WO2021132648A1 - Oligonucléotide anti-sens inhibant l'expression de ｒｅｃｑｌ, et application associée - Google Patents

Oligonucléotide anti-sens inhibant l'expression de ｒｅｃｑｌ, et application associée Download PDF

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WO2021132648A1
WO2021132648A1 PCT/JP2020/048964 JP2020048964W WO2021132648A1 WO 2021132648 A1 WO2021132648 A1 WO 2021132648A1 JP 2020048964 W JP2020048964 W JP 2020048964W WO 2021132648 A1 WO2021132648 A1 WO 2021132648A1
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recql
nucleotide sequence
nucleotide
cells
seq
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佐藤　秀昭
高尾 鈴木
香代子 山田
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ルクサナバイオテク株式会社
株式会社ジーンケア研究所
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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
    • A61K31/712—Nucleic acids or oligonucleotides having modified sugars, i.e. other than ribose or 2'-deoxyribose
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • 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

  • the present invention relates to a novel antisense oligonucleotide capable of strongly inhibiting the expression of RecQ-like helicase-1 (hereinafter referred to as "RECQL" in the present specification) and its use. More specifically, the present invention presents an antisense nucleotide that hybridizes with a specific region of RECQL mRNA and inhibits its expression, as well as inhibition of RECQL expression using the same, suppression of proliferation of cells that highly express RECQL, and / Or related to cell death induction, cancer treatment and / or prevention, etc.
  • RECQL RecQ-like helicase-1
  • DNA helicase is an enzyme that has the activity of unwinding double-stranded DNA into a single strand.
  • the RecQ helicase family which shows homology with the E. coli RecQ gene product, is ubiquitous from microorganisms to higher animals and plants, and it has been suggested that it plays an important role in maintaining the stabilization of the genome.
  • RECQL, WRN, BLM, RTS, and RecQ5 have been identified, of which abnormalities in WRN, BLM, and RTS are known to cause autosomal recessive inheritance with progeria and frequent cancers. ing.
  • RECQL and RecQ5 have not been reported to be associated with genetic diseases, and have not played a leading role in helicase research so far.
  • RECQL is highly expressed in cancer cells and actively proliferating cells, while its expression level is low in telogen cells.
  • Futami et al. Show that inhibition of RECQL expression by siRNA causes mitotic catastrophe in many cancer cells, and that administration of RECQL siRNA to cancer-bearing model animals shows remarkable antitumor activity. Found (see, eg, Patent Documents 1 and 2). It is thought that these are because the cell cycle progresses without the DNA damage caused by DNA replication being repaired by RECQL, and as a result, it reaches the M phase and causes mitotic death. They also report that RNAi activity is enhanced by modifying nucleotides at specific positions constituting RECQL siRNA, such as 2'-methoxyation (Patent Document 3).
  • IONIS of the United States has an antisense oligonucleotide that targets RECQL mRNA or primary transcript (hereinafter sometimes abbreviated as "ASO"), especially with a central gap region consisting of 10 DNAs.
  • a "5-10-5" type gapmer ASO consisting of 5'- and 3'-wing regions consisting of 5 nucleotides adjacent to each other, with 2'-nucleotides in both wing regions.
  • ASO modified with O-methoxyethyl (2'-MOE) and all nucleoside linkages replaced with phosphorothioate (PS) binding was comprehensively synthesized for the RECQL gene sequence to express the RECQL gene.
  • a specific ASO group that significantly decreases has been identified (Patent Document 4).
  • the conventional RECQL target 2'-MOE-modified ASO is insufficiently effective in terms of RECQL expression inhibitory activity and in vivo stability, and may require high-dose administration to achieve a therapeutic effect. However, it may cause adverse events from ASO chemistry. Therefore, the development of ASO for RECQL, which is more powerful and stable, is desired.
  • an object of the present invention is to provide an ASO for RECQL which is superior in RECQL expression inhibitory activity and / or in vivo stability to a conventionally known 2'-MOE-modified ASO, and the RECQL gene is used using the ASO. It is to provide a novel means for treating and / or preventing hyperproliferative diseases such as cancer by inhibiting the expression of.
  • 2', 4'-crosslink modified ASO showed higher RECQL expression inhibitory activity than 2'-MOE modified ASO when the target regions overlapped. Based on these findings, we found that 2', 4'-crosslink-modified ASOs targeting specific regions of RECQL mRNA have high RECQL expression inhibitory activity and low in vivo stability and / or low. Since it has toxicity, it is concluded that it can be a therapeutic and / or preventive agent for hyperproliferative diseases such as cancer accompanied by overexpression of RECQL, and the present invention has been completed.
  • a single-stranded oligonucleotide that inhibits the expression of the RECQL gene consist of the nucleotide sequence represented by SEQ ID NO: 1. Containing a nucleotide sequence complementary to 10 or more contiguous nucleotide sequences in a target region consisting of any of the nucleotide sequences selected from the group consisting of The length of the single-stranded oligonucleotide is 10 to 30 nucleotides.
  • the sugar moiety of at least one nucleoside constituting the single-stranded oligonucleotide is modified by cross-linking between the 2'position and the 4'position of the sugar.
  • the target region is the 356 to 370th, 836 to 851st, 1100 to 1114th, 1312-1328th, 2753 to 2964th, 3157 to 3274th and 3386th to the nucleotide sequence represented by SEQ ID NO: 1.
  • the single-stranded oligonucleotide according to [1] which comprises any nucleotide sequence selected from the group consisting of the 3663th nucleotide sequence and a nucleotide sequence in the vicinity thereof.
  • the target region is selected from the group consisting of the 836 to 851th, 1100 to 1114th, 1312 to 1328th, 2753 to 2769th, and 2950 to 3663th nucleotide sequences in the nucleotide sequence represented by SEQ ID NO: 1.
  • the single-stranded oligonucleotide according to [1] which comprises one of the nucleotide sequences to be used and a nucleotide sequence in the vicinity thereof.
  • the target region is the 356 to 370th, 836 to 851st, 1100 to 1114th, 1312-1328th, 2753 to 2769th, 2814 to 2828th, 2950 to the nucleotide sequence represented by SEQ ID NO: 1.
  • each nucleoside constituting the region (3) is not sugar-modified, and the sugar portion of at least one nucleoside constituting the regions (1) and (2) is at the 4'position and the 2'position of the sugar.
  • Modified by cross-linking between Region (1) and region (2) are 2-5 nucleotides in length and region (3) is 7-10 nucleotides in length.
  • the single-stranded oligonucleotide according to any one of [1] to [4].
  • [6] The one according to any one of [1] to [5], wherein the modification by cross-linking between the 4'-position and the 2'-position of the sugar is selected from the group consisting of LNA, AmNA, GuNA and ScpBNA. Main chain oligonucleotide.
  • [7] The single-stranded oligonucleotide according to any one of [1] to [6], wherein at least one of the bonds between adjacent nucleosides is a phosphorothioate bond.
  • [8] The single-stranded oligonucleotide according to any one of [1] to [7], which has a nucleotide length of 15.
  • [9] The single-stranded oligonucleotide according to any one of [1] to [8], which comprises a nucleotide sequence represented by any of the SEQ ID NOs: selected from the group consisting of SEQ ID NOs: 2 to 20.
  • the agent according to [10] which suppresses the proliferation of cells highly expressing RECQL and / or induces cell death.
  • the agent according to [10] or [11] which is used for treating and / or preventing cancer.
  • [15] A method for inhibiting the expression of RECQL, which comprises contacting a subject with high expression of RECQL with the single-stranded oligonucleotide according to any one of [1] to [9].
  • [16] Suppressing the proliferation of RECQL-expressing cells and / or cells, including contacting RECQL-expressing cells with the single-stranded oligonucleotide according to any one of [1] to [9]. How to induce death.
  • a method for treating and / or preventing hyperproliferative diseases which comprises administering an effective amount of the single-stranded oligonucleotide according to any one of [1] to [9] to a mammal.
  • the expression of the RECQL gene can be strongly and stably inhibited, the dose, the number of administrations, and the production cost can be reduced, and the expression of adverse events can be suppressed while suppressing the expression of the RECQL gene.
  • highly expressed cells eg, cancer cells
  • cell death it becomes possible to treat and prevent hyperproliferative diseases such as cancer.
  • FIG. 1 It is a figure which shows the expression inhibitory effect of RECQL mRNA when various antisense oligonucleotides (ASO) (20 nM) designed for RECQL mRNA are introduced into cancer cells by the lipofection method.
  • the expression level of RECQL mRNA is shown as a relative value with the expression level in the control without ASO introduced as 1.
  • ASO antisense oligonucleotide
  • inhibiting the expression of the RECQL gene means that, as a result, when ASO and cells are brought into contact with each other, the expression level of RECQL protein is reduced and the activity of RECQL helicase is increased as compared with the case where ASO is not brought into contact with cells. It is used in the sense of including any aspect of reducing, and includes, for example, degradation of a target RNA by RNase H (eg, by gapmer) and inhibition of protein synthesis by specific and stable hybrid formation with the target RNA.
  • RNase H eg, by gapmer
  • the degree of inhibition of expression is not particularly limited as long as it is statistically significant, but is, for example, 20% or more, preferably 50% or more, more preferably 75%, as compared with the case where cells are not brought into contact with ASO.
  • the ASO can be considered to have the expression inhibitory activity of the RECQL gene.
  • the ASO of the present invention is characterized in that it specifically hybridizes to a specific region of RECQL mRNA.
  • the nucleotide sequence of RECQL mRNA is the nucleotide sequence of human RECQL mRNA represented by SEQ ID NO: 1 (registered as Accession No. NM_002907.3 in the NCBI database) or its non-human mammalian ortholog (for example, mouse RECQL).
  • the nucleotide sequence of mRNA is registered in the NCBI database as Accession No. NM_023042.3), or its gene polymorphism. In the present specification, unless otherwise specified, the nucleotide position, the range of the nucleotide sequence, etc.
  • nucleotide sequence of human RECQL mRNA represented by SEQ ID NO: 1. In that case, the gene is described.
  • Corresponding nucleotides and nucleotide sequences in polymorphisms and non-human mammalian orthologs are also included in the description.
  • the ASO of the present invention is the 2750th to 3663th, 80th to 3663th in the RECQL mRNA consisting of the nucleotide sequence represented by SEQ ID NO: 1 (however, "t” in the nucleotide sequence is read as “u”).
  • Targets a region consisting of any of the nucleotide sequences selected from the group consisting of the 300th, 330th to 500th, 800th to 930th, 1000th to 1350th, and 2250th to 2500th nucleotide sequences, and is continuous in the region.
  • the RECQL mRNA consisting of the nucleotide sequence represented by SEQ ID NO: 1 (however, "t” in the nucleotide sequence is read as “u"), the 2750th to 3663th, the 800th to 930th and the 1000th to 1350th.
  • a region consisting of any of the nucleotide sequences selected from the group consisting of nucleotide sequences is targeted and contains a nucleotide sequence complementary to 10 or more consecutive nucleotide sequences in the region.
  • complementary means not only a sequence that is completely complementary to the target sequence (that is, hybridizes without mismatch), but also as long as it can be hubridized with RECQL mRNA under the physiological conditions of mammalian cells. It may be a sequence containing a mismatch of to a number (eg, 1, 2, 3, 4, 5) nucleotides, preferably 1 or 2 nucleotides. For example, 90% or more, preferably 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, and most preferably 100% identical to the complementary strand sequence of the target nucleotide sequence in RECQL mRNA. Examples include sex sequences.
  • complementarity in individual bases is not limited to forming Watson-Crick base pairs with target bases, but forms Hoogsteen base pairs and Wobble base pairs. Including to do.
  • the "complementary nucleotide sequence” is a nucleotide sequence that hybridizes with the target sequence under stringent conditions.
  • the “stringent condition” is, for example, the condition described in Current Protocols in Molecular Biology, John Wiley & Sons, 6.3.1-6.3.6, 1999, for example, 6 ⁇ SSC (sodium chloride / sodium citrate). ) / Hybridization at 45 ° C, followed by 0.2 ⁇ SSC / 0.1% SDS / one or more washings at 50-65 ° C. Hybridization conditions can be appropriately selected.
  • the regions in the RECQL mRNA targeted by the ASO of the present invention are positions 356 to 370, 836 to 851, 1100-1114, 1312-1328 in the nucleotide sequence represented by SEQ ID NO: 1. It is a region consisting of any nucleotide sequence selected from the group consisting of nucleotide sequences 2753 to 2964, 3157 to 3274, and 3386 to 3663, and a nucleotide sequence in the vicinity thereof.
  • the region in the RECQL mRNA targeted by the ASO of the present invention is the nucleotides 836 to 851, 1312-1328, 2753 to 2964 and 3157 to 3274 in the nucleotide sequence represented by SEQ ID NO: 1. It is a region consisting of any nucleotide sequence selected from the group consisting of sequences and nucleotide sequences in the vicinity thereof.
  • the term "nucleotide sequence in the vicinity" refers to 50 nucleotides or less, preferably 30 nucleotides or less, more preferably 10 nucleotides or less, adjacent to the 5'- and 3'-terminals of each of the regions defined by the nucleotide number. More preferably, it means a nucleotide sequence of 5 nucleotides or less. The same applies to the following.
  • the regions in the RECQL mRNA targeted by the ASO of the present invention are positions 836 to 851, 1100-1114, 1312-1328, 2753 to 2769 in the nucleotide sequence represented by SEQ ID NO: 1. It is a region consisting of one of the nucleotide sequences selected from the group consisting of the 2950th to 3663th nucleotide sequences and the nucleotide sequence in the vicinity thereof. More preferably, the region in the RECQL mRNA targeted by the ASO of the present invention is from the group consisting of the nucleotide sequences 836 to 851, 1312-1328 and 2950 to 3663 in the nucleotide sequence represented by SEQ ID NO: 1.
  • a region consisting of one of the selected nucleotide sequences and a nucleotide sequence in the vicinity thereof.
  • the RECQL expression inhibitory activity was low or the ASO was low in the gapmer type ASO in which the wing region described in Patent Document 4 (International Publication No. 02/068590) was modified with 2'-MOE. Corresponds to the undesigned area.
  • the regions in the RECQL mRNA targeted by the ASO of the present invention are positions 356-370, 836-851, 1100-1114, 1312-in the nucleotide sequence represented by SEQ ID NO: 1.
  • the region in the RECQL mRNA targeted by the ASO of the present invention is the nucleotides 836 to 851, 1312-1328, 2950 to 2964 and 3157 to 3400 in the nucleotide sequence represented by SEQ ID NO: 1. It is a region consisting of any nucleotide sequence selected from the group consisting of sequences and nucleotide sequences in the vicinity thereof.
  • the ASOs of the present invention are 10 or more consecutive (eg, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20), preferably 15 in any of the above-mentioned target regions.
  • a sequence consisting of one or more nucleotides is used as a target sequence, and a nucleotide sequence complementary thereto is included.
  • the length of the ASO of the present invention is not particularly limited, but is, for example, 10 to 30 nucleotides in length, preferably 12 to 30 nucleotides in length, and more preferably 15 to 25 nucleotides in length.
  • Examples of the constituent units of ASO of the present invention include ribonucleotides and deoxyribonucleotides. These nucleotides may be modified (modified nucleotide residues may be referred to as “modified nucleotide residues") or unmodified (unmodified nucleotide residues are referred to as "unmodified nucleotides”. Sometimes referred to as "residue").
  • the nucleotide residue contains sugar, base and phosphoric acid as components.
  • Ribonucleotides have a ribose residue as a sugar and are replaced by adenine (A), guanine (G), cytosine (C), 5-methylcytosine (mC) and uracil (U) (thymine (T)) as bases.
  • the deoxyribonucleotide residue has a deoxyribose residue as a sugar and as bases adenine (dA), guanine (dG), cytosine (dC), 5-methylcytosine (dmC) and Has thymine (dT) (which can also be replaced with uracil (dU)).
  • nucleotides having adenine, guanine, (5-methyl) cytosine, uracil, and thymine may be referred to as adenine nucleotide, guanine nucleotide, (5-methyl) cytosine nucleotide, uracil nucleotide, and thymine nucleotide, respectively.
  • the unmodified nucleotide residues are such that each of the components is, for example, the same or substantially the same as naturally occurring, preferably the same or substantially the same as naturally occurring in the human body. ..
  • the modified nucleotide residue may be modified by, for example, any of the components of the unmodified nucleotide residue.
  • “modification” includes, for example, substitution, addition and / or deletion of the component, substitution, addition and / or deletion of an atom and / or functional group in the component.
  • the modified nucleotide residue include naturally occurring nucleotide residues, artificially modified nucleotide residues, and the like.
  • the naturally occurring modified nucleotide residue for example, Limbach et al. (1994, Summary: the modified nucleosides of RNA, Nucleic Acids Res. 22: 2183 to 2196) can be referred to.
  • examples of the modified nucleotide residue include residues that are substitutes for the nucleotide.
  • Modification of the nucleotide residue includes, for example, modification of a sugar-phosphate skeleton (the skeleton also includes a base) (hereinafter, sugar phosphate skeleton).
  • the ribose residue when the sugar is ribose, for example, the ribose residue can be modified.
  • the ribose residue can modify, for example, the 2'carbon, and specifically, for example, the hydroxyl group bonded to the 2'carbon can be modified with a methyl group, or the hydroxyl group can be replaced with a halogen such as hydrogen or fluoro. .. Further, by substituting the hydroxyl group of the 2'carbon with hydrogen, the ribose residue can be replaced with deoxyribose.
  • the ribose residue can be replaced with, for example, a stereoisomer, and may be replaced with, for example, an arabinose residue.
  • nucleic acid in which the hydroxyl group bonded to the 2'carbon of the sugar is modified with a methoxy group as described above may be referred to as a 2'-O-methyl modified nucleic acid.
  • nucleic acid includes nucleic acid monomers such as nucleotides.
  • the sugar phosphate skeleton may be replaced, for example, with a non-ribose phosphate skeleton having non-ribose residues (including non-deoxyribose residues) and / or non-phosphate, such substitutions. Is also included in the modification of the sugar phosphate skeleton.
  • examples of the non-ribose phosphate skeleton include uncharged compounds of the sugar phosphate skeleton.
  • Substitutes for the nucleotides substituted with the non-ribos phosphate skeleton include, for example, morpholino, cyclobutyl, pyrrolidine and the like.
  • Other examples of the alternative include artificial nucleic acids.
  • BNA crosslinked artificial nucleic acid
  • BNA Bridged Nucleic Acid
  • BNA Locked artificial nucleic acid
  • AmNA in the following formula (I)
  • R is a methyl group
  • GuNA scpBNA
  • ENA S-cEt
  • the specific structure (nucleoside moiety) of BNA that can be used in the present invention is shown below.
  • R is a hydrogen atom, an alkyl group having 1 to 7 carbon atoms which may form a branched or ring, an alkenyl group having 2 to 7 carbon atoms which may form a branched or ring, Represents an aryl group having 3 to 12 carbon atoms which may contain a heteroatom, an aralkyl group having an aryl moiety having 3 to 12 carbon atoms which may contain a heteroatom, or a protective group of an amino group for nucleic acid synthesis.
  • R is a hydrogen atom, a methyl group, an ethyl group, an n-propyl group, an isopropyl group, a phenyl group, or a benzyl group, and more preferably, R is a hydrogen atom or a methyl group.
  • B or Base represents a base.
  • These artificial nucleic acids can be synthesized by referring to, for example, JP-A-2002-241393, JP-A-2000-297097, and the like.
  • the ASO of the present invention is characterized in that the sugar portion of at least one nucleoside is modified by cross-linking between the 2'position and the 4'position of the sugar.
  • the 2', 4'-crosslink modification can increase the binding force to the target RNA and the metabolic stability (nuclease resistance) in vivo due to its crosslink structure.
  • LNA, AmNA, GuNA, and scpBNA are preferable, and AmNA, GuNA, and scpBNA are more preferable because they are superior in nuclease resistance and have low toxicity.
  • the ASO of the present invention comprises two or more crosslinked artificial nucleic acid residues (eg, 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more).
  • the position of the crosslinked artificial nucleic acid residue is not particularly limited as long as it does not adversely affect the RECQL expression inhibitory activity.
  • the wing region Nucleotide residues are modified with crosslinked artificial nucleic acids.
  • a phosphate group can be modified.
  • the phosphate group closest to the sugar residue is called an ⁇ -phosphate group.
  • the ⁇ -phosphate group is negatively charged, and the charge is uniformly distributed over two oxygen atoms unbonded to sugar residues.
  • the two oxygen atoms that are unbonded to the sugar residue in the phosphodiester bond between the nucleotide residues are hereinafter also referred to as “non-linking oxygen”.
  • linking oxygen the two oxygen atoms bonded to the sugar residue are hereinafter referred to as "linking oxygen”. It is preferable that the ⁇ -phosphate group is modified so that it becomes uncharged or the charge distribution in the unbound oxygen becomes asymmetrical, for example.
  • the phosphoric acid group may replace, for example, the unbound oxygen.
  • the oxygen is, for example, any one of S (sulfur), Se (sulfur), B (boron), C (carbon), H (hydrogen), N (nitrogen) and OR (R is an alkyl group or an aryl group). Can be replaced with an atom of, preferably with S.
  • the unbound oxygen may be substituted with either or both, preferably any one or both.
  • the modified phosphate groups include, for example, phosphorothioate, phosphorodithioate, phosphoroselenate, boranophosphate, boranophosphate ester, phosphonate hydrogen, phosphoramidate, alkyl or arylphosphonate, and. Examples thereof include phosphotriesters, and phosphorothioates and phosphorodithioates are preferable.
  • the phosphoric acid group may be replaced with a phosphorus-free linker.
  • the linker include siloxane, carbonate, carboxymethyl, carbamate, amide, thioether, ethylene oxide linker, sulfonate, sulfonamide, thioform acetal, form acetal, oxime, methylene imino, methylene methyl imino, methylene hydrazo, and methylene. Examples thereof include dimethylhydrazo and methyleneoxymethylimino, and preferably methylenecarbonylamino group and methylenemethylimino group.
  • the phosphoric acid group may be replaced with another phosphoric acid-free linker. Examples of such a linker include those described in “Med. Chem. Communi., 2014, 5, 1454-1471”.
  • 1/2 or more, more preferably 2/3 or more of the phosphate groups contained in the ASO of the present invention are modified with one or more of the above phosphate groups, and even more preferably. All phosphate groups are modified.
  • All phosphate groups are modified.
  • 8 or more, preferably 10 or more, more preferably all phosphate groups are, for example, phosphorothioated, phosphorodithioated or the like. Substitution of unbound oxygen at the phosphodiester bond with sulfur atoms is important for improving nuclease resistance and in the tissue distribution of ASO.
  • nuclease resistance is improved because one or more constituent nucleotides are replaced with crosslinked artificial nucleic acids. Therefore, in the ASO of the present invention, desired in vivo stability may be obtained without converting all phosphate groups into PS.
  • At least one nucleotide residue at the 3'end and the 5'end may be modified.
  • the modification may be, for example, either one of the 3'ends and the 5'end, or both.
  • the modification is, for example, as described above, and is preferably performed on the terminal phosphate group.
  • the phosphoric acid group may, for example, modify the whole, or may modify one or more atoms in the phosphoric acid group. In the former case, for example, the entire phosphate group may be substituted or deleted.
  • Modification of the terminal nucleotide residue includes, for example, addition of another molecule.
  • the other molecule include a labeling substance described later and a functional molecule such as a protecting group.
  • the protecting group include S (sulfur), Si (silicon), B (boron), and ester-containing groups.
  • Functional molecules such as the labeling substance can be used, for example, for detecting ASO of the present invention.
  • the other molecule may be added to the phosphate group of the nucleotide residue, or may be added to the phosphate group or the sugar residue via a spacer, for example.
  • the terminal atom of the spacer can be added or substituted, for example, to the bound oxygen of the phosphate group or O, N, S or C of a sugar residue.
  • the binding site of the sugar residue is preferably, for example, C at the 3'position or C at the 5'position, or an atom that binds to these.
  • the spacer can also be added or substituted, for example, to the terminal atom of a nucleotide substitute such as PNA.
  • the spacer is not particularly limited, and is, for example,-(CH 2 ) n -,-(CH 2 ) n N-,-(CH 2 ) n O-,-(CH 2 ) n S-, O (CH 2).
  • CH 2 O) n CH 2 CH 2 OH non-basic sugar, amide, carboxy, amine, oxyamine, oxyimine, thioether, disulfide, thiourea, sulfonamide, morpholino, etc., as well as biotin and fluorescein reagents, etc. Good.
  • the molecule added to the terminal includes, for example, a dye, an intercalating agent (for example, acrydin), a cross-linking agent (for example, solarene, mitomycin C), porphyrin (TPPC4, texaphyllin, sapphirine), and a polycyclic type.
  • an intercalating agent for example, acrydin
  • a cross-linking agent for example, solarene, mitomycin C
  • porphyrin texaphyllin, sapphirine
  • a polycyclic type for example, a dye, an intercalating agent (for example, acrydin), a cross-linking agent (for example, solarene, mitomycin C), porphyrin (TPPC4, texaphyllin, sapphirine), and a polycyclic type.
  • Aromatic hydrocarbons eg phenazine, dihydrophenazine
  • artificial endonucleases eg EDTA
  • lipophilic carriers eg cholesterol, cholic acid, adamantan acetic acid, 1-pyrenebutyric acid, dihydrotestosterone, 1,3-bis- O (hexadecyl) glycerol, geranyloxyhexyl group, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid, myristic acid, O3- (oleoic) lithocholic acid, O3- (oleoyl) Cholic acid, dimethoxytrityl, or phenoxazine) and peptide complexes (eg, antennapedia peptide, Tat peptide), alkylating agents, phosphates, amino, mercapto, PEG (e
  • the 5'end may be modified with, for example, a phosphate group or a phosphate group analog.
  • the phosphoric acid group is, for example, 5'monophosphoric acid ((HO) 2 (O) PO-5'), 5'diphosphoric acid ((HO) 2 (O) POP (HO) (O) -O- 5'), 5'triphosphate ((HO) 2 (O) PO- (HO) (O) POP (HO) (O) -O-5'), 5'-guanosine cap (7-methylated or Unmethylated, 7m-GO-5'-(HO) (O) PO- (HO) (O) POP (HO) (O) -O-5'), 5'-adenosine cap (Appp), optional Modified or unmodified nucleotide cap structure (NO-5'-(HO) (O) PO- (HO) (O) POP (HO) (O) -O-5'), 5'monothiophosphate (phosphoroth
  • the base is not particularly limited, and may be, for example, a natural base or a non-natural base.
  • the base may be, for example, naturally derived or synthetic.
  • As the base for example, a general base, a modified analog thereof, a universal base, or the like can be used.
  • Examples of the base include purine bases such as adenine and guanine, and pyrimidine bases such as cytosine, 5-methylcytosine, uracil and thymine.
  • Other examples of the base include inosine, thymine, xanthine, hypoxanthine, nubularine, isoguanisine, tubercidine and the like.
  • the base is, for example, an alkyl derivative such as 2-aminoadenine, 6-methylated purine; an alkyl derivative such as 2-propylated purine; 5-halouracil and 5-halocytosine; 5-propynyl uracil and 5-propynylcitosine; 6 -Azouracil, 6-azocitosine and 6-azotimine; 5-uracil (psoid uracil), 4-thiouracil, 5-halouracil, 5- (2-aminopropyl) uracil, 5-aminoallyl uracil; 8-halogenation, amination, Thiolization, thioalkylation, hydroxylation and other 8-substituted purines; 5-trifluoromethylation and other 5-substituted pyrimidines; 7-methylguanine; 5-substituted pyrimidines; 6-azapyrimidines; N-2, N -6, and O-6 substituted purines (including
  • modified nucleotide residue may include, for example, a residue lacking a base, that is, a base-free sugar phosphate skeleton.
  • modified nucleotide residue for example, the residue described in International Publication No. 2004/080406 can be used, and the present invention can refer to these documents.
  • the ASO of the present invention may be labeled with, for example, a labeling substance.
  • the labeling substance is not particularly limited, and examples thereof include fluorescent substances, dyes, and isotopes.
  • the fluorescent substance include fluorescent groups such as pyrene, TAMRA, fluorescein, Cy3 dye, and Cy5 dye.
  • the dye include Alexa dyes such as Alexa 488.
  • the isotope include stable isotopes and radioactive isotopes, and stable isotopes are preferable. For example, the stable isotope has a low risk of exposure and does not require a dedicated facility, so that it is easy to handle and the cost can be reduced.
  • the stable isotope does not change the physical properties of the labeled compound, and is excellent in properties as a tracer.
  • the stable isotope is not particularly limited, and examples thereof include 2 H, 13 C, 15 N, 17 O, 18 O, 33 S, 34 S and 36 S.
  • the ASO of the present invention sets any of the following nucleotide sequences as a sequence complementary to the target sequence in RECQL mRNA (indicated by the position in the nucleotide sequence represented by SEQ ID NO: 1). Including.
  • tgattaactttccgg (SEQ ID NO: 2) (Target sequence: 356-370) acgttaatagtttca (SEQ ID NO: 3) (target sequence: 836-850) tacgttaatagtttc (SEQ ID NO: 4) (target sequence: 837-851) gtcacataaatcagc (SEQ ID NO: 5) (target sequence: 1100-1114) cgtgatttgttgcag (SEQ ID NO: 6) (Target sequence: 1312-1326) aacgtgatttgttgc (SEQ ID NO: 7) (Target sequence: 1314-1328) cgattgtatgaactt (SEQ ID NO: 8) (Target sequence: 2753-2767) gacgattgtatgaac (SEQ ID NO: 9) (target sequence: 2755-2769) aagatagttatgtca (SEQ ID NO: 10) (
  • the ASO of the present invention is a nucleic acid consisting of any of the above nucleotide sequences.
  • the ASO of the present invention is a nucleic acid consisting of the nucleotide sequence represented by SEQ ID NO: 4, 6, 11, 13 or 14.
  • the ASO of the present invention is: (1) 5'wing region located at the 5'end; A gapmer-type nucleic acid comprising (2) a 3'wing region located at the 3'end; and a deoxygap region located between (3) regions (1) and (2).
  • Gapmer-type ASO is a nucleic acid (wing region) having DNA (deoxygap region) and nucleic acids having been modified or cross-linked on both sides thereof, and the DNA strand is used as the main chain to form the main chain. It forms a heteroduplex nucleic acid with a complementary target RNA, which is degraded by RNase H, which is endogenous to the cell.
  • the constituent nucleotides of the wing region may be RNA or DNA.
  • the 5'and 3'wing regions of the gapmer type ASO of the present invention are independently 2 to 5 nucleotides in length, preferably 3 to 5 nucleotides in length, and more preferably 3 nucleotides in length.
  • the length of the deoxygap region of the gapmer type ASO of the present invention is 7 to 10 nucleotides, preferably 8 to 10 nucleotides, and more preferably 9 nucleotides.
  • the total length of the gapmer type ASO of the present invention is, for example, 12 to 30 nucleotides in length, preferably 12 to 25 nucleotides in length. Therefore, the gapmer type ASO of the present invention can be appropriately adjusted by those skilled in the art under conditions that satisfy all the specified ranges of the wing region length, the deoxy gap region length, and the total length.
  • the gapmer type ASO of the present invention is, for example, a 15-nucleotide length "3-9-3” type gapmer, a "3-10-2” type gapmer, or a “2-10-3” type.
  • Gapmer "4-9-2" type gapmer; 16 nucleotide length "3-10-3” type gapmer, "4-9-3” type gapmer; 17 nucleotide length "4-10-3” type gapmer "4-9-4" type gapmer; 18 nucleotide length "4-10-4" type gapmer, "5-9-4" type gapmer; 19 nucleotide length "5-10” type gapmer It is preferably a "-4" type gapmer, a "5-9-5" type gapmer; or a 20 nucleotide length "5-10-5" type gapmer.
  • the sugar portion of at least one nucleoside constituting the 5'and 3'wing regions is modified by cross-linking between the 4'position and the 2'position of the sugar. ..
  • the cross-linking modification include the above-mentioned modification with the cross-linked artificial nucleic acid. It is preferably LNA, AmNA, GuNA and scpBNA, and more preferably AmNA, GuNA and scpBNA.
  • the Gapmer-type ASO of the present invention is an artificial nucleic acid in which two or more (eg, 2, 3, 4, 5) nucleotide residues constituting each of the 5'and 3'wing regions are crosslinked. It has been replaced.
  • the DNA residues constituting the deoxygap region of the gapmer type ASO of the present invention are not sugar-modified.
  • the gapmer type ASO of the present invention may be modified with a base of at least one nucleoside constituting the 5'and 3'wing regions.
  • Examples of the base modification include any of the above modifications.
  • gapmer type ASO of the present invention can be subjected to base modification of the deoxygap region and dual modification of the wing region in order to reduce toxicity. Such modifications are described, for example, in WO 2018/155450.
  • the Gapmer-type ASO of the present invention is a nucleic acid having the nucleotide sequence shown in Table 1 and a sugar, base or phosphate group modification (in the nucleotide sequence below, T is U). May be good).
  • the ASO of the present invention can be produced by a chemical synthesis method known per se.
  • the phosphoramidite method and the H-phosphonate method can be mentioned.
  • the chemical synthesis method can be carried out using, for example, a commercially available automatic nucleic acid synthesizer, and when amidite is used, for example, as a commercially available amidite, RNA Phosphoramidites (2'-O-TBDMSi, trade name, 3) Senri Pharmaceutical Co., Ltd.), ACE amidite and TOM amidite, CEE amidite, CEM amidite, TEM amidite and the like can be used.
  • ASO of the present invention can specifically hybridize to RECQL mRNA and inhibit the expression of the RECQL gene. Therefore, the present invention also provides an expression inhibitor of the RECQL gene, which comprises the ASO of the present invention.
  • the RECQL gene expression inhibitor of the present invention is introduced into a subject having a high expression of RECQL, for example, by contacting the ASO of the present invention alone or with a pharmacologically acceptable carrier. be able to.
  • the contact step can be performed by administering the RECQL gene expression inhibitor of the present invention to the animal.
  • the subject is a culture of cells, tissues or organs derived from animals, this can be carried out by adding the RECQL gene expression inhibitor of the present invention to the medium of the culture.
  • the expression inhibitor of the RECQL gene of the present invention may further contain a reagent for introducing a nucleic acid.
  • the reagents for introducing nucleic acids include atelocollagen; liposomes; nanoparticles; lipofectin, lipofectamine, DOGS (transfectum), DOPE, DOTAP, DDAB, DHDEAB, HDEAB, polybrene, or poly (ethyleneimine) (PEI). ) And the like can be used.
  • the ASO of the present invention can be introduced into target cells by, for example, a calcium ion enrichment (CEM) method in which calcium chloride is added to a medium.
  • CEM calcium ion enrichment
  • the present invention also provides an agent for suppressing cell proliferation and / or inducing cell death of cells highly expressing the RECQL gene containing the ASO of the present invention.
  • the cell proliferation inhibitor and / or cell death inducer of the present invention can be brought into contact with a subject highly expressing RECQL in the same manner as described above.
  • the drug containing the ASO of the present invention as an active ingredient can be used for the treatment and / or prevention of hyperproliferative diseases.
  • hyperproliferative diseases include cancer and the like.
  • the cancer is not particularly limited as long as RECQL is highly expressed, and may be, for example, a cancer derived from epithelial cells, but may be a non-epithelial sarcoma or a blood cancer.
  • gastrointestinal cancer eg, esophageal cancer, gastric cancer, duodenal cancer, colon cancer (colon cancer, rectal cancer), liver cancer (hepatocellular carcinoma, bile duct cancer) Cancer), bile sac cancer, bile duct cancer, pancreatic cancer, anal cancer
  • urinary cancer eg, kidney cancer, urinary tract cancer, bladder cancer, prostate cancer, penis cancer, testis (Testicle) cancer
  • chest cancer eg, breast cancer, lung cancer (non-small cell lung cancer, small cell lung cancer)
  • genital cancer eg, uterine cancer (cervical cancer, uterine body cancer)
  • Ovarian cancer vulgar cancer, vaginal cancer
  • head and neck cancer eg, maxillary cancer, pharyngeal cancer, laryngeal cancer, tongue cancer, thyroid cancer
  • skin cancer eg, (Bass cell carcinoma, spinous cell carcinoma
  • the ASO of the present invention may be used alone or may be formulated as a pharmaceutical composition together with a pharmaceutically acceptable carrier.
  • Pharmaceutically acceptable carriers include, for example, excipients such as sucrose and starch, binders such as cellulose and methyl cellulose, disintegrants such as starch and carboxymethyl cellulose, lubricants such as magnesium stearate and aerodyl, citric acid, etc.
  • Fragrances such as menthol, preservatives such as sodium benzoate and sodium hydrogen sulfite, stabilizers such as citric acid and sodium citrate, suspending agents such as methylcellulose and polyvinylpyrrolidone, dispersants such as surfactants, water, physiology Diluting agents such as saline solution, base wax and the like can be mentioned, but the present invention is not limited thereto.
  • the reagent of the present invention may further contain a reagent for introducing a nucleic acid.
  • a nucleic acid introduction reagent the same reagents as those described above can be used.
  • the medicament of the present invention may be a pharmaceutical composition in which the ASO of the present invention is encapsulated in liposomes.
  • Liposomes are microclosed vesicles having an internal phase surrounded by one or more lipid bilayers, which can usually retain water-soluble substances in the internal phase and fat-soluble substances in the lipid bilayer.
  • the ASO of the present invention may be retained in the liposome internal phase or in the lipid bilayer.
  • the liposome used in the present invention may be a monolayer membrane or a multilayer membrane, and the particle size can be appropriately selected in the range of, for example, 10 to 1000 nm, preferably 50 to 300 nm. Considering the deliverability to the target tissue, the particle size can be, for example, 200 nm or less, preferably 100 nm or less.
  • Examples of the method for encapsulating a water-soluble compound such as an oligonucleotide in liposomes include a lipid film method (vortex method), a reverse phase evaporation method, a surfactant removal method, a freeze-thaw method, and a remote loading method. Any known method can be appropriately selected without limitation.
  • the medicament of the present invention can be administered orally or parenterally to mammals (eg, humans, rats, mice, guinea pigs, rabbits, sheep, horses, pigs, cows, monkeys). However, it is desirable to administer it parenterally.
  • mammals eg, humans, rats, mice, guinea pigs, rabbits, sheep, horses, pigs, cows, monkeys.
  • Suitable formulations for parenteral administration include aqueous and non-aqueous isotonic sterile injections.
  • This may contain an antioxidant, a buffer, an antibacterial agent, an isotonic agent, and the like.
  • examples thereof include aqueous and non-aqueous sterile suspensions, which may include suspending agents, solubilizing agents, thickeners, stabilizers, preservatives and the like.
  • the pharmaceutical product can be encapsulated in a container in a unit dose or a plurality of doses like an ampoule or a vial.
  • the active ingredient and a pharmaceutically acceptable carrier can be freeze-dried and stored in a state where it can be dissolved or suspended in a suitable sterile vehicle immediately before use.
  • the content of ASO of the present invention in the pharmaceutical composition is, for example, about 0.1 to 100% by weight of the entire pharmaceutical composition.
  • the dose of the medicament of the present invention varies depending on the purpose of administration, the administration method, the type and severity of the target disease, and the situation of the administration target (gender, age, weight, etc.).
  • the single dose of ASO of the present invention is 2 nmol / kg or more and 50 nmol / kg or less, and in the case of local administration, 1 pmol / kg or more and 10 nmol / kg or less is desirable.
  • Such amounts can be administered at intervals of, for example, 1 to 6 months, preferably 2 to 4 months, more preferably about 3 months.
  • the ASO of the present invention is remarkably superior in RECQL expression inhibitory activity and in vivo stability as compared with the conventionally known ASO for RECQL, it can exert a therapeutic and / or preventive effect with a small dose and number of administrations. It is possible to improve the QOL of patients, reduce medical expenses, and suppress the occurrence of adverse events.
  • the medicament of the present invention may contain other active ingredients as long as the combination with the ASO of the present invention does not cause an unfavorable interaction.
  • active ingredients for example, various compounds having a therapeutic effect on cancer can be appropriately blended.
  • other active ingredients include alkylating agents (eg, mustards, nitrosoureas), metabolic antagonists (eg, folic acid, pyrimidines, purines), anti-neoplastic antibiotics (eg, anthracyclines), Even if it contains hormone-like drugs (eg, anti-estrogen drugs, anti-androgens, LH-RH agonists, progesterone, estradiol), platinum preparations, topoisomerase inhibitors (eg, topoisomerase I inhibitors, topoisomerase II inhibitors), etc.
  • alkylating agents eg, mustards, nitrosoureas
  • metabolic antagonists eg, folic acid, pyrimidines, purines
  • concomitant agents can be formulated together with the drug of the present invention and administered as a single preparation, or they can be formulated separately from the drug of the present invention and the same as or by a different route from the drug of the present invention. , Can be administered simultaneously or at different times.
  • the dose of these concomitant drugs may be the amount normally used when the drug is administered alone, or may be reduced from the amount normally used.
  • the amide BNA (AmNA) represented by the formula (a) was used as the sugar-modified nucleoside.
  • Base is 5-methylcytosine-1-yl group, thymine-1-yl group, adenine-9-yl group or guanine-9-yl group, and Me is methyl.
  • Oligonucleotides containing amide BNA were synthesized with reference to the method described in WO 2011/052436.
  • Oligonucleotides containing amide BNA were synthesized on a 0.2 ⁇ mol scale using an automatic nucleic acid synthesizer (nS-8 type, manufactured by GeneDesign, Inc.). Chain length extension Height is standard phosphoramidite protocol (solid carrier: CPG, sulfurization for phosphorothioatetization (PS) skeleton formation is DDTT (((dimethylamino-methylidene) amino) -3H-1,2,4 -It was carried out using dithiazaoline-3-thione) etc.).
  • Oligonucleotides containing amide BNA are those in which the terminal 5'-hydroxyl group is not protected by DMTr (4,4'-dimethoxytrityl) groups and the 3'-position is supported on the solid phase. Obtained. Subsequently, the target product was cut out from the solid phase carrier by base treatment, the solvent was distilled off, and the obtained crude product was purified by reverse phase HPLC to obtain the target product. The purity and structure of each of the obtained oligonucleotides were confirmed by LC-MS (manufactured by Waters).
  • Example 2 Design of antisense oligonucleotide
  • the antisense oligonucleotide was designed to target the mRNA of human RECQL (hRECQL) (GenBank: NM_002907.3 (SEQ ID NO: 1)).
  • a region such as a loop structure that is easily accessible to antisense oligonucleotides was selected based on the prediction of the secondary structure of mRNA in which thermodynamically stable complementary binding was calculated from the primary sequence of mRNA.
  • Inversely complementary sequences of mRNA were selected in this region, and sequences containing CG, TCC, and TGC sequences expressing toxicity with antisense were excluded from those sequences.
  • a candidate sequence was selected by evaluating the homology between the sequence and genes other than hRECQL using GGGenome (GGGenome: gggenome.dbcls.jp/).
  • An oligonucleotide having a base sequence complementary to the candidate sequence selected as described above was designed as an antisense oligonucleotide.
  • the antisense oligonucleotide was made into a 15-mer, and an artificial nucleic acid region containing a sugar-modified nucleoside was provided at the 5'end and 3'end, and a natural nucleic acid region containing a natural nucleoside (DNA) was provided at the center. More specifically, 3 bases on the 5'end side (5'wing region) are sugar-modified nucleosides, then 9 bases (gap region) are natural nucleosides (DNA), and then 3 bases on the 3'end side (3'wings).
  • a 3-9-2-1 type gapmer was designed in which two bases on the central side of the region) are sugar-modified nucleosides and one base at the 3'end is DNA.
  • the designed and prepared antisense oligonucleotides are listed in Tables 2-1 and 2-2.
  • Table 2 shows the names of the antisense oligonucleotides (“oligonucleotide names”) along with the 5'end and 3'end of the sequence of the target region (each indicated by the base position of SEQ ID NO: 1).
  • hRECQL-268-AmNA is a 15-mer antisense oligonucleotide containing AmNA in which the 268th position of the nucleotide sequence of SEQ ID NO: 1 is the 5'end of the target region.
  • hRECQL-pn L
  • p is the number of the base position corresponding to the 5'end of the target region in SEQ ID NO: 1.
  • N is a sugar-modified nucleoside (artificial nucleic acid) ("AmNA” in Table 1)
  • AmNA artificial nucleic acid
  • L is the length of the antisense oligonucleotide.
  • the 836 position of the base sequence of SEQ ID NO: 1 is the 5'end of the target region, contains AmNA, and is a 15-mer.
  • the sequence of the antisense oligonucleotide is represented in the direction from 5'to 3'(5' ⁇ 3'), for example, when the antisense oligonucleotide is 15 meters long in hRECQL-836, the nucleotide sequence of SEQ ID NO: 1 In the target mRNA sequence based on 5'-tgaaactattaacgt-3'(SEQ ID NO: 97), which is the DNA base sequence from the 836 position to the 850 position of SEQ ID NO: 1, which is extended from the 836 position to the 3'terminal side of 15 bases.
  • the nucleotide sequence (5'-acgttaatagtttca-3') (SEQ ID NO: 3) of the antisense oligonucleotide hRECQL-836-AmNA (15) designed in this way is the region from position 836 to position 850 of SEQ ID NO: 1.
  • the sequence is complementary to the base sequence (5'-tgaaactattaacgt-3') (SEQ ID NO: 97).
  • Example 3 Suppression of hRECQL mRNA expression in human cervical cancer cells
  • Example 2 mRNA expression suppression analysis of various antisense oligonucleotides
  • the antisense oligonucleotide prepared in Example 2 was examined for the mRNA expression inhibitory activity of hRECQL in vitro in human cervical cancer cells.
  • the antisense oligonucleotides that were prepared and whose expression was suppressed were appropriately numbered (reference numbers LX-A0050 to LX-A0065 and LX-A0254 to LX-A0312; Tables 2-1 and 2-2, respectively. reference). Those to which no oligonucleotide was added were used as controls.
  • negative control oligonucleotides containing AmNA also referred to as Negative Control; NC: 5 (Y) ⁇ A (Y) ⁇ T (Y) ⁇ t ⁇ t ⁇ c ⁇ g ⁇ a ⁇ a ⁇ g ⁇ t ⁇ a ⁇ 5 (Y) ⁇ T (Y) ⁇ c (LX-A0030; array Number 99); 5 (Y) ⁇ A (Y) ⁇ T (Y) ⁇ t ⁇ a ⁇ g ⁇ c ⁇ t ⁇ a ⁇ g ⁇ t ⁇ a ⁇ 5 (Y) ⁇ T (Y) ⁇ c (LX-A0069; array Number 100); and 5 (Y) ⁇ A (Y) ⁇ T (Y) ⁇ t ⁇ t ⁇ a ⁇ g ⁇ a ⁇ g ⁇ t ⁇ c ⁇ 5 (Y) ⁇ T (Y) ⁇ T (Y) ⁇
  • RecQL siRNA (QL-19 described in WO 2017/022650): Sense strand GU (M) U (M) C (M) AGC CACU (M) U (M) C (M) AGC (M) U (M) U (M) tt (SEQ ID NO: 102) Antisense strand AAGCUGAAGUGGU (M) CU (M) GAAC (M) tt (SEQ ID NO: 103) (In the above sequence, t indicates deoxythymidine and (M) indicates 2'-methoxy form.) was also used for comparison.
  • HeLa-S3 cells (American Type Culture Collection: ATCC) were used as human cervical cancer cells. Each antisense oligonucleotide is incorporated into HeLa-S3 cells using a commercially available transfection reagent (ThermoFisher Scientific, lipofectamine RNAiMAX), and the mRNA expression level is measured by the qRT-PCR method to measure the knockdown activity (knockdown activity). Suppression of mRNA expression) was investigated. The procedure is shown below. HeLa-S3 cells in logarithmic growth phase at 3.0 ⁇ 10 4 cells / well in 24-well plate wells (including Dulbecco's Modified Eagle's Medium (DMEM) containing 10% fetal bovine serum (FBS)). Sown.
  • DMEM Dulbecco's Modified Eagle's Medium
  • FBS fetal bovine serum
  • each antisense oligonucleotide was added into the wells to a final concentration of 20 nM and incubated for 24 hours. After incubation, cells were harvested and total RNA was extracted using an RNA extraction reagent (MACHEREY-NAGEL, Nucleo ZOL).
  • a reverse transcription reaction and a PCR amplification reaction were carried out using a nucleic acid amplification reaction reagent (QIAGEN, QuantiFast Probe RT-PCR kit) using the corresponding total RNA as a template.
  • the nucleic acid amplification reaction was carried out by temperature cycling at 50 ° C. for 10 minutes ⁇ 95 ° C. for 5 minutes ⁇ [(95 ° C. for 10 seconds ⁇ 60 ° C.
  • the amount of human actin mRNA in the housekeeping gene was also quantified at the same time, and the amount of hRECQL mRNA relative to the amount of actin mRNA was evaluated.
  • the amount of mRNA by each antisense oligonucleotide or oligonucleotide is shown as a relative value when the amount of mRNA of the oligonucleotide-free cell is 1.
  • primer sets used are as follows: (Primer set for hRECQL detection) TaqMan Gene Expression Assay Hs00262956_m1_4331182 (ThermoFisher Scientific) (Primer set for detecting human actin) Pre-Developed TaqMan Assay Reagents Human ACTB (applied biosystems)
  • FIGS. 1, 2 and 3 The results of the mRNA expression suppression analysis are shown in FIGS. 1, 2 and 3. Antisense oligonucleotides were found that had lower mRNA levels than oligonucleotide-free cells (“control”) and Negative Control-supplemented cells, i.e., suppressed mRNA expression. Table 1 summarizes the antisense oligonucleotides that showed high knockdown activity (inhibition of mRNA expression).
  • HeLa-S3 cells are used as human cervical cancer cells, and each antisense oligonucleotide is incorporated into HeLa-S3 cells using a commercially available transfection reagent (ThermoFisher Scientific, lipofectamine RNAiMAX), and the qRT-PCR method is used. The expression level of mRNA was measured in 1 and the knockdown activity (inhibition of mRNA expression) was examined. The amount of antisense oligonucleotide added to HeLa-S3 cells was set to either 1 nM, 5 nM or 20 nM. In FIG.
  • HeLa-S3 cells were used as human cervical cancer cells, and each antisense oligonucleotide was used by the CEM method (“Ca 2+ enrichment for medium” method using calcium ion enriched medium: Nucleic Acids Research, 2015, Vol.43, It was incorporated into human cervical cancer cells using e128), the expression level of mRNA was measured by the qRT-PCR method, and the knockdown activity (suppression of mRNA expression) was examined. The procedure is shown below. HeLa-S3 cells in logarithmic growth phase at 4.0 ⁇ 10 4 cells / well containing Dulbecco's Modified Eagle's Medium (DMEM) (low glucose) containing 24-well plate wells (10% fetal bovine serum (FBS)).
  • DMEM Dulbecco's Modified Eagle's Medium
  • FBS fetal bovine serum
  • each oligonucleotide was added into the wells with 9 mM calcium chloride to a final concentration of 100 nM, 500 nM or 2000 nM and incubated for 48 hours. After incubation, cells were harvested and total RNA was extracted using an RNA extraction reagent (MACHEREY-NAGEL, Nucleo ZOL).
  • a reverse transcription reaction and a PCR amplification reaction were carried out using a nucleic acid amplification reaction reagent (QIAGEN, QuantiFast Probe RT-PCR kit) using the corresponding total RNA as a template. The nucleic acid amplification reaction was carried out by temperature cycling at 50 ° C. for 10 minutes ⁇ 95 ° C.
  • FIGS. 8 and 9 the results of 500 nM and 2000 nM are shown in order from the left side for various antisense oligonucleotides.
  • antisense oligonucleotide-supplemented cells showed dose-dependent knockdown activity (inhibition of mRNA expression) of antisense oligonucleotides. ..
  • the antisense oligonucleotide showed higher suppression of mRNA expression than RECQL siRNA when added at any concentration.
  • Example 4 Human ovarian cancer cells (ES-2 cells, SK-OV-3 cells, TOV-112D cells, OVCAR-3 cells), human colon cancer cells (LoVo cells, HCT-15 cells), humans In vitro suppression of hRECQL mRNA expression in gastric cancer cells (MKN45 cells))
  • About antisense oligonucleotides of hRECQL-837-AmNA (15), hRECQL-1312-AmNA (15), hRECQL-2950-AmNA (15), hRECQL-3158-AmNA (15) and hRECQL-3260-AmNA (15) Human ovarian cancer cells, human colon cancer cells, and human gastric cancer cells were examined for knockdown activity (inhibition of mRNA expression).
  • ES-2 cells (American Type Culture Collection: ATCC) were used as human ovarian cancer cells.
  • a 24-well plate of wells (McCoy'5A medium containing 10% fetal bovine serum (FBS) (modified)) at 3.0 x 10 4 cells / well in the logarithmic growth phase. (Including) sown in.
  • FBS fetal bovine serum
  • each antisense oligonucleotide was incorporated into ES-2 cells using a commercially available transfection reagent (ThermoFisher Scientific, Lipofectamine RNAiMAX), and the mRNA expression level was measured by the qRT-PCR method.
  • Knockdown activity (inhibition of mRNA expression) was investigated.
  • the amount of antisense oligonucleotide added to ES-2 cells was 20 nM. The results are shown in FIG.
  • SK-OV-3 cells (American Type Culture Collection: ATCC) were used as human ovarian cancer cells. SK-OV-3 cells in logarithmic growth phase at 3.0 x 10 4 cells / well in 24-well plate wells (McCoy '5A medium containing 10% fetal bovine serum (FBS) (modified)) ) Including). After 24 hours, each antisense oligonucleotide was incorporated into SK-OV-3 cells using a commercially available transfection reagent (ThermoFisher Scientific, Lipofectamine RNAiMAX), and the expression level of mRNA was determined by qRT-PCR. The knockdown activity (inhibition of mRNA expression) was examined by measurement. The amount of antisense oligonucleotide added to the SK-OV-3 cells was 20 nM. The results are shown in FIG.
  • TOV-112D cells (American Type Culture Collection: ATCC) were used as human ovarian cancer cells. Logarithmic growth phase TOV-112D cells at 3.0 x 10 4 cells / well in 24-well plate wells (including Dulbecco's Modified Eagle's Medium (DMEM) containing 10% fetal bovine serum (FBS)) Sown. After 24 hours, each antisense oligonucleotide was incorporated into TOV-112D cells using a commercially available transfection reagent (ThermoFisher Scientific, Lipofectamine RNAiMAX), and the mRNA expression level was measured by the qRT-PCR method. , Knockdown activity (inhibition of mRNA expression) was investigated. The amount of antisense oligonucleotide added to TOV-112D cells was 20 nM. The results are shown in FIG.
  • OVCAR-3 cells (American Type Culture Collection: ATCC) were used as human ovarian cancer cells. At 4.0 x 10 4 cells / well of logarithmic growth phase OVCAR-3 cells in 24-well plate wells (Roswell Park Memorial Institute 1640 medium (RPMI-1640) containing 10% fetal bovine serum (FBS)). (Including) sown in. After 24 hours, each antisense oligonucleotide was incorporated into OVCAR-3 cells using a commercially available transfection reagent (ThermoFisher Scientific, Lipofectamine RNAiMAX), and the mRNA expression level was measured by the qRT-PCR method. , Knockdown activity (inhibition of mRNA expression) was investigated. The amount of antisense oligonucleotide added to OVCAR-3 cells was 20 nM. The results are shown in FIG.
  • LoVo cells (National Institute of Physical and Chemical Research: RIKEN) were used as human colorectal cancer cells. LoVo cells in the logarithmic growth phase were seeded at 4.0 ⁇ 10 4 cells / well in 24-well plate wells (including Eagle's Minimal Essential Medium (EMEM) containing 10% fetal bovine serum (FBS)). After 24 hours, each antisense oligonucleotide was incorporated into LoVo cells using a commercially available transfection reagent (ThermoFisher Scientific, Lipofectamine RNAiMAX), the expression level of mRNA was measured by qRT-PCR, and knocking was performed. The down activity (inhibition of mRNA expression) was examined. The amount of antisense oligonucleotide added to LoVo cells was 20 nM. The results are shown in FIG.
  • HCT-15 cells (American Type Culture Collection: ATCC) were used as human colorectal cancer cells. At 4.0 x 10 4 cells / well of logarithmic growth phase HCT-15 cells in 24-well plate wells (Roswell Park Memorial Laboratory 1640 medium (RPMI-1640) containing 10% fetal bovine serum (FBS)). (Including) sown in. After 24 hours, each antisense oligonucleotide was incorporated into HCT-15 cells using a commercially available transfection reagent (ThermoFisher Scientific, Lipofectamine RNAiMAX), and the mRNA expression level was measured by the qRT-PCR method. , Knockdown activity (inhibition of mRNA expression) was investigated. The amount of antisense oligonucleotide added to HCT-15 cells was 20 nM. The results are shown in FIG.
  • MKN45 cells Human Science Promotion Foundation: JCRB
  • JCRB Human Science Promotion Foundation
  • 4.0 x 10 4 cells / well of logarithmic growth phase, 24-well plate wells (including RPMI-1640 medium containing 10% fetal bovine serum (FBS)) Sown inside.
  • FBS fetal bovine serum
  • each antisense oligonucleotide was incorporated into MKN45 cells using a commercially available transfection reagent (ThermoFisher Scientific, Lipofectamine RNAiMAX), the expression level of mRNA was measured by qRT-PCR, and knocking was performed. The down activity (inhibition of mRNA expression) was examined.
  • the amount of antisense oligonucleotide added to MKN45 cells was 20 nM. The results are shown in FIG.
  • FIG. 10-16 shows the results of 20 nM for various antisense oligonucleotides.
  • knockdown activity inhibition of mRNA expression was observed in antisense oligonucleotide-added cells as compared with oligonucleotide-free cells (“control”) and Negative Control-added cells.
  • hRECQL-837-AmNA (15) (LX-A0051), hRECQL-1312-AmNA (15) (LX-A0059), hRECQL-2950-AmNA (15) (LX-A0292), hRECQL-3158-AmNA ( 15) (LX-A0299) and hRECQL-3260-AmNA (15) (LX-A0300) showed higher mRNA expression inhibitory activity than RECQL siRNA.
  • the RECQL is highly expressed in actively proliferating cells such as cancer cells and contributes to the maintenance of genome stabilization of the cells.
  • the expression of the RECQL gene is inhibited in these cells, the genome is impaired. It is thought to stabilize and induce mitotic and mitotic cell death. Therefore, the antisense oligonucleotide of the present invention having high RECQL expression inhibitory activity and excellent in vivo stability can be used for the treatment and / or prevention of hyperproliferative diseases such as cancer. , Extremely useful.

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Abstract

L'invention fournit un oligonucléotide simple brin inhibant l'expression d'un gène ＲＥＣＱＬ. Lequel oligonucléotide simple brin contient une séquence de nucléotides complémentaire d'au moins 10 séquences de nucléotides consécutives, dans une région cible constituée d'une séquence de nucléotides choisi dans un groupe constitué des ２７５０ à ３６６３ème, des ８０ à ３００ième, des ３３０ à ５００ème, des ８００ à ９３０ème, des １０００ à １３５０ème et des ２２５０ à ２５００ème séquences de nucléotides dans un acide nucléiques codant RECQL, et constituée de séquences de nucléotides représentées par SEQ ID N° : 1. La longueur de l'oligonucléotide simple brin est de 10 à 30 nucléotides. La fraction sucre d'au moins un nucléotide configurant ledit oligonucléotide simple brin, est modifiée par réticulation entre la position 2' et la position 4'.
PCT/JP2020/048964 2019-12-27 2020-12-25 Oligonucléotide anti-sens inhibant l'expression de ｒｅｃｑｌ, et application associée WO2021132648A1 (fr)

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Cited By (1)

* Cited by examiner, † Cited by third party
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WO2024004779A1 (fr) * 2022-06-30 2024-01-04 株式会社ジーンケア研究所 Petit arn interférent ciblant le gène de l'hélicase recql1

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WO2002068590A2 (fr) * 2001-02-23 2002-09-06 Isis Pharmaceuticals, Inc. Modulation antisens de l'expression de recql
WO2004100990A1 (fr) * 2003-05-19 2004-11-25 Genecare Research Institute Co., Ltd. Inducteur d'apoptose pour cellule cancereuse
WO2006054625A1 (fr) * 2004-11-19 2006-05-26 Genecare Research Institute Co., Ltd. Agent cytostatique specifique aux cellules cancereuses
WO2010150159A1 (fr) * 2009-06-25 2010-12-29 International Centre For Genetic Engineering And Biotechnology (Icgeb) Agent anticancéreux, spécifique aux tumeurs du cerveau à mécanisme de suppression du recq1
WO2011052436A1 (fr) * 2009-10-29 2011-05-05 国立大学法人大阪大学 Nucléoside et nucléotide artificiels pontés
JP2012219085A (ja) * 2011-04-13 2012-11-12 Shiga Univ Of Medical Science 頭頸部癌及び食道癌用抗癌剤及び増強剤
JP2016521753A (ja) * 2013-06-12 2016-07-25 オンコイミューニン，インコーポレイティド オリゴヌクレオチドの全身性イン−ビボ送達

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002068590A2 (fr) * 2001-02-23 2002-09-06 Isis Pharmaceuticals, Inc. Modulation antisens de l'expression de recql
WO2004100990A1 (fr) * 2003-05-19 2004-11-25 Genecare Research Institute Co., Ltd. Inducteur d'apoptose pour cellule cancereuse
WO2006054625A1 (fr) * 2004-11-19 2006-05-26 Genecare Research Institute Co., Ltd. Agent cytostatique specifique aux cellules cancereuses
WO2010150159A1 (fr) * 2009-06-25 2010-12-29 International Centre For Genetic Engineering And Biotechnology (Icgeb) Agent anticancéreux, spécifique aux tumeurs du cerveau à mécanisme de suppression du recq1
WO2011052436A1 (fr) * 2009-10-29 2011-05-05 国立大学法人大阪大学 Nucléoside et nucléotide artificiels pontés
JP2012219085A (ja) * 2011-04-13 2012-11-12 Shiga Univ Of Medical Science 頭頸部癌及び食道癌用抗癌剤及び増強剤
JP2016521753A (ja) * 2013-06-12 2016-07-25 オンコイミューニン，インコーポレイティド オリゴヌクレオチドの全身性イン−ビボ送達

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2024004779A1 (fr) * 2022-06-30 2024-01-04 株式会社ジーンケア研究所 Petit arn interférent ciblant le gène de l'hélicase recql1

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