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WO2023005843A1 - Procédé de préparation d'arn, procédé de synthèse de protéine et solution de réaction de transcription - Google Patents

Procédé de préparation d'arn, procédé de synthèse de protéine et solution de réaction de transcription Download PDF

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WO2023005843A1
WO2023005843A1 PCT/CN2022/107406 CN2022107406W WO2023005843A1 WO 2023005843 A1 WO2023005843 A1 WO 2023005843A1 CN 2022107406 W CN2022107406 W CN 2022107406W WO 2023005843 A1 WO2023005843 A1 WO 2023005843A1
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molar concentration
final molar
nucleic acid
rna
optionally
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PCT/CN2022/107406
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English (en)
Chinese (zh)
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何苗
魏民志
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上海兆维科技发展有限公司
上海兆维生物工程有限公司
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Publication of WO2023005843A1 publication Critical patent/WO2023005843A1/fr

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    • 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
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • 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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  • the disclosure relates to the technical field of genetic engineering, in particular, to a method for preparing RNA, a method for synthesizing protein, and a transcription reaction solution.
  • In vitro synthetic RNA is mainly obtained through in vitro transcription using DNA as a template, and is commonly used to synthesize in vitro transcription using RNA polymerase using linearized plasmid DNA or PCR amplification products as templates.
  • the main process is to use DNA containing sequences such as T7 promoter (TAATACGACTCACTATAGGG) or SP6 promoter (ATTTAGGTGACACTATAG) as a template under the condition of T7 or SP6 RNA polymerase, and use NTP as a substrate to synthesize a strand complementary to the template DNA.
  • T7 promoter TAATACGACTCACTATAGGG
  • SP6 promoter ATTTAGGTGACACTATAG
  • NTP NTP as a substrate to synthesize a strand complementary to the template DNA.
  • mRNA easy and fast access to large quantities of mRNA molecules.
  • the in vitro transcription reaction solution includes buffer solution containing DTT and magnesium ions, DNA template, NTP and RNA polymerase, and is incuba
  • mRNA therapy uses mRNA as a preparation to treat diseases.
  • mRNA is a direct template for directing protein biosynthesis. It can be used to treat gene defect diseases or tissue repair through the expression of functional proteins, and it can also be used for immunotherapy through the expression of antigens, antibodies or receptors. That is to use the natural structure or chemically modified mRNA molecules to enter the cytoplasm, and use the own translation system in the cytoplasm to produce the proteins needed by the body.
  • mRNA does not enter the nucleus, does not change genomic DNA, has only transient activity, and can be degraded through physiological metabolism.
  • the production of mRNA is relatively simple, low-cost, and can express almost any desired protein.
  • mRNA drugs greatly shorten and reduce the cycle and cost of new drug development, making mRNA drugs have great advantages.
  • IVTT in vitro transcribed
  • dsRNA when dsRNA enters cells, from the extracellular environment to the cytoplasm, dsRNA is regarded as an invader, and all cells will respond to dsRNA through sensors, activating, for example, RNA-specific oligoadenylate synthase (OAS), Adenosine deaminase (ADAR) and RNA-activated protein kinase (PKR), etc.
  • OFAS RNA-specific oligoadenylate synthase
  • ADAR Adenosine deaminase
  • PPKR RNA-activated protein kinase
  • dsRNA also activates cytoplasmic sensors, such as retinoic acid-inducible gene I (RIG-I) and melanoma differentiation-associated protein 5 (MDA5), leading to the secretion of different cytokines, including type I interferons, interleukin-6 (IL -6) and tumor necrosis factor-a (TNF-a).
  • cytoplasmic sensors such as retinoic acid-inducible gene I (RIG-I) and melanoma differentiation-associated protein 5 (MDA5), leading to the secretion of different cytokines, including type I interferons, interleukin-6 (IL -6) and tumor necrosis factor-a (TNF-a).
  • the present disclosure provides a method for preparing RNA, comprising:
  • the raw materials include DNA template, RNA polymerase, NTPs, buffer solution containing magnesium ions and nucleic acid denaturant;
  • the nucleic acid denaturant includes at least one of organic solvent, sugar, sugar alcohol, alkaloid and protein denaturant.
  • the organic solvent includes methanol, ethanol, propanol, isopropanol, pentanol, polyethylene glycol, formamide, 1,2,3,4,5-pentapentyl alcohol, 1,2 ,3,4,5,6-Hexaol, prop-2-en-1-ol, 3,7-dimethylhept-2,6-dien-1-ol, 2-propyn-1-ol Alcohol, cyclohexane-1,2,3,4,5,6-hexaol, 2-(2-propyl)-5-methyl-cyclohexane-1-ol, dimethyl sulfoxide, methyl At least one of butyl sec-butyl sulfoxide, n-propyl sulfoxide, n-butyl sulfoxide, tetramethylene sulfoxide, triethanolamine and ethylene glycol;
  • the sugar includes at least one of trehalose and mannose
  • the sugar alcohol includes at least one of sorbitol and xylitol
  • the alkaloids include betaine;
  • the protein denaturant includes at least one of urea, guanidine hydrochloride, guanidine isothiocyanate, phenol, sulfite and thiosulfate.
  • the organic solvent includes at least one of ethanol, formamide and dimethyl sulfoxide.
  • the organic solvent includes ethanol and formamide.
  • the nucleic acid denaturant includes at least one of trehalose, betaine, urea, guanidine isothiocyanate, guanidine hydrochloride, sorbitol, xylitol and mannose.
  • the nucleic acid denaturant includes at least one of trehalose, betaine, urea and guanidine isothiocyanate.
  • the nucleic acid denaturant includes trehalose and urea.
  • the final molar concentration of the nucleic acid denaturant added is 1.0 mM-10.0 M, wherein the final molar concentration is the ratio of the amount of solute to the total volume of the raw material.
  • the nucleic acid denaturant includes trehalose with a final molar concentration of 1.0mM-10.0M, betaine with a final molar concentration of 1.0mM-10.0M, urea with a final molar concentration of 1.0mM-10.0M, Guanidine isothiocyanate with a final molar concentration of 1.0mM-10.0M, guanidine hydrochloride with a final molar concentration of 1.0mM-10.0M, sorbitol with a final molar concentration of 1.0mM-10.0M, and a final molar concentration of 1.0mM-10.0 At least one of M xylitol and mannose with a final molar concentration of 1.0mM-10.0M; wherein the final molar concentration is the ratio of the amount of solute to the total volume of the raw material.
  • the nucleic acid denaturant includes trehalose with a final molar concentration of 2.5mM-4.0M, betaine with a final molar concentration of 2.5mM-4.0M, urea with a final molar concentration of 2.5mM-4.0M, Guanidine isothiocyanate with a final molar concentration of 2.5mM-4.0M, guanidine hydrochloride with a final molar concentration of 2.5mM-4.0M, sorbitol with a final molar concentration of 2.5mM-4.0M, and a final molar concentration of 2.5mM-4.0 At least one of M xylitol and mannose with a final molar concentration of 2.5mM-4.0M.
  • the nucleic acid denaturant includes trehalose with a final molar concentration of 0.5M-1.2M, betaine with a final molar concentration of 0.5M-1.2M, urea with a final molar concentration of 0.5M-1.2M, Guanidine isothiocyanate with a final molar concentration of 0.5M-1.2M, guanidine hydrochloride with a final molar concentration of 0.5M-1.2M, sorbitol with a final molar concentration of 0.5M-1.2M, and a final molar concentration of 0.5M-1.2 At least one of M xylitol and mannose with a final molar concentration of 0.5M-1.2M.
  • the nucleic acid denaturant includes trehalose with a final molar concentration of 5.0mM-0.5M, guanidine isothiocyanate with a final molar concentration of 5.0mM-0.5M, and sorbitol with a final molar concentration of 5.0mM-0.5M.
  • the final molar concentration is at least one of 5.0mM-0.5M xylitol.
  • the nucleic acid denaturant includes mannose at a final molar concentration of 2.5mM-0.25M.
  • the nucleic acid denaturant includes betaine at a final molar concentration of 25.0 mM-2.5M.
  • the nucleic acid denaturant includes urea with a final molar concentration of 40.0mM-4.0M.
  • the percentage of the organic solvent to the total volume of the raw material is 0.1-70.0%.
  • the percentage of the organic solvent to the total volume of the raw material is 0.5-50.0%.
  • the organic solvent accounts for 10.0-15.0% of the total volume of the raw material.
  • the nucleic acid denaturant includes ethanol and urea; the percentage of ethanol in the total volume of the raw materials is 1.5-15.0%; the final molar concentration of the urea in the system after the raw materials are mixed 40.0mM-1.2M; wherein, the final molar concentration is the ratio of the amount of the solute to the total volume of the raw material.
  • the nucleic acid denaturant includes ethanol, formamide and trehalose; the ethanol and the formamide respectively account for 0.5-15.0% of the total volume of the raw material; the trehalose is in The final molar concentration in the system after the raw materials are mixed is 5.0mM-2.0M; wherein, the final molar concentration is the ratio of the amount of solute to the total volume of the raw materials.
  • the nucleic acid denaturant includes ethanol, formamide and trehalose; the ethanol and the formamide respectively account for 0.5-5.0% of the total volume of the raw material; the trehalose is in The final molar concentration in the system after the raw materials are mixed is 5.0mM-0.5M; wherein, the final molar concentration is the ratio of the amount of solute to the total volume of the raw materials.
  • the starting material further includes a 5' cap structure or a 5' cap analog.
  • the 5' cap structure includes G[5']ppp[5']G,m 7 G[5']ppp[5']G,m 3 2,2,7 G[5']ppp[5']G,m 2 7,3'-o G[5']ppp[5']G(3'-ARCA),m 2 7,2'-o GpppG(2'-ARCA), m 2 7,2'-o GppspG D1( ⁇ -S-ARCA D1),m 2 7,2'-o GppspG D2( ⁇ -S-ARCA D2)and m 2 7,3'-o Gppp(m 2 '-o )ApG, m 7 Gppp(m 2'-o )ApG, m 7 Gppp(m 2'-o )ApG, m 7 Gppp(m 2'-o )ApU.
  • the amount of dsRNA is reduced by 2-99% in the RNA synthesized with the nucleic acid denaturant.
  • the method further includes adding DNase after the transcription reaction for 5-60 minutes.
  • the present disclosure also provides a method for synthesizing protein, comprising: preparing RNA by using any one of the RNA preparation methods described above; and then synthesizing protein using the RNA as a template.
  • the protein is a protein for therapeutic use or a protein for vaccine use.
  • the present disclosure also provides a transcription reaction solution, including DNA template, RNA polymerase, NTPs, a buffer containing magnesium ions, and a nucleic acid denaturant;
  • the nucleic acid denaturant includes an organic solvent, sugar, sugar alcohol, alkaloid, and protein denaturation at least one of the agents.
  • the organic solvent includes methanol, ethanol, propanol, isopropanol, pentanol, polyethylene glycol, formamide, 1,2,3,4,5-pentapentyl alcohol, 1,2 ,3,4,5,6-Hexaol, prop-2-en-1-ol, 3,7-dimethylhept-2,6-dien-1-ol, 2-propyn-1-ol Alcohol, cyclohexane-1,2,3,4,5,6-hexaol, 2-(2-propyl)-5-methyl-cyclohexane-1-ol, dimethyl sulfoxide, methyl at least one of butyl sec-butyl sulfoxide, n-propyl sulfoxide, n-butyl sulfoxide, tetramethylene sulfoxide, triethanolamine and ethylene glycol.
  • the sugar includes at least one of trehalose and mannose.
  • the sugar alcohol includes at least one of sorbitol and xylitol.
  • the alkaloid comprises betaine.
  • the protein denaturant includes at least one of urea, guanidine hydrochloride, guanidine isothiocyanate, phenol, sulfite and thiosulfate.
  • the added volume of the nucleic acid denaturant is 0.1-70.0% of the total reaction volume.
  • the volume percentage of the organic solvent to the total volume of the transcription reaction solution is 0.1-70.0%.
  • the final molar concentration of the nucleic acid denaturant added is 1.0 mM-10.0 M, wherein the final molar concentration is the ratio of the amount of solute to the total volume of the transcription reaction solution.
  • the nucleic acid denaturant includes trehalose with a final molar concentration of 1.0mM-10.0M, betaine with a final molar concentration of 1.0mM-10.0M, urea with a final molar concentration of 1.0mM-10.0M, Guanidine isothiocyanate with a final molar concentration of 1.0mM-10.0M, guanidine hydrochloride with a final molar concentration of 1.0mM-10.0M, sorbitol with a final molar concentration of 1.0mM-10.0M, and a final molar concentration of 1.0mM-10.0 At least one of M xylitol and mannose with a final molar concentration of 1.0 mM-10.0 M; wherein the final molar concentration is the ratio of the amount of solute to the total volume of the transcription reaction solution.
  • the nucleic acid denaturant includes ethanol and urea; the percentage of the ethanol in the total volume of the transcription reaction solution is 0.5-15.0%; the final molar concentration of the urea in the transcription reaction solution 40.0mM-1.2M.
  • the nucleic acid denaturant includes ethanol and urea; the percentage of the ethanol in the total volume of the transcription reaction solution is 1.5-15.0%; the final molar concentration of the urea in the transcription reaction solution 40.0mM-1.2M.
  • the nucleic acid denaturant includes ethanol, formamide and trehalose; the ratios of the ethanol and the formamide to the total volume of the transcription reaction solution are both 0.5-15.0%; the seaweed The final molar concentration of sugar in the transcription reaction solution is 5.0mM-2.0M.
  • the nucleic acid denaturant includes ethanol, formamide and trehalose; the ratios of the ethanol and the formamide to the total volume of the transcription reaction solution are both 0.5-5.0%; the seaweed The final molar concentration of sugar in the transcription reaction solution is 5.0mM-0.5M.
  • FIG. 1 shows a graph of dsRNA content after IVT reaction provided by Examples 51-56 of the present disclosure and Comparative Example 1.
  • FIG. 2 shows a graph of dsRNA content after IVT reaction provided in Examples 57-59 of the present disclosure and Comparative Example 1.
  • FIG. 2 shows a graph of dsRNA content after IVT reaction provided in Examples 57-59 of the present disclosure and Comparative Example 1.
  • FIG. 3 shows the content diagram of IFN ⁇ in mice provided by Examples 56 and 59 of the present disclosure and Comparative Example 1.
  • FIG. 4 shows the content diagram of EPO in mice provided by Examples 56 and 59 of the present disclosure and Comparative Example 1.
  • the disadvantages of this method are also obvious: 1) the yield after separation and purification is very low, especially when the length of the IVT product is at least more than 3000nt, because for long-fragment RNA, its elution The peak is broad, and the overlap between ssRNA and dsRNA is serious; 2) RNA is easily degraded, especially for IVT products with a length of at least 3000 nt, because long RNA is degraded by shearing force through tightly packed column materials; 3) HPLC purification and amplification The scale is limited, and there are certain limitations to large-scale purification of mRNA; HPLC instruments and high operating costs are not suitable for industrial amplification.
  • the RNA purification method mentioned in the CN109072232 patent can remove the double-stranded RNA in the single-stranded RNA by contacting the RNA preparation with the cellulose material.
  • This method requires later purification, and the RNA yield after purification is limited. loss; and one cycle can only remove 90% dsRNA, but when the second and third purification cycles are performed, the dsRNA removal increases to 95% and 97%, but three cycles of purification will inevitably result in lower recovery.
  • WO 2013/102203A1 reported a method for RNase III to degrade dsRNA, but RNase III often causes partial degradation of ssRNA, especially long ssRNA. This may be caused by RNaseIII-catalyzed hydrolysis of the double-stranded secondary structure contained in ssRNA.
  • the unit “M” refers to mol/L; "mM” refers to mmol/L; ssRNA (single-stranded RNA) refers to single-stranded ribonucleic acid, and dsRNA (double-stranded RNA) refers to double-stranded ribose Nucleic acid; IVT (In Vitro Transcription) refers to in vitro transcription; NTPs (Nucleoside triphosphates) refers to nucleoside triphosphates. Protein denaturants refer to substances that can denature proteins.
  • RNA polymerase refers to the RNA polymerase that DNA is a template, polymerase can be selected from but not limited to T7 RNA polymerase, T3 RNA polymerase, SP6 RNA polymerase.
  • nucleic acid denaturation refers to the unraveling of the double helix structure of nucleic acid, the breaking of hydrogen bonds, and the loss of its natural secondary structure, tertiary structure, and quaternary structure in the presence of some extreme environments or some chemical agents. Structural process, but does not involve cleavage of internucleotide phosphodiester bonds.
  • the extreme environment that denatures nucleic acid refers to high temperature, radiation, strong acid, strong alkali conditions, etc.; chemical reagents that denature nucleic acid include organic salts and organic solvents, and these reagents are called nucleic acid denaturants.
  • melting temperature refers to the temperature at which the absorbance increases to half its maximum value, known as the melting temperature or melting point of DNA.
  • Tm melting temperature
  • the double helix structure disintegrates, and the two strands separate to form random coils.
  • a series of physical and chemical properties also change: the ultraviolet absorption value in the 260nm region increases (color enhancement effect), the viscosity decreases, and the buoyant density decreases.
  • the characteristic of DNA denaturation is explosive, and the denaturation occurs in a very narrow range.
  • the Tm value of DNA is generally between 70 and 85°C. When the DNA dilute salt solution contains nucleic acid denaturants, the Tm value will decrease.
  • DNA template refers to a DNA sequence containing an RNA promoter, sources of which include, but are not limited to, PCR and plasmid DNA.
  • transcription and “in vitro transcription” refer to the process in which RNA polymerase uses DNA as a template, recognizes a promoter sequence, and generates RNA.
  • 5' cap structure is a structure comprising a nucleoside 5' triphosphate coupled to a 5' uncapped RNA (eg, an uncapped 5' diphosphate RNA).
  • Cap structures can be natural including but not limited to N7-methylguanosine caps, or non-natural including but not limited to anti-retro-incorporated cap analogs ARCAs.
  • RNA capping can be obtained by enzymatic methods, such as vaccinia virus capping enzyme system or yeast capping enzyme system, and can also be obtained by in vitro transcription reactions containing cap analogs such as including but not limited to ARCA, m 2 7,3'- o Gppp(m 2'-o )ApG, m 7 Gppp(m 2'-o )ApG, m 7 Gppp(m 2'-o )ApU, etc.
  • cap analogs such as including but not limited to ARCA, m 2 7,3'- o Gppp(m 2'-o )ApG, m 7 Gppp(m 2'-o )ApG, m 7 Gppp(m 2'-o )ApU, etc.
  • Some embodiments of the present disclosure provide a method for preparing RNA, which includes mixing raw materials and performing transcription reaction.
  • the raw materials include DNA template, RNA polymerase, NTPs, buffer solution containing magnesium ions and nucleic acid denaturant;
  • the nucleic acid denaturant includes at least one of organic solvent, sugar, sugar alcohol, alkaloid and protein denaturant.
  • any composition described herein may comprise different salt forms, different forms of a single ingredient or a combination.
  • RNA synthesis ends are prepared from transcription, effectively inhibiting the generation of dsRNA, and significantly improving the purity of ssRNA in the system after in vitro transcription, thereby improving the in vivo stability and translation of ssRNA Efficiency and reduced immunogenicity of ssRNA, easy to operate.
  • magnesium ions in a buffer containing magnesium ions combine with NTPs and a DNA template to form a complex so that the promoter in the DNA template is recognized by RNA polymerase.
  • RNA is synthesized by performing IVT under the catalysis of RNA polymerase.
  • the generation of dsRNA can be effectively inhibited from the synthesis end of the RNA prepared by transcription, and the purity of ssRNA in the system after in vitro transcription can be significantly improved, thereby improving the in vivo stability and translation efficiency of ssRNA and reducing the Immunogenicity of ssRNA, easy to operate.
  • the final molar concentration of the nucleic acid denaturant added is 1.0 mM-10.0 M, wherein the final molar concentration is the ratio of the amount of the solute to the total volume of the raw material.
  • the final molar concentration of the nucleic acid denaturant added can be 10mM-5M, 100mM-1M, 200mM-800mM, 1mM-10mM or 10mM-100mM.
  • the percentage of the total amount of nucleic acid denaturant added to the total volume of the raw material is 0.1-70.0%.
  • the percentage of the total amount of nucleic acid denaturant added to the total volume of the raw material is 0.3-70.0%, 0.5-70.0%, 0.5-65%, 1-60%, 10-50% or 20-40%.
  • organic solvents include methanol, ethanol, propanol, isopropanol, pentanol, polyethylene glycol, formamide, 1,2,3,4,5-pentapentanol, 1, 2,3,4,5,6-Hexaol, prop-2-en-1-ol, 3,7-dimethylhept-2,6-dien-1-ol, 2-propyn-1 -alcohol, cyclohexane-1,2,3,4,5,6-hexaol, 2-(2-propyl)-5-methyl-cyclohexane-1-ol, dimethylsulfoxide, At least one of methyl sec-butyl sulfoxide, n-propyl sulfoxide, n-butyl sulfoxide, tetramethylene sulfoxide, triethanolamine and ethylene glycol.
  • the above-mentioned organic solvents can effectively inhibit the production of dsRNA from the synthesis end of the RNA prepared by transcription, thereby significantly improving the purity of ssRNA in the system after in vitro transcription. It can be understood that, in other embodiments of the present disclosure, the organic solvent may not be limited to the above-mentioned solvents.
  • the organic solvent includes at least one of ethanol, formamide, and dimethyl sulfoxide. It is believed that, without being bound by theory, ethanol, formamide or dimethyl sulfoxide, as nucleic acid denaturants, are added to the reaction system before the IVT reaction, which can effectively inhibit the generation of dsRNA (dsRNA content in the post-transcription system is below 1.0%) ), thereby significantly improving the purity of ssRNA in the system after in vitro transcription.
  • organic solvents include ethanol and formamide. The cooperation of ethanol and formamide can effectively inhibit the generation of dsRNA (the content of dsRNA in the post-transcription system is below 0.5%), and significantly improve the purity of ssRNA in the system after in vitro transcription.
  • the sugar includes at least one of trehalose and mannose.
  • the sugar alcohol includes at least one of sorbitol and xylitol.
  • the alkaloid includes betaine.
  • the protein denaturant includes at least one of urea, guanidine hydrochloride, guanidine isothiocyanate, phenol, sulfite, and thiosulfate. It is believed that, without being limited by theory, the above-mentioned substances can effectively inhibit the production of dsRNA from the synthesis end of RNA prepared by transcription, thereby significantly improving the purity of ssRNA in the system after in vitro transcription.
  • the nucleic acid denaturant includes at least one of trehalose, betaine, urea, guanidine isothiocyanate, guanidine hydrochloride, sorbitol, xylitol and mannose.
  • the nucleic acid denaturant includes at least one of trehalose, betaine, urea, and guanidine isothiocyanate. It is believed that, without being bound by theory, the above-mentioned substances can effectively inhibit the production of dsRNA from the synthesis end of RNA prepared by transcription (the content of dsRNA in the post-transcription system is below 1.0%), thereby significantly improving the purity of ssRNA in the system after in vitro transcription. It can be understood that, in other embodiments of the present disclosure, the nucleic acid denaturant may not be limited to the above substances.
  • the nucleic acid denaturants include trehalose and urea. It is believed that, without being limited by theory, the cooperation of trehalose and urea can effectively inhibit the generation of dsRNA (the content of dsRNA in the post-transcription system is below 0.2%), thereby significantly improving the purity of ssRNA in the system after in vitro transcription.
  • the nucleic acid denaturant includes trehalose with a final molar concentration of 1.0mM-10.0M, betaine with a final molar concentration of 1.0mM-10.0M, and urea with a final molar concentration of 1.0mM-10.0M , guanidine isothiocyanate with a final molar concentration of 1.0mM-10.0M, guanidine hydrochloride with a final molar concentration of 1.0mM-10.0M, sorbitol with a final molar concentration of 1.0mM-10.0M, and a final molar concentration of 1.0mM- At least one of 10.0M xylitol and mannose with a final molar concentration of 1.0mM-10.0M; wherein the final molar concentration is the ratio of the amount of solute to the total volume of the raw material.
  • the nucleic acid denaturant includes trehalose
  • the final molar concentration of trehalose in the system after the raw materials are mixed can be 1.0mM, 2.5mM, 5.0mM, 25.0mM, 40.0mM, 0.25M, 0.5M, 1.2M , 2.5M, 4.0M and 10.0M and so on.
  • the above final molar concentration can further inhibit the production of dsRNA and improve the purity of ssRNA in the system after in vitro transcription.
  • the nucleic acid denaturant includes trehalose with a final molar concentration of 2.5mM-4.0M, betaine with a final molar concentration of 2.5mM-4.0M, urea with a final molar concentration of 2.5mM-4.0M, Guanidine isothiocyanate with a concentration of 2.5mM-4.0M, guanidine hydrochloride with a final molar concentration of 2.5mM-4.0M, sorbitol with a final molar concentration of 2.5mM-4.0M, and guanidine hydrochloride with a final molar concentration of 2.5mM-4.0M At least one of xylitol and mannose with a final molar concentration of 2.5mM-4.0M.
  • the nucleic acid denaturant includes trehalose with a final molar concentration of 0.5M-1.2M, betaine with a final molar concentration of 0.5M-1.2M, urea with a final molar concentration of 0.5M-1.2M, Guanidine isothiocyanate with a concentration of 0.5M-1.2M, guanidine hydrochloride with a final molar concentration of 0.5M-1.2M, sorbitol with a final molar concentration of 0.5M-1.2M, and guanidine hydrochloride with a final molar concentration of 0.5M-1.2M At least one of xylitol and mannose with a final molar concentration of 0.5M-1.2M.
  • the nucleic acid denaturant includes trehalose with a final molar concentration of 5.0mM-0.5M, guanidine isothiocyanate with a final molar concentration of 5.0mM-0.5M, sorbitol with a final molar concentration of 5.0mM-0.5M and The molar concentration is at least one of 5.0mM-0.5M xylitol.
  • the nucleic acid denaturant includes mannose at a final molar concentration of 2.5mM-0.25M.
  • the nucleic acid denaturant includes betaine at a final molar concentration of 25.0 mM-2.5M.
  • the nucleic acid denaturant includes urea at a final molar concentration of 40.0 mM-4.0M.
  • the percentage of the organic solvent in the total volume of the raw material is 0.1-70.0%. In some embodiments, the percentage of the organic solvent in the total volume of the raw material can be, for example, 0.3-70.0%, 0.5-70.0% , 0.5-65%, 1-60%, 10-50% or 20-40%, such as 0.1%, 0.5%, 1.5%, 5.0%, 10.0%, 15.0%, 30.0%, 50.0% and 70.0%, etc. .
  • the above volume percentage of the organic solvent can further inhibit the production of dsRNA and improve the purity of ssRNA in the system after in vitro transcription. It should be noted that, in other embodiments of the present application, the percentage of the organic solvent in the total volume of the raw material may not be limited to the above volume percentage.
  • the organic solvent accounts for 0.5-50.0% of the total volume of the raw material. In some embodiments, the organic solvent accounts for 10.0-15.0% of the total volume of the raw material.
  • the nucleic acid denaturant may only include one of organic solvents, sugars, sugar alcohols, alkaloids, and protein denaturants, and may include several of organic solvents, sugars, sugar alcohols, alkaloids, and protein denaturants. species; also includes organic solvents, sugars, sugar alcohols, alkaloids and protein denaturants.
  • the nucleic acid denaturant may only include an organic solvent, may only include at least one of trehalose, betaine, urea, guanidine isothiocyanate, guanidine hydrochloride, sorbitol, xylitol, and mannose, or At least one of trehalose, betaine, urea, guanidine isothiocyanate, guanidine hydrochloride, sorbitol, xylitol and mannose and an organic solvent may be included at the same time.
  • the nucleic acid denaturant includes ethanol and urea; the percentage of ethanol in the total volume of the raw materials is 0.5-15.0%; the final molar concentration of urea in the system after the raw materials are mixed is 40.0mM-1.2M; Among them, the final molar concentration is the ratio of the amount of the solute substance to the total volume of the raw material.
  • the nucleic acid denaturant includes ethanol and urea; the percentage of ethanol in the total volume of the raw materials is 1.5-15.0%; the final molar concentration of urea in the system after the raw materials are mixed is 40.0mM-1.2M; Among them, the final molar concentration is the ratio of the amount of the solute substance to the total volume of the raw material.
  • the percentage of ethanol to the total volume of the feedstock may be, for example, 0.5-14%, 2.0-14.0%, 4.0-12.0% or 5.0-10.0%, such as 0.5%, 1.0%, 1.5%, 5.0%, 10.0% and 15.0%, etc., or an interval value between any two of the above point values.
  • the final molar concentration of urea in the system after the raw materials are mixed can be, for example, 50.0mM-1.0M, 80.0mM-0.8M or 100.0mM-0.5M, such as 40.0mM, 0.12M, 0.4M and 1.2M etc., or an interval value between any two point values above.
  • ethanol and urea cooperate with each other to synergistically inhibit the production of dsRNA, and the dsRNA content in the post-transcription system is as low as 0.02%, thereby further significantly improving the purity of ssRNA in the post-transcription system in vitro. 99.98%.
  • the nucleic acid denaturant includes ethanol, formamide and trehalose; the ethanol and the formamide respectively account for 0.5-15.0% of the total volume of the raw material; the trehalose
  • the final molar concentration in the system after the raw materials are mixed is 5.0mM-2.0M; wherein, the final molar concentration is the ratio of the amount of solute to the total volume of the raw materials.
  • the nucleic acid denaturant includes ethanol, formamide and trehalose; the percentages of ethanol and formamide in the total volume of the raw materials are both 0.5-5.0%; the final molar concentration of trehalose in the system after the raw materials are mixed It is 5.0mM-0.5M; wherein, the final molar concentration is the ratio of the amount of the solute substance to the total volume of the raw material.
  • the percentage of ethanol to the total volume of the feedstock may be, for example, 1.0-13.0%, 2.5-11.0%, or 4.0-8.0%, such as 0.5%, 1.5%, 3.5%, 4.0%, 5.0%, 6.0% , 7.0%, 8.0%, 9.0%, 10.0%, 11.0%, 12.0%, 13.0%, 14.0%, 15.0%, etc., or the interval value between any two point values above.
  • formamide may be, for example, 1.0-13.0%, 2.5-11.0%, or 4.0-8.0%, such as 0.5%, 1.5%, 3.5%, 4.0%, 5.0%, 6.0% of the total volume of the feedstock %, 7.0%, 8.0%, 9.0%, 10.0%, 11.0%, 12.0%, 13.0%, 14.0%, 15.0%, etc., or the interval value between any two point values above.
  • the percentage of ethanol to the total volume of the feedstock and the percentage of formamide to the total volume of the feedstock can be the same or different.
  • the final molar concentration of trehalose in the system after the raw materials are mixed can be, for example, 5.0mM-2.0M, 10.0mM-1.5M, 50.0mM-1.0M or 80.0mM-0.5M, such as 5.0mM, 15mM, 30mM, 50mM, 0.1M, 0.2M, 0.5M, 1M, 1.5M or 2.0M, etc., or the interval value between any two point values above.
  • ethanol, formamide, and trehalose cooperate to inhibit the production of dsRNA in a post-transcriptional system, compared to ethanol, formamide, or trehalose acting alone as nucleic acid denaturants
  • the dsRNA content is as low as 0.02%, which significantly improves the purity of ssRNA in the system after in vitro transcription as high as 99.98%.
  • a DNA template refers to a DNA sequence containing an RNA promoter, and its sources include, but are not limited to, PCR and plasmid DNA.
  • the DNA template may contain a T7 promoter (TAATACGACTCACTATAGGG) or an SP6 promoter (ATTTAGGTGACACTATAG).
  • T7 promoter TAATACGACTCACTATAGGG
  • SP6 promoter ATTTAGGTGACACTATAG
  • the RNA promoter contained in the DNA template is not limited to the above-mentioned promoters.
  • the final mass concentration of the DNA template in the system after the raw materials are mixed is 1-500 ng/ ⁇ L.
  • the final mass concentration of the DNA template in the system after the raw materials are mixed can be, for example, 10-400 ng/ ⁇ L, 50-300 ng/ ⁇ L or 100-200 ng/ ⁇ L, such as 1 ng/ ⁇ L, 5 ng/ ⁇ L, 25 ng/ ⁇ L, ⁇ L, 50ng/ ⁇ L, 100ng/ ⁇ L, 200ng/ ⁇ L, 500ng/ ⁇ L, etc., or the interval value between any two point values above.
  • the RNA polymerase can be selected from natural or non-natural RNA polymerase.
  • the RNA polymerase can be selected from T7 RNA polymerase, T3 RNA polymerase or SP6 RNA polymerase. It should be noted that, in other embodiments of the present disclosure, the RNA polymerase is not limited to the above-mentioned RNA polymerase.
  • the RNA polymerase is T7 RNA polymerase
  • the final concentration of T7 RNA polymerase in the system after the raw materials are mixed is 0.5-50U/ ⁇ L.
  • the final concentration of T7 RNA polymerase in the system after the raw materials are mixed can be, for example, 1.0-45U/ ⁇ L, 5.0-40U/ ⁇ L or 10.0-30U/ ⁇ L, such as 0.5U/ ⁇ L, 2.5U/ ⁇ L , 10U/ ⁇ L, 20U/ ⁇ L, 40U/ ⁇ L, and 50U/ ⁇ L, etc., or the interval value between any two point values above.
  • NTPs are nucleoside triphosphates, and NTPs can be natural or unnatural nucleoside triphosphates.
  • NTPs may include ATP (adenosine triphosphate), CTP (cytidine triphosphate), GTP (guanosine triphosphate) and UTP (uridine triphosphate) and modified NTPs such as 5- Me-CTP, N1-Me-pUTP, 5-OMe-UTP, etc. It should be noted that, in other embodiments of the present disclosure, NTPs are not limited to the above nucleoside triphosphates.
  • the final concentrations of ATP, CTP, GTP, UTP and modified nucleoside triphosphates in the system after the raw materials are mixed are each independently 0.5-20 mM.
  • the final concentrations of ATP, CTP, GTP, UTP and modified nucleoside triphosphates in the system after the raw materials are mixed can be 1-18mM, 5-15mM or 8-12mM, such as 0.5mM, 1mM, 5mM, 8mM, 10mM, 16mM and 20mM etc.
  • a buffer containing magnesium ions is a key factor to maintain a stable pH in the RNA synthesis system.
  • the magnesium ion-containing buffer may comprise MgCl 2 , Tris-HCl (tris-hydrochloride), spermidine, and DTT (dithiothreitol). It should be noted that, in the embodiments of the present disclosure, the substances in the buffer solution containing magnesium ions are not limited to the above substances.
  • the final concentration of magnesium ions in the system after the raw materials are mixed is 2-70 mM.
  • the final concentration of magnesium ions in the system after the raw materials are mixed can be, for example, 5-65mM, 10-60mM or 20-50mM, such as 2mM, 5mM, 20mM, 46mM, 60mM and 70mM, etc., or any of the above The interval value between two point values.
  • the raw material of magnesium ions can be selected from soluble salts containing magnesium ions such as MgCl 2 , MgSO 4 , Mg(AC) 2 and the like.
  • the pH of Tris-HCl is 6.0-9.0; as an example, the pH of Tris-HCl can be 6.0, 7.2, 7.9, 8.3, and 9.0, etc., or any two points above interval value between.
  • the final concentration of Tris-HCl in the system after the raw materials are mixed is 10-100 mM.
  • the final concentration of Tris-HCl in the system after the raw materials are mixed can be 10mM, 20mM, 40mM, 80mM and 100mM, etc., or an interval value between any two point values mentioned above.
  • the final concentration of spermidine in the system after the raw materials are mixed is 0.1-5 mM.
  • the final concentration of spermidine may be 0.1 mM, 0.5 mM, 2 mM, 5 mM, etc., or an interval value between any two of the above-mentioned point values.
  • the final concentration of DTT in the system after the raw materials are mixed is 1-50 mM.
  • the final concentration of DTT in the system after the raw materials are mixed can be 1 mM, 5 mM, 10 mM, 20 mM, 50 mM, etc., or an interval value between any two point values mentioned above.
  • the feedstock also includes inorganic pyrophosphatase, nuclease inhibitors, and nuclease-free water.
  • the final concentration of the inorganic pyrophosphatase in the system after the raw materials are mixed is 0.0001-0.1 U/ ⁇ L.
  • the final concentration of inorganic pyrophosphatase in the system after the raw materials are mixed can be 0.0001U/ ⁇ L, 0.001U/ ⁇ L, 0.005U/ ⁇ L, 0.01U/ ⁇ L, 0.05U/ ⁇ L and 0.1U/ ⁇ L etc., or an interval value between any two point values above.
  • the final concentration of the nuclease inhibitor in the system after the raw materials are mixed is 0.1-5 U/ ⁇ L.
  • the final concentration of the nuclease inhibitor in the system after mixing the raw materials is 0.1U/ ⁇ L, 0.5U/ ⁇ L, 1U/ ⁇ L, 2U/ ⁇ L and 5U/ ⁇ L, etc., or any two of the above-mentioned point values interval value between.
  • the temperature of the transcription reaction is 20-60° C.
  • the time of the transcription reaction is 15 min-10 h.
  • the temperature of the transcription reaction may be 20° C., 25° C., 37° C., 50° C., 60° C., etc., or an interval value between any two points above.
  • the time of the transcription reaction can be 15 min, 30 min, 2 h, 6 h, 10 h, etc., or an interval value between any two point values mentioned above.
  • the above-mentioned transcription reaction temperature and transcription reaction time can keep the transcription reaction going on stably.
  • the starting material further includes a 5' cap structure or a 5' cap analog.
  • the 5' cap structure includes G[5']ppp[5']G,m 7 G[5']ppp[5']G,m 3 2,2,7 G[5']ppp[5']G,m 2 7,3'-o G[5']ppp[5']G(3'-ARCA),m 2 7,2'-o GpppG(2'-ARCA), m 2 7,2'-o GppspG D1( ⁇ -S-ARCA D1),m 2 7,2'-o GppspG D2( ⁇ -S-ARCA D2)and m 2 7,3'-o Gppp(m 2 '-o )ApG, m 7 Gppp(m 2'-o )ApG, m 7 Gppp(m 2'-o )ApG, m 7 Gppp(m 2'-o )ApU. It is believed, without being bound by theory, that the 5' cap directly acquires the capped mRNA by means of co-transcription
  • the method further includes adding DNA nuclease after the transcription reaction for 5-60 minutes.
  • DNase is also referred to as "deoxyribonuclease”.
  • the transcription reaction after the transcription reaction, it also includes adding DNase I enzyme (Deoxyribonuclease I" or “Deoxyribonuclease I” that does not contain RNase contamination, which is a kind of enzyme that can digest single-stranded or double-stranded DNA to produce single deoxyribonuclease. nucleotide or single-stranded or double-stranded oligodeoxynucleotide endonuclease) at 25-45°C to digest the original DNA template for 5-60min.
  • DNase I enzyme Deoxyribonuclease I
  • Deoxyribonuclease I or “Deoxyribonuclease I” that does not contain RNase contamination
  • the temperature for digesting the original DNA template can be 25°C, 30°C, 37°C, 42°C, 45°C, etc., or an interval value between any two of the above-mentioned point values.
  • the time for digesting the original DNA template can be 5min, 10min, 20min, 25min, 30min, 40min and 60min, etc., or the interval value between any two of the above-mentioned point values.
  • the above-mentioned temperature and time for digesting the original DNA template can digest the original DNA template well, which is conducive to improving the purity of ssRNA in the system after in vitro transcription.
  • the amount of dsRNA in the RNA synthesized with the nucleic acid denaturant is reduced by 2%-99%, for example, by 5%-95%. %, 20%-90% or 50%-85%, such as at least a reduction of 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99%.
  • Some embodiments of the present disclosure also provide a method for synthesizing protein, comprising preparing RNA by using the above-mentioned RNA preparation method; and then synthesizing protein using RNA as a template. It is believed that without being bound by theory, the RNA prepared by the RNA preparation method provided above in the present disclosure is directly used as a template to synthesize proteins, since the RNA preparation method provided in the present disclosure effectively inhibits the production of dsRNA so that the prepared RNA The purity of the ssRNA is high, which is beneficial to improve the translation efficiency of the protein.
  • the synthesized protein may be a protein for therapeutic use or a protein for vaccine use.
  • Proteins for therapeutic use can be used to treat gene defect diseases or tissue repair through the expression of functional proteins, and proteins for vaccine use can be used for immunotherapy through the expression of antigens, antibodies or receptors.
  • the present disclosure also provides a transcription reaction liquid, including DNA template, RNA polymerase, NTPs, a buffer solution containing magnesium ions, and a nucleic acid denaturant;
  • the nucleic acid denaturant includes organic solvents, sugars, sugar alcohols, alkaloids, and protein denaturants at least one of .
  • organic solvents include methanol, ethanol, propanol, isopropanol, pentanol, polyethylene glycol, formamide, 1,2,3,4,5-pentapentanol, 1, 2,3,4,5,6-Hexaol, prop-2-en-1-ol, 3,7-dimethylhept-2,6-dien-1-ol, 2-propyn-1 -alcohol, cyclohexane-1,2,3,4,5,6-hexaol, 2-(2-propyl)-5-methyl-cyclohexane-1-ol, dimethylsulfoxide, At least one of methyl sec-butyl sulfoxide, n-propyl sulfoxide, n-butyl sulfoxide, tetramethylene sulfoxide, triethanolamine and ethylene glycol.
  • the sugar includes at least one of trehalose and mannose.
  • the sugar alcohol includes at least one of sorbitol and xylitol.
  • the alkaloids include betaines.
  • the protein denaturant includes at least one of urea, guanidine hydrochloride, guanidine isothiocyanate, phenol, sulfite, and thiosulfate.
  • the volume of the nucleic acid denaturant added is 0.1-70.0% of the total reaction volume.
  • the percentage of the volume of the organic solvent to the total volume of the transcription reaction solution is 0.1-70.0%.
  • the nucleic acid denaturant includes trehalose with a final molar concentration of 1.0mM-10.0M, betaine with a final molar concentration of 1.0mM-10.0M, and urea with a final molar concentration of 1.0mM-10.0M , guanidine isothiocyanate with a final molar concentration of 1.0mM-10.0M, guanidine hydrochloride with a final molar concentration of 1.0mM-10.0M, sorbitol with a final molar concentration of 1.0mM-10.0M, and a final molar concentration of 1.0mM- At least one of 10.0M xylitol and mannose with a final molar concentration of 1.0mM-10.0M; wherein the final molar concentration is the ratio of the amount of the solute to the total volume of the transcription reaction solution.
  • the nucleic acid denaturant includes ethanol and urea; the percentage of ethanol in the total volume of the transcription reaction solution is 1.5-15.0%; the final molar concentration of urea in the transcription reaction solution is 40.0mM-1.2M.
  • the nucleic acid denaturant includes ethanol, formamide and trehalose; the percentages of ethanol and formamide in the total volume of the transcription reaction solution are both 0.5-5.0%; the amount of trehalose in the transcription reaction solution The final molar concentration is 5.0mM-0.5M.
  • nucleic acid denaturants are added to the transcription reaction solution of IVT to effectively reduce dsRNA pollutants produced by IVT from the synthesis end of transcription, and the mRNA produced by the IVT method of the present disclosure has higher purity and reduces dsRNA to obtain RNA compositions more suitable for therapeutic use.
  • Conventional methods remove dsRNA contaminants through post-purification, but the present disclosure fundamentally avoids the production of dsRNA during IVT transcription, and also avoids post-purification steps.
  • the present disclosure can effectively improve the purity of mRNA and reduce the yield of dsRNA by using the mRNA prepared by the above method. Ensure high-purity, high-efficiency mRNA preparations. Avoid causing cellular pathways, reduce inflammatory responses, and improve the effectiveness of mRNA therapy and diagnosis. For example, it can reduce the animal's own defense mechanism, reduce the level of inflammatory cytokines, and reduce adverse reactions.
  • the synthetic end of RNA is prepared from transcription, effectively inhibiting the generation of dsRNA, and significantly improving the purity of ssRNA in the system after in vitro transcription, thereby improving the in vivo stability and translation efficiency of ssRNA and reducing the Immunogenicity of ssRNA, easy to operate.
  • This embodiment provides a transcription reaction solution and a method for preparing RNA.
  • T7 RNA polymerase Mix T7 RNA polymerase, inorganic pyrophosphatase and nuclease inhibitor to prepare a mixed enzyme system; wherein, the concentration of T7 RNA polymerase is 400U/ ⁇ L, and the concentration of inorganic pyrophosphatase is 0.1U/ ⁇ L ⁇ L, the concentration of nuclease inhibitor is 20U/ ⁇ L.
  • the configuration of the transcription reaction solution 2 ⁇ L of magnesium ion-containing buffer (10X), 1 ⁇ L of 200mM ATP, 1 ⁇ L of 200mM GTP, 1 ⁇ L of 200mM CTP, 1 ⁇ L of 200mM UTP, 1 ⁇ L of mixed enzyme, 1 ⁇ L of 1 ⁇ g/ ⁇ L DNA
  • ethanol i.e., nucleic acid denaturant
  • nuclease-free water was added to 20 ⁇ L to prepare a transcription reaction solution; wherein the DNA template was the DNA template for synthesizing erythropoietin (EPO, Erythropoietin (human)) mRNA
  • EPO Erythropoietin
  • the above-mentioned DNA template contains a T7 promoter, and the sequence of the above-mentioned DNA template is referred to Chromosome 7-NC_000007.14.
  • Embodiment 2-59 and comparative example 1 are identical to Embodiment 2-59 and comparative example 1
  • Embodiment 2-59 and comparative example 1 respectively provide a kind of transcription reaction liquid and a kind of preparation method of RNA
  • the reaction liquid system of embodiment 2-59 and comparative example 1 is similar to embodiment 1, please refer to embodiment 1,
  • Examples 2-56 and Comparative Example 1 differ from Example 1 in that the nucleic acid denaturant is different, see Table 1 for details;
  • Examples 57-59 are different from Example 1 in the nucleic acid denaturant, see Table 2 for details.
  • nucleic acid denaturants without organic solvents refers to nucleic acid denaturants that remove organic solvents.
  • Comparative Example 2 respectively provides a transcription reaction solution and a method for preparing RNA.
  • the transcription reaction solution system and method of Comparative Example 2 are similar to those of Example 5.
  • the difference between Comparative Example 2 and Example 5 is that the nucleic acid denaturant is not added in the transcription reaction solution.
  • the nucleic acid denaturant is It is added to the system after adding DNase I enzyme to digest the original DNA template. details as follows:
  • T7 RNA polymerase Mix T7 RNA polymerase, inorganic pyrophosphatase and nuclease inhibitor to prepare a mixed enzyme system; wherein, the concentration of T7 RNA polymerase is 400U/ ⁇ L, and the concentration of inorganic pyrophosphatase is 0.1U/ ⁇ L ⁇ L, the concentration of nuclease inhibitor is 20U/ ⁇ L.
  • RNA product After the transcription reaction solution was transcribed at 37°C for 2 hours, 1 ⁇ L of 1U/ ⁇ L DNase I enzyme was added to digest the original DNA template for 30 minutes at 37°C, and then 10.0 ⁇ L of ethanol was added to obtain the RNA product.
  • Comparative Examples 3-11 respectively provide a transcription reaction solution and a method for preparing RNA.
  • the nucleic acid denaturant of Comparative Examples 3-11 is also added to the system after adding DNase I enzyme to digest the original DNA template.
  • the reaction solution system and method of Comparative Example 3-11 are similar to those of Comparative Example 2.
  • the difference between Comparative Example 3-11 and Comparative Example 2 is that the nucleic acid denaturant is different. See Table 3 for details.
  • nucleic acid denaturants without organic solvents refers to nucleic acid denaturants that remove organic solvents.
  • the embodiment provides another transcription reaction solution and a method for preparing RNA.
  • the DNA template is the DNA template of Enhanced Green Fluorescent Protein (EGFP, Enhanced Green Fluorescent Protein) mRNA
  • the DNA template includes a T7 promoter
  • the promoter sequence is TAATACGACTCACTATAAGG.
  • Examples 61-71 and Comparative Example 12 respectively provide a transcription reaction solution and a method for preparing RNA.
  • the reaction solution systems of Examples 61-71 and Comparative Example 12 are similar to Example 60. Please refer to Example 60.
  • the difference between Examples 61-71 and Comparative Example 12 and Example 60 is that the nucleic acid denaturant is different, see Table 4 for details.
  • the dsRNA content was detected on the RNA obtained from the transcription reaction solution and the RNA preparation method provided in Examples 1-59 and Comparative Examples 1-11.
  • the test results are shown in Table 5.
  • the detection method of dsRNA content adopts sandwich ELISA (double antibody sandwich enzyme-linked immunosorbent assay) method; Detection method is as follows:
  • the establishment of the standard curve the absorbance value at 450nm of the dsRNA standard sample Standard1-8 with different concentrations (ng/mL) was measured respectively with a microplate reader, and the standard curve of the relationship between the concentration and the absorbance value (OD 450 ) was established.
  • nucleic acid denaturant was added to the system after IVT reaction, but the content of dsRNA could not be effectively reduced.
  • Example 4 3 ⁇ L or 10 ⁇ L of ethanol was added separately to the system before the IVT reaction, and the dsRNA content was 0.9% and 0.8% respectively;
  • Example 44 and Example 45 were added separately to the system before the IVT reaction For 3 ⁇ L or 10 ⁇ L of 8M urea, the dsRNA content was 0.9% and 0.2%, respectively; in Example 56, 3.0 ⁇ L of ethanol and 3.0 ⁇ L of 8M urea were added to the system before the IVT reaction, and the dsRNA content was only 0.02%.
  • dsRNA content after adding ethanol and urea to the system before the IVT reaction and then performing the transcription reaction is significantly lower than that after adding ethanol or urea alone and then performing the transcription reaction in the system before the IVT reaction. Therefore, ethanol and urea cooperate with each other to inhibit the production of dsRNA synergistically.
  • Example 3 1.0 ⁇ L of ethanol was added separately to the system before the IVT reaction, and the dsRNA content was 1.1%; in Example 30, 10 ⁇ L of 1.0 M trehalose was added separately to the system before the IVT reaction, and the dsRNA content was 1.0%; 48 Add 1.0 ⁇ L of formamide separately to the system before IVT reaction, and the dsRNA content is 0.6%; Example 59 Add 1.0 ⁇ L of formamide, 1.0 ⁇ L of ethanol and 10 ⁇ L of 1M trehalose to the system before IVT reaction at the same time , dsRNA content is only 0.02%.
  • dsRNA content after adding formamide, ethanol and trehalose to the system before the IVT reaction and then performing the transcription reaction is significantly lower than that after adding formamide, ethanol or trehalose to the system before the IVT reaction and then performing the transcription reaction dsRNA content. Therefore, ethanol, formamide and trehalose cooperate with each other to inhibit the production of dsRNA synergistically.
  • RNA obtained from the transcription reaction solutions and RNA preparation methods provided in Examples 51-59 and Comparative Examples 1-11 were purified by HPLC (High Performance Liquid Chromatography) to remove dsRNA.
  • HPLC High Performance Liquid Chromatography
  • the dsRNA content of the RNA obtained in Examples 51-59 and Comparative Examples 1-11 after HPLC purification was detected.
  • the test results are shown in Table 6, Figure 1 and Figure 2.
  • Example 56 it can be seen from Table 6 that in Example 56, 3.0 ⁇ L ethanol and 3.0 ⁇ L 8M urea were added to the system before the IVT reaction, the dsRNA content before HPLC purification was 0.02%, and the dsRNA content after HPLC purification was also 0.02%, that is, no The purification step can achieve the effect after HPLC purification, and the operation is simple.
  • Example 59 1.0 ⁇ L of formamide, 1.0 ⁇ L of ethanol, and 10 ⁇ L of 1M trehalose were added to the system before the IVT reaction, the dsRNA content after HPLC purification was 0.02%, and the dsRNA content after HPLC purification was also 0.02%, that is, no The purification step can achieve the effect after HPLC purification, and the operation is simple.
  • RNA after IVT in Examples 56, 59 and Comparative Example 1 The translation efficiency and immunogenicity of RNA after IVT in Examples 56, 59 and Comparative Example 1 were detected.
  • the RNAs after IVT of Examples 56, 59 and Comparative Example 1 were enzymatically capped and embedded in lipid nanoparticles (LNPs), and injected intraperitoneally into mice at a dose of 3 ⁇ g/animal (per group of 6). After 2 hours, 6 hours and 24 hours, the serum samples of the mice were collected, and the ELISA (enzyme linked immunosorbent assay, enzyme-linked immunosorbent assay) method was used to measure the alpha interferon (IFN ⁇ ) and erythropoietin (EPO) in the mice. ), the detection results are shown in Table 7, Table 8, Figure 3 and Figure 4.
  • IFN ⁇ alpha interferon
  • EPO erythropoietin
  • Example 56 and Example 59 are significantly lower than the level of IFN ⁇ in the mouse body in Comparative Example 1, so Example 56 and Example 59 can be significantly reduced compared to Comparative Example 1 Immunogenicity induced by dsRNA.
  • Example 56 and Example 59 are significantly higher than the level of EPO in the mouse body in Comparative Example 1, so Example 56 and Example 59 can be significantly improved relative to Comparative Example 1
  • the translation efficiency of mRNA promotes protein expression.
  • the present disclosure can effectively inhibit the generation of dsRNA by adding a nucleic acid denaturant to the system before the IVT reaction.
  • the present disclosure can significantly reduce dsRNA content and effectively improve mRNA purity by adding one or more nucleic acid denaturants .
  • the disclosure provides a method for preparing RNA, a method for synthesizing protein, and a transcription reaction solution.
  • the disclosure prepares RNA synthesis ends from transcription by adding a nucleic acid denaturant to the system before the in vitro transcription reaction, effectively inhibiting the generation of dsRNA, significantly Improve the purity of ssRNA in the system after in vitro transcription, thereby improving the in vivo stability and translation efficiency of ssRNA and reducing the immunogenicity of ssRNA, and the operation is simple, so it has excellent practical performance.

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Abstract

L'invention concerne un procédé de préparation d'ARN, un procédé de synthèse de protéine et une solution de réaction de transcription. Le procédé de préparation d'ARN comprend le mélange de matières premières, puis la réalisation d'une réaction de transcription. Les matières premières comprennent une matrice d'ADN, une ARN polymérase, des NTP, une solution tampon contenant des ions magnésium et un dénaturant d'acide nucléique. Le dénaturant d'acide nucléique comprend au moins l'un des éléments suivants : un solvant organique, un sucre, un alcool de sucre, un alcaloïde et un dénaturant de protéine. Le dénaturant d'acide nucléique est ajouté au système avant une réaction de transcription in vitro, l'extrémité de synthèse de l'ARN est préparée au moyen de la transcription, la génération d'ARNdb est efficacement inhibée, et la pureté de l'ARNsb dans un système transcrit in vitro est améliorée, afin que la stabilité in vivo et l'efficacité de traduction de l'ARNsb soient améliorées, et que l'immunogénicité de l'ARNsb soit réduite.
PCT/CN2022/107406 2021-07-27 2022-07-22 Procédé de préparation d'arn, procédé de synthèse de protéine et solution de réaction de transcription WO2023005843A1 (fr)

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