Method and composition for producing cap1-mrna

Abstract

The invention refers to a method and a composition for synthesizing capped mRNA under conditions allowing for co-transcriptional mRNA capping and methylation of capped mRNA within the same reaction, thereby generating a synthesized capped mRNA, wherein the synthesized capped mRNA has a cap1-type structure.

Description

[0001] METHODS AND COMPOSITIONS FOR PRODUCING CAPPED mRNA

[0002] FIELD OF THE INVENTION

[0003] The invention lies in the field of mRNA synthesis during in vitro transcription (IVT), a procedure allowing for template-directed synthesis of RNA molecules including capping of the synthesized mRNA for increased mRNA stability.

[0004] BACKGROUND

[0005] In nature, eukaryotic messenger RNAs (mRNAs) and many viral RNAs include a 5'-cap structure ("capO") found at the 5' end of the RNA that consists of a N7-methylguanosine nucleoside linked to the 5'-terminal nucleoside of the pre-mRNA via a 5'-5' triphosphate linkage. The "capO" structure is critical for recognition by the translation factor elF4E protein and mRNA translation into proteins by ribosomes, as well as for protection from degradation by 5' exonucleases. Moreover, RNA capping is important for other processes, such as RNA splicing and export from the nucleus.

[0006] Recent studies have revealed the role of a cap 2'0 methylation as an identifier of self RNA to avoid recognition of mRNA by the cellular innate immunity machinery. Such 2'0 methylation is located at the first (+1) nucleotide adjacent to the cap structure at the 5' end of the RNA and is thus called a "capl" structure.

[0007] Owing to the importance of different cap structures for recognition processes in the cell, it becomes clear that an uncapped transcript does not adequately represent a eukaryotic mRNA and that correctly capped RNAs are desirable, e.g., when performing experiments to assess the function of mRNAs in the cellular context. Furthermore, altering the cap structure bears potential to increase mRNA stability and translational efficiency - two properties important in the production of synthetic functional mRNA and its therapeutic applications.

[0008] Preparation of capped mRNA is often achieved by in vitro transcription (IVT) of a DNA template, e.g., using bacteriophage DNA-dependent ribonucleic acid (RNA) polymerases to synthesize template- directed mRNA transcripts. IVT produces uncapped, 5'-triphosphorylated RNA. Such uncapped RNA can be capped during the transcription reaction in a process called "co-transcriptional capping." Alternatively, capO mRNA can be produced by treating uncapped, 5'-triphosphorylated RNA post- transcriptionally, using a series of enzymatic steps, in a process called "enzymatic capping" (see, e.g., Muttach et al., J. Org Chem. (2017) 13: 2819-2832). A common method for enzymatic capping includes a series of three enzymatic reactions in which the 5'-triphosphate terminus of a primary transcript is first cleaved to a diphosphate by RNA triphosphatase, then capped with GMP by RNA guanylyltransferase, and methylated at the N7 position of guanine by RNA (guanine-7)- methyltransferase. In co-transcriptional capping, a cap analog is added to the RNA during the IVT reaction in order to generate capO-type mRNA. CapO-type mRNAs, whether produced by enzymatic or co-transcriptional capping, can be subsequently methylated at the 2'0 position of the nucleotide 3' to the N7-methylguanosine, thereby generating an mRNA having a capl structure. Such protocols are, however, laborious and time consuming.

[0009] The present invention is based in part, on the surprising discovery that capl-type capped mRNA can be achieved in a single reaction, wherein co-transcriptional capping is combined with 2'0 methylation at the +1 position of the RNA in one single reaction. Accordingly, the present invention advantageously enables the production of capped mRNA having a capl structure from DNA in a single reaction step, making the synthesis of capl faster and more economical. Moreover, the yield of capped mRNA having a capl structure is increased using the present inventive methods compared to the two-step protocol widely used in the prior art.

[0010] SUMMARY OF THE INVENTION

[0011] The present invention is defined by the appended claims.

[0012] In a first aspect, the present invention refers to a method of synthesizing capped mRNA. The method includes combining (i) a nucleic acid template comprising a target sequence to be transcribed and an RNA polymerase promoter site operably linked upstream to the target sequence, (ii) an RNA polymerase capable of binding the promoter site, (iii) a transcription buffer, (iv) ribonucleoside triphosphates (NTPs), (v) a dinucleotide cap analog according to Formula I wherein R1is selected from methyl, ethyl, propyl, butyl, benzyl, substituted benzyl, C5-Cg heterocyclyl, C5-Cg substituted heterocyclyl; R2is H, OH or O-CH3; R3is H, OH or O-CH3; R4, R5and R6are independently selected from the group consisting of O, S, Se; R7and R8are OH; n is 1, 2, or 3; and B is a non-modified or modified purine or pyrimidine base, (vi) a 2'-O-methyltransferase, and (vii) S- adenosylmethionine (SAM) to produce a composition; and incubating the composition under conditions allowing for co-transcriptional mRNA capping and methylation of capped mRNA within the same reaction, thereby generating a synthesized capped mRNA, wherein the synthesized capped mRNA has a capl-type structure.

[0013] The components of the composition are combined in a single vessel.

[0014] The composition produced is an IVT reaction mixture. In an embodiment, an in vitro transcription (IVT) of the nucleic acid template is performed during the incubation step.

[0015] In an embodiment, the composition may further comprise an RNase inhibitor. In an embodiment, the composition may further comprise a pyrophosphatase. In an embodiment, the composition may further comprise polyethylene glycol (PEG). In a preferred embodiment, the composition may further comprise an RNase inhibitor, a pyrophosphatase, and / or PEG.

[0016] In another embodiment, at least one of R2or R3of the dinucleotide cap analog is O-CH3. Preferably, R2may be OH and R3may be O-CH3.

[0017] In an embodiment, IVT, co-transcriptional mRNA capping and methylation of capped mRNA may be performed simultaneously.

[0018] In an embodiment, the methylation of capped mRNA is performed without prior mRNA purification.

[0019] The RNA polymerase may be selected from the group comprising T7, SP6, or T3, Vsw-3, Kll, T3D, T5, T8, (02.5, (06 RNA polymerase or a functional variant thereof, or any combination thereof. In a preferred embodiment, the RNA polymerase is a T7, SP6, or T3 RNA polymerase. Alternatively, the RNA polymerase is a T7 RNA polymerase.

[0020] The GTP may be present in a ratio GTP: dinucleotide cap analog from 1:1 to 1:10. The GTP may be present in a ratio GTP: dinucleotide cap analog from 1:1 to 1:8. The GTP may be present in a ratio GTP: dinucleotide cap analog of 1:4.

[0021] The ribonucleotides may comprise ATP, CTP, UTP and GTP, or any modified ribonucleotide. The ribonucleotides may alternatively be ATP, CTP, UTP and GTP.

[0022] The nucleic acid template may be a DNA template selected from the group consisting of a plasmid, a PCR product, cDNA, a double-stranded (ds)oligonucleotide, or a dsDNA comprising covalently closed ends. The nucleic acid template may be a plasmid. The nucleic acid template may be a PCR product. The nucleic acid template may be a cDNA. The nucleic acid template may be a (ds)oligonucleotide. The nucleic acid template may be a dsDNA comprising covalently closed ends. In an embodiment, the nucleic acid template is a combination of two or more types of nucleic acid template selected from the group consisting of a plasmid, a PCR product, cDNA, a double-stranded (ds)oligonucleotide, or a dsDNA comprising covalently closed ends.

[0023] In an embodiment, the nucleic acid template is immobilized on a solid support.

[0024] The nucleic acid template may further comprise a plurality of T nucleotides downstream of the target sequence to be transcribed. In an embodiment, the plurality of T nucleotides comprises modified nucleotides or analogues thereof.

[0025] The composition produced in the method may further comprise a poly-A polymerase.

[0026] The transcription buffer can include a buffering agent, magnesium salts and optionally other salts, a reducing agent and / or additional components. The transcription buffer may comprise MgCL. The transcription buffer may comprise DTT. The transcription buffer may comprise spermidine. The transcription buffer may comprise NaCI. In another embodiment, the transcription buffer comprises MgCh, DTT and spermidine. In another embodiment, the transcription buffer comprises NaCI, MgCL, DTT and / or spermidine.

[0027] The 2'0-methyl transferase may be derived from vaccinia virus.

[0028] In an embodiment, the yield of mRNA comprising capl-type structure is in the range of 95-100%. In other words, the %-amount of synthesized mRNA comprising capl-type structure is in the range of 95-100%. In yet other words, in an embodiment, greater than 95% of the synthesized mRNA comprises a capl-type structure.

[0029] The 2'-O-methyltransferase and / or RNA polymerase may be immobilized on a solid support.

[0030] In an embodiment, the method is an in vitro method.

[0031] In another aspect, the invention refers to a composition for use in the synthesis of capl-type mRNA from a nucleic acid template, the composition comprising RNA polymerase, transcription buffer, ribonucleoside triphosphates (NTPs), dinucleotide capping primer according to Formula I wherein wherein R1is selected from methyl, ethyl, propyl, butyl, benzyl, substituted benzyl, C5-Cg heterocyclyl, C5-Cg substituted heterocyclyl; R2is H, OH or O-CH3; R3is H, OH or O-CH3; R4, R5and R6are independently selected from the group consisting of O, S, Se; R7and R8are OH; n is 1, 2, or 3; and B is a non-modified or modified purine or pyrimidine base; the composition further comprises a 2'-O- methyltransferase, and S-adenosylmethionine (SAM) in one vessel.

[0032] In an embodiment, the composition is suitable for long term storage. In an embodiment, the composition is stable for about 1-10 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 4 months, 1-5 months, 1-6 months.

[0033] In an embodiment, the composition may further comprise an RNase inhibitor. In an embodiment, the composition may further comprise a pyrophosphatase. In an embodiment, the composition may further comprise polyethylene glycol (PEG). In a preferred embodiment, the composition may further comprise an RNase inhibitor, a pyrophosphatase, and / or PEG.

[0034] In another embodiment, at least one of R2or R3of the dinucleotide cap analog is O-CH3. Preferably, R2may be OH and R3may be O-CH3.

[0035] The RNA polymerase may be selected from the group comprising T7, SP6, or T3, Vsw-3, Kll, T3D, T5, T8, (02.5, (06 RNA polymerase or a functional variant thereof, or any combination thereof. In a preferred embodiment, the RNA polymerase is a T7, SP6, or T3 RNA polymerase. Alternatively, the RNA polymerase is a T7 RNA polymerase.

[0036] The GTP may be present in a ratio GTP: dinucleotide cap analog from 1:1 to 1:10. The GTP may be present in a ratio GTP: dinucleotide cap analog from 1:1 to 1:8. The GTP may be present in a ratio GTP: dinucleotide cap analog of 1:4.

[0037] The ribonucleotides may comprise ATP, CTP, UTP and GTP, or any modified ribonucleotide. The ribonucleotides may alternatively be ATP, CTP, UTP and GTP.

[0038] The composition may further comprise a poly-A polymerase.

[0039] The transcription buffer comprises a buffering agent, magnesium salts and optionally other salts, reducing agent and / or additional components. The transcription buffer may comprise MgCL. The transcription buffer may comprise DTT. The transcription buffer may comprise spermidine. The transcription buffer may comprise NaCI. In another embodiment, the transcription buffer comprises MgCL, DTT and spermidine. In another embodiment, the transcription buffer comprises NaCI, MgCL, DTT and / or spermidine.

[0040] The 2'0-methyl transferase may be derived from vaccinia virus.

[0041] In yet another aspect, the present invention refers to the use of the above described composition for the synthesis of mRNA therapeutics. In another aspect, the invention refers to the use of the above described composition for the synthesis of mRNA in a bioreactor.

[0042] In another aspect, the invention refers to the use of the above described composition in an RNA printer.

[0043] In another aspect, the invention refers to the of the above described composition for the synthesis of mRNA in a continuous flow device or a flow reactor.

[0044] DETAILED DESCRIPTION

[0045] 1. Definitions

[0046] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

[0047] Numerical ranges include the numbers defining the range. All numbers are understood to encompass the midpoints of integers above and below the integer, i.e., number 2 encompasses 1.5- 2.5. The number 2.5 encompasses 2.45-2.55 etc. When sample values are provided, unless otherwise specified, for example, each individually can represent an intermediate value in a range of values, and together can represent an extreme value in the range.

[0048] As used herein, the articles "a", "an", and "the" relate equivalently to a meaning as singular or plural unless the context dictates otherwise.

[0049] As used herein and as conventionally understood by those skilled in the relevant art, a "nucleotide" comprises a base, a sugar and one or more phosphate groups. The base (also referred to as a "nitrogenous base" or a "nucleobase") is typically a purine or pyrimidine. The sugar is typically a five- carbon ribose (as in ribonucleotides) or a 2-deoxyribose (as in deoxyribonucleotides), which is bound via a glycosidic linkage to the base. Nucleotides typically have one, two or three phosphate groups (mono-, di- or tri-phosphates). Generally, the phosphate groups form a chemical bond at the 5- carbon position of the sugar, although they can also bond at the 2 or 3-carbon positions of the sugar group. Cyclic nucleotides form when a phosphate group is bound to two hydroxyl groups on the sugar. A "nucleoside" comprises a nucleobase and sugar. A nucleotide can thus also be called a nucleoside mono-, di- or tri-phosphate.

[0050] As used herein, the term "RNA product" refers to the mRNA product of an IVT reaction.

[0051] As used herein, the terms "polynucleotide", "oligonucleotide" and "nucleic acid' are used interchangeably and refer to single stranded and double stranded polymers or oligomers of nucleotide monomers or modified monomers, including ribonucleotides (RNA) and 2'- deoxyribonucleotides (DNA) linked by internucleotide phosphodiester bond linkages or modifications thereof. A polynucleotide may be composed entirely of deoxyribonucleotides, entirely of ribonucleotides or chimeric mixtures thereof.

[0052] As used herein, the term "messenger RNA" (mRNA) refers to any polynucleotide which encodes at least one peptide or polypeptide of interest and which is capable of being translated to produce the encoded peptide or polypeptide of interest in vitro, in vivo, in situ or ex vivo. An mRNA has been transcribed from a DNA sequence by an RNA polymerase enzyme, and interacts with a ribosome to synthesize genetic information encoded by DNA. mRNA, within the meaning of this disclosure, is synthesized in a cell-free environment by in vitro transcription (IVT). As used herein, the term "DNA template" is interchangeable with the term "nucleic acid template", "polynucleotide template" or "in vitro transcription template" and refers to a preferably doublestranded deoxyribonucleic acid (dsDNA) suitable for use in an IVT reaction for the production of mRNA, i.e., the DNA template can be transcribed in an IVT reaction. DNA templates have a promoter (e.g., a T7, T3 or SP6 promoter) recognized by a cognate RNA polymerase upstream of the region that is transcribed. The skilled person will appreciate that nucleic acid templates can take many forms that are useful in the compositions and methods provided herein, including but not limited to linearized plasmid DNA, synthetic oligonucleotides, PCR products, cDNA and the like, provided they contain a functional promoter region in the correct orientation. Double stranded linear DNA with blunt or 5'-protruding ends or 3'-protruding ends may be used as template for in vitro transcription. In some cases, the DNA template can be partially double stranded, as provided in Cazenave C and Uhlenbeck OC (1994) "RNA Template-Directed RNA Synthesis by T7 RNA Polymerase", Proc. Natl. Acad. Sci. USA 91:6972-6976. In some embodiments, an IVT template encodes a 5' untranslated region, contains an open reading frame, and encodes a 3' untranslated region and a poly(A / T) tail. The particular nucleotide sequence composition and length of an IVT template will depend on the mRNA of interest encoded by the template. For example, the template can be double-stranded and have a coding and non-coding nucleic acid strand, wherein the mRNA is the equivalent of the coding strand (also often referred to as sense strand or plus strand) and is transcribed from the non-coding template strand (also often referred to as antisense or minus strand).

[0053] A "5' untranslated region (UTR)" refers to a region of an mRNA that is directly upstream (i.e., 5') from the start codon (i.e., the first codon of an mRNA transcript translated by a ribosome) and that does not encode a protein or peptide.

[0054] A "3' untranslated region (UTR)" refers to a region of an mRNA that is directly downstream (i.e., 3') from the stop codon (i.e., the codon of an mRNA transcript that signals a termination of translation) and that does not encode a protein or peptide.

[0055] An "open reading frame" is a continuous stretch of nucleic acid beginning with a start codon and ending with a stop codon and encodes a protein or peptide. Nucleic acid can be DNA or RNA.

[0056] A "poly(A) tail or stretch" is a region of the mRNA that is downstream, e.g., directly downstream (i.e., 3'), from the 3' UTR that contains multiple, consecutive adenosine monophosphates. A poly(A) tail may contain 10 to 300 adenosine monophosphates. It is noted that the complementary template nucleic acid strand in this region comprises poly(T) tail or stretch.

[0057] As used herein, the term "in vitro transcription" (IVT) refers to a cell-free reaction in which at least partially double-stranded DNA (dsDNA) template is transcribed by a DNA-directed RNA polymerase to produce a mRNA product that contains the genetic information of the template.

[0058] "IVT mRNA" disclosed herein may function as mRNA but are distinguished from wild-type mRNA in their functional and / or structural design features which serve to overcome existing problems of effective polypeptide production. IVT mRNA may be structurally modified or chemically modified. For example, the polynucleotide "ATCG" may be chemically modified to "AT-5meC-G". The same polynucleotide may be structurally modified from "ATCG" to "ATCCCG".

[0059] As used herein, when referring to a polynucleotide, the terms "chemical modification" or, as appropriate, "chemically modified" or alternatively "modified base" refer to modifications with respect to adenosine (A), guanosine (G), uridine (U), thymidine (T) or cytidine (C) ribo- or deoxyribnucleosides and / or the internucleoside linkages in one or more of their position, pattern, percent or population. The modifications may be one or more various distinct modifications. In some embodiments, the regions may contain one, two, or more (optionally different) nucleoside or nucleotide modifications and / or internucleoside linkages. In some embodiments, a modified polynucleotide introduced to a cell may exhibit reduced degradation in the cell as compared to an unmodified polynucleotide.

[0060] As used herein, the term "buffer" or "buffering agent", refers to an agent that allows a solution to resist changes in pH when acid or alkali is added to the solution. Examples of suitable buffering agents that may be used in the methods and compositions of the invention include, for example, Tris, HEPES, TAPS, MOPS, tricine, or MES.

[0061] The term "reaction buffer" refers to the composition of components where the respective enzymatic reaction takes place. In addition to buffering agent, the reaction buffer may also contain one or more of salts, detergents, antifoaming agents, cofactors, reducing agents, additives, etc.

[0062] The term "cap" refers to a non-extendible dinucleotide that facilitates translation or localization, and / or prevents degradation of an RNA transcript when incorporated at the 5' end of an RNA transcript, typically having an m7GpppG or m7GpppA structure.

[0063] The term "dinucleotide cap analog" as used in the present application, refers to a non-extendible dinucleotide that facilitates translation or localization, and / or prevents degradation of an RNA transcript when incorporated at the 5' end of an RNA transcript, typically having an m7GpppG or m7GpppA structure, for example, m7G(5')p3(5')G, in which a guanine nucleotide (G) is linked via its 5'OH to the triphosphate bridge. In some dinucleotide cap analogs the 3'-OH group is replaced with hydrogen or OCHa.The dinucleotide cap analog used in the present invention refers to a compound of Formula I, disclosed herein, such as for example, 3'-OMe-m7G(5')p3G (ARCA).

[0064] The term "capO" is interchangeable with "capO-type", "cap-0", "capO structure" or"cap-0 structure" and refers to an (eukaryotic) mRNA cap structure at its 5'-terminus. The cap consists of 7- methylguanosine (m7G) and a triphosphate bridge, ppp (ps), linking the 5'OH of m7G to the 5'OH of the 5'-terminal nucleotide, N, denoted m7G(5')pppN (or m7G(5')p3N, or m7GpppN) (capO structure).

[0065] The term "capl" is interchangeable with "capl-type", "cap-1", "capl structure" or "cap-1 structure", and refers to a capped mRNA having an additional methylation on the 2'0 position of the initiating nucleotide, i.e. the first nucleotide of the transcribed mRNA. Capl refers to m7GpppNm-, where Nm denotes any nucleotide with a 2'0 methylation (capl structure).

[0066] The "Me" is equivalent to "-CH3", i.e. "-OCH3" or "-OMe" and is used as synonyms, and letter "m" denotes a methyl group bound to oxygen atom. Similarly, "Nm" denotes any nucleotide (N) with a 2'0 methylation.

[0067] As used herein, the term "ARCA" or "anti-reverse cap analog" refers to a modified cap analog in which the 3'0H group of the m7G is replaced with OCH3 (please see the structural formula below). The structure is represented as m27,3 OGpppG or m27,3 O(5')Gppp(5')G. Alternatively, it refers to a modified cap analog in which the 2'0H group is replaced with 0CH3. The structure is represented as m27-2' °GpppG or m27-2' °(5')Gppp(5')G.

[0068] ARCA structure

[0069]

[0070] 2. Figures

[0071] Fig. 1 depicts Mass Spectrometry (MS) analysis of 25 nt RNA vs 26 nt ARCA-RNA (ARCA dinucleotide is incorporated instead of a conventional nucleotide, therefore the +1 nt length). 26 nt IVT product ARCA-RNA mass spectrum (top) compared to uncapped RNA (without ARCA, bottom) spectrum. The difference in highlighted m / z value depicted on the right hand side directly above and below the horizontal line, is 293.1, corresponding to ARCA incorporation.

[0072] Fig. 2 shows MS of 26 nt ARCA-RNA vs 2'-0Me-ARCA-RNA. IVT product ARCA-RNA mass spectrum (top) compared to ARCA-RNA treated with 2OMt and SAM (bottom). The difference in m / z value is 14, corresponding to a methyl group.

[0073] Fig. 3 MS of Sequential reaction. ARCA-RNA (top) compared to a sequential reaction product of IVT and 2OMt treatment (bottom), as described in the method. The difference in m / z value between major peaks corresponds to 2'0-methyl functionality.

[0074] Fig. 4 MS of Add-on reaction. ARCA-RNA (top) compared to the Add-on reaction product of IVT and 2OMt treatment (bottom), as described in Example 2. The difference in m / z value between major peaks corresponds to 2'0-methyl functionality.

[0075] Fig. 5 MS of One-pot reaction. ARCA-RNA (top) compared to the One-pot reaction product of IVT and 2OMt treatment of the present invention. The difference in m / z value between major peaks corresponds to 2'0-methyl functionality.

[0076] Fig. 6 provides a general investigational approach scheme, where the differences between Sequential, Add-on and One-pot reaction are provided. The generalized workflow scheme depicts Sequential addition of 2OMt with an intermediary IVT product purification, Add-on reaction with 2OMt and SAM added to the IVT reaction, and One-pot approach, combining all components at once to produce a capl structure in the same reaction mixture from the beginning.

[0077] Fig. 7 One-pot capping and methylation analysis. Mass spectrometry data analysis comparing the relative abundance of species of corresponding m / z values. Capping efficiency is determined by comparing capped RNA and uncapped RNA. Methylation efficiency is determined by comparing capped RNA and capped, methylated RNA. The methylated fraction is determined by comparing the capped RNA fraction to capped, methylated RNA fraction of the mass spectrum.

[0078] Fig. 8 Comparison of capping efficiency and ARCA methylation.

[0079] 3. Methods

[0080] The method of the invention is based on an in vitro transcription reaction. In vitro transcription (IVT) is a procedure that allows for template-directed synthesis of RNA molecules of any sequence of lengths varying from short oligonucleotides to several kilobases in pg to mg quantities. IVT is based on the engineering of a template that includes a promoter sequence (e.g. from the T7 coliphage) upstream of the sequence of interest followed by transcription using a cognate RNA polymerase. By way of example only, a T7 promoter, for instance, covers the sequence ranging from -17 to +6 with +1 being the first nucleotide of the transcribed region. In IVT, a DNA-dependent ribonucleic acid (RNA) polymerase synthesizes template-derived mRNA transcripts. For in vitro transcripts, transcription termination usually intervenes by "run off," that is when the polymerase falls off at the very end of the template. For PCR and oligo templates transcription termination is defined by the ends of the template products. For plasmid-based templates, this is achieved by linearizing the plasmid by restriction enzyme digestion downstream from the sequence of interest, e.g. as described in Thermo Fisher, 2012, "DNA Template Preparation for in vitro Transcription".

[0081] Generally, there are two options for adding a cap structure to uncapped RNA transcripts generated from IVT; namely post-transcriptional capping and co-transcriptional capping. In post-transcriptional capping, uncapped RNA from IVT is subjected to a dedicated enzymatic capping reaction, i.e., the transcription reaction and the capping reaction occur sequentially, and most often in separate reactions requiring different buffering conditions. The resulting capped mRNA has a cap-0 structure. In co-transcriptional capping, cap analogues are added directly to the IVT reaction, i.e., mRNA cap is incorporated into the mRNA by the RNA polymerase as it transcribes the template nucleic acid. Accordingly, transcription and capping take place in the same reaction vessel and do not require, e.g., buffer exchange or "clean up," which often results in lower yields. Incorporation of dinucleotide cap analogs m7GpppN at the 5'-end by RNA polymerases directly yields the respective 5'-capped mRNA (capO structure that does not contain the 2'-0Me group in the first transcribed nucleotide.). Co-transcriptional capping with m7GpppNmN-derived trinucleotide generates the cap-1 structure (m7GpppNm, Nm is a 2'-O-methylated nucleotide), in which the first transcribed nucleotide has the 2'-O-methyl group. Independent from their way of synthesis, capO transcripts can be further enzymatically converted to mRNAs with a cap-1 structure in a subsequent reaction (please see the exemplary structure below).

[0082] Exemplary CapO, Capl and Cap2 structures:

[0083] Therefore, conventional mRNA synthesis consists of at least two reactions, transcription comprising co-transcriptional capping and the 2'0 methylation of the +1 nucleotide making the process error- prone and time consuming. Prior art additionally recommends to purify capped mRNA prior to setting the methylation reaction. The present invention provides an improved method to synthesize capped mRNA having a capl structure compared to methods known in the art.

[0084] Provided herein is a method for synthesizing capped mRNA, wherein transcription, co-transcriptional capping and 2'0 methylation is performed in one reaction. In other words, a "one pot" solution is provided for the synthesis of capped mRNA from DNA, the cap having a capl structure.

[0085] In yet other words, the present invention provides for a simple and rapid method of capped mRNA synthesis with a maximum yield of capped mRNA (capl structure) of preferably 99% to 100%. Thus, the present method achieves higher yields of successfully capped mRNA (capl structure) then methods known from the art.

[0086] Thus, in a first aspect, the invention relates to a method of synthesizing capped mRNA. The method comprises providing a nucleic acid template comprising a target sequence to be transcribed and an RNA polymerase promoter site operably linked upstream to the target sequence. The method comprises providing nucleic acid template, an RNA polymerase capable of binding the promoter site, a transcription buffer, ribonucleotide triphosphates (NTPs), a 2'-O-methyl transferase, S-adenosyl methionine (SAM) and a dinucleotide cap analog according to Formula I wherein R1is selected from methyl, ethyl, propyl, butyl, benzyl, substituted benzyl, C5-Cg heterocyclyl, C5-Cg substituted heterocyclyl; R2is H, OH or O-CH3; R3is H, OH or O-CH3; R4, R5and R6are independently selected from the group consisting of O, S, Se; R7and R8are OH; n is 1, 2, or 3; and B is a non-modified or modified purine or pyrimidine base. An in vitro transcription (IVT) of the nucleic acid template is performed under conditions allowing for co-transcriptional mRNA capping and methylation of capped mRNA within the same reaction to generate a synthesized capped mRNA, wherein the synthesized capped mRNA has a capl-type structure.

[0087] Preferably, IVT, co-transcriptional mRNA capping and methylation of capped mRNA are performed simultaneously. In some instances, certain components are added again during the process, e.g., in a fed-batch reaction. In some instances, NTPs, SAM and / or reaction buffer are added during the process.

[0088] RNA in vitro transcription reactions are usually performed as batch reactions in which all components are combined and then incubated to allow the synthesis of RNA molecules until the reaction terminates. Additionally, fed-batch reactions were developed to increase the efficiency of the RNA in vitro transcription reaction (Kern et al. (1997) Biotechnol. Prog. 13: 747-756; Kern et al. (1999) Biotechnol. Prog. 15: 174-184). In a fed-batch system, all components are combined, but then additional amounts of some of the reagents are added over time (e.g., NTPs, magnesium salts, etc.) to maintain constant reaction conditions. The one pot solution of the invention thus allows for large scale batch and fed-batch reactions.

[0089] In addition, the use of a transcription bioreactor for the synthesis of RNA molecules by in vitro transcription has been reported (WO 95 / 08626). The bioreactor is configured such that reactants are delivered via a feed line to the reactor core and RNA products are removed by passing through an ultrafiltration membrane (having a nominal molecular weight cut-off, e.g., 100,000 Da) to the exit stream.

[0090] A. Nucleic acid template

[0091] Target sequence

[0092] The nucleic acid template comprises a target sequence to be transcribed and an RNA polymerase promoter site operably linked upstream to the target sequence. In a preferred embodiment, the nucleic acid template is a linear template DNA. In an embodiment, the nucleic acid template for transcription is a DNA template selected from the group comprising a plasmid, a PCR product, cDNA, a double-stranded oligonucleotide or a double-stranded DNA comprising covalently closed ends, comprising at least one covalently closed end, dumbbell-shaped DNA, circular DNA, doggybone DNA (dbDNA) and the like. Preferably, the nucleic acid template for transcription is a DNA template selected from the group consisting of a plasmid, a PCR product, cDNA, a double-stranded oligonucleotide or a double-stranded DNA comprising covalently closed ends. In an embodiment, the nucleic acid template for transcription is a plasmid. In an embodiment, the nucleic acid template for transcription is a PCR product. In an embodiment, the nucleic acid template for transcription is a cDNA. In an embodiment, the nucleic acid template for transcription is a (ds)oligonucleotide. In an embodiment, the nucleic acid template for transcription is a dsDNA with covalently closed ends.

[0093] The target sequence may be any sequence comprising an RNA coding sequence and / or an open reading frame encoding at least one peptide or polypeptide. In an embodiment, the target sequence may be any sequence comprising an RNA coding sequence and / or an open reading frame encoding at least one polypeptide or protein of interest. The particular nucleotide sequence composition and length of the nucleic acid template depends on the mRNA of interest encoded by the template. In some embodiments, the DNA template includes a 5' and / or 3' untranslated region. In some embodiments, the nucleic acid template comprises in addition to an RNA polymerase promoter site a 5' untranslated region, an open reading frame, a 3' untranslated region and a poly A tail. In some embodiments, the nucleic acid template comprises in addition to an RNA polymerase promoter site an open reading frame and a poly A tail.

[0094] In some embodiments, the DNA sequence to be transcribed may be optimized to facilitate more efficient transcription and / or translation. For example, the DNA sequence may be optimized regarding cis-regulatory elements (e.g., TATA box, termination signals, and protein binding sites), artificial recombination sites, chi sites, CpG dinucleotide content, negative CpG islands, GC content, polymerase slippage sites, and / or other elements relevant to transcription; the DNA sequence may be optimized regarding cryptic splice sites, mRNA secondary structure, stable free energy of mRNA, repetitive sequences, RNA instability motif, and / or other elements relevant to mRNA processing and stability; the DNA sequence may be optimized regarding codon usage bias, codon adaptability, internal chi sites, ribosomal binding sites (e.g., IRES), premature polyA sites, Shine-Dalgarno (SD) sequences, and / or other elements relevant to translation; and / or the DNA sequence may be optimized regarding codon context, codon-anticodon interaction, translational pause sites, and / or other elements relevant to protein folding. Optimization methods known in the art may be used in the present invention, e.g., GeneOptimizer by ThermoFisher

[0095] In an embodiment, the nucleic acid template for transcription is a linearized plasmid that typically has the promoter for in vitro transcription immediately upstream from a polylinker for cloning the sequence to be transcribed. The plasmid may be a plasmid cloning vector having one or more promoters for in vitro transcription upstream of multiple cloning sites (MCS) bearing the target sequence to be transcribed. An exemplary plasmid useful in the compositions and methods provided herein is pBlusecript (Stratagene) or a pGEM (Promega). Alternatively, the plasmid may be a plasmid expression vector comprising a DNA fragment including an RNA polymerase promoter (e.g., T7) immediately 5' of the sequence to be transcribed. Exemplary plasmids are pUC18, pUC19 and pBR322. In an embodiment, the nucleic acid template is a linearized pUC18 or a pUC19 or a pBR322 plasmid comprising a DNA fragment including an RNA polymerase promoter (e.g., T7) immediately 5' of the sequence to be transcribed. A specific embodiment is plasmid pTZ19R / Hincll, wherein the phage fl ori and the T7 polymerase promoter are inserted into pUC19, linearized by Hindi restriction.

[0096] In another embodiment, the nucleic acid template for transcription is a PCR product that has the promoter (e.g., T7 promoter) as part of the 5' -oligonucleotide used in the PCR reaction.

[0097] In another embodiment, the nucleic acid template for transcription is a double-stranded oligonucleotide that carries the T7 promoter sequence and the template to be transcribed.

[0098] In some embodiments a double-stranded oligonucleotide template may be synthesized de novo by chemical or enzymatic synthesis using methods known in the art such as those described for example in WO2017062343 Al.

[0099] In another embodiment, the nucleic acid template for transcription is a cDNA that carries the T7 promoter sequence and the template to be transcribed.

[0100] RNA polymerase promoter site

[0101] The promoter comprised in the nucleic acid template for controlling in vitro transcription can be any promoter for any DNA dependent RNA polymerase. Promoter sequences are nucleic acid sequences (either naturally occurring, produced synthetically or a product of a restriction digest) that are specifically recognized by an RNA polymerase that recognizes and binds to that sequence and initiates the process of transcription whereby RNA transcripts are generated.

[0102] Preferably, the promoter sequence has high binding affinity for its respective RNA polymerase, preferably bacteriophage-derived RNA polymerases. In an embodiment, the promoter is a bacteriophage-derived promoter. In preferred embodiments the promoter is a T7, T3 or SP6 RNA polymerase promoter. In an embodiment, the promoter is a T7 RNA polymerase promoter. In an embodiment, the promoter is a T3 RNA polymerase promoter. In an embodiment, the promoter is an SP6 RNA polymerase promoter. In another embodiment, the promoter is derived from E. coli. In yet another embodiment, the promoter is a G-initiating promoter. In yet another embodiment, the promoter is an A-initiating promoter.

[0103] The RNA polymerase promotor site is linked upstream to the target sequence. Linking such promoter site to a target sequence is routine in the art.

[0104] Poly(A / T) seguence

[0105] In an embodiment, the nucleic acid template further comprises a plurality of thymidine nucleotides downstream of the target sequence to be transcribed. In an embodiment, the nucleic acid template is a DNA template comprising a plurality of thymidine nucleotides downstream of the target sequence to be transcribed. This means that the double-stranded DNA template contains a poly (A / T) sequence downstream of the target sequence (i.e. the coding / sense strand comprises a poly A sequence and the non-coding / antisense or template strand comprises a poly T sequence). One conventional method of integration of poly (A / T) stretches into a DNA template is molecular cloning by adding the poly-A / T stretch to the end of the IVT template sequence to be transcribed. Other methods are known to the person skilled in the art. The nucleic acid template strand may comprise at least 50 T nucleotides, at least 60 T nucleotides, at least 70 T nucleotides, at least 80 T nucleotides, at least 90 T nucleotides, at least 100 T nucleotides, at least 110 T nucleotides, at least 120 T nucleotides, at least 130 T nucleotides, at least 140 T nucleotides, at least 150 T nucleotides, at least 160 T nucleotides, at least 170 T nucleotides, at least 180 T nucleotides, at least 190 T nucleotides, at least 200 T nucleotides, at least 225 T nucleotides, at least 250 T nucleotides, at least 275 T nucleotides, at least 300 T nucleotides, at least 325 T nucleotides, at least 350 T nucleotides, at least 375 T nucleotides, at least 400 T nucleotides, at least 425 T nucleotides, at least 450 T nucleotides, at least 475 T nucleotides, at least 500 T nucleotides, or more.

[0106] In an embodiment, the poly (A / T) stretch comprises cytosine residues (see, for example Legnini, I., Alles, J., Karaiskos, N. et al., FLAM-seq: full-length mRNA sequencing reveals principles of poly(A) tail length control, Nat Methods 16, 879-886 (2019)).

[0107] In some embodiments adenine in poly-A / T stretch may be modified (see, for example Strzelecka D. et al., RNA. 2020 Dec; 26(12): 1815-1837. Phosphodiester modifications in mRNA poly(A) tail prevent deadenylation without compromising protein expression).

[0108] In some embodiments, the poly-A / T stretch comprises poly-A / T analogs (see, for example Perzanowska O., et al 2022. Chemically Modified Poly(A) Analogs Targeting PABP: Structure Activity Relationship and Translation Inhibitory Properties. Chemistry - A European Journal Volume 28, Issue 42 e202201115).

[0109] Nucleic acid template immobilized on a solid support

[0110] In an embodiment, the nucleic acid template is immobilized on a solid support. In an embodiment, the nucleic acid template is a DNA template, and the DNA template is immobilized on a solid support. The immobilization of the DNA template allows repeated use of the template and reduces contamination of the RNA product by residual DNA. In addition, immobilization makes the use of DNA expendable for the removal of the DNA template from the final RNA product.

[0111] In an embodiment, enzymes used in a method of synthesizing capped mRNA are immobilized on a solid support. Enzyme immobilization methods are known in the art (e.g. as described in WO2016193226A1). In an embodiment, RNA polymerase is immobilized on a solid support. In an embodiment, 2'-O-methyltransferase is immobilized on a solid support.

[0112] According to the invention, the term "solid support" refers to each undissolved support that is capable of immobilizing a DNA molecule or enzyme or other reaction component on its surface. The solid support material is non-degradable and can be selected from agarose, modified agarose, sepharose, thiopropyl-sepharose, sephadex™, polystyrene, latex, cellulose and ferro- or ferrimagnetic particles, acrylamide, nitrocellulose, glass, gold, polyethylene vinyl acetate, polypropylene, polymethacrylate, polyethylene, polyethylene oxide, polysilicates, polycarbonates, Teflon, fluorocarbons, nylon, silicon rubber, polyanhydrides, polyglycolic acid, polyactic acid, polyorthoesters, functionalized silane, polypropylfumerate, collagen, glycosaminoglycans, polyamino acids, or any combination thereof.

[0113] A solid support may have different shapes, preferable the shape is a slide, a membrane, a matrix, a plate, a chip, a resin or a bead. A solid support may further have holes or depressions to perform reactions at defined locations in an arrayed format on or within the solid support. Reactions on a solid support may be carried out in the presence of one or more additives. Such additives may help to keep beads in suspension or otherwise increase the fidelity of enzymes acting in close proximity to the solid support. The additive may be a chemical compound, a polymer, a polysaccharide, a protein, a chaperon, or any mixture thereof.

[0114] Methods and strategies for choosing appropriate solid supports and for coupling DNA molecules to said solid supports are known in the art (see, for example, Arndt-Jovin et al. 1975. EUR. J. Biochem. 54 (2): 41 1-8; Kerrigan et al., 2001. Current Protocols in Molecular Biology. 24: 12.10.1-12.10.18; WO 1995 / 08626).

[0115] The immobilization of the DNA template on the solid support can be by means of a covalent or non- covalent bond. Preferably, immobilization of the DNA template is produced by a non-covalent bond. For example, immobilization of a DNA template on a solid support can occur through a non-covalent biotin-streptavidin interaction. The non-coding strand of the DNA template can be modified with a biotin group at the 5 'end, whose function is to immobilize the DNA strand in a solid support matrix comprising the streptavidin protein. The complementary RNA coding strand of the DNA template may remain non-immobilized. It is also possible to immobilize the DNA templates on solid supports by other types of non-covalent bonds, for example poly(A) -poly(T) and poly(G) -poly(C) interactions. With equal preference, immobilization of the DNA template occurs via a covalent bond, for example an ester bond or derivative thereof. In an embodiment, the DNA template is covalently coupled to the solid support material. In general, before coupling, the solid support may contain active groups such as NHS, carbodiimide, etc. to allow the coupling reaction with the DNA molecule. The DNA molecule can be coupled to the solid support by direct coupling (for example using functional groups such as amino, sulfhydryl, carboxyl, hydroxyl, aldehyde and ketone groups). The link to the solid support material may involve spacers to optimize the spatial separation of the DNA template from the support. The spacer can be provided by inserting additional nucleotides at the 5 'end of the DNA template. A DNA template may be modified with a ligand for immobilization to a solid support at the 5'-end, the 3'-end, or at an internal nucleotide of a DNA strand. In some instances, the DNA template may be immobilized on the solid support with the 3' -end of the non-coding strand. The immobilization of the DNA template allows the repeated usage of the template and reduces the contamination of the RNA product by residual DNA. In some instances, the DNA template can be a double-stranded duplex or a unit that comprises a double-stranded promoter region upstream of a single-stranded RNA coding region. In some examples, the DNA template may be modified with a ligand for immobilization to a solid support at the 5' end, the 3' end, or at an internal nucleotide of a DNA strand.

[0116] In preferred embodiments, the solid support is selected from the group comprising a microfluidic chip, a column, a cartridge, a membrane, an array, a bead, a nanoparticle or a resin. Preferably, the solid support is selected from the group consisting of a microfluidic chip, a column, an array, a bead, a nanoparticle or a resin.

[0117] More preferably, the solid support is a bead, wherein the bead is of any convenient dimensions and is constructed from any number of known materials. In some examples, the bead may be monodisperse. Examples of such materials include inorganic materials, natural polymers, and synthetic polymers. Specific examples of these materials include cellulose, cellulose derivatives, acrylic resins, glass; silica gel, polystyrene, gelatin, polyvinylpyrrolidone, copolymers of vinyl and acrylamide, polystyrene cross-linked with divinylbenzene, polyacrylamide, latex gel, polystyrene dextran, rubber, silicon, plastic, nitrocellulose, cellulose, natural sponge, silica gel, glass, metal, plastic, cellulose, methacrylate beads, cross-linked dextran (e.g., Sephadex™) and agarose gels (Sepharose). In a specific embodiment, the solid support is a bead, wherein the bead is a magnetic bead or a sepharose bead.

[0118] In some embodiments, the magnetic beads may comprise microparticles or nanoparticles. In some examples, the magnetic beads may contain iron oxide. For example, magnetic nanoclusters as described in patent application No. GB2210796.5 which is hereby incorporated by reference may be used. For example, the magnetic beads may be selected from Dynabeads™ MyOne™ Silane, Dynabeads™ MyOne™ Carboxylic Acid, Dynabeads™ M-270™ Carboxylic Acid, Dynabeads™, Oligo(dT)25 magnetic beads (all available from Thermo Fisher Scientific), SpeedBead™ (General Electric, Boston, MA), BioMagPlus COOH™ and ProMag 1 COOH™ (both Bangs Laboratories, INC Fishers), 4.4 pm fluorescent ferromagnetic beads or 2.0 pm ferromagnetic beads (both available from Spherotech INC Lake Forest, IL), 2 pm beads designated WHM-S001™ or 2 pm beads designated WHM-S002™ (both available from Creative Diagnostics, New York, NY), Silicon Hydroxyl Magnetic Microspheres or Carboxyl Magnetic Microspheres or Oligo(dT) Magnetic Microspheres (available at different nm or pm sizes from VDO Biotech, Suzhou, China), Carboxyl Adembeads (available at 100 nm, 200 nm, 300 nm or 500 nm from Ademtech, France) etc.

[0119] In yet another embodiment, the DNA template is immobilized on nanoparticles. Nanoparticles include, but are not limited to, metal (e.g., gold, silver, copper and platinum), semiconductor (e.g., CdSe, CdS, and CdS coated with ZnS) and magnetic (e.g., ferromagnetic) colloidal materials. Methods to attach DNA templates to the nanoparticles are known in the art. In another embodiment, nanoparticles are attached to a substrate. Nanoparticles with or without immobilized DNA template can be attached to substrates as described in, e.g., Grabar et al., Analyt. Chem., 67, 73-743 (1995); Bethell et al., J. Electroanal. Chem., 409, 137 (1996); Bar et al., Langmuir, 12, 1172 (1996); Colvin et al., J. Am. Chem. Soc., 114, 5221 (1992). Naked nanoparticles may be first attached to the substrate and DNA templates can be attached to the immobilized nanoparticles.

[0120] In embodiments, the solid support comprises a reactive group selected from the group consisting of thiol, haloacetyl, pyridyl disulfide, epoxy, maleimide, hydroxyl, silanol, carboxyl, saccharide moieties and mixtures thereof, even more preferably the solid support comprises a reactive group selected from the group consisting of thiol, haloacetyl, pyridyl disulfide, maleimide and mixtures thereof.

[0121] In some embodiments the solid support comprises an affinity tag or a binding moiety for immobilization of a template or one or more enzymes used in RNA processing steps (e.g. a capping enzyme, RNA polymerase, restriction enzyme, 2'-O-methyltransferase, poly(A) polymerase, pyrophosphatase etc.). In some embodiments the solid support may comprise a streptavidin tag to immobilize a biotinylated target such as a biotinylated DNA template or enzyme.

[0122] In some embodiments, the particles with immobilized template nucleic acid and / or enzymes are subjected to manual workflows, i.e. the particles and solutions are manipulated by the user. In embodiments, the particles with immobilized nucleic acid template and / or enzymes are subjected to automated workflows, using dedicated devices. In some embodiments where magnetic particles are used as solid support automation may include providing a device capable of magnetic separation. Examples of such devices are provided in WO2022081519 Al and W02020002598 Al.

[0123] Concentration of nucleic acid template in the final reaction mixture

[0124] According to a preferred embodiment of the invention, the concentration of the linear DNA template in the reaction mixture of the method of the invention is in a range from about 1 to 100 nM, 1 to 50 nM, 1 to 30 nM, 1 to 20 nM, or about 1 to 10 nM. Even more preferred the concentration of the DNA template is from about 10 to 30 nM. Most preferred the concentration of the DNA template is about 20 nM.

[0125] In case plasmid DNA is used as DNA template, the concentration of the DNA template in the final reaction mixture of the method of the invention is between 1 ng / ml to 100 pg / ml, preferably between 1 pg / ml to 3 pg / ml, most preferably 2.5 pg / ml.

[0126] B. Composition compounds added to the nucleic acid template

[0127] According to the inventive method, the nucleic acid template comprising the target sequence to be transcribed, an RNA polymerase promoter site operably linked upstream to the target sequence, an RNA polymerase capable of binding the promoter site, a transcription buffer, rNTPs, a dinucleotide cap analog according to Formula I, a 2'0-methyltransferase and S-adenosylmethionine are combined in a single vessel, in order to yield the final reaction mixture based on which IVT, co-transcriptional capping and methylation of the capped mRNA is performed.

[0128] RNA polymerase capable of binding the promoter site

[0129] The RNA polymerase may be any DNA-dependent RNA polymerase capable of binding to the promoter sequence within the linearized nucleic acid template. The RNA polymerase recognizes and specifically binds to the target sequence at the promoter site from where the RNA synthesis is initiated. In preferable embodiments, the RNA polymerase is a bacteriophage-derived RNA polymerase, preferably selected from the group comprising T7, T3, Vsw-3, Kll, T3D, T5, T8, (02.5, (06, and SP6 RNA polymerases, their variants, chimeras or mutants thereof. In a further embodiment, the RNA polymerase is selected from the group consisting of a T7 RNA polymerase, a T3 RNA polymerase, and a SP6 RNA polymerase. RNA polymerases may be modified by exchanging, inserting or deleting amino acids of the RNA polymerase sequence. Variants may be obtained by evolving an RNA polymerase, optimizing the RNA polymerase amino acid and / or nucleic acid sequence and / or by using other methods known in the art.

[0130] In an embodiment, the RNA polymerase is a T7 RNA polymerase.

[0131] In some examples, engineered variants of RNA polymerases may be used such as thermostable variants disclosed in W02010016621 Al, US11072808 B2, WO2017123748 Al or WO2018236617 Al. An RNA polymerase may also be evolved for selective incorporation of the capped GTP analogue over GTP as disclosed in W02019005539 Al or W02019005540 Al. Alternatively, RNA polymerase may be a chimeric enzyme as disclosed in US11072808 or evolved to have improved properties, such as increased thermostability, increased transcription efficiency, 3' homogeneity, run-on transcripts, double-stranded contaminants, as disclosed in WO2019 / 199807, US11066686, CN112831484 or US11485960. In one embodiment, T7 RNA polymerase may comprise at least one mutation such as, but not limited to, Ml, I4M, A7T, N9, DIO, G47, E63V, V64D, A65E, D66Y, T75, T76N, A83, K93T, E108, 1109, A113, 1117, C125R, S128R, V134, A136, V137, D147, N165S, E167, K172, R173, G175R, H176L, Y178H, K179, K180, F182L, L196F, G198V, H205, K206, D208Y, E222K, V227, S228A, H230, Q239R, T243N, G259D, P266, M267I, G280C, 1281, R291, V297, H300R, Y312, 1320, A323, A327, K333, W344, V340, C347, E350, D351, A354, E356D, R357, L360P, M362, T375, K378, A383V, V384, Y385C, D388, K389, R394, 1396, S397, M401, Q404, N410S, V426, A428, L446, K450R, P451T, G452V, K454, K461, E484A, M490, C492, S495, C510, A513, C515, H523L, H524N, C530, L534, G542V, F546, G555, E565K, V567, K577E, K577M, A584, D591, N601S, G618, S684Y, N601, L621, Y623, S633, M635, Y639, K642, V650, T654, Q656, D660, G664, L665, S684, V685, V689, M696, L699I, A702, K711, K713E, C723, A724, T729, P730, K740, N748D, Q754R, E775K, V783, H784, Q786, D787, G788, V795, 1810, A827V, M832, D834, T835, C839, A843, D847, F849, A850, D851N, S856, A863, L864F, A866 and / or E877.

[0132] In another embodiment, the RNA polymerase is a T3 RNA polymerase. In yet another embodiment, the RNA polymerase is an SP6 RNA polymerase. Preferably, the RNA polymerase is a T7 RNA polymerase. In addition, in an embodiment the RNA polymerase is a variant of the T7, T3 or SP6 RNA polymerase. In another embodiment, the RNA polymerase is derived from E. coli. In one embodiment, the RNA polymerase is a single subunit RNA polymerase of viral origin. In an embodiment, the RNA polymerase is derived from recombinant source. In one embodiment the RNA polymerase may be a chimeric enzyme and may be fused to another entity (such as a linker, domain, subunit of another enzyme, a tag etc.).

[0133] In an embodiment, the nucleic acid template comprises a target sequence to be transcribed and an RNA polymerase promoter site operably linked upstream to the target sequence, wherein the RNA polymerase promoter is a T7 RNA polymerase promoter and the RNA polymerase used is a T7 RNA polymerase.

[0134] In an embodiment, the nucleic acid template comprises a target sequence to be transcribed and an RNA polymerase promoter site operably linked upstream to the target sequence, wherein the RNA polymerase promoter is a T3 RNA polymerase promoter and the RNA polymerase used is a T3 RNA polymerase.

[0135] In an embodiment, the nucleic acid template comprises a target sequence to be transcribed and an RNA polymerase promoter site operably linked upstream to the target sequence, wherein the RNA polymerase promoter is an SP6 RNA polymerase promoter and the RNA polymerase used is an SP6 RNA polymerase.

[0136] In a specific embodiment, the invention relates to a method of synthesizing capped mRNA, comprising combining, in a single vessel: a nucleic acid template comprising a target sequence to be transcribed and a T7 RNA polymerase promoter site operably linked upstream to the target sequence, a T7 RNA polymerase or functional variant thereof, a transcription buffer, ribonucleoside triphosphates (NTPs), a dinucleotide cap analog according to Formula I, wherein R1is selected from methyl, ethyl, propyl, butyl, benzyl, substituted benzyl, C5-Cg heterocyclyl, C5-Cg substituted heterocyclyl; R2is H, OH or O-CH3; R3is H, OH or O-CH3; R4, R5and R6are independently selected from the group consisting of O, S, Se; R7and R8 are OH; n is 1, 2, or 3; and B is a non-modified or modified purine or pyrimidine base, a 2'-O-methyl transferase, and

[0137] S-adenosyl methionine (SAM), to produce an IVT reaction mixture incubating the IVT reaction mixture under conditions allowing for co-transcriptional mRNA capping and methylation of capped mRNA to generate a synthesized capped mRNA having a capl- type structure.

[0138] In a specific embodiment, the invention relates to a method of synthesizing capped mRNA, comprising combining, in a single vessel: a nucleic acid template comprising a target sequence to be transcribed and a T3 RNA polymerase promoter site operably linked upstream to the target sequence, a T3 RNA polymerase or functional variant thereof, a transcription buffer, ribonucleoside triphosphates (NTPs), a dinucleotide cap analog according to Formula I, wherein R1is selected from methyl, ethyl, propyl, butyl, benzyl, substituted benzyl, C5-Cg heterocyclyl, C5-Cg substituted heterocyclyl; R2is H, OH or O-CH3; R3is H, OH or O-CH3; R4, R5and R6are independently selected from the group consisting of O, S, Se; R7and R8are OH; n is 1, 2, or 3; and B is a non-modified or modified purine or pyrimidine base, a 2'-O-methyl transferase, and

[0139] S-adenosyl methionine (SAM), to produce an in vitro transcription (IVT) reaction mixture

[0140] Incubating the IVT reaction mixture under conditions allowing for co-transcriptional mRNA capping and methylation of capped mRNA to generate a synthesized capped mRNA, having a capl-type structure.

[0141] In a specific embodiment, the invention relates to a method of synthesizing capped mRNA, comprising combining, in a single vessel: a nucleic acid template comprising a target sequence to be transcribed and a SP6 RNA polymerase promoter site operably linked upstream to the target sequence, a SP6 RNA polymerase or functional variant thereof, a transcription buffer, ribonucleoside triphosphates (NTPs), a dinucleotide cap analog according to Formula I, wherein R1is selected from methyl, ethyl, propyl, butyl, benzyl, substituted benzyl, C5-Cg heterocyclyl, C5-Cg substituted heterocyclyl; R2is H, OH or O-CH3; R3is H, OH or O-CH3; R4, R5and R6are independently selected from the group consisting of O, S, Se; R7and R8are OH; n is 1, 2, or 3; and B is a non-modified or modified purine or pyrimidine base, a 2'-O-methyl transferase, and

[0142] S-adenosyl methionine (SAM) to produce an IVT reaction mixture incubating the IVT reaction mixture under conditions allowing for co-transcriptional mRNA capping and methylation of capped mRNA to generate a synthesized capped mRNA, having a capl-type structure.

[0143] Concentration of the RNA Polymerase

[0144] Preferably, the concentration of the RNA polymerase in the final reaction mixture is from about 1 to 100 nM, 1 to 90 nM, 1 to 80 nM, 1 to 70 nM, 1 to 60 nM, 1 to 50 nM, 1 to 40 nM, 1 to 30 nM, 1 to 20 nM, or about 1 to 10 nM. Even more preferred, the concentration of the RNA polymerase is from about 10 to 100 nM, about 10 to 80 nM, about 20 to 70 nM, or 50 to 70 nM. Most preferably, the RNA polymerase has a final concentration of 60 nM

[0145] Most preferred is an RNA polymerase concentration in the final reaction mixture of about 60 nM. The person skilled in the art will understand that the choice of the RNA polymerase concentration is influenced by the concentration of the DNA template. Therefore, in specific embodiments the concentration of the RNA polymerase in the final reaction mixture is between 1 and 1000 U / pg template DNA, preferably between 100 and 300 U / pg DNA, particularly if plasmid DNA is used as template DNA.

[0146] Transcription buffer

[0147] In an embodiment, the transcription buffer comprises a buffering agent, magnesium, reducing agents such as dithiothreitol (DTT) and / or additional components. In an embodiment, the transcription buffer comprises magnesium ions, dithiothreitol (DTT) and polyamine additives. In some embodiments polyamine is spermidine. In some examples, the transcription buffer comprises detergent.

[0148] Common buffer systems or buffering agents used in RNA in vitro transcription include 4-(2-hydroxy- ethyl)-l-piperazineethanesulfonic acid (HEPES) and tris(hydroxymethyl)amino- methane (Tris), other non-limiting examples include TAPS (tris(hydroxymethyl)methylamino]propanesulfonic acid), MOPS (3-(N-morpholino)propanesulfonic acid), tricine, or MES (2-Morpholinoethanesulphonic acid). The pH of the buffer is commonly adjusted to a pH value of 6 to 9. In some embodiments, a reaction buffer has a pH of 7-9.

[0149] The transcription buffer comprises magnesium salt and / or other salts, a buffering agent, reducing agent, and / or other ingredients. In some embodiments, other salts are ammonium sulfate, sodium bicarbonate, sodium citrate, sodium acetate, potassium phosphate, sodium phosphate and / or sodium chloride.

[0150] In some embodiments, the transcription buffer comprises magnesium (Mg2+; e.g., a magnesium salt), preferably MgCL. In some embodiments, magnesium salt is magnesium acetate. The concentration of magnesium present in the final reaction can be, for example, 1-60 mM, preferably 2-50 mM, more preferably 5-40 mM, even more preferably 10-30 mM. In a specific embodiment, the final reaction composition comprises magnesium at a concentration of 10 mM. In a specific embodiment, the final reaction mixture comprises MgCL at a concentration of 15 mM. In a specific embodiment, the final reaction mixture comprises MgCL at a concentration of 20 mM. In a specific embodiment, the final reaction mixture comprises MgCL at a concentration of 25 mM. In a specific embodiment, the final reaction mixture comprises MgCL at a concentration of 30 mM. In a specific embodiment, the final reaction mixture comprises magnesium at 12 mM. In a specific embodiment, the final reaction mixture comprises magnesium at 8 mM. In a specific embodiment, the final reaction mixture comprises magnesium at 6 mM. In some embodiments, the molar ratio of rNTP to magnesium ions (Mg2+; e.g., MgCL) present in the IVT reaction is from 1:1 to 1:5. For example, the molar ratio of NTP to magnesium ion in the final reaction may be 1:1, 1:2, 1:3, 1:4 or 1:5.

[0151] In some embodiments, the transcription buffer comprises a reducing agent. In some embodiments, a reducing agent comprises dithithreitol (DTT) and / or 2 -mercaptoethanol. In an embodiment, the reducing agent is DTT. The concentration of DTT present in the final reaction can be, for example, at least 1 mM, at least 5 mM, or at least 10 mM, or 1-50 mM, preferably 1-30 mM, more preferably 5- 10 mM, even more preferably 7-15 mM. In a specific embodiment, the final reaction mixture comprises DTT at a concentration of 10 mM. In a specific embodiment, the final reaction mixture comprises DTT at a concentration of 5 mM.

[0152] In some embodiments, the transcription buffer comprises spermidine. The concentration of spermidine present in the final reaction can be, for example, 0.1-10 mM, preferably 0.1-5 mM, more preferably 0.5-4 mM, even more preferably 2-5 mM. In a specific embodiment, the final reaction mixture comprises spermidine at a concentration of 2 mM. In a specific embodiment, the final reaction mixture comprises spermidine at a concentration of 1 mM.

[0153] In some embodiments, the transcription buffer comprises a detergent. In some embodiments, the detergent includes Triton X-100, sodium dodecyl sulfate (SDS), CHAPS (3-[(3- cholamidopropyl)dirnethylammonio]-l-propanesulfonate), ethyl trimethyl ammonium bromide, nonyl phenoxypolyethoxylethanol (NP-40), Poloxamer 188 and / or polyethylene glycol (PEG). In some embodiments, the transcription buffer comprises Tris as a buffering agent. The concentration of Tris used in the final reaction can be, for example, at least 10 mM, at least 20 mM, at least 30 mM, at least 40 mM, at least 50 mM, at least 60 mM, at least 70 mM, at least 80 mM, at least 90 mM, at least 100 mM or at least 110 mM. In an example, the final concentration of Tris in the final reaction is 40 mM. In an example, the final concentration of Tris in the final reaction is 50 mM.

[0154] In some embodiments, the transcription buffer optionally further comprises additional components. In some embodiments, additional component is NaCI. In some embodiments NaCI concentration in the final reaction is from 5 mM to 50 mM. In some embodiments NaCI concentration in the final reaction is 10 mM. In some embodiments NaCI concentration in the final reaction is 20 mM.

[0155] In an embodiment, transcription buffer composition could be: 40 mM Tris-HCI, 27.5 mM MgCL, 10 mM DTT, 2 mM spermidine. In a different embodiment, transcription buffer composition is: 40 mM Tris-HCI, 6 mM MgCL, 20 mM NaCI, 10 mM DTT, 2 mM spermidine. In another embodiment, transcription buffer composition is: 40 mM Tris-HCI, 8 mM MgCL, 25 mM NaCI, 2 mM spermidine. In another embodiment, transcription buffer composition is: 40 mM Tris-HCI, 6 mM MgCL, 10 mM DTT, 2 mM spermidine. In another embodiment, transcription buffer composition is: 40 mM Tris-HCI, 6 mM MgCL, 10 mM NaCI, 2 mM spermidine. In another embodiment, transcription buffer composition is: 40 mM Tris-HCI, 6 mM MgCL, 10 mM DTT, 2 mM spermidine. In another embodiment, transcription buffer composition is: 40 mM Tris-HCI, 46 mM MgCL, 10 mM DTT, 2 mM spermidine. In another embodiment, transcription buffer composition is: 40 mM Tris-HCI, 6 mM MgCL, 10 mM DTT, 2 mM spermidine. In another embodiment, transcription buffer composition is: 40 mM Tris-HCI, 80 mM MgCL, 50 mM mM NaCI, 10 mM DTT, 2 mM spermidine. In another embodiment, transcription buffer composition is: 40 mM Tris-HCI, 12 mM MgCL, 10 mM DTT, 2 mM spermidine.

[0156] Ribonucleoside triphosphates (NTPs) In an embodiment, the ribonucleoside triphosphates (rNTPs) comprise ATP, CTP, UTP, GTP and any modified ribonucleotide. The modification may be one or more of a backbone modification, a sugar modification and / or a nucleobase modification. A backbone modification is a modification, in which phosphates of the backbone of the ribonucleotides are chemically modified. A sugar modification in connection with the present invention is a chemical modification of the sugar of the ribonucleotides. Furthermore, a base modification in connection with the present invention is a chemical modification of the base moiety of the ribonucleotides. In this context ribonucleotide modifications are applicable for transcription and / or translation

[0157] According to an embodiment of the invention, a part or all of at least one ribonucleoside triphosphate in the reaction mixture is replaced with a modified nucleoside triphosphate. The ribonucleoside triphosphates provided herein include unmodified or modified ATP, modified or unmodified UTP, modified or unmodified GTP, and / or modified or unmodified CTP. In some embodiments, the rNTPs of in the reaction mixture comprise unmodified ATP. In some embodiments, the rNTPs of in the reaction mixture comprise modified ATP. In some embodiments, the rNTPs of in the reaction mixture comprise an unmodified UTP. In some embodiments, the rNTPs of in the reaction mixture comprise a modified UTP. In some embodiments, the rNTPs of in the reaction mixture comprise unmodified GTP. In some embodiments, the rNTPs of in the reaction mixture comprise modified GTPs. In some embodiments, the rNTPs of in the reaction mixture comprise unmodified CTPs. In some embodiments, the rNTPs of in the reaction mixture comprise modified CTPs.

[0158] In a preferred embodiment of the invention, said modified nucleoside triphosphate is selected from the group consisting of pseudouridine-5'-triphosphate, l-methylpseudouridine-5'-triphosphate, 2- thiouridine-5'-triphosphate, 4-thiouridine-5'-triphosphate and 5-methylcytidine-5'-triphosphate. According to an embodiment of the invention, a part or all of UTP in the reaction mixture is replaced with a modified UTP selected from the group consisting of pseudouridine-5'-triphosphate, 1- methylpseudouridine-5'-triphosphate, 2-thiouridine-5'-triphosphate, 4-thiouridine-5'-triphosphate. According to another embodiment of the invention, a part or all of CTP in the reaction mixture is replaced with 5-methylcytidine-5'-triphosphate.

[0159] Alternatively, the ribonucleotide modifications may be selected from base modifications, which are preferably selected from 2-amino-6-chloropurineriboside-5'-triphosphate, 2-Aminopurine-riboside- 5'-triphosphate; 2-aminoadenosine-5'-triphosphate, 2'-Amino-2'-deoxycytidine-triphosphate, 2- thiocytidine-5'-triphosphate, 2-thiouridine-5'-triphosphate, 2'-Fluorothymidine-5'-triphosphate, 2'-O- Methyl inosine-5'-triphosphate 4-thiouridine-5'-triphosphate, 5-aminoallylcytidine-5'-triphosphate, 5-aminoallyluridine-5'-triphosphate, 5-bromocytidine-5'-triphosphate, 5-bromouridine-5'- triphosphate, 5-Bromo-2'-deoxycytidine-5'-triphosphate, 5-Bromo-2'-deoxyuridine-5'-triphosphate, 5-iodocytidine-5'-triphosphate, 5-lodo-2'-deoxycytidine-5'-triphosphate, 5-iodouridine-5'- triphosphate, 5-lodo-2'-deoxyuridine-5'-triphosphate, 5-methylcytidine-5'-triphosphate, 5- methyluridine-5'-triphosphate, 5-Propynyl-2'-deoxycytidine-5'-triphosphate, 5-Propynyl-2'- deoxyuridine-5'-triphosphate, 6-azacytidine-5'-triphosphate, 6-azauridine-5'-triphosphate, 6- chloropurineriboside-5'-triphosphate, 7-deazaadenosine-5'-triphosphate, 7-deazaguanosine-5'- triphosphate, 8-azaadenosine-5'-triphosphate, 8-azidoadenosine-5'-triphosphate, benzimidazole- riboside-5'-triphosphate, Nl-methyladenosine-5'-triphosphate, Nl-methylguanosine-5'-triphosphate, N6-methyladenosine-5'-triphosphate, 06-methylguanosine-5'-triphosphate, pseudouridine-5'- triphosphate, or puromycin-5'-triphosphate, xanthosine-5'-triphosphate. Particular preference is given to nucleotides for base modifications selected from the group of base-modified nucleotides consisting of 5-methylcytidine-5'-triphosphate, 7-deazaguanosine-5'-triphosphate, 5-bromocytidine- 5'-triphosphate, and pseudouridine-5'-triphosphate.

[0160] Modified ribonucleosides may be selected from pyridin-4-one ribonucleoside, 5-aza-uridine, 2-thio-5- aza-uridine, 2-thiouridine, 4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxyuridine, 3- methyluridine, 5-carboxymethyl-uridine, 1-carboxymethyl-pseudouridine, 5-propynyl-uridine, 1- propynyl-pseudouridine, 5-taurinomethyluridine, 1-taurinomethyl-pseudouridine, 5-taurinomethyl- 2-thio-uridine, l-taurinomethyl-4-thio-uridine, 5-methyl-uridine, 1-methyl-pseudouridine, 4-thio-l- methyl-pseudouridine, 2-thio-l-methyl-pseudouridine, 1-methyl-l-deaza-pseudouridine, 2-thio-l- methyl-l-deaza-pseudouridine, dihydrouridine, dihydropseudouridine, 2-thio-dihydrouridine, 2-thio- dihydropseudouridine, 2-methoxyuridine, 2-methoxy-4-thio-uridine, 4-methoxy-pseudouridine, and 4-methoxy-2-thio-pseudouridine, 5-aminoallyluridine, 5-iodo-uridine, 5,6-dihydrouridine, a-thio- uridine, 4-thio-uridine, 6-aza-uridine, 5-hydroxy-uridine, deoxy-thymidine, 5-methyl-uridine.

[0161] Modified ribonucleosides may be selected from 5-aza-cytidine, pseudoisocytidine, 3-methyl-cytidine, N4-acetylcytidine, 5-formylcytidine, N4-methylcytidine, 5-hydroxymethylcytidine, 1-methyl- pseudoisocytidine, pyrrolo-cytidine, pyrrolo-pseudoisocytidine, 2-thio-cytidine, 2-thio-5-methyl- cytidine, 4-thio-pseudoisocytidine, 4-thio-l-methyl-pseudoisocytidine, 4-thio-l-methyl-l-deaza- pseudoisocytidine, 1-methyl-l-deaza-pseudoisocytidine, zebularine, 5-aza-zebularine, 5-methyl- zebularine, 5-aza-2-thio-zebularine, 2-thio-zebularine, 2-methoxy-cytidine, 2-methoxy-5-methyl- cytidine, 4-methoxy-pseudoisocytidine, and 4-methoxy-l-methyl-pseudoisocytidine, 6-aza-cytidine, 2-thio-cytidine, a-thio-cytidine, Pseudo-iso-cytidine.

[0162] Modified ribonucleosides may be selected from 2-aminopurine, 2,6-diaminopurine, 7-deaza-adenine, 7-deaza-8-aza-adenine, 7-deaza-2-aminopurine, 7-deaza-8-aza-2-aminopurine, 7-deaza-2,6- diaminopurine, 7-deaza-8-aza-2,6-diaminopurine, 1-methyladenosine, N6-methyladenosine, N6- isopentenyladenosine, N6-(cis-hydroxyisopentenyl)adenosine, 2-methylthio-N6-(cis- hydroxyisopentenyl) adenosine, N6-glycinylcarbamoyladenosine, N6-threonylcarbamoyladenosine, 2-methylthio-N6-threonyl carbamoyladenosine, N6,N6-dimethyladenosine, 7-methyladenine, 2- methylthio-adenine, and 2-methoxy-adenine, a-thio-adenosine, 8-azido-adenosine.

[0163] Modified ribonucleosides may be selected from inosine, 1-methyl-inosine, wyosine, wybutosine, 7- deaza-guanosine, 7-deaza-8-aza-guanosine, 6-thio-guanosine, 6-thio-7-deaza-guanosine, 6-thio-7- deaza-8-aza-guanosine, 7-methyl-guanosine, 6-thio-7-methyl-guanosine, 7-methylinosine, 6- methoxy-guanosine, 1-methylguanosine, N2-methylguanosine, N2,N2-dimethylguanosine, 8-oxo- guanosine, 7-methyl-8-oxo-guanosine, l-methyl-6-thio-guanosine, N2-methyl-6-thio-guanosine, and N2,N2-dimethyl-6-thio-guanosine, a-thio-guanosine, 6-methyl-guanosine, 5-methyl-cytdine, 8-oxo- guanosine, 7-deaza-guanosine.

[0164] In some embodiments, the ribonucleotide can be modified on the major groove face and can include replacing hydrogen on C-5 of uracil with a methyl group or a halo group.

[0165] In specific embodiments, a modified ribonucleoside is 5'-O-(l-Thiophosphate)-Adenosine, 5'-O-(l- Thiophosphate)-Cytidine, 5'-O-(l-Thiophosphate)-Guanosine, 5'-O-(l-Thiophosphate)-Uridine or 5'- O-(1-Thiophosphate)-Pseudouridine.

[0166] Further modified nucleotides have been described previously (see, e.g., WO 2013 / 052523).

[0167] Concentration of rN TPs

[0168] According to a preferred embodiment of the invention, the total rNTP concentration in the reaction mixture is between 1 and 200 mM, preferably between 2 and 100 mM, preferably between 10 and 50 mM, and most preferably between 15 and 25 mM. According to the invention, the term "total ribonucleotide concentration" means the total concentration of rNTPs, i.e., the sum of the concentrations of ATP, GTP, CTP, UTP, optionally any modified rNTP and the dinucleotide cap analog present initially in the reaction when the various components of the reaction have been assembled in the final volume for carrying out the reaction. Naturally, as the reaction proceeds, the nucleotides will be incorporated into the RNA molecule and consequently the total nucleotide concentration will be progressively reduced from its initial value.

[0169] In this context it is particularly preferred that the single ribonucleotides ATP, GTP, CTP, UTP, and any modified ATP, GTP, CTP, UTP, are provided in a concentration between 0.1 and 20 mM, preferably between 1 and 10, preferably between 3 and 7 mM, more preferably between 4 mM and 6 mM, and most preferably in a concentration of 5 mM.

[0170] The concentration of GTP is preferably reduced compared to the other ribonucleotides (ATP, CTP and UTP). In an embodiment of the invention, GTP is present in a ratio GTP:dinucleotide cap analog from 1:1 to 1:10. In an embodiment of the invention, GTP is present in a ratio GTP:dinucleotide cap analog from 1:1 to 1:8, from 1:1 to 1:7, from 1:1 to 1:6, from 1:1 to 1:5, from 1:1 to 1:4, from 1:1 to 1:3, from 1:1 to 1:2. In a preferred embodiment of the invention, GTP is present in a ratio GTP:dinucleotide cap analog from 1:3 to 1:5. In an even more preferred embodiment of the invention, GTP is present in a ratio GTP:dinucleotide cap analog of 1:4.

[0171] In this context it is particularly preferred that the single ribonucleotide GTP and any modified GTP, is provided in a concentration between 0.1 and 10 mM, preferably between 0.1 and 5 mM, more preferably between 0.5 mM and 2 mM, and most preferably in a concentration of 1 mM.

[0172] Dinucleotide cap analog

[0173] The dinucleotide cap analog is a compound of Formula I: wherein

[0174] R1is selected from methyl, ethyl, propyl, butyl, benzyl, substituted benzyl, C5-Cs heterocyclyl, C5-Cs substituted heterocyclyl;

[0175] R2is H, OH or O-CH3;

[0176] R3is H, OH or O-CH3;

[0177] R4, R5and R6are independently selected from the group consisting of O, S, Se;

[0178] R7and R8are OH; n is 1, 2, or 3; and

[0179] B is a non-modified or modified purine or pyrimidine base.

[0180] A „dinucleotide cap analog" is a dinucleotide consisting of an outer cap nucleoside, such as 7-methyl- guanosine (m7G), and the nucleotide corresponding to the first nucleotide of the primary transcript (e.g., G) depicted as "B" in Formula I. Therefore, an exemplary cap analog is the m7G(5')ppp(5')G, also referred to as m7GpppG. This cap analog is often used because the primary nucleotide (i.e., the most 5' nucleotide) of most, but not all, primary RNA transcripts synthesized using phage RNA polymerase transcription systems is guanosine.

[0181] The dinucleotide cap analogs are enzymatically incorporated at the mRNA 5'-end by the RNA polymerase, e.g., bacteriophage T7 RNA polymerase during in vitro transcription reaction, i.e., co- transcriptionally.

[0182] The most commonly used cap analogue is m7GpppG but several modified or alternative cap analogues are also accepted by RNA polymerases. Therefore, this route can also be used to install non-natural dinucleotides at the 5'-end that are accessible for a further chemical reaction.

[0183] In a preferred embodiment, the dinucleotide cap analog is an anti-reverse cap analog (ARCA). Thus, in a preferred embodiment, the dinucleotide cap analog is methylated at the 2' -OH or at the 3'-OH of the N7-methylguanosine ribose (m27,3 OGpppN or m27,2 OGpppN). This prevents elongation at the "wrong" 3'-OH and thus prevents yielding mRNA with the cap in reverse orientation as ARCA caps are exclusively incorporated in the correct orientation.

[0184] In an embodiment, the dinucleotide cap analog has Formula I, wherein one of R2or R3of the dinucleotide cap analog is O-CH3. In a preferable embodiment, the dinucleotide has Formula I, wherein R2is OH and R3is O-CH3. In another preferable embodiment, the dinucleotide has Formula I, wherein R2is O-CH3 and R3is OH.

[0185] The dinucleotide cap analog may preferably have a 3'-O-methyl ribose modification or a 2'-O-methyl ribose modification and may have three, four or five phosphate moieties, i.e., m27,3 OGpppN or rr^7,2 -°GpppN, m27,3 OGppppN or m27,2 OGppppN, m27,3 OGpppppN or m27,2 OGpppppN.

[0186] In a preferred embodiment, B (N) is guanosine.

[0187] In a specific embodiment, the dinucleotide cap analog is an anti-reverse cap analog (ARCA) having the structural formula of m27,3 OGpppG also abbreviated as 3'-O-Me-m7G(5')pppG(5'):

[0188] Concentration of dinucleotide cap analogs

[0189] According to a preferred embodiment of the invention, the concentration of the dinucleotide cap analog in the reaction mixture is between 0.1 and 100 mM, between 1 and 50 mM, preferably between 1 and 10 mM, and most preferably between 3 and 5 mM. In a specific embodiment, the dinucleotide cap analog is present in the reaction mixture at a concentration of 4 mM. In a specific embodiment, the dinucleotide cap analog is present in the reaction mixture at a concentration of 5 mM.

[0190] According to a preferred embodiment of the invention, the total rNTP concentration in the reaction mixture is between 1 and 100 mM, preferably between 10 and 50 mM, and most preferably between 15 and 25 mM. According to the invention, the term total ribonucleotide concentration means the total concentration of rNTPs, e.g. the sum of the concentrations of ATP, GTP, CTP, UTP, optionally any modified rNTP and the dinucleotide cap present initially in the reaction when the various components of the reaction have been assembled in the final volume for carrying out the reaction. Naturally, as the reaction proceeds, the nucleotides will be incorporated into the RNA molecule and consequently the total nucleotide concentration will be progressively reduced from its initial value.

[0191] The concentrations of dinucleotide cap analogs and nucleoside triphosphates present in the IVT reaction can vary, as described for the rNPTs before. In some embodiments, the NTP and dinucleotide cap analogs are present in equimolar concentrations in the reaction. That means the molar ratio of dinucleotide cap analog to rNTP is 1:1. In some embodiments, the molar ratio of the dinucleotide cap analog to nucleoside triphosphate in the reaction is greater than 1:1. For example, the molar ratio of cap analog to nucleoside triphosphate in the reaction is 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1. In some embodiments, the molar ratio of the dinucleotide cap analog to nucleoside triphosphate in the reaction is less than 1:1. In some embodiments, the molar ratio of the dinucleotide cap analog to nucleoside triphosphate in the reaction is less than 4:5. In an embodiment, the molar ratio of the dinucleotide cap analog to nucleoside triphosphate ranges from 1:1 to 8:1. In an embodiment, the molar ratio of the dinucleotide cap analog to nucleoside triphosphate ranges from 0.2:1 to 8:1. In preferred embodiments, the molar ratio of the dinucleotide cap analog to ATP is 1:1. In preferred embodiments, the molar ratio of the dinucleotide cap analog to CTP is 1:1. In preferred embodiments, the molar ratio of the dinucleotide cap analog to UTP is 1:1. In preferred embodiments, the molar ratio of the dinucleotide cap analog to a modified ATP, CTP or UTP is 1:1. In preferred embodiments, the molar ratio of the dinucleotide cap analog to GTP ranges from 1:1 to 8:1. In a specific embodiment, the dinucleotide cap analog is present at a ratio dinucleotide cap analog to GTP of 4:1.

[0192] 2'-O-methyltransferase

[0193] "2'-O-methyltransferase" is used interchangeable with "2'-O-ribonucleoside methyltransferase" or "20 MT".

[0194] Cap-0 transcripts can be enzymatically converted to cap-1 in vitro. According to the invention, mRNA cap 2'-0-methyltransferase adds a methyl group at the 2'-0 position of the first nucleotide adjacent to the cap structure at the 5' end of the RNA. The enzyme utilizes S-adenosylmethionine (SAM) as a methyl donor to methylate capped RNA (Cap-0) resulting in a Cap-1 structure. It has been reported that such capl structure may result in a higher translational-competency (Kuge et al. (1998) Nucl. Acids Res. 26(13): 3208-3214.) and cellular stability and a reduced activation of cellular pro- inflammatory cytokines, as compared, e.g., to other cap analog structures known in the art.

[0195] In an embodiment, the 2'-O-methyltransferase is of viral origin. In preferable embodiments, the 2'-O- methyltransferase is derived from a Vaccinia virus. In embodiments, the 2'-O-methyltransferase is a Vaccinia virus cap-specific ribonucleoside 2'-O-methyltransferase, functional mutant, chimera or variant thereof. In an embodiment, 2'-O-methyltransferase is derived from SARS-CoV-2. In some embodiments, the 2'-O-methyltransferase is RNA polymerase exhibiting 2'-O-methyltransferase activity (see for example "An RNA cap (nucleoside-2'-O-)-methyltransferase in the flavivirus RNA polymerase NS5: crystal structure and functional characterization", Egloff et al., 2002, The EMBO Journal.)

[0196] The 2'-O-methyltransferase transfers a methyl group from the co-substrate SAM to the target molecule.

[0197] Concentrations of 2'O-methyltransferase The concentration of 2'-O-methyltransferase in the final reaction is between 10 and 100 Units. Preferably, the concentration of 2'-O-methyltransferase in the final reaction is between 25 and 75 Units. In a preferred embodiment, the concentration of 2'-O-methyltransferase in the final reaction is about 50 Units. In a preferred embodiment, the concentration of 2'-O-methyltransferase in the final reaction is 50 Units.

[0198] S-adenosylmethionine (SAM)

[0199] As used herein, "S-adenosylmethionine", "S-Adenosyl-L-methionine", "SAM", and "AdoMet" are used interchangeably and refer to a compound having formula C15H22N6O5. The 2'-O-methyltransferase transfers a methyl group from the co-substrate S-adenosyl-L-methionine to first nucleotide adjacent to the cap structure at the 5' end of the mRNA.

[0200] Concentrations of S-adenosylmethionine

[0201] The concentration of S-adenosylmethionine in the final reaction is between 0.05 and 1 mM. Preferably, the concentration of S-adenosylmethionine in the final reaction is 0.2-0.3 mM. In preferred embodiments, the concentration of S-adenosylmethionine in the final reaction is about 0.25 mM. In preferred embodiments, the concentration of S-adenosylmethionine in the final reaction is 0.25 mM.

[0202] Poly (A) polymerase

[0203] In some embodiments, the composition optionally further comprises a poly(A) polymerase.

[0204] If the target sequence comprised in the nucleic acid template does not contain a poly(d(A / T)) sequence transcribed into a poly(A) tail, a poly(A) tail may also be added post transcriptionally by enzymatic polyadenylation using a poly(A) polymerase. Poly(A) polymerase catalyzes the templateindependent addition of AMP from ATP to the 3' end of RNA.

[0205] A poly(A) tail may contain 10 to 300 adenosine monophosphates. For example, a poly(A) tail may contain 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290 or 300 adenosine monophosphates. In some embodiments, a poly(A) tail contains 50 to 170 adenosine monophosphates. In preferred embodiments, the poly(A) tail contains between 120 and 150 adenosine monophosphates, preferably 120 adenosine monophosphates.

[0206] The poly(A) polymerase may be modified or unmodified. In a preferred embodiment, the modified or unmodified poly(A) polymerase is of bacterial origin. In a more preferred embodiment, the modified or unmodified poly(A) polymerase is derived from Escherichia coli.

[0207] C. Conditions allowing for co-transcriptional mRNA capping and methylation of capped mRNA within the same reaction

[0208] The method of the invention is performed under conditions allowing for co-transcriptional mRNA capping and methylation of capped mRNA within the same reaction.

[0209] The skilled person knows how to adjust the parameters, such as nucleic acid template, SAM and magnesium ion concentrations, respectively, buffer composition, pH, temperature and incubation times in order to perform the reactions. Reaction temperature, for instance, is dependent on the used enzyme, e.g., 37°C for T7 RNA polymerase. Additional components to improve performance, e.g. DMSO, pyrophosphatase and / or RNase inhibitor can be included. Concentration ranges of the single components are described above.

[0210] D. One pot reaction

[0211] According to the invention, the in vitro transcription of the DNA template, the capping of the transcribed mRNA and the 2'0-methylation of the first ribonucleotide of the transcribed mRNA is all performed within one single reaction. Thais means, IVT, co-transcriptional capping and methylation of capped mRNA is performed simultaneously. Therefore, methylation of the capped mRNA is performed without prior mRNA purification.

[0212] Thus, the present invention provides a method of performing an IVT reaction, comprising contacting a DNA template with the RNA polymerase in the presence of nucleoside triphosphates, a dinucleotide cap analog and buffer under conditions that result in the production of capped mRNA. The presence of the 2'-O-methyltransferase and SAM within the IVT reaction mixture allows for methylation of the first nucleotide of the mRNA next to the cap structure within the same reaction. In other words, the method of the invention is performed with all components, i.e., the nucleic acid template, the RNA polymerase, the transcription buffer, the rNTPs, the dinucleotide cap analog, the 2'-O- methyltransferase and SAM present in only one reaction mixture.

[0213] In an embodiment, the disclosed method is an in vitro method. In vitro transcription may also be performed in a living system, of the IVT components are delivered to a cell.

[0214] Surprisingly, the inventive method disclosed herein results in more than 95% of the RNA transcripts produced comprising a functional cap-1 structure. In some embodiments, greater than 96% of the RNA transcripts produced comprise a functional cap-1 structure. In some embodiments, greater than 97% of the RNA transcripts produced comprise a functional cap-1 structure. In some embodiments, greater than 98% of the RNA transcripts produced comprise a functional cap-1 structure. In some embodiments, greater than 99% of the RNA transcripts produced comprise a functional cap-1 structure. In a preferred embodiment, 100% of the RNA transcripts produced comprise a functional cap-1 structure.

[0215] Importantly, the methylation of capped mRNA is performed without prior mRNA purification.

[0216] 4. Use of the mRNAs produced with the inventive method

[0217] For example, RNA transcripts produced by the present method can be used to produce polypeptides of interest, e.g., therapeutic proteins, vaccine antigens, and the like. In some embodiments, the RNA transcript is a therapeutic RNA. A therapeutic mRNA is an mRNA encoding a therapeutic protein (the term 'protein1encompasses peptides). Therapeutic proteins mediate various effects in a host cell or in a subject to treat a disease or ameliorate the signs and symptoms of a disease. For example, a therapeutic protein can replace a deficient or abnormal protein, enhance the function of an endogenous protein, or provide a new function to the cell (e.g., inhibit or activate endogenous cellular activity), or can serve as a delivery agent for another therapeutic compound (e.g., antibodydrug conjugate). Therapeutic mRNA can be used, for example, for the following diseases and conditions: bacterial infection, viral infection, parasitic infection, cell proliferation. It can be useful in the treatment of disorders, genetic disorders, and autoimmune disorders. Other diseases and conditions are encompassed herein. Thus, a protein of interest encoded by an mRNA when provided herein can be essentially any protein.

[0218] 5. Compositions

[0219] In a second aspect, the invention provides for a composition for use in the synthesis of capl-type mRNA from a nucleic acid template. The composition comprises an RNA polymerase, a transcription buffer, ribonucleoside triphosphates (rNTPs), a dinucleotide cap analog according to Formula I, a 2'0- methyltransferase and S-adenosylmethionine. The composition comprising all ingredients is provided in only one vessel.

[0220] In an embodiment, the RNA polymerase is capable of binding a promoter site operably linked upstream to a target sequence comprised on a nucleic acid template.

[0221] The dinucleotide cap analog comprised in the composition is a dinucleotide cap analog according to Formula I wherein

[0222] R1is selected from methyl, ethyl, propyl, butyl, benzyl, substituted benzyl, C5-Cg heterocyclyl, C5-Cg substituted heterocyclyl;

[0223] R2is H, OH or O-CH3;

[0224] R3is H, OH or O-CH3;

[0225] R4, R5and R6are independently selected from the group consisting of O, S, Se;

[0226] R7and R8are OH; n is 1, 2, or 3; and B is a non-modified or modified purine or pyrimidine base.

[0227] RNA polymerase capable of binding the promoter site

[0228] The RNA polymerase may be any DNA-dependent RNA polymerase capable of binding to the promoter sequence within the linearized nucleic acid template. The RNA polymerase recognizes and specifically binds to the target sequence at the promoter site from where the RNA synthesis is initiated. In preferable embodiments, the RNA polymerase is a bacteriophage-derived RNA polymerase, preferably selected from the group comprising T7, T3, Vsw-3, Kll, T3D, T5, T8, (02.5, (06, and SP6 RNA polymerases, their variants, chimeras or mutants thereof. In a further embodiment, the RNA polymerase is selected from the group consisting of a T7 RNA polymerase, a T3 RNA polymerase, and a SP6 RNA polymerase. RNA polymerases may be modified by exchanging, inserting or deleting amino acids of the RNA polymerase sequence. Variants may be obtained by evolving an RNA polymerase, optimizing the RNA polymerase amino acid and / or nucleic acid sequence and / or by using other methods known in the art.

[0229] In an embodiment, the RNA polymerase is a T7 RNA polymerase. In some examples, engineered variants of RNA polymerases may be used such as thermostable variants disclosed in W02010016621 Al, US11072808 B2, WO2017123748 Al or WO2018236617 Al. An RNA polymerase may also be evolved for selective incorporation of the capped GTP analogue over GTP as disclosed in W02019005539 Al or W02019005540 Al. Alternatively, RNA polymerase may be a chimeric enzyme as disclosed in US11072808 or evolved to have improved properties, such as increased thermostability, increased transcription efficiency, 3' homogeneity, run-on transcripts, double-stranded contaminants, as disclosed in WO2019 / 199807, US11066686, CN112831484 or US11485960. In one embodiment, T7 RNA polymerase may comprise at least one mutation such as, but not limited to, Ml, I4M, A7T, N9, DIO, G47, E63V, V64D, A65E, D66Y, T75, T76N, A83, K93T, E108, 1109, A113, 1117, C125R, S128R, V134, A136, V137, D147, N165S, E167, K172, R173, G175R, H176L, Y178H, K179, K180, F182L, L196F, G198V, H205, K206, D208Y, E222K, V227, S228A, H230, Q239R, T243N, G259D, P266, M267I, G280C, 1281, R291, V297, H300R, Y312, 1320, A323, A327, K333, W344, V340, C347, E350, D351, A354, E356D, R357, L360P, M362, T375, K378, A383V, V384, Y385C, D388, K389, R394, 1396, S397, M401, Q404, N410S, V426, A428, L446, K450R, P451T, G452V, K454, K461, E484A, M490, C492, S495, C510, A513, C515, H523L, H524N, C530, L534, G542V, F546, G555, E565K, V567, K577E, K577M, A584, D591, N601S, G618, S684Y, N601, L621, Y623, S633, M635, Y639, K642, V650, T654, Q656, D660, G664, L665, S684, V685, V689, M696, L699I, A702, K711, K713E, C723, A724, T729, P730, K740, N748D, Q754R, E775K, V783, H784, Q786, D787, G788, V795, 1810, A827V, M832, D834, T835, C839, A843, D847, F849, A850, D851N, S856, A863, L864F, A866 and / or E877.

[0230] In another embodiment, the RNA polymerase is a T3 RNA polymerase. In yet another embodiment, the RNA polymerase is an SP6 RNA polymerase. Preferably, the RNA polymerase is a T7 RNA polymerase. In addition, in an embodiment the RNA polymerase is a variant of the T7, T3 or SP6 RNA polymerase. In another embodiment, the RNA polymerase is derived from E. coli. In one embodiment, the RNA polymerase is a single subunit RNA polymerase of viral origin. In an embodiment, the RNA polymerase is derived from recombinant source. In one embodiment the RNA polymerase may be a chimeric enzyme and may be fused to another entity (such as a linker, domain, subunit of another enzyme, a tag etc.).

[0231] The composition disclosed herein can be used for the synthesis of mRNA in a bioreactor. In a particular embodiment of the invention, the composition is used for a transcription reaction carried out in a transcription reactor (bioreactor). The bioreactor is configured such that reactants are delivered via a feed line to the reactor core and RNA products are removed by passing through an ultrafiltration membrane (having a nominal molecular weight cut-off, e.g., 100,000 daltons) to the exit stream. The ultrafiltration membrane may be composed of a low protein-binding polymer matrix and serves to selectively retain both the RNA polymerase and the immobilized DNA templates in the reactor core. The transcription reactor is operated in either semi-batch or continuous-flow modes. In general, bioreactors are set up to produce either short RNA species (from approximately twenty to approximately one hundred nucleotides in length) or RNA species greater than one hundred nucleotides in length.

[0232] In an embodiment, bioreactor operates in a continuous mode, and is configured for continuous synthesis of an RNA molecule by in vitro transcription using reactants disclosed herein. The apparatus comprises a reaction module, a control module, and a mRNA separation. The reaction module, the control module, and the mRNA separation system are in operable communication with one another. The bioreactor includes at least one inlet connected to a supply line that feeds (supplies) the reactants for the reaction to the bioreactor. In an aspect, the apparatus includes a plurality of supply lines each of which independently supplies a reactant for the reaction to the bioreactor. In addition, the bioreactor includes an outlet from which the reaction product is transported to the mRNA separation system. The control module comprises at least one sensor and provides feedback control of the reaction mixture in the reaction module as well as feedback control of the reaction product in the RNA separation system. The control module receives and analyzes data received from the at least one sensor. In an aspect, the sensor comprises a plurality of sensors that independently monitor nucleotide triphosphates, capping molecules, RNA polymerase, buffer, magnesium levels, or a combination thereof in the reaction mixture. When the concentration of the reactant falls below the threshold level, the reactant is supplied to the bioreactor. As described above, only basic bioreactor structure is provided, the skilled person would understand that various improvements and modifications to the bioreactor are possible for the compositions and methods described herein to be suitable for use in bioreactors.

[0233] The composition disclosed herein can be used in an automated apparatus, device or platform for RNA production. In an embodiment automated RNA synthesis device can be an RNA printer™.

[0234] The composition disclosed herein can be used for the synthesis of mRNA in a continuous flow device or a flow reactor. The flow device may be a liquid flow device. A flow device may comprise one or more bioreactors. The flow device may be a continuous flow device. The flow reactor may be a liquid flow reactor. The flow reactor may be a continuous flow reactor.

[0235] Concentration of RNA Polymerase

[0236] Preferably, the concentration of the RNA polymerase in the final reaction mixture is from about 1 to 100 nM, 1 to 90 nM, 1 to 80 nM, 1 to 70 nM, 1 to 60 nM, 1 to 50 nM, 1 to 40 nM, 1 to 30 nM, 1 to 20 nM, or about 1 to 10 nM. Even more preferred, the concentration of the RNA polymerase is from about 10 to 100 nM, about 10 to 80 nM, about 20 to 70 nM, or 50 to 70 nM. Most preferably, the RNA polymerase has a final concentration of 60 nM

[0237] Most preferred is an RNA polymerase concentration in the final reaction mixture of about 60 nM. The person skilled in the art will understand that the choice of the RNA polymerase concentration is influenced by the concentration of the DNA template. Therefore, in specific embodiments the concentration of the RNA polymerase in the final reaction mixture is between 1 and 1000 U / pg template DNA, preferably between 100 and 300 U / pg DNA, particularly if plasmid DNA is used as template DNA.

[0238] Transcription buffer

[0239] Common buffer systems used in RNA in vitro transcription include 4-(2-hydroxy- ethyl)- 1- piperazineethanesulfonic acid (HEPES) and tris(hydroxymethyl)amino- methane (Tris). The pH value of the buffer is commonly adjusted to a pH value of 6 to 8.5.

[0240] In an embodiment, the transcription buffer comprises a buffering agent, magnesium, dithiothreitol (DTT) and / or additional components. In an embodiment, the transcription buffer comprises magnesium ions, dithiothreitol (DTT) and polyamine additives. In some embodiments polyamine is spermidine. In some examples, the transcription buffer comprises detergents.

[0241] The transcription buffer comprises magnesium salt and / or other salts, a buffering agent, DTT, and / or other ingredients.

[0242] The buffering agent may be Tris, TES, Tricine, TAPS, and / or HEPPS. In some embodiments, the transcription buffer comprises magnesium (Mg2+; e.g., a magnesium salt), preferably MgCL. The concentration of magnesium present in the final reaction can be, for example, 1-60 mM, preferably 2-50 mM, more preferably 5-40 mM, even more preferably 10-30 mM. In a specific embodiment, the final reaction composition comprises magnesium at a concentration of 10 mM. In a specific embodiment, the final reaction mixture comprises MgCL at a concentration of 15 mM. In a specific embodiment, the final reaction mixture comprises MgCL at a concentration of 20 mM. In a specific embodiment, the final reaction mixture comprises MgCL at a concentration of 25 mM. In a specific embodiment, the final reaction mixture comprises MgCL at a concentration of 30 mM.

[0243] In some embodiments, the molar ratio of rNTP to magnesium ions (Mg2+; e.g., MgCL) present in the IVT reaction is from 1:1 to 1:5. For example, the molar ratio of NTP to magnesium ion in the final reaction may be 1:1, 1:2, 1:3, 1:4 or 1:5.

[0244] In some embodiments, the transcription buffer comprises DTT. The concentration of DTT present in the final reaction can be, for example, at least 1 mM, at least 5 mM, or at least 10 mM, or 1-50 mM, preferably 1-30 mM, more preferably 5-10 mM, even more preferably 7-15 mM. In a specific embodiment, the final reaction mixture comprises DTT at a concentration of 10 mM. In a specific embodiment, the final reaction mixture comprises DTT at a concentration of 5 mM.

[0245] In some embodiments, the transcription buffer comprises spermidine. The concentration of spermidine present in the final reaction can be, for example, 0.1-10 mM, preferably 0.1-5 mM, more preferably 0.5-4 mM, even more preferably 2-5 mM. In a specific embodiment, the final reaction mixture comprises spermidine at a concentration of 2 mM. In a specific embodiment, the final reaction mixture comprises spermidine at a concentration of 1 mM.

[0246] In some embodiments, the transcription buffer comprises Tris as a buffering agent. The concentration of Tris used in the final reaction can be, for example, at least 10 mM, at least 20 mM, at least 30 mM, at least 40 mM, at least 50 mM, at least 60 mM, at least 70 mM, at least 80 mM, at least 90 mM, at least 100 mM or at least 110 mM.

[0247] Ribonucleoside triphosphates (NTPs)

[0248] In an embodiment, the ribonucleoside triphosphates (rNTPs) comprise ATP, CTP, UTP, GTP and any modified ribonucleotide. The modification may be one or more of a backbone modification, a sugar modification and / or a nucleobase modification. A backbone modification is a modification, in which phosphates of the backbone of the ribonucleotides are chemically modified. A sugar modification in connection with the present invention is a chemical modification of the sugar of the ribonucleotides. Furthermore, a base modification in connection with the present invention is a chemical modification of the base moiety of the ribonucleotides. In this context ribonucleotide modifications are applicable for transcription and / or translation

[0249] According to an embodiment of the invention, a part or all of at least one ribonucleoside triphosphate in the reaction mixture is replaced with a modified nucleoside triphosphate. The ribonucleoside triphosphates provided herein include unmodified or modified ATP, modified or unmodified UTP, modified or unmodified GTP, and / or modified or unmodified CTP. In some embodiments, the rNTPs of in the reaction mixture comprise unmodified ATP. In some embodiments, the rNTPs of in the reaction mixture comprise modified ATP. In some embodiments, the rNTPs of in the reaction mixture comprise an unmodified UTP. In some embodiments, the rNTPs of in the reaction mixture comprise a modified UTP. In some embodiments, the rNTPs of in the reaction mixture comprise unmodified GTP. In some embodiments, the rNTPs of in the reaction mixture comprise modified GTPs. In some embodiments, the rNTPs of in the reaction mixture comprise unmodified CTPs. In some embodiments, the rNTPs of in the reaction mixture comprise modified CTPs.

[0250] In a preferred embodiment of the invention, said modified nucleoside triphosphate is selected from the group consisting of pseudouridine-5'-triphosphate, l-methylpseudouridine-5'-triphosphate, 2- thiouridine-5'-triphosphate, 4-thiouridine-5'-triphosphate and 5-methylcytidine-5'-triphosphate. According to an embodiment of the invention, a part or all of UTP in the reaction mixture is replaced with a modified UTP selected from the group consisting of pseudouridine-5'-triphosphate, 1- methylpseudouridine-5'-triphosphate, 2-thiouridine-5'-triphosphate, 4-thiouridine-5'-triphosphate. According to another embodiment of the invention, a part or all of CTP in the reaction mixture is replaced with 5-methylcytidine-5'-triphosphate.

[0251] Alternatively, the ribonucleotide modifications may be selected from base modifications, which are preferably selected from 2-amino-6-chloropurineriboside-5'-triphosphate, 2-Aminopurine-riboside- 5'-triphosphate; 2-aminoadenosine-5'-triphosphate, 2'-Amino-2'-deoxycytidine-triphosphate, 2- thiocytidine-5'-triphosphate, 2-thiouridine-5'-triphosphate, 2'-Fluorothymidine-5'-triphosphate, 2'-O- Methyl inosine-5'-triphosphate 4-thiouridine-5'-triphosphate, 5-aminoallylcytidine-5'-triphosphate, 5-aminoallyluridine-5'-triphosphate, 5-bromocytidine-5'-triphosphate, 5-bromouridine-5'- triphosphate, 5-Bromo-2'-deoxycytidine-5'-triphosphate, 5-Bromo-2'-deoxyuridine-5'-triphosphate, 5-iodocytidine-5'-triphosphate, 5-lodo-2'-deoxycytidine-5'-triphosphate, 5-iodouridine-5'- triphosphate, 5-lodo-2'-deoxyuridine-5'-triphosphate, 5-methylcytidine-5'-triphosphate, 5- methyluridine-5'-triphosphate, 5-Propynyl-2'-deoxycytidine-5'-triphosphate, 5-Propynyl-2'- deoxyuridine-5'-triphosphate, 6-azacytidine-5'-triphosphate, 6-azauridine-5'-triphosphate, 6- chloropurineriboside-5'-triphosphate, 7-deazaadenosine-5'-triphosphate, 7-deazaguanosine-5'- triphosphate, 8-azaadenosine-5'-triphosphate, 8-azidoadenosine-5'-triphosphate, benzimidazole- riboside-5'-triphosphate, Nl-methyladenosine-5'-triphosphate, Nl-methylguanosine-5'-triphosphate, N6-methyladenosine-5'-triphosphate, 06-methylguanosine-5'-triphosphate, pseudouridine-5'- triphosphate, or puromycin-5'-triphosphate, xanthosine-5'-triphosphate. Particular preference is given to nucleotides for base modifications selected from the group of base-modified nucleotides consisting of 5-methylcytidine-5'-triphosphate, 7-deazaguanosine-5'-triphosphate, 5-bromocytidine- 5'-triphosphate, and pseudouridine-5'-triphosphate.

[0252] Modified ribonucleosides may be selected from pyridin-4-one ribonucleoside, 5-aza-uridine, 2-thio-5- aza-uridine, 2-thiouridine, 4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxyuridine, 3- methyluridine, 5-carboxymethyl-uridine, 1-carboxymethyl-pseudouridine, 5-propynyl-uridine, 1- propynyl-pseudouridine, 5-taurinomethyluridine, 1-taurinomethyl-pseudouridine, 5-taurinomethyl- 2-thio-uridine, l-taurinomethyl-4-thio-uridine, 5-methyl-uridine, 1-methyl-pseudouridine, 4-thio-l- methyl-pseudouridine, 2-thio-l-methyl-pseudouridine, 1-methyl-l-deaza-pseudouridine, 2-thio-l- methyl-l-deaza-pseudouridine, dihydrouridine, dihydropseudouridine, 2-thio-dihydrouridine, 2-thio- dihydropseudouridine, 2-methoxyuridine, 2-methoxy-4-thio-uridine, 4-methoxy-pseudouridine, and 4-methoxy-2-thio-pseudouridine, 5-aminoallyluridine, 5-iodo-uridine, 5,6-dihydrouridine, a-thio- uridine, 4-thio-uridine, 6-aza-uridine, 5-hydroxy-uridine, deoxy-thymidine, 5-methyl-uridine.

[0253] Modified ribonucleosides may be selected from 5-aza-cytidine, pseudoisocytidine, 3-methyl-cytidine, N4-acetylcytidine, 5-formylcytidine, N4-methylcytidine, 5-hydroxymethylcytidine, 1-methyl- pseudoisocytidine, pyrrolo-cytidine, pyrrolo-pseudoisocytidine, 2-thio-cytidine, 2-thio-5-methyl- cytidine, 4-thio-pseudoisocytidine, 4-thio-l-methyl-pseudoisocytidine, 4-thio-l-methyl-l-deaza- pseudoisocytidine, 1-methyl-l-deaza-pseudoisocytidine, zebularine, 5-aza-zebularine, 5-methyl- zebularine, 5-aza-2-thio-zebularine, 2-thio-zebularine, 2-methoxy-cytidine, 2-methoxy-5-methyl- cytidine, 4-methoxy-pseudoisocytidine, and 4-methoxy-l-methyl-pseudoisocytidine, 6-aza-cytidine, 2-thio-cytidine, a-thio-cytidine, Pseudo-iso-cytidine.

[0254] Modified ribonucleosides may be selected from 2-aminopurine, 2,6-diaminopurine, 7-deaza-adenine, 7-deaza-8-aza-adenine, 7-deaza-2-aminopurine, 7-deaza-8-aza-2-aminopurine, 7-deaza-2,6- diaminopurine, 7-deaza-8-aza-2,6-diaminopurine, 1-methyladenosine, N6-methyladenosine, N6- isopentenyladenosine, N6-(cis-hydroxyisopentenyl)adenosine, 2-methylthio-N6-(cis- hydroxyisopentenyl) adenosine, N6-glycinylcarbamoyladenosine, N6-threonylcarbamoyladenosine, 2-methylthio-N6-threonyl carbamoyladenosine, N6,N6-dimethyladenosine, 7-methyladenine, 2- methylthio-adenine, and 2-methoxy-adenine, a-thio-adenosine, 8-azido-adenosine.

[0255] Modified ribonucleosides may be selected from inosine, 1-methyl-inosine, wyosine, wybutosine, 7- deaza-guanosine, 7-deaza-8-aza-guanosine, 6-thio-guanosine, 6-thio-7-deaza-guanosine, 6-thio-7- deaza-8-aza-guanosine, 7-methyl-guanosine, 6-thio-7-methyl-guanosine, 7-methylinosine, 6- methoxy-guanosine, 1-methylguanosine, N2-methylguanosine, N2,N2-dimethylguanosine, 8-oxo- guanosine, 7-methyl-8-oxo-guanosine, l-methyl-6-thio-guanosine, N2-methyl-6-thio-guanosine, and N2,N2-dimethyl-6-thio-guanosine, a-thio-guanosine, 6-methyl-guanosine, 5-methyl-cytdine, 8-oxo- guanosine, 7-deaza-guanosine.

[0256] In some embodiments, the ribonucleotide can be modified on the major groove face and can include replacing hydrogen on C-5 of uracil with a methyl group or a halo group.

[0257] In specific embodiments, a modified ribonucleoside is 5'-O-(l-Thiophosphate)-Adenosine, 5'-O-(l- Thiophosphate)-Cytidine, 5'-O-(l-Thiophosphate)-Guanosine, 5'-O-(l-Thiophosphate)-Uridine or 5'- O-(1-Thiophosphate)-Pseudouridine.

[0258] Further modified nucleotides have been described previously (see, e.g., WO 2013 / 052523).

[0259] Concentration of rN TPs

[0260] According to a preferred embodiment of the invention, the total rNTP concentration in the reaction mixture is between 1 and 200 mM, preferably between 2 and 100 mM, preferably between 10 and 50 mM, and most preferably between 15 and 25 mM. According to the invention, the term "total ribonucleotide concentration" means the total concentration of rNTPs, i.e., the sum of the concentrations of ATP, GTP, CTP, UTP, optionally any modified rNTP and the dinucleotide cap analog present initially in the reaction when the various components of the reaction have been assembled in the final volume for carrying out the reaction. Naturally, as the reaction proceeds, the nucleotides will be incorporated into the RNA molecule and consequently the total nucleotide concentration will be progressively reduced from its initial value.

[0261] In this context it is particularly preferred that the single ribonucleotides ATP, GTP, CTP, UTP, and any modified ATP, GTP, CTP, UTP, are provided in a concentration between 0.1 and 20 mM, preferably between 1 and 10, preferably between 3 and 7 mM, more preferably between 4 mM and 6 mM, and most preferably in a concentration of 5 mM.

[0262] The concentration of GTP is preferably reduced compared to the other ribonucleotides (ATP, CTP and UTP). In an embodiment of the invention, GTP is present in a ratio GTP:dinucleotide cap analog from 1:1 to 1:10. In an embodiment of the invention, GTP is present in a ratio GTP:dinucleotide cap analog from 1:1 to 1:8, from 1:1 to 1:7, from 1:1 to 1:6, from 1:1 to 1:5, from 1:1 to 1:4, from 1:1 to 1:3, from 1:1 to 1:2. In a preferred embodiment of the invention, GTP is present in a ratio GTP:dinucleotide cap analog from 1:3 to 1:5. In an even more preferred embodiment of the invention, GTP is present in a ratio GTP:dinucleotide cap analog of 1:4.

[0263] In this context it is particularly preferred that the single ribonucleotide GTP and any modified GTP, is provided in a concentration between 0.1 and 10 mM, preferably between 0.1 and 5 mM, more preferably between 0,5 mM and 2 mM, and most preferably in a concentration of 1 mM.

[0264] Dinucleotide cap analog

[0265] The dinucleotide cap analog is a compound of Formula I: wherein

[0266] R1is selected from methyl, ethyl, propyl, butyl, benzyl, substituted benzyl, C5-Cg heterocyclyl, C5-Cg substituted heterocyclyl;

[0267] R2is H, OH or O-CH3;

[0268] R3is H, OH or O-CH3;

[0269] R4, R5and R6are independently selected from the group consisting of O, S, Se;

[0270] R7and R8are OH; n is 1, 2, or 3; and

[0271] B is a non-modified or modified purine or pyrimidine base.

[0272] A „dinucleotide cap analog" is a dinucleotide consisting of an outer cap nucleoside, such as 7-methyl- guanosine (m7G), and the nucleotide corresponding to the first nucleotide of the primary transcript (e.g., G) depicted as "B" in Formula I. Therefore, an exemplary cap analog is the m7G(5')ppp(5')G, also referred to as m7GpppG. This cap analog is often used because the primary nucleotide (i.e., the most 5' nucleotide) of most, but not all, primary RNA transcripts synthesized using phage RNA polymerase transcription systems is guanosine.

[0273] The dinucleotide cap analogs are enzymatically incorporated at the mRNA 5'-end by the RNA polymerase, e.g., bacteriophage T7 RNA polymerase during in vitro transcription reaction, i.e., co- transctiptionally.

[0274] The most commonly used cap analogue is m7GpppG but several modified or alternative cap analogues are also accepted by RNA polymerases. Therefore, this route can also be used to install non-natural dinucleotides at the 5'-end that are accessible for a further chemical reaction.

[0275] In a preferred embodiment, the dinucleotide cap analog is an anti-reverse cap analog (ARCA). Thus, in a preferred embodiment, the dinucleotide cap analog is methylated at the 2' -OH or at the 3'-OH of the N7-methylguanosine ribose (m27,3 OGpppN or m27,2 OGpppN). This prevents elongation at the "wrong" 3'-OH and thus prevents yielding mRNA with the cap in reverse orientation as ARCA caps are exclusively incorporated in the correct orientation.

[0276] In an embodiment, the dinucleotide cap analog has Formula I, wherein one of R2or R3of the dinucleotide cap analog is O-CH3. In a preferable embodiment, the dinucleotide has Formula I, wherein R2is OH and R3is O-CH3. In another preferable embodiment, the dinucleotide has Formula I, wherein R2is O-CH3 and R3is OH.

[0277] The dinucleotide cap analog may preferably have a 3'-O-methyl ribose modification or a 2'-O-methyl ribose modification and may have three, four or five phosphate moieties, i.e., m27,3 OGpppN or m27,2'- °GpppN, m27,3 OGppppN or m27,2 OGppppN, m27,3 OGpppppN or rri27'2 OGpppppN.

[0278] In a preferred embodiment, B (N) is guanosine.

[0279] In a specific embodiment, the dinucleotide cap analog is an anti-reverse cap analog (ARCA) having the structural formula of m27,3 OGpppG also abbreviated as 3'-O-Me-m7G(5')pppG(5'):

[0280] Concentration of dinudeotide cap analogs

[0281] According to a preferred embodiment of the invention, the concentration of the dinucleotide cap analog in the reaction mixture is between 0.1 and 100 mM, between 1 and 50 mM, preferably between 1 and 10 mM, and most preferably between 3 and 5 mM. In a specific embodiment, the dinucleotide cap analog is present in the reaction mixture at a concentration of 4 mM. In a specific embodiment, the dinucleotide cap analog is present in the reaction mixture at a concentration of 5 mM.

[0282] According to a preferred embodiment of the invention, the total rNTP concentration in the reaction mixture is between 1 and 100 mM, preferably between 10 and 50 mM, and most preferably between 15 and 25 mM. According to the invention, the term total ribonucleotide concentration means the total concentration of rNTPs, e.g. the sum of the concentrations of ATP, GTP, CTP, UTP, optionally any modified rNTP and the dinucleotide cap present initially in the reaction when the various components of the reaction have been assembled in the final volume for carrying out the reaction. Naturally, as the reaction proceeds, the nucleotides will be incorporated into the RNA molecule and consequently the total nucleotide concentration will be progressively reduced from its initial value.

[0283] The concentrations of dinucleotide cap analogs and nucleoside triphosphates present in the IVT reaction can vary, as described for the rNPTs before. In some embodiments, the NTP and dinucleotide cap analogs are present in equimolar concentrations in the reaction. That means the molar ratio of dinucleotide cap analog to rNTP is 1:1. In some embodiments, the molar ratio of the dinucleotide cap analog to nucleoside triphosphate in the reaction is greater than 1:1. For example, the molar ratio of cap analog to nucleoside triphosphate in the reaction is 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1. In some embodiments, the molar ratio of the dinucleotide cap analog to nucleoside triphosphate in the reaction is less than 1:1. In some embodiments, the molar ratio of the dinucleotide cap analog to nucleoside triphosphate in the reaction is less than 4:5. In an embodiment, the molar ratio of the dinucleotide cap analog to nucleoside triphosphate ranges from 1:1 to 8:1. In an embodiment, the molar ratio of the dinucleotide cap analog to nucleoside triphosphate ranges from 0.2:1 to 8:1. In preferred embodiments, the molar ratio of the dinucleotide cap analog to ATP is 1:1. In preferred embodiments, the molar ratio of the dinucleotide cap analog to CTP is 1:1. In preferred embodiments, the molar ratio of the dinucleotide cap analog to UTP is 1:1. In preferred embodiments, the molar ratio of the dinucleotide cap analog to a modified ATP, CTP or UTP is 1:1. In preferred embodiments, the molar ratio of the dinucleotide cap analog to GTP ranges from 1:1 to

[0284] 8:1. In a specific embodiment, the dinucleotide cap analog is present at a ratio dinucleotide cap analog to GTP of 4:1.

[0285] 2'-O-methyltransferase

[0286] "2'-O-methyltransferase" is used interchangeable with "2'-O-ribonucleoside methyltransferase"

[0287] Cap-0 transcripts can be enzymatically converted to cap-1 in vitro. According to the invention, mRNA cap 2'-O-methyltransferase adds a methyl group at the 2'-0 position of the first nucleotide adjacent to the cap structure at the 5' end of the RNA. The enzyme utilizes S-adenosylmethionine (SAM) as a methyl donor to methylate capped RNA (Cap-0) resulting in a Cap-1 structure. It has been reported that such capl structure may result in a higher translational-competency (Kuge et al. (1998) Nucl. Acids Res. 26(13): 3208-3214.) and cellular stability and a reduced activation of cellular pro- inflammatory cytokines, as compared, e.g., to other cap analog structures known in the art.

[0288] In an embodiment, the 2'-O-methyltransferase is of viral origin. In preferable embodiments, the 2'-O- methyltransferase is derived from a Vaccinia virus. In embodiments, the 2'-O-methyltransferase is a Vaccinia virus cap-specific ribonucleoside 2'-O-methyltransferase, functional mutant, chimera or variant thereof. In an embodiment, 2'-O-methyltransferase is derived from SARS-CoV-2. In some embodiments, the 2'-O-methyltransferase is RNA polymerase exhibiting 2'-O-methyltransferase activity (see for example "An RNA cap (nucleoside-2'-O-)-methyltransferase in the flavivirus RNA polymerase NS5: crystal structure and functional characterization", Egloff et al., 2002, The EMBO Journal.)

[0289] The 2'-O-methyltransferase transfers a methyl group from the co-substrate SAM to the target molecule.

[0290] Concentrations of 2'0-methyltransferase

[0291] The concentration of 2'-O-methyltransferase in the final reaction is between 10 and 100 Units. Preferably, the concentration of 2'-O-methyltransferase in the final reaction is between 25 and 75 Units. In a preferred embodiment, the concentration of 2'-O-methyltransferase in the final reaction is about 50 Units. In a preferred embodiment, the concentration of 2'-O-methyltransferase in the final reaction is 50 Units.

[0292] S-adenosylmethionine (SAM)

[0293] As used herein, "S-adenosylmethionine", "S-Adenosyl-L-methionine", "SAM", and "AdoMet" are used interchangeably and refer to a compound having formula C15H22N6O5. The 2'-O-methyltransferase transfers a methyl group from the co-substrate S-adenosyl-L-methionine to first nucleotide adjacent to the cap structure at the 5' end of the mRNA.

[0294] Concentrations of S-adenosylmethionine

[0295] The concentration of S-adenosylmethionine in the final reaction is between 0.05 and ImM. Preferably, the concentration of S-adenosylmethionine in the final reaction is 0.2-0.3 mM. In preferred embodiments, the concentration of S-adenosylmethionine in the final reaction is about 0.25 mM. In preferred embodiments, the concentration of S-adenosylmethionine in the final reaction is 0.25 mM.

[0296] Poly (A) polymerase

[0297] In some embodiments, the composition optionally further comprises a poly(A) polymerase.

[0298] If the target sequence comprised in the nucleic acid template does not contain a poly(d(A / T)) sequence transcribed into a poly(A) tail, a poly(A) tail may also be added post transcriptionally by enzymatic polyadenylation using a poly(A) polymerase. Poly(A) polymerase catalyzes the templateindependent addition of AMP from ATP to the 3' end of RNA.

[0299] A poly(A) tail may contain 10 to 300 adenosine monophosphates. For example, a poly(A) tail may contain 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290 or 300 adenosine monophosphates. In some embodiments, a poly(A) tail contains 50 to 170 adenosine monophosphates. In preferred embodiments, the poly(A) tail contains between 120 and 150 adenosine monophosphates, preferably 120 adenosine monophosphates.

[0300] The poly(A) polymerase may be modified or unmodified. In a preferred embodiment, the modified or unmodified poly(A) polymerase is of bacterial origin. In a more preferred embodiment, the modified or unmodified poly(A) polymerase is derived from Escherichia coll.

[0301] The present composition may also be provided as a stock composition, which may be diluted shortly before usage.

[0302] Storage

[0303] In an embodiment, the composition is suitable for long term storage. In an embodiment, the composition is stable for about 1-10 days, including 1 day including 5-24 hours, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 4 months, 1-5 months, 1-6 months. The composition being stable means that the components are still reactive and a reaction can be initiated after the time of storage with no significant loss of product yield. In an embodiment, the composition is stored at appropriate temperatures, comprising temperatures below 8°C.

[0304] Use of the composition for synthesis ofmRNA therapeutics.

[0305] The composition of the invention disclosed herein may be used for synthesis of mRNA therapeutics.

[0306] In an embodiment, mRNA therapeutics comprise compositions for treating cancer. In an embodiment, mRNA therapeutics comprise compositions for treating and / or prevention of infectious disease. In an embodiment, mRNA therapeutics comprise an infectious disease vaccine. In an embodiment, mRNA therapeutics comprise compositions for allergy tolerization. In an embodiment, mRNA therapeutics comprise compositions for protein replacement therapy.

[0307] In an embodiment, mRNA therapeutics delivery is enhanced by LNPs, polymeric nanoparticles, cationic nanoemulsions or other delivery systems (see for example Qin, S., Tang, X., Chen, Y. et al., mRNA-based therapeutics: powerful and versatile tools to combat diseases, Sig Transduct Target Ther 7, 166 (2022)).

[0308] Use of the composition in a bioreactor for RNA in vitro transcription

[0309] The composition according to the invention disclosed herein may be used in a bioreactor for mRNA production. In an embodiment, such bioreactor allows for repetitive use of DNA templates in several RNA production processes. In an embodiment, in such bioreactor DNA templates are immobilized on solid support.

[0310] By using the composition of the invention, mRNA manufacturing is accelerated since all reaction components are present in only one vessel. This decreases the risk of contamination and saves time in adjusting the reaction settings.

[0311] Using the inventive composition disclosed herein with a bioreactor is preferably suitable for a use under regulated conditions (GMP) and suitable for pharmaceutical applications (e.g. pharmaceutical mRNA production). The bioreactor may allow a continuous production or repeated batch production of a liquid mRNA composition.

[0312] Exemplary bioreactors are described in W02020002598 Al, W02022261050A1 or WO2022081519 Al.

[0313] In an embodiment, the bioreactor is an automated system, such as an RNA printer™ (see, for example WO2019122371, W02020002598 Al or Sheridan, C., mRNA printers kick-start personalized medicines for all, Nat Biotechnol 40, 1160-1162 (2022)).

[0314] Use of the composition in a continuous flow device

[0315] The composition according to the invention disclosed herein may be used in a continuous flow device or flow reactor for mRNA production. Methods of continuous flow in vitro transcription are described e.g., in EP1165826 Bl, US20230265477 Al or references therein.

[0316] By using the composition of the invention, mRNA manufacturing is accelerated since all reaction components are present in only one vessel. This decreases the risk of contamination and saves time in adjusting the reaction settings.

[0317] Using the inventive composition disclosed herein with a continuous flow device is preferably suitable for a use under regulated conditions (GMP) and suitable for pharmaceutical applications (e.g. pharmaceutical mRNA production). The continuous flow device may allow for a continuous production or repeated batch production of a liquid mRNA composition. In such device, mRNA is continuously synthesized, while the mRNA product is removed from the flow chamber and a fresh composition according to the invention is flowing into the flow chamber.

[0318] Exemplary continuous flow devices are described in US20200340028 or US20230265476.

[0319] In some embodiments method of present invention can be used in bioreactors for continuous or semi-batch process for synthesizing RNA molecules. The bioreactor contains immobilized DNA used as a template for the transcription reaction. The immobilization of the DNA template allows a repeated usage of the template and reduces the contamination of the desired RNA product by residual DNA. During the transcription reaction, required reaction components, such as enzymes, SAM, NTPs, reaction buffer, etc., may be supplied to the bioreactor. After transcription, the produced RNA molecules can be released batch by batch or continuously to be captured and separated from other soluble components of the transcription reaction and to be moved into other compartments of the bioreactor for purification and other processing. EXAMPLES

[0320] Aspects of the present teachings may be further understood in light of the following examples, which should not be construed as limiting the scope of the present teachings in any way.

[0321] Example 1: 2'0-Methyltransferase one-pot reaction in IVT

[0322] Experiment outline

[0323] The experimental system was based on synthesizing a 25 nt RNA molecule from a linearized template pDNA (pTZ19R (Thermo Scientific cat. no. SD0141) digested with Hindi restriction endonuclease (Thermo Scientific cat. no. FD0494)). Three sets of IVT reactions were performed based on a general co-transcriptional capping IVT protocol as provided below:

[0324] Co-transcriptional IVT followed by purification of the product and subsequent reaction with 2OMt and SAM (Sequential reaction).

[0325] Co-transcriptional IVT followed by the addition of 2OMt and SAM to the reaction mixture (Add-on reaction).

[0326] Co-transcriptional IVT added with 2OMt and SAM from the start (One-pot reaction).

[0327] For all three reactions, the concentration of the reaction components wase the same. The IVT reaction was performed in lx MessageMachine™ buffer as the transcription buffer (Thermo Scientific cat. no. AM1344) containing 35 ng / pL of the pTZ19R / Hincll template. ATP, CTP, and UTP were added to a final concentration of 5 mM each, and GTP was added at 1 mM. A dinucleotide cap analog (here ARCA, Thermo Scientific cat. no. AM8045) was added at a final concentration of 4 mM, yielding a GTP-ARCA ratio of 1:4. T7 RNA polymerase was added as a last component, in the form of Message Machine™ T7 Enzyme Mix (Thermo Scientific cat. no. AM1344). This master mix contained RNase inhibitor. To achieve methylation of capped RNA and thus to convert capO into capl structure, the reaction additionally comprised 2'0-methyltransferase (2OMt, Thermo Fisher Scientific) to a final concentration of 2.5 U / pL and S-adenosyl methionine (SAM, Thermo Scientific) to a final concentration of 0.25 mM.

[0328] The three reactions were performed in appropriate reagent omissions to yield uncapped RNA, ARCA- RNA, and 2'0-methyl-ARCA-RNA.

[0329] Sequential reaction

[0330] IVT reaction was performed in lx MessageMachine™ buffer as the transcription buffer (Thermo Scientific cat. no. AM1344) containing 35 ng / pL of the pTZ19R / Hincll template. ATP, CTP, and UTP were added to a final concentration of 5 mM, and GTP was added at 1 mM. A dinucleotide cap analog (ARCA, Thermo Scientific cat. no. AM8045) was added at a final concentration of 4 mM, yielding a GTP-ARCA ratio of 1:4. T7 RNA polymerase was added as a last component, in the form of Message Machine™ T7 Enzyme Mix (Thermo Scientific cat. no. AM1344). This master mix contained RNase inhibitor, and the reaction was incubated at 37 °C for 2 hours.

[0331] The generated capped mRNA was purified enzymatically. The first step was to use the RNA polyphosphatase (BioSearch Technologies cat. no. RP8092H) according to the manufacturer's recommendations to convert 5' triphosphates of uncapped mRNA molecules into monophosphates. After purification using Collibri™ DNA Library Cleanup Kit (Thermo Scientific cat. no. A38584096) according to manufacturer's recommendations on purifying RNA, terminator 5'-phosphate- dependent exonuclease (BioSearch Technologies cat. no. TER51020) was used based on manufacturer's recommendations to remove uncapped RNA. The purification was performed using Collibri™ DNA Library Cleanup Kit (Thermo Scientific cat. no. A38584096) according to manufacturer's recommendations on purifying RNA.

[0332] To achieve methylation of capped RNA and thus to convert capO into capl structure, 2'0- methyltransferase (20Mt, Thermo Fisher Scientific) was added to the purified mRNA to a final concentration of 2.5 U / pL and S-adenosyl methionine (SAM, Thermo Scientific) to a final concentration of 0.25 mM. The reaction was incubated at 37 °C for one hour. The purification was performed using Collibri™ DNA Library Cleanup Kit (Thermo Scientific cat. no. A38584096) according to manufacturer's recommendations on purifying RNA. Integrity of mRNA as well as capping efficiency is confirmed by mass spectrometry as provided in Figs. 1-5.

[0333] Add-on reaction

[0334] The Add-on reaction was performed similarly to the Sequential reaction, however, without purifying the capped mRNA as an intermediate step. In the Add-on reaction, the 2'0-methyltransferase and the S-adenosyl methionine (SAM) were directly added to the IVT reaction after capping reaction has been completed.

[0335] After adding 2'0-methyltransferase and the S-adenosyl methionine the reaction was incubated at 37 °C for one hour. The purification was performed using Collibri™ DNA Library Cleanup Kit (Thermo Scientific cat. no. A38584096) according to manufacturer's recommendations on purifying RNA.

[0336] One-pot reaction

[0337] The IVT reaction was performed in lx MessageMachine™ buffer as the transcription buffer (Thermo Scientific cat. no. AM1344) containing 35 ng / pL of the pTZ19R / Hincll template. ATP, CTP, and UTP were added to a final concentration of 5 mM, and GTP was added at 1 mM. A dinucleotide cap analog (ARCA, Thermo Scientific cat. no. AM8045) was added at a final concentration of 4 mM, yielding a GTP-ARCA ratio of 1:4. The reaction additionally comprised 2'0-methyltransferase (2OMt, Thermo Fisher Scientific) to a final concentration of 2.5 U / pL and S-adenosyl methionine (SAM, Thermo Scientific) to a final concentration of 0.25 mM. T7 RNA polymerase was added last in the form of Message Machine T7 Enzyme Mix (Thermo Scientific cat. no. AM1344) to initiate the reaction, which was incubated at 37 °C for 2 hours. This master mix contained RNase inhibitor.

[0338] All three reactions were enzymatically purified to remove the plasmid DNA template by adding 1 pL of Turbo DNase (Thermo Scientific cat. no. AM2238) and incubating for 10 minutes at 37 °C, followed by inactivation at 65 °C in the presence of 50 mM EDTA (Thermo Scientific cat. no. AM9260G).

[0339] The reaction products were purified using magnetic bead clean-up kit (Thermo Scientific cat. no. A43190024) according to the manufacturer's instructions.

[0340] Example 2 - Analysis of produced mRNA

[0341] Resulting products obtained in Example 1 were analyzed using capillary electrophoresis and then subject to LC-MS analysis. Liquid chromatography was carried out on a Vanquish Horizon UHPLC System using two-phase separation (mobile phase A: 10 mM triethylamine+100 mM hexafluoroisopropanol; mobile phase B: 1:1 mobile phase A+methanol) on a DNAPac HPLC column (4 pm, 2.1x100 mm, Thermo Scientific cat. no. 088923). Mass spectrometry was carried out on a Q Exactive™ Plus Hybrid Quadrupole-Orbitrap™ Mass Spectrometer (Thermo Scientific). Results are represented in FIG 1-5.

[0342] The reaction products were detected as m / z values described in FIGS 1-5. Fig. 1: RNA vs ARCA-RNA. Top spectrum depicts the distinct peaks characteristic of ARCA-capped 26 nt mRNA. Bottom spectrum depicts the characteristic peaks of the same 25 nt mRNA molecule without ARCA. The highlighted m / z difference between species is 293, corresponding to ARCA molecular weight.

[0343] Fig. 2: ARCA-RNA vs 2'-OMe-ARCA-RNA. Top spectrum depicts ARCA-RNA with its characteristic m / z 8887 (only single charge species are observed herein). Bottom spectrum shows an m / z peak at 8901, an increase of m / z 14 corresponding to a methyl group.

[0344] Fig. 3: ARCA-RNA vs 2'-OMe-ARCA-RNA from sequential reaction. Top spectrum depicts ARCA-RNA with its characteristic m / z 8887 (only single charge species are observed herein). Bottom spectrum shows an m / z peak at 8901, an increase of m / z 14 corresponding to a methyl group.

[0345] Fig. 4: ARCA-RNA vs 2'-OMe-ARCA-RNA from add-on reaction. Top spectrum depicts ARCA-RNA with its characteristic m / z 8887 (only single charge species are observed herein). Bottom spectrum shows an m / z peak at 8901, an increase of m / z 14 corresponding to a methyl group.

[0346] Fig. 5: ARCA-RNA vs 2'-OMe-ARCA-RNA from one-pot reaction. Top spectrum depicts ARCA-RNA with its characteristic m / z 8887 (only single charge species are observed herein). Bottom spectrum shows an m / z peak at 8901, an increase of m / z 14 corresponding to a methyl group.

[0347] The best results of capped mRNA having a capl structure could be achieved with the one-pot system containing the co-transcriptional IVT mix added with 2OMt and SAM. Relative abundances from mass spectrum offered representative data depicted in FIG 7. The capping efficiency corresponds to the values observed in the literature. Capped fraction equals to the capping efficiency, corresponding to the produced ARCA-RNA. Interestingly, 100% methylation efficiency is observed, as no unmethylated species are observed in the mass spectrum (fig. 5). The capped, methylated fraction is therefore equal to the capping efficiency, as 100% of cap is methylated.

[0348] The results are concurrent with ARCA capping efficiency (%Cap) reported in literature, additionally demonstrating 100% methylation efficiency. The present invention allows to obtain a cap-1 structure on the nascent mRNA molecules by having a 2'0-methyltransferase and SAM present in the reaction. Such effect presents the means to obtain an mRNA moiety more readily suitable for direct transfection with potentially better outcomes.

[0349] Example 3 - comparison of capping efficiency and ARCA methylation using different reaction strategies

[0350] Three sets of IVT reactions with different modes / strategies (sequential reaction, Add-on reaction and one-pot reaction) were performed based on a general co-transcriptional capping IVT protocol as described above in Example 1 with slight modification as described further. In the methylation step of the sequential reaction workflow two enzymes were compared. 2'-Omethyltransferase (Thermo Scientific) with the reaction composed of: 10X Capping buffer (Thermo Scientific, lOx buffer composition: 400mM Tris-HCI, 275 mM MgCL, 100 mM DTT, 20 mM Spermidine), S- adenosylmethionine (New England Biolabs, Catalog No.: B9003S), RiboLock RNase Inhibitor (Thermo Scientific, Catalog No.: EO0382), Water, nuclease-free (Thermo Scientific, Catalog No.: R0581), as well as, mRNA Cap 2'-O-Methyltransferase (New England Biolabs, Catalog No.: M0366S) with the reaction composed of: 10X Capping buffer (New England Biolabs, Catalog No.: B2080A), S- adenosylmethionine, (New England Biolabs, Catalog No.: B9003S), RiboLock RNase Inhibitor (Thermo Scientific, Catalog No.: EO0382), Water, nuclease-free (Thermo Scientific, Catalog No.: R0581). The reaction products were analyzed using LC-MS, the results are provided in Fig. 8 (reaction with NEB 2OMt enzyme is denoted "competitor"). Capping efficiency (%) pertains to the mass spectrometry data, i.e., the ratio of RNA and ARCA-RNA species. ARCA methylation (%) pertains to mass spectrometry data, i.e., the ratio of ARCA-RNA and 2'-0Me-ARCA-RNA. As can be seen from the figure 8, highest capping and ARCA methylation efficiency was achieved in One-pot reaction workflow settings.

Claims

Claims1. A method of synthesizing capped mRNA, comprising(a) combining, in a single vessel:(i) a nucleic acid template comprising a target sequence to be transcribed and an RNA polymerase promoter site operably linked upstream to the target sequence,(ii) an RNA polymerase capable of binding the promoter site,(iii) a transcription buffer,(iv) ribonucleoside triphosphates (NTPs),(v) a dinucleotide cap analog according to Formula IwhereinR1is selected from methyl, ethyl, propyl, butyl, benzyl, substituted benzyl, Cs-Ce heterocyclyl, C5-C6 substituted heterocyclyl;R2is H, OH or O-CH3;R3is H, OH or O-CH3;R4, R5and R6are independently selected from the group consisting of O, S, Se;R7and R8are OH; n is 1, 2, or 3; andB is a non-modified or modified purine or pyrimidine base,(vi) a 2'-O-methyltransferase, and(vii) S-adenosylmethionine (SAM), to produce an IVT reaction mixture(b) incubating the IVT reaction mixture under conditions allowing for co- transcriptional mRNA capping and methylation of capped mRNA, to generate a synthesized capped mRNA having a capl-type structure.

2. The method of claim 1, wherein the IVT reaction mixture further comprises an RNase inhibitor, a pyrophosphatase, and / or PEG.

3. The method of claim 1 or 2, wherein at least one of R2or R3of the dinucleotide cap analog is O-CH3, preferably, wherein R2is OH and R3is O-CH3.

4. The method of any of claims 1-3, wherein transcription, mRNA capping and methylation of capped mRNA occur simultaneously.

5. The method of any of claims 1-4, wherein the RNA polymerase is selected from the group consisting of: T7, SP6, or T3, Vsw-3, Kll, T3D, T5, T8, (D2.5, (D6 RNA polymerase, functional variants thereof, and any combination thereof; wherein preferably the RNA polymerase is a T7, SP6, or T3 RNA polymerase, most preferably, the RNA polymerase is a T7 RNA polymerase.

6. The method of any of claims 1-5, wherein GTP is present in a ratio GTP: dinucleotide cap analog from 1:1 to 1:10.

7. The method of any of claims 1-6, wherein the ribonucleotides comprise ATP, CTP, UTP and GTP, or any modified ribonucleotide.

8. The method of any of claims 1-7, wherein the nucleic acid template is a DNA template selected from the group consisting of: a plasmid, a PCR product, a cDNA, a double-stranded (ds)oligonucleotide, and a dsDNA comprising covalently closed ends, or any combination thereof.

9. The method of claim 8, wherein the DNA template is immobilized on a solid support.

10. The method of claim 8 or 9, wherein the DNA template further comprises a plurality of T nucleotides downstream of the target sequence to be transcribed, optionally wherein the plurality of T nucleotides comprises modified nucleotides or analogues thereof.

11. The method of any of claims 1-10, wherein the IVT reaction mixture further comprises a poly-A polymerase.

12. The method of any of claims 1-11, wherein the transcription buffer comprises MgCl2, NaCI, DTT and / or spermidine.

13. The method of any of claims 1-12, wherein the 2'0-methyl transferase is derived from vaccinia virus.

14. The method of any of claims 1-13, wherein greater than 95% of the synthesized mRNA comprises a capl-type structure.

15. The method of any of claims 1-14, wherein the 2'-O-methyltransferase and / or RNA polymerase is immobilized on a solid support.

16. A composition for use in the synthesis of capl-type mRNA from a nucleic acid template, the composition comprising(i) an RNA polymerase(ii) a transcription buffer(iii) ribonucleoside triphosphates (NTPs)(iv) a dinucleotide capping primer according to Formula IwhereinR1is selected from methyl, ethyl, propyl, butyl, benzyl, substituted benzyl, C5-C6 heterocyclyl, C5-C6 substituted heterocyclyl;R2is H, OH or O-CH3;R3is H, OH or O-CH3;R4, R5and R6are independently selected from the group consisting of O, S, Se;R7and R8are OH; n is 1, 2, or 3; andB is a non-modified or modified purine or pyrimidine base,(v) 2'-O-methyl transferase, and(vi) S-adenosyl methionine (SAM) in a single vessel.

17. The composition according to claim 16, wherein the composition further comprises one or more of an RNase inhibitor, a pyrophosphatase, or PEG.

18. The composition according to claim 16 or 17, wherein at least one of R2or R3of the dinucleotide cap analog is O-CH3, preferably, wherein R2is OH and R3is O-CH3.

19. The composition according to any one of claims 16-18, wherein the RNA polymerase is selected from the group consisting of T7, SP6, or T3, Vsw-3, Kll, T3D, T5, T8, (D2.5, (D6 RNA polymerase, and a functional variants thereof, or any combination thereof;preferably the RNA polymerase is a T7, SP6, or T3 RNA polymerase, most preferably, the RNA polymerase is a T7 RNA polymerase.

20. The composition according to any one of claims 16-19, wherein GTP is present in a ratio GTP: dinucleotide cap analog from 1:1 to 1:10.

21. The composition according to any one of claims 16-20, wherein the ribonucleotides are selected from the group consisting of ATP, CTP, UTP and GTP, and a modified ribonucleotide, or any combination thereof .

22. The composition according to any one of claims 16-21, wherein the composition further comprises a poly-A polymerase.

23. The composition according to any one of claims 16-22, wherein the transcription buffer comprises MgCl2, NaCI, a reducing agent, and / or spermidine.

24. The composition according to any one of claims 16-23, wherein the 2'0-methyl transferase is derived from vaccinia virus.

25. Use of the composition according to claim 16 for the synthesis of mRNA therapeutics.

26. Use of the composition according to claim 16 for the synthesis of mRNA in a bioreactor.

27. Use of the composition according to claim 16 for the synthesis of mRNA in a continuous flow device or a flow reactor.

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