Novel double-stranded RNA based on periostin RNA sequence and use thereof

Abstract

Disclosed herein is a double-stranded RNA having a first strand and a second strand complementary to the first strand, wherein the first strand has a main sequence comprising 19-23 bases in which the 5'-terminal base is guanine (G) or cytosine (C), and an additional sequence comprising 2-4 bases added to the 3'-terminal side of the main sequence, wherein the main sequence is determined from among base sequences encoding a signal peptide region of periostin.

Description

Novel double-stranded RNA based on periostin RNA sequence and its use

[0001] The present disclosure relates to double-stranded RNA, compositions containing the double-stranded RNA, and methods of using the same. Specifically, the present disclosure relates to double-stranded RNA used to suppress or inhibit tumor cell growth or metastasis, and compositions comprising the double-stranded RNA. This application claims priority to Japanese Patent Application No. 2023-219517, filed December 26, 2023, the entire contents of which are incorporated herein by reference.

[0002] Cancer is one of the diseases that accounts for a large proportion of deaths in modern people. Periostin (POSTN or PN), a type of extracellular matrix protein, is known to be a gene involved in cancer diseases. Periostin has been suggested to be involved in the pathology of vascular restenosis, inflammation, angiogenesis, age-related macular degeneration, and retinal degeneration, in addition to cancer.

[0003] Under normal conditions, periostin is expressed in various tissues, such as the periosteum, periodontal ligament, heart, and skin. It has been reported that the expression level of periostin increases in various diseases and that splicing variants present in the C-terminal domain of periostin are altered. International Publication WO2019 / 146621 discloses a pharmaceutical composition targeting a periostin splicing variant.

[0004] International Publication WO2019 / 146621

[0005] However, antibody drugs and the like are expensive and it is difficult to maintain consistent quality. Therefore, the present inventors focused on nucleic acid drugs, particularly the signal peptide region of periostin. Nucleic acid drugs can be mass-produced by organic synthesis, and it is easy to control the consistency of quality.

[0006] A main object of the present disclosure is to provide a technique for suppressing or inhibiting cell proliferation in which periostin expression is involved.

[0007] The double-stranded RNA disclosed herein has a first strand and a second strand complementary to the first strand. The first strand has a main sequence consisting of 19 to 23 bases, the 5'-terminal base of which is guanine (G) or cytosine (C), and an additional sequence consisting of 2 to 4 bases added to the 3'-terminal side of the main sequence. Here, the main sequence is determined from a base sequence encoding a periostin signal peptide region.

[0008] The double-stranded RNA can function at least as small interfering RNA (siRNA). That is, such double-stranded RNA is predicted to induce RNA interference (RNAi). This effect is due to the suppression of at least periostin expression, thereby inhibiting the proliferation of cells in which periostin is involved.

[0009] In one aspect of the double-stranded RNA disclosed herein, the second strand has a main sequence complementary to the first strand and an additional sequence consisting of 2 to 4 bases added to the 3'-end of the complementary main sequence. Such double-stranded RNA can function favorably as siRNA, thereby more reliably inhibiting cell proliferation associated with periostin.

[0010] In one aspect of the double-stranded RNA disclosed herein, at least three of the five bases on the 3'-end of the main sequence are adenine (A) and / or uracil (U), thereby more sufficiently suppressing periostin expression and inhibiting cell proliferation associated with periostin.

[0011] In one embodiment of the double-stranded RNA disclosed herein, the nucleotide sequence encoding the periostin signal peptide region is any of the following nucleotide sequences: CCCTTTTTACCCATGTTTT (SEQ ID NO: 1); CTTTTTACCCATGTTTTCT (SEQ ID NO: 2); GCTGCTTATTGTTAACCCT (SEQ ID NO: 3); CTTATTGTTAACCCTATAA (SEQ ID NO: 4); CCCATGTTTTCTCTACTAT (SEQ ID NO: 18); CCATGTTTTCTCTACTATT (SEQ ID NO: 19); CTCTACTATTGCTGCTTAT (SEQ ID NO: 20); CTATTGCTGCTTATTGTTA (SEQ ID NO: 21); CTGCTTATTGTTAACCCTA (SEQ ID NO: 22); CCCTATAAACGCCAACAAT (SEQ ID NO: 23); and CTATAAACGCCAACAATCA (SEQ ID NO: 24). Such double-stranded RNA more specifically suppresses periostin expression, thereby inhibiting the proliferation of cells with increased periostin expression and reducing the effect on normal cells.

[0012] In one embodiment of the double-stranded RNA disclosed herein, the base sequence constituting the additional sequence is thymine-thymine (TT), which can improve the stability of the double-stranded RNA.

[0013] The present disclosure provides a composition capable of inhibiting the proliferation of at least one type of cell. One embodiment of the composition disclosed herein comprises a first strand and a second strand complementary to the first strand, the first strand comprising a main sequence consisting of 19 to 23 bases, the 5'-terminal base of which is guanine (G) or cytosine (C), and an additional sequence consisting of 2 to 4 bases added to the 3'-terminal side of the main sequence. The main sequence comprises double-stranded RNA determined from a base sequence encoding the signal peptide region of periostin. When supplied to cells, such a composition suppresses at least periostin expression and thereby inhibits the proliferation of cells in which periostin is involved.

[0014] In one embodiment of the composition disclosed herein, the cell type whose proliferation is inhibited by the composition is tumor cells, thereby enabling more reliable inhibition of cell proliferation.

[0015] One embodiment of the composition disclosed herein comprises a peptide fragment having cell membrane permeability that allows a foreign substance to be introduced into the cytoplasm by passing through the cell membrane from the outside of the cell, thereby facilitating the introduction of double-stranded RNA into a target cell.

[0016] The present disclosure provides a method for inhibiting the proliferation of at least one type of cell. One embodiment of the method disclosed herein comprises the steps of (1) preparing a composition disclosed herein and (2) supplying the composition to target cells in vitro. This allows for inhibition of cell proliferation in which periostin is involved.

[0017] In one embodiment of the method disclosed herein, the biological species of the cells is the same as the biological species containing periostin, thereby enabling more reliable inhibition of cell proliferation in which periostin is involved.

[0018] 1 is a schematic diagram showing the human periostin (POSTN) gene and its signal peptide region; 2 is a graph showing the cell viability of neuroblastoma cells in Samples 1 to 5 and a Comparative Example; 3 is a graph showing the cell viability of neuroblastoma cells at different addition amounts for Samples 1 to 4 and a Comparative Example; 4 is a graph showing the cell viability of breast cancer cells in Samples 1 to 4 and a Comparative Example; and 5 is a graph showing the cell viability of neuroblastoma cells in Samples 6 to 12 and a Comparative Example.

[0019] <Definition of Terms> The technology disclosed herein is described in detail below. Matters other than those specifically mentioned in this specification (e.g., the structure of double-stranded RNA) that are necessary for implementing this technology (e.g., general matters such as methods for synthesizing polynucleotides, cell culture techniques, and constructs mainly composed of peptides or nucleic acids) can be understood as design matters of a person skilled in the art based on conventional technology in the fields of cell engineering, physiology, medicine, pharmacology, organic chemistry, biochemistry, genetic engineering, protein engineering, molecular biology, genetics, etc. The technology disclosed herein can be implemented based on the content disclosed in this specification and common general technical knowledge in the relevant field.

[0020] As used herein, the term "polynucleotide" refers to a polymer in which multiple (two or more) nucleotides are linked by phosphodiester bonds, and is not limited by the number of nucleotides. For example, a "polynucleotide" herein also encompasses those containing both deoxyribonucleotides and nucleotides. Furthermore, as used herein, the term "artificially designed polynucleotide" refers to a polynucleotide whose nucleotide chain (full length) does not exist alone in nature, but is artificially synthesized by chemical synthesis or biosynthesis (i.e., production based on genetic engineering).

[0021] As used herein, the terms "first strand" and "second strand" refer to one being a sense strand (or coding strand or passenger strand) and the other being an antisense strand (or template strand or non-coding strand or guide strand). That is, if the first strand is a sense strand, the second strand refers to an antisense strand. Also, if the second strand is a sense strand, the first strand refers to an antisense strand. The first strand and the second strand may be completely complementary to each other, or may be at least partially complementary. That is, they may be capable of hybridizing at least under physiological conditions.

[0022] In the present specification, unless the notation "5'" and "3'" is used, the left side of a base sequence always indicates the 5'-terminal side and the right side indicates the 3'-terminal side. Furthermore, in the present specification, the term "amino acid residue" includes the N-terminal amino acid and the C-terminal amino acid of a peptide chain, unless otherwise specified. Furthermore, in the amino acid sequences described in the present specification, the left side always indicates the N-terminal side and the right side indicates the C-terminal side.

[0023] As used herein, the term "tumor" is broadly interpreted and refers to tumors in general (typically malignant tumors), including carcinomas and sarcomas, as well as lesions of the blood or hematopoietic tissues (leukemia, lymphoma, etc.). Furthermore, "tumor cells" are synonymous with "cancer cells," and refer to cells that form such tumors, typically cells that have reached the stage of abnormal proliferation independent of surrounding normal tissues (so-called cancerous cells). Therefore, unless otherwise specified, any cell that is classified as a tumor cell (cancer cell) rather than a normal cell is referred to as a tumor cell, regardless of its origin or properties.

[0024] In this specification, when a numerical range is described as "A to B (where A and B are any numerical values)," it means "A or more and B or less," and also encompasses the meanings of "greater than A and less than B," "greater than A and B or less," and "greater than A and less than B."

[0025] <Periostin> As used herein, "periostin" is also referred to as osteoblast-specific factor 2 (OSF-2). However, the term is intended to encompass all synonyms, including naturally occurring periostin and variants thereof. The biological species from which periostin is derived is not particularly limited, but is preferably the same as the animal species of the cells to which the double-stranded RNA or composition disclosed herein is to be supplied. For example, when the double-stranded RNA or composition disclosed herein is to be supplied to human-derived cells, the main sequence may preferably be a nucleotide sequence based on the nucleotide sequence of human periostin (POSTN or PN). Note that although human-derived periostin is described as a preferred example here, the present technology can also be applied to periostin derived from biological species including mammals other than humans and other animal species.

[0026] Periostin cross-links various extracellular matrices to form fibrotic cell nests. It has also been reported that periostin expression levels are increased and that splicing variants are altered in various diseases. Diseases associated with increased periostin expression levels are those characterized by abnormally proliferating cells. Specific examples of diseases associated with increased periostin expression levels include breast cancer, cholangiocarcinoma, pancreatic cancer, malignant melanoma, glioblastoma, treatment-resistant breast cancer cells undergoing epithelial-mesenchymal transition, heart failure, bronchial asthma, knee osteoarthritis, atopic dermatitis, and idiopathic interstitial pneumonia. Diseases associated with increased periostin expression levels also include age-related macular degeneration, choroidal neovascularization, retinal degeneration, neovascular glaucoma, and diabetic retinopathy, which are characterized by abnormal proliferation of ocular angiogenesis. These diseases are associated with uncontrolled proliferation of abnormal cells and abnormal angiogenesis. That is, the double-stranded RNA and composition disclosed herein can act favorably on the abnormally proliferative cells in which the expression level of periostin is increased due to the above-mentioned diseases, and inhibit their proliferation.

[0027] The nucleotide sequence of periostin is available from international databases. Examples of international databases include the National Center for Biotechnology Information (NCBI), the European Nucleotide Archive (ENA), the DNA Data Bank of Japan (DDBJ), UniPlot, and Ensembl. Specifically, the nucleotide sequence of periostin is provided by NCBI under accession number NM_001135936.2. Information on the signal peptide region of periostin is also available from the above-mentioned international databases.

[0028] Periostin consists of approximately 751 amino acid residues. It is composed of a signal peptide, a cysteine-rich domain (EMI domain), a tetrahomologous repeat domain (fasciclin I (FAS1)), and a C-terminal domain. Periostin has alternative splicing variants in the C-terminal domain, and specific isoforms are formed depending on various diseases.

[0029] The amino acid sequence of SEQ ID NO: 6 consists of 21 amino acid residues and represents the amino acid sequence of the signal peptide of human periostin. The nucleotide sequence of SEQ ID NO: 5 consists of 63 nucleotides and represents the nucleotide sequence of the signal peptide of human periostin.

[0030] <Double-stranded RNA> The double-stranded RNA of the present disclosure is double-stranded RNA consisting of a first strand and a second strand complementary to the first strand. Hereinafter, the first strand will be described in detail as the sense strand and the second strand as the antisense strand. The sense strand has a main sequence consisting of 19 to 23 bases, the 5'-terminal base of which is guanine (G) or cytosine (C), and an additional sequence consisting of 2 to 4 bases added to the 3'-terminal side of the main sequence. The main sequence is determined from the base sequence encoding the signal peptide region of periostin.

[0031] The main sequence is typically composed of a polynucleotide, which is a polymer of ribonucleotides. In other words, the main sequence is composed of RNA. That is, the base sequence of the main sequence is typically represented by the four letters A (adenine), U (uracil), G (guanine), and C (cytosine), or the four letters a, u, g, and c. However, in the attached sequence listing, uracil may be represented by T (thymine).

[0032] The main sequence of the sense strand may be a part of the nucleotide sequence encoding the signal peptide region of periostin. This allows the double-stranded RNA to function as an siRNA (small interfering RNA) targeting periostin. Furthermore, because the nucleotide sequence of the signal peptide region of periostin is located upstream of mRNA, the double-stranded RNA may be able to effectively suppress periostin expression when functioning as an siRNA.

[0033] The double-stranded RNA disclosed herein can at least function as an siRNA. That is, such double-stranded RNA is predicted to induce RNA interference (RNAi). RNAi is a gene silencing process in which short double-stranded RNA such as siRNA suppresses gene expression in a sequence-specific manner. When siRNA is introduced into cells, it forms a complex called RISC (RNA-induced silencing complex) with intracellular proteins. RISC binds to a homologous sequence in mRNA transcribed from a target gene (here, the periostin gene) and specifically cleaves the mRNA, thereby inhibiting translation.

[0034] The main sequence is preferably selected from nucleotide sequences encoding the signal peptide region of periostin, but one or more nucleotides (e.g., two nucleotides) may be substituted with other nucleotides, deleted, and / or added (inserted) within the scope in which the effects of the present technology are exhibited.

[0035] The proportion of the nucleotide sequence encoding the periostin signal peptide region in the main sequence is, for example, preferably 45% or more, and may be 60% or more, 75% or more, 90% or more, or 100% or more, when the entire main sequence is taken as 100%.

[0036] The 5' end of the main sequence is preferably guanine or cytosine. Because guanine and cytosine have stronger binding strength with complementary strands than adenine and uracil, the stability of the 5' end of the sense strand (i.e., the 3' end of the antisense strand) is increased. In other words, the stability of the 5' end of the antisense strand is relatively reduced. Although the details of the mechanism are unclear, RISC, an RNAi-related protein, tends to preferentially incorporate the strand with the more energetically unstable 5' end between the sense strand and the antisense strand. Therefore, by having guanine or cytosine at the 5' end of the main sequence, the antisense strand can be more easily incorporated into RISC, thereby more effectively inducing RNAi. This allows the double-stranded RNA to function favorably as siRNA.

[0037] Of the five bases on the 3'-end of the main sequence, adenine and / or uracil may account for 40% or more (i.e., 2 or more bases), preferably 60% or more (i.e., 3 or more bases), 80% or more (i.e., 4 or more bases), or even 100% (i.e., 5 bases). This makes the 5'-end of the antisense strand relatively less stable than the 3'-end. As a result, the antisense strand is more easily incorporated into RISC, allowing for more effective induction of RNAi.

[0038] The GC content of the entire main sequence (the total proportion of G and C in the entire base sequence constituting the main sequence) is not particularly limited, but may be, for example, 20% to 60% or less, preferably 30% to 50% or less, or may be 30% to 45% or less. The GC content is a parameter related to the binding strength between the antisense strand incorporated into RISC and RNA having the main sequence, the ease of cleavage of RNA, etc. The above GC content allows the RNAi effect to be efficiently exerted.

[0039] As shown in FIG. 1 , the main sequence can be selected from 19 to 23 bases starting from G or C in the signal peptide region of the gene encoding human periostin (POSTN) (for example, SEQ ID NOS: 1 to 2 and 18 to 19 are located upstream of the signal peptide region, and SEQ ID NOS: 5 to 6 and 20 to 24 are located downstream of the signal peptide region). For example, the main sequence may have the following base sequence: CCCUUUUUACCCAUGUUUU (SEQ ID NO: 39); CUUUUUACCCAUGUUUUCU (SEQ ID NO: 40); GCUGCUUAUUGUUAACCCU (SEQ ID NO: 41); CUUAUUGUUAACCCUAUAA (SEQ ID NO: 42); CCCAUGUUUUCUCUACUAU (SEQ ID NO: 43); CCAUGUUUUCUCUACUAUU (SEQ ID NO: 44); CUCUACUAUUGCUGCUUAU (SEQ ID NO: 45); CUAUUGCUGCUUAUUGUUA (SEQ ID NO: 46); CUGCUUAUUGUUAACCCUA (SEQ ID NO: 47); The nucleotide sequences shown in SEQ ID NOs: 39 to 49 can be either CCCUAUAAACGCCAACAAU (SEQ ID NO: 48); or CUAUAAACGCCAACAAUCA (SEQ ID NO: 49). The nucleotide sequences shown in SEQ ID NOs: 39 to 49 are all composed of RNA. The nucleotide sequences shown in SEQ ID NOs: 39 to 49 are all specific to the periostin gene and can avoid the risk of inhibiting translation of mRNA in host cells having a nucleotide sequence similar to the target sequence (so-called off-target effect). Furthermore, double-stranded RNAs having the nucleotide sequences shown in SEQ ID NOs: 39 to 49 as their main sequences significantly suppress the proliferation of abnormally proliferating cells even at low concentrations, and can therefore avoid nonspecific inhibition of expression, nonspecific inhibition of cell growth, stress on cells, and the like.

[0040] The nucleotide sequence shown in SEQ ID NO: 1 (DNA sequence corresponding to the RNA sequence of SEQ ID NO: 39) is the 7th to 25th nucleotides of the nucleotide sequence encoding human periostin (i.e., the sequence from the initiation codon to the termination codon). The nucleotide sequence shown in SEQ ID NO: 2 (DNA sequence corresponding to the RNA sequence of SEQ ID NO: 40) is the 9th to 27th nucleotides of the nucleotide sequence encoding human periostin. The nucleotide sequence shown in SEQ ID NO: 3 (DNA sequence corresponding to the RNA sequence of SEQ ID NO: 41) is the 36th to 54th nucleotides of the nucleotide sequence encoding human periostin. The nucleotide sequence shown in SEQ ID NO: 4 (DNA sequence corresponding to the RNA sequence of SEQ ID NO: 42) is the 40th to 58th nucleotides of the nucleotide sequence encoding human periostin. The nucleotide sequence shown in SEQ ID NO: 18 (DNA sequence corresponding to the RNA sequence of SEQ ID NO: 43) is the 16th to 34th nucleotides of the nucleotide sequence encoding human periostin. The nucleotide sequence shown in SEQ ID NO: 19 (DNA sequence corresponding to the RNA sequence of SEQ ID NO: 44) is the 17th to 35th nucleotides of the nucleotide sequence encoding human periostin. The nucleotide sequence shown in SEQ ID NO: 20 (DNA sequence corresponding to the RNA sequence of SEQ ID NO: 45) is the 26th to 44th nucleotide sequence of the nucleotide sequence encoding human periostin. The nucleotide sequence shown in SEQ ID NO: 21 (DNA sequence corresponding to the RNA sequence of SEQ ID NO: 46) is the 31st to 49th nucleotide sequence of the nucleotide sequence encoding human periostin. The nucleotide sequence shown in SEQ ID NO: 22 (DNA sequence corresponding to the RNA sequence of SEQ ID NO: 47) is the 37th to 55th nucleotide sequence of the nucleotide sequence encoding human periostin. The nucleotide sequence shown in SEQ ID NO: 23 (DNA sequence corresponding to the RNA sequence of SEQ ID NO: 48) is the 51st to 69th nucleotide sequence of the nucleotide sequence encoding human periostin. The nucleotide sequence shown in SEQ ID NO: 24 (DNA sequence corresponding to the RNA sequence of SEQ ID NO: 49) is the 53rd to 71st nucleotide sequence of the nucleotide sequence encoding human periostin. The nucleotide sequences shown in SEQ ID NOs: 1 to 4 and 18 to 24 are the nucleotide sequences of parts of the signal peptide region of human periostin. In the attached sequence listing, the nucleotide sequence shown in SEQ ID NO: 5 is the nucleotide sequence from base 1 to base 63 of the nucleotide sequence encoding human periostin, and is the entire nucleotide sequence of the signal peptide region of human periostin.

[0041] Double-stranded RNAs comprising the main sequences shown in SEQ ID NOs: 1 to 4 and 18 to 24 can suppress or inhibit the proliferation of at least one type of cell by supplying them to the cell. Typically, by supplying them to tumor cells (e.g., neuroblastoma, breast cancer, etc.), the proliferation of the tumor cells can be suppressed or inhibited. Periostin is expressed at low levels in normal cells other than tumor cells, but is overexpressed in tumor cells. Therefore, even if the double-stranded RNA disclosed herein is supplied to normal cells, the amount of periostin present in normal cells is relatively small, and therefore the double-stranded RNA is thought to have little effect.

[0042] <Additional Sequence> The sense strand of the double-stranded RNA disclosed herein may have an additional sequence consisting of 2 to 4 bases added to the 5'-end or 3'-end of the main sequence. Preferably, the additional sequence is added to the 3'-end of the main sequence. The addition of an additional sequence can more effectively induce RNAi.

[0043] The additional sequence is composed of a polynucleotide (dimer, trimer, or tetramer). The polynucleotide constituting the additional sequence may be composed of only ribonucleotides, only deoxynucleotides, or both ribonucleotides and deoxynucleotides. That is, the sense strand and the antisense strand may be entirely RNA, or may be chimeric polynucleotides of RNA and DNA. The additional sequence may also contain modified deoxyribonucleotides, modified ribonucleotides, other known nucleotide analogs, and the like.

[0044] The base sequence constituting the additional sequence is not particularly limited, but preferably contains at least one base of adenine, uracil, or thymine. From the viewpoint of improving the stability of the double-stranded RNA, the base sequence constituting the additional sequence is more preferably TT (thymine-thymine).

[0045] <Sense strand and antisense strand> The sense strand is composed of, for example, a base sequence of 21 to 27 bases, and may be composed of 21 to 25 bases, or 21 to 23 bases. In a preferred example, it is composed of 21 bases, including a main sequence of 19 bases and an additional sequence of 2 bases. In such an example, RNAi can be effectively induced.

[0046] The antisense strand has a base sequence complementary to the main sequence of the sense strand. This allows the antisense strand to hybridize with the sense strand, forming a double-stranded structure. The base sequence of the antisense strand may also be partially complementary to the main sequence of the sense strand. That is, one or more bases (e.g., two bases) of the antisense strand may be substituted, deleted, and / or added (inserted) with other bases. As long as the sense strand and the antisense strand can hybridize at least under physiological conditions, they can function as siRNA. The complementary base sequence is typically composed of a ribonucleotide polymer (RNA).

[0047] In the double-stranded RNA of the present disclosure, the sense strand or antisense strand is typically composed of chemically unmodified ribonucleotides (RNA). However, the double-stranded RNA of the present disclosure may also contain DNA, chemically modified DNA or RNA, other known nucleotide analogs, etc., to the extent that the technology of the present disclosure is not significantly impaired. That is, one or more bases (e.g., two bases) in the sense strand or antisense strand may be substituted with chemically modified RNA (or DNA) such as methylated or pseudouridylated. Examples of chemically modified RNA include pseudouridine, N1-methylpseudouridine, 5-methylcytosine, or inosine. For example, one or more bases (e.g., two bases) of uridine in the double-stranded RNA of the present disclosure can be substituted with pseudouridine.

[0048] In the double-stranded RNA of the present disclosure, the antisense strand may be composed of a main sequence complementary to the sense strand and an additional sequence consisting of 2 to 4 bases added to the 5'-end or 3'-end of the complementary main sequence. From the viewpoint of improving the function of the siRNA, the additional sequence may be added to the 3'-end of the complementary base sequence. In a preferred example, when the additional sequence of the sense strand is added to the 3'-end of the main sequence, the additional sequence of the antisense strand is added to the 3'-end of the complementary base sequence. The configuration of the additional sequence in the antisense strand may be the same as the configuration of the additional sequence in the sense strand described above. Typically, the base sequence of the additional sequence in the antisense strand is the same as the additional sequence in the sense strand to which it hybridizes, but it may also be a different base sequence.

[0049] The antisense strand is composed of, for example, a base sequence of 21 to 27 bases, and may be composed of 21 to 25 bases, or 21 to 23 bases. The antisense strand is composed of a base sequence of the same length as the sense strand, and all or part of the base sequence excluding the additional sequence is composed of a base sequence complementary to the main sequence of the sense strand. In a preferred example, the antisense strand is composed of a base sequence of the same length as the sense strand, and all of the base sequence excluding the additional sequence is composed of a base sequence complementary to the main sequence of the sense strand.

[0050] <Method for Producing Double-Stranded RNA> The sense strand and antisense strand constituting the double-stranded RNA disclosed herein can be produced according to a general chemical synthesis method. For example, they can be synthesized using a commercially available DNA / RNA automatic synthesizer. Alternatively, the sense strand and antisense strand may be synthesized in vitro or in vivo based on genetic engineering techniques. The synthesized sense strand and antisense strand are preferably purified, and can be purified, for example, by HPLC or the like.

[0051] The double-stranded RNA disclosed herein can be produced, for example, by annealing (hybridizing) a sense strand and an antisense strand. Annealing can be performed according to conventional methods. For example, annealing can be performed by mixing equal amounts of the sense strand and the antisense strand in a solvent, heating at 90°C for 1 to 5 minutes, and then cooling to 4°C to room temperature. Examples of such solvents that can be used include distilled water, pure water, ultrapure water, and buffers (e.g., HEPES-KOH buffer at pH 7.4, PBS, etc.). To prevent active RNase (RNA degrading enzyme) from being mixed into the solvent, solvents that have been treated with, for example, DEPC or autoclaved are preferably used.

[0052] <Composition> The composition disclosed herein contains the double-stranded RNA described above. In addition to the double-stranded RNA described above, the composition may contain various pharmaceutically acceptable carriers depending on the intended use. Preferred carriers include those commonly used in medicines as diluents, excipients, etc. The carriers vary depending on the intended use and form of the composition. Typical examples include water, physiological buffer solutions, various organic solvents, etc. The carrier may also be an aqueous solution of an appropriate concentration of alcohol (e.g., ethanol), glycerol, a non-drying oil such as olive oil, or liposomes. Examples of secondary components that may be contained in the pharmaceutical composition include various fillers, extenders, binders, humectants, surfactants, dyes, fragrances, etc. The composition may also contain carriers used in conventional drug delivery systems (DDS).

[0053] The form of the composition disclosed herein is not particularly limited. For example, typical composition forms include solutions, suspensions, emulsions, aerosols, foams, granules, powders, tablets, capsules, and ointments. Furthermore, for use in injections, etc., the composition can be made into a lyophilized product or granulated product that is dissolved in physiological saline or an appropriate buffer solution (e.g., PBS) immediately before use to prepare a medicinal solution. Furthermore, the process of preparing various forms of drugs (compositions) using double-stranded RNA (main component) and various carriers (secondary components) can be based on conventionally known methods. Since such formulation methods do not characterize the present disclosure, detailed explanations are omitted. For example, a detailed source of information regarding formulations is "Comprehensive Medicinal Chemistry," edited by Corwin Hansch, published by Pergamon Press (1990).

[0054] The compositions disclosed herein inhibit the proliferation of at least one type of cell. The cells whose proliferation is inhibited are cells involved in the expression of periostin, such as tumor cells (e.g., sarcoma, neuroblastoma, retinoblastoma, embryonal tumor, etc.), fibroblasts, epithelial cells, mesothelial cells, osteoblasts, etc. Among these, the compositions disclosed herein preferably inhibit the proliferation of abnormally proliferating cells such as tumor cells. That is, the double-stranded RNA and compositions disclosed herein can be preferably used as antitumor agents (anticancer agents) that suppress the proliferation of tumor cells.

[0055] One embodiment of the composition disclosed herein includes, in addition to the double-stranded RNA described above, a peptide fragment (cell-penetrating peptide, CPP) that has cell membrane permeability and can pass through the cell membrane from the outside of a cell to introduce a foreign substance into the cytoplasm. The peptide fragment is directly or indirectly linked to the double-stranded RNA of the present disclosure to construct a construct of the peptide fragment and double-stranded RNA. Generally, double-stranded RNA is negatively charged and therefore cannot pass through the cell membrane. However, for example, by directly or indirectly linking the double-stranded RNA disclosed herein to the N-terminus and / or C-terminus of the peptide fragment, the construct of the peptide fragment and the double-stranded RNA can be introduced into the cytoplasm. The number of amino acid residues in the peptide fragment is not limited as long as cell membrane permeability is not impaired.

[0056] When the peptide fragment and the double-stranded RNA are indirectly bound, for example, a linker is placed between the peptide fragment and the double-stranded RNA. The type of linker is not particularly limited. Typically, it is a peptidic linker, a non-peptidic linker, or the like. Furthermore, the method for binding the peptide fragment and the double-stranded RNA is not particularly limited, and can be carried out according to various conventionally known scientific techniques.

[0057] One embodiment of the composition disclosed herein comprises a peptide fragment and the double-stranded RNA of the present disclosure. However, the double-stranded RNA does not necessarily have to be bound to the N- or C-terminus of the peptide fragment. In such an embodiment, the double-stranded RNA and the peptide fragment may form a complex, for example, through electrical or molecular interaction. Such a complex is more easily introduced into eukaryotic cells, thereby enabling efficient introduction of the double-stranded RNA. Nucleic acids such as double-stranded RNA are typically negatively charged. Therefore, the peptide fragment used preferably has a high proportion of basic amino acids and is positively charged. Furthermore, the proportion of the peptide fragment in this case may be 5 to 100 times, preferably 40 to 60 times, the molar ratio of the double-stranded RNA.

[0058] <Method for producing the composition disclosed herein and use thereof> The present disclosure may provide a method for inhibiting the proliferation of at least one type of cell using the composition disclosed herein. The method disclosed herein includes the steps of preparing the composition disclosed herein and supplying the composition to a target cell.

[0059] In the preparation step, for example, as described above, the composition disclosed herein may be prepared by a conventionally known method.

[0060] In the supplying step, the composition disclosed herein is supplied to at least one type of cell (e.g., tumor cells) in vivo or ex vivo. The animal species of the supplied cells is not particularly limited and may be, for example, mammals, birds, amphibians, reptiles, fish, etc. Preferably, the animal species from which periostin, which is the basis of the main sequence of the double-stranded RNA contained in the composition, is derived is the same as the animal species of the target cells. The type of target cells is also not particularly limited and is preferably tumor cells, more preferably neuroblastoma or breast cancer cells. Note that although cells other than tumor cells may be present at the destination of the composition, the composition may be supplied only to the target cells (i.e., tumor cells).

[0061] The method of administering the composition may be similar to methods conventionally used in animal treatments and is not particularly limited. The composition can be used in vivo in a manner and dosage appropriate for its form and purpose. For example, as a liquid formulation, it can be administered in a desired amount to the affected area (e.g., malignant tumor tissue, virus-infected tissue, inflammatory tissue, etc.) of a patient or animal (i.e., living body) by intravenous, intralymphatic, intramuscular, subcutaneous, intradermal, or intraperitoneal injection. Alternatively, a solid form such as a tablet, or a gel or aqueous jelly such as an ointment, can be administered directly to a specific tissue (e.g., an affected area such as a tissue or organ containing tumor cells, inflammatory cells, etc.). Alternatively, a solid form such as a tablet can be administered orally. For oral administration, encapsulation or application of a protective (coating) material is preferred to prevent degradation by digestive enzymes in the digestive tract.

[0062] The amount of the composition to be supplied in vivo is not particularly limited. For example, the lower limit of the amount of double-stranded RNA per kg of animal may be 0.01 mg or more, 0.05 mg or more, or 0.1 mg or more. The upper limit of the amount of double-stranded RNA per kg of animal may be, for example, 10 mg or less, 5 mg or less, or 1 mg or less. The amount of the composition to be supplied in vitro is not particularly limited. In the culture medium of the subject to be supplied, such as cells, the lower limit of the double-stranded RNA concentration may be, for example, 1 nM or more, 5 nM or more, or 10 nM or more. The upper limit of the double-stranded RNA concentration in such culture medium may be, for example, 10 μM or less, 5 μM or less, 2 μM or less, 1 μM or less, or 100 nM or less.

[0063] The compositions disclosed herein can be delivered to the interior of target cells by known transfection methods. Examples include chemical gene transfer methods using cationic molecules (e.g., commercially available transfection reagents), physical transfer methods such as microinjection and electroporation, and biological gene transfer methods using viruses. Alternatively, as described above, the compositions may be delivered to the interior of cells using cell membrane-permeable peptide fragments.

[0064] Below, several test examples relating to the technology disclosed herein will be described, but it is not intended that the technology disclosed herein be limited to those shown in these test examples.

[0065] <Preparation of double-stranded RNA> Polynucleotides having the base sequences shown in SEQ ID NOS: 8 to 17 and 25 to 38 were artificially synthesized. The base sequences of each polynucleotide are shown in Table 1. In each polynucleotide, the "TT" (additional sequence) at the 3' end is DNA, and the remaining sequence (main sequence) is composed of RNA. The obtained polynucleotides were annealed with a sense strand and an antisense strand having complementary sequences to prepare double-stranded RNAs used in Samples 1 to 12 shown in Table 1. Each of the double-stranded RNAs shown in Samples 1 to 12 was dissolved in PBS to give an RNA concentration of 2 mM, preparing an RNA solution.

[0066]

[0067] As shown in Table 1, the sense strand of the double-stranded RNA of Sample 1 consisted of a main sequence consisting of SEQ ID NO: 18 (a part of the nucleotide sequence encoding the periostin signal peptide) and an additional sequence consisting of TT added to the 3'-end of the main sequence. The sense strand of the double-stranded RNA of Samples 2 to 4 consisted of a main sequence consisting of SEQ ID NOs: 19 to 21 (a part of the nucleotide sequence encoding the periostin signal peptide) and an additional sequence consisting of TT added to the 3'-end of the main sequence. The sense strand of the double-stranded RNA of Sample 5 consisted of a main sequence consisting of SEQ ID NO: 7 (a randomly artificially generated sequence) and an additional sequence consisting of TT added to the 3'-end of the main sequence. The antisense strand in each example consisted of a sequence complementary to the main sequence and an additional sequence consisting of TT added to the 3'-end of the main sequence.

[0068] <Cell proliferation test of human neuroblastoma cells> Human neuroblastoma cells, the SK-N-SH strain, were used as tumor cells. SK-N-SH cells were pre-cultured in a culture medium containing 10% FBS (fetal bovine serum) + E-MEM (Fujifilm Wako Pure Chemical Industries, Ltd., Cat. No. 051-07615) + 1% MEM non-essential amino acid solution (Fujifilm Wako Pure Chemical Industries, Ltd., Cat. No. 139-15651). Note that 0.5% penicillin-streptomycin (Fujifilm Wako Pure Chemical Industries, Ltd., Cat. No. 168-23191) was added to the culture medium only during pre-culture, but was not added during the following culture and evaluation.

[0069] On day 1, the SK-N-SH cells that had adhered to the culture plate were washed with PBS, and then a 0.25% trypsin / EDTA solution was added and incubated at 37°C for 2 minutes. After this incubation, the above-mentioned culture medium was added to inactivate the trypsin. The cells were then precipitated by centrifugation at 150 x g for 5 minutes. After removing the supernatant resulting from centrifugation, the above-mentioned culture medium was added to the precipitate (cell pellet), and approximately 5 x 10 ４ A cell suspension of 5 × 10 cells / mL was prepared. One commercially available 96-well plate was prepared, and the cell suspension was added to each well at a concentration of 5 × 10 ３ The cells were seeded at 100 μL per well and incubated at 37°C, 5% CO ２ and incubated overnight under

[0070] (Sample 1) On the second day, 3 μL of 2 mM siRNA solution prepared with PBS was mixed with 75 μL of Opti-MEM™ to prepare Solution A. Furthermore, 4.5 μL of Lipofectamine™ RNAiMAX was mixed with 75 μL of Opti-MEM™ to prepare Solution B. Next, equal amounts of Solution A and Solution B were mixed to prepare Solution C, which was then incubated at room temperature for 5 minutes. The prepared Solution C was added to wells containing cultured SK-N-SH cells at 11 μL / well (final concentration of double-stranded RNA: 4 μM). The mixture was then incubated at 37°C, 5% CO ２ The mixture was incubated under reduced pressure for 3 days.

[0071] Cell proliferation was evaluated using Cell Counting Kit-8 (CCK-8, Dojin Kagaku Kenkyusho). On day 5 (day 3 after siRNA addition), the 96-well plate in which SK-N-SH cells had been cultured was removed, 10 μL of CCK-8 was added to each well, and the cells were incubated at 37°C, 5% CO ２ The wells were incubated for 1.5 hours under reduced pressure. The absorbance at 450 nm of each well was measured. The absorbance was calculated as the average of the absorbances of three wells. In addition, a blank well was prepared containing only the culture medium and CCK-8 reagent. The absorbance of Sample 1 minus the absorbance of the blank was used as the measured value for Sample 1.

[0072] (Samples 2 to 4, Sample 5) Samples 2 to 4 were prepared in the same manner as Sample 1, except that the double-stranded RNA in Sample 1 was replaced with the double-stranded RNA in Samples 2 to 4 shown in Table 1. Sample 5 was prepared in the same manner as Sample 1, except that the double-stranded RNA in Sample 1 was replaced with the double-stranded RNA in Sample 5 shown in Table 1.

[0073] Comparative Example In the comparative example, the procedure was the same as that of Sample 1, except that a PBS solution was used instead of the RNA solution in Sample 1. That is, in the comparative example, no double-stranded RNA was introduced.

[0074] Untreated wells were prepared in the same manner as Sample 1, except that the RNA solution and Lipofectamine™ RNAiMAX were not added. The cell viability in each test example is shown in Figure 2 as a percentage, with the measured value for the untreated well set at 100%.

[0075] As shown in Figure 2, Samples 1 to 4 had a significantly lower cell viability than the comparative example. These test results suggest that the double-stranded RNAs of Samples 1 to 4 have the function of inhibiting the proliferation of tumor cells (human neuroblastoma cells). Furthermore, the base sequence of SEQ ID NO: 7 is a random sequence with an ATCG content approximately equivalent to that of the base sequences of SEQ ID NOs: 1 to 4. Sample 5, which contained SEQ ID NO: 7 as the main sequence, did not affect cell proliferation. Therefore, these sequences of SEQ ID NOs: 1 to 4 are specific to the periostin gene. Therefore, the double-stranded RNAs of Samples 1 to 4 can avoid off-target effects, do not affect other organs, and are expected to be useful in clinical applications.

[0076] <Cell proliferation test of human neuroblastoma cells using low-concentration double-stranded RNA> Double-stranded RNA for samples 1 to 4 shown in Table 1 was prepared. Each double-stranded RNA shown in samples 1 to 4 was dissolved in PBS to a 2 mM RNA concentration to prepare an RNA solution. This was then further diluted 10-fold with PBS to prepare a low-concentration RNA solution with an RNA concentration of 200 μM. A test similar to the cell proliferation test of human neuroblastoma cells was performed except for the use of the low-concentration RNA solution. Specifically, the final concentration of double-stranded RNA added to wells containing cultured SK-N-SH cells was adjusted to 0.4 μM. The cell viability in each test example was expressed as a percentage of the measured value for untreated wells, which was set at 100%.

[0077] Figure 3 is a graph comparing cell viability when the final concentration of added double-stranded RNA was 4.0 μM and 0.4 μM. As shown in Figure 3, the cell viability of Samples 1 to 4 decreased. Furthermore, the cell viability of Samples 1 to 4 was significantly lower than that of the comparative example. This indicates that the double-stranded RNAs of Samples 1 to 4 have the ability to inhibit the proliferation of tumor cells (neuroblastoma cells) even at low concentrations. Because the double-stranded RNAs of Samples 1 to 4 have sufficient tumor cell growth inhibitory activity even at low concentrations, they can avoid nonspecific expression inhibition and nonspecific cell growth inhibition, making them highly promising for clinical applications. Among these, the double-stranded RNAs of Samples 3 and 4 exhibited particularly excellent inhibitory effects on the proliferation of human neuroblastoma cells. Furthermore, the double-stranded RNA of Sample 4 had the same or better cell-inhibiting activity even at one-tenth the concentration.

[0078] Additionally, double-stranded RNA was prepared for each of Samples 6 to 12 shown in Table 1. The double-stranded RNAs shown in Samples 6 to 12 were dissolved in PBS to a 2 mM RNA concentration to prepare an RNA solution. This was then further diluted 10-fold with PBS to prepare a low-concentration RNA solution with an RNA concentration of 200 μM. Tests were performed in the same manner as in the cell proliferation test for human neuroblastoma cells, except for the use of the low-concentration RNA solution. Specifically, in the tests for Samples 6 to 12, the final concentration of double-stranded RNA added to the wells containing SK-N-SH cells was adjusted to 0.4 μM. The cell viability in each test example was expressed as a percentage of the measured value for the untreated well, which was set at 100%. In the comparative examples, sterilized ultrapure water was used instead of the sample (no double-stranded RNA was added in these comparative examples).

[0079] Figure 5 shows the cell viability of human neuroblastoma cells after 3 days of transfection with the double-stranded RNAs shown in Samples 6 to 12. The double-stranded RNAs in Samples 6 to 12 were able to inhibit tumor cell (neuroblastoma cell) proliferation even at low concentrations. Therefore, nonspecific inhibition of expression and nonspecific inhibition of cell proliferation can be avoided, and clinical application is highly anticipated. Note that the tests using the double-stranded RNAs shown in Samples 6 to 12 were conducted independently on a different day from the tests using the double-stranded RNAs shown in Samples 1 to 5 above.

[0080] <Cell proliferation test of human breast cancer cells> The same procedure as in the cell proliferation test of human neuroblastoma cells was performed except that the human breast cancer cell line MDA-MB-231 was used as the tumor cells. The cell viability in each test example is expressed as a percentage, with the measured value for the untreated well taken as 100%, and is shown in Figure 4.

[0081] 4, the cell viability of Samples 1 to 4 was reduced, and was significantly lower than that of the comparative example. This suggests that the double-stranded RNAs of Samples 1 to 4 have the function of inhibiting the proliferation of tumor cells (breast cancer cells).

[0082] Although specific examples of the technology disclosed herein have been described in detail above, these are merely examples and do not limit the scope of the claims. The technology described in the claims includes various modifications and alterations of the specific examples exemplified above.

[0083] In the technology disclosed herein, each component and each process mentioned herein may be omitted or combined as appropriate, unless a particular problem arises. This specification also includes the disclosures described in the following sections.

[0084] Item 1: A double-stranded RNA having a first strand and a second strand complementary to the first strand, wherein the first strand has a main sequence consisting of 19 to 23 bases, the 5'-terminal base of which is guanine (G) or cytosine (C), and an additional sequence consisting of 2 to 4 bases added to the 3'-terminal side of the main sequence, wherein the main sequence is determined from a base sequence encoding a signal peptide region of periostin.

[0085] Item 2: The double-stranded RNA according to Item 1, wherein the second strand has a main sequence complementary to the first strand and an additional sequence consisting of 2 to 4 bases added to the 3'-end of the complementary main sequence.

[0086] Item 3: The double-stranded RNA according to Item 1 or 2, wherein at least three of the five bases on the 3'-terminal side of the main sequence are adenine (A) and / or uracil (U).

[0087] Item 4: The nucleotide sequence encoding the periostin signal peptide region is any of the following nucleotide sequences: CCCTTTTTACCCATGTTTT (SEQ ID NO: 1); CTTTTTACCCATGTTTTCT (SEQ ID NO: 2); GCTGCTTATTGTTAACCCT (SEQ ID NO: 3); CTTATTGTTAACCCTATAA (SEQ ID NO: 4); CCCATGTTTTCTCTACTAT (SEQ ID NO: 18); CCATGTTTTCTCTACTATT (SEQ ID NO: 19); CTCTACTATTGCTGCTTAT (SEQ ID NO: 20); CTATTGCTGCTTATTGTTA (SEQ ID NO: 21); CTGCTTATTGTTAACCCTA (SEQ ID NO: 22); Item 4. The double-stranded RNA according to any one of Items 1 to 3, consisting of either CCCTATAAACGCCAACAAT (SEQ ID NO: 23); and CTATAAACGCCAACAATCA (SEQ ID NO: 24).

[0088] Item 5: The double-stranded RNA according to any one of Items 1 to 4, wherein the base sequence constituting the additional sequence is thymine-thymine (TT).

[0089] Item 6: A composition that inhibits the proliferation of at least one type of cell, comprising the double-stranded RNA of any one of Items 1 to 5.

[0090] Item 7: The composition of Item 6, wherein the cells are tumor cells.

[0091] Item 8: The composition according to Item 5 or 6, wherein the composition comprises a peptide fragment having cell membrane permeability that can pass through the cell membrane from the outside of the cell and introduce a foreign substance into the cytoplasm.

[0092] Item 9: A method for suppressing the proliferation of at least one type of cell, comprising: preparing the composition according to any one of Items 6 to 8; and supplying the composition to the cell in vitro or in vivo.

[0093] Item 10: The method according to any one of Items 7 to 9, wherein the animal species of the cells and the periostin are the same.

[0094] As described above, the double-stranded RNA disclosed herein can inhibit (or suppress) cell proliferation. Therefore, by using the double-stranded RNA, it is possible to provide a composition (e.g., an antitumor agent) that inhibits the proliferation of at least one type of cell (e.g., cells involved in diseases caused by abnormal cell proliferation, such as tumor cells).

Claims

1. A double-stranded RNA having a first strand and a second strand complementary to the first strand, wherein the first strand has a main sequence consisting of 19 to 23 bases, the 5'-terminal base of which is guanine (G) or cytosine (C), and an additional sequence consisting of 2 to 4 bases added to the 3'-terminal side of the main sequence, wherein the main sequence is determined from a base sequence encoding a signal peptide region of periostin.

2. The double-stranded RNA according to claim 1, wherein the second strand has a main sequence complementary to the first strand and an additional sequence consisting of 2 to 4 bases added to the 3' end of the complementary main sequence.

3. The double-stranded RNA according to claim 1, wherein at least three of the five bases on the 3'-terminal side of the main sequence are adenine (A) and / or uracil (U).

4. The nucleotide sequence encoding the periostin signal peptide region is any of the following nucleotide sequences: CCCTTTTTACCCATGTTTT (SEQ ID NO: 1); CTTTTTACCCATGTTTTCT (SEQ ID NO: 2); GCTGCTTATTGTTAACCCT (SEQ ID NO: 3); CTTATTGTTAACCCTATAA (SEQ ID NO: 4); CCCATGTTTTCTCTACTAT (SEQ ID NO: 18); CCATGTTTTCTCTACTATT (SEQ ID NO: 19); CTCTACTATTGCTGCTTAT (SEQ ID NO: 20); CTATTGCTGCTTATTGTTA (SEQ ID NO: 21); CTGCTTATTGTTAACCCTA (SEQ ID NO: 22); The double-stranded RNA of claim 1 , consisting of either CCCTATAAACGCCAACAAT (SEQ ID NO: 23); or CTATAAACGCCAACAATCA (SEQ ID NO: 24).

5. The double-stranded RNA according to claim 1, wherein the base sequence constituting the additional sequence is thymine-thymine (TT).

6. A composition for inhibiting the proliferation of at least one type of cell, comprising the double-stranded RNA according to any one of claims 1 to 4.

7. The composition of claim 6, wherein the cell is a hyperproliferative cell.

8. The composition according to claim 6, comprising a peptide fragment having cell membrane permeability that can pass through the cell membrane from the outside of the cell and introduce a foreign substance into the cytoplasm.

9. A method for inhibiting the proliferation of at least one type of cell, comprising the steps of: providing a composition according to claim 6; and delivering said composition to said cells in vitro.

10. The method according to claim 7, wherein the biological species of the cells is the same as the biological species containing the periostin.

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