EP3861108A1 - Procédés et compositions pour augmenter l'efficacité de coiffage d'un arn transcrit - Google Patents

Procédés et compositions pour augmenter l'efficacité de coiffage d'un arn transcrit

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Publication number
EP3861108A1
EP3861108A1 EP19839450.4A EP19839450A EP3861108A1 EP 3861108 A1 EP3861108 A1 EP 3861108A1 EP 19839450 A EP19839450 A EP 19839450A EP 3861108 A1 EP3861108 A1 EP 3861108A1
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EP
European Patent Office
Prior art keywords
guanosine
amino acid
seq
analog
positions
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.)
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EP19839450.4A
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German (de)
English (en)
Inventor
Roy BIJOYITA
Jennifer Ong
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New England Biolabs Inc
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New England Biolabs Inc
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Publication of EP3861108A1 publication Critical patent/EP3861108A1/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)
    • C12N9/1241—Nucleotidyltransferases (2.7.7)
    • C12N9/1247—DNA-directed RNA polymerase (2.7.7.6)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y207/00—Transferases transferring phosphorus-containing groups (2.7)
    • C12Y207/07—Nucleotidyltransferases (2.7.7)
    • C12Y207/07006—DNA-directed RNA polymerase (2.7.7.6)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/31—Chemical structure of the backbone
    • C12N2310/317—Chemical structure of the backbone with an inverted bond, e.g. a cap structure

Definitions

  • Eukaryotic mRNAs have a cap structure at their 5'-termini.
  • the cap consists of 7-methylguanosine (m 7 G) and a triphosphate bridge, ppp (p3), linking the 5 ⁇ H of m 7 G to the 5 ⁇ H of the 5'-terminal nucleotide, N, denoted m 7 G(5')pppN (m 7 G(5')p3N) (Cap 0 structure).
  • the cap structure imparts various biological functions to the capped RNA (Ramanathan et al., (2016) Nucleic Acids Research, 44, 7511-7526). These include mRNA processing and transport; imparting stability to the mRNA molecule; increasing translation efficiency; and acceleration of multiple interactions between the mRNA and the cellular machinery including the translation apparatus, immune receptors and effectors.
  • RNA synthesized via in vitro transcription can be capped during the transcription reaction (in a process called "co-transcriptional capping") or afterwards (i.e., post-transcriptionally using a series of enzymatic steps) (see, e.g., Muttach et al., J. Org Chem. (2017) 13: 2819-2832).
  • co-transcriptional capping a cap analog is included in the IVT reaction along with the four ribonucleotide triphosphates.
  • Cap analogs are synthetic analogs of the N 7 -methylated guanosine triphosphate cap and can add a natural or unnatural cap to the RNA (see Muttach, supra).
  • Co-transcriptional capping methods are limited, however, because the cap analogue competes with GTP as initiator nucleotide in the reaction and, as such, not all mRNA obtained from such IVT is capped.
  • This problem can be mitigated in part using an optimized ratio of cap analog to GTP (i.e., by lowering the GTP concentration) or by digesting uncapped (i.e., triphosphorylated) RNA with a phosphatase which dephosphorylates the 5' end of the RNA (see Muttach, supra).
  • the former solution decreases the overall efficiency of the reaction and the latter solution adds work.
  • neither method solves the problem in a practical way and consequently producing large quantities of homogenously capped RNA remains technically challenging.
  • a method for capping an RNA in an in vitro transcription reaction that includes the steps of:
  • RNA polymerase that comprises: (i) an amino acid sequence that is at least 80% sequence identical to SEQ ID NO:l; and (ii) an amino acid substitution at one or more positions corresponding to positions 388 and 567 of SEQ ID NO:l, to produce a reaction mix; and (b) incubating the reaction mix under conditions suitable for in vitro transcription of the DNA
  • cap analog has the following formula:
  • R3 is selected from guanosine, adenosine, cytidine, uridine, guanosine analog, adenosine analog, cytidine analog, uridine analog;
  • R4 is (NIP) X N 2 wherein Ni and N are ribonucleosides, and Ni is the same or different from N ;
  • p is, independently for each position, a phosphate group, a phosphorothioate, phosphorodithioate, alkylphosphonate, arylphosphonate, or an N-phosphoramidate linkage;
  • x is 0-8 where the ribonucleosides Ni in (Ni-p) x are the same or different from each other if X> 2.
  • R1 and R2 groups are independently selected from O-alkyl (O-methyl), halogen, a tag, hydrogen or a hydroxyl.
  • the tag may be joined directly to the ribose or may be joined via a linker group.
  • the tag may be an affinity binding group such as biotin, desthiobiotin or an oligonucleotide.
  • the tag may be a reactive group.
  • the tag may be a detectable label such as a fluorescent molecule.
  • the sugars in Ni and N may be independently, for each position, selected from ribose and deoxyribose, and may comprise modifications including 2' -O-alkyl, 2'-0-methoxyethyl, 2'-0 allyl, 2'-0 alkylamine, 2'-fluororibose, and 2' -deoxyribose; and/or the bases in Ni and N are independently, for each position, selected from adenine, uridine, guanine, or cytidine or analogs of adenine, uridine, guanine, or cytidine, and nucleotide modifications can be selected from N s -methyladenine, N 1 -methyladenine, N s -2'-0- dimethyladenosine, pseudouridine, N 1 -methylpseudouridine, 5-iodouridine, 4-thiouridine, 2-thiouridine, 5- methyluridine, pseudoisocytosine
  • the cap may be its presence in the mixture as a salt or solvated form.
  • the cap may be a single stereoisomer or plurality of stereoisomers of one or more of the compounds described by Formula 1 or a salt or salts thereof.
  • the cap is added for in vitro transcription. It is well known in the art that an RNA polymerase is agnostic about the cap so that although a variety of caps are described herein, they represent examples and are not intended to be inclusive. The examples the describe the use of m7G-p3-Ai p G 2P G 3P. .. only for convenience and is not intended to be limiting.
  • RNA polymerase variants are provided that are preferably thermostable. More specifically, the RNA polymerase variant may include (i) an amino acid sequence is at least 80% sequence identity to SEQ ID NO:l; and (ii) an amino acid substitution at one or more positions corresponding to 388, and 567 of SEQ ID NO:l.
  • the RNA polymerase may additionally include an amino acid substitution of at least one position or two or all 4 positions corresponding to positions selected from the group consisting of 109, 205, 534, and 618 of SEQ ID NO:l. More specifically, the RNA polymerase variant may include a mutation or mutations corresponding to D388E and/or V567P.
  • the RNA polymerase variant may additionally include an amino acid substitutions at one or more positions or at least 10 positions corresponding to positions selected from the group consisting of: 75, 83, 108, 206, 227, 281, 297, 312, 323, 327, 333, 340, 354, 362, 375, 428, 446, 454, 461, 495, 510, 584, 591, 642, 711, 724, 740, 788, 832, 834, 835, 843, 847, 849, 856, 863, 866 and 877 of SEQ ID NO:l.
  • one or more or ten or more substitutions may include T75Q, A83K, E108L, K206P, V227I, I281P, V297I, Y312D, A323I, A327P, K333P, V340E, A354Q, M362P, T375K, T375N, A428P, L446F, K454P, K461R, S495N, C510Q, A584K, D591E, K642R, K711R, A724P, K740R, G788A, M832F, D834E, T835L, A843Q, D847E, F849V, S856T, A863P, A866K and E877R.
  • RNA polymerase variants may be used in the methods and kits described herein.
  • kits contain separately or together a cap or cap analog according to Formula 1 and an RNA polymerase variant described above plus instructions for use including for achieving at least 95% capping, using an incubation temperature in the range of at least 30°C to about 70°C.
  • the polymerase variant may be fused to an exogenous DNA binding domain.
  • FIG. 1 schematically illustrates how transcription is initiated from a DNA template showing one of the two promoters recognized by T7RNA polymerase. These are duplexes having a 5' TAATACGACTCACTATA (SEQ ID NO:2) sequence (used in FIGs. 1, 2D, 3A, 3B) and the Class II promoter sequence 5' TAATACGACTCACTATT (SEQ ID NO:4) (used in FIG. 3C).
  • FIG. 1 shows how conventional co-transcriptional capping methods can result in a mixture of capped products (which have a 5'- m 7 G -ppp) and uncapped products (which have a 5'-ppp).
  • the RNA polymerase binds the promoter sequence in the DNA template (represented in gray) and initiates transcription from the second strand in a 5' direction at a sequence downstream of the promoter represented by the +1 nucleotide in this schematic diagram.
  • the RNA synthesized from this template by the RNA polymerase in the presence of standard nucleotides only has a sequence of ppp-A +i G + 2....(rest of the RNA).
  • the RNA transcript When the RNA is synthesized from this template by the RNA polymerase in the presence of the trinucleotide cap and standard nucleotides, the RNA transcript should have a 5' sequence of m 7 G -A +I G+2. (rest of the RNA).
  • commercial T7 RNA polymerases provide inefficient 5' m 7 G incorporation resulting in a heterogeneous population of transcripts where some of the RNA transcripts are capped with 5' m 7 GAG and some are uncapped 5'-ppp-AG.
  • FIG. 2A-2D shows chromatograms from liquid chromatography-mass spectrometry (LC-MS) analyses of capped synthetic RNA molecules created by transcription of a 25-base pair template with promoter sequence of (SEQ ID NO:2) T AAT ACG ACT C ACT AT A -A +I G +2 G +3.. N +25 .
  • Wild-type T7 RNA polymerase (WT-T7) was used for transcription at 37°C and a variant of WT-T7 (M20) was used for transcription at 37°C and 50°C.
  • WT-T7 Wild-type T7 RNA polymerase
  • M20 variant of WT-T7
  • transcription product was a 25-mer RNA that was capped (Cap-RNA) or if capping was incomplete,
  • the X-axis denotes the mass of the RNA products detected and Y-axis denotes the intensity of each RNA species.
  • Capping efficiency was measured using the following formula: (intensity of capped peaks)/[(intensity of capped peaks) + (intensity of the ppp peaks)].
  • thermostable polymerase M20 which is a thermostable variant of T7 RNA polymerase that is characterized by amino acid substitutions at positions corresponding to 388 and 567 of the WT-T7 sequence among other mutations (see, e.g., US Patent Application Serial No. 15/594,090, which is incorporated by reference herein) initiates transcription using a cap analog with 100% efficiency at 37°C and 50°C, as opposed to the WT- T7 polymerase, which initiates transcription using a cap analog with only 92% efficiency.
  • FIG. 2A shows the capping efficiency using WT-T7 (reaction temperature 37°C) where peaks were observed corresponding to capped RNA and also to phosphorylated uncapped RNA. 92% of the RNA was capped and 8% was phosphorylated. Several peaks are shown for capped transcripts because of the know phenomenon of addition of a single nucleotide at the 3' end. Flere a 26 th nucleotide may be added at the 3' end of the 25- nucleotide transcript to generate two additional peaks corresponding to an addition of C or an addition of G.
  • WT-T7 reaction temperature 37°C
  • FIG. 2B shows the capping efficiency of using M20-T7 RNA polymerase (reaction temperature 37°C) where peaks were observed corresponding to capped RNA only and none to phosphorylated uncapped RNA. 100% of the RNA was capped.
  • FIG. 2C shows the capping efficiency of using M20-T7 RNA polymerase (reaction temperature 50°C) where peaks were observed corresponding to capped RNA only and none to phosphorylated uncapped RNA. 100% of the RNA was capped.
  • FIG. 2D shows the 5' sequence of the promoter and first 2 nucleotides of a 25-nucleotide transcript associated with a tri-nucleotide cap and a third nucleotide and the products of transcription with (1) 5' cap m 7 G pAG and (2) 5' triphosphorylated AG.
  • FIGs.3A-3C provide capping efficiencies using an m 7 G -ppp-A +i G trinucleotide cap during transcription from templates that contained varying promoter sequences for initiation of transcription or varying the nucleotide at the +3 position on the sequence to be transcribed.
  • the mutant M20-T7 RNA polymerase showed improved capping efficiency regardless of changes in the promoter sequence or in changes to the nucleotide at the +3-position compared with WT-T7.
  • M20-T7 RNA polymerase has greater capping efficiency than the commercial T7 mutant RNA polymerase (Toyobo, Osaka, Japan).
  • FIGs. 3A-3C demonstrate that the commercially available "Toyobo" variant of T7 RNA polymerase (Toyobo, Osaka, Japan) initiates transcription using a cap analog with less than 90% efficiency at 37°C and 50°C (FIG. 3A) compared with the thermostable T7 polymerase M20.
  • Thermostable T7 RNA polymerase M20 initiates transcription using a cap analog with close to 100% efficiency at 37°C and 50°C (FIG. 3B).
  • FIG. 3A provides in tabular form, the capping efficiency comparing T7-WT RNA polymerase (37°C) and Toyobo mutant T7 RNA polymerase (37°C and 50°C) for the same DNA template as FIG. 2A-2C namely a promoter sequence of TAATACGACTCACTATA (SEQ ID NO:2) with an adjacent 25 nucleotides.
  • the transcript sequence starts at the AGG adjacent to the promoter sequence.
  • FIG. 3B provides the capping efficiencies observed using the same promoter sequence as in FIG. 3A but where the transcription start site for the 25 nucleotides adjacent to the promoter on the DNA template is AGA instead of AGG.
  • the results for WT-T7 (37°C) and M20-T7 RNA polymerase (37°C and 50°C) show that M20-T7 RNA polymerase transcribes this template as efficiently as the template in FIG. 3A.
  • FIG. 3C provides the capping efficiencies observed using a different promoter sequence from FIG. 3A and FIG. 3B namely with a promoter sequence of TAATACGACTCACTATT (SEQ ID NO:4) and where the adjacent 25 nucleotides of DNA template for transcription start with AGG.
  • the results for WT-T7 (37°C) and M20-T7 RNA polymerase (37°C and 50°C) show that M20-T7 RNA polymerase transcribes this template as efficiently as the template in FIG. 3A and 3B.
  • FIG. 4A-4C shows that capping efficiency of a 1.7kb functional mRNA is increased when transcription is done with the RNA polymerase variant M20. This data shows that the M20 polymerase initiates transcription of a 1.7Kb mRNA using a cap analog with almost 100% efficiency at 37°C and 50°C.
  • FIG. 4A is a schematic representation of the process involved in measuring the capping efficiency of a 1.7kb long mRNA.
  • the capped RNA is subjected to gel electrophoresis, RNaseH-mediated fragmentation to resolve the 5' end into capped and uncapped products and then subjected to MS analyses for evaluating the capping efficiency.
  • FIG. 4B Gel electrophoresis analyses of the RNase-H treated mRNA showing the presence and separation of the capped and the uncapped (ppp) 5' RNA fragments.
  • FIG. 4C Increased capping efficiency was observed with M20 RNA polymerase variant at 37°C and 50°C as compared to WT-T7 when m 7 G-ppp-A-p-G polynucleotide cap was used in the reaction.
  • Capping efficiency is measured using the following formula: (intensity of capped peaks)/[(intensity of capped peaks) + (intensity of the ppp peaks)].
  • RNA polymerase variants for increasing co-transcriptional capping efficiency of in vitro transcribed RNA is provided using an engineered RNA polymerase variant.
  • Present embodiments utilize any of multiple types of polynucleotide caps as substrates for the polymerase variant for IVT of DNA templates to form capped RNA molecules.
  • IVTT in vitro transcription
  • DNA template refers to a dsDNA molecule that is transcribed in an IVT reaction.
  • DNA templates have a promoter (e.g., a T7, T3 or SP6 promoter) recognized by the RNA polymerase upstream of the region that is transcribed.
  • a promoter e.g., a T7, T3 or SP6 promoter
  • RNA product refers to the product of an IVT reaction.
  • the RNA product of IVT contains a mixture of RNA molecules and, depending on how the transcription is done, may contain double- stranded RNA (dsRNA) molecules.
  • dsRNA double- stranded RNA
  • the molecular events that generate dsRNA molecules in IVT reactions is unknown, but they can be detected using an antibody that is specific for dsRNA or liquid chromatography (e.g., HPLC), for example.
  • variant protein 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%, at least 90%, at least 95%, at least 97%, at least 98% or at least 99% identical to the naturally occurring amino acid sequence.
  • the variant may have one or more amino acid substitutions relative to a wild-type protein.
  • a variant protein may include a "fusion” protein.
  • fusion protein refers to a protein composed of a plurality of polypeptide components that are unjoined in their native state. Fusion proteins may be a combination of two, three or even four or more different proteins.
  • polypeptide includes fusion proteins, including, but not limited to, a fusion of two or more heterologous amino acid sequences, a fusion of a polypeptide with: a heterologous targeting sequence, a linker, an epitope tag, a detectable fusion partner, such as a fluorescent protein, b- galactosidase, luciferase, etc., and the like.
  • a fusion protein may have one or more heterologous domains added to the N-terminus, C-terminus, and or the middle portion of the protein. If two parts of a fusion protein are "heterologous", they are not part of the same protein in its natural state.
  • buffering agent refers to an agent that allows a solution to resist changes in pH when acid or alkali is added to the solution.
  • suitable non-naturally occurring buffering agents include, for example, Tris, HEPES,
  • TAPS TAPS
  • MOPS tricine
  • MES MES
  • pharmaceutical acceptable excipient is any solvent that is compatible with administration to a living mammalian organism via transdermal, oral, intravenous, or other administration means used in the art.
  • pharmaceutical acceptable excipients include those described for example in US 2017/0119740.
  • non-naturally occurring refers to a composition that does not exist in nature.
  • any protein described herein may be non-naturally occurring, where the term "non-naturally occurring” refers to a protein that has an amino acid sequence and/or a post-translational modification pattern that is different from the protein in its natural state.
  • a non-naturally occurring protein may have one or more amino acid substitutions, deletions or insertions at the N-terminus, the C-terminus and/or between the N- and C-termini of the protein.
  • a “non-naturally occurring” protein may have 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%, at least 90%, at least 95%, at least 97%, at least 98% or at least 99% identical to the naturally occurring amino acid sequence.
  • a non-naturally occurring protein may contain an N-terminal methionine or may lack one or more post-translational modifications (e.g., glycosylation, phosphorylation, etc.) if it is produced by a different (e.g., bacterial) cell.
  • non-naturally occurring refers to a nucleic acid that contains: a) a sequence of nucleotides that is different from a nucleic acid in its natural state (i.e., having less than 100% sequence identity to a naturally occurring nucleic acid sequence), b) one or more non-naturally occurring nucleotide monomers (which may result in a non-natural backbone or sugar that is not G, A, T or C) and/or c) 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 of the nucleic acid.
  • modifications e.g., an added label or other moiety
  • non-naturally occurring refers to: a) a combination of components that are not combined by nature, e.g., because they are at different locations, in different cells or different cell compartments; b) a combination of components that have relative concentrations that are not found in nature; c) a combination that lacks something that is usually associated with one of the components in nature; d) a combination that is in a form that is not found in nature, e.g., dried, freeze dried, crystalline, aqueous; and/or e) a combination that contains a component that is not found in nature.
  • a preparation may contain a "non-naturally occurring" buffering agent (e.g., Tris, HEPES, TAPS, MOPS, tricine or MES), a detergent, a dye, a reaction enhancer or inhibitor, an oxidizing agent, a reducing agent, a solvent or a preservative that is not found in nature.
  • a buffering agent e.g., Tris, HEPES, TAPS, MOPS, tricine or MES
  • a detergent e.g., Tris, HEPES, TAPS, MOPS, tricine or MES
  • a dye e.g., Tris, HEPES, TAPS, MOPS, tricine or MES
  • a reaction enhancer or inhibitor e.g., an oxidizing agent, a reducing agent, a solvent or a preservative that is not found in nature.
  • cap analog includes natural caps such as 7 mG and any compound of the general formula
  • R3 is selected from guanosine, adenosine, cytidine, uridine, guanosine analog, adenosine analog, cytidine analog, uridine analog;
  • R4 is (NIP) X N 2 wherein Ni and N are ribonucleosides, and Ni is the same or different from N ;
  • p is, independently for each position, a phosphate group, a phosphorothioate, phosphorodithioate, alkylphosphonate, arylphosphonate, or an N-phosphoramidate linkage;
  • x is 0-8 where the ribonucleosides Ni in (Ni-p) x are the same or different from each other if X> 2.
  • R1 and R2 groups are independently selected from O-alkyl (O-methyl), halogen, a tag, hydrogen or a hydroxyl.
  • Cap analogs are added at the 5' end of an RNA transcript in a process called co-transcriptional capping to yield a 5' capped RNA (see, e.g., Muttach, supra).
  • Cap analogs 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 ⁇ H to the triphosphate bridge. In some dinucleotide cap analogs the 3'-OH group is replaced with hydrogen or OCH 3 (U.S.
  • Dinucleotide cap analogs include m 7 G(5')p 3 G, 3'-OMe-m 7 G(5')p 3 G (ARCA).
  • the term "cap analog” also includes trinucleotide cap analogs (defined below) as well as other, longer, molecules (e.g., cap analog that have four, five or six or more nucleotides joined to the triphosphate bridge).
  • the sugars in Ni and N 2 may be independently, for each position, selected from ribose and deoxyribose, and may comprise modifications including 2' -O-alkyl, 2'-0-methoxyethyl, 2'-0 allyl, 2'-0 alkylamine, 2'-fluororibose, and 2' -deoxyribose; and/or the bases in Ni and N 2 may be independently, for each position, selected from adenine, uridine, guanine, or cytidine or analogs of adenine, uridine, guanine, or cytidine, and nucleotide modifications can be selected from N s -methyladenine, N 1 -methyladenine, N s -2'-0- dimethyladenosine, pseudouridine, N 1 -methylpseudouridine, 5-iodouridine, 4-thiouridine, 2-thiouridine, 5- methyluridine,
  • trinucleotide cap analogs e.g., m 7 G(5')p 3 ApG, m 7 G(5')p 3 AmpG (Am is adenine with a 2'OMe-ribose), m 7 G(5')p 3 m s AmpG (m s A is N s -methyladenine), and m 7 G(5')p 3 m 6 ApG are disclosed by Ishikawa, et al., Nucleic Acid Symp. Ser., 2009 53:129- 30, and many others are described in US 2018/0105551, which publications are incorporated by reference herein.
  • RNA in an IVT reaction co- transcriptionally, i.e., using a cap analog and achieving at least 95% capping efficiency.
  • the method may comprise (a) combining rNTPs, or modified rNTPs, a DNA template, a cap analog and a RNA polymerase that comprises: (i) an amino acid sequence is at least 80% sequence identity to SEQ ID NO:l; and (ii) an amino acid substitution at one or more positions corresponding to positions 388 and 567 of SEQ ID NO:l, to produce a reaction mix; and (b) incubating the reaction mix under conditions suitable for IVT of the DNA template to produce a capped RNA copy of the template.
  • the capping results in a product that is almost completely capped (e.g., at least 90%, at least 98% or at least 99% capped) and, as such, the RNA product can potentially be used without any post-transcriptional enzymatic steps.
  • the polymerase may be thermostable and, as such, the reaction can be done at a temperature that is in the range of 30°C to 70°C, e.g., a temperature of 37°C, a temperature of 50°C or a temperature in the range of 50°C to 65°C.
  • the RNA polymerase (M20) used in the method may have an amino acid sequence with at least 80% sequence identity (e.g., at least 90%, at least 90%, at least 95%, at least 97%, at least 98% or at least 99% identity) to SEQ ID NO:l; and (ii) may comprise one or more (e.g., at least two, at least three, at least five, or at least ten) amino acid substitutions at one or more positions corresponding to positions 75, 83, 108, 109, 205, 206, 227, 281, 297, 312, 323, 327, 333, 340, 354, 362, 375, 388, 428, 446, 454, 461, 495,
  • SEQ ID NO:l (WT-T7), shown below:
  • variants include RNA polymerases with an amino acid substitution at one or more (e.g., at least two, three, four, five or six) positions corresponding to positions selected from 109, 205, 388, 534, 567 and 618 of SEQ ID NO:l.
  • the polymerase may comprise an amino acid substitution at one or both positions corresponding to positions 388 and 567.
  • the RNA polymerase having an amino acid sequence with at least 80% sequence identity (e.g., at least 90%, at least 95%, at least 97%, at least 98% or at least 99% identity) to SEQ ID NO:l; and may include one or more (e.g., at least two, at least three, at least five, or at least ten) of the following amino acid substitutions: T75Q, A83K, E108L, K206P, V227I, I281P, V297I, Y312D, A323I, A327P, K333P, V340E, A354Q, M362P, T375K, T375N, A428P , L446F, K454P, K461R, S495N, C510Q, A584K, D591E, K642R, K711R, A724P, K740R, G788A, M832F, D834E, T835L, A843
  • the variant RNA polymerase may include one or more (e.g., one, two, three, four, five or all six) of the following amino acid substitutions: I109L, FI205S, D388E, L534V, V567P and G618Q, wherein the amino acid substitutions are at positions that correspond to positions in SEQ ID NO:l, as well as well as optionally one or more (e.g., at least two, at least three, at least five, or at least ten) of the following amino acid substitutions: T75Q, A83K, E108L, K206P, V227I, I281P, V297I, Y312D, A323I, A327P, K333P, V340E, A354Q, M362P, T375K, T375N, A428P , L446F, K454P, K461R, S495N, C510Q, A584K, D591E, K642R, K711R
  • the variant RNA polymerase may contain any or all of the features described in US 2017/0247670.
  • the variant RNA polymerase may also be a variant SP6 RNA polymerase or variant T3 RNA polymerase all of which are closely related in sequence function and properties.
  • the method for optimizing the efficiency of capping of an RNA with a cap analog includes forming a mixture of reagents with a DNA template, wherein the reagent mixture includes a mixture of rNTPs, and/or modified nucleotides, a cap analog and an RNA polymerase variant of the type described in US Patent Application Serial No.
  • M20 has at least 80% sequence identity (e.g., at least 90%, at least 95%, or 100% sequence identity) with SEQ ID NO:l, one or both mutations corresponding to position 388 and 567 in SEQ ID NO:l, and potentially other mutations up to and including any, some or all of the mutations described above.
  • At least 95% of the transcript formed in the reaction mixture containing the variant RNA polymerase was capped.
  • the efficiency of capping of a newly formed transcript using the reaction mixture was shown to be significantly greater when the RNA polymerase is mutant T7 compared with WT-T7. Confirmation of improved efficiency of co-transcriptional capping to provide at least 95% capping was also demonstrated using Mass spectrometry (Mass Spec) where the capped to uncapped RNA having 5'ppp was compared.
  • RNA polynucleotide caps can be in a salt or solvated form.
  • RNA polynucleotide caps can be single stereoisomers or a plurality of stereoisomers of one or more of the compounds described by Formula 1 or a salt or salts thereof.
  • the modified cap may include a tag optional attached on the R1 and/or R2 groups by a linker, Examples of tags include detectable labels for example those detected by fluorescence or by color facilitating the detection and quantitation of RNA after transcription.
  • the modified cap may include a binding moiety (such as biotin, desthiobiotin, digoxigenin; groups that form an irreversible bond with a protein tag (benzylguanine or benzylchoropyrimidine (SNAP-tag); benzylcytosine (CLIP-tag); haloalkane (FlaloTag)) or the like to facilitate enrichment leading to for example, identification by size or mass.
  • a binding moiety such as biotin, desthiobiotin, digoxigenin; groups that form an irreversible bond with a protein tag (benzylguanine or benzylchoropyrimidine (SNAP-tag); benzylcytosine (CLIP-tag); haloalkane (Flal
  • One or more components of the transcription reaction may be labeled with a detectable label (such as anthraniloyl group, Alexa Fluor dyes; coumarin dyes, BODIPY dyes, Quantum Dots, ATTO dyes) or marker so that the RNA after can be identified, for example, by size, mass, affinity capture or color.
  • a detectable label such as anthraniloyl group, Alexa Fluor dyes; coumarin dyes, BODIPY dyes, Quantum Dots, ATTO dyes
  • the detectable label is a fluorescent dye; and the affinity capture label is biotin or others.
  • the DNA template may have a sequence consistent with naturally occurring or synthetic mRNA, tRNA, guide RNA, small nuclear RNA (snRNA), small nucleolar RNA (snoRNA), small cajal body-specific RNA (scaRNA).
  • the transcribed RNA may include one or more modified nucleoside monophosphates, one or more modified sugars in addition to the cap polynucleotide structure (Formula 1).
  • the cap polynucleotide can have a structure that resembles either a Cap 0 structure (no methylation of 2OFI of +1 ribose), a Cap 1 structure (methylation of 2OFI of +1 ribose), or a Cap 2 structure (methylation of 2OFI of +2 ribose) similar to natural cap structures.
  • thermostable T7 RNA polymerase variant mediated transcription reaction other enzymes, including natural or mutated variants that may be utilized include, for example, SP6 and T3 RNA polymerases and RNA polymerases from other sources including thermostable RNA polymerases.
  • Kits including, the cap analog and the polymerase variant for performing transcription are also contemplated with one or more of the following reagents: modified or unmodified cap analog, one or more unmodified NTPs, one or more modified NTPs, an RNA polymerase or variant, other enzymes, a reaction buffer, magnesium and a DNA template.
  • the kit may also include instructions for incubating the reaction at a temperature in the range of 30°C to 70°C, e.g., a temperature of 37°C, a temperature of 50°C or a temperature in the range of 37°C-50°C or 50°C to 65°C.
  • the RNA product may encode a protein, e.g., a therapeutic protein or a protein expected to alter the cells into which it is introduced and, as such, the RNA molecules in the RNA product may have a 5' untranslated region (5' UTR), one or more coding sequences, and a 3' translated region (3' UTR), where the 3' and 5' UTRs facilitate translation of the one or more coding sequence to produce a protein within the cells.
  • the RNA product may be a therapeutic RNA.
  • the RNA product may be a guide RNA, a short hairpin RNA, a siRNA, a microRNA, a long noncoding RNA, or a protein coding RNA (which may encode a recombinant protein or a protein that is native to the cells).
  • the RNA product may contain modified nucleotides (triphosphates for which can be added to the IVT reaction).
  • modified nucleotides may be incorporated into the IVT RNA. Incorporation of modified nucleotides can increase in translation efficiency of the RNA and increased stability of the RNA.
  • RNA product may be altered during or after the transcription reaction, e.g., to decrease the rate at which the RNA products are degraded in the cells.
  • the RNA product may contain capped RNAs (see, for example: WO 2016/090262; WO 2014/152673; WO 2009/149253;
  • RNAs with poly A tails of varying length and labeled RNAs can also be produced.
  • the method may further comprise testing or using the RNA product (e.g., administering the RNA to a mammalian cell that in vitro (i.e., grown in culture), ex vivo or in vivo, without performing post -transcriptional enzymatic step that removes ppp-G and adds a m 7 G-ppp to the 5' end of the RNA product.
  • testing or using the RNA product e.g., administering the RNA to a mammalian cell that in vitro (i.e., grown in culture), ex vivo or in vivo, without performing post -transcriptional enzymatic step that removes ppp-G and adds a m 7 G-ppp to the 5' end of the RNA product.
  • the IVT may be done using natural NTPs, i.e., GTP, CTP, UTP and ATP to produce a product that does not contain modified nucleosides.
  • natural NTPs i.e., GTP, CTP, UTP and ATP
  • the IVT may be done using NTPs corresponding to G, C, U and A in the absence of pseudo-uridine triphosphate to produce a product that does not contain pseudo-uridine.
  • 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). In these embodiments, the RNA product may be transfected into the cells. In other embodiments, the cells into which the RNA product is introduced may be in vivo (cells that are part of a mammal). In these embodiments, the introducing may be done by administering the RNA product to a subject in vivo.
  • the cells into which the RNA product is introduced may 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 making a formulation are also provided.
  • the method may comprise combining an RNA product made by transcribing a template DNA as described above with a pharmaceutically acceptable excipient to produce a formulation.
  • the method comprises (a) transcribing a template DNA with the RNA polymerase using the method described above to produce a capped RNA product with or without modifications; and (b) combining the RNA product with a pharmaceutically acceptable excipient; wherein the method is done in the absence of a post-transcriptional capping step.
  • the method may include administering the formulation to a mammalian subject in an effective therapeutic dose, where the subject may be a human or any non-human animal (e.g., mouse, rat, rabbit, dog, cat, cattle, swine, sheep, horse or primate).
  • the capped RNA may be administered to non-mammalian subject or eukaryotic or prokaryotic cells in vivo or in vivo.
  • the capped RNA can either be naked or formulated with a suitable excipient for administration 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
  • the formulations may include
  • RNA in virus particles encapsulating the RNA in virus particles.
  • capped RNA product can be delivered into the cells by packaging them into nanoparticles such as cationic lipids and polymers, non-viral carriers like protamine. Direct introduction of the RNA into the cell using transfection, microinjection, electroporation, sonoporation can also be implemented.
  • the delivery (localized or systemic) and the packaging of the RNA (with or without modifications) can be performed at temperatures optimal for the delivery approach or the formulation used (such as those described in: US 9,629,804 B2; US 2012/0251618 Al; WO 2014/152211; US 2016/0038432 Al; US 2016/0032316 Al; US 9,597,413 B2; US 2012/0258176).
  • compositions provided here can be used for the in vitro synthesis of capped RNA products encoding proteins such as antigens for vaccines, for cancer immunotherapies (such as those described in: US 8,217,016 B2; US 2012/0009221 Al; US 2013/0202645 Al; US 9,587,003 B2; Sahin et.
  • Embodiment 1 A method for capping an RNA in an IVT reaction, comprising:
  • RNA polymerase that comprises: (i) an amino acid sequence that is at least 80% sequence identical to SEQ ID NO:l; and (ii) an amino acid substitution at one or more positions corresponding to positions 388 and 567 of SEQ ID NO:l, to produce a reaction mix; and
  • Embodiment 2 The method of embodiment 1, wherein the cap analog is a dinucleotide cap analog.
  • Embodiment 3. The method of embodiment 1, wherein the cap analog is a trinucleotide cap analog.
  • Embodiment 4. The method of any prior embodiment, wherein the cap analog is of the formula
  • R3 is selected from guanosine, adenosine, cytidine, uridine, guanosine analog, adenosine analog, cytidine analog, uridine analog;
  • R4 is (NIP) X N 2 wherein Ni and N 2 are ribonucleosides, and Ni is the same or different from N 2 ;
  • p is, independently for each position, a phosphate group, a phosphorothioate, phosphorodithioate, alkylphosphonate, arylphosphonate, or an N-phosphoramidate linkage;
  • x is 0-8 where the ribonucleosides Ni in (Ni-p) x are the same or different from each other if X> 2.
  • R1 and R2 groups are independently selected from O-alkyl (O-methyl), halogen, a tag, hydrogen or a hydroxyl.
  • Embodiment 5 The method of embodiment 4, wherein the sugars in Ni and N 2 are independently, for each position, selected from ribose and deoxyribose, and may comprise modifications including 2' -O-alkyl, 2'-0- methoxyethyl, 2'-0 allyl, 2'-0 alkylamine, 2'-fluororibose, and 2' -deoxyribose; and/or the bases in Ni and N 2 are independently, for each position, selected from adenine, uridine, guanine, or cytidine or analogs of adenine, uridine, guanine, or cytidine, and nucleotide modifications can be selected from N s -methyladenine, N 1 - methyladenine, N 6 -2'-0-dimethyladenosine, pseudouridine, N ⁇ methylpseudouridine, 5-iodouridine, 4- thiouridine, 2-thiouridine, 5-
  • Embodiment 6 The method of any prior embodiment, wherein the RNA polymerase comprises an amino acid substitution at positions corresponding to positions 388 and 567 of SEQ ID NO:l.
  • Embodiment 7 The method of any prior embodiment, wherein the RNA polymerase further comprises an amino acid substitution of at least one position corresponding to positions selected from 109, 205, 534, and 618 of SEQ ID NO:l.
  • Embodiment 8 The method of any prior embodiment, wherein the RNA polymerase further comprises an amino acid substitution of at least three two positions corresponding to positions selected from 109, 205, 534 and 618 of SEQ ID NO:l.
  • Embodiment 9 The method of any prior embodiment, wherein the RNA polymerase further comprises an amino acid substitution at positions corresponding to positions 109, 205, 534 and 618 of SEQ ID NO:l.
  • Embodiment 10 The method of any prior embodiment, wherein the RNA polymerase comprises an amino acid sequence that is at least 95% sequence identical to SEQ ID NO:l.
  • Embodiment 11 The method according to any of the previous embodiments, wherein the capping efficiency is greater than 95%.
  • Embodiment 12 The method according to embodiment 9, wherein incubating in step (b) further comprises: performing in vitro transcription at a temperature of at least 30°C to about 70°C.
  • Embodiment 13 A composition comprising:
  • RNA polymerase that comprises: (i) an amino acid sequence that is at least 80% sequence identical to SEQ ID NO:l; and (ii) an amino acid substitution at one or more positions corresponding to positions 388 and 567 of SEQ ID NO:l.
  • Embodiment 14 The composition of embodiment 13, wherein the cap analog is of the formula
  • R3 is selected from guanosine, adenosine, cytidine, uridine, guanosine analog, adenosine analog, cytidine analog, uridine analog;
  • R4 is (Nip) x N wherein Ni and N are ribonucleosides, and Ni is the same or different from N ;
  • p is, independently for each position, a phosphate group, a phosphorothioate, phosphorodithioate, alkylphosphonate, arylphosphonate, or an N-phosphoramidate linkage;
  • x is 0-8 where the ribonucleosides Ni in (Ni-p) x are the same or different from each other if X> 2.
  • R1 and R2 groups are independently selected from O-alkyl (O-methyl), halogen, a tag, hydrogen or a hydroxyl;
  • Embodiment 15 The composition of embodiment 12 or 13, wherein the sugars in Ni and N are independently, for each position, selected from ribose and deoxyribose, and may comprise modifications including 2' -O-alkyl, 2'-0-methoxyethyl, 2'-0 allyl, 2'-0 alkylamine, 2'-fluororibose, and 2' -deoxyribose; and/or the bases in Ni and N are independently, for each position, 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 ⁇ methyladenine, N 6 -2'-0-dimethyladenosine
  • Embodiment 16 The composition of any of embodiments 12 or 14, wherein the cap analog is a dinucleotide cap analog or trinucleotide cap analog.
  • Embodiment 17 The composition of any of embodiments 12-15, wherein the RNA polymerase comprises an amino acid substitution at positions corresponding to positions 388 and 567 of SEQ ID NO:l.
  • Embodiment 18 The composition of any of embodiments 12-16, wherein the RNA polymerase further comprises an amino acid substitution of at least one position corresponding to positions selected from 109, 205, 534, and 618 of SEQ ID NO:l.
  • Embodiment 19 The composition of any of embodiments 11-17, wherein the RNA polymerase comprises an amino acid substitution of at least two or three positions corresponding to positions selected from 109, 205, 534 and 618 of SEQ ID NO:l.
  • Embodiment 20 The composition of any of embodiments 12-18, wherein the RNA polymerase further comprises an amino acid substitution at positions corresponding to positions 109, 205, 534 and 618 of SEQ ID NO:l.
  • Embodiment 21 The composition of any of embodiments 12-18, further comprising a nucleic acid template.
  • Embodiment 22 A kit comprising:
  • RNA polymerase that comprises: (i) an amino acid sequence is at least 80% sequence identity to SEQ ID NO:l; and (ii) an amino acid substitution at one or more positions corresponding to positions 388 and 567 of SEQ ID NO:l; and (iii) instructions for use including for achieving at least 95% capping, using an incubation temperature in the range of at least 30°C to about 70°C.
  • Embodiment 23 The kit of embodiment 22, wherein the cap analog is of the formula: wherein:
  • R3 is selected from guanosine, adenosine, cytidine, uridine, guanosine analog, adenosine analog, cytidine analog, uridine analog;
  • R4 is (NIP) X N 2 wherein Ni and N are ribonucleosides, and Ni is the same or different from N ;
  • p is, independently for each position, a phosphate group, a phosphorothioate, phosphorodithioate, alkylphosphonate, arylphosphonate, or an N-phosphoramidate linkage;
  • x is 0-8 where the ribonucleosides Ni in (Ni-p) x are the same or different from each other if X> 2.
  • R1 and R2 groups are independently selected from O-alkyl (O-methyl), halogen, a tag, hydrogen or a hydroxyl.
  • Embodiment 24 The kit according to embodiment 22 or 23, wherein the sugars in Ni and N are independently, for each position, selected from ribose and deoxyribose, and may comprise modifications including 2' -O-alkyl, 2'-0-methoxyethyl, 2'-0 allyl, 2'-0 alkylamine, 2'-fluororibose, and 2' -deoxyribose; and/or the bases in Ni and N are independently, for each position, 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 ⁇ methyladenine, N 6 -2'-0-dimethyladenosine, pseudouridine, N ⁇ methylpseudouridine, 5- iodouridine, 4-thiouridine, 2-thiouridine, 5-methylur
  • Embodiment 24 The kit of any of embodiment 22 -23, wherein the cap analog is a dinucleotide cap analog or trinucleotide cap analog.
  • Embodiment 25 The kit of any of embodiment 22-24, wherein the RNA polymerase comprises an amino acid substitution at positions corresponding to positions 388 and 567 of SEQ ID NO:l.
  • Embodiment 26 The kit of any of embodiment 22-25, wherein the RNA polymerase further comprises an amino acid substitution of at least one position corresponding to positions selected from 109, 205, 534, and 618 of SEQ ID NO:l.
  • Embodiment 27 The kit of any of embodiment 22-26, wherein the RNA polymerase further comprises an amino acid substitution of at least three two positions corresponding to positions selected from 109, 205, 534 and 618 of SEQ ID NO:l.
  • Embodiment 28 The kit of any of embodiment 22-27, wherein the RNA polymerase further comprises an amino acid substitution at positions corresponding to positions 109, 205, 534 and 618 of SEQ ID NO:l.
  • IVT and synthesis of IVT RNA controls (absent caps)
  • IVT reactions were performed according to the description provided by New England Biolabs, Ipswich, MA, catalog 2017/2018 with WT-T7 and with T7 RNA polymerase from Toyobo using the optimized protocol from Toyobo and T7 RNA variants (M20) using the protocol described for WT-T7 RNA polymerase (New England Biolabs Inc, Ipswich, MA).
  • the double stranded DNA templates for FIG. 1, 2A-2D and 3A-3C were generated by annealing two single-stranded DNA oligonucleotides.
  • 4A-C was double stranded plasmid DNA linearized using restriction endonuclease Notl at a site downstream of the T7 promoter (New England Biolabs Inc, Ipswich, MA,). Reactions were performed at 37°C or 50°C for 1 hour.
  • the RNA products of IVT RNAs were processed through a spin column (Norgen Biotek Corp., Ontario, or MEGACLEARTM, Thermo Fisher Scientific, Waltham, Mass.) to remove unincorporated nucleotides before mass-spectrometry analyses.
  • IVT and synthesis of IVT RNA controls (with caps by co-transcriptional capping)
  • RNA polymerase variants were generated with RNA polymerase variants in presence of trinucleotide caps (Trilink BioTechnologies, San Diego, CA) were processed through a spin column (MEGACLEARTM, Thermo Fisher Scientific, Waltham, Mass.) to remove unincorporated nucleotides.
  • MEGACLEARTM Thermo Fisher Scientific, Waltham, Mass.
  • a 25mer oligonucleotide whose sequence is complementary to the 5' end of the transcribed capped RNA was annealed to the RNA and the annealed oligonucleotide-RNA hybrid was then subjected to RNaseH (New England Biolabs Inc, Ipswich, MA) digestion.
  • reaction products were then separated by gel electrophoresis so that the 25mer double stranded capped RNA was detected in one band on the gel and the uncapped products were observed in a second band as shown in FIG. 4B.
  • the nucleic acid in both bands were separately extracted and subjected to liquid chromatography mass spectroscopy (LC-MS) analyses. Capping efficiency was determined using the following formula (intensity of capped peaks)/[(intensity of capped peak) + (intensity of the ppp peak)].

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Abstract

L'invention concerne des procédés et des compositions pour le coiffage d'ARN dans un mélange de transcription in vitro (TIV) qui comprennent un variant d'ARN polymérase thermostable et un analogue de coiffe, de sorte que, lorsqu'un modèle d'ADN est ajouté au mélange et que le mélange est ensuite incubé dans des conditions de transcription in vitro , un ARN coiffé est produit. Les procédés décrivent des rendements de coiffage élevés à des températures élevées ainsi qu'à des températures standard pour la TIV. L'ARN polymérase utilisé est un variant thermostable de l'ARN polymérase T7, appelé polymérase M20, qui comprend des substitutions aux positions 388 et 567, notamment D388E et V567P.
EP19839450.4A 2018-10-04 2019-10-04 Procédés et compositions pour augmenter l'efficacité de coiffage d'un arn transcrit Withdrawn EP3861108A1 (fr)

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