WO2013051435A1 - Agent thérapeutique, agent de thérapie génique et méthode de prévention de l'invasion de granulocytes éosinophiles - Google Patents

Agent thérapeutique, agent de thérapie génique et méthode de prévention de l'invasion de granulocytes éosinophiles Download PDF

Info

Publication number
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
Authority
WO
WIPO (PCT)
Prior art keywords
sirna
gene
expression
therapeutic agent
seq
Prior art date
Application number
PCT/JP2012/074623
Other languages
English (en)
Japanese (ja)
Inventor
元彦 鈴木
Original Assignee
公立大学法人名古屋市立大学
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 公立大学法人名古屋市立大学 filed Critical 公立大学法人名古屋市立大学
Publication of WO2013051435A1 publication Critical patent/WO2013051435A1/fr

Links

Images

Classifications

    • 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

L'invention concerne un agent thérapeutique pour une sinusite à éosinophiles et une otite moyenne à éosinophiles ; un agent de thérapie génique ; une méthode de prévention de l'invasion d'un granulocyte éosinophile. L'invention concerne un agent thérapeutique qui peut être utilisé pour le traitement d'une sinusite à éosinophiles et/ou une otite moyenne à éosinophiles, qui comporte un ARNsi capable d'inhiber l'expression d'un gène STAT-6, un ARNsi capable d'inhiber l'expression d'un gène GATA-3 et/ou un ARNsi capable d'inhiber l'expression d'un gène IL-5 comme principe actif.
PCT/JP2012/074623 2011-10-04 2012-09-26 Agent thérapeutique, agent de thérapie génique et méthode de prévention de l'invasion de granulocytes éosinophiles WO2013051435A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
JP2011-219928 2011-10-04
JP2011219928A JP2013079210A (ja) 2011-10-04 2011-10-04 治療剤、遺伝子治療剤及び好酸球の浸潤抑制方法

Publications (1)

Publication Number Publication Date
WO2013051435A1 true WO2013051435A1 (fr) 2013-04-11

Family

ID=48043597

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/JP2012/074623 WO2013051435A1 (fr) 2011-10-04 2012-09-26 Agent thérapeutique, agent de thérapie génique et méthode de prévention de l'invasion de granulocytes éosinophiles

Country Status (2)

Country Link
JP (1) JP2013079210A (fr)
WO (1) WO2013051435A1 (fr)

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
RU2559593C1 (ru) * 2014-08-06 2015-08-10 Государственное бюджетное образовательное учреждение дополнительного профессионального образования "Новокузнецкий государственный институт усовершенствования врачей" Министерства здравоохранения Российской Федерации Способ лечения обострений хронических воспалительных заболеваний пазух носа

Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004045543A2 (fr) * 2002-11-14 2004-06-03 Dharmacon, Inc. Arnsi fonctionnel et hyperfonctionnel
WO2005011610A2 (fr) * 2003-07-30 2005-02-10 The Board Of Trustees Of The Leland Stanford Junior University Methodes et composes d'inhibition de l'agregation de proteines neurodegeneratives
JP2007537157A (ja) * 2004-03-30 2007-12-20 イッサム リサーチ ディベロップメント カンパニー オブ ザ ヘブリュ ユニバーシティ オブ エルサレム アレルギー性反応に関与する細胞を標的化するための二重特異性抗体、ならびにその組成物および使用
JP2009536518A (ja) * 2006-05-11 2009-10-15 ホールサム バイオファーム ピーティーワイ エルティーディー アレルギー疾患を治療する短鎖干渉ｒｎａ
WO2010104959A1 (fr) * 2009-03-11 2010-09-16 Promedior, Inc. Procédés de traitement et de diagnostic pour troubles d'hypersensibilité
WO2010107958A1 (fr) * 2009-03-19 2010-09-23 Merck Sharp & Dohme Corp. INHIBITION INDUITE PAR ARN INTERFÉRENCE DE L'EXPRESSION DU GÈNE TRANSDUCTEUR DE SIGNAL ET ACTIVITATEUR DE TRANSCRIPTION 6 (STAT6) AU MOYEN D'UN ACIDE NUCLÉIQUE INTERFÉRENT COURT (ANsi)
WO2010107957A2 (fr) * 2009-03-19 2010-09-23 Merck Sharp & Dohme Corp. Inhibition induite par arn interférence d'une expression génique (gata3) d'une protéine de liaison gata au moyen d'un acide nucléique interférent court

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004045543A2 (fr) * 2002-11-14 2004-06-03 Dharmacon, Inc. Arnsi fonctionnel et hyperfonctionnel
WO2005011610A2 (fr) * 2003-07-30 2005-02-10 The Board Of Trustees Of The Leland Stanford Junior University Methodes et composes d'inhibition de l'agregation de proteines neurodegeneratives
JP2007537157A (ja) * 2004-03-30 2007-12-20 イッサム リサーチ ディベロップメント カンパニー オブ ザ ヘブリュ ユニバーシティ オブ エルサレム アレルギー性反応に関与する細胞を標的化するための二重特異性抗体、ならびにその組成物および使用
JP2009536518A (ja) * 2006-05-11 2009-10-15 ホールサム バイオファーム ピーティーワイ エルティーディー アレルギー疾患を治療する短鎖干渉ｒｎａ
WO2010104959A1 (fr) * 2009-03-11 2010-09-16 Promedior, Inc. Procédés de traitement et de diagnostic pour troubles d'hypersensibilité
WO2010107958A1 (fr) * 2009-03-19 2010-09-23 Merck Sharp & Dohme Corp. INHIBITION INDUITE PAR ARN INTERFÉRENCE DE L'EXPRESSION DU GÈNE TRANSDUCTEUR DE SIGNAL ET ACTIVITATEUR DE TRANSCRIPTION 6 (STAT6) AU MOYEN D'UN ACIDE NUCLÉIQUE INTERFÉRENT COURT (ANsi)
WO2010107957A2 (fr) * 2009-03-19 2010-09-23 Merck Sharp & Dohme Corp. Inhibition induite par arn interférence d'une expression génique (gata3) d'une protéine de liaison gata au moyen d'un acide nucléique interférent court

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
JUN'ICHIRO OHORI ET AL.: "Eosinophil Infiltration Mechanisms in Chronic Rhinosinusitis", PRACTICA OTO-RHINO- LARYNGOLOGICA, vol. 104, no. 7, July 2011 (2011-07-01), pages 465 - 473 *
PELIKAN Z.: "Role of nasal allergy in chronic secretory otitis media.", CURR ALLERGY ASTHMA REP., vol. 99, no. 2, 2009, pages 107 - 13, XP055064563 *
TAKENO S ET AL.: "Pathological mechanisms and clinical features of eosinophilic chronic rhinosinusitis in the Japanese population.", ALLERGOL INT., vol. 59, no. 3, 2010, pages 247 - 56, XP055064560 *
YUICHI KURONO: "Tokushu Kosankyu Kanren no Byohen 1. Kosankyu Soron", OTOLARYNGOLOGY-HEAD AND NECK SURGERY, vol. 82, no. 10, 2010, pages 653 - 659 *

Also Published As

Publication number Publication date
JP2013079210A (ja) 2013-05-02

Similar Documents

Publication Publication Date Title
JP6492053B2 (ja) 修飾ｔｇｆ−ベータオリゴヌクレオチド
EP2978844B1 (fr) Oligonucléotides de tgf-bêta2 modifiés
CN116726203A (zh) 编码免疫调节多肽的mrna的组合及其用途
US20180180630A1 (en) Methods for treating inflammatory bowel disease
TW200526238A (en) Combination therapy for treatment of ocular neovascular disorders
EP3210611A2 (fr) Procédés de traitement de troubles inflammatoire vasculaires
ES2732351T3 (es) ARNip y su uso en métodos y composiciones para el tratamiento y/o la prevención de afecciones oculares
US20190144865A1 (en) Modified tgf-beta oligonucleotide for use in a method of preventing and/or treating an ophthalmic disease
RU2663100C2 (ru) Ми-рнк и их применение в способах и композициях для лечения и/или профилактики глазных состояний
JP2016535592A (ja) ＯＲＡＩ１遺伝子の発現を阻害するための方法及び組成物におけるｓｉＲＮＡ及びその使用
TW201420105A (zh) 黏膜治癒促進劑
WO2013051435A1 (fr) Agent thérapeutique, agent de thérapie génique et méthode de prévention de l'invasion de granulocytes éosinophiles
WO2023051822A1 (fr) Oligonucléotide de ciblage pour le traitement de maladies associées à pcsk9
JP2023503804A (ja) Ｉｌ－３４アンチセンス薬剤、およびこれを使用する方法
WO2017136435A1 (fr) Composés permettant de moduler l'expression de fc-epsilon-ri-bêta et utilisations correspondantes
RU2653766C2 (ru) КиРНК И ИХ ИСПОЛЬЗОВАНИЕ В СПОСОБАХ И КОМПОЗИЦИЯХ ДЛЯ ЛЕЧЕНИЯ И/ИЛИ ПРОФИЛАКТИКИ ГЛАЗНЫХ ЗАБОЛЕВАНИЙ
JP2016540499A (ja) ＰＤＫ１遺伝子の発現を阻害するための方法及び組成物におけるｓｉＲＮＡ及びその使用
US20240093197A1 (en) Il-34 antisense agents and methods of using same
WO2017195042A1 (fr) Méthodes de traitement de maladies associées à des cellules ilc3
EP3565571B1 (fr) Formulations d'alicaforsen
WO2012105467A1 (fr) Composition d'acide oligonucléique et agent antiallergique
WO2023092060A1 (fr) Commutateurs d'arnm dépendant de microarn pour des thérapies à base d'arnm spécifiques de tissu
WO2022243299A1 (fr) Agents anti-sens il-34 et leurs procédés d'utilisation
IT202100002573A1 (it) Small interfering rna (sirna) per la terapia della sindrome megacisti-microcolon-ipoperistalsi intestinale (mmihs) causata da mutazione del gene actg2
TW201631157A (zh) 用以治療癌症之短干擾核糖核酸分子

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 12837707

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 12837707

Country of ref document: EP

Kind code of ref document: A1