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WO2024163689A1 - In vitro transcription methods and compounds for use therein - Google Patents

In vitro transcription methods and compounds for use therein Download PDF

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Publication number
WO2024163689A1
WO2024163689A1 PCT/US2024/013902 US2024013902W WO2024163689A1 WO 2024163689 A1 WO2024163689 A1 WO 2024163689A1 US 2024013902 W US2024013902 W US 2024013902W WO 2024163689 A1 WO2024163689 A1 WO 2024163689A1
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WIPO (PCT)
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ome
hours
cap analogue
cap
concentration
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French (fr)
Inventor
Robert Dempcy
Dan Liu
Fengmei PI
Cheng-Hsien Wu
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Genscript Usa Inc
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Genscript Usa Inc
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Priority to EP24750991.2A priority Critical patent/EP4658772A1/en
Publication of WO2024163689A1 publication Critical patent/WO2024163689A1/en
Anticipated expiration legal-status Critical
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y207/00—Transferases transferring phosphorus-containing groups (2.7)
    • C12Y207/07—Nucleotidyltransferases (2.7.7)
    • C12Y207/07006—DNA-directed RNA polymerase (2.7.7.6)
    • 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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1241—Nucleotidyltransferases (2.7.7)
    • C12N9/1247—DNA-directed RNA polymerase (2.7.7.6)
    • 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
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/26—Preparation of nitrogen-containing carbohydrates
    • C12P19/28—N-glycosides
    • C12P19/30—Nucleotides
    • C12P19/34—Polynucleotides, e.g. nucleic acids, oligoribonucleotides

Definitions

  • the present disclosure relates generally to methods for synthesizing mRNA capping analogues and methods and compositions for in vitro transcription.
  • mRNA is a well-defined molecule with structure of 5’ Cap, 5’ end untranslated region (UTR), open reading frame sequence coding for a gene(s) of interest, and 3’ end UTR, and poly A tail.
  • UTR 5’ end untranslated region
  • Preparation of capped mRNA through in vitro synthesis may have significant importance for both fundamental scientific research and new therapeutics development. Several factors go into generating an mRNA that may have high expression levels, stability, and functionality.
  • An mRNA molecule may be flanked with 5’end and 3’end untranslated regions (UTR 2 ).
  • a 5’-UTR serves as the entry site for ribosomes to initiate translation, and a 3’ UTR plays an important role in translation termination and post transcriptional modification, which may influence the expression and half-life of mRNA.
  • a poly A tail of mRNA may make the RNA molecule more stable and prevent mRNA degradation. Additionally, a poly A tail may allow the mature messenger RNA to be exported from nucleus and translated into a protein by ribosomes in the cytoplasm.
  • An mRNA cap is a highly methylated modification at the 5’ end of mRNA, which may protect mRNA from degradation, recruit complexes involved in mRNA processing, and mark cellular mRNA to avoid recognition by the immune system.
  • the predominant 5’ cap structure is an inverted 7-methylguanosine nucleotide linked by a 5’ -5' triphosphate bond to the first transcribed nucleotide.
  • the 7- methylguanosine is methylated at its 7 nitrogen position, and may be referred to as m7 G or 7m G.
  • This cap structure may be expressed as 5’ m7 GpppNi (pN) x , where N is any nucleotide and x is 0 or any number.
  • the first nucleotide has a 2’ hydroxy group in its ribose sugar
  • the first nucleotide has a 2’- o-methyl modification in the ribose
  • this structure may comprise, from 5’ end to 3’ end, m7 G 5 ’pppNi 2 ’" OMe (pN)x, where N is any nucleotide and x may be any integer.
  • the 2’ hydroxy group of the first and second ribose to the m7 G are methylated.
  • This structure may comprise, from 5’ end to 3’ end, m7 G 5 ’pppNi 2 ’" OMe pN2 2 ’” OMe (pN)x, where N is any nucleotide and x may be any integer. See, e.g., Perry RP, “RNA processing comes of age”, J Cell Biol. (1981 Dec);91 (3 Pt 2):28s-38s, incorporated herein by reference in its entirety.
  • Capping may improve properties of the mRNA, such as, but not limited to, its stability and its translational efficiency. See, e.g., Banerjee AK, “5'-terminal cap structure in eucaryotic messenger ribonucleic acids”, Microbiol Rev. (1980 Jun);
  • each capping process may be carried out by enzymes. See, e g., Perry. These processes may be time-consuming, inefficient, and expensive to perform in vitro.
  • embodiments of the present disclosure may include methods for in vitro transcription of a DNA template into RNA, comprising providing a mixture comprising a buffer substance, ribonucleoside triphosphates (NTPs), one or more magnesium salts in a concentration of from about 2 mM to about 60 mM, the DNA template, a recombinant RNA polymerase, and a cap analogue comprising the structure of Formula (I), (II), or (III): wherein R is H or CH3; and Bi is A or N6-methyl-adenine (m 6 A), and B2 is A, U, G, or C; wherein
  • NTPs ribonucleoside triphosphates
  • R 1 is OCH3 and R 2 is OH or H, or
  • R 1 is OH and R 2 is H, or
  • R 1 is H and R 2 is H or OCH3, or
  • R 1 and R 2 are each OCH3,
  • B1 is A or N6-methyl-adenine (m 6 A), and B2 is A, II, G, or C, wherein R is H or CH 3 ; and Bi is A or N6-methyl-adenine (m 6 A), and B2 is A, U, G, or 0; and incubating the reaction mixture at from about 15°C to about 35°C, optionally from about 18°C to about 31 °C, for from about 1 hour to about 12 hours, thereby producing the RNA.
  • buffer substance may be Tris base, HEPES, or Tris-HCl.
  • the concentration of buffer substance may be from about 1 mM to about 100 mM, from about 1 mM to about 90 mM, from about 1 mM to about 80 mM, from about 1 mM to about 70 mM, from about 1 mM to about 60 mM, from about 1 mM to about 50 mM, from about 1 mM to about 40 mM, from about 1 mM to about 30 mM, from about 1 mM to about 20 mM, from about 1 mM to about 10 mM, from about 1 mM to about 5 mM, from about 10 mM to about 20 mM, from about 10 mM to about 30 mM, from about 10 mM to about 40 mM, from about 10 mM to about 50 mM, from about 20 mM to about 50 mM, from about 30 mM to about 50 mM, from about 35 mM to about 45 mM, from about 35 mM
  • the concentration of NTPs may be from about 1 mM to about 50 mM, from about 1 mM to about 40 mM, from about 1 mM to about 30 mM, from about 1 mM to about 20 mM, from about 1 mM to about 10 mM, from about 1 mM to about 5 mM, from about 2 mM to about 10 mM, from about 3 mM to about 10 mM, from about 3 mM to about 9 mM, from about 3 mM to about 8 mM, from about 3 mM to about 7 mM, from about 3 mM to about 6 mM, from about 3 mM to about 5 mM, from about 3 mM to about 4 mM, from about 4 mM to about 10 mM, from about 5 mM to about 10 mM, from about 6 mM to about 10 mM, from about 7 mM to about 10 mM, from about 8 mM to about 10 mM
  • the concentration of one or more magnesium salts may be from about 2 mM to about 50 mM, from about 2 mM to about 40 mM, from about 2 mM to about 30 mM, from about 2 mM to about 40 mM, from about 2 mM to about 30 mM, from about 2 mM to about 20 mM, from about 2 mM to about 10 mM, from about 2 mM to about 5 mM, from about 5 mM to about 50 mM, from about 10 mM to about 45 mM, from about 15 mM to about 40 mM, from about 20 mM to about 35 mM, from about 20 mM to about 30 mM, from about 20 mM to about 25 mM, from about 22 mM to about 28 mM, or from about 25 mM to about 30 mM.
  • the concentration of DNA template may be from about 0.001 ⁇ g/ ⁇ l to about 2 ⁇ g/ ⁇ l, from about 0.001 ⁇ g/ ⁇ l to about 1.5 ⁇ g/ ⁇ l, from about 0.001 ⁇ g/ ⁇ l to about 1 ⁇ g/ ⁇ l, from about 0.01 ⁇ g/ ⁇ l to about 2 ⁇ g/ ⁇ l, from about 0.01 ⁇ g/ ⁇ l to about 1.5 ⁇ g/ ⁇ l, from about 0.01 ⁇ g/ ⁇ l to about 1 ⁇ g/ ⁇ l, from about 0.01 ⁇ g/ ⁇ l to about 0.5 ⁇ g/ ⁇ l, from about 0.01 ⁇ g/ ⁇ l to about 0.1 ⁇ g/ ⁇ l, from about 0.01 ⁇ g/ ⁇ l to about 0.05 ⁇ g/ ⁇ l, from about 0.02 ⁇ g/ ⁇ l to about 0.04 ⁇ g/ ⁇ l, from about 0.02 ⁇ g/ ⁇ l to about 0.1 ⁇ g/ ⁇ l, from about 0.03 ⁇ g/ ⁇ l to about
  • the concentration of recombinant RNA polymerase may be from about 0.1 U/ ⁇ l to about 2 U/ ⁇ l, from about 0.5 U/ ⁇ l to about 2 U/ ⁇ l, from about 1 U/ ⁇ l to about 2 U/ ⁇ l, from about 1 U/ ⁇ l to about 1 .5 U/ ⁇ l, or from about 1 .5 U/ ⁇ l to about 2 U/ ⁇ l.
  • the mixture may further comprise an antioxidant.
  • antioxidant may be dithiothreitol (DTT) in a concentration of from about 1 mM to about 50 mM, from about 2 mM to about 50 mM, from about 3 mM to about 50 mM, from about 4 mM to about 50 mM, from about 5 mM to about 50 mM, from about 6 mM to about 50 mM, from about 7 mM to about 50 mM, from about 8 mM to about 50 mM, from about 9 mM to about 50 mM, from about 10 mM to about 50 mM, from about 10 mM to about 40 mM, from about 15 mM to about 30 mM, from about 15 mM to about 25 mM, from about 15 mM to about 20 mM, from about 20 mM to about 50 mM, from about 30 mM to about 50 mM, or from about 40 mM to about 50 mM.
  • DTT dithiothreitol
  • the mixture may further comprise an RNase inhibitor in a concentration of from about 0.001 U/ ⁇ l to about 5 U/ ⁇ l, from about 0.001 U/ ⁇ l to about 4 U/ ⁇ l, from about 0.001 U/ ⁇ l to about 3 U/ ⁇ l, from about 0.001 U/ ⁇ l to about 2 U/ ⁇ l, from about 0.001 U/ ⁇ l to about 1 U/ ⁇ l, from about 0.01 U/ ⁇ l to about 5 U/ ⁇ l, from about 0.01 U/ ⁇ l to about 4 U/ ⁇ l, from about 0.01 U/ ⁇ l to about 3 U/ ⁇ l, from about 0.01 U/ ⁇ l to about 2 U/ ⁇ l, from about 0.01 U/ ⁇ l to about 1 U/ ⁇ l, from about 0.01 U/ ⁇ l to about 0.5 U/ ⁇ l, from about 0.01 U/ ⁇ l to about 0.1 U/ ⁇ l, from about 0.01 U/ ⁇ l to about 0.05 U/ ⁇ l, from about 0.01 U/ ⁇ l to about 0.04 U/ ⁇ l,
  • cap analogue may be in a concentration of from about 0.5 mM to about 50 mM, from about 0.5 mM to about 40 mM, from about 0.5 mM to about 30 mM, from about 0.5 mM to about 20 mM, from about 0.5 mM to about 10 mM, from about 0.5 mM to about 5 mM, from about 1 mM to about 10 mM, from about 2 mM to about 10 mM, from about 3 mM to about 10 mM, from about 3 mM to about 9 mM, from about 3 mM to about 8 mM, from about 3 mM to about 7 mM, from about 3 mM to about 6 mM, from about 3 mM to about 5 mM, from about 3 mM to about 4 mM, from about 4 mM to about 10 mM, from about 5 mM to about 10 mM, from about 6 mM to about 10 mM, from about 5
  • the mixture may further comprise polyamine.
  • polyamine may be spermine, spermidine, or a combination thereof.
  • the concentration of the polyamine may be from about 0.1 mM to about 5 mM, from about 0.2 mM to about 4.9 mM, from about 0.2 mM to about 4.8 mM, from about 0.2 mM to about 4.7 mM, from about 0.2 mM to about 4.6 mM, from about 0.2 mM to about 4.5 mM, from about 0.2 mM to about 4.4 mM, from about 0.2 mM to about 4.3 mM, from about 0.2 mM to about 4.2 mM, from about 0.2 mM to about 4.1 mM, from about 0.2 mM to about 4 mM, from about 0.2 mM to about
  • the mixture may further comprise a pyrophosphatase in a concentration of from about 0.01 mU/ ⁇ l to about 2 mU/ ⁇ l, from about 0.01 mU/ ⁇ l to about 1.5 mU/ ⁇ l, from about 0.01 mU/ ⁇ l to about 1 mU/ ⁇ l, from about 0.1 mU/ ⁇ l to about 2 mU/ ⁇ l, from about 0.1 mU/ ⁇ l to about 1.5 mU/ ⁇ l, from about 0.1 mU/ ⁇ l to about 1 mU/ ⁇ l, from about 0.1 mU/ ⁇ l to about 0.9 mU/ ⁇ l, from about 0.1 mU/ ⁇ l to about 0.8 mU/ ⁇ l, from about 0.1 mU/ ⁇ l to about 0.7 mU/ ⁇ l, from about 0.1 mU/ ⁇ l to about 0.6 mU/ ⁇ l, from about 0.1 mU/ ⁇ l to about 0.5 mU/ ⁇ l, from about 0.01 mU/ ⁇ l
  • incubating the reaction mixture may be performed at from about 15°C to about 35°C, from about 16°C to about 35°C, from about 17°C to about 35°C, from about 18°C to about 35°C, from about 18°C to about 34°C, from about 18°C to about 33°C, from about 18°C to about 32°C, from about 18°C to about 31 °C, from about 18°C to about 30°C, from about 18°C to about 29°C, from about 18°C to about 28°C, from about 18°C to about 27°C, from about 18°C to about 26°C, from about 18°C to about 25°C, from about 18°C to about 24°C, from about 18°C to about 23°C, from about 18°C to about 22°C, from about 18°C to about 21 °C, from about 18°C to about 20°C, from about 18°C to about 19°C, from about 25°C to about 26°C, from about 25°C to about 26°C,
  • incubating the reaction mixture may be performed for from about 1 hour to about 12 hours, from about 1 hour to about 11 hours, from about
  • I hour to about 10 hours from about 1 hour to about 9 hours, from about 1 hour to about 8 hours, from about 1 hour to about 7 hours, from about 1 hour to about 6 hours, from about 1 hour to about 5 hours, from about 1 hour to about 4 hours, from about 1 hour to about 3 hours, from about 1 hour to about 2 hours, from about 2 hours to about 12 hours, from about 3 hours to about 12 hours, from about 4 hours to about 12 hours, from about 5 hours to about 12 hours, from about 6 hours to about 12 hours, from about "I hours to about 12 hours, from about 8 hours to about 12 hours, from about 9 hours to about 12 hours, from about 10 hours to about 12 hours, from about
  • incubating the reaction mixture may be performed at about 31 °C for from about 1 hour to about 5 hours, from about 1 hour to about 4.5 hours, from about 1 hour to about 4 hours, from about 1 hour to about 3.5 hours, from about 1 hour to about 3 hours, from about 1 hour to about 2.5 hours, from about 1 hour to about 2 hours, from about 1 hour to about 1 .5 hours, from about 0.5 hour to about 1 hour, from about 0.5 hour to about 1.5 hours, about 1 hour, about 2 hours, about 3 hours, about 4 hours, or about 5 hours.
  • DNA template may comprise a promoter operably linked to a nucleic acid comprising a 5’ untranslated region (5’ UTR), an open reading frame (ORF) encoding the RNA of interest, a 3’ UTR, and a poly A region, wherein the promoter comprises a sequence of TAATACGACTCACTATAX1X2X3 (SEQ ID NO: 16), wherein Xi is A or G, X2 is A or G, and X3 is A, T, G, or C, wherein the 5’ UTR is selected from SEQ ID NO: 1 , 3, 5, or 9, and the 3’ UTR is selected from SEQ ID NO: 2, 4, 6, or 8, wherein the poly A region comprises at least 60 adenine bases (As).
  • 5’ UTR 5’ untranslated region
  • ORF open reading frame
  • promoter may comprise a sequence selected from SEQ ID NO: 1
  • 5’ UTR and 3’ UTR may be respectively SEQ ID NO: 1 and 2, 1 and 4, 1 and 6, 3 and 2, 3 and 4, 3 and 6, 3 and 8, 5 and 2, 5 and 4, 5 and 6, 7 and 2, 7 and 4, and 6, 7 and 8, 9 and 2, 9 and 4, 9 and 6, or 9 and 8.
  • 5’ UTR and 3’ UTR may be respectively SEQ ID NO: 1 and 2, 1 and 4, 3 and 2, 1 and 6, 7 and 4, 9 and 2, or 3 and 6.
  • poly A region may comprise from about 60 to about 200 As, from about 60 to about 190 As, from about 60 to about 180 As, from about 60 to about 170 As, from about 60 to about 160 As, from about 60 to about 150 As, from about 60 to about 140 As, from about 60 to about 130 As, from about 60 to about 120 As, from about 60 to about 110 As, from about 60 to about 100 As, from about 70 to about 190 As, from about 80 to about 180 As, from about 90 to about 170 As, from about 100 to about 160 As, from about 100 to about 150 As, from about 100 to about 140 As, from about 100 to about 130 As, from about 100 to about 120 As, about 100 As, about 110 As, about 120 As, about 130 As, about 140 As, or about 150 As.
  • recombinant RNA polymerase may be selected from wild type T7 RNA polymerase or a variant thereof.
  • cap analogue may be one selected from the group consisting of m 7 GpppA2’OmepA2’Ome, m 7 GpppA2’OmepU2'Ome, m 7 GpppA2’OmepG2’Ome, m 7 GpppA2o me pC2’Ome, m 7 Gpppm 6 A2o me pA2’Ome, m 7 Gpppm 6 A2o me pU2Ome, m 7 Gpppm 6 A2o me pG2’Ome, m 7 Gpppm 6 A2’OmepC2’Ome, m 7 G3’OmePPpA2’OmepA2’Ome, m 7 Gso me pppAzo me pU2’Ome, m 7 G3'OmePPpA2’OmepG2’Ome, m 7 G3o me pppAzo me pC2
  • cap analogue may bind to -1 and/or +1 nucleotide of the promoter.
  • embodiments of the present disclosure may include reaction mixtures for in vitro transcription of a DNA template into RNA, comprising a buffer substance in a concentration of from about 45 mM to about 55 mM, an RNase inhibitor in a concentration of from about 0.01 U/ ⁇ l to about 0.03 U/ ⁇ l, NTPs in a concentration of from about 3 mM to about 5 mM, a cap analogue in a concentration of from about 6 mM to about 8 mM, one or more magnesium salts in a concentration of from about 20 mM to about 30 mM, a polyamine in a concentration of from about 1 .5 mM to about 2.5 mM, a DNA template in a concentration of from about 0.01 ⁇ g/ ⁇ l to about 0.05 ⁇ g/ ⁇ l, a pyrophosphatase in a concentration of from about 0.1 mU/ ⁇ l to about 0.5 mU/ ⁇ l, and an RNA polymerase in a concentration of from
  • Ri is OCH3 and R 2 is OH or H, or
  • R 1 is OH and R 2 is H, or
  • Ri is H and R 2 is H or OCH3, or
  • Ri and R 2 are each OCH3,
  • B1 is A or N6-methyl-adenine (m 6 A), and B2 is A, U, G, or C
  • R is H or CH 3 ; and Bi is A or N6-methyl-adenine (m 6 A), and B2 is A, II, G, or C.
  • incubating the reaction mixture may be performed at from about 15°C to about 35°C for from about 1 hour to about 12 hours.
  • incubating may comprise incubating the reaction mixture at from about 18°C to about 31 °C.
  • incubating may comprise incubating the reaction mixture at about 31 °C for about 3 hours.
  • embodiments of the present disclosure may include methods for in vitro transcription of a DNA template into RNA, comprising providing (1 ) a DNA template comprises a promoter operably linked to a nucleic acid comprising a 5’ untranslated region (5’ UTR), an open reading frame (ORF) encoding the RNA of interest, a 3’ UTR, and a poly A region, and (2) a cap analogue comprises the structure of Formula (I), (II), or (III): wherein R is H or CH 3 ; and Bi is A or N6-methyl-adenine (m 6 A), and B2 is A, U, G, or C, wherein
  • Ri is OCH 3 and R 2 is OH or H, or
  • R 1 is OH and R 2 is H, or
  • R 1 is H and R 2 is H or OCH3, or
  • R 1 and R 2 are each OCH 3 ,
  • B1 is A or N6-methyl-adenine (m 6 A)
  • B2 is A, U, G, or C
  • the promoter comprises a sequence of TAATACGACTCACTATAX1X2X3 (SEO ID NO: 16), wherein A at position 17 is -1 nucleotide and Xi at position 18 is +1 nucleotide, when Xi is G, X2 and X3 are each A, T, G, or C, then Bi is A and B2 is G, when Xi is A, X2 and X3 are each A, T, G, or C, then Bi is A and B2 is A, when Xi is C, X2 and X3 are each A, T, G, or C, then Bi is A and B2 is C, and when Xi is T, X2 and X3 are each A, T, G, or C, then Bi is A and B2 is C, and when Xi is T, X2 and X3 are each A, T
  • promoter may comprise a sequence selected from SEQ ID NO: 10, 11 , 12, 13, and 14.
  • the reaction mixture may comprise buffer substance in a concentration of from about 35 mM to about 45 mM, RNase inhibitor in a concentration of from about 0.01 U/ ⁇ l to about 0.03 U/ ⁇ l, NTPs in a concentration of from about 20 mM to about 40 mM, cap analogue in a concentration of from about 6 mM to about 8 mM, one or more magnesium salts in a concentration of from about 20 mM to about 30 mM, polyamine in a concentration of from about 1.5 mM to about 2.5 mM, DNA template in a concentration of from about 0.01 ⁇ g/ ⁇ l to about 0.05 ⁇ g/ ⁇ l, pyrophosphatase in a concentration of from about 0.1 mU/ ⁇ l to about 0.5 mU/ ⁇ l, and RNA polymerase in a concentration of from about 1 U/ ⁇ l to about 2 U/ ⁇ l.
  • buffer substance in a concentration of from about 35 mM to about 45 mM
  • RNase inhibitor in a concentration
  • embodiments of the present disclosure may include reaction mixtures for in vitro transcription of a DNA template into RNA, comprising a DNA template and a cap analogue, wherein the cap analogue comprises the structure of Formula (I), (II), or (III): wherein R is H or CH3; and Bi is A or N6-methyl-adenine (m 6 A), and B2 is A, U, G, or C; wherein
  • R 1 is OCH3 and R 2 is OH or H, or
  • R 1 is OH and R 2 is H, or
  • R 1 is H and R 2 is H or OCH3, or
  • R 1 and R 2 are each OCH 3 ,
  • B1 is A or N6-methyl-adenine (m 6 A), and B2 is A, U, G, or C, wherein R is H or CH 3 ; and Bi is A or N6-methyl-adenine (m 6 A), and B2 is A, U, G, or
  • the DNA template comprises a promoter; and wherein the cap analogue binds to at least -1 and +1 nucleotides of the promoter, or binds to at least+1 and +2 nucleotides of the promoter.
  • embodiments of the present disclosure may include methods for synthesizing a cap analogue, comprising the steps of:
  • embodiments of the present disclosure may include methods for synthesizing a cap analogue, comprising the steps of:
  • first oxidizer and the second oxidizer may be each (1 S)-(+)-(camphorsulfonyl)oxaziridine (CSO) or iodine.
  • divalent metal salt may be MnCI 2 or ZnCI 2 .
  • embodiments of the present disclosure may include cap analogues comprising formula (I): wherein R is H or CH3; and Bi is A or N6-methyl-adenine (m 6 A), and B2 is A, U, G, or C.
  • R is H
  • Bi is A
  • B2 is A
  • U, G, or C in which cap analogue of formula (I) may be selected from the group consisting of m 7 GpppA2’OmepA2’Ome, m 7 GpppA2’OmepU2'Ome, m 7 GpppA2’OmepG2’Ome, and m 7 GpppA2’OmepC2'Ome.
  • R is H
  • Bi is m 6 A
  • B2 is A
  • U, G, or C in which cap analogue of formula (I) may be selected from the group consisting of m 7 Gpppm 6 A2o me pA2’Ome, m 7 Gpppm 6 A2o me PU2Ome, rn 7 Gppprn 6 A2’omepG2’ome, and m 7 Gpppm 6 A2o me pC2’Ome.
  • R is CH 3
  • Bi is A
  • B2 is A, U, G, or C
  • cap analogue of formula (I) may be selected from the group consisting of m 7 G3 o me PPPA2 Ome pA2’Ome, m 7 G3 o me PPPA2 Ome pU2’Ome, m 7 G3o me PPPA2 OmepG2’0me, and m 7 G3’OmePPpA2’Ome pC2’Ome.
  • R is CH 3
  • Bi is m 6 A
  • B2 is A, U, G, or C
  • cap analogue of formula (I) may be selected from the group consisting of m 7 G3o me pppm 6 A2’o me pA2’Ome, m 7 G3o me pppm 6 A2’o me pU2’Ome, m 7 G3'OmePPpm 6 A2’OmepG2'Ome, and m 7 G3o me pppm 6 A2’o me pC2’Ome.
  • embodiments of the present disclosure may include cap analogues comprising formula (II): wherein
  • R 1 is OCH 3 and R 2 is OH or H, or
  • R 1 is OH and R 2 is H, or
  • R 1 is H and R 2 is H or OCH3, or
  • R 1 and R 2 are each OCH 3 ,
  • B1 is A or N6-methyl-adenine (m 6 A), and B2 is A, U, G, or C.
  • R 1 is OCH 3 and R 2 is OH
  • Bi is A
  • B2 is A, U, G, or C, in which cap analogue of formula (II) may be selected from the group consisting of m 7 G2’o me pppAs’o me pA2’Ome, m 7 G2o me PPPA2Ome pU2’Ome, m 7 G2o me PPPA2OmepG2’Ome, and m 7 G2o me PPPA2Ome pCs’Ome.
  • R 1 is OCH 3 and R 2 is OH
  • Bi is m 6 A
  • B2 is A
  • U, G, or C in which cap analogue of formula (II) may be selected from the group consisting of m 7 G2 o me pppm 6 A2’o me pAs’Ome, m 7 G2o me pppm 6 A2’o me PU2Ome, m 7 G2'OmePPpm 6 A2’OmepG2'Ome, and m 7 G2 o me pppm 6 A2’o me pCs’Ome.
  • Ri is OH and R 2 is H
  • Bi is A
  • B2 is A, II, G, or C
  • cap analogue of formula (II) may be selected from the group consisting of m 7 G3’HPPpA2’OmepA2’Ome, m 7 G3’HPPpA2’OmepU2’Ome, rn 7 G3HpppA2’omepG2’ome, and m 7 G3’HPPpA2’OmepC2’Ome.
  • R 1 is OH and R 2 is H
  • Bi is m 6 A and B2 is A
  • U, G, or C in which cap analogue of formula (II) may be selected from the group consisting of m 7 G3 Hpppm 6 A2’o me PA2 Ome, m 7 G3 Hpppm 6 A2’o me pU2’Ome, m 7 G3Hpppm 6 A2’o mepG2’0me, and m 7 G3Hpppm 6 A2’o me PC2 Ome.
  • embodiments of the present disclosure may include cap analogues comprising formula (III): wherein R is H or CH 3 ; and Bi is A or N6-methyl-adenine (m 6 A), and B2 is A, U, G, or C.
  • R is H
  • Bi is A
  • B2 is A
  • R is H
  • Bi is m 6 A
  • B2 is A
  • U, G, or C in which the cap analogue of formula (III) may be selected from the group consisting of m 7 Gppspm 6 A2o me pA2’Ome, m 7 Gppspm 6 A2o me pU2’Ome, m 7 Gppspm 6 A20mepG2’0me, and m 7 Gppspm 6 A2o me pC2’Ome.
  • R is CH3
  • Bi is A
  • B2 is A, U, G, or C
  • the cap analogue of formula (III) may be selected from the group consisting of m 7 G3o me ppspA2’Ome pA2’Ome, m 7 G3o me ppspA2’o me PU2Ome, m 7 G3'OmePPspA2’OmepG2’Ome, and m 7 G3o me ppspA2’Ome PC2Ome.
  • R is CH 3
  • Bi is m 6 A
  • B2 is A, U, G, or C
  • the cap analogue of formula (III) may be selected from the group consisting of m 7 G3o me ppspm 6 A2’o me pA2’Ome, m 7 G3o me ppspm 6 A2’o me pU2Ome, rn 7 G3’omeppsprn 6 A2’omepG2’ome, and m 7 G3o me ppspm 6 A2’o me pC2’Ome.
  • embodiments of the present disclosure may include methods for synthesizing a cap analogue, comprising the steps of:
  • FIG. 1 shows use of cap analogues to initiate in vitro transcription at -1 position in accordance with one embodiment of the present disclosure.
  • FIG. 2 shows a process of preparing cap-2 analogue synthetic intermediates in accordance with one embodiment of the present disclosure.
  • FIG. 3 shows a process of preparing cap-2 analogues in accordance with another embodiment of the present disclosure.
  • FIG. 4 shows a process of preparing additional cap-2 analogues in accordance with another embodiment of the present disclosure.
  • FIG. 5 shows expression levels of mRNA prepared by using various cap analogues in accordance with another embodiment of the present disclosure.
  • FIG. 6 shows a process of preparing additional cap-2 analogues in accordance with another embodiment of the present disclosure.
  • FIG. 7 shows a process of preparing additional cap-2 analogues in accordance with another embodiment of the present disclosure.
  • FIG. 8 shows a process of preparing additional cap-2 analogues in accordance with another embodiment of the present disclosure.
  • FIG. 9 shows in vivo expression levels of mRNA prepared by using various cap analogues in accordance with another embodiment of the present disclosure.
  • FIG. 10 shows biodistribution of mRNA prepared by using various cap analogues in accordance with one embodiment of the present disclosure.
  • FIG. 11 shows effect of mRNA prepared by using various cap analogues on mouse body weight in accordance with one embodiment of the present disclosure.
  • Methods and compositions for in vitro synthesis of mRNA are provided. Aspects of the disclosed methods and compositions may be used singly or in any combination. The disclosed methods and compositions may improve efficiency of mRNA synthesis and may provide mRNA with improved properties.
  • transcripts may be prone to the activity of DCP2, a major cellular decapping enzyme, which, in cooperation with its cellular partner DCP1 , releases m 7 GDP and the monophosphorylated RNA chain.
  • DCP2 a major cellular decapping enzyme
  • DXO decapping exoribonuclease
  • DXO may remove the entire cap structure by hydrolyzing the phosphodiester bond between the first and second transcribed nucleotide, resulting in the release of m 7 GpppN and monophosphorylated RNA, and may continue to degrade RNA as an exoribonuclease in the 5’-3’ direction.
  • DXO decapping activity was shown to be inhibited by cap-1 2’-O- methylation, suggesting its potential role in distinguishing ‘self from ‘non-self’ RNAs.
  • RNA capped with cap-2 resistance of RNA capped with cap-2 to DXO-mediated decapping and degradation was observed.
  • 2’-O-methylation of the second transcribed nucleotide and N6-methylation of adenosine as the first transcribed nucleotide serve as determinants defining transcripts as ‘self and contribute to transcript immune evasion.
  • cap-2 mRNA in vitro efficiently at large scale may be essential to translate these findings into biopharmaceutical applications.
  • mRNAs ranging from about 100b to about 20Kb, from about 200b to about 19Kb, from about 300b to about 18Kb, from about 400b to about 17Kb, from about 500b to about 16Kb, from about 600b to about 15Kb, from about 700b to about 14Kb, from about 800b to about 13Kb, from about 900b to about 12Kb, from about 1 Kb to about 11 Kb, from about 1 Kb to about 10Kb, from about 1 Kb to about 9Kb, from about 1 Kb to about 8Kb, from about 1 Kb to about 7Kb, from about 1 Kb to about 6Kb, from about 1 Kb to about 5Kb, from about 1 Kb to about 4Kb, from about 1 Kb to about 3Kb, from about 1 Kb to about 2Kb, from about 50b to about
  • mRNAs ranging from about 100b to about 20Kb, from about 200b to about 19Kb, from about 300b to about 18Kb, from about 400
  • mRNA synthesized using the disclosed compositions and/or methods may have many applications, including, but not limited to, uses in fundamental scientific research, uses in development of pharmacologies, uses in development of diagnostics, uses in development of therapeutics, uses as pharmacologies, uses as diagnostics, uses as therapeutics, or any combination thereof.
  • Reagents used in said methods may include: a linear DNA template with a promoter sequence that has a high binding affinity for its respective RNA polymerase; ribonucleoside triphosphates (NTPs) for the four bases (adenine, cytosine, guanine and uracil); a cap analogue; other modified nucleotides; DNA- dependent RNA polymerase (e.g., T7, T3 or SP6 RNA polymerase); ribonuclease (RNase) inhibitor to inactivate any contaminating Rnase; pyrophosphatase to degrade pyrophosphate, which inhibits transcription; MgCL and/or Mg(0Ac)2, which supplies Mg 2+ as a cofactor for the RNA polymerase; antioxidants (e.g. DTT); polyamines, such as spermidine; and a buffer to maintain a suitable pH value.
  • NTPs ribonucleoside triphosphates
  • Common buffer systems used in RNA in vitro transcription may include 4-(2- hydroxy-ethyl)-1 -piperazineethanesulfonic acid (HEPES) and tris(hydroxymethyl)aminomethane (Tris).
  • the pH value of the buffer may be commonly adjusted to a pH value of 6 to 8.5.
  • Some commonly used transcription buffers may contain 80 mM HEPES/KOH, pH 7.5 and 40 mM Tris/HCI, pH 7.5.
  • the transcription buffer may also contain a magnesium salt, such as MgCL and/or Mg(OAc)2 commonly in a range between 5-50 mM.
  • Magnesium ions may be an essential component in an RNA in vitro transcription buffer system because free Mg 2+ may function as cofactor in the catalytic center of the RNA polymerase and may be critical for the RNA polymerization reaction. In diffuse binding, fully hydrated Mg ions may also interact with the RNA product via nonspecific long-range electrostatic interactions.
  • RNA in vitro transcription reactions may be performed as batch reactions in which all components are combined and then incubated to allow the synthesis of RNA molecules until the reaction terminates.
  • fed-batch reactions were developed to increase the efficiency of the RNA in vitro transcription reaction (Kern et al. (1997) Biotechnol. Prog. 13: 747-756; Kern et al. (1999) Biotechnol. Prog. 15: 174- 184).
  • all components are combined, but then additional amounts of some of the reagents are added over time (e.g., NTPs, MgCL and/or Mg(OAc)2) to maintain constant reaction conditions.
  • RNA in vitro transcription may relate to a process wherein RNA is synthesized in a cell-free system (/n vitro ⁇ DNA, particularly plasmid DNA, is used as template for the generation of RNA transcripts.
  • RNA may be obtained by DNA-dependent in vitro transcription of an appropriate DNA template, which according to the present disclosure, may be preferably a linearized plasmid DNA template.
  • the promoter for controlling in vitro transcription can be any promoter for any DNA-dependent RNA polymerase.
  • DNA-dependent RNA polymerases are the T7, T3, and SPG RNA polymerases.
  • a DNA template for in vitro RNA transcription may be obtained, for example, by cloning of a nucleic acid, in particular cDNA corresponding to the respective RNA to be in vitro transcribed, and introducing it into an appropriate vector for in vitro transcription, for example, into plasmid DNA.
  • DNA template may be linearized with a suitable restriction enzyme before it is transcribed in vitro.
  • the cDNA may be obtained by reverse transcription of mRNA or chemical synthesis.
  • the DNA template for in vitro RNA synthesis may also be obtained by gene synthesis.
  • reagents used in in vitro transcription may include:
  • RNA polymerase such as bacteriophage-encoded RNA polymerases
  • NTPs ribonucleoside triphosphates
  • RNA-dependent RNA polymerase capable of binding to the promoter sequence within the linearized DNA template (e.g. T7, T3 or SPG RNA polymerase);
  • Rnase ribonuclease
  • pyrophosphatase to degrade pyrophosphate, which may inhibit transcription
  • MgCh and/or magnesium acetate (Mg(C2HsO2)2) (Mg(0Ac)2), which supplies Mg 2+ ions as a co-factor for the polymerase;
  • a buffer to maintain a suitable pH value which can also contain antioxidants (e.g., DTT), amines, such as, betaine and/or polyamines, such as, spermidine at optimal concentrations.
  • antioxidants e.g., DTT
  • amines such as, betaine and/or polyamines, such as, spermidine at optimal concentrations.
  • RNA in vitro transcription in the method of RNA in vitro transcription according to the present disclosure, no reagents, which are only required for the in vitro translation of the transcribed RNA to protein, but not for RNA in vitro transcription are used.
  • the mixture used for RNA in vitro transcription may not contain any proteinogenic amino acid or tRNA. Further, the mixture may not contain any proteinogenic amino acid, tRNA or a cell extract containing ribosomes.
  • co-transcription refers to mRNA prepared with a cap structure through one step in vitro transcription reaction using RNA polymerase, e.g., T7 RNA polymerase.
  • RNA polymerase e.g., T7 RNA polymerase.
  • one or more traditional post transcriptional capping method may need to prepare an uncapped RNA through in vitro transcription (IVT) with RNA polymerase, and then add Cap using capping enzyme, e.g., vaccinia capping enzyme, with the aid of 2’-O-Methyltransferase to add methylation at the +1 base of the mRNA.
  • nucleic acid means any DNA- or RNA-molecule and is used synonymous with polynucleotide. Furthermore, modifications or derivatives of the nucleic acid as defined herein are explicitly included in the general term “nucleic acid.” For example, peptide nucleic acid (RNA) is also included in the term “nucleic acid.”
  • Nucleic acid template provides the nucleic acid sequence that is transcribed into the RNA by the process of in vitro transcription and which therefore comprises a nucleic acid sequence which is complementary to the RNA sequence that is transcribed therefrom.
  • the nucleic acid template comprises a promoter to which the RNA polymerase used in the in vitro transcription process binds with high affinity.
  • the nucleic acid template may be a linearized plasmid DNA template.
  • the linear template DNA may be obtained by contacting plasmid DNA with a restriction enzyme under suitable conditions so that the restriction enzyme cuts the plasmid DNA at its recognition site(s) and disrupts the circular plasmid structure.
  • the plasmid DNA is preferably cut immediately after the end of the sequence that is to be transcribed into RNA.
  • the linear template DNA comprises a free 5' end and a free 3' end, which are not linked to each other. If the plasmid DNA contains only one recognition site for the restriction enzyme, the linear template DNA has the same number of nucleotides as the plasmid DNA.
  • the linear template DNA has a smaller number of nucleotides than the plasmid DNA.
  • the linear template DNA is then the fragment of the plasmid DNA that contains the elements necessary for in vitro transcription, which is a promotor element for RNA transcription and the template DNA element.
  • the open reading frame (ORF) of the linear template DNA may determine the sequence of the transcribed RNA by the rules of base-pairing.
  • nucleic acid template may be selected from a synthetic double stranded DNA construct, a single-stranded DNA template with a double-stranded DNA region comprising the promoter to which the RNA polymerase binds, a cyclic double-stranded DNA template with promoter and terminator sequences or a linear DNA template amplified by PCR or isothermal amplification.
  • the concentration of nucleic acid template comprised in the in vitro transcription mixture described herein may be in a range from about 1 to about 200 nM, from about 10 nM to about 150 nM, from about 20 nM to about 140 nM, from about 30 nM to about 130 nM, from about 40 nM to about 120 nM, from about 50 nM to about 110 nM, from about 60 nM to about 100 nM, from about 65 nM to about 90 nM, from about 65 nM to about 80 nM, from about 65 nM to about 75 nM, from about 65 nM to about 70 nM, from about 70 nM to about 75 nM, about 1 to about 40 nM, about 1 to about 30 nM, about 1 to about 20 nM, or about 1 to about 10 nM. Even more preferred the concentration of the nucleic acid template may be from about 10 to about 30 nM.
  • RNA is the usual abbreviation for ribonucleic acid. It is a nucleic acid molecule, i.e., a polymer consisting of nucleotide monomers. These nucleotides are usually adenosine-monophosphate (AMP), uridine-monophosphate (UMP), guanosine-monophosphate (GMP) and cytidine-monophosphate (CMP) monomers or analogues thereof, which are connected to each other along a so-called backbone.
  • AMP adenosine-monophosphate
  • UMP uridine-monophosphate
  • GMP guanosine-monophosphate
  • CMP cytidine-monophosphate
  • RNA sequence is formed by phosphodiester bonds between the sugar, i.e., ribose, of a first and a phosphate moiety of a second, adjacent monomer.
  • the specific order of the monomers i.e., the order of the bases linked to the sugar/phosphate- backbone, is called the RNA sequence.
  • RNA may be obtainable by transcription of a DNA sequence, e.g., inside a cell. In eukaryotic cells, transcription is typically performed inside the nucleus or the mitochondria. In vivo, transcription of DNA usually results in so-called premature RNA which has to be processed into so- called messenger-RNA, usually abbreviated as mRNA.
  • Processing of the premature RNA comprises a variety of different posttranscriptional-modifications, such as, splicing, 5'-capping, polyadenylation, export from the nucleus or the mitochondria and the like.
  • the sum of these processes is also called maturation of RNA.
  • the mature messenger RNA usually provides nucleotide sequence that may be translated into an amino acid sequence of a particular peptide or protein.
  • a mature mRNA comprises a 5'-cap, optionally a 5' UTR, an open reading frame, optionally a 3 'UTR, and a poly(A) sequence.
  • RNA molecules such as viral RNA, retroviral RNA and replicon RNA, small interfering RNA (siRNA), antisense RNA, CRISPR/Cas9 guide RNA, ribozymes, aptamers, riboswitches, immunostimulating RNA, transfer RNA (tRNA), ribosomal RNA (rRNA), small nuclear RNA (snRNA), small nucleolar RNA (snoRNA), microRNA (miRNA), and Piwi- interacting RNA (piRNA).
  • tRNA transfer RNA
  • rRNA ribosomal RNA
  • snRNA small nuclear RNA
  • snoRNA small nucleolar RNA
  • miRNA microRNA
  • piRNA Piwi- interacting RNA
  • Dicarboxylic acid or salt thereof is an organic acid having two carboxyl groups ( — COOH).
  • the term includes linear saturated dicarboxylic acids having the general formula HO2C — (CH2)n — CO2H such as oxalic acid, malonic acid, succinic acid, glutaric acid, adipic acid, pimelic acid, suberic acid, azelaic acid and sebacic acid. It also includes unsaturated dicarboxylic acids having at least one double bond such as maleic acid and fumaric acid as well as substituted dicarboxylic acids having at least one additional functional group such as malic acid, tartaric acid, cichoric acid and dimercaptosuccinic acid.
  • the salt of the dicarboxylic acid comprises the dicarboxylic acid anion and a suitable cation such as Na + , K + , Ca 2+ or Mg 2+ .
  • Tricarboxylic acid or salt thereof is an organic acid having three carboxyl groups ( — COOH).
  • tricarboxylic acids include citric acid, isocitric acid, aconitic acid, trimesic acid, nitrilotriacetic acid and propane-1 , 2,3- tricarboxylic acid.
  • citric acid (3-carboxy-3-hydroxypentane-1 ,5-dioic acid) is used.
  • the salt of the tricarboxylic acid comprises the tricarboxylic acid anion and a suitable cation, such as Na + , K + , Ca 2+ or Mg 2+ .
  • the reaction mixture for RNA in vitro transcription comprises magnesium citrate, a buffer substance, ribonucleoside triphosphates, a nucleic acid template and RNA polymerase.
  • Buffer substance is a weak acid or base used to maintain the acidity (pH) of a solution near a chosen value after the addition of another acid or base. Hence, the function of a buffer substance is to prevent a rapid change in pH when acids or bases are added to the solution.
  • Suitable buffer substances for use in the present invention include Tris (2-amino-2-hydroxymethyl-propane-1 ,3-diol) and HEPES (2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid).
  • the buffer substance may further comprise an acid or a base for adjusting the pH, such as HCI in case of Tris (Tris-HCI) and KOH in case of HEPES (HEPES-KOH).
  • an acid or a base for adjusting the pH such as HCI in case of Tris (Tris-HCI) and KOH in case of HEPES (HEPES-KOH).
  • citric acid is used to adjust the pH of the buffer substance, preferably of Tris base, so that no other acid has to be added.
  • the pH of the buffer substance is adjusted with an acid or a base such as HCI and KOH and the salt of the dicarboxylic or tricarboxylic acid, preferably citrate, is present in the reaction mixture in addition to the pH-adjusted buffer substance.
  • the concentration of the buffer substance within the mixture for in vitro transcription described herein may be about 10 to about 100 mM, about 10 to about 80 mM, about 10 to about 50 mM, about 10 to about 40 mM, about 10 to about 30 mM or about 10 to about 20 mM.
  • the concentration of the buffer substance is 40 mM.
  • the buffer has a pH value from about 6 to about 8.5, from about 6.5 to about 8.0, from about 7.0 to about 7.5, even more preferred of about 7.5 or about 8.0.
  • Ribonucleoside triphosphates The ribonucleoside triphosphates (NTPs) GTP, ATP, CTP and UTP are the monomers that are polymerized during the in vitro transcription process. They may be provided with a monovalent or divalent cation as counterion.
  • the monovalent cation is selected from the group consisting of Li + , Na + , K + , NH4 + or tris(hydroxymethyl)-aminomethane (Tris).
  • the divalent cation is selected from the group consisting of Mg 2+ , Ba 2+ and Mn 2+ . More preferably, the monovalent cation is Na + or tris(hydroxymethyl)-aminomethane (Tris).
  • NTP concentration may be from about 1 mM to about 50 mM, from about 1 mM to about 40 mM, from about 1 mM to about 30 mM, from about 1 mM to about 20 mM, from about 1 mM to about 10 mM, from about 1 mM to about 5 mM, from about 2 mM to about 10 mM, from about 3 mM to about 10 mM, from about 3 mM to about 9 mM, from about 3 mM to about 8 mM, from about 3 mM to about 7 mM, from about 3 mM to about 6 mM, from about 3 mM to about 5 mM, from about 3 mM to about 4 mM, from about 4 mM to about 10 mM, from about 5 mM to about 10 mM, from about 6 mM to about 10 mM, from about 7 mM to about 10 mM, from about 8 mM to about 10 mM, from about
  • a part or all of at least one ribonucleoside triphosphate in the in vitro transcription reaction mixture is replaced with a modified nucleoside triphosphate as defined below.
  • Modified nucleoside triphosphate The term “modified nucleoside triphosphate” as used herein refers to chemical modifications comprising backbone modifications as well as sugar modifications or base modifications. These modified nucleoside triphosphates are also termed herein as (nucleotide) analogues.
  • the modified nucleoside triphosphates as defined herein are nucleotide analogues/modifications, e g., backbone modifications, sugar modifications or base modifications.
  • a backbone modification in connection with the present invention is a modification, in which phosphates of the backbone of the nucleotides are chemically modified.
  • a sugar modification in connection with the present invention is a chemical modification of the sugar of the nucleotides.
  • a base modification in connection with the present invention is a chemical modification of the base moiety of the nucleotides.
  • nucleotide analogues or modifications are preferably selected from nucleotide analogues which are applicable for transcription and/or translation.
  • modified nucleosides and nucleotides which may be used in the context of the present invention, can be modified in the sugar moiety.
  • the 2' hydroxyl group (OH) can be modified or replaced with a number of different “oxy” or “deoxy” substituents.
  • alkoxy or aryloxy — OR, e.
  • “Deoxy” modifications include hydrogen, amino (e.g., NH2; alkylamino, dialkylamino, heterocyclyl, arylamino, diaryl amino, heteroaryl amino, diheteroaryl amino, or amino acid); or the amino group can be attached to the sugar through a linker, wherein the linker comprises one or more of the atoms C, N, and O.
  • the sugar group can also contain one or more carbons that possess the opposite stereochemical configuration than that of the corresponding carbon in ribose.
  • a modified nucleotide can include nucleotides containing, for instance, arabinose as the sugar.
  • the phosphate backbone may further be modified in the modified nucleosides and nucleotides.
  • the phosphate groups of the backbone can be modified by replacing one or more of the oxygen atoms with a different substituent.
  • the modified nucleosides and nucleotides can include the full replacement of an unmodified phosphate moiety with a modified phosphate as described herein. Examples of modified phosphate groups include, but are not limited to, phosphorothioate, phosphoroselenates, borano phosphates, borano phosphate esters, hydrogen phosphonates, phosphoroam idates, alkyl or aryl phosphonates and phosphotriesters.
  • Phosphorodithioates have both non-linking oxygens replaced by sulfur.
  • the phosphate linker can also be modified by the replacement of a linking oxygen with nitrogen (bridged phosphoroam idates), sulfur (bridged phosphorothioates) and carbon (bridged methylene-phosphonates).
  • the modified nucleosides and nucleotides which may be used in the present disclosure, can further be modified in the nucleobase moiety.
  • nucleobases found in RNA include, but are not limited to, adenine, guanine, cytosine and uracil.
  • nucleosides and nucleotides described herein can be chemically modified on the major groove face.
  • the major groove chemical modifications can include an amino group, a thiol group, an alkyl group, or a halo group.
  • the nucleotide analogues/modifications may be selected from base modifications, which are preferably selected from 2-amino-6-chloropurineriboside-5'-triphosphate, 2- Aminopurine-riboside-5'-triphosphate; 2-aminoadenosine-5'-triphosphate, 2'-Amino-2'- deoxycytidine-triphosphate, 2-thiocytidine-5'-triphosphate, 2-thiouridine-5'- triphosphate, 2'-Fluorothymidine-5’-triphosphate, 2 -O-Methyl inosine-5'-triphosphate
  • 4-thiouridine-5'-triphosphate 5-aminoallylcytidine-5'-triphosphate, 5-aminoallyluridine- 5'-triphosphate, 5-bromocytidine-5’-triphosphate, 5-bromouridine-5'-triphosphate, 5- Bromo-2'-deoxycytidine-5'-triphosphate, 5-Bromo-2'-deoxyuridine-5'-triphosphate, 5- iodocytidine-5'-triphosphate, 5-lodo-2'-deoxycytidine-5'-triphosphate, 5-iodouridine-5'- triphosphate, 5-lodo-2'-deoxyuridine-5'-triphosphate, 5-methylcytidine-5'-triphosphate,
  • 5-methyluridine-5'-triphosphate 5-Propynyl-2'-deoxycytidine-5'-triphosphate, 5- Propynyl-2'-deoxyuridine-5’-triphosphate, 6-azacytidine-5'-triphosphate, 6-azauridine- 5'-triphosphate, 6-chloropurineriboside-5 -triphosphate, 7-deazaadenosine-5'- triphosphate, 7-deazaguanosine-5'-triphosphate, 8-azaadenosine-5'-triphosphate, 8- azidoadenosine-5'-triphosphate, benzimidazole-riboside-5'-triphosphate, N1- methyladenosine-5'-triphosphate, N1-methylguanosine-5'-triphosphate, N6- methyladenosine-5'-triphosphate, O6-methylguanosine-5’-triphosphate, pseudouridine-5'-triphosphate, or puromycin-5'
  • nucleotides for base modifications selected from the group of base-modified nucleotides consisting of 5-methylcytidine- 5'-triphosphate, 7-deazaguanosine-5’-triphosphate, 5-bromocytidine-5'-triphosphate, and pseudouridine-5 -triphosphate.
  • modified nucleosides may include pyridin-4-one ribonucleoside, 5-aza-uridine, 2-thio-5-aza-uridine, 2-thiouridine, 4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxyuridine, 3-methyluridine, 5-carboxymethyl-uridine, 1- carboxymethyl-pseudouridine, 5-propynyl-uridine, 1 -propynyl-pseudouridine, 5- taurinomethyluridine, 1-taurinomethyl-pseudouridine, 5-taurinomethyl-2 -thio-uridine, 1- taurinomethyl-4-thio-uridine, 5-methyl-uridine, 1-methyl-pseudouridine, 4-thio-1- methyl-pseudouridine, 2-th io-1 -methyl-pseudouridine, 1 -methyl-1 -deaza- pseudouridine
  • modified nucleosides may include 5-aza-cytidine, pseudoisocytidine, 3-methyl-cytidine, N4-acetylcytidine, 5-formylcytidine, N4- methylcytidine, 5-hydroxymethylcytidine, 1 -methyl-pseudoisocytidine, pyrrolo-cytidine, pyrrolo-pseudoisocytidine, 2-thio-cytidine, 2-thio-5-methyl-cytidine, 4-thio- pseudoisocytidine, 4-thio-1 -methyl-pseudoisocytidine, 4-th io-1 -methyl-1 -deaza- pseudoisocytidine, 1 -methyl-1 -deaza-pseudoisocytidine, zebularine, 5-aza-zebularine, 5-methyl-zebularine, 5-aza-2-thio-zebularine
  • modified nucleosides may include 2-am inopurine, 2,
  • 6-diaminopurine 7-deaza-adenine, 7-deaza-8-aza-adenine, 7 -deaza-2 -am inopurine,
  • modified nucleosides may include inosine, 1 -methylinosine, wyosine, wybutosine, 7-deaza-guanosine, 7-deaza-8-aza-guanosine, 6-thio- guanosine, 6-thio-7-deaza-guanosine, 6-thio-7-deaza-8-aza-guanosine, 7-methyl- guanosine, 6-thio-7-methyl-guanosine, 7-methylinosine, 6-methoxy-guanosine, 1- methylguanosine, N2-methylguanosine, N2,N2-dimethylguanosine, 8-oxo-guanosine, 7-methyl-8-oxo-guanosine, l-methyl-6-thio-guanosine, N2-methyl-6-thio-guanosine, and N2, N2-dimethyl-6-thio-guanosine.
  • the nucleotide can be modified on the major groove face and can include replacing hydrogen on C-5 of uracil with a methyl group or a halo group.
  • a modified nucleoside is 5'-O-(l-Thiophosphate)- Adenosine, 5'-O-(1-Thiophosphate)-Cytidine, 5'-O-(1-Thiophosphate)-Guanosine, 5'- O-(1-Thiophosphate)-Uridine or 5'-O-(1-Thiophosphate)-Pseudouridine.
  • the modified nucleotides may include nucleoside modifications selected from 6-aza-cytidine, 2-thio-cytidine, a-thio-cytidine, Pseudo-iso-cytidine, 5-aminoallyl-uridine, 5-iodo-uridine, N1-methyl-pseudouridine, 5,6-dihydrouridine, a-thio-uridine, 4-thio-uridine, 6-aza-uridine, 5-hydroxy-uridine, deoxy-thymidine, 5-methyl-uridine, Pyrrolo-cytidine, inosine, a-thio-guanosine, 6- methyl-guanosine, 5-methyl-cytidine, 8-oxo-guanosine, 7 -deaza-guanosine, N1- methyl-adenosine, 2-amino-6-Chloro-purine, N6-methyl-2-amino-purine, P
  • a magnesium salt comprises a magnesium cation and a suitable anion, such as a chloride or an acetate anion.
  • the magnesium salt is magnesium chloride.
  • the initial free Mg 2+ concentration may be from about 1 to about 100 mM, about 1 to about 75 mM, about 1 to about 50 mM, about 1 to about 25 mM, or about 1 to about 10 mM.
  • the initial free Mg 2+ concentration is from about 5 to about 50 mM, about 10 to about 45 mM, about 15 to about 40 mM, or about 16 to about 37 mM, for example, about 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, or 37 mM.
  • the person skilled in the art may understand that the choice of the Mg 2+ concentration may be influenced by the initial total NTP concentration, meaning that a higher Mg 2+ concentration may need to be used, if a higher total NTP concentration is used in the in vitro transcription mixture.
  • the concentration of magnesium salt may be from about 2 mM to about 50 mM, from about 2 mM to about 40 mM, from about 2 mM to about 30 mM, from about 2 mM to about 40 mM, from about 2 mM to about 30 mM, from about 2 mM to about 20 mM, from about 2 mM to about 10 mM, from about 2 mM to about 5 mM, from about 5 mM to about 50 mM, from about 10 mM to about 45 mM, from about 15 mM to about 40 mM, from about 20 mM to about 35 mM, from about 20 mM to about 30 mM, from about 20 mM to about 25 mM, from about 22 mM to about 28 mM, or from about 25 mM to about 30 mM.
  • RNA polymerase is an enzyme that catalyses transcription of DNA template into RNA.
  • Suitable RNA polymerases for use in the present disclosure may include T7, T3, SP6 and E. coli RNA polymerase.
  • T7 RNA polymerase may be used.
  • the RNA polymerase for use in the present disclosure may be recombinant RNA polymerase, meaning that it is added to the RNA in vitro transcription reaction as a single component and not as part of a cell extract that contains other components in addition to the RNA polymerase.
  • T7 RNA polymerase may be used.
  • the RNA polymerase for use in the present disclosure may be recombinant RNA polymerase, meaning that it is added to the RNA in vitro transcription reaction as a single component and not as part of a cell extract that contains other components in addition to the RNA polymerase.
  • a skilled person knows that the choice of the RNA polymerase depends on the promoter present in the DNA template, which has to be bound by
  • the concentration of the RNA polymerase in the in vitro transcription mixture(s) described herein may be from about 0.001 ⁇ g/ ⁇ l to about 2 ⁇ g/ ⁇ l, from about 0.001 ⁇ g/ ⁇ l to about 1.5 ⁇ g/ ⁇ l, from about 0.001 ⁇ g/ ⁇ l to about 1 ⁇ g/ ⁇ l, from about 0.01 ⁇ g/ ⁇ l to about 1 ⁇ g/ ⁇ l, from about 0.01 ⁇ g/ ⁇ l to about 0.5 ⁇ g/ ⁇ l, from about 0.01 ⁇ g/ ⁇ l to about 0.5 ⁇ g/ ⁇ l, from about 0.01 ⁇ g/ ⁇ l to about 0.1 ⁇ g/ ⁇ l, from about 0.01 ⁇ g/ ⁇ l to about 0.05 ⁇ g/ ⁇ l, from about 0.1 ⁇ g/ ⁇ l to about 1 ⁇ g/ ⁇ l, from about 0.1 ⁇ g/ ⁇ l to about 0.9 ⁇ g/ ⁇ l, from about 0.1 ⁇ g/ ⁇ l to about 0.8 ⁇ g/ ⁇
  • the concentration of the RNA polymerase in the in vitro transcription mixture(s) described herein may be from about 0.1 U/ ⁇ l to about 2 U/ ⁇ l, from about 0.5 U/ ⁇ l to about 2 U/ ⁇ l, from about 1 U/ ⁇ l to about 2 U/ ⁇ l, from about 1 U/ ⁇ l to about 1.5 U/ ⁇ l, or from about 1 .5 U/ ⁇ l to about 2 U/ ⁇ l.
  • concentration of DNA template may be from about 0.1 U/ ⁇ l to about 2 U/ ⁇ l, from about 0.5 U/ ⁇ l to about 2 U/ ⁇ l, from about 1 U/ ⁇ l to about 2 U/ ⁇ l, from about 1 U/ ⁇ l to about 1.5 U/ ⁇ l, or from about 1 .5 U/ ⁇ l to about 2 U/ ⁇ l.
  • concentration of the RNA polymerase in the in vitro transcription mixture(s) described herein may be from about 0.1 U/ ⁇ l to about 2 U/ ⁇ l, from about 0.5 U/ ⁇ l to about 2 U/ ⁇ l, from about 1
  • Pyrophosphatase is an acid anhydride hydrolase that hydrolyses diphosphate bonds. In the in vitro transcription reaction, it may serve to hydrolyze the bonds within the diphosphate released upon incorporation of the ribonucleoside triphosphates into the nascent RNA chain.
  • the concentration of the pyrophosphatase in the in vitro transcription mixture(s) described herein may be from about 0.01 mU/ ⁇ l to about 2 mU/ ⁇ l, from about 0.01 mU/ ⁇ l to about 1.5 mU/ ⁇ l, from about 0.01 mU/ ⁇ l to about 1 mU/ ⁇ l, from about 0.1 mU/ ⁇ l to about 2 mU/ ⁇ l, from about 0.1 mU/ ⁇ l to about 1.5 mU/ ⁇ l, from about 0.1 mU/ ⁇ l to about 1 mU/ ⁇ l, from about 0.1 mU/ ⁇ l to about 0.9 mU/ ⁇ l, from about 0.1 mU/ ⁇ l to about 0.8 mU/ ⁇ l, from about 0.1 mU/ ⁇ l to about 0.7 mU/ ⁇ l, from about 0.1 mU/ ⁇ l to about 0.6 mU/ ⁇ l, from about 0.1 mU/ ⁇ l to about 0.5 mU/ ⁇ l,
  • a 5' cap is typically a modified nucleotide, particularly a guanine nucleotide, added to the 5' end of an RNA molecule.
  • 5' cap may be added using a 5'-5'-triphosphate linkage.
  • a 5' cap may be methylated, e.g., m7GpppN, wherein N is the terminal 5' nucleotide of the nucleic acid carrying the 5' cap, typically the 5'-end of an RNA.
  • the naturally occurring 5' cap may include m7GpppN.
  • a 5' cap structure may be formed by a cap analogue.
  • Cap analogue refers to a non-extendable di-nucleotide or tri-nucleotide that has cap functionality which means that it facilitates translation or localization, and/or prevents degradation of the RNA molecule when incorporated at the 5' end of the RNA molecule.
  • Capped mRNA without 5’ end triphosphate structure reduces its immunogenicity side effect.
  • Non-extendable means that the cap analogue will be incorporated only at the 5' terminus because it does not have a 5' triphosphate and therefore cannot be extended in the 3' direction by a template-dependent RNA polymerase.
  • cap analogues may include a cap-2 analogue with the structure of Formula (I): wherein R is H or CH 3 ; and Bi is A or N6-methyl-adenine (m 6 A), and B2 is A, II, G, or C.
  • the cap-2 analogues may include the following: m 7 GpppA2’OmepA2’Ome, m 7 GpppA2’OmepU2’Ome, m 7 GpppA2’OmepG2’Ome, m 7 GpppA2’OmepC2’Ome, m 7 Gppprn 6 A2’omepA2’o me, m 7 Gpppm 6 A2o me pUzo me, rn 7 Gppprn 6 A2’omepG2’ome, and m 7 Gpppm 6 A2’OmepC2’Ome.
  • the cap-2 analogues may include the following: m 7 G3'OmePPpA2’Ome PA2 Ome, m 7 G3 o me PPPA2 Ome pU2’Ome, m 7 G3o me PPPA2OmepG2’Ome, m 7 G3o me PPPA2Ome PC2Ome, m 7 G3’OmePPpm 6 A2’OmepA2’Ome, m 7 G3’OmePPpm 6 A2’OmepU2’Ome, m 7 G3'OmePPpm 6 A2’OmepG2'Ome, and m 7 G3 o me pppm 6 A2’o me pC2’Ome.
  • cap analogues may include cap-2 analogues with the structure of Formula (II):
  • R 1 is OCH 3 and R 2 is OH or H, or
  • R 1 is OH and R 2 is H, or
  • R 1 is H and R 2 is H or OCH3, or
  • R 1 and R 2 are each OCH3,
  • Bi is A or N6-methyl-adenine (m 6 A), and B2 is A, U, G, or C.
  • the cap-2 analogues may include the following: m 7 G2 o me PPPA2 Ome pU2’Ome, m 7 G2 o me PPPA2 OmepG2’0me, m 7 G2’Ome pppA2’Ome pC2’Ome, m 7 G2 o me pppm 6 A2’o me pA2’Ome, m 7 G2 o me pppm 6 A2’o me PU2 Ome, m 7 G2'OmePPpm 6 A2’OmepG2'Ome, and m 7 G2 o me pppm 6 A2’o me pC2’Ome.
  • the cap-2 analogues may include the following: m 7 G3’HPPpA2’OmepA2’Ome, m 7 G3 HpppA2’o me pU2’Ome, m 7 G3'HpppA2’OmepG2’Ome, m 7 G3’HPPpA2’OmepC2’Ome, m 7 G3 Hpppm 6 A2’o me PA2 Ome, m 7 G3'Hpppm 6 A2’OmepU2’Ome, m 7 G3Hpppm 6 A2’omepG2’ome, and m 7 G3Hpppm 6 A2’o me PC2 Ome.
  • cap analogue may be added with an initial concentration in the range of about 1 to about 20 mM, about 1 to about 17.5 mM, about 1 to about 15 mM, about 1 to about 12.5 mM, about 1 to about 10 mM, about 1 to about 7.5 mM, about 1 to about 5 mM or about 1 to about 2.5 mM. Even more preferred the cap analogue may be added with an initial concentration of about 5 to about 20 mM, about 7.5 to about 20 mM, about 10 to about 20 mM or about 12.5 to about 20 mM.
  • cap analogue may be in a concentration of from about 0.5 mM to about 50 mM, from about 0.5 mM to about 40 mM, from about 0.5 mM to about 30 mM, from about 0.5 mM to about 20 mM, from about 0.5 mM to about 10 mM, from about 0.5 mM to about 5 mM, from about 1 mM to about 10 mM, from about 2 mM to about 10 mM, from about 3 mM to about 10 mM, from about 3 mM to about 9 mM, from about 3 mM to about 8 mM, from about 3 mM to about 7 mM, from about 3 mM to about 6 mM, from about 3 mM to about 5 mM, from about 3 mM to about 4 mM, from about 4 mM to about 10 mM, from about 5 mM to about 10 mM, from about 6 mM to about 10 mM, from about 5
  • Ribonuclease inhibitor A ribonuclease inhibitor inhibits the action of a ribonuclease, which degrades RNA.
  • concentration of the ribonuclease inhibitor in the in vitro transcription mixture(s) described herein may be from about 0.001 U/ ⁇ l to about 5 U/ ⁇ l, from about 0.001 U/ ⁇ l to about 4 U/ ⁇ l, from about 0.001 U/ ⁇ l to about 3 U/ ⁇ l, from about 0.001 U/ ⁇ l to about 2 U/ ⁇ l, from about 0.001 U/ ⁇ l to about 1 U/ ⁇ l, from about 0.01 U/ ⁇ l to about 5 U/ ⁇ l, from about 0.01 U/ ⁇ l to about 4 U/ ⁇ l, from about 0.01 U/ ⁇ l to about 3 U/ ⁇ l, from about 0.01 U/ ⁇ l to about 2 U/ ⁇ l, from about 0.01 U/ ⁇ l to about 1 U/ ⁇ l, from about 0.01 U/ ⁇ l to about 0.5
  • Antioxidant An antioxidant inhibits the oxidation of other molecules.
  • Suitable antioxidants for use in the present disclosure may include, but are not limited to, DTT (dithiothreitol), TCEP (tris(2-carboxyethyl)phosphine), NAC (N-acetylcysteine), beta-mercaptoethanol, glutathione, cysteine and cystine.
  • DTT dithiothreitol
  • TCEP tris(2-carboxyethyl)phosphine
  • NAC N-acetylcysteine
  • beta-mercaptoethanol glutathione
  • cysteine cystine
  • cystine cystine
  • DTT may be used in in vitro transcription reaction.
  • the concentration of the antioxidant, preferably of DTT, in the in vitro transcription mixture(s) described herein may be about 1 to about 50 mM, about 5 to about 48 mM, about 8 to about 47 mM, about 10 to about 46 mM, about 15 to about 45 mM, about 18 to about 44 mM, about 20 to about 43 mM, about 23 to about 42 mM, about 25 to about 41 mM or about 28 to about 40 mM.
  • the concentration may be about 20 mM.
  • the amine to be used in the present invention may be betaine (trimethylglycine).
  • concentration of the amine, preferably of betaine may be about 10 mM to about 2M, preferably it may be about 0.7 M to about 1.3 M.
  • the polyamine may be selected from the group consisting of spermine and spermidine.
  • the concentration of the polyamine may be from about 1 to about 25 mM, about 1 to about 20 mM, about 1 to about 15 mM, about 1 to about 10 mM, about 1 to about 5 mM, or about 1 to about 2.5 mM. Even more preferred the concentration of the polyamine may be about 2 mM. Most preferred may be a concentration of about 2 mM of spermidine.
  • Dnases are enzymes which hydrolyze DNA by catalyzing the hydrolytic cleavage of phosphodiester linkages in the DNA backbone. Suitable Dnases may be isolated from bovine pancreas and may be available from various suppliers such as Sigma-Aldrich, New England Biolabs, Qiagen and ThermoFisher. Preferably, Dnase may be free of any RNAse activity. In the method of the present disclosure, treatment with Dnase may be performed after the RNA in vitro transcription reaction by adding Dnase to reaction mixture used for RNA in vitro transcription.
  • a suitable amount of calcium chloride may be added together with the Dnase to the RNA in vitro transcription mixture.
  • the suitable amount of CaCL may be from about 1 to about 5 mM, preferably from about 2 to about 4 mM and more preferably it may be about 3 mM.
  • DNA may be treated with the Dnase for about 1 to about 5 hours, preferably for about 1 .5 to about 3 hours and more preferably for about 2 hours.
  • Dnase treatment may be preferably performed at a temperature of about 37°C.
  • about 3 mM CaCL and about 200 U/ml Dnase I may be added to RNA in vitro transcription mixture and the resulting mixture may be incubated for about two hours at about 37°C.
  • about 3 mM CaCl2 and about 400 U/ml Dnase I may be added to RNA in vitro transcription mixture and the resulting mixture may be incubated for about two hours at about 37°C.
  • Dnase may be added to RNA in vitro transcription mixture and the resulting mixture may be incubated for about 30 minutes at about 31 °C.
  • Dnase treatment can be stopped by adding EDTA or another chelating agent.
  • Dnase treatment may be stopped by adding EDTA to a final concentration of about 25 mM.
  • Embodiments of the present disclosure may provide a combined solution for in vitro synthesis of mRNA of different sizes ranging from, for example without limitation, about 1 Kb-20Kb, such as 1 Kb-15Kb, or 1 Kb-10Kb, with high capping efficiency, uniform poly A tail, high yields and integrity in a time efficient manner.
  • Embodiments of the present disclosure may include design of T7 promoter sequence in a DNA template to provide high affinity to cap analogue to transcribe a capped mRNA with T7 RNA polymerase in vitro.
  • This DNA template promoter design may include a T7 ⁇ t>6.5 promoter followed by a sequence GG. This design can ensure efficient initiation of transcription to prepare a capped mRNA with high fidelity at 5’ end by one step method.
  • One of the key factors that determines mRNA translation efficiency may be its 5’ end capping.
  • capping is performed using capping enzymes, and although the efficiency of the capping may be high, the process is time consuming and costly.
  • Ishikawa demonstrated the use of trinucleotide cap analogues of m7GpppA*pG structure (wherein A* is adenosine or methylated adenosine derivative) to prepare capped mRNA through one step in vitro transcription.
  • A* is adenosine or methylated adenosine derivative
  • Ishikawa obtained a reporter 5’capped mRNAs carrying A, A m , m6 A, or m6 A m as the first transcribed nucleotide and studied their translational properties in rabbit reticulocyte system.
  • Another study also confirmed the co- transcriptional capping of the mRNA using cap analogue.
  • methods and/or compositions that increase capping efficiency may be provided.
  • methods and/or compositions that increase the capping efficiency to greater than about 50% efficiency, greater than about 55% efficiency, greater than about 60% efficiency, greater than about 65% efficiency, greater than about 70% efficiency, greater than about 75% efficiency, greater than about 80% efficiency, greater than about 85% efficiency, greater than about 90% efficiency, greater than about 95% efficiency, greater than about 96% efficiency, greater than about 96.5% efficiency, greater than about 97% efficiency, greater than about 97.5% efficiency, greater than about 98% efficiency, greater than about 98.5% efficiency, greater than about 99% efficiency, greater than about 99.5% efficiency may be provided as measured by a method of cutting mRNA with Rnase H, followed with measuring the capping efficiency with LC-MS.
  • cap analogues can initiate in vitro transcription to synthesize a capped mRNA in one pot reaction.
  • the first base of methyl-A after the inverted G cap may bind to the -1 position of the DNA template, and the second base of G may bind to +1 position of the DNA template, forming a complex with RNA polymerase to recruit the next ribonucleoside triphosphate (NTP) to elongate the RNA during the transcription process.
  • NTP next ribonucleoside triphosphate
  • a cap-2 analogue m 7 GpppA2-omeG2-ome
  • the first base of methyl-A after inverted G cap binds to -1 position of the DNA template
  • the second base of G binds to +1 position of the DNA template, forming the complex with T7 RNA polymerase to recruit the next NTP to elongate the RNA during the transcription process.
  • a composition comprising one or more cap analogue, as described herein, is provided.
  • a method utilizing one or more cap analogue, as described herein is provided.
  • cap analogues as described herein may be used in conjunction with compositions and/or methods described herein and/or in conjunction with traditional compositions and/or methods.
  • methods and/or compositions described herein may increase mRNA capping efficiency to greater than about 50% efficiency, greater than about 55% efficiency, greater than about 60% efficiency, greater than about 65% efficiency, greater than about 70% efficiency, greater than about 75% efficiency, greater than about 80% efficiency, greater than about 85% efficiency, greater than about 90% efficiency, greater than about 95% efficiency, greater than about 96% efficiency, greater than about 96.5% efficiency, greater than about 97% efficiency, greater than about 97.5% efficiency, greater than about 98% efficiency, greater than about 98.5% efficiency, greater than about 99% efficiency, greater than about 99.5% efficiency, or up to about 100% efficiency.
  • the promoter design in a DNA template may be important for DNA dependent RNA polymerase to initiate in vitro transcription.
  • the DNA template promoter design may include a T7 ⁇ t>6.5 promoter followed with a sequence GG, GA or AGG.
  • this design may enable the highly efficient initiation of transcription to prepare a mRNA with high fidelity at 5’end, by one step method.
  • the promoters may have a sequence of
  • TAATACGACTCACTATAX1X2X3 (SEQ ID NO: 16), wherein Xi is A or G, X2 is A or G, and X3 is A, T, G, or C,
  • At least one promoter sequences in Table 1 may be used to initiate in vitro transcription.
  • the promoters can be added into the plasmid vector through gene synthesis or subcloning.
  • a composition comprising one or more promoter, as described herein is provided.
  • a vector comprising one or more promoter, as described herein is provided.
  • a method utilizing one or more promoter, as described herein is provided.
  • a compositions comprising mRNA comprising one or more promoter, as described herein is provided.
  • a vector comprising mRNA comprising one or more promoter, as described herein is provided.
  • a method utilizing mRNA comprising one or more promoter, as described herein is provided.
  • in vitro transcription may start at -1 position, which may help to form a more favorable complex with T7 RNA polymerase and in turn to produce more full length RNAs.
  • -1 and +1 position of starting site for in vitro transcription allows for more flexibility on the choice of first mRNA base (not including cap base) and leave +2 position open for custom sequence as described in Table 1 for mRNA production.
  • Current general practice for incorporating cap molecule during in vitro transcription uses +1 position for initiation of the mRNA synthesis, which requires the template has the exact sequence of AG or AT following T7 promoter sequence of TATA box.
  • Embodiments of the present disclosure may include a method of using DNA template with regular T7 promoter sequences, which has GG following TATA box of T7 promoter, which does not require specific mutagenesis on DNA template for preparing cap-1 mRNA co-transcriptionally.
  • a method for in vitro transcription of a DNA template into RNA may include providing (1 ) a DNA template comprising a promoter operably linked to a nucleic acid containing a 5’ untranslated region (5’ UTR), an open reading frame (ORF) encoding the RNA of interest, a 3’ UTR, and a poly A region, and (2) a cap analogue containing the structure of Formula (I) or (II): in which wherein R is H or CH 3 ; and Bi is A or N6-methyl-adenine (m 6 A), and
  • B 2 is A, U, G, or C,
  • Ri is OCH3 and R 2 is OH or H, or
  • R 1 is OH and R 2 is H, or
  • R 1 is H and R 2 is H or OCH3, or
  • R 1 and R 2 are each OCH3,
  • Bi is A or N6-methyl-adenine (m 6 A), and B2 is A, II, G, or C. in which the promoter may contain a sequence of TAATACGACTCACTATAX1X2X3 (SEQ ID NO: 16), in which A at position 17 is -1 nucleotide and Xi at position 18 is +1 nucleotide, when Xi is G, X2 and X3 are each A, T, G, or C, then Bi is A or m 6 A and B2 is G, when Xi is A, X2 and X3 are each A, T, G, or C, then Bi is A or m 6 A and B2 is A, when Xi is C, X2 and X3 are each A, T, G, or C, then Bi is A or m 6 A and B2 is C, and when Xi is T, X2 and X3 are each A, T, G, or C, then Bi is A or m 6 A and B2 is C,
  • a method for in vitro transcription of a DNA template into RNA may include providing (1 ) a DNA template comprising a promoter operably linked to a nucleic acid containing a 5’ untranslated region (5’ UTR), an open reading frame (ORF) encoding the RNA of interest, a 3’ UTR, and a poly A region, and (2) a cap analogue, in which the cap analogue binds to -1 and +1 nucleotides of the promoter, and incubating the DNA template and the cap analogue in a reaction mixture, in which the incubating may include incubating the reaction mixture at from about 15°C to about 35°C, preferably from about 18°C to about 31 °C, for an appropriate time preferably from about 1 hour to about 12 hours, thereby producing the RNA.
  • a DNA template comprising a promoter operably linked to a nucleic acid containing a 5’ untranslated region (5’ UTR), an open reading frame (ORF) encoding the
  • a method for in vitro transcription of a DNA template into RNA may include providing (1 ) a DNA template comprising a promoter operably linked to a nucleic acid containing a 5’ untranslated region (5’ UTR), an open reading frame (ORF) encoding the RNA of interest, a 3’ UTR, and a poly A region, and (2) a cap analogue, in which the cap analogue binds to -1 and +1 nucleotides of the promoter, and incubating the DNA template and the cap analogue in a reaction mixture, thereby producing the RNA.
  • a DNA template comprising a promoter operably linked to a nucleic acid containing a 5’ untranslated region (5’ UTR), an open reading frame (ORF) encoding the RNA of interest, a 3’ UTR, and a poly A region
  • ORF open reading frame
  • the reaction mixture comprises NTPs and an RNA polymerase.
  • the reaction mixture may further comprise one or more of the following: a buffer substance, an Rnase inhibitor, a magnesium salt, a polyamine, and a pyrophosphatase.
  • the reaction mixture comprises: a buffer substance in a concentration of from about 45 mM to about 55 mM, an Rnase inhibitor in a concentration of from about 0.01 U/ ⁇ l to about 0.03 U/ ⁇ l, NTPs in a concentration of from about 1 mM to about 10 mM, the cap analogue in a concentration of from about 6 mM to about 8 mM, one or more magnesium salts in a concentration of from about 20 mM to about 30 mM, a polyamine in a concentration of from about 1 .5 mM to about 2.5 mM, the DNA template in a concentration of from about 0.01 ⁇ g/ ⁇ l to about 0.05 ⁇ g/ ⁇ l, a pyrophosphatase in a concentration of from about 0.1 mU/ ⁇ l to about 0.5 mU/ ⁇ l, and an RNA polymerase in a concentration of from about 0.01 ⁇ g/ ⁇ l to about 0.05 ⁇ g/ ⁇ l.
  • mRNA molecule may be flanked with 5’ end and 3’ end untranslated regions (UTR).
  • 5’-UTR may be recognized by ribosome to allow for initiation of translation, and 3’UTR may contain regulatory sequences that can influence the expression and halflife of the mRNA.
  • 3’UTR may contain regulatory sequences that can influence the expression and halflife of the mRNA.
  • Cao, et al. (Cao, et al., “High-throughput 5’UTR engineering for enhanced protein production in non-viral gene therapies”, Nature Communications, (2021 ) 12:4138, pages 1-10, incorporated herein by reference in its entirety) has reported a method of generating artificial 5' UTR 2 through a high-throughput screening process.
  • the combination of artificial selected 5’ UTR, and human hemoglobin or mouse hemoglobin 3’ UTR can be used to generate a construct for highly efficient expressing mRNA sequences.
  • the combination thereof may be selected and used to construct a vector for in vitro transcription (IVT). That may be used to generate mRNA with highly efficient protein expression capability.
  • IVT in vitro transcription
  • one or more UTR set forth in Table 2 (see below, in Example 1 ) is used.
  • one or more UTR set forth in Table 2 may be used in any combination.
  • the UTR 2 may be used in pairs, as set forth in Table 2, with the members of pairs being in the same row.
  • SEQ ID NO. 1 is paired with SEQ ID NO. 2, SEQ ID NO.
  • use of one or more of the UTR 2 set forth in Table 5 may enable high expression efficiency of mRNA when expressed in mammalian cells. In embodiments, use of one or more pairs of the UTR 2 set forth in Table 5 may enable high expression efficiency of mRNA when expressed in mammalian cells.
  • Different 5’ UTR and 3’ UTR pairs may be cloned into vector in an orientation, for example, from 5’ to 3’ direction: a promoter, e.g., T7 promoter (SEQ ID NO: 10), 5’ UTR, Kozak sequence (GCCACC), eGFP coding sequence, and 3’ UTR.
  • Vector can then be sub-cloned into pVAX vector for generating plasmid for mRNA preparation.
  • Example of 5’ UTR and 3’ UTR pairs are shown in Table 2. Table 2
  • the T7 promoter sequences, UTR 2 may be added to the open reading frame coding sequences through gene synthesis, which may be then sub-cloned into the plasmid vector to generate large scale plasmid DNA for in vitro transcription application.
  • a composition comprising one or more UTR, as described herein, is provided.
  • a vector comprising one or more UTR, as described herein, is provided.
  • a method utilizing one or more UTR, as described herein is provided.
  • a compositions comprising mRNA comprising one or more UTR, as described herein is provided.
  • a vector comprising mRNA comprising one or more UTR, as described herein is provided.
  • a method utilizing mRNA comprising one or more UTR, as described herein is provided.
  • Poly-A tail quality may directly impact mRNA expression efficiency.
  • Traditional methods for adding poly A tail to a mRNA product in vitro use poly A polymerase. See, e.g., Cao, G.J. and Sarkar, N., “Identification of the gene for an Escherichia coli poly(A) polymerase”, Proc. Natl. Acad. Sci. USA, (1992) 89(21), 10380-10384, incorporated herein by reference in its entirety.
  • such methods usually generate a product with a broad distribution of poly A tail lengths, with only about 70% of mRNA being tailed.
  • mRNA purity and integrity may be important factors that influence the properties of mRNA, such as, but not limited to, its stability and/or expression efficiency. Purification, promoter sequence, 5’ and/or 3’ UTR sequence(s), and/or transcription conditions may contribute to generating a high quality mRNA.
  • a traditional method for adding poly A tail to a mRNA product in vitro uses poly A polymerase as described in, for example, Cao et al. (Proc.Natl. Acad. Sci. USA.
  • this method may generate poly A products with broad distribution of poly A tail length, e.g., with only about 70% of mRNA polyadenylated.
  • methods and/or compositions may include provision of capped analogues, methods of providing and/or improving length and/or distribution of poly-A tails, provision of effective promoters, provision of effective UTR 2 , such as pairs of UTR 2 , provision of effective transcription conditions, provision of an effective transcription system, provision of effective purification, or any combination thereof.
  • methods and/or compositions that increase the uniformity of length and/or distribution of poly-A tails among the transcribed mRNA molecules may be provided.
  • methods and/or compositions may produce a population of mRNAs wherein greater than about 70% are tailed, wherein at least about 71 % are tailed, wherein at least about 72% are tailed, wherein at least about 73% are tailed, wherein at least about 74% are tailed, wherein at least about 75% are tailed, wherein at least about 76% are tailed, wherein at least about 77% are tailed, wherein at least about 78% are tailed, wherein at least about 79% are tailed, wherein at least about 80% are tailed, wherein at least about 85% are tailed, wherein at least about 90% are tailed, wherein at least about 95% are tailed, wherein at least about 99% are tailed.
  • methods and/or compositions may produce a population of mRNAs wherein the lengths (the number of adenines) of the poly-A tails vary by at most about 70 to about 130 adenines between the mRNA molecules, by at most about 60 to about 120 adenines between the mRNA molecules, by at most about 50 to about 100 adenines between the mRNA molecules, by at most about 40 to about 90 adenines between the mRNA molecules, by at most about 50 to about 80 adenines between the mRNA molecules, by at most about 40 to about 70 adenines between the mRNA molecules, by at most about 30 to about 50 adenines between the mRNA molecules, or by at most about 20 to about 40 adenines between the mRNA molecules.
  • the poly A tail length of mRNA may be measured by a method to digest the mRNA with Rnase T1 , followed by purifying and recovering the poly A fragment with oligo-dT magnetic beads, and then test the poly A fragment length by Bioanalyzer capillary gel electrophoresis.
  • a poly-A tail is added to a DNA template before transcription.
  • a method of adding a poly-A tail to a DNA template prior to transcription may be provided.
  • a poly-A tail is added to a DNA template via Polymerase Chain Reaction (PCR), which is a new way of adding poly A tail comparing to traditional method by gene synthesis to insert into the plasmid vector.
  • PCR Polymerase Chain Reaction
  • addition of a poly-A tail to the DNA template may result in the generation of a more uniformly poly-A-tailed mRNA product, a product with longer poly-A tails, or both, each or both of which may result in more efficient mRNA expression.
  • a composition comprising mRNA comprising a poly-A tail added by PCR, as described herein, is provided.
  • a vector comprising a poly-A tail added to the vector by PCR, as described herein is provided.
  • a pVAX1 or pUC57 vector comprising ampicillin resistant gene, T7 promoter sequences, 5’ UTR and 3’ UTR sequences, mRNA comprising a poly-A tail (e.g., 100A) added by PCR, as described herein, is provided.
  • disclosed methods and/or compositions may produce a population of mRNAs wherein greater than about 70% are tailed, wherein at least about 71 % are tailed, wherein at least about 72% are tailed, wherein at least about 73% are tailed, wherein at least about 74% are tailed, wherein at least about 75% are tailed, wherein at least about 76% are tailed, wherein at least about 77% are tailed, wherein at least about 78% are tailed, wherein at least about 79% are tailed, wherein at least about 80% are tailed, wherein at least about 85% are tailed, wherein at least about 90% are tailed, wherein at least about 95% are tailed, wherein at least about 99% are tailed, wherein about 100% are tailed.
  • disclosed methods and/or compositions may produce a population of mRNAs wherein the lengths (the number of adenines) of the poly-A tails vary by at most about 70 to about 130 adenines between the mRNA molecules, by at most about 60 to about 120 adenines between the mRNA molecules, by at most about 50 to about 100 adenines between the mRNA molecules, by at most about 40 to about 90 adenines between the mRNA molecules, by at most about 50 to about 80 adenines between the mRNA molecules, by at most about 40 to about 70 adenines between the mRNA molecules, by at most about 30 to about 50 adenines between the mRNA molecules, or by at most about 20 to about 40 adenines between the mRNA molecules.
  • Common vectors may be typically maintained at high copy number and may induce transcription and translation of antibiotic resistance gene, indicator gene (such as blue/white screening gene) and inserted fragments, causing instability of certain classes of DNA sequences.
  • indicator gene such as blue/white screening gene
  • polyA sequences may be easily lost in the process of plasmid cloning and replication. If such a sequence occurs, unexpected transcription and translation affected by the inductive activity of upstream and downstream promoters may further increase instability of polyA sequences.
  • common strategies may include segmenting the inserted gene (if the inserted gene is significantly cytotoxic) or directional cloning of the ORF in the “reverse" orientation relative to transcription from the vector’s promoter.
  • these two strategies may be not suitable for gene cloning that contain polyA sequences. Segmentation can eliminate the genotoxicity of cloned genes after being expressed, but this strategy may not solve the problem of instability of polyA sequences because the process of transcription has not been eliminated, while the reverse insertion can only eliminate the influence of the promoter in a certain direction.
  • embodiments of the present disclosure may include modifying vectors by inserting transcriptional terminators at the upstream and downstream of the multiple cloning site, which can effectively interrupt the influence of the upstream and downstream promoters of the multiple cloning site on the inserted gene.
  • This strategy effectively improves the stability of polyA sequences in the process of plasmid cloning and replication, especially for some extremely unstable polyA-containing cassette.
  • the results show that, after adding transcriptional terminators to the upstream and downstream of the multiple cloning site, the positive rate of clones reached 25%-50% (almost 0 before adding terminator), and the number of A bases of polyA tails increased from 70-110 to about 120.
  • the yield of plasmid also increased by 32.5%. Without being limited to a particular theory, this increase may be due to the insertion of a transcriptional terminator restores the activity of the origin of replication (Stueber et al., 1982. Transcription from efficient promoters can interfere with plasmid replication and diminish expression of plasmid specified genes. EMBO J 1 : 1399-1404; the content of which is hereby incorporated by reference in its entirety).
  • transcriptional terminators at the upstream and downstream of the multiple cloning site to facilitate the cloning of cytotoxic sequences or sequences of repeats, such as PolyA tails, has not been reported in the art.
  • a transcriptional termination sequence may be any nucleotide sequence, which when placed transcriptionally downstream of a nucleotide sequence encoding an open reading frame, causes the end of transcription of the open reading frame.
  • Such sequences are known in the art and may be of prokaryotic, eukaryotic or phage origin.
  • terminator sequences include, but are not limited to, PTH- terminator, pET-T7 terminator, T3-Tcp terminator, pBR322-P4 terminator, vesicular stomatitis virus terminator, rrnB-T1 terminator, rrnB-T2 terminator, lambda tO terminator, rrnC terminator, Ttadc transcriptional terminator, and yeast-recognized termination sequences, such as Mata (a-factor) transcription terminator, native a- factor transcription termination sequence, ADR 1 transcription termination sequence, ADH2 transcription termination sequence, and GAPD transcription termination sequence.
  • Mata (a-factor) transcription terminator native a- factor transcription termination sequence
  • ADR 1 transcription termination sequence ADH2 transcription termination sequence
  • GAPD transcription termination sequence GAPD transcription termination sequence
  • transcriptional terminator sequences may be found in the iGEM registry, which is available at: partsregistry.org/Terminators/Catalog.
  • the first transcriptional terminator sequence of a series of 2, 3, 4, 5, 6, 7, or more may be placed directly 3’ to the final nucleotide of a gene of interest (or open reading frame) or at a distance of at least 1 -5, 5-10, 10-15, 15-20, 20-25, 25-30, 30-35, 35-40, 40-45, 45-50, 50-100, 100-150, 150-200, 200-300, 300-400, 400-500, 500-1 ,000 or more nucleotides 3’ to the final nucleotide of a gene of interest (or open reading frame).
  • transcriptional terminator sequences may be varied, for example, transcriptional terminator sequences may be separated by 0, 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 10-15, 15-20, 20-25, 25-30, 30-35, 35-40, 40-45, 45-50 or more nucleotides.
  • a vector including one or more of (i) a poly-A tail, as described herein, (ii) one or more UTR, as described herein, (iii) one or more promoter, as described herein, or (iv) combinations thereof, is provided.
  • the vector may have various uses, including, but not limited to, use for insertion of a target template nucleotide sequence.
  • such a vector with a target template nucleotide sequence may be used for, as non-limiting examples, cloning or transcribing the target template nucleotide sequence.
  • transcription may include in vitro transcription.
  • transcription may be carried out using a T7 RNA polymerase.
  • a vector may be a plasmid or a viral vector, for example but without limitation, pVAX1 and/or pUC57.
  • the integrity of mRNA may affect cellular expression; therefore it may be important to begin translation with high integrity mRNA.
  • the conditions used for transcription may affect the quality of mRNA produced. Some transcription conditions can lead to higher truncated products.
  • the integrity of the mRNA may be one of the critical factors that affects cellular expression, therefore it may be paramount to begin with high integrity mRNA.
  • mRNA integrity There are two aspects to the mRNA integrity. Typically, RNA is prone to quicker degradation than DNA due its chemical instability, therefore the storage conditions impact the quality of the RNA. Additionally, transcription conditions can also lead to higher truncated products and therefore the optimal buffer conditions can be determined empirically to have higher integrity over a wide range of mRNA sizes and also have higher yields.
  • Conditions used for purification may affect the quality of mRNA produced.
  • Trinucleotide cap analogues of m7 GpppA*pG residues in the finished mRNA product could compete with capped mRNA for recruiting ribosome, thus inhibiting the mRNA translation efficiency in cells.
  • Purification after in vitro transcription may be an important step in acquiring final purified mRNA product.
  • the present disclosure further provides a solution to obtain pure mRNA product with minimum contaminant cap analogues, free NTPs, and other proteins, which could interfere and compromise the mRNA product’s performance.
  • mRNA purity refers to full length mRNA species ratio in the crude transcribed mRNA product, which may be quantified by the full length peak ratio in capillary gel electrophoresis.
  • purification as described herein may produce highly pure mRNA product with minimum contaminant analogues, free NTPs, and/or other proteins which could interfere with and/or compromise the mRNA product’s stability and/or translation efficiency.
  • a purification method includes using a silica membrane column to bind nucleic acid, then washing with about 60% to about 80% ethanol in water, preferably about 70% to about 80% ethanol in water, followed by elution in water.
  • methods and/or compositions that increase purity of transcribed mRNA may be provided. In embodiments, methods and/or compositions that result in obtained mRNA with about 79% or greater purity; mRNA with about 79.5% or greater purity; mRNA with about 80% or greater purity; mRNA with about
  • mRNA with about 92% or greater purity mRNA with about 93% or greater purity
  • mRNA with about 94% or greater purity mRNA with about 95% or greater purity
  • mRNA with about 96% or greater purity mRNA with about 97% or greater purity
  • mRNA with about 98% or greater purity mRNA with about 99% or greater purity
  • mRNA with about 100% purity may be provided.
  • the mRNA purity may be measured by capillary gel electrophoresis using Bio analyzer equipment, the target peak area ratio (target length ⁇ 15% ) may be calculated for its purity measurement.
  • methods and/or compositions that increase the purity or integrity of transcribed mRNA may be provided. Obtaining high quality long mRNA from in vitro transcription is a great challenge since long mRNAs tends to degrade more easily.
  • IVT may be performed at from about 15°C to about 35°C, from about 16°C to about 35°C, from about 17°C to about 35°C, from about 18°C to about 35°C, from about 18°C to about 34°C, from about 18°C to about 33°C, from about 18°C to about 32°C, from about 18°C to about 31 °C, from about 18°C to about 30°C, from about 18°C to about 29°C, from about 18°C to about 28°C, from about 18°C to about 27°C, from about 18°C to about 26°C, from about 18°C to about 25°C, from about 18°C to about 24°C, from about 18°C to about 23°C, from about 18°C to about 22°C, from about 18°C to about 21 °C, from about 18°C to about 20°C, from about 18°C to about 19°C, from about 25°C to about 26°C, from about 25°C to about 26°C, from about 25°
  • IVT may be performed for from about 1 hour to about 12 hours, from about 1 hour to about 11 hours, from about 1 hour to about 10 hours, from about 1 hour to about 9 hours, from about 1 hour to about 8 hours, from about 1 hour to about 7 hours, from about 1 hour to about 6 hours, from about 1 hour to about 5 hours, from about 1 hour to about 4 hours, from about 1 hour to about 3 hours, from about 1 hour to about 2 hours, from about 2 hours to about 12 hours, from about 3 hours to about 12 hours, from about 4 hours to about 12 hours, from about 5 hours to about 12 hours, from about 6 hours to about 12 hours, from about 7 hours to about 12 hours, from about 8 hours to about 12 hours, from about 9 hours to about 12 hours, from about 10 hours to about 12 hours, from about 11 hours to about 12 hours, about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, or about 10 hours.
  • IVT may be performed at about 31 °C for from about 1 hour to about 5 hours, from about 1 hour to about 4.5 hours, from about 1 hour to about 4 hours, from about 1 hour to about 3.5 hours, from about 1 hour to about 3 hours, from about 1 hour to about 2.5 hours, from about 1 hour to about 2 hours, from about 1 hour to about 1 .5 hours, from about 0.5 hour to about 1 hour, from about 0.5 hour to about 1 .5 hours, about 1 hour, about 2 hours, about 3 hours, about 4 hours, or about 5 hours.
  • RNA polymerase II messenger RNAs
  • T7 RNAP T7 RNA Polymerase
  • RNA polymerase exists in at least two protein states. The first is referred to as the “abortive complex” and may be associated with transcriptional initiation. The second is a very processive conformation called the “elongation complex.”
  • In vitro transcription can be broken into six steps: 1 ) binding of RNA polymerase to promoter sequence, 2) initiation of transcription, 3) non-processive elongation termed abortive transcription, during which polymerase frequently releases DNA template and short abortive transcripts, 4) conversion of open complex to closed complex, 5) processive elongation, and 6) transcriptional termination.
  • Significant amount of RNA produced during transcription may contain short abortive fragments of ⁇ 2-8 nucleotides in length (Biochemistry 19:3245-3253 (1980); Nucleic Acids Res.
  • RNA polymerases may escape from abortive cycling, at the same time losing sequence-specific contacts with promoter DNA, and forming a processive elongation complex, in which RNA chain may be extended in a sequenceindependent manner (J. Mol. Biol. 183:165-177(1985); Proc. Natl. Acad. Sci. U.S.A. 83:3614-3618(1986); Mol. Cell Biol. 7:3371 -3379 (1987), each of which is incorporated herein by reference in its entirety).
  • the consensus sequence for the most active Class III T7 promoters may encompass 17 bp of sequence upstream, and 6 bp downstream, of the transcription start site (Cell 16:815-25. (1979), incorporated by reference herein in its entirety).
  • the position of the first transcribed nucleotide is commonly referred to as the +1 transcript nucleotide of the RNA, the second transcribed nucleotide as +2 transcript nucleotide and so on (Table 3).
  • two strands may be melted to form transcription bubble and the bottom strand of the duplex (shown 3' to 5' in Table 3) is the template for transcription.
  • the template strand may define the identity of the transcribed nucleotides primarily through Watson- Crick base pairing interactions.
  • the nucleotide encoding the first RNA transcript nucleotide is defined as the +1 nucleotide of the template.
  • the +1 transcript nucleotide is G and the +1 template nucleotide is C.
  • the +4 transcript nucleotide is A and the +4 template nucleotide is T.
  • T7 RNAP can also initiate with short oligonucleotide primers.
  • 13 promoters in the T7 genome can initiate with pppGpG (J. Mol. Biol. 370:256-268 (2007), incorporated by reference herein in its entirety). It has been shown that T7 RNAP can initiate from dinucleotide primers
  • RNA in transcription reaction theoretically to less than 1 .4 mg/mL.
  • the method described herein may not require restricting the concentration of any NTP to achieve both an efficient RNA capping and a higher yield of RNA (about 2 to 10 mg/mL) and thus allowing a production of high quality mRNA at a commercially useful cost.
  • T7 RNA Polymerases at least one modification of the T7 RNA polymerase may be selected from the group consisting of P266L, P270L, P270S, P270A, P270Y, Q744L, Q744P, Q744R, Y639F, H784A, E593G, Y639V, V685A, H784G, S430P, N433T, S633P, F849I and F880Y.
  • at least one modification includes Y639F and H784A.
  • at least one modification includes E593G, Y639V, V685A and H784G.
  • At least one modification includes S430P, N433T, S633P, F849I and F880Y. In some embodiments, at least one modification includes S430P, N433T, S633P, F849I , F880Y and P266L. In some embodiments, at least one modification includes S430P, N433T, S633P, F849I , F880Y, Y639F and H784A. In some embodiments, at least one modification includes S430P, N433T, S633P, F849I , F880Y, P266L, Y639F and H784A.
  • At least one modification includes S430P, N433T, S633P, F849I , F880Y, E593G, Y639V, V685A and H784G. In some embodiments, at least one modification includes S430P, N433T, S633P, F849I , F880Y, P266L, E593G, Y639V, V685A and H784G.
  • At least one modification of the T7 RNA polymerase facilitates initiation-elongation transition. In some embodiments, at least one modification increases promoter clearance. In some embodiments, at least one modification increases stability and/or activity of the polymerase. In some embodiments, at least one modification increases thermos stability of the polymerase.
  • Magnesium ions is an essential component in an RNA in vitro transcription buffer system to initiate the transcription with Cap analogues rather than GTP.
  • Conventional buffer systems for RNA in vitro transcription e.g., HEPES buffer, Tris-HCI buffer
  • HEPES buffer, Tris-HCI buffer may contain high concentrations of free magnesium ions, because free Mg 2+ ions may be required to guarantee a high activity of the RNA polymerase enzyme.
  • Mg 2+ complexes with NTP during the reaction keeping no extra free Mg 2+ ions present in buffer systems is important for ensuring high capping efficiency and high integrity of transcribed mRNA.
  • Mg 2+ can cause problems, especially in the context of high- yield/industrial-scale RNA production.
  • Some of the problems associated with free Mg 2+ ions in the production of RNA may include magnesium-driven precipitates, which may lead to a drop in the free Mg 2+ concentration, resulting in depletion of magnesium ions from the RNA polymerase reaction center. A consequence of that would be a less efficient RNA in vitro transcription.
  • IVT were performed in the presence of increasing Mg concentrations.
  • the concentration of free Mg 2+ after complexing with NTPs and cap analogues were at -12 mM, -8 mM, 0 mM and +8 mM, respectively, in the IVT reaction system.
  • the mRNA yield reduced when Mg 2+ concentration increased, and the mRNA integrities also reduced when Mg 2+ concentration increases in the IVT system.
  • 5’-DMT-2’-O-methyl-guanosine (n-ibu) and 5’-DMT-2’-O-methyl-N6-methyl- adenosine 3’-phosphoramidite were purchased from ChemGenes, Inc., (Wilmington, MA).
  • Chemical Phosphorylation reagent [5’-phosphate amidite (O-DMT-2,2 - sulfonyldiethanol)] was purchased from Biosearch Technologies.
  • TLCs were run with aluminium-backed silica gel 60 F254 plates purchased from Sigma-Aldrich. 1 H and 31 P NMR spectra were taken on a Bruker 300 MHz spectrometer.
  • HPLC chromatograms were obtained on an Agilent 1290 Infinity II system employing a HALO 90A C18 column (4.6 x 150 mm) and gradient elution with 50 mM TEAB buffer, pH 7.8 / acetonitrile. TLC and/or HPLC analyses were routinely used for monitoring reaction completion. UV spectral data were obtained on a VWR UV-3100PL spectrophotometer. Buffer solutions were prepared using water dispensed from a Mill- Q EQ 7000 ultrapure water system. Reported compounds were prepared as previously described: 2a: Sawai et al. (Synthesis and Reactions of Nucleoside 5‘- Diphosphate Imidazolide.
  • WO2022036858 discloses methods of preparing cap-2 analogues using enzymes including phosphohydrolase, guanosyltransferase, and T4 RNA Ligase 1. In contrast, embodiments of the present disclosure may not include enzymes in the preparation of cap-2 analogues. A comparison between the methods disclosed in WO2022036858 and that in the present disclosure is shown below.
  • Reagent 100 mg, 0.152 mmol were dissolved in tetrazole solution (1.0 ml of a 0.45 M solution in anhydrous acetonitrile) and stirred for 2 h. under an argon atmosphere. To this solution was added CSO oxidizer (1 .0 ml of a 0.50 M solution in acetonitrile) and the resulting solution was stirred for 20 min and then the solvents were stripped. The residue was redissolved in a 50/50 mixture of methanol/ammonium hydroxide and the sealed reaction vessel was incubated at 37 C for 2 days.
  • reaction solution was then evaporated to dryness, redissolved in 12 ml of water and loaded onto a DEAE Sephedex A25 column and eluted with a gradient of 0 -> 0.60 M TEAB buffer, pH 7.
  • the pure product fractions were pooled and evaporated to dryness.
  • Excess TEAB buffer salt was removed by performing multiple evaporations from water.
  • Both linearized DNA plasmid and PCR product can be used as DNA template for preparing mRNA by in vitro transcription.
  • plasmid vector containing T7 promoter SEQ ID NO: 10 or 12
  • 5 -UTR SEQ ID NO: 9
  • poly A tail 100A
  • the linearized plasmid was transcribed with T7 RNA polymerase (wild-type), cap analogues (cap-2), 10x transcription buffer, NTPs, and RNase Inhibitor to prepare capped mRNA.
  • the in vitro transcription mixture and conditions were as set forth in Tables 4-6. More specifically, a 10x Buffer containing HEPES buffer was prepared by adding magnesium acetate, spermidine, and DTT and stored at -20°C for use. In the in vitro transcription reaction, the buffer, DNase-free and RNase-free water, NTPs (concentrations as specified in the tables), and cap analogue were added into a reaction tube, followed by adding DNA template, T7 polymerase, RNase inhibitor, and Inorganic pyrophosphatase. The reaction was kept at designated temperature ranging from 20-40°C for 1 to 6 hours for transcription to take place. Then, DNA templates were removed by digestion with DNasel RNase-free enzyme.
  • Cap analogues may be any capped analogues described herein.
  • cap-2 analogues m 7 GpppA20mepG20me and m 7 G3’0mepppA2’0mepG20me were used.
  • m 7 G3OmepppA2 o mepG2’0me a methoxy group (— OCH 3 ) replaces the 3 -OH group closer to the m7G.
  • This modification is analogous to the modification in the anti-reverse cap analogue (ARCA: 3'-O-Me-m7GpppG).
  • Stepinski et al. (Stepinski J, Waddell C, Stolarski R, Darzynkiewicz E, Rhoads RE (2001 ) Synthesis and properties of mRNAs containing the novel "anti-reverse" cap analogues 7-methyl(3’-O-methyl)GpppG and 7-methyl(3'deoxy)GpppG.
  • RNA 7: 1486-1495; the content of which is hereby incorporated by reference in its entirety) has shown that this 3’-0 modification in ARCA prevents the generation of mRNAs capped in the reverse orientation.
  • m 7 G3’omepppA2omepG2ome is referred to as ARCA-cap-2. IVT reactions were also carried out using the AG cap-1 analogue.
  • HEPES buffer was 400 mM HEPES in water, pH 7.5.
  • RNA polymerase can be wild type T7 RNA polymerase or T7 RNA polymerase with mutations to enhance stability and/or the ability to incorporate cap-2 analogues.
  • In vitro transcription can be set up in DNase-free RNase-free plastic tubes ranging from 0.2 mL to 15 mL at a defined temperature with or without shaking.
  • IVT1 Three IVT conditions (IVT1 , IVT2, and IVT3) were tested as specified in
  • Tables 4-6 show IVT1 conditions where equal concentrations of NTPs are used for IVT.
  • Table 5 shows IVT2 conditions where GTP concentrations are reduced as shown.
  • Table 6 shows IVT3 conditions where the concentration of ATP and GTP are reduced as shown.
  • mRNA was purified from IVT reactions using a silica membrane column. Briefly, mRNA from in vitro transcription was mixed with a buffer and ethanol, and added to silica membrane column, followed by washing with 70% ethanol and eluting with water or other storage buffer for mRNA.
  • the amount of silica column purified mRNAs was determined by Nanodrop to calculate the overall yield f IVT reactions, which is expressed as the amount (microgram) of mRNAs obtained per microliter of IVT reaction.
  • RNA/DNA hybrid probe was annealed to the mRNA, which was then digested with RNase H. The duplex containing the probe and 5’ end of the mRNA was then purified for LC-MS analysis.
  • eGFP mRNA thus prepared with promoter SEQ ID NO 10 or 12 was used for expression efficiency assay in A549 cells.
  • A549 cells were seeded the day before in 96-well clear-bottom black plates.
  • eGFP mRNA samples were transfected into the seeded cells in triplicates, at 200 ng/well of mRNA plus 0.4 ⁇ l of lipofectamine MessengerMax with OptiMEM.
  • the cells were incubated with mRNA overnight, then the expression efficiency was assessed based on relative fluorescence intensity of eGFP mRNA treated cells using a plate reader.
  • the values were normalized by relative cell number as tested by Cyquant XTT cell viability assay.
  • the normalized values for mRNAs prepared with cap-2 cap analogues were compared to those prepared with AG cap-1 cap analogue.
  • Plasmid templates encoding eGFP were used to generate mRNAs using the above conditions and procedures.
  • the mRNA yield for IVT reactions is presented in Table 7.
  • the mRNA yield using cap-2 and promoter #1 was 2.14 ⁇ g/ ⁇ l and 2.56 ⁇ g/ ⁇ l with promoter #2 (SEQ ID NO: 10).
  • the mRNA yield using ARCA-cap-2 was 2.08 ⁇ g/ ⁇ l using promoter #1 (SEQ ID NO: 12) and 2.06 ⁇ g/ ⁇ l using promoter #2 (SEQ ID NO: 10).
  • the mRNA yield using cap-2 and promoter #1 was 1.98 ⁇ g/ ⁇ l and 2.62 ⁇ g/ ⁇ l using promoter #2 (SEQ ID NO: 10).
  • the mRNA yield using ARCA-cap-2 and promoter #1 was 1 .8 ⁇ g/ ⁇ l and 2.85 ⁇ g/ ⁇ l using promoter #2 (SEQ ID NO: 10).
  • mRNAs prepared with AG cap-1 had 85-93% purity in the 900-1150 nt range, compared to 89-91 % and 89% respectively for mRNAs prepared with cap-2 and ARCA-cap-2.
  • mRNAs generated using cap-2 and ARCA-cap-2 and IVT1 conditions with either promoter #1 (SEQ ID NO: 12) or promoter #2 (SEQ ID NO: 10) showed poor expression as compared to mRNAs generated using AG cap-1 , e.g., at ⁇ 30% of AG cap-1 levels using promoter #1 (SEQ ID NO: 12) and -10% using promoter #2 (SEQ ID NO: 10).
  • promoter #2 (SEQ ID NO: 12) in modified IVT conditions (IVT2 and IVT3) led to significant increase in mRNA expression, which is consistent with their improved capping efficiencies. For example, relative to AG cap-1 mRNAs, >75% mRNA expression levels were obtained using cap-2 and ARCA-Cap II in IVT2 conditions. Nearly 60% and 80% mRNA expression levels, respectively, were obtained using cap-2 and ARCA-Cap II. Improved expression was also observed by using promoter #2 (SEQ ID NO: 10) in IVT2 and IVT3 conditions, albeit at lower levels.
  • cap-2 and ARCA-Cap II were observed by using cap-2 and ARCA-Cap II in IVT2 conditions and >20% of mRNA expression levels were observed by using cap-2 and ARCA-Cap II in IVT3 conditions.
  • cap reagent analog 11 (m 7 G2 OmepppA2 o mepG2’0me ) (Fig. 6)
  • reaction solution was diluted to a total of 1 .8 ml with a solution of ice-cold 0.2 M sodium perchlorate in acetone and the precipitate that formed was spun-down to a pellet. The supernatant was discarded, and the pellet was washed with 1 .8 ml of ice- cold acetone and again spun-down to a pellet. This acetone wash cycle was repeated four more times and then the product pellet was dried under high vacuum affording intermediate 10 as an off-white solid. This solid was combined with intermediate 5 (9.0 mg, 0.0098 mmol) and MgCL (3.2 mg, 0.025 mmol).
  • 3’-O-Methylguanosine-5’ -phosphate (12) was prepared according to Jemielity et al., RNA, 2003, 9, 1108-1122.
  • the triethylammonium salt of Intermediate 12 (132 mg, 0.28 mmol) was dissolved in 4.0 ml of water and the solution was adjusted to pH 4.0 with acetic acid.
  • Dimethyl sulfate (0.38 ml) was added dropwise over a 10 minute period and the reaction solution was stirred for 4 hours with addition of sodium hydroxide solution in order to maintain the reaction solution between pH 3.75-4.25.
  • reaction solution was extracted with methylene chloride (3 x 16 ml) and the crude product, contained in the aqueous phase, was purified by reverse phase (C18) HPLC employing a TEAB, pH 7.8 - acetonitrile gradient.
  • the pure product fractions were lyophilized to dryness affording a white solid: 110-mg (81 %) yield of intermediate 13 as the triethylammonium salt.
  • the pellet was suspended in 25 ml of ice-cold acetone, vortexed and centrifuged to form a pellet. The supernatant was again discarded and this process was repeated for 3-more cycles. The final pellet was dried under high vacuum affording the 5’-phosphoimidazolide analog of 13 as a white solid: 101-mg yield.
  • mice were injected with ALC0315-LNP formulation encapsulated Firefly luciferase (Flue) mRNA generated by IVT using Cap2 analog (Cap2-Fluc) or Cap1 analog (Cap1-Fluc) generated by IVT.
  • Flue Firefly luciferase
  • Cap2 analog Cap2-Fluc
  • Cap1 analog Cap1-Fluc
  • Cap1 analog (Cap1-AG):
  • FIG. 9 shows that the expression levels of Cap2-Fluc mRNA are similar to that of Cap1-Fluc mRNA. Biodistribution was also measured after 96 hours by bioluminescence.
  • FIG. 10 shows that the expression of Cap1-Fluc mRNA and Cap2-Fluc mRNA were mainly distributed to liver.
  • FIG. 11 shows that injection with Cap1 -Fluc mRNA or Cap2-Fluc mRNA did not affect mouse body weight as compared with that injected with PBS.
  • Advantages of the present disclosure may include (1) in vitro transcription reaction mixtures and conditions that can increase yield, integrity, and purity of mRNAs, (2) DNA templates and cap analogues that bind to -1 and/or +1 nucleotides of promoters for in vitro transcription, thus producing more full length mRNAs, allowing for more flexibility on the choice of first mRNA base, and providing +2 position open for custom sequence, and (3) cap-2 analogues with 2’-O-methylation of the second transcribed nucleotide and N6-methylation of adenosine as the first transcribed nucleotide serving as determinants defining transcripts as ‘self and thus contributing to transcript immune evasion.

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Abstract

A method for in vitro transcription of a DNA template into RNA includes providing a mixture containing a buffer substance, ribonucleoside triphosphates (NTPs), one or more magnesium salts in a concentration of from about 2 mM to about 60 mM, the DNA template, a recombinant RNA polymerase, and a cap analogue comprising the structure of Formula (I), (II), or (III), and incubating the reaction mixture at from about 15°C to about 35°C, optionally from about 18°C to about 31°C, for from about 1 hour to about 12 hours, thereby producing the RNA.

Description

IN VITRO TRANSCRIPTION METHODS AND COMPOUNDS FOR USE THEREIN
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] The present application is an International (PCT) Application claiming priority to U.S. Provisional Patent Application No. 63/482,688, filed on February 1 , 2023, the entire contents of which is hereby incorporated by reference for all purposes.
REFERENCE TO SEQUENCE LISTING SUBMITTED AS A COMPLIANT XML FILE
(.xml)
[0002] Pursuant to the EFS-Web legal framework and 37 C.F.R. § 1.821-825 (see M.P.E.P. § 2442.03(a)), a Sequence Listing in the form of - a compliant XML file (entitled "3000076-010977 Seq Listing ST26.xml" created on January 31 , 2024, and 36,110 bytes in size) is submitted concurrently with the instant application, and the entire contents of the Sequence Listing are incorporated herein by reference.
BACKGROUND
[0003] The present disclosure relates generally to methods for synthesizing mRNA capping analogues and methods and compositions for in vitro transcription.
[0004] mRNA is a well-defined molecule with structure of 5’ Cap, 5’ end untranslated region (UTR), open reading frame sequence coding for a gene(s) of interest, and 3’ end UTR, and poly A tail. Preparation of capped mRNA through in vitro synthesis may have significant importance for both fundamental scientific research and new therapeutics development. Several factors go into generating an mRNA that may have high expression levels, stability, and functionality.
[0005] An mRNA molecule may be flanked with 5’end and 3’end untranslated regions (UTR2). A 5’-UTR serves as the entry site for ribosomes to initiate translation, and a 3’ UTR plays an important role in translation termination and post transcriptional modification, which may influence the expression and half-life of mRNA. A poly A tail of mRNA may make the RNA molecule more stable and prevent mRNA degradation. Additionally, a poly A tail may allow the mature messenger RNA to be exported from nucleus and translated into a protein by ribosomes in the cytoplasm. See, e.g., Sachs A and Wahle E, “Poly(A) tail metabolism and function in eucaryotes”, J Biol Chem, (1993 Nov 5); 268(31 ):22955-8, incorporated herein by reference in its entirety.
[0006] An mRNA cap is a highly methylated modification at the 5’ end of mRNA, which may protect mRNA from degradation, recruit complexes involved in mRNA processing, and mark cellular mRNA to avoid recognition by the immune system. In mammals, the predominant 5’ cap structure is an inverted 7-methylguanosine nucleotide linked by a 5’ -5' triphosphate bond to the first transcribed nucleotide. The 7- methylguanosine is methylated at its 7 nitrogen position, and may be referred to as m7G or 7mG. This cap structure may be expressed as 5’m7GpppNi (pN)x, where N is any nucleotide and x is 0 or any number. In cap-0 structure, the first nucleotide has a 2’ hydroxy group in its ribose sugar, while in cap-1 structure, the first nucleotide has a 2’- o-methyl modification in the ribose, and this structure may comprise, from 5’ end to 3’ end, m7G5’pppNi2’"OMe(pN)x, where N is any nucleotide and x may be any integer. Further, in cap-2 structure, the 2’ hydroxy group of the first and second ribose to the m7G are methylated. This structure may comprise, from 5’ end to 3’ end, m7G5’pppNi2’"OMepN22’"OMe(pN)x, where N is any nucleotide and x may be any integer. See, e.g., Perry RP, “RNA processing comes of age”, J Cell Biol. (1981 Dec);91 (3 Pt 2):28s-38s, incorporated herein by reference in its entirety.
[0007] Capping may improve properties of the mRNA, such as, but not limited to, its stability and its translational efficiency. See, e.g., Banerjee AK, “5'-terminal cap structure in eucaryotic messenger ribonucleic acids”, Microbiol Rev. (1980 Jun);
44(2): 175-205, incorporated herein by reference in its entirety. In vivo, each capping process may be carried out by enzymes. See, e g., Perry. These processes may be time-consuming, inefficient, and expensive to perform in vitro.
[0008] Preparation of capped mRNA through in vitro synthesis may have significant importance for both fundamental scientific research, development of pharmacologic, and development of therapeutics. Several factors may contribute to the generation of mRNA that may have high expression levels, stability, and functionality. There is a need for an efficient in vitro transcriptional method that would allow for more efficient production of capped mRNA that may have high expression levels, stability, functionality, or a combination thereof. BRIEF SUMMARY
[0009] In an aspect, embodiments of the present disclosure may include methods for in vitro transcription of a DNA template into RNA, comprising providing a mixture comprising a buffer substance, ribonucleoside triphosphates (NTPs), one or more magnesium salts in a concentration of from about 2 mM to about 60 mM, the DNA template, a recombinant RNA polymerase, and a cap analogue comprising the structure of Formula (I), (II), or (III):
Figure imgf000004_0001
wherein R is H or CH3; and Bi is A or N6-methyl-adenine (m6A), and B2 is A, U, G, or C;
Figure imgf000004_0002
wherein
R1 is OCH3 and R2 is OH or H, or
R1 is OH and R2 is H, or
R1 is H and R2 is H or OCH3, or
R1 and R2 are each OCH3,
B1 is A or N6-methyl-adenine (m6A), and B2 is A, II, G, or C,
Figure imgf000005_0001
wherein R is H or CH3; and Bi is A or N6-methyl-adenine (m6A), and B2 is A, U, G, or 0; and incubating the reaction mixture at from about 15°C to about 35°C, optionally from about 18°C to about 31 °C, for from about 1 hour to about 12 hours, thereby producing the RNA.
[0010] In another aspect, buffer substance may be Tris base, HEPES, or Tris-HCl.
[0011] In another aspect, the concentration of buffer substance may be from about 1 mM to about 100 mM, from about 1 mM to about 90 mM, from about 1 mM to about 80 mM, from about 1 mM to about 70 mM, from about 1 mM to about 60 mM, from about 1 mM to about 50 mM, from about 1 mM to about 40 mM, from about 1 mM to about 30 mM, from about 1 mM to about 20 mM, from about 1 mM to about 10 mM, from about 1 mM to about 5 mM, from about 10 mM to about 20 mM, from about 10 mM to about 30 mM, from about 10 mM to about 40 mM, from about 10 mM to about 50 mM, from about 20 mM to about 50 mM, from about 30 mM to about 50 mM, from about 35 mM to about 45 mM, from about 35 mM to about 40 mM, from about 40 mM to about 50 mM, from about 45 mM to about 50 mM, from about 45 mM to about 55 mM, from about 15 mM to about 45 mM, from about 15 mM to about 35 mM, from about 15 mM to about 30 mM, or from about 15 mM to about 25 mM.
[0012] In another aspect, the concentration of NTPs may be from about 1 mM to about 50 mM, from about 1 mM to about 40 mM, from about 1 mM to about 30 mM, from about 1 mM to about 20 mM, from about 1 mM to about 10 mM, from about 1 mM to about 5 mM, from about 2 mM to about 10 mM, from about 3 mM to about 10 mM, from about 3 mM to about 9 mM, from about 3 mM to about 8 mM, from about 3 mM to about 7 mM, from about 3 mM to about 6 mM, from about 3 mM to about 5 mM, from about 3 mM to about 4 mM, from about 4 mM to about 10 mM, from about 5 mM to about 10 mM, from about 6 mM to about 10 mM, from about 7 mM to about 10 mM, from about 8 mM to about 10 mM, from about 9 mM to about 10 mM, from about 10 mM to about 50 mM. from about 20 mM to about 50 mM, from about 25 mM to about 50 mM, from about 25 mM to about 45 mM, from about 25 mM to about 40 mM, from about 25 mM to about 35 mM or from about 20 mM to about 30 mM.
[0013] In another aspect, the concentration of one or more magnesium salts may be from about 2 mM to about 50 mM, from about 2 mM to about 40 mM, from about 2 mM to about 30 mM, from about 2 mM to about 40 mM, from about 2 mM to about 30 mM, from about 2 mM to about 20 mM, from about 2 mM to about 10 mM, from about 2 mM to about 5 mM, from about 5 mM to about 50 mM, from about 10 mM to about 45 mM, from about 15 mM to about 40 mM, from about 20 mM to about 35 mM, from about 20 mM to about 30 mM, from about 20 mM to about 25 mM, from about 22 mM to about 28 mM, or from about 25 mM to about 30 mM.
[0014] In another aspect, the concentration of DNA template may be from about 0.001 μg/μl to about 2 μg/μl, from about 0.001 μg/μl to about 1.5 μg/μl, from about 0.001 μg/μl to about 1 μg/μl, from about 0.01 μg/μl to about 2 μg/μl, from about 0.01 μg/μl to about 1.5 μg/μl, from about 0.01 μg/μl to about 1 μg/μl, from about 0.01 μg/μl to about 0.5 μg/μl, from about 0.01 μg/μl to about 0.1 μg/μl, from about 0.01 μg/μl to about 0.05 μg/μl, from about 0.02 μg/μl to about 0.04 μg/μl, from about 0.02 μg/μl to about 0.1 μg/μl, from about 0.03 μg/μl to about 0.1 μg/μl, from about 0.04 μg/μl to about 0.1 μg/μl, from about 0.05 μg/μl to about 0.1 μg/μl, from about 0.06 μg/μl to about 0.1 μg/μl, from about 0.07 μg/μl to about 0.1 μg/μl, from about 0.08 μg/μl to about 0.1 μg/μl, or from about 0.09 μg/μl to about 0.1 μg/μl.
[0015] In another aspect, the concentration of recombinant RNA polymerase may be from about 0.1 U/μl to about 2 U/μl, from about 0.5 U/μl to about 2 U/μl, from about 1 U/μl to about 2 U/μl, from about 1 U/μl to about 1 .5 U/μl, or from about 1 .5 U/μl to about 2 U/μl.
[0016] In another aspect, the mixture may further comprise an antioxidant.
[0017] ln another aspect, antioxidant may be dithiothreitol (DTT) in a concentration of from about 1 mM to about 50 mM, from about 2 mM to about 50 mM, from about 3 mM to about 50 mM, from about 4 mM to about 50 mM, from about 5 mM to about 50 mM, from about 6 mM to about 50 mM, from about 7 mM to about 50 mM, from about 8 mM to about 50 mM, from about 9 mM to about 50 mM, from about 10 mM to about 50 mM, from about 10 mM to about 40 mM, from about 15 mM to about 30 mM, from about 15 mM to about 25 mM, from about 15 mM to about 20 mM, from about 20 mM to about 50 mM, from about 30 mM to about 50 mM, or from about 40 mM to about 50 mM.
[0018] In another aspect, the mixture may further comprise an RNase inhibitor in a concentration of from about 0.001 U/μl to about 5 U/μl, from about 0.001 U/μl to about 4 U/μl, from about 0.001 U/μl to about 3 U/μl, from about 0.001 U/μl to about 2 U/μl, from about 0.001 U/μl to about 1 U/μl, from about 0.01 U/μl to about 5 U/μl, from about 0.01 U/μl to about 4 U/μl, from about 0.01 U/μl to about 3 U/μl, from about 0.01 U/μl to about 2 U/μl, from about 0.01 U/μl to about 1 U/μl, from about 0.01 U/μl to about 0.5 U/μl, from about 0.01 U/μl to about 0.1 U/μl, from about 0.01 U/μl to about 0.05 U/μl, from about 0.01 U/μl to about 0.04 U/μl, from about 0.01 U/μl to about 0.03 U/μl, from about 0.01 U/μl to about 0.02 U/μl, from about 0.1 U/μl to about 5 U/μl, from about 0.1 U/μl to about 4 U/μl, from about 0.1 U/μl to about 3 U/μl, from about 0.1 U/μl to about 2 U/μl, from about 0.1 U/μl to about 1 U/μl, from about 0.5 U/μl to about 5 U/μl, from about 0.5 U/μl to about 4 U/μl, from about 0.5 U/μl to about 3 U/μl, from about 0.5 U/μl to about 2 U/μl, from about 0.5 U/μl to about 1 U/μl, from about 1 U/μl to about 5 U/μl, from about 2 U/μl to about 5 U/μl, from about 3 U/μl to about 5 U/μl, from about 4 U/μl to about 5 U/μl, from about 0.001 U/μl to about 0.005 U/μl, from about 0.001 U/μl to about 0.01 U/μl, from about 0.005 U/μl to about 0.01 U/μl, from about 0.01 U/μl to about 0.05 U/μl, from about 0.01 U/μl to about 0.04 U/μl, from about 0.01 U/μl to about 0.03 U/μl, from about 0.01 U/μl to about 0.02 U/μl, from about 0.02 U/μl to about 0.03 U/μl, from about 0.02 U/μl to about 0.04 U/μl, or from about 0.02 U/μl to about 0.05 U/μl.
[0019] In another aspect, cap analogue may be in a concentration of from about 0.5 mM to about 50 mM, from about 0.5 mM to about 40 mM, from about 0.5 mM to about 30 mM, from about 0.5 mM to about 20 mM, from about 0.5 mM to about 10 mM, from about 0.5 mM to about 5 mM, from about 1 mM to about 10 mM, from about 2 mM to about 10 mM, from about 3 mM to about 10 mM, from about 3 mM to about 9 mM, from about 3 mM to about 8 mM, from about 3 mM to about 7 mM, from about 3 mM to about 6 mM, from about 3 mM to about 5 mM, from about 3 mM to about 4 mM, from about 4 mM to about 10 mM, from about 5 mM to about 10 mM, from about 6 mM to about 10 mM, from about 6 mM to about 9 mM, from about 6 mM to about 8 mM, from about 6 mM to about 7 mM, from about 7 mM to about 8 mM, from about 7 mM to about 9 mM, from about 7 mM to about 10 mM, from about 8 mM to about 9 mM, from about 8 mM to about 10 mM, or from about 9 mM to about 10 mM.
[0020] In another aspect, the mixture may further comprise polyamine.
[0021] In another aspect, polyamine may be spermine, spermidine, or a combination thereof.
[0022] In another aspect, the concentration of the polyamine may be from about 0.1 mM to about 5 mM, from about 0.2 mM to about 4.9 mM, from about 0.2 mM to about 4.8 mM, from about 0.2 mM to about 4.7 mM, from about 0.2 mM to about 4.6 mM, from about 0.2 mM to about 4.5 mM, from about 0.2 mM to about 4.4 mM, from about 0.2 mM to about 4.3 mM, from about 0.2 mM to about 4.2 mM, from about 0.2 mM to about 4.1 mM, from about 0.2 mM to about 4 mM, from about 0.2 mM to about
3.5 mM, from about 0.2 mM to about 3 mM, from about 0.2 mM to about 2.5 mM, from about 0.5 mM to about 2.5 mM, from about 1.0 mM to about 2.5 mM, from about 1 .5 mM to about 2.5 mM, from about 0.2 mM to about 2 mM, from about 0.2 mM to about
1 .5 mM, from about 0.2 mM to about 1 mM, from about 0.2 mM to about 0.9 mM, from about 0.2 mM to about 0.8 mM, from about 0.2 mM to about 0.7 mM, from about 0.2 mM to about 0.6 mM, from about 0.2 mM to about 0.5 mM, from about 0.2 mM to about 0.4 mM, from about 0.2 mM to about 0.3 mM, from about 1 to about 25 mM, about 1 to about 20 mM, about 1 to about 15 mM, about 1 to about 10 mM, about 1 to about 5 mM, or about 1 to about 2.5 mM.
[0023] In another aspect, the mixture may further comprise a pyrophosphatase in a concentration of from about 0.01 mU/μl to about 2 mU/μl, from about 0.01 mU/μl to about 1.5 mU/μl, from about 0.01 mU/μl to about 1 mU/μl, from about 0.1 mU/μl to about 2 mU/μl, from about 0.1 mU/μl to about 1.5 mU/μl, from about 0.1 mU/μl to about 1 mU/μl, from about 0.1 mU/μl to about 0.9 mU/μl, from about 0.1 mU/μl to about 0.8 mU/μl, from about 0.1 mU/μl to about 0.7 mU/μl, from about 0.1 mU/μl to about 0.6 mU/μl, from about 0.1 mU/μl to about 0.5 mU/μl, from about 0.1 mU/μl to about 0.4 mU/μl, from about 0.1 mU/μl to about 0.3 mU/μl, or from about 0.1 mU/μl to about 0.2 mU/μl.
[0024] In another aspect, incubating the reaction mixture may be performed at from about 15°C to about 35°C, from about 16°C to about 35°C, from about 17°C to about 35°C, from about 18°C to about 35°C, from about 18°C to about 34°C, from about 18°C to about 33°C, from about 18°C to about 32°C, from about 18°C to about 31 °C, from about 18°C to about 30°C, from about 18°C to about 29°C, from about 18°C to about 28°C, from about 18°C to about 27°C, from about 18°C to about 26°C, from about 18°C to about 25°C, from about 18°C to about 24°C, from about 18°C to about 23°C, from about 18°C to about 22°C, from about 18°C to about 21 °C, from about 18°C to about 20°C, from about 18°C to about 19°C, from about 25°C to about 26°C, from about 25°C to about 27°C, from about 25°C to about 28°C, from about 25°C to about 29°C, from about 25°C to about 30°C, from about 25°C to about 31 °C, from about 21 °C to about 22°C, from about 21 °C to about 23°C, from about 21 °C to about 24°C, from about 21 °C to about 25°C, about 25°C, about 26°C, about 27°C, about 28°C, about 29°C, about 30°C, about 31 °C, about 32°C, about 33°C, about 34°C, or about 35°C.
[0025] In another aspect, incubating the reaction mixture may be performed for from about 1 hour to about 12 hours, from about 1 hour to about 11 hours, from about
I hour to about 10 hours, from about 1 hour to about 9 hours, from about 1 hour to about 8 hours, from about 1 hour to about 7 hours, from about 1 hour to about 6 hours, from about 1 hour to about 5 hours, from about 1 hour to about 4 hours, from about 1 hour to about 3 hours, from about 1 hour to about 2 hours, from about 2 hours to about 12 hours, from about 3 hours to about 12 hours, from about 4 hours to about 12 hours, from about 5 hours to about 12 hours, from about 6 hours to about 12 hours, from about "I hours to about 12 hours, from about 8 hours to about 12 hours, from about 9 hours to about 12 hours, from about 10 hours to about 12 hours, from about
I I hours to about 12 hours, about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, or about 10 hours.
[0026] In another aspect, incubating the reaction mixture may be performed at about 31 °C for from about 1 hour to about 5 hours, from about 1 hour to about 4.5 hours, from about 1 hour to about 4 hours, from about 1 hour to about 3.5 hours, from about 1 hour to about 3 hours, from about 1 hour to about 2.5 hours, from about 1 hour to about 2 hours, from about 1 hour to about 1 .5 hours, from about 0.5 hour to about 1 hour, from about 0.5 hour to about 1.5 hours, about 1 hour, about 2 hours, about 3 hours, about 4 hours, or about 5 hours.
[0027] In another aspect, DNA template may comprise a promoter operably linked to a nucleic acid comprising a 5’ untranslated region (5’ UTR), an open reading frame (ORF) encoding the RNA of interest, a 3’ UTR, and a poly A region, wherein the promoter comprises a sequence of TAATACGACTCACTATAX1X2X3 (SEQ ID NO: 16), wherein Xi is A or G, X2 is A or G, and X3 is A, T, G, or C, wherein the 5’ UTR is selected from SEQ ID NO: 1 , 3, 5, or 9, and the 3’ UTR is selected from SEQ ID NO: 2, 4, 6, or 8, wherein the poly A region comprises at least 60 adenine bases (As).
[0028] In another aspect, promoter may comprise a sequence selected from SEQ
ID NO: 10-15.
[0029] In another aspect, 5’ UTR and 3’ UTR may be respectively SEQ ID NO: 1 and 2, 1 and 4, 1 and 6, 3 and 2, 3 and 4, 3 and 6, 3 and 8, 5 and 2, 5 and 4, 5 and 6, 7 and 2, 7 and 4, and 6, 7 and 8, 9 and 2, 9 and 4, 9 and 6, or 9 and 8.
[0030] In another aspect, 5’ UTR and 3’ UTR may be respectively SEQ ID NO: 1 and 2, 1 and 4, 3 and 2, 1 and 6, 7 and 4, 9 and 2, or 3 and 6.
[0031] In another aspect, poly A region may comprise from about 60 to about 200 As, from about 60 to about 190 As, from about 60 to about 180 As, from about 60 to about 170 As, from about 60 to about 160 As, from about 60 to about 150 As, from about 60 to about 140 As, from about 60 to about 130 As, from about 60 to about 120 As, from about 60 to about 110 As, from about 60 to about 100 As, from about 70 to about 190 As, from about 80 to about 180 As, from about 90 to about 170 As, from about 100 to about 160 As, from about 100 to about 150 As, from about 100 to about 140 As, from about 100 to about 130 As, from about 100 to about 120 As, about 100 As, about 110 As, about 120 As, about 130 As, about 140 As, or about 150 As.
[0032] In another aspect, recombinant RNA polymerase may be selected from wild type T7 RNA polymerase or a variant thereof.
[0033] ln another aspect, cap analogue may be one selected from the group consisting of m7GpppA2’OmepA2’Ome, m7GpppA2’OmepU2'Ome, m7GpppA2’OmepG2’Ome, m7GpppA2o me pC2’Ome, m7Gpppm6A2o me pA2’Ome, m7Gpppm6A2o me pU2Ome, m7Gpppm6A2o me pG2’Ome, m7Gpppm6A2’OmepC2’Ome, m7G3’OmePPpA2’OmepA2’Ome, m7Gso me pppAzo me pU2’Ome, m7G3'OmePPpA2’OmepG2’Ome, m7G3o me pppAzo me pC2’Ome, m7G3o me pppm6A2'o me pA2’Ome, m7G3o me pppm6A2o me pU2’Ome, m7G3o me pppm6A2’o mepG2'Ome, m7G3o me pppm6A2’o me pC2’Ome, m7G2'OmePPpA2’OmepA2’Ome, m7G2’Ome PPPA2Ome pU2’Ome, m7G2o me PPPA2OmepG2’Ome, m7G2'OmePPpA2’OmepC2’Ome, m7G2’Ome pppm6A2’o me pA2’Ome, m7G2o me pppm6A2’o me pU2’Ome, m7G2’OmepPpm6A2’OmepG2’Ome, m7G2’Ome pppm6A2’o me pC2’Ome, m7G3HpppA2’o me PA2Ome, m7G3’HPPpA2’OmepU2’Ome, m7G3’HPPpA2’OmepG2’Ome, m7G3’HPPpA2’OmepC2’Ome, m7G3Hpppm6A2’o me PA2Ome, m7G3Hpppm6A2’o me pU2’Ome, m7G3Hpppm6A2’o mepG2’0me, m7G3Hpppm6A2’o me pC2’Ome, m7GppspA2o me pA2’Ome, m7GppspA2’OmepU2’Ome, m7GppspA2’OmepG2’Ome, m7GppspA2’OmepC2'Ome,
Figure imgf000012_0001
[0034] In another aspect, cap analogue may bind to -1 and/or +1 nucleotide of the promoter.
[0035] In an aspect, embodiments of the present disclosure may include reaction mixtures for in vitro transcription of a DNA template into RNA, comprising a buffer substance in a concentration of from about 45 mM to about 55 mM, an RNase inhibitor in a concentration of from about 0.01 U/μl to about 0.03 U/μl, NTPs in a concentration of from about 3 mM to about 5 mM, a cap analogue in a concentration of from about 6 mM to about 8 mM, one or more magnesium salts in a concentration of from about 20 mM to about 30 mM, a polyamine in a concentration of from about 1 .5 mM to about 2.5 mM, a DNA template in a concentration of from about 0.01 μg/μl to about 0.05 μg/μl, a pyrophosphatase in a concentration of from about 0.1 mU/μl to about 0.5 mU/μl, and an RNA polymerase in a concentration of from about 0.01 μg/μl to about 0.05 μg/μl, wherein the cap analogue comprises the structure of Formula (I), (II), or (III):
Figure imgf000013_0001
wherein R is H or CH3; and Bi is A or N6-methyl-adenine (m6A), and B2 is A, U, G, or C,
Figure imgf000013_0002
wherein
Ri is OCH3 and R2 is OH or H, or
R1 is OH and R2 is H, or
Ri is H and R2 is H or OCH3, or
Ri and R2 are each OCH3,
B1 is A or N6-methyl-adenine (m6A), and B2 is A, U, G, or C
Figure imgf000014_0001
wherein R is H or CH3; and Bi is A or N6-methyl-adenine (m6A), and B2 is A, II, G, or C.
[0036] In another aspect, incubating the reaction mixture may be performed at from about 15°C to about 35°C for from about 1 hour to about 12 hours.
[0037] In another aspect, incubating may comprise incubating the reaction mixture at from about 18°C to about 31 °C.
[0038] In another aspect, incubating may comprise incubating the reaction mixture at about 31 °C for about 3 hours.
[0039] In an aspect, embodiments of the present disclosure may include methods for in vitro transcription of a DNA template into RNA, comprising providing (1 ) a DNA template comprises a promoter operably linked to a nucleic acid comprising a 5’ untranslated region (5’ UTR), an open reading frame (ORF) encoding the RNA of interest, a 3’ UTR, and a poly A region, and (2) a cap analogue comprises the structure of Formula (I), (II), or (III):
Figure imgf000014_0002
wherein R is H or CH3; and Bi is A or N6-methyl-adenine (m6A), and B2 is A, U, G, or C,
Figure imgf000015_0001
wherein
Ri is OCH3 and R2 is OH or H, or
R1 is OH and R2 is H, or
R1 is H and R2 is H or OCH3, or
R1 and R2 are each OCH3,
B1 is A or N6-methyl-adenine (m6A), and B2 is A, U, G, or C, wherein the promoter comprises a sequence of TAATACGACTCACTATAX1X2X3 (SEO ID NO: 16), wherein A at position 17 is -1 nucleotide and Xi at position 18 is +1 nucleotide, when Xi is G, X2 and X3 are each A, T, G, or C, then Bi is A and B2 is G, when Xi is A, X2 and X3 are each A, T, G, or C, then Bi is A and B2 is A, when Xi is C, X2 and X3 are each A, T, G, or C, then Bi is A and B2 is C, and when Xi is T, X2 and X3 are each A, T, G, or C, then Bi is A and B2 is U,
Figure imgf000015_0002
wherein R is H or CH3; and Bi is A or N6-methyl-adenine (m6A), and B2 is A, U, G, or C, wherein the cap analogue binds to -1 and +1 nucleotides of the promoter, and incubating the DNA template and the cap analogue in a reaction mixture, wherein the incubating comprises incubating the reaction mixture at from about 15°C to about 35°C for from about 1 hour to about 12 hours, thereby producing the RNA.
[0040] In another aspect, promoter may comprise a sequence selected from SEQ ID NO: 10, 11 , 12, 13, and 14.
[0041] In another aspect, the reaction mixture may comprise buffer substance in a concentration of from about 35 mM to about 45 mM, RNase inhibitor in a concentration of from about 0.01 U/μl to about 0.03 U/μl, NTPs in a concentration of from about 20 mM to about 40 mM, cap analogue in a concentration of from about 6 mM to about 8 mM, one or more magnesium salts in a concentration of from about 20 mM to about 30 mM, polyamine in a concentration of from about 1.5 mM to about 2.5 mM, DNA template in a concentration of from about 0.01 μg/μl to about 0.05 μg/μl, pyrophosphatase in a concentration of from about 0.1 mU/μl to about 0.5 mU/μl, and RNA polymerase in a concentration of from about 1 U/μl to about 2 U/μl.
[0042] In an aspect, embodiments of the present disclosure may include reaction mixtures for in vitro transcription of a DNA template into RNA, comprising a DNA template and a cap analogue, wherein the cap analogue comprises the structure of Formula (I), (II), or (III):
Figure imgf000016_0001
wherein R is H or CH3; and Bi is A or N6-methyl-adenine (m6A), and B2 is A, U, G, or C;
Figure imgf000017_0001
wherein
R1 is OCH3 and R2 is OH or H, or
R1 is OH and R2 is H, or
R1 is H and R2 is H or OCH3, or
R1 and R2 are each OCH3,
B1 is A or N6-methyl-adenine (m6A), and B2 is A, U, G, or C,
Figure imgf000017_0002
wherein R is H or CH3; and Bi is A or N6-methyl-adenine (m6A), and B2 is A, U, G, or
C wherein the DNA template comprises a promoter; and wherein the cap analogue binds to at least -1 and +1 nucleotides of the promoter, or binds to at least+1 and +2 nucleotides of the promoter.
[0043] In an aspect, embodiments of the present disclosure may include methods for synthesizing a cap analogue, comprising the steps of:
(a) reacting 5’-DMT-2’-O-methyl guanosine (n-ibu) with an acetic anhydride/pyridine, followed by reacting the product with aqueous acetic acid, to obtain Compound (3):
Figure imgf000018_0001
(b) reacting the Compound (3) with 5'-dimethoxytrityl-N-benzoyl-Adenosine,2'-O- methyl,3'-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite and tetrazole, followed by reaction with a first oxidizer, followed by reaction with aqueous acetic acid, to obtain Compound (4):
Figure imgf000018_0003
(c) reacting the Compound (4) with a chemical phosphorylating phosphoramidite reagent and tetrazole, followed by reaction with a second oxidizer, followed by reaction with ammonium hydroxide to obtain Compound (5):
Figure imgf000018_0002
(d) reacting the Compound (5) with (2a) or (2b) in the presence of a divalent metal salt
Figure imgf000019_0001
to obtain the cap analogue (1a) or (1 b):
Figure imgf000019_0002
[0044] In an aspect, embodiments of the present disclosure may include methods for synthesizing a cap analogue, comprising the steps of:
(a) reacting Compound 3:
Figure imgf000019_0003
with 5'-DMT-2'-O-methyl-
N6-methyl-adenosine 3'-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite and tetrazole, followed by reaction with a first oxidizer, followed by reaction with aqueous
acetic acid, to obtain Compound (6):
Figure imgf000020_0002
(b) reacting the Compound (6) with a chemical phosphorylating phosphoramidite reagent and tetrazole, followed by reaction with a second oxidizer, followed by reaction with ammonium hydroxide to obtain Compound (7):
Figure imgf000020_0001
(c) reacting the Compound (7) with Compound (2b) in the presence of a divalent metal salt: to obtain the cap analogue
Figure imgf000020_0003
Compound (8):
Figure imgf000021_0002
[0045] In another aspect, the first oxidizer and the second oxidizer may be each (1 S)-(+)-(camphorsulfonyl)oxaziridine (CSO) or iodine.
[0046] In another aspect, divalent metal salt may be MnCI2 or ZnCI2.
[0047] In an aspect, embodiments of the present disclosure may include cap analogues comprising formula (I):
Figure imgf000021_0001
wherein R is H or CH3; and Bi is A or N6-methyl-adenine (m6A), and B2 is A, U, G, or C.
[0048] In another aspect, R is H, Bi is A and B2 is A, U, G, or C, in which cap analogue of formula (I) may be selected from the group consisting of m7GpppA2’OmepA2’Ome, m7GpppA2’OmepU2'Ome, m7GpppA2’OmepG2’Ome, and m7GpppA2’OmepC2'Ome.
[0049] In another aspect, R is H, Bi is m6A and B2 is A, U, G, or C, in which cap analogue of formula (I) may be selected from the group consisting of m7Gpppm6A2o me pA2’Ome, m7Gpppm6A2o me PU2Ome, rn7Gppprn6A2’omepG2’ome, and m7Gpppm6A2o me pC2’Ome.
[0050] In another aspect, R is CH3, Bi is A and B2 is A, U, G, or C, in which cap analogue of formula (I) may be selected from the group consisting of m7G3 o me PPPA2 Ome pA2’Ome, m7G3 o me PPPA2 Ome pU2’Ome, m7G3o me PPPA2 OmepG2’0me, and m7G3’OmePPpA2’Ome pC2’Ome.
[0051] In another aspect, R is CH3, Bi is m6A and B2 is A, U, G, or C, in which cap analogue of formula (I) may be selected from the group consisting of m7G3o me pppm6A2’o me pA2’Ome, m7G3o me pppm6A2’o me pU2’Ome, m7G3'OmePPpm6A2’OmepG2'Ome, and m7G3o me pppm6A2’o me pC2’Ome.
[0052] In an aspect, embodiments of the present disclosure may include cap analogues comprising formula (II):
Figure imgf000022_0001
wherein
R1 is OCH3 and R2 is OH or H, or
R1 is OH and R2 is H, or
R1 is H and R2 is H or OCH3, or
R1 and R2 are each OCH3,
B1 is A or N6-methyl-adenine (m6A), and B2 is A, U, G, or C. [0053] In another aspect, R1 is OCH3 and R2 is OH, Bi is A and B2 is A, U, G, or C, in which cap analogue of formula (II) may be selected from the group consisting of m7G2’o me pppAs’o me pA2’Ome, m7G2o me PPPA2Ome pU2’Ome, m7G2o me PPPA2OmepG2’Ome, and m7G2o me PPPA2Ome pCs’Ome.
[0054] In another aspect, R1 is OCH3 and R2 is OH, Bi is m6A and B2 is A, U, G, or C, in which cap analogue of formula (II) may be selected from the group consisting of m7G2 o me pppm6A2’o me pAs’Ome, m7G2o me pppm6A2’o me PU2Ome, m7G2'OmePPpm6A2’OmepG2'Ome, and m7G2 o me pppm6A2’o me pCs’Ome.
[0055] In another aspect, Ri is OH and R2 is H, Bi is A and B2 is A, II, G, or C, in which cap analogue of formula (II) may be selected from the group consisting of m7G3’HPPpA2’OmepA2’Ome, m7G3’HPPpA2’OmepU2’Ome, rn7G3HpppA2’omepG2’ome, and m7G3’HPPpA2’OmepC2’Ome.
[0056] In another aspect, R1 is OH and R2 is H, Bi is m6A and B2 is A, U, G, or C, in which cap analogue of formula (II) may be selected from the group consisting of m7G3 Hpppm6A2’o me PA2 Ome, m7G3 Hpppm6A2’o me pU2’Ome, m7G3Hpppm6A2’o mepG2’0me, and m7G3Hpppm6A2’o me PC2 Ome.
[0057] ln an aspect, embodiments of the present disclosure may include cap analogues comprising formula (III):
Figure imgf000024_0001
wherein R is H or CH3; and Bi is A or N6-methyl-adenine (m6A), and B2 is A, U, G, or C.
[0058] In another aspect, R is H, Bi is A and B2 is A, U, G, or 0, in which the cap analogue of formula (III) may be selected from the group consisting of m7GppspA2o me pA2’Ome, m7GppspA2’OmepU2'Ome, rn7GppspA20mepG2’0me, and m7GppspA2o me pC2’Ome.
[0059] In another aspect, R is H, Bi is m6A and B2 is A, U, G, or C, in which the cap analogue of formula (III) may be selected from the group consisting of m7Gppspm6A2o me pA2’Ome, m7Gppspm6A2o me pU2’Ome, m7Gppspm6A20mepG2’0me, and m7Gppspm6A2o me pC2’Ome.
[0060] In another aspect, R is CH3, Bi is A and B2 is A, U, G, or C, in which the cap analogue of formula (III) may be selected from the group consisting of m7G3o me ppspA2’Ome pA2’Ome, m7G3o me ppspA2’o me PU2Ome, m7G3'OmePPspA2’OmepG2’Ome, and m7G3o me ppspA2’Ome PC2Ome.
[0061] In another aspect, R is CH3, Bi is m6A and B2 is A, U, G, or C, in which the cap analogue of formula (III) may be selected from the group consisting of m7G3o me ppspm6A2’o me pA2’Ome, m7G3o me ppspm6A2’o me pU2Ome, rn7G3’omeppsprn6A2’omepG2’ome, and m7G3o me ppspm6A2’o me pC2’Ome.
[0062] In another aspect, embodiments of the present disclosure may include methods for synthesizing a cap analogue, comprising the steps of:
(a) reacting Compound (12) with dimethyl sulfate to
Figure imgf000025_0001
obtain Compound (13)
Figure imgf000025_0002
(b) reacting the Compound (13) with imidazole, triphenylphosphine, 2, 2’- dithiopyridine, followed by reaction with MnCL and bis-triethylammonium thiophosphate to obtain Compound (14)
Figure imgf000025_0003
(c) reacting Compound (5)
Figure imgf000026_0001
with triphenylphosphine, imidazole, and 2, 2’-dithiopyridine to obtain Compound (15)
Figure imgf000026_0002
, and
(d) reacting the Compound (15) with the Compound (14)
Figure imgf000026_0003
in the presence of dimethylformamide and ZnCL to obtain Compound (16)
Figure imgf000026_0004
BRIEF DESCRIPTION OF THE DRAWINGS
[0063] FIG. 1 shows use of cap analogues to initiate in vitro transcription at -1 position in accordance with one embodiment of the present disclosure.
[0064] FIG. 2 shows a process of preparing cap-2 analogue synthetic intermediates in accordance with one embodiment of the present disclosure.
[0065] FIG. 3 shows a process of preparing cap-2 analogues in accordance with another embodiment of the present disclosure.
[0066] FIG. 4 shows a process of preparing additional cap-2 analogues in accordance with another embodiment of the present disclosure.
[0067] FIG. 5 shows expression levels of mRNA prepared by using various cap analogues in accordance with another embodiment of the present disclosure.
[0068] FIG. 6 shows a process of preparing additional cap-2 analogues in accordance with another embodiment of the present disclosure.
[0069] FIG. 7 shows a process of preparing additional cap-2 analogues in accordance with another embodiment of the present disclosure.
[0070] FIG. 8 shows a process of preparing additional cap-2 analogues in accordance with another embodiment of the present disclosure.
[0071] FIG. 9 shows in vivo expression levels of mRNA prepared by using various cap analogues in accordance with another embodiment of the present disclosure.
[0072] FIG. 10 shows biodistribution of mRNA prepared by using various cap analogues in accordance with one embodiment of the present disclosure.
[0073] FIG. 11 shows effect of mRNA prepared by using various cap analogues on mouse body weight in accordance with one embodiment of the present disclosure.
DETAILED DESCRIPTION
[0074] Provided herein are methods and compositions for in vitro synthesis of mRNA. Methods and compositions for in vitro synthesis of capped mRNA are provided. Aspects of the disclosed methods and compositions may be used singly or in any combination. The disclosed methods and compositions may improve efficiency of mRNA synthesis and may provide mRNA with improved properties.
[0075] Independent of the type of cap modification tested to date (e.g., cap-0 and cap-1 ), transcripts may be prone to the activity of DCP2, a major cellular decapping enzyme, which, in cooperation with its cellular partner DCP1 , releases m7GDP and the monophosphorylated RNA chain. Another protein, DXO (decapping exoribonuclease), may serve as a quality control factor to eliminate aberrantly capped transcripts and acts as a decapping enzyme. In contrast to DCP2, DXO may remove the entire cap structure by hydrolyzing the phosphodiester bond between the first and second transcribed nucleotide, resulting in the release of m7GpppN and monophosphorylated RNA, and may continue to degrade RNA as an exoribonuclease in the 5’-3’ direction. Interestingly, DXO decapping activity was shown to be inhibited by cap-1 2’-O- methylation, suggesting its potential role in distinguishing ‘self from ‘non-self’ RNAs.
[0076] Recent work from Drazkowska et al. showed that 2’-O-methylation of the second transcribed nucleotide regulates protein biosynthesis in a cell-dependent manner (Drazkowska K, Tomecki R, Warminski M, Baran N, Cysewski D, Depaix A, Kasprzyk R, Kowalska J, Jemielity J, Sikorski PJ. 2'-O-Methylation of the second transcribed nucleotide within the mRNA 5' cap impacts the protein production level in a cell-specific manner and contributes to RNA immune evasion. Nucleic Acids Res. 2022 Sep 9;50(16):9051 -9071 , incorporated herein by reference in its entirety). Moreover, resistance of RNA capped with cap-2 to DXO-mediated decapping and degradation was observed. Additionally, 2’-O-methylation of the second transcribed nucleotide and N6-methylation of adenosine as the first transcribed nucleotide serve as determinants defining transcripts as ‘self and contribute to transcript immune evasion.
[0077] Preparing cap-2 mRNA in vitro efficiently at large scale may be essential to translate these findings into biopharmaceutical applications.
[0078] The disclosed methods and compositions may be used singly or in any combination to perform in vitro synthesis of mRNA of different sizes, such as, but not limited to, mRNAs ranging from about 100b to about 20Kb, from about 200b to about 19Kb, from about 300b to about 18Kb, from about 400b to about 17Kb, from about 500b to about 16Kb, from about 600b to about 15Kb, from about 700b to about 14Kb, from about 800b to about 13Kb, from about 900b to about 12Kb, from about 1 Kb to about 11 Kb, from about 1 Kb to about 10Kb, from about 1 Kb to about 9Kb, from about 1 Kb to about 8Kb, from about 1 Kb to about 7Kb, from about 1 Kb to about 6Kb, from about 1 Kb to about 5Kb, from about 1 Kb to about 4Kb, from about 1 Kb to about 3Kb, from about 1 Kb to about 2Kb, from about 50b to about 200b, from about 60b to about 190b, from about 70b to about 180b, from about 80b to about 160b, from about 90b to about 100b, from about 90b to about 110b, from about 90b to about 120b, from about 90b to about 130b, from about 90b to about 140b, from about 90b to about 150b, from about 100b to about 140b, from about 110b to about 130b, or from about 110b to about 120b.
[0079] mRNA synthesized using the disclosed compositions and/or methods may have many applications, including, but not limited to, uses in fundamental scientific research, uses in development of pharmacologies, uses in development of diagnostics, uses in development of therapeutics, uses as pharmacologies, uses as diagnostics, uses as therapeutics, or any combination thereof.
[0080] Methods for RNA in vitro transcription are known in the art (see for example Geall et al. (2013) Semin. Immunol. 25(2): 152-159; Brunelle et al. (2013) Methods Enzymol. 530: 101 -14). Reagents used in said methods may include: a linear DNA template with a promoter sequence that has a high binding affinity for its respective RNA polymerase; ribonucleoside triphosphates (NTPs) for the four bases (adenine, cytosine, guanine and uracil); a cap analogue; other modified nucleotides; DNA- dependent RNA polymerase (e.g., T7, T3 or SP6 RNA polymerase); ribonuclease (RNase) inhibitor to inactivate any contaminating Rnase; pyrophosphatase to degrade pyrophosphate, which inhibits transcription; MgCL and/or Mg(0Ac)2, which supplies Mg2+ as a cofactor for the RNA polymerase; antioxidants (e.g. DTT); polyamines, such as spermidine; and a buffer to maintain a suitable pH value.
[0081] Common buffer systems used in RNA in vitro transcription may include 4-(2- hydroxy-ethyl)-1 -piperazineethanesulfonic acid (HEPES) and tris(hydroxymethyl)aminomethane (Tris). The pH value of the buffer may be commonly adjusted to a pH value of 6 to 8.5. Some commonly used transcription buffers may contain 80 mM HEPES/KOH, pH 7.5 and 40 mM Tris/HCI, pH 7.5. [0082] The transcription buffer may also contain a magnesium salt, such as MgCL and/or Mg(OAc)2 commonly in a range between 5-50 mM. Magnesium ions (Mg2+) may be an essential component in an RNA in vitro transcription buffer system because free Mg2+ may function as cofactor in the catalytic center of the RNA polymerase and may be critical for the RNA polymerization reaction. In diffuse binding, fully hydrated Mg ions may also interact with the RNA product via nonspecific long-range electrostatic interactions.
[0083] RNA in vitro transcription reactions may be performed as batch reactions in which all components are combined and then incubated to allow the synthesis of RNA molecules until the reaction terminates. In addition, fed-batch reactions were developed to increase the efficiency of the RNA in vitro transcription reaction (Kern et al. (1997) Biotechnol. Prog. 13: 747-756; Kern et al. (1999) Biotechnol. Prog. 15: 174- 184). In a fed-batch system, all components are combined, but then additional amounts of some of the reagents are added over time (e.g., NTPs, MgCL and/or Mg(OAc)2) to maintain constant reaction conditions.
[0084] For the sake of clarity and readability the following definitions are provided. Any technical feature mentioned in these definitions may be read on each and every embodiment of the invention. Additional definitions and explanations may be specifically provided in the context of these embodiments.
[0085] In vitro transcription: The terms “in vitro transcription" or “RNA in vitro transcription” may relate to a process wherein RNA is synthesized in a cell-free system (/n vitro\ DNA, particularly plasmid DNA, is used as template for the generation of RNA transcripts. RNA may be obtained by DNA-dependent in vitro transcription of an appropriate DNA template, which according to the present disclosure, may be preferably a linearized plasmid DNA template. The promoter for controlling in vitro transcription can be any promoter for any DNA-dependent RNA polymerase. Particular non-limiting examples of DNA-dependent RNA polymerases are the T7, T3, and SPG RNA polymerases. A DNA template for in vitro RNA transcription may be obtained, for example, by cloning of a nucleic acid, in particular cDNA corresponding to the respective RNA to be in vitro transcribed, and introducing it into an appropriate vector for in vitro transcription, for example, into plasmid DNA. In a preferred embodiment of the present disclosure, DNA template may be linearized with a suitable restriction enzyme before it is transcribed in vitro. The cDNA may be obtained by reverse transcription of mRNA or chemical synthesis. Moreover, the DNA template for in vitro RNA synthesis may also be obtained by gene synthesis.
[0086] For example, reagents used in in vitro transcription may include:
1 ) a linearized DNA template with a promoter sequence that has a high binding affinity for its respective RNA polymerase such as bacteriophage-encoded RNA polymerases;
2) ribonucleoside triphosphates (NTPs) for the four bases (adenine, cytosine, guanine and uracil);
3) optionally a cap analogue as defined below;
4) a DNA-dependent RNA polymerase capable of binding to the promoter sequence within the linearized DNA template (e.g. T7, T3 or SPG RNA polymerase);
5) optionally a ribonuclease (Rnase) inhibitor to inactivate any contaminating Rnase;
6) optionally a pyrophosphatase to degrade pyrophosphate, which may inhibit transcription;
7) MgCh and/or magnesium acetate (Mg(C2HsO2)2) (Mg(0Ac)2), which supplies Mg2+ ions as a co-factor for the polymerase;
8) a buffer to maintain a suitable pH value, which can also contain antioxidants (e.g., DTT), amines, such as, betaine and/or polyamines, such as, spermidine at optimal concentrations.
[0087] In embodiments, in the method of RNA in vitro transcription according to the present disclosure, no reagents, which are only required for the in vitro translation of the transcribed RNA to protein, but not for RNA in vitro transcription are used. In particular, the mixture used for RNA in vitro transcription may not contain any proteinogenic amino acid or tRNA. Further, the mixture may not contain any proteinogenic amino acid, tRNA or a cell extract containing ribosomes.
[0088] The term “co-transcription” used herein refers to mRNA prepared with a cap structure through one step in vitro transcription reaction using RNA polymerase, e.g., T7 RNA polymerase. In contrast, one or more traditional post transcriptional capping method may need to prepare an uncapped RNA through in vitro transcription (IVT) with RNA polymerase, and then add Cap using capping enzyme, e.g., vaccinia capping enzyme, with the aid of 2’-O-Methyltransferase to add methylation at the +1 base of the mRNA.
[0089] Nucleic acid: The term “nucleic acid” means any DNA- or RNA-molecule and is used synonymous with polynucleotide. Furthermore, modifications or derivatives of the nucleic acid as defined herein are explicitly included in the general term “nucleic acid.” For example, peptide nucleic acid (RNA) is also included in the term “nucleic acid.”
[0090] Nucleic acid template: The nucleic acid template provides the nucleic acid sequence that is transcribed into the RNA by the process of in vitro transcription and which therefore comprises a nucleic acid sequence which is complementary to the RNA sequence that is transcribed therefrom. In addition to the nucleic acid sequence, which is transcribed into the RNA, the nucleic acid template comprises a promoter to which the RNA polymerase used in the in vitro transcription process binds with high affinity.
[0091] Preferably, the nucleic acid template may be a linearized plasmid DNA template. The linear template DNA may be obtained by contacting plasmid DNA with a restriction enzyme under suitable conditions so that the restriction enzyme cuts the plasmid DNA at its recognition site(s) and disrupts the circular plasmid structure. The plasmid DNA is preferably cut immediately after the end of the sequence that is to be transcribed into RNA. Hence, the linear template DNA comprises a free 5' end and a free 3' end, which are not linked to each other. If the plasmid DNA contains only one recognition site for the restriction enzyme, the linear template DNA has the same number of nucleotides as the plasmid DNA. If the plasmid DNA contains more than one recognition site for the restriction enzyme, the linear template DNA has a smaller number of nucleotides than the plasmid DNA. The linear template DNA is then the fragment of the plasmid DNA that contains the elements necessary for in vitro transcription, which is a promotor element for RNA transcription and the template DNA element. The open reading frame (ORF) of the linear template DNA may determine the sequence of the transcribed RNA by the rules of base-pairing.
[0092] In other embodiments, nucleic acid template may be selected from a synthetic double stranded DNA construct, a single-stranded DNA template with a double-stranded DNA region comprising the promoter to which the RNA polymerase binds, a cyclic double-stranded DNA template with promoter and terminator sequences or a linear DNA template amplified by PCR or isothermal amplification.
[0093] According to a preferred embodiment of the present disclosure, the concentration of nucleic acid template comprised in the in vitro transcription mixture described herein may be in a range from about 1 to about 200 nM, from about 10 nM to about 150 nM, from about 20 nM to about 140 nM, from about 30 nM to about 130 nM, from about 40 nM to about 120 nM, from about 50 nM to about 110 nM, from about 60 nM to about 100 nM, from about 65 nM to about 90 nM, from about 65 nM to about 80 nM, from about 65 nM to about 75 nM, from about 65 nM to about 70 nM, from about 70 nM to about 75 nM, about 1 to about 40 nM, about 1 to about 30 nM, about 1 to about 20 nM, or about 1 to about 10 nM. Even more preferred the concentration of the nucleic acid template may be from about 10 to about 30 nM. Most preferred the concentration of the nucleic acid template may be about 40, 50, 60, 70, 80, 90, or 100 nM.
[0094] RNA, mRNA: RNA is the usual abbreviation for ribonucleic acid. It is a nucleic acid molecule, i.e., a polymer consisting of nucleotide monomers. These nucleotides are usually adenosine-monophosphate (AMP), uridine-monophosphate (UMP), guanosine-monophosphate (GMP) and cytidine-monophosphate (CMP) monomers or analogues thereof, which are connected to each other along a so-called backbone. The backbone is formed by phosphodiester bonds between the sugar, i.e., ribose, of a first and a phosphate moiety of a second, adjacent monomer. The specific order of the monomers, i.e., the order of the bases linked to the sugar/phosphate- backbone, is called the RNA sequence. Usually RNA may be obtainable by transcription of a DNA sequence, e.g., inside a cell. In eukaryotic cells, transcription is typically performed inside the nucleus or the mitochondria. In vivo, transcription of DNA usually results in so-called premature RNA which has to be processed into so- called messenger-RNA, usually abbreviated as mRNA. Processing of the premature RNA, e.g., in eukaryotic organisms, comprises a variety of different posttranscriptional-modifications, such as, splicing, 5'-capping, polyadenylation, export from the nucleus or the mitochondria and the like. The sum of these processes is also called maturation of RNA. The mature messenger RNA usually provides nucleotide sequence that may be translated into an amino acid sequence of a particular peptide or protein. Typically, a mature mRNA comprises a 5'-cap, optionally a 5' UTR, an open reading frame, optionally a 3 'UTR, and a poly(A) sequence.
[0095] In addition to messenger RNA, several non-coding types of RNA exist, which may be involved in regulation of transcription and/or translation, and immunostimulation. The term “RNA” further encompasses RNA molecules, such as viral RNA, retroviral RNA and replicon RNA, small interfering RNA (siRNA), antisense RNA, CRISPR/Cas9 guide RNA, ribozymes, aptamers, riboswitches, immunostimulating RNA, transfer RNA (tRNA), ribosomal RNA (rRNA), small nuclear RNA (snRNA), small nucleolar RNA (snoRNA), microRNA (miRNA), and Piwi- interacting RNA (piRNA).
[0096] Dicarboxylic acid or salt thereof: A dicarboxylic acid is an organic acid having two carboxyl groups ( — COOH). The term includes linear saturated dicarboxylic acids having the general formula HO2C — (CH2)n — CO2H such as oxalic acid, malonic acid, succinic acid, glutaric acid, adipic acid, pimelic acid, suberic acid, azelaic acid and sebacic acid. It also includes unsaturated dicarboxylic acids having at least one double bond such as maleic acid and fumaric acid as well as substituted dicarboxylic acids having at least one additional functional group such as malic acid, tartaric acid, cichoric acid and dimercaptosuccinic acid. The salt of the dicarboxylic acid comprises the dicarboxylic acid anion and a suitable cation such as Na+, K+, Ca2+ or Mg2+.
[0097] Tricarboxylic acid or salt thereof: A tricarboxylic acid is an organic acid having three carboxyl groups ( — COOH). Examples of tricarboxylic acids include citric acid, isocitric acid, aconitic acid, trimesic acid, nitrilotriacetic acid and propane-1 , 2,3- tricarboxylic acid. In the buffer system and methods of the present invention preferably citric acid (3-carboxy-3-hydroxypentane-1 ,5-dioic acid) is used. The salt of the tricarboxylic acid comprises the tricarboxylic acid anion and a suitable cation, such as Na+, K+, Ca2+ or Mg2+. Preferably, sodium or magnesium citrate is used. If magnesium citrate is added to the RNA in vitro transcription reaction, it may be not necessary to add a magnesium salt to the reaction, since the magnesium ions within the magnesium citrate may serve as the cofactor for the RNA polymerase. Hence, in this case the reaction mixture for RNA in vitro transcription comprises magnesium citrate, a buffer substance, ribonucleoside triphosphates, a nucleic acid template and RNA polymerase.
[0098] Buffer substance: A buffer substance is a weak acid or base used to maintain the acidity (pH) of a solution near a chosen value after the addition of another acid or base. Hence, the function of a buffer substance is to prevent a rapid change in pH when acids or bases are added to the solution. Suitable buffer substances for use in the present invention include Tris (2-amino-2-hydroxymethyl-propane-1 ,3-diol) and HEPES (2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid). The buffer substance may further comprise an acid or a base for adjusting the pH, such as HCI in case of Tris (Tris-HCI) and KOH in case of HEPES (HEPES-KOH). In a preferred embodiment of the present invention citric acid is used to adjust the pH of the buffer substance, preferably of Tris base, so that no other acid has to be added. In an alternative embodiment the pH of the buffer substance is adjusted with an acid or a base such as HCI and KOH and the salt of the dicarboxylic or tricarboxylic acid, preferably citrate, is present in the reaction mixture in addition to the pH-adjusted buffer substance.
[0099] The concentration of the buffer substance within the mixture for in vitro transcription described herein may be about 10 to about 100 mM, about 10 to about 80 mM, about 10 to about 50 mM, about 10 to about 40 mM, about 10 to about 30 mM or about 10 to about 20 mM. Preferably, the concentration of the buffer substance is 40 mM.
[00100] Preferably the buffer has a pH value from about 6 to about 8.5, from about 6.5 to about 8.0, from about 7.0 to about 7.5, even more preferred of about 7.5 or about 8.0.
[00101] Ribonucleoside triphosphates: The ribonucleoside triphosphates (NTPs) GTP, ATP, CTP and UTP are the monomers that are polymerized during the in vitro transcription process. They may be provided with a monovalent or divalent cation as counterion. Preferably the monovalent cation is selected from the group consisting of Li+, Na+, K+, NH4+ or tris(hydroxymethyl)-aminomethane (Tris). Preferably, the divalent cation is selected from the group consisting of Mg2+, Ba2+ and Mn2+. More preferably, the monovalent cation is Na+ or tris(hydroxymethyl)-aminomethane (Tris).
[00102] NTP concentration may be from about 1 mM to about 50 mM, from about 1 mM to about 40 mM, from about 1 mM to about 30 mM, from about 1 mM to about 20 mM, from about 1 mM to about 10 mM, from about 1 mM to about 5 mM, from about 2 mM to about 10 mM, from about 3 mM to about 10 mM, from about 3 mM to about 9 mM, from about 3 mM to about 8 mM, from about 3 mM to about 7 mM, from about 3 mM to about 6 mM, from about 3 mM to about 5 mM, from about 3 mM to about 4 mM, from about 4 mM to about 10 mM, from about 5 mM to about 10 mM, from about 6 mM to about 10 mM, from about 7 mM to about 10 mM, from about 8 mM to about 10 mM, from about 9 mM to about 10 mM, from about 10 mM to about 50 mM. from about 20 mM to about 50 mM, from about 25 mM to about 50 mM, from about 25 mM to about 45 mM, from about 25 mM to about 40 mM, from about 25 mM to about 35 mM or from about 20 mM to about 30 mM.
[00103] According to a preferred embodiment of the invention, a part or all of at least one ribonucleoside triphosphate in the in vitro transcription reaction mixture is replaced with a modified nucleoside triphosphate as defined below.
[00104] Modified nucleoside triphosphate: The term “modified nucleoside triphosphate” as used herein refers to chemical modifications comprising backbone modifications as well as sugar modifications or base modifications. These modified nucleoside triphosphates are also termed herein as (nucleotide) analogues.
[00105] In this context, the modified nucleoside triphosphates as defined herein are nucleotide analogues/modifications, e g., backbone modifications, sugar modifications or base modifications. A backbone modification in connection with the present invention is a modification, in which phosphates of the backbone of the nucleotides are chemically modified. A sugar modification in connection with the present invention is a chemical modification of the sugar of the nucleotides. Furthermore, a base modification in connection with the present invention is a chemical modification of the base moiety of the nucleotides. In this context nucleotide analogues or modifications are preferably selected from nucleotide analogues which are applicable for transcription and/or translation.
[00106] Sugar Modifications
[00107] The modified nucleosides and nucleotides, which may be used in the context of the present invention, can be modified in the sugar moiety. For example, the 2' hydroxyl group (OH) can be modified or replaced with a number of different “oxy” or “deoxy” substituents. Examples of “oxy” -2' hydroxyl group modifications include, but are not limited to, alkoxy or aryloxy ( — OR, e.g., R=H, alkyl, cycloalkyl, aryl, aralkyl, heteroaryl or sugar); polyethyleneglycols (PEG), — O(CH2CH2O)nCH2CH2OR; “locked” nucleic acids (LNA) in which the 2' hydroxyl is connected, e.g., by a methylene bridge, to the 4' carbon of the same ribose sugar; and amino groups ( — 0-amino, wherein the amino group, e.g., NRR, can be alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, or diheteroaryl amino, ethylene diamine, polyamino) or aminoalkoxy.
[00108] “Deoxy” modifications include hydrogen, amino (e.g., NH2; alkylamino, dialkylamino, heterocyclyl, arylamino, diaryl amino, heteroaryl amino, diheteroaryl amino, or amino acid); or the amino group can be attached to the sugar through a linker, wherein the linker comprises one or more of the atoms C, N, and O.
[00109] The sugar group can also contain one or more carbons that possess the opposite stereochemical configuration than that of the corresponding carbon in ribose. Thus, a modified nucleotide can include nucleotides containing, for instance, arabinose as the sugar.
[00110] Backbone Modifications
[00111] The phosphate backbone may further be modified in the modified nucleosides and nucleotides. The phosphate groups of the backbone can be modified by replacing one or more of the oxygen atoms with a different substituent. Further, the modified nucleosides and nucleotides can include the full replacement of an unmodified phosphate moiety with a modified phosphate as described herein. Examples of modified phosphate groups include, but are not limited to, phosphorothioate, phosphoroselenates, borano phosphates, borano phosphate esters, hydrogen phosphonates, phosphoroam idates, alkyl or aryl phosphonates and phosphotriesters. Phosphorodithioates have both non-linking oxygens replaced by sulfur. The phosphate linker can also be modified by the replacement of a linking oxygen with nitrogen (bridged phosphoroam idates), sulfur (bridged phosphorothioates) and carbon (bridged methylene-phosphonates).
[00112] Base Modifications
[00113] The modified nucleosides and nucleotides, which may be used in the present disclosure, can further be modified in the nucleobase moiety. Examples of nucleobases found in RNA include, but are not limited to, adenine, guanine, cytosine and uracil. For example, the nucleosides and nucleotides described herein can be chemically modified on the major groove face. In some embodiments, the major groove chemical modifications can include an amino group, a thiol group, an alkyl group, or a halo group.
[00114] In particularly preferred embodiments of the present invention, the nucleotide analogues/modifications may be selected from base modifications, which are preferably selected from 2-amino-6-chloropurineriboside-5'-triphosphate, 2- Aminopurine-riboside-5'-triphosphate; 2-aminoadenosine-5'-triphosphate, 2'-Amino-2'- deoxycytidine-triphosphate, 2-thiocytidine-5'-triphosphate, 2-thiouridine-5'- triphosphate, 2'-Fluorothymidine-5’-triphosphate, 2 -O-Methyl inosine-5'-triphosphate
4-thiouridine-5'-triphosphate, 5-aminoallylcytidine-5'-triphosphate, 5-aminoallyluridine- 5'-triphosphate, 5-bromocytidine-5’-triphosphate, 5-bromouridine-5'-triphosphate, 5- Bromo-2'-deoxycytidine-5'-triphosphate, 5-Bromo-2'-deoxyuridine-5'-triphosphate, 5- iodocytidine-5'-triphosphate, 5-lodo-2'-deoxycytidine-5'-triphosphate, 5-iodouridine-5'- triphosphate, 5-lodo-2'-deoxyuridine-5'-triphosphate, 5-methylcytidine-5'-triphosphate,
5-methyluridine-5'-triphosphate, 5-Propynyl-2'-deoxycytidine-5'-triphosphate, 5- Propynyl-2'-deoxyuridine-5’-triphosphate, 6-azacytidine-5'-triphosphate, 6-azauridine- 5'-triphosphate, 6-chloropurineriboside-5 -triphosphate, 7-deazaadenosine-5'- triphosphate, 7-deazaguanosine-5'-triphosphate, 8-azaadenosine-5'-triphosphate, 8- azidoadenosine-5'-triphosphate, benzimidazole-riboside-5'-triphosphate, N1- methyladenosine-5'-triphosphate, N1-methylguanosine-5'-triphosphate, N6- methyladenosine-5'-triphosphate, O6-methylguanosine-5’-triphosphate, pseudouridine-5'-triphosphate, or puromycin-5'-triphosphate, xanthosine-5’- triphosphate. Particular preference is given to nucleotides for base modifications selected from the group of base-modified nucleotides consisting of 5-methylcytidine- 5'-triphosphate, 7-deazaguanosine-5’-triphosphate, 5-bromocytidine-5'-triphosphate, and pseudouridine-5 -triphosphate.
[00115] In some embodiments, modified nucleosides may include pyridin-4-one ribonucleoside, 5-aza-uridine, 2-thio-5-aza-uridine, 2-thiouridine, 4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxyuridine, 3-methyluridine, 5-carboxymethyl-uridine, 1- carboxymethyl-pseudouridine, 5-propynyl-uridine, 1 -propynyl-pseudouridine, 5- taurinomethyluridine, 1-taurinomethyl-pseudouridine, 5-taurinomethyl-2 -thio-uridine, 1- taurinomethyl-4-thio-uridine, 5-methyl-uridine, 1-methyl-pseudouridine, 4-thio-1- methyl-pseudouridine, 2-th io-1 -methyl-pseudouridine, 1 -methyl-1 -deaza- pseudouridine, 2-th io-1 -methyl-1 -deaza-pseudouridine, dihydrouridine, dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-dihydropseudouridine, 2- methoxyuridine, 2-methoxy-4-thio-uridine, 4-methoxy-pseudouridine, and 4-methoxy- 2-thio-pseudouridine.
[00116] In some embodiments, modified nucleosides may include 5-aza-cytidine, pseudoisocytidine, 3-methyl-cytidine, N4-acetylcytidine, 5-formylcytidine, N4- methylcytidine, 5-hydroxymethylcytidine, 1 -methyl-pseudoisocytidine, pyrrolo-cytidine, pyrrolo-pseudoisocytidine, 2-thio-cytidine, 2-thio-5-methyl-cytidine, 4-thio- pseudoisocytidine, 4-thio-1 -methyl-pseudoisocytidine, 4-th io-1 -methyl-1 -deaza- pseudoisocytidine, 1 -methyl-1 -deaza-pseudoisocytidine, zebularine, 5-aza-zebularine, 5-methyl-zebularine, 5-aza-2-thio-zebularine, 2-thio-zebularine, 2-methoxy-cytidine, 2- methoxy-5-methyl-cytidine, 4-methoxy-pseudoisocytidine, and 4-methoxy-1 -methyl- pseudoisocytidine.
[00117] In other embodiments, modified nucleosides may include 2-am inopurine, 2,
6-diaminopurine, 7-deaza-adenine, 7-deaza-8-aza-adenine, 7 -deaza-2 -am inopurine,
7-deaza-8-aza-2-am inopurine, 7-deaza-2, 6-diaminopurine, 7-deaza-8-aza-2,6- diaminopurine, 1 -methyladenosine, N6-methyladenosine, N6-isopentenyladenosine, N6-(cis-hydroxyisopentenyl)adenosine, 2-methylthio-N6-(cis-hydroxyisopentenyl) adenosine, N6-glycinylcarbamoyladenosine, N6-threonylcarbamoyladenosine, 2- methylthio-N6-threonyl carbamoyladenosine, N6,N6-dimethyladenosine, 7- methyladenine, 2-methylthio-adenine, and 2-methoxy-adenine.
[00118] In other embodiments, modified nucleosides may include inosine, 1 -methylinosine, wyosine, wybutosine, 7-deaza-guanosine, 7-deaza-8-aza-guanosine, 6-thio- guanosine, 6-thio-7-deaza-guanosine, 6-thio-7-deaza-8-aza-guanosine, 7-methyl- guanosine, 6-thio-7-methyl-guanosine, 7-methylinosine, 6-methoxy-guanosine, 1- methylguanosine, N2-methylguanosine, N2,N2-dimethylguanosine, 8-oxo-guanosine, 7-methyl-8-oxo-guanosine, l-methyl-6-thio-guanosine, N2-methyl-6-thio-guanosine, and N2, N2-dimethyl-6-thio-guanosine.
[00119] In some embodiments, the nucleotide can be modified on the major groove face and can include replacing hydrogen on C-5 of uracil with a methyl group or a halo group.
[00120] In specific embodiments, a modified nucleoside is 5'-O-(l-Thiophosphate)- Adenosine, 5'-O-(1-Thiophosphate)-Cytidine, 5'-O-(1-Thiophosphate)-Guanosine, 5'- O-(1-Thiophosphate)-Uridine or 5'-O-(1-Thiophosphate)-Pseudouridine.
[00121] In further specific embodiments the modified nucleotides may include nucleoside modifications selected from 6-aza-cytidine, 2-thio-cytidine, a-thio-cytidine, Pseudo-iso-cytidine, 5-aminoallyl-uridine, 5-iodo-uridine, N1-methyl-pseudouridine, 5,6-dihydrouridine, a-thio-uridine, 4-thio-uridine, 6-aza-uridine, 5-hydroxy-uridine, deoxy-thymidine, 5-methyl-uridine, Pyrrolo-cytidine, inosine, a-thio-guanosine, 6- methyl-guanosine, 5-methyl-cytidine, 8-oxo-guanosine, 7 -deaza-guanosine, N1- methyl-adenosine, 2-amino-6-Chloro-purine, N6-methyl-2-amino-purine, Pseudo-iso- cytidine, 6-Chloro-purine, N6-methyl-adenosine, a-thio-adenosine, 8-azidoadenosine, 7-deaza-adenosine.
[00122] Magnesium salt: A magnesium salt comprises a magnesium cation and a suitable anion, such as a chloride or an acetate anion. Preferably, the magnesium salt is magnesium chloride. In the in vitro transcription mixture(s) described herein. Preferably, the initial free Mg2+ concentration may be from about 1 to about 100 mM, about 1 to about 75 mM, about 1 to about 50 mM, about 1 to about 25 mM, or about 1 to about 10 mM. Even more preferred the initial free Mg2+ concentration is from about 5 to about 50 mM, about 10 to about 45 mM, about 15 to about 40 mM, or about 16 to about 37 mM, for example, about 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, or 37 mM. The person skilled in the art may understand that the choice of the Mg2+ concentration may be influenced by the initial total NTP concentration, meaning that a higher Mg2+ concentration may need to be used, if a higher total NTP concentration is used in the in vitro transcription mixture. In some embodiments, the concentration of magnesium salt may be from about 2 mM to about 50 mM, from about 2 mM to about 40 mM, from about 2 mM to about 30 mM, from about 2 mM to about 40 mM, from about 2 mM to about 30 mM, from about 2 mM to about 20 mM, from about 2 mM to about 10 mM, from about 2 mM to about 5 mM, from about 5 mM to about 50 mM, from about 10 mM to about 45 mM, from about 15 mM to about 40 mM, from about 20 mM to about 35 mM, from about 20 mM to about 30 mM, from about 20 mM to about 25 mM, from about 22 mM to about 28 mM, or from about 25 mM to about 30 mM.
[00123] RNA polymerase: RNA polymerase is an enzyme that catalyses transcription of DNA template into RNA. Suitable RNA polymerases for use in the present disclosure may include T7, T3, SP6 and E. coli RNA polymerase. Preferably, T7 RNA polymerase may be used. Also preferably, the RNA polymerase for use in the present disclosure may be recombinant RNA polymerase, meaning that it is added to the RNA in vitro transcription reaction as a single component and not as part of a cell extract that contains other components in addition to the RNA polymerase. A skilled person knows that the choice of the RNA polymerase depends on the promoter present in the DNA template, which has to be bound by the suitable RNA polymerase. Preferably, the concentration of the RNA polymerase in the in vitro transcription mixture(s) described herein may be from about 0.001 μg/μl to about 2 μg/μl, from about 0.001 μg/μl to about 1.5 μg/μl, from about 0.001 μg/μl to about 1 μg/μl, from about 0.01 μg/μl to about 1 μg/μl, from about 0.01 μg/μl to about 0.5 μg/μl, from about 0.01 μg/μl to about 0.5 μg/μl, from about 0.01 μg/μl to about 0.1 μg/μl, from about 0.01 μg/μl to about 0.05 μg/μl, from about 0.1 μg/μl to about 1 μg/μl, from about 0.1 μg/μl to about 0.9 μg/μl, from about 0.1 μg/μl to about 0.8 μg/μl, from about 0.1 μg/μl to about 0.7 μg/μl, from about 0.1 μg/μl to about 0.6 μg/μl, from about 0.1 μg/μl to about 0.5 μg/μl, from about 0.1 μg/μl to about 0.4 μg/μl, from about 0.1 μg/μl to about 0.3 μg/μl, or from about 0.1 μg/μl to about 0.2 μg/μl. Optionally, the concentration of the RNA polymerase in the in vitro transcription mixture(s) described herein may be from about 0.1 U/μl to about 2 U/μl, from about 0.5 U/μl to about 2 U/μl, from about 1 U/μl to about 2 U/μl, from about 1 U/μl to about 1.5 U/μl, or from about 1 .5 U/μl to about 2 U/μl. A person skilled in the art can understand that the choice of RNA polymerase concentration may be influenced by the concentration of DNA template.
[00124] Pyrophosphatase: A pyrophosphatase is an acid anhydride hydrolase that hydrolyses diphosphate bonds. In the in vitro transcription reaction, it may serve to hydrolyze the bonds within the diphosphate released upon incorporation of the ribonucleoside triphosphates into the nascent RNA chain. Preferably, the concentration of the pyrophosphatase in the in vitro transcription mixture(s) described herein may be from about 0.01 mU/μl to about 2 mU/μl, from about 0.01 mU/μl to about 1.5 mU/μl, from about 0.01 mU/μl to about 1 mU/μl, from about 0.1 mU/μl to about 2 mU/μl, from about 0.1 mU/μl to about 1.5 mU/μl, from about 0.1 mU/μl to about 1 mU/μl, from about 0.1 mU/μl to about 0.9 mU/μl, from about 0.1 mU/μl to about 0.8 mU/μl, from about 0.1 mU/μl to about 0.7 mU/μl, from about 0.1 mU/μl to about 0.6 mU/μl, from about 0.1 mU/μl to about 0.5 mU/μl, from about 0.1 mU/μl to about 0.4 mU/μl, from about 0.1 mU/μl to about 0.3 mU/μl, or from about 0.1 mU/μl to about 0.2 mU/μl,. Even more preferred the concentration of the pyrophosphatase may be about 0.3 mU/μl or may be about 0.5 mU/μl.
[00125] 5'-Cap structure: A 5' cap is typically a modified nucleotide, particularly a guanine nucleotide, added to the 5' end of an RNA molecule. Preferably, 5' cap may be added using a 5'-5'-triphosphate linkage. A 5' cap may be methylated, e.g., m7GpppN, wherein N is the terminal 5' nucleotide of the nucleic acid carrying the 5' cap, typically the 5'-end of an RNA. The naturally occurring 5' cap may include m7GpppN.
[00126] A 5' cap structure may be formed by a cap analogue.
[00127] Cap analogue: A cap analogue refers to a non-extendable di-nucleotide or tri-nucleotide that has cap functionality which means that it facilitates translation or localization, and/or prevents degradation of the RNA molecule when incorporated at the 5' end of the RNA molecule. Capped mRNA without 5’ end triphosphate structure reduces its immunogenicity side effect. Non-extendable means that the cap analogue will be incorporated only at the 5' terminus because it does not have a 5' triphosphate and therefore cannot be extended in the 3' direction by a template-dependent RNA polymerase.
[00128] In some embodiments, cap analogues may include a cap-2 analogue with the structure of Formula (I):
Figure imgf000042_0001
wherein R is H or CH3; and Bi is A or N6-methyl-adenine (m6A), and B2 is A, II, G, or C.
[00129] When R = H, the cap-2 analogues may include the following: m7GpppA2’OmepA2’Ome, m7GpppA2’OmepU2’Ome, m7GpppA2’OmepG2’Ome, m7GpppA2’OmepC2’Ome, m7Gppprn6A2’omepA2’o me, m7Gpppm6A2o me pUzo me, rn7Gppprn6A2’omepG2’ome, and m7Gpppm6A2’OmepC2’Ome.
[00130] When R = CH3, the cap-2 analogues may include the following: m7G3'OmePPpA2’Ome PA2 Ome, m7G3 o me PPPA2 Ome pU2’Ome, m7G3o me PPPA2OmepG2’Ome, m7G3o me PPPA2Ome PC2Ome, m7G3’OmePPpm6A2’OmepA2’Ome, m7G3’OmePPpm6A2’OmepU2’Ome, m7G3'OmePPpm6A2’OmepG2'Ome, and m7G3 o me pppm6A2’o me pC2’Ome.
[00131] In some embodiments, cap analogues may include cap-2 analogues with the structure of Formula (II):
(ii)
Figure imgf000043_0001
, in which
R1 is OCH3 and R2 is OH or H, or
R1 is OH and R2 is H, or
R1 is H and R2 is H or OCH3, or
R1 and R2 are each OCH3,
Bi is A or N6-methyl-adenine (m6A), and B2 is A, U, G, or C.
[00132] When Ri is OCH3 and R2 is OH, the cap-2 analogues may include the following:
Figure imgf000043_0002
m7G2 o me PPPA2 Ome pU2’Ome, m7G2 o me PPPA2 OmepG2’0me, m7G2’Ome pppA2’Ome pC2’Ome, m7G2 o me pppm6A2’o me pA2’Ome, m7G2 o me pppm6A2’o me PU2 Ome, m7G2'OmePPpm6A2’OmepG2'Ome, and m7G2 o me pppm6A2’o me pC2’Ome.
[00133] When R1 is OH and R2 is H, the cap-2 analogues may include the following: m7G3’HPPpA2’OmepA2’Ome, m7G3 HpppA2’o me pU2’Ome, m7G3'HpppA2’OmepG2’Ome, m7G3’HPPpA2’OmepC2’Ome, m7G3 Hpppm6A2’o me PA2 Ome, m7G3'Hpppm6A2’OmepU2’Ome, m7G3Hpppm6A2’omepG2’ome, and m7G3Hpppm6A2’o me PC2 Ome.
[00134] Preferably, cap analogue may be added with an initial concentration in the range of about 1 to about 20 mM, about 1 to about 17.5 mM, about 1 to about 15 mM, about 1 to about 12.5 mM, about 1 to about 10 mM, about 1 to about 7.5 mM, about 1 to about 5 mM or about 1 to about 2.5 mM. Even more preferred the cap analogue may be added with an initial concentration of about 5 to about 20 mM, about 7.5 to about 20 mM, about 10 to about 20 mM or about 12.5 to about 20 mM. In some embodiments, cap analogue may be in a concentration of from about 0.5 mM to about 50 mM, from about 0.5 mM to about 40 mM, from about 0.5 mM to about 30 mM, from about 0.5 mM to about 20 mM, from about 0.5 mM to about 10 mM, from about 0.5 mM to about 5 mM, from about 1 mM to about 10 mM, from about 2 mM to about 10 mM, from about 3 mM to about 10 mM, from about 3 mM to about 9 mM, from about 3 mM to about 8 mM, from about 3 mM to about 7 mM, from about 3 mM to about 6 mM, from about 3 mM to about 5 mM, from about 3 mM to about 4 mM, from about 4 mM to about 10 mM, from about 5 mM to about 10 mM, from about 6 mM to about 10 mM, from about 6 mM to about 9 mM, from about 6 mM to about 8 mM, from about 6 mM to about 7 mM, from about 7 mM to about 8 mM, from about 7 mM to about 9 mM, from about 7 mM to about 10 mM, from about 8 mM to about 9 mM, from about 8 mM to about 10 mM, or from about 9 mM to about 10 mM.
[00135] Ribonuclease inhibitor: A ribonuclease inhibitor inhibits the action of a ribonuclease, which degrades RNA. Preferably, the concentration of the ribonuclease inhibitor in the in vitro transcription mixture(s) described herein may be from about 0.001 U/μl to about 5 U/μl, from about 0.001 U/μl to about 4 U/μl, from about 0.001 U/μl to about 3 U/μl, from about 0.001 U/μl to about 2 U/μl, from about 0.001 U/μl to about 1 U/μl, from about 0.01 U/μl to about 5 U/μl, from about 0.01 U/μl to about 4 U/μl, from about 0.01 U/μl to about 3 U/μl, from about 0.01 U/μl to about 2 U/μl, from about 0.01 U/μl to about 1 U/μl, from about 0.01 U/μl to about 0.5 U/μl, from about 0.01 U/μl to about 0.1 U/μl, from about 0.01 U/μl to about 0.05 U/μl, from about 0.01 U/μl to about 0.04 U/μl, from about 0.01 U/μl to about 0.03 U/μl, from about 0.01 U/μl to about 0.02 U/μl, from about 0.1 U/μl to about 5 U/μl, from about 0.1 U/μl to about 4 U/μl, from about 0.1 U/μl to about 3 U/μl, from about 0.1 U/μl to about 2 U/μl, from about 0.1 U/μl to about 1 U/μl, from about 0.5 U/μl to about 5 U/μl, from about 0.5 U/μl to about 4 U/μl, from about 0.5 U/μl to about 3 U/μl, from about 0.5 U/μl to about 2 U/μl, from about 0.5 U/μl to about 1 U/μl, from about 1 U/μl to about 5 U/μl, from about 2 U/μl to about 5 U/μl, from about 3 U/μl to about 5 U/μl, from about 4 U/μl to about 5 U/μl from about 0.001 U/μl to about 0.005 U/μl, from about 0.001 U/μl to about 0.01 U/μl, from about 0.005 U/μl to about 0.01 U/μl, from about 0.01 U/μl to about 0.05 U/μl, from about 0.01 U/μl to about 0.04 U/μl, from about 0.01 U/μl to about 0.03 U/μl, from about 0.01 U/μl to about 0.02 U/μl, from about 0.02 U/μl to about 0.03 U/μl, from about 0.02 U/μl to about 0.04 U/μl, or from about 0.02 U/μl to about 0.05 U/μl.
[00136] Antioxidant: An antioxidant inhibits the oxidation of other molecules.
Suitable antioxidants for use in the present disclosure may include, but are not limited to, DTT (dithiothreitol), TCEP (tris(2-carboxyethyl)phosphine), NAC (N-acetylcysteine), beta-mercaptoethanol, glutathione, cysteine and cystine. Preferably, DTT may be used in in vitro transcription reaction.
[00137] The concentration of the antioxidant, preferably of DTT, in the in vitro transcription mixture(s) described herein may be about 1 to about 50 mM, about 5 to about 48 mM, about 8 to about 47 mM, about 10 to about 46 mM, about 15 to about 45 mM, about 18 to about 44 mM, about 20 to about 43 mM, about 23 to about 42 mM, about 25 to about 41 mM or about 28 to about 40 mM. Preferably, the concentration may be about 20 mM.
[00138] Amine: Preferably, the amine to be used in the present invention may be betaine (trimethylglycine). The concentration of the amine, preferably of betaine, may be about 10 mM to about 2M, preferably it may be about 0.7 M to about 1.3 M.
[00139] Polyamine: Preferably, the polyamine may be selected from the group consisting of spermine and spermidine. Preferably the concentration of the polyamine may be from about 1 to about 25 mM, about 1 to about 20 mM, about 1 to about 15 mM, about 1 to about 10 mM, about 1 to about 5 mM, or about 1 to about 2.5 mM. Even more preferred the concentration of the polyamine may be about 2 mM. Most preferred may be a concentration of about 2 mM of spermidine.
[00140] Dnase: Dnases are enzymes which hydrolyze DNA by catalyzing the hydrolytic cleavage of phosphodiester linkages in the DNA backbone. Suitable Dnases may be isolated from bovine pancreas and may be available from various suppliers such as Sigma-Aldrich, New England Biolabs, Qiagen and ThermoFisher. Preferably, Dnase may be free of any RNAse activity. In the method of the present disclosure, treatment with Dnase may be performed after the RNA in vitro transcription reaction by adding Dnase to reaction mixture used for RNA in vitro transcription. Preferably, a suitable amount of calcium chloride may be added together with the Dnase to the RNA in vitro transcription mixture. The suitable amount of CaCL may be from about 1 to about 5 mM, preferably from about 2 to about 4 mM and more preferably it may be about 3 mM. DNA may be treated with the Dnase for about 1 to about 5 hours, preferably for about 1 .5 to about 3 hours and more preferably for about 2 hours.
Dnase treatment may be preferably performed at a temperature of about 37°C. In one embodiment, about 3 mM CaCL and about 200 U/ml Dnase I may be added to RNA in vitro transcription mixture and the resulting mixture may be incubated for about two hours at about 37°C. In another embodiment, about 3 mM CaCl2 and about 400 U/ml Dnase I may be added to RNA in vitro transcription mixture and the resulting mixture may be incubated for about two hours at about 37°C. In another embodiment, Dnase may be added to RNA in vitro transcription mixture and the resulting mixture may be incubated for about 30 minutes at about 31 °C. Dnase treatment can be stopped by adding EDTA or another chelating agent. Preferably, Dnase treatment may be stopped by adding EDTA to a final concentration of about 25 mM. [00141] Embodiments of the present disclosure may provide a combined solution for in vitro synthesis of mRNA of different sizes ranging from, for example without limitation, about 1 Kb-20Kb, such as 1 Kb-15Kb, or 1 Kb-10Kb, with high capping efficiency, uniform poly A tail, high yields and integrity in a time efficient manner. Embodiments of the present disclosure may include design of T7 promoter sequence in a DNA template to provide high affinity to cap analogue to transcribe a capped mRNA with T7 RNA polymerase in vitro. This DNA template promoter design may include a T7 <t>6.5 promoter followed by a sequence GG. This design can ensure efficient initiation of transcription to prepare a capped mRNA with high fidelity at 5’ end by one step method.
[00142] 5’ end capping of mRNA
[00143] One of the key factors that determines mRNA translation efficiency may be its 5’ end capping. Typically, capping is performed using capping enzymes, and although the efficiency of the capping may be high, the process is time consuming and costly. There is therefore a need for an efficient co-transcriptional method that allows for quicker production of higher efficiency capped mRNA. This need was partially addressed by Ishikawa M., Ishikawa, et al., “Preparation of eukaryotic mRNA having differently methylated adenosine at the 5'-terminus and the effect of the methyl group in translation”, Nucleic Acids Symposium Series, Volume 53, Issue 1 , September- October 2009, pages 129-130. Ishikawa demonstrated the use of trinucleotide cap analogues of m7GpppA*pG structure (wherein A* is adenosine or methylated adenosine derivative) to prepare capped mRNA through one step in vitro transcription. By using these molecules, Ishikawa obtained a reporter 5’capped mRNAs carrying A, Am, m6A, or m6Am as the first transcribed nucleotide and studied their translational properties in rabbit reticulocyte system. Another study also confirmed the co- transcriptional capping of the mRNA using cap analogue. Sikorski, P.J., et al., 2020, “The identity and methylation status of the first transcribed nucleotide in eukaryotic mRNA 5' cap modulates protein expression in living cells”, Nucleic acids research, 48(4), pp.1607-1626.
[00144] In embodiments, methods and/or compositions that increase capping efficiency may be provided. In embodiments, methods and/or compositions that increase the capping efficiency to greater than about 50% efficiency, greater than about 55% efficiency, greater than about 60% efficiency, greater than about 65% efficiency, greater than about 70% efficiency, greater than about 75% efficiency, greater than about 80% efficiency, greater than about 85% efficiency, greater than about 90% efficiency, greater than about 95% efficiency, greater than about 96% efficiency, greater than about 96.5% efficiency, greater than about 97% efficiency, greater than about 97.5% efficiency, greater than about 98% efficiency, greater than about 98.5% efficiency, greater than about 99% efficiency, greater than about 99.5% efficiency may be provided as measured by a method of cutting mRNA with Rnase H, followed with measuring the capping efficiency with LC-MS.
[00145] In embodiments, cap analogues can initiate in vitro transcription to synthesize a capped mRNA in one pot reaction. In embodiments, the first base of methyl-A after the inverted G cap may bind to the -1 position of the DNA template, and the second base of G may bind to +1 position of the DNA template, forming a complex with RNA polymerase to recruit the next ribonucleoside triphosphate (NTP) to elongate the RNA during the transcription process. For example, as shown in FIG. 1 , when a cap-2 analogue (m7GpppA2-omeG2-ome), is utilized in the IVT, the first base of methyl-A after inverted G cap binds to -1 position of the DNA template, the second base of G binds to +1 position of the DNA template, forming the complex with T7 RNA polymerase to recruit the next NTP to elongate the RNA during the transcription process.
[00146] In embodiments, a composition comprising one or more cap analogue, as described herein, is provided. In embodiments, a method utilizing one or more cap analogue, as described herein, is provided. In embodiments, cap analogues as described herein may be used in conjunction with compositions and/or methods described herein and/or in conjunction with traditional compositions and/or methods. In embodiments, methods and/or compositions described herein may increase mRNA capping efficiency to greater than about 50% efficiency, greater than about 55% efficiency, greater than about 60% efficiency, greater than about 65% efficiency, greater than about 70% efficiency, greater than about 75% efficiency, greater than about 80% efficiency, greater than about 85% efficiency, greater than about 90% efficiency, greater than about 95% efficiency, greater than about 96% efficiency, greater than about 96.5% efficiency, greater than about 97% efficiency, greater than about 97.5% efficiency, greater than about 98% efficiency, greater than about 98.5% efficiency, greater than about 99% efficiency, greater than about 99.5% efficiency, or up to about 100% efficiency.
[00147] Promoters
[00148] The promoter design in a DNA template may be important for DNA dependent RNA polymerase to initiate in vitro transcription. In embodiments, where a T7 RNA polymerase (a single subunit polymerase derived from the T7 bacteriophage) may be used, the DNA template promoter design may include a T7 <t>6.5 promoter followed with a sequence GG, GA or AGG. In embodiments, this design may enable the highly efficient initiation of transcription to prepare a mRNA with high fidelity at 5’end, by one step method.
[00149] In embodiments, the promoters may have a sequence of
TAATACGACTCACTATAX1X2X3 (SEQ ID NO: 16), wherein Xi is A or G, X2 is A or G, and X3 is A, T, G, or C,
[00150] In embodiments, where a T7 RNA polymerase may be used, at least one promoter sequences in Table 1 may be used to initiate in vitro transcription. The promoters can be added into the plasmid vector through gene synthesis or subcloning.
Table 1
Figure imgf000049_0001
[00151] In embodiments, at least one of SEQ ID NO: 10, SEQ ID NO: 11 , SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, or SEQ ID NO: 15 may be used to initiate //? vitro transcription. [00152] In embodiments, a composition comprising one or more promoter, as described herein, is provided. In embodiments, a vector comprising one or more promoter, as described herein, is provided. In embodiments, a method utilizing one or more promoter, as described herein, is provided. In embodiments, a compositions comprising mRNA comprising one or more promoter, as described herein, is provided. In embodiments, a vector comprising mRNA comprising one or more promoter, as described herein, is provided. In embodiments, a method utilizing mRNA comprising one or more promoter, as described herein, is provided.
[00153] In some embodiments, in vitro transcription may start at -1 position, which may help to form a more favorable complex with T7 RNA polymerase and in turn to produce more full length RNAs. Application of using -1 and +1 position of starting site for in vitro transcription allows for more flexibility on the choice of first mRNA base (not including cap base) and leave +2 position open for custom sequence as described in Table 1 for mRNA production. Current general practice for incorporating cap molecule during in vitro transcription uses +1 position for initiation of the mRNA synthesis, which requires the template has the exact sequence of AG or AT following T7 promoter sequence of TATA box. Embodiments of the present disclosure may include a method of using DNA template with regular T7 promoter sequences, which has GG following TATA box of T7 promoter, which does not require specific mutagenesis on DNA template for preparing cap-1 mRNA co-transcriptionally.
[00154] In some embodiment, a method for in vitro transcription of a DNA template into RNA, may include providing (1 ) a DNA template comprising a promoter operably linked to a nucleic acid containing a 5’ untranslated region (5’ UTR), an open reading frame (ORF) encoding the RNA of interest, a 3’ UTR, and a poly A region, and (2) a cap analogue containing the structure of Formula (I) or (II):
Figure imgf000050_0001
in which wherein R is H or CH3; and Bi is A or N6-methyl-adenine (m6A), and
B2 is A, U, G, or C,
(ii)
Figure imgf000051_0001
, in which
Ri is OCH3 and R2 is OH or H, or
R1 is OH and R2 is H, or
R1 is H and R2 is H or OCH3, or
R1 and R2 are each OCH3,
Bi is A or N6-methyl-adenine (m6A), and B2 is A, II, G, or C. in which the promoter may contain a sequence of TAATACGACTCACTATAX1X2X3 (SEQ ID NO: 16), in which A at position 17 is -1 nucleotide and Xi at position 18 is +1 nucleotide, when Xi is G, X2 and X3 are each A, T, G, or C, then Bi is A or m6A and B2 is G, when Xi is A, X2 and X3 are each A, T, G, or C, then Bi is A or m6A and B2 is A, when Xi is C, X2 and X3 are each A, T, G, or C, then Bi is A or m6A and B2 is C, and when Xi is T, X2 and X3 are each A, T, G, or C, then Bi is A or m6A and B2 is U, in which the cap analogue binds to -1 and +1 nucleotides of the promoter, and incubating the DNA template and the cap analogue in a reaction mixture, in which the incubating may include incubating the reaction mixture at from about 15°C to about 35°C for from about 1 hour to about 12 hours, thereby producing the RNA. [00155] The promoter may contain a sequence selected from SEQ ID NO: 10, 11 , 12, 13, and 14.
[00156] In some embodiment, a method for in vitro transcription of a DNA template into RNA, may include providing (1 ) a DNA template comprising a promoter operably linked to a nucleic acid containing a 5’ untranslated region (5’ UTR), an open reading frame (ORF) encoding the RNA of interest, a 3’ UTR, and a poly A region, and (2) a cap analogue, in which the cap analogue binds to -1 and +1 nucleotides of the promoter, and incubating the DNA template and the cap analogue in a reaction mixture, in which the incubating may include incubating the reaction mixture at from about 15°C to about 35°C, preferably from about 18°C to about 31 °C, for an appropriate time preferably from about 1 hour to about 12 hours, thereby producing the RNA.
[00157] In some embodiment, a method for in vitro transcription of a DNA template into RNA, may include providing (1 ) a DNA template comprising a promoter operably linked to a nucleic acid containing a 5’ untranslated region (5’ UTR), an open reading frame (ORF) encoding the RNA of interest, a 3’ UTR, and a poly A region, and (2) a cap analogue, in which the cap analogue binds to -1 and +1 nucleotides of the promoter, and incubating the DNA template and the cap analogue in a reaction mixture, thereby producing the RNA.
[00158] In some embodiment, the reaction mixture comprises NTPs and an RNA polymerase. In some embodiment, the reaction mixture may further comprise one or more of the following: a buffer substance, an Rnase inhibitor, a magnesium salt, a polyamine, and a pyrophosphatase.
[00159] In some embodiment, the reaction mixture comprises: a buffer substance in a concentration of from about 45 mM to about 55 mM, an Rnase inhibitor in a concentration of from about 0.01 U/μl to about 0.03 U/μl, NTPs in a concentration of from about 1 mM to about 10 mM, the cap analogue in a concentration of from about 6 mM to about 8 mM, one or more magnesium salts in a concentration of from about 20 mM to about 30 mM, a polyamine in a concentration of from about 1 .5 mM to about 2.5 mM, the DNA template in a concentration of from about 0.01 μg/μl to about 0.05 μg/μl, a pyrophosphatase in a concentration of from about 0.1 mU/μl to about 0.5 mU/μl, and an RNA polymerase in a concentration of from about 0.01 μg/μl to about 0.05 μg/μl.
[00160] 5’ UTR2 and 3’ UTR2
[00161] mRNA molecule may be flanked with 5’ end and 3’ end untranslated regions (UTR). 5’-UTR may be recognized by ribosome to allow for initiation of translation, and 3’UTR may contain regulatory sequences that can influence the expression and halflife of the mRNA. Several different combinations of 5’ and 3’ UTR can enable high expression efficiency of mRNA when expressed in mammalian cells.
[00162] Cao, et al. (Cao, et al., “High-throughput 5’UTR engineering for enhanced protein production in non-viral gene therapies”, Nature Communications, (2021 ) 12:4138, pages 1-10, incorporated herein by reference in its entirety) has reported a method of generating artificial 5' UTR2 through a high-throughput screening process.
[00163] In embodiments, the combination of artificial selected 5’ UTR, and human hemoglobin or mouse hemoglobin 3’ UTR can be used to generate a construct for highly efficient expressing mRNA sequences. The combination thereof may be selected and used to construct a vector for in vitro transcription (IVT). That may be used to generate mRNA with highly efficient protein expression capability. In embodiments, one or more UTR set forth in Table 2 (see below, in Example 1 ) is used. In embodiments, one or more UTR set forth in Table 2 may be used in any combination. In embodiments, the UTR2 may be used in pairs, as set forth in Table 2, with the members of pairs being in the same row. In embodiments, SEQ ID NO. 1 is paired with SEQ ID NO. 2, SEQ ID NO. 3 is paired with SEQ ID NO. 2, SEQ ID NO. 1 is paired with SEQ ID NO. 4, SEQ ID NO. 1 is paired with SEQ ID NO. 6, SEQ ID NO. 3 is paired with SEQ ID NO. 6, and/or SEQ ID NO. 9 is paired with SEQ ID NO. 2. In embodiments, use of one or more of the UTR2 set forth in Table 5 may enable high expression efficiency of mRNA when expressed in mammalian cells. In embodiments, use of one or more pairs of the UTR2 set forth in Table 5 may enable high expression efficiency of mRNA when expressed in mammalian cells.
[00164] Different 5’ UTR and 3’ UTR pairs may be cloned into vector in an orientation, for example, from 5’ to 3’ direction: a promoter, e.g., T7 promoter (SEQ ID NO: 10), 5’ UTR, Kozak sequence (GCCACC), eGFP coding sequence, and 3’ UTR. Vector can then be sub-cloned into pVAX vector for generating plasmid for mRNA preparation. Example of 5’ UTR and 3’ UTR pairs are shown in Table 2. Table 2
Figure imgf000054_0001
Figure imgf000055_0001
Figure imgf000056_0001
[00165] In embodiments, the T7 promoter sequences, UTR2 may be added to the open reading frame coding sequences through gene synthesis, which may be then sub-cloned into the plasmid vector to generate large scale plasmid DNA for in vitro transcription application. A composition comprising one or more UTR, as described herein, is provided. In embodiments, a vector comprising one or more UTR, as described herein, is provided. In embodiments, a method utilizing one or more UTR, as described herein, is provided. In embodiments, a compositions comprising mRNA comprising one or more UTR, as described herein, is provided. In embodiments, a vector comprising mRNA comprising one or more UTR, as described herein, is provided. In embodiments, a method utilizing mRNA comprising one or more UTR, as described herein, is provided.
[00166] Poly A tails
[00167] Poly-A tail quality (such as length and uniformity of length and distribution) may directly impact mRNA expression efficiency. Traditional methods for adding poly A tail to a mRNA product in vitro use poly A polymerase. See, e.g., Cao, G.J. and Sarkar, N., “Identification of the gene for an Escherichia coli poly(A) polymerase”, Proc. Natl. Acad. Sci. USA, (1992) 89(21), 10380-10384, incorporated herein by reference in its entirety. However, such methods usually generate a product with a broad distribution of poly A tail lengths, with only about 70% of mRNA being tailed.
[00168] In addition to capping and poly-A tail length and distribution, mRNA purity and integrity may be important factors that influence the properties of mRNA, such as, but not limited to, its stability and/or expression efficiency. Purification, promoter sequence, 5’ and/or 3’ UTR sequence(s), and/or transcription conditions may contribute to generating a high quality mRNA. [00169] A traditional method for adding poly A tail to a mRNA product in vitro uses poly A polymerase as described in, for example, Cao et al. (Proc.Natl. Acad. Sci. USA.
89, 10380-10384). But this method may generate poly A products with broad distribution of poly A tail length, e.g., with only about 70% of mRNA polyadenylated.
[00170] In embodiments, methods and/or compositions may include provision of capped analogues, methods of providing and/or improving length and/or distribution of poly-A tails, provision of effective promoters, provision of effective UTR2, such as pairs of UTR2, provision of effective transcription conditions, provision of an effective transcription system, provision of effective purification, or any combination thereof.
[00171] In embodiments, methods and/or compositions that increase the uniformity of length and/or distribution of poly-A tails among the transcribed mRNA molecules may be provided. In embodiments, methods and/or compositions may produce a population of mRNAs wherein greater than about 70% are tailed, wherein at least about 71 % are tailed, wherein at least about 72% are tailed, wherein at least about 73% are tailed, wherein at least about 74% are tailed, wherein at least about 75% are tailed, wherein at least about 76% are tailed, wherein at least about 77% are tailed, wherein at least about 78% are tailed, wherein at least about 79% are tailed, wherein at least about 80% are tailed, wherein at least about 85% are tailed, wherein at least about 90% are tailed, wherein at least about 95% are tailed, wherein at least about 99% are tailed.
[00172] In embodiments, methods and/or compositions may produce a population of mRNAs wherein the lengths (the number of adenines) of the poly-A tails vary by at most about 70 to about 130 adenines between the mRNA molecules, by at most about 60 to about 120 adenines between the mRNA molecules, by at most about 50 to about 100 adenines between the mRNA molecules, by at most about 40 to about 90 adenines between the mRNA molecules, by at most about 50 to about 80 adenines between the mRNA molecules, by at most about 40 to about 70 adenines between the mRNA molecules, by at most about 30 to about 50 adenines between the mRNA molecules, or by at most about 20 to about 40 adenines between the mRNA molecules. The poly A tail length of mRNA may be measured by a method to digest the mRNA with Rnase T1 , followed by purifying and recovering the poly A fragment with oligo-dT magnetic beads, and then test the poly A fragment length by Bioanalyzer capillary gel electrophoresis. [00173] In embodiments, a poly-A tail is added to a DNA template before transcription. In embodiments, a method of adding a poly-A tail to a DNA template prior to transcription may be provided. In embodiments, a poly-A tail is added to a DNA template via Polymerase Chain Reaction (PCR), which is a new way of adding poly A tail comparing to traditional method by gene synthesis to insert into the plasmid vector. In embodiments, addition of a poly-A tail to the DNA template may result in the generation of a more uniformly poly-A-tailed mRNA product, a product with longer poly-A tails, or both, each or both of which may result in more efficient mRNA expression.
[00174] In embodiments, a composition comprising mRNA comprising a poly-A tail added by PCR, as described herein, is provided. In embodiments, a vector comprising a poly-A tail added to the vector by PCR, as described herein, is provided. In embodiments, a pVAX1 or pUC57 vector comprising ampicillin resistant gene, T7 promoter sequences, 5’ UTR and 3’ UTR sequences, mRNA comprising a poly-A tail (e.g., 100A) added by PCR, as described herein, is provided.
[00175] In embodiments, disclosed methods and/or compositions may produce a population of mRNAs wherein greater than about 70% are tailed, wherein at least about 71 % are tailed, wherein at least about 72% are tailed, wherein at least about 73% are tailed, wherein at least about 74% are tailed, wherein at least about 75% are tailed, wherein at least about 76% are tailed, wherein at least about 77% are tailed, wherein at least about 78% are tailed, wherein at least about 79% are tailed, wherein at least about 80% are tailed, wherein at least about 85% are tailed, wherein at least about 90% are tailed, wherein at least about 95% are tailed, wherein at least about 99% are tailed, wherein about 100% are tailed.
[00176] In embodiments, disclosed methods and/or compositions may produce a population of mRNAs wherein the lengths (the number of adenines) of the poly-A tails vary by at most about 70 to about 130 adenines between the mRNA molecules, by at most about 60 to about 120 adenines between the mRNA molecules, by at most about 50 to about 100 adenines between the mRNA molecules, by at most about 40 to about 90 adenines between the mRNA molecules, by at most about 50 to about 80 adenines between the mRNA molecules, by at most about 40 to about 70 adenines between the mRNA molecules, by at most about 30 to about 50 adenines between the mRNA molecules, or by at most about 20 to about 40 adenines between the mRNA molecules.
[00177] Bacterial studies have demonstrated that the sequence of repeats, e.g., CTG.CAG, as well as the mode and level of plasmid replication and transcription may play a role in expansion and deletion of repeats. The frequency of deletion of cloned repeats may increase up to 20-fold upon induction of the lacZ promoter, which drives transcription of inserts in pUC19 (Bowater et al., 1997. Transcription increases the deletion frequency of long CTG GAG triplet repeats from plasmids in Escherichia coli. Nucleic Acids Res 25: 2861-2868; the content of which is hereby incorporated by reference in its entirety). Common vectors may be typically maintained at high copy number and may induce transcription and translation of antibiotic resistance gene, indicator gene (such as blue/white screening gene) and inserted fragments, causing instability of certain classes of DNA sequences. As an extreme repeat and extremely low GC ratio sequence, polyA sequences may be easily lost in the process of plasmid cloning and replication. If such a sequence occurs, unexpected transcription and translation affected by the inductive activity of upstream and downstream promoters may further increase instability of polyA sequences. In order to avoid the initiation activity of similar promoters in vectors for the inserted sequence, common strategies may include segmenting the inserted gene (if the inserted gene is significantly cytotoxic) or directional cloning of the ORF in the “reverse" orientation relative to transcription from the vector’s promoter. However, these two strategies may be not suitable for gene cloning that contain polyA sequences. Segmentation can eliminate the genotoxicity of cloned genes after being expressed, but this strategy may not solve the problem of instability of polyA sequences because the process of transcription has not been eliminated, while the reverse insertion can only eliminate the influence of the promoter in a certain direction. Therefore, embodiments of the present disclosure may include modifying vectors by inserting transcriptional terminators at the upstream and downstream of the multiple cloning site, which can effectively interrupt the influence of the upstream and downstream promoters of the multiple cloning site on the inserted gene. This strategy effectively improves the stability of polyA sequences in the process of plasmid cloning and replication, especially for some extremely unstable polyA-containing cassette. The results show that, after adding transcriptional terminators to the upstream and downstream of the multiple cloning site, the positive rate of clones reached 25%-50% (almost 0 before adding terminator), and the number of A bases of polyA tails increased from 70-110 to about 120. In addition, after adding terminators upstream and downstream of the inserted cassette, the yield of plasmid also increased by 32.5%. Without being limited to a particular theory, this increase may be due to the insertion of a transcriptional terminator restores the activity of the origin of replication (Stueber et al., 1982. Transcription from efficient promoters can interfere with plasmid replication and diminish expression of plasmid specified genes. EMBO J 1 : 1399-1404; the content of which is hereby incorporated by reference in its entirety). Using transcriptional terminators at the upstream and downstream of the multiple cloning site to facilitate the cloning of cytotoxic sequences or sequences of repeats, such as PolyA tails, has not been reported in the art.
[00178] A transcriptional termination sequence may be any nucleotide sequence, which when placed transcriptionally downstream of a nucleotide sequence encoding an open reading frame, causes the end of transcription of the open reading frame. Such sequences are known in the art and may be of prokaryotic, eukaryotic or phage origin. Examples of terminator sequences include, but are not limited to, PTH- terminator, pET-T7 terminator, T3-Tcp terminator, pBR322-P4 terminator, vesicular stomatitis virus terminator, rrnB-T1 terminator, rrnB-T2 terminator, lambda tO terminator, rrnC terminator, Ttadc transcriptional terminator, and yeast-recognized termination sequences, such as Mata (a-factor) transcription terminator, native a- factor transcription termination sequence, ADR1 transcription termination sequence, ADH2 transcription termination sequence, and GAPD transcription termination sequence. A non-exhaustive listing of transcriptional terminator sequences may be found in the iGEM registry, which is available at: partsregistry.org/Terminators/Catalog. The first transcriptional terminator sequence of a series of 2, 3, 4, 5, 6, 7, or more may be placed directly 3’ to the final nucleotide of a gene of interest (or open reading frame) or at a distance of at least 1 -5, 5-10, 10-15, 15-20, 20-25, 25-30, 30-35, 35-40, 40-45, 45-50, 50-100, 100-150, 150-200, 200-300, 300-400, 400-500, 500-1 ,000 or more nucleotides 3’ to the final nucleotide of a gene of interest (or open reading frame). The number of nucleotides between tandem transcriptional terminator sequences may be varied, for example, transcriptional terminator sequences may be separated by 0, 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 10-15, 15-20, 20-25, 25-30, 30-35, 35-40, 40-45, 45-50 or more nucleotides.
[00179] Vectors [00180] In embodiments, a vector including one or more of (i) a poly-A tail, as described herein, (ii) one or more UTR, as described herein, (iii) one or more promoter, as described herein, or (iv) combinations thereof, is provided. In embodiments, the vector may have various uses, including, but not limited to, use for insertion of a target template nucleotide sequence. In embodiments, such a vector with a target template nucleotide sequence may be used for, as non-limiting examples, cloning or transcribing the target template nucleotide sequence. In embodiments, transcription may include in vitro transcription. In embodiments, transcription may be carried out using a T7 RNA polymerase. In embodiments, a vector may be a plasmid or a viral vector, for example but without limitation, pVAX1 and/or pUC57.
[00181] mRNA purity
[00182] The integrity of mRNA may affect cellular expression; therefore it may be important to begin translation with high integrity mRNA. The conditions used for transcription may affect the quality of mRNA produced. Some transcription conditions can lead to higher truncated products.
[00183] The integrity of the mRNA may be one of the critical factors that affects cellular expression, therefore it may be paramount to begin with high integrity mRNA. There are two aspects to the mRNA integrity. Typically, RNA is prone to quicker degradation than DNA due its chemical instability, therefore the storage conditions impact the quality of the RNA. Additionally, transcription conditions can also lead to higher truncated products and therefore the optimal buffer conditions can be determined empirically to have higher integrity over a wide range of mRNA sizes and also have higher yields.
[00184] Conditions used for purification may affect the quality of mRNA produced. For example, Trinucleotide cap analogues of m7GpppA*pG residues in the finished mRNA product could compete with capped mRNA for recruiting ribosome, thus inhibiting the mRNA translation efficiency in cells. Purification after in vitro transcription may be an important step in acquiring final purified mRNA product.
[00185] The present disclosure further provides a solution to obtain pure mRNA product with minimum contaminant cap analogues, free NTPs, and other proteins, which could interfere and compromise the mRNA product’s performance. mRNA purity refers to full length mRNA species ratio in the crude transcribed mRNA product, which may be quantified by the full length peak ratio in capillary gel electrophoresis.
[00186] In embodiments, purification as described herein may produce highly pure mRNA product with minimum contaminant analogues, free NTPs, and/or other proteins which could interfere with and/or compromise the mRNA product’s stability and/or translation efficiency. In embodiments, a purification method includes using a silica membrane column to bind nucleic acid, then washing with about 60% to about 80% ethanol in water, preferably about 70% to about 80% ethanol in water, followed by elution in water.
[00187] Other conventional purification methods with LiCI precipitation, or affinity based magnetic beads purification can be applied for the same purpose. In embodiments, the purification methods as described herein may be used in conjunction with compositions and/or methods described herein and/or in conjunction with traditional compositions and/or methods.
[00188] In embodiments, methods and/or compositions that increase purity of transcribed mRNA may be provided. In embodiments, methods and/or compositions that result in obtained mRNA with about 79% or greater purity; mRNA with about 79.5% or greater purity; mRNA with about 80% or greater purity; mRNA with about
80.5% or greater purity; mRNA with about 81 % or greater purity; mRNA with about
81 .5% or greater purity; mRNA with about 82% or greater purity; mRNA with about
82.5% or greater purity; mRNA with about 83% or greater purity; mRNA with about
83.5% or greater purity; mRNA with about 84% or greater purity; mRNA with about
84.5% or greater purity; mRNA with about 85% or greater purity; mRNA with about
85.5% or greater purity; mRNA with about 86% or greater purity; mRNA with about
86.5% or greater purity; mRNA with about 87% or greater purity; mRNA with about
87.5% or greater purity; mRNA with about 88% or greater purity; mRNA with about
88.5% or greater purity; mRNA with about 89% or greater purity; mRNA with about
89.5% or greater purity; mRNA with about 90% or greater purity; mRNA with about
91 % or greater purity; mRNA with about 92% or greater purity; mRNA with about 93% or greater purity; mRNA with about 94% or greater purity; mRNA with about 95% or greater purity; mRNA with about 96% or greater purity; mRNA with about 97% or greater purity; mRNA with about 98% or greater purity; mRNA with about 99% or greater purity; or mRNA with about 100% purity may be provided. The mRNA purity may be measured by capillary gel electrophoresis using Bio analyzer equipment, the target peak area ratio (target length ± 15% ) may be calculated for its purity measurement.
[00189] In embodiments, methods and/or compositions that increase the purity or integrity of transcribed mRNA may be provided. Obtaining high quality long mRNA from in vitro transcription is a great challenge since long mRNAs tends to degrade more easily.
[00190] In some embodiments, IVT may be performed at from about 15°C to about 35°C, from about 16°C to about 35°C, from about 17°C to about 35°C, from about 18°C to about 35°C, from about 18°C to about 34°C, from about 18°C to about 33°C, from about 18°C to about 32°C, from about 18°C to about 31 °C, from about 18°C to about 30°C, from about 18°C to about 29°C, from about 18°C to about 28°C, from about 18°C to about 27°C, from about 18°C to about 26°C, from about 18°C to about 25°C, from about 18°C to about 24°C, from about 18°C to about 23°C, from about 18°C to about 22°C, from about 18°C to about 21 °C, from about 18°C to about 20°C, from about 18°C to about 19°C, from about 25°C to about 26°C, from about 25°C to about 27°C, from about 25°C to about 28°C, from about 25°C to about 29°C, from about 25°C to about 30°C, from about 25°C to about 31 °C, from about 21 °C to about 22°C, from about 21 °C to about 23°C, from about 21 °C to about 24°C, from about 21 °C to about 25°C, about 25°C, about 26°C, about 27°C, about 28°C, about 29°C, about 30°C, about 31 °C, about 32°C, about 33°C, about 34°C, or about 35°C.
[00191] In some embodiments, IVT may be performed for from about 1 hour to about 12 hours, from about 1 hour to about 11 hours, from about 1 hour to about 10 hours, from about 1 hour to about 9 hours, from about 1 hour to about 8 hours, from about 1 hour to about 7 hours, from about 1 hour to about 6 hours, from about 1 hour to about 5 hours, from about 1 hour to about 4 hours, from about 1 hour to about 3 hours, from about 1 hour to about 2 hours, from about 2 hours to about 12 hours, from about 3 hours to about 12 hours, from about 4 hours to about 12 hours, from about 5 hours to about 12 hours, from about 6 hours to about 12 hours, from about 7 hours to about 12 hours, from about 8 hours to about 12 hours, from about 9 hours to about 12 hours, from about 10 hours to about 12 hours, from about 11 hours to about 12 hours, about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, or about 10 hours. [00192] In some embodiments, IVT may be performed at about 31 °C for from about 1 hour to about 5 hours, from about 1 hour to about 4.5 hours, from about 1 hour to about 4 hours, from about 1 hour to about 3.5 hours, from about 1 hour to about 3 hours, from about 1 hour to about 2.5 hours, from about 1 hour to about 2 hours, from about 1 hour to about 1 .5 hours, from about 0.5 hour to about 1 hour, from about 0.5 hour to about 1 .5 hours, about 1 hour, about 2 hours, about 3 hours, about 4 hours, or about 5 hours.
[00193] T7 RNA polymerase-mediated transcription
[00194] In eukaryotes, transcription of messenger RNAs (mRNAs) is done by RNA polymerase II. This is a complicated multi-subunit enzyme with complex regulation. To carry out large scale transcription in vitro, researchers commonly use single subunit phage polymerases derived from T7, T3, SP6, K1 -5, K1 E, K1 F or K11 bacteriophages. This family of polymerases uses simple, minimal promoter sequences of ~17 nucleotides, which may not require accessory proteins and may have minimal constraints of the initiating nucleotide sequence. While this application focuses on T7 RNA Polymerase (T7 RNAP), one skilled in the art would understand that the present disclosure may be practiced with other RNA polymerases.
[00195] T7 RNA polymerase (RNAP) exists in at least two protein states. The first is referred to as the “abortive complex” and may be associated with transcriptional initiation. The second is a very processive conformation called the “elongation complex.” In vitro transcription can be broken into six steps: 1 ) binding of RNA polymerase to promoter sequence, 2) initiation of transcription, 3) non-processive elongation termed abortive transcription, during which polymerase frequently releases DNA template and short abortive transcripts, 4) conversion of open complex to closed complex, 5) processive elongation, and 6) transcriptional termination. Significant amount of RNA produced during transcription may contain short abortive fragments of ~2-8 nucleotides in length (Biochemistry 19:3245-3253 (1980); Nucleic Acids Res.
9:31 -45 (1981 ); Nucleic Acids Res. 15:8783-8798 (1987); Biochemistry 27:3966-3974 (1988), each of which is incorporated herein by reference in its entirety). After synthesis of about 10-14 bases, RNA polymerases may escape from abortive cycling, at the same time losing sequence-specific contacts with promoter DNA, and forming a processive elongation complex, in which RNA chain may be extended in a sequenceindependent manner (J. Mol. Biol. 183:165-177(1985); Proc. Natl. Acad. Sci. U.S.A. 83:3614-3618(1986); Mol. Cell Biol. 7:3371 -3379 (1987), each of which is incorporated herein by reference in its entirety).
Table 3
Figure imgf000065_0001
[00196] The consensus sequence for the most active Class III T7 promoters may encompass 17 bp of sequence upstream, and 6 bp downstream, of the transcription start site (Cell 16:815-25. (1979), incorporated by reference herein in its entirety). The position of the first transcribed nucleotide is commonly referred to as the +1 transcript nucleotide of the RNA, the second transcribed nucleotide as +2 transcript nucleotide and so on (Table 3). During transcription, two strands may be melted to form transcription bubble and the bottom strand of the duplex (shown 3' to 5' in Table 3) is the template for transcription. For transcript nucleotides +3 and beyond, the template strand may define the identity of the transcribed nucleotides primarily through Watson- Crick base pairing interactions. Here, the nucleotide encoding the first RNA transcript nucleotide is defined as the +1 nucleotide of the template. In the example shown in Table 3, the +1 transcript nucleotide is G and the +1 template nucleotide is C. Likewise the +4 transcript nucleotide is A and the +4 template nucleotide is T.
[00197] It is known that T7 RNAP can also initiate with short oligonucleotide primers. For example, it is known that 13 promoters in the T7 genome can initiate with pppGpG (J. Mol. Biol. 370:256-268 (2007), incorporated by reference herein in its entirety). It has been shown that T7 RNAP can initiate from dinucleotide primers
(Biochemistry 24:5716-5723 (1985), incorporated by reference herein in its entirety). Axelrod et al. showed that an uncapped GpA dinucleotide could initiate from +1 and +2 template nucleotides that were 2 '-deoxycytidine and 2'-deoxythymidine, respectively (“CT" template). Their reaction conditions were 200 micromolar (pM) dimer and 100 pM ATP, CTP, GTP and UTP. Their reaction mixture also contained 100 pM 3' dATP, 3' dCTP 3' dUTP or 50 pM 3' dGTP. They observed only GpA initiated RNAs and not a mixture of GpA initiated RNAs and 5' triphosphate RNAs from GTP initiation. This is likely due to the reaction conditions employed. 100 pM GTP is well below the 2 mM Kd of T7 polymerase for the first initiating guanosine (J. Mol. Biol. (2007) 370, 256-268, incorporated by reference herein in its entirety). Since GTP competes for initiation with the initiating oligonucleotide, using a low GTP concentration favours GpA initiation but may result in low transcription yield (maximum calculated yield estimated to be <150 ug/mL of reaction). When initiating transcription on “CT” template with ApG, CpG, UpG or GpG, they observed formation of RNA transcripts with an additional untemplated 5' nucleotide (A, C, U or G, respectively).
[00198] Ishikawa et al. showed that capped initiating oligonucleotide trimers of the structure m7GpppApG, m7Gpppm6ApG, m7GpppA2omepG or m7Gpppm6A2omepG could initiate transcription on template with 2'-deoxycytidine residues at template positions +1 and +2 (“CC” template; Nucleic Acids Symposium Series No. 53: 129 (2009), incorporated by reference herein in its entirety). The authors state, “The different result from the case of using m7G5'pppG may be caused from base pairing between additional adenosine (N1 ) in m7G5'pppN1 pG and 2'-deoxythymidine in T7 promoter at -1 position." This method clearly differs from the method described in the present disclosure where the +1 and +2 nucleotides of the initiating capped oligonucleotide trimer pair with the +1 and +2 of the template nucleotides. Ishikawa et al. used 6 mM initiating oligonucleotide trimer, 0.9 mM GTP and 7.5 mM each of ATP, CTP and UTP. The authors used a greater than 6 fold excess of the capped initiating oligonucleotide primer, the most expensive nucleotide component of transcription reaction, over competing GTP to drive the transcription reaction toward capped RNA over pppRNA, which increases the total cost of synthesized RNA. On the other hand, a low concentration of GTP (0.9 mM) limits the total yield of the RNA in transcription reaction (theoretically to less than 1 .4 mg/mL). On the contrary the method described herein may not require restricting the concentration of any NTP to achieve both an efficient RNA capping and a higher yield of RNA (about 2 to 10 mg/mL) and thus allowing a production of high quality mRNA at a commercially useful cost.
[00199] T7 RNA Polymerases [00200] In some embodiments, at least one modification of the T7 RNA polymerase may be selected from the group consisting of P266L, P270L, P270S, P270A, P270Y, Q744L, Q744P, Q744R, Y639F, H784A, E593G, Y639V, V685A, H784G, S430P, N433T, S633P, F849I and F880Y. In some embodiments, at least one modification includes Y639F and H784A. In some embodiments, at least one modification includes E593G, Y639V, V685A and H784G. In some embodiments, at least one modification includes S430P, N433T, S633P, F849I and F880Y. In some embodiments, at least one modification includes S430P, N433T, S633P, F849I , F880Y and P266L. In some embodiments, at least one modification includes S430P, N433T, S633P, F849I , F880Y, Y639F and H784A. In some embodiments, at least one modification includes S430P, N433T, S633P, F849I , F880Y, P266L, Y639F and H784A. In some embodiments at least one modification includes S430P, N433T, S633P, F849I , F880Y, E593G, Y639V, V685A and H784G. In some embodiments, at least one modification includes S430P, N433T, S633P, F849I , F880Y, P266L, E593G, Y639V, V685A and H784G.
[00201] In some embodiments, at least one modification of the T7 RNA polymerase facilitates initiation-elongation transition. In some embodiments, at least one modification increases promoter clearance. In some embodiments, at least one modification increases stability and/or activity of the polymerase. In some embodiments, at least one modification increases thermos stability of the polymerase.
[00202] Magnesium ions in in vitro transcription
[00203] Magnesium ions (Mg2+) is an essential component in an RNA in vitro transcription buffer system to initiate the transcription with Cap analogues rather than GTP. Conventional buffer systems for RNA in vitro transcription (e.g., HEPES buffer, Tris-HCI buffer) may contain high concentrations of free magnesium ions, because free Mg2+ ions may be required to guarantee a high activity of the RNA polymerase enzyme. During in vitro transcription process, Mg2+ complexes with NTP during the reaction, keeping no extra free Mg2+ ions present in buffer systems is important for ensuring high capping efficiency and high integrity of transcribed mRNA. Therefore, higher concentration of Mg2+ can cause problems, especially in the context of high- yield/industrial-scale RNA production. Some of the problems associated with free Mg2+ ions in the production of RNA may include magnesium-driven precipitates, which may lead to a drop in the free Mg2+ concentration, resulting in depletion of magnesium ions from the RNA polymerase reaction center. A consequence of that would be a less efficient RNA in vitro transcription. To assess the impact of Mg concentration on mRNA IVT yield and integrity, IVT were performed in the presence of increasing Mg concentrations. By keeping final Mg concentration at 16.5 mM, 21 mM, 29 mM, and 37 mM, respectively, in the IVT reaction, the concentration of free Mg2+ after complexing with NTPs and cap analogues were at -12 mM, -8 mM, 0 mM and +8 mM, respectively, in the IVT reaction system. The mRNA yield reduced when Mg2+ concentration increased, and the mRNA integrities also reduced when Mg2+ concentration increases in the IVT system.
EXAMPLES
EXAMPLE 1
[00204] Synthesis of cap analogues
[00205] General.
[00206] 5’-DMT-2’-O-methyl-guanosine (n-ibu) and 5’-DMT-2’-O-methyl-N6-methyl- adenosine 3’-phosphoramidite were purchased from ChemGenes, Inc., (Wilmington, MA). Chemical Phosphorylation reagent [5’-phosphate amidite (O-DMT-2,2 - sulfonyldiethanol)] was purchased from Biosearch Technologies. TLCs were run with aluminium-backed silica gel 60 F254 plates purchased from Sigma-Aldrich. 1H and 31 P NMR spectra were taken on a Bruker 300 MHz spectrometer. HPLC chromatograms were obtained on an Agilent 1290 Infinity II system employing a HALO 90A C18 column (4.6 x 150 mm) and gradient elution with 50 mM TEAB buffer, pH 7.8 / acetonitrile. TLC and/or HPLC analyses were routinely used for monitoring reaction completion. UV spectral data were obtained on a VWR UV-3100PL spectrophotometer. Buffer solutions were prepared using water dispensed from a Mill- Q EQ 7000 ultrapure water system. Reported compounds were prepared as previously described: 2a: Sawai et al. (Synthesis and Reactions of Nucleoside 5‘- Diphosphate Imidazolide. A Non-enzymatic Capping Agent for 5‘-Monophosphorylated Oligo ribonucleotides in Aqueous Solution, J. Org. Chem., 1999, 64, 5836-5840); 2b and 9: Jemielity et al. (Novel "anti-reverse" cap analogues with superior translational properties, RNA, 2003, 9, 1108-1122). The contents of which are hereby incorporated by reference in their entireties. [00207] Synthesis of cap-2 analogues
[00208] Preparation of intermediate 3, (FIG. 2).
[00209] To a solution of 5’-DMT-2’-O-methyl guanosine (n-ibu) (1.0 g, 1.49 mmol) in 20 ml of anhydrous pyridine was added 3.4 ml of a solution consisting of 9:1 , acetic anhydride/anhydrous THF. The resulting solution was stirred under an argon atmosphere for 22 hr. before the solution was evaporated to dryness.
[00210] The residue was then stirred in 10 ml. of 80% aqueous acetic acid for 2 hr. The solvents were stripped under reduced pressure and the residue was purified by silica gel chromatography eluting with a gradient of 0 ->10% methanol in ethyl acetate. The pure product fractions were pooled and evaporated affording a white foam: 590- mg (96%) yield; TLC (ethyl acetate / methanol [9:1 ]) Rf = 0.48; 'H NMR (dimethyl-d6 sulfoxide) 5 12.10 (1 H, br s), 11.68 (1 H br s), 8.33 (1 H, s), 5.88 (1 H, d, J = 9.0 Hz), 5.44 (1 H, d, J = 6.0 Hz), 5.31 (1 H, t, J = 6.0 Hz), 4.56 (1 H, m), 4.14 (1 H, br t), 3.63 (2H, t, J = 3.0 Hz), 3.26 (3H, s), 2.77 (1 H, m), 2.13 (3H, s), 1.14 & 1.11 (6H, 2 x s); mass spectrum (ESI+) m/z 410.16 (M + H+).
[00211] Preparation of intermediate 4, (FIG. 3).
[00212] Intermediate 3 (585 mg, 1.43 mmol) and 5'-dimethoxytrityl-N-benzoyl- Adenosine,2'-O-methyl,3'-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite (1 .27 g, 1 .48 mmol) were dissolved in 40 ml of anhydrous acetonitrile. Tetrazole (6.0 ml of a 0.45 M solution in anhydrous acetonitrile) was added and the resulting solution was stirred for 18 h. under an argon atmosphere. To this solution was added (1 S)-(+)- (camphorsulfonyl)oxaziridine (CSO) oxidizer (6.0 ml of a 0.50 M solution in acetonitrile) and the resulting solution was stirred for 16 h. The solvent was stripped and the residue was stirred in 10 ml of 80% aqueous acetic acid for 16 h. The solution was evaporated to dryness under reduced pressure and the residue was purified by silica gel chromatography eluting with a mobile-phase gradient of 0 -> 15% methanol in ethyl acetate. The pure fractions were pooled and evaporated affording an amorphous solid: 865-mg (67%) yield; TLC (ethyl acetate / methanol [9:1]) Rf = 0.21 ; 31 P NMR (dimethyl-d6 sulfoxide) 5 -2.11 & -2.21 (1 P,2 x s, diasteriomeric pair); mass spectrum (ESI+) m/z 910.42 (M + H+).
[00213] Preparation of intermediate 5 (FIG. 3). [00214] Intermediate 4 (124 mg, 0.136 mmol) and “Chemical Phosphorylating Reagent” (197 mg, 0.30 mmol) were stirred in 3.0 ml of 0.45M tetrazole in anhydrous acetonitrile for 4.0 hr. at room temperature under an argon atmosphere. HPLC analysis indicated that the coupling reaction was complete and 3.0 ml of CSO oxidizer solution (0.50 M in anhydrous acetonitrile) was added directly to the reaction solution. Stirring was continued for 1 .0 hr. before the solvent was stripped under reduced pressure.
[00215] To the residue was added 20 ml of 1/1 , concentrated ammonium hydroxide/methanol. The reaction vessel was sealed and allowed to sit at 37 C for 2 days. The solvents were stripped the residue was mixed with 20 ml of water and then filtered. The filtrate was applied to a DEAE-Sephadex A25 column and eluted with a gradient of 0 -> 0.70 M TEAB buffer, pH 7.8. The pure product fractions were pooled and evaporated to dryness. TEAB salt was removed by multiple evaporations from water. The final residue was dissolved in a minimum volume of 10% aqueous DMF and the triethylammonium salt of the product was precipitated by addition of acetonitrile: 57-mg (45%) yield. 'H NMR (D2O) 5 8.31 (1 H, s), 8.02 (1 H, s), 7.43 (1 H, s), 5.96 (1 H d, J = 5.1 Hz), 5.8 (1 H, d, J = 5.1 Hz), 4.83 (1 H, m), 4.53 (1 H, t, J = 4.4 Hz), 4.35 (3H, m), 4.22 (1 H, m), 4.10 (2H, m), 3.97 (2H, m), 3.37 (3H, s), 3.34 (3H, s), 3.08 & 1.16 (q and t, triethylammonium counter-ion); 31 P NMR (D2O) 5 0.26 (1 P, s), - 0.78 (1 P, s); mass spectrum (ESI) m/z 720.2.
[00216] Preparation of intermediate 1a (FIG. 3).
[00217] A mixture of intermediate 5 (12.8 mg, 0.014 mmol), 2a (16.7 mg, 0.027 mmol), and MnCI2 (2.5 mg, 0.020 mmol) was stirred in anhydrous DMSO (257 pl) for 18 hr at room temperature. EDTA (75 pl of a 0.5 M solution in water) was added and the solution was diluted to 2.0 ml with water. This solution was applied to a DEAE- Sephadex A25 column and eluted with a gradient of 0 -> 0.70 M TEAB buffer, pH 7.8. The product fractions were pooled and evaporated to dryness under reduced pressure. TEAB buffer salt was eliminated by multiple evaporations from water. The residue was further purified by reverse-phase (C18) high pressure liquid chromatography: 4.7-umol (34%) yield; mass spectrum (ESI) m/z 1 ,159.8.
[00218] Preparation of intermediate 1b (FIG. 3). [00219] A mixture of intermediate 5 (7.0 mg, 0.076 mmol), 2a (7.0 mg, 0.011 mmol), and MnCI2 (2.5 mg, 0.016 mmol) was stirred in anhydrous DMSO (200 pl) for 18 hr. at room temperature. EDTA (50ul of a 0.5 M solution in water) was added and the solution was diluted to 5.0 ml with water and then lyophilized to dryness. The crude product was purified by reverse-phase (C18) high pressure liquid chromatography: 5.9-umol (78%) yield; mass spectrum (ESI) m/z 1 ,173.2.
[00220] WO2022036858 discloses methods of preparing cap-2 analogues using enzymes including phosphohydrolase, guanosyltransferase, and T4 RNA Ligase 1. In contrast, embodiments of the present disclosure may not include enzymes in the preparation of cap-2 analogues. A comparison between the methods disclosed in WO2022036858 and that in the present disclosure is shown below.
Figure imgf000071_0001
Figure imgf000072_0001
Figure imgf000073_0001
[00221] Synthesis of N6-methyl-adenine (m6A)-cap-2 analogues
[00222] Preparation of intermediate 7 (FIG. 4).
[00223] Intermediate 3 (51 mg, 0.125 mmol) and 5'-DMT-2'-O-methyl-N6-methyl- adenosine 3'-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite (100 mg, 0.125 mmol) were dissolved in tetrazole solution (1.0 ml of a 0.45 M solution in anhydrous acetonitrile) and stirred for 2 h. under an argon atmosphere. To this solution was added CSO oxidizer (1 .0 ml of a 0.50 M solution in acetonitrile) and the resulting solution was stirred for 20 min. The solvent was stripped and the residue was stirred in 1.0 ml of 80% aqueous acetic acid for 16 h. at room temperature. The solution was evaporated to dryness under reduced pressure and the residue was purified by silica gel chromatography eluting with a mobile-phase gradient of 0 -> 20% methanol in ethyl acetate. The pure fractions of intermediate 6 were pooled and evaporated affording an amorphous solid: 80 mg (87%) yield.
[00224] Intermediate 6 (80 mg, 0.109 mmol) and Chemical Phosphorylation
Reagent (100 mg, 0.152 mmol) were dissolved in tetrazole solution (1.0 ml of a 0.45 M solution in anhydrous acetonitrile) and stirred for 2 h. under an argon atmosphere. To this solution was added CSO oxidizer (1 .0 ml of a 0.50 M solution in acetonitrile) and the resulting solution was stirred for 20 min and then the solvents were stripped. The residue was redissolved in a 50/50 mixture of methanol/ammonium hydroxide and the sealed reaction vessel was incubated at 37 C for 2 days. The reaction solution was then evaporated to dryness, redissolved in 12 ml of water and loaded onto a DEAE Sephedex A25 column and eluted with a gradient of 0 -> 0.60 M TEAB buffer, pH 7. The pure product fractions were pooled and evaporated to dryness. Excess TEAB buffer salt was removed by performing multiple evaporations from water. The triethylammonium salt form of intermediate 7 was obtained as an amorphous solid: 28 mg yield; TLC (n-butanol/water/acetic acid, [5:3:1]) Rr= 0.13; mass spectrum (ESI) m/z 734.2.
[00225] Preparation of Cap reagent analogue 8 (Fig. 4).
[00226] A mixture of the triethylammonium salt form of intermediate 7 (6.9 mg, 0.007 mmol), the triethylammonium salt form of 2b (10.0 mg, 0.016 mmol), and MnCI2 (2.0 mg, 0.016 mmol) was stirred in anhydrous DMSO (200 pl) for 40 hr at room temperature. EDTA (100 pl of a 0.5 M solution in water) was added and the solution was diluted to 5.0 ml with water and lyophilized to dryness. The residue was purified by reverse-phase (C18) high pressure liquid chromatography: 6.0 mg (54%) yield; mass spectrum (ESI) m/z 1 ,187.6.
EXAMPLE 2
[00227] In vitro Transcription
[00228] Both linearized DNA plasmid and PCR product can be used as DNA template for preparing mRNA by in vitro transcription. To test in vitro transcription conditions, plasmid vector containing T7 promoter (SEQ ID NO: 10 or 12), 5 -UTR (SEQ ID NO: 9), eGFP coding sequences, 3’-UTR (SEQ ID NO: 2), and poly A tail (100A) was linearized by the BspQ1 or Bbs1 restriction enzyme, and purified using magnetic beads. The linearized plasmid was transcribed with T7 RNA polymerase (wild-type), cap analogues (cap-2), 10x transcription buffer, NTPs, and RNase Inhibitor to prepare capped mRNA.
[00229] The in vitro transcription mixture and conditions were as set forth in Tables 4-6. More specifically, a 10x Buffer containing HEPES buffer was prepared by adding magnesium acetate, spermidine, and DTT and stored at -20°C for use. In the in vitro transcription reaction, the buffer, DNase-free and RNase-free water, NTPs (concentrations as specified in the tables), and cap analogue were added into a reaction tube, followed by adding DNA template, T7 polymerase, RNase inhibitor, and Inorganic pyrophosphatase. The reaction was kept at designated temperature ranging from 20-40°C for 1 to 6 hours for transcription to take place. Then, DNA templates were removed by digestion with DNasel RNase-free enzyme. [00230] Cap analogues may be any capped analogues described herein. For IVT reactions described in Tables 4-6, cap-2 analogues m7GpppA20mepG20me and m7G3’0mepppA2’0mepG20me were used. For these examples, 7GpppA2’0mepG20me i referred to here as cap-2. In m7G3OmepppA2 o mepG2’0me, a methoxy group (— OCH3) replaces the 3 -OH group closer to the m7G. This modification is analogous to the modification in the anti-reverse cap analogue (ARCA: 3'-O-Me-m7GpppG). Stepinski et al. (Stepinski J, Waddell C, Stolarski R, Darzynkiewicz E, Rhoads RE (2001 ) Synthesis and properties of mRNAs containing the novel "anti-reverse" cap analogues 7-methyl(3’-O-methyl)GpppG and 7-methyl(3'deoxy)GpppG. RNA 7: 1486-1495; the content of which is hereby incorporated by reference in its entirety) has shown that this 3’-0 modification in ARCA prevents the generation of mRNAs capped in the reverse orientation. m7G3’omepppA2omepG2ome is referred to as ARCA-cap-2. IVT reactions were also carried out using the AG cap-1 analogue.
[00231] HEPES buffer was 400 mM HEPES in water, pH 7.5. RNA polymerase can be wild type T7 RNA polymerase or T7 RNA polymerase with mutations to enhance stability and/or the ability to incorporate cap-2 analogues. In vitro transcription can be set up in DNase-free RNase-free plastic tubes ranging from 0.2 mL to 15 mL at a defined temperature with or without shaking.
[00232] Three IVT conditions (IVT1 , IVT2, and IVT3) were tested as specified in
Tables 4-6. Table 4 shows IVT1 conditions where equal concentrations of NTPs are used for IVT. Table 5 shows IVT2 conditions where GTP concentrations are reduced as shown. Table 6 shows IVT3 conditions where the concentration of ATP and GTP are reduced as shown.
Table 4. IVT1 conditions
Figure imgf000075_0001
Figure imgf000076_0001
Figure imgf000077_0001
[00233] mRNA was purified from IVT reactions using a silica membrane column. Briefly, mRNA from in vitro transcription was mixed with a buffer and ethanol, and added to silica membrane column, followed by washing with 70% ethanol and eluting with water or other storage buffer for mRNA.
[00234] The amount of silica column purified mRNAs was determined by Nanodrop to calculate the overall yield f IVT reactions, which is expressed as the amount (microgram) of mRNAs obtained per microliter of IVT reaction.
[00235] The integrity of mRNAs prepared by the methods of the present disclosure were subjected to integrity analysis by Agilent Bioanalyzer to assess purity based on size. Agilent RNA Nano 6000 kit was used. The purity of analyzed mRNA was analyzed through smear analysis to calculate the ratio of target length ± 10% mRNA populations.
[00236] Capping efficiencies of the mRNAs were examined by RNase H digest and LC-MS as described by Beverly et al. (“Label-free analysis of mRNA capping efficiency using RNase H probes and LC-MS.” Anal Bioanal Cham. 2016
Jul;408(18):5021 -30, the content of which is hereby incorporated by reference in its entirety). Briefly, a short biotinylated RNA/DNA hybrid probe was annealed to the mRNA, which was then digested with RNase H. The duplex containing the probe and 5’ end of the mRNA was then purified for LC-MS analysis.
[00237] eGFP mRNA thus prepared with promoter SEQ ID NO 10 or 12 was used for expression efficiency assay in A549 cells. A549 cells were seeded the day before in 96-well clear-bottom black plates. eGFP mRNA samples were transfected into the seeded cells in triplicates, at 200 ng/well of mRNA plus 0.4 μl of lipofectamine MessengerMax with OptiMEM. The cells were incubated with mRNA overnight, then the expression efficiency was assessed based on relative fluorescence intensity of eGFP mRNA treated cells using a plate reader. The values were normalized by relative cell number as tested by Cyquant XTT cell viability assay. And the normalized values for mRNAs prepared with cap-2 cap analogues were compared to those prepared with AG cap-1 cap analogue.
Results
[00238] Plasmid templates encoding eGFP were used to generate mRNAs using the above conditions and procedures. The mRNA yield for IVT reactions is presented in Table 7.
Cap-2:
Figure imgf000078_0001
ARCA-Cap 2:
Figure imgf000078_0002
[00239] Using IVT1 conditions and promoter #2 (SEQ ID NO: 10), the mRNA yield using cap-2 (8.07 μg/μl) or ARCA-cap-2 (7.65 μg/μl) was similar to that of using AG cap-1 (6.99 μg/μl). The mRNA yield using promoter #1 (SEQ ID NO: 12) was lower using cap-2 (2.12 μg/μl) and ARCA-cap-2 (2.09 μg/μl) than that using promoter #2 (SEQ ID NO: 10).
[00240] Using IVT2 conditions, the mRNA yield using cap-2 and promoter #1 (SEQ ID NO: 12) was 2.14 μg/μl and 2.56 μg/μl with promoter #2 (SEQ ID NO: 10). The mRNA yield using ARCA-cap-2 was 2.08 μg/μl using promoter #1 (SEQ ID NO: 12) and 2.06 μg/μl using promoter #2 (SEQ ID NO: 10). [00241] Using IVT3 conditions, the mRNA yield using cap-2 and promoter #1 (SEQ ID NO: 12) was 1.98 μg/μl and 2.62 μg/μl using promoter #2 (SEQ ID NO: 10). The mRNA yield using ARCA-cap-2 and promoter #1 (SEQ ID NO: 12) was 1 .8 μg/μl and 2.85 μg/μl using promoter #2 (SEQ ID NO: 10).
[00242] Table 7. IVT reaction yield with modified IVT conditions.
Table 7. Yield (μg/μl) of IVT reactions using different cap analogues and templates
Figure imgf000079_0001
[00243] The integrity of IVT mRNAs capped with the cap-2 or ARCA-cap-2 was compared to those capped with AG cap-1 on an Agilent Bioanalyzer using the RNA Nano 6000 kit. The results are shown in Table 8. With promoter #1 (SEQ ID NO: 12) template, mRNAs prepared with AG cap-1 had 71 -78% purity in the 900-1150nt range. In comparison, mRNAs prepared with cap-2 and ARCA-cap-2 had 82-83% and 80-84% purity in the same range. With promoter #2 (SEQ ID NO: 10) template, mRNAs prepared with AG cap-1 had 85-93% purity in the 900-1150 nt range, compared to 89-91 % and 89% respectively for mRNAs prepared with cap-2 and ARCA-cap-2.
[00244] Table 8. mRNA integrity (900-1150 nt) from IVT reactions with modified conditions.
Figure imgf000079_0002
[00245] Capping efficiencies of the mRNAs were examined by RNase H digest and LC-MS as described above. The AG cap-1 cap analog can be efficiently co- transcriptionally incorporated into IVT mRNAs using IVT1 conditions, with >80% capping efficiency. Using the same IVT1 conditions, co-transcriptional capping with cap-2 is of low efficiency. As shown in Table 9, using IVT1 conditions and promoter #1 (SEQ ID NO: 12), cap-2 and ARCA-cap-2 had 34.6% and 14.4% capping efficiency, respectively. Compared with that used promoter #1 (SEQ ID NO: 12), the capping efficiency was better using promoter #1 (SEQ ID NO: 10) with cap-2 (44.4%) but worse for ARCA-Cap II (2.3%). Using modified IVT conditions, efficiency of co- transcriptional capping of mRNAs improved. Using promoter #1 (SEQ ID NO: 12) and cap-2, the capping efficiencies were 69.6% and 63.0% in IVT2 and IVT3 conditions, respectively. Using promoter #2 (SEQ ID NO: 10), additional improvement was observed, e.g., 71.2% and 67.5% in IVT2 and IVT3 conditions, respectively. Similarly, using ARCA-cap-2 and promoter #2 (SEQ ID NO: 12), the efficiencies were 62.6% (IVT2) and 67.9% (IVT3). While capping efficiencies improved for ARCA-cap-2 using promoter #1 (SEQ ID NO: 10)in modified IVT2 (20.5%) and IVT3 (17.45%) conditions as compared with that in IVT1 (2.30%), the efficiency was less than that using cap-2 in IVT2 (71 .2%) and IVT3 (67.5%).
Table 9. Capping efficiencies of different mRNAs
Figure imgf000080_0001
[00246] The mRNAs were also transfected into A549 cells to determine their translatability. A549 cells were seeded the day before and examined 24 hours later using a plate reader. As shown in FIG. 5, consistent with their poor capping efficiencies, mRNAs generated using cap-2 and ARCA-cap-2 and IVT1 conditions with either promoter #1 (SEQ ID NO: 12) or promoter #2 (SEQ ID NO: 10) showed poor expression as compared to mRNAs generated using AG cap-1 , e.g., at <30% of AG cap-1 levels using promoter #1 (SEQ ID NO: 12) and -10% using promoter #2 (SEQ ID NO: 10). Using promoter #2 (SEQ ID NO: 12) in modified IVT conditions (IVT2 and IVT3) led to significant increase in mRNA expression, which is consistent with their improved capping efficiencies. For example, relative to AG cap-1 mRNAs, >75% mRNA expression levels were obtained using cap-2 and ARCA-Cap II in IVT2 conditions. Nearly 60% and 80% mRNA expression levels, respectively, were obtained using cap-2 and ARCA-Cap II. Improved expression was also observed by using promoter #2 (SEQ ID NO: 10) in IVT2 and IVT3 conditions, albeit at lower levels. For example, relative to AG cap-1 mRNAs, >40% of mRNA expression levels were observed by using cap-2 and ARCA-Cap II in IVT2 conditions and >20% of mRNA expression levels were observed by using cap-2 and ARCA-Cap II in IVT3 conditions.
EXAMPLE 3
[00247] A. Preparation of cap reagent analog 11 (m7G2 OmepppA2 o mepG2’0me ) (Fig. 6)
[00248] Intermediate 9 (16 mg, 0.028 mmol) combined with triphenylphosphine (28 mg, 0.11 mmol), imidazole (13 mg, 0.19 mmol) and 2, 2’-dithiopyridine (25 mg, 0.11 mmol). The mixture was dissolved in anhydrous DMSO (146 ul) and triethylamine (19 ul) was added. The resulting solution was stirred at room temperature for 45 minutes and then diluted with 1 .8 ml of ice-cold 0.2 M sodium perchlorate in acetone. The reaction solution was diluted to a total of 1 .8 ml with a solution of ice-cold 0.2 M sodium perchlorate in acetone and the precipitate that formed was spun-down to a pellet. The supernatant was discarded, and the pellet was washed with 1 .8 ml of ice- cold acetone and again spun-down to a pellet. This acetone wash cycle was repeated four more times and then the product pellet was dried under high vacuum affording intermediate 10 as an off-white solid. This solid was combined with intermediate 5 (9.0 mg, 0.0098 mmol) and MgCL (3.2 mg, 0.025 mmol). The mixture was dissolved in anhydrous DMSO (160 ul) and the resulting solution was incubated at 37 degrees Celsius for 18 hours. EDTA (400 ul of a 0.5 M solution) was added and the resulting solution was then diluted to 5.0 ml with water and lyophilzed to dryness. The crude reaction product was dissolved in water and purified by reverse-phase (C18) high pressure liquid chromatography: 5.3 umol (54%) yield of 11 ; mass spectrum (ESI) m/z 1 ,172.
[00249] B. Preparation of rn7G3’0meppspA2’0mepG2’0me (16)
[00250] B-1. Preparation of intermediate 13 and 14 (Fig. 7)
[00251] 3’-O-Methylguanosine-5’ -phosphate (12) was prepared according to Jemielity et al., RNA, 2003, 9, 1108-1122. The triethylammonium salt of Intermediate 12 (132 mg, 0.28 mmol) was dissolved in 4.0 ml of water and the solution was adjusted to pH 4.0 with acetic acid. Dimethyl sulfate (0.38 ml) was added dropwise over a 10 minute period and the reaction solution was stirred for 4 hours with addition of sodium hydroxide solution in order to maintain the reaction solution between pH 3.75-4.25. After the 4-hour reaction period the reaction solution was extracted with methylene chloride (3 x 16 ml) and the crude product, contained in the aqueous phase, was purified by reverse phase (C18) HPLC employing a TEAB, pH 7.8 - acetonitrile gradient. The pure product fractions were lyophilized to dryness affording a white solid: 110-mg (81 %) yield of intermediate 13 as the triethylammonium salt.
[00252] Intermediate 13 (104 mg, 0.18 mmol) was combined with imidazole (85 mg, 1.25 mmol), triphenylphosphine (188 mg, 0.74 mmol) and 2, 2’-dithiopyridine (164 mg, 0.72 mmol). The mixture was dissolved in 2.0 ml of dry DMSO containing triethylamine (130 ul). The resulting solution was stirred at room temperature for 4.0 hr. The reaction solution was then diluted to 25 ml with ice-cold 0.2 M sodium perch lorate/acetone solution. The precipitate that formed was spun down to a pellet and the supernatant was discarded. The pellet was suspended in 25 ml of ice-cold acetone, vortexed and centrifuged to form a pellet. The supernatant was again discarded and this process was repeated for 3-more cycles. The final pellet was dried under high vacuum affording the 5’-phosphoimidazolide analog of 13 as a white solid: 101-mg yield.
[00253] The 5’-phosphoimidazolide analog of 13 (101 mg) was combined with MnCI2 (3.0 mg, 0.024 mmol) and stirred in 4.0 ml of 0.25 M bis-triethylammonium thiophosphate in dimethylformamide at room temperature for 1.0 hour. The reaction solution was then diluted to 50 ml with water. The crude product was purified by ionexchange chromatography (DEAE Sephedex A25 resin) employing a gradient of 0 ->0.70 M TEAB, pH 8.3 buffer. The pure product fractions were pooled and evaporated from water several times to remove volatile salt species. The purified product (the bis-triethylammonium salt form of 14) was lyophilized to afford a white solid: 63-mg (87% yield); mass spectrum (ESI) m/z 486.
[00254] B-2. Preparation of intermediate 15 (Fig. 8)
[00255] Intermediate 5 (28 mg, 0.030 mmol) was combined with triphenylphosphine (49 mg, 0.19 mmol), imidazole (22 mg, 0.32 mmol), 2, 2’-dithiopyridine (42 mg, 0.18 mmol). The mixture was dissolved in anhydrous DMSO (250 ul) containing triethylamine (33 ul). The reaction was stirred at RT for 30 minutes and then diluted with 15 ml of ice-cold 0.2 M sodium perchlorate/acetone solution. The precipitate that formed was spun down to a pellet and the supernatant was discarded. The pellet was washed with 15 ml of ice-cold acetone and again spun down to a pellet. The supernatant was discarded and this process was repeated three more times. Finally, the pellet was dried under high vacuum to afford a white solid: 20-mg (82%) yield of intermediate 15 in the disodium salt form.
[00256] B-3. Preparation of cap reagent analog 16 (Fig. 8)
[00257] Intermediate 15 (20 mg, 0.025 mmol) and intermediate 14 (9.0 mg, 0.015 mmol) were combined and stirred in anhydrous dimethylformamide (145 ul), containing anhydrous zinc chloride (22 mg), for 18 hours at 37 C. EDTA (400 ul of a 0.5 M solution) was added and the solution was diluted to 5.0 ml with water and lyophilized to dryness. The powder was redissolved in water and the two forms of cap analog 16 (diastereomers) were isolated together by reverse-phase (C18) HPLC employing a 50 mM TEAB, pH 8.0 - acetonitrile gradient. The pure product fractions were pooled and lyophilzed to dryness affording a white solid: 2.0 umol yield (determined spectrophotometrically at 255 nm; extinction coefficient = 30,540); mass spectrum (ESI) m/z 1188.
EXAMPLE 4
[00258] To test in vivo protein expression of mRNA generated by IVT using cap-2 analogs, mice were injected with ALC0315-LNP formulation encapsulated Firefly luciferase (Flue) mRNA generated by IVT using Cap2 analog (Cap2-Fluc) or Cap1 analog (Cap1-Fluc) generated by IVT. Cap1 analog and Cap2 analog used for this example are shown below:
Cap1 analog (Cap1-AG):
Figure imgf000084_0001
[00259] Mice injected with PBS served as negative control. The expression of Flue protein was examined by IVIS living imaging for over 96 hours post injection. FIG. 9 shows that the expression levels of Cap2-Fluc mRNA are similar to that of Cap1-Fluc mRNA. Biodistribution was also measured after 96 hours by bioluminescence. FIG. 10 shows that the expression of Cap1-Fluc mRNA and Cap2-Fluc mRNA were mainly distributed to liver. FIG. 11 shows that injection with Cap1 -Fluc mRNA or Cap2-Fluc mRNA did not affect mouse body weight as compared with that injected with PBS.
[00260] Advantages of the present disclosure may include (1) in vitro transcription reaction mixtures and conditions that can increase yield, integrity, and purity of mRNAs, (2) DNA templates and cap analogues that bind to -1 and/or +1 nucleotides of promoters for in vitro transcription, thus producing more full length mRNAs, allowing for more flexibility on the choice of first mRNA base, and providing +2 position open for custom sequence, and (3) cap-2 analogues with 2’-O-methylation of the second transcribed nucleotide and N6-methylation of adenosine as the first transcribed nucleotide serving as determinants defining transcripts as ‘self and thus contributing to transcript immune evasion. [00261] All references cited in this specification are herein incorporated by reference as though each reference was specifically and individually indicated to be incorporated by reference. The citation of any reference is for its disclosure prior to the filing date and should not be construed as an admission that the present disclosure is not entitled to antedate such reference by virtue of prior invention.
[00262] It will be understood that each of the elements described above, or two or more together may also find a useful application in other types of methods differing from the type described above. Without further analysis, the foregoing will so fully reveal the gist of the present disclosure that others can, by applying current knowledge, readily adapt it for various applications without omitting features that, from the standpoint of prior art, fairly constitute essential characteristics of the generic or specific aspects of this disclosure set forth in the appended claims. The foregoing embodiments are presented by way of example only; the scope of the present disclosure is to be limited only by the following claims.

Claims

1 . A method for in vitro transcription of a DNA template into RNA, comprising providing a mixture comprising a buffer substance, ribonucleoside triphosphates (NTPs), one or more magnesium salts in a concentration of from about 2 mM to about 60 mM, the DNA template, a recombinant RNA polymerase, and a cap analogue comprising the structure of Formula (I). (II), or (III):
(I)
Figure imgf000086_0001
wherein R is H or CH3; and Bi is A or N6-methyl-adenine (m6A), and B2 is A, U, G, or C;
Figure imgf000086_0002
wherein
Ri is OCH3 and R2 is OH or H, or
R1 is OH and R2 is H, or R1 is H and R2 is H or OCH3, or
R1 and R2 are each OCH3,
B1 is A or N6-methyl-adenine (m6A), and B2 is A, U, G, or C,
(in)
Figure imgf000087_0001
wherein R is H or CH3; and Bi is A or N6-methyl-adenine (m6A), and B2 is A, U, G, or C; and incubating the reaction mixture at from about 15°C to about 35°C, optionally from about 18°C to about 31 °C, for from about 1 hour to about 12 hours, thereby producing the RNA.
2. The method of claim 1 , wherein the buffer substance is Tris base, HEPES, or Tris- HCI.
3. The method of claim 1 or 2, wherein the concentration of the buffer substance is from about 1 mM to about 100 mM, from about 1 mM to about 90 mM, from about 1 mM to about 80 mM, from about 1 mM to about 70 mM, from about 1 mM to about 60 mM, from about 1 mM to about 50 mM, from about 1 mM to about 40 mM, from about 1 mM to about 30 mM, from about 1 mM to about 20 mM, from about 1 mM to about 10 mM, from about 1 mM to about 5 mM, from about 10 mM to about 20 mM, from about 10 mM to about 30 mM, from about 10 mM to about 40 mM, from about 10 mM to about 50 mM, from about 20 mM to about 50 mM, from about 30 mM to about 50 mM, from about 35 mM to about 45 mM, from about 35 mM to about 40 mM, from about 40 mM to about 50 mM, from about 45 mM to about 50 mM, from about 45 mM to about 55 mM, from about 15 mM to about 45 mM, from about 15 mM to about 35 mM, from about 15 mM to about 30 mM, or from about 15 mM to about 25 mM.
4. The method of any one of claims 1-3, wherein the concentration of the NTPs is from about 1 mM to about 50 mM, from about 1 mM to about 40 mM, from about 1 mM to about 30 mM, from about 1 mM to about 20 mM, from about 1 mM to about 10 mM, from about 1 mM to about 5 mM, from about 2 mM to about 10 mM, from about 3 mM to about 10 mM, from about 3 mM to about 9 mM, from about 3 mM to about 8 mM, from about 3 mM to about 7 mM, from about 3 mM to about 6 mM, from about 3 mM to about 5 mM, from about 3 mM to about 4 mM, from about 4 mM to about 10 mM, from about 5 mM to about 10 mM, from about 6 mM to about 10 mM, from about 7 mM to about 10 mM, from about 8 mM to about 10 mM, from about 9 mM to about 10 mM, from about 10 mM to about 50 mM. from about 20 mM to about 50 mM, from about 25 mM to about 50 mM, from about 25 mM to about 45 mM, from about 25 mM to about 40 mM, from about 25 mM to about 35 mM or from about 20 mM to about 30 mM.
5. The method of any one of claims 1-4, wherein the concentration of the one or more magnesium salts is from about 2 mM to about 50 mM, from about 2 mM to about 40 mM, from about 2 mM to about 30 mM, from about 2 mM to about 40 mM, from about 2 mM to about 30 mM, from about 2 mM to about 20 mM, from about 2 mM to about 10 mM, from about 2 mM to about 5 mM, from about 5 mM to about 50 mM, from about 10 mM to about 45 mM, from about 15 mM to about 40 mM, from about 20 mM to about 35 mM, from about 20 mM to about 30 mM, from about 20 mM to about 25 mM, from about 22 mM to about 28 mM, or from about 25 mM to about 30 mM.
6. The method of any one of claims 1-5, wherein the concentration of the DNA template is from about 0.001 μg/μl to about 2 μg/μl, from about 0.001 μg/μl to about 1.5 μg/μl, from about 0.001 μg/μl to about 1 μg/μl, from about 0.01 μg/μl to about 2 μg/μl, from about 0.01 μg/μl to about 1.5 μg/μl, from about 0.01 μg/μl to about 1 μg/μl, from about 0.01 μg/μl to about 0.5 μg/μl, from about 0.01 μg/μl to about 0.1 μg/μl, from about 0.01 μg/μl to about 0.05 μg/μl, from about 0.02 μg/μl to about 0.04 μg/μl, from about 0.02 μg/μl to about 0.1 μg/μl, from about 0.03 μg/μl to about 0.1 μg/μl, from about 0.04 μg/μl to about 0.1 μg/μl, from about 0.05 μg/μl to about 0.1 μg/μl, from about 0.06 μg/μl to about 0.1 μg/μl, from about 0.07 μg/μl to about 0.1 μg/μl, from about 0.08 μg/μl to about 0.1 μg/μl, or from about 0.09 μg/μl to about 0.1 μg/μl.
7. The method of any one of claims 1-6, wherein the concentration of the recombinant RNA polymerase is from about 0.1 U/μl to about 2 U/μl, from about 0.5 U/μl to about 2 U/μl, from about 1 U/μl to about 2 U/μl, from about 1 U/p I to about 1 .5 U/p I, or from about 1 .5 U/μl to about 2 U/μl.
8. The method of any one of claims 1-7, wherein the mixture further comprises an antioxidant.
9. The method of claim 8, the antioxidant is dithiothreitol (DTT) in a concentration of from about 1 mM to about 50 mM, from about 2 mM to about 50 mM, from about 3 mM to about 50 mM, from about 4 mM to about 50 mM, from about 5 mM to about 50 mM, from about 6 mM to about 50 mM, from about 7 mM to about 50 mM, from about 8 mM to about 50 mM, from about 9 mM to about 50 mM, from about 10 mM to about 50 mM, from about 10 mM to about 40 mM, from about 15 mM to about 30 mM, from about 15 mM to about 25 mM, from about 15 mM to about 20 mM, from about 20 mM to about 50 mM, from about 30 mM to about 50 mM, or from about 40 mM to about 50 mM.
10. The method of any one of claims 1 -9, wherein the mixture further comprises an RNase inhibitor in a concentration of from about 0.001 U/μl to about 5 U/μl, from about 0.001 U/μl to about 4 U/μl, from about 0.001 U/μl to about 3 U/μl, from about 0.001 U/μl to about 2 U/μl, from about 0.001 U/μl to about 1 U/μl, from about 0.01 U/μl to about 5 U/μl, from about 0.01 U/μl to about 4 U/μl, from about 0.01 U/μl to about 3 U/μl, from about 0.01 U/μl to about 2 U/μl, from about 0.01 U/μl to about 1 U/μl, from about 0.01 U/μl to about 0.5 U/μl, from about 0.01 U/μl to about 0.1 U/μl, from about 0.01 U/μl to about 0.05 U/μl, from about 0.01 U/μl to about 0.04 U/μl, from about 0.01 U/μl to about 0.03 U/μl, from about 0.01 U/μl to about 0.02 U/μl, from about 0.1 U/μl to about 5 U/μl, from about 0.1 U/μl to about 4 U/μl, from about 0.1 U/μl to about 3 U/μl, from about 0.1 U/μl to about 2 U/μl, from about 0.1 U/μl to about 1 U/μl, from about 0.5 U/μl to about 5 U/μl, from about 0.5 U/μl to about 4 U/μl, from about 0.5 U/μl to about 3 U/μl, from about 0.5 U/μl to about 2 U/μl, from about 0.5 U/μl to about 1 U/μl, from about 1 U/μl to about 5 U/μl, from about 2 U/μl to about 5 U/μl, from about 3 U/μl to about 5 U/μl, from about 4 U/μl to about 5 U/μl, from about 0.001 U/μl to about 0.005 U/μl, from about 0.001 U/μl to about 0.01 U/μl, from about 0.005 U/μl to about 0.01 U/μl, from about 0.01 U/μl to about 0.05 U/μl, from about 0.01 U/μl to about 0.04 U/μl, from about 0.01 U/μl to about 0.03 U/μl, from about 0.01 U/μl to about 0.02 U/μl, from about 0.02 U/μl to about 0.03 U/μl, from about 0.02 U/μl to about 0.04 U/μl, or from about 0.02 U/μl to about 0.05 U/μl.
11 . The method of any one of claims 1 -10, wherein the cap analogue is in a concentration of from about 0.5 mM to about 50 mM, from about 0.5 mM to about 40 mM, from about 0.5 mM to about 30 mM, from about 0.5 mM to about 20 mM, from about 0.5 mM to about 10 mM, from about 0.5 mM to about 5 mM, from about 1 mM to about 10 mM, from about 2 mM to about 10 mM, from about 3 mM to about 10 mM, from about 3 mM to about 9 mM, from about 3 mM to about 8 mM, from about 3 mM to about 7 mM, from about 3 mM to about 6 mM, from about 3 mM to about 5 mM, from about 3 mM to about 4 mM, from about 4 mM to about 10 mM, from about 5 mM to about 10 mM, from about 6 mM to about 10 mM, from about 6 mM to about 9 mM, from about 6 mM to about 8 mM, from about 6 mM to about 7 mM, from about 7 mM to about 8 mM, from about 7 mM to about 9 mM, from about 7 mM to about 10 mM, from about 8 mM to about 9 mM, from about 8 mM to about 10 mM, or from about 9 mM to about 10 mM.
12. The method of any one of claims 1 -11 , wherein the mixture further comprises a polyamine.
13. The method of claim 12, wherein the polyamine is spermine, spermidine, or a combination thereof.
14. The method of claim 12 or 13, wherein the concentration of the polyamine is from about 0.1 mM to about 5 mM, from about 0.2 mM to about 4.9 mM, from about 0.2 mM to about 4.8 mM, from about 0.2 mM to about 4.7 mM, from about 0.2 mM to about 4.6 mM, from about 0.2 mM to about 4.5 mM, from about 0.2 mM to about 4.4 mM, from about 0.2 mM to about 4.3 mM, from about 0.2 mM to about 4.2 mM, from about 0.2 mM to about 4.1 mM, from about 0.2 mM to about 4 mM, from about 0.2 mM to about
3.5 mM, from about 0.2 mM to about 3 mM, from about 0.2 mM to about 2.5 mM, from about 0.5 mM to about 2.5 mM, from about 1.0 mM to about 2.5 mM, from about 1 .5 mM to about 2.5 mM, from about 0.2 mM to about 2 mM, from about 0.2 mM to about
1 .5 mM, from about 0.2 mM to about 1 mM, from about 0.2 mM to about 0.9 mM, from about 0.2 mM to about 0.8 mM, from about 0.2 mM to about 0.7 mM, from about 0.2 mM to about 0.6 mM, from about 0.2 mM to about 0.5 mM, from about 0.2 mM to about 0.4 mM, from about 0.2 mM to about 0.3 mM, from about 1 to about 25 mM, about 1 to about 20 mM, about 1 to about 15 mM, about 1 to about 10 mM, about 1 to about 5 mM, or about 1 to about 2.5 mM.
15. The method of any one of claims 1 -14, wherein the mixture further comprises a pyrophosphatase in a concentration of from about 0.01 mU/μl to about 2 mU/μl, from about 0.01 mU/μl to about 1.5 mU/μl, from about 0.01 mU/μl to about 1 mU/μl, from about 0.1 mU/μl to about 2 mU/μl, from about 0.1 mU/μl to about 1 .5 mU/μl, from about 0.1 mU/μl to about 1 mU/μl, from about 0.1 mU/μl to about 0.9 mU/μl, from about 0.1 mU/μl to about 0.8 mU/μl, from about 0.1 mU/μl to about 0.7 mU/μl, from about 0.1 mU/μl to about 0.6 mU/μl, from about 0.1 mU/μl to about 0.5 mU/μl, from about 0.1 mU/μl to about 0.4 mU/μl, from about 0.1 mU/μl to about 0.3 mU/μl, or from about 0.1 mU/μl to about 0.2 mU/μl.
16. The method of any one of claims 1 -15, wherein the incubating the reaction mixture is performed at from about 15°C to about 35°C, from about 16°C to about 35°C, from about 17°C to about 35°C, from about 18°C to about 35°C, from about 18°C to about 34°C, from about 18°C to about 33°C, from about 18°C to about 32°C, from about 18°C to about 31 °C, from about 18°C to about 30°C, from about 18°C to about 29°C, from about 18°C to about 28°C, from about 18°C to about 27°C, from about 18°C to about 26°C, from about 18°C to about 25°C, from about 18°C to about 24°C, from about 18°C to about 23°C, from about 18°C to about 22°C, from about 18°C to about 21 °C, from about 18°C to about 20°C, from about 18°C to about 19°C, from about 25°C to about 26°C, from about 25°C to about 27°C, from about 25°C to about 28°C, from about 25°C to about 29°C, from about 25°C to about 30°C, from about 25°C to about 31 °C, from about 21 °C to about 22°C, from about 21 °C to about 23°C, from about 21 °C to about 24°C, from about 21 °C to about 25°C, about 25°C, about 26°C, about 27°C, about 28°C, about 29°C, about 30°C, about 31 °C, about 32°C, about 33°C, about 34°C, or about 35°C.
17. The method of any one of claims 1 -16, wherein the incubating the reaction mixture is performed for from about 1 hour to about 12 hours, from about 1 hour to about 11 hours, from about 1 hour to about 10 hours, from about 1 hour to about 9 hours, from about 1 hour to about 8 hours, from about 1 hour to about 7 hours, from about 1 hour to about 6 hours, from about 1 hour to about 5 hours, from about 1 hour to about 4 hours, from about 1 hour to about 3 hours, from about 1 hour to about 2 hours, from about 2 hours to about 12 hours, from about 3 hours to about 12 hours, from about 4 hours to about 12 hours, from about 5 hours to about 12 hours, from about 6 hours to about 12 hours, from about 7 hours to about 12 hours, from about 8 hours to about 12 hours, from about 9 hours to about 12 hours, from about 10 hours to about 12 hours, from about 11 hours to about 12 hours, about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, or about 10 hours.
18. The method of any one of claims 1 -17, wherein the incubating the reaction mixture is performed at about 31 °C for from about 1 hour to about 5 hours, from about 1 hour to about 4.5 hours, from about 1 hour to about 4 hours, from about 1 hour to about 3.5 hours, from about 1 hour to about 3 hours, from about 1 hour to about 2.5 hours, from about 1 hour to about 2 hours, from about 1 hour to about 1 .5 hours, from about 0.5 hour to about 1 hour, from about 0.5 hour to about 1 .5 hours, about 1 hour, about 2 hours, about 3 hours, about 4 hours, or about 5 hours.
19. The method of any one of claims 1 -18, wherein the DNA template comprises a promoter operably linked to a nucleic acid comprising a 5’ untranslated region (5’ UTR), an open reading frame (ORF) encoding the RNA of interest, a 3’ UTR, and a poly A region, wherein the promoter comprises a sequence of TAATACGACTCACTATAX1X2X3 (SEQ ID NO: 16), wherein Xi is A or G, X2 is A or G, and X3 is A, T, G, or C, wherein the 5’ UTR is selected from SEQ ID NO: 1 , 3, 5, or 9, and the 3’ UTR is selected from SEQ ID NO: 2, 4, 6, or 8, wherein the poly A region comprises at least 60 adenine bases (As).
20. The method of claim 19, wherein the promoter comprises a sequence selected from SEQ ID NO: 10-15.
21 . The method of claim 19 or 20, wherein the 5’ UTR and the 3’ UTR are respectively SEQ ID NO: 1 and 2, 1 and 4, 1 and 6, 3 and 2, 3 and 4, 3 and 6, 3 and 8, 5 and 2, 5 and 4, 5 and 6, 7 and 2, 7 and 4, 7 and 6, 7 and 8, 9 and 2, 9 and 4, 9 and 6, or 9 and 8.
22. The method of claim 21 , wherein the 5’ UTR and the 3’ UTR are respectively SEQ ID NO: 1 and 2, 1 and 4, 3 and 2, 1 and 6, 7 and 4, 9 and 2, or 3 and 6.
23. The method of any one of claims 19-22, wherein the poly A region comprises from about 60 to about 200 As, from about 60 to about 190 As, from about 60 to about 180 As, from about 60 to about 170 As, from about 60 to about 160 As, from about 60 to about 150 As, from about 60 to about 140 As, from about 60 to about 130 As, from about 60 to about 120 As, from about 60 to about 110 As, from about 60 to about 100 As, from about 70 to about 190 As, from about 80 to about 180 As, from about 90 to about 170 As, from about 100 to about 160 As, from about 100 to about 150 As, from about 100 to about 140 As, from about 100 to about 130 As, from about 100 to about 120 As, about 100 As, about 110 As, about 120 As, about 130 As, about 140 As, or about 150 As.
24. The method of any one of claims 1 -23, wherein the recombinant RNA polymerase is selected from wild type T7 RNA polymerase or a variant thereof.
25. The method of any one of claims 1 -24, wherein the cap analogue is one selected from the group consisting of
Figure imgf000093_0001
Figure imgf000094_0001
m7G3’0meppspm6A2’0mepC20me.
26. The method of any one of claims 19-25, wherein the cap analogue binds to -1 and/or +1 nucleotide of the promoter.
27. A reaction mixture for in vitro transcription of a DNA template into RNA, comprising a buffer substance in a concentration of from about 45 mM to about 55 mM, an RNase inhibitor in a concentration of from about 0.01 U/μl to about 0.03 U/μl,
NTPs in a concentration of from about 3 mM to about 5 mM, a cap analogue in a concentration of from about 6 mM to about 8 mM, one or more magnesium salts in a concentration of from about 20 mM to about 30 mM, a polyamine in a concentration of from about 1.5 mM to about 2.5 mM, a DNA template in a concentration of from about 0.01 μg/μl to about 0.05 μg/μl, a pyrophosphatase in a concentration of from about 0.1 mU/μl to about 0.5 mU/μl, and an RNA polymerase in a concentration of from about 0.01 μg/μl to about 0.05 μg/μl, wherein the cap analogue comprises the structure of Formula (I), (II), or (III):
(I)
Figure imgf000095_0001
wherein R is H or CH3; and Bi is A or N6-methyl-adenine (m6A), and B2 is A, U, G, or C, (II)
Figure imgf000096_0002
wherein
R1 is OCH3 and R2 is OH or H, or
R1 is OH and R2 is H, or
R1 is H and R2 is H or OCH3, or
R1 and R2 are each OCH3,
B1 is A or N6-methyl-adenine (m6A), and B2 is A, U, G, or C,
(in)
Figure imgf000096_0001
wherein R is H or CH3; and Bi is A or N6-methyl-adenine (m6A), and B2 is A, U, G, or C.
28. The reaction mixture of claims 27, wherein the buffer substance is Tris base, HEPES, or Tris-HCl.
29. The reaction mixture of claim 27 or 28, further comprising an antioxidant.
30. The reaction mixture of any one of claims 27-29, wherein the cap analogue is one selected from the group consisting of m7GpppA2’OmepA2’Ome, m7GpppA2o me pU2Ome, m7GpppA2’OmepG2’Ome, m7GpppA2’OmepC2’Ome, m7Gpppm6A2o me pA2’Ome, m7Gpppm6A2o me pU2Ome, m7Gpppm6A2’OmepG2’Ome, m7Gpppm6A2'OmepC2Ome, m7G3'OmepPpA2’OmepA2’Ome, m7G3’Ome pppA2’Ome pU2’Ome, m7G3o me PPPA2OmepG2’0me, m7G3o me pPpA2’OmepC2’Ome, m7G3o me pppm6A2’o me pA2’Ome, m7G3o me pppm6A2’o me pU2’Ome, m7G3’OmepPpm6A2’OmepG2’Ome, m7G3o me pppm6A2’o me pC2’Ome, rn7G2'OmepPpA2’OmepA2’Ome, m7G2o me PPPA2Ome pU2’Ome, m7G2o me PPPA2OmepG2’0me, m7G2’Ome PPPA2Ome PC2Ome, m7G2o me pppm6A2o me pA2’Ome, m7G2o me pppm6A2’o me pU2’Ome, m7G2’o me pppm6A2’o mepG2'0me, m7G2’OmepPpm6A2’OmepC2’Ome, m7G3HpppA2’OmepA2'Ome, m7G3’HPPpA2’OmepU2’Ome, m7G3'HpppA2’OmepG2’Ome, m7G3’HPPpA2’OmepC2’Ome, m7G3Hpppm6A2’o me PA2Ome, m7G3’HPPPm6A2’OmepU2’Ome, m7G3’HPPpm6A2’OmepG2’Ome, m7G3Hpppm6A2’o me PC2Ome, m7GppspA2o me pA2’Ome, m7GppspA2’OmepU2'Ome,
Figure imgf000098_0001
31 . The reaction mixture of any one of claims 27-30, wherein the one or more magnesium salts is MgCl2 and/or magnesium acetate (Mg(C2H3O2)2) (Mg(OAc)2).
32. The reaction mixture of any one of claims 27-31 , wherein the polyamine is spermine, spermidine, or a combination thereof.
33. The reaction mixture of any one of claims 27-32, wherein the DNA template comprises a promoter operably linked to a nucleic acid comprising a 5’ untranslated region (5' UTR), an open reading frame (ORF) encoding the RNA of interest, a 3’ UTR, and a poly A region, wherein the promoter comprises a sequence of TAATACGACTCACTATAX1X2X3 (SEQ ID NO: 16), wherein Xi is A or G, X2 is A or G, and X3 is A, T, G, or C, wherein the 5’ UTR is selected from SEQ ID NO: 1 , 3, 5, or 9, and the 3’ UTR is selected from SEQ ID NO: 2, 4, 6, or 8, wherein the poly A region comprises at least 60 adenine bases (As).
34. The reaction mixture of claim 33, wherein the promoter comprises a sequence selected from SEQ ID NO: 10-15.
35. The reaction mixture of claim 33 or 34, wherein the 5’ UTR and the 3’ UTR are respectively SEQ ID NO: 1 and 2, 1 and 4, 1 and 6, 3 and 2, 3 and 4, 3 and 6, 3 and 8, 5 and 2, 5 and 4, 5 and 6, 7 and 2, 7 and 4, 7 and 6, 7 and 8, 9 and 2, 9 and 4, 9 and 6, or 9 and 8.
36. The reaction mixture of claim 35, wherein the 5’ UTR and the 3’ UTR are respectively SEQ ID NO: 1 and 2, 1 and 4, 3 and 2, 1 and 6, 7 and 4, 9 and 2, or 3 and 6.
37. The reaction mixture of any one of claims 33-36, wherein the poly A region comprises from about 60 to about 200 As, from about 60 to about 190 As, from about 60 to about 180 As, from about 60 to about 170 As, from about 60 to about 160 As, from about 60 to about 150 As, from about 60 to about 140 As, from about 60 to about 130 As, from about 60 to about 120 As, from about 60 to about 110 As, from about 60 to about 100 As, from about 70 to about 190 As, from about 80 to about 180 As, from about 90 to about 170 As, from about 100 to about 160 As, from about 100 to about 150 As, from about 100 to about 140 As, from about 100 to about 130 As, from about 100 to about 120 As, about 100 As, about 110 As, about 120 As, about 130 As, about 140 As, or about 150 As.
38. The reaction mixture of any one of claims 27-37, wherein the recombinant RNA polymerase is selected from wild type T7 RNA polymerase or a variant thereof.
39. A method for in vitro transcription of a DNA template into RNA, comprising providing the reaction mixture of any one of claims 27-38, and incubating the reaction mixture at from about 15°C to about 35°C for from about
1 hour to about 12 hours, thereby producing the RNA.
40. The method of claim 39, wherein the incubating comprises incubating the reaction mixture at from about 18°C to about 31 °C.
41 . The method of claim 39, wherein the incubating comprises incubating the reaction mixture at about 31 °C for about 3 hours.
42. A method for in vitro transcription of a DNA template into RNA, comprising providing (1 ) a DNA template comprises a promoter operably linked to a nucleic acid comprising a 5’ untranslated region (5’ UTR), an open reading frame (ORF) encoding the RNA of interest, a 3’ UTR, and a poly A region, and
(2) a cap analogue comprises the structure of Formula (I), (II), or (III):
(i)
Figure imgf000100_0001
wherein R is H or CH3; and Bi is A or N6-methyl-adenine (m6A), and B2 is A, U, G, or C,
(II)
Figure imgf000100_0002
wherein
Ri is OCH3 and R2 is OH or H, or
R1 is OH and R2 is H, or
R1 is H and R2 is H or OCH3, or
R1 and R2 are each OCH3,
B1 is A or N6-methyl-adenine (m6A), and B2 is A, U, G, or C, wherein the promoter comprises a sequence of
TAATACGACTCACTATAX1X2X3 (SEQ ID NO: 16), wherein A at position 17 is -1 nucleotide and Xi at position 18 is +1 nucleotide, when Xi is G, X2 and X3 are each A, T, G, or C, then Bi is A and B2 is G, when Xi is A, X2 and X3 are each A, T, G, or C, then Bi is A and B2 is A, when Xi is C, X2 and X3 are each A, T, G, or C, then Bi is A and B2 is C, and when Xi is T, X2 and X3 are each A, T, G, or C, then Bi is A and B2 is U,
(III)
Figure imgf000101_0001
wherein R is H or CH3; and Bi is A or N6-methyl-adenine (m6A), and B2 is A, U, G, or C; wherein the cap analogue binds to -1 and +1 nucleotides of the promoter, and incubating the DNA template and the cap analogue in a reaction mixture, wherein the incubating comprises incubating the reaction mixture at from about 15°C to about 35°C for from about 1 hour to about 12 hours, thereby producing the RNA.
43. The method of claim 42, wherein the promoter comprises a sequence selected from SEQ ID NO: 10, 11 , 12, 13, and 14.
44. The method of claim 42 or 43, wherein the 5’ UTR and the 3’ UTR are respectively SEQ ID NO: 1 and 2, 1 and 4, 1 and 6, 3 and 2, 3 and 4, 3 and 6, 3 and 8, 5 and 2, 5 and 4, 5 and 6, 7 and 2, 7 and 4, 7 and 6, 7 and 8, 9 and 2, 9 and 4, 9 and 6, or 9 and 8.
45. The method of claim 44, wherein the 5’ UTR and the 3’ UTR are respectively SEQ ID NO: 1 and 2, 1 and 4, 3 and 2, 1 and 6, 7 and 4, 9 and 2, or 3 and 6.
46. The method of any one of claims 42-45, wherein the poly A region comprises from about 60 to about 200 As, from about 60 to about 190 As, from about 60 to about 180 As, from about 60 to about 170 As, from about 60 to about 160 As, from about 60 to about 150 As, from about 60 to about 140 As, from about 60 to about 130 As, from about 60 to about 120 As, from about 60 to about 110 As, from about 60 to about 100 As, from about 70 to about 190 As, from about 80 to about 180 As, from about 90 to about 170 As, from about 100 to about 160 As, from about 100 to about 150 As, from about 100 to about 140 As, from about 100 to about 130 As, from about 100 to about 120 As, about 100 As, about 110 As, about 120 As, about 130 As, about 140 As, or about 150 As.
47. The method of any one of claims 42-46, wherein the cap analogue is selected from the group consisting of m7GpppA2 o me pA2’Ome, m7GpppA2’OmepU2'Ome, m7GpppA2’OmepG2’Ome, m7GpppA2’OmepC2'Ome, m7Gpppm6A2o me pA2’Ome, m7Gpppm6A2o me pU2'Ome, m7Gpppm6A2’OmepG2’Ome, m7Gpppm6A2o me pC2’Ome, m7G3o me PPPA2Ome pA2’Ome, m7G3o me PPPA2 Ome pU2’Ome, m7G3'OmePPpA2’OmepG2’Ome, m7G3’Ome PPPA2Ome pC2’Ome, m7G3o me pppm6A2’o me pA2’Ome, m7G3o me pppm6A2’o me pU2’Ome, m7G3’OmePPpm6A2’OmepG2’Ome, m7G3'OmePPpm6A2’OmepC2’Ome, m7G2’o me PPpA2’Ome pA2’Ome, m7G2 o me PPPA2 Ome pU2’Ome, m7G2 o me PPPA2 OmepG2’Ome, m7G2’Ome PPPA2 Ome pC2’Ome, m7G2’OmePPpm6A2’OmepA2’Ome, m7G2'OmePPpm6A2’OmepU2’Ome, m7G2’OmePPpm6A2’OmepG2’Ome, m7G2’OmePPpm6A2’OmepC2’Ome, m7G3 HpppA2’o me pA2’Ome, m7G3’HpppA2’OmepU2’Ome, m7G3'HpppA2’OmepG2’Ome, m7G3 HpppA2’o me PC2 Ome, m7G3 Hpppm6A2’o me pA2’Ome, m7G3 Hpppm6A2’o me pU2’Ome, m7G3 Hpppm6A2’o mepG2’Ome, m7G3’Hpppm6A2’OmepC2’Ome, m7GppspA2’OmepA2’Ome, m7GppspA2’OmepU2’Ome, m7GppspA2’OmepG2’Ome, m7GppspA2’OmepC2'Ome, m7Gppspm6A2’0mepA2’0me, m7Gppspm6A2o me pU2’Ome, m7Gppsprn6A2’0mepG2’0me, m7Gppspm6A2 o me pC2’Ome, m7G3 o me PPspA2’Ome pA2’Ome, m7G3 o me ppspA2’Ome pU2’Ome, m7G3 o me ppspA2’OmepG2’Ome, m7G3’OmePPspA2’Ome pC2’Ome, m7G3'OmePPspm6A2’OmepA2’Ome, m7G3'OmePPspm6A2’OmepU2’Ome, rn7G3’omeppspm6A2’omepG2’ome, and m7G3'OmePPspm6A2’OmepC2’Ome..
48. The method of any one of claims 42-47, wherein the reaction mixture comprises a buffer substance in a concentration of from about 35 mM to about 45 mM, an RNase inhibitor in a concentration of from about 0.01 U/μl to about 0.03 U/μl,
NTPs in a concentration of from about 20 mM to about 40 mM, the cap analogue in a concentration of from about 6 mM to about 8 mM, one or more magnesium salts in a concentration of from about 20 mM to about
30 mM, a polyamine in a concentration of from about 1.5 mM to about 2.5 mM, the DNA template in a concentration of from about 0.01 μg/μl to about 0.05 μg/μl, a pyrophosphatase in a concentration of from about 0.1 mU/μl to about 0.5 mU/μl, and an RNA polymerase in a concentration of from about 1 U/μl to about 2 U/μl.
49. The method of claim 48, wherein the RNA polymerase is selected from wild type T7 RNA polymerase or a variant thereof.
50. The method of claim 42, wherein the incubating comprises incubating the reaction mixture at from about 18°C to about 31 °C.
51 . The method of claim 50, wherein the incubating comprises incubating the reaction mixture at about 31 °C for about 3 hours.
52. The method of any one of claims 19-26 and 42-51 , wherein the DNA template further comprises at least one transcriptional terminator located upstream and/or downstream of the open reading frame (ORF).
53. A reaction mixture for in vitro transcription of a DNA template into RNA, comprising a DNA template and a cap analogue, wherein the cap analogue comprises the structure of Formula (I), (II), or (III):
Figure imgf000105_0001
wherein R is H or CH3; and Bi is A or N6-methyl-adenine (m6A), and B2 is A, U, G, or C;
Figure imgf000105_0002
wherein
R1 is OCH3 and R2 is OH or H, or
R1 is OH and R2 is H, or
R1 is H and R2 is H or OCH3, or
R1 and R2 are each OCH3,
B1 is A or N6-methyl-adenine (m6A), and B2 is A, U, G, or C,
(in)
Figure imgf000106_0001
wherein R is H or CH3; and Bi is A or N6-methyl-adenine (m6A), and B2 is A, U, G, or C; wherein the DNA template comprises a promoter; and wherein the cap analogue binds to at least -1 and +1 nucleotides of the promoter, or binds to at least+1 and +2 nucleotides of the promoter.
54. A method for synthesizing a cap analogue, comprising the steps of:
(a) reacting 5’-DMT-2’-O-methyl guanosine (n-ibu) with an acetic anhydride/pyridine, followed by reacting the product with aqueous acetic acid, to obtain Compound (3):
Figure imgf000106_0002
(b) reacting the Compound (3) with 5'-dimethoxytrityl-N-benzoyl-Adenosine,2'- O-methyl,3'-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite and tetrazole, followed by reaction with a first oxidizer, followed by reaction with aqueous acetic acid, to obtain Compound (
Figure imgf000107_0003
(c) reacting the Compound (4) with a chemical phosphorylating phosphoram idite reagent and tetrazole, followed by reaction with a second oxidizer, followed by reaction with ammonium hydroxide to obtain Compound (5):
Figure imgf000107_0001
(d) reacting the Compound (5) with (2a) or (2b) in the presence of a divalent metal salt
Figure imgf000107_0002
to obtain the cap analogue (1 a) or (1 b):
Figure imgf000108_0001
55. A method for synthesizing a cap analogue, comprising the steps of:
(a) reacting Compound 3:
Figure imgf000108_0003
methyl-N6-methyl-adenosine 3'-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoram idite and tetrazole, followed by reaction with a first oxidizer, followed by reaction with aqueous acetic acid, to obtain Compound (6):
Figure imgf000108_0002
(b) reacting the Compound (6) with a chemical phosphorylating phosphoram idite reagent and tetrazole, followed by reaction with a second oxidizer, followed by reaction with ammonium hydroxide to obtain Compound (7):
Figure imgf000109_0001
(c) reacting the Compound (7) with Compound (2b) in the presence of a divalent metal salt:
Figure imgf000109_0003
to obtain the cap analogue Compound (8):
Figure imgf000109_0002
56. The methods of claim 54 or 55, wherein the first oxidizer and the second oxidizer are each (1 S)-(+)-(camphorsulfonyl)oxaziridine (CSO) or iodine.
57. The methods of any one of claims 54-56, wherein the divalent metal salt is MnCL or ZnCL.
58. A cap analogue comprising formula (I):
Figure imgf000110_0001
wherein R is H or CH3; and Bi is A or N6-methyl-adenine (m6A), and B2 is A, U, G, or C.
59. The cap analogue of claim 58, wherein R is H, Bi is A and B2 is A, U, G, or C.
60. The cap analogue of claim 59, wherein the cap analogue is selected from the group consisting of m7GpppA2’OmepA2’Ome, m7GpppA2’OmepU2' Ome m7GpppA2’OmepG2’Ome, and m7GpppA2’OmepC2' Ome.
61 . The cap analogue of claim 58, wherein R is H, Bi is m6A and B2 is A, U, G, or C.
62. The cap analogue of claim 61 , wherein the cap analogue is selected from the group consisting of m7Gpppm6A2o me pA2’Ome, m7Gpppm6A2o me p U2 me, m7Gpppm6A2o mepG2’Ome, and m7Gpppm6A2’OmepC2'Ome.
63. The cap analogue of claim 58, wherein R is CH3, Bi is A and B2 is A, U, G, or C.
64. The cap analogue of claim 63, wherein the cap analogue is selected from the group consisting of m7G3 o me PPPA2 Ome pA2’Ome, m7G3o me PPPA2Ome pU2’Ome, m7Gso me PPPA2OmepGs’Ome, and m7G3o me PPPA2Ome PC2Ome.
65. The cap analogue of claim 58, wherein R is CH3, Bi is m6A and Bs is A, U, G, or C.
66. The cap analogue of claim 65, wherein the cap analogue is selected from the group consisting of
Figure imgf000111_0001
67. A cap analogue comprising formula (II):
(II)
Figure imgf000111_0003
, wherein
R1 is OCH3 and R2 is OH or H, or
R1 is OH and R2 is H, or
R1 is H and R2 is H or OCH3, or
R1 and R2 are each OCH3,
B1 is A or N6-methyl-adenine (m6A), and B2 is A, U, G, or C.
68. The cap analogue of claim 67, wherein Ri is OCH3 and R2 is OH, Bi is A and Bs is A, U, G, or C.
69. The cap analogue of claim 68, wherein the cap analogue is selected from the group consisting of:
Figure imgf000111_0002
m7G2o me PPPA2Ome pC2’Ome.
70. The cap analogue of claim 67, wherein Ri is OCH3 and R2 is OH, Bi is m6A and B2 is A, U, G, or C.
71 . The cap analogue of claim 70, wherein the cap analogue is selected from the group consisting of: m7G2’o me pppm6A2’o me pA2’Ome, m7G2o me pppm6A2’o me pU2’Ome, m7G2o me pppm6A2’o mepG2'Ome, and m7G2o me pppm6A2o me pC2’Ome.
72. The cap analogue of claim 67, wherein R1 is OH and R2 is H, Bi is A and B2 is A, U, G, or C.
73. The cap analogue of claim 72, wherein the cap analogue is selected from the group consisting of: m7G3’HpppA2’OmepA2’Ome, m7G3’HPPpA2’OmepU2’Ome, rn7G3HpppA2’omepG2’ome, and m7G3’HPPpA2’OmepC2’Ome.
74. The cap analogue of claim 67, wherein R1 is OH and R2 is H, Bi is m6A and B2 is A, U, G, or C.
75. The cap analogue of claim 74, wherein the cap analogue is selected from the group consisting of: m7G3Hpppm6A2’o me pA2’Ome, m7G3Hpppm6A2’o me pU2’Ome, m7G3HPPpm6A2’omepG2’ome, and m7G3Hpppm6A2’o me pC2’Ome.
76. A cap analogue comprising formula (III): (Ill
Figure imgf000113_0001
) wherein R is H or CH3; and Bi is A or N6-methyl-adenine (m6A), and B2 is A, U, G, or C.
77. The cap analogue of claim 76, wherein R is H, Bi is A and B2 is A, U, G, or C.
78. The cap analogue of claim 77, wherein the cap analogue is selected from the group consisting of m7GppspA2o me pA2’Ome, m7GppspA2’OmepU2’Ome, m7GppspA20mepG2’0me, and m7GppspA2 o me pC2’Ome.
79. The cap analogue of claim 76, wherein R is H, Bi is m6A and B2 is A, U, G, or C.
80. The cap analogue of claim 78, wherein the cap analogue is selected from the group consisting of m7Gppspm6A2o me pA2’Ome, m7Gppspm6A2o me pU2’Ome, m7Gppsprn6A20mepG2’0me, and m7Gppspm6A2o me pC2’Ome.
81 . The cap analogue of claim 76, wherein R is CH3, Bi is A and B2 is A, U, G, or C.
82. The cap analogue of claim 81 , wherein the cap analogue is selected from the group consisting of m7G3o me ppspA2’Ome pA2’Ome, m7G3o me ppspA2’Ome pU2’Ome,
Figure imgf000114_0002
83. The cap analogue of claim 76, wherein R is CH3, Bi is m6A and B2 is A, U, G, or C.
84. The cap analogue of claim 83, wherein the cap analogue is selected from the group consisting of
Figure imgf000114_0001
85. A method for synthesizing a cap analogue, comprising the steps of:
(a) reacting Compound (12) with dimethyl sulfate
Figure imgf000114_0003
to obtain Compound (13)
Figure imgf000114_0004
(b) reacting the Compound (13) with imidazole, triphenylphosphine, 2, 2’- dithiopyridine, followed by reaction with MnCI2and bis-triethylammonium thiophosphate to obtain Compound (14)
Figure imgf000114_0005
(c) reacting Compound (5
Figure imgf000115_0002
) with triphenylphosphine, imidazole, and 2, 2’-dithiopyridine to obtain
Compound (15) , and
Figure imgf000115_0003
(d) reacting the Compound (15) with the Compound (14)
Figure imgf000115_0004
in the presence of dimethylformamide and ZnCI2 to obtain Compound (16)
Figure imgf000115_0001
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