Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
  • A61K31/7088—Compounds having three or more nucleosides or nucleotides
  • A61K31/7105—Natural ribonucleic acids, i.e. containing only riboses attached to adenine, guanine, cytosine or uracil and having 3'-5' phosphodiester links
• • 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
  • A61K47/54—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 compound
  • A61K47/549—Sugars, nucleosides, nucleotides or nucleic acids
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
  • A61P35/02—Antineoplastic agents specific for leukemia
• • 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
• • 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

Definitions

• This disclosure relates to the use of nucleic acid delivery structures in the treatment of hematopoietic tumors.
• DDS drug delivery system
• RAS antisense oligonucleotides
• siRNA siRNA
• an artificial nucleic acid which is characterized by a structure in which a hydrophilic group is linked to the 3' end of the artificial nucleic acid, which is composed of a structural unit in which a base is bound to a ring structure selected from ribose and deoxyribose, and a linking structure that links the two structural units, forms micelles or nanoparticles when annealed with RNA, that the micelles or nanoparticles act as vesicles that deliver RNA, and that a composition in which miR-143 is made into nanoparticles using a cationic artificial nucleic acid is effective in suppressing the growth of colon cancer, a solid cancer (Patent Document 1).
• Hematopoietic tumors such as leukemia, are diseases in which white blood cells or immature cells proliferate on their own. Unlike solid cancers, it is said that treatment with surgical techniques alone is difficult. Some hematopoietic tumors, particularly RAS-mutated multiple myeloma, are known to recur repeatedly and have a poor prognosis. The inventors have found that miR-143 and its derivatives are effective against RAS-mutated cancers (Patent Document 2).
• RAS-mutated multiple myeloma is known to be one of the hematopoietic tumors typified by leukemia, but it is prone to recurrence and has a poor prognosis.
• miR-143 which was effective against solid cancers, is expected to be effective against such blood cancers, but it is difficult to introduce nucleic acids into blood cancer cells that are suspended in water, and a new nucleic acid drug delivery system was needed.
• the purpose of this disclosure is to provide a nucleic acid delivery structure that is effective in treating hematopoietic tumors, a method for producing the same, a medicine for hematopoietic tumors, and a method for treating hematopoietic tumors.
• micelles or nanoparticles composed of cationic artificial nucleic acids and delivery nucleotides, the surface of which is modified with ligands, suppress the proliferation of cells derived from hematopoietic tumors, and thus completed the present disclosure.
• nucleic acid delivery structure having an association structure in which a nucleic acid analog represented by the following formula (1) and a target nucleic acid consisting of a microRNA that controls the network of the cancer gene KRAS are associated by electrostatic interaction in the treatment of hematopoietic tumors.
• N represents a cationic artificial nucleic acid
• H represents a hydrophilic polymer
• S1 represents a spacer 1
• S2 represents a spacer 2
• L represents a ligand
• s represents 0 or 1
• t represents 0 or 1
• N has a skeleton structure including a structural unit in which a base is bound to a hexose selected from ribose and deoxyribose, a linking structure linking two of the structural units, and a cationic group
• the cationic artificial nucleic acid can be associated with the phosphate group of the nucleic acid to be delivered by electrostatic interaction with the cationic group.
• nucleic acid delivery structure characterized in that the cationic group has a pKa in the range of 6 to 9, in the treatment of hematopoietic tumors.
• nucleic acid delivery structure according to [1] or [2] above, wherein the cationic group, in a cationic state, has a partial structure selected from the group consisting of the following formulas (C1) to (C7):
• R 1 to R 3 are hydrogen or an alkyl group having 1 to 10 carbon atoms, and R 1 to R 3 may be the same or different.
• Ring is a cyclic compound composed of 4 to 8 carbon atoms, and may be a heterocyclic ring in which one or more of the carbon atoms are substituted with a heteroatom selected from nitrogen, oxygen, and sulfur.
• nucleic acid delivery structure according to any one of [1] to [3] in the treatment of hematopoietic tumors, wherein the linking structure of the cationic artificial nucleic acid (N) has at least one structure selected from the following formulas (L1) to (L4) in a cationic state.
• X + is a functional group containing the cationic group
• Z is O or S
• W is -O- or -NR4-
• R4 is hydrogen or an alkyl group having 1 to 10 carbon atoms
• * represents a bond to the adjacent structural unit.
• nucleic acid delivery structure according to any one of [1] to [4] in the treatment of hematopoietic tumors, wherein the cationic artificial nucleic acid has a nucleotide skeleton represented by the following formula (N1):
• N1 nucleotide skeleton represented by the following formula (N1):
• X + is a functional group containing the cationic group
• Base is a base
• R5 is H or OH
• * means a bond to the phosphate of the adjacent nucleotide backbone, at least one of the 5'-end or 3'-end is bonded to the hydrophilic polymer, and when it is not bonded to the hydrophilic polymer, it is hydrogen.
• nucleic acid delivery structure according to [5], wherein X + in a cationic state can be an ammonium cation represented by the following formula (F1), in the treatment of hematopoietic tumors.
• R 1 to R 3 are hydrogen or an alkyl group having 1 to 10 carbon atoms and may be the same or different from each other, m is an integer of 0 to 10, and n is an integer of 0 or 1.
• nucleic acid delivery structure in the treatment of hematopoietic tumors, characterized in that the hydrophilic polymer is selected from polyethylene glycol, polyvinyl alcohol, polyglutamic acid, polyvinylpyrrolidone, polyacrylamide, polyethyleneimine, polyalkylacrylate, polyoxazoline, polyacrylamide, poly(carboxybetaine methacrylate), poly(sulfobetaine methacrylate), poly(2-methacryloyloxyethylphosphocholine), hyaluronic acid, chitosan, dextran, and derivatives thereof.
• the hydrophilic polymer is selected from polyethylene glycol, polyvinyl alcohol, polyglutamic acid, polyvinylpyrrolidone, polyacrylamide, polyethyleneimine, polyalkylacrylate, polyoxazoline, polyacrylamide, poly(carboxybetaine methacrylate), poly(sulfobetaine methacrylate), poly(2-methacryloyloxyethy
• nucleic acid delivery structure characterized in that the hydrophilic polymer (H) has a polyethylene glycol backbone represented by the following formula (A1), in the treatment of hematopoietic tumors. (wherein p is an integer from 1 to 20.)
• nucleic acid delivery structure characterized in that the formula (A1) is linked via a phosphate diester group, in the treatment of hematopoietic tumors.
• the spacer 2 (S2) is a phosphodiester bond or a phosphodiester bond containing a triazole represented by the following formula (S21):
• Ka represents an amide bond containing a methylene group having 1 to 20 carbon atoms, a compound containing an aromatic group having 6 to 12 carbon atoms, or a direct bond; q is 0 or 1; when q is 0, the 1,2,3-triazole ring is bonded to the ligand.
• nucleic acid delivery structure according to any one of [1] to [11] in the treatment of hematopoietic tumors, characterized in that the ligand (L) is selected from glucose, mannose, galactose, sucrose, maltose, and lactose.
• nucleic acid delivery structure according to any one of [1] to [12] in the treatment of hematopoietic tumors, the nucleic acid delivery structure being a nanoscale structure in which a plurality of the nucleic acid delivery structures are assembled.
• nucleic acid delivery structure in the treatment of hematopoietic tumors, characterized in that the association structure is a vesicle or micelle in which the hydrophilic polymer and ligand are located on the outside and the association structure is located on the inside.
• nucleic acid delivery structure according to any one of [1] to [14] in the treatment of hematopoietic tumors, characterized in that the nucleic acid to be delivered is selected from miR-143 or its analogs.
• [16] Use of the nucleic acid delivery structure according to [15] in the treatment of hematopoietic tumors, wherein the miR-143 is selected from the group consisting of SEQ-1 to SEQ-23.
• [18] A method for treating hematopoietic tumors, comprising administering to a patient the nucleic acid delivery structure described in [1] to [16].
• a method for inhibiting the proliferation of cancerous cells in hematopoietic tumors comprising administering to the cancerous cells a nucleic acid delivery structure having an association structure in which a nucleic acid analog represented by the following formula (1) and a target nucleic acid comprising a microRNA that controls the network of the cancer gene KRAS are associated by electrostatic interaction:
• N represents a cationic artificial nucleic acid
• H represents a hydrophilic polymer
• S1 represents a spacer 1
• S2 represents a spacer 2
• L represents a ligand
• s represents 0 or 1
• t represents 0 or 1
• N has a skeleton structure including a structural unit in which a base is bound to a hexose selected from ribose and deoxyribose, a linking structure linking two of the structural units, and a cationic group
• the cationic artificial nucleic acid can be associated with the phosphate group of the nucleic acid to be delivered
• a nucleic acid delivery structure used for the treatment of hematopoietic tumors characterized in that the nucleic acid delivery structure has an association structure in which a nucleic acid analog represented by the following formula (1) and a target nucleic acid consisting of a microRNA that controls the network of the cancer gene KRAS are associated by electrostatic interaction:
• N represents a cationic artificial nucleic acid
• H represents a hydrophilic polymer
• S1 represents a spacer 1
• S2 represents a spacer 2
• L represents a ligand
• s represents 0 or 1
• t represents 0 or 1
• N has a skeleton structure including a structural unit in which a base is bound to a hexose selected from ribose and deoxyribose, a linking structure linking two of the structural units, and a cationic group
• the cationic artificial nucleic acid can be associated with the phosphate group of the nucleic acid to be delivered by electrostatic interaction with
• the present disclosure provides a nucleic acid delivery structure that is effective in treating hematopoietic tumors, a method for producing the same, a medicine for hematopoietic tumors, and a method for treating hematopoietic tumors.
• FIG. 1 is a diagram showing an outline of the nucleic acid analog and nucleic acid delivery construct of the present disclosure; (a) a diagram showing the sequence design for Glu-RION-miR143#12; (b) a diagram showing the generation of a delivery construct of Glu-RION-miR143#12; (c) a diagram showing the particle size distribution of Glu-RION-miR143#12; and (d) a diagram showing the stability in the blood after administration of Glu-RION-miR143#12 to mice.
• FIG. 1 shows an example of a synthesis scheme for a cationic artificial nucleic acid.
• FIG. 1 shows an example of a synthesis scheme.
• FIG. 5d This is a graph showing the survival rate of DLD-1 cells when miR-143#12 was treated with RION (control) or Glu-RION for 48 hours. The control had no inhibitory effect.
• This figure shows the effect of Glu-RION-miR143#12 on various hematopoietic tumor cells.
• Various cells were treated for 72 hours to examine the cell proliferation inhibitory effect (Fig. 5a, c, e).
• RPMI8226 cells were treated with and without Glu-RION-miR143#12, and observed under a fluorescent microscope. Hoechst 33342 was used as the fluorescent reagent (Fig. 5d).
• FIG. 5c shows the effect of Glu-RION-miR143#12 on various hematopoietic tumor cells.
• Various cells were treated for 72 hours to examine the cell proliferation inhibitory effect (Fig. 5a, c, e).
• PrMI8226 cells were treated with and without Glu-RION-miR143#12 and observed under a fluorescent microscope. The presence of glucose transporters was confirmed on hematopoietic tumor cells.
• NB4 cells were stained with Hoechst 33342 and then observed under a fluorescent microscope (Fig. 5f).
• FIG. 1 shows the results of Western blot analysis investigating the expression status of downstream factors of the RAS network by Glu-RION-miR143#12 in RPMI8226 cells.
• FIG. 1 shows the results of Western blot analysis investigating the expression status of downstream factors of the RAS network by Glu-RION-miR143#12 in NB4, HL-60, and Jurkat cells. This figure shows the effect of Glu-RION-miR143#12 on the growth of human peripheral lymphocytes, in relation to the presence or absence of stimulation with concanavalin A.
• the present disclosure describes micelles or nanoparticles obtained from a composition of a nucleic acid analog represented by the following formula (1) and miR-143 and its analogs, which are used in the treatment of hematopoietic tumors.
• Hematopoietic tumors The micelles or nanoparticles used in the present disclosure are used for the treatment of hematopoietic tumors.
• hematopoietic tumors refer to tumors that are broadly classified into leukemia, malignant lymphoma, and multiple myeloma, and are said to be tumors in which the K-RAS network is abnormal in many cases. Unlike solid cancers, it is said that treatment of any of these cancers by surgical techniques alone is difficult in many cases.
• “treating hematopoietic tumors” refers to killing or inhibiting the proliferation of tumor cells that are collectively present in the hematopoietic organs or suspended in blood or body fluids by controlling the K-RAS network.
• Leukemia is a general term for diseases in which white blood cells, or cells in an immature stage, multiply involuntarily. Leukemia can be broadly divided into acute leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia, adult T-cell leukemia, and adult T-cell leukemia.
• Acute leukemia is further classified into acute myeloid leukemia and acute lymphocytic leukemia, and the diagnostic criteria known as the FAB classification (French-American-British Classification) include morphological findings, and acute myeloid leukemia is reclassified into acute promyelocytic leukemia, acute myelomonocytic leukemia, acute monocytic leukemia, acute erythroleukemia, and acute megakaryoblastic leukemia.
• Malignant lymphoma is a disease in which lymphocytes in the blood become cancerous, and occurs mainly in lymphatic tissues such as lymph nodes, spleen and tonsils, but can also occur in organs other than lymphatic tissues, such as the stomach, intestines, thyroid gland, lungs, liver, skin, bone marrow and brain.
• Malignant lymphomas are classified into more than 50 types based on histological pathology, and are broadly divided into Hodgkin's lymphoma and non-Hodgkin's lymphoma.
• Non-Hodgkin's lymphoma is classified into two types, B cell and T/NK cell, and is further divided into more specific histological types.
• Plasma cells are lymphocytes, a type of white blood cell, that differentiate from B cells, and when plasma cells become cancerous and abnormal (myeloma cells), they lose the ability to attack foreign substances.
• lymphocytes a type of white blood cell, that differentiate from B cells, and when plasma cells become cancerous and abnormal (myeloma cells), they lose the ability to attack foreign substances.
• plasma cell tumors There are several types of plasma cell tumors, and in addition to multiple myeloma, other known types include plasmacytoma and macroglobulinemia.
• the nucleic acid to be delivered in the present disclosure is a microRNA that controls the network of the cancer gene KRAS.
• MicroRNA (hereinafter referred to as "miRNA") is an endogenous non-coding RNA of about 20 to 25 bases encoded on the genome. miRNA is first transcribed from the miRNA gene on the genomic DNA as a primary transcript (Primary miRNA, Pri-miRNA) of about several hundred to several thousand bases in length, and then processed to become a pre-miRNA (precusor miRNA) having a hairpin structure of about 60 to 110 bases.
• Primary miRNA Primary miRNA
• precusor miRNA precusor miRNA
• RNA-143 may be referred to as "miR143".
• miRNAs More than 1,000 types of miRNAs are known in humans and mice, and each regulates the expression of multiple target genes, and it has been suggested that they are involved in various vital phenomena such as cell proliferation and differentiation, and are involved in the onset and progression of cancer, cardiovascular disease, neurodegenerative disease, psychiatric disease, chronic inflammatory disease, etc. Many researchers have pointed out that miRNAs are deeply involved in the proliferation of cancer cells, and research and development of miRNAs as nucleic acid drugs is being conducted. RAS forms a vast network by controlling more than 10 downstream signals. miR-143 is an inhibitory miRNA that controls the K-RAS network in a multifaceted manner and can suppress cancer growth.
• miR-143s are known to suppress cancer, and the inventors have proposed the following miR-143s (SEQ-1 to SEQ-23) (Tables 1 and 2 below: Japanese Patent No. 6730717).
• the miR-143 analogs disclosed herein refer to microRNAs that have a similar sequence and/or structure to these miR-143s and have the effect of controlling the K-RAS network in a multifaceted manner.
• A, U, G, and C represent RNA containing adenine, uracil, guanine, or cytosine, respectively; dT and dG represent DNA containing thymine or guanine, respectively; RNAf represents 2'-FRNA; RNAm represents 2'-OMeRNA; ⁇ represents -P(S)OH-; * represents -P(O)OH-; and Ab (Abasic) represents the group shown below:
• S-1 SEQ ID NO: 1
• S-17 SEQ ID NO: 2
• S-18 SEQ ID NO: 3
• S-19 SEQ ID NO: 4
• AS-4 SEQ ID NO: 5
• AS-7 SEQ ID NO: 6
• AS-30 SEQ ID NO: 7
• AS-31 SEQ ID NO: 8
• AS-3 SEQ ID NO: 9
• AS-10 SEQ ID NO: 10
• AS-12 SEQ ID NO: 11
• AS-13 SEQ ID NO: 12
• AS-50 SEQ ID NO: 15
• AS-51 SEQ ID NO: 16
• AS-52 SEQ ID NO: 17
• AS-55 SEQ ID NO: 18
• AS-56 SEQ ID NO: 19
• AS-57 SEQ ID NO: 20
• SEQ-8 sense strand S-18, antisense strand AS-12 in the table above is preferred for hematopoietic tumors, which are the target of this disclosure.
• the nucleic acid analog of the present disclosure is composed of a cationic artificial nucleic acid and a hydrophilic polymer bound to the cationic artificial nucleic acid.
• the nucleic acid analog is preferably used as a carrier for delivering a nucleic acid to be delivered (hereinafter, may be referred to as a "nucleic acid to be delivered") to a target site.
• the nucleic acid to be delivered of the present disclosure is miR-143 or a derivative of the miR-143.
• the nucleic acid analog of the present disclosure can be represented by the following formula (1).
• N represents a cationic artificial nucleic acid
• H represents a hydrophilic polymer
• S1 represents a spacer 1
• S2 represents a spacer 2
• L represents a ligand
• s represents 0 or 1
• t represents 0 or 1.
• the cationic artificial nucleic acid represented by N has a structural unit in which a base is bound to a hexose selected from ribose and deoxyribose, a linking structure linking the two structural units, and a backbone structure consisting of a cationic group.
• the base sequence of the cationic artificial nucleic acid can be appropriately designed in consideration of the base complementarity, the strength of electrostatic interaction, and the like, depending on the base sequence of the nucleic acid to be delivered.
• the cationic group in a cationic state, has a partial structure selected from the group consisting of the following formulas (C1) to (C7).
• R 1 to R 3 are hydrogen or an alkyl group having 1 to 10 carbon atoms, and R 6 and R 7 may be the same or different.
• Ring is a cyclic compound consisting of 4 to 8 carbon atoms, and may be a heterocyclic ring in which one or more of the carbon atoms are substituted with a heteroatom selected from nitrogen, oxygen, and sulfur.
• Cationic artificial nucleic acids can associate with other nucleotides through electrostatic interactions between the phosphate group and the cationic group.
• other nucleotides refers to nucleotides such as DNA and RNA, or their analogs, and when a nucleic acid analog is used as a carrier, refers to the nucleotides or their analogs that make up the nucleic acid to be delivered.
• the pKa of the cationic group is higher than the pKa of the phosphate group of other nucleotides because of the electrostatic interaction with the phosphate group of other nucleotides.
• the cationic group Under pH conditions that are lower than the pKa of the cationic group of the nucleic acid analog and higher than the pKa of the phosphate group of other nucleotides, the cationic group is positively charged and the phosphate group is negatively charged, so these groups electrostatically bond.
• the pKa of the phosphate group of a nucleotide is generally less than 1
• the pKa of the cationic group is 1 or more, preferably 3 or more, and more preferably 6.0 or more.
• the upper limit of the pKa of the cationic group is not particularly limited, but is 12 or less, preferably 11 or less, and more preferably 9 or less. From the viewpoint of the strength of the electrostatic interaction with the phosphate group of other nucleotides and the structure of the cationic artificial nucleic acid, the pKa of the cationic group is preferably within the range of 6 to 9.
• bases examples include adenine, guanine, cytosine, thymine, uracil, N-methyladenine, N-benzoyladenine, 2-methylthioadenine, 2-aminoadenine, 7-methylguanine, N-isobutyrylguanine, 5-fluorocytosine, 5-bromocytosine, 5-methylcytosine, 4-N-methylcytosine, 4-N,N-dimethylcytosine, 5-fluorouracil, 5-bromouracil, 5-chlorouracil, and 5,6-dihydrouracil.
• the linking structure preferably has at least a structure selected from the following formulae (L1) to (L4) in a cationic state.
• X + is a functional group containing a cationic group of the above formulae (C1) to (C7), Z is O or S, W is -O- or -NR4- , where R4 is hydrogen or an alkyl group having 1 to 10 carbon atoms, and * represents a bond to the adjacent structural unit above.
• a cationic state refers to the case where the cationic group is assumed to be positively charged, and depending on the environmental conditions in which the nucleic acid analogue is placed (such as the pH and type of solvent), the cationic group may not be positively charged.
• the cationic group when the cationic group is ammonium, examples of a state that is not positively charged include a primary amine (which becomes primary ammonium when cationized), a secondary amine (which becomes secondary ammonium when cationized), and a tertiary amine (which becomes tertiary ammonium when cationized).
• Examples of cationic artificial nucleic acids include those having a nucleotide skeleton with a nucleotide unit in which a base is bound to ribose or deoxyribose. In addition, those having a morpholino skeleton with a structure in which a base is bound to morpholine are also shown.
• Base represents a base
• R5 represents H or OH
• * represents a bond to the phosphate of the adjacent nucleotide backbone, and at least one of the 5'-end or 3'-end is bonded to the hydrophilic polymer, and when it is not bonded to the hydrophilic polymer, it is hydrogen.
• nucleotide backbone examples include the following.
• X + in a cationic state is preferably an ammonium cation represented by the following formula (F1). (wherein R 1 to R 3 are hydrogen or an alkyl group having 1 to 10 carbon atoms and may be the same or different from each other, m is an integer of 0 to 10, and n is an integer of 0 or 1.)
• the strength of electrostatic interaction with the phosphate group of other nucleotides is primary amine (R 1 to R 3 are all hydrogen) ⁇ secondary amine (two of R 1 to R 3 are hydrogen, the others are alkyl groups) ⁇ tertiary amine (one of R 1 to R 3 is hydrogen, the others are alkyl groups) ⁇ quaternary ammonium (R 1 to R 3 are all alkyl groups).
• R 1 to R 3 are all hydrogen
• secondary amine two of R 1 to R 3 are hydrogen, the others are alkyl groups
• tertiary amine one of R 1 to R 3 is hydrogen, the others are alkyl groups
• quaternary ammonium R 1 to R 3 are all alkyl groups.
• structures having quaternary ammonium as the cationic group have a property of being easily structurally collapsed with a decrease in the pH of the environment.
• quaternary ammonium is particularly preferable as the cationic group of X + .
• the pKa of the cationic group varies depending on the structure, but is generally 6.1 to 7.9 for primary amines, 6.9 to 7.0 for secondary amines, 8.0 to 8.6 for tertiary amines, and 8.0 to 9.0 for quaternary ammonium groups.
• examples of those having a morpholino skeleton include a structure represented by the following formula (M1).
• Base represents a base. * represents a bond to phosphorus of the adjacent morpholino skeleton, and at least one of the 5'-end and 3'-end is bonded to the hydrophilic polymer, and when it is not bonded to the hydrophilic polymer, it is hydrogen.
• Examples of such structures having a morpholino skeleton include the following.
• the number of structural units (degree of polymerization) in which the ring structure and base that constitute the cationic artificial nucleic acid are bonded can be appropriately set depending on conditions such as the ring structure, the type of cationic group, and the type and length (number of bases) of the nucleic acid to be delivered.
• the degree of polymerization of the cationic artificial nucleic acid is about 5 to 100, and preferably about 10 to 50.
• the degree of polymerization (number of bases) of the nucleic acid to be delivered is short, such as about 20 bases, it is preferable that the degree of polymerization of the cationic artificial nucleic acid, which is the carrier, is also about the same as that of the nucleic acid to be delivered.
• the degree of polymerization of the cationic artificial nucleic acid may be different from the degree of polymerization of the nucleic acid to be delivered.
• the nucleic acid to be delivered is a long nucleic acid such as mRNA
• the degree of polymerization of the cationic artificial nucleic acid may be smaller than the degree of polymerization of the nucleic acid to be delivered.
• cationic groups are introduced into some or all of the linking structures that connect the constituent units. From the viewpoint of forming structures such as vesicles and micelles, which will be described later, it is preferable that the ratio of the linking structures into which cationic groups have been introduced is 50% or more, more preferably 80% or more, and particularly preferably 100% (all of the linking structures) relative to the total number of linking structures. If the ratio of the number of linking structures into which cationic groups have been introduced is low relative to the total number of linking structures, the negative charge of the delivery target nucleic acid will be dominant in the association structure formed between the cationic artificial nucleic acid and the delivery target nucleic acid, making it difficult to form a structure.
• cationic groups are continuously introduced into one of the 3' or 5' ends, or if cationic groups are introduced into discrete linking structures, such as every other structure, it is possible that a structure will be formed, but these structures are considered to be unstable because the pH response changes depending on the introduction ratio of the cations. For this reason, it is preferable that the ratio of linking structures into which cationic groups have been introduced is high.
• H represents a hydrophilic polymer.
• Various polymers can be used as the hydrophilic polymer depending on the application of the nucleic acid analog.
• the hydrophilic polymer is preferably one having biocompatibility.
• the hydrophilic polymer is preferably one that is neutral and does not easily interact electrically (attract, repel) with the nucleic acid analog.
• Such hydrophilic polymers are preferably selected from polyethylene glycol, polyvinyl alcohol, polyglutamic acid, polyvinylpyrrolidone, polyacrylamide, polyethyleneimine, polyalkylacrylate, polyoxazoline, polyacrylamide, poly(carboxybetaine methacrylate), poly(sulfobetaine methacrylate), poly(2-methacryloyloxyethylphosphocholine), hyaluronic acid, chitosan, dextran, and derivatives thereof.
• hydrophilic polymers examples include the following:
• the type of monomer constituting the hydrophilic polymer and the number of monomer units (degree of polymerization) can be appropriately set depending on conditions such as the type and molecular weight of the nucleic acid to be delivered, the molecular weight of the artificial nucleic acid, and the surrounding environment of the target site to be delivered.
• Representative types of monomers include oligomers of ethylene glycol, propylene glycol, and butylene glycol units, and the degree of polymerization of the hydrophilic polymer is about 2 to 100, preferably about 2 to 50, and more preferably about 2 to 10.
• these oligomers of hydrophilic polymers may be repeated two or more times via a phosphate diester group or the like.
• the number of repetitions via a phosphate diester bond may be three or more, preferably four or more, depending on the molecular weight of the artificial nucleic acid, complementarity with the nucleic acid to be delivered, and the balance with hydrophobicity when associated through electrostatic interaction, and the number of repetitions is at most 100 or less, preferably 30 or less, and more preferably 10 or less. If the degree of polymerization of the hydrophilic polymer or the number of repetitions of the hydrophilic polymer is low, it becomes difficult to form the structure described below, and the structure is likely to be eliminated as a foreign body in the body, and the degradation stability and blood retention are likely to be low. Conversely, if the degree of polymerization of the hydrophilic polymer is too high, the size of the structure is too large, and the delivery efficiency of the nucleic acid to be delivered to the target site is likely to be low.
• polyethylene glycol is particularly preferred as the hydrophilic polymer in terms of blood retention, etc.
• polyethylene glycol polyethylene glycol of 10 to 100K may be used in some cases, but it is preferable that the polyethylene glycol has a polyethylene glycol skeleton represented by the following formula (A1). (wherein p is an integer from 1 to 20.)
• A1 can also be used as a polymer having a nucleic acid base or a phosphate derivative (-PO(OH)-, -PS(OH)-, PO(SH)-) as a monomer unit.
• the number of repetitions q of ethylene glycol in the monomer unit A1 is 1 to 20, preferably 3 to 10, the monomer structures may be the same or different, and the degree of polymerization of the monomer unit is 1 to 10, preferably 2 to 10, more preferably 2 to 8.
• phosphate derivative monomer units include ethylene glycol phosphate derivative units, diethylene glycol phosphate derivative units, triethylene glycol phosphate derivative units, and hexaethylene glycol phosphate derivative units. These derivative units can also be used as monomers for polymers.
• the degree of polymerization of the phosphate derivative unit depends on the size of the artificial nucleic acid, but is 1 to 10, preferably 2 to 10, and more preferably 2 to 8, when the number of bases of the artificial nucleic acid is 10 to 30.
• the monomer unit is most preferably a triethylene glycol phosphate derivative or a hexaethylene glycol phosphate derivative.
• the type and degree of polymerization of the monomer of the hydrophilic polymer described above, particularly the degree of polymerization of the ethylene glycol phosphate derivative monomer can be selected from the viewpoints of ease of formation of the structure described below, difficulty of being recognized as a foreign substance in the body, low decomposition stability and low blood retention, etc.
• the surface of the structure may be modified with various ligands.
• the ligands include immunoglobulins, carbohydrates, peptides, proteins, aptamers, and the like.
• the main ligands and their application fields expected from research on lipid nanoparticles and the like are as follows. In this disclosure, despite the following predictions, it has been found that when glucose is used as a ligand, it is also effective against hematopoietic tumors of suspended cells.
• Glucose Tumor research, drug delivery to brain capillary endothelial cells
• Mannose Efficient formation of giant liposomes
• Galactose Study of galactose receptors in macrophages, targeted delivery of galactose to liver cells
• Sucrose Cancer treatment with doxorubicin
• Maltose Transport of doxorubicin in cancer treatment
• Lactose Study of liposome size and stability
• Oligosaccharides Design of therapeutic inhibitors
• Lectins Pulmonary drug delivery Tomato lectin, wheat germ agglutinin: Oral administration of insulin
• NCL-aptamer Chemotherapy with cisplatin for a wide range of cancers sgc8 aptamer: For leukemia
• NX 1838 Specific binding to VEGF on cancer cells
• Anti-CD44 Selective targeting of cancer cells
• DAG-NX213 Specificity to VEGF that promotes angiogenesis
• the cationic artificial nucleic acid and the hydrophilic polymer may be bonded in various bonding modes or may be bonded via a linker.
• the linker these may be used alone or in combination, and may be bonded via an ester bond, an ether bond, a disulfide bond, or the like of an alkyl chain.
• these linkers are preferably bonded to the hydroxyl group at the 5' end or 3' end or both of these ends of the cationic artificial nucleic acid, and bonded via the cationic artificial nucleic acid and the hydrophilic polymer.
• the cationic artificial nucleic acid and the hydrophilic polymer are bonded via a linker, it is expected that the charge can be adjusted because the linker has a different structure from the hydrophilic polymer.
• a phosphoramidite it is preferable because it behaves as a negative charge like a polymer made of polyethylene glycol. Furthermore, it is advantageous in that the induction of PEG antibodies can be suppressed.
• Spacer 1 has a structure linking a cationic artificial nucleic acid and a hydrophilic polymer.
• Spacer 1 (S1) may have a bond represented by the following formula (S11). (In the formula, R 6 and R 7 represent methylene groups having 1 to 12 carbon atoms, and R 6 and R 7 may be the same or different.)
• Spacer 2 When the ligand is an aldose and the corresponding phosphoramidite reagent is unstable, a spacer 22 can be used.
• the spacer 22 has a structure that connects the hydrophilic polymer and the ligand. It is preferable that the spacer 2 (S2) is a phosphodiester bond or a phosphodiester bond containing a triazole as shown in formula (S21).
• Ka represents an amide bond containing a methylene group having 1 to 20 carbon atoms, a compound containing an aromatic group having 6 to 12 carbon atoms, or a direct bond, and q is 0 or 1, and when q is 0, the 1,2,3-triazole ring is bonded to a ligand.
• Spacer 1 and spacer 2 can have any structure in the nucleic acid analog and can be provided as necessary.
• FIG. 1 is a schematic diagram illustrating the use of a nucleic acid analog as a carrier.
• the nucleic acid analog has a cationic group and a hydrophilic polymer as a primary structure
• the target nucleic acid such as a natural nucleic acid
• An association structure is formed by electrostatic interaction with the cationic group of the nucleic acid analog, resulting in a complex (ion complex) composed of the nucleic acid analog as a carrier and the nucleic acid to be delivered.
• the cationic group and the phosphate group of the nucleic acid to be delivered can be associated by electrostatic interaction. That is, not only the nucleic acid to be delivered that has perfect complementarity (100% match), but also the nucleic acid to be delivered that has, for example, 80% or 90% complementarity, an association structure is formed by the electrostatic interaction to become a complex. Therefore, it becomes possible to deliver the nucleic acid to be delivered, which is a non-complementary strand, to the target site.
• the degree of complementarity between the cationic artificial nucleic acid and the nucleic acid to be delivered is preferably 50% or more, more preferably 80% or more, and particularly preferably 100%.
• nanoscale nucleic acid delivery structure In an aqueous environment such as blood, the hydrophilic polymer segments of this complex associate with each other to form a nanoscale nucleic acid delivery structure (hereinafter sometimes simply referred to as "structure").
• nanoscale structures include micelles and vesicles, in which the segments of the associated structure are located on the inside and the hydrophilic polymer segments are located on the outside (see “Nanostructure Formation” in the figure).
• These micelles and vesicles form spherical structures with a hollow space in the center, and drugs such as low-molecular-weight compounds can be encapsulated in this hollow space. This makes it possible to deliver not only the target nucleic acid but also low-molecular-weight drugs.
• the diameter of the hollow space is approximately 50 to 500 nm.
• a micelle has a structure in which a hydrophilic polymer segment is located at the outermost shell of a spherical structure, and an association structure segment is located facing the hollow part at the center of the spherical structure.
• a vesicle has a bilayer structure in which two complexes are associated via an association structure segment, and one hydrophilic polymer segment of this bilayer is located at the outermost shell of the spherical structure, and the other hydrophilic polymer segment is located facing the hollow part at the center of the spherical structure.
• Such structures have high degradation stability because the nucleic acid to be delivered is located inside the spherical structure.
• the above structure has excellent retention in the blood because it has hydrophilic polymer segments in the outermost shell.
• the structure is then enveloped in an endosome at the target site, such as a cell, and taken up into the cytoplasm, after which it escapes the endosome and releases the nucleic acid to be delivered or a small molecule drug into the cytoplasm or nucleus (see “Biochemical Evaluation” in the figure).
• nucleic acid analogs disclosed herein are effective as carriers for delivering the anti-onco-microRNA microRNA-143 as the target nucleic acid, targeting the cancer-promoting gene K-Ras and its network.
• the nucleic acid delivery structure may have a surface modified with various ligands. By modifying the surface of the structure with such ligands, it is possible to impart targeting properties to the structure.
• the nucleic acid delivery structure is referred to as "Reversibly Ionic Oligonucleotide-based Nanoparticles" and may be written as RION or RIO.
• a nucleic acid analog can be produced by various methods, including a method in which a cationic artificial nucleic acid and a hydrophilic polymer are separately synthesized and then bonded together. That is, the method for producing a nucleic acid analog is as follows: (i) a cationic nucleic acid synthesis step of synthesizing a cationic artificial nucleic acid; (ii) a hydrophilic polymerizing step of synthesizing a hydrophilic polymer; (iii) a binding step of binding the cationic artificial nucleic acid to a hydrophilic polymer.
• the 3' or 5' of the nucleic acid may be fixed, a cationic artificial nucleic acid may be synthesized first, a hydrophilic polymer may be synthesized, and then the ligand may be bound to the cationic artificial nucleic acid, or the ligand may be fixed first, a hydrophilic polymer may be synthesized, and then the nucleic acid may be synthesized after the hydrophilic polymer, and a spacer may be inserted between the hydrophilic polymer and the nucleic acid.
• the nucleic acid produced in this way is anionic nucleic acid, but the nucleic acid produced by the above reaction may also be cationized. Further details will be provided below.
• a-1) Two-step synthesis method I nucleic acid is sulfurized (S-modified) by automatic nucleic acid synthesis, and then a hydrophilic polymer phosphoramidite (e.g., ethylene glycol phosphoramidite) is linked to this to synthesize an oligoPS-hydrophilic polymer, which is then reacted with a Br compound to introduce a cationic group into the nucleic acid (TEG-PS oligo and HEG-PS oligo systems in the examples described below).
• a hydrophilic polymer phosphoramidite e.g., ethylene glycol phosphoramidite
• the cationic artificial nucleic acid can be synthesized by a method including the steps of introducing a thiophosphate ester into a linking structure and reacting a bromo compound having a cationic group with the thiophosphate ester to introduce the cationic group into the linking structure, as described in the Examples below.
• thiophosphate can be synthesized by the known phosphoramidite method.
• a nucleoside or nucleotide protected with a 4,4'-dimethoxytrityl (DMTr) group at its 5' end is supported on a solid phase (supporting step).
• the DMTr group is deprotected with a deprotecting reagent such as dichloroacetic acid (deprotecting step), and the nucleoside or nucleotide is coupled with a phosphoramidite nucleotide in the presence of an activating agent such as 4,5-dicyanoimidazole (coupling step).
• a deprotecting reagent such as dichloroacetic acid
• an activating agent such as 4,5-dicyanoimidazole
• the phosphite is converted to a thiophosphate by a sulfurizing agent such as (N,N-dimethylaminomethylidene)amino-3H-1,2,4-dithiazoline-3-thione (DDTT) (sulfurizing step).
• a sulfurizing agent such as (N,N-dimethylaminomethylidene)amino-3H-1,2,4-dithiazoline-3-thione (DDTT)
• DDTT sulfurizing step
• the phosphite is converted to a phosphoric acid diester by an oxidizing agent containing iodine, pyridine, or the like (oxidizing step).
• oxidizing step By repeating this step, a synthetic nucleic acid containing a thiophosphate in the linking structure can be produced.
• By changing the type of base in the phosphoramidite it is possible to produce a synthetic nucleic acid having a desired sequence.
• a sulfurization step instead of an oxid
• the resulting synthetic nucleic acid is reacted with a bromo compound having an amine or ammonium group.
• the bromo compound include 3-bromo-1-propylamine hydrobromide, 2-bromo-N,N-diethylethylamine hydrobromide, and (3-bromopropyl)trimethylammonium bromide.
• the reaction between the synthetic nucleic acid and the bromo compound can be carried out in a phosphate buffer solution or the like, and the reaction conditions can be appropriately set, but for example, the pH can be within the range of 5 to 7, the reaction temperature can be 30 to 60° C., and the reaction time can be 10 to 50 hours.
• nucleic acid is converted to boranophosphate (B) by automatic nucleic acid synthesis, and then a hydrophilic polymer phosphoramidite (e.g., ethylene glycol phosphoramidite) is linked to this to synthesize an oligo B-hydrophilic polymer ( Figure 2), and then an amino compound is reacted by iodine oxidation to introduce a cationic group (Strategy 2 in Figure 3).
• a hydrophilic polymer phosphoramidite e.g., ethylene glycol phosphoramidite
• a cationic artificial nucleic acid is synthesized by automatic nucleic acid synthesis, and then a hydrophilic portion is introduced by a click reaction (the PEG-PMO system in the examples described later).
• this method involves synthesizing a cationic artificial nucleic acid having a cationic group in the backbone, and then linking it to a hydrophilic azide compound such as azide polyethylene glycol by a click reaction.
• a diisopropylamidophosphoryl compound and a nucleotide monomer are reacted with a nucleotide supported on a solid phase ("1. Coupling” in the diagram). Some of the hydroxyl groups are protected with protecting groups ("2. Capping"), and then oxidized or boronized with an oxidizing agent or boronizing agent ("3. Oxidation or Boronation"). An amino group compound is reacted by iodine oxidation to introduce a cationic group.
• Figure 3 shows an example of a synthesis scheme for nucleic acid analogs.
• a scheme is shown for synthesizing a nucleic acid analog having a nucleotide backbone as a cationic artificial nucleic acid and polyethylene glycol as a hydrophilic polymer.
• phosphoramidite synthesis section of the figure, phosphoramidite nucleotides are synthesized in the order of compounds 1 to 3 by a known method. Meanwhile, phosphoramidite polyethylene glycol is synthesized in the order of compounds 4 to 6.
• nucleic acid analog is synthesized according to the scheme shown in "Strategy 1: Synthesis of cation-introduced oligonucleic acid” in the figure.
• the phosphoramidite nucleotide and phosphoramidite polyethylene glycol synthesized according to the above scheme, and a phosphoramidite disulfide having a disulfide bond and a phosphoramidite in the molecule are used as raw materials.
• reactions such as protection with a protecting group and deprotection are carried out to bind a hydrophilic polymer to the 5' position of the cationic artificial nucleic acid.
• a cationic artificial nucleic acid may be synthesized in one step by solid-phase synthesis.
• Patent Document 1 and the like can be used as a reference for the method of producing a cationic artificial nucleic acid.
• the nucleic acid delivery structure is formed by associating the nucleic acid to be delivered with the nucleic acid analog to form a complex (association step). When there is complementarity between the cationic group of the nucleic acid analog and the phosphate group of the nucleic acid to be delivered, the two are bound by annealing to form a double strand.
• Annealing is performed by raising the temperature to a predetermined temperature and then lowering the temperature. For annealing, raising the temperature to 80°C or higher is preferable, and raising the temperature to 90°C or higher is more preferable.
• the time for holding the elevated temperature is preferably 5 minutes or more, more preferably 10 minutes or more. Thereafter, the temperature is lowered to 50°C or lower, preferably 30°C or lower, and the temperature is held for 10 minutes or more, preferably 30 minutes or more.
• the complexes self-aggregate in an aqueous solvent such as water or an aqueous solution to form structures such as micelles or vesicles.
• concentration of the complexes to form the structures is approximately 25 to 2500 ⁇ M, and a range of 100 to 1000 ⁇ M is more preferable.
• the nucleic acid delivery method of the present disclosure includes an administration step of administering the nucleic acid delivery structure to incorporate the nucleic acid delivery structure into a target site, and a release step of releasing the nucleic acid within the target site.
• the structure bound to the nucleic acid to be delivered is administered to humans or other mammals.
• the drug containing the structure is preferably administered into the blood, but can be appropriately determined depending on the disease to be treated and the type of hydrophilic polymer.
• the target site is a cell
• the administered structure is incorporated into the cytoplasm via an endosome.
• the nucleic acid to be delivered is released from the structure incorporated into the cell, which is the target site.
• the structure collapses in the acidic environment within the endosome, making it easier to release the nucleic acid to be delivered.
• a highly pH-responsive cationic group such as quaternary ammonium
• the structure collapses in the acidic environment within the endosome, making it easier to release the nucleic acid to be delivered.
• DLD-1 (culture line of human colon cancer cells), ASF4-1 (culture line of human fibroblast cells), PRMI8226 (culture line of human multiple myeloma cells), NB4 (culture line of human acute promyelocytic leukemia pre-B cells), HL-60 (culture line of human promyelocytic leukemia cells) and Jrukat cells (cell line derived from human leukemia T cells) used in the evaluation were obtained from the JCRB Cell Bank of the National Institutes of Biomedical Innovation, Health and Nutrition.
• RPMI-1640 medium (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) was used to culture DLD-1, PRMI8226, NB4, HL-60 and Jrukat cells.
• Eagle's minimum essential medium was used to culture ASF4-1 cells. All media were supplemented with heat-inactivated fetal bovine serum (Nichirei Biosciences) to make the content 10 (v/v)%, and the cells were incubated in an atmosphere of 95% air and 5% carbon dioxide.
• Cell Evaluation All cells were seeded in 12-well plates at 0.25 x 105 cells/well one day before sample processing. Samples were administered so that the concentration of miR-143#12 was 0.5-10 nM. After 48 or 72 hours of culture, each cell was evaluated. Cell proliferation was evaluated by counting the number of live cells using the trypan blue dye exclusion method. mRNA expression levels and protein expression levels were analyzed by collecting cells after culture.
• the particle size distribution was determined by dynamic light scattering using a Zetasizer (Beckman Coulter Inc.) For the measurement, a 10 ⁇ M PBS solution (pH 7.4) of the nucleic acid to be delivered (miR-143 of the present disclosure) was prepared and used as a sample.
• qRT-PCR of mRNA to evaluate the expression levels of NRAS and KRAS mRNA was performed using qPCT Thunderbird® Next XYBR® qPCR Mix (Toyobo). The primers for NRAS, KRAS, and GAPDH are as follows: GADPH was used as an internal control. Relative expression levels were calculated using the ⁇ Ct method.
• NRAS sense strand 5'-CCT CCT CAC TTG GCT GTC TG-3' (SEQ ID NO:27)
• NRAS antisense strand 5'-TCA CGT TTG CGG TTT GGT TC-3' (SEQ ID NO:28)
• KRAS sense strand 5'-TGG TGG TGT GCC AAG ACA TT-3' (SEQ ID NO: 29)
• KRAS antisense strand 5'-CAC CTC ACC ATG CCA TCT CA-3' (SEQ ID NO: 30)
• GAPDH sense strand 5'-TCT AGA CGG CAG GTC AGG TCC ACC-3' (SEQ ID NO: 31)
• GAPDH antisense strand 5'-CCACCC ATG GCA AAT TCC ATG GCA-3' (SEQ ID NO: 32)
• the cell lysate was prepared with a lysis buffer consisting of 10 mM Tris-HCl (pH 7.4), 1% NP-40, 0.1% deoxycholic acid, 0.1% SDS, 2% protease inhibitor cocktail, 2% phosphatase inhibitor cocktail II, and 2% phosphate inhibitor cocktail III (Sigma-Aldrich), and left on ice for 20 minutes. After centrifugation at 13,000 rpm (16,200 x g) for 20 minutes at 4°C, the supernatant was used as a protein sample. Protein content was measured using a DC protein assay kit (Bio-Rad).
• Two micrograms of dissolved protein was separated by dodecyl sulfate polyacrylamide gel using 10.0% or 12.5% polyacrylamide gels and electroblotted onto Immobilon-P membranes with polyvinylidene difluoride (PVDF) (Merck Millipore). After blocking nonspecific binding with 20% PVDF in Can Get Signal (registered trademark: Toyobo) in distilled water for 1 hour, the Immobilon-P membranes were incubated overnight at 4°C with primary antibodies using Get Signal Solution 1 (Toyobo). The next day, the membranes were washed three times with Tris-buffered saline (TBS) containing 0.1% Tween 20 (TBS-T).
• TBS Tris-buffered saline
• Immunoblots were visualized using Immobilon Forte Western horseradish peroxidase (HRP) substrate (Millipore). Immunoblot images were acquired using an ImageQuant LAS4000 biomolecular imager (GE Healthcare Life Sciences, Pittsburgh, PA, USA). Densitometry analysis was performed using image analysis software ImageQuant Total Lab-7 (GE Healthcare Life Sciences).
• HRP horseradish peroxidase
• the primary antibodies used were: Anti-AKT, ERK1/2, PARP, Cyclin D1, ERK5, SOS1, GLUT1, and GLUT4 (Cell Signaling Technology); anti-KRAS (LifeSpan BioScience, Seattle, WA, USA); anti-Total (T)-RAS, including KRAS, HRAS, NRAS (abcam). anti- ⁇ -actin (Sigma-Aldrich); anti- ⁇ -Tubulin (Medical & Biological Laboratories, Inc., Tokyo, Japan).
• G guide strand of miR-143
• P artificial nucleic acid of the present disclosure
• AS antisense strand of siRNA control
• S sense strand of siRNA control
• N RNA n:DNA
• Mf 2'-F-RNA *: Anionic skeleton of phosphoric diester
• ⁇ -P(S)OH- +: cationic backbone of phosphate
• X hexaethylene glycol
• Y linker (spacer 2 of the present application)
• Z Acetylene derivative
• Glu Glucose Underline: Mismatched base
• sequences in the above table and their sequence numbers in the sequence listing are as follows: P#1 (SEQ ID NO:21), P#2 (SEQ ID NO:22), P#3 (SEQ ID NO:23), AS#1 (SEQ ID NO:24), S#1 (SEQ ID NO:25), S#2 (SEQ ID NO:26)
• the artificial nucleic acid P#2 was synthesized using an automatic nucleic acid synthesizer. After linking the nucleotide sequence described in P#2 with a thiophosphate diester, 6 units of hexaethylene glycol were introduced with a phosphate group by the phosphoramidite method, and a triple bond structure shown in the following chemical formula was constructed at the end to obtain P#2 in which the artificial nucleic acid was linked with a thiophosphate diester.
• 1-azidoglucose was synthesized by the method described in WO 2016/152980.
• nucleic acid P#2 synthesized above linked with a thiophosphate diester was dissolved in PBS (pH 7.4).
• nucleic acid P#2 (10 nmol, 1 equivalent) was mixed with 50 mM sodium ascorbate solution (10 equivalents), 50 mM copper (II) sulfate (10 equivalents), and 50 mM 1-azido glucose in PBS (pH 7.4) and reacted for 15 minutes.
• the sample after the reaction was purified by high-performance liquid chromatography to obtain nucleic acid P#3 linked with an artificial nucleic acid with a thiophosphate diester.
• Nucleic acid P#3 (4 nmol) in which an artificial nucleic acid is linked with a thiophosphate diester was reacted with 4 M 2-(diethylamino)ethyl bromide hydrobromide (Waco, Osaka, Japan, 2 ⁇ L, 8 mmol) in PBS at 45°C for 24 hours. After the reaction, the sample was dialyzed against distilled water for 3 to 5 days and purified by freeze-drying to obtain P#3.
• Glu-RION miR-143 analog G#12 (sometimes referred to as "miR-143G#12") and the nucleic acid analog P#3 obtained in Production Example 1 were mixed in a ratio of 1:5 at 98°C for 15 minutes and then incubated at 25°C for 10 minutes.
• Glu-RION-miR143#12 was obtained by annealing at 45°C for 50 min.
• miR-143 analog G#12 and the nucleic acid analog P#3 obtained in Production Example 1 were mixed in a molar ratio of 1:5 and annealed. The results are shown in Figure 1c. It can be seen that miR-143G#12 and the nucleic acid analog P#3 were annealed and self-assembled to form RION-miR143#12 as nanoparticles.
• the IC 50 of Glu-RION-miR143#12 was 3.3 nM, and it suppressed the expression of KRAS, Sos-1, Akt, and ERK1/2, which are the target genes of miR-143.
• the cell death of DLD-1 was found to be apoptosis because cleaved PARP-1 was observed. Therefore, it is considered that the introduction of nucleic acids into cancer cells via glucose transporters is useful.
• Example 1 We investigated the characteristics of human KRAS mutant hematopoietic tumor cells and the growth-suppressing effect of delivery of miR-143#12 using Glu-RION-miR143#12.
• RPMI8226 KRASG12A
• NB4 KRASA18D
• HL-60 NRASQ61L
• Jurkat wild type
• Glu-RION-miR143#12 induced cell death concentration-dependently at 1-10 nM. Therefore, Glu-RION-miR143#12, which is a nucleic acid introduced into PRMI8226 cells, is a useful nucleic acid introduction technique because it allows nucleic acid introduction at low concentrations.
• the cell proliferation inhibitory effect was also confirmed in HL-60 cells and NB4 cells, but apoptosis was also observed in NB4 cells (Figure 5d). It was confirmed that glucose transporters (GLUT1 and GLUT4) were expressed in hematopoietic tumor cells (Figure 5g).
• Example 2 The effect of delivering miR-143#12 to hematopoietic tumors using Glu-RION-miR143#12 was examined.
• the expression levels of RAS and RAS-related proteins Sos-1, ERK1/2, ERK5, Akt
• FIG. 6a This result indicates that by using Glu-RION-miR143#12, the expression of the target genes of miR-143, TRAS, Akt, ERK5, ERK1/2, was suppressed by miR-143#12. This is thought to be because miR-143#12 was delivered into the cells and the expression of the protein was suppressed by RNA interference.
• a glucose transporter which is one of the nutrient transmitters, is present, and it is thought that the ligand present on the surface of RION recognizes this transmitter, and the nucleic acid is efficiently taken up into the floating cells.
• Example 3 Using Glu-RION-miR143#12, we investigated the difference between the stimulation of peripheral lymphocytes in the steady state of miR-143#12 with and without concanavalin A. The results are shown in Figure 7. The survival rate of peripheral lymphocytes increased when concanavalin A was not used, but when concanavalin A was used, there was a tendency for the proliferation of peripheral lymphocytes to be suppressed. This proliferation suppression is thought to be due to the arrest of the cell cycle.

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