Compositions and methods for reduction of double-stranded RNA

Abstract

Described herein are methods and compositions for reduction and removal of double-stranded RNA (dsRNA) molecules produced during synthesis of nucleic acid molecules, in particular synthesis of RNA molecules via an in vitro transcription reaction.

Description

[0001] PC073148A

[0002] COMPOSITIONS AND METHODS FOR REDUCTION OF DOUBLE-STRANDED RNA

[0003] CROSS-REFERENCE TO RELATED APPLICATIONS

[0004] This application claims the benefit under 35 U.S.C. § 119(e) of U.S. Provisional Patent Application Serial No. 63 / 700,069, filed September 27, 2024, the disclosure of which is hereby incorporated by reference in its entirety.

[0005] FIELD

[0006] The present compositions and methods relate to improved processes for reduction and removal of double-stranded RNA (dsRNA) created during synthesis of nucleic acid molecules, in particular synthesis of RNA molecules via in vitro transcription (IVT).

[0007] BACKGROUND

[0008] In vitro transcription (IVT) can be used to manufacture mRNA for vaccines and therapeutic applications. T7 RNA polymerase (T7 RNAP), a DNA-dependent RNA polymerase from the T7 bacteriophage, is often used in IVT to manufacture target mRNA. In addition to T7 RNAP, an IVT reaction often includes each of the four NTPs, a linear DNA template (often a linear plasmid DNA template), a pyrophosphatase enzyme and a buffer system.

[0009] Although T7 RNAP has robust activity, it is also known to generate various kinds of aberrant byproducts, including double stranded RNA (dsRNA). dsRNA is of particular interest because it is known to produce an immune response and reduce the efficacy of the target therapeutic. T7 RNAP has shown both RNA-dependent as well as pDNA template-independent RNA polymerase activity [Baiersdorfer et al., Mol Ther Nucleic Acids 15 (2019)]. In addition, promoter independent transcription of anti-sense RNA has been shown as a new mode of dsRNA synthesis in IVT [Mu et al., Nucleic Acids Res 46 (2018)]. Removing the dsRNA impurity from mRNA generated by IVT using standard purification methods has been challenging and unsuccessful, especially with large-scale mRNA manufacturing platforms [Baiersdorfer et al., Mol Ther Nucleic Acids 15 (2019); Weissman et al., Methods Mol Biol 969 (2013)].

[0010] SUMMARY OF THE INVENTION

[0011] In some embodiments, disclosed herein are methods of reducing the amount of double-stranded RNA (dsRNA) produced in an in vitro transcription (IVT) reaction, the methods comprising: (a) obtaining a DNA template; (b) contacting the DNA template with an in vitro transcription (IVT) reaction system to obtain a composition, wherein the IVT reaction system comprises a monovalent salt, spermidine, dithiothreitol (DTT), and dimethylsulfoxide (DMSO); and (c) incubating the composition at a temperature between about 15 °C and 45 °C to obtain an IVT reaction, thereby reducing the amount of dsRNA produced in the IVT reaction. In some embodiments, incubating the composition occurs for a time period between about 30 minutes and 64 days. In some embodiments, the IVT reaction system further comprises a buffer, a 5’ cap analog, ATP, CTP, GTP, UTP, a magnesium ion, an RNase inhibitor, pyrophosphatase, and an RNA polymerase. In some embodiments, the monovalent salt is selected from the group consisting of KCI, NaCI, RbCI, CsCI, NaCIC , NaF, NaBr, and NH4CL In some embodiments, the monovalent salt is present in an amount between about 10 mM and about 150 mM. In some embodiments, the spermidine is present in an amount between about 0.2 mM and about 10 mM. In some embodiments, the dithiothreitol (DTT) is present in an amount between about 15 mM and about 25 mM. In some embodiments, the dimethylsulfoxide (DMSO) is present in an amount between about 4% and about 15%. In some embodiments, the DNA template is selected from the group consisting of a linearized DNA template, a circular DNA template, and a synthetic DNA template. In some embodiments, disclosed herein are methods of reducing the amount of doublestranded RNA (dsRNA) produced in an in vitro transcription (IVT) reaction, the methods comprising: (a) obtaining a DNA template; (b) contacting the DNA template with an in vitro transcription (IVT) reaction system to obtain a composition, wherein the IVT reaction system comprises a monovalent salt, spermidine, dithiothreitol (DTT), and dimethylsulfoxide (DMSO); (c) incubating the composition at a temperature between about 15 °C and 45 °C to obtain an IVT reaction, (d) contacting the IVT reaction with a deoxyribonuclease (DNase) to obtain a digested IVT reaction; (e) contacting the digested IVT reaction with a protease to obtain an inactivated reaction; and (f) contacting the inactivated reaction with a chelating agent to obtain a chelated reaction, thereby reducing the amount of dsRNA produced in the IVT reaction. In some embodiments, the protease is proteinase K. In some embodiments, the chelating agent is ethylenediaminetetraacetic acid (EDTA). In some embodiments, disclosed herein are methods of reducing the amount of double-stranded RNA (dsRNA) produced in an in vitro transcription (IVT) reaction, the methods comprising: (a) obtaining a DNA template; (b) contacting the DNA template with an in vitro transcription (IVT) reaction system to obtain a composition, wherein the IVT reaction system comprises a monovalent salt, spermidine, dithiothreitol (DTT), and dimethylsulfoxide (DMSO); (c) incubating the composition at a temperature between about 15 °C and 45 °C to obtain an IVT reaction, (d) contacting the IVT reaction with a deoxyribonuclease (DNase) to obtain a digested IVT reaction; (e) contacting the digested IVT reaction with a protease to obtain an inactivated reaction; (f) contacting the inactivated reaction with a chelating agent to obtain a chelated reaction; and (g) precipitating an mRNA molecule in the chelated reaction with lithium chloride to obtain a purified mRNA, thereby reducing the amount of dsRNA produced in the IVT reaction. In some embodiments, disclosed herein are methods of reducing the amount of double-stranded RNA (dsRNA) produced in an in vitro transcription (IVT) reaction, the methods comprising: (a) obtaining a DNA template; (b) contacting the DNA template with an in vitro transcription (IVT) reaction system to obtain a composition, wherein the IVT reaction system comprises a monovalent salt, spermidine, dithiothreitol (DTT), and dimethylsulfoxide (DMSO); (c) incubating the composition at a temperature between about 15 °C and 45 °C to obtain an IVT reaction, (d) contacting the IVT reaction with a deoxyribonuclease (DNase) to obtain a digested IVT reaction; (e) contacting the digested IVT reaction with a protease to obtain an inactivated reaction; (f) contacting the inactivated reaction with a chelating agent to obtain a chelated reaction; (g) precipitating an mRNA molecule in the chelated reaction with lithium chloride to obtain a purified mRNA; (h) incubating a cellulose-based chromatographic medium with a buffer comprising dimethylsulfoxide (DMSO), wherein the DMSO is present in an amount between about 2.5% and 25%; (i) contacting the purified mRNA with the cellulose-based chromatographic medium to obtain a bound cellulose-based chromatographic medium; and (j) centrifuging the bound cellulose-based chromatographic medium, thereby reducing the amount of dsRNA produced in the IVT reaction. In some embodiments, the amount of dsRNA produced in the IVT reaction is reduced by at least 10% when compared to an amount of dsRNA produced by a standard method of RNA molecule synthesis.

[0012] In some embodiments, disclosed herein are methods of reducing the amount of double-stranded RNA (dsRNA) produced in an in vitro transcription (IVT) reaction, the methods comprising: (a) obtaining a DNA template; (b) contacting the DNA template with an in vitro transcription (IVT) reaction system to obtain a composition, wherein the IVT reaction system comprises a monovalent salt, spermidine, dithiothreitol (DTT), and dihydrolevoglucosenone; and (c) incubating the composition at a temperature between about 15 °C and 45 °C to obtain an IVT reaction, thereby reducing the amount of dsRNA produced in the IVT reaction. In some embodiments, incubating the composition occurs for a time period between about 30 minutes and 64 days. In some embodiments, the IVT reaction system further comprises a buffer, a 5’ cap analog, ATP, CTP, GTP, UTP, a magnesium ion, an RNase inhibitor, pyrophosphatase, and an RNA polymerase. In some embodiments, the monovalent salt is selected from the group consisting of KCI, NaCI, RbCI, CsCI, NaCIC , NaF, NaBr, and NH4CL In some embodiments, the monovalent salt is present in an amount between about 10 mM and about 150 mM. In some embodiments, the spermidine is present in an amount between about 0.2 mM and about 10 mM. In some embodiments, the dithiothreitol (DTT) is present in an amount between about 15 mM and about 25 mM. In some embodiments, the dihydrolevoglucosenone is present in an amount between about 4% and about 15%. In some embodiments, the DNA template is selected from the group consisting of a linearized DNA template, a circular DNA template, and a synthetic DNA template. In some embodiments, disclosed herein are methods of reducing the amount of doublestranded RNA (dsRNA) produced in an in vitro transcription (IVT) reaction, the methods comprising: (a) obtaining a DNA template; (b) contacting the DNA template with an in vitro transcription (IVT) reaction system to obtain a composition, wherein the IVT reaction system comprises a monovalent salt, spermidine, dithiothreitol (DTT), and dihydrolevoglucosenone; (c) incubating the composition at a temperature between about 15 °C and 45 °C to obtain an IVT reaction; (d) contacting the IVT reaction with a deoxyribonuclease (DNase) to obtain a digested IVT reaction; (e) contacting the digested IVT reaction with a protease to obtain an inactivated reaction; and (f) contacting the inactivated reaction with a chelating agent to obtain a chelated reaction, thereby reducing the amount of dsRNA produced in the IVT reaction. In some embodiments, the protease is proteinase K. In some embodiments, the chelating agent is ethylenediaminetetraacetic acid (EDTA). In some embodiments, disclosed herein are methods of reducing the amount of double-stranded RNA (dsRNA) produced in an in vitro transcription (IVT) reaction, the methods comprising: (a) obtaining a DNA template; (b) contacting the DNA template with an in vitro transcription (IVT) reaction system to obtain a composition, wherein the IVT reaction system comprises a monovalent salt, spermidine, dithiothreitol (DTT), and dihydrolevoglucosenone; (c) incubating the composition at a temperature between about 15 °C and 45 °C to obtain an IVT reaction; (d) contacting the IVT reaction with a deoxyribonuclease (DNase) to obtain a digested IVT reaction; (e) contacting the digested IVT reaction with a protease to obtain an inactivated reaction; (f) contacting the inactivated reaction with a chelating agent to obtain a chelated reaction; and (g) precipitating an mRNA molecule in the chelated reaction with lithium chloride to obtain a purified mRNA, thereby reducing the amount of dsRNA produced in the IVT reaction. In some embodiments, disclosed herein are methods of reducing the amount of double-stranded RNA (dsRNA) produced in an in vitro transcription (IVT) reaction, the methods comprising: (a) obtaining a DNA template; (b) contacting the DNA template with an in vitro transcription (IVT) reaction system to obtain a composition, wherein the IVT reaction system comprises a monovalent salt, spermidine, dithiothreitol (DTT), and dihydrolevoglucosenone; (c) incubating the composition at a temperature between about 15 °C and 45 °C to obtain an IVT reaction; (d) contacting the IVT reaction with a deoxyribonuclease (DNase) to obtain a digested IVT reaction; (e) contacting the digested IVT reaction with a protease to obtain an inactivated reaction; (f) contacting the inactivated reaction with a chelating agent to obtain a chelated reaction; (g) precipitating an mRNA molecule in the chelated reaction with lithium chloride to obtain a purified mRNA; (h) incubating a cellulose-based chromatographic medium with a buffer comprising dimethylsulfoxide (DMSO), wherein the DMSO is present in an amount between about 2.5% and 25%; (i) contacting the purified mRNA with the cellulose-based chromatographic medium to obtain a bound cellulose-based chromatographic medium; and (j) centrifuging the bound cellulose-based chromatographic medium, thereby reducing the amount of dsRNA produced in the IVT reaction. In some embodiments, the amount of dsRNA produced in the IVT reaction is reduced by at least 10% when compared to an amount of dsRNA produced by a standard method of RNA molecule synthesis.

[0013] In some embodiments, disclosed herein are methods of reducing the amount of double-stranded RNA (dsRNA) produced in an in vitro transcription (IVT) reaction, the methods comprising: (a) obtaining a DNA template; (b) contacting the DNA template with an in vitro transcription (IVT) reaction system to obtain a composition, wherein the IVT reaction system comprises an (organo)sulfur oxoacid; and (c) incubating the composition at a temperature between about 15 °C and 45 °C to obtain an IVT reaction, thereby reducing the amount of dsRNA produced in the IVT reaction. In some embodiments, In some embodiments, incubating the composition occurs for a time period between about 30 minutes and 64 days. In some embodiments, the (organo)sulfur oxoacid comprises a tetrahedral or trigonal pyramidal geometry. In some embodiments, the (organo)sulfur oxoacid comprises a sulfonic acid or sulfonic acid derivative. In some embodiments, the sulfonic acid comprises an ethanesulfonate. In some embodiments, the ethanesulfonate is selected from the group consisting of taurine (2-aminoethanesulfonic acid), PIPES (piperazine-N,N'-bis(2-ethanesulfonic acid), and MES (2-(N-morpholino)ethanesulfonic acid). In some embodiments, the taurine is present in an amount between about 0.01 M and about 2.5 M. In some embodiments, the PIPES is present in an amount between about 10 mM and about 180 mM. In some embodiments, the sulfonic acid comprises HEPPSO (2-hydroxy-3- [4-(2-hydroxyethyl)piperazin-1-yl]propane-1 -sulfonic acid). In some embodiments, the HEPPSO is present in an amount between about 20 mM and about 180 mM. In some embodiments, the MES is present in an amount between about 10 mM and about 180 mM. In some embodiments, the sulfonic acid derivate is selected from the group consisting of a polyanionic synthetic sulfonic acid polymer (SSAP), ethylene dimethanesulfonate, and polystyrene sulfonate. In some embodiments, the (organo)sulfur oxoacid comprises a sulfuric acid or sulfuric acid derivative. In some embodiments, the (organo)sulfur oxoacid is selected from the group consisting of magnesium sulfate, ammonium sulfate, and a heparin derivative. In some embodiments, the heparin derivative comprises enoxaparin. In some embodiments, the IVT reaction system further comprises a 5’ cap analog, ATP, CTP, GTP, UTP, a magnesium ion, an RNase inhibitor, pyrophosphatase, and an RNA polymerase. In some embodiments, the IVT reaction system further comprises a buffer. In some embodiments, the buffer is selected from the group consisting of Tris and HEPES. In some embodiments, the DNA template is selected from the group consisting of a linearized DNA template, a circular DNA template, and a synthetic DNA template. In some embodiments, disclosed herein are methods of reducing the amount of double-stranded RNA (dsRNA) produced in an in vitro transcription (IVT) reaction, the methods comprising: (a) obtaining a DNA template; (b) contacting the DNA template with an in vitro transcription (IVT) reaction system to obtain a composition, wherein the IVT reaction system comprises an (organo)sulfur oxoacid; (c) incubating the composition at a temperature between about 15 °C and 45 °C to obtain an IVT reaction, (d) contacting the IVT reaction with a deoxyribonuclease (DNase) to obtain a digested IVT reaction; (e) contacting the digested IVT reaction with a protease to obtain an inactivated reaction; and (f) contacting the inactivated reaction with a chelating agent to obtain a chelated reaction. In some embodiments, the protease is proteinase K. In some embodiments, the chelating agent is ethylenediaminetetraacetic acid (EDTA). In some embodiments, the amount of dsRNA produced in the IVT reaction is reduced by at least 10% when compared to an amount of dsRNA produced by a standard method of RNA molecule synthesis.

[0014] In some embodiments, disclosed herein are compositions for in vitro transcription (IVT) comprising: (a) a DNA template; and (b) an in vitro transcription (IVT) reaction system comprising a buffer, a 5’ cap analog, ATP, CTP, GTP, UTP, a magnesium ion, an RNase inhibitor, pyrophosphatase, and an RNA polymerase, wherein the IVT reaction system further comprises: i. 40 mM potassium chloride (KCI); ii. 0.53 mM spermidine; iii. 20 mM dithiothreitol (DTT); and iv. 10% dimethylsulfoxide (DMSO) or 10% dihydrolevoglucosenone. In some embodiments, the buffer is selected from the group consisting of T ris and HEPES. In some embodiments, the buffer comprises Tris-HCI and PIPES (piperazine-N,N'-bis(2-ethanesulfonic acid). In some embodiments, the DNA template is selected from the group consisting of a linearized DNA template, a circular DNA template, and a synthetic DNA template.

[0015] In some embodiments, disclosed herein are compositions for in vitro transcription (IVT) comprising: (a) a DNA template; and (b) an in vitro transcription (IVT) reaction system comprising a buffer, a 5’ cap analog, ATP, CTP, GTP, UTP, a magnesium ion, an RNase inhibitor, pyrophosphatase, and an RNA polymerase, wherein the IVT reaction system further comprises: i. 0.53 mM spermidine; ii. 20 mM dithiothreitol (DTT); and iii. 0.5 M taurine. In some embodiments, the DNA template is selected from the group consisting of a linearized DNA template, a circular DNA template, and a synthetic DNA template.

[0016] In some embodiments, disclosed herein are methods of reducing an amount of double-stranded RNA (dsRNA) produced in an in vitro transcription (IVT) reaction, wherein the methods comprise: (a) obtaining a DNA template; (b) contacting the DNA template with an in vitro transcription (IVT) reaction system to obtain a composition, wherein the IVT reaction system comprises spermidine, dithiothreitol (DTT), and dihydrolevoglucosenone; and (c) incubating the composition at a temperature between about 15 °C and 45 °C to obtain an IVT reaction, thereby reducing the amount of dsRNA produced in the IVT reaction. In some embodiments, incubating the composition occurs for a time period between about 30 minutes and 64 days. In some embodiments, the IVT reaction system further comprises a buffer, a 5’ cap analog, ATP, CTP, GTP, UTP, a magnesium ion, an RNase inhibitor, pyrophosphatase, and an RNA polymerase. In some embodiments, the spermidine is present in an amount between about 0.2 mM and about 10 mM. In some embodiments, the dithiothreitol (DTT) is present in an amount between about 15 mM and about 25 mM. In some embodiments, the dihydrolevoglucosenone is present in an amount between about 4% and about 15%. In some embodiments, the DNA template is selected from the group consisting of a linearized DNA template, a circular DNA template, and a synthetic DNA template. In some embodiments, the methods disclosed herein further comprise: (d) contacting the IVT reaction with a deoxyribonuclease (DNase) to obtain a digested IVT reaction; (e) contacting the digested IVT reaction with a protease to obtain an inactivated reaction; and (f) contacting the inactivated reaction with a chelating agent to obtain a chelated reaction. In some embodiments, the protease is proteinase K. In some embodiments, the chelating agent is ethylenediaminetetraacetic acid (EDTA). In some embodiments, the methods disclosed herein further comprise: (g) precipitating an mRNA molecule in the chelated reaction with lithium chloride to obtain a purified mRNA. In some embodiments, the methods disclosed herein further comprise: (h) incubating a cellulose-based chromatographic medium with a buffer comprising dimethylsulfoxide (DMSO), wherein the DMSO is present in an amount between about 2.5% and 25%; (i) contacting the purified mRNA with the cellulose-based chromatographic medium to obtain a bound cellulose-based chromatographic medium; and (j) centrifuging the bound cellulose-based chromatographic medium. In some embodiments, the amount of dsRNA produced in the IVT reaction is reduced by at least 10% when compared to an amount of dsRNA produced by a standard method of RNA molecule synthesis. In some embodiments, the IVT reaction system further comprises a pH between 2.5 and 8 and a tris buffer in a range from 20 mM to 150 mM.

[0017] In some embodiments, disclosed herein are methods of reducing an amount of double-stranded RNA (dsRNA) produced in an in vitro transcription (IVT) reaction, the methods comprising: (a) obtaining a DNA template; (b) contacting the DNA template with an in vitro transcription (IVT) reaction system to obtain a composition, wherein the IVT reaction system comprises a monovalent salt, spermidine, dithiothreitol (DTT), and dimethylsulfoxide (DMSO), and further wherein the IVT reaction system comprises a pH between 2.5 and 8 and a tris buffer in a range from 20 mM to 150 mM; and (c) incubating the composition at a temperature between about 15 °C and 45 °C to obtain an IVT reaction, thereby reducing the amount of dsRNA produced in the IVT reaction. In some embodiments, disclosed herein are methods of reducing an amount of double-stranded RNA (dsRNA) produced in an in vitro transcription (IVT) reaction, the methods comprising: (a) obtaining a DNA template; (b) contacting the DNA template with an in vitro transcription (IVT) reaction system to obtain a composition, wherein the IVT reaction system comprises spermidine, dithiothreitol (DTT), and dihydrolevoglucosenone, and further wherein the IVT reaction system comprises a pH between 2.5 and 8 and a tris buffer in a range from 20 mM to 150 mM; and (c) incubating the composition at a temperature between about 15 °C and 45 °C to obtain an IVT reaction, thereby reducing the amount of dsRNA produced in the IVT reaction. In some embodiments, the amount of dsRNA produced in the IVT reaction is reduced by at least 10% when compared to an amount of dsRNA produced by a standard method of RNA molecule synthesis.

[0018] DETAILED DESCRIPTION

[0019] RNA technology has tremendous potential to prevent and treat diseases. To achieve that potential, high-quality RNA compositions (including RNA compositions for vaccine or therapeutic purposes) are needed. In vitro transcription (IVT) can be carried out to synthesize RNA from a DNA template using a variety of natural or engineered RNA polymerases, including SP6, T7 or T3 RNA polymerases. Traditionally, the RNA molecules are made using batch mode in vitro transcription where the reaction components are combined and then incubated for a few hours. Another approach to produce RNA molecules is by fed-batch in vitro transcription where any of the reaction components are added at certain time intervals. Another approach to produce RNA molecules is by continuous-flow in vitro transcription (CF-IVT). CF-IVT can be achieved by contacting a constantly or near constantly flowing solution of reactants (in vitro transcription reaction system) with stationary non-consumables / catalysts (e.g., RNA polymerase, DNA template, or combination of both).

[0020] Regardless of method of RNA synthesis, the undesired byproduct double-stranded RNA (dsRNA) is often produced. dsRNA can cause immunogenic reactions and reduce protein expression, which can result in a reduction of efficacy of the RNA composition and, while dsRNA can be reduced in subsequent purification steps, this often results in a reduction of RNA yield and / or RNA integrity [Baiersdorfer et al., Mol Ther Nucleic Acids 15 (2019)]. Therefore improved manufacturing processes and purification steps to address the presence of dsRNA in RNA compositions are needed.

[0021] Disclosed herein are compositions and methods to reduce the presence of dsRNA in vaccines and therapeutic compositions comprising RNA molecules. RNA molecules, e.g., mRNA (including modified mRNA molecules and / or self-amplifying RNA (saRNA)), can be produced using IVT processes (including batch, fed-batch, and continuous flow processes) that are useful for producing clinical grade RNA, such as mRNA, of high purity and potency, consistently, reproducibly, and in compliance with current good manufacturing practices (cGMP). The methods to reduce the presence of dsRNA may be used with a DNA template in an in vitro transcription (IVT) reaction to generate the RNA molecule and can be applied to a wide variety of constructs with varying 5’ UTRS, coding sequence lengths, 3’ UTRs, and 3’ ends. In some aspects, the methods to reduce the presence of dsRNA include methods to reduce the formation of dsRNA during the IVT reaction. In some aspects, the dsRNA is removed via chromatographic or filtration methods after the IVT reaction (including e.g., oligo(dT), hydroxyapatite, phenyl boronate, ion exchange, hydrophobic interaction, tangential flow filtration, single-pass tangential flow filtration). In some aspects, enzymatic capping is used for 5' capping of the RNA molecule after prevention of formation of dsRNA or after removal of dsRNA from the vaccines and therapeutic compositions comprising RNA molecules.

[0022] Certain Definitions

[0023] Throughout this application, the term “about” is used according to its plain and ordinary meaning in the area of cell and molecular biology to indicate that a value includes the inherent variation or standard deviation of error for the measurement or quantitation method being employed to determine the value. For example, in some aspects, the term “about” may encompass a range of values that are within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11 %, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1 %, or less of the measurement or quantitation.

[0024] The use of the word “a” or “an” when used in conjunction with the term “comprising” may mean “one,” but it is also consistent with the meaning of “one or more,” “at least one,” and “one or more than one.”

[0025] The phrase “and / or” means “and” or “or”. To illustrate, A, B, and / or C includes: A alone, B alone, C alone, a combination of A and B, a combination of A and C, a combination of B and C, or a combination of A, B, and C. In other words, “and / or” operates as an inclusive or.

[0026] The phrase “essentially all” is defined as “at least 95%”; if essentially all members of a group have a certain property, then at least 95% of members of the group have that property. In some instances, essentially all means equal to any one of, at least any one of, or between any two of 95, 96, 97, 98, 99, or 100 % of members of the group have that property.

[0027] The compositions and methods for their use can “comprise,” “consist essentially of,” or “consist of” any of the ingredients or steps disclosed throughout the specification. Throughout this specification, unless the context requires otherwise, the words “comprising” (and any form of comprising, such as “comprise” and “comprises”), “having” (and any form of having, such as “have” and “has”), “including” (and any form of including, such as “includes” and “include”) or “containing” (and any form of containing, such as “contains” and “contain”) are inclusive or open- ended and will be understood to imply the inclusion of a stated step or element or group of steps or elements but not the exclusion of any other step or element or group of steps or elements. It is contemplated that aspects described herein in the context of the term “comprising” may also be implemented in the context of the term “consisting of’ or “consisting essentially of.” Compositions and methods “consisting essentially of” any of the ingredients or steps disclosed limits the scope of the claim to the specified materials or steps which do not materially affect the basic and novel characteristic of the claimed disclosure. The words “consisting of” (and any form of consisting of, such as “consist of’ and “consists of’) means including, and limited to, whatever follows the phrase “consisting of.” Thus, the phrase “consisting of’ indicates that the listed elements are required or mandatory, and that no other elements may be present.

[0028] Reference throughout this specification to “one embodiment,” “an embodiment,” “a particular embodiment,” “a related embodiment,” “a certain embodiment,” “an additional embodiment,” “a further embodiment,” “some embodiments”, “one aspect,” “an aspect,” “a particular aspect,” “a related aspect,” “a certain aspect,” “an additional aspect,” “a further aspect,” “some aspects” or combinations thereof means that a particular feature, structure or characteristic described in connection with the aspect is included in at least one aspect of the present disclosure. Thus, the appearances of the foregoing phrases in various places throughout this specification are not necessarily all referring to the same aspect. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in one or more aspects.

[0029] The terms “inhibiting” or “reducing” or any variation of these terms includes any measurable decrease or complete inhibition to achieve a desired result. The terms “improve,” “promote,” or “increase” or any variation of these terms includes any measurable increase to achieve a desired result or production of a protein or molecule.

[0030] As used herein, the terms “reference,” “standard,” or “control” describe a value relative to which a comparison is performed. For example, an agent, subject, population, sample, or value of interest is compared with a reference, standard, or control agent, subject, population, sample, or value of interest. A reference, standard, or control may be tested and / or determined substantially simultaneously and / or with the testing or determination of interest for an agent, subject, population, sample, or value of interest and / or may be determined or characterized under comparable conditions or circumstances to the agent, subject, population, sample, or value of interest under assessment.

[0031] The term “DNA,” as used herein, means a nucleic acid molecule that includes deoxyribonucleotide residues (such as containing the nucleotide base(s) adenine (A), cytosine (C), guanine (G) and / or thymine (T)). For example, DNA can contain all, or a majority of, deoxyribonucleotide residues. As used herein, the term “deoxyribonucleotide” means a nucleotide lacking a hydroxyl group at the 2' position of a p-D-ribofuranosyl group. Without any limitation, DNA can encompass double stranded DNA, antisense DNA, single stranded DNA, isolated DNA, synthetic DNA, DNA that is recombinantly produced, and modified DNA.

[0032] The term “RNA,” as used herein, means a nucleic acid molecule that includes ribonucleotide residues (such as containing the nucleotide base(s) adenine (A), cytosine (C), guanine (G) and / or uracil (U) or N1 -methylpseudouridine). For example, RNA can contain all, or a majority of, ribonucleotide residues. As used herein, the term “ribonucleotide” means a nucleotide with a hydroxyl group at the 2' position of a p-D-ribofuranosyl group. In one aspect, RNA can be messenger RNA (mRNA) that relates to an RNA transcript which encodes a peptide or protein. As known to those of skill in the art, mRNA generally contains a 5' untranslated region (5'-UTR), a polypeptide coding region, and a 3' untranslated region (3 -UTR). Without any limitation, RNA can encompass double stranded RNA, antisense RNA, single stranded RNA, isolated RNA, synthetic RNA, RNA that is recombinantly produced, circular RNA, self-amplifying RNA (saRNA), guide RNA (gRNA), and modified RNA (modRNA).

[0033] The term “RNA drug substance,” as used herein, means a purified RNA that is solubilized in any form of aqueous solution appropriate to permit subsequent encapsulation of the RNA within encapsulating agents as described below.

[0034] The term “drug substance RNA,” as used herein, means the RNA component of the RNA drug substance.

[0035] The term “RNA drug product,” as used herein, means a purified RNA that has been encapsulated in any form of encapsulating agents (e.g., lipid nanoparticles) described herein and forms a colloidal dispersion (e.g., RNA-loaded LNP dispersion) and where the resulting colloidal dispersion has been adjusted and purified to stabilize the encapsulated RNA.

[0036] As contemplated herein, without any limitations, RNA can be used as a therapeutic modality to treat and / or prevent a number of conditions in mammals, including humans. Methods contemplated comprise administration of the RNA described herein to a mammal, such as a human. For example, in one aspect, such methods of use for RNA include an antigen-coding RNA vaccine to induce robust neutralizing antibodies and accompanying / concomitant T-cell response to achieve protective immunization with preferably minimal vaccine doses. The RNA administered is preferably in vitro transcribed RNA. In some aspects, the RNA is administered to edit, repair, restore the function of, or reduce the level of a gene or protein in a mammal, such as a human.

[0037] An “isolated RNA” is defined as an RNA molecule that can be recombinant or has been isolated from total genomic nucleic acid. A “modified RNA” or “modRNA” refers to an RNA molecule, e.g., an mRNA molecule, having at least one addition, deletion, substitution, and / or alteration of one or more nucleotides as compared to naturally occurring RNA. Such alterations can refer to the addition of non-nucleotide material to internal RNA nucleotides, or to the 5' and / or 3' end(s) of RNA. In one aspect, such modRNA contains at least one modified nucleotide, such as an alteration to the base of the nucleotide. For example, a modified nucleotide can replace one or more uridine and / or cytidine nucleotides. For example, these replacements can occur for every instance of uridine and / or cytidine in the RNA sequence, or can occur for only select uridine and / or cytidine nucleotides. Such alterations to the standard nucleotides in RNA can include nonstandard nucleotides, such as chemically synthesized nucleotides or deoxynucleotides. For example, at least one uridine nucleotide can be replaced with N1-methylpseudouridine in an RNA sequence. Other altered nucleotides are known to those of skill in the art. Such altered RNAs are considered analogs of naturally-occurring RNA. In some aspects, the RNA is produced by in vitro transcription using a DNA template, where DNA refers to a nucleic acid that contains deoxyribonucleotides. In some aspects, the RNA can be replicon RNA (replicon), in particular self-replicating RNA, or self-amplifying RNA (saRNA). In some aspects, the RNA can be a guide RNA (gRNA) or circular RNA.

[0038] As used herein, a “protein,” “polypeptide,” or “peptide” refers to a molecule comprising at least two amino acid residues. As used herein, the term “wild-type” or “native” refers to the endogenous version of a molecule that occurs naturally in an organism. In some aspects, wild-type versions of a protein or polypeptide are employed, however, in many aspects of the disclosure, a modified protein or polypeptide is employed to generate an immune response. The terms described above may be used interchangeably. A “modified protein” or “modified polypeptide” or a “variant” refers to a protein or polypeptide whose chemical structure, particularly its amino acid sequence, is altered with respect to the wild-type protein or polypeptide. In some aspects, a modified / variant protein or polypeptide has at least one modified activity or function (recognizing that proteins or polypeptides may have multiple activities or functions). It is specifically contemplated that a modified / variant protein or polypeptide may be altered with respect to one activity or function yet retain a wild-type activity or function in other respects, such as immunogenicity. Where a protein is specifically mentioned herein, it is in general a reference to a native (wild-type) or recombinant (modified) protein. The protein may be isolated directly from the organism of which it is native, produced by recombinant DNA / exogenous expression methods, produced by solid-phase peptide synthesis (SPPS), or other in vitro methods. In particular aspects, there are isolated nucleic acid segments and recombinant vectors incorporating nucleic acid sequences that encode a polypeptide (e.g., an antigen or fragment thereof). The term “recombinant” may be used in conjunction with a polypeptide or the name of a specific polypeptide, and this generally refers to a polypeptide produced from a nucleic acid molecule that has been manipulated in vitro or that is a replication product of such a molecule.

[0039] The term “isolated” can refer to a nucleic acid or polypeptide that is substantially free of cellular material, bacterial material, viral material, or culture medium (e.g., when produced by recombinant DNA techniques) of their source of origin, or chemical precursors or other chemicals (e.g., when chemically synthesized). Moreover, an isolated compound refers to one that can be administered to a subject as an isolated compound; in other words, the compound may not simply be considered “isolated” if it is adhered to a column or embedded in an agarose gel. Moreover, an “isolated nucleic acid fragment” or “isolated peptide” is a nucleic acid or protein fragment that is not naturally occurring as a fragment and / or is not typically in the functional state and / or that is altered or removed from the natural state through human intervention. For example, a DNA naturally present in a living animal is not “isolated,” but a synthetic DNA, or a DNA partially or completely separated from the coexisting materials of its natural state is “isolated.” An isolated nucleic acid can exist in substantially purified form, or can exist in a non-native environment such as, for example, a cell into which the nucleic acid has been delivered.

[0040] All patents, published patent applications, other publications, and databases referred to herein are incorporated by reference in their entirety with respect to the related technology.

[0041] DNA Template

[0042] In some aspects, the methods for reducing dsRNA include an RNA molecule, e.g., mRNA, synthesized from a sample that includes a linear DNA template. The DNA template includes a sequence coding for a gene of interest that encodes, e.g., a peptide or polypeptide of interest. In some aspects, the DNA template includes an RNA polymerase promoter sequence operably linked to the sequence coding for a gene of interest. In some aspects, the DNA template includes an RNA polymerase promoter sequence operably linked to the respective RNA polymerase gene sequence, which is operably linked to a subgenomic promoter, which is operably linked to a sequence coding for a gene of interest. For example, in some preferred aspects, the DNA template includes an RNA-dependent RNA polymerase (RdRp) promoter sequence operably linked to the RdRp gene sequence, which is operably linked to a subgenomic promoter, which is operably linked to a sequence coding for a gene of interest. In some aspects, a DNA template lacks a plasmid backbone.

[0043] In some aspects, the linear DNA template is a linearized plasmid DNA used as the template for in vitro transcription. In some aspects, cells, e.g., bacterial cells, e.g., E. coli, e.g., DH10B cells, are transfected with the plasmid DNA template. The transfected cells are cultured to replicate the plasmid DNA which is then isolated and purified. In some aspects, the linear DNA template is synthesized in a cell-free environment. In some aspects, the linear DNA template is synthesized by rolling circle amplification (RCA). In some aspects, RCA includes an amplification target circle (ATC) that forms a template on which new DNA is made, thereby extending the initial sequence as a continuous sequence of repeated sequences complementary to the circle but generating only about several thousand copies per hour. In some aspects, the linear DNA template is provided by exponential RCA, including hyperbranched RCA (also termed ramification amplification).

[0044] In some aspects, the linear DNA template is provided by a cell-free process for synthesizing DNA that includes contacting a DNA template with at least one polymerase in the presence of nucleotides to form a reaction mixture, wherein the DNA template is amplified by strand displacement replication, and wherein further nucleotides are supplied to the reaction mixture continuously or at intervals during the process. In some aspects, the linear DNA template is provided by PCR.

[0045] In some aspects, the DNA template also includes an RNA polymerase promoter sequence, e.g., a T7 promoter, located 5' to and operably linked to the gene of interest. In some aspects, the DNA template includes an RNA polymerase promoter sequence operably linked to the respective RNA polymerase gene sequence, which is operably linked to a subgenomic promoter, which is operably linked to a sequence coding for a gene of interest. In some preferred aspects, the DNA template includes an RNA-dependent RNA polymerase (RdRp) promoter sequence, located 5' to and operably linked to a subgenomic promoter, which is operably linked to a sequence coding for a gene of interest. As used herein, the phrase “operably linked” refers to a functional connection between two or more molecules, constructs, transcripts, entities, moieties or the like. For example, a gene of interest operably linked to an RNA polymerase promoter allows transcription of the gene of interest. Any RNA polymerase or variants thereof may be used in the methods described here. The RNA polymerase may be selected from, but is not limited to, a phage RNA polymerase, e.g., a T7 RNA polymerase, a T3 RNA polymerase, a SP6 RNA polymerase, and / or mutant polymerases such as, but not limited to, polymerases able to incorporate modified nucleic acids.

[0046] As used herein, “gene of interest” refers to a polynucleotide which encodes a polypeptide or protein of interest. Depending on the context, the gene of interest refers to a deoxyribonucleic acid, e.g., a gene of interest in a DNA template which may be transcribed to an RNA molecule, or a ribonucleic acid, e.g., a gene of interest in an RNA molecule which may be translated to produce the encoded polypeptide of interest in vitro, in vivo, in situ or ex vivo. As described in more detail below, a polypeptide of interest includes but is not limited to, biologies, antibodies, vaccines, therapeutic proteins or peptides, etc.

[0047] Flanking Regions: Untranslated Regions (UTRs)

[0048] In some aspects, the methods for reducing dsRNA include an RNA molecule synthesized from a sample having a linear DNA template, the DNA template includes an RNA polymerase promoter sequence operably linked to a sequence coding for a gene of interest and a 5' untranslated region (UTR) and / or a 3' UTR. In some aspects, the DNA template includes an RNA polymerase promoter sequence operably linked to the respective RNA polymerase gene sequence, which is operably linked to a subgenomic promoter, which is operably linked to a sequence coding for a gene of interest. For example, in some aspects, the RNA molecule is synthesized from a sample having a linear DNA template, the DNA template includes an RNA-dependent RNA polymerase (RdRp) promoter sequence, located 5' to and operably linked to a subgenomic promoter, which is operably linked to a sequence coding for a gene of interest and a 5' untranslated region (UTR) and / or a 3' UTR.

[0049] The DNA template and RNA molecule may include UTRs. Untranslated regions (UTRs) of a gene are transcribed but not translated. The 5' UTR starts at the transcription start site and continues to the start codon but does not include the start codon; whereas, the 3' UTR starts immediately following the stop codon and continues until the transcriptional termination signal. The regulatory features of a UTR may be incorporated into the polynucleotides, primary constructs and / or mRNA of the present invention to enhance the stability of the molecule. The specific features may also be incorporated to ensure controlled down-regulation of the transcript in case they are misdirected to undesired organs sites.

[0050] Natural 5' UTRs bear features which play roles in translation initiation. They harbor signatures like Kozak sequences which are commonly known to be involved in the process by which the ribosome initiates translation of many genes. Kozak sequences have the consensus CCR(A / G)CCAUGG, where R is a purine (adenine or guanine) three bases upstream of the start codon (AUG), which is followed by another ‘G’. 5' UTR also have been known to form secondary structures which are involved in elongation factor binding. By engineering the features typically found in abundantly expressed genes of specific target organs, one may enhance the stability and protein production of the polynucleotides, primary constructs. For example, use of 5' UTR from other tissue-specific mRNA to improve expression in that tissue is possible for muscle (MyoD, Myosin, Myoglobin, Myogenin, Herculin), for endothelial cells (Tie-1 , CD36), etc.

[0051] Other non-UTR sequences may be incorporated into the 5' (or 3' UTR) UTRs. For example, introns or portions of introns sequences may be incorporated into the flanking regions of the polynucleotides, primary constructs or mRNA described here. Incorporation of intronic sequences may increase protein production as well as mRNA levels. Cap-dependent translation involves recruitment of the pre-initiation complex (PIC) to the 5' end of an mRNA followed by scanning to find an AUG initiation codon in an optimum sequence context. AUG recognition promotes scanning cessation, release of most initiation factors, and recruitment of the large ribosomal subunit to initiate elongation. Efficient recognition of an initiation codon depends on its surrounding sequence. In some aspects, the sequence CRCCaugG (R = purine, A or G) may provide optimal context for AUG recognition in eukaryotes.

[0052] 3' UTRs are known to have stretches of Adenosines and Uridines embedded in them. These AU rich signatures are particularly prevalent in genes with high rates of turnover. Based on their sequence features and functional properties, the AU rich elements (AREs) may be separated into three classes: Class I AREs include several dispersed copies of an AUUUA motif within U-rich regions. C-Myc and MyoD include class I AREs. Class II AREs possess two or more overlapping UUAUUUA(U / A)(U / A) nonamers. Molecules including this type of AREs include GM-CSF and TNF-alpha. Class III ARES are less well defined. These U rich regions do not include an AUUUA motif c-Jun and Myogenin are two well-studied examples of this class. Most proteins binding to the AREs are known to destabilize the messenger, whereas members of the ELAV family, most notably HuR, have been documented to increase the stability of mRNA. HuR binds to AREs of all the three classes. Engineering the HuR specific binding sites into the 3' UTR of nucleic acid molecules may lead to HuR binding and thus, stabilization of the message in vivo. Introduction, removal or modification of 3' UTR AREs may be used to modulate the stability of polynucleotides, and primary constructs.

[0053] When engineering specific polynucleotides, and / or primary constructs, one or more copies of an ARE may be introduced to make polynucleotides, and / or primary constructs less stable and thereby curtail translation and decrease production of the resultant protein. Likewise, AREs may be identified and removed or mutated to increase the intracellular stability and thus increase translation and production of the resultant protein. Transfection experiments may be conducted in relevant cell lines, using polynucleotides, and / or primary constructs and protein production may be assayed at various time points post-transfection. For example, cells may be transfected with different ARE-engineering molecules and by using an ELISA kit to the relevant protein and assaying protein produced at 6 hour, 12 hour, 24 hour, 48 hour, and 7 days post-transfection.

[0054] Poly(A) tail

[0055] In some aspects, the methods for reducing dsRNA disclosed herein include an RNA molecule synthesized from a sample having a DNA template, the DNA template having an RNA polymerase promoter sequence operably linked to a sequence coding for a gene of interest and a poly(A) tail sequence of 20-100 nucleotides. The poly(A) tail can prevent degradation of the RNA molecule in a cell. Accordingly, in some aspects, the plasmid DNA template includes a sequence coding for a poly(A) tail located 3' to the gene of interest. As used herein, “poly(A) tail” refers to a chain of adenine nucleotides. In some aspects, the poly(A) tail includes 5-300 adenine nucleotides in length, e.g., at least, at most, or about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 225, 230, 235, 240, 245, 250, 255, 260, 265, 270, 275, 280, 285, 290, 295, or 300 adenine nucleotides in length, or any range or value derivable therein. In preferred aspects, the DNA template includes a poly(A) tail that includes about 40 adenines. In preferred aspects, the DNA template includes a poly(A) tail that includes about 80 adenines. In some aspects, the poly(A) tail is encoded in the DNA template. In other aspects, the poly(A) tail is added to the RNA molecule by enzymatic treatment with a Poly(A) polymerase. In some aspects, the RNA molecule does not include a poly(A) tail.

[0056] In some aspects, immediately downstream of the poly(A) tail coding sequence on the plasmid DNA template is a recognition site for a restriction endonuclease to linearize the plasmid. Linearization of the plasmid can mitigate transcriptional readthrough.

[0057] In some aspects, following linearization, the plasmid DNA template is filtered into an appropriate solvent, e.g., water, HEPES, and EDTA. In a preferred aspect, the solvent includes 10 mM HEPES, 0.1 mM EDTA, and the like. Filtration occurs via, e.g., ultrafiltration, diafiltration, or, e.g., tangential flow ultrafiltration / diafiltration.

[0058] The linear DNA template may be purified before use as a template for in vitro transcription. For example, the linear DNA template may be purified chromatographically or by ethanol precipitation.

[0059] In vitro Transcription

[0060] In vitro transcription (IVT) (whether fed-batch, continuous-flow, or otherwise) refers to a procedure that allows for DNA-directed synthesis of RNA molecules of any sequence, ranging in size from short oligonucleotides to several kilobases. In some aspects, in vitro transcription involves engineering of a DNA template to include a bacteriophage promoter sequence (e.g., from the T7 coliphage) upstream of the sequence of interest followed by transcription using the corresponding RNA polymerase. In some aspects, the resulting RNA molecules are subsequently modified (e.g., by capping, splicing, the addition of a poly(A) tail, etc.).

[0061] The methods described herein for reducing dsRNA during synthesis of an RNA molecule, e.g., mRNA, include contacting a DNA template with an in vitro transcription (IVT) reaction system. In some aspects, the IVT reaction system includes an RNA polymerase and ribonucleotides, which may be natural and / or modified ribonucleotides. In some aspects, the IVT reaction system includes a transcription buffer, nucleoside triphosphates (NTPs), an RNase inhibitor and an RNA polymerase. The NTPs may be selected from, but are not limited to, those described herein including natural and unnatural (modified, such as, for example, N1-methylpseudouridine-5’- triphosphate) NTPs.

[0062] The methods described herein for reducing dsRNA during synthesis of an RNA molecule, e.g., mRNA, reduce dsRNA when compared to the amount of dsRNA produced during standard methods of RNA molecule synthesis using standard or modified nuclewotides and including cap analogs, enzymatic capping, or uncapped molecules. For example, the methods described herein reduce dsRNA when compared to in vitro transcription (IVT) reactions performed using an IVT reaction system lacking one or more of a monovalent salt, spermidine, dithiothreitol (DTT), and dimethylsulfoxide (DMSO). In some aspects, the methods described herein reduce dsRNA when compared to IVT reactions performed using an IVT reaction system lacking monovalent salts, including IVT reaction systems lacking potassium chloride (KCI), sodium chloride (NaCI), rubidium chloride (RbCI), caesium chloride (CsCI), sodium perchlorate (NaCIC ), sodium fluoride (NaF), sodium bromide (NaBr), and ammonium chloride (NH4CI). In some aspects, the methods described herein reduce dsRNA when compared to IVT reactions performed using an IVT reaction system lacking spermidine. In some aspects, the methods described herein reduce dsRNA when compared to IVT reactions performed using an IVT reaction system lacking DTT. In some aspects, the methods described herein reduce dsRNA when compared to IVT reactions performed using an IVT reaction system lacking DMSO. In some aspects, the methods described herein reduce dsRNA when compared to in vitro transcription (IVT) reactions performed using an IVT reaction system lacking one or more of a monovalent salt, spermidine, dithiothreitol (DTT), and dihydrolevoglucosenone. In some aspects, the methods described herein reduce dsRNA when compared to IVT reactions performed using an IVT reaction system lacking dihydrolevoglucosenone. In some aspects, the methods described herein reduce dsRNA when compared to IVT reactions performed using an IVT reaction system lacking an (organo)sulfur oxoacid, including IVT reaction systems lacking an (organo)sulfur oxoacid comprising a tetrahedral or trigonal pyramidal geometry, a sulfonic acid (such as an ethanesulfonate, including an ethanesulfonate selected from the group consisting of taurine, (2-aminoethanesulfonic acid), PIPES (piperazine-N,N'-bis(2-ethanesulfonic acid), and MES (2-(N-morpholino)ethanesulfonic acid)) or sulfonic acid derivative, a polyanionic synthetic sulfonic acid polymer (SSAP), an ethylene dimethanesulfonate, and polystyrene sulfonate, a sulfuric acid or sulfuric acid derivative, a magnesium sulfate, ammonium sulfate, and / or a heparin derivative. In some aspects, the methods described herein reduce dsRNA when compared to IVT reactions performed using an IVT reaction system lacking one or more of spermidine, dithiothreitol (DTT) and taurine. In some aspects, the methods described herein reduce dsRNA when compared to IVT reactions performed using an IVT reaction system lacking taurine.

[0063] In some aspects, the methods described herein for reducing dsRNA during synthesis of an RNA molecule result in at least a 1 % decrease of dsRNA when compared to standard methods of RNA molecule synthesis. In some aspects, the methods described herein for reducing dsRNA during synthesis of an RNA molecule result in at least a 2% decrease of dsRNA when compared to standard methods of RNA molecule synthesis. In some aspects, the methods described herein for reducing dsRNA during synthesis of an RNA molecule result in at least a 3% decrease of dsRNA when compared to standard methods of RNA molecule synthesis. In some aspects, the methods described herein for reducing dsRNA during synthesis of an RNA molecule result in at least a 4% decrease of dsRNA when compared to standard methods of RNA molecule synthesis. In some aspects, the methods described herein for reducing dsRNA during synthesis of an RNA molecule result in at least a 5% decrease of dsRNA when compared to standard methods of RNA molecule synthesis. In some aspects, the methods described herein for reducing dsRNA during synthesis of an RNA molecule result in at least a 10% decrease of dsRNA when compared to standard methods of RNA molecule synthesis. In some aspects, the methods described herein for reducing dsRNA during synthesis of an RNA molecule result in at least a 15% decrease of dsRNA when compared to standard methods of RNA molecule synthesis. In some aspects, the methods described herein for reducing dsRNA during synthesis of an RNA molecule result in at least a 20% decrease of dsRNA when compared to standard methods of RNA molecule synthesis. In some aspects, the methods described herein for reducing dsRNA during synthesis of an RNA molecule result in at least a 25% decrease of dsRNA when compared to standard methods of RNA molecule synthesis. In some aspects, the methods described herein for reducing dsRNA during synthesis of an RNA molecule result in at least a 30% decrease of dsRNA when compared to standard methods of RNA molecule synthesis. In some aspects, the methods described herein for reducing dsRNA during synthesis of an RNA molecule result in at least a 40% decrease of dsRNA when compared to standard methods of RNA molecule synthesis. In some aspects, the methods described herein for reducing dsRNA during synthesis of an RNA molecule result in at least a 50% decrease of dsRNA when compared to standard methods of RNA molecule synthesis. In some aspects, the methods described herein for reducing dsRNA during synthesis of an RNA molecule result in at least a 55% decrease of dsRNA when compared to standard methods of RNA molecule synthesis. In some aspects, the methods described herein for reducing dsRNA during synthesis of an RNA molecule result in at least a 6%, 7%, 8%, 9%, 10%, 11 %, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21 %, 22%, 23%, 24%, 25%, 26%,

[0064] 27%, 28%, 29%, 30%, 31 %, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41 %, 42%, 43%,

[0065] 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51 %, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%,

[0066] 61 %, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71 %, 72%, 73%, 74%, 75%, 76%, 77%,

[0067] 78%, 79%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%,

[0068] 95%, 96%, 97%, 98%, or 99% decrease of dsRNA when compared to standard methods of RNA molecule synthesis.

[0069] In some aspects, the methods described herein for reducing dsRNA during synthesis of an RNA molecule result in at least a 1.5-fold reduction of dsRNA when compared to standard methods of RNA molecule synthesis. In some aspects, the methods described herein for reducing dsRNA during synthesis of an RNA molecule result in at least a 2-fold reduction of dsRNA when compared to standard methods of RNA molecule synthesis. In some aspects, the methods described herein for reducing dsRNA during synthesis of an RNA molecule result in at least a 3- fold reduction of dsRNA when compared to standard methods of RNA molecule synthesis. In some aspects, the methods described herein for reducing dsRNA during synthesis of an RNA molecule result in at least a 4-fold reduction of dsRNA when compared to standard methods of RNA molecule synthesis. In some aspects, the methods described herein for reducing dsRNA during synthesis of an RNA molecule result in at least a 5-fold reduction of dsRNA when compared to standard methods of RNA molecule synthesis. In some aspects, the methods described herein for reducing dsRNA during synthesis of an RNA molecule result in at least a 6- fold reduction of dsRNA when compared to standard methods of RNA molecule synthesis. In some aspects, the methods described herein for reducing dsRNA during synthesis of an RNA molecule result in at least a 7-fold reduction of dsRNA when compared to standard methods of RNA molecule synthesis. In some aspects, the methods described herein for reducing dsRNA during synthesis of an RNA molecule result in at least a 8-fold reduction of dsRNA when compared to standard methods of RNA molecule synthesis. In some aspects, the methods described herein for reducing dsRNA during synthesis of an RNA molecule result in at least a 9- fold reduction of dsRNA when compared to standard methods of RNA molecule synthesis. In some aspects, the methods described herein for reducing dsRNA during synthesis of an RNA molecule result in at least a 10-fold reduction of dsRNA when compared to standard methods of RNA molecule synthesis. In some aspects, the methods described herein for reducing dsRNA during synthesis of an RNA molecule result in at least a 11 -fold reduction of dsRNA when compared to standard methods of RNA molecule synthesis. In some aspects, the methods described herein for reducing dsRNA during synthesis of an RNA molecule result in at least a 12- fold reduction of dsRNA when compared to standard methods of RNA molecule synthesis. In some aspects, the methods described herein for reducing dsRNA during synthesis of an RNA molecule result in at least a 13-fold reduction of dsRNA when compared to standard methods of RNA molecule synthesis. In some aspects, the methods described herein for reducing dsRNA during synthesis of an RNA molecule result in at least a 14-fold reduction of dsRNA when compared to standard methods of RNA molecule synthesis. In some aspects, the methods described herein for reducing dsRNA during synthesis of an RNA molecule result in at least a 15- fold reduction of dsRNA when compared to standard methods of RNA molecule synthesis. In some aspects, the methods described herein for reducing dsRNA during synthesis of an RNA molecule result in at least a 16-fold reduction of dsRNA when compared to standard methods of RNA molecule synthesis. In some aspects, the methods described herein for reducing dsRNA during synthesis of an RNA molecule result in at least a 17-fold reduction of dsRNA when compared to standard methods of RNA molecule synthesis. In some aspects, the methods described herein for reducing dsRNA during synthesis of an RNA molecule result in at least a 18- fold reduction of dsRNA when compared to standard methods of RNA molecule synthesis. In some aspects, the methods described herein for reducing dsRNA during synthesis of an RNA molecule result in at least a 19-fold reduction of dsRNA when compared to standard methods of RNA molecule synthesis. In some aspects, the methods described herein for reducing dsRNA during synthesis of an RNA molecule result in at least a 20-fold reduction of dsRNA when compared to standard methods of RNA molecule synthesis.

[0070] In some aspects, the methods described herein for reducing dsRNA during synthesis of an RNA molecule result in at least a 10 pg / ug (pg of dsRNA per ug of total RNA) decrease of dsRNA when compared to standard methods of RNA molecule synthesis. In some aspects, the methods described herein for reducing dsRNA during synthesis of an RNA molecule result in at least a 20 pg / ug decrease of dsRNA when compared to standard methods of RNA molecule synthesis. In some aspects, the methods described herein for reducing dsRNA during synthesis of an RNA molecule result in at least a 30 pg / ug decrease of dsRNA when compared to standard methods of RNA molecule synthesis. In some aspects, the methods described herein for reducing dsRNA during synthesis of an RNA molecule result in at least a 40 pg / ug decrease of dsRNA when compared to standard methods of RNA molecule synthesis. In some aspects, the methods described herein for reducing dsRNA during synthesis of an RNA molecule result in at least a 50 pg / ug decrease of dsRNA when compared to standard methods of RNA molecule synthesis. In some aspects, the methods described herein for reducing dsRNA during synthesis of an RNA molecule result in at least a 100 pg / ug decrease of dsRNA when compared to standard methods of RNA molecule synthesis. In some aspects, the methods described herein for reducing dsRNA during synthesis of an RNA molecule result in at least a 200 pg / ug decrease of dsRNA when compared to standard methods of RNA molecule synthesis. In some aspects, the methods described herein for reducing dsRNA during synthesis of an RNA molecule result in at least a 300 pg / ug decrease of dsRNA when compared to standard methods of RNA molecule synthesis. In some aspects, the methods described herein for reducing dsRNA during synthesis of an RNA molecule result in at least a 400 pg / ug decrease of dsRNA when compared to standard methods of RNA molecule synthesis. In some aspects, the methods described herein for reducing dsRNA during synthesis of an RNA molecule result in at least a 500 pg / ug decrease of dsRNA when compared to standard methods of RNA molecule synthesis. In some aspects, the methods described herein for reducing dsRNA during synthesis of an RNA molecule result in at least a 1000 pg / ug decrease of dsRNA when compared to standard methods of RNA molecule synthesis. In some aspects, the methods described herein for reducing dsRNA during synthesis of an RNA molecule result in at least a 1500 pg / ug decrease of dsRNA when compared to standard methods of RNA molecule synthesis. In some aspects, the methods described herein for reducing dsRNA during synthesis of an RNA molecule result in at least a 2000 pg / ug decrease of dsRNA when compared to standard methods of RNA molecule synthesis. In some aspects, the methods described herein for reducing dsRNA during synthesis of an RNA molecule result in at least a 2500 pg / ug decrease of dsRNA when compared to standard methods of RNA molecule synthesis. In some aspects, the methods described herein for reducing dsRNA during synthesis of an RNA molecule result in at least a 3000 pg / ug decrease of dsRNA when compared to standard methods of RNA molecule synthesis. In some aspects, the methods described herein for reducing dsRNA during synthesis of an RNA molecule result in at least a 3500 pg / ug decrease of dsRNA when compared to standard methods of RNA molecule synthesis. In some aspects, the methods described herein for reducing dsRNA during synthesis of an RNA molecule result in at least a 4000 pg / ug decrease of dsRNA when compared to standard methods of RNA molecule synthesis. In some aspects, the methods described herein for reducing dsRNA during synthesis of an RNA molecule result in at least a 4500 pg / ug decrease of dsRNA when compared to standard methods of RNA molecule synthesis.

[0071] In some aspects, the decrease of dsRNA is quantified by ELISA. In some aspects, the decrease of dsRNA is quantified by J2 antibody-based ELISA. In some aspects, the decrease of dsRNA is quantified by dot blot. In some aspects, the decrease of dsRNA is quantified by chromatography or spectrophotometry. In some aspects, the decrease of dsRNA is quantified by ultra-performance liquid chromatography I high performance liquid chromatography with ultraviolet detection (UPLC / HPLC-UV). In some aspects, the decrease of dsRNA is quantified by UV spectrophotometry. In some aspects, the decrease of dsRNA is quantified by quantitative PCR (qPCR).

[0072] RNA Polymerase

[0073] In some aspects, the RNA polymerase used to generate the mRNA transcript may also be referred to as a “DNA-dependent RNA polymerase” which transcribes DNA into RNA molecules. Exemplary RNA polymerases include bacteriophage T7, T3, Syn5, and SP6 RNA polymerases, or variants thereof (including thermostable / thermophilic variants), which may be used to transcribe the mRNA, self-amplifying RNA, or guide RNA from a DNA template. RNA polymerases represent the primary machinery that drives transcription. RNA polymerases have been isolated and purified sufficiently that they are useful for producing RNA in vitro. In some aspects, the RNA polymerase is a T7 RNA polymerase, which refers to a monomeric T7 bacteriophage-encoded DNA directed RNA polymerase that catalyzes the formation of RNA in the 5' to 3' direction. The wild-type T7 RNA polymerase includes 883 amino acids. It is homologous to T3 RNA polymerase and somewhat homologous to SP6 RNA polymerase.

[0074] In some aspects, the RNA polymerase includes an engineered T7 RNA polymerase variant, such as a variant that allows for selective incorporation of a 5’ cap analog (e.g. m7G(5')ppp(5')m7G, m7G(5’)ppp(5’)(2’OMeA)pG, or m7G(5')ppp(5')(2'OMeA)pU cap analogs) over GTP at the initiation of in vitro transcription. For example, in some aspects, the RNA polymerase has been modified to preferentially accept a cap (also referred to as an RNA cap, an RNA 7- methylguanosine cap or an RNA m7G cap) or cap analog (e.g., the “Anti Reverse Cap Analog” (3'-O-Me-m7G(5')ppp(5')G; “ARCA”), or a methylated cap analog with one or more nucleotides at the transcription initiation site (e.g., m7G(5')ppp(5')N, wherein N is any nucleotide) to begin transcription during transcription initiation. The 5' cap is an altered nucleotide on the 5' end of some eukaryotic primary transcripts such as precursor messenger RNA. The typical cap structure includes a 7-methylguanosine (m7G) linked to the first nucleotide of the transcript via a 5'-5' triphosphate bridge. Cap analogs may include, for example, one, two or more methyl (or other substitution) groups at specific positions. Exemplary 5’ cap analogs include, but are not limited to, m7G(5')ppp(5')m7G, 3'-O-Me-m7G(5')ppp(5')G, m7G(5')ppp(5')G, G(5')ppp(5')G, m7G(5')ppp(5')A, G(5')ppp(5')A, m7G(5’)ppp(5’)(2’OMeA)pG, m7(3’OMeG)(5’)ppp(5’)(2’OMeA)pG, m7G(5')ppp(5')(2'OMeA)pU, or m7(3’OMeG)(5’)ppp(5’)m6(2’OMeA)pG. Cap molecules or 5’ cap analogs may be added either upfront in the IVT reaction or after the synthesis of m RNA, by enzymatic capping. Cap molecules added upfront in the IVT reaction can make the mRNA production more straightforward.

[0075] Ribonucleotides

[0076] In the methods described herein, the IVT reaction system includes nucleotides (for example, nonmodified ribonucleoside triphosphates or modified ribonucleoside triphosphates). The nucleotides may be selected from any one of natural nucleotides, e.g., A, G, C, and U ribonucleotides; modified nucleotides (such as, for example, N1-methylpseudouridine-5’- triphosphate); or a combination thereof. In some aspects, the ribonucleotides are Tris buffered, such as a 100 mM aqueous solution of ribonucleotide titrated to pH 7.3-7.5 with Tris base. In some aspects, the ribonucleotides are in sodium salt.

[0077] Modified nucleobases which may be incorporated into modified nucleosides and nucleotides and be present in the RNA molecules generated by the IVT reaction system include, for example, m5C (5- methylcytidine), m5U (5-methyluridine), m6A (N6-methyladenosine), s2U (2- thiouridine), Um (2'-0-methyluridine), mlA (1-methyladenosine); m2A (2- methyladenosine); Am (2-1-O-methyladenosine); ms2m6A (2-methylthio-N6- methyladenosine); i6A (N6- isopentenyladenosine); ms2i6A (2-methylthio- N6isopentenyladenosine); io6A (N6-(cis- hydroxyisopentenyl)adenosine); ms2io6A (2- methylthio-N6-(cis-hydroxyisopentenyl) adenosine); g6A (N6- glycinylcarbamoyladenosine); t6A (N6-threonyl carbamoyladenosine); ms2t6A (2- methylthio-N6-threonyl carbamoyladenosine); m6t6A (N6-methyl-N6- threonylcarbamoyladenosine); hn6A(N6-hydroxynorvalylcarbamoyl adenosine); ms2hn6A (2- methylthio-N6-hydroxynorvalyl carbamoyladenosine); Ar(p) (2'-0- ribosyladenosine (phosphate)); I (inosine); mil (1-methylinosine); m'lm (l,2'-0- dimethylinosine); m3C (3-methylcytidine); Cm (2T- O-methylcytidine); s2C (2- thiocytidine); ac4C (N4-acetylcytidine); £5C (5-fonnylcytidine); m5Cm (5,2-0- dimethylcytidine); ac4Cm (N4acetyl2TOmethylcytidine); k2C (lysidine); mIG (1- methylguanosine); m2G (N2-methylguanosine); m7G (7-methylguanosine); Gm (2'-0- methylguanosine); m22G (N2,N2-dimethylguanosine); m2Gm (N2,2'-0- dimethylguanosine); m22Gm (N2,N2,2'-0-trimethylguanosine); Gr(p) (2'-0- ribosylguanosine (phosphate)); yW (wybutosine); o2yW (peroxywybutosine); OHyW (hydroxywybutosine); OHyW* (undermodified hydroxywybutosine); imG (wyosine); mimG (methylguanosine); Q (queuosine); oQ (epoxyqueuosine); galQ (galtactosyl- queuosine); manQ (mannosyl-queuosine); preQo (7-cyano- 7-deazaguanosine); preQi (7- aminomethyl-7-deazaguanosine); G* (archaeosine); D (dihydrouridine); m5Um (5,2'-0- dimethyluridine); s4U (4-thiouridine); m5s2U (5-methyl-2- thiouridine); s2Um (2-thio-2'- O-methyluridine); acp3U (3-(3-amino-3-carboxypropyl)uridine); ho5U (5- hydroxyuridine); mo5U (5-methoxyuridine); cmo5U (uridine 5-oxyacetic acid); mcmo5U (uridine 5-oxyacetic acid methyl ester); chm5U (5-(carboxyhydroxymethyl)uridine)); mchm5U (5- (carboxyhydroxymethyl)uridine methyl ester); mcm5U (5-methoxycarbonyl methyluridine); mcm5Um (S-methoxycarbonylmethyl-2-O-methyluridine); mcm5s2U (5- methoxycarbonylmethyl- 2-thiouridine); nm5s2U (5-aminomethyl-2-thiouridine); mnm5U (5-methylaminomethyluridine); mnm5s2U (5-methylaminomethyl-2-thiouridine); mnm5se2U (5-methylaminomethyl-2- selenouridine); ncm5U (5-carbamoylmethyl uridine); ncm5Um (5-carbamoylmethyl-2'-0- methyluridine); cmnm5U (5- carboxymethylaminomethyluridine); cnmm5Um (5-carboxymethy 1 aminomethyl-2-L- Omethyluridine); cmnm5s2U (5-carboxymethylaminomethyl-2-thiouridine); m62A (N6,N6-dimethyladenosine); Tm (2'-0-methylinosine); m4C (N4-methylcytidine); m4Cm (N4,2-0-dimethylcytidine); hm5C (5-hydroxymethylcytidine); m3U (3-methyluridine); cm5U (5- carboxymethyluridine); m6Am (N6,T-0-dimethyladenosine); rn62Am (N6,N6,0-2- trimethyladenosine); m2'7G (N2,7-dimethylguanosine); m2'2'7G (N2,N2,7- trimethylguanosine); m3Um (3,2T-0-dimethyluridine); m5D (5-methyldihydrouridine); f5Cm (5-formyl-2'-0- methylcytidine); mIGm (l,2'-0-dimethylguanosine); m'Am (1 ,2-0- dimethyl adenosine) irinomethyluridine); tm5s2U (S-taurinomethyl-2 -thiouridine)); imG- 14 (4-demethyl guanosine); imG2 (isoguanosine); ac6A (N6-acetyladenosine), hypoxanthine, inosine, 8-oxo-adenine, 7- substituted derivatives thereof, dihydrouracil, pseudouracil, 2-thiouracil, 4-thiouracil, 5- aminouracil, 5-(Ci-C6)-alkyluracil, 5-methyluracil, 5-(C2-Ce)-alkenyluracil, 5-(C2-Ce)- alkynyluracil, 5-(hydroxymethyl)uracil, 5-chlorouracil, 5-fluorouracil, 5-bromouracil, 5- hydroxycytosine, 5-(Ci-C6 )- alkylcytosine, 5-methylcytosine, 5-(C2-C6)-alkenylcytosine, 5-(C2- C6)-alkynylcytosine, 5- chlorocytosine, 5-fluorocytosine, 5-bromocytosine, N2-dimethylguanine, 7-deazaguanine, 8-azaguanine, 7-deaza-7-substituted guanine, 7-deaza-7-(C2- C6)alkynylguanine, 7-deaza- 8-substituted guanine, 8-hydroxyguanine, 6-thioguanine, 8- oxoguanine, 2-aminopurine, 2-amino-6-chloropurine, 2,4-diaminopurine, 2,6-diaminopurine, 8- azapurine, substituted 7-deazapurine, 7-deaza-7-substituted purine, 7-deaza-8-substituted purine, hydrogen (abasic residue), m5C, m5U, m6A, s2U, W, or 2'-0-methyl-U. Additional exemplary modified nucleotides include any one of N1-methylpseudouridine; pseudouridine, N6- methyladenosine, 5-methylcytidine, and 5-methyluridine.

[0078] In some aspects, the RNA molecule may include phosphoramidate, phosphorothioate, and / or methylphosphonate linkages.

[0079] In some aspects, the RNA molecule does not include modified nucleotides, e.g., does not include modified nucleobases, and all of the nucleotides in the RNA molecule are conventional standard ribonucleotides A, U, G and C, with the exception of an optional 5' cap that may include, for example, 7-methylguanosine. In other aspects, the RNA may include a 5' cap comprising a 7'- methylguanosine, and the first 1 , 2 or 3 5' ribonucleotides may be methylated at the 2' position of the ribose.

[0080] Exemplary In Vitro Transcription Reaction Systems

[0081] In some aspects, the in vitro transcription reaction system includes the following: an RNA polymerase, e.g., a T7 RNA polymerase, DNA template; nucleoside triphosphates (NTPs); magnesium; and a buffer such as, e.g., HEPES or Tris (or both HEPES and Tris). In some aspects, the in vitro transcription reaction system does not include RNA polymerase. In some aspects, the in vitro transcription reaction system does not include DNA template. In some aspects, the in vitro transcription reaction system includes neither RNA polymerase nor DNA template. In some aspects, the RNA polymerase is stationary. In some aspects, the DNA template is stationary. In some aspects, both RNA polymerase and DNA template are stationary. In some aspects, the in vitro transcription reaction system is flowing across a substrate containing stationary RNA polymerase and / or DNA template with a residence time within the substrate of between 1 second and 3 hours at a temperature between about 20 °C and 50 °C. In some aspects, the total time that the in vitro transcription reaction system is flowing across a substrate, i.e., the total amount of time the in vitro transcription reaction proceeds, is between 1 second and 365 days. In some aspects, the total amount of time the in vitro transcription reaction proceeds is more than 365 days.

[0082] In some aspects, the in vitro transcription reaction system includes the RNA polymerase, e.g., a T7 RNA polymerase, at a final concentration of 1000-44000 U / mL, e.g., at least, at most, or about 1000, 1050, 1100, 1150, 1200, 1250, 1300, 1350, 1400, 1450, 1500, 1550, 1600, 1650, 1700,

[0083] 1750, 1800, 1850, 1900, 1950, 2000, 2050, 2100, 2150, 2200, 2250, 2300, 2350, 2400, 2450,

[0084] 2500, 2550, 2600, 2650, 2700, 2750, 2800, 2850, 2900, 2950, 3000, 3050, 3100, 3150, 3200,

[0085] 3250, 3300, 3350, 3400, 3450, 3500, 3550, 3600, 3650, 3700, 3750, 3800, 3850, 3900, 3950,

[0086] 4000, 4050, 4100, 4150, 4200, 4250, 4300, 4350, 4400, 4450, 4500, 4550, 4600, 4650, 4700,

[0087] 4750, 4800, 4850, 4900, 4950, 5000, 5050, 5100, 5150, 5200, 5250, 5300, 5350, 5400, 5450,

[0088] 5500, 5550, 5600, 5650, 5700, 5750, 5800, 5850, 5900, 5950, 6000, 6050, 6100, 6150, 6200,

[0089] 6250, 6300, 6350, 6400, 6450, 6500, 6550, 6600, 6650, 6700, 6750, 6800, 6850, 6900, 6950,

[0090] 7000, 7050, 7100, 7150, 7200, 7250, 7300, 7350, 7400, 7450, 7500, 7550, 7600, 7650, 7700, 7750, 7800, 7850, 7900, 7950, 8000, 8050, 8100, 8150, 8200, 8250, 8300, 8350, 8400, 8450, 8500, 8550, 8600, 8650, 8700, 8750, 8800, 8850, 8900, 8950, 9000, 9050, 9100, 9150, 9200, 9250, 9300, 9350, 9400, 9450, 9500, 9550, 9600, 9650, 9700, 9750, 9800, 9850, 9900, 9950, 10000, 10050, 10100, 10150, 10200, 10250, 10300, 10350, 10400, 10450, 10500, 10550, 10600, 10650, 10700, 10750, 10800, 10850, 10900, 10950, 11000, 11050, 11100, 11150, 11200, 11250, 11300, 11350, 11400, 11450, 11500, 11550, 11600, 11650, 11700, 11750, 11800, 11850, 11900, 11950, 12000, 12500, 13000, 13500, 14000, 14500, 15000, 15500, 16000, 16500, 17000, 17500, 18000, 18500, 19000, 19500, 20000, 20500, 21000, 21500, 22000, 22500, 23000, 23500, 24000, 24500, 25000, 25500, 26000, 26500, 27000, 27500, 28000, 28500, 29000, 29500, 30000, 30500, 31000, 31500, 32000, 32500, 33000, 33500, 34000, 34500, 35000, 35500, 36000, 36500, 37000, 37500, 38000, 38500, 39000, 39500, 40000, 40500, 41000, 41500, 42000, 42500, 43000, 43500, or 44000 U / mL, or any range or value derivable therein. In some aspects, the in vitro transcription reaction system includes a T7 RNA polymerase at either 8U / uL, 10U / uL, 13U / uL, or 15U / uL. In some aspects, the in vitro transcription reaction system includes an RNA polymerase, e.g., a T7 RNA polymerase, at a final concentration of 7000 U / mL. In some aspects, the in vitro transcription reaction system includes an RNA polymerase, e.g., a T7 RNA polymerase, at a final concentration of 8000 U / mL. In some aspects, the in vitro transcription reaction system includes an RNA polymerase, e.g., a T7 RNA polymerase, at a final concentration of 14000 U / mL. In some aspects, the in vitro transcription reaction system includes an RNA polymerase, e.g., a T7 RNA polymerase, at a final concentration of 17000 U / mL. In some aspects, the in vitro transcription reaction system includes an RNA polymerase, e.g., a T7 RNA polymerase, at a final concentration of 25000 U / mL. In some aspects, the in vitro transcription reaction system includes an RNA polymerase, e.g., a T7 RNA polymerase, at a final concentration of 40000 U / mL.

[0091] In some aspects, the in vitro transcription reaction system includes the DNA template at a final concentration of, e.g., at least, at most, or about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16,

[0092] 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 ,

[0093] 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59, 60, 61 , 62, 63, 64, 65, 66,

[0094] 67, 68, 69, 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 ,

[0095] 92, 93, 94, 95, 96, 97, 98, 99, 100, 101 , 102, 103, 104, 105, 106, 107, 108, 109, 110, 111 , 112,

[0096] 113, 114, 115, 116, 117, 118, 119, 120, 121 , 122, 123, 124, 125, 126, 127, 128, 129, 130, 131 ,

[0097] 132, 133, 134, 135, 136, 137, 138, 139, 140, 141 , 142, 143, 144, 145, 146, 147, 148, 149, 150,

[0098] 151 , 152, 153, 154, 155, 156, 157, 158, 159, or 160 nM, or any range or value derivable therein.

[0099] In some aspects, the in vitro transcription reaction system includes the DNA template at a final concentration of 40 nM. In some aspects, the in vitro transcription reaction system includes the DNA template at a final concentration of 144 nM. In some aspects, the in vitro transcription reaction system includes the DNA template at a final concentration of 5-24 nM DNA, e.g., at least, at most, or about 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, or 24 nM, or any range or value derivable therein. In some aspects, the in vitro transcription reaction system includes the DNA template at a final concentration of 36 to 144 nM DNA, e.g., at least, at most, or about 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59, 60, 61 , 62, 63, 64, 65, 66, 67, 68, 69, 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99, 100, 101 , 102, 103, 104, 105, 106, 107, 108, 109, 110, 111 , 112, 113, 114, 115, 116, 117, 118, 119, 120, 121 , 122, 123, 124, 125, 126, 127, 128, 129, 130, 131 , 132, 133, 134, 135, 136, 137, 138, 139, 140, 141 , 142, 143, or 144 nM, or any range or valuable derivable therein. In some aspects, the in vitro transcription reaction system includes the DNA template at a final concentration of, e.g., at least, at most, or about 0.01 , 0.02, 0.025, 0.03, 0.04, 0.05, 0.06, 0.07, 0.075, 0.08, 0.09, 0.1 , 0.11 , 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.20, 0.21 , 0.22, 0.23, 0.24, 0.25, 0.26, 0.27, 0.28, 0.29, 0.30, 0.31 , 0.32, 0.33, 0.34, 0.35, 0.36, 0.37, 0.38, 0.39, 0.40, 0.41 , 0.42, 0.43, 0.44, 0.45, 0.46, 0.47, 0.48, 0.49, or 0.50 mg / mL, or any range or value derivable therein. In some aspects, the in vitro transcription reaction system includes the DNA template at a final concentration of 0.025 mg / mL. In some aspects, the in vitro transcription reaction system includes the DNA template at a final concentration of 0.05 mg / mL. In some aspects, the in vitro transcription reaction system includes the DNA template at a final concentration of 0.075 mg / mL. In some aspects, the in vitro transcription reaction system includes the DNA template at a final concentration of 0.1 mg / mL.

[0100] In some aspects, the in vitro transcription reaction system includes each nucleoside triphosphate (NTP) at a final concentration of, e.g., at least, at most, or about 0.4, 0.8, 1 .0, 1 .25, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, or 26 mM, or any range or value derivable therein. In some aspects, the in vitro transcription system includes each nucleoside triphosphate (NTP) at a starting concentration of, e.g., at least, at most, or about 0.4, 0.8, 1.0, 1.25, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, or 26 mM. In some aspects, the in vitro transcription reaction system includes the nucleoside triphosphates (NTPs) at a final concentration of about 8 mM each. In some aspects, the in vitro transcription reaction system includes the nucleoside triphosphate ATP at a final concentration of, e.g., at least, at most, or about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, or 26 mM, or any range or value derivable therein. In some aspects, the in vitro transcription reaction system includes the nucleoside triphosphate CTP at a final concentration of, e.g., at least, at most, or about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, or 26 mM, or any range or value derivable therein. In some aspects, the in vitro transcription reaction system includes the nucleoside triphosphate GTP at a final concentration of, e.g., at least, at most, or about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, or 26 mM, or any range or value derivable therein. In some aspects, the in vitro transcription reaction system includes the nucleoside triphosphate UTP or modified UTP at a final concentration of, e.g., at least, at most, or about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, or 26 mM, or any range or value derivable therein. In certain aspects the concentration of NTPs in the reaction is 0.4mM, 0.8mM, 1 mM, 1.25mM, 3.mM, 5mM, 6mM, 7mM, 7.5mM, 8mM, 8.5mM, 9mM, 9.5mM, or 10mM.

[0101] In some aspects, the in vitro transcription reaction system includes a 5’ cap analog. Exemplary 5’ cap analogs include, but are not limited to, m7G(5')ppp(5')m7G, 3'-O-Me-m7G(5')ppp(5')G, m7G(5')ppp(5')G, G(5')ppp(5')G, m7G(5')ppp(5')A, G(5')ppp(5')A, m7G(5’)ppp(5’)(2’OMeA)pG, m7(3'OMeG)(5')ppp(5')(2'OMeA)pG, m7G(5')ppp(5')(2'OMeA)pU, or m7(3’OMeG)(5’)ppp(5’)m6(2’OMeA)pG. In some aspects, the in vitro transcription reaction system includes a 5’ cap analog at a final concentration of, e.g., at least, at most, or about 0.4, 0.8, 1.0, 1.25, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, or 26 mM, or any range or value derivable therein. In some aspects, the in vitro transcription system includes a 5’ cap analog at a starting concentration of, e.g., at least, at most, or about 0.4, 0.8, 1.0, 1.25, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, or 26 mM. In some aspects, the in vitro transcription reaction system includes a 5’ cap analog at a final concentration of about 4 mM. In certain aspects the concentration of 5’ cap analog in the reaction is 0.4mM, 0.8mM, 1 mM, 1.25mM, 3.mM, 5mM, 6mM, 7mM, 7.5mM, 8mM, 8.5mM, 9mM, 9.5mM, or 10mM.

[0102] In some aspects, the IVT reaction system includes magnesium ion, for example, as a magnesium salt, such as any one of magnesium sulfate, magnesium chloride and magnesium acetate. In some aspects, the in vitro transcription reaction system includes the magnesium at a final concentration of, e.g., at least, at most, or about 12, 13, 14, 15, 16, 16.5, 17, 18, 19, 20, 21 , 22,

[0103] 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47,

[0104] 48, 49, 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59, 60, 61 , 62, 63, 64, 65, 66, 67, 68, 69, 70, 71 , 72,

[0105] 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97,

[0106] 98, 99, 100, 101 , 102, 103, 104, 105, 106, 107, 108, 109, 110, 111 , 112, 113, 114, 115, 116, 117, 118, 119, 120, 121 , 122, 123, 124, 125, 126, 127, 128, 129, 130, 131 , 132, 133, 134, 135, 136,

[0107] 137, 138, 139, 140, 141 , 142, 143, 144, 145, 146, 147, 148, 149, 150, 151 , 152, 153, 154, 155,

[0108] 156, 157, 158, 159, 160, 161 , 162, 163, 164, 165, 166, 167, 168, 169, 170, 171 , 172, 173, 174,

[0109] 175, 176, 177, 178, 179, 180, 181 , 182, 183, 184, 185, 186, 187, 188, 189, 190, 191 , 192, 193,

[0110] 194, 195, 196, 197, 198, 199, 200, 201 , 202, 203, 204, 205, 206, 207, 208, 209, 210, 211 , 212, 213, 214, 215, 216, 217, 218, 219, or 220 mM, or any range or value derivable therein. In some aspects, the in vitro transcription reaction system includes magnesium acetate at 30 mM. In some aspects, the in vitro transcription reaction system includes magnesium acetate at 40 mM. In some aspects, the in vitro transcription reaction system includes magnesium acetate at 16.5 mM. In some aspects, the in vitro transcription reaction system includes magnesium acetate at 33 mM. In some aspects, the in vitro transcription reaction system includes magnesium acetate at 36 mM. In some aspects, the in vitro transcription reaction system includes magnesium acetate at 50 mM. In some aspects, the in vitro transcription reaction system includes magnesium acetate at 110 mM. In some aspects, the Mg:NTP ratio can be maintained at a ratio of, e.g., at least, at most, or about, 0, 0.8, 0.9, 1.0, 1.1 , 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1 , or 2.2 mM Mg / mM NTP, or any range or value derivable therein. In some aspects, one or more reaction components are added during in vitro transcription by occasional bolus feeds, semi-continuous feeds, or continuous feeds. Bolus feeds can be delivered at intervals of, e.g., at least, at most, or about, 0, 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, or 35 minutes, or any range or value derivable therein. These components can include, but are not limited to, one or more NTPs and a cation such as magnesium. These components can be combined into a single feed, or they can be delivered separately in the form of multiple feeds. In some aspects, a continuous feed of at least 1 NTP can be delivered at flow rates of, e.g, at least, at most, or about 0, 0.5, 1 , 1.5, 2, 2.5, 3, 3.5, or 4 mL / L / min. In some aspects, a continuous feed of a cation such as magnesium can be delivered at concentrations of, e.g., at least, at most, or about 0, 0.01 , 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.11 , 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5, 0.55, 0.6, 0.65, 0.7, 0.75, 0.8, 0.85, 0.9, 0.95, or 1 .0 mM / min, or any range or value derivable therein

[0111] In some aspects, the in vitro transcription (IVT) reaction system includes a buffer. Exemplary buffers for the in vitro transcription reaction system may include Tris and / or HEPES. In some aspects, the in vitro transcription reaction system includes the buffer at a pH of, e.g., at least, at most, or about 7, 7.1 , 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8, 8.1 , 8.2, 8.3, 8.4, or 8.5, or any range or value derivable therein. In some aspects, the buffer is Tris-HCI, pH 8.0. In some aspects, the in vitro transcription reaction system includes 40 mM Tris HCI, pH 8.0. In some aspects, the in vitro transcription reaction system includes 40 mM Tris HCI, pH 7.0. In some aspects, the in vitro transcription reaction system includes PIPES (piperazine-N,N'-bis(2-ethanesulfonic acid) pH 7.0, HEPPSO (2-hydroxy-3-[4-(2-hydroxyethyl)piperazin-1-yl]propane-1 -sulfonic acid), or MES (2-(N- morpholino)ethanesulfonic acid). Alternative buffers for the IVT reaction system include 40 mM Tris pH 7.5, 80 mM HEPES. In some aspects, the in vitro transcription reaction system does not include PIPES. In some aspects, the in vitro transcription reaction system includes PIPES and Tris.

[0112] In some aspects, an RNase inhibitor is included in the in vitro transcription reaction system. The RNase inhibitor may reduce RNase-induced degradation during the transcription reaction. For example, murine RNase inhibitor may be utilized at a final concentration of 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, or 1200 U / mL. In some aspects, the in vitro transcription reaction system comprises RNase inhibitor at a final concentration of 100 U / mL. In some aspects, the in vitro transcription reaction system comprises RNase inhibitor at a final concentration of 1000 U / mL.

[0113] In some aspects, a pyrophosphatase is included in the in vitro transcription reaction system. The pyrophosphatase may cleave the inorganic pyrophosphate generated following each nucleotide incorporation into two units of inorganic phosphate, which may reduce the likelihood of magnesium co-precipitating with pyrophosphate to form magnesium pyrophosphate. Pyrophosphatase in certain aspects may be diluted in pyrophosphatase buffer and present in the reaction at concentrations of 0.01 mU / uL, 0.02mU / uL, 0.05mU / uL, 0.08mU / uL, 0.1 mll / uL, 0.2mll / uL, 0.8mll / uL, or 2mll / uL. In some aspects, the in vitro transcription reaction system includes an inorganic pyrophosphatase at a final concentration of 0.25 U / mL. In some aspects, the in vitro transcription reaction system includes an inorganic pyrophosphatase at a final concentration of 0.5 U / mL. In some aspects, the in vitro transcription reaction system includes an inorganic pyrophosphatase at a final concentration of 1 U / mL. In some aspects, the in vitro transcription reaction system includes an inorganic pyrophosphatase at a final concentration of 2 U / mL. In some aspects, the in vitro transcription reaction system includes an inorganic pyrophosphatase at a final concentration of 3 U / mL. In some aspects, the in vitro transcription reaction system includes an inorganic pyrophosphatase at a final concentration of 6 U / mL.

[0114] In some aspects, the in vitro transcription reaction system includes a polyamine. Exemplary polyamines include spermine, putrescene, and spermidine. In some aspects, 0.2 mM spermidine is included. In some aspects, 1 mM spermidine is included. In some aspects, 2.0 mM spermidine is included. In some aspects, 2.15 mM spermidine is included. In some aspects 0.53 mM spermidine is included. In some aspects, 10 mM spermidine is included.

[0115] In some aspects, the IVT reaction system includes a reducing reagent, such as, for example, DTT (dithiothreitol), e.g., at least, at most, or about 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, or 40 mM, or any range or value derivable therein. In some aspects, the reducing agent is selected from the group consisting of dithiothreitol (DTT), dithioerythritol (DTE), Tris(2- carboxyethyl)phosphine (TCEP) and beta-mercaptoethanol. In some aspects, the IVT reaction system includes 1 mM DTT. In some aspects, the IVT reaction system includes 5 mM DTT. In some aspects, the IVT reaction system includes 10 mM DTT. In some aspects, the IVT reaction system includes 20 mM DTT. In some aspects, the IVT reaction system includes 25 mM DTT.

[0116] In some aspects, the IVT reaction system includes a monovalent salt. In some aspects, the monovalent salt is selected from the group consisting of KCI, NaCI, RbCI, CsCI, NaCIOzi, NaF, NaBr, and NH4CL In some aspects, the monovalent salt is present in the IVT reaction system in an amount between about 10 mM and about 150 mM. In some aspects, the IVT reaction system includes about 10, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, or 150 mM potassium chloride (KCI). In some aspects, the IVT reaction system includes about 10, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, or 150 mM sodium chloride (NaCI). In some aspects, the IVT reaction system includes about 10, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, or 150 mM cesium chloride (CsCI). In some aspects, the IVT reaction system includes about 10, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, or 150 mM rubidium chloride (RbCI). In some aspects, the IVT reaction system includes about 10, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, or 150 mM ammonium chloride (NH4CI).

[0117] In some aspects, the IVT reaction system includes dimethylsulfoxide (DMSO). In some aspects, the DMSO is present in the IVT reaction system in an amount between about 4% and about 15%. In some aspects, the IVT reaction system includes about 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, or 15% DMSO. In some aspects, the IVT reaction system includes 5% DMSO. In some aspects, the IVT reaction system includes 10% DMSO.

[0118] In some aspects, the IVT reaction system includes dihydrolevoglucosenone (CYRENE™). In some aspects, the dihydrolevoglucosenone is present in the IVT reaction system in an amount between about 4% and about 15%. In some aspects, the IVT reaction system includes about 0.1 , 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.25, 1.5, 1.75, 2.0, 2.5, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, or 15% dihydrolevoglucosenone. In some aspects, the IVT reaction system includes 5% dihydrolevoglucosenone. In some aspects, the IVT reaction system includes 10% dihydrolevoglucosenone.

[0119] In some aspects, the in vitro transcription reaction proceeds, for example, at about 37°C for about 4 hours or about 240 minutes, e.g., at least, at most, or about 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 225, 230, 235, or 240 minutes, or any range or value derivable therein. In some preferred aspects, the in vitro transcription reaction proceeds, for example, at less than 50°C for less than 4 hours, such as for example, at least, at most, or about 50°C, 49°C, 48°C, 47°C, 46°C, 45°C, 44°C, 43°C, 42°C, 41 °C, 40°C, 39°C, 38°C, 37°C, 36°C, 35°C, 34°C, 33°C, 32°C, 31 °C, 30°C, 29°C, 28°C, 27°C, 26°C, 25°C, 24°C, 23°C, 22°C, 21 °C, or about 20°C, for at least, at most, or about 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 225, 230, 235, or 240 minutes, or any range or value derivable therein. In some aspects, the in vitro transcription reaction proceeds at about 36°C for about 120 minutes. In some aspects, the in vitro transcription reaction proceeds at about 36°C for about 150 minutes. In some aspects, the in vitro transcription reaction proceeds at about 37°C for about 120 minutes. In some aspects, the in vitro transcription reaction proceeds at about 37°C for about 150 minutes.

[0120] In some aspects, the in vitro transcription reaction proceeds at about 37°C, at greater than 120 minutes and less than 360 minutes, preferably greater than 120 minutes and less than 300 minutes, more preferably greater than 120 minutes and less than 260 minutes. In some preferred aspects, the in vitro transcription reaction proceeds at about 37°C for about 150 minutes.

[0121] In some aspects, the in vitro transcription reaction proceeds, for example, at about 37°C for about 12 days, e.g., at least, at most, or about 0.1 , 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , or 12 days, or any range or value derivable therein. In some aspects, the in vitro transcription reaction proceeds, for example, at about 37°C for about 365 days, e.g., at least, at most, or about 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 220, 240, 260, 280, 300, 320, 340, 360, or 365 days or any range or value derivable therein. In some aspects, the in vitro transcription reaction proceeds, for example, at about 37°C for any number of days less than 0.1 days. In some aspects, the in vitro transcription reaction proceeds, for example, at about 37°C for any number of days greater than 365 days. In some aspects, the in vitro transcription reaction proceeds, for example, at less than 50°C for less than 365 days, such as for example, at least, at most, or about 50°C, 49°C, 48°C, 47°C, 46°C, 45°C, 44°C, 43°C, 42°C, 41 °C, 40°C, 39°C, 38°C, 37°C, 36°C, 35°C, 34°C, 33°C, 32°C, 31 °C, 30°C, 29°C, 28°C, 27°C, 26°C, 25°C, 24°C, 23°C, 22°C, 21 °C, or about 20°C, for at least, at most, or about 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 220, 240, 260, 280, 300, 320, 340, 360, or 365 days or any range or value derivable therein.

[0122] In some aspects, the IVT reactions described herein result in high yields of highly pure RNA. In some aspects, yields per in vitro transcription reaction may be at least 0.3 mg of RNA per mL starting volume of IVT reaction to about 20 mg of RNA per mL starting volume of IVT reaction. For example, in some aspects, the total yield of RNA molecule may be at least, at most, or about 0.3 mg RNA / mL, 0.4 mg RNA / mL, 0.5 mg RNA / mL, 0.6 mg RNA / mL, 0.7 mg RNA / mL, 0.8 mg RNA / mL, 0.9 mg RNA / mL, 1.0 mg RNA / mL, 2 mg RNA / mL, 3 mg RNA / mL, 4 mg RNA / mL, preferably at least 5 mg RNA / mL, 6 mg RNA / mL, 7 mg RNA / mL, 8 mg RNA / mL, 9 mg RNA / mL, 10 mg RNA / mL, 11 mg RNA / mL, 12 mg RNA / mL, 13 mg RNA / mL, 14 mg RNA / mL, 15 mg RNA / mL, 16 mg RNA / mL, 17 mg RNA / mL, 18 mg RNA / mL, 19 mg RNA / mL, or 20 mg RNA / mL starting volume of IVT reaction, or any range or value derivable therein. In preferred aspects, the total yield per in vitro transcription reaction of RNA molecule produced having at least 90% of the intended full length transcript may be at least 2 mg RNA / mL, 3 mg RNA / mL, 4 mg RNA / mL, preferably at least 5 mg RNA / mL, 6 mg RNA / mL, 7 mg RNA / mL, 8 mg RNA / mL, 9 mg RNA / mL, 10 mg RNA / mL, 11 mg RNA / mL, 12 mg RNA / mL, 13 mg RNA / mL, 14 mg RNA / mL, 15 mg RNA / mL, 16 mg RNA / mL, 17 mg RNA / mL, 18 mg RNA / mL, 19 mg RNA / mL, or 20 mg RNA / mL starting volume of IVT reaction. In some aspects, the total yield per in vitro transcription reaction of RNA molecule produced having at least 90% of the intended full length transcript is at least 17 mg RNA / mL starting volume of IVT reaction.

[0123] In some aspects, the IVT reactions described herein result in high productivity of RNA. Productivity in the case of in vitro transcription refers to the amount of RNA produced per volume of reactor per unit time. In some aspects, productivity of the IVT reaction may be at least 0.1 mg / mL / h to about 1000 mg / mL / h, where volume refers to the reaction volume. For example, in some aspects, the productivity of the IVT reaction may be at least, at most, or about 0.1 mg / mL / h, 0.2 mg / mL / h, 0.3 mg / mL / h, 0.4 mg / mL / h, 0.5 mg / mL / h, 0.6 mg / mL / h, 0.7 mg / mL / h, 0.8 mg / mL / h, 0.9 mg / mL / h, 1.0 mg / mL / h, 2 mg / mL / h, 3 mg / mL / h, 4 mg / mL / h, 5 mg / mL / h, 6 mg / mL / h, 7 mg / mL / h, 8 mg / mL / h, 9 mg / mL / h, 10 mg / mL / h, 11 mg / mL / h, 12 mg / mL / h, 13 mg / mL / h, 14 mg / mL / h, 15 mg / mL / h, 16 mg / mL / h, 17 mg / mL / h, 18 mg / mL / h, 19 mg / mL / h, 20 mg / mL / h, 25 mg / mL / h, 30 mg / mL / h, 35 mg / mL / h, 40 mg / mL / h, 45 mg / mL / h, 50 mg / mL / h, 55 mg / mL / h, 60 mg / mL / h, 65 mg / mL / h, 70 mg / mL / h, 75 mg / mL / h, 80 mg / mL / h, 85 mg / mL / h, 90 mg / mL / h, 95 mg / mL / h, 100 mg / mL / h, 150 mg / mL / h, 200 mg / mL / h, 250 mg / mL / h, 300 mg / mL / h, 350 mg / mL / h, 400 mg / mL / h, 450 mg / mL / h, 500 mg / mL / h, 600 mg / mL / h, 700 mg / mL / h, 800 mg / mL / h, 900 mg / mL / h, or 1000 mg / mL / h, or any range or value derivable therein, where volume refers to the reaction volume. In some aspects, the productivity of the IVT reaction may be any value less than 0.1 mg / mL / h, where volume refers to the reaction volume. In some aspects, the productivity of the IVT reaction may be any value greater than 1000mg / mL / h, where volume refers to the reaction volume. In some preferred aspects, the productivity of the IVT reaction producing RNA having at least 90% of the intended full length transcript is at least 30 mg / mL / h, where volume refers to the reaction volume.

[0124] In some aspects, the IVT reactions described herein are carried out in a reaction volume under specified conditions. In some aspects, the reaction volume ranges from at least 0.0001 , 0.001 , 0.01 , 0.1 , 1 , 10, 100, or 200 liters or more, or any volume in between. In some aspects, the volume of the IVT reaction is at least 10L, 30L or 50L. In some aspects, the volume of the IVT reaction is at least 0.01 L. In some aspects, the volume of the IVT reaction is at least 0.08L. In some aspects, the volume of the IVT reaction is at least 0.1 L. In some aspects, the volume of the IVT reaction is at least 100L.

[0125] In some aspects, following an IVT reaction using a DNA template and an RNA polymerase as described here, a first composition that includes an uncapped RNA molecule is produced. In some aspects, the RNA molecule includes the coding sequence for a gene of interest and a poly(A) tail. As used herein, the RNA molecule includes an mRNA. The RNA molecule may include modifications, such as, modified nucleotides.

[0126] As used herein, an “RNA molecule” produced by in vitro transcription may be referred to as an “RNA transcript” or an “in vitro transcribed RNA.” An “RNA molecule,” an “RNA transcript,” or “in vitro transcribed RNA” may encompass any one of modified mRNA “modRNA,” unmodified mRNA, self-amplifying RNA (saRNA), and guide RNA (gRNA).

[0127] In some aspects, the methods of reducing dsRNA presence in RNA molecules synthesized by contacting a DNA sample with an in vitro transcription reaction system described herein produce a first composition having an uncapped RNA molecule. In some aspects, at least 30% of the RNA molecules in the first composition includes uncapped RNA molecules. In some aspects, the first composition includes at least, at most, or about 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 71 %, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or any range or value derivable therein, uncapped RNA molecules.

[0128] RNA Molecule

[0129] The RNA molecule produced by the methods described herein may be a non-coding and / or a coding RNA. A non-coding RNA (ncRNA) molecule includes a functional RNA molecule that is not translated into a peptide or polypeptide. Non-coding RNA molecules may include highly abundant and functionally important RNAs such as transfer RNA (tRNA) and ribosomal RNA (rRNA), as well as RNAs such as snoRNAs, microRNAs, siRNAs, snRNAs, guideRNAs, circularRNAs, exRNAs, piRNAs, and long ncRNAs. In a preferred aspect, the RNA molecule is an mRNA molecule that includes a modified nucleotide (herein referred to as a “modified RNA molecule” or “modified mRNA molecule” or “modRNA”). In some preferred aspects, the RNA molecule is a self-amplifying RNA molecule.

[0130] Coding RNA includes a functional RNA molecule that may be translated into a peptide or polypeptide. In some aspects, the coding RNA molecule includes at least one open reading frame coding for at least one peptide or polypeptide. The coding RNA molecule may include one (monocistronic), two (bicistronic) or more (multicistronic) open reading frames (ORFs). The coding RNA molecule may be a messenger RNA (mRNA) molecule, viral RNA molecule or selfamplifying RNA molecule (saRNA, also referred to as a replicon). Preferably, the RNA molecule is an mRNA molecule.

[0131] The RNA molecule may encode more than one protein, e.g., two, three, four, five, ten or more polypeptides. Alternatively, or in addition, one RNA molecule may also encode more than one antigen, e.g., a bicistronic, or tricistronic RNA molecule that encodes different or identical antigens.

[0132] The sequence of the RNA molecule may be codon optimized or de-optimized for expression in a desired host, such as a human cell.

[0133] The sequence of the RNA molecule may be modified if desired, for example to increase the efficacy of expression or replication of the RNA, or to provide additional stability or resistance to degradation. For example, the RNA sequence may be modified with respect to its codon usage, for example, to increase translation efficacy and half-life of the RNA.

[0134] In some aspects, the RNA molecules may include one or more structural and / or chemical modifications or alterations which impart useful properties to the polynucleotide including, in some aspects, the lack of a substantial induction of the innate immune response of a cell into which the polynucleotide is introduced. As used herein, a “structural” feature or modification is one in which two or more linked nucleotides are inserted, deleted, duplicated, inverted or randomized in an RNA molecule without significant chemical modification to the nucleotides themselves. Because chemical bonds will necessarily be broken and reformed to effect a structural modification, structural modifications are of a chemical nature and hence are chemical modifications. However, structural modifications will result in a different sequence of nucleotides. For example, the polynucleotide “ATCG” may be chemically modified to “AT-5meC-G”. The same polynucleotide may be structurally modified from “ATCG” to “ATCCCG”. Here, the dinucleotide “CC” has been inserted, resulting in a structural modification to the polynucleotide.

[0135] In some aspects, the RNA molecule may include one or more modified nucleotides in addition to any 5' cap structure. Naturally occurring nucleoside modifications are known in the art.

[0136] In some aspects, the RNA molecule produced by the in vitro transcription reactions described herein includes from at least, at most, or about 20 to at least, at most, or about 100,000 nucleotides, or any range or value derivable therein (e.g., from 30 to 50, from 30 to 100, from 30 to 250, from 30 to 500, from 30 to 1 ,000, from 30 to 1 ,500, from 30 to 3,000, from 30 to 5,000, from 30 to 7,000, from 30 to 10,000, from 30 to 25,000, from 30 to 50,000, from 30 to 70,000, from 100 to 250, from 100 to 500, from 100 to 1 ,000, from 100 to 1 ,500, from 100 to 3,000, from 100 to 5,000, from 100 to 7,000, from 100 to 10,000, from 100 to 25,000, from 100 to 50,000, from 100 to 70,000, from 100 to 100,000, from 500 to 1 ,000, from 500 to 1 ,500, from 500 to 2,000, from 500 to 3,000, from 500 to 5,000, from 500 to 7,000, from 500 to 10,000, from 500 to 25,000, from 500 to 50,000, from 500 to 70,000, from 500 to 100,000, from 1 ,000 to 1 ,500, from 1 ,000 to 2,000, from 1 ,000 to 3,000, from 1 ,000 to 5,000, from 1 ,000 to 7,000, from 1 ,000 to 10,000, from 1 ,000 to 25,000, from 1 ,000 to 50,000, from 1 ,000 to 70,000, from 1 ,000 to 100,000, from 1 ,500 to 3,000, from 1 ,500 to 5,000, from 1 ,500 to 7,000, from 1 ,500 to 10,000, from 1 ,500 to 25,000, from 1 ,500 to 50,000, from 1 ,500 to 70,000, from 1 ,500 to 100,000, from 2,000 to 3,000, from 2,000 to 5,000, from 2,000 to 7,000, from 2,000 to 10,000, from 2,000 to 25,000, from 2,000 to 50,000, from 2,000 to 70,000, and from 2,000 to 100,000). In preferred aspects, the RNA molecule includes at least 100 nucleotides. For example, in some aspects, the RNA has a length between 100 and 15,000 nucleotides; between 7,000 and 16,000 nucleotides; between 8,000 and 15,000 nucleotides; between 9,000 and 12,500 nucleotides; between 11 ,000 and 15,000 nucleotides; between 13,000 and 16,000 nucleotides. In some aspects, the RNA has a length between about 1 ,600 nucleotides and 9,600 nucleotides. In preferred aspects, the RNA molecule that is the polynucleotide product of the in vitro transcription reaction described herein includes a gene of interest and a poly(A) tail. In some aspects, the RNA molecule further includes a 5' UTR and a 3' UTR.

[0137] In some aspects, the modified mRNA molecule encodes a single polypeptide antigen or, optionally, two or more of polypeptide antigens linked together in a way that each of the sequences retains its identity (e.g., linked in series) when expressed as an amino acid sequence. The polypeptide(s) generated from the modified mRNA may then be produced as a fusion polypeptide or engineered in such a manner to result in separate polypeptide or peptide sequences. In preferred aspects, the modified mRNA molecule encodes a single polypeptide of interest.

[0138] In some preferred aspects, the RNA molecule is a saRNA. “Self-amplifying RNA,” “self-replicating RNA,” and “replicon” refer to RNA with the ability to replicate itself. Self-amplifying RNA molecules may be produced by using replication elements derived from, e.g., alphaviruses, and substituting the structural viral polypeptides with a nucleotide sequence encoding a polypeptide of interest. A self-amplifying RNA molecule is typically a positive-strand molecule that may be directly translated after delivery to a cell, and this translation provides an RNA-dependent RNA polymerase which then produces both antisense and sense transcripts from the delivered RNA. The delivered RNA leads to the production of multiple daughter RNAs. These daughter RNAs, as well as collinear subgenomic transcripts, may be translated themselves to provide in situ expression of an encoded gene of interest, e.g., a viral antigen, or may be transcribed to provide further transcripts with the same sense as the delivered RNA which are translated to provide in situ expression of the antigen. The overall result of this sequence of transcriptions is an amplification in the number of the introduced saRNAs and so the encoded gene of interest, e.g., a viral antigen, becomes a major polypeptide product of the cells.

[0139] In some aspects, the self-amplifying RNA includes at least one or more genes selected from any one of viral replicases, viral proteases, viral helicases and other nonstructural viral proteins. In some aspects, the self-amplifying RNA may also include 5'- and 3 -end tractive replication sequences, and optionally a heterologous sequence that encodes a desired amino acid sequence (e.g., an antigen of interest). A subgenomic promoter that directs expression of the heterologous sequence may be included in the self-amplifying RNA. Optionally, the heterologous sequence (e.g., an antigen of interest) may be fused in frame to other coding regions in the self-amplifying RNA and / or may be under the control of an internal ribosome entry site (IRES).

[0140] In preferred aspects, the self-amplifying RNA molecule is not encapsulated in a virus-like particle. Self-amplifying RNA molecules described herein may be designed so that the self-amplifying RNA molecule cannot induce production of infectious viral particles. This may be achieved, for example, by omitting one or more viral genes encoding structural proteins that are necessary to produce viral particles in the self-amplifying RNA. For example, when the self-amplifying RNA molecule is based on an alpha virus, such as Sindbis virus (SINV), Semliki Forest virus and Venezuelan equine encephalitis virus (VEEV), one or more genes encoding viral structural proteins, such as capsid and / or envelope glycoproteins, may be omitted.

[0141] In some aspects, a self-amplifying RNA molecule described herein encodes (i) an RNA- dependent RNA polymerase that may transcribe RNA from the self-amplifying RNA molecule and (ii) a polypeptide of interest, e.g., a viral antigen. In some aspects, the polymerase may be an alphavirus replicase e.g., including alphavirus protein nsP4. In some aspects, the self-amplifying RNA molecules described herein may include one or more modified nucleotides (e.g., pseudouridine, N6-methyladenosine, 5- methylcytidine, 5-methyluridine).

[0142] The saRNA construct may encode at least one non-structural protein (nsP), disposed 5' or 3' of the sequence encoding at least one peptide or polypeptide of interest. Preferably, the sequence encoding at least one nsP is disposed 5' of the sequences encoding the peptide or polypeptide of interest. Thus, preferably the sequence encoding at least one nsP is disposed at the 5' end of the RNA construct. In some aspects, at least one non-structural protein encoded by the RNA construct may be the RNA polymerase nsP4. Preferably, the saRNA construct encodes nsP1 , nsP2, nsP3 and nsP4. As is known in the art, nsP1 is the viral capping enzyme and membrane anchor of the replication complex (RC). nsP2 is an RNA helicase and the protease responsible for the ns polyprotein processing. nsP3 interacts with several host proteins and may modulate protein poly- and mono-ADP-ribosylation. nsP4 is the core viral RNA-dependent RNA polymerase. In some aspects, the polymerase may be an alphavirus replicase, e.g., comprising one or more of alphavirus proteins nsP1 nsP2, nsP3 and nsP4.

[0143] Whereas natural alphavirus genomes encode structural virion proteins in addition to the non- structural replicase polypeptide, in some aspects, the self-amplifying RNA molecules do not encode alphavirus structural proteins. In some aspects, the self-amplifying RNA may lead to the production of genomic RNA copies of itself in a cell, but not to the production of RNA that includes virions. Without being bound by theory or mechanism, the inability to produce these virions means that, unlike a wild-type alphavirus, the self-amplifying RNA molecule cannot perpetuate itself in infectious form. The alphavirus structural proteins which are necessary for perpetuation in wildtype viruses are absent from self-amplifying RNAs of the present disclosure and their place is taken by gene(s) encoding the protein of interest, such that the subgenomic transcript encodes the immunogen rather than the structural alphavirus virion proteins.

[0144] In some aspects, the self-amplifying RNA molecule may have two open reading frames. The first (5') open reading frame encodes a replicase; the second (3') open reading frame encodes a polypeptide comprising an antigen of interest. In some aspects the RNA may have additional (e.g., downstream) open reading frames, e.g., to encode further antigens or to encode accessory polypeptides.

[0145] Optionally, self-amplifying RNA molecules described herein may also be designed to induce production of infectious viral particles that are attenuated or virulent, or to produce viral particles that are capable of a single round of subsequent infection.

[0146] When delivered to a vertebrate cell, a self-amplifying RNA molecule may lead to the production of multiple daughter RNAs by transcription from itself (or from an antisense copy of itself). The self-amplifying RNA may be directly translated after delivery to a cell, and this translation provides an RNA-dependent RNA polymerase which then produces transcripts from the delivered RNA, thereby producing multiple daughter RNAs. These RNA molecules are antisense relative to the delivered RNA and may be translated themselves to provide in situ expression of a gene product, or may be transcribed to provide further transcripts with the same sense as the delivered RNA which are translated to provide in situ expression of the gene product.

[0147] In some aspects, the saRNA molecule is alphavirus-based. Alphaviruses include a set of genetically, structurally, and serologically related arthropod-borne viruses of the Togaviridae family. Exemplary viruses and virus subtypes within the alphavirus genus include Sindbis virus, Semliki Forest virus, Ross River virus, and Venezuelan equine encephalitis virus. As such, the self-amplifying RNA described herein may incorporate an RNA replicase derived from any one of Semliki Forest virus (SFV), Sindbis virus (SINV), Venezuelan equine encephalitis virus (VEEV), Ross-River virus (RRV), or other viruses belonging to the alphavirus family. In some aspects, the self-amplifying RNA described herein may incorporate sequences derived from a mutant or wildtype virus sequence, e.g., the attenuated TC83 mutant of VEEV has been used in saRNAs.

[0148] Alphavirus-based saRNAs are (+)-stranded saRNAs that may be translated after delivery to a cell, which leads to translation of a replicase (or replicase- transcriptase). The replicase is translated as a polyprotein which auto-cleaves to provide a replication complex which creates genomic (-)-strand copies of the (+)-strand delivered RNA. These (-)-strand transcripts may themselves be transcribed to give further copies of the (+)-stranded parent RNA and also to give a subgenomic transcript which encodes the desired gene product. T ranslation of the subgenomic transcript thus leads to in situ expression of the desired gene product by the infected cell. Suitable alphavirus saRNAs may use a replicase from a Sindbis virus, a Semliki Forest virus, an eastern equine encephalitis virus, a Venezuelan equine encephalitis virus, or mutant variants thereof.

[0149] In some aspects, the self-amplifying RNA molecule is derived from or based on a virus other than an alphavirus, preferably, a positive-stranded RNA virus, and more preferably a picornavirus, flavivirus, rubivirus, pestivirus, hepacivirus, calicivirus, or coronavirus. Suitable wild-type alphavirus sequences are well-known and are available from sequence depositories, such as the American Type Culture Collection, Rockville, Md. Representative examples of suitable alphaviruses include Aura (ATCC VR-368), Bebaru virus (ATCC VR-600, ATCC VR-1240), Cabassou (ATCC VR-922), Chikungunya virus (ATCC VR-64, ATCC VR-1241 ), Eastern equine encephalomyelitis virus (ATCC VR-65, ATCC VR-1242), Fort Morgan (ATCC VR-924), Getah virus (ATCC VR-369, ATCC VR-1243), Kyzylagach (ATCC VR-927), Mayaro (ATCC VR- 66), Mayaro virus (ATCC VR-1277), Middleburg (ATCC VR-370), Mucambo virus (ATCC VR-580, ATCC VR-1244), Ndumu (ATCC VR-371 ), Pixuna virus (ATCC VR- 372, ATCC VR-1245), Ross River virus (ATCC VR-373, ATCC VR-1246), Semliki Forest (ATCC VR-67, ATCC VR-1247), Sindbis virus (ATCC VR-68, ATCC VR-1248), Tonate (ATCC VR-925), Triniti (ATCC VR-469), Una (ATCC VR-374), Venezuelan equine encephalomyelitis (ATCC VR-69, ATCC VR-923, ATCC VR-1250 ATCC VR- 1249, ATCC VR-532), Western equine encephalomyelitis (ATCC VR- 70, ATCC VR- 1251 , ATCC VR-622, ATCC VR-1252), Whataroa (ATCC VR-926), and Y-62-33 (ATCC VR-375).

[0150] In some aspects, the self-amplifying RNA molecules described herein are larger than other types of RNA (e.g., mRNA). Typically, the self-amplifying RNA molecules described herein include at least about 4 kb. For example, the self-amplifying RNA may include at least, at most, or about 5 kb, 6 kb, 7 kb, 8 kb, 9 kb, 10 kb, 11 kb, or 12 kb or more, or any range or value derivable therein. In certain examples, the self-amplifying RNA is about 4 kb to about 12 kb, about 5 kb to about 12 kb, about 6 kb to about 12 kb, about 7 kb to about 12 kb, about 8 kb to about 12 kb, about 9 kb to about 12 kb, about 10 kb to about 12 kb, about 11 kb to about 12 kb, about 5 kb to about 11 kb, about 5 kb to about 10 kb, about 5 kb to about 9 kb, about 5 kb to about 8 kb, about 5 kb to about 7 kb, about 5 kb to about 6 kb, about 6 kb to about 12 kb, about 6 kb to about 11 kb, about 6 kb to about 10 kb, about 6 kb to about 9 kb, about 6 kb to about 8 kb, about 6 kb to about 7 kb, about 7 kb to about 11 kb, about 7 kb to about 10 kb, about 7 kb to about 9 kb, about 7 kb to about 8 kb, about 8 kb to about 11 kb, about 8 kb to about 10 kb, about 8 kb to about 9 kb, about 9 kb to about 11 kb, about 9 kb to about 10 kb, or about 10 kb to about 11 kb.

[0151] In some aspects, the self-amplifying RNA molecule may encode a single polypeptide antigen or, optionally, two or more of polypeptide antigens linked together in a way that each of the sequences retains its identity (e.g., linked in series) when expressed as an amino acid sequence. The polypeptides generated from the self-amplifying RNA may then be produced as a fusion polypeptide or engineered in such a manner to result in separate polypeptide or peptide sequences.

[0152] In some aspects, the self-amplifying RNA described herein may encode one or more polypeptide antigens that include a range of epitopes. Preferably epitopes capable of eliciting either a helper T-cell response or a cytotoxic T-cell response or both. In some aspects, the RNA molecule has a 3' poly(A) tail, that is, a stretch of consecutive adenosine residues, that may be attached to the 3' end of the RNA. The poly(A) tail may increase the half-life of the RNA molecule. The RNA molecule may further include a poly(A) polymerase recognition sequence (e.g., AAUAAA) near its 3' end. In some aspects, the 3' poly(A) tail has a stretch of at least 10 consecutive adenosine residues and at most 300 consecutive adenosine residues. Preferably, the RNA molecule includes at least 20 consecutive adenosine residues and at most 40 consecutive adenosine residues. In some preferred aspects, the RNA molecule includes about 40 consecutive adenosine residues. In some aspects, the RNA molecule includes about 80 consecutive adenosine residues. Poly(A) tails may play key regulatory roles in enhancing translation efficiency and regulating the efficiency of mRNA quality control and degradation. Short sequences or hyper-polyadenylation may signal for RNA degradation. Exemplary designs include a poly(A) tails of about 40 As, about 80 As. In some aspects, the RNA molecule further includes an endonuclease recognition site sequence immediately downstream of the poly(A) tail sequence.

[0153] In some aspects, the RNA molecule produced by the in vitro transcription reactions described herein is purified, e.g., including filtration that may occur via, e.g., ultrafiltration, diafiltration, or, e.g., tangential flow ultrafiltration / diafiltration.

[0154] Capping of RNA Molecule

[0155] In some aspects, the methods described herein further include capping uncapped RNA molecules by contacting the uncapped RNA molecules with a capping reaction system, which includes any one of guanylyltransferase (e.g., vaccinia capping enzyme or faustovirus capping enzyme), s-adenosyl-L-methionine (SAM), guanosine triphosphate (GTP), and 2'-O- methyltransferase, and any combination thereof, to produce a capped RNA molecule. In some aspects, the 5' end of the RNA is capped with a modified ribonucleotide with the structure m7G(5')ppp(5')N (cap 0 structure) or a derivative thereof, which may be incorporated during RNA synthesis (co-transcriptional capping) or may be performed enzymatically after RNA transcription (post-transcriptional capping). In some aspects, the 5' end of the RNA molecule is capped with a modified ribonucleotide via an enzymatic reaction after RNA transcription. In some aspects, capping is performed after purification, e.g., tangential flow filtration, of the RNA molecule.

[0156] An exemplary enzymatic reaction for capping may include use of Vaccinia Virus Capping Enzyme (VCE) that includes mRNA triphosphatase, guanylyltransferase and guanine-7- methytransferase, which catalyzes the construction of N7-monomethylated cap 0 structures). Cap 0 structure plays an important role in maintaining the stability and translational efficacy of the RNA molecule. The 5' cap of the RNA molecule may be further modified by a 2’-O- Methyltransferase which results in the generation of a cap 1 structure (m7Gppp [m2 ’-O] N), which may further increase translation efficacy.

[0157] In some aspects, the RNA molecule may be enzymatically capped at the 5' end using Vaccinia or Faustovirus guanylyltransferase, guanosine triphosphate and S-adenosyl-L-methionine to yield cap 0 structure. An inverted 7-methylguanosine cap is added via a 5' to 5' triphosphate bridge. Alternatively, use of a 2'-O-methyltransferase with Vaccinia or Faustovirus guanylyltransferase yields the cap 1 structure where in addition to the cap 0 structure, the 2'-OH group is methylated on the first transcribed nucleotide. S-adenosyl-L-methionine (SAM) is a cofactor utilized as a methyl transfer reagent. In some aspects, RNase inhibitor is not included in the enzymatic capping reaction. In another aspect, the enzymatic capping reaction step is performed under constant mixing. In another aspect, the RNA molecule is not co-transcriptionally capped.

[0158] Non-limiting examples of 5' cap structures are those which, among other things, have enhanced binding of cap binding polypeptides, increased half-life, reduced susceptibility to 5' endonucleases and / or reduced 5' de-capping, as compared to synthetic 5' cap structures known in the art (or to a wild-type, natural or physiological 5' cap structure). For example, recombinant Vaccinia Virus or Faustovirus Capping Enzyme and recombinant 2'-O-methyltransferase enzyme may create a canonical 5'-5'-triphosphate linkage between the 5'-terminal nucleotide of an mRNA and a guanine cap nucleotide wherein the cap guanine includes an N7 methylation and the 5'- terminal nucleotide of the mRNA includes a 2'-O-methyl. Such a structure is termed the Cap1 structure. This cap results in a higher translational-competency and cellular stability and a reduced activation of cellular pro-inflammatory cytokines, as compared, e.g., to other 5' cap analog structures known in the art. Cap structures include, but are not limited to, m7G(5')ppp(5')N (cap 0) and m7G(5')ppp(5')Nm (cap 1 ). Cap 0 is a N7-methyl guanosine connected to the 5' nucleotide through a 5' to 5' triphosphate linkage, typically referred to as m7G cap or m7Gppp or m7G(5')ppp(5')N. In the cell, the cap 0 structure is essential for efficient translation of the mRNA that carries the cap. An additional methylation on the 2'-0 position of the initiating nucleotide generates Cap 1 , sometimes referred to as m7GpppNm- or m7G(5')ppp(5')Nm, wherein Nm denotes any nucleotide with a 2'-0 methylation. In some aspects, the 5' terminal cap includes a cap analog. Exemplary 5’ cap analogs include, but are not limited to, m7G(5')ppp(5')m7G, 3'-O- Me-m7G(5')ppp(5')G, m7G(5')ppp(5')G, G(5')ppp(5')G, m7G(5')ppp(5')A, G(5')ppp(5')A, m7G(5’)ppp(5’)(2’OmeA)pG, m7(3'OMeG)(5')ppp(5')(2'OMeA)pG, m7G(5’)ppp(5’)(2’0meA)pll, or m7(3’OMeG)(5’)ppp(5’)m6(2’OmeA)pG. In some aspects, a 5' terminal cap may include a guanine analog. Exemplary guanine analogs include, but are not limited to, inosine, N1-methyl- guanosine, 2'fluoro-guanosine, 7-deaza-guanosine, 8-oxo-guanosine, 2-amino-guanosine, LNA- guanosine, and 2-azido-guanosine.

[0159] In some aspects, the capping region may include a single cap or a series of nucleotides forming the cap. In this aspect the capping region may be from 1 to 10, e.g., at least, at most, or about 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides, or any range or value therein, e.g., 2-9, 3-8, 4-7, 1-5, 5- 10, or at least 2, or 10 or fewer nucleotides in length. In some aspects, the cap is absent.

[0160] In some aspects, the first and second operational regions may range from 3 to 40, e.g., at least, at most, or about 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, or 40 nucleotides, or any range or value derivable therein, e.g., 5-30, 10-20, 15, or at least 4, or 30 or fewer nucleotides in length and may comprise, in addition to a Start and / or Stop codon, one or more signal and / or restriction sequences.

[0161] In some aspects, the self-amplifying RNA molecules described herein have a 5' cap (e.g., a 7- methylguanosine). This cap may enhance in vivo translation of the RNA. In some aspects, the self-amplifying RNA may include (in addition to any 5' cap structure) one or more nucleotides having a modified nucleobase. In some aspects, the RNA molecule includes only phosphodiester linkages between nucleosides. In some aspects, the RNA molecule may include phosphoramidate, phosphorothioate, and / or methyl phosphonate linkages.

[0162] In some aspects, the modified mRNA molecules described herein have a 5' cap (e.g., a 7- methylguanosine). This cap may enhance in vivo translation of the RNA. In some aspects, the modified mRNA may include (in addition to any 5' cap structure) one or more nucleotides having a modified nucleobase. In some aspects, the RNA molecule includes only phosphodiester linkages between nucleosides. In some aspects, the RNA molecule may include phosphoramidate, phosphorothioate, and / or methyl phosphonate linkages.

[0163] In one aspect, the capping reaction system can includes enzymatic 5' capping that is performed as follows. The final 1X buffer conditions can include the following: at least, at most, or about 50 mM Tris HCI, pH 8, 5 mM KCI, 1 mM MgCI2, 0.5 mM GTP, 0.2 mM S-adenosyl-methionine and 1 mM dithiothreitol. In some aspects, the final 1X buffer does not include dithiothreitol.

[0164] In some aspects, the capping reaction occurs in the vessel in which the IVT reaction was performed. In such aspects, the IVT reaction is diluted between 3-fold and 10-fold, e.g., at least, at most, or about 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold, before the capping reaction. In some aspects, the IVT reaction is diluted with Tris pH 7.0 buffer. In some aspects, the capping reaction occurs in a separate reactor.

[0165] To degrade residual DNA template from the IVT reaction, DNase I can be added. In some aspects, DNase I is added at a concentration between at least, at most, or about 1 U / μg of DNA to 10 U / μg of DNA, e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 U / μg of DNA, or any range or value derivable therein. In addition to DNase I, CaCh can be added as a co-factor for DNase I at a concentration between at least, at most, or about 0.1 mM to 4 mM, e.g., 0.1 , 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1 , 1.1 , 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1 , 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1 , 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, or 4.0 mM, or any range or value derivable therein.

[0166] In some aspects, pyrophosphatase is added into the capping reaction. Pyrophosphatase assists with degrading pyrophosphate, which is the inhibitory by-product that is generated by the IVT reaction or by the capping reaction.

[0167] In some aspects, the capping reaction is conducted under 37 °C for 1 minute to 2 hours, e.g., at least, at most, or about 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22,

[0168] 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47,

[0169] 48, 49, 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59, 60, 61 , 62, 63, 64, 65, 66, 67, 68, 69, 70, 71 , 72,

[0170] 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97,

[0171] 98, 99, 100, 101 , 102, 103, 104, 105, 106, 107, 108, 109, 110, 111 , 112, 113, 114, 115, 116, 117, 118, 119, or 120 minutes, or any range or value derivable therein. In some aspects, the capping reaction is conducted at a temperature greater than 20°C and less than 50°C, e.g., 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, or 50°C.

[0172] In some aspects, the step of capping the uncapped RNA molecules results in at least, at most, or about 50%, 55%, 60%, 65%, 70%, 71 %, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% capped RNA molecules of the total of RNA molecules (capped and uncapped), or any range or value derivable therein. Purity may be determined as described herein, e.g., via reverse phase HPLC or Fragment analyzer or Bioanalyzer chip-based electrophoresis and measured by, e.g., peak area of full-length RNA molecule relative to total peak.

[0173] Purification

[0174] The term “purified” when used in relation to a nucleic acid such as a “purified nucleic acid” refers to one that is separated from at least one contaminant. A “contaminant” is any substance that makes another unfit, impure or inferior.

[0175] Thus, a purified nucleic acid is present in a form or setting different from that in which it is found in nature, or a form or setting different from that which existed prior to subjecting it to a treatment and / or purification method.

[0176] Through purification of RNA, the RNA can be solubilized in an aqueous solution appropriate to permit subsequent encapsulation of the RNA within encapsulating agents (e.g., LNPs) and the solution is thus called “RNA drug substance.”

[0177] In some aspects, the RNA molecules produced by the methods described herein may be contacted with DNase I and CaCh to enzymatically digest DNA template following the in vitro transcription reaction.

[0178] In some aspects, the RNA molecules produced by the methods described herein may be contacted with EDTA and proteinase K. The EDTA may quench any cationic metal species, including magnesium, and the proteinase K may digest proteins present in the IVT product, reducing their size.

[0179] In some aspects, the methods described herein may be contacted with DNase I and CaCh followed by EDTA and proteinase K.

[0180] In some aspects, the methods described herein do not include contacting the RNA molecules produced by the methods described herein with DNase I and CaCh to enzymatically digest DNA template following the in vitro transcription reaction.

[0181] In some aspects, the methods described herein do not include contacting the RNA molecules produced by the methods described herein with EDTA and proteinase K to digest proteins present in the IVT product.

[0182] In some aspects, the methods described herein do not include contacting the RNA molecules produced by the methods described herein with DNase I and CaCh followed by EDTA and proteinase K.

[0183] In some aspects, the RNA molecules produced by the methods described herein may be followed by continuous purification steps, e.g., an affinity chromatography step, an ion exchange chromatography step, a hydrophobic interaction chromatography (HIC) step, a ceramic hydroxyapatite (CHA) chromatography step, a phenyl boronate chromatography step, and / or a filtration step (e.g., ultrafiltration, diafiltration, tangential flow ultrafiltration / diafiltration, or singlepass variants thereof).

[0184] In some aspects, the RNA molecules produced by the methods described herein may be purified by methods including chromatographic adsorption (e.g., hydrogen bonding, hydrophobic interaction, ion-exchange, diol bonding, or metal affinity) or filtration (e.g., ultrafiltration, diafiltration, tangential flow ultrafiltration / diafiltration, or single-pass variants thereof).

[0185] In some aspects, components of the IVT product (e.g., NTPs, 5’ cap analog, RNA polymerase, template DNA), having been separated from the in vitro transcribed RNA molecule through the above-mentioned continuous purification methods, can then be recovered and returned to the IVT reaction.

[0186] In some aspects, at least a portion of the IVT product is adsorbed on a substrate such as a resin bead or monolith. The RNA may bind to the substrate while impurities flow past the substrate or the impurities may bind to the substrate while RNA flows past the substrate. The mode of interaction may be based on hydrogen bonding, hydrophobic interaction, ion-exchange, diol bonding, or metal affinity, or any combination of these.

[0187] In some aspects, the RNA molecule binds to an affinity substrate while the DNA template flows through and is removed. In some aspects, the RNA molecule binds to an affinity substrate while RNA polymerase flows through and is removed.

[0188] In some aspects, the chromatographic method involves poly(A) capture-based affinity purification, i.e., oligo(dT) purification. For example, a polythymidine ligand may be immobilized to a derivatized chromatography resin or monolithic column. The mechanism of purification may involve hybridization of the poly(A) tail of the RNA molecule to the oligonucleotide ligand, wherein the DNA template will not bind. In preferred aspects, the RNA molecules that do not include Poly(A) stretches (e.g., abortive transcripts and other truncates formed during in vitro transcription) will not bind to the resin and will not form a duplex with the affinity ligand. Polyadenylated RNA may subsequently be eluted from the resin utilizing a low ionic strength buffer or a competitive binding oligonucleotide solution.

[0189] In some aspects, adsorbed RNA may be eluted using a solution that prepares the RNA solution for further purification, encapsulation in encapsulating agents (e.g. LNPs), or formulation operations.

[0190] In some aspects, the adsorption of RNA may occur on packed resin bead columns or a monolith. The process may operate with 1 or more columns in parallel and 1 or more columns in series. The columns may be operated independently or in a multi-column format where the effluent from one column is fed to the inlet of another column. After the first column is loaded, the IVT product continues loading to the next column while the first loaded column is further processed and eluted.

[0191] In some aspects, 2 or more columns with different modes of adsorption are used in series. The RNA may bind to 1 or more of the columns and RNA polymerase or other IVT product components may bind to 1 or more of the columns. In some cases, the RNA polymerase and / or other IVT product components may be eluted from the chromatography column and recycled for repeated use in the IVT step.

[0192] In some aspects, the amount of RNA loaded on the column is controlled by in-line, at-line, or offline concentration measurements. In some aspects, the measurements are by HPLC, NMR, Raman spectroscopy, or UV spectroscopy.

[0193] In some aspects, the IVT product may be mixed with additional buffer solutions (e.g., through inline mixing, filtration, or surge vessel) before loading on the chromatographic step.

[0194] In some aspects, where the RNA is bound to the chromatographic step, 1 or more wash steps may be applied to remove additional impurities or exchange the RNA into a new buffer solution.

[0195] In some aspects, at least a portion of the IVT product is filtered. The IVT product may be filtered via ultrafiltration and / or diafiltration and / or single-pass variants of these to remove at least some impurities from the IVT product and / or to change buffer solution for at least a portion of IVT product to produce a concentrated RNA solution as a retentate.

[0196] In some aspects, filtration may be used to prepare the purified RNA in a solution that is appropriate for subsequent encapsulation in encapsulating agents (e.g. LNPs) and formulation operations.

[0197] In some aspects, the RNA polymerase and / or other IVT product components may be recovered from the IVT product filtration permeate and recycled for repeated use in the IVT step.

[0198] In some aspects, the amount of RNA passed through the filter is controlled by in-line, at-line, or offline concentration measurements. In some aspects, the measurements are by HPLC, NMR, Raman spectroscopy, or UV spectroscopy.

[0199] In some aspects, the IVT product may be mixed with additional buffer solutions (e.g., through inline mixing or surge vessel) before passing through the subsequent filtration step.

[0200] In some aspects, the RNA purified by the methods described above may be mixed with additional buffer solutions (e.g., through in-line mixing, filtration, or surge vessel) to prepare the solution for subsequent encapsulation in encapsulating agents (e.g. LNPs) and formulation.

[0201] In some aspects, both “ultrafiltration” and “diafiltration” refer to a membrane filtration process. Ultrafiltration typically uses membranes having pore sizes of at least, at most, exactly, or between (inclusive or exclusive) any two of 0.001 , 0.002, 0.003, 0.004, 0.005, 0.006, 0.007, 0.008, 0.009, 0.01 , 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, and 0.1 pm. In some aspects, ultrafiltration membranes are typically classified by molecular weight cutoff (MWCO) rather than pore size. For example, the MWCO may be at least, at most, exactly, or between (inclusive or exclusive) any two of 30 kDa, 40 kDa, 50 kDa, 60 kDa, 70 kDa, 80 kDa, 90 kDa, 100 kDa, 110 kDa, 120 kDa,

[0202] 130 kDa, 140 kDa, 150 kDa, 160 kDa, 170 kDa, 180 kDa, 190 kDa, 200 kDa, 210 kDa, 220 kDa,

[0203] 230 kDa, 240 kDa, 250 kDa, 260 kDa, 270 kDa, 280 kDa, 290 kDa, 300 kDa, 310 kDa, 320 kDa,

[0204] 330 kDa, 340 kDa, 350 kDa, 360 kDa, 370 kDa, 380 kDa, 390 kDa, 400 kDa, 500 kDa, 600 kDa,

[0205] 700 kDa, 800 kDa, 900 kDa, 1000 kDa, 2000 kDa, 3000 kDa, 4000 kDa, 5000 kDa, 6000 kDa, 7000 kDa, 8000 kDa, 9000 kDa, and 10000 kDa. A skilled artisan will understand that filtration membranes may comprise different suitable materials, including, e.g., polymers, cellulose, ceramic, etc., depending upon the application. In some aspects, membrane filtration may be more desirable for continuous and / or large volume purification process.

[0206] In some aspects, the RNA molecule has a clinical grade purity. In some aspects, the purity of the RNA molecule is between about 60% and about 100%. In some aspects, the purity of the RNA molecule is between about 80% and 99%. In some aspects, the purity of the RNA molecule is between about 90% and about 99%. In some aspects, the purified mRNA has a clinical grade purity without further purification. In some aspects, the clinical grade purity is achieved through a method including adsorption or filtration purification.

[0207] In some aspects, the clinical grade purity is achieved with purification methods such as high- performance liquid chromatography (HPLC) purification, ligand or binding based purification, and / or ion exchange chromatography.

[0208] In some aspects, the methods of purifying the RNA molecule removes short or long abortive RNA species, double-stranded RNA (dsRNA), residual template DNA, residual RNA polymerase, residual solvent, and / or residual salt. In some aspects, the short abortive transcript contaminants comprise less than 15 bases. In some aspects, the short abortive transcript contaminants comprise about 8-12 bases.

[0209] In some embodiments, the method of purifying the RNA molecule also removes RNase inhibitor and inorganic yeast pyrophosphatase.

[0210] In some embodiments, the method of purifying the RNA molecule may include, but is not limited to, phenol / chloroform extraction and / or precipitation with either alcohol (ethanol, isopropanol) in the presence of monovalent cations or lithium chloride for nucleic acid clean-up, quality assurance and quality control.

[0211] Additional, non-limiting examples of RNA purification procedures include AGENCOURT® beads (BECKMAN COULTER GENOMICS), poly-T beads, LNATM oligo-T capture probes (EXIQON INC), HPLC based purification methods such as, but not limited to, strong anion exchange HPLC, weak anion exchange HPLC, reverse phase HPLC (RP-HPLC), and hydrophobic interaction chromatography-HPLC (HIC-HPLC), size exclusion chromatography, and silica-based affinity chromatography and polyacrylamide gel electrophoresis.

[0212] RNA purification can also be performed using a variety of commercially available kits including, but not limited to SV Total Isolation System (PROMEGA) and In vitro Transcription Cleanup and Concentration Kit (NORGEN BIOTEK). In some aspects, 1 , 2, 3, 4, 5, or more of the foregoing purification methods may be excluded

[0213] Preferably, purified RNA material is substantially free of one or more impurities or contaminants including the linear DNA template and / or reverse complement transcription products described herein and for instance is at least 90%, 91 %, 92%, 93%, 94%, 95%, 96%, or 97% pure; more preferably, at least 98% pure, and more preferably still at least 99% pure.

[0214] Characterization and Analysis of the RNA Molecule

[0215] In some aspects, the RNA molecules produced by the methods described herein may be analyzed and characterized using various methods. Analysis may be performed before or after capping. Alternatively, analysis may be performed before or after purification via poly(A) capturebased affinity, TFF, SPTFF, or other chromatographic and filtration purification methods. In another aspect, analysis may be performed before or after additional purification steps, e.g., anion exchange chromatography and the like. RNA transcript integrity may be determined using electrophoresis (e.g., using the fragment analyzer capillary or Bioanalyzer chip systems) or through a reverse phase HPLC method. In other aspects, RNA purity is analyzed using analytical reverse phase HPLC. Capping efficiency may be analyzed using, e.g., total nuclease digestion followed by LC-UV or LC-MS quantitation of the dinucleotide cap species vs. uncapped GTP species. The level of residual DNA template (resDNA) can be measured using quantitative polymerase chain reaction (qPCR). The concentration of residual NTPs and / or 5’ cap analogs can be measured using anion-exchange chromatography on an HPLC-UV system. Next- Generation Sequencing (also referred to as massively parallel sequencing) which refers to nonSanger sequencing technologies enables the determination of nucleic acid order which can be used to confirm the sequence identity of the mRNA transcripts as well as determine the location and frequency of sequence variants within said transcripts. In vitro efficacy may be analyzed by, e.g., transfecting RNA molecules into a human cell line. Protein expression of the polypeptide of interest may be quantified using methods such as ELISA or flow cytometry. Immunogenicity may be analyzed by, e.g., transfecting RNA molecules into cell lines that indicate innate immune stimulation, e.g., PBMCs. Cytokine induction may be analyzed using, e.g., methods such as ELISA to quantify a cytokine, e.g., Interferon-a.

[0216] The RNA molecules produced by the methods described herein may produce an RNA molecule that is at least 30% full-length transcript, or at least, at most, or about 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 71 %, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% full-length transcript, or any range or value derivable therein. Purity may be determined as described herein, e.g., via reverse phase HPLC or Bioanalyzer chip-based electrophoresis and measure by, e.g., peak area of full-length RNA molecule relative to total peak.

[0217] Genes of Interest

[0218] The DNA template and resulting RNA molecules described herein include a gene of interest. The gene of interest encodes a polypeptide of interest selected from, e.g., biologies, antibodies, vaccines, therapeutic polypeptides or peptides, cell penetrating peptides, secreted polypeptides, plasma membrane polypeptides, cytoplasmic or cytoskeletal polypeptides, intracellular membrane bound polypeptides, nuclear polypeptides, polypeptides associated with human disease, targeting moieties or those polypeptides encoded by the human genome for which no therapeutic indication has been identified but which nonetheless have utility in areas of research and discovery. The sequence for a particular gene of interest is readily identified by one of skill in the art using public and private databases, e.g., GenBank.

[0219] In some aspects, the RNA molecule includes a coding region for an antigen preferably derived from a pathogen associated with infectious disease which are preferably selected from antigens derived from the pathogens Acinetobacter baumannii, Anaplasma genus, Anaplasma phagocytophilum, Ancylostoma braziliense, Ancylostoma duodenale, Arcanobacterium haemolyticum, Ascaris lumbricoides, Aspergillus genus, Astroviridae, Babesia genus, Bacillus anthracis, Bacillus cereus, Bartonella henselae, BK virus, Blastocystis hominis, Blastomyces dermatitidis, Bordetella pertussis, Borrelia burgdorferi, Borrelia genus, Borrelia spp, Brucella genus, Brugia malayi, Bunyaviridae family, Burkholderia cepacia and other Burkholderia species, Burkholderia mallei, Burkholderia pseudomallei, Caliciviridae family, Campylobacter genus, Candida albicans, Candida spp, Chlamydia trachomatis, Chlamydophila pneumoniae, Chlamydophila psittaci, CJD prion, Clonorchis sinensis, Clostridium botulinum, Clostridium difficile, Clostridium perfringens, Clostridium perfringens, Clostridium spp, Clostridium tetani, Coccidioides spp, coronaviruses, Corynebacterium diphtheriae, Coxiella burnetii, Crimean- Congo hemorrhagic fever virus, Cryptococcus neoformans, Cryptosporidium genus, Cytomegalovirus (CMV), Dengue viruses (DENV-1 , DENV-2, DENV-3 and DENV-4), Dientamoeba fragilis, Ebolavirus (EBOV), Echinococcus genus, Ehrlichia chaffeensis, Ehrlichia ewingii, Ehrlichia genus, Entamoeba histolytica, Enterococcus genus, Enterovirus genus, Enteroviruses, mainly Coxsackie A virus and Enterovirus 71 (EV71 ), Epidermophyton spp, Epstein-Barr Virus (EBV), Escherichia coli O157:H7, 0111 and 0104:H4, Fasciola hepatica and Fasciola gigantica, FFI prion, Filarioidea superfamily, Flaviviruses, Francisella tularensis, Fusobacterium genus, Geotrichum candidum, Giardia intestinalis, Gnathostoma spp, GSS prion, Guanarito virus, Haemophilus ducreyi, Haemophilus influenzae, Helicobacter pylori, Henipavirus (Hendra virus Nipah virus), Hepatitis A Virus, Hepatitis B Virus (HBV), Hepatitis C Virus (HCV), Hepatitis D Virus, Hepatitis E Virus, Herpes simplex virus 1 and 2 (HSV-1 and HSV-2), Histoplasma capsulatum, HIV (Human immunodeficiency virus), Hortaea werneckii, Human bocavirus (HBoV), Human herpesvirus 6 (HHV-6) and Human herpesvirus 7 (HHV-7), Human metapneumovirus (hMPV), Human papillomavirus (HPV), Human parainfluenza viruses (HPIV), Japanese encephalitis virus, JC virus, Junin virus, Kingella kingae, Klebsiella granulomatis, Kuru prion, Lassa virus, Legionella pneumophila, Leishmania genus, Leptospira genus, Listeria monocytogenes, Lymphocytic choriomeningitis virus (LCMV), Machupo virus, Malassezia spp, Marburg virus, Measles virus, Metagonimus yokagawai, Microsporidia phylum, Molluscum contagiosum virus (MCV), Mumps virus, Mycobacterium leprae and Mycobacterium lepromatosis, Mycobacterium tuberculosis, Mycobacterium ulcerans, Mycoplasma pneumoniae, Naegleria fowleri, Necator americanus, Neisseria gonorrhoeae, Neisseria meningitidis, Nocardia asteroides, Nocardia spp, Onchocerca volvulus, Orientia tsutsugamushi, Orthomyxoviridae family (including Influenza such as avian influenza and human influenza), Paracoccidioides brasiliensis, Paragonimus spp, Paragonimus westermani, Parvovirus B19, Pasteurella genus, Plasmodium genus, Pneumocystis jirovecii, Poliovirus, Rabies virus, Respiratory syncytial virus (RSV), Rhinovirus, rhinoviruses, Rickettsia akari, Rickettsia genus, Rickettsia prowazekii, Rickettsia rickettsii, Rickettsia typhi, Rift Valley fever virus, Rotavirus, Rubella virus, Sabia virus, Salmonella genus, Sarcoptes scabiei, Coronavirus (e.g., SARS-CoV-2), Schistosoma genus, Shigella genus, Sin Nombre virus, Hantavirus, Sporothrix schenckii, Staphylococcus genus, Staphylococcus genus, Streptococcus agalactiae, Streptococcus pneumoniae, Streptococcus pyogenes, Strongyloides stercoralis, Taenia genus, Taenia solium, Tick-borne encephalitis virus (TBEV), Toxocara canis or Toxocara cati, Toxoplasma gondii, Treponema pallidum, Trichinella spiralis, Trichomonas vaginalis, Trichophyton spp, Trichuris trichiura, Trypanosoma brucei, Trypanosoma cruzi, Ureaplasma urealyticum, Varicella zoster virus (VZV), Varicella zoster virus (VZV), Variola major or Variola minor, vCJD prion, Venezuelan equine encephalitis virus, Vibrio cholerae, West Nile virus, Western equine encephalitis virus, Wuchereria bancrofti, Yellow fever virus, Yersinia enterocolitica, Yersinia pestis, and Yersinia pseudotuberculosis.

[0220] In some aspects, the RNA molecules of the present disclosure encode a viral polypeptide or fragment thereof, including naturally occurring or engineered variants thereof, for prophylaxis against a virus in humans.

[0221] Entrapment of RNA in Nanoparticles and Processing to Drug Product

[0222] In some aspects, the in vitro transcribed RNA molecules within the RNA drug substance of the present disclosure may be encapsulated to form colloidal dispersions (e.g. RNA-loaded LNP dispersion) comprising at least one encapsulating agent. In one aspect, the encapsulating agent comprises one or more lipids, a lipid nanoparticle (LNP), lipoplexes, one or more polymers, polymeric particles, polyplexes, monolithic delivery systems, or a combination thereof. In some aspects, 1 , 2, 3, 4, 5, or more of the foregoing elements may be excluded as an encapsulating agent.

[0223] In one aspect, the encapsulating agent is a lipid, and produced is an RNA-loaded LNP dispersion. Without intending to be bound by any theory, it is believed that the cationic or cationically ionizable lipid or lipid-like material and / or the cationic polymer combine together with the nucleic acid to form colloidally stable dispersions.

[0224] A lipid may be a naturally occurring lipid or a synthetic lipid. However, a lipid is usually a biological substance. Biological lipids are well known in the art, and include for example, neutral fats, phospholipids, phosphoglycerides, steroids, terpenes, lysolipids, glycosphingolipids, glucolipids, sulphatides, lipids with ether and ester-linked fatty acids and polymerizable lipids, and combinations thereof. As encapsulating agent, a lipid is a substance that is insoluble or partially insoluble in water and extractable with an organic solvent. Compounds other than those specifically described herein are understood by one of skill in the art as lipids and are encompassed by the compositions and methods of the present disclosure. A lipid component and a non-lipid may be attached to one another, either covalently or non-covalently.

[0225] In some aspects, LNPs may be designed to protect RNA molecules with unmodified and / or modified nitrogenous bases and various sizes (e.g., mRNA, modified mRNA [modRNA], saRNA, gRNA and / or circRNA) from extracellular RNases and / or may be engineered for systemic delivery of the RNA to target cells. In some aspects, such LNPs may be particularly useful to deliver RNA molecules when RNA molecules are intravenously administered to a subject in need thereof. In some aspects, such LNPs may be particularly useful to deliver RNA molecules when RNA molecules are intramuscularly administered to a subject in need thereof. In some aspects, such LNPs may be particularly useful to deliver RNA molecules when RNA molecules are intradermally administered to a subject in need thereof. In some aspects, such LNPs may be particularly useful to deliver RNA molecules when RNA molecules are intranasally administered to a subject in need thereof.

[0226] In one aspect, the RNA in the RNA drug substance is at a concentration of < 1 mg / mL. In another aspect, the RNA is at a concentration of at least or at least about 0.05 mg / mL. In another aspect, the RNA is at a concentration of at least or at least about 0.5 mg / mL. In another aspect, the RNA is at a concentration of at least or at least about 1 mg / mL. In another aspect, the RNA concentration is from or from about 0.05 mg / mL to about 0.5 mg / mL. In another aspect, the RNA is at a concentration of at least 10 mg / mL. In another aspect, the RNA is at a concentration of at least 50 mg / mL. In some aspects, the RNA is or is not at a concentration of at least, at most, exactly, between (inclusive or exclusive) any two of, or about 0.05 mg / mL, 0.5 mg / mL, 1 mg / mL, 10 mg / mL, 50 mg / mL, 75 mg / mL, 100 mg / mL, 150 mg / mL, 200 mg / mL, 250 mg / mL, 300 mg / mL, 400 mg / mL, or more.

[0227] The present disclosure provides for an RNA drug substance and a lipid preparation mixture or compositions thereof comprising at least one RNA encoding, e.g., an antigen complexed with, encapsulated in, and / or formulated with one or more lipids, and forming lipid nanoparticles (LNPs), liposomes, lipoplexes and / or nanoliposomes. In some aspects, the composition comprises a lipid nanoparticle.

[0228] A lipid nanoparticle or LNP refers to particles of any morphology generated when a cationic lipid and optionally one or more further lipids are combined, e.g., in an aqueous environment and / or in the presence of RNA. In some aspects, lipid nanoparticles are included in a formulation that may be used to deliver an active agent or therapeutic agent, such as a nucleic acid (e.g., mRNA) to a target site of interest (e.g., cell, tissue, organ, tumor, and the like). In some aspects, the lipid nanoparticles of the present disclosure comprise a nucleic acid (e.g., mRNA). Such lipid nanoparticles typically comprise a cationic lipid and one or more excipients, e.g., one or more neutral lipids, charged lipids, steroids, polymer conjugated lipids, or combinations thereof. In some aspects, the LNPs comprise at least one cationic (e.g., ionizable) lipid, at least one neutral (e.g., non-cationic) lipid, at least one structural lipid (e.g., a steroid), and / or at least one polymer conjugated lipid (e.g., a polyethylene glycol (PEG)-modified lipid). In some aspects, 1 , 2, 3, or more of the foregoing excipients may be excluded from the LNPs.

[0229] In some aspects, the LNPs comprise 20-60 mol% cationic (e.g., ionizable) lipid(s). For example, the LNPs may comprise 20-50 mol%, 20-40 mol%, 20-30 mol%, 30-60 mol%, 30-50 mol%, 30- 40 mol%, 40-60 mol%, 40-50 mol%, or 50-60 mol% cationic (e.g., ionizable) lipid(s). In some aspects, the LNPs comprise or do not comprise at least, at most, exactly, or between (inclusive or exclusive) any two of 20 mol%, 30 mol%, 40 mol%, 50, or 60 mol% cationic (e.g., ionizable) lipid(s). In some aspects, the LNPs comprise 45 to 55 mole percent (mol%) cationic (e.g., ionizable) lipid(s). For example, LNPs may comprise or not comprise at least, at most, exactly, or between (inclusive or exclusive) any two of 45, 46, 47, 48, 49, 50, 51 , 52, 53, 54, or 55 mol% cationic (e.g., ionizable) lipid(s).

[0230] In some aspects, the LNPs comprise 5-25 mol% neutral (e.g., non-cationic) lipid(s). For example, the LNPs may comprise 5-20 mol%, 5-15 mol%, 5-10 mol%, 10-25 mol%, 10-20 mol%, 10-25 mol%, 15-25 mol%, 15-20 mol%, or 20-25 mol% neutral (e.g., non-cationic) lipid(s). In some aspects, the LNPs are or are not at least, at most, exactly, or between (inclusive or exclusive) any two of 5 mol%, 10 mol%, 15 mol%, 20 mol%, or 25 mol% neutral (e.g., non-cationic) lipid(s). In some aspects, the LNPs comprise 5 to 15 mol% neutral (e.g., non-cationic) lipid(s). For example, LNPs may comprise at least, at most, exactly, or between (inclusive or exclusive) any two of 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, or 15 mol% neutral (e.g., non-cationic) lipid(s).

[0231] In some aspects, the LNPs comprise 25-55 mol% structural lipid(s) (e.g., a steroid). For example, the LNPs may comprise 25-50 mol%, 25-45 mol%, 25-40 mol%, 25-35 mol%, 25-30 mol%, 30- 55 mol%, 30-50 mol%, 30-45 mol%, 30-40 mol%, 30-35 mol%, 35-55 mol%, 35-50 mol%, 35-45 mol%, 35-40 mol%, 40-55 mol%, 40-50 mol%, 40-45 mol%, 45-55 mol%, 45-50 mol%, or 50-55 mol% structural lipid(s) (e.g., a steroid). In some aspects, the LNPs are or are not at least, at most, exactly, or between (inclusive or exclusive) any two of 25 mol%, 30 mol%, 35 mol%, 40 mol%, 45 mol%, 50 mol%, or 55 mol% structural lipid(s) (e.g., a steroid). In some aspects, the LNPs comprise 35 to 40 mol% structural lipid(s) (e.g., a steroid). For example, LNPs may comprise at least, at most, exactly, or between (inclusive or exclusive) any two of 35, 36, 37, 38, 39, or 40 mol% structural lipid(s) (e.g., a steroid).

[0232] In some aspects, the LNPs comprise 0.5-15 mol% polymer conjugated lipid(s) (e.g., a polyethylene glycol (PEG)-conjugated lipid). For example, the lipid nanoparticles (LNPs) may comprise 0.5-10 mol%, 0.5-5 mol%, 1-15 mol%, 1-10 mol%, 1-5 mol%, 2-15 mol%, 2-10 mol%, 2-5 mol%, 5-15 mol%, 5-10 mol%, or 10-15 mol% polymer conjugated lipid(s) (e.g., a PEG- conjugated lipid). In some aspects, the LNPs are or are not at least, at most, exactly, or between (inclusive or exclusive) any two of 0.5 mol%, 1 mol%, 2 mol%, 3 mol%, 4 mol%, 5 mol%, 6 mol%, 7 mol%, 8 mol%, 9 mol%, 10 mol%, 11 mol%, 12 mol%, 13 mol%, 14 mol%, or 15 mol% polymer conjugated lipid(s) (e.g., a polyethylene glycol (PEG)-conjugated lipid). In some aspects, the LNPs comprise 1 to 2 mol% polymer conjugated lipid(s) (e.g., a polyethylene glycol (PEG)- conjugated lipid). For example, LNPs may comprise at least, at most, exactly, or between (inclusive or exclusive) any two of 1 , 1.5, or 2 mol% polymer conjugated lipid(s) (e.g., a PEG- conjugated lipid).

[0233] In some aspects, the LNPs comprise 20-75 mol% cationic (e.g., ionizable) lipid(s) (e.g., at least, at most, exactly, or between (inclusive or exclusive) any two of 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, and 75%), 0.5-25 mol% neutral (e.g., non-cationic) lipid(s) (e.g., at least, at most, exactly, or between (inclusive or exclusive) of 0.5%, 2.25%, 4%, 5.75%, 7.5%, 9.25%, 11 %, 12.75%, 14.5%, 16.25%, 18%, 19.75%, 21.5%, 23.25%, and 25%), 5-55 mol% structural lipid(s) (e.g., a sterol) e.g., non-cationic) lipid(s) (e.g., at least, at most, exactly, or between (inclusive or exclusive) of 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, and 55%), and 0.5-20 mol% polymer conjugated lipid(s) (e.g., a polyethylene glycol (PEG)-modified lipid) (e.g., at least, at most, exactly, or between (inclusive or exclusive) of 0.5%, 2%, 3.5%, 5%, 6.5%, 8%, 9.5%, 11 %, 12.5%, 14%, 15.5%, 17%, 18.5%, and 20%). In some aspects, 1 , 2, 3, or more of the lipids may be excluded from the LNPs.

[0234] In some non-limiting aspects, the molar lipid ratio is 50 / 10 / 38.5 / 1.5 (mol% cationic lipid / neutral lipid / structural lipid / polymer conjugated lipid), 60 / 7.5 / 31 / 1.5 (mol% cationic lipid / neutral lipid / structural lipid / polymer conjugated lipid), 57.5 / 7.5 / 31.5 / 3.5 (mol% cationic lipid / neutral lipid / structural lipid / polymer conjugated lipid), 57.2 / 7.1 / 34.3 / 1.4 (mol% cationic lipid / neutral lipid / structural lipid / polymer conjugated lipid), 40 / 15 / 40 / 5 (mol% cationic lipid / neutral lipid / structural lipid / polymer conjugated lipid), 50 / 10 / 35 / 4.5 / 0.5 (mol% cationic lipid / neutral lipid / structural lipid / polymer conjugated lipid), 50 / 10 / 35 / 5 (mol% cationic lipid / neutral lipid / structural lipid / polymer conjugated lipid), 40 / 10 / 40 / 10 (mol% cationic lipid / neutral lipid / structural lipid / polymer conjugated lipid), 35 / 15 / 40 / 10 (mol% cationic lipid / neutral lipid / structural lipid / polymer conjugated lipid), or 52 / 13 / 30 / 5 (mol% cationic lipid / neutral lipid / structural lipid / polymer conjugated lipid).

[0235] In some aspects, the active agent or therapeutic agent, such as a nucleic acid (e.g., mRNA), may be encapsulated in the lipid portion of the lipid nanoparticle and / or an aqueous space enveloped by some or all of the lipid portion of the lipid nanoparticle, thereby protecting it from enzymatic degradation or other undesirable effects induced by the mechanisms of the host organism or cells, e.g., an adverse immune response. The nucleic acid (e.g., mRNA) or a portion thereof may also be associated and complexed with the lipid nanoparticle. A lipid nanoparticle may comprise any lipid capable of forming a particle to which the nucleic acids are attached, and / or in which the one or more nucleic acids are encapsulated.

[0236] In some aspects, provided RNA molecules (e.g., mRNA, modRNA, saRNA, gRNA and / or circRNA) may be formulated with LNPs. In some aspects, the lipid nanoparticles may or may not have a mean diameter of or of about 1 to 500 nm (e.g., at least, at most, exactly, or between (inclusive or exclusive) of 1 , 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, or 500 nm). In some aspects, the lipid nanoparticles have a mean diameter of or of from about 30 nm to about 150 nm, about 40 nm to about 150 nm, about 50 nm to about 150 nm, about 60 nm to about 130 nm, about 70 nm to about 110 nm, about 70 nm to about 100 nm, about 80 nm to about 100 nm, about 90 nm to about 100 nm, about 70 to about 90 nm, about 80 nm to about 90 nm, about 70 nm to about 80 nm, or at least, at most, exactly, or between (inclusive or exclusive) of 30 nm, 35 nm, 40 nm, 45 nm, 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 95 nm, 100 nm, 105 nm, 110 nm, 115 nm, 120 nm, 125 nm, 130 nm, 135 nm, 140 nm, 145 nm, or 150 nm, and are substantially non-toxic. The term “mean diameter” refers to the mean hydrodynamic diameter of particles as measured by dynamic laser light scattering (DLS) with data analysis using the so-called cumulant algorithm, which provides as results the so-called Z-average with the dimension of a length, and the polydispersity index (PI), which is dimensionless (Koppel, D., J. Chem. Phys. 57, 1972, pp 4814-4820, ISO 13321 ). Here, “mean diameter,” “diameter,” or “size” for particles is used synonymously with the value of the Z-average.

[0237] LNPs described herein may exhibit a polydispersity index less than or less than about 0.5, 0.4, 0.3, or 0.2 or less. By way of example, the LNPs may or may not exhibit a polydispersity index of at least, at most, exactly, or between (inclusive or exclusive) of 0.1 , 0.11 , 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.2, 0.21 , 0.22, 0.23, 0.24, 0.25, 0.26, 0.27, 0.28, 0.29, 0.3, 0.31 , 0.32, 0.33, 0.34, 0.35, 0.36, 0.37, 0.38, 0.39, 0.4, 0.41 , 0.42, 0.43, 0.44, 0.45, 0.46, 0.47, 0.48, 0.49, or 0.5. The polydispersity index is, in some aspects, calculated based on dynamic light scattering measurements by the so-called cumulant analysis referred to in the definition of “average diameter.” Under certain prerequisites, it may be taken as a measure of the size distribution of an ensemble of nanoparticles.

[0238] In some aspects, an LNP of the disclosure comprises or does not comprise a molar ratio of positively chargeable nitrogen of tertiary amine in the cationic lipid to negatively charged phosphates of mRNA backbone (known as N:P ratio) of or of from about 2:1 to about 30:1 , e.g., at least, at most, exactly, or between (inclusive or exclusive) of 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, 21:1, 22:1, 23:1, 24:1, 25:1, 26:1, 27:1, 28:1, 29:1, or 30:1. In some aspects, an LNP of the disclosure comprises an N:P ratio of or of about 6:1. In some aspects, an LNP of the disclosure comprises an N:P ratio of or of about 3:1.

[0239] In some aspects, an LNP of the disclosure comprises or does not comprise a wt / wt ratio of the cationic lipid component to the RNA of or of from about 5:1 to about 100:1 , e.g., at least, at most, exactly, or between (inclusive or exclusive) of 5:1 , 6:1, 7:1 , 8:1, 9:1 , 10:1 , 11:1, 12:1 , 13:1 , 14:1 , 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, 21:1, 22:1, 23:1, 24:1, 25:1, 26:1, 27:1, 28:1, 29:1, 30:1, 31:1, 32:1, 33:1, 34:1, 35:1, 36:1, 37:1, 38:1, 39:1, 40:1, 41:1, 42:1, 43:1, 44:1, 45:1, 46:1, 47:1, 48:1, 49:1, 50:1, 51:1, 52:1, 53:1, 54:1, 55:1, 56:1, 57:1, 58:1, 59:1, 60:1, 61:1, 62:1, 63:1, 64:1, 65:1, 66:1, 67:1, 68:1, 69:1, 70:1, 71:1, 72:1, 73:1, 74:1, 75:1, 76:1, 77:1, 78:1, 79:1, 80:1, 81:1, 82:1, 83:1, 84:1, 85:1, 86:1, 87:1, 88:1, 89:1, 90:1, 91:1, 92:1, 93:1, 94:1, 95:1, 96:1, 97:1, 98:1, 99:1, or 100:1. In some aspects, an LNP of the disclosure comprises a wt / wt ratio of the ionizable cationic lipid component to the RNAof or of about 20:1. In some aspects, an LNP of the disclosure comprises a wt / wt ratio of the ionizable cationic lipid component to the RNA of or of about 10:1.

[0240] In certain aspects, nucleic acids (e.g., RNA molecules), when present in provided LNPs, are resistant in aqueous solution to degradation with a nuclease. In some aspects, LNPs are livertargeting lipid nanoparticles. In some aspects, LNPs are cationic lipid nanoparticles comprising one or more cationic lipids (e.g., those described herein). In some aspects, cationic LNPs may comprise at least one cationic lipid, at least one polymer conjugated lipid, and at least one helper lipid (e.g., at least one neutral lipid).

[0241] In certain aspects, the mixture of RNA and lipid preparations or compositions thereof may have at least, at most, exactly, between (inclusive or exclusive) of, or about 1%, 2%, 3%, 4% 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%,

[0242] 41 %, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51 %, 52%, 53%, 54%, 55%, 56%, 57%,

[0243] 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%,

[0244] 75%, 76%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %,

[0245] 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of a particular lipid, lipid type, or non-lipid component such as lipid-like materials and / or cationic polymers and / or an adjuvant, antigen, peptide, polypeptide, sugar, nucleic acid or other material disclosed herein or as would be known to one of skill in the art.

[0246] LNPs described herein can be generated using components, compositions, and methods as are generally known in the art, see for example PCT / US2016 / 052352; PCT / US2016 / 068300; PCT / US2017 / 037551 ; PCT / US2015 / 027400; PCT / US2016 / 047406; PCT / US2016000129; PCT / US2016 / 014280; PCT / US2016 / 014280; PCT / US2017 / 038426; PCT / US2014 / 027077; PCT / US2014 / 055394; PCT / US2016 / 52117; PCT / US2012 / 069610; PCT / US2017 / 027492; PCT / US2016 / 059575 and PCT / US2016 / 069491 all of which are incorporated by reference herein in their entirety. For example, methods of preparing LNPs may involve obtaining a colloid from at least one cationic or cationically ionizable lipid or lipid-like material and / or at least one cationic polymer and mixing the colloid with nucleic acid to obtain nucleic acid particles. The term “colloid” as used herein relates to a type of mixture in which dispersed particles do not settle out. The insoluble particles in the mixture are microscopic, with particle sizes between 1 and 1000 nanometers. The mixture may be termed a colloid or a colloidal dispersion. Sometimes the term “colloid” refers only to the particles in the mixture and not the entire dispersion.

[0247] While methods for preparing a colloid containing an organic solvent are described herein, other methods having organic solvent-free characteristics may also be used according to the present disclosure.

[0248] In some aspects, an RNA-loaded LNP dispersion may be produced by inline mixing of an RNA solution or adjusted RNA solution described herein (e.g., an RNA drug substance) and a lipid preparation described herein (comprising, e.g., at least one cationic lipid and optionally one or more other lipid components, in an organic solvent) under conditions such that a sudden change in solubility of lipid component(s) is triggered, which drives the lipids towards self-assembly in the form of LNPs. In some aspects, suitable buffering agents comprise tris, histidine, citrate, acetate, phosphate, and / or succinate. In some aspects, 1 , 2, 3, or more of the foregoing buffering agents are excluded. The pH of a liquid formulation relates to the pKa of the encapsulating agent (e.g., cationic lipid). The pH of the acidifying buffer may be at least half a pH scale less than the pKa of the encapsulating agent (e.g., cationic lipid), and the pH of the final buffer may be at least half a pH scale greater than the pKa of the encapsulating agent (e.g., cationic lipid). In some aspects, properties of a cationic lipid are chosen such that nascent formation of particles occurs by association with an oppositely charged backbone of a nucleic acid (e.g., RNA). In this way, particles are formed around the nucleic acid, which, for example, in some aspects, may result in greater encapsulation efficiency than is achieved in the absence of interactions between nucleic acids and at least one of the lipid components. In certain aspects, nucleic acids, when present in the lipid nanoparticles, are resistant in aqueous solution to degradation with a nuclease.

[0249] Lipid nanoparticles comprising nucleic acids and their method of preparation are disclosed in, e.g., U.S. Patent Publication Nos. 2004 / 0142025, 2007 / 0042031 and PCT Pub. Nos. WO 2013 / 016058 and WO 2013 / 086373, the full disclosures of which are herein incorporated by reference in their entirety for all purposes.

[0250] Some aspects described herein relate to compositions, methods and uses involving more than one, e.g., 2, 3, 4, 5, 6 or even more nucleic acid species, such as RNA species. In an LNP formulation, it is possible that each nucleic acid species is separately formulated as an individual LNP formulation. In that case, each individual LNP formulation will comprise one nucleic acid species. The individual LNP formulations may be present as separate entities, e.g., in separate containers. Such formulations are obtainable by providing each nucleic acid species separately (typically each in the form of a nucleic acid-containing solution) together with suitable cationic or cationically ionizable lipids or lipid-like materials and cationic polymers that allow the formation of LNPs. Respective particles will contain exclusively the specific nucleic acid species that is being provided when the particles are formed (individual particulate formulations).

[0251] The resulting dispersion following the formation of LNPs can be filtered as liquid feed in a single pass mode through a single pass tangential flow filtration (SPTFF) system and recovering the retentate (containing the LNPs) and permeate from the system in separate containers without recirculation through the SPTFF system, thereby filtering the liquid feed.

[0252] Embodiments of the disclosure are further described in the following numbered embodiments:

[0253] E1 ) A method of reducing an amount of double-stranded RNA (dsRNA) produced in an in vitro transcription (IVT) reaction, the method comprising: a) obtaining a DNA template; b) contacting the DNA template with an in vitro transcription (IVT) reaction system to obtain a composition, wherein the IVT reaction system comprises a monovalent salt, spermidine, dithiothreitol (DTT), and dimethylsulfoxide (DMSO); and c) incubating the composition at a temperature between about 15 °C and 45 °C to obtain an IVT reaction, thereby reducing the amount of dsRNA produced in the IVT reaction.

[0254] E2) The method of embodiment 1 , wherein incubating the composition occurs for a time period between about 30 minutes and 64 days.

[0255] E3) The method of embodiment 1 or 2, wherein the IVT reaction system further comprises a buffer, a 5’ cap analog, ATP, CTP, GTP, UTP, a magnesium ion, an RNase inhibitor, pyrophosphatase, and an RNA polymerase.

[0256] E4) The method of any one of embodiments 1 to 3, wherein the monovalent salt is selected from the group consisting of KCI, NaCI, RbCI, CsCI, NaCIO4, NaF, NaBr, and NH4CL

[0257] E5) The method of any one of embodiments 1 to 4, wherein the monovalent salt is present in an amount between about 10 mM and about 150 mM. E6) The method of any one of embodiments 1 to 5, wherein the spermidine is present in an amount between about 0.2 mM and about 10 mM.

[0258] E7) The method of any one of embodiments 1 to 6, wherein the dithiothreitol (DTT) is present in an amount between about 15 mM and about 25 mM.

[0259] E8) The method of any one of embodiments 1 to 7, wherein the dimethylsulfoxide (DMSO) is present in an amount between about 4% and about 15%.

[0260] E9) The method of any one of embodiments 1 to 8, wherein the DNA template is selected from the group consisting of a linearized DNA template, a circular DNA template, and a synthetic DNA template.

[0261] E10) The method of any one of embodiments 1 to 9, further comprising: d) contacting the IVT reaction with a deoxyribonuclease (DNase) to obtain a digested IVT reaction; e) contacting the digested IVT reaction with a protease to obtain an inactivated reaction; and f) contacting the inactivated reaction with a chelating agent to obtain a chelated reaction.

[0262] E11 ) The method of embodiment 10, wherein the protease is proteinase K.

[0263] E12) The method of embodiment 10 or 11 , wherein the chelating agent is ethylenediaminetetraacetic acid (EDTA).

[0264] E13) The method of any one of embodiments 10 to 12, further comprising: g) precipitating an mRNA molecule in the chelated reaction with lithium chloride to obtain a purified mRNA.

[0265] E14) The method of embodiment 13, further comprising: h) incubating a cellulose-based chromatographic medium with a buffer comprising dimethylsulfoxide (DMSO), wherein the DMSO is present in an amount between about 2.5% and 25%; i) contacting the purified mRNA with the cellulose-based chromatographic medium to obtain a bound cellulose-based chromatographic medium; and j) centrifuging the bound cellulose-based chromatographic medium.

[0266] E15) The method of any one of embodiments 1 to 14, wherein the amount of dsRNA produced in the IVT reaction is reduced by at least 10% when compared to an amount of dsRNA produced by a standard method of RNA molecule synthesis.

[0267] E16) A method of reducing an amount of double-stranded RNA (dsRNA) produced in an in vitro transcription (IVT) reaction, the method comprising: a) obtaining a DNA template; b) contacting the DNA template with an in vitro transcription (IVT) reaction system to obtain a composition, wherein the IVT reaction system comprises a monovalent salt, spermidine, dithiothreitol (DTT), and dihydrolevoglucosenone; and c) incubating the composition at a temperature between about 15 °C and 45 °C to obtain an IVT reaction, thereby reducing the amount of dsRNA produced in the IVT reaction.

[0268] E17) The method of embodiment 16, wherein incubating the composition occurs for a time period between about 30 minutes and 64 days.

[0269] E18) The method of embodiment 16 or 17, wherein the IVT reaction system further comprises a buffer, a 5’ cap analog, ATP, CTP, GTP, UTP, a magnesium ion, an RNase inhibitor, pyrophosphatase, and an RNA polymerase.

[0270] E19) The method of any one of embodiments 16 to 18, wherein the monovalent salt is selected from the group consisting of KCI, NaCI, RbCI, CsCI, NaCIO4, NaF, NaBr, and NH4CL

[0271] E20) The method of any one of embodiments 16 to 19, wherein the monovalent salt is present in an amount between about 10 mM and about 150 mM.

[0272] E21 ) The method of any one of embodiments 16 to 20, wherein the spermidine is present in an amount between about 0.2 mM and about 10 mM.

[0273] E22) The method of any one of embodiments 16 to 21 , wherein the dithiothreitol (DTT) is present in an amount between about 15 mM and about 25 mM.

[0274] E23) The method of any one of embodiments 16 to 22, wherein the dihydrolevoglucosenone is present in an amount between about 4% and about 15%.

[0275] E24) The method of any one of embodiments 16 to 23, wherein the DNA template is selected from the group consisting of a linearized DNA template, a circular DNA template, and a synthetic DNA template.

[0276] E25) The method of embodiment 16, further comprising: d) contacting the IVT reaction with a deoxyribonuclease (DNase) to obtain a digested IVT reaction; e) contacting the digested IVT reaction with a protease to obtain an inactivated reaction; and f) contacting the inactivated reaction with a chelating agent to obtain a chelated reaction.

[0277] E26) The method of embodiment 25, wherein the protease is proteinase K. E27) The method of embodiment 25 or 26, wherein the chelating agent is ethylenediaminetetraacetic acid (EDTA).

[0278] E28) The method of any one of embodiments 25 to 27, further comprising: g) precipitating an mRNA molecule in the chelated reaction with lithium chloride to obtain a purified mRNA.

[0279] E29) The method of embodiment 28, further comprising: h) incubating a cellulose-based chromatographic medium with a buffer comprising dimethylsulfoxide (DMSO), wherein the DMSO is present in an amount between about 2.5% and 25%; i) contacting the purified mRNA with the cellulose-based chromatographic medium to obtain a bound cellulose-based chromatographic medium; and j) centrifuging the bound cellulose-based chromatographic medium.

[0280] E30) The method of any one of embodiments 16 to 29, wherein the amount of dsRNA produced in the IVT reaction is reduced by at least 10% when compared to an amount of dsRNA produced by a standard method of RNA molecule synthesis.

[0281] E31 ) A method of reducing an amount of double-stranded RNA (dsRNA) produced in an in vitro transcription (IVT) reaction, the method comprising: a) obtaining a DNA template; b) contacting the DNA template with an in vitro transcription (IVT) reaction system to obtain a composition, wherein the IVT reaction system comprises an (organo)sulfur oxoacid; and c) incubating the composition at a temperature between about 15 °C and 45 °C to obtain an IVT reaction, thereby reducing the amount of dsRNA produced in the IVT reaction.

[0282] E32) The method of embodiment 31 , wherein incubating the composition occurs for a time period between about 30 minutes and 64 days.

[0283] E33) The method of embodiment 31 , wherein the (organo)sulfur oxoacid comprises a tetrahedral or trigonal pyramidal geometry.

[0284] E34) The method of embodiment 31 , wherein the (organo)sulfur oxoacid comprises a sulfonic acid or sulfonic acid derivative.

[0285] E35) The method of embodiment 34, wherein the sulfonic acid comprises an ethanesulfonate.

[0286] E36) The method of embodiment 35, wherein the ethanesulfonate is selected from the group consisting of taurine (2-aminoethanesulfonic acid), PIPES (piperazine-N,N'-bis(2-ethanesulfonic acid), and MES (2-(N-morpholino)ethanesulfonic acid). E37) The method of embodiment 36, wherein the taurine is present in an amount between about 0.01 M and about 2.5 M.

[0287] E38) The method of embodiment 36, wherein the PIPES is present in an amount between about 10 mM and about 180 mM.

[0288] E39) The method of embodiment 34, wherein the sulfonic acid comprises HEPPSO (2- hydroxy-3-[4-(2-hydroxyethyl)piperazin-1-yl]propane-1 -sulfonic acid).

[0289] E40) The method of embodiment 39, wherein the HEPPSO is present in an amount between about 20 mM and about 180 mM.

[0290] E41 ) The method of embodiment 36, wherein the MES is present in an amount between about 10 mM and about 180 mM.

[0291] E42) The method of embodiment 34, wherein the sulfonic acid derivate is selected from the group consisting of a polyanionic synthetic sulfonic acid polymer (SSAP), ethylene dimethanesulfonate, and polystyrene sulfonate.

[0292] E43) The method of embodiment 31 , wherein the (organo)sulfur oxoacid comprises a sulfuric acid or sulfuric acid derivative.

[0293] E44) The method of embodiment 31 , wherein the (organo)sulfur oxoacid is selected from the group consisting of magnesium sulfate, ammonium sulfate, and a heparin derivative.

[0294] E45) The method of embodiment 44, wherein the heparin derivative comprises enoxaparin.

[0295] E46) The method of any one of embodiments 31 to 45, wherein the IVT reaction system further comprises a 5’ cap analog, ATP, CTP, GTP, UTP, a magnesium ion, an RNase inhibitor, pyrophosphatase, and an RNA polymerase.

[0296] E47) The method of any one of embodiments 31 to 44, wherein the IVT reaction system further comprises a buffer.

[0297] E48) The method of embodiment 47, wherein the buffer is selected from the group consisting of Tris and HEPES.

[0298] E49) The method of any one of embodiments 31 to 48, wherein the DNA template is selected from the group consisting of a linearized DNA template, a circular DNA template, and a synthetic DNA template.

[0299] E50) The method of any one of embodiments 31 to 49, further comprising: d) contacting the IVT reaction with a deoxyribonuclease (DNase) to obtain a digested IVT reaction; e) contacting the digested IVT reaction with a protease to obtain an inactivated reaction; and f) contacting the inactivated reaction with a chelating agent to obtain a chelated reaction.

[0300] E51 ) The method of embodiment 50, wherein the protease is proteinase K.

[0301] E52) The method of embodiment 50 or 51 , wherein the chelating agent is ethylenediaminetetraacetic acid (EDTA).

[0302] E53) The method of any one of embodiments 31 to 52, wherein the amount of dsRNA produced in the IVT reaction is reduced by at least 10% when compared to an amount of dsRNA produced by a standard method of RNA molecule synthesis.

[0303] E54) A composition for in vitro transcription (IVT) comprising: a) a DNA template; and b) an in vitro transcription (IVT) reaction system comprising a buffer, a 5’ cap analog, ATP, CTP, GTP, UTP, a magnesium ion, an RNase inhibitor, pyrophosphatase, and an RNA polymerase, wherein the IVT reaction system further comprises: i. 40 mM potassium chloride (KCI);

[0304] II. 0.53 mM spermidine; ill. 20 mM dithiothreitol (DTT); and iv. 10% dimethylsulfoxide (DMSO) or 10% dihydrolevoglucosenone.

[0305] E55) The composition of embodiment 54, wherein the buffer is selected from the group consisting of Tris and HEPES.

[0306] E56) The composition of embodiment 54, wherein the buffer comprises Tris-HCI and PIPES (piperazine-N,N'-bis(2-ethanesulfonic acid).

[0307] E57) The composition of any one of embodiments 54 to 56, wherein the DNA template is selected from the group consisting of a linearized DNA template, a circular DNA template, and a synthetic DNA template.

[0308] E58) A composition for in vitro transcription (IVT) comprising: a) a DNA template; and b) an in vitro transcription (IVT) reaction system comprising a buffer, a 5’ cap analog, ATP, CTP, GTP, UTP, a magnesium ion, an RNase inhibitor, pyrophosphatase, and an RNA polymerase, wherein the IVT reaction system further comprises: i. 0.53 mM spermidine;

[0309] II. 20 mM dithiothreitol (DTT); and ill. 0.5 M taurine. E59) The composition of embodiment 58, wherein the DNA template is selected from the group consisting of a linearized DNA template, a circular DNA template, and a synthetic DNA template.

[0310] E60) A method of reducing an amount of double-stranded RNA (dsRNA) produced in an in vitro transcription (IVT) reaction, the method comprising:

[0311] (a) obtaining a DNA template;

[0312] (b) contacting the DNA template with an in vitro transcription (IVT) reaction system to obtain a composition, wherein the IVT reaction system comprises spermidine, dithiothreitol (DTT), and dihydrolevoglucosenone; and

[0313] (c) incubating the composition at a temperature between about 15 °C and 45 °C to obtain an IVT reaction, thereby reducing the amount of dsRNA produced in the IVT reaction.

[0314] E61 ) The method of embodiment 60, wherein incubating the composition occurs for a time period between about 30 minutes and 64 days.

[0315] E62) The method of embodiment 60 or 61 , wherein the IVT reaction system further comprises a buffer, a 5’ cap analog, ATP, CTP, GTP, UTP, a magnesium ion, an RNase inhibitor, pyrophosphatase, and an RNA polymerase.

[0316] E63) The method of any one of embodiments 60 to 62, wherein the spermidine is present in an amount between about 0.2 mM and about 10 mM.

[0317] E64) The method of any one of embodiments 60 to 63, wherein the dithiothreitol (DTT) is present in an amount between about 15 mM and about 25 mM.

[0318] E65) The method of any one of embodiments 60 to 64, wherein the dihydrolevoglucosenone is present in an amount between about 4% and about 15%.

[0319] E66) The method of any one of embodiments 60 to 65, wherein the DNA template is selected from the group consisting of a linearized DNA template, a circular DNA template, and a synthetic DNA template.

[0320] E67) The method of embodiment 60, further comprising:

[0321] (d) contacting the IVT reaction with a deoxyribonuclease (DNase) to obtain a digested IVT reaction;

[0322] (e) contacting the digested IVT reaction with a protease to obtain an inactivated reaction; and

[0323] (f) contacting the inactivated reaction with a chelating agent to obtain a chelated reaction.

[0324] E68) The method of embodiment 67, wherein the protease is proteinase K. E69) The method of embodiment 67 or 68, wherein the chelating agent is ethylenediaminetetraacetic acid (EDTA).

[0325] E70) The method of any one of embodiments 67 to 69, further comprising:

[0326] (g) precipitating an mRNA molecule in the chelated reaction with lithium chloride to obtain a purified mRNA.

[0327] E71 ) The method of embodiment 70, further comprising:

[0328] (h) incubating a cellulose-based chromatographic medium with a buffer comprising dimethylsulfoxide (DMSO), wherein the DMSO is present in an amount between about 2.5% and 25%;

[0329] (i) contacting the purified mRNA with the cellulose-based chromatographic medium to obtain a bound cellulose-based chromatographic medium; and

[0330] (j) centrifuging the bound cellulose-based chromatographic medium.

[0331] E72) The method of any one of embodiments 60 to 71 , wherein the amount of dsRNA produced in the IVT reaction is reduced by at least 10% when compared to an amount of dsRNA produced by a standard method of RNA molecule synthesis.

[0332] E73) The method of any one of embodiments 60 to 72, wherein the IVT reaction system further comprises a pH between 2.5 and 8 and a tris buffer in a range from 20 mM to 150 mM.

[0333] E74) A method of reducing an amount of double-stranded RNA (dsRNA) produced in an in vitro transcription (IVT) reaction, the method comprising:

[0334] (a) obtaining a DNA template;

[0335] (b) contacting the DNA template with an in vitro transcription (IVT) reaction system to obtain a composition, wherein the IVT reaction system comprises a monovalent salt, spermidine, dithiothreitol (DTT), and dimethylsulfoxide (DMSO), and further wherein the IVT reaction system comprises a pH between 2.5 and 8 and a tris buffer in a range from 20 mM to 150 mM; and

[0336] (c) incubating the composition at a temperature between about 15 °C and 45 °C to obtain an IVT reaction, thereby reducing the amount of dsRNA produced in the IVT reaction.

[0337] E75) A method of reducing an amount of double-stranded RNA (dsRNA) produced in an in vitro transcription (IVT) reaction, the method comprising:

[0338] (a) obtaining a DNA template;

[0339] (b) contacting the DNA template with an in vitro transcription (IVT) reaction system to obtain a composition, wherein the IVT reaction system comprises spermidine, dithiothreitol (DTT), and dihydrolevoglucosenone, and further wherein the IVT reaction system comprises a pH between 2.5 and 8 and a tris buffer in a range from 20 mM to 150 mM; and

[0340] (c) incubating the composition at a temperature between about 15 °C and 45 °C to obtain an IVT reaction, thereby reducing the amount of dsRNA produced in the IVT reaction.

[0341] E76) The method of any one of embodiments 1 to 53 or 60 to 76, or the composition of any one of embodiments 54 to 59, wherein the IVT reaction system further comprises a pH between 2.5 and 8 and a tris buffer in a range from 20 mM to 150 mM.

[0342] EXAMPLES

[0343] Below are examples of specific aspects for carrying out the present invention. The examples are offered for illustrative purposes only, and are not intended to limit the scope of the present invention in any way. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperatures, etc.), but some experimental error and deviation should, of course, be allowed for.

[0344] The practice of the present invention will employ, unless otherwise indicated, conventional methods of protein chemistry, biochemistry, recombinant DNA techniques and pharmacology, within the skill of the art.

[0345] EXAMPLE 1

[0346] INFLUENCE OF MONOVALENT SALTS ON DOUBLE-STRANDED RNA MITIGATION

[0347] Monovalent salts have been shown to exert a strong inhibitory influence on T7 RNA Polymerase (RNAP) activity, mainly attributed to chloride ions competing with plasmid DNA (pDNA) template for anion-binding sites on the RNA Polymerase [Maslak et al., Biochemistry 33(22) (1994)]. T7 RNAP does several rounds of abortive transcription before generating a full-length transcript and abortive transcripts have been shown to contribute to dsRNA formation [Baiersdorfer et al., Mol Ther Nucleic Acids 15 (2019); Martin et al., Biochemistry 27(11 ) (1988). Although some studies have suggested an alternate role for monovalent salts at high salt concentration on abortive transcript formation, this example assessed the effect of monovalent salts on tightening transcription initiation by T7 RNAP in reducing dsRNA formation [Maslak et al., Biochemistry 33(22) (1994)]. To this end, the impact of following monovalent salts on reducing dsRNA during IVT (without impacting other product qualities) was investigated: Potassium Chloride (KCI), Sodium Chloride (NaCI), Rubidium Chloride (RbCI), Cesium Chloride (CsCI) and Ammonium Chloride (NH4CI).

[0348] The reactions were carried out in 1 mL tubes with 0.1 mg / mL linearized 13.9 kb pDNA (encoding a 9.5 kb mRNA); 2 mM CLEANCAP™; 9 mM each of ATP, CTP, GTP, and UTP; 36 mM Mg acetate; 100 U / mL RNase inhibitor; 0.1 U / mL pyrophosphatase; 10,000 U / mL T7 RNAP; and 40 mM of T ris (pH 8.0). The reaction was incubated at 36 C for 170 mins. After IVT, DNase I (8000 U / mg DNA) was added to digest the DNA template, which was followed by 10 minutes of ProK (0.5 U / mL) digestion and EDTA (75 mM) quenching. LiCI-precipitated samples were analyzed for concentration using SoloVPE (or NanoDrop), RNA integrity with Fragment Analysis (FA), and dsRNA by J2 antibody-based ELISA. This IVT process is adopted from IVT processes described in PCT Patent Application Publication No. WO 2023-214339, the IVT processes of which are incorporated by reference herein. Table 1 describes various monovalent salt concentrations used in each experiment and corresponding data obtained.

[0349] Table 1 : Monovalent salt (KCI, NaCI) One Factor at a Time

[0350] NMT = No More Than; dsRNA (pg / ug) = pg of dsRNA per ug of total RNA

[0351] As the concentration of KCI or NaCI increased from 0 mM to 50 mM in IVT buffer (Table 1 ) the IVT yields decreased from 6.1 G / L to 0.8 G / L with no reduction in dsRNA. Similar results were also observed with CsCI, RbCI (Table 2) & NH4CI (Table 3). Overall, these results confirm the inhibitory action of monovalent salts on T7 RNAP activity. Whereas the dsRNA levels remained unaltered with all concentrations of monovalent salts tested, these results suggest that the reduced T7 RNAP activity has no direct role in reducing dsRNA. It is predicted that the monovalent salts may have reduced abortive transcript formation however, the higher dsRNA observed could be due to turn around transcription or anti-sense transcription.

[0352] Table 2: Monovalent salt (CaCI, RbCI) One Factor at a Time

[0353] NMT = No More Than; dsRNA (pg / ug) = pg of dsRNA per ug of total RNA

[0354] Table 3: Monovalent salt (NH4CI) One Factor at a Time dsRNA (pg / ug) = pg of dsRNA per ug of total RNA

[0355] EXAMPLE 2

[0356] EFFECT OF SPERMIDINE, DTT & MONOVALENT SALTS ON DSRNA MITIGATION dsRNA can be formed during IVT by 3’-extension of the run-off transcript folding back on the same RNA molecule. Reducing 3’-extension by aiding the T7 RNAP to drop off from the plasmid DNA template after transcribing the 3’ UTR might reduce dsRNA formed by this mechanism. Polyamines, including spermidine, may enable T7 RNAP dissociation from the plasmid DNA template as well as inhibit RNA synthesis by exogenous RNA [Frugier et al., Nucleic Acids Res 22 (1994); Kartje et al., J Biol Chem 296 (2021 )]. Additionally, dithiothreitol (DTT) is a reducing agent commonly added in enzyme preparations for reducing protein oxidation. Although studies have shown that adding up to 10 mM DTT in IVT doesn’t impact transcription, there are no reports on the effect of higher DTT concentration on transcription efficiency [Kartje et al., J Biol Chem 296 (2021 )]. This example tested if adding spermidine in IVT buffer will reduce dsRNA formed by 3’-extended run-off transcripts. This example also evaluated the combined effect of spermidine, DTT and monovalent salts on reducing dsRNA.

[0357] Reactions were carried out at the 1 mL scale as described in Example 1 above, with the exception of one factor at a time conditions wherein various concentrations of spermidine were tested (Table 4), or a combination of spermidine, DTT and 40 mM (Table 5) or 25 mM (Table 6) of either monovalent salt. A spermidine concentration of 2.15 mM was used in combination with 40 mM of monovalent salts and 0.53 mM with 25 mM of monovalent salts, respectively. DTT was used at 20 mM concentration.

[0358] As shown in Table 4, increasing the concentration of spermidine in IVT buffer from 1.07 mM to 8.6 mM significantly reduced mRNA yields with no reduction in dsRNA and with little impact on RNA integrity. It can be inferred that higher concentration of monovalent salts and spermidine both independently reduce IVT yields by negatively impacting T7 RNAP activity. However, a combination of spermidine and DTT had no impact on IVT yields but slightly reduced dsRNA (Table 5, condition A). Interestingly when either one of the monovalent salts was combined with 2.15 mM spermidine and 20 mM DTT at a 40 mM concentration there was a significant reduction in mRNA yield as well as dsRNA (Table 5, conditions L-O). To increase the mRNA yield while keeping the dsRNA levels reduced, the spermidine concentration was reduced from 2.15 mM to 0.53 mM and the monovalent salt concentration was reduced from 40 mM to 25 mM while keeping the same DTT concentration. As shown in Table 6 below, reducing both spermidine and monovalent salt concentration in combination with DTT significantly increased IVT yield with a slight increase in dsRNA. Results from these experiments suggest an overall inhibitory influence of spermidine and monovalent salts on IVT yields and the dsRNA impurity.

[0359] Table 4: Effect of Spermidine / DTT on dsRNA impurity NMT = No More Than; NT = Not Tested; dsRNA (pg / ug) = pg of dsRNA per ug of total RNA

[0360] Table 5: Combination effect of Spermidine / DTT with monovalent sa t on dsRNA impurity dsRNA (pg / ug) = pg of dsRNA per ug of total RNA Table 6: Combination effect of decreased Spermidine / DTT with monovalent salt on dsRNA impurity EXAMPLE 3

[0361] COMBINING DMSO WITH SPERMIDINE, DTT & MONOVALENT SALT IN IVT BUFFER REDUCES DSRNA WHILE MAINTAINING MRNA YIELD AND INTEGRITY

[0362] DMSO may increase IVT yields by altering T7 RNAP structure and transcription rate by synthesizing both abortive and full-length transcripts [Chen et al., Biochem Biophys Res Commun 333 (2005)]. This example tests whether combining monovalent salts and spermidine with DMSO would selectively increase mRNA yield of full-length transcripts while reducing dsRNA. This example tests a range of DMSO concentrations in IVT buffer without adding monovalent salts, spermidine or DTT as well as combining all four together.

[0363] Reactions were carried out at a 1 mL scale as described in Example 1 with one factor at a time conditions for DMSO alone or with a combination of 0.53 mM of spermidine, 20 mM DTT and 40 mM of each monovalent salts.

[0364] The addition of either 5 or 10% DMSO in IVT buffer increased RNA yield by approximately 20% (Table 7) with no reduction in dsRNA. However, when 10% DMSO was combined with 0.53 mM spermidine and 20 mM DTT, there was significant reduction (55%) in dsRNA levels while maintaining mRNA yield and integrity (Table 8, Condition C). This data suggests spermidine significantly reduces dsRNA in the presence of DMSO. Next, a combination of either of the monovalent salts with spermidine, DTT, and DMSO was evaluated. These combinations saw a further reduction in dsRNA (55%) with no impact on mRNA yield or integrity (Table 8, Conditions D - H). A combination of monovalent salt, spermidine, DTT and DMSO in IVT buffer reduced dsRNA levels to approximately 600 pg / μg from NMT 3000 pg / μg observed in platform control. Since buffer pH plays a significant role in all enzymatic reactions, altering the IVT buffer pH and Tris concentration to further reduce dsRNA levels were tested. As shown in Table 9, both reducing pH (Table 9-A) and increasing Tris concentration (Table 9-B) significantly reduced dsRNA in combination with spermidine and DTT. Next, KCI and Tris concentrations were titrated in conjunction with a lower buffer pH (7.0). Doubling KCI concentration alone from 40 mM to 80 mM with low buffer pH significantly reduced dsRNA (Table 9-C) with no negative impact on mRNA yield and integrity. Additionally, when Tris concentration was increased from 40 mM to 100 mM and combined with 40 mM KCI, a similar decrease in dsRNA was seen with no impact on other product qualities (PQs) (Table 9-D). However, when KCI concentration was increased to 80 mM (Table 9-E) or 120 mM (Table 9-F) with 100 mM Tris, a significant decrease in dsRNA was seen with no alterations in other PQs. All KCI titrations with lower pH and higher Tris concentrations were performed with Spermidine, DTT and DMSO.

[0365] Table 7: Combination effect of DMSO on dsRNA impurity

[0366] Table 8: Combination effect of DMSO, Spermidine / DTT with monovalent salt on dsRNA impurity NMT = No More Than; dsRNA (pg / ug) = pg of dsRNA per ug of total RNA

[0367] Table 9: Combination effect of DMSO, Spermidine / DTT, monovalent salt and lower pH on dsRNA impurity dsRNA (pg / ug) = pg of dsRNA per ug of total RNA EXAMPLE 4 DIHYDROLEVOGLUCOSENONE (CYRENE) ADDITION TO IVT BUFFER INHIBITS TRANSCRIPTION

[0368] Dimethylsulfoxide (DMSO) has been shown to have no acute oral toxicity and is thought to have low mutagenicity and ecotoxicity. However, DMSO presents the environmental issue of forming sulphur dioxide gas upon incineration, which can contribute to acid rain [Camp et aL, RSC Med Chem 11 (1 ) (2020)]. Dihydrolevoglucosenone (CYRENE™), is a polar yet aprotic solvent produced from cellulose [Sherwood et aL, Chem Commun 50(68) (2014)]. CYRENE has one carbonyl group and two ether groups granting it good hydrogen bond accepting capacity. CYRENE is derived from waste biomass, is biodegradable and has low mutagenicity with no acute oral toxicity [Zhang et aL, ACS Sustainable Chem Eng 4 (2016); Camp et aL, RSC Med Chem 11 (1 ) (2020)]. Recent studies have demonstrated the suitability of CYRENE as a replacement solvent for DMSO in antibacterial susceptibility testing. This example tested whether CYRENE has a similar influence to DMSO on T7 RNAP and increases the transcription efficiency resulting in an environmentally greener manufacturing process.

[0369] Reactions were carried out at the 1 mL scale as described in Example 1 above, with exception of one factor at a time conditions wherein various concentrations of Cyrene were tested (Table 10) and CLEANCAP™ was used at 1 mM for all reactions. Since CLEANCAP™ concentration has a negative impact on mRNA yield, and magnesium could alleviate this inhibition, CYRENE concentrations with a lower and higher dose of magnesium in the IVT buffer were tested. In addition to this these reactions did not include spermidine, DTT or any of the monovalent salts.

[0370] Surprisingly, including CYRENE in the IVT buffer reduced IVT yields in a dose dependent manner with maximum inhibition at 10% (Table 10-C, J). These results suggest an exact opposite influence of CYRENE on T7 RNAP as compared to DMSO. In addition to this only 5% CYRENE showed 50% reduction in dsRNA with significantly reduced mRNA yields and no impact on RNA integrity (Table 10-D-K). Whereas higher mRNA yields were observed with higher magnesium concentration for all tested CYRENE concentrations, the RNA integrity was negatively impacted (Table 10-1, P). Overall, these results suggest an inhibitory role for CYRENE on transcription with a potential to reduce dsRNA.

[0371] Table 10: Effect of Cyrene on reducing dsRNA in IVT dsRNA (pg / ug) = pg of dsRNA per ug of total RNA

[0372] EXAMPLE 5

[0373] CYRENE IN COMBINATION WITH SPERMIDINE AND DTT REDUCED DSRNA

[0374] Polyamines have been shown to stimulate DNA dependent RNA synthesis [N Moussatche, Biochim Biophys Acta 826(2-3) (1985)]. In addition to this spermidine has been specifically shown to stimulate RNA synthesis by acting on the elongation phase of RNA synthesis and had no effect on initiation phase [Jain et al., Mol Cell Biochem 78(1) (1987)]. Also, DTT is shown to reduce protein oxidation and preserve enzyme function. This example tested whether including both spermidine and DTT in the IVT buffer along with CYRENE may improve transcription efficiency while reducing dsRNA.

[0375] Reactions were carried out at the 1-mL scale as described in Example 1 above, with exception of one factor at a time conditions wherein various concentrations of CYRENE were tested (Table 11 ). In addition, these reactions included spermidine (0.53 mM) & DTT (20 mM) along with 36 mM Mg+2.

[0376] Combining spermidine and DTT with CYRENE did increase mRNA yield, reduce dsRNA and maintain integrity (Table 11-A-E). The most significant reduction in dsRNA (3-fold) was observed with 5% CYRENE (Table 11-C) along with a 3-fold reduction in mRNA yield. These results suggest that including spermidine and DTT in the IVT buffer with CYRENE improves T7 RNAP transcription efficiency. dsRNA (pg / ug) = pg of dsRNA per ug of total RNA EXAMPLE 6

[0377] INCREASING MAGNESIUM CONCENTRATION IN IVT BUFFER INCREASES MRNA YIELD WITH CYRENE WHILE REDUCING DSRNA

[0378] T7 RNA Polymerase requires magnesium to function, and previous studies have recommended using 6 mM MgCh above the nucleotide concentration for optimal transcriptional efficiency [Milligan et aL, Methods Enzymol 180 (1989)]. Additionally, magnesium forms precipitates with pyrophosphates during transcription and may alter the available free Mg+2for T7 RNAP function [Kartje et aL, J Biol Chem 296 (2021 )]. Since previous studies with CYRENE showed significant transcription inhibition, this example assessed whether including higher magnesium concentration in the IVT buffer would alleviate this transcription inhibition.

[0379] Reactions were carried out at the 1 mL scale as described in Example 1 above, with exception of one factor at a time conditions wherein various concentrations of CYRENE were tested (Table 12 & 13). Additionally, these reactions included spermidine (0.53 mM) and DTT (20 mM) along with 46 mM Mg+2(Table 12) and 60 mM Mg+2(Table 13).

[0380] As shown in Table 12, increasing Mg+2concentration to 46 mM alone doubled the mRNA yields for all tested concentrations of CYRENE compared to 36 mM Mg+2and similar CYRENE concentrations as in Table 11 . Interestingly, a 2-fold reduction in mRNA yield was observed with 5% CYRENE as in the earlier experiment (Table 11 ) with similar reduction in dsRNA and no impact on RNA integrity. In the next set of experiments, Mg+2concentration was further increased to 60 mM in IVT buffer in combination with various concentrations of CYRENE. This higher Mg+2concentration significantly increased IVT yields when compared to 36 mM Mg+2(3-fold) (Table 11 ) or (1 .5-fold) when compared to 46 mM Mg+2(Table 12). Although higher mRNA yields were obtained with 5% CYRENE (Table 13-D) and significantly reduced dsRNA, the RNA integrity reduced by 10% when compared to either 36 mM or 46 mM Mg+2concentrations (Table 11 & 12). Overall, these conditions reduced dsRNA levels to <500 pg / ug mRNA with 5% CYRENE and 60 mM Mg+2along with spermidine and DTT.

[0381] Table 12: Effect of higher Mg+2concentration (46 mM) and CYRENE in reducing dsRNA in IVT dsRNA (pg / ug) = pg of dsRNA per ug of total RNA

[0382] Table 13: Effect of higher Mg+2concentration (60 mM) and CYRENE in reducing dsRNA in IVT dsRNA (pg / ug) = pg of dsRNA per ug of total RNA

[0383] EXAMPLE 7

[0384] DECREASING PH OF IVT BUFFER INCREASES YIELD WITH CYRENE WHILE REDUCING DSRNA

[0385] Proton concentration influences enzyme activity and titrating pH may be advantageous in improving mRNA yield and integrity. Since reduced mRNA yields and integrity were obtained with CYRENE even at 60 mM Mg+2concentration, this example tested a range of different pH values with Tris and HEPES IVT buffers. The goal was to improve T7 RNAP activity in terms of mRNA yield and integrity while keeping dsRNA low in the IVT process.

[0386] Reactions were carried out at the 1 mL scale as described in Example 1 above, with exception of one factor at a time conditions wherein various ranges of pH were tested (Table 14) with both Tris and HEPES buffer. Additionally, these reactions included spermidine (0.53 mM) & DTT (20 mM) along with 60 mM Mg+2and CYRENE at 5% concentration.

[0387] Reducing Tris buffer pH from 8.0 to 7.0 significantly increased RNA yield and reduced dsRNA with no considerable change in RNA integrity (Table 14-A-C). Although a more pronounced increase in mRNA yield was seen with reduced pH in HEPES buffer, the dsRNA levels were slightly increased and so was the RNA integrity (Table 14-D-E). Whereas earlier studies have shown a pH about 8.0 to benefit transcription, current results in the presence of CYRENE suggest an alternate mechanism. Overall, by using a 40 mM Tris buffer at pH 7.0 in combination with 60 mM Mg+2, 5% CYRENE, 0.53 mM Spermidine and 20 mM DTT it was possible to reduce the dsRNA levels to under 500 pg / μg mRNA with good mRNA yield and comparable integrity. dsRNA (pg / ug) = pg of dsRNA per ug of total RNA

[0388] This example also tested the effect of lowering pH of Tris-based IVT buffers and saw a reduction in dsRNA while maintaining both mRNA yield and integrity, without the need to increase Mg2+ concentration. Both lowering pH and increasing Tris Concentration of IVT buffer further reduced dsRNA while maintaining IVT yields with monovalent salt treatment.

[0389] Based on recent studies highlighting the significance of lower pH on transcription rate [Rosa et al., Biotechnol Bioeng 119 (2022); Kern & Davis, Biotechnology Progress 13 (1997)] it was hypothesized that combining a lower pH with increased Tris concentration in the IVT buffer could selectively enhance full-length transcripts and reduce dsRNA. Experiments were conducted using a range of CYRENE concentrations without adding extra Mg2+. This approach aims to maintain IVT yields under CYRENE-treated conditions while avoiding the negative impact on RNA integrity associated with higher Mg2+concentrations. The results from these experiments should provide valuable insights for creating the optimal conditions in producing high quality transcripts with minimal dsRNA impurity.

[0390] Reactions were carried out at a 1 mL scale as described in Example 1 with OFAT conditions wherein pH 7.0 and different concentration of Tris (40 or 100 mM) with either 2.5% or 5% of CYRENE along with 36 mM Mg2+.

[0391] It was discovered that lowering the pH in IVT buffer was effective in maintaining mRNA yield when using either 2.5 % or 5% CYRENE, while also reducing dsRNA impurities. Notably, with 5% CYRENE, the dsRNA was reduced to below 500 pg / ug with only a 17% reduction in mRNA yield, and this was achieved without the additional of extra Mg2+(Table 15, condition 28-B). This adjustment also resulted in a slight improvement in RNA integrity. Furthermore, at a lower pH with increased Tris concentration, the mRNA yield was maintained with a slight improvement in RNA integrity (Table 15, condition 28-B). Under CYRENE-treated conditions, although the mRNA yield was a bit lower compared to 40 mM Tris, there was a significant decrease in dsRNA and an enhancement in RNA integrity (Table 15, condition 11-H and 11-1). This suggests that optimizing pH and Tris concentration can improve the IVT process without relying on increased Mg2+concentration.

[0392] Table 15: Effect of lower pH and increased Tris concentration, combined with CYRENE in reducing dsRNA in IVT (Tris pH 7.0 136 mM Mg2+10.53 mM spermidine 120 mM DTT) dsRNA (pg / ug) = pg of dsRNA per ug of total RNA

[0393] EXAMPLE 8

[0394] DSRNA MITIGATED IVT BUFFER USING VARIOUS RNA CONSTRUCTS

[0395] This example evaluated whether the IVT buffer developed by combining monovalent salt, spermidine, DTT and DMSO could be used with different plasmid DNA (pDNA) constructs. To achieve this goal, a 5.1 kb plasmid DNA construct (encoding a 2.1 kb modRNA) and a 3.2 kb plasmid DNA construct (encoding a 0.9 kb mRNA for the light chain of a monoclonal antibody) (mAb-LC) were used for manufacturing mRNA using an IVT process with the IVT buffer described above.

[0396] Reactions were carried out at the 1 mL scale with 2.5 mM of CLEANCAP™ molecule with (or without) 10% DMSO, 0.53 mM of spermidine, 20 mM DTT and 40 mM of each monovalent salt. All reactions used the same process parameters as the reference protocol, except for the one factor at a time condition, and all reactions used the same pDNA. The experimental conditions used for each reaction are in the table below.

[0397] As shown in Table 16 below, both constructs (modRNA in conditions A & B; mAb-LC in conditions C & H) gave comparable mRNA yield and integrity with the new IVT buffer while reducing dsRNA. A more robust decrease in dsRNA occurred with the modRNA pDNA (compare condition A to condition B; 15-fold) than the mAb-LC (compare condition C to condition H; 2.5-fold). These results confirm the dsRNA lowering capability of the new IVT buffer across various pDNA constructs for mRNA manufacturing using an IVT process.

[0398] Table 16: General applicability of dsRNA mitigated IVT buffer on modRNA (conditions A & B) and mAb-LC (conditions C & H) dsRNA (pg / ug) = pg of dsRNA per ug of total RNA

[0399] EXAMPLE 9

[0400] DEMONSTRATING EFFECTIVENESS OF DSRNA REDUCING IVT BUFFERS WITH AN ALTERNATIVE ANTIGEN

[0401] The goal of this experiment was to evaluate whether the IVT buffers developed by combining monovalent salt, spermidine, DTT, and DMSO or by combining CYRENE, spermidine and DTT could reduce dsRNA levels with an alternative antigen construct. To achieve this goal, the test buffers were evaluated with a qualified scaled down model for manufacturing.

[0402] IVT reactions were carried out at the 8 mL AMBR15 scale (IVT platform process #2) either with a combination of 40 mM of monovalent salt, 0.53 mM of spermidine, 20 mM DTT and 10% DMSO or 5% CYRENE in combination with 0.53 mM of spermidine, and 20 mM DTT. In addition to the dsRNA mitigation buffer components listed above, the starting reaction mixture contained 11 mM of ATP (Final 24 mM), 9 mM CTP (Final 22 mM), 1 mM GTP (Final 16 mM), and 4 mM UTP (Final 13 mM), 2.5 mM CLEANCAPTM-AG, 40 mM Tris at pH 8.0, 25 mM Mg acetate (Final 60 mM), 100 U / mL RIBOLOCK™, 0.1 mg / mL of a 5.0 kb plasmid DNA construct (encoding a 2.0 kb RNA), 10,000 U / mL T7 polymerase, and 0.25 U / mL pyrophosphatase. The reactions were incubated at 37 °C for 180 minutes with starting agitation of 420 rpm. After IVT, DNase I (2,000 U / mg DNA) was added to digest the DNA template, which was followed by 10 minutes of ProK (0.5 U / mL) digestion and EDTA (75 mM) quenching. LiCI-precipitated samples were analyzed for concentration using SOLOVPE™ (or NANODROP™), RNA integrity with Fragment Analysis (FA), and dsRNA by J2 antibody-based ELISA. All reactions used the same process parameters as the reference protocol, except for the one factor at a time condition, and all reactions used the same pDNA. The experimental conditions used for each reaction can be found in the table below.

[0403] As shown in Table 17 below, a significant reduction in dsRNA levels was observed using monovalent salt with the alternative antigen construct in the IVT buffer spermidine, DTT, and DMSO at both pH 7.0 and 8.0 (Table 17-B & E). Additionally, there was an increase in mRNA yield with the test buffer, however, the RNA integrity remained unaltered compared to control platform condition (Table 17-A & D). As expected, the test buffer combining CYRENE, spermidine, and DTT also gave significant reduction in dsRNA with the alternative antigen construct (Table 18-C). However, with Cyrene treatment, there was about a 40% reduction in mRNA yield with no change in RNA integrity (Table 18, A-C). Results from this study clearly highlight the effectiveness of both test buffers in reducing dsRNA even while using scale-up qualified manufacturing model.

[0404] Table 17: Effectiveness of an IVT buffer containing monovalent salt on dsRNA reduction with an alternative antigen construct dsRNA (pg / ug) = pg of dsRNA per ug of total RNA Table 18: Effectiveness of an IVT buffer containing CYRENE on reducing dsRNA with an alternative antigen construct dsRNA (pg / ug) = pg of dsRNA per ug of total RNA

[0405] EXAMPLE 10 SCALABILITY OF IVT BUFFER DEVELOPED USING MONOVALENT SALTS

[0406] This example tested the new IVT buffer developed by combining 40 mM KCI (monovalent salt), 0.53 mM spermidine, 20 mM DTT and 10% DMSO in AMBR 250 for reducing dsRNA at manufacturing scale. This was done using an IVT process with the above buffer with no other modifications.

[0407] An IVT reaction was carried out at 80 mL scale in the AMBR 250 Modular bioreactor system. The reaction was incubated at 36 °C for 170 minutes with starting agitation of 290 rpm. The starting reaction mixture contained 9 mM of each NTPs (including regular UTP), 4 mM CLEANCAP™- AU, 40 mM Tris at pH 8.0, 36 mM Mg acetate, 0.53 mM spermidine, 20 mM DTT, 10% DMSO, 40 mM of KCI, 100 U / mL RIBOLOCK™, 0.1 mg / mL of a 13.9 kb pDNA (encoding a 9.5 kb selfamplifying mRNA), 10000 U / mL T7 polymerase, and 0.1 U / mL pyrophosphatase. After IVT, DNase I (8000 U / mg DNA) was added to digest the DNA template, which was followed by 10 minutes of ProK (0.5 U / mL) digestion and EDTA (75 mM) quenching. A fraction of this reaction was also purified by using Oligo dT purification. LiCI-precipitated samples were analyzed for concentration using SOLOVPE™ (or NANODROP™), RNA integrity with Fragment Analysis (FA), and dsRNA by J2 antibody-based ELISA

[0408] As shown in the table below, post-IVT mRNA yield was at 10 g / L with RNA integrity of about 63%, with a significant reduction in dsRNA levels of <500 pg / μg RNA (Table 19-A). Further, the saRNA drug substance obtained after platform purification process showed a further reduction of dsRNA (342 pg / ug) and an RNA integrity of 84%. These results confirm the dsRNA lowering ability of the new IVT buffer which combines monovalent salt, spermidine, DTT and DMSO as additional components. Additionally, this example successfully demonstrated the scalability of IVT process using this buffer for reducing dsRNA.

[0409] Table 19: Manufacturing scale evaluation of new IVT buffer with monovalent salt, spermidine, DTT and DMSO dsRNA (pg / ug) = pg of dsRNA per ug of total RNA

[0410] EXAMPLE 11 DSRNA REMOVAL USING CELLULOSE CHROMATOGRAPHY

[0411] Cellulose chromatography may efficiently remove dsRNA from IVT mRNA, which can be an alternative to ion-pair reversed phase HPLC which is currently being used to purify IVT mRNA. Although, the cellulose chromatography efficiently removes dsRNA it uses 16% ethanol in binding buffer, which poses a challenge for manufacturing. The examples above demonstrate that including DMSO in IVT buffer in combination with monovalent salts, spermidine and DTT helped reduce dsRNA formation in IVT. Accordingly, replacing 16% ethanol with 10% DMSO may eliminate dsRNA from IVT material purified with cellulose chromatography.

[0412] An IVT reaction was carried out at 1 mL scale as described above in Example 1 , using standard IVT platform buffer and IVT conditions. At the end of IVT, 300 μg of LiCI purified IVT mRNA was loaded onto cellulose packed microcentrifuge columns. Three chromatography buffers were evaluated: 10 mM HEPES (pH 7.2), 0.1 mM EDTA, 125 mM NaCI and either 16% Ethanol (Buffer 1 ) or 16% Isopropanol (Buffer 2) or 10% DMSO (Buffer 3). Cellulose fibers were suspended in each buffer at a concentration of 0.2 G / mL and incubated for 10 minutes with shaking. 1 .4 G of cellulose fibers from the suspension were transferred to a spin column and centrifuged, the flow- through was discarded and 500 μl of each buffer (Buffers 1 - 3) was added to the respective columns, incubated for 5 minutes with shaking, and then centrifuged. Subsequently, 300 μg of LiCI purified IVT mRNA was loaded onto each column and incubated for 30 minutes followed by centrifugation. Following centrifugation, the flowthrough was reapplied to the column and incubated for 30 minutes. At the end of 30-minute incubation the columns were spun, and mRNA was precipitated from the flow-through using 3 M NaOAc (pH 5.5) and 1 volume of isopropanol. In the purified samples, mRNA concentration was measured using NANODROP™, RNA integrity with Fragment Analysis (FA), and dsRNA by J2 antibody-based ELISA.

[0413] Cellulose chromatography purified mRNA had similar mRNA yield and integrity as platform process generated material (Table 20). Interestingly, using 10% DMSO in chromatography buffer significantly reduced dsRNA with about 80% recovery of IVT material (Table 20-C). mRNA purified using both Isopropanol and DMSO containing buffers showed similar integrity and dsRNA reduction in final drug substance (DS) (Table 20-B, C). Higher recovery and lower dsRNA was observed with Buffer 1 compared to the other 2 buffers (Table 20-A). These results suggest using 10% DMSO in chromatography buffer for mRNA purification using cellulose chromatography significantly removes dsRNA with no impact on RNA integrity.

[0414] Table 20: Purifying IVT mRNA with cellulose chromatography using either Ethanol, Isopropanol or DMSO dsRNA (pg / ug) = pg of dsRNA per ug of total RNA

[0415] EXAMPLE 12

[0416] TAURINE ADDITION DURING IVT REDUCES DSRNA FORMATION

[0417] A predominant pathway of dsRNA formation in in vitro transcription (IVT) is the result of the RNA- dependent RNA polymerase activity of phage T7 RNA polymerase. In this pathway, the runoff transcripts can anneal to complementary sequences in cis or trans to form RNA duplexes that can rebind to the RNA polymerase, which extends the 3’ end using RNA as a template. Since RNA duplex is not the preferred substrate for T7 RNA polymerase, RNA-dependent synthesis is inefficient relative to promoter-driven DNA-dependent synthesis. A potential strategy to reduce dsRNA formation in IVT is to create a weakly competitive environment by inclusion of competitor molecules that, ideally, prevent the less desirable RNA template, but not the preferred DNA template, from binding the RNA polymerase.

[0418] RNA polymerases require nucleic acid templates to transcribe. The structural framework of nucleic acids, including DNA and RNA, is formed by a sugar phosphate backbone. Phosphate and sulfate groups share similar physico-chemical properties such as overall size, broad geometry, polyprotic Lewis basicity, and the electronegativity of central atoms [Lima et al., J R Soc Interface 19(193) (2022)]. Additionally, heparin (containing sulfate groups), polyethanesulfonate, and Congo red dye (containing sulfonate groups) were hypothesized to bind to bacterial RNA polymerase at or near the site used for DNA binding, to inhibit RNA synthesis [Chamberlin et al., J Biol Chem 248(6) (1973)]. This example tested whether certain (organo)sulfur oxoacid compounds, such as sulfonate or sulfate groups, could reduce dsRNA formation in IVT processes by means of weak competition for phosphate-binding sites in phage T7 RNA polymerase. Therefore, the addition of taurine (2-aminoethanesulfonic acid), an organic amino sulfonic acid and stabilizing osmolyte widely distributed in animal tissues, was first tested for the purpose of dsRNA reduction during IVT.

[0419] Osmolytes are small organic solutes that are taken up or synthesized by cells in response to changes is osmotic pressure. Osmolytes belong to diverse chemical families including methylamines, polyhydric alcohols, and amino acids. Most osmolytes stabilize protein structures, except protein denaturants such as urea and guanidine hydrochloride. Due to the general RNA- secondary-structure disruptive characteristic of stabilizing osmolytes [Lambert et al., J Mol Biol 370(5) (2007)], other stabilizing osmolytes (TMAO, sorbitol, trehalose, sucrose, PEG 1000, PEG 6000, 1 ,2-propanediol, betaine, proline, glycine, and ectoine) were also included in this experiment for comparison with Taurine. The denaturing osmolyte urea, which is a known chaotrope and was shown to reduce dsRNA in IVT [Piao et al., Mol Ther Nucleic Acids 29 (2022)], was also included as a comparison. Additionally, a non-organic ammonium sulfate salt was also included in this example.

[0420] A 9.5 kb self-amplifying RNA (saRNA) was synthesized by IVT. A mixture of linearized 13.9 kb DNA template (0.05 mg / mL), rNTPs (5 mM each), CLEANCAP™ AU (4 mM), taurine or other compounds (at specified concentrations below), reaction buffer (40 mM HEPES, pH 7.3, 10 mM DTT, 2 mM spermidine, 0.002% triton X-100, 16.5 mM magnesium acetate), T7 RNA polymerase (4000 U / mL), RIBOLOCK™ (1000 U / mL), inorganic pyrophosphatase (2 U / mL) was prepared in a final volume of 1 mL. The IVT reaction was incubated for 150 minutes at 37 °C. For the experiment testing the effects of proline and urea (Table 21 ), DNase I (8000 U / mg DNA) was added to digest the DNA template after IVT, which was followed by 10 minutes of ProK (0.5 U / mL) digestion and EDTA (75 mM) quenching. To simplify operational complexities, for experiments performed later (Tables 22-24), only EDTA (75 mM) was added to quench the IVT reaction before LiCI precipitation, which is known to precipitate RNA, not DNA or proteins. LiCI-precipitated samples were analyzed in house for SOLOVPE™ (or NANODROP™) concentration, Fragment Analysis (FA) integrity, and dsRNA ELISA analyses.

[0421] As shown in one-factor-at-a-time (OFAT) analysis of Tables 21-24, when testing the addition of 12 different stabilizing osmolytes to IVT reactions using a relatively lower-yield process, it was found that methylamines (TMAO) and polyhydric alcohols (sorbitol, trehalose, sucrose and PEGs) generally increased dsRNA levels with the exception of 1 ,2-Propanediol, a synthetic osmolyte, while amino acids / amino acid derivatives (betaine, proline, glycine, ectoine, and taurine) generally reduced dsRNA levels to different extents. All amino acids / amino acid derivative treatments also had the added benefit of increased integrity, likely due to weak chelation of Mg2+ions. Among the amino acids / amino acid derivatives tested, the only amino sulfonic acid, taurine, stood out due to its potency on dsRNA reduction yet with less severe impairment on mRNA yield, under the conditions tested. The addition of the chaotropic urea, which was known to reduce dsRNA, was included as a comparison. A non-organic ammonium sulfate salt was also tested, which aborted transcription in general, likely due to increased ionic strength of the solution. dsRNA (pg / ug) = pg of dsRNA per ug of total RNA

[0422] Table 23: Osmolyte (Sucrose, TMAO, and Betaine) and ammonium sulfate salt OFAT dsRNA (pg / ug) = pg of dsRNA per ug of total RNA

[0423] Table 24: Osmolyte (Glycine, ectoine, taurine, 1,2-propanediol, PEG 1000, PEG 6000) OFAT dsRNA (pg / ug) = pg of dsRNA per ug of total RNA

[0424] EXAMPLE 13

[0425] GENERAL APPLICABILITY OF TAURINE TREATMENT ACROSS DIFFERENT IVT CONDITIONS AND OPTIMIZATION FOR HIGHER-YIELD TRANSCRIPTION

[0426] To improve mRNA yield and to test the general applicability of taurine treatment across different IVT conditions, a one-factor-at-a-time (OFAT) experiment was performed varying pH, [MgOAc], and the presence or absence of spermidine / DTT with or without 0.5 M taurine. The reasons for varying these variables have been justified in detail in earlier examples of this application. The ideal outcome of this experiment would be the reduction of dsRNA impurities while maintaining or improving other product quality attributes (mRNA yield and integrity).

[0427] A 9.5 kb self-amplifying RNA (saRNA) was synthesized by IVT. A mixture of a 13.9 kb linearized DNA template (0.1 mg / mL), rNTPs (9 mM each), CLEANCAP™ AU (1 mM), taurine (0 or 0.5 M), reaction buffer (40 mM Tris, pH 8.0, 20 mM DTT, 0.53 mM spermidine, 36 mM magnesium acetate), T7 RNA polymerase (10000 U / mL), RIBOLOCK™ (100 U / mL), inorganic pyrophosphatase (0.25 U / mL) was prepared in a final volume of 1 mL, unless otherwise specified in conditions below. IVT reaction was incubated for 170 minutes at 36 °C. After IVT, EDTA (75 mM) was added to quench the IVT reaction. LiCI-precipitated samples were analyzed in house for SOLOVPE™ concentration, FA integrity, and dsRNA ELISA analyses

[0428] As shown in Table 25, under the conditions tested, lower pH and higher concentration of magnesium acetate (MgOAc) improved mRNA yield. Additionally, lower pH, the addition of spermidine / DTT, and increased concentration of magnesium acetate reduced dsRNA formation. Despite these variations in IVT parameters, 0.5 M taurine consistently reduced dsRNA and improved integrity, while having minimal negative effects on mRNA yield, demonstrating the general applicability of taurine treatment across various IVT conditions. Although the dsRNA- reducing effect of taurine is dampened with higher [MgOAc], which, on its own reduces dsRNA but also decreases integrity, the addition of taurine partially rescued the lower integrity at higher [Mg2+],

[0429] Table 25: Taurine treatment across different IVT conditions with varied pH, [Mg2+] and the presence or absence of spermidine / DTT dsRNA (pg / ug) = pg of dsRNA per ug of total RNA

[0430] EXAMPLE 14 SULFONATE-CONTAINING BUFFERING SPECIES IN IVT

[0431] Many biological buffers (e.g., Good’s buffers) are compounds containing sulfonate groups, but this example focused on the ability of alternative buffering species at pH 6.5 and pH 7.0 to reduce dsRNA.

[0432] A 9.5 kb self-amplifying RNA (saRNA) was synthesized by IVT. A mixture of a 13.9 kb linearized DNA template (0.1 mg / mL), rNTPs (9 mM each), CLEANCAP™ AU (1 mM), reaction buffer (40 mM Tris, pH 7.0, 20 mM DTT, 0.53 mM spermidine, 36 mM magnesium acetate), T7 RNA polymerase (10000 U / mL), RIBOLOCK™ (100 U / mL), inorganic pyrophosphatase (0.25 U / mL) was prepared in a final volume of 0.2 mL, unless otherwise specified in conditions below. The IVT reaction was incubated for 170 minutes at 36 °C. After IVT, EDTA (75 mM) was added to quench the IVT reaction. LiCI-precipitated samples were analyzed in house for SOLOVPE™ concentration, FA integrity, and dsRNA ELISA analyses. As shown in Tables 26 and 27, most buffering species tested are suitable for IVT reactions, except for phosphate, sodium citrate, ADA, and maleate, which are known to chelate Mg2+to varying degrees and therefore inhibit transcription reaction. All tested buffering species reduce dsRNA at increasing concentrations. Compared to T ris-HCI, which is the most prevalent buffering species used in IVT, several sulfonate-containing buffering species, such as PIPES, MES, and HEPPSO, exhibited more potency in dsRNA reduction.

[0433] Table 26: Buffering species (Phosphate, PIPES, imidazole, Tris, HEPES at pH 6.5 or 7.0) OFAT dsRNA (pg / ug) = pg of dsRNA per ug of total RNA

[0434] Table 27: Buffering species (Tris, sodium citrate, MES, MOPS, ADA, Maleate, ACES, Tricine, HEPPSO, BIS-TRIS, and PIPES at pH 6.5, 7.0, or 7.5) OFAT

[0435] EXAMPLE 15

[0436] COMBINATION OF PIPES AND TRIS RESULTED IN MORE EFFICIENT TRANSCRIPTION THAN THESE COMPONENTS ALONE

[0437] This example explored an extended range of concentration of PIPES (pH 7.0) and the effects of combining Tris and PIPES with the goal of reducing dsRNA byproducts while maintaining mRNA yield and integrity.

[0438] A 9.5 kb self-amplifying RNA (saRNA) was synthesized by IVT. A mixture of a 13.9 kb linearized DNA template (0.1 mg / mL), rNTPs (9 mM each), CLEANCAP™ AU (1 mM), reaction buffer (40 mM Tris, pH 7.0, 20 mM DTT, 0.53 mM spermidine, 36 mM magnesium acetate), T7 RNA polymerase (10000 U / mL), RIBOLOCK™ (100 U / mL), inorganic pyrophosphatase (0.25 U / mL) was prepared in a final volume of 1 mL, unless otherwise specified in conditions below. The IVT reaction was incubated for 170 minutes at 36 °C. After IVT, EDTA (75 mM) was added to quench the IVT reaction. LiCI-precipitated samples were analyzed in house for SOLOVPE™ concentration, FA integrity, and dsRNA ELISA analyses.

[0439] As shown in Table 28, compared to 40 mM Tris (pH 7.0), 120 mM PIPES (pH 7.0) alone resulted in <500 pg / ug dsRNA without negative impact on mRNA yield. Additionally, adding 20 or 40 mM Tris to PIPES buffer alleviates the yield-inhibitory effect of using PIPES alone at higher concentrations while adding to the dsRNA reducing effect. The benefits of adding Tris to PIPES were especially apparent at pH 6.5, which reduced both mRNA yield and dsRNA more potently. 40 mM Tris with 120 mM PIPES at pH 7.0 and 40 mM Tris with 80 mM PIPES at pH 6.5 produced the most favorable results among all conditions.

[0440] Table 28: IVT reactions with varying combinations of Tris and PIPES buffers

[0441] *NT : Not tested; dsRNA (pg / ug) = pg of dsRNA per ug of total RNA

[0442] EXAMPLE 16

[0443] THE EFFECT OF TAURINE IS DEPENDENT ON THE BUFFERING SPECIES

[0444] This example tested the effects of taurine treatment in the background of different buffering species with the goal of reducing dsRNA impurities while maintaining other product quality attributes.

[0445] A 9.5 kb self-amplifying RNA (saRNA) was synthesized by IVT. A mixture of a 13.9 kb linearized DNA template (0.1 mg / mL), rNTPs (9 mM each), CLEANCAP™ AU (1 mM), taurine (0 or 0.5 M), reaction buffer (40 mM Tris-HCI, pH 7.0, 20 mM DTT, 0.53 mM spermidine, 36 mM magnesium acetate), T7 RNA polymerase (10000 U / mL), RIBOLOCK™ (100 U / mL), inorganic pyrophosphatase (0.25 U / mL) was prepared in a final volume of 1 mL, unless otherwise specified in conditions below. The IVT reaction was incubated for 170 minutes at 36 °C. After IVT, EDTA (75 mM) was added to quench the IVT reaction. LiCI-precipitated samples were analyzed in house for SOLOVPE™ concentration, FA integrity, and dsRNA ELISA analyses. As shown in Table 29, the effect of taurine is dependent on the buffering species and their concentrations, though in all cases further reduces dsRNA.

[0446] Table 29: The effect of taurine is dependent on the buffering species and their concentrations dsRNA (pg / ug) = pg of dsRNA per ug of total RNA

[0447] EXAMPLE 17

[0448] MGS04 AS AN IVT COFACTOR CHOICE

[0449] One way to introduce sulfur oxoacid into an IVT reaction is to replace magnesium acetate with magnesium sulfate as cofactor. This example evaluated the performance of MgSO4 versus MgOAc as a cofactor for IVT reactions.

[0450] A 9.5 kb self-amplifying RNA (saRNA) was synthesized by IVT. A mixture of a 13.9 kb linearized DNA template (0.1 mg / mL), rNTPs (9 mM each), CLEANCAP™ AU (1 mM), reaction buffer (40 mM Tris, pH 7.0, 20 mM DTT, 0.53 mM spermidine, 36 mM magnesium acetate), T7 RNA polymerase (10000 U / mL), RIBOLOCK™ (100 U / mL), inorganic pyrophosphatase (0.25 U / mL) was prepared in a final volume of 1 mL, unless otherwise specified in conditions below. The IVT reaction was incubated for 170 minutes at 36 °C. After IVT, EDTA (75 mM) was added to quench the IVT reaction. LiCI-precipitated samples were analyzed in house for SOLOVPE™ concentration, FA integrity, and dsRNA ELISA analyses.

[0451] As shown in Table 30, MgSO4 performed better than MgOAc in terms of dsRNA reduction and integrity in Tris buffer background.

[0452] EXAMPLE 18

[0453] DEMONSTRATING GENERAL APPLICABILITY OF THE PIPES METHODS FOR DSRNA REDUCTION USING A DIFFERENT RNA CONSTRUCT

[0454] The goal of this example was to evaluate whether the PIPES methods could be applicable to reduce dsRNA with other RNA molecules. To this end, a 0.9 kb mRNA for the light chain of a monoclonal antibody (mRNA-mAb-LC) was produced using PIPES buffer compositions shown above to be effective in reducing dsRNA for a self-amplifying mRNA construct. mRNA-mAb-LC was synthesized by IVT. A mixture of a 3.2 kb linearized DNA template (0.05 mg / mL), rNTPs (9 mM each), CLEANCAP™ AG (4 mM), reaction buffer (40 mM Tris, pH 8.0, 0 mM DTT, 0 mM spermidine, 36 mM magnesium acetate), T7 RNA polymerase (14000 U / mL), RIBOLOCK™ (100 U / mL), and inorganic pyrophosphatase (0.25 U / mL) was prepared in a final volume of 1 mL, unless otherwise specified in conditions below. The IVT reaction was incubated for 150 minutes at 37 °C. After IVT, EDTA (75 mM) was added to quench the IVT reaction. LiCI- precipitated samples were analyzed in house for SOLOVPE™ concentration, FA integrity, dsRNA ELISA, and 5’ capping analyses. A residual NTP assay was performed on post-EDTA undiluted (Neat) samples.

[0455] As shown in Table 31 , 40 mM Tris-HCI + 120 mM PIPES (pH 7.0) reduced dsRNA without impacting mRNA yield for this RNA construct. Additionally, some impairment in transcription that was observed with 120 mM PIPES (pH 7.0) without spermidine / DTT (Condition Q) was rescued by the addition of 0.53 mM spermidine + 20 mM DTT (Condition R) or 40 mM Tris (Condition S). Furthermore, residual CLEANCAP™ data suggested that less CLEANCAP™ molecules were consumed in PIPES buffer than in Tris buffer despite mRNA products having similar 5’ capping % (shown in Table 31). This observation may reveal a previously unknown property of PIPES in IVT that can reduce CLEANCAP™ usage and therefore cost of goods in IVT mRNA manufacturing.

[0456] Table 31 : General applicability of PIPES method to reduce dsRNA on mRNA-mAb-LC construct dsRNA (pg / ug) = pg of dsRNA per ug of total RNA

[0457] The examples and embodiments described herein are for illustrative purposes only and various modifications or changes suggested to person skilled in the art are to be included within the spirit and purview of this application and scope of the appended claims.

Claims

1. CLAIMS1. A method of reducing an amount of double-stranded RNA (dsRNA) produced in an in vitro transcription (IVT) reaction, the method comprising:(a) obtaining a DNA template;(b) contacting the DNA template with an in vitro transcription (IVT) reaction system to obtain a composition, wherein the IVT reaction system comprises a monovalent salt, spermidine, dithiothreitol (DTT), and dimethylsulfoxide (DMSO); and(c) incubating the composition at a temperature between about 15 °C and 45 °C to obtain an IVT reaction, thereby reducing the amount of dsRNA produced in the IVT reaction.

2. The method of claim 1 , wherein incubating the composition occurs for a time period between about 30 minutes and 64 days.

3. The method of claim 1 or 2, wherein the IVT reaction system further comprises a buffer, a 5’ cap analog, ATP, CTP, GTP, UTP, a magnesium ion, an RNase inhibitor, pyrophosphatase, and an RNA polymerase.

4. The method of any one of claims 1 to 3, wherein the monovalent salt is selected from the group consisting of KCI, NaCI, RbCI, CsCI, NaCIC , NaF, NaBr, and NH4CL5. The method of any one of claims 1 to 4, wherein the monovalent salt is present in an amount between about 10 mM and about 150 mM.

6. The method of any one of claims 1 to 5, wherein the spermidine is present in an amount between about 0.2 mM and about 10 mM.

7. The method of any one of claims 1 to 6, wherein the dithiothreitol (DTT) is present in an amount between about 15 mM and about 25 mM.

8. The method of any one of claims 1 to 7, wherein the dimethylsulfoxide (DMSO) is present in an amount between about 4% and about 15%.

9. The method of any one of claims 1 to 8, wherein the DNA template is selected from the group consisting of a linearized DNA template, a circular DNA template, and a synthetic DNA template.

10. The method of any one of claims 1 to 9, further comprising:(d) contacting the IVT reaction with a deoxyribonuclease (DNase) to obtain a digested IVT reaction;(e) contacting the digested IVT reaction with a protease to obtain an inactivated reaction; and(f) contacting the inactivated reaction with a chelating agent to obtain a chelated reaction.11 . The method of claim 10, wherein the protease is proteinase K.

12. The method of claim 10 or 11 , wherein the chelating agent is ethylenediaminetetraacetic acid (EDTA).

13. The method of any one of claims 10 to 12, further comprising:(g) precipitating an mRNA molecule in the chelated reaction with lithium chloride to obtain a purified mRNA.

14. The method of claim 13, further comprising:(h) incubating a cellulose-based chromatographic medium with a buffer comprising dimethylsulfoxide (DMSO), wherein the DMSO is present in an amount between about 2.5% and 25%;(i) contacting the purified mRNA with the cellulose-based chromatographic medium to obtain a bound cellulose-based chromatographic medium; and(j) centrifuging the bound cellulose-based chromatographic medium.

15. The method of any one of claims 1 to 14, wherein the amount of dsRNA produced in the IVT reaction is reduced by at least 10% when compared to an amount of dsRNA produced by a standard method of RNA molecule synthesis.

16. A method of reducing an amount of double-stranded RNA (dsRNA) produced in an in vitro transcription (IVT) reaction, the method comprising:(a) obtaining a DNA template;(b) contacting the DNA template with an in vitro transcription (IVT) reaction system to obtain a composition, wherein the IVT reaction system comprises a monovalent salt, spermidine, dithiothreitol (DTT), and dihydrolevoglucosenone; and(c) incubating the composition at a temperature between about 15 °C and 45 °C to obtain an IVT reaction,thereby reducing the amount of dsRNA produced in the IVT reaction.

17. The method of claim 16, wherein incubating the composition occurs for a time period between about 30 minutes and 64 days.

18. The method of claim 16 or 17, wherein the IVT reaction system further comprises a buffer, a 5’ cap analog, ATP, CTP, GTP, UTP, a magnesium ion, an RNase inhibitor, pyrophosphatase, and an RNA polymerase.

19. The method of any one of claims 16 to 18, wherein the monovalent salt is selected from the group consisting of KCI, NaCI, RbCI, CsCI, NaCIC , NaF, NaBr, and NH4CL20. The method of any one of claims 16 to 19, wherein the monovalent salt is present in an amount between about 10 mM and about 150 mM.21 . The method of any one of claims 16 to 20, wherein the spermidine is present in an amount between about 0.2 mM and about 10 mM.

22. The method of any one of claims 16 to 21 , wherein the dithiothreitol (DTT) is present in an amount between about 15 mM and about 25 mM.

23. The method of any one of claims 16 to 22, wherein the dihydrolevoglucosenone is present in an amount between about 4% and about 15%.

24. The method of any one of claims 16 to 23, wherein the DNA template is selected from the group consisting of a linearized DNA template, a circular DNA template, and a synthetic DNA template.

25. The method of claim 16, further comprising:(d) contacting the IVT reaction with a deoxyribonuclease (DNase) to obtain a digested IVT reaction;(e) contacting the digested IVT reaction with a protease to obtain an inactivated reaction; and(f) contacting the inactivated reaction with a chelating agent to obtain a chelated reaction.

26. The method of claim 25, wherein the protease is proteinase K.

27. The method of claim 25 or 26, wherein the chelating agent is ethylenediaminetetraacetic acid (EDTA).

28. The method of any one of claims 25 to 27, further comprising:(g) precipitating an mRNA molecule in the chelated reaction with lithium chloride to obtain a purified mRNA.

29. The method of claim 28, further comprising:(h) incubating a cellulose-based chromatographic medium with a buffer comprising dimethylsulfoxide (DMSO), wherein the DMSO is present in an amount between about 2.5% and 25%;(i) contacting the purified mRNA with the cellulose-based chromatographic medium to obtain a bound cellulose-based chromatographic medium; and(j) centrifuging the bound cellulose-based chromatographic medium.

30. The method of any one of claims 16 to 29, wherein the amount of dsRNA produced in the IVT reaction is reduced by at least 10% when compared to an amount of dsRNA produced by a standard method of RNA molecule synthesis.31 . A method of reducing an amount of double-stranded RNA (dsRNA) produced in an in vitro transcription (IVT) reaction, the method comprising:(a) obtaining a DNA template;(b) contacting the DNA template with an in vitro transcription (IVT) reaction system to obtain a composition, wherein the IVT reaction system comprises an (organo)sulfur oxoacid; and(c) incubating the composition at a temperature between about 15 °C and 45 °C to obtain an IVT reaction, thereby reducing the amount of dsRNA produced in the IVT reaction.

32. The method of claim 31 , wherein incubating the composition occurs for a time period between about 30 minutes and 64 days.

33. The method of claim 31 , wherein the (organo)sulfur oxoacid comprises a tetrahedral or trigonal pyramidal geometry.

34. The method of claim 31 , wherein the (organo)sulfur oxoacid comprises a sulfonic acid or sulfonic acid derivative.

35. The method of claim 34, wherein the sulfonic acid comprises an ethanesulfonate.

36. The method of claim 35, wherein the ethanesulfonate is selected from the group consisting of taurine (2-aminoethanesulfonic acid), PIPES (piperazine-N,N'-bis(2- ethanesulfonic acid), and MES (2-(N-morpholino)ethanesulfonic acid).

37. The method of claim 36, wherein the taurine is present in an amount between about 0.01 M and about 2.5 M.

38. The method of claim 36, wherein the PIPES is present in an amount between about 10 mM and about 180 mM.

39. The method of claim 34, wherein the sulfonic acid comprises HEPPSO (2-hydroxy-3-[4- (2-hydroxyethyl)piperazin-1-yl]propane-1 -sulfonic acid).

40. The method of claim 39, wherein the HEPPSO is present in an amount between about 20 mM and about 180 mM.41 . The method of claim 36, wherein the MES is present in an amount between about 10 mM and about 180 mM.

42. The method of claim 34, wherein the sulfonic acid derivate is selected from the group consisting of a polyanionic synthetic sulfonic acid polymer (SSAP), ethylene dimethanesulfonate, and polystyrene sulfonate.

43. The method of claim 31 , wherein the (organo)sulfur oxoacid comprises a sulfuric acid or sulfuric acid derivative.

44. The method of claim 31 , wherein the (organo)sulfur oxoacid is selected from the group consisting of magnesium sulfate, ammonium sulfate, and a heparin derivative.

45. The method of claim 44, wherein the heparin derivative comprises enoxaparin.

46. The method of any one of claims 31 to 45, wherein the IVT reaction system further comprises a 5’ cap analog, ATP, CTP, GTP, UTP, a magnesium ion, an RNase inhibitor, pyrophosphatase, and an RNA polymerase.

47. The method of any one of claims 31 to 44, wherein the IVT reaction system further comprises a buffer.

48. The method of claim 47, wherein the buffer is selected from the group consisting of T ris and HEPES.

49. The method of any one of claims 31 to 48, wherein the DNA template is selected from the group consisting of a linearized DNA template, a circular DNA template, and a synthetic DNA template.

50. The method of any one of claims 31 to 49, further comprising:(d) contacting the IVT reaction with a deoxyribonuclease (DNase) to obtain a digested IVT reaction;(e) contacting the digested IVT reaction with a protease to obtain an inactivated reaction; and(f) contacting the inactivated reaction with a chelating agent to obtain a chelated reaction.51 . The method of claim 50, wherein the protease is proteinase K.

52. The method of claim 50 or 51 , wherein the chelating agent is ethylenediaminetetraacetic acid (EDTA).

53. The method of any one of claims 31 to 52, wherein the amount of dsRNA produced in the IVT reaction is reduced by at least 10% when compared to an amount of dsRNA produced by a standard method of RNA molecule synthesis.

54. The method of any one of claims 31 to 53, wherein the IVT reaction system further comprises a pH between 2.5 and 8 and a tris buffer in a range from 20 mM to 150 mM.

55. A composition for in vitro transcription (IVT) comprising:(a) a DNA template; and(b) an in vitro transcription (IVT) reaction system comprising a buffer, a 5’ cap analog, ATP, CTP, GTP, UTP, a magnesium ion, an RNase inhibitor, pyrophosphatase, and an RNA polymerase, wherein the IVT reaction system further comprises: i. 40 mM potassium chloride (KCI);II. 0.53 mM spermidine; ill. 20 mM dithiothreitol (DTT); and iv. 10% dimethylsulfoxide (DMSO) or 10% dihydrolevoglucosenone.

56. The composition of claim 55, wherein the buffer is selected from the group consisting of Tris and HEPES.

57. The composition of claim 55, wherein the buffer comprises Tris-HCI and PIPES (piperazine-N,N'-bis(2-ethanesulfonic acid).

58. The composition of any one of claims 55 to 57, wherein the DNA template is selected from the group consisting of a linearized DNA template, a circular DNA template, and a synthetic DNA template.

59. A composition for in vitro transcription (IVT) comprising:(a) a DNA template; and(b) an in vitro transcription (IVT) reaction system comprising a buffer, a 5’ cap analog, ATP, CTP, GTP, UTP, a magnesium ion, an RNase inhibitor, pyrophosphatase, and an RNA polymerase, wherein the IVT reaction system further comprises: i. 0.53 mM spermidine;II. 20 mM dithiothreitol (DTT); and ill. 0.5 M taurine.

60. The composition of claim 59, wherein the DNA template is selected from the group consisting of a linearized DNA template, a circular DNA template, and a synthetic DNA template.61 . A method of reducing an amount of double-stranded RNA (dsRNA) produced in an in vitro transcription (IVT) reaction, the method comprising:(a) obtaining a DNA template;(b) contacting the DNA template with an in vitro transcription (IVT) reaction system to obtain a composition, wherein the IVT reaction system comprises spermidine, dithiothreitol (DTT), and dihydrolevoglucosenone; and(c) incubating the composition at a temperature between about 15 °C and 45 °C to obtain an IVT reaction, thereby reducing the amount of dsRNA produced in the IVT reaction.

62. The method of claim 61 , wherein incubating the composition occurs for a time period between about 30 minutes and 64 days.

63. The method of claim 61 or 62, wherein the IVT reaction system further comprises a buffer, a 5’ cap analog, ATP, CTP, GTP, UTP, a magnesium ion, an RNase inhibitor, pyrophosphatase, and an RNA polymerase.

64. The method of any one of claims 61 to 63, wherein the spermidine is present in an amount between about 0.2 mM and about 10 mM.

65. The method of any one of claims 61 to 64, wherein the dithiothreitol (DTT) is present in an amount between about 15 mM and about 25 mM.

66. The method of any one of claims 61 to 65, wherein the dihydrolevoglucosenone is present in an amount between about 4% and about 15%.

67. The method of any one of claims 61 to 66, wherein the DNA template is selected from the group consisting of a linearized DNA template, a circular DNA template, and a synthetic DNA template.

68. The method of claim 61 , further comprising:(d) contacting the IVT reaction with a deoxyribonuclease (DNase) to obtain a digested IVT reaction;(e) contacting the digested IVT reaction with a protease to obtain an inactivated reaction; and(f) contacting the inactivated reaction with a chelating agent to obtain a chelated reaction.

69. The method of claim 68, wherein the protease is proteinase K.

70. The method of claim 68 or 69, wherein the chelating agent is ethylenediaminetetraacetic acid (EDTA).71 . The method of any one of claims 68 to 70, further comprising:(g) precipitating an mRNA molecule in the chelated reaction with lithium chloride to obtain a purified mRNA.

72. The method of claim 71 , further comprising:(h) incubating a cellulose-based chromatographic medium with a buffer comprising dimethylsulfoxide (DMSO), wherein the DMSO is present in an amount between about 2.5% and 25%;(i) contacting the purified mRNA with the cellulose-based chromatographic medium to obtain a bound cellulose-based chromatographic medium; and(j) centrifuging the bound cellulose-based chromatographic medium.

73. The method of any one of claims 61 to 72, wherein the amount of dsRNA produced in the IVT reaction is reduced by at least 10% when compared to an amount of dsRNA produced by a standard method of RNA molecule synthesis.

74. The method of any one of claims 61 to 73, wherein the IVT reaction system further comprises a pH between 2.5 and 8 and a tris buffer in a range from 20 mM to 150 mM.

75. A method of reducing an amount of double-stranded RNA (dsRNA) produced in an in vitro transcription (IVT) reaction, the method comprising:(a) obtaining a DNA template;(b) contacting the DNA template with an in vitro transcription (IVT) reaction system to obtain a composition, wherein the IVT reaction system comprises a monovalent salt, spermidine, dithiothreitol (DTT), and dimethylsulfoxide (DMSO), and further wherein the IVT reaction system comprises a pH between 2.5 and 8 and a tris buffer in a range from 20 mM to 150 mM; and(c) incubating the composition at a temperature between about 15 °C and 45 °C to obtain an IVT reaction, thereby reducing the amount of dsRNA produced in the IVT reaction.

76. A method of reducing an amount of double-stranded RNA (dsRNA) produced in an in vitro transcription (IVT) reaction, the method comprising:(a) obtaining a DNA template;(b) contacting the DNA template with an in vitro transcription (IVT) reaction system to obtain a composition, wherein the IVT reaction system comprises spermidine, dithiothreitol (DTT), and dihydrolevoglucosenone, and further wherein the IVT reaction system comprises a pH between 2.5 and 8 and a tris buffer in a range from 20 mM to 150 mM; and(c) incubating the composition at a temperature between about 15 °C and 45 °C to obtain an IVT reaction, thereby reducing the amount of dsRNA produced in the IVT reaction.

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