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WO2011111874A1 - Arn du type en forme d'haltère et à perméation de membrane cellulaire et son procédé de fabrication - Google Patents

Arn du type en forme d'haltère et à perméation de membrane cellulaire et son procédé de fabrication Download PDF

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WO2011111874A1
WO2011111874A1 PCT/JP2011/056492 JP2011056492W WO2011111874A1 WO 2011111874 A1 WO2011111874 A1 WO 2011111874A1 JP 2011056492 W JP2011056492 W JP 2011056492W WO 2011111874 A1 WO2011111874 A1 WO 2011111874A1
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strand
modified
cell membrane
cell
dbrna
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PCT/JP2011/056492
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Japanese (ja)
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阿部洋
伊藤嘉浩
阿部奈保子
烏田美和子
中嶋裕子
須賀晶子
高橋政代
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独立行政法人理化学研究所
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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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/14—Type of nucleic acid interfering N.A.
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/35—Nature of the modification
    • C12N2310/351—Conjugate
    • C12N2310/3513—Protein; Peptide
    • 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
    • C12N2310/50—Physical structure
    • C12N2310/53—Physical structure partially self-complementary or closed
    • C12N2310/531—Stem-loop; Hairpin
    • 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
    • C12N2310/50—Physical structure
    • C12N2310/53—Physical structure partially self-complementary or closed
    • C12N2310/532—Closed or circular

Definitions

  • the present invention relates to a cell membrane-permeable dumbbell-type single-stranded circular RNA and a method for producing the same.
  • RNA interference methods are roughly classified into a method using chemically synthesized double-stranded RNA and a method using a plasmid vector.
  • RNA interference technology using the latter plasmid vector is widely used as biotechnology and is mainly used for basic biology experiments.
  • RNA interference technology is applied to drug development, it seems to be desirable to use the former chemically synthesized double-stranded RNA because the method using a plasmid vector has a problem of safety to the human body.
  • chemically synthesized double-stranded RNA has in vivo instability, that is, it has a problem that it is easily degraded by intracellular enzymes (nucleases).
  • RNA strand into which a non-natural nucleic acid has been introduced has been developed in order to increase the stability of double-stranded RNA in cells.
  • the stability is increased, but the biological activity is lowered. It was.
  • the toxicity given to non-natural nucleic acids is unknown, and its application to pharmaceuticals has been difficult.
  • RNA used for RNA interference is double-stranded RNA having a blunt end or protruding end, RNA having a hairpin structure in which one of double-stranded RNA ends is in a loop shape (Patent Document 1), There is a circular RNA having a stem composed of about 19 base pairs and two loops and having a chemically modified polynucleotide (Patent Document 2). Further, although an RNA-DNA chimera dumbbell-type nucleic acid is disclosed (Patent Document 3), this nucleic acid is not used for RNA interference, and the RNA portion of the dumbbell-type nucleic acid is cleaved by an enzyme in the cell to produce anti-antibody.
  • Patent Document 3 discloses that a nucleic acid has a dumbbell-type structure, and thus has high resistance to nucleolytic enzymes in cells, and stably exists in cells until ribonuclease H acts. It is disclosed that only when a portion is excised, a single-stranded oligonucleotide containing an antisense DNA portion is released into the cell, so that the antisense effect per dose is considered to be increased.
  • an oligonucleotide is synthesized linearly to form a stem portion and a hairpin loop portion, and one place of the target circular dumbbell oligonucleotide (the above hairpin)
  • a circular dumbbell-shaped circular nucleic acid is prepared by obtaining a nick dumbbell-shaped oligonucleotide to which the end of the loop portion is not bonded and ligating the 5 ′ end with ligase (Patent Literature). 3).
  • RNA interference action in a sustained and sustained manner.
  • An object of the present invention is to provide a dumbbell-type single-stranded circular RNA having cell membrane permeability and a method for producing the same.
  • the present inventors can impart cell membrane permeability to the DbRNA by binding a cell membrane permeable peptide to a loop portion of a dumbbell-shaped single-stranded circular RNA (hereinafter referred to as “DbRNA”). Furthermore, it was found that the loop portion of DbRNA is cleaved in the cell to generate a functional double-stranded RNA, which causes the RNA interference effect and the control of the biological action of the target gene, thereby completing the present invention. It came. That is, the present invention includes the following inventions.
  • [1] comprising a sense strand sequence, an antisense strand sequence complementary to the sense strand sequence, and two loop sequences that are the same or different that bind both strands between the sense strand and the antisense strand,
  • the sense strand and the antisense strand pair to form a stem, and one or two of the loop sequences are modified with one or more cell membrane-permeable peptides,
  • a cell membrane-permeable peptide-modified dumbbell-shaped single-stranded circular RNA that has a two-loop sequence in a cell and generates a functional double-stranded RNA.
  • the cell membrane-permeable peptide-modified dumbbell-type single-stranded circular RNA according to any one of [1] to [3] is added to a human-derived cell, and the RNA is added without using another intracellular delivery system.
  • a method of suppressing expression of a gene encoding the protein in vitro which comprises introducing into the cell, thereby disabling target RNA in the cell and inhibiting translation into the protein.
  • the cell membrane-permeable peptide-modified dumbbell-type single-stranded circular RNA according to any one of [1] to [3] is added to a non-human animal, plant, or cell thereof, and the RNA is delivered to another intracellular delivery system.
  • a method for suppressing the expression of a gene encoding the protein which comprises introducing the protein into the cell without using the protein, thereby disabling target RNA in the cell and inhibiting translation into the protein.
  • DbRNA to which the cell membrane permeability was provided and its manufacturing method can be provided.
  • DbRNA can be produced by modifying the loop portion with a cell membrane permeable peptide and can be taken up into the cell by itself. Therefore, it is not necessary to use other intracellular delivery systems such as conventional viral vectors and cationic liposomes whose safety is questioned.
  • it is specifically recognized by in vivo enzyme dicer in the incorporated cells, and the loops on both sides are cleaved to become functional RNA duplexes, so that the RNA interference effect and the biological action of the target gene Can cause control.
  • This specification includes the contents described in the specification and / or drawings of Japanese Patent Application No. 2010-054740 which is the basis of the priority of the present application.
  • FIG. 1 shows the flow of synthesis of TAT peptide-modified DbRNA via maleimide modification.
  • FIG. 2 shows the structure (a) and sequence (b) of the TAT peptide modified DbRNA. Among the sequences shown in (b), the underlined sequence is a sequence forming a loop part, and the site shown in bold indicates the amino modification site.
  • FIG. 3 shows the analysis result of maleimide modification reaction of DbRNA by reverse phase HPLC. Peak 1: maleimide; peak 2: amino-modified DbRNA; peak 3: maleimide-modified DbRNA. (0% -80% CH3CN / 50 mM TEAA buffer)
  • FIG. 4 shows the analysis result of TAT peptide-modified DbRNA by 10% denaturing PAGE.
  • FIG. 5 shows the structure and sequence of siRNA used as a control and DbRNA without TAT peptide modification (Db23).
  • FIG. 6 shows the evaluation result of the RNA interference effect by comparing the expression level of luciferase.
  • each sample is as follows from the left side of the figure: buffer only (control); siRNA mixed with transfection reagent (25 nM siRNA w / TR) (positive control); DbRNA mixed with transfection reagent (25 nM) Db23 w / TR (positive control); siRNA only (200 nM siRNA w / o TR); DbRNA only (200 nM Db23 w / o TR); DbRNA mixed with TAT peptide (TAT + 200 nM) SiRNA mixed with TAT peptide (TAT + 200 nM siRNA); TAT peptide modified DbRNA (25 nM TAT-Db23); TAT peptide modified DbRNA (100 nM TAT-Db23); Plastid qualified DbRNA (200nM TAT-Db23).
  • FIG. 7 shows the flow of synthesis of TAT peptide-modified DbRNA via iodoacetyl modification.
  • FIG. 8 shows the results of ion-exchange HPLC analysis of amino-modified DbRNA, iodoacetylated DbRNA and TAT peptide-modified DbRNA.
  • FIG. 9 shows the analysis result of TAT peptide-modified DbRNA by 10% denaturing PAGE. 1: amino modified DbRNA; 2: iodoacetylated DbRNA; 3: TAT peptide modified DbRNA.
  • FIG. 10 shows the structure (a) and sequence (b) of TAT-dbGSK.
  • FIG. 11 shows the result of double staining of BrdU / GS (glutamine synthetase) in retinal tissues with TAT-Db23 (200 nM) added (control) (A) or TAT-dbGSK (200 nM) added (B). Shown: BrdU (red) labels the nuclei of proliferated cells (arrows), GS (green) is a marker for Mueller glial cells.
  • DbRNA includes a stem formed by pairing a sense strand sequence homologous to a base sequence of a target RNA or a part thereof and an antisense strand sequence complementary to the sense strand sequence, the sense strand and the antisense strand It includes two loops formed by a base sequence that is between and does not form a complementary strand.
  • the length of the stem is not particularly limited, and the length may be determined according to the type or structure of the target RNA. For example, the length is 19 to 31 base pairs, preferably 21 to 25 base pairs, more preferably 22 to 24. It consists of base pairs, more preferably 23 base pairs.
  • the length of the loop is preferably 2 to 20 bases, more preferably 6 to 12 bases.
  • the entire single-stranded circular RNA is preferably formed of 42 to 102 bases.
  • the base sequences and lengths of the two loop portions may be the same or different.
  • the sense strand of DbRNA has a sequence complementary to the target RNA (for example, mRNA or its precursor RNA).
  • the base sequence of the DbRNA loop is not particularly limited, and examples thereof include UUCAGAGA, UGUGCUGUC (M. Miyagishi et al., Oligonucleotides 2003, 13: 1-7) and the like.
  • the loop of DbRNA is modified with a cell membrane permeable peptide.
  • modification refers to binding of a protein, peptide, chemical substance, compound or the like having a specific function to a nucleic acid sequence constituting a DbRNA loop.
  • modification and “binding” are used interchangeably.
  • the “cell membrane permeable peptide” means a peptide having a function of imparting a cell membrane permeability to the protein or the like by modifying the protein, peptide, or low molecular weight compound with the peptide and introducing the protein into the cell.
  • cell membrane permeable peptides generally include basic peptides rich in arginine and lysine, and are not particularly limited, and include known peptides having this function, such as HIV-1 TAT peptide, HIV-1 Rev peptide. , FHV Coat peptide, BMV Gag peptide, HTLV-II Rex peptide, CCMV Gag peptide, P22 N peptide, ⁇ N peptide, ⁇ 21N peptide, yeast PRP6 peptide, human U2AF peptide, Antennapedia peptide, VP22 peptide and the like.
  • it is an HIV-1 TAT peptide.
  • the “TAT peptide” is a peptide sequence containing a large amount of basic amino acids derived from amino acid residues 48 to 60 of the TAT protein that is a transcriptional activator of HIV-1 (human immunodeficiency virus type 1). N-terminal) - 48 Gly-Arg-Lys -Lys-Arg-Arg-Gln-Arg-Arg-Arg-Pro-Pro-Gln 60 - having a (C-terminal) (SEQ ID NO: 1).
  • the “TAT peptide” includes an amino acid sequence having at least 10 consecutive amino acids in the amino acid sequence of SEQ ID NO: 1 and having the cell membrane permeation function.
  • the “TAT peptide” includes an amino acid sequence in which one or more amino acids are deleted, substituted, inserted or added in the amino acid sequence of SEQ ID NO: 1, and has the above-mentioned cell membrane permeation function.
  • Cell membrane permeable peptides may be obtained from naturally occurring sources, or may be produced using genetic engineering techniques or chemical synthesis.
  • the cell membrane permeable peptide is bound to the loop of DbRNA. Since the loop part is cleaved and removed by enzymes such as Dicer in the cell, the stem acts as a functional double-stranded RNA, that is, siRNA or miRNA, when it exerts RNA interference action or biological action of the target gene.
  • the bound cell membrane-penetrating peptide hardly affects the action.
  • the cell membrane permeable peptide may be bound to only one loop in one DbRNA, or may be bound to both loops.
  • One or a plurality of cell membrane permeable peptides may be bound to one loop.
  • “Plurality” means 2 or more, 3 or more, 4 or more, 5 or more, or more.
  • the binding site of the cell membrane permeable peptide in the loop is not particularly limited, but a site away from the stem is considered preferable.
  • the number and binding position of cell membrane permeable peptides bound to the loop are appropriately determined within a range that does not interfere with the desired RNA interference action or the control action of the biological action of the target gene.
  • the cell membrane permeable peptide is bound to one of the loops at the most distal position from the stem.
  • the cell membrane permeable peptide is bound to the loop of DbRNA using a covalent bond or a non-covalent bond as described in detail in the following DbRNA production method and Examples.
  • the loop of DbRNA may be chemically modified. For example, when modified with polyethylene glycol having a molecular weight of about 2000 to 5000, the stability of DbRNA in vivo can be improved.
  • the cell membrane-permeable peptide-modified DbRNA of the present invention is a known intracellular delivery system (for example, electroporation method, microinjection method, lipofection method, calcium phosphate method, in particular viral vectors and cationic liposomes) for introducing nucleic acid into cells. Etc.) can be introduced into the cell without using. Introduction of the cell membrane-permeable peptide-modified DbRNA of the present invention into a cell can be confirmed by comparing the expression level of the target RNA in the cell with the expression level of the cell into which the DbRNA of the present invention has not been introduced.
  • the method for producing a cell membrane-permeable peptide-modified DbRNA of the present invention comprises synthesizing a first strand and a second strand, each having a base sequence comprising a base sequence constituting the DbRNA divided into two at arbitrary locations, 5 'end base of the first strand base sequence and 3' end base of the second strand base sequence, and 3 'end base of the first strand base sequence and 5' end of the second strand base sequence. Including simultaneously ligating the bases with a ligase. Furthermore, it further includes binding one or more cell membrane permeable peptides in the base sequence constituting the loop of the first strand and / or the second strand.
  • the sense strand and antisense strand constituting the stem in DbRNA are designed from the base sequence of the target gene so that the target gene can be suppressed.
  • a plurality of sense strands and antisense strands may be prepared and the suppression efficiency may be confirmed respectively, but for example, a design based on an siRNA design algorithm or the like may be used (reference: JA Jaeger et al.). al., Methods in Enzymology (1989) 183: 281-306; DH Mathews et al., J. Mol. Biol. (1999) 288: 911-940).
  • the target gene is not particularly limited, and examples thereof include a gene that causes human disease or a gene that is specific to bacteria or viruses that cause human infectious diseases, a gene that regulates cell growth (eg, GSK3 ⁇ gene), and the like.
  • the sequence constituting the loop in DbRNA is not particularly limited as described above, and can be selected from, for example, the sequence UUCAGAGA or UGUGCUGUC. In the sequence constituting the loop, a modified base is introduced at a position where the cell membrane-permeable peptide is bound.
  • One or more modified bases can be introduced depending on the number of cell membrane permeable peptides to be bound.
  • the modified base those known to be useful for internal modification of nucleic acids can be used.
  • biotin-modified bases and / or amino-modified bases known to be useful for internal modification of nucleic acids eg, Biotin-dT, Amino Modifier C2 dT, Amino Modifier C6 dT, Amino Modifier C6 dA, Amino Modifier C6 dC). Etc.
  • the modified base preferably has a spacer having an appropriate length.
  • the spacer can be a hydrocarbon having 3 to 20 carbon atoms, preferably 5 to 12 carbon atoms, preferably a linear saturated hydrocarbon.
  • the modified base can be introduced by insertion or substitution at any position of the sequence UUCAGAGA or UGUGCUGUC. As described above, the position and number of modified bases to be introduced are determined and designed as appropriate as long as the cell membrane-permeable peptide added to the loop does not interfere with the desired RNA interference action or the control action of the target gene biological action. can do.
  • the first strand and the second strand are designed by dividing the base sequence of the DbRNA designed to have the above-described stem and loop configuration into two at arbitrary locations, and synthesized separately.
  • the “arbitrary portion” is not particularly limited, and can be selected from the portions constituting the stem and / or the loop.
  • the base sequence constituting one loop is the 5 ′ end of each strand and Located at the 3 'end or at the 3' and 5 'ends, respectively.
  • the sense strand and the antisense strand are inserted in each base sequence of the first strand or the second strand with the base sequence constituting the loop interposed therebetween. There are sequences that make up.
  • Nucleic acid synthesis methods include various methods such as an in vitro transcription synthesis method, a method using a plasmid and a viral vector, and a method using a PCR cassette, and are not particularly limited. However, the purity is high, mass synthesis is possible, and in vivo use is safe.
  • the chemical synthesis method is preferable from the viewpoint of high property and possible chemical modification.
  • the chemical synthesis method includes an H-phosphonate method and a phosphoramidite method, and is not particularly limited, and a commercially available automatic nucleic acid synthesizer can be used.
  • the ends of base sequences that do not form complementary strands at both ends of the sense strand and the antisense strand are ligated with ligase (eg, T4 RNA ligase, T4 DNA ligase, etc.) to form two loops simultaneously.
  • ligase eg, T4 RNA ligase, T4 DNA ligase, etc.
  • the reaction conditions may be incubated at a low temperature for 20 hours in a buffer containing, for example, polyethylene glycol (PEG), BSA and the like.
  • PEG polyethylene glycol
  • BSA polyethylene glycol
  • the synthesized DbRNA can be recovered and purified by a conventional method (for example, high performance liquid chromatography, PAGE method, etc.).
  • the purity of the recovered and purified DbRNA can be increased by decomposing RNA that has not been cyclized by exonuclease treatment. Binding of the cell membrane-permeable peptide to DbRNA can be appropriately performed based on a known technique with respect to the modified base introduced into the sequence forming the loop. For example, when the introduced base residue is a biotin-modified base, DbRNA and the cell membrane-permeable peptide can be bound by modifying the C-terminus of the cell membrane-permeable peptide with streptavidin.
  • the C-terminus is modified with a thiol group or the C-terminus is modified by modifying the end of the spacer with a maleimide group, iodoacetyl group or thiol group. It can be coupled with a cell membrane permeable peptide modified with a maleimide group or an iodoacetyl group.
  • the single-stranded circular RNA may be chemically modified with polyethylene glycol (PEG) or the like, and the chemical modification is preferably performed on the loop portion.
  • both ends of the PEG are modified to introduce functional groups reactive with the amino group of the base, such as, for example, formyl group, N-hydroxysuccinimide ester group.
  • DbRNA conjugated with cell membrane permeable peptide can be obtained by, for example, precipitation or heat denaturation with ammonium sulfate, PEG, antibody, etc., followed by centrifugation; chromatography step, eg, ion exchange chromatography step, gel filtration chromatography step, reverse phase Chromatography step, hydroxylapatite chromatography step, and affinity chromatography step; isoelectric focusing; gel electrophoresis; and combinations thereof, as well as other known techniques, can be appropriately collected and purified.
  • RNA interference is also referred to as RNA interference (RNAi), and is a phenomenon in which a small RNA molecule having a sequence complementary to a target RNA binds to the target RNA and degrades it or suppresses translation.
  • RNAi RNA interference
  • the cell membrane-permeable peptide-modified DbRNA of the present invention has a target RNA 24 hours after cell introduction in a cell into which the DbRNA has been introduced, assuming that the expression of the target RNA in a control cell (in which DbRNA is not introduced) is 1. It is preferable that the expression is 0.4 or less.
  • the expression of the target RNA is performed by, for example, transforming a reporter gene (luciferase, ⁇ -galactosidase, ⁇ -glucuronidase, green fluorescence protein (GFP), etc.) into a cell, using the reporter gene mRNA as a target RNA, and expressing the target RNA.
  • a reporter gene luciferase, ⁇ -galactosidase, ⁇ -glucuronidase, green fluorescence protein (GFP), etc.
  • the cell membrane-permeable peptide-modified DbRNA of the present invention is an intracellular delivery system that has been conventionally used in the introduction of nucleic acids into cells in vitro and in vivo, such as an electroporation method, a microinjection method, a lipofection method, calcium phosphate. It can be introduced into cells without using a method, particularly a viral vector or cationic liposome, and the target RNA can be disabled and the translation into protein can be continuously inhibited. As the cells, human cells or non-human cells can be used.
  • a sample containing cell membrane-permeable peptide-modified DbRNA is prepared by dialysis, pH adjustment, etc. so as to be compatible with the living body.
  • the method for introduction into animals or plants is not particularly limited, and includes, for example, local administration, intravenous administration, and a method using a gene gun.
  • a method that does not use microorganisms is preferable from the viewpoint of safety. .
  • siRNA RNA induced silencing complex
  • the cell membrane-permeable peptide-modified DbRNA of the present invention can be used in plants, animals (eg, mammals including humans, pets, livestock, etc.), and their cells, and has a wide range of applications particularly in the fields of medicine and agriculture. Be expected.
  • the cell membrane-permeable peptide-modified DbRNA of the present invention is used for various purposes such as elucidation of the function of a specific gene or protein at the plant or animal level or plant or animal cell level, for example, elucidation of the function by a knockout method. can do.
  • this invention contains the pharmaceutical composition which contains cell membrane permeation
  • the amount of the cell membrane-permeable peptide-modified DbRNA added to the pharmaceutical composition can be adjusted according to the type and purpose of the composition, and is 0.01% by weight, 0.1% by weight, for example, with respect to the total amount of the composition. 0.5%, 1%, 3%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% , 90% by weight and 100% by weight, but not limited thereto.
  • Examples of the dosage form of the pharmaceutical composition of the present invention include liquid preparations (solutions, suspensions, emulsions, etc.), solid preparations (lyophilized preparations etc. prepared at the time of use), and the like.
  • Parenteral administration is preferable, and includes, for example, local administration directly administered to the affected area, transmucosal administration such as pulmonary administration, nasal administration, intravenous administration and the like.
  • the pharmaceutical composition of the present invention may contain excipients (eg, physiological saline, sterilized water, Ringer's solution), buffers, isotonic agents, stabilizers, etc., depending on the formulation, dosage form, etc. .
  • the dosage of the pharmaceutical composition of the present invention can be changed according to the sex, weight, age, severity, symptoms, etc. of the patient.
  • the application of the pharmaceutical composition of the present invention is not particularly limited, but for example, treatment of diseases such as cancer, for example, suppression of functions of genes and proteins specifically expressed in cancer cells, causative bacteria or viruses of infectious diseases Examples thereof include suppression of functions of genes and proteins that are specifically expressed, and promotion or inhibition of cell proliferation.
  • diseases such as cancer
  • suppression of functions of genes and proteins specifically expressed in cancer cells for example, suppression of functions of genes and proteins specifically expressed in cancer cells, causative bacteria or viruses of infectious diseases Examples thereof include suppression of functions of genes and proteins that are specifically expressed, and promotion or inhibition of cell proliferation.
  • TAT peptide The sequence of the TAT peptide, which is a cell membrane-permeable peptide used in this example, is shown below. (N-terminal) 47 Tyr-Gly-Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg57-Cys (C-terminal) (SEQ ID NO: 2) This is a cysteine introduced at the C-terminus of the amino acid sequence of residues 47-57 of wild type HIV-1 TAT protein. The synthesis of the peptide was requested from PH Japan. Synthesis of TAT peptide-modified DbRNA via maleimide modification The flow of synthesis of TAT peptide-modified DbRNA via maleimide modification is shown in FIG.
  • TAT peptide-modified DbRNA is synthesized by reacting DbRNA whose loop portion is amino-modified with a maleimide group, and further reacting the maleimide group with a thiol group contained in the cysteine of the TAT peptide.
  • the structure and sequence of the TAT peptide-modified DbRNA are shown in FIGS. 2 (a) and (b).
  • the underlined sequence is a sequence that forms a loop portion, and the site shown in bold indicates the amino modification site. Synthesis of DbRNA in which the loop portion is amino-modified can be performed based on the method described in JP 2008-278784 A.
  • RNA amidite used TBDMS protector (Proligo), and 5 'phosphorylation used Chemical Phosphorylation Reagent (Glen Research).
  • Amino-Modifier C6-dT was introduced into the amino modification site of the loop part. Deprotection was performed by PAGE purification according to a conventional method.
  • the enzyme reaction was performed using 2 ⁇ M RNA double strand, 2.0 units / ⁇ l T4 RNA ligase, 0.006% BSA, 25% PEG 6000, 50 mM Tris-HCl (pH 7.5), 10 mM MgCl 2 , 10 mM DTT, 1 mM ATP. With a reaction scale of 25 ⁇ l. Specifically, first, in 100 mM Tris-HCl (pH 7.5), 20 mM MgCl 2 , 20 mM DTT, 2 mM ATP buffer (2 ⁇ buffer) so that the 5 ′ phosphorylated RNA duplex becomes 4 ⁇ M.
  • a maleimide compound is reacted with the amino group of DbRNA in which the loop portion is amino-modified to synthesize maleimide-modified DbRNA.
  • Sulfo-SMCC Sulfo-N-Succinimidyl 4- (Maleimidemethyl) cyclohexane-1-carboxylate
  • the loop portion was amino-modified DbRNA was dissolved in 5 mM Sulfo-SMCC, 75 mM NaB buffer (pH 8.5) and shaken at room temperature for 3 hours. Thereafter, the reaction mixture was concentrated with Amicon Ultra-0.5 (Millipore) to remove unreacted Sulfo-SMCC, followed by isopropanol precipitation, and DbRNA whose loop portion was modified with maleimide was recovered. The progress of the reaction was confirmed by reverse phase HPLC (FIG. 3).
  • the maleimide group of DbRNA in which the loop part was modified with maleimide was reacted with the thiol group of the TAT peptide (included in cysteine at the C-terminal) to synthesize TAT peptide-modified DbRNA.
  • the loop part was dissolved in 1 mM TAT peptide, 0.01 M NaB buffer (pH 8.5) so that the DbRNA modified with maleimide was 3.69 ⁇ M, and shaken at room temperature for 12 hours. After the reaction, the reaction solution was directly applied to 10% denatured PAGE to confirm the progress of the reaction (FIG. 4).
  • TAT peptide-modified DbRNA is referred to as “TAT-Db23” in the present specification and drawings.
  • siRNA and DbRNA not modified with the TAT peptide (hereinafter referred to as “Db23”) were used as controls.
  • the structure and sequence of siRNA and Db23 are shown in FIG.
  • siRNA and Db23 can be prepared using RNA having the sequences shown in SEQ ID NOs: 5 and 6 and SEQ ID NOs: 7 and 8, respectively, based on a known technique (Japanese Patent Laid-Open No. 2008-278784).
  • RNA mix was adjusted so that various RNA + Opti-MEM (and transfection reagent (Lipofectamine 2000) as necessary) were 50 ⁇ L / well in total.
  • RNA mix and cells were mixed at 50 ⁇ L / well, allowed to stand for 10 minutes, and then seeded in a 96-well plate. Thereafter, incubation was performed at 37 ° C. under 5% CO 2 for 2.5 hours, and DMEM medium supplemented with 20% FBS and 1 ⁇ penicillin-streptomycin was added to each well.
  • the luciferase expression level was quantified by solubilizing the cells using the Dual-luciferase Reporter Assay System (Promega) according to the attached protocol (conditions—reagent volume: 30 ⁇ l; delay time: 2 seconds; read) time: 10 seconds; instrument: Wallac ARVO SX 1420 Multilabel Counter).
  • buffer only control
  • siRNA or Db23 mixed with transfection reagent positive control
  • siRNA or Db23 only siRNA or Db23 mixed with TAT peptide were also evaluated.
  • the emission intensity of firefly luciferase was corrected by the emission intensity of Renilla luciferase which is an internal standard. The results are shown in FIG.
  • RNA interference The expression level of luciferase when each sample is added is shown relative to the expression level of luciferase when the buffer alone (control) is added. From this result, it was shown that siRNA or Db23 introduced alone without using a transfection reagent does not show RNA interference, and therefore, RNA alone does not cause intracellular introduction. Moreover, since siRNA or Db23 only mixed with the TAT peptide does not show RNA interference action, it was shown that RNA is not taken up into cells only by mixing the TAT peptide and RNA. On the other hand, TAT-Db23 did not show RNA interference at 25 nM, but showed RNA interference at 100 nM and 200 nM.
  • the RNA can be introduced into cells without a transfection reagent. It can be said that 200 nM TAT-Db23 exhibits almost the same effect as positive control Db23. As described above, the cell membrane permeability was successfully imparted to DbRNA by adding the TAT peptide.
  • TAT peptide-modified DbRNA via iodoacetyl modification The flow of synthesis of TAT peptide-modified DbRNA via iodoacetyl modification is shown in FIG.
  • the TAT peptide-modified DbRNA via iodoacetyl modification is synthesized by iodoacetylating DbRNA having a loop modified with amino and then reacting the iodoacetyl group with the thiol group contained in the cysteine of the TAT peptide.
  • DbRNA whose amino acid is modified in the loop portion (prepared in the same manner as in Example 1 above) is dissolved in 20 ⁇ L of 0.1 M sodium borate buffer so that the concentration is 50 ⁇ M, and 2 ⁇ L of 50 mM iodoacetyl NHS ester is dissolved. In addition, the mixture was allowed to stand at room temperature for 3 hours. Thereafter, 4 ⁇ L of 3M NaOAc (pH 5.2) and 80 ⁇ L of 2-propanol were added, mixed, incubated at ⁇ 30 ° C. for 1 hour, centrifuged (20,000 g, 4 ° C., 30 minutes), and iodoacetylated DbRNA was added. Collected (yield 79%).
  • TAT peptide modification of iodoacetylated DbRNA was performed. Specifically, it was dissolved in 25 ⁇ L of 2M TEAA buffer so that the iodoacetylated DbRNA was 1 ⁇ M, 350 ⁇ L formamide and 0.5 ⁇ L 5 mM TAT peptide were added, and the mixture was stirred overnight at room temperature. Thereafter, 4 ⁇ L of 3M NaOAc (pH 5.2) and 80 ⁇ L of 2-propanol were added, mixed, incubated at ⁇ 30 ° C. for 1 hour, centrifuged (20,000 g, 4 ° C., 30 minutes), and TAT peptide-modified DbRNA was added. It was collected.
  • TAT peptide-modified DbRNA was purified by HPLC using an ion exchange column (TOSOH TSK gel DEAE-2SW column No. M0009, Part No. 07168) (0-80% 1M sodium perchlorate, 20 mM Tris- Cl (pH 6.8), 50% formamide / 20 mM Tris-Cl (pH 6.8), 50% formamide) (88% yield).
  • the amino-modified DbRNA, iodoacetylated DbRNA and TAT peptide-modified DbRNA were each analyzed by HPLC using an ion exchange column (TOSOH TSK gel DEAE-2SW column No. M0009, Part No.
  • TAT peptide-modified DbRNA (lane 3) is TAT peptide-modified because the band is shifted to the polymer side compared to amino-modified DbRNA (lane 1) and iodoacetylated DbRNA (lane 2). I was able to confirm.
  • TAT peptide-modified DbRNA targeting GSK3 ⁇ gene 5′-phosphorylated RNA used as a raw material for TAT peptide-modified DbRNA targeting GSK3 ⁇ gene was synthesized based on a known method (Japanese Patent Laid-Open No. 2008-278784). ).
  • the structure and sequence of a TAT peptide-modified DbRNA targeting the GSK3 ⁇ gene are shown in FIGS. 10 (a) and (b).
  • the underlined sequence is a sequence forming a loop portion, and the site shown in bold indicates the amino modification site.
  • two RNA sequences SEQ ID NOs: 9 and 10 shown in FIG.
  • RNA amidite TBDMS protector (Proligo) and 5 ′ phosphorylation were performed using Chemical Phosphorylation Reagent (Glen Research). Amino-Modifier C6-dT was introduced into the amino modification site of the loop part. Deprotection was performed by PAGE purification according to a conventional method. Synthesis of TAT peptide-modified DbRNA using the RNA sequence was performed via iodoacetyl modification, as in Example 2 above.
  • TAT-dbGSK The obtained TAT peptide-modified DbRNA targeting the GSK3 ⁇ gene is hereinafter referred to as “TAT-dbGSK” in the present specification and drawings.
  • GSK3 ⁇ gene expression inhibition experiment by TAT-dbGSK Inhibition of GSK3 ⁇ gene expression in retinal cells promotes cell proliferation due to activation of the Wnt / ⁇ -catenin pathway, resulting in regeneration of retinal tissue. (Osakada et al. The Journal of Neuroscience (2007) 27 (15): 4210-4219).
  • the inhibitory effect of TAT-dbGSK on GSK3 ⁇ gene expression was evaluated using retinal cell proliferation as an index depending on whether or not TAT-dbGSK was added to the retinal tissue culture system.
  • the cells were cultured for 4 days at 35.5 ° C. with 5% CO 2 .
  • the insert with the neuronal retina was transferred to an empty 6-well dish, and 10 ⁇ L of TAT-db23 or TAT-dbGSK solution diluted with HBSS was applied onto the neuronal retina twice every 30 minutes. I put it.
  • the insert was returned to the dish containing the culture solution again, and fixed on 3 days at 4 ° C. using Superfix (KURABO) on the 4th day of culture, and the tissue was embedded in paraffin to prepare a section.
  • KURABO Superfix
  • rat anti-BrdU serotec, OBT0030
  • mouse anti-Glutamine synthetase Mcillipore
  • Alexa Fluor 546 goat anti-rat IgG and Alexa Fluor 8 were used as secondary antibodies.
  • FIG. Compared with the case where TAT-db23 (control) was added, more BrdU-labeled (red) cells were observed in the retinal tissue to which TAT-dbGSK was added (arrows in the figure). This result suggests that TSK-dbGSK RNA interference action inhibited GSK3 ⁇ gene expression and promoted retinal cell proliferation. From the above, it was revealed that TAT-dbGSK effectively causes RNA interference in target cells without using other compounds that impart cell membrane permeability.
  • DbRNA that generates functional double-stranded RNA such as siRNA or miRNA can be introduced into a cell without using a viral vector or a cationic liposome, and causes RNA control in the cell safely and efficiently. Can do. Therefore, the present invention is expected as a new therapeutic agent for diseases such as cancer. All publications, patents and patent applications cited herein are incorporated herein by reference in their entirety.

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Abstract

La présence invention concerne un ARNDb auquel on a conféré une aptitude à la perméation de membrane cellulaire, ainsi que son procédé de fabrication. L'ARNDb de l'invention est obtenu par l'utilisation d'un peptide à perméation de membrane cellulaire afin de modifier le segment en boucle de l'ARNDb.
PCT/JP2011/056492 2010-03-11 2011-03-11 Arn du type en forme d'haltère et à perméation de membrane cellulaire et son procédé de fabrication WO2011111874A1 (fr)

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WO2016100401A1 (fr) * 2014-12-15 2016-06-23 Dicerna Pharmaceuticals, Inc. Acides nucléiques double brin modifiés par un ligand
EP4095248A1 (fr) * 2021-05-25 2022-11-30 Max-Planck-Gesellschaft zur Förderung der Wissenschaften e.V. Arn circulaires pour silençage génique
JP2023055874A (ja) * 2015-12-21 2023-04-18 スツラ セラポーティクス エルティーディー ステープルまたはステッチペプチドに複合された生物活性化合物

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MEADE, B.R. ET AL.: "Exogenous siRNA delivery using peptide transduction domains/cell penetrating peptides", ADV.DRUG DELIV.REV., vol. 59, no. 2-3, 2007, pages 134 - 140 *
MIWAKO UDA ET AL.: "Maku Tokano o Yusuru Shushoku Dumbbell-gata RNA ni yoru RNA Kanshoho no Kaihatsu", CSJ: THE CHEMICAL SOCIETY OF JAPAN KOEN YOKOSHU, vol. 90, no. 3, 12 March 2010 (2010-03-12), pages 785 *
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Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2016100401A1 (fr) * 2014-12-15 2016-06-23 Dicerna Pharmaceuticals, Inc. Acides nucléiques double brin modifiés par un ligand
EP3569711A1 (fr) * 2014-12-15 2019-11-20 Dicerna Pharmaceuticals, Inc. Acides nucléiques à double brin modifiés par ligands
JP2023055874A (ja) * 2015-12-21 2023-04-18 スツラ セラポーティクス エルティーディー ステープルまたはステッチペプチドに複合された生物活性化合物
US11944688B2 (en) 2015-12-21 2024-04-02 Sutura Therapeutics Ltd Biologically active compounds
EP4095248A1 (fr) * 2021-05-25 2022-11-30 Max-Planck-Gesellschaft zur Förderung der Wissenschaften e.V. Arn circulaires pour silençage génique
WO2022248572A1 (fr) * 2021-05-25 2022-12-01 MAX-PLANCK-Gesellschaft zur Förderung der Wissenschaften e.V. Arn circulaire pour le silençage génique

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