RNA nucleic acid molecule, lipid-nucleic acid complex, pharmaceutical composition, and method for inducing expression of gene of interest

Abstract

Provided are: an RNA nucleic acid molecule capable of controlling the induction of expression of a gene of interest according to the presence or absence of a targeting miRNA; and others.　An RNA nucleic acid molecule according to the present disclosure comprises a sequence encoding a gene of interest, a PolyA sequence, and a degradation-inducing sequence, wherein the sequence encoding the gene of interest, the PolyA sequence, and the degradation-inducing sequence are arranged in this order from the 5'-end toward the 3'-end, and the degradation-inducing sequence includes a target sequence for an miRNA. In the absence of an miRNA capable of binding to the target sequence, hybridization of the miRNA to the target sequence does not occur, and thus the degradation of the RNA nucleic acid molecule can be induced. In the presence of the miRNA capable of binding to the target sequence, hybridization of the miRNA to the target sequence occurs, and thus the degradation of the RNA nucleic acid molecule can be suppressed and the expression of the gene of interest can be induced.

Description

RNA nucleic acid molecule, lipid nucleic acid complex, pharmaceutical composition, and method for inducing the expression of a target gene

[0001] This disclosure relates to RNA nucleic acid molecules, lipid nucleic acid complexes, pharmaceutical compositions, and methods for inducing the expression of a target gene.

[0002] MicroRNAs (miRNAs) are known to be specifically expressed in desired cells and are attracting attention as biomarkers for various diseases, including cancer. Furthermore, the development of technologies that enable cell-specific editing of target genes using miRNA activity as an indicator is being explored (Patent Document 1).

[0003] International Publication No. 2015 / 105172

[0004] Therefore, this disclosure aims to provide an RNA nucleic acid molecule capable of controlling the expression of a target gene in response to miRNA.

[0005] To achieve the aforementioned objective, the RNA nucleic acid molecule of this disclosure comprises a sequence encoding a target gene, a PolyA sequence, and a degradation induction sequence, wherein the sequence encoding the target gene, the PolyA sequence, and the degradation induction sequence are arranged in this order from the 5' end to the 3' end, and the degradation induction sequence comprises a target sequence of miRNA.

[0006] The DNA nucleic acid molecules of this disclosure encode the RNA nucleic acid molecules of this disclosure.

[0007] The lipid nucleic acid complex of this disclosure comprises the RNA nucleic acid molecule of this disclosure and a lipid.

[0008] The pharmaceutical compositions of this disclosure comprise an RNA nucleic acid molecule of this disclosure, a DNA nucleic acid molecule of this disclosure, a vector of this disclosure, and / or a lipid nucleic acid complex of this disclosure, and a pharmaceutically acceptable carrier.

[0009] The method for inducing the expression of a target gene described herein involves introducing the RNA nucleic acid molecule described herein, the DNA nucleic acid molecule described herein, the vector described herein, and / or the lipid nucleic acid complex described herein into cells expressing a target miRNA, thereby inducing the expression of the target gene.

[0010] The screening method of this disclosure selects cells expressing a target miRNA by introducing the RNA nucleic acid molecule, DNA nucleic acid molecule, vector, and / or lipid nucleic acid complex of this disclosure into the cells.

[0011] According to this disclosure, it is possible to provide RNA nucleic acid molecules that can control the expression of a target gene in response to miRNA.

[0012] Figure 1 is a schematic diagram showing the estimated mechanism by which the RNA nucleic acid molecules of this disclosure induce the expression of a target gene in cells containing the target miRNA. Figure 2 is a graph showing the results of the luciferase assay in Example 1. Figure 3 is a graph showing the results of the semi-quantitative PCR method in Example 1. Figure 4 is a graph showing the results of the luciferase assay in Example 1. Figure 5 is a schematic diagram showing the predicted secondary structures of various RNA nucleic acid molecules in Example 1. Figure 6 is a schematic diagram showing the predicted secondary structures of various RNA nucleic acid molecules in Example 1. Figure 7 is a graph showing the results of the luciferase assay in Example 1. Figure 8 is a graph showing the results of the dual luciferase assay and qPCR assay in Example 1. Figure 9 is a schematic diagram showing the predicted secondary structures of various RNA nucleic acid molecules in Example 1. Figure 10 is a graph showing the results of the luciferase assay and qPCR assay in Example 1. Figure 11 is a schematic diagram showing the predicted secondary structures of various RNA nucleic acid molecules in Example 1. Figure 12 is a graph showing the results of the dual luciferase assay and the qPCR assay in Example 1. Figure 13 is a graph showing the results of the luciferase assay in Example 1. Figure 14 is a graph showing the results of the luciferase assay in Example 1. Figure 15 is a graph showing the results of the luciferase assay in Example 1. Figure 16 is a schematic diagram of the method for testing miRNA activity in Example 1. Figure 17 is a graph showing the flow cytometry analysis results in Example 1. Figure 18 is a graph showing the results of the dual luciferase assay in Example 1.

[0013] The following provides a detailed explanation of this disclosure, including examples. Unless otherwise specified, each disclosure may refer to the explanations of other disclosures.

[0014] <Definitions> In this specification, “nucleic acid,” “polynucleotide,” or “oligonucleotide” means a polymer of deoxyribonucleotide (DNA), ribonucleotide (RNA), and / or modified nucleotides. The nucleic acid may be a single-stranded nucleic acid molecule or a double-stranded nucleic acid molecule. The polynucleotide may consist of natural nucleotides, modified or artificial nucleotides, or both.

[0015] In this specification, “RNA” means natural or unnatural ribonucleic acid. The RNA is a polymer composed of ribonucleotides. The RNA may be modified (modified nucleic acid) or unmodified. Examples of such modifications include methylation, pseudouridine, and thiolation. In this disclosure, it is preferable that the uridine in the RNA is pseudouridine. The pseudouridine may be partial or whole uridine. The RNA may be linear or circular, for example.

[0016] In this specification, “DNA” means natural or unnatural deoxyribonucleic acid. The DNA is a polymer composed of deoxyribonucleotides. The DNA may be modified (modified nucleic acid) or unmodified. Examples of such modifications include methylation, pseudouridineation, and thiolation.

[0017] As used herein, "microRNA (miRNA)" refers to a non-coding RNA having a length of approximately 18 to 25 nucleotides. The miRNA is known to regulate post-transcriptional expression of genes. miRNA is transcribed by RNA polymerase II as part of a capped, polyadenylated pri-miRNA. The pri-miRNA is cleaved by the Drosha ribonuclease III enzyme to produce a pre-miRNA having a stem-loop structure. After the production, it is further cleaved by Dicer ribonuclease to generate a mature miRNA. The mature miRNA interacts with a plurality of predetermined proteins to form an RNA-induced silencing complex (RISC). The RISC recognizes a target mRNA through incomplete base pairing with the miRNA, and causes translational inhibition and destabilization of the target mRNA. Hereinafter, as used herein, pri-miRNA and pre-miRNA are also collectively referred to as miRNA precursors. The miRNA can also be referred to as "microRNA", "miR" and the like.

[0018] As used herein, "mir-X" refers to the miRNA precursor numbered X, and "miR-X" refers to the mature form (miRNA) of the miRNA numbered X. As used herein, when two miRNAs originate from opposite arms of the same miRNA precursor, the two miRNAs are indicated using the suffixes "-3p" or "-5p". Hereinafter, unless otherwise specified, when the miRNA precursor comprises two miRNAs, said "miR-X" refers to both the -3p and -5p miRNAs. In addition, as used herein, when the relative expression levels of two miRNAs are known, and the expression level of one miRNA is lower than that of the other miRNA, the miRNA with lower expression level is indicated by an asterisk after the name. Information such as the nucleotide sequences of each "mir-X" and "miR-X" can be obtained with reference to miRBase (http: / / www.mirbase.org / ).

[0019] As used herein, the term "miRNA mimic" refers to a double-stranded RNA molecule that mimics the function of a specific endogenous miRNA, wherein part or all of the polynucleotide is chemically modified. Also as used herein, the term "X mimic" refers to a miRNA mimic designed to mimic the function of miR-X. Since the X mimic functions in cells in the same manner as endogenous miR-X, introducing the X mimic into cells with low expression / activity of miR-X allows investigation of the properties of the RNA nucleic acid molecule of the present specification as if cells with high expression / activity of miR-X were used. Various miRNA mimics are commercially available.

[0020] As used herein, the term "miRNA inhibitor" refers to a single-stranded DNA molecule that suppresses the function of a specific endogenous miRNA, wherein part or all of the polynucleotide is chemically modified. Also as used herein, the term "X inhibitor" refers to a miRNA inhibitor designed to suppress the function of miR-X. Since the X inhibitor suppresses the function of endogenous miR-X in cells, introducing the X inhibitor into cells with high expression / activity of miR-X allows investigation of the properties of the RNA nucleic acid molecule of the present specification as if cells with low expression / activity of miR-X were used. Various miRNA inhibitors are commercially available.

[0021] As used herein, the term "polyA" refers to adenosine added to mRNA by polyadenylation. polyA is known to contribute to protein synthesis, mRNA stabilization, and the like. Said polyA can be added, for example, by performing in vitro transcription using a DNA strand with consecutive thymine nucleotides (having polyd(T)) as a template.

[0022] In this specification, "stem-loop structure" means a structure in a single-stranded nucleic acid in which partially complementary base pairs form a complementary strand (stem structure) through interactions between the base pairs, and a non-complementary sequence sandwiched between the complementary base pairs forms a loop structure. The stem-loop structure is also called a hairpin structure or hairpin-loop structure. Furthermore, in this specification, "sequence x is capable of forming a stem-loop" means that all or part of the polynucleotide constituting sequence x can form one or more stem-loop structures, either alone or together with other sequences (as a whole).

[0023] In this specification, “gene expression” means the translation of mRNA into a polypeptide or protein, or post-translational modification of a polypeptide or protein.

[0024] In this specification, "gene expression induction" means that the expression of a target gene is induced, and may also mean a change from a state in which the target gene is not expressed to a state in which it is expressed. In this disclosure, the induction of gene expression is also referred to, for example, as the state in which an ON switch is functioning. The expression of the target gene can be evaluated, for example, by measuring the expression level of the protein of the target gene and / or the function or amount of the protein of the target gene using luminescence or ELISA, or by measuring the expression level of the mRNA of the target gene using quantitative PCR, in accordance with Example 1 described below.

[0025] In this specification, "hybridize" can be detected, for example, by various hybridization assays under stringent conditions. The hybridization assay is not particularly limited, and methods such as those described in Sambrook et al., "Molecular Cloning: A Laboratory Manual 2nd Ed." [Cold Spring Harbor Laboratory Press (1989)] may be employed.

[0026] In this specification, "stringent conditions" may be, for example, low-stringent conditions, medium-stringent conditions, or high-stringent conditions. "Low-stringent conditions" are, for example, 5×SSC, 5×Denhardt solution, 0.5% SDS, 50% formamide, and 32°C. "Medium-stringent conditions" are, for example, 5×SSC, 5×Denhardt solution, 0.5% SDS, 50% formamide, and 42°C. "High-stringent conditions" are, for example, 5×SSC, 5×Denhardt solution, 0.5% SDS, 50% formamide, and 50°C. The degree of stringency can be set by those skilled in the art by appropriately selecting conditions such as temperature, salt concentration, probe concentration and length, ionic strength, and time. The "stringent conditions" can also be those described in Sambrook et al.'s "Molecular Cloning: A Laboratory Manual 2nd Ed." [Cold Spring Harbor Laboratory Press (1989)], for example.

[0027] In this specification, “complementary” means that one polynucleotide can form a nucleotide pair, i.e., a base pair, with another polynucleotide.

[0028] In this specification, “vector” (expression vector) means a recombinant plasmid or virus containing a nucleic acid molecule that is delivered to a cell in vitro or in vivo.

[0029] In this specification, "lipid" means a molecule having polar groups (hydrophilic groups) and nonpolar groups (hydrophobic groups) (amphiphilic molecules).

[0030] In this specification, "protein," "peptide," or "polypeptide" means a polymer composed of unmodified amino acids (natural amino acids), modified amino acids, and / or artificial amino acids. The polypeptide is, for example, a peptide having a length of 10 amino acids or more.

[0031] In this specification, "lipid nanoparticles" (LNPs) refer to nanoparticles with a diameter of approximately 10 nm to 1000 nm, mainly composed of lipids. These lipid nanoparticles can deliver nucleic acids through mechanisms such as cellular uptake, intracellular transport, endosomal release, and endosomal escape, and are used as nonviral drug delivery systems (DDS).

[0032] In this specification, "binding molecule" is a molecule capable of binding to a predetermined molecule. Examples of the binding molecule include nucleic acid molecules, proteins, sugar chains, etc., that can bind to the predetermined molecule. Specific examples of the binding molecule include aptamers, antibodies, receptors, ligands, etc., that can bind to the predetermined molecule. The binding molecule may be, for example, a known binding molecule capable of binding to the predetermined molecule, or a newly prepared binding molecule using methods such as the SELEX method or phage display method. The binding molecule may be, for example, an aptamer, antibody, receptor, or ligand capable of binding to an aging marker.

[0033] In this specification, “antibody” means a protein comprising one or more polypeptides substantially or partially encoded by an immunoglobulin gene or a fragment of an immunoglobulin gene. Examples of such antibodies include polyclonal antibodies and monoclonal antibodies. Examples of such antibody isotypes include IgG (e.g., IgG1, IgG2, IgG3, IgG4, etc.), IgM, IgA (e.g., IgA1, IgA2, etc.), IgE, IgD, IgY, etc. Examples of such antibody origins include mammals such as mice, rats, hamsters, rabbits, goats, cattle, horses, camels, and alpacas; birds such as chickens and ostriches; and cartilaginous fish such as sharks. Examples of such antibodies include heavy chain antibodies (VHH antibodies) derived from camelids, immunoglobulin neoantigen receptors (IgNARs) derived from cartilaginous fish, and antibody fragments (e.g., Fab, Fab', F(ab')). ２The antibodies may be single-domain antibodies (nanobody, etc.), recombinant antibodies (e.g., scFv, disulfide-bonded Fv (dsFv), diabody, minibody, etc.). The antibodies may also be antibody-like molecules (e.g., affibody, anticalin, DARPins, monobody, etc.) produced by molecular biological techniques such as phage display and / or by protein engineering techniques using existing protein motifs.

[0034] In this specification, “label” means a label used to distinguish a target molecule or substance from other molecules or substances. Such labels include, for example, fluorescent labels such as fluorescent dyes or fluorescent substances (e.g., fluorescein, fluorescein isothiocyanate, rhodamine); chemiluminescent labels such as luciferin and aequorin; luminescent substances such as luminol and acridinium derivatives; electroluminescent substances such as ruthenium complexes; and enzymatic labels such as horseradish peroxidase, alkaline phosphatase, β-galactosidase (β-gal), glucose oxidase, and luciferase. ３ H, １４ C, ３２ P, ３５ S, １２５ Examples include labeling with radioactive isotopes (RI), such as I.

[0035] In this specification, “target gene” means identified and isolated, recovered from components in their natural state, identified and isolated, and / or recovered from components in their natural state.

[0036] In this specification, “treatment” means therapeutic treatment and / or preventive treatment. In this specification, “treatment” means the treatment, cure, prevention, suppression, remission (remission), or improvement of a disease, condition, or disorder, or the cessation, suppression, reduction, or delay of the progression of a disease, condition, or disorder. In this specification, “prevention” means a reduction in the likelihood of developing a disease or condition, or a delay in the development of a disease or condition. The “treatment” may be, for example, treatment of a patient who develops the disease in question, or treatment of a model animal of the disease in question.

[0037] In this specification, “subject” means an animal or cells, tissues, or organs derived from an animal. The term “subject” is used in particular to include humans. The term “animal” means humans and non-human animals. Examples of non-human animals include mammals such as mice, rats, rabbits, dogs, cats, cattle, horses, pigs, monkeys, dolphins, and sea lions. In this specification, “patient” means a subject receiving preventive or therapeutic treatment. The term “subject” includes, for example, a patient in addition to a healthy person.

[0038] Sequence information for proteins or nucleic acids (e.g., DNA or RNA) encoding them, as described herein, can be obtained from sources such as the Protein Data Bank, UniProt, or GenBank. RNA nucleic acid sequences can also be obtained from the corresponding DNA base sequences using appropriate sequence conversion software.

[0039] The following explanation of this disclosure will be based on examples, but this disclosure is not limited to the following examples and can be modified and implemented as desired. Furthermore, the descriptions in this disclosure and each embodiment are mutually interchangeable unless otherwise specified. In this specification, when the expression "~" is used, it is used to mean including the numerical or physical values ​​before and after it. Also, in this specification, the expression "A and / or B" includes "A only," "B only," and "both A and B."

[0040] <RNA nucleic acid molecule> In one embodiment, the present disclosure provides an RNA nucleic acid molecule capable of controlling the expression of a target gene. The RNA nucleic acid molecule of the present disclosure comprises a sequence encoding a target gene, a PolyA sequence, and a degradation induction sequence, wherein the sequence encoding the target gene, the PolyA sequence, and the degradation induction sequence are arranged in this order from the 5' end to the 3' end, and the degradation induction sequence comprises a target sequence of miRNA.

[0041] The inventors conceived the idea that the expression of a target gene could be controlled in cells expressing a target miRNA by utilizing miRNA expression. Through diligent research, the inventors discovered that the expression of a target gene can be induced in cells containing the target miRNA by using an RNA nucleic acid molecule containing a sequence encoding the target gene, a PolyA sequence, and a degradation induction sequence, thus establishing this disclosure. Specifically, the method for inducing the expression of a target gene in cells containing the target miRNA using the RNA nucleic acid molecule of this disclosure is presumed to be induced by the mechanism described below, as shown in Figure 1. This disclosure is not limited in any way to the mechanism described below. The RNA nucleic acid molecule of this disclosure, as shown in Figure 1, includes the sequence encoding the target gene, the PolyA sequence, and a degradation induction sequence containing the target sequence of the miRNA. As shown in Figure 1(A), in cells that do not have a target miRNA, the target miRNA does not hybridize to the miRNA target sequence, so the degradation-inducing sequence can form a higher-order structure such as a stem-loop structure. When the RNA nucleic acid molecule of this disclosure forms a higher-order structure, a nuclease recognizes the higher-order structure and degrades the RNA nucleic acid molecule, or degrades the 3' region of the RNA nucleic acid molecule, shortening the length of the RNA nucleic acid molecule and making it unstable, thereby degrading the entire RNA nucleic acid molecule. For this reason, in cells that do not have a target miRNA, the degradation of the RNA nucleic acid molecule of this disclosure is induced or promoted, so the expression of the target gene in the RNA nucleic acid molecule is not induced (OFF state). On the other hand, as shown in Figure 1(B), in cells that have a target miRNA, the target miRNA hybridizes to the target sequence of the miRNA in the RNA nucleic acid molecule of this disclosure. When hybridization occurs in the RNA nucleic acid molecule of this disclosure, cleavage occurs in the central part of the miRNA complementary sequence. As a result, the RNA nucleic acid molecule of this disclosure is rescued from degradation by the nuclease, and the expression of the target gene in the RNA nucleic acid molecule is induced (ON state).Therefore, according to this disclosure, in cells expressing a specific miRNA, the degradation of the RNA nucleic acid molecule of this disclosure is suppressed, and the expression of the target gene can be induced. Furthermore, in this disclosure, the mechanism by which the expression of the target gene changes from an OFF state to an ON state in the RNA nucleic acid molecule in the presence of the target miRNA is also referred to as an ON switch.

[0042] The target gene may be, for example, a protein-coding gene, a disease-causing gene, or a genome editing-related gene. The protein may be, for example, an antibody or an antibody variant (e.g., scFv, Fab, Fab'). ２Examples include T cell receptors, chimeric antigen receptors, growth factors or their receptors, receptors for ligands in vivo, apoptosis regulatory proteins, fluorescent proteins, luminescent proteins, accessory proteins that assist in fluorescence, luminescence, or coloration, cytokines, chemokines, transcription factors, etc. Examples of growth factors include EFG, FGF, IGF, TGF, VEGF, etc. The aforementioned fluorescent proteins include, for example, blue fluorescent proteins such as Sirius and EBFP; cyan fluorescent proteins such as mTurquoise, TagCFP, AmCyan, mTFP1, MidoriishiCyan, and CFP; green fluorescent proteins such as TurboGFP, AcGFP, TagGFP, Azami-Green, ZsGreen, EmGFP, EGFP, GFP2, and Hyper; and TagYFP, EYFP, Venus, YFP, PhiYFP, PhiYFP-m, TurboYFP, and ZsYell. Examples include yellow fluorescent proteins such as ow and mBanana; orange fluorescent proteins such as KusabiraOrange and mOrange; red fluorescent proteins such as TurboRFP, DsRed-Express, DsRed2, TagRFP, DsRed-Monomer, AsRed2, and mStrawberry; and near-infrared fluorescent proteins such as TurboFP602, mRFP1, JRed, KillerRed, mCherry, HcRed, KeimaRed, mRasberry, and mPlum. Examples of the luminescent proteins include aequorin and luciferase. Examples of the auxiliary proteins include phosphatase, peroxidase, β-galactosidase, and β-lactamase. The disease-causing genes mentioned above include, for example, normal genes in diseases caused by gene dysfunction, specifically, VEGF-A for myocardial infarction. The apoptosis-regulating proteins include, for example, apoptosis-inducing proteins such as Bim-EL, Bax, FADD, and caspases, apoptosis-inhibiting proteins such as Bcl-xL and BCL-2, and toxin-antitoxin system (TA system) related proteins such as toxins and antitoxins.The genome editing-related genes mentioned above include, for example, Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9, Cas10, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, and Csm Examples include CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) enzymes such as Csm5, Csm6, Cmr1, Cmr3, Cmr4, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, and Csf4; Cre enzymes; etc.

[0043] The sequence encoding the target gene may, for example, be codon-optimized. The codon-optimized sequence encoding the target gene can be designed, for example, using algorithms such as GeneArt (Life Technologies) or DNA2.0 (MenloParkCA), depending on the target cell described later.

[0044] The sequence encoding the target gene may, for example, have a sequence encoding a signal peptide at its 5' end.

[0045] The length of the polyA sequence is, for example, 20 nucleotides or more, preferably 20 to 400 nucleotides, 20 to 300 nucleotides, 40 to 200 nucleotides, 60 to 150 nucleotides, 60 to 120 nucleotides, 80 to 120 nucleotides, etc. The length of the polyA sequence is preferably short so that the degradation of the RNA nucleic acid molecule occurs quickly in cells that do not express miRNA, for example, 80 to 120 nucleotides.

[0046] In the RNA nucleic acid molecule of this disclosure, the degradation induction sequence includes a target sequence for miRNA. The target sequence can be designed, for example, according to the miRNA expressed by the cell into which the RNA nucleic acid molecule of this disclosure is introduced. That is, if it is desired to induce the expression of the target gene in the cell, the target sequence can be a nucleotide sequence that can hybridize with the miRNA expressed by the cell or a miRNA that is active in the cell. If it is not desired to induce the expression of the target gene in the cell, the target sequence can be a nucleotide sequence that can hybridize with the miRNA not expressed by the cell or a miRNA that is inactive in the cell. Therefore, in the RNA nucleic acid molecule of this disclosure, for example, if it is desired to induce the expression of the target gene in cell A and suppress the expression of the target gene in cell B, cell-specific expression of the target gene can be induced by adopting a target sequence for a miRNA that is expressed or active in cell A but not expressed or is inactive in cell B. The target sequence of the miRNA can also be defined as, for example, the target sequence of a miRNA that is expressed or active in the target cell.

[0047] The cells are not particularly limited and include, for example, cells derived from mammals (e.g., humans, or non-human animals such as mice, rats, rabbits, dogs, cats, cattle, horses, pigs, monkeys, dolphins, sea lions, and other mammals, as well as birds). The cells may be cells isolated from a species, or cultured cells of isolated cells. The cells may include, for example, keratinizing epithelial cells (e.g., keratinized epidermal cells), mucosal epithelial cells (e.g., epithelial cells of the tongue surface), exocrine gland epithelial cells (e.g., mammary gland cells), hormone-secreting cells (e.g., adrenal medullary cells), metabolic and storage cells (e.g., hepatocytes), luminal epithelial cells constituting the interface (e.g., type I alveolar cells), luminal epithelial cells of the inner chain canal (e.g., vascular endothelial cells), ciliated cells with transporting ability (e.g., airway epithelial cells), extracellular matrix secretory cells (e.g., fibroblasts), and contractile cells. Examples include smooth muscle cells, blood and immune system cells (e.g., T lymphocytes (T cells), NK cells, macrophages, dendritic cells, monocytes), sensory cells (e.g., rod cells), autonomic nervous system neurons (e.g., cholinergic neurons), supporting cells of sensory organs and peripheral neurons (e.g., accompanying cells), central nervous system neurons and glial cells (e.g., astrocytes), pigment cells (e.g., retinal pigment epithelial cells), and their progenitor cells (e.g., tissue progenitor cells, hematopoietic stem cells). The aforementioned cells may be one type or multiple types.

[0048] The target cells are preferably cells that make up tissues such as muscle tissue, vascular tissue, and liver; blood cells; etc. The blood cells are preferably T cells and monocytes. The cells that make up the tissue and the blood cells may be one type or multiple types.

[0049] The target sequence is not particularly limited, for example, as long as it can hybridize with the miRNA, and can be a nucleotide sequence that is completely or partially complementary to the miRNA. If the target sequence is a partially complementary nucleotide sequence, the 5' and / or 3' nucleotides or nucleotide sequences of the target sequence may, for example, be a nucleotide sequence complementary to the miRNA nucleotide sequence, or a nucleotide sequence containing mismatched nucleotides with respect to the miRNA nucleotide sequence. If the target sequence is a partially complementary nucleotide sequence, it is preferable that the 3' nucleotide sequence of the target sequence is complementary to, for example, a nucleotide sequence complementary to the miRNA nucleotide sequence. In the RNA nucleic acid molecule of this disclosure, if the 3' nucleotide sequence of the target sequence is a nucleotide sequence complementary to the miRNA nucleotide sequence, the RNA nucleic acid molecule is stabilized, for example, in the presence of the miRNA. In the RNA nucleic acid molecule of this disclosure, for example, when RNA cleavage occurs within the target sequence of the miRNA in a miRNA-dependent manner, a portion of the target sequence of the miRNA remains downstream of the polyA sequence. In this case, if the 3' base sequence of the target sequence is a base sequence complementary to the base sequence of the miRNA, the RNA nucleic acid molecule of this disclosure can shorten the length of the portion of the target sequence remaining downstream of the polyA sequence, for example, and is therefore presumed to be able to suppress the destabilization of the RNA nucleic acid molecule caused by the sequence downstream of the polyA sequence. The number of mismatched bases between the miRNA and the target sequence should be within the range in which the miRNA can hybridize to the target sequence. If the target sequence contains mismatched bases with the miRNA, the number of mismatched bases is, for example, 1 to 10 bases, 1 to 9 bases, 1 to 8 bases, 1 to 7 bases, 1 to 6 bases, 1 to 5 bases, 1 to 4 bases, 1 to 3 bases, 1 to 2 bases, or 1 base in a base sequence complementary to the miRNA. In this disclosure, the numerical range of the number of bases, etc., means disclosing all positive integers belonging to that range. That is, for example, the statement "1 to 5" means the disclosure of all "1, 2, 3, 4, 5" (the same applies hereinafter).The ratio of the number of mismatched bases in the target sequence (mismatch rate) is, for example, 50% or less, 45% or less, 40% or less, 35% or less, 30% or less, 25% or less, 20% or less, 15% or less, 10% or less, 5% or less, 4% or less, 3% or less, 2% or less, or 1% or less.

[0050] When the target sequence contains a mismatched base with the miRNA sequence, the target sequence has, for example, a nucleotide sequence having insertion, addition, substitution, and / or deletion of one or several nucleotides relative to the nucleotide sequence complementary to the miRNA. The "one or several" is, for example, 1 to 10 bases, 1 to 9 bases, 1 to 8 bases, 1 to 7 bases, 1 to 6 bases, 1 to 5 bases, 1 to 4 bases, 1 to 3 bases, 1 to 2 bases, or 1 base. The target sequence may contain one type of mutation or multiple types of mutations relative to the nucleotide sequence complementary to the miRNA.

[0051] The target sequence has, for example, a nucleotide sequence having a deletion of one or several nucleotides in the nucleotide sequence complementary to the miRNA. The above "one or several" can, for example, use the aforementioned examples, and is preferably 1 to 6, 2 to 6, or 3 to 6. Thereby, the RNA nucleic acid molecule of the present disclosure can, for example, enhance the expression level (E ON ) of the target gene when miRNA is present relative to the target sequence, and can improve the ratio (E OFF ) of the expression level (E ON ) of the target gene when miRNA is present relative to the target sequence to the expression level (E ON ) / E OFF ) of the target gene when miRNA is absent relative to the target sequence. When there are two or more deletion mutations, each deletion mutation may be continuous or discontinuous. The position of the deletion mutation is, for example, any position in the target sequence, and may be the 5' end, the 3' end, and / or a region other than these. The position of the deletion mutation is, for example, on the 5' end side of the nucleotide sequence complementary to the miRNA, and as a specific example, it is the one or several bases at the 5' end in the nucleotide sequence complementary to the miRNA.

[0052] The target sequence, for example, has a nucleotide sequence having one or several base substitutions in a nucleotide sequence complementary to the miRNA. "One or several" can be, for example, based on the examples given above, preferably 1 to 6, 1 to 4, or 1 to 2. Thus, the RNA nucleic acid molecule of this disclosure, for example, controls the expression level (E) of the target gene in the presence of the miRNA relative to the target sequence. ON ) can be enhanced, and the expression level of the target gene in the absence of miRNA for the target sequence (E OFF The expression level of the target gene in the presence of miRNA relative to the target sequence (E ON ) ratio (E ON / E OFF ) can be improved. If there are two or more substitution mutations, each substitution mutation may be consecutive or discontinuous. The location of the substitution mutation is, for example, any location in the target sequence, such as the 5' end, the 3' end and / or other regions. The location of the substitution mutation is, for example, in the nucleotide sequence complementary to the miRNA, with reference to the 3' end base (position 1), the bases at positions 1, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 and / or 24, preferably positions 1, 13, 14, 15, 16, 17, 18, 19 and / or 23, and more preferably positions 1, 14, 15, 16, 17 and / or 18. If there are two or more substitution mutations, the locations of the substitution mutations are, for example, in the nucleotide sequence complementary to the miRNA, with reference to the 3' end base (position 1), the bases at positions 14 and 15. Furthermore, the substitution mutation is, for example, one in which U or G is substituted with A or C.

[0053] If the target sequence contains mismatched bases with the miRNA sequence, the RNA nucleic acid molecule of this disclosure increases the expression level of the target gene compared to, for example, an RNA nucleic acid molecule in which the target sequence of the miRNA does not contain mismatched bases with the miRNA sequence, i.e., an RNA nucleic acid molecule containing a base sequence complementary to the miRNA (control nucleic acid molecule). The RNA nucleic acid molecule that does not contain mismatched bases with the miRNA sequence is an RNA nucleic acid molecule having the same composition except for the absence of mismatched bases. Specifically, in the presence of the miRNA, the RNA nucleic acid molecule of this disclosure exhibits an expression level of the target gene that is, for example, more than 1, 1.02 to 4, 1.03 to 3.5, 1.04 to 3, 1.05 to 2.5, or 1.1 to 2.41 times higher than the expression level of the target gene of the control nucleic acid molecule.

[0054] If the target sequence contains mismatched bases with the miRNA sequence, the RNA nucleic acid molecule of this disclosure, for example, compared to an RNA nucleic acid molecule in which the target sequence of the miRNA does not contain mismatched bases with the miRNA sequence, i.e., an RNA nucleic acid molecule containing a base sequence complementary to the miRNA (control nucleic acid molecule), the expression level of the target gene (E) in the absence of the miRNA. ＯＦＦ The expression level of the target gene in the presence of the miRNA (E) ＯＮ ) ratio (E ＯＮ / E ＯＦＦ ) increases. An RNA nucleic acid molecule that does not contain mismatched bases with the sequence of the miRNA is an RNA nucleic acid molecule that has the same composition as the one described above except that it does not contain the mismatched bases. Specifically, the RNA nucleic acid molecule of this disclosure, for example, increases the expression level of the target gene (E) in the absence of the miRNA compared to the control nucleic acid molecule. ＯＦＦ The expression level of the target gene in the presence of the miRNA (E) ＯＮ ) ratio (E ＯＮ / E ＯＦＦ ) are, for example, greater than 1, 1.02 to 4 times, 1.03 to 3.5 times, 1.04 to 3 times, 1.05 to 2.5 times, or 1.1 to 2.43 times.

[0055] The length of the target sequence of the miRNA can be designed, for example, according to the length of the miRNA, and may be the same as or different from the length of the miRNA. If the length of the target sequence of the miRNA is different from the length of the miRNA, the length of the target sequence of the miRNA may be, for example, 1 to 10 nucleotides, 1 to 8 nucleotides, 1 to 6 nucleotides, 1 to 5 nucleotides, 1 to 4 nucleotides, 1 to 3 nucleotides, 1 to 2 nucleotides, or 1 nucleotide shorter than the length of the miRNA, based on the length of the miRNA. Specific examples of target sequence lengths for the miRNA include, for example, 8 to 30 nucleotides, 10 to 28 nucleotides, 12 to 26 nucleotides, 12 to 24 nucleotides, 14 to 24 nucleotides, 14 to 22 nucleotides, 16 to 22 nucleotides, 14 to 20 nucleotides, or 16 to 20 nucleotides. In the RNA nucleic acid molecule of this disclosure, if the length of the miRNA target sequence is made shorter than the length of the miRNA, the RNA nucleic acid molecule is stabilized in the presence of the miRNA. In the RNA nucleic acid molecule of this disclosure, for example, if RNA cleavage occurs within the miRNA target sequence in a miRNA-dependent manner, a portion of the miRNA target sequence remains downstream of the polyA sequence. In this case, if the length of the miRNA target sequence is shortened, the RNA nucleic acid molecule of this disclosure can, for example, shorten the length of the portion of the target sequence remaining downstream of the polyA sequence, and is therefore presumed to be able to suppress the destabilization of the RNA nucleic acid molecule caused by the sequence downstream of the polyA sequence.

[0056] The degradation-inducing sequence may, for example, include a target sequence for one type of miRNA, or it may include separate target sequences for multiple types of miRNA. When the degradation-inducing sequence includes target sequences for multiple types of miRNA, the RNA nucleic acid molecule of this disclosure can, for example, specifically induce the expression of the target gene in the target cells by combining multiple types of miRNA. Furthermore, the degradation-inducing sequence may, for example, include different target sequences for one type of miRNA. That is, the degradation-inducing sequence may, for example, include multiple types of target sequences that can hybridize to one type of miRNA.

[0057] The miRNA may be, for example, a miRNA expressed in cells (target cells) intended for the introduction of the RNA nucleic acid molecule, and can also be a miRNA that is highly expressed or highly active in target cells. Examples of miRNAs that are highly expressed in target cells include those that show a high expression level of 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, or higher compared to expression in cells other than target cells. The miRNA can be selected from at least 10,000 types of miRNAs. The miRNA may be selected, for example, from miRNAs registered in a database. Examples of the aforementioned databases include miRBase (http: / / www.mirbase.org), Rfam (https: / / rfam.org / search), miRIAD (https: / / www.miriad-database.org / ), and FANTOM5 (https: / / fantom.gsc.riken.jp / 5 / suppl / De_Rie_et_al_2017 / ).

[0058] The expression of the aforementioned miRNA can be measured using, for example, microarrays, next-generation sequencers, real-time PCR, in situ hybridization, etc. (see Reference 1 below). Specifically, when measuring using a next-generation sequencer, a commercially available miRNA measurement kit is used to perform next-generation sequencing analysis and calculate the miRNA expression level in terms of TPM (transcripts per million). A TPM value of 10,000 or more indicates high expression, a TPM value of 1,000 to 10,000 indicates moderate expression, and a TPM value of 1,000 or less indicates low expression. For this reason, as the aforementioned miRNA, for example, a miRNA with a TPM value of 10,000 or more in the target cells can be used. Reference 1: Liu J, Jennings SF, Tong W, Hong H. Next generation sequencing for profiling expression of miRNAs: technical progress and applications in drug development. J Biomed Sci Eng. 2011 Oct;4(10):666-676. doi: 10.4236 / jbise.2011.410083. PMID: 22457835; PMCID: PMC3312786.

[0059] The activity of the miRNA can be evaluated by its relative expression level, calculated by dividing the intensity of luminescence produced when a Fluc-mRNA with the miRNA target sequence positioned within the 5'UTR is introduced into a cell by the luminescence intensity when a Fluc-mRNA without the target sequence is introduced into a cell (e.g., a target cell) (miRNA activity test). In the miRNA activity test, a relative expression level of 0.25 or less indicates high activity, a relative expression level greater than 0.25 but less than 0.8 indicates moderate activity, and a relative expression level of 0.8 or higher indicates low activity. Therefore, if the relative expression level of the target miRNA in the activity test is 0.25 or less, the target miRNA can be evaluated as having high activity or activity. On the other hand, if the relative expression level of the target miRNA in the activity test is 0.8 or higher, the target miRNA can be evaluated as having low activity or no activity. The activity test of the miRNA can be specifically carried out, for example, by the methods described in Reference Examples 1 to 3 below.

[0060] When selecting a miRNA based on its activity in the target cells, the target sequence of the miRNA has, for example, a base sequence that can hybridize with a miRNA that exhibits high activity in the target cells. Specifically, the target sequence of the miRNA may have a sequence that can hybridize with a miRNA whose relative expression level is 0.5 or less, 0.4 or less, 0.3 or less, 0.2 or less, or 0.1 or less in the miRNA activity test. The relative expression level is, for example, a measurement obtained by performing the miRNA activity test using Fluc-mRNA in which the target sequence of the target miRNA is positioned within the 5'UTR and target cells.

[0061] When inducing the expression of the target gene in the aforementioned cells, the combination of the cells and the miRNA may include, for example, the following combinations. Alternatively, the combination of the cells and the miRNA may refer to, for example, the combination described in U.S. Patent Application No. 2023 / 0086537.・Pluripotent stem cells: hsa-miR-302a, hsa-miR-302b, hsa-miR-302c, hsa-miR-302d, hsa-miR-367 , hsa-5201, hsa-miR-92b, hsa-miR-106a, hsa-miR-18b, hsa-miR-20b, hsa-miR-19b-2 , hsa-miR-92a-2, hsa-miR-363, hsa-miR-20a, hsa-miR-17, hsa-miR-18a, hsa-miR-19 a, hsa-miR-19b-1, hsa-miR-373, hsa-miR-330, hsa-miR-520c, hsa-miR-182, hsa-miR -183, hsa-miR-96, hsa-miR-92a-1, hsa-miR-92a-2, hsa-miR-141, hsa-miR-200c, hs a-miR-27a, hsa-miR-7-1, hsa-miR-7-2, hsa-miR-7-3, hsa-miR-374a, hsa-miR-106b, hsa-miR-93, hsa-miR-25, hsa-miR-584, hsa-miR-374b, hsa-miR-21, hsa-miR-212, hs a-miR-371a, hsa-miR-371b, hsa-miR-372, hsa-miR-200b, hsa-miR-200a, hsa-miR-42 • Cardiomyocytes: hsa-miR-1, hsa-miR-133 • Neurons: hsa-miR-124-3p • Liver cells or hepatocytes: has-miR-122-5p • Vascular endothelial cells: hsa-miR-126-3p • Skeletal muscle cells: hsa-miR-125b-5p, hsa-miR-199a-5p • T cells: hsa-miR-142-3p, hsa-miR-155-5p • Monocytes: has-miR-223-3p

[0062] The RNA nucleic acid molecule of this disclosure may induce or suppress the degradation of the RNA nucleic acid molecule, that is, control the degradation of the RNA nucleic acid molecule in a miRNA-dependent manner, depending on the presence or absence of a miRNA capable of binding to the target sequence. Specifically, if there is no miRNA capable of binding to the target sequence, the RNA nucleic acid molecule of this disclosure may be configured to induce degradation of the RNA nucleic acid molecule, for example, if the miRNA does not hybridize to the target sequence of the miRNA. Alternatively, if there is a miRNA capable of binding to the target sequence, the RNA nucleic acid molecule of this disclosure may be configured such that, for example, if the miRNA hybridizes to the target sequence of the miRNA, the degradation of the RNA nucleic acid molecule is suppressed and the expression of the target gene is induced.

[0063] The decomposition-inducing sequence includes, for example, a sequence capable of forming a higher-order structure of the stem-loop structure. When the decomposition-inducing sequence forms the stem-loop structure, it is preferable that the stem-loop structure is formed on the 3' end of the decomposition-inducing sequence.

[0064] In the RNA nucleic acid molecule of this disclosure, if the degradation-inducing sequence has a sequence capable of forming a stem-loop structure, the RNA nucleic acid molecule functions, for example, as follows: If there is no miRNA capable of binding to the target sequence of the miRNA, the miRNA does not hybridize to the target sequence of the miRNA, and therefore the degradation-inducing sequence is able to form a stem-loop structure without, for example, inhibition of higher-order structure formation. In this case, in the RNA nucleic acid molecule of this disclosure, the degradation-inducing sequence can induce degradation of the RNA nucleic acid molecule, for example, by forming the stem-loop structure. As a result, the RNA nucleic acid molecule of this disclosure can suppress the expression of the target gene, for example. In the RNA nucleic acid molecule of this disclosure, it is presumed that the degradation-inducing sequence inhibits the binding of poly(A) binding proteins to the RNA nucleic acid molecule by forming the stem-loop structure, thereby inducing degradation by endonucleases. On the other hand, if a miRNA capable of binding to the target sequence of the miRNA exists, the miRNA hybridizes to the target sequence of the miRNA, causing a cleavage in the central part of the miRNA complementary sequence. In this case, in the RNA nucleic acid molecule of this disclosure, the degradation-inducing sequence is rescued from degradation by the nuclease because, for example, the region containing the stem loop from the central part to the 3' end of the miRNA complementary sequence is removed due to the lack of formation of the stem-loop structure, thus suppressing the degradation of the RNA nucleic acid molecule. As a result, it is presumed that the RNA nucleic acid molecule of this disclosure can induce the expression of the target gene, for example.

[0065] In the aforementioned degradation-inducing sequence, the sequence capable of forming the stem-loop structure may be, for example, a sequence that has been confirmed to form the stem-loop structure, or a sequence that has been estimated to form the stem-loop structure by estimating the secondary structure of a desired sequence using secondary structure prediction software (for example, RNAfold WebServer: http: / / rna.tbi.univie.ac.at / / cgi-bin / RNAWebSuite / RNAfold.cgi), etc. When using RNAfold WebServer as the secondary structure prediction site, for example, default parameters can be used.

[0066] The sequences capable of forming the aforementioned stem-loop structure may, for example, refer to known sequences capable of forming stem-loop structures. Specific examples include sequences containing 3'-prime-end Associated nuclease Binder (pAB) sequences, tetra-stem-loop (TL) sequences, histone stem-loop (HSL) sequences, and Fast Decay Stem (FDS) sequences, or modified sequences thereof. Examples of each sequence are shown below. The FDS sequences may include, for example, FDS #1 to #5 below, as well as the FDS sequences described in Reference 2 below. Furthermore, the sequences capable of forming the aforementioned stem-loop structure may be designed, for example, by estimating the secondary structure described above. pAB sequence (SEQ ID NO: 1): 5'-GUUGCACCAGAGGUACACUCUGUA-3' HSL sequence (SEQ ID NO: 2): 5'-AAAAAGGCUCUUUUCAGAGCCACCCA-3' Tetrastem loop sequence (SEQ ID NO: 3): 5'-CCGGGCGCAAGCCUUAAG-3' FDS sequence: FDS#1 (SEQ ID NO: 4): 5'-UCCUAUUUAUAAGGA-3' FDS#2 (SEQ ID NO: 5): 5'-AUGAGAUGGUGCAAUCGUUACUAUUUCAU-3' FDS#3 (SEQ ID NO: 6): 5'-GAAGCGCCAUGUAAAUGGUCUUC-3' FDS#4 (SEQ ID NO: 7): 5'-GUUUGUUGCACGGCAGUAGCGAGAGAC-3' FDS#5 (SEQ ID NO: 8): 5'-UAUCUCUUUUUAUCAUAAUGAAGAAGAUG-3' Reference 2: Rabani M, Kertesz M, Segal E. Computational prediction of RNA structural motifs involved in post-transcriptional regulatory processes. Methods Mol Biol. 2011;714:467-79.

[0067] The modified sequence is, for example, a sequence having one or more base insertions, additions, substitutions, and / or deletions in the base sequence of each sequence, and is capable of forming a stem-loop structure. Preferably, the modified sequence is a sequence having one or more base substitutions and / or deletions in the base sequence of each sequence, and is capable of forming a stem-loop structure. More preferably, the modified sequence is a sequence having one or more base deletions in the base sequence of each sequence, and is capable of forming a stem-loop structure. For example, the modified sequence is a sequence having 50% or more identity with respect to the base sequence of each sequence, and is capable of forming a stem-loop structure. Preferably, the deletion is, for example, a deletion from the 5' end base, a deletion from the 3' end base, and / or a deletion of a base pair that forms the stem in the stem-loop structure. The substitution is preferably one in which, for example, in the base sequence of each of the sequences, a mismatched base pair in the stem region of the stem-loop structure is changed to a base pair that can form a base pair.

[0068] In the modified sequence, "capable of forming a stem-loop structure" means, for example, that the modified sequence can form a stem-loop structure on its own, or that the modified sequence and other sequences in the decomposition-inducing sequence, specifically the sequence at the 5' end of the modified sequence and / or the sequence at the 3' end of the modified sequence, can form a stem-loop structure together.

[0069] In the modified sequence, "one or several" means, for example, that the modified sequence is in a range capable of forming a stem-loop structure, and specific examples include 1 to 30, 1 to 25, 1 to 20, 1 to 18, 1 to 15, 1 to 12, 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 to 2, or 1.

[0070] In the modified sequence, the "identity" is defined as, for example, a range in which the modified sequence can form a stem-loop structure, and specific examples include 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more. The "identity" can be determined, for example, by aligning two base sequences or amino acid sequences (the same applies hereinafter). The alignment can be calculated, for example, using BLAST, FASTA, etc. with default parameters.

[0071] Preferably, the modified sequence has reduced binding ability to Histone RNA hairpin-binding protein or stem-loop binding protein (SLBP) compared to the original sequence. Specifically, the modified sequence has reduced binding ability to SLBP compared to the original sequence (B ｂ The binding ability of the modified sequence to SLBP (B) ａ ) ratio (B ａ / B ｂThe sequence is such that the ×100 (%) is 80% or less, 75% or less, 70% or less, 65% or less, 60% or less, 55% or less, 50% or less, 45% or less, 40% or less, 35% or less, 30% or less, 25% or less, 20% or less, 15% or less, 10% or less, or 5% or less. The ability to bind to SLBP can be implemented, for example, in the same manner as in Reference 3 below. A modified sequence with reduced ability to bind to SLBP can be designed, for example, by substituting an adenine located near the 5' end of the sequence capable of forming the stem-loop structure in the original sequence with another base (e.g., uracil, guanine, cytosine). The adenines present in the vicinity are, for example, adenines within 1 to 10 bases, 1 to 7 bases, 1 to 5 bases, 1 to 4 bases, 1 to 3 bases, 1 to 2 bases, or 1 base, relative to the 5' end base of the sequence capable of forming the stem-loop structure (base 0). Reference 3: Dominski Z, Yang XC, Kaygun H, Dadlez M, Marzluff WF. A 3' exonuclease that specifically interacts with the 3' end of histone mRNA. Mol Cell. 2003 Aug;12(2):295-305. doi: 10.1016 / s1097-2765(03)00278-8. PMID: 14536070.

[0072] In the degradation induction sequence, some or all of the sequence of the miRNA target sequence may constitute, for example, some or all of the stem-loop structure. That is, if the degradation induction sequence forms a stem-loop structure, some or all of the sequence of the miRNA target sequence may be configured to form the stem-loop structure, or some or all of the sequence of the miRNA target sequence may be configured to form the stem-loop structure together with other sequences in the degradation induction sequence. The RNA nucleic acid molecule of this disclosure can shorten the length of the degradation induction sequence by, for example, using some or all of the sequence of the miRNA target sequence as a sequence that forms the stem-loop structure. As a result, the RNA nucleic acid molecule of this disclosure can reduce the expression level of the target gene (E) in the presence of miRNA relative to the target sequence. ONThis can enhance the miRNA. The sequence of the stem-loop structure containing the miRNA target sequence can be designed, for example, by estimating the aforementioned secondary structure for the sequence containing the miRNA target sequence.

[0073] The arrangement of the target sequence of the miRNA and the sequence capable of forming the stem-loop structure is not particularly limited, and is sufficient if, for example, the formation of the stem-loop structure in the degradation-inducing sequence can be controlled depending on the presence or absence of the miRNA. If the target sequence of the miRNA does not constitute part of the stem-loop structure, the target sequence of the miRNA is, for example, the 5' end or 3' end of the sequence capable of forming the stem-loop structure, preferably the 5' end of the sequence capable of forming the stem-loop structure. If the target sequence of the miRNA constitutes part of the stem-loop structure, the target sequence of the miRNA may constitute, for example, the 5' end of the sequence capable of forming the stem-loop structure and part of the sequence on the 5' end of the sequence capable of forming the stem-loop structure, or the 3' end of the sequence capable of forming the stem-loop structure and part of the sequence on the 3' end of the sequence capable of forming the stem-loop structure.

[0074] The stem-loop structure, as described above, includes a stem region and a loop region. The length of the stem region can be, for example, 2 to 50 nucleotides, 2 to 40 nucleotides, 2 to 30 nucleotides, or 2 to 20 nucleotides. When the length of the stem region of the stem-loop structure becomes relatively shorter, that is, when the number of nucleotides forming base pairs in the stem region becomes relatively smaller, the expression level of the target gene in the absence of miRNA for the target sequence (E OFF The expression level of the target gene in the presence of miRNA relative to the target sequence (E ON ) ratio (E ON / E OFF Since the relative size of the stem region is large, the length of the stem region is preferably 2 to 35 bases, 2 to 30 bases, 2 to 25 bases, 2 to 20 bases, 2 to 15 bases, or 2 to 10 bases. The length of the stem region is, for example, the length of the stem region E ON / E OFFHowever, the length is 2 times or more, 3 times or more, 4 times or more, 5 times or more, 6 times or more, 7 times or more, 8 times or more, 9 times or more, or 10 times or more. The length of the stem region is, for example, E ON / E OFF However, the length is 2 to 20 times, 3 to 15 times, 4 to 14 times, 5 to 13 times, 6 to 12 times, 7 to 11 times, 8 to 10 times, or 9 to 10 times. The length of the stem region refers to the number of base pairs that are formed in the stem region. The length of the loop region can be, for example, 4 to 20 bases, 4 to 15 bases, 4 to 12 bases, 4 to 10 bases, 4 to 8 bases, 4 to 6 bases, or 4 to 5 bases.

[0075] In the degradation-inducing sequence, the number of stem-loop structures can be, for example, 1 to 10, 1 to 7, 1 to 5, 1 to 4, 1 to 3, 1 to 2, or 1. For example, when the number of stem-loop structures in the degradation-inducing sequence is relatively small, the expression level of the target gene in the presence of miRNA for the target sequence (E ON Because the relative size of the stem-loop structure becomes larger and it is less affected by the limitations of packaging DDS such as lipid nanoparticles, the number of stem-loop structures is preferably 1 to 3, 1 to 2, or 1.

[0076] The RNA nucleic acid molecule of this disclosure may have its degradation induced or suppressed by using, for example, a binding sequence to which an RNA-binding protein (RNABP) can bind as the degradation-inducing sequence, and controlling the binding of the RNA-binding protein to the binding sequence in a miRNA-dependent manner to the target sequence. The RNABP may be, for example, a protein that induces the degradation of the bound RNA nucleic acid molecule upon binding to the binding sequence (degradation-inducing RNABP).

[0077] Examples of the degradation-inducible RNABPs include heteronuclear ribonucleoprotein (hnRNP), CNOT (CCR4-NOT transcription complex, subunit 4), AU-binding factor 1 (AUF1), and tristeraproline (TTP). Examples of the hnRNPs include hnRNPs A1, hnRNPs A2, and hnRNPs B1, which are hnRNPs capable of catalyzing deadenylation reactions. The hnRNPs, AUF1, and TTP can destabilize RNA molecules by recognizing RNA nucleic acid molecules and recruiting the CCR4-CAF1-NOT deadenylation enzyme complex, thereby inducing deadenylation and inducing the degradation of the RNA molecules.

[0078] If the degradation-inducing sequence includes a binding sequence for the degradation-inducible RNABP, the degradation-inducing sequence may be a sequence that controls, for example, the binding of RNABP to the RNA nucleic acid molecule, more specifically, to the degradation-inducing sequence, in a miRNA-dependent manner to the target sequence. For example, if there is no miRNA capable of binding to the target sequence of the miRNA, the miRNA does not hybridize to the target sequence of the miRNA, and therefore, for example, the RNABP can bind to the degradation-inducing sequence. In this case, in the RNA nucleic acid molecule of this disclosure, it is presumed that, for example, the binding of RNABP to the degradation-inducing sequence causes a deadenylation reaction of the RNA nucleic acid molecule, thereby inducing the degradation of the RNA nucleic acid molecule. On the other hand, if there is a miRNA capable of binding to the target sequence of the miRNA, the miRNA hybridizes to the target sequence of the miRNA, and therefore, for example, the degradation-inducing sequence forms a double-stranded structure between the miRNA and the target sequence, and the RNABP cannot bind. In this case, the RNA nucleic acid molecule of the present disclosure is presumed to be able to suppress the degradation of the RNA nucleic acid molecule because, for example, binding to the RNABP degradation-inducing sequence does not occur, and as a result, the deadenylation reaction of the RNA nucleic acid molecule does not occur.

[0079] If the degradation-inducing sequence includes the hnRNP binding sequence, and no miRNA capable of binding to the target sequence is present, the miRNA will not hybridize to its target sequence. Therefore, the degradation-inducing sequence can bind to the hnRNP, and the binding of the hnRNP can induce the degradation of the RNA nucleic acid molecule. Furthermore, if a miRNA capable of binding to the target sequence is present, the miRNA will hybridize to its target sequence. Therefore, the degradation-inducing sequence cannot bind to the hnRNP, and the degradation of the RNA nucleic acid molecule can be suppressed.

[0080] The binding sequence for the degradation-inducible RNABP may be, for example, an AU-rich element (ARE) sequence (e.g., a UUAUUUAUU motif), an hnRNP binding sequence, etc. The ARE sequence may be, for example, a sequence with a [UUUA] motif as a repeating unit. The number of repeats in the repeating unit may be, for example, 1 to 20, 1 to 8, 1 to 4, or 2 to 4. The repeating unit may be, for example, 1, 2, 3, 6, or 9. The ARE sequence may also be UU. The binding sequence for the hnRNP may be, for example, a sequence with a [UAASUUAU] (S=G or C) motif as a repeating unit. The number of repeats in the repeating unit may be, for example, 1 to 20, 1 to 4, or 2 to 4.

[0081] The binding sequence for the degradation-inducible RNABP may be, for example, the modified sequence described above. In this case, the modified sequence consists of a base sequence having one or more base insertions, additions, substitutions, and / or deletions in the base sequence of the binding sequence, and is a sequence to which the degradation-inducible RNABP can bind. Preferably, the modified sequence consists of a base sequence having one or more base substitutions and / or deletions in the base sequence of the binding sequence, and is a sequence to which the degradation-inducible RNABP can bind. More preferably, the modified sequence consists of a base sequence having one or more base deletions in the base sequence of the binding sequence, and is a sequence to which the degradation-inducible RNABP can bind. For example, the modified sequence consists of a base sequence having 50% or more identity with respect to the base sequence of the binding sequence, and is a sequence to which the degradation-inducible RNABP can bind. The terms "one or several" and "identity" in the modified sequence can be applied to the description of the modified sequence of the sequence capable of forming a stem-loop structure, by substituting "sequence capable of forming a stem-loop structure" with "sequence to which degradation-inducible RNABP can bind."

[0082] In the modified sequence, "binding to degradation-inducible RNABP" means, for example, that the degradation-inducible RNABP may be binding to the modified sequence, or that the degradation-inducible RNABP may be binding to a sequence formed by the modified sequence and other sequences in the degradation-inducible sequence, specifically the sequence at the 5' end of the modified sequence and / or the sequence at the 3' end of the modified sequence.

[0083] The lengths of the decomposition induction sequences can be, for example, 20 to 200 nucleotides, 22 to 180 nucleotides, 24 to 160 nucleotides, 26 to 140 nucleotides, 26 to 120 nucleotides, 26 to 110 nucleotides, 26 to 100 nucleotides, 26 to 90 nucleotides, 28 to 80 nucleotides, 28 to 70 nucleotides, 28 to 60 nucleotides, 28 to 50 nucleotides, 35 to 50 nucleotides, or 30 to 40 nucleotides.

[0084] The aforementioned degradation-inducing sequence may be, for example, a sequence that does not satisfy one or more of the following conditions (1) to (3): (1) a sequence of 20 or more bases that specifically recognizes the PolyA sequence; (2) a sequence that specifically recognizes the 5'UTR in the sequence encoding the target gene; (3) a sequence of 100 bases or more.

[0085] The length of the RNA nucleic acid molecules of this disclosure is not particularly limited and can be determined, for example, according to the length of each sequence. Examples of RNA nucleic acid molecules of this disclosure include lengths of 100 to 13,000 nucleotides, 100 to 12,000 nucleotides, 100 to 11,000 nucleotides, 100 to 10,000 nucleotides, 1,000 to 8,000 nucleotides, 1,000 to 5,000 nucleotides, 1,000 to 3,000 nucleotides, and so on.

[0086] In the RNA nucleic acid molecule of this disclosure, the arrangement of each component is not particularly limited, and is arranged, for example, in such a way that the expression of the target gene can be controlled depending on the presence or absence of miRNA. Specifically, in the RNA nucleic acid molecule of this disclosure, for example, the sequence encoding the target gene, the PolyA sequence, and the degradation induction sequence may be arranged in this order from the 5' end to the 3' end.

[0087] The RNA nucleic acid molecules of this disclosure may include, for example, a cap structure or a cap analogue at their 5' end. When the RNA nucleic acid molecules of this disclosure include a cap structure or a cap analogue at their 5' end, the number of bases between the cap structure or the cap analogue and the sequence encoding the target gene may be 0 to 100 bases, 0 to 50 bases, etc. The cap structure may be, for example, a 5' guanosine cap structure produced using RNA cap analogues such as 3'-O-Me-m7G(5')ppp(5')G[ARCA cap], G(5')ppp(5')A, G(5')ppp(5')G, m7G(5')ppp(5')A, m7G(5')ppp(5')G, m7G(5')ppp(5')G-2'-O-methyl. The aforementioned 5'-guanosine cap structure can be generated using commercially available products sold by companies such as New England BioLabs.

[0088] The RNA nucleic acid molecules of this disclosure may include an internal ribosome entry site (IRES). The IRES may be, for example, a natural IRES or a non-natural IRES. The natural IRES may be, for example, an IRES present in an RNA molecule of an organism (including an organism that can reproduce using multiple cells, such as a virus). The IRES may be, for example, a known IRES that can be appropriately selected and used. When the RNA nucleic acid molecule of this disclosure includes an IRES, the IRES may be, for example, located at the 5' end of the target gene.

[0089] The RNA nucleic acid molecules of this disclosure may have a structure similar to that of mRNA, for example. In this case, the RNA nucleic acid molecules of this disclosure may include one or more regions or portions that act or function as untranslated regions (UTRs) to enhance the stability of the RNA nucleic acid molecule, for example. Specifically, the RNA nucleic acid molecules of this disclosure may include untranslated regions (UTRs) at their ends, such as a 5'UTR and a 3'UTR. The 5'UTR is a region of mRNA located immediately upstream (5') of the start codon (the first codon of the mRNA transcript translated by the ribosome). The 3'UTR is a region of mRNA located immediately downstream (3') of the stop codon (the codon of the mRNA transcript that signals the end of translation).

[0090] The UTR sequence may be a known sequence. The 5'UTR may include, for example, a Kozak sequence. The 5'UTR may be, for example, a 5'UTR from a different cell species than the target cell, or a synthetic 5'UTR. The 5'UTR may be, for example, a 5'UTR derived from a vector plasmid such as pUC19. The RNA nucleic acid molecule of this disclosure may use, for example, an internal ribosome entry site (IRES) instead of a 5'UTR. The 5'UTR preferably does not have, for example, the target sequence of the miRNA. The 5'UTR preferably does not form, or has difficulty forming, the stem-loop structure. The 5'UTR may be, for example, a sequence that does not contain a start codon. The 5'UTR may be, for example, a 5'UTR optimized using machine learning, as described in Reference 16 below. Reference 16: Tang, Xiaoshan et al. “A novel deep generative model for mRNA vaccine development: Designing 5' UTRs with N1-methyl-pseudouridine modification.” Acta pharmaceutica Sinica. B vol. 14,4 (2024): 1814-1826. doi:10.1016 / j.apsb.2023.11.003

[0091] The length of the 5'UTR is, for example, 20 base pairs or more, and specific examples include 30 to 200 base pairs, 40 to 150 base pairs, and 50 to 100 base pairs.

[0092] The 3'UTR may include, for example, AU-rich elements (AREs) such as class I ARE, class II ARE, and class III ARE, or the 3'UTR in the mRNA of the rabies glycoprotein (rabies G mRNA 3'UTR). The 3'UTR may be, for example, a 3'UTR from a different cell species than the target cell, or a synthetic 3'UTR. The UTR may include, for example, regulatory factors. Examples of regulatory factors include promoters, enhancers, internal ribosome entry sites, introns, and poly-U sequences.

[0093] If the RNA nucleic acid molecule of the present disclosure has a structure similar to mRNA, the RNA nucleic acid molecule of the present disclosure may include, for example, the cap structure or cap analogue, the 5'UTR, the sequence encoding the target gene, the 3'UTR, the PolyA sequence, and the degradation induction sequence in this order from the 5' end.

[0094] In the RNA nucleic acid molecule of this disclosure, the sequence encoding the target gene, the PolyA sequence, and the degradation induction sequence, or the cap structure or cap analogue, the 5'UTR, the sequence encoding the target gene, the 3'UTR, the PolyA sequence, and the degradation induction sequence are each directly or indirectly linked, preferably directly. The direct linkage is such that the 3' ribonucleotide residue of each sequence is covalently linked to the 5' ribonucleotide residue of the other sequence. The indirect linkage is such that the 3' ribonucleotide residue of each sequence is covalently linked to the 5' ribonucleotide residue of the other sequence via a linker sequence. The length of the linker sequence may be, for example, 1 to 10 nucleotides. The linker sequence is not particularly limited as long as it is a linker used in the art.

[0095] The RNA nucleic acid molecules of this disclosure may be unmodified RNA nucleic acid molecules or modified RNA nucleic acid molecules. If the RNA nucleic acid molecules of this disclosure are unmodified RNA nucleic acid molecules, they consist of unmodified nucleotide residues, for example, native nucleotide residues. If the RNA molecules of this disclosure are modified RNA nucleic acid molecules, they consist of modified nucleotide residues, or modified nucleotide residues and unmodified nucleotide residues. If the RNA nucleic acid molecules of this disclosure consist of modified nucleotide residues and unmodified nucleotide residues, the proportion of the modified nucleotide residues is 1% or more, 5% or more, 10% or more, 20% or more, 25% or more, 30% or more, 35% or more, 40% or more, 45% or more, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, or 75% or more, relative to the total length of the RNA nucleic acid molecule of this disclosure. The proportion of the unmodified nucleotide residues is 25% or more, 30% or more, 35% or more, 40% or more, 45% or more, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 80% or more, 90% or more, 95% or more, and 99% or more, relative to the total length of the RNA nucleic acid molecule of this disclosure.

[0096] In the nucleotide residue, the base is not particularly limited and may be a natural base or a non-natural base. For example, a common base, a modified analog thereof, etc., can be used.

[0097] Examples of the aforementioned bases include purine bases such as adenine and guanine, and pyrimidine bases such as cytosine, uracil, and thymine. Other examples of the aforementioned bases include inosine, thymine, xanthine, hypoxanthine, nubularine, isoguanisine, and tubercidine. Examples of the aforementioned bases include alkyl derivatives such as 2-aminoadenine and 6-methylated purine; alkyl derivatives such as 2-propylated purine; 5-halouracil and 5-halocytosine; 5-propynyluracil and 5-propynylcytosine; 6-azouracil, 6-azocytosine, and 6-azothimine; 5-uracil (pseudouracil), 4-thiouracil, 5-halouracil, 5-(2-aminopropyl)uracil, and 5-aminoallylu Racil; 8-halo-, amin-, thio-, thio-alkyl-, hydroxy-, and other 8-substituted purines; 5-trifluoromethyl- and other 5-substituted pyrimidines; 7-methylguanine; 5-substituted pyrimidines; 6-azapyrimidines; N-2, N-6, and O-6 substituted purines (including 2-aminopropyladenine); 5-propynyluracil and 5-propynylcytosine; dihydrouracil; 3-deaza-5-azacytosine; 2- Aminopurine; 5-alkyluracil; 7-alkylguanine; 5-alkylcytosine; 7-deazaadenine; N6,N6-dimethyladenine; 2,6-diaminopurine; 5-amino-allyl-uracil; N3-methyluracil; substituted 1,2,4-triazole; 2-pyridinone; 5-nitroindole; 3-nitropyrrole; 5-methoxyuracil; uracil-5-oxyacetic acid; 5-methoxycarbonylmethyluracil; 5-methyl- Examples include 2-thiouracil; 5-methoxycarbonylmethyl-2-thiouracil; 5-methylaminomethyl-2-thiouracil; 3-(3-amino-3-carboxypropyl)uracil; 3-methylcytosine; 5-methylcytosine; N4-acetylcytosine; 2-thiocytosine; N6-methyladenine; N6-isopentyladenine; 2-methylthio-N6-isopentenyladenine; N-methylguanine; and O-alkylated bases.The purines and pyrimidines include, for example, those disclosed in U.S. Patent No. 3,687,808, "Concise Encyclopedia of Polymer Science and Engineering," pp. 858–859, edited by Kroschwitz JI, John Wiley & Sons, 1990, and English et al., Angewandte Chemie, International Edition, 1991, Vol. 30, p. 613.

[0098] The nucleotide residue may or may not be modified. Examples of modified nucleotide residues include 5-methyluridine (m5U), 2-thiouridine (s2U), 5-methoxyuridine (mo5U), 2'-O-methyluridine (Um), pseudouridine (ψ), N1-methylpseudridine (m1ψ), 5-methylcytosine (m5C), N6-methyladenosine (m6A), and N1-methyladenosine (m1A). When the base is U, the modified base is preferably N1-methylpseudridine or pseudouridine, and more preferably N1-methylpseudridine.

[0099] The modified nucleotide residues may also include, for example, residues that delete a base, i.e., a baseless ribophosphate skeleton. The modified nucleotide residues may be, for example, those described in International Publication No. 2004 / 080406.

[0100] The RNA nucleic acid molecules of this disclosure may include, for example, a labeling substance. Examples of the labeling substance include fluorescent substances, dyes, isotopes, etc. Examples of the labeling substance include fluorophores such as pyrene, TAMRA, fluorescein, Cy3 dyes, and Cy5 dyes, and examples of the dye include Alexa dyes such as Alexa 488. Examples of the isotope include stable isotopes and radioactive isotopes, and stable isotopes are preferred. Stable isotopes have advantages such as low risk of exposure, no need for dedicated facilities, excellent handling properties, and reduced costs. Furthermore, stable isotopes do not alter the physical properties of the labeled compound and have excellent tracer properties. Examples of stable isotopes include, ２ H, １３ C, １５ N, １７ O, １８ O, ３３ S, ３４ S, ３６ S is one example.

[0101] The RNA nucleic acid molecules of this disclosure can be produced, for example, by an in vitro transcription system based on the base sequence constituting the RNA nucleic acid molecule. In this case, the RNA nucleic acid molecules of this disclosure can be synthesized, for example, using a DNA nucleic acid molecule encoding the RNA nucleic acid molecule of this disclosure as a template, and using a phage RNA polymerase derived from a T7 phage, T3 phage, SP6 phage, etc. The in vitro transcription may be carried out, for example, by the methods described in International Publication No. 2014 / 152027, International Publication No. 2018 / 053209, International Publication No. 2019 / 036682, etc.

[0102] <DNA Nucleic Acid Molecules> In another embodiment, the Disclosure provides DNA nucleic acid molecules that encode the RNA nucleic acid molecules of the Disclosure. The DNA nucleic acid molecules of the Disclosure encode the RNA nucleic acid molecules of the Disclosure. The RNA nucleic acid molecules of the Disclosure can be produced, for example, by in vitro transcription, using the DNA nucleic acid molecules of the Disclosure. Furthermore, the vectors of the Disclosure, as described below, can be produced, for example, using the DNA nucleic acid molecules of the Disclosure.

[0103] The DNA nucleic acids of this disclosure may be functionally ligated to a vector, such as a plasmid vector.

[0104] The DNA nucleic acid in this disclosure may be a single-stranded nucleic acid molecule or a double-stranded nucleic acid molecule.

[0105] The DNA nucleic acid of this disclosure can be designed by replacing the base sequence of the RNA nucleic acid molecule of this disclosure with the base sequence of the corresponding deoxynucleotide. The nucleic acid of this disclosure may also be obtained by, for example, the following method: First, cDNA is synthesized using the RNA nucleic acid molecule of this disclosure and reverse transcriptase. The obtained cDNA can be amplified using appropriate primers, and the base sequence information of the DNA can be determined from the amplified fragment. Alternatively, the base sequence encoding the RNA nucleic acid molecule of this disclosure can be obtained by chemical synthesis from the obtained base sequence information.

[0106] The DNA nucleic acids described herein can be synthesized, for example, by genetic engineering or organic synthesis methods, and can also be referred to as synthetic DNA such as cDNA.

[0107] <Vectors> In another embodiment, the Disclosure provides a vector comprising the DNA nucleic acid molecule of the Disclosure. The vector of the Disclosure comprises the DNA nucleic acid molecule of the Disclosure. The vector of the Disclosure allows for the suitable production of the RNA nucleic acid molecule of the Disclosure by genetic engineering methods. Furthermore, the vector of the Disclosure allows for the expression of the RNA nucleic acid molecule of the Disclosure, for example, in vivo.

[0108] The vector of this disclosure, for example, has the DNA nucleic acid molecule of this disclosure inserted into it. The vector can also be described as having the DNA nucleic acid molecule functionally linked to it. The vector means a nucleic acid molecule that can transport, for example, an inserted polynucleotide such as a gene into a target such as a cell.

[0109] The vector of this disclosure may, for example, contain a polynucleotide encoding the RNA nucleic acid molecule so as to be able to express the RNA nucleic acid molecule encoded by the polynucleotide of the DNA nucleic acid molecule of this disclosure, and its composition is not particularly limited.

[0110] The vectors of this disclosure can be prepared, for example, by inserting a polynucleotide encoding the RNA nucleic acid molecule, i.e., the DNA nucleic acid molecule of this disclosure, into a skeletal vector (hereinafter also referred to as the "basic vector"). The type of vector is not particularly limited and can be appropriately determined, for example, depending on the type of host (host cell) or the target of the vector administration. Specifically, when synthesizing the expression vector by genetic engineering, the synthesis of the vector first involves, for example, designing and synthesizing a DNA nucleic acid molecule encoding the RNA nucleic acid molecule. The design and synthesis can be carried out, for example, by PCR using a vector containing the DNA nucleic acid molecule encoding the RNA nucleic acid molecule as a template and primers designed to synthesize a desired nucleic acid region. Then, a recombinant vector (vector) can be obtained by ligating the obtained DNA nucleic acid molecule into a suitable vector. The vector may be introduced into a host, for example, to amplify the vector. In this case, the vector can be used to produce transformants, for example, by introducing it into a host (Sambrook J. et al., Molecular Cloning, A Laboratory Manual (4th edition) (Cold Spring Harbor Laboratory Press (2012))).

[0111] The host used for transformation is not particularly limited as long as it can amplify the vector, and examples include non-human hosts such as microorganisms, animal cells, insect cells, or cultured cells thereof, isolated human cells or their cultured cells, mammalian cells, etc. Examples of prokaryotes include bacteria such as Escherichia coli and Pseudomonas putida. Examples of eukaryotes include yeasts such as Saccharomyces cerevisiae. Examples of animal cells include HEK293A cells, HEK293T cells, CHO cells, COS cells, Vero cells, HeLa cells, L929 cells, BALB / c3T3 cells, C127 cells, NIH3T3 cells, etc., and examples of insect cells include Sf9, Sf21, etc.

[0112] The vectors of this disclosure include viral vectors and non-viral vectors. When transforming a host using the vector, a non-viral vector such as a plasmid vector can be used. Examples of such vectors include plasmid vectors such as binary vectors. Examples of such vectors include pETDuet-1, pQE-80L, and pUCP26Km. When transforming bacteria such as E. coli, examples of expression vectors include pETDuet-1 vector (Novagen), pQE-80L (QIAGEN), pBR322, pB325, pAT153, and pUC8. When transforming yeast, examples of expression vectors include pYepSec1, pMFa, and pYES2. When transforming insect cells, examples of expression vectors include pAc and pVL. When performing transformation on the aforementioned mammalian cells, the expression vectors include, for example, pCDM8 and pMT2PC.

[0113] Examples of the aforementioned viral vectors include retroviral vectors, lentiviral vectors, adenovirus vectors, adeno-associated virus vectors, vaccinia virus vectors, poxvirus vectors, herpes simplex virus vectors, mouse leukemia virus vectors, and hybrid viral vectors.

[0114] The vectors of this disclosure preferably have a regulatory sequence that regulates the expression of a DNA nucleic acid molecule encoding the RNA nucleic acid molecule. Examples of the regulatory sequence include a promoter, terminator, enhancer, polyadenylation signal sequence, origin of replication sequence (ORI), etc. The arrangement of the regulatory sequence in the vector is not particularly limited. In the vector, the regulatory sequence only needs to be arranged so as to functionally regulate the expression of the DNA nucleic acid molecule encoding the RNA nucleic acid molecule and the expression of the antigen peptide encoded therein, and can be arranged according to known methods. The regulatory sequence may, for example, utilize a sequence already present in the vector, or the regulatory sequence may be further inserted into the vector, or the regulatory sequence present in the basic vector may be replaced with another regulatory sequence.

[0115] If the vector is a viral vector, it can be prepared, for example, by introducing all the nucleic acid molecules necessary for the production of the viral vector into the host. Specifically, the viral vector may be prepared by introducing the remaining nucleic acid molecules necessary for the production of the viral vector into a host that has already been introduced some of the nucleic acid molecules necessary for the production of the viral vector. Alternatively, the viral vector may be prepared, for example, by introducing the nucleic acid molecules to be delivered by the viral vector, and optionally the remaining nucleic acid molecules necessary for the production of viral particles, into a host cell (packaging cell) that has already been introduced some or all of the nucleic acid molecules necessary for the production of viral particles. Examples of packaging cells include cells into which retrovirus Gag and / or Pol and / or envelope proteins have been introduced, and cells into which the adenovirus E1A and / or E1B regions have been introduced. As the packaging cells, commercially available cells suitable for viral vector production may be used, for example, Plat-E cells (CosmoBio, #RV-101) and Lenti-X293T cells (Takara, #Z2180N) can be used.

[0116] <Lipid-Nucleic Acid Complexes> In another embodiment, the present disclosure provides lipid-nucleic acid complexes. The lipid-nucleic acid complexes of the present disclosure comprise a nucleic acid molecule of the present disclosure and a lipid. The lipid-nucleic acid complexes of the present disclosure are expected to facilitate the delivery of the nucleic acid molecule of the present disclosure to target cells.

[0117] Examples of the lipids include lipid nanoparticles, liposomes, and extracellular vesicles (EVs). When the lipids are lipid nanoparticles, the lipid nucleic acid complex is a nucleic acid lipid nanoparticle. In the lipid nucleic acid complex of this disclosure, it is preferable that the nucleic acid molecule is encapsulated in the lipid, for example.

[0118] The lipid nanoparticles may include, for example, cationic lipids, non-cationic lipids, sterols (cholesterol), PEG lipids, etc. Examples of non-cationic lipids include neutral lipids, anionic lipids, amphiphilic lipids, etc. Specific examples of each lipid constituting the lipid nanoparticles, their composition, and methods for producing them can be found, for example, in International Publication No. 2009 / 127060.

[0119] The lipid nucleic acid complex may be modified or altered, for example, to target specific cells. The specific cells are, for example, target cells in the RNA nucleic acid molecules of this disclosure. The lipid nucleic acid complex may be further targeted, for example, to a specific type or class of cells among the target cells. The nucleic acid lipid complex of this disclosure can be specifically transported to a specific type or class of cells among the target cells, for example, by the targeting. Targeting of the lipid nucleic acid complex can be carried out, for example, by adding a specific cell-specific molecule (e.g., protein, glycan, etc.), preferably a binding molecule that can bind to a cell surface protein specific to the specific cell, to the lipid or lipid nanoparticles. In this case, the lipid nucleic acid complex further includes, for example, a binding molecule that can bind to the target cells, and the lipid or lipid nanoparticles have the binding molecule outside the lipid. The specific molecule can be appropriately set, for example, depending on the type of cell. Specifically, if the particular cell is a T cell, the specific molecule may be, for example, the cell surface proteins of the T cell, such as CD3, CD4, CD5, and CD8 (References 11-15). Reference 11: Tombacz, Istvan et al. “Highly efficient CD4+ T cell targeting and genetic recombination using engineered CD4+ cell-homing mRNA-LNPs.” Molecular therapy: the journal of the American Society of Gene Therapy vol. 29,11 (2021): 3293-3304. doi:10.1016 / j.ymthe.2021.06.004 Reference 12: Rurik, Joel G et al. “CAR T cells produced in vivo to treat cardiac injury.” Science (New York, NY) vol. 375,6576 (2022): 91-96. doi:10.1126 / science.abm0594 Reference 13: Tilsed, Caitlin M et al. “IL7 increases targeted lipid nanoparticle-mediated mRNA expression in T cells in vitro and in vivo by enhancing T cell protein translation.” Proceedings of the National Academy of Sciences of the United States of America vol. 121,13 (2024): e2319856121. doi:10.1073 / pnas.2319856121 Reference 14: Robinson, Elise R., et al. "CD3 and CD8 targeting of ionizable lipid nanoparticles for in vivo mRNA delivery to T cells." Cancer Research 82.12_Supplement (2022): 5372-5372. Reference 15: Adams, Gregor, et al. "In vivo engineering of CAR T cells using a novel targeted LNP-mRNA technology." J Immunother Cancer 11.suppl 1 (2023): A1-731.

[0120] Examples of the cationic lipids include cationic lipids containing primary, secondary, or tertiary amines, such as 1,2-dioleyloxy-N,N-dimethylaminopropane (DODMA), 1,2-distearyloxy-N,N-dimethylaminopropane (DSDMA), 3-(N-(N',N'-dimethylaminoethane)-carbamoyl)cholesterol (DC-Chol), and 2,3-dioleyloxy-N-[2(spermine-carboxamide)ethyl]-N,N-dimethyl-1-propaneamine. Umtrifluoroacetate (DOSPA), dioctadecylamideglycylspermine (DOGS), 1,2-dilinoleyloxy-N,N-dimethyl-3-aminopropane (DLinDMA), 2-(2,2-di((9Z,12Z)-octadeca-9,12-dien-1-yl)-1,3-dioxolan-4-yl)-N,N-dimethylethanolamine (Dlin-KC2-DMA), (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl Examples include 4-(dimethylamino)butanoate (Dlin-MC3-DMA), 3,6-bis(4-(bis(2-hydroxydodecyl)amino)butyl)piperazine-2,5-dione (cKK-E12), heptadecan-9-yl 8-((2-hydroxyethyl)(6-oxo-6-(undecyloxy)hexyl)amino)octanoate (SM-102), [(4-hydroxybutyl)azandiyl]bis(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-0315), and mixtures thereof. Furthermore, the cationic lipids include, for example, cationic lipids containing quaternary ammonium compounds, such as N-(1-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA), N-(1,2-dimyristiloxypropane-3-yl)-N,N-dimethyl-N-hydroxyethylammonium bromide (DMRIE), N,N-dioleyl-N,N-dimethylammonium chloride (DODAC), N,N-distearyl-N,N-dimethylammonium bromide (DDAB), N-(1-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP), and mixtures thereof.Alternatively, a mixture of a cationic lipid containing a primary, secondary, or tertiary amine and a cationic lipid containing a quaternary ammonium can be used.

[0121] The aforementioned noncationic lipids are 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dilinoleoyl-sn-glycero-3-phosphocholine, 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-diundecanoyl-sn-glycero-3-phosphocholine (DUPC), and 1-palmitoyl-2-oleoyl- Examples include sn-glycero-3-phosphocholine (POPC), egg yolk phosphatidylcholine (EPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt (DOPG), sphingomyelin, and mixtures thereof.

[0122] Examples of PEG-modified lipids include PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, PEG-modified dialkylglycerol, DMG-PEG, PEG-c-DOMG (also called PEG-DOMG), PEG-DSG, PEG-DPG, and mixtures thereof.

[0123] Examples of the sterols mentioned above include cholesterol, fecosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatidine, ursolic acid, alpha-tocopherol, and mixtures thereof.

[0124] <Pharmaceutical Compositions> In another embodiment, the Disclosure provides pharmaceutical compositions. The pharmaceutical compositions of the Disclosure comprise a nucleic acid molecule and / or a lipid nucleic acid complex of the Disclosure and a pharmaceutically acceptable carrier. The pharmaceutical compositions of the Disclosure are expected to induce the expression of a target gene in cells expressing a target miRNA, thereby enabling suitable treatment for specific diseases.

[0125] The pharmaceutical compositions of this disclosure may further include pharmaceutically acceptable carriers. Examples of such carriers include suspensions for administering the active ingredient, solubilizers, stabilizers, isotonic agents, preservatives, anti-adsorption agents, surfactants, diluents, media, pH adjusters, analgesics, buffers, sulfur-containing reducing agents, antioxidants, etc., and can be appropriately added insofar as they do not interfere with the effects of this disclosure.

[0126] The suspending agent is not particularly limited and includes, for example, methylcellulose, polysorbate 80, hydroxyethylcellulose, gum arabic, tragacanth powder, sodium carboxymethylcellulose, polyoxyethylene sorbitan monolaurate, and the like.

[0127] The aforementioned solution additive is not particularly limited and includes, for example, polyoxyethylene hydrogenated castor oil, polysorbate 80, nicotinamide, polyoxyethylene sorbitan monolaurate, macrogol, castor oil fatty acid ethyl ester, and the like.

[0128] The stabilizer is not particularly limited and examples include dextran 40, methylcellulose, gelatin, sodium sulfite, sodium metasulfate, and the like.

[0129] The isotonic agent is not particularly limited, and examples include D-mannitol and sorbitol.

[0130] The preservative is not particularly limited and examples include methyl parahydroxybenzoate, ethyl parahydroxybenzoate, sorbic acid, phenol, cresol, chlorocresol, and the like.

[0131] The adsorption inhibitor is not particularly limited and includes, for example, human serum albumin, lecithin, dextran, ethylene oxide propylene oxide copolymer, hydroxypropyl cellulose, methylcellulose, hydrogenated castor oil, polyethylene glycol, and the like.

[0132] The sulfur-containing reducing agent is not particularly limited and can be, for example, N-acetylcysteine, N-acetylhomocysteine, thioxytoic acid, thiodiglycol, thioethanolamine, thioglycerol, thiosorbitol, thioglycolic acid and its salts, sodium thiosulfate, glutathione, thioalkanoic acid having 1 to 7 carbon atoms, or any other substance having a sulfohydryl group.

[0133] The antioxidant is not particularly limited, and examples include erythorbic acid, dibutylhydroxytoluene, butylhydroxyanisole, α-tocopherol, tocopherol acetate, L-ascorbic acid and its salts, L-ascorbic acid palmitate, L-ascorbic acid stearate, sodium bisulfite, sodium sulfite, triamyl gallate, propyl gallate, or chelating agents such as ethylenediaminetetraacetate (EDTA), sodium pyrophosphate, and sodium metaphosphate.

[0134] The pharmaceutical composition of the present invention may further contain, as appropriate, commonly added components such as inorganic salts like sodium chloride, potassium chloride, calcium chloride, sodium phosphate, potassium phosphate, and sodium bicarbonate; organic salts like sodium citrate, potassium citrate, and sodium acetate; and sugars like glucose.

[0135] The pharmaceutical compositions of this disclosure may be used, for example, in vitro or in vivo. The pharmaceutical compositions of this disclosure may also be used, for example, as research reagents or as pharmaceuticals. In the former case, the pharmaceutical compositions of this disclosure may also be called test reagents or test kits.

[0136] The subjects to whom the pharmaceutical compositions of this disclosure are administered are not particularly limited. When the pharmaceutical compositions of this disclosure are used in vivo, the subjects (subjects to administration) include, for example, humans and non-human animals. Examples of non-human animals include mammals such as mice, rats, rabbits, dogs, cats, cattle, horses, pigs, monkeys, dolphins, and sea lions. When the pharmaceutical compositions of this disclosure are used in vitro, the subjects to administration include, for example, cells, tissues, organs, etc., where examples of cells include cells collected from living organisms, cultured cells, etc., and examples of tissues or organs include tissues (living tissues) or organs collected from living organisms, etc. The aforementioned cells are any cells that express miRNA, and include, for example, immune cells such as T cells, B cells, NK cells, and dendritic cells; somatic cells such as hepatocytes (hepatocytes), epithelial cells, and fibroblasts; stem cells such as embryonic stem (ES) cells, induced pluripotent stem (iPS) cells, embryonic cancer (EC) cells, and embryonic germ stem (EG) cells; progenitor cells such as hematopoietic progenitor cells, neural progenitor cells, and adipocyte progenitor cells; and so on.

[0137] When the pharmaceutical composition of this disclosure is used in vivo, the recipient may be a healthy person not suffering from a specific disease, a person who may be suffering from a specific disease, or a patient suffering from a specific disease, but it is preferable that the recipient is someone for whom treatment of a specific disease is desired.

[0138] The conditions for use (administration conditions) of the pharmaceutical composition disclosed herein are not particularly limited, and the administration form, timing of administration, dosage, etc. can be appropriately set depending on the type of active ingredient in the pharmaceutical composition, the type of target for administration, etc.

[0139] Methods of administering the pharmaceutical composition of this disclosure include, for example, intracerebral administration, intrathecal administration, intramuscular administration, subcutaneous administration, intravenous administration, and the like.

[0140] The dosage of the pharmaceutical composition disclosed herein is the amount that can induce the expression of the target gene in the recipient, i.e., the effective dose. The dosage can be appropriately determined, for example, by the age, weight, symptoms, etc., of the recipient.

[0141] The number of administrations of the pharmaceutical composition disclosed herein is one or more times. The number of administrations is, for example, two, three, four, five or more times. The number of administrations may be determined as appropriate while confirming the preventive effect on the target of administration. When multiple administrations are performed, the administration interval can be determined as appropriate while confirming the therapeutic effect on the target of administration, and examples include once a day, once a week, once every two weeks, once a month, once every three months, once every six months, etc.

[0142] The pharmaceutical compositions disclosed herein can prevent or alleviate at least one symptom caused by a specific disease in the subject of administration. The prevention of such symptoms can be evaluated subjectively or objectively, and specific examples include self-assessment by the subject of administration; assessment by a physician; QOL (Quality of Life) assessment; and assessment of delay in the progression of symptoms of the specific disease or alleviation of symptoms of the specific disease. The objective assessment may be conducted by animals or by humans.

[0143] <First Method for Inducing Expression> In another embodiment, the present disclosure provides a method for inducing the expression of a target gene (hereinafter also referred to as the "first method for inducing expression"). The first method for inducing expression of the present disclosure induces the expression of a target gene by introducing the nucleic acid molecule and / or the lipid nucleic acid complex of the present disclosure into cells expressing a target miRNA. The first method for inducing expression of the present disclosure is expected to suitably induce the expression of a target gene in cells expressing a target miRNA. The first method for inducing expression of the present disclosure can be described by reference to the descriptions of the nucleic acid molecule, lipid nucleic acid complex and pharmaceutical composition of the present disclosure.

[0144] The introduction method is not particularly limited and can be carried out by known methods. The introduction method can be appropriately set according to the type of cell, for example. Examples of the introduction method include introduction by gene gun such as a particle gun, calcium phosphate method, polyethylene glycol method, lipofection method using liposomes, electroporation method, ultrasonic nucleic acid introduction method, DEAE-dextran method, direct injection method using microglass tubes, hydrodynamic method, cationic liposome method, method using introduction aids, method via Agrobacterium, protoplast method, etc. Examples of liposomes include lipofectamine and cationic liposomes, and examples of introduction aids include atelocollagen, nanoparticles and polymers.

[0145] After the introduction, the cells may be cultured. The culture method can be carried out by known methods and can be appropriately set according to the type of cells.

[0146] <Second Method for Inducing Expression> In another embodiment, the present disclosure provides a method for inducing the expression of a target gene in cells expressing a target miRNA of interest (hereinafter also referred to as the "second method for inducing expression"). The second method for inducing expression of the present disclosure includes the step of administering to a subject the nucleic acid molecule and / or the lipid nucleic acid complex of the present disclosure. The second method for inducing expression of the present disclosure is expected to suitably induce the expression of a target gene in cells expressing a target miRNA of interest. The second method for inducing expression of the present disclosure can be described by reference to the descriptions of the nucleic acid molecule, lipid nucleic acid complex, pharmaceutical composition and the first method for inducing expression of the present disclosure.

[0147] <Screening Method> In another embodiment, the Disclosure provides a screening method (hereinafter also referred to as the "Screening Method") for selecting cells expressing a target miRNA. The Screening Method of the Disclosure selects cells expressing a target miRNA by introducing the RNA nucleic acid molecule of the Disclosure, the DNA nucleic acid molecule of the Disclosure, the vector of the Disclosure, and / or the lipid nucleic acid complex of the Disclosure into cells. The Screening Method of the Disclosure is expected to be able to distinguish between cells expressing a specific miRNA and cells that do not express the specific miRNA. The Screening Method of the Disclosure can be described by reference to the descriptions of the nucleic acid molecule, lipid nucleic acid complex, pharmaceutical composition, first expression induction method, and second expression induction method of the Disclosure.

[0148] <Treatment Method> In another embodiment, the present disclosure provides a method for treating a disease (hereinafter also referred to as the "treatment method"). The treatment method of the present disclosure comprises the step of administering to a patient a nucleic acid molecule and / or a lipid nucleic acid complex of the present disclosure, wherein the target gene is the patient's disease-causing gene and / or genome editing-related gene, and the miRNA is a miRNA expressed in the patient's disease-causing cell. The treatment method of the present disclosure is expected to allow for suitable treatment of a specific disease. The treatment method of the present disclosure can be described by reference to the descriptions of the nucleic acid molecule, lipid nucleic acid complex, pharmaceutical composition, first expression induction method and second expression induction method of the present disclosure.

[0149] <Use> In another embodiment, the Disclosure relates to the use of the nucleic acid molecules and / or lipid nucleic acid complexes of the Disclosure for inducing the expression of a target gene in cells expressing a target miRNA. The use of the nucleic acid molecules and / or lipid nucleic acid complexes of the Disclosure can be made by reference to the descriptions of the nucleic acid molecules, lipid nucleic acid complexes, pharmaceutical compositions, first expression induction method, second expression induction method, and treatment method of the Disclosure.

[0150] Next, examples of the present disclosure will be described. However, the present disclosure is not limited to the following examples. Commercial reagents were used according to their protocols unless otherwise specified. Note that "mol / l" may also be denoted as "M".

[0151] [Example 1] (1) Examination of degradation induction sequences We investigated whether the stem-loop structure is important for the degradation induction sequence of RNA nucleic acid molecules. Specifically, we first constructed an RNA nucleic acid molecule containing the target sequence and pAB sequence of miR-122-5p (5'-UGGAGUGUGACAAUGGUGUUUG-3': SEQ ID NO: 9). The RNA nucleic acid molecule was designed so that from the 5' end to the 3' end, it would have a cap structure, a 5' UTR, a sequence encoding the target gene, firefly luciferase (FLuc, SEQ ID NO: 10), a 3' UTR, a poly(A) (90 bases) sequence, a target sequence, and a pAB sequence (SEQ ID NO: 1) (122-5p ON(pAB) (SEQ ID NO: 15)). Next, for the 122-5p ON (pAB) sequence, instead of the pAB sequence, a histone stem-loop (HSL) sequence, shown underlined and capable of forming a stem-loop structure similar to the pAB sequence, was applied to construct an RNA nucleic acid molecule having an HSL sequence (HSL+0 (SEQ ID NO: 16)). RNA nucleic acid molecules were also constructed in which five or ten sequences, shown in bold and underlined, were inserted between the 3' end of the target sequence and the 5' end of the HSL sequence (HSL+5 (SEQ ID NO: 17), HSL+10 (SEQ ID NO: 18)). Table 1 below shows the various degradation-inducing sequences (target sequence and pAB sequence or HSL sequence) of the RNA nucleic acid molecules, and Table 2 below shows the full-length sequences of each RNA nucleic acid molecule. An RNA nucleic acid molecule (Control), similar to the above RNA nucleic acid molecules except for the absence of degradation-inducing sequences, was also constructed in the same manner.

[0152]

[0153]

[0154]

[0155] The expression of target genes by each RNA nucleic acid molecule shown in Table 1 was investigated. Specifically, first, human alveolar basal epithelial adenocarcinoma cells (A549 cells; cells with low activity of endogenous miR-122-5p) were introduced using Lipofectamine® RNAiMAX Reagent (Thermo Fisher Scientific) with either 122-5p mimic (mirVana® miRNA mimic, Assay name: hsa-miR-122-5p, Assay ID: MC11012, Thermo Fisher Scientific) or a negative control (NC mimic; mirVana® miRNA Mimic, Negative Control #1 (Cat No: 4464058, Thermo Fisher Scientific)). The introduction of the miRNA mimic was carried out according to the protocol to achieve a final concentration of 10 nM miRNA mimic. 24 hours after the introduction of the miRNA mimic, each RNA nucleic acid molecule constructed above was introduced using Lipofectamine® MessengerMAX® (Thermo Fisher Scientific). The 122-5p mimic or NC mimic was introduced into the A549 cells, which had been introduced using a protocol (96-well plate format) with a scientific (Ph.A. Scientific) product. After introduction, a luciferase assay was performed using the ONE-Glo® EX Luciferase Assay System (Promega) according to the protocol. Luminescence in the luciferase assay was measured using a GloMax® (Promega) luminescence analyzer with an exposure time set to 0.1–1 seconds / well. These results are shown in Figure 2.

[0156] Figure 2 is a graph showing the results of the luciferase assay. In Figure 2, the vertical axis represents the relative value of the luminescence intensity (with the luminescence intensity of Control set to 1), and the horizontal axis represents the type of RNA nucleic acid molecule. In Figure 2, the numerical values ​​at the top of the graph show the fold change in the presence of 122-5p mimic (122-5p mimic) compared to the absence of 122-5p mimic (NC mimic). As shown in Figure 2, it was found that the luminescence intensity of luciferase increased in the presence of miR-122-5p compared to the absence of miR-122-5p, not only for RNA nucleic acid molecules with pAB sequences but also for RNA nucleic acid molecules with HSL sequences. These results suggest that, when the degradation-inducing sequence of the RNA nucleic acid molecule presents is capable of forming a stem-loop structure, the RNA nucleic acid molecule presents can express the target gene, firefly luciferase, in the presence of a miRNA capable of binding to the target sequence of the RNA nucleic acid molecule presents, and thus function as an ON switch.

[0157] (2) Investigation of RNA nucleic acid molecule degradation The degradation of RNA nucleic acid molecules in the absence of target miRNA was investigated. Specifically, semi-quantitative PCR was performed on 122-5p ON (pAB) constructed in Example 1 (1) above. Specifically, in the semi-quantitative PCR method, first, 122-5p mimic or negative control (NC mimic) was introduced into A549 cells in the same manner as in Example 1 (1) above. 24 hours after the introduction, 2.5 μg / well of the RNA nucleic acid molecule (122-5p ON (pAB)) constructed in Example 1 (1) above was introduced into the A549 cells using Lipofectamine® MessengerMAX®. Six hours after the introduction, total RNA was extracted from the cells according to the protocol using TRIzol RNA Isolation Reagents (Thermo Fisher Scientific) and RNA Clean & Concentrator™-5 (ZYMO RESEARCH). After extraction, the total RNA was reverse transcribed according to the protocol using SuperScript™ IV Reverse Transcriptase (Thermo Fisher Scientific) and a primer that recognizes the 3' UTR sequence of RNA nucleic acid molecules (SEQ ID NO: 19). The amount of total RNA used as a template was equal for each sample. After reverse transcription, the obtained cDNA was diluted 10-fold and used as a template for PCR using Platinum™ SuperFi™ PCR Master Mix (Thermo Fisher Scientific) and the following primer set (final concentration 0.2 μM) under the thermal cycler conditions shown in Table 3 below. In the aforementioned PCR, the PCR product was separated every 10 cycles and quantified using the Qubit™ 1X dsDNA HS Assay Kit (Thermo Fisher Scientific). The degradation of the RNA nucleic acid molecule (Control), which is similar to the RNA nucleic acid molecule except that it does not have a degradation-inducing sequence, was also investigated using the same method. These results are shown in Figure 3.

[0158] - Primer that recognizes the 3' UTR sequence of RNA nucleic acid molecules (SEQ ID NO: 19) 5'-GCTTTATTCAAAGACCAAGAGG-3' - PCR primer set Forward for FLuc: 5'-GCAAGACCATGACCGAGAAG-3' (SEQ ID NO: 20) Reverse for FLuc: 5'-CCCTTCTTGGCCTTGATCAG-3' (SEQ ID NO: 21)

[0159]

[0160] Figure 3 is a graph showing the results of semi-quantitative PCR. In Figure 3, the vertical axis represents the concentration of RNA nucleic acid molecules (ng / μl), and the horizontal axis represents the number of PCR cycles. As shown in Figure 3, it was found that the concentration of RNA nucleic acid molecules decreased in the absence of miR-122-5p compared to the presence of miR-122-5p. From these results, it was found that RNA nucleic acid molecules are degraded in the absence of the target miRNA.

[0161] (3) Examination of each loop structure In this example 1 (3), the polyA sequence of the various RNA nucleic acid molecules used was set to 109 bases. Stem-loop binding proteins (SLBPs) are known to bind to stem-loops and suppress the degradation of histone mRNA by human 3' exonuclease (3'hExo) (see reference 4 below). It is also known that introducing a mutation into a part of the HSL sequence reduces the ability of SLBPs to bind to stem-loops (see reference 4 below). Therefore, we investigated whether the function of 3'hExo could be maximized by suppressing the ability of SLBPs to bind to stem-loops. Specifically, we constructed an RNA nucleic acid molecule (122-5p ON (HSL) (SEQ ID NO: 26)) that is similar to the RNA nucleic acid molecule with an HSL sequence constructed in Example 1 (1) above (122-5p ON (HSL+0)), but with a PolyA length of 109 bases. Next, we constructed an HSL mutation (SEQ ID NO: 27) in which a mutation was introduced into a part of the HSL sequence, and an HSL deletion (SEQ ID NO: 28) in which a part of the HSL sequence was deleted to the extent that a stem-loop was formed. We also constructed an RNA nucleic acid molecule in which a tetraloop sequence, which has been reported as a sequence that does not bind SLBP, was applied instead of the pAB sequence of 122-5p ON (pAB) constructed in Example 1(1) (TL, SEQ ID NO: 29). Furthermore, we constructed an RNA nucleic acid molecule in which the six sequences shown in bold and underlined were inserted between the 3' end of the target sequence and the 5' end of the TL sequence (TL+linker, SEQ ID NO: 30). Table 4 below shows the degradation induction sequences of various RNA nucleic acid molecules, and Table 5 below shows the full-length sequences of each RNA nucleic acid molecule. An RNA nucleic acid molecule (Control) that is the same as the above RNA nucleic acid molecule except that it does not have a degradation induction sequence was also constructed in the same way. Reference 4: Dominski Z, Yang XC, Kaygun H, Dadlez M, Marzluff WF. A 3' exonuclease that specifically interacts with the 3' end of histone mRNA. Mol Cell. 2003 Aug;12(2):295-305. doi: 10.1016 / s1097-2765(03)00278-8. PMID: 14536070.

[0162]

[0163]

[0164] The various RNA nucleic acid molecules mentioned above were introduced into cells and subjected to luciferase assays using the same method as in Example 1(1). The results are shown in Figure 4.

[0165] Figure 4 is a graph showing the results of the luciferase assay. In Figure 4, the vertical axis represents the relative value of luminescence intensity (with the luminescence intensity of Control set to 1), and the horizontal axis represents the type of RNA nucleic acid molecule. In Figure 4, the numerical values ​​at the top of the graph represent the fold change in the presence of 122-5p mimic compared to the absence of 122-5p mimic. As shown in Figure 4, HSL mutation and HSL deletion were found to improve fold change compared to HSL. Furthermore, TL was found to have a greater effect on improving fold change compared to HSL. From these results, it was found that these can be used as degradation induction sequences instead of pAB sequences.

[0166] (4) Examination of pAB sequence modification We investigated whether modifying the pAB sequence would improve the expression of the target gene. Specifically, we constructed RNA nucleic acid molecules with point mutations introduced into the pAB sequence of 122-5p ON (pAB) constructed in Example 1 (1) above, or RNA nucleic acid molecules with shortened pAB sequences (Mutant #1 to #6 (SEQ ID NOs. 37 to 42)). Table 6 below shows the degradation induction sequences of the various RNA nucleic acid molecules constructed, and Tables 7 and 8 below show the full-length sequences of each RNA nucleic acid molecule. RNA nucleic acid molecules (Control) that are the same as the above RNA nucleic acid molecules except that they do not have degradation induction sequences were constructed in the same way. Furthermore, the expected secondary structures of the various RNA nucleic acid molecules constructed are shown in Figures 5 and 6. Figure 5 shows the expected secondary structure of the RNA nucleic acid molecule with point mutations introduced into the pAB sequence, and Figure 6 shows the expected secondary structure of the RNA nucleic acid molecule with shortened pAB sequence.

[0167]

[0168]

[0169]

[0170] The various RNA nucleic acid molecules mentioned above were introduced into cells and subjected to luciferase assays using the same method as in Example 1(1). The results are shown in Figure 7.

[0171] Figure 7 is a graph showing the results of the luciferase assay. In Figure 7, the vertical axis represents the relative value of the luminescence intensity (with the luminescence intensity of Control set to 1), and the horizontal axis represents the type of RNA nucleic acid molecule. In Figure 7, the numerical values ​​at the top of the graph represent the fold change in the presence of 122-5p mimic compared to the absence of 122-5p mimic. As shown in Figure 7, it was found that all degradation-inducing sequences that form one or two stem-loops, except for #6, showed high fold change. On the other hand, it was suggested that fold change decreases when the stem portion of the stem-loop structure becomes longer (base pairs are continuous without mismatches), such as in #6 vs. #5 (the same applies to #1 vs. pAB and pAB vs. #2).

[0172] (5) Examination of target gene expression and degradation of various RNA nucleic acid molecules The expression of target genes and degradation of various RNA nucleic acid molecules were examined. Specifically, first, various RNA nucleic acid molecules having degradation-inducing sequences shown in Table 9 below were constructed using the same method as in Example (1) above (A109 / pAB (SEQ ID NO: 46), A109 / pAB del7C-24A (SEQ ID NO: 47), TL (SEQ ID NO: 29), and A90 / TL (SEQ ID NO: 48)). The sequences of the various RNA nucleic acid molecules are shown in Table 10 below. Next, 122-5p mimic or negative control (NC mimic) was introduced into A549 cells seeded in a 12-well plate. Twenty-four hours after the introduction, various RNA nucleic acid molecules (1 μg / well) having degradation-inducing sequences shown in Table 9 below, and RLuc mRNA (sea licorice luciferase, internal standard, 0.1 μg / well) were introduced into the A549 cells using Lipofectamine® MessengerMAX®. Six hours after the introduction, the introduced cells were detached using the cell detachment agent Accutase (Nacalai Tesque). After detachment, the cells were washed with RPMI-1640 medium containing 10% FBS. After washing, the sample was divided into two equal parts; one sample was used for a dual luciferase assay, and the other for a qPCR assay. The sample for the dual luciferase assay was centrifuged to obtain a cell pellet. The pellet was subjected to a dual luciferase assay using the Dual-Glo® Luciferase Assay System (Promega). The sample for the qPCR assay was centrifuged to obtain a cell pellet. Total RNA was extracted and reverse transcribed from the pellet in the same manner as in Example (2). After reverse transcription, the obtained cDNA was diluted 10-fold and used as a template to perform PCR using Power SYBR® Green PCR Master Mix (Thermo Fisher Scientific) and the following primer set (final concentration 0.2 μM) under the thermal cycler conditions shown in Table 11 below.In the aforementioned PCR, the StepOnePlus® Real-Time PCR System (Thermo Fisher Scientific) was used as the real-time PCR instrument. For both the dual luciferase assay and the qPCR assay, fold change was calculated using RLuc as the internal standard. These results are shown in Figure 8. PCR primer set Forward for FLuc: 5'-GCAAGACCATGACCGAGAAG-3' (SEQ ID NO: 20) Reverse for FLuc: 5'-CCCTTCTTGGCCTTGATCAG-3' (SEQ ID NO: 21) Forward for RLuc: 5'-CGAAGAGGGCGAGAAAATGG-3' (SEQ ID NO: 43) Reverse for RLuc: 5'-ACTCCTCAGGCTCCAGTTTC-3' (SEQ ID NO: 44).

[0173]

[0174]

[0175]

[0176] Figure 8 is a graph showing the results of the dual luciferase assay and the qPCR assay. Figure 8(A) is a graph showing the results of the dual luciferase assay. In Figure 8(A), the vertical axis shows the luminescence intensity standardized with RLuc set to 1, and the horizontal axis shows the type of RNA nucleic acid molecule. Figure 8(B) is a graph showing the results of the qPCR assay. In Figure 8(B), the vertical axis shows the relative value of mRNA amount with RLuc set to 1, and the horizontal axis shows the type of RNA nucleic acid molecule. In Figure 8, the numerical values ​​at the top of the graph show the fold change in the presence of 122-5p mimic compared to the absence of 122-5p mimic. As shown in Figure 8, compared to A109 / pAB, it was found that fold change was improved in A109 / pAB del7C-24A, TL, or A90 / TL in both the dual luciferase assay and the qPCR assay results.

[0177] (6) Examination of RNA nucleic acid molecules having the 21-5p target sequence We also investigated whether RNA nucleic acid molecules having target sequences other than miR-122-5p could function as an ON switch. Specifically, we constructed an RNA nucleic acid molecule (21-5p ON (pAB) (SEQ ID NO: 54)) in which the target sequence of miR-122-5p ON constructed in Example 1 (1) above was replaced with the target sequence of miR-21-5p (5'-UAGCUUAUCAGACUGAUGUUGA-3': SEQ ID NO: 49). Furthermore, various RNA nucleic acid molecules were constructed by applying a 4-base addition sequence (a sequence capable of forming a stem-loop structure in conjunction with the target sequence), a TL sequence, or an HSL sequence instead of the pAB sequence of 21-5p ON(pAB) (SEQ ID NO: 55), 21-5p ON(TL) (SEQ ID NO: 56), and 21-5p ON(HSL) (SEQ ID NO: 57)). Table 12 below shows the various degradation induction sequences of the RNA nucleic acid molecules, and Table 13 below shows the full-length sequences of each RNA nucleic acid molecule. Figure 9 shows the expected secondary structures of the various constructed RNA nucleic acid molecules.

[0178]

[0179]

[0180] The aforementioned RNA nucleic acid molecules and 12-5p inhibitor (mirVana® Inhibitor, Assay name: hsa-miR-12-5p, Assay ID: MH10206, Thermo Fisher Scientific, final concentration after adding to culture medium: 10 nM) were introduced into A549 cells (cells with high activity of endogenous miR-21-5p) using Lipofectamine® MessengerMAX® (Thermo Fisher Scientific) according to the protocol. After introduction, a dual luciferase assay and a qPCR assay were performed in the same manner as in Example 1(5). The results are shown in Figure 10.

[0181] Figure 10 is a graph showing the results of the dual luciferase assay and the qPCR assay. Figure 10(A) is a graph showing the results of the dual luciferase assay. In Figure 10(A), the vertical axis shows the luminescence intensity standardized with RLuc set to 1, and the horizontal axis shows the type of RNA nucleic acid molecule. Figure 10(B) is a graph showing the results of the qPCR assay. In Figure 10(B), the vertical axis shows the relative value of mRNA amount with RLuc set to 1, and the horizontal axis shows the type of RNA nucleic acid molecule. In Figure 10, the values ​​at the top of the graph show the fold change in the absence of the 21-5p inhibitor (i.e., in the state where endogenous miR-21-5p is active) compared to the presence of the 21-5p inhibitor (i.e., in the state where endogenous miR-21-5p is inactive). As shown in Figure 10, it was found that pAB and TL show a similar degree of fold change. Furthermore, there is a high correlation (R) between the fold change of the expression (translation) level and mRNA level of the target gene. ２ It was found that this indicates = 0.98.

[0182] (7) Examination of RNA nucleic acid molecules having the 205-5p target sequence We also investigated whether RNA nucleic acid molecules having target sequences other than miR-122-5p and miR-21-5p could function as an ON switch. Specifically, we constructed an RNA nucleic acid molecule (205-5p ON (pAB) (SEQ ID NO: 63)) in which the target sequence of 122-5p ON (pAB) constructed in Example 1 (1) above was replaced with the target sequence of miR-205-5p (5'-UCCUUCAUUCCACCGGAGUCUG-3': SEQ ID NO: 58). Furthermore, various RNA nucleic acid molecules were constructed by applying a 4-base addition sequence (a sequence capable of forming a stem-loop structure in conjunction with the target sequence, a 4nt sequence), a TL sequence, or an HSL sequence instead of the pAB sequence of 205-5p ON(pAB) (SEQ ID NO: 64), 205-5p ON(TL) (SEQ ID NO: 65), and 205-5p ON(HSL) (SEQ ID NO: 66)). Table 14 below shows the various degradation induction sequences of the RNA nucleic acid molecules, and Table 15 below shows the full-length sequences of each RNA nucleic acid molecule. Figure 11 shows the expected secondary structures of the various constructed RNA nucleic acid molecules.

[0183]

[0184]

[0185] The various RNA nucleic acid molecules described above were introduced into A549 cells (cells with low activity of endogenous miR-205-5p) using the same method as in Example 1(1). After introduction, a dual luciferase assay and a qPCR assay were performed using the same method as in Example 1(5). Note that 205-5p mimic (mirVana® miRNA mimic, Assay name: has-miR-205-5p, Assay ID: MC11015, Thermo Fisher Scientific) was used instead of 122-5p mimic. These results are shown in Figure 12.

[0186] Figure 12 is a graph showing the results of the dual luciferase assay and the qPCR assay. Figure 12(A) is a graph showing the results of the dual luciferase assay. In Figure 12(A), the vertical axis shows the luminescence intensity standardized with RLuc set to 1, and the horizontal axis shows the type of RNA nucleic acid molecule. Figure 12(B) is a graph showing the results of the qPCR assay. In Figure 12(B), the vertical axis shows the relative value of mRNA amount with RLuc set to 1, and the horizontal axis shows the type of RNA nucleic acid molecule. In Figure 12, the numerical values ​​at the top of the graph show the fold change in the presence of 205-5p mimic compared to the absence of 205-5p mimic. As shown in Figure 12, it was found that fold change improved in 4nt, TL, and HSL compared to pAB. Furthermore, there was a high correlation (R) between the fold change of the expression (translation) amount of the target gene and the mRNA amount. ２ It was found that this corresponds to 0.96.

[0187] (8) Examination of the application of known mRNA degradation induction sequences to degradation induction sequences We examined whether known mRNA degradation induction sequences function as an ON switch when applied to the degradation induction sequence of RNA nucleic acid molecules. Specifically, we applied known mRNA degradation induction sequences in place of the pAB sequence of 122-5p ON (pAB) constructed in Example 1 (1) above. The known mRNA degradation induction sequences applied were AU Rich Element (ARE) sequences, hnRNPs binding motifs (UAASUUAU, S=C or G), or Fast Decay Stem (FDS) sequences (ARE sequence application: SEQ ID NOs. 74-76, hnRNPs binding motif application: SEQ ID NOs. 77-79, FDS sequence application: SEQ ID NO. 80). The ARE sequence is known to be recognized by various RNA-binding proteins and is a sequence related to mRNA stability. Among known ARE sequences, the UUAUUAUU motif has been reported to be important in promoting mRNA degradation (see references 5 and 6 below). The hnRNPs-binding motif has been reported to be recognized by RNA-binding proteins hnRNPs and to promote mRNA degradation via de-adenylation by CCR4-NOT (see reference 7 below). The FDS sequence is a motif predicted as a result of analyzing the common structure of mRNAs that degrade rapidly (see reference 8 below). For the ARE sequence, RNA nucleic acid molecules with 0, 5, or 10 adenines attached to the 3' end were prepared (ARE+A0 (SEQ ID NO: 74), ARE+A5 (SEQ ID NO: 75), ARE+A10 (SEQ ID NO: 76)). Furthermore, RNA nucleic acid molecules were prepared in which the hnRNPs binding motif was given 0, 5, or 10 adenines at the 3' end (hnRNPs binding motif + A0 (SEQ ID NO: 77), hnRNPs binding motif + A5 (SEQ ID NO: 78), hnRNPs binding motif + A10 (SEQ ID NO: 79)). Table 16 below shows the various degradation induction sequences of the RNA nucleic acid molecules, and Tables 17 and 18 below show the full-length sequences of each RNA nucleic acid molecule.Reference 5: Zubiaga, A M et al. “The nonamer UUAUUUAUU is the key AU-rich sequence motif that mediates mRNA degradation.” Molecular and cellular biology vol. 15,4 (1995): 2219-30. doi:10.1128 / MCB.15.4.2219 Reference 6: Siegel, David A et al. “Massively parallel analysis of human 3' UTRs reveals that AU-rich element length and registration predict mRNA destabilization.” G3 (Bethesda, Md.) vol. 12,1 (2022): jkab404. doi:10.1093 / g3journal / jkab404 Reference 7: Geissler, Rene et al. “A widespread sequence-specific mRNA decay pathway mediated by hnRNPs A1 and A2 / B1.” Genes & development vol. 30,9 (2016): 1070-85. doi:10.1101 / gad.277392.116 Reference 8: Rabani, Michal et al. “Computational prediction of RNA structural motifs involved in post-transcriptional regulatory processes.” Methods in molecular biology (Clifton, N.J.) vol. 714 (2011): 467-79. doi:10.1007 / 978-1-61779-005-8_28.

[0188]

[0189]

[0190]

[0191] The various RNA nucleic acid molecules mentioned above were introduced into cells and subjected to luciferase assays using the same method as in Example 1(1). The results are shown in Figure 13.

[0192] Figure 13 is a graph showing the results of the luciferase assay. In Figure 13, the vertical axis represents the relative value of the luminescence intensity (Control = 1), and the horizontal axis represents the type of RNA nucleic acid molecule. In Figure 13, the numerical values ​​at the top of the graph represent the fold change in the presence of 122-5p mimic compared to the absence of 122-5p mimic. As shown in Figure 13, it was found that ARE+A5 and hnRNPs binding motif+A5 showed improved fold change compared to pAB. From these results, it was found that even when these sequences other than pAB are used as degradation induction sequences, the target gene, firefly luciferase, can be expressed and function as an ON switch.

[0193] (9) Examination of modification of FDS sequences We also examined whether FDS sequences other than the FDS sequence used in Example 1(8) could function as an ON switch in the degradation induction sequence. Specifically, based on the motifs conserved in the rapidly degrading mRNA described in Reference 8, we modified the FDS sequence of the RNA nucleic acid molecule having the FDS sequence constructed in Example 1(8) and constructed RNA nucleic acid molecules (FDS #2 to #5: Sequence IDs 85 to 88). Table 19 below shows the degradation induction sequences of the various RNA nucleic acid molecules constructed, and Table 20 below shows the full-length sequences of each RNA nucleic acid molecule.

[0194]

[0195]

[0196] The various RNA nucleic acid molecules mentioned above were introduced into cells and subjected to luciferase assays using the same method as in Example 1(1). The results are shown in Figure 14.

[0197] Figure 14 is a graph showing the results of the luciferase assay. In Figure 14, the vertical axis represents the relative value of the luminescence intensity (=1), and the horizontal axis represents the type of RNA nucleic acid molecule. In Figure 14, the numerical values ​​at the top of the graph represent the fold change in the presence of 122-5p mimic compared to the absence of 122-5p mimic. As shown in Figure 14, it was found that in all cases of FDS #2 to #5, the fold change was improved to the same extent as or greater than that of pAB. From these results, it was found that even when these sequences other than pAB are used as degradation induction sequences, the target gene, firefly luciferase, can be expressed and function as an ON switch.

[0198] (10) Examination of modification of the target sequence portion of the miRNA of the degradation induction sequence We investigated whether the expression of the target gene would be improved by modifying the target sequence portion of the miRNA of the degradation induction sequence.

[0199] In the presence of miRNA, the target sequence portion of the miRNA in the degradation induction sequence is cleaved between approximately the 10th and 11th base pairs from the 3' end. At this time, the 5' end of the target sequence portion remains downstream of the polyA sequence at 3', and this remaining portion is thought to work in a way that reduces the amount of translation. Here, a mutation that shortens the 5' end of the target sequence portion is thought to shorten the remaining portion and, as a result, increase the amount of translation. Furthermore, a mutation in the target sequence portion that results in a higher proportion of A in the remaining portion is thought to increase the amount of translation. Also, according to reference 9, the addition of C to the polyA site increases the amount of translation, so a mutation in the target sequence portion that results in a higher proportion of C in the remaining portion is also thought to increase the amount of translation. Furthermore, according to reference 10, a mutation to A at the 3' end of the target sequence portion of the miRNA can be expected to improve cleavage efficiency. Therefore, this point was confirmed by modifying the target sequence portion of the miRNA in the degradation induction sequence. Reference 9: Li, CY et al. “Cytidine-containing tails robustly enhance and prolong protein production of synthetic mRNA in cell and in vivo.” Molecular Therapy Nucleic Acids 30 (2022): 300-310. Reference 10: Kim, D. et al. “Rules for functional microRNA targeting.” BMB Reports 50 (2017): 554-559.

[0200] Specifically, RNA nucleic acid molecules were first constructed. These RNA nucleic acid molecules were designed so that, from the 5' end to the 3' end, they consisted of a cap structure, a 5' UTR, a sequence encoding the target gene, firefly luciferase, a 3' UTR, a polyA sequence (90 nucleotides or 109 nucleotides long), a miRNA target sequence (target sequence of miR-126-3p, miR-125b-5p, or miR-199a-5p), and a pAB sequence. The combinations of the polyA sequence and the target sequence in these RNA nucleic acid molecules are shown in Tables 21 to 24 below. In Tables 21 to 24 below, the underlined and bolded bases indicate bases in which substitution mutations have been introduced in the base sequence complementary to the miRNA. The underlined bases indicate pAB sequences. Furthermore, in Tables 21-24 below, for example, M18U>C indicates that in the nucleotide sequence complementary to the miRNA, the 18th base from the 3' end is substituted from U to C. In addition, in Tables 21-24 below, TAIL-X indicates that in the nucleotide sequence complementary to the miRNA, X consecutive bases are deleted from the 5' end. The nucleotide sequences of various RNA nucleic acid molecules are shown in Tables 25-31 below. The sequences of miR-126-3p, miR-125b-5p, and miR-199a-5p are shown in Table 32 below.

[0201]

[0202]

[0203]

[0204]

[0205]

[0206]

[0207]

[0208]

[0209]

[0210]

[0211]

[0212]

[0213] Next, we investigated the expression of target genes by each RNA nucleic acid molecule. Specifically, each RNA nucleic acid molecule shown in Tables 21 to 24 above was introduced into cells and luciferase assays were performed using the same method as in Example 1(1), but with A172 cells (human glioblastoma cells; cells with low activity of all of miR-126-3p, miR-125b-5p, and miR-199a-5p) instead of A549 cells, and with 126-3p mimic (Assay ID: MC12841, Themo Fisher Scientific), miR-125b-5p mimic (Assay ID: MC10148, Themo Fisher Scientific), or miR-199a-5p mimic (Assay ID: MC10893, Themo Fisher Scientific) instead of 122-5p mimic. These results are shown in Tables 21 to 24 above.

[0214] Table 21 shows the relative values ​​of luminescence intensity in the absence or presence of 126-3p mimic (with the luminescence intensity of Control set to 1), and the expression level ratio (Fold change) in the presence / absence of miRNA. As shown in Table 21, it was found that introducing mutations into the target sequence of miRNA resulted in expression levels and expression levels exceeding those of unmodified RNA nucleic acid molecules.

[0215] Table 22 shows the relative values ​​of luminescence intensity in the absence or presence of 125b-5p mimic (with the luminescence intensity of Control set to 1), and the ratio of expression levels in the presence / absence of miRNA (Fold change). As shown in Table 22, it was found that introducing mutations into the target sequence of miRNA resulted in expression levels and expression ratios exceeding those of unmodified RNA nucleic acid molecules.

[0216] Table 23 shows the relative values ​​of luminescence intensity in the absence or presence of 199a-5p mimic (with the luminescence intensity of Control set to 1), and the ratio of expression levels in the presence / absence of miRNA (Fold change). As shown in Table 23, it was found that introducing mutations into the target sequence of miRNA resulted in expression levels and expression ratios exceeding those of unmodified RNA nucleic acid molecules.

[0217] Table 24 shows the relative values ​​of luminescence intensity in the absence or presence of 199a-5p miRNA (with the luminescence intensity of Control set to 1), and the ratio of expression levels in the presence / absence of miRNA (Fold change). As shown in Table 24, it was found that introducing mutations into the target sequence of miRNA resulted in expression levels and expression ratios exceeding those of unmodified RNA nucleic acid molecules.

[0218] From the above, it was found that introducing mutations into the target sequence of miRNA can enhance the expression of the target gene contained in the mRNA, and also enhance the ratio of expression levels in the presence / absence of the gene. In other words, it was found that introducing mutations into the target sequence of miRNA can induce the expression of the target gene more specifically in cells where the target miRNA is present. Furthermore, it was confirmed that the amount of translation (expression) of the target gene can be increased by introducing mutations that shorten the 5' end of the target sequence portion of the miRNA from the location of each mutation substitution. In addition, the amount of translation (expression) could be increased by introducing substitution mutations into the target sequence portion such that the remaining portion has a higher proportion of A. For this reason, it was estimated that the amount of translation (expression) of the miRNA target sequence can be increased by introducing mutations at position 1 and positions 13-24, relative to the 3' end base, in a base sequence complementary to the miRNA.

[0219] (11) Examination of introducing mutations into the target sequence of 122-5p We investigated whether the expression of the target gene would be improved by modifying the target sequence portion of miR-122-5p in the degradation induction sequence. Specifically, we first constructed an RNA nucleic acid molecule. The RNA nucleic acid molecule was designed so that, from the 5' end to the 3' end, it would consist of a cap structure, a 5' UTR, a sequence encoding the target gene, firefly luciferase, a 3' UTR, a poly(A) sequence (90 bases long), the target sequence of miR-122-5p, and a pAB sequence. These RNA nucleic acid molecules are shown in Table 33 below. In Table 33 below, the bases shown in underline and bold indicate the bases into which substitution mutations have been introduced in the base sequence complementary to miRNA. The bases shown in underline indicate the pAB sequence. Also, in Table 33 below, Tail-4 indicates that four consecutive bases are deleted from the 5' end in the base sequence complementary to miR-122-5p. Furthermore, in Table 33 below, for example, U12>A indicates that in the nucleotide sequence complementary to miR-122-5p, the 12th nucleotide from the 3' end is substituted from U to A. The nucleotide sequences of various RNA nucleic acid molecules are shown in Table 34 below. The sequence of miR-122-5p is 5'-UGGAGUGUGACAAUGGUGUUUG-3' (Sequence ID 9).

[0220]

[0221]

[0222] Next, we investigated the expression of target genes by each RNA nucleic acid molecule. Specifically, using the same method as in Example 1(1) above, 122-5p mimic (or NC mimic) and each RNA nucleic acid molecule shown in Table 33 were introduced into A549 cells (cells with low activity of endogenous miR-122-5p), and a luciferase assay was performed. The results are shown in Figure 15.

[0223] Figure 15 is a graph showing the results of the luciferase assay. In Figure 15, the vertical axis represents the relative value of the luminescence intensity (with the luminescence intensity of Control set to 1), and the horizontal axis represents the type of RNA nucleic acid molecule. In Figure 15, the numerical values ​​at the top of the graph represent the fold change in the presence of 122-5p mimic (122-5p mimic) compared to the absence of 122-5p mimic (NC mimic). As shown in Figure 15, it was found that for all RNA nucleic acid molecules, the luminescence intensity of luciferase increased in the presence of miR-122-5p compared to the absence of miR-122-5p. Furthermore, it was found that for all RNA nucleic acid molecules, the luminescence intensity increased in the presence of miR-122-5p compared to the unmutated molecule (Tail-4 (shown as WT in Figure 15)). Furthermore, it was found that U13>C showed improved fold change compared to the unmutated form (Tail-4 (shown as WT in Figure 15)).

[0224] These results show that the RNA nucleic acid molecule of this disclosure can express the target gene, firefly luciferase, and function as an ON switch in the presence of a miRNA that can bind to the target sequence of the RNA nucleic acid molecule of this disclosure, even if the target sequence is not perfectly complementary to the miRNA. Furthermore, it was found that introducing mutations into the target sequence of the miRNA can enhance the expression of the target gene contained in the mRNA.

[0225] (12) Examination of the application of the UUUA repeat sequence to the EGFP gene We investigated whether an RNA nucleic acid molecule could function as an ON switch when applied to the Enhanced Green Fluorescent Protein (EGFP) gene. The UUUA repeat sequence was applied as the degradation induction sequence. Specifically, first, an RNA nucleic acid molecule was constructed. The RNA nucleic acid molecule was designed so that, from the 5' end to the 3' end, it would consist of a cap structure, a 5' UTR, a sequence encoding the target gene EGFP, a 3' UTR, a poly(A) (95 bases) sequence, the target sequence of miRNA-206 (5'-UGGAAUGUAAGGAAGUGUGUGG-3'; Sequence ID No. 144), and a UUUA repeat sequence. Table 35 below shows the various degradation induction sequences of the RNA nucleic acid molecule, and Table 36 below shows the base sequences of the various RNA nucleic acid molecules. Furthermore, as a control, RNA nucleic acid molecules without degradation-inducing sequences (Control-EGFP, Normal mRNA) were constructed.

[0226]

[0227]

[0228] Next, the expression of target genes by each RNA nucleic acid molecule was investigated using a flow cytometer, as shown in Figure 16. Specifically, first, using Lipofectamine® MessengerMAX Reagent (Thermo Fisher Scientific), 0.5 pmol of 206 mimic (Assay ID: MC10409, Thermo Fischer Sicentifc) (or negative control (NC mimic)), 100 ng of the aforementioned RNA nucleic acid molecule (or Control-EGGFP), and mRNA encoding iRFP670 as a reference (100 ng) were introduced into HEK293FT cells (cells with low miR-206 activity) according to the protocol (24-well plate format). 24 hours after introduction, the cells were washed with PBS, treated with 0.25% trypsin-EDTA, and incubated at 37°C under 5% CO2. ２The cells were incubated for 5 minutes. Subsequently, the cells were subjected to a CytoFLEX S flow cytometer (Beckman Coulter) (525 / 40 nm and 660 / 10 nm emission filters), and the data were analyzed using FlowJo 10.6.2 (BD Biosciences). The viable cell population was gated for iRFP670 positivity, and its EGFP intensity was normalized by iRFP670 intensity. These results are shown in Figure 17.

[0229] Figure 17 is a graph showing the results of flow cytometry analysis. In Figure 17(A), the vertical axis shows the relative value of the EGFP fluorescence level (Control-EGFP is set to 1), and the horizontal axis shows the type of RNA nucleic acid molecule. In Figure 17(B), the vertical axis shows the fold change (ON / OFF ratio) in the presence of 206 mimic compared to the absence of 206 mimic, and the horizontal axis shows the type of RNA nucleic acid molecule. As shown in Figure 17, it was found that in RNA nucleic acid molecules having one or more repeat sequences (UUUA sequences), the EGFP emission level improved in the presence of miR-206 compared to the absence of miR-206. Furthermore, it was found that fold change improved in the case of longer repeat sequences (UUUA sequences). From these results, it was found that the RNA nucleic acid molecules described herein can be applied to the EGFP gene, and that UUUA repeat sequences can be used as degradation induction sequences.

[0230] (13) Examination of the application of the UUUA repeat sequence to the NLuc gene We investigated whether an RNA nucleic acid molecule could function as an ON switch when applied to the NanoLuc (NLuc) gene. Similar to Example 1 (12) above, an RNA nucleic acid molecule containing the UUUA repeat sequence was constructed using a sequence encoding NLuc as the target gene instead of EGFP, and the target sequence of miRNA-21-5p (5'-UAGCUUAUCAGACUGAUGUUGA-3'; Sequence ID No. 49) instead of miR-206. Table 37 below shows the various degradation induction sequences of the RNA nucleic acid molecule, and Table 38 below shows the base sequences of the various RNA nucleic acid molecules. In addition, an RNA nucleic acid molecule without a degradation induction sequence (Control-NLuc, Normal mRNA) was constructed as a control.

[0231]

[0232]

[0233] Next, the expression of target genes by each RNA nucleic acid molecule was investigated using a dual luciferase assay. Specifically, in the same manner as in Example 1 (12), 2 pmol of 21-5p inhibitor (Assay ID: MH10206, Thermo Fischer Scientific) (or negative control (NC inhibitor, Cat No. 4464084, Thermo Fischer Scientific)), each RNA nucleic acid molecule employing NLuc as the target gene, and reference mRNA encoding FLuc were transfected into HeLa cells (cells with high activity of miR-21-5p). 24 hours after the transfection, the luminescence levels of NLuc and FLuc were measured using a GloMax® Navigator Microplate Luminometer (Promega). These results are shown in Figure 18.

[0234] Figure 18 is a graph showing the results of a dual luciferase assay. In Figure 18(A), the vertical axis shows the relative value of NLuc expression level corrected for FLuc, and the horizontal axis shows the type of RNA nucleic acid molecule. In Figure 18(B), the vertical axis shows the fold change (ON / OFF ratio) in the absence of 21-5p inhibitor (i.e., in the state where endogenous miR-21-5p is active) compared to the presence of 21-5p inhibitor (i.e., in the state where endogenous miR-21-5p is inactive), and the horizontal axis shows the type of RNA nucleic acid molecule. As shown in Figure 18, in RNA nucleic acid molecules having one or more repeat sequences (UUUA sequences), the expression level of NLuc was found to be improved in the absence of the 21-5p inhibitor (i.e., in the presence of miR-21-5p) compared to the presence of the 21-5p inhibitor (i.e., in the absence of miR-21-5p). These results indicate that the RNA nucleic acid molecules described herein are applicable to NLuc and that UUUA repeat sequences can be used as degradation induction sequences.

[0235] (14) Investigation of the function of RNA nucleic acid molecules having the 142-3p target sequence in T cells We investigated whether RNA nucleic acid molecules having the miR-142-3p target sequence and a sequence in which U is mutated to A would function in T cells. Specifically, similar to the RNA nucleic acid molecule constructed in Example 1(1) above, we constructed an RNA nucleic acid molecule (142-3p ON (pAB): SEQ ID NO: 169) that has a target sequence completely complementary to miR-142-3p (5'-UGUAGUGUUUCCUACUUUAUGGA-3': SEQ ID NO: 168) and a PolyA length of 109 bases. Furthermore, we synthesized an RNA nucleic acid molecule in which U of the miR-142-3p target sequence was substituted to A (SEQ ID NO: 170). Furthermore, we also constructed an RNA nucleic acid molecule (Non-coding ON (pAB): SEQ ID NO: 171) in which the target sequence is not complementary to any known human miRNA. In addition, RNA nucleic acid molecules similar to the 142-3p ON (pAB) described above, but with a TL sequence (SEQ ID NO: 3) or an FDS#2 sequence (SEQ ID NO: 5) instead of the pAB sequence (SEQ ID NO: 172, 174), and RNA nucleic acid molecules in which the U of the target sequence of these miR-142-3p was substituted with A were synthesized (SEQ ID NO: 173, 175). The various degradation induction sequences of these RNA nucleic acid molecules are shown in Table 39 below. In Table 39 below, the underlined and bolded bases indicate the bases in which substitution mutations have been introduced in the base sequence complementary to miRNA. The underlined bases indicate the pAB sequence, TL sequence, or FDS#2 sequence. Also in Table 39 below, for example, M19U>A indicates that in the base sequence complementary to miR-142-3p, the 19th base from the 3' end is substituted from U to A. The base sequences of the various RNA nucleic acid molecules are shown in Tables 40-41 below. As a control, FLuc-mRNA (Control-FLuc) that does not contain either the miRNA target sequence or the degradation-inducing sequence was prepared, and Renilla luciferase (RLuc)-mRNA was prepared as a reference.

[0236]

[0237]

[0238]

[0239] Next, the expression of target genes by each RNA nucleic acid molecule shown in Table 39 was investigated. Specifically, each RNA nucleic acid molecule shown in Table 39 was introduced into Jurkat cells (a human leukemia T cell-derived cell line) by electroporation (using a Neon Transfection System (Invitrogen, MPK5000) and 10 μL of the Neon Transfection kit, at 1350 V, 10 m sec, and 3 pulses). After introduction, a dual luciferase assay was performed using the Dual-Glo® Luciferase Assay System (Promega) according to the attached protocol. The expression levels of FLuc were normalized so that the Control-FLuc expression level was 1 after correcting for the RLuc expression level as an internal standard. These results are shown in the "Relative Luminescence Intensity" column of Table 39.

[0240] As shown in Table 39 above, RNA nucleic acid molecules possessing the target sequence of miR-142-3p and the pAB sequence were found to significantly increase FLuc expression in T cells compared to RNA nucleic acid molecules whose target sequences are not complementary to any known human miRNAs. Furthermore, it was found that mutating U in the target sequence, which is perfectly complementary to miR-142-3p, to A increased FLuc expression. In addition, it was found that RNA nucleic acid molecules possessing the target sequence of miR-142-3p similarly increased FLuc expression in T cells when they possessed not only pAB but also TL and FDS#2. Furthermore, it was found that mutating U in the target sequence, which is perfectly complementary to miR-142-3p, to A increased the expression of the target gene.

[0241] (15) Investigation of the function of RNA nucleic acid molecules with the 155-5p target sequence in T cells We investigated whether RNA nucleic acid molecules with the miR-155-5p target sequence function in T cells. Specifically, similar to the RNA nucleic acid molecule constructed in Example 1(1) above, we constructed an RNA nucleic acid molecule (155-5p ON (pAB): SEQ ID NO: 184) with the miR-155-5p (5'-UUAAUGCUAAUCGUGAUAGGGGUU-3': SEQ ID NO: 183) target sequence and a PolyA length of 109 bases. This RNA nucleic acid molecule is shown in Table 42 below. The underlined bases indicate the pAB sequence. The base sequence of the RNA nucleic acid molecule is shown in Table 43 below. As a control, FLuc-mRNA (Control-FLuc) that does not contain either the miRNA target sequence or the degradation induction sequence was prepared, and Renilla luciferase (RLuc)-mRNA was prepared as a reference.

[0242]

[0243]

[0244] Next, we investigated the expression of target genes using the RNA nucleic acid molecules shown in Table 42. Specifically, the RNA nucleic acid molecules shown in Table 42 were introduced into human peripheral blood-derived T cells (activated by culturing for 3 days in ImmunoCult-XF T Cell Expansion Medium (Stemcell Technologies #10981) supplemented with ImmunoCult Human CD3 / CD28 T Cell Activator (Stemcell Technologies #10971)) using electroporation (Neon Transfection System (Invitrogen, MPK5000) and 10 μL of Neon Transfection kit, 1325 V, 10 msec, 3 pulses). In addition, the RNA nucleic acid molecules shown in Table 42 were introduced into HepG2 cells (human liver cancer-derived cell line; cells with low miR-155-5p activity) using Lipofectamine® MessengerMAX (Thermo Fisher Scientific) according to the protocol (96-well plate format). A dual luciferase assay was performed using the same method as in Example 1(5) above. The results (relative luminescence intensity in T cells or HepG2 cells) are shown in Table 42.

[0245] As shown in Table 42 above, RNA nucleic acid molecules containing the miR-155-5p target sequence showed higher expression levels of the target gene in T cells compared to human liver cancer-derived cell line HepG2 cells. As shown in Reference Examples 2-3 below, miR-155-5p exhibited high activity in T cells while showing moderate activity in HepG2 cells, indicating that the miR-155-5p target sequence can be used as a miRNA when targeting T cells.

[0246] (16) Examination of introducing mutations in the target sequence of 155-5p We investigated whether the expression of the target gene would be improved by modifying the target sequence portion of miR-155-5p with a mutation. Specifically, RNA nucleic acid molecules were synthesized in which the U in the target sequence of miR-155-5p in 155-5p ON (pAB) (SEQ ID NO: 184) constructed in Example 1 (15) above was mutated to C or A (SEQ ID NOs: 186-187). The degradation induction sequences of these RNA nucleic acid molecules are shown in Table 44 below. In Table 44 below, the bases shown in underline and bold indicate the bases in which substitution mutations have been introduced in the base sequence complementary to miRNA. The bases shown in underline indicate the pAB sequence. Also in Table 44 below, for example, M18U>C indicates that in the base sequence complementary to miR-155-5p, the 18th base from the 3' end is substituted from U to C. The base sequences of various RNA nucleic acid molecules are shown in Table 45 below. As a control, FLuc-mRNA (Control-FLuc) that does not contain either the miRNA target sequence or the degradation-inducing sequence was prepared, and Renilla luciferase (RLuc)-mRNA was prepared as a reference.

[0247]

[0248]

[0249] Next, the expression of target genes by each RNA nucleic acid molecule shown in Table 44 was investigated. Dual luciferase assays were performed in HepG2 cells (human liver cancer-derived cell line; cells with low miR-155-5p activity) in the presence or absence of 155-5p mimic (mirVana® miRNA mimic, Assay name: hsa-miR-155-5p, Assay ID: MC28440, Themo Fisher Scientific) using the same method as in Example 1(5). These results are shown in Table 44.

[0250] As shown in Table 44 above, it was found that mutating U, a target sequence completely complementary to miR-155-5p, to C or A increases the expression level of the target gene.

[0251] [Reference Example 1] The activity of the miRNA targeted in Example 1 was confirmed (miRNA activity test).

[0252] The activity of various miRNAs in PAEC (human pulmonary artery endothelial cells), A172 cells (human glioblastoma cells), and SkMC (human skeletal muscle cells) was measured as follows. First, FLuc-mRNAs were constructed by positioning the target sequence of the miRNA within the 5'UTR so that, from the 5' end to the 3' end, the sequence consisted of a cap structure, a 5'-linker sequence (5'-AGGUUCCUUAAUCGCGGAUCC-3' (SEQ ID NO: 161)), the target sequence of each miRNA, the 3'-linker sequence, the sequence encoding the target gene FLuc, the 3'UTR, and a poly(A) (120 bases) sequence (SEQ ID NOs: 190-195). Table 46 below shows the target sequences and 3'-linker sequences of the miRNAs used, and the sequences of the FLuc-mRNAs are shown in Tables 47-48 below. In addition, as a control, mRNA encoding FLuc without the target sequence of the miRNA was constructed (Control).

[0253]

[0254]

[0255]

[0256] Next, the expression of the target gene by FLuc-mRNA in which the target sequence of the miRNA was positioned within the 5'UTR in the aforementioned cells was investigated using a dual luciferase assay. Specifically, FLuc-mRNA (or Control) in which the target sequence of the miRNA was positioned within the 5'UTR, and RLuc-mRNA as a reference, were introduced into PAEC, A-172 cells, and SkMCs using Lipofectamine® MessengerMAX® (Thermo Fisher Scientific) according to the protocol. 18 to 22 hours after introduction, the luminescence levels of FLuc and RLuc were measured using a GloMax® Navigator Microplate Luminometer (Promega). The luminescence level of FLuc in each sample was divided by the luminescence level of RLuc, and the quotient was normalized by Control to calculate the relative expression level (Control = 1). The results are shown in Table 49 below.

[0257]

[0258] As shown in Table 49 above, in PAEC, the relative expression level of FLuc by RNA nucleic acid molecules with the target sequences of miR-21-5p and miR-126-3p positioned in the 5' UTR was low, and the activity of miR-21-5p and miR-126-3p was high in PAEC. Similarly, as shown in Table 49 above, the activity of miR-21-5p, let-7a-5p, let-7b-5p, mi-125b-5p, and miR-199a-5p was high in SkMC. On the other hand, in A172 cells, the relative expression level of FLuc by RNA nucleic acid molecules with the target sequence of miR-126-3p positioned in the 5' UTR was high, and the activity of miR-126-3p was low in A172 cells.

[0259] [Reference Example 2] The activity of various miRNAs in HepG2 cells (human liver cancer-derived cell line) was measured using the same method as in Reference Example 1. As in Reference Example 1, FLuc-mRNAs were constructed with the target sequences of each miRNA placed within the 5'UTR (Sequence IDs 196-197). Table 50 below shows the target sequences and 3'-linker sequences of the miRNAs used, and Table 51 below shows the sequences of the FLuc-mRNAs. The results of the dual luciferase assay are shown in Table 52 below.

[0260]

[0261]

[0262]

[0263] As shown in Table 52 above, in HepG2 cells, the relative expression levels of FLuc by RNA nucleic acid molecules with the target sequences of miR-142-3p and miR-155-5p positioned within the 5' UTR were high or moderate, and the activity of miR-142-3p and miR-155-5p was low or moderate in HepG2 cells.

[0264] [Reference Example 3] The activity of various miRNAs in human T cells and human monocytes was measured. Similar to Reference Example 1, FLuc-mRNAs were constructed in which the target sequence of each miRNA was placed within the 5' UTR. Table 53 below shows the target sequences and 3'-linker sequences of the miRNAs used, and Table 54 below shows the sequences of the FLuc-mRNAs.

[0265]

[0266]

[0267] Next, the expression of the target gene in the aforementioned cells using FLuc-mRNA in which the target sequence of the miRNA was positioned within the 5'UTR was investigated by a dual luciferase assay. Specifically, FLuc-mRNA (or Control) in which the target sequence of the miRNA was positioned within the 5' UTR, and RLuc-mRNA as a reference, were introduced into human peripheral blood-derived T cells (activated by 3 days of culture in ImmunoCult-XF T Cell Expansion Medium (Stemcell Technologies #10981) supplemented with ImmunoCult Human CD3 / CD28 T Cell Activator (Stemcell Technologies #10971)) using electroporation (Neon Transfection System (Invitrogen, MPK5000) and 10 μL of Neon Transfection kit, 1325 V, 10 msec, 3 pulses), and into human monocytes using electroporation (1800 V, 10 msec, 3 pulses). Eighteen to twenty-two hours after the introduction described above, the luminescence levels of FLuc and RLuc were measured using a GloMax® Navigator Microplate Luminometer (Promega). The luminescence level of FLuc in each sample was divided by the luminescence level of RLuc, and the quotient was normalized by Control to calculate the relative expression level (Control = 1). The results are shown in Table 55 below.

[0268]

[0269] As shown in Table 55 above, in human T cells, the relative expression level of FLuc mediated by RNA nucleic acid molecules containing the target sequences of miR-142-3p and miR-155-5p within the 5' UTR was low, and the activity of miR-142-3p and miR-155-5p was high in human T cells. Furthermore, the relative expression level of FLuc mediated by RNA nucleic acid molecules containing the target sequence of miR-223-3p within the 5' UTR was moderate, and the activity of miR-223-3p was moderate in human T cells. In addition, in human monocytes, the relative expression level of FLuc mediated by RNA nucleic acid molecules containing the target sequence of miR-155-5p within the 5' UTR was moderate, and the activity of miR-155-5p was moderate in human monocytes. Furthermore, in human monocytes, the relative expression level of FLuc mediated by RNA nucleic acid molecules with the miR-223-3p target sequence positioned within the 5' UTR was low, while the activity of miR-223-3p was high in human monocytes.

[0270] [Example 2] (1) Examination of RNA nucleic acid molecules having the 223-3p target sequence and RNA nucleic acid molecules having the same mutant sequence We also examined whether RNA nucleic acid molecules having the miR-223-3p target sequence and RNA nucleic acid molecules having the same mutant sequence function as an ON switch. Specifically, we first constructed RNA nucleic acid molecules. The RNA nucleic acid molecule was designed so that from the 5' end to the 3' end, it would have a cap structure, a 5' UTR, a sequence encoding the target gene, firefly luciferase (SEQ ID NO: 10), a 3' UTR, a poly(A) sequence (109 nucleotides long), the miR-223-3p target sequence, and an FDS#2 sequence. These RNA nucleic acid molecules are shown in Table 56 below. In Table 56 below, the underlined and bolded bases indicate bases in which substitution mutations have been introduced in the base sequence complementary to miRNA. The underlined bases indicate the FDS#2 sequence. Furthermore, in Table 56 below, for example, M21G>A indicates that in the nucleotide sequence complementary to miR-223-3p, the 21st base from the 3' end is substituted from G to A. In addition, in Table 56 below, TAIL-X indicates that in the nucleotide sequence complementary to miRNA, X consecutive bases are deleted from the 5' end. The nucleotide sequences of various RNA nucleic acid molecules are shown in Tables 57-58 below. The sequence of miR-223-3p is 5'-UGUCAGUUUGUCAAAUACCCCA-3' (Sequence ID 201).

[0271]

[0272]

[0273]

[0274] Next, the expression of target genes by each RNA nucleic acid molecule was investigated. Specifically, each RNA nucleic acid molecule shown in Table 56 was introduced into A172 cells (cells with low activity of endogenous miR-223-3p) using the same method as in Example 1(1). After introduction, a dual luciferase assay was performed using the same method as in Example 1(5). Note that 223-3p mimic (mirVana® miRNA mimic, Assay name: has-miR-223-3p, Assay ID: MC12301, Thermo Fisher Scientific) was used instead of 122-5p mimic. These results are shown in Table 56.

[0275] Table 56 shows the relative values ​​of luminescence intensity in the absence or presence of 223-3p mimic (with the luminescence intensity of Control set to 1), and the expression level ratio (Fold change) in the presence / absence of miRNA. As shown in Table 56, it was found that introducing mutations into the target sequence of miRNA resulted in expression levels and expression levels exceeding those of unmodified RNA nucleic acid molecules.

[0276] (2) Examination of RNA nucleic acid molecules with known UTRs We also examined whether RNA nucleic acid molecules with high-performance UTRs described in publicly available papers function as ON switches. Specifically, we first constructed RNA nucleic acid molecules. The RNA nucleic acid molecule was designed so that from the 5' end to the 3' end, it would have a cap structure, the 5' UTR described in Reference 16 below, the sequence encoding the target gene, firefly luciferase (SEQ ID NO: 10), the 3' UTR described in Reference 17 below, a poly(A) sequence (117 nucleotides long), the target sequence of miR-142-3p (5'-UGUAGUGUUUCCUACUUUAUGGA-3': SEQ ID NO: 168), and a pAB sequence or FDS#2 sequence. These RNA nucleic acid molecules are shown in Table 59 below. In Table 59 below, the underlined and bolded bases indicate bases in which substitution mutations have been introduced in the base sequence complementary to miRNA. The underlined bases indicate pAB sequences or FDS#2 sequences. Furthermore, in Table 59 below, for example, M19U>A indicates that in the nucleotide sequence complementary to miR-142-3p, the 19th nucleotide from the 3' end is substituted from U to A. The nucleotide sequences of various RNA nucleic acid molecules are shown in Table 60 below. The sequence of the 5'UTR is 5'-ACTTATATTTCTCTGACAAAGTCACAGACACAGACACGCAGAGCAGAGTG-3' ("7.21", SEQ ID NO: 214), and the sequence of the 3'UTR is 5'-GAGCTGGCCGTCCTTTCAACGATCCAAGTCCTGAAGATCACCTCCCCTTGGGGGGTTCTTTTTGAAAAAAAA-3' (derived from Rabies Virus G mRNA, SEQ ID NO: 215).Reference 16: Tang, Xiaoshan et al. “A novel deep generative model for mRNA vaccine development: Designing 5' UTRs with N1-methyl-pseudouridine modification.” Acta pharmaceutica Sinica. B vol. 14,4 (2024): 1814-1826. doi:10.1016 / j.apsb.2023.11.003 Reference 17: Palusa, Saiprasad et al. “The 3' untranslated region of the rabies virus glycoprotein mRNA specifically interacts with cellular PCBP2 protein and promotes transcript stability.” PloS one vol. 7,3 (2012): e33561. doi:10.1371 / journal.pone.0033561.

[0277]

[0278]

[0279] A dual luciferase assay was performed using Jurkat cells (cells with high miR-142-3p activity) in the same manner as in Example 1 (14). The expression levels of FLuc were corrected using the expression level of RLuc as an internal standard, and then normalized so that the Control level was 1. These results are shown in the relative luminescence intensity in Table 59.

[0280] As shown in Table 59 above, RNA nucleic acid molecules using UTRs with the miR-142-3p target sequence, as described in publicly available papers, showed high activity in Jurkat cells.

[0281] While the present disclosure has been described above with reference to embodiments and examples, the present disclosure is not limited to the above embodiments and examples. Various modifications to the structure and details of the present disclosure are possible, which will be understood by those skilled in the art within the scope of the present disclosure.

[0282] The patents, patent applications, and documents cited herein are incorporated herein by reference in the same manner as their contents are specifically described herein.

[0283] This application claims priority based on Japanese Patent Application No. 2025-039399, filed on 12 March 2025, and Japanese Patent Application No. 2025-082812, filed on 16 May 2025, and incorporates all of their disclosures herein.

[0284] <Note> Some or all of the above embodiments and examples may be described as follows, but are not limited to the following. <RNA nucleic acid molecule> (Note 1) An RNA nucleic acid molecule comprising a sequence encoding a target gene, a PolyA sequence, and a degradation induction sequence, wherein the sequence encoding the target gene, the PolyA sequence, and the degradation induction sequence are arranged in this order from the 5' end to the 3' end, the degradation induction sequence comprises a target sequence of miRNA, and in the absence of miRNA capable of binding to the target sequence, the miRNA does not hybridize with the target sequence of miRNA, the RNA nucleic acid molecule can be degraded, and in the presence of miRNA capable of binding to the target sequence, the miRNA hybridizes with the target sequence of miRNA, the degradation of the RNA nucleic acid molecule can be suppressed, and the expression of the target gene can be induced. (Note 2) The RNA nucleic acid molecule as described in Note 1, wherein, in the absence of a miRNA capable of binding to the target sequence, the miRNA does not hybridize with the target sequence of the miRNA, the degradation-inducing sequence is capable of forming a higher-order structure, preferably a stem-loop structure, and the degradation of the RNA nucleic acid molecule can be induced by the formation of the higher-order structure, preferably the stem-loop structure, and in the presence of a miRNA capable of binding to the target sequence, if the miRNA hybridizes with the target sequence of the miRNA, the degradation-inducing sequence is unable to form the higher-order structure, preferably the stem-loop structure, and the degradation of the RNA nucleic acid molecule can be suppressed. (Note 3) An RNA nucleic acid molecule comprising a sequence encoding a target gene, a PolyA sequence, and a degradation induction sequence, wherein the sequence encoding the target gene, the PolyA sequence, and the degradation induction sequence are arranged in this order from the 5' end to the 3' end, the degradation induction sequence comprises a target sequence of miRNA, and is capable of forming a higher-order structure, preferably a stem-loop structure. (Note 4) The RNA nucleic acid molecule according to Note 2 or 3, wherein the target sequence of miRNA is capable of forming the higher-order structure, preferably a stem-loop structure.(Note 5) The RNA nucleic acid molecule according to Note 2 or 3, wherein a portion of the target sequence of the miRNA is capable of forming the higher-order structure, preferably a stem-loop structure. (Note 6) The RNA nucleic acid molecule according to any one of Notes 2 to 5, wherein the degradation induction sequence comprises 1 to 3 higher-order structures, preferably a stem-loop structure. (Note 7) The RNA nucleic acid molecule according to any one of Notes 2 to 6, wherein the stem-loop structure comprises a stem region and a loop region, and the length of the stem region is 2 to 35 nucleotides. (Note 8) The RNA nucleic acid molecule according to any one of Notes 1 to 7, wherein the degradation induction sequence comprises a binding sequence for heteronuclear ribonucleoprotein (hnRNP), CNOT (CCR4-NOT transcription complex, subunit 4), AU-rich element binding / degradation factor 1 (AUF1), and tristetraproline (TTP). (Note 9) The RNA nucleic acid molecule according to Note 8, wherein, in the absence of a miRNA capable of binding to the target sequence, the miRNA does not hybridize with the target sequence of the miRNA, the degradation-inducing sequence can bind to the hnRNP, CNOT, AUF1, and / or TTP, and by such binding, the degradation of the RNA nucleic acid molecule can be promoted, and in the presence of a miRNA capable of binding to the target sequence, if the miRNA hybridizes with the target sequence of the miRNA, the degradation-inducing sequence cannot bind to the hnRNP, CNOT, AUF1, and / or TTP, and the degradation of the RNA nucleic acid molecule can be suppressed. (Note 10) The RNA nucleic acid molecule according to any one of Notes 1 to 9, wherein the length of the target sequence of the miRNA is 18 to 25 nucleotides. (Note 11) The RNA nucleic acid molecule according to any one of Notes 1 to 10, wherein the target sequence of the miRNA includes mismatched bases with the sequence of the miRNA. (Note 12) The RNA nucleic acid molecule according to any one of Notes 1 to 11, wherein the target sequence of the miRNA has a base sequence in which one or more bases are inserted, added, substituted, and / or deleted from a base sequence complementary to the miRNA. (Note 13) The RNA nucleic acid molecule according to Note 11 or 12, wherein the target sequence of the miRNA has a base sequence in which one or more bases are deleted from a base sequence complementary to the miRNA.(Note 14) The RNA nucleic acid molecule according to any one of Notes 11 to 13, wherein the target sequence of the miRNA has a nucleotide sequence in which one or several bases are deleted from the 5' end in a nucleotide sequence complementary to the miRNA. (Note 15) The RNA nucleic acid molecule according to any one of Notes 11 to 14, wherein the target sequence of the miRNA has a nucleotide sequence in which one or several bases are substituted in a nucleotide sequence complementary to the miRNA. (Note 16) The RNA nucleic acid molecule according to any one of Notes 11 to 15, wherein the target sequence of the miRNA has a nucleotide sequence in which bases are substituted from the group consisting of position 1 and positions 13 to 24, with respect to the base at the 3' end. (Note 17) The RNA nucleic acid molecule according to any one of Notes 11 to 16, wherein the expression level of the target gene is increased compared to an RNA nucleic acid molecule in which the target sequence of the miRNA has a nucleotide sequence complementary to the miRNA. (Note 18) The expression level of the target gene (E) in the absence of the miRNA is compared to an RNA nucleic acid molecule in which the target sequence of the miRNA is a complementary base sequence to the miRNA. ＯＦＦ The expression level of the target gene in the presence of the miRNA (E) ＯＮ ) ratio (E ＯＮ / E ＯＦＦ(Note 19) An RNA nucleic acid molecule according to any one of the appendices 11 to 17, wherein the miRNA increases. (Note 19) An RNA nucleic acid molecule according to any one of the appendices 1 to 18, wherein the target sequence of the miRNA has a base sequence that can hybridize with a miRNA with high miRNA activity in the target cell. (Note 20) An RNA nucleic acid molecule according to appendice 19, wherein the target cell is a cell constituting a tissue selected from the group consisting of muscle tissue, vascular tissue, and liver, or a blood cell. (Note 21) An RNA nucleic acid molecule according to appendice 20, wherein the blood cell is at least one of a T cell and a monocyte. (Note 22) An RNA nucleic acid molecule according to any one of the appendices 1 to 21, wherein the miRNA is selected from the group consisting of miR-142-3p, miR-155-5p, miR-223-3p, miR-122-5p, and miR-126-3p. (Note 23) The RNA nucleic acid molecule according to any one of Notes 19 to 22, wherein the target cell is a T cell and the miRNA is at least one of miR-142-3p and miR-155-5p, the target cell is a monocyte and the miRNA is miR-223-3p, the target cell is a cell constituting the liver and the miRNA is miR-122-5p, and / or the target cell is a vascular endothelial cell and the miRNA is miR-126-3p. (Note 24) The RNA nucleic acid molecule according to any one of Notes 1 to 23, wherein the target sequence of the miRNA has a base sequence that can hybridize with a miRNA whose relative expression level of FLuc is 0.25 or less in the miRNA activity test. (Note 25) The RNA nucleic acid molecule according to any one of Notes 1 to 24, wherein the length of the degradation induction sequence is 26 to 150 bases. (Note 26) The RNA nucleic acid molecule described in any of Notes 1 to 25, wherein the degradation induction sequence is a sequence that does not satisfy (1) to (3) below: (1) a sequence of 20 or more bases that specifically recognizes the PolyA sequence; (2) a sequence that specifically recognizes the 5'UTR in the sequence encoding the target gene; (3) a sequence of 100 bases or more. (Note 27) The RNA nucleic acid molecule described in any of Notes 1 to 26, wherein the target gene is selected from the group consisting of protein-coding genes, disease-causing genes, and genome editing-related genes.(Note 28) The RNA nucleic acid molecule according to Note 27, wherein the protein is selected from the group consisting of growth factors or their receptors, fluorescent proteins, luminescent proteins, antibodies or antibody modifiers, T cell receptors, chimeric antigen receptors, and apoptosis regulatory proteins. (Note 29) The RNA nucleic acid molecule according to any one of Notes 1 to 28, wherein the length of the polyA sequence is 20 nucleotides or more. (Note 30) The RNA nucleic acid molecule according to any one of Notes 1 to 29, wherein the miRNA is a miRNA expressed in cells intended for the introduction of the RNA nucleic acid molecule. (Note 31) The RNA nucleic acid molecule according to any one of Notes 1 to 30, comprising a cap structure or cap analogue at the 5' end. (Note 32) The RNA nucleic acid molecule according to any one of Notes 1 to 31, comprising an internal ribosome entry site (IRES). <DNA nucleic acid molecule> (Note 33) A DNA nucleic acid molecule encoding the RNA nucleic acid molecule according to any one of Notes 1 to 32. <Vector> (Note 34) A vector comprising the DNA nucleic acid molecule described in Note 33. (Note 35) The vector described in Note 34, wherein the vector is a viral vector or a plasmid vector. <Lipid nucleic acid complex> (Note 36) A lipid nucleic acid complex comprising the RNA nucleic acid molecule described in any of Notes 1 to 32 and a lipid. (Note 37) The lipid nucleic acid complex described in Note 36, wherein the lipid is a lipid nanoparticle, and the lipid nucleic acid complex is a nucleic acid lipid nanoparticle. <Pharmaceutical composition> (Note 38) A pharmaceutical composition comprising the RNA nucleic acid molecule described in any of Notes 1 to 32, the DNA nucleic acid molecule described in Note 33, the vector described in Note 34 or 35, and / or the lipid nucleic acid complex described in Note 36 or 37, and a pharmaceutically acceptable carrier. <Method for inducing the expression of a target gene> (Appendix 39) A method for inducing the expression of a target gene, comprising introducing an RNA nucleic acid molecule described in any of Appendix 1 to 32, a DNA nucleic acid molecule described in Appendix 33, a vector described in Appendix 34 or 35, and / or a lipid nucleic acid complex described in Appendix 36 or 37 into cells expressing a target miRNA.<Screening Method> (Note 40) A screening method for selecting cells expressing a target miRNA by introducing an RNA nucleic acid molecule described in any of Notes 1 to 32, a DNA nucleic acid molecule described in Note 33, a vector described in Note 34 or 35, and / or a lipid nucleic acid complex described in Note 36 or 37 into cells. <Treatment Method> (Note 41) A method for treating a disease, comprising the step of administering to a patient an RNA nucleic acid molecule described in any of Notes 1 to 32, a DNA nucleic acid molecule described in Note 33, a vector described in Note 34 or 35, a lipid nucleic acid complex described in Note 36 or 37, and / or a pharmaceutical composition described in Note 38, wherein the target gene is the disease-causing gene and / or genome editing-related gene of the patient, and the miRNA is a miRNA expressed in the disease-causing cells of the patient. (Note 42) A method for inducing the expression of a target gene in cells expressing a target miRNA, comprising the step of administering to a target an RNA nucleic acid molecule described in any of Notes 1 to 32, a DNA nucleic acid molecule described in Note 33, a vector described in Note 34 or 35, and / or a lipid nucleic acid complex described in Note 36 or 37. <Use> (Note 43) Use of an RNA nucleic acid molecule described in any of Notes 1 to 32, a DNA nucleic acid molecule described in Note 33, a vector described in Note 34 or 35, and / or a lipid nucleic acid complex described in Note 36 or 37 for inducing the expression of a target gene in cells expressing a target miRNA.

[0285] As explained above, this disclosure provides an RNA nucleic acid molecule capable of controlling the induction of target gene expression depending on the presence or absence of target miRNA. For this reason, this disclosure is extremely useful, for example, in the medical field.

Claims

1. An RNA nucleic acid molecule comprising a sequence encoding a target gene, a PolyA sequence, and a degradation induction sequence, wherein the sequence encoding the target gene, the PolyA sequence, and the degradation induction sequence are arranged in this order from the 5' end to the 3' end, the degradation induction sequence includes a target sequence of miRNA, and the RNA nucleic acid molecule can be degraded if, in the absence of a miRNA capable of binding to the target sequence, the miRNA does not hybridize to the target sequence, and the RNA nucleic acid molecule can be degraded if, in the presence of a miRNA capable of binding to the target sequence, the miRNA hybridizes to the target sequence, thereby suppressing the degradation of the RNA nucleic acid molecule and inducing the expression of the target gene.

2. The RNA nucleic acid molecule according to claim 1, wherein, in the absence of a miRNA capable of binding to the target sequence, the miRNA does not hybridize with the target sequence of the miRNA, the degradation-inducing sequence is capable of forming a higher-order structure, and the formation of the higher-order structure can induce the degradation of the RNA nucleic acid molecule, and in the presence of a miRNA capable of binding to the target sequence, if the miRNA hybridizes with the target sequence of the miRNA, the degradation-inducing sequence is unable to form the higher-order structure, and the degradation of the RNA nucleic acid molecule can be suppressed.

3. An RNA nucleic acid molecule comprising a sequence encoding a target gene, a PolyA sequence, and a degradation induction sequence, wherein the sequence encoding the target gene, the PolyA sequence, and the degradation induction sequence are arranged in this order from the 5' end to the 3' end, and the degradation induction sequence comprises a target sequence of miRNA and is capable of forming a higher-order structure.

4. The RNA nucleic acid molecule according to claim 2 or 3, wherein the target sequence of the miRNA is capable of forming a stem-loop structure.

5. The RNA nucleic acid molecule according to claim 2 or 3, wherein a portion of the target sequence of the miRNA is capable of forming a stem-loop structure.

6. The RNA nucleic acid molecule according to any one of claims 2 to 5, wherein the degradation-inducing sequence comprises one to three stem-loop structures.

7. The RNA nucleic acid molecule according to any one of claims 4 to 6, wherein the stem-loop structure includes a stem region and a loop region, and the length of the stem region is 2 to 35 nucleotides.

8. The RNA nucleic acid molecule according to any one of claims 1 to 7, wherein the degradation-inducing sequence includes a binding sequence for heteronuclear ribonucleoprotein (hnRNP), CNOT (CCR4-NOT transcription complex, subunit 4), AU-rich element binding / degradation factor 1 (AUF1), and tristetraproline (TTP).

9. The RNA nucleic acid molecule according to claim 8, wherein, in the absence of a miRNA capable of binding to the target sequence, the miRNA does not hybridize with the target sequence of the miRNA, the degradation-inducing sequence is capable of binding to the hnRNP, CNOT, AUF1, and / or TTP, and such binding promotes the degradation of the RNA nucleic acid molecule, and in the presence of a miRNA capable of binding to the target sequence, if the miRNA hybridizes with the target sequence of the miRNA, the degradation-inducing sequence is not capable of binding to the hnRNP, CNOT, AUF1, and / or TTP, and the degradation of the RNA nucleic acid molecule is suppressed.

10. The RNA nucleic acid molecule according to any one of claims 1 to 9, wherein the length of the target sequence of the miRNA is 18 to 25 nucleotides.

11. The RNA nucleic acid molecule according to any one of claims 1 to 10, wherein the target sequence of the miRNA includes a mismatch base with the sequence of the miRNA.

12. The RNA nucleic acid molecule according to any one of claims 1 to 11, wherein the target sequence of the miRNA has a base sequence having one or more base insertions, additions, substitutions, and / or deletions relative to a base sequence complementary to the miRNA.

13. The RNA nucleic acid molecule according to claim 11 or 12, wherein the target sequence of the miRNA has a nucleotide sequence having one or more base deletions in a nucleotide sequence complementary to the miRNA.

14. The RNA nucleic acid molecule according to any one of claims 11 to 13, wherein the target sequence of the miRNA has a nucleotide sequence having a deletion of one or more nucleotides from the 5' end in a nucleotide sequence complementary to the miRNA.

15. The RNA nucleic acid molecule according to any one of claims 11 to 14, wherein the target sequence of the miRNA has a base sequence having one or several base substitutions in a base sequence complementary to the miRNA.

16. The RNA nucleic acid molecule according to any one of claims 11 to 15, wherein the target sequence of the miRNA has a base sequence complementary to the miRNA, in which a base substitution is made between the base at the 3' end and a base selected from the group consisting of positions 13 to 24.

17. The RNA nucleic acid molecule according to any one of claims 11 to 16, wherein the expression level of the target gene is increased compared to an RNA nucleic acid molecule in which the target sequence of the miRNA is a base sequence complementary to the miRNA.

18. Compared to an RNA nucleic acid molecule in which the target sequence of the miRNA is a base sequence complementary to the miRNA, the expression level of the target gene in the absence of the miRNA (E ＯＦＦ The expression level of the target gene in the presence of the miRNA (E) ＯＮ ) ratio (E ＯＮ / E ＯＦＦ The RNA nucleic acid molecule according to any one of claims 11 to 17, wherein ) increases.

19. The RNA nucleic acid molecule according to any one of claims 1 to 18, wherein the target sequence of the miRNA has a base sequence that can hybridize with a miRNA with high activity in the target cell.

20. The RNA nucleic acid molecule according to claim 19, wherein the target cell is a cell constituting a tissue selected from the group consisting of muscle tissue, vascular tissue, and liver, or a blood cell.

21. The RNA nucleic acid molecule according to claim 20, wherein the blood cell is a monocyte.

22. The RNA nucleic acid molecule according to any one of claims 1 to 21, wherein the miRNA is selected from the group consisting of miR-223-3p, miR-122-5p, and miR-126-3p.

23. The RNA nucleic acid molecule according to any one of claims 19 to 22, wherein the target cell is a monocyte and the miRNA is miR-223-3p, the target cell is a cell constituting the liver and the miRNA is miR-122-5p, and / or the target cell is a vascular endothelial cell and the miRNA is miR-126-3p.

24. The RNA nucleic acid molecule according to any one of claims 1 to 23, wherein the target sequence of the miRNA has a base sequence that can hybridize with a miRNA in which the relative expression level of FLuc is 0.25 or less in an activity test of the miRNA.

25. The RNA nucleic acid molecule according to any one of claims 1 to 24, wherein the length of the degradation-inducing sequence is 26 to 150 base pairs.

26. The RNA nucleic acid molecule according to any one of claims 1 to 25, wherein the degradation-inducing sequence is a sequence that does not satisfy the following (1) to (3): (1) a sequence of 20 or more bases that specifically recognizes the PolyA sequence; (2) a sequence that specifically recognizes the 5'UTR in the sequence encoding the target gene; (3) a sequence of 100 or more bases.

27. The RNA nucleic acid molecule according to any one of claims 1 to 26, wherein the target gene is selected from the group consisting of protein-coding genes, disease-causing genes, and genome editing-related genes.

28. The RNA nucleic acid molecule according to claim 27, wherein the protein is selected from the group consisting of growth factors or their receptors, fluorescent proteins, luminescent proteins, antibodies or antibody modifiers, T cell receptors, chimeric antigen receptors, and apoptosis regulatory proteins.

29. The RNA nucleic acid molecule according to any one of claims 1 to 28, wherein the length of the polyA sequence is 20 nucleotides or more.

30. The RNA nucleic acid molecule according to any one of claims 1 to 29, wherein the miRNA is a miRNA expressed in a cell intended for the introduction of the RNA nucleic acid molecule.

31. An RNA nucleic acid molecule according to any one of claims 1 to 30, comprising a cap structure or a cap analogue at its 5' end.

32. An RNA nucleic acid molecule according to any one of claims 1 to 31, comprising an internal ribosome entry site (IRES).

33. A DNA nucleic acid molecule encoding an RNA nucleic acid molecule according to any one of claims 1 to 32.

34. A vector comprising the DNA nucleic acid molecule described in claim 33.

35. The vector according to claim 34, wherein the vector is a viral vector or a plasmid vector.

36. A lipid nucleic acid complex comprising an RNA nucleic acid molecule according to any one of claims 1 to 32 and a lipid.

37. The lipid nucleic acid complex according to claim 36, wherein the lipid is a lipid nanoparticle and the lipid nucleic acid complex is a nucleic acid lipid nanoparticle.

38. A pharmaceutical composition comprising an RNA nucleic acid molecule according to any one of claims 1 to 32, a DNA nucleic acid molecule according to claim 33, a vector according to claim 34 or 35, and / or a lipid nucleic acid complex according to claim 36 or 37, and a pharmaceutically acceptable carrier.

39. A method for inducing the expression of a target gene, comprising introducing an RNA nucleic acid molecule according to any one of claims 1 to 32, a DNA nucleic acid molecule according to claim 33, a vector according to claim 34 or 35, and / or a lipid nucleic acid complex according to claim 36 or 37 into cells expressing a target miRNA.

40. A screening method for selecting cells expressing a target miRNA by introducing an RNA nucleic acid molecule according to any one of claims 1 to 32, a DNA nucleic acid molecule according to claim 33, a vector according to claim 34 or 35, and / or a lipid nucleic acid complex according to claim 36 or 37 into cells.

41. A method for treating a disease, comprising the step of administering to a patient an RNA nucleic acid molecule according to any one of claims 1 to 32, a DNA nucleic acid molecule according to claim 33, a vector according to claim 34 or 35, a lipid nucleic acid complex according to claim 36 or 37, and / or a pharmaceutical composition according to claim 38, wherein the target gene is the patient's disease-causing gene and / or a genome editing-related gene, and the miRNA is a miRNA expressed in the patient's disease-causing cell.

42. A method for inducing the expression of a target gene in cells expressing a target miRNA, comprising the step of administering to a target an RNA nucleic acid molecule according to any one of claims 1 to 32, a DNA nucleic acid molecule according to claim 33, a vector according to claim 34 or 35, and / or a lipid nucleic acid complex according to claim 36 or 37.

43. Use of an RNA nucleic acid molecule according to any one of claims 1 to 32, a DNA nucleic acid molecule according to claim 33, a vector according to claim 34 or 35, and / or a lipid nucleic acid complex according to claim 36 or 37 for inducing the expression of a target gene in cells expressing a target miRNA.

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