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WO2025159163A1 - Nouvel arn double brin à base de séquence d'arn et utilisation associée - Google Patents

Nouvel arn double brin à base de séquence d'arn et utilisation associée

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Publication number
WO2025159163A1
WO2025159163A1 PCT/JP2025/002078 JP2025002078W WO2025159163A1 WO 2025159163 A1 WO2025159163 A1 WO 2025159163A1 JP 2025002078 W JP2025002078 W JP 2025002078W WO 2025159163 A1 WO2025159163 A1 WO 2025159163A1
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Prior art keywords
double
stranded rna
sequence
strand
cells
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Pending
Application number
PCT/JP2025/002078
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English (en)
Japanese (ja)
Inventor
徹彦 吉田
菜穂子 ベイリー小林
エリック ネルソン ベイリー
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Toagosei Co Ltd
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Toagosei Co Ltd
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Publication of WO2025159163A1 publication Critical patent/WO2025159163A1/fr
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/713—Double-stranded nucleic acids or oligonucleotides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing

Definitions

  • This disclosure relates to double-stranded RNA, compositions containing the double-stranded RNA, and methods of using them. Specifically, it relates to double-stranded RNA used to suppress or inhibit tumor cell proliferation or metastasis, and compositions comprising the double-stranded RNA.
  • This application claims priority to Japanese Patent Application No. 2024-008379, filed January 24, 2024, the entire contents of which are incorporated herein by reference.
  • Amyloid precursor protein (or amyloid beta precursor protein, hereafter referred to as "APP") is a membrane protein expressed in many tissues. APP has a variety of functions, such as nerve cell formation and signal transduction. Under normal circumstances, APP may play an important role in the growth and repair of various cells.
  • JP 2022-515193 A discloses a double-stranded ribonucleic acid drug that targets the APP gene.
  • WO 2012/093732 discloses an antibody that targets the signal peptide region of APP.
  • APP has also been suggested to be involved in the proliferation and invasion of tumor cells.
  • APP is known to be overexpressed in breast cancer and prostate cancer.
  • the primary objective of this disclosure is to provide technology for suppressing or inhibiting cell proliferation involving APP gene expression.
  • the double-stranded RNA disclosed herein consists of 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.
  • the main sequence is a part of the base sequence encoding amyloid precursor protein, and includes at least a part of the base sequence encoding the signal peptide region of amyloid precursor protein.
  • the above-mentioned double-stranded RNA can function at least as small interfering RNA (siRNA).
  • siRNA small interfering RNA
  • RNAi RNA interference
  • 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 involving APP.
  • At least three of the seven bases on the 3' end of the main sequence are adenine (A) and/or uracil (U). This more effectively suppresses APP expression, thereby inhibiting the proliferation of cells in which APP is involved.
  • the base sequence comprising at least a part of the base sequence encoding the signal peptide region of APP is the following base sequence: GCTGGAGGTACCCACTGAT (SEQ ID NO: 1); GCGCTGGAGGTACCCACTGAT (SEQ ID NO: 2); CCGGTTTGGCACTGCTCCT (SEQ ID NO: 11); GTTTGGCACTGCTCCTGCT (SEQ ID NO: 12); GGCGCTGGAGGTACCCACT (SEQ ID NO: 13); GGCGCTGGAGGTACCCACTGA (SEQ ID NO: 14); and CGCTGGAGGTACCCACTGA (SEQ ID NO: 15);
  • Such double-stranded RNA more specifically suppresses the expression of amyloid precursor protein, thereby making it possible to inhibit the proliferation of cells in which the expression level of APP is increased.
  • the base sequence constituting the additional sequence is thymine-thymine (TT). This improves the stability of the double-stranded RNA.
  • 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, 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.
  • the main sequence is part of a base sequence encoding amyloid precursor protein, and comprises double-stranded RNA including at least a portion of a base sequence encoding the signal peptide region of amyloid precursor protein.
  • the cell type whose proliferation is inhibited by the composition is tumor cells. This allows for more reliable inhibition of cell proliferation.
  • composition disclosed herein contains a peptide fragment that has cell membrane permeability, allowing it to pass through the cell membrane from the outside of the cell and introduce a foreign substance into the cytoplasm. This makes it easier to introduce double-stranded RNA into the target cell.
  • 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 makes it possible to inhibit the proliferation of cells in which APP is involved.
  • the biological species of the cells is the same as the biological species containing APP. This makes it possible to more reliably inhibit the proliferation of cells in which APP is involved.
  • 1 is a schematic diagram showing the human APP gene and its signal peptide region.
  • 1 is a graph showing cell viability of neuroblastoma cells in Samples 1 and 2 and a comparative example.
  • 1 is a graph showing the cell viability of neuroblastoma at different addition amounts for Samples 1 and 2 and a comparative example.
  • 1 is a graph showing cell viability of neuroblastoma cells in Samples 3 to 7 and a comparative example.
  • 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.
  • polynucleotides herein also include those containing both deoxyribonucleotides and nucleotides.
  • artificially designed polynucleotides refer to polynucleotides whose nucleotide chains (full length) do not exist alone in nature, but are artificially synthesized by chemical synthesis or biosynthesis (i.e., production based on genetic engineering).
  • first strand and second strand refer to one being the sense strand (or coding strand or passenger strand) and the other being the antisense strand (or template strand or non-coding strand or guide strand). That is, if the first strand is the sense strand, the second strand refers to the antisense strand. Also, if the second strand is the sense strand, the first strand refers to the antisense strand.
  • the first strand and second strand may be fully complementary to each other, or may be at least partially complementary. That is, they may be capable of hybridizing at least under physiological conditions.
  • the left side of a base sequence always indicates the 5'-end and the right side indicates the 3'-end.
  • amino acid residue includes the N-terminal amino acid and C-terminal amino acid of a peptide chain, unless otherwise specified.
  • the left side always indicates the N-terminal side and the right side indicates the C-terminal side.
  • tumor is interpreted broadly 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.).
  • tumor cells are synonymous with “cancer cells” and refer to cells that form such tumors, typically cells that have begun to grow abnormally independent of surrounding normal tissue (so-called cancerous cells). Therefore, unless otherwise specified, cells that are classified as tumor cells (cancer cells) rather than normal cells are referred to as tumor cells, regardless of their origin or properties.
  • amyloid precursor protein is also referred to as amyloid ⁇ precursor protein or APP.
  • amyloid precursor protein is also referred to as amyloid ⁇ precursor protein or APP.
  • the biological species from which the amyloid precursor protein is derived is not particularly limited. However, it is preferably the same as the animal species to which the double-stranded RNA or composition disclosed herein is to be delivered. For example, when delivering the double-stranded RNA or composition disclosed herein to human-derived cells, it is preferable to use a base sequence based on the base sequence of human amyloid precursor protein as the main sequence.
  • human-derived amyloid precursor protein is described as a preferred example. However, the present technology can also be applied to amyloid precursor proteins derived from biological species, including mammals other than humans and other animal species.
  • APP is known to be involved in Alzheimer's disease and various other diseases and disorders. It has also been suggested that APP is overexpressed in tumor cells. In addition to the aforementioned Alzheimer's disease, APP is also involved in Down's syndrome, amyloidosis, rheumatoid arthritis, neurodegenerative diseases, cancer, and other cancers. It has also been suggested that APP is involved in cancers such as prostate cancer, breast cancer, colon cancer, thyroid cancer, lung cancer, nasopharyngeal cancer, gastrointestinal cancer, and leukemia. It is also involved in the inflammatory responses and cell death associated with these diseases and disorders, as well as the proliferation, migration, and infiltration of abnormal cells.
  • APP expression is increased in cells associated with the above diseases and disorders.
  • the double-stranded RNA and compositions disclosed herein act favorably on cells in which APP expression levels are increased due to the above diseases and disorders, and can inhibit their proliferation.
  • the nucleotide sequence of APP can be obtained from international databases.
  • international databases include NCBI (National Center for Biotechnology Information), ENA (European Nucleotide Archive), DDBJ (DNA Data Bank of Japan), UniPlot, Ensembl, etc.
  • NCBI National Center for Biotechnology Information
  • ENA European Nucleotide Archive
  • DDBJ DNA Data Bank of Japan
  • UniPlot UniPlot
  • Ensembl etc.
  • the nucleotide sequence of human APP is provided by NCBI under accession number NM_000484.4, etc.
  • Information on the signal peptide region of APP can also be obtained from the above-mentioned international databases.
  • APP consists of approximately 770 amino acid residues.
  • APP is a type I membrane protein. From the N-terminus, APP is primarily composed of a signal peptide region, an extracellular region, an A ⁇ domain, and a cytoplasmic region. The extracellular region contains, from the N-terminus, an E1 domain (growth factor-like domain (GFLD) and copper-binding domain (CuBD)), an acidic domain (AcD), a serine protease inhibitor domain (KPI), and an E2 domain (central APP domain (CAPPD)). Part of the A ⁇ domain is contained in the transmembrane region. The intracellular region contains an AID domain and an AICD domain. APP also exists in several isoforms.
  • Figure 1 schematically illustrates the above domains and the signal peptide region of human APP. It has been suggested that APP mutations may be involved in various diseases and disorders. Targeting the signal peptide region may be useful for treating or preventing diseases and disorders related to human APP gene expression.
  • the amino acid sequence shown in SEQ ID NO: 3 consists of 17 amino acid residues and represents the amino acid sequence of the signal peptide of human APP.
  • the base sequence shown in SEQ ID NO: 4 consists of 51 bases and represents the base sequence of the signal peptide of human APP.
  • the double-stranded RNA of the present disclosure is a double-stranded RNA having a first strand and a second strand complementary to the first strand.
  • the first strand will be referred to as the sense strand and the second strand as the antisense strand, as will be described in detail.
  • 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 a portion of the base sequence encoding APP, and includes at least a portion of the base sequence encoding the signal peptide region of APP.
  • the main sequence is typically composed of a polynucleotide, which is a polymer of ribonucleotides.
  • 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.
  • uracil can also be represented by T (thymine).
  • the main sequence of the sense strand can be, for example, a base sequence including a portion of the base sequence encoding the signal peptide region of APP. This allows the double-stranded RNA to function as an siRNA (small interfering RNA) targeting APP. Furthermore, because the base sequence of the APP signal peptide region is located upstream of the mRNA, when the double-stranded RNA functions as an siRNA, it can effectively suppress the expression of APP.
  • RNAi RNA interference
  • siRNAi is a gene silencing process in which short double-stranded RNAs such as siRNAs suppress gene expression in a sequence-specific manner.
  • siRNAs When siRNAs are introduced into cells, they form a complex with intracellular proteins called RISC (RNA-induced silencing complex). RISC binds to homologous sequences in mRNA transcribed from the target gene (here, the APP gene) and specifically cleaves the mRNA, thereby inhibiting translation.
  • RISC RNA-induced silencing complex
  • the main sequence is preferably selected to include the signal peptide region of APP or a base sequence encoding the signal peptide region of APP, but one or more bases (e.g., two bases) may be substituted, deleted, and/or added (inserted) to other bases as long as the effects of the present technology are achieved.
  • one or more bases e.g., two bases
  • the proportion of the APP signal peptide region or the base sequence encoding the APP signal peptide region in the main sequence there are no particular limitations on the proportion of the APP signal peptide region or the base sequence encoding the APP signal peptide region in the main sequence.
  • the entire main sequence is taken as 100%, it is preferably 5% or more, but may also be 10% or more, 15% or more, 90% or more, or 100% or more.
  • 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 5' end of the sense strand (i.e., the 3' end of the antisense strand) is more stable. In other words, the 5' end of the antisense strand is relatively less stable. While the details of this mechanism are unclear, RISC, an RNAi-related protein, tends to preferentially incorporate the sense strand or antisense strand, whichever has the more energetically unstable 5' end.
  • the antisense strand can be more easily incorporated into RISC, more effectively inducing RNAi. This allows the double-stranded RNA to function effectively as siRNA.
  • Adenine and/or uracil may account for 40% or more, 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) of the five bases on the 3' end of the main sequence. Furthermore, adenine and/or uracil preferably account for 3 or more bases (i.e., 40% or more) of the seven bases on the 3' end of the main sequence. 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, enabling more effective induction of RNAi.
  • 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, 50% to 75%, preferably 55% to 70%, or may be 20% to 80%.
  • the GC content is a parameter related to the binding strength between the antisense strand taken up by RISC and the RNA comprising the main sequence, as well as the ease of cleavage of the RNA. With this GC content, the effect of RNAi can be efficiently exerted.
  • the main sequence can be selected from 19 to 23 bases from G or C of the gene encoding human APP.
  • the main sequence can be the following base sequence: GCUGGAGGUACCCACUGAU (SEQ ID NO: 9); GCGCUGGAGGUACCCACUGAU (SEQ ID NO: 10); CCGGUUUGGCACUGCUCCU (SEQ ID NO: 26); GUUUGGCACUGCUCCUGCU (SEQ ID NO: 27); GGCGCUGGAGGUACCCACU (SEQ ID NO: 28); GGCGCUGGAGGUACCCACUGA (SEQ ID NO: 29); and CGCUGGAGGUACCCACUGA (SEQ ID NO: 30);
  • the base sequences shown in SEQ ID NOs: 9 to 10 and 26 to 30 are all composed of RNA.
  • Double-stranded RNA having any of the base sequences shown in SEQ ID NOs: 9, 10, and 26 to 30 as its main sequence significantly suppresses the proliferation of abnormally proliferating cells even at low concentrations, and can therefore avoid nonspecific inhibition of expression, nonspecific inhibition of cell proliferation, stress on cells, and the like.
  • the base sequence shown in SEQ ID NO: 1 is the 51st to 69th bases of the base sequence encoding human APP (i.e., the sequence from the start codon to the stop codon).
  • the base sequence shown in SEQ ID NO: 2 is the 49th to 69th bases of the base sequence encoding human APP.
  • the base sequences shown in SEQ ID NOs: 1 and 2 are base sequences that include part of the signal peptide region of human APP.
  • the base sequence shown in SEQ ID NO: 11 (the DNA sequence corresponding to the RNA sequence shown in SEQ ID NO: 26) is the 8th to 26th bases of the base sequence encoding human APP.
  • the base sequence shown in SEQ ID NO: 12 (the DNA sequence corresponding to the RNA sequence shown in SEQ ID NO: 27) is the 11th to 29th bases of the base sequence encoding human APP.
  • the base sequence shown in SEQ ID NO: 13 (the DNA sequence corresponding to the RNA sequence shown in SEQ ID NO: 28) is the 48th to 66th bases of the base sequence encoding human APP.
  • the base sequence shown in SEQ ID NO: 14 (the DNA sequence corresponding to the RNA sequence shown in SEQ ID NO: 29) is the 48th to 68th bases of the base sequence encoding human APP.
  • the base sequence shown in SEQ ID NO: 15 (a DNA sequence corresponding to the RNA sequence shown in SEQ ID NO: 30) is the 50th to 68th base sequence of the base sequence encoding human APP.
  • the base sequences shown in SEQ ID NOs: 11 and 12 are the base sequences of part of the signal peptide region of human APP.
  • the base sequences shown in SEQ ID NOs: 13 to 15 are the base sequences including part of the signal peptide region of human APP.
  • Double-stranded RNAs composed of the main sequences shown in SEQ ID NOs: 9-10 and SEQ ID NOs: 26-30 can suppress or inhibit the proliferation of at least one type of cell by supplying them to the cell.
  • tumor cells e.g., neuroblastoma
  • the proliferation of the tumor cells can be suppressed or inhibited.
  • APP 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 APP present in normal cells is relatively small, and therefore the double-stranded RNA is thought to have little effect.
  • 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.
  • the additional sequence is added to the 3'-end of the main sequence.
  • the additional sequence is composed of polynucleotides (dimers, trimers, or tetramers).
  • the polynucleotides that make up the additional sequence may be composed of only ribonucleotides, only deoxynucleotides, or both ribonucleotides and deoxynucleotides.
  • the sense strand and 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, etc.
  • the base sequence constituting the additional sequence is not particularly limited, but preferably contains at least one base: adenine, uracil, or thymine. Furthermore, from the perspective of improving the stability of the double-stranded RNA, it is more preferable that the base sequence constituting the additional sequence is TT (thymine-thymine).
  • 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, the sense strand is composed of 21 to 23 bases, consisting of a main sequence of 19 to 21 bases and an additional sequence of 2 bases. In such an example, RNAi can be effectively induced.
  • 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. If the sense strand and the antisense strand can hybridize at least under physiological conditions, they can function as siRNA.
  • the complementary base sequence portion is typically composed of a ribonucleotide polymer (RNA).
  • the sense strand or antisense strand is typically composed of chemically unmodified ribonucleotides (RNA).
  • 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 it does not significantly impair the technology of the present disclosure. 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.
  • chemically modified RNA include pseudouridine, N1-methylpseudouridine, 5-methylcytosine, and inosine.
  • one or more bases (e.g., two bases) of uridine in the double-stranded RNA of the present disclosure can be substituted with pseudouridine.
  • the antisense strand may have a main sequence complementary to the sense strand and an additional sequence consisting of 2 to 4 bases added to the 5' or 3' end of the complementary main sequence.
  • the additional sequence is preferably added to the 3' end of the complementary base 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.
  • 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.
  • the antisense strand is composed of a base sequence of, for example, 21 to 27 bases, and can 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 that is complementary to the main sequence of the sense strand.
  • 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 that is complementary to the main sequence of the sense strand.
  • the sense and antisense 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 sense and antisense strands may be synthesized in vitro or in vivo based on genetic engineering techniques. The synthesized sense and antisense strands are preferably purified, and can be purified, for example, by HPLC.
  • 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 in accordance with conventional methods.
  • annealing can be performed by mixing equal amounts of the sense strand and antisense strand in a solvent, heating at 90°C for 1 to 5 minutes, and then cooling to 4°C to room temperature.
  • 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.).
  • buffers e.g., HEPES-KOH buffer at pH 7.4, PBS, etc.
  • solvents that have been treated with, for example, DEPC or autoclaved are preferably used.
  • compositions disclosed herein contain the double-stranded RNA described above.
  • the compositions may 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.
  • the carriers may also include aqueous solutions of alcohol (e.g., ethanol) at appropriate concentrations, glycerol, non-drying oils such as olive oil, or liposomes.
  • Secondary components that may be contained in pharmaceutical compositions include various fillers, extenders, binders, humectants, surfactants, dyes, fragrances, etc.
  • the compositions may also contain carriers used in conventional drug delivery systems (DDS).
  • DDS drug delivery systems
  • compositions disclosed herein are not particularly limited. Typical composition forms include solutions, suspensions, emulsions, aerosols, foams, granules, powders, tablets, capsules, and ointments. Furthermore, for injections and the like, the compositions may be freeze-dried or granulated, so that they can be 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 (minor components) may conform to conventionally known methods. Since such formulation methods do not characterize the present disclosure, detailed explanations are omitted. A source of detailed information regarding formulations is, for example, *Comprehensive Medicinal Chemistry*, edited by Corwin Hansch, published by Pergamon Press (1990).
  • the composition disclosed herein inhibits the proliferation of at least one type of cell.
  • the cells whose proliferation is inhibited are cells involved in the expression of APP, such as tumor cells (e.g., neuroblastoma, breast cancer, lung cancer, lymphoma, prostate cancer, colon cancer, thyroid cancer, lung cancer, nasopharyngeal cancer, gastrointestinal cancer, etc.), liver cells, eye cells, brain cells, etc.
  • tumor cells e.g., neuroblastoma, breast cancer, lung cancer, lymphoma, prostate cancer, colon cancer, thyroid cancer, lung cancer, nasopharyngeal cancer, gastrointestinal cancer, etc.
  • liver cells e.g., eye cells, brain cells, etc.
  • the composition disclosed herein preferably inhibits the proliferation of tumor cells.
  • the double-stranded RNA and composition disclosed herein can be preferably used as antitumor agents (anticancer agents) that suppress the proliferation of tumor cells.
  • composition disclosed herein includes, in addition to the double-stranded RNA described above, a peptide fragment (cell-penetrating peptide, CPP) with cell membrane permeability that allows it to pass through the cell membrane from outside the cell and introduce foreign substances into the cytoplasm.
  • the peptide fragment is directly or indirectly bound (linked) to the double-stranded RNA disclosed herein to construct a construct of the peptide fragment and double-stranded RNA.
  • double-stranded RNA is negatively charged and therefore cannot pass through the cell membrane.
  • 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.
  • 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.
  • 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.
  • composition disclosed herein comprises a peptide fragment and the double-stranded RNA disclosed herein.
  • the double-stranded RNA does not have to be bound to the N- or C-terminus of the peptide fragment.
  • the double-stranded RNA and the peptide fragment may form a complex, for example, through electrical or molecular interaction. This complex is more easily introduced into eukaryotic cells, allowing for 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 the molar amount of the double-stranded RNA, preferably 40 to 60 times.
  • the present disclosure may provide a method for inhibiting the proliferation of at least one type of cell using the composition disclosed herein, which includes the steps of preparing a composition of the present disclosure and delivering the composition to a target cell.
  • composition disclosed herein may be prepared by a conventionally known method, for example, as described above.
  • the composition disclosed herein is supplied to at least one type of cell (e.g., tumor cells) in a living body (in vivo) or outside the body (in vitro).
  • the animal species of the supplied cells is not particularly limited and may be, for example, mammals, birds, amphibians, reptiles, fish, etc.
  • the animal species from which the APP that forms 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, but is preferably tumor cells, more preferably neuroblastoma 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).
  • the method of administering the composition is not particularly limited and may be similar to methods conventionally used in animal treatment.
  • the composition can be administered in vivo in a manner and dosage appropriate for its form and purpose.
  • a liquid it can be administered in the desired amount to the affected area (e.g., malignant tumor tissue, virus-infected tissue, inflammatory tissue, etc.) of a patient or animal (i.e., a living organism) by intravenous, intralymphatic, intramuscular, subcutaneous, intradermal, or intraperitoneal injection.
  • a solid form such as a tablet, or a gel or aqueous jelly such as an ointment
  • a solid form such as a tablet, or a gel or aqueous jelly such as an ointment
  • the affected area e.g., the affected area of a tissue or organ containing tumor cells, inflammatory cells, etc.
  • a solid form such as a tablet can be administered orally.
  • encapsulation or the application of a protective (coating) material is preferred to prevent degradation by digestive enzymes in the digestive tract.
  • the amount of the composition to be supplied is not particularly limited.
  • 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 is not particularly limited.
  • 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 ⁇ M or less, 5 ⁇ M or less, 2 ⁇ M or less, 1 ⁇ M or less, or 100 nM or less.
  • compositions disclosed herein can be delivered to the interior of target cells using known transfection methods. Examples include chemical gene transfer methods using cationic molecules (such as commercially available transfection reagents), physical transfer methods such as microinjection and electroporation, and biological gene transfer methods using viruses. Furthermore, as mentioned above, the compositions may also be delivered to the interior of cells using cell membrane-permeable peptide fragments.
  • the sense strand of the double-stranded RNA of Sample 1 is composed of a main sequence consisting of SEQ ID NO: 1 (a base sequence including a portion of the base sequence encoding the signal peptide region of APP) 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 and 3-7 shown in Table 1 is composed of a main sequence consisting of SEQ ID NO: 2 and SEQ ID NOs: 11-15 (a base sequence including a portion of the base sequence encoding the signal peptide region of APP) and an additional sequence consisting of TT added to the 3' end of the main sequence.
  • the antisense strand in each example is composed of a sequence complementary to the main sequence and an additional sequence consisting of TT added to the 3' end of that sequence.
  • SK-N-SH cells were pre-cultured in a culture medium containing 10% fetal bovine serum (FBS) + 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.
  • FBS fetal bovine serum
  • E-MEM Fejifilm Wako Pure Chemical Industries, Ltd., Cat. No. 051-07615
  • MEM non-essential amino acid solution Ferjifilm Wako Pure Chemical Industries, Ltd., Cat. No. 139-15651
  • the SK-N-SH cells that had adhered to the culture plate were washed with PBS, then a 0.25% trypsin/EDTA solution was added and the cells were 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 centrifuged at 150 x g for 5 minutes to precipitate. The supernatant resulting from the centrifugation was removed, and the above-mentioned culture medium was added to the precipitate (cell pellet) to prepare a cell suspension of approximately 5 x 10 4 cells/mL. A commercially available 96-well plate was prepared, and the cell suspension was seeded into each well at 5 x 10 3 cells/100 ⁇ L/well, followed by overnight incubation at 37°C and 5% CO 2 .
  • RNA solution adjusted to 2 mM with PBS was mixed with 75 ⁇ L of Opti-MEMTM to prepare Solution A.
  • Opti-MEMTM was mixed with 75 ⁇ L of Opti-MEMTM to prepare Solution B.
  • 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 ⁇ L/well (final double-stranded RNA concentration: 4 ⁇ M). The cells were then incubated at 37°C under 5 % CO2 for 3 days.
  • Cell proliferation was evaluated using Cell Counting Kit-8 (CCK-8, Dojin Kagaku Kenkyusho).
  • CCK-8 Cell Counting Kit-8
  • the 96-well plate in which SK-N-SH cells had been cultured was removed, 10 ⁇ L of CCK-8 was added to each well, and the plate was incubated at 37°C under 5 % CO2 for 2 hours.
  • the absorbance at 450 nm for each well was measured.
  • the absorbance was calculated as the average of three wells.
  • a blank well containing only the culture medium and CCK-8 reagent was set up.
  • the measured value for Sample 1 was calculated by subtracting the absorbance of the blank from the absorbance of Sample 1.
  • Samples 2 and 3 to 7 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 and 3 to 7 shown in Table 1.
  • the comparative example was the same as Sample 1, except that a PBS solution was used instead of the RNA solution in Sample 1. In other words, no double-stranded RNA was introduced in the comparative example.
  • Untreated wells were prepared in the same manner as Sample 1, except that the RNA solution and LipofectamineTM RNAiMAX were not added.
  • the cell viability in each test example is shown in Figure 2, expressed as a percentage of the measured value for the untreated well, which is set at 100%.
  • Figure 2 shows the results of a test using the double-stranded RNA shown in Table 1, in which neuroblastoma cells were treated with double-stranded RNA whose main sequence was a base sequence (SEQ ID NOs: 1-2) that included a portion of the base sequence encoding the APP signal peptide region.
  • the cell viability of samples 1 and 2 was reduced, and was significantly lower than that of the comparative example. Based on these test results, it is believed that the double-stranded RNA of samples 1 and 2 has the function of inhibiting the proliferation of tumor cells (neuroblastoma cells). Furthermore, although not shown in detail, the sequences of samples 1 and 2 are specific to the APP gene. For this reason, the double-stranded RNA of samples 1 and 2 can avoid off-target effects, does not affect other organs, and is therefore expected to be suitable for clinical application.
  • RNA concentration test of human neuroblastoma cells using low concentrations of double-stranded RNA The double-stranded RNAs used in Samples 1 and 2 shown in Table 1 were prepared. Each of the double-stranded RNAs shown in Samples 1 and 2 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 ⁇ M. A test similar to the cell proliferation test for human neuroblastoma cells was performed, except that the low-concentration RNA solution was used.
  • the final concentration of double-stranded RNA added to the wells in which SK-N-SH cells were cultured was adjusted to 0.4 ⁇ M.
  • the cell viability in each test example was expressed as a percentage, with the measured value for the untreated wells set at 100%.
  • Figure 3 is a graph comparing cell viability when the final concentration of added double-stranded RNA was 4.0 ⁇ M and 0.4 ⁇ M. As shown in Figure 3, the cell viability of Samples 1 and 2 decreased. Furthermore, the cell viability of Samples 1 and 2 was significantly lower than that of the comparative example. This demonstrates that the double-stranded RNA of Samples 1 and 2 was able to inhibit the proliferation of tumor cells (neuroblastoma cells) even at low concentrations. Because the double-stranded RNA of Samples 1 and 2 was able to sufficiently inhibit the proliferation of tumor cells even at low concentrations, it is possible to avoid nonspecific expression inhibition and nonspecific cell proliferation inhibition, and clinical applications are highly anticipated. Furthermore, the double-stranded RNA of Samples 1 and 2 had the same or greater cell inhibition function even at one-tenth the concentration. The above tests were conducted on the same day.
  • double-stranded RNA was prepared for samples 3 to 7 shown in Table 1.
  • Each double-stranded RNA shown in samples 3 to 7 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 ⁇ M.
  • the test was conducted in the same manner as the cell proliferation test for human neuroblastoma cells. That is, in the tests for samples 3 to 7, the final concentration of double-stranded RNA added to the wells in which SK-N-SH cells were cultured was adjusted to 0.4 ⁇ M.
  • the cell viability in each test example was expressed as a percentage, with the measured value for the untreated well being taken as 100%.
  • sterilized ultrapure water was used instead of the sample (no double-stranded RNA was added in these comparative examples).
  • Figure 4 shows the cell viability of human neuroblastoma cells after three days of transfection with the double-stranded RNAs shown in samples 3 to 7.
  • the double-stranded RNAs in samples 3 to 7 were able to inhibit the proliferation of tumor cells (neuroblastoma cells) even at low concentrations. Therefore, they are able to avoid nonspecific inhibition of expression and nonspecific inhibition of cell proliferation, and are expected to be useful in clinical applications. Note that tests using the double-stranded RNAs shown in samples 3 to 7 were conducted independently on a different day from the tests using the double-stranded RNAs shown in samples 1 and 2 above.
  • Item 1 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 a part of a base sequence encoding an amyloid precursor protein and includes at least a part of a base sequence encoding a signal peptide region of the amyloid precursor protein.
  • G guanine
  • C cytosine
  • Item 2 The double-stranded RNA described in Item 1, wherein the second strand is composed of 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.
  • Item 3 The double-stranded RNA according to Item 1 or 2, wherein at least three of the seven bases on the 3'-terminal side of the main sequence are adenine (A) and/or uracil (U).
  • A adenine
  • U uracil
  • the base sequence comprising at least a part of the base sequence encoding the signal peptide region of the amyloid precursor protein is the following base sequence: GCTGGAGGTACCCACTGAT (SEQ ID NO: 1); GCGCTGGAGGTACCCACTGAT (SEQ ID NO: 2); CCGGTTTGGCACTGCTCCT (SEQ ID NO: 11); GTTTGGCACTGCTCCTGCT (SEQ ID NO: 12); GGCGCTGGAGGTACCCACT (SEQ ID NO: 13); GGCGCTGGAGGTACCCACTGA (SEQ ID NO: 14); and CGCTGGAGGTACCCACTGA (SEQ ID NO: 15); Item 4.
  • the double-stranded RNA according to any one of Items 1 to 3, which consists of any one of:
  • 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).
  • TT thymine-thymine
  • Item 6 A composition that inhibits the proliferation of at least one type of cell, comprising the double-stranded RNA described in any one of Items 1 to 5.
  • Item 7 The composition described in Item 6, wherein the cells are tumor cells.
  • Item 8 The composition according to Item 6 or 7, comprising 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.
  • Item 9 A method for inhibiting the proliferation of at least one type of cell, comprising: A preparation step of preparing the composition according to any one of items 6 to 8; and providing said composition to said cells in vitro or in vivo.
  • Item 10 The method according to Item 9, wherein the biological species of the cells is the same as the biological species containing the amyloid precursor protein.
  • the double-stranded RNA disclosed herein can inhibit (or suppress) cell proliferation. Therefore, by using this double-stranded RNA, it is possible to provide a composition (e.g., an anti-tumor agent) that inhibits the proliferation of at least one type of cell (e.g., tumor cells).
  • a composition e.g., an anti-tumor agent

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Abstract

Est divulgué un ARN double brin qui a un premier brin et un second brin complémentaire du premier. Le premier brin comprend : une séquence principale qui comprend 19 à 23 nucléotides et dans laquelle le nucléotide à l'extrémité 5' est la guanine (G) ou la cytosine (C) ; et une séquence supplémentaire qui comprend 2 à 4 nucléotides et est ajoutée au côté d'extrémité 3' de la séquence principale. La séquence principale comprend une partie d'une séquence nucléotidique codant pour une protéine précurseur amyloïde, la partie comprenant au moins une partie d'une séquence nucléotidique codant pour une région peptidique de signal de la protéine précurseur amyloïde.
PCT/JP2025/002078 2024-01-24 2025-01-23 Nouvel arn double brin à base de séquence d'arn et utilisation associée Pending WO2025159163A1 (fr)

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20060110440A1 (en) * 2004-10-22 2006-05-25 Kiminobu Sugaya Method and system for biasing cellular development
WO2009113579A1 (fr) * 2008-03-11 2009-09-17 学校法人埼玉医科大学 Molécule d'acide nucléique bicaténaire adaptée pour la prévention ou le traitement du cancer, inhibiteur de prolifération de cellules cancéreuses, et préparation pharmaceutique

Patent Citations (2)

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Publication number Priority date Publication date Assignee Title
US20060110440A1 (en) * 2004-10-22 2006-05-25 Kiminobu Sugaya Method and system for biasing cellular development
WO2009113579A1 (fr) * 2008-03-11 2009-09-17 学校法人埼玉医科大学 Molécule d'acide nucléique bicaténaire adaptée pour la prévention ou le traitement du cancer, inhibiteur de prolifération de cellules cancéreuses, et préparation pharmaceutique

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SOBOL ANNA, GALLUZZO PAOLA, WEBER MEGAN J., ALANI SARA, BOCCHETTA MAURIZIO: "­­Depletion of Amyloid Precursor Protein (APP) Causes G0 Arrest in Non‐Small Cell Lung Cancer (NSCLC) Cells", JOURNAL OF CELLULAR PHYSIOLOGY, vol. 230, no. 6, 1 June 2015 (2015-06-01), US , pages 1332 - 1341, XP093339960, ISSN: 0021-9541, DOI: 10.1002/jcp.24875 *
ZHANG DONGQING, ZHOU CHANGKUO, LI YAN, GAO LIJIAN, PANG ZHIPENG, YIN GANG, SHI BENKANG: "Amyloid precursor protein is overexpressed in bladder cancer and contributes to the malignant bladder cancer cell behaviors", INTERNATIONAL JOURNAL OF UROLOGY, vol. 25, no. 9, 1 September 2018 (2018-09-01), JP , pages 808 - 816, XP093339958, ISSN: 0919-8172, DOI: 10.1111/iju.13726 *

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