Structure for nucleic acid delivery, method for producing structure for nucleic acid delivery, and medicine

Abstract

The present disclosure relates to a structure for nucleic acid delivery, said structure being characterized by having an association structure in which a nucleic acid analog represented by formula (1) and a nucleic acid to be delivered are associated by electrostatic interaction. (In the formula: N represents a cationic artificial nucleic acid including constituent units in which a base is bonded to a ring structure selected from ribose, deoxyribose, and morpholine, and a linking structure that has a cationic group and links two constituent units; H represents a hydrophilic polymer; S1 represents a spacer 1; S2 represents a spacer 2; L represents a ligand selected from C5 or lower saccharides and derivatives thereof, alcohols, and carboxylic acids; s represents 0 or 1; t represents 0 or 1; and the cationic artificial nucleic acid may be associated by electrostatic interaction between the cationic group and a phosphate group of the nucleic acid to be delivered).

Description

Nucleic acid delivery structure, method for producing nucleic acid delivery structure, and medicine

[0001] The present disclosure relates to a nucleic acid delivery structure, a method for producing a nucleic acid delivery structure, and a pharmaceutical.

[0002] In the field of gene therapy, diseases are treated by delivering oligonucleotides such as DNA and RNA to target sites such as cells to suppress gene expression or perform gene recombination. In such treatments, drug delivery system (DDS) technology, which efficiently delivers nucleic acids to target sites, is crucial. Various DDS methods have been developed to date. Furthermore, DDSs are also being actively applied in the fields of nanomaterials and artificial cells as nucleic acid analysis, diagnostic tracers, molecular machines, and molecular computers.

[0003] The inventors have discovered that nucleic acids characterized by a structure in which a hydrophilic group is linked to the 3' end of an artificial nucleic acid consisting of a structural unit in which a base is bound to a ring structure selected from ribose and deoxyribose and a linking structure connecting the two structural units form micelles or nanoparticles when annealed with RNA, that the micelles or nanoparticles act as vesicles for delivering RNA, and further that a composition in which miR-143 is made into nanoparticles using a cationic artificial nucleic acid is effective in suppressing the growth of colon cancer, a solid cancer (Patent Document 1).

[0004] "Transporters" are membrane proteins present in the cell membrane along with channels and receptors. However, unlike channels, they do not have a water channel. Instead, they are carriers that allow substances to pass through the cell membrane by switching and resetting the orientation of their substrate-binding sites between inside and outside the cell each time they are transported. Two types of transporters are known to date: the ABC (ATP-binding cassette) family, which transports substances using ATP energy, and the SLC (solute carrier). The development of drugs targeting these transporters has been proposed (Non-Patent Document 1). A detailed review of transporters in cancer cells has also been published, explaining that the transporters expressed vary depending on the cell type (Non-Patent Document 2).

[0005] Conventionally, methods have been known in which nanoparticles are loaded with targeting ligands to guide them to their targets. A recent example is one in which lipid nanoparticles target T cells by using antibodies that recognize T cells (Patent Document 2). Peptide ligands are also known as ligands that provide targeted binding to cell surface proteins (Patent Document 3). Furthermore, in nucleic acid delivery systems, ligands may be attached to the 3' or 5' end of the nucleic acid to be delivered (Patent Document 4).

[0006] International Publication No. 2022 / 230990 International Publication No. 2019 / 131770 Special Publication No. 2020-502120 Special Publication No. 2021-535227

[0007] Takahiko Anzai, Transporters: Aiming for Systemic Control from the Kidney, Japanese Journal of Pediatric Nephrology Vol. 26 No. 1. Zeribe Chike Nwosu, Mun Gu Song, Marina Pasca di Magliano, Costas A. Lyssiotis, Sung Eun Kim, “Nutrient transporters: connecting cancer metabolism to therapeutic opportunities”, Oncogene. 2023 March; 42(10): 711-724. doi:10.1038 / s41388-023-02593-x.

[0008] Some tumors are known to specifically express furanose-type transporters. We provide a drug delivery method for delivering nucleic acids for tumor control to tumors that have such transporters.

[0009] The inventors discovered that micelles or nanoparticles composed of cationic artificial nucleic acids and delivery nucleotides, whose surfaces are modified with ligands, recognize and bind to nutrient transporters specifically expressed in tumor cells, making them more susceptible to endocytosis, and thus completed the present disclosure.

[0010] [1] A nucleic acid delivery structure, characterized in that a nucleic acid analog represented by the following formula (1) and a nucleic acid to be delivered have an associated structure formed by electrostatic interaction: (Here, N represents a cationic artificial nucleic acid including a structural unit having a base bonded to a ring structure selected from ribose, deoxyribose, and morpholine, and a linking structure having a cationic group that links two of the structural units; H represents a hydrophilic polymer; S1 represents spacer 1, S2 represents spacer 2; L represents a ligand selected from a sugar having 5 or less carbon atoms and a derivative thereof, an alcohol having 5 or less carbon atoms, or a carboxylic acid having 5 or less carbon atoms; s represents 0 or 1, and t represents 0 or 1; and the cationic artificial nucleic acid can associate with the phosphate group of the nucleic acid to be delivered through electrostatic interaction with the cationic group. The structural unit having a base bonded to a ring structure may be selected from ribose and deoxyribose.)

[0011] [2] A nucleic acid delivery structure, characterized in that a nucleic acid analog represented by the following formula (1) and a nucleic acid to be delivered have an associated structure formed by electrostatic interaction: (Here, N represents a cationic artificial nucleic acid including a structural unit having a base bonded to a ring structure selected from ribose and deoxyribose, and a linking structure having a cationic group that links the two structural units; H represents a hydrophilic polymer; S1 represents spacer 1, S2 represents spacer 2; L represents a ligand selected from a sugar having 5 or less carbon atoms and a derivative thereof, an alcohol having 5 or less carbon atoms, or a carboxylic acid having 5 or less carbon atoms; s represents 0 or 1, t represents 0 or 1; and the cationic artificial nucleic acid can associate with the phosphate group of the nucleic acid to be delivered through electrostatic interaction with the cationic group. The structural unit having a base bonded to a ring structure may be selected from ribose and deoxyribose.)

[0012] [3] The nucleic acid delivery structure according to [1] or [2], wherein the cationic group has a pKa value in the range of 6 to 9.

[0013] [4] The nucleic acid delivery structure according to any one of [1] to [3], wherein the cationic group has, in a cationic state, a partial structure selected from the group consisting of the following formulas (C1) to (C7): (where R １ ~R ３ represents hydrogen or an alkyl group having 1 to 10 carbon atoms, and R １ ~R ３ may be the same or different. The alkyl group having 1 to 10 carbon atoms may have a substituent. Ring is a cyclic compound composed of 4 to 8 carbon atoms, and may be a heterocyclic ring in which one or more of the carbon atoms is substituted with a heteroatom selected from nitrogen, oxygen, and sulfur.

[0014] [5] The nucleic acid delivery structure according to any one of [1] to [4], characterized in that the linking structure of the cationic artificial nucleic acid (N) has at least a structure selected from the following formulas (L1) to (L4) in a cationic state: (where X ＋ is a functional group containing the cationic group, Z represents O or S, and W represents —O— or —NR ４ -, where R ４ represents hydrogen or an alkyl group having 1 to 10 carbon atoms. The alkyl group having 1 to 10 carbon atoms may have a substituent. * represents a bond to the adjacent structural unit.

[0015] [6] The nucleic acid delivery structure according to any one of [1] to [5], wherein the cationic artificial nucleic acid (N) has a nucleotide skeleton represented by the following formula (N1): (where X ＋ is a functional group containing the cationic group, Base is a base, and R ５ represents H or OH. * represents a bond to the phosphate of the adjacent nucleotide backbone, and at least one of the bonds at the 5'-end or 3'-end is bonded to the hydrophilic polymer or S1 of formula (1), and when not bonded to the hydrophilic polymer, it is hydrogen.)

[0016] [7] The functional group (X ＋) is capable of becoming an ammonium cation represented by the following formula (F1) in a cationic state. (where R １ ~R ３ represents hydrogen or an alkyl group having 1 to 10 carbon atoms, and may be the same or different from each other, m represents an integer of 0 to 10, and n represents an integer of 0 or 1. The alkyl group having 1 to 10 carbon atoms may have a substituent.

[0017] [8] The nucleic acid delivery structure according to any one of [1] to [7], wherein the hydrophilic polymer (H) is selected from polyethylene glycol, polyvinyl alcohol, polyglutamic acid, polyvinylpyrrolidone, polyacrylamide, polyethyleneimine, polyalkyl acrylate, polyoxazoline, polyacrylamide, poly(carboxybetaine methacrylate), poly(sulfobetaine methacrylate), poly(2-methacryloyloxyethylphosphocholine), hyaluronic acid, chitosan, dextran, and derivatives thereof.

[0018] [9] The nucleic acid delivery structure according to [8], wherein the hydrophilic polymer (H) has a polyethylene glycol backbone represented by the following formula (A1): (wherein p is an integer from 1 to 20.)

[0019]

[10] The nucleic acid delivery structure according to [9], wherein the formula (A1) is bonded to any of S1, S2, N and L in the formula (1) via a phosphate diester group.

[0020]

[11] The nucleic acid delivery structure according to any one of [1] to

[10] , wherein the spacer 1 (S1) has a bond represented by the following formula (S11): (wherein R ６ , R ７ represents a methylene group having 1 to 12 carbon atoms, and R ６ , R ７ may be the same or different. The methylene group having 1 to 12 carbon atoms may be substituted.

[0021]

[12] The nucleic acid delivery structure according to any one of [1] to

[11] , wherein the spacer 2 (S2) is a phosphate diester bond or a phosphate diester bond containing a triazole represented by the following formula (S21): (In the formula, Ka represents an amide bond containing a methylene group having 1 to 20 carbon atoms, an aromatic group having 6 to 12 carbon atoms, or a direct bond; q is 0 or 1; when q is 0, Ka bonds to a ligand. The methylene group having 1 to 20 carbon atoms and the aromatic residue having 6 to 12 carbon atoms may have a substituent.)

[0022]

[13] The nucleic acid delivery structure according to any one of [1] to

[12] above, wherein the ligand (L) is selected from the group consisting of the following formula (L5): (wherein X represents carbon, oxygen, or sulfur; R ８ is hydrogen, hydroxyl group, -OR １３ or a purine base and a pyrimidine base or a derivative thereof, R ８ The wavy bond indicates that the bond is an α or β bond, and R ９ , R １０ , R １１、 R １２ is C １ ~C ２０ alkyl group, hydrogen, halogen, hydroxyl group, or OR １３ Or -R １４ represents OH, and R ９ , R １０ , R １１、 R １２ may be the same or different. １３ is C １ ~C ２０ is an alkyl group of the formula R １４ is C １ ~C ２０ represents an alkylene group of the formula:

[0023]

[14] The nucleic acid delivery structure according to any one of [1] to

[13] , characterized in that it is a nanoscale structure in which a plurality of the nucleic acid delivery structures are associated.

[0024]

[15] The nucleic acid delivery structure according to

[14] , characterized in that the associated structure is a vesicle or micelle in which the hydrophilic polymer and the ligand are located on the outside and the associated structure is located on the inside.

[0025]

[16] The nucleic acid delivery structure according to any one of [1] to

[15] , wherein the nucleic acid to be delivered is a microRNA or an analog thereof.

[0026]

[17] A method for producing a nucleic acid delivery structure according to any one of [1] to

[16] above, comprising the steps of: binding the hydrophilic polymer to the 3' end of a cationic artificial nucleic acid; binding a ligand to the hydrophilic polymer to produce the nucleic acid analog; and annealing the nucleic acid analog and a nucleic acid to be delivered to produce the nucleic acid delivery structure.

[0027]

[18] A pharmaceutical for treating hematopoietic tumors, comprising the nucleic acid delivery structure described in any one of [1] to

[16] above.

[0028]

[19] A nucleic acid analogue represented by the following formula (1): (Here, N represents a cationic artificial nucleic acid including a structural unit in which a base is bound to a ring structure selected from ribose, deoxyribose, and morpholine, and a linking structure having a cationic group that links two of the structural units; H represents a hydrophilic polymer; S1 represents spacer 1, S2 represents spacer 2; L represents a ligand selected from a saccharide having 5 or less carbon atoms and a derivative thereof, an alcohol having 5 or less carbon atoms, or a carboxylic acid having 5 or less carbon atoms; s represents 0 or 1, and t represents 0 or 1.)

[0029]

[20] A nucleic acid analogue represented by the following formula (1): (Here, N represents a cationic artificial nucleic acid including a structural unit in which a base is bound to a ring structure selected from ribose and deoxyribose, and a linking structure having a cationic group that links the two structural units; H represents a hydrophilic polymer; S1 represents spacer 1, S2 represents spacer 2; L represents a ligand selected from a saccharide having 5 or less carbon atoms and a derivative thereof, an alcohol having 5 or less carbon atoms, or a carboxylic acid having 5 or less carbon atoms; s represents 0 or 1, and t represents 0 or 1.)

[0030]

[21] A method for producing a nucleic acid analogue represented by the following formula (2), comprising the steps of: binding a monomer comprising H to L' immobilised on a solid phase; and binding a monomer comprising N to the end of the bound monomer. (Here, N represents a cationic artificial nucleic acid including a structural unit in which a base is bound to a ring structure selected from ribose, deoxyribose, and morpholine, and a linking structure having a cationic group that links the two structural units; H represents a hydrophilic polymer; S1 represents spacer 1, S2 represents spacer 2; L' represents a ligand selected from a saccharide having 6 or less carbon atoms and a derivative thereof, an alcohol having 6 or less carbon atoms, or a carboxylic acid having 6 or less carbon atoms; s represents 0 or 1, and t represents 0 or 1.)

[0031]

[22] A method for producing a nucleic acid analogue represented by the following formula (1), comprising the steps of: binding a monomer constituting H to L immobilised on a solid phase; and binding a monomer constituting N to the end of the bound monomer. (Here, N represents a cationic artificial nucleic acid including a structural unit in which a base is bound to a ring structure selected from ribose, deoxyribose, and morpholine, and a linking structure having a cationic group that links two of the structural units; H represents a hydrophilic polymer; S1 represents spacer 1, S2 represents spacer 2; L represents a ligand selected from a sugar having 5 or less carbon atoms and a derivative thereof, an alcohol having 5 or less carbon atoms, or a carboxylic acid having 5 or less carbon atoms; s represents 0 or 1, and t represents 0 or 1; and the cationic artificial nucleic acid can associate with the phosphate group of the nucleic acid to be delivered through electrostatic interaction with the cationic group.)

[0032] According to the present disclosure, it is possible to provide a nucleic acid delivery structure that is effective primarily for treating tumors, a method for producing the same, and a medicament for hematopoietic tumors.

[0033] 1 is a schematic diagram illustrating the use of a nucleic acid analog as a carrier. It is an explanatory diagram showing an example of a method for producing a nucleic acid analog. It is a diagram showing the uptake rate into RPMI8226 for each type of ligand of cationic artificial nucleic acid. It is a diagram investigating the amount of uptake into RPMI8226 cells in the presence and absence of ribose using RIONs in which four types of ligands (dA, rA, rNut, and dNut) are bound to CM-miR143, RIONs made with only CM-miR143, and RIONs made with only artificial nucleic acids. It is a diagram investigating the uptake of the nucleic acid to be delivered into Hela cells using RIONs with rNut and dNut as ligands, RIONs without ligands, and RIONs made with only the nucleic acid to be delivered. 1 is a diagram showing the uptake of nucleic acids to be delivered into U251, RPMI8226, and Miapaca2 cells by RIONs with rA, dA, rNut, and dNut as ligands, and RIONs without a ligand. FIG. 2 is a diagram showing the cell viability of RPMI8226 cells. FIG. 3 is a diagram showing the evaluation of protein expression. FIG. 4 is a diagram showing the cell viability of RPMI8226 cells. FIG. 5 is a diagram showing a microscopic image of stained cell nuclei. FIG. 6 is an explanatory diagram showing an example of a method for producing a nucleic acid delivery structure. FIG. 7 is a graph showing the ratio of the number of red lights to the number of blue lights indicating cell nuclei per unit area in microscopic observation. FIG. 8 is a diagram showing a microscopic image of stained cell nuclei. FIG. 9 is a diagram relating to the evaluation of luciferase activity.

[0034] (Tumor Cells) The present disclosure aims to deliver a nucleic acid that exhibits medicinal effects that control tumors and ultimately lead to a cure to tumor cells occurring in any organ in the body. That is, the nucleic acid needs to be preferentially delivered to a target organ selected from the lung, heart, brain, spleen, lymph node, bone, bone marrow, skeletal muscle, stomach, small intestine, large intestine, kidney, bladder, breast, liver, testis, ovary, uterus, spleen, thymus, brainstem, cerebellum, spinal cord, eye, ear, tongue, or skin, and also needs to be delivered into tumor cells present therein.

[0035] Tumor cells are known to have nutrient transporters for glucose, fructose, lactate, amino acids (glutamine, cysteine, and other linear amino acids), fatty acids, and other nutrients. Transporters are carriers that capture these small molecule nutrients outside the cell, permeate the membrane, and release them into the cell. Therefore, while it was conceivable that they could be targeted to nutrients, it was difficult to imagine them as targets for nanoparticles containing nucleic acids, as in the present application. However, it has been revealed that when ligands on the surface of nanoparticles bind to or interact with transporters in some way, endocytosis occurs, allowing the nanoparticles to be taken up into the cell.

[0036] (Nucleic Acid to be Delivered: Nucleic Acid to be Delivered) In the present disclosure, the nucleic acid to be delivered (hereinafter sometimes referred to as "Nucleic Acid to be Delivered") can be DNA, RNA, or derivatives thereof. Examples of DNA include antisense DNA. Antisense DNA is single-stranded DNA that has a sequence complementary to a target mRNA or microRNA and binds to these RNAs to inhibit their function. Examples of RNA include microRNA. MicroRNA (hereinafter referred to as "miRNA") is an endogenous non-coding RNA of approximately 20 to 25 bases encoded on the genome. miRNA is first transcribed from the miRNA gene on genomic DNA as a primary transcript (Primary miRNA, Pri-miRNA) of approximately several hundred to several thousand bases in length, and then processed to become pre-miRNA (precusor miRNA) with a hairpin structure of approximately 60 to 110 bases. It then moves from the nucleus into the cytoplasm, where it becomes double-stranded miRNA of approximately 20 to 25 bases through splicing. The double-stranded miRNA is incorporated into a protein called RISC and becomes single-stranded miRNA (guide strand, antisense strand), while the more unstable single-stranded miRNA (passenger strand, sense strand) is degraded. The single-stranded miRNA inhibits the translation of the target gene by binding to the mRNA of the target gene with a partially complementary base sequence. "miR-143" is known as a microRNA that controls the network of the oncogene KRAS. Note that, in this specification, "miR-143" may be referred to as "miR143", for example.

[0037] More than 1,000 types of miRNAs are known in humans, mice, and other organisms. Each miRNA regulates the expression of multiple target genes and is involved in various biological phenomena, such as cell proliferation and differentiation. It has also been suggested that miRNAs are involved in the onset and progression of cancer, cardiovascular disease, neurodegenerative diseases, psychiatric disorders, and chronic inflammatory diseases. Many researchers have pointed out that miRNAs are particularly involved in cancer cell proliferation, and research and development of miRNAs as nucleic acid drugs is underway. In this disclosure, the nucleic acid to be delivered may be a nucleic acid that induces RNA interference, such as siRNA. Delivery can also be performed in either single-stranded or double-stranded forms. Cancer-associated microRNAs include miR143, miR43a, and miR145. Other known oncogenic miRNAs (microRNAs) are listed in Tables 1 and 2 of Journals Cancers Volume 7, Issue 4, 10.3390 / cancers7040904. Therefore, chemically modified versions of these microRNAs can serve as nucleic acids to be delivered as pharmaceuticals. Furthermore, the target is not limited to cancer, but also includes microRNA, siRNA, and AS nucleic acids associated with each disease.

[0038] The nucleic acid to be delivered (delivery target nucleic acid) may be stabilized in the blood by chemical modification. Examples of chemical modifications include substituting deoxyribose for the ribose of the RNA nucleoside, substituting fluorine at the 2' position, or substituting 2'-OMe, as well as substituting a portion of the phosphate diester used in the nucleoside bond with a thiophosphate diester. Specific examples include the following structures of pentose (formula (R1) below) and nucleotide (formula (R2) below), which are the building blocks of nucleic acids. Furthermore, the terminals may be stabilized, or the 5' terminal may be modified with a vinyl phosphate group.

[0039] (Nucleic Acid Analog) The nucleic acid analog of the present disclosure will be described below. The nucleic acid analog of the present disclosure comprises at least a cationic artificial nucleic acid, a hydrophilic polymer bound to the cationic artificial nucleic acid, and a ligand. The nucleic acid analog is preferably used as a carrier for delivering a nucleic acid to be delivered (hereinafter sometimes referred to as a "nucleic acid to be delivered") to a target site. The nucleic acid to be delivered of the present disclosure is preferably miR-143 or a derivative of the miR-143. Alternatively, siRNA can also be used. The nucleic acid analog of the present disclosure can be represented by the following formula (1): (Here, N represents a cationic artificial nucleic acid, H represents a hydrophilic polymer, S1 represents spacer 1, S2 represents spacer 2, L represents a ligand, s represents 0 or 1, and t represents 0 or 1.)

[0040] (Cationic Artificial Nucleic Acid) Among the elements constituting a nucleic acid analog, the cationic artificial nucleic acid represented by N has a structural unit in which a base is bound to a ring structure selected from ribose, deoxyribose, and morpholine, and a linking structure having a cationic group that links the two structural units. The structural unit in which a base is bound to a ring structure may be selected from ribose and deoxyribose. The base sequence of the cationic artificial nucleic acid can be appropriately designed depending on the base sequence of the nucleic acid to be delivered, taking into consideration base complementarity, the strength of electrostatic interaction, and the like. Here, structural units in which a base is bound to a ring structure of ribose and deoxyribose are preferred.

[0041] The cationic group, in a cationic state, preferably has a partial structure selected from the group consisting of the following formulae (C1) to (C7). (where R １ ~R ３ represents hydrogen or an alkyl group having 1 to 10 carbon atoms, and R １ ~R ３ may be the same or different. The alkyl group having 1 to 10 carbon atoms may have a substituent. Ring is a cyclic compound consisting of 4 to 8 carbon atoms, and may be a heterocyclic ring in which one or more of the carbon atoms is substituted with a heteroatom selected from nitrogen, oxygen, and sulfur. It may have a substituent.

[0042] The cationic artificial nucleic acid can associate with another nucleotide through electrostatic interaction between the phosphate group and the cationic group of the other nucleotide. Here, "another nucleotide" refers to a nucleotide such as DNA or RNA or its analog, and when a nucleic acid analog is used as a carrier, it refers to a nucleotide or its analog that constitutes the nucleic acid to be delivered.

[0043] The pKa of the cationic group is higher than the pKa of the phosphate group of other nucleotides due to electrostatic interactions with the phosphate group of other nucleotides. Under pH conditions lower than the pKa of the cationic group of the nucleic acid analog but higher than the pKa of the phosphate group of other nucleotides, the cationic group is positively charged and the phosphate group is negatively charged, resulting in electrostatic bonding between these groups. Here, since the pKa of the phosphate group of a nucleotide is generally less than 1, the pKa of the cationic group is 1 or greater, preferably 3 or greater, and more preferably 6.0 or greater. The upper limit of the pKa of the cationic group is not particularly limited, but is 12 or less, preferably 11 or less, and more preferably 9 or less. From the viewpoint of the strength of the electrostatic interaction with the phosphate group of other nucleotides and the structure of the cationic artificial nucleic acid, the pKa of the cationic group is preferably within the range of 6 to 9.

[0044] Examples of the base include adenine, guanine, cytosine, thymine, uracil, N-methyladenine, N-benzoyladenine, 2-methylthioadenine, 2-aminoadenine, 7-methylguanine, N-isobutyrylguanine, 5-fluorocytosine, 5-bromocytosine, 5-methylcytosine, 4-N-methylcytosine, 4-N,N-dimethylcytosine, 5-fluorouracil, 5-bromouracil, 5-chlorouracil, and 5,6-dihydrouracil.

[0045] The linking structure preferably has at least a structure selected from the following formulae (L1) to (L4) in a cationic state. (where X ＋ is a functional group containing a cationic group of the above formulae (C1) to (C7), Z represents O or S, and W represents —O— or —NR ４ -, where R ４represents hydrogen or an alkyl group having 1 to 10 carbon atoms. The alkyl group having 1 to 10 carbon atoms may have a substituent. * represents a bond to the adjacent structural unit described above.)

[0046] Here, "in a cationic state" refers to the case where the cationic group is assumed to be positively charged, whereas the target nucleic acid is normally negatively charged under the same environmental conditions. It is thought that the two may associate through electrostatic interaction to form a hydrophobic state. Depending on the environmental conditions of the nucleic acid analog (such as the pH and type of solvent), the cationic group may not be positively charged. For example, in the case of the above formula (C1), when the cationic group is ammonium, examples of a non-positively charged state include primary amine (primary ammonium when cationized), secondary amine (secondary ammonium when cationized), and tertiary amine (tertiary ammonium when cationized).

[0047] Examples of cationic artificial nucleic acids include those having a nucleotide backbone whose constituent unit is a nucleotide in which a phosphate and a base are bound to a ribose or deoxyribose. Preferred structures are shown below. (wherein, Base represents a base, and R ５ represents H or OH. * represents a bond to the phosphate of the adjacent nucleotide backbone, and at least one of the 5'-end or 3'-end is bound to the hydrophilic polymer or spacer 1(1) described above, and when not bound to the hydrophilic polymer, it is hydrogen. X ＋ is a functional group containing the cationic group.

[0048] Examples of structures having such a nucleotide backbone include the following:

[0049] In the case of (N1), X ＋ is preferably an ammonium cation represented by the following formula (F1) in a cationic state. (where R １ ~R ３represents hydrogen or an alkyl group having 1 to 10 carbon atoms, and may be the same or different from each other, m represents an integer of 0 to 10, and n represents an integer of 0 or 1. The alkyl group having 1 to 10 carbon atoms may have a substituent.

[0050] Here, the strength of the electrostatic interaction with the phosphate group of another nucleotide is determined by the primary amine (R １ ~R ３ are all hydrogen) < secondary amine (R １ ~R ３ Two of them are hydrogen and the others are alkyl groups) < tertiary amine (R １ ~R ３ one of which is hydrogen and the other is an alkyl group) < Quaternary ammonium (R １ ~R ３ are all alkyl groups). Furthermore, as will be shown in the examples below, structures having quaternary ammonium as the cationic group have the property of being more susceptible to structural collapse as the pH of the environment decreases, compared to tertiary amines and the like. This means that when a structure having quaternary ammonium as the cationic group is transferred to the cytoplasm via endosomes, it collapses in response to the weakly acidic pH in the endosome, and is more likely to release the nucleic acid to be delivered. Therefore, X ＋ Among these, quaternary ammonium is particularly preferred as the cationic group. The pKa of the cationic group varies depending on the structure, but is generally 6.1 to 7.9 for primary amines, 6.9 to 7.0 for secondary amines, 8.0 to 8.6 for tertiary amines, and 8.0 to 9.0 for quaternary ammonium.

[0051] The nucleotide skeleton may have a structure in which a base is bonded to morpholine as a constituent unit. Examples of those having a morpholino skeleton include the structure represented by the following formula (M1). (Here, Base represents a base. * represents a bond to phosphorus of the adjacent morpholino skeleton, ribose skeleton, or deoxyribose skeleton, and at least one of the 5'-end or 3'-end is bonded to the above-mentioned hydrophilic polymer or spacer 1(1), and when not bonded to the hydrophilic polymer, it is hydrogen. X ＋ is a functional group containing the cationic group.

[0052] An example of such a structure having a morpholino skeleton is the following formula (M2).

[0053] The number of structural units (degree of polymerization) formed by bonding a ring structure and a base that constitute the cationic artificial nucleic acid can be appropriately set depending on conditions such as the ring structure, the type of cationic group, and the type and length (number of bases) of the nucleic acid to be delivered. Generally, the degree of polymerization of a cationic artificial nucleic acid is about 5 to 100, preferably about 10 to 50. When the nucleic acid to be delivered has a degree of polymerization (number of bases) as short as about 20 bases, it is preferable that the degree of polymerization of the cationic artificial nucleic acid carrier be similar to that of the nucleic acid to be delivered. On the other hand, when delivering a relatively long nucleic acid such as mRNA, the proportion of negative charges relative to the length of the nucleic acid becomes smaller, and the influence of negatively charged portions on the entire nucleic acid becomes smaller. Therefore, the degree of polymerization of the cationic artificial nucleic acid may be different from the degree of polymerization of the nucleic acid to be delivered, as long as it does not interfere with the association of the cationic artificial nucleic acid and the nucleic acid to be delivered. For example, when the nucleic acid to be delivered is a long nucleic acid such as mRNA, the degree of polymerization of the cationic artificial nucleic acid may be smaller than the degree of polymerization of the nucleic acid to be delivered.

[0054] In cationic artificial nucleic acids, cationic groups are introduced into some or all of the linking structures connecting the constituent units. From the perspective of forming structures such as vesicles and micelles, as described below, the proportion of linking structures into which cationic groups have been introduced relative to the total number of linking structures is preferably 50% or more, more preferably 80% or more, and particularly preferably 100% (all linking structures). If the proportion of linking structures into which cationic groups have been introduced relative to the total number of linking structures is low, the negative charge of the target nucleic acid will predominate in the association structure formed between the cationic artificial nucleic acid and the target nucleic acid, making it difficult to form a structure. Alternatively, if cationic groups are introduced consecutively at either the 3' or 5' end, or if cationic groups are introduced into discrete linking structures, such as every other linking structure, it is possible to form a structure. However, these structures are thought to exhibit changes in pH response depending on the proportion of cations introduced, making the structure unstable. For this reason, a high proportion of linking structures into which cationic groups have been introduced is preferred.

[0055] (Hydrophilic Polymer) In formula (1), H represents a hydrophilic polymer (hydrophilic polymer structure, linking group derived from a polymer). Various polymers can be used as the hydrophilic polymer depending on the application of the nucleic acid analog. In particular, when the nucleic acid analog is used as a carrier for delivering nucleic acid to a target site, a hydrophilic polymer having biocompatibility is preferred. Furthermore, since the cationic artificial nucleic acid (N) is cationic, a neutral hydrophilic polymer that is less likely to electrically interact (attract or repel) with the cationic artificial nucleic acid (N) is preferred.

[0056] Such hydrophilic polymers are preferably selected from polyethylene glycol, polyvinyl alcohol, polyglutamic acid, polyvinylpyrrolidone, polyacrylamide, polyethyleneimine, polyalkyl acrylate, polyoxazoline, polyacrylamide, poly(carboxybetaine methacrylate), poly(sulfobetaine methacrylate), poly(2-methacryloyloxyethylphosphocholine), hyaluronic acid, chitosan, dextran, and derivatives thereof.

[0057] Examples of such hydrophilic polymers include those represented by the following formulae (P1) to (P14).

[0058] The type of monomer constituting the hydrophilic polymer and the number of monomer units (degree of polymerization) can be appropriately set depending on conditions such as the type and molecular weight of the nucleic acid to be delivered, the molecular weight of the artificial nucleic acid, and the surrounding environment of the target site to be delivered. Generally, the degree of polymerization of the hydrophilic polymer is approximately 2 to 100, preferably approximately 2 to 50, and more preferably approximately 2 to 10. Furthermore, these hydrophilic polymers may be repeated two or more times via phosphate groups or the like. In relation to the molecular weight of the artificial nucleic acid, the number of repeats of the hydrophilic polymer may be three or more, with the maximum number of repeats being 100 or less, preferably 30 or less, and more preferably 10 or less. If the degree of polymerization of the hydrophilic polymer or the number of repeats of the hydrophilic polymer is low, it becomes difficult to form the structure described below. Furthermore, the hydrophilic polymer is likely to be eliminated as a foreign substance in the body, and the degradation stability and blood retention are likely to be low. Conversely, if the degree of polymerization of the hydrophilic polymer is too high, the size of the structure will be too large, and the delivery efficiency of the target nucleic acid to be delivered to the target site will likely be low.

[0059] In particular, when a nucleic acid analogue is used as a carrier for a drug delivery system (DDS), polyethylene glycol is particularly preferred as the hydrophilic polymer from the viewpoint of blood retention, etc. As the polyethylene glycol, polyethylene glycol of 10 to 100K may be used in some cases, but it is preferable that the polyethylene glycol has a polyethylene glycol skeleton represented by the following formula (A1): (wherein p is an integer from 1 to 20.)

[0060] Furthermore, A1 can also be used by repeatedly and continuously bonding a nucleic acid base or a phosphate ester derivative (-PO(OH)-, -PS(OH)-, PO(SH)-) between them. The number of repetitions q of ethylene glycol in the monomer unit A1 is 1 to 20, preferably 3 to 10, and the monomer structures may be the same or different, with the degree of polymerization of the monomer unit being 1 to 10, preferably 2 to 10, and more preferably 2 to 8. Examples of such phosphate ester derivative monomer units include ethylene glycol phosphate derivative units, diethylene glycol phosphate derivative units, tetraethylene glycol phosphate derivative units, and hexaethylene glycol phosphate derivative units.

[0061] The degree of polymerization of the phosphate derivative unit depends on the size of the artificial nucleic acid, but is 1 to 10, preferably 2 to 10, and more preferably 2 to 8, when the number of bases in the artificial nucleic acid is 10 to 30. The monomer unit is not limited to ethylene glycol (A1), but examples include diethylene glycol, triethylene glycol, and hexaethylene glycol, and the monomer unit may be linked by phosphoramide (-P(OH)-NH-) or phosphate ester (-OPO-O-). The type and degree of polymerization of the monomer of the hydrophilic polymer described above can be selected from the viewpoints of ease of formation of the structure described below, resistance to recognition as a foreign substance in the body, degradation stability, and low blood retention, etc.

[0062] (Ligand) The surface of the structure may be modified with various ligands. Modifying the surface of the structure with such ligands can impart targeting properties to the structure. Examples of the ligand represented by L' in the nucleic acid analogue represented by formula (2) include immunoglobulins, carbohydrates, peptides, proteins, aptamers, etc. Major ligands predicted from research on lipid nanoparticles and their application fields are listed below.

[0063] ・Glucose: Tumor research, drug delivery to brain capillary endothelial cells ・Mannose: Efficient formation of giant liposomes ・Galactose: Study of galactose receptors on macrophages, targeted delivery of galactose to liver cells ・Sucrose: Cancer treatment with doxorubicin ・Maltose: Transport of doxorubicin in cancer treatment ・Lactose: Study of liposome size and stability ・Oligosaccharides: Design of therapeutic inhibitors ・Lectins: Pulmonary drug delivery ・Tomato lectin, wheat germ agglutinin: Oral administration of insulin ・NCL-aptamer: Cisplatin-based chemotherapy for a wide range of cancers ・sgc8 aptamer: For leukemia CEM-CCRF cells ・NX 1838: Specific binding to VEGF on cancer cells ・Anti-CD44: Selective targeting of cancer cells ・DAG-NX213: Specificity for VEGF, which promotes angiogenesis - AS1411: Cytotoxicity against breast cancer cells MCF-7 - Macugen: A therapeutic drug for macular age-related macular degeneration - BOCK: Use for recognizing different binding sites of thrombin - TASSET: Use for recognizing different binding sites of target proteins - xPSM-A9: Use against prostate-specific membrane antigen expressed in prostate cancer cells - IL-4Rα: Inhibition of tumor growth using the tumor microenvironment Furthermore, it has been found that sugars and derivatives thereof having 6 or fewer carbon atoms, alcohols having 6 or fewer carbon atoms, or carboxylic acids having 6 or fewer carbon atoms, sugars and derivatives thereof having 5 or fewer carbon atoms, alcohols having 5 or fewer carbon atoms, or carboxylic acids having 5 or fewer carbon atoms can also be used as ligands.

[0064] Here, the ligand L in formula (1) is selected from saccharides and derivatives thereof having 5 or fewer carbon atoms, alcohols having 5 or fewer carbon atoms, or carboxylic acids having 5 or fewer carbon atoms. It has been discovered that a ligand selected from saccharides and derivatives thereof having 5 or fewer carbon atoms, alcohols having 5 or fewer carbon atoms, or carboxylic acids having 5 or fewer carbon atoms recognizes tumor cells, binds to a transporter in the tumor cells, and transports the nucleic acid nanoparticles of the present disclosure into the cells by endocytosis. Known transporters include nutrient transporters and nucleic acid base transporters such as purine base transporters. The ligand L of the present disclosure is thought to bind to these transporters and induce endocytosis. It is particularly effective against hematopoietic tumor cells.

[0065] Examples of sugars that can be used as ligands include ribose, arabinose, xylose, lyxose, ribulose, xylulose, erythrose, threose, erythrulose, glyceraldehyde, and dihydroxyacetone. Examples of sugar derivatives include those in which the functional groups of these sugars are substituted with substituents. Examples of such substituents include alkyl groups, hydrogen, halogens, hydroxyl groups, and bases such as adenine, thymine, guanine, cytosine, and uracil. Hexose sugars are not included as substituents. Furthermore, the ribose or deoxyribose derivatives of the present invention also include structures in which the oxygen atom in the five-membered ring structure of pentose is replaced with another atom, such as a sulfur atom or a carbon atom. Examples of alcohols include pentyl alcohol, butyl alcohol, propyl alcohol, and their derivatives. Examples of fatty acids include pentenoic acid, butyric acid, propionic acid, fatty acids, and their derivatives. Furthermore, examples of fatty acid derivatives include pyruvic acid and lactic acid. Any substituent may be contained as long as it is recognized by the nutrient transporters and nucleic acid base transporters such as purine bases. When these substituents contain a carbon atom, this carbon atom is not included in the carbon number of the sugars and derivatives thereof having 5 or less carbon atoms, the alcohols having 5 or less carbon atoms, or the carboxylic acids having 5 or less carbon atoms.

[0066] Preferred examples of the ligand include the following pentose sugar represented by the following formula (L5) and derivatives thereof. (wherein X represents carbon, oxygen, or sulfur; R ８ is hydrogen, hydroxyl group, -OR １３ or a purine base and a pyrimidine base or a derivative thereof, R ８ The wavy bond indicates that the bond is an α or β bond, and R ９ , R １０ , R １１、 R １２ is C １ ~C ２０ alkyl group, hydrogen, halogen, hydroxyl group, or OR １４ Or -R １５ represents OH, and R ９ , R １０ , R １１ , R １２ , R １４ , R １５ may be the same or different. １４ is C １ ~C ２０ is an alkyl group of the formula R １５ is C １ ~C ２０ represents an alkylene group represented by the formula: １ ~C ２０ and alkyl groups of C １ ~C ２０ The alkylene group may have a substituent.

[0067] Examples of purine bases and pyrimidine bases include adenine, guanine, cytosine, thymine, and uracil, as well as derivatives of these bases, such as N-methyladenine, N-benzoyladenine, 2-methylthioadenine, 2-aminoadenine, 7-methylguanine, N-isobutyrylguanine, 5-fluorocytosine, 5-bromocytosine, 5-methylcytosine, 4-N-methylcytosine, 4-N,N-dimethylcytosine, 5-fluorouracil, 5-bromouracil, 5-chlorouracil, and 5,6-dihydrouracil.

[0068] More specifically, examples of the ligand include ribose derivatives and furan (furanose) derivatives. Particularly preferred are ribose, deoxyribose, 1-deoxy-D-ribofuranose, 1-deoxy-L-ribofuranose, 1,2-deoxy-D-ribofuranose, 1,2-deoxy-L-ribofuranose, nucleosides (adenosine, thymidine, guanosine, cytidine, uridine), and deoxynucleosides (deoxyadenosine, deoxythymidine, deoxyguanosine, deoxycytidine, deoxyuridine).

[0069] (Spacer 1) The cationic artificial nucleic acid and the hydrophilic polymer may be bonded via spacer 1 (1). Various bonding modes can be used for (S1). Examples of such bonds include an ester bond (-C(=O)-O-), an ether bond (-O-), a disulfide bond (-S-S-), a phosphoramidide (-P(OH)-NH-), and a phosphate ester (-OPO-O-). These may be used alone or in combination, and may be bonded via an ester bond, an ether bond, a disulfide bond, or the like, of an alkyl chain. When the cationic artificial nucleic acid has a nucleotide backbone, spacer 1 is preferably bonded to the hydroxyl group at the 5' end or 3' end or both of these ends of the cationic artificial nucleic acid, and bonded via the cationic artificial nucleic acid and the hydrophilic polymer. When the cationic artificial nucleic acid and the hydrophilic polymer are bonded via spacer 1, spacer 1 is expected to regulate the charge because of its different structure from the hydrophilic polymer. Furthermore, when a phosphoramidite group or a phosphate ester group is used, it behaves as a negative charge similar to a polymer made of polyethylene glycol, which is preferable. Furthermore, it is advantageous in that the induction of PEG antibodies is suppressed. Furthermore, when a phosphoramidite group is used, it is industrially advantageous in that the entire process from the nucleic acid sequence to the hydrophilic polymer moiety can be synthesized using an automated nucleic acid synthesizer.

[0070] Spacer 1 has a structure that links a cationic artificial nucleic acid and a hydrophilic polymer. Spacer 1 (S1) may have a bond represented by the following formula (S11). (wherein R ６, R ７ represents a methylene group having 1 to 12 carbon atoms, and R ６ , R ７ may be the same or different. The methylene group having 1 to 12 carbon atoms may be substituted.

[0071] (Spacer 2) Spacer 2 has a structure that links the hydrophilic polymer and the ligand. Spacer 2 (S2) is preferably a phosphate diester bond or a phosphate diester bond containing a triazole represented by formula (S21). In the formula, Ka represents an amide bond containing a methylene group having 1 to 20 carbon atoms, a compound containing an aromatic group having 6 to 12 carbon atoms, or a direct bond; q = 0 or 1; when q = 0, Ka binds to a ligand. The methylene group having 1 to 20 carbon atoms and the aromatic residue having 6 to 12 carbon atoms may have a substituent. Spacer 1 and spacer 2 have any structure in the nucleic acid analog and can be provided as needed. In formula (1), when spacer 1 is absent, t = 0, and when spacer 2 is absent, s = 0.

[0072] (Structure for Nucleic Acid Delivery) The nucleic acid analog of the present disclosure can be particularly preferably used as a carrier for delivering a nucleic acid to be delivered to a target site. FIG. 1 is a schematic diagram illustrating the use of a nucleic acid analog as a carrier. As shown in this figure, the nucleic acid analog ("Sense" in FIG. 1) has a cationic group and a hydrophilic polymer as a primary structure, while the nucleic acid to be delivered, such as a natural nucleic acid ("Anti Sense" in FIG. 1), is anionic. An association structure is formed between this anionic nucleic acid to be delivered and the cationic group of the nucleic acid analog through electrostatic interaction, resulting in a complex (ion complex: "RION" in FIG. 1) composed of the nucleic acid analog as a carrier and the nucleic acid to be delivered.

[0073] Here, when there is complementarity between the target nucleic acid and the cationic artificial nucleic acid, a double strand is formed by base-to-base hydrogen bonds. However, the present disclosure is advantageous in that even when the complementarity is low, the cationic group and the phosphate group of the target nucleic acid can associate through electrostatic interactions. That is, not only target nucleic acids with perfect complementarity (100% match), but also target nucleic acids with, for example, 80% or 90% complementarity, form an association structure and form a complex through the above-mentioned electrostatic interactions. This makes it possible to deliver target nucleic acids that are non-complementary strands to the target site. Although it depends on the type of cationic group, the degree of complementarity between the cationic artificial nucleic acid and the target nucleic acid is preferably 50% or more, more preferably 80% or more, and particularly preferably 100%.

[0074] In an aqueous environment such as blood, the hydrophilic polymer segments of this complex associate with each other to form a nanoscale nucleic acid delivery structure (hereinafter sometimes simply referred to as "structure"). Examples of such nanoscale structures include micelles and vesicles, in which the associated structural segments are located on the inside and the hydrophilic polymer segments are located on the outside. These micelles and vesicles form spherical structures with a hollow core, which can encapsulate drugs such as low-molecular-weight compounds. This makes it possible to deliver not only target nucleic acids but also low-molecular-weight drugs. The diameter of the hollow core is approximately 50 to 500 nm.

[0075] A micelle has a structure in which a hydrophilic polymer segment is located at the outermost shell of a spherical structure, and an association structure segment is located facing the hollow space at the center of the spherical structure. On the other hand, a vesicle has a bilayer structure in which two complexes are associated via an association structure segment, and one hydrophilic polymer segment of this bilayer is located at the outermost shell of the spherical structure, and the other hydrophilic polymer segment is located facing the hollow space at the center of the spherical structure. For this reason, it is preferable to encapsulate a hydrophobic drug or the like in the hollow space of a micelle, and it is preferable to encapsulate a hydrophilic drug or the like in the hollow space of a vesicle.

[0076] Such structures have high degradation stability because the target nucleic acid is located inside the spherical structure. Furthermore, because the structure has a hydrophilic polymer segment in the outermost shell, it has excellent blood retention. The structure is then encapsulated in an endosome in the target cell or other cell and taken up into the cytoplasm, after which it escapes the endosome and releases the target nucleic acid or small molecule drug into the cytoplasm or nucleus. Examples of uses for such structures include drug delivery carriers for various diseases. Therefore, by using the nucleic acid delivery structure of the present invention, the target nucleic acid or small molecule drug is less likely to be targeted by immune attack compared to when the target nucleic acid or small molecule drug is administered directly. Furthermore, while drugs encapsulated in LNP (Lipid Nanoparticle) accumulate in the liver by binding to apolipoprotein E (ApoE), the nucleic acid delivery structure of the present invention, which does not undergo LNP treatment, has the advantage of suppressing liver accumulation. It is believed to be extremely useful in medicine because it has a long blood retention time and suppresses liver accumulation, allowing for efficient delivery of target nucleic acids and small molecule drugs to target tumor cells.

[0077] The surface of the nucleic acid delivery structure is modified with various ligands. By modifying the surface of the structure with such ligands, it is possible to impart targeting properties to the structure. For convenience, the nucleic acid delivery structure is referred to as "Reversibly Ionic Oligonucleotide-based Nanoparticles" and may be abbreviated as RION or RIO.

[0078] (Method for Producing Nucleic Acid Analogs) Next, a method for producing nucleic acid analogs will be described. Nucleic acid analogs can be produced by various methods, including a method in which a cationic artificial nucleic acid and a hydrophilic polymer are separately synthesized and then linked together. That is, a method for producing nucleic acid analogs includes: (i) a cationic nucleic acid synthesis step for synthesizing a cationic artificial nucleic acid; (ii) a hydrophilic polymerization step for synthesizing a hydrophilic polymer; and (iii) a linking step for linking the cationic artificial nucleic acid and the hydrophilic polymer. More specifically, when using an automated nucleic acid synthesizer, the 3' or 5' end of the nucleic acid can be fixed, and the cationic artificial nucleic acid can be synthesized first, followed by the synthesis of a hydrophilic polymer and then linking the hydrophilic polymer to a ligand. Alternatively, the ligand can be fixed first, the hydrophilic polymer can be synthesized, and then the nucleic acid can be synthesized after that, and a spacer can be inserted between the hydrophilic polymer and the nucleic acid. The nucleic acid produced in this manner is anionic nucleic acid, but the nucleic acid produced by the above reaction can also be cationized. This will be described in further detail.

[0079] (a) Two-Step Synthesis Method Cationic artificial nucleic acids can be synthesized through a two-step reaction (two-step synthesis method). There are three main types of two-step synthesis methods. These are explained below in order. (a-1) Two-Step Synthesis Method I In this method, a nucleic acid is sulfurized (S-modified) by automated nucleic acid synthesis, and then a hydrophilic polymer phosphoramidite (e.g., ethylene glycol phosphoramidite) is linked to the sulfurized nucleic acid to synthesize an oligo-PS hydrophilic polymer (first step). A Br compound is then reacted to introduce a cationic group into the nucleic acid (second step). In the case of a nucleotide backbone, cationic artificial nucleic acids can be synthesized by a method including the steps of introducing a thiophosphate ester into the linking structure and reacting a bromo compound having a cationic group with the thiophosphate ester to introduce the cationic group into the linking structure.

[0080] (a-1-1) Introduction of Thiophosphates Thiophosphates can be synthesized by the well-known phosphoramidite method. Briefly, the phosphoramidite method involves supporting a nucleoside or nucleotide whose 5' end is protected with a 4,4'-dimethoxytrityl (DMTr) group on a solid phase (supporting step). Next, the DMTr group is deprotected with a deprotecting reagent such as dichloroacetic acid (deprotecting step), followed by coupling with a phosphoramidite nucleotide in the presence of an activating agent such as 4,5-dicyanoimidazole (coupling step). The phosphite is then converted to a thiophosphate using a sulfurizing agent such as (N,N-dimethylaminomethylidene)amino-3H-1,2,4-dithiazoline-3-thione (DDTT) (sulfurizing step). Alternatively, the phosphite is converted to a phosphate diester using an oxidizing agent containing iodine, pyridine, or the like (oxidizing step). By repeating this process, synthetic nucleic acids containing a thiophosphate in the linking structure can be produced. By changing the type of base in the phosphoramidite, it is possible to produce a synthetic nucleic acid having a desired sequence. Note that by performing a sulfurization step instead of an oxidation step, it is also possible to introduce a thiophosphate ester only at the desired position in the linkage structure that constitutes the nucleotide backbone.

[0081] (a-1-2) Reaction of a Bromo Compound Having a Cationic Group with a Thiophosphate Ester Next, the resulting synthetic nucleic acid is reacted with a bromo compound having an amine or ammonium group. Examples of bromo compounds include 3-bromo-1-propylamine hydrobromide, 2-bromo-N,N-diethylethylamine hydrobromide, and (3-bromopropyl)trimethylammonium bromide. The reaction of the synthetic nucleic acid with the bromo compound can be carried out in a phosphate buffer solution or the like. The reaction conditions can be appropriately set, but for example, the pH can be within the range of 5 to 7, the reaction temperature can be 30 to 60°C, and the reaction time can be 10 to 50 hours.

[0082] (a-2) Two-Step Synthesis Method II In this method, nucleic acids are converted to boranophosphates (B-forms) by automated nucleic acid synthesis, and then a hydrophilic polymer phosphoramidite (e.g., ethylene glycol phosphoramidite) is linked to the converted oligo-B-form hydrophilic polymer (first step). Then, an amino compound is reacted with the converted oligo by iodine oxidation to introduce a cationic group (second step). In this method, two cationic groups can be introduced to one phosphate group (double cation introduction) by using a cationic phosphoramidite (described below).

[0083] (a-3) Two-Step Synthesis Method III In this method, a cationic artificial nucleic acid is synthesized by automated nucleic acid synthesis (first step), and then a hydrophilic moiety is introduced by a click reaction (second step). That is, a cationic artificial nucleic acid having a cationic group in the backbone is synthesized, and then a hydrophilic azide compound such as azide polyethylene glycol is linked to the cationic artificial nucleic acid by a click reaction.

[0084] (b) One-step synthesis method On the other hand, cationic artificial nucleic acids can also be synthesized in one step. This method is roughly a one-step synthesis of a cationic hydrophilic polymer by automated nucleic acid synthesis of cationic phosphoramidites and ethylene glycol phosphoramidites. This method is also basically a similar scheme to the phosphoramidite method shown in the two-step synthesis method, but there are some differences.

[0085] First, a diisopropylamidophosphorous acid compound and a nucleotide monomer are reacted with a nucleotide supported on a solid phase. Some hydroxyl groups are protected with protecting groups, and then oxidized or boronated with an oxidizing agent or boronating agent. An amino group compound is reacted with the nucleotide by iodine oxidation to introduce a cationic group. Next, phosphoramidite nucleotides and phosphoramidite polyethylene glycols are synthesized by known methods.

[0086] Furthermore, the synthesized phosphoramidite nucleotide, phosphoramidite polyethylene glycol, and phosphoramidite disulfide having a disulfide bond and a phosphoramidite in the molecule are used as raw materials. Then, reactions such as protection with a protecting group and deprotection are performed to bind a hydrophilic polymer to the 5' position of the cationic artificial nucleic acid. The cationic artificial nucleic acid may also be synthesized in one step by solid-phase synthesis. The ligand introduced in this disclosure is characterized by its ease of mass synthesis and availability, since a series of reactions can be performed, from attaching a spacer and a hydrophilic group to the cationic artificial nucleic acid to binding the ligand. Patent Document 1 and other documents can be referenced for methods of producing cationic artificial nucleic acids.

[0087] Another method for producing a nucleic acid analog is to sequentially bind monomers from the 3'-end side by solid-phase synthesis in one step. That is, this method is a method for producing a nucleic acid analog represented by the following formula (2), which includes the steps of binding a monomer constituting H to L' immobilized on a solid phase, and binding a monomer constituting N to the end of the bound monomer. (Here, N represents a cationic artificial nucleic acid including a structural unit in which a base is bound to a ring structure selected from ribose and deoxyribose, and a linking structure having a cationic group that links the two structural units; H represents a hydrophilic polymer; S1 represents spacer 1, S2 represents spacer 2; L' represents a ligand selected from a saccharide having 6 or less carbon atoms and a derivative thereof, an alcohol having 6 or less carbon atoms, or a carboxylic acid having 6 or less carbon atoms; s represents 0 or 1, and t represents 0 or 1.)

[0088] Furthermore, there is provided the following production method: A method for producing a nucleic acid analogue represented by the following formula (1), comprising the steps of: binding a monomer constituting H to L immobilized on a solid phase; and binding a monomer constituting N to the end of the bound monomer. (Here, N represents a cationic artificial nucleic acid comprising a structural unit in which a base is bonded to a ring structure selected from ribose, deoxyribose, and morpholine, and a linking structure having a cationic group that links the two structural units; H represents a hydrophilic polymer; S1 represents spacer 1, S2 represents spacer 2; L represents a ligand selected from a sugar and a derivative thereof having 5 or fewer carbon atoms, an alcohol having 5 or fewer carbon atoms, or a carboxylic acid having 5 or fewer carbon atoms; s represents 0 or 1, t represents 0 or 1; and the cationic artificial nucleic acid can associate through electrostatic interaction between the phosphate group of the nucleic acid to be delivered and the cationic group.) In all cases, N is preferably a cationic artificial nucleic acid comprising a structural unit in which a base is bonded to a ring structure selected from ribose and deoxyribose, and a linking structure having a cationic group that links the two structural units.

[0089] In this method, for example, as shown in Figure 2, a ligand (adenosine is shown as an example of ligand L' in Figure 2) is first immobilized on a solid phase, and monomers constituting a hydrophilic polymer are linked via phosphodiester bonds using an automated nucleic acid synthesizer. Next, monomers (phosphoramidite nucleotides) constituting a cationic artificial nucleic acid are sequentially linked to the ends of the hydrophilic polymer, optionally via a spacer. For example, a method for producing a nucleic acid analog may include the steps of: introducing a phosphoramidite group at the 3-position of the pentose (ribose) of a ligand (adenosine); coupling the ligand to a solid phase via the introduced phosphoramidite group using an automated nucleic acid synthesizer; protecting functional groups such as the 5-position of the pentose of the ligand with a protecting group; stabilizing the phosphite; deprotecting the aforementioned 5-position protecting group and repeating the same cycle using a triethylene glycol phosphoramidite reagent to form a polymer; binding a spacer to the polymer as needed; and synthesizing a nucleic acid sequence from the 3' end to the 5' end. The monomer linkage direction is 3'→5'. Nucleic acid analogs can also be synthesized by this method. Alternatively, conventional nucleic acid synthesis may be performed, followed by cationization of the nucleic acid after the automated synthesis series is complete.

[0090] This one-step synthesis method is simpler than the two-step synthesis method because it allows the entire cationic artificial nucleic acid analog to be synthesized automatically by binding the cationic artificial nucleic acid to the ligand using an automated synthesizer. This one-step synthesis method can be performed using ligands other than pentoses. However, when a pentose is used as the ligand, commercially available phosphoramidite reagents can be used, which has the advantage of allowing for easier synthesis than when a hexose such as glucose is used as the ligand. Examples of phosphoramidite reagents that can be used include adenosine 3'-phosphoramidite reagent (Glen Research), oxyadenosine 3'-phosphoramidite reagent (Glen Research), Abasic II Phosphoramidite reagent (Glen Research), and dSpacer CE Phosphoramidite reagent (Glen Research). For example, when the hexose glucose is used as a ligand, glycosyl phosphoramidite is used. (dx.doi.org / 10.1021 / jo102584g | J. Org. Chem. 2011, 76, 2648-2659, M. Adinolfi et al. / Tetrahedran 58 (2002) 6697-6704, Mol Divers (2011) 15: 751-757 DOI10.1007 / s11030-011-9305-6)

[0091] (Method for manufacturing nucleic acid delivery structure) Next, a method for manufacturing a nucleic acid delivery structure will be described. The nucleic acid delivery structure is formed by associating a nucleic acid to be delivered with a nucleic acid analog to form a complex (association step). The association of the nucleic acid analog with the nucleic acid to be delivered causes the two to bind by annealing to form a double strand. The ratio of nucleic acid analog to nucleic acid to be delivered during annealing is not particularly limited, but a nucleic acid analog:nucleic acid to be delivered ratio of 1:1 to 1:10 is preferred. Annealing is performed by raising the temperature to a predetermined temperature and then lowering the temperature. For annealing, raising the temperature to 80°C or higher is preferred, and raising the temperature to 90°C or higher is more preferred. The time for maintaining the elevated temperature is preferably 5 minutes or longer, more preferably 10 minutes or longer. The temperature is then lowered to 50°C or lower, preferably 30°C or lower, and maintained at that temperature for 10 minutes or longer, preferably 30 minutes or longer.

[0092] In view of the electrostatic interaction between the cationic group of the nucleic acid analog and the phosphate group of the nucleic acid to be delivered, this step is preferably carried out under pH conditions that are lower than the pKa of the cationic group of the nucleic acid analog and higher than the pKa of the phosphate group of the nucleic acid to be delivered. That is, under such pH conditions, the cationic group is positively charged and the phosphate group is negatively charged, and these groups electrostatically bond. Although such pH conditions vary depending on the pKa of the cationic group, a pH of about 2 to 7 is preferred, and a pH of about 3 to 6 is more preferred.

[0093] Next, multiple complexes are associated to form a higher-order structure (aggregate formation step). In this step, the complexes self-aggregate in an aqueous solvent such as water or an aqueous solution to form structures such as micelles or vesicles. The concentration of the complexes to form the structures is approximately 25 to 2500 μM, and more preferably within the range of 100 to 1000 μM.

[0094] (Nucleic Acid Delivery Method) The nucleic acid delivery method of the present disclosure includes an administration step of administering the nucleic acid delivery structure described above to incorporate the nucleic acid delivery structure into a target site, and a release step of releasing the nucleic acid within the target site. In the administration step, the structure described above bound to the nucleic acid to be delivered is administered to a human or other mammal. The drug containing the structure is preferably administered into the bloodstream, but this can be determined appropriately depending on the disease to be treated, the type of hydrophilic polymer, and other factors. When the target site is a cell, the administered structure is incorporated into the cytoplasm via endosomes. In the release step, the nucleic acid to be delivered is released from the structure incorporated into the cell, which is the target site. Structures with highly pH-responsive cationic groups, such as quaternary ammonium, undergo structural collapse in the acidic environment within the endosome, making it easier to release the nucleic acid to be delivered. Furthermore, as described above, it is also possible to encapsulate a small molecule drug in the hollow portion of the structure and release it within the target site. This method allows the delivery of the nucleic acid to be delivered or a small molecule drug to the target site.

[0095] Ligands are exposed on the surface of the nucleic acid delivery structure used in the present disclosure. These ligands recognize nutrient transporters or nucleic acid base transporters on the surface of target tumor cells, bind to the transporters, and are taken up into the cells as endosomes by endocytosis. Since different types of nutrient transporters are expressed depending on the type of tumor cell and tumor stage, ligands can be selected depending on the target tumor. Furthermore, these ligands are not recognized by nutrient transporters in immune cells such as macrophages, allowing them to remain in the blood for longer.

[0096] The present disclosure will be specifically described below based on examples, but these are not intended to limit the scope of the present disclosure. In the following examples, "%" is based on mass (mass percent) unless otherwise specified.

[0097] 1. Production of RION Unless otherwise specified, the nucleic acid delivery structure of the present disclosure can be produced by mixing the nucleic acid to be delivered with a cationic artificial nucleic acid corresponding to the nucleic acid to be delivered obtained in a production example of the present disclosure in a ratio of 1:5, and annealing at 98°C for 15 minutes, 25°C for 50 minutes, and 45°C for 50 minutes. In this specification, the nucleic acid delivery structure may be abbreviated as RION, and the ligand-attached RION may be abbreviated as L-RION. The sequences of the nucleic acids used in this example are shown in Table 1.

[0098] The nucleic acids to be delivered in this example were CM-miRNA-143 (SEQ IDs 1 and 2) and luciferase siRNA (SEQ IDs 13 and 14). SEQ IDs 2 and 14 were conjugated to the 5' end with the fluorescent dye Cy5 (ThermoFisher) to track nucleic acid delivery by RION. Artificial nucleic acids corresponding to the antisense strand of miRNA-143 are SEQ IDs 3 to 12, and of these, the cationic artificial nucleic acids used for RION formation are SEQ IDs 4, 6, 8, 10, and 12. Artificial nucleic acids corresponding to the antisense strand of luciferase siRNA are SEQ IDs 15 to 24, and of these, the cationic artificial nucleic acids used for RION formation are SEQ IDs 16, 18, 20, 22, and 24. A list of nucleic acid sequences is shown in the table below.

[0099]

[0100] In the above table, the meaning of each symbol is as follows: AS: Antisense strand S: Sense strand N (uppercase): RNA (A: base is adenine, G: base is guanine, C: base is cytosine, U: base is uracil) n (lowercase): DNA (a: base is adenine, g: base is guanine, c: base is cytosine, t: base is thymine) N ｆ : 2'-F-RNA N ｍ : 2'-OMeRNA X: -(OCH ２ CH ２ ) ３ O- *: -P(O)OH- ＾ : -P(S)OH- (represents a structure in which two structures are conjugated) ＋: -P(S-(CH ２ ) ２ -N(CH ２ CH ３ ) ２ )OH- S: -O(CH ２ ) ６ -S-S-(CH ２ ) ６ O-(abbreviated as C6SS) 1: 2-deoxyribose 2: 1',2'-dideoxyribose

[0101] <Nucleic Acid Synthesis> The linking structure of the nucleic acid, spacer, hydrophilic polymer, and ligand can be produced using the respective phosphoramidite reagents in an automatic nucleic acid synthesizer, and synthesis was outsourced to Gene Design Inc. (Osaka, Japan).

[0102] <Conferring Cationicity> Nucleic acid analogs (SEQ IDs 6, 8, 10, and 12) were prepared from SEQ IDs 5, 7, 9, and 11, each of which had been pre-bound to a hydrophilic polymer, by reacting them with bromo compounds (2000 equivalents) in PBS (pH 7.4) at 45°C for 24 hours. After the reaction, the mixture was purified by dialysis against distilled water for 3 days using a Float-A-Lyzer G2, CE, 1 ml, 3.5-5 KDa (Repligen, Waltham, MA, US). After purification, the mixture was concentrated using a lyophilizer to obtain the desired compound. 2-Bromo-N,N-diethylethylamine hydrobromide was purchased from Merck (Darmstadt, Germany).

[0103] Nucleic acid analogs (SEQ IDs 4, 16, 18, 20, 22, and 24) were prepared from SEQ IDs 3, 15, 17, 19, 21, and 23, each of which had been pre-conjugated to a hydrophilic polymer, by reaction with bromo compounds (2000 equivalents) in PBS (pH 6.5) at 45°C for 24 hours. After the reaction, the reaction mixture was purified by dialysis against distilled water for 3 days using a Float-A-Lyzer G2, CE, 1 ml, 3.5-5 kDa (Repligen, Waltham, MA, US). After purification, the mixture was concentrated using a lyophilizer to obtain the desired compound. 2-Bromo-N,N-diethylethylamine hydrobromide was purchased from Merck (Darmstadt, Germany). Nucleic acid analogs (SEQ IDs 3 and 5) were obtained from Gene Design Inc. as described above. IDs 9, 13, 14, 15, 17, 19, 21, and 23 were also synthesized according to the synthesis method of IDs 3 and 5, so as to have the nucleic acid sequences and terminal modifications shown in Tables 1 and 2.

[0104] <Conversion into RION> The nucleic acid to be delivered was prepared so that the ratio of AS-1:AS-2 or AS-3:AS-4 was 9:1. Each nucleotide was dissolved in sterile water and then mixed at a weight ratio of AS:S = 1:5. The mixture was then incubated at 98°C for 15 minutes, 25°C for 50 minutes, and 45°C for 50 minutes to obtain an annealing mixture (AN1-10 in the table below). The association was confirmed by native PAGE. The L-RIONs AN1 to AN10 used in this example are summarized in the table below.

[0105]

[0106] <Native PAGE> 5-10 pmol of the annealing mixture adjusted to 10 μM was applied to a 20 wt% acrylamide gel and electrophoresed for 25 minutes. After electrophoresis, Cy5 fluorescence was detected using an Alliance Q9 (UVITEC, Cambridge, UK). After photography, nucleic acids were stained with SYBrGold for 5 minutes, and fluorescence from SYBR Gold Nucleic Acid Gel Stain (Thermo Fisher Scientific, Waltham, MA) was detected. Markers, 100 bp DNA Ladder (Takara Bio Inc., Shiga, Japan) and 20 bp DNA Ladder (Takara Bio Inc., Shiga, Japan), were applied to both ends of the sample, confirming that the target nucleic acid marked with Cy5 had been incorporated into the RION.

[0107] (1) Production Example 1: Production of rA-miRNA-RION (AN2) The cationic artificial nucleic acid was prepared using an automated nucleic acid synthesizer. The 3-position of adenosine (formula (R3)) was coupled to a solid phase using a 3'-phosphoramidite reagent (Glen Research). This was followed by protection of unreacted residues, stabilization of the phosphite, and deprotection of the 5-position. A similar cycle was repeated using a triethylene glycol phosphoramidite reagent to form a hexamer. Thiol-Modifier C6 S-S (Glen Research) was then attached as a spacer. A nucleic acid sequence identical to that of SEQ ID3 was synthesized from the 3' end to the 5' end to obtain SEQ ID 5. This was then combined with a bromo compound (2000 equivalents) to obtain the cationic artificial nucleic acid SEQ ID 6. SEQ ID 1 and SEQ ID 2 were mixed at a ratio of 9:1 to obtain the nucleic acid to be delivered. The target nucleic acid was annealed to form an RION at a ratio of 1 part delivery target nucleic acid to 5 parts cationic artificial nucleic acid, yielding rA-miRNA-RION (AN2).

[0108] (2) Production Example 2: Production of dA-miRNA-RION (AN3) dA-miRNA-RION (AN3) was obtained in the same manner as in Production Example 1, except that SEQ ID 8 obtained using a 3'-phosphoramidite reagent of deoxyadenosine (formula (R4) below) (manufactured by Glen Research) was used instead of the 3'-phosphoramidite reagent of adenosine in Production Example 1.

[0109] (3) Production Example 3: Production of rNut-miRNA-RION (AN4) rNut-miRNA-RION (AN4) was obtained in the same manner as in Production Example 1, except that SEQ ID 10 obtained using Abasic II Phosphoramidite reagent (manufactured by Glen Research) (see formula (R5) below) was used instead of the adenosine 3'-phosphoamidite reagent in Production Example 1.

[0110] (4) Production Example 4: Production of dNut-miRNA-RION (AN5) dNut-miRNA-RION (AN5) was obtained by the same procedure as in Production Example 1, except that SEQ ID 12 obtained using dSpacer CE Phosphoramidite reagent (manufactured by Glen Research) (see formula (R6) below) was used instead of the adenosine 3'-phosphoamidite reagent in Production Example 1.

[0111] (5) Production Example 5: Production of non-miRNA-RION (AN1) Non-miR143-RION (AN1) was obtained in the same manner as in Production Example 1, except that SEQ ID 4 obtained without using a ligand in Production Example 1 was used.

[0112] (6) Production Example 6: Production of rA-siRNA-RION (AN7) In Production Example 1, the nucleic acid sequence of SEQ ID3 was synthesized from the 3' end to the 5' end without using Thiol-Modifier C6 S-S (Glen Research), to obtain SEQ ID 15. As in Production Example 1, this was combined with a bromo compound (2000 equivalents) to obtain cationic artificial nucleic acid SEQ ID 16. As for the nucleic acid to be delivered, SEQ ID 13 and SEQ ID 14 were mixed at a ratio of 9:1 to obtain RION (AN7) as the nucleic acid to be delivered.

[0113] (7) Production Example 7: Production of dA-siRNA-RION (AN8) RION (AN8) was obtained in the same manner as in Production Example 18, except that SEQ ID 18 obtained using a deoxyadenosine 3'-phosphoramidite reagent (manufactured by Glen Research) was used instead of the adenosine 3'-phosphoramidite reagent in Production Example 6.

[0114] (8) Production Example 8: Production of rNut-siRNA-RION (AN9) rNut-siRNA-RION (AN9) was obtained in the same manner as in Production Example 6, except that SEQ ID 20 obtained using Abasic II Phosphoramidite reagent (manufactured by Glen Research) was used instead of the adenosine 3'-phosphoamidite reagent in Production Example 6.

[0115] (9) Production Example 9: Production of dNut-siRNA-RION (AN10) dNut-siRNA-RION (AN10) was obtained in the same manner as in Production Example 1, except that SEQ ID 22 obtained using dSpacer CE Phosphoramidite reagent (manufactured by Glen Research) was used instead of the adenosine 3'-phosphoamidite reagent in Production Example 6.

[0116] (10) Production Example 10: Production of non-siRNA-RION (AN6) Non-siRNA-RION (AN6) was obtained in the same manner as in Production Example 1, except that the adenosine phosphoramidite reagent in Production Example 6 was not used.

[0117] (Cells) HeLa cells, RAW264.7 cells, U251MG cells, and Miapaca2 cells were purchased from the Japanese Collection Research Bioresources Cell Bank. HeLa cells were cultured in 10% heat-inactivated FBS-containing D-MEM (Waco Inc., Osaka, Japan) at 5% CO ２ RAW264.7, RPMI8226, and AsPC-1 were cultured in RPMI-1640 medium (Waco Inc., Osaka, Japan) containing 10% heat-inactivated FBS under 5% CO ２ U251MG and Miapaca2 cells were cultured under conditions of 10% heat-inactivated FBS, E-MEM (Waco Inc., Osaka, Japan), and 5% CO. ２ The cells were cultured under the conditions of 100°C, 37°C.

[0118] (Confirmation of Cellular Uptake of RION) All cells were cultured at 0.25 × 10 cells per day before sample treatment. ５ Cells were seeded onto a 12-well plate at 100-200 nM per well and cultured overnight. AN1-10 RIONs were then added to the medium (final AS chain concentration: 100-200 nM). After 24 hours at 37°C, the cell nuclei were stained with Hoechst 3342 and observed under a confocal laser microscope. The area stained with red light (Cy5 / Hoechst) was compared to the area stained with blue light. When the nucleic acid to be delivered bound to the red dye by RION entered the cell, the cell was stained red. Furthermore, the nuclear staining dye (blue light) easily entered the cell. This allowed for quantitative analysis of the uptake of nucleic acids delivered by RION into the cell.

[0119] (1) Example 1 The uptake of the target nucleic acid miRNA into RPMI8226 was compared for each of the RIONs in Production Examples 1 to 5. The results are shown in the table below and in FIG.

[0120] These results indicate that RIONs with ligands (rA, dA, rNut, dNut in the table) were able to take up more of the target nucleic acid into cells than RIONs without ligands (RIONs).

[0121] (2) Example 2 The difference in uptake of the target nucleic acid depending on the presence or absence of free ribose was investigated under the same conditions as in Example 1. The results are shown in the table below and in FIG.

[0122] Figure 4 shows the amount of nucleic acid uptake into PRMI8266 cells using RIONs in which four types of ligands, dA, rA, rNut, and dNut, were bound to CM-miR143 ("dNut-RION" to "rA-RION" in the figure), and RIONs without a ligand ("none-RION" in the figure), in the presence ("+" in the figure) and absence ("-" in the figure). In the absence of ribose ("-"), the amount of nucleic acid uptake into cells was increased for RIONs with ligands compared to RIONs without a ligand (none-RION). When the type of ligand was the same, the amount of uptake was lower in the presence of free ribose than in the absence of free ribose. This suggests the possibility of competition for the ribose transporter.

[0123] (3) Example 3 The uptake of target nucleic acids into HeLa cells was investigated using RIONs (100 nM, 200 nM) with rNut or dNut as ligands, and RIONs without ligands (non) using the same method as in Example 1. The results are shown in the table below and in Figure 5.

[0124] (4) Example 4 The uptake of the nucleic acid to be delivered into RAW cells was investigated using RAW cells instead of HeLa cells in Example 3. The results are shown in the table below.

[0125] In Table 6, No detected (ND) indicates that no uptake of RION was observed.

[0126] FIG. 5 shows the results of investigating the uptake of the target nucleic acid into HeLa cells using RIONs with rNut or dNut as ligands, RIONs without a ligand, and the target nucleic acid alone.

[0127] Comparing Example 3 and Example 4, as shown in Table 6, the uptake of the nucleic acid to be delivered into RAW cells, which are macrophage-like cells, was significantly low, and therefore the nucleic acid delivery structure of the present disclosure is expected to migrate to the target without being phagocytosed by macrophages.

[0128] (5) Example 5: The nucleic acid to be delivered in Example 1 was changed to siRNA, and Miapaca2 cells and U251 cells were used instead of RPMI8226 cells to investigate the ability of each of the RIONs in Preparation Examples 6 to 10 to deliver the nucleic acid to cells. The results are shown in the table below and in Figure 6.

[0129]

[0130] Figure 6 also shows the results for RPMI8226 (Example 1) cells to clarify the differences in the uptake of target nucleic acids into cell types between RIONs with rA, dA, rNut, and dNut as ligands and RIONs without ligands. As shown in this figure, when RPMI8226 cells and Miapaca2 cells were used, the presence of a ligand promoted the uptake of target nucleic acids into the cells compared to the absence of a ligand (Non). In U251MG cells, the uptake of target nucleic acids into the cells was promoted when the ligand was rA or dA compared to the absence of a ligand (Non). Furthermore, when it comes to the type of ligand, rA and dA tend to have a higher nucleic acid uptake than rNut and dNut.

[0131] (6) Example 6 The cationic artificial nucleic acids used in this example are shown in Table 8 below.

[0132]

[0133] In Table 8 above, the meaning of each symbol is as follows: G: Guide strand P: Passenger strand AS: Antisense strand S: Sense strand N (uppercase): RNA (A: base is adenine, G: base is guanine, C: base is cytosine, U: base is uracil) n (lowercase): DNA (a: base is adenine, g: base is guanine, c: base is cytosine, t: base is thymine) N ｆ : 2'-F-RNA Nm : 2'-OMe-RNA *: Anionic backbone of phosphodiester ＾ : -P(S)OH- (represents a structure in which two structures are conjugated) ＋ : Phosphate cationic backbone X: Triethylene glycol Y: Linker (disulfide linker) dT: Deoxythymidine S: -O(CH ２ ) ６ -S-S-(CH ２ ) ６ O- (abbreviated as C6SS)

[0134] <Synthesis of Cationic Artificial Nucleic Acids> Cationic artificial nucleic acids were prepared using an automated nucleic acid synthesizer. The 3-position of an adenosine 3'-phosphoramidite reagent (Glen Research) was coupled to a solid phase, followed by protection of unreacted residues, stabilization of the phosphite, and deprotection of the 5-position. A similar cycle was repeated using a triethylene glycol phosphoramidite reagent to form a hexamer. Thiol-Modifier C6 S-S (Glen Research) was then attached as a spacer. The nucleic acid sequences were then synthesized from the 3' to 5' ends to obtain SEQ IDs 27, 29, 33, 35, 37, and 39. These were then synthesized using a bromo compound (2000 equivalents) to obtain cationic artificial nucleic acids SEQ IDs 28, 30, 34, 36, 38, and 40.

[0135] SEQ ID 25 and SEQ ID 26 were mixed at a ratio of 9:1 to prepare the nucleic acid to be delivered. The nucleic acid to be delivered and the cationic artificial nucleic acid were mixed at a molar ratio of 1:5, and the mixture was annealed at 98°C for 15 minutes, 25°C for 50 minutes, and 45°C for 50 minutes to form RIONs, yielding non-miRNA-RION (AN11) and rA-miRNA-RION (AN12). AN11 to AN16 are summarized in Table 9 below.

[0136]

[0137] [Functional Evaluation of the Nucleic Acid Delivery Structure According to the Present Invention in Suspension Cells of RPMI8226 (Multiple Myeloma)] RPMI8226 cells were purchased from the Japanese Collection Research Bioresources Cell Bank. RPMI8226 cells were cultured in RPMI-1640 (Waco Inc., Osaka, Japan) containing 10% heat-inactivated FBS under 5% CO ２ The cells were cultured under the conditions of 100°C, 37°C.

[0138] <Measurement of cell viability by cellular uptake of RION> All cells were cultured at 0.25 × 10 ５ Cells / well were seeded onto a 96-well plate and cultured overnight. After overnight incubation, the reagent was added to the cells to achieve the desired final concentration. 48 hours later, WST-8 reagent was added to the medium, and the absorbance was measured to assess cell viability. The results are shown in Figure 7. Cell viability was expressed as a percentage of viable cells, relative to the untreated (non-treated) control. In the synthesis described above, the RION without ligand binding was designated Non-RION143 (AN11).

[0139] The cell viability was 15% for AN12 (20 nM), and compared to 90% at 1000 nM for AN11 without a ligand, the cell viability decreased at a 50-fold lower concentration, suggesting that the improved cellular uptake by the ligand resulted in a significant inhibition of cell viability.

[0140] <Protein Expression Evaluation> Furthermore, AN12 and AN13, which has no target sequence (nontarget), were used as control RIONs to evaluate their effect on the oncogene KRAS (Figure 8). GAPDH was used as an internal control. AN12 reduced the expression of the oncogene KRAS. On the other hand, no reduction in expression was observed with the control AN13. This demonstrated that AN2 delivered the guide strand of miR143 into cells and induced RNA interference.

[0141] [Evaluation of microRNA143 function in transporter knockout] Cells were seeded in a 6-well plate, and 100 nM of siRNA-SLC29A / Lipofectamine, which knocks out the cell surface nucleoside transporter, was added and cultured for 48 hours. The cells were then seeded again in a 96-well plate. Untreated cells (wild type (WT)) were used as a control. The synthesized AN2 was added, and after 48 hours of culture, cell viability was determined using WST8 reagent. The results are shown in Figure 9.

[0142] When A-RION143 was added after the addition of siRNA-SLC29A, which knocks out the nucleoside transporter on the cell surface, the cell viability increased by approximately 10% to 72% compared to WT (cells not treated with nucleoside transporter knockout).

[0143] <Confirmation of Cellular Uptake of RION> Cells were cultured at 0.25 × 10 cells per day before sample treatment. ５ Cells were seeded onto a 96-well plate at 100 cells / well and cultured overnight. AN2 RION was then added to the medium (final G chain concentration: 200 nM). After incubation at 37°C for 24 hours, the cell nuclei were stained with Hoechst 3342 and observed under a confocal laser microscope. The results are shown in Figure 10.

[0144] Observation with a confocal laser microscope showed that AN2 was taken up into cells via the nucleoside transporter when the nucleoside transporter was knocked out.

[0145] [Evaluation of nucleic acid delivery constructs according to the present invention prepared using different hydrophilic polymers] Nucleic acid delivery constructs were prepared using the same synthesis method as above, except that the hydrophilic polymer was changed to the following (see Figure 11). For A-t6-RION (At6, AN13), A-t9-RION (At9, AN14), and A-t12-RION (At12, AN15), the hydrophilic polymer moiety consisted of 6 repeating units, 9 repeating units, and 12 repeating units of TEG (Chemical Formula 30), respectively. Furthermore, in A-t6-RION DNA (AN16), the ribose in the nucleic acid analog of the sense strand was changed to deoxyribose.

[0146]

[0147] <Confirmation of Cellular Uptake of RION> All cells were cultured at 0.25 × 10 cells per day before sample treatment. ５ Cells were seeded onto a 96-well plate at 1000 cells / well and cultured overnight. AN14 RION was then added to the medium (final AS chain concentration: 200 nM). After 24 hours at 37°C, the cell nuclei were stained with Hoechst 3342 and observed under a confocal laser microscope. The area stained with blue light and the area stained with red light (Cy5 / Hoechst) were compared. When the nucleic acid to be delivered bound to the red dye by RION entered the cell, the cell was stained red. Furthermore, the nuclear staining dye was distributed in the cell nucleus. This allowed for quantitative analysis of the uptake of nucleic acid delivered by RION into the cell.

[0148] The ratio of the number of red light particles to the number of blue light particles, which indicate cell nuclei per unit area, observed under a confocal laser scanning microscope is shown in the graph of Figure 12. The results of the confocal laser scanning microscope observation are shown in Figure 13.

[0149] Among the different hydrophilic polymer lengths, AN14 was the most well-internalized. It is believed that the length of the hydrophilic polymer affects the interaction between the ligand and receptor, and there is an optimal particle size for uptake, i.e., the number of repeating hydrophilic polymer segments. The particle sizes (hydrodynamic diameters) of the above RIONs were 158 nm (AN13), 218 nm (AN14), 84 nm (AN15), and 233 nm (AN16). Furthermore, AN16, which uses deoxyribose instead of ribose in the sense strand of the nucleic acid analog, was less well-internalized than AN13. This suggests that AN13 is more easily internalized by cells. Particle size was measured using a dynamic light scattering analyzer (Delsa Max, Beckman).

[0150] [Evaluation of Luciferase Activity] Furthermore, the luciferase activity of the synthesized A-t9-RION (At9, AN14), the ligand-unbound t6-RION (t6), and the non-RIONized NT was evaluated. The activity was evaluated by the following procedure.

[0151] Miapaca-2-Luc cells were purchased from the Japanese Collection Research Bioresources Cell Bank. The cells were cultured in 10% heat-inactivated FBS-containing DMEM (Waco Inc., Osaka, Japan) under conditions of 5% CO and 37°C.

[0152] <Evaluation of suppression of luciferase activity of RION> One day before sample treatment, cells were seeded onto a 24-well plate at 0.25 × 10 cells / well and cultured overnight, and then added to the cells to achieve the respective final concentrations. 96 hours later, each well was washed with PBS, and the plate was frozen at −40°C and then returned to room temperature, and the amount of luciferase luminescence was quantified according to the protocol of the luciferase reagent (manufactured by Promega Corporation).

[0153] The results are shown in Figure 14. At9 (AN14) most strongly inhibited luciferase activity. This suggests that receptor recognition of A (adenine) at the nanoparticle interface is related to the interfacial freedom of the length of the hydrophilic moiety. As the hydrophilic moiety of At9 becomes longer than that of At6, the degree of freedom of A at the interface increases, resulting in improved receptor recognition. On the other hand, At12, which has a longer hydrophilic moiety, exhibits even greater interfacial freedom, but exhibits reduced receptor recognition. This suggests that the hydrophilic moiety is designed to optimize the multivalent effect of A and receptor recognition. Here, the multivalent effect refers to the apparent strengthening of the interaction with the receptor due to the multipoint formation of weak interactions such as hydrogen bonds. In other words, it is thought that the placement of multiple A at the interface improves uptake through the multivalent effect of interactions with multiple receptors.

Claims

1. A nucleic acid delivery structure, characterized in that it has an association structure in which a nucleic acid analogue represented by the following formula (1) and a nucleic acid to be delivered are associated by electrostatic interaction: (Here, N represents a cationic artificial nucleic acid including a structural unit in which a base is bound to a ring structure selected from ribose, deoxyribose, and morpholine, and a linking structure having a cationic group that links two of the structural units; H represents a hydrophilic polymer; S1 represents spacer 1, S2 represents spacer 2; L represents a ligand selected from a sugar having 5 or less carbon atoms and a derivative thereof, an alcohol having 5 or less carbon atoms, or a carboxylic acid having 5 or less carbon atoms; s represents 0 or 1, and t represents 0 or 1; and the cationic artificial nucleic acid can associate with the phosphate group of the nucleic acid to be delivered through electrostatic interaction with the cationic group.) 2. A nucleic acid delivery structure, characterized in that it has an association structure in which a nucleic acid analogue represented by the following formula (1) and a nucleic acid to be delivered are associated by electrostatic interaction: (Here, N represents a cationic artificial nucleic acid including a structural unit in which a base is bound to a ring structure selected from ribose and deoxyribose, and a linking structure having a cationic group that links two of the structural units; H represents a hydrophilic polymer; S1 represents spacer 1, S2 represents spacer 2; L represents a ligand selected from a sugar having 5 or less carbon atoms and a derivative thereof, an alcohol having 5 or less carbon atoms, or a carboxylic acid having 5 or less carbon atoms; s represents 0 or 1, t represents 0 or 1; and the cationic artificial nucleic acid can associate with the phosphate group of the nucleic acid to be delivered through electrostatic interaction with the cationic group.) 3. The nucleic acid delivery structure according to claim 1 or 2, wherein the cationic group has a pKa value in the range of 6 to 9.

4. The nucleic acid delivery structure described in claim 1 or 2, characterized in that the cationic group, in a cationic state, has a partial structure selected from the group consisting of the following formulas (C1) to (C7): (where R １ ~R ３ represents hydrogen or an alkyl group having 1 to 10 carbon atoms, and R １ ~R ３ may be the same or different. Ring is a cyclic compound composed of 4 to 8 carbon atoms, and may be a heterocyclic ring in which one or more of the carbon atoms is substituted with a heteroatom selected from nitrogen, oxygen, and sulfur.

5. A nucleic acid delivery structure according to claim 1 or 2, characterized in that the linking structure of the cationic artificial nucleic acid (N) has at least one structure selected from the following formulas (L1) to (L4) in a cationic state: (where X ＋ is a functional group containing the cationic group, Z represents O or S, and W represents —O— or —NR ４ -, where R ４ represents hydrogen or an alkyl group having 1 to 10 carbon atoms. * represents a bond to the adjacent structural unit.) 6. The nucleic acid delivery structure according to claim 1 or 2, characterized in that the cationic artificial nucleic acid (N) has a nucleotide skeleton represented by the following formula (N1): (where X ＋ is a functional group containing the cationic group, Base is a base, and R ５ represents H or OH. * represents a bond to the phosphate of the adjacent nucleotide backbone, and at least one of the bonds at the 5'-end or 3'-end is bonded to the hydrophilic polymer or S1 of formula (1), and when not bonded to the hydrophilic polymer, it is hydrogen.) 7. The functional group (X ＋ 7. The nucleic acid delivery structure according to claim 6, wherein, in a cationic state, the structure can become an ammonium cation represented by the following formula (F1): (where R １ ~R ３ represents hydrogen or an alkyl group having 1 to 10 carbon atoms, and may be the same or different from each other, m represents an integer of 0 to 10, and n represents an integer of 0 or 1.

8. The nucleic acid delivery structure described in claim 1 or 2, characterized in that the hydrophilic polymer (H) is selected from polyethylene glycol, polyvinyl alcohol, polyglutamic acid, polyvinylpyrrolidone, polyacrylamide, polyethyleneimine, polyalkyl acrylate, polyoxazoline, polyacrylamide, poly(carboxybetaine methacrylate), poly(sulfobetaine methacrylate), poly(2-methacryloyloxyethylphosphocholine), hyaluronic acid, chitosan, dextran, and derivatives thereof.

9. The nucleic acid delivery structure according to claim 8, characterized in that the hydrophilic polymer (H) has a polyethylene glycol backbone represented by the following formula (A1): (wherein p is an integer from 1 to 20.) 10. The nucleic acid delivery structure according to claim 9, characterized in that the formula (A1) is bonded to any of S1, S2, N and L in the formula (1) via a phosphate diester group.

11. The nucleic acid delivery structure according to claim 1 or 2, characterized in that the spacer 1 (S1) has a bond represented by the following formula (S11): (wherein R ６ , R ７ represents a methylene group having 1 to 12 carbon atoms, and R ６ , R ７ may be the same or different.) 12. A nucleic acid delivery structure according to claim 1 or 2, characterized in that spacer 2 (S2) is a phosphate diester bond or a phosphate diester bond containing a triazole shown in the following formula (S21). (In the formula, Ka represents an amide bond containing a methylene group having 1 to 20 carbon atoms, an aromatic group having 6 to 12 carbon atoms, or a direct bond; q is 0 or 1; when q is 0, Ka is bonded to a ligand.) 13. The nucleic acid delivery structure according to claim 1 or 2, characterized in that the ligand (L) is selected from the group represented by the following formula (L5): (wherein X represents carbon, oxygen, or sulfur; R ８ is hydrogen, hydroxyl group, -OR １３ or a purine base and a pyrimidine base or a derivative thereof, R ８ The wavy bond indicates that the bond is an α or β bond, and R ９ , R １０ , R １１、 R １２ is C １ ~C ２０ alkyl group, hydrogen, halogen, hydroxyl group, or OR １３ Or -R １４ represents OH, and R ９ , R １０ , R １１、 R １２ may be the same or different. １３ is C １ ~C ２０ is an alkyl group of the formula R １４ is C １ ~C ２０ represents an alkylene group of the formula:

14. The nucleic acid delivery structure according to claim 1 or 2, characterized in that it is a nanoscale structure in which a plurality of the nucleic acid delivery structures are associated.

15. The nucleic acid delivery structure according to claim 14, characterized in that the assembly structure is a vesicle or micelle in which the hydrophilic polymer and the ligand are located on the inside and the outside.

16. The nucleic acid delivery structure according to claim 1 or 2, characterized in that the nucleic acid to be delivered is microRNA or an analog thereof.

17. A method for producing a nucleic acid delivery structure according to claim 1 or 2, comprising the steps of: binding the hydrophilic polymer to the 3' end of a cationic artificial nucleic acid; binding a ligand to the hydrophilic polymer to produce the nucleic acid analog; and annealing the nucleic acid analog with a nucleic acid to be delivered to produce the nucleic acid delivery structure.

18. A pharmaceutical for treating hematopoietic tumors, comprising the nucleic acid delivery structure described in claim 1 or 2.

19. A nucleic acid analogue represented by the following formula (1): (Here, N represents a cationic artificial nucleic acid including a structural unit in which a base is bound to a ring structure selected from ribose, deoxyribose, and morpholine, and a linking structure having a cationic group that links two of the structural units; H represents a hydrophilic polymer; S1 represents spacer 1, S2 represents spacer 2; L represents a ligand selected from a saccharide having 5 or less carbon atoms and a derivative thereof, an alcohol having 5 or less carbon atoms, or a carboxylic acid having 5 or less carbon atoms; s represents 0 or 1, and t represents 0 or 1.) 20. A nucleic acid analogue represented by the following formula (1): (Here, N represents a cationic artificial nucleic acid including a structural unit in which a base is bound to a ring structure selected from ribose and deoxyribose, and a linking structure having a cationic group that links the two structural units; H represents a hydrophilic polymer; S1 represents spacer 1, S2 represents spacer 2; L represents a ligand selected from a saccharide having 5 or less carbon atoms and a derivative thereof, an alcohol having 5 or less carbon atoms, or a carboxylic acid having 5 or less carbon atoms; s represents 0 or 1, and t represents 0 or 1.) 21. A method for producing a nucleic acid analogue represented by the following formula (2), comprising the steps of: binding a monomer comprising H to L' immobilised on a solid phase; and binding a monomer comprising N to the end of the bound monomer. (Here, N represents a cationic artificial nucleic acid including a structural unit in which a base is bound to a ring structure selected from ribose, deoxyribose, and morpholine, and a linking structure having a cationic group that links the two structural units; H represents a hydrophilic polymer; S1 represents spacer 1, S2 represents spacer 2; L' represents a ligand selected from a saccharide having 6 or less carbon atoms and a derivative thereof, an alcohol having 6 or less carbon atoms, or a carboxylic acid having 6 or less carbon atoms; s represents 0 or 1, and t represents 0 or 1.) 22. A method for producing a nucleic acid analogue represented by the following formula (1), comprising the steps of: binding a monomer constituting H to L immobilised on a solid phase; and binding a monomer constituting N to the end of the bound monomer. (Here, N represents a cationic artificial nucleic acid including a structural unit in which a base is bound to a ring structure selected from ribose, deoxyribose, and morpholine, and a linking structure having a cationic group that links two of the structural units; H represents a hydrophilic polymer; S1 represents spacer 1, S2 represents spacer 2; L represents a ligand selected from a sugar having 5 or less carbon atoms and a derivative thereof, an alcohol having 5 or less carbon atoms, or a carboxylic acid having 5 or less carbon atoms; s represents 0 or 1, and t represents 0 or 1; and the cationic artificial nucleic acid can associate with the phosphate group of the nucleic acid to be delivered through electrostatic interaction with the cationic group.)

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