Optimized tailing of messenger RNA

Abstract

The present invention relates to a method of tailing an in vitro transcriptional (IVT) messenger RNA (mRNA) comprising a modified ribonucleotide, said 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.

Description

Technical Field

[0001] The present invention generally relates 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 for reducing the percentage of untailed IVT mRNA when adding a tail individually at the 3'-end using a tailing polymerase after in vitro synthesis of the mRNA. Background Art

[0002] Messenger RNA (mRNA) is becoming increasingly important as a therapeutic agent. mRNA therapies can restore normal levels of endogenous proteins or provide exogenous therapeutic proteins without permanently altering the genomic sequence or entering the nucleus. mRNA therapies utilize the cell's own protein production and processing machinery to express therapeutic peptides, polypeptides, or proteins, offering flexibility in customized dosages and formulations and being widely applicable to any disease or disorder treatable by providing an exogenous protein.

[0003] The expression level of the protein encoded by mRNA can significantly affect the efficacy and therapeutic benefit of mRNA therapies. Efficient expression or production of protein from mRNA intracellularly, as well as the stability of the mRNA itself, depends on multiple factors, including the presence of a cap at the 5'-end and an appropriately sized tail at the 3'-end of the mRNA.

[0004] The process of manufacturing mRNA for use in therapies typically involves in vitro transcription of mRNA from a DNA template. Capping and tailing can occur cotranscriptionally. Alternatively, in vitro transcribed (IVT) mRNA can be capped and / or tailed individually in subsequent enzymatic reactions.

[0005] Methods and compositions for tailing IVT mRNA have been developed previously. For example, WO 2021 / 163134 describes methods for purifying high-quality mRNA suitable for clinical use. The disclosed methods involve capping and tailing mRNA in a reaction buffer with a pH below 8.0 and an MgCl2 concentration below 1.25 mM. WO 2006 / 029350 describes methods and compositions capable of labeling and amplifying targeted RNA molecules. These methods can involve enzymatic addition of a polyA tail in a separate reaction.

[0006] Achieving high tailing efficiency using existing methods, especially for IVT mRNA containing modified ribonucleotides, remains challenging. Thus, there is a need for methods and compositions capable of improving the tailing efficiency of IVT mRNA. Summary of the Invention

[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 the tailing conditions can significantly improve the tailing efficiency. In particular, the inventors have found that using a reaction buffer containing an alkali metal salt at 30 mM or lower (such as 5 mM or lower) and a reducing agent at 5 mM or higher unexpectedly results in higher tailing efficiency for mRNA containing modified ribonucleotides. This is particularly useful when manufacturing mRNA for therapeutic use.

[0008] Unless otherwise specified, the concentrations of the reaction buffer components provided herein for describing the tailing conditions (e.g., in the context of a tailing method or reaction) refer to the working concentrations (i.e., 1x concentration). Those skilled in the art will understand that higher concentrations (typically 10x) are used for storing the reaction buffer, and thus the reaction buffer prepared for storage may need to be diluted for use in the methods or processes described herein.

[0009] The inventors hypothesize that the presence of modified ribonucleotides (such as N1-methylpseudouridine) in mRNA may neutralize the phosphate backbone of IVT mRNA. This can lead to the formation of complex secondary structures in IVT mRNA. These secondary structures may interfere with the access of the tailing polymerase to the 3'-end of IVT mRNA and initiate tailing. Reducing the concentration of the alkali metal salt present in the reaction buffer reduces its ionic strength. Without wishing to be bound by any particular theory, the lower concentration of the alkali metal salt in the reaction buffer of the present invention may reduce the formation of secondary structures in IVT mRNA, thereby improving the tailing efficiency.

[0010] The inventors further hypothesize that the secondary structure of the nascent tail may interfere with the activity of the tailing polymerase, resulting in a higher percentage of untailed IVT mRNA and tailed IVT mRNA with a tail length longer than desired. Without wishing to be bound by any particular theory, adding a reducing agent may reduce the disulfide bonds in the tailing polymerase, potentially leading to conformational changes that enable the enzyme to cope with the more complex secondary structures formed by the nascent tail, further improving the tailing efficiency and resulting in most IVT mRNA having the desired tail length.

[0011] In one aspect, the present 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 containing an alkali metal salt at 30 mM or lower and a reducing agent at 5 mM or higher. In some embodiments, the IVT mRNA contains a 5' cap. In some embodiments, the 5' cap is added in a separate reaction containing a reaction buffer different from the reaction buffer used for tailing the IVT mRNA.

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

[0013] In some embodiments, the concentration of the reducing agent in the reaction buffer is from 5 mM to 50 mM. In some embodiments, the concentration of the reducing agent in the reaction buffer is from 5 mM to 20 mM. In certain embodiments, the concentration of the reducing agent in the reaction buffer is 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. In certain 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, using the tailing method according to the present invention, at least 93% of the IVT mRNA is tailed. 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 contains from about 100 to about 800 ribonucleotides. In some embodiments, the mRNA tail contains from about 100 to about 500 ribonucleotides. In some embodiments, the mRNA tail contains from about 100 to about 250 ribonucleotides. In some embodiments, the mRNA tail contains about 100 or about 200 ribonucleotides.

[0019] In some embodiments, the reaction buffer is maintained at a pH between about pH 7 and about pH 8. In certain embodiments, the reaction buffer is maintained at a pH of about 7.5.

[0020] In some embodiments, the reaction buffer is maintained at a 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 between about 5 mM and about 100 mM. In some embodiments, the buffering reagent is present at a concentration between about 10 mM and 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 contains divalent cations. In some embodiments, the divalent cations are selected from Mg 2+ and Mn 2+ . In some embodiments, the divalent cations are present at a concentration between about 5 mM and about 20 mM. In certain embodiments, the concentration of the divalent cations is between about 5 mM and about 10 mM. In particular embodiments, the concentration of the divalent cations is 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 uridine at the 3' end. In certain embodiments, the IVT mRNA does not contain N1-methylpseudouridine at the 3’ end.

[0023] In some embodiments, the poly(A) polymerase is a polyadenylation polymerase. In some embodiments, the poly(A) polymerase is a bacterial poly(A) polymerase or a yeast poly(A) polymerase. In certain embodiments, the poly(A) polymerase is an Escherichia coli poly(A) polymerase.

[0024] In some embodiments, the reaction buffer includes an appropriate concentration of ATP. In some embodiments, ATP is present at a concentration between about 0.1 mM and about 10 mM.

[0025] In a further aspect, the invention relates to compositions comprising polyadenylated IVT mRNA obtainable by the polyadenylation method of the invention. These compositions are characterized by a low or undetectable content of non-polyadenylated IVT mRNA, and a narrow size distribution of the polyadenylated IVT mRNA. For example, in some embodiments, a composition comprising polyadenylated IVT mRNA obtainable by the polyadenylation method of the invention comprises less than 5% (e.g., 2% or less) of non-polyadenylated IVT mRNA, as determined by the area under the curve of each species in a capillary gel electrophoresis profile. Additionally, the average tail length typically approaches the desired tail length, e.g., within about 25%, about 20%, about 15%, about 10%, or about 5% of the desired tail length (as determined, for example, by capillary gel electrophoresis).

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

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

[0028] In some embodiments, the concentration of the reducing agent in the reaction buffer is from 5 mM to 50 mM. In some embodiments, the concentration of the reducing agent in the reaction buffer is from 5 mM to 20 mM. In certain embodiments, the concentration of the reducing agent in the reaction buffer is 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. In certain embodiments, the alkali metal salt is NaCl.

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

[0032] In some embodiments, the reaction buffer contains Tris, HEPES, MOPS, acetate, citrate, or phosphate as a buffering reagent. In some embodiments, the buffering reagent is present at a concentration between about 5 mM and about 100 mM. In some embodiments, the buffering reagent is present at a concentration between about 10 mM and 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 contains divalent cations. In some embodiments, the divalent cations are selected from Mg 2+ and Mn 2+ . In some embodiments, the reaction buffer contains MgCl2 or MnCl2.

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

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

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

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

[0038] In some embodiments, the concentration of the reducing agent in the reaction buffer is from 50 mM to 500 mM. In some embodiments, the concentration of the reducing agent in the reaction buffer is from 50 mM to 200 mM. In certain embodiments, the concentration of the reducing agent in the reaction buffer is 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. In certain embodiments, the alkali metal salt is NaCl.

[0041] In some embodiments, the reaction buffer has a pH between about pH 7 and about pH 8. In certain embodiments, the reaction buffer has a pH of about 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 between about 50 mM and about 1000 mM. In some embodiments, the buffering reagent is present at a concentration between about 100 mM and 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 contains divalent cations. In some embodiments, the divalent cations are selected from Mg 2+ and Mn 2+ . In some embodiments, the divalent cations comprise MgCl2 or MnCl2.

[0044] In some embodiments, the divalent cation is present at a concentration between about 50 mM and about 200 mM. In certain embodiments, the concentration of the divalent cation is about 100 mM.

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

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

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

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

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

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

[0051] In some embodiments, the reaction buffer contains divalent cations (e.g., Mg 2+ ). In some embodiments, the divalent cations are present at a concentration between about 5 mM and about 20 mM. In certain embodiments, the concentration of the divalent cations is about 10 mM.

[0052] Other features, objects, and advantages of the invention will become apparent from the following detailed description, drawings, and examples. However, it should be understood that although the detailed description, drawings, and examples indicate embodiments of the invention, they are given by way of illustration only and not by way of limitation. Various changes and modifications will be apparent to those skilled in the art. BRIEF DESCRIPTION OF THE DRAWINGS

[0053] Embodiments of the invention will be described by way of example with reference to the following drawings.

[0054] Figures 1-3 illustrate that a reaction buffer containing 30 mM or lower alkali metal salt and 5 mM or higher reducing agent improves the 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 represents the length of each IVT mRNA (expressed as the number of ribonucleotides). The y-axis shows the relative fluorescence units (RFU). In panel A of Figures 1-3, the 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, the 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, the IVT mRNA was tailed in a reaction buffer containing 5 mM alkali metal salt and 10 mM reducing agent (buffer 8 containing 10 mM DTT in Table 2). Using the control buffer resulted in a clearly distinguishable additional peak (marked by an arrow) before the main peak, indicating the presence of a large amount of untailed IVT mRNA (see panel A of Figures 1-3). Reducing the alkali metal salt concentration significantly decreased the size of the additional peak before the main peak (see the arrow in panel B of Figures 1-3), and shortened the average tail length, as the main peak in panel B shifted to the left compared to the main peak in panel A. When a reducing agent was further added to the buffer containing a reduced alkali metal salt concentration, the additional peak was no longer distinguishable, and the average tail length was further shortened (see panel C of Figures 1-3). In fact, the main peak in panel C shifted to the left compared to the main peaks in panels A and B.

[0055] Figure 4Explanation: An optimized reaction buffer containing 5 mM or less of an alkali metal salt and 5 mM or more of a reducing agent improves the tailing efficiency, regardless of the ribonucleotides or their ribonucleotide sequences at the 3'-end of the IVT mRNA. Seven different IVT mRNAs were prepared from a DNA template plasmid with a backbone (I) cut with HindIII (labeled "HindIII-cut template") or a DNA template plasmid with a backbone (II) cut with BspQI (labeled "BspQI-cut template"). Tailing of each IVT mRNA was carried out in a reaction buffer containing 250 mM alkali metal salt and 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 containing DTT provided in Table 2). For IVT mRNAs 1 - 6, the desired tail length was 500 nucleotides, and for IVT mRNA 7, the desired tail length was 200 nucleotides. The IVT-mRNA generated from the Hind-III-cut template has a modified ribonucleotide (N1-methylpseudouridine) at the 3'-end. The bar graph shows the percentage of the untailed IVT mRNA of each construct after tailing in the control buffer ("C") or buffer 8 containing DTT ("O"). For each IVT mRNA, the tailing efficiency was improved when the tailing reaction was carried out in buffer 8 containing DTT compared to that in the control buffer. The percentage of the untailed mRNA in the in vitro transcribed mRNA from the BspQI-cut template was lower than that from the HindIII-cut template.

[0056] Figure 5 Explanation: The optimized reaction buffer of the present invention can be used for large-scale (each batch > 1 g of IVT mRNA) tailing of IVT mRNA containing modified ribonucleotides, where the resulting average tail length is close to the desired length. Using capillary gel electrophoresis, ten batches of 10 g of tailed IVTmRNA prepared with the unoptimized control buffer (buffer "C") described in Table 2 were compared with four batches of 16 g prepared with buffer 8 containing 10 mM DTT (buffer "O"). The desired tail length was 200 nucleotides. For most of the tested batches (9 out of 10), the average tail length achieved using buffer "C" was 45% - 50% longer than the desired tail length. In contrast, using buffer "O", for three out of the four tested batches, the average tail length was only about 5% longer than the desired tail length. Even for the worst-performing batch, when using buffer "O", the average tail length was only about 25% longer than the desired tail length. The nucleic acid sequence of the IVT mRNA does not affect the tailing efficiency using buffer "O". Definition

[0057] To make the present invention easier to understand, certain terms are first defined below. Additional definitions of the following terms and other terms are set forth throughout the specification.

[0058] Unless the context clearly dictates otherwise, as used in this specification and the appended claims, the singular forms "a / an" and "the" include plural referents. For example, "ribonucleotide" is understood to represent one or more ribonucleotides. Thus, the terms "a or an", "one or more", and "at least one" may be used interchangeably herein.

[0059] Unless specifically stated or obvious from the context, as used herein, the term "or" is understood to be inclusive and encompasses "or" as well as "and". Further, when used herein, "and / or" is regarded as a specific disclosure of each of the two designated features or components with or without the other. Thus, the term "and / or" as used in phrases such as "A and / or B" herein is intended to include "A and B", "A or B", "A" (alone), and "B" (alone). Similarly, the term "and / or" as used in phrases such as "A, B, and / or C" is intended to cover each of the following aspects: 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).

[0060] It is understood that wherever the language "comprising" is used in this application, other similar aspects described in terms of "consisting of" and / or "consisting essentially of" are also provided.

[0061] As used herein, the term "about" refers to the accuracy interval that a person skilled in the art will understand still ensures the technical effect of the feature being discussed. This term indicates a deviation of ±10% from the indicated value. In some embodiments, the deviation is ±5% of the indicated value. In certain embodiments, the deviation is ±1% of the indicated value.

[0062] As used herein, the term "mRNA" refers to a polyribonucleotide encoding at least one polypeptide. The mRNA as used herein encompasses modified and unmodified RNA. The mRNA may contain one or more coding regions and non-coding regions (e.g., 5' untranslated region and 3' untranslated region). The mRNA can be purified from natural sources, produced using recombinant expression systems and optionally purified, in vitro transcribed, or chemically synthesized. The present invention particularly relates to in vitro transcribed (IVT) mRNA. In appropriate cases, for example, in the case of chemically synthesized molecules, the mRNA may contain nucleoside analogs, such as analogs having chemically modified bases or sugars, backbone modifications, etc. Unless otherwise indicated, mRNA sequences are presented in the 5' to 3' direction. A typical mRNA contains 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.

[0063] As used herein, the term "sequence-optimized" is used to describe 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' UTRs and 3' UTRs that are not typically associated with naturally occurring or wild-type nucleic acids. As used herein, the terms "codon optimization" and "codon-optimized" refer to the modification of the codon composition of a naturally occurring or wild-type nucleic acid encoding a peptide, polypeptide, or protein without changing its amino acid sequence, thereby improving the protein expression of the nucleic acid. In the context of the present invention, "codon optimization" can also refer to the process of obtaining one or more optimized nucleotide sequences by removing suboptimal nucleotide sequences from a list of nucleotide sequences using a filter, for example, by filtering based on guanine-cytosine content, codon adaptation index, presence of unstable nucleic acid sequences or motifs, and / or the presence of pause sites and / or terminator signals.

[0064] As used herein, the term "substantially" refers to a qualitative condition exhibiting all or nearly all of the range or degree of a desired characteristic or property. One of ordinary skill in the biological arts will understand that biological and chemical phenomena rarely, if ever, go to completion and / or proceed to completion or are achieved or avoid absolute results. Thus, the term "substantially" is used herein to capture the potential lack of completion inherent in many biological and chemical phenomena.

[0065] As used herein, the term "template DNA" (or "DNA template") refers to a DNA molecule comprising a nucleic acid sequence encoding an mRNA transcript that will be synthesized by in vitro transcription. The template DNA serves as a template for in vitro transcription to produce the mRNA transcript encoded by the template DNA. The template DNA contains all the elements required for in vitro transcription, particularly a promoter element for binding a DNA-dependent RNA polymerase (such as, for example, T3, T7, or SP6 RNA polymerase), which is operably linked to the DNA sequence encoding the desired mRNA transcript. In addition, the template DNA may contain primer binding sites at the 5' and / or 3' of the DNA sequence encoding the mRNA transcript to, for example, determine the identity of the DNA sequence encoding the mRNA transcript by PCR or DNA sequencing. The "template DNA" in the context of the present invention can be a linear or circular DNA molecule. As used herein, the term "template DNA" can refer to a DNA vector, such as plasmid DNA, that contains the nucleic acid sequence encoding the desired mRNA transcript.

[0066] As used herein, the term "tailing efficiency" refers to the effectiveness of the tailing polymerase in adding a tail to the IVT mRNA. Typically, an effective tailing reaction results in an average tail length of the IVT mRNA that is close to the desired tail length. The desired tail length is related to the average tail length expected under optimal tailing conditions. Thus, increasing the tailing efficiency generally results in a lower percentage of untailed IVT mRNA and an average tail length that is close to the indicated desired tail length.

[0067] Unless otherwise defined herein, technical and scientific 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 art to which this application belongs. Although methods and materials similar or equivalent to those described herein can also be used in the practice or testing of this disclosure, exemplary methods and materials are described below. In case of conflict, the present specification, including definitions, shall control.

[0068] Generally, the nomenclatures and techniques used in connection with cell and tissue culture, molecular biology, virology, immunology, microbiology, genetics, analytical chemistry, synthetic organic chemistry, medicine and medicinal chemistry, and protein and nucleic acid chemistry and hybridization as described herein are those well-known and commonly used in the art. Enzymatic reactions and purification techniques are performed according to the manufacturer's instructions as commonly practiced in the art or as described herein. Further, unless the context otherwise requires, singular terms shall include the plural, and plural terms shall include the singular.

[0069] Throughout this specification and the examples, the words "have", "comprise" or variations thereof (such as "has", "having", "comprises", "comprising") shall be understood to mean including the stated integers or groups of integers, but not excluding any other integers or groups of integers.

[0070] All publications and other references cited herein are hereby incorporated by reference in their entirety. Although many documents are cited herein, such citation does not mean that any of these documents constitutes a part of the common general knowledge in the art. Detailed Description

[0071] 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 the tailing efficiency. In particular, it has been 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 results in higher tailing efficiency, even when the IVT mRNA has a terminal modified ribonucleotide at the 3'-end.

[0072] The inventors have also found that the tailing efficiency can be improved if the DNA template has a 3'-terminal residue that does not encode the modified ribonucleotide of the IVT mRNA. Thus, in some aspects, the present invention also relates to a method for tailing in vitro transcribed (IVT) messenger RNA (mRNA) containing modified ribonucleotides, the method comprising: (i) providing an untailed IVT mRNA that does not contain the modified ribonucleotide at the 3'-end; and (ii) adding a reaction buffer and a tailing polymerase. IVT mRNA In vitro transcription

[0073] Various methods for synthesizing mRNA by in vitro transcription (IVT) are described in U.S. Patent Publication No. US2018 / 0258423 and International Patent Publication No. WO 2021 / 168052A1, which are incorporated herein by reference, and these methods can be used to practice the present invention. Briefly, IVT is typically carried out with a reaction mixture that contains a DNA template containing a promoter, a pool of ribonucleotide triphosphates, a buffer system (which may include 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 adding DNase I, which digests the DNA template. The exact conditions will vary depending on the specific application. DNA template

[0074] A typical DNA template according to the present invention contains a promoter sequence, such as a T3, T7, or SP6 promoter, followed by the nucleotide sequence of the desired mRNA. The nucleotide sequence typically contains a 5' untranslated region (5'UTR), a coding region for the polypeptide of interest, and a 3' untranslated region (3'UTR).

[0075] In some embodiments, the nucleotide sequence contains a 5'UTR that is different from the 5'UTR present in the naturally occurring mRNA encoding the polypeptide of interest.

[0076] In some embodiments, the nucleotide sequence contains a 3'UTR that is different from the 3'UTR present in the naturally occurring mRNA encoding the polypeptide of interest.

[0077] For example, suitable 5' and 3'UTRs are described in WO 2012 / 075040, which is incorporated herein by reference.

[0078] In certain embodiments, the 5’ and / or 3’UTR sequences can be derived from stable mRNAs (e.g., globin, actin, GAPDH, tubulin, histone, or citric acid cycle enzymes) to increase the stability of the mRNA. For example, the 5’UTR sequence can include a partial sequence or a fragment of the CMV immediate early 1 (IE1) gene to improve the nuclease resistance of the mRNA and / or extend its half-life. Inclusion of the sequence encoding human growth hormone (hGH) or a fragment thereof into the 3’ end or untranslated region of the mRNA is also contemplated. Exemplary 5’UTRs include sequences derived from the CMV immediate early 1 (IE1) gene (U.S. 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. Publication No. 2016 / 0151409 (incorporated herein by reference).

[0079] 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. Additionally, 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. Publication Nos. 2017 / 0029847, 2016 / 0304883, 2016 / 0235864, and 2016 / 0166710, each of which is incorporated herein by reference).

[0080] In certain embodiments, the 5'UTR is derived from the ribosomal protein large 32 (L32) gene (U.S. Publication No. 2017 / 0029847, ibid.).

[0081] In certain embodiments, the 5'UTR is derived from the 5'UTR of the hydroxysteroid (17-beta) dehydrogenase 4 gene (HSD17B4) (U.S. Publication No. 2016 / 0166710, ibid.).

[0082] In certain embodiments, the 5'UTR is derived from the 5'UTR of the ATP5A1 gene (U.S. Publication No. 2016 / 0166710, ibid.).

[0083] In some embodiments, an internal ribosome entry site (IRES) is used in place of the 5'UTR.

[0084] IVT mRNA is typically transcribed from a DNA template linearized using a restriction enzyme. In this context, any restriction enzyme can be used (see, e.g., Roberts et al. (2015) Nucl. Acids Res. [Nucleic Acids Research] 43; D1:D298-D299). Generally, the restriction enzyme is a type II restriction enzyme, such as a type IIP or 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.

[0085] In some embodiments, the restriction site used to linearize the DNA template is selected such that the resulting IVT mRNA does not include a modified ribonucleotide as the terminal nucleotide at the 3' end. Thus, in some aspects, the present invention also relates to a method for generating an in vitro transcribed (IVT) messenger RNA (mRNA) comprising a modified ribonucleotide, wherein the method comprises: (i) Prepare a DNA template, wherein the 3'-terminal residue of the DNA template does not encode the modified ribonucleotide of the IVT mRNA; and (ii) Transcribe the DNA template with an RNA polymerase in an in vitro transcription reaction containing the modified ribonucleotide.

[0086] In some embodiments, the DNA template is a circular vector containing a restriction site. In some embodiments, step (i) includes cleaving the circular vector at the restriction site to generate the 3'-terminal 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. 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 3'-terminal residue. Ribonucleotide

[0087] According to the present invention, the IVT mRNA is a modified RNA, wherein the modification refers to a chemical or biological modification including a backbone modification, a sugar modification or a base modification. The backbone modification is a modification that chemically modifies the phosphate of the RNA nucleotide backbone (e.g., phosphorothioate and 5'-N-phosphoramidite bond). The sugar modification is a chemical modification of the sugar of the RNA nucleotide (e.g., 2'-fluororibose, ribose, 2'-deoxyribose, arabinose and hexose). The base modification is a chemical modification of the base moiety of the RNA nucleotide.

[0088] In particular embodiments, the IVT mRNA contains modified ribonucleotides, such as ribonucleotide analogs (e.g., adenosine analogs, guanosine analogs, cytidine analogs and / or uridine analogs). The presence of the modified ribonucleotides may make the mRNA more stable and / or less immunogenic than a control mRNA having the same sequence but containing only naturally occurring ribonucleotides.

[0089] Modified ribonucleotides typically replace naturally occurring nucleotides. Thus, the IVT mRNAs of the invention contain both unmodified and modified ribonucleotides. Such IVT mRNAs can be prepared by including modified ribonucleosides (typically replacing the naturally occurring ribonucleosides (e.g., N1-methylpseudouridine replacing uridine)) in the IVT reaction mixture. This results in 100% of the naturally occurring ribonucleotides in the IVT mRNA being replaced by the corresponding modified ribonucleotides (e.g., 100% of uridine being replaced by N1-methyl-pseudouridine). In some embodiments, only a portion of the naturally occurring ribonucleosides (e.g., at least 1%, 5%, 10%, 15%, 20%, or 25% of the naturally occurring ribonucleosides) are replaced by modified ribonucleosides. In some embodiments, one or more of the naturally occurring ribonucleosides are replaced by modified ribonucleosides. For example, two or more ribonucleosides can be modified ribonucleosides (e.g., uridine can be replaced by 2-thio-uridine, and cytidine can be replaced by 5-methylcytidine). For example, 25% of uridine can be replaced by 2-thio-uridine and / or 25% of the cytidine residues can be replaced by 5-methylcytidine.

[0090] In some embodiments, the modified ribonucleoside contains at least one modification selected from a modified sugar and a modified nucleobase, relative to the corresponding naturally occurring ribonucleoside.

[0091] Modified ribonucleosides can be modified uridine, cytidine, adenosine, or guanosine. Some exemplary chemical modifications of ribonucleosides in an mRNA molecule include, for example, pyridin-4-one ribonucleoside, 5-aza-uridine, 2-thio-5-aza-uridine, 2-thio-uridine, 4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxyuridine, 3-methyluridine, 5-carboxymethyluridine, 1-carboxymethyl-pseudouridine, 5-propynyl-uridine, 1-propynyl-pseudouridine, 5-tauromethyluridine, 1-tauromethyl-pseudouridine, 5-tauromethyl-2-thio-uridine, 1-tauromethyl-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-thio-dihydrouridine, 2-thio-dihydropseudouridine, 2-methoxyuridine, 2-methoxy-4-thio-uridine, 4-methoxy-pseudouridine, 4-methoxy-2-thio-pseudouridine, 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-pseudoisocytidine, 4-thio-1-methyl-pseudoisocytidine, 4-thio-1-methyl-1-deaza-pseudoisocytidine, 1-methyl-1-deazapseudoisocytidine, 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 -isopentenyladenosine, N 6 -(cis-hydroxyisopentenyl)adenosine, 2-methylthio-N 6 -(cis-hydroxyisopentenyl)adenosine, N 6 -glycylcarbamoyladenosine, 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.

[0092] In some embodiments, the modified ribonucleoside is a modified uridine selected from: 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-halouridine (e.g., 5-iodouridine or 5-bromouridine), 3-methyluridine, 5-methoxy-uridine, uridine-5-oxyacetic acid, methyl uridine-5-oxyacetate, 5-carboxymethyl-uridine, 1-carboxymethyl-pseudouridine, 5-carboxymethyloxyuridine, methyl 5-carboxymethyloxy-uridine, 5-methoxycarbonylmethyluridine, 5-methoxycarbonylmethyl-2-thio-uridine, 5-aminomethyl-2-thiouridine, 5-methylaminomethyluridine, 5-methylaminomethyl-2-thio-uridine, 5-methylaminomethyl-2-selenouridine, 5-carbamoylmethyl-uridine, 5-carboxymethylaminomethyl-uridine, 5-carboxymethylaminomethyl-2-thio-uridine, 5-propynyl-uridine, 1-propynyl-pseudouridine, 5-tauromethyl-uridine, 1-tauromethylpseudouridine, 5-tauromethyl-2-thio-uridine, 1-tauromethyl-4-thio-pseudouridine, 5-methyl-uridine (m 5U, for example, having a nucleobase deoxythymidine), 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)pseudouridine, 5-(isopentenylaminomethyl)uridine, 5-(isopentenylaminomethyl)-2-thio-uridine, α-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-arabinouridine, 2'-F-uridine, 2'-OH-arabinouridine, 5-(2-methoxycarbonylviny)uridine, and 5-[3-(1-E-propenylamino)uridine.

[0093] 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.

[0094] In some embodiments, the modified ribonucleoside is a modified cytidine selected from: 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 ,2'-O-trimethylcytidine, 1-thio-cytidine, 2'-F-arabinocytidine, 2'-F-cytidine, and 2'-OH-arabinocytidine.

[0095] In some embodiments, the modified ribonucleoside is a modified pyrimidine ribonucleoside. In some embodiments, the modified ribonucleoside is selected from pseudouridine, N1-methylpseudouridine, 5-methylcytidine, 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).

[0096] In some embodiments, the modified ribonucleoside is a modified purine ribonucleoside. In some embodiments, the modified ribonucleoside is a modified adenosine selected from: 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-azidoadenine, 7-deaza-2-aminopurine, 7-deaza-8-azido-2-aminopurine, 7-deaza-2,6-diaminopurine, 7-deaza-8-azido-2,6-diaminopurine, 1-methyladenosine, 2-methyladenine, N 6 -methyladenosine, 2-methylthio-N 6 -methyladenosine, N 6 -isopentenyladenosine, 2-methylthio-N 6 -isopentenyladenosine, N 6 -(cis-hydroxyisopentenyl)adenosine, 2-methylthio-N 6-(cis-Hydroxyisopentenyl)adenosine, N 6 -Glycylcarbamoyladenosine, N 6 -Threonylcarbamoyladenosine, N 6 -Methyl-N 6 -Threonylcarbamoyladenosine, 2-Methylthio-N 6 -Threonylcarbamoyladenosine, N 6 , N 6 -Dimethyladenosine, N 6 -Hydroxy-norvalylcarbamoyladenosine, 2-Methylthio-N 6 -Hydroxy-norvalylcarbamoyladenosine, N 6 -Acetyladenosine, 7-Methyladenine, 2-Methylthio-adenine, 2-Methoxyadenine, α-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-Arabino-adenosine, 2'-F Adenosine, 2'-OH-Arabino-adenosine, and N 6 -(19-Amino-pentaoxanonadecyl)adenosine.

[0097] In some embodiments, the modified ribonucleoside is a modified guanosine selected from the following: inosine, 1-Methylinosine, wyosine, Methylwyosine, 4-Demethylwyosine, Isowyosine, Wybutosine, Peroxywybutosine, Hydroxywybutosine, Under-modified Hydroxywybutosine, 7-Deaza-guanosine, Spongosine, Epoxyspongosine, Galactosylspongosine, Mannosylspongosine, 7-Cyano-7-deaza-guanosine, 7-Aminomethyl-7-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 , N 2,7 -Dimethylguanosine, 8-Oxo-guanosine, 7-Methyl-8-oxo-guanosine, 1-Methylguanosine, N 2 -Methyl-6-thio-guanosine, N 2 , N 2-Dimethyl-6-thio-guanosine, α-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 -Methylguanosine, 2'-F-arabinoguanosine, and 2'-F guanosine.

[0098] In some embodiments, the modified ribonucleoside is a ribonucleoside analog selected from: 2-aminoadenosine, inosine, pyrrolopyrimidine, 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. For discussion of 5-methylcytidine, pseudouridine, and 2-thio-uridine and their incorporation into mRNA, see, e.g., U.S. Patent No. 8,278,036 or WO 2011 / 012316.

[0099] In some embodiments, the modified ribonucleoside is selected from pseudouridine, N1-methylpseudouridine, 2-thiouridine, 4'-thiouridine, 5-methylcytidine, 2-thio-l-methyl-1-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-methoxypseudouridine, 4-thio-l-methyl-pseudouridine, 4-thiopseudouridine, 5-aza-uridine, dihydropseudouridine, 5-methyluridine, 5-methoxyuridine, and 2'-O-methyluridine.

[0100] In some embodiments, the IVT mRNA can be an RNA in which 25% of the uridine residues are 2-thio-uridine and 25% of the cytidine residues are 5-methylcytidine. The teachings of using such modified RNAs are disclosed in U.S. Patent Publication US2012 / 0195936 and International Publication WO2011 / 012316, which are hereby incorporated by reference in their entireties. In some embodiments, the IVT mRNA can be an RNA in which 100% of the uridine residues are N1-methylpseudouridine (sometimes also referred to as 1-methylpseudouridine). Polyadenylation conditions Alkali metal salt

[0101] According to the present invention, the concentration of the alkali metal salt in the reaction buffer is about 30 mM or lower, such as 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 concentration of the alkali metal salt in the reaction buffer is about 25 mM or lower, such as about 1 mM to about 25 mM, or about 5 mM to about 25 mM. In some embodiments, the concentration of the alkali metal salt in the reaction buffer is about 20 mM or lower, such as about 1 mM to about 20 mM, or about 5 mM to about 20 mM. In some embodiments, the concentration of the alkali metal salt in the reaction buffer is about 15 mM or lower, such as about 1 mM to about 15 mM, or about 5 mM to about 15 mM. In some embodiments, the concentration of the alkali metal salt in the reaction buffer is about 10 mM or lower, such as about 1 mM to about 10 mM, or about 5 mM to about 10 mM.

[0102] In some embodiments, the concentration of the alkali metal salt in the reaction buffer is about 5 mM or lower, such as about 1 mM to about 5 mM. In certain embodiments, the concentration of the alkali metal salt is about 5 mM. In other embodiments, the concentration of the alkali metal salt is about 4 mM, about 3 mM, about 2 mM, or about 1 mM.

[0103] 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.

[0104] In some embodiments, the alkali metal salt in the reaction buffer is NaCl and the concentration is about 5 mM or lower, such as about 1 mM to about 5 mM, such as about 4 mM, about 3 mM, or about 2 mM. In certain embodiments, the alkali metal salt in the reaction buffer is NaCl and the concentration is about 5 mM.

[0105] In some embodiments, the alkali metal salt in the reaction buffer is KCl and the concentration is about 5 mM or lower, such as about 1 mM to about 5 mM, such as about 4 mM, about 3 mM, or about 2 mM. In certain embodiments, the alkali metal salt in the reaction buffer is KCl and the concentration is about 5 mM. Reducing agent

[0106] The inventors have found that when the reaction buffer contains an alkali metal salt of 30 mM or lower (e.g., 5 mM or lower), the addition of a reducing agent can further improve the polyadenylation efficiency. It has been found that a reducing agent with a concentration of at least 5 mM is effective for this purpose. Thus, in some embodiments, the reaction buffer contains a reducing agent.

[0107] In some embodiments, the concentration of the reducing agent is from about 5 mM to about 50 mM. In some embodiments, the concentration of the reducing agent is from about 5 mM to about 20 mM. In some embodiments, the concentration of the reducing agent is about 5 mM, about 10 mM, about 15 mM, or about 20 mM. In certain embodiments, the concentration of the reducing agent is about 10 mM.

[0108] 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).

[0109] In certain embodiments, the reducing agent is DTT. In some embodiments, the reaction buffer contains DTT at a concentration of from about 5 mM to about 50 mM. In certain embodiments, the reaction buffer contains DTT at a concentration of about 10 mM. pH

[0110] In some embodiments, the reaction buffer is maintained at a pH between about 6 and about 8.5. In some embodiments, the reaction buffer is maintained at a pH between about 7 and pH 8 (e.g., between about 7.2 and 7.8). In some embodiments, the reaction buffer is maintained at 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 is maintained at a pH of about 7.5.

[0111] In some embodiments, the reaction buffer maintains the pH (e.g., pH 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 the 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 between about 5 mM and about 100 mM. In some embodiments, the buffering reagent is present at a concentration between about 10 mM and about 50 mM. In certain embodiments, the buffering reagent is present at a concentration of about 50 mM.

[0112] For example, about 50 mM Tris-HCl can be used to maintain the pH of the reaction buffer between 7 and 8 (e.g., about 7.5). Poly(A) polymerase

[0113] According to the present invention, tailing is performed after in vitro transcription (IVT) of the target mRNA. Thus, there is no co-transcriptional tailing during IVT. According to the present invention, the untailed IVT mRNA is added to the reaction buffer, and vice versa. A tailing polymerase is added to initiate the tailing reaction.

[0114] In some embodiments, the concentration of the poly(A) polymerase in the poly(A) reaction mixture is from about 20 mg / g to about 75 mg / g. In some embodiments, the concentration of the poly(A) polymerase in the reaction mixture is from 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 concentration of the poly(A) polymerase in the reaction mixture is from about 25 mg / g to about 35 mg / g. At the same mass concentration, the reaction mixture containing shorter mRNA contains more molecules than the mixture containing longer mRNA. Thus, in some embodiments, the concentration of the poly(A) polymerase in the reaction mixture is adjusted according to the length of the IVT mRNA to be polyadenylated. For example, for IVT mRNA up to 1 kb in length, a poly(A) polymerase concentration of about 40 mg / g to about 75 mg / g may be suitable. For IVT mRNA 1-2 kb in length, the concentration can be from about 30 mg / g to about 40 mg / g. For IVT mRNA 2-4 kb in length, the concentration can be from about 20 mg / g to about 30 mg / g.

[0115] In some embodiments, the poly(A) polymerase is poly(A) polymerase. In some embodiments, the poly(A) polymerase is a bacterial poly(A) polymerase or a yeast poly(A) polymerase. In some embodiments, the bacterial poly(A) polymerase is Escherichia coli poly(A) polymerase.

[0116] In some embodiments, the poly(A) polymerase is poly(C) polymerase. Divalent cation

[0117] In some embodiments, the reaction buffer contains divalent cations. The presence of divalent cations can maintain the activity of the polymerase during polyadenylation.

[0118] In some embodiments, the divalent cations are present at a concentration of from about 1 mM to about 20 mM. In some embodiments, the concentration of the divalent cations is between about 5 mM and about 20 mM. The inventors have found that a divalent cation concentration of 5 mM or higher is particularly effective in achieving high polyadenylation efficiency. In some embodiments, the concentration of the divalent cations is about 5 mM, about 6 mM, about 7 mM, about 8 mM, about 9 mM, or about 10 mM.

[0119] In one specific embodiment, the concentration of the divalent cations is about 5 mM. In another specific embodiment, the concentration of the divalent cations is about 10 mM. In yet another specific embodiment, the concentration of the divalent cations is about 15 mM. In additional specific embodiments, the concentration of the divalent cations is about 20 mM.

[0120] In some embodiments, the divalent cation is selected from Mg 2+ and Mn 2+ . The choice of a particular divalent cation in the reaction buffer may depend on the tailing polymerase. For example, poly(A) polymerase typically can use Mg 2+ or Mn 2+ as a cofactor. Using Mg 2 + may more effectively maintain the activity of E. coli poly(A) polymerase in the reaction buffer, while for yeast poly(A) polymerase, Mn 2+ may be more effective.

[0121] The concentration of the divalent cation can vary depending on the particular tailing polymerase and the desired tail length. For example, it has been found that a concentration of about 10 mM Mg 2+ effectively maintains the activity of the polymerase during the tailing reaction to provide IVT mRNA with a poly(A) tail of a length of about 200 nucleotides. To obtain a longer tail, a higher concentration may be required.

[0122] Typically, the divalent cation is added to the reaction buffer in the form of a salt (e.g., MgCl2 or MnCl2). ATP concentration

[0123] In some embodiments, the tailing reaction comprises ATP at a final concentration of about 0.1 mM to about 10 mM. For example, the final concentration of ATP 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.

[0124] At the same mass concentration, a reaction mixture containing shorter mRNA contains more molecules than a reaction mixture containing longer mRNA. The shorter the mRNA, the more ATP is required to tail substantially all of the IVT mRNA in a single reaction. Thus, in some embodiments, the molar concentration of ATP is adjusted according to the length of the IVT mRNA. Typically, both the concentration of the tailing polymerase and the molar concentration of ATP in the tailing reaction are adjusted taking into account the length of the IVT mRNA.

[0125] For example, for IVT mRNA up to 1 kb in length, an ATP concentration of approximately 2 mM may be appropriate. For IVT mRNA 1 - 2 kb in length, the ATP concentration can be adjusted to approximately 0.8 mM. For IVT mRNA 2 - 4 kb in length, the concentration can be adjusted to approximately 0.4 mM. Tail length

[0126] 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 resulting tail length of the IVT mRNA. Altering the concentration of the tailing polymerase, divalent cation, and / or ATP may increase or decrease the tail length. Similarly, increasing or decreasing the reaction time may affect the total length of the tail. For example, increasing the concentration of the tailing polymerase and / or ATP may result in longer tails being added to the IVT mRNA.

[0127] In one particular embodiment, the tail structure of the mRNA comprises a polyA tail. In another particular embodiment, the tail structure of the mRNA comprises a polyC tail. In some embodiments, the tail structure comprises at least 50 adenosine or cytosine ribonucleotides. In a typical embodiment, the length of the tail structure is approximately 100 - 500 ribonucleotides. For example, a tail length of approximately 200 nucleotides (e.g., polyA tail) has been shown to stabilize IVT mRNA in vivo.

[0128] The polyA or polyC tail at the 3' end of the IVT mRNA typically comprises 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, at least 500 adenosine or cytosine ribonucleotides.

[0129] In some embodiments, the tail structure comprises a combination of polyA and polyC stretches 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 at the 3' end of the IVT mRNA produced according to the present invention comprises between 100 and 500 adenosine ribonucleotides.

[0130] In some embodiments, the polyA tail comprises at least two polyA sequences separated from each other by a nucleotide sequence comprising 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 or consisting thereof. In some embodiments, the nucleotide sequence does not contain 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 from 1 to about 200 nucleotides, 10 to 90, 20 to 85, 30 to 80, 40 to 80, 50 to 75 or 55 to 85 nucleotides, wherein the nucleotide sequence does not contain more than 10, 9, 8, 7, 6, 5, 4, 3 or 2 consecutive adenine nucleotides.

[0131] 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, for example, as described in WO 2016 / 091391, which is incorporated herein by reference. Poly(A) addition efficiency

[0132] The methods of the present invention provide tailing conditions that improve the tailing efficiency. These conditions allow the tailing polymerase to more efficiently add a tail to the IVT mRNA containing modified ribonucleotides. In some embodiments, the tailing efficiency is increased by at least 5% (e.g., about 10%) relative 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 increased by about 10%, about 20%, about 30%, about 40% or about 50% relative to the control.

[0133] In methods for tailing IVT mRNA comprising modified ribonucleotides, an increase in tailing efficiency generally results in a decrease in the percentage of untailed IVT mRNA. 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, in the manufacture of mRNA for therapeutic use, a preparation in which at least 80% (e.g., at least 90% or 95%) of the IVT mRNA is tailed is acceptable. In some embodiments, the methods for tailing IVT comprising modified ribonucleotides according to the invention result in a preparation in which untailed IVT mRNA is not detected. As demonstrated in the examples, untailed mRNA can be detected by capillary gel electrophoresis. Alternatively, RNase H digestion combined with ultra-high pressure liquid chromatography (UHPLC) separation and liquid chromatography-mass spectrometry (LC-MS) detection can be used to determine the percentage of tailed IVT mRNA.

[0134] Typically, a decrease in the percentage of untailed IVT mRNA results in the average tail length of the tailed IVT mRNA approaching the desired tail length. For example, as illustrated herein, when using an unoptimized reaction buffer, the average tail length may be 45%-50% longer than the desired length. In contrast, the inventors have observed that when using the optimized reaction buffer of the invention, the average tail length is at most about 30%-25% longer than the desired tail length, and more typically only about 5% longer than the desired length. In one embodiment, the desired tail length is at least 100 nucleotides (e.g., 100-500 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%, about 25%, about 20%, about 15%, about 10%, or about 5% of that value.

[0135] 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 and the average tail length is 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 and the average tail length is within 20% of the desired length. A suitable assay for measuring the polyA tail length using a minor groove binding dye and one or more ribonucleases is described in WO 2022 / 232499. Exemplary poly(A) addition conditions

[0136] In particular embodiments, the invention relates to a method for tailing an IVT mRNA comprising modified ribonucleotides, the method comprising adding the IVT mRNA to a tailing polymerase in a reaction buffer comprising a buffer (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 2+ , e.g., MgCl2) at a concentration of about 10 mM, and a reducing agent (e.g., DTT) at a concentration of about 10 mM. If desired, the pH of the reaction buffer can be adjusted to about 7.5. Optional capping step

[0137] IVT mRNA with a methylated 5' cap structure is efficiently translated in vivo. The IVT process can include co-transcriptionally added cap analogs. Alternatively, a 5' cap structure can be added enzymatically after completion of the IVT reaction. At least 90% of the IVT mRNA subjected to enzymatic capping can contain a cap 1 structure.

[0138] Several types of 5’ caps are known. The 7-methylguanosine cap (also referred to as “m7G” or “cap 0”) contains guanosine linked to the first transcribed nucleotide by a 5'-5'-triphosphate bond. The 5’ cap is typically added as follows: First, an RNA terminal phosphatase removes a terminal phosphate group from the 5’ nucleotide, leaving two terminal phosphates; then guanosine triphosphate (GTP) is added to the terminal phosphate via a guanylyltransferase, generating a 5'5'5 triphosphate bond; then, the 7-nitrogen of guanine is methylated by a methyltransferase. Examples of cap structures include, but are not limited to, m7G(5')ppp, (5'(A,G(5')ppp(5')A, and G(5')ppp(5')G. Additional cap structures are described in U.S. Publication Nos. US2016 / 0032356 and US 2018 / 0125989, which are incorporated herein by reference.

[0139] During co-transcriptional capping, a cap analogue is included in the IVT reaction mixture. The cap analogue can be incorporated as the first “base” into the nascent RNA strand. The cap analogue can be cap 0, cap 1, cap 2, m 6 Am, or a chemical cap analogue. For example, the following chemical cap analogues can be used to generate a 5’-guanosine cap structure 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).

[0140] The vaccinia virus capping enzyme can be used to generate the cap 0 structure: m7G(5’)ppp(5’)G. The vaccinia virus capping enzyme and a 2'-O methyltransferase can be used to generate the cap 1 structure to generate: m7G(5')ppp(5')G-2'-O-methyl. The cap 2 structure can be generated from the cap 1 structure, followed by 2’-O-methylation of the 5’ penultimate nucleotide using a 2’-O methyltransferase. The cap 3 structure can be generated from the cap 2 structure, followed by 2’-O-methylation of the 5’ antepenultimate nucleotide using a 2’-O methyltransferase.

[0141] 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 (guanylyl transferase) and guanine. Suitable capping enzymes can be derived from vaccinia virus (vaccinia virus guanylyl transferase). Typically, the capping step also includes adding guanine methyltransferase and 2'-O-methyltransferase. Capping can be performed separately, for example, after in vitro transcription. The capping step is typically performed before polyadenylation of the IVT mRNA.

[0142] 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 polyadenylation polymerase. Reactants that can form part of the capping buffer (e.g., reducing agents and / or divalent cations) are typically consumed during the capping reaction. Adding the reaction buffer of the present invention provides components such as reducing agents at concentrations suitable for the polyadenylation reaction.

[0143] In certain embodiments, the IVT mRNA can comprise a 5' cap having the following structure: Purification

[0144] In some embodiments, the IVT mRNA is purified before polyadenylation according to the present invention. In some embodiments, the IVT mRNA is purified after polyadenylation. In some embodiments, the IVT mRNA is capped before adding the tail. In some embodiments, the capped IVT mRNA is purified before polyadenylation.

[0145] Various methods can be used to purify mRNA before and / or after capping and / or polyadenylation. In some embodiments, mRNA is purified by precipitation and centrifugation. In some embodiments, mRNA is purified by filtration using, for example, forward flow filtration or tangential flow filtration (TFF).

[0146] Suitable purification methods include those described in published U.S. application numbers US2016 / 0040154, US2015 / 0376220, US2018 / 0251755, US2018 / 0251754, US2020 / 0095571, US2021 / 0388338, and US2021 / 0002635, as well as U.S. Provisional Application No. 63 / 086,095 filed on October 1, 2020, all of which are incorporated herein by reference and which methods can be used to practice the present invention. Reaction buffer

[0147] The present invention also relates to a 1x concentration reaction buffer comprising an alkali metal salt at 30 mM or lower and a reducing agent at 5 mM or higher. As described herein, it has been found that such a reaction buffer is particularly suitable for use in a method of tailing IVT mRNA comprising modified ribonucleotides. Although the buffer was developed specifically to address the reduced efficiency observed when tailing IVT mRNA comprising modified ribonucleotides in prior art reaction buffers, the inventors have found that the reaction buffer of the present invention can also be used to tail IVT mRNA that does not comprise modified ribonucleotides.

[0148] The following paragraphs describe in more detail the composition of the 1x reaction buffer. Those skilled in the art will generally understand that reaction buffers are typically provided at a 10x concentration, for example, for shipping and storage. The present invention expressly includes embodiments of a 10x concentration reaction buffer, for example, for shipping and storage. For example, as a 10x stock solution, the components of the reaction buffer will be concentrated 10x. For example, a 10x stock solution will contain an alkali metal salt at 300 mM or lower and a reducing agent at 50 mM or higher, etc. Alkali metal salt

[0149] In some embodiments, the concentration of the alkali metal salt in the reaction buffer is from about 1 mM to about 30 mM or from about 5 mM to about 30 mM, such as from about 5 mM to about 27.5 mM. In some embodiments, the concentration of the alkali metal salt in the reaction buffer is about 25 mM or lower, such as from about 1 mM to about 25 mM, or from about 5 mM to about 25 mM. In some embodiments, the concentration of the alkali metal salt in the reaction buffer is about 20 mM or lower, such as from about 1 mM to about 20 mM, or from about 5 mM to about 20 mM. In some embodiments, the concentration of the alkali metal salt in the reaction buffer is about 15 mM or lower, such as from about 1 mM to about 15 mM, or from about 5 mM to about 15 mM. In some embodiments, the concentration of the alkali metal salt in the reaction buffer is about 10 mM or lower, such as from about 1 mM to about 10 mM, or from about 5 mM to about 10 mM.

[0150] In particular embodiments, the concentration of the alkali metal salt in the reaction buffer is about 5 mM or lower, such as from about 1 mM to about 5 mM. In certain embodiments, the concentration of the alkali metal salt is about 5 mM. In other embodiments, the concentration of the alkali metal salt is about 4 mM, about 3 mM, about 2 mM, or about 1 mM.

[0151] 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.

[0152] In some embodiments, the alkali metal salt in the reaction buffer is NaCl and the concentration is about 30 mM or lower (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 the concentration is about 5 mM to about 30 mM.

[0153] In particular embodiments, the alkali metal salt in the reaction buffer is NaCl and the concentration is about 5 mM or lower, 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 the concentration is about 5 mM.

[0154] In some embodiments, the alkali metal salt in the reaction buffer is KCl and the concentration is about 30 mM or lower (e.g., between about 1 mM and about 30 mM, e.g., 25 mM, 20 mM, 15 mM, or 10 mM). In some embodiments, the alkali metal salt in the reaction buffer is KCl and the concentration is about 5 mM to about 30 mM.

[0155] In particular embodiments, the alkali metal salt in the reaction buffer is KCl and the concentration is about 5 mM or lower, 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 the concentration is about 5 mM. Reducing agent

[0156] In some embodiments, the concentration of the reducing agent is about 5 mM or higher, e.g., about 5 mM to about 50 mM. In some embodiments, the concentration of the reducing agent is about 5 mM to about 20 mM. In some embodiments, the concentration of the reducing agent is about 5 mM, about 10 mM, about 15 mM, or about 20 mM. In certain embodiments, the concentration of the reducing agent is about 10 mM.

[0157] 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).

[0158] 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. pH

[0159] In some embodiments, the reaction buffer has a pH between about 6 and about 8.5. In some embodiments, the reaction buffer has a pH between about 7 and pH 8 (e.g., between about 7.2 and 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 certain embodiments, the reaction buffer has a pH of about 7.5.

[0160] In some embodiments, the reaction buffer contains Tris, HEPES, MOPS, acetate, citrate, or phosphate as a buffering reagent. In some embodiments, the reaction buffer contains sodium acetate or sodium citrate as a buffering reagent. In certain embodiments, the reaction buffer includes Tris-HCl as a buffering reagent. In some embodiments, the buffering reagent is present at a concentration between about 5 mM and about 100 mM. In some embodiments, the buffering reagent is present at a concentration between about 10 mM and about 50 mM. In certain embodiments, the buffering reagent is present at a concentration of about 50 mM.

[0161] For example, about 50 mM of Tris-HCl can be used to maintain the pH of the reaction buffer between 7 and 8 (e.g., about 7.5). Divalent cation

[0162] In some embodiments, the reaction buffer contains divalent cations. In some embodiments, the divalent cations are selected from Mg 2+ and Mn 2+ . In some embodiments, the reaction buffer contains MgCl2 or MnCl2.

[0163] In some embodiments, the divalent cations are present at a concentration between about 1 mM and about 20 mM. In some embodiments, the concentration of the divalent cations is between about 5 mM and about 20 mM. The inventors have found that a divalent cation concentration of 5 mM or higher is particularly effective in achieving high tailing efficiency. In some embodiments, the concentration of the divalent cations is 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.

[0164] In one particular embodiment, the concentration of the divalent cations is about 5 mM. In another particular embodiment, the concentration of the divalent cations is about 10 mM. In yet another particular embodiment, the concentration of the divalent cations is about 15 mM. In additional particular embodiments, the concentration of the divalent cations is about 20 mM.

[0165] The choice and concentration of divalent cations can vary, e.g., depending on the specific poly(A) polymerase used with the buffer. For example, it has been found that a concentration of 5 - 20 mM Mg 2+ (e.g., about 10 mM Mg 2+ ) effectively maintains the activity of E. coli poly(A) polymerase during the polyadenylation reaction described herein. Exemplary reaction buffer

[0166] An exemplary reaction buffer for use in the methods described herein contains a buffering reagent (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 2+ , e.g., MgCl2) at a concentration of about 10 mM, and a reducing agent (e.g., DTT) at a concentration of about 10 mM. If desired, the pH of the reaction buffer can be adjusted to about 7.5.

[0167] For example, the reaction buffer of the present invention can have the composition shown in Table 1 below. In a typical embodiment, the pH of the buffer is about 7.5. Table 1 Component Concentration 1x / 10x Tris-HCl 50 mM / 500 mM NaCl 5 mM / 50 mM <![CDATA[MgCl2]]> 10 mM / 100 mM DTT 10 mM / 100 mM

[0168] The reaction buffer is used at 1x concentration for polyadenylating IVT mRNA. The reaction buffer can be shipped or stored at 10x concentration. Methods of manufacturing mRNA

[0169] The present invention also relates to methods of manufacturing mRNA, which include synthesizing mRNA by in vitro transcription; and polyadenylating the in vitro transcribed (IVT) mRNA using the methods described herein, i.e., by adding the IVT mRNA to a poly(A) 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).

[0170] In some embodiments, the mRNA is synthesized in batches. The inventors have used the tailing methods described herein to tail 2 mg and 5 mg IVT mRNA batches. The inventors have found that corresponding conditions can be used to tail approximately 1 g and approximately 16 g IVT mRNA batches. Thus, in some embodiments, the batch comprises at least 1 mg IVT mRNA (e.g., 2 mg, 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg, or 9 mg). In some embodiments, the batch comprises at least 10 mg IVT mRNA (e.g., 11 mg, 12 mg, 13 mg, 14 mg, 15 mg, 16 mg, 17 g, 18 g, 19 g, or 20 g). In some embodiments, the batch comprises at least 100 mg 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 IVT mRNA (e.g., 5 g, 10 g, 15 g, 20 g, or 25 g). In additional embodiments, the batch comprises at least 50 g IVT mRNA (e.g., 75 g, 100 g, 150 g, 200 g, or 250 g).

[0171] In other embodiments, the batch comprises at least 0.5 kg 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 method of the invention.

[0172] The inventors unexpectedly found that the presence of a modified ribonucleotide at the 3'-terminal residue of the IVT mRNA can significantly reduce the tailing efficiency. Thus, the present invention also relates to a method for generating (or manufacturing) an in vitro transcribed (IVT) messenger RNA (mRNA) comprising a modified ribonucleotide, wherein the method comprises: (i) preparing a DNA template, wherein the 3'-terminal 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 comprising the modified ribonucleotide. Since the DNA template is prepared such that the 3'-terminal residue of the DNA template does not encode a modified ribonucleotide, the IVT mRNA obtained in step (ii) does not include a modified ribonucleotide as the 3'-terminal residue and can be tailed more efficiently. Compositions comprising tailed IVT mRNA

[0173] The present invention also relates to compositions comprising polyadenylated IVT mRNA obtainable by the methods described herein. In some embodiments, at least 80% of the IVT mRNA is polyadenylated. 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 polyadenylated. For example, in the manufacture of mRNA for therapeutic use, a formulation in which at least 80% of the IVT mRNA is polyadenylated is acceptable.

[0174] In some embodiments, in the compositions obtainable by the polyadenylation method of the present invention, substantially all of the IVT mRNA is polyadenylated. Thus, the compositions of the present invention comprising polyadenylated IVT mRNA are characterized by the absence of detectable amounts of non-polyadenylated IVT mRNA. A suitable method for determining the absence of non-polyadenylated IVT mRNA is capillary gel electrophoresis. As demonstrated in the examples, the method for polyadenylating IVT mRNA described herein produces a composition comprising polyadenylated IVT mRNA that is characterized by the absence of a peak corresponding to non-polyadenylated IVT mRNA when analyzed by capillary gel electrophoresis.

[0175] Furthermore, the compositions are typically characterized by a narrow size distribution of the polyadenylated IVT mRNA. A decrease in the percentage of non-polyadenylated IVT mRNA generally results in an average tail length of the polyadenylated IVT mRNA approaching the desired tail length. For example, the desired tail length can be from 100 to 500 nucleotides, and at least 80% of the IVT mRNA in the composition has 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 mRNA in the composition has 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 mRNA in the composition has 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 mRNA in the composition has 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 mRNA in the composition has 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 mRNA in the composition has the desired tail length. Examples

[0176] The following examples are included for illustrative purposes only and are not intended to limit the scope of the invention. Example 1. Preparation of in vitro transcribed mRNA

[0177] Prepare in vitro transcribed (IVT) mRNA as described in Example 1 of WO 2021 / 168052, which is incorporated herein by reference. Briefly, for each gram of transcribed mRNA, prepare a reaction containing the following in RNase-free water: a linearized 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 a buffer reagent. Then incubate the reaction mixture at 37 °C for 60 to 90 min. In Examples 2-5, UTP was replaced with N1-methylpseudouridine triphosphate to prepare IVT mRNA containing modified ribonucleotides. DNase I was added to terminate the reaction, and the reaction mixture was incubated at 37 °C for an additional 15 minutes. Purify the resulting IVT mRNA.

[0178] Then cap the purified IVT mRNA by mixing it with GTP (1.0 mM), S-adenosylmethionine, an 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, 12.5 mM MgCl2). Incubate the resulting reaction mixture at 37 °C for 30 to 90 minutes.

[0179] Poly(A) tailing is typically carried out by adding poly(A) polymerase, ATP, and a tailing reaction buffer (10x: 500 mM Tris-HCl (pH 7.5), 2.5 M NaCl, 100 mM MgCl2) to the reaction and incubating the reaction mixture at 37 °C for 20 to 60 minutes. Example 2. Optimization of the reaction buffer for tailing

[0180] It was observed that the tailing efficiency was reduced when the mRNA molecule included modified ribonucleotides (such as N1-methylpseudouridine) compared to the tailing efficiency obtained with mRNA molecules that did not contain modified ribonucleotides. This example demonstrates that optimizing the concentration of alkali metal salts in the reaction buffer for tailing in vitro transcribed (IVT) mRNA containing modified ribonucleotides can improve the tailing efficiency.

[0181] To verify this hypothesis, reaction buffers containing different concentrations of buffering reagents and alkali metal salts were used to tail IVT mRNA with a tailing polymerase. Poly(A) tailing polymerase and ATP were added to each test reaction buffer. The IVT mRNA was prepared as described in Example 1 and included modified ribonucleotides (N1-methylpseudouridine). Reaction conditions were selected to achieve a target tail length of approximately 500 ribonucleotides.

[0182] Table 2 summarizes the test conditions and the resulting tailing efficiency and tail length. The molar concentrations of the buffering reagent, alkali metal salt, and divalent cation listed are the concentrations in 1x reaction buffer. Table 2

[0183] Buffers 3, 7, and 10 were internal replicates used to determine run variability. As a control, a reaction buffer that had previously been shown to be effective in tailing reactions of 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 as the buffering reagent, 2.5 M alkali metal salt NaCl, and 100 mM MgCl2. The pH of the control buffer was 7.5.

[0184] Capillary gel electrophoresis was used to evaluate the tailing of the mRNA. Briefly, a Standard Sensitivity RNA Analysis Kit (15 nt) was purchased from Agilent and used for capillary electrophoresis runs on a Fragment Analyzer instrument (Agilent) with a twelve-capillary array. After 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 into each well of a 96-well plate. A molecular weight indicator ladder was prepared by mixing 2 μl of a Standard Sensitivity RNA ladder with 22 μl of the diluent marker. Sample injection was at 5.0 kV for 4 seconds and sample separation was at 8.0 kV for 60.0 min. The electropherogram of each sample was processed by ProSize2 software (Advanced Analytical) to yield a tabulation of the sizes (nt) and abundances of the fragments present in the sample.

[0185] As can be seen from Table 2, when the IVT mRNA containing modified ribonucleotides was tailed using a control buffer containing 250 mM NaCl, approximately 8% of the IVT mRNA remained untailed. Notably, the average tail length of approximately 1200 ribonucleotides far exceeded the target tail length of 500 ribonucleotides. Relative to the control buffer, all 12 tested conditions increased the tailing efficiency by at least 4-fold. The key parameter appears to be the alkali metal salt of the reaction, as reducing the alkali metal salt concentration 7 to 8-fold to approximately 30 mM increased the tailing efficiency relative to the reaction buffer used as a control. When the concentration of the alkali metal salt was reduced 50-fold to 5 mM while keeping the concentration of the buffer reagent at 50 mM unchanged, the tailing efficiency increased by more than 5-fold - see the control buffer and Buffer 8 in Table 2. When Buffer 8 was used, only 1.4% of the IVT mRNA remained untailed. Additionally, under these conditions, when tailing occurred, an average tail length of 450 ribonucleotides was achieved.

[0186] The overall reduction in the ionic strength of the reaction buffer led to a significant decrease in untailed IVT mRNA. However, in some cases, this decrease was accompanied by a shortening of the average tail length. For example, in addition to reducing the alkali metal salt concentration 50-fold, reducing the buffer reagent concentration 10-fold did not provide additional benefits - see Buffer 1 and Buffer 8 in Table 2. When Buffer 1 was used as the reaction buffer, the average tail length of 241 ribonucleotides was less than half of the required target length of 500 ribonucleotides.

[0187] This example demonstrates that optimizing the alkali metal salt concentration in the reaction buffer used for tailing the IVT mRNA containing modified ribonucleotides to 30 mM or lower (e.g., 5 mM or lower) increases the efficiency of the tailing reaction. Given that using Buffer 8 for the tailing reaction resulted in the lowest percentage of untailed IVT mRNA and an average tail length close to the required target length, this buffer composition was used as the basis for subsequent experiments. Example 3. Addition of Reducing Agent

[0188] This example shows that the tailing efficiency can be further increased by adding a reducing agent to the reaction buffer containing 30 mM or lower of an alkali metal salt.

[0189] As described in Example 2, 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 the modified ribonucleotide (N1-methylpseudouridine) (see Table 2), were tailed in either control buffer or buffer 8. Additionally, each IVT mRNA was also tailed in a modified version of buffer 8 that further included a reducing agent (DTT). The target tail length was 200 nucleotides. The resulting tailed IVT mRNAs were analyzed by capillary gel electrophoresis. Figures 1, 2, and 3 summarize the results obtained for mRNA A, mRNA B, and mRNA C, respectively. For each IVT mRNA, using the control buffer resulted in a clearly distinguishable additional peak prior to the main peak, indicating the presence of a large amount of untailed IVT mRNA (see panels A of Figures 1, 2, and 3). As observed in Example 2, compared to the control buffer, using buffer 8 significantly reduced the percentage of untailed IVT mRNA, as evidenced by a significant decrease in the size of the additional peak prior to 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 distinguishable, indicating a further reduction in the amount of untailed IVT mRNA (see panel C of Figures 1, 2, and 3).

[0190] Notably, despite using a relatively high concentration of the reducing agent (10 mM at 1x) in buffer 8, the performance of the polyA polymerase was not negatively affected. This was unexpected because while commercially available reaction buffers sometimes contain a reducing agent, it is typically present at a much lower concentration (e.g., 0.2 mM at 1x).

[0191] Furthermore, improvements due to a decrease in the concentration of alkali metal salts in the reaction buffer and a further increase in tailing efficiency achieved by adding a reducing agent to the reaction buffer were observed, which were independent of the ribonucleotide sequence of the IVT mRNA. In fact, similar improvements were observed for each of mRNA A, mRNA B, and mRNA C.

[0192] This example demonstrates that tailing efficiency can be further increased by adding a reducing agent to a reaction buffer that contains a reduced amount of alkali metal salts compared to the control buffer. The improvements observed were independent of the ribonucleotide sequence of the IVT mRNA. Example 4. Concentration of the reducing agent

[0193] This example demonstrates that tailing efficiency can be increased by adding a reducing agent at 5 mM or higher to a reaction buffer that contains 5 mM or less of an alkali metal salt.

[0194] To determine the appropriate concentration range of the reducing agent, the experiment described in Example 3 was repeated using different 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. Table 3 summarizes the results. Table 3

[0195] At 0 mM, an additional peak was observed before the main peak, indicating the presence of a large amount of untailed IVT mRNA. When the reducing agent was added to Buffer 8, the additional peak was no longer distinguishable, which was consistent with previous observations. No additional peaks were observed in the range of 5 - 50 mM reducing agent concentration.

[0196] This example demonstrates that the tailing efficiency can be improved by adding 5 mM or higher reducing agent to a reaction buffer containing 5 mM or lower alkali metal salts. Example 5. Improved tailing efficiency in the absence of modified ribonucleotides at the 3'-end

[0197] This example demonstrates that even when modified ribonucleotides are present as the terminal ribonucleotide at the 3'-end of the IVT mRNA, using the optimized reaction buffer (Buffer 8 containing DTT) identified in Example 3 improves tailing. The tailing efficiency is highest when the IVT mRNA does not include modified ribonucleotides at the 3'-end. Thus, this example also demonstrates that tailing of IVT mRNA containing modified ribonucleotides can be improved by providing untailed IVT mRNA that does not contain modified ribonucleotides at the 3'-end.

[0198] 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 plasmid with backbone (I) was linearized with HindIII, and the template plasmid with backbone (II) was linearized with BspQI. The IVT reaction including the linearized template DNA was carried out as described in Example 1. Modified uridine (N1-methylpseudouridine) was included in the reaction mixture instead of UTP. HindIII cleavage of the template produced IVT mRNA with modified ribonucleotides as the final ribonucleotides at the 3'-end. BspQI cleavage of the template produced IVT mRNA that did not include modified ribonucleotides at the 3'-end. The resulting IVT mRNA was tailed in the control buffer described in Example 2, or in Buffer 8 additionally containing 10 mM DTT described in Example 3.

[0199] FromFigure 4 It can be seen that, compared with the mRNA generated from the BspQ1 cleavage template, the polyadenylation efficiency of the IVT mRNA synthesized from the HindIII cleavage template and containing modified ribonucleotides at the 3' end is reduced.

[0200] Surprisingly, even in the presence of modified uridine at the 3' end, when buffer 8 containing 10 mM DTT is used in the polyadenylation reaction, only 10%-15% of the IVT mRNA remains non-polyadenylated. This is advantageous compared to the results obtained using the control buffer ( Figure 4 in which the polyadenylated HindIII-cleaved mRNA in the control buffer (buffer "C") was compared with the corresponding mRNA polyadenylated in buffer 8 containing 10 mM DTT (buffer "O")). Generally, when using the control buffer described in Example 2, the polyadenylation efficiency is approximately two-fold lower. In some cases, when using the control buffer, more than 30% of the IVT mRNA with modified ribonucleotides at the 3' end remains non-polyadenylated. Different from Example 3, the overall polyadenylation efficiency varies depending on the ribonucleotide sequence of the IVT mRNA. Sequence optimization can further improve the polyadenylation efficiency.

[0201] Notably, when the IVT mRNA is prepared from the BspQI cleavage template and thus has no modified ribonucleotides at the 3' end, using the control buffer results in up to 20% of the IVT mRNA remaining non-polyadenylated. In contrast, even when using the most challenging ribonucleotide sequences, when buffer 8 containing 10 mM DTT is used, the non-polyadenylated IVT mRNA does not exceed 5%. In fact, when buffer 8 containing 10 mM DTT is used, non-polyadenylated IVT mRNA is not detected in 3 out of 4 test samples.

[0202] 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 the polyadenylation efficiency, regardless of the ribonucleotides at the 3' end of the IVT mRNA or the mRNA ribonucleotide sequence. The polyadenylation efficiency is highest when the IVT mRNA does not include modified ribonucleotides at the 3' end. Thus, this example also demonstrates that the polyadenylation of IVT mRNA containing modified ribonucleotides can be improved by providing non-polyadenylated IVT mRNA that does not contain modified ribonucleotides at the 3' end. Interestingly, compared with the non-optimized reaction buffer, the optimized reaction buffer (buffer 8 containing DTT) even improves the polyadenylation efficiency of the IVT mRNA with modified ribonucleotides at the 3' end. Example 6. Use of an Optimized Reaction Buffer in the Case of Unmodified IVT mRNA

[0203] 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 consisting only of unmodified ribonucleotides.

[0204] In parallel with the experiment described in Example 4, a tailing reaction was performed using IVT mRNA prepared only with unmodified naturally occurring ribonucleotides (ATP, GTP, CTP, and UTP). The tailing conditions were otherwise as described in Example 4. The concentration of the reducing agent in Buffer 8 was varied as described therein. The resulting tailed unmodified IVT mRNA was analyzed by capillary gel electrophoresis. Table 4 summarizes the results. Table 4

[0205] Under the test conditions of Example 4, no additional peaks were discerned in any of the samples containing unmodified IVT mRNA. Thus, this example demonstrates that the reaction buffer of the present invention can also be used for tailing IVT mRNA consisting only of unmodified ribonucleotides. Example 7. Large-scale tailing of modified IVT mRNA

[0206] This example demonstrates that the optimized reaction buffer of the present invention can be used for large-scale ( > 1 g IVT mRNA per batch) tailing of IVT mRNA containing modified ribonucleotides.

[0207] Four IVT mRNAs (mRNA 1 - 4) with different nucleic acid sequences and lengths (1941, 1941, 1989, and 1995 nucleotides, respectively) before tailing were tailed using the optimized reaction buffer identified in Example 4 (Buffer 8 containing 10 mM DTT). Each batch contained approximately 16 g of untailed IVT mRNA containing modified ribonucleotides (N1-methylpseudouridine instead of uridine). The IVT mRNA was prepared as described in Example 1. The tailing conditions were selected to add a polyA tail to the IVT mRNA to a length of 200 nucleotides (desired tail length).

[0208] Capillary gel electrophoresis was used to determine the average length of the added polyA tail and the presence of untailed mRNA. Table 5 summarizes the results. Table 5 mRNA Tail length (nt) Undoped mRNA detected 1 208 No 2 203 No 3 205 No 4 254 No

[0209] 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.

[0210] Using capillary gel electrophoresis, the corresponding polyadenylation reactions of ten 10 μg batches carried out using the unoptimized control buffer described in Table 2 were compared with four 16 μg batches prepared in this example. The average length of the added poly-A tail was plotted on Figure 5 the graph shown. The average tail length obtained in the polyadenylation reaction using the unoptimized control buffer was close to 300 nucleotides. As can be seen from Figure 5 , for most of the test batches (9 out of 10), the average tail length obtained using the unoptimized control buffer (labeled buffer "C") was 45%-50% longer than the desired tail length. In contrast, using the optimized reaction buffer of the present invention (labeled buffer "O"), the average tail length of three out of four test batches was only about 5% longer than the desired tail length. Even for the worst-performing batch, when using the optimized reaction buffer, the average tail length was only about 25% longer than the desired tail length.

[0211] This example demonstrates that the optimized reaction buffer of the present invention can be used for large-scale (>1 μg) polyadenylation of IVT mRNA batches containing modified ribonucleotides. The resulting average tail length is close to the desired length (typically within 5% of the desired value). This example further confirms that when using the optimized reaction buffer of the present invention, the nucleic acid sequence of the IVT mRNA does not affect the polyadenylation efficiency.

Claims

1. A method for tailing an 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. The method according to claim 1, wherein the IVT mRNA comprises a 5' cap.

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

4. The method according to any one of claims 1-3, wherein the concentration of the alkali metal salt in the reaction buffer is from about 1 mM to about 30 mM.

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

6. The method according to any one of the preceding claims, wherein the concentration of the reducing agent in the reaction buffer is 5 mM to 50 mM.

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

8. The method according to claim 7, wherein the concentration of the reducing agent in the reaction buffer is about 10 mM.

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

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

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

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

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

14. The method according to any one of the preceding claims, wherein the modified ribonucleotide is modified uridine.

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

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

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

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

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

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

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

22. The method according to any one of the preceding claims, wherein the mRNA tail comprises about 100 to about 800 ribonucleotides.

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

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

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

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

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

5.

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

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

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

32. The method according to any one of the preceding claims, wherein the reaction buffer maintains the activity of the polyadenylation polymerase by providing divalent cations.

33. The method according to claim 32, wherein the divalent cation is selected from Mg 2+ and Mn 2+ .

34. The method according to claim 32 or 33, wherein the divalent cations are present at a concentration of from about 5 mM to about 20 mM.

35. The method according to claim 34, wherein the concentration of the divalent cations is from about 5 mM to about 10 mM.

36. The method according to claim 35, wherein the concentration of the divalent cations is about 10 mM.

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

38. The method according to any one of the preceding claims, wherein the IVT mRNA does not contain modified uridine at the 3'-end.

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

40. The method according to any one of the preceding claims, wherein the polyadenylation polymerase is poly(A) polymerase.

41. The method according to claim 40, wherein the poly(A) polymerase is a bacterial poly(A) polymerase or a yeast poly(A) polymerase.

42. The method according to claim 41, wherein the poly(A) polymerase is Escherichia coli poly(A) polymerase.

43. The method according to any one of claims 40 - 42, wherein the reaction buffer comprises an appropriate concentration of ATP.

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

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

46. The method according to claim 45, wherein the concentration of the alkali metal salt in the reaction buffer is from about 1 mM to about 30 mM.

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

48. The reaction buffer according to any one of claims 45 - 47, wherein the concentration of the reducing agent in the reaction buffer is from 5 mM to 50 mM.

49. The reaction buffer according to claim 48, wherein the concentration of the reducing agent in the reaction buffer is from 5 mM to 20 mM.

50. The reaction buffer according to claim 49, wherein the concentration of the reducing agent in the reaction buffer is about 10 mM.

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

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

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

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

55. The reaction buffer according to any one of claims 45 - 54, wherein the reaction buffer has a pH of from about pH 7 to about pH 8.

56. The reaction buffer according to claim 55, wherein the reaction buffer has a pH of about pH 7.

5.

57. The reaction buffer according to claim 55 or 56, wherein the reaction buffer contains Tris, HEPES, MOPS, acetate, citrate, or phosphate as a buffering reagent.

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

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

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

61. The reaction buffer according to any one of claims 45 - 60, wherein the reaction buffer contains divalent cations.

62. The reaction buffer according to claim 61, wherein the divalent cation is selected from Mg 2+ and Mn 2+ .

63. The reaction buffer according to claim 62, wherein the reaction buffer contains MgCl2 or MnCl2.

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

65. The reaction buffer according to claim 64, wherein the concentration of the divalent cations is about 10 mM.

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

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

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

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

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

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

72. The reaction buffer according to claim 71, wherein the concentration of the reducing agent in the reaction buffer is about 100 mM.

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

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

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

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

77. The reaction buffer according to any one of claims 67-76, wherein the reaction buffer has a pH of about pH 7 to about pH 8.

78. The reaction buffer according to claim 77, wherein the reaction buffer has a pH of about pH 7.

5.

79. The reaction buffer according to claim 77 or 78, wherein the reaction buffer comprises Tris, HEPES, MOPS, acetate, citrate, or phosphate as a buffering reagent.

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

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

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

83. The reaction buffer according to any one of claims 67-82, wherein the reaction buffer comprises divalent cations.

84. The reaction buffer according to claim 83, wherein the divalent cation is selected from Mg 2+ and Mn 2+ .

85. The reaction buffer according to claim 84, wherein the divalent cations comprise MgCl2 or MnCl2.

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

87. The reaction buffer according to claim 86, wherein the concentration of the divalent cations is about 100 mM.

88. A method for generating in vitro transcribed (IVT) messenger RNA (mRNA) comprising modified ribonucleotides, the method comprising: (i) preparing a DNA template, wherein the 3'-terminal residue of the DNA template does not encode the modified ribonucleotide of the IVT mRNA; and (ii) transcribing the DNA template with an RNA polymerase in an in vitro transcription (IVT) reaction comprising the modified ribonucleotide.

89. The method according to claim 88, wherein the DNA template is a circular vector comprising a restriction site.

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

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

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

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

94. The method according to claim 93, wherein the concentration of the alkali metal salt in the reaction buffer is 5 mM or lower.

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

96. The method according to claim 95, wherein the reaction buffer comprises an alkali metal salt at 30 mM or lower and a reducing agent at 5 mM or higher.

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

98. The method according to claim 96 or 97, wherein the concentration of the alkali metal salt in the reaction buffer is 5 mM or lower.

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

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

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

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