Optimized tailing of messenger RNA

Abstract

The present invention relates to a method for tailing in vitro transcribed (IVT) messenger RNA (mRNA) containing modified ribonucleotides, the method comprising adding the IVT mRNA to a tailing polymerase in a reaction buffer containing up to 30 mM of an alkali metal salt and at least 5 mM of a reducing agent.

Description

[Technical Field]

[0001] The present invention relates generally to methods and compositions for improving the tailing efficiency of in vitro transcribed (IVT) messenger RNA (mRNA) containing modified ribonucleotides. In particular, the present invention relates to methods and compositions that reduce the percentage of untailed IVT mRNA when a tailing polymerase is used to separately add a tail to the 3' end after in vitro synthesis of the mRNA. [Background technology]

[0002] Messenger RNA (mRNA) as a therapeutic agent is becoming increasingly important. mRNA therapy can restore normal levels of endogenous proteins or provide exogenous therapeutic proteins without permanently altering the genome sequence or entering the cell's nucleus. mRNA therapy utilizes the cell's own protein production and processing machinery to express therapeutic peptides, polypeptides, or proteins, is flexible for individualized dosing and formulation, and is broadly applicable to any disease or condition treatable through the delivery of exogenous proteins.

[0003] The expression level of a protein encoded by an mRNA can significantly affect the efficacy and therapeutic effect of mRNA therapy. Effective expression or production of a protein from an mRNA in a cell and the stability of the mRNA itself depend on various factors, including the presence of a cap at the 5' end and a tail of appropriate size at the 3' end of the mRNA.

[0004] The process of producing mRNA for use in therapy typically involves in vitro transcription of mRNA from a DNA template. Capping and tailing can occur co-transcriptionally. Alternatively, in vitro transcribed (IVT) mRNA can be separately capped and / or tailed in subsequent enzymatic reactions.

[0005] Methods and compositions for tailing IVT mRNA have been previously developed. For example, International Publication No. 2021 / 163134 describes a method for purifying high-quality mRNA suitable for clinical use. The disclosed method involves capping and tailing mRNA in a reaction buffer having a pH of less than 8.0 and a MgCl concentration of less than 1.25 mM. International Publication No. 2006 / 029350 describes methods and compositions that allow tagging and amplification of target RNA molecules. These methods may involve enzymatic addition of a polyA tail in a separate reaction. Summary of the Invention [Problem to be solved by the invention]

[0006] In particular, it remains difficult to achieve high tailing efficiency for IVT mRNA containing modified ribonucleotides using existing methods. Therefore, there is a need for methods and compositions that can increase the tailing efficiency of IVT mRNA. [Means for solving the problem]

[0007] The present invention relates to a method for tailing in vitro transcribed (IVT) mRNA containing modified ribonucleotides. The present invention is based on the discovery that adjusting tailing conditions can significantly improve tailing efficiency. In particular, the inventors have surprisingly discovered that using a reaction buffer containing 30 mM or less (e.g., 5 mM or less) of alkali metal salt and 5 mM or more of reducing agent results in higher tailing efficiency of mRNA containing modified ribonucleotides. This is particularly useful when producing mRNA for therapeutic use.

[0008] Unless otherwise specified, concentrations of reaction buffer components provided herein to describe tailing conditions (e.g., in connection with a tailing method or reaction) refer to working concentrations (i.e., 1x concentration). Those skilled in the art will understand that higher concentrations (typically 10x) are used for storage of reaction buffers, and therefore, reaction buffers prepared for storage may require dilution for use in methods or processes described herein.

[0009] The present inventors hypothesized that the presence of modified ribonucleotides, such as N1-methylpseudouridine, in mRNA may neutralize the phosphate backbone of IVT mRNA. This may result in the formation of complex secondary structures in IVT mRNA. These secondary structures may interfere with tailing polymerases approaching the 3' end of the IVT mRNA and initiating tailing. Reducing the concentration of alkali metal salts in the reaction buffer reduces its ionic strength. Without wishing to be bound by any particular theory, a lower concentration of alkali metal salts in the reaction buffer of the present invention may reduce the formation of secondary structures in IVT mRNA, resulting in increased tailing efficiency.

[0010] The inventors further hypothesized that the secondary structure of the nascent tail interferes with the activity of the tailing polymerase, resulting in a high percentage of untailed and tailed IVT mRNAs with longer tails than desired. Without wishing to be bound by any particular theory, the addition of a reducing agent may reduce disulfide bonds in the tailing polymerase, potentially resulting in a conformational change that allows the enzyme to handle the more complex secondary structure formed by the nascent tail, further increasing tailing efficiency and resulting in a majority of IVT mRNAs with the desired tail length.

[0011] In one aspect, the invention relates to a method for tailing in vitro transcribed (IVT) mRNA containing modified ribonucleotides, the method comprising adding the IVT mRNA to a tailing polymerase in a reaction buffer comprising 30 mM or less of an alkali metal salt and 5 mM or more of a reducing agent. In some embodiments, the IVT mRNA comprises a 5' cap. In some embodiments, the 5' cap is added in a separate reaction comprising a reaction buffer different from the reaction buffer used to tail the IVT mRNA.

[0012] In some embodiments, the alkali metal salt in the reaction buffer has a concentration of about 1 mM to about 30 mM. In some embodiments, the alkali metal salt in the reaction buffer has a concentration of about 5 mM or less. In some embodiments, the alkali metal salt in the reaction buffer has a concentration of about 1 mM to about 5 mM.

[0013] In some embodiments, the reducing agent in the reaction buffer has a concentration of 5 mM to 50 mM. In some embodiments, the reducing agent in the reaction buffer has a concentration of 5 mM to 20 mM. In certain embodiments, the reducing agent in the reaction buffer has a concentration of about 10 mM.

[0014] In some embodiments, the reducing agent is selected from dithiothreitol (DTT), 2-mercaptoethanol (2-ME), and tris(2-carboxyethyl)phosphine (TCEP). In certain embodiments, the reducing agent is dithiothreitol (DTT).

[0015] In some embodiments, the alkali metal salt is NaCl or KCl, hi particular embodiments, the alkali metal salt is NaCl.

[0016] In some embodiments, the modified ribonucleotide is selected from pseudouridine, N1-methylpseudouridine, 5-methylcytidine, and 5-methoxyuridine. In some embodiments, the modified ribonucleotide is a modified uridine. In some embodiments, the modified uridine is N1-methylpseudouridine.

[0017] In some embodiments, at least 93% of the IVT mRNA is tailed using a tailing method according to the present invention. In some embodiments, at least 94% of the IVT mRNA is tailed. In some embodiments, at least 95% of the IVT mRNA is tailed. In some embodiments, at least 96% of the IVT mRNA is tailed. In some embodiments, at least 97% of the IVT mRNA is tailed. In some embodiments, at least 98% of the IVT mRNA is tailed. In some embodiments, at least 99% of the IVT mRNA is tailed. In some embodiments, 100% of the IVT mRNA is tailed.

[0018] In some embodiments, the mRNA tail comprises about 100 to about 800 ribonucleotides. In some embodiments, the mRNA tail comprises about 100 to about 500 ribonucleotides. In some embodiments, the mRNA tail comprises about 100 to about 250 ribonucleotides. In some embodiments, the mRNA tail comprises about 100 or about 200 ribonucleotides.

[0019] In some embodiments, the reaction buffer maintains a pH of about pH 7 to about pH 8. In certain embodiments, the reaction buffer maintains a pH of about pH 7.5.

[0020] In some embodiments, the reaction buffer maintains pH with a buffering reagent. In some embodiments, the buffering reagent is selected from Tris, HEPES, MOPS, acetate, citrate, and phosphate. In some embodiments, the buffering reagent is present at a concentration of about 5 mM to about 100 mM. In some embodiments, the buffering reagent is present at a concentration of about 10 mM to about 50 mM. In certain embodiments, the buffering reagent is present at a concentration of about 50 mM.

[0021] In some embodiments, the reaction buffer comprises a divalent cation. In some embodiments, the divalent cation is Mg 2+ and Mn 2+ In some embodiments, the divalent cation is present at a concentration of about 5 mM to about 20 mM. In certain embodiments, the divalent cation is at a concentration of about 5 mM to about 10 mM. In certain embodiments, the divalent cation is at a concentration of about 10 mM.

[0022] In some embodiments, the IVT mRNA does not contain modified ribonucleotides at the 3' end. In some embodiments, the IVT mRNA does not contain modified uridines at the 3' end. In certain embodiments, the IVT mRNA does not contain N1-methylpseudouridine at the 3' end.

[0023] In some embodiments, the tailing polymerase is a polyA polymerase. In some embodiments, the polyA polymerase is a bacterial polyA polymerase or a yeast polyA polymerase. In certain embodiments, the polyA polymerase is an E. coli polyA polymerase.

[0024] In some embodiments, the reaction buffer contains a suitable concentration of ATP, hi some embodiments, the ATP is present at a concentration of about 0.1 mM to about 10 mM.

[0025] In a further aspect, the present invention relates to compositions comprising tailed IVT mRNA obtainable by the tailing method of the present invention. These compositions are characterized by low or undetectable amounts of untailed IVT mRNA and a narrow size distribution of the tailed IVT mRNA. For example, in some embodiments, compositions comprising tailed IVT mRNA obtainable by the tailing method of the present invention contain less than 5% (e.g., 2% or less) untailed IVT mRNA as determined by the area under the various curves in a capillary gel electropherogram. Furthermore, the average tail length is typically close to the desired tail length, e.g., within about 25%, about 20%, about 15%, about 10%, or about 5% of the desired tail length (e.g., as determined by capillary gel electrophoresis).

[0026] In a further aspect, the present invention relates to a reaction buffer for use in a method for tailing in vitro transcribed (IVT) mRNA, comprising 30 mM or less of an alkali metal salt and 5 mM or more of a reducing agent. The reaction buffer is optimized for tailing IVT mRNA containing modified ribonucleotides. The inventors have found that this reaction buffer can also be used with IVT mRNA composed solely of unmodified ribonucleotides.

[0027] In some embodiments, the alkali metal salt in the reaction buffer has a concentration of about 1 mM to about 30 mM. In some embodiments, the alkali metal salt in the reaction buffer has a concentration of about 5 mM or less. In some embodiments, the alkali metal salt in the reaction buffer has a concentration of about 1 mM to about 5 mM.

[0028] In some embodiments, the reducing agent in the reaction buffer has a concentration of 5 mM to 50 mM. In some embodiments, the reducing agent in the reaction buffer has a concentration of 5 mM to 20 mM. In certain embodiments, the reducing agent in the reaction buffer has a concentration of about 10 mM.

[0029] In some embodiments, the reducing agent is selected from dithiothreitol (DTT), 2-mercaptoethanol (2-ME), and tris(2-carboxyethyl)phosphine (TCEP). In certain embodiments, the reducing agent is dithiothreitol (DTT).

[0030] In some embodiments, the alkali metal salt in the reaction buffer is NaCl or KCl, hi certain embodiments, the alkali metal salt is NaCl.

[0031] In some embodiments, the reaction buffer has a pH of about pH 7 to about pH 8. In certain embodiments, the reaction buffer has a pH of about pH 7.5.

[0032] In some embodiments, the reaction buffer comprises Tris, HEPES, MOPS, acetate, citrate, or phosphate as a buffering reagent. In some embodiments, the buffering reagent is present at a concentration of about 5 mM to about 100 mM. In some embodiments, the buffering reagent is present at a concentration of about 10 mM to about 50 mM. In certain embodiments, the buffering reagent is present at a concentration of about 50 mM.

[0033] In some embodiments, the reaction buffer comprises a divalent cation. In some embodiments, the divalent cation is Mg 2+ and Mn 2+ In some embodiments, the reaction buffer comprises MgCl or MnCl.

[0034] In some embodiments, the divalent cations in the reaction buffer are present at a concentration of about 5 mM to about 20 mM. In certain embodiments, the divalent cations are at a concentration of about 10 mM.

[0035] In a further aspect, the present invention also relates to a composition comprising in vitro transcribed (IVT) mRNA in a reaction buffer as defined in the preceding paragraph.

[0036] In yet another aspect, the present invention relates to a reaction buffer for use in a method for tailing in vitro transcribed (IVT) mRNA, comprising 300 mM or less of an alkali metal salt and 50 mM or more of a reducing agent, the reaction buffer being suitable for transport and storage and diluted 10-fold prior to use.

[0037] In some embodiments, the alkali metal salt in the reaction buffer has a concentration of about 50 mM or less.

[0038] In some embodiments, the reducing agent in the reaction buffer has a concentration of 50 mM to 500 mM. In some embodiments, the reducing agent in the reaction buffer has a concentration of 50 mM to 200 mM. In certain embodiments, the reducing agent in the reaction buffer has a concentration of about 100 mM.

[0039] In some embodiments, the reducing agent is selected from dithiothreitol (DTT), 2-mercaptoethanol (2-ME), and tris(2-carboxyethyl)phosphine (TCEP). In certain embodiments, the reducing agent is dithiothreitol (DTT).

[0040] In some embodiments, the alkali metal salt is NaCl or KCl, hi particular embodiments, the alkali metal salt is NaCl.

[0041] In some embodiments, the reaction buffer has a pH of about pH 7 to about pH 8. In certain embodiments, the reaction buffer has a pH of about pH 7.5.

[0042] In some embodiments, the reaction buffer comprises Tris, HEPES, MOPS, acetate, citrate, or phosphate as a buffering reagent. In some embodiments, the buffering reagent is present at a concentration of about 50 mM to about 1000 mM. In some embodiments, the buffering reagent is present at a concentration of about 100 mM to about 500 mM. In certain embodiments, the buffering reagent is present at a concentration of about 500 mM.

[0043] In some embodiments, the reaction buffer comprises a divalent cation. In some embodiments, the divalent cation is Mg 2+ and Mn 2+ In some embodiments, the divalent cation comprises MgCl or MnCl.

[0044] In some embodiments, the divalent cations are present at a concentration of about 50 mM to about 200 mM, hi certain embodiments, the divalent cations are at a concentration of about 100 mM.

[0045] The inventors have surprisingly found that tailing IVT mRNA comprising modified ribonucleotides can be improved by providing a non-tailed IVT mRNA that does not comprise modified ribonucleotides at the 3' end. Thus, in a further aspect, the present invention provides a method for producing in vitro transcribed (IVT) mRNA comprising modified ribonucleotides, the method comprising: (i) preparing a DNA template, wherein the terminal 3' residue of the DNA template does not encode a modified ribonucleotide of an IVT mRNA; (ii) transcribing a DNA template using an RNA polymerase in an in vitro transcription (IVT) reaction containing modified ribonucleotides; The present invention relates to a method comprising:

[0046] In some embodiments, the DNA template is a circular vector containing a restriction site. In some embodiments, step (i) comprises cleaving the circular vector at the restriction site to obtain the terminal 3' residue of the DNA template that does not encode the modified ribonucleotide of the IVT mRNA. In some embodiments, the restriction site is cleaved by BspQI.

[0047] In some embodiments, the method for producing IVT mRNA further comprises tailing the IVT mRNA. In some embodiments, the tailing step comprises adding the IVT mRNA to a tailing polymerase in a reaction buffer comprising 30 mM or less of an alkali metal salt (e.g., NaCl) and 5 mM or more of a reducing agent (e.g., DTT). In some embodiments, the alkali metal salt in the reaction buffer has a concentration of 5 mM or less.

[0048] In a further aspect, the present invention provides a method for tailing in vitro transcribed (IVT) mRNA comprising modified ribonucleotides, the method comprising: (i) providing an untailed IVT mRNA that does not contain modified ribonucleotides at the 3' end; (ii) adding reaction buffer and tailing polymerase; The present invention relates to a method comprising:

[0049] In some embodiments, the reaction buffer comprises 30 mM or less of an alkali metal salt (e.g., NaCl) and 5 mM or more of a reducing agent (e.g., DTT). In certain embodiments, the alkali metal salt in the reaction buffer has a concentration of about 5 mM or less.

[0050] In some embodiments, the reducing agent in the reaction buffer has a concentration of 5 mM to 50 mM. In some embodiments, the reducing agent in the reaction buffer has a concentration of 5 mM to 20 mM. In certain embodiments, the reducing agent in the reaction buffer has a concentration of about 10 mM.

[0051] In some embodiments, the reaction buffer contains a divalent cation (e.g., Mg 2+ In some embodiments, the divalent cations are present at a concentration of about 5 mM to about 20 mM. In particular embodiments, the divalent cations are at a concentration of about 10 mM.

[0052] Other features, objects, and advantages of the present invention will be apparent from the following detailed description, drawings, and embodiments. It should be understood, however, that the detailed description, drawings, and embodiments, while indicating embodiments of the present invention, are given by way of illustration only, not limitation. Various changes and modifications will be apparent to those skilled in the art.

[0053] Embodiments of the invention will now be described, by way of example only, with reference to the following drawings, in which: [Brief explanation of the drawings]

[0054] [Figure 1A-1B]We show that a reaction buffer containing 30 mM or less of alkali metal salt and 5 mM or less of reducing agent improves tailing efficiency and significantly reduces the percentage of untailed IVT mRNA containing modified ribonucleotides. Figures 1-3 show the separation of IVT mRNA A (Figure 1), mRNA B (Figure 2), and mRNA C (Figure 3) by capillary electrophoresis. The x-axis shows the length of each IVT mRNA as a number of ribonucleotides. The y-axis shows relative fluorescence units (RFU). In panel A of Figures 1-3, IVT mRNA was tailed in a reaction buffer containing 250 mM alkali metal salt and no reducing agent (control buffer in Table 2). In panel B of Figures 1-3, IVT mRNA was tailed in a reaction buffer containing 5 mM alkali metal salt and no reducing agent (buffer 8 in Table 2). In panel C, IVT mRNA was tailed in a reaction buffer containing 5 mM alkali metal salt and 10 mM reducing agent (buffer 8 in Table 2, containing 10 mM DTT). Use of a control buffer resulted in a clearly identifiable additional peak preceding the main peak (indicated by an arrow), indicating a significant amount of untailed IVT mRNA (see panel A in Figures 1-3). Reducing the alkali metal salt concentration significantly reduced the size (see arrow in panel B in Figures 1-3) and average tail length of the additional peak preceding the main peak, as the main peak shifted to the left in panel B compared to the main peak in panel A. When reducing agent was further added to the buffer containing the reduced alkali metal salt concentration, the additional peak was no longer discernible, and the average tail length further decreased (see panel C in Figures 1-3). Indeed, the main peak shifted to the left in panel C compared to the main peak in panels A and B. [Figure 1C]We show that a reaction buffer containing 30 mM or less of alkali metal salt and 5 mM or less of reducing agent improves tailing efficiency and significantly reduces the percentage of untailed IVT mRNA containing modified ribonucleotides. Figures 1-3 show the separation of IVT mRNA A (Figure 1), mRNA B (Figure 2), and mRNA C (Figure 3) by capillary electrophoresis. The x-axis shows the length of each IVT mRNA as a number of ribonucleotides. The y-axis shows relative fluorescence units (RFU). In panel A of Figures 1-3, IVT mRNA was tailed in a reaction buffer containing 250 mM alkali metal salt and no reducing agent (control buffer in Table 2). In panel B of Figures 1-3, IVT mRNA was tailed in a reaction buffer containing 5 mM alkali metal salt and no reducing agent (buffer 8 in Table 2). In panel C, IVT mRNA was tailed in a reaction buffer containing 5 mM alkali metal salt and 10 mM reducing agent (buffer 8 in Table 2, containing 10 mM DTT). Use of a control buffer resulted in a clearly identifiable additional peak preceding the main peak (indicated by an arrow), indicating a significant amount of untailed IVT mRNA (see panel A in Figures 1-3). Reducing the alkali metal salt concentration significantly reduced the size (see arrow in panel B in Figures 1-3) and average tail length of the additional peak preceding the main peak, as the main peak shifted to the left in panel B compared to the main peak in panel A. When reducing agent was further added to the buffer containing the reduced alkali metal salt concentration, the additional peak was no longer discernible, and the average tail length further decreased (see panel C in Figures 1-3). Indeed, the main peak shifted to the left in panel C compared to the main peak in panels A and B. [Figure 2A]We show that a reaction buffer containing 30 mM or less of alkali metal salt and 5 mM or less of reducing agent improves tailing efficiency and significantly reduces the percentage of untailed IVT mRNA containing modified ribonucleotides. Figures 1-3 show the separation of IVT mRNA A (Figure 1), mRNA B (Figure 2), and mRNA C (Figure 3) by capillary electrophoresis. The x-axis shows the length of each IVT mRNA as a number of ribonucleotides. The y-axis shows relative fluorescence units (RFU). In panel A of Figures 1-3, IVT mRNA was tailed in a reaction buffer containing 250 mM alkali metal salt and no reducing agent (control buffer in Table 2). In panel B of Figures 1-3, IVT mRNA was tailed in a reaction buffer containing 5 mM alkali metal salt and no reducing agent (buffer 8 in Table 2). In panel C, IVT mRNA was tailed in a reaction buffer containing 5 mM alkali metal salt and 10 mM reducing agent (buffer 8 in Table 2, containing 10 mM DTT). Use of a control buffer resulted in a clearly identifiable additional peak preceding the main peak (indicated by an arrow), indicating a significant amount of untailed IVT mRNA (see panel A in Figures 1-3). Reducing the alkali metal salt concentration significantly reduced the size (see arrow in panel B in Figures 1-3) and average tail length of the additional peak preceding the main peak, as the main peak shifted to the left in panel B compared to the main peak in panel A. When reducing agent was further added to the buffer containing the reduced alkali metal salt concentration, the additional peak was no longer discernible, and the average tail length further decreased (see panel C in Figures 1-3). Indeed, the main peak shifted to the left in panel C compared to the main peak in panels A and B. [Figure 2B-2C]We show that a reaction buffer containing 30 mM or less of alkali metal salt and 5 mM or less of reducing agent improves tailing efficiency and significantly reduces the percentage of untailed IVT mRNA containing modified ribonucleotides. Figures 1-3 show the separation of IVT mRNA A (Figure 1), mRNA B (Figure 2), and mRNA C (Figure 3) by capillary electrophoresis. The x-axis shows the length of each IVT mRNA as a number of ribonucleotides. The y-axis shows relative fluorescence units (RFU). In panel A of Figures 1-3, IVT mRNA was tailed in a reaction buffer containing 250 mM alkali metal salt and no reducing agent (control buffer in Table 2). In panel B of Figures 1-3, IVT mRNA was tailed in a reaction buffer containing 5 mM alkali metal salt and no reducing agent (buffer 8 in Table 2). In panel C, IVT mRNA was tailed in a reaction buffer containing 5 mM alkali metal salt and 10 mM reducing agent (buffer 8 in Table 2, containing 10 mM DTT). Use of a control buffer resulted in a clearly identifiable additional peak preceding the main peak (indicated by an arrow), indicating a significant amount of untailed IVT mRNA (see panel A in Figures 1-3). Reducing the alkali metal salt concentration significantly reduced the size (see arrow in panel B in Figures 1-3) and average tail length of the additional peak preceding the main peak, as the main peak shifted to the left in panel B compared to the main peak in panel A. When reducing agent was further added to the buffer containing the reduced alkali metal salt concentration, the additional peak was no longer discernible, and the average tail length further decreased (see panel C in Figures 1-3). Indeed, the main peak shifted to the left in panel C compared to the main peak in panels A and B. [Figure 3A-3B]We show that a reaction buffer containing 30 mM or less of alkali metal salt and 5 mM or less of reducing agent improves tailing efficiency and significantly reduces the percentage of untailed IVT mRNA containing modified ribonucleotides. Figures 1-3 show the separation of IVT mRNA A (Figure 1), mRNA B (Figure 2), and mRNA C (Figure 3) by capillary electrophoresis. The x-axis shows the length of each IVT mRNA as a number of ribonucleotides. The y-axis shows relative fluorescence units (RFU). In panel A of Figures 1-3, IVT mRNA was tailed in a reaction buffer containing 250 mM alkali metal salt and no reducing agent (control buffer in Table 2). In panel B of Figures 1-3, IVT mRNA was tailed in a reaction buffer containing 5 mM alkali metal salt and no reducing agent (buffer 8 in Table 2). In panel C, IVT mRNA was tailed in a reaction buffer containing 5 mM alkali metal salt and 10 mM reducing agent (buffer 8 in Table 2, containing 10 mM DTT). Use of a control buffer resulted in a clearly identifiable additional peak preceding the main peak (indicated by an arrow), indicating a significant amount of untailed IVT mRNA (see panel A in Figures 1-3). Reducing the alkali metal salt concentration significantly reduced the size (see arrow in panel B in Figures 1-3) and average tail length of the additional peak preceding the main peak, as the main peak shifted to the left in panel B compared to the main peak in panel A. When reducing agent was further added to the buffer containing the reduced alkali metal salt concentration, the additional peak was no longer discernible, and the average tail length further decreased (see panel C in Figures 1-3). Indeed, the main peak shifted to the left in panel C compared to the main peak in panels A and B. [Figure 3C]We show that a reaction buffer containing 30 mM or less of alkali metal salt and 5 mM or less of reducing agent improves tailing efficiency and significantly reduces the percentage of untailed IVT mRNA containing modified ribonucleotides. Figures 1-3 show the separation of IVT mRNA A (Figure 1), mRNA B (Figure 2), and mRNA C (Figure 3) by capillary electrophoresis. The x-axis shows the length of each IVT mRNA as a number of ribonucleotides. The y-axis shows relative fluorescence units (RFU). In panel A of Figures 1-3, IVT mRNA was tailed in a reaction buffer containing 250 mM alkali metal salt and no reducing agent (control buffer in Table 2). In panel B of Figures 1-3, IVT mRNA was tailed in a reaction buffer containing 5 mM alkali metal salt and no reducing agent (buffer 8 in Table 2). In panel C, IVT mRNA was tailed in a reaction buffer containing 5 mM alkali metal salt and 10 mM reducing agent (buffer 8 in Table 2, containing 10 mM DTT). Use of a control buffer resulted in a clearly identifiable additional peak preceding the main peak (indicated by an arrow), indicating a significant amount of untailed IVT mRNA (see panel A in Figures 1-3). Reducing the alkali metal salt concentration significantly reduced the size (see arrow in panel B in Figures 1-3) and average tail length of the additional peak preceding the main peak, as the main peak shifted to the left in panel B compared to the main peak in panel A. When reducing agent was further added to the buffer containing the reduced alkali metal salt concentration, the additional peak was no longer discernible, and the average tail length further decreased (see panel C in Figures 1-3). Indeed, the main peak shifted to the left in panel C compared to the main peak in panels A and B. [Figure 4]We demonstrate that an optimized reaction buffer containing 5 mM or less of alkali metal salt and 5 mM or more of reducing agent improves tailing efficiency, regardless of the ribonucleotide at the 3' end of the IVT mRNA or its ribonucleotide sequence. Seven different IVT mRNAs were prepared from either a DNA template plasmid in which the backbone (I) was cleaved with HindIII (labeled "HindIII-cleaved template") or a DNA template plasmid in which the backbone (II) was cleaved with BspQI (labeled "BspQI-cleaved template"). Tailing of each IVT mRNA was performed in a reaction buffer containing 250 mM alkali metal salt but no reducing agent (labeled "Buffer C," corresponding to the control buffer provided in Table 2) or in a reaction buffer containing 5 mM alkali metal salt and 10 mM reducing agent (labeled "Buffer O," corresponding to Buffer 8 provided in Table 2 with DTT). The desired tail lengths were 500 nucleotides for IVT mRNAs 1–6 and 200 nucleotides for IVT mRNA 7. IVT-mRNA produced using Hind-III-cleaved templates had a modified ribonucleotide (N1-methylpseudouridine) at the 3' end. The bar graph shows the percentage of untailed IVT mRNA for each construct after tailing in control buffer ("C") or Buffer 8 containing DTT ("O"). For each IVT mRNA, performing the tailing reaction in Buffer 8 containing DTT improved the tailing efficiency compared to performing the tailing reaction in control buffer. In vitro transcribed mRNA from BspQI-cleaved templates had a lower percentage of untailed mRNA compared to mRNA transcribed from Hind-III-cleaved templates. [Figure 5]We demonstrate that our optimized reaction buffer can be used to tail IVT mRNA containing modified ribonucleotides on a large scale (>1 g per batch of IVT mRNA), with the resulting average tail length approaching the desired length. Using capillary gel electrophoresis, we compared ten 10 g batches of tailed IVT mRNA prepared using the non-optimized control buffer (Buffer "C") listed in Table 2 with four 16 g batches prepared using Buffer 8 (Buffer "O") containing 10 mM DTT. The desired tail length was 200 nucleotides. The average tail length achieved with Buffer "C" was 45-50% longer than the desired tail length for the majority of the batches tested (9 out of 10). In contrast, using Buffer "O," the average tail length was only approximately 5% longer than the desired tail length for three out of four tested batches. Even in the worst-performing batch, using Buffer "O," the average tail length was only approximately 25% longer than the desired tail length. The nucleic acid sequence of IVT mRNA did not affect the tailing efficiency with buffer "O." DETAILED DESCRIPTION OF THE INVENTION

[0055] definition To facilitate understanding of the present invention, certain terms are first defined below. Further definitions for these and other terms are set forth throughout the specification.

[0056] As used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. For example, "a ribonucleotide" is understood to refer to one or more ribonucleotides. Thus, the terms "a" (or "an"), "one or more," and "at least one" can be used interchangeably herein.

[0057] Unless specifically stated or clear from the context, as used herein, the term "or" is understood to be inclusive, including both "or" and "and." Furthermore, "and / or," as used herein, should be interpreted as a specific disclosure of each of the two specified features or components, with or without the other. Thus, when the term "and / or" is used herein in phrases such as "A and / or B," it is intended to include "A and B," "A or B," "A" (alone) and "B" (alone). Similarly, when the term "and / or" is used in phrases such as "A, B and / or C," it is intended to encompass each of the following embodiments: A, B and C; A, B or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0058] Whenever an embodiment is described herein with the language "comprising," it is understood that similar embodiments that are otherwise described in terms of "consisting of" and / or "consisting essentially of" are also provided.

[0059] As used herein, the term "about" refers to an interval of accuracy that a person skilled in the art would understand to still ensure the technical effect of the target feature. This term indicates a deviation of ±10% from the indicated numerical value. In some embodiments, the deviation is ±5% of the indicated numerical value. In certain embodiments, the deviation is ±1% of the indicated numerical value.

[0060] As used herein, the term "mRNA" refers to a polyribonucleotide that encodes at least one polypeptide. As used herein, mRNA encompasses both modified and unmodified RNA. mRNA may contain one or more coding and non-coding regions (e.g., 5' and 3' untranslated regions). mRNA may be purified from natural sources, produced using recombinant expression systems, optionally purified, in vitro transcribed, or chemically synthesized. The present invention particularly relates to in vitro transcribed (IVT) mRNA. Where appropriate, e.g., in the case of chemically synthesized molecules, mRNA may contain nucleoside analogs, such as analogs with chemically modified bases or sugars, backbone modifications, and the like. Unless otherwise indicated, mRNA sequences are presented in the 5' to 3' direction. A typical mRNA includes a 5' cap, a 5' untranslated region (5' UTR), a protein-coding region, a 3' untranslated region (3' UTR), and a 3' tail. In some embodiments, the tail structure is a poly(C) tail. More typically, the tail structure is a polyA tail.

[0061] As used herein, the term "sequence-optimized" refers to a nucleotide sequence that has been modified relative to a naturally occurring or wild-type nucleotide sequence. Such modifications can include, for example, codon optimization and / or the use of 5' and 3' UTRs not normally associated with that naturally occurring or wild-type nucleic acid. As used herein, the terms "codon optimization" and "codon-optimized" refer to modifications of the codon composition of a naturally occurring or wild-type nucleic acid encoding a peptide, polypeptide, or protein that do not alter its amino acid sequence, thereby improving protein expression of the nucleic acid. In the context of the present invention, "codon optimization" can also refer to the process of filtering out suboptimal nucleotide sequences from a list of nucleotide sequences, such as by guanine-cytosine content, codon adaptability index, the presence of destabilizing nucleic acid sequences or motifs, and / or the presence of pause sites and / or termination signals, thereby arriving at one or more optimized nucleotide sequences.

[0062] As used herein, the term "substantially" refers to the qualitative condition of exhibiting the entire or nearly full extent or degree of a desired characteristic or property. Those skilled in the art of biology will understand that biological and chemical events rarely, if ever, proceed to completion and / or perfection or achieve or avoid an absolute result. Thus, the term "substantially" is used herein to capture the potential lack of perfection inherent in many biological and chemical events.

[0063] As used herein, the term "template DNA" (or "DNA template") refers to a DNA molecule containing a nucleic acid sequence encoding an mRNA transcript to be synthesized by in vitro transcription. The template DNA is used as a template for in vitro transcription to produce an mRNA transcript encoded by the template DNA. The template DNA contains all elements necessary for in vitro transcription, in particular a promoter element operably linked to the DNA sequence encoding the desired mRNA transcript for binding of a DNA-dependent RNA polymerase, such as T3, T7, or SP6 RNA polymerase. Furthermore, the template DNA may contain primer binding sites 5' and / or 3' to the DNA sequence encoding the mRNA transcript, so that the identity of the DNA sequence encoding the mRNA transcript can be determined, for example, by PCR or DNA sequencing. In the context of the present invention, "template DNA" may be a linear or circular DNA molecule. As used herein, the term "template DNA" may refer to a DNA vector, such as a plasmid DNA, containing a nucleic acid sequence encoding the desired mRNA transcript.

[0064] As used herein, the term "tailing efficiency" refers to how effective a tailing polymerase is in adding a tail to IVT mRNA. Typically, an efficient tailing reaction results in IVT mRNA with an average tail length close to the desired tail length. The desired tail length correlates with the expected average tail length under optimal tail length conditions. Therefore, an increase in tailing efficiency usually results in both a lower percentage of untailed IVT mRNA and an average tail length close to the desired tail length indicated.

[0065] Unless otherwise defined herein, scientific and technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs and as commonly used in the technical field to which this application belongs. Exemplary methods and materials are described below, although methods and materials similar or equivalent to those described herein may also be used in the practice or testing of this disclosure. In case of conflict, the present specification, including definitions, will control.

[0066] Generally, the nomenclature used in connection with and techniques of cell and tissue culture, molecular biology, virology, immunology, microbiology, genetics, analytical chemistry, synthetic organic chemistry, medicinal and pharmaceutical chemistry, and protein and nucleic acid chemistry and hybridization described herein are those well known and commonly used in the art. Enzymatic reactions and purification techniques are performed according to manufacturer's specifications, as commonly accomplished in the art, or as described herein. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular.

[0067] Throughout this specification and the embodiments, the words "have" and "comprise" or variations thereof, such as "has," "having," "comprises," or "including," are understood to mean the inclusion of a stated integer or group of integers, but not the exclusion of any other integer or group of integers.

[0068] All publications and other reference materials referred to herein are hereby incorporated by reference in their entirety. Although a number of documents are cited in this specification, such citation does not constitute an admission that any of those documents forms part of the common general knowledge in the art.

[0069] The present invention relates to a method for tailing in vitro transcribed (IVT) messenger RNA (mRNA) containing modified ribonucleotides. The present invention is based on the discovery that adjusting the tailing conditions can significantly improve tailing efficiency. In particular, we found that using a reaction buffer containing 5 mM or less of an alkali metal salt and 5 mM or more of a reducing agent can achieve higher tailing efficiency, even when the IVT mRNA has terminally modified ribonucleotides at the 3' end.

[0070] The inventors have further found that tailing efficiency can be improved if the DNA template has terminal 3' residues that do not encode modified ribonucleotides of the IVT mRNA. Thus, in some aspects, the present invention provides a method for tailing in vitro transcribed (IVT) messenger RNA (mRNA) that contains modified ribonucleotides, comprising: (i) providing an untailed IVT mRNA that does not contain modified ribonucleotides at the 3' end; (ii) adding reaction buffer and tailing polymerase; The present invention also relates to a method comprising:

[0071] IVT mRNA In vitro transcription Various methods for synthesizing mRNA via in vitro transcription (IVT) are described in U.S. Patent Application Publication No. 2018 / 0258423 and International Publication No. WO 2021 / 168052 A1 (incorporated herein by reference and can be used to practice the present invention). Briefly, IVT is typically performed using a reaction mixture containing a DNA template containing a promoter, a pool of ribonucleotide triphosphates, a buffer system (which may contain DTT and magnesium ions), and a suitable RNA polymerase (e.g., T3, T7, or SP6 RNA polymerase). The DNA template is typically linearized with a suitable restriction enzyme prior to the IVT reaction. The IVT reaction can be terminated by the addition of DNase I, which digests the DNA template. The exact conditions will vary depending on the specific application.

[0072] DNA template A typical DNA template according to the present invention comprises a promoter sequence, such as a T3, T7, or SP6 promoter, followed by the nucleotide sequence of the desired mRNA, which typically includes a 5' untranslated region (5' UTR), a coding region for the polypeptide of interest, and a 3' untranslated region (3' UTR).

[0073] In some embodiments, the nucleotide sequence comprises a 5' untranslated region (5'UTR) that differs from the 5'UTR present in the naturally occurring mRNA encoding the polypeptide of interest.

[0074] In some embodiments, the nucleotide sequence comprises a 3' untranslated region (3'UTR) that differs from the 3'UTR present in the naturally occurring mRNA encoding the polypeptide of interest.

[0075] For example, suitable 5' and 3' UTRs are described in WO 2012 / 075040, which is incorporated herein by reference.

[0076] In certain embodiments, the 5' and / or 3' UTR sequences may be derived from stable mRNAs (e.g., globin, actin, GAPDH, tubulin, histones, or citric acid cycle enzymes) to enhance mRNA stability. For example, the 5' UTR sequence may include a subsequence of the CMV immediate-early 1 (IE1) gene or a fragment thereof to improve nuclease resistance and / or improve mRNA half-life. It is also contemplated to include a sequence encoding human growth hormone (hGH) or a fragment thereof in the 3' end or untranslated region of the mRNA. Exemplary 5' UTRs include sequences derived from the CMV immediate-early 1 (IE1) gene (U.S. Patent Application Publication Nos. 2014 / 0206753 and 2015 / 0157565, each of which is incorporated herein by reference) or the sequence provided in Example 1 of U.S. Patent Application Publication No. 2016 / 0151409, incorporated herein by reference.

[0077] In various embodiments, the 5'UTR can be derived from the 5'UTR of a TOP gene. TOP genes are typically characterized by the presence of a 5'-terminal oligopyrimidine (TOP) tract. Furthermore, most TOP genes are characterized by growth-related translational regulation. However, TOP genes with tissue-specific translational regulation are also known. In certain embodiments, the 5'UTR derived from the 5'UTR of a TOP gene lacks a 5'TOP motif (oligopyrimidine tract) (e.g., U.S. Patent Application Publication Nos. 2017 / 0029847, 2016 / 0304883, 2016 / 0235864, and 2016 / 0166710, each of which is incorporated herein by reference).

[0078] In certain embodiments, the 5'UTR is derived from the ribosomal protein large 32 (L32) gene (US Patent Application Publication No. 2017 / 0029847, supra).

[0079] In certain embodiments, the 5'UTR is derived from the 5'UTR of the hydroxysteroid (17-b) dehydrogenase 4 gene (HSD17B4) (US Patent Application Publication No. 2016 / 0166710, supra).

[0080] In certain embodiments, the 5'UTR is derived from the 5'UTR of the ATP5A1 gene (US Patent Application Publication No. 2016 / 0166710, supra).

[0081] In some embodiments, an internal ribosome entry site (IRES) is used in place of the 5'UTR.

[0082] IVT mRNA is typically transcribed from a DNA template that is linearized using a restriction enzyme. In this regard, any restriction enzyme (see, e.g., Roberts et al. (2015) Nucl. Acids Res. 43; D1: D298-D299) can be used. Generally, the restriction enzyme is a type II restriction enzyme, such as a type IIP or type IIS restriction enzyme. In some embodiments, the restriction enzyme is EcoRI, BciVI, Spel, Xbal, Ndel, Aflll, Sacl, Kpnl, Smal, BamHI, Sail, Sbfl, Pstl, BspQI, or Hindlll.

[0083] In some embodiments, the restriction site for linearizing the DNA template is selected such that the resulting IVT mRNA does not contain a modified ribonucleotide as the terminal nucleotide at the 3' end. Thus, in some aspects, the invention provides a method for producing in vitro transcribed (IVT) messenger RNA (mRNA) containing modified ribonucleotides, comprising: (i) preparing a DNA template, wherein the terminal 3' residue of the DNA template does not encode a modified ribonucleotide of an IVT mRNA; (ii) transcribing a DNA template using an RNA polymerase in an in vitro transcription reaction containing modified ribonucleotides; The present invention also relates to a method comprising:

[0084] In some embodiments, the DNA template is a circular vector containing a restriction site. In some embodiments, step (i) comprises cleaving the circular vector at the restriction site to obtain a terminal 3' residue of the DNA template that does not encode a modified ribonucleotide of the IVT mRNA. In some embodiments, the restriction site is cleaved by BspQI. Thus, in some embodiments, a suitable restriction enzyme for preparing the DNA template in step (i) is BspQI. In some embodiments, the IVT mRNA does not contain N1-methylpseudouridine as the terminal 3' residue.

[0085] Ribonucleotides According to the present invention, IVT mRNA is modified RNA, and the term "modification" refers to a chemical or biological modification, including backbone modification, sugar modification, or base modification. Backbone modification refers to a chemical modification of the phosphate of the backbone of RNA nucleotides (e.g., phosphorothioate and 5'-N-phosphoramidite linkage). Sugar modification refers to a chemical modification of the sugar of RNA nucleotides (e.g., 2'-fluororibose, ribose, 2'-deoxyribose, arabinose, and hexose). Base modification refers to a chemical modification of the base moiety of RNA nucleotides.

[0086] In certain embodiments, the IVT mRNA comprises modified ribonucleotides, such as ribonucleotide analogs (e.g., adenosine analogs, guanosine analogs, cytidine analogs, and / or uridine analogs). The presence of modified ribonucleotides may render the mRNA more stable and / or less immunogenic than a control mRNA having the same sequence but containing only naturally occurring ribonucleotides.

[0087] Modified ribonucleotides typically substitute for naturally occurring nucleotides. Thus, the IVT mRNA of the present invention contains both unmodified and modified ribonucleotides. Such IVT mRNA can be prepared by including modified ribonucleosides in the IVT reaction mixture in place of typically naturally occurring ribonucleosides (e.g., N1-methylpseudouridine instead of uridine). This results in an IVT mRNA in which 100% of the naturally occurring ribonucleotides are substituted with the corresponding modified ribonucleotides (e.g., 100% of the uridines are substituted with N1-methylpseudouridine). In some embodiments, a portion of the naturally occurring ribonucleosides (e.g., at least 1%, 5%, 10%, 15%, 20%, or 25% of the naturally occurring ribonucleosides) are substituted with modified ribonucleosides. In some embodiments, one or more naturally occurring ribonucleosides are substituted with modified ribonucleosides. For example, two or more of the ribonucleosides can be modified ribonucleosides (e.g., uridine can be substituted with 5-thio-uridine and cytidine can be substituted with 2-methylcytidine). For example, 25% of the uridine residues can be substituted with 2-thio-uridine and / or 25% of the cytidine residues can be substituted with 5-methylcytidine.

[0088] In some embodiments, a modified ribonucleoside comprises at least one modification relative to a corresponding naturally occurring ribonucleoside selected from a modified sugar and a modified nucleobase.

[0089] The modified ribonucleoside can be a modified uridine, cytidine, adenosine, or guanosine. Some exemplary chemical modifications of ribonucleosides in mRNA molecules include, for example, pyridin-4-one ribonucleosides, 5-aza-uridine, 2-thio-5-aza-uridine, 2-thiouridine, 4-thiopseudouridine, 2-thiopseudouridine, 5-hydroxyuridine, 3-methyluridine, 5-carboxymethyluridine, 1-carboxymethyl-pseudouridine, 5-propynyl-uridine, 1-propynyl-pseudouridine, 5-taurinomethyluridine, 1- Taurinomethyl-pseudouridine, 5-taurinomethyl-2-thio-uridine, 1-taurinomethyl-4-thio-uridine, 5-methyl-uridine, 1-methyl-pseudouridine, 4-thio-1-methyl-pseudouridine, 2-thio-1-methyl-pseudouridine, 1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-1-deaza-pseudouridine, dihydrouridine, dihydropseudouridine, 2-thiodihydrouridine, 2-thio-dihydropseudouridine 2-Methoxyuridine, 2-Methoxy-4-thio-uridine, 4-Methoxy-pseudouridine, 4-Methoxy-2-thiopseudouridine, 5-Aza-cytidine, Pseudoisocytidine, 3-Methyl-cytidine, N4-Acetylcytidine, 5-Formylcytidine, N4-Methylcytidine, 5-Hydroxymethylcytidine, 1-Methyl-pseudoisocytidine, Pyrrolo-cytidine, Pyrrolo-pseudoisocytidine, 2-Thio-cytidine, 2-Thio-5-methyl-cytidine, 4-Thio-cytidine pseudoisocytidine, 4-thio-1-methyl-pseudoisocytidine, 4-thio-1-methyl-1-deaza-pseudoisocytidine, 1-methyl-1-deaza-pseudoisocytidine, zebularine, 5-aza-zebularine, 5-methyl-zebularine, 5-aza-2-thio-zebularine, 2-thio-zebularine, 2-methoxy-cytidine, 2-methoxy-5-methyl-cytidine, 4-methoxy-pseudoisocytidine, 4-methoxy-1-methyl-pseudoisocytidine, 2-aminopurine, 2,6-Diaminopurine, 7-Deazaadenine, 7-Deaza-8-Aza-Adenine, 7-Deaza-2-aminopurine, 7-Deaza-8-Aza-2-aminopurine, 7-Deaza-2,6-Diaminopurine, 7-Deaza-8-Aza-2,6-Diaminopurine, 1-Methyladenosine, N, 6 -methyladenosine, N 6 -Isopentenyl adenosine, N 6 -(cis-Hydroxyisopentenyl)adenosine, 2-methylthio-N 6 -(cis-hydroxyisopentenyl)adenosine, N 6 -Glycinylcarbamoyl adenosine, N 6 -Threonylcarbamoyladenosine, 2-methylthio-N 6 -Threonylcarbamoyladenosine, N 6 ,N 6 -dimethyladenosine, 7-methyladenine, 2-methylthioadenine, 2-methoxyadenine, inosine, 1-methyl-inosine, wyosine, wybutosine, 7-deazaguanosine, 7-deaza-8-aza-guanosine, 6-thioguanosine, 6-thio-7-deazaguanosine, 6-thio-7-deaza-8-aza-guanosine, 7-methyl-guanosine, 6-thio-7-methylguanosine, 7-methylinosine, 6-methoxyguanosine, 1-methylguanosine, N 2 -methylguanosine, N 2 ,N 2 -dimethylguanosine, 8-oxo-guanosine, 7-methyl-8-oxo-guanosine, 1-methyl-6-thio-guanosine, N 2 -methyl-6-thio-guanosine and N 2 ,N 2 -dimethyl-6-thio-guanosine.

[0090] In some embodiments, the modified ribonucleoside is pseudouridine, pyridin-4-one ribonucleoside, 5-aza-uridine, 6-aza-uridine, 2-thio-5-aza-uridine, 2-thio-uridine, 4-thio-uridine, 4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxyuridine, 5-aminoallyl-uridine, 5-halo-uridine (e.g., 5-iodouridine or 5-bromouridine), 3-methyluridine, 5-methoxy-uridine, uridine-5-oxyacetic acid, uridine-5-oxyacetic acid methyl ester, 5-carboxymethyl-uridine, 1-carboxymethyl-pseudouridine, 5-carboxyhydroxymethyluridine, 5-carboxyhydroxymethyluridine, 5-carboxymethyl-uridine, 5-carboxymethyl-uridine, 5-carboxyhydroxymethyluridine, 5-carboxymethyl-uridine, 5-carboxymethyl-uridine, 5-carboxyhydroxymethyluridine, 5-carboxymethyl-uridine, 5-carboxymethyl-uridine, 5-carboxyhydroxymethyluridine, 5-carboxymethyl-uridine, 5-carboxymethyl-uridine, 5-carboxyhydroxymethyluridine, 5-carboxymethyl-uridine, 5-carboxymethyl-uridine, 5-carboxyhydroxymethyluridine, 5-carboxyhydroxymethyluridine, 5-carboxyhydroxymethyluridine, 5-carboxyhydroxymethyluridine, 5-carboxyhydroxymethyluridine, 5-hydroxymethyl- ... 1-uridine methyl ester, 5-methoxycarbonylmethyluridine, 5-methoxycarbonylmethyl-2-thiouridine, 5-aminomethyl-2-thiouridine, 5-methylaminomethyluridine, 5-methylaminomethyl-2-thiouridine, 5-methylaminomethyl-2-selenouridine, 5-carbamoylmethyluridine, 5-carboxymethylaminomethyluridine, 5-carboxymethylaminomethyl-2-thiouridine, 5-propynyluridine, 1-propynyl-pseudouridine, 5-taurinomethyluridine, 1-taurinomethylpseudouridine, 5-taurinomethyl-2-thiouridine, 1-taurinomethyl-4-thiopseudouridine, 5-methyluridine (m 5U, e.g., having the nucleobase deoxythymine), 1-methyl-pseudouridine, 5-methyl-2-thio-uridine, 1-methyl-4-thio-pseudouridine, 4-thio-1-methyl-pseudouridine, 3-methyl-pseudouridine), 2-thio-1-methyl-pseudouridine, 1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-1-deaza-pseudouridine, dihydrouridine, dihydropseudouridine, 5,6-dihydrouridine, 5-methyl-dihydrouridine, 2-thio-dihydrouridine, 2-thio-dihydropseudouridine, 2-methoxy-uridine, 2-methoxy-4-thio-uridine, 4-methoxy-pseudouridine, 4-methoxy-2-thio-pseudouridine, N1-methyl-pseudouridine, 3-(3-amino-3-carboxypropyl)uridine, 1-methyl-3-(3-amino-3-carboxypropyl)uridine, Propyl)pseudouridine, 5-(isopentenylaminomethyl)uridine, 5-(isopentenylaminomethyl)-2-thio-uridine, alpha-thio-uridine, 2'-O-methyluridine, 5,2'-O-dimethyluridine, 2'-O-methyl-pseudouridine, 2-thio-2'-O-methyluridine, 5-methoxycarbonylmethyl-2'-O-methyluridine, 5-carbamoylmethyl-2'-O-methyluridine , 5-carboxymethylaminomethyl-2'-O-methyluridine, 3,2'-O-dimethyluridine, 5-(isopentenylaminomethyl)-2'-O-methyluridine, 1-thio-uridine, deoxythymidine, 2'-F-ara-uridine, 2'-F-uridine, 2'-OH-ara-uridine, 5-(2-carbomethoxyvinyl)uridine, and 5-[3-(1-E-propenylamino)uridine]uridine.

[0091] In some embodiments, the modified uridine is selected from N1-methylpseudouridine, pseudouridine, 2-thiouridine, 4'-thiouridine, 2-thio-1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-pseudouridine, 2-thio-5-aza-uridine, 2-thio-dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-pseudouridine, 4-methoxy-2-thio-pseudouridine, 4-methoxy-pseudouridine, 4-thio-1-methyl-pseudouridine, 4-thio-pseudouridine, 5-aza-uridine, dihydropseudouridine, 5-methyluridine, 5-methyluridine, 5-methoxyuridine, and 2'-O-methyluridine. In some embodiments, the modified uridine is N1-methylpseudouridine.

[0092] In some embodiments, the modified ribonucleoside is 5-aza-cytidine, 6-aza-cytidine, pseudoisocytidine, 3-methylcytidine, N 4 -acetylcytidine, 5-formyl-cytidine, N 4 -methylcytidine, 5-methylcytidine, 5-halocytidine (e.g., 5-iodocytidine), 5-hydroxymethylcytidine, 1-methyl-pseudoisocytidine, pyrrolo-cytidine, pyrrolo-pseudoisocytidine, 2-thio-cytidine, 2-thio-5-methylcytidine, 4-thio-pseudoisocytidine, 4-thio-1-methyl-pseudoisocytidine, 4-thio-1-methyl-1-deaza-pseudoisocytidine, 1 -methyl-1-deaza-pseudoisocytidine, zebularine, 5-aza-zebularine, 5-methyl-zebularine, 5-aza-2-thiozebularine, 2-thio-zebularine, 2-methoxycytidine, 2-methoxy-5-methylcytidine, 4-methoxypseudoisocytidine, 4-methoxy-1-methyl-pseudoisocytidine, lysidine, α-thio-cytidine, 2'-O-methylcytidine, 5,2'-O-dimethylcytidine, N 4 -acetyl-2'-O-methylcytidine, N 4 ,2'-O-dimethylcytidine, 5-formyl-2'-O-methylcytidine, N 4 ,N 4, a modified cytidine selected from 2'-O-trimethylcytidine, 1-thio-cytidine, 2'-F-ara-cytidine, 2'-F-cytidine and 2'-OH-ara-cytidine.

[0093] In some embodiments, the modified ribonucleoside is a modified pyrimidine ribonucleoside. In some embodiments, the modified ribonucleoside is selected from the group consisting of pseudouridine, N1-methylpseudouridine, 5-methylcytosine, 5-methoxyuridine, and any combination thereof. In some embodiments, both cytidine and uracil are replaced with modified nucleosides (e.g., N1-methylpseudouridine and 5-methylcytidine).

[0094] In some embodiments, the modified ribonucleoside is a modified purine ribonucleoside. In some embodiments, the modified ribonucleoside is 2-aminopurine, 2,6-diaminopurine, 2-amino-6-halopurine (e.g., 2-amino-6-chloropurine), 6-halopurine (e.g., 6-chloropurine), 2-amino-6-methylpurine, 8-azidoadenosine, 7-deaza-adenine, 7-deaza-8-azaadenine, 7-deaza-2-aminopurine, 7-deaza-8-aza-2-aminopurine, 7-deaza-2,6-diaminopurine, 7-deaza-8-aza-2,6-diaminopurine, 1-methyladenosine, 2-methyladenine, N 6 -Methyladenosine, 2-methylthio-N 6 -methyladenosine, N 6 -Isopentenyl adenosine, 2-methylthio-N 6 -Isopentenyl adenosine, N 6 -(cis-Hydroxyisopentenyl)adenosine, 2-methylthio-N 6 -(cis-hydroxyisopentenyl)adenosine, N 6 -Glycinylcarbamoyl adenosine, N 6 -Threonylcarbamoyladenosine, N 6 -methyl-N 6 -Threonylcarbamoyladenosine, 2-methylthio-N6 -Threonylcarbamoyladenosine, N 6 ,N 6 -Dimethyladenosine, N 6 -Hydroxynorvalylcarbamoyladenosine, 2-methylthio-N 6 -hydroxynorvalylcarbamoyl adenosine, N 6 -acetyladenosine, 7-methyladenine, 2-methylthio-adenine, 2-methoxyadenine, alpha-thio-adenosine, 2-O-methyladenosine, N 6 ,2'-O-dimethyladenosine,N 6 ,N 6 ,2'-O-trimethyladenosine, 1,2'-O-dimethyladenosine, 2'-O-ribosyladenosine (phosphate), 2-amino-N 6 -methylpurine, 1-thio-adenosine, 8-azido-adenosine, 2'-F-ara-adenosine, 2'-F-adenosine, 2'-OH-ara-adenosine and N 6 -(19-amino-pentaoxanonadecyl)adenosine.

[0095] In some embodiments, the modified ribonucleoside is inosine, 1-methylinosine, wyosine, methylwyosine, 4-demethylwyosine, isowyosine, wybutosine, peroxywybutosine, hydroxywybutosine, unmodified hydroxywybutosine, 7-deaza-guanosine, queosine, epoxyqueosine, galactosylqueosine, mannosylqueosine, 7-cyano-7-deaza-guanosine, 7-aminomethyl7-deaza-guanosine, archaeosine, 7-deaza-8-aza-guanosine, 6-thio-guanosine, 6-thio-7-deaza-guanosine, 6-thio-7-deaza-8-aza-guanosine, 7-methylguanosine, 6-thio-7-methylguanosine, 7-methylinosine, 6-methoxyguanosine, 1-methylguanosine, N 2 -methyl-guanosine, N 2 ,N 2 -Dimethylguanosine, N 2,7 -Dimethylguanosine, N 2 ,N2,7 -Guanosine, 8-oxo-guanosine, 7-methyl-8-oxo-guanosine, 1-methylguanosine, N 2 -methyl-6-thio-guanosine, N 2 ,N 2 -Dimethyl-6-thio-guanosine, alpha-thio-guanosine, 2'-O-methylguanosine, N 2 -methyl-2'-O-methylguanosine, N 2 ,N 2 -dimethyl-2'-O-methylguanosine, 1-methyl-2'-O-methylguanosine, N 2,7 -dimethyl-2'-O-methylguanosine, 2'-O-methylinosine, 1,2'-O-dimethylinosine, 2'-O-ribosylguanosine (phosphate), 1-thio-guanosine, O 6 2'-F-guanosine is a modified guanosine selected from 2'-methylguanosine, 2'-F-araguanosine and 2'-F-guanosine.

[0096] In some embodiments, the modified ribonucleoside is a ribonucleoside analog selected from 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine, 3-methyladenosine, 5-methylcytidine, C-5 propynyl-cytidine, C-5 propynyl-uridine, 2-aminoadenosine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyl-uridine, C5-propynyl-cytidine, C5-methylcytidine, 2-aminoadenosine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, O(6)-methylguanine, pseudouridine (e.g., N1-methylpseudouridine), 2-thiouridine, and 2-thiocytidine. See, for example, U.S. Pat. No. 8,278,036 or WO 2011 / 012316 for a discussion of 5-methylcytidine, pseudouridine, and 2-thiouridine and their incorporation into mRNA.

[0097] In some embodiments, the modified ribonucleoside is selected from pseudouridine, N1-methylpseudouridine, 2-thiouridine, 4'-thiouridine, 5-methylcytosine, 2-thio-l-methyl-l-deaza-pseudouridine, 2-thio-l-methyl-pseudouridine, 2-thio-5-aza-uridine, 2-thio-dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-pseudouridine, 4-methoxy-2-thio-pseudouridine, 4-methoxy-pseudouridine, 4-thio-l-methyl-pseudouridine, 4-thio-pseudouridine, 5-aza-uridine, dihydropseudouridine, 5-methyluridine, 5-methoxyuridine, and 2'-O-methyluridine.

[0098] In some embodiments, the IVT mRNA can be RNA in which 25% of the uridine residues are 2-thiouridine and 25% of the cytidine residues are 5-methylcytidine. Teachings for the use of such modified RNA are disclosed in U.S. Patent Application Publication No. 2012 / 0195936 and WO 2011 / 012316, both of which are incorporated by reference in their entireties. In some embodiments, the IVT mRNA can be RNA in which 100% of the uridine residues are N1-methylpseudouridine (sometimes referred to as 1-methylpseudouridine).

[0099] Tailing Conditions alkali metal salts According to the present invention, the alkali metal salt in the reaction buffer has a concentration of about 30 mM or less, for example, about 1 mM to about 30 mM, about 5 mM to about 30 mM, or about 5 mM to about 27.5 mM. In some embodiments, the alkali metal salt in the reaction buffer has a concentration of about 25 mM or less, for example, about 1 mM to about 25 mM, or about 5 mM to about 25 mM. In some embodiments, the alkali metal salt in the reaction buffer has a concentration of about 20 mM or less, for example, about 1 mM to about 20 mM, or about 5 mM to about 20 mM. In some embodiments, the alkali metal salt in the reaction buffer has a concentration of about 15 mM or less, for example, about 1 mM to about 15 mM, or about 5 mM to about 15 mM. In some embodiments, the alkali metal salt in the reaction buffer has a concentration of about 10 mM or less, for example, about 1 mM to about 10 mM, or about 5 mM to about 10 mM.

[0100] In some embodiments, the alkali metal salt in the reaction buffer has a concentration of about 5 mM or less, e.g., about 1 mM to about 5 mM. In certain embodiments, the alkali metal salt has a concentration of about 5 mM. In other embodiments, the alkali metal salt has a concentration of about 4 mM, about 3 mM, about 2 mM, or about 1 mM.

[0101] In some embodiments, the alkali metal salt in the reaction buffer is NaCl. In some embodiments, the alkali metal salt in the reaction buffer is KCl.

[0102] In some embodiments, the alkali metal salt in the reaction buffer is NaCl and has a concentration of about 5 mM or less, e.g., about 1 mM to about 5 mM, e.g., about 4 mM, about 3 mM, or about 2 mM. In certain embodiments, the alkali metal salt in the reaction buffer is NaCl and has a concentration of about 5 mM.

[0103] In some embodiments, the alkali metal salt in the reaction buffer is KCl and has a concentration of about 5 mM or less, e.g., about 1 mM to about 5 mM, e.g., about 4 mM, about 3 mM, or about 2 mM. In certain embodiments, the alkali metal salt in the reaction buffer is KCl and has a concentration of about 5 mM.

[0104] reducing agent The inventors have found that when the reaction buffer contains 30 mM or less (e.g., 5 mM or less) of an alkali metal salt, the addition of a reducing agent can further increase tailing efficiency. A reducing agent at a concentration of at least 5 mM has been found to be effective for this purpose. Thus, in some embodiments, the reaction buffer contains a reducing agent.

[0105] In some embodiments, the reducing agent has a concentration of about 5 mM to about 50 mM. In some embodiments, the reducing agent has a concentration of about 5 mM to about 20 mM. In some embodiments, the reducing agent has a concentration of about 5 mM, about 10 mM, about 15 mM, or about 20 mM. In certain embodiments, the reducing agent has a concentration of about 10 mM.

[0106] In some embodiments, the reducing agent reduces disulfide bonds. Suitable reducing agents include dithiothreitol (DTT), 2-mercaptoethanol (2-ME), and tris(2-carboxyethyl)phosphine (TCEP).

[0107] In certain embodiments, the reducing agent is DTT. In some embodiments, the reaction buffer comprises DTT at a concentration of about 5 mM to about 50 mM. In certain embodiments, the reaction buffer comprises DTT at a concentration of about 10 mM.

[0108] pH In some embodiments, the reaction buffer maintains a pH of about 6 to about 8.5. In some embodiments, the reaction buffer maintains a pH of about 7 to about 8 (e.g., about 7.2 to 7.8). In some embodiments, the reaction buffer maintains a pH of about 7.0, about 7.1, about 7.2, about 7.3, about 7.4, about 7.5, about 7.6, about 7.7, about 7.8, about 7.9, or about 8.0. In certain embodiments, the reaction buffer maintains a pH of about 7.5.

[0109] In some embodiments, the reaction buffer maintains the pH (e.g., about 7.5) with a buffering reagent selected from Tris, HEPES, MOPS, acetate, citrate, and phosphate. In some embodiments, sodium acetate or sodium citrate is included as a buffering reagent. In certain embodiments, the reaction buffer includes Tris-HCl to maintain the pH during the tailing reaction. In some embodiments, the buffering reagent is present at a concentration of about 5 mM to about 100 mM. In some embodiments, the buffering reagent is present at a concentration of about 10 mM to about 50 mM. In certain embodiments, the buffering reagent is present at a concentration of about 50 mM.

[0110] For example, about 50 mM Tris-HCl can be used to maintain the pH of the reaction buffer at 7-8 (e.g., about 7.5).

[0111] tailing polymerase According to the present invention, tailing is performed after in vitro transcription (IVT) of the mRNA of interest. Therefore, there is no co-transcriptional tailing during IVT. According to the present invention, non-tailed IVT mRNA is added to the reaction buffer or vice versa. A tailing polymerase is added to initiate the tailing reaction.

[0112] In some embodiments, the tailing polymerase in the tailing reaction mixture has a concentration of about 20 mg / g to about 75 mg / g. In some embodiments, the tailing polymerase in the reaction mixture has a concentration of about 20 mg / g to 45 mg / g (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, or 45 mg / g). In some embodiments, the tailing polymerase in the reaction mixture has a concentration of about 25 mg / g to about 35 mg / g. At the same mass concentration, a reaction mixture containing a shorter mRNA will contain more molecules than a mixture containing a longer mRNA. Thus, in some embodiments, the concentration of the tailing polymerase in the reaction mixture is adjusted according to the length of the IVT mRNA to be tailed. For example, a tailing polymerase concentration of 40 mg / g to about 75 mg / g may be suitable for IVT mRNAs up to 1 kb in length. For IVT mRNAs 1 to 2 kb in length, the concentration may be about 30 mg / g to about 40 mg / g. For IVT mRNAs 2 to 4 kb in length, the concentration may be about 20 mg / g to about 30 mg / g.

[0113] In some embodiments, the tailing polymerase is a polyA polymerase. In some embodiments, the polyA polymerase is a bacterial polyA polymerase or a yeast polyA polymerase. In some embodiments, the bacterial polyA polymerase is an Escherichia coli polyA polymerase.

[0114] In some embodiments, the tailing polymerase is a poly-C polymerase.

[0115] divalent cations In some embodiments, the reaction buffer comprises divalent cations. The presence of divalent cations can maintain the activity of the polymerase during tailing.

[0116] In some embodiments, the divalent cations are present at a concentration of about 1 mM to about 20 mM. In some embodiments, the divalent cations are at a concentration of about 5 mM to about 20 mM. The inventors have found divalent cation concentrations of 5 mM or greater to be particularly effective in achieving high tailing efficiency. In some embodiments, the divalent cations are at a concentration of about 5 mM, about 6 mM, about 7 mM, about 8 mM, about 9 mM, or about 10 mM.

[0117] In one particular embodiment, the divalent cations are at a concentration of about 5 mM. In another particular embodiment, the divalent cations are at a concentration of about 10 mM. In yet another particular embodiment, the divalent cations are at a concentration of about 15 mM. In an even more particular embodiment, the divalent cations are at a concentration of about 20 mM.

[0118] In some embodiments, the divalent cation is Mg 2+ and Mn 2+ The choice of a particular divalent cation in the reaction buffer may depend on the tailing polymerase. For example, polyA polymerases typically require Mg as a cofactor. 2+ and Mn 2+ Either Mg or Mg can be used. 2+ The use of Mn may be more effective in maintaining the activity of E. coli polyA polymerase in the reaction buffer, whereas Mn may be more effective for yeast polyA polymerase. 2+ may be more effective.

[0119] The concentration of divalent cations can vary depending on the particular tailing polymerase and the desired tail length. For example, about 10 mM Mg 2+ A concentration of 0.05% has been found to be effective in maintaining polymerase activity during the tailing reaction to provide IVT mRNA with a polyA tail approximately 200 nucleotides long. Higher concentrations may be required to achieve longer tails.

[0120] Divalent cations are typically added to the reaction buffer in the form of salts such as MgCl2 or MnCl2.

[0121] ATP concentration In some embodiments, the tailing reaction contains ATP at a final concentration of about 0.1 mM to about 10 mM. For example, the final ATP concentration can be about 0.1 mM, about 0.2 mM, about 0.3 mM, about 0.5 mM, about 0.6 mM, about 0.7 mM, about 0.8 mM, about 0.9 mM, about 1 mM, about 2 mM, about 3 mM, about 4 mM, about 5 mM, about 6 mM, about 7 mM, about 8 mM, about 9 mM, or about 10 mM. ATP can be included in the reaction buffer. In some embodiments, ATP is added separately to the reaction mixture to initiate the tailing reaction.

[0122] At the same mass concentration, a reaction mixture containing a shorter mRNA contains more molecules than a reaction mixture containing a longer mRNA.The shorter the mRNA, the more ATP is required to tail substantially all of the IVT mRNA in a single reaction.Therefore, in some embodiments, the molar concentration of ATP is adjusted according to the length of the IVT mRNA.Typically, both the concentration of tailing polymerase and the molar concentration of ATP in the tailing reaction are adjusted taking into account the length of the IVT mRNA.

[0123] For example, an ATP concentration of about 2 mM may be suitable for IVT mRNAs up to 1 kb in length. For IVT mRNAs 1-2 kb in length, the ATP concentration may be adjusted to about 0.8 mM. For IVT mRNAs 2-4 kb in length, the concentration may be adjusted to about 0.4 mM.

[0124] Tail length As used herein, the term "tail length" refers to the average number of ribonucleotides added to the IVT mRNA by the tailing polymerase in the reaction buffer of the present invention. The tailing conditions determine the tail length of the resulting IVT mRNA. The tail length can be increased or decreased by changing the concentrations of the tailing polymerase, divalent cations, and / or ATP. Similarly, increasing or decreasing the reaction time can affect the total length of the tail. For example, increasing the concentrations of the tailing polymerase and / or ATP can lead to a longer tail being added to the IVT mRNA.

[0125] In one specific embodiment, the mRNA tail structure comprises a poly-A tail. In another specific embodiment, the mRNA tail structure comprises a poly-C tail. In some embodiments, the tail structure comprises at least 50 adenosine or cytosine ribonucleotides. In typical embodiments, the tail structure is about 100-500 ribonucleotides in length. For example, a tail length of about 200 nucleotides (e.g., a poly-A tail) has been shown to stabilize IVT mRNA in vivo.

[0126] The poly A or poly C tail on the 3' end of the IVT mRNA typically contains at least 50 adenosine or cytosine ribonucleotides, at least 100 adenosine or cytosine ribonucleotides, at least 150 adenosine or cytosine ribonucleotides, at least 200 adenosine or cytosine ribonucleotides, at least 250 adenosine or cytosine ribonucleotides, at least 300 adenosine or cytosine ribonucleotides, at least 350 adenosine or cytosine ribonucleotides, at least 400 adenosine or cytosine ribonucleotides, at least 450 adenosine or cytosine ribonucleotides, or at least 500 adenosine or cytosine ribonucleotides, respectively.

[0127] In some embodiments, the tail structure comprises a combination of poly(A) and poly(C) tails of various lengths as described herein. In some embodiments, the tail structure comprises at least 50%, 55%, 65%, 70%, 75%, 80%, 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, or 99% adenosine ribonucleotides. In some embodiments, the tail structure comprises at least 50%, 55%, 65%, 70%, 75%, 80%, 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, or 99% cytosine ribonucleotides. More typically, the polyA tail on the 3' end of an IVT mRNA produced according to the present invention comprises 100 to 500 adenosine ribonucleotides.

[0128] In some embodiments, the poly-A tail comprises at least two poly-A sequences that are isolated from each other by a nucleotide sequence comprising or consisting of at least 1, 2, 3, 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, or 150 nucleotides, In some embodiments, the nucleotide sequence does not comprise more than 10, 9, 8, 7, 6, 5, 4, 3, or 2 consecutive adenine nucleotides. In some embodiments, the nucleotide sequence separating the first and second polyA sequences comprises between 1 and about 200 nucleotides, between 10 and 90, between 20 and 85, between 30 and 80, between 40 and 80, between 50 and 75, or between 55 and 85 nucleotides, and the nucleotide sequence does not comprise more than 10, 9, 8, 7, 6, 5, 4, 3, or 2 consecutive adenine nucleotides.

[0129] In some embodiments, a portion of the polyA tail is derived from the template DNA, and a portion of the polyA tail is generated by enzymatic polyadenylation, e.g., as described in WO 2016 / 091391, which is incorporated herein by reference.

[0130] Tailing Efficiency The methods of the present invention provide tailing conditions that improve tailing efficiency, allowing the tailing polymerase to be more effective at adding tails to IVT mRNAs containing modified ribonucleotides. In some embodiments, the tailing efficiency is improved by at least 5% (e.g., about 10%) compared to a control (e.g., a non-optimized reaction buffer containing 250 mM NaCl and no reducing agent). In some embodiments, the tailing efficiency is improved by about 10%, about 20%, about 30%, about 40%, or about 50% compared to the control.

[0131] Increased tailing efficiency typically results in a reduction in the percentage of untailed IVT mRNA in methods of tailing IVT mRNA containing modified ribonucleotides. In some embodiments, at least 80% of the IVT mRNA is tailed. More typically, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the IVT mRNA is tailed. In some embodiments, substantially all of the IVT mRNA is tailed. For example, preparations in which at least 80% (e.g., at least 90% or 95%) of the IVT mRNA is tailed are acceptable for producing mRNA for therapeutic use. In some embodiments, methods of tailing IVT containing modified ribonucleotides according to the present invention result in preparations in which untailed IVT mRNA is not detectable. Untailed mRNA can be detected by capillary gel electrophoresis, as demonstrated in the Examples. Alternatively, RNase H digestion coupled with ultra-high pressure liquid chromatography (UHPLC) separation and liquid chromatography mass spectrometry (LC-MS) detection can be used to determine the percentage of IVT mRNA that is tailed.

[0132] Typically, reducing the percentage of untailed IVT mRNA results in tailed IVT mRNA with an average tail length closer to the desired tail length. For example, as exemplified herein, the average tail length can be 45% to 50% longer than the desired length when using a non-optimized reaction buffer. In contrast, the inventors have observed that when using the optimized reaction buffer of the present invention, the average tail length is up to about 30% to 25% longer than the desired tail length, and more typically about 5% shorter than desired. In one embodiment, the desired tail length is at least 100 nucleotides (e.g., 100 to 500 nucleotides). For example, the desired tail length can be a specific value (e.g., 200 nucleotides), and the average length of the tailed IVT mRNA can be within about 30%, 25%, 20%, 15%, 10%, or 5% of the desired length.

[0133] In some embodiments, the tailing efficiency of the tailing reaction is at least 80%, e.g., at least 80% of the IVT mRNA is tailed, with an average tail length within 20% of the desired length. In some embodiments, the tailing efficiency of the tailing reaction is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%. For example, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the IVT mRNA is tailed, with an average tail length within 20% of the desired length. Suitable assays for measuring poly-A tail length using a minor groove-binding dye and one or more ribonucleases are described in WO 2022 / 232499.

[0134] Exemplary Tailing Conditions In certain embodiments, the present invention provides a method for tailing IVT mRNA comprising modified ribonucleotides, wherein the modified ribonucleotides are prepared from a solution of about 50 mM of a buffer (e.g., Tris-HCl), about 5 mM of an alkali metal salt (e.g., NaCl or KCl), about 10 mM of a divalent cation (e.g., Mg 2+ , e.g., MgCl2) and a reducing agent (e.g., DTT) at a concentration of about 10 mM. If necessary, the pH of the reaction buffer can be adjusted to about 7.5.

[0135] Optional Capping Step IVT mRNAs with a methylated 5' cap structure are efficiently translated in vivo. The IVT process may involve a cap analog being added co-transcriptionally. Alternatively, the 5' cap structure can be added enzymatically after the IVT reaction is complete. At least 90% of IVT mRNAs subjected to enzymatic capping may contain the Cap1 structure.

[0136] Several types of 5' caps are known. The 7-methylguanosine cap (also referred to as "m7G" or "cap0") contains a guanosine linked to the first transcribed nucleotide through a 5'-5' triphosphate bond. The 5' cap is typically added as follows: first, an RNA terminal phosphatase removes one of the terminal phosphate groups from the 5' nucleotide, leaving two terminal phosphates; subsequently, guanosine triphosphate (GTP) is added to the terminal phosphate by a guanylyltransferase, generating a 5'5'5 triphosphate bond; and then, the 7-nitrogen of the guanine is methylated by a methyltransferase. Examples of cap structures include, but are not limited to, mG(5')ppp, (5'(A,G(5')ppp(5')A, and G(5')ppp(5')G. Additional cap structures are described in U.S. Patent Application Publication Nos. 2016 / 0032356 and 2018 / 0125989, which are incorporated herein by reference.

[0137] During co-transcriptional capping, a cap analog is included in the IVT reaction mixture. The cap analog can be incorporated as the first "base" in the nascent RNA strand. Cap analogs are classified as cap0, cap1, cap2, m 6 The cap structure may be Am or a chemical cap analog. For example, to generate a 5'-guanosine cap structure, the following chemical cap analogs can be used according to the manufacturer's instructions: 3'-O-Me-m7G(5')ppp(5')G (ARCA cap); G(5')ppp(5')A; G(5')ppp(5')G; m7G(5')ppp(5')A; m7G(5')ppp(5')G; m7G(5')ppp(5')(2'OMeA)pG; m7G(5')ppp(5')(2'OMeA)pU; m7G(5')ppp(5')(2'OMeG)pG (New England BioLabs, Ipswich, MA; TriLink Biotechnologies).

[0138] Vaccinia virus capping enzyme can be used to generate the Cap 0 structure: m7G(5')ppp(5')G. Vaccinia virus capping enzyme and a 2'-O-methyltransferase can both be used to generate the Cap 1 structure: m7G(5')ppp(5')G-2'-O-methyl. A Cap 2 structure can be generated from the Cap 1 structure, followed by 2'-O-methylation of the penultimate 5'-nucleotide using a 2'-O-methyltransferase. A Cap 3 structure can be generated from the Cap 2 structure, followed by 2'-O-methylation of the penultimate 5'-nucleotide using a 2'-O-methyltransferase.

[0139] In some embodiments, the method according to the present invention further comprises a step of capping the IVT mRNA. The capping step may involve adding a capping enzyme (guanylyltransferase) and guanine. A suitable capping enzyme may be derived from vaccinia virus (vaccinia virus guanylyltransferase). Typically, the capping step also includes adding guanine methyltransferase and 2'-O-methyltransferase. Capping may be performed separately, for example, after in vitro transcription. The capping step is generally performed before tailing the IVT mRNA.

[0140] In some embodiments, the reaction buffer of the present invention is added after the IVT mRNA is capped to adjust the reaction conditions before adding the tailing polymerase. Reactants that may form part of the capping buffer (e.g., reducing agents and / or divalent cations) are typically consumed during the capping reaction. Addition of the reaction buffer of the present invention provides components such as reducing agents at concentrations suitable for the tailing reaction.

[0141] In certain embodiments, the IVT mRNA may include a 5' cap having the following structure: [ka]

[0142] purification In some embodiments, the IVT mRNA is purified before it is tailed according to the present invention. In some embodiments, the IVT mRNA is purified after tailing. In some embodiments, the IVT mRNA is capped before adding the tail. In some embodiments, the capped IVT mRNA is purified before tailing.

[0143] Various methods can be used to purify mRNA before or after capping and / or tailing. In some embodiments, mRNA is purified by precipitation and centrifugation. In some embodiments, mRNA is purified by filtration, for example, using normal flow filtration or tangential flow filtration (TFF).

[0144] Suitable purification methods include those described in U.S. Patent Application Publication Nos. 2016 / 0040154, 2015 / 0376220, 2018 / 0251755, 2018 / 0251754, 2020 / 0095571, 2021 / 0388338, and 2021 / 0002635, and U.S. Provisional Patent Application No. 63 / 086,095, filed October 1, 2020, all of which are incorporated herein by reference and may be used to practice the present invention.

[0145] Reaction buffer The present invention also relates to a reaction buffer comprising 30 mM or less of an alkali metal salt and 5 mM or more of a reducing agent at a 1x concentration. As described herein, such a reaction buffer has been found to be particularly suitable for use in a method for tailing IVT mRNA containing modified ribonucleotides. Although the buffer was specifically developed to address the reduced efficiency observed when tailing IVT mRNA containing modified ribonucleotides in prior art reaction buffers, the inventors have found that the reaction buffer of the present invention can also be used for tailing IVT mRNA without modified ribonucleotides.

[0146] The following paragraphs describe the composition of a 1x reaction buffer in more detail. It is generally understood by those skilled in the art that reaction buffers are typically provided at 10x concentrations, for example, for transport and storage. The present invention expressly includes embodiments of 10x reaction buffers, for example, for transport and storage. For example, as a 10x stock solution, the components of the reaction buffer are 10x more concentrated; for example, a 10x stock solution would contain 300mM or less of an alkali metal salt and 50mM or more of a reducing agent.

[0147] alkali metal salts In some embodiments, the alkali metal salt in the reaction buffer has a concentration of about 1 mM to about 30 mM or about 5 mM to about 30 mM, for example, about 5 mM to about 27.5 mM. In some embodiments, the alkali metal salt in the reaction buffer has a concentration of about 25 mM or less, for example, about 1 mM to about 25 mM or about 5 mM to about 25 mM. In some embodiments, the alkali metal salt in the reaction buffer has a concentration of about 20 mM or less, for example, about 1 mM to about 20 mM or about 5 mM to about 20 mM. In some embodiments, the alkali metal salt in the reaction buffer has a concentration of about 15 mM or less, for example, about 1 mM to about 15 mM or about 5 mM to about 15 mM. In some embodiments, the alkali metal salt in the reaction buffer has a concentration of about 10 mM or less, for example, about 1 mM to about 10 mM or about 5 mM to about 10 mM.

[0148] In certain embodiments, the alkali metal salt in the reaction buffer has a concentration of about 5 mM or less, for example, about 1 mM to about 5 mM. In certain embodiments, the alkali metal salt has a concentration of about 5 mM. In other embodiments, the alkali metal salt has a concentration of about 4 mM, about 3 mM, about 2 mM, or about 1 mM.

[0149] In some embodiments, the alkali metal salt in the reaction buffer is NaCl. In some embodiments, the alkali metal salt in the reaction buffer is KCl.

[0150] In some embodiments, the alkali metal salt in the reaction buffer is NaCl and has a concentration of about 30 mM or less (e.g., 25 mM, 20 mM, 15 mM, or 10 mM). In some embodiments, the alkali metal salt in the reaction buffer is NaCl and has a concentration of about 5 mM to about 30 mM.

[0151] In certain embodiments, the alkali metal salt in the reaction buffer is NaCl and has a concentration of about 5 mM or less, e.g., about 1 mM to about 5 mM, e.g., about 4 mM, about 3 mM, or about 2 mM. In certain embodiments, the alkali metal salt in the reaction buffer is NaCl and has a concentration of about 5 mM.

[0152] In some embodiments, the alkali metal salt in the reaction buffer is KCl and has a concentration of about 30 mM or less (e.g., about 1 mM to about 30 mM, such as about 25, 20 mM, 15 mM, or 10 mM). In some embodiments, the alkali metal salt in the reaction buffer is KCl and has a concentration of about 5 mM to about 30 mM.

[0153] In certain embodiments, the alkali metal salt in the reaction buffer is KCl and has a concentration of about 5 mM or less, e.g., about 1 mM to about 5 mM, e.g., about 4 mM, about 3 mM, or about 2 mM. In certain embodiments, the alkali metal salt in the reaction buffer is KCl and has a concentration of about 5 mM.

[0154] reducing agent In some embodiments, the reducing agent has a concentration of about 5 mM or more, e.g., about 5 mM to about 50 mM. In some embodiments, the reducing agent has a concentration of about 5 mM to about 20 mM. In some embodiments, the reducing agent has a concentration of about 5 mM, about 10 mM, about 15 mM, or about 20 mM. In certain embodiments, the reducing agent has a concentration of about 10 mM.

[0155] In some embodiments, the reducing agent reduces disulfide bonds. Suitable reducing agents include dithiothreitol (DTT), 2-mercaptoethanol (2-ME), and tris(2-carboxyethyl)phosphine (TCEP).

[0156] In certain embodiments, the reducing agent is DTT. In some embodiments, the reaction buffer contains DTT at a concentration of at least 5 mM, e.g., about 5 mM to about 50 mM. In certain embodiments, the reaction buffer contains DTT at a concentration of about 10 mM.

[0157] pH In some embodiments, the reaction buffer has a pH of about 6 to about 8.5. In some embodiments, the reaction buffer has a pH of about 7 to about 8 (e.g., about 7.2 to 7.8). In some embodiments, the reaction buffer has a pH of about 7.0, about 7.1, about 7.2, about 7.3, about 7.4, about 7.5, about 7.6, about 7.7, about 7.8, about 7.9, or about 8.0. In some embodiments, the reaction buffer has a pH of about 7.5.

[0158] In some embodiments, the reaction buffer comprises Tris, HEPES, MOPS, acetate, citrate, or phosphate as a buffering reagent. In some embodiments, the reaction buffer comprises sodium acetate or sodium citrate as a buffering reagent. In certain embodiments, the reaction buffer comprises Tris-HCl as a buffering reagent. In some embodiments, the buffering reagent is present at a concentration of about 5 mM to about 100 mM. In some embodiments, the buffering reagent is present at a concentration of about 10 mM to about 50 mM. In certain embodiments, the buffering reagent is present at a concentration of about 50 mM.

[0159] For example, about 50 mM Tris-HCl can be used to maintain the pH of the reaction buffer at 7-8 (e.g., about 7.5).

[0160] divalent cations In some embodiments, the reaction buffer comprises a divalent cation. In some embodiments, the divalent cation is Mg 2+ and Mn 2+ In some embodiments, the reaction buffer comprises MgCl or MnCl.

[0161] In some embodiments, the divalent cations are present at a concentration of about 1 mM to about 20 mM. In some embodiments, the divalent cations are at a concentration of about 5 mM to about 20 mM. The inventors have found divalent cation concentrations of 5 mM or greater to be particularly effective in achieving high tailing efficiency. In some embodiments, the divalent cations are at a concentration of about 1 mM, about 2 mM, about 3 mM, about 4 mM, about 5 mM, about 6 mM, about 7 mM, about 8 mM, about 9 mM, or about 10 mM.

[0162] In one particular embodiment, the divalent cations are at a concentration of about 5 mM. In another particular embodiment, the divalent cations are at a concentration of about 10 mM. In yet another particular embodiment, the divalent cations are at a concentration of about 15 mM. In an even more particular embodiment, the divalent cations are at a concentration of about 20 mM.

[0163] The choice and concentration of divalent cations can vary depending on, for example, the particular tailing polymerase in which the buffer is used. For example, 5-20 mM Mg 2+ (e.g., about 10 mM Mg 2+ ) has been found to be effective in maintaining the activity of E. coli poly A polymerase during the tailing reaction described herein.

[0164] Exemplary Reaction Buffers An exemplary reaction buffer for use in the methods described herein contains a buffering agent (e.g., Tris-HCl) at a concentration of about 50 mM, an alkali metal salt (e.g., NaCl or KCl) at a concentration of about 5 mM, a divalent cation (e.g., Mg) at a concentration of about 10 mM, and a phosphate buffer (e.g., phosphate buffer). 2+ , e.g., MgCl2) and a reducing agent (e.g., DTT) at a concentration of about 10 mM. If necessary, the pH of the reaction buffer can be adjusted to about 7.5.

[0165] For example, a reaction buffer of the present invention may have the composition shown below in Table 1. In a typical embodiment, the pH of the buffer is about 7.5.

[0166] [Table 1]

[0167] The reaction buffer is used for tailing IVT mRNA at 1x concentration. The reaction buffer can be shipped or stored at 10x concentration.

[0168] How to produce mRNA The present invention also relates to methods for producing mRNA, comprising synthesizing mRNA by in vitro transcription and tailing the in vitro transcribed (IVT) mRNA using the methods described herein, i.e., by adding the IVT mRNA to a tailing polymerase in a reaction buffer containing 30 mM or less of an alkali metal salt and 5 mM or more of a reducing agent. Typically, the IVT mRNA contains modified ribonucleotides (e.g., N1-methylpseudouridine).

[0169] In some embodiments, mRNA is synthesized in batches. The inventors have used the tailing method described herein to tail 2 mg and 5 mg batches of IVT mRNA. The inventors have found that, using corresponding conditions, it is possible to tail approximately 1 g and approximately 16 g batches of IVT mRNA. Thus, in some embodiments, a batch contains at least 1 mg of IVT mRNA (e.g., 2 mg, 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg, or 9 mg). In some embodiments, a batch contains at least 10 mg of IVT mRNA (e.g., 11 mg, 12 mg, 13 mg, 14 mg, 15 mg, 16 mg, 17 mg, 18 mg, 19 g, or 20 g). In some embodiments, a batch contains at least 100 mg of IVT mRNA (e.g., 200 mg, 300 mg, 400 mg, 500 mg, 600 mg, 700 mg, 800 mg, or 900 mg). In other embodiments, the batch comprises at least 1 g of IVT mRNA (e.g., 5 g, 10 g, 15 g, 20 g, or 25 g). In further embodiments, the batch comprises at least 50 g of IVT mRNA (e.g., 75 g, 100 g, 150 g, 200 g, or 250 g).

[0170] In other embodiments, the batch contains at least 0.5 kg of IVT mRNA (e.g., 0.75 kg, 1 kg, or 5 kg). In some embodiments, 10 kg, 50 kg, 100 kg, 1000 kg, or more of IVT mRNA is synthesized in a single batch and then tailed according to the methods of the invention.

[0171] The present inventors have surprisingly found that the presence of modified ribonucleotides at the terminal 3' residue of IVT mRNA can dramatically reduce tailing efficiency. Therefore, the present invention also relates to a method for generating (or producing) in vitro transcribed (IVT) messenger RNA (mRNA) containing modified ribonucleotides, the method comprising: (i) preparing a DNA template, wherein the terminal 3' residue of the DNA template does not encode the modified ribonucleotide of the IVT mRNA; and (ii) transcribing the DNA template in an in vitro transcription reaction containing modified ribonucleotides. Because the DNA template is prepared in such a way that the terminal 3' residue of the DNA template does not encode the modified ribonucleotide, the IVT mRNA obtained in step (ii) does not contain modified ribonucleotides as the 3'-terminal residue and can be tailed more efficiently.

[0172] Compositions Comprising Tailed IVT mRNA The present invention also relates to compositions comprising tailed IVT mRNA obtainable by the methods described herein. In some embodiments, at least 80% of the IVT mRNA is tailed. More typically, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the IVT mRNA is tailed. For example, preparations in which at least 80% of the IVT mRNA is tailed are acceptable for producing mRNA for therapeutic use.

[0173] In some embodiments, substantially all of the IVT mRNA is tailed in the composition obtained by the tailing method of the present invention.Therefore, the composition of the present invention containing tailed IVT mRNA is characterized by the absence of detectable amounts of untailed IVT mRNA.A suitable method for determining the absence of untailed IVT mRNA is capillary gel electrophoresis.As demonstrated in the examples, the tailing IVT mRNA method described herein results in a composition containing tailed IVT mRNA characterized by the absence of peaks corresponding to untailed IVT mRNA when analyzed by capillary gel electrophoresis.

[0174] Furthermore, the compositions are typically characterized by a narrow size distribution of the tailed IVT mRNA. Reducing the percentage of untailed IVT mRNA results in tailed IVT mRNA with an average tail length closer to the desired tail length. For example, the desired tail length can be 100-500 nucleotides, and at least 80% of the IVT mRNAs in the composition have the desired tail length. In some embodiments, the desired tail length is at least 100 nucleotides, and at least 80% (e.g., at least 90% or at least 95%) of the IVT mRNAs in the composition have the desired tail length. In some embodiments, the desired tail length is at least 150 nucleotides, and at least 80% (e.g., at least 90% or at least 95%) of the IVT mRNAs in the composition have the desired tail length. In some embodiments, the desired tail length is about 200 nucleotides, and at least 80% (e.g., at least 90% or at least 95%) of the IVT mRNAs in the composition have the desired tail length. In some embodiments, the desired tail length is about 250 nucleotides, and at least 80% (e.g., at least 90% or at least 95%) of the IVT mRNAs in the composition have the desired tail length. In some embodiments, the desired tail length is about 500 nucleotides, and at least 80% (e.g., at least 90% or at least 95%) of the IVT mRNAs in the composition have the desired tail length. [Example]

[0175] The following examples are included for illustrative purposes only and are not intended to limit the scope of the invention.

[0176] Example 1. Preparation of in vitro transcribed mRNA In vitro transcribed (IVT) mRNA was prepared as described in Example 1 of WO 2021 / 168052, incorporated herein by reference. Briefly, for each gram of transcribed mRNA, a reaction mixture containing a linear double-stranded DNA plasmid with an RNA polymerase-specific promoter, an RNA polymerase (e.g., SP6 polymerase or T7 polymerase), an RNase inhibitor, pyrophosphatase, NTPs, DTT, and buffer reagents was prepared in RNase-free water. The reaction mixture was then incubated at 37°C for 60-90 minutes. In Examples 2-5, UTP was replaced with N1-methylpseudouridine triphosphate to prepare IVT mRNA containing modified ribonucleotides. DNase I was added to stop the reaction, and the reaction mixture was incubated at 37°C for an additional 15 minutes. The resulting IVT mRNA was purified.

[0177] The purified IVT mRNA was then capped by mixing it with GTP (1.0 mM), S-adenosylmethionine, RNase inhibitor, 2'-O-methyltransferase, and guanylyltransferase in a suitable reaction buffer (e.g., 10x buffer containing 500 mM Tris-HCl (pH 8.0), 60 mM KCl, and 12.5 mM MgCl). The resulting reaction mixture was incubated at 37 °C for 30–90 min.

[0178] PolyA tailing was typically performed by adding polyA polymerase, ATP, and tailing reaction buffer (10x: 500 mM Tris-HCl (pH 7.5), 2.5 M NaCl, 100 mM MgCl) to the reaction and incubating the reaction mixture at 37 °C for 20–60 min.

[0179] Example 2. Optimization of reaction buffer for tailing When mRNA molecules contain modified ribonucleotides such as N1-methylpseudouridine, tailing efficiency is observed to be reduced compared to the tailing efficiency obtained with mRNA molecules that do not contain modified ribonucleotides. This example shows that optimizing the concentration of alkali metal salts in the reaction buffer used to tail in vitro transcribed (IVT) mRNA containing modified ribonucleotides can improve tailing efficiency.

[0180] To test this hypothesis, reaction buffers containing different concentrations of buffering reagents and alkali metal salts were used for tailing IVT mRNA with tailing polymerase. PolyA tailing polymerase and ATP were added to each of the test reaction buffers. IVT mRNA was prepared as described in Example 1 and contained modified ribonucleotides (N1-methylpseudouridine). Reaction conditions were selected to result in a target tail length of approximately 500 ribonucleotides.

[0181] The conditions tested and the resulting tailing efficiencies and tailing lengths are summarized in Table 2. The listed molar concentrations of buffering reagents, alkali metal salts and divalent cations are those found in 1x reaction buffer.

[0182] [Table 2]

[0183] Buffers 3, 7, and 10 were internal replicates to determine run variability. As a control, a reaction buffer previously proven effective in tailing reactions with IVT mRNA without modified ribonucleotides was included. This buffer was previously described in WO 2021 / 168052. At 10x, it consisted of 500 mM Tris-HCl, 2.5 M alkali metal salts NaCl, and 100 mM MgCl2 as buffering reagents. The pH of the control buffer was 7.5.

[0184] Capillary gel electrophoresis was used to assess mRNA tailing. Briefly, a standard sensitivity RNA analysis kit (15 nt) was purchased from Agilent and used for capillary electrophoresis runs on a Fragment Analyzer instrument equipped with a 12-capillary array (Agilent). During gel priming, 300 ng of total RNA was mixed with a diluent marker at a 1:11 (RNA:marker) ratio, and 24 μL was loaded per well into a 96-well plate. A molecular weight indicator ladder was prepared by mixing 2 μl of standard sensitivity RNA ladder with 22 μL of diluent marker. Sample injection was at 5.0 kV for 4 seconds, and sample separation was at 8.0 kV for 60.0 minutes. Electropherograms for each sample were processed through ProSize 2 software (Advanced Analytical) to generate tabulated sizes (nt) and abundances of fragments present in the sample.

[0185] As can be seen from Table 2, when tailing IVT mRNA containing modified ribonucleotides using the control buffer containing 250 mM NaCl, approximately 8% of the IVT mRNA remained untailed. Notably, the average tail length of approximately 1,200 ribonucleotides far exceeded the target tail length of 500 ribonucleotides. All 12 test conditions improved tailing efficiency by at least fourfold compared to the control buffer. The key parameter appeared to be the alkali metal salt in the reaction, as reducing the alkali metal salt concentration from 7-8 fold to approximately 30 mM improved tailing efficiency compared to the reaction buffer used as a control. When the alkali metal salt concentration was reduced from 50-fold to 5 mM, but the buffer reagent concentration was not changed to 50 mM (see control buffer and buffer 8 in Table 2), tailing efficiency improved by more than fivefold. When buffer 8 was used, only 1.4% of the IVT mRNA remained untailed. Furthermore, under these conditions, when tailing occurred, an average tail length of 450 ribonucleotides was achieved.

[0186] An overall reduction in the ionic strength of the reaction buffer resulted in a significant reduction in untailed IVT mRNA. However, in some instances, this reduction was accompanied by a decrease in the average tail length. For example, a 50-fold reduction in alkali metal salt concentration plus a 10-fold reduction in the concentration of buffering reagents did not provide any additional benefit (see Buffer 1 and Buffer 8 in Table 2). At 241 ribonucleotides, when Buffer 1 was used as the reaction buffer, the average tail length was less than half the desired target length of 500 ribonucleotides.

[0187] This example demonstrates that optimizing the concentration of alkali metal salt in the reaction buffer used to tail IVT mRNA containing modified ribonucleotides to 30 mM or less (e.g., 5 mM or less) improves the efficiency of the tailing reaction. Given that performing the tailing reaction with Buffer 8 resulted in the lowest percentage of untailed IVT mRNA and an average tail length close to the desired target length, this buffer composition was used as the benchmark for subsequent experiments.

[0188] Example 3. Addition of a reducing agent This example shows that tailing efficiency can be further improved by adding a reducing agent to the reaction buffer containing up to 30 mM of an alkali metal salt.

[0189] Three different IVT mRNAs (mRNA A, consisting of 1256 ribonucleotides; mRNA B, consisting of 1268 ribonucleotides; and mRNA C, consisting of 1205 ribonucleotides), each containing a modified ribonucleotide (N1-methylpseudouridine), were tailed in control buffer or buffer 8 as described in Example 2 (see Table 2). In addition, each IVT mRNA was also tailed in a modified version of buffer 8 that further contained a reducing agent (DTT). The target tail length was 200 nucleotides. The resulting tailed IVT mRNAs were analyzed by capillary gel electrophoresis. The results obtained for mRNA A, mRNA B, and mRNA C are summarized in Figures 1, 2, and 3, respectively. For each IVT mRNA, the use of the control buffer resulted in a clearly identifiable additional peak preceding the main peak, indicating a significant amount of non-tailed IVT mRNA (see panel A in Figures 1–3). As observed in Example 2, the use of Buffer 8 significantly reduced the percentage of untailed IVT mRNA compared to the control buffer, as seen by the dramatic reduction in the size of the additional peak preceding the main peak (see Panel B of Figures 1, 2 and 3). When a reducing agent was added to Buffer 8, the additional peak was no longer discernible, indicating that the amount of untailed IVT mRNA was further reduced (see Panel C of Figures 1, 2 and 3).

[0190] Notably, despite the use of a relatively high concentration of reducing agent in Buffer 8 (10 mM in 1x), polyA polymerase performance was not adversely affected. This is surprising because, although commercially available reaction buffers may contain reducing agents, the reducing agents are typically present at much lower concentrations (e.g., 0.2 mM in 1x).

[0191] Furthermore, regardless of the ribonucleotide sequence of the IVT mRNA, improvements were observed due to a decrease in the alkali metal salt concentration in the reaction buffer, and further increased tailing efficiency was achieved by adding a reducing agent to the reaction buffer. Indeed, similar improvements were observed for each of mRNA A, mRNA B, and mRNA C.

[0192] This example demonstrates that tailing efficiency can be further improved by adding a reducing agent to a reaction buffer containing a reduced amount of alkali metal salt compared to a control buffer. The observed improvement is independent of the ribonucleotide sequence of the IVT mRNA.

[0193] Example 4. Concentration of reducing agent This example demonstrates that tailing efficiency can be improved by adding 5 mM or more of a reducing agent to a reaction buffer containing 5 mM or less of an alkali metal salt.

[0194] To determine a suitable concentration range for the reducing agent, the experiment described in Example 3 was repeated using various concentrations of DTT as the reducing agent in Buffer 8. The following concentrations were tested: 0 mM, 5 mM, 10 mM, 15 mM, 25 mM, and 50 mM. The resulting tailed IVT mRNA was analyzed by capillary gel electrophoresis. The results are summarized in Table 3.

[0195] [Table 3]

[0196] At 0 mM, an additional peak preceding the main peak was observed, indicating a significant amount of untailed IVT mRNA. When reducing agent was added to buffer 8, the additional peak was no longer discernible, consistent with previous observations. At reducing agent concentrations ranging from 5 to 50 mM, no additional peak was observed.

[0197] This example demonstrates that tailing efficiency can be improved by adding 5 mM or more of a reducing agent to a reaction buffer containing 5 mM or less of an alkali metal salt.

[0198] Example 5. Improved tailing efficiency without modified ribonucleotides at the 3' end This example shows that the optimized reaction buffer (Buffer 8 with DTT) identified in Example 3 improves tailing even when modified ribonucleotides are present as terminal ribonucleotides at the 3' end of the IVT mRNA. Tailing efficiency is highest when the IVT mRNA does not contain modified ribonucleotides at the 3' end. Therefore, this example also demonstrates that tailing IVT mRNA containing modified ribonucleotides can be improved by providing an untailed IVT mRNA that does not contain modified ribonucleotides at the 3' end.

[0199] Two different plasmid backbones (I) and (II) were prepared. Different template nucleic acids were inserted into the two backbones to evaluate the effect of the ribonucleotide sequence on the tailing efficiency of IVT mRNA. The template-containing plasmid with backbone (I) was linearized with HindIII, and the template-containing plasmid with backbone (II) was linearized with BspQI. IVT reactions containing linear template DNA were performed as described in Example 1. Modified uridine (N1-methylpseudouridine) was included in the reaction mixture instead of UTP. HindIII-cleaved templates yielded IVT mRNA with a modified ribonucleotide as the final ribonucleotide at the 3' end. BspQI-cleaved templates yielded IVT mRNA without a modified ribonucleotide at the 3' end. The resulting IVT mRNA was tailed either in the control buffer described in Example 2 or in buffer 8, which further contained 10 mM DTT, as described in Example 3.

[0200] As can be seen from Figure 4, for IVT mRNA synthesized from HindIII-cleaved templates containing modified ribonucleotides at the 3' end, tailing efficiency was reduced compared to mRNA produced from BspQ1-cleaved templates.

[0201] Surprisingly, even in the presence of modified uridine at the 3' end, only 10% to 15% of the IVT mRNA remained untailed when Buffer 8 containing 10 mM DTT was used in the tailing reaction. This compared favorably with the results obtained using the control buffer (Figure 4 compares HindIII-cleaved mRNA tailed in control buffer (Buffer "C") with the corresponding mRNA tailed in Buffer 8 containing 10 mM DTT (Buffer "O").) In general, tailing efficiency was approximately two-fold lower when using the control buffer described in Example 2. In some instances, more than 30% of the IVT mRNA with modified ribonucleotides at the 3' end remained untailed when the control buffer was used. Unlike Example 3, the overall tailing efficiency varied depending on the ribonucleotide sequence of the IVT mRNA. Sequence optimization can further improve tailing efficiency.

[0202] Notably, when IVT mRNA was prepared from a BspQI-cleaved template and therefore had no modified ribonucleotides at the 3' end, use of the control buffer left up to 20% of the IVT mRNA untailed. In contrast, even with the most challenging ribonucleotide sequence, Buffer 8 containing 10 mM DTT did not result in more than 5% untailed IVT mRNA. In fact, when Buffer 8 containing 10 mM DTT was used, untailed IVT mRNA was not detected in three of the four test samples.

[0203] This example demonstrates that an optimized reaction buffer containing 5 mM or less of an alkali metal salt and 5 mM or more of a reducing agent improves tailing efficiency, regardless of the ribonucleotide at the 3' end of the IVT mRNA or the mRNA ribonucleotide sequence. The tailing efficiency was highest when the IVT mRNA did not contain modified ribonucleotides at the 3' end. Therefore, this example also demonstrates that tailing IVT mRNA containing modified ribonucleotides can be improved by providing an untailed IVT mRNA that does not contain modified ribonucleotides at the 3' end. Interestingly, compared to a non-optimized reaction buffer, the optimized reaction buffer (Buffer 8 containing DTT) further improved the tailing efficiency of IVT mRNA with modified ribonucleotides at the 3' end.

[0204] Example 6. Use of optimized reaction buffer with unmodified IVT mRNA This example demonstrates that a reaction buffer optimized for tailing in vitro transcribed (IVT) mRNA containing modified ribonucleotides can also be used for tailing IVT mRNA composed exclusively of unmodified ribonucleotides.

[0205] In parallel with the experiment described in Example 4, tailing reactions were performed using IVT mRNA prepared using only unmodified, naturally occurring ribonucleotides (ATP, GTP, CTP, and UTP). Tailing conditions were otherwise as described in Example 4. The concentration of reducing agent in Buffer 8 was varied as described therein. The resulting tailed, unmodified IVT mRNA was analyzed by capillary gel electrophoresis. The results are summarized in Table 4.

[0206] [Table 4]

[0207] No additional peaks were discernible in any of the samples containing unmodified IVT mRNA under the test conditions of Example 4. Thus, this example demonstrates that the reaction buffer of the present invention can also be used to tail IVT mRNA composed exclusively of unmodified ribonucleotides.

[0208] Example 7. Large-scale tailing of modified IVT mRNA This example demonstrates that the optimized reaction buffer of the present invention can be used for tailing IVT mRNA with modified ribonucleotides on a large scale (>1 g of IVT mRNA per batch).

[0209] Using the optimized reaction buffer (Buffer 8 containing 10 mM DTT) identified in Example 4, four IVT mRNAs (mRNAs 1-4) with different nucleic acid sequences and lengths (1941, 1941, 1989, and 1995 nucleotides, respectively) were tailed before tailing. Each batch contained approximately 16 g of untailed IVT mRNA containing modified ribonucleotides (N1-methylpseudouridine instead of uridine). The IVT mRNAs were prepared as described in Example 1. Tailing conditions were selected to add a polyA tail to the IVT mRNA with a length of 200 nucleotides (the desired tail length).

[0210] Capillary gel electrophoresis was used to determine the average length of the added polyA tail and the presence of untailed mRNA, and the results are summarized in Table 5.

[0211] [Table 5]

[0212] As can be seen from Table 5, the average length of the polyA tail was close to the desired tail length of 200 nucleotides. No untailed species were detected.

[0213] Using capillary gel electrophoresis, ten 10 g batches of corresponding tailing reactions performed using the non-optimized control buffer listed in Table 2 were compared with four 16 g batches prepared in this example. The average length of the added polyA tail was plotted on the graph shown in Figure 5. The average tail length obtained in the tailing reactions using the non-optimized control buffer was closer to 300 nucleotides. As can be seen in Figure 5, the average tail length achieved with the non-optimized control buffer (labeled "Buffer C") was 45-50% longer than the desired tail length for the majority of the batches tested (9 out of 10). In contrast, using the optimized reaction buffer of the present invention (labeled "Buffer O"), the average tail length was only approximately 5% longer than the desired tail length for three out of four tested batches. Even in the worst-performing batch, using the optimized reaction buffer, the average tail length was only approximately 25% longer than the desired tail length.

[0214] This example demonstrates that the optimized reaction buffer of the present invention can be used to tail batches of IVT mRNA containing modified ribonucleotides on a large scale (>1 g IVT). The average tail lengths obtained were close to the desired length (typically within 5% of the desired value). This example further confirms that the nucleic acid sequence of the IVT mRNA does not affect tailing efficiency when the optimized reaction buffer of the present invention is used.

Claims

1. 1. A method for tailing in vitro transcribed (IVT) messenger RNA (mRNA) containing modified ribonucleotides, the method comprising adding the IVT mRNA to a tailing polymerase in a reaction buffer containing 30 mM or less of an alkali metal salt and 5 mM or more of a reducing agent.

2. 2. The method of claim 1, wherein the IVT mRNA comprises a 5' cap.

3. 3. The method of claim 2, wherein the 5' cap is added in a separate reaction comprising a reaction buffer different from the reaction buffer of claim 1.

4. 4. The method of claim 1, wherein the alkali metal salt in the reaction buffer has a concentration of about 1 mM to about 30 mM.

5. The method of any one of claims 1 to 4, wherein the alkali metal salt in the reaction buffer has a concentration of about 5 mM or less.

6. The method of any one of claims 1 to 5, wherein the reducing agent in the reaction buffer has a concentration of 5 mM to 50 mM.

7. 7. The method of claim 6, wherein the reducing agent in the reaction buffer has a concentration of 5 mM to 20 mM.

8. 8. The method of claim 7, wherein the reducing agent in the reaction buffer has a concentration of about 10 mM.

9. The method according to any one of claims 1 to 8, wherein the reducing agent is selected from dithiothreitol (DTT), 2-mercaptoethanol (2-ME) and tris(2-carboxyethyl)phosphine (TCEP).

10. 10. The method of claim 9, wherein the reducing agent is dithiothreitol (DTT).

11. The method according to any one of claims 1 to 10, wherein the alkali metal salt is NaCl or KCl.

12. 12. The method of claim 11, wherein the alkali metal salt is NaCl.

13. The method of any one of claims 1 to 12, wherein the modified ribonucleotide is selected from pseudouridine, N1-methylpseudouridine, 5-methylcytidine, and 5-methoxyuridine.

14. The method of any one of claims 1 to 13, wherein the modified ribonucleotide is a modified uridine.

15. 15. The method of claim 14, wherein the modified uridine is N1-methylpseudouridine.

16. 16. The method of any one of claims 1 to 15, wherein at least 93% of the IVT mRNA is tailed.

17. 17. The method of claim 16, wherein at least 94% of the IVT mRNA is tailed.

18. 18. The method of claim 17, wherein at least 95% of the IVT mRNA is tailed.

19. 19. The method of claim 18, wherein at least 96% of the IVT mRNA is tailed.

20. 20. The method of claim 19, wherein at least 97% of the IVT mRNA is tailed.

21. 21. The method of claim 20, wherein at least 98% of the IVT mRNA is tailed.

22. 22. The method of any one of claims 1 to 21, wherein the mRNA tail comprises from about 100 to about 800 ribonucleotides.

23. 23. The method of claim 22, wherein the mRNA tail comprises about 100 to about 500 ribonucleotides.

24. 24. The method of claim 23, wherein the mRNA tail comprises about 100 to about 250 ribonucleotides.

25. 25. The method of claim 24, wherein the mRNA tail comprises about 100 or about 200 ribonucleotides.

26. 26. The method of any one of claims 1 to 25, wherein the reaction buffer maintains a pH of about pH 7 to about pH 8.

27. 27. The method of claim 26, wherein the reaction buffer maintains a pH of about pH 7.

5.

28. 28. The method of claim 26 or 27, wherein the reaction buffer maintains the pH with a buffering reagent selected from Tris, HEPES, MOPS, acetate, citrate and phosphate.

29. 29. The method of claim 28, wherein the buffering reagent is present at a concentration of about 5 mM to about 100 mM.

30. 30. The method of claim 29, wherein the buffering reagent is present at a concentration of about 10 mM to about 50 mM.

31. 31. The method of claim 30, wherein the buffering reagent is present at a concentration of about 50 mM.

32. 32. The method of any one of claims 1 to 31, wherein the reaction buffer maintains the activity of the tailing polymerase by providing divalent cations.

33. The divalent cation is Mg ２＋ and Mn ２＋ 33. The method of claim 32, wherein the

34. 34. The method of claim 32 or 33, wherein the divalent cation is present at a concentration of about 5 mM to about 20 mM.

35. 35. The method of claim 34, wherein the divalent cation is at a concentration of about 5 mM to about 10 mM.

36. 36. The method of claim 35, wherein the divalent cation is at a concentration of about 10 mM.

37. 37. The method of any one of claims 1 to 36, wherein the IVT mRNA does not contain modified ribonucleotides at the 3' end.

38. 38. The method of any one of claims 1 to 37, wherein the IVT mRNA does not contain a modified uridine at the 3' end.

39. 39. The method of any one of claims 1 to 38, wherein the IVT mRNA does not contain N1-methylpseudouridine at the 3' end.

40. 40. The method of any one of claims 1 to 39, wherein the tailing polymerase is polyA polymerase.

41. 41. The method of claim 40, wherein the polyA polymerase is a bacterial polyA polymerase or a yeast polyA polymerase.

42. 42. The method of claim 41, wherein the polyA polymerase is E. coli polyA polymerase.

43. The method of any one of claims 40 to 42, wherein the reaction buffer contains a suitable concentration of ATP.

44. 44. The method of claim 43, wherein the ATP is present at a concentration of about 0.1 mM to about 10 mM.

45. A reaction buffer for use in a method for tailing in vitro transcribed (IVT) messenger RNA (mRNA), comprising 30 mM or less of an alkali metal salt and 5 mM or more of a reducing agent.

46. 46. ​​The method of claim 45, wherein the alkali metal salt in the reaction buffer has a concentration of about 1 mM to about 30 mM.

47. 47. The reaction buffer of claim 45 or 46, wherein the alkali metal salt in the reaction buffer has a concentration of about 5 mM or less.

48. 48. The reaction buffer of any one of claims 45 to 47, wherein the reducing agent in the reaction buffer has a concentration of 5 mM to 50 mM.

49. 49. The reaction buffer of claim 48, wherein the reducing agent in the reaction buffer has a concentration of 5 mM to 20 mM.

50. 50. The reaction buffer of claim 49, wherein the reducing agent in the reaction buffer has a concentration of about 10 mM.

51. 51. The reaction buffer of any one of claims 45 to 50, wherein the reducing agent is selected from dithiothreitol (DTT), 2-mercaptoethanol (2-ME) and tris(2-carboxyethyl)phosphine (TCEP).

52. 52. The reaction buffer of claim 51, wherein the reducing agent is dithiothreitol (DTT).

53. 53. The reaction buffer of any one of claims 45 to 52, wherein the alkali metal salt is NaCl or KCl.

54. 54. The reaction buffer of claim 53, wherein the alkali metal salt is NaCl.

55. 55. The reaction buffer of any one of claims 45 to 54, having a pH of about pH 7 to about pH 8.

56. 56. The reaction buffer of claim 55, having a pH of about pH 7.

5.

57. 57. The reaction buffer of claim 55 or 56, comprising Tris, HEPES, MOPS, acetate, citrate or phosphate as a buffering reagent.

58. 58. The reaction buffer of claim 57, wherein the buffering reagent is present at a concentration of about 5 mM to 100 mM.

59. 59. The reaction buffer of claim 58, wherein the buffering reagent is present at a concentration of about 10 mM to about 50 mM.

60. 60. The reaction buffer of claim 59, wherein the buffering reagent is present at a concentration of about 50 mM.

61. 61. The reaction buffer of any one of claims 45 to 60, comprising a divalent cation.

62. The divalent cation is Mg ２＋ and Mn ２＋ 62. The reaction buffer of claim 61, selected from:

63. MgCl ２ or MnCl ２ 63. The reaction buffer of claim 62, comprising:

64. 64. The reaction buffer of any one of claims 45 to 63, wherein the divalent cations are present at a concentration of about 5 mM to about 20 mM.

65. 65. The reaction buffer of claim 64, wherein the divalent cation is at a concentration of about 10 mM.

66. 66. A composition comprising in vitro transcribed (IVT) messenger RNA (mRNA) in a reaction buffer according to any one of claims 45 to 65.

67. 1. A reaction buffer for use in a method for tailing in vitro transcribed (IVT) messenger RNA (mRNA), comprising 300 mM or less of an alkali metal salt and 50 mM or more of a reducing agent, the reaction buffer being diluted 10-fold before use.

68. 68. The reaction buffer of claim 67, wherein the alkali metal salt in the reaction buffer has a concentration of about 10 mM to about 300 mM.

69. 69. The reaction buffer of claim 67 or 68, wherein the alkali metal salt in the reaction buffer has a concentration of about 50 mM or less.

70. 70. The reaction buffer of claim 69, wherein the reducing agent in the reaction buffer has a concentration of 50 mM to 500 mM.

71. 71. The reaction buffer of claim 70, wherein the reducing agent in the reaction buffer has a concentration of 50 mM to 200 mM.

72. 72. The reaction buffer of claim 71, wherein the reducing agent in the reaction buffer has a concentration of about 100 mM.

73. 73. The reaction buffer of any one of claims 67 to 72, wherein the reducing agent is selected from dithiothreitol (DTT), 2-mercaptoethanol (2-ME) and tris(2-carboxyethyl)phosphine (TCEP).

74. 74. The reaction buffer of claim 73, wherein the reducing agent is dithiothreitol (DTT).

75. 75. The reaction buffer of any one of claims 67 to 74, wherein the alkali metal salt is NaCl or KCl.

76. 76. The reaction buffer of claim 75, wherein the alkali metal salt is NaCl.

77. 77. The reaction buffer of any one of claims 67 to 76, having a pH of about pH 7 to about pH 8.

78. 78. The reaction buffer of claim 77, having a pH of about pH 7.

5.

79. 79. The reaction buffer of claim 77 or 78, comprising Tris, HEPES, MOPS, acetate, citrate or phosphate as a buffering reagent.

80. 80. The reaction buffer of claim 79, wherein the buffering reagent is present at a concentration of about 50 mM to 1000 mM.

81. 81. The reaction buffer of claim 80, wherein the buffering reagent is present at a concentration of about 100 mM to about 500 mM.

82. 82. The reaction buffer of claim 81, wherein the buffering reagent is present at a concentration of about 500 mM.

83. 83. The reaction buffer of any one of claims 67 to 82, comprising a divalent cation.

84. The divalent cation is Mg ２＋ and Mn ２＋ 84. The reaction buffer of claim 83, selected from:

85. The divalent cation is MgCl ２ or MnCl ２ 85. The reaction buffer of claim 84, comprising:

86. 86. The reaction buffer of any one of claims 83 to 85, wherein the divalent cations are present at a concentration of about 50 mM to about 200 mM.

87. 87. The reaction buffer of claim 86, wherein the divalent cations are at a concentration of about 100 mM.

88. 1. A method for producing in vitro transcribed (IVT) messenger RNA (mRNA) comprising modified ribonucleotides, comprising: (i) preparing a DNA template, wherein the terminal 3′ residue of the DNA template does not encode a modified ribonucleotide of the IVT mRNA; (ii) transcribing the DNA template using an RNA polymerase in an in vitro transcription (IVT) reaction that includes the modified ribonucleotides; A method comprising:

89. 89. The method of claim 88, wherein the DNA template is a circular vector containing a restriction site.

90. 90. The method of Claim 89, wherein step (i) comprises cleaving the circular vector at the restriction site to obtain the terminal 3' residue of the DNA template that does not encode a modified ribonucleotide of the IVT mRNA.

91. 91. The method of claim 89 or 90, wherein the restriction site is cleaved by BspQI.

92. 92. The method of any one of claims 88-91, further comprising tailing the IVT mRNA.

93. 93. The method of claim 92, wherein the step of tailing comprises adding the IVT mRNA to a tailing polymerase in a reaction buffer comprising 30 mM or less of an alkali metal salt and 5 mM or more of a reducing agent.

94. 94. The method of claim 93, wherein the alkali metal salt in the reaction buffer has a concentration of 5 mM or less.

95. 1. A method for tailing in vitro transcribed (IVT) messenger RNA (mRNA) containing modified ribonucleotides, comprising: (i) providing a non-tailed IVT mRNA that does not contain said modified ribonucleotides at the 3' end; (ii) adding a reaction buffer and a tailing polymerase; A method comprising:

96. 96. The method of claim 95, wherein the reaction buffer comprises 30 mM or less of an alkali metal salt and 5 mM or more of a reducing agent.

97. 97. The method of claim 96, wherein the alkali metal salt in the reaction buffer has a concentration of about 1 mM to about 30 mM.

98. 98. The method of claim 96 or 97, wherein the alkali metal salt in the reaction buffer has a concentration of 5 mM or less.

99. 99. The method of any one of claims 95 to 98, wherein the reducing agent in the reaction buffer has a concentration of 5 mM to 20 mM.

100. 100. The method of any one of claims 95 to 99, wherein the reaction buffer comprises a divalent cation.

101. 101. The method of claim 100, wherein the divalent cation is present at a concentration of about 5 mM to about 20 mM.

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