WO2022086140A1 - 5'-캡핑된 rna 합성용 올리고뉴클레오티드 - Google Patents
5'-캡핑된 rna 합성용 올리고뉴클레오티드 Download PDFInfo
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- WO2022086140A1 WO2022086140A1 PCT/KR2021/014625 KR2021014625W WO2022086140A1 WO 2022086140 A1 WO2022086140 A1 WO 2022086140A1 KR 2021014625 W KR2021014625 W KR 2021014625W WO 2022086140 A1 WO2022086140 A1 WO 2022086140A1
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- alkyl
- hydroxy
- oxy
- phosphoryl
- rna
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Images
Classifications
-
- 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
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- 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
- C07H21/02—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
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/67—General methods for enhancing the expression
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
Definitions
- the present invention relates to novel oligonucleotides used in the synthesis of 5'-capped RNA.
- 5'-capping of RNA is the first step of pre-mRNA processing in eukaryotic cells, and is a process in which a cap is placed on the 5' end of the transcribed RNA.
- a 5' cap is formed in which 7-methylguanosine (m 7 G) is linked through a 5' triphosphate bond, which is methyltransferase enzyme in vivo.
- eukaryotic mRNA including methylation of the 2' hydroxyl group of the first ribose sugar at the 5' end (cap-1) and methylation of the 2' hydroxyl group of the second ribose sugar (cap-2).
- Cap-1 methylation of the 2' hydroxyl group of the first ribose sugar at the 5' end
- cap-22 methylation of the 2' hydroxyl group of the second ribose sugar
- capped mRNA encoding a specific gene can be artificially expressed by transfecting eukaryotes or microinjecting them into cells or embryos.
- RNA is not only rapidly degraded, but also has immunogenicity, and protein translation efficiency is sharply reduced. Therefore, it is very important that 5' capping is properly performed to synthesize mRNA in vitro.
- RNA synthesis it has been found that the process of first in vitro transcription of RNA having a 5'-triphosphate group and then performing 5' capping using a capping enzyme is not only expensive but also inefficient. Accordingly, a synthetic method was developed for preparing an oligonucleotide having a 5'-capping structure and using it as a primer to initiate in vitro transcription.
- WO2008/016473 and WO2013/059475 disclose dinucleotide mRNA cap analogs for synthesizing 5'-capped RNA, and WO2017/053297 synthesizing 5'-capped RNA Trinucleotide mRNA cap analogs for
- RNA cap analogs that enable large-scale synthesis of RNA.
- RNA therapeutics or vaccines are provided, if a cap structure that can maintain mRNA stability in vivo, improve translation efficiency, or reduce side effects such as immunogenicity when applied as an actual RNA therapeutic agent or vaccine is provided, universal utility in the field of RNA therapeutics or vaccines is achieved it could be
- Another object of the present invention is to provide an RNA molecule prepared using the oligonucleotide primer for RNA capping and uses thereof.
- the present inventors provide a novel oligonucleotide primer for RNA capping, a method for preparing the same, and a method for using the same in order to achieve the above object.
- all terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. All patent and non-patent publications mentioned throughout this disclosure are incorporated by reference in their entirety.
- the present invention provides a compound represented by the following formula (1), a stereoisomer thereof, or a salt thereof:
- B 1 and B 2 are each independently a natural, unnatural or modified nucleoside base
- X 1 is —OH, —O(C 1-4 alkyl), or —halo
- X 2 is -H or X 1 combined to form an LNA ring ⁇ wherein at least one H of the LNA ring is -(C 1-4 alkyl), -OH, or -O(C 1-4 alkyl) may be substituted with ⁇ ;
- Y 1 is —O(C 1-4 alkyl), —O(C 1-4 alkyl)O(C 1-4 alkyl), —CH 2 O(C 1-4 alkyl), or -halo;
- Y 2 is -H or Y 1 combined to form an LNA ring ⁇ wherein at least one H of the LNA ring is -(C 1-4 alkyl), -OH, or -O(C 1-4 alkyl) may be substituted with ⁇ ;
- Z 1 and Z 2 are each independently -OH, -O(C 1-4 alkyl), -O(C 1-4 alkyl)O(C 1-4 alkyl), -CH 2 O(C 1-4 alkyl) ), or -halo;
- Z 3 is -H or Z 1 combined to form an LNA ring ⁇ wherein at least one H of the LNA ring is -(C 1-4 alkyl), -OH, or -O(C 1-4 alkyl) may be substituted with ⁇ ;
- n is an integer from 0 to 3;
- n is an integer from 1 to 10 ⁇ wherein, when m is 2 or more, each B 2 , Z 1 , and Z 2 may be different from each other ⁇ ;
- R 1 and R 2 are each independently —H or —(C 1-4 alkyl);
- R 3 is -(C 1-4 alkyl).
- nucleoside base not only natural nucleoside bases but also non-natural or modified nucleoside bases can be used.
- the base rings most commonly found in naturally occurring nucleosides are the purine and pyrimidine rings.
- the naturally occurring purine ring is, for example, adenine ( ), guanine ( ), and N 6 -methyladenine ( ) is included.
- the naturally occurring pyrimidine ring is, for example, cytosine ( ), thymine ( ), 5-methylcytosine ( ), uracil ( ) is included.
- the unnatural or modified nucleoside base is one of the natural NTPs (eg ATP, UTP, CTP and GTP) or an artificial artificial recognizable by RNA polymerase as a replacement for another specific NTP. It may be a base derived from a nucleoside analog with a base (Loakes, D., Nucleic Acids Res., 29:2437-2447 (2001); Crey-Desbiolles, C., et al., Nucleic Acids Res., 33:1532-1543 (2005); Kincaid, K., et al., Nucleic Acids Res., 33:2620-2628 (2005); Preparata, FP, Oliver, JS, J. Comput. Biol. 753 -765 (2004); and Hill, F., et al., Proc Natl Acad. Sci. U S A, 95:4258-4263 (1998), et al.).
- NTPs eg ATP, UTP, CTP and
- the unnatural or modified nucleoside base is a halogen-substituted purine (eg, 6-fluoropurine), a halogen-substituted pyrimidine, N 6 -ethyladenine, N 4 - It may be a base derived from (alkyl)-cytosine, 5-ethylcytosine, etc., but is not limited thereto.
- B 1 and B 2 are each independently , , , , , or can be
- X 1 is —OH
- X 2 may be -H.
- Y 1 is —O(C 1-4 alkyl) or —O(C 1-4 alkyl)O(C 1-4 alkyl), or —halo;
- Y 2 may be combined with -H or Y 1 to form an LNA ring ⁇ here, at least one H of the LNA ring may be substituted with -(C 1-4 alkyl) ⁇ .
- Z 1 is —OH or —O(C 1-4 alkyl);
- Z 2 is —OH
- Z 3 may be -H.
- n may be 0 or 1.
- m may be 1.
- R 1 and R 2 are each independently —H
- R 3 may be -(C 1-4 alkyl).
- the compound represented by Formula 1 may be any one selected from the group consisting of the following compounds:
- the compound represented by Formula 1 may be at least one selected from the group consisting of the following compounds:
- the compound represented by Formula 1 may be an oligonucleotide primer for RNA capping.
- stereoisomer means a compound of the present invention or a salt thereof having the same chemical formula or molecular formula but sterically different. Each of these stereoisomers and mixtures thereof are also included within the scope of the present invention. Also, unless otherwise noted, a solid bond (-) connecting an asymmetric carbon atom is wedge-shaped indicating the absolute configuration of the stereocenter. or wedge-dotted join may include
- salt refers to a salt commonly used in the pharmaceutical industry. Specifically, it may be a base addition salt.
- inorganic ionic salts made of, for example, calcium, potassium, sodium or magnesium; amino acid salts prepared from arginine, lysine, histidine and the like; and amine salts prepared from trimethylamine, triethylamine, ammonia, pyridine, picoline, and the like, but the types of salts in the present invention are not limited by these listed salts.
- oligonucleotide primer for RNA capping may be used to prepare an RNA molecule comprising the same. Accordingly, the present invention provides an RNA molecule comprising an oligonucleotide primer for RNA capping represented by the formula (1).
- the oligonucleotide primer for RNA capping according to the present invention can be extended by RNA polymerase through incorporation of NTP onto the 3'-end.
- In vitro transcription can be initiated under the control of a promoter in a transcription system containing essential components such as a DNA template (eg DNA plasmid), RNA polymerase, nucleoside 5'-triphosphate and an appropriate buffer.
- the oligonucleotide primer is complementary to the DNA template at the initiation site.
- a promoter refers to a specific region of a dsDNA template that induces and controls the initiation of transcription of a specific DNA sequence (eg, a gene).
- the promoter is located on the same strand and upstream (toward the 5' region of the sense strand) on the DNA.
- a promoter is typically immediately adjacent to, or partially overlaps with, the DNA sequence to be transcribed. Nucleotide positions within the promoter are designed relative to the transcriptional start site at which transcription of the DNA begins (position +1).
- the initiating oligonucleotide primer is complementary to the initiation site of the promoter sequence, which, in certain embodiments, is at positions +1 and +2 and, in the case of an initiating tetramer, at positions +1, +2 and +3 .
- RNA molecules thus formed include, but are not limited to, mRNA, small nuclear RNA (snRNA), small nucleolar RNA (snoRNA), and small casal body-specific RNA (scaRNA).
- snRNA small nuclear RNA
- snoRNA small nucleolar RNA
- scaRNA small casal body-specific RNA
- the RNA molecule is mRNA and may include one or more coding sequences (CDS). Additionally, the RNA molecule may comprise a polyA sequence and/or a polyadenylation signal.
- the polyA sequence may consist entirely or predominantly of adenine nucleotides or analogs or derivatives thereof.
- the polyA sequence may be a tail located adjacent to the 3' untranslated region of the nucleic acid.
- the present invention also comprises the steps of (S-1) mixing a DNA template, an oligonucleotide primer for RNA capping represented by Chemical Formula 1 and RNA polymerase; and (S-2) incubating the mixture to perform transcription of the polynucleotide template.
- RNA molecules comprising the oligonucleotide primer for RNA capping represented by Formula 1 provided in the present invention may be used to synthesize an RNA molecule comprising the same.
- the DNA template may be double-stranded linear DNA, partially double-stranded linear DNA, circular double-stranded DNA, DNA plasmid, PCR amplicon, in addition to modified modifications capable of reacting appropriately with RNA polymerase It may be a nucleic acid.
- the synthesis of the RNA molecule is performed in vitro.
- single subunit phage polymerases derived from T7, T3, SP6, K1-5, K1E, K1F or K11 bacteriophages can be used.
- This class of polymerases has a simple minimal promoter sequence of -17 nucleotides with minimal constraints of the initiating nucleotide sequence without the need for helper proteins.
- T7 RNA polymerase T7 RNA polymerase
- T7 RNAP T7 RNA polymerase
- T7 RNAP initiates RNA synthesis in the absence of primers.
- the first step in initiation is called de novo RNA synthesis, in which RNA polymerase recognizes a specific sequence on a DNA template and a first pair of nucleotide triphosphates complementary to the template residues at positions +1 and +2. , and catalyzes the formation of a phosphodiester bond to form a dinucleotide.
- the 5'-capped RNA molecule according to the present invention as described above can be usefully utilized for medicinal purposes.
- the 5'-capped RNA molecule prepared according to the present invention can be utilized as a nucleic acid therapeutic agent or vaccine.
- the nucleic acid therapeutic agent or vaccine may be used as an RNA vaccine (a vaccine for preventing cancer or infectious disease).
- the nucleic acid therapeutic or vaccine can be administered to a subject and translated in vivo to produce a desired peptide.
- the 5'-capped RNA molecule can be introduced into a cell to produce a protein capable of treating a medical condition of the cell or having a therapeutic effect on the cell.
- the nucleic acid therapeutic agent or vaccine may further include a carrier capable of introducing the RNA molecule into a target cell together with the RNA molecule.
- the present invention provides a peptide translated from the above-described 5'-capped RNA molecule.
- the present invention provides a cell into which the above-described 5'-capped RNA molecule is introduced.
- the cell may be a somatic cell of a subject, or a cell line that can be cultured in vitro.
- the present invention provides a peptide produced in a cell into which a 5'-capped RNA molecule has been introduced.
- compositions for RNA therapeutics or vaccines may be formulated for administration by injection, or by other suitable routes known to those skilled in the art for treating a particular condition.
- injectable compositions for parenteral administration typically contain the active compound in suitable solutions and/or pharmaceutical carriers such as sterile physiological saline.
- the compositions may also be formulated as suspensions in lipids or phospholipids, liposome suspensions, or aqueous emulsions.
- compositions and/or formulations for RNA therapeutics or vaccines are known to those skilled in the art (see Remington's Pharmaceutical Sciences (19 th Ed., Williams & Wilkins, 1995)).
- the composition to be administered will contain a specified amount of the compound selected in an amount that is pharmaceutically safe and effective to increase expression of the protein of interest in the target cell or tissue.
- the pharmaceutical composition contains at least 0.1% (w/v) of a compound as described above, in some embodiments, the pharmaceutical composition contains greater than 0.1% of the compound, and in some embodiments, the pharmaceutical composition comprises: It contains about 10% or less, in some embodiments, the pharmaceutical composition contains about 5% or less, and in some embodiments, the pharmaceutical composition contains about 1% (w/v) or less.
- the choice of suitable concentration depends on factors such as the desired dose, frequency, and method of delivery of the active agent.
- the dosage When treating a subject, such as a mammal or human, the dosage is determined based on factors such as the subject's weight and general health, the condition being treated, the severity of symptoms, and the like. Determine the dosage and concentration that will produce the desired benefit, while avoiding any undesirable side effects.
- a typical dosage of a subject compound is in the range of about 0.0005 to 500 mg/day for human patients, and in some embodiments is in the range of about 1 to 100 mg/day.
- higher dose regimens include, for example, 50 to 100, 75 to 100, or 50 to 75 mg/day
- lower dosage regimens include, for example, 1 to 50, 25 to 50, or 1 to 25 mg/day.
- the novel oligonucleotide according to the present invention is utilized for the synthesis of 5'-capped RNA to improve the RNA production process (eg, synthesis yield, synthesis scale, purity, etc.), and the efficacy of a nucleic acid therapeutic or vaccine using the same (eg, , RNA stability and / or protein expression efficiency) improvement and side effects reduction (eg, immunogenicity reduction) effect. Accordingly, the present invention can be usefully applied in the field of nucleic acid therapeutics or vaccines.
- the mixture was diluted with DCM, and a 10% sodium thiosulfate pentahydrate solution was added thereto, and the organic layer and the aqueous layer were separated.
- the organic layer was dehydrated with Na 2 SO 4 , concentrated under reduced pressure, and separated by column chromatography to obtain the title compound (5.78 g) as a yellow solid.
- Step 3 ((2R,3R,4R,5R)-3-(((((2R,3S,4R,5R)-5-(2-amino-6-oxo-1,6-dihydro-9H-) Purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(hydroxy)phosphoryl)oxy)-5-(6-amino-9H-purin-9-yl) -4-Fluorotetrahydrofuran-2-yl)methyl dihydrogen phosphate.
- 2-triethylamine salt 2-triethylamine salt
- reaction solution was cooled to 0 ⁇ 5 °C, aqueous ammonia (42.2 mL, 1083 mmol) was slowly added thereto. After slowly raising the temperature to room temperature, it was heated to 50 ⁇ 55 °C for 23 hours. The reaction solution was washed with DCM (2 times with 150 mL), and the aqueous layer was purified with DEAE Sephadex resin to obtain the title compound (1.98 g) as a white solid.
- reaction solution was cooled to -10 ⁇ -20 °C, and 0.1M sodium percholate in acetone (108 mL, 10.83 mmol) was added dropwise.
- acetone 108 mL, 10.83 mmol
- the resulting solid was filtered and dried to obtain the title compound (2.33 g) as a white solid.
- Example 1 2-Amino-9-((2R,3R,4S,5R)-5-(((((((((((2R,3R,4R,5R)-3-(((((2R) ,3S,4R,5R)-5-(2-amino-6-oxo-1,6-dihydro-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl) Methoxy)(hydroxy)phosphoyl)oxy)-5-(6-amino-9H-purin-9-yl)-4-fluorotetrahydrofuran-2-yl)methoxy)(hydroxy)phosphoyl )oxy)(hydroxy)phosphoyl)oxy)(hydroxy)phosphoyl)oxy)(hydroxy)phosphoyl)oxy)methyl)-3,4-dihydroxytetrahydrofuran-2-yl)-7-methyl-6-oxo-6, Preparation of 9-dihydro-1H-purine-7-ium.
- Example 2 2-Amino-9-((2R,3R,4S,5R)-5-((((((((((((2R,3R,4R,5R)-3-(((((2R) ,3S,4R,5R)-5-(2-amino-6-oxo-1,6-dihydro-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl) Methoxy)(hydroxy)phosphoryl)oxy)-5-(6-amino-9H-purin-9-yl)-4-(2-methoxyethoxy)tetrahydrofuran-2-yl)methoxy) (hydroxy)phosphoryl)oxy)(hydroxy)phosphoryl)oxy)(hydroxy)phosphoryl)oxy)methyl)-3,4-dihydroxytetrahydrofuran-2-yl)-7-methyl- Preparation of 6-oxo-6,9-dihydro-1H-purine-7-ium.3-trieth
- Example 7 2-Amino-9-((2S,3S,4R,5S)-5-((((((((((((2R,3R,4R,5R)-3-(((((2R) ,3S,4R,5R)-5-(5-amino-7-oxo-6,7-dihydro-3H-[1,2,3]triazolo[4,5-d]pyrimidin-3-yl )-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(hydroxy)phosphoryl)oxy)-5-(6-amino-9H-purin-9-yl)-4-fluoro Tetrahydrofuran-2-yl)methoxy)(hydroxy)phosphoryl)oxy)(hydroxy)phosphoryl)oxy)hydroxy)phosphoryl)oxy)methyl)-3-hydroxy-4-methoxytetra Preparation of hydrofuran-2-yl)-7-methyl-6-oxo-6,9-dihydro-1H-purin
- Example 8 2-Amino-9-((2S,3S,4R,5S)-5-(((((((((((2R,3R,4R,5R)-3-(((((2R) ,3S,4R,5R)-5-(5-amino-7-oxo-6,7-dihydro-3H-[1,2,3]triazolo[4,5-d]pyrimidin-3-yl )-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(hydroxy)phosphoryl)oxy)-5-(6-amino-9H-purin-9-yl)-4-fluoro Tetrahydrofuran-2-yl)methoxy)(hydroxy)phosphoryl)oxy)(hydroxy)phosphoryl)oxy)hydroxy)phosphoryl)oxy)methyl)-3,4-dihydroxytetrahydrofuran Preparation of -2-yl)-7-methyl-6-oxo-6,9-dihydro-1H-purine
- Example 10 2-Amino-9-((2S,3S,4R,5S)-5-(((((((((((2R,3R,4R,5R)-5-(2-amino-6) -oxo-1,6-dihydro-9H-purin-9-yl)-3-((((2R,3S,4R,5R)-5-(2-amino-6-oxo-1,6- Dihydro-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(hydroxy)phosphoryl)oxy)-4-fluorotetrahydrofuran-2- yl)methoxy)(hydroxy)phosphoryl)oxy)(hydroxy)phosphoryl)oxy)hydroxy)phosphoryl)oxy)methyl)-3-hydroxy-4-methoxytetrahydrofuran-2-yl Preparation of )-7-methyl-6-oxo-6,9-dihydro-1H-purine-7-ium.3-
- Example 11 2-amino-9-((2S,3S,4R,5S)-5-(((((((((((2R,3R,4R,5R)-5-(2-amino-6) -oxo-1,6-dihydro-9H-purin-9-yl)-3-((((2R,3S,4R,5R)-5-(2-amino-6-oxo-1,6- Dihydro-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(hydroxy)phosphoryl)oxy)-4-fluorotetrahydrofuran-2- yl)methoxy)(hydroxy)phosphoryl)oxy)(hydroxy)phosphoryl)oxy)hydroxy)phosphoryl)oxy)methyl)-3,4-dihydroxytetrahydrofuran-2-yl)- Preparation of 7-methyl-6-oxo-6,9-dihydro-1H-purin-7-ium.3-tri
- Example 12 2-Amino-9-((2S,3S,4R,5S)-5-((((((((2R,3R,4R,5R)-3-(((((2R,3S) ,4R,5R)-5-(2-amino-6-oxo-1,6-dihydro-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy )(hydroxy)phosphoryl)oxy)-5-(6-amino-9H-purin-9-yl)-4-fluorotetrahydrofuran-2-yl)methoxy)(hydroxy)phosphoryl)oxy )(hydroxy)phosphoryl)oxy)methyl)-3,4-dihydroxytetrahydrofuran-2-yl)-7-methyl-6-oxo-6,9-dihydro-1H-purine-7- Ium.3 Preparation of triethylamine salt (oligonucleotide 12)
- plasmid gene synthesis having T7 RNA promoter, 5'UTR, EGFP, 3'UTR, and poly A sequences was performed from Bionics. Then, in order to change the promoter sequence according to the cap material, PCR (polymerase After chain reaction), a plasmid gene was prepared through cloning using XbaI (Takara catalog # 1093A) and BamHI (Takara catalog # 1010A) restriction enzymes in the existing plasmid gene.
- a GFP DNA transcription template (SEQ ID NO: 3) was obtained using the AccuPrep PCR purification kit (Bioneer catalog # K-3037).
- the transcription reaction mixture was incubated at 37 °C for 5 hours. To the reaction was added 40 mM Tris.HCl (pH 7.5), 8 mM magnesium chloride, 5 mM DTT and 5000 U/mL recombinant DNase I (Takara Cat. #2270A), and incubated at 37° C. for 1 hour. The resulting mRNA was purified using the Zymoly Research RNA clean & concentrator-25 kit (catalog # 1017) according to the manufacturer's instructions.
- mRNA was synthesized was confirmed by electrophoresis using a 1% agarose gel to which Lonza GelStarTM Nucleic Acid gel Stain (catalog #5 0535) was added (FIG. 1). UV analysis of the synthesized mRNA was measured using a Thermo Scientific Nanodrop one UV-vis spectrometer. As a result, it was confirmed that GFP mRNA containing poly A (120) of about 1066 nt was generated.
- the transcription reaction mixture was incubated at 37 °C for 5 hours. To the reaction was added 40 mM Tris.HCl (pH 7.5), 8 mM magnesium chloride, 5 mM DTT and 5000 U/mL recombinant DNase I (Takara Cat. #2270A), and incubated at 37° C. for 1 hour. The resulting mRNA was purified using the Zymoly Research RNA clean & concentrator-25 kit (catalog # 1017) according to the manufacturer's instructions.
- mRNA was synthesized was confirmed by electrophoresis using a 1% agarose gel to which Lonza GelStarTM Nucleic Acid gel Stain (catalog #5 0535) was added (FIG. 1). UV analysis of the synthesized mRNA was measured using a Thermo Scientific Nanodrop one UV-vis spectrometer. As a result, it was confirmed that the dimeric ARCA capping GFP mRNA containing poly A (120) of about 1067 nt was generated.
- Tris HCl (pH 7.9), 25 mM magnesium chloride, 2 mM spermidine, 2 mM ATP, 2 mM UTP, 2 mM CTP, 2 mM GTP, 3.2 mM
- Example compound 5% DMSO (Sigma-Aldrich catalog #472301-500ML), 5 mg/mL GFP DNA transcription template, 800 U/mL recombinant RNase inhibitory protein (Takara catalog #2316A), 2 U/mL yeast inorganic fatigue Phosphatase (Thermo Scientific catalog # EF0221) and 2500 U/mL T7 RNA polymerase (Dynbio catalog # dy1670) were mixed.
- the transcription reaction mixture was incubated at 37 °C for 5 hours. To the reaction was added 40 mM Tris.HCl (pH 7.5), 8 mM magnesium chloride, 5 mM DTT and 5000 U/mL recombinant DNase I (Takara Cat. #2270A), and incubated at 37° C. for 1 hour. The resulting mRNA was purified using the Zymoly Research RNA clean & concentrator-25 kit (catalog # 1017) according to the manufacturer's instructions.
- mRNA was synthesized was confirmed by electrophoresis using a 1% agarose gel to which Lonza GelStarTM Nucleic Acid gel Stain (catalog #5 0535) was added (FIG. 1). UV analysis of the synthesized mRNA was measured using a Thermo Scientific Nanodrop one UV-vis spectrometer. As a result, it was confirmed that the example capping GFP mRNA containing poly A (120) of about 1067 nt was generated.
- Hela human cervix cancer cell line
- Hela cells were cultured in DMEM supplemented with 10% FBS and 1% penicillin/streptomycin at 37° C. under an atmosphere of 5% CO 2 . 1 ⁇ 10 6 cells/well of Hela cells were plated in 6-well plates. The next day, cells were transfected using transfection reagent (messengerMAX lipofectamine; Invitrogen catalog # LMRNA003): in 125 ⁇ L of complex medium (Opti-MEM; Life technologies) in tube A, according to the transfection reagent manufacturer's instructions. Dilute 7.5 ⁇ L of transfection reagent and incubate at room temperature for 10 min.
- transfection reagent messengerMAX lipofectamine
- Opti-MEM complex medium
- tube B prepare by diluting 5 ⁇ g of mRNA prepared in 125 ⁇ L of Opti-MEM.
- the solutions of tube A and tube B were mixed and incubated for 5 minutes at room temperature.
- the cell culture medium was replaced with DMEM containing 10% FBS without penicillin/streptomycin.
- the cells were transfected using the incubated mixed solution.
- 5% CO 2 After incubation at 37 ° C. for 3 to 4 hours under an atmosphere, the cell culture medium was replaced with DMEM supplemented with 10% FBS and 1% penicillin/streptomycin. After 96 hours of transfection, the expression of the fluorescent protein was confirmed using a fluorescence microscope.
- mRNA prepared using the example compounds showed excellent fluorescence activity.
- mRNA without cap as a control showed no fluorescence activity.
- the mRNA prepared using the Example compounds all showed excellent protein expression rates (FIG. 2).
- the culture medium was removed and washed once with PBS. After treatment with 200 ⁇ L of cell lysis buffer (RIPA + phosphatase inhibitor + PMSF), the cells were lysed at 4 °C for 10 minutes. After recovering the cell lysate, centrifugation was performed at 12000 rpm, 4° C., for 10 minutes. After recovering the supernatant, it was mixed with 5x SDS-sample loading buffer and then boiled at 100° C. for 5 to 10 minutes. Running at 120V, 10 minutes, 170V, 1 hour on 10% SDS PAGE-gel. Transfer was performed at 30V for 1 hour using Xcell 2 blot Module (Invitrogen).
- cell lysis buffer (RIPA + phosphatase inhibitor + PMSF)
- Blocking was performed for 1 hour at room temperature using 5% BSA-PBST.
- the rabbit anti-GFP antibody was diluted 1:1000 in 5% BSA-PBST, and then reacted at 4°C overnight. Then, it was washed 3 times with PBST for 10 minutes.
- HRP-conjugated rabbit secondary antibody was diluted 1:10000 in 2.5% BSA-PBST and then treated at room temperature for 1 hour. Then, it was washed 3 times for 10 minutes with PBST. After ECL treatment, it was confirmed by chemiluminescence and the expression level of GFP compared to ⁇ -actin was analyzed (Table 1).
- the culture medium was removed and washed once with PBS. After trypsin-EDTA treatment, it was incubated for about 2-3 minutes at 37 °C until the cells started to fall off. After neutralization with a culture medium (HG DMEM + 10% FBS + 1% penicillin/streptomycin), it was centrifuged for 2 minutes at 1200 rpm at room temperature. After removing the supernatant, 1 mL of PBS was dispensed, and the clumped cells were released by pipetting. GFP signal (FITC) was measured by FACS (BD FACSDiva 8.0.3) (FSC, SSC confirmation, Table 2).
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Abstract
Description
Control | Non cap | ARCA | 실시예 1 | 실시예 6 | 실시예 7 | |
상대값 | 1.0 | 1.11 | 4.09 | 8.67 | 8.30 | 4.51 |
Materials | % parent-P3 | Relative |
Negative | 0.4 | 1.0 |
Non cap | 4.9 | 12.3 |
실시예 1 | 35.6 | 89.0 |
실시예 6 | 23.2 | 58 |
실시예 7 | 24.3 | 60.8 |
실시예 8 | 12.2 | 30.5 |
ARCA | 9.4 | 23.5 |
Claims (24)
- 하기 화학식 1로 표시되는 화합물, 이의 입체이성질체, 또는 이의 염:[화학식 1]상기 화학식에서,B1 및 B2는 각각 독립적으로 천연, 비천연 또는 변형된 뉴클레오시드 염기이고;X1은 -OH, -O(C1-4알킬), 또는 -할로이고;X2는 -H 또는 X1과 결합하여 LNA 고리를 형성하는 것이고 {여기서, 상기 LNA 고리의 하나 이상의 H는 -(C1-4알킬), -OH, 또는 -O(C1-4알킬)로 치환될 수 있음};Y1은 -O(C1-4알킬), -O(C1-4알킬)O(C1-4알킬), -CH2O(C1-4알킬), 또는 -할로이고;Y2는 -H 또는 Y1과 결합하여 LNA 고리를 형성하는 것이고 {여기서, 상기 LNA 고리의 하나 이상의 H는 -(C1-4알킬), -OH, 또는 -O(C1-4알킬)로 치환될 수 있음};Z1 및 Z2는 각각 독립적으로 -OH, -O(C1-4알킬), -O(C1-4알킬)O(C1-4알킬), -CH2O(C1-4알킬), 또는 -할로이고;Z3는 -H 또는 Z1과 결합하여 LNA 고리를 형성하는 것이고 {여기서, 상기 LNA 고리의 하나 이상의 H는 -(C1-4알킬), -OH, 또는 -O(C1-4알킬)로 치환될 수 있음};n은 0 내지 3의 정수이고;m은 1 내지 10의 정수이고 {여기서, m이 2 이상인 경우 각각의 B2, Z1 및 Z2는 서로 다를 수 있음};R1 및 R2는 각각 독립적으로 -H 또는 -(C1-4알킬)이고;R3는 -(C1-4알킬)이다.
- 제 1 항에 있어서,상기 비천연 또는 변형된 뉴클레오시드 염기는 상기 천연 뉴클레오시드 염기의 이성질체이거나 상기 천연 뉴클레오시드 염기의 하나 이상의 -H가 -(C1-4알킬), -O(C1-4알킬) 또는 -할로로 치환, 하나 이상의 =CH-가 =N-으로 치환, 및 하나 이상의 =O가 =S로 치환으로 이루어진 군으로부터 선택된 1 이상으로 치환된 것인;화합물, 이의 입체이성질체, 또는 이의 염.
- 제 1 항에 있어서,X1은 -OH이고;X2는 -H인;화합물, 이의 입체이성질체, 또는 이의 염.
- 제 1 항에 있어서,Y1은 -O(C1-4알킬) 또는 -O(C1-4알킬)O(C1-4알킬), 또는 -할로이고;Y2는 -H 또는 Y1과 결합하여 LNA 고리를 형성하는 것인 {여기서, 상기 LNA 고리의 하나 이상의 H는 -(C1-4알킬)로 치환될 수 있음};화합물, 이의 입체이성질체, 또는 이의 염.
- 제 1 항에 있어서,Z1은 -OH 또는 -O(C1-4알킬)이고;Z2는 -OH이고;Z3는 -H인;화합물, 이의 입체이성질체, 또는 이의 염.
- 제 1 항에 있어서,n은 0 또는 1인;화합물, 이의 입체이성질체, 또는 이의 염.
- 제 1 항에 있어서,m은 1인;화합물, 이의 입체이성질체, 또는 이의 염.
- 제 1 항에 있어서,R1 및 R2는 각각 독립적으로 -H이고;R3는 -(C1-4알킬)인;화합물, 이의 입체이성질체, 또는 이의 염.
- 제 1 항에 있어서,상기 화합물, 이의 입체이성질체, 또는 이의 염은 RNA 캡핑용 올리고뉴클레오티드 프라이머인,화합물, 이의 입체이성질체, 또는 이의 염.
- 제 1 항 내지 제 11 항 중 어느 한 항에 따른 RNA 캡핑용 올리고뉴클레오티드 프라이머를 포함하는 RNA 분자.
- 제 12 항에 있어서,RNA 캡핑용 올리고뉴클레오티드 프라이머는 RNA 분자의 5' 상단에 부착된 것인 RNA 분자.
- 제 12 항에 있어서,RNA 분자는 1개 이상의 코딩 서열(coding sequence; CDS)을 포함하는 mRNA인 RNA 분자.
- (S-1) DNA 주형, 제 1 항 내지 제 11 항 중 어느 한 항에 따른 RNA 캡핑용 올리고뉴클레오티드 프라이머 및 RNA 폴리머라제를 혼합하는 단계; 및(S-2) 상기 혼합물을 인큐베이션하여 상기 폴리뉴클레오티드 주형의 전사를 수행하는 단계;를 포함하는 제 12 항에 따른 RNA 분자의 합성 방법.
- 제 15 항에 있어서,RNA 분자의 합성은 in vitro에서 수행되는 것인 합성 방법.
- 제 12 항에 따른 RNA 분자로부터 번역된 펩티드.
- 제 12 항에 따른 RNA 분자가 도입된 세포.
- 제 18 항에 따른 세포로부터 생산된 펩티드.
- 제 12 항에 따른 RNA 분자를 포함하는 핵산 치료제.
- 제 20 항에 있어서,RNA 분자를 표적 세포로 도입할 수 있는 전달체를 포함하는 핵산 치료제.
- 제 12 항에 따른 RNA 분자를 포함하는 백신.
- 제 22 항에 있어서,RNA 분자를 표적 세포로 도입할 수 있는 전달체를 포함하는 백신.
- 제 22 항에 있어서,암 또는 감염성 질환을 예방하기 위한 백신.
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CN113957108A (zh) * | 2021-09-09 | 2022-01-21 | 上海兆维科技发展有限公司 | 一种加帽rna的合成方法以及加帽rna转录反应液 |
KR102692464B1 (ko) * | 2022-03-31 | 2024-08-07 | 한미정밀화학주식회사 | mRNA 캡 유사체 및 이의 용도 |
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CA3198727A1 (en) | 2022-04-28 |
IL302215A (en) | 2023-06-01 |
US20230382943A1 (en) | 2023-11-30 |
KR102366490B1 (ko) | 2022-02-23 |
JP2023549592A (ja) | 2023-11-28 |
KR102366490B9 (ko) | 2024-08-26 |
CN116438306A (zh) | 2023-07-14 |
EP4234567A4 (en) | 2024-09-11 |
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