Improved method for in vitro transcription

Abstract

Disclosed herein is a method for producing a transcribed RNA product with improved yield and reduced dsRNA impurities.

Description

Technical Field

[0001] This application generally relates to methods for in vitro transcription.

Background Art

[0002] RNA-based therapeutics and vaccines are at the forefront of modern medicine, offering hope to people suffering from genetic diseases and those fearing deadly pathogens. For example, messenger RNA (mRNA) can be used in protein replacement therapies to treat protein deficiencies such as cystic fibrosis or diseases caused by defective proteins. When a gene has a mutation that stops protein production or produces a defective protein, mRNA pharmaceuticals can provide a healthy version of the missing protein.

[0003] RNA-based vaccines have emerged as a new class of RNA pharmaceuticals. RNA vaccines can be developed more rapidly than traditional vaccines in response to a pandemic of infectious diseases, as demonstrated by the first two vaccines to obtain Emergency Use Authorization from the FDA for the prevention of COVID-19, a deadly viral infection caused by SARS-CoV-2.

[0004] One challenge in the development of RNA-based therapeutics and vaccines is the robust and efficient production of mRNA with low impurity content, such as double-stranded RNA (dsRNA), and high yields. Double-stranded RNA is an abnormal byproduct of in vitro transcription (IVT) enzyme reactions. This induces an immune response, inhibits protein translation, and thus reduces the safety / efficacy of mRNA therapeutics and vaccines. Therefore, there is an urgent need to address the removal of dsRNA either at the IVT level or in downstream purification steps in the manufacturing process. Another problem that has not been addressed in IVT manufacturing is the method of increasing the yield of mRNA per given IVT volume. In manufacturing, there are many challenges related to materials (e.g., cost, limited supplies), the ability to comply with manufacturing and quality control standards (GMP) for pharmaceuticals and pharmaceutical excipients (e.g., limited slots available, significant time and labor required for single run preparation), and equipment (e.g., availability, validation, handling). Maximizing the amount of mRNA produced at a given scale leads to reduced material costs, fewer expensive GMP runs, time, and labor, and in some cases, eliminates the need to validate larger-scale equipment and processes. Therefore, there is an urgent need to develop an mRNA manufacturing process that reliably and efficiently produces mRNA with improved yields and reduced dsRNA.

Summary of the Invention

[0005] Methods are described herein for producing transcription RNA products with improved yields and reduced dsRNA impurities.

[0006] Accordingly, the present application relates to the result that a combination of increased nucleoside triphosphates (NTPs), Mg 2+ , and time results in high yields (measured in g / L) of mRNA, and under such conditions, the level of double-stranded RNA (dsRNA), a common impurity in in vitro transcription (IVT) reactions, is significantly reduced.

[0007] In another aspect, the present application also relates to the unexpected result that the addition of an organic solvent can reduce dsRNA by one digit or more.

[0008] In another aspect, the present application also relates to the unexpected result that increasing the salt concentration in the linear DNA (L.DNA) stock before the IVT reaction results in a reduction of dsRNA without inhibiting the yield in the IVT reaction using wild-type and mixed wild-type NTPs.

[0009] In one aspect, the present application is (a) reacting a transcription reaction mixture comprising a buffer containing Mg 2+ , a linear DNA (L.DNA) template, ribonucleoside triphosphates (rNTPs), optionally an RNA capping reagent, and an RNA polymerase, wherein the molar concentration of Mg2+ is 2 to 15 mM higher than the total molar concentration of all rNTPs plus any RNA capping reagent, and the RNA polymerase / L.DNA template ratio is 0.25 to 3; (b) stopping the transcription reaction by digesting the L.DNA template with deoxyribonuclease (DNase) or quenching the RNA polymerase with EDTA, a method for generating a transcribed RNA product, comprising providing a method in which single-stranded transcribed RNA is produced at a yield of about 1 g to about 25 g per liter of the solution of the transcription reaction before adding DNase in step (b).

[0010] Accordingly, it is an object of the present invention that the applicant secures rights and discloses any disclaimer of any known product, process, or method herein, and that the present invention does not encompass previously known products, processes for making products, or methods of using products.

Brief Description of the Drawings

[0011] The following detailed description is given by way of example and is not intended to limit the invention to only the specific embodiments described, but can be best understood in conjunction with the accompanying drawings.

[0012]

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Mode for Carrying Out the Invention

[0013] Various configurations of the subject technology will be readily apparent to those skilled in the art from the present disclosure, and it is understood that the various configurations of the subject technology are shown and described by way of example. Of course, the subject technology has other configurations and capabilities of different configurations, and some details of all of them can be modified in various other respects without departing from the scope of the subject technology. Therefore, the summary, drawings, and detailed description are to be regarded essentially as illustrative and not restrictive.

[0014] Definitions Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Refer to several terms defined to have the following meanings in this specification and the following claims.

[0015] The terms used herein are for the purpose of describing particular embodiments only and are not intended to be limiting. As used herein, the singular forms "a", "an", and "the" are intended to include the plural forms as well, unless the context clearly dictates otherwise.

[0016] "Any" or "optionally" means that the event or situation described below may or may not occur, and the description includes instances where the event or situation occurs and instances where it does not.

[0017] The term "about", when used before a numerical designation, indicates an approximation that can vary by, for example, (+) or (-) 10%, 5%, 1%, or any sub-range or sub-value therebetween, including ranges, such as for temperature, time, amount, concentration, and the like. Preferably, when used with respect to an amount, the term "about" means that the amount can vary by + / - 10%.

[0018] "Comprising" or "comprises" is intended to mean that the compositions and methods include the recited elements, but not to the exclusion of others. When used to define compositions and methods, "consisting essentially of" means excluding any other elements of any essential significance to the combination for the stated purpose. Thus, a composition consisting essentially of the elements defined herein will not exclude other materials or steps that do not substantially affect the basic and novel characteristics of the claimed invention. "Consisting of" means excluding other components to the extent of trace components and substantial method steps. Embodiments defined by each of these transitional terms are within the scope of the present disclosure.

[0019] The terms "isolated," "isolating," "purified," and the like, when applied to a nucleic acid or protein, denote that the nucleic acid or protein is substantially free of other cellular components that naturally associate with it in its native state. This can be, for example, in a homogeneous state and can be either a dry solution or an aqueous solution. Purity and homogeneity are typically measured using analytical chemistry techniques such as polyacrylamide gel electrophoresis or high performance liquid chromatography. A nucleic acid that is the predominant species present in a preparation is substantially purified.

[0020] As used herein, when possible, the terms "nucleic acid", "nucleic acid molecule", "nucleic acid oligomer", "oligonucleotide", "nucleic acid sequence", "nucleic acid fragment", and "polynucleotide" are used interchangeably and may have various lengths, and include polymeric forms of covalently linked nucleotides, either deoxyribonucleotides or ribonucleotides, or analogs, derivatives, or modifications thereof, but are not intended to be limited thereto. Various polynucleotides may have various three-dimensional structures and may perform various functions, known or unknown. Non-limiting examples of polynucleotides include genes, gene fragments, exons, introns, intergenic DNA (including but not limited to heterochromatic DNA), messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of a sequence, isolated RNA of a sequence, nucleic acid probes, and primers. Polynucleotides useful in the methods of the present disclosure may include natural nucleic acid sequences and variants thereof, artificial nucleic acid sequences, or combinations of these sequences.

[0021] Polynucleotides typically consist of a specific sequence of four nucleotide bases: adenine (A), cytosine (C), guanine (G), and thymine (T) (uracil (U) in place of thymine (T) if the polynucleotide is RNA). Thus, the term "polynucleotide sequence" is an alphanumeric representation of a polynucleotide molecule, and alternatively, this term may be applied to the polynucleotide molecule itself. This alphanumeric representation can be entered into a database in a computer having a central processing unit and used for bioinformatics applications such as functional genomics and homology searches. Polynucleotides may optionally contain one or more non-standard nucleotides, nucleotide analogs, and / or modified nucleotides.

[0022] "Nucleic acid" refers to nucleotides (e.g., deoxyribonucleotides or ribonucleotides) and their polymers, or their complements, or nucleosides (e.g., deoxyribonucleosides or ribonucleosides) in any of single-stranded, double-stranded, or multi-stranded forms. In a plurality of embodiments, "nucleic acid" does not include nucleosides. Terms such as "polynucleotide", "oligonucleotide", "oligo" refer to a linear sequence of nucleotides in their ordinary and customary meanings. The term "nucleoside" refers to a glycosylamine containing a nucleobase and a 5-carbon sugar (ribose or deoxyribose) in its ordinary and customary meanings. Non-limiting examples of nucleosides include cytidine, uridine, adenosine, guanosine, thymidine, and inosine. The term "nucleotide" refers to a single unit, i.e., a monomer, of a polynucleotide in its ordinary and customary meanings. Nucleotides can be ribonucleotides, deoxyribonucleotides, or modified forms thereof. Examples of polynucleotides contemplated herein include single-stranded DNA and double-stranded DNA, single-stranded RNA and double-stranded RNA, and hybrid molecules having mixtures of single-stranded and double-stranded DNA and RNA. Examples of nucleic acids, e.g., polynucleotides contemplated herein, include any type of RNA, e.g., mRNA, siRNA, miRNA, and guide RNA, and any type of DNA, genomic DNA, plasmid DNA, and minicircle DNA, and any fragments thereof. The term "double-stranded" in relation to a polynucleotide refers to double-strandedness in its ordinary and customary meanings. Nucleic acids can be linear or branched. For example, a nucleic acid can be a linear chain of nucleotides, or a nucleic acid can be branched, e.g., such that the nucleic acid contains one or more arms or branches of nucleotides. Optionally, branched nucleic acids branch iteratively to form higher-order structures such as dendrimers.

[0023] This term encompasses nucleic acids containing known nucleotide analogs or modified backbone residues or linkages, which are synthetic, naturally occurring, and non-naturally occurring, and which have binding properties similar to those of a reference nucleic acid and are metabolized in a manner similar to a reference nucleotide. Examples of such analogs include, but are not limited to, phosphodiester derivatives such as phosphoramidates, phosphorodiamidates, phosphorothioates (also known as phosphorothioates having a double bond sulfur replacing an oxygen in phosphoric acid), phosphorodithioates, phosphonocarboxylic acids, phosphonocarboxylates, phosphonoacetic acid, phosphonoglycolic acid, methyl phosphonate, boranophosphonate, or O-methyl phosphoramidite linkages (see Eckstein, OLIGONUCLEOTIDES AND ANALOGUES: A PRACTICAL APPROACH, Oxford University Press), as well as modifications to nucleotide bases such as 5-methylcytidine or pseudouridine, and peptide nucleic acid backbones and linkages. Other analog nucleic acids include those having a positive backbone, a non-ionic backbone, modified sugars, and non-ribose backbones (e.g., phosphorodiamidate morpholino oligos or locked nucleic acids (LNA) known in the art), including those described in U.S. Patent Nos. 5,235,033 and 5,034,506, and Chapters 6 and 7, ASC Symposium Series 580, CARBOHYDRATE MODIFICATIONS IN ANTISENSE RESEARCH, edited by Sanghui & Cook. Nucleic acids containing one or more carbocyclic sugars are also included within the definition of nucleic acids. Modification of the ribose-phosphate backbone can be carried out for various reasons, e.g., to increase the stability and half-life of such molecules in a physiological environment or as probes on biochips. Mixtures of natural nucleic acids and analogs can be produced, or mixtures of different nucleic acid analogs and mixtures of natural nucleic acids and analogs can be produced. In a plurality of embodiments, the inter-nucleotide linkages in DNA are phosphodiesters, phosphodiester derivatives, or a combination of both.

[0024] As used herein, the term "expression" is used according to its plain and ordinary meaning and refers to any step involved in the production of a polypeptide, including but not limited to transcription, post-transcriptional modification, translation, post-translational modification, and secretion. Expression can be detected using conventional techniques for detecting proteins (e.g., ELISA, Western blotting, flow cytometry, immunofluorescence, immunohistochemistry, etc.).

[0025] As used herein, the term "transcription" generally refers to the process of copying a segment of DNA into RNA, and a segment of DNA transcribed into an RNA molecule that encodes a protein produces mRNA. In multiple embodiments, the segment of DNA copied into the RNA molecule is called non-coding RNA.

[0026] As used herein, the terms "inhibitor", "repressor", or "antagonist", or "downregulator" are used according to their plain and ordinary meaning and refer to a substance that can detectably decrease the expression or activity of a given gene or protein. An antagonist can decrease expression or activity by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more compared to a control in the absence of the antagonist. In some cases, the expression or activity is 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold or less than the expression or activity in the absence of the antagonist.

[0027] As used herein, the term "polymerase" generally refers to an enzyme that catalyzes the synthesis of DNA or RNA whose sequence is complementary to the original template. In multiple embodiments, an RNA polymerase is an enzyme that synthesizes RNA from a DNA template.

[0028] As used herein, the term "template" generally refers to an antisense DNA strand. In multiple embodiments, a cell uses the antisense strand as a template for generating messenger RNA (mRNA) that directs protein synthesis. In multiple embodiments, the term "linear DNA (L.DNA) template" generally refers to a DNA antisense strand that has been uncoiled or linearized by the use of a restriction enzyme or is a PCR amplicon.

[0029] As used herein, the term "DNase" generally refers to deoxyribonuclease, an enzyme that catalyzes the hydrolytic cleavage of phosphodiester bonds that link nucleotides in the DNA backbone.

[0030] As used herein, terms such as "transcribed RNA product", "in vitro transcribed RNA", "in vitro synthesized RNA" generally refer to mRNA synthesized or prepared using a method that includes in vitro transcription of one or more DNA templates by RNA polymerase. In multiple embodiments, the RNA synthesized in vitro encodes (or presents the coding sequence of) at least one protein or polypeptide. In some embodiments, the RNA encodes at least one protein that can produce a biological or biochemical effect when repeatedly or continuously introduced into human or animal cells (e.g., mammalian cells). In some embodiments, the present disclosure includes an RNA composition comprising or consisting of in vitro synthesized RNA encoding one protein or polypeptide. In multiple embodiments, the present disclosure includes an RNA composition comprising or consisting of a mixture of multiple different in vitro synthesized ssRNA or mRNA, each of which encodes a different protein. Other embodiments of the present disclosure include an RNA composition comprising or consisting of in vitro synthesized ssRNA that does not encode a protein or polypeptide but instead presents the sequence of at least one long non-coding RNA (ncRNA). Still other embodiments include reaction mixtures, kits, and methods that include or use an RNA composition.

[0031] As used herein, the terms "substantially free of dsRNA," "virtually free of dsRNA," "essentially free of dsRNA," "practically free of dsRNA," "extremely free of dsRNA," or "absolutely free of dsRNA" each mean that less than about 0.5%, 0.1%, 0.05%, 0.01%, 0.001%, or 0.0002% of the mass of the RNA in the processed ssRNA composition is dsRNA of a size greater than about 40 base pairs.

[0032] In a plurality of embodiments, one or more in vitro transcribed RNAs are substantially free of uncapped RNAs bearing 5'-triphosphate groups (which are considered one type of contaminant RNA molecule herein). As used herein, an RNA product contains at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.99% less uncapped RNA bearing 5'-triphosphate groups.

[0033] As used herein, the term "salt addition" refers to adding sodium chloride (NaCl) or an equivalent salt to L.DNA prior to adding the L.DNA to an in vitro transcription vessel (IVT).

[0034] As used herein, the term "quench" refers to the process of inactivating any unreacted reagent.

[0035] Methods are described herein for producing transcription RNA products with improved yields and reduced dsRNA impurities.

[0036] Accordingly, the present application provides increased nucleoside triphosphates (NTPs), Mg 2+and combinations of time result in high yields (measured in g / L) of mRNA, and under such conditions, the level of double-stranded RNA (dsRNA), a common impurity in in vitro transcription (IVT) reactions, is significantly reduced.

[0037] In vitro transcription In vitro transcription (IVT) generally consists of reactions typically performed in a single temperature-controlled vessel. The IVT reaction requires important components but can be enhanced by the use of one or more additives and by controlling various reaction conditions. The process generally involves the use of a manipulated DNA template combined with RNA polymerase and nucleoside triphosphates in a reaction buffer. When required by the RNA polymerase necessary for the reaction, Mg 2+ ions are also part of the reaction mixture. When the IVT reaction is carried out for a sufficient time, the reaction stops and the crude reaction transcript can be separated and purified. The various components, additives, conditions, and other characteristics of the IVT reaction are described in detail below.

[0038] Buffer Any suitable buffer composition can be used in the IVT process of the present disclosure. Generally, the buffer system selected for the IVT process can mimic the biological environment of the enzymes used in the process and further facilitate the transcription reaction. Suitable buffers include, but are not limited to, phosphate buffered saline (PBS), 2-(N-morpholino)ethanesulfonic acid (MES), 2-amino-2-hydroxymethyl-propane-1,3-diol hydrochloride (Tris or Tris-HCl), 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), 2-bis(2-hydroxyethyl)amino-2-(hydroxymethyl)-1,3-propanediol (bis-Tris), N-(2-hydroxy-1,1-bis(hydroxymethyl)ethyl)glycine (tricine), 3-morpholinopropane-1-sulfonic acid (MOPS), acetate, citrate, citrate saline (SSC), phosphate, N-cyclohexyl-2-aminoethanesulfonic acid (CHES), borate, [tris(hydroxymethyl)methylamino]propanesulfonic acid (TAPS), 2-(bis(2-hydroxyethyl)amino)acetic acid (bicine), 3-[N-tris(hydroxymethyl)methylamino]-2-hydroxypropanesulfonic acid (TAPSO), 2-[[1,3-dihydroxy-2-(hydroxymethyl)propan-2-yl]amino]ethanesulfonic acid (TES), or piperazine-N,N'-bis(2-ethanesulfonic acid (PIPES).

[0039] The buffer can also be at any suitable concentration. In a plurality of embodiments, the buffer is at a concentration in the range of about 50 mM to about 2000 mM, about 75 mM to about 1800 mM, about 80 mM to about 1700 mM, about 85 mM to about 1600 mM, about 90 mM to about 1500 mM, about 95 mM to about 1400 mM, about 100 mM to about 1300 mM, about 125 mM to about 1200 mM, about 150 mM to about 1100 mM, about 175 mM to about 1000 mM, about 200 mM to about 900 mM, about 250 mM to about 800 mM, about 275 mM to about 700 mM, about 300 mM to about 600 mM, about 325 mM to about 550 mM, about 350 mM to about 525 mM, about 325 mM to about 575 mM, about 350 mM to about 450 mM, or about 200 mM to about 600 mM.

[0040] In multiple embodiments, the buffer can have a concentration of about 100 mM, about 110 mM, about 120 mM, about 130 mM, about 140 mM, about 150 mM, about 160 mM, about 170 mM, about 180 mM, about 190 mM, about 200 mM, about 210 mM, about 220 mM, about 230 mM, about 240 mM, about 250 mM, about 260 mM, about 270 mM, about 280 mM, about 290 mM, about 300 mM, about 310 mM, about 320 mM, about 330 mM, about 340 mM, about 350 mM, about 360 mM, about 370 mM, about 375 mM, about 376 mM, about 377 mM, about 378 mM, about 379 mM, about 380 mM, about 381 mM, about 382 mM, about 383 mM, about 384 mM, about 385 mM, about 386 mM, about 387 mM, about 388 mM, about 389 mM, about 390 mM, about 391 mM, about 392 mM, about 392 mM, about 393 mM, about 394 mM, about 395 mM, about 396 mM, about 397 mM, about 398 mM, about 399 mM, about 400 mM, about 401 mM, about 402 mM, about 403 mM, about 404 mM, about 405 mM, about 406 mM, about 407 mM, about 408 mM, about 409 mM, about 410 mM, about 411 mM, about 412 mM, about 413 mM, about 414 mM, about 415 mM, about 416 mM, about 417 mM, about 418 mM, about 419 mM, about 420 mM, about 421 mM, about 422 mM, about 422 mM, about 423 mM, about 424 mM, about 425 mM, about 430 mM, about 440 mM, about 450 mM, about 460 mM, about 470 mM, about 480 mM, about 490 mM, about 500 mM, about 510 mM, about 520 mM, about 530 mM, about 540 mM, about 550 mM, about 560 mM, about 570 mM, about 580 mM, about 590 mM, or about 600 mM.

[0041] Nucleoside triphosphate (NTP) RNA synthesis is catalyzed by RNA polymerase, which covalently attaches the free -OH group on the 3’ carbon of the growing nucleotide chain to the α-phosphate on the 5’ carbon of the next NTP, releasing the β-phosphate group and γ-phosphate group as pyrophosphate (PPi). This results in the formation of a phosphodiester bond between the two NTPs. The release of PPi provides the energy necessary for the reaction to occur. Typically, the NTPs in a transcription reaction are the four natural ribonucleoside triphosphates, adenosine triphosphate (ATP), uridine triphosphate (UTP), guanosine triphosphate (GTP), and cytidine triphosphate (CTP). Generally, when an in vitro transcription reaction is described in this disclosure as being carried out with NTPs without further description, it is understood that such a reaction is carried out in the presence of ATP, UTP, GTP, and CTP. However, the IVT reaction can also be carried out in the presence of one or more modified nucleoside triphosphates. For a given base type (i.e., A, U, G, C) of an RNA transcript prepared by IVT, the transcript can be prepared in any desired molar ratio of NTP or modified NTP to that base type. For example, the U bases of in vitro transcribed RNA can contain 50% natural uridine and 50% 5-methoxyuridine.

[0042] In multiple embodiments, the IVT reactions of the present disclosure include unnatural, modified, and chemically modified nucleotides, including any such nucleotides known in the art. Nucleotides can be artificially modified either in the base moiety or the sugar moiety. In nature, most polynucleotides include nucleotides that are unmodified or natural nucleotides, including the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C), and uracil (U). These bases are typically fixed to ribose at the 1' position. The use of RNA polynucleotides containing chemically modified nucleotides has been shown to improve RNA expression, expression rate, half-life, and / or the concentration of the expressed protein. RNA polynucleotides containing chemically modified nucleotides are also useful for optimizing protein localization, thereby avoiding detrimental biological reactions such as immune responses and / or degradation pathways.

[0043] In multiple embodiments, one or more nucleoside triphosphates can be chemically modified.

[0044] Examples of modified or chemically modified nucleotides include 5-hydroxycytidine, 5-alkylcytidine, 5-hydroxyalkylcytidine, 5-carboxycytidine, 5-formylcytidine, 5-alkoxycytidine, 5-alkynylcytidine, 5-halocytidine, 2-thiocytidine, N4-alkylcytidine, N4-aminocytidine, N4-acetylcytidine, and N4,N4-dialkylcytidine.

[0045] Examples of modified or chemically modified nucleotides include 5-hydroxycytidine, 5-methylcytidine, 5-hydroxymethylcytidine, 5-carboxycytidine, 5-formylcytidine, 5-methoxycytidine, 5-propynylcytidine, 5-bromocytidine, 5-iodocytidine, 2-thiocytidine, N4-methylcytidine, N4-aminocytidine, N4-acetylcytidine, and N4,N4-dimethylcytidine.

[0046] Examples of modified or chemically modified nucleotides include 5-hydroxyuridine, 5-alkyluridine, 5-hydroxyalkyluridine, 5-carboxyuridine, 5-carboxyalkyl ester uridine, 5-formyluridine, 5-alkoxyuridine, 5-alkynyluridine, 5-halouridine, 2-thiouridine, and 6-alkyluridine.

[0047] Examples of modified or chemically modified nucleotides include 5-hydroxyuridine, 5-methyluridine, 5-hydroxymethyluridine, 5-carboxyuridine, 5-carboxymethyl ester uridine, 5-formyluridine, 5-methoxyuridine (also referred to herein as "5MeOU"), 5-propynyluridine, 5-bromouridine, 5-fluorouridine, 5-iodouridine, 2-thiouridine, and 6-methyluridine.

[0048] Examples of modified or chemically modified nucleotides include 5-methoxycarbonylmethyl-2-thiouridine, 5-methylaminomethyl-2-thiouridine, 5-carbamoylmethyluridine, 5-carbamoylmethyl-2'-O-methyluridine, 1-methyl-3-(3-amino-3-carboxypropyl)pseudouridine, 5-methylaminomethyl-2-selenouridine, 5-carboxymethyluridine, 5-methyldihydrouridine, 5-taurinomethyluridine, 5-taurinomethyl-2-thiouridine, 5-(isopentenylaminomethyl)uridine, 2'-O-methylpseudouridine, 2-thio-2'O-methyluridine, and 3,2'-O-dimethyluridine.

[0049] Examples of modified or chemically modified nucleotides include N6-methyladenosine, 2-aminoadenosine, 3-methyladenosine, 8-azaadenosine, 7-deazaadenosine, 8-oxoadenosine, 8-bromoadenosine, 2-methylthio-N6-methyladenosine, N6-isopentyladenosine, 2-methylthio-N6-isopentyladenosine, N6-(cis-hydroxyisopentenyl)adenosine, 2-methylthio-N6-(cis-hydroxyisopentenyl)adenosine, N6-glycinylcarbamoyladenosine, N6-threonylcarbamoyl-adenosine, N6-methyl-N6-threonylcarbamoyl-adenosine, 2-methylthio-N6-threonylcarbamoyl-adenosine N6,N6-dimethyladenosine, N6-hydroxynorvalylcarbamoyladenosine, 2methylthio-N6-hydroxynorvalylcarbamoyl-adenosine, N6-acetyl-adenosine, 7-methyl-adenine, 2-methylthio-adenine, 2-methoxy-adenine, alpha-thio-adenosine, 2'-O-methyl-adenosine, N6,2'-O-dimethyl-adenosine, N6,N6,2'-O-trimethyl-adenosine, 1,2'-O-dimethyl-adenosine, 2'-O-ribosyladenosine, 2-amino-N6-methyl-purine, 1-thio-adenosine, 2'-F-ara-adenosine, 2'-F-adenosine, 2'-OH-ara-adenosine, and N6-(19-amino-pentaoxanonadecyl)-adenosine.

[0050] Examples of modified or chemically modified nucleotides include N1-alkylguanine, N2-alkylguanine, thienoguanine, 7-deazaguanine, 8-oxoguanine, 8-bromoguanine, O6-alkylguanine, xanthosine, inosine, and N1-alkylinosine.

[0051] Examples of modified or chemically modified nucleotides include N1-methylguanine, N2-methylguanine, thienoguanine, 7-deazaguanine, 8-oxoguanine, 8-bromoguanine, O6-methylguanine, xanthosine, inosine, and N1-methylinosine.

[0052] Examples of modified or chemically modified nucleotides include pseudouridine. Examples of pseudouridine include N1-alkylpseudouridine, N1-cycloalkylpseudouridine, N1-hydroxypseudouridine, N1-hydroxyalkylpseudouridine, N1-phenylpseudouridine, N1-phenylalkylpseudouridine, N1-aminoalkylpseudouridine, N3-alkylpseudouridine, N6-alkylpseudouridine, N6-alkoxypseudouridine, N6-hydroxypseudouridine, N6-hydroxyalkylpseudouridine, N6-morpholinopseudouridine, N6-phenylpseudouridine, and N6-halopseudouridine. Examples of pseudouridine include N1-alkyl-N6-alkylpseudouridine, N1-alkyl-N6-alkoxypseudouridine, N1-alkyl-N6-hydroxypseudouridine, N1-alkyl-N6-hydroxyalkylpseudouridine, N1-alkyl-N6-morpholinopseudouridine, N1-alkyl-N6-phenylpseudouridine, and N1-alkyl-N6-halopseudouridine. In these examples, the alkyl, cycloalkyl, and phenyl substituents may be unsubstituted or further substituted with an alkyl, halo, haloalkyl, amino, or nitro substituent.

[0053] Examples of pseudouridine include N1-methylpseudouridine (also referred to herein as "N1MPU"), N1-ethylpseudouridine, N1-propylpseudouridine, N1-cyclopropylpseudouridine, N1-phenylpseudouridine, N1-aminomethylpseudouridine, N3-methylpseudouridine, N1-hydroxypseudouridine, and N1-hydroxymethylpseudouridine.

[0054] Examples of nucleic acid monomers include modified nucleotides and chemically modified nucleotides, including any such nucleotides known in the art.

[0055] Examples of modified and chemically modified nucleotide monomers include any such nucleotides known in the art, such as 2'-O-methyl ribonucleotides, 2'-O-methyl purine nucleotides, 2'-deoxy-2'-fluoro ribonucleotides, 2'-deoxy-2'-fluoro pyrimidine nucleotides, 2'-deoxy ribonucleotides, 2'-deoxy purine nucleotides, universal base nucleotides, 5-C-methyl-nucleotides, and inverted deoxy abasic monomer residues.

[0056] Examples of modified and chemically modified nucleotide monomers include 3'-terminal stabilized nucleotides, 3'-glyceryl nucleotides, 3'-inverted abasic nucleotides, and 3'-inverted thymidine.

[0057] Examples of modified and chemically modified nucleotide monomers include locked nucleic acid nucleotides (LNA), 2'-O,4'-C-methylene-(D-ribofuranosyl) nucleotides, 2'-methoxyethoxy (MOE) nucleotides, 2'-methyl-thio-ethyl, 2'-deoxy-2'-fluoro nucleotides, and 2'-O-methyl nucleotides. In an exemplary embodiment, the modified monomer is a locked nucleic acid nucleotide (LNA).

[0058] Examples of modified and chemically modified nucleotide monomers include 2',4'-constrained 2'-O-methoxyethyl (cMOE) and 2'-O-ethyl (cEt) modified DNA.

[0059] Examples of modified and chemically modified nucleotide monomers include 2'-amino nucleotides, 2'-O-amino nucleotides, 2'-C-allyl nucleotides, and 2'-O-allyl nucleotides.

[0060] Examples of modified and chemically modified nucleotide monomers include N6-methyl adenosine nucleotides.

[0061] Examples of modified and chemically modified nucleotide monomers include nucleotide monomers having a modified base such as 5-(3-amino)propyluridine, 5-(2-mercapto)ethyluridine, 5-bromouridine, 8-bromoguanosine, or 7-deazadenosine.

[0062] Examples of modified and chemically modified nucleotide monomers include 2'-O-aminopropyl-substituted nucleotides.

[0063] Examples of modified and chemically modified nucleotide monomers include those in which the 2'-OH group of the nucleotide is substituted with 2'-R, 2'-OR, 2'-halogen, 2'-SR, or 2'-amino, where R can be H, alkyl, alkenyl, or alkynyl.

[0064] The above examples of base modifications can be combined with additional modifications of the nucleoside or nucleotide structure, including sugar modifications and linkage modifications. Certain modified or chemically modified nucleotide monomers can be found in nature.

[0065] Preferred nucleotide modifications include N1-methylpseudouridine and 5-methoxyuridine.

[0066] 5’mRNA cap Only RNA molecules bearing a Cap structure are active in Cap-dependent translation, and "decapping" of mRNA results in almost complete loss of their template activity for protein synthesis (Nature, 255:33-37, (1975); J. Biol. Chem., vol. 253:5228-5231, (1978), and Proc. Natl. Acad. Sci. USA, 72:1189-1193, (1975)).

[0067] Another element of eukaryotic mRNA is the presence of 2'-O-methylnucleoside residues at transcription position 1 (Cap1) and, optionally, at transcription positions 1 and 2 (Cap2). 2'-O-methylation of mRNA provides higher efficiency of mRNA translation in vivo (see Proc. Natl. Acad. Sci. USA, 77:3952-3956 (1980)) and further improves the nuclease stability of mRNA with a 5'-cap. mRNA with Cap1 (and Cap2) is a distinctive landmark that allows cells to recognize the 5' end of authentic mRNA and, optionally, to distinguish it from transcripts issued from infectious genetic elements (Nucleic Acid Research 43: 482-492 (2015)).

[0068] Some examples of 5'-cap structures and methods for preparing mRNA containing it are provided in International Publication No. WO 2015 / 051169, International Publication No. WO 2015 / 061491, U.S. Patent Application No. 2018 / 0273576, and U.S. Patents Nos. 8,093,367, 8,304,529, and U.S. Patent No. 10,487,105. Such methods can include co-transcriptional capping, in which an RNA capping reagent that hybridizes to a linear DNA template at or near the transcription start site is included in the in vitro transcription reaction mixture. Another method is to perform capping post-transcriptionally through the use of enzymes that can add a cap or methylate specific nucleotides.

[0069] In multiple embodiments, the RNA transcripts produced by the methods provided herein further comprise a 5' cap. Any 5' cap can be included in such RNA molecules. In further embodiments, the RNA cap can be selected from m7GpppA, m7GpppC, unmethylated cap analogs (e.g., GpppG), dimethylated cap analogs (e.g., m2,7GpppG), trimethylated cap analogs (e.g., m2,2,7GpppG), dimethylated symmetric cap analogs (e.g., m7Gpppm7G), or anti-reverse cap analogs (e.g., ARCA, m7,2’OmeGpppG, m72’dGpppG, m7,3’OmeGpppG, m7,3’dGpppG, and their tetraphosphate derivatives) (see, e.g., Jemielity, J. et al., RNA 9: 1108-1122 (2003)). In additional embodiments, the RNA cap can be an ARCA cap (3’-OMe-m7G(5’)pppG). The RNA cap can be mCAP (m7G(5’)ppp(5’)G, N7-methyl-guanosine-5’-triphosphate-5’-guanosine). The RNA cap can be resistant to hydrolysis.

[0070] Magnesium ion (Mg 2+ ) Generally, enzymes with nucleotidyl transferase activity employ a general two-metal ion mechanism to carry out the NTP condensation reaction when NTP is added to the growing nucleotide chain. (Svetlov, Vladimir, and Evgeny Nudler. “Basic mechanism of transcription by RNA polymerase II.” Biochimica et Biophysica Acta vol. 1829,1 (2013): 20-8). For RNA polymerases used in DNA-dependent RNA transcription, a central feature of this mechanism is the use of two magnesium cations coordinated by at least two aspartate residues located in the active site. According to this general model, the first magnesium (A) facilitates the deprotonation of the RNA 3’OH, promotes the 3’O-attack on the substrate NTP α-phosphate, and then a new phosphodiester bond and the leaving group, pyrophosphate (PPi) are formed. Thus, magnesium cations play an essential role in the transcription reaction.

[0071] For IVT, any suitable water-soluble Mg 2+ salt can be used. For example, Mg 2+ can be in the form of aqueous MgC2H3O2 (i.e., magnesium acetate or MgOAc), MgCl2, MgI2, MgBr2, and Mg(NO3)2. In embodiments of the IVT method of the present disclosure, the concentration of Mg 2+ in the transcription reaction is determined as an amount relative to the total concentration of NTP (plus the start cap, if present). The concentration of Mg 2+ may exceed at least 2 mM, 3 mM, 4 mM, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, 10 mM, 11 mM, 12 mM, 13 mM, 14 mM, 15 mM, 16 mM, 17 mM, 18 mM, 19 mM, 20 mM or more of the total concentration of NTP in the reaction mixture (plus the concentration of the start cap, if present). The concentration of Mg 2+All tested concentrations were found to be suitable for achieving an increase in reaction yield, and no upper limit was found for this parameter.

[0072] DNA-dependent RNA polymerase In the IVT method of the present disclosure, any suitable DNA-dependent RNA polymerase can be used. One of ordinary skill in the art will understand that each RNA polymerase requires a promoter sequence that specifically matches on the complementary L.DNA strand to direct the RNA polymerase to the location where transcription is to be initiated. To synthesize RNA, particularly large amounts of RNA, a bacteriophage DNA-dependent RNA polymerase (enzyme) is used to catalyze the transcription of RNA from a DNA template. In a plurality of embodiments, a phage RNA polymerase is used. In a plurality of embodiments, the RNA polymerase can be, but is not limited to, T7, T3, or SP6. Bacteriophage T7 RNA polymerase is a "prototype" of other DNA-dependent RNA polymerases such as T3, SP6, and mitochondrial DNA-dependent RNA polymerase. It is also considered one of the simplest enzymes that catalyze RNA synthesis. In a plurality of embodiments, the RNA polymerase is T7 polymerase. In a plurality of embodiments, the RNA polymerase is T7 polymerase. In a plurality of embodiments, the RNA polymerase is SP6 polymerase. In a plurality of embodiments, the RNA polymerase is Escherichia coli polymerase.

[0073] In multiple embodiments, the amount of RNA polymerase in the transcription reaction mixture is from about 0.0125 μg / μL to about 0.15 μg / μL. In multiple embodiments, the amount of RNA polymerase in the transcription reaction mixture is about 0.0125 μg / μL, about 0.0250 μg / μL, about 0.0375 μg / μL, about 0.0500 μg / μL, about 0.0625 μg / μL, about 0.0750 μg / μL, about 0.0875 μg / μL, about 0.1000 μg / μL, 0.1125 μg / μL, about 0.1250 μg / μL, about 0.1375 μg / μL, about 0.1500 μg / μL, about 0.1625 μg / μL, about 0.1750 μg / μL, about 0.1875 μg / μL, or about 0.2000 μg / μL. In multiple embodiments, the range may be any interval listed between the amounts listed herein.

[0074] In multiple embodiments, the mass ratio of RNA polymerase to linear DNA template is from 0.25 to 3.0. In multiple embodiments, the mass ratio of RNA polymerase to linear DNA template is 0.25, 0.30, 0.35, 0.40, 0.45, 0.50, 0.55, 0.60, 0.65, 0.70, 0.75, 0.80, 0.85, 0.90, 0.95, 1.0, 1.05, 1.10, 1.15, 1.20, 1.25, 1.30, 1.35, 1.40, 1.45, 1.50, 1.55, 1.60, 1.65, 1.70, 1.75, 1.80, 1.85, 1.90, 1.95, 2.0, 2.05, 2.10, 2.15, 2.20, 2.25, 2.30, 2.35, 2.40, 2.45, 2.50, 2.55, 2.60, 2.65, 2.70, 2.75, 2.80, 2.85, 2.90, 2.95, or 3.0. In multiple embodiments, the range may be any interval listed between the mass ratios listed herein.

[0075] DNA linearization As used herein, "L.DNA" refers to linear DNA. Generally, DNA linearization is a method of producing an RNA transcript of a specified length. The DNA plasmid used as a template is linearized by a restriction enzyme downstream of the insert. In several embodiments, a restriction enzyme that generates a 5' overhang may be used, which is preferred for a 3' overhang. Since multiple RNA polymerases tend to "read through" transcription, circular plasmid templates produce long heterogeneous RNA transcripts in higher amounts than linear templates. Therefore, the DNA plasmid must be completely linearized to ensure efficient synthesis of a specific RNA transcript length. In multiple embodiments, this application refers to "L.DNA template", which refers to plasmid DNA used as a linearized template.

[0076] In a plurality of embodiments, the amount of L.DNA template in the transcription reaction mixture is from about 0.01 mg / mL to about 0.5 mg / mL. In a plurality of embodiments, the amount of L.DNA template in the transcription reaction mixture is from about 0.01 mg / mL to about 0.3 mg / mL. In a plurality of embodiments, the amount of L.DNA template in the transcription reaction mixture is about 0.01 mg / mL, about 0.02 mg / mL, about 0.03 mg / mL, about 0.04 mg / mL, about 0.05 mg / mL, about 0.06 mg / mL, about 0.07 mg / mL, about 0.08 mg / mL, about 0.09 mg / mL, about 0.10 mg / mL, about 0.11 mg / mL, about 0.12 mg / mL, about 0.13 mg / mL, about 0.14 mg / mL, about 0.15 mg / mL, about 0.16 mg / mL, about 0.17 mg / mL, about 0.18 mg / mL, about 0.19 mg / mL, about 0.20 mg / mL, about 0.21 mg / mL, about 0.22 mg / mL, about 0.23 mg / mL, about 0.24 mg / mL, about 0.25 mg / mL, about 0.26 mg / mL, about 0.27 mg / mL, about 0.28 mg / mL, about 0.29 mg / mL, about 0.30 mg / mL, about 0.31 mg / mL, about 0.32 mg / mL, about 0.33 mg / mL, about 0.34 mg / mL, about 0.35 mg / mL, about 0.36 mg / mL, about 0.37 mg / mL, about 0.38 mg / mL, about 0.39 mg / mL, about 0.40 mg / mL, about 0.41 mg / mL, about 0.42 mg / mL, about 0.43 mg / mL, about 0.44 mg / mL, about 0.45 mg / mL, about 0.46 mg / mL, about 0.47 mg / mL, about 0.48 mg / mL, about 0.49 mg / mL, or about 0.50 mg / mL. In a plurality of embodiments, the range may be any interval listed between the amounts listed herein.

[0077] Reaction vessel Aspects of the present application contemplate efficient large-scale methods. Generally, variables such as the type of reaction vessel or reaction process become more important to meet the need for efficient methods. Two types of processes are described herein: (1) batch processes and (2) continuous processes, each of which offers advantages and disadvantages.

[0078] A batch process refers to a process involving a series of steps that follow a specific order. Batch processing involves the processing of bulk materials within a group throughout each step of the process. The processing of subsequent batches must wait until the current processing is completed. Batch processing has the initial advantage of lower initial setup costs, but the overall cost of processing increases. Furthermore, verified modeling studies confirm that the dynamics of in vitro transcription and co-transcriptional capping are equal for batch and continuous processing. In multiple embodiments, the batch process reaction vessel can be a batch reactor.

[0079] A continuous process refers to a single unit of product flowing between all steps of the process without any interruption in time, matter, or degree. With respect to in vitro transcription as particularly envisioned herein, the continuous flow mode offers advantages such as improved space-time yield, speed, and capacity, and reduced lead time. In multiple embodiments, the continuous process reaction vessel can be a continuous stirred tank reactor.

[0080] Reaction temperature In aspects of the present application, the reaction temperature plays an important role in the in vitro transcription process. Generally, as the temperature increases, the reaction rate increases and the average kinetic energy of the reactants increases. Further, typical in vitro transcription reactions are carried out at room temperature or 37°C. The transcription rate significantly decreases when carried out at lower temperatures. Without being bound by any one theory, lowering the reaction temperature slows down the progress of the polymerase, thereby preventing the polymerase from being displaced by a secondary structure or a string of one particular nucleotide. In a plurality of embodiments, the reaction temperature during the method of generating a transcribed RNA product is about 30°C, about 31°C, about 32°C, about 33°C, about 34°C, about 35°C, about 36°C, about 37°C, about 38°C, about 39°C, or about 40°C. In a plurality of embodiments, the reaction temperature during the method of generating a transcribed RNA product is 30°C - 32°C, 31°C - 33°C, 32°C - 34°C, 33°C - 35°C, 34°C - 36°C, 35°C - 37°C, 36°C - 38°C, 37°C - 39°C, or 38°C - 40°C.

[0081] Reaction time As used herein, "reaction time" refers to the incubation time of the reaction until the reaction stops. In aspects of the present application, the reaction time plays an important role in the quality and quantity of RNA produced in the in vitro transcription reaction. For example, a typical reaction time contemplated herein is 4 hours, although the time can be optimized for each RNA. For transcripts shorter than 4 kb, the reaction or incubation time may be about 2 - 3 hours. For transcripts over 4 kb, the reaction time may be only 2 hours to minimize heat exposure that can cause RNA degradation. Although typical reaction times may vary in duration, extending the overnight incubation is not recommended because at low nucleoside triphosphate concentrations, T7 RNA polymerase exhibits RNase activity.

[0082] In multiple embodiments, the reaction time for in vitro transcription contemplated herein is from about 20 minutes to about 240 minutes. In multiple embodiments, the transcription reaction time is at least 20 minutes before stopping the reaction. In multiple embodiments, the reaction time is at least 40 minutes before stopping the reaction. In multiple embodiments, the reaction time is from about 20 minutes to 240 minutes before stopping the reaction. In multiple embodiments, the reaction time is from about 40 minutes to 240 minutes before stopping the reaction. In multiple embodiments, the reaction time is from about 40 minutes to 60 minutes before stopping the reaction.

[0083] In multiple embodiments, the reaction time for in vitro transcription contemplated herein is at least 5 minutes, at least 10 minutes, at least 15 minutes, at least 20 minutes, at least 25 minutes, at least 30 minutes, at least 35 minutes, at least 40 minutes, at least 45 minutes, at least 50 minutes, at least 55 minutes, at least 60 minutes, at least 65 minutes, at least 70 minutes, at least 75 minutes, at least 80 minutes, at least 85 minutes, at least 90 minutes, at least 95 minutes, at least 100 minutes, at least 105 minutes, at least 115 minutes, at least 120 minutes, at least 125 minutes, at least 130 minutes, at least 135 minutes, at least 140 minutes, at least 145 minutes, at least 150 minutes, at least 155 minutes, at least 160 minutes, at least 165 minutes, at least 170 minutes, at least 175 minutes, at least 180 minutes, at least 185 minutes, at least 190 minutes, at least 195 minutes, at least 200 minutes, at least 205 minutes, at least 215 minutes, at least 220 minutes, at least 225 minutes, at least 230 minutes, at least 235 minutes, at least 240 minutes, at least 245 minutes, at least 250 minutes, at least 255 minutes, at least 260 minutes, at least 265 minutes, at least 270 minutes, at least 275 minutes, at least 280 minutes, at least 285 minutes, at least 290 minutes, at least 295 minutes, or at least 300 minutes. In multiple embodiments, the reaction time may be at any interval from 10 minutes to 300 minutes. In multiple embodiments, the reaction time may be up to 480 minutes (8 hours).

[0084] In vitro transcription termination As used herein, "in vitro transcription termination" refers to the process of terminating or stopping a reaction. Transcription termination occurs when the transcribed RNA polymerase releases the DNA template and the processed RNA. Termination is necessary to prevent inappropriate transcription of downstream nucleotides and to recycle the polymerase. There are multiple methods for stopping transcription known in the art, such as specific sequences, alternative polymerases, and degradation of the template. For example, when a plasmid is linearized, T7 polymerase has a tendency to read-through transcription or "run-off."

[0085] This application contemplates digesting or degrading the template as a method of stopping the reaction. In multiple embodiments, this application contemplates a method of reducing DNA contamination. In multiple embodiments, in vitro transcription can be terminated by adding DNase I. In multiple embodiments, DNase I is used to remove all genomic DNA that results in purified RNA. In multiple embodiments, EDTA is used in combination with DNase I to stop in vitro transcription.

[0086] Separation of crude product and other purification methods This application contemplates highly purified mRNA. In the art, there are many techniques and methods for purifying RNA products from complex mixtures, also referred to as "crude products" in the art. Some of these methods include, but are not limited to, precipitation, solvent extraction, ultracentrifugation, polyacrylamide gel electrophoresis, and liquid chromatography (e.g., reverse phase ion pair HPLC, ion exchange HPLC, affinity chromatography, and size exclusion chromatography).

[0087] In one embodiment, the method herein purifies the RNA product by precipitation. RNA has a negatively charged backbone and is highly soluble in water due to its polar nature. In multiple embodiments, the cation used in combination with ice-cold ethanol as a co-solvent can form an ionic bond with the negatively charged backbone, thereby reducing the solubility of the RNA such that the RNA selectively precipitates out of the solution. In multiple embodiments, the cation and its salt may be, but are not limited to, ammonium acetate and lithium chloride. The choice of cation depends on the size and concentration of the RNA to be precipitated.

[0088] In multiple embodiments, the method herein purifies the RNA product by solvent extraction. In multiple embodiments, a guanidinium thiocyanate-phenol-chloroform solvent system is used to isolate and extract the RNA. In multiple embodiments, the crude mixture is incubated with an equimolar mixture of phenol and chloroform. Guanidinium thiocyanate is also a ribonuclease inhibitor. As used herein, guanidinium thiocyanate denatures the protein, allowing the protein to be separated by the organic phase, and the RNA product is dissolved and thus extracted by the aqueous phase.

[0089] In multiple embodiments, the method herein purifies the RNA product by ultracentrifugation. Ultracentrifugation is particularly useful for isolating large molecules such as ribosomes and ribosomal subunits.

[0090] In multiple embodiments, the methods herein utilize polyacrylamide gel electrophoresis (PAGE). Polyacrylamide gel electrophoresis is a method of separating large amounts of RNA products that can be applied to various RNA sizes using a minimal setup and cost-effective reagents. In multiple embodiments, polyacrylamide gel electrophoresis uses an electric field applied to a gel that moves molecules based on size. A polymer mesh is then used to separate the molecules based on size. In multiple embodiments, the desired RNA product is isolated by removing the band from the gel. The gel is then treated such that the RNA product diffuses into solution, and the RNA product is extracted with an ethanol solution.

[0091] In embodiments, the methods herein utilize liquid chromatography. In multiple embodiments, liquid chromatography can include, but is not limited to, normal phase column chromatography (e.g., silica solid support), reversed-phase ion-pair high-performance liquid chromatography (RP-IP-HPLC), ion-exchange high-performance liquid chromatography (IE-HPLC), ion-exchange fast protein liquid chromatography (IE-FPLC), affinity chromatography, and size-exclusion chromatography.

[0092] In multiple embodiments, reversed-phase ion-pair high-performance liquid chromatography utilizes lipophilic cations to separate RNA products. In multiple embodiments, a quaternary ammonium compound forms an ion pair with the negatively charged sugar-phosphate backbone to obtain an ion pair complex. The ion pair is lipophilic and then interacts with the nonpolar stationary phase of the column. The desired RNA product is then eluted and separated with an organic solvent gradient. In multiple embodiments, the organic solvent is acetonitrile.

[0093] In multiple embodiments, ion-exchange high-performance liquid chromatography utilizes a stationary phase containing cationic groups to generate an ion pair with the negatively charged backbone of RNA. The desired RNA product is then eluted and separated using a salt gradient.

[0094] In multiple embodiments, affinity chromatography is a separation method based on specific binding interactions between an immobilized ligand and its binding partner. In multiple embodiments, the immobilized ligand includes a ligand chemically bonded or linked to a solid support. In multiple embodiments, a crude mixture passes through a column and molecules having specific binding affinity for the ligand bind. When impurities in the crude are eluted, the bound molecules (analytes) are stripped from the support and purified from the original sample. In multiple embodiments, RNA products may be tagged with specific sequences to create affinity targets. In further embodiments, the tagged RNA products may, in combination with a compound-activated ribozyme, cleave the RNA product of interest from the stationary phase. In multiple embodiments, the affinity column is an oligodeoxythymidine ligand bound to a solid support.

[0095] In multiple embodiments, size exclusion chromatography is a separation method based on the different sizes of molecules or the hydrodynamic radius of molecules. In multiple embodiments, the stationary phase is porous with a specific size to exclude larger molecules and allow smaller molecules that fit within the pores to be retained. With respect to in vitro transcription, size exclusion chromatography can separate plasmid DNA from the desired RNA product. In some embodiments, the crude mixture can be first washed with phenol to extract exogenous proteins from the transcription mixture to achieve maximum purification.

[0096] Additive In aspects of the present application, several additives are used herein. In multiple embodiments, additives can include, but are not limited to, pyrophosphatase, RNase inhibitor, solvent, calcium chloride (CaCl2), and dithiothreitol (DTT).

[0097] Pyrophosphatase As used herein, "pyrophosphatase" also refers to diphosphatase. A pyrophosphatase is an enzyme that is an acid anhydrase hydrolase that hydrolyzes a diphosphate bond. Pyrophosphatase is used in in vitro transcription reactions for synthesizing large-scale RNA products because it prevents pyrophosphate from precipitating with magnesium ions, thereby improving the rate of the in vitro transcription reaction. In a plurality of embodiments, the pyrophosphatase is an inorganic pyrophosphatase.

[0098] RNase inhibitor This application addresses the problems in the manufacture of compositions having mRNA known to those of skill in the art, including the sensitivity and instability of the molecule. There are several factors that contribute to instability and sensitivity: (1) the presence of RNases (e.g., 5'-exonuclease, 3'-exonuclease, and endonuclease), (2) RNA is susceptible to the effects of electrophilic addition, alkylation, and oxidation, and (3) the hydrolysis rate increases when the pH of the solution exceeds 6.

[0099] As used herein, "RNase inhibitor" or "RI" refers to a ribonuclease inhibitor. An RNase inhibitor is a macromolecule with a size of about 49 kDa and is rich in both cysteine and leucine compared to typical proteins. Due to its very repetitive and rich leucine content, a tight complex can be formed. The crystal structure of the RNase inhibitor and RNase A complex suggests that the interaction is mostly electrostatic in nature for protein-protein interactions. During in vitro transcription, the newly transcribed mRNA is protected from nuclease attack by the RNase inhibitor.

[0100] In multiple embodiments, the RNase inhibitor is added to the in vitro transcription mixture. In multiple embodiments, the RNase inhibitor is added to the in vitro transcription mixture together with another additive. In multiple embodiments, the RNase inhibitor is added together with inorganic pyrophosphatase. In multiple embodiments, the RNase inhibitor is guanidinium thiocyanate. In multiple embodiments, the RNase inhibitor is guanidinium isothiocyanate. In multiple embodiments, the RNase inhibitor can be used in an amount of about 0.10 μg / μL, about 0.11 μg / μL, about 0.12 μg / μL, about 0.13 μg / μL, about 0.14 μg / μL, about 0.15 μg / μL, about 0.16 μg / μL, about 0.17 μg / μL, about 0.18 μg / μL, about 0.19 μg / μL, about 0.20 μg / μL, about 0.21 μg / μL, about 0.22 μg / μL, about 0.23 μg / μL, about 0.24 μg / μL, about 0.25 μg / μL, about 0.26 μg / μL, about 0.27 μg / μL, about 0.28 μg / μL, about 0.29 μg / μL, or about 0.30 μg / μL.

[0101] Solvent In aspects of the present application, additives such as organic solvents are contemplated.

[0102] In multiple embodiments, the solvent is selected from polar protic solvents. In multiple embodiments, the polar protic solvent is selected from the group consisting of water, methanol, ethanol, and isopropanol. In multiple embodiments, the solvent is selected from polar aprotic solvents. In multiple embodiments, the polar aprotic solvent is selected from acetonitrile. In multiple embodiments, the solvent is selected from methanol (MeOH), ethanol (EtOH), isopropanol (i-PrOH), acetonitrile (CH3CN or MeCN), and combinations thereof.

[0103] In a plurality of embodiments, the concentration of ethanol used as a solvent in the reaction mixture for RNA transcription is from about 1% v / v to about 10% v / v. In a plurality of embodiments, the concentration of ethanol used as a solvent in the reaction mixture for RNA transcription is about 1% v / v, about 2% v / v, about 3% v / v, about 4% v / v, about 5% v / v, about 6% v / v, about 7% v / v, about 8% v / v, about 9% v / v, or about 10% v / v.

[0104] In a plurality of embodiments, the concentration of methanol used as a solvent in the reaction mixture for RNA transcription is from about 1% v / v to about 10% v / v. In a plurality of embodiments, the concentration of methanol used as a solvent in the reaction mixture for RNA transcription is about 1% v / v, about 2% v / v, about 3% v / v, about 4% v / v, about 5% v / v, about 6% v / v, about 7% v / v, about 8% v / v, about 9% v / v, or about 10% v / v.

[0105] In a plurality of embodiments, the concentration of isopropanol used as a solvent in the reaction mixture for RNA transcription is from about 1% v / v to about 10% v / v. In a plurality of embodiments, the concentration of isopropanol used as a solvent in the reaction mixture for RNA transcription is about 1% v / v, about 2% v / v, about 3% v / v, about 4% v / v, about 5% v / v, about 6% v / v, about 7% v / v, about 8% v / v, about 9% v / v, or about 10% v / v.

[0106] In a plurality of embodiments, the concentration of acetonitrile used as a solvent in the reaction mixture for RNA transcription is from about 1% v / v to about 8% v / v. In a plurality of embodiments, the concentration of acetonitrile used as a solvent in the reaction mixture for RNA transcription is about 1% v / v, about 2% v / v, about 3% v / v, about 4% v / v, about 5% v / v, about 6% v / v, about 7% v / v, or about 8% v / v.

[0107] CaCl 2 Role As used herein, "DNase I" refers to deoxyribonuclease I, an endonuclease that nonspecifically cleaves single-stranded and double-stranded DNA. It hydrolyzes phosphodiester bonds to produce mono- and oligodeoxyribonucleotides having 5'-phosphate and 3'-OH groups. Calcium chloride that provides Ca 2+ is an additive that has been found to be required for DNase I activity using Mg 2+ . Without being bound by any one theory, Ca 2+ ions play an important role in the structural integrity of DNase I, while other metal cations such as Mg 2+ and Mn 2+ bind to the DNA substrate itself (Pan, C.Q. and Lazarus, R.A. Protein Science 1999, 8, 1780-1788). In the crystal structure of bovine DNase I, "there are two distinct Ca 2+ binding sites that stabilize two surface loops, as well as an additional metal ion binding site in the active site." (Pan and Lazarus, 1999 citing Oefner C. and Suck D. J Mol. Biol.1986,192,605-632.) Thus, the presence of calcium chloride has been found to play an essential role in in vitro transcription as contemplated herein.

[0108] DTT As used herein, "DTT" refers to dithiothreitol or Cleland's reagent, an additive contemplated herein for in vitro transcription. DTT is a reducing agent used to reduce protein disulfide bonds and prevent the formation of intra- and intermolecular disulfide bonds formed between cysteine residues of a protein. In multiple embodiments, DTT is used to prevent dimer formation. In multiple embodiments, DTT is used as an additive to improve the separation of proteins during electrophoresis by denatured proteins.

[0109] Guanylidine hydrochloride As used herein, "GnCl" refers to guanidine hydrochloride, a denaturant used in in vitro transcription to improve the processive transcription activity of the T7 RNA polymerase enzyme.

[0110] Embodiments In one aspect, the present application is a. Reacting a transcription reaction mixture comprising Mg 2+ , a linear DNA (L.DNA) template, ribonucleoside triphosphates (rNTPs), optionally an RNA capping reagent, and a buffer containing an RNA polymerase, wherein the molar concentration of Mg2+ is 2-15 mM higher than the total molar concentration of all rNTPs plus any RNA capping reagent, and the RNA polymerase / linear DNA template ratio is 0.25-3; b. Stopping the transcription reaction by digesting the L.DNA template with deoxyribonuclease (DNase) or quenching the enzyme with ethylenediaminetetraacetic acid (EDTA), a method for producing a transcribed RNA product, Providing a method in which single-stranded transcribed RNA is produced in an amount of about 1 g / L to about 25 g / L, cleaved in the transcription reaction, before adding DNase in step (b).

[0111] In one embodiment, the transcription reaction in step (b) is stopped by digesting the L.DNA template with DNase.

[0112] In one embodiment, the reaction in step (b) is stopped by quenching the enzyme with EDTA.

[0113] In one embodiment, the L.DNA template is produced as a salt-added L.DNA template in a solution containing 50 mM to 1200 mM NaCl before being introduced into the transcription reaction mixture.

[0114] In one embodiment, the salt-added L.DNA template is added to a solution containing 200 mM to 1000 mM NaCl before being introduced into the transcription reaction mixture.

[0115] In one embodiment, the transcription reaction mixture of step (a) further comprises one or more selected from the group consisting of RNase inhibitors and inorganic pyrophosphatase.

[0116] In one embodiment, the pH of the reaction mixture ranges from 6.5 to 8.0.

[0117] In one embodiment, the temperature between steps a) and b) ranges from 30°C to 40°C.

[0118] In one embodiment, the L.DNA template in the transcription reaction mixture is from about 0.01 mg / mL to about 0.3 mg / mL mM.

[0119] In one embodiment, the RNA polymerase is T7 polymerase.

[0120] In one embodiment, the T7 polymerase KU activity per 1 mg of L.DNA is 125 KU or more of T7 polymerase activity.

[0121] In one embodiment, the transcription reaction mixture of step (a) is reacted for at least 20 minutes before stopping the transcription reaction of step (b).

[0122] In one embodiment, the transcription reaction mixture of step (a) is reacted for at least 40 minutes before stopping the reaction by adding DNase I of step (b).

[0123] In one embodiment, the transcription reaction mixture of step (a) is reacted for 40 to 240 minutes before stopping the reaction of step (b).

[0124] In one embodiment, the transcription reaction mixture of step (a) is reacted for 40 to 60 minutes before stopping the reaction of step (b).

[0125] In one embodiment, the transcription mixture of step (a) further comprises a transcription initiation RNA capping reagent.

[0126] In one embodiment, the RNA cap is an anti-reverse cap analog (ARCA cap).

[0127] In one embodiment, the method further includes a post-transcriptional capping step.

[0128] In one embodiment, the buffer of step (a) further includes one or more buffers selected from tris(hydroxymethyl)aminomethane (TRIS) and (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES).

[0129] In one embodiment, Mg 2+ has a molar concentration that is 5 - 15 mM higher than the total molar concentration of all rNTPs plus the molar concentration of any optional RNA capping reagent.

[0130] In one embodiment, Mg 2+ has a molar concentration that is about 7 - 10 mM higher than the total molar concentration of all rNTPs plus the molar concentration of any optional RNA capping reagent.

[0131] In one embodiment, the RNA polymerase is T7 polymerase at 0.0125 - 0.15 μg / μL.

[0132] In one embodiment, the reaction produces single-stranded transcribed RNA at 3 - 20 g / L.

[0133] In one embodiment, the reaction produces single-stranded transcribed RNA at 5 - 16 g / L.

[0134] In one embodiment, the amount of single-stranded transcribed RNA is measured after purification of the transcription mixture after step (b) through a silica column.

[0135] In one embodiment, the amount of single-stranded transcribed RNA is measured after purification of the transcription mixture after step (b) through affinity column chromatography. In one embodiment, the affinity column chromatography is an oligodeoxymine ligand bound to a solid support.

[0136] In one embodiment, the purification of the transcription mixture after step (b) through a silica column is carried out before the purification through affinity column chromatography.

[0137] In one embodiment, the RNA transcription mixture in step (a) further comprises one or more solvents selected from the group consisting of EtOH at a concentration of 1-10% v / v, i-PrOH at a concentration of 1-10% v / v, MeOH at a concentration of 1-10% v / v, and acetonitrile at a concentration of 1-8% v / v, provided that the total concentration of EtOH, i-PrOH, MeOH, and acetonitrile does not exceed 10% v / v of the RNA transcription mixture in step (a).

[0138] In one embodiment, the RNA transcription mixture in step (a) contains 1-10% (v / v) of EtOH.

[0139] In one embodiment, the RNA transcription mixture contains 5% (v / v) of EtOH.

[0140] In one embodiment, the RNA transcription mixture in step (a) contains 1-10% (v / v) of acetonitrile.

[0141] In one embodiment, the RNA transcription mixture contains 5% (v / v) of acetonitrile.

[0142] In one embodiment, the RNA transcription mixture in step (a) contains 1-10% (v / v) of MeOH.

[0143] In one embodiment, the RNA transcription mixture contains 7% (v / v) of MeOH.

[0144] In one embodiment, the RNA transcription mixture in step (a) contains 1-10% (v / v) of iPrOH.

[0145] In one embodiment, the RNA transcription mixture contains 5% (v / v) of iPrOH.

[0146] In one embodiment, the L-DNA template contains an open reading frame encoding a vaccine antigen, an enzyme, an antibody, a receptor, tRNA, and / or a protein.

[0147] In one embodiment, the total concentration of rNTPs is at least 8 mM.

[0148] In one embodiment, a yield of RNA transcript greater than about 5 g / L has a reduced amount of dsRNA compared to other identical transcription reactions with a yield less than about 5 g / L.

[0149] In one aspect, the present application is a. loading a buffer containing Mg 2+ and nuclease-free water into the reactor; b. adding a solution containing the L-DNA template with 200-1000 mM NaCl added; c. adding rNTPs (defined as wild-type and analogs) and optionally an RNA cap (as defined herein); d. adding RNA polymerase and mixing the resulting transcription solution, wherein the RNA polymerase / DNA template mass ratio is 0.25-3; e. adding DNase to the transcription solution, a method for generating a transcription product, after steps (a)-(b) are completed, the molar concentration of Mg 2+ is 2-15 mM higher than the total molar concentration of all rNTPs plus any RNA cap, and 1-25 g of single-stranded transcribed RNA / L per liter of the liquid of the transcription reaction is produced before adding DNase in step (b), a method is provided.

[0150] In one embodiment, a yield of RNA transcript greater than about 5 g / L has a reduced amount of dsRNA compared to other identical transcription reactions with a yield less than about 5 g / L.

[0151] In one aspect, the present application is a. Mg 2+A step of reacting with a reaction mixture containing a buffer solution containing an L.DNA template, rNTP, an optional RNA cap, and RNA polymerase, to which 50 to 1200 mM of NaCl has been previously added, wherein the RNA polymerase / L.DNA template mass ratio is 0.25 to 3, and one or more solvents are selected from the group consisting of EtOH at a concentration of 1 to 10% v / v, i-PrOH at a concentration of 1 to 10% v / v, MeOH at a concentration of 1 to 10% v / v, and acetonitrile at a concentration of 1 to 8% v / v, provided that the total concentration of EtOH, i-PrOH, MeOH, and acetonitrile does not exceed 10 ± 1% v / v of the RNA transcription mixture, b. A step of adding DNase to the transcription reaction mixture, a method for producing a transcription RNA product having reduced dsRNA, The molar concentration of Mg 2+ in the transcription reaction mixture is 2 to 15 mM higher than the total molar concentration of all rNTP and said optional RNA cap. A method is provided.

[0152] In one embodiment, the amount of dsRNA is reduced compared to a separately identical transcription reaction in which the transcription reaction mixture does not have added EtOH, i-PrOH, MeOH, and / or acetonitrile.

Examples

[0153] The following examples are provided for illustrative purposes and are not intended to limit the scope of the claims provided herein. All literature citations in these examples and throughout this specification are hereby incorporated by reference into this specification for all legal purposes thereby served.

[0154] Example 1 In vitro transcription (IVT) for mRNA synthesis The IVT reaction vessel was loaded with nuclease-free water and a 10×IVT buffer containing 400 mM TRIS, pH 7.5, and various amounts of MgOAc as listed in Table 2 below. Dithiothreitol, a salt-added linearized plasmid DNA (L. DNA) template, an rNTP mixture (these rNTPs contain ATP, CTP, GTP, UTP, and / or modified NTPs), and any capping reagent (for the reactants described as used) were added to the reaction vessel and equilibrated at 37 ± 2 °C. Then, T7 RNA polymerase, RNase inhibitor (RI), and pyrophosphatase (IPPase) were added, and the resulting mixture was incubated at about 37 °C for 20 - 240 minutes. The amounts of reagents used in this reaction other than those listed above are listed in Table 1. Following the transcription reaction, a DNase I enzyme was added in a buffer containing TRIS, MgCl2, and CaCl2 at about 37 °C to perform a DNase reaction. The reactants were stirred for about 15 - 20 minutes. The DNase reaction was quenched by adding EDTA (ethylenediaminetetraacetic acid). The resulting mixture was purified using silica column purification. The yield was calculated via UV spectrophotometry at A260. The presence of dsRNA impurities was determined via the dot blot method described in Example 2.

[0155] When additives that reduce dsRNA such as ethanol and acetonitrile were used, they were added after heating nuclease-free water and 10×IVT buffer to 37 °C and immediately before adding L.DNA.

[0156] Table 1 shows the amounts of each raw material, additive, and enzyme added to perform a 200 μL IVT reaction that yielded a total of 5 g / L or 1 mg of mRNA. This reaction is hereinafter referred to as the 5 g / L reaction. The results of the reaction in Table 1 are shown in Figure 1.

Table 1

[0157] Specifically, T7 RNA polymerase was procured from Roche, and each lot of the protein has a constant concentration (1.0 mg / mL), but has various amounts of specific activity (the specificity of volumetric activity is ≧1000 KU / mL, and the specific activity corresponds to ≧1000 KU / mgP).

[0158] For the purpose of achieving the desired target yields of 5 g / L, 10 g / L, and 15 g / L, three reactions were carried out with various parameters. Table 2 illustrates the main differences between the conventional (5 g / L IVT reaction conditions) and the other two high-yield reaction conditions (under which yields as high as 10 g / L and 15 g / L were achieved). Unless otherwise specified in Table 2, the reagent amounts in Table 1 above did not vary across these different methods.

Table 2

[0159] Post-transcriptional capping and co-transcriptional capping A. Enzymatic capping of IVT mRNA The messenger RNA transcripts of the present disclosure can be capped by any suitable means including post-transcriptional enzymatic capping or co-transcriptional capping. For enzymatic capping, the scaled-up version (50-fold scale) of New England BioLabs® (NEB)'s one-step capping and 2'-O-methylation reaction, which was suitable for treating up to 1 mg of IVT transcript, was used. Based on the assumption that the transcript length is shorter than 100 nt, 10 μg of RNA in 20 μL of reaction was recommended. However, for mRNA transcripts, which are generally longer (about 1,000 - 15,000 nt), a higher substrate-to-reaction volume was tolerated. Before initiating the capping reaction, the RNA was denatured at 65 °C for 5 minutes and then immediately placed on ice to relax any secondary structures. For a total 1 mL capping reaction, 1 mg of denatured RNA in 700 μL of nuclease-free water was combined with 100 μL (10×) of capping buffer, 50 μL (10 mM) of GTP, 50 μL (4 mM) of SAM, 50 μL (10 U / μL) of vaccinia capping enzyme, and 50 μL of mRNA cap 2'-O-methyltransferase (50 U / μL) and incubated at 37 °C for 1 hour. The resulting capped mRNA was eluted using nuclease-free water, re-purified on an RNeasy column, and then quantified by Nanodrop. The mRNA was also visualized on a denaturing gel by electrophoresis of 500 ng of purified product per lane after denaturation and immediately placing on ice to remove secondary structures.

[0160] B. Co-transcriptional capping For co-transcriptional capping using the anti-reverse cap analog (ARCA) cap, a scaled-up version of the New England Biolab’s® (NEB) protocol was used. 10 μg of RNA was mixed with 100 μL of 2× ARCA / NTP mixture, 20 μL of T7 RNA polymerase mixture, and 1.7 μL of IPPase and RI. Nuclease-free water was added to bring the total volume to 200 μL. The reaction mixture was mixed well and incubated at 37 °C for 30 minutes. After performing the DNase I reaction, the mRNA was purified and quantified.

[0161] Silica purification A 1 mg RNA sample was diluted with a mixture of nuclease-free water, β-mercaptoethanol, guanidinium thiocyanate, and ethanol. The mixture was loaded onto a Nucleospin® Blood XL column and centrifuged at 4000 x g for 2 minutes at ambient temperature. The column was washed twice with an equal volume of a solution containing ethanol and guanidinium thiocyanate, and the purified mRNA was eluted with WFI. Silica purification was used to evaluate the concentration of IVT reactions in the range of 200 μL to 20 liters. In vitro transcribed RNA was analyzed for dsRNA impurities using dot blot as described in Example 2.

[0162] Oligo dT purification To measure the yield, the IVT pool was further purified using a BIA Separations oligo-dT column. The oligo-dT column contains dT 18 oligomers as ligands, which hybridize with the mRNA polyA tail. The binding and washing conditions used a high-salt sodium phosphate / NaCl buffer and were eluted with WFI. Impurities such as enzymes, free NTP, digested DNA, and incomplete mRNA were removed, and the purified mRNA was collected in the WFI elution fraction. The method used for oligo dT purification is further described in US Patent Application Publication No. 2019 / 0203199, the entire content of which is incorporated herein by reference.

[0163] (i). By increasing the NTP concentration alone, the yield does not improve Using the above process of Example 1 on a 200 μl scale (Table 1), using a 11000 nt replicon as the target, and using 0.075 mg / mL of L. DNA supplemented with 200 mM NaCl (5 mM of each NTP, 1.5 mM RNA cap, 30 mM magnesium acetate (7 mM excess), 0.025 μg / μL of T7 polymerase, 40 mM Tris, pH 7.6, 10 mM DTT, 0.25 U / μL of RNase inhibitor, and 0.002 U / μL of PPase), as shown in Figure 1, a target yield of 5 g / L (±1 g / L yield variation) was obtained after 50 minutes. This shows the IVT yield over the reaction time for the reaction with the target at 5 g / L. Similar results were obtained with L.DNA of various sizes (Table 3).

[0164] Table 3 lists different mRNA types tested using the old (5 g / L) and new (10 or 15 g / L) conditions of the present disclosure. The mRNAs tested covered various sizes, chemistries, self-replicating vs. non-replicating, capped vs. uncapped, in addition to vaccines and therapeutic targets, regardless of the presence or absence of a poly(A) tail.

Table 3

[0165] Analysis of the reaction mixture against the targeted 5 g / L reaction conditions revealed that NTP was depleted from the IVT reaction mixture, suggesting that the yield might be improved by increasing NTP. In a 200 μL scale reaction, a 11000 nt replicon was used as the target, and 0.075 mg / mL of L.DNA was added with 200 mM NaCl, 10 mM of each NTP, 1.5 mM RNA cap, 30 mM magnesium acetate (deficient by 11.5 mM), 0.025 μg / μL of T7 polymerase, 40 mM pH 7.6, 10 mM DTT, 0.25 μg / μL of RNase inhibitor (RI), and 0.002 U / μL of pyrophosphatase (PPase). Using the above process of Example 1, only 0.6 g / L was obtained after 60 minutes, but the target was 10 g / L. Similar results were obtained with L.DNA of various sizes.

[0166] This proved that simply increasing the NTP concentration did not improve the IVT reaction yield.

[0167] (ii). Mg 2+ Increasing the concentration and the NTP concentration do not achieve the yield of the IVT target reaction at the 60-minute point Without being bound by any theory, the low yield obtained after increasing NTP was hypothesized to be due to depletion of Mg from the solution because each NTP molecule was bound to an Mg 2+ atom. Therefore, while increasing each NTP to 10 mM, Mg 2+ was also increased. 2+The concentration was increased from 30 mM to 50 mM, which was in excess of 8.5 mM. In a 200 μL scale reaction, using an 11000 nt replicon as the target, 200 mM NaCl, 10 mM of each NTP, 1.5 mM RNA cap, 50 mM magnesium acetate (8.5 mM excess), 0.025 μg / μL T7 polymerase, 40 mM Tris pH7.6, 10 mM DTT, 0.25 μg / μL RI, and 0.075 mg / mL L.DNA with 0.002 U / μL PPase added, using the process of Example 1, unexpectedly only 7.1 g / L was obtained after 60 minutes. Similar results were obtained with other L.DNAs of various sizes.

[0168] Therefore, Mg 2+ Even by adjusting the reaction conditions to further increase the concentration, the target IVT reaction yield was not achieved.

[0169] (iii). Long reaction times with increasing NTP and Mg 2+ concentrations result in an increase in the yield Both NTP and Mg 2+ To investigate whether the expected IVT yield could be achieved by increasing both concentrations, the time course of the reaction was studied.

[0170] On a 200 μL scale, using the above process of Example 1 with an 11000 nt replicon as the encoded transcript, 200 mM NaCl, 10 mM of each NTP, 1.5 mM RNA cap, 50 mM magnesium acetate (8.5 mM excess), 0.025 μg / μL T7 polymerase, 40 mM Tris pH7.6, 10 mM DTT, 0.25 μg / μL RI, and 0.075 mg / mL L.DNA with 0.002 U / μL PPase added, and extending the IVT time from 60 minutes to 90 minutes, a yield of 9.5 g / L was obtained. Similar results were obtained with L.DNAs of various sizes.

[0171] Similarly, using a 200 μL scale of the transcript encoded with a 11000 nt replicon, 200 mM NaCl, 15 mM each NTP, 1.5 mM RNA cap, 65 mM magnesium acetate (3.5 mM excess), 0.025 μg / μL T7 polymerase, 40 mM Tris pH7.6, 10 mM DTT, 0.25 μg / μL RI, and 0.002 U / μL PPase in 0.075 mg / mL L, the above process of Example 1 was used and a yield of 15 g / L was obtained after 240 minutes. Similar results were obtained with L.DNA of various sizes.

[0172] Larger scale of 10 g / L: The above process of Example 1 was applied to a 50 mL scale IVT volume using a 11000 nt replicon as the encoded transcript, with 200 mM NaCl, 10 mM each NTP, 1.5 mM RNA cap, 50 mM magnesium acetate (8.5 mM excess), 0.025 μg / μL T7 polymerase, 40 mM Tris, pH7.6, 10 mM DTT, 0.25 μg / μL RI, and 0.002 U / μL PPase in 0.075 mg / mL L.DNA, and a yield of 10.5 g / L was obtained in 90 minutes.

[0173] Larger scale of 15 g / L: Similarly, applying to a 50 mL scale IVT using a 11000 nt replicon as the encoded transcript, with 200 mM NaCl, 15 mM each NTP, 1.5 mM RNA cap, 65 mM magnesium acetate (3.5 mM excess), 0.025 μg / μL T7 polymerase, 40 mM Tris pH7.6, 10 mM DTT, 0.25 μg / μL RI, and 0.002 U / μL PPase in 0.075 mg / mL L.DNA, using the above process of Example 1, a yield of 15 g / L was obtained after 240 minutes. Similar results were obtained with L.DNA of various sizes.

[0174] Table 4 shows an overview of the above experiments and applicable results (yield and dsRNA level).

Table 4

[0175] Figure 2a shows the yield over time of the reaction conducted at a target of 10 g / L, and Figure 2b shows the yield over time of the reaction conducted at a target of 15 g / L. For both conditions, it can be seen that the yield continues to increase beyond a reaction time of 60 minutes.

[0176] Conclusion: The IVT yield was determined by the amount of NTP (building block), Mg 2+ concentration, and time. An increase in NTP alone was not effective in achieving a high yield. An increase in NTP must be accompanied by an increase in Mg 2+ concentration and time. Mg 2+ must be maintained higher than the total molar concentration of all NTPs (including caps), and since the reaction with a higher yield has a slower kinetics, it is necessary to increase the time. To increase the IVT yield, an increase in the NTP / Mg 2+ / time trio is required, and the absence of any of this trio will result in the inability to achieve the target yield.

[0177] Example 2 dsRNA Reduction Quantification of dsRNA by dot blot The mRNA sample (100 ng) was spotted onto each mRNA Biodyne® pre-cut modified nylon membrane (Thermo Scientific, catalog number 77016) (0.45 µm, 8 × 12 cm). The membrane was blocked by incubating it with 5% non-fat dry milk in TBS-T buffer (50 mM HCl, 150 mM NaCl (pH 7.4), and 0.05% Tween® -20) for 1 hour, and then incubated with the primary antibody anti-dsRNA mAB J2 (English and Scientific Consulting Kft., Hungary, J2 monoclonal antibody (mAb), mouse, IgG2a, batch number J2-1507, 1.0 mg / mL). After a 1-hour incubation time, the membrane was washed using TBS-T buffer, 7 minutes each (4 × 7 minutes). The membrane was then incubated with the secondary antibody (Life Technologies, goat anti-mouse IgG (H+L), HRP conjugate, catalog number 16066) for 1 hour at room temperature, followed by washing 6 times with TBS-T (6 × 5 minutes) and then once with TBS (5 minutes). The resulting membrane was incubated with ECL reagent (SUPERSIGNAL WEST PICO AND FEMTO MIX, Thermo Scientific, catalog numbers 34080 and 34095) for 3 - 4 minutes and exposed under white light inside the Chemidoc-It 2 Imaging System. The intensity of the sample dots per mRNA loading was compared to the dot intensities generated from a series of poly(I:C) standards or reference samples. Poly(I:C) forms double-stranded RNA, where one strand is inosinic acid and the other is cytidylic acid.

[0178] Quantification of dsRNA by ELISA Samples of mRNA produced by IVT were diluted in water, prepared in buffer, and then transferred to Nunc MaxiSorp flat bottom plates (Invitrogen 44 - 2404 - 21) pre - coated with a dsRNA - specific antibody (primary antibody, GenScript A1 monoclonal antibody (mAB), mouse, IgG2a, full - length antibody, kappa light chain (GenScript lot number US3177EG180 - 1 / P7EH011)). The plates were blocked with TBST, 1% BSA buffer in advance to avoid non - specific binding and reduce background noise. Then the plates were washed with TBST and dsRNA was detected using an HRP - conjugated primary antibody. The primary antibody binds specifically to dsRNA. Then the plates were washed 3 times with TBST and then treated with a TMB substrate that binds to HRP to induce a color change. After 10 minutes at ambient temperature, the reaction was quenched using sulfuric acid. Absorbance values of all signals were measured at 450 nm. The absorbance values of the samples per mRNA load were compared with the absorbance values from a series of poly(I:C) standards or a series of dsRNA reference standards. Poly I:C forms double - stranded RNA, one strand being inosinic acid and the other being cytidylic acid.

[0179] (iv). When the yield of IVT increases, dsRNA also decreases NTP, Mg 2+ By increasing the concentration and the time to achieve a higher yield, dsRNA impurities are also reduced 5 - to 10 - fold.

[0180] The general process outlined in Example 1 was applied to IVT reactions targeting yields of 10 g / L and 15 g / L at 200 μL (small - scale) and 50 mL (large - scale) using the parameters outlined in Table 5 below. Silica gel purification was then followed by dot - blot to evaluate the amount of dsRNA, an important impurity in the IVT reaction.

Table 5

[0181] As shown in FIGS. 3a and 3b, at both small and large scales, at both 10 g / L and 15 g / L, 5-10 times less dsRNA was shown compared to the 5 g / L IVT reaction.

[0182] (v). Using an organic solvent additive in in vitro transcription further reduces dsRNA The in vitro transcription procedure used in Example 1 was modified by adding a solvent such as ethanol, acetonitrile, isopropanol, or methanol. After heating the WFI and buffer to 37° C., the additive was added immediately before adding the L.DNA. The additive did not affect the time of the IVT reaction. Table 6 below shows the dsRNA yields and levels when ethanol and acetonitrile were added to the IVT reaction.

Table 6

[0183] Various amounts of ethanol, acetonitrile, methanol, and isopropanol were tested in IVT. When ethanol and acetonitrile were added to the IVT at concentrations of 1-11% v / v, a significant decrease in dsRNA levels was brought about. Ethanol and ACN decreased the dsRNA levels 5-10 times independently of the starting levels of dsRNA or the target IVT conditions (quantified by the dot blot performed in Example 2) (FIG. 4A, compare lanes 2 and 3 to lane 1, and lanes 5 and 6 to lane 4). Acetonitrile showed a better decrease in dsRNA than ethanol (FIG. 4A, lanes 2 vs 3 and 5 vs 6). The additive showed a decrease in dsRNA whether the mRNA was a large replicon with a lot of wt UTP and thus a lot of dsRNA (FIG. 4B, lane 3 vs 2), or a small mRNA with a lot of N1-methylpseudouridine UTP and thus little dsRNA (FIG. 4B, lane 5 vs 4).

[0184] The yields for dsRNA and the effects of additives were synergistic as illustrated in Figure 4A in lanes 1, 4 - 5, and 6. The results in lane 4 showed an increase in the yield effect for dsRNA, and the results in lanes 5 - 6 showed an increase in yield at 5 - 10 g / L while adding ethanol and acetonitrile. The resulting decrease in dsRNA was 25 - 60 - fold. This was confirmed by a Sandwich Elisa assay using a dsRNA antibody for capture and detection as described in Figure 5.

[0185] Other additives such as isopropanol and methanol showed similar levels for the reduction of IVT dsRNA but different levels compared to ethanol and acetonitrile (AcN) to each other (Figure 6). The higher the %v / v of the additives tested, the lower the IVT yield. For acetonitrile, no significant yield was observed above 8% v / v, while for ethanol, a lower yield of 10% was observed (Figure 7).

[0186] Thus, the addition of organic solvents such as ethanol, acetonitrile, methanol, and isopropanol surprisingly decreased dsRNA without negatively affecting the high - yield IVT reaction conditions described in Example 1.

[0187] (vi). Using salt-added L.DNA in in vitro transcription reduces dsRNA The addition of NaCl to L.DNA before addition to the IVT vessel (referred to as "salt addition") was tested as another possible way to reduce dsRNA in the IVT product. This effect was variable based on mRNA chemistry, size, and molecule. For self - replicating mRNA made with unmodified NTPs, any salt addition level contributed to the decrease in dsRNA level, and a significant effect was seen with NaCl salt addition above 200 mM (Figure 8). It was observed that NaCl salt addition above 1200 mM resulted in inhibition of the IVT yield.

[0188] A combination of high yield, organic additives, and DNA salt addition was applied to a large-scale production scale of in vitro transcription in 4 L, and 25 g of the final total product was obtained. The reaction conditions and an overview of the double-stranded RNA and the product are listed in Table 7. [Table 7]

[0189] (vii). Effects of equimolar and non-equimolar conditions of in vitro transcription on dsRNA impurities To thoroughly study the methodology presented herein, a study evaluating the effects of equimolar and non-equimolar in vitro transcription was conducted in a direct study. Seven conditions were studied, and the reference method refers to the conditions presented in U.S. Patent No. 10,653,712. [Table 8]

[0190] Explanation of conditions Condition 1 : An equimolar amount of NTP (5 mM) containing 1.5 mM of capping reagent, 30 mM of MgOAc, and 0.0375 μg / μL of T7 polymerase. Condition 2 : (Reference condition "equimolar") An equimolar amount of NTP (7.5 mM) containing 1.5 mM of capping reagent, 40 mM of MgOAc, and 0.0375 μg / μL of T7 polymerase. Condition 3 : An equimolar amount of NTP (12 mM) containing 1.5 mM of capping reagent, 65 mM of MgOAc, 0.0750 μg / μL of T7 polymerase, and no ethanol. Condition 4 : (Reference condition "alpha") 30 mM of GTP, 15 mM of ATP, 7.5 mM of CTP and UTP, 1.5 mM of capping reagent, 40 mM of MgOAc, 0.0750 μg / μL of T7 polymerase, and 5-fold amount of iPP at 0.01 U / μL. Condition 5:(Modify the reference condition "alpha" to the current experimental method) 30 mM GTP, 15 mM ATP, CTP, and UTP, 1.5 mM capping reagent, 80 mM MgOAc, 0.0750 μg / μL T7 polymerase, and 5-fold amount of iPP at 0.01 U / μL. Condition 6 :(Modify condition 5) A non-equimolar amount using 10 mM GTP and 5 mM ATP, CTP, and UTP, 1.5 mM capping reagent, 30 mM MgOAc, and 0.0375 μg / μL T7 polymerase. Condition 7 :(Modify the reference condition "alpha" to the current experimental methodology) 24 mM GTP, 12 mM ATP, CTP, UTP, 1.5 mM capping reagent, 70 mM MgOAc, 0.0750 μg / μL T7 polymerase, and 5-fold amount of iPP at 0.01 U / μL.

[0191] Referring to the results of performing in vitro transcription reactions using the various conditions described above, some results are shown in FIGS. 9A - 9C. FIG. 9C, which shows the percentage of full-length transcripts resulting from IVT, indicates that the reaction fails completely when there is no sufficient Mg 2+ present. The results for condition 5 in FIG. 9C suggest that there is sufficient Mg 2+ present but not in excess, resulting in a slight decrease in purity. Further, there is a direct correlation between the Mg / NTP ratio (FIG. 9A) and the yield (FIG. 9B), and an indirect correlation between the Mg / NTP ratio (FIG. 9A) and the full-length % (FIG. 9C) (data plots not shown). Condition 4 is a comparative condition labeled "alpha" according to Table 1 of U.S. Patent No. 10,653,712. It is also clear that using equimolar amounts of NTPs does not necessarily increase the yield. This is most evident in the comparison between condition 1 and condition 6, or condition 3 and condition 5 in FIG. 9B. Finally, there is no significant increase in yield during the 2 - 4 hour execution of the reaction.

[0192] Repeat the previous experiments with conditions 3 and 5, as well as new experimental condition 7, to provide a more direct analysis of the experimental conditions relative to the reference conditions. In the repeated experiments, Mg2+ A direct correlation was again observed between the / NTP ratio (Figure 10A) and the yield (Figure 10B), as well as Mg 2+ An indirect correlation was observed between the / NTP ratio (Figure 10A) and the proportion of full-length transcripts (Figure 10C) (data plots not shown). Referring to Figure 10D, Condition 7 (both runs) has less dsRNA than Condition 3.

[0193] In further experiments, Conditions 3 and 7 were scaled up to a 12 g / L target reaction, and the salt addition and the presence of ethanol were varied for each. Explanation of the conditions: Condition 3 : Equimolar NTP (12 mM) containing 1.5 mM capping reagent, 65 mM MgOAc, 0.0750 μg / μL T7 polymerase, 0.002 U / μL amount of iPP, and no ethanol. Condition 3 + salt addition : Equimolar NTP (12 mM) containing 1.5 mM capping reagent, 65 mM MgOAc, 0.0750 μg / μL T7 polymerase, 0.002 U / μL amount of iPP, salt addition (82 mM), and no ethanol. Condition 3 + salt addition + ethanol : Equimolar NTP (12 mM) containing 1.5 mM capping reagent, 65 mM MgOAc, 0.0750 μg / μL T7 polymerase, 0.002 U / μL amount of iPP, salt addition (82 mM), and 3% v / v ethanol. Condition 7 : (Reference condition “alpha” modified with current experimental method) 24 mM GTP, 12 mM ATP, CTP, UTP, 1.5 mM capping reagent, 70 mM MgOAc, 0.0750 μg / μL T7 polymerase, and 0.002 U / μL amount of iPP. Condition 7 + salt + ethanol : (Reference condition “alpha” modified with current experimental method) 24 mM GTP, 12 mM ATP, CTP, UTP, 1.5 mM capping reagent, 70 mM MgOAc, 0.0750 μg / μL T7 polymerase, 0.002 U / μL amount of iPP, salt addition (82 mM), and 3% v / v ethanol.

[0194] The results of the large-scale IVT reactions indicate that the yield is not affected by varying the molar amount of NTP (equimolar vs. non-equimolar) (e.g., FIGS. 11A-11C). Further, FIG. 11D shows that Comparative Condition 7 had a lower amount of dsRNA, but in the presence of salt addition and ethanol, the results were no longer observed (e.g., see the last column).

[0195] As a conclusion, when the NTP up to the Mg 2+ concentration was greater than 1 (i.e., the Mg 2+ was insufficient), the reaction failed. In contrast to the reference method, non-equimolar amounts of NTP do not increase the yield but appear to decrease the presence of dsRNA. However, adding NaCl to the L.DNA and introducing ethanol eliminates any further benefit from using non-equimolar amounts of NTP.

[0196] (viii). Effects of denaturant on solvent on dsRNA impurities in in vitro transcription This application shows a correlation between the yield and the level of dsRNA. However, other studies have reported that a specific range of denaturants promotes the processive transcription activity of T7 RNA polymerase (e.g., see Das, M. and Dasguta D. FEBS Letters 1998, 427, 337-340). Further studies were conducted to fully investigate the methodology presented herein by identifying the effect of the denaturant (guanidine hydrochloride) on the dsRNA impurity level and comparing it with the effect of the solvent (e.g., ethanol).

[0197] Figure 12 shows the percentage of dsRNA in the mRNA sample relative to the control, prepared using high-yield IVT conditions without any denaturant or solvent. Addition of 5% ethanol or 60 mM guanidine hydrochloride to the IVT mixture resulted in a ~33 - 35% reduction in dsRNA: relative levels of 65.2% and 66.6% respectively. When both 5% ethanol and 60 mM guanidine hydrochloride were added to the IVT reaction mixture, there was a further reduction in dsRNA (by ~70%): a relative level of 29.2% of dsRNA. No significant difference in mRNA yield was observed between these samples (data not shown).

[0198] The results indicate that dsRNA reduction using a denaturant such as guanidine hydrochloride results in a reduction equivalent to that of the solvent. Furthermore, the results indicate that there was an added benefit when the denaturant was used in combination with either the high-yield conditions and solvents described herein as additives in any of the embodiments described herein.

[0199] The examples and embodiments described herein are for illustrative purposes only, and in some embodiments, various modifications or changes should be included within the scope of the present disclosure and the appended claims.

Claims

1. a. Mg ２＋ Reacting a transcription reaction mixture containing a buffer solution comprising linear DNA (L.DNA) template, ribonucleoside triphosphate (rNTP), optionally an RNA capping reagent, and RNA polymerase, wherein the molar concentration of Mg ２＋ is 2 to 15 mM higher than the total molar concentration of all rNTPs plus any RNA capping reagent, and the RNA polymerase / L.DNA template ratio is from 0.25 to 3; b. a step of stopping the transcription reaction by digesting the L.DNA template with deoxyribonuclease (DNase) or quenching the enzyme with ethylenediaminetetraacetic acid (EDTA), A method for producing a transcribed RNA product, comprising: The method, wherein single-stranded transcribed RNA is produced at a production rate of about 1 g to about 25 g per liter of the liquid of the transcription reaction before adding DNase in step (b).

2. The method according to claim 1, wherein the L.DNA template is a salt-added L.DNA template in a solution containing 50 mM to 1200 mM of NaCl before being introduced into the transcription reaction mixture.

3. The method according to claim 1, wherein the transcription reaction mixture in step (a) further comprises one or more of the group consisting of an RNase inhibitor and inorganic pyrophosphatase.

4. The method according to claim 1, wherein the pH of the reaction mixture is in the range of 6.5 to 8.

0.

5. The method according to claim 1, wherein the temperature between steps a) and b) is in the range of about 30 °C to about 40 °C.

6. The method according to claim 1, wherein the L.DNA template in the transcription reaction mixture is about 0.01 mg / mL to about 0.3 mg / mL.

7. The method according to claim 1, wherein the RNA polymerase is T7 polymerase.

8. The method according to claim 7, wherein the T7 polymerase KU activity per mg of L.DNA is a T7 polymerase activity of 125 KU or more.

9. The method according to claim 1, wherein the transcription reaction mixture in step (a) reacts for at least 20 minutes before stopping the transcription reaction in step (b).

10. The method according to claim 1, wherein the transcription mixture in step (a) further comprises a transcription initiation RNA capping reagent. Mg ２＋ The method according to claim 1, wherein the molar concentration of Mg is 5 to 15 mM higher than the total molar concentration of all rNTPs plus the molar concentration of any RNA capping reagent.

11.

12. The method according to claim 1, wherein the RNA polymerase is T7 polymerase at 0.0125 to 0.15 μg / μL.

13. The method according to claim 1, wherein the reaction produces 3 to 20 g / L of single-stranded transcribed RNA.

14. The method according to claim 1, wherein the amount of single-stranded transcribed RNA is measured after purification of the transcription mixture after step (b) through a silica column.

15. The method according to claim 1, wherein the amount of single-stranded transcribed RNA is measured after purification of the transcription mixture after step (b) through affinity column chromatography.

16. The method according to claim 15, wherein the purification of the transcription mixture after step (b) through a silica column is carried out before the purification through an affinity column chromatography.

17. The method according to claim 1, wherein the RNA transcription mixture in step (a) further comprises one or more solvents selected from the group consisting of EtOH at a concentration of 1 to 10% v / v, i-PrOH at a concentration of 1 to 10% v / v, MeOH at a concentration of 1 to 10% v / v, and acetonitrile at a concentration of 1 to 8% v / v, provided that the total concentration of EtOH, i-PrOH, MeOH, and acetonitrile does not exceed 10% v / v of the RNA transcription mixture in step (a).

18. The method according to claim 17, wherein the RNA transcription mixture in step (a) contains 1 to 10% (v / v) of EtOH.

19. The method according to claim 17, wherein the RNA transcription mixture in step (a) contains 1 to 10% (v / v) of acetonitrile.

20. The method according to claim 17, wherein the RNA transcription mixture in step (a) contains 1 to 10% (v / v) of MeOH.

21. The method according to claim 17, wherein the RNA transcription mixture in step (a) contains 1 to 10% (v / v) of iPrOH.

22. The method according to claim 1, wherein the L. DNA template contains an open reading frame encoding a vaccine antigen, an enzyme, an antibody, a receptor, tRNA, and / or a protein.

23. The method according to claim 1, wherein the total concentration of rNTPs is at least 8 mM.

24. The method according to claim 1, wherein when the yield of the RNA transcript exceeds about 5 g / L, the amount of dsRNA decreases as compared to the same transcription reaction with a yield of less than about 5 g / L.

25. a. Mg ２＋ loading a buffer solution containing nuclease-free water into a reactor; b. A step of adding a solution containing an L. DNA template added with 200 to 1000 mM NaCl; c. A step of adding rNTPs (defined as wild-type and analogs) and optionally an RNA cap (defined herein); d. A step of adding RNA polymerase and mixing the obtained transcription solution, wherein the RNA polymerase / DNA template mass ratio is 0.25 to 3; e. A method for producing a transcription product, comprising a step of adding DNase to the transcription solution. After steps (a) to (b) are completed, the molar concentration of Mg2+ is 2 to 15 mM higher than the total molar concentration of all rNTPs plus any RNA cap, and 1 to 25 g of single-stranded transcribed RNA / L per liter of the liquid of the transcription reaction is produced before adding DNase in step (b).

26. a. Mg ２＋ Reacting with a reaction mixture comprising a buffer containing L. DNA template, rNTP, any RNA cap, and RNA polymerase, to which 50 to 1200 mM of NaCl has been previously added, wherein the RNA polymerase / L. DNA template mass ratio is 0.25 to 3, and one or more solvents are selected from the group consisting of EtOH at a concentration of 1 to 10% v / v, i-PrOH at a concentration of 1 to 10% v / v, MeOH at a concentration of 1 to 10% v / v, and acetonitrile at a concentration of 1 to 8% v / v, provided that the total concentration of EtOH, i-PrOH, MeOH, and acetonitrile does not exceed 10 ± 1% v / v of the RNA transcription mixture, and b. A method for producing a transcribed RNA product having reduced dsRNA, comprising the step of adding DNase to the transcription reaction mixture. The molar concentration of Mg in the transcription reaction mixture ２＋ is 2 to 15 mM higher than the total molar concentration of all rNTPs and said optional RNA cap, a method.

27. The method according to claim 25, wherein a yield of RNA transcript exceeding about 5 g / L results in a reduced amount of dsRNA compared to other identical transcription reactions, and the yield is less than about 5 g / L.

28. The method according to claim 26, wherein the amount of dsRNA is reduced compared to transcription reactions that are identical in other respects, and the transcription reaction mixture excludes added EtOH, i-PrOH, MeOH, and acetonitrile.

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