IVT byproduct reduction mediated by host metabolites

Abstract

The present invention relates to the production of RNA, in particular to in vitro transcription. Specifically, the present invention relates to methods for decreasing the content of double stranded RNA (dsRNA) during RNA in vitro transcription (IVT).

Description

[0001] Foreignfiling_text P25-429

[0002] 1

[0003] IVT byproduct reduction mediated by host metabolites

[0004] The present invention relates to the production of RNA, in particular to in vitro transcription. Specifically, the present invention relates to methods for decreasing the amount of double stranded RNA (dsRNA) during RNA production by in vitro transcription (IVT).

[0005] 5 Background of the invention

[0006] Production of RNA in vitro or IVT is gaining significance in the biopharmaceutical industry due to the recent expansion in the applications of RNA in therapeutic and preventive medicine. As an example, IVT is currently the most important manufacturing strategy for mRNA vaccines. In addition, therapies based on mRNA have demonstrated great potential in diverse areas of medicine, such as cancer and infectious 10 diseases.

[0007] IVT is one among numerous steps of the manufacturing process; however, it determines the overall quality and yield of the final product. For this reason, industrial-scale production of RNA using IVT has been the subject of several optimization attempts.

[0008] One drawback of IVT is the occurrence of dsRNA as a by-product. This presents a challenge since, parallel 15 to reducing the purity and total amount of the desired mRNA yield, dsRNA can affect the quality and even the utility of the RNA final product, or negatively impact the efficacy and safety of mRNA-based vaccines and therapies. For in vivo applications, dsRNA has been shown to be highly immunogenic and trigger undesired cellular immune responses.

[0009] A typical IVT represents a highly complex interplay of 2-3 proteins, DNA template, NTPs, ions, spermidine, 0 reducing agents, chelating agents, detergent, and buffers to ensure a stable pH. During incubation, the absolute amount of NTPs is reduced and single stranded RNA product as well as double stranded (ds)RNA byproduct are synthesized. Moreover, the RNA products form a broad spectrum of secondary structures, which are affected by ion concentrations and temperature .

[0010] To facilitate the in vitro reaction, single subunit RNA polymerases (RNAP) are used. Thus, the most 5 frequently applied RNA polymerase is derived from Escherichia coli (E. coli) infecting phage T7. Based on this RNAP, several studies reported optimization of IVT conditions towards e.g. lower dsRNA, higher yields and efficient addition of co-transcriptional capping analogs.

[0011] However, current IVT reaction conditions do not resemble the natural physiological environment of T7 RNAP. Thus, it can be rationalized, that dsRNA formation could be an non-natural result of the IVT reaction. 0 During the infection by T7 phage, the host metabolism is reprogrammed to support viral propagation.

[0012] Thereby, naturally occurring metabolic pathways are hijacked.

[0013] Current approaches to remove dsRNA from IVT are predominantly related to analytical purification such as chromatography-based purification methods. These may be effective in reducing dsRNA content, but 5 represent an additional step in the workflow, require costly and highly specialized equipment, and can create problems when upscaling the IVT.

[0014] Alternative concepts are based on the use of mutated T7 RNA polymerases. Point mutations were introduced to decrease the formation of dsRNA byproducts. However, the introduction of mutations can be accompanied with decreased IVT yields, decreased synthesis fidelity, or decreased co-transcriptional Foreignfiling_text P25-429

[0015] 2 capping efficiency. Moreover, engineered enzymes represent major cost-drivers in manufacturing of RNA therapeutics.

[0016] Alternative approaches have been proposed involving altering the reaction conditions. Such alternatives have focused around the effects of magnesium concentrations in the IVT reaction. For example, it has been 5 suggested that lowering or increasing Mg concentration in the reaction can reduce the formation of dsRNA.

[0017] However, Mg plays an important role in transcription to modulate enzymatic activity. This means that manipulating Mg concentrations requires costly and cumbersome fine-tuning to avoid the side effects of impairing the transcription reaction and the total yield of RNA.

[0018] Other approaches involve using chaotropic agents in the initial reaction mixture. Some of these substances 10 are classified as toxic or detrimental in bioprocessing, making them unsuitable for pharmaceutical production. Conversely, others demonstrate minimal dsRNA reduction or can result in significant losses in RNA yield while only achieving a moderate reduction in dsRNA content.

[0019] The above-mentioned approaches have shown varying degrees of effectiveness, but they constrain the cost-effectiveness and time efficiency of the essentially uncomplicated and practical IVT procedure.

[0020] To address these challenges, methods are required that are able to reduce the content of dsRNA in IVT reactions but require no additional post-IVT purification steps, mutated enzymes, complex real-time monitoring settings, or the addition of compounds that negatively affect the amount, quality or integrity of the product RNA, thereby avoiding reducing the inherent advantages in time, cost and simplicity of IVT. 0 Furthermore, it was another object to reduce the content of dsRNA in IVT reactions as outlined and optionally additionally avoiding a reduced yield of the produced RNA or even increasing the yield of the produced RNA. RNA produced by the method exhibits significantly improved purity profiles, leads to substantial reductions in production costs, and provides mRNA therapeutics with enhanced therapeutic efficacy. 5 Summary of the invention

[0021] It was unexpectedly found that supplementing a standard in vitro transcription (IVT) reaction with small molecules related to Escherichia coli metabolism reduces the formation of double-stranded RNA (dsRNA). Multiple metabolites associated with different metabolic pathways, including but not limited to glycolysis, the tricarboxylic acid (TCA) cycle, the pentose phosphate pathway or DNA replication were identified to 0 produce this effect. Addition of the selected metabolites to IVT reactions at various concentrations resulted in a significant reduction in dsRNA formation.

[0022] Hence, in one aspect, the invention relates to a method for producing RNA by in vitro transcription, comprising adding a compound involved in a metabolic pathways of the originating host of a RNA 5 polymerase to a reaction mix comprising a template DNA, RNA polymerases and ribonucleotides.

[0023] In another aspect, the invention relates to a method for reducing the content of double stranded RNA in a produced RNA during in vitro transcription, the method comprising adding to a reaction mix comprising a template DNA, an RNA polymerase and ribonucleotides, a compound involved in a metabolic pathways of 0 the host of the RNA polymerase. Foreignfiling_text P25-429

[0024] 3

[0025] In one embodiment the RNA polymerase is a single subunit RNA polymerase derived from Escherichia coli infecting phage T7 and the originating host is Escherichia coli.

[0026] 5 In another aspect, the invention relates to a method for reducing the content of double stranded RNA in a produced RNA during in vitro transcription, the method comprising adding to a reaction mix comprising a template DNA, an RNA polymerase and ribonucleotides, a compound involved in a metabolic pathways of the originating host of the RNA polymerase or a pharmaceutically acceptable salt, co-crystal, polymorph, solvate, hydrate, tautomer, stereoisomer and isotopically labeled derivative thereof.

[0027] In another aspect, the invention relates to the use of a compound involved in a metabolic pathway of the originating host of an RNA polymerase to decrease the content of double stranded RNA (dsRNA) during RNA in vitro transcription.

[0028] 15 In another aspect, the invention relates to a kit for RNA in vitro transcription, comprising a buffer system, ribonucleotides, a RNA polymerase and a compound involved in the metabolic pathways of the originating host of the RNA polymerase.

[0029] Brief description of the figures

[0030] Fig. 1 shows a bar graph of the relative dsRNA formation by IVT reactions supplemented with metabolites 0 involved in protein biosynthesis in E. coli compared to standard IVT (Std). Byproduct formation was quantified by anti-dsRNA ELISA.

[0031] Fig. 2 shows a bar graph of relative product concentration representing stable mRNA product yields of IVT reactions supplemented with metabolites involved in protein biosynthesis in E. coli compared to standard 5 IVT (Std). Product synthesis was quantified by A260 after standardized silica extraction.

[0032] Fig. 3 shows a bar graph of relative dsRNA formation by IVT reactions supplemented with metabolites involved in replication in E. coli compared to standard IVT (Std). Byproduct formation was quantified by anti-dsRNA ELISA.

[0033] Fig. 4 shows a bar graph of relative product concentration representing stable mRNA product yields of IVT reactions supplemented with metabolites involved in replication in E. coli compared to standard IVT (Std) . Product synthesis was quantified by A260 after standardized silica extraction.

[0034] 35 Fig. 5 shows a bar graph of relative dsRNA formation by IVT reactions supplemented with metabolites involved in glycolysis in E. coli compared to standard IVT (Std). Byproduct formation was quantified by anti- dsRNA ELISA. IVT supplementation was done with either 150 mM pyruvate (pyruvate) or 15 mM (low pyruvate). Foreignfiling_text P25-429

[0035] 4

[0036] Fig. 6 shows a bar graph of the relative product concentration representing stable mRNA product yields of IVT reactions supplemented with metabolites involved in glycolysis in E. coli compared to standard IVT (Std). Product synthesis was quantified by A260 after standardized silica extraction. IVT supplementation was done with either 150 mM pyruvate (pyruvate) or 15 mM (low pyruvate).

[0037] Fig. 7 shows is a bar graph of relative dsRNA formation by IVT reactions supplemented with metabolites involved in glycolysis linkage of the TCA cycle in E. coli compared to standard IVT (Std). Byproduct formation was quantified by anti-dsRNA ELISA.

[0038] 10 Fig. 8 shows relative product concentration representing stable mRNA product yields of IVT reactions supplemented with metabolites involved in glycolysis linkage of the TCA cycle in E. coli compared to standard IVT (Std). Product synthesis was quantified by A260 after standardized silica extraction.

[0039] Fig. 9 shows relative dsRNA formation by IVT reactions supplemented with metabolites involved in the 15 TCA cycle in E. coli compared to standard IVT (Std). Byproduct formation was quantified by anti-dsRNA ELISA.

[0040] Fig. 10 shows relative product concentration representingmRNA product yields of IVT reactions supplemented with metabolites involved in the TCA cycle of E. coli compared to standard IVT (Std). Product 0 synthesis was quantified by A260 after standardized silica extraction.

[0041] Fig. 11 shows relative dsRNA formation by IVT reactions supplemented with metabolites involved in the uric acid cycle, anaerobic metabolism, or pathophysiological nutrition of E. coli compared to standard IVT (Std). Byproduct formation was quantified by anti-dsRNA ELISA.

[0042] Fig. 12 shows a bar graph of relative product concentration representing mRNA product yields of IVT reactions supplemented with metabolites involved in the uric acid cycle, anaerobic metabolism or pathophysiological nutrition of E. coli compared to standard IVT (Std) is representing yield. Product synthesis was quantified by A260 after standardized silica extraction. 0 Detailed description of the invention

[0043] As used herein, the term “compound involved in prokaryotic metabolism” refers to any chemical entity that directly or indirectly participates in, is produced by, is consumed by, is modified by, is coupled to, or modulates one or more enzymatic reaction(s) or transport process(es) that together constitute a metabolic pathway in a prokaryotic organism. Metabolic pathways include, without limitation, the glycolysis, the citric 5 acid cycle, the protein biosynthesis, the replication, the pentose phosphate pathway, the anaerobic metabolism or the uric acid cycle.

[0044] Such a compound includes, without limitation, substrates, intermediates, end-products, cofactors, prosthetic groups, electron carriers, energy carriers, allosteric effectors, enzyme inhibitors and activators, transportable metabolites, signaling molecules, and other species that alter metabolic flux or pathway 0 regulation. The term encompasses compounds that are naturally occurring in E. coli, compounds Foreignfiling_text P25-429

[0045] 5 exogenously introduced into E. coli, and compounds generated in situ (e.g., by engineered enzymes), and further encompasses synthetic analogs, derivatives, prodrugs, salts, esters, tautomers, stereoisomers, isotopically labeled variants, and covalent or non-covalent conjugates thereof. The term also covers such compounds whether located intra- or extracellularly, whether free or protein-bound, and whether present in 5 wild-type, mutant or genetically engineered strains, or in cell-free reaction systems. Examples include, without limitation, metabolites such as glucose, glycerol, pyruvate, phosphoenolpyruvate, acetyl-CoA, citrate, succinate, fumarate, malate, oxaloacetate, amino acids (e.g., glutamate, serine), nucleotides, nucleosides, nucleotide base (e.g., ATP, ADP), NAD+ / NADH, NADP+ / NADPH, FAD, FAD+ / FADH, FADH+ / FADH2and related analogs.

[0046] As used herein, the term “compound involved in a metabolic pathways of the originating host of the RNA polymerase” refers to any chemical entity that directly or indirectly participates in, is produced by, is consumed by, is modified by, is coupled to, or modulates one or more enzymatic reaction(s) or transport process(es) that together constitute a metabolic pathway in the originating host of the RNA polymerase.

[0047] 15 As used herein, the term “originating host of the RNA polymerase” refers to the organism in which the nucleotide sequence encoding the RNA polymerase naturally occurs or from which that nucleotide sequence was isolated, or, alternatively, an organism (cellular or viral) that is the direct source sequence for an RNA polymerase variant used in the invention. The term expressly includes bacteriophages (for example T7-, SP6- and related phage RNAPs), bacteria (for example Escherichia coli, Salmonella spp.), 0 and other organisms that naturally encode single-subunit or multi-subunit RNAPs, and further includes progenitor, descendant, homologous, orthologous, paralogous or engineered derivatives that are derived from, substantially based upon, or predominantly sequence / structure / functionally derived from an RNAP originally found in such host. 5 In one embodiment the originating host of the RNA polymerase is Escherichia coli, in particular wherein the host is E. coli and the RNA polymerase is a single subunit RNA polymerases derived from E. coli-infecting bacteriophage, in particular bacteriophage T7. Hence, the term “compound involved in a metabolic pathways of the originating host of the RNA polymerase” refers to any chemical entity that directly or indirectly participates in, is produced by, is consumed by, is modified by, is coupled to, or modulates one 0 or more enzymatic reaction(s) or transport process(es) that together constitute a metabolic pathway in Escherichia coli.

[0048] In one embodiment the originating host of the RNA polymerase is Salmonella, in particular wherein the host is Salmonella and the RNA polymerase is a single subunit RNA polymerases derived from Salmonella- 5 infecting bacteriophage, in particular bacteriophage SP6. Hence, the “compound involved in a metabolic pathways of the originating host of the RNA polymerase” refers to any chemical entity that directly or indirectly participates in, is produced by, is consumed by, is modified by, is coupled to, or modulates one or more enzymatic reaction(s) or transport process(es) that together constitute a metabolic pathway in Salmonella. Foreignfiling_text P25-429

[0049] 6

[0050] As used herein, the term “metabolite involved in glycolysis of Escherichia coli” refers to any chemical entity that directly or indirectly participates in, is produced by, is consumed by, or modulates one or more enzymatic reaction(s), transport process(es) or regulatory event(s) that together constitute glycolytic conversion of carbohydrates in Escherichia coli. The term expressly encompasses metabolites of the 5 Embden-Meyerhof-Parnas (EMP) pathway and the Entner-Doudoroff (ED) pathway as carried out in E. coli, and any natural, engineered or variant forms of those pathways, including associated uptake and phosphorylation systems (e.g., the phosphotransferase system) and other glycolytic-like routes operative in E. coli. The term includes, without limitation, substrates, pathway intermediates, end-products, cofactors, prosthetic groups, energy carriers, electron carriers, allosteric effectors, enzyme inhibitors and activators, 10 transportable metabolites, and other species that alter glycolytic flux or pathway regulation. The term further encompasses metabolites that are naturally occurring in E. coli, metabolites exogenously introduced into E. coli, and metabolites generated in situ (e.g., by engineered enzymes), and also encompasses synthetic analogs, derivatives, prodrugs, salts, esters, tautomers, stereoisomers, isotopically labeled variants, and covalent or non-covalent conjugates thereof. The term covers such metabolites whether located intra- or 15 extracellularly, whether free or protein-bound, and whether present in wild-type, mutant or genetically engineered strains, or in cell-free reaction systems. Examples include, without limitation, glucose, glucose- 6-phosphate, fructose-6-phosphate, fructose-1 ,6-bisphosphate, glyceraldehyde-3-phosphate, dihydroxyacetone phosphate, 6-phosphogluconate, 2-keto-3-deoxy-6-phosphogluconate (KDPG), 1 ,3- bisphosphoglycerate, 3-phosphoglycerate, 2-phosphoglycerate, phosphoenolpyruvate, pyruvate, 0 adenosine triphosphate (ATP), adenosine diphosphate (ADP), nicotinamide adenine dinucleotide oxidized (NAD+), nicotinamide adenine dinucleotide reduced (NADH), nicotinamide adenine dinucleotide, niacin or nicotinamide.

[0051] As used herein, the term “metabolites or cofactors of the Embden-Meyerhof-Parnas pathway” refers to 5 any chemical entity that directly or indirectly participates in, is produced by, is consumed by, or modulates one or more enzymatic reaction(s), transport process(es) or regulatory event(s) that together constitute the Embden-Meyerhof-Parnas (EMP) glycolytic pathway in Escherichia coli, including, without limitation, substrates, pathway intermediates, end-products, cofactors, prosthetic groups, electron carriers, energy carriers, allosteric effectors, enzyme inhibitors and activators, and transportable metabolites. 0 The term encompasses metabolites and cofactors that are naturally occurring in E. coli, exogenously supplied to E. coli, or generated in situ (for example by engineered enzymes), and further encompasses synthetic analogs, derivatives, prodrugs, salts, esters, tautomers, stereoisomers, isotopically labeled variants, and covalent or non-covalent conjugates thereof. The term covers such species whether located intra- or extracellularly, whether free or protein-bound, and whether present in wild-type, mutant or 5 genetically engineered strains, or in cell-free reaction systems.

[0052] Examples, without limitation, include glucose, glucose-6-phosphate, fructose-6-phosphate, fructose-1 ,6- bisphosphate, glyceraldehyde-3-phosphate, dihydroxyacetone phosphate, 1 ,3-bisphosphoglycerate, 3- phosphoglycerate, 2-phosphoglycerate, phosphoenolpyruvate, pyruvate, adenosine triphosphate, adenosine diphosphate, nicotinamide adenine dinucleotide oxidized, nicotinamide adenine dinucleotide 0 reduced, niacin or nicotinamide. Foreignfiling_text P25-429

[0053] 7

[0054] As used herein, the term “metabolites or cofactors of the Entner-Doudoroff pathway” refers to any chemical entity that directly or indirectly participates in, is produced by, is consumed by, or modulates one or more enzymatic reaction(s), transport process(es) or regulatory event(s) that together constitute the Entner- Doudoroff (ED) pathway as carried out in Escherichia coli, and any natural, engineered or variant forms of 5 that pathway. The term expressly includes substrates, pathway intermediates, end-products, cofactors, prosthetic groups, electron carriers, energy carriers, allosteric effectors, enzyme inhibitors and activators, transportable metabolites, and other species that alter ED pathway flux or regulation. The term further encompasses metabolites and cofactors that are naturally occurring in E. coli, exogenously supplied to E. coli, or generated in situ (for example by engineered enzymes), and also encompasses synthetic analogs, 10 derivatives, prodrugs, salts, esters, tautomers, stereoisomers, isotopically labeled variants, and covalent or non-covalent conjugates thereof. The term covers such species whether located intra- or extracellularly, whether free or protein-bound, and whether present in wild-type, mutant or genetically engineered strains, or in cell-free reaction systems.

[0055] Examples, without limitation, include glucose, glucose-6-phosphate, 6-phosphogluconate, 2-keto-3-deoxy- 15 6-phosphogluconate, glyceraldehyde-3-phosphate, 1 ,3-bisphosphoglycerate, 3-phosphoglycerate, 2- phosphoglycerate, phosphoenolpyruvate, pyruvate, adenosine triphosphate, adenosine diphosphate, nicotinamide adenine dinucleotide oxidized, nicotinamide adenine dinucleotide, niacin or nicotinamide.

[0056] As used herein, the term “metabolite or cofactor involved in the citric acid cycle of Escherichia coli” refers 0 to any chemical entity that directly or indirectly participates in, is produced by, is consumed by, or modulates one or more enzymatic reaction(s), transport process(es), electron transfer event(s) or regulatory event(s) that together constitute the citric acid cycle (TCA cycle, Krebs cycle) in Escherichia coli.

[0057] The term includes, without limitation, substrates, pathway intermediates, end-products, cofactors, prosthetic groups, electron carriers, energy carriers, allosteric effectors, enzyme inhibitors and activators, 5 transportable metabolites, metal ion cofactors, and other species that alter TCA flux or regulation. The term further encompasses species that are naturally occurring in E. coli, exogenously supplied to E. coli, or generated in situ (for example by engineered enzymes), and also encompasses synthetic analogs, derivatives, prodrugs, salts, esters, tautomers, stereoisomers, isotopically labeled variants, and covalent or non-covalent conjugates thereof. The term covers such species whether located intra- or extracellularly, 0 whether free or protein-bound, and whether present in wild-type, mutant or genetically engineered strains, or in cell-free reaction systems.

[0058] Examples, without limitation, include acetyl-coa, citrate, cis-aconitate, isocitrate, 2-oxoglutarate, succinyl- CoA, succinate, fumarate, malate, oxaloacetate, nicotinamide adenine dinucleotide oxidized and nicotinamide adenine dinucleotide reduced, flavin adenine dinucleotide oxidized, flavin adenine 5 dinucleotide reduced, coenzyme A, guanosine diphosphate, guanosine triphosphate, adenosine triphosphate, adenosine diphosphate, thiamine, thiamine monophosphate or thamine pyrophosphate.

[0059] As used herein, the term “metabolite or cofactor involved in DNA replication of Escherichia coli” refers to any chemical entity that directly or indirectly participates in, is produced by, is consumed by, or modulates 0 one or more enzymatic reaction(s), binding event(s), transport process(es), energy transfer(s) or regulatory event(s) that together contribute to DNA replication, primer synthesis, lagging / leading strand synthesis, Foreignfiling_text P25-429

[0060] 8

[0061] Okazaki fragment processing, ligation, or the biosynthesis and supply of nucleotides required for those processes in Escherichia coli. The term expressly includes substrates, pathway intermediates, end-products, cofactors, prosthetic groups, metal ion cofactors, energy carriers, electron carriers, allosteric effectors, enzyme inhibitors and activators, transportable metabolites, and other species that alter 5 replication flux, fidelity or regulation.

[0062] The term encompasses such metabolites and cofactors whether they are naturally occurring in E. coli, exogenously supplied to E. coli, or generated in situ (for example by engineered enzymes), and further encompasses synthetic analogs, derivatives, prodrugs, salts, esters, tautomers, stereoisomers, isotopically labeled variants, and covalent or non-covalent conjugates thereof. The term covers such species whether 10 located intra- or extracellularly, whether free or protein-bound, and whether present in wild-type, mutant or genetically engineered strains, or in cell-free reaction systems.

[0063] Examples, without limitation, include adenine, guanine, cytosine, uracil, thymine, adenosine, guanosine, cytidine, uridine, thymidine, deoxyadenosine, deoxyguanosine, deoxycytidine, deoxythymidine, phosphoribosyl pyrophosphate, ribose-5-phosphate, glucose-6-phosphate, 6-phosphogluconate, 15 carbamoyl phosphate, aspartate, glycine, glutamine, formyl-tetrahydrofolate, 5,10-methylene- tetra hydrofol ate, tetrahydrofolate, orotic acid, orotidine-5'-monophosphate, uridine monophosphate, uridine diphosphate, uridine triphosphate, cytidine monophosphate, cytidine diphosphate, cytidine triphosphate, inosine monophosphate, adenosine monophosphate, guanosine monophosphate, adenosine diphosphate, guanosine diphosphate, adenosine triphosphate, guanosine triphosphate, deoxyadenosine triphosphate, 0 deoxyguanosine triphosphate, deoxycytidine triphosphate, deoxythymidine triphosphate, nicotinamide adenine dinucleotide phosphate oxidized, nicotinamide adenine dinucleotide phosphate reduced, nicotinamide adenine dinucleotide oxidized, nicotinamide adenine dinucleotide reduced, S- adenosylmethionine, decarboxylated S-adenosylmethionine, L-ornithine, putrescine, cyclic guanosine monophosphate, cyclic adenosine monophosphate, cyclic di-guanosine monophosphate, cyclic di- 5 adenosine monophosphate, cyclic gmp-amp, cyclic cytidine monophosphate or cyclic uridine monophosphate.

[0064] In one aspect, the present invention relates to a method for producing RNA by in vitro transcription, comprising adding to a reaction mix comprising a template DNA, an RNA polymerase and ribonucleotides, 0 a compound involved in prokaryotic metabolism or a pharmaceutically acceptable derivative, salt, cocrystal, polymorph, solvate, hydrate, tautomer, stereoisomer and isotopically labeled derivative thereof.

[0065] In another aspect, the invention relates to a method for reducing the content of double stranded RNA (dsRNA) in a produced RNA during in vitro transcription, comprising adding to a reaction mix comprising a 5 template DNA, an RNA polymerase and ribonucleotides, a compound involved in prokaryotic metabolism, or a pharmaceutically acceptable salt, co-crystal, polymorph, solvate, hydrate, tautomer, stereoisomer and isotopically labeled derivative thereof.

[0066] In one embodiment, the compound is involved in a metabolic pathways of the originating host of the RNA polymerase, wherein the RNA polymerase is derived from a bacteriophage, in particular bacteriophage T7, 0 SP6 or T3. Foreignfiling_text P25-429

[0067] 9

[0068] In another aspect, the invention relates to a method for reducing the content of double stranded RNA in a produced RNA during in vitro transcription, the method comprising adding to a reaction mix comprising a template DNA, an RNA polymerase and ribonucleotides, a compound involved in a metabolic pathways of the originating host of the RNA polymerase or a pharmaceutically acceptable salt, co-crystal, polymorph, 5 solvate, hydrate, tautomer, stereoisomer and isotopically labeled derivative thereof.

[0069] In one embodiment, the metabolic pathways is selected from a list consisting of the glycolysis, the citric acid cycle, the DNA replication or the anaerobic metabolism.

[0070] 10 In one embodiment, the RNA polymerase is a single subunit RNA polymerase derived from Escherichia coli infecting phage T7 and the originating host is Escherichia coli.

[0071] In another embodiment the compound involved in a metabolic pathways of the originating host of the RNA polymerase is a compound involved in a metabolic pathways of Escherichia coli.

[0072] In another embodiment the compound involved in a metabolic pathways of the originating host of the RNA polymerase is a compound selected from the list comprising L-serine, glycine, Lys-Lys-Lys, pyridoxine, L- histidine, L-lysine, N(alpha)-acetly-L-lysine, L-tryptophane, L-threonine, L-glutamine, L-tyrosine, creatinine, L-methionine, guanine, cytosine, uracil, adenosine, guanosine, cytidine, uridine, orotic acid, L-ornithine, 0 cyclic guanosine monophosphate, pyruvate, niacin, sodium acetate, nicotinamide, acetyl-CoA, succinate, thiamine, thiamine monophosphate, thiamine pyrophosphate, tartrate, L-citrulline, uric acid, N-acetly glucosamine, ascorbic acid and carnithine.

[0073] In another embodiment the compound involved in a metabolic pathways of the originating host of the RNA 5 polymerase is a compound selected from the list comprising Lys-Lys-Lys, pyridoxine, L-histidine, L-lysine, creatinine, cytidine, uridine, L-ornithine, pyruvate, nicotinamide, acetyl-CoA, succinate, thiamine, thiamine monophosphate, thiamine pyrophosphate, tartrate and ascorbic acid.

[0074] In another embodiment the compound involved in a metabolic pathways of the originating host of the RNA 0 polymerase is a compound selected from the list comprising Lys-Lys-Lys, pyridoxine, L-histidine, cytidine, uridine, L-ornithine, pyruvate, nicotinamide, niacin, thiamine, thiamine monophosphate, thiamine pyrophosphate, acetyl-CoA, succinate, tartrate and ascorbic acid.

[0075] In another embodiment the compound involved in a metabolic pathways of the originating host of the RNA 5 polymerase is a compound selected from the list comprising Lys-Lys-Lys, pyridoxinea and L-histidine.

[0076] In another embodiment the compound involved in a metabolic pathways of the originating host of the RNA polymerase is a compound selected from the list comprising cytidine, uridine and L-ornithine. 0 In another embodiment the compound involved in a metabolic pathways of the originating host of the RNA polymerase is a compound selected from the list comprising pyruvate, nicotinamide and niacine. Foreignfiling_text P25-429

[0077] 10

[0078] In another embodiment the compound involved in a metabolic pathways of the originating host of the RNA polymerase is a compound selected from the list comprising thiamine, thiamine monophosphate and thiamine pyrophosphate.

[0079] In another embodiment the compound involved in a metabolic pathways of the originating host of the RNA polymerase is a compound selected from the list comprising acetyl-CoA and succinate.

[0080] In another embodiment the compound involved in a metabolic pathways of the originating host of the RNA 10 polymerase is tartrate.

[0081] In another embodiment the compound involved in a metabolic pathways of the originating host of the RNA polymerase is ascorbic acid.

[0082] 15 In another embodiment, the compound is a metabolite involved in the glycolysis, preferably in E.coli.

[0083] In another embodiment, the compound involved in a metabolic pathways of Escherichia coli is a metabolite or cofactors of the Embden-Meyerhof-Parnas pathway. 0 In another embodiment, the compound involved in Embden-Meyerhof-Parnas pathway is a compound selected from the group consisting of glucose, glucose-6-phosphate, fructose-6-phosphate, fructose-1 ,6- bisphosphate, glyceraldehyde-3-phosphate, dihydroxyacetone phosphate, 1 ,3-bisphosphoglycerate, 3- phosphoglycerate, 2-phosphoglycerate, phosphoenolpyruvate, pyruvate, adenosine triphosphate, adenosine diphosphate, nicotinamide adenine dinucleotide oxidized, nicotinamide adenine dinucleotide 5 reduced, niacin and nicotinamide.

[0084] In another embodiment, the compound involved in a metabolic pathways of Escherichia coli is a metabolite or cofactors of the Entner-Doudoroff pathway.

[0085] 30 In another embodiment, the compound involved in Entner-Doudoroff pathway is a compound selected from the group consisting of glucose, glucose-6-phosphate, 6-phosphogluconate, 2-keto-3-deoxy-6- phosphogluconate, glyceraldehyde-3-phosphate, 1 ,3-bisphosphoglycerate, 3-phosphoglycerate, 2- phosphoglycerate, phosphoenolpyruvate, pyruvate, adenosine triphosphate, adenosine diphosphate, nicotinamide adenine dinucleotide oxidized, nicotinamide adenine dinucleotide, niacin and nicotinamide.

[0086] In another embodiment, the compound is selected from the group consisting of pyruvate, low pyruvate, niacin, sodium acetate and nicotinamide.

[0087] In another embodiment, the compound is pyruvate or nicotinamide. Foreignfiling_text P25-429

[0088] 11

[0089] In another embodiment, the compound is a metabolite or cofactor involved in the protein biosynthesis, preferably in E. coli.

[0090] In another embodiment, the compound involved in the protein biosynthesis is selected from the group 5 consisting of L-serine, glycine, Lys-Lys-Lys, pyridoxine, L-histidine, L-lysine, N(alpha)-acetly-L-lysine, L- tryptophane, L-threonine, L-glutamine, L-tyrosine, creatinine and L-methionine.

[0091] In another embodiment, the compound is Lys-Lys-Lys, pyridoxine, L-histidine, L-lysine or creatinine.

[0092] 10 In another embodiment, the compound is a metabolite or cofactor involved in the citric acid cycle, preferably in E. coli.

[0093] In another embodiment, the compound involved in the citric acid cycle is selected from the group consisting of acetyl-coa, citrate, cis-aconitate, isocitrate, 2-oxoglutarate, succinyl-CoA, succinate, fumarate, malate, 15 oxaloacetate, nicotinamide adenine dinucleotide oxidized and nicotinamide adenine dinucleotide reduced, flavin adenine dinucleotide oxidized, flavin adenine dinucleotide reduced, coenzyme A, guanosine diphosphate, guanosine triphosphate, adenosine triphosphate, adenosine diphosphate, thiamine, thiamine monophosphate, thamine pyrophosphate. 0 In another embodiment, the compound is selected from the group consisting of acetyl-CoA, succinate, thiamine, thiamine monophosphate and thiamine pyrophosphate.

[0094] In another embodiment, the compound is acetyl-CoA or succinate. 5 In another embodiment, the compound is a metabolite or cofactor involved in DNA replication.

[0095] In another embodiment, the compound is a metabolite or cofactor involved in DNA replication, wherein the compound is selected from the group consisting of adenine, guanine, cytosine, uracil, thymine, adenosine, guanosine, cytidine, uridine, thymidine, deoxyadenosine, deoxyguanosine, deoxycytidine, deoxythymidine, 0 phosphoribosyl pyrophosphate, ribose-5-phosphate, glucose-6-phosphate, 6-phosphogluconate, carbamoyl phosphate, aspartate, glycine, glutamine, formyl-tetrahydrofolate, 5,10-methylene- tetra hydrofol ate, tetrahydrofolate, orotic acid, orotidine-5'-monophosphate, uridine monophosphate, uridine diphosphate, uridine triphosphate, cytidine monophosphate, cytidine diphosphate, cytidine triphosphate, inosine monophosphate, adenosine monophosphate, guanosine monophosphate, adenosine diphosphate, 5 guanosine diphosphate, adenosine triphosphate, guanosine triphosphate, deoxyadenosine triphosphate, deoxyguanosine triphosphate, deoxycytidine triphosphate, deoxythymidine triphosphate, nicotinamide adenine dinucleotide phosphate oxidized, nicotinamide adenine dinucleotide phosphate reduced, nicotinamide adenine dinucleotide oxidized, nicotinamide adenine dinucleotide reduced, S- adenosylmethionine, decarboxylated S-adenosylmethionine, L-ornithine, putrescine, spermidine, cyclic 0 guanosine monophosphate, cyclic adenosine monophosphate, cyclic di-guanosine monophosphate, cyclic Foreignfiling_text P25-429

[0096] 12 di-adenosine monophosphate, cyclic gmp-amp, cyclic cytidine monophosphate, cyclic uridine monophosphate.

[0097] In another embodiment, the compound is selected from the group consisting of guanine, cytosine, uracil, 5 adenosine, guanosine, cytidine, uridine, orotic acid, L-ornithine, cyclic guanosine monophosphate.

[0098] In another embodiment, the compound is cytidine, uridine or L-ornithine.

[0099] In another embodiment, the compound is a metabolite or cofactor involved in anaerobic metabolism.

[0100] In another embodiment, the compound is a metabolite or cofactor involved in anaerobic metabolism, wherein the compound is selected from the group consisting of adenine, guanine, cytosine, uracil, thymine, adenosine, guanosine, cytidine, uridine, thymidine, deoxyadenosine, deoxyguanosine, deoxycytidine, deoxythymidine, phosphoribosyl pyrophosphate, ribose-5-phosphate, glucose-6-phosphate, 6- 15 phosphogluconate, carbamoyl phosphate, aspartate, glycine, glutamine, formyl-tetrahydrofolate, 5,10- methylene-tetrahydrofolate, tetrahydrofolate, orotic acid, orotidine-5'-monophosphate, uridine monophosphate, uridine diphosphate, uridine triphosphate, cytidine monophosphate, cytidine diphosphate, cytidine triphosphate, inosine monophosphate, adenosine monophosphate, guanosine monophosphate, adenosine diphosphate, guanosine diphosphate, adenosine triphosphate, guanosine triphosphate, 0 deoxyadenosine triphosphate, deoxyguanosine triphosphate, deoxycytidine triphosphate, deoxythymidine triphosphate, nicotinamide adenine dinucleotide phosphate oxidized, nicotinamide adenine dinucleotide phosphate reduced, nicotinamide adenine dinucleotide oxidized, nicotinamide adenine dinucleotide reduced, S-adenosylmethionine, decarboxylated S-adenosylmethionine, L-ornithine, putrescine, spermidine, cyclic guanosine monophosphate, cyclic adenosine monophosphate, cyclic di-guanosine 5 monophosphate, cyclic di-adenosine monophosphate, cyclic gmp-amp, cyclic cytidine monophosphate, cyclic uridine monophosphate.

[0101] In another embodiment, the compound is tartrate or ascorbic acid.

[0102] 30 In another embodiment, the compound is selected from the group consisting of tartrate, L-citrulline, uric acid, N-acetly glucosamine, ascorbic acid and carnithine.

[0103] In one embodiment, the present invention refers to a method, wherein the content of dsRNA in the produced RNA is reduced by at least 50%, preferably by at least 70%, more preferably by at least 80%, most 5 preferably at least 90% than when the method is performed without the addition of the compound involved in a metabolic pathways of the originating host of the RNA polymerase.

[0104] In another embodiment, the present invention refers to a method, wherein the yield of the produced RNA is not essentially reduced than when the method is performed without the addition of a compound involved in a metabolic pathways of the originating host of the RNA polymerase, preferably not reduced by more 0 than 5%, more preferably not reduced by more than 10%, most preferably not reduced by more than 20%. Foreignfiling_text P25-429

[0105] 13

[0106] In another embodiment, the present invention refers to a method, wherein the yield of the produced RNA is higher than when the method is performed without the addition of a compound involved in a metabolic pathways of the originating host of the RNA polymerase, preferably at least 2% higher, more preferably at least 5% higher, most preferably at least 10% higher.

[0107] As used therein, the term “yield of the produced RNA” means the absolute amount (mass or molar quantity) of full-length, correctly formed RNA recovered from a defined reaction and measured under specified assay conditions, expressed per reaction or normalized to input template (e.g., ng RNA per reaction or pmol RNA per nmol template).

[0108] In one embodiment, the produced RNA is a mRNA.

[0109] In one embodiment, the present invention refers to a method, wherein the produced RNA has a higher mRNA efficiency than when the method is performed without the addition of a compound involved in a 15 metabolic pathways of the originating host of the RNA polymerase, preferably at least 25% higher, more preferably at least 50% higher, most preferably at least 75% higher. In another embodiment, the mRNA efficiency is preferably at least 100% higher, more preferably at least 500% higher, most preferably at least 1000% higher. 0 As used therein, the term “the produced RNA has a higher mRNA efficiency” refers to improved functional performance of the produced mRNA molecules compared to mRNA produced by the same method without the addition of the compound. Higher mRNA efficiency encompasses one or more of the following characteristics: (i) increased translation efficiency, meaning enhanced conversion of mRNA into protein as measured by protein expression levels; (ii) improved mRNA stability, including resistance to nuclease 5 degradation and extended functional half-life; (iii) enhanced cellular uptake and bioavailability when the mRNA is delivered to target cells; and (iv) increased overall protein yield per unit of mRNA. mRNA efficiency may be quantified by measuring protein expression levels, mRNA half-life, translation rates, cellular uptake percentages, or functional activity of the resulting protein product, with "higher efficiency" indicating a statistically significant improvement in one or more of these parameters compared to the control method 0 performed without the compound addition.

[0110] In another embodiment, the present invention refers to a method, wherein the compound involved in a metabolic pathways of the originating host of the RNA polymerase is added to the reaction mix up to a concentration of between 0.1 mM and 1 M, preferably between 1 mM and 500 mM, more preferably 5 between 1 mM and 200 mM.

[0111] In another aspect the inventions refers to the use of a compound involved in a metabolic pathways of the originating host of an RNA polymerase to decrease the content of dsRNA during in vitro transcription. Foreignfiling_text P25-429

[0112] 14

[0113] In another aspect the inventions refers to a kit for in vitro transcription, comprising a buffer system, ribonucleotides, an RNA polymerase and a compound involved in the metabolic pathways of the originating host of an RNA polymerase reducing intermolecular interactions.

[0114] 5 As used therein, the term “in vitro transcription (IVT)” refers to any method of producing or synthesizing RNA in vitro from a template DNA using an RNA polymerase. Methods for RNA IVT are known in the art. In particular, the skilled person knows how to set up a reaction mix for performing RNA IVT. Briefly, a reaction mix for RNA IVT comprises a template DNA containing the DNA sequence to be transcribed, an RNA polymerase and ribonucleotides.

[0115] In addition to the DNA sequence to be transcribed, the template DNA also comprises a promoter upstream of the sequence to be transcribed that can be recognized by the respective RNA polymerase. A promoter is herein defined as a DNA sequence allowing for the initiation of RNA transcription. Commonly used promoters include the T7 promoter to be used with the T7 or T3 RNA polymerase, the T3 promoter to be 15 used with the T3 RNA polymerase and the SP6 promoter to be used together with the SP6 RNA polymerase. Other than the wild-type form of these promoters, mutated forms thereof may be used.

[0116] In some embodiments, the template DNA further comprises a polyadenosine sequence after the sequence to be transcribed and / or a stop codon. In some embodiments, the template DNA has been linearized or is 0 generated using PCR.

[0117] The skilled person is aware of available methods, databases or other resources to determine essential molecules in the metabolism of prokaryotic organisms. For example, essential metabolic pathways for glycolysis in prokaryotics organisms e.g. E. coli were broadly analyzed and described.

[0118] As defined herein “metabolites” are molecules or single atoms that take part in essential reactions constituting a living organism. As defined herein “metabolic pathways” are essential reactions constituting a living organism. Collectively, metabolites undergo reactions within metabolic pathways. The metabolic pathways dictate or respond to the physical and chemical activities of living organisms and their 30 environment and hence express themselves e.g. in stress response, replication / proliferation, energy conversion, morphologic changes, growth and behavior. Metabolites and associates metabolic pathways include uptake of nutrients, energy consumption, energy storage, conversion of intracellular molecules to store or release energy, molecules that are modified to generate proteins, molecules that are modified to generate nucleic acids, molcules modified to generate ATP, cAMP, NAD, NADH, NADH2, FAD, FADH, 5 FADH2, NADP, NADPH, NADPH2 or derivates. The concentration of metabolits within metabolic pathways may differ during lifetime of prokaryotic organisms e.g. as modulated by environment, proliferation or stress

[0119] A “nucleic acid” is herein defined as macromolecule composed of nucleotides. A “nucleotide” is a monomeric structure of the nucleic acid comprised of three components: a 5-carbon sugar, a phosphate group and a nitrogenous base. The two main classes of nucleic acids are deoxyribonucleic acid (DNA) and Foreignfiling_text P25-429

[0120] 15 ribonucleic acid (RNA). If the sugar is ribose, the polymer is RNA; if the sugar is the ribose derivative deoxyribose, the polymer is DNA. There are several forms of DNA, for example, but not limited to plasmid DNA or genomic DNA. Similarly, RNA can be found in the form of messenger RNA (mRNA), transfer-RNA (t-RNA), catalytical RNA or structural RNA. DNA or RNA molecules can also be synthetically prepared for 5 different functions. In one embodiment, the nucleic acid is DNA or RNA. In one embodiment, the nucleic acid is DNA. In one embodiment, the nucleic acid is RNA.

[0121] A “RNA” in terms of the produced RNA during in vitro transcription is herein defined as a nucleic acid molecule composed of a chain of nucleotides, each consisting of a ribose sugar, a phosphate group, and 10 a nitrogenous base. Different forms and types of RNA are known to the skilled person in the art and are included under the present definition. There are several forms of RNA in terms of the RNA product of the in vitro transcription in the form of linear or circular RNA, for example, but not limited to mRNA, circRNA, self-amplifying (saRNA) I self-replicative RNA, long non-coding RNA, guide RNA, tRNA, asRNA, siRNA, shRNA, ribozymes, riboswitch, piRNA, tracrRNA, microRNA, ribosomal RNA (rRNA), small nuclear RNA 15 (snRNA), small nucleolar RNA (snoRNA), long intergenic non-coding RNA (lincRNA), cytoplasmic RNA, and viral RNA.

[0122] A “template DNA” is herein defined as a nucleic acid molecule composed of a chain of nucleotides, each consisting of a deoxyribose sugar, a phosphate group, and a nitrogenous base. Different forms and types 0 of template DNA are known to the skilled person in the art and are included under the present definition.

[0123] There are several forms of template DNA in the form of linear or circular DNA, for example, but not limited to linear DNA, plasmid DNA, PCR-based DNA, isothermal amplified DNA, genomic DNA, cDNA (complementary DNA), synthetic DNA, mitochondrial DNA, bacterial artificial chromosomes (BACs), viral DNA, and transgenic DNA.

[0124] The term “derivative” relates to a compound that is derived from a parent compound through a chemical modification or transformation, which may involve the alteration of functional groups, the introduction of substituents, or changes in molecular structure. This modification can enhance specific properties of the parent compound, such as increased efficacy, improved stability, increased solubility, or altered 0 pharmacokinetics. Derivatives of compounds can include analogs or esters that retain some biological activity while exhibiting distinct characteristics compared to the parent compound.

[0125] In the present specification, a structural formula of a compound may represent a certain isomer of said compound. It is to be understood, however, that the present disclosure includes all isomers such as 5 geometrical isomers, optical isomers based on an asymmetrical carbon, stereoisomers, tautomers and the like which occur structurally and isomer mixtures and is not limited to the description of the formula. Furthermore, in the present specification, a structural formula of a compound may represent a specific salt and / or solvate of said compound. It is to be understood, however, that the present disclosure includes all salts (e.g., pharmaceutically acceptable salts) and solvates (e.g., hydrates) and is not limited to the 0 description of the specific salt and / or solvate. Foreignfiling_text P25-429

[0126] 16

[0127] Isomers are compounds having the same molecular formula but differ in structure (structural isomers) or in the geometrical (spatial) positioning of the functional groups and / or atoms (stereoisomers). Enantiomers are a pair of stereoisomers which are non- superimposable mirror-images of each other. A racemic mixture or racemate contains a pair of enantiomers in equal amounts and is denoted by the prefix (plus or minus).

[0128] 5 Diastereomers are stereoisomers which are non-superimposable and which are not mirror-images of each other. "Tautomers" are structural isomers of the same chemical substance that spontaneously and reversibly interconvert into each other, even when pure, due to the migration of individual atoms or groups of atoms; i.e., the tautomers are in a dynamic chemical equilibrium with each other. An example of tautomers are the isomers of the keto-enol-tautomerism. "Conformers" are stereoisomers that can be 10 interconverted just by rotations about formally single bonds, and include - in particular - those leading to different 3-dimentional forms of (hetero)cyclic rings, such as chair, half-chair, boat, and twist-boat forms of cyclohexane.

[0129] The term "polymorph" refers to a crystalline form of a compound (or a salt, hydrate, or solvate thereof). All 15 polymorphs have the same elemental composition. Different crystalline forms usually have different X-ray diffraction patterns, infrared spectra, melting points, density, hardness, crystal shape, optical and electrical properties, stability, and solubility. Recrystallization solvent, rate of crystallization, storage temperature, and other factors may cause one crystal form to dominate. Various polymorphs of a compound can be prepared by crystallization under different conditions. Q

[0130] The term "solvate" as used herein refers to an addition complex of a dissolved material in a solvent (such as an organic solvent (e.g., an aliphatic alcohol (such as methanol, ethanol, n- propanol, isopropanol), acetone, acetonitrile, ether, and the like), water or a mixture of two or more of these liquids), wherein the addition complex exists in the form of a crystal or mixed crystal. The amount of solvent contained in the 5 addition complex may be stoichiometric or non-stoichiometric. A hydrate is a solvate wherein the solvent is water.

[0131] The term "pharmaceutically acceptable" refers to the non-toxicity of a material which does not interact with the action of the active component of the pharmaceutical composition.

[0132] Pharmaceutically acceptable salts in the context of the present invention are physiologically acceptable salts of the compounds according to the invention. Salts which are not themselves suitable for pharmaceutical uses but can be used, for example, for isolation, purification or storage of the compounds according to the invention are also included.

[0133] In isotopically labeled derivative of a compound of the invention, one or more atoms are replaced by a corresponding atom having the same number of protons but differing in the number of neutrons. For example, a hydrogen atom may be replaced by a deuterium or tritium atom. Exemplary isotopes which can be used in the present disclosure include deuterium, tritium, 1 1 C, 13C, 14C, 15N, 18F, 32P, 32S, 35S, 0 36CI, and 1251. Foreignfiling_text P25-429

[0134] 17

[0135] In some embodiments, the compound according to the invention is a compound comprising an amino group and / or a carboxyl group. In some embodiments, the compound additionally comprises a phosphate group.

[0136] In some embodiments, the compound comprising an amino group is selected from the group consisting of 5 amino acids, peptides, vitamins, creatine, creatinine, , nucleosides, cyclic guanosine monophosphate, acetylCoA, thiamines, nicotineamide, glucosamines.

[0137] The amino acid to be used as compound according to the invention according to the invention may be any known amino acid such as alpha-, beta- and gamma amino acids, naturally occurring amino acids and 10 unnatural / non-proteinogenic amino acids. Preferably, the amino acid is selected from the group consisting of glycine, histidine, lysine, serine, ornithine, citrulline, methionine, tyrosine, glutamine, trypthophane and derivatives thereof. In a more preferred embodiment, the amino acid to be used as viscosity reducing agent according to the invention is ornithine or a derivative thereof. In some embodiments, the amino acids are poly(amino acids) comprising at least two amino acid monomers, preferably triple lysine, poly(histidine), or 15 poly(ornithine).

[0138] Peptides to be used as compound according to the invention according to the invention may comprise any known amino acid, but preferably comprise at least one amino acid selected from the group consisting of glycine, histidine, lysine, serine, ornithine, citrulline and derivatives thereof. In a more preferred 0 embodiment, the peptide comprises ornithine. In some embodiments, the peptides are between 2 and 200 amino acids in length, preferably between 2 and 140 amino acids.

[0139] In one embodiment, the compound involved in a metabolic pathway or metaboliteis are a vitamin or a pharmaceutically acceptable salt, co-crystal, polymorph, solvate, hydrate, tautomer, stereoisomer and 5 isotopically labeled derivative thereof.

[0140] A “vitamin” is herein defined as an organic compound that is essential in small amounts for normal metabolic functioning and overall health in living organisms. Vitamins are typically obtained through diet, as the body either cannot synthesize them at all or cannot produce them in sufficient quantities. They play crucial roles 30 in various physiological processes, including energy production, immune function, blood clotting, and the maintenance of healthy skin, eyes, and nervous system. Vitamins are classified into two main categories: water-soluble vitamins (such as vitamin C and the B vitamins) and fat-soluble vitamins (such as vitamins A, D, E, and K). Each vitamin has specific functions and is vital for maintaining health and preventing deficiency-related diseases.

[0141] In one embodiment, the compound involved in a metabolic pathway or metaboliteis a vitamin B or vitamin C or a pharmaceutically acceptable salt, co-crystal, polymorph, solvate, hydrate, tautomer, stereoisomer and isotopically labeled derivative thereof. 0 A “vitamin B” is herein defined as a group of water-soluble vitamins that play important roles in cell metabolism and overall health. The B vitamins include several distinct compounds, each with its own Foreignfiling_text P25-429

[0142] 18 specific functions and benefits, such as vitamin B1 (thiamine), which is essential for energy metabolism and the proper functioning of the nervous system, vitamin B2 (riboflavin), important for energy production and the metabolism of fats, drugs, and steroids, vitamin B3 (niacin), which plays a key role in energy production and the synthesis of fatty acids and cholesterol, vitamin B5 (pantothenic acid), involved in the 5 synthesis of coenzyme A crucial for fatty acid metabolism and energy production, vitamin B6 (pyridoxine), essential for amino acid metabolism and neurotransmitter synthesis, vitamin B7 (biotin), important for carbohydrate and fat metabolism, as well as the synthesis of certain proteins, vitamin B9 (folate), crucial for DNA synthesis and cell division, and vitamin B12 (cobalamin), necessary for red blood cell formation, neurological function, and DNA synthesis.

[0143] In one embodiment, the compound involved in a metabolic pathway or metaboliteis a vitamin B or a pharmaceutically acceptable salt, co-crystal, polymorph, solvate, hydrate, tautomer, stereoisomer and isotopically labeled derivative thereof.

[0144] 15 In one embodiment, the compound according to the invention is a vitamin B selected from the group consisting of thiamine, thiamine monophosphate (TMP), thiamine pyrophosphate (TPP), niacin, nicotinamide, pyridoxin, acetylCoA and orotic acid or a pharmaceutically acceptable salt, co-crystal, polymorph, solvate, hydrate, tautomer, stereoisomer and isotopically labeled derivative thereof. 0 In one embodiment, the compound according to the invention is a vitamin B selected from the group consisting of thiamine, thiamine monophosphate (TMP), thiamine pyrophosphate (TPP) and pyridoxin or a pharmaceutically acceptable salt, co-crystal, polymorph, solvate, hydrate, tautomer, stereoisomer and isotopically labeled derivative thereof. 5 In one embodiment, the compound according to the invention is a vitamin B selected from the group consisting of thiamine, thiamine pyrophosphate (TPP), thiamine monophosphate (TMP) and pyridoxin or a pharmaceutically acceptable salt, co-crystal, polymorph, solvate, hydrate, tautomer, stereoisomer and isotopically labeled derivative thereof. 0 In some embodiments, the compound according to the invention comprising a carboxyl group is sodium tartrate, pyruvate, succinate or acetate.

[0145] In one embodiment, the compound according to the invention is selected from the group consisting of glycine, histidine, lysine, serine, ornithine, citrulline, triple lysine, N(alpha)-acetyl-lysine, tyrosine, 5 tryptophane, methionine, glutamine, threonine, poly(histidine), thiamine pyrophosphate, thiamine monophosphate, thiamine, nicotineamide, niacine, pyrrolochinoline chinone, pyridoxine, ascorbic acid, creatine, creatinine, carnitine, pyrrolochinoline chinone, cyclic guanosine monophosphate, guanine, guanosine, orotic acid, cytosine, uracil, carnitine, uric acid, citrulline, N-acetyl glucosamine, tartrate, pyruvate, acetylCoA, succinate, acetate, uridine, cytidine, thymidine or a pharmaceutically acceptable salt, 0 co-crystal, polymorph, solvate, hydrate, tautomer, stereoisomer and isotopically labeled derivative thereof. Foreignfiling_text P25-429

[0146] 19

[0147] In one embodiment, the compound according to the invention is selected from the group consisting of Na- acetyl-L-lysine, lysine, triple lysine or a pharmaceutically acceptable salt, co-crystal, polymorph, solvate, hydrate, tautomer, stereoisomer and isotopically labeled derivative thereof.

[0148] 5 In one embodiment, the compound according to the invention is selected from the group consisting of nucleic acid forming pyrimidines including cytidine, cytosine, thymidine, thymine, uridine, uracile or a pharmaceutically acceptable salt, co-crystal, polymorph, solvate, hydrate, tautomer, stereoisomer and isotopically labeled derivative thereof.

[0149] 10 In one embodiment, the compound according to the invention is selected from the group consisting of glucosamine, acetylCoA, succinate, pyruvate, pyrrolochinoline chinone, thiamine, thiamine monophosphate, thiamine pyrophosphate, nicotineaminde, niacine, orotic acid, cytidine, uridine, creatinine, tartrate, acetate or a pharmaceutically acceptable salt, co-crystal, polymorph, solvate, hydrate, tautomer, stereoisomer and isotopically labeled derivative thereof.

[0150] In one embodiment, the compound according to the invention is selected from the group consisting of acetylCoA, acetate, succinate, pyruvate, methionine, cytidine, uridine, nicotineamide, thiamine, thiamine pyrophosphate, thiamine monophosphate, tartrate, ascorbic acid, carnitine or a pharmaceutically acceptable salt, co-crystal, polymorph, solvate, hydrate, tautomer, stereoisomer and isotopically labeled 0 derivative thereof.

[0151] The method according to the invention is able to decrease the content of dsRNA in the produced RNA. It was unexpectedly found that the addition of a compound reducing intermolecular interactions or a viscosity reducing agent to the IVT reaction mix reduces the occurrence of dsRNA and therefore increases the quality 5 of the obtained RNA.

[0152] Compared to other approaches, the method according to the invention provides an optimized IVT process with improved reduction of dsRNA that does not require additional cumbersome steps or post-IVT procedures and does not impair the transcription reaction.

[0153] In one embodiment, the compound according to the invention is, serine, Lys-Lys-Lys, pyridoxine, histidine, lysine, N(alpha)-acetyl-lysine, creatinine, methionine, cGMP, guanine, cytosine, uracil, orotic acid, adenosine, cytidine, guanosine, uridine, ornithine, nicotineamide, niacine, pyruvate, acetate, thiamine, thiamine monophosphate, thiamine pyrophosphate, acetylCoA, succinate, citrulline, uric acid, tartrate, 5 ascorbic acid, carnitine or a pharmaceutically acceptable salt, co-crystal, polymorph, solvate, hydrate, tautomer, stereoisomer and isotopically labeled derivative thereof, each show advantageous results in reducing the dsRNA content of IVT produced RNA.

[0154] In one embodiment, the compound according to the invention is ascorbic acid, ornithine, thiamine 0 monophosphate (TMP), Lys-Lys-Lys (Lyss), pyridoxine, thiamine, nicotinamide, sodium tartrate, thiamine pyrophosphate, histidine, niacin, lysine, carnitine, orotic acid, succinate, pyruvate, nucleoside riboflavin, Foreignfiling_text P25-429

[0155] 20 acetyl coenzyme A or serine. As demonstrated in the examples below, ascorbic acid, ornithine, thiamine monophosphate (TMP), Lys-Lys-Lys (Lyss), pyridoxine, thiamine, nicotinamide, sodium tartrate, thiamine pyrophosphate, histidine, niacin, lysine, carnitine, orotic acid, riboflavin, acetyl coenzyme A and serine or a pharmaceutically acceptable salt, co-crystal, polymorph, solvate, hydrate, tautomer, stereoisomer and 5 isotopically labeled derivative thereof.

[0156] In one embodiment, the compound according to the invention is ascorbic acid, ornithine, thiamine monophosphate (TMP), Lys-Lys-Lys (Lyss), pyridoxine, thiamine, nicotinamide, sodium tartrate, thiamine pyrophosphate, histidine, niacin, lysine, carnitine, orotic acid, succinate, pyruvate, nucleoside, acetyl 10 coenzyme A or serine. As demonstrated in the examples below, ascorbic acid, ornithine, thiamine monophosphate (TMP), Lys-Lys-Lys (Lyss), pyridoxine, thiamine, nicotinamide, sodium tartrate, thiamine pyrophosphate, histidine, niacin, lysine, carnitine and orotic acid or a pharmaceutically acceptable salt, cocrystal, polymorph, solvate, hydrate, tautomer, stereoisomer and isotopically labeled derivative thereof.

[0157] 15 In one embodiment, the content of dsRNA in the produced RNA is reduced than when the method is performed without the addition of the compound according to the invention, for example by at least 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97% or 100% lower. In one embodiment, the content of dsRNA is reduced by at least 50%, at least 70%, at least 75%, at least 80%, at least 90% or at least 95% . Q

[0158] The content of dsRNA can be measured using any method known in the art, for example via anti-dsRNA- ELISA,a dot blot, dedicated reporter cell lines or by monitoring dsRNA-related immune responses in vivo.

[0159] In one embodiment, the content of dsRNA in the produced RNA is reduced by at least 50% compared to 5 when the method is performed without the addition of a compound involved in a metabolic pathway or metabolite.

[0160] In one embodiment, the compound according to the invention is selected from the group consisting of thiamine, thiamine pyrophosphate, Lys-Lys-Lys (Lyss), ornithine, ascorbic acid, pyruvate, succinate, 0 tartrate, pyridoxine, nicotinamide, thiamine monophosphate (TMP), histidine, creatinine, cytidine, uridine, acetylCoA and lysine or a pharmaceutically acceptable salt, co-crystal, polymorph, solvate, hydrate, tautomer, stereoisomer and isotopically labeled derivative thereof.

[0161] In one embodiment, the content of dsRNA in the produced RNA is reduced by at least 75% compared to 5 when the method is performed without the addition of a compound involved in a metabolic pathway or metabolite.

[0162] In one embodiment, the compound according to the invention is selected from the group consisting of thiamine, Lys-Lys-Lys (Lyss), ornithine, ascorbic acid, pyruvate, succinate, tartrate, pyridoxine, 0 nicotinamide, thiamine monophosphate (TMP) and lysine or a pharmaceutically acceptable salt, co-crystal, polymorph, solvate, hydrate, tautomer, stereoisomer and isotopically labeled derivative thereof. Foreignfiling_text P25-429

[0163] 21

[0164] In one embodiment, the compound according to the invention is selected from the group consisting of pyruvate, tartrate, ascorbic acid, ornithine, thiamine monophosphate (TMP), Lys-Lys-Lys (Lyss), pyridoxine, thiamine and nicotinamide or a pharmaceutically acceptable salt, co-crystal, polymorph, solvate, hydrate, 5 tautomer, stereoisomer and isotopically labeled derivative thereof.

[0165] In one embodiment, the content of dsRNA in the produced RNA is reduced by at least 80% compared to when the method is performed without the addition of the compound involved in a metabolic pathway or metabolite.

[0166] In one embodiment, the compound according to the invention is selected from the group consisting of thiamine, Lys-Lys-Lys (Lyss), ornithine, ascorbic acid, pyruvate, tartrate, pyridoxine, nicotinamid, thiamine monophosphate (TMP) and lysine or a pharmaceutically acceptable salt, co-crystal, polymorph, solvate, hydrate, tautomer, stereoisomer and isotopically labeled derivative thereof.

[0167] In one embodiment, the content of dsRNA in the produced RNA is reduced by at least 90% compared to when the method is performed without the addition of the compound involved in a metabolic pathway or metabolite. 0 In one embodiment, the compound according to the invention is selected from the group consisting of thiamine, Lys-Lys-Lys (Lyss), tartrate, pyruvate, ornithine and ascorbic acid or a pharmaceutically acceptable salt, co-crystal, polymorph, solvate, hydrate, tautomer, stereoisomer and isotopically labeled derivative thereof. 5 In one embodiment, the content of dsRNA in the produced RNA is reduced by at least 95% compared to when the method is performed without the addition of the compound according to the invention.

[0168] In one embodiment, the compound according to the invention is selected from the group consisting of thiamine, pyruvate, Lys-Lys-Lys (Lyss) and ornithine or a pharmaceutically acceptable salt, co-crystal, 30 polymorph, solvate, hydrate, tautomer, stereoisomer and isotopically labeled derivative thereof.

[0169] In one embodiment, the compound involved in a metabolic pathway or metabolite is added to the reaction medium up to a concentration of between 1 mM and 1 M, preferably between 10 mM and 500 mM, more preferably between 50 mM and 300 mM, most preferably of about 150 mM.

[0170] In one embodiment, the compound involved in a metabolic pathway or metaboliteis added to the reaction medium up to a concentration of between 0.1 mM and 1 M, between 1 mM and 500 mM, between 1 mM and 200 mM, between 5 mM and 500 mM, between 5 mM and 200 mM, between 10 mM and 200 mM, between 5 mM and 100 mM, between 10 mM and 100 mM, between 0.1 mM and 50 mM, between 0.1 mM 0 and 40 mM, between 0.1 mM and 30 mM, between 0.1 mM and 20 mM, between 1 mM and 50 mM, between 1 mM and 40 mM, between 1 mM and 30 mM or between 1 mM and 20 mM. Foreignfiling_text P25-429

[0171] 22

[0172] In one embodiment, the compound involved in a metabolic pathway or metabolite is added to the reaction medium up to a concentration of between 0.1 mM and 1 M, between 1 mM and 500 mM or between 1 mM and 200 mM.

[0173] In one embodiment, the compound involved in a metabolic pathway or metaboliteis thiamine or a pharmaceutically acceptable salt, co-crystal, polymorph, solvate, hydrate, tautomer, stereoisomer and isotopically labeled derivative thereof. In another embodiment, thiamine is added to the reaction medium up to a concentration of between 1 mM and 1 M, between 1 mM and 500 mM, between 1 mM and 200 mM, 10 between 1 mM and 150 mM, between 1 mM and 120 mM, between 5 mM and 120 mM, between 5 mM and 100 mM, between 10 mM and 200 mM, between 10 mM and 193 mM or between 10 mM and 100 mM.

[0174] In one embodiment, the the compound involved in a metabolic pathway or metabolite is ornithine or a pharmaceutically acceptable salt, co-crystal, polymorph, solvate, hydrate, tautomer, stereoisomer and 15 isotopically labeled derivative thereof. In another embodiment, ornithine is added to the reaction medium up to a concentration of between 1 mM and 1 M, preferably between 10 mM and 500 mM, more preferably between 10 mM and 300 mM, most preferably of 150 mM.

[0175] In one embodiment, the compound involved in a metabolic pathway or metabolitecis pyruvate or a 0 pharmaceutically acceptable salt, co-crystal, polymorph, solvate, hydrate, tautomer, stereoisomer and isotopically labeled derivative thereof. In another embodiment, pyruvate is added to the reaction medium up to a concentration of between 1 mM and 1 M, between 1 mM and 500 mM, between 1 mM and 200 mM, between 5 mM and 200 mM, between 5 mM and 150 mM, between 10 mM and 200 mM, between 10 mM and 193 mM or between 50 mM and 150 mM.

[0176] In one embodiment, the compound involved in a metabolic pathway or metabolite is tartrate or a pharmaceutically acceptable salt, co-crystal, polymorph, solvate, hydrate, tautomer, stereoisomer and isotopically labeled derivative thereof. In another embodiment, tartrate is added to the reaction medium up to a concentration of between 1 mM and 1 M, between 1 mM and 500 mM, between 1 mM and 200 mM, 0 between 5 mM and 200 mM, between 5 mM and 150 mM, between 10 mM and 200 mM, between 10 mM and 193 mM or between 50 mM and 150 mM.

[0177] In another embodiment, the compound involved in a metabolic pathway or metabolite is thiamine monophosphate (TMP) or a pharmaceutically acceptable salt, co-crystal, polymorph, solvate, hydrate, 5 tautomer, stereoisomer and isotopically labeled derivative thereof. In another embodiment, thiamine monophosphate (TMP) is added to the reaction medium up to a concentration of preferably between 1 mM and 1 M, more preferably between 20 mM and 100 mM, most preferably of 60 mM.

[0178] In one embodiment, compound involved in a metabolic pathway or metabolite is ascorbic acid. In another 0 embodiment, ascorbic acid is added to the reaction medium up to a concentration of between 1 mM and 1 Foreignfiling_text P25-429

[0179] 23

[0180] M, between 1 mM and 500 mM, between 1 mM and 200 mM, between 5 mM and 200 mM, between 5 mM and 150 mM, between 10 mM and 200 mM, between 10 mM and 193 mM or between 10 mM and 150 mM.

[0181] In one embodiment, the compound involved in a metabolic pathway or metabolite is Lys-Lys-Lys (Lyss) or 5 a pharmaceutically acceptable salt, co-crystal, polymorph, solvate, hydrate, tautomer, stereoisomer and isotopically labeled derivative thereof. In another embodiment, Lys-Lys-Lys (Lyss) is added to the reaction medium up to a concentration of between 1 mM and 1 M, between 1 mM and 500 mM, between 1 mM and 200 mM, between 1 mM and 100 mM, between 5 mM and 100 mM, between 5 mM and 96.5 mM, between 5 mM and 75 mM or between 5 mM and 50 mM.

[0182] In one embodiment, compound involved in a metabolic pathway or metabolite is pyridoxine or a pharmaceutically acceptable salt, co-crystal, polymorph, solvate, hydrate, tautomer, stereoisomer and isotopically labeled derivative thereof. In another embodiment, pyridoxine is added to the reaction medium up to a concentration of between 1 mM and 1 M, between 1 mM and 500 mM, between 1 mM and 200 mM, 15 between 1 mM and 150 mM, between 1 mM and 120 mM, between 5 mM and 120 mM, between 5 mM and 100 mM, between 10 mM and 200 mM, between 10 mM and 193 mM or between 10 mM and 100 mM.

[0183] In one embodiment, compound involved in a metabolic pathway or metabolite is nicotinamide or a pharmaceutically acceptable salt, co-crystal, polymorph, solvate, hydrate, tautomer, stereoisomer and 0 isotopically labeled derivative thereof. In another embodiment, nicotinamide is added to the reaction medium up to a concentration of between 1 mM and 1 M, between 1 mM and 500 mM, between 1 mM and 200 mM, between 5 mM and 200 mM, between 10 mM and 200 mM, between 10 mM and 193 mM, between 50 mM and 500 mM, between 50 mM and 200 mM or between 50 mM and 193 mM. 5 In one embodiment, the compound involved in a metabolic pathway or metabolite is lysine or a pharmaceutically acceptable salt, co-crystal, polymorph, solvate, hydrate, tautomer, stereoisomer and isotopically labeled derivative thereof. In another embodiment, lysine is added to the reaction medium up to a concentration of between 1 mM and 1 M, between 1 mM and 500 mM, between 1 mM and 200 mM, between 5 mM and 200 mM, between 10 mM and 200 mM, between 10 mM and 193 mM or between 10 30 mM and 150 mM.

[0184] In one embodiment, the compound involved in a metabolic pathway or metabolite is niacin or a pharmaceutically acceptable salt, co-crystal, polymorph, solvate, hydrate, tautomer, stereoisomer and isotopically labeled derivative thereof. In another embodiment, niacin is added to the reaction medium up 35 to a concentration of between 0.1 mM and 1 M, between 0.1 mM and 500 mM, between 0.1 mM and 100 mM, between 0.1 mM and 20 mM, between 0.5 mM and 20 mM, between 1 mM and 20 mM or between 1 mM and 19.3 mM.

[0185] In one embodiment, the compound involved in a metabolic pathway or metabolite is thiamine 0 pyrophosphate or a pharmaceutically acceptable salt, co-crystal, polymorph, solvate, hydrate, tautomer, stereoisomer and isotopically labeled derivative thereof. In another embodiment, thiamine pyrophosphate Foreignfiling_text P25-429

[0186] 24 is added to the reaction medium up to a concentration of between 0.1 mM and 1 M, between 0.1 mM and 500 mM, between 0.1 mM and 100 mM, between 0.1 mM and 50 mM, between 0.1 mM and 30 mM, between 1 mM and 50 mM, between 1 mM and 30 mM, between 1.4 mM and 30 mM or between 1.4 mM and 27.7 mM.

[0187] In one embodiment, the compound involved in a metabolic pathway or metabolite is glycine or a pharmaceutically acceptable salt, co-crystal, polymorph, solvate, hydrate, tautomer, stereoisomer and isotopically labeled derivative thereof. In another embodiment, glycine is added to the reaction medium up to a concentration of between 1 mM and 1 M, between 1 mM and 500 mM, between 1 mM and 200 mM, between 5 mM and 200 mM, between 10 mM and 200 mM or between 10 mM and 193 mM.

[0188] In one embodiment, the compound involved in a metabolic pathway or metabolite is Na-acetyl-L-lysine or a pharmaceutically acceptable salt, co-crystal, polymorph, solvate, hydrate, tautomer, stereoisomer and 15 isotopically labeled derivative thereof. In another embodiment, Na-acetyl-L-lysine is added to the reaction medium up to a concentration of between 0.1 mM and 1 M, between 0.1 mM and 500 mM, between 0.1 mM and 100 mM, between 0.1 mM and 50 mM, between 0.1 mM and 40 mM, between 1 mM and 50 mM, between 1 mM and 40 mM, between 2 mM and 40 mM or between 2 mM and 38.6 mM. 0 An undesirable effect of some excipients is the reduction of the yield of the produced RNA during IVT. Therefore, in one embodiment, the yield of the produced RNA is not reduced or not essentially reduced than when the method is performed without the addition of a compound according to the invention, for example not reduced by more than 2%, 3%, 4%, 5%, 10%, 15%, 20%, 30%, 40% or 50%. 5 In one embodiment, the compound involved in a metabolic pathway or metabolite is selected from the group consisting of thiamine, Lys-Lys-Lys (Lyss), ornithine, ascorbic acid, pyridoxine, nicotinamid, thiamine monophosphate (TMP), thiamine pyrophosphate, creatinine, pyruvate, acetylCoA, cytidine, uridine, methionine and lysine or a pharmaceutically acceptable salt, co-crystal, polymorph, solvate, hydrate, tautomer, stereoisomer and isotopically labeled derivative thereof.

[0189] In some embodiments, the yield of the produced RNA is higher than when the method is performed without the addition of a compound involved in a metabolic pathway or metabolite, for example at least 2%, 3%, 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% higher. In one embodiment, the yield of the produced RNA is at least 3% higher, at least 5% higher, at least 7.5% higher, at least 10% higher, 35 15% higher or at least 20% higher.

[0190] The content of dsRNA can be measured using any method known in the art, for example by anti-dsRNA ELISA or similar Dot Blot methods. Alternative methods utilize e.g. genetically modified reporter cell lines combined with e.g. luminescent quantification or monitor activity of previously described receptors triggered 0 by immunogenicity of dsRNA. Foreignfiling_text P25-429

[0191] 25

[0192] In some embodiments, the produced RNA leads to increased mRNA efficiency than when the method is performed without the addition of a compound involved in a metabolic pathway or metabolite, for example at least 10%, 50%, 100%, 200%, 300%, 500%, 700%, 800%, 1000%, 1500%, 2000%, 2500%, 3000% or 4000% higher.

[0193] In one embodiment, the compound involved in a metabolic pathway or metabolite is selected from the group consisting ofthiamine, Lys-Lys-Lys (Lyss), ornithine, pyridoxine, pyruvate, succinate, creatinine, acetylCoA, uridine, cytidine, methionine, ascorbic acid, sodium tartrate, lysine, histidine, citrulline and glycine or a pharmaceutically acceptable salt, co-crystal, polymorph, solvate, hydrate, tautomer, stereoisomer and 10 isotopically labeled derivative thereof.

[0194] In one embodiment, the compound according to the invention is selected from the group consisting of thiamine, Lys-Lys-Lys (Lyss), ornithine, pyridoxine, meglumine and ascorbic acid or a pharmaceutically acceptable salt, co-crystal, polymorph, solvate, hydrate, tautomer, stereoisomer and isotopically labeled 15 derivative thereof.

[0195] In one embodiment, the compound according to the invention is selected from the group consisting of thiamine, Lys-Lys-Lys (Lyss) and ornithine or a pharmaceutically acceptable salt, co-crystal, polymorph, solvate, hydrate, tautomer, stereoisomer and isotopically labeled derivative thereof. Q

[0196] In some embodiments, the reaction mix is incubated after addition of compound involved in a metabolic pathway or metabolite, preferably at a temperature between 35 and 47 °C for at least one hour. In a preferred embodiment, the reaction mix is incubated at 37 °C for two hours. 5 In another aspect, the invention relates to the use of a compound involved in a metabolic pathway , preferably a metabolite, to decrease the content of double stranded RNA during RNA in vitro transcription.

[0197] In a further aspect, the invention relates to a method for reducing the content of double stranded RNA (dsRNA) in a produced RNA in in vitro transcribed RNA, comprising adding a compound involved in a 0 metabolic pathway, preferably a metabolite, to a reaction mix comprising a template DNA, an RNA polymerase and ribonucleotides.

[0198] In yet another aspect, the invention relates to a kit for RNA in vitro transcription, comprising a buffer system, ribonucleotides, an RNA polymerase and a acompound involved in a metabolic pathway, preferably a 35 metabolite.

[0199] In addition to the experimental settings disclosed in the example section, the following experiments can be performed to further assess the produced RNA and the influence of the different metabolites:

[0200] Hydrodynamic radius measurements of IVT RNA Foreignfiling_text P25-429

[0201] 26

[0202] To determine the hydrodynamic radius of IVT reactions, excipients are added after DNase treatment. A Dynapro III Plate Reader (Wyatt Technology, Santa Barbara, California USA) is used to determine the hydrodynamic radius. 10 acquisitions of 5 seconds each are accumulated. Measurements are performed in a 384-well-plate with 35 pl volume per well. All samples are measured in triplicates. Temperature is 5 increased from 5 °C to 37 °C in incremental steps of 4 °C. An equilibration step is implemented after reaching each respective temperature level.

[0203] Electrophoresis

[0204] Agarose gel electrophoresis (AGE) is performed by using precast 1 % (w / v) agarose gels with SYBR™ Gold 10 II staining. For separation and imaging, the E-Gel™ EX system (ThermoFisher) is used according to the manufacturer’s instructions. RNA size is contrasted by the E-Gel™ 1 Kb Plus Express Ladder (ThermoFisher). Purified RNAs are diluted 1 :100 and 20 pL are applied for separation. Capillary electrophoresis (CE) is performed by using an automated parallel capillary electrophoresis system (Fragment Analyzer, Agilent). For analysis of IVT mRNA, a corresponding kit is used according to the 15 manufacturer’s instructions (RNA Kit 15N).

[0205] Fluorometric detection of IVT mRNA

[0206] RNA yields of crude IVT solutions are quantified using a fluorometric assay. To this end, the Qubit™ RNA BR Assay (ThermoFisher) is performed according to the manufacturer’s instructions. Q

[0207] Cell culture, transfection, and luciferase quantification

[0208] HeLa cells are cultivated in Dulbecco's Modified Eagle Medium (DMEM) containing 4,500 mg / L glucose (Merck KGaA), supplied with 10% (v / v) fetal bovine serum (FBS) (Merck KGaA) in T75 cell culture flasks. Passaging is performed using PBS (Merck KGaA) and 2 mL Accutase® solution every 2-3 days. For 5 cultivation, a humidified tissue culture incubator at 37 °C and 10% CO2 is used.

[0209] For Flue encoding mRNA analysis, 5,000 cells per well are seeded in 96-well plates. By following the manufacturer’s recommendations, Lipofectamine™ MessengerMax™ (0.3 pL) is used for transient transfection of 0.1 pg RNA per well the next day. After transient transfection, cells are incubated 24 h at 37 °C and 10% CO2. For fluorescent viability analysis, the CellTiter-Fluor™ Cell Viability Assay (Promega) is 30 performed according to the manufacturer’s instructions. In short, each well is supplemented with 5X CellTiter-Fluor™ reagent, mixed by orbital shaking (300-500 rpm), incubated for 30 min at 37 °C and fluorescence is detected by a plate reader. For detection of luciferase-derived luminescence, the ONE-Glo™ Luciferase assay system (Promega) is used by following the manufacturer’s instructions. To this end, 100 pL substrate-containing solution is added to each well, incubated for 5 min at room 5 temperature and luminescence is recorded by a plate reader.

[0210] Examples

[0211] In vitro transcription and mRNA purification

[0212] Synthesis of mRNA was performed by run-off in vitro transcription (IVT). Linear DNA templates were generated by polymerase chain reaction (PCR) and encoded a 3' poly(A)-tail (120 nt). As mRNA models 0 Photinus pyralis (firefly) luciferase (Flue) was selected. For standard IVT with co-transcriptional capping, Foreignfiling_text P25-429

[0213] 27

[0214] DNA templates were incubated in nuclease-free water at 37°C for 2 hours with T7 RNA polymerase, RNase inhibitor, Pyrophosphatase, ATP, GTP, UTP, CTP and Cap analog. Reactions were compiled using the HiScribe™ T7 High Yield RNA Synthesis Kit (New England Biolabs®) and CleanCap® Reagent AG (TriLink) according to the manufacturer’s instructions.

[0215] 5 For optimized IVT reactions, the nuclease-free water was supplemented with 1 M L-Ornithine pH 7.0 (Merck KGaA) to reach a final concentration of 150 mM, or 1 M L-Serine pH 7.0 (Merck KGaA) to reach a final concentration of 150 mM, or 1 M Glycine pH 7.0 (Merck KGaA) to reach a final concentration of 193 mM, or 1 M Pyridoxine pH 7.0 (Merck KGaA) to reach a final concentration of 100 mM, or 1 M Nicotinamide pH 7.0 (Merck KGaA) to reach a final concentration of 193 mM, or 1 M Ascorbic acid pH 7.0 (Merck KGaA) to 10 reach a final concentration of 150 mM, or 1 M Sodium Tartrate pH 7.0 (Merck KGaA) to reach a final concentration of 150 mM, or 1 M Creatinine pH 7.0 (Merck KGaA) to reach a final concentration of 150 mM, or 1 M L-Citrulline pH 7.0 (Merck KGaA) to reach a final concentration of 150 mM, or 1 M L-Lysine pH 6.7 (Merck KGaA) to reach a final concentration of 150 mM, or 1 M L-Carnitine (Merck KGaA) to reach a final concentration of 150 mM, or 0.5 M Lys-Lys-Lys (Lyss) pH 7.0 (Merck KGaA) to reach a final concentration 15 of 50 mM, or 0.5 M L-Histidine pH 7.0 (Merck KGaA) to reach a final concentration of 75 mM, or 0.5 M Thiamine pH 7.0 (Merck KGaA) to reach a final concentration of 100 mM, or 420 mM Thiamine monophosphate pH 7.0 (TMP, Merck KGaA) to reach a final concentration of 60 mM, or 200 mM Na- Acetyl- L-Lysine pH 6.6 (Merck KGaA) to reach a final concentration of 38.6 mM, or 143 mM Thiamine pyrophosphate pH 7.2 (TMP, Merck KGaA) to reach a final concentration of 27.7 mM, or 100 mM Niacin 0 (Nicotinic Acid) pH 7.0 (Merck KGaA) to reach a final concentration of 19.3 mM, or 100 mM acetylcoenzyme A (Merck KGaA) to reach a final concentration of 15 mM, or 75.5 mM Pyrroloquinoline quinone (PQQ, methoxatin, Merck KGaA) to reach a final concentration of 11.3 mM, or 17.5 mM cyclic Guanosine Monophosphate (cGMP, Merck KGaA) to reach a final concentration of 2.6 mM, or 1 M Uridine (Merck KGaA) to reach a final concentration of 150 mM, or 1 M Cytidine (Merck KGaA) to reach a final 5 concentration of 150 mM, or 20 mM Adenosine pH 5.0 (Merck KGaA) to reach a final concentration of 3 mM, or 5 mM Guanosine pH 5.0 (Merck KGaA) to reach a final concentration of 0.75 mM, or 1 M Succinate pH 8.0 (Merck KGaA) to reach a final concentration of 150 mM, or 1 M pyruvate pH 7.0 (Merck KGaA) to reach a final concentration of 150 mM, or 1 mM Uric acid pH 5.0 (Merck KGaA) to reach a final concentration of 0.15 mM, or 1 M N-acetyl glucosamine pH 5.0 (Merck KGaA) to reach a final concentration of 150 mM, 30 1 M sodium acetate pH 8.0 (Merck KGaA) to reach a final concentration of 150 mM, or 0.5 M threonine pH

[0216] 5.0 (Merck KGaA) to reach a final concentration of 75 mM, or 0.25 M glutamine pH 5.0 (Merck KGaA) to reach a final concentration of 37.5 mM, or 5 mM tyrosine pH 5.0 (Merck KGaA) to reach a final concentration of 0.75 mM, or 0.25 M methionine pH 5.0 (Merck KGaA) to reach a final concentration of 37.5 mM.

[0217] For hardly soluble excipients we prepared saturated solutions including Guanine pH 8.5 (Merck KGaA), 35 Cytosine pH 7.5 (Merck KGaA), Uracil pH 6.1 (Merck KGaA), L-Tryptophan pH 7.8 (Merck KGaA), orotic acid (Merck KGaA), collected the supernatant after centrifugation (ca. 1000 g) and added 15 % (v / v) to the IVT.

[0218] Subsequently, residual DNA template was removed by a DNase treatment for 1 hour at 37°C. Further residual reaction components were removed by silica-membrane based RNA extraction in spin columns 0 (RNeasy®, Qiagen) according to the manufacturer’s instructions for crude RNA. Contrasted samples were obtained with the same elution volume. Final mRNA concentrations were determined by A260. Foreignfiling_text P25-429

[0219] 28

[0220] Enzyme-linked Immunosorbent Assay

[0221] The anti-dsRNA ELISA was performed as previously described by Schonborn et al. (Schonborn J, Oberstrass J, Breyel E, Tittgen J, Schumacher J, Lukacs N. Monoclonal antibodies to double-stranded RNA 5 as probes of RNA structure in crude nucleic acid extracts. Nucleic Acids Res. 1991 Jun 1 1 ;19(11):2993- 3000). Microtiter plates were coated using dsRNA-specific monoclonal J2 antibody, and remaining binding sites were saturated with 1 % BSA (w / v). Subsequently, plates were washed using PBS containing 0.5% (v / v) TWEEN® 20. Dilutions of mRNA sample or in-house produced dsRNA standard were added and incubated for 2 hours at room temperature. Unbound RNA was removed by washing with 0.5% (v / v) 10 TWEEN® 20 in PBS. Subsequently, plates were incubated with dsRNA-specific monoclonal K2 antibody for 2 hours at room temperature. For chemiluminescent detection, wells were incubated with horseradish peroxidase (HRP) conjugated goat anti-IgM (mouse) antibody for 1 hours at room temperature. Secondary antibody incubation was followed by a final washing step before imaging. HRP substrate 3, 3', 5,5'- Tetramethylbenzidin (TMB) was added and incubated for 10 min in darkness. After addition of a sulfuric 15 acid-containing stop solution, the absorption was detected at 450 nm.

[0222] Conclusion

[0223] As versatile tools, RNAs enable a broad spectrum of therapeutical applications by encoding proteins for ribosomal synthesis. To enable therapeutic use, synthetic RNAs need to hijack the cellular protein synthesis 0 machinery and pass the immune system.

[0224] In the context of mRNA synthesis (in vitro transcription, IVT), double-stranded (ds)RNA byproduct species are generated. Due to the inscrutable reaction, the actual underlying mechanisms for dsRNA synthesis are unknown. The byproducts are immunogenic and hence important quality attributes. Several attempts towards mRNA manufacturing with low dsRNA formation were reported. However, current solutions e.g. 5 involve additional manufacturing steps, toxic or flameable reagents or mutated enzymes with approximately 2-fold reduced dsRNA formation.

[0225] A typical IVT represents a highly complex interplay of 2-3 proteins, DNA template, NTPs, ions, spermidine, reducing agents, chelating agents, detergent, and buffers to ensure a stable pH. During incubation, the absolute amount of NTPs is reduced and single stranded RNA product as well as double stranded (ds)RNA 0 byproduct are synthesized.

[0226] To facilitate the in vitro reaction, single subunit RNA polymerases (RNAP) are used. Thus, the most frequently applied RNA polymerase is derived from Escherichia coli infecting phage T7. Based on this RNAP, several studies reported optimization of IVT conditions towards e.g. lower dsRNA, higher yields and efficient addition of co-transcriptional capping analogs. Optimizations were focused on the concentrations 5 of individual components, temperature, incubation time, selected buffers, pH, template and salt concentrations. However, recently the addition of chaotropic and toxic substances was reported to counteract dsRNA formation. Based on this approach, IVT yields were mainly diminished but dsRNA levels reduced. The applicability in a pharmaceutical context is limited due to the reduced yields and introduction of e.g. toxic compounds. 0 However, current IVT standard reaction conditions do not resemble the physiological environment of T7 RNAP. Thus, it can be speculated, that dsRNA formation could be an artefact of IVTs. During the infection Foreignfiling_text P25-429

[0227] 29 by T7 phage, the host metabolism is reprogrammed to support viral propagation. Thereby, intrinsic metabolic pathways are hijacked and reprogramed. To assess the potential impact of accumulating metabolites in T7 RNAP activity, we supplemented standard IVT reactions with compounds representing essential metabolic pathways of the T7 RNAP host. Thereby we observed that molecules associated e.g. with glycolysis, citric acid cycle, protein synthesis, replication and pathophysiological nutrition reduced dsRNA levels. By adding the corresponding metabolites to the IVT in various concentrations, dsRNA formation was reduced up to 97%.

[0228] To assess the impact of compounds associated to metabolic pathways on dsRNA formation in IVT, we prepared standard IVTs and added respective stock solutions before reaction start (e.g. by enzyme or template addition). Metabolites were present during the whole incubation time (2 h, 37°C) to achieve a maximum of effectiveness. We rationalized, that supplementation during incubation would decrease maximum effectiveness. Compounds were solved in nuclease-free water (NFW) at 1 M (pH 7) or limit of solubility, respectively. As model, we focused on generation of Firefly luciferase (FLuc) encoding mRNA. Subsequently, mRNA product was confirmed using agarose gel electrophoresis (AGE). To enable accurate dsRNA analysis, we purified product RNAs by silica-membrane extraction. A standardized elution volume was applied to enable yield assessments. Final RNA product concentration was determined by A260 nm and dsRNA levels quantified using Enzyme-Linked Immunosorbent Assay (ELISA).

[0229] First, we analyzed metabolites associated with protein biosynthesis. Strikingly, several molecules significantly reduced dsRNA formation during IVT (Fig. 1 , table 1). Addition of oligopeptide Lys-Lys-Lys resulted in 97% dsRNA reduction. The essential coenzyme for many enzymes in amino acid metabolism, pyridoxine, reduced dsRNA formation by 90%, relative to standard IVTs. Foreignfiling_text P25-429

[0230] 30

[0231] Table 1 Reduced dsRNA formation by IVTs supplemented with E. coli metabolites associated with protein biosynthesis. Relative dsRNA formation in contrast to standard IVTs (Std). Byproduct formation quantified by anti-dsRNA ELISA. Final concentration of respective metabolite in IVT.

[0232] Interestingly, in parallel to dsRNA reduction, supplemented IVTs maintained expected standard yields (Fig. 2). Collectively, the present data indicated, that compounds associated to metabolic pathways and especially protein biosynthesis enabled efficient RNA synthesis by IVT with reduced dsRNA.

[0233] Next, we assessed metabolites associated with replication and transcription. After mRNA purification, we again observed reduced dsRNA formation for several molecules by ELISA (Fig. 3, table 2). Interestingly, metabolites associated with pyrimidine bases (uridine, cytidine) and respective synthesis (L-ornithine) resulted in up to 96% reduced dsRNA formation.

[0234] Table 2 Reduced dsRNA formation by IVTs supplemented with E. coli metabolites associated with replication. Relative dsRNA formation in contrast to standard IVTs (Std). Byproduct formation quantified by anti-dsRNA ELISA. Final concentration of respective metabolite in IVT.

[0235] The majority of used metabolites enabled IVTs with expected standard yields (Fig. 4). This data further clarified that metabolites represent ideal supplements for efficient mRNA synthesis by IVT with reduced dsRNA.

[0236] Next, we focused on molecules associated with cellular energy provision e.g. by cell respiration. To this end, we first assessed molecules associated with commonly described pathways for glycolysis (Entner- Doudoroff, Embden-Meyerhof-Parnas (EMP)). Again, several molecules like nicotinamide, actetate niacine or pyruvate showed significant dsRNA reduction properties up to 96% (Fig. 5, table 3). Foreignfiling_text P25-429

[0237] 31

[0238] Table 3 Reduced dsRNA formation by IVTs supplemented with E. coli metabolites associated with replication. Relative dsRNA formation in contrast to standard IVTs (Std). Byproduct formation quantified by anti-dsRNA ELISA. Final concentration of respective metabolite in IVT.

[0239] All used compounds associated to metabolic pathways enabled IVT yields similar to standard reactions (Fig. 6).

[0240] Pyrroloquinoline quinone (PQQ) is not a direct metabolite of glycolysis. Previous studies reported, that PQQ is increasing glycolytic activity. The increase is achieved by modulating expression of key enzymes like hexokinases, pyruvate kinase and lactate dehydrogenase. Furthermore, the substance is supporting mitochondrial functions, reducing inflammation and other pathways interconnected with glycolysis.

[0241] Glycolysis is linked to citric acid cycle via thiamine derivate cofactors. Here, we further detected dsRNA reduction up to 98% (Fig. 7, table 4).

[0242] Table 4 Reduced dsRNA formation by IVTs supplemented with E. coli metabolites associated with glycolysis and citric acid cycle. Relative dsRNA formation in contrast to standard IVTs (Std). Byproduct formation quantified by anti-dsRNA ELISA. Final concentration of respective metabolite in IVT.

[0243] Again, addition of selected compounds to IVT resulted in yields similar to standard reactions (Fig. 8).

[0244] Subsequently, we further identified molecules associated with TCA cycle (e.g. succinate, acetyl coenzyme A). In presence of the selected compounds, mRNA products were obtained and significant dsRNA reduction observed (Fig. 9, Fig. 10, table 5). Foreignfiling_text P25-429

[0245] 32

[0246] Table 5 Reduced dsRNA formation by IVTs supplemented with E. coli metabolites associated with citric acid cycle. Relative dsRNA formation in contrast to standard IVTs (Std). Byproduct formation quantified by anti-dsRNA ELISA. Final concentration of respective metabolite in IVT.

[0247] Finally, we assessed the impact of molecules associated with anaerobic metabolism, uric acid cycle and pathophysiological nutrition (Fig. 11 , Fig .12, table 6).

[0248] Table 6 Reduced dsRNA formation by IVTs supplemented with E. coli metabolites associated with anaerobic metabolism, uric acid cycle and nutrition. Relative dsRNA formation in contrast to standard IVTs (Std). Byproduct formation quantified by anti-dsRNA ELISA. Final concentration of respective metabolite in IVT.

[0249] Tartrate pathway in E. coli enables anaerobic fermentation of tartrate to succinate. Thus, E. coli can metabolize tartrate to succinate for energy supply under anaerobic conditions (see above). The L-tartrate pathway is based on L-tartrate dehydratase TtdAB and L-tartrate / succinate antiporter TtdT. Vice versa the D-tartrate pathway is based on C4-dicarboxylate carriers DcuB (fumarate / succinate antiporter) and fumarase B (FumB). L-tartrate is taken up by L-tartrate / succinate antiporter, TtdT. L-tartrate is dehydrated to oxaloacetate by TtdAB. Oxaloacetate is converted to malate, and next to fumarate. Finally, fumarate is reduced to succinate by fumarate reductase (FrdABCD). D-tartrate is taken up by the general C4- dicarboxylate carrier, DcuB. In contrast to L-tartrate, D-tartrate does not require a TtdAB dehydratase. It is dehydrated by fumarase (FumB) and subsequently processed through fumarate respiration pathway to succinate. Foreignfiling_text P25-429

[0250] 33

[0251] Furthermore, E. coli exhibits a formate-based reductive pathway for anaerobic uric acid metabolism. Starting from isoxanthine formation and culminating in pyruvate and ammonia formation. Here, involvement of citrulline is currently under investigation.

[0252] Under pathophysiological conditions, E. coli is executing several stress-related metabolic processes. Here, more precisely, N-acetyl glucosamine can be associated to glycolysis. After cellular uptake, it is transformed into Fructose-6-phosphat (F6P). The fructose derivate is relevant as substrates for enzymes related to glycolysis (Phosphofructokinases, see above). Carnitine is metabolized by E. coli under osmotic stress conditions, which could be linked to infection by T7 phage. Furthermore, ascorbic acid is inducing catalase activity in E. coli. The enzyme is used for detoxification from hydrogen peroxide.

[0253] Again, we identified several compounds associated to a metabolic pathway with significant dsRNA reduction properties e.g. tartrate, carnitine or ascorbic acid.

[0254] Collectively, E.coli represents the originating host for physiological T7 RNAP-mediated RNA transcription. The present dataset demonstrated, that compounds associated to various RNAP host metabolic pathways can be added to in vitro transcrtiption. The compounds enable standard IVT reactions with commonly observed yields and significantly reduce unwanted dsRNA byproduct formation. The concept represents a safe, simple and sustainable alternative with high efficiency to commonly used techniques decreasing dsRNA during IVT or by subsequent purification procedures.

Claims

1. Foreignfiling_text P25-42934Claims1. A method for reducing the content of double stranded RNA in a produced RNA during in vitro transcription, the method comprising adding to a reaction mix comprising a template DNA, an RNA polymerase and ribonucleotides, a compound involved in a metabolic pathway of the originating host5 of the RNA polymerase or a pharmaceutically acceptable salt, co-crystal, polymorph, solvate, hydrate, tautomer, stereoisomer and isotopically labeled derivative thereof.

2. The method according to claim 1 , wherein the metabolic pathway is selected from the list consisting of the glycolysis, the citric acid cycle, the DNA replication, the pentose phosphate pathway and the10 anaerobic metabolism.

3. The method according to claims 1 or 2, wherein the RNA polymerase is a single subunit RNA polymerase derived from Escherichia coli infecting phage T7 and the originating host is Escherichia coli.

4. The method according to any of claims 1 to 3, wherein the compound is selected from the group consisting of L-serine, glycine, Lys-Lys-Lys, pyridoxine, L-histidine, L-lysine, N(alpha)-acetly-L-lysine, L-tryptophane, L-threonine, L-glutamine, L-tyrosine, creatinine, L-methionine, guanine, cytosine, uracil, adenosine, guanosine, cytidine, uridine, orotic acid, L-ornithine, cyclic guanosine 0 monophosphate, pyruvate, niacin, sodium acetate, nicotinamide, acetyl-CoA, succinate, thiamine, thiamine monophosphate, thiamine pyrophosphate, tartrate, L-citrulline, uric acid, N-acetly glucosamine, ascorbic acid and carnithine or a pharmaceutically acceptable salt, co-crystal, polymorph, solvate, hydrate, tautomer, stereoisomer and isotopically labeled derivative thereof. 5 5. The method according to any of claims 1 to 4, wherein the compound is selected from the group consisting of Lys-Lys-Lys, pyridoxine, L-histidine, cytidine, uridine, L-ornithine, pyruvate, nicotinamide, niacin, thiamine, thiamine monophosphate, thiamine pyrophosphate, acetyl CoA, succinate, tartrate and ascorbic acid or a pharmaceutically acceptable salt, co-crystal, polymorph, solvate, hydrate, tautomer, stereoisomer and isotopically labeled derivative thereof.

6. The method according to any of claims 1 to 5, wherein the content of dsRNA in the produced RNA is reduced by at least 50% than when the method is performed without the addition of the compound involved in a metabolic pathways of the originating host of the RNA polymerase. 5 7. The method according to any of claims 1 to 6, wherein the yield of the produced RNA is not reduced by more than 20% than when the method is performed without the addition of a compound involved in a metabolic pathways of the originating host of the RNA polymerase.

8. The method according to any of claims 1 to 7, wherein the yield of the produced RNA is higher than 0 when the method is performed without the addition of a compound involved in a metabolic pathways of the originating host of the RNA polymerase.Foreignfiling_text P25-429359. The method according to any of claims 1 to 8, wherein the produced RNA is a mRNA.

10. The method according to claim 9, wherein the produced RNA has a higher mRNA efficiency than when the method is performed without the addition of a compound involved in a metabolic pathways of the originating host of the RNA polymerase.

11. The method according to any of claims 1 to 10, wherein the compound involved in a metabolic pathways of the originating host of the RNA polymerase is added to the reaction mix up to a concentration of between 0.1 mM and 1 M.

12. Use of a compound involved in a metabolic pathways of the originating host of an RNA polymerase to decrease the content of dsRNA during in vitro transcription.

13. Use according to claim 12, wherein the RNA polymerase is a single subunit RNA polymerase derived from Escherichia coli infecting phage T7 and the originating host is Escherichia coli.

14. Use according to claims 12 and 13, wherein the compound is selected from the group consisting of Lys-Lys-Lys, pyridoxine, L-histidine, cytidine, uridine, L-ornithine, pyruvate, nicotinamide, niacin, thiamine, thiamine monophosphate, thiamine pyrophosphate, acetyl CoA, succinate, tartrate and ascorbic acid or a pharmaceutically acceptable salt, co-crystal, polymorph, solvate, hydrate, tautomer, stereoisomer and isotopically labeled derivative thereof.

15. A kit for in vitro transcription, comprising a buffer system, ribonucleotides, an RNA polymerase and a compound involved in the metabolic pathways of the originating host of an RNA polymerase reducing intermolecular interactions.

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