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  • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
  • C07H21/02—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids with ribosyl as saccharide radical
• • 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
  • A61K31/7125—Nucleic acids or oligonucleotides having modified internucleoside linkage, i.e. other than 3'-5' phosphodiesters
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  • 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
• • 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
• • 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
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  • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
  • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
  • C07H21/04—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids with deoxyribosyl as saccharide radical
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  • 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
  • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
  • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
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  • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
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  • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
  • C12P21/00—Preparation of peptides or proteins
  • C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione
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  • 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
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  • C12N15/67—General methods for enhancing the expression
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  • C12N2310/00—Structure or type of the nucleic acid
  • C12N2310/30—Chemical structure
  • C12N2310/31—Chemical structure of the backbone
  • C12N2310/315—Phosphorothioates
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  • C12N2830/00—Vector systems having a special element relevant for transcription
  • C12N2830/50—Vector systems having a special element relevant for transcription regulating RNA stability, not being an intron, e.g. poly A signal

Definitions

• the present invention relates to polynucleotides and pharmaceutical compositions comprising said polynucleotides.
• mRNA messenger RNA
• RNA synthetase RNA synthetase
• DNA DNA as a template
• ribosomes binding of ribosomes to the transcribed single-stranded mRNA to synthesize proteins through translation. and has been communicated.
• This mode of transmission is called the "central dogma” in molecular biology and is a fundamental principle common to both prokaryotes and eukaryotes.
• mRNA which is an intermediate substance of genetic information transmission, has base sequence information and structure for being directly recognized by ribosomes and translated into proteins.
• Non-Patent Document 2 polynucleotides containing sugar-modified nucleotides such as 2'-O-methyl modified RNA, 2'-F-modified RNA, 2'-O-methoxyethyl-modified RNA and crosslinked nucleic acids such as LNA are nucleic acid medicines. It has been shown that it is effective both in reducing the immunoreactivity of the enzyme and in imparting resistance to nucleolytic enzymes (Non-Patent Document 3).
• Non-Patent Document 4 reports that in a clinical trial of an artificial mRNA cancer vaccine for melanoma patients, the incidence of metastasis is significantly reduced after administration of the cancer vaccine is started. , certain achievements in using artificial mRNA as a drug are also being reported.
• these clinically applied artificial mRNAs are produced by IVT.
• Artificial mRNA produced by IVT has the following two problems. First, the introduction position of modified nucleotides introduced for the purpose of reducing immunoreactivity or imparting stability to nucleolytic enzymes cannot be controlled.
• Patent Document 1 discloses a case in which peptide translation ability is attenuated or lost in an artificial mRNA into which 2'-F-modified RNA is introduced by IVT. Second, only modified nucleotides that are recognized as substrates by the RNA synthetase used in IVT can be introduced. Moreover, Patent Document 1 discloses that artificial mRNA containing 2′-O-methyl modified RNA is difficult to prepare by IVT reaction using a general RNA polymerase. Therefore, it is difficult to say that sufficient consideration has been given to the position and type of modified nucleotides in artificial mRNAs produced by introducing modified nucleotides by IVT.
• Non-Patent Documents 6 and 7 A method of artificially synthesizing mRNA using a technique of chemically linking multiple RNAs has been reported (Non-Patent Documents 6 and 7). Using this method, it is possible to introduce sugar-modified nucleotides at any position of the artificial mRNA, including the translated and untranslated regions.
• Patent Documents 2 and 3 disclose the concept of stabilizing mRNA by introducing sugar-modified nucleotides into the untranslated region of mRNA by a method of synthesizing artificial mRNA using a technique of chemically linking multiple RNAs.
• Non-Patent Documents 6 and 7 disclose that the peptide translating ability of artificial mRNA introduced with 2'-O-methyl modified RNA at one site in the translation region of mRNA was confirmed. On the other hand, it has also been disclosed that the peptide translation ability is remarkably attenuated depending on the introduction position of the sugar-modified nucleotide (Non-Patent Documents 6 and 7). In view of the above, in order to achieve sufficiently low immunoreactivity, high stability, and excellent translation ability as an artificial mRNA nucleic acid drug, further knowledge regarding the modification rate, position, and type of modified nucleotides is required.
• the purpose of the present invention is to provide a polynucleotide with excellent translatability.
• the present invention includes the following embodiments. [1] translation region from the start codon to the stop codon, A polynucleotide comprising a 5′ untranslated region and a poly A chain, wherein 65% or more of the nucleotides constituting the poly A chain are sugar-modified nucleotides.
• each modified sugar moiety of the sugar-modified nucleotide is independently selected from any one of the following structures:
• the polynucleotide [4] of [1] or [2] each modified sugar moiety of the sugar-modified nucleotide is independently selected from any one of the following structures:
• nucleotide according to any one of [1] to [8], wherein the nucleotides of the 5' untranslated region are independently selected from 2'-deoxyribonucleotides, spacer-modified nucleotides, and sugar-modified nucleotides.
• the 1st to 6th nucleotides from the 5' end of the 5' untranslated region are sugar-modified nucleotides, and the modified sugar portion of the sugar-modified nucleotide has the following structure: The polynucleotide according to any one of [1] to [9].
• the nucleotides excluding the 1st to 6th nucleotides from the 5' end of the 5' untranslated region contain 2'-deoxyribonucleotides and/or spacer modifications, [1] to [11]. Polynucleotide.
• the 5'-side untranslated region and/or the 3'-side untranslated region contains a spacer modification, preferably the 5'-side untranslated region and/or the 3'-side untranslated region contains a spacer modification, and the spacer modification are each independently selected from any one of the following structures: [In the formula, Rx is ethynyl, a hydrogen atom or OH, M is a hydrogen atom or OH, n1 is 1, 2 or 5; n2 is 1, 2 or 3; ] The polynucleotide according to any one of [1] to [12]. As a spacer modification in [13], the oxygen atom of the 5-membered ring in the leftmost structure may be substituted with NH.
• the first nucleotide of all codons excluding the termination codon is a sugar-modified nucleotide
• the modified sugar portion of the sugar-modified nucleotide has the following structure: The polynucleotide according to any one of [1] to [17]. [19] The polynucleotide according to any one of [1] to [18], wherein the translation region contains 2000 or less codons. [19-1] The polynucleotide according to any one of [1] to [19], wherein the translation region comprises 4 or more and 2000 or less (4-2000) codons.
• the present invention further includes the following embodiments.
• each modified sugar moiety of the sugar-modified nucleotide is independently selected from any one of the following structures: The polynucleotide of any one of [101]-[102].
• each modified sugar moiety of the sugar-modified nucleotide is independently selected from any one of the following structures: The polynucleotide [105] of any one of [101] to [103] The polynucleotide of any one of [101] to [104], wherein the poly A chain comprises at least one phosphate-modified nucleotide. [106] 1st to 2nd nucleotides, 1st to 3rd nucleotides, 1st to 4th nucleotides, or 1st to 5th nucleotides from the 3' end of the poly A chain are linked by phosphorothioate, The polynucleotide of any one of [101]-[105].
• the 1st to 6th nucleotides from the 5' end of the 5' untranslated region are sugar-modified nucleotides, and the modified sugar portion of the sugar-modified nucleotide has the following structure: The polynucleotide of any one of [101]-[108].
• the nucleotides excluding the 1st to 6th nucleotides from the 5' end of the 5' untranslated region contain 2'-deoxyribonucleotides and/or spacer modifications, [101] to [110] according to any one of [101] to [110] Polynucleotide.
• the 5'-side untranslated region and/or the 3'-side untranslated region contains a spacer modification, preferably the 5'-side untranslated region and/or the 3'-side untranslated region contains a spacer modification, and the spacer modification are each independently selected from any one of the following structures: [In the formula, Rx is ethynyl, a hydrogen atom or OH, M is a hydrogen atom or OH, n1 is 1, 2 or 5; n2 is 1, 2 or 3; ] The polynucleotide of any one of [101]-[111]. As a spacer modification in [112], the oxygen atom of the 5-membered ring in the leftmost structure may be substituted with NH.
• the first nucleotide of all codons excluding the termination codon is a sugar-modified nucleotide
• the modified sugar portion of the sugar-modified nucleotide has the following structure: The polynucleotide according to any one of [101] to [116-1].
• the present invention further includes the following embodiments as aspects different from the above [1] to [22].
• the polynucleotide of [201], wherein the nucleotides constituting the poly A chain comprise at least one or more sugar-modified nucleotides.
• each modified sugar moiety of the sugar-modified nucleotide is independently selected from any one of the following structures: The polynucleotide of any one of [201]-[202].
• each modified sugar moiety of the sugar-modified nucleotide is independently selected from any one of the following structures: The polynucleotide of any one of [201]-[203].
• the 1st to 6th nucleotides from the 5' end of the 5' untranslated region are sugar-modified nucleotides, and the modified sugar portion of the sugar-modified nucleotide has the following structure: The polynucleotide of any one of [201]-[208].
• the polynucleotide of [209] further comprising a portion consisting of 1-10 sugar unmodified nucleotides 5' to the 5' end of said 5' untranslated region.
• nucleotides excluding the 1st to 6th nucleotides from the 5' end of the 5' untranslated region contain 2'-deoxyribonucleotides and/or spacer modifications, [201] to [210]. Polynucleotide.
• the 5'-side untranslated region and/or the 3'-side untranslated region contains a spacer modification, preferably the 5'-side untranslated region and/or the 3'-side untranslated region contains a spacer modification, and the spacer modification are each independently selected from any one of the following structures: [In the formula, Rx is ethynyl, a hydrogen atom or OH, M is a hydrogen atom or OH, n1 is 1, 2 or 5; n2 is 1, 2 or 3; ] The polynucleotide of any one of [201]-[211]. As a spacer modification in [212], the oxygen atom of the 5-membered ring in the leftmost structure may be substituted with NH.
• the first nucleotide of all codons excluding the termination codon is a sugar-modified nucleotide
• the modified sugar portion of the sugar-modified nucleotide has the following structure: The polynucleotide of any one of [201]-[216-1].
• [218] The polynucleotide of any one of [201]-[217], wherein the coding region comprises 2000 or less codons.
• [218-1] The polynucleotide of any one of [201]-[218], wherein the coding region comprises 4-2000 codons.
• the present invention further includes the following embodiments.
• [1C] The polynucleotide of any of [1]-[21], [101]-[120] and [201]-[220] or [22], [121] and [221] for treating a disease
• Use of a pharmaceutical composition according to any one of [1D] Use of the polynucleotide of any one of [1]-[21], [101]-[120] and [201]-[220] in the manufacture of a medicament for treating a disease.
• [1E] A polynucleotide according to any one of [1]-[21], [101]-[120] and [201]-[220] for use in the manufacture of a medicament for treating a disease.
• the polynucleotide is translation region from the start codon to the stop codon, It contains a 5′ untranslated region and a poly A chain, and 65% or more of the nucleotides constituting the poly A chain are sugar-modified nucleotides.
• 65% or more of the nucleotides constituting the poly A chain are sugar-modified nucleotides, thereby exhibiting excellent translation ability.
• the polynucleotide of this embodiment contains a translation region and a poly A chain, and the 5' untranslated region, translation region, and poly A chain are arranged from the 5' side to the 3' side of the polynucleotide. is preferred, and the translation region and the poly A chain may be directly linked, and there may be another region or a sequence not included in the poly A chain between them.
• the translation region and the poly A chain are directly linked means that the poly A chain is linked following the termination codon of the translation region. is a chain.
• the poly A chain exists within the 3' untranslated region, and the polynucleotide includes a 5' untranslated region, a translated region, and a 3' untranslated region. In this case, the poly A chain is present at the 3' end of the 3' untranslated region.
• polypeptide in this specification includes proteins
• mRNA small open reading frame
• non- Polynucleotides are understood to have functions equivalent to canonical open reading frames, long noncoding RNAs (lncRNAs), and pri-microRNAs (pri-miRNAs).
• the polynucleotides may be single-stranded polynucleotides or circular polynucleotides in which the ends of the polynucleotides are joined together.
• the polynucleotide of the present embodiment is a combination of multiple nucleotides, and each nucleotide constituting the polynucleotide usually contains a sugar moiety, a base moiety and a phosphate moiety.
• the sugar moiety is the moiety contained in the nucleotide corresponding to the sugar
• the base moiety is the moiety contained in the nucleotide corresponding to the base
• the phosphate moiety is the moiety contained in the nucleotide corresponding to the phosphate. is.
• nucleotides are selected from adenine (A), guanine (G), cytosine (C), uracil (U) or thymine (T) for the base portion, and ribose or 2′-deoxy for the sugar portion. Selected from ribose. Ribose and 2'-deoxyribose are each preferably in the D form.
• a nucleotide consists of a combination of the above base portion and the above sugar portion, and has adenine (A), guanine (G), cytosine (C) or uracil (U) as the base portion and D-ribose as the sugar portion. Ribonucleotides are preferred.
• the nucleotides constituting the polynucleotide of the present embodiment may be ribonucleotides (AUGC) that are unmodified nucleotides, or deoxyribonucleotides (ATGC) that are unmodified nucleotides.
• AUGC ribonucleotides
• ATGC deoxyribonucleotides
• Modified nucleotides having a structure not derived from unmodified nucleotides in at least part of the phosphate moiety may also be used.
• a nucleotide modified at the sugar moiety is referred to as a "sugar-modified nucleotide”
• a nucleotide modified at the base moiety is referred to as a “base-modified nucleotide”
• a nucleotide modified at the phosphate moiety is referred to as a "phosphate-modified nucleotide”.
• “modification” means changing the structure of a sugar moiety, a base moiety, or a phosphate moiety. Structural change by modification is not particularly limited. Modifications include, for example, substitution at any site with any substituent.
• Nucleotides that have either a modified sugar moiety, a modified base moiety, or a modified phosphate moiety are referred to as modified nucleotides, and nucleotides that have no modification in either the sugar moiety, the base moiety, or the phosphate moiety are referred to as unmodified nucleotides.
• the modified nucleotide may have one modified moiety selected from a modified sugar moiety, a modified base moiety, or a modified phosphate moiety, or may have any combination of two modified moieties. may have a modified portion of
• the unmodified sugar moiety is a sugar moiety corresponding to ribose or 2'-deoxyribose, more preferably a sugar moiety corresponding to ribose. That is, in the polynucleotide of the present embodiment, nucleotides other than sugar-modified nucleotides preferably contain a sugar moiety corresponding to ribose or 2'-deoxyribose, and more preferably contain a sugar moiety corresponding to ribose.
• sugar-modified nucleotide is not particularly limited as long as the sugar portion of the nucleotide is modified, but it preferably contains a sugar portion modified at least at the 2'-position. Stability against enzymes can be improved by modifying the 2'-position.
• the sugar moiety modified at least at the 2'-position may be a sugar moiety in which the 2'-position and the 4'-position are bridged.
• Modified sugar moieties include, for example, the following.
• M is R1 , OR1 , R2OR1 , OR2OR1 , SH , SR1 , NH2 , NHR1 , NR12 , N3 , CN, F, Cl, Br or I; each R 1 is independently alkyl or aryl, preferably alkyl having 1 to 6 carbon atoms, more preferably alkyl having 1 to 3 carbon atoms, R 2 is alkylene, preferably alkylene having 1 to 6 carbon atoms]
• M is H or OH, it is an unmodified sugar moiety, and a nucleotide having an unmodified sugar moiety in which M is H is a 2′-deoxyribonucleotide, and M is OH. Nucleotides with unmodified sugar moieties are ribonucleotides.
• alkyl having 1 to 6 carbon atoms include linear or branched alkyl having 1 to 6 carbon atoms.
• Straight-chain alkyls having 1 to 6 carbon atoms include, for example, methyl, ethyl, propyl, butyl, pentyl and hexyl.
• Branched alkyls having 1 to 6 carbon atoms include, for example, isopropyl, isobutyl, sec-butyl, tert-butyl, and methyl-substituted pentyl.
• alkyl having 1 to 3 carbon atoms include methyl, ethyl, propyl and isopropyl.
• aryl includes, for example, optionally substituted phenyl and optionally substituted naphthyl.
• alkylene having 1 to 6 carbon atoms is a group from which one hydrogen atom bonded to a carbon atom of alkyl having 1 to 6 carbon atoms is removed.
• modified sugar moiety refers to a modified sugar structure contained in a sugar-modified nucleotide.
• M of the modified sugar moiety may further include 2-(methoxy)ethoxy, 3-aminopropoxy, 2-[(N,N-dimethylamino)oxy]ethoxy, 3-(N,N-dimethylamino)propoxy, 2- Mention may also be made of [2-(N,N-dimethylamino)ethoxy]ethoxy, 2-(methylamino)-2-oxoethoxy, 2-(N-methylcarbamoyl)ethoxy), and 2-cyanoethoxy.
• Modified sugar moieties further include sugar moieties of the following nucleic acids: Locked Nucleic Acid (LNA) [Tetrahedron Letters, 38, 8735 (1997) and Tetrahedron, 54, 3607 (1998)]; - Ethylene bridged nucleic acid (ENA) [Nucleic Acids Research, 32, e175 (2004)]; ⁇ Constrained Ethyl (cEt) [The Journal of Organic Chemistry 75, 1569 (2010)]; ⁇ Amido-Bridged Nucleic Acid (AmNA) [Chem Bio Chem 13, 2513 (2012)]; ⁇ 2'-O,4'-c-Spirocyclopropylene bridged nucleic acid (scpBNA) [Chem.
• LNA Locked Nucleic Acid
• EDA Ethylene bridged nucleic acid
• cEt ⁇ Constrained Ethyl
• AmNA AmNA
• scpBNA ⁇ 2'-O,4'-c-Spiro
• modified sugar moiety is not particularly limited, it is preferably selected from the following.
• the sugar-modified nucleotide preferably contains a base portion corresponding to a base selected from the group consisting of adenine (A), guanine (G), cytosine (C) and uracil (U), and at least two kinds of bases are used. Kinds are preferred.
• “at least two kinds of bases” means that, for example, one sugar-modified nucleotide contains a base portion corresponding to adenine, and another sugar-modified nucleotide contains a base portion corresponding to guanine. do.
• the sugar-modified nucleotide may be a base-modified nucleotide and/or a phosphate-modified nucleotide (in other words, the sugar-modified nucleotide may further contain a modified base portion and/or a modified phosphate portion). At least one of the sugar-modified nucleotides may contain a modified base portion.
• the base-modified nucleotide is not particularly limited as long as the base portion of the nucleotide is modified.
• Examples of unmodified base moieties include base moieties corresponding to adenine, guanine, cytosine and uracil.
• the modified base portion includes, for example, a base portion in which the oxygen atom of the unmodified base portion is substituted with a sulfur atom, a base portion in which the hydrogen atom of the unmodified base portion is substituted with alkyl having 1 to 6 carbon atoms, halogen, etc.
• modified base portions possessed by base-modified nucleotides include, for example, 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyladenine, 6- methylguanine, 2-propyladenine, 2-propylguanine, 2-thiouracil, 2-thiothymine, 2-thiocytosine, 5-propynyluracil, 5-propynylcytosine, 6-azouracil, 6-azocytosine, 6-azothymine, 5-pseudo Uracil, 4-thiouracil, 8-haloadenine, 8-haloguanine, 8-aminoadenine, 8-aminoguanine, 8-mercaptoadenine, 8-mercaptoguanine, 8-alkylthioadenine, 8-alkylthioguanine, 8-hydroxyadenine, 8 - hydroxyguanine, 5-brom
• the base-modified nucleotide may be a sugar-modified nucleotide and/or a phosphate-modified nucleotide (in other words, the base-modified nucleotide may further contain a modified sugar moiety and/or a modified phosphate moiety).
• the phosphate-modified nucleotide is not particularly limited as long as the phosphate portion (phosphodiester bond) of the nucleotide is modified.
• Modified phosphate moieties include, for example, phosphorothioate, phosphorodithioate, alkylphosphonate, and phosphoramidate linkages.
• the translation region may contain phosphate-modified nucleotides in which the modified phosphate moiety is an optical isomer (Rp, Sp).
• Rp, Sp an optical isomer
• the phosphate-modified nucleotide may be a sugar-modified nucleotide and/or a base-modified nucleotide (in other words, the phosphate-modified nucleotide may further contain a modified sugar portion and/or a modified base portion).
• the polynucleotide of this embodiment includes a translation region.
• the coding region is also called the coding sequence (CDS).
• the translational region is a region composed of a plurality of codons from an initiation codon to a termination codon (or called a termination codon) and translated to synthesize a polypeptide.
• a codon is a unit that encodes each amino acid that constitutes a polypeptide, and is composed of three nucleotides.
• one polynucleotide may contain a plurality of translational regions, and in a polynucleotide containing a plurality of translational regions, the translational region portion in the polynucleotide containing one translational region may be a plurality of may contain the translation region of
• initiation codon is AUG, which encodes methionine, based on the natural codon table.
• Non-canonical start codons other than AUG can also include CUG, GUG, UUG, ACG, AUC, AUU, AAG, AUA, and AGG.
• Stop codons include, for example, UAA, UAG and UGA.
• the type of codons constituting the translation region is not particularly limited, and can be appropriately selected according to the target polypeptide.
• the number (n) of codons constituting the translational region is preferably an integer of 2 to 2000, more preferably an integer of 2 to 1500, still more preferably an integer of 2 to 1000, most preferably 2 to 500 integers. Also, the lower limit of the numerical range may be changed to 5, 10, 50, 100, 200, or the like.
• the number (n) of codons constituting the translation region when the lower limit is changed is preferably an integer of 5 to 2000, 10 to 2000, 50 to 2000, 100 to 2000 or 200 to 2000, more preferably 5 an integer of ⁇ 1500, 10-1500, 50-1500, 100-1500 or 200-1500, more preferably an integer of 5-1000, 10-1000, 50-1000, 100-1000 or 200-1000, Most preferably it is an integer of 5-500, 10-500, 50-500, 100-500 or 200-500.
• the number of nucleotides constituting the translation region is three times the number of codons (n).
• Each codon contains the 1st, 2nd and 3rd nucleotides.
• the first nucleotide is A
• the second nucleotide is U
• the third nucleotide is G.
• the translation region includes n codons, wherein n is a positive integer of 2 or more, and the n codons include the first, second, and third nucleotides, respectively, the n
• the first nucleotide in at least two of the codons is a sugar-modified nucleotide.
• the translation region preferably includes at least two codons in which the first nucleotide of the codons is a sugar-modified nucleotide, and at least two codons in which the first nucleotide in the codons is a sugar-modified nucleotide are It can be a codon at any position in the region.
• the polynucleotide of the present embodiment Since the translation activity is maintained even if the sugar moiety of the first nucleotide in a plurality of codons constituting the translation region is modified, the polynucleotide of the present embodiment has a modification site in the translation region but does not have translation activity. maintain.
• translational activity means the activity of translating mRNA to synthesize a polypeptide.
• the polynucleotides of this embodiment also have excellent stability against enzymes (eg, nucleases). As long as the translation region maintains translation activity, the polynucleotide of this embodiment exhibits excellent translation ability when 65% or more of the nucleotides constituting the poly A chain are sugar-modified nucleotides.
• translational activity is maintained means that a polynucleotide modified at the sugar moiety of the first nucleotide in a plurality of codons has a translation activity of 60% or more compared to an unmodified polynucleotide. indicates that there is The translation activity of the modified polynucleotide is preferably 70% or more, 80% or more, 90% or more, or 100% or more of that of the unmodified polynucleotide.
• At least two of the first nucleotides contained in the codons constituting the translation region may be sugar-modified nucleotides.
• the position of codons containing sugar-modified nucleotides is not particularly limited.
• the percentage of the first nucleotide being a sugar-modified nucleotide is 5% or more, 10% or more, 15% or more, 20% or more, 25% or more, 30% or more, 35% or more, 40% or more, 45% or more, 50%. % or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, or 100%.
• the percentage of 100% means that all of the first nucleotides are sugar-modified nucleotides.
• the substituent at the 2'-position of the sugar moiety of the first nucleotide is preferably fluorine.
• At least one of the second nucleotides contained in the codons constituting the translation region may be a sugar-modified nucleotide, but the sugar moiety of the second nucleotide is not modified. good too.
• the percentage in which the second nucleotide is a sugar-modified nucleotide is 50% or less, 45% or less, 40% or less, 35% or less, 30% or less, 25% or less, 20% or less, 15% or less, 10% or less, 5 % or less, or 0%.
• the percentage of 0% means that not all nucleotides at the second position are sugar-modified nucleotides.
• the substituent at the 2'-position of the sugar moiety of the second nucleotide is preferably fluorine.
• At least one of the third nucleotides contained in the codons constituting the translation region may be a sugar-modified nucleotide.
• the percentage in which the third nucleotide is a sugar-modified nucleotide is 100%, 90% or less, 80% or less, 70% or less, 60% or less, 50% or less, 45% or less, 40% or less, 35% or less, 30%. 25% or less, 20% or less, 15% or less, 10% or less, 5% or less, or 0% or less.
• the 1st, 2nd and 3rd nucleotides of the stop codon may be sugar-modified nucleotides from the viewpoint of improving translation activity. It may be a translation region in which all of the first nucleotides and all of the stop codon nucleotides are sugar modified nucleotides.
• the 1st, 2nd and 3rd nucleotides of the initiation codon may be sugar-modified nucleotides from the viewpoint of improving stability against nucleolytic enzymes.
• the substituents at the 2'-positions of the sugar moieties of the 1st, 2nd and 3rd nucleotides of the initiation codon are preferably fluorine.
• the first nucleotide in all codons other than the termination codon may be a sugar-modified nucleotide.
• all of the substituents at the 2'-position of the sugar moieties of the first nucleotides of all codons other than the termination codon are fluorine.
• the translation region may contain base-modified nucleotides.
• the position of base-modified nucleotides in the translation region is not particularly limited.
• the translational region may contain phosphate-modified nucleotides.
• the position of the phosphate-modified nucleotide in the translation region is not particularly limited, but the phosphate group connecting the first and second nucleotides of the codon is preferably a phosphorothioate bond.
• the polynucleotide of this embodiment includes a 5' untranslated region (5'UTR).
• the 5′ untranslated region is a region located upstream (5′ terminal side) of the translated region and not translated for polypeptide synthesis.
• the number of nucleotides constituting the 5' untranslated region is preferably 1 or more, and may be 6 or more.
• the number of nucleotides constituting the 5′-side untranslated region is preferably 1000 or less, and may be 500 or less, 250 or less, or 100 or less.
• the number of nucleotides constituting the 5′-side untranslated region may be any range selected from the above upper and lower limits, but is preferably an integer of 1 to 1000, more preferably 1 to 500. is an integer of , more preferably an integer of 6-250, and particularly preferably an integer of 6-100.
• the polynucleotide of this embodiment is linked in the order of the 5′ untranslated region and the translated region.
• the 5' untranslated region may contain 2'-deoxyribonucleotides, spacer-modified or sugar-modified nucleotides.
• the positions of these nucleotides are not particularly limited within the 5' untranslated region.
• the 1st, 2nd and 3rd nucleotides from the 5' end may be sugar-modified nucleotides, and the 1st to 6th nucleotides from the 5' end are all sugar-modified nucleotides.
• all nucleotides in the 5' untranslated region may be sugar-modified nucleotides.
• the substituent at the 2'-position of the sugar moiety is preferably a methoxyethoxy group (OCH 2 CH 2 OCH 3 ) or fluorine (F).
• the polynucleotide is translation region from the start codon to the stop codon, A polynucleotide comprising a 5'-untranslated region and a poly A chain, wherein the nucleotides of the 5'-untranslated region are each independently selected from 2'-deoxyribonucleotides, spacer-modified nucleotides, or sugar-modified nucleotides. be.
• nucleotides in the 5'-untranslated region of the polynucleotide are each independently selected from 2'-deoxyribonucleotides, spacer-modified nucleotides, and sugar-modified nucleotides, thereby exhibiting excellent translation ability.
• nucleotides of the 5' untranslated region are composed of 2'-deoxyribonucleotides, spacer-modified or sugar-modified nucleotides, sugar-modified nucleotides are preferably included.
• Polynucleotides of the present embodiment also include those with an appropriate non-sugar-modified nucleotide of 1 to 10 bases added to the original 5' end.
• the polynucleotide of this embodiment may further comprise a 5' cap structure at the original 5' end.
• the 5' cap structure may exist in a form added to the 5' untranslated region. Inclusion of a 5' cap structure tends to improve translational activity.
• the 5′ cap structure in the present application refers to the following structure in which a triline structure is added to 7-methylguanylic acid (m7G).
• 5' cap analogues as disclosed in the following papers can also be used for the 5' cap structure.
• ⁇ ARCA RNA, Vol. 7, pp. 1486-1495 (2001), Cell Cycle, Vol. 17, No. 13, pp. 1624-1636 (2016); LNA: Journal of American Chemical Society, Vol. 131, No. 18, pp. 6364-6365 (2009); - S Cap: RNA, Vol. 14, pp. 1119-1131 (2008); - Nature Reviews Drug Discovery, Vol. 13, pp. 759-780 (2014).
• the 5' untranslated region may contain base-modified nucleotides.
• the position where the base-modified nucleotide exists in the 5'-side untranslated region is not particularly limited.
• Base-modified nucleotides may be sugar-modified nucleotides and/or phosphate-modified nucleotides (in other words, base-modified nucleotides may further comprise modified sugar moieties and/or modified phosphate moieties).
• the 5′-side untranslated region preferably contains the following modified base portions.
• R is an alkyl group having 1 to 6 carbon atoms
• the alkyl group R of the modified base portion is preferably methyl or ethyl.
• the 5' untranslated region may contain phosphate-modified nucleotides.
• the position of the phosphate-modified nucleotide in the 5' untranslated region is not particularly limited.
• Phosphate-modified nucleotides may be sugar-modified nucleotides and/or base-modified nucleotides (in other words, phosphate-modified nucleotides may further comprise modified sugar moieties and/or modified base moieties).
• the 5' untranslated region may contain 2'-deoxyribonucleotide or spacer modifications.
• the position where the 2'-deoxyribonucleotide or spacer modification exists in the 5'-side untranslated region is not particularly limited, but the nucleotide at any position except the 1st to 6th from the 5' end is 2'-deoxyribonucleotide or spacer-modified is preferably included. In this embodiment, it is a preferred embodiment that the translation region does not contain the spacer modification.
• Rx is alkyl having 1 to 6 carbon atoms, alkenyl having 1 to 6 carbon atoms, alkynyl having 1 to 6 carbon atoms, hydrogen atom or OH
• M is R1 , OR1 , R2OR1 , OR2OR1 , SH , SR1 , NH2 , NHR1 , NR12 , N 3 , hydrogen atom, OH, CN, F, Cl, Br or I
• X is O, S or NR1 ; each R 1 is independently alkyl or aryl, preferably alkyl having 1 to 6 carbon atoms, more preferably alkyl having 1 to 3 carbon atoms, R 2 is alkylene, preferably alkylene having 1 to 6 carbon atoms
• Spacer modification is not particularly limited, but the following structures are preferred.
• Rx is ethynyl, a hydrogen atom or OH
• M is a hydrogen atom or OH
• n1 is 1, 2 or 5
• n2 is 1, 2 or 3;
• the polynucleotide of this embodiment includes a poly A chain. 65% or more of the constituent nucleotides of the poly A chain in one aspect of the present embodiment are sugar-modified nucleotides.
• the poly A chain is contained in the 3' untranslated region. At least one poly A chain in the present embodiment is contained in the 3′ untranslated region.
• a poly A chain is a polyadenylic acid composed of two or more AMPs.
• AMP in this application includes nucleotides corresponding to AMP (including, for example, AMP sugar-modified nucleotides, AMP 2'-deoxyribonucleotides, AMP phosphate-modified nucleotides and AMP base-modified nucleotides).
• AMP or nucleotides corresponding to AMP are collectively referred to as AMP in the present application.
• the poly A chain may contain ribonucleotides other than AMP (eg, CMP, GMP, UMP, or nucleotides corresponding to each) as long as it has a polyadenylic acid structure containing two or more AMPs.
• the nucleotide at the 5' end of the poly A chain is understood to be AMP at the start of a sequence in which two or more AMPs are continuous.
• the proportion of ribonucleotides other than AMP among the nucleotides constituting the poly A chain is 40% or less, 30% or less, 20% or less, or 10% or less, 30% or less is preferable, 20% or less is more preferable, and 10% or less is even more preferable.
• 65% or more of the nucleotides in the poly A chain are neither ribonucleotides nor 2'-deoxyribonucleotides.
• poly A chain containing ribonucleotides other than AMP examples include, for example, Nature Medicine, Vol. 23, No. 7, pp. 815-817 (2017), Science, Vol. 361, pp. 701-704 (2016), RN (RNA), Vol. 25, pp. 507-518 (2019).
• a poly A chain also means a sequence in which two or more consecutive regions of AMP present at two or more locations are linked with an arbitrary linker.
• linkers include polyethylene glycol, polypeptides, alkyl chains and the like, but are not particularly limited.
• International Publication No. 2016/011306 discloses a method for linking nucleotides with a specific linker.
• the poly A chain in one aspect of this embodiment may contain 2'-deoxyribonucleotides, spacer-modified or sugar-modified nucleotides.
• the positions of these nucleotides are not particularly limited within the 3' untranslated region.
• the poly A chain of this embodiment may not contain AMP, but the description of the poly A chain described as the above embodiment is also applicable.
• the poly A chain may contain 65% or more, 70% or more, 80% or more, 90% or more, 95% or more, 100% of 2'-deoxyribonucleotides, spacer-modified or sugar-modified nucleotides, and the poly A chain is , 2′-deoxyribonucleotides, spacer-modified or sugar-modified nucleotides.
• nucleotides of the poly A chain are composed of 2'-deoxyribonucleotides, spacer-modified or sugar-modified nucleotides, sugar-modified nucleotides are preferably included.
• the sugar-modified nucleotides may constitute 65% or more of the nucleotides constituting the poly A chain.
• the 3'-untranslated region (3'UTR) is a region located downstream (3'-terminal side) of the translated region and is not translated for polypeptide synthesis.
• the number of nucleotides constituting the 3′ untranslated region is preferably an integer of 2 to 6000, more preferably an integer of 2 to 3000, still more preferably an integer of 2 to 1000, particularly preferably 2 An integer from ⁇ 500.
• the region other than the poly A chain in the 3' untranslated region may be any nucleotide, and each nucleotide in the region other than the poly A chain in the 3' untranslated region may be an unmodified nucleotide, It may be a modified nucleotide.
• the polynucleotide of this embodiment is linked in the order of translation region and 3′-side untranslation region.
• the length of the poly A chain is preferably 2 to 500 bases long, more preferably 2 to 200 bases long, still more preferably 2 to 80 bases long, still more preferably 2 to 40 bases long. , more preferably 3 to 40 bases long, more preferably 5 to 40 bases long, still more preferably 10 to 40 bases long, particularly preferably 20 to 40 bases long.
• 65% or more of the constituent nucleotides of the poly A chain are sugar-modified nucleotides.
• the position of the sugar-modified nucleotide within the polyA chain is not particularly limited.
• the proportion of sugar-modified nucleotides in the poly A chain is preferably 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, or 100%.
• the percentage of 100% means that all of the nucleotides in the poly A chain are sugar-modified nucleotides.
• poly A chain is composed of 2'-deoxyribonucleotides, spacer-modified or sugar-modified nucleotides, it preferably contains sugar-modified nucleotides.
• the poly A chain is composed of 2'-deoxyribonucleotides, spacer-modified and sugar-modified nucleotides, among the nucleotides constituting the poly A chain, sugar-modified nucleotides account for 50% or more and 2'-deoxyribonucleotides account for 30% or less.
• the spacer modification is preferably 20% or less.
• the sugar-modified nucleotides account for 50% or more and the 2'-deoxyribonucleotides account for 50% or less of the nucleotides constituting the poly A chain. is preferred.
• the poly A chain is composed of spacer-modified and sugar-modified nucleotides
• the 1st, 2nd and 3rd nucleotides from the 3' end of the 3' untranslated region may be sugar-modified nucleotides.
• the substituents at the 2'-positions of the sugar moieties of the first, second and third nucleotides from the 3' -end are preferably methoxyethoxy groups ( OCH2CH2OCH3 ).
• modified sugar moieties of sugar-modified nucleotides are preferably, for example, independently selected from any one of the following structures: Each is preferably independently selected from any one of the following structures.
• the poly A chain may contain base-modified nucleotides.
• the position of the base-modified nucleotide in the polyA chain is not particularly limited.
• Base-modified nucleotides may be sugar-modified nucleotides and/or phosphate-modified nucleotides (in other words, base-modified nucleotides may further comprise modified sugar moieties and/or modified phosphate moieties).
• the 3'-untranslated region may contain 2'-deoxyribonucleotide or spacer modifications, preferably in the 3'-untranslated region other than the poly A chain.
• Specific structures of the spacer modification include, for example, those described in the item (Spacer modification) of (5′-side untranslated region) above.
• Polynucleotides of this embodiment also include those in which suitable non-sugar-modified nucleotides of 1 to 10 base lengths are added to the original 3′ end.
• the poly A chain may contain phosphate-modified nucleotides.
• the position of the phosphate-modified nucleotide in the poly A chain is not particularly limited.
• Phosphate-modified nucleotides may be sugar-modified nucleotides and/or base-modified nucleotides (in other words, phosphate-modified nucleotides may contain modified sugar moieties and/or modified base moieties).
• Phosphorothioate is preferred for the modified phosphate moiety contained in the poly A chain. It is desirable that the positions of the phosphorothioate-linked nucleotides in the poly A chain are contiguous from the 3′ end side.
• the percentage of nucleotides linked by phosphorothioate is 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% Above, it is 90% or more, or 100% or more, preferably 50% or more, more preferably 80% or more, and particularly preferably 100%.
• the ratio of 100% means that all the nucleotides of the poly A chain are linked by phosphorothioate.
• Phosphate-modified nucleotides can impart stability to endonuclease, which is one of nucleolytic enzymes, and therefore may be included two or more consecutively from the 5'-end and/or 3'-end of the polynucleotide of the present invention. preferable.
• the polynucleotide of this embodiment may contain the following linking portions.
• R 1 and R 2 are each independently H, OH, F, OCH 2 CH 2 OCH 3 or OCH 3 ; B 1 and B 2 are each independently a base moiety, X 1 is O, S or NH; X2 is O, S, NH or the structure X 3 is OH, SH, or a salt thereof (OH and SH of X 3 may be represented as O - and S - , respectively); However, X 1 and X 2 are not O at the same time.
• the base portion may be an unmodified base portion or a modified base portion.
• the left and right nucleotides in the junction are two nucleotides that constitute the polynucleotide of this embodiment. Translational activity can be maintained even when the linking portion is included.
• the terminal nucleotide D may be unmodified.
• the OH and SH salts of X 3 in the linking moiety include, for example, pharmaceutically acceptable salts.
• Pharmaceutically acceptable salts include, for example, alkali metal salts, alkaline earth metal salts, ammonium salts, organic amine salts, and amino acid salts.
• Alkali metal salts include, for example, sodium, lithium, and potassium salts.
• Alkaline earth metal salts include, for example, calcium salts and magnesium salts.
• connecting portion includes the following. [wherein R 1 , R 2 , B 1 , B 2 and X 3 are as defined above]
• the position where the connecting part exists is not particularly limited.
• the linking portion may be present in any of the translation region, the 5′-side untranslated region, and the 3′-side untranslated region (including the poly A chain). is preferably present at least in the translational region.
• the number of the connecting portions is not particularly limited, and can be appropriately selected according to the length of the polynucleotide.
• the number of connecting parts is, for example, 1 to 200, 1 to 100, 1 to 50, 1 to 20, 1 to 10, 1 to 8, 1 to 6, 1 to 4, 1 ⁇ 3, or 1 or 2.
• the first and second nucleotides of at least one of the multiple codons constituting the translation region may be linked by phosphorothioate.
• the number of phosphorothioate bonds is not particularly limited, and can be appropriately selected according to the length of the polynucleotide.
• the number of phosphorothioate bonds is, for example, 1 to 200, 1 to 100, 1 to 50, 1 to 20, 1 to 10, 1 to 8, 1 to 6, 1 to 4, 1 ⁇ 3, or 1 or 2.
• nucleotides may be linked by phosphorothioates.
• the 1st and 2nd nucleotides from the 5' end of the 5' untranslated region are linked by phosphorothioate means that the 1st nucleotide and the 2nd nucleotide from the 5' end of the 5' untranslated region is synonymous with being linked by phosphorothioate, for example, the first to third nucleotides are linked by phosphorothioate, and the first and second nucleotides are linked by phosphorothioate, and , means that the second and third nucleotides are linked by a phosphorothioate.
• the structure of the 5' side of the 1st nucleotide and the 3' side of the 3rd nucleotide may be arbitrary.
• the 1st to 2nd nucleotides, 1st to 3rd nucleotides, 1st to 4th nucleotides, or 1st to 5th nucleotides from the 3' end of the 3' untranslated region are phosphorothioate may be connected by From the viewpoint of improving translation activity, the 1st to 2nd nucleotides, 1st to 3rd nucleotides, 1st to 4th nucleotides, or 1st to 5th nucleotides from the 3' end of the poly A chain are linked by phosphorothioate. may be Also, all nucleotides of the poly A chain may be linked by phosphorothioate.
• Another embodiment of the present invention relates to a polynucleotide in which the 1st, 2nd and 3rd nucleotides from the 5' end of the 5' untranslated region are sugar-modified nucleotides.
• Another embodiment of the present invention relates to a polynucleotide in which the 1st, 2nd and 3rd nucleotides from the 3' end of the poly A chain are sugar-modified nucleotides.
• the 1st, 2nd and 3rd nucleotides from the 5' end of the 5' untranslated region are sugar-modified nucleotides, It relates to a polynucleotide in which the first, second and third nucleotides from the 3' end of the poly A chain are sugar-modified nucleotides.
• the 1st to 3rd nucleotides, 1st to 4th nucleotides, or 1st to 5th nucleotides from the 5' end of the 5' untranslated region are linked by phosphorothioate. Regarding polynucleotides.
• Another embodiment of the present invention relates to a polynucleotide in which the 1st to 3rd nucleotides, 1st to 4th nucleotides, or 1st to 5th nucleotides from the 3' end of the poly A chain are linked by phosphorothioate. .
• the 1st to 3rd nucleotides, 1st to 4th nucleotides, or 1st to 5th nucleotides from the 5' end of the 5' untranslated region are linked by phosphorothioate
• 1 to 3 nucleotides, 1 to 4 nucleotides, or 1 to 5 nucleotides from the 3' end of the poly A chain are linked by phosphorothioate.
• the 1st, 2nd and 3rd nucleotides from the 5' end of the 5' untranslated region are sugar-modified nucleotides, and 1st from the 5' end of the 5' untranslated region.
• the 1st, 2nd and 3rd nucleotides from the 3' end of the poly A chain are sugar-modified nucleotides, and the 1st to 3rd nucleotides from the 3' end of the poly A chain, Polynucleotides in which nucleotides 1-4, or nucleotides 1-5 are linked by phosphorothioates.
• the 1st, 2nd and 3rd nucleotides from the 5' end of the 5' untranslated region are sugar-modified nucleotides, and 1st from the 5' end of the 5' untranslated region.
• nucleotides 1-5 are linked by a phosphorothioate
• the 1st, 2nd and 3rd nucleotides from the 3' end of the poly A chain are sugar-modified nucleotides, the 1st to 3rd nucleotides from the 3' end of the poly A chain, the 1st to 4th nucleotides, or 1 It relates to polynucleotides in which nucleotides 1-5 are linked by phosphorothioates.
• a translation region, a translation region and a poly A chain may be present, and any one or two of the 5′ untranslated region, the translation region and the poly A chain may be in any combination of suitable aspects.
• regions other than those described as the 5′-side untranslated region, translated region, and poly A chain may be appropriately combined from exemplary embodiments and preferred embodiments. That is, in this specification, all combinations of exemplary embodiments and preferred embodiments in each description described as a 5′-side untranslated region, a translated region, and a poly A chain are described and exemplified as embodiments herein. be.
• the polynucleotide of this embodiment may further comprise a Kozak sequence and/or a Ribosome Binding sequence (RBS).
• RBS Ribosome Binding sequence
• the polynucleotide of this embodiment can be produced, for example, by chemical synthesis.
• the polynucleotide of the present embodiment can be produced by introducing a predetermined sugar-modified nucleotide at a predetermined position while elongating the polynucleotide chain using a known chemical synthesis method.
• Known chemical synthesis methods include, for example, the phosphoramidite method, the phosphorothioate method, the phosphotriester method, and the CEM method (see Nucleic Acids Research, 35, 3287 (2007)).
• ABI3900 high-throughput nucleic acid synthesizer manufactured by Applied Biosystems can also be used.
• a phosphoramidite that is not commercially available can be chemically synthesized and used as a raw material to produce the polynucleotide of the present embodiment.
• a method for synthesizing phosphoramidite (f) as a starting material for base-modified nucleotides is shown below.
• Ra is a hydrogen atom, F, OCH 2 CH 2 OCH 3 or OCH 3
• Rb is a protecting group that can be removed with a fluoride ion such as di-tert-butylsilyl
• Rc is a carbon It is alkyl of numbers 1 to 6
• Rd is a protective group used in solid-phase nucleic acid synthesis, for example, p,p'-dimethoxytrityl group.
• Compound (b) is prepared by reacting compound (a) with, for example, the corresponding silylating agent in a solvent in the presence of a base at a temperature between 0°C and 80°C for 10 minutes to 3 days.
• Solvents include, for example, DMF, DMA, NMP and the like, and these may be used alone or in combination.
• bases include imidazole, triethylamine, diisopropylethylamine and the like.
• Examples of the silylating agent include bis(trifluoromethanesulfonate) di-tert-butylsilyl and the like.
• Compound (c) can be prepared by reacting compound (b) with the corresponding alkylating agent in a solvent in the presence of a base at a temperature between 0°C and 150°C for 10 minutes to 3 days. can. Suitable additives can also accelerate the reaction. Solvents include, for example, DMF, pyridine, dichloromethane, THF, ethyl acetate, 1,4-dioxane, NMP and the like, and these may be used alone or in combination. Examples of the base include aqueous sodium hydroxide solution, potassium carbonate, pyridine, triethylamine, N-ethyl-N,N-diisopropylamine and the like. Examples of alkylating agents include methyl iodide, ethyl iodide, methyl bromide and the like. Additives include, for example, tetrabutylammonium bromide.
• Compound (d) can be produced by reacting compound (c) with a fluorine reagent in a solvent at a temperature between -80°C and 200°C for 10 seconds to 72 hours.
• a base can also be added at this time.
• fluorine reagents include hydrogen fluoride, triethylamine hydrofluoric acid, tetrabutylammonium fluoride (TBAF), and the like.
• bases include triethylamine, N,N-diisopropylethylamine, and the like.
• solvents examples include dichloromethane, chloroform, acetonitrile, toluene, ethyl acetate, THF, 1,4-dioxane, DMF, N,N-dimethylacetamide (DMA), NMP, dimethylsulfoxide (DMSO) and the like.
• Compound (e) can be prepared by reacting compound (d) with the corresponding alkylating agent in a solvent in the presence of a base at a temperature between 0°C and 150°C for 10 minutes to 3 days. can.
• a suitable activating agent can also accelerate the reaction.
• Solvents include, for example, DMF, pyridine, dichloromethane, THF, ethyl acetate, 1,4-dioxane, NMP and the like, and these may be used alone or in combination.
• bases include pyridine, triethylamine, N-ethyl-N,N-diisopropylamine, 2,6-lutidine and the like.
• alkylating agents include trityl chloride, p,p'-dimethoxytrityl chloride and the like.
• activators include 4-dimethylaminopyridine and the like.
• Compound (f) can be produced by reacting compound (e) and compound (g) in a solvent in the presence of a base at a temperature between 0°C and 100°C for 10 seconds to 24 hours.
• Solvents include, for example, dichloromethane, acetonitrile, toluene, ethyl acetate, THF, 1,4-dioxane, DMF, NMP and the like, and these may be used alone or in combination.
• bases include triethylamine, N,N-diisopropylethylamine, pyridine and the like, and these may be used alone or in combination.
• the 5' cap structure can be introduced using known methods (eg, enzymatic methods and chemical synthesis methods).
• known methods include, for example, the methods described in Top. Curr. Chem. (Z) (2017) 375:16 and Beilstein J. Org. Chem. 2017, 13, 2819-2832.
• the ligation method is not particularly limited, and examples thereof include an enzymatic method and a chemical synthesis method.
• Enzymatic ligation includes, for example, ligation using ligase.
• ligases include T4 DNA Ligase, T4 RNA Ligase1, T4 RNA Ligase2, T4 RNA Ligase2, truncated T4 RNA Ligase2, truncated KQ, E. coli DNA Ligase, Taq. They can be mixed and used.
• the 3'-terminal nucleotide A of the polynucleotide unit constituting the 5'-terminal side of the polynucleotide (hereinafter referred to as "5'-terminal polynucleotide unit") and the 3' of the polynucleotide 5'-terminal nucleotide B of the polynucleotide unit constituting the terminal side (hereinafter referred to as "3'-terminal polynucleotide unit”)
• the nucleotide C adjacent to B and the nucleotide D adjacent to said nucleotide C are not modified.
• polydisperse polyethylene glycol may be used to promote the ligation reaction due to the molecular crowding effect.
• polydisperse PEG include PEG4000, PEG6000, PEG8000, PEG10000 and the like, and these can be used alone or in combination.
• Ligation by a chemical synthesis method includes, for example, the 3' end of the 5'-terminal polynucleotide unit (right side below) and the 3'-terminal polynucleotide unit, as shown below.
• a method of condensing the 5′-end (left side below) in the presence of a condensing agent can be mentioned.
• Condensing agents include, for example, 1,3-dicyclohexanecarbodiimide (DCC), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC), carbonyldiimidazole, benzotriazol-1-yloxy tris(dimethylamino)phosphonium hexafluorophosphate, (benzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate, O-(7-azabenzotriazol-1-yl)-N,N,N', N'-tetramethyluronium hexafluorophosphate (HATU), O-(benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HBTU), iodide 2-chloro-1-methylpyridinium, 1H-imidazole-1-carbon
• the condensation reaction is carried out in the presence of a template DNA comprising a nucleotide strand on the 3'-terminal side of the 5'-terminal polynucleotide unit and a nucleotide strand complementary to the nucleotide strand on the 5'-terminal side of the 3'-terminal polynucleotide unit.
• the template DNA is preferably a nucleotide chain having a length of 2 to 50 bases, more preferably 5 to 40 bases, from the 3' end of the 5'-terminal side polynucleotide unit, and from the 5' end of the 3'-terminal side polynucleotide unit.
• base sequence identity is, for example, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, or 100%.
• Additives may be added in the condensation reaction.
• Additives include, for example, 1-hydroxybenzotriazole (HOBt) and 4-dimethylaminopyridine (DMAP).
• HOBt 1-hydroxybenzotriazole
• DMAP 4-dimethylaminopyridine
• a metal salt may be added in the condensation reaction.
• metal salts include zinc (II) chloride, zinc (II) bromide, zinc (II) acetate, nickel (II) chloride, manganese (II) chloride, and the like.
• the condensation reaction may be carried out in the presence of a buffer.
• Buffers include, for example, acetate buffers, Tris buffers, citrate buffers, phosphate buffers, and water.
• the temperature of the condensation reaction is not particularly limited, it may be room temperature to 200°C, for example.
• the condensation reaction time is not particularly limited, but may be, for example, 5 minutes to 100 hours.
• condensation reaction between the 3' end of the 5'-terminal polynucleotide unit (right side below) and the 5' end of the 3'-terminal polynucleotide unit (left side below) include the following. mentioned. [In the formula, R 1 , R 2 , B 1 , B 2 and X 3 are as defined above, and X 4 is a leaving group. ]
• leaving groups include a chloro group, a bromo group, an iodo group, a methanesulfonyl group, a p-toluenesulfonyl group, and a trifluoromethanesulfonyl group.
• the leaving group is not particularly limited, it is preferably a chloro group or a bromo group.
• the ligation of polynucleotide units may be repeated multiple times depending on the length of the target polynucleotide.
• the number of times of ligation is not particularly limited. , 1 to 3 times, or 1 or 2 times.
• B p is a base which may be protected with a protecting group
• B is a base
• Polymer is a solid phase support.
• R 4 is a selectively deprotectable protecting group, such as tert-butyldimethylsilyl group and triethylsilyl group
• R 3 is a protecting group used in solid-phase nucleic acid synthesis, such as p, p '-dimethoxytrityl group
• X a is a nucleic acid sequence
• Y a and Y b are each independently a leaving group, eg a halogen, preferably a chlorine or bromine atom.
• a nucleic acid sequence is a substructure in a nucleic acid that together with each binding compound forms the nucleic acid.
• each B may be the same and may differ. ]
• Step 1 Compound (B) can be produced by reacting compound (A) in a solvent at a temperature between 60° C. and the boiling point of the solvent used for 10 seconds to 3 days.
• solvents include toluene, xylene, 1,2-dichloroethane, 1,4-dioxane, N,N-dimethylformamide (DMF), N-methylpyrrolidone (NMP), 1,2-dichlorobenzene, water, and the like. and these can be used singly or in combination.
• Compound (A) is described, for example, in J. Am. Am. Chem. Soc. (1999), 121, 5661-5665. It can be produced by the method described in .
• B p in compound (A) is not particularly limited, it preferably has any one of the following structures.
• R 6 is a group that constitutes a part of a base-protecting group, and represents, for example, a methyl group, an isopropyl group, a phenyl group that may have a substituent, and the like.
• substituents in the phenyl group which may have a substituent include methyl, isopropyl and tert-butyl.
• Step 2 Compound (C) is prepared by treating compound (B) in a solvent in the presence of 1 to 100 equivalents of an oxidizing agent at a temperature between 0° C. and the boiling point of the solvent used for 10 seconds to 3 days, preferably 1 to It can be prepared by reacting with 100 equivalents of an additive.
• Solvents include, for example, aprotic solvents such as chloroform and dichloromethane, and the like, and these can be used alone or in combination.
• oxidizing agent examples include organic oxidizing agents such as Jones reagent, chromic acid, pyridinium dichromate, ruthenium tetroxide, sodium chlorite, Dess-Martin reagent, and inorganic oxidizing agents such as pyridinium chlorochromate. system oxidizing agents, etc., and these can be used singly or in combination.
• Additives include, for example, pyridine, triethylamine, N,N-diisopropylethylamine and the like, and these can be used alone or in combination.
• Step 3 Compound (D) is obtained by reacting compound (C) in a solvent such as pyridine in the presence of hydroxylamine hydrochloride at a temperature between 0° C. and the boiling point of the solvent used for 10 seconds to 3 days. can be manufactured.
• Step 4 Compound (E) is prepared by reacting compound (D) in a solvent in the presence of 1 to 100,000 equivalents of a deprotecting agent at a temperature between 0°C and the boiling point of the solvent used for 10 seconds to 3 days.
• Solvents include, for example, toluene, xylene, water, and the like, and these can be used alone or in combination.
• Deprotecting agents include, for example, trifluoroacetic acid, trichloroacetic acid, acetic acid, hydrochloric acid and the like, and these can be used alone or in combination.
• Compound (F) can be produced by reacting compound (E) in a solvent in the presence of a reducing agent at a temperature between 0° C. and the boiling point of the solvent used for 10 seconds to 3 days.
• Solvents include, for example, trifluoroacetic acid, trichloroacetic acid, acetic acid, hydrochloric acid, toluene, xylene, toluene, xylene, tetrahydrofuran, methanol, ethanol, 1,4-dioxane, water and the like, and these may be used alone or in combination. can be used.
• reducing agents include sodium borohydride, sodium cyanoborohydride, lithium borohydride, sodium triacetoxyborohydride and the like.
• Step 6 Compound (G) is produced by reacting compound (F) in a solvent in the presence of a catalyst under a hydrogen atmosphere at a temperature between 0°C and the boiling point of the solvent used for 10 seconds to 3 days.
• Solvents include, for example, trifluoroacetic acid, acetic acid, dilute hydrochloric acid, methanol, ethanol, isopropanol, water and the like, and these can be used alone or in combination.
• the catalyst include palladium carbon, ruthenium carbon, and the like.
• Compound (G) can also be produced, for example, by the method described in International Publication No. 2017/123669.
• Step 7 Compound (H) is prepared by dissolving compound (G) in a solvent in the presence of 1 to 100 equivalents of compound (G′) and a base at a temperature between 0° C. and the boiling point of the solvent used for 10 seconds to 3 days. , preferably by reacting with 1 to 1000 equivalents of a base.
• solvents examples include methanol, ethanol, isopropanol, dichloromethane, acetonitrile, toluene, ethyl acetate, tetrahydrofuran (THF), 1,4-dioxane, N,N-dimethylformamide (DMF), N-methylpyrrolidone (NMP), Water and the like can be mentioned, and these can be used alone or in combination.
• bases include pyridine, triethylamine, N-ethyl-N,N-diisopropylamine, 2,6-lutidine and the like, and these can be used alone or in combination.
• a commercial item can be used for a compound (G').
• Step 8 Compound (I) is prepared by reacting compound (H) and p,p'-dimethoxytrityl chloride in a solvent such as pyridine, optionally in the presence of a co-solvent, at a temperature between 0°C and 100°C for 5 minutes. It can be produced by reacting for ⁇ 100 hours.
• a solvent such as pyridine
• Co-solvents include, for example, methanol, ethanol, dichloromethane, chloroform, 1,2-dichloroethane, toluene, ethyl acetate, acetonitrile, diethyl ether, tetrahydrofuran, 1,2-dimethoxyethane, dioxane, N,N-dimethylformamide (DMF ), N,N-dimethylacetamide, N-methylpyrrolidone, triethylamine, N,N-diisopropylethylamine, water and the like, and these can be used alone or in combination.
• DMF N,N-dimethylformamide
• N-methylpyrrolidone triethylamine, N,N-diisopropylethylamine, water and the like, and these can be used alone or in combination.
• Step 9 Compound (J) is prepared by reacting compound (I) in a solvent at a temperature between 0° C. and the boiling point of the solvent used for 10 minutes to 10 days with 1 to 10 equivalents of an additive.
• Solvents include, for example, dichloromethane, acetonitrile, toluene, ethyl acetate, THF, 1,4-dioxane, DMF, DMA, NMP and the like, and these can be used alone or in combination.
• the additive include tetrabutylammonium fluoride, triethylamine trihydrofluoride, and the like, and these can be used alone or in combination.
• Step 10 Compound (K) is prepared by reacting compound (J) with succinic anhydride in a solvent in the presence of 1 to 30 equivalents of a base at a temperature between room temperature and 200° C. for 5 minutes to 100 hours. can do.
• solvents include methanol, ethanol, dichloromethane, chloroform, 1,2-dichloroethane, toluene, ethyl acetate, acetonitrile, diethyl ether, tetrahydrofuran, 1,2-dimethoxyethane, dioxane, N,N-dimethylformamide (DMF).
• N,N-dimethylacetamide, N-methylpyrrolidone, pyridine, water and the like examples include cesium carbonate, potassium carbonate, potassium hydroxide, sodium hydroxide, sodium methoxide, potassium tert-butoxide, triethylamine, diisopropylethylamine, N-methylmorpholine, pyridine, 1,8-diazabicyclo[5.4 .0]-7-undecene (DBU), N,N-dimethyl-4-aminopyridine (DMAP) and the like, and these can be used alone or in combination.
• bases include cesium carbonate, potassium carbonate, potassium hydroxide, sodium hydroxide, sodium methoxide, potassium tert-butoxide, triethylamine, diisopropylethylamine, N-methylmorpholine, pyridine, 1,8-diazabicyclo[5.4 .0]-7-undecene (DBU), N,N-dimethyl-4-a
• Step 11 Compound (L) is compound (K) and a terminally aminated solid phase support in the absence or in a solvent of 1 to 30 equivalents of a base, a condensing agent and, if necessary, 0.01 to Reaction in the presence of 30 equivalents of additive at a temperature between room temperature and 200° C. for 5 minutes to 100 hours, followed by reaction in an acetic anhydride/pyridine solution at a temperature between room temperature and 200° C. for 5 minutes to 100 hours. It can be manufactured by Examples of the solvent include those exemplified in Step 4.
• bases include cesium carbonate, potassium carbonate, potassium hydroxide, sodium hydroxide, sodium methoxide, potassium tert-butoxide, triethylamine, diisopropylethylamine, N-methylmorpholine, pyridine, 1,8-diazabicyclo[5.4 .0]-7-undecene (DBU), N,N-dimethyl-4-aminopyridine (DMAP) and the like, and these can be used alone or in combination.
• DBU 1,8-diazabicyclo[5.4 .0]-7-undecene
• DMAP N,N-dimethyl-4-aminopyridine
• Condensing agents include, for example, 1,3-dicyclohexanecarbodiimide (DCC), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC), carbonyldiimidazole, benzotriazol-1-yloxy tris(dimethylamino)phosphonium hexafluorophosphate, (benzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate, O-(7-azabenzotriazol-1-yl)-N,N,N', N'-tetramethyluronium hexafluorophosphate (HATU), O-(benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HBTU), iodide 2-chloro-1-methylpyridinium and the like.
• DCC 1,3-
• Additives include, for example, 1-hydroxybenzotriazole (HOBt), 4-dimethylaminopyridine (DMAP) and the like, and these can be used alone or in combination.
• the solid-phase support is not particularly limited as long as it is a known aminated solid-phase support for solid-phase synthesis, but for example, CPG modified with a long-chain alkylamino group ( solid phase supports such as controlled pore glass) and PS (polystyrene resin).
• CPG modified with a long-chain alkylamino group solid phase supports such as controlled pore glass
• PS polystyrene resin
• LCAA-CPG long chain alkylamine porous glass
• Step 12 Compound (M) is produced by using compound (L) and extending the corresponding nucleotide chain by a known oligonucleotide chemical synthesis method, followed by elimination from the solid phase, deprotection of the protecting group, and purification. can do. Removal from the solid phase and deprotection can be produced by treating with a base in a solvent or in the absence of a solvent at a temperature between ⁇ 80° C. and 200° C. for 10 seconds to 72 hours after chemical synthesis of the oligonucleotide. can.
• bases include ammonia, methylamine, dimethylamine, ethylamine, diethylamine, isopropylamine, diisopropylamine, piperidine, triethylamine, ethylenediamine, 1,8-diazabicyclo[5.4.0]-7-undecene (DBU), Potassium carbonate and the like can be mentioned, and these can be used alone or in combination.
• the solvent include water, methanol, ethanol, THF, and the like, and these can be used alone or in combination.
• Oligonucleotide purification can be performed using a C18 reversed phase column or an anion exchange column, preferably by a combination of the above two techniques. The purity of the nucleic acid complex after purification is preferably 90% or higher, more preferably 95% or higher.
• Step 13 Compound (N) is reacted with compound (M) in the presence of 1 to 1000 equivalents of compound (O) in a buffer at a temperature between room temperature and 100° C. for 5 minutes to 100 hours.
• buffers include acetate buffers, Tris buffers, citrate buffers, phosphate buffers, water and the like, and these can be used alone or in combination.
• a commercial item can be used for the compound (O).
• B p is a base which may be protected with a protecting group
• B is a base
• R 7 is a protecting group such as a tert-butyldimethylsilyl group or a triethylsilyl group
• Y is For example, a chlorine atom, a bromine atom, a tosylate group
• X b is a nucleic acid sequence.
• each B may be the same and may differ.
• Step 14 Compound (Q) is produced by reacting compound (P) in a solvent in the presence of an additive and a base at a temperature between 0°C and the boiling point of the solvent used for 10 seconds to 3 days.
• Solvents include, for example, dichloromethane, acetonitrile, toluene, ethyl acetate, THF, 1,4-dioxane, DMF, DMA, NMP and the like, and these can be used alone or in combination.
• Additives include, for example, tosyl anhydride, tosyl chloride, thionyl chloride, oxalyl chloride and the like, and these can be used alone or in combination.
• bases examples include pyridine, triethylamine, N-ethyl-N,N-diisopropylamine, potassium carbonate and the like, and these can be used alone or in combination.
• a commercial item can be used for the compound (P).
• Step 15 Compound (R) is prepared by reacting compound (Q) in a solvent in the presence of an azidating agent and optionally a base at a temperature between room temperature and the boiling point of the solvent used for 10 seconds to 3 days.
• Solvents include, for example, dichloromethane, acetonitrile, toluene, ethyl acetate, THF, 1,4-dioxane, DMF, DMA, NMP and the like, and these can be used alone or in combination.
• the azidating agent include sodium azide.
• bases include pyridine, triethylamine, N-ethyl-N,N-diisopropylamine, potassium carbonate and the like, and these can be used alone or in combination.
• Compound (S) can be produced by reacting compound (R) in a solvent in the presence of a silylating agent and a base at a temperature between room temperature and the boiling point of the solvent used for 10 seconds to 3 days.
• Solvents include, for example, dichloromethane, acetonitrile, toluene, ethyl acetate, THF, 1,4-dioxane, DMF, DMA, NMP and the like, and these can be used alone or in combination.
• Silylating agents include, for example, tert-butyldimethylsilyl chloride, tert-butyldimethylsilyl triflate, triethylsilyl chloride and the like.
• bases examples include pyridine, triethylamine, N-ethyl-N,N-diisopropylamine, potassium carbonate, potassium hydroxide, sodium hydroxide, sodium methoxide, potassium tert-butoxide, triethylamine, diisopropylethylamine, N-methylmorpholine. , pyridine, 1,8-diazabicyclo[5.4.0]-7-undecene (DBU), N,N-dimethyl-4-aminopyridine (DMAP) and the like, which are used alone or in combination be able to.
• DBU 1,8-diazabicyclo[5.4.0]-7-undecene
• DMAP N,N-dimethyl-4-aminopyridine
• Step 17 Compound (T) can be produced by reacting compound (S) in a solvent with a reducing agent at a temperature between room temperature and the boiling point of the solvent used for 10 seconds to 3 days.
• solvents include methanol, ethanol, dichloromethane, chloroform, 1,2-dichloroethane, toluene, ethyl acetate, acetonitrile, diethyl ether, tetrahydrofuran, 1,2-dimethoxyethane, dioxane, N,N-dimethylformamide (DMF).
• reducing agents include sodium borohydride, sodium cyanoborohydride, lithium borohydride, sodium triacetoxyborohydride, and palladium carbon under hydrogen atmosphere.
• Step 18 Compound (U) can be produced in the same manner as in step 7 using compound (T).
• Compound (V) can be produced by reacting compound (U) and compound (AA) in a solvent in the presence of a base at a temperature between 0°C and 100°C for 10 seconds to 24 hours.
• Solvents include, for example, dichloromethane, acetonitrile, toluene, ethyl acetate, THF, 1,4-dioxane, DMF, NMP and the like, and these can be used alone or in combination.
• bases include triethylamine, N,N-diisopropylethylamine, pyridine and the like, and these can be used alone or in combination.
• a commercial item can be used for the compound (AA).
• Step 20 Compound (W) can be produced in the same manner as in step 12 using compound (V).
• the polynucleotide of this embodiment When the polynucleotide of this embodiment is produced by ligating a plurality of polynucleotide units, it may partially contain polynucleotide units produced by IVT.
• the method for ligating polynucleotides produced by IVT is not particularly limited, and examples thereof include the enzymatic method and chemical synthesis method described above.
• a method for producing a polynucleotide unit using IVT includes a method in which RNA is transcribed from template DNA having a promoter sequence using RNA polymerase. More specifically, known IVT includes methods described in the following documents. RA, Methods in Molecular Biology (Methods and Protocols), Vol. 703, Chapter 3 (2011); Cardiac Gene Therapy: Methods in Moleculer Biology (Methods and Protocols), Vol. 1521, Chapter 8 (2016); • Journal of Molecular Biology, vol. 249, pp. 398-408 (1995).
• template DNA used for IVT examples include those produced by chemical synthesis, those produced by polymerase chain reaction, plasmid DNA, and those produced by linearizing plasmid DNA with a restriction enzyme. They can be used alone or mixed.
• RNA polymerases include T3 RNA polymerase, T7 RNA polymerase, SP6 RNA polymerase and the like, and these can be used alone or in combination.
• the ribonucleoside triphosphates used for transcription may be modified, and multiple types of ribonucleoside triphosphates may be mixed and used.
• m7G(5')ppp(5')G Trilink, catalog no.
• ⁇ Pharmaceutical composition> relates to a pharmaceutical composition comprising said polynucleotide.
• the pharmaceutical composition of this embodiment By administering the pharmaceutical composition of this embodiment to a patient with a disease, the polynucleotide is translated, the polypeptide encoded by the polynucleotide is synthesized, and the disease is treated.
• supplementing the function or activity with a polypeptide translated from the polynucleotide for a disease characterized by the loss or abnormality of the function or activity of a specific protein is also provided by expressing in vivo a foreign antigenic peptide and its analogue by a polypeptide translated from the polynucleotide.
• a therapeutic method for artificially controlling immune response is also provided by expressing in vivo a specific protein such as a transcription factor or a polypeptide that does not originally exist in the living body by a polypeptide translated from the polynucleotide.
• the function, differentiation, proliferation, etc. of cells can be artificially enhanced. It is also possible to dynamically control and modify tissues and cells.
• a therapeutic method for restoring cell function is also provided.
• Diseases include, but are not limited to, cancer and proliferative diseases, infectious diseases and parasitic diseases, blood and hematopoietic diseases, autoimmune diseases, endocrine, nutritional and metabolic diseases (inborn errors of metabolism ), psychiatric, nervous system diseases, skin and subcutaneous tissue diseases, eye diseases, ear diseases, respiratory diseases, digestive system diseases, renal, urogenital, cardiovascular diseases, cerebrovascular diseases, musculoskeletal diseases Diseases of the system and connective tissue, miscarriages, perinatal diseases, congenital malformations, acquired injuries, and poisoning.
• the pharmaceutical composition may be administered in the form of a prescribed formulation.
• Formulations include, for example, liquid dosage forms for oral or parenteral administration, such as pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups, and elixirs.
• Liquid dosage forms contain, in addition to the active ingredient, inert diluents (e.g., water or other solvents), solubilizers and emulsifiers (e.g., ethyl alcohol, isopropyl alcohol, ethyl carbonate, etc.) commonly used in the art.
• Formulations for oral administration may also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening agents, flavoring agents, and/or perfuming agents.
• Formulations for parenteral administration may include solubilizers such as Cremophor®, alcohols, oils, modified oils, glycols, polysorbates, cyclodextrins, polymers, and combinations thereof.
• Methods of administering the pharmaceutical composition include, for example, local lymph node administration, intratumoral local administration, intramuscular administration, intradermal administration, subcutaneous administration, intratracheal administration, intrathecal administration, intracerebroventricular administration, intraocular administration, and intracerebroventricular administration.
• Intraroom administration coronary artery catheterization, hepatic portal vein catheterization, myocardial catheterization, transurethral catheterization, and intravenous administration.
• the pharmaceutical composition may contain optional ingredients in addition to the polynucleotide.
• Optional components include, for example, solvents, aqueous solvents, non-aqueous solvents, dispersion media, diluents, dispersants, suspension aids, surfactants, isotonic agents, thickeners, emulsifiers, preservatives, lipids, lipidoids.
• one or more pharmaceutically acceptable excipients selected from liposomes, lipid nanoparticles, core-shell nanoparticles, polymers, lipoplexes, peptides, proteins, cells, hyaluronidases, and mixtures thereof mentioned.
• Reagents used in compound synthesis were purchased from Sigma-Aldrich Co., Ltd., Tokyo Chemical Industry Co., Ltd., Wako Pure Chemical Industries, Ltd., and Kanto Chemical Co., Ltd., and were used without purification.
• Anhydrous solvents were prepared by drying over activated molecular sieves 4A for 12 hours or commercially available anhydrous grade solvents were used.
• the reaction was tracked by thin-layer silica gel chromatography (silica gel 70F254 TLC plate-Wako, Wako Pure Chemical Industries, Ltd.).
• Silica gel 60N for flash chromatography sipherical, neutral, particle size 40-50 ⁇ m
• NMR was measured using JEOL ECS 400 MHz (JEOL Ltd.) using deuterated solvents (CDCl 3 , CD 3 OD, DMSO-d 6 ) (Kanto Chemical Co., Ltd.) as measurement solvents.
• Data analysis of the acquired NMR was performed using JEOL Delta (JEOL Ltd.) as software, and the chemical shift values were based on residual signals in deuterated solvents (CDCl 3 : 7.26, CD 3 OD : 3.31, DMSO-d 6 : 2.50) (Organometallics 2010, 29, 2176-2179).
• Step 1 Synthesis of Compound 4 N-(9-((2R,3S,4S,5R)-3-(tert-butyldimethylsilyloxy)-5-((tert-butyldimethylsilyloxy)methyl)-4-hydroxy-tetrahydrofuran-2-yl)-6-oxo- 6,9-dihydro-1H-purin-2-yl)isobutyramide
• Compound 3 obtained by the method described in the literature (J. Am. Chem. Soc., 1999, 121, 5661-5665) was dissolved in 1,2-dichlorobenzene (2.0 mL) and placed in an oil bath (160 °C) for 4 hours.
• Step 2 Synthesis of compound 5 N-(9-((2R,3S,5S)-3-(tert-butyldimethylsilyloxy)-5-((tert-butyldimethylsilyloxy) methyl)-4-(hydroxyimino)-tetrahydrofuran-2-yl)-6-oxo- 6,9-dihydro-1H-purin-2-yl) isobutyramide
• Molecular sieve 3A (powder) (258 mg) was added to a solution of chromic acid (129 mg, 1.29 mmol) in anhydrous dichloromethane (2.0 mL) and cooled on an ice bath.
• Step 3 Synthesis of compound 6 N-(9-((2R,3S,5S)-3-(tert-butyldimethylsilyloxy)-4-(hydroxyimino)-5-(hydroxymethyl)-tetrahydrofuran-2-yl)-6-oxo-6,9-dihydro -1H-purin-2-yl)isobutyramide
• An ice-cooled 90% trifluoroacetic acid aqueous solution (1.0 mL) was added to compound 5 (129 mg, 0.22 mmol), and the mixture was stirred on an ice bath for 30 minutes.
• reaction solution was concentrated under reduced pressure, and the resulting residue was azeotroped three times with toluene and water (1:1, v/v) under reduced pressure.
• Step 4 Synthesis of Compound 7 N-(9-((2R,3S,4S,5S)-4-amino-3-(tert-butyldimethylsilyloxy)-5-(hydroxymethyl)-tetrahydrofuran-2-yl)-6-oxo-6,9-dihydro -1H-purin-2-yl)isobutyramide
• Sodium borohydride (15 mg, 0.38 mmol) was added to a solution of compound 6 (93 mg, 0.19 mmol) in acetic acid (1.9 mL), and the mixture was stirred at room temperature for 1 hour.
• Step 5 Synthesis of compound 8 N-(9-((2R,3S,4S,5S)-4-amino-3-(tert-butyldimethylsilyloxy)-5-(hydroxymethyl)-tetrahydrofuran-2-yl)-6-oxo-6,9-dihydro -1H-purin-2-yl)isobutyramide 10% palladium on carbon (20 mg) was added to a solution of compound 7 (50 mg, 0.10 mmol) in 90% aqueous acetic acid (1.5 mL), and the mixture was stirred at room temperature for 18 hours under a hydrogen atmosphere.
• Step 6 Synthesis of Compound 9 N-(9-((2R,3S,4R,5S)-3-(tert-butyldimethylsilyloxy)-5-(hydroxymethyl)-4-(2,2,2-trifluoroacetamido)-tetrahydrofuran-2-yl)-6 -oxo-6,9-dihydro-1H-purin-2-yl)isobutyramide
• Ethyl trifluoroacetate (0.76 mL) was added to a methanol solution (0.76 mL) of compound 8 (40 mg, 0.076 mmol) of known literature (WO2017/123669) and triethylamine (45 L, 0.38 mmol). Stirred for an hour.
• Step 7 Synthesis of Compound 10 N-(9-((2R,3S,4R,5S)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-3-(tert-butyldimethylsilyloxy)-4-(2,2,2 -trifluoroacetamido)-tetrahydrofuran-2-yl)-6-oxo-6,9-dihydro-1H-purin-2-yl)isobutyramide Dimethoxytrityl chloride (18 mg, 0.053 mmol) was added to a solution of compound 9 (10 mg, 0.017 mmol) in anhydrous pyridine (1 mL), and the mixture was stirred at room temperature for 1.5 hours.
• Step 8 Synthesis of Compound 11 N-(9-((2R,3S,4S,5S)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-3-hydroxy-4-(2,2,2-trifluoroacetamido)- tetrahydrofuran-2-yl)-6-oxo-6,9-dihydro-1H-purin-2-yl)isobutyramide Tetrabutylammonium fluoride (1M tetrahydrofuran solution, 19 ⁇ L, 0.019 mmol) was added to a solution of compound 10 (14 mg, 0.016 mmol) in tetrahydrofuran (1 mL), and the mixture was stirred at room temperature for 1 hour.
• Step 9 Synthesis of compound 12 To a solution of compound 11 (0.90 g, 1.20 mmol), triethylamine (0.42 mL, 3.0 mmol) in acetonitrile (12 mL), succinic anhydride (0.24 g, 2.40 mmol), dimethylaminopyridine (29 mg , 0.24 mmol) was added, and the mixture was stirred at room temperature for 1 hour. After confirming the disappearance of the raw materials by thin-layer chromatography, the reaction solution was concentrated under reduced pressure. The residue was dissolved in ethyl acetate and washed twice with saturated aqueous sodium bicarbonate and then with saturated brine.
• Compound 12 can also be synthesized by obtaining intermediate 6 from starting material 13 below.
• Step 10 Synthesis of Compound 14 N-(9-((2R,3R,5S)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-3-((tert-butyldimethylsilyl)oxy)-4-(hydroxyimino)tetrahydrofuran- 2-yl)-6-oxo-6,9-dihydro-1H-purin-2-yl)isobutyramide Under an argon atmosphere, compound 13 (manufactured by ChemGenes, 5.0 g, 6.5 mmol) was dissolved in dehydrated dichloromethane (50 mL) and stirred while cooling in an ice bath.
• reaction solution was transferred to an eggplant-shaped flask while rinsing with chloroform (containing 1% triethylamine), and concentrated.
• chloroform containing 1% triethylamine
• the residue was added to saturated aqueous sodium bicarbonate, stirred for 15 minutes, and then extracted twice with chloroform.
• the organic layers were combined, washed once with a saturated saline solution, and then dehydrated with anhydrous sodium sulfate. After filtering off the drying agent, the filtrate was concentrated to give compound 14 (4.13 g, diastereomeric mixture, 81% yield in two steps) as an orange foam.
• the amount of compound 12 supported on the solid phase was calculated by the Lambert-Beer formula. That is, the obtained solid phase carrier (2.0 mg) was weighed into a 2 mL volumetric flask, a deblocking reagent was added to bring the total volume to 2 mL, and the mixture was mixed by inversion to obtain a measurement sample. After performing blank measurement using a 3 w/v% trichloroacetic acid/dichloromethane solution, measurement was performed using a measurement sample. Absorbance at 504 nm: 0.377, loading: 24.8 ⁇ mol/g)
• Step 12 Synthesis of Compound 17 N-(9-((3aR,4R,6R,6aR)-6-(hydroxymethyl)-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)-9H-purin- 6-yl)benzamide
• N6-Benzyladenosine Compound 16 (100 g, 269 mmol, 1.0 eq.), acetone (2.70 L), dimethoxypropane (166 mL, 1.35 mol, 5.0 eq.) were placed in a 10 L four-necked flask under an argon atmosphere. were added sequentially.
• reaction mixture was cooled in an ice bath, and saturated aqueous sodium bicarbonate solution (400 mL) was added dropwise over 5 minutes to keep the internal temperature at 3-5°C. neutralized.
• the reaction solution was concentrated under reduced pressure, and distilled water (2.0 L) was added to the residue.
• the solution was extracted with chloroform (1.0 L) three times and the organic layer was dried over anhydrous sodium sulfate. After filtration, the solvent was distilled off under reduced pressure to obtain compound 17 (222 g). The resulting compound 17 was used in the next step without further purification.
• Step 13 Synthesis of Compound 18 ((3aR,4R,6R,6aR)-6-(6-benzamido-9H-purin-9-yl)-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl) methyl methanesulfonate Under an argon atmosphere, compound 17 (222 g) obtained in step 12 and pyridine (520 mL) were added to a 2 L four-necked flask, and the reaction mixture was cooled in an ice bath until the internal temperature reached 4°C to 9°C.
• Methanesulfonyl chloride (25.0 mL, 321 mmol, 1.2 eq.) was added dropwise over 15 minutes and stirred for 2 hours. After confirming the progress of the reaction by LC/MS, distilled water (500 mL) was added to the reaction mixture, and the solution was extracted with ethyl acetate (1.0 L) three times. mL ⁇ 2), saturated aqueous sodium bicarbonate solution (500 mL ⁇ 2) and saturated brine (500 mL ⁇ 2), followed by dehydration over anhydrous sodium sulfate.
• Step 14 Synthesis of Compound 19 N-(9-((3aR,4R,6R,6aR)-6-(azidomethyl)-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)-9H-purin- 6-yl)benzamide
• Compound 18 (150 g) obtained in Step 13 and dehydrated DMF (1.26 L) were added to a 3 L four-necked flask under an argon atmosphere.
• Sodium azide (82.8 g, 1.26 mol, 5.0 eq.) was added to the reaction solution, the temperature was raised to 60°C over 30 minutes, and the mixture was stirred at 60°C for 3 hours and 30 minutes.
• the reaction solution was gradually cooled to room temperature, and distilled water (1.0 L) and ethyl acetate (600 mL) were added. Distilled water (3.0 L) was added to the resulting solution, and the aqueous layer was extracted six times with ethyl acetate (500 mL). The organic layer was washed twice with distilled water (800 mL) and twice with saturated brine (800 mL), and dehydrated over anhydrous sodium sulfate.
• Step 15 Synthesis of Compound 20 N-(9-((3aR,4R,6R,6aR)-2,2-dimethyl-6-((2,2,2-trifluoroacetamido)methyl)tetrahydrofuro[3,4-d][1,3]dioxol -4-yl)-9H-purin-6-yl)benzamide
• Compound 19 obtained in step 14 (55.7 g, 128 mmol, 1.0 eq.) and methanol (1.28 L) were added to a 3 L four-necked flask under an argon atmosphere.
• Step 16 Synthesis of Compound 21 N-(9-((2R,3R,4S,5R)-3,4-dihydroxy-5-((2,2,2-trifluoroacetamido)methyl)tetrahydrofuran-2-yl)-9H-purin-6-yl )benzamide
• Compound 20 (10.0 g, 19.8 mmol, 10 eq.) obtained in Step 15 and distilled water (50.0 mL) were added to a 1 L eggplant flask, and the solution was cooled in an ice bath.
• Step 17 Synthesis of Compound 22 N-(9-((2R,3R,4R,5R)-3-((tert-butyldimethylsilyl)oxy)-4-hydroxy-5-((2,2,2-trifluoroacetamido)methyl)tetrahydrofuran-2-yl )-9H-purin-6-yl)benzamide
• Compound 21 obtained in Step 16 (15.6 g, 33.6 mmol, 1.0 eq.) and dehydrated DMF (111 mL) were added to a 500 mL eggplant flask under an argon atmosphere, and the solution was cooled in an ice bath.
• Step 18 Synthesis of Compound 24 (2R,3R,4R,5R)-5-(6-benzamido-9H-purin-9-yl)-4-((tert-butyldimethylsilyl)oxy)-2-((2,2,2-trifluoroacetamido)methyl )tetrahydrofuran-3-yl (2-cyanoethyl) diisopropylphosphoramidite
• Step 1 Synthesis of Compound 2a N-(9-((3aR,5R,6R,6aS)-2,2-di-tert-butyl-6-methoxytetrahydrofuro[2,3-d][1,3,2]dioxasilol-5-yl)- 9H-purin-6-yl)benzamide
• DMF 300 mL
• di-t-butylsilylbis(trifluoromethanesulfonate) 68.6 g, 156 mmol
• Step 2 Synthesis of Compound 3a N-(9-((3aR,5R,6R,6aS)-2,2-di-tert-butyl-6-methoxytetrahydrofuro[2,3-d][1,3,2]dioxasilol -5-yl)-9H-purin-6-yl)-N-methylbenzamide
• Compound 2a (10.0 g, 19.0 mmol) was dissolved in dichloromethane (50 mL), and tetrabutylammonium bromide (9.20 g, 28.5 mmol) and 1 M aqueous sodium hydroxide solution (50 ml) were added.
• Step 3 Synthesis of Compound 4a N-(9-((2R,3R,4S,5S)-4,5-dihydroxy-3-methoxytetrahydrofuran-2-yl)-9H-purin-6-yl)-N-methylbenzamide
• Compound 3a (6.25 g, 11.6 mmol) was dissolved in tetrahydrofuran (63 mL) and cooled in an ice bath.
• Triethylamine (8.07 ml, 57.9 mmol) and triethylamine trihydrofluoride (1.89 ml, 11.6 mmol) were added, and the mixture was stirred for 1 hour and 5 minutes while cooling in an ice bath.
• Step 4 Synthesis of Compound 5a N-(9-((2R,3R,4S,5S)-5-(bis(4-methoxyphenyl)(phenyl)methoxy)-4-hydroxy-3-methoxytetrahydrofuran-2-yl)-9H-purin-6- yl)-N-methylbenzamide
• Compound 4a (4.25 g, 10.6 mmol) was dissolved in pyridine (43 mL) and stirred in an ice bath.
• 4,4'-Dimethoxytrityl chloride (5.41 g, 20.0 mmol) was added to the reaction mixture, and the mixture was stirred at room temperature for 2 hours and 25 minutes.
• Step 1 Synthesis of Compound 2b N-(9-((4aR,6R,7R,7aS)-2,2-di-tert-butyl-7-fluorotetrahydro-4H-furo[3,2-d][1,3,2]dioxasilin-6 -yl)-9H-purin-6-yl)benzamide
• DMF 300 mL
• Step 2 Synthesis of Compound 3b N-(9-((4aR,6R,7R,7aS)-2,2-di-tert-butyl-7-fluorotetrahydro-4H-furo[3,2-d][1,3,2]dioxasilin-6 -yl)-9H-purin-6-yl)-N-methylbenzamide
• Compound 2b (10.0 g, 19.5 mmol) was dissolved in dichloromethane (50 mL), and tetrabutylammonium bromide (9,41 g, 29.2 mmol) and 1 M aqueous sodium hydroxide solution (50 ml) were added.
• Step 4 Synthesis of Compound 5b N-(9-((2R,3R,4R,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-3-fluoro-4-hydroxytetrahydrofuran-2-yl)-9H-purin -6-yl)-N-methylbenzamide
• Compound 4b (4.93 g, 12.7 mmol) was dissolved in pyridine (49 mL) and stirred in an ice bath.
• 4,4'-Dimethoxytrityl chloride (6.47 g, 29.2 mmol) was added to the reaction solution, and the mixture was stirred at room temperature for 1 hour and 20 minutes.
• Step 1 Synthesis of compound 3c N-(9-((4aR,6R,7R,7aS)-2,2-di-tert-butyl-7-methoxytetrahydro-4H-furo[3,2-d][1,3,2]dioxasilin-6 -yl)-9H-purin-6-yl)-N-ethylbenzamide
• Compound 2a (11.7 g, 22.3 mmol) was dissolved in dichloromethane (58.5 mL), and tetrabutylammonium bromide (10.8 g, 33.4 mmol) and 1 M aqueous sodium hydroxide solution (58.5 ml) were added.
• Step 2 Synthesis of compound 4c N-ethyl-N-(9-((2R,3R,4R,5R)-4-hydroxy-5-(hydroxymethyl)-3-methoxytetrahydrofuran-2-yl)-9H-purin-6-yl)benzamide
• Compound 3c (6.14 g, 11.1 mmol) was dissolved in tetrahydrofuran (61.4 mL) and cooled in an ice bath.
• Triethylamine (7.73 ml, 55.4 mmol) and triethylamine trihydrofluoride (1.81 ml, 11.1 mmol) were added, and the mixture was stirred for 2 hours while cooling in an ice bath.
• Step 3 Synthesis of compound 5c N-(9-((2R,3R,4R,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-hydroxy-3-methoxytetrahydrofuran-2-yl)-9H-purin -6-yl)-N-ethylbenzamide
• Compound 4c (4.58 g, 11.1 mmol) was dissolved in pyridine (46 mL) and stirred in an ice bath.
• 4,4'-Dimethoxytrityl chloride (5.63 g, 16.6 mmol) was added to the reaction mixture, and the mixture was stirred at room temperature for 2 hours.
• Step 1 Synthesis of compound 3d N-(9-((4aR,6R,7R,7aS)-2,2-di-tert-butyl-7-fluorotetrahydro-4H-furo[3,2-d][1,3,2]dioxasilin-6 -yl)-9H-purin-6-yl)-N-ethylbenzamide
• Compound 2b (1.00 g, 1.95 mmol) was dissolved in dichloromethane (5.0 mL), and tetrabutylammonium bromide (0.942 g, 2.92 mmol) and 1 M aqueous sodium hydroxide solution (5.0 ml) were added.
• RNA oligonucleotides are 2'-TOM (triisopropylsilyloxymethyl) protected ⁇ -cyanoethyl phosphoramidites (DMT-2'-O-TOM-rA(Ac), DMT-2'-O-TOM-rG(Ac) , DMT-2'-O-TOM-rC(Ac), DMT-2'-O-TOM-rU) (Glen Research or ChemGenes, respectively) were used, and the DNA oligonucleotides were ⁇ -cyanoethyl phosphoramidite (DMT -dA (Bz), DMT-dG (iBu), DMT-dC (Ac), DMT-T) were used.
• a 0.05 mol/L acetonitrile solution of each phosphoramidite monomer was prepared, and 0.2 ⁇ mol or 0.8 ⁇ mol of the solid-phase carrier was used with a DNA/RNA solid-phase synthesizer (NTS M-2-MX, Nippon Techno Service Co., Ltd.).
• NTS M-2-MX DNA/RNA solid-phase synthesizer
• CPG 1000A (dA-CPG, dG-CPG, Ac-dC-CPG, dT-CPG) (Glen Research) was used as a solid-phase carrier for DNA oligonucleotides, and the condensation time was 2 minutes.
• RNA with a phosphate group at the 5' end uses Universal UnyLinker Support 2000A (ChemGenes) as a solid-phase carrier, condensing the first base for 15 minutes, and then condensing for 3 minutes each. and Phosphorylation of the 5'-terminal hydroxyl group was performed using a chemical phosphorylation reagent (0.05 mol/L acetonitrile solution) (Glen Research or ChemGenes). Compound 15 was used for solid-phase synthesis of RNA oligonucleotides with a 3'-aminoguanosine monomer introduced at the 3' end. The condensation time for the first base was 15 minutes, and after that it was 3 minutes each.
• the following reagents were used in the solid-phase synthesizer.
• the dimethoxytrityl group of the 5' terminal hydroxyl group is removed by using a commercially available deblocking reagent (Deblocking Solution-1, 3 w/v% trichloroacetic acid/dichloromethane solution) (Wako Pure Chemical Industries, Ltd.) and reacting for 10 seconds. went.
• a commercially available activator solution (activator solution 3) (Wako Pure Chemical Industries, Ltd.) was used as a phosphoramidite activator.
• Capping of the unreacted 5′ terminal hydroxyl group was carried out by reacting for 10 seconds using commercially available capping solutions (cap A solution-2 and cap B solution-2) (Wako Pure Chemical Industries, Ltd.).
• a solution containing pyridine, THF, water and iodine (Oxidizer, 0.01 M iodine, 29.2% water, 6.3% pyridine, 64.5% acetonitrile), Honeywell), React for 10 seconds.
• Oxidizer 0.01 M iodine, 29.2% water, 6.3% pyridine, 64.5% acetonitrile
• Honeywell a solution containing pyridine, THF, water and iodine (Oxidizer, 0.01 M iodine, 29.2% water, 6.3% pyridine, 64.5% acetonitrile), Honeywell), React for 10 seconds.
• the dimethoxytrityl group of the 5'-terminal hydroxyl group of the RNA oligonucleotide was deprotected on the solid-phase carrier. All of the synthesized DNA and RNA oligonucleotides were deresinized and deprotected according to standard methods (concentrated ammoni
• DNA oligonucleotides were purified using a cartridge column (MicroPure II Column, LGC Biosearch Technology) according to the manufacturer's protocol.
• RNA oligonucleotides the solution obtained by deresinization was completely dried by concentration using a centrifugal evaporator, and then tetrabutylammonium fluoride (1M tetrahydrofuran solution) (1 mL) was used to remove the TOM-protecting group of the 2' hydroxyl group. (50°C, 10 minutes, then room temperature for 12 hours; or 50°C, 10 minutes, then 35°C for 6 hours).
• Tris-hydrochloride buffer (hereinafter referred to as Tris-HCl) (1 M, pH 7.4) (1 mL) was added to the solution and mixed, and then tetrahydrofuran was removed by concentration using a centrifugal evaporator.
• the resulting solution was processed according to the manufacturer's protocol using a gel filtration column (NAP-25, GE Healthcare) equilibrated with ultrapure water. Fractions containing the resulting RNA oligonucleotides were concentrated using a centrifugal evaporator and then purified using a denaturing polyacrylamide gel (hereinafter referred to as dPAGE).
• RNA fragments using dPAGE An aqueous solution of ammonium persulfate (hereinafter referred to as APS) and N,N,N',N'-tetramethylethylenediamine (hereinafter referred to as TEMED) are added as polymerization agents to an acrylamide gel solution (containing 7M urea as a denaturant) to solidify. (room temperature, 6-12 hours) to form a gel.
• APS ammonium persulfate
• TEMED N,N,N',N'-tetramethylethylenediamine
• RNA pellets were crushed finely and then extracted from the gel with ultrapure water (shaking at room temperature for 6 to 12 hours).
• the structure of the purified oligonucleotide was determined by mass spectrometry using MALDI-TOF MS (Ultraflex III, Bruker Daltonics) (matrix: 3-hydroxypicolinic acid) or analysis using denaturing polyacrylamide gel electrophoresis.
• RNA bands were detected by gel staining (at room temperature for 15 minutes) using SYBR (registered trademark) Green II Nucleic Acid Stain (Lonza) diluted 10,000 times with ultrapure water. : ChemiDoc, BIORAD). The yield of the chemical ligation reaction was calculated by comparing the band intensity of the RNA ligation product using the ligation product isolated and purified by dPAGE as a reference substance.
• RNA ligation product obtained by the chemical ligation reaction was collected as an RNA pellet from the reaction solution by ethanol precipitation (0.3M sodium acetate (pH 5.2)/70% ethanol), and then purified by dPAGE.
• RNA each nucleotide N (capital letter) in Tables 1 to 27-5 below is RNA
• each nucleotide n (lower case) is DNA
• N (M) is 2'-O-methyl modified RNA
• N (F) is 2'-F modified RNA
• N(L) represents LNA
• N(MOE) represents 2'-O-methoxyethyl modified RNA.
• Am6 represents that the base portion is N6-methyladenine.
• DNA may be described as dN.
• p indicates that the 3'-end or 5'-end is phosphorylated.
• ⁇ indicates that the phosphate group connecting the sugar moieties is phosphorothioate.
• the underlined "AUG” represents the start codon
• the underlined "UGA” or "TGA” represents the stop codon.
• Table 1 below shows the sequence information of the compounds (polynucleotides) used in Examples 1 and 2.
• RNA fragment E1-1 (10 nmol and 5' phosphate RNA fragment E1-2 (10 nmol) obtained by solid-phase synthesis
• template DNA1 (10 nmol) in ultrapure aqueous solution (200 ⁇ L, final nucleic acid concentration: 50 ⁇ M ) was prepared in 3 batches.
• 100 ⁇ L of T4 RNA Ligase 2 Reaction Buffer (10X) (New England BioLabs) and 440 ⁇ L of ultrapure water were added, heated at 90°C for 5 minutes, and then incubated for 30 minutes.
• Example 2 (ligation by chemical reaction) 1M A sodium chloride aqueous solution and ultrapure water were added to prepare a 100 mM sodium chloride aqueous solution (180 ⁇ L). After heating the prepared solution at 90° C. for 5 minutes, it was returned to room temperature over 30 minutes. A 100 mM zinc (II) chloride aqueous solution was added to this solution to a final concentration of 5 mM. A 100 mM 1H-imidazole-1-carbonitrile (manufactured by Apollo Scientific)/DMSO solution was added to this solution to a final concentration of 5 mM, mixed, and then left standing on a temperature-controlled heat block (30 ° C., 20 hours).
• RNA ligation product E2 (2.5 nmol, yield 25%).
• Example 3 The structures of rSpacer, dSpacer, Pyrrolidine, Ethynyl-dSpacer, C3, C2, and Spacer9 in Tables 2-1 to 2-60 below are spacer modifications in place of sugar moieties of nucleotides.
• Tables 2-1 to 2-60 below show the sequence information of the compounds (polynucleotides) used in Example 3 and their synthetic methods. Tables 3-1 to 3-14 below show the yield (%) and MS (actual values) of the compound (polynucleotide) of Example 3. Note that MS (actual value) was measured using Agilent Technologies LC (1260 Infinity II)/MSD XT (G6135B). Tables 2-1 to 2-60:
• RNA fragments E217-1, E217-2, E217-3 obtained by solid-phase synthesis, template DNA2 and template DNA3 were used simultaneously, and in the same manner as in Example 1, RNA ligation product E217 (8.9 nmol, yield 45%).
• RNA fragments E218-1, E218-2 and E218-3 obtained by solid-phase synthesis, template DNA2 and template DNA3 were simultaneously used, and in the same manner as in Example 1, RNA ligation product E218 (2.6 nmol, yield 13%).
• RNA fragments E219-1, E219-2, E219-3 obtained by solid-phase synthesis, template DNA2 and template DNA3 were used simultaneously, and in the same manner as in Example 1, RNA ligation product E219 (1.4 nmol, yield 7%).
• Test example 1 Translation reaction test using Hela cell line lysate of mRNA sample
• Tables 5-1 to 5-25 below translation activity in human cell lines was evaluated using 1-Step Human Coupled IVT Kit (manufactured by Thermo Fisher Scientific, Catalog No. 88882).
• each mRNA was diluted with THE RNA storage solution (Thermo Fisher Scientific, Cat. No. AM7001) so that the final concentration of each mRNA was 0.3 ⁇ M. Noted.
• 6*His, His-Tag antibody (Protein Tech Co., catalog number 66005-1-Ig) was diluted to 3 ⁇ g/mL with 0.1M Carbonate buffer (pH 9.4) and applied to a 96-well ELISA plate (Nunc (manufactured by the same company), and allowed to stand overnight at 4°C to prepare a plate on which antibodies were immobilized. Subsequently, the plate was washed with Tris Buffered Saline with Tween 20 (Santa Cruz, Cat. No.
• washing solution diluted with purified water to 1-fold concentration, followed by bovine serum albumin (Wako Pure Pharmaceutical company, catalog number 017-22231) diluted to a final concentration of 3% (hereinafter referred to as blocking solution) was dispensed at 200 ⁇ L per well and allowed to stand at room temperature for 1 hour.
• blocking solution bovine serum albumin (Wako Pure Pharmaceutical company, catalog number 017-22231) diluted to a final concentration of 3%
• HRP Monoclonal ANTI-FLAG M2-Peroxidase Ab produced in mouse (SIGMA, catalog antibody A8592-1MG) diluted 10,000 times with blocking solution was added at 50 ⁇ L per well. Dispensed and allowed to stand at room temperature for 1 hour. After washing the plate with a washing solution, 1-Step Ultra TMB-ELISA (manufactured by Thermo Fisher Scientific, catalog number 34028) was dispensed at 50 ⁇ L per well and allowed to stand at room temperature for several minutes. After that, 50 ⁇ L of 0.5 M sulfuric acid (manufactured by Wako Pure Chemical Industries, Ltd.) was dispensed into each well to stop the reaction.
• 1-Step Ultra TMB-ELISA manufactured by Thermo Fisher Scientific, catalog number 34028
• each mRNA having a sugar modification was added to the HeLa cell lysate, and then converted into a polypeptide encoded by the gene sequence by the translation system of eukaryotic cells. produced.
• Test example 2 In vitro translation reaction test using Hela cell line of mRNA sample
• RPMI medium manufactured by Nacalai Tesque
• fetal bovine serum 10% fetal bovine serum were seeded in a 96-well adherent cell culture plate so that the number of cells per well was 10,000 cells/100 ⁇ L. It was cultured overnight at 37°C and 5% CO2.
• each mRNA having a sugar modification produced a polypeptide encoded by the gene sequence after being added to HeLa cells, and the translation amount Superior to unmodified mRNA.
• Test example 3 In vitro translation reaction test using Hela cell line of mRNA sample, for each mRNA shown in Tables 7-1 to 7-4 below, the persistence of translation activity in vitro was evaluated using the Hela cell line.
• Cell culture and mRNA introduction were performed in the same manner as in Test Example 2, except that the final concentration of each mRNA was adjusted to 30 nM. Remove the culture supernatant from the cells after adding each mRNA and culturing for 4 hours, add 50 ⁇ L per well of RPMI medium (manufactured by Nacalai Tesque) containing 10% fetal bovine serum, and add the cells under 37°C, 5% CO2 conditions. culture was continued.
• RPMI medium manufactured by Nacalai Tesque
• the culture supernatant was removed from the cultured cells, and the cells were lysed in the same manner as in Test Example 2.
• the translation product in the resulting cell lysate was analyzed by the same method as the sandwich ELISA method described in Test Example 1.
• the translation product concentration (nM) in each translation reaction solution quantified using a calibration curve prepared based on the absorbance of the polypeptide standard is shown in Table 7 below.
• each mRNA having a sugar modification produced a polypeptide encoded by the gene sequence after being added to HeLa cells, and the translation amount Superior to unmodified mRNA.
• Test example 4 In vitro translation reaction test using Hela cell line of mRNA sample
• the in vitro translation activity was evaluated using the Hela cell line.
• each mRNA was diluted to 19 ⁇ M with THE RNA Storage Solution (manufactured by Thermo Fisher Scientific, Catalog No. AM7000).
• the Hela cell line was suspended in Opti-MEM I Reduced Serum Media (ThermoFisher Scientific, Catalog No. 31985070) containing bovine serum albumin (Wako Pure Chemical Industries, Ltd., Catalog No. 017-22231) at a final concentration of 1%.
• cells were suspended in RPMI medium (manufactured by Nacalai Tesque) containing 10% fetal bovine serum, and the number of cells per well was 50,000 cells/145 ⁇ L. and cultured at 37°C, 5% CO2. After 3 hours, 8 hours, and 24 hours of culture, the culture supernatant was removed from each of the cells, washed once with ice-cold D-PBS(-) (manufactured by Nacalai Tesque), and treated with 2% protease inhibition.
• RPMI medium manufactured by Nacalai Tesque
• Test example 5 In vitro translation reaction test using human aortic smooth muscle cells for mRNA samples, human aortic smooth muscle cells (Human Aortic Smooth Muscle Cells, Lonza, CC-2571, hereinafter sometimes referred to as hAoSMC) Translation activity in vitro was evaluated. First, hAoSMCs were cultured in SmGM-2 BulletKit medium (Lonza, CC-3182) as described in the manufacturer's manual. /100 ⁇ L of the mixture was seeded in a 96-well adherent cell culture plate and cultured overnight at 37°C under 5% CO2 conditions.
• SmGM-2 BulletKit medium Lionza, CC-3182
• each mRNA having a sugar modification produced a polypeptide encoded by the gene sequence after being added to hAoSMC, and the translation amount superior to mRNAs without
• Test example 6 In vitro translation reaction test using human aortic smooth muscle cells for mRNA samples, for each mRNA shown in Tables 10-1 to 10-5 below, the in vitro translation activity was evaluated using human aortic smooth muscle cells. First, each mRNA was diluted to 19 ⁇ M with THE RNA Storage Solution (manufactured by Thermo Fisher Scientific, Catalog No. AM7000). hAoSMCs were suspended in Opti-MEM I Reduced Serum Media (ThermoFisher Scientific, Cat. No. 31985070) containing bovine serum albumin (Wako Pure Chemical Industries, Ltd., Cat. No. 017-22231) at a final concentration of 1%.
• Opti-MEM I Reduced Serum Media ThermoFisher Scientific, Cat. No. 31985070
• bovine serum albumin Wako Pure Chemical Industries, Ltd., Cat. No. 017-22231
• mRNA having a sugar modification produced a polypeptide encoded by the gene sequence after electroporation into hAoSMC, and its activity was observed in the translation region. superior to mRNA without sugar modifications.
• Test example 7 serum stability test of mRNA sample
• serum nucleic acid stability was evaluated using commercially available mouse serum (Kohjin Bio, Catalog No. 12081001).
• mouse serum was diluted 50-fold with UltraPure DNase/RNase-Free Distilled Water (DW) (Invitrogen, catalog number 10977-015) to prepare a diluted serum solution.
• DW UltraPure DNase/RNase-Free Distilled Water
• Each mRNA was diluted to 5 ⁇ M with THE RNA storage solution (Thermo Fisher Scientific, Catalog No. AM7001).
• 8 ⁇ L of diluted serum solution 10.5 ⁇ L of mixed solution of 2.5 ⁇ L of 6 U/ ⁇ L Ribonuclease Inhibitor (Takara Bio, Catalog No.
• a calibration curve was prepared for each evaluated mRNA, and each mRNA was diluted 4-fold from 4 ⁇ M with THE RNA storage solution to obtain 11-point concentrations, and a dilution series was prepared.
• 2.5 ⁇ L of the sample after the standard curve and enzyme reaction was diluted 1071 times using DW to which Ribonuclease Inhibitor was added at a final concentration of 0.2 U/mL.
• the reaction temperature was 16°C 30 min ⁇ 42°C 30 min ⁇ 85°C 5 min.
• the instrument used was Quantstudio 12K Flex (Applied Biosystems).
• the DNA sequences of the primers and Taqman MGB Probes used are as follows.
• RT primer 5'-TCAGTGGTGGTGGTGGTGGTGTTTG-3' (SEQ ID NO: 431)
• Fw primer 5′-ATCTTGTCGTCGTCGTCCTT-3′
• Rv primer 5'-GAATACAAGCTACTTGTTTCTTTT-3' (SEQ ID NO: 433)
• Taqman MGB Probe 5'-CAGCCACCATG-3' (SEQ ID NO: 434)
• Test example 8 Translation reaction test using Hela cell line lysate of mRNA sample
• Tables 12-1 to 12-7 For each mRNA shown in Tables 12-1 to 12-7 below, translation activity in human cell lines was evaluated using 1-Step Human Coupled IVT Kit (manufactured by Thermo Fisher Scientific, Catalog No. 88882). The translation reaction was carried out in the same manner as in Test Example 1 under the condition that the final mRNA concentration was 1 ⁇ M. The translation product in the reaction solution after the translation reaction was analyzed by the sandwich ELISA method described in Test Example 1, except that the following peptide (manufactured by Cosmo Bio) was used as the translation product polypeptide standard. was carried out.
• Translation product concentration (nM) in each translation reaction solution and relative translation product amount when E30 without sugar modification is 1, quantified using a calibration curve prepared based on the absorbance of the polypeptide standard are listed in Table 12 below.
• each mRNA produced a polypeptide encoded by the gene sequence by the eukaryotic cell translation system after being added to the Hela cell lysate.
• Example 5 Synthesis of mRNA translating VEGF
• the sequence information of the material (polynucleotide) used for the synthesis of mRNA translated into VEGF protein is shown.
• mRNA (VEGF-1, VEGF-2, VEGF-3) was obtained by the following series of operations.
• Step 1 Preparation of linearized plasmid DNA and preparation of RNA fragments by in vitro transcription
• Plasmid DNA used was a commercially available pUC19 vector in which the artificially synthesized gene sequence GN shown in Table 13 was inserted into the EcoRV site and XbaI site (manufactured by Genewith Co., Ltd.). Plasmid DNA was linearized using restriction enzyme XbaI. The final concentration of the reaction solution was 20 ng/ ⁇ L of plasmid DNA, 0.01% BSA, 0.15 U/ ⁇ L of Xba I (Takara 1093A), and 1 ⁇ attachment buffer.
• RNA 5' Pyrophosphohydrolase RppH
• Step 2 Preparation of RNA ligation product by RNA ligation
• N1, N2, N3 Each 5'-terminal polynucleotide fragment (N1, N2, N3) shown in Table 13 obtained by chemical synthesis according to a standard method, a 3'-terminal polynucleotide fragment obtained by in vitro transcription in step 1, and template DNA- 4 was used for ligation with RNA ligase 2.
• the final concentrations are as follows. 5'-end RNA 2 ⁇ M, 3'-end RNA 1 ⁇ M, template DNA 4 ⁇ M, PEG8000 10%, T4 RNA ligase 2 1 U/ ⁇ L (NEB, M0239), 1 x attachment buffer, Murine RNase inhibitor 1 U/ ⁇ L (NEB, M0314).
• Test example 9 Translation reaction of mRNA sample
• Tables 14-1 and 14-2 The mRNA sequence information obtained in Example 5 above is shown in Tables 14-1 and 14-2.
• the in vitro translation activity was evaluated using the Hela cell line.
• Hela cells suspended in RPMI medium manufactured by Nacalai Tesque
• 10% fetal bovine serum were seeded in a 96-well culture plate for adherent cells so that the number of cells per well was 10,000 cells/100 ⁇ L. It was cultured overnight at 37°C under 5% CO2 conditions. After removing the culture supernatant from the cells after overnight culture, 40 ⁇ L per well of RPMI medium containing 10% fetal bovine serum was added, and the final concentrations of each compound were 0.3, 1, 3 and 10 nM.
• Each compound and a final concentration of 0.3% Lipofectamin MessengerMAX Transfection Reagent (manufactured by Thermo Fisher Scientific, catalog number: LMRNA008) were diluted with Optimem (manufactured by Thermo Fisher Scientific, catalog number: 31985-070).
• the mixture was added to each culture plate at 10 ⁇ L per well, and cultured at 37°C, 5% CO2 for 24 hours.
• the culture supernatant was collected from the cells after culturing for 24 hours, and the amount of VEGF protein in the resulting culture supernatant was measured using Human VEGE Quantikine ELISA (manufactured by R&D, catalog number DVE00) according to the manual attached to the kit. .
• the VEGF protein concentration (ng/mL) in each culture supernatant quantified as a result of the measurement is shown in Table 15 below.
• mRNA (IVT-1) was obtained by the following series of operations.
• Step 1 Preparation of linearized DNA
• the plasmid DNA used was a commercially available pUC57 vector in which an artificial synthetic gene sequence GO shown in Table 16 was inserted into the BamHI site and PstI site.
• a PCR reaction was performed using the plasmid DNA as follows. Specifically, plasmid DNA at a final concentration of 250 ng/ ⁇ L, primers P1 and P2 at a final concentration of 250 nM each, and 200 ⁇ L of Primestar MAX (manufactured by Takara Bio Inc., Catalog No. R045B) were mixed and dissolved in Nuclease-free water.
• Primestar MAX manufactured by Takara Bio Inc., Catalog No. R045B
• the resulting reaction solution was electrophoresed on a 3.0% agarose-TAE gel, the relevant band was excised, and the PCR product was purified using NucleoSpin Gel and PCR Clean-up Midi (manufactured by MACHEREY-NAGEL, catalog number 740986.20). Chloroform extraction and ethanol precipitation were performed to obtain PCR DNA.
• Step 2 Preparation of RNA fragment by in vitro transcription
• a transcription reaction was performed using the obtained PCR DNA.
• the final concentration of the reaction solution is as follows: PCR DNA 4 ng/ ⁇ L, ATP, CTP, UTP, GTP each 9 mM, T7 Enzyme 10% attached to MEGAScript T7 Transcription Kit (manufactured by Invitrogen, catalog number AMB13345), MEGAScript T7 10% T7 Reaction Buffer supplied with the Transcription Kit.
• the reaction volume was 400 ⁇ L, and incubation was performed at 37° C. for 6 hours.
• Trubo DNase attached to the MEGAScript T7 Transcription Kit was added in an amount of 1/20 vol of the reaction volume, mixed, and shaken at 37°C for 15 minutes. Crude purification was performed by phenol-chloroform extraction and ethanol precipitation to obtain an RNA fragment. The resulting RNA fragment was capped using the Vaccinia Capping System (manufactured by New England Biolab, catalog number MB2080S) and ScriptCap 2'-O-Methyltransferase Kit (manufactured by CELLSCRIPT, catalog number C-SCMT0625) as follows. reacted.
• the final concentration of the reaction solution is as follows: 500 ng/ ⁇ L RNA fragment, 10% Capping Buffer, 0.5 mM GTP, 0.2 mM SAM, 0.5 U/ ⁇ L Vaccinia capping enzyme, 1 U/ ⁇ L RNase inhibitor, 2.5 U/ ⁇ L 2'-O-Methyltransferase.
• the reaction volume was adjusted to 2000 ⁇ L with Nuclease-free water and allowed to stand at 37°C for 1 hour. Purification was performed by phenol-chloroform extraction and ethanol precipitation to obtain a capped RNA fragment.
• Each nucleotide N (capital letter) in Table 17 represents RNA, N(M) represents 2'-O-methyl modified RNA, and m7Gppp represents the following structural formula.
• Test example 10 Translation reaction test using Hela cell line lysate of mRNA sample
• Tables 18-1 to 18-10 below translation activity in human cell lines was evaluated by the same method as in Test Example 1.
• the translation product concentration (nM) in the translation reaction solution to which 0.3 ⁇ M of each mRNA was added is shown in Table 18 below.
• each mRNA having a sugar modification was added to the HeLa cell lysate, and then converted into a polypeptide encoded by the gene sequence by the translation system of eukaryotic cells. produced.
• Test example 11 In vitro translation reaction test using Hela cell line of mRNA sample
• Tables 19-1 to 19-13 below In vitro translation activity was evaluated using the human Hela cell line in the same manner as in Test Example 2.
• concentrations (nM) of translation products in cell lysates obtained from the cells 5 hours after addition of 3 to 30 nM of each mRNA are shown in Table 19 below.
• each mRNA having a sugar modification produced a polypeptide encoded by the gene sequence after being added to HeLa cells, and the translation amount was Superior to unmodified mRNA.
• Test example 12 In vitro translation reaction test using Hela cell line of mRNA sample, for each mRNA shown in Tables 20-1 to 20-8 below, the persistence of translational activity in vitro was evaluated using the HeLa cell line in the same manner as in Test Example 3. Translation product concentrations (nM) in cell lysates obtained from cells to which 30 nM of each mRNA was added are shown in Table 20 below.
• each mRNA having a sugar modification produced a polypeptide encoded by the gene sequence after being added to HeLa cells, and the translation amount Superior to unmodified mRNA.
• Test example 13 In vitro translation reaction test using Hela cell line of mRNA sample
• Tables 21-1 to 21-4 below the in vitro translation activity was evaluated using the Hela cell line in the same manner as in Test Example 4.
• Translation product concentrations (nM) in cell lysates obtained from cells to which each mRNA was added are shown in Table 21 below.
• Test example 14 Translation reaction test using Hela cell line lysate of mRNA sample
• Tables 22-1 and 22-2 below translation activity in human cell lines was evaluated by the same method as in Test Example 8.
• Table 22 below shows the translation product concentration (nM) in the translation reaction solution to which 1 ⁇ M of each mRNA was added.
• each mRNA produced a polypeptide encoded by the gene sequence by the eukaryotic cell translation system after being added to the Hela cell lysate.
• Test example 15 Intracellular nucleic acid stability test using Hela cell line for mRNA samples, for each mRNA shown in Table 23 below, the intracellular nucleic acid stability was evaluated using the HeLa cell line.
• Cell culture and mRNA introduction were carried out in the same manner as in Test Example 3, with the final concentration of each mRNA adjusted to 30 nM. Remove the culture supernatant from the cells after adding each mRNA and culturing for 4 hours, add 50 ⁇ L per well of RPMI medium (manufactured by Nacalai Tesque) containing 10% fetal bovine serum, and add the cells under 37°C, 5% CO2 conditions. culture was continued.
• RPMI medium manufactured by Nacalai Tesque
• the cells were lysed as follows. Specifically, after removing the culture supernatant from the cells, after washing once with ice-cold D-PBS(-) (manufactured by Nacalai Tesque), 2% protease inhibitor cocktail (for animal cell extracts) 20 ⁇ L of iScript RT-qPCR Sample Preparation Reagent (Bio-Rad, 1708898) containing iScript RT-qPCR Sample Preparation Reagent (Bio-Rad, 1708898) was added per well, and vigorously permeated for 30 seconds to lyse the cells.
• DW was prepared by adding Ribonuclease Inhibitor at a final concentration of 0.2 U/mL as a sample dilution solution.
• a calibration curve was prepared for each evaluated mRNA, and each mRNA was diluted with a cell lysate prepared from nucleic acid-free cells diluted 10-fold with a sample diluent to obtain 11-point concentrations from 1 ⁇ M to 4-fold dilutions. was taken to prepare a dilution series. Each cell lysate to be measured was diluted 10-fold with a specimen diluent.
• the sugar-modified mRNA has improved degradation resistance in cells compared to the mRNA prepared by the IVT method.
• Test example 16 Translation reaction test using Hela cell line lysate of mRNA sample
• Table 24 shows the translation product concentration (nM) in the translation reaction solution to which 0.3 ⁇ M of each mRNA was added.
• each mRNA having a sugar modification produced a polypeptide encoded by the gene sequence by the eukaryotic cell translation system after being added to the Hela cell lysate.
• Test example 17 In vitro translation reaction test using Hela cell line of mRNA sample
• Table 25 shows the concentration (nM) of the translation product in the cell lysate obtained from the cells 5 hours after addition of 3 to 30 nM of each mRNA.
• E222 having a sugar-modified poly A chain length of 5 E223 having a sugar-modified poly A chain length of 10, E65, E224 and E225 having a sugar-modified poly A chain length of 20, sugar-modified poly A E59, which has a chain length of 40, showed superior translating ability compared to mRNAs with unmodified polyA chains of the same length, respectively.
• Test example 18 In vitro translation reaction test using Hela cell line of mRNA sample, for each mRNA shown in Table 26 below, the persistence of translational activity in vitro was evaluated using the Hela cell line in the same manner as in Test Example 3. Translation product concentrations (nM) in cell lysates obtained from cells to which 30 nM of each mRNA was added are shown in Table 26 below.
• each mRNA having a sugar modification produced a polypeptide encoded by the gene sequence after being added to Hela cells, and the translation amount of the sugar increased depending on the length of the poly A chain. Superior to unmodified mRNA.
• Test example 19 Translation reaction test using Hela cell line lysate of mRNA sample
• Tables 27-1 to 27-5 below show the translation product concentration (nM) in the translation reaction solution to which 1 ⁇ M mRNA was added, and the relative translation product amount when E226 or E30, which is mRNA without sugar modification, is set to 1. -5 described.
• each mRNA produced a polypeptide encoded by the gene sequence by the translation system of eukaryotic cells after being added to the HeLa cell lysate, and the translation thereof Quantities were superior to mRNAs without sugar modifications.

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