WO2006123631A1 - Rna-containing composition - Google Patents

Rna-containing composition Download PDF

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Publication number
WO2006123631A1
WO2006123631A1 PCT/JP2006/309699 JP2006309699W WO2006123631A1 WO 2006123631 A1 WO2006123631 A1 WO 2006123631A1 JP 2006309699 W JP2006309699 W JP 2006309699W WO 2006123631 A1 WO2006123631 A1 WO 2006123631A1
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Prior art keywords
rna
carrier
blood
complex
amino acid
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PCT/JP2006/309699
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French (fr)
Japanese (ja)
Inventor
Ayumi Sato
Miwa Hirai
Motoki Takagi
Akira Shimamoto
Atsushi Maruyama
Sung Won Choi
Arihiro Kano
Asako Yamayoshi
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Kyushu University, National University Corporation
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Publication of WO2006123631A1 publication Critical patent/WO2006123631A1/en

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    • 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
    • C12N15/1137—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 against enzymes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/59—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
    • A61K47/60—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A61K47/645—Polycationic or polyanionic oligopeptides, polypeptides or polyamino acids, e.g. polylysine, polyarginine, polyglutamic acid or peptide TAT
    • A61K47/6455—Polycationic oligopeptides, polypeptides or polyamino acids, e.g. for complexing nucleic acids
    • 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
    • C12N15/111—General methods applicable to biologically active non-coding nucleic acids
    • 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.
    • 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
    • C12N2320/00—Applications; Uses
    • C12N2320/30—Special therapeutic applications
    • C12N2320/32—Special delivery means, e.g. tissue-specific

Definitions

  • the present invention relates to an RNA-containing composition that can be administered into blood.
  • a technology for effectively reaching an intended tissue or cell as an active ingredient is one of the important research subjects.
  • a compound having an excellent pharmacological action cannot be expected to have a pharmacological effect unless it reaches the target tissue.
  • active pharmaceutical ingredients usually exert their pharmacological effects by reaching the site of action after being administered into the blood or orally. If the administered drug cannot efficiently reach the site of action, a large amount of drug must be administered. As a result, the risk of side effects due to drug administration increases.
  • Various mechanisms have been devised to deliver drugs to the intended site (Delivery). Such a mechanism is called a drug delivery system (DDS).
  • DDS drug delivery system
  • a drug administered to a living body is affected by the living body as follows. Drugs that escape these effects and eventually reach the site of action provide the expected therapeutic effect. Drugs that do not lead to therapeutic effects as a result are metabolized or excreted.
  • the drug must first be absorbed by the body. Except for local administration, the drug is usually administered to a site away from the affected area, absorbed by the living body, and then diffused into the living body to reach the affected area. Absorption of the drug by the living body is the first barrier for the drug to reach the site of action.
  • the amount of drug absorbed affects the in vivo concentration, depending on the administration method such as oral administration, transdermal administration, enteral administration, or respiratory tract administration.
  • the drug concentration in a living body usually depends on the dose and is not affected by absorption.
  • the site of action is isolated from the bloodstream, the degree of drug transfer from the blood to the site of action is limited.
  • the final working concentration of the drug will be affected.
  • cancer tissue is a tissue with increased angiogenesis and abundant blood flow.
  • the drug needs to penetrate the cell membrane.
  • the site where many pile cancer drugs are applied is inside the cell. In other words, a therapeutic effect is achieved by a drug that has permeated the cell membrane of cancer cells.
  • the process of reaching the site of action after the drug is absorbed by the living body and transferred to the blood can also be positioned as drug absorption.
  • the drug is a foreign substance for the living body. Therefore, drugs that are administered (or absorbed) to the living body are usually removed from the living body. Specifically, the degradation mechanism and excretion mechanism of the living body acts on the drug in the direction of removing it. As a result, generally, the in vivo concentration of a drug administered to a living body decreases with time. If the drug is not accumulative at the site of action, the concentration in the body will match that of the drug. In other words, it means that the effect of the drug decreases with time. Alternatively, it is difficult to expect a high therapeutic effect for a drug that is excreted quickly and cannot maintain its blood concentration, even if the drug has good transferability from blood to the site of action. That is, it can be said that the therapeutic effect by the drug is brought about until the administered drug is removed from the living body.
  • the delivery of a drug by DDS aims to control the influence of a living body on the drug and efficiently deliver the drug to the site of operation. Specifically, efforts have been made to efficiently bring drugs to the site of action by the following approach, for example.
  • Targeting a drug to a site of action A technique for selectively transferring a drug to a specific cell using a substance having a high affinity for the specific cell is known. For example, anticancer agents in which a cytotoxic substance is bound to an antibody are known. This type of treatment is called targeting therapy. Targeting causes the drug to bind to specific cells. As a result, the drug concentration at (or near) the site of action can be kept high. Targeting also prevents the transfer of drugs to the organs responsible for the metabolism of the liver or kidneys. As a result, the drug can be held in the living body for a long time.
  • [0007] Drug protection: If the metabolism of the drug can be prevented, It can exist in the living body for a long time.
  • drugs are often filled into capsules that withstand strong acidity.
  • the administered capsule dissolves in the intestine via the stomach and releases the drug.
  • the role of the capsule is to prevent degradation of the drug under strong acidic conditions in the stomach and increase the amount of absorption.
  • blood concentration can be maintained at a high level by binding a polymer compound to a protein preparation. This method utilizes the phenomenon that protein metabolism by the living body is inhibited by the binding of polymer compounds.
  • RNAi RNA interference
  • C. elegans Non-patent Document 1 / Fire et al., Nature, 391, 806-811, 1998), and RNAi due to double-stranded RNA of 21 bases was subsequently detected in mammalian cells.
  • Non-Patent Document 2 Elbashir et al., Nature, 411, 494-498, 2001.
  • the mechanism of RNAi is currently not fully clarified. The following models have been estimated by various analyses.
  • RNA force SRNaselll-type nucleolytic enzyme introduced into a cell is fragmented into short RNA having a length of about 21-23 bases.
  • the R Naselll-type nucleolytic enzyme that acts at this time is called a dicer.
  • Fragmented RNA forms a complex with multiple proteins such as helicases.
  • the complex of RNA and protein formed at this time is RISC (RNA-induced silencing cmplex).
  • a helicase is an enzyme that has the effect of breaking a double-stranded nucleic acid into a single strand in an ATP-dependent manner. RISC becomes active when its double-stranded RNA is made into single strands by the action of helicase.
  • siRNA small interfering RNA
  • siRNA The gene expression suppression effect by siRNA is very strong. Therefore, it is attracting attention as a new gene expression suppression technology that can replace antisense and ribozyme.
  • the suppressive action of gene expression is applied to various genes. For example, in medicine, attempts have been reported to suppress the expression of genes that cause disease by the action of siRNA. The siRNA targets that have been confirmed to be effective in the medical field are shown below. Infectious agent genes: HIV, HBV, HCV, etc.
  • Oncogene Her2 / neu, EGFR, VEGF, HPV, etc.
  • Patent Document 1 / WO2004 / 100990.
  • RNA is a biological molecule that is extremely susceptible to degradation. The level of protein expression can be controlled by the transcriptional regulation of genes, because RNA is easily degraded. In vivo, mRNA that has finished protein synthesis is rapidly degraded. In addition, short RNAs present in the blood are rapidly excreted in the urine by the kidneys as they are. However, when RNA is used as a medicine, there is a risk that sustained efficacy cannot be expected unless RNA is stably maintained. To address these issues, for example, there are attempts to express siRNA in cells using various vectors.
  • siRNA In order to expect the therapeutic effect of siRNA in vivo, technology to efficiently deliver siRNA to the target tissue is required.
  • the drug when it is desired to transport siRNA to a specific tissue such as cancer, it is preferable that the drug has tissue transferability.
  • currently known vectors for gene transfer into living organisms have low selectivity for cells. Therefore, when administered systemically, it is difficult to target a specific tissue.
  • Non-patent Document 3 ZSoutschek et al., Nature, 432, 173). -178, 2004.
  • hepatic apolipoprotein B expression was suppressed. It would be useful if a carrier having transferability was provided to other tissues.
  • Patent Document 2 Block copolymer of polyethylene glycol and polyaspartic acid + adriamycin
  • Patent Document 3 Thiolated block copolymer such as polyethylene glycol and polylysine + polymer electrolyte
  • Patent Document 4 Block copolymer such as polyethylene glycol and polyglutamic acid + cisbratin
  • Patent Document 5 Block copolymer of polyethylene glycol and polymethacrylic acid + nucleic acid
  • Patent Document 6 Block copolymer of polyethylene glycol and polycation + nucleic acid or anionic protein
  • Non-patent document 4 / Bioconjug Chem. (1998) 9, 29 2-299 .; Non-patent document 5 / Bioconjug Chem. (2000) 11, 520-526 .; Non-patent document Reference 6 / Nat Mater. (2003) 2, 815-820 ⁇ ).
  • Non-Patent Document 1 Fire et al., Nature, 391, 806-811, 1998
  • Non-Patent Document 2 Elbashir et al., Nature, 411, 494-498, 2001
  • Non-Patent Document 3 Soutschek et al., Nature, 432, 173-178, 2004
  • Patent Document 4 Maruyama A, Watanabe H, Ferdous A, Katoh M, Ishihara T, Akaike T. Characterization of interpolyelectrolyte complexes between double-stranded DN A and polylysine comb-type copolymers having hydrophilic side chains.Bioconjug C hem. (1998) 9, 292-299.
  • Non-Special Terms 5 Ferdous A, Akaike T, Maruyama A. Mechanism of intermolecular puri ne-purine-pyrimidine triple helix stabilization by comb-type polylysine graft copolymer at physiologic potassium concentration. Bioconjug Chem. (2000) 11, 520-526.
  • Non-Patent Document 6 Kim WJ, Sato Y, Akaike T, Maruyama A. Cationic comb-type copoly mers for DNA analysis. Nat Mater. (2003) 2, 815—820.
  • Patent Document 1 WO2004 / 100990
  • Patent Document 2 JP-A-7-69900
  • Patent Document 3 JP 2001-146556
  • Patent Document 4 WO2002 / 26241
  • Patent Document 5 JP 2003-113214 A
  • Patent Document 6 JP-A-2004-352972
  • Patent Document 7 Japanese Patent Laid-Open No. 10-158196
  • An object of the present invention is to provide an RNA composition that can be administered into blood.
  • an object of the present invention is to provide a composition that can stably maintain RNA in blood, or a method therefor.
  • an object of the present invention is to provide a use of RNA having improved retention in blood.
  • the present inventors have conducted research on compounds that improve the retention of RNA in blood. It was also found that the retention of RNA in blood is improved by blending with a specific carrier. Furthermore, it has been clarified that the retention of RNA in blood is improved when RNA and a specific carrier are administered separately. That is, the present invention provides the following composition, a method for producing the same, and a method for stabilizing RNA in blood. The present invention also provides use of RNA with improved retention in blood based on the present invention.
  • the present invention provides the following [1] to [41].
  • a composition for administering RNA into blood comprising a complex of RNA and a carrier having a hydrophilic group bonded in a comb shape to a polycationic compound as a side chain and RNA.
  • RNA containing a carrier having a hydrophilic group as a side chain bonded to a polycationic compound in a comb shape as an active ingredient, RNA in blood Yarn and composition for stabilization.
  • composition according to [2], wherein the carrier is administered simultaneously with RNA or before RNA is administered simultaneously with RNA or before RNA.
  • composition according to [1] or [2], wherein the polycationic compound is poly (cationic amino acid).
  • composition according to [1] or [2], wherein the hydrophilic group is at least one selected from the group consisting of glycosaminodarlican, dextran, polyethylene glycol, a polyethylene glycol derivative, and a saccharide.
  • Ratio of the number of cationic amino acid groups constituting the poly (cationic amino acid) (N) to the number of phosphate groups contained in the RNA complexed with the carrier (P) (N / P ratio) ) Is in the range of 0.5 to 40. 4.
  • composition according to [12] which is RNA having a functional RNA power 3 ⁇ 4 NAi effect.
  • a method for stabilizing RNA in blood comprising a step of forming a complex of RNA and a carrier having a hydrophilic group bonded in a comb shape to a polycationic compound as a side chain.
  • a method for stabilizing RNA in blood comprising the following steps.
  • Ratio of the number of cationic amino acid groups constituting the poly (cationic amino acid) (N) and the number of phosphate groups contained in the nucleic acid to be complexed with the carrier (P) ( (N / P ratio) is in the range of 0.5 ⁇ 40 to 40, according to [16].
  • a composition for suppressing excretion of RNA from the kidney comprising a complex of a carrier having a hydrophilic group as a side chain bonded in a comb shape to a polycationic compound and RNA. .
  • RNA kidney A composition for suppressing excretion [22] characterized by being used in combination with RNA, comprising a carrier having a hydrophilic group as a side chain bonded to a polycationic compound in a comb shape as an active ingredient, from RNA kidney A composition for suppressing excretion.
  • composition according to [22], wherein the carrier is administered simultaneously with RNA or before RNA is administered simultaneously with RNA or before RNA.
  • a method for suppressing excretion of RNA administered into blood from the kidney comprising a step of forming a complex of the carrier and RNA to be administered and a step of administering the obtained complex into the blood.
  • a method for suppressing excretion of RNA administered into blood from the kidney comprising the following steps.
  • a step of administering into the blood a carrier having a hydrophilic group bonded in a comb shape as a side chain to a polycationic compound.
  • a composition for inhibiting degradation of RNA by a nuclease comprising a complex of RNA and a carrier having a hydrophilic group as a side chain bonded to a polycationic compound in a comb shape. object.
  • RNA nuclease comprising a carrier having a hydrophilic group as a side chain bonded to a polycationic compound in a comb shape as an active ingredient, characterized by being used in combination with RNA A composition for inhibiting decomposition.
  • composition according to [31] The composition according to [30], wherein the carrier is administered simultaneously with RNA or before RNA.
  • a method for suppressing degradation of RNA administered into blood by nuclease comprising a step of administering.
  • a method for suppressing degradation of RNA administered into blood by nuclease comprising the following steps.
  • a method for analyzing the function of a gene comprising the following steps:
  • a method for analyzing the function of a gene comprising the following steps;
  • RNAi effect The stability of RNA in blood has been improved by the present invention. That is, the present invention has made it possible to directly administer functional RNA, such as siRNA, in which stability and retention in blood are important issues in blood. Inhibition of gene expression by RNAi effect is powerful. Therefore, using RNAi effect to suppress the expression of specific genes is important as a therapeutic strategy for diseases. Stabilization of RNA in the blood is an important issue when using the RNAi effect as a therapeutic strategy.
  • the present invention has realized a composition and method for dramatically improving the stability and retention of RNA in blood. According to the present invention, it can be said that the clinical application ability of RNA that brings about the RNAi effect has greatly advanced.
  • a specific use of the present invention is application as a therapeutic agent for RNAi effect.
  • many anticancer drugs induce cell growth inhibition or cell death by damaging cells. If the molecule that the drug acts on is expressed specifically in cancer cells, the possibility of realizing cancer cell-specific treatment increases.
  • Various helicases are generally genes that have a particularly high expression level in cancer cells and a low expression level in normal cells. Therefore, the effect of suppressing the expression of the helicase gene is unlikely to appear in normal cells. Therefore, if RNA that can suppress helicase expression by the RNAi effect is administered into blood using the present invention, there is a possibility that effective cancer treatment can be realized. According to the composition or RNA administration method based on the present invention, the stability and retention of RNA in blood can be improved. Therefore, the present invention can realize cancer treatment by RNAi effect using helicase as a target molecule.
  • RNA molecules such as siRNA can be administered into blood and stably maintained in blood. Therefore, gene function analysis can be easily performed using a living body.
  • gene functions can be identified through phenotypic changes in the organism. For example, the following phenotypic changes are biological responses, and it is difficult to capture the changes directly at the cellular level. On the other hand, if a living body is used, such a phenotypic change can be detected easily and clearly.
  • Tissue function (respiration, digestion, circulation, endocrine, reproduction, etc.)
  • Physiological reactions physiological reactions such as blood pressure, heart rate, sweating, excretion
  • Nervous system functions of the nervous system such as memory, behavior, and exercise
  • FIG. 6 A graph summarizing the results of examining the inhibitory effect of gene expression after administration of siRNA against the endogenous gene Ubcl3 in the blood of mice.
  • the vertical axis represents the mRNA level (%) relative to the control, and the horizontal axis represents the tissue type.
  • A siRNA alone
  • B siRNA administered 20 minutes after carrier (28K90P) administration
  • C complex of carrier and siRNA (siRNA / 28K90P) administered.
  • FIG. 8 is a diagram showing the results of examining the influence of the PEG graft ratio of a synthesized carrier (PLL-PEG).
  • A siRNA
  • B siRNA
  • the present invention provides a complex of RNA and a carrier having a hydrophilic group bonded in a comb shape to a polycationic compound as a side chain, and RNA for administration into blood. Concerning the composition.
  • the present invention also includes a carrier having, as an active ingredient, a carrier having a hydrophilic group bonded in a comb shape to a polycationic compound as a side chain, which is used in combination with RNA.
  • It relates to a composition for stabilizing A in the blood.
  • the present invention also includes a step of forming a complex of RNA and a carrier having a hydrophilic group bonded in a comb shape to the polycationic compound as a side chain and RNA in the blood. It relates to the method of stylization.
  • this invention relates to the method of stabilizing RNA in the blood including the following processes.
  • the present invention provides a composition for administering RNA into blood, comprising a step of mixing a carrier having a hydrophilic group as a side chain bonded in a comb shape to a polycationic compound with RNA. Regarding the method.
  • the present invention suppresses excretion of RNA from the kidney, comprising a complex of a carrier having a hydrophilic group bonded in a comb-like manner to a polycationic compound as a side chain and RNA.
  • a composition is provided.
  • the present invention also includes a step of forming a complex of RNA and a carrier having a hydrophilic group as a side chain, which binds to a polycationic compound in a comb shape, and the resulting complex in blood.
  • a method for suppressing excretion of RNA administered into blood from the kidney which comprises a step of administering to the blood.
  • the present invention comprises a carrier having, as an active ingredient, a hydrophilic group bonded in a comb shape to a polycationic compound as a side chain, which is used in combination with RNA.
  • a composition for suppressing excretion of kidney from the kidney is provided.
  • the present invention also provides a method for suppressing the excretion of renal strength of RNA administered into blood, comprising the following steps:
  • a step of administering into the blood a carrier having a hydrophilic group bonded in a comb shape as a side chain to a polycationic compound.
  • the present invention suppresses degradation of RNA by a nuclease, which includes a complex of RNA and a carrier having a hydrophilic group bonded in a comb shape to a polycationic compound as a side chain and RNA.
  • a composition is provided.
  • the present invention comprises a step of forming a complex of a carrier having a hydrophilic group bonded in a comb shape with a polycationic compound as a side chain and RNA, and the resulting complex in blood.
  • a method for suppressing degradation of RNA administered into blood by a nuclease comprising a step of administering.
  • the present invention comprises a carrier having a hydrophilic group as a side chain bonded to a polycationic compound in a comb-like manner as an active ingredient, which is used in combination with RNA.
  • a composition for inhibiting degradation of NA by nuclease is provided.
  • the present invention also provides a composition for inhibiting the degradation of RNA administered into blood by nuclease, comprising the following steps.
  • a step of administering into the blood a carrier having a hydrophilic group bonded in a comb shape as a side chain to a polycationic compound.
  • a carrier having a hydrophilic group as a side chain bonded to a cationic compound in a comb shape is used.
  • the cationic compound in the present invention includes, for example, a polymer containing a repeating structure of a structural unit containing a force thione functional group. Such a polymer is called a cationic polymer. That is, the carrier in the present invention can be a compound having a hydrophilic side chain bonded to the main chain in a comb shape with respect to the main chain of the cationic polymer.
  • the cationic functional group constituting the cationic polymer includes an amino group, an imino group, a guanidino group, a biguanide group, and the like.
  • the following polymers can be shown as the cationic polymer constituting the carrier of the present invention. Methods for producing these polymers are known.
  • a compound having a poly (cationic amino acid) structure as a main chain and having a side chain introduced into its amino group is a preferred carrier in the present invention.
  • the poly (cationic amino acid) that can be used as the main chain of the carrier include poly (lysine), poly (ornithine), and poly (ornithine-serine).
  • poly (cationic amino acids) can be synthesized by a generally known polymerization method.
  • poly (lysine) can be obtained by polymerization using ⁇ -carbobenzoxy-lysine_ ⁇ -carboxylic anhydride, primary amine as an initiator.
  • primary amine that is a polymerization initiator
  • polyethylene oxide having a single terminal amino group (molecular weight 200-250,000) can be used.
  • benzyl-serine_ ⁇ _carboxylic anhydride an amino acid polymer containing serine can be synthesized.
  • the molecular weight of the polyamino acid moiety in the polyethylene oxide polyamino acid block copolymer is not limited. The preferred molecular weight is 200-500,000.
  • the carrier in the present invention is a compound in which a hydrophilic group is introduced (bonded) as a side chain into a main chain composed of the polycationic compound.
  • Side chains are introduced by chemical modifications or chemical bonds. More specifically, side chains are introduced by, for example, graft polymerization.
  • examples of hydrophilic groups introduced into the comb shape by graft polymerization are shown below.
  • the comb-like bond of a hydrophilic group means that a hydrophilic group is bonded as a side chain of a repeating unit containing a cationic functional group constituting the main chain.
  • a hydrophilic group is bonded to the cationic amino acid constituting the repeating unit.
  • the side chain composed of the hydrophilic group may be bonded to all or part of the force thione amino acid constituting the main chain.
  • the carrier in the present invention has a hydrophilic group in a part of the cationic amino acid constituting the main chain. Includes combined structures.
  • the ratio of the hydrophilic group to the cationic amino acid constituting the main chain can be measured as an average value by analyzing the structure of the obtained carrier.
  • the side chain introduction rate and structure can be clarified through analysis such as NMR, GPC, or static light scattering.
  • an aldehyde derivative, an amino acid derivative, a carboxymethyl derivative and the like of methoxypolyethylene glycol can be shown as a polyethylene glycol derivative.
  • glycosaminodarlicans include hyaluronic acid, heparin, and chondroitin sulfate.
  • saccharides include dextran and amylose.
  • examples of the synthetic water-soluble polymer include polyacrylamide, hydroxypropyl cellulose (HPC), and hydroxypropylmethyl cellulose (HPMC).
  • the hydrophilic group which is a preferred side chain in the present invention includes dextran (Dex), polyethylene glycol (PEG) or a derivative thereof, hyaluronic acid (HA), hyaluronate (for example, sodium hyaluronate). Salt, potassium hyaluronate).
  • Dex dextran
  • PEG polyethylene glycol
  • HA hyaluronic acid
  • hyaluronate for example, sodium hyaluronate
  • Salt potassium hyaluronate
  • examples of the compounds that can be used as the carrier in the present invention include the following graft copolymers.
  • the method for synthesizing the graft copolymer having poly (cationic amino acid) as the main chain and having a hydrophilic group in the side chain is not limited. Graft copolymers containing these polymers as constituent units can be synthesized by known organic synthesis methods. That is, a side chain may be introduced into the functional group of the main chain polymer by an appropriate bond formation reaction. For example, when a polysaccharide chain is used as a side chain, it is possible to generate an amino bond based on a reaction between a sugar chain reducing end and a polyamino acid amino group. The reducing end of the sugar chain and the amino group of the polyamino acid undergo a Schiff base formation. Can be combined.
  • the reducing end of the polysaccharide can be coupled to an amino group such as poly (lysine).
  • an amino group such as poly (lysine).
  • a method can be used in which the reducing end of a polysaccharide is oxidized with iodine or the like and then carboxylated, then ratatoned and coupled to an amino group.
  • an unspecified portion of the polysaccharide can be bound to poly (lysine) or the like instead of the end of the polysaccharide.
  • a synthesis method therefor a method in which a polysaccharide is oxidized with periodic acid to form an aldehyde group and then reductively aminated with an amino group such as poly (lysine) can be shown.
  • a graft polymer having polyethylene glycol as a hydrophilic side chain can also be synthesized by forming a Schiff base using an aldehyde derivative of methoxypolyethylene glycol, for example.
  • polyethylene glycol can be used as a side chain as it is.
  • a derivative in which one hydroxyl group of polyethylene glycol is protected and the other hydroxyl group is substituted with an appropriate functional group can be used.
  • Any protecting group can be used for protecting the hydroxyl group.
  • the protecting group may include an alkoxy group such as a methoxy group, an alkylthioethylenesulfonyl group, and the like.
  • the functional group for introduction into the other hydroxyl group include an aldehyde group, an amino group, a succinyl group, a carboxyl group, a carboxymethyl group, a thiol group, and a dimethoxytrityl group.
  • the molecular weight of polyethylene glycol or a derivative thereof that can be introduced as a side chain is not particularly limited. Preferred polyethylene glycols or their derivatives have an average molecular weight of 300-100,000, more preferably ⁇ 1,000-20,000
  • the ratio of the hydrophilic group introduced into the carrier is not limited. It can be appropriately selected depending on the main chain constituting the carrier and the type of hydrophilic group to be introduced. Usually, the ratio of the hydrophilic group to the carrier is 10% to 99% by weight of the carrier, preferably 20% to 99% by weight, more preferably 50% to 99% by weight. When the amount is smaller than the range, the effect of solubilization mainly based on the hydrophilic group is reduced. On the other hand, when it is larger than the range to be applied, the electrostatic interaction with RNA also decreases as the cationicity decreases. In the range of the ratio of the hydrophilic group, the hydrophilic group The higher the polymerization rate, the higher the binding to RNA. Whether the polymerization rate of the hydrophilic group is high or not can be examined by measuring the anisotropy of the carrier-RNA complex.
  • RNA into the blood including a complex of a carrier having a hydrophilic group as a side chain and bound to the polycationic compound of the present invention in a comb shape.
  • This composition can be obtained by mixing the carrier with RNA.
  • the amount of carrier used for RNA is not limited. For example, about 0.01-100 ⁇ mol of carrier can be added to l z mol of nucleic acid.
  • the mixing ratio of the carrier and RNA can be adjusted to an appropriate range as long as the stability and retention of RNA in blood can be expected.
  • the ratio of carrier to RNA is the ratio of the number of cationic functional groups on the carrier to the number of phosphate groups on the nucleic acid ((carrier cationic functional group) (N) / [nucleic acid phosphate group] (P)).
  • N can be calculated from the molecular weight and weight of the polycation used to synthesize the carrier, and P depends on the length of the nucleic acid to be incorporated. Therefore, [cationic functional group of carrier] (N) / [phosphate group of nucleic acid] (P) is the structure of polycation used for carrier synthesis, its molecular weight and weight, and the length of RNA nucleic acid to be mixed. You can ask for it.
  • N / P ratio is calculated as follows.
  • a 21-base siRNA is composed of 21 nucleotide residues per strand. Since one phosphate group is contained per nucleotide molecule, it has 20 phosphate groups per strand.
  • 21-base siRNA Since 21-base siRNA is double-stranded, it has 40 phosphate groups per molecule.
  • the carrier and RNA have a low introduction ratio of a hydrophilic group to a cationic polyamino acid (20 mol% or less)
  • the power to condense near the above charge specific force Cationic homologue such as polylysine Polymer and DNA not only condense near charge ratio 1 but also precipitate.
  • the charge ratio is usually in the range of 0.5 to 40, more preferably 2 to 20. In such a range, a sufficient stabilizing effect of RNA in blood can be obtained. That is, the carrier in the present invention functions as a carrier for stabilizing RNA.
  • the N / P ratio is 2 or more, a sufficient amount of the carrier molecule is bound to RNA by electrostatic interaction, and degradation by an enzyme such as nuclease is inhibited.
  • a sufficient amount of the carrier molecule is present, so that the complex is stabilized even if dilution in blood occurs.
  • a composition containing the carrier as an active ingredient may be used in combination with RNA.
  • the combined use means that an RNA stabilizing effect is obtained by administering to the blood a composition containing RNA and the carrier as an active ingredient in order to stabilize RNA in the blood.
  • the administration of RNA into the blood can be performed simultaneously with the composition containing the carrier molecule or after the administration of the composition.
  • RNA administration is preferably performed after 1 minute to 24 hours, more preferably after 1 minute to 2 hours, and even more preferably after 1 minute to 20 minutes after administration of the composition. It can be performed.
  • composition containing the carrier as an active ingredient and the RNA are preferably administered in the blood so that the mixing ratio of the carrier and the RNA is in the blood. Specifically, it is preferable to administer such that the charge ratio is usually in the range of 0.5 to 40, more preferably 2 to 20.
  • compositions containing a sufficient amount of the carrier molecule in blood and RNA are administered separately, an antibody-RNA complex is formed in the blood, and the degradation of RNA by an enzyme such as a nuclease is inhibited.
  • any RNA can be used as the RNA constituting the carrier-RNA complex in the present invention.
  • the length of the RNA forming the complex can be any length of RNA that needs to be administered to a living body.
  • Preferred length of RNA is, for example, 500 bases or less, or 300 bases or less, usually 5 to 200 bases, more specifically 10 to 100 salts.
  • Basic RNA can be used.
  • RNA may be single-stranded or double-stranded. Single-stranded RNA can contain complementary base sequences in the same molecule. Single-stranded RNA containing a complementary base sequence forms a partial double strand by hybridizing complementary base sequences to each other. As a result, structures such as stem loops and stem bulges are formed.
  • RNA having these structures can also be used for complex formation in the present invention.
  • RNA includes a complementary sequence and has a double-stranded structure, either end may be overhanged.
  • the RNA forming the complex includes, in addition to ribonucleotide nucleic acids constituting natural RNA, those substituted with artificial bases and derivatives thereof. Therefore, RNA having inosine (i) can be used for complex formation instead of natural bases a, u, c, and g. Alternatively, a method for artificially synthesizing a nucleic acid derivative in which a phosphate bond is substituted with a thioate bond or a boranophosphate bond is also known. The sugar structure of ribonucleotide nucleic acid can also be modified.
  • RNA derivatives obtained by binding other substances to RNA
  • a compound such as PEG, cholesterol, sugar, a membrane-permeable peptide, or an antibody can be bound to RNA.
  • RNA has been shown to have a variety of functions.
  • the RNAi effect and the antisense effect are gene expression suppression effects possessed by RNA having a base sequence complementary to a gene.
  • ribozymes having various structures have been shown to suppress gene expression in cells.
  • These RNAs having a gene expression-inhibiting action can be administered into blood as a carrier-one RNA complex based on the present invention.
  • RNA having a specific base sequence specifically binds to a high molecular compound such as a protein.
  • RNA having binding activity for substances other than nucleic acids is called aptamer. Aptamers are bound by binding to proteins. Therefore, it may have an effect of regulating its activity.
  • RNA functioning as an abutama can also be used as the RNA-carrier complex of the present invention.
  • RNAs having functions other than the transmission of the genetic code are sometimes called functional RNAs (ftmctional RNAs) in the present invention.
  • functional RNA refers to RNA having a function other than the function of translating the genetic code into an amino acid sequence.
  • the translation function of the genetic code includes transcription of DNA base sequences and transfer of amino acids. Therefore, for example, RNA having the following functions is included in functional RNA.
  • the translation function of the genetic code is usually supported by mRNA and tRNA in the cell.
  • RNA containing the same base sequence as mRNA or tRNA is included in functional RNA if the RNA has a function other than translation.
  • These functional RNAs can be synthesized by ligating DNA encoding the base sequence of the target RNA downstream of an appropriate promoter and transcribing it with RNA polymerase. Transcription to RNA may be carried out in the cell, or it can be synthesized by in vitro transcription reaction provided an appropriate environment.
  • a transcription termination signal can be preferably placed on the 3 ′ side of the coding sequence of the DNA to be a cage.
  • RNAi having RNAi effect [0052] RNAi having RNAi effect:
  • RNAi refers to target RNA by introducing into the cell a double-stranded RNA consisting of a sense RNA consisting of a sequence homologous to the mRNA sequence of the target gene and an antisense RNA consisting of a complementary sequence.
  • RNAi effect is currently considered to include the following mechanism.
  • DICER RNase ⁇ nuclease family
  • siRNA small interfering RNA
  • the siRNA is also included in the double-stranded RNA having RNAi effect in the present invention.
  • the RNA used for RNAi does not need to be completely identical (homologous) to the partial region of the gene whose expression is to be suppressed, but preferably has perfect identity (homology).
  • a gene whose expression is to be suppressed is referred to as a target gene.
  • the double-stranded RNA having the RNAi effect is usually a sense RNA consisting of a sequence homologous to any continuous base sequence in the mRNA of the target gene, and a sequence complementary to the sense RNA It is a double-stranded RNA consisting of antisense RNA consisting of
  • the length of the “arbitrary continuous base sequence” is usually 20 to 30 bases, preferably 21 to 23 bases.
  • the length of the double-stranded RNA in the present invention is particularly Not limited.
  • RNA having an RNAi effect a long double-stranded RNA corresponding to the full length or almost the full length region of the target gene mRNA is decomposed in advance with, for example, DICER, and the degradation product is used as RNA having an RNAi effect. You can also.
  • Such degradation products are expected to contain double-stranded RNA molecules (siRNA) having the RNAi effect. According to this method, a region on mRNA expected to have an RNAi effect does not need to be selected.
  • Double-stranded RNA having an overhang of several bases at the end is known to have a high RNAi effect. Therefore, it is desirable that double-stranded RNA having an RNAi effect has an overhang of several bases at the end.
  • the length of the base that forms this overhang is not particularly limited.
  • the number of overhanging bases is preferably 2 bases.
  • TT two thymines
  • UU two uracils
  • Double-stranded RNA having an RNAi effect includes chimeric molecules in which the base that forms an overhang is DNA.
  • double-stranded RNA refers to RNA containing a structure in which complementary sequences are hybridized to each other.
  • a single-stranded RNA contains a complementary base sequence and has a double-stranded structure by hybridizing with each other, it is included in the double-stranded RNA. That is, single-stranded RNA having a stem-loop structure is included in double-stranded RNA because it contains a double-stranded structure (stem portion).
  • a person skilled in the art can appropriately design double-stranded RNA having an RNAi effect on a target gene based on the base sequence. That is, based on the base sequence of the target gene, any continuous RNA region of mRNA that is a transcription product of the sequence can be selected, and double-stranded RNA corresponding to this region can be prepared.
  • a method for selecting an siRNA sequence having a stronger RNAi effect from an mRNA sequence that is a transcription product of the sequence is also known. For example, a paper published by Reynolds et al. (Reynold et al. Nature biotechnology 22. 326-330 (2004)), a paper published by Ui-Tei et al. (Ui-Tei et al. Nucleic Acids Res. 32. 936- 948 (2004)) and the like, the base sequence necessary for siRNA can be predicted.
  • siRNA can also be designed based on a partial base sequence of a gene.
  • base sequence of siRNA it is sufficient that an arbitrary continuous base sequence to be selected is known.
  • the length of the necessary base sequence is, for example, at least 20 to 30 bases.
  • siRNA can be designed for a target gene whose full-length sequence is not clear. Therefore, two genes that suppress the expression of the gene, such as EST (Expressed Sequence Tag), from a gene fragment whose partial mRNA is already known based on the base sequence of the fragment. Strand RNA can be made.
  • RNA having an antisense effect RNA having an antisense effect:
  • RNA having an antisense effect on a gene can be used.
  • a method for inhibiting (suppressing) the expression of a specific gene a method using an anti-sense technique is known. Inhibition of target gene expression by antisense nucleic acids involves the following mechanisms.
  • antisense nucleic acids inhibit the expression of target genes by inhibiting various processes such as transcription, splicing, or translation (Hirashima and Inoue, Shinsei Kagaku Kogyo Lecture 2) Expression, Japanese Biochemical Society, Tokyo Chemical Dojin, 1993, p.31 9-347).
  • the RNA having an antisense effect used in the present invention includes RNA that can inhibit the expression of a target gene by any of these actions.
  • designing an antisense sequence complementary to the untranslated region near the 5 ′ end of the mRNA of the target gene is thought to be effective in inhibiting gene translation. It is also possible to use an IJ complementary to the coding region or the 3 'non-translation region.
  • RNA containing an antisense sequence of a non-translated region only in the target gene translation region is also included in the RNA having an antisense effect in the present invention.
  • the antisense RNA of the present invention can be synthesized by any method.
  • RNA having a necessary base sequence can be obtained by transcription reaction using RNA polymerase or chemical synthesis.
  • S-oligo phosphorothioate-type oligonucleotide in which 0 (oxygen) in the phosphate ester bond is replaced with S (sulfur) can be used.
  • S oligo By using S oligo Thus, resistance to nuclease degradation can be imparted. Therefore, in the present invention, S oligo is preferred as a functional RNA.
  • the sequence of the antisense RNA is preferably a sequence complementary to the target gene or a part thereof. However, as long as gene expression can be effectively suppressed, the base sequence constituting the antisense RNA may not be completely complementary to the base sequence of the target gene.
  • the transcribed RNA is preferably 90% or more, most preferably 95, relative to the transcript of the target gene. /. It has the above complementarity.
  • the length of the antisense RNA is at least 15 bases, preferably 100 bases or more, more preferably 500 bases or more. is there.
  • RNA having ribozyme activity [0065]
  • RNA having ribozyme activity can also be used.
  • Ribozyme refers to an RNA molecule having catalytic activity. Some ribozymes have various activities. For example, ribozymes can be designed that cleave RNA site-specifically. Some ribozymes have a size of 400 nucleotides or more, such as group I intron type and Ml RNA contained in RNase P. Some have a 40-nucleotide active domain called hammerhead type or hairpin type. (Makoto Koizumi and Eiko Otsuka, Protein Nucleic Acid Enzyme, 1990, 35, 2191).
  • the self-cleaving domain of the hammerhead ribozyme cleaves on the 3 'side of C15 in the sequence G13U14C15.
  • Base pairing between U14 and A9 is important for its cleavage activity. It has also been shown to be cleaved by A15 or U15 instead of C15 (Koizu mi, M. et al., FEBS Lett, 1988, 228, 228.).
  • a ribozyme whose substrate binding site is complementary to the RNA sequence near the target site, it is possible to artificially create a restriction RNA-cleaving ribozyme that recognizes the sequence UC, UU or UA in the target RNA.
  • Hairpin ribozymes are also useful for RNA cleavage. Hairpin ribozymes are found, for example, in the minus strand of satellite RNA of tobacco ring spot virus (Buzayan, JM., Nature, 1986, 323, 349). Target sequence based on hairpin ribozyme Specific RNA-cleaving ribozymes can be created (Kikuchi, Y. & Sasaki, N., Nucl Acids Res, 1991, 19, 6751., Hiroshi Kikuchi, Chemistry and Biology, 1992, 30, 112.). Thus, RNA having ribozyme activity capable of specifically cleaving the transcript of the target gene can be designed and used in the present invention.
  • the composition of the present invention is administered into blood.
  • the administration into blood includes, in addition to administration into blood vessels, mixing the composition of the present invention in the collected blood in advance and then administering the blood to a living body (ex vivo).
  • the expression of a target gene can be suppressed in vitro by adding the composition of the present invention containing siRNA that suppresses the expression of a specific gene to blood collected for blood transfusion.
  • the blood mixed with the composition of the present invention may be not only whole blood but also fractionated whole blood.
  • a carrier-RNA complex can be mixed in advance with blood components such as serum, plasma, platelets, and lymphocytes.
  • blood containing cells with gene expression such as lymphocytes or a fraction thereof is preferred as a blood component to be mixed with the carrier RNA complex.
  • composition of the present invention may be administered alone, or may be administered together with other compounds.
  • Applicants have shown that the action of certain drugs may be enhanced by suppression of gene expression of certain enzymes.
  • a carrier-RNA complex containing siRNA capable of suppressing gene expression of a specific enzyme can be administered together with a drug.
  • the siRNA suppresses the expression of the enzyme gene by administration of the carrier-RNA complex, the action of the administered drug can be enhanced.
  • RNA in blood means extending the residence time of RNA in blood. More specifically, degradation of RNA in the blood The state in which is suppressed is included in the state where RNA is stabilized. RNA is degraded in blood mainly by the action of nucleases. In other words, imparting resistance to nuclease to RNA is included in RNA stabilization.
  • RNA which is a water-soluble low molecular weight compound, is rapidly excreted in urine in the living body. The stabilization of RNA in the blood includes a state where the excretion of RNA into the urine by the kidney is suppressed.
  • the present invention suppresses the excretion of RNA from the kidney, which contains a complex of a carrier having a hydrophilic group bonded in a comb shape to a polycationic compound as a side chain and RNA. It relates to a composition for The present invention also includes a step of forming a complex of a carrier having a hydrophilic group bonded in a comb shape with a polycationic compound as a side chain and RNA, and the resulting complex in blood.
  • a method for suppressing excretion of RNA administered into blood from the kidney comprising a step of administering.
  • the present invention comprises a carrier having, as an active ingredient, a hydrophilic group bonded as a side chain to a polycationic compound, which is used in combination with RNA, as an active ingredient.
  • the present invention relates to a composition for suppressing the excretion of kidney from the kidney.
  • the present invention also provides a method for suppressing the excretion of renal strength of RNA administered into blood, comprising the following steps.
  • a step of administering into the blood a carrier having a hydrophilic group bonded in a comb shape as a side chain to a polycationic compound.
  • a preferable carrier in the present invention is a compound having a poly (cationic amino acid) as a main chain and a hydrophilic group bonded in a comb shape to the poly (cationic amino acid) as a side chain. is there.
  • the present invention suppresses degradation of RNA by a nuclease, which includes a complex of RNA and a carrier having a hydrophilic group bonded in a comb shape to a polycationic compound as a side chain. Relates to a composition.
  • the present invention includes a step of forming a complex of a carrier having a hydrophilic group bonded in a comb shape as a side chain with a polycationic compound and RNA, and the obtained complex in blood.
  • a method for inhibiting degradation of RNA administered into blood by nuclease comprising a step of administering.
  • the present invention provides a polycationic compound characterized by being used in combination with RNA.
  • the present invention relates to a composition for inhibiting degradation of RNase by nuclease, which contains, as an active ingredient, a carrier having a hydrophilic group bonded in a comb shape as a side chain.
  • the present invention also provides a composition for suppressing degradation of RNA administered into blood by nuclease, comprising the following steps.
  • a step of administering into the blood a carrier having a hydrophilic group bonded in a comb shape as a side chain to a polycationic compound.
  • a preferable carrier in the present invention is a compound having a poly (cationic amino acid) as a main chain and a hydrophilic group bonded in a comb shape to the poly (cationic amino acid) as a side chain. is there.
  • the carrier RNA complex of the present invention has improved retention of RNA in blood.
  • RNA administered into blood according to the present invention is effectively delivered to organs or tissues with high blood flow.
  • organs such as the liver and kidney are considered to have a high blood flow.
  • RNA can be delivered to these organs according to the present invention.
  • cancer tissue is a tissue with a lot of blood flow accompanied by neovascularization. Therefore, it is also useful for delivering RNA to cancer.
  • the carrier-RNA complex can be targeted to a specific organ or cell.
  • the introduction of genes for cells that make up the liver is an extremely important issue for the treatment of severe liver disease.
  • hepatic endothelial cells take up hyaluronic acid by receptor-mediated endocytosis.
  • Hyaluronic acid is a component of the extracellular matrix. By this action, hyaluronic acid is removed from the circulatory system extremely efficiently.
  • a carrier containing hyaluronic acid as a constituent is synthesized as the carrier, RNA can be targeted to the liver by utilizing endocytosis of liver endothelial cells.
  • Such a carrier can be obtained, for example, by graft polymerization of hyanolic acid to poly (cationic amino acid).
  • a complex of a carrier having such a structure and RNA is specifically taken up by hepatic sinusoidal endothelial cells.
  • the carrier-RNA complex to be administered into the blood, or the carrier and RNA for forming the complex in the blood are suitable depending on the method of use, purpose of use, and the like. It can be adjusted accordingly.
  • the daily dose is usually about 0.1 ⁇ g / kg to 200 mg / kg, more specifically about 1 ⁇ g / kg daily: 100 mg / kg. It is.
  • the present invention provides a gene functional analysis method comprising the following steps (1) to (4).
  • the present invention also provides a gene function analysis method including the following steps.
  • a preferable carrier in the present invention is a compound having a poly (cationic amino acid) as a main chain and a hydrophilic group bonded in a comb shape to the poly (cationic amino acid) as a side chain. is there.
  • any non-human animal to which the composition of the present invention is administered can be any animal other than humans.
  • common laboratory animals such as mice, rats, monkeys, monkeys, cats, tusks, goats, hidges, or rabbits can be used.
  • various disease model animals can be used to clarify the relationship between diseases and genes.
  • a disease model animal is an animal that has been artificially placed in a pathological state by a special breeding environment, administration of drugs, surgical treatment, or genetic modification.
  • the administration method of the composition of the present invention is not limited.
  • the complex is administered into the blood. Therefore, the complex is usually administered by injection into the blood vessel.
  • the phenotype after administration is observed. If a phenotypic difference is confirmed compared to the control, it can be seen that the difference is due to the effect of RNA applied as a complex.
  • RNA having a function of suppressing the expression of a target gene when administered as a complex, the phenotypic difference can be considered to be caused by the suppression of the expression of the gene. That is, it is possible to identify a phenotype resulting from functional suppression of the target gene.
  • the control can be, for example, the phenotype of an animal administered with only the carrier constituting the complex.
  • a complex of RNA and a carrier containing a base sequence that a non-human animal does not have can be administered as a control.
  • RNA having a greater effect than that RNA can be found.
  • the composition of the present invention can be administered alone, or can be administered together with other components other than the complex.
  • a drug and a complex can be administered together to elucidate the metabolic mechanism of the drug. If suppression of target gene expression enhances (or suppresses) the pharmacological action or side effect of a drug, it indicates that the target gene is associated with the pharmacological action or side effect of the drug.
  • RNA can be verified by administering a substance that induces various disease states to a non-human animal to be a disease model animal and administering the composition of the present invention.
  • a substance that induces various disease states For example, Based on the present invention, suppression of the expression of various target genes can be attempted in a model animal to which a carcinogen is administered. If carcinogenesis can be prevented by suppressing the expression of a gene, the gene is identified as a gene involved in carcinogenesis.
  • composition of the present invention can be administered to a non-human animal transplanted with a cancer tissue collected from a patient. If the transplanted cancer tissue is regressed by administration of the complex, it can be confirmed that the target gene is useful as a therapeutic target. For example, when there are multiple target genes that may be useful for cancer treatment, it is also useful as a method for evaluating which target genes are effective for a patient's cancer.
  • the present invention provides a pharmaceutical composition comprising the carrier RNA complex or a carrier for forming a complex in blood and RNA, and a method for producing the same.
  • RNAs that have been confirmed to be effective as therapeutic agents are known.
  • cell growth can be inhibited by introducing siRNA capable of suppressing specific gene expression into malignant cells such as cancer cells.
  • a pharmaceutical composition can be produced using RNA having such a therapeutic effect.
  • the present invention provides a complex of RNA with a carrier having a hydrophilic group bonded in a comb shape to a polycationic compound as a side chain and RNA, or a complex in blood.
  • a pharmaceutical composition for administration into blood comprising the carrier and RNA, and a pharmaceutically provided carrier.
  • the present invention relates to a complex of RNA and a carrier having a hydrophilic group bonded in a comb shape to a polycationic compound as a side chain and RNA, or the carrier and RNA for forming a complex in blood.
  • the present invention relates to a method for producing a pharmaceutical composition for administration into blood, comprising the step of mixing with a pharmaceutically provided carrier.
  • a preferable carrier in the present invention is a compound having poly (cationic amino acid) as a main chain and a hydrophilic group bonded in a comb shape to the poly (cationic amino acid) as a side chain.
  • pharmaceutically acceptable carriers include, but are not limited to, surfactants, colorants, flavoring agents, preservatives, stabilizers, buffering agents, suspending agents, tonicity agents, fluidity promoters, and the like. However, other commonly used carriers can be used as appropriate.
  • the above-mentioned carrier can be added as necessary according to a conventional method.
  • lactose, mannitol, carmellose sodium examples thereof include droxypropenoresenorerose, hydroxypropinoremethinoresenellose, gelatin, polyoxyethylene hydrogenated castor oil 60, sucrose, carboxymethylcellulose, and inorganic salts.
  • the complex blended with these carriers can be made into a pharmaceutical composition for administration into the blood as an injection in a dissolved state. Alternatively, after drying, it can be dissolved in sterile physiological saline at the time of administration to give an injection.
  • the dosage of the pharmaceutical composition of the present invention can be appropriately determined finally based on the judgment of a doctor in consideration of the type of dosage form, administration method, patient age and weight, patient symptom, and the like. It should be noted that all prior art documents cited in this specification are incorporated herein by reference. Hereinafter, the present invention will be described more specifically based on examples.
  • siRNA delivery system based on the present invention
  • poly-L-lysine-g-dextran having the structure shown in the following formula 1 and poly-L- having the structure shown in the formula 2 are used.
  • Lysine-g-polyethylene glycol (PLL-PEG) was synthesized.
  • Each of these compounds is a compound having a polycationic amino acid as a main chain and a hydrophilic group bonded to the main chain in a comb shape as a side chain.
  • PLL-Dex is a compound having a main chain of poly-L-lysine (PLL) and a side chain of dextran.
  • PLL-PEG is a compound whose main chain is PLL and whose side chain is polyethylene glycol.
  • 7K90D was synthesized as a compound represented by Formula 1.
  • 7K90D is a compound that has PLL (molecular weight 7,000) as the main chain and 7.3 dextran (molecular weight 6000) on the side chain as an average.
  • the weight of dextran (Mn 6000) against 7K90D is 90wt%.
  • the grafting rate indicates what percentage of amino acid residues dextran is bound to all amino acid residues of PLL.
  • PLL with a molecular weight of 7,000 has an average of 55 amino acid residues, and dextran is bound to 13.4% of amino acid residues. This means that an average of 7.3 dextrans are bound to the PLL—molecule.
  • Graffiti The annealing rate can be calculated by 1 H NMR measurement.
  • 28K90D, 7K70D, and 28K70D which are compounds with a molecular weight of 3 ⁇ 4 8,000 and compounds with different grafting rates. The graphing rate for each compound and the numerical value of xyz in the general formula are shown.
  • 7K90D, 7K70D, and 28K70D were synthesized by graft polymerizing 6,000 dextran to a PLL with molecular weights of 7000 (7K) and 28,000 (28k).
  • the PEG solution was added to the PLL solution and stirred at 25 ° C for 1 hour. Thereafter, 1.32 ml of a solution in which 314.2 mg of sodium cyanoborohydride (NaBH3CN, manufactured by Nacalai Testa) was dissolved in 5 ml of water was added and stirred at 25 ° C. for 1 day.
  • the solution was dialyzed using a dialysis membrane (Molecular exclusion limit 50,000, manufactured by Spetatramu Laboratories), and further purified by ultrafiltration (Molecular exclusion limit 50,000, manufactured by Toyo Roshi Kaisha, Ltd.). The resulting solution was lyophilized to give the product. The yield of product was 1.1068 g (yield 79.1%).
  • the PEG introduction rate and structure were confirmed by molecular weight analysis by 1 H NMR, gel permeation chromatography (GPC), and static light scattering.
  • 7K90P, 7 ⁇ 70 ⁇ , and 28 ⁇ 70 ⁇ were synthesized by grafting PEG with molecular weight of 5,000 to PLL with molecular weight of 7000 (7K) and 28,000 (28k).
  • Table 1 shows the physical properties of each compound synthesized as a carrier.
  • siRNA 21 mer The following sequences were used as siRNA (21 mer).
  • the usage amount of 28K90P is calculated as follows. There are 40 phosphate groups in one molecule of siRNA, so there are 40 nmol phosphate groups in 1 nmol of siRNA. Since the N / P ratio is 2, 80 nmol of amino group is required. Therefore, the required weight of the 28K90P PLL part is 20.48 x g at 80 nmol X 128 (molecular weight of lysine unit). Furthermore, the required weight of 28K90P is 20.48 ⁇ g ⁇ 0.1, which means that 10% by weight of PLL is 204.8 ⁇ g.
  • N in the N / P ratio is the number of moles of the amino group of the PLL
  • P is the number of moles of the phosphate group of the siRNA.
  • RNase A Nahon Bon Gene
  • the reaction solution was calcined with disodium ethylenediamine tetraacetate and sodium dodecyl sulfate to a final concentration of 10 mM and 1%, respectively, and siRNA was extracted by phenol / chloroform form treatment. did.
  • the extract was subjected to polyacrylamide electrophoresis, and the genole was stained with SYBR Gold (Invitrogen).
  • the gel was detected with an image analyzer (FMBIOII, HITACHI). The results of electrophoresis are shown in FIG.
  • siRNA and 27mer dsRNA were completely degraded in the absence of 28K90P. However, when it was complexed with 28K90P, it was highly resistant to RNaseA.
  • siRNA and 27mer dsRNA were degraded in plasma in the absence of 28K90P. However, when it formed a complex with 28K90P, it was highly stable with almost no degradation in plasma.
  • N in the N / P ratio is the number of moles of the amino group of the PLL
  • P is the number of moles of the phosphate group of the siRNA.
  • the prepared mixture was administered from the tail vein of a mouse (ICR, 5 weeks old, male), and the fundus oculi was collected over time.
  • the blood was treated with disodium ethylenediamine tetraacetate and sodium dodecyl sulfate to a final concentration of 10 mM and 1%, respectively, and siRNA was extracted by phenol / chloroform form treatment.
  • the extract was subjected to polyacrylamide electrophoresis, and fluorescence-labeled siRNA was detected with an image analyzer (FMBIOII, HITACHI).
  • FMBIOII, HITACHI fluorescence-labeled siRNA was detected with an image analyzer
  • siRNA alone was administered intravenously, it disappeared from the blood approximately 5 minutes after administration (naked in Fig. 3).
  • siRNA mixed with PLL-Dex or PLL-PEG remained in the blood for 30 minutes after administration and up to 90 minutes at the longest.
  • the amount of siRNA in the blood in each direction with an N / P ratio of 4 to 8 was high.
  • NS siRNA non-silencing siRNA that did not affect the expression of Ubcl3.
  • ABI PRISM 7000 Sequence Detection System (Alied Biosystems) was used for quantitative PCR. Primers for RT-PCR and TaqMan probe of Ubcl3 gene and / 3-actin gene were purchased from Applied Biosystems. RT-PCR reaction was performed using QuantiTect Probe RT-PCR Kit (Qiagen) according to the manual. The expression of Ubcl3 mRNA was quantitatively compared using the expression level of ⁇ _actin as a standard. The measurement results are summarized in FIG.
  • Synthesized PLL-PEG (28K90P) was administered from the tail vein of mice (ICR, 5 weeks old, male). After 20 minutes, a mixture of fluorescently labeled siRNA (SEQ ID NOs: 1, 2, 7, and 8) 0.64 nmol and unlabeled siRNA (SEQ ID NOs: 1, 2, 7, and 8) 2.56 nmol with an N / P ratio of 4 As such, it was administered to the tail vein.
  • the ocular fundus was collected over time, and disodium ethylenediamine tetraacetate and sodium dodecyl sulfate were added to the blood to a final concentration of 10 mM and 1%, respectively, and siRNA was extracted by phenol / chloroform form treatment. The extract was subjected to polyacrylamide gel electrophoresis, and fluorescence-labeled siRNA was detected with an image analyzer (FMBIO II, HITACHI). The result of electrophoresis is shown in FIG.
  • siRNA alone When siRNA alone was administered intravenously, it disappeared from the blood 5 minutes after administration (lane (A) in FIG. 7). In contrast, when siRNA alone was administered 20 minutes after intravenous administration of PLL-PEG (28K90P), it remained in the blood for 1.5 hours after administration and for a maximum of 2 hours (lane in Fig. 7). O)).
  • SiRNA 21mer (SEQ ID NO: 1, 2, 7, and 8), 27mer (SEQ ID NO: 3 and 4)) in phosphate buffer 5 pmol was added respectively.
  • the synthesized PLL-PEG 28K90P, 28K70P was added so that the N / P ratio was 0 to 100.
  • the prepared mixture was incubated at 37 ° C for 30 minutes, and then the fluorescence anisotropy was measured (MF-20, Olympus). The measurement results are shown in FIG.
  • RNA can be administered into blood and stably maintained.
  • RNA can be used in gene expression suppression technology using the RNAi effect. Therefore, the present invention can be used for treatment or prevention of diseases or gene function analysis by suppressing gene expression.
  • the therapeutic RNA can be administered into blood based on the present invention, and the stability in blood can be improved.
  • siRNA capable of suppressing the expression of a gene whose function is to be analyzed can be administered into a living body, and phenotypic changes associated with expression suppression can be known. Since effective gene expression suppression can be realized in the living body, it is possible to know physiological changes in the living body and the effects on actual disease model animals. Such knowledge cannot be obtained by suppressing gene expression in cultured cells.
  • RNA can be administered separately from the carrier. Therefore, it is possible to simplify the formulation process, such as formulating the carrier and RNA separately.

Abstract

A composition for use for introducing RNA into the blood, which comprises a complex of a carrier and RNA, where the carrier comprises a polycationic compound having, as a side chain, a hydrophilic group bound to the polycationic compound in a comb-like configuration. The polycationic compound is preferably a poly(cationic amino acid). By using the composition, the excretion of RNA from the kidney can be inhibited, and the RNA retention can be improved. For example, the treatment of cancer can be effected by introducing siRNA having a therapeutic effect on cancer into the blood by utilizing the composition. Further, it is found that the retention of RNA in the blood can also be improved by administering the carrier and RNA separately. The carrier may be, for example, PLL-Dex represented by the formula 1 or PLL-PEG represented by the formula 2.

Description

明 細 書  Specification
RNA含有組成物  RNA-containing composition
技術分野  Technical field
[0001] 本発明は、血液中に投与することができる RNA含有組成物に関する。  [0001] The present invention relates to an RNA-containing composition that can be administered into blood.
背景技術  Background art
[0002] 医薬品開発において、有効成分を目的とする組織あるいは細胞に効果的に到達さ せる技術は重要な研究課題の一つである。たとえば、いかに優れた薬理作用を有す る化合物も、 目的とする組織に到達しなければ、薬理効果は期待できない。局所的 な投与方法を除けば、通常、医薬品の有効成分 (薬物）は、血液中や経口的に投与 された後に、作用部位に到達することによってその薬理効果を発揮する。投与された 薬物が作用部位に効率的に到達できない場合には、大量の薬物を投与しなければ ならない。その結果、薬物投与による副作用の危険が高まる。薬物を目的とする部位 に送達 (Delivery)するために、様々なメカニズムが考え出された。このようなメカニズム は、薬物送達システム（Drug Delivery System ;以下 DDSと省略する）と呼ばれている  [0002] In drug development, a technology for effectively reaching an intended tissue or cell as an active ingredient is one of the important research subjects. For example, a compound having an excellent pharmacological action cannot be expected to have a pharmacological effect unless it reaches the target tissue. Except for local administration methods, active pharmaceutical ingredients (drugs) usually exert their pharmacological effects by reaching the site of action after being administered into the blood or orally. If the administered drug cannot efficiently reach the site of action, a large amount of drug must be administered. As a result, the risk of side effects due to drug administration increases. Various mechanisms have been devised to deliver drugs to the intended site (Delivery). Such a mechanism is called a drug delivery system (DDS).
[0003] 生体に投与された薬物は、生体から次のような影響を受ける。これらの影響を逃れ て最終的に作用部位に到達した薬物によって、期待された治療効果がもたらされる。 結果として治療効果につながらなかった薬物は、代謝あるいは***される。 [0003] A drug administered to a living body is affected by the living body as follows. Drugs that escape these effects and eventually reach the site of action provide the expected therapeutic effect. Drugs that do not lead to therapeutic effects as a result are metabolized or excreted.
一吸収一  One absorption one
薬物はまず生体に吸収されなければならない。局所投与を除けば、薬物は、通常、 患部から離れた部位に投与され、生体に吸収された後に、生体内に拡散することに より、患部に到達する。生体による薬物の吸収は、薬物が作用部位に到達するため の最初の関門となる。たとえば、経口投与、経皮的投与、経腸的投与、あるいは経気 道投与などの投与方法にぉレ、ては、薬物の吸収量がその生体内濃度を左右する。  The drug must first be absorbed by the body. Except for local administration, the drug is usually administered to a site away from the affected area, absorbed by the living body, and then diffused into the living body to reach the affected area. Absorption of the drug by the living body is the first barrier for the drug to reach the site of action. For example, the amount of drug absorbed affects the in vivo concentration, depending on the administration method such as oral administration, transdermal administration, enteral administration, or respiratory tract administration.
[0004] 薬物を静脈内に投与する場合には、通常、生体における薬物濃度は投与量に依 存し、吸収の影響は受けない。ただし、静脈内に投与する場合であっても、作用部位 が血流から隔離されているときには、血液中から作用部位への薬物の移行の程度が 、薬物の最終的な作用濃度を左右することになる。たとえばがん組織は、血管新生が 亢進していて、血流の豊富な組織である。しかし組織を構成する個々の細胞に薬物 が到達するためには、薬物が細胞膜を透過する必要がある。抗体医薬などのように、 細胞表面への結合によって治療効果が得られる場合を除けば、多くの杭がん剤の作 用部位は細胞内部である。つまり、がん細胞の細胞膜を透過した薬物によって治療 効果が達成される。薬物が生体に吸収されて血液中に移行した後、更に作用部位に 到達する過程も、薬物の吸収に位置づけることができる。 [0004] When a drug is administered intravenously, the drug concentration in a living body usually depends on the dose and is not affected by absorption. However, even when administered intravenously, when the site of action is isolated from the bloodstream, the degree of drug transfer from the blood to the site of action is limited. The final working concentration of the drug will be affected. For example, cancer tissue is a tissue with increased angiogenesis and abundant blood flow. However, in order for the drug to reach the individual cells that make up the tissue, the drug needs to penetrate the cell membrane. Except in cases where therapeutic effects are obtained by binding to the cell surface, such as antibody drugs, the site where many pile cancer drugs are applied is inside the cell. In other words, a therapeutic effect is achieved by a drug that has permeated the cell membrane of cancer cells. The process of reaching the site of action after the drug is absorbed by the living body and transferred to the blood can also be positioned as drug absorption.
[0005] 一生体による薬物の代謝  [0005] Metabolism of drugs by a living body
多くの場合、薬物は、生体にとっては異物である。そのため生体に投与（あるいは 吸収）された薬物は、通常、生体から除去される。具体的には、生体が有する分解機 構や***機構は、薬物に対してそれを除去する方向に作用する。その結果、一般に 、生体に投与された薬物の生体内濃度は、時間とともに低下する。もしも薬物が作用 部位に対する蓄積性が無い場合には、生体内濃度は薬物の作用濃度と一致する。 つまり、時間とともに薬物の効果が低下することを意味する。あるいは血液から作用 部位への移行性が良好な薬物であっても、速やかに***され血中濃度を維持できな い薬物では、高い治療効果を期待することは難しい。すなわち薬物による治療効果 は、投与された薬物が生体から除去されるまでの間にもたらされているといってよい。  In many cases, the drug is a foreign substance for the living body. Therefore, drugs that are administered (or absorbed) to the living body are usually removed from the living body. Specifically, the degradation mechanism and excretion mechanism of the living body acts on the drug in the direction of removing it. As a result, generally, the in vivo concentration of a drug administered to a living body decreases with time. If the drug is not accumulative at the site of action, the concentration in the body will match that of the drug. In other words, it means that the effect of the drug decreases with time. Alternatively, it is difficult to expect a high therapeutic effect for a drug that is excreted quickly and cannot maintain its blood concentration, even if the drug has good transferability from blood to the site of action. That is, it can be said that the therapeutic effect by the drug is brought about until the administered drug is removed from the living body.
[0006] DDSによる薬物の送達は、薬物に対する生体の影響を制御し、薬物を効率的に作 用部位に送達することを目的としている。具体的には、たとえば次のようなアプローチ によって薬物を効率的に作用部位に到達させる努力が続けられてきた。  [0006] The delivery of a drug by DDS aims to control the influence of a living body on the drug and efficiently deliver the drug to the site of operation. Specifically, efforts have been made to efficiently bring drugs to the site of action by the following approach, for example.
一薬物の作用部位への標的化：特定の細胞に親和性の高い物質を利用して、薬 物を特定の細胞に選択的に移送する技術が公知である。たとえば抗体に細胞障害 性物質を結合した抗がん剤が公知である。このようなアプローチによる治療方法を、 標的治療 (targeting therapy)と呼ぶ。標的化により、薬物は特定の細胞に結合させら れる。その結果、作用部位 (あるいはその近く）における薬物濃度を高く維持すること ができる。また標的化によって、薬物の肝臓あるいは腎臓などの代謝をつかさどる器 官への移行が妨げられる。その結果、薬物を生体内に長く保持することができる。  Targeting a drug to a site of action: A technique for selectively transferring a drug to a specific cell using a substance having a high affinity for the specific cell is known. For example, anticancer agents in which a cytotoxic substance is bound to an antibody are known. This type of treatment is called targeting therapy. Targeting causes the drug to bind to specific cells. As a result, the drug concentration at (or near) the site of action can be kept high. Targeting also prevents the transfer of drugs to the organs responsible for the metabolism of the liver or kidneys. As a result, the drug can be held in the living body for a long time.
[0007] —薬物の保護:薬物の生体による代謝を防ぐことができれば、投与した薬物をより 長時間、生体内に存在させることができる。たとえば、経口投与においては、しばしば 強酸性に耐えるカプセルに薬物が充填される。投与されたカプセルは胃を経て腸内 で溶解し薬物を放出する。胃内の強酸性条件下による薬物の分解を防ぎ、吸収量を 高めることがカプセルの役割である。また、たんぱく質製剤に高分子化合物を結合す ることによって、血中濃度を高い水準に維持できることも公知である。この方法は生体 によるたんぱく質の代謝が、高分子化合物の結合によって阻害される現象を利用し ている。 [0007] —Drug protection: If the metabolism of the drug can be prevented, It can exist in the living body for a long time. For example, for oral administration, drugs are often filled into capsules that withstand strong acidity. The administered capsule dissolves in the intestine via the stomach and releases the drug. The role of the capsule is to prevent degradation of the drug under strong acidic conditions in the stomach and increase the amount of absorption. It is also known that blood concentration can be maintained at a high level by binding a polymer compound to a protein preparation. This method utilizes the phenomenon that protein metabolism by the living body is inhibited by the binding of polymer compounds.
[0008] さて、遺伝子の発現を効果的に制御することができる技術として、 RNA干渉 (RNA in terferance;RNAi)と呼ばれる現象が明らかにされている。 RNAiは、 2本鎖 RNAが相同 な塩基配列を有する遺伝子の発現を特異的に抑制する現象である。当初、線虫に おいて確認された現象で（非特許文献 1/Fire et al., Nature, 391, 806-811, 1998) 、その後哺乳動物細胞においても 21塩基の 2本鎖 RNAによる RNAiが確認された (非 特許文献 2/Elbashir et al., Nature, 411, 494-498, 2001)。 RNAiのメカニズムは、現 在のところ、完全には明らかにされていない。さまざまな解析によって次のようなモデ ルが推定されている。  [0008] As a technique capable of effectively controlling gene expression, a phenomenon called RNA interference (RNAi) has been clarified. RNAi is a phenomenon that specifically suppresses the expression of genes having double-stranded RNA homologous base sequences. This phenomenon was first confirmed in C. elegans (Non-patent Document 1 / Fire et al., Nature, 391, 806-811, 1998), and RNAi due to double-stranded RNA of 21 bases was subsequently detected in mammalian cells. (Non-Patent Document 2 / Elbashir et al., Nature, 411, 494-498, 2001). The mechanism of RNAi is currently not fully clarified. The following models have been estimated by various analyses.
[0009] すなわち、まず細胞内に導入された 2本鎖 RNA力 SRNaselll型の核酸分解酵素によ つて、 21— 23塩基程度の長さを有する短レ、 RNAに断片化される。このとき作用する R Naselll型の核酸分解酵素は、ダイサー (dicer)と呼ばれている。断片化された RNAは ヘリカーゼなどの複数のたんぱく質と複合体を形成する。このとき形成される RNAとた んぱく質との複合体が RISC(RNA -induced silencing cmplex)である。ヘリカーゼは、 2 本鎖の核酸を ATP依存的に 1本鎖に解きほぐす作用を有する酵素である。 RISCは、 それを構成している 2本鎖 RNAがへリカーゼの作用によって 1本鎖化されると、活性 型となる。続レ、て活性型 RISCが有する 1本鎖 RNAに相補的な塩基配列を含む mRNA が分解される。 RISCを構成する断片化された RNAは、特に siRNA(small interfering R NA)と呼ばれた。し力 現在では、細胞への導入によって遺伝子発現抑制をもたらす 人為的に合成された 2本鎖 RNAも含めて広く siRNAと呼んでいる。  That is, first, a double-strand RNA force SRNaselll-type nucleolytic enzyme introduced into a cell is fragmented into short RNA having a length of about 21-23 bases. The R Naselll-type nucleolytic enzyme that acts at this time is called a dicer. Fragmented RNA forms a complex with multiple proteins such as helicases. The complex of RNA and protein formed at this time is RISC (RNA-induced silencing cmplex). A helicase is an enzyme that has the effect of breaking a double-stranded nucleic acid into a single strand in an ATP-dependent manner. RISC becomes active when its double-stranded RNA is made into single strands by the action of helicase. Subsequently, mRNA containing a base sequence complementary to the single-stranded RNA of active RISC is degraded. The fragmented RNA constituting RISC was specifically called siRNA (small interfering RNA). Currently, it is widely called siRNA, including artificially synthesized double-stranded RNA that causes gene expression suppression by introduction into cells.
[0010] siRNAによる遺伝子発現抑制作用は非常に強力である。そのため、アンチセンスや リボザィムに代わる、新たな遺伝子発現抑制技術として注目されている。 siRNAが有 する遺伝子発現の抑制作用は、さまざまな遺伝子に応用された。たとえば医療にお いては、疾患の原因となる遺伝子の発現を、 siRNAの作用によって抑制する試みが 報告されている。以下に医療分野において効果が確認された siRNAの標的を示す。 感染性病原体の遺伝子： HIV、 HBV、 HCVなど [0010] The gene expression suppression effect by siRNA is very strong. Therefore, it is attracting attention as a new gene expression suppression technology that can replace antisense and ribozyme. Has siRNA The suppressive action of gene expression is applied to various genes. For example, in medicine, attempts have been reported to suppress the expression of genes that cause disease by the action of siRNA. The siRNA targets that have been confirmed to be effective in the medical field are shown below. Infectious agent genes: HIV, HBV, HCV, etc.
がん遺伝子： Her2/neu， EGFR, VEGF, HPVなど  Oncogene: Her2 / neu, EGFR, VEGF, HPV, etc.
本発明者らも、各種のへリカーゼの発現を siRNAによって抑制することによって、が ん細胞にアポトーシスを誘導しうることを明らかにして特許出願している (特許文献 1 /WO2004/100990)。  The present inventors have also filed a patent application by clarifying that apoptosis can be induced in cancer cells by suppressing the expression of various helicases with siRNA (Patent Document 1 / WO2004 / 100990).
[0011] これらの siRNAの医療分野における利用には、なお解決すべき課題が残されている 。課題のひとつは、 siRNAの安定性である。もともと RNAはきわめて分解されやすい生 体分子である。たんぱく質の発現レベルが遺伝子の転写調節によってコントロールで きるのは、 RNAが分解されやすいために他ならなレ、。生体内においては、たんぱく質 合成を終えた mRNAは速やかに分解される。加えて、血液中に存在する短鎖の RNA は、そのままでは腎臓によって速やかに尿中に***される。ところが RNAを医薬とし て利用するときには、 RNAが安定に維持されなければ持続的な薬効が望めない恐れ がある。これらの課題に対して、たとえば各種のベクターを用いて、細胞内で siRNAを 発現させる試みがある。  [0011] Use of these siRNAs in the medical field still has problems to be solved. One challenge is the stability of siRNA. Originally, RNA is a biological molecule that is extremely susceptible to degradation. The level of protein expression can be controlled by the transcriptional regulation of genes, because RNA is easily degraded. In vivo, mRNA that has finished protein synthesis is rapidly degraded. In addition, short RNAs present in the blood are rapidly excreted in the urine by the kidneys as they are. However, when RNA is used as a medicine, there is a risk that sustained efficacy cannot be expected unless RNA is stably maintained. To address these issues, for example, there are attempts to express siRNA in cells using various vectors.
生体内において siRNAの治療効果を期待するためには、 目的とする組織に効率的 に siRNAをデリバリーする技術が必要である。特に、たとえばがんのように、特定の組 織に対して siRNAを運搬したい場合には、薬剤が組織移行性を有することが好ましい 。ところが現在知られている生体への遺伝子導入用ベクターは、細胞に対する選択 性が低い。そのため、全身性に投与した場合には、特定の組織を標的とすることが難 しい。  In order to expect the therapeutic effect of siRNA in vivo, technology to efficiently deliver siRNA to the target tissue is required. In particular, when it is desired to transport siRNA to a specific tissue such as cancer, it is preferable that the drug has tissue transferability. However, currently known vectors for gene transfer into living organisms have low selectivity for cells. Therefore, when administered systemically, it is difficult to target a specific tissue.
[0012] 更に、コレステロールをキャリア一として用レ、、化学修飾した 2本鎖 RNAを組み合わ せて遺伝子の発現抑制を実現した報告もある (非特許文献 3ZSoutschek et al.， Natu re, 432, 173-178， 2004)。この報告においては肝のアポリポプロテイン Bの発現が抑 制された。その他の組織に移行性を有するキャリアーが提供されれば有用である。  [0012] Furthermore, there is a report that uses cholesterol as a carrier and suppresses gene expression by combining chemically modified double-stranded RNA (Non-patent Document 3 ZSoutschek et al., Nature, 432, 173). -178, 2004). In this report, hepatic apolipoprotein B expression was suppressed. It would be useful if a carrier having transferability was provided to other tissues.
[0013] 一方、たとえば、次のようなブロック共重合体と核酸との配合によって、薬剤や核酸 などの運搬に有用なナノミセルを得られることが明らかにされている。しかしこれらの ナノミセルを構成する重合体の構造と、血液中への投与後の挙動との関連性は明ら かでない。 [0013] On the other hand, for example, by combining a block copolymer and a nucleic acid as described below, a drug or a nucleic acid It has been clarified that nano micelles useful for transportation of the above can be obtained. However, the relationship between the structure of the polymer constituting these nanomicelles and the behavior after administration into blood is not clear.
特開平 7-69900 (特許文献 2)：ポリエチレングリコールとポリアスパラギン酸などのブ ロック共重合体 +アドリアマイシン  JP-A-7-69900 (Patent Document 2): Block copolymer of polyethylene glycol and polyaspartic acid + adriamycin
特開 2001-146556 (特許文献 3)：チオール化された、ポリエチレングリコールとポリリ ジンなどのブロック共重合体 +高分子電解質  JP 2001-146556 (Patent Document 3): Thiolated block copolymer such as polyethylene glycol and polylysine + polymer electrolyte
WO2002/26241 (特許文献 4)：ポリエチレングリコールとポリグルタミン酸などのブロ ック共重合体 +シスブラチン  WO2002 / 26241 (Patent Document 4): Block copolymer such as polyethylene glycol and polyglutamic acid + cisbratin
特開 2003-113214 (特許文献 5)：ポリエチレングリコールとポリメタクリル酸などのブ ロック共重合体 +核酸  JP 2003-113214 (Patent Document 5): Block copolymer of polyethylene glycol and polymethacrylic acid + nucleic acid
特開 2004-352972 (特許文献 6)：ポリエチレングリコールとポリカチオンのブロック共 重合体 +核酸または陰イオン性たんぱく質  JP 2004-352972 (Patent Document 6): Block copolymer of polyethylene glycol and polycation + nucleic acid or anionic protein
[0014] 更に本発明者らは、カチオン性のアミノ酸ポリマーに、親水性基を櫛型に結合した 担体が、核酸のハイブリダィゼーシヨンを促進し、 2本鎖あるいは 3本鎖形成の安定化 作用を有することを明らかにしている（非特許文献 4/Bioconjug Chem. (1998) 9， 29 2-299.;非特許文献 5/Bioconjug Chem. (2000) 11， 520-526.；非特許文献 6/Nat Mater. (2003) 2, 815-820·)。この現象に基づいて、カチオン性のアミノ酸ポリマーに 、親水性基を櫛型に結合した担体の、核酸キャリアーとしての有用性が見出されてい る（特許文献 7/特開平 10-158196)。 [0014] Further, the present inventors have shown that a carrier in which a hydrophilic group is bound to a cationic amino acid polymer in a comb shape promotes hybridization of nucleic acids and stabilizes formation of double strands or triple strands. Non-patent document 4 / Bioconjug Chem. (1998) 9, 29 2-299 .; Non-patent document 5 / Bioconjug Chem. (2000) 11, 520-526 .; Non-patent document Reference 6 / Nat Mater. (2003) 2, 815-820 ·). Based on this phenomenon, the usefulness of a carrier in which a hydrophilic group is bound to a cationic amino acid polymer in a comb shape has been found as a nucleic acid carrier (Patent Document 7 / JP-A-10-158196).
[0015] 非特許文献 1 : Fire et al.， Nature, 391， 806-811, 1998 [0015] Non-Patent Document 1: Fire et al., Nature, 391, 806-811, 1998
非特許文献 2 : Elbashir et al.， Nature, 411, 494-498, 2001  Non-Patent Document 2: Elbashir et al., Nature, 411, 494-498, 2001
非特許文献 3 : Soutschek et al.， Nature, 432, 173-178， 2004  Non-Patent Document 3: Soutschek et al., Nature, 432, 173-178, 2004
特許文献 4 : Maruyama A, Watanabe H, Ferdous A, Katoh M, Ishihara T, Akaike T. Characterization of interpolyelectrolyte complexes between double-stranded DN A and polylysine comb-type copolymers having hydrophilic side chains. Bioconjug C hem. (1998) 9， 292-299.  Patent Document 4: Maruyama A, Watanabe H, Ferdous A, Katoh M, Ishihara T, Akaike T. Characterization of interpolyelectrolyte complexes between double-stranded DN A and polylysine comb-type copolymers having hydrophilic side chains.Bioconjug C hem. (1998) 9, 292-299.
非特言午文献 5： Ferdous A, Akaike T, Maruyama A. Mechanism of intermolecular puri ne-purine-pyrimidine triple helix stabilization by comb-type polylysine graft copoly mer at physiologic potassium concentration. Bioconjug Chem. (2000) 11, 520-526. 非特許文献 6： Kim WJ, Sato Y, Akaike T， Maruyama A. Cationic comb-type copoly mers for DNA analysis. Nat Mater. (2003) 2, 815—820. Non-Special Terms 5: Ferdous A, Akaike T, Maruyama A. Mechanism of intermolecular puri ne-purine-pyrimidine triple helix stabilization by comb-type polylysine graft copolymer at physiologic potassium concentration. Bioconjug Chem. (2000) 11, 520-526. Non-Patent Document 6: Kim WJ, Sato Y, Akaike T, Maruyama A. Cationic comb-type copoly mers for DNA analysis. Nat Mater. (2003) 2, 815—820.
特許文献 1 : WO2004/100990  Patent Document 1: WO2004 / 100990
特許文献 2 :特開平 7-69900  Patent Document 2: JP-A-7-69900
特許文献 3：特開 2001-146556  Patent Document 3: JP 2001-146556
特許文献 4： WO2002/26241  Patent Document 4: WO2002 / 26241
特許文献 5：特開 2003-113214  Patent Document 5: JP 2003-113214 A
特許文献 6：特開 2004-352972  Patent Document 6: JP-A-2004-352972
特許文献 7：特開平 10-158196  Patent Document 7: Japanese Patent Laid-Open No. 10-158196
発明の開示  Disclosure of the invention
発明が解決しょうとする課題  Problems to be solved by the invention
[0016] 本発明は、血液中に投与することができる RNA組成物の提供を課題とする。あるい は本発明は、 RNAを血液中で安定に維持しうる組成物、あるいはそのための方法の 提供を課題とする。更に本発明は、血液中における滞留性が改善された RNAの用途 の提供を課題とする。 [0016] An object of the present invention is to provide an RNA composition that can be administered into blood. Alternatively, an object of the present invention is to provide a composition that can stably maintain RNA in blood, or a method therefor. Furthermore, an object of the present invention is to provide a use of RNA having improved retention in blood.
課題を解決するための手段  Means for solving the problem
[0017] 本発明者らは上記の課題を解決するために、 RNAの血中滞留性を向上させる化合 物について研究を重ねた。そして特定の担体との配合によって、 RNAの血液中にお ける滞留性が改善されることを見出した。さらに、 RNAと特定の担体とを別々に投与し た時にも、 RNAの血液中における滞留性は向上することを明らかにした。すなわち本 発明は、以下の組成物、その製造方法、そして RNAの血液中における安定化方法を 提供する。また本発明は、本発明に基づいて血液中における滞留性が改善された R NAの用途を提供する。 [0017] In order to solve the above problems, the present inventors have conducted research on compounds that improve the retention of RNA in blood. It was also found that the retention of RNA in blood is improved by blending with a specific carrier. Furthermore, it has been clarified that the retention of RNA in blood is improved when RNA and a specific carrier are administered separately. That is, the present invention provides the following composition, a method for producing the same, and a method for stabilizing RNA in blood. The present invention also provides use of RNA with improved retention in blood based on the present invention.
本発明は、より具体的には以下の〔1〕〜〔41〕を提供するものである。  More specifically, the present invention provides the following [1] to [41].
〔1〕 ポリカチオン性化合物に対して櫛型に結合している親水性基を側鎖として有す る担体と、 RNAとの複合体を含む、 RNAを血液中に投与するための組成物。 〔2〕 RNAと併用することを特徴とする、ポリカチオン性化合物に対して櫛型に結合し てレ、る親水性基を側鎖として有する担体を有効成分として含有する、 RNAを血液中 で安定化するための糸且成物。 [1] A composition for administering RNA into blood, comprising a complex of RNA and a carrier having a hydrophilic group bonded in a comb shape to a polycationic compound as a side chain and RNA. [2] Use in combination with RNA, containing a carrier having a hydrophilic group as a side chain bonded to a polycationic compound in a comb shape as an active ingredient, RNA in blood Yarn and composition for stabilization.
〔3〕 担体が RNAと同時または、 RNAより前に投与されることを特徴とする、 〔2〕に記 載の組成物。  [3] The composition according to [2], wherein the carrier is administered simultaneously with RNA or before RNA.
〔4〕 ポリカチオン性化合物がポリ（カチオン性アミノ酸）である、〔1〕または〔2〕に記載 の組成物。  [4] The composition according to [1] or [2], wherein the polycationic compound is poly (cationic amino acid).
〔5〕 ポリ（カチオン性アミノ酸）力 ポリ（リジン)である〔4〕に記載の組成物。  [5] Poly (cationic amino acid) force The composition according to [4], which is poly (lysine).
〔6〕 親水性基が、グリコサミノダリカン、デキストラン、ポリエチレングリコール、ポリエ チレングリコール誘導体、及び糖類からなる群から選択される少なくとも 1つである〔1 〕または〔2〕に記載の組成物。 [6] The composition according to [1] or [2], wherein the hydrophilic group is at least one selected from the group consisting of glycosaminodarlican, dextran, polyethylene glycol, a polyethylene glycol derivative, and a saccharide.
[7] ポリカチオンと親水性基とが、グラフト重合している〔1〕または〔2〕に記載の組成 物。  [7] The composition according to [1] or [2], wherein the polycation and the hydrophilic group are graft-polymerized.
〔8〕 担体の 10重量％〜99重量％が親水性基である〔1〕または〔2〕に記載の組成 物。  [8] The composition according to [1] or [2], wherein 10% to 99% by weight of the carrier is a hydrophilic group.
〔9〕 ポリ（カチオン性アミノ酸）を構成するカチオン性アミノ酸基の数 (N)と、担体と 複合体を形成する RNAに含まれるリン酸基の数 (P)との比（N/P比）が、 0. 5〜40 の範囲である、 〔4〕に記載の組成物。  [9] Ratio of the number of cationic amino acid groups constituting the poly (cationic amino acid) (N) to the number of phosphate groups contained in the RNA complexed with the carrier (P) (N / P ratio) ) Is in the range of 0.5 to 40. 4. The composition according to [4].
〔10〕 前記ポリ（カチオン性アミノ酸）を構成するカチオン性アミノ酸基の数 (N)と、担 体と複合体を形成する RNAに含まれるリン酸基の数 (P)との比（N/P比）が、 2〜20 である〔9〕に記載の組成物。  [10] Ratio of the number of cationic amino acid groups (N) constituting the poly (cationic amino acid) to the number of phosphate groups (P) contained in RNA forming a complex with the carrier (N / [9] The composition according to [9], wherein the P ratio is 2 to 20.
〔11〕 RNAが機能性 RNAである〔1〕または〔2〕に記載の組成物。  [11] The composition according to [1] or [2], wherein the RNA is functional RNA.
[12] 機能性 RNA力 ¾NAi効果を有する RNAである〔11〕に記載の組成物。  [12] The composition according to [11], which is RNA having a functional RNA power ¾ NAi effect.
〔13〕 ポリカチオン性化合物に対して櫛型に結合している親水性基を側鎖として有 する担体と RNAとの複合体を形成させる工程を含む、 RNAを血液中で安定化する方 法。  [13] A method for stabilizing RNA in blood, comprising a step of forming a complex of RNA and a carrier having a hydrophilic group bonded in a comb shape to a polycationic compound as a side chain. .
〔14〕 以下の工程を含む、 RNAを血液中で安定化する方法。  [14] A method for stabilizing RNA in blood, comprising the following steps.
( 1 )ポリカチオン性化合物に対して櫛型に結合している親水性基を側鎖として有す る担体を、血液中に投与する工程 (1) having a hydrophilic group as a side chain bonded to the polycationic compound in a comb shape A carrier to be administered into blood
(2) RNAを血液中に投与する工程  (2) The process of administering RNA into blood
[15] RNAを投与する工程が、担体を投与する工程の後または同時に行われること を特徴とする、〔14〕に記載の方法。 [15] The method according to [14], wherein the step of administering RNA is performed after or simultaneously with the step of administering a carrier.
〔16〕 ポリカチオン性化合物力 ポリ（カチオン性アミノ酸)である、 〔13〕または〔14〕 に記載の方法。  [16] The method according to [13] or [14], wherein the compound is a poly (cationic amino acid).
[17] 前記ポリ（カチオン性アミノ酸）を構成するカチオン性アミノ酸基の数 (N)と、前 記担体と複合体を形成するべき核酸に含まれるリン酸基の数 (P)との比 (N/P比）が 、0· 5〜40の範囲であることを特徴とする、〔16〕に記載の方法。  [17] Ratio of the number of cationic amino acid groups constituting the poly (cationic amino acid) (N) and the number of phosphate groups contained in the nucleic acid to be complexed with the carrier (P) ( (N / P ratio) is in the range of 0.5 · 40 to 40, according to [16].
〔18〕 前記ポリ（カチオン性アミノ酸）を構成するカチオン性アミノ酸基の数 (N)と、担 体と複合体を形成する RNAに含まれるリン酸基の数 (P)との比（N/P比）が、 2〜20 である〔17〕に記載の方法。 [18] Ratio of the number of cationic amino acid groups (N) constituting the poly (cationic amino acid) to the number of phosphate groups (P) contained in RNA forming a complex with the carrier (N / The method according to [17], wherein the P ratio is 2 to 20.
〔19〕 ポリカチオン性化合物に対して櫛型に結合している親水性基を側鎖として有 する担体を RNAと混合する工程を含む、 RNAを血液中に投与するための組成物の製 造方法。  [19] Manufacture of a composition for administering RNA into blood, comprising a step of mixing a carrier having a hydrophilic group as a side chain bonded to a polycationic compound in a comb shape with RNA. Method.
〔20〕 ポリカチオン性化合物力 ポリ（カチオン性アミノ酸)である〔19〕に記載の製造 方法。  [20] Polycationic compound strength The production method according to [19], which is poly (cationic amino acid).
〔21〕 ポリカチオン性化合物に対して櫛型に結合している親水性基を側鎖として有 する担体と、 RNAとの複合体を含む、 RNAの腎臓からの***を抑制するための組成 物。  [21] A composition for suppressing excretion of RNA from the kidney, comprising a complex of a carrier having a hydrophilic group as a side chain bonded in a comb shape to a polycationic compound and RNA. .
[22] RNAと併用することを特徴とする、ポリカチオン性化合物に対して櫛型に結合 してレ、る親水性基を側鎖として有する担体を有効成分として含有する、 RNAの腎臓 からの***を抑制するための組成物。  [22] characterized by being used in combination with RNA, comprising a carrier having a hydrophilic group as a side chain bonded to a polycationic compound in a comb shape as an active ingredient, from RNA kidney A composition for suppressing excretion.
〔23〕 担体が RNAと同時または、 RNAより前に投与されることを特徴とする、 〔22〕に 記載の組成物。  [23] The composition according to [22], wherein the carrier is administered simultaneously with RNA or before RNA.
〔24〕 ポリカチオン性化合物力 ポリ（カチオン性アミノ酸)である〔21〕または〔22〕に 記載の組成物。  [24] Polycationic compound strength The composition according to [21] or [22], which is a poly (cationic amino acid).
〔25〕 ポリカチオン性化合物に対して櫛型に結合してレ、る親水性基を側鎖として有 する担体と RNAとの複合体を形成させる工程と、得られた複合体を血液中に投与す る工程を含む、血液中に投与された RNAの腎臓からの***を抑制する方法。 [25] Having a hydrophilic group as a side chain that binds to a polycationic compound in a comb shape. A method for suppressing excretion of RNA administered into blood from the kidney, comprising a step of forming a complex of the carrier and RNA to be administered and a step of administering the obtained complex into the blood.
〔26〕 以下の工程を含む、血液中に投与された RNAの腎臓からの***を抑制する 方法。 [26] A method for suppressing excretion of RNA administered into blood from the kidney, comprising the following steps.
( 1 )ポリカチオン性化合物に対して櫛型に結合している親水性基を側鎖として有す る担体を、血液中に投与する工程  (1) A step of administering into the blood a carrier having a hydrophilic group bonded in a comb shape as a side chain to a polycationic compound.
(2) RNAを血液中に投与する工程  (2) The process of administering RNA into blood
[27] RNAを投与する工程が、担体を投与する工程の後または同時に行われること を特徴とする、〔26〕に記載の方法。  [27] The method according to [26], wherein the step of administering RNA is performed after or simultaneously with the step of administering a carrier.
〔28〕 ポリカチオン性化合物力 ポリ（カチオン性アミノ酸)である〔25〕または〔26〕に 記載の方法。  [28] Polycationic compound power The method according to [25] or [26], which is poly (cationic amino acid).
〔29〕 ポリカチオン性化合物に対して櫛型に結合してレ、る親水性基を側鎖として有 する担体と、 RNAとの複合体を含む、 RNAのヌクレアーゼによる分解を抑制するため の組成物。  [29] A composition for inhibiting degradation of RNA by a nuclease, comprising a complex of RNA and a carrier having a hydrophilic group as a side chain bonded to a polycationic compound in a comb shape. object.
〔30〕 RNAと併用することを特徴とする、ポリカチオン性化合物に対して櫛型に結合 してレ、る親水性基を側鎖として有する担体を有効成分として含有する、 RNAのヌクレ ァーゼによる分解を抑制するための組成物。  [30] According to RNA nuclease, comprising a carrier having a hydrophilic group as a side chain bonded to a polycationic compound in a comb shape as an active ingredient, characterized by being used in combination with RNA A composition for inhibiting decomposition.
〔31〕 担体が RNAと同時または、 RNAより前に投与されることを特徴とする、 〔30〕に 記載の組成物。  [31] The composition according to [30], wherein the carrier is administered simultaneously with RNA or before RNA.
〔32〕 ポリカチオン性化合物力 ポリ（カチオン性アミノ酸)である〔29〕または〔30〕に 記載の組成物。  [32] Polycationic compound strength The composition according to [29] or [30], which is poly (cationic amino acid).
〔33〕 ポリカチオン性化合物に対して櫛型に結合してレ、る親水性基を側鎖として有 する担体と RNAとの複合体を形成させる工程と、得られた複合体を血液中に投与す る工程を含む、血液中に投与された RNAのヌクレアーゼによる分解を抑制する方法。 〔34〕 以下の工程を含む、血液中に投与された RNAのヌクレアーゼによる分解を抑 制する方法。  [33] A step of forming a complex of RNA and a carrier having a hydrophilic group as a side chain by binding to a polycationic compound in a comb shape, and the resulting complex in blood. A method for suppressing degradation of RNA administered into blood by nuclease, comprising a step of administering. [34] A method for suppressing degradation of RNA administered into blood by nuclease, comprising the following steps.
( 1 )ポリカチオン性化合物に対して櫛型に結合している親水性基を側鎖として有す る担体を、血液中に投与する工程 (2) RNAを血液中に投与する工程 (1) A step of administering into the blood a carrier having a hydrophilic group bonded in a comb shape as a side chain to a polycationic compound. (2) The process of administering RNA into blood
[35] RNAを投与する工程が、担体を投与する工程の後または同時に行われること を特徴とする、〔34〕に記載の方法。 [35] The method according to [34], wherein the step of administering RNA is performed after or simultaneously with the step of administering a carrier.
〔36〕 ポリカチオン性化合物力 ポリ（カチオン性アミノ酸)である〔33〕または〔34〕に 記載の方法。  [36] Polycationic compound power The method according to [33] or [34], which is poly (cationic amino acid).
[37] 次の工程を含む、遺伝子の機能解析方法；  [37] A method for analyzing the function of a gene, comprising the following steps:
(1)ポリカチオン性化合物に対して櫛型に結合している親水性基を側鎖として有す る担体と、機能解析の対象遺伝子に相補的な塩基配列を含む 2本鎖 RNAとの複合 体を形成させる工程；  (1) A composite of a carrier having a hydrophilic group as a side chain bonded in a comb shape to a polycationic compound and a double-stranded RNA containing a base sequence complementary to the target gene for functional analysis Forming a body;
(2) (1)の RNA—担体複合体を非ヒト動物の血液中に投与する工程；  (2) A step of administering the RNA-carrier complex of (1) into the blood of a non-human animal;
(3) (1)の RNA—担体複合体を投与された非ヒト動物の表現型を観察し対照と比較す る工程；および  (3) observing the phenotype of the non-human animal administered with the RNA-carrier complex of (1) and comparing it with a control; and
(4)対照と比較して表現型の相違が検出されたときに、前記遺伝子の機能抑制に起 因する表現型が同定される工程。  (4) A step of identifying a phenotype caused by functional suppression of the gene when a phenotypic difference is detected as compared with a control.
〔38〕 次の工程を含む、遺伝子の機能解析方法；  [38] A method for analyzing the function of a gene, comprising the following steps;
(1)ポリカチオン性化合物に対して櫛型に結合している親水性基を側鎖として有す る担体を非ヒト動物の血液中に投与する工程；  (1) A step of administering a carrier having a hydrophilic group bonded in a comb shape as a side chain to a polycationic compound into the blood of a non-human animal;
(2)機能解析の対象遺伝子に相補的な塩基配列を含む 2本鎖 RNAを非ヒト動物の 血液中に投与する工程；  (2) a step of administering a double-stranded RNA containing a base sequence complementary to a target gene for functional analysis into the blood of a non-human animal;
(3) (1)の担体および (2)の RNAを投与された非ヒト動物の表現型を観察し対照と比較 する工程；および  (3) observing the phenotype of the non-human animal administered the carrier of (1) and (2) RNA and comparing it to a control; and
(4)対照と比較して表現型の相違が検出されたときに、前記遺伝子の機能抑制に起 因する表現型が同定される工程。  (4) A step of identifying a phenotype caused by functional suppression of the gene when a phenotypic difference is detected as compared with a control.
〔39〕 RNAを投与する工程が、担体を投与する工程の後または同時に行われること を特徴とする、〔38〕に記載の方法。  [39] The method according to [38], wherein the step of administering RNA is performed after or simultaneously with the step of administering a carrier.
〔40〕 ポリカチオン性化合物力 ポリ（カチオン性アミノ酸)である〔37〕または〔38〕に 記載の方法。  [40] Polycationic compound power The method according to [37] or [38], which is poly (cationic amino acid).
〔41〕 ポリ（カチオン性アミノ酸）を主鎖とし、かつ前記ポリ（カチオン性アミノ酸）に対 して櫛型に結合してレ、る親水性基を側鎖として有する担体と、 RNAとの複合体からな り、前記ポリ（カチオン性アミノ酸）を構成するカチオン性アミノ酸基の数 (N)と、担体 と複合体を形成する RNAに含まれるリン酸基の数 (P)との比（N/P比）が 2〜20であ る複合体。 [41] Poly (cationic amino acid) as a main chain, and against the poly (cationic amino acid) The number of cationic amino acid groups (N) comprising a complex of RNA and a carrier having a hydrophilic group as a side chain bonded to a comb shape and RNA. And the number of phosphate groups (P) contained in RNA forming a complex with the carrier (N / P ratio) is a complex having a ratio of 2-20.
発明の効果  The invention's effect
[0018] 本発明によって RNAの血液中における安定性が改善された。すなわち本発明は、 s iRNAのような安定性ならびに血液中における滞留性の向上が重要な課題となる機能 性 RNAを、血液中に直接投与することを可能とした。 RNAi効果による遺伝子の発現 抑制作用は強力である。したがって RNAi効果を利用して特定の遺伝子の発現を抑 制することは、疾患の治療戦略として重要である。 RNAi効果を治療戦略として利用す るときに、 RNAの血液中における安定化は重要な課題である。本発明は、 RNAの血 液中における安定性、ならびに滞留性を飛躍的に向上させるための組成物、そして 方法を実現した。本発明によって、 RNAi効果をもたらす RNAの臨床応用力、大きく前 進したということができる。  [0018] The stability of RNA in blood has been improved by the present invention. That is, the present invention has made it possible to directly administer functional RNA, such as siRNA, in which stability and retention in blood are important issues in blood. Inhibition of gene expression by RNAi effect is powerful. Therefore, using RNAi effect to suppress the expression of specific genes is important as a therapeutic strategy for diseases. Stabilization of RNA in the blood is an important issue when using the RNAi effect as a therapeutic strategy. The present invention has realized a composition and method for dramatically improving the stability and retention of RNA in blood. According to the present invention, it can be said that the clinical application ability of RNA that brings about the RNAi effect has greatly advanced.
[0019] 本発明の具体的な用途として、 RNAi効果の治療薬としての応用が挙げられる。たと えば多くの抗がん剤は、細胞に障害を与えることによって、細胞増殖の抑制、あるい は細胞死を誘導する。薬剤が作用する分子が、がん細胞に特異的に発現していれ ば、がん細胞特異的な治療を実現できる可能性が高まる。各種のへリカーゼは、一 般に、がん細胞において特に発現レベルが高ぐ正常細胞における発現レベルが低 い遺伝子とされている。したがってへリカーゼ遺伝子の発現抑制における影響は、も ともと正常細胞には現れにくいと考えられる。したがって、本発明を利用してヘリカー ゼの発現を RNAi効果によって抑制しうる RNAを血液中に投与すれば、効果的ながん 治療を実現できる可能性がある。本発明に基づく組成物、あるいは RNAの投与方法 によれば、 RNAの血液中における安定性と滞留性を向上させることができる。したが つて、ヘリカーゼを標的分子とする RNAi効果によるがんの治療を、本発明によって実 現すること力 Sできる。  [0019] A specific use of the present invention is application as a therapeutic agent for RNAi effect. For example, many anticancer drugs induce cell growth inhibition or cell death by damaging cells. If the molecule that the drug acts on is expressed specifically in cancer cells, the possibility of realizing cancer cell-specific treatment increases. Various helicases are generally genes that have a particularly high expression level in cancer cells and a low expression level in normal cells. Therefore, the effect of suppressing the expression of the helicase gene is unlikely to appear in normal cells. Therefore, if RNA that can suppress helicase expression by the RNAi effect is administered into blood using the present invention, there is a possibility that effective cancer treatment can be realized. According to the composition or RNA administration method based on the present invention, the stability and retention of RNA in blood can be improved. Therefore, the present invention can realize cancer treatment by RNAi effect using helicase as a target molecule.
[0020] あるいは、 RNAi効果の強力な遺伝子発現抑制効果は、遺伝子の機能解析におい ても有用なツールである。 RNAi効果によって解析すべき遺伝子発現を抑制すれば、 当該遺伝子の機能を推定することができる。本発明を利用すれば、 siRNAのような RN A分子を血液中に投与し、血液中で安定に維持することができる。そのため、生体を 使って遺伝子機能解析を容易に実施することができる。 RNAを細胞に直接導入する だけでな 生体を利用することによって、生体における表現型の変化を通じて、遺 伝子の機能を同定することができる。たとえば、次のような表現型の変化は、生体反 応であるため、細胞レベルではその変化を直接的に捉えることが困難である。一方、 生体を利用すれば、このような表現型の変化を、容易に、かつ明瞭に検出することが できる。 [0020] Alternatively, the strong gene expression suppression effect of the RNAi effect is a useful tool in the functional analysis of genes. If you suppress the gene expression to be analyzed by the RNAi effect, The function of the gene can be estimated. By utilizing the present invention, RNA molecules such as siRNA can be administered into blood and stably maintained in blood. Therefore, gene function analysis can be easily performed using a living body. By using living organisms that do not simply introduce RNA directly into cells, gene functions can be identified through phenotypic changes in the organism. For example, the following phenotypic changes are biological responses, and it is difficult to capture the changes directly at the cellular level. On the other hand, if a living body is used, such a phenotypic change can be detected easily and clearly.
組織の機能（呼吸、消化、循環、内分泌、生殖などの機能）  Tissue function (respiration, digestion, circulation, endocrine, reproduction, etc.)
生理反応 (血圧、心拍、発汗、***等の生理反応）  Physiological reactions (physiological reactions such as blood pressure, heart rate, sweating, excretion)
神経系（記憶、行動、運動などの神経系の働き）  Nervous system (functions of the nervous system such as memory, behavior, and exercise)
疾患 (疾患モデル動物における症状の変化）  Disease (Changes in symptoms in disease model animals)
薬剤応答 (薬剤の投与に対する生体の応答の変化）  Drug response (changes in the body's response to drug administration)
[0021] あるいはがん細胞の増殖抑制効果のような、細胞を使った実験が可能な場合であ つても、生体中で、 RNAi効果による遺伝子抑制の影響を確認できる意義は大きい。 生体を利用することによって、より、実際の治療に近い環境で、遺伝子抑制の影響を 確認することができる。たとえば、がんを移植した動物における抗がん作用の確認は 、がんとして細胞株のみならず患者から採取されたがん組織を使った実験を可能と する。あるいは、生体を利用することによって、血液中力らがん細胞への RNAの移行 に関する知見を得ることもできる。 [0021] Alternatively, even when an experiment using cells, such as an effect of suppressing the growth of cancer cells, is possible, it is highly meaningful to confirm the influence of gene suppression due to the RNAi effect in vivo. By using living organisms, the effect of gene suppression can be confirmed in an environment closer to actual treatment. For example, confirmation of anticancer effects in animals transplanted with cancer enables experiments using cancer tissues collected from patients as well as cell lines. Alternatively, by using a living body, it is possible to obtain knowledge about the transfer of RNA to cancer cells such as blood force.
図面の簡単な説明  Brief Description of Drawings
[0022] [図 1]合成した担体 PLL-PEG (28K90P)と、 siRNA (21mer)または dsRNA (27mer)の複 合体 (N/P比 =2)の RNaseAに対する分解阻害効果を調べた電気泳動像を示す写真 である。  [0022] [Fig. 1] Electrophoresis image of the degradation inhibitory effect of the synthesized carrier PLL-PEG (28K90P) and siRNA (21mer) or dsRNA (27mer) complex (N / P ratio = 2) on RNaseA It is the photograph which shows.
[図 2]合成した担体 PLL-PEG (28K90P)と、 siRNAまたは dsRNAの複合体 (N/P比 =2) の血漿中での安定性を調べた電気泳動像を示す写真である。  FIG. 2 is a photograph showing an electrophoretic image of the stability of a synthesized carrier PLL-PEG (28K90P) and siRNA or dsRNA complex (N / P ratio = 2) in plasma.
[図 3]各種担体（PLL-Dex)と siRNA (21mer)の複合体（N/P比 =4)をマウスの血液中に 投与後、 0分〜 90分間の siRNAの血中滞留性を比較した電気泳動像を示す写真で ある。 nakedは、 siRNAを担体と混合しないで RNAのまま投与した結果である。 [Figure 3] Comparison of retention of siRNA in blood for 0 to 90 minutes after administration of various carriers (PLL-Dex) and siRNA (21mer) complex (N / P ratio = 4) into mouse blood A photograph showing the electrophoretic image is there. Naked is the result of administering siRNA as it is without mixing with the carrier.
[図 4]各種担体 (PLL-PEG)と siRNA (21mer)の複合体 (N/P比 =8)をマウスの血液中に 投与後、 0分〜 90分間の siRNAの血中滞留性を比較した電気泳動像を示す写真で ある。  [Figure 4] Comparison of retention of siRNA in blood for 0 to 90 minutes after administration of various carriers (PLL-PEG) and siRNA (21mer) complex (N / P ratio = 8) into the blood of mice 2 is a photograph showing an electrophoretic image obtained.
[図 5]合成した担体（PLL-PEG)と dsRNAの複合体 (N/P比 =4)をマウスの血液中に投 与後、 0分〜 90分間の dsRNA (27mer)の血中滞留性を比較した電気泳動像を示す 写真である。  [Fig.5] Retention of dsRNA (27mer) in blood for 0 to 90 minutes after injection of the synthesized carrier (PLL-PEG) and dsRNA complex (N / P ratio = 4) into mouse blood It is the photograph which shows the electrophoresis image which compared these.
[図 6]内在性遺伝子 Ubcl3に対する siRNAをマウスの血液中に投与後の遺伝子発現 の抑制効果を調べた結果をまとめたグラフである。図中、縦軸は対照に対する mRNA のレベル (％)を、横軸は組織の種類を示す。  [FIG. 6] A graph summarizing the results of examining the inhibitory effect of gene expression after administration of siRNA against the endogenous gene Ubcl3 in the blood of mice. In the figure, the vertical axis represents the mRNA level (%) relative to the control, and the horizontal axis represents the tissue type.
[図 7]合成した担体（PLL-PEG)と、 siRNAを時間差でマウスの血液中に投与（NA^b= 4)した後、 1分〜 2. 5時間の siRNAの血中滞留性を比較した電気泳動像を示す写真 である。 (A) siRNA単独投与、 (B)担体（28K90P)投与の 20分後に siRNAを投与、（C) 担体と siRNAの複合体（siRNA/28K90P)を投与、した結果を示す。  [Fig.7] Comparison of retention of siRNA in blood between 1 minute and 2.5 hours after administration of synthesized carrier (PLL-PEG) and siRNA into mouse blood with time difference (NA ^ b = 4) 2 is a photograph showing an electrophoretic image obtained. (A) siRNA alone, (B) siRNA administered 20 minutes after carrier (28K90P) administration, (C) complex of carrier and siRNA (siRNA / 28K90P) administered.
[図 8]合成した担体（PLL-PEG)の PEGグラフト率の影響を調べた結果を示す図である 。（A)、（B) ;グラフト率の異なる担体（28K90P、 28Κ70Ρ)により、（A) siRNA (21mer)、 (B) siRNA (27mer)について蛍光異方性を測定した結果を示す。  FIG. 8 is a diagram showing the results of examining the influence of the PEG graft ratio of a synthesized carrier (PLL-PEG). (A), (B): The results of fluorescence anisotropy measurement for (A) siRNA (21mer) and (B) siRNA (27mer) using carriers (28K90P, 28Κ70Κ) with different graft ratios are shown.
発明を実施するための最良の形態  BEST MODE FOR CARRYING OUT THE INVENTION
[0023] 本発明は、ポリカチオン性化合物に対して櫛型に結合している親水性基を側鎖とし て有する担体と、 RNAとの複合体を含む、 RNAを血液中に投与するための組成物に 関する。 [0023] The present invention provides a complex of RNA and a carrier having a hydrophilic group bonded in a comb shape to a polycationic compound as a side chain, and RNA for administration into blood. Concerning the composition.
[0024] また本発明は、 RNAと併用することを特徴とする、ポリカチオン性化合物に対して櫛 型に結合している親水性基を側鎖として有する担体を有効成分として含有する、 RN [0024] The present invention also includes a carrier having, as an active ingredient, a carrier having a hydrophilic group bonded in a comb shape to a polycationic compound as a side chain, which is used in combination with RNA.
Aを血液中で安定化するための組成物に関する。 It relates to a composition for stabilizing A in the blood.
[0025] また本発明は、ポリカチオン性化合物に対して櫛型に結合している親水性基を側 鎖として有する担体と RNAとの複合体を形成させる工程を含む、 RNAを血液中で安 定化する方法に関する。 [0025] The present invention also includes a step of forming a complex of RNA and a carrier having a hydrophilic group bonded in a comb shape to the polycationic compound as a side chain and RNA in the blood. It relates to the method of stylization.
さらに、本発明は以下の工程を含む、 RNAを血液中で安定化する方法に関する。 ( 1 )ポリカチオン性化合物に対して櫛型に結合している親水性基を側鎖として有す る担体を、血液中に投与する工程 Furthermore, this invention relates to the method of stabilizing RNA in the blood including the following processes. (1) A step of administering into the blood a carrier having a hydrophilic group bonded in a comb shape as a side chain to a polycationic compound.
(2) RNAを血液中に投与する工程  (2) The process of administering RNA into blood
あるいは本発明は、ポリカチオン性化合物に対して櫛型に結合している親水性基を 側鎖として有する担体を RNAと混合する工程を含む、 RNAを血液中に投与するため の組成物の製造方法に関する。  Alternatively, the present invention provides a composition for administering RNA into blood, comprising a step of mixing a carrier having a hydrophilic group as a side chain bonded in a comb shape to a polycationic compound with RNA. Regarding the method.
[0026] 更に本発明は、ポリカチオン性化合物に対して櫛型に結合している親水性基を側 鎖として有する担体と、 RNAとの複合体を含む、 RNAの腎臓からの***を抑制するた めの組成物を提供する。また本発明は、ポリカチオン性化合物に対して櫛型に結合 してレゝる親水性基を側鎖として有する担体と RNAとの複合体を形成させる工程と、得 られた複合体を血液中に投与する工程を含む、血液中に投与された RNAの腎臓から の***を抑制する方法に関する。  [0026] Furthermore, the present invention suppresses excretion of RNA from the kidney, comprising a complex of a carrier having a hydrophilic group bonded in a comb-like manner to a polycationic compound as a side chain and RNA. A composition is provided. The present invention also includes a step of forming a complex of RNA and a carrier having a hydrophilic group as a side chain, which binds to a polycationic compound in a comb shape, and the resulting complex in blood. And a method for suppressing excretion of RNA administered into blood from the kidney, which comprises a step of administering to the blood.
[0027] さらに本発明は、 RNAと併用することを特徴とする、ポリカチオン性化合物に対して 櫛型に結合している親水性基を側鎖として有する担体を有効成分として含有する、 R NAの腎臓からの***を抑制するための組成物を提供する。また本発明は、以下の 工程を含む、血液中に投与された RNAの腎臓力 の***を抑制する方法を提供する  [0027] Further, the present invention comprises a carrier having, as an active ingredient, a hydrophilic group bonded in a comb shape to a polycationic compound as a side chain, which is used in combination with RNA. A composition for suppressing excretion of kidney from the kidney is provided. The present invention also provides a method for suppressing the excretion of renal strength of RNA administered into blood, comprising the following steps:
( 1 )ポリカチオン性化合物に対して櫛型に結合している親水性基を側鎖として有す る担体を、血液中に投与する工程 (1) A step of administering into the blood a carrier having a hydrophilic group bonded in a comb shape as a side chain to a polycationic compound.
(2) RNAを血液中に投与する工程  (2) The process of administering RNA into blood
[0028] 加えて本発明は、ポリカチオン性化合物に対して櫛型に結合している親水性基を 側鎖として有する担体と、 RNAとの複合体を含む、 RNAのヌクレアーゼによる分解を 抑制するための組成物を提供する。あるいは本発明は、ポリカチオン性化合物に対 して櫛型に結合している親水性基を側鎖として有する担体と RNAとの複合体を形成 させる工程と、得られた複合体を血液中に投与する工程を含む、血液中に投与され た RNAのヌクレアーゼによる分解を抑制する方法を提供する。  [0028] In addition, the present invention suppresses degradation of RNA by a nuclease, which includes a complex of RNA and a carrier having a hydrophilic group bonded in a comb shape to a polycationic compound as a side chain and RNA. A composition is provided. Alternatively, the present invention comprises a step of forming a complex of a carrier having a hydrophilic group bonded in a comb shape with a polycationic compound as a side chain and RNA, and the resulting complex in blood. Provided is a method for suppressing degradation of RNA administered into blood by a nuclease, comprising a step of administering.
[0029] さらに本発明は、 RNAと併用することを特徴とする、ポリカチオン性化合物に対して 櫛型に結合している親水性基を側鎖として有する担体を有効成分として含有する、 R NAのヌクレアーゼによる分解を抑制するための組成物を提供する。また本発明は、 以下の工程を含む、血液中に投与された RNAのヌクレアーゼによる分解を抑制する ための組成物を提供する。 [0029] Furthermore, the present invention comprises a carrier having a hydrophilic group as a side chain bonded to a polycationic compound in a comb-like manner as an active ingredient, which is used in combination with RNA. A composition for inhibiting degradation of NA by nuclease is provided. The present invention also provides a composition for inhibiting the degradation of RNA administered into blood by nuclease, comprising the following steps.
( 1 )ポリカチオン性化合物に対して櫛型に結合している親水性基を側鎖として有す る担体を、血液中に投与する工程  (1) A step of administering into the blood a carrier having a hydrophilic group bonded in a comb shape as a side chain to a polycationic compound.
(2) RNAを血液中に投与する工程  (2) The process of administering RNA into blood
[0030] 本発明においては、カチオン性化合物に対して櫛型に結合している親水性基を側 鎖として有する担体が用いられる。本発明におけるカチオン性化合物は、たとえば力 チオン性の官能基を含む構成単位の繰り返し構造を含むポリマーを含む。このような ポリマーはカチオン性ポリマーと呼ばれる。すなわち本発明における担体は、カチォ ン性ポリマーの主鎖に対して、該主鎖に櫛型に結合した親水性側鎖を有する化合物 であることができる。  [0030] In the present invention, a carrier having a hydrophilic group as a side chain bonded to a cationic compound in a comb shape is used. The cationic compound in the present invention includes, for example, a polymer containing a repeating structure of a structural unit containing a force thione functional group. Such a polymer is called a cationic polymer. That is, the carrier in the present invention can be a compound having a hydrophilic side chain bonded to the main chain in a comb shape with respect to the main chain of the cationic polymer.
カチオン性ポリマーを構成するカチオン性の官能基には、アミノ基、イミノ基、グァニ ジノ基、およびビグアニド基等が含まれる。具体的には、たとえば、次のようなポリマ 一を本発明の担体を構成するカチオン性ポリマーとして示すことができる。これらのポ リマーの製造方法は公知である。  The cationic functional group constituting the cationic polymer includes an amino group, an imino group, a guanidino group, a biguanide group, and the like. Specifically, for example, the following polymers can be shown as the cationic polymer constituting the carrier of the present invention. Methods for producing these polymers are known.
ポリエチレンィミン  Polyethyleneimine
ポリビュルァミン  Polybulamine
ポリアリルアミン  Polyallylamine
ポリオノレ二チン  Polionoletinine
ポリリジン  Polylysine
ポリ（ァリルビグアニド- CO-ァリルァミン）  Poly (Arilbiguanide-CO-Allyamine)
ポリ（ァリル- N-力ルバモイルグァニジノ -co-ァリルァミン）  Poly (Aril-N-Force Rubamoylguanidino-co-arlylamin)
カチオン性アミノ酸を含むポリペプチド  Polypeptides containing cationic amino acids
ポリアノレギニン  Polyanoreginin
RGDぺプタイド等  RGD peptide etc.
[0031] 中でも、ポリ（カチオン性アミノ酸）の構造を主鎖として有し、かつそのアミノ基に側 鎖を導入された化合物は、本発明における好ましい担体である。本発明において、 担体の主鎖とすることができるポリ（カチオン性アミノ酸）としては、例えば、ポリ（リジン )、ポリ（オル二チン）、およびポリ（オル二チン—セリン）等を示すことができる。 [0031] Among them, a compound having a poly (cationic amino acid) structure as a main chain and having a side chain introduced into its amino group is a preferred carrier in the present invention. In the present invention, Examples of the poly (cationic amino acid) that can be used as the main chain of the carrier include poly (lysine), poly (ornithine), and poly (ornithine-serine).
[0032] これらのポリ（カチオン性アミノ酸）は、通常公知の重合方法により合成することがで きる。例えば、 ε—カルボベンゾキシ—リジン _Ν—カルボン酸無水物、第 1級ァミン を開始剤として重合させて、ポリ（リジン)を得ることができる。重合開始剤である第 1級 ァミンには、片末端アミノ基のポリエチレンォキシド（分子量 200— 250， 000)等を用 レ、ることができる。あるいはベンジルーセリン _Ν_カルボン酸無水物を利用すれば 、セリンを含むアミノ酸ポリマーを合成することもできる。ポリエチレンォキシドーポリア ミノ酸ブロックコポリマーにおけるポリアミノ酸部分の分子量は、限定されない。好まし レヽ分子量は、 200〜500, 000である。  [0032] These poly (cationic amino acids) can be synthesized by a generally known polymerization method. For example, poly (lysine) can be obtained by polymerization using ε-carbobenzoxy-lysine_Ν-carboxylic anhydride, primary amine as an initiator. For the primary amine that is a polymerization initiator, polyethylene oxide having a single terminal amino group (molecular weight 200-250,000) can be used. Alternatively, by using benzyl-serine_Ν_carboxylic anhydride, an amino acid polymer containing serine can be synthesized. The molecular weight of the polyamino acid moiety in the polyethylene oxide polyamino acid block copolymer is not limited. The preferred molecular weight is 200-500,000.
[0033] 本発明における担体は、上記ポリカチオン性化合物からなる主鎖に、側鎖として、 親水性基が櫛型状に導入 (結合)された化合物である。側鎖は、化学修飾、あるいは 化学結合によって導入される。より具体的には、たとえばグラフト重合によって側鎖が 導入される。本発明において、グラフト重合によって櫛型に導入される親水性基の例 を以下に示す。  [0033] The carrier in the present invention is a compound in which a hydrophilic group is introduced (bonded) as a side chain into a main chain composed of the polycationic compound. Side chains are introduced by chemical modifications or chemical bonds. More specifically, side chains are introduced by, for example, graft polymerization. In the present invention, examples of hydrophilic groups introduced into the comb shape by graft polymerization are shown below.
ポリエチレングリコール（PEG)  Polyethylene glycol (PEG)
PEG誘導体  PEG derivatives
グリコサミノダリカン  Glycosaminodarican
単糖  Monosaccharide
オリゴ糖  oligosaccharide
多糖類等の糖類  Sugars such as polysaccharides
合成水溶性高分子など  Synthetic water-soluble polymers
[0034] 本発明において、親水性基の櫛型の結合とは、主鎖を構成するカチオン官能基を 含む繰り返し単位の側鎖として親水性基が結合されていることを言う。たとえばポリ（ カチオン性アミノ酸）を主鎖とするときには、繰り返し単位を構成するカチオン性ァミノ 酸に親水性基が結合される。このとき、親水性基からなる側鎖は、主鎖を構成する力 チオン性アミノ酸の、すべてに結合される場合と、その一部に結合される場合がある 。本発明における担体は、主鎖を構成するカチオン性アミノ酸の一部に親水性基が 結合された構造を含む。主鎖を構成するカチオン性アミノ酸に対する親水性基の割 合は、得られた担体の構造を解析することによって、平均値として測定することができ る。たとえば、実施例に示すように、 NMR、 GPC、あるいは静的光散乱法などの解 析を通じて、側鎖の導入率と構造を明らかにすることができる。 [0034] In the present invention, the comb-like bond of a hydrophilic group means that a hydrophilic group is bonded as a side chain of a repeating unit containing a cationic functional group constituting the main chain. For example, when poly (cationic amino acid) is used as the main chain, a hydrophilic group is bonded to the cationic amino acid constituting the repeating unit. At this time, the side chain composed of the hydrophilic group may be bonded to all or part of the force thione amino acid constituting the main chain. The carrier in the present invention has a hydrophilic group in a part of the cationic amino acid constituting the main chain. Includes combined structures. The ratio of the hydrophilic group to the cationic amino acid constituting the main chain can be measured as an average value by analyzing the structure of the obtained carrier. For example, as shown in the examples, the side chain introduction rate and structure can be clarified through analysis such as NMR, GPC, or static light scattering.
[0035] より具体的には、たとえばポリエチレングリコール誘導体として、メトキシポリエチレン グリコールのアルデヒド誘導体、アミノ酸誘導体、カルボキシメチル誘導体等を示すこ とができる。またグリコサミノダリカンとしては、例えばヒアルロン酸、へパリン、コンドロ ィチン硫酸等を示すことができる。更に糖類には、例えばデキストランやアミロース等 を示すことができる。あるいは合成水溶性高分子としては、ポリアクリルアミド、ヒドロキ シプロピルセルロース（HPC)、ヒドロキシプロピルメチルセルロース（HPMC)などを 示すことができる。 More specifically, for example, an aldehyde derivative, an amino acid derivative, a carboxymethyl derivative and the like of methoxypolyethylene glycol can be shown as a polyethylene glycol derivative. Examples of glycosaminodarlicans include hyaluronic acid, heparin, and chondroitin sulfate. Furthermore, examples of saccharides include dextran and amylose. Alternatively, examples of the synthetic water-soluble polymer include polyacrylamide, hydroxypropyl cellulose (HPC), and hydroxypropylmethyl cellulose (HPMC).
[0036] 特に、本発明において好ましい側鎖となる親水性基としては、デキストラン (Dex)、 ポリエチレングリコール（PEG)またはその誘導体、またはヒアルロン酸（HA)、ヒアル ロン酸塩（例えば、ヒアルロン酸ナトリウム塩、ヒアルロン酸カリウム塩）である。本発明 における担体として利用することができる化合物としては、以下のようなグラフト共重 合体を示すことができる。  [0036] In particular, the hydrophilic group which is a preferred side chain in the present invention includes dextran (Dex), polyethylene glycol (PEG) or a derivative thereof, hyaluronic acid (HA), hyaluronate (for example, sodium hyaluronate). Salt, potassium hyaluronate). Examples of the compounds that can be used as the carrier in the present invention include the following graft copolymers.
ポリ（リジン）とポリエチレングリコール（PEG)とのグラフト共重合体  Graft copolymer of poly (lysine) and polyethylene glycol (PEG)
ポリ（オノレニチン）と PEGとのグラフト共重合体  Graft copolymer of poly (onorenitine) and PEG
ポリ（オル二チン一セリン）と PEGとのグラフト共重合体  Graft copolymer of poly (ornithine monoserine) and PEG
[0037] たとえば下記式（1)の構造を有するポリ -L -リジンとデキストランのグラフト共重合体 、および式（2)の構造を有するポリ- L -リジンとポリエチレングリコールのグラフト共重 合体は、 RNAとの複合体として血液中に投与したときに、 RNAの血中滞留性が著しく 向上することが確認された。 [0037] For example, a graft copolymer of poly-L-lysine and dextran having the structure of the following formula (1), and a graft copolymer of poly-L-lysine and polyethylene glycol having the structure of the formula (2): It was confirmed that the retention of RNA in blood was significantly improved when administered to blood as a complex with RNA.
Figure imgf000021_0001
Figure imgf000021_0001
(式 1)  (Formula 1)
前記式（1)で示される化合物において、 X=l-1,000、 Y=l-1，000、 ζ=ι-ι,οοο、たと えば X=l-300、 Y=l-300、 Z=l-300、更に具体的には X=l_50、 Y=l_100、 Ζ=10_100で ある化合物を、本発明における好ましい化合物として示すことができる。  In the compound represented by the formula (1), X = l-1,000, Y = l-1,000, ζ = ι-ι, οοο, for example, X = l-300, Y = l-300, Z = l -300, more specifically, a compound in which X = l_50, Y = l_100, and Ζ = 10_100 can be shown as a preferred compound in the present invention.
Figure imgf000021_0002
Figure imgf000021_0002
(式 2)  (Formula 2)
前記式（2)で示される化合物において、 X=1_1,000、 Y=l-1，000、 Z=l_5,000、たと えば X=l-300、 Y=l_300、 Z=10-l，000、更に具体的には X=l_50、 Y=l_200、 Ζ=20-30 0である化合物を、本発明における好ましい化合物として示すことができる。  In the compound represented by the formula (2), X = 1_1,000, Y = l-1,000, Z = l_5,000, for example, X = l-300, Y = l_300, Z = 10-l, 000 More specifically, compounds having X = l_50, Y = l_200, and Ζ = 20-300 can be shown as preferred compounds in the present invention.
[0040] 上記、ポリ（カチオン性アミノ酸）を主鎖とし、親水性基を側鎖にもつグラフト共重合 体の合成方法は限定されない。これらのポリマーを構成単位とするグラフト共重合体 は、公知の有機合成法により合成することができる。すなわち、主鎖重合体の官能基 へ適当な結合生成反応により側鎖を導入すればよい。たとえば、多糖鎖を側鎖とす る場合、糖鎖還元末端とポリアミノ酸のァミノ基との反応に基づくァミノ結合を生成す ること力 Sできる。糖鎖還元末端とポリアミノ酸のァミノ基とは、シッフ塩基形成を経由し て結合させることができる。 [0040] The method for synthesizing the graft copolymer having poly (cationic amino acid) as the main chain and having a hydrophilic group in the side chain is not limited. Graft copolymers containing these polymers as constituent units can be synthesized by known organic synthesis methods. That is, a side chain may be introduced into the functional group of the main chain polymer by an appropriate bond formation reaction. For example, when a polysaccharide chain is used as a side chain, it is possible to generate an amino bond based on a reaction between a sugar chain reducing end and a polyamino acid amino group. The reducing end of the sugar chain and the amino group of the polyamino acid undergo a Schiff base formation. Can be combined.
[0041] また、デキストランまたはヒアルロン酸等の多糖類を親水性の側鎖とする場合は、多 糖の還元末端をポリ（リジン)等のアミノ基とカップリングさせることができる。具体的に は、多糖の還元末端をヨウ素等で酸化しカルボン酸化した後にラタトン化し、アミノ基 とカップリングさせる方法を利用することができる。あるいは、多糖の末端ではなく不 特定の部分をポリ（リジン)等と結合させることもできる。そのための合成方法としては 、多糖を過ヨウ素酸で酸化しアルデヒド基を生成させた後に、ポリ（リジン)等のアミノ 基と還元アミノ化させる方法を示すことができる。  [0041] When a polysaccharide such as dextran or hyaluronic acid is used as a hydrophilic side chain, the reducing end of the polysaccharide can be coupled to an amino group such as poly (lysine). Specifically, a method can be used in which the reducing end of a polysaccharide is oxidized with iodine or the like and then carboxylated, then ratatoned and coupled to an amino group. Alternatively, an unspecified portion of the polysaccharide can be bound to poly (lysine) or the like instead of the end of the polysaccharide. As a synthesis method therefor, a method in which a polysaccharide is oxidized with periodic acid to form an aldehyde group and then reductively aminated with an amino group such as poly (lysine) can be shown.
[0042] さらに、ポリエチレングリコールを親水性の側鎖とするグラフト重合体についても、例 えばメトキシポリエチレングリコールのアルデヒド誘導体を用いて同じくシッフ塩基を 形成することで合成すること力 Sできる。この場合、ポリエチレングリコールをそのまま側 鎖として用いることができる。あるいは、ポリエチレングリコールの一方の水酸基を保 護し、他方の水酸基を適当な官能基に置換した誘導体を用いることもできる。  [0042] Further, a graft polymer having polyethylene glycol as a hydrophilic side chain can also be synthesized by forming a Schiff base using an aldehyde derivative of methoxypolyethylene glycol, for example. In this case, polyethylene glycol can be used as a side chain as it is. Alternatively, a derivative in which one hydroxyl group of polyethylene glycol is protected and the other hydroxyl group is substituted with an appropriate functional group can be used.
水酸基の保護には任意の保護基を利用することができる。保護基としては、たとえ ばメトキシ基等のアルコキシ基、アルキルチオエチレンスルホ二ル基等を示すことが できる。他方の水酸基に導入するための官能基としては、アルデヒド基、アミノ基、サ クシニル基、カルボキシル基、カルボキシメチル基、チオール基、ジメトキシトリチル基 等を示すことができる。側鎖として導入することができるポリエチレングリコールまたは その誘導体の分子量は特に限定されなレ、。好ましいポリエチレングリコールまたはそ の誘導体の平均分子量は 300〜100、 000、より好まし <は 1、 000〜20、 000である  Any protecting group can be used for protecting the hydroxyl group. Examples of the protecting group may include an alkoxy group such as a methoxy group, an alkylthioethylenesulfonyl group, and the like. Examples of the functional group for introduction into the other hydroxyl group include an aldehyde group, an amino group, a succinyl group, a carboxyl group, a carboxymethyl group, a thiol group, and a dimethoxytrityl group. The molecular weight of polyethylene glycol or a derivative thereof that can be introduced as a side chain is not particularly limited. Preferred polyethylene glycols or their derivatives have an average molecular weight of 300-100,000, more preferably <1,000-20,000
[0043] 本発明において、担体に対して導入される親水性基の割合は、限定されない。担 体を構成する主鎖と、導入される親水性基の種類に応じて、適宜選択することができ る。通常は担体に対する親水性基の割合は、担体の 10重量％〜99重量％であり、 好ましくは 20重量％〜99重量％、さらに好ましくは 50重量％〜99重量％である。か 力る範囲より少ない場合には、主に親水性基に基づぐ可溶化の効果が小さくなる。 また力かる範囲より大きい場合には、カチオン性の低下にともなって、 RNAとの静電 気相互作用も低下する。なお、上記親水性基の割合の範囲においては、親水性基 の重合率が高いほど、 RNAとの結合性が高くなる。親水性基の重合率が高いかどう かは、担体一 RNA複合体の異方性を測定することにより調べることができる。 In the present invention, the ratio of the hydrophilic group introduced into the carrier is not limited. It can be appropriately selected depending on the main chain constituting the carrier and the type of hydrophilic group to be introduced. Usually, the ratio of the hydrophilic group to the carrier is 10% to 99% by weight of the carrier, preferably 20% to 99% by weight, more preferably 50% to 99% by weight. When the amount is smaller than the range, the effect of solubilization mainly based on the hydrophilic group is reduced. On the other hand, when it is larger than the range to be applied, the electrostatic interaction with RNA also decreases as the cationicity decreases. In the range of the ratio of the hydrophilic group, the hydrophilic group The higher the polymerization rate, the higher the binding to RNA. Whether the polymerization rate of the hydrophilic group is high or not can be examined by measuring the anisotropy of the carrier-RNA complex.
[0044] 本発明のポリカチオン性化合物に対して櫛型に結合してレ、る親水性基を側鎖とし て有する担体と、 RNAとの複合体を含む、 RNAを血液中に投与するための組成物は 、上記担体を RNAと混合することによって得ることができる。本発明において RNAに 対する担体の使用量は限定されなレ、。例えば、核酸 l z molに対して約 0. 01-100 0 μ molの担体を配合することができる。また担体と RNAの配合比は、 RNAの血液中 における安定性と滞留性の改善が期待できる範囲で、適切な範囲に調節することが できる。 [0044] In order to administer RNA into the blood, including a complex of a carrier having a hydrophilic group as a side chain and bound to the polycationic compound of the present invention in a comb shape. This composition can be obtained by mixing the carrier with RNA. In the present invention, the amount of carrier used for RNA is not limited. For example, about 0.01-100 μmol of carrier can be added to l z mol of nucleic acid. The mixing ratio of the carrier and RNA can be adjusted to an appropriate range as long as the stability and retention of RNA in blood can be expected.
担体と RNAの配合比は、担体のカチオン性の官能基の数と核酸のリン酸基の数と の比、（[担体のカチオン性の官能基] (N)/ [核酸のリン酸基] (P))によって表すことが できる。 Nは担体の合成に使ったポリカチオンの分子量、および重量から算出するこ とができ、また Pは配合する核酸の長さに依存する。したがって、 [担体のカチオン性 の官能基] (N)/ [核酸のリン酸基] (P)はそれぞれ担体の合成に使ったポリカチオンの 構造とその分子量および重量、そして配合する RNA核酸の長さから求めることがで きる。  The ratio of carrier to RNA is the ratio of the number of cationic functional groups on the carrier to the number of phosphate groups on the nucleic acid ((carrier cationic functional group) (N) / [nucleic acid phosphate group] (P)). N can be calculated from the molecular weight and weight of the polycation used to synthesize the carrier, and P depends on the length of the nucleic acid to be incorporated. Therefore, [cationic functional group of carrier] (N) / [phosphate group of nucleic acid] (P) is the structure of polycation used for carrier synthesis, its molecular weight and weight, and the length of RNA nucleic acid to be mixed. You can ask for it.
[0045] 例えば担体のポリカチオンとしてポリ- L-リジン（分子量 25、 856)を、核酸として 21 塩基の siRNAを使用したとき、 N/P比は以下のように計算される。ポリ- L-リジンはリジ ン (分子量 128)が重合したものであり、構成しているリジン残基の数は分子量から求 められる。 25856,128 = 202、つまりこのポリ -！ ^_リジン 1分子あたり 202個のリジン 残基を有する。一方、 21塩基の siRNAは、 1本鎖当たり 21個のヌクレオチド残基より 構成される。ヌクレオチド 1分子につきリン酸基 1個が含まれることから、 1本鎖当たり 2 0個のリン酸基を有する。 21塩基の siRNAは 2本鎖なので、 1分子につき 40個のリン 酸基を有する。上記ポリ -L -リジン 1分子と 21塩基の siRNAl分子を混合したときの N/ Pi を求めると、 N=202、 P=40なので、 202/40 = 5. 1となる。  For example, when poly-L-lysine (molecular weight 25, 856) is used as the carrier polycation and siRNA having 21 bases as the nucleic acid, the N / P ratio is calculated as follows. Poly-L-lysine is a polymer of lysine (molecular weight 128), and the number of lysine residues constituting it can be determined from the molecular weight. 25856,128 = 202, ie this poly-! ^ _Lysine There are 202 lysine residues per molecule. On the other hand, a 21-base siRNA is composed of 21 nucleotide residues per strand. Since one phosphate group is contained per nucleotide molecule, it has 20 phosphate groups per strand. Since 21-base siRNA is double-stranded, it has 40 phosphate groups per molecule. When N / Pi is calculated by mixing the above-mentioned poly-L-lysine molecule and a 21-base siRNA molecule, 202/40 = 5.1 because N = 202 and P = 40.
[0046] 例えば、担体と RNAは、カチオン性ポリアミノ酸への親水性基の導入率が低い（20 mol%以下）場合、上記チャージ比力 付近であれば凝縮する力 ポリリジン等のカチ オン性ホモポリマーと DNAでは、チャージ比 1付近では凝縮するのみならず、沈殿を 形成する。すなわち、本発明における担体と RNAの複合体においては、凝縮を生じ るが、該担体分子の親水性基の存在により沈殿が起こらず、例えばコロイド状粒子と して溶液中に安定に存在するという機能を示す。本発明に係る担体においては、上 記チャージ比は、通常 0. 5〜40の範囲であり、より好ましくは 2〜20である。このよう な範囲において、 RNAの血液中における十分な安定化効果を得ることができる。すな わち本発明における担体は、 RNAの安定化用キャリア一として機能する。 [0046] For example, when the carrier and RNA have a low introduction ratio of a hydrophilic group to a cationic polyamino acid (20 mol% or less), the power to condense near the above charge specific force Cationic homologue such as polylysine Polymer and DNA not only condense near charge ratio 1 but also precipitate. Form. That is, in the complex of the carrier and RNA in the present invention, condensation occurs, but precipitation does not occur due to the presence of the hydrophilic group of the carrier molecule, and for example, it exists stably in solution as colloidal particles. Indicates function. In the carrier according to the present invention, the charge ratio is usually in the range of 0.5 to 40, more preferably 2 to 20. In such a range, a sufficient stabilizing effect of RNA in blood can be obtained. That is, the carrier in the present invention functions as a carrier for stabilizing RNA.
N/P比が 2以上では、 RNAに対して十分量の該担体分子が静電的相互作用により 結合することになり、ヌクレアーゼなどの酵素による分解が阻害される。また、血液中 へ投与した場合、十分量の該担体分子が存在することで、血液中での希釈が起きて も複合体は安定化した状態にある。  When the N / P ratio is 2 or more, a sufficient amount of the carrier molecule is bound to RNA by electrostatic interaction, and degradation by an enzyme such as nuclease is inhibited. In addition, when administered into blood, a sufficient amount of the carrier molecule is present, so that the complex is stabilized even if dilution in blood occurs.
[0047] また本発明においては、上記担体を有効成分として含有する組成物を、 RNAと併 用してもよい。併用とは、 RNAを血液中で安定化するために、 RNAおよび該担体を有 効成分として含有する組成物をともに血液中に投与して RNA安定化効果を得ることを いう。 RNAの血液中への投与は、該担体分子を含む組成物と同時または、該組成物 の投与より後に行うことができる。該組成物の投与より後に行う場合には、該組成物 の投与後、好ましくは 1分〜 24時間後、より好ましくは 1分〜 2時間後、さらに好ましく は 1分〜 20分後に RNAの投与を行うことができる。 [0047] In the present invention, a composition containing the carrier as an active ingredient may be used in combination with RNA. The combined use means that an RNA stabilizing effect is obtained by administering to the blood a composition containing RNA and the carrier as an active ingredient in order to stabilize RNA in the blood. The administration of RNA into the blood can be performed simultaneously with the composition containing the carrier molecule or after the administration of the composition. When it is performed after administration of the composition, RNA administration is preferably performed after 1 minute to 24 hours, more preferably after 1 minute to 2 hours, and even more preferably after 1 minute to 20 minutes after administration of the composition. It can be performed.
該担体を有効成分として含有する組成物と RNAとは、血液中において、上記担体と RNAの配合比となるように血液中に投与することが好ましい。具体的には、チャージ 比が、通常 0. 5〜40の範囲であり、より好ましくは 2〜20となるように投与することが 好ましい。  The composition containing the carrier as an active ingredient and the RNA are preferably administered in the blood so that the mixing ratio of the carrier and the RNA is in the blood. Specifically, it is preferable to administer such that the charge ratio is usually in the range of 0.5 to 40, more preferably 2 to 20.
血液中に十分量の該担体分子を含む組成物と、 RNAを別々に投与した場合にも、 血液中において抗体— RNA複合体が形成され、ヌクレアーゼなどの酵素による RNA の分解が阻害される。  Even when a composition containing a sufficient amount of the carrier molecule in blood and RNA are administered separately, an antibody-RNA complex is formed in the blood, and the degradation of RNA by an enzyme such as a nuclease is inhibited.
[0048] —方、本発明における担体— RNA複合体を構成する RNAとしては、任意の RNAを 用レ、ることができる。たとえば複合体を形成する RNAの長さは、生体への投与が必要 な任意の長さの RNAとすることができる。好ましレ、 RNAの長さとしては、たとえば 500 塩基以下、あるいは 300塩基以下、通常 5〜200塩基、より具体的には 10〜100塩 基程度の RNAを用いることができる。 RNAは、 1本鎖であっても 2本鎖であっても良い 。 1本鎖 RNAは、同じ分子内に相補的な塩基配列を含むことができる。相補的な塩基 配列を含む 1本鎖 RNAは、相補的な塩基配列が互いにハイブリダィズすることによつ て部分的な 2本鎖を形成する。その結果、ステムループゃステムバルジなどの構造が 形成される。これらの構造を複数含むことによって、更に高度な高次構造が形成され る場合もある。これらの構造を有する RNAを、本発明における複合体形成に用いるこ ともできる。 RNAが相補配列を含み、 2本鎖構造を有する場合、いずれかの末端がォ 一バーハングした構造とすることもできる。 [0048] On the other hand, any RNA can be used as the RNA constituting the carrier-RNA complex in the present invention. For example, the length of the RNA forming the complex can be any length of RNA that needs to be administered to a living body. Preferred length of RNA is, for example, 500 bases or less, or 300 bases or less, usually 5 to 200 bases, more specifically 10 to 100 salts. Basic RNA can be used. RNA may be single-stranded or double-stranded. Single-stranded RNA can contain complementary base sequences in the same molecule. Single-stranded RNA containing a complementary base sequence forms a partial double strand by hybridizing complementary base sequences to each other. As a result, structures such as stem loops and stem bulges are formed. By including a plurality of these structures, a higher-order higher-order structure may be formed. RNA having these structures can also be used for complex formation in the present invention. When RNA includes a complementary sequence and has a double-stranded structure, either end may be overhanged.
[0049] また複合体を形成する RNAは、天然の RNAを構成するリボヌクレオチド核酸に加え 、人工的な塩基に置換したものや、その誘導体を含む。したがって、天然の塩基であ る a、 u、 c、および gに代えて、イノシン (i)を有する RNAを複合体形成に用いることがで きる。あるいはリン酸結合をチォエート結合やボラノフォスフェート結合に置換した核 酸誘導体を人工的に合成する方法も公知である。リボヌクレオチド核酸の糖構造を修 飾することもできる。糖構造の修飾方法として、 2' -0-メチル修飾、 2' -フルォロ修飾、 あるいは locked nucleic acid (LNA)修飾等を用いることもできる。また、部分的に DNA を導入した DNA-RNAキメラ分子も公知である。  [0049] The RNA forming the complex includes, in addition to ribonucleotide nucleic acids constituting natural RNA, those substituted with artificial bases and derivatives thereof. Therefore, RNA having inosine (i) can be used for complex formation instead of natural bases a, u, c, and g. Alternatively, a method for artificially synthesizing a nucleic acid derivative in which a phosphate bond is substituted with a thioate bond or a boranophosphate bond is also known. The sugar structure of ribonucleotide nucleic acid can also be modified. As a method for modifying the sugar structure, 2′-0-methyl modification, 2′-fluoro modification, locked nucleic acid (LNA) modification, or the like can be used. In addition, DNA-RNA chimeric molecules into which DNA has been partially introduced are also known.
更に、 RNAに他の物質を結合させた誘導体を、本発明における RNAとして利用する こともできる。たとえば、 PEG,コレステロール、糖、膜透過性ペプチド、抗体などの化 合物を RNAに結合させることができる。これらの物質の結合によって、 RNAの血中滞 留性の向上、もしくは疾患部位への効率的送達を可能とすることができる。  Furthermore, derivatives obtained by binding other substances to RNA can also be used as RNA in the present invention. For example, a compound such as PEG, cholesterol, sugar, a membrane-permeable peptide, or an antibody can be bound to RNA. By combining these substances, it is possible to improve the retention of RNA in blood or to efficiently deliver it to a disease site.
[0050] RNAは、さまざまな機能を有することが明らかにされている。たとえば、 RNAi効果や アンチセンス効果は、遺伝子に相補的な塩基配列を含む RNAが有する、遺伝子発 現の抑制効果である。同様に、さまざまな構造を有するリボザィムが細胞内において 、遺伝子発現を抑制することも明らかにされている。これらの遺伝子発現抑制作用を 有する RNAは、レ、ずれも本発明に基づレ、て担体一 RNA複合体として血液中に投与 することができる。あるいは、特定の塩基配列を有する RNAが、蛋白質などの高分子 化合物に特異的に結合する現象も明らかにされている。核酸以外の物質に対する結 合活性を有する RNAは、ァプタマ一と呼ばれる。ァプタマ一は、蛋白質への結合によ つて、その活性を調節する作用を有する場合がある。アブタマ一として機能する RNA を、本発明の RNA—担体複合体とすることもできる。 [0050] RNA has been shown to have a variety of functions. For example, the RNAi effect and the antisense effect are gene expression suppression effects possessed by RNA having a base sequence complementary to a gene. Similarly, ribozymes having various structures have been shown to suppress gene expression in cells. These RNAs having a gene expression-inhibiting action can be administered into blood as a carrier-one RNA complex based on the present invention. Alternatively, it has been clarified that RNA having a specific base sequence specifically binds to a high molecular compound such as a protein. RNA having binding activity for substances other than nucleic acids is called aptamer. Aptamers are bound by binding to proteins. Therefore, it may have an effect of regulating its activity. RNA functioning as an abutama can also be used as the RNA-carrier complex of the present invention.
[0051] これらの、遺伝暗号の伝達以外の機能を有する RNAを、本発明においては特に機 能性 RNA(ftmctional RNA)と呼ぶ場合がある。本発明において、機能性 RNAとは、遺 伝暗号をアミノ酸配列に翻訳する機能以外の、機能を有する RNAを言う。遺伝暗号 の翻訳機能には、 DNAの塩基配列の転写とアミノ酸の移送が含まれる。したがって、 たとえば以下のような機能を有する RNAは、機能性 RNAに含まれる。 [0051] These RNAs having functions other than the transmission of the genetic code are sometimes called functional RNAs (ftmctional RNAs) in the present invention. In the present invention, functional RNA refers to RNA having a function other than the function of translating the genetic code into an amino acid sequence. The translation function of the genetic code includes transcription of DNA base sequences and transfer of amino acids. Therefore, for example, RNA having the following functions is included in functional RNA.
核酸の切断  Nucleic acid cleavage
蛋白質の合成阻害  Inhibition of protein synthesis
核酸以外の物質に対する結合  Binding to substances other than nucleic acids
なお遺伝暗号の翻訳機能は、細胞内においては、通常、 mRNAと tRNAによって支 えられている。本発明においては、 mRNAや tRNAと同じ塩基配列を含む RNAであつ ても、その RNAが翻訳以外の機能を有する場合には、機能性 RNAに含まれる。これら の機能性 RNAは、 目的とする RNAの塩基配列をコードする DNAを適当なプロモータ 一の下流に連結し、 RNAポリメラーゼによって転写することによって合成することがで きる。 RNAへの転写は、細胞内で行っても良いし、適当な環境を与えれば、 in vitroに おける転写反応によって合成することもできる。铸型となる DNAのコード配列の 3'側に は、好ましくは転写終結シグナルを配置することができる。以下に各種の機能性 RNA について更に具体的に説明する。  The translation function of the genetic code is usually supported by mRNA and tRNA in the cell. In the present invention, even RNA containing the same base sequence as mRNA or tRNA is included in functional RNA if the RNA has a function other than translation. These functional RNAs can be synthesized by ligating DNA encoding the base sequence of the target RNA downstream of an appropriate promoter and transcribing it with RNA polymerase. Transcription to RNA may be carried out in the cell, or it can be synthesized by in vitro transcription reaction provided an appropriate environment. A transcription termination signal can be preferably placed on the 3 ′ side of the coding sequence of the DNA to be a cage. Hereinafter, various functional RNAs will be described in more detail.
[0052] RNAi効果を有する RNA : [0052] RNAi having RNAi effect:
本発明における機能性 RNAとして、遺伝子に対して RNAi (RNA interferance； RNA 干渉)効果を有する二本鎖 RNAを示すことができる。一般的に RNAiとは、標的遺伝 子の mRNA配列と相同な配列からなるセンス RNAおよびこれと相補的な配列からなる アンチセンス RNAとからなる二本鎖 RNAを細胞内に導入することにより、標的遺伝子 mRNAの破壊を誘導し、標的遺伝子の発現が阻害される現象を言う。  As the functional RNA in the present invention, a double-stranded RNA having an RNAi (RNA interferance) effect on a gene can be shown. In general, RNAi refers to target RNA by introducing into the cell a double-stranded RNA consisting of a sense RNA consisting of a sequence homologous to the mRNA sequence of the target gene and an antisense RNA consisting of a complementary sequence. Gene A phenomenon that induces the destruction of mRNA and inhibits the expression of the target gene.
[0053] RNAi効果は、現在のところ、次のようなメカニズムを含むと考えられている。 [0053] The RNAi effect is currently considered to include the following mechanism.
— DICERといわれる酵素（RNase ΠΙ核酸分解酵素ファミリーの一種）と 2本鎖 RNAと の接触；および _ 2本鎖 RNAの DICERによる small interfering RNAまたは siRNAと呼ばれる小さな断 片への分解. — Contact with an enzyme called DICER (RNase ΠΙ nuclease family) and double-stranded RNA; and _ Degradation of double-stranded RNA into small fragments called small interfering RNA or siRNA by DICER.
本発明における RNAi効果を有する 2本鎖 RNAには、この siRNAも含まれる。 RNAiのために使用される RNAは、発現抑制すべき遺伝子の部分領域と完全に同 一（相同）である必要はないが、完全な同一（相同）性を有することが好ましい。以下、 発現抑制の対象とする遺伝子を標的遺伝子と言う。  The siRNA is also included in the double-stranded RNA having RNAi effect in the present invention. The RNA used for RNAi does not need to be completely identical (homologous) to the partial region of the gene whose expression is to be suppressed, but preferably has perfect identity (homology). Hereinafter, a gene whose expression is to be suppressed is referred to as a target gene.
[0054] 本発明において、 RNAi効果を有する 2本鎖 RNAは、通常、標的遺伝子の mRNAに おける任意の連続する塩基配列と相同な配列からなるセンス RNA、および該センス R NAに相補的な配列からなるアンチセンス RNAからなる 2本鎖 RNAである。上記「任意 の連続する塩基配列」の長さは、通常 20〜30塩基であり、好ましくは 21〜23塩基であ る。しかしながら、そのままの長さでは RNAi効果を有さないような長鎖の RNAであって も、細胞において RNAi効果を有する siRNAへ分解されるため、本発明における 2本鎖 RNAの長さは、特に制限されない。  [0054] In the present invention, the double-stranded RNA having the RNAi effect is usually a sense RNA consisting of a sequence homologous to any continuous base sequence in the mRNA of the target gene, and a sequence complementary to the sense RNA It is a double-stranded RNA consisting of antisense RNA consisting of The length of the “arbitrary continuous base sequence” is usually 20 to 30 bases, preferably 21 to 23 bases. However, even a long RNA that does not have an RNAi effect as it is is degraded into siRNA having an RNAi effect in the cell, and therefore the length of the double-stranded RNA in the present invention is particularly Not limited.
[0055] また、標的遺伝子の mRNAの全長もしくはほぼ全長の領域に対応する長鎖二本鎖 R NAを、例えば、予め DICERで分解させ、その分解産物を RNAi効果を有する RNAとし て利用することもできる。このような分解産物には、 RNAi効果を有する二本鎖 RNA分 子 (siRNA)が含まれることが期待される。この方法によれば、 RNAi効果を有することが 期待される mRNA上の領域を、特に選択しなくともよい。  [0055] Also, a long double-stranded RNA corresponding to the full length or almost the full length region of the target gene mRNA is decomposed in advance with, for example, DICER, and the degradation product is used as RNA having an RNAi effect. You can also. Such degradation products are expected to contain double-stranded RNA molecules (siRNA) having the RNAi effect. According to this method, a region on mRNA expected to have an RNAi effect does not need to be selected.
[0056] また、末端に数塩基のオーバーハングを有する二本鎖 RNAは、 RNAi効果が高いこ とが知られている。したがって RNAi効果を有する二本鎖 RNAは、末端に数塩基のォ 一バーハングを有することが望ましレ、。このオーバーハングを形成する塩基の長さは 特に制限されなレ、。オーバーハングの塩基の数は、好ましくは、 2塩基である。本発 明においては例えば、 TT (チミンが 2個）、 UU (ゥラシルが 2個）、その他の塩基のォー バーハングを有する二本鎖 RNAが好ましレ、。たとえばヒトにおいては、 19塩基の二本 鎖 RNAと 2塩基 (TT)のオーバーハングを有する分子は、 RNAi効果が高いとレ、われて いる。 RNAi効果を有する二本鎖 RNAには、オーバーハングを形成する塩基が DNAで あるようなキメラ分子も含まれる。  [0056] Double-stranded RNA having an overhang of several bases at the end is known to have a high RNAi effect. Therefore, it is desirable that double-stranded RNA having an RNAi effect has an overhang of several bases at the end. The length of the base that forms this overhang is not particularly limited. The number of overhanging bases is preferably 2 bases. In the present invention, for example, TT (two thymines), UU (two uracils), and double-stranded RNA having an overhang of other bases are preferred. For example, in humans, molecules with 19-base double-stranded RNA and 2-base (TT) overhangs are reported to have high RNAi effects. Double-stranded RNA having an RNAi effect includes chimeric molecules in which the base that forms an overhang is DNA.
[0057] ここでレ、う 2本鎖 RNAは、相補配列が互いにハイブリダィズした構造を含む RNAを言 う。したがって、先に述べたように 1本鎖 RNA中に相補的な塩基配列を含み、それが 互いにハイブリダィズすることによって 2本鎖構造を取った場合には、 2本鎖 RNAに含 まれる。すなわちステムループ構造を取る 1本鎖 RNAは、 2本鎖構造 (ステム部分）を 含むため、 2本鎖 RNAに含まれる。 [0057] Here, double-stranded RNA refers to RNA containing a structure in which complementary sequences are hybridized to each other. Yeah. Therefore, as described above, when a single-stranded RNA contains a complementary base sequence and has a double-stranded structure by hybridizing with each other, it is included in the double-stranded RNA. That is, single-stranded RNA having a stem-loop structure is included in double-stranded RNA because it contains a double-stranded structure (stem portion).
[0058] 当業者は、標的遺伝子に対して RNAi効果を有する二本鎖 RNAを、その塩基配列を もとに、適宜デザインすることができる。すなわち、標的遺伝子の塩基配列をもとに、 該配列の転写産物である mRNAの任意の連続する RNA領域を選択し、この領域に対 応する二本鎖 RNAを作製することができる。また、該配列の転写産物である mRNA配 列から、より強い RNAi効果を有する siRNA配列を選択する方法も公知である。例えば 、 Reynoldsらが発表した論文（Reynold et al. Nature biotechnology 22. 326-330 (200 4))や、 Ui-Teiらが発表した論文（Ui-Tei et al. Nucleic Acids Res. 32. 936-948 (2004 ))等に基づいて、 siRNAに必要な塩基配列を予測することができる。  [0058] A person skilled in the art can appropriately design double-stranded RNA having an RNAi effect on a target gene based on the base sequence. That is, based on the base sequence of the target gene, any continuous RNA region of mRNA that is a transcription product of the sequence can be selected, and double-stranded RNA corresponding to this region can be prepared. A method for selecting an siRNA sequence having a stronger RNAi effect from an mRNA sequence that is a transcription product of the sequence is also known. For example, a paper published by Reynolds et al. (Reynold et al. Nature biotechnology 22. 326-330 (2004)), a paper published by Ui-Tei et al. (Ui-Tei et al. Nucleic Acids Res. 32. 936- 948 (2004)) and the like, the base sequence necessary for siRNA can be predicted.
[0059] siRNAは、遺伝子の部分的な塩基配列に基づいてデザインすることもできる。 siRNA の塩基配列を特定するためには、選択すべき任意の連続する塩基配列が判明して いればよい。必要な塩基配列の長さは、たとえば、少なくとも 20〜30塩基である。つま り、全長配列が明らかでない標的遺伝子に対して、 siRNAをデザインすることもできる 。従って、 EST (Expressed Sequence Tag)等のように mRNAの一部は判明している力 全長が判明していない遺伝子断片からも、該断片の塩基配列を基に当該遺伝子の 発現を抑制する二本鎖 RNAを作製することができる。  [0059] siRNA can also be designed based on a partial base sequence of a gene. In order to specify the base sequence of siRNA, it is sufficient that an arbitrary continuous base sequence to be selected is known. The length of the necessary base sequence is, for example, at least 20 to 30 bases. In other words, siRNA can be designed for a target gene whose full-length sequence is not clear. Therefore, two genes that suppress the expression of the gene, such as EST (Expressed Sequence Tag), from a gene fragment whose partial mRNA is already known based on the base sequence of the fragment. Strand RNA can be made.
[0060] アンチセンス効果を有する RNA :  [0060] RNA having an antisense effect:
本発明における機能性 RNAとして、遺伝子に対してアンチセンス効果を有する RNA を用レ、ることもできる。特定の遺伝子の発現を阻害 (抑制）する方法として、アンチセ ンス技術を利用する方法が公知である。アンチセンス核酸による標的遺伝子の発現 阻害には、以下のような複数のメカニズムが関与している。  As the functional RNA in the present invention, RNA having an antisense effect on a gene can be used. As a method for inhibiting (suppressing) the expression of a specific gene, a method using an anti-sense technique is known. Inhibition of target gene expression by antisense nucleic acids involves the following mechanisms.
一三重鎖形成による転写開始阻害、  Inhibition of transcription initiation by single triplex formation,
_RNAポリメラーゼによって局部的に開状ループ構造が作られた部位とのハイプリ ッド形成による転写阻害、  _ Transcriptional inhibition due to the formation of a hybrid with the site where an open loop structure was locally created by RNA polymerase,
合成の進みつつある RNAとのハイブリッド形成による転写阻害；、 —イントロンとェキソンとの接合点におけるハイブリッド形成によるスプライシング阻 害. Transcriptional inhibition by hybridization with RNA, which is being synthesized; —Splicing inhibition by hybridization at the junction of intron and exon.
—スプライソソーム形成部位とのハイブリッド形成によるスプライシング阻害；  —Splicing inhibition by hybridization with spliceosome formation site;
_mRNAとのハイブリッド形成による核から細胞質への移行阻害；  Inhibition of nuclear to cytoplasmic hybridization by hybridization with _mRNA;
—キヤッビング部位やポリ (A)付加部位とのハイブリッド形成によるスプライシング阻 一翻訳開始因子結合部位とのハイブリッド形成による翻訳開始阻害；  —Splicing inhibition by hybridization with a cabbing site or poly (A) addition site. Inhibition of translation initiation by hybridization with a translation initiation factor binding site;
開始コドン近傍のリボソーム結合部位とのハイブリッド形成による翻訳阻害； mRNAの翻訳領域やポリソーム結合部位とのハイブリッド形成によるペプチド鎖の 伸長阻害；および  Translation inhibition by hybridization with the ribosome binding site near the initiation codon; inhibition of peptide chain elongation by hybridization with the mRNA translation region and polysome binding site; and
一発現制御領域と転写調節因子との相互作用部位とのハイブリッド形成による遺伝 子発現阻害など  Inhibition of gene expression by hybridization between an expression control region and a site that interacts with a transcriptional regulator
[0061] このようにアンチセンス核酸は、転写、スプライシングまたは翻訳など様々な過程を 阻害することで、標的遺伝子の発現を阻害する（平島および井上著、新生化学実験 講座 2核酸 IV遺伝子の複製と発現、 日本生化学会編、東京化学同人、 1993年、 p.31 9-347)。  [0061] Thus, antisense nucleic acids inhibit the expression of target genes by inhibiting various processes such as transcription, splicing, or translation (Hirashima and Inoue, Shinsei Kagaku Kogyo Lecture 2) Expression, Japanese Biochemical Society, Tokyo Chemical Dojin, 1993, p.31 9-347).
[0062] 本発明で用いられるアンチセンス効果を有する RNAには、これらのいずれかの作用 によって標的遺伝子の発現を阻害しうる RNAが含まれる。一つの態様としては、標的 遺伝子の mRNAの 5'端近傍の非翻訳領域に相補的なアンチセンス配列を設計すれ ば、遺伝子の翻訳阻害に効果的と考えられる。また、コード領域もしくは 3'側の非翻 訳領域に相補的な配歹 IJも使用することができる。このように、標的遺伝子の翻訳領域 だけでな 非翻訳領域の配列のアンチセンス配列を含む RNAも、本発明における アンチセンス効果を有する RNAに含まれる。  [0062] The RNA having an antisense effect used in the present invention includes RNA that can inhibit the expression of a target gene by any of these actions. In one embodiment, designing an antisense sequence complementary to the untranslated region near the 5 ′ end of the mRNA of the target gene is thought to be effective in inhibiting gene translation. It is also possible to use an IJ complementary to the coding region or the 3 'non-translation region. Thus, RNA containing an antisense sequence of a non-translated region only in the target gene translation region is also included in the RNA having an antisense effect in the present invention.
[0063] 本発明におけるアンチセンス RNAは、任意の方法によって合成することができる。  [0063] The antisense RNA of the present invention can be synthesized by any method.
具体的には、 RNAポリメラーゼによる転写反応、あるいは化学合成によって、必要な 塩基配列からなる RNAを得ることができる。合成 RNAオリゴマーとしてアンチセンス RN Aを合成する場合には、リン酸エステル結合部の 0 (酸素）を S (硫黄）に置換した Sオリ ゴ（ホスホロチォエート型オリゴヌクレオチド）とすることができる。 Sオリゴとすることによ つて、ヌクレアーゼ分解に対する耐性を付与することができる。したがって本発明に おいて、 Sオリゴは、機能性 RNAとして好ましい。 Specifically, RNA having a necessary base sequence can be obtained by transcription reaction using RNA polymerase or chemical synthesis. When synthesizing antisense RNA as a synthetic RNA oligomer, S-oligo (phosphorothioate-type oligonucleotide) in which 0 (oxygen) in the phosphate ester bond is replaced with S (sulfur) can be used. . By using S oligo Thus, resistance to nuclease degradation can be imparted. Therefore, in the present invention, S oligo is preferred as a functional RNA.
[0064] アンチセンス RNAの配列は、標的遺伝子またはその一部と相補的な配列であること が好ましい。ただし、遺伝子の発現を有効に抑制できる限り、アンチセンス RNAを構 成する塩基配列は、標的遺伝子の塩基配列に対して完全に相補的でなくてもよい。 転写された RNAは、標的遺伝子の転写産物に対して好ましくは 90%以上、最も好ま しくは 95。/。以上の相補性を有する。アンチセンス RNAを用いて標的遺伝子の発現を 効果的に抑制するには、アンチセンス RNAの長さは少なくとも 15塩基以上であり、好 ましくは 100塩基以上であり、さらに好ましくは 500塩基以上である。  [0064] The sequence of the antisense RNA is preferably a sequence complementary to the target gene or a part thereof. However, as long as gene expression can be effectively suppressed, the base sequence constituting the antisense RNA may not be completely complementary to the base sequence of the target gene. The transcribed RNA is preferably 90% or more, most preferably 95, relative to the transcript of the target gene. /. It has the above complementarity. In order to effectively suppress the expression of a target gene using antisense RNA, the length of the antisense RNA is at least 15 bases, preferably 100 bases or more, more preferably 500 bases or more. is there.
[0065] リボザィム活性を有する RNA :  [0065] RNA having ribozyme activity:
本発明における機能性 RNAとして、リボザィム活性を有する RNAを利用することもで きる。リボザィムとは触媒活性を有する RNA分子を指す。リボザィムには種々の活性 を有するものが存在する。たとえば、 RNAを部位特異的に切断するリボザィムを設計 することもできる。リボザィムには、グループ Iイントロン型や RNase Pに含まれる Ml RN Aのように 400ヌクレオチド以上の大きさのものもある力 ハンマーヘッド型やヘアピン 型と呼ばれる 40ヌクレオチド程度の活性ドメインを有するものもある（小泉誠および大 塚栄子、蛋白質核酸酵素、 1990年、 35、 2191)。  As functional RNA in the present invention, RNA having ribozyme activity can also be used. Ribozyme refers to an RNA molecule having catalytic activity. Some ribozymes have various activities. For example, ribozymes can be designed that cleave RNA site-specifically. Some ribozymes have a size of 400 nucleotides or more, such as group I intron type and Ml RNA contained in RNase P. Some have a 40-nucleotide active domain called hammerhead type or hairpin type. (Makoto Koizumi and Eiko Otsuka, Protein Nucleic Acid Enzyme, 1990, 35, 2191).
[0066] 例えば、ハンマーヘッド型リボザィムの自己切断ドメインは、 G13U14C15という配列 の C15の 3'側を切断する。その切断活性には U14と A9との塩基対形成が重要とされ ている。また、 C15の代わりに A15または U15でも切断されることも示されている（Koizu mi, M.ら著、 FEBS Lett, 1988年、 228、 228.)。基質結合部位が標的部位近傍の RN A配列と相補的なリボザィムを設計すれば、標的 RNA中の UC、 UUまたは UAという配 列を認識する制限酵素的な RNA切断リボザィムを人工的に作り出すことができる（Koi zumi, M.ら著、 FEBS Lett, 1988年、 239, 285.、小泉誠および大塚栄子、蛋白質核酸 酵素、 1990年、 35， 2191·、 Koizumi, M.ら著、 Nucl Acids Res、 1989年、 17， 7059.) 0 [0066] For example, the self-cleaving domain of the hammerhead ribozyme cleaves on the 3 'side of C15 in the sequence G13U14C15. Base pairing between U14 and A9 is important for its cleavage activity. It has also been shown to be cleaved by A15 or U15 instead of C15 (Koizu mi, M. et al., FEBS Lett, 1988, 228, 228.). By designing a ribozyme whose substrate binding site is complementary to the RNA sequence near the target site, it is possible to artificially create a restriction RNA-cleaving ribozyme that recognizes the sequence UC, UU or UA in the target RNA. (Koi zumi, M. et al., FEBS Lett, 1988, 239, 285., Makoto Koizumi and Eiko Otsuka, Protein Nucleic Acid Enzyme, 1990, 35, 2191, Koizumi, M. et al., Nucl Acids Res, (1989, 17, 7059.) 0
[0067] また、ヘアピン型リボザィムも RNAの切断に有用である。ヘアピン型リボザィムは、 例えばタバコリングスポットウィルスのサテライト RNAのマイナス鎖に見出される（Buza yan, JM., Nature, 1986年、 323, 349·)。ヘアピン型リボザィムに基づいて、標的配列 特異的な RNA切断リボザィムを作り出すことができる（Kikuchi, Y. & Sasaki, N.， Nucl Acids Res, 1991, 19， 6751.、菊池洋，化学と生物， 1992, 30， 112.)。このように、標的 遺伝子の転写産物を特異的に切断することができるリボザィム活性を有する RNAを デザインし、本発明に利用することもできる。 [0067] Hairpin ribozymes are also useful for RNA cleavage. Hairpin ribozymes are found, for example, in the minus strand of satellite RNA of tobacco ring spot virus (Buzayan, JM., Nature, 1986, 323, 349). Target sequence based on hairpin ribozyme Specific RNA-cleaving ribozymes can be created (Kikuchi, Y. & Sasaki, N., Nucl Acids Res, 1991, 19, 6751., Hiroshi Kikuchi, Chemistry and Biology, 1992, 30, 112.). Thus, RNA having ribozyme activity capable of specifically cleaving the transcript of the target gene can be designed and used in the present invention.
[0068] 本発明における組成物は、血液中に投与される。血液中への投与とは、血管内へ の投与に加え、採血された血液中へ予め本発明の組成物を混合した後に、当該血 液を生体に投与すること (ex vivo)も含まれる。また、輸血のために採取された血液に 、特定の遺伝子の発現を抑制する siRNAを含む本発明の組成物を加え、生体外で標 的遺伝子の発現を抑制することもできる。本発明の組成物を混合する血液は、全血 のみならず、全血を分画したものであってもよい。たとえば、血清、血漿、血小板、リン パ球などの血液成分に、予め担体— RNA複合体を混合することができる。特に、リン パ球などの遺伝子発現を伴っている細胞を含む血液あるいはその分画は、担体 R NA複合体を混合する血液成分として好ましレ、。  [0068] The composition of the present invention is administered into blood. The administration into blood includes, in addition to administration into blood vessels, mixing the composition of the present invention in the collected blood in advance and then administering the blood to a living body (ex vivo). Further, the expression of a target gene can be suppressed in vitro by adding the composition of the present invention containing siRNA that suppresses the expression of a specific gene to blood collected for blood transfusion. The blood mixed with the composition of the present invention may be not only whole blood but also fractionated whole blood. For example, a carrier-RNA complex can be mixed in advance with blood components such as serum, plasma, platelets, and lymphocytes. In particular, blood containing cells with gene expression such as lymphocytes or a fraction thereof is preferred as a blood component to be mixed with the carrier RNA complex.
[0069] 本発明における組成物は、 目的に応じて、本発明の組成物のみを投与してもよいし 、あるいは他の化合物とともに投与してもよい。たとえば、本出願人は、ある種の薬剤 の作用が、特定の酵素の遺伝子発現の抑制によって増強される場合があることを明 らかにしている。本発明においては、特定の酵素の遺伝子発現を抑制することができ る siRNAを含む担体一RNA複合体を、薬剤とともに投与することもできる。この場合、 担体— RNA複合体の投与によって、 siRNAが酵素遺伝子の発現を抑制すれば、とも に投与された薬剤の作用を増強することができる。  [0069] Depending on the purpose, the composition of the present invention may be administered alone, or may be administered together with other compounds. For example, Applicants have shown that the action of certain drugs may be enhanced by suppression of gene expression of certain enzymes. In the present invention, a carrier-RNA complex containing siRNA capable of suppressing gene expression of a specific enzyme can be administered together with a drug. In this case, if the siRNA suppresses the expression of the enzyme gene by administration of the carrier-RNA complex, the action of the administered drug can be enhanced.
[0070] あるいは、ある薬剤を投与した場合の生体のレスポンス力 特定の遺伝子の発現抑 制によってどのように変化するのかを観察することもできる。たとえば、薬剤代謝に関 連する各種の遺伝子の発現抑制によって、生体におけるある薬剤の代謝に関与する 遺伝子群を同定することもできる。その他、発現抑制によって、薬剤の効果の増強、 あるいは副作用の軽減につながる遺伝子を見出すこともできる。  [0070] Alternatively, it is possible to observe how the response of a living body when a certain drug is administered changes how the expression of a specific gene is suppressed. For example, by suppressing the expression of various genes related to drug metabolism, a group of genes involved in the metabolism of a certain drug in the living body can be identified. In addition, by suppressing expression, it is possible to find genes that enhance the effects of drugs or reduce side effects.
[0071] 本発明によって、血液中における RNAの安定性が増強された担体— RNA複合体が 提供された。本発明において、血液中における RNAの安定化とは、血液中における R NAの滞留時間を延長することを意味する。より具体的には、血液中での RNAの分解 が抑制された状態は、 RNAが安定化された状態に含まれる。 RNAは、血液中におい ては、主にヌクレアーゼの作用によって分解される。つまり、ヌクレーゼに対する耐性 を RNAに与えることは、 RNAの安定化に含まれる。また、水溶性の低分子化合物であ る RNAは、生体においては、速やかに尿中に***される。血液中の RNAの安定化に は、腎臓による尿への RNAの***が抑制された状態も含まれる。 [0071] According to the present invention, there is provided a carrier-RNA complex with enhanced RNA stability in blood. In the present invention, stabilization of RNA in blood means extending the residence time of RNA in blood. More specifically, degradation of RNA in the blood The state in which is suppressed is included in the state where RNA is stabilized. RNA is degraded in blood mainly by the action of nucleases. In other words, imparting resistance to nuclease to RNA is included in RNA stabilization. In addition, RNA, which is a water-soluble low molecular weight compound, is rapidly excreted in urine in the living body. The stabilization of RNA in the blood includes a state where the excretion of RNA into the urine by the kidney is suppressed.
[0072] すなわち本発明は、ポリカチオン性化合物に対して櫛型に結合している親水性基 を側鎖として有する担体と、 RNAとの複合体を含む、 RNAの腎臓からの***を抑制す るための組成物に関する。また本発明は、ポリカチオン性化合物に対して櫛型に結 合している親水性基を側鎖として有する担体と RNAとの複合体を形成させる工程と、 得られた複合体を血液中に投与する工程を含む、血液中に投与された RNAの腎臓 からの***を抑制する方法を提供する。  That is, the present invention suppresses the excretion of RNA from the kidney, which contains a complex of a carrier having a hydrophilic group bonded in a comb shape to a polycationic compound as a side chain and RNA. It relates to a composition for The present invention also includes a step of forming a complex of a carrier having a hydrophilic group bonded in a comb shape with a polycationic compound as a side chain and RNA, and the resulting complex in blood. Provided is a method for suppressing excretion of RNA administered into blood from the kidney, comprising a step of administering.
[0073] さらに本発明は、 RNAと併用することを特徴とする、ポリカチオン性化合物に対して 櫛型に結合している親水性基を側鎖として有する担体を有効成分として含有する、 R NAの腎臓からの***を抑制するための組成物に関する。また本発明は、以下のェ 程を含む、血液中に投与された RNAの腎臓力 の***を抑制する方法を提供する。  [0073] Further, the present invention comprises a carrier having, as an active ingredient, a hydrophilic group bonded as a side chain to a polycationic compound, which is used in combination with RNA, as an active ingredient. The present invention relates to a composition for suppressing the excretion of kidney from the kidney. The present invention also provides a method for suppressing the excretion of renal strength of RNA administered into blood, comprising the following steps.
( 1 )ポリカチオン性化合物に対して櫛型に結合している親水性基を側鎖として有す る担体を、血液中に投与する工程  (1) A step of administering into the blood a carrier having a hydrophilic group bonded in a comb shape as a side chain to a polycationic compound.
(2) RNAを血液中に投与する工程  (2) The process of administering RNA into blood
本発明における好ましい担体は、ポリ（カチオン性アミノ酸）を主鎖とし、かつ前記ポ リ（カチオン性アミノ酸）に対して櫛型に結合している親水性基を側鎖として有するィ匕 合物である。  A preferable carrier in the present invention is a compound having a poly (cationic amino acid) as a main chain and a hydrophilic group bonded in a comb shape to the poly (cationic amino acid) as a side chain. is there.
[0074] 加えて本発明は、ポリカチオン性化合物に対して櫛型に結合している親水性基を 側鎖として有する担体と、 RNAとの複合体を含む、 RNAのヌクレアーゼによる分解を 抑制するための組成物に関する。あるいは本発明は、ポリカチオン性化合物に対し て櫛型に結合している親水性基を側鎖として有する担体と RNAとの複合体を形成さ せる工程と、得られた複合体を血液中に投与する工程を含む、血液中に投与された RNAのヌクレアーゼによる分解を抑制する方法を提供する。  [0074] In addition, the present invention suppresses degradation of RNA by a nuclease, which includes a complex of RNA and a carrier having a hydrophilic group bonded in a comb shape to a polycationic compound as a side chain. Relates to a composition. Alternatively, the present invention includes a step of forming a complex of a carrier having a hydrophilic group bonded in a comb shape as a side chain with a polycationic compound and RNA, and the obtained complex in blood. There is provided a method for inhibiting degradation of RNA administered into blood by nuclease, comprising a step of administering.
[0075] さらに本発明は、 RNAと併用することを特徴とする、ポリカチオン性化合物に対して 櫛型に結合している親水性基を側鎖として有する担体を有効成分として含有する、 R NAのヌクレアーゼによる分解を抑制するための組成物に関する。また本発明は、以 下の工程を含む、血液中に投与された RNAのヌクレアーゼによる分解を抑制するた めの組成物を提供する。 [0075] Furthermore, the present invention provides a polycationic compound characterized by being used in combination with RNA. The present invention relates to a composition for inhibiting degradation of RNase by nuclease, which contains, as an active ingredient, a carrier having a hydrophilic group bonded in a comb shape as a side chain. The present invention also provides a composition for suppressing degradation of RNA administered into blood by nuclease, comprising the following steps.
( 1 )ポリカチオン性化合物に対して櫛型に結合している親水性基を側鎖として有す る担体を、血液中に投与する工程  (1) A step of administering into the blood a carrier having a hydrophilic group bonded in a comb shape as a side chain to a polycationic compound.
(2) RNAを血液中に投与する工程  (2) The process of administering RNA into blood
本発明における好ましい担体は、ポリ（カチオン性アミノ酸）を主鎖とし、かつ前記ポ リ（カチオン性アミノ酸）に対して櫛型に結合している親水性基を側鎖として有するィ匕 合物である。  A preferable carrier in the present invention is a compound having a poly (cationic amino acid) as a main chain and a hydrophilic group bonded in a comb shape to the poly (cationic amino acid) as a side chain. is there.
[0076] 本発明の担体 RNA複合体は、 RNAの血液中の滞留性が向上している。その結果 、本発明にしたがって血液中に投与された RNAは、血流の多い臓器、あるいは組織 に、効果的に送達される。具体的には、肝臓や腎臓などの臓器は、血流の多い臓器 とされている。したがって、本発明によって、これらの臓器に RNAを送達することがで きる。また、がん組織は血管の新生を伴う血流の多い組織である。したがって、がん への RNAの送達にも有用である。  [0076] The carrier RNA complex of the present invention has improved retention of RNA in blood. As a result, RNA administered into blood according to the present invention is effectively delivered to organs or tissues with high blood flow. Specifically, organs such as the liver and kidney are considered to have a high blood flow. Thus, RNA can be delivered to these organs according to the present invention. In addition, cancer tissue is a tissue with a lot of blood flow accompanied by neovascularization. Therefore, it is also useful for delivering RNA to cancer.
[0077] あるいは、前記担体- RNA複合体を特定の臓器や細胞に対して標的化することもで きる。たとえば、肝臓を構成する細胞を対象にした遺伝子の導入は重症肝疾患の治 療にとって極めて重要な課題となっている。肝臓の内皮細胞がレセプターを介したェ ンドサイト一シスにより、ヒアルロン酸を取り込む現象が知られている。ヒアルロン酸は 、細胞外マトリックスの成分である。この作用によってヒアルロン酸は、循環系から極 めて効率よく除去される。前記担体としてヒアルロン酸を構成成分とする担体を合成 すれば、肝臓の内皮細胞のエンドサイト一シスを利用して、 RNAを肝臓に対して標的 化することができる。このような担体は、たとえばポリ（カチオン性アミノ酸）に、ヒアノレ口 ン酸をグラフト重合することによって得ることができる。このような構造を有する担体と R NAとの複合体は、肝類洞内皮細胞に特異的に取り込まれる。  [0077] Alternatively, the carrier-RNA complex can be targeted to a specific organ or cell. For example, the introduction of genes for cells that make up the liver is an extremely important issue for the treatment of severe liver disease. It is known that hepatic endothelial cells take up hyaluronic acid by receptor-mediated endocytosis. Hyaluronic acid is a component of the extracellular matrix. By this action, hyaluronic acid is removed from the circulatory system extremely efficiently. If a carrier containing hyaluronic acid as a constituent is synthesized as the carrier, RNA can be targeted to the liver by utilizing endocytosis of liver endothelial cells. Such a carrier can be obtained, for example, by graft polymerization of hyanolic acid to poly (cationic amino acid). A complex of a carrier having such a structure and RNA is specifically taken up by hepatic sinusoidal endothelial cells.
[0078] 本発明において、血液中に投与される担体一 RNA複合体、または血液中で複合体 を形成するための担体および RNAは、その使用方法、使用目的等により応じて、適 宜調節することができる。例えば、注射投与に用いる場合の投与量は、通常、 1日量 として約 0. 1 μ g/kg〜200mg/kg、より具体的には 1日量約 1 μ g/kg〜： 100mg/kgで ある。 [0078] In the present invention, the carrier-RNA complex to be administered into the blood, or the carrier and RNA for forming the complex in the blood are suitable depending on the method of use, purpose of use, and the like. It can be adjusted accordingly. For example, when used for injection administration, the daily dose is usually about 0.1 μg / kg to 200 mg / kg, more specifically about 1 μg / kg daily: 100 mg / kg. It is.
[0079] 本発明により、 RNAの血液中における滞留性が改善された。その結果、各種の機 能性 RNAを生体に投与し、その機能を生体中で安定に維持できることが確認された 。したがって本発明を利用すれば、機能性 RNAが生体にもたらす影響を明らかにす ることができる。すなわち本発明は、次の工程 (1)〜(4)を含む、遺伝子の機能解析方 法を提供する。  [0079] According to the present invention, the retention of RNA in blood was improved. As a result, it was confirmed that various functional RNAs can be administered to a living body and the function can be stably maintained in the living body. Therefore, by using the present invention, it is possible to clarify the effect of functional RNA on living bodies. That is, the present invention provides a gene functional analysis method comprising the following steps (1) to (4).
(1)ポリカチオン性化合物に対して櫛型に結合している親水性基を側鎖として有す る担体と、機能解析の対象遺伝子に相補的な塩基配列を含む 2本鎖 RNAとの複合 体を形成させる工程；  (1) A composite of a carrier having a hydrophilic group as a side chain bonded in a comb shape to a polycationic compound and a double-stranded RNA containing a base sequence complementary to the target gene for functional analysis Forming a body;
(2) (1)の RNA—担体複合体を非ヒト動物の血液中に投与する工程；  (2) A step of administering the RNA-carrier complex of (1) into the blood of a non-human animal;
(3) (1)の RNA—担体複合体を投与された非ヒト動物の表現型を観察し対照と比較す る工程；および  (3) observing the phenotype of the non-human animal administered with the RNA-carrier complex of (1) and comparing it with a control; and
(4)対照と比較して表現型の相違が検出されたときに、前記遺伝子の機能抑制に起 因する表現型が同定される工程  (4) A step of identifying a phenotype caused by suppression of the function of the gene when a phenotypic difference is detected compared to the control.
[0080] また、本発明は次の工程を含む、遺伝子の機能解析方法を提供する。  [0080] The present invention also provides a gene function analysis method including the following steps.
(1)ポリカチオン性化合物に対して櫛型に結合している親水性基を側鎖として有す る担体を非ヒト動物の血液中に投与する工程；  (1) A step of administering a carrier having a hydrophilic group bonded in a comb shape as a side chain to a polycationic compound into the blood of a non-human animal;
(2)機能解析の対象遺伝子に相補的な塩基配列を含む 2本鎖 RNAを非ヒト動物の 血液中に投与する工程；  (2) a step of administering a double-stranded RNA containing a base sequence complementary to a target gene for functional analysis into the blood of a non-human animal;
(3) (1)の担体および (2)の RNAを投与された非ヒト動物の表現型を観察し対照と比較 する工程；および  (3) observing the phenotype of the non-human animal administered the carrier of (1) and (2) RNA and comparing it to a control; and
(4)対照と比較して表現型の相違が検出されたときに、前記遺伝子の機能抑制に起 因する表現型が同定される工程。  (4) A step of identifying a phenotype caused by functional suppression of the gene when a phenotypic difference is detected as compared with a control.
本発明における好ましい担体は、ポリ（カチオン性アミノ酸）を主鎖とし、かつ前記ポ リ（カチオン性アミノ酸）に対して櫛型に結合している親水性基を側鎖として有するィ匕 合物である。 [0081] 本発明において、本発明の組成物を投与する非ヒト動物としては、ヒト以外の任意 の動物を利用することができる。たとえば、マウス、ラット、サル、ィヌ、ネコ、ゥシ、ャギ 、ヒッジ、あるいはゥサギなどの、一般的な実験動物を利用することができる。あるい は、各種の、疾患モデル動物を利用して、疾患と遺伝子の関係を明らかにすることも できる。疾患モデル動物とは、特殊な飼育環境、薬物の投与、外科的処置、あるいは 遺伝学的な改変などによって、人為的に病的な状態に置かれた動物である。 A preferable carrier in the present invention is a compound having a poly (cationic amino acid) as a main chain and a hydrophilic group bonded in a comb shape to the poly (cationic amino acid) as a side chain. is there. [0081] In the present invention, any non-human animal to which the composition of the present invention is administered can be any animal other than humans. For example, common laboratory animals such as mice, rats, monkeys, monkeys, cats, tusks, goats, hidges, or rabbits can be used. Or, various disease model animals can be used to clarify the relationship between diseases and genes. A disease model animal is an animal that has been artificially placed in a pathological state by a special breeding environment, administration of drugs, surgical treatment, or genetic modification.
本発明において、本発明の組成物の投与方法は限定されない。本発明において は、複合体は血液中に投与される。したがって、通常、複合体は、血管への注射によ つて、投与される。複合体を投与された非ヒト動物は、投与後の表現型が観察される 。対照と比較して表現型の相違が確認された場合には、その相違が複合体として投 与された RNAの影響によるものであることがわかる。  In the present invention, the administration method of the composition of the present invention is not limited. In the present invention, the complex is administered into the blood. Therefore, the complex is usually administered by injection into the blood vessel. In non-human animals that have been administered the complex, the phenotype after administration is observed. If a phenotypic difference is confirmed compared to the control, it can be seen that the difference is due to the effect of RNA applied as a complex.
たとえば、標的遺伝子の発現を抑制する機能を有する RNAを複合体として投与した ときには、表現型の相違は、当該遺伝子の発現抑制によってもたらされたと考えるこ とができる。すなわち、標的遺伝子の機能抑制に起因する表現型を同定することがで きる。  For example, when RNA having a function of suppressing the expression of a target gene is administered as a complex, the phenotypic difference can be considered to be caused by the suppression of the expression of the gene. That is, it is possible to identify a phenotype resulting from functional suppression of the target gene.
[0082] 本発明において、対照とは、たとえば複合体を構成する担体のみを投与した動物 の表現型とすることができる。あるいは、非ヒト動物が有していない塩基配列を含む R NAと担体との複合体を投与して対照とすることもできる。あるいは、一定のレベルの 遺伝子抑制作用を有することが明らかな RNAを含む複合体を投与した非ヒト動物を 対照として用いれば、その RNAよりも、より大きな作用を有する RNAを見出すことがで きる。  [0082] In the present invention, the control can be, for example, the phenotype of an animal administered with only the carrier constituting the complex. Alternatively, a complex of RNA and a carrier containing a base sequence that a non-human animal does not have can be administered as a control. Alternatively, if a non-human animal administered with a complex containing RNA that is clearly known to have a certain level of gene suppression activity, RNA having a greater effect than that RNA can be found.
[0083] 本発明において、本発明の組成物は、単独で投与することもできるし、あるいは複 合体以外の他の成分とともに投与することもできる。たとえば、薬物の代謝メカニズム を解明するために、薬物と複合体とをともに投与することもできる。標的遺伝子の発現 抑制によって、薬物の薬理作用や副作用が増強 (あるいは抑制）されれば、標的遺 伝子が、その薬物の薬理作用や副作用と関連していることがわかる。  [0083] In the present invention, the composition of the present invention can be administered alone, or can be administered together with other components other than the complex. For example, a drug and a complex can be administered together to elucidate the metabolic mechanism of the drug. If suppression of target gene expression enhances (or suppresses) the pharmacological action or side effect of a drug, it indicates that the target gene is associated with the pharmacological action or side effect of the drug.
[0084] その他、非ヒト動物にさまざまな病態を誘導する物質を投与して疾患モデル動物と し、本発明の組成物を投与して、 RNAの治療効果を検証することができる。たとえば、 発がん物質を投与したモデル動物において、本発明に基づいて、さまざまな標的遺 伝子の発現抑制を試みることができる。もしもある遺伝子の発現抑制によって発癌が 防止できれば、その遺伝子は、発癌に関与している遺伝子であると同定される。 [0084] In addition, the therapeutic effect of RNA can be verified by administering a substance that induces various disease states to a non-human animal to be a disease model animal and administering the composition of the present invention. For example, Based on the present invention, suppression of the expression of various target genes can be attempted in a model animal to which a carcinogen is administered. If carcinogenesis can be prevented by suppressing the expression of a gene, the gene is identified as a gene involved in carcinogenesis.
[0085] 更に、患者から採取されたがん組織を移植した非ヒト動物に、本発明の組成物を投 与することもできる。移植したがん組織が複合体の投与によって退縮すれば、当該標 的遺伝子が治療標的として有用であることが確認できる。たとえば、がん治療に有効 な可能性のある複数の標的遺伝子があるとき、患者のがんに対してどの標的遺伝子 が有効なのかを評価する方法としても有用である。  [0085] Furthermore, the composition of the present invention can be administered to a non-human animal transplanted with a cancer tissue collected from a patient. If the transplanted cancer tissue is regressed by administration of the complex, it can be confirmed that the target gene is useful as a therapeutic target. For example, when there are multiple target genes that may be useful for cancer treatment, it is also useful as a method for evaluating which target genes are effective for a patient's cancer.
[0086] さらに本発明は、前記担体 RNA複合体、または血液中で複合体を形成するため の担体および RNAを含む医薬組成物とその製造方法を提供する。治療薬としての有 効性が確認された多くの RNAが公知である。たとえば、がん細胞などの悪性の細胞 に対して、特定の遺伝子発現を抑制しうる siRNAを導入することで、細胞増殖を阻害 できることが知られている。このような治療効果を有する RNAを用いて、本発明に基づ レ、て医薬組成物を製造することができる。  [0086] Furthermore, the present invention provides a pharmaceutical composition comprising the carrier RNA complex or a carrier for forming a complex in blood and RNA, and a method for producing the same. Many RNAs that have been confirmed to be effective as therapeutic agents are known. For example, it is known that cell growth can be inhibited by introducing siRNA capable of suppressing specific gene expression into malignant cells such as cancer cells. Based on the present invention, a pharmaceutical composition can be produced using RNA having such a therapeutic effect.
[0087] すなわち本発明は、ポリカチオン性化合物に対して櫛型に結合している親水性基 を側鎖として有する担体と、 RNAとの複合体、または血液中で複合体を形成するため の該担体および RNA、および薬学的に供される担体とを含む血液中に投与するため の医薬組成物を提供する。あるいは本発明は、ポリカチオン性化合物に対して櫛型 に結合している親水性基を側鎖として有する担体と RNAとの複合体、または血液中 で複合体を形成するための該担体および RNAを薬学的に供される担体と混合するェ 程を含む、血液中に投与するための医薬組成物の製造方法に関する。本発明にお ける好ましい担体は、ポリ（カチオン性アミノ酸）を主鎖とし、かつ前記ポリ（カチオン性 アミノ酸）に対して櫛型に結合している親水性基を側鎖として有する化合物である。 薬学上許容される担体として、例えば界面活性剤、着色料、着香料、保存料、安定 剤、緩衝剤、懸濁剤、等張化剤、流動性促進剤等が挙げられるが、これらに制限され ず、その他常用の担体を適宜使用することができる。  That is, the present invention provides a complex of RNA with a carrier having a hydrophilic group bonded in a comb shape to a polycationic compound as a side chain and RNA, or a complex in blood. Provided is a pharmaceutical composition for administration into blood comprising the carrier and RNA, and a pharmaceutically provided carrier. Alternatively, the present invention relates to a complex of RNA and a carrier having a hydrophilic group bonded in a comb shape to a polycationic compound as a side chain and RNA, or the carrier and RNA for forming a complex in blood. The present invention relates to a method for producing a pharmaceutical composition for administration into blood, comprising the step of mixing with a pharmaceutically provided carrier. A preferable carrier in the present invention is a compound having poly (cationic amino acid) as a main chain and a hydrophilic group bonded in a comb shape to the poly (cationic amino acid) as a side chain. Examples of pharmaceutically acceptable carriers include, but are not limited to, surfactants, colorants, flavoring agents, preservatives, stabilizers, buffering agents, suspending agents, tonicity agents, fluidity promoters, and the like. However, other commonly used carriers can be used as appropriate.
[0088] 本発明の医薬組成物の製剤化にあたっては、常法に従い、必要に応じて上記担体 を添加することができる。具体的には、乳糖、マンニトール、カルメロースナトリウム、ヒ ドロキシプロピノレセノレロース、ヒドロキシプロピノレメチノレセノレロース、ゼラチン、ポリオキ シエチレン硬化ヒマシ油 60、白糖、カルボキシメチルセルロース、無機塩類等を挙げ ることができる。これらの担体と配合された前記複合体は、溶解状態で注射剤として 血液中に投与するための医薬組成物とすることができる。あるいは乾燥させた後に、 投与時に滅菌生理食塩水などで溶解して、注射剤とすることもできる。 [0088] In formulating the pharmaceutical composition of the present invention, the above-mentioned carrier can be added as necessary according to a conventional method. Specifically, lactose, mannitol, carmellose sodium, Examples thereof include droxypropenoresenorerose, hydroxypropinoremethinoresenellose, gelatin, polyoxyethylene hydrogenated castor oil 60, sucrose, carboxymethylcellulose, and inorganic salts. The complex blended with these carriers can be made into a pharmaceutical composition for administration into the blood as an injection in a dissolved state. Alternatively, after drying, it can be dissolved in sterile physiological saline at the time of administration to give an injection.
本発明の医薬組成物の投与量は、剤型の種類、投与方法、患者の年齢や体重、 患者の症状等を考慮して、最終的には医師の判断により適宜決定することができる。 なお、本明細書において引用された全ての先行技術文献は、参照として本明細書 に組み入れられる。以下、実施例に基づいて本発明を更に具体的に説明する。 実施例  The dosage of the pharmaceutical composition of the present invention can be appropriately determined finally based on the judgment of a doctor in consideration of the type of dosage form, administration method, patient age and weight, patient symptom, and the like. It should be noted that all prior art documents cited in this specification are incorporated herein by reference. Hereinafter, the present invention will be described more specifically based on examples. Example
[0089] 本発明に基づく siRNAデリバリーシステム  [0089] siRNA delivery system based on the present invention
1.ポリ- L-リジン- g-デキストラン (PLL-Dex)、およびポリ- L-リジン _g-ポリエチレンダリ コーノレ PLL- PEGの合成  1.Synthesis of poly-L-lysine-g-dextran (PLL-Dex) and poly-L-lysine _g-polyethylene daliconole PLL-PEG
本発明における担体— RNA複合体を得るために、下記式 1に示す構造を有するポ リ -L-リジン- g-デキストラン（PLL- Dex)および、および式 2に示す構造を有するポリ- L -リジン- g -ポリエチレングリコール（PLL-PEG)を合成した。これらの化合物は、いず れもポリカチオン性アミノ酸を主鎖として、この主鎖に対して櫛型に結合している親水 性基を側鎖として有する化合物である。  In order to obtain the carrier-RNA complex in the present invention, poly-L-lysine-g-dextran (PLL-Dex) having the structure shown in the following formula 1 and poly-L- having the structure shown in the formula 2 are used. Lysine-g-polyethylene glycol (PLL-PEG) was synthesized. Each of these compounds is a compound having a polycationic amino acid as a main chain and a hydrophilic group bonded to the main chain in a comb shape as a side chain.
[0090] PLL-Dexは主鎖がポリ- L-リジン（PLL)で、側鎖がデキストランの化合物である。ま た PLL-PEGは主鎖が PLLで、側鎖がポリエチレングリコールの化合物である。本実施 例においては、式 1で示す化合物として「7K90D」を合成した。 7K90Dは、 PLL (分子 量 7,000)を主鎖とし、側鎖として平均 7.3個のデキストラン（分子量 6000)が PLLに結合 している化合物である。 7K90Dに対するデキストラン（Mn 6000)の重量は、 90wt%とな る。  [0090] PLL-Dex is a compound having a main chain of poly-L-lysine (PLL) and a side chain of dextran. PLL-PEG is a compound whose main chain is PLL and whose side chain is polyethylene glycol. In this example, “7K90D” was synthesized as a compound represented by Formula 1. 7K90D is a compound that has PLL (molecular weight 7,000) as the main chain and 7.3 dextran (molecular weight 6000) on the side chain as an average. The weight of dextran (Mn 6000) against 7K90D is 90wt%.
[0091] グラフティング率とは PLLのすベてのアミノ酸残基に対して何％のアミノ酸残基にデ キストランが結合しているかを示している。 7K90Dの場合、分子量 7,000の PLLは平均 55個のアミノ酸残基があり、 13.4%のアミノ酸残基にデキストランが結合している。す なわち、平均 7.3個のデキストランが PLL—分子に結合していることになる。グラフティ ング率は1 H NMRの測定により算出することができる。 7K90D以外に PLLの分子量力 ¾ 8, 000の化合物やグラフティング率が異なる化合物である 28K90D、 7K70D、 28K70D を合成した。各化合物におけるグラフティング率と、一般式中の xyzの数値を示す。 [0091] The grafting rate indicates what percentage of amino acid residues dextran is bound to all amino acid residues of PLL. In the case of 7K90D, PLL with a molecular weight of 7,000 has an average of 55 amino acid residues, and dextran is bound to 13.4% of amino acid residues. This means that an average of 7.3 dextrans are bound to the PLL—molecule. Graffiti The annealing rate can be calculated by 1 H NMR measurement. In addition to 7K90D, we synthesized 28K90D, 7K70D, and 28K70D, which are compounds with a molecular weight of ¾ 8,000 and compounds with different grafting rates. The graphing rate for each compound and the numerical value of xyz in the general formula are shown.
[0092] [0092]
Figure imgf000038_0001
Figure imgf000038_0001
(式 1)  (Formula 1)
グラフティング率  Grafting rate
7K90D 13.4% x= 6.5, y= 7.3, z=36  7K90D 13.4% x = 6.5, y = 7.3, z = 36
28K90D 9.5% x= 9.5, y=20.8, z=36  28K90D 9.5% x = 9.5, y = 20.8, z = 36
7K70D 5.1% x=18.6, y= 2.8， z=36  7K70D 5.1% x = 18.6, y = 2.8, z = 36
28K70D 3.1% x=31.3, y= 6.8, z=36  28K70D 3.1% x = 31.3, y = 6.8, z = 36
[0093] 塩ィ匕ナトリウム（NaCl、和光純薬工業社製） 2.3376 gと四ほう酸ニナトリウム（Na B 0 [0093] Sodium salt sodium (NaCl, manufactured by Wako Pure Chemical Industries, Ltd.) 2.3376 g and disodium tetraborate (Na B 0
2 4 twenty four
、和光純薬工業社製） 2.0122 gに水を加えて 100mlとした。別に塩化ナトリウム 2.337 6 gとほう酸 (H BO、和光純薬工業社製） 0.6183 gに水を加えて 100 mlとした。この溶 (Manufactured by Wako Pure Chemical Industries, Ltd.) Water was added to 2.0122 g to make 100 ml. Separately, water was added to 2.337 6 g of sodium chloride and 0.6183 g of boric acid (H BO, manufactured by Wako Pure Chemical Industries, Ltd.) to make 100 ml. This solution
3 3  3 3
液に先の溶液を pHが 8.5になるように加え、 0.4 M塩化ナトリウムを含む 0.1 Mほう酸緩 衝液 PH8.5を調製した。  The previous solution was added to the solution so that the pH was 8.5, and 0.1 M boric acid buffer PH8.5 containing 0.4 M sodium chloride was prepared.
28K90Dを合成する時には、上記のほう酸緩衝液 10 mlにポリ -L-リジン臭化水素酸 塩 (PLL' HBr、シグマ社製） 201.8 mgおよびデキストラン (Dextran T、アマシャムバイ ォサイエンス社製） 804.7 mgをカ卩え、 40°Cで 4時間、撹拌した。その後、水 5 mlに水素 化シァノホウ酸ナトリウム（NaBH CN、ナカライテスタ社製） 78.6 mgを溶力 た溶液 1.9  When synthesizing 28K90D, poly-L-lysine hydrobromide (PLL 'HBr, manufactured by Sigma) 201.8 mg and dextran (Dextran T, manufactured by Amersham Biosciences) 804.7 mg Was stirred and stirred at 40 ° C for 4 hours. Then, a solution in which 78.6 mg of sodium cyanoborohydride (NaBH CN, manufactured by Nacalai Testa) was dissolved in 5 ml of water 1.9
3  Three
mlを加え、 40°Cで 5日間、撹拌した。この溶液を透析膜 (分子排除限界 50,000、スぺ クトラムラボラトリーズ社製）を用いて透析後、さらに限外ろ過（分子排除限界 50，000、 東洋濾紙社製）で精製した。得られた溶液を凍結乾燥し、生成物を得た。生成物の 収量は 320.5 mg (収率 34.5%)であった。デキストラン導入率と化学構造は1 H— NMR、 ゲル浸透クロマトグラフ (GPC)および、静的光散乱法による分子量解析によって確認 した。 ml was added and stirred at 40 ° C for 5 days. This solution is dialyzed using a dialysis membrane (molecular exclusion limit 50,000, manufactured by Spectra Laboratories), and then ultrafiltered (molecular exclusion limit 50,000, Purified by Toyo Roshi Kaisha, Ltd. The resulting solution was lyophilized to give the product. The yield of product was 320.5 mg (yield 34.5%). Dextran introduction rate and chemical structure were confirmed by 1 H-NMR, gel permeation chromatography (GPC), and molecular weight analysis by static light scattering.
同様に、分子量 7000(7K)と 28，000(28k)の PLLに、分子量 6,000のデキストランをグラ フト重合させ、 7K90D, 7K70D、 28K70Dを合成した。  Similarly, 7K90D, 7K70D, and 28K70D were synthesized by graft polymerizing 6,000 dextran to a PLL with molecular weights of 7000 (7K) and 28,000 (28k).
[0094] 分子量 7000(7k)と 28,000(28k)の PLLに、分子量 5000のポリエチレングリコールをグ ラフト重合させ、式 2の構造を有する 4種類の担体 7k90P， 28k90P, 7k70P,および 28k 70Pを合成した。各化合物におけるグラフ xyzの数値を示 す。  [0094] Polyethylene glycol with a molecular weight of 5000 was grafted onto a PLL with molecular weights of 7000 (7k) and 28,000 (28k) to synthesize four types of carriers 7k90P, 28k90P, 7k70P, and 28k70P having the structure of Formula 2. . The numerical value of the graph xyz for each compound is shown.
Figure imgf000039_0001
Figure imgf000039_0001
(式 2)  (Formula 2)
7k90P 16.7% x= 5.0, Y= 9.1, ζ=114 7k90P 16.7% x = 5.0, Y = 9.1, ζ = 114
28k90P 22.5% χ= 3.4, Υ=49.7, ζ=114  28k90P 22.5% χ = 3.4, Υ = 49.7, ζ = 114
7k70P 6.3% χ=14.9, Υ= 3.4, ζ=114  7k70P 6.3% χ = 14.9, Υ = 3.4, ζ = 114
28k70P 5.1% χ=18.6, Υ=11.2, ζ=114  28k70P 5.1% χ = 18.6, Υ = 11.2, ζ = 114
[0096] 28Κ90Ρを合成する時には、ポリ- L-リジン臭化水素酸塩 (PLい HBrシグマ社製) 200 .3 mgを水 15 mlに溶解し PLL溶液とした。別にポリエチレングリコール（PEG、 日本油 脂社製） 1.2763 gをァセトニトリル (CH CN、和光純薬工業社製） 73 mlに溶解し PEG  [0096] When synthesizing 28Κ90Ρ, 20.3 mg of poly-L-lysine hydrobromide (PL, manufactured by HBr Sigma) was dissolved in 15 ml of water to obtain a PLL solution. Separately, 1.2763 g of polyethylene glycol (PEG, manufactured by Nippon Oil & Fats Co., Ltd.) is dissolved in 73 ml of acetonitrile (CH CN, manufactured by Wako Pure Chemical Industries, Ltd.).
3  Three
溶液とした。 PLL溶液に PEG溶液を加え 25°Cで 1時間、撹拌した。その後、水 5 mlに 水素化シァノホウ酸ナトリウム（NaBH3CN、ナカライテスタ社製） 314.2 mgを溶力 た 溶液 1.32 mlをカ卩え、 25°Cで 1日間、撹拌した。反応溶液をロータリーエバポレーター で 30 ml程度になるまで濃縮した。その溶液を透析膜 (分子排除限界 50,000、スぺタト ラムラボラトリーズ社製)を用いて透析後、さらに限外ろ過（分子排除限界 50,000、東 洋濾紙社製）で精製した。得られた溶液を凍結乾燥し、生成物を得た。生成物の収 量は 1.1068 g (収率 79.1%)であった。 PEG導入率と構造は1 H_NMR、ゲル浸透クロマ トグラフ (GPC)、および静的光散乱法による分子量解析によって確認した。 It was set as the solution. The PEG solution was added to the PLL solution and stirred at 25 ° C for 1 hour. Thereafter, 1.32 ml of a solution in which 314.2 mg of sodium cyanoborohydride (NaBH3CN, manufactured by Nacalai Testa) was dissolved in 5 ml of water was added and stirred at 25 ° C. for 1 day. Rotary evaporator Concentrate to about 30 ml. The solution was dialyzed using a dialysis membrane (Molecular exclusion limit 50,000, manufactured by Spetatramu Laboratories), and further purified by ultrafiltration (Molecular exclusion limit 50,000, manufactured by Toyo Roshi Kaisha, Ltd.). The resulting solution was lyophilized to give the product. The yield of product was 1.1068 g (yield 79.1%). The PEG introduction rate and structure were confirmed by molecular weight analysis by 1 H NMR, gel permeation chromatography (GPC), and static light scattering.
同様に、分子量 7000(7K)と 28，000(28k)の PLLに、分子量 5、 000の PEGをグラフト重 合させ、 7K90P、 7Κ70Ρ、 28Κ70Ρを合成した。  Similarly, 7K90P, 7Κ70Ρ, and 28Κ70Ρ were synthesized by grafting PEG with molecular weight of 5,000 to PLL with molecular weight of 7000 (7K) and 28,000 (28k).
担体として合成した各化合物の物性は、表 1のとおりである。  Table 1 shows the physical properties of each compound synthesized as a carrier.
1] 1]
Figure imgf000041_0001
Figure imgf000041_0001
. RNaseAに対する安定性の向上  . Improved stability against RNaseA
siRNA (21 mer)としては、以下の配列を用いた。  The following sequences were used as siRNA (21 mer).
guu cag acc acu uca gcu u 3'オーバーハング： dTdT (DNA) (配列番号： 1) aag cug aag ugg ucu gaa c 3，オーバーハング： dTdT (DNA) (配列番号： 2) aag uuc aga cca cuu cag c、 3'オーバーハング： dTdT (DNA) (配列番号： 7) gcu gaa gug guc uga acu u、 3'オーバーハング： dTdT (DNA) (配列番号： 8) また、ブラントエンド dsRNA (27 mer)としては、以下の配列を用いた。 guu cag acc acu uca gcu u 3 'overhang: dTdT (DNA) (SEQ ID NO: 1) aag cug aag ugg ucu gaa c 3, overhang: dTdT (DNA) (SEQ ID NO: 2) aag uuc aga cca cuu cag c, 3 'overhang: dTdT (DNA) (SEQ ID NO: 7) gcu gaa gug guc uga acu u, 3' overhang: dTdT (DNA) (SEQ ID NO: 8) Also, blunt end The following sequences were used as dsRNA (27 mer).
aaa agu uca gac cac uuc age uug aaa (目列 ¾ "亏： 3)  aaa agu uca gac cac uuc age uug aaa (eye sequence ¾ "亏: 3)
uuu caa gcu gaa gug guc uga acu uuu (酉己列番号： 4)  uuu caa gcu gaa gug guc uga acu uuu (酉 Self column number: 4)
[0099] ここで、 21mer siRNA 1 nmolに対して、 N/P比が 2になるように 28K90Pを混合する時 、 28K90Pの使用量は以下のように算出する。 1分子の siRNAに 40個のリン酸基がある ので、 siRNA 1 nmolにリン酸基は 40 nmolある。 N/P比が 2であるため、アミノ基は 80 n mol必要である。従って、 28K90Pの PLL部分の必要重量は 80 nmol X 128 (リジンュニ ットの分子量)で 20.48 x gになる。更に、 28K90Pの必要重量は、 10重量％が PLLであ ること力ゝら、 20.48 μ g÷ 0.1で 204.8 μ gとなる。 [0099] Here, when 28K90P is mixed so that the N / P ratio is 2 with respect to 1merol of 21mer siRNA, the usage amount of 28K90P is calculated as follows. There are 40 phosphate groups in one molecule of siRNA, so there are 40 nmol phosphate groups in 1 nmol of siRNA. Since the N / P ratio is 2, 80 nmol of amino group is required. Therefore, the required weight of the 28K90P PLL part is 20.48 x g at 80 nmol X 128 (molecular weight of lysine unit). Furthermore, the required weight of 28K90P is 20.48 μg ÷ 0.1, which means that 10% by weight of PLL is 204.8 μg.
[0100] リン酸緩衝液に siRNA (21 mer)と 27 merのブラントエンド dsRNAを 20 pmolそれぞれ カロえた。次いで、合成した PLL-PEG (28K90P)を N/P比が 2となるように加えた。ここで N/P比の Nは PLLのァミノ基のモル数を、 Pは siRNAのリン酸基のモル数を表す。更に 、 RNaseA (二ツボンジーン社製)を 1 μ g加え、全量を 50 μ 1にした。 37°C、 30分保温 後、反応溶液にエチレンジァミン四酢酸ニナトリウム、ドデシル硫酸ナトリウムをそれ ぞれ終濃度 10 mM、 1 %となるようにカロえ、フエノール/クロ口ホルム処理にて siRNAを 抽出した。抽出液をポリアクリルアミド電気泳動し、ゲノレを SYBR Gold (Invitrogen社製 )にて染色した。そのゲルをイメージアナライザー（FMBIOII、 HITACHI)で検出した。 電気泳動の結果を図 1に示した。 [0100] 20 pmol each of siRNA (21 mer) and 27 mer blunt-end dsRNA was added to the phosphate buffer. Subsequently, the synthesized PLL-PEG (28K90P) was added so that the N / P ratio was 2. Here, N in the N / P ratio is the number of moles of the amino group of the PLL, and P is the number of moles of the phosphate group of the siRNA. Further, 1 μg of RNase A (Nihon Bon Gene) was added to make the total amount 50 μl. After incubating at 37 ° C for 30 minutes, the reaction solution was calcined with disodium ethylenediamine tetraacetate and sodium dodecyl sulfate to a final concentration of 10 mM and 1%, respectively, and siRNA was extracted by phenol / chloroform form treatment. did. The extract was subjected to polyacrylamide electrophoresis, and the genole was stained with SYBR Gold (Invitrogen). The gel was detected with an image analyzer (FMBIOII, HITACHI). The results of electrophoresis are shown in FIG.
siRNAおよび、 27mer dsRNAは、いずれも 28K90Pが存在しない条件では完全分解 された。し力 ながら、 28K90Pと複合体を形成すると RNaseAに対して高い耐性を示し た。  Both siRNA and 27mer dsRNA were completely degraded in the absence of 28K90P. However, when it was complexed with 28K90P, it was highly resistant to RNaseA.
[0101] 蛍光標識した siRNA 48 pmolと非標識 siRNA 192 pmolを混合した。次レ、で、合成し た PLL-PEG (28K90P)を N/P比が 2となるように加えた。更に、マウスより採血した血漿 を 135 μ ΐ加え、全量を 150 μ ΐにした。 37°C、 48時間保温後、反応溶液にエチレンジ アミン四酢酸ニナトリウム、ドデシノレ硫酸ナトリウムをそれぞれ終濃度 10 mM、 1 %とな るように加え、フエノール/クロ口ホルム処理にて siRNAを抽出した。抽出液をポリアタリ ルアミド電気泳動し、蛍光標識 siRNAをイメージアナライザー（FMBIOII、 HITACHI) で検出した。電気泳動の結果を図 2に示した。 [0101] 48 pmol of fluorescently labeled siRNA and 192 pmol of unlabeled siRNA were mixed. Next, the synthesized PLL-PEG (28K90P) was added so that the N / P ratio was 2. Furthermore, 135 μΐ of plasma collected from mice was added to make the total volume 150 μΐ. After incubating at 37 ° C for 48 hours, disodium ethylenediamine tetraacetate and sodium dodecinole sulfate were added to the reaction solution to a final concentration of 10 mM and 1%, respectively, and siRNA was extracted by phenol / chloroform form treatment. . Extract the extract into polyatari Luamide electrophoresis was performed, and fluorescence-labeled siRNA was detected with an image analyzer (FMBIOII, HITACHI). The results of electrophoresis are shown in FIG.
siRNAおよび、 27mer dsRNAは、いずれも 28K90Pが存在しない条件では血漿中で 分解された。し力 ながら、 28K90Pと複合体を形成すると血漿中でほとんど分解を受 けずに高い安定を示した。  Both siRNA and 27mer dsRNA were degraded in plasma in the absence of 28K90P. However, when it formed a complex with 28K90P, it was highly stable with almost no degradation in plasma.
[0102] 3.担体一 RNA複合体の血液中における滞留性 [0102] 3. Carrier-retention of RNA complex in blood
蛍光標識した siRNA 0.64 nmolと非標識 siRNA 2.56 nmolを混合した。次いで、 1で 合成した各種 PLL_Dex、およびび PLL-PEGを N/P比 4または 8となるように加えた。こ こで N/P比の Nは PLLのァミノ基のモル数を、 Pは siRNAのリン酸基のモル数を表す。 調製した混合液をマウス (ICR、 5週令、雄）の尾静脈より投与し、経時的に眼底採血 した。血液にエチレンジァミン四酢酸ニナトリウム、ドデシル硫酸ナトリウムをそれぞれ 終濃度 10 mM、 1 %となるようにカロえ、フエノール/クロ口ホルム処理にて siRNAを抽出 した。抽出液をポリアクリルアミド電気泳動し、蛍光標識 siRNAをイメージアナライザー (FMBIOII, HITACHI)で検出した。電気泳動の結果を図 3(PLL_Dex)、および図 4(P しし一 PEG)こ した。  Fluorescently labeled siRNA 0.64 nmol and unlabeled siRNA 2.56 nmol were mixed. Next, various PLL_Dex and PLL-PEG synthesized in 1 were added so that the N / P ratio was 4 or 8. Here, N in the N / P ratio is the number of moles of the amino group of the PLL, and P is the number of moles of the phosphate group of the siRNA. The prepared mixture was administered from the tail vein of a mouse (ICR, 5 weeks old, male), and the fundus oculi was collected over time. The blood was treated with disodium ethylenediamine tetraacetate and sodium dodecyl sulfate to a final concentration of 10 mM and 1%, respectively, and siRNA was extracted by phenol / chloroform form treatment. The extract was subjected to polyacrylamide electrophoresis, and fluorescence-labeled siRNA was detected with an image analyzer (FMBIOII, HITACHI). The results of electrophoresis were shown in Fig. 3 (PLL_Dex) and Fig. 4 (P and PEG).
[0103] siRNA単独を静脈内投与したところ、投与後約 5分で血液中から消失した（図 3の na ked)。これに対し、 PLL-Dexまたは PLL-PEGと混合して投与した siRNAは、投与後 30 分、長いものでは 90分まで血液中に滞留していた。また、 N/P比が 4より 8の方力 各 時間における血中 siRNA量が多力つた。  [0103] When siRNA alone was administered intravenously, it disappeared from the blood approximately 5 minutes after administration (naked in Fig. 3). In contrast, siRNA mixed with PLL-Dex or PLL-PEG remained in the blood for 30 minutes after administration and up to 90 minutes at the longest. In addition, the amount of siRNA in the blood in each direction with an N / P ratio of 4 to 8 was high.
図 3と同様の条件で 27mer dsRNAを 28K90Pと混合して、マウスに投与した。その結 果、 2時間以上の高い血中滞留性を示した（図 5)。  Under the same conditions as in FIG. 3, 27mer dsRNA was mixed with 28K90P and administered to mice. As a result, it showed high retention in blood for over 2 hours (Fig. 5).
[0104] 4.遺伝子発現の抑制効果 [0104] 4. Suppression of gene expression
内在性遺伝子 Ubcl3に対する siRNAとしては、以下の配列を用いた。  The following sequences were used as siRNAs against the endogenous gene Ubcl3.
gua cgu uuc aug acc aaa a、 3'オーバーハング： dTdT (DNA) (配列番号： 5) uuu ugg uca uga aac gua c、 3'オーバーハング： dTdT (DNA) (配列番号： 6) 内在性遺伝子 Ubcl3に対する siRNA 6.4 nmolと、滞留性が最も優れていた PLL-PE G (28K90P)を N/P比 4となるように混合し、マウス（ICR、 5週令、雄）の尾静脈より投与 した。 24時間後に肺、肝臓を回収し、 RNeasy Kit (キアゲン)を用いてメーカーのプロト コールに従レ、、 RNAを抽出した。また、コントローノレとして、 Ubcl3の発現に影響を及 ぼさなレ、 Non-silencing (NS) siRNAを用いて同様の処理を行った。定量的 PCRには、 ABI PRISM 7000 Sequence Detection System (A卯 lied Biosystems)を用いた。 Ubcl3 遺伝子および、 /3 -ァクチン遺伝子の RT-PCR用プライマーおよび、 TaqManプローブ を、 Applied Biosystemsより購入した。 RT-PCR反応を QuantiTect Probe RT- PCR Kit (Qiagen)を用いて、そのマニュアルに従って行った。 Ubcl3 mRNAの発現を、 β _ァ クチンの発現量を標準として用いて定量比較した。測定結果を図 6にまとめた。 gua cgu uuc aug acc aaa a, 3 'overhang: dTdT (DNA) (SEQ ID NO: 5) uuu ugg uca uga aac gua c, 3' overhang: dTdT (DNA) (SEQ ID NO: 6) endogenous gene Ubcl3 SiRNA (6.4 nmol) and PLL-PE G (28K90P), which had the best retention, were mixed at an N / P ratio of 4 and administered from the tail vein of mice (ICR, 5 weeks old, male). After 24 hours, collect the lungs and liver and use the RNeasy Kit (Qiagen) Following the call, RNA was extracted. In addition, as a control, the same treatment was performed using a non-silencing (NS) siRNA that did not affect the expression of Ubcl3. ABI PRISM 7000 Sequence Detection System (Alied Biosystems) was used for quantitative PCR. Primers for RT-PCR and TaqMan probe of Ubcl3 gene and / 3-actin gene were purchased from Applied Biosystems. RT-PCR reaction was performed using QuantiTect Probe RT-PCR Kit (Qiagen) according to the manual. The expression of Ubcl3 mRNA was quantitatively compared using the expression level of β_actin as a standard. The measurement results are summarized in FIG.
[0105] NS siRNAを投与したマウスの各組織における遺伝子発現量を 100%とし、 Ubcl3遺 伝子に対する siRNAを投与したマウスでの発現量を求めた。その結果、肝臓におい て約 80%の遺伝子発現の抑制効果が得られた。肺にぉレ、ても 30%程度の抑制効果 が観察された。以上の結果をまとめると、ポリ- L-リジン _g-デキストラン (PLL_Dex)、あ るいはポリ- L-リジン- g-ポリエチレングリコール (PLL-PEG)と siRNAとの混合体は、 siR NAの血中滞留性を飛躍的に上昇させ、特に肝臓での遺伝子発現を強く抑制するこ とが判明した。 [0105] The gene expression level in each tissue of mice administered NS siRNA was taken as 100%, and the expression level in mice administered siRNA against the Ubcl3 gene was determined. As a result, about 80% of the gene expression suppression effect was obtained in the liver. A suppression effect of about 30% was observed in the lungs. To summarize the above results, poly-L-lysine _g-dextran (PLL_Dex) or a mixture of poly-L-lysine-g-polyethylene glycol (PLL-PEG) and siRNA is in siRNA blood. It was found that the retention was drastically increased and gene expression in the liver was strongly suppressed.
[0106] 5. RNAの血液中における滞留性 (担体と別投与した場合）  [0106] 5. Retention of RNA in blood (when administered separately from carrier)
合成した PLL-PEG (28K90P)をマウス（ICR、 5週齢、雄）の尾静注より投与した。 20 分後、蛍光標識した siRNA (配列番号： 1と 2, 7と 8) 0.64nmolと非標識 siRNA (配列番 号： 1と 2， 7と 8) 2.56nmolの混合物を N/P比 4となるように尾静注に投与した。経時的 に眼底採血し、血液にエチレンジァミン四酢酸ニナトリウム、ドデシル硫酸ナトリウム をそれぞれ終濃度 10mM、 1%となるように加え、フエノール/クロ口ホルム処理にて siRN Aを抽出した。抽出液をポリアクリルアミドゲル電気泳動し、蛍光標識 siRNAをイメージ アナライザー（FMBIO II, HITACHI)で検出した。電気泳動の結果を図 7に示した。  Synthesized PLL-PEG (28K90P) was administered from the tail vein of mice (ICR, 5 weeks old, male). After 20 minutes, a mixture of fluorescently labeled siRNA (SEQ ID NOs: 1, 2, 7, and 8) 0.64 nmol and unlabeled siRNA (SEQ ID NOs: 1, 2, 7, and 8) 2.56 nmol with an N / P ratio of 4 As such, it was administered to the tail vein. The ocular fundus was collected over time, and disodium ethylenediamine tetraacetate and sodium dodecyl sulfate were added to the blood to a final concentration of 10 mM and 1%, respectively, and siRNA was extracted by phenol / chloroform form treatment. The extract was subjected to polyacrylamide gel electrophoresis, and fluorescence-labeled siRNA was detected with an image analyzer (FMBIO II, HITACHI). The result of electrophoresis is shown in FIG.
siRNA単独を静脈投与したところ、投与後 5分で血液中から消失した（図 7のレーン ( A))。これに対し、 PLL-PEG (28K90P)を静脈投与した 20分後に siRNA単独を投与し た場合には、投与後 1.5時間、長いもので 2時間まで、血液中に滞留していた（図 7の レーンお)）。  When siRNA alone was administered intravenously, it disappeared from the blood 5 minutes after administration (lane (A) in FIG. 7). In contrast, when siRNA alone was administered 20 minutes after intravenous administration of PLL-PEG (28K90P), it remained in the blood for 1.5 hours after administration and for a maximum of 2 hours (lane in Fig. 7). O)).
[0107] 6.担体-共重合体の結合性評価 [0107] 6. Evaluation of carrier-copolymer bonding
リン酸緩衝液に siRNA (21mer (配列番号： 1と 2, 7と 8) , 27mer (配列番号： 3と 4) 0.2 5pmolをそれぞれ加えた。次いで、合成した PLL-PEG (28K90P， 28K70P)を N/P比 0 〜100となるように加えた。調製した混合液を 37°Cで 30分インキュベートした後、蛍光 異方性を測定した（MF-20, Olympus)。測定結果を図 8に示した。 SiRNA (21mer (SEQ ID NO: 1, 2, 7, and 8), 27mer (SEQ ID NO: 3 and 4)) in phosphate buffer 5 pmol was added respectively. Subsequently, the synthesized PLL-PEG (28K90P, 28K70P) was added so that the N / P ratio was 0 to 100. The prepared mixture was incubated at 37 ° C for 30 minutes, and then the fluorescence anisotropy was measured (MF-20, Olympus). The measurement results are shown in FIG.
[0108] 双方とも、蛍光異方性値力 比の上昇とともに増加し、 PLL-PEGと結合することで 異方性が大きくなることを示した。次に、 PLL_g-PEGのグラフト率の影響を 28k90P， 28 k70Pを比較することで検討した。双方とも、グラフト率が高い 28k90Pでより低い N/P比 力 並進拡散時間が上昇し、 28k70Pよりも 28k90Pの方が siRNAとの結合性が高いこ とが示唆された。 [0108] Both increased with increasing fluorescence anisotropy power ratio, and showed that anisotropy was increased by binding to PLL-PEG. Next, we investigated the effect of the graft ratio of PLL_g-PEG by comparing 28k90P and 28k70P. In both cases, the lower N / P specific translational diffusion time increased at 28k90P, which had a high graft rate, suggesting that 28k90P has higher siRNA binding than 28k70P.
産業上の利用可能性  Industrial applicability
[0109] 本発明によって、 RNAを血液中に投与し、安定に維持することができる。 RNAは、た とえば、 RNAi効果を利用した遺伝子の発現抑制技術に利用することができる。したが つて、遺伝子の発現抑制による、疾患の治療や予防、あるいは遺伝子の機能解析な どに、本発明を利用することができる。治療や予防に利用した場合には、本発明に基 づいて、治療用の RNAを血液中に投与し、その血液中における安定性を向上させる こと力 Sできる。あるいは、遺伝子の機能解析においては、機能を解析すべき遺伝子の 発現を抑制しうる siRNAを生体中に投与し、発現抑制に伴う表現型の変化を知ること ができる。生体に対して、効果的な遺伝子発現抑制を実現できるので、生体の生理 的な変化や、実際の疾患モデル動物に対する影響を知ることができる。このような知 見は、培養細胞における遺伝子発現抑制では得ることができない。 [0109] According to the present invention, RNA can be administered into blood and stably maintained. For example, RNA can be used in gene expression suppression technology using the RNAi effect. Therefore, the present invention can be used for treatment or prevention of diseases or gene function analysis by suppressing gene expression. When used for treatment or prevention, the therapeutic RNA can be administered into blood based on the present invention, and the stability in blood can be improved. Alternatively, in gene function analysis, siRNA capable of suppressing the expression of a gene whose function is to be analyzed can be administered into a living body, and phenotypic changes associated with expression suppression can be known. Since effective gene expression suppression can be realized in the living body, it is possible to know physiological changes in the living body and the effects on actual disease model animals. Such knowledge cannot be obtained by suppressing gene expression in cultured cells.
また、本発明の方法によれば、 RNAを担体と別々に投与することができる。したがつ て、担体と RNAを別々に製剤化する等、製剤過程の簡便化を可能とすることができる  Moreover, according to the method of the present invention, RNA can be administered separately from the carrier. Therefore, it is possible to simplify the formulation process, such as formulating the carrier and RNA separately.

Claims

請求の範囲  The scope of the claims
[I] ポリカチオン性化合物に対して櫛型に結合している親水性基を側鎖として有する担 体と、 RNAとの複合体を含む、 RNAを血液中に投与するための組成物。  [I] A composition for administering RNA into blood, comprising a complex of a carrier having a hydrophilic group bonded in a comb shape to a polycationic compound as a side chain and RNA.
[2] RNAと併用することを特徴とする、ポリカチオン性化合物に対して櫛型に結合してい る親水性基を側鎖として有する担体を有効成分として含有する、 RNAを血液中で安 定化するための組成物。  [2] characterized in that it is used in combination with RNA, comprising as an active ingredient a carrier having a hydrophilic group bonded in a comb shape to a polycationic compound as a side chain, RNA is stable in blood A composition for converting to
[3] 担体が RNAと同時または、 RNAより前に投与されることを特徴とする、請求項 2に記 載の組成物。  [3] The composition according to claim 2, wherein the carrier is administered simultaneously with RNA or before RNA.
[4] ポリカチオン性化合物がポリ（カチオン性アミノ酸）である、請求項 1または 2に記載 の組成物。  [4] The composition according to claim 1 or 2, wherein the polycationic compound is poly (cationic amino acid).
[5] ポリ（カチオン性アミノ酸）力 ポリ（リジン)である請求項 4に記載の組成物。  5. The composition according to claim 4, wherein the composition is poly (cationic amino acid) force poly (lysine).
[6] 親水性基が、グリコサミノダリカン、デキストラン、ポリエチレングリコール、ポリエチレ ングリコール誘導体、及び糖類からなる群から選択される少なくとも 1つである請求項 [6] The hydrophilic group is at least one selected from the group consisting of glycosaminodarlican, dextran, polyethylene glycol, polyethylene glycol derivatives, and saccharides.
1または 2に記載の,袓成物。 The product according to 1 or 2.
[7] ポリカチオンと親水性基とが、グラフト重合している請求項 1または 2に記載の組成 物。 [7] The composition according to claim 1 or 2, wherein the polycation and the hydrophilic group are graft-polymerized.
[8] 担体の 10重量％〜99重量％が親水性基である請求項 1または 2に記載の組成物  8. The composition according to claim 1 or 2, wherein 10% to 99% by weight of the carrier is a hydrophilic group.
[9] ポリ（カチオン性アミノ酸）を構成するカチオン性アミノ酸基の数 (N)と、担体と複合 体を形成する RNAに含まれるリン酸基の数（P)との比（N/P比）が、 0. 5〜40の範 囲である、請求項 4に記載の組成物。 [9] Ratio (N / P ratio) of the number of cationic amino acid groups (N) constituting the poly (cationic amino acid) and the number of phosphate groups (P) contained in the RNA that forms a complex with the carrier 5. The composition according to claim 4, wherein is in the range of 0.5-40.
[10] 前記ポリ（カチオン性アミノ酸）を構成するカチオン性アミノ酸基の数 (N)と、担体と 複合体を形成する RNAに含まれるリン酸基の数 (P)との比（N/P比） 、 2〜20であ る請求項 9に記載の組成物。 [10] Ratio (N / P) of the number of cationic amino acid groups (N) constituting the poly (cationic amino acid) and the number of phosphate groups (P) contained in RNA forming a complex with the carrier 10. The composition according to claim 9, wherein the ratio is 2-20.
[I I] RNAが機能性 RNAである請求項 1または 2に記載の組成物。  [I I] The composition according to claim 1 or 2, wherein the RNA is a functional RNA.
[12] 機能性 RNA力 NAi効果を有する RNAである請求項 11に記載の組成物。  [12] The composition according to claim 11, which is RNA having NAi effect.
[13] ポリカチオン性化合物に対して櫛型に結合している親水性基を側鎖として有する担 体と RNAとの複合体を形成させる工程を含む、 RNAを血液中で安定化する方法。 [13] A method for stabilizing RNA in blood, comprising a step of forming a complex of RNA and a carrier having a hydrophilic group bonded in a comb shape to a polycationic compound as a side chain.
[14] 以下の工程を含む、 RNAを血液中で安定化する方法。 [14] A method for stabilizing RNA in blood, comprising the following steps.
( 1 )ポリカチオン性化合物に対して櫛型に結合している親水性基を側鎖として有す る担体を、血液中に投与する工程  (1) A step of administering into the blood a carrier having a hydrophilic group bonded in a comb shape as a side chain to a polycationic compound.
(2) RNAを血液中に投与する工程  (2) The process of administering RNA into blood
[15] RNAを投与する工程が、担体を投与する工程の後または同時に行われることを特 徴とする、請求項 14に記載の方法。  [15] The method according to claim 14, wherein the step of administering RNA is performed after or simultaneously with the step of administering a carrier.
[16] ポリカチオン性化合物力 ポリ（カチオン性アミノ酸）である、請求項 13または 14に 記載の方法。 [16] The method according to claim 13 or 14, wherein the compound is a poly (cationic amino acid).
[17] 前記ポリ（カチオン性アミノ酸）を構成するカチオン性アミノ酸基の数 (N)と、前記担 体と複合体を形成するべき核酸に含まれるリン酸基の数 (P)との比 (N/P比）力 0. [17] Ratio of the number of cationic amino acid groups (N) constituting the poly (cationic amino acid) to the number of phosphate groups (P) contained in the nucleic acid to form a complex with the carrier (P) N / P ratio) force 0.
5〜40の範囲であることを特徴とする、請求項 16に記載の方法。 The method according to claim 16, characterized in that it is in the range of 5-40.
[18] 前記ポリ（カチオン性アミノ酸）を構成するカチオン性アミノ酸基の数 (N)と、担体と 複合体を形成する RNAに含まれるリン酸基の数 (P)との比（N/P比） 、 2〜20であ る請求項 17に記載の方法。 [18] Ratio of the number of cationic amino acid groups constituting the poly (cationic amino acid) (N) to the number of phosphate groups (P) contained in RNA forming a complex with the carrier (N / P The method according to claim 17, wherein the ratio is 2-20.
[19] ポリカチオン性化合物に対して櫛型に結合している親水性基を側鎖として有する担 体を RNAと混合する工程を含む、 RNAを血液中に投与するための組成物の製造方 法。 [19] A method for producing a composition for administering RNA into blood, comprising a step of mixing a carrier having, as a side chain, a hydrophilic group bonded in a comb shape to a polycationic compound with RNA. Law.
[20] ポリカチオン性化合物力 ポリ（カチオン性アミノ酸）である請求項 19に記載の製造 方法。  [20] The method according to [19], wherein the polycationic compound power is poly (cationic amino acid).
[21] ポリカチオン性化合物に対して櫛型に結合している親水性基を側鎖として有する担 体と、 RNAとの複合体を含む、 RNAの腎臓からの***を抑制するための組成物。  [21] A composition for inhibiting excretion of RNA from the kidney, comprising a complex of a carrier having, as a side chain, a hydrophilic group bonded in a comb shape to a polycationic compound, and RNA .
[22] RNAと併用することを特徴とする、ポリカチオン性化合物に対して櫛型に結合してい る親水性基を側鎖として有する担体を有効成分として含有する、 RNAの腎臓からの ***を抑制するための組成物。 [22] It is used in combination with RNA, and contains as an active ingredient a carrier having a hydrophilic group as a side chain bonded in a comb shape to a polycationic compound, and excretion of RNA from the kidney A composition for inhibiting.
[23] 担体が RNAと同時または、 RNAより前に投与されることを特徴とする、請求項 22に 記載の組成物。 [23] The composition according to claim 22, wherein the carrier is administered simultaneously with RNA or before RNA.
[24] ポリカチオン性化合物力 ポリ（カチオン性アミノ酸）である請求項 21または 22に記 載の組成物。 [24] The composition according to claim 21 or 22, wherein the composition is a poly (cationic amino acid).
[25] ポリカチオン性化合物に対して櫛型に結合している親水性基を側鎖として有する担 体と RNAとの複合体を形成させる工程と、得られた複合体を血液中に投与する工程 を含む、血液中に投与された RNAの腎臓からの***を抑制する方法。 [25] A step of forming a complex of RNA and a carrier having a hydrophilic group bonded in a comb shape to the polycationic compound as a side chain, and administering the obtained complex into blood A method for suppressing excretion of RNA administered into blood from the kidney, comprising a step.
[26] 以下の工程を含む、血液中に投与された RNAの腎臓からの***を抑制する方法。  [26] A method for suppressing excretion of RNA administered into blood from the kidney, comprising the following steps.
( 1 )ポリカチオン性化合物に対して櫛型に結合している親水性基を側鎖として有す る担体を、血液中に投与する工程  (1) A step of administering into the blood a carrier having a hydrophilic group bonded in a comb shape as a side chain to a polycationic compound.
(2) RNAを血液中に投与する工程  (2) The process of administering RNA into blood
[27] RNAを投与する工程力 担体を投与する工程の後または同時に行われることを特 徴とする、請求項 26に記載の方法。  [27] Process capability of administering RNA The method according to claim 26, which is performed after or simultaneously with the process of administering a carrier.
[28] ポリカチオン性化合物力 ポリ（カチオン性アミノ酸）である請求項 25または 26に記 載の方法。 [28] The method according to claim 25 or 26, wherein the compound is a poly (cationic amino acid).
[29] ポリカチオン性化合物に対して櫛型に結合している親水性基を側鎖として有する担 体と、 RNAとの複合体を含む、 RNAのヌクレアーゼによる分解を抑制するための組成 物。  [29] A composition for suppressing degradation of RNA by a nuclease, comprising a complex of a carrier having a hydrophilic group bonded in a comb shape to a polycationic compound as a side chain and RNA.
[30] RNAと併用することを特徴とする、ポリカチオン性化合物に対して櫛型に結合してい る親水性基を側鎖として有する担体を有効成分として含有する、 RNAのヌクレアーゼ による分解を抑制するための組成物。  [30] Suppresses degradation of RNA by nuclease, containing as an active ingredient a carrier having a hydrophilic group as a side chain bonded to a polycationic compound in a comb shape, characterized by being used in combination with RNA Composition to do.
[31] 担体が RNAと同時または、 RNAより前に投与されることを特徴とする、請求項 30に 記載の組成物。  [31] The composition according to claim 30, wherein the carrier is administered simultaneously with RNA or before RNA.
[32] ポリカチオン性化合物力 ポリ（カチオン性アミノ酸）である請求項 29または 30に記 載の組成物。  [32] The composition according to claim 29 or 30, wherein the composition is a poly (cationic amino acid).
[33] ポリカチオン性化合物に対して櫛型に結合している親水性基を側鎖として有する担 体と RNAとの複合体を形成させる工程と、得られた複合体を血液中に投与する工程 を含む、血液中に投与された RNAのヌクレアーゼによる分解を抑制する方法。  [33] A step of forming a complex of RNA and a carrier having a hydrophilic group bonded in a comb shape to a polycationic compound as a side chain, and administering the obtained complex into blood A method for suppressing degradation of RNA administered into blood by nuclease, comprising a step.
[34] 以下の工程を含む、血液中に投与された RNAのヌクレアーゼによる分解を抑制す る方法。  [34] A method for suppressing degradation of RNA administered into blood by a nuclease, comprising the following steps.
( 1 )ポリカチオン性化合物に対して櫛型に結合している親水性基を側鎖として有す る担体を、血液中に投与する工程 (2) RNAを血液中に投与する工程 (1) A step of administering into the blood a carrier having a hydrophilic group bonded in a comb shape as a side chain to a polycationic compound. (2) The process of administering RNA into blood
[35] RNAを投与する工程が、担体を投与する工程の後または同時に行われることを特 徴とする、請求項 34に記載の方法。 [35] The method according to claim 34, wherein the step of administering RNA is performed after or simultaneously with the step of administering a carrier.
[36] ポリカチオン性化合物力 ポリ（カチオン性アミノ酸）である請求項 33または 34に記 載の方法。 [36] The method according to claim 33 or 34, wherein the compound is a poly (cationic amino acid).
[37] 次の工程を含む、遺伝子の機能解析方法；  [37] A method for analyzing the function of a gene, comprising the following steps:
(1)ポリカチオン性化合物に対して櫛型に結合している親水性基を側鎖として有す る担体と、機能解析の対象遺伝子に相補的な塩基配列を含む 2本鎖 RNAとの複合 体を形成させる工程；  (1) A composite of a carrier having a hydrophilic group as a side chain bonded in a comb shape to a polycationic compound and a double-stranded RNA containing a base sequence complementary to the target gene for functional analysis Forming a body;
(2) (1)の RNA—担体複合体を非ヒト動物の血液中に投与する工程；  (2) A step of administering the RNA-carrier complex of (1) into the blood of a non-human animal;
(3) (1)の RNA—担体複合体を投与された非ヒト動物の表現型を観察し対照と比較す る工程；および  (3) observing the phenotype of the non-human animal administered with the RNA-carrier complex of (1) and comparing it with a control; and
(4)対照と比較して表現型の相違が検出されたときに、前記遺伝子の機能抑制に起 因する表現型が同定される工程。  (4) A step of identifying a phenotype caused by functional suppression of the gene when a phenotypic difference is detected as compared with a control.
[38] 次の工程を含む、遺伝子の機能解析方法；  [38] A method for analyzing the function of a gene, comprising the following steps:
(1)ポリカチオン性化合物に対して櫛型に結合している親水性基を側鎖として有す る担体を非ヒト動物の血液中に投与する工程；  (1) A step of administering a carrier having a hydrophilic group bonded in a comb shape as a side chain to a polycationic compound into the blood of a non-human animal;
(2)機能解析の対象遺伝子に相補的な塩基配列を含む 2本鎖 RNAを非ヒト動物の 血液中に投与する工程；  (2) a step of administering a double-stranded RNA containing a base sequence complementary to a target gene for functional analysis into the blood of a non-human animal;
(3) (1)の担体および (2)の RNAを投与された非ヒト動物の表現型を観察し対照と比較 する工程；および  (3) observing the phenotype of the non-human animal administered the carrier of (1) and (2) RNA and comparing it to a control; and
(4)対照と比較して表現型の相違が検出されたときに、前記遺伝子の機能抑制に起 因する表現型が同定される工程。  (4) A step of identifying a phenotype caused by functional suppression of the gene when a phenotypic difference is detected as compared with a control.
[39] RNAを投与する工程が、担体を投与する工程の後または同時に行われることを特 徴とする、請求項 38に記載の方法。  [39] The method according to claim 38, wherein the step of administering RNA is performed after or simultaneously with the step of administering a carrier.
[40] ポリカチオン性化合物力 ポリ（カチオン性アミノ酸）である請求項 37または 38に記 載の方法。 [40] The method according to claim 37 or 38, wherein the compound is a poly (cationic amino acid).
[41] ポリ（カチオン性アミノ酸）を主鎖とし、かつ前記ポリ（カチオン性アミノ酸）に対して 櫛型に結合している親水性基を側鎖として有する担体と、 RNAとの複合体からなり、 前記ポリ（カチオン性アミノ酸）を構成するカチオン性アミノ酸基の数 (N)と、担体と複 合体を形成する RNAに含まれるリン酸基の数 (P)との比（N/P比）が 2〜20である複 合体。 [41] poly (cationic amino acid) as a main chain and with respect to the poly (cationic amino acid) Composed of a complex of RNA and a carrier having a hydrophilic group bonded in a comb shape as a side chain, the number of cationic amino acid groups (N) constituting the poly (cationic amino acid), a carrier and a complex. A complex having a ratio (N / P ratio) of 2 to 20 with the number of phosphate groups (P) contained in RNA forming the complex.
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