WO2005078084A1 - Acide oligonucléique double brin portant un complexe de polyion, procédé pour produire celui-ci et composition pharmaceutique contenant celui-ci - Google Patents

Acide oligonucléique double brin portant un complexe de polyion, procédé pour produire celui-ci et composition pharmaceutique contenant celui-ci Download PDF

Info

Publication number
WO2005078084A1
WO2005078084A1 PCT/JP2004/011957 JP2004011957W WO2005078084A1 WO 2005078084 A1 WO2005078084 A1 WO 2005078084A1 JP 2004011957 W JP2004011957 W JP 2004011957W WO 2005078084 A1 WO2005078084 A1 WO 2005078084A1
Authority
WO
WIPO (PCT)
Prior art keywords
group
polyion complex
complex according
integer
peg
Prior art date
Application number
PCT/JP2004/011957
Other languages
English (en)
Japanese (ja)
Inventor
Kazunori Kataoka
Keiji Itaka
Shigeto Fukushima
Naoki Kanayama
Original Assignee
Toudai Tlo, Ltd.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Toudai Tlo, Ltd. filed Critical Toudai Tlo, Ltd.
Priority to JP2005517894A priority Critical patent/JPWO2005078084A1/ja
Publication of WO2005078084A1 publication Critical patent/WO2005078084A1/fr

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
    • C08G73/00—Macromolecular compounds obtained by reactions forming a linkage containing nitrogen with or without oxygen or carbon in the main chain of the macromolecule, not provided for in groups C08G12/00 - C08G71/00
    • C08G73/02—Polyamines
    • C08G73/028—Polyamidoamines
    • 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
    • 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
    • A61K47/69—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 conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6905—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 conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a colloid or an emulsion
    • A61K47/6907—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 conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a colloid or an emulsion the form being a microemulsion, nanoemulsion or micelle
    • 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
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy

Definitions

  • the present invention relates to a double-stranded oligo nucleic acid delivery system for gene knockdown.
  • RNA interference small double-stranded RNA molecules
  • RISC RNA-induced silencing complex
  • RNA DNA hybrid oligonucleic acids As a nucleic acid molecule that causes RNAi, in addition to siRNA, short hairpin RNA (shRNA), which is an RNA molecule with a hairpin structure, has the same effect, and gene knockdown is limited to RNA molecules like conventional antisense DNA And double-stranded oligo DNA (dsODN), The possibility that RNA DNA hybrid oligonucleic acids are also effective is being investigated. As a phenomenon similar to RNAi, several hundred small endogenous small RNA families have been discovered in various cells, and are involved in a wide range of life phenomena through time and site-specific gene expression control. It has become clear.
  • microRNA microRNA
  • the double-stranded oligonucleic acid is directly administered to cells and tissues, it cannot be stably present due to (i) nucleolytic enzymes (nucleases) in the living body or cells, and (ii) the nucleic acid itself has a negative charge. For this reason, the function of the double-stranded oligonucleic acid cannot be sufficiently exhibited by the reason that cell uptake hardly occurs.
  • siRNA which is one of double-stranded oligonucleic acids
  • a cationic transfection reagent based on cationic lipid such as Oligofectamine (Invitrogen) or RNAiFect (QIAGEN).
  • Oligofectamine Invitrogen
  • RNAiFect QIAGEN
  • siRNA delivery based on these lipids is susceptible to serum components and proteins, and such a system is not practical when RNAi is considered for clinical applications. Therefore, there is an urgent need to develop a delivery system that can stably deliver double-stranded oligonucleic acids such as siRNA to target cells and tissues without losing their functions.
  • An object of the present invention is to provide a delivery system useful for delivering a double-stranded oligonucleic acid functioning in gene knockdown to cells or tissues.
  • the present inventors have conducted intensive studies to solve the above problems, and as a result, have found that efficient delivery can be achieved by employing a polyion complex for delivery of a double-stranded oligonucleic acid to cells and tissues,
  • the present invention has been completed. That is, the present invention is as follows.
  • a polyion complex comprising a block copolymer comprising a non-chargeable segment and a chargeable segment, and a double-stranded oligonucleic acid.
  • R 1 a and R lb each independently represents a hydrogen atom or an unsubstituted or substituted linear or branched C physician 12 alkyl group
  • a L 1 and L 2 are linking groups
  • R 2a, R 2b, R 2 ⁇ : and R 2d represents a methylene group or Echire emissions groups independently
  • R 3 represents a hydrogen atom, a protective group, hydrophobic group or polymerizable group
  • R 4 Is the same as R or R 5d or is an initiator residue
  • R 5a , R 5b , R 5e and R 5d are each independently a hydroxyl group, an oxybenzyl group, one NH— (CH 2 ) a —X group
  • X is independently an amine compound residue containing one or more of primary, secondary, tertiary amines or quaternary ammonium salts, or a compound residue that is not an amine.
  • a is an integer of 1 to 5
  • "and! 61> are each independently a hydrogen atom or a protecting group, wherein the protecting group is usually used as a protecting group for an amino group.
  • Z Boc, acetyl, trifluoroacetyl, etc., where m is an integer of 5 to 20,000 Yes, n is an integer from 2 to 5,000, y is an integer from 0 to 5,000, z is an integer from 0 to 5,000, but y + z shall not be greater than n )
  • R 1 a or a linear or branched C 2 alkyl group R lb is substituted, wherein the substituents ⁇ Se evening Ichiru formyl group, Shiano group, a formyl group, a carboxyl group, an amino group , C alkoxycarbonyl group, C 2 _ 7 Ashiruami de groups, the same or properly different tri - a C i-6 alkyl siloxy group, a siloxy group, or silylamino group (5) polyion complex according.
  • L 1 is one (CH 2 ) b — NH—, where b is;
  • R 2 a, R 2b, R 2 c and R 2d is a methylene group (5) to (9) Noise Re polyion complex according to any.
  • R 7 a, R 7b and R 7 e each independently Represents a hydrogen atom or a methyl group
  • dl, d2 and d3 each independently represent an integer of 1 to 5
  • el, e2 and e3 each independently represent an integer of 1 to 5
  • f represents an integer of 0 to 15;
  • R 8 a and R 8 b are each independently a hydrogen atom or a protecting group, wherein the protecting group Z which is used as a protecting group of the normal amino group and group, B oc group, Asechiru group, tri Furuoroasechiru group And g represents an integer of 0 to 15. )
  • R 5a and R 5b is one NH— (CH 2 ) a — (NR 7 a (CH 2 ) d1 ) e1 — NHR 8a (5), (6), ( (7) The polyion complex according to any one of (8), (10) and (11).
  • (1 3) 85% or more of the total of R 5c and R 5d is — NH_ (CH 2 ) a — (NR 7a (CH 2 ) d1 ) e1 — NHR 8a (5), (6), (7) , (9), the polyion complex according to any one of (10) and (11).
  • R 5a and R 5b At least 85% of the total of R 5a and R 5b is —NH— (CH 2 ) 3 — NH— (CH 2 ) 3 — NH 2 (5), (6), (7), (8 ), The polyion complex according to any one of (10) and (11).
  • R 3 is Asechiru group, Akuriroiru group or a methacryloyl group (5), (6), (7), (8), (1 0), (1 1), (1 2), (14 ) And the polyion complex according to any one of (16).
  • R 4 is - is NH- R 9, wherein R 9 is Chokukusarima other which is unsubstituted or substituted branched (: Bok ⁇ represents an alkyl group (5), (6), (7)
  • Bok ⁇ represents an alkyl group (5), (6), (7)
  • the polyion complex according to any one of (9), (10), (11), (13), (15) and (16).
  • Figure 1 shows the PEG-PLL / dsODN, PEG.DPT / dsODN, and PEG-DPT / siRNA components.
  • FIG. 3 shows the rex formation by electrophoresis.
  • FIG. 2 is a view showing the results of measuring the particle size of PEG-PLL / dsODN complex and PEG-DPT / dsODN complex by dynamic light scattering measurement.
  • FIG. 3 is a graph showing the effect of suppressing luciferase gene expression using a PEG-PLL / siRNA complex.
  • FIG. 4 is a view showing the PEG-DET / siRNA complex, the PEG-DPT / siRNA complex, and the effect of suppressing luciferase gene expression.
  • FIG. 5 is a diagram showing the luciferase gene expression-suppressing effect of siRNA-loaded polyion complex after serum incubation.
  • FIG. 6 shows the knockdown effect of the endogenous gene LaminA / C by the siRNA-supporting polyion complex.
  • FIG. 7 is a diagram showing the knockdown effect of the endogenous gene ATX by the siRNA-supporting polyion complex.
  • the present invention relates to an electrostatically-coupled polymer (polymer) capable of suppressing gene expression in a target tissue or cell by forming a nucleic acid that causes gene knockdown with a block copolymer to form a polyion complex. It relates to a micelle pharmaceutical composition.
  • the block copolymer used in the present invention is composed of a non-chargeable segment and a chargeable segment, and the chargeable segment can form a polyion complex with the nucleic acid for gene knockdown.
  • non-chargeable segment examples include polyethylene glycol (PEG), polyalkylene glycol such as polypropylene glycol, polyalkylene oxide, polysaccharide, polyacrylamide, polysubstituted acrylamide, polyacrylamide, polysubstituted acrylamide, and polyvinylpyrrolidone.
  • PEG polyethylene glycol
  • polyalkylene glycol such as polypropylene glycol, polyalkylene oxide
  • polysaccharide polyacrylamide, polysubstituted acrylamide, polyacrylamide, polysubstituted acrylamide
  • polyvinylpyrrolidone examples include various segments derived from acrylates, polymer acrylates, or derivatives thereof.
  • Preferred molecular weights of the uncharged segments are from 200 to 100,000, more preferably from 500 to 200,000, particularly preferably from 1,000 to 50,000.
  • the chargeable segment examples include a polyamino acid or a derivative thereof or a salt thereof, and more specifically, a polyamino acid such as polylysine (PLL), polyarginine, or polyhistidine; or an ester of polyaspartic acid or polyglutamic acid. Or those having a chargeable group in the side chain of the acid amide. In addition, esters of polyacrylic acid, polymethacrylic acid and the like or those having a chargeable group in the side chain of acid amide can also be mentioned. Preferred molecular weights of the charged segments are from 200 to 1,000,000, more preferably from 500 to 200,000, particularly preferably from 1,000 to 50,000.
  • each of 13 and 1 ⁇ 111 independently represents a hydrogen atom or an unsubstituted or substituted linear or branched C i-i 2 alkyl group
  • 12 alkyl include methyl, ethyl, n-propyl, iso-propyl, n-butyl, sec-butyl, tert-butyl, n-pentyl, n-hexyl, decyl, pendecyl and the like.
  • the Asetaru formyl group as a substituent when it is substituted Shiano group, formyl group, carboxyl group, amino groups, C I 6 alkoxycarbonyl groups, C 2 - 7 Ashiruami de group, same or lay Is a different tree C ⁇ 6 alkylsiloxy group, siloxy group or silylamino group.
  • the substituent is an acetalized formyl group, it can be hydrolyzed under acidic and mild conditions to convert to another formyl group (_CH ⁇ : or aldehyde group).
  • Such a formyl group, or a carboxyl group or an amino group can be present, for example, in the shell of a polyion complex micelle of the copolymer according to the present invention and a nucleic acid or an anionic protein, and via these groups.
  • a formyl group, or a carboxyl group or an amino group can be present, for example, in the shell of a polyion complex micelle of the copolymer according to the present invention and a nucleic acid or an anionic protein, and via these groups.
  • F (ab ') 2, F (ab), etc. an antibody or a fragment thereof having specific binding properties (F (ab ') 2, F (ab), etc.) and other proteins capable of conferring the micelles with a function or targeting property.
  • a PEG segment having such a functional group at one end is, for example, a block described in WO96 / 32243, WO966 / 332233, WO97 / 06202. It can be conveniently formed
  • the PEG segment portion and the poly (amino acid or derivative thereof) segment portion formed in this manner can take any linking mode according to the method for producing the copolymer of the above general formula (I) or (II). However, they may be linked by any linking group as long as the object of the present invention is met.
  • the method for producing the above copolymer is not particularly limited, one method is, for example, using a PEG derivative having an amino group at a terminal, and adding (3-benzyl-L-aspartate) to the amino terminal.
  • ⁇ -Z_L-lysine and other protected amino acids such as carboxylic anhydride (NCA) are polymerized to synthesize a block copolymer, and then the side chain is converted to obtain the copolymer of the present invention.
  • the structure of the copolymer is represented by the general formula (I), and the linking group L 1 has a force that becomes a structure derived from the terminal structure of the used PEG derivative or the like, preferably — (CH 2 ) b — NH—, and b is an integer of 1 to 5.
  • the copolymer of the present invention can be produced by a method of synthesizing a poly (amino acid or a derivative thereof) segment portion and then bonding the segment to a PEG segment portion.
  • the structure may be the same as that of the compound, but may be the structure of general formula (II).
  • the linking group L 2 is not particularly limited. Is preferably — (CH 2 ) c —CO—, and c is an integer of 1 to 5.
  • R 5a , R 5b , R 5c and R 5d in the general formula (I) or (II) can be each independently a hydroxyl group, an oxybenzyl group, a —NH_ (CH 2 ) a —X group, moiety (typically R 5 a and R 5 b total 85% or more, 85 preferably 95% or more, more preferably 98% or more, particularly preferably 100%, of the total R 5 c and R 5 d % or more, preferably 95% or more, more preferably 98% or more, particularly preferably 100%) - NH- (CH 2) a possible _X a group, _NH- (CH 2) a - (NR 7 a ( CH 2) d1) e1 - it is NHR 8 a, or one NH- (CH 2) 3- H- ( CH 2) 3 - is preferably NH 2 (R 7a, dl, el, R 8a About later). Note that a is an integer of 1 to 5.
  • R 6a and R 6b in the general formula (I) or ( ⁇ ⁇ ⁇ ) can be each independently a hydrogen atom or a protecting group, but it is preferable that most of them are hydrogen atoms.
  • the protecting group means a Z group, a Boc group, an acetyl group, a trifluoroacetyl group or the like which is usually used as a protecting group for an amino group.
  • X is not particularly limited as long as the copolymer satisfies the conditions of the present invention, but is independently an amine compound residue containing one or more of primary, secondary, tertiary amine or quaternary ammonium salts. It is a compound residue that is not a certain force or an amine, for example, a residue represented by the following formula.
  • X 2 is a hydrogen atom or CI- e alkyl or Amino alkyl group
  • R 7 a, R 7b and R 7c independently represents a hydrogen atom or a methyl group
  • dl, d 2 and d 3 is an integer of 1-5 their respective independently
  • el, e 2 and e 3 is an integer of 1 to 5 independently
  • f is an integer from 0 to 1 5
  • R 8 a and R 8 b each independently represent a protecting group such as a Z group, a Boc group, an acetyl group, a trifluoroacetyl group or the like which is usually used as a protecting group for a hydrogen atom or an amino group; It can be an integer of 15.
  • the method of introducing these residues into the side chain of the polyamino acid structure is particularly effective in the case of a polyaspartic acid structure, for example, in the case of a poly (//) as described in Japanese Patent No. It can be conveniently prepared by the ester-to-amide exchange reaction by aminolysis of the 3-benzyl-L-aspartate) moiety. Alternatively, benzyl ester can be converted to polyaspartic acid or polyglutamic acid by catalytic reduction, hydrolysis with an acid, alkali or the like, and then the compound having these residues can be bound using a condensing agent or the like. Can be manufactured.
  • the block copolymer represented by the general formula (I) or (II) may form a salt thereof.
  • the salt-forming counterions are C 1 —, Br—, I—, (1 S 0 4) -, N0 3 -, (1Z2 C_ ⁇ 3) -, (1 Bruno 3 P0 4) -, CH 3 C_rei_0 one, CF 3 C_ ⁇ _ ⁇ -, CH 3 S0 3 -, CF 3 S 0 3 — and the like.
  • R 2a and R 2b represents both methylene groups and E styrene group
  • R 2 e and R 2d is a methylene group
  • the repeating units of the aspartic acid derivative and the glutamic acid derivative can be present in the form of blocks or can be present at random.
  • a block copolymer obtained by reacting dipropylene triamine with a polyethylene glycol-poly () 3-benziru L-aspartate) block copolymer to effect aminolysis Is one of the particularly preferred block copolymers in the present invention.
  • a block copolymer having the structure represented by the following formula (IV), which can be obtained by reacting the above-mentioned polyethylene glycol-poly (/ 3-benzyl-L-spartate) block copolymer with spermine is also one of the particularly preferred block copolymers in the present invention.
  • PEG-SPM is obtained by cutting double-stranded oligonucleic acids (see 2. below), single-stranded oligonucleic acids such as ssDNA and antisense RNA, and circular nucleic acids such as plasmid DNA, for example, by cutting circular nucleic acids. It can also form polyion complex (PIC) micelles with the linear nucleic acid obtained.
  • R 3 in the general formula (I) represents a hydrogen atom, a protecting group, a hydrophobic group or a polymerizable group.
  • the hydrophobic group include derivatives such as benzene, naphthylene, anthracene, and pyrene.
  • the polymerizable group include a methacryloyl group and an acryloyl group.
  • the copolymer is used as a so-called macromer. For example, after forming a PIC micelle, it can be crosslinked via these polymerizable groups using other comonomers as necessary.
  • Methods for introducing these protecting groups, hydrophobic groups, and polymerizable groups into the terminal of the copolymer include a method using an acid halide, a method using an acid anhydride, a method using an active ester, and the like. The method used in is mentioned.
  • R 4 in the general formula ( II ) can be independently a hydroxyl group, an oxybenzyl group, or an NH— (CH 2 ) a _X group, similarly to R 5e or R 5d .
  • R 4 is an initiator residue.
  • a block copolymer is produced by polymerizing NCA of a protected amino acid using a low molecular initiator to synthesize a poly (amino acid or its derivative) segment and then bonding it to a PEG segment, structure derived from the initiator, i.e. one NH- R 9 is a by straight chain or branched where R 9 is unsubstituted or substituted C ⁇ 2.
  • An alkyl group can also be taken.
  • the chain length of the PEG segment and the chain length of the poly (amino acid or derivative thereof) segment are defined.
  • M and n are each an integer of 5 to 20,000 for m, preferably 10 to 5,000, particularly preferably 40. And n is an integer of 2 to 5,000, more preferably 5 to 1,000, and particularly preferably 10 to 200.
  • the copolymer of the general formula (I) or (II) is preferably It is not limited as long as it forms a PIC micelle with a nucleic acid or an anionic protein. Therefore, in this specification, for convenience, they are referred to as polyethylene glycol, polyamino acid, etc. However, the term “poly” is used as a concept that also includes what is called “oligo”.
  • each component can be either randomly distributed or distributed in a block shape, and is written in order in the notation, but is not limited thereto.
  • the polyion complex of the present invention is in the form of a polymer micelle, it is considered that a double-stranded oligonucleic acid is supported on the core of the micelle and an uncharged segment is present on the shell.
  • preferred block copolymers include, in addition to PEG-DPT and PEG-SPM described above, polyethylene glycol having a primary amino group at one terminal and ⁇ ⁇ _Z—L_lysine N carboxylic anhydride.
  • PEG-PLL polyethylene glycol-polylysine block copolymer
  • PEG-DET poly (ethylene daricol) -block-poly (/ 3-benzyl-L-aspartate) block Block copolymer
  • PEG-PBLA copolymer
  • PEG-PBLA-PLL (Z) polyethylene glycol-block-poly (/ 3-benzyl-L-aspartate) -block-Poly (N ⁇ -ZL-lysine) triblock copolymer
  • PEG-PBLA-PLL (Z) Triblock copolymer
  • PIC micelles containing PEG-DET are characterized by low cytotoxicity.
  • the nucleic acid electrostatically carried in the polymer micelles comprising the block copolymer is a double-stranded oligonucleic acid for gene knockdown, It is not limited to that type.
  • “gene knockdown” is a generic term for suppressing gene expression using an oligonucleic acid
  • the knockdown nucleic acids include siRNA, shRNA, dsDNA, and RNA-DNA hybrid oligonucleic acid.
  • at least one of the double-stranded oligonucleic acids including
  • siRNA shRNA
  • RNA Chihiro RNA Chihiro
  • siRNA short interference RNA, small interfering RNA
  • shRNA short hairpin RNA
  • the sense strand and the antisense strand are connected via a loop, and the expression of the knockdown effect is further processed through intracellular processing to form siRNA.
  • miRNAs miRNAs
  • the length of the siRNA is 10-: 100 nucleotides (nt), preferably 10-50 nt, more preferably 18-25 nt.
  • antisense DNA a single-stranded oligo DNA
  • RNAi double-stranded oligo RNA is taken into the RISC complex, and it is thought that high knockdown efficiency is exhibited through dissociation and activation of siRNA.
  • the details of the mechanism are still unknown, and it has been reported that a similar knockdown effect can be obtained with oligo DNA if it is effectively delinoriated into cells. Therefore, dsODN and RNA-DNA high It is presumed that the pride oligonucleic acid may also have a gene knockdown function in cells. These oligonucleic acids can be similarly supported on polymer micelles.
  • the method of producing a polyion complex of these nucleic acids and a block copolymer is based on the fact that the nucleic acids are electrostatically bound to the block copolymer by mixing the respective solutions at an appropriate mixing ratio. And That is, when a nucleic acid and a block copolymer are mixed, a negative ion of the nucleic acid and a positive charge of the block copolymer form a polyion complex in the form of micelles.
  • the mixing ratio between the nucleic acid and the block copolymer can be represented by the ratio (N / P ratio) between the phosphate group in the double-stranded oligonucleic acid molecule and the cation in the block copolymer.
  • the N / P ratio is a quantity defined by the following equation, and unless otherwise noted, the N / P ratio refers to this quantity.
  • N / P ratio [Total number of nucleotides in double-stranded oligonucleic acid in solution]
  • the N / P ratio is not limited as long as a polyion complex can be formed, and the uncharged segment or the charged segment contained in the block copolymer is not limited.
  • the An appropriate N / P ratio in the present invention can be appropriately selected by those skilled in the art.
  • oligonucleic acid by supporting a double-stranded oligonucleic acid on the polymer block copolymer obtained as described above, it can be used as a pharmaceutical composition for gene therapy.
  • the double-stranded oligonucleic acid to be supported on the polymer block copolymer the above-described double-stranded oligonucleic acid such as siRNA, shRNA, dsODN, and RNA-DNA hybrid oligonucleic acid can be used. Or can be used in an appropriate combination.
  • the administration form of the pharmaceutical composition of the present invention include injections, and include ordinary intravenous, intraarterial, etc. Can be administered systemically, or locally in muscles, joints, subcutaneous, or intradermal. Further, it is also possible to adopt a dosage form using a catheter.
  • the powder is usually provided in the form of a unit dose ampoule or a multi-dose container, and may be a powder which can be redissolved in a suitable carrier for use, for example, sterile pyrogen-free water.
  • these dosage forms may contain additives generally used in pharmaceutical preparations.
  • the dose varies depending on the purpose of treatment, the form of micelles, the age of the subject to be administered, the route of administration, and the number of administrations, and can vary widely.
  • the amount of double-stranded oligonucleic acid contained in the pharmaceutical composition of the present invention is Those skilled in the art can appropriately set the values.
  • an effective amount administered as a combination of an effective amount of the pharmaceutical composition of the present invention with a suitable diluent and a pharmacologically acceptable carrier can be from 10 g / kg body weight per serving:! OOOO g. Yes, given every 3 to 4 weeks.
  • a polyion complex (PIC) was prepared using double-stranded oligonucleic acid siRNA and dsODN, and its formation was confirmed, and its physical properties and gene knockdown ability were evaluated.
  • PIC polyion complex
  • SiRNA samples targeting GL3, GFP and LaminA / C were purchased from QIAGEN.
  • SEQ ID NO: 1 the base in parentheses is overhang.
  • dsODN purchases the sense (sense) and antisense (antisense) chains of oligo (01igo) DNA (21 bps) (Sigma) having the same nucleotide sequence as GL3 targeting siRNA, and anneals both. It was prepared as follows. After confirming the formation of double strand by electrophoresis (PAGE), Used for the experiment.
  • RNAiFect an siRNA transfection reagent, was purchased from QIAGEN. PIC carrying dsODN and siRNA was prepared by adding and mixing each block copolymer solution at a volume ratio of 2: 1 to each oligonucleic acid solution.
  • ⁇ —Z—L_lysine N-carboxylic anhydride was polymerized using polyethylene glycol having a primary amino group at one end as an initiator.
  • the reaction solution was dropped into cold ether, and the polyethylene glycol-poly ( ⁇ -ZL-lysine) block copolymer (PEG-PLL (Z)) produced and collected by filtration.
  • PEG-PLL (Z) was dissolved in trifluoroacetic acid, deprotection was performed using HBr acetic acid, and ether reprecipitation, dialysis, and lyophilization yielded a polyethylene glycol-polylysine block copolymer (PEG-PLL). .
  • Polyethylene glycol having an average molecular weight of 12,000 with methoxy at one terminal and aminopropyl at one terminal is dissolved in methylene chloride, and / 3-benzyl-L-aspartate-N-carbonic anhydride (BLA-NCA) is ⁇ , ⁇ - Dissolve in a mixed solvent of dimethylformamide (DMF) and methylene chloride, add, react at 40 for 2 days, acetylate the ⁇ -terminal with acetic anhydride, and use polyethylene glycol-block-poly () 3-benzyl -L-aspartate) (PEG-PBLA) was obtained. NMR analysis revealed that the degree of polymerization of the PBLA moiety was 68.
  • a block copolymer having a molecular weight of PEG of 12,000 and a degree of polymerization of the PBLA portion of 68 may be described as PEG-PBLA (12-68) (the number 12 in parentheses indicates a molecular weight of 12,000, and 68 indicates a molecular weight of 12,000. Represents the degree of polymerization).
  • PEG-block-poly (3-benzyl-L-aspartate) (PEG-PBLA) was dissolved in benzene, freeze-dried, and then dissolved in dry dimethylformamide (DMF) under an argon atmosphere.
  • DMF dry dimethylformamide
  • 50 times equivalent of dipropylene triamine dried and purified by distillation was added, and the mixture was stirred at 40 under argon atmosphere for 24 hours.
  • reaction is added dropwise to 10% acetic acid, dialyzed against 0.1N-HC1 using a dialysis membrane with a molecular weight cutoff of 3500, and the dialysis membrane solution is collected, concentrated, and lyophilized to form a PEG-DPT block copolymer.
  • PEG-PBLA PEG-block-poly (3-benzyl-L-aspartate)
  • PEG-PBLA (12-68) was dissolved in benzene and freeze-dried, and then dissolved in dry dimethylformamide (DMF) under an argon atmosphere.
  • DMF dry dimethylformamide
  • a solution of spermine dissolved in benzene, freeze-dried and dehydrated in dry DMF was added, and the mixture was stirred at 40 in an argon atmosphere at 24 hours.
  • the amount of spermine was used 50 times equivalent to benzyl ester.
  • the reaction solution was added dropwise to 10% acetic acid, dialyzed against 0.1N-HC1 using a dialysis membrane with a molecular weight cutoff of 6-8000, and the solution in the dialysis membrane was collected and lyophilized to obtain the following formula (IV) Was obtained as a white solid.
  • PEG-PBLA (12-68) was dissolved in benzene and freeze-dried, and then dissolved in dry dimethylformamide (DMF) under an argon atmosphere.
  • diethylenetriamine dried and purified by distillation, was added in an amount of 50 times the equivalent of benzyl ester, and the mixture was stirred at 40 in an argon atmosphere for 24 hours.
  • the reaction solution is added dropwise to 10% acetic acid, dialyzed against 0.1N-HC1 using a dialysis membrane with a molecular weight cutoff of 3500, and the dialysis membrane inner solution is recovered and freeze-dried.
  • the PEG-DET block copolymer was obtained as a white solid.
  • Polyethylene Dalicol-block-Poly (/ 3-benzyl -L-aspartate) -block-Po Li (N ⁇ -ZL-lysine) triploc copolymer (PEG-PBLA-PLL (Z)) was synthesized, and the N-terminal was acetylated using acetic anhydride.
  • the molecular weight of PEG was 12000, and the degrees of polymerization of PBLA and PLL (Z) were 36 and 50, respectively (denoted as 12-36-50).
  • This polymer was dissolved in benzene and freeze-dried, and then dissolved in dry dimethylformamide (DMF) under an argon atmosphere.
  • DMF dry dimethylformamide
  • Fig. 1 The results are shown in Figure 1.
  • the left panel shows the results of electrophoresis of complex (PEG-PLL (12-48) / dsODN) when PEG-PLL carries dsODN
  • the middle panel shows the results when dsODN is carried on PEG-DPT.
  • electrophoresis of complex PEG-DPT (l2-68) / dsODN
  • the right panel shows the results of electrophoresis of the complex (PEG-DPT (l2-68) / siRNA) when PEG-DPT was loaded with siRNA.
  • Each lane is as follows.
  • the formed aggregate has a so-called core-shell micelle-like structure having a PEG layer on the outer shell and a polycation block and a polyion complex of double-stranded oligonucleic acid in the inner core. It was shown to be an aggregate of the form Na It can be easily inferred that almost the same aggregate is formed even when siRNA having the same sequence as dsODN is used.
  • Example 2
  • GL3 firefly
  • RL emisshiiyuuga
  • HuH-7 cells were seeded in a 24-well dish at 2.5 ⁇ 10 4 cells / well, incubated in DMEM containing 10% serum for 24 hours, and then an experiment was performed.
  • the medium was replaced with Opti-MEM (Invitrogen), and pDNA (Promega) encoding two luciferase genes, pGL3 and pRL, were simultaneously transfected using LipofectAMINE (Invitrogen).
  • Opti-MEM Invitrogen
  • pDNA Promega
  • encoding two luciferase genes pGL3 and pRL
  • the expression of the two luciferases was quantified using the DuaHuciferase reporter assay system (Promega).
  • the selective suppression of GL3 expression by GL3 targeting siRNA was evaluated based on the relative expression level of GL3 using the RL expression level as a standard, and the expression of GL3 and RL in mock cells without siRNA administration was evaluated. The values are shown based on the quantitative ratio (GL3 / RL).
  • each block copolymer of PEG-DET, PEG DPT, and PEG APM PLL was used. As shown in Fig. 4, good suppression of luciferase gene expression was observed with each block copolymer.
  • PEG-DPT was able to suppress gene expression even more than PEG-PLL, and especially when N / P ratio was 20, the suppression was close to 80%, which was more effective than RNAiFect.
  • siRNA complex for the purpose of evaluating the stability of siRNA complex, one of double-stranded oligonucleic acids, in serum, the complex prepared as described above was incubated in 50% serum, and then gene knockout was performed. Used for down experiments.
  • the method was performed by transfection of HuL-7 cells with pDNA encoding two luciferase genes, pGL3 and pRL, as in Example 2, followed by selective knockdown of GL3. .
  • Two types of siRNA complex PEG_DPT / siRNA and PEG-PLL / siRNA, were used.
  • As an incubation in the serum before administration an equal volume of serum was added to the complex solution, and incubated at 37 for 30 minutes before use in the experiment.
  • the gene knockdown was evaluated in the same manner as in Example 2 based on the relative expression level of GL3 based on the expression level of RL.
  • the micelle structure which is obtained by complexing the block copolymer and siRNA and has the outer shell covered by PEG and encapsulating the siRNA in the inner core, has a very stable knockdown function even in a physiological environment. It was shown to be effective to exert.
  • siRNA delivery ifi endogenous gene by a block copolymer in order to confirm that the same effect can also be achieved for knockdown of siRNA delivery ifi endogenous gene by a block copolymer, an siRNA using LaminA / C, a kind of nuclear protein, as an overnight target was used. The knockdown of LaminA / C mRNA was evaluated by quantitative PCR.
  • HuH-7 cells and 293T cells were each seeded on a 6-well dish, incubated in DMEM containing 10% serum for 24 hours, and then an experiment was performed. The medium was exchanged, 1 ml of DMEM containing 10% serum was added to each well, and the previously prepared siRNA complex (PEG-DPT / siRNA, PEG-PLL / siRNA) was added at 4 g / well as siRNA. .
  • Example 5 When the effect of incubation of the complex in serum was examined in the same manner as in Example 2, almost the same expression suppression was obtained after incubation with PEG-DPT, whereas the suppression efficiency was significantly reduced with RNAiFect. However, its stability in a physiological environment was confirmed.
  • Example 5 When the effect of incubation of the complex in serum was examined in the same manner as in Example 2, almost the same expression suppression was obtained after incubation with PEG-DPT, whereas the suppression efficiency was significantly reduced with RNAiFect. However, its stability in a physiological environment was confirmed.
  • ATX Autotaxin
  • Example 4 knockdown of ATX mRNA was evaluated by a quantitative PCR method.
  • a polyion complex was formed by mixing a double-stranded oligonucleic acid with a block copolymer consisting of an uncharged segment and a charged segment.
  • Efficient gene knockdown including knockdown of endogenous genes, by complexing siRNA, one of the double-stranded oligonucleic acids, with a block copolymer consisting of uncharged and charged segments Down was possible.
  • the knockdown effect was nucleotide sequence-specific, and it was confirmed that the knockdown effect was RNAi due to effective delivery of the siRNA into the cells. Knockdown efficiency was very good, even after incubation in serum, unlike commercial lipid-based siRNA transfection reagents.
  • the present invention is to be a very practical system capable of stably carrying double-stranded oligonucleic acids such as siRNA without losing their functions even in a physiological environment. Became clear. Therefore, the PIC of the present invention is very useful as a clinically applicable delivery system required for treating diseases using double-stranded oligonucleic acid. Industrial potential
  • the present invention provides an excellent delivery system for a double-stranded oligonucleic acid such as an siRNA to a tissue or a cell.
  • the double-stranded oligonucleic acid-carrying polycomplex of the present invention has high stability in a physiological environment and is a drug delivery system that can be applied clinically. Functions as a stem.
  • the polyion complex of the present invention is used as a pharmaceutical composition, it is possible to specifically knock down a target gene in a tissue or a cell. Sequence listing free text
  • SEQ ID NO: 1 siRNA sequence

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Medicinal Chemistry (AREA)
  • Genetics & Genomics (AREA)
  • General Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Epidemiology (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • General Engineering & Computer Science (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Molecular Biology (AREA)
  • Biotechnology (AREA)
  • Biomedical Technology (AREA)
  • Physics & Mathematics (AREA)
  • Biophysics (AREA)
  • Dispersion Chemistry (AREA)
  • Nanotechnology (AREA)
  • Plant Pathology (AREA)
  • Microbiology (AREA)
  • Polymers & Plastics (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Biochemistry (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Medicinal Preparation (AREA)

Abstract

Complexe de polyion couplé de façon électrostatique comprenant un copolymère séquencé composé de segments non chargés et de segments chargés, le copolymère séquencé portant un acide oligonucléique double brin pour l'inactivation fonctionnelle (knockdown) de gènes ; et composition pharmaceutique comprenant le complexe de polyion.
PCT/JP2004/011957 2004-02-13 2004-08-13 Acide oligonucléique double brin portant un complexe de polyion, procédé pour produire celui-ci et composition pharmaceutique contenant celui-ci WO2005078084A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
JP2005517894A JPWO2005078084A1 (ja) 2004-02-13 2004-08-13 ２本鎖オリゴ核酸を担持したポリイオンコンプレックスおよびその製造法とそれを含む医薬組成物

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
JP2004037322 2004-02-13
JP2004-037322 2004-02-13

Publications (1)

Publication Number Publication Date
WO2005078084A1 true WO2005078084A1 (fr) 2005-08-25

Family

ID=34857757

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/JP2004/011957 WO2005078084A1 (fr) 2004-02-13 2004-08-13 Acide oligonucléique double brin portant un complexe de polyion, procédé pour produire celui-ci et composition pharmaceutique contenant celui-ci

Country Status (2)

Country Link
JP (1) JPWO2005078084A1 (fr)
WO (1) WO2005078084A1 (fr)

Cited By (17)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006085664A1 (fr) * 2005-02-10 2006-08-17 The University Of Tokyo Polymere chargeable de polycations et utilisation comme vecteur d'acides nucleiques
WO2007099661A1 (fr) * 2006-03-01 2007-09-07 The University Of Tokyo complexe de micelles polymeres contenant de l'acide nucleique
WO2007099660A1 (fr) * 2006-03-01 2007-09-07 The University Of Tokyo complexe de micelles polymeres contenant de l'acide nucleique
EP2087912A1 (fr) * 2006-11-22 2009-08-12 The University of Tokyo Support d'arnsi sensible à l'environnement utilisant une micelle polymérique à pont disulfure
JP2010163364A (ja) * 2009-01-13 2010-07-29 Univ Of Tsukuba 四級化アミノ基を有する架橋ポリマー由来のポリマー微粒子と核酸の複合体
WO2010093036A1 (fr) * 2009-02-13 2010-08-19 国立大学法人東京大学 Poly(acides aminés) cationiques et leurs utilisations
WO2010100781A1 (fr) * 2009-03-06 2010-09-10 国立大学法人東京大学 Composition pour l'administration d'acide nucléique
WO2010106700A1 (fr) * 2009-03-17 2010-09-23 国立大学法人東京大学 Régulateur de la charge électrique d'une protéine et complexe micellaire polymère à l'intérieur duquel est encapsulée une protéine
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
WO2011010691A1 (fr) * 2009-07-22 2011-01-27 国立大学法人東京大学 Complexe polyionique comprenant une substance inhibant l'expression de la phd2
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
WO2011105402A1 (fr) * 2010-02-23 2011-09-01 ナノキャリア株式会社 Acide polyaminé cationique à chaîne courte et son utilisation
WO2012005376A1 (fr) * 2010-07-09 2012-01-12 国立大学法人 東京大学 Composition d'administration d'acides nucléiques, composition d'excipient, composition pharmaceutique contenant la composition d'administration d'acides nucléiques ou la composition d'excipient et méthode d'administration d'acides nucléiques
WO2013162041A1 (fr) * 2012-04-27 2013-10-31 国立大学法人 東京大学 Composition pharmaceutique de type structure unitaire pour une administration d'acide nucléique
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
TWI764022B (zh) * 2018-07-30 2022-05-11 中央研究院 凝聚複合物的治療性奈米粒子及其治療細菌的用途
AU2021263769B2 (en) * 2020-04-29 2024-05-02 Medimmune, Llc Compositions and methods for delivering pharmaceutically active agents

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH08188541A (ja) * 1995-01-10 1996-07-23 Res Dev Corp Of Japan 静電結合型高分子ミセル薬物担体とその薬剤
WO2000003694A1 (fr) * 1998-07-17 2000-01-27 Mirus Corporation Inversion de charges de complexes de polyions
JP2001146556A (ja) * 1999-11-19 2001-05-29 Nano Career Kk コア−シェル構造のポリイオンコンプレックスミセル
JP2003503569A (ja) * 1999-06-25 2003-01-28 クリスティアン プランク 核酸を細胞に輸送するためのコポリマー

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP4535229B2 (ja) * 2003-05-08 2010-09-01 国立大学法人 東京大学 ポリエチレングリコール−ポリカチオンブロック共重合体

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH08188541A (ja) * 1995-01-10 1996-07-23 Res Dev Corp Of Japan 静電結合型高分子ミセル薬物担体とその薬剤
WO2000003694A1 (fr) * 1998-07-17 2000-01-27 Mirus Corporation Inversion de charges de complexes de polyions
JP2003503569A (ja) * 1999-06-25 2003-01-28 クリスティアン プランク 核酸を細胞に輸送するためのコポリマー
JP2001146556A (ja) * 1999-11-19 2001-05-29 Nano Career Kk コア−シェル構造のポリイオンコンプレックスミセル

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
ITAKA K. ET AL.: "Polyion complex micelles from plasmid DNA and poly(ethylene glycol).poly(-lysine) block copolymer as serum-tolerable polyplex system: physicochemical properties of micelles relevant to gene transfection efficiency", BIOMATERIALS, vol. 24, 2003, pages 4495 - 4506, XP004446090 *
KAKIZAWA Y. ET AL.: "Block copolymer micelles for delivery of gene and related compounds", ADVANCD DRUG DELIVERY REVIEWS, vol. 54, 2002, pages 203 - 222, XP001150717 *

Cited By (44)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7829657B2 (en) 2005-02-10 2010-11-09 The University Of Tokyo Polycationically charged polymer and the use of the same as a carrier for nucleic acid
WO2006085664A1 (fr) * 2005-02-10 2006-08-17 The University Of Tokyo Polymere chargeable de polycations et utilisation comme vecteur d'acides nucleiques
WO2007099661A1 (fr) * 2006-03-01 2007-09-07 The University Of Tokyo complexe de micelles polymeres contenant de l'acide nucleique
WO2007099660A1 (fr) * 2006-03-01 2007-09-07 The University Of Tokyo complexe de micelles polymeres contenant de l'acide nucleique
US8592385B2 (en) 2006-03-01 2013-11-26 The University Of Tokyo Polymer micelle complex including nucleic acid
JP5277439B2 (ja) * 2006-03-01 2013-08-28 国立大学法人 東京大学 核酸内包高分子ミセル複合体
JP5277440B2 (ja) * 2006-03-01 2013-08-28 国立大学法人 東京大学 核酸内包高分子ミセル複合体
EP2087912A1 (fr) * 2006-11-22 2009-08-12 The University of Tokyo Support d'arnsi sensible à l'environnement utilisant une micelle polymérique à pont disulfure
EP2087912A4 (fr) * 2006-11-22 2013-07-31 Univ Tokyo Support d'arnsi sensible à l'environnement utilisant une micelle polymérique à pont disulfure
JP2010163364A (ja) * 2009-01-13 2010-07-29 Univ Of Tsukuba 四級化アミノ基を有する架橋ポリマー由来のポリマー微粒子と核酸の複合体
JP4582821B2 (ja) * 2009-02-13 2010-11-17 国立大学法人 東京大学 カチオン性のポリ（アミノ酸）およびその使用
JPWO2010093036A1 (ja) * 2009-02-13 2012-08-16 国立大学法人 東京大学 カチオン性のポリ（アミノ酸）およびその使用
WO2010093036A1 (fr) * 2009-02-13 2010-08-19 国立大学法人東京大学 Poly(acides aminés) cationiques et leurs utilisations
US8546487B2 (en) 2009-02-13 2013-10-01 The University Of Tokyo Cationic poly (amino acids) and uses thereof
WO2010100781A1 (fr) * 2009-03-06 2010-09-10 国立大学法人東京大学 Composition pour l'administration d'acide nucléique
US9051354B2 (en) 2009-03-17 2015-06-09 The University Of Tokyo Protein charge regulator and protein-encapsulating polymer micelle complex
WO2010106700A1 (fr) * 2009-03-17 2010-09-23 国立大学法人東京大学 Régulateur de la charge électrique d'une protéine et complexe micellaire polymère à l'intérieur duquel est encapsulée une protéine
JP5472288B2 (ja) * 2009-03-17 2014-04-16 国立大学法人 東京大学 タンパク質の電荷調節剤、及びタンパク質内包高分子ミセル複合体
US8668933B2 (en) 2009-03-31 2014-03-11 The University Of Tokyo Polyion complex of double-stranded ribonucleic acid
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
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
US8791086B2 (en) 2009-07-22 2014-07-29 The University Of Tokyo Polyion complex comprising PHD2 expression inhibiting substance
JP2011026219A (ja) * 2009-07-22 2011-02-10 Univ Of Tokyo Ｐｈｄ２発現抑制物質搭載ポリイオンコンプレックス
WO2011010691A1 (fr) * 2009-07-22 2011-01-27 国立大学法人東京大学 Complexe polyionique comprenant une substance inhibant l'expression de la phd2
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
JP5661626B2 (ja) * 2009-07-23 2015-01-28 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
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
WO2011105402A1 (fr) * 2010-02-23 2011-09-01 ナノキャリア株式会社 Acide polyaminé cationique à chaîne courte et son utilisation
US8853167B2 (en) 2010-02-23 2014-10-07 Nanocarrier Co., Ltd. Short-chain cationic polyamino acid and use thereof
US9314529B2 (en) 2010-07-09 2016-04-19 The University Of Tokyo Nucleic acid delivery composition and carrier composition, pharmaceutical composition using the same, and method for nucleic acid delivery
CN102971002A (zh) * 2010-07-09 2013-03-13 国立大学法人东京大学 核酸递送用组合物及载体组合物、使用其的药物组合物以及核酸递送方法
WO2012005376A1 (fr) * 2010-07-09 2012-01-12 国立大学法人 東京大学 Composition d'administration d'acides nucléiques, composition d'excipient, composition pharmaceutique contenant la composition d'administration d'acides nucléiques ou la composition d'excipient et méthode d'administration d'acides nucléiques
JPWO2013162041A1 (ja) * 2012-04-27 2015-12-24 国立大学法人 東京大学 核酸デリバリー用ユニット構造型医薬組成物
WO2013162041A1 (fr) * 2012-04-27 2013-10-31 国立大学法人 東京大学 Composition pharmaceutique de type structure unitaire pour une administration d'acide nucléique
US9808480B2 (en) 2012-04-27 2017-11-07 Nanocarrier Co., Ltd. Unit structure-type pharmaceutical composition for nucleic acid delivery
JP2018008989A (ja) * 2012-04-27 2018-01-18 国立大学法人 東京大学 核酸デリバリー用ユニット構造型医薬組成物
US11020418B2 (en) 2012-04-27 2021-06-01 Nanocarrier Co., Ltd. Unit structure-type pharmaceutical composition for nucleic acid delivery
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
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
TWI764022B (zh) * 2018-07-30 2022-05-11 中央研究院 凝聚複合物的治療性奈米粒子及其治療細菌的用途
AU2021263769B2 (en) * 2020-04-29 2024-05-02 Medimmune, Llc Compositions and methods for delivering pharmaceutically active agents

Also Published As

Publication number Publication date
JPWO2005078084A1 (ja) 2008-01-10

Similar Documents

Publication Publication Date Title
Tan et al. Polycation architecture and assembly direct successful gene delivery: micelleplexes outperform polyplexes via optimal DNA packaging
Malamas et al. Design and evaluation of new pH-sensitive amphiphilic cationic lipids for siRNA delivery
EP2087912B1 (fr) Support de parni utilisant une micelle polymérique à pont disulfure
Chiper et al. Self-aggregating 1.8 kDa polyethylenimines with dissolution switch at endosomal acidic pH are delivery carriers for plasmid DNA, mRNA, siRNA and exon-skipping oligonucleotides
JP5061349B2 (ja) ポリカチオン荷電性ポリマー及び核酸のキャリヤーとしての使用
WO2005078084A1 (fr) Acide oligonucléique double brin portant un complexe de polyion, procédé pour produire celui-ci et composition pharmaceutique contenant celui-ci
US20080112916A1 (en) CHEMICALLY MODIFIED POLYCATION POLYMER FOR siRNA DELIVERY
CN108026527A (zh) 确定的多偶联寡核苷酸
KR101590652B1 (ko) 합성 중합체를 사용하여 유전자 사일런싱에 활성이 있는 핵산을 전달하기 위한 수단
WO2014066912A1 (fr) Nanoparticules polymères
WO2004099287A1 (fr) Copolymere sequence polyethyleneglycol/polycation
CN114302715A (zh) 用于体内药物递送的脂质纳米颗粒及其用途
EP2399948A1 (fr) Poly(acides aminés) cationiques et leurs utilisations
BR112014016562B1 (pt) Estrutura de oligo-rna de dupla hélice, nanopartícula e método de preparação
Miyazaki et al. Guanidine-phosphate interactions stabilize polyion complex micelles based on flexible catiomers to improve mRNA delivery
RU2617059C2 (ru) Способ получения амфифильных блок-сополимеров N,N-диметиламиноэтилметакрилата для доставки нуклеиновых кислот в живые клетки
CN116744979A (zh) 包含甘露糖的脂质纳米颗粒或其应用
Xu et al. Efficient siRNA delivery using PEG-conjugated PAMAM dendrimers targeting vascular endothelial growth factor in a CoCl2-induced neovascularization model in retinal endothelial cells
Goyal et al. 1, 4-Butanediol diglycidyl ether (BDE)-crosslinked PEI-g-imidazole nanoparticles as nucleic acid-carriers in vitro and in vivo
AU2014284835B2 (en) Dengue virus-specific siRNA, double helix oligo-RNA structure comprising siRNA, and composition for suppressing proliferation of dengue virus comprising RNA structure
US20110287547A1 (en) Nucleic acid delivery compositions and methods
US8945927B2 (en) Polymers for delivering molecules of interest
Ganbold et al. Efficient in vivo siRNA delivery by stabilized d-peptide-based lipid nanoparticles
He et al. Reductive triblock copolymer micelles with a dynamic covalent linkage deliver antimiR-21 for gastric cancer therapy
KR102701681B1 (ko) 이중가닥 올리고뉴클레오티드 및 이를 포함하는 코로나바이러스감염증-19〔covid-19〕치료용 조성물

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AE AG AL AM AT AU AZ BA BB BG BR BW BY BZ CA CH CN CO CR CU CZ DE DK DM DZ EC EE EG ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MA MD MG MK MN MW MX MZ NA NI NO NZ OM PG PH PL PT RO RU SC SD SE SG SK SL SY TJ TM TN TR TT TZ UA UG US UZ VC VN YU ZA ZM ZW

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): GM KE LS MW MZ NA SD SL SZ TZ UG ZM ZW AM AZ BY KG KZ MD RU TJ TM AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HU IE IT LU MC NL PL PT RO SE SI SK TR BF BJ CF CG CI CM GA GN GQ GW ML MR NE SN TD TG

121 Ep: the epo has been informed by wipo that ep was designated in this application
WWE Wipo information: entry into national phase

Ref document number: 2005517894

Country of ref document: JP

NENP Non-entry into the national phase

Ref country code: DE

WWW Wipo information: withdrawn in national office

Country of ref document: DE

122 Ep: pct application non-entry in european phase