WO2006123631A1 - Composition contenant de l'arn - Google Patents

Composition contenant de l'arn 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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English (en)
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 NUC
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Kyushu University NUC
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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 nucleic acids [NA]
    • 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.

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Abstract

L'invention porte sur une composition destinée à être utilisée pour introduire de l'ARN dans le sang et qui comprend un complexe d'un excipient et d'ARN. L'excipient comprend un composé polycationique possédant, comme chaîne latérale, un groupe hydrophile lié au composé polycationique dans une configuration de type peigne. Le composé polycationique est, de préférence, un amino-acide polycationique. Lors de l'utilisation de la composition, l'excrétion de l'ARN du rein peut être inhibée, la rétention de l'ARN se trouvant ainsi améliorée. Par exemple, on peut traiter le cancer en introduisant l'ARNsi, qui a un effet thérapeutique sur le cancer, dans le sang en utilisant la composition. D'autre part, il s'est avéré que la rétention de l'ARN dans le sang peut également être améliorée par administration séparée de l'excipient et de l'ARN. L'excipient peut-être, par exemple, PLL-Dex représenté par la formule (1) ou PLL-PEG représenté par la formule (2).
PCT/JP2006/309699 2005-05-16 2006-05-16 Composition contenant de l'arn Ceased WO2006123631A1 (fr)

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WO2011010714A1 (fr) * 2009-07-23 2011-01-27 国立大学法人東京大学 Polymère anionique, complexe polyions utilisant un polymère anionique, composite de polymère ternaire, et composition pharmaceutique
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WO2018234564A1 (fr) * 2017-06-22 2018-12-27 Paris Sciences Et Lettres - Quartier Latin Copolymère de type peigne clivable par oxydo-réduction pour une adhérence régulée entre des cellules et des substrats
WO2019044937A1 (fr) * 2017-08-31 2019-03-07 国立大学法人 東京大学 Complexe polyionique unitaire chargé d'acide nucléique
EP3766519A4 (fr) * 2017-10-05 2021-12-01 Kawasaki Institute of Industrial Promotion Composition régulant la pharmacocinétique dans le corps

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JP2010233499A (ja) * 2009-03-31 2010-10-21 Univ Of Tokyo 二本鎖リボ核酸ポリイオンコンプレックス
CN102449152A (zh) * 2009-03-31 2012-05-09 国立大学法人东京大学 双链核糖核酸的聚离子复合物
US8668933B2 (en) 2009-03-31 2014-03-11 The University Of Tokyo Polyion complex of double-stranded ribonucleic acid
WO2010114013A1 (fr) * 2009-03-31 2010-10-07 国立大学法人 東京大学 Complexe polyionique d'acide ribonucléique double brin
WO2011010714A1 (fr) * 2009-07-23 2011-01-27 国立大学法人東京大学 Polymère anionique, complexe polyions utilisant un polymère anionique, composite de polymère ternaire, et composition pharmaceutique
JPWO2011010714A1 (ja) * 2009-07-23 2013-01-07 国立大学法人 東京大学 アニオン性ポリマー、該アニオン性ポリマーを用いたポリイオンコンプレックスおよび三元系ポリマー複合体、ならびに薬学組成物
US8450282B2 (en) 2009-07-23 2013-05-28 The University Of Tokyo Anionic polymer, polyion complex and ternary polymer composite using anionic polymer, and pharmaceutical composition
JPWO2018216792A1 (ja) * 2017-05-26 2020-03-26 公益財団法人川崎市産業振興財団 血中におけるｒｎａの安定性の改善剤および投与方法
WO2018216792A1 (fr) * 2017-05-26 2018-11-29 公益財団法人川崎市産業振興財団 Agent permettant d'améliorer la stabilité de l'arn dans le sang et son procédé d'administration
JP7237825B2 (ja) 2017-05-26 2023-03-13 公益財団法人川崎市産業振興財団 血中におけるｒｎａの安定性の改善剤および投与方法
WO2018234564A1 (fr) * 2017-06-22 2018-12-27 Paris Sciences Et Lettres - Quartier Latin Copolymère de type peigne clivable par oxydo-réduction pour une adhérence régulée entre des cellules et des substrats
CN111050752A (zh) * 2017-08-31 2020-04-21 国立大学法人东京大学 搭载核酸的单元型聚离子复合物
US11324835B2 (en) 2017-08-31 2022-05-10 Kawasaki Institute Of Industrial Promotion Nucleic acid-loaded unit polyion complex
WO2019044937A1 (fr) * 2017-08-31 2019-03-07 国立大学法人 東京大学 Complexe polyionique unitaire chargé d'acide nucléique
EP3766519A4 (fr) * 2017-10-05 2021-12-01 Kawasaki Institute of Industrial Promotion Composition régulant la pharmacocinétique dans le corps
US11957708B2 (en) 2017-10-05 2024-04-16 Kawasaki Institute Of Industrial Promotion Composition controlling pharmacokinetics in the body
US12582669B2 (en) 2017-10-05 2026-03-24 Kawasaki Institute Of Industrial Promotion Composition controlling pharmacokinetics in the body

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