WO2013051435A1 - Therapeutic agent, gene therapy agent, and method for preventing invasion of eosinophil - Google Patents

Therapeutic agent, gene therapy agent, and method for preventing invasion of eosinophil Download PDF

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WO2013051435A1
WO2013051435A1 PCT/JP2012/074623 JP2012074623W WO2013051435A1 WO 2013051435 A1 WO2013051435 A1 WO 2013051435A1 JP 2012074623 W JP2012074623 W JP 2012074623W WO 2013051435 A1 WO2013051435 A1 WO 2013051435A1
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sirna
gene
expression
therapeutic agent
seq
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PCT/JP2012/074623
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French (fr)
Japanese (ja)
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元彦 鈴木
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公立大学法人名古屋市立大学
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/713—Double-stranded nucleic acids or oligonucleotides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A61P11/02—Nasal agents, e.g. decongestants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses
    • A61P27/16—Otologicals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/14—Type of nucleic acid interfering N.A.

Definitions

  • the present invention relates to eosinophilic sinusitis and eosinophilic otitis media using knockdown of STAT-6 gene, GATA-3 gene and / or interleukin 5 (hereinafter also referred to as IL-5) gene. And a method for suppressing eosinophil infiltration.
  • Sinusitis is a disease that causes inflammation in the sinuses.
  • Treatment for chronic sinusitis includes (1) drug therapy and (2) surgical therapy.
  • macrolide therapy in drug therapy has shown a significant effect on the control of sinusitis caused by neutrophils.
  • the safety and effectiveness of surgery have improved with the spread of endoscopic sinus surgery in surgical therapy.
  • refractory sinusitis that is resistant to drug therapy including macrolide therapy and is easily relapsed even after surgery.
  • One typical example of such intractable sinusitis is sinusitis involving eosinophils (recently called eosinophilic sinusitis).
  • eosinophils infiltrate the sinus tissue, and activated eosinophils are said to damage the sinus tissue. Postoperative recurrence is said to be over 50% in 5 years. Most eosinophilic sinusitis is associated with eosinophilic otitis media. Eosinophilic otitis media is also resistant to treatment and is a clinical problem. Both eosinophilic sinusitis and eosinophilic otitis media are said to induce Th2 cells and Th2 cytokines and participate in the pathogenesis.
  • RNA interference is one of the therapeutic techniques that are being clinically applied.
  • RNA interference is a phenomenon in which mRNA having a base sequence complementary to double-stranded RNA is degraded, and this can be used to suppress the expression of a specific gene.
  • siRNA that is a short interfering RNA is suitable when introducing double-stranded RNA into a mammalian cell or living body. I was ascertained.
  • Patent Document 1 discloses a staple-type oligonucleotide and a medicine containing the same as an active ingredient. Specifically, transcription factor inhibitor, antisense oligonucleotide or siRNA, more specifically arthritis, dermatitis, nephritis, hepatitis, renal failure, cystitis, prostatitis, urethritis, ulcerative colitis or clone Disclosed is a preventive, therapeutic or ameliorating agent for diseases, rheumatoid arthritis or osteoarthritis, atopic dermatitis, contact dermatitis, psoriasis, skin ulcers or pruritus.
  • Patent Document 2 discloses a drug that suppresses nerve fiber degeneration containing siRNA as an active ingredient.
  • International Publication WO2005 / 113014 discloses a pharmaceutical composition for treating JC virus infection, comprising a vector incorporating a gene capable of forming siRNA.
  • IL-5 is a cytokine involved in the differentiation, proliferation and induction of eosinophils.
  • the present inventor has obtained the knowledge that each of the above siRNAs has an effect of suppressing IL-5 production. Therefore, it was reasonably speculated that by using each of the above siRNAs, it was possible to treat an eosinophilic disease in which eosinophil infiltration was observed.
  • the present inventor has completed the present invention. It is an object of the present invention to provide a useful therapeutic agent for eosinophilic sinusitis, eosinophilic otitis media, a gene therapeutic agent, and a method for suppressing infiltration of eosinophils.
  • the first invention of the present application for solving the above-described problems is as follows. It contains siRNA that suppresses the expression of the STAT-6 gene, siRNA that suppresses the expression of the GATA-3 gene and / or siRNA that suppresses the expression of the IL-5 gene as an active ingredient, and contains eosinophilic sinusitis and / or acidophilic acid It is a therapeutic agent used for the treatment of bulbar otitis media.
  • the second invention of the present application for solving the above-described problems is as follows.
  • the therapeutic agent according to the first invention, wherein the siRNA that suppresses the expression of the active ingredient STAT-6 gene is an siRNA having a homology of 80% or more with the siRNA having the chain shown in SEQ ID NO: 1 or SEQ ID NO: 14. It is.
  • the third invention of the present application for solving the above problem is The siRNA that suppresses the expression of the active ingredient GATA-3 gene is 80% or more of the siRNA having the chain of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17 or SEQ ID NO: 18.
  • the therapeutic agent according to the first or second invention which is a siRNA having the homology of:
  • the fourth invention of the present application is: 4.
  • the invention according to any one of the first to third inventions, wherein the siRNA that suppresses the expression of the active ingredient IL-5 gene is an siRNA having a homology of 80% or more with the siRNA having the strand shown in SEQ ID NO: 4. It is a therapeutic agent.
  • the fifth invention of the present application for solving the above-mentioned problems is A gene containing a vector incorporating the gene capable of forming siRNA according to any one of the first to fourth inventions as an active ingredient, and used for the treatment of eosinophilic sinusitis and / or eosinophilic otitis media It is a therapeutic agent.
  • the sixth invention of the present application for solving the above-described problems is as follows. This is a non-therapeutic method for suppressing eosinophil infiltration by knocking down STAT-6 gene, GATA-3 gene and / or IL-5 gene.
  • the first to fourth inventions of the present application provide useful therapeutic agents for eosinophilic sinusitis and eosinophilic otitis media.
  • the first invention of the present application contains siRNA as an active ingredient and enables treatment at the gene level. That is, it is considered possible to treat from the basis of the pathogenesis of eosinophilic sinusitis and eosinophilic otitis media. It is considered that symptoms and pathological conditions of eosinophilic sinusitis and eosinophilic otitis media can be reduced, initial onset, deterioration can be prevented, or relapse cases can be prevented / reduced. Furthermore, it can be expected that eosinophilic sinusitis and eosinophilic otitis media can be curatively treated.
  • the therapeutic agent of the present invention is considered to be a safer therapeutic agent compared with a therapeutic agent containing decoy. While siRNA acts in the cytoplasm, decoy binds to DNA, i.e., acts in the nucleus, which is considered to increase the risk.
  • the fifth invention of the present application provides a useful gene therapy agent.
  • gene therapy there is an embodiment using a vector. Therefore, a gene therapy agent comprising as an active ingredient a siRNA that suppresses the expression of the STAT-6 gene, an siRNA that suppresses the expression of the GATA-3 gene, or a vector incorporating a gene capable of forming an siRNA that suppresses the expression of the IL-5 gene is useful. It is.
  • the gene therapeutic agent is considered to have the same merit as the above-described therapeutic agent.
  • the sixth invention of the present application provides a useful infiltration suppressing method.
  • the infiltration suppression method of the present invention is useful as a tool for elucidating the relationship between eosinophil infiltration and, for example, STAT-6 gene, GATA-3 gene and / or IL-5 gene knockdown. It is expected that elucidation of the kinetics of eosinophils and elucidation of the kinetics of cells / tissues undergoing infiltration will be promoted by utilizing the infiltration suppressing method of the present invention.
  • 3 is a graph showing suppression of STAT-6 gene expression by STAT-6 siRNA.
  • 2 is a graph showing suppression of IL-5 production by STAT-6 siRNA. It is a graph which shows the production
  • 2 is a graph showing suppression of IL-5 production by GATA-3 siRNA.
  • 2 is a graph showing suppression of IL-5 production by IL-5 siRNA.
  • 2 is a graph showing suppression of sinus eosinophil count by STAT-6 siRNA.
  • Eosinophilic sinusitis is sinusitis in which eosinophil infiltration is observed.
  • Eosinophilic otitis media is otitis media with eosinophil infiltration.
  • the therapeutic agent of the present invention contains, as an active ingredient, siRNA that suppresses the expression of the STAT-6 gene, siRNA that suppresses the expression of the GATA-3 gene, and / or siRNA that suppresses the expression of the IL-5 gene.
  • the therapeutic agent of the present invention is used for the treatment of eosinophilic sinusitis and / or eosinophilic otitis media.
  • the STAT-6 gene is a gene belonging to the STAT family. STAT-6 is considered to be an essential transcription factor for Th2 cell differentiation. Examples of siRNAs of the STAT-6 gene include, for example, “Darcan-Nicolaisen Y, Meinicke H, Gabriele Fels G, et al. Small interfering RNA against transcription factor STAT6hibihibits allergic air 7501-7508. ”.
  • GATA-3 belongs to the GATA family and is a regulatory factor for Th2 cells that produce IL-4, IL-5, and IL-13.
  • Examples of siRNA of the GATA-3 gene include, for example, “Xin Y, Yan Y, Hai-yan HE, et al. GATA3 siRNA inhibits the binding of NFAT1 to interleukin-13promoter in human T cells Chin Med J 2010; 123 (6): 739-744 ".
  • IL-5 is a cytokine involved in the differentiation, proliferation and induction of eosinophils.
  • IL-5 is a kind of Th2 cytokine produced by Th2 cells.
  • the IL-5 gene is a gene encoding IL-5.
  • STAT-6 genes, GATA-3 genes, and IL-5 genes are known, and the full-length nucleotide sequences of the genes can be obtained from various papers and databases.
  • an NCBI database can be used.
  • An example of the STAT-6 gene is “http://www.ncbi.nlm.nih.gov/nuccore/NM_001178078.1”
  • an example of the GATA-3 gene is “http://www.ncbi.nlm.nih.gov”. / nuccore / 50541957 ”
  • an example of IL-5 gene is disclosed in“ http://www.ncbi.nlm.nih.gov/nuccore/NM_000879.2 ”.
  • Gene expression refers to the flow of transcription from gene to mRNA and translation from mRNA to protein. Suppressing gene expression is broadly classified into expression suppression due to mRNA degradation and expression suppression due to protein degradation. Since the therapeutic agent of the present invention contains siRNA as an active ingredient, it is considered that when the therapeutic agent of the present invention is used, gene expression is suppressed by degradation of the mRNA of the target gene.
  • siRNA which is an active ingredient, is a short chain that has a function of suppressing post-transcriptional expression of a gene by degrading the mRNA of the target gene when introduced into a living body or cell. It refers to heavy chain RNA.
  • siRNA is used for the purpose of knocking down a target gene.
  • the degree of knockdown may be the degree of knockdown that exhibits a partial therapeutic effect. For example, it suffices to be able to reduce symptoms and pathologies of eosinophilic sinusitis and eosinophilic otitis media, prevent initial onset, prevent worsening, and prevent or reduce relapse cases. It is preferable that eosinophilic sinusitis and eosinophilic otitis media can be radically treated.
  • the siRNA is composed of a sense strand consisting of a sequence homologous to the mRNA of the target gene and an antisense strand consisting of a complementary sequence thereto.
  • it is a “homologous” or “complementary” sequence if the target gene can be knocked down. That is, even when the mRNA of the target gene does not match or correspond to the sequence, it may correspond to the siRNA of the present invention. Furthermore, it is good also as a form containing the said siRNA.
  • the sense strand and the antisense strand may have a protruding portion that does not form a double strand on the 3 'end side and / or 5' end side.
  • siRNA which has the structure where one end was closed
  • shRNA siRNA
  • it may be modified with a nucleic acid molecule chain, peptide, sugar, phosphate, liposome, protein, polymer or the like. These modifications are considered to make knockdown more powerful, increase the efficiency of siRNA introduction into living organisms and cells, reduce side effects, and the like.
  • the length of the sense strand and the antisense strand of the siRNA is not particularly limited as long as the effect of the therapeutic agent of the present invention is exerted, but is preferably 10 to 30 bases, more preferably 15 to 25 bases, 19 More preferably it is ⁇ 23 bases.
  • the ribonucleotide may be a corresponding deoxyribonucleotide.
  • the term “corresponding” refers to the same base species (adenine, guanine, cytosine) although the structures of the sugar moieties are different. In the case of uracil, thymine corresponds.
  • siRNA of the present invention can be appropriately prepared by a known method.
  • a long double-stranded RNA may be prepared, and siRNA may be prepared using Dicer or the like, or may be prepared using a reaction that extends a nucleic acid chain.
  • siRNA that suppresses the expression of the STAT-6 gene, the siRNA that suppresses the expression of the GATA-3 gene, and the siRNA that suppresses the expression of the IL-5 gene are not particularly limited as long as each target gene can be knocked down. It is preferable.
  • the siRNA that suppresses the expression of the STAT-6 gene has a strand described in SEQ ID NO: 1 to be described later, or a strand described in SEQ ID NO: 14 below (that is, a siRNA having a complementary sequence).
  • the siRNA that suppresses the expression of the GATA-3 gene the strand described in SEQ ID NO: 2, the strand described in SEQ ID NO: 3, or the following SEQ ID NOs: 15-18 (candidate sequences of SEQ ID NO: 15-18 are disclosed in the paper)
  • siRNA having a chain described in SEQ ID NO: 4 (the sequence described in SEQ ID NO: 4 is not disclosed in the paper), 80% or more, preferably 90% or more, more preferably 95% or more, more preferably 97% or more.
  • 100% homology is particularly preferred.
  • the homology can be calculated using BLAST.
  • the number of bases may be increased or decreased within the homology range.
  • Sequence number 14 5'-AAGCAGGAAGAACUCAAGUUU-3 ' Sequence number 15: 5'-CUACAAGCUUCACAAUAUU-3 ' Sequence number 16: 5'-GCUUCACAAUAUUAACAGA-3 ' SEQ ID NO: 17: 5'-ACCGAAAAAUGUCUAGCAA-3 ' SEQ ID NO: 18: 5′-CUGGAGGACUUCCCCAAGA-3 ′
  • the siRNA of the present invention may have a structure in which several bases are substituted, deleted, or added in the strand described in each SEQ ID NO.
  • the number of bases to be substituted, deleted or added is preferably 6 or less, more preferably 4 or less, and still more preferably 2 or less.
  • siRNA which is an active ingredient contained in the therapeutic agent of the present invention, is homologous to other genes that are not STAT-6 gene, GATA-3 gene, or IL-5 gene. If not only the STAT-6 gene, GATA-3 gene and IL-5 gene but also other genes are homologous, there is a risk of knocking down to the other genes.
  • the therapeutic agent of the present invention may contain a plurality of siRNAs having different sequences.
  • the therapeutic agent of the present invention can be made into a composition using a siRNA that is an active ingredient, a substance that promotes absorption of the siRNA into a living body, a cell, and the like, a pharmaceutically acceptable carrier, and the like.
  • the pharmaceutically acceptable carrier includes various organic or inorganic carrier substances conventionally used as pharmaceutical materials, excipients, lubricants, binders, disintegrants in solid preparations; solvents in liquid preparations, dissolution aids Agents, suspending agents, isotonic agents, buffers, soothing agents and the like. Further, if necessary, preparation additives such as preservatives, antioxidants, colorants, sweeteners and the like can be used.
  • excipients include lactose, sucrose, D-mannitol, D-sorbitol, starch, pregelatinized starch, dextrin, crystalline cellulose, low-substituted hydroxypropylcellulose, sodium carboxymethylcellulose, gum arabic, pullulan, light Anhydrous silicic acid, synthetic aluminum silicate, magnesium aluminate metasilicate and the like can be mentioned.
  • lubricant examples include magnesium stearate, calcium stearate, talc, colloidal silica and the like.
  • Preferred examples of the binder include pregelatinized starch, sucrose, gelatin, gum arabic, methylcellulose, carboxymethylcellulose, sodium carboxymethylcellulose, crystalline cellulose, sucrose, D-mannitol, trehalose, dextrin, pullulan, hydroxypropylcellulose, hydroxy Examples thereof include propylmethylcellulose and polyvinylpyrrolidone.
  • disintegrant examples include lactose, sucrose, starch, carboxymethyl cellulose, carboxymethyl cellulose calcium, croscarmellose sodium, carboxymethyl starch sodium, light anhydrous silicic acid, low substituted hydroxypropyl cellulose and the like.
  • the solvent include water for injection, physiological saline, Ringer's solution, alcohol, propylene glycol, polyethylene glycol, sesame oil, corn oil, olive oil, cottonseed oil and the like.
  • solubilizer examples include polyethylene glycol, propylene glycol, D-mannitol, trehalose, benzyl benzoate, ethanol, trisaminomethane, cholesterol, triethanolamine, sodium carbonate, sodium citrate, sodium salicylate, sodium acetate. Etc.
  • suspending agent examples include surfactants such as stearyltriethanolamine, sodium lauryl sulfate, laurylaminopropionic acid, lecithin, benzalkonium chloride, benzethonium chloride, glyceryl monostearate; for example, polyvinyl alcohol, polyvinyl Examples include hydrophilic polymers such as pyrrolidone, sodium carboxymethylcellulose, methylcellulose, hydroxymethylcellulose, hydroxyethylcellulose, and hydroxypropylcellulose; polysorbates, polyoxyethylene hydrogenated castor oil, and the like.
  • isotonic agent include sodium chloride, glycerin, D-mannitol, D-sorbitol, glucose and the like.
  • buffer solutions such as phosphate, acetate, carbonate and citrate.
  • Benzyl alcohol etc. are mentioned as a suitable example of a soothing agent.
  • preservative examples include p-hydroxybenzoates, chlorobutanol, benzyl alcohol, phenethyl alcohol, dehydroacetic acid, sorbic acid and the like.
  • antioxidant examples include sulfite and ascorbate.
  • Suitable examples of the colorant include water-soluble edible tar dyes (eg, edible dyes such as edible red Nos. 2 and 3, edible yellow Nos. 4 and 5, edible blue Nos. 1 and 2, and water-insoluble lake dyes ( Examples include aluminum salts of the aforementioned water-soluble edible tar pigments), natural pigments (eg, ⁇ -carotene, chlorophyll, bengara, etc.).
  • water-soluble edible tar dyes eg, edible dyes such as edible red Nos. 2 and 3, edible yellow Nos. 4 and 5, edible blue Nos. 1 and 2
  • water-insoluble lake dyes examples include aluminum salts of the aforementioned water-soluble edible tar pigments), natural pigments (eg, ⁇ -carotene, chlorophyll, bengara, etc.).
  • sweeteners include saccharin sodium, dipotassium glycyrrhizinate, aspartame, stevia and the like.
  • composition can be produced by a method commonly used in the field of pharmaceutical technology, for example, a method described in Japanese Pharmacopoeia.
  • the dosage form of the therapeutic agent of the present invention is not particularly limited, but liquid, emulsion, gel, powder, or ointment is preferable. It is also preferable to use a liposome or the like to form a dosage form in which a drug delivery system is constructed.
  • the therapeutic agent of the present invention is preferably a nasal solution, an injection containing a liquid used for intravenous injection or local administration, and a coating agent.
  • the therapeutic agent of the present invention is also preferably administered directly to the affected area.
  • the administration schedule of the therapeutic agent of the present invention is determined by a doctor in consideration of symptoms and health conditions.
  • the therapeutic agent of the present invention and other drugs can be used in combination.
  • Conventional surgical treatment and administration of the therapeutic agent of the present invention may be used in combination.
  • the therapeutic agent of the present invention may be administered during or immediately after surgery, or the therapeutic agent may be administered after a certain time has elapsed after surgery.
  • the gene therapy agent of the present invention contains, as an active ingredient, a vector in which a gene capable of forming the above-described active ingredient siRNA is incorporated.
  • the vector is not particularly limited as long as the effect of the gene therapy agent of the present invention is exerted, but an adenovirus vector, a herpes virus vector, a vaccinia virus vector, a retrovirus vector, a lentivirus vector, a vector other than a virus, a modified vector thereof, or the like is used. can do.
  • the gene incorporated into the vector is a gene capable of forming the above-mentioned active ingredient siRNA.
  • a gene encoding the sense strand and the antisense strand of the siRNA can be incorporated.
  • a gene encoding shRNA can also be incorporated. If the effects of the present invention are exhibited, for example, a dsRNA gene capable of generating the siRNA of the present invention by the action of Dicer may be incorporated into a vector. In some cases, a gene encoding Dicer can be incorporated.
  • a publicly known method can be appropriately employed for gene integration into the vector.
  • a method using a restriction enzyme and ligase may be used.
  • siRNA structure, preparation, use, administration, and therapeutic effect of the gene therapy agent of the present invention are the same as those of the above-mentioned therapeutic agent.
  • the infiltration suppression method of the present invention suppresses infiltration of eosinophils by knocking down the STAT-6 gene, GATA-3 gene and / or IL-5 gene.
  • the infiltration suppressing method of the present invention provides a method for treating eosinophilic sinusitis and eosinophilic otitis media.
  • these treatment methods it is preferable to perform knockdown using siRNA which is the active ingredient described above. Therefore, it is preferable to use the therapeutic agents and gene therapeutic agents described above for the treatment of diseases.
  • the infiltration suppressing method of the present invention is useful in non-therapeutic applications.
  • Use as a research tool is preferred.
  • it is useful as a tool for elucidating the relationship between STAT-6 gene, GATA-3 gene and / or IL-5 gene knockdown and eosinophil infiltration. It is expected that elucidation of the kinetics of eosinophils and elucidation of the kinetics of cells / tissues undergoing infiltration will be promoted by utilizing the infiltration suppressing method of the present invention.
  • the target of gene knockdown may be an organ, tissue, cell, a disease model animal, or a system that reproduces a disease in vitro. May be.
  • nasal polyps derived from patients with sinusitis nasal polyps forming the nasal polyps, sinus tissues, and middle ear tissues are targeted.
  • eosinophilic sinusitis eosinophilic otitis media, for example, involved in the migration and activation of eosinophils or by means of suppressing substances released from eosinophils It is thought that it can be treated.
  • cytokines IL-2, IL-3, IL-4, IL-6, IL-8, IL-10, IL-12, IL-13, IL-16, IL-18, TGF- ⁇ , TGF- ⁇ , GM-CSF, TNF- ⁇ , TNF- ⁇ ), lipid mediator (leukotriene, PGE2, PAF), adhesion molecule (ICAM-1, VLA-4, LFA-1), chemokine receptor (CCR3) , CCR-1), major basic protein (MBP), eosinophilic protein protein (ECP), eosinophil-derived neurotoxin (EDN), eosinophil peroxidase, chemokine, etc.
  • cytokines IL-2, IL-3, IL-4, IL-6, IL-8, IL-10, IL-12, IL-13, IL-16, IL-18, TGF- ⁇ , TGF- ⁇ , GM-CSF, TNF- ⁇ , TNF- ⁇
  • lipid mediator leu
  • siRNA is effective in the treatment of sinusitis and otitis media involving neutrophils.
  • siRNA that suppresses the expression of IL-6 gene can be used to suppress neutrophils and treat sinusitis and otitis media.
  • Nasal cells were previously reported ("Draheim, R., U. Egerland, and C. Rundfeldt. 2004. Anti-inflammatory potential of the selective phosphodiesterase 4 inhibitor N- (3,5-dichloro-pyrid-4-yl)-[1- (4-fluorobenzyl) -5-hydroxy-indole-3-yl] -gly oxylic acid amide (AWD 12-281), in Human cell preparations.
  • ALD 12-281 ALD 12-281
  • Prostaglandin E (2) suppresses staphylococcal enterotoxin-induced eosinophilia-associated cellular responses dominantly through an E-prostanoid 2-mediated pathway in nasal polyps. J Allergy Clin Immunol 123 (4): 868-74 e13. Prepared from nasal polyps of eosinophilic sinusitis patients.
  • the nose was cut into small pieces. 1 g of the finely cut nasal folds are placed in 4 ml of cultured medium A (the composition of the cultured medium A is RPMI1640 medium containing 2 mg / ml protease, 1.5 mg / ml collagenase, 0.75 mg / ml hyaluronidase, 0.05 mg / ml DNase) The cells were cultured at 37 ° C. for 2 hours.
  • the composition of the cultured medium A is RPMI1640 medium containing 2 mg / ml protease, 1.5 mg / ml collagenase, 0.75 mg / ml hyaluronidase, 0.05 mg / ml DNase
  • the cell suspension cultured using a 70 ⁇ m cell strainer was filtered, and then medium B (the composition of the medium B was RPMI1640 containing 2% FCS, 2 mmol / L glutamine, 100 U / ml penicillin, 100 ⁇ g / ml streptomycin)
  • the filtered cells were washed twice using (medium).
  • the cells were suspended in cultured medium C (the composition of the cultured medium C was RPMI1640 medium containing 10% FCS, 2 mmol / L glutamine, 100 U / ml penicillin, 100 ⁇ g / ml streptomycin).
  • siRNA sensitization For a siRNA specific to the STAT-6 gene having the chain described in SEQ ID NO: 1 (hereinafter also referred to as STAT-6 siRNA), the GATA-3 gene having the chain described in SEQ ID NO: 2 and SEQ ID NO: 3 Specific siRNA (hereinafter, the siRNA having the strand shown in SEQ ID NO: 2 is also referred to as GATA-3 siRNA-1, and the siRNA having the strand shown in SEQ ID NO: 3 is also referred to as GATA-3 siRNA-2), SEQ ID NO: 4 below.
  • siRNA specific to the IL-5 gene having the chain described in (hereinafter also referred to as IL-5 siRNA) and siRNA (hereinafter also referred to as control siRNA) which is a control having the chain described in SEQ ID NO: 5. And tested. Moreover, the control (No siRNA) which does not use siRNA as another control was also produced.
  • Sequence number 1 5'-AGACCUGUCCAUUCGCUCA-3 ' Sequence number 2: 5'-ACAGACCCCUGACUAUGAA-3 '(Position 1609) Sequence number 3: 5'-AAUCCAGACCAGAAACCGAAA-3 ' Sequence number 4: 5'-CAAGUGCAUUGGUGAAAGA-3 '(Position 121) Sequence number 5: 5'-UUCUCCGAACGUGUCACGU-3 '
  • siRNA (2 ⁇ g) and GeneSilencer reagent (20 ⁇ l) were mixed with 50 ⁇ l of RPMI 1640 medium and left at room temperature for 30 minutes. After the indwelling, this mixed solution was added to 400 ⁇ l of the nasal cell suspension in the 12-well plate (the medium in which the nasal cell was suspended was RPMI 1640 medium). After 4 hours, the same amount of medium D (the composition of medium D is 20% FBS, 20 ng / ml GM-CSF (PeproTech), 20 RPMI 1640 medium containing ng / ml IL-4) was added to the nasal mucosa cell suspension.
  • the composition of medium D is 20% FBS, 20 ng / ml GM-CSF (PeproTech), 20 RPMI 1640 medium containing ng / ml IL-4
  • the nasal mucosa cells sensitized with siRNA were cultured using a 48-well plate.
  • Staphylococcal enterotoxin B (SEB, 100 ng / ml), anti-HLA antibody, and anti-ICAM-1 antibody were added to 1 ⁇ 10 6 cells / ml of nasal sputum cells, and cultured at 37 ° C., 5% CO 2 / air mixture.
  • RNA was degraded using 10 ⁇ U of DNase® I (30 minutes at 37 ° C.), then extracted with phenol: chloroform (3: 1), coagulated with ethanol, and washed with 70% ethanol. Then, it melt
  • First-strand cDNA was prepared using SuperScript Preamplification System (Invitrogen).
  • Real-time PCR was performed using SYBR Green PCR Master mix (Stratagene) and 100 nM gene-specific primers. PCR conditions were 95 ° C. for 10 minutes, 95 ° C. for 30 seconds, 58 ° C. for 1 minute, and 72 ° C. for 30 seconds (40 cycles). A GAPDH primer was used as a control. The primer sequences used in the examples are shown below.
  • Sequence number 6 STAT-6 forward: 5'-CCTCGTCACCAGTTGCTT-3 ' Sequence number 7: STAT-6 reverse: 5'-TCCAGTGCTTTCTGCTCC-3 ' Sequence number 8: GATA-3 forward: 5'-GCGGGCTCTATCACAAAATGA-3 ' Sequence number 9: GATA-3 reverse: 5'-GCTCTCCTGGCTGCAGACAGC-3 ' Sequence number 10: IL-5 forward: 5'-GCTTCTGCATTTGAGTTTGCTAGCT-3 ' Sequence number 11: IL-5 reverse: 5'-TGGCCGTCAATGTATTTCTTTATTAAG-3 ' Sequence number 12: GAPDH forward: 5'-TGATGACATCAAGAAGGTGGTGAA-3 ' Sequence number 13: GAPDH reverse: 5'-TCCTTGGAGGCCATGTAGGCCAT-3 '
  • IL-5 and RANTES in the culture supernatant were measured by EIA.
  • IL-5 was measured using Opt EIA sets (BD Biosciences).
  • RANTES was measured using a DuoSet ELISA development kit (R & D Systems).
  • ⁇ result ⁇ Nasal polyps were collected from patients with eosinophilic sinusitis and nasal polyps, and nasal polyps were isolated from the nasal polyps and cultured.
  • STAT-6 siRNA, GATA-3 siRNA, or IL-5 siRNA was introduced into nasal mucosa cells and the effect was examined.
  • a control siRNA introduction group and a siRNA non-introduction group were prepared.
  • the production amount of IL-5 was suppressed to 35 pg / ml by STAT-6 siRNA (no siRNA group was 289 pg / ml, and control siRNA group was 311 pg / ml). The above results are shown in FIG.
  • the amount of RANTES produced was suppressed to 82 pg / ml by STAT-6 siRNA (the no siRNA group was 1350 pg / ml, and the control siRNA group was 1476 pg / ml). The above results are shown in FIG.
  • IL-5 production was suppressed to 29 pg / ml by GATA-3 siRNA-1. In addition, IL-5 production was suppressed to 59 pg / ml by GATA-3 siRNA-2. (The no siRNA test was 272 pg / ml, and the control siRNA test was 282 pg / ml). The above results are shown in FIG.
  • IL-5 siRNA suppressed IL-5 production to 56 pg / ml.
  • the no siRNA group was 312 pg / ml, and the control siRNA test was 336 pg / ml).
  • the above results are shown in FIG. Each of these tests was carried out 5 times, each test result was described as an average value, and FIGS. 2 to 5 were prepared.
  • Th2 In sinusitis involving eosinophils, Th2 is also involved.
  • STAT-6 is considered to be an essential transcription factor for Th2 cell differentiation. In fact, in STAT-6-deficient mice, differentiation into Th2 cells is suppressed and eosinophil infiltration is hardly observed.
  • IL-5 is a cytokine that is deeply involved in the differentiation, proliferation, and induction of eosinophils.
  • siRNA that suppresses the expression of the GATA-3 gene and siRNA that suppresses the expression of the IL-5 gene are considered to suppress the eosinophilic infiltration of the sinuses as well as the siRNA that suppresses the expression of the STAT-6 gene. . It is reasonably speculated that these siRNAs are useful for the treatment of eosinophilic otitis media.
  • RANTES regulated up activation normal Tcell expressed and secreted
  • siRNA that suppresses STAT-6 gene expression suppresses nasal eosinophil infiltration. It is done.
  • the present invention provides a useful therapeutic agent for eosinophilic sinusitis and eosinophilic otitis media, a gene therapeutic agent, and a method for suppressing eosinophil infiltration.

Abstract

[Problem] To provide: a therapeutic agent for eosinophilic sinusitis and eosinophilic otitis media; a gene therapy agent; and a method for preventing the invasion of an eosinophil. [Solution] A therapeutic agent which can be used for the treatment of eosinophilic sinusitis and/or eosinophilic otitis media, which comprises siRNA capable of inhibiting the expression of a STAT-6 gene, siRNA capable of inhibiting the expression of a GATA-3 gene and/or siRNA capable of inhibiting the expression of an IL-5 gene as an active ingredient.

Description

治療剤、遺伝子治療剤及び好酸球の浸潤抑制方法Therapeutic agent, gene therapeutic agent, and method for suppressing eosinophil infiltration
　本発明は、ＳＴＡＴ－６遺伝子、ＧＡＴＡ－３遺伝子及び／又はインターロイキン５（以下、ＩＬ－５とも称する。）遺伝子のノックダウンを利用する、好酸球性副鼻腔炎、好酸球性中耳炎の治療、並びに好酸球の浸潤抑制方法に関する。 The present invention relates to eosinophilic sinusitis and eosinophilic otitis media using knockdown of STAT-6 gene, GATA-3 gene and / or interleukin 5 (hereinafter also referred to as IL-5) gene. And a method for suppressing eosinophil infiltration.
　副鼻腔炎とは、副鼻腔に炎症が生じる疾患である。慢性副鼻腔炎に対する治療としては（１）薬物療法と（２）手術療法がある。薬物療法における近年のマクロライド療法の発見は、好中球等によってひきおこされた副鼻腔炎の制御に著しい効果を示した。また手術療法における内視鏡下副鼻腔手術の普及によって手術の安全性や有効性が向上した。しかし、マクロライド療法を含む薬物療法に抵抗性で、手術をしても易再発性の難治性副鼻腔炎も数多く存在する。その難治性副鼻腔炎の代表的な一つが好酸球が関与する副鼻腔炎である（好酸球性副鼻腔炎と最近呼ばれている）。多くの好酸球が副鼻腔組織に浸潤し、活性化した好酸球が副鼻腔組織を障害すると言われている。術後再発は５年間で５０％以上と言われている。好酸球性副鼻腔炎の多くは好酸球性中耳炎を合併している。好酸球性中耳炎も治療に対して抵抗性を示し、臨床上問題となっている。好酸球性副鼻腔炎、好酸球性中耳炎ともにＴｈ２細胞、Ｔｈ２サイトカインが誘導され、病態の形成に関与していると言われている。 Sinusitis is a disease that causes inflammation in the sinuses. Treatment for chronic sinusitis includes (1) drug therapy and (2) surgical therapy. The recent discovery of macrolide therapy in drug therapy has shown a significant effect on the control of sinusitis caused by neutrophils. In addition, the safety and effectiveness of surgery have improved with the spread of endoscopic sinus surgery in surgical therapy. However, there are many refractory sinusitis that is resistant to drug therapy including macrolide therapy and is easily relapsed even after surgery. One typical example of such intractable sinusitis is sinusitis involving eosinophils (recently called eosinophilic sinusitis). Many eosinophils infiltrate the sinus tissue, and activated eosinophils are said to damage the sinus tissue. Postoperative recurrence is said to be over 50% in 5 years. Most eosinophilic sinusitis is associated with eosinophilic otitis media. Eosinophilic otitis media is also resistant to treatment and is a clinical problem. Both eosinophilic sinusitis and eosinophilic otitis media are said to induce Th2 cells and Th2 cytokines and participate in the pathogenesis.
　薬物療法や手術療法の他、臨床応用が進められている治療手法の一つにＲＮＡ干渉（以下、ＲＮＡｉとも称する。）がある。ＲＮＡ干渉は二重鎖ＲＮＡと相補的な塩基配列をもつｍＲＮＡが分解される現象で、これを利用して特定の遺伝子発現を抑制することが可能となる。 In addition to drug therapy and surgical therapy, RNA interference (hereinafter also referred to as RNAi) is one of the therapeutic techniques that are being clinically applied. RNA interference is a phenomenon in which mRNA having a base sequence complementary to double-stranded RNA is degraded, and this can be used to suppress the expression of a specific gene.
　当該ＲＮＡｉを引き起こす二重鎖ＲＮＡ（以下、ｄｓＲＮＡとも称する。）の導入の検討がなされ、二重鎖ＲＮＡを哺乳類の細胞や生体に導入する場合、短い干渉ＲＮＡであるｓｉＲＮＡが好適であることが突き止められた。 When introduction of double-stranded RNA (hereinafter also referred to as dsRNA) that causes RNAi has been studied, and siRNA that is a short interfering RNA is suitable when introducing double-stranded RNA into a mammalian cell or living body. I was ascertained.
　また、遺伝子治療としてベクターにｓｉＲＮＡを形成しうる遺伝子を組み込み、当該ベクターを投与する技術の臨床応用も進められている。 Also, as a gene therapy, a clinical application of a technique for incorporating a gene capable of forming siRNA into a vector and administering the vector has been promoted.
国際公開ＷＯ２００５／０３０９６０号　上記特許文献１には、ステイプル型オリゴヌクレオチドおよびそれを有効成分とする医薬が開示されている。具体的には転写因子阻害剤、アンチセンスオリゴヌクレオチドまたはｓｉＲＮＡであり、さらに具体的には関節炎、皮膚炎、腎炎、肝炎、腎不全、膀胱炎、前立腺炎、尿道炎、潰瘍性大腸炎またはクローン病、慢性関節リウマチまたは変形性関節症、アトピー性皮膚炎、接触性皮膚炎、乾癬、皮膚潰瘍または褥そうの予防・治療・改善剤を開示する。International Publication WO 2005/030960 Patent Document 1 discloses a staple-type oligonucleotide and a medicine containing the same as an active ingredient. Specifically, transcription factor inhibitor, antisense oligonucleotide or siRNA, more specifically arthritis, dermatitis, nephritis, hepatitis, renal failure, cystitis, prostatitis, urethritis, ulcerative colitis or clone Disclosed is a preventive, therapeutic or ameliorating agent for diseases, rheumatoid arthritis or osteoarthritis, atopic dermatitis, contact dermatitis, psoriasis, skin ulcers or pruritus. 国際公開ＷＯ２００８／０２９４９３号　上記特許文献２にはｓｉＲＮＡを有効成分として含む神経線維変性を抑制する薬剤の開示がある。International Publication WO 2008/029493 Patent Document 2 discloses a drug that suppresses nerve fiber degeneration containing siRNA as an active ingredient. 国際公開ＷＯ２００５／１１３０１４号　上記特許文献３には、ＪＣウィルス感染症の治療のための医薬組成物であって、ｓｉＲＮＡを形成しうる遺伝子を組み込んだベクターを含む医薬組成物を開示する。International Publication WO2005 / 113014 The above-mentioned patent document 3 discloses a pharmaceutical composition for treating JC virus infection, comprising a vector incorporating a gene capable of forming siRNA.
　副鼻腔炎に対する治療法としては、従来、薬物療法や手術療法が中心となってきた。しかし、当該治療法では治療後再発する症例も多く、本願発明者は更に有効な副鼻腔炎の治療について検討した。 Conventionally, pharmacotherapy and surgical treatment have been the main treatment methods for sinusitis. However, there are many cases of recurrence after the treatment, and the inventor of the present application examined a more effective treatment for sinusitis.
　第一に、従来の副鼻腔炎の治療法では、発症機序の根本から治療できていなかった。本願発明者は、発症機序の根本に働きかけない治療ゆえに、治療後再発する症例が多くなると考えた。 First of all, conventional sinusitis treatment has not been able to treat from the basis of the onset mechanism. The inventor of the present application considered that there are many cases of recurrence after treatment because the treatment does not work on the root of the onset mechanism.
　難治性副鼻腔炎の発症機序には好酸球が関与していることが報告されているが、難治性副鼻腔炎の発症機序の根本からの治療を目指し鋭意研究を重ねた結果、本願発明者はＳＴＡＴ－６、ＧＡＴＡ－３、ＩＬ－５遺伝子の発現を抑えることが有効であることを見出した。更には、ＳＴＡＴ－６、ＧＡＴＡ－３、ＩＬ－５遺伝子の発現を抑えるｓｉＲＮＡを利用することが有効であることを見出した。 It has been reported that eosinophils are involved in the pathogenesis of intractable sinusitis, but as a result of intensive research aimed at the fundamental treatment of the pathogenesis of intractable sinusitis, The present inventor has found that it is effective to suppress the expression of the STAT-6, GATA-3, and IL-5 genes. Furthermore, it has been found that it is effective to use siRNA that suppresses the expression of STAT-6, GATA-3, and IL-5 genes.
　ＩＬ－５は好酸球の分化、増殖、誘導に関与するサイトカインである。鋭意研究の結果、本願発明者は上記各ｓｉＲＮＡはＩＬ－５産生を抑制する効果を有するという知見を得た。よって、上記各ｓｉＲＮＡを利用することで、好酸球の浸潤を認める好酸球性疾患の治療が可能であると合理的に推測された。 IL-5 is a cytokine involved in the differentiation, proliferation and induction of eosinophils. As a result of intensive studies, the present inventor has obtained the knowledge that each of the above siRNAs has an effect of suppressing IL-5 production. Therefore, it was reasonably speculated that by using each of the above siRNAs, it was possible to treat an eosinophilic disease in which eosinophil infiltration was observed.
　以上の知見に基づき、本願発明者は本発明を完成させた。有用な好酸球性副鼻腔炎、好酸球性中耳炎の治療剤、遺伝子治療剤、並びに、好酸球の浸潤抑制方法を提供することを本発明が解決すべき課題とする。 Based on the above findings, the present inventor has completed the present invention. It is an object of the present invention to provide a useful therapeutic agent for eosinophilic sinusitis, eosinophilic otitis media, a gene therapeutic agent, and a method for suppressing infiltration of eosinophils.
　（第１発明）
　上記課題を解決するための本願第１発明は、
　ＳＴＡＴ－６遺伝子の発現を抑えるｓｉＲＮＡ、ＧＡＴＡ－３遺伝子の発現を抑えるｓｉＲＮＡ及び／又はＩＬ－５遺伝子の発現を抑えるｓｉＲＮＡを有効成分として含有し、好酸球性副鼻腔炎及び／又は好酸球性中耳炎の治療に用いる治療剤である。 (First invention)
The first invention of the present application for solving the above-described problems is as follows.
It contains siRNA that suppresses the expression of the STAT-6 gene, siRNA that suppresses the expression of the GATA-3 gene and / or siRNA that suppresses the expression of the IL-5 gene as an active ingredient, and contains eosinophilic sinusitis and / or acidophilic acid It is a therapeutic agent used for the treatment of bulbar otitis media.
　（第２発明）
　上記課題を解決するための本願第２発明は、
　前記有効成分であるＳＴＡＴ－６遺伝子の発現を抑えるｓｉＲＮＡが、配列番号１又は配列番号１４に記載の鎖を有するｓｉＲＮＡと８０％以上の相同性があるｓｉＲＮＡである第１発明に記載の治療剤である。 (Second invention)
The second invention of the present application for solving the above-described problems is as follows.
The therapeutic agent according to the first invention, wherein the siRNA that suppresses the expression of the active ingredient STAT-6 gene is an siRNA having a homology of 80% or more with the siRNA having the chain shown in SEQ ID NO: 1 or SEQ ID NO: 14. It is.
　（第３発明）
　上記課題を解決するための本願第３発明は、
　前記有効成分であるＧＡＴＡ－３遺伝子の発現を抑えるｓｉＲＮＡが、配列番号２、配列番号３、配列番号１５、配列番号１６、配列番号１７又は配列番号１８に記載の鎖を有するｓｉＲＮＡと８０％以上の相同性があるｓｉＲＮＡである第１発明又は第２発明に記載の治療剤である。 (Third invention)
The third invention of the present application for solving the above problem is
The siRNA that suppresses the expression of the active ingredient GATA-3 gene is 80% or more of the siRNA having the chain of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17 or SEQ ID NO: 18. The therapeutic agent according to the first or second invention, which is a siRNA having the homology of:
　（第４発明）
　上記課題を解決するための本願第４発明は、
　前記有効成分であるＩＬ－５遺伝子の発現を抑えるｓｉＲＮＡが、配列番号４に記載の鎖を有するｓｉＲＮＡと８０％以上の相同性があるｓｉＲＮＡである第１発明～第３発明のいずれかに記載の治療剤である。 (Fourth invention)
In order to solve the above problems, the fourth invention of the present application is:
4. The invention according to any one of the first to third inventions, wherein the siRNA that suppresses the expression of the active ingredient IL-5 gene is an siRNA having a homology of 80% or more with the siRNA having the strand shown in SEQ ID NO: 4. It is a therapeutic agent.
　（第５発明）
　上記課題を解決するための本願第５発明は、
　第１発明～第４発明のいずれかに記載のｓｉＲＮＡを形成しうる遺伝子を組み込んだベクターを有効成分として含有し、好酸球性副鼻腔炎及び／又は好酸球性中耳炎の治療に用いる遺伝子治療剤である。 (Fifth invention)
The fifth invention of the present application for solving the above-mentioned problems is
A gene containing a vector incorporating the gene capable of forming siRNA according to any one of the first to fourth inventions as an active ingredient, and used for the treatment of eosinophilic sinusitis and / or eosinophilic otitis media It is a therapeutic agent.
　（第６発明）
　上記課題を解決するための本願第６発明は、
　ＳＴＡＴ－６遺伝子、ＧＡＴＡ－３遺伝子及び／又はＩＬ－５遺伝子をノックダウンする非治療的な好酸球の浸潤抑制方法である。 (Sixth invention)
The sixth invention of the present application for solving the above-described problems is as follows.
This is a non-therapeutic method for suppressing eosinophil infiltration by knocking down STAT-6 gene, GATA-3 gene and / or IL-5 gene.
　（第１発明～第４発明）
　本願第１発明～第４発明により、有用な好酸球性副鼻腔炎、好酸球性中耳炎の治療剤が提供される。 (First invention to fourth invention)
The first to fourth inventions of the present application provide useful therapeutic agents for eosinophilic sinusitis and eosinophilic otitis media.
　従来の好酸球性副鼻腔炎の薬物療法や手術療法では、治療後５～６年程度で副鼻腔炎が再発することが多かった。また、従来の好酸球性中耳炎の治療は根本的な治療でないという課題があった。本願発明によれば、好酸球性副鼻腔炎、好酸球性中耳炎の発症機序の根本からの治療が可能であると考えられる。 In conventional drug therapy and surgical treatment for eosinophilic sinusitis, sinusitis often recurs about 5 to 6 years after treatment. Further, there has been a problem that conventional treatment of eosinophilic otitis media is not a fundamental treatment. According to the present invention, it is considered possible to treat from the fundamental pathogenesis of eosinophilic sinusitis and eosinophilic otitis media.
　本願第１発明はｓｉＲＮＡを有効成分として含むものであり、遺伝子レベルでの治療が可能となる。即ち、好酸球性副鼻腔炎、好酸球性中耳炎の発症機序の根本からの治療が可能であると考えられる。好酸球性副鼻腔炎や好酸球性中耳炎の症状や病態を軽減、初発の予防、悪化の予防、又は再発症例の予防・軽減をすることができると考えられる。更には、好酸球性副鼻腔炎、好酸球性中耳炎が根治治療できると期待できる。 The first invention of the present application contains siRNA as an active ingredient and enables treatment at the gene level. That is, it is considered possible to treat from the basis of the pathogenesis of eosinophilic sinusitis and eosinophilic otitis media. It is considered that symptoms and pathological conditions of eosinophilic sinusitis and eosinophilic otitis media can be reduced, initial onset, deterioration can be prevented, or relapse cases can be prevented / reduced. Furthermore, it can be expected that eosinophilic sinusitis and eosinophilic otitis media can be curatively treated.
　また、本発明の治療剤は、デコイを含む治療剤と比べて安全性の高い治療剤であると考えられる。ｓｉＲＮＡが細胞質で作用するのに対しデコイはＤＮＡと結合する、即ち、核内において作用するため危険性が高くなると考えられる。 Also, the therapeutic agent of the present invention is considered to be a safer therapeutic agent compared with a therapeutic agent containing decoy. While siRNA acts in the cytoplasm, decoy binds to DNA, i.e., acts in the nucleus, which is considered to increase the risk.
　（第５発明）
　本願第５発明により、有用な遺伝子治療剤が提供される。遺伝子治療の態様として、ベクターを利用する態様がある。よって、ＳＴＡＴ－６遺伝子の発現を抑えるｓｉＲＮＡ、ＧＡＴＡ－３遺伝子の発現を抑えるｓｉＲＮＡ、ＩＬ－５遺伝子の発現を抑えるｓｉＲＮＡを形成しうる遺伝子を組み込んだベクターを有効成分とする遺伝子治療剤は有用である。当該遺伝子治療剤は上記した治療剤と同様のメリットがあると考えられる。 (Fifth invention)
The fifth invention of the present application provides a useful gene therapy agent. As an embodiment of gene therapy, there is an embodiment using a vector. Therefore, a gene therapy agent comprising as an active ingredient a siRNA that suppresses the expression of the STAT-6 gene, an siRNA that suppresses the expression of the GATA-3 gene, or a vector incorporating a gene capable of forming an siRNA that suppresses the expression of the IL-5 gene is useful. It is. The gene therapeutic agent is considered to have the same merit as the above-described therapeutic agent.
　（第６発明）
　本願第６発明により、有用な浸潤抑制方法が提供される。本発明の浸潤抑制方法は、例えば、ＳＴＡＴ－６遺伝子、ＧＡＴＡ－３遺伝子及び／又はＩＬ－５遺伝子のノックダウンと、好酸球の浸潤との関係を解明するツールとして有用である。本発明の浸潤抑制方法を利用することで好酸球の動態の解明、浸潤を受ける細胞・組織等の動態の解明が進むことが期待される。 (Sixth invention)
The sixth invention of the present application provides a useful infiltration suppressing method. The infiltration suppression method of the present invention is useful as a tool for elucidating the relationship between eosinophil infiltration and, for example, STAT-6 gene, GATA-3 gene and / or IL-5 gene knockdown. It is expected that elucidation of the kinetics of eosinophils and elucidation of the kinetics of cells / tissues undergoing infiltration will be promoted by utilizing the infiltration suppressing method of the present invention.
　特に、好酸球副鼻腔炎、好酸球性中耳炎は症状が重度であり、難治療性の疾患であるので、当該疾患の治療だけでなく予防の要望もある。好酸球と副鼻腔や中耳における細胞・組織等の相互関係が解明されれば、他の観点からの治療薬の開発や、疾患を予防する生活態度の解明が進むと考えられる。 Especially, since eosinophil sinusitis and eosinophilic otitis media are severely symptomatic and difficult to treat, there is a demand not only for treatment of the disease but also for prevention. If the relationship between eosinophils and cells / tissues in the paranasal sinuses and middle ear is elucidated, the development of therapeutic agents from other viewpoints and the elucidation of lifestyles to prevent diseases will progress.
ＳＴＡＴ－６ｓｉＲＮＡによる、ＳＴＡＴ－６遺伝子の発現抑制を示すグラフである。3 is a graph showing suppression of STAT-6 gene expression by STAT-6 siRNA. ＳＴＡＴ－６ｓｉＲＮＡによる、ＩＬ－５の産生抑制を示すグラフである。2 is a graph showing suppression of IL-5 production by STAT-6 siRNA. ＳＴＡＴ－６ｓｉＲＮＡによる、ＲＡＮＴＥＳの産生抑制を示すグラフである。It is a graph which shows the production | generation suppression of RANTES by STAT-6siRNA. ＧＡＴＡ－３ｓｉＲＮＡによる、ＩＬ－５の産生抑制を示すグラフである。2 is a graph showing suppression of IL-5 production by GATA-3 siRNA. ＩＬ‐５ｓｉＲＮＡによる、ＩＬ－５の産生抑制を示すグラフである。2 is a graph showing suppression of IL-5 production by IL-5 siRNA. ＳＴＡＴ－６ｓｉＲＮＡによる、副鼻腔好酸球数の抑制を示すグラフである。2 is a graph showing suppression of sinus eosinophil count by STAT-6 siRNA.
　以下に、本発明の実施形態を、最良の形態を含めて説明する。
　好酸球性副鼻腔炎とは好酸球の浸潤を認める副鼻腔炎である。好酸球性中耳炎とは好酸球の浸潤を認める中耳炎である。 Embodiments of the present invention will be described below including the best mode.
Eosinophilic sinusitis is sinusitis in which eosinophil infiltration is observed. Eosinophilic otitis media is otitis media with eosinophil infiltration.
　〔治療剤〕
　本発明の治療剤は、ＳＴＡＴ－６遺伝子の発現を抑えるｓｉＲＮＡ、ＧＡＴＡ－３遺伝子の発現を抑えるｓｉＲＮＡ、及び／又はＩＬ－５遺伝子の発現を抑えるｓｉＲＮＡを有効成分として含有する。本発明の治療剤は、好酸球性副鼻腔炎及び／又は好酸球性中耳炎の治療に用いる。 [Therapeutic agent]
The therapeutic agent of the present invention contains, as an active ingredient, siRNA that suppresses the expression of the STAT-6 gene, siRNA that suppresses the expression of the GATA-3 gene, and / or siRNA that suppresses the expression of the IL-5 gene. The therapeutic agent of the present invention is used for the treatment of eosinophilic sinusitis and / or eosinophilic otitis media.
　Ｓｔａｔとは、シグナル伝達兼転写活性化因子であり、複数のファミリーを有する。ＳＴＡＴ－６遺伝子はＳＴＡＴファミリーに属する遺伝子である。ＳＴＡＴ－６はＴｈ２細胞の分化に必須の転写因子と考えられている。ＳＴＡＴ－６遺伝子のｓｉＲＮＡとして、例えば、「Darcan-Nicolaisen Y, Meinicke H, Gabriele Fels G, et al. Small interfering RNA against transcription factor STAT6 inhibits allergic airway inflammation and hyperreactivity in mice The Journal of Immunology, 2009, 182: 7501-7508.」に記載のｓｉＲＮＡがある。 Stat is a signal transduction and transcriptional activator and has multiple families. The STAT-6 gene is a gene belonging to the STAT family. STAT-6 is considered to be an essential transcription factor for Th2 cell differentiation. Examples of siRNAs of the STAT-6 gene include, for example, “Darcan-Nicolaisen Y, Meinicke H, Gabriele Fels G, et al. Small interfering RNA against transcription factor STAT6hibihibits allergic air 7501-7508. ”.
　ＧＡＴＡ－３はＧＡＴＡファミリーに属し、ＩＬ－４、ＩＬ－５、ＩＬ－１３を産生するＴｈ２細胞への制御因子である。ＧＡＴＡ－３遺伝子のｓｉＲＮＡとして、例えば、「Xin Y, Yan Y, Hai-yan HE, et al. GATA3 siRNA inhibits the binding of NFAT1 to　interleukin-13promoter in human T cells Chin Med J 2010;123(6):739-744」に記載のｓｉＲＮＡがある。 GATA-3 belongs to the GATA family and is a regulatory factor for Th2 cells that produce IL-4, IL-5, and IL-13. Examples of siRNA of the GATA-3 gene include, for example, “Xin Y, Yan Y, Hai-yan HE, et al. GATA3 siRNA inhibits the binding of NFAT1 to interleukin-13promoter in human T cells Chin Med J 2010; 123 (6): 739-744 ".
　ＩＬ－５は好酸球の分化、増殖、誘導に関与するサイトカインである。また、ＩＬ－５はＴｈ２細胞が産生するＴｈ２サイトカインの一種である。ＩＬ－５遺伝子は、当該ＩＬ－５をコードする遺伝子である。 IL-5 is a cytokine involved in the differentiation, proliferation and induction of eosinophils. IL-5 is a kind of Th2 cytokine produced by Th2 cells. The IL-5 gene is a gene encoding IL-5.
　ＳＴＡＴ－６遺伝子、ＧＡＴＡ－３遺伝子、ＩＬ－５遺伝子は多数公知となっており、遺伝子の全長の塩基配列は種々の論文やデータベース等から得ることができる。例えば、ＮＣＢＩのデータベースを利用することができる。ＳＴＡＴ－６遺伝子の一例は「http://www.ncbi.nlm.nih.gov/nuccore/NM_001178078.1」、ＧＡＴＡ－３遺伝子の一例は「http://www.ncbi.nlm.nih.gov/nuccore/50541957」、ＩＬ－５遺伝子の一例は「http://www.ncbi.nlm.nih.gov/nuccore/NM_000879.2」に開示されている。 Many STAT-6 genes, GATA-3 genes, and IL-5 genes are known, and the full-length nucleotide sequences of the genes can be obtained from various papers and databases. For example, an NCBI database can be used. An example of the STAT-6 gene is “http://www.ncbi.nlm.nih.gov/nuccore/NM_001178078.1”, and an example of the GATA-3 gene is “http://www.ncbi.nlm.nih.gov”. / nuccore / 50541957 ”, an example of IL-5 gene is disclosed in“ http://www.ncbi.nlm.nih.gov/nuccore/NM_000879.2 ”.
　遺伝子の発現とは、遺伝子からｍＲＮＡへの転写、ｍＲＮＡから蛋白質への翻訳という流れを指す。遺伝子の発現を抑えるとは、大別して、ｍＲＮＡの分解による発現抑制と、蛋白質の分解による発現抑制がある。本発明の治療剤はｓｉＲＮＡを有効成分として含むので、本発明の治療剤を使用すると、上記標的遺伝子のｍＲＮＡの分解によって遺伝子の発現が抑えられると考えられる。 Gene expression refers to the flow of transcription from gene to mRNA and translation from mRNA to protein. Suppressing gene expression is broadly classified into expression suppression due to mRNA degradation and expression suppression due to protein degradation. Since the therapeutic agent of the present invention contains siRNA as an active ingredient, it is considered that when the therapeutic agent of the present invention is used, gene expression is suppressed by degradation of the mRNA of the target gene.
　本発明の治療剤において、有効成分であるｓｉＲＮＡとは、生体内や細胞内等に導入されたとき、上記標的遺伝子のｍＲＮＡの分解により遺伝子の転写後発現を抑制する機能を持つ短鎖の二重鎖ＲＮＡをいう。ｓｉＲＮＡは標的遺伝子をノックダウンする目的で使用する。当該ノックダウンの程度は、部分的な治療効果を奏するノックダウンの程度でもよい。例えば、好酸球性副鼻腔炎や好酸球性中耳炎の症状や病態を軽減、初発の予防、悪化の予防、再発症例の予防・軽減をすることができればよい。好酸球性副鼻腔炎、好酸球性中耳炎が根治治療できることが好ましい。 In the therapeutic agent of the present invention, siRNA, which is an active ingredient, is a short chain that has a function of suppressing post-transcriptional expression of a gene by degrading the mRNA of the target gene when introduced into a living body or cell. It refers to heavy chain RNA. siRNA is used for the purpose of knocking down a target gene. The degree of knockdown may be the degree of knockdown that exhibits a partial therapeutic effect. For example, it suffices to be able to reduce symptoms and pathologies of eosinophilic sinusitis and eosinophilic otitis media, prevent initial onset, prevent worsening, and prevent or reduce relapse cases. It is preferable that eosinophilic sinusitis and eosinophilic otitis media can be radically treated.
　上記ｓｉＲＮＡは、上記標的遺伝子のｍＲＮＡと相同な配列からなるセンス鎖と、これと相補的な配列からなるアンチセンス鎖からなる。本発明の治療剤においては、上記標的遺伝子のノックダウンをすることができれば「相同」、「相補」配列である。即ち、標的遺伝子のｍＲＮＡと配列が一致・対応しない場合であっても、本発明のｓｉＲＮＡに該当する場合がある。更に、当該ｓｉＲＮＡを含む形態としても良い。本発明の治療剤の効果を奏する限り、当該センス鎖、アンチセンス鎖は３’末端側及び／又は５’末端側に二重鎖を形成しない突出部位を有していても良い。また、一方の端が閉じた構造であるｓｉＲＮＡ、例えば、ヘアピン構造を有するｓｉＲＮＡ（ｓｈＲＮＡ）としてもよい。また、本発明の治療剤の効果を奏する限り、核酸分子鎖、ペプチド、糖、リン酸、リポゾーム、蛋白質、ポリマー等によって修飾されても良い。これらの修飾により、ノックダウンをより強力にしたり、生体や細胞へのｓｉＲＮＡの導入効率を上げたり、副作用を減少させたりすること等ができると考えられる。上記ｓｉＲＮＡのセンス鎖、アンチセンス鎖の長さは本発明の治療剤の効果を奏する限り特に限定されないが、１０～３０塩基であることが好ましく、１５～２５塩基であることがより好ましく、１９～２３塩基であることが更に好ましい。 The siRNA is composed of a sense strand consisting of a sequence homologous to the mRNA of the target gene and an antisense strand consisting of a complementary sequence thereto. In the therapeutic agent of the present invention, it is a “homologous” or “complementary” sequence if the target gene can be knocked down. That is, even when the mRNA of the target gene does not match or correspond to the sequence, it may correspond to the siRNA of the present invention. Furthermore, it is good also as a form containing the said siRNA. As long as the effect of the therapeutic agent of the present invention is exhibited, the sense strand and the antisense strand may have a protruding portion that does not form a double strand on the 3 'end side and / or 5' end side. Moreover, it is good also as siRNA which has the structure where one end was closed, for example, siRNA (shRNA) which has a hairpin structure. Further, as long as the effect of the therapeutic agent of the present invention is exhibited, it may be modified with a nucleic acid molecule chain, peptide, sugar, phosphate, liposome, protein, polymer or the like. These modifications are considered to make knockdown more powerful, increase the efficiency of siRNA introduction into living organisms and cells, reduce side effects, and the like. The length of the sense strand and the antisense strand of the siRNA is not particularly limited as long as the effect of the therapeutic agent of the present invention is exerted, but is preferably 10 to 30 bases, more preferably 15 to 25 bases, 19 More preferably it is ˜23 bases.
　本発明のｓｉＲＮＡは全てのヌクレオチドがリボヌクレオチドでなくても良い。本発明の効果を奏する限り、リボヌクレオチドは対応するデオキシリボヌクレオチドであっても良い。上記対応するとは、糖部分の構造は異なるものの、同一の塩基種（アデニン、グアニン、シトシン）であることを指す。ウラシルの場合は、チミンが対応する。 In the siRNA of the present invention, not all nucleotides may be ribonucleotides. As long as the effects of the present invention are exhibited, the ribonucleotide may be a corresponding deoxyribonucleotide. The term “corresponding” refers to the same base species (adenine, guanine, cytosine) although the structures of the sugar moieties are different. In the case of uracil, thymine corresponds.
　本発明のｓｉＲＮＡは公知の手法により適宜調製することができる。例えば、長鎖の二重鎖ＲＮＡを用意し、Ｄｉｃｅｒ等によりｓｉＲＮＡを調製しても良いし、核酸鎖を伸長する反応を利用して調製することも可能である。 The siRNA of the present invention can be appropriately prepared by a known method. For example, a long double-stranded RNA may be prepared, and siRNA may be prepared using Dicer or the like, or may be prepared using a reaction that extends a nucleic acid chain.
　ＳＴＡＴ－６遺伝子の発現を抑えるｓｉＲＮＡ、ＧＡＴＡ－３遺伝子の発現を抑えるｓｉＲＮＡ、ＩＬ－５遺伝子の発現を抑えるｓｉＲＮＡは、各標的遺伝子をノックダウンできる限り特段に限定されないが、以下のｓｉＲＮＡとすることが好ましい。 The siRNA that suppresses the expression of the STAT-6 gene, the siRNA that suppresses the expression of the GATA-3 gene, and the siRNA that suppresses the expression of the IL-5 gene are not particularly limited as long as each target gene can be knocked down. It is preferable.
　ＳＴＡＴ－６遺伝子の発現を抑えるｓｉＲＮＡについては後述する配列番号１に記載の鎖、又は下記配列番号１４に記載の鎖を有する（即ち、更に相補的な配列を備えている。）ｓｉＲＮＡ、
　ＧＡＴＡ－３遺伝子の発現を抑えるｓｉＲＮＡについては後述する配列番号２に記載の鎖、配列番号３に記載の鎖、又は下記配列番号１５～１８（当該配列番号１５～１８の候補配列は論文で開示されていない。）に記載の鎖を有するｓｉＲＮＡ、
　ＩＬ－５遺伝子の発現を抑えるｓｉＲＮＡについては後述する配列番号４（当該配列番号４に記載の配列は論文に開示されていない。）に記載の鎖を有するｓｉＲＮＡ、
と８０％以上の相同性があることが好ましく、９０％以上の相同性があることがより好ましく、９５％以上の相同性があることがより好ましく、９７％以上の相同性があることが更に好ましく、１００％の相同性があることが特に好ましい。相同性については、ＢＬＡＳＴを利用して計算可能である。当該相同性の範囲内であれば、塩基数は増減しても良い。本発明において、相同性は、センス鎖又はアンチセンス鎖の一方を用いて計算することとする。 The siRNA that suppresses the expression of the STAT-6 gene has a strand described in SEQ ID NO: 1 to be described later, or a strand described in SEQ ID NO: 14 below (that is, a siRNA having a complementary sequence).
For the siRNA that suppresses the expression of the GATA-3 gene, the strand described in SEQ ID NO: 2, the strand described in SEQ ID NO: 3, or the following SEQ ID NOs: 15-18 (candidate sequences of SEQ ID NO: 15-18 are disclosed in the paper) A siRNA having the strand according to claim 1,
For siRNA that suppresses the expression of IL-5 gene, siRNA having a chain described in SEQ ID NO: 4 (the sequence described in SEQ ID NO: 4 is not disclosed in the paper),
80% or more, preferably 90% or more, more preferably 95% or more, more preferably 97% or more. Preferably, 100% homology is particularly preferred. The homology can be calculated using BLAST. The number of bases may be increased or decreased within the homology range. In the present invention, the homology is calculated using either the sense strand or the antisense strand.
配列番号１４：5’-AAGCAGGAAGAACUCAAGUUU-3’
配列番号１５：5’-CUACAAGCUUCACAAUAUU-3’
配列番号１６：5’-GCUUCACAAUAUUAACAGA-3’
配列番号１７：5’-ACCGAAAAAUGUCUAGCAA-3’
配列番号１８：5’-CUGGAGGACUUCCCCAAGA-3’ Sequence number 14: 5'-AAGCAGGAAGAACUCAAGUUU-3 '
Sequence number 15: 5'-CUACAAGCUUCACAAUAUU-3 '
Sequence number 16: 5'-GCUUCACAAUAUUAACAGA-3 '
SEQ ID NO: 17: 5'-ACCGAAAAAUGUCUAGCAA-3 '
SEQ ID NO: 18: 5′-CUGGAGGACUUCCCCAAGA-3 ′
　本発明のｓｉＲＮＡには、各配列番号に記載の鎖において数塩基が置換、欠失、付加されたものを有する物であっても良い。当該置換、欠失、付加される塩基数は６以下であることが好ましく、４以下であることがより好ましく、２以下であることが更に好ましい。 The siRNA of the present invention may have a structure in which several bases are substituted, deleted, or added in the strand described in each SEQ ID NO. The number of bases to be substituted, deleted or added is preferably 6 or less, more preferably 4 or less, and still more preferably 2 or less.
　本発明の治療剤に含有する有効成分であるｓｉＲＮＡがＳＴＡＴ－６遺伝子、ＧＡＴＡ－３遺伝子、及びＩＬ－５遺伝子でない他の遺伝子と相同であるか調べることが好ましい。ＳＴＡＴ－６遺伝子、ＧＡＴＡ－３遺伝子、ＩＬ－５遺伝子のみならず、他の遺伝子とも相同である場合、当該他の遺伝子までノックダウンするおそれがある。 It is preferable to examine whether siRNA, which is an active ingredient contained in the therapeutic agent of the present invention, is homologous to other genes that are not STAT-6 gene, GATA-3 gene, or IL-5 gene. If not only the STAT-6 gene, GATA-3 gene and IL-5 gene but also other genes are homologous, there is a risk of knocking down to the other genes.
　本発明の治療剤には、異なる配列からなる複数のｓｉＲＮＡを含んでも良い。 The therapeutic agent of the present invention may contain a plurality of siRNAs having different sequences.
　本発明の治療剤は、有効成分であるｓｉＲＮＡの他、生体や細胞等への当該ｓｉＲＮＡの吸収を促進する物質や薬学的に許容される担体等を用いて組成物とすることができる。ここで、薬学的に許容される担体とは、製剤素材として慣用の各種有機あるいは無機担体物質含み、固形製剤における賦形剤、滑沢剤、結合剤、崩壊剤；液状製剤における溶剤、溶解補助剤、懸濁化剤、等張化剤、緩衝剤、無痛化剤などを含む。また必要に応じて、防腐剤、抗酸化剤、着色剤、甘味剤などの製剤添加物を用いることもできる。 The therapeutic agent of the present invention can be made into a composition using a siRNA that is an active ingredient, a substance that promotes absorption of the siRNA into a living body, a cell, and the like, a pharmaceutically acceptable carrier, and the like. Here, the pharmaceutically acceptable carrier includes various organic or inorganic carrier substances conventionally used as pharmaceutical materials, excipients, lubricants, binders, disintegrants in solid preparations; solvents in liquid preparations, dissolution aids Agents, suspending agents, isotonic agents, buffers, soothing agents and the like. Further, if necessary, preparation additives such as preservatives, antioxidants, colorants, sweeteners and the like can be used.
　賦形剤の好適な例としては、乳糖、白糖、Ｄ－マンニトール、Ｄ－ソルビトール、デンプン、α化デンプン、デキストリン、結晶セルロース、低置換度ヒドロキシプロピルセルロース、カルボキシメチルセルロースナトリウム、アラビアゴム、プルラン、軽質無水ケイ酸、合成ケイ酸アルミニウム、メタケイ酸アルミン酸マグネシウムなどが挙げられる。 Preferable examples of excipients include lactose, sucrose, D-mannitol, D-sorbitol, starch, pregelatinized starch, dextrin, crystalline cellulose, low-substituted hydroxypropylcellulose, sodium carboxymethylcellulose, gum arabic, pullulan, light Anhydrous silicic acid, synthetic aluminum silicate, magnesium aluminate metasilicate and the like can be mentioned.
　滑沢剤の好適な例としては、ステアリン酸マグネシウム、ステアリン酸カルシウム、タルク、コロイドシリカなどが挙げられる。 Preferable examples of the lubricant include magnesium stearate, calcium stearate, talc, colloidal silica and the like.
　結合剤の好適な例としては、α化デンプン、ショ糖、ゼラチン、アラビアゴム、メチルセルロース、カルボキシメチルセルロース、カルボキシメチルセルロースナトリウム、結晶セルロース、白糖、Ｄ－マンニトール、トレハロース、デキストリン、プルラン、ヒドロキシプロピルセルロース、ヒドロキシプロピルメチルセルロース、ポリビニルピロリドンなどが挙げられる。 Preferred examples of the binder include pregelatinized starch, sucrose, gelatin, gum arabic, methylcellulose, carboxymethylcellulose, sodium carboxymethylcellulose, crystalline cellulose, sucrose, D-mannitol, trehalose, dextrin, pullulan, hydroxypropylcellulose, hydroxy Examples thereof include propylmethylcellulose and polyvinylpyrrolidone.
　崩壊剤の好適な例としては、乳糖、白糖、デンプン、カルボキシメチルセルロース、カルボキシメチルセルロースカルシウム、クロスカルメロースナトリウム、カルボキシメチルスターチナトリウム、軽質無水ケイ酸、低置換度ヒドロキシプロピルセルロースなどが挙げられる。 Preferable examples of the disintegrant include lactose, sucrose, starch, carboxymethyl cellulose, carboxymethyl cellulose calcium, croscarmellose sodium, carboxymethyl starch sodium, light anhydrous silicic acid, low substituted hydroxypropyl cellulose and the like.
　溶剤の好適な例としては、注射用水、生理的食塩水、リンゲル液、アルコール、プロピレングリコール、ポリエチレングリコール、ゴマ油、トウモロコシ油、オリーブ油、綿実油などが挙げられる。 Favorable examples of the solvent include water for injection, physiological saline, Ringer's solution, alcohol, propylene glycol, polyethylene glycol, sesame oil, corn oil, olive oil, cottonseed oil and the like.
　溶解補助剤の好適な例としては、ポリエチレングリコール、プロピレングリコール、Ｄ－マンニトール、トレハロース、安息香酸ベンジル、エタノール、トリスアミノメタン、コレステロール、トリエタノールアミン、炭酸ナトリウム、クエン酸ナトリウム、サリチル酸ナトリウム、酢酸ナトリウムなどが挙げられる。 Preferable examples of the solubilizer include polyethylene glycol, propylene glycol, D-mannitol, trehalose, benzyl benzoate, ethanol, trisaminomethane, cholesterol, triethanolamine, sodium carbonate, sodium citrate, sodium salicylate, sodium acetate. Etc.
　懸濁化剤の好適な例としては、ステアリルトリエタノールアミン、ラウリル硫酸ナトリウム、ラウリルアミノプロピオン酸、レシチン、塩化ベンザルコニウム、塩化ベンゼトニウム、モノステアリン酸グリセリンなどの界面活性剤；例えばポリビニルアルコール、ポリビニルピロリドン、カルボキシメチルセルロースナトリウム、メチルセルロース、ヒドロキシメチルセルロース、ヒドロキシエチルセルロース、ヒドロキシプロピルセルロースなどの親水性高分子；ポリソルベート類、ポリオキシエチレン硬化ヒマシ油などが挙げられる。
等張化剤の好適な例としては、塩化ナトリウム、グリセリン、Ｄ－マンニトール、Ｄ－ソルビトール、ブドウ糖などが挙げられる。 Suitable examples of the suspending agent include surfactants such as stearyltriethanolamine, sodium lauryl sulfate, laurylaminopropionic acid, lecithin, benzalkonium chloride, benzethonium chloride, glyceryl monostearate; for example, polyvinyl alcohol, polyvinyl Examples include hydrophilic polymers such as pyrrolidone, sodium carboxymethylcellulose, methylcellulose, hydroxymethylcellulose, hydroxyethylcellulose, and hydroxypropylcellulose; polysorbates, polyoxyethylene hydrogenated castor oil, and the like.
Preferable examples of the isotonic agent include sodium chloride, glycerin, D-mannitol, D-sorbitol, glucose and the like.
　緩衝剤の好適な例としては、リン酸塩、酢酸塩、炭酸塩、クエン酸塩などの緩衝液などが挙げられる。 Favorable examples of the buffer include buffer solutions such as phosphate, acetate, carbonate and citrate.
　無痛化剤の好適な例としては、ベンジルアルコールなどが挙げられる。 Benzyl alcohol etc. are mentioned as a suitable example of a soothing agent.
　防腐剤の好適な例としては、パラオキシ安息香酸エステル類、クロロブタノール、ベンジルアルコール、フェネチルアルコール、デヒドロ酢酸、ソルビン酸などが挙げられる。 Preferable examples of the preservative include p-hydroxybenzoates, chlorobutanol, benzyl alcohol, phenethyl alcohol, dehydroacetic acid, sorbic acid and the like.
　抗酸化剤の好適な例としては、亜硫酸塩、アスコルビン酸塩などが挙げられる。 Preferable examples of the antioxidant include sulfite and ascorbate.
　着色剤の好適な例としては、水溶性食用タール色素（例、食用赤色２号および３号、食用黄色４号および５号、食用青色１号および２号などの食用色素、水不溶性レーキ色素（例、前記水溶性食用タール色素のアルミニウム塩など）、天然色素（例、β－カロチン、クロロフィル、ベンガラなど）などが挙げられる。 Suitable examples of the colorant include water-soluble edible tar dyes (eg, edible dyes such as edible red Nos. 2 and 3, edible yellow Nos. 4 and 5, edible blue Nos. 1 and 2, and water-insoluble lake dyes ( Examples include aluminum salts of the aforementioned water-soluble edible tar pigments), natural pigments (eg, β-carotene, chlorophyll, bengara, etc.).
　甘味剤の好適な例としては、サッカリンナトリウム、グリチルリチン酸二カリウム、アスパルテーム、ステビアなどが挙げられる。 Favorable examples of sweeteners include saccharin sodium, dipotassium glycyrrhizinate, aspartame, stevia and the like.
　また、組成物は、製剤技術分野において慣用の方法、例えば日本薬局方に記載の方法等により製造することができる。 In addition, the composition can be produced by a method commonly used in the field of pharmaceutical technology, for example, a method described in Japanese Pharmacopoeia.
　本発明の治療剤の剤型は特に限定されないが、液状、乳化物、ゲル状、粉状、又は軟膏状が好ましい。また、リポソーム等を利用して、ドラッグデリバリーシステムを構築した剤型とすることも好ましい。本発明の治療剤は、点鼻剤、静脈注射や局所投与に用いる液剤を含む注射剤、塗布剤とすることが好ましい。本発明の治療剤は、患部に直接投与することも好ましい。 The dosage form of the therapeutic agent of the present invention is not particularly limited, but liquid, emulsion, gel, powder, or ointment is preferable. It is also preferable to use a liposome or the like to form a dosage form in which a drug delivery system is constructed. The therapeutic agent of the present invention is preferably a nasal solution, an injection containing a liquid used for intravenous injection or local administration, and a coating agent. The therapeutic agent of the present invention is also preferably administered directly to the affected area.
　本発明の治療剤の投与方法・投与量・投与期間・投与間隔等の投与スケジュールは、症状や健康状態を加味し、医師により決定される。本発明の治療剤と他の薬剤を併用することもできる。従来の手術治療と、本発明の治療剤の投与を併用しても良い。例えば、手術中もしくは直後に本発明の治療剤を投与しても良いし、手術後一定の時間が経過してから治療剤を投与しても良い。 The administration schedule of the therapeutic agent of the present invention, such as the administration method, dosage, administration period, and administration interval, is determined by a doctor in consideration of symptoms and health conditions. The therapeutic agent of the present invention and other drugs can be used in combination. Conventional surgical treatment and administration of the therapeutic agent of the present invention may be used in combination. For example, the therapeutic agent of the present invention may be administered during or immediately after surgery, or the therapeutic agent may be administered after a certain time has elapsed after surgery.
　〔遺伝子治療剤〕
　本発明の遺伝子治療剤は、上記した有効成分であるｓｉＲＮＡを形成しうる遺伝子を組み込んだベクターを有効成分として含有する。 [Gene therapy]
The gene therapy agent of the present invention contains, as an active ingredient, a vector in which a gene capable of forming the above-described active ingredient siRNA is incorporated.
　当該ベクターは本発明の遺伝子治療剤の効果を奏する限り特に限定されないが、アデノウィルスベクター、ヘルペスウィルスベクター、ワクシニアウィルスベクター、レトロウィルスベクター、レンチウィルスベクター、ウィルス以外のベクター及びこれらの改変ベクター等を利用することができる。 The vector is not particularly limited as long as the effect of the gene therapy agent of the present invention is exerted, but an adenovirus vector, a herpes virus vector, a vaccinia virus vector, a retrovirus vector, a lentivirus vector, a vector other than a virus, a modified vector thereof, or the like is used. can do.
　当該ベクターに組み込まれる遺伝子は、上記した有効成分であるｓｉＲＮＡを形成しうる遺伝子である。例えば、当該ｓｉＲＮＡのセンス鎖及びアンチセンス鎖をコードする遺伝子を組み込むことができる。また、ｓｈＲＮＡをコードする遺伝子を組み込むこともできる。本発明の効果を奏するのであれば、例えば、Ｄｉｃｅｒの働きにより本発明のｓｉＲＮＡを生成し得るｄｓＲＮＡの遺伝子をベクターに組み込んでも良い。場合により、Ｄｉｃｅｒをコードする遺伝子を組み込むこともできる。 The gene incorporated into the vector is a gene capable of forming the above-mentioned active ingredient siRNA. For example, a gene encoding the sense strand and the antisense strand of the siRNA can be incorporated. A gene encoding shRNA can also be incorporated. If the effects of the present invention are exhibited, for example, a dsRNA gene capable of generating the siRNA of the present invention by the action of Dicer may be incorporated into a vector. In some cases, a gene encoding Dicer can be incorporated.
　ベクターへの遺伝子の組み込みは、公知の手法を適宜採用することができる。例えば、制限酵素とリガーゼを利用する方法等を利用しても良い。 A publicly known method can be appropriately employed for gene integration into the vector. For example, a method using a restriction enzyme and ligase may be used.
　本発明の遺伝子治療剤におけるｓｉＲＮＡの構造、製剤、用途、投与、治療効果に関しては、上記治療剤と同様である。 The siRNA structure, preparation, use, administration, and therapeutic effect of the gene therapy agent of the present invention are the same as those of the above-mentioned therapeutic agent.
　〔浸潤抑制方法〕
　本発明の浸潤抑制方法は、ＳＴＡＴ－６遺伝子、ＧＡＴＡ－３遺伝子及び／又はＩＬ－５遺伝子をノックダウンすることにより、好酸球の浸潤を抑制する。 [Infiltration suppression method]
The infiltration suppression method of the present invention suppresses infiltration of eosinophils by knocking down the STAT-6 gene, GATA-3 gene and / or IL-5 gene.
　即ち、本発明の浸潤抑制方法は好酸球性副鼻腔炎、好酸球性中耳炎の治療方法を提供する。これらの治療方法においては上記した有効成分であるｓｉＲＮＡを用いてノックダウンを行うことが好ましい。よって、疾患の治療に上記した治療剤、遺伝子治療剤を用いることが好ましい。 That is, the infiltration suppressing method of the present invention provides a method for treating eosinophilic sinusitis and eosinophilic otitis media. In these treatment methods, it is preferable to perform knockdown using siRNA which is the active ingredient described above. Therefore, it is preferable to use the therapeutic agents and gene therapeutic agents described above for the treatment of diseases.
　また、本発明の浸潤抑制方法は非治療的な用途においても有用である。リサーチツールとしての活用が好ましい。例えば、ＳＴＡＴ－６遺伝子、ＧＡＴＡ－３遺伝子及び／又はＩＬ－５遺伝子のノックダウンと、好酸球の浸潤との関係を解明するツールとして有用である。本発明の浸潤抑制方法を利用することで好酸球の動態の解明、浸潤を受ける細胞・組織等の動態の解明が進むことが期待される。 Also, the infiltration suppressing method of the present invention is useful in non-therapeutic applications. Use as a research tool is preferred. For example, it is useful as a tool for elucidating the relationship between STAT-6 gene, GATA-3 gene and / or IL-5 gene knockdown and eosinophil infiltration. It is expected that elucidation of the kinetics of eosinophils and elucidation of the kinetics of cells / tissues undergoing infiltration will be promoted by utilizing the infiltration suppressing method of the present invention.
　本発明の非治療的な浸潤抑制方法において遺伝子のノックダウンを受ける対象は器官、組織、細胞、であって良いし、疾患モデル動物であって良いし、Ｉｎ　ｖｉｔｒｏで疾患を再現した系であっても良い。特に、副鼻腔炎患者由来の鼻ポリープ、当該鼻ポリープを形成する鼻茸、副鼻腔組織、中耳組織が対象になる。 In the non-therapeutic invasion suppression method of the present invention, the target of gene knockdown may be an organ, tissue, cell, a disease model animal, or a system that reproduces a disease in vitro. May be. In particular, nasal polyps derived from patients with sinusitis, nasal polyps forming the nasal polyps, sinus tissues, and middle ear tissues are targeted.
　〔その他〕
　本願発明者の知見に基づけば、例えば好酸球の遊走、活性化に関与したり、好酸球が遊離する物質を抑制する手段によっても、好酸球性副鼻腔炎、好酸球性中耳炎の治療ができると考えられる。当該物質として、例えば、サイトカイン（ＩＬ－２，ＩＬ－３，ＩＬ－４，ＩＬ－６，ＩＬ－８，ＩＬ－１０，ＩＬ－１２，ＩＬ－１３,ＩＬ－１６，ＩＬ－１８，ＴＧＦ－α,ＴＧＦ－β,ＧＭ－ＣＳＦ，ＴＮＦ－α,ＴＮＦ－β）、脂質メディエーター（ロイコトリエン、ＰＧＥ２、ＰＡＦ）、接着分子（ＩＣＡＭ－１、ＶＬＡ－４、ＬＦＡ－１）、ケモカイン受容体（ＣＣＲ３、ＣＣＲ－１）、顆粒蛋白（ｍａｊｏｒ　ｂａｓｉｃ　ｐｒｏｔｅｉｎ（ＭＢＰ），ｅｏｓｉｎｏｐｈｉｌｉｃ　ｃａｔｉｏｎｉｃ　ｐｒｏｔｅｉｎ（ＥＣＰ），ｅｏｓｉｎｏｐｈｉｌ－ｄｅｒｉｖｅｄ　ｎｅｕｒｏｔｏｘｉｎ（ＥＤＮ），ｅｏｓｉｎｏｐｈｉｌ　ｐｅｒｏｘｉｄａｓｅ）、ケモカイン（ｅｏｔａｘｉｎ）等を挙げることができる。 [Others]
Based on the knowledge of the present inventor, eosinophilic sinusitis, eosinophilic otitis media, for example, involved in the migration and activation of eosinophils or by means of suppressing substances released from eosinophils It is thought that it can be treated. Examples of the substance include cytokines (IL-2, IL-3, IL-4, IL-6, IL-8, IL-10, IL-12, IL-13, IL-16, IL-18, TGF- α, TGF-β, GM-CSF, TNF-α, TNF-β), lipid mediator (leukotriene, PGE2, PAF), adhesion molecule (ICAM-1, VLA-4, LFA-1), chemokine receptor (CCR3) , CCR-1), major basic protein (MBP), eosinophilic protein protein (ECP), eosinophil-derived neurotoxin (EDN), eosinophil peroxidase, chemokine, etc.
　また、本願発明者の知見に基づけば、好中球が関与する副鼻腔炎、中耳炎の治療においても、ｓｉＲＮＡの利用が有効であると考えられる。例えば、ＩＬ－６遺伝子の発現を抑えるｓｉＲＮＡを利用することで、好中球を抑制し、副鼻腔炎や中耳炎の治療ができると考えられる。 Also, based on the knowledge of the present inventors, it is considered that the use of siRNA is effective in the treatment of sinusitis and otitis media involving neutrophils. For example, it is considered that siRNA that suppresses the expression of IL-6 gene can be used to suppress neutrophils and treat sinusitis and otitis media.
　以下に、本発明の実施例を説明する。本発明の技術的範囲は下記の実施例に限定されない。 Hereinafter, examples of the present invention will be described. The technical scope of the present invention is not limited to the following examples.
　〔鼻茸細胞の準備〕
　鼻茸細胞は以前から報告されている方法（「Draheim, R., U. Egerland,
and C. Rundfeldt. 2004. Anti-inflammatory potential of the selective
phosphodiesterase 4 inhibitor N-(3,5-dichloro-pyrid-4-yl)-[1-(4-fluorobenzyl)-5-hydroxy-indole-3-yl]-gly oxylic acid amide (AWD 12-281), in human cell preparations. J Pharmacol Exp Ther 308(2):555-63.」、「Okano, M., T. Fujiwara, T. Haruna, S. Kariya, S. Makihara, T. Higaki, and K. Nishizaki. 2009. Prostaglandin E(2) suppresses staphylococcal enterotoxin-induced eosinophilia-associated cellular responses dominantly through an E-prostanoid 2-mediated pathway in nasal polyps. J Allergy Clin Immunol 123(4):868-74 e13.」）を用いて、好酸球性副鼻腔炎患者の鼻茸より準備した。 [Preparation of nasal cell]
Nasal cells were previously reported ("Draheim, R., U. Egerland,
and C. Rundfeldt. 2004. Anti-inflammatory potential of the selective
phosphodiesterase 4 inhibitor N- (3,5-dichloro-pyrid-4-yl)-[1- (4-fluorobenzyl) -5-hydroxy-indole-3-yl] -gly oxylic acid amide (AWD 12-281), in Human cell preparations. J Pharmacol Exp Ther 308 (2): 555-63., Okano, M., T. Fujiwara, T. Haruna, S. Kariya, S. Makihara, T. Higaki, and K. Nishizaki 2009. Prostaglandin E (2) suppresses staphylococcal enterotoxin-induced eosinophilia-associated cellular responses dominantly through an E-prostanoid 2-mediated pathway in nasal polyps. J Allergy Clin Immunol 123 (4): 868-74 e13. Prepared from nasal polyps of eosinophilic sinusitis patients.
　患者より鼻茸を採取後、鼻茸を細かく切った。当該細かく切った鼻茸１ｇを４ｍｌの培養ミディウムＡ（当該培養ミディウムＡの組成は、2mg/ml protease、1.5mg/ml collagenase、0.75mg/ml hyaluronidase、0.05mg/ml DNaseを含むRPMI1640培地）に入れ、３７℃の状態で２時間培養した。次いで、７０μｍ　ｃｅｌｌ　ｓｔｒａｉｎｅｒを用いて培養した細胞懸濁液をろ過した後、ミディウムＢ（当該ミディウムＢの組成は、2% FCS、2mmol/L glutamine、100U/ml penicillin、100μg/ml streptomycinを含むRPMI1640培地）を用いて２回、ろ過した細胞を洗浄した。その後、培養ミディウムＣ（当該培養ミディウムＣの組成は、10% FCS、2mmol/L glutamine、100U/ml penicillin、100μg/ml streptomycinを含むRPMI1640培地）で細胞を懸濁状態にした。 After collecting a nose from the patient, the nose was cut into small pieces. 1 g of the finely cut nasal folds are placed in 4 ml of cultured medium A (the composition of the cultured medium A is RPMI1640 medium containing 2 mg / ml protease, 1.5 mg / ml collagenase, 0.75 mg / ml hyaluronidase, 0.05 mg / ml DNase) The cells were cultured at 37 ° C. for 2 hours. Subsequently, the cell suspension cultured using a 70 μm cell strainer was filtered, and then medium B (the composition of the medium B was RPMI1640 containing 2% FCS, 2 mmol / L glutamine, 100 U / ml penicillin, 100 μg / ml streptomycin) The filtered cells were washed twice using (medium). Thereafter, the cells were suspended in cultured medium C (the composition of the cultured medium C was RPMI1640 medium containing 10% FCS, 2 mmol / L glutamine, 100 U / ml penicillin, 100 μg / ml streptomycin).
　〔ｓｉＲＮＡの感作〕
　下記配列番号１に記載の鎖を有するＳＴＡＴ－６遺伝子に特異的なｓｉＲＮＡ（以下、ＳＴＡＴ－６ｓｉＲＮＡとも称する。）、下記配列番号２及び配列番号３に記載の鎖を有するＧＡＴＡ－３遺伝子に対して特異的なｓｉＲＮＡ（以下、配列番号２に記載の鎖を有するｓｉＲＮＡをＧＡＴＡ－３ｓｉＲＮＡ－１、配列番号３に記載の鎖を有するｓｉＲＮＡをＧＡＴＡ－３ｓｉＲＮＡ－２とも称する。）、下記配列番号４に記載の鎖を有するＩＬ－５遺伝子に特異的なｓｉＲＮＡ（以下、ＩＬ－５ｓｉＲＮＡとも称する。）、下記配列番号５に記載の鎖を有するコントロールであるｓｉＲＮＡ（以下、コントロールｓｉＲＮＡとも称する。）を用いて試験した。また、もう一つのコントロールとしてｓｉＲＮＡを使用しないコントロール（Ｎｏ　ｓｉＲＮＡ）も作製した。 [SiRNA sensitization]
For a siRNA specific to the STAT-6 gene having the chain described in SEQ ID NO: 1 (hereinafter also referred to as STAT-6 siRNA), the GATA-3 gene having the chain described in SEQ ID NO: 2 and SEQ ID NO: 3 Specific siRNA (hereinafter, the siRNA having the strand shown in SEQ ID NO: 2 is also referred to as GATA-3 siRNA-1, and the siRNA having the strand shown in SEQ ID NO: 3 is also referred to as GATA-3 siRNA-2), SEQ ID NO: 4 below. SiRNA specific to the IL-5 gene having the chain described in (hereinafter also referred to as IL-5 siRNA) and siRNA (hereinafter also referred to as control siRNA) which is a control having the chain described in SEQ ID NO: 5. And tested. Moreover, the control (No siRNA) which does not use siRNA as another control was also produced.
配列番号１：5’-AGACCUGUCCAUUCGCUCA-3’
配列番号２：5’-ACAGACCCCUGACUAUGAA-3’（Ｐｏｓｉｔｉｏｎ１６０９）
配列番号３：5’-AAUCCAGACCAGAAACCGAAA-3’
配列番号４：5’-CAAGUGCAUUGGUGAAAGA-3’（Ｐｏｓｉｔｉｏｎ１２１）
配列番号５：5’-UUCUCCGAACGUGUCACGU-3’ Sequence number 1: 5'-AGACCUGUCCAUUCGCUCA-3 '
Sequence number 2: 5'-ACAGACCCCUGACUAUGAA-3 '(Position 1609)
Sequence number 3: 5'-AAUCCAGACCAGAAACCGAAA-3 '
Sequence number 4: 5'-CAAGUGCAUUGGUGAAAGA-3 '(Position 121)
Sequence number 5: 5'-UUCUCCGAACGUGUCACGU-3 '
　ｓｉＲＮＡ（２μｇ）、ＧｅｎｅＳｉｌｅｎｃｅｒ　ｒｅａｇｅｎｔ（２０μｌ）を５０μｌのＲＰＭＩ１６４０培地に混ぜ３０分間、室温にて留置した。留置後、この混合液を１２穴プレート中にある４００μｌの鼻茸細胞の懸濁液に加えた（当該鼻茸細胞を懸濁した培地はＲＰＭＩ１６４０培地である。）。４時間後、同量のミディアムＤ（当該ミディアムＤの組成は、20% FBS, 20 ng/ml GM-CSF (PeproTech)、20
ng/ml IL-4を含んだＲＰＭＩ１６４０培地）を鼻茸細胞懸濁液に加えた。 siRNA (2 μg) and GeneSilencer reagent (20 μl) were mixed with 50 μl of RPMI 1640 medium and left at room temperature for 30 minutes. After the indwelling, this mixed solution was added to 400 μl of the nasal cell suspension in the 12-well plate (the medium in which the nasal cell was suspended was RPMI 1640 medium). After 4 hours, the same amount of medium D (the composition of medium D is 20% FBS, 20 ng / ml GM-CSF (PeproTech), 20
RPMI 1640 medium containing ng / ml IL-4) was added to the nasal mucosa cell suspension.
　〔培養〕
　４８ウェルプレートを用いてｓｉＲＮＡを感作させた鼻茸細胞を培養した。 〔culture〕
The nasal mucosa cells sensitized with siRNA were cultured using a 48-well plate.
　１×１０６個／ｍｌの鼻茸細胞にＳｔａｐｙｌｏｃｏｃｃａｌ　ｅｎｔｅｒｏｔｏｘｉｎＢ（ＳＥＢ、１００ｎｇ／ｍｌ）、抗ＨＬＡ抗体、抗ＩＣＡＭ－１抗体を加え、３７℃、５％ＣＯ２／ａｉｒ　ｍｉｘｔｕｒｅの状態で培養した。 Staphylococcal enterotoxin B (SEB, 100 ng / ml), anti-HLA antibody, and anti-ICAM-1 antibody were added to 1 × 10 6 cells / ml of nasal sputum cells, and cultured at 37 ° C., 5% CO 2 / air mixture.
　〔Ｒｅａｌ－ｔｉｍｅ　ＰＣＲ〕
　ＴＲＩｚｏｌ（Ｉｎｖｉｔｒｏｇｅｎ）を用いて、鼻茸細胞よりＴｏｔａｌ　ＲＮＡを抽出した（「Suzuki, M., X. Zheng, X. Zhang, M. Li, C. Vladau, T. E. Ichim, H. Sun, L. R. Min, B. Garcia, and W. P. Min. 2008. Novel vaccination for allergy through gene silencing of CD40 using small interfering RNA. J Immunol 180(12):8461-9.」に記載の手法を採用した。）。 [Real-time PCR]
Total RNA was extracted from nasal cells using TRIzol (Invitrogen) (“Suzuki, M., X. Zheng, X. Zhang, M. Li, C. Vladau, TE Ichim, H. Sun, LR Min, B Garcia, and WP Min. 2008. Novel vaccination for allergy through gene silencing of CD40 using small interfering RNA. J Immunol 180 (12): 8461-9.
　10 U of DNase I を用いて２０μｇＲＮＡを分解（３７℃下で３０分間）、次いでｐｈｅｎｏｌ：ｃｈｌｏｒｏｆｏｒｍ（３：１）で抽出、次いでｅｔｈａｎｏｌで凝結、７０％ｅｔｈａｎｏｌで洗浄した。その後、２０μｌの水（ＲＮａｓｅが存在しない水）を用いて溶解した。 20 μg RNA was degraded using 10 μU of DNase® I (30 minutes at 37 ° C.), then extracted with phenol: chloroform (3: 1), coagulated with ethanol, and washed with 70% ethanol. Then, it melt | dissolved using 20 microliters water (water which RNase does not exist).
　ＳｕｐｅｒＳｃｒｉｐｔ　Ｐｒｅａｍｐｌｉｆｉｃａｔｉｏｎ　Ｓｙｓｔｅｍ（Ｉｎｖｉｔｒｏｇｅｎ）を用いてＦｉｒｓｔ－ｓｔｒａｎｄ　ｃＤＮＡを作製した。 First-strand cDNA was prepared using SuperScript Preamplification System (Invitrogen).
　Ｒｅａｌ－ｔｉｍｅ　ＰＣＲはＳＹＢＲ　Ｇｒｅｅｎ　ＰＣＲ　Ｍａｓｔｅｒ　ｍｉｘ（Ｓｔｒａｔａｇｅｎｅ）と１００ｎＭの遺伝子特異的プライマーを用いて施行した。ＰＣＲのコンディションは９５℃１０分、９５℃３０秒、５８℃１分、７２℃３０秒（４０サイクル）とした。コントロールとしてはＧＡＰＤＨプライマーを使用した。以下、本実施例で使用したプライマーの配列を示す。 Real-time PCR was performed using SYBR Green PCR Master mix (Stratagene) and 100 nM gene-specific primers. PCR conditions were 95 ° C. for 10 minutes, 95 ° C. for 30 seconds, 58 ° C. for 1 minute, and 72 ° C. for 30 seconds (40 cycles). A GAPDH primer was used as a control. The primer sequences used in the examples are shown below.
配列番号６：STAT-6 forward： 5’-CCTCGTCACCAGTTGCTT-3’
配列番号７：STAT-6 reverse： 5’-TCCAGTGCTTTCTGCTCC-3’
配列番号８：GATA-3 forward： 5’-GCGGGCTCTATCACAAAATGA-3’ 
配列番号９：GATA-3 reverse：5’-GCTCTCCTGGCTGCAGACAGC-3’ 
配列番号１０：IL-5 forward：5’-GCTTCTGCATTTGAGTTTGCTAGCT-3’ 
配列番号１１：IL-5 reverse: 5’-TGGCCGTCAATGTATTTCTTTATTAAG-3’
配列番号１２：GAPDH forward： 5’-TGATGACATCAAGAAGGTGGTGAA-3’
配列番号１３：GAPDH reverse： 5’-TCCTTGGAGGCCATGTAGGCCAT-3’ Sequence number 6: STAT-6 forward: 5'-CCTCGTCACCAGTTGCTT-3 '
Sequence number 7: STAT-6 reverse: 5'-TCCAGTGCTTTCTGCTCC-3 '
Sequence number 8: GATA-3 forward: 5'-GCGGGCTCTATCACAAAATGA-3 '
Sequence number 9: GATA-3 reverse: 5'-GCTCTCCTGGCTGCAGACAGC-3 '
Sequence number 10: IL-5 forward: 5'-GCTTCTGCATTTGAGTTTGCTAGCT-3 '
Sequence number 11: IL-5 reverse: 5'-TGGCCGTCAATGTATTTCTTTATTAAG-3 '
Sequence number 12: GAPDH forward: 5'-TGATGACATCAAGAAGGTGGTGAA-3 '
Sequence number 13: GAPDH reverse: 5'-TCCTTGGAGGCCATGTAGGCCAT-3 '
　〔ＩＬ－５、ＲＡＮＴＥＳの測定〕
　培養上清中のＩＬ－５、ＲＡＮＴＥＳをＥＩＡにて測定した。ＩＬ－５はＯｐｔ　ＥＩＡ　ｓｅｔｓ（ＢＤ　Ｂｉｏｓｃｉｅｎｃｅｓ）を用いて測定した。ＲＡＮＴＥＳはＤｕｏＳｅｔ　ＥＬＩＳＡ　ｄｅｖｅｌｏｐｍｅｎｔ　ｋｉｔ　（Ｒ＆Ｄ　Ｓｙｓｔｅｍｓ）を用いて測定した。 [Measurement of IL-5, RANTES]
IL-5 and RANTES in the culture supernatant were measured by EIA. IL-5 was measured using Opt EIA sets (BD Biosciences). RANTES was measured using a DuoSet ELISA development kit (R & D Systems).
　〔感作、免疫と治療〕
　８週齢の雄ＢＡＬＢ／ｃマウスを用いて以下の試験を行った。１０μｇ卵白アルブミン（ｏｖａｌｂｕｍｉｎ、以下、ＯＶＡとも称する。）と２ｍｇのＡｌ（ＯＨ）３を試験開始時（以下、試験開始日をＤａｙ０とも称する。試験開始から経た日数も同様に記載する。）とＤａｙ１４の２回腹腔内に投与した。Ｄａｙ２１からＤａｙ２７まで毎日ＯＶＡ（６００μｇ）を点鼻投与した。治療としてＤａｙ２１からＤａｙ２７まで毎日、上記ＯＶＡ点鼻の５時間前に１００μｇのＳｔａｔ－６ｓｉＲＮＡ、もしくはコントロールｓｉＲＮＡを投与した。 [Sensitization, immunity and treatment]
The following tests were performed using 8-week-old male BALB / c mice. 10 μg ovalbumin (ovalbumin, hereinafter also referred to as OVA) and 2 mg of Al (OH) 3 at the start of the test (hereinafter, the test start date is also referred to as Day 0. The number of days passed from the start of the test is also described) Day 14 Was administered intraperitoneally twice. OVA (600 μg) was administered nasally every day from Day 21 to Day 27. As a treatment, 100 μg of Stat-6 siRNA or control siRNA was administered daily from Day 21 to Day 27, 5 hours before the OVA nasal drop.
　〔病理〕
　上記「感作、免疫と治療」の手順で処置したマウスの頭部を切り離して１０％ｆｏｒｍａｌｉｎで固定した後、脱灰して、薄片に切り分けた。鼻組織を３μＭの厚さで切った後、Ｌｕｎａ染色で染めた。鼻中隔に存在する好酸球の数をカウントした。強拡（１０×４０）で認められる好酸球数を測定した。 [Pathology]
The head of the mouse treated by the procedure of “sensitization, immunization and therapy” was cut off and fixed with 10% formalin, then decalcified and cut into slices. The nasal tissue was cut at a thickness of 3 μM and dyed with Luna staining. The number of eosinophils present in the nasal septum was counted. The number of eosinophils observed with strong expansion (10 × 40) was measured.
　〔統計学的解析〕
　One-way ANOVA（Newman-Keuls Test）を用いて解析した。p < 0.05の場合、統計学的に有意と判断した。 (Statistical analysis)
One-way ANOVA (Newman-Keuls Test) was used for analysis. When p <0.05, it was judged to be statistically significant.
　〔結果〕
　好酸球性副鼻腔炎、鼻ポリープを認める患者より鼻ポリープを採取、鼻ポリープより鼻茸細胞を分離、培養した。鼻茸細胞にＳＴＡＴ－６ｓｉＲＮＡ、ＧＡＴＡ－３ｓｉＲＮＡ、又はＩＬ－５ｓｉＲＮＡを導入してその効果を検討した。コントロール群としてコントロールｓｉＲＮＡ導入群とｓｉＲＮＡ非導入群（Ｎｏ　ｓｉＲＮＡ）を作製した。 〔result〕
Nasal polyps were collected from patients with eosinophilic sinusitis and nasal polyps, and nasal polyps were isolated from the nasal polyps and cultured. STAT-6 siRNA, GATA-3 siRNA, or IL-5 siRNA was introduced into nasal mucosa cells and the effect was examined. As a control group, a control siRNA introduction group and a siRNA non-introduction group (No siRNA) were prepared.
　ＳＴＡＴ－６ｓｉＲＮＡを導入した鼻茸細胞において、ＳＴＡＴ－６遺伝子の発現は有意に低下（コントロールｓｉＲＮＡ群、Ｎｏ　ｓｉＲＮＡ群に対して１２．２％であった。）していた。（図１）。また、ＧＡＴＡ－３ｓｉＲＮＡを導入した鼻茸細胞において、ＧＡＴＡ－３遺伝子の発現は有意に低下（コントロールｓｉＲＮＡ群、Ｎｏ　ｓｉＲＮＡ群に対してＧＡＴＡ－３ｓｉＲＮＡ－１群は１０．４％、ＧＡＴＡ－３ｓｉＲＮＡ－２は２１．２％であった。）していた。また、ＩＬ－５ｓｉＲＮＡを導入した鼻茸細胞において、ＩＬ－５遺伝子の発現は有意に低下（コントロールｓｉＲＮＡ群に対して１２．８％、Ｎｏ　ｓｉＲＮＡ群に対して１３．１％であった。）していた。これらの試験は各５連行い、平均値にて各試験結果を記載した。ＳＴＡＴ－６ｓｉＲＮＡ試験の結果は図１に示した。一方、ＧＡＴＡ－３ｓｉＲＮＡ及びＩＬ－５ｓｉＲＮＡの試験結果の図示は省略する。 In nasal cells that introduced STAT-6 siRNA, the expression of the STAT-6 gene was significantly decreased (12.2% compared to the control siRNA group and the No siRNA group). (FIG. 1). In addition, the expression of the GATA-3 gene was significantly decreased in the nasal sputum cells into which GATA-3 siRNA was introduced (the GATA-3 siRNA-1 group was 10.4% of the control siRNA group and the No siRNA group, and GATA-3 siRNA-2). Was 21.2%). In addition, IL-5 gene expression was significantly decreased in nasal mucosa cells into which IL-5 siRNA had been introduced (12.8% for the control siRNA group and 13.1% for the No siRNA group). It was. Each of these tests was performed 5 times, and each test result was described as an average value. The results of the STAT-6 siRNA test are shown in FIG. On the other hand, illustration of test results of GATA-3 siRNA and IL-5 siRNA is omitted.
　コントロール群と比較して、ＳＴＡＴ－６ｓｉＲＮＡ、ＧＡＴＡ－３ｓｉＲＮＡ－１、ＧＡＴＡ－３ｓｉＲＮＡ－２及びＩＬ－５ｓｉＲＮＡは有意に鼻茸細胞より産生されるＩＬ－５、及び／又は、ＲＡＮＴＥＳを抑制した（図２～図５）。 Compared with the control group, STAT-6 siRNA, GATA-3 siRNA-1, GATA-3 siRNA-2 and IL-5 siRNA significantly suppressed IL-5 and / or RANTES produced from nasal mucosa cells (FIG. 2). To FIG.
　ＳＴＡＴ－６ｓｉＲＮＡによって、ＩＬ－５の産生量は３５ｐｇ／ｍｌまで抑制された（ｎｏ　ｓｉＲＮＡ群は２８９ｐｇ／ｍｌ、ｃｏｎｔｒｏｌ　ｓｉＲＮＡ群は３１１ｐｇ／ｍｌであった。）。以上の結果は図２に示す。ＳＴＡＴ－６ｓｉＲＮＡによって、ＲＡＮＴＥＳの産生量は８２ｐｇ／ｍｌまで抑制された（ｎｏ　ｓｉＲＮＡ群は１３５０ｐｇ／ｍｌ、ｃｏｎｔｒｏｌ　ｓｉＲＮＡ群は　１４７６ｐｇ／ｍｌであった。）。以上の結果は図３に示す。 The production amount of IL-5 was suppressed to 35 pg / ml by STAT-6 siRNA (no siRNA group was 289 pg / ml, and control siRNA group was 311 pg / ml). The above results are shown in FIG. The amount of RANTES produced was suppressed to 82 pg / ml by STAT-6 siRNA (the no siRNA group was 1350 pg / ml, and the control siRNA group was 1476 pg / ml). The above results are shown in FIG.
　ＧＡＴＡ－３ｓｉＲＮＡ－１によって、ＩＬ－５の産生量は２９ｐｇ／ｍｌまで抑制された。また、ＧＡＴＡ－３ｓｉＲＮＡ－２によって、ＩＬ－５の産生量は５９ｐｇ／ｍｌまで抑制された。（ｎｏ　ｓｉＲＮＡ試験は２７２ｐｇ／ｍｌ、ｃｏｎｔｒｏｌ　ｓｉＲＮＡ試験は２８２ｐｇ／ｍｌであった。）。以上の結果は図４に示す。 IL-5 production was suppressed to 29 pg / ml by GATA-3 siRNA-1. In addition, IL-5 production was suppressed to 59 pg / ml by GATA-3 siRNA-2. (The no siRNA test was 272 pg / ml, and the control siRNA test was 282 pg / ml). The above results are shown in FIG.
　ＩＬ－５ｓｉＲＮＡによって、ＩＬ－５の産生量は５６ｐｇ／ｍｌまで抑制された。（ｎｏ　ｓｉＲＮＡ群は３１２ｐｇ／ｍｌ、ｃｏｎｔｒｏｌ　ｓｉＲＮＡ試験は３３６ｐｇ／ｍｌであった。）。以上の結果は図５に示す。これらの試験は各５連行い、平均値にて各試験結果を記載、並びに図２～図５を作成した。 IL-5 siRNA suppressed IL-5 production to 56 pg / ml. (The no siRNA group was 312 pg / ml, and the control siRNA test was 336 pg / ml). The above results are shown in FIG. Each of these tests was carried out 5 times, each test result was described as an average value, and FIGS. 2 to 5 were prepared.
　ＯＶＡを感作、点鼻したマウスにおいて副鼻腔に好酸球浸潤を認めた。ＳＴＡＴ－６ｓｉＲＮＡの投与は有意に副鼻腔の好酸球浸潤を抑制した（図６）。ＳＴＡＴ－６ｓｉＲＮＡ投与では６個、ｎｏ　ｓｉＲＮＡでは２６個、ｃｏｎｔｒｏｌ　ｓｉＲＮＡでは２８個であった。これらの試験は各５連行い、平均値にて各試験結果を記載、並びに図６を作成した。 In mice sensitized and nasalized with OVA, infiltration of eosinophils was observed in the sinuses. Administration of STAT-6 siRNA significantly suppressed sinus eosinophil infiltration (FIG. 6). The number of STAT-6 siRNA administered was 6, that of no siRNA was 26, and that of control siRNA was 28. Each of these tests was performed five times, each test result was described as an average value, and FIG. 6 was created.
　ヘルパーＴ細胞は大きくＴｈ１とＴｈ２に分類される。好酸球が関与する副鼻腔炎では、Ｔｈ２も関与している。ＳＴＡＴ－６はＴｈ２細胞の分化に必須の転写因子と考えられている。実際ＳＴＡＴ－６欠損マウスでは，Ｔｈ２細胞への分化が抑制され，好酸球浸潤がほとんどみられない。 Helper T cells are roughly classified into Th1 and Th2. In sinusitis involving eosinophils, Th2 is also involved. STAT-6 is considered to be an essential transcription factor for Th2 cell differentiation. In fact, in STAT-6-deficient mice, differentiation into Th2 cells is suppressed and eosinophil infiltration is hardly observed.
　ＩＬ－５は好酸球の分化、増殖、誘導に深く関与するサイトカインである。上述の試験結果より、ＧＡＴＡ－３遺伝子の発現を抑えるｓｉＲＮＡ、及びＩＬ－５遺伝子の発現を抑えるｓｉＲＮＡもＳＴＡＴ－６遺伝子の発現を抑えるｓｉＲＮＡ同様に副鼻腔の好酸球浸潤を抑制すると考えられる。また、これらのｓｉＲＮＡが好酸球性中耳炎の治療に有用であることが合理的に推測される。ＲＡＮＴＥＳ（ｒｅｇｕｌａｔｅｄ　ｕｐｏｎ　ａｃｔｉｖａｔｉｏｎ　ｎｏｒｍａｌ　Ｔｃｅｌｌ　ｅｘｐｒｅｓｓｅｄ　ａｎｄ　ｓｅｃｒｅｔｅｄ）も好酸球遊走に関与するケモカインであるが、上述の試験結果よりＳＴＡＴ‐６遺伝子の発現を抑えるｓｉＲＮＡは副鼻腔の好酸球浸潤を抑制すると考えられる。 IL-5 is a cytokine that is deeply involved in the differentiation, proliferation, and induction of eosinophils. Based on the above test results, siRNA that suppresses the expression of the GATA-3 gene and siRNA that suppresses the expression of the IL-5 gene are considered to suppress the eosinophilic infiltration of the sinuses as well as the siRNA that suppresses the expression of the STAT-6 gene. . It is reasonably speculated that these siRNAs are useful for the treatment of eosinophilic otitis media. RANTES (regulated up activation normal Tcell expressed and secreted) is also a chemokine involved in eosinophil migration, but the above test results suggest that siRNA that suppresses STAT-6 gene expression suppresses nasal eosinophil infiltration. It is done.
　本発明により、有用な好酸球性副鼻腔炎、好酸球性中耳炎の治療剤、遺伝子治療剤、並びに、好酸球の浸潤抑制方法が提供される。 The present invention provides a useful therapeutic agent for eosinophilic sinusitis and eosinophilic otitis media, a gene therapeutic agent, and a method for suppressing eosinophil infiltration.

Claims (6)

  1. ＳＴＡＴ－６遺伝子の発現を抑えるｓｉＲＮＡ、ＧＡＴＡ－３遺伝子の発現を抑えるｓｉＲＮＡ及び／又はＩＬ－５遺伝子の発現を抑えるｓｉＲＮＡを有効成分として含有し、
    　好酸球性副鼻腔炎及び／又は好酸球性中耳炎の治療に用いることを特徴とする治療剤。     SiRNA that suppresses the expression of the STAT-6 gene, siRNA that suppresses the expression of the GATA-3 gene and / or siRNA that suppresses the expression of the IL-5 gene as an active ingredient,
    A therapeutic agent for use in the treatment of eosinophilic sinusitis and / or eosinophilic otitis media.
  2. 前記有効成分であるＳＴＡＴ－６遺伝子の発現を抑えるｓｉＲＮＡが、配列番号１又は配列番号１４に記載の鎖を有するｓｉＲＮＡと８０％以上の相同性があるｓｉＲＮＡであることを特徴する請求項１に記載の治療剤。 The siRNA that suppresses the expression of the STAT-6 gene, which is the active ingredient, is an siRNA having a homology of 80% or more with the siRNA having a strand shown in SEQ ID NO: 1 or SEQ ID NO: 14. The therapeutic agent described.
  3. 前記有効成分であるＧＡＴＡ－３遺伝子の発現を抑えるｓｉＲＮＡが、配列番号２、配列番号３、配列番号１５、配列番号１６、配列番号１７又は配列番号１８に記載の鎖を有するｓｉＲＮＡと８０％以上の相同性があるｓｉＲＮＡであることを特徴する請求項１又は請求項２に記載の治療剤。 The siRNA that suppresses the expression of the active ingredient GATA-3 gene is 80% or more of the siRNA having the chain of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17 or SEQ ID NO: 18. The therapeutic agent according to claim 1 or 2, which is an siRNA having the homology of:
  4. 前記有効成分であるＩＬ－５遺伝子の発現を抑えるｓｉＲＮＡが、配列番号４に記載の鎖を有するｓｉＲＮＡと８０％以上の相同性があるｓｉＲＮＡであることを特徴する請求項１～請求項３のいずれかに記載の治療剤。 The siRNA that suppresses the expression of the active ingredient IL-5 gene is an siRNA having a homology of 80% or more with the siRNA having the strand shown in SEQ ID NO: 4. The therapeutic agent in any one.
  5. 請求項１～請求項４のいずれかに記載のｓｉＲＮＡを形成しうる遺伝子を組み込んだベクターを有効成分として含有し、
    　好酸球性副鼻腔炎及び／又は好酸球性中耳炎の治療に用いることを特徴とする遺伝子治療剤。     A vector incorporating a gene capable of forming siRNA according to any one of claims 1 to 4 as an active ingredient,
    A gene therapy agent for use in the treatment of eosinophilic sinusitis and / or eosinophilic otitis media.
  6. ＳＴＡＴ－６遺伝子、ＧＡＴＡ－３遺伝子及び／又はＩＬ－５遺伝子をノックダウンすることを特徴とする非治療的な好酸球の浸潤抑制方法。
          A non-therapeutic method for suppressing eosinophil infiltration, comprising knocking down a STAT-6 gene, a GATA-3 gene and / or an IL-5 gene.
PCT/JP2012/074623 2011-10-04 2012-09-26 Therapeutic agent, gene therapy agent, and method for preventing invasion of eosinophil WO2013051435A1 (en)

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