Double-stranded RNA targeting psap and use thereof

Abstract

The present disclosure provides a technique for suppressing the proliferation of cells. A double-stranded RNA disclosed herein comprises a first strand and a second strand. The first strand comprises a main sequence comprising 19-23 bases. The second strand comprises a complementary sequence that complementarily binds to the main sequence. The main sequence is a part of the nucleotide sequence encoding prosaposin and comprises the nucleotide sequence represented by SEQ ID NO: 1.

Description

Double-stranded RNA targeting PSAP and its use

[0001] The present disclosure relates to double-stranded RNAs targeting prosaposin (PSAP) and uses thereof. This application claims priority to Japanese Patent Application No. 2024-147396, filed on August 29, 2024, the entire contents of which are incorporated herein by reference.

[0002] Miyahara et al., "Prosaposin, a tumor-secreted protein, promotes pancreatic cancer progression by decreasing tumor-infiltrating lymphocytes," Cancer Science, vol. 113 (2022), pp. 2548-2559, describes the involvement of PSAP in the progression of pancreatic ductal carcinoma (PDAC). According to this literature, PDAC cell lines express high amounts of PSAP. However, it also describes that knocking down PSAP expression in PDAC cell lines did not affect cell proliferation or migration ability.

[0003] Miyahara et al., “Prosaposin, tumor-secreted protein, promotes pancreatic cancer progression by decreasing tumor-infiltrating lymphocytes”, Cancer Science, vol. 113 (2022), pp. 2548-2559

[0004] The present inventors have been developing double-stranded RNAs to provide nucleic acid drugs capable of inhibiting cell proliferation. The double-stranded RNAs capable of inhibiting cell proliferation can be used, for example, as antitumor agents.

[0005] One aspect of the present technology provides a double-stranded RNA. In some embodiments, the double-stranded RNA comprises a first strand and a second strand. The first strand comprises a main sequence consisting of 19 to 23 bases. The second strand comprises a complementary sequence that binds complementarily to the main sequence. The main sequence is composed of a portion of the base sequence encoding prosaposin and comprises the base sequence: CUUGGACUGAAAGA (SEQ ID NO: 1). The double-stranded RNA is capable of inhibiting cell proliferation.

[0006] In some embodiments, the base at the 5' end of the main sequence is guanine (G) or cytosine (C).

[0007] In some embodiments, at least two of the five bases on the 3'-terminal side of the main sequence are adenine (A) and / or uracil (U).

[0008] In some embodiments, the main sequence consists of any of the following base sequences: CGGUCCUUGGACUGAAAGA (SEQ ID NO: 2); GGUCCUUGGACUGAAAGAA (SEQ ID NO: 3); GUCCUUGGACUGAAAGAAU (SEQ ID NO: 4); and CUUGGACUGAAAGAAUGCA (SEQ ID NO: 5).

[0009] In some embodiments, the first strand includes a first additional sequence added to the 3'-end of the main sequence, and the base sequence constituting the first additional sequence is thymine-thymine (TT), thereby improving the stability of the double-stranded RNA.

[0010] In some embodiments, the second strand includes a second additional sequence added to the 3'-end of the complementary sequence, and the base sequence constituting the second additional sequence is thymine-thymine (TT), thereby improving the stability of the double-stranded RNA.

[0011] In one aspect of the present technology, a composition comprising the double-stranded RNA disclosed herein is provided, which is capable of inhibiting the proliferation of at least one type of cell.

[0012] In some embodiments, the composition is capable of inhibiting tumor cell proliferation.

[0013] In some embodiments, the composition comprises a peptide fragment having cell membrane permeability that allows a foreign substance to be introduced into the cytoplasm through the cell membrane from the outside of the cell, thereby enabling efficient introduction of double-stranded RNA into the cell.

[0014] In one aspect of the present technology, there is provided a method for inhibiting proliferation of at least one cell, comprising providing a composition as disclosed herein and administering the composition to the cell. In some embodiments, the cell and the prosaposin are of the same species.

[0015] FIG. 1 is a graph showing the cell viability for each example.

[0016] <Definition of Terms> Matters other than those specifically mentioned in this specification (e.g., the structure of double-stranded RNA) that are necessary for carrying out the present technology (e.g., general matters such as methods for synthesizing polynucleotides, cell culture techniques, constructs mainly composed of peptides or nucleic acids, etc.) 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 carried out based on the contents disclosed in this specification and common general technical knowledge in the relevant field.

[0017] 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, an "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).

[0018] As used herein, the terms "first strand" and "second strand" refer to one being the sense strand (which may also be referred to as the coding strand or passenger strand) and the other being the antisense strand (which may also be referred to as the template strand, non-coding strand, or guide strand). That is, when the first strand is the sense strand, the second strand refers to the antisense strand. Also, when the second strand is the sense strand, the first strand refers to the antisense strand.

[0019] 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.

[0020] 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.

[0021] 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 includes 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."

[0022] <Prosaposin (PSAP)> PSAP is the precursor protein of saposin A (SAP-A), saposin B (SAP-B), saposin C (SAP-C), and saposin D (SAP-D). Saposins A to D are generated by decomposition of PSAP through processing. Saposins A to D are localized in lysosomes and contribute to the hydrolysis of specific glycosphingolipids.

[0023] It is known that PSAP may be associated with cancer progression. However, according to Miyahara et al., "Prosaposin, a tumor-secreted protein, promotes pancreatic cancer progression by decreasing tumor-infiltrating lymphocytes," Cancer Science, vol. 113 (2022), pp. 2548-2559, knocking down PSAP expression in pancreatic ductal carcinoma cell lines using commercially available siRNA did not suppress cell proliferation.

[0024] In the course of developing nucleic acid drugs capable of inhibiting cell proliferation, the present inventors investigated double-stranded RNAs that could function as siRNAs targeting PSAP. As a result, they found that double-stranded RNAs targeting a region containing a specific base sequence (CUUGGACUGAAAGA (SEQ ID NO: 1)) could inhibit cell proliferation. Details are described below.

[0025] <Double-stranded RNA> The double-stranded RNA comprises a first strand and a second strand. In some embodiments, the first strand comprises a main sequence and a first additional sequence. The second strand comprises a complementary sequence that binds complementarily to the main sequence of the first strand, and a second additional sequence. The first strand and the second strand are hybridized to the main sequence at the complementary sequence. The first strand and the second strand may be artificially synthesized polynucleotides. Note that in some embodiments, the first additional sequence and the second additional sequence may be omitted.

[0026] The double-stranded RNA disclosed herein can function as a small interfering RNA (siRNA) targeting PSAP. That is, the double-stranded RNA disclosed herein can induce RNA interference (RNAi). RNAi is a gene silencing process in which short double-stranded RNAs such as siRNAs suppress gene expression in a sequence-specific manner. When siRNAs are introduced into cells, they form a complex called an RNA-induced silencing complex (RISC) with intracellular proteins. RISC binds to a homologous sequence in mRNA that has a base sequence encoding the target protein (here, PSAP) and specifically cleaves the mRNA. This inhibits translation of the target protein.

[0027] <First Strand> The main sequence of the first strand may be composed of a polynucleotide, which is a polymer of ribonucleotides. In other words, the main sequence may be 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).

[0028] The main sequence of the first strand may be a portion of the base sequence encoding PSAP. That is, it may have the same sequence as a portion of the base sequence encoding PSAP. Preferably, the main sequence contains the base sequence CUUGGACUGAAAGA (SEQ ID NO: 1), which is a portion of the base sequence encoding PSAP. The base sequence shown in SEQ ID NO: 1 may be composed of RNA.

[0029] The nucleotide sequence of PSAP can be obtained from international databases. For example, international databases include the National Center for Biotechnology Information (NCBI), the European Nucleotide Archive (ENA), the DNA Data Bank of Japan (DDBJ), UniPlot, Ensembl, etc. Specifically, the nucleotide sequence of human PSAP is provided by NCBI under accession number NM_002778.4, etc. SEQ ID NO: 14 shows the nucleotide sequence of the mRNA of human PSAP disclosed in accession number NM_002778.4. SEQ ID NO: 15 shows the amino acid sequence of human PSAP based on the CDS of the mRNA disclosed in accession number NM_002778.4. Based on the information of accession number NM_002778.4, the nucleotide sequence shown in SEQ ID NO: 1 is the nucleotide sequence from bases 58 to 71 of the nucleotide sequence encoding human PSAP (the sequence from the start codon to the stop codon; CDS). At the time of filing, data on three transcript variants of human PSAP (accession numbers NM_002778.4, NM_001042466.3, and NM_001035930.1) had been registered with NCBI. The nucleotide sequence shown in SEQ ID NO: 1 is common to the three transcript variants.

[0030] In the present specification, the biological species from which PSAP is derived is not particularly limited. The base sequence shown in SEQ ID NO: 1 is widely conserved not only in humans but also in primates, such as gorillas, chimpanzees, orangutans, black gibbons, and siamangs.

[0031] The number of bases constituting the main sequence may be, for example, from 19 to 23, or from 19 to 21. The main sequence may also be composed of 19 bases.

[0032] In some embodiments, the base at the 5' end of the main sequence is guanine (G) or cytosine (C). Because guanine and cytosine have stronger binding strength with complementary strands than adenine and uracil, the stability of the 5' end of the first strand (i.e., the 3' end of the second strand) is increased. In other words, the stability of the 5' end of the second 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, when the 5' end of the main sequence is guanine or cytosine, the antisense strand (here, the second strand) is more easily incorporated into RISC, which can more effectively induce RNAi. This allows the double-stranded RNA to function favorably as siRNA.

[0033] In some embodiments, at least two of the five bases on the 3'-end of the main sequence are adenine and / or uracil, preferably three or more, more preferably four or more. This makes the 5'-end of the second strand less stable than the 3'-end. As a result, the second strand is more easily incorporated into RISC, allowing for more efficient induction of RNAi.

[0034] 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 70% or less, preferably 30% to 60% or less, or may be 40% to 55% 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.

[0035] In some preferred embodiments, the main sequence may be any of the following base sequences: CGGUCCUUGGACUGAAAGA (SEQ ID NO: 2); GGUCCUUGGACUGAAAGAA (SEQ ID NO: 3); GUCCUUGGACUGAAAGAAU (SEQ ID NO: 4); and CUUGGACUGAAAGAAUGCA (SEQ ID NO: 5). The base sequences shown in SEQ ID NOs: 2 to 5 are all composed of RNA. The base sequences shown in SEQ ID NOs: 2 to 5 are all identical to at least a portion of the base sequence encoding human PSAP. The base sequences shown in SEQ ID NOs: 2 to 5 all contain the base sequence shown in SEQ ID NO: 1. Double-stranded RNAs having the base sequences shown in SEQ ID NOs: 2 to 5 as their main sequences can significantly inhibit cell proliferation.

[0036] The base sequence shown in SEQ ID NO: 2 is the 53rd to 71st bases of the base sequence encoding human PSAP (i.e., the sequence from the initiation codon to the termination codon). The base sequence shown in SEQ ID NO: 3 is the 54th to 72nd bases of the base sequence encoding human PSAP (i.e., the sequence from the initiation codon to the termination codon). The base sequence shown in SEQ ID NO: 4 is the 55th to 73rd bases of the base sequence encoding human PSAP. The base sequence shown in SEQ ID NO: 5 is the 58th to 76th bases of the base sequence encoding human PSAP. SEQ ID NOs: 16 to 20 are DNA sequences corresponding to the RNA sequences of SEQ ID NOs: 1 to 5.

[0037] The main sequence may have one or more bases (e.g., two bases) substituted with other bases, deleted, and / or added (inserted) to it, as long as the effects of the present technology are maintained. The main sequence may also contain nucleotide analogs such as modified ribonucleotides, as long as the effects of the present technology are maintained.

[0038] The first strand may include a first additional sequence added to the 5'-end and / or 3'-end of the main sequence. Preferably, the first additional sequence is added to the 3'-end of the main sequence. Addition of the first additional sequence to the main sequence can more effectively induce RNAi.

[0039] The first additional sequence may be composed of nucleotides or polynucleotides (dimers, trimers, or tetramers). The polynucleotides constituting the first additional sequence may be composed of only ribonucleotides, only deoxyribonucleotides, or both ribonucleotides and deoxyribonucleotides. That is, the first strand may be entirely RNA, or may be a chimeric polynucleotide of RNA and DNA.

[0040] The base sequence constituting the first additional sequence is, for example, composed of 1 to 4 bases, preferably 2 to 4 bases, and more preferably 2 bases. The first additional sequence preferably contains at least one base, such as adenine, uracil, or thymine. From the viewpoint of improving the stability of double-stranded RNA, the base sequence constituting the first additional sequence is preferably TT (thymine-thymine). The first additional sequence may protrude from the 5'-end or 3'-end of the second strand. In some embodiments, the first additional sequence may be an overhang in the siRNA. When the first additional sequence is added to both the 5'-end and the 3'-end of the main sequence, the total number of bases may be, for example, 2 to 4.

[0041] The first 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 embodiment, the base sequence of the first strand is composed of 21 to 23 bases, consisting of 19 to 21 bases of the main sequence and 2 bases of the first additional sequence. In such an embodiment, RNAi can be effectively induced.

[0042] The first strand may contain nucleotide analogs such as modified RNA or DNA, as long as the effects of the present technology are achieved. Examples of modifications include methylation, pseudouridylation, fluorination, and deamination. Examples of nucleotide analogs include pseudouridine, N1-methylpseudouridine, 5-methylcytosine, 5-fluorouracil, inosine, and acyclic artificial nucleic acids. For example, one or more (e.g., two to five) of the bases constituting the first strand may be substituted with a nucleotide analog. The first strand may also have a ligand that binds to a receptor specifically expressed in the target cells. The ligand may be added, for example, to the 5'-end and / or 3'-end of the first strand. The ligand may be, for example, N-acetylgalactosamine (GalNAc) or a derivative thereof. Having GalNAc as the ligand may improve delivery to hepatocytes.

[0043] <Second strand> The complementary sequence of the second strand has a base sequence that binds complementarily to the main sequence of the first strand. This allows the first strand and the second strand to hybridize and form a double-stranded structure. The complementary sequence can be composed of a polynucleotide, which is a polymer of ribonucleotides. In other words, the complementary sequence can be composed of RNA.

[0044] In this specification, "complementarily bind" typically refers to the formation of a base pair between adenine and uracil or thymine linked by a hydrogen bond, and / or the formation of a base pair between guanine and cytosine linked by a hydrogen bond. However, in the case of modified bases, this may also include cases where they can form base pairs with other bases through hydrogen bonds.

[0045] The complementary sequence preferably has a base sequence complementary to the entire main sequence. That is, it is preferable that each base constituting the main sequence forms a base pair with a base contained in the complementary sequence. This allows the bases to be complementarily bound to form a stable double-stranded structure, which can facilitate the induction of RNAi.

[0046] As long as the effects of the present technology are achieved, the complementary sequence may bind partially complementarily to the bases constituting the main sequence. That is, the complementary sequence may not form base pairs with some of the bases constituting the main sequence. For example, the complementary sequence may not form base pairs with one or more (e.g., two) of the bases constituting the main sequence.

[0047] The number of bases constituting the complementary sequence may be, for example, the same as the number of bases constituting the main sequence. In a preferred embodiment, the number of bases constituting the complementary sequence is the same as the number of bases constituting the main sequence, and the bases constituting the main sequence and the bases constituting the complementary sequence are all complementary to each other. This allows the formation of a stable double-stranded structure, which can facilitate the induction of RNAi.

[0048] As long as the effects of the present technology are achieved, the number of bases constituting the complementary sequence may be greater than the number of bases constituting the main sequence. For example, the complementary sequence may have one or more (e.g., 2 to 4) bases that do not form base pairs with the main sequence at the 5'-end. Furthermore, for example, one or more (e.g., 2 to 4, loop structure, etc.) bases that do not form base pairs with the main sequence may be located between the complementary sequence and the main sequence.

[0049] As long as the effects of the present technology are achieved, the number of bases constituting the complementary sequence may be less than the number of bases constituting the main sequence, for example, the number of bases constituting the complementary sequence may be one or two bases less than the number of bases constituting the main sequence.

[0050] The second strand may contain a second additional sequence added to the 5'-end and / or 3'-end of the complementary sequence. From the viewpoint of improving the function of siRNA, when the first additional sequence is added to the 3'-end of the main sequence in the first strand, it is preferable that the second additional sequence be added to the 3'-end of the complementary sequence.

[0051] The second additional sequence may be composed of nucleotides or polynucleotides (dimers, trimers, or tetramers). The polynucleotides constituting the second additional sequence may be composed of only ribonucleotides, only deoxyribonucleotides, or both ribonucleotides and deoxyribonucleotides. That is, the second strand may be entirely RNA, or may be a chimeric polynucleotide of RNA and DNA.

[0052] The base sequence constituting the second additional sequence is, for example, composed of 2 to 4 bases, preferably 2 bases. The second additional sequence 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 second additional sequence is preferably TT (thymine-thymine).

[0053] Although not particularly limited, the second strand may be composed of, for example, a base sequence of 21 to 27 bases, 21 to 25 bases, or 21 to 23 bases. In a preferred embodiment, the second strand is composed of 21 to 23 bases, consisting of 19 to 21 bases of the complementary sequence and 2 bases of the second additional sequence. In such an embodiment, RNAi can be effectively induced. In some embodiments, the second additional sequence may be an overhang in the siRNA. Note that when the second additional sequence is added to both the 5'-end and the 3'-end of the main sequence, the total number of bases may be, for example, 2 to 4 bases.

[0054] The second strand may contain nucleotide analogs, ligands, etc., such as modified RNA or DNA, as long as the effects of the present technology are achieved. Examples of the types of modifications, nucleotide analogs, and ligands may be the same as those for the first strand described above. For example, one or more (e.g., two to five bases) of the bases constituting the second strand may be substituted with nucleotide analogs.

[0055] <Method for Producing Double-Stranded RNA> The first and second strands constituting the double-stranded RNA disclosed herein can be produced according to common chemical synthesis methods. For example, they can be synthesized using a commercially available DNA / RNA automatic synthesizer. Alternatively, the first and second strands may be synthesized in vitro or in vivo based on genetic engineering techniques. The synthesized first and second strands are preferably purified, and can be purified, for example, by HPLC or the like.

[0056] The double-stranded RNA disclosed herein can be produced, for example, by annealing (hybridizing) the first strand and the second strand. Annealing can be performed according to conventional methods. For example, annealing can be performed by mixing equal amounts of the first strand and the second 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.

[0057] <Other Embodiments of Double-Stranded RNA> The double-stranded RNA disclosed herein may include a double-stranded RNA in which the first strand and the second strand form a partially double-stranded structure via a loop structure. That is, the double-stranded RNA may also be used as an shRNA (short hairpin RNA) in another embodiment. The shRNA is an RNA in which a main sequence, its complementary sequence, and a loop sequence for forming the loop structure are present on a single strand. The shRNA has a loop structure, which allows the main sequence and its complementary sequence to hybridize and form a partially double-stranded structure. This allows the shRNA to be processed by Dicer, an enzyme present in cells, to form the siRNA of the above-mentioned embodiment.

[0058] The structure of the shRNA may be the same as that of conventionally known shRNAs. The length of the shRNA may be, for example, 50 to 80 bases. The length of the loop sequence may be, for example, 19 to 29 bases. The shRNA may be incorporated into a vector (e.g., a lentiviral expression vector). By using the shRNA, RNAi can be stably induced in cells, and viral proliferation can be stably suppressed.

[0059] <Composition> The composition disclosed herein contains the double-stranded RNA described above. In addition to the double-stranded RNA described above, the composition may also contain various pharmaceutically acceptable carriers depending on the intended use. Preferred carriers include those commonly used in pharmaceuticals 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. Furthermore, such carriers may include aqueous solutions of alcohol (e.g., ethanol) at appropriate concentrations, glycerol, non-drying oils such as olive oil, lipid nanoparticles (LNPs), liposomes, etc. Secondary components that may be contained in the pharmaceutical composition include various fillers, bulking agents, binders, wetting agents, surfactants, dyes, fragrances, etc. The composition may also include carriers used in conventional drug delivery systems (DDS).

[0060] 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).

[0061] The compositions disclosed herein inhibit the proliferation of at least one type of cell. Examples of cells whose proliferation can be inhibited include cells expressing mRNA containing a nucleotide sequence encoding PSAP. Examples of cells expressing mRNA containing a nucleotide sequence encoding PSAP include brain cells, retinal cells, and tumor cells (e.g., adrenocortical carcinoma, bile duct cancer, bladder cancer, bone tumor, brain tumor, breast cancer, cervical cancer, colon cancer, esophageal cancer, gallbladder cancer, gastric cancer, head and neck cancer, kidney cancer, liver cancer, lung cancer, lymphoma, myeloma, neuroblastoma, uterine fibroids, ovarian cancer, pancreatic cancer, prostate cancer, rhabdoid tumor, sarcoma, skin cancer, testicular tumor, thyroid cancer, and uterine cancer). The compositions disclosed herein inhibit, for example, the proliferation of tumor cells. Therefore, the double-stranded RNA and compositions disclosed herein can be suitably used, for example, as cell proliferation inhibitors (anticancer agents) that inhibit the proliferation of tumor cells.

[0062] In some embodiments, the composition includes, in addition to the double-stranded RNA described above, a peptide fragment (cell-penetrating peptide, hereinafter referred to as "CPP") that has cell membrane permeability and can pass through the cell membrane from the outside of the cell to introduce a foreign substance into the cytoplasm. A construct comprising a CPP and a double-stranded RNA is constructed by directly or indirectly binding (linking) the CPP to the double-stranded RNA disclosed herein. Generally, double-stranded RNA has a negative charge and therefore does not easily pass through the cell membrane. However, for example, by directly or indirectly linking (linking) the double-stranded RNA disclosed herein to the N-terminus and / or C-terminus of the CPP, a construct comprising a CPP and a double-stranded RNA can be efficiently introduced into the cytoplasm. The number of amino acid residues in the CPP is not limited as long as cell membrane permeability is not significantly impaired.

[0063] When a CPP and a double-stranded RNA are indirectly bound to each other, for example, a linker is placed between the CPP and the double-stranded RNA. The type of linker is not particularly limited. Typically, it is a peptide linker, a non-peptide linker, or the like. Furthermore, the method for binding a CPP and a double-stranded RNA is not particularly limited, and can be carried out according to various conventionally known scientific techniques.

[0064] One embodiment of the composition disclosed herein contains a CPP and the double-stranded RNA of the present disclosure. However, the double-stranded RNA does not have to be bound to the N- or C-terminus of the CPP. For example, the double-stranded RNA and the CPP may form a complex 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 CPP used preferably has a high proportion of basic amino acids and is positively charged. Furthermore, the molar ratio of the CPP in this case may be 5 to 100 times that of the double-stranded RNA, preferably 40 to 60 times.

[0065] Known CPPs can be used (see, for example, van Asbeck et al., "Molecular Parameters of siRNA-Cell Penetrating Peptide Nanocomplexes for Efficient Cellular Delivery," ACS Nano., Vol. 7, No. 5, pp. 3797-3807, 2013).

[0066] <Method of Use> 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 a composition of the present disclosure and administering the composition to a cell in vitro or in vivo.

[0067] In the preparation step, for example, as described above, the composition disclosed herein may be prepared by a conventionally known method.

[0068] In the administration step, the composition prepared in the preparation step is administered to at least one type of cell (e.g., tumor cell, etc.) in vivo or ex vivo. The biological species of such cells is not particularly limited and may be, for example, mammals, birds, amphibians, reptiles, fish, etc. Preferably, the biological species from which the PSAP targeted by the main sequence of the double-stranded RNA contained in the composition is derived is the same as the biological species of the target cell. The target cell is, for example, a cell expressing mRNA containing the above-mentioned nucleotide sequence encoding PSAP. In one example, such a cell may be a tumor cell (e.g., neuroblastoma). The cell type to which the composition is administered may be one type alone or two or more types.

[0069] 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.

[0070] The amount of the composition administered in vivo is not particularly limited. For example, the amount of double-stranded RNA per 1 kg of an animal may be 0.01 mg or more, 0.05 mg or more, or 0.1 mg or more. The amount of double-stranded RNA per 1 kg of an animal may be, for example, 10 mg or less, 5 mg or less, 2 mg or less, or 1 mg or less.

[0071] The amount of the composition to be administered in vitro is not particularly limited. For example, the double-stranded RNA concentration in the culture medium of a subject containing cells can be 1 nM or more, 10 nM or more, or 100 nM or more. The double-stranded RNA concentration in the culture medium can be, for example, 5 μM or less, 2 μM or less, 1 μM or less, or 500 nM or less.

[0072] 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 CPPs.

[0073] 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.

[0074] <Preparation of double-stranded RNA> Polynucleotides having the base sequences shown in SEQ ID NOS: 6 to 13 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 or complementary 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 of Samples 1 to 4 shown in Table 1. Each of the double-stranded RNAs shown in Samples 1 to 4 was dissolved in sterile water (DNase- and RNase-free) to an RNA concentration of 200 μM, to prepare an RNA solution.

[0075]

[0076] (Example 1) <Cell proliferation test> Human neuroblastoma cells, SK-N-SH cells, were used as eukaryotic 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 subsequent culture or evaluation.

[0077] 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 commercially available 96-well plate was prepared, and the cell suspension was added to each well at 5 × 10 cells / mL. ３ The cells were seeded at 100 μL per well and incubated at 37°C, 5% CO ２ and incubated overnight under

[0078] On the second day, 1 μL of an RNA solution containing 200 μM sample 1 was mixed with 25 μL of Opti-MEM (Thermo Fisher Scientific) to prepare solution A. Furthermore, 1.5 μL of Lipofectamine RNAiMAX (Thermo Fisher Scientific) was mixed with 25 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, so that the concentration of sample 1 in the culture medium in the wells was 0.4 μM. The cells were then incubated at 37°C, 5% CO ２ SK-N-SH cells were incubated under HCl for 72 hours.

[0079] The cell amount in each well was evaluated using Cell Counting Kit-8 (CCK-8, Dojin Kagaku Kenkyusho). On day 5 (day 3 after siRNA addition), 10 μL of CCK-8 reagent was added to each well in which SK-N-SH cells had been cultured, and the cells were incubated at 37°C and 5% CO ２ The wells were incubated for 2 hours under a constant temperature. Thereafter, the absorbance at 450 nm of each well was measured. The absorbance was calculated as the average value of 3 wells. In addition, a blank well was prepared containing only the culture medium and the CCK-8 reagent. The measured value of Sample 1 was calculated by subtracting the absorbance of the blank from the absorbance of Sample 1. The measured value of Example 1 was calculated relative to the measured value of Reference Example described below as the reference value. The cell viability of Example 1 was calculated using this relative value.

[0080] (Example 2) The same procedure as in Example 1 was carried out except that Sample 1 was changed to Sample 2.

[0081] Example 3 The same procedure as in Example 1 was carried out except that Sample 1 was changed to Sample 3.

[0082] Example 4 The same procedure as in Example 1 was carried out except that Sample 1 was changed to Sample 4.

[0083] Reference Example In the Reference Example, the procedure was the same as in Example 1, except that sterile water was used instead of the RNA solution. That is, in the Reference Example, no double-stranded RNA was added.

[0084] Figure 1 is a graph showing the cell viability of each example. The cell viability of the Reference Example is shown as 100%. As shown in Figure 1, the cell viability of Examples 1 to 4 was lower than that of the Reference Example. In other words, it can be seen that cell proliferation was inhibited in Examples 1 to 4.

[0085] 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.

[0086] The double-stranded RNA disclosed herein can inhibit cell proliferation, and therefore, by using the double-stranded RNA, a composition (e.g., an antitumor agent) that inhibits the proliferation of at least one type of cell (e.g., tumor cell) can be provided.

[0087] Specific embodiments of the technology disclosed herein include those described in the following paragraphs. Item 1: A double-stranded RNA comprising a first strand and a second strand, wherein the first strand comprises a main sequence consisting of 19 to 23 bases, and the second strand comprises a complementary sequence that binds complementarily to the main sequence, wherein the main sequence is composed of a part of a base sequence encoding prosaposin and comprises the base sequence CUUGGACUGAAAGA (SEQ ID NO: 1). Item 2: The double-stranded RNA according to Item 1, wherein the base at the 5' end of the main sequence is guanine (G) or cytosine (C). Item 3: The double-stranded RNA according to Item 1 or 2, wherein at least two of the five bases on the 3' end of the main sequence are adenine (A) and / or uracil (U). Item 4: The double-stranded RNA according to any one of Items 1 to 3, wherein the main sequence consists of any one of the following base sequences: CGGUCCUUGGACUGAAAGA (SEQ ID NO: 2); GGUCCUUGGACUGAAAGAA (SEQ ID NO: 3); GUCCUUGGACUGAAAGAAU (SEQ ID NO: 4); and CUUGGACUGAAAGAAUGCA (SEQ ID NO: 5). Item 5: The double-stranded RNA according to any one of Items 1 to 4, wherein the first strand comprises a first additional sequence added to the 3'-end of the main sequence, and the base sequences constituting the first additional sequence and the second additional sequence are thymine-thymine (TT). Item 6: The double-stranded RNA according to any one of Items 1 to 5, wherein the second strand comprises a second additional sequence added to the 3'-end of the complementary sequence, and the base sequence constituting the second additional sequence is thymine-thymine (TT). Item 7: A composition comprising the double-stranded RNA according to any one of Items 1 to 6, which suppresses the proliferation of at least one type of cell. Item 8: The composition according to Item 7, wherein the cell is a tumor cell. Item 9: The composition according to Item 7 or 8, which comprises a peptide fragment having cell membrane permeability that is capable of passing through the cell membrane from the outside of the cell and introducing a foreign substance into the cytoplasm. Item 10: A method for suppressing the proliferation of at least one type of cell, comprising the steps of: preparing the composition according to any one of Items 7 to 9; and administering the composition to the cell.Item 11: The method according to Item 10, wherein the biological species of the cells and the biological species of the prosaposin are the same.

Claims

1. A double-stranded RNA comprising a first strand and a second strand, wherein the first strand comprises a main sequence consisting of 19 to 23 bases, and the second strand comprises a complementary sequence that binds complementarily to the main sequence, wherein the main sequence is composed of a portion of the base sequence encoding prosaposin and comprises the base sequence: CUUGGACUGAAAGA (SEQ ID NO: 1).

2. The double-stranded RNA according to claim 1, wherein the base at the 5' end of the main sequence is guanine (G) or cytosine (C).

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

4. The double-stranded RNA according to claim 1, wherein the main sequence consists of any one of the following base sequences: CGGUCCUUGGACUGAAAGA (SEQ ID NO: 2); GGUCCUUGGACUGAAAGAA (SEQ ID NO: 3); GUCCUUGGACUGAAAGAAU (SEQ ID NO: 4); and CUUGGACUGAAAGAAUGCA (SEQ ID NO: 5).

5. The double-stranded RNA according to claim 1, wherein the first strand comprises a first additional sequence added to the 3' end of the main sequence, and the base sequence constituting the first additional sequence is thymine-thymine (TT).

6. The double-stranded RNA according to claim 1, wherein the second strand comprises a second additional sequence added to the 3' end of the complementary sequence, and the base sequence constituting the second additional sequence is thymine-thymine (TT).

7. A composition comprising the double-stranded RNA according to any one of claims 1 to 6, which inhibits the proliferation of at least one type of cell.

8. The composition of claim 7, wherein the cells are tumor cells.

9. The composition according to claim 7, 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.

10. A method for inhibiting the proliferation of at least one type of cell, comprising the steps of: providing a composition according to claim 7; and administering said composition to said cells in vitro.

11. The method of claim 10, wherein the biological species of the cells and the biological species of the prosaposin are the same.