Oligo-nucleic acid nanoparticle

Abstract

To increase the amount of oligo-nucleic acid transported into cytoplasm by efficiently interacting a cell internalization enhancer with a target cell.SOLUTION: An oligo-nucleic acid nanoparticle according to one aspect of the present invention comprises: a core constituted by a dendritic polymer; a plurality of oligo-nucleic acids bonded to the core; one or a plurality of hydrophilic polymers bonded to the plurality of oligo-nucleic acids; and one or a plurality of cell internalization enhancers bonded to the one or plurality of hydrophilic polymers. The bonds between the core and the oligo-nucleic acids, the bonds between the oligo-nucleic acids and the hydrophilic polymer(s), and the bonds between the hydrophilic polymer(s) and the cell internalization enhancer(s) are each independently direct bonds or bonds facilitated by a linker. The cell internalization enhancer is located on the surface of the oligo-nucleic acid nanoparticle.SELECTED DRAWING: Figure 1

Description

Technical Field

[0001] The present invention relates to oligonucleic acid nanoparticles.

Background Art

[0002] Nucleic acid pharmaceuticals that can directly control the expression of various gene products expressed in cells can be therapeutic agents for diseases that cannot be treated with conventional pharmaceuticals, and thus their medical applications are highly expected. However, nucleic acid pharmaceuticals have a large molecular weight of the nucleic acid molecule itself, have many negative charges, and are highly hydrophilic, and thus cannot spontaneously penetrate cell membranes. As methods for transporting nucleic acid molecules into the cytoplasm, which is the location where their action is expressed, a method of modifying nucleic acid molecules with cell internalization promoters such as hydrophobic molecules and sugars, and a method of encapsulating nucleic acid molecules in functional nanoparticles are known.

[0003] For example, Patent Document 1 and Non-Patent Document 1 disclose a method of preparing a nanostructure by covalently bonding a nucleic acid molecule and a cell internalization promoter to a polymer and transporting the nucleic acid molecule into the cytoplasm.

Prior Art Documents

Patent Documents

[0004]

Patent Document 1

Non-Patent Documents

[0005]

Non-Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0006] In the technologies of Patent Document 1 and Non-Patent Document 1, there was room for improvement in efficiently interacting a cell internalization promoter with target cells. The main object of the present invention is to efficiently interact a cell internalization promoter with target cells to improve the amount of oligonucleic acid transported into the cytoplasm.

Means for Solving the Problems

[0007] As a result of intensive studies, the present inventors have developed a technique capable of efficiently transporting oligonucleic acid into cells. The present invention provides an oligonucleic acid nanoparticle, which is a functional nanoparticle having a structure suitable for reducing non-specific interaction with biological components and containing a cell internalization promoter, and a method for producing the same.

[0008] That is, one aspect of the present invention provides an oligonucleic acid nanoparticle composed of a single molecule, which includes a core composed of a dendritic polymer, and a plurality of oligonucleic acids, one or more hydrophilic polymers, and one or more cell internalization promoters disposed around the core. The oligonucleic acids are preferably covalently bound to the core, the hydrophilic polymers are preferably covalently bound to the oligonucleic acids, and the cell internalization promoters are preferably covalently bound to the hydrophilic polymers. Another aspect of the present invention provides an oligonucleic acid nanoparticle composed of a single molecule, which includes a core composed of a dendritic polymer, and a plurality of oligonucleic acids, one or more spacers, one or more hydrophilic polymers, and one or more cell internalization promoters disposed around the core. The oligonucleic acids or spacers are preferably covalently bound to the core, the hydrophilic polymers are preferably covalently bound to the spacers, and the cell internalization promoters are preferably covalently bound to the hydrophilic polymers. In another aspect of the present invention, the reactive functional groups of the dendritic polymer forming the core are also used for binding to a capping agent in addition to the oligonucleic acids or spacers. In these aspects, since the hydrophilic polymers are located outside the spatial spread (radius of gyration) of the oligonucleic acids and the cell internalization promoters are located on the surface of the oligonucleic acid nanoparticles, the oligonucleic acid nanoparticles can efficiently interact with target cells, and the non-specific interaction between the oligonucleic acid nanoparticles and biological components other than target cells can be reduced.

[0009] That is, the present invention is as follows. [1] A core composed of a dendritic polymer, a plurality of oligonucleic acids bound to the core, one or more hydrophilic polymers bound to the plurality of oligonucleic acids, and one or more cell internalization promoters bound to the one or more hydrophilic polymers, an oligonucleic acid nanoparticle comprising: The binding between the core and the oligonucleic acid, the binding between the oligonucleic acid and the hydrophilic polymer, and the binding between the hydrophilic polymer and the intracellular uptake promoter are each independently a direct binding or a binding via a linker. An oligonucleic acid nanoparticle in which the intracellular uptake promoter is located on the surface of the oligonucleic acid nanoparticle. [2] The oligonucleic acid nanoparticle according to [1], wherein the direct binding, or the binding between the linker and the core, the oligonucleic acid, the hydrophilic polymer, or the intracellular uptake promoter is by a covalent bond, a metal coordination, or a host-guest interaction. [3] The oligonucleic acid nanoparticle according to [1], wherein the direct binding, or the binding between the linker and the core, the oligonucleic acid, the hydrophilic polymer, or the intracellular uptake promoter is by a covalent bond or a metal coordination. [4] The oligonucleic acid nanoparticle according to [1], wherein the direct binding, or the binding between the linker and the core, the oligonucleic acid, the hydrophilic polymer, or the intracellular uptake promoter is by a covalent bond. [5] The oligonucleic acid nanoparticle according to any one of [1] to [4], wherein at least a part of the reactive functional groups of the dendritic polymer is capped with a capping agent. [6] A core composed of a dendritic polymer, a plurality of oligonucleic acids bound to the core, one or more hydrophilic polymers bound to the core via a spacer, one or more intracellular uptake promoters bound to the one or more hydrophilic polymers, an oligonucleic acid nanoparticle comprising: The binding between the core and the oligonucleic acid, the binding between the core and the spacer, the binding between the spacer and the hydrophilic polymer, and the binding between the hydrophilic polymer and the intracellular uptake promoter are each independently a direct binding or a binding via a linker. An oligonucleic acid nanoparticle in which the intracellular uptake promoter is located on the surface of the oligonucleic acid nanoparticle. [7] The oligonucleotide nanoparticle according to [6], wherein the direct bond, or the bond between the linker and the core, the oligonucleotide, the spacer, the hydrophilic polymer, or the cell internalization promoter is by covalent bond, metal coordination or host-guest interaction. [8] The oligonucleotide nanoparticle according to [6], wherein the direct bond, or the bond between the linker and the core, the oligonucleotide, the spacer, the hydrophilic polymer, or the cell internalization promoter is by covalent bond or metal coordination. [9] The oligonucleotide nanoparticle according to [6], wherein the direct bond, or the bond between the linker and the core, the oligonucleotide, the spacer, the hydrophilic polymer, or the cell internalization promoter is by covalent bond.

[10] The oligonucleotide nanoparticle according to any one of [6] to [9], wherein at least a part of the reactive functional groups of the dendritic polymer is capped with a capping agent.

[11] The oligonucleotide nanoparticle according to [5] or

[10] , wherein the capping agent is one or more molecules selected from the group consisting of hydrophilic molecules and hydrophobic molecules.

[12] The oligonucleotide nanoparticle according to

[11] , wherein the capping agent is a hydrophilic molecule.

[13] The oligonucleotide nanoparticle according to

[11] , wherein the capping agent is one or more hydrophilic molecules selected from the group consisting of electrically neutral hydrophilic molecules, polar molecules that are protonated under acidic conditions, anionic molecules, and cationic molecules.

[14] The oligonucleotide nanoparticle according to

[11] , wherein the capping agent is one or more hydrophilic molecules selected from the group consisting of electrically neutral hydrophilic molecules, polar molecules that are protonated under acidic conditions, and anionic molecules.

[15] The oligonucleotide nanoparticle according to

[11] , wherein the capping agent is a hydrophobic molecule.

[16] The oligonucleotide nanoparticle according to

[11] , wherein the capping agent is one or more molecules selected from the group consisting of aliphatic compounds, aromatic compounds, trialkylamines, and steroids.

[17] The oligonucleic acid nanoparticle according to

[11] , wherein the capping agent is an aliphatic compound.

[18] The oligonucleic acid nanoparticle according to any one of [1] to

[17] , wherein the dendritic polymer is a dendrigraft or a dendrimer.

[19] The oligonucleic acid nanoparticle according to any one of [1] to

[17] , wherein the monomers in the dendritic polymer are bonded to each other by an amide bond, an ester bond, or a glycoside bond.

[20] The oligonucleic acid nanoparticle according to any one of [1] to

[17] , wherein the monomers in the dendritic polymer are bonded to each other by an amide bond or an ester bond.

[21] The oligonucleic acid nanoparticle according to any one of [1] to

[17] , wherein the dendritic polymer is poly-L-lysine dendrigraft, polyamidoamine dendrimer, or 2,2-bis(hydroxyl-methyl)propionic acid dendrimer.

[22] The oligonucleic acid nanoparticle according to any one of [1] to

[21] , wherein the oligonucleic acid is a gene expression regulator.

[23] The oligonucleic acid nanoparticle according to

[22] , wherein the gene expression regulator is a molecule that suppresses the expression of mRNA.

[24] The oligonucleic acid nanoparticle according to

[22] , wherein the gene expression regulator is an RNA interference-inducing nucleic acid or an antisense nucleic acid.

[25] The oligonucleic acid nanoparticle according to any one of [6] to

[10] , wherein the spacer is one or more spacers selected from the group consisting of polyethylene glycol, poly(2-alkyl-2-oxazoline), polypeptide, and polypeptoid.

[26] The oligonucleic acid nanoparticle according to any one of [6] to

[10] , wherein the spacer is polyethylene glycol or a cationic polypeptide.

[27] The oligonucleic acid nanoparticle according to any one of [1] to

[26] , wherein the hydrophilic polymer is one or more hydrophilic polymers selected from the group consisting of polyethylene glycol, poly(2-alkyl-2-oxazoline), polypeptide, and polypeptoid.

[28] The oligonucleic acid nanoparticles according to any one of [1] to

[26] , wherein the hydrophilic polymer is at least one aqueous polymer selected from the group consisting of polyethylene glycol, poly(2-methyl-2-oxazoline), EK peptide, and polysarcosine.

[29] The oligonucleic acid nanoparticles according to any one of [1] to

[28] , wherein the intracellular uptake promoter is at least one intracellular uptake promoter selected from the group consisting of polypeptides, aptamers, antibodies or fragments thereof, sugar chains, lipids, and other low molecular weight compounds.

[30] The oligonucleic acid nanoparticles according to any one of [1] to

[28] , wherein the intracellular uptake promoter is a low molecular weight compound having a molecular weight of 2000 or less other than hydrophobic molecules, polycations, polypeptides, aptamers, antibodies or fragments thereof, sugars or sugar chains, and lipids.

[31] The oligonucleic acid nanoparticles according to any one of [1] to

[28] , wherein the intracellular uptake promoter is a polypeptide.

[32] The oligonucleic acid nanoparticles according to any one of [1] to

[28] , wherein the intracellular uptake promoter is an aptamer.

[33] The oligonucleic acid nanoparticles according to any one of [1] to

[28] , wherein the intracellular uptake promoter is an antibody or a fragment thereof.

[34] The oligonucleic acid nanoparticles according to any one of [1] to

[28] , wherein the intracellular uptake promoter is a sugar chain.

[35] The oligonucleic acid nanoparticles according to any one of [1] to

[28] , wherein the intracellular uptake promoter is a lipid.

[36] A pharmaceutical composition comprising the oligonucleic acid nanoparticles according to any one of [1] to

[35] as an active ingredient.

[37] Containing the oligonucleic acid nanoparticles according to any one of [1] to

[35] as an active ingredient, A therapeutic or prophylactic agent for a disease selected from the group consisting of inborn errors of metabolism, congenital endocrine diseases, single-gene diseases, neurodegenerative diseases, neurological diseases, muscular diseases, meningitis, encephalitis, encephalopathy, lysosomal diseases, malignant neoplasms, fibrosis, inflammatory diseases, immunodeficiency diseases, autoimmune diseases, and infectious diseases.

[38] Administering an oligonucleic acid nanoparticle according to any one of [1] to

[35] in a therapeutically effective amount, A method for treating and / or preventing a disease selected from the group consisting of inborn errors of metabolism, congenital endocrine diseases, single-gene diseases, neurodegenerative diseases, neurological diseases, muscular diseases, meningitis, encephalitis, encephalopathy, lysosomal diseases, malignant neoplasms, fibrosis, inflammatory diseases, immunodeficiency diseases, autoimmune diseases, and infectious diseases.

[39] Use of an oligonucleic acid nanoparticle according to any one of [1] to

[35] for the manufacture of a therapeutic agent and / or a prophylactic agent for a disease selected from the group consisting of inborn errors of metabolism, congenital endocrine diseases, single-gene diseases, neurodegenerative diseases, neurological diseases, muscular diseases, meningitis, encephalitis, encephalopathy, lysosomal diseases, malignant neoplasms, fibrosis, inflammatory diseases, immunodeficiency diseases, autoimmune diseases, and infectious diseases.

[40] An oligonucleic acid nanoparticle according to any one of [1] to

[35] for use in the treatment and / or prevention of a disease selected from the group consisting of inborn errors of metabolism, congenital endocrine diseases, single-gene diseases, neurodegenerative diseases, neurological diseases, muscular diseases, meningitis, encephalitis, encephalopathy, lysosomal diseases, malignant neoplasms, fibrosis, inflammatory diseases, immunodeficiency diseases, autoimmune diseases, and infectious diseases.

[41] An oligonucleic acid nanoparticle according to any one of [1] to

[35] , in combination with one or more therapeutic agents and / or one or more prophylactic agents for the disease, wherein the disease is selected from the group consisting of inborn errors of metabolism, congenital endocrine diseases, single-gene diseases, neurodegenerative diseases, neurological diseases, muscular diseases, meningitis, encephalitis, encephalopathy, lysosomal diseases, malignant neoplasms, fibrosis, inflammatory diseases, immunodeficiency diseases, autoimmune diseases, and infectious diseases, a medicament.

[42] An oligonucleic acid nanoparticle according to any one of [1] to

[35] for treating a disease in combination with one or more therapeutic agents and / or one or more prophylactic agents for the disease, The disease is an oligonucleotide nanoparticle selected from the group consisting of inborn errors of metabolism, congenital endocrine diseases, single-gene diseases, neurodegenerative diseases, nerve diseases, muscle diseases, meningitis, encephalitis, encephalopathy, lysosomal diseases, malignant neoplasms, fibrosis, inflammatory diseases, immunodeficiency diseases, autoimmune diseases, and infectious diseases.

[43] (a1) A step of binding a plurality of oligonucleotides to a core composed of a dendritic polymer; (a2) A step of binding a hydrophilic polymer to the oligonucleotide; (a3) A step of binding a cell internalization promoter to the hydrophilic polymer, which is a method for producing the oligonucleotide nanoparticle according to any one of [1] to [4].

[44] (b1) A step of binding one or more spacers to a core composed of a dendritic polymer; (b2) A step of binding a plurality of oligonucleotides to the core; (b3) A step of binding a hydrophilic polymer to the spacer; (b4) A step of binding a cell internalization promoter to the hydrophilic polymer, which is a method for producing the oligonucleotide nanoparticle according to any one of [6] to [9].

[45] (a1) A step of binding a plurality of oligonucleotides to a core composed of a dendritic polymer; (a2) A step of binding a hydrophilic polymer to the oligonucleotide; (a3) A step of binding a cell internalization promoter to the hydrophilic polymer; (a4) A step of binding a capping agent to the core, which is a method for producing the oligonucleotide nanoparticle according to [5].

[46] (b1) A step of binding one or more spacers to a core composed of a dendritic polymer; (b2) A step of binding a plurality of oligonucleotides to the core; (b3) A step of binding a hydrophilic polymer to the spacer; (b4) A step of binding a cell internalization promoter to the hydrophilic polymer; (a5) A step of binding a capping agent to the core, which is a method for producing the oligonucleotide nanoparticle according to

[10] .

Advantages of the Invention

[0010] According to the present invention, since the intracellular uptake promoter can efficiently interact with target cells, oligonucleotides can be efficiently transported into cells, and thus the amount of oligonucleotides transported into the cytoplasm can be improved.

[0011] In addition, according to the present invention, since non-specific interactions with other biological components other than target cells, such as biological components in the reticuloendothelial system, can be reduced, pharmacokinetics can be improved (i.e., blood residence time can be extended), and the amount of oligonucleotides transported into the cytoplasm can be improved.

[0012] Furthermore, according to the present invention, structural defects of self-assembled nanoparticles, which are representative of conventional functional nanoparticles, can be avoided. For example, functional nanoparticles using liposomes or micelles have unstable structures and can be destroyed by organic solvents, surfactants, dilution, shear stress, or interactions with biological components. In addition, it has been difficult to precisely control the size of these particles to less than 50 nm. In contrast, the oligonucleotide nanoparticles according to one aspect of the present invention have a stable structure and their size can be easily controlled.

Brief Description of the Drawings

[0013]

Figure 1

Figure 2

Figure 3

Figure 4

Modes for Carrying Out the Invention

[0014] Hereinafter, preferred embodiments of the present invention will be described in detail.

[0015] The oligonucleic acid nanoparticles according to one aspect of the present invention (hereinafter also referred to as oligonucleic acid nanoparticles according to the first aspect) include a core composed of a dendritic polymer, a plurality of oligonucleic acids bound to the core, one or more hydrophilic polymers bound to the plurality of oligonucleic acids, and one or more intracellular uptake promoters bound to the one or more hydrophilic polymers. As used herein, the oligonucleic acid nanoparticles mean nanoparticles composed of a single molecule formed by the binding of an oligonucleic acid and other molecules. Here, the nanoparticles mean particles having a size of 10 to 200 nm. In this field, there is abundant knowledge regarding the behavior of nanoparticles in vivo.

[0016] As used herein, the dendritic polymer means a polymer that branches dendritically from the center and has a regular branching structure. The dendritic polymer may be a dendrimer, a dendron, or a dendrigraft. A dendrimer is generally a three-dimensionally highly branched molecule having a dendritic structure and has a substantially spherical shape. A dendron has a structure in which at least one functional group at the center of the dendrimer is not branched. Dendrimers and dendrons have a regular branched structure, and their repeating units are called "generations". In the case of a dendrigraft, molecular chains are comb-like bound to the side chains on the main molecular chain, and further molecular chains are comb-like bound to the side chains of the comb-like molecular chains, thereby forming a radially expanded structure. In the case of a dendrigraft, the comb-like repeating unit is called a "generation".

[0017] The generation of the dendritic polymer is preferably from the 3rd generation to the 20th generation. For example, in the case of a polyamidoamine (PAMAM) dendrimer having an ethylenediamine core, the generation is preferably from the 5th generation to the 20th generation, more preferably from the 5th generation to the 10th generation. In the case of a polylysine dendrigraft, the generation is preferably from the 3rd generation to the 6th generation, more preferably from the 3rd generation to the 5th generation. In the case of a 2,2-bis(hydroxyl-methyl)propionic acid (Bis-MPA) dendrimer, the generation is preferably from the 4th generation to the 20th generation, more preferably from the 4th generation to the 10th generation.

[0018] The dendritic polymer constitutes the core of the oligonucleic acid nanoparticle, and its average diameter (in other words, average particle size) is preferably 5 nm or more, more preferably 5 nm to 25 nm, and even more preferably 5 nm to 15 nm. In this specification, the average particle size of the dendritic polymer (core) means the average particle size in the particle size distribution obtained by dynamic light scattering.

[0019] The monomers in the above dendritic polymer may be bonded by, for example, bonding modes such as single bond, double bond, triple bond, carbon-silicon bond, amide bond, glycoside bond, ester bond, ether bond, urethane bond, acetal bond, phosphate ester bond, thioether bond, thioester bond, disulfide bond, triazole bond, hydrazone bond, hydrazide bond, imine or oxime bond, urea or thiourea bond, amidine bond, sulfonamide bond, etc., but the bonding modes are not limited to these. Any of these bonding modes can be used, but those in which the bond is cleaved by an enzyme or under in vivo environments such as acidic conditions or a reducing environment are preferred from the viewpoint of safety. Examples of preferred bonding modes are amide bond, ester bond, or glycoside bond, but are not limited thereto.

[0020] Examples of suitable dendritic polymers include, but are not limited to, polylysine dendrimers, polylysine dendrigrafts, PAMAM dendrimers, Bis-MPA dendrimers, or glucose dendrimers. The dendritic polymer may be, for example, a poly-L-lysine dendrimer or a poly-L-lysine dendrigraft.

[0021] As used herein, an oligonucleic acid is a polymer having nucleotides consisting of a base, a sugar, and a phosphate as repeating units. The type of oligonucleic acid is not particularly limited, and the oligonucleic acid nanoparticles may contain one or more types of oligonucleic acids. Examples of oligonucleic acids include single-stranded or double-stranded ones consisting of, for example, RNA, DNA, or a combination thereof, including oligonucleic acids in which RNA and DNA are mixed in the same strand. The nucleotides contained in the oligonucleic acid may be natural-type nucleotides, chemically modified non-natural-type nucleotides, or nucleotides to which molecules such as amino groups, thiol groups, or fluorescent compounds are added. The oligonucleic acid may be a non-natural-type oligonucleic acid, and the range of non-natural-type oligonucleic acids includes artificial molecules having an action of controlling gene expression similar to that of natural-type oligonucleic acids, such as peptide nucleic acids (PNA) having a peptide structure in the main chain and morpholino nucleic acids having a morpholine ring in the main chain.

[0022] The function or action of the oligonucleic acid is not limited, but examples of the oligonucleic acid include antisense nucleic acids, sgRNA, RNA editing nucleic acids, miRNA, siRNA, saRNA, shRNA, or dicer substrate RNA.

[0023] The oligonucleic acid may be, for example, a gene expression regulator. A gene expression regulator is a compound that increases or decreases the synthesis of a specific gene product. Examples of gene products include, for example, mRNA or its precursor, miRNA or its precursor, ncRNA, enzyme, antibody, or other proteins. Examples of gene expression regulators include, for example, molecules that positively or negatively control the expression of mRNA (i.e., promote or suppress the expression), or molecules that edit RNA or DNA. Such gene expression regulators include, for example, nucleic acids that induce RNA interference (RNAi) such as miRNA, siRNA (RNAi-inducing nucleic acids), antisense nucleic acids, miRNA inhibitors, RNA activating nucleic acids, RNA editing-inducing nucleic acids, or genome editing-inducing nucleic acids, but the gene expression regulators are not limited thereto.

[0024] The length of the oligonucleic acid may be, for example, 4 to 200 bases (pairs), 7 to 100 bases (pairs), or 12 to 30 bases (pairs).

[0025] The number of oligonucleic acids in the oligonucleic acid nanoparticles is not particularly limited as long as it is plural, and may be, for example, 2 or more, 6 or more, 10 or more, 15 or more, 17 or more, 18 or more, 20 or more, 25 or more, 30 or more, 31 or more, 35 or more, or 50 or more, and may be 400 or less, 200 or less, or 100 or less. When the oligonucleic acid is covalently bound to the dendritic polymer, the number of oligonucleic acids may be, for example, 0.5% or more, 1% or more, or 2% or more of the reactive functional groups of the dendritic polymer, preferably 3% or more or 5% or more of the reactive functional groups of the dendritic polymer. The number of oligonucleic acids in the oligonucleic acid nanoparticles can be determined, for example, by measuring the concentration of the dendritic polymer and the concentration of the oligonucleic acid in the solution containing the oligonucleic acid nanoparticles, and calculating the ratio of the oligonucleic acid to the dendritic polymer from these values. The concentration of the dendritic polymer in the solution containing the oligonucleic acid nanoparticles can be measured, for example, by high performance liquid chromatography (HPLC). The concentration of the oligonucleic acid can be determined, for example, from the absorption at 260 nm measured using an ultraviolet-visible spectrophotometer.

[0026] Oligonucleotides can be produced by known methods. Oligonucleotides can be produced, for example, by solid-phase synthesis or liquid-phase synthesis using the phosphoramidite method or the triester method, either with an automated nucleic acid synthesizer or manually.

[0027] In the present specification, the hydrophilic polymer is a hydrophilic molecule for shielding the oligonucleotide from the outside world and exposing the cell internalization promoter on the surface of the oligonucleotide nanoparticle. A hydrophilic molecule means a molecule that easily forms a hydrogen bond with water and has a property of being easily soluble in water or easily miscible with water. The hydrophilic molecule may be a charged molecule or a non-charged highly polar molecule. The charged group of the charged molecule may be a positively charged group (cation), a negatively charged group (anion), or a combination thereof. When the polymer (i.e., stealth polymer) for shielding the oligonucleotide from the outside world is hydrophilic, it can be advantageous in terms of suppressing aggregation of oligonucleotide nanoparticles, improving solubility, avoiding phagocytosis by the reticuloendothelial system, avoiding non-specific interactions with biological components, and improving pharmacokinetics (i.e., extending blood retention time). Examples of the hydrophilic polymer include polyethylene glycol (PEG), poly(2-alkyl-2-oxazoline), polypeptide, polypeptoid, or polybetaine, but the hydrophilic polymer is not limited thereto. The oligonucleotide nanoparticle may contain one or more hydrophilic polymers. The hydrophilic polymer is preferably one or more selected from the group consisting of PEG, poly(2-methyl-2-oxazoline) (pMeOx), polysarcosine (pSar), and EK peptide. The EK peptide is a peptide in which glutamic acid and lysine are arranged alternately.

[0028] One hydrophilic polymer may have a plurality of segments. Examples of the hydrophilic polymer having a plurality of segments include, but are not limited to, a polymer formed by binding an EK peptide and PEG. The hydrophilic polymer may have a linear structure or a branched structure.

[0029] As an example, when the hydrophilic polymer is PEG, the number average molecular weight of the hydrophilic polymer may be 500 or more, 1000 or more, 2000 or more, 3400 or more, 5000 or more, 6000 or more, 8000 or more, or 10000 or more. When the hydrophilic polymer is pMeOx or pSar, the number average molecular weight of the hydrophilic polymer may be 1000 or more, 2000 or more, 4000 or more, 7000 or more, 10000 or more, 15000 or more, or 20000 or more. When the hydrophilic polymer is an EK peptide, the EK peptide may be an EK peptide containing an alternating sequence of glutamic acid and lysine of 1 or more, 2 or more, 5 or more, 7 or more, 10 or more, 15 or more, or 20 or more. In this specification, the number average molecular weight is a value determined by an end-group quantification method using nuclear magnetic resonance (NMR) or size exclusion chromatography (SEC).

[0030] The number of hydrophilic polymers may be less than, more than, or the same as the number of oligonucleotides or cell internalization promoters. The number of hydrophilic polymers bound to one oligonucleotide may be one or plural, but is preferably one. There may also be hydrophilic polymers to which the cell internalization promoter is not bound. When the hydrophilic polymer is bound to the oligonucleotide by a covalent bond, the number of hydrophilic polymers may be, for example, 1 or more, 2 or more, or 1% or more of the reactive functional groups of the dendritic polymer, preferably 2% or more, more preferably 3% or more, or 5% or more of the reactive functional groups of the dendritic polymer. Also, the hydrophilic polymer is preferably bound to 40% or more, 60% or more, 80% or more, or 85% or more of the oligonucleotide. The number of hydrophilic polymers in the oligonucleotide nanoparticles can be calculated, for example, from the ratio of the oligonucleotide alone separated by SEC and the oligonucleotide bound to the hydrophilic polymer after cleaving the bond between the oligonucleotide and the dendritic polymer.

[0031] In this specification, a cell internalization promoter is a molecular species that induces the internalization of a substance bound to the cell internalization promoter into a target cell by interacting specifically or non-specifically with the target cell. In the oligonucleotide nanoparticles according to this aspect, by binding a cell internalization promoter to a hydrophilic polymer, the oligonucleotide can be efficiently transported into the target cell as compared with the case where the cell internalization promoter is not included (for example, when an oligonucleotide is used alone). As will be described later, the cell internalization promoter is located on the surface of the oligonucleotide nanoparticles.

[0032] Examples of the cell internalization promoter include, but are not limited to, substances that interact with cell surface receptors, substances that interact with membrane transporters, substances that interact with cell adhesion factors, or other substances that interact with the cell membrane surface. The cell internalization promoter may be, for example, a substance that interacts with integrin, which is a cell adhesion factor present on the cell membrane surface, a substance that interacts with epithelial cell adhesion molecule, a substance that interacts with nucleolin, a substance that interacts with vimentin, which is a cytoskeletal molecule, a substance that interacts with prostate-specific membrane antigen, a substance that interacts with a cell surface receptor such as epidermal growth factor receptor, somatostatin receptor, mannose receptor, asialoglycoprotein receptor, folate receptor, or a substance that interacts with a transporter such as glucose transporter, non-selective monoamine transporter.

[0033] Examples of the cell internalization promoter include, for example, hydrophobic molecules, polycations, polypeptides, aptamers, antibodies or fragments thereof, sugars or sugar chains, lipids, or other low-molecular compounds, but the cell internalization promoter is not limited thereto. Other low-molecular compounds are compounds having a molecular weight of 2000 or less other than hydrophobic molecules, polycations, polypeptides, aptamers, antibodies or fragments thereof, sugars or sugar chains, and lipids. The oligonucleic acid nanoparticles may contain one or more cell internalization promoters. The cell internalization promoter is preferably one or more selected from the group consisting of polypeptides, aptamers, sugars or sugar chains, and the above-mentioned other low-molecular compounds. The cell internalization promoter is more preferably a polypeptide, an aptamer, or the above-mentioned other low-molecular compound.

[0034] The molecular weight of the polypeptide may be, for example, 50 kDa or less, 15 kDa or less, 6 kDa or less, 2 kDa or less, or 1 kDa or less, but is not limited thereto. The molecular weight of the polypeptide can be determined, for example, by mass spectrometry.

[0035] In the present specification, an antibody or a fragment thereof refers to a scaffold protein having a function of specifically binding to a specific factor, and includes immunoglobulins such as IgA, IgD, IgE, IgG, IgM, fragmented antibodies such as F(ab)'2, Fab', Fab, scFv, single domain antibodies such as shark VNAR, camelid VHH, and antibody mimetics such as affibody, affilin, monobody, and alphabody, but is not limited thereto.

[0036] Specific examples of the cell internalization promoter include polypeptides represented by the following formulas (I) to (IV). The polypeptide represented by formula (I) is cRGDfK (molecular weight: 603.7 Da, Pharmaceutics, 2018, 10, 2), which is a kind of cyclic peptide ligand (cRGD) containing an arginine-glycine-aspartic acid (RGD) sequence. cRGDfK is integrin α V β 3interacts with. cRGDs other than cRGDfK can also be used as cell internalization promoters. The polypeptide shown in formula (II) interacts with integrin α V β 6 and is c(avb6) (molecular weight: 1046.2, ACS Omega, 2018, 3, 2428 - 2436). The polypeptide shown in formula (III) is GE11 (molecular weight: 1539.7 Da) that interacts with the epidermal growth factor receptor. The polypeptide shown in formula (IV) is an octreotide derivative (OCT; molecular weight: 1577.8 Da) that interacts with the somatostatin receptor. As cRGD, commercially available products can be used. The peptides shown in formulas (II) - (IV) can be easily obtained by well-known synthetic methods.

[0037]

Chemical formula

Chemical formula

[0038] Specific examples of other cell internalization promoters include low-molecular-weight compounds shown in the following formulas (V) - (VII). The low-molecular-weight compound shown in formula (V) is folic acid that interacts with the folate receptor. The low-molecular-weight compound shown in formula (VI) is DUPA that interacts with the prostate-specific membrane antigen. The low-molecular-weight compound shown in formula (VII) is indatraline (IND) that interacts with the non-selective monoamine transporter.

[0039]

Chemical formula

[0040] Specific examples of other cell internalization promoters include sugars represented by the following formulas (VIII) to (XII). The sugar represented by formula (VIII) is glucose (Glu) that interacts with the glucose transporter. The sugar represented by formula (IX) is mannose (Man) that interacts with the mannose receptor. The sugars represented by formulas (X) and (XI) are N-acetylgalactosamine (GalNAc) and galactose (Gal) that interact with the asialoglycoprotein receptor. The sugar represented by formula (XII) is N-acetylglucosamine (GlcNAc) that interacts with vimentin, a cytoskeletal molecule.

[0041]

Chemical Structure

[0042] Other cell internalization promoters include, for example, aptamers having the base sequences represented by SEQ ID NOs: 1 to 6 shown in the following table. Examples of DNA aptamers that interact with nucleolin include AS1411 shown in SEQ ID NO: 1 (Oncotarget, 2015, 6(26), 22270-22281) and FAN-1524dI shown in SEQ ID NO: 2 (Scientific Reports, 2016, 6, 1-12). Examples of aptamers that interact with epithelial cell adhesion molecule include EpCAM Aptamer shown in SEQ ID NO: 3 (Molecular Cancer Therapeutics, 2015, 14(10), 2279-2291) and EpCAM Aptamer shown in SEQ ID NO: 4 (Theranostics, 2015, 5(10), 1083-1097). Examples of aptamers that interact with the transferrin receptor include FB4 shown in SEQ ID NO: 5 (Proc Natl Acad Sci USA., 2008, 105(41), 15908-15913) and GS24 shown in SEQ ID NO: 6 (Mol Ther Nucleic Acids, 2014, 3(1), e144).

[0043]

Table 1

[0044] The number of cell internalization promoters in the oligo-nucleic acid nanoparticles may be, for example, 1 or more, 2 or more, 6 or more, 11 or more, 18 or more, 25 or more, 26 or more, 27 or more, or 30 or more, from the viewpoint of efficiently interacting the cell internalization promoter with the target cells to improve the intracellular transport efficiency of the oligo-nucleic acid nanoparticles, and may be 400 or less, 200 or less, or 100 or less. The number of cell internalization promoters in the oligo-nucleic acid nanoparticles can be determined, for example, by measuring the concentration of the dendritic polymer and the concentration of the cell internalization promoter in the solution containing the oligo-nucleic acid nanoparticles, and calculating the ratio of the cell internalization promoter to the dendritic polymer from these values. The concentration of the dendritic polymer and the concentration of the cell internalization promoter can be measured, for example, by HPLC or an ultraviolet-visible spectrophotometer. The number of cell internalization promoters bound to one hydrophilic polymer may be one or plural.

[0045] As described above, an oligo-nucleic acid is bound to at least a part of the dendritic polymer, more specifically, the reactive functional groups of the dendritic polymer (these are terminal functional groups). In one embodiment, at least a part or all of the unreacted reactive functional groups not bound to the oligo-nucleic acid may be capped with a capping agent. Capping of the reactive functional group, in other words, is to reduce the reactivity of the reactive functional group by bonding. The capping agent protects the dendritic polymer from various interactions or chemical reactions by binding to the reactive functional group of the dendritic polymer. For example, the capping agent protects the dendritic polymer from electrostatic interaction, decomposition reaction, condensation reaction, addition reaction, etc.

[0046] In addition, the capping agent can add a function or activity that the dendritic polymer does not originally have to the dendritic polymer by binding to the dendritic polymer. Examples of such capping agents include molecules that improve stealth properties, molecules that interact with lipid bilayers, molecules having proton buffering ability, etc., but the capping agent is not limited to these.

[0047] The capping agent may be, for example, one or two molecules selected from the group consisting of a) hydrophilic molecules and b) hydrophobic molecules.

[0048] The above a) hydrophilic molecules may be a-1) electrically neutral hydrophilic molecules, a-2) polar molecules that are protonated under acidic conditions, a-3) anionic molecules, or a-4) cationic molecules. The a) hydrophilic molecules may be the same type of molecules as the hydrophilic polymer or different types of molecules from the hydrophilic polymer. In this specification, "electrically neutral" refers to the case where the number of cations and anions is equal or the difference in the number of cations and anions is within 10% based on the number of the more numerous charged groups.

[0049] Examples of the above a-1) electrically neutral hydrophilic molecules include molecules having hydrophilic groups such as hydroxyl group, alkoxy group, oxime group, ester group, amide group, imide group, alkoxyamide group, carbonyl group, sulfonyl group, nitro group, pyrrolidone group, zwitterions such as betaine, PEG, or alkoxypolyethylene glycol such as methoxypolyethylene glycol, but the hydrophilic molecules are not limited thereto.

[0050] The above a-2) polar molecules that are protonated under acidic conditions are molecules that have different charges in an acidic environment such as inside endosomes and under physiological conditions such as in blood and interstitial fluid. Polar molecules that are protonated under acidic conditions refer to molecules having an acid dissociation constant (pK a ) of 7.4 or less, preferably 5.0 to 7.4. Examples of polar molecules that are protonated under acidic conditions include molecules having polar groups such as tertiary amino group, diethyltriamine (DET) group (-NH-CH 2 -CH 2 -NH-CH 2 -CH 2 -NH 2 ), morpholino group, thiomorpholino group, imidazolyl group, pyridyl group, carboxy group, etc., but polar molecules that are protonated under acidic conditions are not limited thereto.

[0051] The above-mentioned anionic molecule (a-3) is a molecule with a negative ionic valence under physiological conditions. Examples include molecules having functional groups such as carboxy groups, sulfo groups, phosphate groups, and phosphate ester groups, but anionic molecules are not limited to these.

[0052] The above-mentioned cationic molecule (a-4) is a molecule with a positive ionic valence under physiological conditions. Examples include molecules having functional groups such as primary amino groups, secondary amino groups, tertiary amino groups, and guanidino groups, but cationic molecules are not limited to these.

[0053] The above-mentioned hydrophobic molecule (b) means a molecule that hardly forms a hydrogen bond with water and has a low affinity for water. The hydrophobic molecule may be a non-polar molecule or a molecule with a partition coefficient of 2.0 or more. Examples of hydrophobic molecules include aliphatic compounds, molecules having hydrophobic groups such as trialkylamine aromatic groups, cholesterol, or steroids, but hydrophobic molecules are not limited to these.

[0054] In this specification, "bond" refers to a direct or indirect, irreversible bond. An irreversible bond refers to a bond in which the reaction does not proceed reversibly, that is, a bond that does not dissociate by a reverse reaction once formed or a bond in which dissociation by a reverse reaction can be ignored.

[0055] The binding between the dendritic polymer (core) and the oligonucleic acid, the binding between the oligonucleic acid and the hydrophilic polymer, and the binding between the hydrophilic polymer and the intracellular uptake promoter are each independently a direct bond (i.e., a bond without a linker) or a bond via a linker. The linker is not particularly limited and may be a known linker such as PEG or an alkyl linker (e.g., a hexyl linker). The linker may consist of one type of linker or may be a linker formed by binding two or more types of linkers. When the linker is PEG, its number average molecular weight may be, for example, 1000 or less, 800 or less, 600 or less, or 300 or less.

[0056] A direct bond between a dendritic polymer (core) and an oligonucleic acid, between the oligonucleic acid and a hydrophilic polymer, or between the hydrophilic polymer and a cell internalization promoter, or a bond between the linker and the dendritic polymer, oligonucleic acid, hydrophilic polymer, or cell internalization promoter may be, for example, a covalent bond formed by a chemical reaction such as a nucleophilic addition reaction, nucleophilic substitution reaction, electrophilic substitution reaction, etc. between functional groups, a metal coordination bond such as the bond between ammonia and platinum, or a host-guest interaction such as the bond between biotin and avidin. From the viewpoint of achieving high structural stability and controlling the size of the oligonucleic acid nanoparticles, the bond is preferably a covalent bond.

[0057] Examples of covalent bonds include single bonds, double bonds, triple bonds, amide bonds, glycoside bonds, ester bonds, ether bonds, urethane bonds, acetal bonds, phosphate ester bonds, thioether bonds, thioester bonds, disulfide bonds, triazole bonds, hydrazone bonds, hydrazide bonds, imine or oxime bonds, urea or thiourea bonds, amidine bonds, sulfonamide bonds, or bonds formed by an inverse electron demand Diels-Alder reaction, but covalent bonds are not limited to these.

[0058] An amide bond is formed between a carboxy group and an amino group. An amide bond is formed, for example, using a conventional amide bond formation reaction that occurs between a preferably protected amino group and an activated carboxylic acid (such as an ester activated with N-hydroxysuccinimide).

[0059] A disulfide bond (-S-S-) is formed, for example, by thiol exchange between a component having a thiol group (also called a mercaptan group) (-SH) and an activated thiol group of another component.

[0060] A thioether bond (-S-) is formed, for example, using a conventional thioether bond formation reaction that occurs between a thiol group and a maleimide group.

[0061] The triazole bond is formed between an azide group and a carbon-carbon triple bond. The triazole bond is formed, for example, by a so-called click reaction such as Huisgen cyclization using a metal catalyst or strain-promoted alkyne-azide cycloaddition without using a metal catalyst.

[0062] Metal coordination is a bonding mode in which a metal ion and a ligand bind by forming a complex. Examples of metal ions include, but are not limited to, ions of metal elements such as platinum group elements, manganese, cobalt, copper, and gadolinium. Examples of ligands include, but are not limited to, ammonia, pyridine, bipyridine, ethylenediamine, ethylenediaminetetraacetic acid, acetylacetonate, or derivatives thereof.

[0063] Host-guest interaction is an interaction between a host molecule, which is a molecule that provides a space capable of selectively recognizing a specific molecule, and a guest molecule, which is a molecule that is accepted therein. Examples of host molecules include, but are not limited to, cyclodextrin, calixarene, cavitand, crown ether, cryptand, cucurbituril, calixarene, avidin, and streptavidin. Examples of guest molecules include, but are not limited to, adamantane, diamantane, cholesterol, naphthalene, and biotin.

[0064] The single molecules constituting the oligonucleic acid nanoparticles according to this aspect may be free forms or pharmaceutically acceptable salts. The single molecules constituting the oligonucleic acid nanoparticles may be either solvates (e.g., hydrates, ethanol solvates, propylene glycol solvates) or non-solvates. The pharmaceutically acceptable salts may be either acid addition salts or base addition salts. Examples of acid addition salts include salts with organic acids such as formate, acetate, trifluoroacetic acid (TFA), propionate, succinate, lactate, malate, adipate, citrate, tartrate, methanesulfonate, fumarate, maleate, p-toluenesulfonate, ascorbate, etc.; salts with inorganic acids such as hydrochloride, hydrobromide, sulfate, nitrate, phosphate, etc. Examples of base addition salts include alkali metal salts such as sodium salt, potassium salt, etc.; alkaline earth metal salts such as calcium salt, magnesium salt, etc.; ammonium salt; trimethylamine salt; triethylamine salt; aliphatic amine salts such as dicyclohexylamine salt, ethanolamine salt, diethanolamine salt, triethanolamine salt, procaine salt, etc.; aralkylamine salts such as N,N-dibenzylethylenediamine salt; heterocyclic aromatic amine salts such as pyridine salt, picoline salt, quinoline salt, isoquinoline salt, etc.; quaternary ammonium salts such as tetramethylammonium salt, tetraethylammonium salt, benzyltrimethylammonium salt, benzyltriethylammonium salt, benzyltributylammonium salt, methyltrioctylammonium salt, tetrabutylammonium salt, etc.; basic amino acid salts such as arginine salt, lysine salt, etc.

[0065] Next, the structure of the oligo-nucleic acid nanoparticles according to this aspect will be described with reference to FIG. 1. FIG. 1 is a schematic diagram showing one embodiment of the oligo-nucleic acid nanoparticles according to this aspect. The oligo-nucleic acid nanoparticles 100 include a core 50 (dendritic polymer) located at the center of the oligo-nucleic acid nanoparticles 100, a plurality of oligo-nucleic acids 11, a hydrophilic polymer 12, and a cell internalization promoter 13 arranged around the core 50. The oligo-nucleic acid 11 is bound to the core 50 via a linker 31. The hydrophilic polymer 12 is bound to the oligo-nucleic acid 11 to shield the oligo-nucleic acid 11 from the outside world, and exposes the cell internalization promoter 13 bound to the hydrophilic polymer 12 on the surface of the oligo-nucleic acid nanoparticles 100. A capping agent 21 is also bound to the core 50. In an aqueous solution, the oligo-nucleic acid 11 and the hydrophilic polymer 12 extend substantially radially from the core 50, so that the oligo-nucleic acid nanoparticles 100 take a substantially spherical shape. In FIG. 1, the oligo-nucleic acid 11 is bound to the core 50 via the linker 31. However, as described above, the oligo-nucleic acid 11 may be directly bound to the core 50. Further, the oligo-nucleic acid 11 and the hydrophilic polymer 12, and the hydrophilic polymer 12 and the cell internalization promoter 13 may be bound via any of the above-mentioned linkers.

[0066] The average particle diameter of the oligo-nucleic acid nanoparticles 100 is preferably 10 to 100 nm, more preferably 15 to 45 nm. In this specification, the average particle diameter of the oligo-nucleic acid nanoparticles means the average particle diameter in the particle size distribution obtained by dynamic light scattering. Since the oligo-nucleic acid nanoparticles 100 have a dendritic polymer as the core 50, the size can be easily controlled and precise design is possible.

[0067] For the oligonucleic acid nanoparticle 100 to be transported into cells, the intracellular uptake promoter 13 needs to interact with the cells. From the perspective of improving the transport efficiency of the oligonucleic acid nanoparticle 100 into cells, it is preferable that the density of the intracellular uptake promoter 13 is high. According to the oligonucleic acid nanoparticle 100, the intracellular uptake promoter 13 is bound to the core 50 composed of a highly branched dendritic polymer via the oligonucleic acid 11 and the hydrophilic polymer 12, so that a high density of the intracellular uptake promoter 13 can be achieved, and thus the intracellular uptake promoter 13 can efficiently interact with the target cells. Further, in order to efficiently shield the oligonucleic acid 11 from the outside world by the binding of the hydrophilic polymer 12, it is preferable that the oligonucleic acid 11 is bound to the core 50 having a small free mobility at the polymer terminal and taking a three-dimensionally distinct spatial form. From this perspective as well, the oligonucleic acid nanoparticle 100 having a dendritic polymer as the core 50 is preferable compared to the case having a linear polymer as the core.

[0068] In the oligo-nucleic acid nanoparticle 100, since the hydrophilic polymer 12 is bound to the oligo-nucleic acid 11, the hydrophilic polymer 12 and the cell internalization promoter 13 bound thereto are located outside the spatial spread (radius of gyration) of the oligo-nucleic acid 11, and the cell internalization promoter 13 can be located on the surface (i.e., the outermost shell) of the oligo-nucleic acid nanoparticle 100. When the cell internalization promoter 13 is located on the surface of the oligo-nucleic acid nanoparticle 100, the oligo-nucleic acid nanoparticle 100 can interact efficiently with target cells. In addition, since the hydrophilic polymer 12 is located outside the spatial spread of the oligo-nucleic acid 11, an effect of reducing non-specific interaction between the oligo-nucleic acid nanoparticle 100 and biological components other than target cells can be expected. The fact that the cell internalization promoter 13 is located on the surface of the oligo-nucleic acid nanoparticle 100 can be confirmed by evaluating the binding activity by methods such as surface plasmon resonance (SPR) method and enzyme-linked immunosorbent assay (ELISA) method. From the viewpoint of exposing the cell internalization promoter 13 on the surface of the oligo-nucleic acid nanoparticle 100, the hydrophilic polymer 12, the oligo-nucleic acid 11, and the cell internalization promoter 13 are preferably bound such that the hydrophilic polymer 12 and the cell internalization promoter 13 are present at positions further away from the spatial spread of the oligo-nucleic acid 11. For example, as shown in FIG. 1, the hydrophilic polymer 12 is preferably bound to the end of the oligo-nucleic acid 11 that is not bound to the core 50. Further, although it depends on the shape of the hydrophilic polymer 12, the cell internalization promoter 13 is preferably bound to the end of the hydrophilic polymer 12 that is not bound to the oligo-nucleic acid 11.

[0069] The oligo-nucleic acid nanoparticle according to one aspect of the present invention (hereinafter, also referred to as the oligo-nucleic acid nanoparticle according to the second aspect) includes a core composed of a dendritic polymer, a plurality of oligo-nucleic acids bound to the core, one or more hydrophilic polymers bound to the core via a spacer, and one or more cell internalization promoters bound to the one or more hydrophilic polymers.

[0070] The oligonucleic acid nanoparticles according to this aspect are different from the oligonucleic acid nanoparticles according to the first aspect in that the hydrophilic polymer to which the cell internalization promoter is bound is not bound to the oligonucleic acid, but is bound to the core via a spacer. The details of the dendrimer, oligonucleic acid, hydrophilic polymer, and cell internalization promoter are the same as those of the oligonucleic acid nanoparticles according to the first aspect and are as described above.

[0071] In the present specification, the spacer is a polymer for linking the core and the hydrophilic polymer, and the hydrophilic polymer and the cell internalization promoter bound thereto are disposed outside the spatial spread (radius of gyration) of the oligonucleic acid. The spacer is not particularly limited and may be a polar molecule or a non-polar molecule, and may have a positive charge or a negative charge. Examples of the spacer include PEG, pMeOx, cationic or anionic polypeptides, polypeptoids, and alkyl chains, but the spacer is not limited thereto. From the viewpoint of exposing the cell internalization promoter on the surface of the oligonucleic acid nanoparticles, the spacer is preferably a spacer having a rigid structure or a cationic spacer. The spacer is preferably PEG or a cationic polypeptide. The oligonucleic acid nanoparticles may contain one or more spacers as the spacer.

[0072] The length of the spacer is appropriately adjusted according to the length of the oligonucleic acid and the size of the hydrophilic polymer so that the cell internalization promoter can be located on the surface of the oligonucleic acid nanoparticles. The length of the spacer is preferably sufficiently long compared to the length of the oligonucleic acid.

[0073] The number of spacers may be, for example, 1 or more, 2 or more, or 1% or more of the reactive functional groups of the dendrimer, preferably 2% or more, more preferably 3% or more or 5% or more of the reactive functional groups of the dendrimer. The number of spacers in the oligonucleic acid nanoparticles can be determined, for example, by binding a fluorescent dye to the terminal functional group of the spacer and dividing the fluorescent dye concentration in the solution containing the obtained oligonucleic acid nanoparticles by the concentration of the dendrimer.

[0074] In one embodiment, at least a part or all of the unreacted reactive functional groups not bound to the oligo nucleic acid or the spacer may be capped with a capping agent. Details of the capping agent are as described above.

[0075] The binding between the dendritic polymer (core) and the oligo nucleic acid, the binding between the core and the spacer, the binding between the spacer and the hydrophilic polymer, and the binding between the hydrophilic polymer and the cell internalization promoter are each independently a direct bond (i.e., a bond not through a linker) or a bond through a linker. Details of the linker are as described above. The direct bond between the core and the oligo nucleic acid, between the core and the spacer, between the spacer and the hydrophilic polymer, or between the hydrophilic polymer and the cell internalization promoter, or the binding between the above linker and the dendritic polymer, oligo nucleic acid, spacer, hydrophilic polymer, or cell internalization promoter may be, for example, a covalent bond, a metal coordination bond, or a host-guest interaction. Details of these bonds or interactions are as described above. From the viewpoint of achieving high structural stability and controlling the size of the oligo nucleic acid nanoparticles, the bond is preferably a covalent bond.

[0076] The single molecules constituting the oligo nucleic acid nanoparticles according to this aspect may be free bodies or pharmaceutically acceptable salts. The single molecules constituting the oligo nucleic acid nanoparticles may be either solvates (e.g., hydrates, ethanol solvates, propylene glycol solvates) or non-solvates. Details of the pharmaceutically acceptable salts are as described above.

[0077] Next, the structure of the oligo-nucleic acid nanoparticle according to this aspect will be described with reference to FIG. 2. FIG. 2 is a schematic diagram showing one aspect of the oligo-nucleic acid nanoparticle according to this aspect. The oligo-nucleic acid nanoparticle 200 includes a core 50 (dendritic polymer) located at the center of the oligo-nucleic acid nanoparticle 200, and a plurality of oligo-nucleic acids 11, spacers 14, hydrophilic polymers 12, and intracellular uptake promoters 13 arranged around the core 50. The oligo-nucleic acid 11 is bound to the core 50 via a linker 31. The hydrophilic polymer 12 is bound to the core 50 via a spacer 14, shields the oligo-nucleic acid 11 from the outside world, and exposes the intracellular uptake promoter 13 bound to the hydrophilic polymer 12 on the surface of the oligo-nucleic acid nanoparticle 200. A capping agent 21 is also bound to the core 50. In an aqueous solution, the oligo-nucleic acid 11, spacer 14, and hydrophilic polymer 12 extend substantially radially from the core 50, so that the oligo-nucleic acid nanoparticle 200 has a substantially spherical shape. In FIG. 2, the oligo-nucleic acid 11 is bound to the core 50 via a linker 31, but as described above, the oligo-nucleic acid 11 may be directly bound to the core 50. Further, the spacer 14 and the hydrophilic polymer 12, and the hydrophilic polymer 12 and the intracellular uptake promoter 13 may be bound via any of the above linkers.

[0078] The average particle diameter of the oligo-nucleic acid nanoparticle 200 may be the same as that of the oligo-nucleic acid nanoparticle 100, and the details are as described above.

[0079] According to the oligo-nucleic acid nanoparticle 200, since the intracellular uptake promoter 13 is bound to the core 50 composed of a highly branched dendritic polymer via the spacer 14 and the hydrophilic polymer 12, a high density of the intracellular uptake promoter 13 can be achieved, and thus the intracellular uptake promoter 13 can efficiently interact with target cells.

[0080] In the oligo nucleic acid nanoparticle 200, since the hydrophilic polymer 12 is bound to the core 50 via the spacer 14, when the length of the spacer 14 is sufficiently long compared to the length of the oligo nucleic acid 11, the hydrophilic polymer 12 and the cell internalization promoter 13 bound thereto are located outside the spatial spread (radius of gyration) of the oligo nucleic acid 11, and the cell internalization promoter 13 can be located on the surface (i.e., the outermost shell) of the oligo nucleic acid nanoparticle 200. When the cell internalization promoter 13 is located on the surface of the oligo nucleic acid nanoparticle 200, the oligo nucleic acid nanoparticle 200 can easily interact efficiently with the target cell. Further, when the hydrophilic polymer 12 is located outside the spatial spread of the oligo nucleic acid 11, an effect of reducing non-specific interaction between the oligo nucleic acid nanoparticle 200 and biological components other than the target cell can be expected. When the spacer 14 has a rigid structure or is cationic and can form a complex with the oligo nucleic acid 11, the hydrophilic polymer 12 and the cell internalization promoter 13 bound thereto are more likely to be located outside the spatial spread of the oligo nucleic acid 11.

[0081] The present invention also provides a method for producing an oligo nucleic acid nanoparticle according to the first aspect. That is, one aspect of the present invention is (a1) a step of binding a plurality of oligo nucleic acids to a core composed of a dendritic polymer; (a2) a step of binding a hydrophilic polymer to the oligo nucleic acid; (a3) a step of binding a cell internalization promoter to the hydrophilic polymer, and is a method for producing an oligo nucleic acid nanoparticle. In one embodiment, the method for producing an oligo nucleic acid nanoparticle may further include (a4) a step of binding a capping agent to the core. Thereby, an oligo nucleic acid nanoparticle in which at least a part of the reactive functional groups of the dendritic polymer is capped with the capping agent can be produced.

[0082] The core, the oligonucleic acid, the oligonucleic acid and the hydrophilic polymer, and the hydrophilic polymer and the intracellular uptake promoter can each be independently bound directly or via a linker. For example, step (a1) may include a step of binding a plurality of linkers to the core and a step of binding an oligonucleic acid to the linker.

[0083] Steps (a1) to (a4) can be carried out in this order, but it is not essential to carry them out in this order. For example, step (a4) may be carried out before step (a1), simultaneously with step (a1), or between step (a1) and step (a2). Further, step (a3) may be carried out before step (a2), and in step (a2), the hydrophilic polymer bound with the intracellular uptake promoter may be bound to the oligonucleic acid.

[0084] As an example, the oligonucleic acid nanoparticles can be produced by a method including, in this order, a step of binding a plurality of capping agents and linkers to a core composed of a dendritic polymer, a step of binding an oligonucleic acid to the linker, a step of binding a hydrophilic polymer to the oligonucleic acid, and a step of binding an intracellular uptake promoter to the hydrophilic polymer.

[0085] The present invention also provides a method for producing the oligonucleic acid nanoparticles according to the second aspect. That is, one aspect of the present invention is (b1) a step of binding one or more spacers to a core composed of a dendritic polymer, (b2) a step of binding a plurality of oligonucleic acids to the core, (b3) a step of binding a hydrophilic polymer to the spacer, (b4) a step of binding an intracellular uptake promoter to the hydrophilic polymer, which is a method for producing oligonucleic acid nanoparticles. In one embodiment, the method for producing oligonucleic acid nanoparticles may further include (b5) a step of binding a capping agent to the core. Thereby, oligonucleic acid nanoparticles in which at least a part of the reactive functional groups of the dendritic polymer are capped with the capping agent can be produced.

[0086] The core, the oligo nucleic acid, the core and the spacer, the spacer and the hydrophilic polymer, and the hydrophilic polymer and the intracellular uptake promoter can each be independently bound directly or via a linker. For example, step (b1) may include a step of binding a plurality of linkers to the core and a step of binding an oligo nucleic acid to the linker.

[0087] Steps (b1) to (b5) can be carried out in this order, but it is not essential to carry them out in this order. For example, steps (b1) and (b2) may be carried out simultaneously, or step (b5) may be carried out before step (b1), simultaneously with step (b1), between steps (b1) and (b2), or between steps (b2) and (b3). Further, step (b4) may be carried out before step (b3), and in step (b3), the hydrophilic polymer bound with the intracellular uptake promoter may be bound to the spacer. Further, step (b3) may be carried out before step (b1), and in step (b1), one or more spacers bound with the hydrophilic polymer may be bound to the core.

[0088] Any of the steps of the above method for producing the oligo nucleic acid nanoparticles according to the first or second aspect can be carried out using a known method. Examples of the known method include a method of reacting an amino group and a carboxy group using an activating group to form an amide bond, a method of reacting thiol groups using an activating group to form a disulfide bond, a method of reacting a thiol group and a maleimide group to form a thioether bond, a method of forming a triazole bond from an azide group and an alkynyl group by utilizing click chemistry using a catalyst or an activating group, and a method of forming a bond by utilizing an inverse electron demand Diels-Alder reaction from a highly electron-deficient heterocycle such as tetrazine and triazine and a compound having a strained carbon multiple bond such as norbornene, trans-cyclooctene, and cyclooctyne.

[0089] The oligo nucleic acid nanoparticles according to the above aspect may be produced by a known method other than the method according to the above aspect.

[0090] One aspect of the present invention is a pharmaceutical composition containing an oligonucleic acid nanoparticle according to the above aspect as an active ingredient. The pharmaceutical composition contains pharmaceutically acceptable additives. As used herein, "pharmaceutically acceptable" refers to being acceptable to mammals from a pharmacological or toxicological perspective. That is, a "pharmaceutically acceptable" substance is physiologically acceptable and typically does not cause allergic reactions or other harmful or toxic reactions when administered to mammals. A "pharmaceutically acceptable" substance means a substance that has been approved by a generally recognized regulatory agency or listed in a generally recognized pharmacopoeia for use in mammals, more specifically humans. A "pharmaceutically acceptable additive" means a pharmacologically inert material used together with the oligonucleic acid nanoparticle to formulate the pharmaceutical composition.

[0091] The additive may be liquid or solid. The additive is selected with the intended mode of administration in mind so as to obtain a pharmaceutical composition with a desired dosage, consistency, etc. The additive is not particularly limited, and examples include water, physiological saline, other aqueous solvents, various carriers such as aqueous or oily substrates, excipients, binders, pH adjusters, disintegrants, absorption promoters, lubricants, colorants, flavoring agents, fragrances, and the like. The blending ratio of the additive can be appropriately set based on the range commonly employed in the pharmaceutical field.

[0092] The pharmaceutical composition may be, for example, a sterile composition for injection. The sterile composition for injection can be prepared according to normal pharmaceutical operations (for example, dissolving or suspending the active ingredient in a solvent such as water for injection, natural vegetable oil, etc.). As the aqueous liquid for injection, for example, physiological saline, glucose, or an isotonic solution containing other auxiliary agents (for example, D-sorbitol, D-mannitol, lactose, sucrose, sodium chloride, etc.) is used. The aqueous liquid for injection may further contain appropriate solubilizing aids such as alcohol (e.g., ethanol), polyalcohol (e.g., propylene glycol or polyethylene glycol), nonionic surfactant (e.g., polysorbate 80TM or HCO-50). Also, the aqueous liquid for injection may contain various known materials in the art such as buffers (for example, phosphate buffer or sodium acetate buffer), soothing agents (for example, benzalkonium chloride, procaine hydrochloride, etc.), stabilizers (for example, human serum albumin or polyethylene glycol), preservatives (for example, benzyl alcohol, phenol, etc.), antibacterial agents, dispersants, antioxidants, etc. The injection may be, for example, a freeze-dried preparation.

[0093] The oligonucleic acid nanoparticles or pharmaceutical composition according to the above aspect of the present invention can be used for the treatment and / or prevention of diseases involving specific gene products. Diseases involving specific gene products include, for example, inborn errors of metabolism, congenital endocrine diseases, single-gene diseases, neurodegenerative diseases, nerve diseases, muscle diseases, meningitis, encephalitis, encephalopathy, lysosomal diseases, malignant neoplasms, fibrosis, inflammatory diseases, immunodeficiency diseases, autoimmune diseases, or infectious diseases, but the diseases are not limited thereto. Therefore, one aspect of the present invention is a therapeutic or prophylactic agent for the above diseases containing oligonucleic acid nanoparticles as an active ingredient.

[0094] Another aspect of the present invention is a method for treating and / or preventing the above-mentioned disease, which includes administering a therapeutically effective amount of oligonucleic acid nanoparticles to a human or a non-human animal. The human may be a human in need of treatment, i.e., a patient. Non-human animals include warm-blooded mammals such as primates; birds; domestic animals or livestock such as cats, dogs, sheep, goats, cows, horses, pigs; experimental animals such as mice, rats, guinea pigs; fish; insects; zoo animals; and wild animals. The administration methods may include, but are not limited to, oral, sublingual, intravenous, intraarterial, subcutaneous, intradermal, intraperitoneal, intramuscular, intrathecal, intraventricular, intranasal, transmucosal, rectal, ophthalmic, intraocular, transpulmonary, transdermal, intra-articular, topical (skin), intradermal follicular, intravaginal, intrauterine, intratumoral, or intralymphatic administration, or combinations thereof.

[0095] Another aspect of the present invention is oligonucleic acid nanoparticles for use in treating and / or preventing the above-mentioned disease. Another aspect of the present invention is the use of oligonucleic acid nanoparticles for manufacturing a therapeutic and / or prophylactic agent against the above-mentioned disease.

[0096] The oligonucleic acid nanoparticles or pharmaceutical compositions according to the above aspects of the present invention can also be used in combination with one or more other drugs. The other drugs may be one or more therapeutic and / or prophylactic agents against diseases involving the specific gene products described above. For example, when the target disease is a malignant neoplasm, examples of other drugs include pharmaceuticals that can be used in chemotherapy. That is, one aspect of the present invention is oligonucleic acid nanoparticles for treating a disease in combination with one or more therapeutic and / or prophylactic agents against the above-mentioned disease. Another aspect of the present invention is a pharmaceutical comprising a combination of an oligonucleic acid nanoparticle or a pharmaceutical composition and one or more therapeutic and / or prophylactic agents against the above-mentioned disease. However, since the present invention is a platform technology capable of efficiently transporting oligonucleic acids into cells and can be used for any disease for which oligonucleic acids can be applied as therapeutic or prophylactic agents, the other drugs are not limited to specific drugs.

[0097] The administration timing of the oligonucleic acid nanoparticles or pharmaceutical composition and the other drugs used in combination therewith is not limited, and these may be administered simultaneously or at appropriate intervals to humans or non-human animals. Alternatively, the other drugs may be formulated into the pharmaceutical composition according to the above aspect to prepare a combined preparation. The dosage and formulation amount of the other drugs can be appropriately determined based on the clinically used dosages. In addition, the mixing ratio of the oligonucleic acid nanoparticles or pharmaceutical composition and the other drugs can be appropriately determined according to the administration subject, administration route, target disease, symptoms, combination of other drugs, etc.

Examples

[0098] The present invention will be described in detail below with reference to examples and test examples, but the present invention is not limited to these examples. In the following text, “%” means weight % unless otherwise specified.

[0099] <Synthesis of Oligonucleic Acid> The siRNAs shown in Table 2 were prepared. A thiol group was bonded to the 3'-end of the sense strand RNA of the siRNA via spacer18 (hexaethylene glycol) and C6 linker (hexyl). An amino group was bonded to the 5'-end of the sense strand RNA of the siRNA via C6 linker. These nucleic acids were manufactured by Gene Design Co., Ltd.

Table 2

[0100] siRNA and trisodium ethylenediaminetetraacetate (EDTA 3Na) were dissolved in 10 mM phosphate buffered saline (PBS) at pH 7.4, and dithiothreitol (DTT) was further added (final concentration: EDTA 0.5 mM, DTT 40 mM). This solution was heated at 25 °C for 6 hours and then purified 6 times by ultrafiltration (molecular weight cut-off 10 kDa) using PBS. The nucleic acid concentration of the obtained solution was determined from the measured absorbance at 260 nm using an ultraviolet-visible spectrophotometer (Tecan, Infinite M200 PRO).

[0101] <Example 1. Production of cRGD-binding oligonucleotide nanoparticles 1> (A) Synthesis of NH2-siRNA-DBCO To 58 μL of a 5.74 mM PBS solution of the siRNA shown in Table 3, 41.9 μL of 10 mM PBS at pH 7.0 and 66.6 μL of a 100 mM DMSO solution of sulfo DBCO-PEG4-maleimide (manufactured by Broadpharm) were added, and the mixture was stirred at 4 °C for 14 hours to react the SH group of the siRNA with the maleimide group of sulfo DBCO-PEG4-maleimide to obtain NH2-siRNA-DBCO. After adding 300 μL of PBS to the reaction solution and mixing, it was purified 6 times by ultrafiltration (Merck, Amicon Ultra, molecular weight cut-off 10 kDa) using PBS. Pure water was added to the recovered aqueous solution to adjust the volume of the solution to 60 μL.

[0102] (B) Synthesis of azide-PEG12 AF6 DGL G4 As the dendritic polymer, the fourth-generation polylysine dendrograft having an amino group on the surface (DGL G4, number of reactive functional groups: 366) manufactured by COLCOM was used. To 4 μL of a 50 mg / mL dimethyl sulfoxide (DMSO) solution of DGL G4, 1.91 μL of an 8 mM DMSO solution of Alexa Fluor 647 NHS ester (manufactured by Thermo Fisher Scientific) and 2.34 μL of a 10 v / v% DMSO solution of triethylamine (TEA) were added, and the mixture was stirred at room temperature for 5 hours. Next, 6.13 μL of a 50 mM DMSO solution of Azido-PEG12-NHS ester (manufactured by Broadpharm) was added to this reaction solution, and the mixture was further stirred at room temperature for 14 hours. Then, 11.2 μL of a 200 mM DMSO solution of m-dPEG (registered trademark)12-NHS ester (manufactured by Quanta BioDesign) was added, and the mixture was further stirred at room temperature for 8 hours. By reacting the amino group of DGL G4 with the N-hydroxysuccinimide (NHS) groups of Azido-PEG12-NHS ester, the anionic fluorescent dye Alexa Fluor 647 NHS ester, and m-dPEG12-NHS ester in the above manner, the nanoparticle compound azide-PEG12 AF6 DGL G4 was obtained. After adding 1000 μL of pure water to the reaction solution and mixing, it was purified 6 times by ultrafiltration (Amicon Ultra, molecular weight cut-off 30 kDa) using pure water. Pure water was added to the recovered aqueous solution to adjust the volume of the solution to 75 μL.

[0103] (C) Synthesis of NH2-siRNA-PEG12 AF6 DGL G4 To 10 μL of the aqueous solution of azide-PEG12 AF6 DGL G4 shown in (B), 15.6 μL of the PBS solution of NH2-siRNA-DBCO shown in (A) and 3.77 μL of DMSO were added, and the mixture was stirred at room temperature. Then, 1.99 μL of 3 M aqueous sodium chloride solution was added 30 minutes after the start of stirring, 2.22 μL was added 1 hour after the start of stirring, and 4.09 μL was added 2 hours after the start of stirring, and then the mixture was further stirred at room temperature for 14 hours. In this way, the azide group of azide-PEG12 AF6 DGL G4 was reacted with the DBCO group (dibenzocyclooctyne group) of NH2-siRNA-DBCO. The reaction solution was purified by gel filtration using Hiprep 16 / 60 Sephacryl S-200 HR (manufactured by Cytiva) (eluent: PBS). The fraction containing DGL G4 bound with siRNA was collected, the solvent was replaced with 100 mM PBS using ultrafiltration (Amicon Ultra, molecular weight cut-off 50 kDa), and then the volume was adjusted to 100 μL.

[0104] (D) Synthesis of N3-PEG2000-siRNA-PEG12 AF6 DGL G4 To 50 μL of the PBS solution of NH2-siRNA-PEG12 AF6 DGL G4 shown in (C), 19.4 μL of DMSO and 13.9 μL of a 50 mM DMSO solution of N3-PEG-NHS (Biopharma PEG Scientific, number average molecular weight of PEG 2000) were added, and the amino group of NH2-siRNA-PEG12 AF6 DGL G4 was reacted with the NHS group of N3-PEG-NHS by stirring at room temperature for 17 hours. The reaction solution was purified by gel filtration using Hiprep 16 / 60 Sephacryl S-200 HR (eluent: PBS). The fraction containing DGL G4 bound with siRNA was collected, concentrated by ultrafiltration (Amicon Ultra, molecular weight cut-off 50 kDa), and the volume of the solution was adjusted to 100 μL.

[0105] (E) Synthesis of cRGD-DBCO To 65.4 μL of a 200 mM DMSO solution of Cyclo(-RGDfK) (manufactured by Chemscene), 43.6 μL of a 300 mM DMSO solution of DBCO-NHCO-PEG4-NHS (manufactured by BroadPharm) and 5.5 μL of TEA were added, and the mixture was stirred at 25 °C for 25 hours to react the amino group of Cyclo(-RGDfK) with the NHS group of DBCO-NHCO-PEG4-NHS. The reaction solution was concentrated by removing the solvent under reduced pressure while heating at 45 °C, and purified by reverse-phase HPLC (column: Waters Xbridge Peptide BEH C18, 300 Å, 4.6×100 mm; eluent A: 0.1 v / v% trifluoroacetic acid (TFA) / acetonitrile (90 / 10; v / v); eluent B: 0.1 v / v% TFA / acetonitrile (10 / 90; v / v)). After removing the solvent under reduced pressure while heating the recovered solution at 45 °C, DMSO was added to adjust the concentration to 50 mM.

[0106] (F) Synthesis of (cRGD-PEG2000-siRNA-PEG12 AF6 DGL G4) To 100 μL of the PBS solution of N3-PEG2000-siRNA-PEG12 AF6 DGL G4 shown in (D), 0.26 μL of DMSO and 10.9 μL of a 4 mM DMSO solution of cRGD-DBCO shown in (E) were added, and the mixture was stirred at room temperature for 15 hours to react the azide group of N3-PEG2000-siRNA-PEG12 AF6 DGL G4 with the DBCO group of cRGD-DBCO. The reaction solution was purified using a Zeba (registered trademark) Spin Desalting Column (manufactured by Thermo Fisher Scientific, molecular weight fractionation: 40 kDa). Subsequently, purification was performed 3 times by ultrafiltration (Amicon Ultra, molecular weight cut-off 50 kDa) using PBS, and the volume of the solution was adjusted to 85 μL to obtain a PBS solution of the oligonucleic acid nanoparticle cRGD-PEG2000-siRNA-PEG12 AF6 DGL G4.

[0107] <Example 2. Production of cRGD-conjugated oligonucleic acid nanoparticle 2> (A) Synthesis of azide-PEG12 AF6 DGL G4 According to (B) of Example 1, azide-PEG12 AF6 DGL G4 was synthesized. However, the addition amount of the 50 mM DMSO solution of azido-PEG12-NHS ester was changed from 6.13 μL to 12.3 μL.

[0108] (B) Synthesis of NH2-siRNA-PEG12 AF6 DGL G4 To 10 μL of the aqueous solution of azide-PEG12 AF6 DGL G4 shown in (A), 31.3 μL of the PBS solution of NH2-siRNA-DBCO shown in (A) of Example 1 and 6.02 μL of DMSO were added, and the mixture was stirred at room temperature. Then, 2.86 μL of 3 M aqueous sodium chloride solution was added 30 minutes after the start of stirring, 3.56 μL was added 1 hour after the start of stirring, and 6.53 μL was added 2 hours after the start of stirring, and then the mixture was further stirred at room temperature for 14 hours. The reaction solution was purified by gel filtration using Hiprep 16 / 60 Sephacryl S-200 HR (eluent: PBS). The fraction containing DGL G4 bound with siRNA was collected, and the solvent was replaced with 100 mM PBS using ultrafiltration (Amicon Ultra, molecular weight cut-off 50 kDa), and then the volume was adjusted to 100 μL.

[0109] (C) Synthesis of N3-PEG2000-siRNA-PEG12 AF6 DGL G4 Using NH2-siRNA-PEG12 AF6 DGL G4 shown in (B), N3-PEG2000-siRNA-PEG12 AF6 DGL G4 was synthesized according to (D) of Example 1. However, instead of adding 19.4 μL of DMSO and 13.9 μL of the 50 mM DMSO solution of N3-PEG-NHS, 5.56 μL of DMSO and 27.8 μL of the 50 mM DMSO solution of N3-PEG-NHS were added.

[0110] (D) Synthesis of cRGD-PEG2000-siRNA-PEG12 AF6 DGL G4 Using N3-PEG2000-siRNA-PEG12 AF6 DGL G4 shown in (C) and cRGD-DBCO shown in (E) of Example 1, cRGD-PEG2000-siRNA-PEG12 AF6 DGL G4 was synthesized according to (F) of Example 1.

[0111] <Test Example 1. Evaluation of Oligonucleotide Nanoparticles> The concentrations of the oligonucleotide, cell internalization promoter, and fluorescent molecule in the oligonucleotide nanoparticle samples used in each of the following test examples were determined as follows. From these concentrations, the number of oligonucleotides, hydrophilic polymers, cell internalization promoters, and fluorescent molecules bound to one dendrimer was calculated. Note that the number of hydrophilic polymers was regarded as the same as the number of cell internalization promoters. The calculation results are shown in each of the following test examples.

[0112] The concentrations of the oligonucleotide and fluorescent molecule were determined from the measured absorption values at the following wavelengths using an ultraviolet-visible spectrophotometer: 260 nm for siRNA and 651 nm for Alexa Fluor 647.

[0113] The concentrations of the dendritic polymer and the polypeptide-based intracellularization promoter were quantified by amino acid quantitative analysis (AQC method) as follows. First, 30 μL of an aqueous solution of the oligonucleic acid nanoparticle sample and 300 μL of constant-boiling hydrochloric acid were added to a sealable glass bottle, sealed, and hydrolyzed by heating at 110 °C for 24 hours. After hydrolysis, the solvent was removed under reduced pressure while heating the reaction solution at 45 °C. AQC (manufactured by Adipogen Life Sciences) was dissolved in acetonitrile (ultra-dehydrated) at 60 °C and adjusted to 3 mg / mL. To the glass bottle containing the dried oligonucleic acid nanoparticle sample, 30 μL of 20 mM hydrochloric acid, 90 μL of 0.2 M borate buffer (pH 8.8), and 30 μL of 3 mg / mL AQC acetonitrile solution were added, stirred, and allowed to stand at 60 °C. After standing for 10 minutes, the solvent was removed under reduced pressure while heating at 45 °C. The obtained solid was dissolved in 150 μL of eluent A and then filter-filtered (Merck Ultrafree;-MC, GV, 0.22 μm). The obtained filtrate was analyzed by reverse-phase HPLC (column: AccQ-Tag Column, 60 Å, 4 μm 3.9 × 150 mm, eluent A: AccQ-Tag Eluent A / water (1 / 9; v / v), eluent B: water / acetonitrile (1 / 1; v / v)), and the concentration of DGL G4 was quantified from the integrated value of the peak of the lysine residue, and the concentration of cRGDfK was quantified from the integrated value of each specific peak. The AccQ-Tag Column and AccQ-Tag Eluent A were purchased from Waters Corporation.

[0114] <Test Example 2. Cell Evaluation of cRGD Ligand-Bound Oligonucleic Acid Nanoparticles> Human glioblastoma cell line (U-87MG) was seeded in a 96-well plate and cultured in a 37 °C, 5% CO 2 incubator using DMEM medium containing 10% FBS. The next day, the medium was changed, and samples were added to each well to transfect the cells, and then incubated at 37 °C, 5% CO 2They were cultured in an incubator. The siRNA concentrations of the samples at the time of transfection were 0.01 μM, 0.1 μM, or 1 μM. 48 hours after transfection, the cells were washed with PBS, and then the fluorescence intensity was measured (excitation wavelength 640 nm, fluorescence wavelength 675 nm). Furthermore, the siRNA concentration was converted to the concentration of fluorescent molecules based on the ratio of the number of siRNA to the number of fluorescent molecules (number of siRNA / number of fluorescent molecules). After approximating that the fluorescence intensity was directly proportional to the concentration of fluorescent molecules, the fluorescence intensity when the concentration of fluorescent molecules was 2 nM, 20 nM, or 200 nM was calculated. The samples used are shown in Table 3, and the results obtained are shown in Figure 3.

[0115]

Table 3

[0116] Human glioblastoma cell line (U-87MG) was seeded in a 96-well plate and cultured in DMEM medium containing 10% FBS in an incubator at 37 °C and 5% CO 2 The medium was changed the next day, and samples were added to each well to transfect the cells, and then cultured at 37 °C and 5% CO 2It was cultured in an incubator. The siRNA concentration of the sample at the time of transfection was 1 μM. For the control group, PBS was added instead of the sample. 48 hours after transfection, mRNA was extracted using the RNeasy Mini Kit (manufactured by Qiagen), and cDNA was synthesized from a fixed amount of mRNA using the High Capacity RNA-to-cDNA Kit (Applied Biosystems (registered trademark)). Subsequently, using the obtained cDNA as a template, quantitative RT-PCR was performed using PowerUp SYBR Green Master Mix (Applied Biosystems). As the ATP5B primers, the primers of SEQ ID NO: 9 and SEQ ID NO: 10 shown in Table 4 below were used, and as the GAPDH primers, the primers of SEQ ID NO: 11 and SEQ ID NO: 12 shown in Table 4 below were used. The PCR conditions (temperature and time) were as follows. It was designed with 1 second at 95°C and 30 seconds at 60°C as one cycle, and 40 cycles were performed. Based on the results of quantitative RT-PCR, the value of "expression level of ATP5B / expression level of GAPDH (internal standard gene)" was calculated, and the calculation result for the control group was compared with the calculation result for the sample addition group. The samples used are shown in Table 3, and the obtained results are shown in Figure 4.

[0117]

Table 4

[0118] <Oligonucleotide synthesis> Prepare the siRNA shown in Table 5. A thiol group is bonded to the 3'-end of the sense strand RNA of the siRNA via spacer18 (hexaethylene glycol) and a C6 linker.

[0119]

Table 5

[0120] <Example 3. Production of cRGD-conjugated oligonucleotide nanoparticles 3> (A) Synthesis of bis(azide-PEG3)-PEG5k-COOH To 50.0 μL of a 100 mM DMSO solution of AMINO-PEG4-BIS-PEG3-AZIDE (Conju-Probe), 400 μL of a 2.5 mM DMSO solution of NHS-PEG-COOH (manufactured by Biopharma PEG Scientific, molecular weight 5000), and 10.5 μL of a 10 v / v% DMSO solution of TEA were added, and the amino group of AMINO-PEG4-BIS-PEG3-AZIDE was reacted with the NHS group of NHS-PEG-COOH by stirring overnight at room temperature. After adding 1000 μL of PBS to the reaction solution and mixing, it was purified 6 times by ultrafiltration (Amicon Ultra, molecular weight cut-off 3 kDa) using pure water. After removing the solvent by lyophilization, 500 μL of DMSO was added to obtain a 2.0 mM DMSO solution of bis(azide-PEG3)-PEG5k-COOH.

[0121] (B) Synthesis of bis(azide-PEG3) SPDP AF6 DGL G4 To 3.0 μL of a 50 mg / mL DMSO solution of DGL G4, add 2.30 μL of a 60 mM DMSO solution of PEG12-SPDP (manufactured by Thermo Fisher Scientific), 1.44 μL of an 8 mM DMSO solution of Alexa Fluor® 647 NHS ester, and 2.13 μL of a 10 v / v% DMSO solution of TEA, and stir at room temperature for 8 hours. Next, add 172.3 μL of a DMSO solution of bis(azide-PEG3)-PEG5k-COOH shown in (A), 3.45 μL of a 200 mM DMSO solution of NHS, and 3.45 μL of a 200 mM DMSO solution of EDC-HCl, and stir overnight at room temperature. Then, add 8.41 μL of a 200 mM DMSO solution of m-dPEG®12-NHS ester and stir at room temperature for an additional 8 hours to react the amino group of DGL G4 with the COOH group of bis(azide-PEG3)-PEG5k-COOH and the NHS groups of Alexa Fluor647 NHS ester, m-dPEG12-NHS ester, and PEG12-SPDP. After adding 1000 μL of PBS to the reaction solution and mixing, purify 6 times by ultrafiltration (Amicon Ultra, molecular weight cut-off 100 kDa) using pure water, and adjust the volume of the recovered aqueous solution to 50 μL by adding PBS to obtain a PBS solution of bis(azide-PEG3) SPDP AF6 DGL G4.

[0122] (C) Synthesis of bis(azide-PEG3) siRNA AF6 DGL G4 To 10 μL of the PBS solution of bis(azide-PEG3) SPDP AF6 DGL G4 shown in (B), add 5.94 μL of DMSO, 10.7 μL of the 6 mM PBS solution of the siRNA shown in Table 5, and 3.03 μL of 3 M aqueous NaCl solution, and stir overnight at room temperature to react the pyridyldisulfide group of bis(azide-PEG3) SPDP AF6 DGL G4 with the SH group of the siRNA. The reaction solution is purified by gel filtration using Hiprep 16 / 60 Sephacryl S-200 HR (eluent: PBS), and the fraction containing DGL G4 bound with siRNA is collected. The collected solution is concentrated by ultrafiltration (Amicon Ultra, molecular weight cut-off 50 kDa), and the volume of the solution is adjusted to 100 μL to obtain a PBS solution of bis(azide-PEG3) siRNA AF6 DGL G4.

[0123] (D) Synthesis of cRGD-PEG2k-DBCO To 55.0 μL of the 300 mM DMSO solution of Cyclo(-RGDfK), add 330.0 μL of the 10 mM DMSO solution of DBCO-PEG2k-NHS (manufactured by Biopharma PEG Scientific) and 4.60 μL of TEA, and stir overnight at room temperature to react the amino group of Cyclo(-RGDfK) with the NHS group of DBCO-PEG2k-NHS. The reaction solution is purified by gel filtration using Sephadex LH-20 (manufactured by Cytiva) (eluent: DMSO), and the fraction containing PEG bound with cRGDfK is collected. After removing the solvent from the collected solution by lyophilization, DMSO is added and adjusted to a concentration of 50 mM to obtain a DMSO solution of cRGD-PEG2k-DBCO.

[0124] (E) Synthesis of bis(cRGD-PEG2k) siRNA AF6 DGL G4 To 40.0 μL of the PBS solution of bis(azide-PEG3) siRNA AF6 DGL G4 shown in (C), 1.47 μL of the DMSO solution of cRGD-PEG2k-DBCO shown in (D) and 2.97 μL of DMSO are added, and the mixture is stirred overnight at room temperature to react the azide group of bis(azide-PEG3) siRNA AF6 DGL G4 with the DBCO group of cRGD-PEG2k-DBCO. The reaction solution is purified using a Zeba (registered trademark) Spin Desalting Column (molecular weight fractionation: 40 kDa). Further purification is performed 3 times by ultrafiltration (Amicon Ultra, molecular weight cut-off 100 kDa) using PBS, and the volume of the solution is adjusted to 100 μL to obtain a PBS solution of the oligonucleic acid nanoparticle bis(cRGD-PEG2k) siRNA AF6 DGL G4.

[0125] <Example 4. Production of cRGD-conjugated oligonucleic acid nanoparticle 4> (A) Synthesis of azide-CP1 SPDP AF6 DGL4 To 3.0 μL of a 50 mg / mL DMSO solution of DGL G4, 2.30 μL of a 60 mM DMSO solution of PEG12-SPDP, 1.44 μL of an 8 mM DMSO solution of Alexa Fluor® 647 NHS ester, and 1.81 μL of a 10 v / v% DMSO solution of TEA are added, and the mixture is stirred at room temperature for 8 hours. Subsequently, 45.9 μL of a 5 mM DMSO solution of peptide CP1 shown in the following formula (XIII) and 1.15 μL of a 200 mM DMSO solution of HATU (manufactured by Fujifilm Wako Pure Chemical Corporation) are added, and the mixture is stirred overnight at room temperature. Then, 8.41 μL of a 200 mM DMSO solution of m-dPEG®12-NHS ester is added, and the mixture is further stirred at room temperature for 8 hours to react the amino group of DGL G4 with the COOH group of CP1 and the NHS groups of Alexa Fluor647 NHS ester, m-dPEG12-NHS ester, and PEG12-SPDP. The reaction solution is purified by gel filtration using Sephadex LH-20 (eluent: DMSO), and the fraction containing DGL G4 bound with CP1 is collected. After removing the solvent by freeze-drying the collected solution and then adding DMSO to adjust the concentration to 32.2 mM, a DMSO solution of azide-CP1 SPDP AF6 DGL4 can be obtained.

[0126]

Chemical formula

[0127] (B) Synthesis of azide-CP1 siRNA AF6 DGL4 To 10.0 μL of the DMSO solution of azide-CP1 SPDP AF6 DGL4 shown in (A), add 11.3 μL of the 6 mM PBS solution of the siRNA shown in Table 5, 3.09 μL of the 3 M aqueous NaCl solution, and 8.99 μL of DMSO, and stir overnight at room temperature to react the pyridyldisulfide group of azide-CP1 SPDP AF6 DGL4 with the SH group of the siRNA. The reaction solution is purified by gel filtration using Hiprep 16 / 60 Sephacryl S-200 HR (eluent: PBS), and the fraction containing DGL G4 bound with the siRNA is collected. The collected solution is concentrated by ultrafiltration (Amicon Ultra, molecular weight cut-off 50 kDa), and the volume of the solution is adjusted to 100 μL to obtain a PBS solution of azide-CP1 siRNA AF6 DGL4.

[0128] (C) Synthesis of azide-CP2 siRNA AF6 DGL4 To 50.0 μL of the PBS solution of azide-CP1 siRNA AF6 DGL4 shown in (B), add 12.5 μL of TFA and stir at 4 °C for 2 hours to deprotect the tert-butyl ether group of the Ser side chain and the Boc group of the Lys side chain contained in CP1, thereby converting CP1 to CP2 shown by the following formula (XIV). Add 1000 μL of PBS to the reaction solution, perform purification 3 times by ultrafiltration (Amicon Ultra, molecular weight cut-off 100 kDa), and adjust the volume of the solution to 100 μL to obtain a PBS solution of azide-CP2 siRNA AF6 DGL4.

[0129] [Chemical formula]

[0130] (D) Synthesis of cRGD-PEG5k-DBCO cRGD-PEG5k-DBCO can be synthesized according to (D) of Example 3. However, instead of 330.0 μL of a 10 mM DMSO solution of DBCO-PEG2k-NHS, 1000 μL of a 2 mM DMSO solution of DBCO-PEG5k-NHS is used. The amount of the 300 mM DMSO solution of Cyclo(-RGDfK) is changed from 55.0 μL to 33.3 μL, and the amount of TEA is changed from 4.6 μL to 2.79 μL.

[0131] (E) Synthesis of cRGD-PEG5k-CP2 siRNA AF6 DGL G4 To 80.0 μL of the PBS solution of azide-CP2 siRNA AF6 DGL4 shown in (C), 3.86 μL of a 50 mM DMSO solution of cRGD-PEG5k-DBCO shown in (D) and 5.01 μL of DMSO are added, and the mixture is stirred overnight at room temperature to react the azide group of azide-CP2 siRNA AF6 DGL4 with the DBCO group of cRGD-PEG5k-DBCO. The reaction solution is purified using a Zeba (registered trademark) Spin Desalting Column (molecular weight fractionation: 40 kDa). Further purification is performed 3 times by ultrafiltration (Amicon Ultra, cut-off molecular weight 100 kDa) using PBS, and the volume of the solution is adjusted to 100 μL to obtain a PBS solution of the oligonucleic acid nanoparticle cRGD-PEG5k-CP2 siRNA AF6 DGL G4.

Industrial Applicability

[0132] The oligonucleic acid nanoparticle according to one aspect of the present invention can improve the amount of the oligonucleic acid transported into the cytoplasm, and thus can be used as a pharmaceutical composition or a medicine for treating or preventing diseases.

Explanation of Symbols

[0133] 11... oligonucleic acid, 12... hydrophilic polymer, 13... intracellular agentization promoter, 14... spacer, 21... capping agent, 31... linker, 50... core, 100, 200... oligonucleic acid nanoparticles.

Claims

1. A core composed of a dendritic polymer, a plurality of oligo nucleic acids bound to the core, one or more hydrophilic polymers bound to the plurality of oligo nucleic acids, and one or more intracellularization promoters bound to the one or more hydrophilic polymers, wherein the oligo nucleic acid nanoparticle, the binding between the core and the oligo nucleic acid, the binding between the oligo nucleic acid and the hydrophilic polymer, and the binding between the hydrophilic polymer and the intracellularization promoter are each independently a direct binding or a binding via a linker, the intracellularization promoter is located on the surface of the oligo nucleic acid nanoparticle.

2. The oligo nucleic acid nanoparticle according to claim 1, wherein the direct binding, or the binding between the linker and the core, the oligo nucleic acid, the hydrophilic polymer, or the intracellularization promoter is by a covalent bond, metal coordination, or host-guest interaction.

3. The oligo nucleic acid nanoparticle according to claim 1, wherein the direct binding, or the binding between the linker and the core, the oligo nucleic acid, the hydrophilic polymer, or the intracellularization promoter is by a covalent bond or metal coordination.

4. The oligo nucleic acid nanoparticle according to claim 1, wherein the direct binding, or the binding between the linker and the core, the oligo nucleic acid, the hydrophilic polymer, or the intracellularization promoter is by a covalent bond.

5. The oligo nucleic acid nanoparticle according to any one of claims 1 to 4, wherein at least a part of the reactive functional groups of the dendritic polymer is capped with a capping agent.

6. A core composed of a dendritic polymer, a plurality of oligo nucleic acids bound to the core, one or more hydrophilic polymers bound to the core via a spacer, and one or more intracellularization promoters bound to the one or more hydrophilic polymers, wherein the oligo nucleic acid nanoparticle, the binding between the core and the oligo nucleic acid, the binding between the core and the spacer, the binding between the spacer and the hydrophilic polymer, and the binding between the hydrophilic polymer and the intracellularization promoter are each independently a direct binding or a binding via a linker, the intracellularization promoter is located on the surface of the oligo nucleic acid nanoparticle.

7. The oligonucleic acid nanoparticle according to claim 6, wherein the direct bond, or the bond between the linker and the core, the oligonucleic acid, the spacer, the hydrophilic polymer, or the cell internalization promoter is by a covalent bond, a metal coordination, or a host-guest interaction.

8. The oligonucleic acid nanoparticle according to claim 6, wherein the direct bond, or the bond between the linker and the core, the oligonucleic acid, the spacer, the hydrophilic polymer, or the cell internalization promoter is by a covalent bond or a metal coordination.

9. The oligonucleic acid nanoparticle according to claim 6, wherein the direct bond, or the bond between the linker and the core, the oligonucleic acid, the spacer, the hydrophilic polymer, or the cell internalization promoter is by a covalent bond.

10. The oligonucleic acid nanoparticle according to any one of claims 6 to 9, wherein at least a part of the reactive functional groups of the dendritic polymer is capped with a capping agent.

11. The oligonucleic acid nanoparticle according to claim 5 or 10, wherein the capping agent is one or more molecules selected from the group consisting of hydrophilic molecules and hydrophobic molecules.

12. The oligonucleic acid nanoparticle according to claim 11, wherein the capping agent is a hydrophilic molecule.

13. The oligonucleic acid nanoparticle according to claim 11, wherein the capping agent is one or more hydrophilic molecules selected from the group consisting of electrically neutral hydrophilic molecules, polar molecules that are protonated under acidic conditions, anionic molecules, and cationic molecules.

14. The oligonucleic acid nanoparticle according to claim 11, wherein the capping agent is one or more hydrophilic molecules selected from the group consisting of electrically neutral hydrophilic molecules, polar molecules that are protonated under acidic conditions, and anionic molecules.

15. The oligonucleic acid nanoparticle according to claim 11, wherein the capping agent is a hydrophobic molecule.

16. The oligonucleic acid nanoparticle according to claim 11, wherein the capping agent is one or more molecules selected from the group consisting of aliphatic compounds, aromatic compounds, trialkylamines, and steroids.

17. The oligonucleic acid nanoparticle according to claim 11, wherein the capping agent is an aliphatic compound.

18. The oligonucleic acid nanoparticle according to any one of claims 1 to 17, wherein the dendritic polymer is a dendrigraft or a dendrimer.

19. The oligonucleic acid nanoparticle according to any one of claims 1 to 17, wherein the monomers in the dendritic polymer are linked by an amide bond, an ester bond, or a glycoside bond.

20. The oligonucleic acid nanoparticle according to any one of claims 1 to 17, wherein the monomers in the dendritic polymer are linked by an amide bond or an ester bond.

21. The oligonucleic acid nanoparticle according to any one of claims 1 to 17, wherein the dendritic polymer is a poly-L-lysine dendrograft, a polyamidoamine dendrimer, or a 2,2-bis(hydroxymethyl)propionic acid dendrimer.

22. The oligonucleic acid nanoparticle according to any one of claims 1 to 21, wherein the oligonucleic acid is a gene expression regulator.

23. The oligonucleic acid nanoparticle according to claim 22, wherein the gene expression regulator is a molecule that suppresses the expression of mRNA.

24. The oligonucleic acid nanoparticle according to claim 22, wherein the gene expression regulator is an RNA interference-inducing nucleic acid or an antisense nucleic acid.

25. The oligonucleic acid nanoparticle according to any one of claims 6 to 10, wherein the spacer is one or more spacers selected from the group consisting of polyethylene glycol, poly(2-alkyl-2-oxazoline), polypeptide, and polypeptoid.

26. The oligonucleic acid nanoparticle according to any one of claims 6 to 10, wherein the spacer is polyethylene glycol or a cationic polypeptide.

27. The oligonucleic acid nanoparticle according to any one of claims 1 to 26, wherein the hydrophilic polymer is one or more hydrophilic polymers selected from the group consisting of polyethylene glycol, poly(2-alkyl-2-oxazoline), polypeptide, and polypeptoid.

28. The oligonucleic acid nanoparticle according to any one of claims 1 to 26, wherein the hydrophilic polymer is one or more aqueous polymers selected from the group consisting of polyethylene glycol, poly(2-methyl-2-oxazoline), EK peptide, and polysarcosine.

29. The oligonucleic acid nanoparticle according to any one of claims 1 to 28, wherein the intracellular uptake promoter is one or more intracellular uptake promoters selected from the group consisting of polypeptide, aptamer, antibody or fragment thereof, sugar chain, lipid, and other low molecular weight compounds.

30. The oligonucleic acid nanoparticles according to any one of claims 1 to 28, wherein the intracellular uptake promoter is a low molecular weight compound having a molecular weight of 2000 or less other than a hydrophobic molecule, a polycation, a polypeptide, an aptamer, an antibody or a fragment thereof, a sugar or a sugar chain, and a lipid.

31. The oligonucleic acid nanoparticles according to any one of claims 1 to 28, wherein the intracellular uptake promoter is a polypeptide.

32. The oligonucleic acid nanoparticles according to any one of claims 1 to 28, wherein the intracellular uptake promoter is an aptamer.

33. The oligonucleic acid nanoparticles according to any one of claims 1 to 28, wherein the intracellular uptake promoter is an antibody or a fragment thereof.

34. The oligonucleic acid nanoparticles according to any one of claims 1 to 28, wherein the intracellular uptake promoter is a sugar chain.

35. The oligonucleic acid nanoparticles according to any one of claims 1 to 28, wherein the intracellular uptake promoter is a lipid.

36. A pharmaceutical composition comprising the oligonucleic acid nanoparticles according to any one of claims 1 to 35 as an active ingredient.

37. Comprising the oligonucleic acid nanoparticles according to any one of claims 1 to 35 as an active ingredient, A therapeutic or prophylactic agent for a disease selected from the group consisting of inborn errors of metabolism, congenital endocrine diseases, single-gene diseases, neurodegenerative diseases, neurological diseases, muscle diseases, meningitis, encephalitis, encephalopathy, lysosomal diseases, malignant neoplasms, fibrosis, inflammatory diseases, immunodeficiency diseases, autoimmune diseases, and infectious diseases.

38. Comprising administering a therapeutically effective amount of the oligonucleic acid nanoparticles according to any one of claims 1 to 35, A method for treating and / or preventing a disease selected from the group consisting of inborn errors of metabolism, congenital endocrine diseases, single-gene diseases, neurodegenerative diseases, neurological diseases, muscle diseases, meningitis, encephalitis, encephalopathy, lysosomal diseases, malignant neoplasms, fibrosis, inflammatory diseases, immunodeficiency diseases, autoimmune diseases, and infectious diseases.

39. Use of the oligonucleic acid nanoparticles according to any one of claims 1 to 35 for producing a therapeutic and / or prophylactic agent for a disease selected from the group consisting of inborn errors of metabolism, congenital endocrine diseases, single-gene diseases, neurodegenerative diseases, neurological diseases, muscle diseases, meningitis, encephalitis, encephalopathy, lysosomal diseases, malignant neoplasms, fibrosis, inflammatory diseases, immunodeficiency diseases, autoimmune diseases, and infectious diseases.

40. The oligonucleic acid nanoparticles according to any one of claims 1 to 35 for use in the treatment and / or prevention of a disease selected from the group consisting of inborn errors of metabolism, congenital endocrine disorders, single-gene diseases, neurodegenerative diseases, neurological diseases, muscular diseases, meningitis, encephalitis, encephalopathy, lysosomal diseases, malignant neoplasms, fibrosis, inflammatory diseases, immunodeficiency diseases, autoimmune diseases, and infectious diseases.

41. A combination comprising the oligonucleic acid nanoparticles according to any one of claims 1 to 35 and one or more therapeutic agents and / or one or more prophylactic agents for a disease, wherein the disease is selected from the group consisting of inborn errors of metabolism, congenital endocrine disorders, single-gene diseases, neurodegenerative diseases, neurological diseases, muscular diseases, meningitis, encephalitis, encephalopathy, lysosomal diseases, malignant neoplasms, fibrosis, inflammatory diseases, immunodeficiency diseases, autoimmune diseases, and infectious diseases, a medicament.

42. The oligonucleic acid nanoparticles according to any one of claims 1 to 35 for treating a disease in combination with one or more therapeutic agents and / or one or more prophylactic agents for the disease, wherein the disease is selected from the group consisting of inborn errors of metabolism, congenital endocrine disorders, single-gene diseases, neurodegenerative diseases, neurological diseases, muscular diseases, meningitis, encephalitis, encephalopathy, lysosomal diseases, malignant neoplasms, fibrosis, inflammatory diseases, immunodeficiency diseases, autoimmune diseases, and infectious diseases, the oligonucleic acid nanoparticles.

43. (a1) A step of binding a plurality of oligonucleic acids to a core composed of a dendritic polymer; (a2) A step of binding a hydrophilic polymer to the oligonucleic acid; (a3) A step of binding a cell internalization promoter to the hydrophilic polymer, a method for producing the oligonucleic acid nanoparticles according to any one of claims 1 to 4.

44. (b1) A step of binding one or a plurality of spacers to a core composed of a dendritic polymer; (b2) A step of binding a plurality of oligonucleic acids to the core; (b3) A step of binding a hydrophilic polymer to the spacer; (b4) A step of binding a cell internalization promoter to the hydrophilic polymer, a method for producing the oligonucleic acid nanoparticles according to any one of claims 6 to 9.

45. (a1) A step of binding a plurality of oligonucleic acids to a core composed of a dendritic polymer; (a2) A step of binding a hydrophilic polymer to the oligonucleic acid; (a3) A step of binding a cell internalization promoter to the hydrophilic polymer; (a4) binding a capping agent to the core, and a method for producing an oligo-nucleic acid nanoparticle according to claim 5, comprising the step of.

46. (b1) binding one or more spacers to a core composed of a dendritic polymer; (b2) binding a plurality of oligo-nucleic acids to the core; (b3) binding a hydrophilic polymer to the spacer; (b4) binding a cell internalization promoter to the hydrophilic polymer; (a5) binding a capping agent to the core, and a method for producing an oligo-nucleic acid nanoparticle according to claim 10, comprising the step of.

Images