Use of structure for nucleic acid delivery in treatment of hematopoietic tumor

Abstract

The present disclosure is a structure for nucleic acid delivery to be used for the treatment of hematopoietic tumors, the 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 comprising a microRNA that controls a network of cancer genes KRAS are associated by electrostatic interaction. (Here, N represents a cationic artificial nucleic acid, H represents a hydrophilic polymer, S1 represents a spacer 1, S2 represents a spacer 2, and L represents a ligand; N has a constituent unit in which a base is bonded to a hexose selected from ribose and deoxyribose, a connection structure connecting two constituent units, and a backbone structure composed of cationic groups; and the cationic artificial nucleic acid can be associated by electrostatic interaction between a phosphate group of the nucleic acid to be delivered and the cationic groups.)

Description

Use of nucleic acid delivery constructs in the treatment of hematopoietic tumors

[0001] The present disclosure relates to the use of nucleic acid delivery constructs in the treatment of hematopoietic malignancies.

[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] On the other hand, drug discovery targeting RAS, a cancer-promoting gene associated with 30% of all cancers, has been widely pursued over the past 40 years. However, the RAS protein has a narrow pocket for small molecules, making it difficult to design inhibitors, and cancer cells acquire resistance when controlling a single RAS gene with antisense oligonucleotides (AS-ODN) or siRNA. Therefore, no effective therapeutic drug has yet been developed.

[0004] 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).

[0005] Hematopoietic tumors, such as leukemia, are diseases in which white blood cells or immature cells proliferate independently. Unlike solid cancers, treatment with surgical techniques alone is said to be difficult. Some hematopoietic tumors, particularly RAS-mutated multiple myeloma, are known to recur repeatedly and have a poor prognosis. The inventors have discovered that miR-143 and its derivatives are effective against RAS-mutated cancers (Patent Document 2).

[0006] International Publication No. WO 2022 / 230990 International Publication No. WO 2017 / 179660

[0007] RAS-mutated multiple myeloma is known to be one of the hematopoietic tumors typified by leukemia, but it is prone to recurrence and has a poor prognosis. Although miR-143, which was effective against solid cancers, is expected to be effective against such blood cancers, nucleic acid delivery to suspended cells of blood cancer is difficult, and a new nucleic acid drug delivery system was needed.

[0008] An object of the present disclosure is to provide a nucleic acid delivery structure that is effective in treating hematopoietic tumors, a method for producing the same, a medicine for hematopoietic tumors, and a method for treating hematopoietic tumors.

[0009] The inventors have discovered that micelles or nanoparticles comprising cationic artificial nucleic acids and delivery nucleotides, the surfaces of which are modified with ligands, suppress the proliferation of cells derived from hematopoietic tumors, and have thereby completed the present disclosure.

[0010] [1] Use of a nucleic acid delivery structure in the treatment of hematopoietic tumors, the nucleic acid delivery structure having an assembly structure in which a nucleic acid analog represented by the following formula (1) and a nucleic acid to be delivered, which is a microRNA that controls the network of the oncogene KRAS, are associated by electrostatic interaction: (wherein 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, t represents 0 or 1, N has a backbone structure consisting of a structural unit in which a base is bound to a hexose selected from ribose and deoxyribose, a linking structure connecting two of the structural units, and a cationic group, and the cationic artificial nucleic acid can associate with the phosphate group of the nucleic acid to be delivered by electrostatic interaction with the cationic group.)

[0011] [2] Use of the nucleic acid delivery structure according to [1], wherein the cationic group has a pKa value in the range of 6 to 9, in the treatment of hematopoietic tumors.

[0012] [3] Use of the nucleic acid delivery structure according to [1] or [2] above, wherein the cationic group, in a cationic state, has a partial structure selected from the group consisting of the following 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. 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.

[0013] [4] Use of the nucleic acid delivery structure according to any one of [1] to [3] in the treatment of hematopoietic tumors, wherein the linking structure of the cationic artificial nucleic acid (N) has at least one structure selected from the following formulas (L1) to (L4) in a cationic state: (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.)

[0014] [5] Use of the nucleic acid delivery structure according to any one of [1] to [4] in the treatment of hematopoietic tumors, wherein the cationic artificial nucleic acid has a nucleotide backbone 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, and when it is not bonded to the hydrophilic polymer, it is hydrogen.)

[0015] [6] The X ＋ is capable of becoming an ammonium cation represented by the following formula (F1) in a cationic state, for use in the treatment of hematopoietic tumors. (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.

[0016] [7] Use of the nucleic acid delivery structure according to [1] in the treatment of hematopoietic tumors, wherein the hydrophilic polymer 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.

[0017] [8] Use of the nucleic acid delivery structure according to [7], characterized in that the hydrophilic polymer (H) has a polyethylene glycol backbone represented by the following formula (A1), in the treatment of hematopoietic tumors. (wherein p is an integer from 1 to 20.)

[0018] [9] Use of the nucleic acid delivery structure according to [8], wherein the formula (A1) is linked via a phosphate diester group, in the treatment of hematopoietic tumors.

[0019]

[10] Use of the nucleic acid delivery structure according to any one of [1] to [9] in the treatment of hematopoietic tumors, 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.)

[0020]

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

[10] in the treatment of hematopoietic tumors, wherein the spacer 2 (S2) is a phosphodiester bond or a phosphodiester 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, a compound containing an aromatic group having 6 to 12 carbon atoms, or a direct bond; q is 0 or 1; when q is 0, the 1,2,3-triazole ring is bonded to the ligand.)

[0021]

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

[11] in the treatment of hematopoietic tumors, wherein the ligand (L) is selected from glucose, mannose, galactose, sucrose, maltose, and lactose.

[0022]

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

[12] in the treatment of hematopoietic tumors, wherein the nucleic acid delivery structure is a nanoscale structure in which a plurality of the nucleic acid delivery structures are associated.

[0023]

[14] Use of the nucleic acid delivery structure according to

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

[0024]

[15] Use of the nucleic acid delivery structure according to any one of [1] to

[14] in the treatment of hematopoietic tumors, wherein the nucleic acid to be delivered is selected from miR-143 or analogs thereof.

[0025]

[16] Use of the nucleic acid delivery structure according to

[15] in the treatment of hematopoietic tumors, wherein the miR-143 is selected from the group consisting of SEQ-1 to SEQ-23.

[0026]

[17] Use of a pharmaceutical comprising the nucleic acid delivery structure according to any one of [1] to

[16] above in the treatment of hematopoietic tumors.

[0027]

[18] A method for treating hematopoietic tumors, comprising administering to a patient the nucleic acid delivery structure described in [1] to

[16] above.

[0028]

[19] A method for inhibiting the proliferation of cancerous cells in hematopoietic tumors, comprising administering to the cancerous cells a nucleic acid delivery structure having an association structure in which a nucleic acid analog represented by the following formula (1) and a nucleic acid to be delivered, the nucleic acid being a microRNA that controls the network of the oncogene KRAS, are associated by electrostatic interaction: (wherein 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, t represents 0 or 1, N has a backbone structure consisting of a structural unit in which a base is bound to a hexose selected from ribose and deoxyribose, a linking structure connecting two of the structural units, and a cationic group, and the cationic artificial nucleic acid can associate with the phosphate group of the nucleic acid to be delivered by electrostatic interaction with the cationic group.)

[0029]

[20] A nucleic acid delivery structure used for the treatment of hematopoietic tumors, characterized in that it has an association structure in which a nucleic acid analog represented by the following formula (1) and a nucleic acid to be delivered, which is composed of a microRNA that controls the network of the oncogene KRAS, are associated by electrostatic interaction: (wherein 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, t represents 0 or 1, N has a backbone structure consisting of a structural unit in which a base is bound to a hexose selected from ribose and deoxyribose, a linking structure connecting two of the structural units, and a cationic group, and the cationic artificial nucleic acid can associate with the phosphate group of the nucleic acid to be delivered by electrostatic interaction with the cationic group.)

[0030] According to the present disclosure, it is possible to provide a nucleic acid delivery structure effective for treating hematopoietic tumors, a method for producing the same, a medicine for hematopoietic tumors, and a method for treating hematopoietic tumors.

[0031] 1 shows an outline of the nucleic acid analogs and nucleic acid delivery constructs of the present disclosure. (a) A diagram showing the sequence design for Glu-RION-miR143#12. (b) A diagram showing the generation of a delivery construct for Glu-RION-miR143#12. (c) A diagram showing the particle size distribution of Glu-RION-miR143#12. (d) A diagram showing the stability of Glu-RION-miR143#12 in the blood after administration to mice. A diagram showing an example of a synthesis scheme for cationic artificial nucleic acids. A diagram showing an example of a synthesis scheme. A diagram showing the survival rate of DLD-1 cells when miR-143#12 was treated with RION (control) and Glu-RION for 48 hours. The control had no inhibitory effect. A diagram showing the effect of Glu-RION-miR143#12 on various hematopoietic tumor cells. Various cells were treated for 72 hours to examine their cell growth inhibitory effects (Figures 5a, c, and e). PRMI8226 cells were treated with and without Glu-RION-miR143#12 and observed under a fluorescent microscope. Hoechst 33342 was used as the fluorescent reagent (Figure 5d). Using the gene transfer reagent Lipofectamine instead of Glu-RION did not have any cell growth inhibitory effect on NB4 and HL-60 cells (Figure 5c). This figure shows the effect of Glu-RION-miR143#12 on various hematopoietic tumor cells. Various cells were treated for 72 hours to examine their cell growth inhibitory effects (Figures 5a, c, and e). PRMI8226 cells were treated with and without Glu-RION-miR143#12 and observed under a fluorescent microscope. The presence of glucose transporters was confirmed on hematopoietic tumor cells. NB4 cells were stained with Hoechst 33342 and then observed under a fluorescence microscope (Fig. 5f). Figure 5f shows the results of Western blot analysis investigating the expression of downstream factors of the RAS network mediated by Glu-RION-miR143#12 in PRMI8226 cells. Figure 5f shows the results of Western blot analysis investigating the expression of downstream factors of the RAS network mediated by Glu-RION-miR143#12 in NB4, HL-60, and Jurkat cells.This figure shows the effect of Glu-RION-miR143#12 on the growth of human peripheral lymphocytes, with or without stimulation with concanavalin A.

[0032] The micelles or nanoparticles obtained from a composition of a nucleic acid analog represented by the following formula (1) and miR-143 or its analogs, which are used in the treatment of hematopoietic tumors according to the present disclosure, are described below.

[0033] (Hematopoietic Tumors) The micelles or nanoparticles used in the present disclosure are used in the treatment of hematopoietic tumors. In the present disclosure, "hematopoietic tumors" refer to tumors broadly classified as leukemia, malignant lymphoma, and multiple myeloma, and in many cases, it is said that there is an abnormality in the K-RAS network. Unlike solid cancers, it is said that treatment of these cancers by surgical techniques alone is difficult in many cases. In the present disclosure, "treating hematopoietic tumors" refers to controlling the K-RAS network, thereby leading to the death or growth inhibition of tumor cells present collectively in the hematopoietic organs or suspended in blood or body fluids.

[0034] Leukemia is a general term for diseases in which white blood cells, or immature cells, multiply involuntarily. Leukemia is broadly classified into acute leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia, adult T-cell leukemia, and adult T-cell leukemia. Acute leukemia is further classified into acute myeloid leukemia and acute lymphocytic leukemia. In addition, the diagnostic criteria known as the FAB classification (French-American-British Classification) take morphological findings into account and reclassify acute myeloid leukemia into acute promyelocytic leukemia, acute myelomonocytic leukemia, acute monocytic leukemia, acute erythroleukemia, and acute megakaryoblastic leukemia.

[0035] Malignant lymphoma is a disease in which lymphocytes in the blood become cancerous, and occurs mainly in lymphatic tissues such as lymph nodes, spleen, and tonsils, but can also occur in organs other than lymphatic tissues, such as the stomach, intestinal tract, thyroid gland, lungs, liver, skin, bone marrow, and brain. Malignant lymphomas are classified into more than 50 types histopathologically, and are broadly divided into Hodgkin's lymphoma and non-Hodgkin's lymphoma. Non-Hodgkin's lymphoma is classified into two types, B-cell and T / NK cell, and is further divided into more specific histopathological types.

[0036] Multiple myeloma occurs when plasma cells, a type of blood cell, become cancerous. Plasma cells are lymphocytes, a type of white blood cell, that differentiate from B cells. When plasma cells become cancerous and become abnormal cells (myeloma cells), they lose their ability to attack foreign substances. There are several types of plasma cell tumors, including multiple myeloma, plasmacytoma, and macroglobulinemia.

[0037] (Nucleic acid to be delivered: miR-143 and its analogs) The nucleic acid to be delivered in the present disclosure is a microRNA that controls the network of the oncogene KRAS. 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) having a hairpin structure of approximately 60 to 110 bases. It then migrates from the nucleus into the cytoplasm, where it becomes a double-stranded miRNA of approximately 20 to 25 bases through splicing. Double-stranded miRNA is incorporated into a protein called RISC, where it becomes single-stranded miRNA (guide strand, antisense strand), while the more unstable single-stranded miRNA (passenger strand, sense strand) is degraded. Single-stranded miRNA inhibits the translation of target genes by binding to the mRNA of the target gene with which it has a partially complementary base sequence. In this specification, "miR-143" may be referred to as "miR143."

[0038] More than 1,000 types of miRNAs are known in humans and mice, each of which 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 deeply involved in cancer cell proliferation, and research and development of miRNAs as nucleic acid drugs is underway. RAS forms a vast network by controlling more than 10 downstream signals. miR-143 is an inhibitory miRNA that multifacetedly regulates the K-RAS network and can suppress cancer growth. Many miR-143s are known to suppress cancer, and the following miR-143s (SEQ-1 to SEQ-23) have been proposed, primarily by the inventors (Tables 1 and 2 below: Patent No. 6730717). The miR-143 analogs of the present disclosure refer to microRNAs that have a similar sequence and / or structure to these miR-143s and have the effect of regulating the K-RAS network in multiple ways.

[0039]

[0040] (In the sequences, A, U, G, and C represent RNA containing adenine, uracil, guanine, or cytosine, respectively; dT and dG represent DNA containing thymine or guanine, respectively; RNAf represents 2'-FRNA; RNAm represents 2'-OMeRNA; ^ represents -P(S)OH-; * represents -P(O)OH-; and Ab (Abasic) represents the group shown below: The VP- at the 5' end of AS-42 and the Um at the end of the oligonucleotide constitute a group shown below, and the formula is: =CH-P(=O)(OH) ２ corresponds to the 5'-end modification (VP-).

[0041] The sequences in the above table and their SEQ ID NOs in the Sequence Listing are as follows: S-1 (SEQ ID NO: 1), S-17 (SEQ ID NO: 2), S-18 (SEQ ID NO: 3), S-19 (SEQ ID NO: 4), AS-4 (SEQ ID NO: 5), AS-7 (SEQ ID NO: 6), AS-30 (SEQ ID NO: 7), AS-31 (SEQ ID NO: 8), AS-3 (SEQ ID NO: 9), AS-10 (SEQ ID NO: 10), AS-12 (SEQ ID NO: 11), AS-13 (SEQ ID NO: 12), AS-42 (SEQ ID NO: 13), AS-47 (SEQ ID NO: 14), AS-50 (SEQ ID NO: 15), AS-51 (SEQ ID NO: 16), AS-52 (SEQ ID NO: 17), AS-55 (SEQ ID NO: 18), AS-56 (SEQ ID NO: 19), and AS-57 (SEQ ID NO: 20).

[0042] Of the above, SEQ-8 ​​(sense strand S-18, antisense strand AS-12) in the table above is preferred for hematopoietic tumors, which are the target of the present disclosure.

[0043] (Nucleic Acid Analog) The nucleic acid analog of the present disclosure will be described below. The nucleic acid analog of the present disclosure is composed of a cationic artificial nucleic acid and a hydrophilic polymer bound to the cationic artificial nucleic acid. The nucleic acid analog is preferably used as a carrier for delivering a nucleic acid to be delivered (hereinafter 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 miR-143 or a derivative of the miR-143. The nucleic acid analog of the present disclosure can be represented by the following formula (1): (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.)

[0044] (Cationic Artificial Nucleic Acid) Among the components 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 hexose selected from ribose and deoxyribose, a linking structure connecting the two structural units, and a backbone structure consisting of a cationic group. The base sequence of the cationic artificial nucleic acid can be appropriately designed 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.

[0045] The cationic group has, in a cationic state, 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 ６ , R ７ 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.

[0046] 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.

[0047] 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.

[0048] 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.

[0049] 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 ４ represents hydrogen or an alkyl group having 1 to 10 carbon atoms. * represents a bond to the adjacent structural unit.)

[0050] Here, "in a cationic state" refers to the case where the cationic group is assumed to be positively charged, and 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 a primary amine (primary ammonium when cationized), a secondary amine (secondary ammonium when cationized), and a tertiary amine (tertiary ammonium when cationized).

[0051] Examples of cationic artificial nucleic acids include those having a nucleotide backbone whose constituent unit is a nucleotide in which a base is bound to ribose or deoxyribose. Also included are those having a morpholino backbone whose constituent unit is a structure in which a base is bound to morpholine. (wherein, Base represents a base, and R ５represents 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, and when it is not bound to the hydrophilic polymer, it is hydrogen.)

[0052] Examples of structures having such a nucleotide backbone include the following:

[0053] In the above case, X ＋ is preferably 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.

[0054] 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.

[0055] On the other hand, examples of compounds having a morpholino skeleton include structures represented by the following formula (M1). (Here, Base represents a base. * represents a bond to phosphorus of the adjacent morpholino skeleton, and at least one of the 5'-end and 3'-end is bonded to the hydrophilic polymer, and when not bonded to the hydrophilic polymer, it represents hydrogen.)

[0056] Examples of such structures having a morpholino skeleton include the following.

[0057] 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.

[0058] 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.

[0059] (Hydrophilic Polymer) In formula (1), H represents a hydrophilic polymer. Various polymers can be used as the hydrophilic polymer depending on the application of the nucleic acid analog, etc. In particular, when the nucleic acid analog is used as a carrier for delivering the nucleic acid to a target site, the hydrophilic polymer is preferably biocompatible. Furthermore, since the nucleic acid analog is cationic, the hydrophilic polymer is preferably neutral and does not easily interact electrically (attract or repel) with the nucleic acid analog.

[0060] 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.

[0061] Examples of such hydrophilic polymers include the following:

[0062] 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. Typical examples of the monomer include oligomers of ethylene glycol, propylene glycol, and butylene glycol units. 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. These hydrophilic polymer oligomers may be repeated two or more times via a phosphate diester group or the like. The number of repeats via a phosphate diester bond may be three or more, preferably four or more, depending on the molecular weight of the artificial nucleic acid and the balance with the complementarity with the nucleic acid to be delivered and the hydrophobicity when associated through electrostatic interaction. The maximum number of repeats is 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. In addition, the hydrophilic polymer is likely to be eliminated as a foreign substance in the body, resulting in poor degradation stability and poor blood retention. Conversely, if the degree of polymerization of the hydrophilic polymer is too high, the size of the structure will be too large, and the efficiency of delivery of the target nucleic acid to the target site will likely be low.

[0063] 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.)

[0064] Furthermore, A1 can also be used as a polymer containing nucleic acid bases or phosphate derivatives (-PO(OH)-, -PS(OH)-, PO(SH)-) as monomer units. The number of repeating ethylene glycol units q in the monomer unit A1 is 1 to 20, preferably 3 to 10. The monomer structures may be the same or different, and the degree of polymerization of the monomer unit is 1 to 10, preferably 2 to 10, and more preferably 2 to 8. Examples of such phosphate ester derivative monomer units include ethylene glycol phosphate ester derivative units, diethylene glycol phosphate ester derivative units, triethylene glycol phosphate ester derivative units, and hexaethylene glycol phosphate ester derivative units. These derivative units can also be used as monomers for further polymers. The degree of polymerization of the phosphate ester derivative units 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 artificial nucleic acid has 10 to 30 bases. The monomer unit is most preferably a triethylene glycol phosphate derivative or a hexaethylene glycol phosphate derivative. The type and degree of polymerization of the monomer of the hydrophilic polymer described above, particularly the degree of polymerization of the ethylene glycol phosphate derivative monomer, can be selected from the viewpoints of ease of formation of the structure described below, difficulty in being recognized as a foreign substance in the body, low degradation stability, low blood retention, etc.

[0065] (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 ligands include immunoglobulins, carbohydrates, peptides, proteins, and aptamers. The main ligands predicted from research on lipid nanoparticles and their application fields are as follows. In the present disclosure, despite the following predictions, it has been found that when glucose is used as a ligand, it is also effective against hematopoietic tumors in suspension cells.

[0066] ・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: Used to recognize different binding sites of thrombin ・TASSET: Used to recognize different binding sites of target proteins ・xPSM-A9: Used against prostate-specific membrane antigen expressed in prostate cancer cells ・IL-4Rα: Inhibition of tumor growth by utilizing the tumor microenvironment

[0067] (Spacer 1)

[0068] The cationic artificial nucleic acid and the hydrophilic polymer may be bonded via various bonding modes or via a linker. Examples of such bonds include an ester bond (-C(=O)-O-), an ether bond (-O-), a disulfide bond (-S-S-), a phosphoramidite (-P(OH)-NH-), and a phosphate ester (-OPO-O-). These linkers 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, these linkers are preferably bonded to the hydroxyl group at the 5' end or 3' end or both of these ends of the cationic artificial nucleic acid, and bonded via the cationic artificial nucleic acid and the hydrophilic polymer. When the cationic artificial nucleic acid and the hydrophilic polymer are bonded via a linker, the linker has a different structure from the hydrophilic polymer, and therefore it is expected that the charge can be adjusted. Furthermore, when a phosphoramidite is used, this is preferred because it behaves as a negative charge, similar to a polymer made of polyethylene glycol. Furthermore, it is advantageous in that the induction of PEG antibodies is suppressed. Furthermore, when using phosphoramidites, the ability to synthesize everything from the nucleic acid sequence to the hydrophilic polymer moiety using automated nucleic acid synthesis is an industrial advantage. 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.)

[0069] (Spacer 2) When the ligand is an aldose and the corresponding phosphoramidite reagent is unstable, spacer 22 can be used. Spacer 22 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, and q is 0 or 1. When q is 0, the 1,2,3-triazole ring is bonded to the ligand. Spacer 1 and spacer 2 have any structure in the nucleic acid analog and can be provided as needed.

[0070] (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 has a cationic group and a hydrophilic polymer as its primary structure, and the nucleic acid to be delivered, such as a natural nucleic acid, 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) composed of the nucleic acid analog as a carrier and the nucleic acid to be delivered.

[0071] Here, when there is complementarity between the target nucleic acid and the cationic group, a double strand is formed by base-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 become 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%.

[0072] 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 the "structure"). These 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 (see "Nanostructure Formation" in the figure). These micelles and vesicles form spherical structures with a hollow core, which can encapsulate drugs such as low-molecular-weight compounds. This enables the delivery of not only target nucleic acids but also low-molecular-weight drugs. The diameter of the hollow core is approximately 50-500 nm.

[0073] 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.

[0074] Such structures have high degradation stability because the target nucleic acid is located inside the spherical structure. Furthermore, because the above structures have hydrophilic polymer segments in the outermost shell, they have excellent blood retention. The structures are then encapsulated in endosomes at the target site, such as cells, and taken up into the cytoplasm. They then escape the endosomes and release the target nucleic acid or small molecule drug into the cytoplasm or nucleus (see "Biochemical Evaluation" in the figure).

[0075] Examples of uses of such structures include drug delivery carriers for various diseases, etc. For example, in the Examples described below, it is shown that the nucleic acid analogs of the present disclosure are effective as carriers for delivering the anti-onco-microRNA microRNA-143 as a target nucleic acid, targeting the cancer-promoting gene K-Ras and its network.

[0076] The surface of the nucleic acid delivery structure may be 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.

[0077] (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.

[0078] (a) Two-Step Synthesis Method Cationic artificial nucleic acids can be synthesized in two steps (two-step synthesis method). There are three main types of two-step synthesis methods. These methods are explained below. (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 nucleic acid to synthesize an oligo-PS hydrophilic polymer. A Br compound is then reacted to introduce a cationic group into the nucleic acid (as in the TEG-PS oligo and HEG-PS oligo systems in the Examples described below). 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, as described in the Examples described below.

[0079] (a-1-2) 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.

[0080] (a-1-2) 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 between the synthetic nucleic acid and 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.

[0081] (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 hydrophilic polymer phosphoramidites (e.g., ethylene glycol phosphoramidites) are linked to the boranophosphates to synthesize oligo-B-form hydrophilic polymers (Figure 2). This is then reacted with an amino compound via iodine oxidation to introduce a cationic group (Strategy 2 in Figure 3). This method also allows the introduction of two cationic groups into one phosphate group (double cation introduction) by using a cationic phosphoramidite, as described below.

[0082] (a-3) Two-Step Synthesis Method III In this method, a cationic artificial nucleic acid is synthesized by automated nucleic acid synthesis, and then a hydrophilic moiety is introduced by a click reaction (the PEG-PMO system in the Examples described below). That is, this method involves synthesizing a cationic artificial nucleic acid having a cationic group in the backbone, and then linking it to a hydrophilic azide compound such as azide polyethylene glycol by a click reaction.

[0083] (b) One-Step Synthesis Method The method described above is a two-step synthesis method in which a synthetic nucleic acid containing a thiophosphate ester is prepared and then reacted with a bromo compound. However, it is also possible to synthesize cationic artificial nucleic acids in one step. Figure 2 shows the scheme of this method. In summary, this method is a one-step synthesis of a cationic hydrophilic polymer by automated nucleic acid synthesis using cationic phosphoramidites and ethylene glycol phosphoramidites (Strategy 1 in Figures 2 and 3). This method is also basically similar in scheme to the phosphoramidite method described in the two-step synthesis method, but there are some differences.

[0084] First, a diisopropylamidophosphorous compound and a nucleotide monomer are reacted with a nucleotide supported on a solid phase ("1. Coupling" in the diagram). Some hydroxyl groups are protected with protecting groups ("2. Capping"), and then oxidized or borated with an oxidizing agent or boronating agent ("3. Oxidation or Boronation"). An amino group compound is reacted with iodine oxidation to introduce a cationic group.

[0085] An example of a synthesis scheme for nucleic acid analogs is shown in Figure 3. This example shows a scheme for synthesizing a nucleic acid analog having a nucleotide backbone as a cationic artificial nucleic acid and polyethylene glycol as a hydrophilic polymer. In the "phosphoramidite synthesis" section of the figure, phosphoramidite nucleotides are synthesized in the order of compounds 1 to 3 using a known method. Meanwhile, phosphoramidite polyethylene glycol is synthesized in the order of compounds 4 to 6.

[0086] Next, a nucleic acid analog is synthesized according to the scheme shown in "Strategy 1: Synthesis of a cation-incorporated oligonucleotide" in the figure. Specifically, the phosphoramidite nucleotide and phosphoramidite polyethylene glycol synthesized according to the above scheme, and a phosphoramidite disulfide having a disulfide bond and a phosphoramidite in the molecule are used as raw materials. Then, reactions such as protection with a protecting group and deprotection are carried out to bind a hydrophilic polymer to the 5' position of the cationic artificial nucleic acid. Alternatively, a cationic artificial nucleic acid may be synthesized in one step by solid-phase synthesis, as shown in the scheme shown in "Strategy 2: Synthesis of a cation-incorporated oligonucleotide" in the figure. Patent Document 1 and other documents can be used as references for methods of producing cationic artificial nucleic acids.

[0087] (Method for manufacturing nucleic acid delivery construct) Next, a method for manufacturing a nucleic acid delivery construct will be described. The nucleic acid delivery construct is formed by associating a nucleic acid to be delivered with a nucleic acid analog to form a complex (association step). If there is complementarity between the cationic group of the nucleic acid analog and the phosphate group of the nucleic acid to be delivered, they are bound 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.

[0088] 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.

[0089] 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.

[0090] (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, facilitating the release of the nucleic acid to be delivered. Furthermore, as described above, it is also possible to encapsulate a small molecule drug within 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.

[0091] 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.

[0092] (Cells) The DLD-1 (human colon cancer cell line), ASF4-1 (human fibroblast cell line), PRMI8226 (human multiple myeloma cell line), NB4 (human acute promyelocytic leukemia pre-B cell line), HL-60 (human promyelocytic leukemia cell line), and Jrukat cells (human leukemia T cell-derived cell line) used in the evaluation were obtained from the JCRB Cell Bank of the National Institutes of Biomedical Innovation, Health, and Nutrition. RPMI-1640 medium (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) was used to culture DLD-1, PRMI8226, NB4, HL-60, and Jrukat cells. Eagle's minimum essential medium was used to culture ASF4-1 cells. All media were supplemented with heat-inactivated fetal bovine serum (Nichirei Biosciences) to a concentration of 10 (v / v), and the cells were incubated in an atmosphere of 95% air and 5% carbon dioxide.

[0093] (Cell Evaluation) All cells were cultured at 0.25 x 10 cells per well one day before sample processing. ５ Cells were seeded at 12-well plate at 1000 cells / well. Samples were administered so that the concentration of miR-143#12 was 0.5-10 nM. After 48 or 72 hours of culture, cell evaluation was performed. Cell proliferation was evaluated by counting the number of viable cells using the trypan blue dye exclusion method. After culture, cells were collected and analyzed for mRNA expression and protein expression levels.

[0094] (Particle size distribution) The particle size distribution was determined by dynamic light scattering using a Zetasizer (Beckman Coulter Inc.) For the measurement, a 10 μM PBS solution (pH 7.4) of the nucleic acid to be delivered (miR-143 of the present disclosure) was prepared and used as a sample.

[0095] RNA was isolated from cultured cells using NucLeoSpin® miRNA (TaKaRa). RNA concentration and purity were assessed using UV spectroscopy. RNU6B was used as an internal reference standard. In addition, qRT-PCR of mRNA to evaluate the expression levels of NRAS and KRAS was performed using qPCT Thunderbird® Next XYBR® qPCR Mix (Toyobo). Primers for NRAS, KRAS, and GAPDH were as follows: GADPH was used as an internal control; relative expression levels were calculated using the ΔΔCt method. NRAS sense strand: 5'-CCT CCT CAC TTG GCT GTC TG-3' (SEQ ID NO: 27) NRAS antisense strand: 5'-TCA CGT TTG CGG TTT GGT TC-3' (SEQ ID NO: 28) KRAS sense strand: 5'-TGG TGG TGT GCC AAG ACA TT-3' (SEQ ID NO: 29) KRAS antisense strand: 5'-CAC CTC ACC ATG CCA TCT CA-3' (SEQ ID NO: 30) GAPDH sense strand: 5'-TCT AGA CGG CAG GTC AGG TCC ACC-3' (SEQ ID NO: 31) GAPDH antisense strand: 5'-CCACCC ATG GCA AAT TCC ATG GCA-3 '(SEQ ID NO: 32)

[0096] (Western Blot) Cell lysates were prepared in a lysis buffer consisting of 10 mM Tris-HCl (pH 7.4), 1% NP-40, 0.1% deoxycholic acid, 0.1% SDS, 2% protease inhibitor cocktail, 2% phosphatase inhibitor cocktail II, and 2% phosphate inhibitor cocktail III (Sigma-Aldrich) and left on ice for 20 minutes. After centrifugation at 13,000 rpm (16,200 x g) for 20 minutes at 4°C, the supernatant was used as a protein sample. Protein content was measured using a DC protein assay kit (Bio-Rad). Two micrograms of dissolved protein was separated by 10.0% or 12.5% ​​polyacrylamide gel electroblotting onto Immobilon-P membranes of polyvinylidene difluoride (PVDF) (Merck Millipore). Nonspecific binding was blocked for 1 hour with 20% PVDF in distilled water using Can Get Signal (registered trademark: Toyobo). The Immobilon-P membranes were then incubated overnight at 4°C with primary antibodies using Get Signal Solution 1 (Toyobo). The next day, the membranes were washed three times with Tris-buffered saline (TBS) containing 0.1% Tween 20 (TBS-T). Immunoblots were visualized using Immobilon Forte Western horseradish peroxidase (HRP) substrate (Millipore). Immunoblot images were acquired using an ImageQuant LAS4000 biomolecular imager (GE Healthcare Life Sciences, Pittsburgh, PA, USA). Densitometry analysis was performed using the image analysis software ImageQuant Total Lab-7 (GE Healthcare Life Sciences).

[0097] The primary antibodies used were as follows: anti-AKT, ERK1 / 2, PARP, Cyclin D1, ERK5, SOS1, GLUT1, and GLUT4 (Cell Signaling Technology); anti-KRAS (LifeSpan BioScience, Seattle, WA, USA); anti-Total (T)-RAS including KRAS, HRAS, and NRAS (abcam); anti-β-actin (Sigma-Aldrich); and anti-α-Tubulin (Medical & Biol., Tokyo, Japan).

[0098] G: guide strand of miR-143 P: artificial nucleic acid of the present disclosure AS: antisense strand of siRNA (control) S: sense strand of siRNA (control) N: RNA n: DNA Mf: 2'-F-RNA *: anionic backbone of phosphodiester ^: -P(S)OH- +: cationic backbone of phosphate X: hexaethylene glycol Y: linker (spacer 2 of the present application) Z: acetylene derivative Glu: glucose Underline: mismatched base

[0099] The sequences in the above table and their SEQ ID NOs are as follows: P#1 (SEQ ID NO: 21), P#2 (SEQ ID NO: 22), P#3 (SEQ ID NO: 23), AS#1 (SEQ ID NO: 24), S#1 (SEQ ID NO: 25), S#2 (SEQ ID NO: 26).

[0100] (Production Example 1: Synthesis of P#2) The base sequence of the artificial nucleic acid (ID4, P#3) for delivering miR-143 and its derivatives was designed based on the base sequence of the nucleic acid to be delivered. The miR-143 and its derivatives used in this example were nucleic acids G#12 (same as AS-4).

[0101] The artificial nucleic acid P#2 was synthesized using an automated nucleic acid synthesizer. The nucleotide sequence described in P#2 was linked using a thiophosphate diester, and then six units of hexaethylene glycol were introduced via phosphate groups using the phosphoramidite method. A triple bond structure represented by the following chemical formula was then constructed at the end of the linkage, yielding P#2, in which the artificial nucleic acid was linked via a thiophosphate diester. Separately, 1-azidoglucose was synthesized using the method described in WO 2016 / 152980.

[0102]

[0103] The artificial nucleic acid P#2 synthesized above, in which the artificial nucleic acid was linked via a thiophosphate diester, was dissolved in PBS (pH 7.4). Next, nucleic acid P#2 (10 nmol, 1 equivalent) was mixed with 50 mM sodium ascorbate solution (10 equivalents), 50 mM copper(II) sulfate (10 equivalents), and 50 mM 1-azidoglucose in PBS (pH 7.4), and the mixture was allowed to react for 15 minutes. The reaction sample was purified by high-performance liquid chromatography to obtain nucleic acid P#3, in which the artificial nucleic acid was linked via a thiophosphate diester.

[0104] Nucleic acid P#3 (4 nmol), in which an artificial nucleic acid was linked via a thiophosphate diester, was reacted with 4 M 2-(diethylamino)ethyl bromide hydrobromide (Waco, Osaka, Japan, 2 μL, 8 mmol) in PBS at 45°C for 24 hours. After the reaction, the sample was dialyzed against distilled water for 3 to 5 days and purified by lyophilization to obtain P#3.

[0105] (Production Example 2) P#3 was obtained in the same manner as in Production Example 1, except that the artificial nucleic acid in Production Example 1 was one in which the terminal triple bond was linked with a thiophosphate diester having the structure shown in the chemical formula below.

[0106] (Production Example 3: Glu-RION) miR-143 analog G#12 (sometimes referred to as "miR-143G#12") and the nucleic acid analog P#3 obtained in Production Example 1 were annealed at a 1:5 ratio at 98°C for 15 minutes, 25°C for 50 minutes, and 45°C for 50 minutes to obtain Glu-RION-miR143#12.

[0107] (Measurement of particle size distribution) miR-143 analog G#12 and nucleic acid analog P#3 obtained in Production Example 1 were mixed at a molar ratio of 1:5 and annealed. The results are shown in Figure 1c. It can be seen that miR-143G#12 and nucleic acid analog P#3 annealed and self-assembled to form RION-miR143#12 nanoparticles.

[0108] (Reference Example 1) The effect of Glu-RION-miR143#12 on human colon cancer DLD-1 cells was investigated and compared with RION-Control (Control). The results are shown in Figure 4 and Table 4. Glu-RION-miR143#12 showed improved cellular uptake compared to the miR143 without glucose addition (Figures 4a and 4b). RION-miR143#12 enters cells by passive transport, but when Glu-RION-miR#143 is used, it is thought that cellular uptake is improved via the glucose transporter expressed in the cells. The IC of Glu-RION-miR143#12 ５０ The concentration of miR-143 was 3.3 nM, and it suppressed the expression of the target genes KRAS, Sos-1, Akt, and ERK1 / 2. The cell death of DLD-1 cells was determined to be apoptosis, as cleaved PARP-1 was observed. Therefore, it is thought that delivery via glucose transporters is useful for nucleic acid delivery to cancer cells.

[0109] Example 1: Characterization of human KRAS mutant hematopoietic tumor cells and the growth-suppressing effect of miR-143#12 delivery using Glu-RION-miR143#12 were investigated. The hematopoietic tumor cells used were RPMI8226 (KRASG12A), NB4 (KRASA18D), HL-60 (NRASQ61L), and Jurkat (wild type). These are suspension cells into which nucleic acid introduction is difficult using existing transfection methods. The results are shown in Figure 5 and Table 4. The IC50 for RPMI8226 was 6.9 nM, the IC50 for NB4 was 6.4 nM, and the IC50 for HL60 was 7.3 nM.

[0110] The effect of Glu-RION-miR143#12 was confirmed in PRMI8226 cells. However, neither Glu-RION nor miR-143#12, nor miR-143#12 plus the gene transfer reagent Lipofectamine, demonstrated any inhibitory effect on cell proliferation (Figure 5b). While lipofection reagents are widely used as general gene transfer reagents, their transfection efficiency is known to be extremely low in suspension cells such as PRMI8226 cells. Figure 5b shows that miR143#12 transfection did not induce cell death, even at a nucleic acid concentration of 200 nM. In contrast, Figure 5c shows that Glu-RION-miR143#12 induced cell death in a concentration-dependent manner at 1-10 nM. Therefore, Glu-RION-miR143#12, which was introduced into PRMI8226 cells, is a useful nucleic acid transfer technique because it allows nucleic acid transfer at low concentrations. Cell proliferation inhibitory effects were also confirmed in HL-60 and NB4 cells, and apoptosis was also observed in NB4 cells (Figure 5d). Expression of glucose transporters (GLUT1 and GLUT4) was confirmed in hematopoietic tumor cells (Figure 5g).

[0111] Example 2: The effect of miR-143#12 delivery to hematopoietic tumors was investigated using Glu-RION-miR143#12. Expression levels of RAS and RAS-related proteins (Sos-1, ERK1 / 2, ERK5, and Akt) were determined in RPMI8226 cells by Western blot analysis. The results are shown in Figure 6a. These results demonstrate that the use of Glu-RION-miR143#12 suppressed the expression of miR-143 target genes TRAS, Akt, ERK5, and ERK1 / 2. This is thought to be due to the delivery of miR-143#12 into the cells, resulting in the suppression of protein expression by RNA interference. RPMI8226 cells contain a glucose transporter, which is one of the nutrient transmitters, and it is thought that the ligand present on the surface of RION recognizes this transmitter, allowing nucleic acids to be efficiently taken up into the suspension cells.

[0112] Figure 6b shows the evaluation of mRNA expression levels. miRNAs inhibit protein translation by binding to the untranslated regions of mRNA; in other words, it is known that the action of miRNA does not reduce mRNA expression levels. Analysis of mRNA NRAS or KRAS expression levels did not reveal any significant reduction in expression levels. This suggests that the inhibitory effects of Glu-RION-miR143#12 on protein expression and cell proliferation in RPMI8226 cells are due to the action of miRNA143#12. Figures 6c and 6d show the evaluation of protein expression levels of Glu-RION143#12 in NB4 and HL60. As a result, decreased expression of miR143#12 target proteins, Sos-1, T-RAS, Akt, ERK5, and ERK1 / 2, was observed in NB4 cells, while decreased expression of TRAS, Akt, and ERK1 / 2 was observed in HL-60 cells. This suggests that cell growth inhibition (Figure 5e) is induced by the efficacy of miR143#12. Meanwhile, Figure 6e evaluates protein expression levels in Jurkat cells. Decreased expression of miR143#12 target proteins, Sos-1, T-RAS, Akt, ERK5, and ERK1 / 2, was observed in Jurkat cells. However, cell growth inhibition was not induced in Jurkat cells (Figure 6e), suggesting that RAS is not a driver gene for this cancer.

[0113] Example 3: Using Glu-RION-miR143#12, we investigated the difference between the use and absence of concanavalin A in the stimulation of peripheral lymphocytes in the steady state of miR-143#12. The results are shown in Figure 7. The survival rate of peripheral lymphocytes increased when concanavalin A was not used, but when concanavalin A was used, a tendency for peripheral lymphocyte proliferation to be suppressed was observed. This suppression of proliferation is thought to be due to arrest of the cell cycle.

[0114]

Claims

1. Use of a nucleic acid delivery structure in the treatment of hematopoietic tumors, characterized in that the structure has an association structure in which a nucleic acid analog represented by the following formula (1) and a target nucleic acid for delivery selected from miR-143 or an analog thereof are associated by electrostatic interaction, the nucleic acid analog being a microRNA that controls the network of the cancer gene KRAS: 【Chemistry 1】 (wherein N represents a cationic artificial nucleic acid, H represents a hydrophilic polymer selected from polyethylene glycol, polyvinyl alcohol, polyglutamic acid, polyvinylpyrrolidone, polyacrylamide, polyethyleneimine, polyalkylacrylate, polyoxazoline, polyacrylamide, poly(carboxybetaine methacrylate), poly(sulfobetaine methacrylate), poly(2-methacryloyloxyethylphosphocholine), hyaluronic acid, chitosan, dextran, and derivatives thereof, S1 represents spacer 1, S2 represents spacer 2, L represents a ligand selected from glucose, mannose, galactose, sucrose, maltose, and lactose, s represents 0 or 1, and t represents 0 or 1; N has 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, The cationic artificial nucleic acid can be associated with the phosphate group of the nucleic acid to be delivered by electrostatic interaction with the cationic group.

2. 2. Use of the nucleic acid delivery structure according to claim 1, wherein the cationic group has a pKa value in the range of 6 to 9, in the treatment of hematopoietic tumors.

3. 2. Use of the nucleic acid delivery structure according to claim 1, 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): 【Chemistry 2】 (Here, R １ ~R ３ represents hydrogen or an alkyl group having 1 to 10 carbon atoms, and R1 to R3 may be the same or different. Ring is a cyclic compound composed of 4 to 8 carbon atoms, and may be a heterocyclic ring in which one or more of the carbon atoms are substituted with a heteroatom selected from nitrogen, oxygen, and sulfur.

4. Use of the nucleic acid delivery structure according to claim 1, characterized in that the linking structure of the cationic artificial nucleic acid has at least a structure selected from the following formulas (L1) to (L4) in a cationic state. 【Chemistry 3】 (Here, X ＋ is a functional group containing the cationic group, Z is O or S, and W is -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.)

5. 2. Use of the nucleic acid delivery structure according to claim 1, characterized in that the cationic artificial nucleic acid has a nucleotide backbone represented by the following formula (N1): 【Chemistry 4】 (Here, X ＋ is a functional group containing the cationic group, Base is a base, and R5 is H or OH. * means a bond to the phosphate of the adjacent nucleotide backbone, and at least one of the 5'-end or 3'-end is bonded to the hydrophilic polymer, and when it is not bonded to the hydrophilic polymer, it is hydrogen.)

6. The X ＋ The use of the nucleic acid delivery structure according to claim 5, characterized in that, in a cationic state, it can become an ammonium cation represented by the following formula (F1): 【Chemistry 5】 (wherein R1 to R3 represent 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.)

7. The hematopoietic tumor expresses a glucose transporter on cells, 2. Use of the nucleic acid delivery structure according to claim 1, in the treatment of hematopoietic tumors, wherein the ligand is glucose.

8. 2. Use of the nucleic acid delivery structure according to claim 1, characterized in that the hydrophilic polymer has a polyethylene glycol backbone represented by the following formula (A1): 【Chemistry 6】 (wherein p is an integer from 1 to 20.)

9. 9. Use of the nucleic acid delivery structure according to claim 8, characterized in that the compound of formula (A1) is linked via a phosphate diester group, in the treatment of hematopoietic tumors.

10. 2. Use of the nucleic acid delivery structure according to claim 1, characterized in that the spacer 1 has a bond represented by the following formula (S11): 【Chemistry 7】 (In the formula, R6 and R7 represent methylene groups having 1 to 12 carbon atoms, and R6 and R7 may be the same or different.)

11. Use of the nucleic acid delivery structure according to claim 1 for the treatment of hematopoietic tumors, characterized in that the spacer 2 is a phosphodiester bond or a phosphodiester bond containing a triazole shown in the following formula (S21): 【Chemistry 8】 (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 is 0 or 1; and when q is 0, Ka is bonded to a ligand.)

12. Use of the nucleic acid delivery structure according to claim 1, characterized in that a plurality of the nucleic acid delivery structures are associated to form a nanoscale structure.

13. Use of the nucleic acid delivery structure described in claim 12 for the treatment of hematopoietic tumors, characterized in that the association structure is a vesicle or micelle in which the hydrophilic polymer and ligand are located on the inside and the outside.

14. 2. Use of the nucleic acid delivery structure according to claim 1 in the treatment of hematopoietic tumors, wherein the miR-143 is selected from the group consisting of SEQ-1 to SEQ-23 below. 【Table 1】 【Table 2】 (In the sequences, A, U, G, and C represent RNA containing adenine, uracil, guanine, or cytosine, respectively; dT and dG represent DNA containing thymine or guanine, respectively; RNAf represents 2'-FRNA; RNAm represents 2'-OMeRNA; ^ represents -P(S)OH-; * represents -P(O)OH-; and Ab (Abasic) represents the group shown below: 【Chemistry 9】 【Chemistry 10】 The VP- at the 5' end of AS-42 and the Um at the end of the oligonucleotide constitute a group represented by the following formula: =CH-P(=O)(OH) ２ corresponds to 5'-end modification (VP-).

15. 2. Use of a medicine comprising the nucleic acid delivery structure according to claim 1 in the treatment of hematopoietic tumors.

16. A method for treating hematopoietic tumors, comprising administering the nucleic acid delivery structure according to claim 1 to a patient.

17. A method for inhibiting the proliferation of cancerous cells in hematopoietic tumors, comprising administering to the cancerous cells a nucleic acid delivery structure having an association structure in which a nucleic acid analog represented by the following formula (1) and a target nucleic acid for delivery selected from miR-143 or an analog thereof are associated by electrostatic interaction with each other, the nucleic acid analog being a microRNA that controls the network of an oncogene KRAS: 【Chemistry 1】 (wherein N represents a cationic artificial nucleic acid, H represents a hydrophilic polymer selected from polyethylene glycol, polyvinyl alcohol, polyglutamic acid, polyvinylpyrrolidone, polyacrylamide, polyethyleneimine, polyalkylacrylate, polyoxazoline, polyacrylamide, poly(carboxybetaine methacrylate), poly(sulfobetaine methacrylate), poly(2-methacryloyloxyethylphosphocholine), hyaluronic acid, chitosan, dextran, and derivatives thereof, S1 represents spacer 1, S2 represents spacer 2, L represents a ligand selected from glucose, mannose, galactose, sucrose, maltose, and lactose, s represents 0 or 1, and t represents 0 or 1; N has 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, The cationic artificial nucleic acid can be associated with the phosphate group of the nucleic acid to be delivered by electrostatic interaction with the cationic group.

18. A nucleic acid delivery structure used for the treatment of hematopoietic tumors, the nucleic acid delivery structure comprising a nucleic acid analog represented by the following formula (1) and a nucleic acid to be delivered selected from miR-143 or an analog thereof, the nucleic acid analog being composed of a microRNA that controls the network of the cancer gene KRAS, and having an association structure formed by electrostatic interaction: 【Chemistry 1】 (wherein N represents a cationic artificial nucleic acid, H represents a hydrophilic polymer selected from polyethylene glycol, polyvinyl alcohol, polyglutamic acid, polyvinylpyrrolidone, polyacrylamide, polyethyleneimine, polyalkylacrylate, polyoxazoline, polyacrylamide, poly(carboxybetaine methacrylate), poly(sulfobetaine methacrylate), poly(2-methacryloyloxyethylphosphocholine), hyaluronic acid, chitosan, dextran, and derivatives thereof, S1 represents spacer 1, S2 represents spacer 2, L represents a ligand selected from glucose, mannose, galactose, sucrose, maltose, and lactose, s represents 0 or 1, and t represents 0 or 1; N has 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, The cationic artificial nucleic acid can be associated with the phosphate group of the nucleic acid to be delivered by electrostatic interaction with the cationic group.

Images