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EP4284922A1 - Enzyme de coiffage de faustovirus, compositions d'enzyme de coiffage d'arnm, procédés et nécessaires - Google Patents

Enzyme de coiffage de faustovirus, compositions d'enzyme de coiffage d'arnm, procédés et nécessaires

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Publication number
EP4284922A1
EP4284922A1 EP21707503.5A EP21707503A EP4284922A1 EP 4284922 A1 EP4284922 A1 EP 4284922A1 EP 21707503 A EP21707503 A EP 21707503A EP 4284922 A1 EP4284922 A1 EP 4284922A1
Authority
EP
European Patent Office
Prior art keywords
fce
seq
variant
positions
amino acid
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
EP21707503.5A
Other languages
German (de)
English (en)
Inventor
Mehul Ganatra
Siu-Hong Chan
Chrispher H. TARON
G. Brett ROBB
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
New England Biolabs Inc
Original Assignee
New England Biolabs Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by New England Biolabs Inc filed Critical New England Biolabs Inc
Publication of EP4284922A1 publication Critical patent/EP4284922A1/fr
Pending legal-status Critical Current

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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)
    • C12N9/1241—Nucleotidyltransferases (2.7.7)
    • 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)

Definitions

  • mRNA as a therapeutic modality may supplement functional therapeutic proteins that are not antigens, for example, erythropoietin, CFTR, or genome editing proteins (e.g., CRISPR-Cas9, meganucleases).
  • Manufacturing mRNA may be cell-free and scalable. Once the sequence of a desired antigen is provided, the time required to produce clinical batches of vaccine might be weeks instead of months. Such rapid production may limit or even avert widespread outbreaks.
  • mRNA alternatives to a number of protein replacement regimens are envisioned.
  • RNA vaccine Production of stable mRNA capable of efficient translation upon introduction to a subject may require an appropriate cap structure, such as a Cap 0 structure (m7Gppp5 N) at the 5’ end. Capping by a capping enzyme may be desired or even required for production of an effective RNA vaccine. For example, a suitable cap structure may impact the stability and translatability of an RNA vaccine.
  • an RNA capping enzyme may include an FCE variant having (a) an amino acid sequence at least 90% identical to positions 1 to 878 of SEQ ID NO: 1, and/or (b) a substitution relative to SEQ ID NO: 1 at a position selected from positions corresponding to positions 215, 337, 572, 648, and 833 (e.g., aposition selected from positions corresponding to position 215, 337, and 572) of SEQ ID NO: 1.
  • An FCE variant may comprise a second substitution at a position (i) other than the position of the first substitution and (ii) corresponding to position 215, 337, 572, 648, or 833 of SEQ ID NO: 1.
  • An FCE variant in some embodiments, may comprise a third substitution at a position (iii) other than the position of the first and second substitutions and (iv) corresponding to position 215, 337, 572, 648, or 833 of SEQ ID NO: 1.
  • An FCE variant in some embodiments, may comprise a fourth substitution at a position (v) other than the position of the first, second and third substitutions and (vi) corresponding to position 215, 337, 572, 648, or 833 of SEQ ID NO: 1.
  • an FCE variant may have an amino acid sequence that is at least 90% identical, but not 100% identical to SEQ ID NO: 1. In some embodiments, an FCE variant may have an amino acid sequence that is at least 90% identical, but not 100% identical to SEQ ID NO: 1.
  • An FCE variant (a) may have an amino acid sequence (a) at least 90% identical to SEQ ID NO: 26, and/or (b) may have an amino acid other than asparagine at a position selected from positions corresponding to positions X215, X337, X572, X648, and X833 of SEQ ID NO: 26.
  • an FCE variant may include additional peptides (e.g. , for sorting, processing, and/or purification of the catalytically active portion of the molecule).
  • an FCE variant may comprise a purification tag and/or a sorting signal.
  • an FCE variant may comprise, in an N-terminal to C-terminal direction, (a) a purification tag or sorting signal peptide, and (b)(i) an amino acid sequence at least 90% identical to positions 1 to 878 of SEQ ID NO: 1, and/or (ii) a substitution relative to SEQ ID NO: 1 at a position selected from positions corresponding to positions 215, 337, 572, 648, and 833.
  • an FCE variant may further comprise an insertion (e.g. , a sorting signal or a purification tag) on the N-terminal side of the position corresponding to position 1 of SEQ ID NO: 1.
  • an FCE variant may further comprise an insertion (e.g. , a sorting signal or a purification tag) on the C-terminal side of the position corresponding to position 878 of SEQ ID NO: 1.
  • compositions may include an FCE variant (e.g., any of the foregoing FCE variants) and a polynucleotide, wherein the polynucleotide comprises ribonucleotides and deoxyribonucleotides.
  • a composition may comprise an FCE variant (e.g., any of the foregoing FCE variants) and a polyribonucleotide.
  • a composition may optionally comprise, for example, a cap, an NTP, a modified NTP, a buffer, S-adenosylmethionine, and/or an RNase inhibitor, according to some embodiments.
  • an FCE variant transcript may comprise a transcript (e.g., polynucleotide transcript comprising RNA) encoding an amino acid sequence having (a) at least 90% identical to positions 1 to 878 of SEQ ID NO: 1, and (b) a substitution relative to SEQ ID NO: 1 at a position selected from positions corresponding to positions 215, 337, 572, 648, and 833 (e.g., a position selected from positions corresponding to position 215, 337, and 572) of SEQ ID NO: 1, and (c) optionally, a cap.
  • a transcript e.g., polynucleotide transcript comprising RNA
  • An amino acid sequence encoded by an FCE variant transcript may comprise a second substitution at a position (i) other than the position of the first substitution and (ii) corresponding to position 215, 337, 572, 648, or 833 of SEQ ID NO: 1.
  • An amino acid sequence encoded by an FCE variant transcript may comprise a third substitution at a position (iii) other than the position of the first and second substitutions and (iv) corresponding to position 215, 337, 572, 648, or 833 of SEQ ID NO: 1.
  • An amino acid sequence encoded by an FCE variant transcript may comprise a fourth substitution at a position (v) other than the position of the first, second and third substitutions and (vi) corresponding to position 215, 337, 572, 648, or 833 of SEQ ID NO: 1.
  • An FCE variant transcript may comprise in a 5’ to 3’ direction, (I) a nucleotide sequence encoding a purification tag or a sorting signal peptide, and (II) an FCE variant transcript comprising, for example, a transcript encoding an amino acid sequence having (A) at least 90% identical to positions 1 to 878 of SEQ ID NO: 1, and (B) a substitution relative to SEQ ID NO: 1 at a position selected from positions corresponding to positions 215, 337, 572, 648, and 833 of SEQ ID NO: 1, and (C) optionally, a cap, wherein the purification tag or sorting signal peptide is operably linked to the FCE variant encoded by (II).
  • an FCE variant transcript may comprise in a 5’ to 3’ direction, (I) an FCE variant transcript comprising, for example, a transcript encoding an amino acid sequence having (A) at least 90% identical to positions 1 to 878 of SEQ ID NO: 1, and (B) a substitution relative to SEQ ID NO: 1 at a position selected from positions corresponding to positions 215, 337, 572, 648, and 833 of SEQ ID NO: 1, and (C) optionally, a cap, and (II) a nucleotide sequence encoding a purification tag or a sorting signal peptide, wherein the purification tag or sorting signal peptide is operably linked to the FCE variant encoded by (I).
  • an FCE variant transcript comprising, for example, a transcript encoding an amino acid sequence having (A) at least 90% identical to positions 1 to 878 of SEQ ID NO: 1, and (B) a substitution relative to SEQ ID NO: 1 at a position selected from positions corresponding to positions 215, 337,
  • an FCE variant transcript may encode an amino acid sequence further comprising an insertion (e.g., a sorting signal or a purification tag) on the N-terminal side of the position corresponding to position 1 of SEQ ID NO: 1 and/or on the C-terminal side of the position corresponding to position 878 of SEQ ID NO: 1.
  • an FCE variant transcript comprises (C) a cap (e.g., a natural cap, a dinucleotide cap, or a modified cap).
  • the present disclosure further relates to cells and cell-based and cell-free methods of producing FCE variants and FCE variant transcripts.
  • a cell may comprise one or more FCE variants and or one or more FCE variant transcripts.
  • a cell may comprise an FCE variant transcript may comprise a polynucleotide (e.g., polynucleotide transcript comprising RNA) encoding an amino acid sequence having (a) at least 90% identical to positions 1 to 878 of SEQ ID NO: 1, and (b) a substitution relative to SEQ ID NO: 1 at a position selected from positions corresponding to positions 215, 337, 572, 648, and 833 (e.g., a position selected from positions corresponding to position 215, 337, and 572) of SEQ ID NO: 1, and (c) optionally, a cap.
  • a cell may comprise a genomic or an extra-genomic polynucleotide (e.g.
  • a DNA expression vector or cassette having a sequence encoding an FCE variant having (a) an amino acid sequence at least 90% identical to positions 1 to 878 of SEQ ID NO: 1, and/or (b) a substitution relative to SEQ ID NO: 1 at a position selected from positions corresponding to positions 215, 337, 572, 648, and 833 (e.g., a position selected from positions corresponding to position 215, 337, and 572) of SEQ ID NO: 1.
  • a cell in some embodiments, may be a eukaryotic cell, for example, a yeast cell.
  • a capping method may comprise, for example, contacting (a) an FCE variant having (i) an amino acid sequence at least 90% identical to positions 1 to 878 of SEQ ID NO: 1, and (ii) a substitution at a position selected from positions corresponding to positions 215, 337, 572, 648, and 833 of SEQ ID NO: 1, (b) a target RNA (e.g., an uncapped RNA), and (c) one or more of a cap, an NTP, and a modified NTP, and optionally (d) a buffer, S-adenosylmethionine, and/or an RNase inhibitor, to form a capped target RNA.
  • a target RNA e.g., an uncapped RNA
  • a buffer, S-adenosylmethionine, and/or an RNase inhibitor e.g., a buffer, S-adenosylmethionine, and/or an RNase inhibitor
  • contacting may comprise contacting at a temperature in the range of 37 °C - 60°C and/or for a time in the range of seconds to hours (e.g., 60 seconds to 16 hours).
  • a target RNA may or may not be capped (e.g., before contacting).
  • a method may comprise contacting a target RNA (e.g., a capped target RNA) with a decapping enzyme to form an uncapped target RNA, for example, prior to contacting the target RNA with the FCE variant.
  • a method may further comprise contacting the capped target RNA with one or more pharmaceutically acceptable additives.
  • a method of producing an FCE variant may comprise, for example, contacting (a) an FCE variant transcript comprising an RNA encoding an amino acid sequence having (i) an amino acid sequence at least 90% identical to positions 1 to 878 of SEQ ID NO: 1, and (ii) a substitution at a position selected from positions corresponding to positions 215, 337, 572, 648, and 833 of SEQ ID NO: 1 with (b) an expression system (e.g., a cell-based or cell-free expression system).
  • an expression system e.g., a cell-based or cell-free expression system.
  • FIGURES 1A and IB show general maps of yeast expression vectors.
  • FIGURE 1A shows a general map of pD912 (ATUM, formerly DNA 2.0) and
  • FIGURE IB shows a general map of pKLMF-EK (New England Biolabs) used to assemble plasmids containing FCE, a- mating factor and MBP-fusion constructs respectively.
  • FIGURE 2 shows schematically the linear integrative expression cassettes that were prepared by SacI-HF restriction digestion or PCR from the assembled plasmids as a DNA template.
  • the assembled plasmids and linear cassettes have the following design: GAP or LAC4 promoter, followed by the a-mating factor pre-pro domain (secretion) or malE gene (cytoplasmic), FCE with carboxy-terminal His-tag; AOX1 terminator sequence (TAOXI) or K.
  • lactis LAC4 transcription terminator TT
  • zeocin resistance gene under control of the ILV5 promoter PILVS + Zeo r
  • amdS fungal acetamidase selectable marker gene expressed from the yeast ADH1 promoter
  • Promoter fragment contains the sequence of the actual promoter).
  • pKLMF-EK also contains an ampicillin resistance gene (Ap R ) for selection in E. coli.
  • FIGURES 3A and 3B show effective expression of FCE constructs in Pichia pastoris and Kluyveromyces lactis.
  • FIGURE 3 A shows secreted expression of FCE wild type (WT) and individual FCE N-glycan variants in P. pastoris cells transformed with constructs containing GAP promoter. Transformants were grown in Buffered Minimal Glycerol medium, BMGY with 1% glycerol at 30°C for 48 hours. The spent culture medium was harvested, concentrated and buffer exchanged. After overnight digestion with Endo Hf, the spent media was analyzed by SDS-PAGE on a 4-20% polyacrylamide gel followed by western blotting with a His-tag antibody.
  • FIGURE 3B shows cytoplasmic expression of MBP-FCE in K. lactis cells transformed with a construct containing the K. lactis LAC4 promoter. The transformants were grown in yeast extract peptone (YEP) medium with 2% galactose at 30°C for 48 hours.
  • YEP yeast extract peptone
  • Cells were harvested, and cell lysates were prepared by sonication and analyzed by SDS-PAGE on a 4-20% polyacrylamide gel, followed by western blotting with a His-tag antibody.
  • FIGURE 4 shows the activity of expressed FCE proteins.
  • FIGURE 4 A shows spent culture media from P. pastoris cells treated with Endo Hf.
  • FIGURE 4B shows cell lysates from K. lactis cells. The activity was assayed using an in vitro mRNA capping assay as described in Examples section V.
  • FIGURE 5 shows secreted expression of FCE WT, FCE (N215Q/ N337Q/ N572Q) and FCE (N215Q/ N337Q/ N572Q/ N648Q/ N833Q) mutants in Pichia pastoris cells transformed with constructs containing the GAP promoter.
  • Transformants were grown in Buffered Minimal Glycerol medium, BMGY with 1% glycerol at 30°C for 48 hours. Spent culture media were harvested, concentrated, buffer exchanged and purified using NEB Express Ni Spin Columns.
  • the load (spent culture media) and elution fractions were analyzed by SDS-PAGE on a 4-20% polyacrylamide gel and stained using Simply Blue Safe Stain (Thermofisher).
  • Lanes 1,2 spent culture medium and elution fraction of FCE-WT
  • Lanes 3,4 spent culture medium and elution fraction of FCE (N215Q/ N337Q/ N572Q) mutant
  • Lanes 5,6 spent culture medium and elution fraction of FCE (N215Q/ N337Q/ N572Q/ N648Q/ N833Q) mutant
  • Lanes 7,8 spent culture medium and elution fraction of control P. pastoris MutS (empty strain)
  • M Color Prestained Protein Standard, Broad Range (NEB).
  • FIGURE 6 shows the activity of the concentrated and buffer exchanged spent culture media, from P. pastoris cells, of FCE WT, FCE (N215Q/ N337Q/ N572Q), FCE (N215Q/ N337Q/ N572Q/ N648Q/ N833Q) and control P. pastoris MutS (empty strain).
  • FIGURE 7 A and FIGURE 7B represent the secreted expression of FCE wild type (WT), (N215Q/ N337Q/ N572Q) and (N215Q/ N337Q/ N572Q/ N648Q/ N833Q) N-glycan mutants in Pichia pastoris cells transformed with constructs containing GAP promoter and control P. pastoris MutS (empty strain).
  • the transformants were grown in Buffered Minimal Glycerol medium, BMGY with 1% glycerol at 30°C for 48 hours.
  • the spent culture medium was harvested, concentrated and buffer exchanged.
  • FIG. 7A is a Simply Blue Safe Stained gel.
  • FIG. 7B is a corresponding Western blot.
  • SEQ ID NO: 1 illustrates an amino acid sequence of an FCE variant having a C-terminal 8xHis tag (879-886) in which positions 215, 337, 572, 648, and/or 833 may be glycosylated or replaced with any other amino acid e.g., glutamine);
  • SEQ ID NO: 2 illustrates an amino acid sequence of an FCE variant having a region with a maltose binding protein (MBP), Linker, and enterokinase (EK) cleavage sequence (DDDK) (1-388) and a C-terminal 8xHis tag (1267-1274);
  • MBP maltose binding protein
  • EK enterokinase
  • SEQ ID NO: 3 illustrates a forward primer for amplification of an FCE variant in which positions 1-20 overlap with the signal peptide sequence in the pD912 vector (SEQ ID NO: 24 or 26);
  • SEQ ID NO: 4 illustrates a reverse primer for amplification of an FCE variant in which positions 1-20 overlap with the TAOXI sequence in the pD912 vector (SEQ ID NO: 24 or 26);
  • SEQ ID NO: 5 illustrates a forward primer for amplification of a pD912 vector fragment
  • SEQ ID NO: 6 illustrates a reverse primer for amplification of a pD912 vector fragment
  • SEQ ID NO: 7 illustrates a forward primer adapted (e.g., at positions 11-13) for modifying the codon corresponding to position 215 of SEQ ID NO: 1 from asparagine to glutamine;
  • SEQ ID NO: 8 illustrates a reverse primer adapted for modifying the codon corresponding to position 215 of SEQ ID NO: 1 from asparagine to glutamine;
  • SEQ ID NO: 9 illustrates a forward primer adapted (e.g., at positions 11-13) for modifying the codon corresponding to position 337 of SEQ ID NO: 1 from asparagine to glutamine;
  • SEQ ID NO: 10 illustrates a reverse primer adapted for modifying the codon corresponding to position 337 of SEQ ID NO: 1 from asparagine to glutamine;
  • SEQ ID NO: 11 illustrates a forward primer adapted (e.g., at positions 11-13) for modifying the codon corresponding to position 572 of SEQ ID NO: 1 from asparagine to glutamine
  • SEQ ID NO: 12 illustrates a reverse primer adapted for modifying the codon corresponding to position 572 of SEQ ID NO: 1 from asparagine to glutamine;
  • SEQ ID NO: 13 illustrates a forward primer adapted (e.g., at positions 11-13) for modifying the codon corresponding to position 648 of SEQ ID NO: 1 from asparagine to glutamine;
  • SEQ ID NO: 14 illustrates a reverse primer adapted for modifying the codon corresponding to position 648 of SEQ ID NO: 1 from asparagine to glutamine;
  • SEQ ID NO: 15 illustrates a forward primer adapted (e.g., at positions 11-13) for modifying the codon corresponding to position 833 of SEQ ID NO: 1 from asparagine to glutamine;
  • SEQ ID NO: 16 illustrates a reverse primer adapted for modifying the codon corresponding to position 833 of SEQ ID NO: 1 from asparagine to glutamine;
  • SEQ ID NO: 17 illustrates a forward primer for amplification of an FCE variant in which positions 1-20 overlap with the malE sequence in the pKLMF-EK vector;
  • SEQ ID NO: 18 illustrates a reverse primer for amplification of an FCE variant in which positions 1-20 overlap with multiple cloning site sequence in the pKLMF-EK vector;
  • SEQ ID NO: 19 illustrates a forward primer for amplification of a pKLMF-EK vector fragment
  • SEQ ID NO: 20 illustrates a reverse primer for amplification of a pKLMF-EK vector fragment
  • SEQ ID NO: 21 illustrates a forward primer for amplification of an assembled linear expression cassette of pKLMF-EK-FCE
  • SEQ ID NO: 22 illustrates a reverse primer for amplification of an assembled linear expression cassette of pKLMF-EK-FCE
  • SEQ ID NO: 23 illustrates a substrate RNA for in vitro capping reactions
  • SEQ ID NO: 24 illustrates a nucleotide sequence of an expression plasmid, namely pD912-FCE(N215Q/ N337Q/ N572Q) expression plasmid;
  • SEQ ID NO: 25 illustrates a fully processed mature FCE variant protein with asparagine to glutamine substitutions at positions corresponding to positions 215, 337, and 572 of SEQ ID NO: 1;
  • SEQ ID NO: 26 illustrates a nucleotide sequence of an expression plasmid, namely pD912-FCE (N215Q/ N337Q/ N572Q N648Q/ N833Q) expression plasmid;
  • SEQ ID NO: 27 illustrates a fully processed mature FCE variant protein with asparagine to glutamine substitutions at positions corresponding to positions 215, 337, 572, 648, AND 833 of SEQ ID NO: 1; and
  • SEQ ID NO: 28 illustrates an amino acid sequence of an FCE variant in which positions 215, 337, 572, 648 and/or 833 may comprise any amino acid (e.g., optionally, any amino acid other than asparagine.
  • Sources of commonly understood terms and symbols may include: standard treatises and texts such as Kornberg and Baker, DNA Replication, Second Edition (W.H. Freeman, New York, 1992); Lehninger, Biochemistry, Second Edition (Worth Publishers, New York, 1975); Strachan and Read, Human Molecular Genetics, Second Edition (Wiley-Liss, New York, 1999); Eckstein, editor, Oligonucleotides and Analogs: A Practical Approach (Oxford University Press, New York, 1991); Gait, editor, Oligonucleotide Synthesis: A Practical Approach (IRL Press, Oxford, 1984); Singleton, et al., Dictionary of Microbiology and Molecular biology, 2d ed., John Wiley and Sons, New York (1994), and Hale & Markham, the Harper Collins Dictionary of Biology, Harper Perennial, N.Y. (1991) and the like.
  • a protein refers to one or more proteins, i.e., a single protein and multiple proteins.
  • the claims can be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for use of such exclusive terminology as “solely,” “only” and the like in connection with the recitation of claim elements or use of a “negative” limitation.
  • Numeric ranges are inclusive of the numbers defining the range. All numbers should be understood to encompass the midpoint of the integer above and below the integer i.e., the number 2 encompasses 1.5-2.5. The number 2.5 encompasses 2.45-2.55 etc. When sample numerical values are provided, each alone may represent an intermediate value in a range of values and together may represent the extremes of a range unless specified.
  • an active FCE capping enzyme has at least detectable RNA-triphosphatase activity, at least detectable RNA guanylyltransferase activity, or at least detectable RNA N7- guanine methyltransferase activity.
  • Techniques for detecting TPase activity include, for example, combining the subject enzyme with y- 32 P- poly(A) RNA, separating reaction products using thin layer chromatography, excising Pi spots and subjecting to scintillation counting to measure Pi (and indirectly pp-poly(A) RNA) release from ppp-poly(A) RNA.
  • Techniques for detecting GTase activity include an enzyme-GMP intermediate assay in which, for example, the subject enzyme is combined with a- 32 P-GTP, reaction products are separated by SDS-PAGE, and the enzyme-GMP covalent intermediate formed (if any) is detected (e.g., by autoradiography).
  • This activity can also be assessed in a cap formation reaction in which, for example, the subject enzyme is combined with a- 32 P-GTP and poly(A) RNA and the reaction products are analyzed by TCA precipitation, filter binding, and scintillation counting (measuring the amount of Gppp-poly(A) RNA).
  • Techniques for detecting MTase activity include combining a radiolabeled capped poly(A) RNA (e.g., a- 32 P- GTP with poly(A) RNA) and VCE to produce G*ppp-poly(A) RNA, contacting that product with SAM and the subject enzyme, digesting with Pl nuclease, separating reaction products by thin layer chromatography, and analyzing excised spots by scintillation counting to measure the amount of m7GpppA from poly(A) RNA (JBC (1989) 264:9690-9695).
  • a radiolabeled capped poly(A) RNA e.g., a- 32 P- GTP with poly(A) RNA
  • VCE e.g., VCE
  • MTase activity may also be detected by combining the subject enzyme with GpppA (New England Biolabs, Inc.) and 3 H-S-adenosyl methionine, separating reaction products by thin layer chromatography, and analyzing excised bands by scintillation counting to measure amount of m7GpppA formed (RNA (2008) 14: 2297-2304).
  • buffer and “buffering agent” refer to a chemical entity or composition that itself resists and, when present in a solution, allows such solution to resist changes in pH when such solution is contacted with a chemical entity or composition having a higher or lower pH (e.g., an acid or alkali).
  • suitable non- naturally occurring buffering agents include, for example, Tris, HEPES, TAPS, MOPS, tricine, or MES.
  • cap refers to a natural cap, such as 7 mG, and to a compound of the general formula R3p3Nl-[p-N](x), where R3 is a guanine, adenine, cytosine, uridine or analogs thereof (e.g., N 7 -methylguanosine; m 7 G), ps is a triphosphate linkage, N1 and Nx are ribonucleosides, x is 0-8 and p is, independently for each position, a phosphate group, a phosphorothioate, a phosphorodithioate, an alkylphosphonate, an arylphosphonate, or a N-phosphoramidate linkage.
  • R3 is a guanine, adenine, cytosine, uridine or analogs thereof (e.g., N 7 -methylguanosine; m 7 G)
  • ps is a triphosphate linkage
  • R3 may have an added label at the 2’ or 3’ position of the ribose, and, in some embodiments, the label may be an oligonucleotide, a detectable label such as a fluorophore, or a capture moiety such as biotin or desthiobiotin, where the label may be optionally linked to the ribose of the nucleotide by a linker, for example.
  • a cap may have a cap 0 structure, a cap 1 structure or a cap 2 structure (e.g., as reviewed in Ramanathan, Nucleic Acids Res. 2016 44: 7511-7526), depending on which enzymes and/or whether SAM is present in the capping reaction.
  • Caps include dinucleotide cap analogs, e.g., of formula m 7 G(5')p3(5')G, in which a guanine nucleotide (G) is linked via its 5 'OH to the triphosphate bridge.
  • G guanine nucleotide
  • some dinucleotide caps the 3'-OH group is replaced with hydrogen or OCH3 (U.S. 7,074,596; Kore, Nucleosides, Nucleotides, and Nucleic Acids, 2006, 25: 15 307-14; and Kore, Nucleosides, Nucleotides, and Nucleic Acids, 2006, 25: 337-40).
  • Dinucleotide caps include m 7 G(5')p3G, 3'-OMe-m 7 G(5')p3G (ARC A).
  • Caps also include trinucleotide cap analogs (defined below) as well as other, longer, molecules (e.g., cap that have four, five or six or more nucleotides joined to the triphosphate bridge).
  • the 2’ and 3’ groups on the ribose of the m 7 G may be independently selected O-alkyl (e.g., O-methyl), halogen, a linker, hydrogen or a hydroxyl and the sugars 20 in N1 and NX may be independently selected from ribose, deoxyribose, 2’ -O-alkyl, 2’-O- methoxy ethyl, 2’-O-allyl, 2’-O-alkylamine, 2’ -fluororibose, and 2’ -deoxyribose.
  • O-alkyl e.g., O-methyl
  • halogen e.g., halogen, a linker, hydrogen or a hydroxyl
  • the sugars 20 in N1 and NX may be independently selected from ribose, deoxyribose, 2’ -O-alkyl, 2’-O- methoxy ethyl, 2’-O-allyl
  • N1 and NX may independently (for each position) comprise a base selected from adenine, uridine, guanine, or cytidine or analogs of adenine, uridine, guanine, or cytidine, and nucleotide modifications can be selected from N 6 -methyladenine, N 1 -methyladenine,N 6 -2’-Odimethyladenosine, pseudouridine, N 1 -methylpseudouridine, 5 -iodouridine, 4-thiouridine, 2-thiouridine, 5- methyluridine, pseudoisocytosine, 5 -methoxycytosine, 2-thiocytosine, 5 -hydroxycytosine, N 4 - methylcytosine, 5-hydroxymethylcytosine, hypoxanthine, Nl-methylguanine, O 6 - methylguanine, 1-methyl-guanosine, N 2 -methylguanosine, N 2 ,N 2 -dimethyl-gu
  • capping refers to the addition of a cap onto the 5’ end of an RNA.
  • Caps may be added at the 5' end of an RNA (e.g., an uncapped RNA transcript) chemically or enzymatically apart from transcription or co-transcriptionally to yield a 5' capped RNA. Capping may or may not be reversible.
  • decapping enzyme refers to an enzyme that removes a cap from an RNA, leaving the RNA with a 5’ monophosphate, but otherwise unchanged. Decapping enzymes may have pyrophosphohydrolase activity. Examples of decapping enzymes include enzymes in the Nudix hydrolase family (e.g., RppH, DCP1/DCP2 complex, NUDT16, African swine fever virus decapping enzyme), DXO family (e.g., Dxolp, Railp), histidine triad family (e.g., DCPS, Fhit), and Apa-H-like phosphatase. Examples of decapping enzymes are described in Kramer and McLennan, 2019, WIREs RNA 10(l)el511.
  • expression system refers to systems for producing a protein from a polynucleotide template comprising components to produce the protein according to an RNA template (e.g., enzymes, amino acids, an energy source), (optionally) components to produce the RNA template according to another RNA template or a DNA template (e.g., enzymes, nucleotides, an energy source).
  • An expression system may comprise a bacterial (e.g., Escherichia coli) or yeast (e.g., Kluyveromyces lactis or Pichia pastoris) expression system in which the protein is encoded by an RNA or DNA template within an expression cassette, a plasmid or other expression vector.
  • An expression system may comprise a viral expression system in which the protein is encoded by an RNA or DNA template (e.g., in an expression cassette) within a viral genome or viral expression vector.
  • cell-free expression systems may include or comprise cell extracts of Escherichia coli S30, rabbit reticulocytes or wheat germ, PUREEXPRESS® (New England Biolabs, Ipswich, MA), an insect cell extract system (e.g. , Promega # LI 101), or HeLa cell lysate-based protein expression systems (e.g., Thermo Fisher Scientific # 88882).
  • An expression cassette may comprise, in some embodiments, an expression control sequence (e.g.
  • promoter a coding sequence encoding the gene product (e.g., protein) of interest (e.g., a vaccinia capping enzyme fusion), and/or one or more termination sequences (e.g., terminators).
  • a coding sequence encoding the gene product (e.g., protein) of interest e.g., a vaccinia capping enzyme fusion
  • termination sequences e.g., terminators
  • promoter may comprise any promoter operative in a desired expression system, including, for example, a GAP promoter, an AOX1 promoter, a LAC4 promoter, a P350 hybrid promoter, a T7 promoter, a T5 promoter, a Ptac promoter, a Ptrc promoter, ParaB AD promoter, a PrhaBAD promoter, a Tet promoter or a PhoA phosphate-starvation promoter.
  • FCE refers to a single-chain enzyme having RNA capping activity and having the amino acid sequence of positions 1 to 878 of SEQ ID NO:1.
  • FCE variant refers to a non-naturally occurring, single-chain enzyme having (a) RNA capping activity and (b) less than 100% amino acid sequence identity to a naturally occurring single-chain RNA capping enzyme and/or a non-naturally occurring chemical modification (e.g., a polypeptide fused to its amino terminal or carboxy terminal end or other chemical modification).
  • a variant amino acid sequence may have at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98% or at least 99% identity to the amino acid sequence of FCE. Sequence differences may include insertions or deletions extending and/or shortening the N- and/or C-terminal ends.
  • An FCE variant may have an amino acid sequence having less than 100% identity to positions 1 to 878 of SEQ ID NO: 1.
  • An FCE variant may have, for example, an amino acid sequence having one or more substitutions with respect to positions 1 to 878 of SEQ ID NO: 1 and having at least 90%, at least 92%, at least 94%, at least 96%, or at least 98% identity with positions 1 to 878 of SEQ ID NO: 1.
  • An FCE variant may have, for example, an amino acid sequence having one or more substitutions with respect to SEQ ID NO: 1 and having at least 90%, at least 92%, at least 94%, at least 96%, or at least 98% identity with SEQ ID NO: 1 or SEQ ID NO: 2.
  • An FCE variant may have an amino acid sequence having less than 100% identity to positions 1 to 878 of SEQ ID NO: 1.
  • An FCE variant may have, for example, an amino acid sequence having one or more substitutions with respect to positions 389 to 1266 of SEQ ID NO: 2 and having at least 90%, at least 92%, at least 94%, at least 96%, or at least 98% identity with positions 389 to 1266 of SEQ ID NO: 2.
  • FCE variants may comprise one or more substitutions that impact glycosylation of the protein.
  • an FCE variant may comprise one or more substitutions at one or more positions selected from positions corresponding to N215, N337, N572, N648, and N833 of SEQ ID NO: 1 or selected from positions corresponding to N603, N725, N960, N1036, and N1221 of SEQ ID NO: 2.
  • Substitutions at positions corresponding to N215, N337, N572, N648, and N833 of SEQ ID NO: 1 and positions corresponding to N603, N725, N960, N1036, and N1221 of SEQ ID NO: 2 may be a deletion or any amino acid other than asparagine, but may be selected to retain one or more properties of the asparagine replaced.
  • replacement amino acids for asparagine may be glutamine.
  • an FCE variant may comprise an amino acid sequence having (a) at least 90% identity to positions 1-214 of SEQ ID NO:1, (b) at least 90% identity to positions 216-336 of SEQ ID NO:1, (c) at least 90% identity to positions 338- 571 of SEQ ID NO:1, (d) at least 90% identity to positions 573-647 of SEQ ID NO:1, (e) at least 90% identity to positions 649-832 of SEQ ID NO:1, (f) at least 90% identity to positions 834-878 of SEQ ID NO:1, and (g) a substitution at a position corresponding to position 215, 337, 572, 648, or 833 of SEQ ID NO: 1 (e.g., a deletion or any amino acid other than asparagine).
  • an FCE variant may comprise an amino acid sequence having (a) at least 90% identity to positions 389-602 of SEQ ID NO:2, (b) at least 90% identity to positions 604-724 of SEQ ID NO:2, (c) at least 90% identity to positions 726-959 of SEQ ID NO:2, (d) at least 90% identity to positions 961-1035 of SEQ ID NO:2, (e) at least 90% identity to positions 1037-1220 of SEQ ID NO:2, (f) at least 90% identity to positions 1222- 1266 of SEQ ID NO:2, and (g) a substitution at a position corresponding to position 603, 725, 960, 1036, or 1221 of SEQ ID NO: 2 (e.g. , a deletion or any amino acid other than asparagine).
  • an FCE variant may comprise a polypeptide having the amino acid sequence of SEQ ID NO:25 or SEQ ID NO:27.
  • in vitro transcription refers to a cell- free reaction in which a DNA template is copied by a DNA-directed RNA polymerase (typically a bacteriophage polymerase) to produce a product that comprises one or more RNA molecules that have been copied from the template.
  • a DNA-directed RNA polymerase typically a bacteriophage polymerase
  • modified nucleotide refers to nucleotides having a modification on the sugars (e.g., 2'-fluororibose, ribose, 2'-deoxyribose, arabinose, and hexose); and/or in the phosphate groups (e.g., phosphorothioates and 5'-N- phosphoramidite linkages); and/or in the nucleotide base (e.g., as described in US 8,383,340; WO 2013/151666; US 9,428,535 B2; US 2016/0032316).
  • sugars e.g., 2'-fluororibose, ribose, 2'-deoxyribose, arabinose, and hexose
  • phosphate groups e.g., phosphorothioates and 5'-N- phosphoramidite linkages
  • nucleotide base e.g., as described in US 8,383,340
  • non-naturally occurring refers to a polynucleotide, polypeptide, carbohydrate, lipid, or composition that does not exist in nature.
  • a polynucleotide, polypeptide, carbohydrate, lipid, or composition may differ from naturally occurring polynucleotides polypeptides, carbohydrates, lipids, or compositions in one or more respects.
  • a polymer e.g. , a polynucleotide, polypeptide, or carbohydrate
  • the component building blocks e.g., nucleotide sequence, amino acid sequence, or sugar molecules.
  • 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).
  • buffering agents e.g., a detergent, a dye, a solvent or a preservative
  • polymerase refers to an enzyme that synthesizes a polynucleotide from NTPs with or without a template.
  • enzymes include T3 RNA polymerase, T7 RNA polymerase, SP6 polymerase, among others and variants thereof including thermostable variants (e.g., International Application No. PCT/US2017/013179 and US Application Serial No. 15/594,090).
  • a “single-chain RNA capping enzyme” refers to a capping enzyme in which a single polypeptide chain as a monomer displays RNA triphosphatase (TPase), guanylyltransferase (GTase) and guanine-N7 methyltransferase (N7 MTase) activities.
  • TPase RNA triphosphatase
  • GTase guanylyltransferase
  • N7 MTase guanine-N7 methyltransferase
  • Faustovirus, mimivirus and moumouvirus capping enzymes are examples of single-chain RNA capping enzymes.
  • VCE is a heterodimer and, as such, is not a single-chain RNA capping enzyme.
  • a “substitution” at a position in a comparator amino acid sequence refers to any difference at that position relative to the corresponding position in a reference sequence, including a deletion, an insertion, and a different amino acid, where the comparator and reference sequences are at least 80% identical to each other.
  • a substitution in a comparator sequence, in addition to being different than the reference sequence, may differ from all corresponding positions in naturally occurring sequences that are at least 80% identical to the comparator sequence.
  • transcript refers to a polynucleotide template for a polypeptide.
  • a transcript may comprise RNA (e.g., ssRNA), a cap or cap analog, and/or a poly A tail.
  • a transcript may be capable of translation in a cell (e.g., a bacterial cell and/or a yeast cell).
  • a transcript may be or comprise mRNA.
  • a fusion transcript may comprise polynucleotide templates for two or more polypeptides in a single polynucleotide.
  • uncapped refers to a condition of an RNA in which it does not have a cap structure at its 5’ end.
  • Uncapped RNA typically has a triphosphoryl, di-phosphoryl, mono-phosphoryl or a hydroxyl group at the 5’ end.
  • RNAs Production of stable mRNA capable of efficient translation upon introduction to a subject may require an appropriate cap structure.
  • a cap may avoid triggering the innate immune response observed upon introduction of uncapped (5 ’-triphosphate) RNAs (Pichlmair, et al., Science 2006 314: 997-1001; Diamond, et al., Cytokine & Growth Factor Reviews, 2014 25: 543-550).
  • it may be desirable to add a cap to synthetic RNA in many therapeutic applications (e.g., protein replacement therapy as well as prophylactic or therapeutic vaccination).
  • Vaccinia virus like most viruses, has a robust set of mechanisms to co-opt host cell machinery for the production of viral proteins.
  • One such tool is the vaccinia capping enzyme, which forms a Cap 0 structure (m7Gppp5 N) at the 5’ end of uncapped RNA molecules through its RNA triphosphatase, guanylyltransferase, and guanine methyltransferase activities.
  • capping viral transcripts allows them to be transcribed by the infected cells.
  • Other transcripts may be capped rapidly in vitro in the presence of the vaccinia capping enzyme, reaction buffer, GTP, and the methyl donor, SAM. Production of active vaccinia capping enzyme for cell-free vaccine production can be challenging.
  • VCE may impede production (e.g., high capacity production) and use of the enzyme.
  • efforts to express the vaccinia virus DIR gene in bacteria and yeast as a means to produce and recover the 97 kDa subunit result in poor yields, possibly due, at least in part, to the insolubility and/or hydrophobicity of the 97 kDa subunit.
  • in vitro assembly of the small and large subunits into a whole protein may yield an enzyme with little to no catalytic activity. Separately produced subunits may not be present in an appropriate ratio or conformation to efficiently or productively bind to one another and/or bind to substrates. Accordingly, a need has arisen for alternatives to VCE for efficient enzymatic capping or RNA molecules.
  • the present disclosure relates to RNA capping enzymes from Faustovirus and variants thereof (e.g., variants across strains of Faustovirus and variants from related viruses) and kits including these enzymes.
  • the present disclosure further relates to methods of making and using such enzymes.
  • the present disclosure provides FCE and variants thereof.
  • An FCE variant may comprise a non-naturally occurring amino acid sequence that is at least 90% identical (e.g., at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical) to positions 1-878 of SEQ ID NO: 1 or at least 90% identical (e.g., at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical) to SEQ ID NO: 1 or at least 90% identical (e.g., at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical) to positions 389 to 1266 of SEQ ID NO: 2 or at least 90% identical (e.g., at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical) to SEQ ID NO: 2.
  • An FCE variant may be fused to one or more peptides (e.g. , sorting signals, His, MBP or other purification tags) or polypeptides (e.g., other enzymes, linkers or spacers).
  • an FCE variant may be immobilized, for example, to a solid support (e.g., magnetic, agarose, polystyrene, polyacrylamide, chitin).
  • a composition may include one or more FCE variants, one or more substrates of the one or more FCE variants (e.g., uncapped RNA, GDP), one or more intermediates or products (e.g. , inorganic phosphate, inorganic diphosphate) of the one or more FCE variants, one or more transcripts of one or more FCE variants (e.g. , a capped FCE variant transcript), and any combination thereof.
  • a composition may include, according to some embodiments, an FCE variant and one or more additional components that support storage, transportation, activity and/or use of such FCE variant.
  • a composition may comprise an FCE variant and an uncapped ribonucleic acid (e.g., an uncapped therapeutic RNA), dNTPs, rNTPs, primers, other enzymes (e.g. , decapping enzymes, polymerases, or other enzymes), buffering agents (e.g., a storage buffer, a reaction buffer), or combinations thereof.
  • Uncapped RNA may be synthesized using solid-phase oligonucleotide synthesis chemistry or by transcribing a DNA template using a polymerase (e.g., a bacteriophage polymerase) in an in vitro transcription reaction, for example.
  • a composition may comprise SAM and/or a cap 2’0 methyltransferase enzyme (2’OMTase).
  • a composition may comprise one or more additives (e.g. glycerol), salts (e.g. KC1), reducing agents, chelating agents (e.g., EDTA), detergents, and/or denaturants (e.g., caffeine, urea), among others.
  • a composition comprising dNTPs may include one, two, three or all four of dATP, dTTP, dGTP and dCTP.
  • a composition comprising rNTPs may include one, two, three of all four or rATP, rUTP, rGTP and rCTP.
  • a composition may further comprise one or more modified nucleotides.
  • a composition may comprise one or more modified nucleotides.
  • a composition may optionally comprise one or more primers (random primers, bump primers, exonuclease-resistant primers, chemically-modified primers, custom sequence primers, or combinations thereof).
  • Compositions optionally may comprise one or more of the components set forth below for kits.
  • a composition may be glycerol-free, may be dry (e.g., as a result of lyophilization), and/or may be aqueous.
  • a composition may be formulated for delivery to a subject (e.g., a human subject, a non-human animal subject).
  • a composition for example, a composition including one or more products of an FCE variant, may be free of materials (e.g., enzymes) derived from non-human animals according to some embodiments.
  • a capping method may comprise contacting a singlechain RNA capping enzyme (e.g., FCE, an FCE variant) with one or more of a target RNA (e.g., an uncapped therapeutic RNA), an NTP, a modified NTP, a cap, S- adenosylmethionine (SAM), and a buffering agent to form a reaction mix.
  • a target RNA e.g., an uncapped therapeutic RNA
  • NTP e.g., an uncapped therapeutic RNA
  • NTP e.g., a modified NTP
  • a cap e.g., S- adenosylmethionine (SAM), and a buffering agent
  • SAM S- adenosylmethionine
  • This contact may be at a sufficient temperature and for a sufficient time to form a capped target RNA.
  • the contact may be at a temperature (e.g. , constant or varying) in the range of 37
  • a single-chain RNA capping enzyme for a capping method may comprise an amino acid sequence that is at least 90% identical (e.g., at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical) to positions 1-878 of SEQ ID NO: 1 or at least 90% identical (e.g., at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical) to positions SEQ ID NO: 1 or at least 90% identical (e.g., at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical) to positions 389 to 1266 of SEQ ID NO: 2 or at least 90% identical (e.g., at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical) to SEQ ID NO: 2.
  • a capping method may further comprise monitoring the appearance capped target RNA.
  • Capped RNA may be monitored/detected by denaturing urea polyacrylamide gel electrophoresis, radiometric assays, capillary electrophoresis, or mass spectrometry -based methods (e.g., as provided by Beverly, M., Dell, A., Parmar, P., and Houghton, L. 2016. Label-free analysis of mRNA capping efficiency using RNase H probes and LC-MS. Analytical and bioanalytical chemistry 408:5021-5030).
  • a capping method may comprise (a) contacting a polymerase with one or more of a polynucleotide template (DNA or RNA) encoding a target RNA, rNTPs and/or modified rNTPs, and a buffer to form a transcription product comprising the target RNA and (b) contacting a single-chain RNA capping enzyme (e.g., FCE, an FCE variant) with one or more of the transcription products, an NTP, a modified NTP, a cap, S- adenosylmethionine (SAM), and a buffering agent to form a reaction mix.
  • This contact may be at a sufficient temperature and for a sufficient time to form a capped target RNA.
  • the contact may be at a temperature (e.g. , constant or varying) in the range of 37°C -60°C and/or for a time in the range of second to hours (e.g., from 60 seconds to 16 hours).
  • a capping method may further comprise contacting the capped RNA with a one or more pharmaceutically acceptable additives (e.g., excipients, diluents, and/or carriers), including, for example, fluids, solvents, dispersion media, wetting agents, crowding agents, micelles, lipidoids, liposomes, polymers, lipoplexes, peptides, proteins, salts, surface active agents, isotonic agents, thickeners, emulsifiers, preservatives, stabilizers, solubilizers, buffers, sugars, starches, cellulose, waxes, glycols, polyols, polyesters, polycarbonates, poly anhydrides, hyaluronidase, nanoparticles (e.g., lipid nanoparticles, core-shell nanoparticles, and/or nanoparticle mimics), and combinations thereof.
  • a pharmaceutically acceptable additives e.g., excipients, diluents, and/or carriers
  • pharmaceutically acceptable additives protect, preserve, and/or stabilize a capped RNA during manufacture, storage, and/or administration to a subject.
  • pharmaceutical acceptable additives include those described in U.S. Patent Publication No. 2017/0119740.
  • a capping method may further comprise contacting the capped RNA with one or more additives selected from lipidoids, liposomes, polymers, lipoplexes, peptides, proteins, cells transfected with HCMV RNA vaccines (e.g., for transplantation into a subject), hyaluronidase, nanoparticles (e.g., lipid nanoparticles, core-shell nanoparticles, and/or nanoparticle mimics).
  • Capped RNAs may be formulated for delivery and/or delivered to a eukaryotic organism. Examples of subjects that may receive a capped RNA include humans and nonhuman animals (e.g., mouse, rat, rabbit, dog, cat, cattle, swine, sheep, horse or primate). Capped RNAs may be delivered to plants or plant cells, according to some embodiments, to confer or augment resistance to or tolerance of an environmental condition (e.g., drought, salt) and/or to prevent, mitigate or treat herbivory, pathogen infection, or the effects thereof. Capped RNA also may be delivered to one or more yeast cells.
  • an environmental condition e.g., drought, salt
  • the present disclosure provides methods for preparing a capped RNA dosage form comprising, contacting an uncapped RNA with an FCE variant to form a capped RNA, and contacting the capped RNA with one or more pharmaceutically acceptable additives, binders, buffers, coatings, colors, controlled release agents, delivery agents (e.g., liposomes, propellants), diluents, disintegrants, dyes, excipients, fillers, lipids, lubricants, salts, sorbants, stabilizers, and/or other agents to produce an RNA dosage form.
  • a capped RNA may be combined (e.g., in a single dosage form or delivered concurrently or in sequence with one or more other active pharmaceutical agents.
  • a capped RNA and/or its encoded translation product(s) may function in a subject as an active pharmaceutical agent, according to some embodiments.
  • a capped RNA e.g., a capped RNA dosage form
  • Capped RNA can either be naked or formulated in a suitable form for delivery to a subject, e.g., a human.
  • Formulations can include liquid formulations (solutions, suspensions, dispersions), topical formulations (gels, ointments, drops, creams), liposomal formulations (such as those described in: US 9,629,804 B2; US 2012/0251618 Al; WO 2014/152211; US 2016/0038432 Al).
  • the cells into which the RNA product is introduced may be in vitro (i.e., cells that have been cultured in vitro on a synthetic medium). Accordingly, the RNA product may be transfected into the cells.
  • the cells into which the RNA product is introduced may be in vivo (cells that are part of a mammal).
  • the cells into which the RNA product is introduced may be present ex vivo (cells that are part of a tissue, e.g. , a soft tissue that has been removed from a mammal or isolated from the blood of a mammal).
  • Methods for production of an FCE variant may comprise, for example, contacting a polynucleotide encoding such FCE variant with an expression system (e.g., a bacterial expression system, a yeast expression system, an insect expression system, a mammalian expression system, a viral expression system or a cell-free expression system).
  • an expression system e.g., a bacterial expression system, a yeast expression system, an insect expression system, a mammalian expression system, a viral expression system or a cell-free expression system.
  • a method of producing an FCE variant may comprise contacting an uncapped FCE variant transcript with a capping enzyme (e.g., vaccinia capping enzyme, FCE, an FCE variant) to form a capped FCE variant transcript.
  • a capping enzyme e.g., vaccinia capping enzyme, FCE, an FCE variant
  • a method may further include contacting a capped FCE variant transcript with an expression system to form FCE protein.
  • An FCE variant protein may be produced, according to some embodiments, by constructing an expression plasmid compatible to E. coli or yeast expression systems under the control of an appropriate promoter.
  • the plasmid can be introduced into the cells via transformation and the resultant E. coli or yeast strain can be cultured using appropriate methods.
  • the expression of the FCE variant protein can be induced by subjecting the culture to appropriate conditions in case of inducible promoters or by following appropriate culture conditions for auto-induced promoters. Cultivation conditions (e.g., time, temperature, media composition) may be maintained or adjusted as needed to express the FCE protein variant. Cultures may be harvested, for example, by centrifugation or tangential flow filtration.
  • Harvested cells may be stored at low temperatures or lysed using an appropriate method such as sonication or mechanical sheering.
  • Lysates may be clarified, for example, by centrifugation or tangential flow filtration.
  • the FCE variant protein may be purified from the clarified lysate or spent culture medium, for example, by chromatographic methods.
  • an FCE variant protein may be produced by contacting an FCE variant protein expression DNA construct operably linked to an expression control sequence (e.g., an appropriate promoter) to an in vitro transcription/translation system such as PURExpress In vitro Protein Synthesis Kit (New England Biolabs, Inc.) or TnT Quick Coupled Transcription/Translation System (Promega).
  • an FCE variant protein can be produced by contacting an FCE variant protein expression DNA construct under the control of an appropriate promoter to a cell-free protein synthesis system derived from organisms such as E. coli (e.g., NEB Express Cell-free E. coli Protein Synthesis System (New England Biolabs, Inc.), rabbit, wheat germ, insect, or human.
  • Reaction conditions e.g., time, temperature, reaction composition
  • Expressed variant protein may be purified by appropriate methods (e.g., chromatographic methods).
  • kits including an FCE variant may include an FCE variant and an uncapped ribonucleic acid, dNTPs, rNTPs, primers, other enzymes (e.g., decapping enzymes, polymerases, or other enzymes), buffering agents, or combinations thereof.
  • An FCE variant may be included in a storage buffer (e.g., comprising glycerol and a buffering agent).
  • a kit may include a reaction buffer which may be in concentrated form, and the buffer may contain additives (e.g. glycerol), salt (e.g. KC1), reducing agent, EDTA or detergents, among others.
  • a kit comprising dNTPs may include one, two, three or all four of dATP, dTTP, dGTP and dCTP.
  • a kit comprising rNTPs may include one, two, three of all four or rATP, rUTP, rGTP and rCTP.
  • a kit may further comprise one or more modified nucleotides.
  • a kit may optionally comprise one or more primers (random primers, bump primers, exonuclease-resistant primers, chemically-modified primers, custom sequence primers, or combinations thereof).
  • One or more components of a kit may be included in one container for a single step reaction, or one or more components may be contained in one container, but separated from other components for sequential use or parallel use. The contents of a kit may be formulated for use in a desired method or process.
  • a kit contains: (i) an FCE variant; and (ii) a buffer.
  • An FCE variant may have a lyophilized form or may be included in a buffer (e.g. , a storage buffer or a reaction buffer in concentrated form).
  • a kit may contain an FCE variant in a mastermix suitable for receiving and capping a template ribonucleic acid.
  • An FCE variant may be a purified enzyme so as to contain no other detectable enzyme activities.
  • the reaction buffer in (ii) and/or storage buffers containing an FCE variant in (i) may include non-ionic, ionic e.g. anionic or zwitterionic surfactants, denaturants, and/or crowding agents.
  • a kit may include an FCE variant and the reaction buffer in a single tube or in different tubes.
  • a subject kit may further include instructions for using the components of the kit to practice a desired method.
  • the instructions may be recorded on a suitable recording medium.
  • instructions may be printed on a substrate, such as paper or plastic, etc.
  • the instructions may be present in the kits as a package insert, in the labeling of the container of the kit or components thereof (i.e., associated with the packaging or subpackaging) etc.
  • Instructions may be present as an electronic storage data file residing on a suitable computer readable storage medium (e.g., a CD-ROM, a flash drive). Instructions may be provided remotely using, for example, cloud or internet resources with a link or other access instructions provided in or with a kit.
  • EXAMPLE 1A Construction of P. pastoris FCE Expression Vectors for Secreted Expression
  • Histidine tag was amplified by PCR using the forward and reverse primers, respectively:
  • the forward and reverse primers were engineered to contain sequences that overlap the pD912(GAP) vector (lower case) (FIGURE 1A).
  • the NEBuilder Assembly Tool was used for primer and assembly design.
  • the 2661 bp FCE gene was amplified from the plasmid pFCE-CHis8 containing the full-length gene and 8X Histidine tag, using Q5 High-Fidelity 2X Master Mix (New England Biolabs).
  • the pD912(GAP) was prepared from pD912(AOX) vector by replacing 462 bp long AOX1 promoter sequence with 483 bp long DNA fragment containing Pichia pastoris GAP promoter.
  • the 3826 bp vector fragment was amplified from the plasmid pD912(GAP) by PCR using the forward and reverse primers, respectively:
  • This integrative expression vector contains the mating factor alpha secretion leader for extracellular expression, the GAP1 promoter which initiates and terminates transcription and the zeocin resistance gene which allows for selection of transformants by growth on zeocin- containing medium.
  • the two fragments were joined using NEBuilder HiFi DNA Assembly Master Mix (New England Biolabs). 2 pl of the reaction was transformed into 50 pl of NEB 5-alpha Competent E. coli (High Efficiency) cells, plated on LB-zeocin (25 pg/mL) plates and incubated overnight at 37 °C resulting in a P. pastoris expression vector pD912(GAP)- FCE(WT)-8His (SEQ ID NO:1) (FIGURE 2).
  • N-linked glycosylation sites were first identified using the NetNGlyc 1.0 server (http://www.cbs.dtu.dk/services/NetNGlyc/). The prediction results indicated 5 potential sites at amino acid sequence positions 215, 337, 572, 648, and 833. Positions 215, 337 and 572 scored above the N-glycosylation threshold potential. In light of these predictions, 2 variant constructs were generated, one with N215Q, N337Q, N572Q, N648Q, and N833Q substitutions, and the other with N215Q, N337Q and N572Q substitutions.
  • the Q5 Site Directed Mutagenesis Kit (New England Biolabs, Inc.) was used for construction of the variant expression vectors. The NEBaseChanger tool was used for primer design. The primers for each variant are listed below:
  • the forward primers contained the nucleotide sequence encoding the glutamine residue (lower case).
  • the FCE gene containing a single mutated N-linked site was amplified from the plasmid pD912(GAP)-FCE-8His using Q5 High-Fidelity 2X Master Mix (New England Biolabs).
  • the pD912 (PGAP) vector fragment containing the S. cerevisae tz-mating factor pre-pro signal sequence, was also amplified by Q5 High-Fidelity 2X Master Mix using the primers described above. Each FCE fragment was joined to the pD912 (GAP) vector fragment using NEBuilder HiFi DNA Assembly Master Mix.
  • a plasmid containing three N-glycan variants was created by amplifying the 708 bp fragment from N337 to N572 and assembling the resulting PCR product into the pD912(GAP)-FCE(N215Q)-8His plasmid using NEBuilder HiFi DNA Assembly Mix. This resulted in the plasmid, pD912(GAP)-FCE(N215Q/ N337Q/ N572Q)-8His (SEQ ID NO:24).
  • a plasmid containing all five N-glycan variants was created by amplifying the 557 bp fragment from N648 to N833 and assembling the resulting PCR product into the pD912(GAP)-FCE(N215Q/ N337Q/ N572Q)-8His plasmid using NEBuilder HiFi DNA Assembly Mix. This resulted in the plasmid, pD912(GAP)-FCE(N215Q/ N337Q/ N572Q/ N648Q/ N833Q)-8His (SEQ ID NO:24).
  • Double variants of N215Q/ N337Q and N215Q/ N572Q were constructed using the Q5 Site Directed Mutagenesis Kit as described above resulting in the plasmids, pD912(GAP)-FCE(N215Q/ N337Q)-8His and pD912(GAP)-FCE(N215Q/ N572Q)-8His.
  • the forward and reverse primers were engineered to contain sequences that overlap the pKLMF-EK vector (New England Biolabs) (lower case) (FIGURE IB).
  • the NEBuilder Assembly Tool was used for primer and assembly design.
  • the 2661 bp FCE gene was amplified from the plasmid pFCE-CHis8 (Siuhong Chan) containing the full-length gene and 8x Histidine tag, using Q5 High-Fidelity 2X Master Mix (New England Biolabs).
  • the 10028 bp vector fragment was amplified from the plasmid (CT867) pKLMF- A313V-EK-LongerLinker (unoptimized) by Q5 High-Fidelity 2X Master Mix using the forward and reverse primers, respectively:
  • CTCGAGAAAAGAGAGGCTGAAGCT SEQ ID NO: 19
  • CTTGTCATCGTCATCCCCGAG SEQ ID NO: 20
  • This integrative expression vector contains the malE gene which encodes for maltose binding protein (MBP), the LAC4 promoter which initiates and terminates transcription and the acetamidase gene which allows for selection of transformants by growth on acetamide- containing medium.
  • MBP maltose binding protein
  • LAC4 the LAC4 promoter which initiates and terminates transcription
  • acetamidase gene which allows for selection of transformants by growth on acetamide- containing medium.
  • K. lactis a- mating factor pre-pro signal sequence has been replaced with the malE gene.
  • MBP-fusion proteins will not be directed to the secretory pathway but instead will be retained in the yeast cytosol.
  • the two fragments were joined using NEBuilder HiFi DNA Assembly Master Mix (New England Biolabs).
  • the assembled linear expression cassette (FIGURE 2) was amplified by PCR using the forward and reverse primers, respectively (SEQ ID NO:2):
  • Pichia pastoris aoxl A (MutS) (ATUM, formerly DNA 2.0) and Kluyveromyces lactis GG799 (New England Biolabs) strains were for each relevant experiment. Electrocompetent cells were prepared using the lithium acetate/DTT method (Wu and Letch worth, 2004). Electroporation conditions were 1.5 KV, 25 pF and 200 Ohm using a 0.2 cm cuvette followed by selection of transformants by growth on yeast peptone dextrose (YPD) agar medium supplemented with 1 M sorbitol and 500 pg/mL Zeocin (Teknova) (P. pastoris) and yeast carbon base (YCB) supplemented with 5 mM acetamide (K. lactis) and incubated for 3- 4 days at 30°C.
  • YPD yeast peptone dextrose
  • YPD yeast peptone dextrose
  • YCB yeast carbon base
  • P. pastoris expression plasmids were linearized by .She I- HF restriction digestion and the purified products were used to transform electrocompetent P. pastoris MutS cells.
  • Pichia pastoris constructs containing GAP promoter
  • transformants were grown at 30°C in 5 mL of BMGY-Buffered Glycerol Complex Medium (Teknova) (1% yeast extract, 2% tryptone, 1.34% yeast nitrogen base (YNB) without amino acids with ammonium sulfate, 0.0004% biotin, 1% glycerol as the carbon source, 100 mM potassium phosphate, pH 6.0). After 48 hours, the spent culture media was harvested.
  • BMGY-Buffered Glycerol Complex Medium Teknova
  • yeast extract 2% tryptone
  • yeast nitrogen base yeast nitrogen base
  • the spent culture media (P. pastoris constructs) were buffer-exchanged in 50 mM Tris-HCl, pH 7.5 buffer containing 300 mM NaCl and concentrated ten-fold using Vivaspin 30 kDa MWCO filters (Sartorius). To assess the extent of glycosylation, the concentrated spent culture media were subject to Endo Hf (New England Biolabs) digestions under native conditions in the presence of lx GlycoBuffer 3 (50 mM sodium acetate, pH 6.0).
  • FCE proteins wild-type and N-glycan variants were purified from the concentrated spent cultures using NEBExpress Ni Spin Columns (New England Biolabs). The columns were washed twice with 50 mM Tris-HCl, pH 7.5 buffer containing 300 mM NaCl and 5 mM imidazole then once with 50 mM Tris-HCl, pH 7.5 buffer containing 300 mM NaCl and 10 mM imidazole. The purified protein was eluted with 50 mM Tris-HCl, pH 7.5 buffer containing 300 mM NaCl and 500 mM imidazole.
  • cell lysates K. lactis constructs
  • cells were resuspended in 50 mM Tris- HCl, pH 7.5 buffer containing 300 mM NaCl and sonicated (Qsonica).
  • the soluble cell lysate was pre-cleared by centrifugation at 16000 x g for 15 minutes at 4°C.
  • the cell lysates and spent culture media were analyzed by SDS-PAGE on 4-20% polyacrylamide gel, followed by western blotting with a His-tag antibody (Thermo Fisher Scientific).
  • Reactions were diluted in nuclease-free water to reach a final substrate concentration of 5 nM before capillary electrophoresis on either an Applied Biosystems 3130x1 Genetic Analyzer (16 capillary array) or an Applied Biosystems 3730x1 Genetic Analyzer (96 capillary array) using GeneScan 120 LIZ dye Size Standard (Applied Biosystems). Reaction products were analyzed using PeakScanner software (Thermo Fisher Scientific).
  • the full-length FCE is also expressed as a single polypeptide in the K. lactis cytoplasm (FIGURE 3B). All recombinants displayed mRNA capping activity both before and after EndoHf digestion (FIGURE 4 A and FIGURE 4B).
  • the load (spent culture media) and elution fractions were analyzed by SDS-PAGE.
  • the eluted FCE (N215Q/ N337Q/ N572Q) mutant shows a significant decrease in glycosylation as observed by the improved band resolution as compared to wild type.
  • the purified FCE (N215Q/ N337Q/ N572Q/ N648Q/ N833Q) mutant shows further improved band resolution, indicating a further decrease in glycosylation (FIGURE 5).
  • the concentrated spent culture media from all 3 recombinants showed mRNA capping activity (FIGURE 6).

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Abstract

La présente divulgation se rapporte à des compositions, à des nécessaires et à des procédés de fabrication de vaccins à ARN ayant une structure de coiffe appropriée. Des systèmes, un appareil, des compositions et/ou des procédés peuvent comprendre et/ou utiliser, dans certains modes de réalisation, des enzymes de coiffage d'ARN à chaîne unique non naturelles. Dans certains modes de réalisation, une enzyme de coiffage d'ARN peut comprendre un variant de FCE ayant (a) une séquence d'acides aminés au moins à 90 % identique aux positions 1 à 878 de SEQ ID NO : 1, et/ou (b) une ou plusieurs substitutions par rapport à SEQ ID NO : 1 à une position choisie parmi des positions correspondant aux positions 215, 337, 572, 648 et 833 (par exemple, une position choisie parmi des positions correspondant à la position 215, 337 et 572) de SEQ ID NO : 1.
EP21707503.5A 2021-01-27 2021-01-27 Enzyme de coiffage de faustovirus, compositions d'enzyme de coiffage d'arnm, procédés et nécessaires Pending EP4284922A1 (fr)

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US6312926B1 (en) * 1998-08-14 2001-11-06 University Of Medicine & Dentistry Of New Jersey mRNA capping enzymes and uses thereof
US7074596B2 (en) 2002-03-25 2006-07-11 Board Of Supervisors Of Louisiana State University And Agricultural And Mechanical College Synthesis and use of anti-reverse mRNA cap analogues
EP1893635A4 (fr) * 2005-06-17 2009-02-25 Biorexis Pharmaceutical Corp Librairies de proteines de fusion transferine a ancrage
DE102006061015A1 (de) 2006-12-22 2008-06-26 Curevac Gmbh Verfahren zur Reinigung von RNA im präparativen Maßstab mittels HPLC
JP2014511687A (ja) 2011-03-31 2014-05-19 モデルナ セラピューティクス インコーポレイテッド 工学操作された核酸の送達および製剤
KR20190099538A (ko) 2011-10-03 2019-08-27 모더나 세라퓨틱스, 인코포레이티드 변형된 뉴클레오사이드, 뉴클레오타이드, 및 핵산, 및 이들의 용도
CN104411338A (zh) 2012-04-02 2015-03-11 现代治疗公司 用于产生与人类疾病相关的生物制剂和蛋白质的修饰多核苷酸
WO2014160243A1 (fr) 2013-03-14 2014-10-02 The Trustees Of The University Of Pennsylvania Purification et évaluation de la pureté de molécules d'arn synthétisées comprenant des nucléosides modifiés
WO2014152211A1 (fr) 2013-03-14 2014-09-25 Moderna Therapeutics, Inc. Formulation et administration de compositions de nucléosides, de nucléotides, et d'acides nucléiques modifiés
KR102096796B1 (ko) 2013-10-22 2020-05-27 샤이어 휴먼 지네틱 테라피즈 인크. 메신저 rna의 전달을 위한 지질 제형
WO2015073691A1 (fr) 2013-11-14 2015-05-21 The Board Of Trustees Of The Leland Stanford Junior University Procédés pour traiter le cancer par activation de la signalisation bmp
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WO2021041267A1 (fr) * 2019-08-23 2021-03-04 New England Biolabs, Inc. Procédé de coiffage d'arn enzymatique

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