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WO2025207837A1 - Enzyme de coiffage d'arnm viral et procédés d'utilisation associés - Google Patents

Enzyme de coiffage d'arnm viral et procédés d'utilisation associés

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Publication number
WO2025207837A1
WO2025207837A1 PCT/US2025/021656 US2025021656W WO2025207837A1 WO 2025207837 A1 WO2025207837 A1 WO 2025207837A1 US 2025021656 W US2025021656 W US 2025021656W WO 2025207837 A1 WO2025207837 A1 WO 2025207837A1
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WIPO (PCT)
Prior art keywords
rna
capping enzyme
enzyme
seq
marseillevirus
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PCT/US2025/021656
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English (en)
Inventor
Rachel GOMEZ
Matthias Strieker
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Natures Toolbox Inc
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Natures Toolbox Inc
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Publication of WO2025207837A1 publication Critical patent/WO2025207837A1/fr
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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)
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6816—Hybridisation assays characterised by the detection means
    • 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)
    • C12Y207/0705—Nucleotidyltransferases (2.7.7) mRNA guanylyltransferase (2.7.7.50)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/67—General methods for enhancing the expression
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/00022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • 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
    • C12N2795/00—Bacteriophages
    • C12N2795/00011—Details
    • C12N2795/10011—Details dsDNA Bacteriophages
    • C12N2795/10211—Podoviridae
    • C12N2795/10222—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes

Definitions

  • the present invention is directed to the production of mRNA transcripts.
  • novel systems and compositions to facilitate the high-efficiency addition of a cap to the 5’ end of an uncapped RNA in in vitro production systems.
  • Such a capped transcript can be represented as m 7 G(5')ppp(5')Ni(pN)x — OH(3'), or more simply, as m 7 GpppNi(pN) x , where m 7 G represents the 7-methylguanosine cap nucleoside, ppp represents the triphosphate bridge between the 5' carbons of the cap nucleoside and the first nucleotide of the primary RNA transcript, and Ni(pN)x — OH(3') represents the primary RNA transcript, of which Ni is the most 5 '-nucleotide.
  • RNAs that have a 5 '-triphosphate are reported to activate the innate immune response.
  • it is highly desirable to add a cap to synthetic RNA in many therapeutic applications e g., protein replacement therapy as well as prophylactic or therapeutic vaccination).
  • the present invention includes systems, methods, compositions, and kits for capping RNA oligonucleotides, preferably synthetic RNAs generated in vitro.
  • the capping reactions may comprise a capping enzyme from the Mar seillevirus genus q/viruses, and preferably a capping enzyme from Marseillevirus marseillevirus, or a fragment or variant thereof.
  • a capping enzyme from the Mar seillevirus genus q/viruses and preferably a capping enzyme from Marseillevirus marseillevirus, or a fragment or variant thereof.
  • One advantage of the Marseillevirus marseillevirus capping enzyme described herein is the ability to express the protein and purify it from the soluble fraction of E. coli cells. Unlike the traditional Vaccinia capping enzyme (VCE), Marseillevirus marseillevirus capping enzyme is a single polypeptide chain with each of the three domains needed for addition of a CapO onto RNA.
  • the invention include systems, methods, compositions, and kits to generate all or part of the enzymatic reactions are involved in capping of an uncapped mRNA transcript, generally including wherein: (1) a RNA triphosphatase cleaves the 5 '-triphosphate of mRNA to a diphosphate; (2) a RNA guanyltransferase catalyzes joining of GTP to the 5'- diphosphate of the most 5' nucleotide; and (3) a guanine-7-methyltransferase, using S-adenosyl- methionine as a co-factor, catalyzes methylation of the 7-nitrogen of guanine in the cap nucleotide.
  • the present invention includes a method of contacting an RNA sample comprising an uncapped RNA with a capping enzyme of the invention comprising an amino acid sequence according to SEQ ID NO: 1, or a sequence having between 80% to 99% or more sequence identity with SEQ ID NO. 1.
  • the method may be performed in an in vitro transcription system, and preferably a cell-free expression/RNA production system as described herein.
  • the present invention includes a method of contacting an RNA sample comprising an uncapped RNA with a capping enzyme of the invention comprising a nucleotide sequence according to SEQ ID NO: 2, encoding a capping enzyme, or a sequence having between 80% to 99% or more sequence identity with the nucleotide sequence according to SEQ ID NO. 2.
  • the method may be performed in an in vitro transcription system, and preferably a cell-free expression/RNA production system as described herein.
  • the present invention includes an isolated nucleotide sequence according to SEQ ID NO: 2 encoding a capping enzyme, or a sequence having between 80% to 99% or more sequence identity with the nucleotide sequence according to SEQ ID NO.
  • this sequence can be operably linked with a promoter forming an expression vector.
  • a cell such as a bacterial cell can be transformed by the expression vector, and express a protein, which can further be isolated according according to SEQ ID NO: 1, or a fragment or variant thereof, or a sequence having between 80% to 99% or more sequence identity with SEQ ID NO. 1.
  • the present invention provides an expression vector encoding a capping enzyme, or a fragment or variant thereof.
  • the expression vector may include an expression cassette encoding all or a part of a nucleotide sequence according to SEQ ID NO. 2, operably linked to a promoter, which generates a capping enzyme comprising an amino acid sequence SEQ ID NO: 1, or a fragment or variant thereof.
  • may expression vector include an expression cassette encoding a nucleotide sequence encoding all or a part of a nucleotide sequence according to SEQ ID NO.
  • the expression vector of the invention may be heterologously expressed in a cell, such a prokaryotic or eukaryotic cell and further isolated as noted above.
  • FIG. 1A-C Show initial expression of M. mar sei lievirus capping enzyme (MMCE) in of E. coli culture (strain BL21(DE3)).
  • MMCE M. mar sei lievirus capping enzyme
  • A gel was run from cell lysates. Samples were taken preinduction and 5 hours post-induction and demonstrate soluble and insoluble fractions. MMCE is ⁇ 94 kDa.
  • B show the purification of MMCE from thawed pellet. A desalted protein concentration was measured by UV-Vis using an extinction coefficient 0.867 and showed a concentration of 0.0564 mg/mL and a yield of 0.6 mg/L.
  • C sows Western Blot of MMCE incubated at different temperatures indicating variable yields due to incubation temperature.
  • FIG. 2A-B (A) SDS-PAGE gel demonstrating expression of MMCE in ArcticExpress(DE3) cells with positive and negative controls and incubated overnight at 37° C. (B) Western blot confirming expression of MMCE in ArcticExpress(DE3) cells.
  • FIG. 3A-B demonstrates purification of MMCE from 3L culture of ArcticExpress(DE3) cells utilizing AKTA purification protocols.
  • (C) shows MMCE Cap intermediates relative to concentration of RNA and MMCE enzyme. Notably, as enzyme concentration increases, MMCE converts GTP to GDP, which is then converted to Cap-0. Unmethylated Cap-0 concentrations are relatively low and stable across enzyme concentration.
  • the inventive technology includes systems, methods and compositions for capping RNA oligonucleotides, and preferably capping RNA oligonucleotides in vitro.
  • the invention may comprise methods and compositions including the RNA sample comprising an uncapped target RNA that can be contacted with an RNA guanylyltransferase capping enzyme from a virus of the genus Marseillevirus or a fragment or variant thereof, under conditions wherein a Cap-0 RNA is synthesized in vitro.
  • the said capping enzyme from a Marseillevirus virus may include a capping enzyme from Marseillevirus marseillevirus virus, or a variant thereof.
  • the RNA capping enzyme can include a functional fragment of the amino acid sequence according to SEQ ID NO: 1, or nucleotide sequence SEQ ID NO. 2.
  • RNA capping enzyme comprising an amino acid sequence that is between 80% to 99% or more identical to SEQ ID NO: 1, or nucleotide sequence SEQ ID NO. 2.
  • the RNA capping enzyme may further include a tag, such as a His-6 tag to facilitate isolation and purification according to know methos within the art.
  • guanosine triphosphate (GTP) or modified GTP, and a buffering agent may also be contacted to facilitate the synthesis of Cap-0 RNA is synthesized in vitro.
  • the method may include methylating the Cap-0 RNA forming a Cap-1 RNA.
  • the Cap-0 RNA may be contacted with methyltransferase, such as a guanine-7-methyltransferase, and a methyl donor, such as S-adenosyl methionine (SAM).
  • SAM S-adenosyl methionine
  • An exemplary SAM being provided at Accession No. AAG58487, or a sequence having between 80% -99% or more sequence identity.
  • the efficiency determined by yield of capped RNA may be improved by adjusting the temperature and ratio of capping enzyme and uncapped RNA oligonucleotides.
  • Uncapped RNA of the invention may also specifically include a primary RNA transcript or an RNA that has a 5'-diphosphate is selected from the group consisting of: prokaryotic mRNA; uncapped eukaryotic primary mRNA; RNA from an in vitro transcription reaction using an RNA polymerase; RNA from an in vitro replication reaction using a replicase; RNA from an in vivo transcription reaction, wherein the RNA polymerase is expressed in a prokaryotic or eukaryotic cell that contains a DNA template that is functionally joined downstream of an RNA polymerase promoter that binds the RNA polymerase; RNA from an in vivo replication reaction using a replicase; RNA from an RNA amplification reaction; eukaryotic small nuclear (snRNA); and micro RNA (miRNA) and the like.
  • prokaryotic mRNA RNA from an in vitro transcription reaction using an RNA polymerase
  • an uncapped target RNA may be less than 200 nt in length, at least 200 nt in length (e.g., at least 300 nt, at least 500 nt or at least 1,000 nt) and may encode a polypeptide such as a therapeutic protein or vaccine.
  • Target RNA having secondary structure including therapeutic RNA can be capped more efficiently using method of this disclosure.
  • the capping method and compositions described herein may produce (e.g., may co-transcriptionally produce) approximately 99% Cap-0 RNA in vitro in thirty-minutes or less.
  • the components and/or combinations thereof may be RNase-free, and contacting may optionally further comprise one or more RNase inhibitors.
  • Cap-0 RNA from uncapped target RNA oligonucleotides may be performed in vitro, such as in a bioreactor, a microfluidics surface, a reaction tube or other reaction vessel configured to produce mRNA or other macromolecules such as proteins for therapeutic or industrial purposes. Examples may include in vitro transcription systems, cell-free expression systems as generally described or incorporated herein.
  • RNA capping enzyme from virus of the genus Marseillevirus may include a capping enzyme from M. marseillevirus virus.
  • RNA capping enzyme comprising an amino acid sequence that is between 80% to 99% identical to SEQ ID NO: 1, or a nucleotide sequence that is between 80% to 99% identical to SEQ ID NO: 2.
  • the RNA capping enzyme may further include a tag, such as a His-6 tag to facilitate isolation and purification according to methods known in the art.
  • a composition may further comprise a DNA template, a polymerase (e.g., a bacteriophage polymerase) and ribonucleotides, for transcribing the DNA template to form the uncapped target RNA.
  • a single uncapped target RNA may be at least 200 nt in length (at least 300 nt, at least 500 nt or at least 1,000 nt) and may encode a polypeptide such as a therapeutic protein or vaccine.
  • kits for producing capped RNA, and preferably mRNA transcripts in vitro may include an RNA guanylyltransferase capping enzyme from a virus of the genus Marseillevirus, or a fragment or variant thereof, wherein the enzyme is in a storage buffering agent; and a reaction buffering agent.
  • additional aspects may include a kit for producing capped RNA, and preferably mRNA transcripts in a cell-free expression system comprising, a reaction mixture having cell-free reaction components necessary for in vitro macromolecule synthesis, including an RNA guanylyltransferase capping enzyme from a Marseillevirus virus, or a fragment or variant thereof; and a storage buffering agent.
  • the capping enzyme from a Marseillevirus virus may include a capping enzyme from Marseillevirus marseillevirus virus.
  • RNA capping enzyme comprising an amino acid sequence that is between 80% to 99% identical to SEQ ID NO. 1, or a nucleotide sequence that is between 80% to 99% identical to SEQ ID NO: 2.
  • the RNA capping enzyme may further include a tag, such as a His-6 tag to facilitate isolation and purification according to methods known in the art.
  • the kit of the invention may further include a DNA template polymerase, such as a T7 bacteriophage polymerase and ribonucleotides, for transcribing a template polynucleotide encoding an uncapped target RNA, as well as a methyltransferase, such as a guanine-7- methyltransferase '-O-methyltransferase enzyme, and a methyl donor, such as S-adenosyl methionine (SAM).
  • a kit may be RNase-free and may optionally comprise one or more RNase inhibitors.
  • a kit may further comprise a DNA template, a polymerase (e.g., a bacteriophage polymerase) and ribonucleotides, for transcribing the DNA template to form the uncapped target RNA.
  • a single uncapped target RNA may be at least 200 nt in length (at least 300 nt, at least 500 nt or at least 1,000 nt) and may encode a polypeptide such as a therapeutic protein or vaccine.
  • an RNA capping enzyme may include a peptide having an amino acid sequence according to SEQ ID NO: 1, or a fragment thereof, which may preferably be isolated. In some embodiments, an RNA capping enzyme may include an amino acid sequence that is between 80% to 99% identical to SEQ ID NO: 1. In other embodiments, an RNA capping enzyme may include an nucleotide sequence encoding an RNA capping enzyme sequence that is that is between 80% to 99% identical to SEQ ID NO: 1. In some embodiments, an expression vector having a nucleotide sequence, operably linked to a promoter, encoding an RNA capping enzyme comprises an amino acid sequence according to SEQ ID NO: 1, or a fragment thereof.
  • an expression vector having a nucleotide sequence, operably linked to a promoter, encoding an RNA capping enzyme comprises an amino acid sequence that is between 80% to 99% identical to SEQ ID NO: 1.
  • the invention provides for efficient capping of RNA substrate and may support capping with less enzyme added to a capping reaction, producing more capped RNA (as a percentage of the RNA in the reaction) using the same amount of enzyme, terminating the reaction earlier, and/or capping RNAs that have secondary structure at the 5' end more efficiently.
  • Disclosed reaction conditions may be varied, including, without limitation, reaction temperature, reaction duration, reaction component concentrations (e.g., SAM, inorganic pyrophosphatase, NTPs, transcript template), and enzymes (e.g., capping enzymes, polymerases, and etc.).
  • uncapped RNA in the reaction mix may be prepared by solid-phase oligonucleotide synthesis chemistry (see, e g., Li, et al J. Org. Chem. 2012 77: 9889-9892), in which case the RNA in the sample may be may have a length in the range of 10-500 bases, e.g., 20-200 bases).
  • the product may also be a fusion protein composed of more than one immunogen, e.g., a fusion protein that consist of two or more epitopes, peptides or proteins derived from the same or different virusproteins, wherein the epitopes, peptides or proteins may be linked by linker sequences.
  • a fusion protein composed of more than one immunogen, e.g., a fusion protein that consist of two or more epitopes, peptides or proteins derived from the same or different virusproteins, wherein the epitopes, peptides or proteins may be linked by linker sequences.
  • the in vitro produce mRNA configured to be translated to form a peptide, and preferably in a host organism, such as a mammal or human subject in need thereof.
  • a peptide is a polymer of amino acid monomers. Usually, the monomers are linked by peptide bonds.
  • the term “peptide” does not limit the length of the polymer chain of amino acids. In some embodiments of the present invention a peptide may for example contain less than 50 monomer units. Longer peptides are also called polypeptides, typically having 50 to 600 monomeric units, more specifically 50 to 300 monomeric units.
  • IVT systems include in vitro recombinant cell-free expression systems, which refers to the cell-free synthesis of polypeptides in a reaction mixture or solution comprising biological extracts and/or defined cell-free reaction components, such as the exemplary system described by Koglin et al., in PCT/US2020/028005 and PCT/US2021/027774 (incorporated herein by reference).
  • the reaction mixture may optionally comprise a template, or genetic template, for production of the macromolecule, e.g., DNA, mRNA, etc.; monomers for the macromolecule to be synthesized, e.g., amino acids, nucleotides, etc.; and such co-factors, enzymes and other reagents that are necessary for the synthesis, e.g., ribosomes, tRNA, polymerases, transcriptional factors, etc.
  • the recombinant cell-free synthesis reaction, and/or cellular adenosine triphosphate (ATP) energy regeneration system components may be performed/added as batch, continuous flow, or semi-continuous flow.
  • Humbert et al. in PCT/US2018/0121121, and PCT/US2021/027774 (previously identified as incorporated by reference) may be used as an in vitro platform to produce synthetic mRNAs.
  • lysate-based in vitro systems are challenged by limited stability of typical E. coli enzymes, by the activity of most metabolic processes (nucleotide recycling) and the presence of nucleases and proteases and insufficient ATP regeneration.
  • the in vitro synthesis of the mRNA may be performed in hollow fiber reactors using a continuous flow system, as well as other traditional bioreactors known in the art.
  • the inner chamber (hollow fibers) of the bioreactor provides additional nucleotides in flow
  • the outer chamber holds the RNA polymerase and each linear DNA template.
  • the present inventors demonstrate that the total turnover of the RNAP is at least 50 fold higher than in batch reaction and, coupled with modifications to selected enzyme, produce cleaner mRNA without smear.
  • RNA biosynthesis may be the mid ppb range.
  • the resulting mRNA is stable as a powder and does not contain traces of any components from the manufacturing process. It is ready to ship requiring only reduced volumes without the need of hard-to-monitor and expensive shipping conditions.
  • the output of the cell-free expression system may be a product, RNA, or other macromolecule such as a peptide or fragment thereof that may be isolated or purified.
  • solation or purification of a of a target protein wherein the target protein is at least partially separated from at least one other component in the reaction mixture for example, by organic solvent precipitation, such as methanol, ethanol or acetone precipitation, organic or inorganic salt precipitation such as trichloroacetic acid (TCA) or ammonium sulfate precipitation, nonionic polymer precipitation such as polyethylene glycol (PEG) precipitation, pH precipitation, temperature precipitation, immunoprecipitation, chromatographic separation such as adsorption, ion-exchange, affinity and gel exclusion chromatography, chromatofocusing, isoelectric focusing, high performance liquid chromatography (HPLC), gel electrophoresis, dialysis, microfiltration, and the like.
  • organic solvent precipitation such as methanol, ethanol or acetone precipitation
  • codon usage preferences may be used in the design of nucleic acid molecules encoding the proteins and chimeras of the invention in order to optimize expression in a particular host cell system. All nucleotide sequences described in the invention may be codon optimized for expression in a particular organism, or for increases in production yield. Codon optimization generally improves the protein expression by increasing the translational efficiency of a gene of interest. The functionality of a gene may also be increased by optimizing codon usage within the custom designed gene.
  • a codon of low frequency in a species may be replaced by a codon with high frequency, for example, a codon UUA of low frequency may be replaced by a codon CUG of high frequency for leucine.
  • Codon optimization may increase mRNA stability and therefore modify the rate of protein translation or protein folding. Further, codon optimization may customize transcriptional and translational control, modify ribosome binding sites, or stabilize mRNA degradation sites.
  • a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions), the complementary (or complement) sequence, and the reverse complement sequence, as well as the sequence explicitly indicated.
  • degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and/or deoxyinosine residues (see e.g., Batzer et al., Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985); and Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)).
  • alterations in a polynucleotide that result in the production of a chemically equivalent amino acid at a given site, but do not affect the functional properties of the encoded polypeptide are well known in the art.
  • Constant amino acid substitutions are those substitutions that are predicted to interfere least with the properties of the reference polypeptide. In other words, conservative amino acid substitutions substantially conserve the structure and the function of the reference protein.
  • a codon for the amino acid alanine, a hydrophobic amino acid may be substituted by a codon encoding another less hydrophobic residue, such as glycine, or a more hydrophobic residue, such as valine, leucine, or isoleucine.
  • a codon encoding another less hydrophobic residue such as glycine
  • a more hydrophobic residue such as valine, leucine, or isoleucine.
  • changes which result in substitution of one negatively charged residue for another such as aspartic acid for glutamic acid, or one positively charged residue for another, such as lysine for arginine or histidine, can also be expected to produce a functionally equivalent protein or polypeptide.
  • Exemplary conservative amino acid substitutions are known by those of ordinary skill in the art.
  • sequence identity in the context of two nucleic acid or polypeptide sequences includes reference to the residues in the two sequences which are the same when aligned.
  • sequence identity in reference to proteins it is recognized that residue positions which are not identical often differ by conservative amino acid substitutions, where amino acid residues are substituted for other amino acid residues with similar chemical properties (e.g., charge or hydrophobicity) and therefore do not change the functional properties of the molecule.
  • sequences differ in conservative substitutions
  • percent sequence identity may be adjusted upwards to correct for the conservative nature of the substitution.
  • Sequences which differ by such conservative substitutions are to have "sequence similarity" or “similarity”. Means for making this adjustment are well-known to those of skill in the art. Typically this involves scoring a conservative substitution as a partial rather than a full mismatch, thereby increasing the percentage sequence identity. Thus, for example, where an identical amino acid is given a score of 1 and a non-conservative substitution is given a score of zero, a conservative substitution is given a score between zero and 1. The scoring of conservative substitutions is calculated, e.g., according to the algorithm of Henikoff S and Henikoff JG. [Amino acid substitution matrices from protein blocks. Proc. Natl. Acad. Sci. U.S.A. 1992, 89(22): 10915- 9]
  • a “polymerase” refers to an enzyme that catalyzes the polymerization of nucleotides.
  • DNA polymerase catalyzes the polymerization of deoxyribonucleotides.
  • Known DNA polymerases include, for example, Pyrococcus furiosus (Pfu) DNA polymerase, E. coli DNA polymerase I, T7 DNA polymerase and Thermus aquaticus (Taq) DNA polymerase, among others.
  • RNA polymerase catalyzes the polymerization of ribonucleotides.
  • the foregoing examples of DNA polymerases are also known as DNA-dependent DNA polymerases.
  • RNA-dependent DNA polymerases also fall within the scope of DNA polymerases.
  • Reverse transcriptase which includes viral polymerases encoded by retroviruses, is an example of an RNA-dependent DNA polymerase.
  • RNA polymerase include, for example, T3 RNA polymerase, T7 RNA polymerase, SP6 RNA polymerase and E. coli RNA polymerase, among others.
  • the foregoing examples of RNA polymerases are also known as DNA-dependent RNA polymerase.
  • the polymerase activity of any of the above enzymes can be determined by means well known in the art.
  • reaction mixture refers to a solution containing reagents necessary to carry out a given reaction.
  • a cell-free expression system “reaction mixture” or “reaction solution” typically contains a crude or partially purified extract, (such as from bacteria, plant cells, microalgae, fungi, or mammalian cells) nucleotide translation template, and a suitable reaction buffer for promoting cell-free protein synthesis from the translation template.
  • the CF reaction mixture can include an exogenous RNA translation template.
  • the CF reaction mixture can include a DNA expression template encoding an open reading frame operably linked to a promoter element for a DNA-dependent RNA polymerase.
  • the CF reaction mixture can also include a DNA-dependent RNA polymerase to direct transcription of an RNA translation template encoding the open reading frame.
  • additional NTPs and divalent cation cofactor can be included in the CF reaction mixture.
  • a reaction mixture is referred to as complete if it contains all reagents necessary to enable the reaction, and incomplete if it contains only a subset of the necessary reagents.
  • the term “transformation” or “genetically modified” refers to the transfer of one or more nucleic acid molecule(s) into a cell, preferably through an expression vector.
  • a microorganism is “transformed” or “genetically modified” by a nucleic acid molecule transduced into the bacteria or cell or organism when the nucleic acid molecule becomes stably replicated.
  • the term “transformation” or “genetically modified” encompasses all techniques by which a nucleic acid molecule can be introduced into a cell or organism, such as a bacterium.
  • promoter refers to a region of DNA that may be upstream from the start of transcription, and that may be involved in recognition and binding of RNA polymerase and other proteins to initiate transcription.
  • a promoter may be operably linked to a coding sequence for expression in a cell, or a promoter may be operably linked to a nucleotide sequence encoding a signal sequence which may be operably linked to a coding sequence for expression in a cell.
  • operably linked when used in reference to a regulatory sequence and a coding sequence, means that the regulatory sequence affects the expression of the linked coding sequence.
  • regulatory sequences refer to nucleotide sequences that influence the timing and level/amount of transcription, RNA processing or stability, or translation of the associated coding sequence. Regulatory sequences may include promoters; translation leader sequences; introns; enhancers; stem-loop structures; repressor or binding sequences; termination sequences; polyadenylation recognition sequences; etc. Particular regulatory sequences may be located upstream and/or downstream of a coding sequence operably linked thereto. Also, particular regulatory sequences operably linked to a coding sequence may be located on the associated complementary strand of a double-stranded nucleic acid molecule.
  • modified nucleotides refers to any noncanonical nucleoside, nucleotide or corresponding phosphorylated versions thereof.
  • Modified nucleotides may include one or more backbone or base modifications. Examples of modified nucleotides include dl, dU, 8- oxo-dG, dX, and THF. Additional examples of modified nucleotides include the modified nucleotides disclosed in U.S. Patent Publication Nos. US20170056528A1, US20160038612A1, US2015/0167017A1, and US20200040026A1. Modified nucleotides may include naturally or non-naturally occurring nucleotides.
  • a polymer may differ from a naturally occurring polymer with respect to the molecule(s) to which it is linked.
  • a “non-naturally occurring” protein may differ from naturally occurring proteins in its secondary, tertiary, or quaternary structure, by having a chemical bond (e.g., a covalent bond including a peptide bond, a phosphate bond, a disulfide bond, an ester bond, and ether bond, and others) to a polypeptide (e.g., a fusion protein), a lipid, a carbohydrate, or any other molecule.
  • a chemical bond e.g., a covalent bond including a peptide bond, a phosphate bond, a disulfide bond, an ester bond, and ether bond, and others
  • a “non-naturally occurring” polynucleotide or nucleic acid may contain one or more other modifications (e.g., an added label or other moiety) to the 5'-end, the 3'-end, and/or between the 5'- and 3'-ends (e.g., methylation) of the nucleic acid.
  • a “non-naturally occurring” composition may differ from naturally occurring compositions in one or more of the following respects: (a) having components that are not combined in nature, (b) having components in concentrations not found in nature, (c) omitting one or components otherwise found in naturally occurring compositions, (d) having a form not found in nature, e.g., dried, freeze dried, crystalline, aqueous, and (e) having one or more additional components beyond those found in nature (e.g., buffering agents, a detergent, a dye, a solvent or a preservative).
  • All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.
  • single uncapped RNA target species refers to a mixture of target RNA molecules that have essentially the same sequence. Transcripts made by in vitro transcription and RNA oligonucleotides made by solid-phase synthesis are examples of single uncapped RNA target species. It is recognized that a certain amount of the RNA products in such a mixture may be truncated. Single uncapped RNA target species may sometimes contain modified nucleotides (e.g., noncanonical nucleotides that are not found in nature). Preparations of RNA from a cell contain a complex mixture of naturally occurring RNA molecules having different sequences; such preparations do not contain only targeted uncapped RNA species but also contain a wide variety of non-target RNAs. In some embodiments, the targeted uncapped RNA species is a single species of RNA.
  • target RNA refers to a polyribonucleotide of interest.
  • a polyribonucleotide may be or comprise a therapeutic RNA or precursor thereof (e.g., an uncapped precursor of a capped therapeutic RNA).
  • a target RNA may arise from cellular transcription or in vitro transcription.
  • a target RNA may be present in a mixture, for example, an in vitro transcription reaction mixture, a cell, or a cell lysate.
  • a target RNA may be uncapped.
  • a target RNA may be contacted with a decapping enzyme, for example, as a co-treatment with or pre-treatment before capping.
  • variant refers to a protein that has an amino acid sequence that is different from a naturally occurring amino acid sequence (i.e., having less than 100% sequence identity to the amino acid sequence of a naturally occurring protein) but that is at least 80%, at least 85%, between 80% to 99% , at least 95%, at least 97%, at least 98% or at least 99% identical to the naturally occurring amino acid sequence.
  • fragment refers to a portion of a peptide or nucleotide sequence that still retains the activity of the whole.
  • a “bioreactor” may be any form of enclosed apparatus configured to maintain an environment conducive to the production of macromolecules in vitro.
  • a bioreactor may be configured to run on a batch, continuous, or semi-continuous basis, for example by a feeder reaction solution. Examples of a bioreactor and conditions for synthesis of RNA or other macromolecules have been previously described in by Koglin, et al. PCT/US2021/027774 (the bioreactor system, apparatus, and methods of use being incorporated herein by reference).

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Abstract

L'invention concerne des systèmes, des procédés, des compositions et des kits pour le coiffage d'oligonucléotides d'ARN, de préférence des ARN synthétiques générés in vitro utilisant une enzyme de coiffage du genre Marseillevirus des virus et, de préférence une enzyme de coiffage de Marseillevirus marseillevirus ou un fragment ou un variant associé.
PCT/US2025/021656 2024-03-28 2025-03-26 Enzyme de coiffage d'arnm viral et procédés d'utilisation associés Pending WO2025207837A1 (fr)

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