Method for producing nucleic acids

Abstract

Methods of producing RNA molecules in an in vitro transcription reaction are described. Also described are methods of producing mRNA molecules from a cyclic double-stranded DNA template using an in vitro transcription reaction system, wherein the in vitro transcription reaction system lacks a polyamine. An in vitro transcription reaction system comprising enzymatic 5'capping and oligomeric (dT) purification is also described.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of U.S. Provisional Patent Application Serial No. 63 / 499,276 filed on May 1, 2023 and U.S. Provisional Patent Application Serial No. 63 / 338,878 filed on May 5, 2022 under 35 U.S.C. §119(e), the disclosures of which are hereby incorporated by reference in their entirety. Technical Field

[0003] The present invention relates to an improved method for the synthesis of nucleic acid molecules, in particular the cell-free enzymatic synthesis of DNA and the synthesis of RNA molecules via in vitro transcription (IVT). Background Art

[0004] There is a need in the art for improved and efficient in vitro nucleic acid amplification methods, particularly DNA molecules and templates for IVT, without the use of plasmids and bacterial fermentation (e.g., cell-free production of DNA molecules). For example, such improved methods can facilitate faster response times to changes in viral strains during a pandemic or seasonal viral changes, as traditional multi-step methods for generating DNA templates from plasmids produced by bacterial fermentation can be very time consuming. Summary of the invention

[0005] In some embodiments, disclosed herein is a method for producing an mRNA molecule, comprising: (a) obtaining a composition comprising: a circular double-stranded DNA (dsDNA) template, a primer, a deoxyribonucleoside triphosphate (dNTP), and a DNA polymerase; (b) incubating the composition at a temperature between about 30°C and 45°C for a period of time between about three and twenty-four hours to obtain an incubated composition; and (c) contacting the incubated composition with an in vitro transcription reaction system comprising an RNA polymerase and ribonucleotides under conditions sufficient for in vitro transcription to produce mRNA molecules, wherein the contacting occurs for about 120 minutes to about 260 minutes, wherein the contacting occurs at a temperature between 25°C and 45°C, wherein the in vitro transcription reaction system further comprises a buffer, magnesium ions, and a reducing agent, and wherein the in vitro transcription reaction system lacks a polyamine. In some embodiments, the method further comprises contacting the incubated composition with a restriction endonuclease to obtain a digested composition before contacting the incubated composition with the in vitro transcription reaction system. In some embodiments, the contact of the composition of the incubation with the restriction endonuclease occurs in the same reaction vessel in which the composition is incubated. In some embodiments, the contact of the composition of the incubation with the in vitro transcription reaction occurs in the same reaction vessel in which the composition is incubated. In some embodiments, the method for producing mRNA molecules disclosed herein does not include a thermal denaturation reaction before incubating the composition. In some embodiments, the method for producing mRNA molecules disclosed herein does not include a thermal inactivation reaction before performing an in vitro transcription reaction. In some embodiments, the method for producing mRNA molecules disclosed herein further includes: (d) using a capture method selected from oligo (dT) magnetic beads, resins, and monoliths to capture mRNA molecules.

[0006] In some embodiments, disclosed herein is a method for producing an mRNA molecule, comprising: (a) obtaining a composition comprising: a circular double-stranded DNA (dsDNA) template, a primer, a deoxyribonucleoside triphosphate (dNTP), and a DNA polymerase; (b) incubating the composition at a temperature between about 30°C and 45°C for a period of time between about 3 and 8 hours to obtain an incubated composition; and (c) contacting the incubated composition with an in vitro transcription reaction system comprising an RNA polymerase and ribonucleotides under conditions sufficient for in vitro transcription to produce mRNA molecules, wherein the contacting occurs for about 120 minutes to about 260 minutes, wherein the contacting occurs at a temperature between 25°C and 45°C, wherein the in vitro transcription reaction system further comprises a buffer, magnesium ions, and a reducing agent, and wherein the in vitro transcription reaction system lacks a polyamine. In some embodiments, the period of time is about eighteen to twenty-four hours. In some embodiments, disclosed herein is a method for producing mRNA molecules, comprising: (a) obtaining a composition comprising: a circular double-stranded DNA (dsDNA) template, a primer, a deoxyribonucleoside triphosphate (dNTP) and a DNA polymerase; (b) incubating the composition at a temperature between about 30°C and 45°C for a period of about three to twenty-four hours to obtain an incubated composition; and (c) contacting the incubated composition with an in vitro transcription reaction system comprising an RNA polymerase and ribonucleotides under conditions sufficient for in vitro transcription to produce mRNA molecules, wherein the contacting occurs for about 120 minutes to about 260 minutes, wherein the contacting occurs at a temperature between 25°C and 45°C, wherein the in vitro transcription reaction system further comprises a buffer, magnesium ions and a reducing agent, wherein the in vitro transcription reaction system lacks polyamines, and wherein the DNA polymerase is phi29 DNA polymerase.

[0007] In some embodiments, disclosed herein is a method for producing an mRNA molecule, comprising: (a) obtaining a composition comprising: a circular double-stranded DNA (dsDNA) template, a primer, a deoxyribonucleoside triphosphate (dNTP), and a DNA polymerase; (b) incubating the composition at a temperature between about 30°C and 45°C for a period of about three to twenty-four hours to obtain an incubated composition; and (c) contacting the incubated composition with an in vitro transcription reaction system comprising an RNA polymerase and ribonucleotides under conditions sufficient for in vitro transcription to produce mRNA molecules, wherein the contact occurs for about 120 minutes to about 260 minutes, wherein the contact occurs at a temperature between 25°C and 45°C, wherein the in vitro transcription reaction system further comprises a buffer, magnesium ions, and a reducing agent, wherein the in vitro transcription reaction system lacks a polyamine, and wherein the contact further comprises supplementing the in vitro transcription reaction system with ribonucleotides. In some embodiments, the supplementation comprises a continuous feed of ribonucleotides. In some embodiments, the supplementation comprises a semi-continuous feed of ribonucleotides. In some embodiments, the supplementation comprises bolus feeding of ribonucleotides.

[0008] In some embodiments, disclosed herein is a method for producing mRNA molecules, comprising: (a) obtaining a composition comprising: a circular double-stranded DNA (dsDNA) template, a primer, a deoxyribonucleoside triphosphate (dNTP) and a DNA polymerase; (b) incubating the composition at a temperature between about 30°C and 45°C for a period of about three to twenty-four hours to obtain an incubated composition; and (c) contacting the incubated composition with an in vitro transcription reaction system comprising RNA polymerase and ribonucleotides under conditions sufficient for in vitro transcription to produce mRNA molecules, wherein the contact occurs for about 120 minutes to about 260 minutes, wherein the contact occurs at a temperature between 25°C and 45°C, wherein the in vitro transcription reaction system further comprises a buffer, magnesium ions and a reducing agent, wherein the in vitro transcription reaction system lacks polyamines, and wherein the contact further comprises supplementing the in vitro transcription reaction system with magnesium ions. In some embodiments, the supplement is selected from continuous feeding, semi-continuous feeding and bolus feeding of magnesium ions. In some embodiments, the contact further comprises stirring. In some embodiments, the stirring is selected from: about 1.3 to about 71.7 W / m 3 The invention relates to a power / volume ratio between about 1 and 2, a mixing time between about 1.3 and about 12.1 seconds, and an impeller tip speed between about 0.1 and about 0.4 m / s.

[0009] In some embodiments, disclosed herein is a method for producing an mRNA molecule, comprising: (a) obtaining a composition comprising: a circular double-stranded DNA (dsDNA) template, a primer, a deoxyribonucleoside triphosphate (dNTP), and a DNA polymerase; (b) incubating the composition at a temperature between about 30°C and 45°C for a period of about three to twenty-four hours to obtain an incubated composition; and (c) contacting the incubated composition with an in vitro transcription reaction system comprising an RNA polymerase and ribonucleotides under conditions sufficient for in vitro transcription to produce mRNA molecules, wherein the contact occurs for about 120 minutes to about 260 minutes, wherein the contact occurs at a temperature between 25°C and 45°C, wherein the in vitro transcription reaction system further comprises a buffer, magnesium ions, and a reducing agent, wherein the in vitro transcription reaction system lacks polyamines, and wherein the in vitro transcription reaction system further comprises a pyrophosphatase. In some embodiments, the in vitro transcription reaction system further comprises a ribonuclease inhibitor. In some embodiments, the in vitro transcription reaction system further comprises acetate ions. In some embodiments, the buffer is selected from TRIS and HEPES. In some embodiments, the ribonucleotide is present in an amount between about 16 and about 50mM. In some embodiments, the RNA polymerase is present in an amount between about 4000 and about 12000U / mL. In some embodiments, the pyrophosphatase is present in an amount between about 0.25 and about 8.0U / mL. In some embodiments, the buffer comprises a starting pH between about 7.5 and about 8.5. In some embodiments, the magnesium ion is present in an amount between about 12.8 and about 110mM. In some embodiments, the magnesium ion and ribonucleotide are present with a magnesium:ribonucleotide ratio of about 0.8 to about 2.2mM Mg / mM NTP. In some embodiments, the buffer does not contain dithiothreitol (DTT).

[0010] In some embodiments, disclosed herein is a method for producing an mRNA molecule, comprising: (a) obtaining a composition comprising: a plurality of linear double-stranded DNA (dsDNA) fragments, a 5' exonuclease, a DNA polymerase, and a DNA ligase; (b) incubating the composition at a temperature between about 45°C and 55°C to obtain a circularized dsDNA template; and (c) contacting the circularized dsDNA template with a rolling circle amplification (RCA) reaction mixture to obtain an amplification template, the rolling circle amplification reaction mixture comprising: a primer or a primase, a deoxyribonucleoside triphosphate ( dNTP) and DNA polymerase; (d) contacting the amplified template with a restriction enzyme to obtain a digested template; and (e) contacting the digested template with an in vitro transcription reaction system comprising RNA polymerase and ribonucleotides under conditions sufficient for in vitro transcription to produce mRNA molecules, wherein the contact occurs for about 120 minutes to about 260 minutes, wherein the contact occurs at a temperature between 25°C and 45°C, wherein the in vitro transcription reaction system further comprises a buffer and magnesium ions, and wherein steps (a) to (e) are performed sequentially in a single reaction vessel. In some embodiments, the circularized dsDNA template is present at a concentration of at least 0.5ng / mL RCA reaction mixture. In some embodiments, the plurality of linear dsDNA fragments include a target gene, a promoter sequence, a 5'UTR, a 3'UTR, and a poly A sequence. In some embodiments, the target gene encodes an RNA molecule between 1.0kb and 12.0kb. In some embodiments, the target gene lacks a homopolymer sequence greater than 5 base pairs. In some embodiments, the mRNA molecule is capped after in vitro transcription. In some embodiments, the mRNA molecules are enzymatically capped by a vaccinia capping enzyme. In some embodiments, the mRNA molecules are incubated at 30° C. or less for 60 minutes or less prior to capping with a vaccinia capping enzyme.

[0011] In some embodiments, disclosed herein is a method for producing an mRNA molecule, comprising: (a) obtaining a composition comprising: a plurality of linear double-stranded DNA (dsDNA) fragments, a 5' exonuclease, a DNA polymerase, and a DNA ligase; (b) incubating the composition at a temperature between about 45°C and 55°C to obtain a circularized dsDNA template; and (c) contacting the circularized dsDNA template with a rolling circle amplification (RCA) reaction mixture to obtain an amplified template, the rolling circle amplification reaction mixture comprising: a primer or a primase, deoxyribonucleoside triphosphates (dNTPs), and a DNA polymerase; (d) contacting the amplified template with a restriction enzyme to obtain a digested template. ;And (e) under conditions sufficient for in vitro transcription to produce mRNA molecules, the digested template is contacted with an in vitro transcription reaction system comprising RNA polymerase and ribonucleotides, wherein the contact occurs for about 120 minutes to about 260 minutes, wherein the contact occurs at a temperature between 25°C and 45°C, wherein the in vitro transcription reaction system further comprises a buffer and magnesium ions, wherein the steps (a) to (e) are performed sequentially in a single reaction vessel, and wherein the contact of the digested template with the in vitro transcription reaction system further comprises supplementing the in vitro transcription reaction system with ribonucleotides, wherein the supplementation comprises increasing the concentration of a single ribonucleotide from an initial amount to a final amount. In some embodiments, the supplementation comprises a method selected from the following: continuous feeding of ribonucleotides, semi-continuous feeding of ribonucleotides, and bolus feeding of ribonucleotides. In some embodiments, the initial amount is between 1mM and 11mM, and the final amount is between 11mM and 26mM. In some embodiments, the initial amount of ATP is about 11mM, the initial amount of CTP is about 9mM, the initial amount of GTP is about 1mM, and the initial amount of pUTP is about 4mM. In some embodiments, the final amount of ATP is about 24mM, the final amount of CTP is about 22mM, the final amount of GTP is about 16mM, and the final amount of pUTP is about 13mM. In some embodiments, the supplementation further includes bolus feeding of nucleotides every five minutes in the in vitro transcription reaction. In some embodiments, the contact of the digested template with the in vitro transcription reaction system further includes supplementing the in vitro transcription reaction system with magnesium ions, wherein the supplementation includes increasing the concentration of magnesium ions from the initial amount to the final amount. In some embodiments, the supplementation includes a method selected from the following: continuous feeding of magnesium ions, semi-continuous feeding of magnesium ions, and bolus feeding of magnesium ions. In some embodiments, the supplementation of magnesium ions to the in vitro transcription reaction system further includes adding magnesium ions at least four times during the in vitro transcription reaction. In some embodiments, the initial amount of magnesium ions is about 25 mM, and the final amount of magnesium ions is about 60 mM. In some embodiments, the supplementation further comprises bolus feeding of magnesium ions every 15 minutes within the first 60 minutes of the in vitro transcription reaction.

[0012] In some embodiments, the method for producing mRNA molecules disclosed herein includes using a capture method selected from oligo (dT) magnetic beads, resins and monolithic columns to capture mRNA molecules produced by in vitro transcription reactions. In some embodiments, the method further includes washing oligo (dT) magnetic beads, resins and monolithic columns after capturing mRNA. In some embodiments, the washing solution includes a buffer selected from the following: Tris, HEPES, NaPi, KCl, NaCl, urea, arginine and EDTA.

[0013] In some embodiments, disclosed herein is a method of producing an mRNA molecule, comprising contacting a composition comprising a DNA template with an in vitro transcription reaction system comprising an RNA polymerase and ribonucleotides under conditions sufficient for in vitro transcription to produce an mRNA molecule, wherein the contacting occurs for about 120 minutes to about 260 minutes, wherein the contacting occurs at a temperature between 25°C and 45°C, wherein the in vitro transcription reaction system further comprises a buffer and magnesium ions, and wherein the in vitro transcription reaction system lacks a polyamine and a reducing agent. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 is an agarose gel image of plasmid dsDNA used as template in the RCA reaction.

[0015] Figure 2 are agarose gel images of non-linearized RCA DNA without and with heat denaturation prior to the RCA reaction.

[0016] Figure 3 are agarose gel images of RCA DNA with and without various purification steps. Lane 1 is undigested, unpurified RCA DNA; Lane 2 is undigested and purified RCA DNA; Lane 3 is RCA DNA linearized with SapI without purification before or after linearization (for sequential single-vessel one-pot reactions); Lane 4 is RCA DNA linearized with SapI without purification before linearization, but purified after linearization; Lane 5 is RCA DNA purified before linearization with SapI (buffer added); Lane 6 is RCA DNA purified before linearization with SapI (buffer added) and also purified after linearization; Lane 7 is pDNA digested with SapI and purified after linearization; Lane 8 is pDNA digested with SapI without purification.

[0017] Figure 4Is an agarose gel image of an RCA reaction using RCA DNA from a previous RCA reaction as a template. Shown are 3 sequential RCA reactions using the previous reaction as a template for the next reaction (5ng template). The initial reaction (lanes 2 and 3) uses pDNA as a template. Lanes 2, 4 and 6 are uncut RCA DNA, and lanes 3, 5 and 7 are restricted digested with SapI to show linearization.

[0018] Figure 5 is an agarose gel image of RNA products from IVT reactions. RNA yields are indicated below each lane. Lane 1 shows sequential single-vessel "one-pot" RNA; Lane 2 shows RNA from RCA DNA template that was linearized and then purified (standard RCA method); Lanes 3-4 used undigested RCA DNA template that was not purified and purified; Lane 5 shows RNA using pDNA template that was purified after linearization; Lane 6 shows RNA using pDNA template that was not purified after linearization (one-pot pDNA).

[0019] Figure 6 RNA integrity by Bioanalyzer fragment analysis of mRNA generated from unselected and oligo(dT)-selected pDNA or RCA DNA templates is shown. Figure 5 The samples in lanes 1, 5, and 6 are related. pDNA-CA09 indicates IVT using a plasmid template; pDNA-CA09 one-pot indicates IVT using a plasmid template that was not purified after linearization; RCA-CA09 one-pot indicates IVT in a sequential single-vessel one-pot reaction.

[0020] Figure 7 The effects of Mg and ATP+CTP+GTP+pUTP on RNA concentration are shown.

[0021] Figure 8 The effects of Mg and ATP+CTP+GTP+pUTP on RNA integrity are shown.

[0022] Fig. 9 Shown are contour plots of the DNase and calcium two-factor interaction on RNA integrity after 60 minutes of DNA digestion.

[0023] Fig.10 Shown are contour plots of the two-way interaction of magnesium and manganese for the presence of contaminant residual DNA after 45 minutes of DNA digestion.

[0024] Fig.11 An electropherogram depicting the output of residual protein is shown.

[0025] Fig.12is a bar graph demonstrating that increasing feed frequency improves method performance.

[0026] Fig.13 is a bar graph depicting the yield, integrity and 5'-capping results from Experiment 2.

[0027] Fig.14 is a description of the use of plasmid DNA (pDNA) and synthetic DNA (USTAT), from 15 and Bar graph of yield, integrity and 5'-capping results for bolus feed versus continuous feed at 250 scale.

[0028] Fig.15 The bar graph and scatter plot depict the combination of the three methods, demonstrating the effect of agitation on the presence and yield of impure residual DNA, respectively.

[0029] Fig.16 The bar graph and scatter plot depict the combination of the three methods, demonstrating the effect of power per volume on the presence of residual DNA and yield, respectively.

[0030] Fig.17 is a bar graph depicting two methods demonstrating the effect of pyrophosphatase on the presence of contaminating residual DNA.

[0031] Fig.18 is a bar graph depicting the effect of different template concentrations on yield in a batch IVT method.

[0032] Fig.19 is a bar graph depicting different incubation temperatures and times in the batch IVT method.

[0033] Fig. 20 is a bar graph depicting different incubation times and template concentrations for different concentrations of long RNA molecules in the bulk IVT method.

[0034] Fig.21 is a bar graph depicting incubation temperatures below 37°C for 2 or 3 hour incubations for long RNA molecules in the bulk IVT method.

[0035] Fig. 22 is a bar graph depicting the effect of each NTP on cap incorporation.

[0036] Fig.23 is a bar graph depicting the effect of magnesium acetate titration.

[0037] Fig.24 is a bar graph depicting different cap analog concentrations in the IVT method with 0.4 mM GTP feed.

[0038] Fig.25 is a bar graph depicting different cap analog concentrations in the IVT method with 0.8 mM GTP feed.

[0039] Fig.26 is a bar graph depicting different cap analog concentrations in the IVT method with 1 mM GTP feed.

[0040] Fig.27A -B shows RCA DNA yields using plasmid template concentrations from 0.5–5 ng / mL RCA reactions ( Fig.27A ), and an agarose gel of the RCA DNA products obtained using these template concentrations after linearization with BspQI ( Fig.27B ).

[0041] Fig.28A -B shows the RCA DNA yield when the reaction temperature was varied from 27–30°C ( Fig.28A ), and agarose gels of RCA DNA products generated at these temperatures after linearization with BspQI ( Fig.28B ).

[0042] Fig.29 Agarose gels of RCA DNA products before and after digestion with different concentrations of the restriction enzyme BspQI are shown. In all cases, the major product was the expected 5.2 kb.

[0043] Fig. 30A -B shows the results of RCA DNA products used as templates for small-scale IVT reactions. Commercially available RCA preparations were compared with optimized USTAT reactions to produce RCA DNA templates ( Fig. 30A ), which is used to transcribe RNA in the IVT reaction ( Fig. 30B ). Include linearized and purified plasmid DNA as a control for IVT. Measure RNA integrity by fragment analysis.

[0044] Fig.31A -B shows the results at 250 mL scale using USTAT RCA DNA and linearized, purified plasmid DNA as IVT FB V1 template. RNA yield and integrity results are shown in Fig.31A Integrity was measured using fragment analysis. The % capped mRNA of products generated using either plasmid DNA or RCA DNA templates was measured and Fig.31B shown.

[0045] Fig.32A -D shows analysis of next generation sequencing data from a linearized plasmid control, linearized, unpurified plasmid, and USTAT RCA DNA. Fig.32Ashows similar coverage between sequences in each sample, and Fig.32B A high percentage of sequences without any insertions or deletions between samples is shown. Fig.32C shows the sequence coverage across the transcription template, and Fig.32D Shows that sequence variants were not observed in the sequence template.

[0046] Fig.33 Shown is the expression of GFP protein over time in HEK293 cells following transfection with RNA transcribed from a plasmid DNA control template or a USTAT RCA DNA template. Either 0.5 ug or 0.05 ug of RNA encoding GFP was incubated in the transfection mixture.

[0047] Fig.34A -B shows the results of RCA DNA template in a small-scale IVT reaction. A commercially available reaction buffer was compared to an optimized USTAT reaction to produce RCA DNA, and the reaction yields were shown in Figure 2. Fig.34A The DNA is used as a template in the IVT reaction ( Fig.34B ). RNA integrity was measured using fragment analysis (FA).

[0048] Fig.35 Figure 1 is a graph of IVT results for transcripts of various sizes using USTAT RCA DNA as template. USTATRCA reactions were primed with either random primers or primase. Linearized plasmid template was included as a control. RNA integrity was measured using FA and yield was measured by UV.

[0049] Fig.36 Figure 1 is an IVT result graph using assembled DNA template for USTAT. The assembled DNA template was treated with or without T5 exonuclease digestion prior to USTAT. A plasmid DNA template for USTAT was included as a control. RNA integrity was measured using FA.

[0050] Fig.37 is a graph showing that increasing the frequency of Mg addition during the IVT reaction resulted in improved RNA quality.

[0051] Fig.38 Figure 2 is a graph showing that the addition of magnesium during the IVT reaction resulted in significant effects on RNA quality attributes and yield.

[0052] Fig.39 is a graph showing comparison of BV1 and FB V2 across seven RNA constructs.

[0053] Detailed Description

[0054] mRNA technology has great potential for treating diseases. In order to realize this potential, high-quality mRNA for vaccine or therapeutic purposes is needed. One of three bacteriophage RNA polymerases SP6, T7 or T3 can be used for in vitro transcription (IVT) to synthesize RNA from a DNA template. Traditionally, RNA molecules are made using batch mode in vitro transcription, in which the reaction components are combined and then incubated for several hours. Another method for producing RNA molecules is by batch fed in vitro transcription, in which any reaction components are added at certain time intervals.

[0055] Disclosed herein are batch and fed-batch methods for producing RNA molecules, such as mRNA, including modified mRNA molecules and / or self-amplifying RNA (saRNA). The methods can be used to consistently and reproducibly produce clinical-grade RNA (e.g., mRNA) of high purity and potency, and in compliance with current good manufacturing practices (cGMP). The methods use a DNA template in an in vitro transcription (IVT) reaction to generate RNA molecules, and can be applied to a wide variety of constructs with different 5'UTRs, coding sequence lengths, 3'UTRs, and 3' ends. In some aspects, RNA molecules are purified via chromatographic methods, such as by using an oligo (dT) matrix. In some aspects, enzymatic capping is used for 5' capping of RNA molecules.

[0056] Also disclosed herein are methods for various "one-pot" reactions for producing RNA molecules (e.g., mRNA, including modified mRNA molecules, unmodified mRNA molecules, and / or saRNA), which can be used to consistently and reproducibly produce clinical-grade RNA (e.g., mRNA) of high purity and potency, and in compliance with current good manufacturing practices (cGMP). One or more steps of the method (e.g., amplification, linearization, IVT, and / or capping) are performed in the same reaction vessel without the need for an intermediate nucleic acid purification process between steps. In some aspects, all steps of the method steps (e.g., amplification, linearization, IVT, and / or capping) are performed in the same reaction vessel without the need for an intermediate nucleic acid purification process between steps.

[0057] Specific Definitions

[0058] Throughout this application, the term "about" is used in accordance with its ordinary meaning in the field of cell and molecular biology to illustrate that a value includes the standard deviation of the inherent variation or error of the measurement or quantitative method used to determine the value. For example, in some aspects, the term "about" can encompass a range of values ​​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 quantification.

[0059] When used in conjunction with the term "comprising," the use of the word "a" or "an" can mean "one," but it is also consistent with the meaning of "one or more," "at least one," and "one or more than one."

[0060] The phrase "and / or" means "and" or "or". By way of example, 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" serves as an inclusive or.

[0061] The phrase "substantially all" is defined as "at least 95%"; if substantially all members of a group have a certain property, then at least 95% of the members of the group have that property. In some cases, substantially all means that any one, at least any one, or between any two of 95%, 96%, 97%, 98%, 99%, or 100% of the members of the group have that property.

[0062] Compositions and methods of use thereof may "comprise," "consist essentially of," or "consist of" any ingredient or step disclosed throughout this specification. Throughout this specification, unless the context requires otherwise, the words "comprising" (and any forms of including, such as "comprise" and "comprises"), "having" (and any forms of having, such as "have" and "has"), "including" (and any forms of including, such as "include" and "includes"), or "containing" (and any forms of containing, such as "contains" and "contain") are inclusive or open-ended, and will be understood to indicate 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 disclosed ingredients or steps, limiting the scope of the claims to specific materials or steps that do not materially affect the basic and novel characteristics of the claimed disclosure. The word "consisting of" (and any form of consisting of, such as "consisting of" and "consists of") is meant to include and be limited to whatever follows the phrase "consisting of." Thus, the phrase "consisting of" indicates that the listed elements are required or mandatory, and no other elements may be present.

[0063] Throughout this specification, references to "one embodiment", "an embodiment", "specific embodiment", "related embodiment", "an embodiment", "additional embodiment", "further embodiment", "some embodiments", "one aspect", "an aspect", "specific aspect", "related aspect", "an aspect", "additional aspect", "further aspect", "some aspects" or combinations thereof mean that the particular features, structures or characteristics described in association with the aspect are included in at least one aspect of the present disclosure. Therefore, the above phrases appearing in various places throughout this specification do not necessarily all refer to the same aspect. Further, the particular features, structures or characteristics may be combined in one or more aspects in any suitable manner.

[0064] The terms "inhibit" or "reduce" or any variation of these terms, include any measurable decrease or complete inhibition to achieve a desired result. The terms "improve", "enhance" or "increase" or any variation of these terms, include any measurable increase to achieve a desired result or production of a protein or molecule.

[0065] As used herein, the terms "reference," "standard," or "control" describe a value to which a relative comparison is made. For example, an agent, subject, population, sample, or value of interest is compared to a reference, standard, or control agent, subject, population, sample, or value of interest. Testing or determination of a reference, standard, or control can be performed substantially simultaneously and / or together with testing or determination of an agent, subject, population, sample, or value of interest, and / or can be determined or characterized under conditions or circumstances comparable to the agent, subject, population, sample, or value of interest being evaluated.

[0066] As used herein, the term "DNA" means a nucleic acid molecule comprising deoxyribonucleotide residues (e.g., containing the nucleotide bases adenine (A), cytosine (C), guanine (G) and / or thymine (T)). For example, DNA may contain all or most of the deoxyribonucleotide residues. As used herein, the term "deoxyribonucleotide" means a nucleotide lacking a hydroxyl group at the 2' position of the β-D-ribofuranosyl group. Without any limitation, DNA may encompass double-stranded DNA, antisense DNA, single-stranded DNA, isolated DNA, synthetic DNA, recombinantly produced DNA, and modified DNA.

[0067] As used herein, the term "RNA" means a nucleic acid molecule comprising ribonucleotide residues (e.g., containing the nucleotide bases adenine (A), cytosine (C), guanine (G) and / or uracil (U) or N-1-methyl pseudouridine). For example, RNA may contain all or most of the ribonucleotide residues. As used herein, the term "ribonucleotide" means a nucleotide having a hydroxyl group at the 2' position of the β-D-ribofuranosyl group. In one aspect, RNA may be a messenger RNA (mRNA), which relates to an RNA transcript encoding a peptide or protein. As known to those skilled 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 may include double-stranded RNA, antisense RNA, single-stranded RNA, isolated RNA, synthetic RNA, recombinantly produced RNA, circular RNA, self-amplifying RNA (saRNA), and modified RNA (modRNA).

[0068] As contemplated herein, without any limitation, RNA can be used as a therapeutic modality for treating and / or preventing a variety of disorders in mammals (including humans). The contemplated methods include administering the RNA described herein to a mammal, such as a human. For example, in one aspect, such methods of using RNA include RNA vaccines encoding antigens to induce powerful neutralizing antibodies and accompanying / concomitant T cell responses, with preferably minimal vaccine doses to achieve protective immunity. The RNA administered is preferably RNA transcribed in vitro.

[0069] "Isolated RNA" is defined as an RNA molecule that can be recombined or has been separated from the total genomic nucleic acid. "Modified RNA" or "modRNA" refers to an RNA molecule (e.g., an mRNA molecule) having at least one addition, deletion, substitution, and / or change of one or more nucleotides compared to naturally occurring RNA. Such changes may be directed to the addition of non-nucleotide substances to the 5' and / or 3' ends of internal RNA nucleotides or RNA. In one aspect, such modRNA contains at least one modified nucleotide, such as a change to a nucleotide base. For example, the modified nucleotide may replace one or more uridine and / or cytidine nucleotides. For example, these replacements may occur in each uridine and / or cytidine instance in the RNA sequence, or may occur only in selected uridine and / or cytidine nucleotides. Such changes to standard nucleotides in RNA may include non-standard nucleotides, such as chemically synthesized nucleotides or deoxynucleotides. For example, at least one uridine nucleotide in the RNA sequence may be replaced by 1-methyl pseudouridine. Other such changed nucleotides are known to those skilled in the art. Such changed RNA is considered an analog of naturally occurring RNA. In some aspects, the RNA is produced by in vitro transcription using a DNA template, wherein DNA refers to a nucleic acid containing deoxyribonucleotides. In some aspects, the RNA can be a replicon RNA (replicon), particularly a self-replicating RNA, or a self-amplifying RNA (saRNA).

[0070] As used herein, "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 an endogenous version of a molecule naturally occurring in an organism. In some aspects, a wild type version of a protein or polypeptide is used, however, in many aspects of the present disclosure, a modified protein or polypeptide is used to induce an immune response. The above terms can be used interchangeably. "Modified protein" or "modified polypeptide" or "variant" refers to a protein or polypeptide whose chemical structure (particularly its amino acid sequence) is changed relative to a wild type protein or polypeptide. In some aspects, a modified / variant protein or polypeptide has at least one modified activity or function (it should be recognized that a protein or polypeptide may have multiple activities or functions). It is specifically envisioned that a modified / variant protein or polypeptide may be changed in one activity or function, but retains the activity or function of the wild type in other aspects (e.g., immunogenicity). When a protein is specifically mentioned herein, it is generally referred to as a natural (wild type) or recombinant (modified) protein. Proteins can be isolated directly from their native organisms, 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 fragments and recombinant vectors that incorporate a nucleic acid sequence encoding a polypeptide (e.g., an antigen or fragment thereof). The term "recombinant" can be used in conjunction with the name of a polypeptide or a specific polypeptide, and this generally refers to a polypeptide produced from a nucleic acid molecule that has been subjected to in vitro manipulation of the nucleic acid or as a replication product of such a molecule.

[0071] 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 from which it was derived (e.g., when produced by recombinant DNA technology), or chemical precursors or other chemicals (e.g., when chemically synthesized). In addition, an isolated compound refers to a compound that can be administered to a subject as an isolated compound; in other words, if the compound is attached to a column or embedded in an agarose gel, it cannot simply be considered "isolated." In addition, an "isolated nucleic acid fragment" or "isolated peptide" is a non-naturally occurring fragment and / or a nucleic acid or protein fragment that is not normally in a functional state and / or has been altered or removed from its natural state by human intervention. For example, DNA naturally occurring in living animals is not "isolated," but synthetic DNA, or DNA that is partially or completely separated from materials coexisting with its natural state, is "isolated." An isolated nucleic acid can be present in a substantially purified form, or can be present in a non-natural environment, such as, for example, a cell into which the nucleic acid is delivered.

[0072] All patents, published patent applications, other publications, and databases mentioned herein are incorporated by reference in their entirety into the relevant art.

[0073] DNA template

[0074] In some respects, the method for producing RNA molecules (e.g., mRNA) includes providing a sample comprising a linear DNA template. The DNA template includes a sequence encoding a target gene, and the target gene encodes, for example, a target peptide or polypeptide. In some respects, the DNA template includes an RNA polymerase promoter sequence operably connected to the sequence encoding the target gene. In some respects, the DNA template includes an RNA polymerase promoter sequence operably connected to a corresponding RNA polymerase gene sequence, and the RNA polymerase promoter sequence is operably connected to a subgenomic promoter, and the subgenomic promoter is operably connected to the sequence encoding the target gene. For example, in some preferred aspects, the DNA template includes an RNA-dependent RNA polymerase (RdRp) promoter sequence operably connected to a RdRp gene sequence, and the RdRp promoter sequence is operably connected to a subgenomic promoter, and the subgenomic promoter is operably connected to the sequence encoding the target gene. In some respects, the DNA template lacks a plasmid backbone.

[0075] In some aspects, linear DNA template is a linearized plasmid DNA used as an in vitro transcription template. In some aspects, with plasmid DNA template transfection cells, such as bacterial cells, such as Escherichia coli, such as DH10B cells. Cultivate the cells of transfection to replicate plasmid DNA, and then separate and purify the plasmid DNA. 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 the amplification target loop (ATC) forming the template, and new DNA is prepared on the template, so that the initial sequence is extended to the continuous sequence of the repetitive sequence complementary to the ring, but only about thousands of copies are produced per hour. In some aspects, linear DNA template is provided by index RCA, which includes super branch RCA (also referred to as branch amplification).

[0076] In other aspects, the linear DNA template is provided by a cell-free DNA synthesis method, the method comprising contacting the 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 more nucleotides are continuously or intermittently supplied to the reaction mixture during the method.

[0077] In some aspects, the DNA template also includes an RNA polymerase promoter sequence, such as a T7 promoter, which is located at the 5' end of the target gene and is operably connected to the target gene. In some aspects, the DNA template includes an RNA polymerase promoter sequence that is operably connected to a corresponding RNA polymerase gene sequence, and the RNA polymerase promoter sequence is operably connected to a subgenomic promoter, which is operably connected to the sequence encoding the target gene. In some preferred aspects, the DNA template includes an RNA-dependent RNA polymerase (RdRp) promoter sequence, which is located at the 5' end of the subgenomic promoter and is operably connected to a subgenomic promoter, which is operably connected to the sequence encoding the target gene. As used herein, the phrase "operably connected" refers to the functional connection between two or more molecules, constructs, transcripts, entities, parts, etc. For example, the target gene operably connected to the RNA polymerase promoter allows the transcription of the target gene. Any RNA polymerase or its variant can be used for methods described herein. The RNA polymerase may be selected from, but is not limited to, a bacteriophage RNA polymerase, such as T7 RNA polymerase, T3 RNA polymerase, SP6 RNA polymerase, and / or a mutant polymerase, such as, but not limited to, a polymerase capable of incorporating a modified nucleic acid.

[0078] As used herein, "target gene" refers to a polynucleotide encoding a target polypeptide or protein. Depending on the context, a target gene refers to a deoxyribonucleic acid, such as a target gene in a DNA template that can be transcribed into an RNA molecule, or a ribonucleic acid, such as a target gene in an RNA molecule that can be translated to produce an encoded target polypeptide in vitro, in vivo, in situ or ex vivo. As described in more detail below, target polypeptides include, but are not limited to, biologics, antibodies, vaccines, therapeutic proteins or peptides, and the like.

[0079] Flanking region: untranslated region (UTR)

[0080] In some aspects, the method for producing RNA molecules includes (a) providing a sample with a linear DNA template, the DNA template includes an RNA polymerase promoter sequence, which is operably connected to the sequence encoding the target gene and a 5' untranslated region (UTR) and / or a 3'UTR. In some aspects, the DNA template includes an RNA polymerase promoter sequence operably connected to a corresponding RNA polymerase gene sequence, the RNA polymerase gene sequence is operably connected to a subgenomic promoter, and the subgenomic promoter is operably connected to the sequence encoding the target gene. For example, in some aspects, the method for producing RNA molecules includes (a) providing a sample with a linear DNA template, the DNA template includes an RNA dependent RNA polymerase (RdRp) promoter sequence, which is located at the 5' end of the subgenomic promoter and is operably connected to the subgenomic promoter, and the subgenomic promoter is operably connected to the sequence encoding the target gene and a 5' untranslated region (UTR) and / or a 3'UTR.

[0081] DNA templates and RNA molecules can include UTRs. The untranslated region (UTR) of a gene is transcribed but not translated. The 5'UTR starts from the transcription start site and continues to the start codon, but does not include the start codon; while the 3'UTR starts immediately after the stop codon and continues to the transcription termination signal. The regulatory features of the UTR can be incorporated into the polynucleotides, primary constructs and / or mRNA of the present invention to enhance the stability of the molecule. Specific features can also be incorporated to ensure that the transcripts are controlled down-regulated to prevent them from being misdirected to unwanted organ sites.

[0082] Natural 5'UTR has the characteristic of playing a role in translation initiation. They have characteristics such as Kozak sequence, which is well known to be involved in the method by which ribosomes initiate translation of many genes. Kozak sequence has a consensus sequence CCR (A / G) CCAUGG, where R is a purine (adenine or guanine) three bases upstream of the start codon (AUG), followed by another "G". 5'UTR is also known to form a secondary structure involved in the binding of elongation factors. By engineering the characteristics usually found in genes expressed in large quantities in specific target organs, the stability and protein production of polynucleotides, primary constructs can be enhanced. For example, for muscle (MyoD, myosin, myoglobin, myogenin, myogenic factor (Herculin)), endothelial cells (Tie-1, CD36), etc., 5'UTR from other tissue-specific mRNAs can be used to improve expression in the tissue.

[0083] Other non-UTR sequences can be incorporated into 5' (or 3' UTR) UTR. For example, an intron or a portion of an intron sequence can be incorporated into the flanking region of the polynucleotide, primary construct or mRNA herein. The incorporation of an intron sequence can increase protein production and mRNA levels. Cap-dependent translation involves recruiting the pre-transcription initiation complex (PIC) to the 5' end of the mRNA, followed by scanning to find the AUG start codon in the optimal sequence environment. AUG recognition promotes scanning stop, the release of most initiation factors, and the recruitment of large ribosomal subunits to initiate extension. The effective recognition of the start codon depends on the sequence around it. In some preferred aspects, the DNA template includes the start codon GGG. In some preferred aspects, the DNA template includes the start codon AGA. In some aspects, the sequence CRCCaugG (R = purine, A or G) can provide the best environment for AUG recognition in eukaryotes.

[0084] 3'UTRs are known to have segments of adenosine and uridine embedded in them. These AU-rich features are particularly common in genes with high turnover rates. Based on their sequence characteristics and functional properties, AU-rich elements (AREs) can be divided into three categories: Class I AREs include several dispersed copies of the AUUUA motif within the U-rich region. C-Myc and MyoD include Class I AREs. Class II AREs have two or more overlapping UUAUUUA (U / A) (U / A) nonamers. Molecules that include this type of ARE include GM-CSF and TNF-α. Class III ARES are less well defined. These U-rich regions do not include the AUUUA motif, and c-Jun and myogenin are two examples of this type that have been studied more deeply. Most proteins that bind to AREs are known to destabilize the messenger, while members of the ELAV family (most notably HuR) have been shown to increase the stability of mRNA. HuR can bind to all three types of AREs. Engineering HuR-specific binding sites into the 3'UTR of nucleic acid molecules can result in HuR binding and thereby stabilizing the messenger in vivo. The introduction, removal or modification of the 3' UTRARE can be used to modulate the stability of polynucleotides and primary constructs.

[0085] When engineering specific polynucleotides and / or primary constructs, one or more copies of ARE can be introduced to make the polynucleotides and / or primary constructs more unstable, thereby reducing translation and reducing the production of product proteins. Similarly, ARE can be identified and removed or mutated to increase intracellular stability, and thus increase the translation and production of product proteins. Polynucleotides and / or primary constructs can be used in related cell lines for transfection experiments, and the production of proteins can be measured at different time points after transfection. For example, cells can be transfected with different ARE engineered molecules, and the protein produced can be measured at 6 hours, 12 hours, 24 hours, 48 ​​hours and 7 days after transfection by using an ELISA kit for the related protein.

[0086] Poly(A) tail

[0087] In some aspects, the method for producing RNA molecules disclosed herein includes providing a sample with a DNA template, wherein the DNA template has an RNA polymerase promoter sequence operably connected to a sequence encoding a target gene and a poly (A) tail sequence of 20-100 nucleotides. The poly (A) tail can prevent the degradation of RNA molecules in cells. Therefore, in some aspects, the plasmid DNA template includes a sequence encoding a poly (A) tail located at the 3' end of the target gene. As used herein, "poly (A) tail" refers to an adenine nucleotide chain. In some aspects, the poly (A) tail comprises 5-300 adenine nucleotides in length, for example, 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, 200, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221 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 poly A polymerase. In some aspects, the RNA molecule does not include the poly (A) tail.

[0088] In some aspects, immediately downstream of the poly(A) tail coding sequence on the plasmid DNA template is a restriction endonuclease recognition site to linearize the plasmid. Linearization of the plasmid can reduce transcriptional read-through.

[0089] In some aspects, after linearization, the plasmid DNA template is filtered into a suitable solvent, such as water, HEPES and EDTA. In preferred aspects, the solvent includes 10 mM HEPES, 0.1 mM EDTA, etc. Filtration is performed via, for example, ultrafiltration, diafiltration, or, for example, tangential flow ultrafiltration / diafiltration.

[0090] The linear DNA template can be purified prior to use as a template for in vitro transcription. For example, the linear DNA template can be purified by chromatography or ethanol precipitation.

[0091] In vitro transcription

[0092] In vitro transcription (IVT) refers to a process that allows the synthesis of RNA molecules of any sequence under the guidance of DNA, ranging in size from short oligonucleotides to several kilobases. In some aspects, in vitro transcription involves engineering a DNA template to include a phage promoter sequence (e.g., from T7 E. coli phage) upstream of the target sequence, followed by transcription using a corresponding RNA polymerase. In some aspects, the resulting RNA molecule is then modified (e.g., by capping, splicing, adding a poly (A) tail, etc.).

[0093] Methods for producing RNA molecules (e.g., mRNA) described herein include contacting a DNA template with an in vitro transcription (IVT) reaction system. In some aspects, the IVT reaction system comprises an RNA polymerase and ribonucleotides, which may be natural and / or modified ribonucleotides. In some aspects, the IVT reaction system comprises 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 non-natural (modified, such as, for example, N-1-methylpseudouridine triphosphate) NTPs.

[0094] RNA polymerase

[0095] In some aspects, the RNA polymerase used to produce mRNA transcripts 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 can be used to transcribe mRNA or self-amplify RNA from a DNA template. RNA polymerases represent the primary mechanism driving transcription. RNA polymerases have been fully isolated and purified so that they can be used to produce RNA in vitro. In some aspects, the RNA polymerase is a T7 RNA polymerase, which refers to a monomeric T7 bacteriophage-encoded DNA-guided RNA polymerase that catalyzes the formation of RNA in the 5' to 3' direction. Wild-type T7 RNA polymerase includes 883 amino acids. It is homologous to the T3 RNA polymerase and has some homology to the SP6 RNA polymerase.

[0096] In some aspects, the RNA polymerase comprises an engineered T7 RNA polymerase variant, such as a variant that allows for selective incorporation of an m7G(5')ppp(5')m7G cap analog over GTP during initiation of transcription in vitro. 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 a cap analog (e.g., an "anti-reverse cap analog" (3'-O-Me-m7G(5')ppp(5')G; "ARCA"), or a methylated cap analog having one or more nucleotides at the transcription start site (e.g., m7G(5')ppp(5')N, where N is any nucleotide) to initiate transcription during transcription initiation. The 5' cap is an altered nucleotide at the 5' end of some eukaryotic primary transcripts (e.g., precursor messenger RNA). A typical cap structure comprises a 7-methylguanosine (m7G) cap connected to the first nucleotide of the transcript via a 5'-5' triphosphate bridge. 7 G). Cap analogs can include, for example, one, two or more methyl (or other substitution) groups at specific positions. Cap molecules can be added before the IVT reaction or after mRNA synthesis by enzymatic capping. Cap molecules added before the IVT reaction can make mRNA production more direct.

[0097] Ribonucleotide

[0098] In the methods described herein, the IVT reaction system comprises nucleotides (e.g., non-modified ribonucleoside triphosphates or modified ribonucleoside triphosphates). The nucleotides can be selected from any of the natural nucleotides, such as A, G, C, and U ribonucleotides; modified nucleotides (such as, for example, N-1-methylpseudouridine triphosphate); or combinations thereof. In some aspects, the ribonucleotides are Tris buffered, such as a 100 mM ribonucleotide aqueous solution titrated to pH 7.3-7.5 with Tris base. In some aspects, the ribonucleotides are present in the form of sodium salts.

[0099] Modified nucleobases that can be incorporated into modified nucleosides and nucleotides and are present in RNA molecules produced 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-N6 isopentenyladenosine); io6A (N6-(cis-hydroxyisopentenyl)adenosine); ms2io6A (2-methylthio-N6 -(cis-hydroxyisopentenyl)adenosine); g6A (N6-glycylaminoformyladenosine); t6A (N6-threonylaminoformyladenosine); ms2t6A (2-methylthio-N6-threonylaminoformyladenosine); m6t6A (N6-methyl-N6-threonylaminoformyladenosine); hn6A (N6-hydroxynorvalylaminoformyladenosine); ms2hn6A (2-methylthio-N6-hydroxynorvalylaminoformyladenosine); Ar(p) (2'-0-riboadenosine (phosphate)); I (inosine); mil (1-methylinosine); m'lm (1,2'-0-dimethylinosine); m3C (3-methylcytidine); Cm (2T-0-methylcytidine); s2C (2-thiocytidine); ac4C (N4-acetylcytidine); ￡5C (5-formylcytidine); m5Cm (5,2-0-dimethylcytidine); ac4Cm (N4-acetyl 2TO-methylcytidine); k2C (lysine); mlG (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 (whitingoside); o2yW (peroxywhitingoside); OHyW (hydroxy whitinoside); OHyW* (unmodified hydroxywhitinoside); imG (whitinoside); mimG (methylguanosine); Q (quercetin); oQ (epoxyquercetin); galQ (galactosylquercetin); manQ (mannosylquercetin); preQo (7-cyano-7-deazaguanosine); preQi (7-aminomethyl-7-deazaguanosine); G* (archaurine); 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-hydroxyacetic acid); mcmo5U (uridine-5-hydroxyacetic acid methyl ester); chm5U (5-(carboxyhydroxymethyl)uridine); mchm5U (5-(carboxyhydroxymethyl)uridine methyl ester); mcm5U (5-methoxycarbonylmethyluridine); 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-seleno-uridine); ncm5U (5-carbamoylmethyluridine); ncm5Um (5-carbamoylmethyl-2'-0-methyluridine); cmnm5U (5-carboxymethylaminomethyluridine); cnmm5Um (5-carboxymethylaminomethyl-2-LO-methyluridine); 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, TO-dimethyladenosine); rn62Am (N6, N6, 0-2-trimethyladenosine); m2'7G (N2,7-dimethyl methylguanosine); m2'2'7G (N2,N2,7-trimethylguanosine); m3Um (3,2T-0-dimethyluridine); m5D (5-methyldihydrouridine); f5Cm (5-formyl-2'-0-methylcytidine); mlGm (l,2'-0-dimethylguanosine); m'Am (1,2-0-dimethyladenosine) methyleneuridine); tm5s2U (S-taurine methyl-2-thiouridine); imG-14 (4-demethylguanosine); imG2 (isoguanosine);ac6A (N6-acetyladenosine), hypoxanthine, inosine, 8-oxoadenine, 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-chloro Cytosine, 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 of N-1-methylpseudouridine, pseudouridine, N6-methyladenosine, 5-methylcytidine, and 5-methyluridine. ;

[0100] In some aspects, the RNA molecules can include phosphoramidite, phosphorothioate, and / or methylphosphonate bonds.

[0101] In some aspects, the RNA molecule does not include modified nucleotides, e.g., does not include modified nucleobases, and all nucleotides in the RNA molecule are conventional standard ribonucleotides A, U, G, and C, except for an optional 5' cap, which may include, e.g., 7-methylguanosine. In other aspects, the RNA may include a 5' cap comprising 7'-methylguanosine, and the first 1, 2, or 3 5' ribonucleotides may be methylated at the 2' position of the ribose.

[0102] Exemplary in vitro transcription reaction system

[0103] In some aspects, the in vitro transcription reaction system comprises the following: an RNA polymerase, such as T7 RNA polymerase, a DNA template; nucleoside triphosphates (NTPs); magnesium; and a buffer, such as HEPES or Tris (or both HEPES and Tris).

[0104] In some aspects, the in vitro transcription reaction system comprises an RNA polymerase, such as T7 RNA polymerase at a final concentration of 1000-44000 U / mL, such as at least, at most, or about 1000, 1050, 1100, 1150, 1200, 1250, 1300, 1350, 1400, 1450, 1500, 1550, 1600, 1650, 1700, 1750, 1800, 1850, 1900, 1950, 2000, 2050, 2100, 2150, 2200, 2250, 2300, 2350, 2400, 2450, 2500, 2550, 2600, 2650, 2700, 2750, 2800, 2850, 2900, 2950, ​​3000, 3100, 3200 050、3100、3150、3200、3250、3300、3350、3400、3450、3500、3550、3600、3650、3700、3750、3800、3850、3900、3950、4000、4050、4100、4150、4200、4250、4300、4350、4400、4450、4500、4550、4600、4650、4700、4750、4800、4850、4900、4950、5000、5050、5100、5150、5200、5250、5300、5350、5400、5450 , 5500, 5550, 5600, 5650, 5700, 5750, 5800, 5850, 5900, 5950, 6000, 6050, 6100, 6150, 6200, 6250, 6300, 6350, 6400, 6450, 6500, 6550, 6600, 6650, 6700, 6750, 6800, 6850, 6900, 6950, 7000, 7050, 7100, 7150, 7200, 7250, 7300, 7350, 7400, 7450, 7500, 7550, 7600, 7650, 7700, 7750, 7800, 7850, 79 00、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、115 50、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、330 In some aspects, the in vitro transcription reaction system comprises an RNA polymerase, such as a T7 RNA polymerase, having a final concentration of 7000U / mL. In some aspects, the in vitro transcription reaction system comprises an RNA polymerase, such as T7 RNA polymerase, at a final concentration of 8000U / mL. In some aspects, the in vitro transcription reaction system comprises an RNA polymerase, such as T7 RNA polymerase, at a final concentration of 14000U / mL. In some aspects, the in vitro transcription reaction system comprises an RNA polymerase, such as T7 RNA polymerase, at a final concentration of 17000U / mL. In some aspects, the in vitro transcription reaction system comprises an RNA polymerase, such as T7 RNA polymerase, at a final concentration of 25000U / mL. In some aspects, the in vitro transcription reaction system comprises an RNA polymerase, such as T7 RNA polymerase, at a final concentration of 40000U / mL.

[0105] In some aspects, the in vitro transcription reaction system comprises a 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, 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, 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 In some aspects, the in vitro transcription reaction system comprises a DNA template having a final concentration of 40 nM. In some aspects, the in vitro transcription reaction system comprises a DNA template having a final concentration of 144 nM. In some aspects, the in vitro transcription reaction system comprises a DNA template at a final concentration of 5-24 nM DNA, such as 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 derivative therein.In some aspects, the in vitro transcription reaction system comprises a DNA template at a final concentration of 36 to 144 nM DNA, such as 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 value derivative therein. In some aspects, the in vitro transcription reaction system comprises a DNA template at a final concentration of, for example, 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, 0.50, 0.51, 0.52, 0.53, 0.54, 0.55, 0.56, 0.57, 0.58, 0.59, In some aspects, the in vitro transcription reaction system comprises a DNA template having a final concentration of 0.025 mg / mL. In some aspects, the in vitro transcription reaction system comprises a DNA template having a final concentration of 0.05 mg / mL. In some aspects, the in vitro transcription reaction system comprises a DNA template having a final concentration of 0.075 mg / mL. In some aspects, the in vitro transcription reaction system comprises a DNA template at a final concentration of 0.1 mg / mL.

[0106] In some aspects, the in vitro transcription reaction system comprises each nucleoside triphosphate (NTP) at a final concentration of, for example, 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 derived therefrom. In some aspects, the in vitro transcription system comprises each nucleoside triphosphate (NTP) at a starting concentration of, for example, 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 comprises a nucleoside triphosphate (NTP) having a final concentration of about 8 mM each. In some aspects, the in vitro transcription reaction system comprises a nucleoside triphosphate ATP having a final concentration of, for example, 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 derived therefrom. In some aspects, the in vitro transcription reaction system comprises a nucleoside triphosphate CTP having a final concentration of, for example, 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 derived therefrom. In some aspects, the in vitro transcription reaction system comprises a nucleoside triphosphate GTP at a final concentration of, for example, 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 derived therefrom. In some aspects, the in vitro transcription reaction system comprises UTP or a nucleoside triphosphate-modified UTP at a final concentration of, for example, 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 derived therefrom. In certain aspects, the concentration of NTPs in the reaction is 0.4 mM, 0.8 mM, 1 mM, 1.25 mM, 3 mM, 5 mM, 6 mM, 7 mM, 7.5 mM, 8 mM, 8.5 mM, 9 mM, 9.5 mM, or 10 mM.

[0107] In some aspects, the IVT reaction system comprises magnesium ions, e.g., as a magnesium salt, e.g., any of magnesium chloride and magnesium acetate. In some aspects, the in vitro transcription reaction system comprises 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, 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, 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, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, In some aspects, the in vitro transcription reaction system comprises 30 mM magnesium acetate. In some aspects, the in vitro transcription reaction system comprises 40 mM magnesium acetate. In some aspects, the in vitro transcription reaction system comprises 16.5 mM magnesium acetate. In some aspects, the in vitro transcription reaction system comprises 33 mM magnesium acetate. In some aspects, the in vitro transcription reaction system comprises 36 mM magnesium acetate. In some aspects, the in vitro transcription reaction system comprises 50 mM magnesium acetate.In some aspects, the in vitro transcription reaction system comprises 110 mM magnesium acetate. In some aspects, the Mg:NTP ratio can be maintained at, for example, 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 ratio, or any range or value derived therefrom. In some aspects, one or more reaction components are added during in vitro transcription by occasional bolus feeding, semi-continuous feeding, or continuous feeding. Bolus feeds can be delivered at intervals of, for example, 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 derived therefrom. These components may include, but are not limited to, one or more NTPs and cations, such as magnesium. These components can be combined into a single feed, or they can also be delivered separately in the form of multiple feeds. In some aspects, a continuous feed of at least 1 NTP can be delivered at a flow rate of, for example, 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, the continuous feed of a cation (e.g., magnesium) can be delivered at a concentration of, for example, 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 in any range or value derivative therein.

[0108] In some aspects, the in vitro transcription (IVT) reaction system comprises a buffer. Exemplary buffers for IVT reaction systems may include Tris and / or HEPES. In some aspects, the in vitro transcription reaction system comprises a buffer having a pH of, for example, 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 derived therefrom. In some aspects, the buffer is Tris-HCl at pH 8.0. In some aspects, the in vitro transcription reaction system comprises 40 mM Tris-HCl at pH 8.0. An alternative buffer for the IVT reaction system comprises 40 mM Tris at pH 7.5, 80 mM HEPES. In some aspects, the in vitro transcription reaction system does not comprise HEPES. In some aspects, the in vitro transcription reaction system comprises HEPES and Tris. In some aspects, the buffer does not comprise dithiothreitol (DTT).

[0109] In some respects, the in vitro transcription reaction system comprises an RNase inhibitor. The RNase inhibitor can reduce the degraded RNase induction in the transcription reaction method. For example, mouse RNase inhibitor can be utilized with an ultimate density 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 1200U / mL. In some respects, the in vitro transcription reaction system comprises an RNase inhibitor that ultimate density is 100U / mL. In some respects, the in vitro transcription reaction system comprises an RNase inhibitor that ultimate density is 1000U / mL.

[0110] In some aspects, pyrophosphatase is included in the in vitro transcription reaction system. Pyrophosphatase can be each nucleotide mixed with the inorganic pyrophosphate produced after the splitting into two inorganic phosphate units, which can reduce the possibility of magnesium and pyrophosphate coprecipitation to form magnesium pyrophosphate. In some aspects, pyrophosphatase can be diluted in pyrophosphatase buffer, and with 0.01mU / uL, 0.02mU / uL, 0.05mU / uL, 0.08mU / uL, 0.1mU / uL, 0.2mU / uL, 0.8mU / uL or 2mU / uL concentration is present in the reaction. In some aspects, the in vitro transcription reaction system comprises an inorganic pyrophosphatase with a final concentration of 0.25U / mL. In some aspects, the in vitro transcription reaction system comprises an inorganic pyrophosphatase with a final concentration of 0.5U / mL. In some aspects, the in vitro transcription reaction system comprises an inorganic pyrophosphatase with a final concentration of 1U / mL. In some aspects, the in vitro transcription reaction system comprises an inorganic pyrophosphatase with a final concentration of 2U / mL. In some aspects, the in vitro transcription reaction system comprises an inorganic pyrophosphatase with a final concentration of 3U / mL. In some aspects, the in vitro transcription reaction system comprises an inorganic pyrophosphatase with a final concentration of 6U / mL.

[0111] In some aspects, the in vitro transcription reaction system comprises polyamines. Exemplary polyamines include spermine, putrescene and spermidine. In some aspects, 1mM spermidine is included. In some aspects, 2.0mM spermidine is included. In some aspects, 2.15mM spermidine is included. In some aspects, the in vitro transcription reaction system lacks polyamines. In some aspects, the in vitro transcription reaction system lacks spermidine.

[0112] In some aspects, the IVT reaction system comprises a reducing agent, such as, for example, DTT (dithiothreitol), for example, 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 derived therefrom. In some aspects, the reducing agent is selected from dithiothreitol (DTT), dithioerythritol (DTE), tris (2-carboxyethyl) phosphine (TCEP), and β-mercaptoethanol. In some aspects, the IVT reaction system comprises 1 mM DTT. In some aspects, the IVT reaction system comprises 5 mM DTT. In some aspects, the IVT reaction system comprises 10 mM DTT. In some aspects, the IVT reaction system comprises 20 mM DTT. In some aspects, the IVT reaction system lacks a reducing agent. In some aspects, the IVT reaction system does not comprise DTT. In some aspects, the IVT reaction system does not comprise added DTT beyond the protective amount of DTT present in the T7 RNA polymerase stock solution.

[0113] In some aspects, the in vitro transcription reaction is carried out, e.g., 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 derivative therein. In some preferred aspects, the in vitro transcription reaction is carried out at, for example, 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, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 210, 220, 230, 240, 250, 260, 270, 280, 270, 280, 29 ... In some aspects, the in vitro transcription reaction is carried out at about 36°C for about 120 minutes. In some aspects, the in vitro transcription reaction is carried out at about 36°C for about 150 minutes. In some aspects, the in vitro transcription reaction is carried out at about 37°C for about 120 minutes. In some aspects, the in vitro transcription reaction is performed at about 37°C for about 150 minutes.

[0114] In some aspects, the in vitro transcription reaction is carried out at about 37°C for a period of 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 is carried out at about 37°C for about 150 minutes.

[0115] As disclosed herein, a reaction system that is performed at about 37°C for less than 4 hours results in high yields of highly pure RNA. In some aspects, the yield of each in vitro transcription reaction can be at least 0.3 mg RNA per mL IVT reaction starting volume to about 20 mg RNA per mL IVT reaction starting volume. For example, in some aspects, the total yield of RNA molecules can be at least, up to, 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, and 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 of the IVT reaction starting volume, or any range or value derivative therein. In preferred aspects, the total yield of RNA molecules with at least 90% of the expected full-length transcript produced by each in vitro transcription reaction can 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 IVT reaction starting volume. In some aspects, the total yield of RNA molecules with at least 90% of the expected full-length transcript produced by each in vitro transcription reaction is at least 17 mg RNA / mL IVT reaction starting volume.

[0116] In some aspects, after an IVT reaction using a DNA template and an RNA polymerase as described herein, a first composition comprising an uncapped RNA molecule is produced. In some aspects, the RNA molecule comprises a coding sequence of a target gene and a poly (A) tail. As used herein, RNA molecules include mRNA. The RNA molecule may comprise modifications, such as modified nucleotides. As used herein, "RNA molecules" produced by in vitro transcription may be referred to as "RNA transcripts". "RNA molecules" and "RNA transcripts" may include any of modified mRNA "modRNA", unmodified mRNA, and self-amplifying RNA (saRNA). In preferred aspects, the RNA molecule is saRNA.

[0117] In some aspects, the method for producing RNA molecules by contacting a DNA sample with an in vitro transcription reaction system as described herein produces a first composition with uncapped RNA molecules. In some aspects, at least 30% of the RNA molecules in the first composition include 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% of uncapped RNA molecules, or any range or value derived therefrom.

[0118] RNA molecules

[0119] The RNA molecules produced by the methods described herein can be non-coding and / or coding RNA. Non-coding RNA (ncRNA) molecules include functional RNA molecules that are not translated into peptides or polypeptides. Non-coding RNA molecules may include high abundance and functionally important RNAs, such as transfer RNA (tRNA) and ribosomal RNA (rRNA), and RNAs such as snoRNA, microRNA, siRNA, snRNA, guide RNA, circular RNA, exRNA and piRNA and long ncRNA. In preferred aspects, RNA molecules are mRNA molecules (referred to herein as "modified RNA molecules" or "modified mRNA molecules") comprising modified nucleotides. In some preferred aspects, RNA molecules are self-amplifying RNA molecules.

[0120] Coding RNA includes functional RNA molecules that can be translated into peptides or polypeptides. In some aspects, the coding RNA molecule includes at least one open reading frame encoding at least one peptide or polypeptide. The coding RNA molecule may include one (monocistron), two (bicistrons) or more (polycistronic) open reading frames (ORFs). The coding RNA molecule may be a messenger RNA (mRNA) molecule, a viral RNA molecule or a self-amplifying RNA molecule (saRNA, also referred to as a replicon). Preferably, the RNA molecule is mRNA.

[0121] RNA molecules can encode more than one protein, such as two, three, four, five, ten or more polypeptides. Alternatively, or additionally, one RNA molecule can also encode more than one antigen, such as a bicistronic or tricistronic RNA molecule encoding different or identical antigens.

[0122] The sequence of the RNA molecule may be codon optimized or de-optimized for expression in a desired host, such as human cells.

[0123] If desired, the sequence of the RNA molecule can be modified, for example, to improve the efficiency of RNA expression or replication, or to provide additional stability or resistance to degradation. For example, the RNA sequence can be modified with respect to its codon usage, for example, to improve the translation efficiency and half-life of the RNA.

[0124] In some aspects, RNA molecules may include one or more structures and / or chemical modifications or changes, which give the polynucleotide useful properties, in some aspects, including lacking the substantial induction of the innate immune response of the cell introducing the polynucleotide. As used herein, "structure" feature or modification refers to inserting, deleting, duplicating, inverting or randomizing two or more connected nucleotides in the RNA molecule, without significant chemical modification to the nucleotide itself. Due to the fact that chemical bonds are inevitably destroyed and re-formed to achieve structural modification, structural modification has chemical properties, and is therefore chemically modified. However, structural modification will result in different nucleotide sequences. For example, polynucleotide "ATCG" can be chemically modified to "AT-5meC-G". The same polynucleotide can be structurally modified to "ATCCCG" from "ATCG". Here, dinucleotide "CC" is inserted, resulting in structural modification of polynucleotides.

[0125] In some aspects, the RNA molecule may comprise one or more modified nucleotides in addition to any 5' cap structure. Naturally occurring nucleoside modifications are known in the art.

[0126] In some aspects, the RNA molecules produced by the in vitro transcription reactions described herein comprise from at least, up to, or about 20 to at least, up to, or about 100,000 nucleotides, or any range or value derivative 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 1 100 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, In some embodiments, the RNA molecule comprises at least 100 nucleotides. In some embodiments, the RNA molecule comprises at least 100 nucleotides. For example, in some aspects, the RNA is between 100 and 15,000 nucleotides in length; 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 length of the RNA is 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 comprises the target gene and a poly (A) tail. In some aspects, the RNA molecule further comprises a 5'UTR and a 3'UTR.

[0127] In some aspects, the mRNA molecule of modification encodes a single polypeptide antigen, or alternatively encodes two or more polypeptide antigens, which are linked together in the following manner, i.e., each sequence retains its nature (e.g., tandem connection) when expressed as an amino acid sequence. Then, the polypeptide produced by the mRNA of modification can be produced as a fusion polypeptide, or engineered in this manner to produce a single polypeptide or peptide sequence. In preferred aspects, the mRNA molecule of modification encodes a single polypeptide of interest.

[0128] In some preferred aspects, the RNA molecule is saRNA. "Self-amplifying RNA", "self-amplifying RNA" and "replicon" refer to RNA with self-replication ability. Self-amplifying RNA molecules can be produced by using replication elements derived from, for example, alpha viruses and replacing viral structural polypeptides with nucleotide sequences encoding polypeptides of interest. Self-amplifying RNA molecules are typically positive-strand molecules that can be directly translated after delivery to cells, and this translation provides RNA-dependent RNA polymerases that subsequently produce both antisense and sense transcripts from the delivered RNA. The delivered RNA results in the production of multiple daughter RNAs. These daughter RNAs, as well as colinear subgenomic transcripts, can be self-translated to provide in situ expression of the encoded target gene (e.g., viral antigen), or can be transcribed to provide further transcripts with the same meaning as the delivered RNA, which are translated to provide in situ expression of the antigen. The overall result of this series of transcriptions is the amplification of the number of introduced saRNAs, and thus the encoded target gene (e.g., viral antigen) becomes the main polypeptide product of the cell.

[0129] In some aspects, the self-amplification RNA comprises at least one or more genes selected from any one of viral replicase, viral protease, viral helicase and other non-structural viral proteins. In some aspects, the self-amplification RNA may also include 5'- and 3'-end traction replication sequences, and optionally a heterologous sequence encoding a desired amino acid sequence (e.g., target antigen). A subgenomic promoter for guiding heterologous sequence expression may be included in the self-amplification RNA. Alternatively, the heterologous sequence (e.g., target antigen) may be fused in-frame with other coding regions in the self-amplification RNA and / or may be under the control of an internal ribosome entry site (IRES).

[0130] In preferred aspects, the self-amplifying RNA molecules are not encapsulated in virus-like particles. The self-amplifying RNA molecules described herein can be designed so that the self-amplifying RNA molecules cannot induce the production of infectious viral particles. This can be achieved by, for example, omitting one or more viral genes encoding structural proteins necessary for the production of viral particles in the self-amplifying RNA. For example, when the self-amplifying RNA molecules are based on alpha viruses, such as Sindbis virus (SINV), Semliki Forest virus and Venezuelan equine encephalitis virus (VEEV), one or more genes encoding viral structural proteins (e.g., capsid and / or envelope glycoproteins) can be omitted.

[0131] In some aspects, the self-amplifying RNA molecules described herein encode (i) an RNA-dependent RNA polymerase that can transcribe RNA from the self-amplifying RNA molecule and (ii) a target polypeptide, such as a viral antigen. In some aspects, the polymerase can be an alphavirus replicase, such as an alphavirus protein nsP4. In some aspects, the self-amplifying RNA molecules described herein can include one or more modified nucleotides (e.g., pseudouridine, N6-methyladenosine, 5-methylcytidine, 5-methyluridine).

[0132] The saRNA construct may encode at least one nonstructural protein (nsP) located at the 5' or 3' end of the sequence encoding at least one peptide or polypeptide of interest. Preferably, the sequence encoding at least one nsP is located at the 5' end of the sequence encoding the peptide or polypeptide of interest. Therefore, the sequence encoding at least one nsP is preferably located at the 5' end of the RNA construct. In some aspects, at least one nonstructural protein encoded by the RNA construct may be an RNA polymerase nsP4. Preferably, the saRNA construct encodes nsP1, nsP2, nsP3 and nsP4. As known in the art, nsP1 is a membrane anchor (membraneanchor) of the viral capping enzyme and replication complex (RC). nsP2 is an RNA helicase and a protease responsible for ns polyprotein processing. nsP3 interacts with several host proteins and can regulate protein poly-ADP-ribosylation and mono-ADP-ribosylation. nsP4 is a core viral RNA-dependent RNA polymerase. In some aspects, the polymerase may be an alpha virus replicase, for example comprising one or more alpha virus proteins nsP1, nsP2, nsP3 and nsP4.

[0133] Although the natural alphavirus genome encodes virion structural proteins in addition to encoding non-structural replicase polypeptides, in some aspects, the self-amplifying RNA molecules do not encode alphavirus structural proteins. In some aspects, the self-amplifying RNA can lead to the production of its own genomic RNA copies in the cell, but no viral particles containing the RNA are produced. Without being bound by theory or mechanism, the inability to produce these viral particles means that, unlike wild-type alphaviruses, self-amplifying RNA molecules cannot self-perpetuate in an infectious form. Alphavirus structural proteins necessary for perpetuation in wild-type viruses are not present in the self-amplifying RNA of the present disclosure, and their positions are occupied by genes encoding target proteins, so that the subgenomic transcripts encode immunogens rather than alphavirus virion structural proteins.

[0134] 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, for example, to encode further antigens or to encode additional polypeptides.

[0135] Alternatively, the self-amplifying RNA molecules described herein can also be designed to induce the production of attenuated or virulent infectious viral particles, or to produce viral particles capable of a single round of subsequent infection.

[0136] When delivered to vertebrate cells, self-amplifying RNA molecules can be transcribed from themselves (or from their own antisense copies) to cause the production of multiple progeny RNAs. Self-amplifying RNA can be directly translated after delivery to cells, and this translation provides RNA-dependent RNA polymerase, which then produces transcripts from the RNA delivered, thereby producing multiple progeny RNAs. These RNA molecules are antisense relative to the RNA delivered, and can be translated by themselves to provide the in situ expression of gene products, or can be transcribed to provide further transcripts with the same meaning as the RNA delivered, and the transcripts are translated to provide the in situ expression of gene products.

[0137] In some aspects, saRNA molecules are based on alphaviruses. Alphaviruses include a group of genetically, structurally, and serologically related arthropod-borne viruses of the Togaviridae family. Exemplary viruses and subtypes of viruses in the genus Alphavirus include Sindbis virus, Semliki Forest virus, Ross River virus, and Venezuelan equine encephalitis virus. Therefore, the self-amplifying RNA described herein may include an RNA replicase derived from any 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 include a sequence derived from a mutant or wild-type viral sequence, such as an attenuated TC83 mutant of VEEV that has been used for saRNA.

[0138] Alphavirus-based saRNA is (+) chain saRNA, which can be translated after delivery to cells, which results in the translation of replicase (or replicase-transcriptase). Replicase is translated into polyprotein, which automatically cleaves to provide a replication complex, which produces a genome (-) chain copy of the (+) chain delivery RNA. These (-) chain transcripts themselves can be transcribed to produce further copies of the (+) chain parent RNA, and also produce subgenomic transcripts encoding the desired gene product. Therefore, the translation of subgenomic transcripts results in the in situ expression of the desired gene product by infected cells. Suitable alphavirus saRNA can use replicase from Sindbis virus, Semliki Forest virus, Eastern equine encephalitis virus, Venezuelan equine encephalitis virus or its mutant variants.

[0139] In some aspects, the self-amplifying RNA molecule is derived from or based on a virus other than an alphavirus, preferably a positive-strand RNA virus, and more preferably a picornavirus, a flavivirus, a rubella virus, a pestivirus, a hepatitis virus, a calicivirus, or a coronavirus. Suitable wild-type alphavirus sequences are well known and available from sequence depositories, such as the American Type Culture Collection, Rockville, Md. Representative examples of suitable alphaviruses include Aura (ATCC VR-368), Bebaru (ATCC VR-600, ATCC VR-1240), Kabaso (ATCC VR-922), Chikungunya (ATCC VR-64, ATCC VR-1241), Eastern Equine Encephalitis (ATCC VR-65, ATCC VR-1242), Fort Morgan (ATCC VR-924), Geta (ATCC VR-369, ATCC VR-1243), Kuzilagachi (ATCC VR-927), Mayaro (ATCC VR-66), Mayaro (ATCC VR-1277), Middelburg (ATCC VR-370), Mukumbu (ATCC VR-580, ATCC VR-1244), Ndum (ATCC VR-371), Pishuna (ATCC VR-372, ATCC VR-1245), Ross River (ATCC VR-373), and VR-373, ATCC VR-1246), Semliki Forest virus (ATCC VR-67, ATCC VR-1247), Sindbis virus (ATCC VR-68, ATCC VR-1248), Tonate (ATCC VR-925), Trinity (ATCC VR-469), Una (ATCC VR-374), Venezuelan equine encephalitis virus (ATCC VR-69, ATCC VR-923, ATCC VR-1250, ATCC VR-1249, ATCC VR-532), Western equine encephalitis virus (ATCC VR-70, ATCC VR-1251, ATCC VR-622, ATCC VR-1252), Vodaro River (ATCC VR-926), and Y-62-33 (ATCC VR-375).

[0140] 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 4kb. For example, the self-amplifying RNA may include at least, at most, or about 5kb, 6kb, 7kb, 8kb, 9kb, 10kb, 11kb, or 12kb or more, or any range or value derived therefrom. In some instances, the self-amplifying RNA is about 4kb to about 12kb, about 5kb to about 12kb, about 6kb to about 12kb, about 7kb to about 12kb, about 8kb to about 12kb, about 9kb to about 12kb, about 10kb to about 12kb, about 11kb to about 12kb, about 5kb to about 11kb, about 5kb to about 10kb, about 5kb to about 9kb, about 5kb to about 8kb, about 5kb to about 7kb, about 5kb to about 6kb, about 6kb to about 7kb, about 5kb to about 6kb, about 6kb to about 6kb. 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.

[0141] In some aspects, the self-amplifying RNA molecule can encode a single polypeptide antigen, or alternatively, two or more polypeptide antigens linked together in a manner such that each sequence retains its identity when expressed as an amino acid sequence (e.g., linked in series). The polypeptide produced by the self-amplifying RNA can then be produced as a fusion polypeptide, or engineered in such a manner to produce a separate polypeptide or peptide sequence.

[0142] In some aspects, the self-amplification RNA described herein can encode one or more polypeptide antigens comprising a series of epitopes. Preferably, the epitope can trigger 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, i.e., a continuous adenosine residue, which can be attached to the 3' end of the RNA. The poly (A) tail can increase the half-life of the RNA molecule. The RNA molecule can also 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 section of at least 10 continuous adenosine residues and up to 300 continuous adenosine residues. Preferably, the RNA molecule includes at least 20 continuous adenosine residues and up to 40 continuous adenosine residues. In some preferred aspects, the RNA molecule includes about 40 continuous adenosine residues. In some aspects, the RNA molecule includes about 80 continuous adenosine residues. The poly (A) tail can play a key regulatory role in enhancing translation efficiency and regulating the efficiency of mRNA quality control and degradation. Short sequences or high polyadenylation can send RNA degradation signals. Exemplary designs include poly (A) tails of about 40 A, about 80 A. In some aspects, the RNA molecule further comprises a nuclease recognition site sequence immediately downstream of the poly (A) tail sequence.

[0143] In some aspects, RNA molecules produced by the in vitro transcription reactions described herein are purified, e.g., including filtration, which can be performed, e.g., by ultrafiltration, diafiltration, or, e.g., tangential flow ultrafiltration / diafiltration.

[0144] RNA molecule capping

[0145] In some aspects, the method for producing RNA molecules described herein further includes capping the uncapped RNA molecules by contacting the uncapped RNA molecules with a capping reaction system, wherein the capping reaction system comprises any one of guanylate transferase, s-adenosine-L-methionine (SAM), guanosine triphosphate (GTP) and 2'-O-methyltransferase and any combination thereof, to produce capped RNA molecules. In some aspects, the 5' end of the RNA is capped with a modified ribonucleotide having the structure m7G (5') ppp (5') N (O-cap structure) or a derivative thereof, which can be incorporated during RNA synthesis (co-transcriptional capping) or can be performed enzymatically after RNA transcription (capping after transcription). In some preferred 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 the RNA molecule is purified (e.g., tangential flow filtration).

[0146] An exemplary enzymatic reaction for capping can include the use of vaccinia virus capping enzyme (VCE), which comprises mRNA triphosphatase, guanylate transferase, and guanine-7-methyltransferase, which catalyzes the construction of N7-monomethylated type O cap structure. Type O cap structure plays an important role in maintaining the stability and translation efficacy of RNA molecules. The 5' cap of RNA molecules can be further modified by 2'-O-methyltransferase, which results in the generation of type 1 cap structure (m7Gppp[m2'-O]N), which can further improve translation efficacy.

[0147] In some aspects, RNA molecules can be enzymatically capped at the 5' end using vaccinia guanylate transferase, guanosine triphosphate and S-adenosyl-L-methionine to produce a type 0 cap structure. An inverted 7-methylguanosine cap is added via a 5' to 5' triphosphate bridge. Alternatively, a type 1 cap structure is produced using a 2'-O-methyltransferase with vaccinia guanylate transferase, wherein in addition to the type 0 cap structure, the 2'-OH group on the first transcribed nucleotide is methylated. S-adenosyl-L-methionine (SAM) is a cofactor used as a methyl transfer agent. In some preferred aspects, RNase inhibitors are not included in the enzymatic capping reaction. In preferred aspects, the enzymatic capping reaction step is performed under continuous mixing. On the other hand, the RNA molecules are not co-transcriptionally capped.

[0148] Non-limiting examples of 5' cap structures are, in particular, structures that have stronger cap-binding polypeptide binding, longer half-life, lower sensitivity to 5' endonucleases, and / or lower 5' decapping than synthetic 5' cap structures known in the art (or compared to wild-type, natural or physiological 5' cap structures). For example, a recombinant vaccinia virus capping enzyme and a recombinant 2'-O-methyltransferase can generate a typical 5'-5'-triphosphate bond between the 5'-terminal nucleotide of the mRNA and the guanine cap nucleotide, wherein the cap guanine comprises N7 methylation and the 5'-terminal nucleotide of the mRNA comprises a 2'-O-methyl group. Such structures are referred to as type 1 cap structures. The cap can result in higher translational capacity and cellular stability and lower cellular proinflammatory cytokine activation compared to, for example, other 5' cap analog structures known in the art. Cap structures include, but are not limited to, 7mG(5')ppp(5')N,pN2p (type 0 cap) and 7mG(5')ppp(5')N1mpNp (type 1 cap). The 0-type cap is an N7-methylguanosine attached to the 5' nucleotide via a 5' to 5' triphosphate bond, commonly referred to as an m7G cap or m7Gppp. In cells, the 0-type cap structure is essential for the efficient translation of the mRNA carrying the cap. Additional methylation at the 2'-O position of the start nucleotide produces a 1-type cap, or m7GpppNm-, where Nm represents any nucleotide with 2'-O methylation. In some aspects, the 5' end cap includes a cap analog, for example, the 5' end cap may include a guanine analog. Exemplary guanine analogs include, but are not limited to, inosine, N1-methylguanosine, 2'fluoroguanosine, 7-deazaguanosine, 8-oxoguanosine, 2-aminoguanosine, LNAguanosine, and 2-azidoguanosine.

[0149] In some aspects, the capping region can include a single cap or a series of nucleotides that form a cap. In this regard, the capping region can be from 1 to 10, such as at least, at most, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides, or any range or value therein, such as 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.

[0150] In some aspects, the length of the first and second operational regions can be from 3 to 40, such as at least, up to 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 derived therefrom, such as 5-30, 10-20, 15 or at least 4 or 30 or fewer nucleotides in length, and in addition to the start and / or stop codons, may also contain one or more signal and / or restriction sequences.

[0151] In some aspects, the self-amplification RNA molecule described herein has a 5' cap (e.g., 7-methylguanosine). The cap can enhance the in vivo translation of RNA. In some aspects, the self-amplification RNA can include (except any 5' cap structure) one or more nucleotides with modified core bases. In some aspects, the RNA molecule only includes the phosphodiester bond between nucleosides. In some aspects, the RNA molecule can include phosphoramide, thiophosphate and / or methylphosphonate bond.

[0152] In some aspects, the modified mRNA molecules described herein have a 5' cap (e.g., 7-methylguanosine). The cap can enhance the in vivo translation of RNA. In some aspects, the modified mRNA may include (except any 5' cap structure) one or more nucleotides with modified core bases. In some aspects, the RNA molecule only includes a phosphodiester bond between nucleosides. In some aspects, the RNA molecule may include phosphoramide, phosphorothioate and / or methylphosphonate bonds.

[0153] In one aspect, the capping reaction system comprises enzymatic 5' capping performed as follows. The final 1X buffer conditions comprise the following: at least, at most, or about 50 mM Tris HCl, pH 8, 5 mM KCl, 1 mM MgCl2, 0.5 mM GTP, 0.2 mM S-adenosyl-methionine, and 1 mM dithiothreitol. In some aspects, the final 1X buffer does not comprise dithiothreitol.

[0154] In some aspects, the capping reaction occurs in the vessel in which the IVT reaction is performed. In such aspects, the IVT reaction is diluted between 3-fold and 10-fold, such as at least, at most, or about 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold, prior to the capping reaction. In some aspects, the IVT reaction is diluted with Tris buffer at pH 7.0.

[0155] To degrade residual DNA template from the IVT reaction, DNase I can be added. In some aspects, the concentration of DNase I added is at least, at most, or about 1 U / μg DNA to 10 U / μg DNA, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 U / μg DNA, or any range or value derivable therein. In addition to DNase I, CaCl2 can be added as a cofactor for DNase I at a concentration of at least, at most, or about 0.1 mM to 4 mM, for example, 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 derivative therein.

[0156] In some aspects, a pyrophosphatase is added to the capping reaction. The pyrophosphatase helps to degrade pyrophosphate, which is an inhibitory byproduct produced by the IVT reaction or the capping reaction.

[0157] In some aspects, the capping reaction is carried out at 37° C. for 30 minutes to 2 hours, e.g., at least, up to, or about 30, 31, 32, 33, 34, 35, 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, 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, 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, In some aspects, the capping reaction is performed 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.

[0158] In some aspects, the step of capping the uncapped RNA molecules results in the capped RNA molecules accounting for at least, up to, 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% of the total RNA molecules (capped and uncapped), or any range or value derived therefrom. Purity can be determined as described herein, for example, via reverse phase HPLC or fragment analyzer or Bioanalyzer chip-based electrophoresis, and measured by, for example, the peak area of ​​the full-length RNA molecule relative to the total peak.

[0159] purification

[0160] In some aspects, RNA molecules produced by the methods described herein can be contacted with DNase I and CaCl2 after the in vitro transcription reaction to enzymatically digest the DNA template. In some aspects, such as during large-scale (reaction volume exceeding 10 mL) operations, the IVT reaction can include the addition of DNase I and CaCl2 and additional treatment with EDTA and proteinase K. EDTA can quench any cationic metal species, including magnesium, and proteinase K can digest proteins present in the IVT reaction to reduce their size.

[0161] In some aspects, the methods described herein do not include contacting the RNA molecules produced by the methods described herein with DNase I to enzymatically digest the DNA template following the in vitro transcription reaction.

[0162] In some respects, linear DNA template is removed from in vitro transcription reaction system, for example, DNA template is separated from RNA molecule via chromatography. In some respects, RNA molecule is combined with affinity matrix, and DNA template flows through and is removed. In some respects, affinity purification based on poly (A) capture is oligo (dT) purification. For example, poly-thymidine ligand can be fixed on the chromatographic resin of derivatization. Purification mechanism can relate to the hybridization of poly (A) tail and oligonucleotide ligand of RNA molecule, wherein DNA template can not combine. In preferred aspects, RNA molecule (failed transcript and other truncated copies formed during in vitro transcription) that does not include poly (A) extension will not be bound to resin, and will not form double-strand with affinity ligand. Can utilize low ionic strength buffer or competitive binding oligonucleotide solution to elute polyadenylated RNA from resin subsequently.

[0163] Preferably, the purified material is substantially free of one or more impurities or contaminants, including the linear DNA template and / or the reverse complementary transcription product described herein, and is, for example, at least 90%, 91%, 92%, 93%, 94%, 95%, 96% or 97% pure; more preferably, at least 98% pure, and more preferably, at least 99% pure.

[0164] The production method of RNA molecules may include additional purification steps after in vitro transcription, such as ion exchange chromatography steps, hydrophobic interaction chromatography (HIC) steps, ceramic hydroxyapatite (CHA) chromatography steps and / or ultrafiltration / diafiltration steps. In a preferred aspect, the synthesis of RNA molecules of a given sequence is carried out in a large-scale synthetic manner.

[0165] As used herein, the term "large scale" refers to the reaction yield of the RNA molecule in the milligram level, preferably at least one gram. In some aspects, the in vitro transcription reaction is carried out in a bioreactor for large-scale synthesis of RNA. Therefore, the bioreactor may be suitable for performing the method described herein. In some aspects, the bioreactor is suitable for performing a batch method (wherein all components for in vitro transcription are supplied at the beginning of the reaction), a batch feeding method (wherein the in vitro transcription components are initially provided, and additional in vitro transcription components are supplied in a continuous or incremental manner in the method, including or not including regular harvest products) or a continuous (also referred to as a perfusion) method (wherein components (such as templates and / or one or more enzymes) are fixed, and other components and / or products are added and removed continuously, stepwise or intermittently throughout the entire method). In some aspects, the bioreactor for synthesizing RNA molecules of a given sequence (preferably on a large scale) is a modularly designed in vitro transcription reactor system, which is included in a reaction module for performing in vitro RNA transcription reactions in a sequence-optimized reaction mixture, a capture module for temporarily capturing transcribed RNA molecules, and a control module for controlling the reaction mixture component feed into the reaction module. The reaction module preferably comprises a filtration membrane for separating nucleotides from the reaction mixture, and the control of the feeding of the reaction mixture components by the control module is based on the measured nucleotide concentration.

[0166] As used herein, the term "bioreactor" refers to a chamber or test tube or column in which an in vitro transcription reaction is performed under specific conditions. The bioreactor can be thermally regulated to accurately maintain a specific temperature, typically between 25°C and 45°C. In some aspects, the bioreactor maintains a temperature of about 25°C. In some aspects, the bioreactor maintains a temperature of about 30°C. In some aspects, the bioreactor maintains a temperature of about 37°C. The volume range of the bioreactor can be from at least 1, 10, 100 or 200 liters or more, or a volume between any two. 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 100L. In some aspects, the DNA template is obtained by fermentation of cells grown during the initial growth phase (or growth phase), and the length or shortness of the growth time depends on the needs of the practitioner and the requirements of the cells themselves. In some aspects, the period of cell growth is sufficient to reach a predetermined cell density. In some aspects, the period of cell growth is sufficient to reach the following predetermined cell density: about 1x 10 6 cells / ml, about 5x 10 6 cells / ml, about 1x 10 7 cells / ml, about 5x 10 7 cells / ml, about 1x 10 8 cells / mL or approximately 5 x 10 8 In some aspects, the cells are grown for a period of time sufficient to achieve a predetermined optical density (OD 600): about 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. In some aspects, the time period of cell growth is enough to reach the following cell density, i.e., the given percentage of the maximum cell density that the cell will eventually reach when it can be undisturbed. For example, the time period of cell growth can be enough to reach the required viable cell density, i.e., 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 99% of the maximum cell density. In some aspects, the cell growth is made until the increase of the cell density per day of cultivation is no more than 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or 1%. In some aspects, the cell growth is made until the increase of the cell density per day of cultivation is no more than 5%. In some aspects, a determined time period of cell growth is made. For example, depending on the starting concentration of the cell culture and the intrinsic growth rate of the cells, the cells can be grown for 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 days or more, preferably from 4 to 10 days. The cell culture can be stirred or shaken during the method. In some aspects, the bioreactor is configured to include stirring. The bioreactor can be a stirring unit with a variable stirring rate. In some aspects, the bioreactor comprises a stirring speed (or corresponding power / volume, tip speed, or mixing time) of at least 0, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, or 1,000 RPM. In some aspects, the bioreactor comprises a stirring speed of 150 RPM (equivalent to 1.3 W / m 3 In some aspects, the bioreactor comprises an agitation speed of 250 RPM (equivalent to 5.9 W / m 3 In some aspects, the bioreactor comprises an agitation speed of 600 RPM (equivalent to 71.7 W / m 3In some aspects, the bioreactor comprises a mixing time of at least 1.0, 1.2, 1.3, 1.5, 2.0, 5.0, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, or 60 seconds. In some aspects, the bioreactor comprises an impeller outer edge speed of at least 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, or 0.9 m / s. In some aspects, the bioreactor can be configured with an inflow or feed line and an outlet. In some preferred aspects, the bioreactor comprises a filter membrane for separating small molecules and macromolecules from the reaction mixture, particularly for separating nucleotides, pyrophosphates, and other low molecular weight components from large (e.g., greater than at least 7,000 nt) mRNA products in the reaction mixture. Introducing a filter membrane (e.g., an ultrafiltration membrane) in such a flow system can be used to separate high molecular weight components (e.g., polypeptides and / or polynucleotides) from low molecular weight components (e.g., nucleotides and pyrophosphates). In some aspects, the filter membrane selectively retains fixed DNA template, RNA polymerase and synthetic RNA molecules in the reactor core of the reaction module, and smaller molecules (such as nucleoside triphosphates (NTPs)) can enter the independent smaller compartment of the reaction module through the filter membrane, i.e., the filter compartment. The nucleotide concentration in the separation fluid including low molecular weight components can be measured then, such as by spectral analysis. Alternatively, nucleotide concentration can be measured by an online HPLC system. In this reactor system, NTP mixture is applied so that nucleotide concentration can be measured in real time during the in vitro transcription reaction to monitor the progress of the in vitro transcription reaction. In some aspects, the bioreactor includes static or stirring tube reactions. In some aspects, the bioreactor includes Sartorius AMBR system, including but not limited to AMBR15, AMBR250 modularization and AMBR250 HT system. In some aspects, the bioreactor includes EppendorfBioBlu system. In some aspects, the bioreactor includes the orifice plate reaction utilizing Tecan liquid handler. In some aspects, the bioreactor includes a container controlled by Mettler Toledo EasyMax102 or 402 synthesis workstations.

[0167] Characterization and analysis of RNA molecules

[0168] Various methods can be used to analyze and characterize the RNA molecules produced by the methods described herein. The analysis can be performed before or after capping. Alternatively, the analysis can be performed before or after affinity purification based on poly (A) capture. In another aspect, the analysis can be performed before or after additional purification steps (such as anion exchange chromatography, etc.). For example, the integrity of the RNA transcript can be determined using electrophoresis (using a fragment analyzer capillary or Bioanalyzer chip system) or by a reversed-phase HPLC method. The fragment analyzer can automatically perform capillary electrophoresis and HPLC in some cases. In other aspects, analytical reversed-phase HPLC is used to analyze RNA template purity, respectively. For example, total nuclease digestion can be used and then LC-UV or LC-MS quantification can be performed on dinucleotide cap species and uncapped GTP species to analyze capping efficiency. In vitro efficacy can be analyzed by, for example, transfecting RNA molecules into human cell lines. Protein expression of the target polypeptide can be quantified using methods such as ELISA or flow cytometry. Immunogenicity can be analyzed by, for example, transfecting RNA molecules into cell lines (such as PBMC) indicating innate immune stimulation. Cytokine induction can be analyzed using methods such as ELISA to quantify cytokines, such as interferon-α.

[0169] The methods of producing RNA molecules described herein can produce RNA molecules of at least 30% full-length transcripts, or at least, up to 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 transcripts, or any range or value derived therefrom. Purity can be determined as described herein, for example, via reverse phase HPLC or fragment analyzer or Bioanalyzer chip-based electrophoresis, and measured by, for example, the peak area of ​​the full-length RNA molecule relative to the total peak.

[0170] Target gene

[0171] The DNA templates and resulting RNA molecules of the present invention comprise a target gene. The target gene encodes a target polypeptide selected from, for example, biological products, antibodies, vaccines, therapeutic polypeptides or peptides, cell penetrating peptides, secretory polypeptides, plasma membrane polypeptides, cytoplasmic or cytoskeletal polypeptides, intracellular membrane-bound polypeptides, nuclear polypeptides, polypeptides associated with human diseases, targeting moieties, or those encoded by the human genome, which have not yet been identified for therapeutic indications, but are still useful in the field of research and discovery. The sequence of a specific target gene can be easily identified by those skilled in the art using public and private databases (e.g., GenBank).

[0172] In some aspects, the RNA comprises a coding region comprising an antigen, the antigen preferably being derived from a pathogen associated with an infectious disease, preferably selected from antigens derived from the following pathogens: Acinetobacterbaumannii, Anaplasma, Anaplasma phagocytophilum, Ancylostoma braziliense, Ancylostoma duodenale, Arcanobacterium haemolyticum, Ascaris lumbricoides, Aspergillus, Astroviridae, Babesia, Bacillus anthracis, Bacillus cereus, Bartonella henselae, BK virus, Blastocystis hominis, Blastomyces dermatitis dermatitidis), Bordetella pertussis, Borrelia burgdorferi, Borrelia, Borrelia species, Brucella, Brugia malayi, Bunyaviridae, Burkholderia cepacia and other Burkholderia species, Burkholderia mallei, Burkholderia pseudomallei, Caliciviridae, Campylobacter, Candida albicans, Candida species, Chlamydia trachomatis trachomatis), Chlamydophilapneumoniae, Chlamydophilapsittaci, CJD prions, Clonorchissinensis, Clostridium botulinum, Clostridium difficile, Clostridium perfringens, Clostridium perfringens, Clostridium spp., Clostridium tetanitetani), Coccidioides species, coronaviruses, Corynebacterium diphtheriae, Coxiella burnetii, Crimean-Congo hemorrhagic fever virus, Cryptococcus neoformans, Cryptosporidium, cytomegalovirus (CMV), dengue viruses (DENV-1, DENV-2, DENV-3, and DENV-4), Dientamoeba fragilis, Ebola virus (EBOV), Echinococcus, Ehrlichia chaffeensis, Ehrlichia ewingii, Ehrlichia, Entamoeba histolytica histolytica), Enterococcus, Enterovirus, Enterovirus (primarily coxsackie A and enterovirus 71 (EV71)), Epidermophyton species, Epstein-Barr virus (EBV), Escherichia coli O157:H7, 0111, and 0104:H4, Fasciola hepatica and Fasciolagigantica, FFI prions, Filarial superfamily, Flavivirus, Francisella tularensis, Fusobacterium, Geotrichum candidum, Giardia intestinalis, Gnathostoma species, GSS prions, Guanarito virus, Haemophilus ducreyi ducreyi), Haemophilus influenzae, Helicobacter pylori, Henipaviruses (Hendra virus, Nipah virus), Hepatitis A virus, Hepatitis B virus (HBV), Hepatitis C virus (HCV), Hepatitis D virus, Hepatitis E virus, Herpes simplex virus type 1 and type 2 (HSV-1 and HSV-2), Histoplasma capsulatum, HIV (human immunodeficiency virus), Hortaea vinnickewerneckii), human bocavirus (HBoV), human herpesvirus 6 (HHV-6) and human herpesvirus 7 (HHV-7), human metapneumovirus (hMPV), human papillomavirus (HPV), human parainfluenza virus (HPIV), Japanese encephalitis virus, JC virus, Junin virus, Kingella kingae, Klebsiella granulomatis, kuru prion, Lassa virus, Legionella pneumophila pneumophila, Leishmania, Leptospira, Listeria monocytogenes, lymphocytic choriomeningitis virus (LCMV), Machupo virus, Malassezia species, Marburg virus, measles virus, Metagonimus yokagawa, Microsporidia, Molluscum contagiosum virus (MCV), mumps virus, Mycobacterium leprae and Mycobacterium lepromatosis, Mycobacterium tuberculosis, Mycobacterium ulcerans ulcerans), Mycoplasma pneumoniae, Naegleria fowleri, Necatora americanus, Neisseria gonorrhoeae, Neisseria meningitidis, Nocardia asteroides, Nocardia species, Onchocerca volvulus, Orientiatsutsugamushi, Orthomyxoviridae (including influenza viruses such as avian and human influenza), Paracoccidioides brasiliensis, Paragonimus species, Paragonimus westermaniwestermani), parvovirus B19, Pasteurella, Plasmodium, Pneumocystis jirovecii, poliovirus, rabies virus, respiratory syncytial virus (RSV), Rhinovirus, Rhinovirus, Rickettsia akari, Rickettsia, Rickettsia prowazekii, Rickettsia rickettsii, Rickettsia typhi, Rift Valley fever virus, Rotavirus, Rubella virus, Sabia virus, Salmonella, Sarcoptes scabiei), coronaviruses (e.g., SARS-CoV-2), Schistosoma, Shigella, Sin Nombre virus, Hantavirus, Sporothrix schenckii, Staphylococcus, Staphylococcus, Streptococcus agalactiae, Streptococcus pneumoniae, Streptococcus pyogenes, Strongyloides stercoralis, Taenia, Taenia solium, Tick-borne encephalitis virus (TBEV), Toxocara canis or Toxocara cati, Toxoplasma gondii, Treponemapallidum, Trichinella spiralis, Trichomonas vaginalis, Trichophyton species, Trichuris trichiura, Trypanosoma brucei, Trypanosoma cruzi, Ureaplasma urealyticum, varicella zoster virus (VZV), varicella zoster virus (VZV), smallpox major or smallpox minor (Variola major),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.

[0173] In some aspects, the RNA molecules of the present disclosure encode viral polypeptides or fragments thereof, including naturally occurring or engineered variants thereof, for preventing viruses in humans. In some aspects, the viral polypeptides do not include coronavirus polypeptides. In some aspects, the viral polypeptides do not include severe acute respiratory syndrome (SARS) viral polypeptides. In some aspects, the viral polypeptides do not include SARS-CoV-2 polypeptides.

[0174] Thus, in some aspects, the RNA molecules of the present disclosure do not encode coronavirus polypeptides or fragments thereof, including naturally occurring or engineered variants thereof. In some aspects, the RNA molecules of the present disclosure do not encode SARS virus polypeptides or fragments thereof, including naturally occurring or engineered variants thereof. In some aspects, the RNA molecules of the present disclosure do not encode SARS-CoV-2 virus polypeptides or fragments thereof, including naturally occurring or engineered variants thereof.

[0175] In further aspects, the RNA molecules of the present disclosure are not used to prevent human coronaviruses. In some aspects, the RNA molecules of the present disclosure are not used to prevent human SARS viruses. In some aspects, the RNA molecules of the present disclosure are not used to prevent human SARS-CoV-2. Example

[0176] The following are examples of specific aspects of the present invention. These examples are provided for illustrative purposes only and are not intended to limit the scope of the present invention in any way. Every effort has been made to ensure the accuracy of the numbers used (e.g., amounts, temperatures, etc.), but of course some experimental errors and deviations should be allowed.

[0177] The practice of the present invention will employ, unless otherwise indicated, conventional methods of protein chemistry, biochemistry, recombinant DNA techniques and pharmacology, which are within the skill of the art.

[0178] Example 1: Creation of dsDNA minicircles by assembling synthetic fragments

[0179] The 3'UTR and poly(A) sequence were amplified by PCR from plasmid pVV-0513 containing the mod flu-HA transcription cassette. The kanamycin resistance vector plasmid PFE-pUC-Kan3 was linearized by digestion. HiFiDNA Assembly Cloning Kit (NEW ENGLAND ) The two fragments (3'UTR-pA and T7p-5'UTR) were assembled into a linearized pUC-Kan backbone to generate plasmid pREG5. A fragment containing regulatory sequences (3'UTR, poly(A), T7 promoter, 5'UTR) was excised from pREG5.

[0180] The adjustment sequence is The obtained synthetic antigen sequences were assembled to produce a dsDNA minimal circular template.

[0181] Following assembly, the reaction was treated with T5 exonuclease to remove any remaining linear fragments, leaving only the circular dsDNA to serve as template for RCA.

[0182] T5 exonuclease was removed by digestion with heat-labile proteinase K and subsequent heat inactivation of proteinase K.

[0183] Example 2: RCA reaction using circular double-stranded DNA as the starting template

[0184] In some aspects, further simplified methods for producing mRNA utilize synthetically constructed circular double-stranded DNA that is isothermally amplified by multistrand replacement rolling circle amplification (RCA) via Phi29 DNA polymerase or a mutant thereof.

[0185] Figure 1 1 to 5 ng of circular supercoiled dsDNA template (containing a transcribable cassette) as shown in the figure can be used in a rolling circle amplification (RCA) reaction (Table 1) to generate 10 5 The template is amplified by multiple times to produce dsDNA concatemers.

[0186] Table 1: Use of EQUIPHI29 TM RCA reaction of DNA polymerase

[0187]

[0188] The mixture is gently mixed with or without heat denaturation and then incubated at 30-45°C for 3-6 hours to overnight. TM The polymerase can be heat inactivated at 65°C for 10 minutes. Preferably, EQUIPHI29 TMThe polymerase was not heat inactivated. The reaction was able to achieve 1.5-2 mg / mL DNA.

[0189] The RCA DNA concatemers generated by the above reaction are shown in Figure 2 agarose gel images in (without and with heat denaturation), and Figure 3 Lanes 1-2 in (without and with purification after RCA).

[0190] These experiments showed that the RCA reaction can be performed at temperatures of 30-45°C for 3-6 hours or overnight. Additionally, BSA can be removed and RCA templates for IVT reactions can be successfully generated using different dNTP concentrations and Phi29 polymerase. Finally, the heat denaturation step before RCA incubation and the heat inactivation, pre-linearization after RCA can be omitted from the RCA reaction method without affecting the integrity of the RNA produced from the RCA generated template. Removal of these two steps can make commercial manufacturing easier and allow isothermal methods to be performed while still producing surprisingly high quality mRNA compared to plasmid DNA, as shown in the following examples.

[0191] Example 3: RCA DNA digestion without purification

[0192] Additionally, in some aspects, the RCA DNA generated from the circular dsDNA template in Example 2 can be enzymatically digested by one or more restriction endonucleases to generate linearized DNA fragments for use as in vitro transcription reaction templates in a "one-pot" reaction without the need to purify the RCA DNA prior to enzymatic digestion. Conventional reactions require purification of the DNA template, reconstitution of the DNA template in a buffer, and then enzymatic digestion of the DNA template. As described herein, the entire reaction (i.e., amplification and linearization) can be performed in the same reaction vessel without loss of material because there is no need to transfer to different containers for different reaction steps.

[0193] RCA DNA can be digested with restriction endonucleases to produce linear dsDNA without the need for DNA purification or additional buffering agents. 2-3 U of SapI (10 U / μL, NEB TM , #R0659) into an RCA reaction vessel and incubate at 37°C in a heating block for 8 hours to overnight. Figure 3 As shown, lanes 3 and 4 (unpurified before linearization) are compared to lanes 5 and 6 (purified), and the cleavage products can be visualized using agarose gel electrophoresis.

[0194] Example 4: RCA reaction using RCA DNA as starting template

[0195] Optionally, in some aspects, sequential multi-step RCA reactions are used to achieve increases of up to 10 from as little as 1-5 ng of template DNA starting material. 10 The continuous multi-step RCA reaction uses the RCADNA from the previous RCA reaction as the starting template for further rolling circle amplification rounds.

[0196] The RCA DNA concatemers generated from Example 3 were used as templates for a second RCA reaction using the RCA DNA as a starting template. The second RCA reaction was identical to the reaction described in Table 1, except that 5 ng of RCA DNA (RCA DNA template 1) obtained from the reaction in Example 3 was used as a template for RCA instead of a circular dsDNA plasmid (plasmid template) to produce RCA DNA reaction template 2. The RCA DNA concatemers generated from the second RCA reaction were as follows: Figure 4 Agarose gel images of lane 4 (uncleaved template) and lane 5 (restriction digested linearized template) are shown.

[0197] The RCA DNA reaction template 2 generated by the second RCA reaction was then used as a template for the third RCA reaction. The third RCA reaction was identical to the second RCA reaction, except that 5 ng of RCA DNA reaction template 2 was used instead of RCA DNA reaction template 1 as a template for RCA to produce RCA reaction template 3. The RCA DNA concatemers generated from the second RCA reaction were Figure 4 Agarose gel images of lane 6 (uncleaved template) and lane 7 (restriction digested linearized template) are shown.

[0198] These results indicate that RCA reactions using RCA DNA as a starting template can be repeated up to three times without loss of yield or quality.

[0199] Example 5: IVT using linearized DNA as template

[0200] Linearized DNA generated from circular dsDNA (plasmid DNA or minicircles) or RCADNA templates can be used as a template for in vitro transcription.

[0201] The linearized RCA DNA from Examples 2-4 was used as an IVT template to generate mRNA. The linearized RCA DNA was purified using ethanol / 2.5M NH4CH3CO2 precipitation and resuspended in nuclease-free water. The RCA DNA was then used as a template for the IVT reaction as shown in Table 2 below (Trilink protocol). RCA DNA was supplemented at a final concentration of 0.05 mg / mL.

[0202] Table 2: IVT reaction using linear RCA DNA as template

[0203]

[0204] The reaction was vortexed gently and incubated at 37°C for 120 minutes. DNaseI and CaCl2 were added and the reaction mixture was incubated at 37°C. The reaction was purified by LiCl precipitation. The reaction mixture was centrifuged, the resulting pellet was washed with ethanol, and the reaction was further centrifuged. The final pellet was resuspended in water and the RNA concentration was measured on a nanodrop. Figure 5 As shown, the product can be visualized using agarose gel electrophoresis without further purification.

[0205] Example 6: Purification of mRNA produced from linearized RCA DNA template to increase RNA integrity

[0206] In some aspects, the in vitro transcription reaction mixture produced according to Example 5 can be purified by oligo(dT) affinity capture to further enrich for full-length mRNA products. Using oligo(dT) capture to purify mRNA generated from linearized RCA DNA templates can improve RNA integrity to equal or exceed the integrity of RNA generated from linearized plasmid DNA.

[0207] By using oligo(dT) magnetic beads (DYNABEADS TM Oligo(dT)25, INVITROGEN TM ) capture, full-length transcripts can be enriched from polyadenylated mRNA generated from linearized RCA DNA. The RNA is resuspended in an equal volume of binding buffer (Tris-HCl, pH 7.5; LiCl; EDTA) and mixed with magnetic beads in an equal volume of binding buffer. The mixture is incubated with rotation at room temperature, and the unbound portion is removed. The beads are washed twice with wash buffer B (Tris-HCl, pH 7.5; LiCl; EDTA), and the RNA is then eluted with Tris-HCl, pH 7.5. In some aspects, the RNA is then precipitated in LiCl, followed by centrifugation at 4°C. The resulting precipitate is then washed with ethanol and further centrifuged at 4°C, and the final precipitate is resuspended in water.

[0208] The integrity of the mRNA was assessed by fragment analysis. Oligo(dT)-selected mRNA transcribed from plasmid DNA was used as a control. Figure 6As shown, after oligo(dT) selection, the RNA integrity of mRNA from RCA DNA template was improved and equal to that obtained from RNA templated from plasmid DNA. Therefore, the oligo(dT) purification step after the IVT reaction is a creative solution to enrich the RNA yield with high integrity.

[0209] Example 7: Sequential, single-vessel reaction to generate uncapped mRNA

[0210] RCA reaction of circular dsDNA (plasmid or minicircle), followed by restriction enzyme linearization and IVT can be performed sequentially in a single reaction vessel to generate mRNA without any prior purification steps. The method allows DNA-directed synthesis of RNA molecules of any sequence, ranging in size from short oligonucleotides to several kilobases.

[0211] In some aspects, DNA (plasmid or DNA generated by RCA reaction) can be linearized and used as template for IVT reaction, which is a step-wise reaction in a single vessel without purification or volume transfer. For RCA single vessel reaction, the template can be as little as 1 ng of circular supercoiled dsDNA. No heat denaturation of template DNA is required. RCA reaction components for such step-wise single vessel "one pot" RCA reaction are shown in Table 1 above.

[0212] The reaction mixture was vortexed gently and incubated at 30°C for 25 hours. The RCA reaction was then digested with restriction endonucleases in the same vessel to produce linear dsDNA without DNA purification or additional buffering reagents. 2-3U of SapI (10U / μL, NEB, #R0659) per microgram of RCA DNA was pipetted into the RCA reaction vessel and incubated at 37°C for 8 hours to overnight. Figure 3 As shown in lane 3 in , the cleavage products of the repeated reactions can be visualized by agarose gel electrophoresis without any purification.

[0213] The entire digestion reaction (linearized plasmid DNA or linearized RCA DNA) can be used in the same container for the subsequent IVT step without purification. Add the IVT reagents directly to the enzymatic digestion reaction according to Table 2 above. Adjust the DNA concentration to 50 ng / μL for IVT using nuclease-free water. Mix the reagents gently, and incubate the reaction at 37°C for 120 minutes. Add DNaseI and CaCl2 to a final concentration of 5U DNaseI / μg RCA DNA and 10mM CaCl2, and incubate the reaction at 37°C for an additional 40 minutes. Add 500mM EDTA and 0.6U / μL Proteinase K to a concentration of 50mM EDTA and 0.001U / μL Proteinase K, and incubate the reaction at 37°C for an additional 10 minutes.

[0214] The reaction can be purified by LiCl precipitation. The mixture is centrifuged at 4°C, the resulting pellet is washed with ethanol and further centrifuged at 4°C, and the final pellet is resuspended in water and heated at 50°C for 10 minutes to dissolve the RNA into solution. The concentration is measured on a nanodrop, and typically 2.0 to 5.0 mg / mL of uncapped RNA is produced.

[0215] The integrity of the resulting RNA was assessed using fragment analysis. RNA generated using linearized plasmid DNA was used as a control. After capture and purification with oligo(dT), RNA integrity was further improved compared to precipitation alone.

[0216] Example 8: In vitro transcription reaction

[0217] The transcription buffer components were varied to identify the levers that had the greatest impact on in vitro transcription (IVT) performance and product quality attributes. The reaction components and concentration ranges studied are shown in Table 3 below.

[0218] The reaction was carried out in a high-throughput small-scale model. The list of constant parameters throughout the experiment can be found in Table 37. RNA samples were purified by precipitation and concentration was measured by UV. RNA integrity was determined by a fragment analyzer (FA). Parameter ranges were confirmed based on RNA yield ≥ 1.85 mg / mL, model adaptability and economic considerations. Table 3 below shows reagents and their research ranges. A two-level factorial design experiment (DoE) was generated by 16 of the 64 possible high and low combinations of each factor. Two repeated center point experiments were also run.

[0219] Table 3: DOE1 factors and ranges

[0220]

[0221] *Note: One unit represents the actual number of units in a coded unit.

[0222] A model capable of detecting all main effects and two-factor interactions was generated for UV concentration and integrity. A p-value < 0.05 was used to determine whether a parameter was significant.

[0223] The ranges of RNA concentration and integrity observed throughout the experiments are listed in Table 4. The values ​​of integrity and concentration were acceptable across the range tested, indicating that the method can be safely operated within this range.

[0224] Table 4: DOE1 Response

[0225] response unit Minimum Maximum average value Standard Deviation UV Concentration mg / mL 3.96 4.41 4.14 1.11 Completeness ％ 80.97 92.33 87.58 3.75

[0226] The fit statistics for the concentration and integrity models are shown in Table 5. 2 A relatively strong integrity model was developed based on the analysis of the values.

[0227] Table 5: DOE1 model overview

[0228] Model <![CDATA[R 2 ]]> <![CDATA[Adjusted R 2 > <![CDATA[Predicted R 2 > UV Concentration 0.6666 0.5777 0.3396 Completeness 0.9136 0.9034 0.8838

[0229] In the UV concentration model shown in Table 6, only the PEG-8000 concentration and the interaction term between Tris and spermidine were statistically significant. The coefficients for each of these parameters were very small, so changing each parameter over the tested range resulted in negligible changes in concentration. Therefore, these terms were determined to be statistically significant but not actually significant over their tested range.

[0230] Table 6: DOE1 UV concentration model

[0231]

[0232] In the integrity model, Tris and spermidine are statistically significant. However, Tris concentration (20-60mM) is actually not significant because the coefficient estimates of the data are within the FA determination variability. Surprisingly, the addition of spermidine is associated with a significant decrease in integrity. Spermidine is generally considered to be a key component of in vitro transcription reactions [Kartje et al., J Biol Chem 296 (2021)]. Spermidine is a polyamine that is thought to help polymerase separate from DNA molecules and initiate RNA synthesis on new DNA molecules. As shown in Tables 6 and 7, spermidine can be removed from the reaction for the purpose of enhancing integrity without significantly affecting concentration.

[0233] Table 7: DOE1 integrity model

[0234] parameter p-value Statistically significant? Coefficient estimates Actually significant? A–Tris 0.0053 Y -0.9300 N B – Spermidine <0.0001 Y -3.78 Y intercept N / A N / A 4.14 N / A

[0235] Interestingly, the DTT concentration was not statistically significant over the range tested. DTT is a reducing agent that is often included in IVT reactions to protect enzymes (e.g., T7 polymerase) from protein oxidation [Kartje et al., J Biol Chem 296 (2021)]. The T7 polymerase used in these reactions contained a small amount of DTT to protect the enzyme for long-term storage, but additional DTT did not appear to be required to protect T7 during the reaction progress.

[0236] As shown herein, the method can be run without spermidine, which is intended to improve integrity. Further, a streamlined IVT matrix free of spermidine, DTT, Triton, DMSO, and PEG-8000 can be used in the method, resulting in simplified manufacturing operations, improved intermediate solution stability, and reduced costs.

[0237] Example 9: Enhanced in vitro transcription reaction

[0238] The following factors were evaluated to determine the most important factors in IVT and to determine which factors deserve further optimization studies: concentration of each NTP, T7 concentration, pyrophosphatase concentration, initial pH, Mg / NTP ratio, time, and temperature. The Mg / NTP ratio was chosen as a factor rather than Mg concentration because of the interdependence between Mg and NTP levels. NTPs are known to chelate Mg ions, and if a certain Mg / NTP ratio threshold is exceeded, mRNA production will be significantly reduced [Kartje et al., J Biol Chem 296:100175 (2021); Young et al., Biotechnol Bioeng 56(2):210-220 (1997); Thomen et al., Biophysical Journal 95(5)2423-2433 (2008)].

[0239] As in Example 8, ranges were determined based on the results of the IVT reactions and then further adjusted to ensure response in the model and minimize cost.

[0240] Reactions were performed in a small-scale high-throughput model with varying levels of ATP, CTP, GTP, and pUTP; T7 concentration; pyrophosphatase concentration; initial reaction pH and Mg acetate. Mg acetate was varied with NTP concentration, with ratios ranging from 0.8-2.2. This equates to an operating range of 12.8-110 mM Mg. For simplicity, the ratio of ATP:CTP:GTP:pUTP was kept at 1:1:1:1; for example, 16 mM total NTPs is equivalent to 4 mM ATP, 4 mM CTP, 4 mM GTP, and 4 mM pUTP. Reactions were incubated at different temperatures for different times. All other consistent parameters can be found in Table 41. RNA samples were purified by precipitation and concentrations were measured by UV.

[0241] The levels of each parameter tested are described in Table 8. The design consisted of 16 of the 128 possible high / low combinations for each parameter and contained 2 replicate center points.

[0242] Table 8: DOE2 factors and ranges

[0243]

[0244] *Note: One unit represents the actual number of units in a coded unit.

[0245] A model for UV concentration was generated in Design Expert, which described all main effects and two-factor interactions. A p-value < 0.05 was used to determine whether each parameter was significant.

[0246] The performance of the UV concentration model generated in Design Expert is shown in Table 9. Predicted R 2 Very low and with correction R 2 This indicates that the model has limited ability to predict concentrations.

[0247] Table 9: DOE2 model overview

[0248] Model <![CDATA[R 2 ]]> <![CDATA[Adjusted R 2 > <![CDATA[Predicted R 2 <!-- 35 -->]]> UV Concentration 0.7204 0.6330 0.4300

[0249] Table 10 shows one possible model for the data. The midpoint control (consisting of the "target" range recorded in Table 8) has a higher concentration than the rest of the conditions. This results in a significant curvature in the data and A 2 This is indicated by the presence of high coefficients in the term. Many different models are possible, including quadratic models for any of the parameters tested (possibly due to the fact that all factors were concentrated in the midpoint control). While most parameters could be incorporated into a quadratic model, plans were made to test pyrophosphatase, initial pH, and time in additional experiments. The remaining parameters were continued for further optimization.

[0250] Table 10: DOE2 UV concentration model

[0251] parameter p-value Statistically significant? Coefficient estimates Actually significant? A – Total NTP 0.0627 N -0.6879 Y B–T7 0.0282 Y 0.8701 Y F – Time 0.0255 Y 0.8806 Y G-Temperature 0.0330 Y 0.8418 Y <![CDATA[A 2 ]]> 0.0005 Y -4.39 Y intercept N / A N / A 6.67 N / A

[0252] RNA was successfully produced in all tested ranges of total NTPs, T7, pyrophosphatase, initial pH, Mg ratio, time, and temperature.

[0253] Example 10: Improvements to enhance in vitro transcription reactions

[0254] Based on the experimental results described in the aforementioned embodiment, it was determined that NTP levels, T7 RNA polymerase concentration, Mg concentration and temperature will be further optimized. The purpose of this experiment is to optimize the set value of each parameter in these parameters for RNA concentration. Mg is tested as a separate parameter, rather than the combined Mg:NTP ratio, to read the separate impact of Mg and NTP concentrations.

[0255] Reactions were performed in a high-throughput small-scale model with different levels of ATP, CTP, GTP and pUTP, T7 concentrations and magnesium acetate concentrations. For simplicity, the ratio of ATP:CTP:GTP:pUTP was fixed at 1:1:1:1. For example, a total NTP concentration of 20 mM means 5 mM each of ATP, CTP, GTP and pUTP. A further description of fixed parameters can be found in Table 41. All parameters of variation are shown in Table 11. A modified full factorial DOE design was used to test parameters within a specified range. The design consists of 6 centers, 8 axes and 16 factor points. In order to simplify the experiment, the 4th and 15th runs were run at 37°C and 30°C, respectively, rather than at set points outside the design space. RNA samples were purified by precipitation and concentrations were estimated by UV. Percent integrity was determined by a fragment analyzer (FA).

[0256] Table 11: DOE3 design

[0257]

[0258] *Note: One unit represents the actual number of units in a coded unit.

[0259] Based on the data collected in this experiment, a model for optimizing RNA concentration was generated in Jmp and Design Expert. As shown in Table 12, R 2 , adjusted R 2 and the predicted R 2 is very high, which indicates that the model fits well.

[0260] Table 12: DOE3 model performance

[0261] Model <![CDATA[R 2 ]]> <![CDATA[Adjusted R 2 > <![CDATA[Predicted R 2 > UV Concentration 0.9090 0.8680 0.7506

[0262] The UV concentration model of Design Expert can be seen in Table 13. All four test parameters are statistically significant as p < 0.05 for each parameter. The prediction analyzer was generated in Jmp. The ranges shown in each of the prediction curves are significant and cannot be explained by experimental variability. The interaction terms of total NTP with Mg and Mg with temperature were confirmed.

[0263] Table 13: DOE3 model for UV concentration

[0264] parameter p-value Statistically significant? Coefficient estimates A – Total NTP 0.0002 Y -0.7294 B–T7 0.0047 Y 0.5053 C–Mg <0.0001 Y 0.9381 D – Temperature 0.0002 Y 0.8317 AC 0.0001 Y 0.9286 CD 0.0089 Y 0.5642 <![CDATA[A 2 ]]> <0.0001 Y -0.8826 <![CDATA[C 2 ]]> <0.0001 Y -0.7149 <![CDATA[D 2 ]]> 0.0047 Y -0.9357 intercept N / A N / A 5.33

[0265] A prediction analyzer was generated in Jmp to predict at what level of each parameter the mRNA concentration would be maximized. The consensus analysis indicated that total ATP, CTP, GTP, and pUTP were optimal at approximately 28 mM, T7 was optimal at higher concentrations, Mg was maximized at approximately 33-36 mM, and temperature was optimal at approximately 36 degrees Celsius.

[0266] The best performing condition in this experiment is called "DOE3 F1", which contains 36mM total ATP / CTP / GTP / pUTP; 14,000U / mL T7 and 36mM Mg. The reaction temperature is 37 degrees Celsius. This condition produces an mRNA concentration of 6.9mg / mL and an integrity of 89%. However, multiple successful runs using different parameters show that the method is robust and can be run over a wide range.

[0267] Example 11: T7 and Temperature

[0268] Based on the previous examples, it was confirmed that the reaction yield was optimized at higher levels of T7 RNA polymerase. For this reason, the effect of higher T7 levels was investigated in this experiment. Additionally, it would be interesting to further investigate temperature as a parameter in IVT reactions. Further, it is possible that lowering the temperature may improve integrity.

[0269] The experiments were performed in a high throughput small scale model. The conditions named DOE3Opt described in Table 37 were run as controls in two single factorial (OFAT) experiments and are labeled as Condition A in Tables 14 and 15. In the remaining conditions, T7 or temperature was manipulated.

[0270] RNA samples were purified by precipitation and concentration was measured by UV. Percent integrity was determined by fragment analyzer (FA). As T7 concentration increased, the resulting mRNA concentration remained comparable within the experimental variation range. When T7 concentration increased from 17000U / mL to 40000U / mL, integrity was observed to have decreased by 6%. These results are shown in Table 14 and Table 15.

[0271] Table 14: OFAT results for T7 concentrations

[0272]

[0273] When the temperature was reduced from 36°C to 32°C, the concentration and integrity were not significantly affected; all results were within the expected range of assay variation. These results are shown in Table 15.

[0274] Table 15: OFAT results for temperature

[0275]

[0276] This experiment demonstrates that the method is capable of producing high-quality mRNA over the range of temperatures and T7 polymerases tested.

[0277] Example 12: Linearization of plasmid DNA concentration

[0278] This experiment determined whether the starting DNA concentration affected integrity and yield and evaluated a range of 0.025–0.075 mg / mL DNA.

[0279] This experiment was performed in a high throughput small scale model. The control (represented as condition B in Table 16) was the method represented as DOE3 F1 in Table 37. The remaining conditions had the same parameters, but the level of linearized plasmid DNA was different. RNA samples were purified by precipitation and concentration was measured by UV. Percent integrity was determined by a fragment analyzer (FA).

[0280] Table 16 shows the results of this experiment. When the DNA concentration was varied between 0.025 and 0.075 mg / mL, the mRNA concentration was affected. When the DNA concentration was increased from 0.025 mg / mL to 0.05 mg / mL, the mRNA concentration increased significantly, although increasing the DNA concentration from 0.05 mg / mL to 0.075 mg / mL did not have such a large effect on the resulting mRNA concentration. Within this range, integrity remained within the assay variability, indicating that no impact on integrity was observed.

[0281] Table 16: OFAT results for DNA

[0282]

[0283]

[0284] Example 13: Effect of Mg and Total NTP on IVT Performance

[0285] The ANOVA model shown in Table 17 contains a significant interaction term between total NTP and Mg (labeled as AC). Confirm that the model is meaningful. Additionally, further confirmation studies are needed to supplement data. The purpose of this experiment is to determine the impact of the ATP+CTP+GTP+pUTP summation varying from 28-44mM. At each NTP level, four different Mg / NTP levels were assessed: 1.1, 1.3, 1.4 and 1.6. The experiment was performed in a small-scale high-throughput model with different levels of NTP and different levels of Mg, as shown in Table 17. All other parameters were taken from the condition DOE3 Opt listed in Table 37. RNA samples were purified by precipitation, and concentration was estimated by UV. Percent integrity was determined by a fragment analyzer (FA).

[0286] Table 17: Different parameters in Mg / NTP experiments

[0287] ATP+CTP+GTP+pUTP(mM) Mg / NTP ratio Mg(mM) 28 1.1 32 28 1.3 36 28 1.4 40 28 1.6 44 36 1.1 41 36 1.3 46 36 1.4 51 36 1.6 57 44 1.1 50 44 1.3 57 44 1.4 63 44 1.6 69

[0288] Effects of Mg concentration and total ATP+CTP+GTP+pUTP on IVT reaction performance Fig. 9 AB. Addition of NTPs above 28 mM significantly increased RNA concentration. However, it was observed that increasing Mg levels too high while keeping NTP concentration constant also affected RNA concentration.

[0289] Example 14: Effect of divalent metals on residual DNA

[0290] This DOE experiment was conducted to study the effects of divalent metals: calcium, magnesium, and manganese in the DNA digestion step. Table 18 shows the four-factor DOE design with pivot points. The concentration range of calcium was from the standard protocol (THERMO DNase I) was obtained, using 1x concentration as low concentration and 10x concentration as high concentration. The scope of magnesium uses low concentration and high concentration of 1mM and 10mM respectively, to include the magnesium loss caused by pyrophosphate complexation. The low and high scope of manganese imitates the scope of calcium. During DNase digestion, time course samples were collected at 30 minutes, 45 minutes and 60 minutes.

[0291] An automated microbial reactor system was used and method FB V1 using DNase I digestion was used as a baseline. The reactions completed in this experiment used a modified method FB V1 with a pyrophosphatase concentration of 0.05 U / mL to determine residual DNA trends. Calcium, magnesium, manganese and DNase enzyme concentrations were varied within the DOE range and added to the DNase I enzyme addition. Table 18 below provides the DOE concentrations studied. The experimental outputs were RNA yield, integrity and residual DNA.

[0292] Table 18: Different parameters and DOE designs

[0293]

[0294] The results of this DOE design showed that the DNase I enzyme concentration and calcium concentration range had no effect on the residual DNA trend at the 30- and 45-minute time points, but did affect the integrity at the 60-minute mark ( Fig. 9 ). Manganese and magnesium had no significant effect on the growth of the cells at 30 min (data not shown) and 45 min ( Fig.10 ) has an impact on the residual DNA trends at both time points. Fig.10 The contour plots in show that the low points of manganese and magnesium concentrations result in the lowest residual DNA impurities.

[0295] Example 15: Proteinase K analysis

[0296] After IVT is complete and prior to downstream purification, protein degradation with a protease such as proteinase K (ProK) is an ideal method to further inhibit the IVT reaction and reduce the risk of off-target behavior of proteins such as DNase I. In addition, degradation of larger proteins facilitates robust removal by purification methods such as UF / DF, chromatography, etc. Proteinase K has been described as an enzyme commonly used to purify RNA [Tullis and Rubin, Anal Biochem 107(1):260-264(1980)]. A large number of IVT reactions ("ConfOpt") were assembled and DNase digestions were performed under "ConfOpt" conditions according to the confirmed set points described in Table 37. DNase digestion parameters included 1 mM calcium chloride, 2000 DNase I (U / mg pDNA), DNase digestion time of 45 minutes, and DNase digestion temperature of 36°C. This large mixture was divided into smaller aliquots and further digested with proteinase K using the parameters described in Table 19. Digestion steps (THERMO The recommended range of ProK for proteinase K at 50°C was about 1.5-30 U / mL, which was extended to a lower limit of 0.5 U / mL and modified to run at a reaction temperature of 36°C for 45 minutes to prevent damage to mRNA.

[0297] Prior to ProK digestion, EDTA was used to reduce free CaCl2 levels within the reaction, as CaCl2 can potentially inhibit the ability of ProK to digest DNase [Tullis and Rubin, Anal Biochem 107(1):260-264 (1980)]. 50 mM EDTA was sufficient to quench the IVT reaction described in Example 8, so 50 mM EDTA was used as the starting point for the experiment. After the incubation period, samples were snap frozen and residual protein was determined on a Perkin Elmer LabChip. Integrity was also measured by a fragment analyzer (FA).

[0298] Table 19: ProK digestion conditions

[0299]

[0300] The integrity results are listed in Table 19. Adjusting the level of proteinase K did not appear to affect integrity. Complete protein digestion was confirmed by residual protein assay across all ProK conditions. The conditions listed in rows A, B, and C of Table 19 ("ConfOpt") were run on the residual protein assay and the resulting electropherograms are shown in Table 19. Fig.11As shown. The conditions in rows D, E, and F ("FBV1") were obtained using a fed-batch process labeled FB V1, as described in Table 65. A sample without ProK digestion was run as a control. This control contained peaks at approximately 19 seconds and 23 seconds, which correspond to DNase I and T7, respectively. These peaks were no longer visible for samples dosed with 1.5 U / mL ProK, indicating that the digestion was successful. Further, this indicates that the ProK range of 1.5 U / mL and above is sufficient for digestion, with no impact on integrity within the concentration range tested. Residual protein analysis was also performed on the conditions in rows D, E, and F, and the resulting electropherograms showed similar results (data not shown), with the same DNase I and T7 peaks observed at 19 seconds and 23 seconds, respectively, in the control sample, but no peaks were detected at these residence times for samples dosed with 0.5, 1.0, and 1.5 U / mL ProK.

[0301] Example 16: General applicability of IVT to various RNA constructs

[0302] The purpose of this experiment was to evaluate whether the IVT bulk method developed herein is applicable to other RNA molecules of various sizes and compositions. To this end, RNA#2 was made using Method BV1. The effect of spermidine on the performance of the method was also evaluated. The data shown in Table 20 below demonstrate that Method BV1 produced high quality RNA. In addition, as shown in Table 20, removal of spermidine improved RNA capping efficiency, which further demonstrates that spermidine can be removed from the IVT method.

[0303] Table 20: General applicability of method BV1 to RNA#2

[0304]

[0305] Next, the modified method BV1 (described in Table 37) was evaluated using RNA #3 (about 5.7 times larger compared to RNA #2). The modified method BV1 is modular and can be easily customized to accommodate both enzymatic and co-transcriptional methods. Here, the co-transcriptional method was implemented using a capping molecule, but capping molecule analogs can also be used (Table 21). The RNA samples were purified by precipitation and the IVT yield (measured by UV), integrity (measured by fragment analyzer), 5' cap (measured by LC-UV), and residual DNA (measured by quantitative polymerase chain reaction) were determined (Table 22). This example demonstrates the high capping efficiency and RNA integrity using larger RNA constructs and the flexibility of the modified method BV1 in making a variety of RNAs.

[0306] Table 21: Experimental parameters

[0307]

[0308] RNA yield was positively correlated with IVT temperature, and increasing the temperature to 36°C increased RNA yield by approximately 18-fold. Furthermore, all conditions produced sufficiently high RNA integrity of >70% using large constructs.

[0309] Table 22: Determination of the effect of IVT temperature on IVT performance using modified method BV1

[0310]

[0311] Next, the effects of IVT time (150–210 min) and pyrophosphatase concentration (0.02-0.5 U / mL) were evaluated. As shown in Tables 23–26 below, increasing IVT time resulted in >20% increase in RNA yield, however, a slight decrease in integrity was also observed. In addition, capping efficiency was evaluated for some conditions, and all conditions had capping efficiencies >90%, indicating that the modified method BV1 is robust in producing high-quality RNA.

[0312] Table 23: Effect of IVT time and 0.5 U / mL pyrophosphatase concentration on reaction performance

[0313]

[0314] Table 24: Effect of IVT time and 0.25 U / mL pyrophosphatase concentration on reaction performance

[0315]

[0316] Table 25: Effect of IVT time and 0.1 U / mL pyrophosphatase concentration on reaction performance

[0317]

[0318] Table 26: Effect of IVT time and 0.02 U / mL pyrophosphatase concentration on reaction performance

[0319]

[0320] The concentration of the capping molecule was also evaluated. A concentration range of 2-9 mM was tested, and as shown in Table 27, a dose-dependent response between RNA concentration and the capping molecule was observed.

[0321] Table 27: Effect of capping molecule concentration on reaction performance

[0322]

[0323] In order to prove that the improved method BV1 is scalable, More than 250 parallel bioreactor systems A 100 mL scale IVT run was performed on . As shown in Table 28, the data is comparable to the 1 mL scale and also shows very low residual DNA impurities.

[0324] Table 28: Scale-up IVT of RNA#3 in the AMBr 250 system

[0325]

[0326] To further improve the integrity of mRNA, POROS TM Oligo(dT)25(THERMO ) and oligo(dT)18 monolithic column (SARTORIUSBIA ) affinity chromatography step for purified mRNA. Both steps bound mRNA in 10mM Tris / 500mM KCl / 0.1mM EDTA, pH 7.2 and eluted with 10mM Tris / 0.1mM EDTA, pH 7.2. As shown in Table 29 below, mRNA integrity increased from 61% for non-affinity purified material to 79% when purified by the affinity step.

[0327] Table 29: mRNA affinity chromatography

[0328]

[0329] The data in this example demonstrate the general applicability and scalability of Method BV1 to multiple RNA constructs of different sizes. This method was applied to RNA#3 and demonstrated similar product quality and low impurities.

[0330] Example 17: Development of a Fed-Batch Process

[0331] The goal of this experiment was to develop a batch feeding method starting from the above method BV1 by feeding in a bolus, semi-continuous and / or continuous manner [Kern and Davis, Biotechnol Prog 13(6):747-56(1997)]. Here, an automated microbial reactor system was used to determine the feeding method. The experimental design is shown in Table 30 below, and the initial baseline NTP was adapted from Henderson et al. [Henderson et al., CurrProtoc 1(2):e39(2021)]. In addition, a method using different initial GTP concentrations was also used [mMESSAGE Kit User Guide (Pub. No. 1340MRev.G) THERMO FISHER] was used to evaluate the effect on capping efficiency while maintaining the overall concentration by feeding [Kern and Davis, Biotechnol Prog 13(6):747-56 (1997)].

[0332] Table 30: Overview of fed-batch evaluation experiments

[0333]

[0334]

[0335] An automated microbial reactor system was used and Method BV1 was modified by reducing the pyrophosphatase concentration to 0.25 U / mL and increasing the temperature to 37°C, respectively. This modified Method BV1 was used as a baseline in these experiments. The experimental outputs were RNA yield, integrity, capping efficiency, and residual NTPs.

[0336] like Fig.12 As shown, RNA integrity was similar across all conditions. However, the high-frequency NTP feeding schedule with 5-minute intervals had better yields compared to its 10-minute and 25-minute counterparts. In addition, reducing the initial GTP concentration improved the capping efficiency of all feeding schedules (up to 93%). The batch feeding method with GTP concentrations of 1 and 2.5 mM had better capping efficiency than the batch method. Next, residual NTPs after 150 minutes of IVT were evaluated, and some NTPs were low (data not shown). To this end, subsequent experiments were completed to evaluate increasing NTP concentrations.

[0337] In this second fed-batch development experiment, the BV1 process was used as a baseline control. The best fed-batch process (capping efficiency up to 93%, condition H in Table 30 above) was further used in subsequent experiments, where the NTP concentration was adjusted to Fig. 9 Similar ranges as described in A-9B and adjustment of DNA template concentration to extend the results of Example 12: Linearized plasmid DNA (pDNA), which showed improved yield with increasing pDNA concentration. The experimental design is shown in Table 31 below.

[0338] Table 31: Experiment 2 reaction conditions to evaluate higher NTP and DNA concentrations

[0339]

[0340] The results of this experiment are as follows Fig.13As shown. When ATP / CTP was increased to 12 mM and the final GTP concentration was increased to 10 mM, the yield and 5' cap percentage increased to 11.1 mg / mL and 97%, respectively. Therefore, condition H (listed in Table 31) with 0.1 g / L plasmid DNA, 12 mM final ATP and CTP, and 10 mM final GTP continued to be further developed as method FB V1.

[0341] Example 18: Scale-up, continuous feed simulation and synthetic DNA

[0342] The purpose of this experiment was to demonstrate the scalability of method FB V1 and to show that bolus feeding or semi-continuous feeding developed herein is technically equivalent to continuous feeding by producing similar high-quality RNA in method FB V1. A further goal was to demonstrate that method FB V1 has general applicability in that it produces comparable high-quality RNA across scales (using pDNA or synthetically produced DNA, such as from the integrated sequential template amplification and transcription [USTAT] method). NTP feeding was achieved by bolus feeding or semi-continuous feeding and continuous feeding using an automated microbial reactor system.

[0343] like Fig.14 As shown, bolus feeding or semi-continuous feeding produced RNA of similar quality and yield to their continuous feeding counterparts. The data also showed that the continuous feeding method produced high quality RNA of similar quality using either pDNA or synthetically produced DNA (USTAT). In addition, method FB V1 can be scaled up across multiple scales, such as Fig.14 Taken together, these data demonstrate the general scalability and applicability of method FB V1 to a variety of DNA template sources.

[0344] Example 19: Implementation of Mg bolus injection in IVT reactions

[0345] like Figure 7 As shown, both high and low Mg levels affect RNA concentration. Importantly, the data not only indicate that there is an optimal operating range for magnesium concentration, but also that the strategy for addition during IVT can be optimized.

[0346] As more NTP is added to the reaction system over time, the Mg:NTP ratio may not remain consistent. In addition, the Mg formed throughout the reaction method 2+ and pyrophosphate precipitation can reduce the amount of Mg available to enzymes such as T7, such as Fig.17As shown in and reported elsewhere [Kartje et al., J Biol Chem 296 (2021)]. For these reasons, stepwise delivery of Mg was explored to improve RNA quality attributes without negatively affecting yield and to maintain a more consistent Mg:NTP ratio throughout the reaction protocol.

[0347] A reaction system for stepwise feeding of a mixture of NTP and MgCl2 has been described, wherein the reaction system based on the reaction from CIMacPrimaS TM The three feed stages were initiated by online measurement of residual NTP by HPLC [Skok et al., Chem Ing Tech 94(12):1-9(2022)]. Unlike those previously described systems, the method described here is not divided into multiple feed stages and does not rely on the CIMac PrimaS TM Measurements on HPLC system. Additionally, the feed consisted of magnesium acetate and further optimized NTP levels and the overall benefit on the capping percentage was shown.

[0348] IVT reactions were assembled with varying starting levels of ATP, CTP, GTP, and pUTP and magnesium acetate. Throughout the reaction method, combined feeds of ATP, CTP, GTP, and pUTP were used to achieve the total levels listed in Table 32. Some conditions fed magnesium acetate as a bolus every 35 minutes, while other conditions were "front loaded" with a target amount of magnesium acetate. Samples were precipitated prior to analytical measurements. Concentration was measured by UV, integrity by a fragment analyzer (FA), and percent capping by LC-UV methods.

[0349] As shown in Table 32, the conditions that were preloaded with Mg had lower capping percentages than the corresponding conditions that received a Mg bolus. In one pair of conditions, the Mg bolus appeared to improve capping by about 15%. Each pair had similar mRNA yields within experimental and analytical variation, but this may be because the Mg:NTP ratio remained consistent across all conditions tested.

[0350] Table 32: Effect of Mg bolus injection on IVT performance

[0351]

[0352] This example demonstrates the benefits of stepwise feeding of Mg. For example, in this example, stepwise feeding of Mg improved 5' capping, indicating that stepwise delivery of Mg is a strategy to improve capping. The Mg bolus approach can also demonstrate that stepwise feeding is required to maintain yield at different Mg:NTP target ratios.

[0353] Example 20: Capping molecule concentration and scale-up

[0354] This example explores the feasibility of reducing the concentration of the capping molecule to 2.5 mM while still maintaining 95% capping. As shown in Example 19: Mg bolus injection is implemented in the IVT reaction, stepwise feeding of Mg can promote capping. For this reason, the starting Mg was also investigated in this example while the ending Mg was kept unchanged at 36 mM. An additional goal was to determine whether the Mg feeding strategy is scalable and can be performed with continuous feeding.

[0355] The present embodiment includes two independent experiments. One experiment was carried out in a 15mL reactor, and the other experiment was carried out in a 100mL reactor. The constant parameters in each experiment are listed in Table 38, and the variable parameters are shown in Table 33. In the 8mL system, ATP / CTP / GTP / modified UTP feeds were delivered once every 5 minutes, and a separate MgAc feed was delivered once every 15 minutes. In the 90mL system, a combined feed of NTP and MgAc was delivered continuously. Concentration was measured by UV method, integrity was measured by fragment analyzer (FA), and capping percentage was assessed by LC-UV determination.

[0356] As shown in Table 33, all reactions performed well at the 8 mL scale. mRNA yields and fragment analyzer (FA) integrity across all conditions tested were within the assay and analytical variability. A slight increase in capping of approximately 3% was observed between conditions B and F, which was only slightly above the expected assay variability. Since the concentration of 5' caps is generally high, the contribution of bolus Mg to the cap percentage is not significant, but it may still contribute to the robustness of the overall method if GTP is overfed. Based on these results, it would be feasible to reduce the concentration of the capping molecule below 2.5 mM.

[0357] Table 33: 8mL Evaluation of Capping Molecules and Mg

[0358]

[0359] The reaction concentration and integrity of Condition B were compared to the performance of the 90 mL condition (Table 34). The RNA concentration and integrity were within the experimental variation, indicating that the method can be scaled up and that the combined Mg:NTP feed can simplify operation. It also provides evidence that Mg bolus injection is similar to continuous feed containing Mg.

[0360] Table 34: Comparison of performance between AMBr15 and EasyMax

[0361]

[0362] As shown herein, the capping molecule can be used at 2.5 mM (and possibly lower), and the method can be run with a 5 minute bolus, with continuous feeding, with separate Mg and NTP feeds, and / or a combined Mg / NTP feed.

[0363] Example 21: High NTP Feed

[0364] This example explores the highest NTP level that can be fed at 36 mM front-loaded magnesium acetate. In Example 10, an interaction term between NTP and Mg was observed. For this reason, it is of interest to determine the robustness of the method at various NTP concentrations.

[0365] This example was performed in an automated microbioreactor system with an 8 mL starting volume. All constant parameters are listed in Table 38. Different amounts of feed containing ATP, CTP, GTP, and pUTP were added every 5 minutes using a liquid handler to reach the total levels listed in Table 35. Concentration was measured by UV, integrity by FA, ​​and percent capping was assessed by LC-UV assay.

[0366] The method showed robustness over a wide range of NTP concentrations. Each reaction produced large amounts of mRNA in the range of 45–79 mM total NTP. In Example 10, the batch method showed a lower optimal NTP range. In summary, these results indicate that the fed-batch method can handle higher total NTPs with high yields and improved product quality characteristics.

[0367] Table 35: High NTP Feed Results

[0368]

[0369] Example 22: Effect of stirring on residual DNA

[0370] For patient safety, impurities in drug substances need to be well controlled and minimized. One of the more challenging impurities to remove from IVT methods is residual DNA (resDNA). The effects of many parameters on resDNA have been studied. Here, agitation and mixing parameters are identified and shown to be key factors affecting resDNA.

[0371] In this experiment, three methods were performed with RNA#1 in an automated microbial reactor system. Method FB V1 with DNase digestion was used as a baseline to study the effect of mixing parameters on resDNA impurities. These reactions were modified from method FB V1 in terms of stirring rate and pyrophosphatase concentration. Fig.15 and Fig.16The three methods shown in the Figure 1 evaluated the stirring range described by multiple suppliers at three different pyrophosphatase levels. The stirring rate range studied was 150–600 RPM ( Fig.15 ), which is related to the power per unit volume ( Fig.16 ), mixing time and impeller outer edge speed ranges are synonymous. These ranges are listed in Table 36 below. These four mixing measures and other mixing factors not listed can be similarly used to reduce the level of residual DNA impurities.

[0372] Table 36: Mixing parameter ranges studied

[0373] Stirring (RPM) <![CDATA[Power / Volume (W / m 3 )]]> Mixing time(S) Impeller outer edge speed (m / s) 150–600 1.3–71.7 1.3–12.1 0.1–0.4

[0374] The results of this experiment highlight the effect of agitation rate on residual DNA impurities, as residual DNA levels consistently decreased in all three methods with increasing agitation rate, or power per volume, mixing time, or impeller tip speed. Therefore, agitation rate can be intentionally adjusted to reduce residual DNA impurities in IVT reactions. This example demonstrates that the range of agitation rates investigated corresponds to multiple parameters that affect the mixing profile, including parameters such as power per volume, impeller tip speed, and mixing time, and that these mixing parameters can be similarly adjusted to reduce resDNA impurities.

[0375] Table 37: Batch processing parameters

[0376]

[0377]

[0378] Table 38: Fed-Batch Method Parameters

[0379]

[0380]

[0381] Example 23: Plasmid

[0382] Several plasmids were used in the following examples. The GFP plasmid (5854 bp) includes the coding sequence of GFP (green fluorescent protein) and the BsaI restriction endonuclease recognition site immediately downstream of the poly (A) tail sequence. The self-amplification (SA) plasmid (13,826 bp) includes the NSP and RSV F protein and the LguI restriction endonuclease recognition site immediately downstream of the poly (A) tail sequence. The B globin plasmid (5101 bp) encodes the RSV F protein and the LguI restriction endonuclease recognition site immediately downstream of the poly (A) tail sequence segment. Both the GFP plasmid and the B globin plasmid use the 5' and 3' UTRs of β-globin. The poly (A) tail of the GFP plasmid RNA is 96 A (segmented), and the poly (A) tail of the B globin and SA plasmids is 80 A. The E. coli strain containing the desired plasmids was grown in animal component-free LB liquid medium containing kanamycin, and the resulting cell bodies were purified by Plasmid Giga Kit Process to isolate and purify plasmid DNA. Confirm the homogeneity of plasmid DNA by agarose gel electrophoresis.

[0383] Plasmid linearization was completed by incubation at 37 degrees Celsius for 18 hours. Plasmid GFP was linearized with 1.5U / ug BsaI restriction enzyme, SA plasmid was linearized with 0.5U / ug LguI, and B globin plasmid was linearized with 1.5U / ug LguI. Linearization was confirmed by agarose gel electrophoresis.

[0384] All in vitro transcription reaction components (except RiboLock, pyrophosphatase, and T7 RNA polymerase) were thawed on ice for 10-15 minutes, the reaction components were mixed, and the reaction was incubated for 2 or 3 hours. After the reaction was completed, the DNA template was removed by treatment with 6U / uL or 8U / uL DNase I and 1-2mM CaCl2 for 30 minutes. T7 RNA polymerase and pyrophosphatase (both THERMO FISHER ). ATP, CTP, UTP, N1-methyl pseudouridine (P-UTP) and GTP (THERMO FISHER ) were stored at recommended temperature and used as needed. DTT and spermidine (both SIGMA ) as needed.

[0385] RNA was precipitated with lithium chloride overnight in a -80°C freezer. The precipitate was then washed three times with ice-cold 100% ethanol and then nuclease-free water was added to dry the precipitate. RNA quantification and yield were reported as mg / mL IVT reaction (NANODROP )calculate.

[0386] Example 24: NTP concentration

[0387] Different concentrations of NTPs in the IVT reactions were investigated, such as 5mM (total 20mM NTP), 6mM (total 24mM NTP), 7mM (total 28mM NTP), and 8mM (total 32mM NTP) to cover a wider range of NTPs. Cap analogs were pre-added in the reaction to produce capped mRNA in this batch mode IVT method development. The IVT method described by Henderson et al. [Henderson et al., Curr Protoc 1(2):e39(2021)] has 5mM of each NTP and 4mM CleancapAG (3OMe). For different NTP concentrations, the single NTP / CleancapAG (3OMe) ratio was maintained at 1.25. The optimal concentration of magnesium, the cofactor of T7 RNA polymerase, can be determined to maintain ≥90% RNA integrity and ≥90% cap incorporation, as well as the maximum achievable yield. Another hypothesis tested was whether the Mg+2 concentration should be 4-6mM higher than the total NTPs. Therefore, different concentrations of MgOAC2 were studied to find a baseline concentration that ran comparably across different NTP concentrations. During the IVT reaction, as T7 RNA polymerase incorporates NTPs, pyrophosphate is released, which forms a complex with magnesium and precipitates in the reaction [Kern & Davis, Biotech progress 13 (6): 747-756 (1997)]. Pyrophosphatase can be added to the reaction to break down pyrophosphate into monophosphates, making magnesium free and available for the reaction. Some research groups have demonstrated that pyrophosphatase in IVT reactions increases RNA yields [Cunningham & Ofengand, Biotechniques 9 (6): 713-714 (1990)], while other research groups have shown that pyrophosphatase has no effect in increasing RNA yields [Samnuan et al., F1000 Research 11: 333 (2022)]. In view of these different observations, this example was tested with lower concentrations of pyrophosphatase to study a wider range. To obtain the maximum RNA yield from the different IVT conditions, the GFP template concentration was kept at 0.1ug / uL. The T7 RNA polymerase concentration range studied was 8U / uL and 10U / uL, and the reaction contained 1U / uL of Ribolock RNase Inhibitor (THERMOFISHER ), 10 mM DTT, 2 mM spermidine, and the reaction incubation temperature was 37 degrees Celsius, as shown in Table 39. The yield, capping percentage, and RNA integrity percentage of the reaction are shown in Table 40.

[0388] Table 39: IVT method parameters

[0389]

[0390] Table 40: IVT method results

[0391]

[0392] A 7 mM NTP concentration and a 5.6 mM cap concentration were chosen to pursue a maximum target yield of 9.2 mg / mL. MgOAC2 at 32 mM was chosen because it performed comparably for different NTP concentrations. Mg+2 was 4 mM higher than total NTPs, and its RNA integrity and cap incorporation remained above 90% under most IVT conditions.

[0393] Example 25: DTT, spermidine and pyrophosphatase concentrations

[0394] DTT is a reducing agent that prevents enzyme oxidation in the reaction. DTT concentrations of 5, 10, and 20 mM were studied to determine the optimal concentration. Spermidine is a polyamine that helps T7 RNA polymerase dissociate from the DNA template and quickly transfer to another DNA template for RNA synthesis, thereby improving transcription efficiency. To examine the effect of spermidine on RNA properties, concentrations of 0 mM and 2 mM were selected. Pyrophosphatase concentrations of 0.01 mU / uL and 0.02 mU / uL were studied, as shown in Table 41. The yield, capping % and RNA integrity % of the reaction are shown in Table 42. Different RNase inhibitor concentrations in the control reaction were also studied, as shown in Table 43.

[0395] Table 41: IVT method parameters (DTT, spermidine, pyrophosphatase)

[0396] IVT ingredients unit The different concentrations studied T7 RNA polymerase, AOF U / uL 8,10 DTT mM 5,10,20 Spermidine mM 0,2 Pyrophosphatase, AOF mU / uL 0.01,0.02

[0397] Table 42: IVT method results

[0398]

[0399]

[0400] Table 43: IVT method parameters (RNase inhibitor concentration)

[0401]

[0402] 80% of the reactions showed higher yields when using 10U / uL T7 RNA polymerase (see Table 42), therefore, 10U / uL T7 RNA polymerase was selected. There was not much difference in RNA quality attributes when using different concentrations of DTT. However, since DTT helps maintain enzyme activity in the reaction, a DTT concentration of 5mM was selected. The presence of spermidine showed increased yield at a lower pyrophosphatase concentration of 0.01mU / uL, but did not show the same effect on yield at a pyrophosphatase concentration of 0.02mU / uL. A spermidine concentration of 2mM was selected. The control IVT reaction without RibolockRNase inhibitor showed lower RNA integrity. The Ribolock concentration was reduced to 0.08U / uL because it was as effective as 1U / uL in maintaining RNA integrity. Similar results were observed at reduced Triton X-100 levels (see Table 45).

[0403] Example 26: Cap and pyrophosphatase concentration

[0404] Different concentrations of cap and pyrophosphatase were studied to determine the minimum cap concentration for the developed bulk IVT method and whether pyrophosphatase increases RNA yield while maintaining high cap incorporation and RNA integrity. The cap analog concentrations studied (mM) were 1, 2, 2.5, 3, 3.5, 4, 4.5, and 5.6, and the pyrophosphatase concentrations studied (mU / uL) were 0.01, 0.02, 0.05, 0.1, 0.2, and 2. The yield, % capping, and % RNA integrity of the reactions are shown in Table 44.

[0405] Table 44: IVT method results

[0406]

[0407]

[0408] RNA integrity decreased with increasing pyrophosphatase. RNA yield increased when increasing pyrophosphatase from 0.01mU / uL to 0.02mU / uL. However, yields were consistent when increasing pyrophosphatase from 0.02mU / uL to 2mU / uL, suggesting that the reaction conditions were balanced with respect to magnesium and pyrophosphatase. Capping efficiency decreased with decreasing cap concentrations. A cap concentration of 3mM was equally effective at varying pyrophosphatase concentrations up to 0.1mU / uL and still maintained >90% capping %. Under most conditions, a decrease in yield was observed with decreasing cap concentrations.

[0409] Example 27: Development of a batch-mode IVT method for RNA molecules

[0410] Using the guidance of the previous examples, a batch mode IVT reaction was developed containing: Tris HCl buffer (at a concentration of 40 mM and pH 8), Ribolock (0.08 U / uL), cap analog (3 mM or 4 mM), ATP (7 mM), CTP (7 mM), GTP (7 mM), P-UTP / UTP (7 mM), Mg(OAc)2 (32 mM), DTT (5 mM), spermidine (2 mM), DNA template (0.1 ug / uL), AOF pyrophosphatase (0.02 mU / uL), AOF T7 RNA polymerase (10 U / uL), with static reaction agitation and run at 37°C for 120 minutes. The results of the control reaction using reduced Triton X-100 levels (100-fold lower than those shown in Table 43) are shown in Table 45.

[0411] Table 45: IVT method results

[0412]

[0413] Example 28: Template concentration

[0414] To explore the optimal template concentration, a range of template concentrations were studied. The optimal template concentration can help reduce downstream DNA impurities. The developed IVT method has a DNA template concentration of 0.1ug / uL. The results of these experiments are shown in Fig.18 The results show that increasing the template concentration does not lead to a large difference in yield. Therefore, the template concentration can be reduced to 0.05ug / uL.

[0415] Example 29: Incubation temperature and time

[0416] To investigate whether the incubation temperature might have any effect on the synthesis of intact RNA, different temperatures and incubation times for IVT were investigated. Fig.19 As shown, a decrease in RNA yield was observed as the incubation temperature of IVT was reduced to 25°C. At 30°C, 2 and 3 hours of incubation time, ~90% RNA integrity was maintained. However, at 20°C, cap incorporation was minimal (approximately 50%).

[0417] Example 30: Implementation of a batch-mode IVT method on long RNA molecules

[0418] The above batch mode IVT method was tested on long RNA molecules (~9.48KB self-amplifying RNA) with incubation times of 2 or 3 hours at 37°C and template concentrations of 0.05, 0.075 or 0.1 ug / uL. No capping molecules were added to the reactions except for the parameters tested. Fig. 20As shown, at 37°C, the RNA integrity of large RNA molecules (~9.48KB) is about 60% to 70% integrity, and the DNA concentrations of 0.05ug / uL, 0.075ug / uL and 0.1ug / uL are consistent with the results for yield and RNA integrity. Therefore, a lower DNA concentration of 0.05ug / uL was selected. In addition, longer incubation times lead to reduced RNA integrity. Therefore, lower incubation temperatures were tested.

[0419] Example 31: Testing Lower IVT Incubation Temperatures and Times

[0420] Lower incubation temperatures were explored to determine if they would yield greater amounts of intact RNA molecules. Temperatures of 20°C, 25°C, and 30°C were tested at a template concentration of 0.05 ug / uL over a 2 or 3 hour incubation time. Fig.21 As shown, compared with 37℃( Fig. 20 ). Compared with 2 hours of incubation time, lowering the incubation temperature of large RNA molecules resulted in higher RNA integrity. At 25°C and 30°C, RNA integrity was ≤77%. 25°C was selected as the incubation temperature. In addition, increasing the incubation time to 3 hours resulted in slightly higher RNA yields compared to an incubation time of 2 hours.

[0421] Example 32: Testing Mg2+ concentration to increase RNA integrity

[0422] In order to achieve the goal of ≥80% RNA integrity, magnesium titration was performed to balance the concentration of pyrophosphatase and NTP in the reaction for long RNA molecules. In addition, two incubation times were tested to obtain optimal yield and RNA integrity. As shown in Table 46, when the magnesium concentration was reduced to 26mM, RNA integrity increased by 2-4%. Mg2+ at a concentration of 28mM is a selected concentration for long RNA molecules because it achieves ≥80% RNA integrity and RNA yield is still optimal. In addition, a 3-hour incubation was selected to ensure that a yield of ≥5mg / mL was obtained.

[0423] Table 46: Mg2+ titration results

[0424]

[0425]

[0426] Example 33: Pyrophosphatase titration and yield

[0427] Pyrophosphatase titration studies were performed to test whether RNA yield could be increased while maintaining RNA integrity. Pyrophosphatase concentrations of 0.02, 0.05, 0.08, 0.1, 0.15, 0.2, and 2 mM were tested at 25°C, with an incubation time of 3 hours and a Mg2+ concentration of 26 or 28 mM. As shown in Table 47, increasing the pyrophosphatase concentration in the reaction resulted in an increase in RNA yield of ˜1-2 mg at certain concentrations. However, increasing the concentration resulted in a decrease in RNA integrity; therefore, the selected pyrophosphatase concentration was 0.02 mU / uL.

[0428] Table 47: Pyrophosphatase titration results

[0429]

[0430] Example 34: Batch Mode IVT Method for Long RNA Molecules

[0431] The guidance of the previous examples was used to develop a batch mode IVT reaction for long RNA molecules, which contained: TrisHCl buffer (concentration of 40mM and pH 8), Ribolock (0.08U / uL), ATP (7mM), CTP (7mM), GTP (7mM), UTP (7mM), Mg(OAc)2 (28mM), DTT (5mM), spermidine (2mM), DNA template (0.05ug / uL), AOF pyrophosphatase (0.02mU / uL), AOF T7 RNA polymerase (10U / uL), and was run at 25°C for 180 minutes.

[0432] Example 35: Feed IVT Development – ​​Increasing NTP Concentration to Increase RNA Yield

[0433] In addition to reducing the cap concentration, the goal of the fed-batch IVT method is to increase RNA yield with approximately 1 mM cap. To increase yield, the NTP concentration in the batch mode IVT was increased. As shown in Table 48, increasing the NTP concentration resulted in reduced capping efficiency in the batch mode IVT while still maintaining RNA integrity. Since the target yield was ~12.5 mg / mL, a concentration of 9.5 mM of each NTP was selected for the fed-batch IVT. The following examples explore Mg2+, T7, and pyrophosphatase titrations to increase RNA yield and balance the reaction.

[0434] Table 48: Results of increasing NTP concentrations

[0435]

[0436] Example 36: Effect of specific NTPs on capping efficiency

[0437] To determine if any of the four NTPs had an effect on cap incorporation, two different ratios of NTP to cap were tested. The two ratios of NTP to cap tested were 1.25 and 3.5. For all other NTPs not tested in this sample, the NTP concentration was kept at 9.5 mM and the cap concentration was 1 mM. The cap analog refers to CleancapAG (3OMe). Fig. 22 As shown, decreasing the GTP concentration resulted in higher cap incorporation than decreasing the concentration of all other NTPs. For both NTP to cap ratios tested, cap incorporation was ≥90%. For all other NTPs, cap incorporation decreased with NTP concentration. This suggests that the initial concentration of GTP affects the capping percentage. Therefore, the GTP feed was selected to proceed.

[0438] Example 37: Increasing RNA yield

[0439] Before investigating the feeding strategy, IVT was further optimized to increase yield using a baseline 1 mM cap concentration. To increase RNA yield, a range of magnesium acetate concentrations were investigated, followed by various concentrations of pyrophosphatase and T7 RNA polymerase. In this example, the NTP concentration was 9.5 mM and the cap concentration was 1 mM. Fig.23 As shown, RNA integrity decreased slightly with increasing magnesium concentration. RNA yields ranged from 6-7.5 mg / mL. 36 mM magnesium was chosen as the equilibrium concentration in the reaction to avoid any negative impact on RNA integrity.

[0440] To further increase the yield, various concentrations of T7 RNA polymerase and pyrophosphatase were tested. As shown in Table 49, with the increase of pyrophosphatase, the RNA yield under most conditions increased. However, in the case of 0.1mU / uL pyrophosphatase with 13U / uL and 15U / uL T7 RNA polymerase, the yield decreased slightly. Therefore, 0.08mU / uL pyrophosphatase was selected. Using 15U / uL and 13U / uL T7 RNA polymerase and 0.08mU / uL pyrophosphatase, the yield achieved was ≤10mg / mL. However, 15U / uL was selected to ensure that the maximum possible yield was achieved.

[0441] Table 49: T7 RNA polymerase and pyrophosphatase at different concentrations

[0442]

[0443] Example 38: Determining the amount of GTP used during a time interval

[0444] To determine the amount of GTP consumed at different time intervals in the developed IVT method, reactions were performed using the following GTP concentrations: 0.4 mM, 0.8 mM, and 1 mM. Reactions were stopped at different time points and tested for NTP residual. In the first reaction, 0.4 mM GTP was added to the developed IVT method. Reactions were stopped at 4, 5, 8, 10, 12, or 15 minutes. After 4 minutes of reaction, residual GTP was still present. Therefore, a 4 minute GTP feeding interval with a feeding concentration of 0.4 mM GTP was selected to be tested in the feeding IVT study.

[0445] In the second reaction, 0.8 mM GTP was added to the developed IVT method. The reactions were stopped at 5, 8, 10, 12, or 15 minutes and residual GTP was measured. At 8 minutes into the reaction, residual GTP was still present. Therefore, 5 and 8 minute GTP feed intervals with a feed concentration of 0.8 mM GTP were selected to be tested in the fed IVT study.

[0446] In the third reaction, 1 mM GTP was added to the developed IVT method. The reactions were stopped at 5, 8, 10, 12, or 15 minutes and residual GTP was measured. At 8 minutes into the reaction, residual GTP was still present. Therefore, 5 and 8 minute GTP feed intervals with a feed concentration of 1 mM GTP were selected to be tested in the fed IVT study.

[0447] Example 39: Determination of the amount of GTP:cap analog

[0448] In the following examples, different GTP to cap ratios were tested to determine how low the ratio of the concentration of the cap analog to the amount of GTP fed in the reaction could be. As preferred for ARCA cap analog G, the baseline GTP / cap ratio was 0.25. In this example, three different GTP concentrations were tested, and the cap concentration range was 0.1 mM to 4 mM. As shown in Table 50, the lowest cap concentration showing ≥90% capping percentage in the reaction was 0.4 mM. A cap of 0.4 mM and any concentration above 0.4 mM allowed ≥90% capping. In addition, 0.4 mM, 0.8 mM and 1 mM GTP concentrations allowed ~90% cap incorporation. Therefore, different GTP:cap ratios and different GTP feed concentrations were tested in the following studies.

[0449] Table 50: Different ratios of GTP / cap

[0450]

[0451]

[0452] Example 40: Different GTP feed concentrations

[0453] In the following examples, different GTP feed concentrations were tested. In the first study, 0.4 mM GTP was fed every 4 minutes. In total, 9.5 mM GTP was added during the IVT process. Different concentrations of the cap analog were tested to determine the optimal conditions. Fig.24 As shown, increasing cap analogs resulted in higher cap incorporation. Cap concentrations of ≥ 0.7 mM and 0.4 mM GTP feed were baseline conditions to achieve > 90% capping, 90% RNA integrity, and ~ 9 mg / mL yield.

[0454] In the second study, 0.8 mM GTP was fed every 5 or 8 min. Different concentrations of the cap analogs were also tested. Fig.25 As shown, feeding 0.8 mM GTP every 8 minutes resulted in higher yields and increased cap incorporation compared to a 5 minute feed. In this study, feeding 0.8 mM GTP every 8 minutes and ≥1 mM cap was the baseline condition to achieve ∼90% RNA integrity, ∼90% cap incorporation, and ∼7.5-10 mg / mL yield.

[0455] In the third study, 1 mM GTP was fed every 5 or 8 minutes. Again, different concentrations of the cap analog were tested. Fig.26 As shown, again, an 8-minute feed resulted in slightly higher yields compared to a 5-minute feed. From these results, the baseline optimal condition is to feed 0.7 mM cap analog and 1 mM GTP every 8 minutes to obtain a yield of ≥7 mg / mL while still maintaining ∼90% RNA integrity and ∼90% cap incorporation.

[0456] Example 41: RCA reactions with different RCA template concentrations

[0457] In the following examples, as little as 0.5 to five (0.5-5) nanograms of circular supercoiled double-stranded DNA (dsDNA) template (comprising a transcribable cassette) was used in a rolling circle amplification (RCA) reaction (described in Table 51) to generate 10 5 –10 6 times the template amplification to produce dsDNA concatemers, such as Fig.27A As shown, and after linearization, Fig.27B shown.

[0458] Table 51: RCA reaction using phi29 DNA polymerase

[0459]

[0460]

[0461] The above mixture is mixed with or without heat denaturation and then incubated at 30°C for 25-30 hours. After incubation, the phi29 polymerase can be heat-inactivated at 65°C for 10 minutes. Preferably, the polymerase is not heat-inactivated. The reaction can achieve 0.5-0.85 mg / mL DNA.

[0462] As shown herein, the RCA reaction can be carried out at a temperature of 27-30°C for 25-30 hours (see Figure 28). Additionally, BSA can be removed, the RCA buffer composition can be adjusted, and different dNTP concentrations and phi29 polymerase can be used to successfully generate RCA templates for in vitro transcription reactions. Finally, both the heat denaturation step before RCA incubation and the heat inactivation step after RCA (but before linearization) can be omitted from the RCA reaction method. Removing these two steps simplifies commercial manufacturing and allows isothermal methods, while still producing mRNA of amazing quality compared to plasmid DNA, as shown below.

[0463] Example 42: RCA DNA digestion without purification

[0464] In the following examples, RCA DNA generated from a circular dsDNA template can be enzymatically digested by one or more restriction endonucleases to generate linearized DNA fragments for use as templates for in vitro transcription reactions without the need to purify the RCA DNA prior to enzymatic digestion. Conventional reactions require purification of the DNA template, reconstitution of the DNA template in a buffer, and then enzymatic digestion of the DNA template. As described herein, the entire reaction (i.e., amplification and linearization) can be performed in the same reaction vessel without material loss because there is no need to transfer to different containers for different reaction steps.

[0465] RCA DNA can be digested with restriction endonucleases to produce linear dsDNA without the need for DNA purification or additional buffer reagents. 0.8-2.2 U of BspQI (10 U / μL, NEB TM #0712) into the RCA reaction vessel and incubate overnight at 37°C in a heating block. Fig.29 As shown, the cleavage products can be visualized by agarose gel electrophoresis.

[0466] Example 43: In vitro transcription using linearized DNA template without purification

[0467] Linearized RCA DNA or linearized plasmid DNA can be used as a template for in vitro transcription without purification after digestion with restriction endonucleases. Typically, DNA templates need to be purified and resuspended in buffer before use in IVT reactions. Surprisingly, this purification step can be removed without affecting the yield or quality of RNA in IVT reactions.

[0468] Linearized RCA DNA prepared according to the above examples will be used as an IVT template to generate mRNA without post-digestion purification. Additionally, unpurified linearized plasmid DNA can be included as a template for IVT. Linearized DNA can also be purified using ethanol / 2.5M NH4CH3CO2 precipitation and resuspended in nuclease-free water. Each DNA sample will be used as a template in an IVT reaction using the parameters shown in Table 52. DNA will be supplemented to a final concentration of 0.1 mg / mL.

[0469] Table 52: USTAT v1 IVT reactions using linear RCA DNA as template

[0470]

[0471] The above reaction will be vortexed gently and incubated at 37°C for 150 minutes. DNase I and 1M CaCl2 will be added and the reaction mixture will be incubated at 37°C. The product will then be purified by LiCl precipitation. The concentration will be measured on the nanodrop and RNA integrity will be assessed using fragment analysis. RNA produced using linearized, purified plasmid DNA will be used as a control.

[0472] Example 44: Integrated Sequential Template Amplification and Transcription

[0473] Linearized RCA DNA or linearized plasmid DNA can be used as a template for in vitro transcription without purification after digestion with restriction enzymes. Typically, DNA templates need to be purified and resuspended in buffer before use in IVT reactions. Surprisingly, this purification step can be removed without affecting the yield or quality of RNA in IVT reactions.

[0474] RCA reactions of circular dsDNA (plasmid or synthetic), followed by restriction enzyme linearization and IVT can be performed sequentially in a single reaction vessel to generate mRNA without any prior purification steps. The method allows DNA-directed synthesis of RNA molecules of any sequence, ranging in size from short oligonucleotides to several kilobases.

[0475] The DNA generated by the RCA reaction can be linearized and used as a template for an IVT reaction, which is a step-wise reaction in a single vessel without purification or volume transfer, called integrated sequential template amplification and transcription (USTAT). The template for this single-vessel reaction can be as little as 0.5 ng of circular supercoiled dsDNA per mL RCA reaction without any impact on RNA yield or quality. No heat denaturation of the template DNA is required. The RCA reaction components for this type of step-wise single-vessel RCA reaction are shown in Table 51.

[0476] Assemble the RCA reaction as shown in Table 51, using 0.5 ng of plasmid DNA per mL reaction volume. Similar reactions using commercially available buffer conditions are included for comparison. The reaction mixture was gently vortexed and incubated at 30°C for 25-30 hours. The RCA reaction was then digested with restriction endonucleases in the same container to produce linearized dsDNA without DNA purification or additional buffer reagents. 1.0 U BspQI (10 U / μL, NEB, #R0712) per microgram of RCA DNA was transferred into the RCA reaction vessel and incubated at 37°C for 20-25 hours.

[0477] The product of the digestion reaction can be used in the subsequent IVT step without purification. The linearized RCA DNA was used directly in the IVT reaction as shown in Table 52. The linearized plasmid DNA template was used as a control. The reaction was gently vortexed and incubated at 37°C for 150 minutes. DNase I and 1M CaCl2 were added and the reaction mixture was incubated at 37°C. The product was purified by LiCl precipitation and the concentration was measured using Nanodrop. RNA integrity was assessed using fragment analysis. The results are shown in Figure 30.

[0478] Example 45: Combination of USTAT and IVT Reaction Improvement

[0479] Integrated sequential template amplification and transcription (USTAT) produces higher quality RNA products when combined with IVT reaction improvements.

[0480] The RCA reaction was performed, the product was digested, and the DNA was used as an IVT template without further purification as described in Example 44. In a second aspect, the plasmid DNA was linearized using 2U BspQI per microgram DNA and used as an IVT template without purification. The linearized purified plasmid DNA template for IVT was included as a control.

[0481] IVT reactions using the above DNA templates were performed using one of the following three IVT reaction conditions: USTAT v1 IVT reactions shown in Table 52, bulk IVT reactions (BV1) shown in Table 37, or fed-batch IVT reactions (FBV1) shown in Table 38. The reactions may optionally include components for co-transcriptional capping and / or modified NTPs. RCA DNA or plasmid DNA was supplemented to a final concentration of 0.05-0.1 mg / mL.

[0482] RNA samples were purified by precipitation and concentrations were measured by UV. The integrity of the resulting RNA was assessed using fragment analysis, and the fraction of capped mRNA was assessed. The results of comparing the RCADNA template with a linearized plasmid template (purified control or unpurified) in IVT using USTAT V1, BV1 or FB V1 IVT methods are shown in Table 53.

[0483] Table 53: Comparison of RCA DNA template and linearized plasmid

[0484]

[0485] Example 46: Scalability of USTAT reactions

[0486] The volume of the integrated sequential template amplification and transcription (USTAT) reaction can be increased to enable production of larger amounts of RNA.

[0487] RCA DNA amplification reactions were performed at a 100 mL scale and the products were linearized using BspQI as described in Example 44. DNA was supplemented at 0.1 mg / mL into the FB V1 IVT reactions shown in Table 38. Reactions using linearized purified plasmid DNA template were included as controls. RNA samples were purified by precipitation and concentrations were determined by UV. RNA integrity was determined by FA and the percentage of capping was assessed by LC-UV assay. The results are shown in Figure 31.

[0488] Example 47: Next Generation Sequencing of Linearized USTAT RCA DNA

[0489] Next generation sequencing can be used to assess the sequence fidelity of the DNA amplified in the RCA reaction relative to the plasmid template. The sequences from linearized and purified plasmid DNA, linearized but unpurified plasmid DNA, and linearized RCA DNA were assessed for insertion / deletion or any sequence variants. No variants were observed within the transcription template, as shown in Figure 32.

[0490] Example 48: USTAT RNA Expression in Cell Culture

[0491] RNA produced using USTAT can be transfected into HEK293 cells, resulting in protein expression similar to that observed using RNA derived from a plasmid DNA template.

[0492] USTAT RCA DNA or linearized plasmid control DNA (each containing a transcription cassette encoding GFP) was used as template in the IVT reaction as shown in Table 37. Reactions were run in duplicate to produce uncapped as well as co-transcriptionally capped RNA. RNA was purified using lithium chloride precipitation, resuspended in water and the concentration was measured using a nanodrop. A total of 5 μg RNA was transfected per well, of which 0.5 μg or 0.05 μg was capped RNA and the remainder was uncapped RNA. Transfected cells were allowed to recover for 1 hour and then analyzed by FACS (BD FACS ELO DYNAMICS). TM ) to evaluate GFP expression. The number of cells expressing GPF in each 5000 counts was Fig.33 Control transfections using uncapped RNA alone showed no measurable GFP expression.

[0493] Example 49: USTAT reactions using variable size templates

[0494] The USTAT reaction can be used to produce RNA of various sizes, from 1.4 kb to over 10 kb. A set of plasmids containing transcription cassettes for RNA of varying sizes were used as templates in the USTAT RCA, as described in Example 44: Integrated Sequential Template Amplification and Transcription. The linearized DNA was added to an IVT reaction consisting of the components in Table 37. The RNA was purified using lithium chloride precipitation and resuspended in water. The yields and integrity of reactions using constructs with variable size IVT transcript templates are shown in Table 54. RCA DNA yields, RNA yields, and RNA integrity are shown. A plasmid DNA template was included as a control. RNA integrity was measured using FA.

[0495] Table 54: Comparison of reactions using IVT transcript templates of varying sizes

[0496]

[0497] Example 50: Single-step template assembly and USTAT

[0498] Circular dsDNA templates can be assembled from linear dsDNA fragments for use in USTAT reactions without any clean-up or purification steps prior to DNA amplification. This allows template assembly, DNA amplification, linearization, and IVT to be performed sequentially in a single vessel without the need for purification steps. Linear dsDNA fragments can be generated by in vitro DNA synthesis, enzymatic digestion of plasmid fragments, or by PCR.

[0499] Linear dsDNA fragments containing the target gene (GOI) sequence can be obtained from a commercial supplier (IDT) or generated by PCR. The fragments are separated from the plasmid by using enzymatic digestion or a second group of fragments are generated by PCR, which includes a promoter sequence, 5' and 3' UTR and a poly A sequence. As an alternative, a single dsDNA fragment is amplified from the plasmid by PCR to be cyclized in the next step. Linear dsDNA is reacted with an enzyme and a buffer (NEBuilder HiFi assembly mixture or the like) in combination, and the resulting circular dsDNA is directly combined with the RCA reaction components, as shown in Table 51, using 0.5ng DNA per milliliter of reaction volume. This reaction is used to generate mRNA, as shown in Example 44.

[0500] The linearized RCA DNA was used directly in the IVT reaction as shown in Table 52. The reaction was gently vortexed and incubated at 37°C for 150 minutes, followed by purification by LiCl precipitation. RNA concentration was measured using NanoDrop, and RNA integrity was assessed using fragment analysis. The results are shown in Table 55.

[0501] Table 55: Comparison of minicircle USTAT template and plasmid DNA

[0502]

[0503] Example 51: Buffer-modified USTAT

[0504] The components of the buffer formulation for integrated sequential template amplification and transcription (USTAT) can be adjusted to optimize mRNA product quality. In the following examples, the effects of improvements to the reaction components and reduced amounts of DNA template addition on mRNA integrity and yield were evaluated.

[0505] RCA reactions were assembled as per Table 56, using 0.05 ng of plasmid DNA per mL of reaction volume. Reactions using commercially available buffer compositions were included as controls. The reaction mixtures were gently vortexed and incubated at 30°C for 22-30 hours. The RCA reactions were then digested with restriction endonucleases in the same vessel to produce linearized dsDNA without DNA purification or additional buffering reagents. 1.0 U of BspQI (NEW ENGLAND BIOLABS) per mL of RCA reaction was pipetted into the RCA reaction vessel and incubated at 37°C for 20-25 hours. RCA DNA yields were as follows: Fig.34A The linearized DNA was used directly as an IVT template.

[0506] Table 56: USTAT RCA reaction and buffer composition options

[0507]

[0508] DNA was supplemented to the BV1 IVT reaction at 0.1 mg / mL as shown in Table 37. RNA samples were purified by precipitation and concentrations were determined by UV. RNA integrity was determined by FA and the results are shown in Table 37. Fig.34B shown.

[0509] Example 52: USTAT with different priming options and template lengths

[0510] Priming options were evaluated using dsDNA templates for USTAT producing mRNAs of varying lengths. A set of plasmids containing transcription cassettes for RNAs of various sizes ranging from 1.4 kb to 11 kb were used as templates for USTAT RCA. The reactions were assembled as shown in Table 56 and included either random primers or 10 ug / mL priming enzyme to create a priming site for phi29 DNA polymerase. Linearized RCA DNA and corresponding linearized plasmid template controls were used as templates for BV1 IVT as described in Example 51. RNA samples were purified by precipitation and concentrations determined by UV. RNA integrity was determined by FA and the results are shown in Table 56. Fig.35 shown.

[0511] Example 53: Improvement of USTAT synthetic template assembly

[0512] Linear dsDNA fragments can be assembled and used directly as templates in the USTAT reaction to produce mRNA, which provides the opportunity for the generation of fully synthetic templates. Several improvements to the synthesis strategy have been introduced. The design parameters of the template sequence have been modified, including recoding the target gene to eliminate homopolymer sequences larger than 5 bp. This modification will reduce the occurrence of insertions or deletions generated by phi29 at homopolymer sequences. Second, because the assembled dsDNA is used as a template for USTAT without the need for exonuclease treatment or purification steps, the assembly design has been adjusted to remove sequences downstream of the T7 promoter and reduce any linear fragments from potential transcription through the IVT step.

[0513] Linear dsDNA fragments containing the gene of interest and 5'UTR sequence were obtained from a commercial supplier. A second fragment containing the T7 RNA polymerase promoter, 3'UTR and poly A sequence was digested from the plasmid DNA. The fragments were assembled using a reaction mixture containing DNA polymerase, T5 exonuclease and Taq ligase according to a previously described method [Gibson et al., Nat Methods 6 (2009)]. For comparison, each assembly reaction was performed with and without the T5 exonuclease digestion step, and the assembled DNA was directly mixed with the USTAT components, as shown in Table 56, using 0.05 ng DNA per mL reaction. The linearized RCA DNA was used as a template for BV1 IVT, as shown in Example 51. The RNA sample was purified by precipitation and the concentration was determined by UV. The RNA integrity was determined by FA, ​​and the results are shown in Table 56. Fig.36 shown.

[0514] Example 54: Post-transcriptional enzymatic capping

[0515] Post-transcriptional enzymatic capping by vaccinia capping enzyme is applicable to longer RNA molecules as well as RNAs of all other sizes. Uncapped RNA prepared by the in vitro transcription method described in Example 37 was subjected to post-transcriptional enzymatic capping with vaccinia capping enzyme as recommended by the manufacturer (NEWENGLAND BIOLABS) to generate type 1 capped RNA. RNA was also pretreated at different temperatures to understand the effect of pretreatment temperature on RNA integrity % and capping %. These results are shown in Table 57.

[0516] Table 57: Effects of pre-treatment of RNA at different temperatures

[0517] serial number Preheat RNA temperature RNA integrity % Cap % Uncap58 N / A 81.3 N / A VCE1 65℃, on ice for 5 minutes (control) 69.9 100 VCE2 37°C, 5 min 73.9 100 VCE3 25℃, 5 minutes 74.4 100

[0518] Table 57 shows that preheating RNA at a low temperature of 25°C had little effect on the % loss of RNA integrity. Cap incorporation was maintained at >90% across all conditions tested.

[0519] To understand whether the RNA integrity percentage can be maintained at a lower post-enzymatic capping reaction temperature, uncapped RNA was subjected to post-transcriptional enzymatic capping at incubation temperatures of 25° C. and 30° C. for 30 minutes and 60 minutes, respectively. These results are shown in Table 58.

[0520] Table 58: Effect of time and RNA pretreatment

[0521]

[0522]

[0523] Table 58 shows that lower reaction temperatures of 25°C and 30°C and an incubation time of 30 minutes resulted in less loss of RNA integrity %. At an incubation temperature of 30°C for 60 minutes, the loss of RNA integrity % was greater.

[0524] Example 55: Exploration of buffers in IVT reactions

[0525] HEPES can be used as a buffer in IVT reactions to make RNA [Kern and Davis, Biotechnol Prog 15:174-84 (1999)]. To evaluate the effect of HEPES on the BV1 method, an OFAT (one-time one-factor) study was performed. Mimicking the range explored above with Tris, this experiment tested HEPES concentrations of 20-60 mM and pH values ​​of 7.5-8.5. Tris at pH 8.0 was not added to the reaction, but HEPES was used instead, but all other parameters for BV1 described in Table 37 remained consistent. As shown in Table 59, IVT yields may decrease slightly at higher pH and higher HEPES concentrations. However, all conditions tested were effective for the IVT reaction and produced intact mRNA.

[0526] Table 59: Single Factor Analysis of HEPES and pH

[0527]

[0528] Additional studies were conducted to determine if the buffering agent Tris could be delivered via other raw materials rather than stock solutions. Tris-buffered NTPs were investigated as alternatives to sodium-stabilized NTPs. These Tris NTPs were titrated to a pH of 7.3-7.5 with an undetermined amount of Tris base (THERMO FISHER). NaOH was added to the experimental conditions to keep the starting pH constant. Method BV1 described in Table 37 was run in a high-throughput manner. Condition A, shown in Table 60, used sodium-stabilized NTPs, while Conditions BE used Tris NTPs. The use of Tris NTPs was associated with a slight decrease in FA, but the product quality was still excellent. Surprisingly, Condition B, which did not have any additional Tris beyond that provided in the NTPs, performed most similarly to the control. This suggests that if a single IVT raw material has sufficient buffering capacity, additional buffer may not always be required to stabilize the reaction system.

[0529] Table 60: Tris NTP and Tris stock solutions

[0530]

[0531] Example 56: Application of IVT Platform Batch Method

[0532] The IVT methods described herein are generally applicable to a wide range of mRNA constructs. The methods described herein accelerate method development and enable faster delivery of life-saving products. To demonstrate the successful application of the methods, method BV1 (as described in Table 37) was used to generate unique mRNA molecules. For these reactions, the RNase inhibitor was increased from 100U / mL to 1000U / mL to reduce the risk of RNase contamination in the plasmid DNA preparation, but all other parameters were matched to BV1 (as described in Table 37). A series of OFAT (single factor) studies were performed in a series of small-scale high-throughput IVT reactions. Under each condition, the 5'UTR, coding sequence, or sequence length was varied. As shown in Table 61, the IVT BV1 method effectively produced mRNA in 13 different construct designs.

[0533] Table 61: Evaluation of the BV1 method with respect to 5'UTR, coding sequence and coding sequence length

[0534]

[0535]

[0536] The BV1 method was further evaluated in a separate set of 7 plasmid DNA constructs. No modifications were made to the BV1 method when performing this evaluation. The data shown in Table 62 further confirm that BV1 can successfully produce high-quality mRNA on constructs with a wide variety of coding sequences. In addition, the mRNA generated by BV1 was of high quality, with high FA integrity values ​​and 5' capping percentages of >80%.

[0537] Table 62: Evaluation of the BV1 method on different coding sequences

[0538] Construct Name (kb) Yield (g / L starting volume) Completeness (%) 5' cap (%) RNA1(2.13) 8.8 94 83 RNA5(2.16) 8.5 91 84 RNA6(1.87) 9.2 92 87 RNA7(2.16) 8.8 79 83 RNA8(1.87) 9.0 94 83 RNA9(2.16) 8.8 92 84 RNA10(1.87) 9.5 87 84

[0539] Example 57: NTP Enhancement in IVT Fed-Batch Reactions

[0540] In order to enhance the IVT method to be generally applicable to a variety of constructs (e.g., size, UTR, etc.), the batch feeding method was further improved by strengthening the NTP level. The purpose of this experiment was to determine the NTP level that can be fed while maintaining the production of high-quality mRNA (e.g., high integrity and capping efficiency) and yield. The NTP was significantly increased, and as shown in Table 63, high-quality mRNA (i.e., integrity>90% and capping efficiency>85%) and RNA yield>10g / L starting volume can be maintained while the NTP is increased. In addition, although the data also shows the limitation of Mg concentration under high NTP, as shown by the lower yield (e.g., 10.6v14.5g / L, conditions J and K, respectively), some conditions with increased Mg concentration (e.g., K) surprisingly showed a decrease in capping efficiency. This observation is consistent with the data in Table 63, which shows that the increase in initial Mg level affects the capping efficiency.

[0541] Table 63: NTP enhancement affects yield, integrity and 5' capping

[0542]

[0543]

[0544] Example 58: Mg addition strategy in IVT reaction

[0545] To investigate the effect of Mg concentration in IVT reactions, Mg addition strategies were evaluated in a fed-batch process with enhanced NTP concentrations. In this study, the conditions in row J of Table 63 (referred to as "Method FBV1.1") were used as the baseline method for this example. Specific details of the method are listed in Table 65. Two Mg addition strategies were evaluated here: 1) adding the total volume of Mg at the start of the IVT reaction ("front loading"); 2) starting with a lower Mg concentration at the start of the IVT reaction and feeding the remaining Mg to reach a specified total concentration or final concentration. Fig.37 It was shown that increasing Mg levels to 50 mM significantly increased mRNA yield. However, the increase in yield unexpectedly resulted in a decrease in capping efficiency when increased Mg levels were front-loaded. Surprisingly, feeding some Mg during the IVT reaction improved capping efficiency. Importantly, increasing the frequency of Mg addition resulted in a surprising further increase in 5' capping efficiency.

[0546] Next, we further investigated the effect of total Mg concentration on RNA quality attributes and yield. Fig.38As shown, increasing the front-loaded Mg results in a significant reduction in the 5' capping efficiency of RNA, as well as minimal negative effects on RNA integrity and yield. However, starting from a lower initial Mg concentration and supplementing the remaining Mg by feeding significantly improves the capping efficiency. Interestingly, starting from a lower Mg concentration and feeding the remaining Mg to the total amount leads to further improvement in the capping efficiency, as shown in conditions where the total Mg concentration is 60mM but the starting Mg concentration is different. In summary, these data show that the feeding strategy developed in this study significantly improves RNA quality attributes and yield. As described herein, increasing the front-loaded Mg concentration level has a negative impact on RNA quality. However, the feed Mg concentration improves the capping efficiency, and has little or no effect on integrity and yield. In these data, 4 Mg bolus feeds are used to reach the total concentration (e.g., for conditions starting with 36mM Mg, 4 feeds of 3.5mM each are added at equally spaced time points during IVT to reach a total concentration of 50mM, and similarly, starting from 25mM and ending with 60mM leads to 4 feeds, each 8.75mM). Fig.38 The dashed line in shows the general trend.

[0547] Example 59: Effects of IVT incubation time, capping molecule concentration, and GTP concentration on IVT performance

[0548] The purpose of this experiment was to investigate the effects of capping molecule concentration, final GTP concentration, and increased IVT time on yield, integrity, and 5' capping in an NTP-enhanced fed-batch process. In this example, an automated microbial reactor system was used, and method FB V1.2 was used as the baseline method (details are provided in Table 65). The modifications to each condition in method FBV1.2 are summarized in Table 64 below. As shown in Table 64, an increase in yield was observed at an IVT time of 180 minutes compared to the same conditions run at 150 minutes. Additionally, when targeting a final GTP concentration of 16 mM, the yield increased to above 16 g / L compared to 13.5 mM. No effect on integrity was observed at 180 minutes compared to 150 minutes. Additionally, no significant effect on 5' capping was observed between 150 and 180 minutes. A dose-dependent relationship was observed between the percentage of capped RNA and the concentration of the capping molecule, with 4 mM 5' capping analog producing the highest percentage of capped RNA. Overall, these data demonstrate the success of NTP fortification and the importance of IVT component feeding strategies (e.g., Mg) to enhance RNA quality attributes and yield within the target IVT time of 180 minutes. This method can therefore be modified (e.g., modifying the total IVT incubation time) to accommodate a range of RNA construct sizes.

[0549] Table 64: Effects of capping molecule concentration, final GTP and total IVT time on yield, integrity and 5' capping

[0550]

[0551]

[0552] Example 60: Application of IVT Platform Fed-Batch Method

[0553] In this example, method FB V2 was used to demonstrate the general applicability, method robustness, and successful application of this fed-batch method in various RNA constructs in different backgrounds. The specific details of this FB V2 method are listed in Table 65. Fig.39 As shown, all seven RNA constructs exhibited significantly increased yield (>~16 g / L), RNA integrity (>~80%), and capping efficiency (>93%) in FB V2 compared to their BV1 counterparts.

[0554] Table 65: Fed-Batch IVT Method Parameters

[0555]

[0556]

[0557] Similarly, method FB V2 was tested in several constructs with different coding sequence lengths. As construct length increases, it is speculated that there will be more opportunities for premature termination of T7 transcription and mRNA degradation. Therefore, for long constructs, it is of interest to determine whether the mRNA generated by FB V2 has acceptable integrity. For all constructs up to 6 kb, the integrity was above 80%, as shown in Table 66 below. This suggests that FB V2 is a suitable platform method for a wide range of constructs.

[0558] Table 66: Effect of construct length in Method FB V2

[0559]

[0560] Example 61: Effect of Residual Nucleotides on Residual DNA Impurities

[0561] In this example, the effect of enhancing NTPs on residual DNA during IVT batch feeding was determined. FB V1 was adjusted with 0.05U / mL pyrophosphatase and 150RPM to produce a reaction with high residual DNA. The studies were performed using an automated bioreactor system. Tables 67-69 show the effect of adjusting NTPs on the adjusted FB V1. When ATP levels were increased from 12-22mM, the level of residual DNA showed a downward trend. When CTP and pUTP levels were increased to 12-22mM and 9-15mM, respectively, further reductions in residual DNA were shown. The results of this experiment indicate that higher residual levels of at least one NTP can be used to improve residual DNA impurities.

[0562] Table 67: Effect of residual ATP on product quality

[0563]

[0564] Table 68: Effect of residual CTP on product quality

[0565]

[0566]

[0567] Table 69: Effect of residual pUTP on product quality

[0568]

[0569] Example 62: Oligo(dT) mRNA Affinity Studies

[0570] To further improve the integrity of mRNA, POROS TM Oligo(dT)25(THERMO ) and oligo(dT)18 monolithic column (SARTORIUSBIA ) and an affinity chromatography step for purified mRNA. Both steps bound mRNA in 10 mM Tris / 500 mM KCl / 0.1 mM EDTA, pH 7.2 and eluted with 10 mM Tris / 0.1 mM EDTA, pH 7.2. As shown in Table 70 below, mRNA integrity increased from 61% for non-affinity purified material to 79% when purified by the affinity step.

[0571] Table 70: mRNA affinity chromatography

[0572]

[0573] The mRNA affinity chromatography step was further developed by evaluating a range of buffer compositions (e.g., NaPi, Tris, HEPES, KCl, NaCl, EDTA, etc.) at various salt concentrations to optimize integrity improvement and overall recovery. TM Oligo(dT)25(THERMO ) and oligo(dT)18 monolithic column (SARTORIUSBIA ) Treatment between columns produced similar integrity improvements for buffers containing HEPES, Tris, NaPi, KCl, and NaCl buffers.

[0574] Table 71: Effect of buffer composition on mRNA integrity and recovery

[0575]

[0576]

[0577] In order to further improve the integrity and determine the removal of impurities related to the method (e.g., fragments, residual DNA, LMS, etc.), a washing study using a series of salts (e.g., NaCl, KCl, etc.), denaturants (urea, arginine, etc.) and chelating agents (e.g., EDTA, citrate, etc.) was explored by high-throughput screening on a 96-well plate containing oligo (dT) 25 resin using a 2000 nucleotide modRNA. The oligo (dT) resin was filled with 10mM Tris / 500mM KCl / 0.1mM EDTA, pH 7.2. The mRNA was bound to the column and washed with 10mM Tris / 500mM KCl / 0.1mM EDTA, pH 7.2. The resin was removed from the column and transferred to a 96-well plate by an automatic liquid handling system. Each well was washed with a Tris buffer having a pH of 7.2, the buffer containing the following range of salts, as shown in Table 72 below.

[0578] Table 72: Range of tested washing solutions

[0579]

[0580] Each condition was eluted using 10 mM Tris, 1 mM EDTA, pH 7.2. The high yield elution pool was analyzed for residual DNA. The conditions with high residual DNA log reduction values ​​(LRV) were analyzed for mRNA integrity by fragment analyzer, as shown in Table 73 below:

[0581] Table 73: Residual DNA analysis and mRNA integrity

[0582]

[0583] From the plate screening, it was determined that KCl, NaCl, arginine, and urea provided sufficient residual DNA removal, maintained or improved mRNA integrity, and reduced DS-LMS in some cases. These conditions were scaled to RoboColumns using the wash solution ranges shown in Table 74. The results of residual DNA analysis and mRNA integrity are shown in Table 75.

[0584] Table 74: Range of wash solutions tested in the RoboColumn

[0585]

[0586] Table 75: Residual DNA analysis and mRNA integrity in RoboColumns

[0587]

[0588]

[0589] The conditions that resulted in a greater reduction in residual DNA were scaled up to resin and monolithic columns for mRNAs of 2000 nucleotides (as shown in Table 76) and 10000 nucleotides (as shown in Table 77). DS-LMS was observed to reduce, and integrity was maintained or improved across a variety of conditions. NaCl and KCl were observed to have similar performance, and only KCl was continued to be used in research. From the plate screening, EDTA demonstrated a potential improvement in the method, and therefore the EDTA design branch was added to the experiment.

[0590] Table 76: Residual DNA and mRNA Analysis of 2000 nucleotide mRNA

[0591]

[0592] Table 77: Residual DNA and mRNA Analysis of 10,000 nucleotide mRNA

[0593]

[0594]

[0595] For the 2000 nucleotide mRNA, a reduction of 2 log reduction value (LRV) was observed under all conditions. For the 10,000 nucleotide mRNA, an LRV of 1.3-1.5 was observed under all conditions. Although the reduction in LRV calculations was less for the 10,000 nucleotide mRNA, the absolute level of residual DNA in the elution pool was the same, regardless of mRNA length. The lower LRV may be a product of the significantly lower residual DNA in the 10,000 nucleotide load. The lower residual DNA in the load is inherent to the IVT method, which utilizes less DNA template in the 2000 nucleotide method. For the 2000 nucleotide mRNA, some DS-LMS reduction was observed under the 1000 mM urea condition. Across the mRNA length, the 1000 mM urea condition for both the resin and the monolithic column resulted in one of the highest integrity improvements. Generally, for the 10,000 nucleotide mRNA, similar integrity improvements were observed between the monolithic column and the resin. Condition A resulted in samples that could not return mRNA integrity data. Condition G was added to the resin arm of the experiment to compare the NaPi and NaCl to the Tris and KCl methods in terms of integrity improvement. Results showed equivalent integrity improvement between the conditions.

[0596] Double-stranded RNA (dsRNA) was measured in the loading and elution pools of 10,000 nucleotides of mRNA for Condition AG, as shown in Table 78 below. Some reduction was observed, but not significantly. It is well known that dsRNA from the IVT mixture consists of truncated / failed species or turnover transcripts that form complementary base pairs with other mRNAs. In both cases, the dsRNA may have a poly (A) tail and therefore still bind. The slight reduction under different conditions, as measured by the method, suggests that removal of non-poly (A) tail impurities also reduces some dsRNA. The further reduction observed under urea + EDTA conditions suggests that disruption of hydrogen bonds between failed / truncated species and complementary mRNA may be the key. For double-stranded nucleic acids in oligonucleotides, the relationship between temperature and hydrogen bonding is well known, specifically, as temperature increases, hydrogen bonds between complementary base pairs of double-stranded species weaken, resulting in conversion to single-stranded species. Future experiments anticipate that optimizing the wash steps for buffer composition and temperature will result in removal of double-stranded species while retaining the target mRNA.

[0597] Table 78: Presence of dsRNA

[0598]

[0599] Targeting POROS TM Oligo(dT)25(THERMO ) and oligo(dT)18 monolithic column (SARTORIUSBIA ) for mRNAs ranging in length from 2000 to 10,000 nucleotides showed that a buffer composed of a combination of Tris, HEPES, NaPi, KCl, NaCl, urea, arginine, and EDTA could improve integrity and / or remove residual DNA.

[0600] The examples and embodiments described herein are for illustrative purposes only, and various modifications or changes suggested by those skilled in the art should be included within the spirit and scope of this application and the scope of the appended claims.

Claims

1. A method for producing an mRNA molecule, comprising: (a) obtaining a composition comprising: a. Circular double-stranded DNA (dsDNA) template, b. Primers, c. deoxyribonucleoside triphosphates (dNTPs), and d. DNA polymerase; (b) incubating the composition at a temperature between about 30° C. and 45° C. for a period of time between about 3 and 30 hours to obtain an incubated composition; as well as (c) contacting the incubated composition with an in vitro transcription reaction system comprising an RNA polymerase and ribonucleotides under conditions sufficient for in vitro transcription to produce mRNA molecules, wherein the contacting occurs for about 120 minutes to about 260 minutes, wherein the contacting occurs at a temperature between 25°C and 45°C, The in vitro transcription reaction system further comprises a buffer and magnesium ions, and The in vitro transcription reaction system lacks polyamines.

2. The method of claim 1, further comprising contacting the incubated composition with a restriction endonuclease to obtain a digested composition before contacting the incubated composition with an in vitro transcription reaction system.

3. The method of claim 2, wherein the contacting of the incubated composition with the restriction endonuclease occurs in the same reaction vessel in which the composition is incubated.

4. The method of any one of claims 1 to 3, wherein the contacting of the incubated composition with the in vitro transcription reaction system occurs in the same reaction vessel in which the composition is incubated.

5. The method of any one of claims 1 to 4, wherein the method does not comprise a heat denaturation reaction prior to incubating the composition.

6. The method of any one of claims 1 to 5, wherein the method does not include a heat inactivation reaction prior to performing the in vitro transcription reaction.

7. The method of any one of claims 1 to 6, further comprising: (d) capturing mRNA molecules using a capture method selected from the group consisting of oligo(dT) magnetic beads, resins, and monolithic columns.

8. The method of any one of claims 1-7, wherein the time period is between about three and eight hours.

9. The method of any one of claims 1-7, wherein the time period is between about eighteen and twenty-four hours.

10. The method of any one of claims 1-9, wherein the DNA polymerase is phi29 DNA polymerase.

11. The method of any one of claims 1-10, wherein the contacting further comprises supplementing the in vitro transcription reaction system with ribonucleotides.

12. The method of claim 11, wherein the replenishment comprises a continuous feed of ribonucleotides.

13. The method of claim 11, wherein the replenishment comprises a semi-continuous feed of ribonucleotides.

14. The method of claim 11, wherein the supplementation comprises bolus injection of feed ribonucleotides.

15. The method of any one of claims 1-14, wherein the contacting further comprises supplementing the in vitro transcription reaction system with magnesium ions.

16. The method of claim 15, wherein the supplementation is selected from the group consisting of continuous feeding, semi-continuous feeding, and bolus feeding of magnesium ions.

17. The method of any one of claims 1-17, wherein the contacting further comprises stirring.

18. The method of claim 17, wherein the stirring is selected from the range of about 1.3 to about 71.7 W / m 3 , a power / volume between about 1.3 and about 12.1 seconds, and an impeller tip speed between about 0.1 and about 0.4 m / s.

19. The method of any one of claims 1 to 18, wherein the in vitro transcription reaction system further comprises pyrophosphatase.

20. The method of any one of claims 1 to 19, wherein the in vitro transcription reaction system further comprises a ribonuclease inhibitor.

21. The method of any one of claims 1 to 20, wherein the in vitro transcription reaction system further comprises acetate ions.

22. The method of any one of claims 1-21, wherein the buffer is selected from the group consisting of TRIS and HEPES.

23. The method of any one of claims 1-22, wherein the ribonucleotides are present in an amount between about 16 and about 50 mM.

24. The method of any one of claims 1-23, wherein the RNA polymerase is present in an amount between about 4000 and about 12000 U / mL.

25. The method of claim 19, wherein the pyrophosphatase is present in an amount between about 0.25 and about 8.0 U / mL.

26. The method of any one of claims 1-25, wherein the buffer comprises a starting pH between about 7.5 and about 8.

5.

27. The method of any one of claims 1-26, wherein the magnesium ion is present in an amount between about 12.8 and about 110 mM.

28. The method of any one of claims 1-27, wherein the magnesium ions and ribonucleotides are present in a magnesium:ribonucleotide ratio of about 0.8 to about 2.2 mM Mg / mM NTP.

29. The method of any one of claims 1-28, wherein the in vitro transcription reaction system lacks a reducing agent.

30. The method of claim 29, wherein the reducing agent is dithiothreitol (DTT).

31. A method for producing an mRNA molecule, comprising: (a) obtaining a composition comprising: a. Multiple linear double-stranded DNA (dsDNA) fragments, b.5' exonuclease, c. DNA polymerase, and d. DNA ligase; (b) incubating the composition at a temperature between about 45° C. and 55° C. to obtain a circularized dsDNA template; and (c) contacting the circularized dsDNA template with a rolling circle amplification (RCA) reaction mixture, the rolling circle amplification reaction mixture comprising: a. Primer or primase, b. deoxyribonucleoside triphosphates (dNTPs), and c.DNA polymerase To obtain a template for amplification; (d) contacting the amplified template with a restriction enzyme to obtain a digested template; as well as (e) contacting the digested template with an in vitro transcription reaction system comprising an RNA polymerase and ribonucleotides under conditions sufficient for in vitro transcription to produce mRNA molecules, wherein the contacting occurs for about 120 minutes to about 260 minutes, wherein the contacting occurs at a temperature between 25°C and 45°C, The in vitro transcription reaction system further comprises a buffer and magnesium ions, and wherein steps (a) to (e) are performed sequentially in a single reaction vessel.

32. The method of claim 31, wherein the circularized dsDNA template is present at a concentration of at least 0.5 ng / mL RCA reaction mixture.

33. The method of claim 31, wherein the plurality of linear dsDNA fragments comprises a gene of interest, a promoter sequence, a 5'UTR, a 3'UTR, and a poly A sequence.

34. The method of claim 33, wherein the gene of interest encodes an RNA molecule between 1.0 kb and 12.0 kb.

35. The method of claim 33, wherein the gene of interest lacks homopolymer sequences greater than 5 base pairs.

36. The method of claim 31, wherein the mRNA molecule is capped after in vitro transcription.

37. The method of claim 36, wherein the mRNA molecule is enzymatically capped by a vaccinia virus capping enzyme.

38. The method of claim 37, wherein the mRNA molecules are incubated at 30°C or less for 60 minutes or less prior to capping by a vaccinia virus capping enzyme.

39. The method of claim 31, wherein the contacting of the digested template with the in vitro transcription reaction system further comprises supplementing the in vitro transcription reaction system with ribonucleotides, wherein the supplementing comprises increasing the concentration of a single ribonucleotide from an initial amount to a final amount.

40. The method of claim 39, wherein the supplementation comprises a method selected from the group consisting of continuous feeding of ribonucleotides, semi-continuous feeding of ribonucleotides, and bolus feeding of ribonucleotides.

41. The method of claim 39, wherein the initial amount is between 1 mM and 11 mM and the final amount is between 11 mM and 26 mM.

42. The method of claim 41, wherein the initial amount of ATP is about 11 mM, the initial amount of CTP is about 9 mM, the initial amount of GTP is about 1 mM, and the initial amount of pUTP is about 4 mM.

43. The method of claim 42, wherein the final amount of ATP is about 24 mM, the final amount of CTP is about 22 mM, the final amount of GTP is about 16 mM, and the final amount of pUTP is about 13 mM.

44. The method of claim 43, wherein the supplementing further comprises bolus injection of feed nucleotides every five minutes in the in vitro transcription reaction.

45. The method of claim 31, wherein the contacting of the digested template with the in vitro transcription reaction system further comprises supplementing the in vitro transcription reaction system with magnesium ions, wherein the supplementing comprises increasing the concentration of magnesium ions from an initial amount to a final amount.

46. ​​The method of claim 45, wherein the supplementation comprises a method selected from the group consisting of continuous feeding of magnesium ions, semi-continuous feeding of magnesium ions, and bolus feeding of magnesium ions.

47. The method of claim 46, wherein supplementing the in vitro transcription reaction system with magnesium ions further comprises adding magnesium ions at least four times during the in vitro transcription reaction.

48. The method of claim 45, wherein the initial amount of magnesium ions is about 25 mM and the final amount of magnesium ions is about 60 mM.

49. The method of claim 48, wherein the supplementation further comprises bolus feeding of magnesium ions every 15 minutes for the first 60 minutes of the in vitro transcription reaction.

50. The method of any one of claims 31-49, further comprising: (f) capturing mRNA molecules using a capture method selected from the group consisting of oligo(dT) magnetic beads, resins, and monolithic columns.

51. The method of claim 50, further comprising washing the capture process with a wash solution after capturing the mRNA.

52. The method of claim 51, wherein the wash solution comprises a buffer selected from the group consisting of Tris, HEPES, NaPi, KCl, NaCl, urea, arginine, and EDTA.

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