Methods for RNA production

Abstract

The invention relates to a method for producing RNA by in vitro transcription, the method comprising adding a compound reducing intermolecular interactions to a reaction mix comprising a template DNA, an RNA polymerase and ribonucleotides.

Description

[0001] Foreignfiling text P24-168

[0002] - 1 -

[0003] METHODS FOR RNA PRODUCTION

[0004] Field of the invention

[0005] The present invention relates to the production of RNA, in particular to in vitro transcription.

[0006] 5 Specifically, the present invention relates to methods for decreasing the content of double stranded RNA (dsRNA) during RNA in vitro transcription (IVT).

[0007] Technical background

[0008] 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, with several IVT-produced SARS-CoV-2 vaccines having received clinical approval in recent years. In addition, therapies based on mRNA have demonstrated great potential in diverse areas of medicine such as cancer and infectious diseases.

[0009] 15 The relatively simple IVT procedure itself 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.

[0010] One important drawback of IVT is the occurrence of dsRNA as a by-product. This presents

[0011] 20 a challenge since, parallel 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.

[0012] Current approaches to overcome the formation of dsRNA during IVT are predominantly related to analytical purification such as chromatography-based purification methods. These may be effective in reducing dsRNA content, but represent an additional step in the workflow, require costly and highly specialized equipment, and can give rise to problems when upscaling the IVT.

[0013] Other approaches center on methods for monitoring the IVT and the efficacy of its enzymatic

[0014] 30 reactions in real time in order to adjust the conditions accordingly. These approaches generally involve the costly and time-consuming use of RNA aptamers, fluorescence dyes Foreignfiling text P24-168

[0015] - 2 - and additional HPLC procedures, thus increasing manufacturing costs, as well as compromising the integrity and quality of the RNA product.

[0016] Alternative approaches to purification and real-time monitoring have been proposed involving altering the reaction conditions. Such alternatives have focused around the effects

[0017] 5 of magnesium concentrations in the IVT reaction. For example, it has been suggested that lowering or increasing Mg concentration in the reaction can reduce the formation of dsRNA. However, Mg plays an important role in transcription as it modulates enzymatic activity. This means that manipulating Mg concentrations requires costly and cumbersome fine-tuning in order to avoid the side effects of impairing the transcription reaction and therefore the total yield of RNA.

[0018] Other approaches involve using chaotropic agents in the initial reaction mixture. Some of these substances 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

[0020] 15 certainly constrain the cost-effectiveness and time efficiency of the essentially uncomplicated and practical IVT procedure.

[0021] Objective problem to be solved

[0022] To address these challenges, methods are required that are able to reduce the content of

[0023] 20 dsRNA in IVT reactions but require no additional post-IVT purification steps, complex realtime monitoring settings, or the addition of compounds that negatively affect the amount, quality or integrity of the product RNA, thereby avoiding cutting short the inherent advantages in time, cost and simplicity of IVT. 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.

[0024] Summary of the invention

[0025] In one aspect, the invention relates to a method for producing RNA by in vitro transcription, comprising adding a compound reducing intermolecular interactions or a viscosity reducing agent to a reaction mix comprising a template DNA, an RNA polymerase and

[0026] 30 ribonucleotides. Foreignfiling text P24-168

[0027] - 3 - ln another aspect, the invention relates to the use of a compound reducing intermolecular interactions or a viscosity reducing agent to decrease the content of double stranded RNA (dsRNA) during RNA in vitro transcription.

[0028] In another aspect, the invention relates to a method for reducing the content of double

[0029] 5 stranded RNA (dsRNA) in a produced RNA in in vitro transcribed RNA, comprising adding a compound reducing intermolecular interactions or a viscosity reducing agent to a reaction mix comprising a template DNA, an RNA polymerase and ribonucleotides.

[0030] 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, the method comprising adding to a reaction mix comprising a template DNA, an RNA polymerase and ribonucleotides, a compound reducing intermolecular interactions or a viscosity reducing agent or a pharmaceutically acceptable salt, co-crystal, polymorph, solvate, hydrate, tautomer, stereoisomer and isotopically labeled derivative thereof.

[0031] In another aspect, the invention relates to a kit for RNA in vitro transcription, comprising a

[0032] 15 buffer system, ribonucleotides, a RNA polymerase and a compound reducing intermolecular interactions or a viscosity reducing agent.

[0033] Brief description of the figures

[0034] Fig. 1 shows the viscosity of purified RNAs and impacts of excipients. As models, a 2000 nt

[0035] 20 (8.8 mg / mL, TE, pH 7.0), 4000 nt (5.68 mg / mL, TE, pH 7.0) and 6000 nt (7.53 mg / mL, TE, pH 7.0) mRNAs were used. Depending on the mRNA size, addition of ornithine (Orn), or thiamine monophosphate (TMP) affected viscosity.

[0036] Fig. 2 shows the impacts of excipients on crude IVT reactions. After incubation, model IVTs were supplemented with Orn, or TMP (150 mM), respectively. As reference the IVT was performed using a Firefly luciferase (FLuc) encoding template, a, Excipient addition decreased the hydrodynamic radius ( ), especially at lower temperatures, b, At 20°C, addition of excipients decreased the dynamic viscosity of crude IVT reactions.

[0037] Fig. 3 shows the product concentration and size of optimized IVT reactions with excipients. As model, Firefly luciferase (FLuc) encoding mRNA was manufactured and purified by silica

[0038] 30 membrane. Reactions were performed according to standard procedures for IVT in presence of excipients in selected concentrations, respectively. Analysis was performed using three individual reactions, a, Product concentration of individual reactions was quantified by A260 Foreignfiling text P24-168

[0039] - 4 -

[0040] (n = 3 analytical replicates). Mean mRNA concentration, error bars indicate standard deviation, b, Agarose gel electrophoresis (AGE) of optimized IVT reactions indicates product size and integrity.

[0041] Fig. 4 shows exact product integrity of optimized IVT reactions with excipients in capillary

[0042] 5 electrophoresis (CE). As model, FLuc encoding mRNA was manufactured and purified by silica membrane. Reactions were performed according to standard procedures for IVT in presence of excipients in selected concentrations, respectively. Analysis was performed using three individual reactions. Representative analysis of three analytical replicates.

[0043] Fig. 5 demonstrates product dsRNA content and translation of optimized IVT reactions with excipients. As model, Firefly luciferase (FLuc) encoding mRNA was manufactured and purified by silica membrane, a-c For dsRNA quantification, three individual reactions were pooled (a, c) or analyzed separately (b) by ELISA. Mean dsRNA content of analytical replicates with error bars indicating standard deviation, d, In cellulo analysis of IVT products by transient transfection of HeLa cells. For transfection, purified IVT products of three independent reactions were pooled. Resulting protein biosynthesis was contrasted by

[0044] 15 recording FLuc-derived luminescence. Mean luminescence (n = 3) with error bars indicating standard deviation.

[0045] Fig. 6 shows optimized IVT reactions with excipients for Cas9 encoding mRNAs. Reactions were performed according to standard procedures for IVT in presence of excipients in selected concentrations, respectively. IVT products were purified by silica membranes to

[0046] 20 ensure accurate characterization, a, Product concentration of three individual reactions quantified by A260 (n = 3 analytical replicates). Mean mRNA concentration, error bars indicate standard deviation, b For dsRNA quantification, individual reactions analyzed by ELISA. Mean dsRNA content of analytical replicates with error bars indicating standard deviation.

[0047] Fig. 7 shows product integrity of optimized IVT reactions with excipients in CE for Cas9 encoding mRNAs. Reactions were performed according to standard procedures for IVT in presence of excipients in selected concentrations, respectively. Analysis was performed using three individual reactions. Representative analysis of three analytical replicates.

[0048] Fig. 8 shows optimized IVT reactions with excipients for human erythropoietin (hEPO) encoding mRNAs. Reactions were performed according to standard procedures for IVT in

[0049] 30 presence of excipients in selected concentrations, respectively. IVT products were purified by silica membranes to ensure accurate characterization, a, Product concentration of three individual reactions quantified by A260 (n = 3 analytical replicates). Mean mRNA Foreignfiling text P24-168

[0050] - 5 - concentration, error bars indicate standard deviation, b For dsRNA quantification, individual reactions analyzed by ELISA. Mean dsRNA content of analytical replicates with error bars indicating standard deviation.

[0051] Fig. 9 shows product integrity of optimized IVT reactions with excipients in CE for hEPO

[0052] 5 encoding mRNAs. Reactions were performed according to standard procedures for IVT in presence of excipients in selected concentrations, respectively. Analysis was performed using three individual reactions. Representative analysis of three analytical replicates.

[0053] Fig. 10 shows the product concentration and dsRNA content of optimized IVT reactions with further excipients (panel I). As model, Firefly luciferase (FLuc) encoding mRNA was manufactured, a, b, Reactions were performed according to standard procedures for IVT in presence of excipients in selected concentrations, respectively. For analysis, three individual reactions were pooled and purified by silica membrane. Product concentration of individual reaction pools quantified by A260 (n = 3 analytical replicates). Mean mRNA concentration, error bars indicate standard deviation. For dsRNA quantification, pooled reactions analyzed by ELISA. Mean dsRNA content of analytical replicates with error bars indicating standard

[0054] 15 deviation.

[0055] Fig. 11 shows the product concentration and dsRNA content of optimized IVT reactions with further excipients (panel II). As model, Firefly luciferase (FLuc) encoding mRNA was manufactured, a, b, Reactions were performed according to standard procedures for IVT in presence of excipients in selected concentrations, respectively. For analysis, three individual

[0056] 20 reactions were pooled and purified by silica membrane. Product concentration of individual reaction pools quantified by A260 (n = 3 analytical replicates). Mean mRNA concentration, error bars indicate standard deviation. For dsRNA quantification, pooled reactions analyzed by ELISA. Mean dsRNA content of analytical replicates with error bars indicating standard deviation.

[0057] Fig. 12 shows the product concentration and dsRNA content of optimized IVT reactions with further excipients (panel III). As model, Firefly luciferase (FLuc) encoding mRNA was manufactured, a, b, Reactions were performed according to standard procedures for IVT in presence of excipients in selected concentrations, respectively. For analysis, three individual reactions were pooled and purified by silica membrane. Product concentration of individual reaction pools quantified by A260 (n = 3 analytical replicates). Mean mRNA concentration,

[0058] 30 error bars indicate standard deviation. For dsRNA quantification, pooled reactions analyzed by ELISA. Mean dsRNA content of analytical replicates with error bars indicating standard deviation. Foreignfiling text P24-168

[0059] - 6 -

[0060] Fig. 13 shows the product concentration and dsRNA content of optimized IVT reactions with further excipients (panel IV). As model, Firefly luciferase (FLuc) encoding mRNA was manufactured, a, b, Reactions were performed according to standard procedures for IVT in presence of excipients in selected concentrations, respectively. For analysis, three individual

[0061] 5 reactions were pooled and purified by silica membrane. Product concentration of individual reaction pools quantified by A260 (n = 3 analytical replicates). Mean mRNA concentration, error bars indicate standard deviation. For dsRNA quantification, pooled reactions analyzed by ELISA. Mean dsRNA content of analytical replicates with error bars indicating standard deviation.

[0062] Fig. 14 shows the product concentration and dsRNA content of optimized IVT reactions with further excipients (panel V). As model, Firefly luciferase (FLuc) encoding mRNA was manufactured, a, b, Reactions were performed according to standard procedures for IVT in presence of excipients in selected concentrations, respectively. For analysis, three individual reactions were pooled and purified by silica membrane. Product concentration of individual reaction pools quantified by A260 (n = 3 analytical replicates). Mean mRNA concentration,

[0063] 15 error bars indicate standard deviation. For dsRNA quantification, pooled reactions analyzed by ELISA. Mean dsRNA content of analytical replicates with error bars indicating standard deviation, PPQ = pyrroloquinoline quinone.

[0064] Fig. 15 shows the product concentration and dsRNA content of optimized IVT reactions based on thiamine addition. As model, Firefly luciferase (FLuc) encoding mRNA was

[0065] 20 manufactured. Reactions were performed according to standard procedures for IVT in presence of various thiamine concentrations. For analysis, three individual reactions were pooled and purified by silica membrane. Product concentration of individual reaction pools quantified by A260 (n = 3 analytical replicates). Mean mRNA concentration, error bars indicate standard deviation. For dsRNA quantification, pooled reactions analyzed by ELISA. Mean dsRNA content of analytical replicates with error bars indicating standard deviation.

[0066] Fig. 16 shows the product concentration and dsRNA content of optimized IVT reactions based on Lyss addition. As model, Firefly luciferase (FLuc) encoding mRNA was manufactured. Reactions were performed according to standard procedures for IVT in presence of various Lyss concentrations. For analysis, three individual reactions were pooled and purified by silica membrane. Product concentration of individual reaction pools

[0067] 30 quantified by A260 (n = 3 analytical replicates). Mean mRNA concentration, error bars indicate standard deviation. For dsRNA quantification, pooled reactions analyzed by ELISA. Mean dsRNA content of analytical replicates with error bars indicating standard deviation. Foreignfiling text P24-168

[0068] - 7 -

[0069] Fig. 17 shows the product concentration and dsRNA content of optimized IVT reactions based on meglumine addition. As model, Firefly luciferase (FLuc) encoding mRNA was manufactured. Reactions were performed according to standard procedures for IVT in presence of various meglumine concentrations. For analysis, three individual reactions were

[0070] 5 pooled and purified by silica membrane. Product concentration of individual reaction pools quantified by A260 (n = 3 analytical replicates). Mean mRNA concentration, error bars indicate standard deviation. For dsRNA quantification, pooled reactions analyzed by ELISA. Mean dsRNA content of analytical replicates with error bars indicating standard deviation.

[0071] Fig. 18 shows the product concentration and dsRNA content of optimized IVT reactions based on ascorbic acid addition. As model, Firefly luciferase (FLuc) encoding mRNA was manufactured. Reactions were performed according to standard procedures for IVT in presence of various ascorbic acid concentrations. For analysis, three individual reactions were pooled and purified by silica membrane. Product concentration of individual reaction pools quantified by A260 (n = 3 analytical replicates). Mean mRNA concentration, error bars indicate standard deviation. For dsRNA quantification, pooled reactions analyzed by ELISA.

[0072] 15 Mean dsRNA content of analytical replicates with error bars indicating standard deviation.

[0073] Fig. 19 shows the product concentration and dsRNA content of optimized IVT reactions based on nicotinamide addition. As model, Firefly luciferase (FLuc) encoding mRNA was manufactured. Reactions were performed according to standard procedures for IVT in presence of various nicotinamide concentrations. For analysis, three individual reactions

[0074] 20 were pooled and purified by silica membrane. Product concentration of individual reaction pools quantified by A260 (n = 3 analytical replicates). Mean mRNA concentration, error bars indicate standard deviation. For dsRNA quantification, pooled reactions analyzed by ELISA. Mean dsRNA content of analytical replicates with error bars indicating standard deviation.

[0075] Fig. 20 shows the product concentration and dsRNA content of optimized IVT reactions based on lysine addition. As model, Firefly luciferase (FLuc) encoding mRNA was manufactured. Reactions were performed according to standard procedures for IVT in presence of various lysine concentrations. For analysis, three individual reactions were pooled and purified by silica membrane. Product concentration of individual reaction pools quantified by A260 (n = 3 analytical replicates). Mean mRNA concentration, error bars indicate standard deviation. For dsRNA quantification, pooled reactions analyzed by ELISA. Mean

[0076] 30 dsRNA content of analytical replicates with error bars indicating standard deviation. Foreignfiling text P24-168

[0077] - 8 -

[0078] Fig. 21 shows the product concentration and dsRNA content of optimized IVT reactions based on pyridoxine addition. As model, Firefly luciferase (FLuc) encoding mRNA was manufactured. Reactions were performed according to standard procedures for IVT in presence of various pyridoxine concentrations. For analysis, three individual reactions were pooled and purified by silica membrane. Product concentration of individual reaction pools

[0079] 5 quantified by A260 (n = 3 analytical replicates). Mean mRNA concentration, error bars indicate standard deviation. For dsRNA quantification, pooled reactions analyzed by ELISA. Mean dsRNA content of analytical replicates with error bars indicating standard deviation.

[0080] Fig. 22 shows the product concentration and dsRNA content of optimized IVT reactions based on niacin addition. As model, Firefly luciferase (FLuc) encoding mRNA was manufactured. Reactions were performed according to standard procedures for IVT in presence of various niacin concentrations. For analysis, three individual reactions were pooled and purified by silica membrane. Product concentration of individual reaction pools quantified by A260 (n = 3 analytical replicates). Mean mRNA concentration, error bars indicate standard deviation. For dsRNA quantification, pooled reactions analyzed by ELISA. Mean dsRNA content of analytical replicates with error bars indicating standard deviation.

[0081] 15

[0082] Fig. 23 shows the product concentration and dsRNA content of optimized IVT reactions based on thiamine pyrophosphate addition. As model, Firefly luciferase (FLuc) encoding mRNA was manufactured. Reactions were performed according to standard procedures for IVT in presence of various thiamine pyrophosphate concentrations. For analysis, three individual reactions were pooled and purified by silica membrane. Product concentration of

[0083] 20 individual reaction pools quantified by A260 (n = 3 analytical replicates). Mean mRNA concentration, error bars indicate standard deviation. For dsRNA quantification, pooled reactions analyzed by ELISA. Mean dsRNA content of analytical replicates with error bars indicating standard deviation.

[0084] Fig. 24 shows the product concentration and dsRNA content of optimized IVT reactions based on glycine addition. As model, Firefly luciferase (FLuc) encoding mRNA was manufactured. Reactions were performed according to standard procedures for IVT in presence of various glycine concentrations. For analysis, three individual reactions were pooled and purified by silica membrane. Product concentration of individual reaction pools quantified by A260 (n = 3 analytical replicates). Mean mRNA concentration, error bars indicate standard deviation. For dsRNA quantification, pooled reactions analyzed by ELISA. Mean

[0085] 30 dsRNA content of analytical replicates with error bars indicating standard deviation. Foreignfiling text P24-168

[0086] - 9 -

[0087] Fig. 25 shows the product concentration and dsRNA content of optimized IVT reactions based on sorbitol addition. As model, Firefly luciferase (FLuc) encoding mRNA was manufactured. Reactions were performed according to standard procedures for IVT in presence of various sorbitol concentrations. For analysis, three individual reactions were pooled and purified by silica membrane. Product concentration of individual reaction pools

[0088] 5 quantified by A260 (n = 3 analytical replicates). Mean mRNA concentration, error bars indicate standard deviation. For dsRNA quantification, pooled reactions analyzed by ELISA. Mean dsRNA content of analytical replicates with error bars indicating standard deviation.

[0089] Fig. 26 shows the product concentration and dsRNA content of optimized IVT reactions based on Na-Acetyl-L-lysine addition. As model, Firefly luciferase (FLuc) encoding mRNA was manufactured. Reactions were performed according to standard procedures for IVT in presence of various Na-Acetyl-L-lysine concentrations. For analysis, three individual reactions were pooled and purified by silica membrane. Product concentration of individual reaction pools quantified by A260 (n = 3 analytical replicates). Mean mRNA concentration, error bars indicate standard deviation. For dsRNA quantification, pooled reactions analyzed by ELISA. Mean dsRNA content of analytical replicates with error bars indicating standard

[0090] 15 deviation.

[0091] Fig. 27 In cellulo analysis of Flue encoding mRNA products generated by optimized IVT using various excipients (l / ll). For transient transfection of HeLa cells, purified IVT products of three independent reactions were pooled. After 24 h, FLuc-derived bioluminescence was recorded to contrast protein biosynthesis. Mean luminescence (n = 3) with error bars

[0092] 20 indicating standard deviation.

[0093] Fig. 28 In cellulo analysis of Flue encoding mRNA products generated by optimized IVT using various excipients (ll / ll). For transient transfection of HeLa cells, purified IVT products of three independent reactions were pooled. After 24 h, FLuc-derived bioluminescence was recorded to contrast protein biosynthesis. Mean luminescence (n = 3) with error bars indicating standard deviation.

[0094] Fig. 29 In cellulo analysis of Flue encoding mRNA products generated by optimized IVT with various concentrations of thiamine (a) or Lyss (b). Previously oberserved differences in dsRNA levels are reflected in functional protein signals. For transient transfection of HeLa cells, purified IVT products of three independent reactions were pooled. After 24 h, FLuc-

[0095] 30 derived bioluminescence was recorded to contrast protein biosynthesis. Mean luminescence (n = 3) with error bars indicating standard deviation. Foreignfiling text P24-168

[0096] - 10 -

[0097] Detailed description of the invention

[0098] 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, a compound reducing intermolecular interactions or a

[0099] 5 pharmaceutically acceptable derivative, salt, co-crystal, polymorph, solvate, hydrate, tautomer, stereoisomer and isotopically labeled derivative thereof.

[0100] 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 template DNA, an RNA polymerase and ribonucleotides, a compound reducing intermolecular interactions or a viscosity reducing agent, or a pharmaceutically acceptable salt, co-crystal, polymorph, solvate, hydrate, tautomer, stereoisomer and isotopically labeled derivative thereof.

[0101] 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

[0102] 15 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.

[0103] 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

[0104] 20 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 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.

[0105] 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.

[0106] As used herein, the term “compound reducing intermolecular interactions” refers to any agent capable of reducing intermolecular interactions between two molecules, in particular

[0107] 30 between two nucleic acid molecules. Foreignfiling text P24-168

[0108] - 11 -

[0109] As defined herein “intermolecular interactions” are forces that exist between individual molecules in spatial proximity. Collectively, intermolecular interactions dictate the physical and chemical properties of molecule mixtures and hence express themselves e.g. in boiling points, melting points, solubility, and viscosity. Intermolecular interactions include hydrogen bonds, van der Waals forces including dipole-induced interactions and London dispersion

[0110] 5 forces and ionic interactions. Diffusion is another phenomenon which is affected by the sum of occurring intermolecular interactions. Based on the random movement of molecules (Brownian motion), is describes the distribution of molecules from a spatial compartment of higher concentration to a spatial compartment of lower concentration. Hereby, the strength of intermolecular interactions directly affects the rate of diffusion.

[0111] The skilled person is aware of available methods to determine the strength of intermolecular interactions, such as viscometry or boiling point measurements. For example, high viscosity is based on strong intermolecular forces, while low viscosity is the result of reduced intermolecular interactions.

[0112] As used herein, the term “compound reducing intermolecular interactions” may refer to a

[0113] 15 single compound reducing intermolecular interactions or a mixture of different compounds. That is, in some embodiments, only a single compound is added to the reaction mix. In other embodiments, two or more compounds are added to the react mixture, e.g., two, three, four, five or more compounds reducing intermolecular interactions are added.

[0114] In some embodiments, the compound reducing intermolecular interactions is a viscosity

[0115] 20 reducing agent.

[0116] As used herein, the term “viscosity reducing agent” may refer to substance or compound that is capable of reducing the viscosity of a solution, in particular of a solution comprising a nucleic acid. That is, in some embodiments, only a single viscosity reducing agent is added to the reaction mix. In other embodiments, two or more viscosity reducing agents are added to the react mixture, e.g., two, three, four, five or more viscosity reducing agents are added.

[0117] 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 ribonucleic acid (RNA). If the sugar is ribose, the

[0118] 30 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), Foreignfiling text P24-168

[0119] - 12 - catalytical RNA or structural RNA. DNA or RNA molecules can also be synthetically prepared for 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.

[0120] A “RNA” in terms of the produced RNA during in vitro transcription is herein defined as a

[0121] 5 nucleic acid molecule composed of a chain of nucleotides, each consisting of a ribose sugar, a phosphate group, and 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) / self- replicative RNA, long non-coding RNA, guide RNA, tRNA, asRNA, siRNA, shRNA, ribozymes, riboswitch, piRNA, tracrRNA, microRNA, ribosomal RNA (rRNA), small nuclear RNA (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

[0123] 15 base. Different forms and types of template DNA are known to the skilled person in the art and are included under the present definition. 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] 20

[0125] As defined herein, “viscosity” refers to the resistance of a substance (typically a liquid) to flow. Viscosity is related to the concept of shear force; it can be understood as the effect of different layers of the fluid exerting shearing force on each other, or on other surfaces, as they move against each other. There are several ways to express viscosity. The units of viscosity are Ns / m2, known as Pascal-seconds (Pas). Viscosity can be “kinematic” or “absolute”. Kinematic viscosity is a measure of the rate at which momentum is transferred through a fluid. It is measured in Stokes (St). The kinematic viscosity is a measure of the resistive flow of a fluid under the influence of gravity. When two fluids of equal volume and differing viscosity are placed in identical capillary viscometers and allowed to flow by gravity, the more viscous fluid takes longer than the less viscous fluid to flow through the capillary. If, for example, one fluid takes 200 seconds (s) to complete its flow and another fluid takes

[0126] 30 400 s, the second fluid is called twice as viscous as the first on a kinematic viscosity scale. The dimension of kinematic viscosity is length2 / time. Commonly, kinematic viscosity is expressed in centiStokes (cSt). The SI unit of kinematic viscosity is mm2 / s, which is equal to Foreignfiling text P24-168

[0127] - 13 -

[0128] 1 cSt. The “absolute viscosity,” sometimes called “dynamic viscosity” or “simple viscosity,” is the product of kinematic viscosity and fluid density. Absolute viscosity is expressed in units of centipoise (cP). The SI unit of absolute viscosity is the milliPascal-second (mPas), where 1 cP=1 mPas.

[0129] 5 Viscosity may be measured by using, for example, a viscometer at a given shear rate or multiple shear rates. An “extrapolated zero-shear” viscosity can be determined by creating a best fit line of the four highest-shear points on a plot of absolute viscosity versus shear rate, and linearly extrapolating viscosity back to zero-shear. Alternatively, for a Newtonian fluid, viscosity can be determined by averaging viscosity values at multiple shear rates. Viscosity can also be measured using a microfluidic viscometer at single or multiple shear rates (also called flow rates), wherein absolute viscosity is derived from a change in pressure as a liquid flows through a channel. Viscosity equals shear stress over shear rate. Viscosities measured with microfluidic viscometers can, in some embodiments, be directly compared to extrapolated zero-shear viscosities, for example those extrapolated from viscosities measured at multiple shear rates using a cone and plate viscometer.

[0130] 15 “Shear rate” herein refers to the rate of change of velocity at which one layer of fluid passes over an adjacent layer. The velocity gradient is the rate of change of velocity with distance from the plates. This simple case shows the uniform velocity gradient with shear rate (v1 -v2) / h in units of (cm / sec) / (cm)=1 / sec. Hence, shear rate units are reciprocal seconds or, in general, reciprocal time. For a microfluidic viscometer, change in pressure and flow rate are related to shear rate. “Shear rate” is to the speed with which a material is deformed.

[0131] 20 Formulations containing nucleic acids and excipients are typically measured at shear rates ranging from about 0.5 s-1to about 200 s-1when measured using a cone and plate viscometer and a spindle appropriately chosen by one skilled in the art to accurately measure viscosities in the viscosity range of the sample of interest (i.e., a sample of 20 cP is most accurately measured on a CPE40 spindle affixed to a DV2T viscometer (Brookfield)); greater than about 20 s-1to about 3,000 s-1when measured using a microfluidic viscometer.

[0132] The person skilled in the art is familiar with the viscosity measurement using a microfluidic viscometer. As microfluidic viscometer the RheoSense mVROC microfluidic viscometer (mVROC™ Technology), especially with the parameters described above can be used. Detailed specifications, methods and setting can be found in the 901003.5.1- mVROC_User’s_Manual.

[0133] 30

[0134] Preferably, viscosity is measured at 20 °C using a microfluidic viscometer. More preferably the viscosity of purified RNA is measured using a RheoSense mVROC microfluidic Foreignfiling text P24-168

[0135] - 14 - viscometer at 20 °C. Most preferably the viscosity is measured at 20 °C using a RheoSense mVROC microfluidic viscometer and using a 250 pl syringe, a shear rate of 1500 s-1 or 1000 s-1 and a volume of 60 to 80 pl. Alternatively, the viscosity, e.g., of crude IVT reactions, is measured by a rolling-ball Anton Paar Lovis 2000 ME viscometer. Rolling-ball measurements are preferably performed at 20 °C operating temperature using a

[0136] 5 Polychlortrifluorethylen (PCTFE) capillary with preferably 1 .62 mm diameter, matching steel balls, with maximal 6 determinations and 0.5% Lovis average run time maximal deviation.

[0137] The person skilled in the art is also familiar with viscosity measurement using a rolling-ball viscometer. As rolling-ball viscometer the Anton Paar Lovis 2000 ME viscometer can be used. Rolling-ball measurements are preferably performed at 20 °C operating temperature using a Polychlortrifluorethylen (PCTFE) capillary with preferably 1 .62 mm diameter, matching steel balls, with maximal 6 determinations and 0.5% Lovis average run time maximal deviation. Detailed specifications, methods and setting can be found in the Lovis 2000M / ME_User’s_Manual.

[0138] For classical “Newtonian” fluids, viscosity is essentially independent of shear rate. For “non¬

[0139] 15 Newtonian fluids,” however, viscosity either decreases or increases with increasing shear rate, e.g., the fluids are “shear thinning” or “shear thickening”, respectively. In the case of concentrated (i.e., high-concentration) liquid compositions comprising a nucleic acid, this may manifest as pseudoplastic shear-thinning behavior, i.e., a decrease in viscosity with shear rate.

[0140] 20 The viscosity of liquid compositions is reduced when at least one of the methods described above shows a reduction in viscosity.

[0141] In one embodiment, the viscosity reducing agents reduces the viscosity of a solution comprising a nucleic acid by at least 2%, 3%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70% or 75% compared to an identical composition not comprising the viscosity reducing agent.

[0142] 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,

[0143] 30 improved stability, or altered pharmacokinetics. Derivatives of compounds can include analogs or esters that retain some biological activity while exhibiting distinct characteristics compared to the parent compound. Foreignfiling text P24-168

[0144] - 15 -

[0145] 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 geometrical isomers, optical isomers based on an asymmetrical carbon, stereoisomers, tautomers and the like which occur structurally and isomer mixtures

[0146] 5 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 description of the specific salt and / or solvate.

[0147] 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). Diastereomers are stereoisomers which are non-superimposable and which are not mirror-images of each

[0148] 15 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 interconverted just by rotations about formally single bonds, and include - in particular - those leading to different

[0149] 20 3-dimentional forms of (hetero)cyclic rings, such as chair, half-chair, boat, and twist-boat forms of cyclohexane.

[0150] The term "polymorph" refers to a crystalline form of a compound (or a salt, hydrate, or solvate thereof). All 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.

[0151] The term "solvate" as used herein refers to an addition complex of a dissolved material in a

[0152] 30 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 Foreignfiling text P24-168

[0153] - 16 - mixed crystal. The amount of solvent contained in the addition complex may be stoichiometric or non-stoichiometric. A hydrate is a solvate wherein the solvent is water.

[0154] 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.

[0155] 5

[0156] 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.

[0157] 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, 11 C, 13C, 14C, 15N, 18F, 32P, 32S, 35S, 36CI, and 1251.

[0158] 15 In some embodiments, the compound reducing intermolecular interactions or the viscosity reducing agent is a compound comprising an amino group and / or a carboxyl group. In some embodiments, the compound additionally comprises a phosphate group.

[0159] In some embodiments, the compound comprising an amino group is selected from the group consisting of amino acids, peptides, vitamins, meglumine, spermine, creatine, histone

[0160] 20 peptides, cyclic guanosine monophosphate.

[0161] The amino acid to be used as compound reducing intermolecular interactions or viscosity reducing agent according to the invention may be any known amino acid such as alpha-, beta- and gamma amino acids, naturally occurring amino acids and unnatural / non- proteinogenic amino acids. Preferably, the amino acid is selected from the group consisting of glycine, histidine, lysine, serine, ornithine, citrulline 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 poly(ornithine).

[0162] 30 Peptides to be used as compound reducing intermolecular interactions or viscosity reducing agent 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, Foreignfiling text P24-168

[0163] - 17 - lysine, serine, ornithine, citrulline and derivatives thereof. In a more preferred 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.

[0164] In one embodiment, the compound reducing intermolecular interactions or the viscosity

[0165] 5 reducing agent is a vitamin or a pharmaceutically acceptable salt, co-crystal, polymorph, solvate, hydrate, tautomer, stereoisomer and isotopically labeled derivative thereof.

[0166] 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 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.

[0167] 15

[0168] In one embodiment, the compound reducing intermolecular interactions or the viscosity reducing agent is a vitamin B, vitamin C or vitamin E or a pharmaceutically acceptable salt, co-crystal, polymorph, solvate, hydrate, tautomer, stereoisomer and isotopically labeled derivative thereof.

[0169] 20 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 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 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.

[0170] 30 Foreignfiling text P24-168

[0171] - 18 -

[0172] In one embodiment, the compound reducing intermolecular interactions or the viscosity reducing agent is a vitamin B or a pharmaceutically acceptable salt, co-crystal, polymorph, solvate, hydrate, tautomer, stereoisomer and isotopically labeled derivative thereof.

[0173] In one embodiment, the compound reducing intermolecular interactions or the viscosity

[0174] 5 reducing agent is a vitamin B selected from the group consisting of thiamine, thiamine monophosphate (TMP), thiamine pyrophosphate (TPP), riboflavin, niacin, nicotinamide, pyridoxin, acetylCoA and orotic acid or a pharmaceutically acceptable salt, co-crystal, polymorph, solvate, hydrate, tautomer, stereoisomer and isotopically labeled derivative thereof.

[0175] In one embodiment, the compound reducing intermolecular interactions or the viscosity reducing agent 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.

[0176] 15 In one embodiment, the compound reducing intermolecular interactions or the viscosity reducing agent is a vitamin B selected from the group consisting of thiamine, thiamine monophosphate (TMP) and pyridoxin or a pharmaceutically acceptable salt, co-crystal, polymorph, solvate, hydrate, tautomer, stereoisomer and isotopically labeled derivative thereof.

[0177] 20 In some embodiments, the compound reducing intermolecular interactions or the viscosity reducing agent comprising a carboxyl group is sodium tartrate. In some embodiments, the viscosity reducing agent is benzenesulfonic acid.

[0178] In one embodiment, the compound reducing intermolecular interactions or the viscosity reducing agent is selected from the group consisting of glycine, histidine, lysine, serine, ornithine, citrulline, triple lysine, poly(histidine), poly(ornithine), thiamine pyrophosphate, nicotineamide, pyridoxine, ascorbic acid, meglumine, spermine, histamine, creatine, histone, cyclic guanosine monophosphate, cyclic cytidine monosphosphate sodium tartrate and benzenesulfonic acid or a pharmaceutically acceptable salt, co-crystal, polymorph, solvate, hydrate, tautomer, stereoisomer and isotopically labeled derivative thereof.

[0179] 30 In one embodiment, the compound reducing intermolecular interactions or the viscosity reducing agent is selected from the group consisting of beta-carotene, lycopene, riboflavin, niacin, sorbitol, mannitol, Fmoc-lysine, Na-acetyl-L-lysine, lysine, flunixin meglumine, vitamin Foreignfiling text P24-168

[0180] - 19 -

[0181] E, polyethylenimines, camphorsulfonic acid, nitrilotriacetic acid and histone solution or a pharmaceutically acceptable salt, co-crystal, polymorph, solvate, hydrate, tautomer, stereoisomer and isotopically labeled derivative thereof.

[0182] The method according to the invention is able to decrease the content of dsRNA in the

[0183] 5 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 of the obtained RNA.

[0184] 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.

[0185] In one embodiment, the compound reducing intermolecular interactions or the viscosity reducing agent is meglumine, ascorbic acid, ornithine, thiamine monophosphate (TMP), Lys- Lys-Lys (Lyss), pyridoxine, thiamine, nicotinamide, sodium tartrate, thiamine pyrophosphate, histidine, niacin, lysine, carnitine, orotic acid, vitamin E, Fmoc-Lys(Ac)-OH, riboflavin, acetyl

[0186] 15 coenzyme A, serine, glycine, sorbitol, Na-acetyl-L-lysine, citrulline, folic acid, histone H3 peptide, poly-histidine, creatine, nitrilotriacetic acid, lycopene, mannitol, beta-carotene, guanine, cytosine, uracil, biotin, pantothenic acid, pyridoxal-5'-phosphate, tryptophan, pyroloquinoline quinone (methoxatin), cGMP and melatonin or a pharmaceutically acceptable salt, co-crystal, polymorph, solvate, hydrate, tautomer, stereoisomer and isotopically labeled derivative thereof, each show advantageous results in reducing the

[0187] 20 dsRNA content of IVT produced RNA.

[0188] In one embodiment, the compound reducing intermolecular interactions or the viscosity reducing agent is meglumine, ascorbic acid, ornithine, thiamine monophosphate (TMP), Lys- Lys-Lys (Lyss), pyridoxine, thiamine, nicotinamide, sodium tartrate, thiamine pyrophosphate, histidine, niacin, lysine, carnitine, orotic acid, vitamin E, Fmoc-Lys(Ac)-OH, riboflavin, acetyl coenzyme A or serine. As demonstrated in the examples below, meglumine, ascorbic acid, ornithine, thiamine monophosphate (TMP), Lys-Lys-Lys (Lyss), pyridoxine, thiamine, nicotinamide, sodium tartrate, thiamine pyrophosphate, histidine, niacin, lysine, carnitine, orotic acid, vitamin E, Fmoc-Lys(Ac)-OH, riboflavin, acetyl coenzyme A and serine or a pharmaceutically acceptable salt, co-crystal, polymorph, solvate, hydrate, tautomer,

[0189] 30 stereoisomer and isotopically labeled derivative thereof. Foreignfiling text P24-168

[0190] - 20 - ln one embodiment, the compound reducing intermolecular interactions or the viscosity reducing agent is meglumine, ascorbic acid, ornithine, thiamine monophosphate (TMP), Lys- Lys-Lys (Lyss), pyridoxine, thiamine, nicotinamide, sodium tartrate, thiamine pyrophosphate, histidine, niacin, lysine, carnitine, orotic acid, vitamin E, Fmoc-Lys(Ac)-OH, riboflavin, acetyl coenzyme A or serine. As demonstrated in the examples below, meglumine, ascorbic acid,

[0191] 5 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, co-crystal, polymorph, solvate, hydrate, tautomer, stereoisomer and isotopically labeled derivative thereof.

[0192] 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 reducing intermolecular interactions or the viscosity reducing agent, 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%.

[0193] 15 The content of dsRNA can be measured using any method known in the art, for example via anti-dsRNA-ELISA or a dot blot.

[0194] In one embodiment, the content of dsRNA in the produced RNA is reduced by at least 75% compared to when the method is performed without the addition of the compound reducing intermolecular interactions or the viscosity reducing agent.

[0195] 20

[0196] In one embodiment, the compound reducing intermolecular interactions or the viscosity reducing agent is selected from the group consisting of thiamine, Lys-Lys-Lys (Lyss), ornithine, ascorbic acid, meglumine, pyridoxine, nicotinamid, thiamine monophosphate (TMP) and lysine or a pharmaceutically acceptable salt, co-crystal, polymorph, solvate, hydrate, tautomer, stereoisomer and isotopically labeled derivative thereof.

[0197] In one embodiment, the compound reducing intermolecular interactions or the viscosity reducing agent is selected from the group consisting of meglumine, ascorbic acid, ornithine, thiamine monophosphate (TMP), Lys-Lys-Lys (Lyss), pyridoxine, thiamine and nicotinamide or a pharmaceutically acceptable salt, co-crystal, polymorph, solvate, hydrate, tautomer, stereoisomer and isotopically labeled derivative thereof.

[0198] 30 Foreignfiling text P24-168

[0199] - 21 -

[0200] 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 reducing intermolecular interactions or the viscosity reducing agent.

[0201] In one embodiment, the compound reducing intermolecular interactions or the viscosity

[0202] 5 reducing agent is selected from the group consisting of thiamine, Lys-Lys-Lys (Lyss), ornithine, ascorbic acid, meglumine, pyridoxine, nicotinamid, thiamine monophosphate (TMP) and lysine or a pharmaceutically acceptable salt, co-crystal, polymorph, solvate, hydrate, tautomer, stereoisomer and isotopically labeled derivative thereof.

[0203] 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 reducing intermolecular interactions or the viscosity reducing agent.

[0204] In one embodiment, the compound reducing intermolecular interactions or the viscosity reducing agent is selected from the group consisting of thiamine, Lys-Lys-Lys (Lyss), ornithine and ascorbic acid or a pharmaceutically acceptable salt, co-crystal, polymorph,

[0205] 15 solvate, hydrate, tautomer, stereoisomer and isotopically labeled derivative thereof.

[0206] 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 reducing intermolecular interactions or the viscosity reducing agent.

[0207] 20 In one embodiment, the compound reducing intermolecular interactions or the viscosity reducing agent 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.

[0208] In one embodiment, the compound reducing intermolecular interactions or the viscosity reducing agent 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.

[0209] In one embodiment, the compound reducing intermolecular interactions or the viscosity reducing agent is added to the reaction medium up to a concentration of between 0.1 mM

[0210] 30 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 and 40 Foreignfiling text P24-168

[0211] - 22 - 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.

[0212] In one embodiment, the compound reducing intermolecular interactions or the viscosity reducing agent is added to the reaction medium up to a concentration of between 0.1 mM

[0213] 5 and 1 M, between 1 mM and 500 mM or between 1 mM and 200 mM.

[0214] In one embodiment, the compound reducing intermolecular interactions or the viscosity reducing agent is 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, 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.

[0215] In one embodiment, the compound reducing intermolecular interactions or the viscosity

[0216] 15 reducing agent is ornithine or a pharmaceutically acceptable salt, co-crystal, polymorph, solvate, hydrate, tautomer, stereoisomer and 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 10mM and 500 mM, more preferably between 10 mM and 300 mM, most preferably of 150 mM.

[0217] 20 In one embodiment, the compound reducing intermolecular interactions or the viscosity reducing agent is meglumine or a pharmaceutically acceptable salt, co-crystal, polymorph, solvate, hydrate, tautomer, stereoisomer and isotopically labeled derivative thereof. In another embodiment, meglumine 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.

[0218] In another embodiment, the compound reducing intermolecular interactions or the viscosity reducing agent is thiamine monophosphate (TMP) or a pharmaceutically acceptable salt, co-crystal, polymorph, solvate, hydrate, tautomer, stereoisomer and isotopically labeled derivative thereof. In another embodiment, thiamine monophosphate (TMP) is added to the

[0219] 30 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. Foreignfiling text P24-168

[0220] - 23 - ln one embodiment, the compound reducing intermolecular interactions or the viscosity reducing agent is ascorbic acid. In another embodiment, ascorbic acid 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 10 mM and 150

[0221] 5 mM.

[0222] In one embodiment, the compound reducing intermolecular interactions or the viscosity reducing agent is Lys-Lys-Lys (Lyss) or 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.

[0223] In one embodiment, the compound reducing intermolecular interactions or the viscosity reducing agent is pyridoxine or a pharmaceutically acceptable salt, co-crystal, polymorph,

[0224] 15 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, 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.

[0225] 20

[0226] In one embodiment, the compound reducing intermolecular interactions or the viscosity reducing agent is nicotinamide or a pharmaceutically acceptable salt, co-crystal, polymorph, solvate, hydrate, tautomer, stereoisomer and 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.

[0227] In one embodiment, the compound reducing intermolecular interactions or the viscosity reducing agent is lysine or a pharmaceutically acceptable salt, co-crystal, polymorph, solvate, hydrate, tautomer, stereoisomer and isotopically labeled derivative thereof. In

[0228] 30 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 Foreignfiling text P24-168

[0229] - 24 -

[0230] 5 mM and 200 mM, between 10 mM and 200 mM, between 10 mM and 193 mM or between 10 mM and 150 mM.

[0231] In one embodiment, the compound reducing intermolecular interactions or the viscosity reducing agent is niacin or a pharmaceutically acceptable salt, co-crystal, polymorph,

[0232] 5 solvate, hydrate, tautomer, stereoisomer and isotopically labeled derivative thereof. In another embodiment, niacin 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 20 mM, between 0.5 mM and 20 mM, between 1 mM and 20 mM or between 1 mM and 19.3 mM.

[0233] In one embodiment, the compound reducing intermolecular interactions or the viscosity reducing agent is thiamine pyrophosphate or a pharmaceutically acceptable salt, co-crystal, polymorph, solvate, hydrate, tautomer, stereoisomer and isotopically labeled derivative thereof. In another embodiment, thiamine pyrophosphate 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

[0234] 15 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.

[0235] In one embodiment, the compound reducing intermolecular interactions or the viscosity reducing agent is glycine or a pharmaceutically acceptable salt, co-crystal, polymorph, solvate, hydrate, tautomer, stereoisomer and isotopically labeled derivative thereof. In

[0236] 20 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.

[0237] In one embodiment, the compound reducing intermolecular interactions or the viscosity reducing agent is sorbitol or a pharmaceutically acceptable salt, co-crystal, polymorph, solvate, hydrate, tautomer, stereoisomer and isotopically labeled derivative thereof. In another embodiment, sorbitol 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.

[0238] In one embodiment, the compound reducing intermolecular interactions or the viscosity

[0239] 30 reducing agent is Na-acetyl-L-lysine or a pharmaceutically acceptable salt, co-crystal, polymorph, solvate, hydrate, tautomer, stereoisomer and isotopically labeled derivative thereof. In another embodiment, Na-acetyl-L-lysine is added to the reaction medium up to a Foreignfiling text P24-168

[0240] - 25 - 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.

[0241] 5 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 reducing intermolecular interactions or the viscosity reducing agent, for example not reduced by more than 2%, 3%, 4%, 5%, 10%, 15%, 20%, 30%, 40% or 50%.

[0242] In one embodiment, the compound reducing intermolecular interactions or the viscosity reducing agent is selected from the group consisting of thiamine, Lys-Lys-Lys (Lyss), ornithine, ascorbic acid, meglumine, pyridoxine, nicotinamid, thiamine monophosphate (TMP) and lysine or a pharmaceutically acceptable salt, co-crystal, polymorph, solvate, hydrate, tautomer, stereoisomer and isotopically labeled derivative thereof.

[0243] 15 In some embodiments, the yield of the produced RNA is higher than when the method is performed without the addition of a compound reducing intermolecular interactions or the viscosity reducing agent, 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, 15% higher or at least 20% higher.

[0244] 20

[0245] 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.

[0246] In some embodiments, the produced RNA leads to increased mRNA efficiency than when the method is performed without the addition of a compound reducing intermolecular interactions or the viscosity reducing agent, for example at least 10%, 50%, 100%, 200%, 300%, 500%, 700%, 800%, 1000%, 1500%, 2000%, 2500%, 3000% or 4000% higher.

[0247] In one embodiment, the compound reducing intermolecular interactions or the viscosity reducing agent is selected from the group consisting of thiamine, Lys-Lys-Lys (Lyss),

[0248] 30 ornithine, pyridoxine, meglumine, ascorbic acid, sodium tartrate, lysine, histidine, citrulline and glycine or a pharmaceutically acceptable salt, co-crystal, polymorph, solvate, hydrate, tautomer, stereoisomer and isotopically labeled derivative thereof. Foreignfiling text P24-168

[0249] - 26 -

[0250] In one embodiment, the compound reducing intermolecular interactions or the viscosity reducing agent 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

[0251] 5 derivative thereof.

[0252] In one embodiment, the compound reducing intermolecular interactions or the viscosity reducing agent 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.

[0253] In some embodiments, the reaction mix is incubated after addition of the compound reducing intermolecular interactions, 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.

[0254] In another aspect, the invention relates to the use of a compound reducing intermolecular

[0255] 15 interactions, preferably a viscosity reducing agent, to decrease the content of double stranded RNA during RNA in vitro transcription.

[0256] 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 reducing intermolecular interactions, preferably a viscosity reducing agent, to a

[0257] 20 reaction mix comprising a template DNA, an RNA polymerase and ribonucleotides.

[0258] 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 compound reducing intermolecular interactions, preferably a viscosity reducing agent.

[0259] Examples

[0260] Viscosity of purified RNA

[0261] A concentrated nuclease-free TE-Buffer was diluted by addition of nuclease-free water to result in a 1x TE-Buffer. pH was adjusted to pH 7.0 using HCI and NaOH if necessary.

[0262] 30 Excipient solutions of 150 mM Ornithine or thiamine monophosphate were prepared in TE- Buffer pH 7.0, respectively. The pH was adjusted using HCI or NaOH, if necessary. A concentrated mRNA solution containing the desired excipients was prepared using Foreignfiling text P24-168

[0263] - 27 - centrifugal filters (Amicon®, 30 kDA MWCO) to exchange the original buffer with a buffer containing the relevant excipients and to reduce the volume of the solution. The mRNA was subsequently diluted to 8800 pg / mL, 5680 pg / mL and 7530 pg / mL, respectively. mRNA Concentration was determined using fluorescence spectroscopy. For fluorescence spectroscopy an assay (Quant-IT™ RNA XR Assay Kit, Thermo Fisher Scientific) was

[0264] 5 utilized. The fluorophores were excitated at 644nm and fluorescence was measured at 673 nm using a Spark® Multimode Platereader (Tecan). The assay was performed and interpreted according to the manufacturer's instructions.

[0265] The mVROC™ Technology (Rheo Sense, San Ramon, California USA) was used for viscosity measurements. Measurements were performed using a 250 pl syringe and a shear rate of 1500 s1. A volume of 80 pl was used. All samples were measured as triplicates.

[0266] In vitro transcription and mRNA purification

[0267] 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)-

[0268] 15 tail (120 nt). As mRNA models Photinus pyralis (firefly) luciferase (Flue), Streptococcus pyogenes Cas9 with C-terminal nuclear localization sequence (NLS) and human erythropoietin (hEPO) were selected. For standard IVT with co-transcriptional capping, 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

[0269] 20 England Biolabs®) and CleanCap® Reagent AG (TriLink) according to the manufacturer’s instructions. 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 150 mM, or 1 M Pyridoxine pH 7.0 (Merck KGaA) to reach a final concentration of 150 mM, or 1 M Nicotinamide pH 7.0 (Merck KGaA) to reach a final concentration of 150 mM, or 1 M Meglumine pH 7.0 (Merck KGaA) to reach a final concentration of 150 mM, or 1 M Ascorbic acid pH 7.0 (Merck KGaA) to 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 L-Citrulline pH 7.0 (Merck KGaA) to reach a final concentration of 150 mM, or 1 M Sorbitol (Parteck®SI 200, D-Glucitol) pH 6.0 (Merck KGaA) to reach a final concentration of 150 mM, or 1 M L-Lysine pH 6.7 (Merck KGaA) to reach a

[0270] 30 final concentration of 150 mM, or 1 M D-Mannitol pH 6.3 (Merck KGaA) to reach a final concentration of 150 mM, or 1 M Pantothenic acid pH 7.2 (Merck KGaA) to reach a final concentration of 150 mM, or 1 M pyridoxal 5'-phosphate (Merck KGaA) to reach a final Foreignfiling text P24-168

[0271] - 28 - 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 of 75 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 75 mM, or 1 M Thiamine pH 6.5 (Merck KGaA) to reach a final concentration of 193 mM, 150 mM, 100 mM,

[0272] 5 50 mM 10 mM, or 0.5 M (-)-Riboflavin pH 6.5 (Merck KGaA) to reach a final concentration of 75 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 30 mM, or 150 mM Creatine pH 7.0 (Merck KGaA) to reach a final concentration of 22.5 mM, or 143 mM Thiamine pyrophosphate pH 7.2 (TMP, Merck KGaA) to reach a final concentration of 21.45 mM, or 100 mM Niacin (Nicotinic Acid) pH 7.0 (Merck KGaA) to reach a final concentration of 15 mM, or 100 mM acetyl-coenzyme A (Merck KGaA) to reach a final concentration of 15 mM, or 90 mM biotin (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 0.5 mM human histone H3 peptide (H3F3B(3021), Merck KGaA) to reach a final concentration of

[0273] 15 0.075 mM, or 100x BME Vitamins solution pH 6.0 (Merck KGaA) to reach a final concentration of 15x, or 500 mg / mL Vitamin E (Merck KGaA) to reach a final concentration of 75 mg / mL. For hardly soluble excipients we prepared saturated solutions including poly- Histidine (Merck KGaA), folic acid (Merck KGaA), nitrilotriacetic acid pH 8.4 (Merck KGaA), Lycopene (Merck KGaA), Fmoc-lysine-(Ac)-OH (Merck KGaA), p-carotene (Merck KGaA), Guanine pH 8.5 (Merck KGaA), Cytosine pH 7.5 (Merck KGaA), Uracil pH 6.1 (Merck KGaA),

[0274] 20 L-Tryptophan pH 7.8 (Merck KGaA), melatonin (Merck KGaA), orotic acid (Merck KGaA) pH 6.8, collected the supernatant after centrifugation (ca. 1000 g) and added 15 % (v / v) to the IVT. The mixture of Guanine, Cytosine and Uracil was prepared as 1 :1 :1 (v / v).

[0275] 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 (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.

[0276] Hydrodynamic radius measurements of IVT RNA

[0277] To determine the hydrodynamic radius of IVT reactions, excipients were added after DNase

[0278] 30 treatment. A Dynapro III Plate Reader (Wyatt Technology, Santa Barbara, California USA) was used to determine the hydrodynamic radius. 10 acquisitions of 5 seconds each were accumulated. Measurements were performed in a 384-well-plate with 35 pl volume per well. Foreignfiling text P24-168

[0279] - 29 -

[0280] All samples were measured in triplicates. Temperature was increased from 5 °C to 37 °C in incremental steps of 4 °C. An equilibration step was implemented after reaching each respective temperature level.

[0281] Viscosity measurements of IVT RNA

[0282] 5

[0283] To measure the viscosity of IVT reactions, excipients were added after DNase treatment. The viscosity was determined by a rolling-ball viscometer Lovis 2000 ME (Anton Paar, Ostfildern-Scharnhausen, Germany). Measurements were performed at 20 °C operating temperature using a Polychlortrifluorethylen (PCTFE) capillary with 1.62 mm diameter, matching steel balls, with maximal 6 determinations and 0.5% Lovis average run time maximal deviation.

[0284] Electrophoresis

[0285] Agarose gel electrophoresis (AGE) was performed by using precast 1 % (w / v) agarose gels

[0286] 15 with SYBR™ Gold II staining. For separation and imaging, the E-Gel™ EX system (ThermoFisher) was used according to the manufacturer’s instructions. RNA size was contrasted by the E-Gel™ 1 Kb Plus Express Ladder (ThermoFisher). Purified RNAs were diluted 1 :100 and 20 pL were applied for separation. Capillary electrophoresis (CE) was performed by using an automated parallel capillary electrophoresis system (Fragment Analyzer, Agilent). For analysis of IVT mRNA, a corresponding kit was used according to

[0287] 20 the manufacturer’s instructions (RNA Kit 15N).

[0288] Fluorometric detection of IVT mRNA

[0289] RNA yields of crude IVT solutions were quantified using a fluorometric assay. To this end, the Qubit™ RNA BR Assay (ThermoFisher) was performed according to the manufacturer’s instructions.

[0290] Enzyme-linked Immunosorbent Assay

[0291] 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

[0292] 30 antibodies to double-stranded RNA 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 Foreignfiling text P24-168

[0293] - 30 - 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) TWEEN® 20 in PBS. Subsequently, plates were incubated with dsRNA- specific monoclonal K2 antibody for 2 hours at room temperature. For chemiluminescent

[0294] 5 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 acid-containing stop solution, the absorption was detected at 450 nm.

[0295] Cell culture, transfection, and luciferase quantification

[0296] HeLa cells were 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 was performed using PBS (Merck KGaA) and 2 mL Accutase®-solution every 2-3 days. For cultivation, a humidified tissue culture incubator

[0297] 15 at 37 °C and 10% CO2 was used.

[0298] For Flue encoding mRNA analysis, 5,000 cells per well were seeded in 96 well plates. By following the manufacturer’s recommendations, Lipofectamine™ MessengerMax™ (0.3 pL) was used for transient transfection of 0.1 pg RNA per well at the next day. After transient transfection, cells were incubated 24 h at 37 °C and 10% CO2. For fluorescent viability

[0299] 20 analysis, the CellTiter-Fluor™ Cell Viability Assay (Promega) was performed according to the manufacturer’s instructions. In short, each well was supplemented with 5X CellTiter- Fluor™ reagent, mixed by orbital shaking (300-500 rpm), incubated for 30 min at 37°C and fluorescence detected by a plate reader. For detection of luciferase-derived luminescence, the ONE-Glo™ Luciferase assay system (Promega) was used by following the manufacturer’s instructions. To this end, 100 pL substrate containing solution were added to each well, incubated for 5 min at room temperature and luminescence recorded by a plate reader.

[0300] Conclusion

[0301] As versatile tools, RNAs are ideal for a broad range of applications in research and therapy.

[0302] 30 RNAs are mainly synthesized by an enzymatic reaction termed in vitro transcription (IVT). The common reaction represents a complex interplay of RNA polymerase, template DNA, nucleoside triphosphates (NTPs), magnesium ions, reducing agents, spermidine and other Foreignfiling text P24-168

[0303] - 31 - optional components like pyrophosphatase, RNase inhibitors and co-transcriptional capping analogs. Additionally, to actual product RNAs, immunogenic double-stranded RNA (dsRNA) byproducts are generated. Due to the inscrutable reaction, the actual underlying mechanisms for dsRNA synthesis are unknown. To counteract byproduct formation, we describe the implementation of pharmaceutical excipients formerly used for protein

[0304] 5 formulations e.g. as viscosity reducing agents.

[0305] For protein-based pharmaceuticals, excipients are widely used for stabilization or viscosity reduction. In contrast to crude IVT mRNAs, proteins are typically formulated in a purified state. Thus, we first focused on the impact of excipients on the viscosity of purified RNAs. To this end, we supplemented three RNA models of various size with exemplary excipients. Previous studies have shown that protein formulations do not display strong intermolecular interactions at the low excipient concentration levels used for purified RNAs in this context. Hence, we directly excluded potential interference with the proteins used in IVT reactions.

[0306] Example excipients ornithine, or thiamine monophosphate (TMP) affected the viscosity of all purified model RNAs (Fig.1). Hence, the results demonstrated that the selected formulation

[0307] 15 can influence intermolecular interactions of purified RNAs. Next, we assigned the excipients and corresponding concentrations to unpurified IVT reaction mixes. After reaction stop, model IVTs were supplemented with excipients. We first assessed the excipients impact on the reaction’s intermolecular interactions by measuring the hydrodynamic radius ( ). All three excipients clearly decreased up to approx. 2-fold and especially at lower temperatures (Fig. 2a). The results indicated that the addition of excipients affected

[0308] 20 interactions in IVT reactions. For viscosity measurements, we performed a rolling-ball analysis. Again, all excipients strongly decreased the viscosity of crude IVT reactions (Fig. 2b). In sum, the data demonstrated that excipients enable a decrease of intermolecular interactions in the context of crude IVT reactions.

[0309] Next, we focused on excipient impact on actual RNA synthesis. As initial model, we focused on generation of Firefly luciferase (FLuc) encoding mRNA. We prepared IVT reaction mixes supplemented with the previously elaborated excipient levels and started the reactions by incubation at 37°C. To enable accurate analysis, we purified the product RNAs by silica- membrane extraction. The obtained product concentration indicated, that the augmented IVT reactions resulted in typical yields and products (Fig. 3a). Gel electrophoresis further indicated that a specific product at the expected size was generated (Fig. 3b). For improved

[0310] 30 product integrity analysis, RNAs were analyzed in capillary electrophoresis (CE). Here, the augmented IVTs resulted in a distinct peak at the expected product size (Fig. 4). The present data demonstrated that the excipients allowed typical RNA synthesis by IVT. Foreignfiling text P24-168

[0311] - 32 -

[0312] To determine the content of dsRNA byproducts, generated RNAs were analyzed by ELISA. All three excipients resulted in strongly decreased dsRNA byproduct formation (Fig. 5a-c). Due to its immunogenic character, high dsRNA contents are decreasing the efficiency of RNA pharmaceuticals in cellulo. To demonstrate, that the reduced dsRNA content is

[0313] 5 reflected in improved protein synthesis, we transiently transfected HeLa cells with the generated product RNAs. RNA translation was followed by recording FLuc-derived luminescence. All excipient augmented IVT reactions resulted in clearly increased FLuc levels (Fig. 5d). Collectively, the used excipients improved the IVT reaction performance by clearly counteracting the formation of dsRNA byproduct.

[0314] As alternative model, we assigned the optimized IVT conditions on generation of Cas9 encoding mRNA. Again, we guaranteed accurate analysis, by purifying product RNAs using silica-membrane extraction. Similar to FLuc-encoding mRNAs, product concentration indicated that the augmented IVT reactions resulted in typical yields (Fig. 6a). Corroborated by ELISA, all three excipients resulted again in strongly decreased dsRNA byproducts (Fig 6b). Integrity analysis by CE, again demonstrated that optimized IVTs resulted in a distinct

[0315] 15 peak at the expected product size (Fig. 7).

[0316] As alternative model, we assigned the optimized IVT conditions on generation of human erythropoietin (hEPO) encoding mRNA. Again, we guaranteed accurate analysis, by purifying product RNAs using silica-membrane extraction. Similar to FLuc-encoding or Cas9 encoding mRNAs, product concentration indicated that the augmented IVT reactions

[0317] 20 resulted in typical yields (Fig. 8a). Corroborated by ELISA, all three excipients resulted again in strongly decreased dsRNA byproducts (Fig 8b). Integrity analysis by CE, again demonstrated that optimized IVTs resulted in a distinct peak at the expected product size (Fig. 9).

[0318] After validating robustness of the dsRNA reducing effect of ornithine and TMP, we identified further molecules with similar chemical structures. As previously demonstrated, we manufactured FLuc encoding mRNAs in presence of these potential excipients. Again, we analyzed final product concentration and dsRNA content by ELISA. Interestingly, we observed that ascorbic acid, sodium tartrate and Lys-Lys-Lys reduced the overall yield of manufactured mRNA. Thereby, we detected dsRNA reduction by the use of serine, glycine, meglumine, ascorbic acid, sodium tartrate, citrulline, Lys-Lys-Lys, and thiamine. The amino

[0319] 30 acids serine, glycine and citrulline resulted in dsRNA reduction. In contrast thiamine resulted in approx. 30-fold reduced dsRNA and Lys-Lys-Lys in approx. 45-fold reduced dsRNA (Fig. 10). Foreignfiling text P24-168

[0320] - 33 -

[0321] Based on the strong dsRNA reduction of Lys-Lys-Lys and thiamine, we rationalized further compounds with similar chemical structures. Again, we manufactured FLuc encoding mRNAs in presence of identified compounds. We analyzed final product concentration and dsRNA content by ELISA. Like previous results, we observed that pyridoxine reduced the

[0322] 5 total amount of manufactured mRNA (Fig. 11 a). We detected dsRNA reduction by using thiaminepyrophosphate, pyridoxine, cGMP, nicotinamide and histidine. Relative to controls, pyridoxine resulted in approx. 10-fold reduced dsRNA, nicotinamide in approx. 4-fold and histidine in 3-fold reduced dsRNA (Fig. 11).

[0323] Further guided by the dsRNA reductions observed for pyridoxine, nicotinamide and histidine, we selected addtional excipients based on similar chemical properties. To ensure comparability, we again manufactured FLuc encoding mRNAs in presence of the additionally identified compounds. Again, we analyzed final product concentration and dsRNA content by ELISA (Fig. 12). Comparable to previous results, we observed that lysine reduced the total amount of manufactured mRNA (Fig.12b). We detected dsRNA reduction by using niacin, a BME vitamin solution and lysine (Fig. 12). Relative to controls, lysine resulted in

[0324] 15 approx. 4.5-fold reduction of dsRNA (Fig. 12b).

[0325] Additionally, we analyzed the impact of nucleotide bases and riboflavin (vitamin B2). To maintain comparability, we again manufactured FLuc encoding mRNAs in presence of the additionally identified compounds. We analyzed final product concentration and dsRNA content by ELISA (Fig. 13). For riboflavin, we detected a perceptible reduction in dsRNA

[0326] 20 byproduct formation. The addition nucleotide bases (guanine, cytosine, uracil) resulted relative to controls in similar product and dsRNA levels.

[0327] Following our previous results we analyzed the impact of other vitamins, amino acids or similar substances. Again, we manufactured FLuc encoding mRNAs in presence of the rationalized compounds. We analyzed final product concentration and dsRNA content by ELISA (Fig. 14). For carnitine, we detected a perceptible reduction in dsRNA byproduct formation (Fig. 14a). The addition of a saturated solution of orotic acid resulted in reduced product and dsRNA levels, relative to controls (Fig. 14b)

[0328] Next, we focused on the dsRNA reduction effect for various concentrations of the previously selected excipients. First, we analyzed excipients with high solubility and dominant dsRNA

[0329] 30 reduction.

[0330] For addition of thiamine, we observed a step-wise decrease in dsRNA from 10 mM to 100 mM (Fig. 15). Strinkingly, the product concentration was not affected by addition of thiamine Foreignfiling text P24-168

[0331] - 34 - up to 100 mM. For 150 to 193 mM, a strong reduction in dsRNA was accompanied by reduced product concentration. Based on highest dsRNA reduction and stable yield, 100 mM represented the optimal concentration. Relative to controls, the dsRNA reduction was approx. 43-fold or 97.7% (Fig. 15).

[0332] 5 The addition of Lyss resulted in a step-wise decrease in dsRNA from 5 mM to 96.5 mM (Fig. 16). Product concentration was not affected by addition of Lyss up to 50 mM. For 75 mM to 96.5 mM, a strong reduction in dsRNA and reduced product concentration were detected. Based on highest dsRNA reduction and stable yield, 50 mM represented the optimal concentration. Relative to controls, the dsRNA reduction was approx. 26-fold or 96.3% (Fig. 16).

[0333] Furthermore, addition of meglumine resulted in a step-wise decrease in dsRNA from 10 mM to 193 mM (Fig. 17). Product concentration was not affected by meglumine up to 150 mM. For 193 mM, a strong reduction in dsRNA and reduced product concentration were observed. Based on highest dsRNA reduction and stable yield, 150 mM represented the optimal concentration. Relative to controls, the dsRNA reduction was approx. 10-fold or

[0334] 15 89.7% (Fig. 17).

[0335] Addition of ascorbic acid resulted in a step-wise decrease in dsRNA from 10 mM to 193 mM (Fig. 18). Product concentration was not affected by addition of ascorbic acid up to 150 mM. For 193 mM, dsRNA could not be detected and a reduced product concentration was observed. Rationalized by high dsRNA reduction and stable yield, 150 mM represented the

[0336] 20 optimal concentration. Relative to controls, the dsRNA reduction was approx. 14-fold or 92.9% (Fig. 18).

[0337] For addition of nicotinamide, we observed a step-wise decrease in dsRNA from 10 mM to 193 mM (Fig. 19). Strinkingly, the product concentration was not affected by addition of nicotinamide up to 193 mM. Based on highest dsRNA reduction and stable yield, 193 mM represented the optimal concentration. Relative to controls, the dsRNA reduction was approx. 9-fold or 88.8% (Fig. 19).

[0338] The addition of lysine resulted in a step-wise decrease in dsRNA from 10 mM to 193 mM (Fig. 20). Product concentration was not affected by addition of lysine up to 100 mM. For 150 mM to 193 mM, a strong reduction in dsRNA and reduced product concentration were

[0339] 30 determined. Based on highest dsRNA reduction and stable yield, 100 mM represented the optimal concentration. Relative to controls, the dsRNA reduction was approx. 4-fold or 76.6% (Fig. 20). Foreignfiling text P24-168

[0340] - 35 -

[0341] Furthermore, addition of pyridoxine resulted in a step-wise decrease in dsRNA from 10 mM to 100 mM (Fig. 21). Product concentration was not affected by pyridoxine up to 100 mM. For150 mM to 193 mM, a strong reduction in dsRNA and reduced product concentration were observed. Based on highest dsRNA reduction and stable yield, 100 mM represented

[0342] 5 the optimal concentration. Relative to controls, the dsRNA reduction was approx. 8-fold or 88.2% (Fig. 21).

[0343] Next, we investigated the impact of various concentrations of excipients that showed reduced solubility. For addition of niacin, we observed a step-wise decrease in dsRNA from 1 mM to 19.3 mM (Fig. 22). Strinkingly, the product concentration was not reduced by addition of niacin. For 10 to 19.3 mM, a clear reduction in dsRNA was observed. Based on highest dsRNA reduction and stable yield, 19.3 mM represented the optimal concentration. Relative to controls, the dsRNA reduction was approx. 1.5-fold or 38.2% (Fig. 22). Addition of thiamine pyrophosphate resulted in a step-wise decrease in dsRNA from 21 .5 mM to 27.7 mM (Fig. 23). Product concentration was not affected by addition of thiamine pyrophosphate up to 21.5 mM. Based on high dsRNA reduction and moderate reduction in product

[0344] 15 concentration, 27.7 mM represented the optimal concentration. Relative to controls, the dsRNA reduction was approx. 2-fold or 51 .7% (Fig. 23).

[0345] Finally, we focused on excipients that showed no effect on dsRNA in previous experiments. Addition of highly soluble glycine and sorbitol resulted in dsRNA levels and product concentrations similar to controls for 10 mM to 193 mM (Fig. 24, Fig.25). For Na-Acetyl-L-

[0346] 20 lysine with lower solubility, dsRNA levels and product concentrations were similar to controls for 2 mM up to 38.6 mM (Fig. 26).

[0347] To further assess, if the reduced dsRNA content of optimized IVTs is reflected in mRNA efficiency, we analyzed protein biosynthesis in cellulo. \Ne transiently transfected HeLa cells with Fluc-encoding mRNA products derived from various optimized IVTs. Relative to controls, IVT optimization by citrulline (150 mM), glycine (150 mM) or serine (150 mM) resulted in unobtrusive bioluminescence (Fig. 27a). In contrast, the previously elaborated optimization by a BME vitamins solution (15x) or lysine (150 mM) showed that the decreased dsRNA levels were translated into increased FLuc bioluminescence (Fig. 12, Fig 27b). Moreover, also for IVT optimization using meglumine (150 mM), ascorbic acid (150 mM), sodium tartrate (150 mM), pyridoxine (150 mM), nicotinamide (150 mM) or histidine (75 mM)

[0348] 30 the previously observed descrease in dsRNA was accompanied by significantly increased FLuc-derived bioluminescence (Fig. 28). Foreignfiling text P24-168

[0349] - 36 -

[0350] In previous experiments, a step-wise decrease in dsRNA content with increasing thiamine concentration was observed in the range of 10 mM to 100 mM (Fig. 15). Vice versa, a corresponding step-wise increase in FLuc-derived bioluminescence was observed from 10 mM to 100 mM (Fig. 29a). Furthermore, for Lyss a similar step-wise decrease in dsRNA content was observed from 5 mM to 75 mM (Fig. 16). Again, the reduced dsRNA was also

[0351] 5 reflected in the FLuc-derived bioluminescence from 5 to 75 mM (Fig. 29b). In sum, the data demonstrated, that dsRNA reduction based on our optimized IVT products is correlated with the functional biosynthesis of encoded FLuc.

[0352] 30

Claims

Foreignfiling_text P24-168Claims1 . A method for reducing the content of double stranded RNA (dsRNA) 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 reducing5 intermolecular interactions 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 compound reducing intermolecular interactions is a viscosity reducing agent.

3. The method according to claim 1 or 2, wherein the compound is selected from the group consisting of meglumine, ascorbic acid, ornithine, thiamine monophosphate (TMP), Lys-Lys- Lys (Lyss), pyridoxine, thiamine, nicotinamide, sodium tartrate, thiamine pyrophosphate, histidine, niacin, lysine, carnitine, orotic acid, vitamin E, Fmoc-Lys(Ac)-OH, riboflavin, acetyl coenzyme A or serine or a pharmaceutically acceptable salt, co-crystal, polymorph, solvate, hydrate, tautomer, stereoisomer and isotopically labeled derivative thereof.

154. The method according to any of claims 1 to 3, wherein the compound is selected from the group consisting of thiamine, Lys-Lys-Lys (Lyss), ornithine, ascorbic acid, meglumine, pyridoxine, nicotinamid, thiamine monophosphate (TMP) and lysine or a pharmaceutically acceptable salt, co-crystal, polymorph, solvate, hydrate, tautomer, stereoisomer and isotopically labeled derivative thereof.

205. The method according to any of claims 1 to 4, wherein the compound is selected from the group consisting of thiamine, Lys-Lys-Lys (Lyss), ornithine 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 claim 1 or 2, wherein the compound is a vitamin, preferably a vitamin B or a pharmaceutically acceptable derivative, salt, co-crystal, polymorph, solvate, hydrate, tautomer, stereoisomer and isotopically labeled derivative thereof.

7. The method according to claim 6, wherein the vitamin 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,30 tautomer, stereoisomer and isotopically labeled derivative thereof.Foreignfiling text P24-168- 38 -8. The method according to any of claims 1 to 7, 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 preferably at least 90% than when the method is performed without the addition of the compound reducing intermolecular interactions.5 9. The method according to any of claims 1 to 8, wherein the yield of the produced RNA is not essentially reduced than when the method is performed without the addition of a compound reducing intermolecular interactions, preferably not reduced by more than 5%, more preferably not reduced by more than 10%, most preferably not reduced by more than 20%.

10. The method according to any of claims 1 to 9, wherein the yield of the produced RNA is higher than when the method is performed without the addition of a compound reducing intermolecular interactions, preferably at least 2% higher, more preferably at least 5% higher, most preferably at least 10% higher.11 . The method according to any of claims 1 to 10, wherein the produced RNA is a mRNA.

12. The method according to claim 11 , wherein the produced RNA has a higher mRNA efficiency15 than when the method is performed without the addition of a compound reducing intermolecular interactions, preferably at least 100% higher, more preferably at least 500% higher, most preferably at least 1000% higher.

13. The method according to any of claims 1 to 12, wherein the compound reducing intermolecular interactions is added to the reaction mix up to a concentration of between 0.120 mM and 1 M, preferably between 1 mM and 500 mM, more preferably between 1 mM and 200 mM.

14. Use of a compound reducing intermolecular interactions to decrease the content of dsRNA during in vitro transcription.

15. A kit for in vitro transcription, comprising a buffer system, ribonucleotides, an RNA polymerase and a compound reducing intermolecular interactions.30

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