or a mixture of both. Suppositories are prepared using a carrier such as cocoa butter, hydrogenated fat or carboxylic acid. The spray is prepared using a carrier that does not irritate the recipient's oral cavity and airway mucosa, and that facilitates absorption by dispersing the active ingredient as fine particles.

Specific examples of the carrier include lactose and glycerin. Depending on the properties of the degranulation inhibitor of the present invention and the carrier used, preparations such as aerosols and dry powders are possible. In these parenteral preparations, the components exemplified as additives for oral preparations can also be added.

The dose or frequency of administration varies depending on the intended therapeutic effect, administration method, treatment period, age, body weight, etc., but is usually 10 μg / kg to 20 mg / kg per day for an adult.

The therapeutic agent containing the degranulation inhibitor of the present invention as an active ingredient, such as a substance that inhibits the process of biosynthesis of microRNA from a microRNA precursor, is used as a nucleic acid-expressing vector and nucleic acid therapeutic agent. It can also be produced by blending with a base [Nature Genet., 8, (42 (1994)].

The base used in the therapeutic agent of the present invention may be any base as long as it is usually used in injections, salt water such as distilled water, sodium chloride or a mixture of sodium chloride and an inorganic salt, mannitol, Examples thereof include a solution of lactose, dextran, glucose and the like, an amino acid solution such as glycine and arginine, an organic acid solution or a mixed solution of a salt solution and a glucose solution, and the like. In addition, according to a conventional method, these bases are mixed with an osmotic pressure adjusting agent, a pH adjusting agent, a vegetable oil such as sesame oil and soybean oil, or an auxiliary such as a surfactant such as lecithin or a nonionic surfactant. An injection may be prepared as a suspension or dispersion. These injections can be prepared as preparations for dissolution at the time of use by operations such as pulverization and freeze-drying. The therapeutic agent of the present invention can be used for treatment as it is in the case of a liquid just before the treatment, or in the case of a solid, dissolved in the above sterilized base if necessary.

When the substance that inhibits the process of biosynthesis of microRNA from a microRNA precursor is a nucleic acid (for example, siRNA), the vector that expresses the nucleic acid can be the recombinant virus vector prepared in 3 above. More specifically, a retrovirus vector, a lentivirus vector, etc. can be mentioned.

For example, a viral vector can be prepared by preparing a complex by combining the above nucleic acid with a polylysine-conjugated antibody specific for an adenovirus hexon protein and binding the resulting complex to an adenovirus vector. The virus vector stably reaches the target cell, is taken up into the cell by endosomes, is degraded in the cell, and the nucleic acid can be efficiently expressed.

In addition, viral vectors based on Sendai virus (-) strand RNA virus have been developed (WO97 / 16538, WO97 / 16539), and Sendai virus incorporating the nucleic acid is prepared using the Sendai virus. be able to.

The nucleic acid can also be transferred by non-viral nucleic acid transfer method. For example, calcium phosphate coprecipitation method [Virology, 52, 456-467 (1973); Science, 209, 1414-1422 (1980)], microinjection method [Proc. Natl. Acad. Sci. USA, 77, 5399-5403-5 ( 1980); Proc. Natl. Acad. Sci. USA, 77, 7380-7384 (1980); Cell, 27, 223-231 (1981); Nature, 294, 92-94 (1981)], liposome-mediated membrane Fusion-Interventional Transfer [Proc. Natl. Acad. Sci. USA, 84, 13 7413-7417 (1987); Biochemistry, 28, 9508-9514 (1989); J. Biol. Chem., 264, 12126-12129 (1989) ); Hum. Gene Ther., 3, 267-275, (1992); Science, 249, 1285-1288 (1990); Circulation, 83, 2007-2011 (1992)] or direct DNA uptake and receptor-mediated DNA Transfer method [Science, 247, 1465-1468 (1990); J. Biol. Chem., 266, 14338-14342 (1991); Proc.Natl.Acad.Sci. USA, 87, 3655-3659 (1991); J Biol. Chem., 264, 16985-16987 (1989); BioTechniques, 11, 474-485 (1991); Proc. Natl. Acad. Sci. USA, 87, 3410-3414 (1990); Proc. Natl. Acad .Sc i. USA, 88, 4255-4259 (1991); Proc.Natl.Acad.Sci. USA, 87, 4033-4037 (1990); Proc.Natl.Acad.Sci. USA, 88, 8850-8854 (1991) Hum. Gene Ther., 3, 147-154 (1991)].

Membrane fusion-mediated transfer via liposomes allows the nucleic acid to be taken up and expressed locally in the tissue by administering the liposome preparation directly to the target tissue [Hum. Gene Ther., 3, 399 (1992)]. Direct DNA uptake techniques are preferred for targeting DNA directly to the lesion.

Receptor-mediated DNA transfer can be performed, for example, by binding DNA (typically in the form of a covalently closed supercoiled plasmid) to a protein ligand via polylysine. The ligand is selected based on the presence of the corresponding ligand receptor on the cell surface of the target cell or tissue. The ligand-DNA conjugate can be injected directly into the blood vessel, if desired, and can be directed to a target tissue where receptor binding and internalization of the DNA-protein complex occurs. To prevent intracellular destruction of DNA, adenovirus can be co-infected to disrupt endosomal function.

Hereinafter, the present invention will be described specifically by way of examples. However, the present invention is not limited to these examples.

1. SiRNA that inhibits the process of biosynthesis of microRNA
As a substance that inhibits the process of biosynthesis of microRNA, we focused on siRNA of human Dicer1 gene, which is one of the essential factors for biosynthesis of microRNA, and examined whether this actually reduces Dicer1 gene expression. .

HeLa cells are seeded at a density of 2x10 ⁵ per well in a 6-well plate, and siRNA for human Dicer1 gene is lipofection method, specifically, Lipofectamine2000 (Invitrogen) is used to achieve a final concentration of 20 nM. Introduced to be. For the siRNA sequence for human Dicer1 gene, Dicer-siRNA-1 having SEQ ID NO: 1 as a target sequence and Dicer-siRNA-2 having SEQ ID NO: 2 as a target sequence were purchased from Qiagen. Lipofection followed the method described in the instructions attached to the product. Moreover, the cell which carried out lipofection to the HeLa cell, without adding siRNA was set as control.

Two days after the introduction of siRNA, total RNA was extracted from each cell using RNeasy (Qiagen), and cDNA was synthesized using SuperScriptIII reverse transcriptase (Invitrogen). Each experimental procedure followed the method described in the instructions attached to the product.

The cDNA synthesized above was used as a template for the PCR reaction, and Dicer1-specific PCR amplification was performed by SYBR-Green® PCR using ABI7900HT® Fast (Applied Biosystems) to quantify the amount of mRNA. The amount of mRNA in the sample was evaluated as a relative ratio when the amount of Dicer1 mRNA was 1 in the control cells.

As a result, the relative expression level of Dicer1 mRNA in Dicer-siRNA-1-introduced cells was 0.38, and the relative expression level of Dicer1 in Dicer-siRNA-2-introduced cells was 0.24, and expression of Dicer1 gene by siRNA introduction into Dicer1 It was confirmed that the amount was reduced.

2. Effects on degranulation in human mast cells that inhibit the process of biosynthesis of microRNAs (1)
SiRNA for human Dicer1 gene was introduced into LAD2, a human mast cell line, and the effect on degranulation was examined.
LAD2 is a recently established human mast cell line that is well known to retain the properties of human mast cells [Leuk. Res., 27, 671 (2003); Leuk. Res., 27, 677 (2003)]. LAD2 was obtained from National Institute of Allergy and Infectious Diseases, National Institutes of Health (Bethesda, MD 20892-1881, USA), and cultured in Stem Pro-34 medium [manufactured by Invitrogen] containing 100 ng / mL SCF.

LAD2 is seeded in a 6-well plate at 5x10 ⁵ per well, and siRNA (Dicer-siRNA-1) for human Dicer1 gene is lipofection method, specifically using Gene Silencer (Genlantis) The final concentration was 25 nM. As the siRNA sequence for human Dicer1 gene, Dicer-siRNA-1 (manufactured by Qiagen) having SEQ ID NO: 1 as a target sequence was used. Lipofection followed the method described in the instructions attached to the product.

Two days after the introduction of siRNA by the lipofection method, 1.0 μg / mL human myeloma IgE (manufactured by Cosmo Bio) was added and cultured overnight in an incubator at 37 ° C. with 5% CO ₂ concentration. The next day, the medium was removed by centrifugation, and Tyrode buffer (126.1 mmol / L NaCl, 4.0 mmol / L KCl, 1.0 mmol / L CaCl ₂ , 0.6 mmol / L MgCl ₂ , 0.6 mmol / L KH ₂ PO _4. After washing with 10 mM HEPES, 5.6 mmol / L D-glucose, 0.1% bovine serum albumin, pH 7.4), add 1.5 mL of Tyrode's buffer to suspend the cells. 100 μL was dispensed per unit. Subsequently, a rabbit anti-human IgE antibody (manufactured by Dako) was added to a final concentration of 10 μg / mL and incubated for 20 minutes in an incubator at 37 ° C. with 5% CO ₂ concentration to induce degranulation. The supernatant was collected by centrifugation, and the degree of degranulation was measured by measuring β-hexosaminidase activity in the supernatant. The β-hexosaminidase activity was determined by using 4 mmol / L p-nitrophenyl N-acetyl-β-glucosaminide (Sigma) dissolved in 40 mmol / L citrate buffer (pH 4.5) in 50 μL of the collected supernatant. ), Incubate at 37 ° C for 1 hour, and then measure the absorbance at 405 nm of the sample to which 100 µL of 0.2 mol / L glycine (pH 10.7) has been added using a plate reader 1420 ARVOsx (Perkin Elmer). It was evaluated by. Further, the total β-hexosaminidase activity in LAD2 was measured by adding Triton X-100 having a final concentration of 1% instead of the rabbit anti-human IgE antibody and conducting the same experiment. The percentage of degranulation was calculated as the percentage of β-hexosaminidase activity in the supernatant with respect to the total β-hexosaminidase activity, and the percentage of degranulation in the negative control (Gene Silencer only) for each. The relative degranulation activity was calculated when 1.0 was 1.0.

In addition, 7 days after the introduction of the siRNA, human myeloma IgE (manufactured by Cosmo Bio) was added to a final concentration of 1.0 μg / mL and cultured overnight in an incubator at 37 ° C. with 5% CO ₂ concentration. The following day, degranulation was induced by adding rabbit anti-human IgE antibody, and the degranulation rate was measured. For each, the degranulation relative activity when the degranulation rate in the negative control group (Gene Silencer only) was 1.0 Was calculated.

Table 1 shows the results of degranulation relative activity on the next day (that is, after 3 days and 8 days) after 2 days and 7 days after the introduction of the siRNA, respectively.

Furthermore, in order to investigate the universality of siRNA against human Dicer1 gene, siRNA (SEQ ID NO: 1) as a target sequence (Dicer-siRNA-1) and siRNA (SEQ ID NO: 2) as a target sequence (Dicer-siRNA-2) are LAD2 The cells were introduced into cells, and degranulation activity was measured using a 6-well plate.

LAD2 is seeded in a 6-well plate at 5x10 ⁵ per well, and siRNA is introduced by lipofection method, specifically Gene Silencer (Genlantis) to a final concentration of 30 nM. did. Lipofection followed the method described in the instructions attached to the product.

Six days after introduction of siRNA by the lipofection method, 1.0 μg / mL human myeloma IgE (manufactured by Cosmo Bio) was added and cultured overnight in an incubator at 37 ° C. with 5% CO ₂ concentration. The next day, the medium was removed by centrifugation, and after washing with Tyrode buffer, 1.5 mL of Tyrode buffer was added to suspend the cells, and 100 μL per well was dispensed into a 96-well plate. Subsequently, a rabbit anti-human IgE antibody (manufactured by Dako) was added to a final concentration of 15 μg / mL and incubated for 20 minutes in an incubator at 37 ° C. with 5% CO ₂ concentration to induce degranulation. The supernatant was collected by centrifugation, and the degree of degranulation was measured by measuring β-hexosaminidase activity in the supernatant. The β-hexosaminidase activity was determined by using 4 mmol / L p-nitrophenyl N-acetyl-β-glucosaminide (Sigma) dissolved in 40 mmol / L citrate buffer (pH 4.5) in 50 μL of the collected supernatant. ), Incubate at 37 ° C for 1 hour, and then measure the absorbance at 405 nm of the sample to which 100 µL of 0.2 mol / L glycine (pH 10.7) has been added using a plate reader 1420 ARVOsx (Perkin Elmer). It was evaluated by. Further, the total β-hexosaminidase activity in LAD2 was measured by adding Triton X-100 having a final concentration of 1% instead of the rabbit anti-human IgE antibody and conducting the same experiment. The percentage of degranulation was calculated as the percentage of β-hexosaminidase activity in the supernatant with respect to the total β-hexosaminidase activity, and the percentage of degranulation in the negative control (Gene Silencer only) for each. The relative degranulation activity was calculated when 1.0 was 1.0.

Table 2 shows the results of degranulation relative activity in which each siRNA was introduced.

From these experimental results, it was confirmed that the rate of degranulation was reduced by the introduction of siRNA for Dicer1 gene.

3. Effects on degranulation in human mast cells that inhibit the process of biosynthesis of microRNAs (2)
LAD2 introduced with siRNA against human Dicer1 gene was stimulated with other than anti-IgE antibody, and the influence on degranulation was examined.
LAD2 is seeded in a 6-well plate at 5x10 ⁵ per well, and siRNA (Dicer-siRNA-1) for human Dicer1 gene is lipofection method, specifically using Gene Silencer (Genlantis) The final concentration was 30 nM. As the siRNA sequence for human Dicer1 gene, Dicer-siRNA-1 (manufactured by Qiagen) having SEQ ID NO: 1 as a target sequence was used. Lipofection followed the method described in the instructions attached to the product.

Three days after introduction of siRNA by the lipofection method, the medium is removed by centrifugation, washed with Tyrode buffer, 1.5 mL of Tyrode buffer is added, the cells are suspended, and 1 is added to a 96-well plate. Dispense 100 μL per well. Subsequently, compound 48/80 (manufactured by Sigma-Aldrich) was added to a final concentration of 0.5 μg / mL, and incubated for 20 minutes in an incubator at 37 ° C. with 5% CO ₂ concentration to induce degranulation. The supernatant was collected by centrifugation, and the degree of degranulation was measured by measuring β-hexosaminidase activity in the supernatant. The β-hexosaminidase activity was determined by using 4 mmol / L p-nitrophenyl N-acetyl-β-glucosaminide (Sigma) dissolved in 40 mmol / L citrate buffer (pH 4.5) in 50 μL of the collected supernatant. ), Incubate at 37 ° C for 1 hour, and then measure the absorbance at 405 nm of the sample to which 100 µL of 0.2 mol / L glycine (pH 10.7) has been added using a plate reader 1420 ARVOsx (Perkin Elmer). It was evaluated by. Further, the total β-hexosaminidase activity in LAD2 was measured by adding Triton X-100 having a final concentration of 1% instead of Compound 48/80 and conducting the same experiment. The percentage of degranulation was calculated as the percentage of β-hexosaminidase activity in the supernatant with respect to the total β-hexosaminidase activity, and the percentage of degranulation in the negative control (Gene Silencer only) for each. The relative degranulation activity was calculated when 1.0 was 1.0.

Also, 7 days after the introduction of the siRNA, degranulation was measured by inducing degranulation by adding compound 48/80, and the degranulation ratio in the negative control group (Gene-Silencer only) was 1.0 for each. The relative degranulation activity was calculated.

Table 3 shows the results of degranulation relative activity 3 days and 7 days after the introduction of the siRNA, respectively.

From this experimental result, it was confirmed that the ratio of degranulation by compound 48/80 stimulation was reduced by siRNA introduction to Dicer1 gene.

Claims

A mast cell degranulation inhibitor containing as an active ingredient a substance that inhibits the process of biosynthesis of microRNA from a microRNA precursor.
The degranulation inhibitor according to claim 1, wherein the substance that inhibits the process of biosynthesis of microRNA from a microRNA precursor is a substance that suppresses the expression of Dicer1 gene.
The degranulation inhibitor according to claim 2, wherein the substance that suppresses the expression of Dicer1 gene is a nucleic acid.
4. The degranulation inhibitor according to claim 3, wherein the nucleic acid is siRNA.
A mast cell degranulation inhibitor containing, as an active ingredient, a vector that expresses siRNA that suppresses the expression of Dicer1 gene.
The degranulation inhibitor according to claim 4 or 5, wherein the siRNA is a siRNA having a base sequence represented by any one of SEQ ID NOs: 1 to 5 as a target sequence.
A method for suppressing degranulation of mast cells, comprising using a substance that inhibits the process of biosynthesis of microRNA from a microRNA precursor.
The method for inhibiting degranulation according to claim 7, wherein the substance that inhibits the process of biosynthesis of microRNA from a microRNA precursor is a substance that suppresses the expression of Dicer1 gene.
The method for inhibiting degranulation according to claim 8, wherein the substance that suppresses the expression of Dicer1 gene is a nucleic acid.
The method for suppressing degranulation according to claim 9, wherein the nucleic acid is siRNA.
A method for inhibiting degranulation of mast cells, characterized by using a vector that expresses siRNA that suppresses the expression of Dicer1 gene.
The method for suppressing degranulation according to claim 10 or 11, wherein the siRNA is a siRNA having a base sequence represented by any of SEQ ID NOs: 1 to 5 as a target sequence.
A therapeutic agent for a disease caused by an abnormality in mast cells, comprising the degranulation inhibitor according to any one of claims 1 to 6 as an active ingredient.
The therapeutic agent according to claim 13, wherein the disease caused by an abnormality in mast cells is a disease selected from the group consisting of atopic dermatitis, asthma, chronic obstructive pulmonary disease, and allergic disease.
A method for treating a disease caused by abnormal mast cells, comprising administering an effective amount of the degranulation inhibitor according to any one of claims 1 to 6 to a subject in need thereof.
The treatment method according to claim 15, wherein the disease caused by abnormality of mast cells is a disease selected from the group consisting of atopic dermatitis, asthma, chronic obstructive pulmonary disease, and allergic disease.
Use of the degranulation inhibitor according to any one of claims 1 to 6 for the manufacture of a therapeutic agent for a disease caused by an abnormality of mast cells.
The use according to claim 17, wherein the disease caused by abnormality of mast cells is a disease selected from the group consisting of atopic dermatitis, asthma, chronic obstructive pulmonary disease and allergic disease.