Production of messenger RNA using KP34 polymerase

Abstract

The present disclosure provides methods and compositions for improving in vitro transcription (IVT) of messenger RNA using Klebsiella phage KP34 RNA polymerase. In particular, the present disclosure provides a method for producing messenger RNA, comprising: (a) providing a DNA template comprising a nucleic acid sequence encoding an mRNA transcript for expression of a polypeptide or protein; and (b) contacting the DNA template with Klebsiella phage KP34 RNA polymerase under conditions suitable for IVT of the mRNA transcript.

Description

[Technical Field]

[0001] Sequence Listing This specification refers to a Sequence Listing (submitted electronically on March 7, 2024 under the .xml file name "2024-03-07 PAT23003-WO-PCT Sequence Listing"). This .xml file was created on March 7, 2024 and is 47KB in size. The entire contents of the Sequence Listing are incorporated herein by reference.

[0002] The present invention relates generally to methods and compositions for improving in vitro transcription (IVT) of messenger RNA (mRNA). In particular, the present invention relates to the use of Klebsiella phage KP34 polymerase in the production of mRNA. [Background technology]

[0003] Messenger RNA (mRNA) is becoming increasingly important as a therapeutic agent. mRNA therapy can be used to restore normal levels of endogenous proteins or to provide exogenous therapeutic proteins (e.g., vaccine antigens or antibodies) without permanently altering the genome sequence or entering the cell's nucleus. mRNA therapy utilizes a cell's own protein production and processing machinery to express therapeutic peptides, polypeptides, or proteins, is amenable to customized dosing and formulation, and can be broadly applied to any disease or condition that can be treated by providing exogenous proteins.

[0004] The process of producing mRNA for therapeutic use typically involves in vitro transcription (IVT) of mRNA from a DNA template using a phage-derived DNA-dependent RNA polymerase. This synthesis process generally produces transcription by-products in addition to the desired mRNA transcript. For example, T7 RNA polymerase produces double-stranded RNA (dsRNA) during IVT. Double-stranded RNA (dsRNA) is undesirable because it leads to inefficient translation of the administered mRNA product, induces cytokines, and triggers interferon (IFN)-mediated inflammatory immune responses.

[0005] For example, activation of dsRNA-dependent enzymes such as oligoadenylate synthetase (OAS), RNA-specific adenosine deaminase (ADAR), and RNA-activated protein kinase (PKR) can trigger cellular responses, inhibiting protein synthesis and potentially reducing the efficacy of mRNA therapy. dsRNA can also stimulate cellular pathogen sensors, such as Toll-like receptor 3 (TLR3), retinoic acid-inducible gene I (RIG-I), and melanoma differentiation-associated protein 5 (MDA5), leading to the secretion of various cytokines, including type I interferon, interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α).

[0006] Because dsRNA is highly immunogenic, there are obstacles to using mRNA containing dsRNA contamination.Therefore, it is desirable to eliminate or significantly reduce the amount of dsRNA from IVT mRNA for many reasons, such as limiting cytokine induction and reducing protein synthesis.

[0007] Kariko et al. (Nucleic Acids Res. 2011;39(21);e142) used high-performance liquid chromatography (HPLC) to remove dsRNA from mRNA transcripts prepared by in vitro transfection using T7 polymerase. In in vitro transfection experiments, HPLC-purified mRNA was up to 1,000-fold more potent at inducing the expression of mRNA-encoded proteins. However, HPLC-based purification is not easily scalable, is expensive, and often requires the use of toxic organic solvents that must be subsequently removed by additional purification steps. Therefore, efforts have been made to develop alternative purification methods. For example, Baiersdoerfer et al. (Mol Ther Nucleic Acids. 2019;15;26-35) described a cellulose-based chromatography method for removing dsRNA contaminants. This method avoids the use of toxic organic solvents, is more cost-effective, and does not face the same scalability issues as previous HPLC-based methods. The use of higher concentrations of ethanol further improved the efficiency of dsRNA removal using cellulose-based methods, however, these concentrations resulted in reduced recovery of single-stranded in vitro transcribed mRNA (Kwon et al., Arch Pharm Res. 2022;45(4);245-262).

[0008] To avoid the time-consuming and expensive purification of mRNA after IVT, efforts have focused on reducing the formation of transcription by-products such as dsRNA. For example, SP6 RNA polymerase produces less dsRNA than T7 RNA polymerase (see, e.g., WO 2022 / 082001 and WO 2018 / 157153). Recently, DNA-dependent RNA polymerases phylogenetically distant from T7 and SP6 have been discovered (Xia et al., RNA Biol. 2022;19(1);1130-1142). These enzymes produce much less dsRNA than the short RNA transcripts (less than 100 bases) of T7 and SP6 RNA polymerases during IVT, but often at reduced yields compared to T7 and SP6.

[0009] Therefore, there is a need for methods that can be used at scale to produce mRNA (typically greater than 500 bases in length) for expressing therapeutic polypeptides or proteins while producing only negligible amounts of transcription by-products. Summary of the Invention [Means for solving the problem]

[0010] The present invention is based on the discovery that purified Klebsiella phage KP34 RNA polymerase can be used to produce mRNA in place of the commonly used RNA polymerases T7 and SP6. One obstacle to using this polymerase to produce mRNA has been low mRNA yields. The inventors found that recombinantly expressed Klebsiella phage KP34 RNA polymerase in bacterial cells forms enzymatically inactive aggregates. By removing these aggregates or avoiding their formation in the first place (e.g., by genetic engineering), the yield of long RNA transcripts (≥500 ribonucleotides) encoding polypeptides and proteins during IVT can be dramatically improved. The inventors also found that by optimizing the nucleic acid sequence 3′ adjacent to the KP34 core promoter, mRNA yields during in vitro synthesis can be further improved, reaching levels comparable to those of SP6. Furthermore, compared to T7 and SP6, Klebsiella phage KP34 RNA polymerase produces significantly fewer transcription by-products, such as dsRNA, making it particularly suitable for producing mRNA for therapeutic use.

[0011] Accordingly, the present invention relates to methods for producing mRNA, comprising: (a) providing a DNA template comprising a nucleic acid sequence encoding an mRNA transcript for expression of a polypeptide or protein, and (b) contacting the DNA template with Klebsiella phage KP34 RNA polymerase under conditions suitable for IVT of the mRNA transcript. In some embodiments, the mRNA transcript is for expressing a therapeutic polypeptide or protein (e.g., for therapeutic use).

[0012] In certain embodiments, the Klebsiella phage KP34 RNA polymerase provided in step (b) is recombinantly expressed in Escherichia coli (E. coli) cells and purified to remove enzymatically inactive aggregates. In some embodiments, enzymatically inactive aggregates are removed from an affinity-purified preparation comprising Klebsiella phage KP34 RNA polymerase. In some embodiments, enzymatically inactive aggregates are removed by size-exclusion chromatography (e.g., gel filtration). In some embodiments, the Klebsiella phage KP34 RNA polymerase contains less than 5% enzymatically inactive aggregates (e.g., less than 4%, less than 3%, less than 2%, or less than 1%).

[0013] In some embodiments, the amino acid sequence of the Klebsiella phage KP34 RNA polymerase is at least 90% identical (e.g., at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical, or identical to) the amino acid sequence of SEQ ID NO: 1. In some embodiments, the Klebsiella phage KP34 RNA polymerase is present at a concentration ranging from 0.01 to 0.5 mg / mL. In some embodiments, the mRNA transcript comprises at least 500 ribonucleotides, e.g., at least 600, 700, 800, 900, 1000 ribonucleotides.

[0014] In some embodiments, the amount of dsRNA contained in the mRNA transcript obtained in step (b) is below the detection limit. The presence of dsRNA produced during IVT can be assessed by dot blot assay using an anti-dsRNA monoclonal antibody (mAb), such as J2 mAb, K1 mAb, or K2 mAb. In certain embodiments, the dot blot assay uses J2 mAb. Alternatively, the presence of dsRNA produced during IVT can be assessed by ELISA using anti-dsRNA mAbs, such as J2 and K1 mAb, or K1 and K2 mAb. In certain embodiments, the ELISA uses J2 and K1 mAb.

[0015] In some embodiments, the amount of dsRNA produced in step (b) is at least 10-fold lower (e.g., 20-fold, 50-fold, or 100-fold lower) than a corresponding method using SP6 RNA polymerase or T7 RNA polymerase instead of Klebsiella phage KP34 RNA polymerase. The amount of dsRNA can be determined by ELISA using anti-dsRNA mAbs, such as J2 and K1 mAbs or K1 and K2 mAbs, typically J2 and K1 mAbs. In some embodiments, the amount of dsRNA is determined by dot blot assay using anti-dsRNA mAbs, such as J2 mAb, K1 mAb, or K2 mAb, typically J2 mAb.

[0016] In some embodiments, 10% or less by weight of the mRNA transcripts obtained in step (b) contain non-template nucleic acids. In some embodiments, the mRNA transcripts obtained in step (b) contain less than 10% by weight of dsRNA (e.g., less than 5%, less than 3%, or less than 1%). In some embodiments, the amount of dsRNA is determined by ELISA using antibodies J2 and K1, or K1 and K2, typically J2 and K1.

[0017] In some embodiments, less than 10% by weight of the mRNA transcripts obtained in step (b) are abortive transcripts. In some embodiments, the abortive transcripts comprise fewer than 20 nucleotides. For example, in some embodiments, the abortive transcripts have a length of 2-19 nucleotides, e.g., 5-15 nucleotides. In some embodiments, the abortive transcripts are detectable by gel electrophoresis.

[0018] In some embodiments, the method involves synthesizing at least 1 mg of mRNA (eg, at least 10 mg, at least 100 mg, or at least 1 g) in a single batch.

[0019] In some embodiments, the DNA template comprises a Klebsiella phage KP34 promoter sequence operably linked to a nucleic acid sequence encoding an mRNA transcript, hi some embodiments, the promoter sequence comprises the nucleic acid sequence set forth in SEQ ID NO:5.

[0020] In some embodiments, the promoter sequence is optimized to improve the yield of mRNA transcript. In some embodiments, the yield of mRNA transcript obtained in step b) is equivalent to the yield achieved by a corresponding method using SP6 RNA polymerase or T7 RNA polymerase instead of Klebsiella phage KP34 RNA polymerase. In some embodiments, the promoter sequence comprises the nucleic acid sequence set forth in SEQ ID NO: 7, 8, 9, or 41. In some embodiments, the promoter sequence comprises the nucleic acid sequence set forth in SEQ ID NO: 7 or 8. In some embodiments, the promoter sequence comprises the nucleic acid sequence set forth in SEQ ID NO: 19 or 20. In some embodiments, the promoter sequence comprises the nucleic acid sequence set forth in SEQ ID NO: 26, 32, or 35, e.g., SEQ ID NO: 26.

[0021] In some embodiments, the DNA template is at a concentration of 0.05 mg / mL to 0.5 mg / mL. In some embodiments, the DNA template is linear or linearized.

[0022] In some embodiments, IVT is performed in the presence of magnesium chloride (MgCl). In some embodiments, the concentration of MgCl is greater than 20 mM. In some embodiments, the concentration of MgCl is about 25 mM.

[0023] In some embodiments, IVT is performed in the presence of sodium chloride (NaCl). In some embodiments, the concentration of NaCl is less than 20 mM. In some embodiments, the concentration of NaCl is about 0.5 mM.

[0024] In some embodiments, IVT is performed in the presence of a buffer. In some embodiments, the buffer is selected from Tris, HEPES, ammonium sulfate, sodium bicarbonate, sodium citrate, sodium acetate, potassium phosphate, and sodium phosphate. In some embodiments, the buffer is Tris-HCl. In some embodiments, Tris-HCl is present at a concentration of less than 40 mM. In some embodiments, Tris-HCl is present at a concentration of less than 25 mM.

[0025] In some embodiments, IVT is performed in the presence of unmodified ribonucleotides. In some embodiments, IVT is performed in the presence of modified ribonucleotides.

[0026] In some embodiments, the modified ribonucleotide comprises a modified nucleoside. In some embodiments, the modified nucleoside is selected from 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine, 3-methyladenosine, 5-methylcytidine, C-5 propynyl-cytidine, C-5 propynyl-uridine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-methylcytidine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, O(6)-methylguanine, pseudouridine, 2-thiouridine, and 2-thiocytidine. In some embodiments, the modified nucleoside is pseudouridine, N1-methylpseudouridine, 5-methylcytidine, or 5-methoxyuridine, typically N1-methylpseudouridine.

[0027] In some embodiments, IVT is performed in the presence of ribonucleotides, with each ribonucleotide being present at a concentration of 0.1 mM to 10 mM.

[0028] In some embodiments, IVT is performed at a pH of 7.0 to 7.7. In some embodiments, the pH is about 7.5.

[0029] In some embodiments, the IVT is performed at a temperature between 37° C. and 42° C. In some embodiments, the temperature is 37° C.

[0030] In some embodiments, the IVT reaction is carried out over a period of 30 minutes to 6 hours.

[0031] In some embodiments, the IVT is terminated by the addition of DNase I and DNase I buffer.

[0032] In some embodiments, the method for producing mRNA according to the invention further comprises purifying the mRNA transcript obtained in step (b) from Klebsiella phage KP34 RNA polymerase (and optionally other reactants and enzymes present after completion of IVT). In some embodiments, the step of purifying the mRNA transcript comprises (i) cellulose chromatography and / or (ii) a method other than HPLC using a buffer system comprising triethylammonium acetate and / or acetonitrile.

[0033] The present invention also relates to compositions obtainable by the methods for producing mRNA disclosed herein. For example, the present invention provides a composition comprising an mRNA transcript for expressing a polypeptide or protein and Klebsiella phage KP34 RNA polymerase, wherein the composition contains less than 1% dsRNA by weight and less than 10% of the mRNA transcripts by weight are abortive transcripts.

[0034] The present invention also includes mRNA obtained using the methods of the present invention. Such mRNA can be distinguished from prior art mRNA preparations in that it does not contain residual cellulose or organic solvents (i.e., components used to purify IVT mRNA produced by other RNA polymerases, such as T7). Such mRNA can also contain substantially smaller amounts of contaminating transcription by-products, such as dsRNA, or other by-products of IVT that can be difficult to remove by purification.

[0035] The present invention also relates to pharmaceutical compositions comprising mRNA obtained by the methods for producing mRNA disclosed herein (especially mRNA encoding a therapeutic polypeptide or protein), and their therapeutic uses, e.g., in methods for treating or preventing a disease or disorder in a subject.

[0036] Other features, objects, and advantages of the present invention will be apparent from the following detailed description, drawings, and embodiments. It should be understood, however, that the detailed description, drawings, and embodiments, while indicating embodiments of the present invention, are given by way of example only, not by way of limitation. Various changes and modifications will be apparent to those skilled in the art.

[0037] Embodiments of the present invention will now be described, by way of example only, with reference to the following drawings, in which: [Brief explanation of the drawings]

[0038] [Figure 1A-1B] Affinity-based purification of Klebsiella phage KP34 RNA polymerase is illustrated in Figure 1A. An SDS-PAGE gel is shown. From left to right, the following samples were loaded onto the gel: E. coli whole-cell lysate ("Lysate"), pellet fraction resuspended in loading buffer for IMAC ("Ni Load"), flow-through collected after loading ("Ni FT"), fraction collected after washing the loaded IMAC column ("Ni Wash"), and various elution fractions collected by adding increasing concentrations of imidazole to elution buffer ("Ni Elution" labeled 1A6, 1A7, 1A8, 1A9, 1A10, 1A11, 1A12), and SeeBlue™ prestained protein standards (the molecular weights of the protein standards are indicated in kDa to the right of the gel image). The band corresponding to Klebsiella phage KP34 RNA polymerase ("KP34") is indicated by an arrow. Figure 1B shows a chromatogram of the eluate from a Superdex™ 75 gel filtration column. UV absorbance was detected at 280 nm. Elution volume (mL) is plotted against UV absorbance (mAU). Elutions containing buffers containing increasing concentrations of imidazole are indicated by vertical lines at the bottom of the graph. The main peak corresponding to purified KP34 is indicated by an arrow. [Figure 2]This figure illustrates the lack of RNA-dependent 3'-end extension of mRNA transcripts when Klebsiella phage KP34 RNA polymerase is used for in vitro transcription (IVT). In an RNA-dependent RNA polymerase (RdRp) assay, T7, SP6, and KP34 RNA polymerases (RNAP) were tested at concentrations of 0 μM (negative control), 0.1 μM, 0.2 μM, or 0.6 μM. The polymerase was incubated with 0.4 μM of a 50-base synthetic RNA (RNA50) as a template. The template was allowed to self-anneal. Upon annealing of the internal region of complementarity, the formation of a stretch of dsRNA occurs in cis by forming a loop, allowing self-templated 3'-end extension. The template was incubated in the presence of unmodified ATP, CTP, GTP, and UTP, as described in Example 5. After incubation, each sample was separated on a gel to determine whether 3'-end extension had occurred. RNA was detected using SYBR Gold staining. The first lane, labeled "M," corresponds to a molecular weight ladder, with molecular weights indicated in nucleotides (nt) to the left of the gel image. A representative band corresponding to RNA 50 is indicated by a line. A representative band containing 3'-extended RNA is enclosed by two lines. [Figure 3] This figure illustrates that comparable mRNA yields are obtained from IVT reactions performed using either SP6 RNA polymerase or Klebsiella phage KP34 RNA polymerase. Four DNA templates were tested (labeled as sequences 2, 3, 4, or 5). Templates ranged in size from approximately 1100 bp to approximately 4700 bp. The control DNA template (labeled as sequence 1 in Figure 3) corresponds to DNA template number 1 specified in Example 3. The average IVT yields from SP6 (left bar) and KP34 (right bar) for each sequence tested are shown. Error bars indicate standard deviation. [Figure 4]This figure illustrates that IVT using Klebsiella phage KP34 RNA polymerase results in undetectable amounts of dsRNA in dot blots. IVT was performed with SP6 RNA polymerase ("SP6") or Klebsiella phage KP34 RNA polymerase ("KP34") in the presence of unmodified or modified ribonucleotides, as described in Example 2. The resulting mRNA transcripts are labeled unmodified or modified accordingly (denoted as "Unmodified" or "Mod"). 100 ng, 200 ng, or 400 ng of RNA in a 2 μL sample volume was blotted onto a nitrocellulose membrane. 1 ng, 20 ng, or 40 ng of dsRNA controls were used as references. Anti-dsRNA monoclonal antibody J2 was used as the primary antibody. Anti-mouse IgG HRP was used as the secondary antibody. Signals were detected after a 1-minute exposure. The amount of dsRNA in samples prepared with Klebsiella phage KP34 RNA polymerase was several-fold lower than the amount of dsRNA produced when SP6 RNA polymerase was used for IVT under identical conditions. [Figure 5A-5B]Figure 5A illustrates the removal of enzymatically inactive aggregates from affinity-based purification of Klebsiella phage KP34 RNA polymerase. Figure 5A shows a chromatogram of the eluate from a Superdex™ 200 gel filtration column. UV absorbance was detected at 280 nm (light gray, dashed line) and 400 nm (dark gray, solid line). Elution volume (mL) is plotted against UV absorbance (mAU). Two peaks, labeled I and II, were identified. Both peaks corresponded to purified Klebsiella phage KP34 RNA polymerase. The peak I fraction eluted earlier, consistent with its larger particle size than the peak II fraction. The collected elution fractions are indicated by vertical lines at the bottom of the graph and were analyzed by SDS-PAGE. Figure 5B shows a representative SDS-PAGE gel confirming that both Peak I and Peak II fractions contain Klebsiella phage KP34 RNA polymerase. From left to right, the following samples were loaded onto the gel: SeeBlue™ prestained protein standards (the molecular weights of the protein standards are indicated in kDa to the left of the gel image), the crude sample loaded onto the gel filtration column ("SEC load"), and various elution fractions collected during the gel filtration process ("SEC 1A9," "SEC 1A11," "SEC 1B1," "SEC 1B3," "SEC 1B5," "SEC 1C2," "SEC 1C4," "SEC 1C6," "SEC 1C8," and "SEC 1C10"). The elution fractions enclosed by a solid black border correspond to Peak I fractions. The elution fractions enclosed by a dashed border correspond to Peak II fractions. The band corresponding to KP34 is indicated by an arrow. [Figure 6]This figure illustrates the amount of abortive transcripts formed by purified Klebsiella phage KP34 RNA polymerase during IVT reactions using DNA templates containing different promoter sequences. The bar graph represents the amount of peak area per μg of sample analyzed by liquid chromatography-mass spectrometry (LC-MS) as described in Example 11. Values ​​are the sum of all peaks from 6.5 to 12 minutes from duplicate samples. Sample IDs correspond to those in Table 11. For comparison, the amount of abortive transcripts formed by purified SP6 and T7 RNA polymerases during IVT was also measured (labeled "SP6" and "T7" accordingly). DNA templates containing the optimized KP34 promoter sequence of SEQ ID NO:26 or SEQ ID NO:35 (sample IDs 4 and 9) showed particularly low levels of short abortive transcripts in IVT. [Figure 7A] Figure 7A illustrates the results from a screen for solubility tags designed to improve the expression and solubility of Klebsiella phage KP34 RNA polymerase during recombinant expression in Escherichia coli (E. coli). In Figure 7A, each bar represents the protein concentration (mg / mL) of a fusion protein containing Klebsiella phage KP34 RNA polymerase and the indicated tag, purified by immobilized metal ion affinity chromatography (IMAC), and desalted. Fusion proteins marked with * and ** yielded statistically significantly higher concentrations (p-values ​​of 0.05 and <0.05, respectively) compared to the wild-type KP34 enzyme (WT). The level of WT protein is indicated by the dashed line. For comparison, SP6 RNA polymerase was expressed and purified using the same process. [Figure 7B]Figure 7 illustrates the results from a screen for solubility tags designed to improve expression and solubility of Klebsiella phage KP34 RNA polymerase during recombinant expression in Escherichia coli (E. coli). Fusion proteins that resulted in significantly higher yields were tested for their solubility compared to WT, as summarized in the bar graphs shown in Figure 7B. The fold increase in solubility compared to WT is shown in each bar. Highly expressed fusion proteins had higher solubility compared to WT. [Figure 8] Figure 7A illustrates the results of in vitro transcription (IVT) reactions using each of the polymerase fusion proteins tested in the lysis tag screen shown in Figure 7A. IVT reactions were performed as described in Example 3. The resulting RNA concentrations are shown in ng / μL. Despite normalizing for protein concentration prior to the IVT assay, the fusion proteins were expressed at significantly higher levels, had improved solubility compared to the WT, and also tended to yield more mRNA for the reaction. DETAILED DESCRIPTION OF THE INVENTION

[0039] definition In order that the present invention may be more readily understood, certain terms are first defined below. Additional definitions for these and other terms are set forth throughout the specification.

[0040] As used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. For example, "a ribonucleotide" is understood to refer to one or more ribonucleotides. Thus, the terms "a" (or "an"), "one or more," and "at least one" can be used interchangeably herein.

[0041] Unless specifically stated otherwise or clear from the context, as used herein, the term "or" is understood to be inclusive, including both "or" and "and." Furthermore, as used herein, "and / or" should be considered a specific disclosure of each of the two particular features or components, regardless of the presence or absence of other features or components. Thus, when the term "and / or" is used herein in phrases such as "A and / or B," it is intended to include "A and B," "A or B," "A" (alone) and "B" (alone). Similarly, when the term "and / or" is used in phrases such as "A, B and / or C," it is intended to encompass each of the following 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).

[0042] Throughout the specification and embodiments, the words "have" and "comprise," or variations such as "has," "having," "comprises," or "comprising," are understood to mean the inclusion of a specified integer or group of integers, but not the exclusion of any other integer or group of integers. It is further understood that whenever the words "comprising" or "having" are described herein along with their grammatical equivalents, similar embodiments separately described with the terms "consisting of" and / or "consisting essentially of" are also provided.

[0043] As used herein, the term "about" refers to an interval of accuracy that a person skilled in the art would understand to ensure that the technical effect of the target feature is still ensured. This term indicates a deviation of ±10% from the indicated numerical value. In some embodiments, the deviation is ±5% of the indicated numerical value. In some embodiments, the deviation is ±1% of the indicated numerical value.

[0044] As used herein, the term "mRNA" refers to a polyribonucleotide that encodes at least one polypeptide. As used herein, mRNA encompasses both modified and unmodified RNA. mRNA can contain one or more coding and non-coding regions (e.g., 5' and 3' untranslated regions). mRNA can be purified from natural sources, produced using recombinant expression systems, optionally purified, in vitro transcribed, or chemically synthesized. The present invention particularly relates to in vitro transcribed mRNA. Optionally, for example, in the case of chemically synthesized molecules, mRNA can contain nucleoside analogs, such as analogs with chemically modified bases or sugars, backbone modifications, etc. mRNA sequences are presented in the 5' to 3' direction unless otherwise indicated. A typical mRNA includes a 5' cap, a 5' untranslated region (5' UTR), a protein coding region, a 3' untranslated region (3' UTR), and a 3' tail. In some embodiments, the tail structure is a poly(C) tail. More typically, the tail structure is a polyA tail.

[0045] As used herein, the terms "Klebsiella phage KP34 RNA polymerase," "KP34 RNA polymerase," "KP34 polymerase," and "KP34" are used interchangeably and all refer to a DNA-dependent RNA polymerase obtainable from Klebsiella phage (e.g., an RNA polymerase having the amino acid sequence set forth in SEQ ID NO: 1).

[0046] As used herein to describe the amino acid sequence of Klebsiella phage KP34 RNA polymerase, the term "naturally occurring" refers to the wild-type or native amino acid sequence. In some embodiments, the Klebsiella phage KP34 RNA polymerase disclosed herein can include one or more amino acid substitutions, deletions, insertions, and / or additions relative to the naturally occurring amino acid sequence to make the protein more suitable for use in the methods and compositions of the invention. Without wishing to be bound by any particular theory, the inventors believe that the polymerase function of such modified enzymes will be essentially the same or have improved polymerase activity compared to the wild-type or native enzyme.

[0047] As used herein, the term "sequence-optimized" is used to describe a nucleotide sequence that has been modified compared to a naturally occurring or wild-type nucleotide sequence. In the case of a sequence-optimized mRNA, such modifications can include, for example, codon optimization and / or the use of 5'UTRs and 3'UTRs not normally 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 altering its amino acid sequence, thereby improving protein expression of the nucleic acid. In the context of the present invention, "codon optimization" can also refer to the process of arriving at one or more optimized nucleotide sequences by filtering out suboptimal nucleotide sequences from a list of nucleotide sequences, for example, by sorting by guanine-cytosine (GC) content, codon adaptation index (CAI), the presence of unstable nucleic acid sequences or motifs, and / or the presence of pause sites and / or termination signals.

[0048] As used herein, the term "template DNA" (or "DNA template") refers to a DNA molecule containing a nucleic acid sequence encoding an RNA transcript to be synthesized by IVT. Template DNA is used as a template for IVT to produce an mRNA transcript encoded by the template DNA. Template DNA contains all elements required for IVT, in particular, a promoter element for binding DNA-dependent RNA polymerase, operably linked to a DNA sequence encoding the desired mRNA transcript. Furthermore, template DNA may contain primer binding sites 5' and / or 3' of the DNA sequence encoding the mRNA transcript for determining the identity of the DNA sequence encoding the mRNA transcript, for example, by PCR or DNA sequencing. In the context of the present invention, "template DNA" may be a linear or circular DNA molecule. As used herein, the term "template DNA" may refer to a DNA vector, such as a plasmid DNA, containing a nucleic acid sequence encoding the desired mRNA transcript.

[0049] The terms "short abortive transcript" and "abortive transcript" are used interchangeably herein. They refer to transcripts generated upon failure of transcription initiation by RNA polymerase. Abortive transcripts are commonly observed during in vitro transcription (IVT) reactions. Abortive transcripts typically contain fewer than approximately 20 nucleotides. During in vitro mRNA synthesis, RNA polymerase (RNAP) recognizes its cognate promoter, resulting in local melting of the double-stranded DNA template, forming a transcription "initiation complex." Transcription during this phase is characterized by the repetitive synthesis and release of 2–6 nucleotides, termed "abortive cycling," which is common to all RNAPs. Abortive transcripts are present in in vitro reactions, even at saturating nucleotide concentrations, but their length varies among different RNAPs. After synthesizing approximately 8–12 nucleotides, the polymerase undergoes extensive structural rearrangements, dissociates from the promoter (promoter clearance), and begins processive RNA synthesis, forming an "elongation complex" until transcription terminates. Because the initiation complex is unstable compared to the elongation complex, abortive transcripts are repeatedly released until the polymerase engages in productive transcription to produce a full-length transcript.

[0050] As used herein, the term "truncated transcript" refers to any transcript generated during elongation that is shorter than the full-length mRNA molecule encoded by the DNA template, for example, as a result of premature termination of transcription. In some embodiments, a truncated transcript may be less than 90% of the length of the full-length mRNA molecule transcribed from the target molecule, e.g., less than 80%, 70%, 60%, 50%, 40%, 30%, 20%, or 10%. The length of an mRNA molecule encoding a full-length polypeptide or protein is at least 90% of the length of the theoretical transcript. In practice, the length of a theoretical transcript may differ from the length measured using a particular assay. Thus, in some embodiments, the term "full-length mRNA" refers to the length measured as characterized by a particular assay, for example, gel electrophoresis and detection using ultraviolet light, i.e., ultraviolet absorption spectroscopy with separation by capillary gel electrophoresis. In embodiments using such particular assays, an mRNA transcript transcribed from a DNA template is considered full-length if it is at least 95% (e.g., at least 96%, 97%, 98%, or 99%) of the theoretical length of a corresponding reference mRNA that expresses the full-length polypeptide or protein encoded by the mRNA transcript.

[0051] As used herein, the term "double-stranded RNA" or "dsRNA" refers to RNA produced during IVT, which contains two complementary strands of ribonucleic acid base-paired to each other. During IVT, dsRNA is generated in cis by the formation of a loop of a full-length RNA containing an internal region of complementarity. In addition, abortive transcripts are generated during the initiation phase of IVT, allowing the 3' end of the full-length RNA to prime complementary RNA synthesis in trans from the primary transcript. Another mechanism for dsRNA generation is promoter-independent transcription of full-length antisense RNA.

[0052] As used herein, the term "batch" refers to the quantity or amount of mRNA synthesized at one time, e.g., produced according to a single manufacturing order during the same manufacturing cycle. A batch can refer to the amount of mRNA synthesized in one reaction using a single enzyme aliquot and / or a single DNA template aliquot for sequential synthesis under a set of conditions. In some embodiments, a batch includes mRNA produced from a reaction in which not all reagents and / or components are replenished and / or supplemented as the reaction progresses. The term "batch" does not refer to mRNA synthesized at different times that are combined to achieve a desired amount.

[0053] As used herein, "expression" of a nucleic acid sequence refers to one or more of the following events: (1) production of an RNA template from a DNA sequence (e.g., by transcription); (2) processing of the RNA transcript (e.g., by splicing, editing, 5' cap formation, and / or 3' end formation); (3) translation of the RNA into a polypeptide or protein; and / or (4) post-translational modification of the polypeptide or protein. As used herein, the terms "expression" and "production," and grammatical equivalents, are used interchangeably.

[0054] As used herein, the term "therapeutic" refers to any agent, drug, or composition that can be used to treat or prevent a disease, illness, condition, or disorder of bodily function.

[0055] As used herein, the term "substantially" refers to a qualitative condition exhibiting a total or near-total extent or degree of a desired characteristic or property. Those skilled in the art of biology will understand that biological and chemical phenomena rarely, if ever, proceed to completion and / or perfection or achieve or avoid absolute results. Thus, the term "substantially" is used herein to capture the potential lack of perfection inherent in many biological and chemical phenomena.

[0056] As used herein, the term "in vitro" refers to events that occur not within a multicellular organism, but in an artificial environment, e.g., in a test tube or reaction vessel, in a cell culture, etc. For example, as used herein, the term "in vitro transcription" or "in vitro synthesis" refers to the transcription or synthesis of RNA that occurs outside of cells and / or in the absence of cell lysate, typically in a test tube or reaction vessel (e.g., a bioreactor). "In vitro transcription" or "in vitro synthesis" typically involves the use of recombinantly produced and purified enzymatic components (e.g., KP34 RNA polymerase).

[0057] As used herein, the term "in vivo" refers to events that occur within a multicellular organism, such as a human or non-human animal. In the context of cell-based systems, the term may be used to refer to events that occur within living cells (as opposed to, for example, in vitro systems).

[0058] As used herein, the term "isolated" refers to substances and / or entities that (1) are separated from at least some of the components with which they are associated when originally produced (whether in nature and / or in an experimental setting) and / or (2) are produced, prepared, and / or manufactured by the hand of man.

[0059] Unless otherwise defined herein, scientific and technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains, and as commonly used in the technical field to which this application pertains. Exemplary methods and materials are described below; however, methods and materials similar or equivalent to those described herein can also be used to practice or test the present disclosure. In case of conflict, the present specification, including definitions, will control.

[0060] Generally, the nomenclatures and techniques used in connection with cell and tissue culture, molecular biology, virology, immunology, microbiology, genetics, analytical chemistry, synthetic organic chemistry, medicinal and pharmaceutical chemistry, and protein and nucleic acid chemistry and hybridization described herein are well known and commonly used in the art. Enzymatic reactions and purification techniques are performed according to manufacturer's specifications, as commonly accomplished in the art or as described herein. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular.

[0061] All publications and other reference materials referred to herein are incorporated herein by reference in their entirety. Although certain documents are cited herein, this citation does not constitute an admission that any of these documents form part of the common general knowledge in the art.

[0062] Detailed Description of the Invention The present invention relates to methods for producing mRNA using Klebsiella phage KP34 RNA polymerase under conditions suitable for IVT of mRNA transcripts. The present invention is based, in part, on the discovery that Klebsiella phage KP34 RNA polymerase produces fewer transcription by-products, such as dsRNA, than other RNA polymerases commonly used for IVT, such as T7 and SP6.

[0063] In particular, the present invention provides methods for producing mRNA for therapeutic use, comprising: (a) providing a DNA template comprising a nucleic acid sequence encoding an mRNA transcript for expressing a therapeutic polypeptide or protein; and (b) contacting the DNA template with Klebsiella phage KP34 RNA polymerase under conditions suitable for IVT of the mRNA transcript.

[0064] Klebsiella phage KP34 RNA polymerase Klebsiella phage KP34 RNA polymerase is a DNA-dependent RNA polymerase that is only distantly related to polymerases from other known phages, such as T7 and SP6.

[0065] The naturally occurring Klebsiella phage KP34 RNA polymerase (NCBI Reference Sequence: YP_003347629.1) has the following amino acid sequence: [ka] It has.

[0066] Klebsiella phage KP34 RNA polymerases suitable for use in the present invention can be modified enzymes with substantially the same or improved polymerase activity as naturally occurring Klebsiella phage KP34 RNA polymerases. Thus, in some embodiments, KP34 RNA polymerases can be modified from SEQ ID NO: 1, for example, to include one or more amino acid substitutions, deletions, insertions, and / or additions relative to SEQ ID NO: 1. In some embodiments, suitable KP34 RNA polymerases have an amino acid sequence that is about 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1. In certain embodiments, the amino acid sequence of the KP34 RNA polymerase is at least about 90% (eg, at least about 91%, 92%, 93%, 94%, or 95%) identical to the amino acid sequence of SEQ ID NO:1.

[0067] In some embodiments, a suitable KP34 RNA polymerase may be a truncated protein (N-terminal, C-terminal, or internal) but retains polymerase activity. In some embodiments, a suitable KP34 RNA polymerase is a fusion protein. For example, a KP34 RNA polymerase may contain one or more tags to facilitate isolation, purification, or dissolution of the enzyme. Suitable tags may be located at the N-terminus, C-terminus, and / or internally. Typically, the tag is located at the N-terminus. Non-limiting examples of suitable tags include calmodulin-binding protein (CBP); Fasciola hepatica 8 kDa antigen (Fh8); FLAG tag peptide; glutathione-S-transferase (GST); histidine tags (e.g., hexahistidine tag (His6)); maltose-binding protein (MBP); N-utilization substance (NusA); small ubiquitin-like modifier (SUMO) fusion tags; streptavidin-binding peptide (STREP); tandem affinity purification (TAP); and thioredoxin (TrxA). Other tags may also be used in the present invention. These and other fusion tags are described, for example, in Costa et al., Frontiers in Microbiology, 5 (2014); 63 and PCT / US16 / 57044, the contents of which are incorporated herein by reference in their entireties. In some embodiments, a His tag is located at the N-terminus of KP34.

[0068] In certain embodiments, a suitable Klebsiella phage KP34 RNA polymerase is modified for purification by affinity chromatography, for example, as described in Example 1. A suitable modified Klebsiella phage KP34 RNA polymerase for use in the present invention has the following amino acid sequence: [ka] It has.

[0069] In some embodiments, Klebsiella phage KP34 RNA polymerase for use in the methods of the invention can be prepared recombinantly as described in Example 1 using an expression plasmid containing the following coding sequence optimized for expression in E. coli: [ka] [ka]

[0070] Expression and purification In some embodiments, Klebsiella phage KP34 RNA polymerase suitable for use in the methods for producing mRNA described herein can be recombinantly expressed in bacterial cells such as Escherichia coli.

[0071] In some embodiments, Klebsiella phage KP34 RNA polymerase is purified from crude bacterial extracts, for example, by affinity purification. For example, the recombinant protein may be fused to an affinity tag (e.g., a His tag) to facilitate purification. The affinity-purified enzyme can be used directly in IVT. Typically, the presence of the tag does not interfere with polymerase activity. For example, as shown in the Examples, a tag added to the N-terminus of Klebsiella phage KP34 RNA polymerase does not interfere with polymerase activity.

[0072] In another embodiment, KP34 is purified using hydrophobic interaction chromatography (HIC). For example, KP34 can be purified using an HIC column containing a butyl ligand, such as Capto™ butyl. Purification by HIC avoids the need to add an affinity tag to purify recombinant proteins.

[0073] The present inventors have found that overexpression of Klebsiella phage KP34 RNA polymerase in bacterial cells can result in aggregation. These aggregates have been found to be enzymatically inactive. Therefore, in some embodiments, the isolated Klebsiella phage KP34 RNA polymerase may be further purified to remove enzymatically inactive aggregates. For example, enzymatically inactive aggregates may be removed by gel filtration (e.g., of an affinity-purified preparation).

[0074] In certain embodiments, recombinantly expressed Klebsiella phage KP34 RNA polymerase is isolated from bacterial extracts (e.g., using affinity purification) followed by chromatography, such as size exclusion chromatography (e.g., gel purification), to remove enzymatically inactive aggregates. In some embodiments, a gel filtration column (e.g., Superdex™ 200) is used to remove enzymatically inactive aggregates.

[0075] In some embodiments, Klebsiella phage KP34 RNA polymerase for use in the methods of the invention contains less than 5% enzymatically inactive aggregates (e.g., less than 4%, 3%, 2%, or 1%). In some embodiments, Klebsiella phage KP34 RNA polymerase contains less than 1% enzymatically inactive aggregates. In some embodiments, Klebsiella phage KP34 RNA polymerase is substantially free of enzymatically inactive aggregates. The presence of aggregates can be determined, for example, by gel filtration or Western blot.

[0076] In some embodiments, at least 95% (e.g., at least 96%, 97%, or 98%) of the Klebsiella phage KP34 RNA polymerase for use in the methods of the invention is in monomeric form, hi some embodiments, 99% of the Klebsiella phage KP34 RNA polymerase is in monomeric form.

[0077] In some embodiments, Klebsiella phage KP34 RNA polymerase for use in the methods of the invention is provided as a fusion protein containing a lysis tag, e.g., to improve recombinant expression in E. coli. In typical embodiments, the lysis tag is added to the N-terminus of Klebsiella phage KP34 RNA polymerase. As provided herein, suitable lysis tags include InfB7, DxsN, P17, SmbP, and T7A3.

[0078] DNA template Klebsiella phage KP34 RNA polymerase is a DNA-dependent RNA polymerase. DNA-dependent RNA polymerase initiates transcription by contacting a suitable promoter sequence in a DNA template. Thus, a typical DNA template for use in the methods of the present invention comprises a Klebsiella phage KP34 promoter sequence, e.g., a promoter, operably linked to a nucleic acid sequence encoding an mRNA transcript.

[0079] promoter Any promoter that can be recognized by Klebsiella phage KP34 RNA polymerase can be used in the present invention. An exemplary Klebsiella phage KP34 core promoter comprises the following sequence: 5'-TAATGTTACAGGAGTA-3' (SEQ ID NO: 4). Alternatively, an exemplary Klebsiella phage KP34 core promoter comprises the following sequence: 5'-ATGTTACAGGAGTA-3' (SEQ ID NO: 5).

[0080] A KP34 promoter suitable for the present invention may be at least about 70%, 75%, 80%, 85%, 90%, 95%, or 99% (e.g., about 70%, 75%, 80%, 85%, 90%, 95%, or 99%) identical to SEQ ID NO:4. Alternatively, a suitable KP34 promoter may be at least about 70%, 75%, 80%, 85%, 90%, 95%, or 99% (e.g., about 70%, 75%, 80%, 85%, 90%, 95%, or 99%) identical to SEQ ID NO:5. In some embodiments, promoters homologous to SEQ ID NO:4 or 5 may be used in practicing the present invention. In some embodiments, a KP34 promoter suitable for the present invention may include one or more additional nucleotides 5' and / or 3' to any one of the promoter sequences described herein.

[0081] In some embodiments, the nucleic acid sequence 3' adjacent to the core promoter sequence is optimized to improve mRNA yield during in vitro synthesis. For example, the KP34 promoter may contain three guanines 3' adjacent to the core promoter sequence of SEQ ID NO: 4 or 5. Thus, an exemplary Klebsiella phage KP34 promoter contains the following sequence: TAATGTTACAGGAGTAGGG (SEQ ID NO: 6). The inventors have observed that this promoter sequence worked particularly well when the 3' flanking sequence of SEQ ID NO: 6 was A or GA.

[0082] In some embodiments, the additional nucleotides 3' adjacent to the KP34 core promoter (e.g., SEQ ID NO: 4 or 5) are GGA, GGGA, or GGGGA. Thus, an exemplary KP34 promoter can include one of the following nucleic acid sequences, with the minimal core promoter sequence (SEQ ID NO: 5) shown in bold: [ka] [ka] or [ka]

[0083] The inventors have observed that nucleic acid sequences further downstream of the core promoter sequence may also affect function. Thus, in some embodiments, additional nucleotides 3' to the KP34 core promoter (e.g., SEQ ID NO: 4 or 5) include a G n AN1N2N3N4W, where n is 2, 3, or 4; 1~4 is any one of A, C, G, or T, and W is A or T. In some embodiments, the additional nucleotides 3' to the KP34 core promoter (e.g., SEQ ID NO: 4 or 5) are G n AN1N2N3N4N5WV, where n is 2, 3, or 4; 1~5 is any one of A, C, G, or T, W is A or T, and V is C or T. In some embodiments, N 1~5are independently selected from C, A, and G, and W is A or T. In some embodiments, N1 is C or G, N2 is A, N3 is A, C, or G, and N4 and N5 are independently selected from A and G. Exemplary nucleic acid sequences 3' adjacent to the core promoter can be GGGGACAAGATC (SEQ ID NO:37), GGAGACAGATC (SEQ ID NO:38), GGAGAGAGATC (SEQ ID NO:39), or GGAGACAGTTT (SEQ ID NO:40). Typically, the nucleic acid sequence 3' adjacent to the core promoter is GGGGACAAGATC (SEQ ID NO:37) or GGAGACAGTTT (SEQ ID NO:40), e.g., SEQ ID NO:40. Thus, in some embodiments, the KP34 promoter comprises the following nucleic acid sequence, with the minimal core promoter sequence (SEQ ID NO:5) shown in bold: [ka]

[0084] Similarly, the sequences set forth in SEQ ID NOs: 38, 39, or 40 may be combined with a KP34 promoter sequence such as that set forth in SEQ ID NO: 5, for example, as set forth in SEQ ID NOs: 42-44. Further exemplary sequences are set forth in SEQ ID NOs: 45-48.

[0085] In some embodiments, the KP34 promoter may comprise one of the following nucleic acid sequences, with the minimal core promoter sequence (SEQ ID NO: 5) shown in bold: [ka] (wherein n is 1 to 5, i.e., G, GG, GGG, GGGG, or GGGGG). In certain embodiments, n is 1 or 3 (i.e., G or GGG). In certain embodiments, N1N2N3N4 is CAGA. Thus, an exemplary KP34 promoter sequence is: [ka] or SEQ ID NO: 12 [ka] Further exemplary promoter sequences include: [ka] Further exemplary promoter sequences include: [ka] [ka] or [ka] Includes:

[0086] 5' and 3' untranslated regions A nucleotide sequence encoding an mRNA transcript for expressing a polypeptide or protein typically includes a 5' untranslated region (5'UTR), a coding region for the polypeptide of interest, and a 3' untranslated region (3'UTR).

[0087] In some embodiments, the 5' untranslated region contains one or more elements that affect mRNA stability or translation, such as an iron-responsive element. In some embodiments, the 5' untranslated region can be about 50-500 nucleotides in length.

[0088] In some embodiments, the 3' untranslated region comprises one or more of a polyadenylation signal, a binding site for a protein that affects the stability of the location of the mRNA within the cell, or one or more binding sites for an miRNA. In some embodiments, the 3' untranslated region can be 50 to 500 or more nucleotides in length.

[0089] In some embodiments, the nucleotide sequence comprises a 5'UTR that differs from the 5'UTR present in the naturally occurring mRNA encoding the polypeptide of interest.

[0090] In some embodiments, the nucleotide sequence comprises a 3'UTR that differs from the 3'UTR present in the naturally occurring mRNA encoding the polypeptide of interest.

[0091] For example, suitable 5' and 3' UTRs are described in WO 2012 / 075040, which is incorporated herein by reference.

[0092] In certain embodiments, the 5' and / or 3' UTR sequences may be derived from stable mRNAs (e.g., globin, actin, GAPDH, tubulin, histones, or citric acid cycle enzymes) to enhance mRNA stability. For example, the 5' UTR sequence may include a partial sequence or fragment thereof of the CMV immediate-early 1 (IE1) gene to improve nuclease resistance and / or improve mRNA half-life. It is also contemplated to include a sequence encoding human growth hormone (hGH) or a fragment thereof in the 3' end or untranslated region of the mRNA. Exemplary 5' UTRs include sequences derived from the CMV immediate-early 1 (IE1) gene (see U.S. Patent Application Publication Nos. 2014 / 0206753 and 2015 / 0157565, each of which is incorporated herein by reference), or the sequence set forth in Example 1 of U.S. Patent Application Publication No. 2016 / 0151409, incorporated herein by reference.

[0093] In various embodiments, the 5'UTR can be derived from the 5'UTR of a TOP gene. TOP genes are typically characterized by the presence of a 5'-terminal oligopyrimidine (TOP) tract. Furthermore, most TOP genes are characterized by growth-related translational regulation. However, TOP genes with tissue-specific translational regulation are also known. In certain embodiments, the 5'UTR derived from the 5'UTR of a TOP gene lacks a 5'TOP motif (oligopyrimidine tract) (e.g., U.S. Patent Application Publication Nos. 2017 / 0029847, 2016 / 0304883, 2016 / 0235864, and 2016 / 0166710, each of which is incorporated herein by reference).

[0094] In one embodiment, the 5'UTR is derived from the ribosomal protein large 32 (L32) gene (U.S. Patent Application Publication No. 2017 / 0029847, supra).

[0095] In one embodiment, the 5'UTR is derived from the 5'UTR of the hydroxysteroid (17-b) dehydrogenase 4 gene (HSD17B4) (US Patent Application Publication No. 2016 / 0166710, supra).

[0096] In one embodiment, the 5'UTR is derived from the 5'UTR of the ATP5A1 gene (US Patent Application Publication No. 2016 / 0166710, supra).

[0097] In some embodiments, an internal ribosome entry site (IRES) is used in place of the 5'UTR.

[0098] A 5'UTR for use in the present invention may comprise one of the nucleic acid sequences shown in Table 1. The nucleotides immediately adjacent 3' to the KP34 core promoter are shown in bold.

[0099] [Table 1]

[0100] For example, the sequence shown in Table 1 can be combined with the KP34 promoter to form the following sequence, with the minimal core promoter sequence (SEQ ID NO: 5) shown in bold: [ka] [ka]

[0101] An exemplary 5'UTR for use in the present invention has the following sequence: [ka] The underlined 5' nucleic acid sequence is present in the sequences of Table 1 (except SEQ ID NO: 18, which contains further modified sequences). For example, the underlined 5' nucleic acid sequence of SEQ ID NO: 21 is present in the KP34 promoter sequence of SEQ ID NO: 33 in Table 10 (SEQ ID NOs: 32, 34, and 35 contain further modified sequences).

[0102] Linearization of the mold In vitro transcribed mRNA is typically transcribed from a linearized DNA template using a restriction enzyme. In this context, any restriction enzyme (see, e.g., Roberts et al. (2015) Nucl. Acids Res. 43; D1: D298-D299) can be used. Generally, the restriction enzyme is a type II restriction enzyme, such as a type IIP or type IIS restriction enzyme. In some embodiments, the restriction enzyme is EcoRI, BciVI, Spel, Xbal, Ndel, Aflll, Sacl, Kpnl, Smal, BamHI, Sail, Sbfl, Pstl, BspQI, or Hindlll.

[0103] In some embodiments, the linearized DNA template has blunt ends.

[0104] Array Optimization In some embodiments, the transcribed DNA sequence may be optimized to facilitate more efficient transcription and / or translation. For example, the DNA sequence may be optimized with respect to cis-regulatory elements (e.g., TATA boxes, termination signals, and protein binding sites), artificial recombination sites, Chi sites, CpG dinucleotide content, cryptic CpG islands, GC content, polymerase slippage sites, and / or other elements related to transcription; the DNA sequence may be optimized with respect to cryptic splice sites, mRNA secondary structure, mRNA stable free energy, repetitive sequences, RNA instability motifs, and / or other elements related to mRNA processing and stability; the DNA sequence may be optimized with respect to codon usage bias, codon adaptability, internal Chi sites, ribosome binding sites (e.g., IRES), premature polyA sites, Shine-Dalgarno (SD) sequences, and / or other elements related to translation; and / or the DNA sequence may be optimized with respect to codon context, codon-anticodon interactions, translational pause sites, and / or other elements related to protein folding. Suitable sequence optimization methods are described in WO 2021 / 226461 A1, which is incorporated herein by reference.

[0105] In vitro transcription (IVT) Various methods for synthesizing mRNA by IVT are described in U.S. Patent Application Publication No. 2018 / 0258423 and International Publication No. WO 2021 / 168052A1 (incorporated herein by reference) and can be used to practice the present invention. Briefly, IVT is typically carried out using a reaction mixture containing a DNA template, a pool of ribonucleotide triphosphates, a buffering reagent (which may include DTT), and one or more salts (e.g., MgCl and NaCl). A typical IVT reaction buffer may also include spermidine. The exact conditions will vary depending on the particular application.

[0106] Template concentration The concentration of the DNA template in the IVT reaction ranges from 0.05 mg / mL to 0.5 mg / mL. In some embodiments, the concentration of the DNA template is 0.05 mg / mL, 0.06 mg / mL, 0.07 mg / mL, 0.08 mg / mL, 0.09 mg / mL, 0.1 mg / mL, 0.11 mg / mL, 0.12 mg / mL, 0.13 mg / mL, 0.14 mg / mL, 0.15 mg / mL, 0.16 mg / mL, 0.17 mg / mL, 0.18 mg / mL, 0.19 mg / mL, 0.2 mg / mL, 0.21 mg / mL, 0.22 mg / mL, 0.23 mg / mL, 0.24 mg / mL, 0.25 mg / mL, 0.26 mg / mL, 0.27 mg / mL, 0.28 mg / mL, 0.29 mg / mL, 0.30 mg / mL, 0.31 mg / mL, 0.32 mg / mL, 0.33 mg / mL, 0.34 mg / mL, 0.35 mg / mL, 0.36 mg / mL, 0.37 mg / mL, 0.38 mg / mL, 0.39 mg / mL, 0.40 mg / mL, 0.41 mg / mL, 0.42 mg / mL, 0.43 mg / mL, 0.44 mg / mL, 0.45 mg / mL, 0.46 mg / mL, 0.47 mg / mL, 0.48 mg / mL, 0.49 mg / mL, 0.50 mg / mL, 0.51 mg / mL, 0.52 mg / mL, 0.53 mg / mL, 0.54 mg / mL, 0.55 mg / mL, 0.56 mg / mL, 0. .27mg / mL, 0.28mg / mL, 0.29mg / mL, 0.3mg / mL, 0.31mg / mL, 0.32mg / mL, 0.33mg / mL, 0.34mg / mL, 0.35mg / mL, 0.36mg / mL, 0.37mg / mL, 0.38mg / mL, 0. 39mg / mL, 0.4mg / mL, 0.41mg / mL, 0.42mg / mL, 0.43mg / mL, 0.44mg / mL, 0.45mg / mL, 0.46mg / mL, 0.47mg / mL, 0.48mg / mL, 0.49mg / mL, or 0.5mg / mL.

[0107] Polymerase concentration The concentration of Klebsiella phage KP34 RNA polymerase in the IVT reaction ranges from 0.01 to 0.5 mg / mL. In some embodiments, Klebsiella phage KP34 The concentrations of RNA polymerase are 0.01mg / mL, 0.02mg / mL, 0.03mg / mL, 0.04mg / mL, 0.05mg / mL, 0.06mg / mL, 0.07mg / mL, 0.08mg / mL, 0.09mg / mL, 0.1mg / mL, 0.11mg / mL, 0.12m g / mL, 0.13mg / mL, 0.14mg / mL, 0.15mg / mL, 0.16mg / mL, 0.17mg / mL, 0.18mg / mL, 0.19mg / mL, 0.2mg / mL, 0.21mg / mL, 0.22mg / mL, 0.23mg / mL, 0.24mg / mL, 0.25m g / mL, 0.26 mg / mL, 0.27 mg / mL, 0.28 mg / mL, 0.29 mg / mL, 0.3 mg / mL, 0.31 mg / mL, 0.32 mg / mL, 0.33 mg / mL, 0.34 mg / mL, 0.35 mg / mL, 0.36 mg / mL, 0.37 mg / mL, 0.38 mg / mL, 0.39 mg / mL, 0.4 mg / mL, 0.41 mg / mL, 0.42 mg / mL, 0.43 mg / mL, 0.44 mg / mL, 0.45 mg / mL, 0.46 mg / mL, 0.47 mg / mL, 0.48 mg / mL, 0.49 mg / mL, or 0.5 mg / mL.

[0108] Buffer Reagents IVT is typically performed in the presence of a buffer. In some embodiments, the buffer is selected from Tris, HEPES, ammonium sulfate, sodium bicarbonate, sodium citrate, sodium acetate, potassium phosphate, and sodium phosphate. In one embodiment, the buffer is Tris-HCl. In further embodiments, Tris-HCl is present at a concentration of less than 40 mM. In some embodiments, Tris-HCl is present at a concentration of about 25 mM.

[0109] pH The pH of the reaction mixture may be about 6-8.5, 6.5-8.0, 7.0-7.5, and in some embodiments, the pH is 7.5.

[0110] temperature In some embodiments, IVT is performed at a temperature of about 37° C. to about 42° C. In some embodiments, IVT is performed at a temperature of about 37° C., 38° C., 39° C., 40° C., 41° C., or 42° C.

[0111] cofactor Most polymerases contain divalent cations as cofactors. Thus, in some embodiments, the IVT contains divalent cations, such as Mn 2+ or Mg 2+ It is carried out in the presence of

[0112] In some embodiments, IVT is performed in the presence of magnesium chloride (MgCl). The inventors have found that Klebsiella phage KP34 RNA polymerase has high activity in the presence of MgCl concentrations greater than 20 mM. Thus, in some embodiments, the MgCl concentration is greater than 20 mM. In some embodiments, the MgCl concentration is greater than 20 mM and less than 40 mM (e.g., less than 30 mM). In some embodiments, the MgCl concentration is about 25 mM.

[0113] salt Salt is typically included in the reaction mixture to provide optimal conditions for IVT. In some embodiments, IVT is performed in the presence of sodium chloride (NaCl). In some embodiments, the NaCl concentration is less than 20 mM. In some embodiments, the NaCl concentration is 0.05 mM to 15 mM. In some embodiments, the NaCl concentration is 0.1 mM to 10 mM. In some embodiments, the NaCl concentration is 0.1 mM to 5 mM. In some embodiments, the NaCl concentration is 0.1 mM to 2 mM. In some embodiments, the NaCl concentration is 0.1 mM to 1 mM. In some embodiments, the NaCl concentration is about 0.5 mM.

[0114] Ribonucleotides In some embodiments, the concentration of each ribonucleotide (e.g., ATP, UTP, GTP, and CTP) in the reaction mixture is about 0.1 mM to about 10 mM, e.g., about 1 mM to about 10 mM, about 2 mM to about 10 mM, about 3 mM to about 10 mM, about 1 mM to about 8 mM, about 1 mM to about 6 mM, about 3 mM to about 10 mM, about 3 mM to about 8 mM, about 3 mM to about 6 mM, about 4 mM to about 7 mM, about 4 mM to about 6 mM, or about 4 mM to about 5 mM. In some embodiments, each ribonucleotide is present in the reaction mixture at about 5 mM.

[0115] In some embodiments, the total concentration of ribonucleotides (e.g., ATP, GTP, CTP, and UTP combined) used in the reaction is about 1 mM to about 40 mM. In some embodiments, the total concentration of ribonucleotides (e.g., ATP, GTP, CTP, and UTP combined) used in the reaction is about 1 mM to about 30 mM, about 1 mM to about 28 mM, about 1 mM to about 25 mM, or about 1 mM to about 20 mM.

[0116] In some embodiments, the total ribonucleotide concentration is less than about 30 mM. In some embodiments, the total ribonucleotide concentration is less than about 25 mM. In some embodiments, the total ribonucleotide concentration is less than about 20 mM. In some embodiments, the total ribonucleotide concentration is less than about 15 mM. In some embodiments, the total ribonucleotide concentration is less than about 10 mM.

[0117] modified RNA In some embodiments, mRNA transcripts are synthesized with one or more modifications (i.e., as modified mRNA), where modification refers to a chemical or biological modification including a backbone modification, a sugar modification, or a base modification. A backbone modification is a chemical modification of the phosphate of the backbone of an RNA nucleotide (e.g., phosphorothioate and 5'-N-phosphoramidite linkage). A sugar modification is a chemical modification of the sugar of an RNA nucleotide (e.g., 2'-fluororibose, ribose, 2'-deoxyribose, arabinose, and hexose). A base modification is a chemical modification of the base moiety of an RNA nucleotide.

[0118] In certain embodiments, the modified mRNA comprises modified ribonucleotides, such as ribonucleotide analogs (e.g., adenosine analogs, guanosine analogs, cytidine analogs, and / or uridine analogs). The presence of modified ribonucleotides can stabilize the mRNA and / or make it less immunogenic compared to a control mRNA having the same sequence but containing only naturally occurring ribonucleotides.

[0119] Modified ribonucleotides typically substitute for naturally occurring nucleotides. Thus, in one aspect, the in vitro transcribed mRNA of the present invention contains both unmodified and modified ribonucleotides. Such in vitro transcribed mRNA can typically be prepared by including modified ribonucleosides (e.g., N1-methylpseudouridine instead of uridine) in the IVT reaction mixture instead of naturally occurring ribonucleosides. This results in an in vitro transcribed mRNA in which 100% of the naturally occurring ribonucleotides are replaced with the corresponding modified ribonucleotides (e.g., 100% of the uridines are replaced with N1-methylpseudouridine). In some embodiments, only a small portion of the naturally occurring ribonucleosides (e.g., at least 1%, 5%, 10%, 15%, 20%, or 25% of the naturally occurring ribonucleosides) are replaced with modified ribonucleosides. In some embodiments, one or more naturally occurring ribonucleosides are replaced with modified ribonucleosides. For example, two or more ribonucleosides may be modified ribonucleosides (e.g., uridine may be replaced with 2-thio-uridine and cytidine may be replaced with 5-methylcytidine), e.g., 25% of the uridine may be replaced with 2-thio-uridine and / or 25% of the cytidine residues may be replaced with 5-methylcytidine.

[0120] In some embodiments, a modified ribonucleoside comprises at least one modification relative to a corresponding naturally occurring ribonucleoside selected from a modified sugar and a modified nucleobase.

[0121] The modified ribonucleoside can be a modified uridine, cytidine, adenosine, or guanosine. Some exemplary chemical modifications of ribonucleosides in mRNA molecules include, for example, pyridin-4-one ribonucleosides, 5-aza-uridine, 2-thio-5-aza-uridine, 2-thiouridine, 4-thiopseudouridine, 2-thiopseudouridine, 5-hydroxyuridine, 3-methyluridine, 5-carboxymethyluridine, 1-carboxymethyl-pseudouridine, 5-propynyl-uridine, 1-propynyl-pseudouridine, and 5-taurinomethyluridine. , 1-taurinomethyl-pseudouridine, 5-taurinomethyl-2-thio-uridine, 1-taurinomethyl-4-thio-uridine, 5-methyl-uridine, 1-methyl-pseudouridine, 4-thio-1-methyl-pseudouridine, 2-thio-1-methyl-pseudouridine, 1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-1-deaza-pseudouridine, dihydrouridine, dihydropseudouridine, 2-thiodihydrouridine, 2-thio-dihydropseudouridine Lysine, 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-deaza-pseudoisocytidine, zebularine, 5-aza-zebularine, 5-methyl-zebularine, 5-aza-2-thio-zebularine, 2-thio-zebularine, 2-methoxy-cytidine, 2-methoxy-5-methyl-cytidine, 4-methoxy-pseudoisocytidine, 4-methoxy-1-methyl-pseudoisocytidine, 2-aminopurine, 2,6-Diaminopurine, 7-Deazaadenine, 7-Deaza-8-Aza-Adenine, 7-Deaza-2-aminopurine, 7-Deaza-8-Aza-2-aminopurine, 7-Deaza-2,6-Diaminopurine, 7-Deaza-8-Aza-2,6-Diaminopurine, 1-Methyladenosine, N, 6 -methyladenosine, N 6 -Isopentenyl adenosine, N 6 -(cis-Hydroxyisopentenyl)adenosine, 2-methylthio-N 6 -(cis-hydroxyisopentenyl)adenosine, N 6 -Glycinylcarbamoyl adenosine, N 6 -Threonylcarbamoyladenosine, 2-methylthio-N 6 -Threonylcarbamoyladenosine, N 6 ,N 6 -dimethyladenosine, 7-methyladenine, 2-methylthioadenine, 2-methoxyadenine, inosine, 1-methyl-inosine, wyosine, wybutosine, 7-deazaguanosine, 7-deaza-8-aza-guanosine, 6-thioguanosine, 6-thio-7-deazaguanosine, 6-thio-7-deaza-8-aza-guanosine, 7-methyl-guanosine, 6-thio-7-methylguanosine, 7-methylinosine, 6-methoxyguanosine, 1-methylguanosine, N 2 -methylguanosine, N 2 ,N 2 -dimethylguanosine, 8-oxo-guanosine, 7-methyl-8-oxo-guanosine, 1-methyl-6-thio-guanosine, N 2 -methyl-6-thio-guanosine, and N 2 ,N 2 -dimethyl-6-thio-guanosine.

[0122] In some embodiments, the modified ribonucleoside is pseudouridine, pyridin-4-one ribonucleoside, 5-aza-uridine, 6-aza-uridine, 2-thio-5-aza-uridine, 2-thio-uridine, 4-thio-uridine, 4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxyuridine, 5-aminoallyl-uridine, 5-halo-uridine (e.g., 5-iodouridine or 5-bromouridine), 3-methyluridine, 5-methoxy-uridine, uridine-5-oxyacetic acid, uridine-5-oxyacetic acid methyl ester, 5-carboxymethyl-uridine, 1-carboxymethyl-pseudouridine, 5-carboxyhydroxymethyluridine, 5-carboxyhydroxymethyluridine, 5-carboxymethyl-uridine, 5-carboxymethyl-uridine, 5-carboxyhydroxymethyluridine, 5-carboxymethyl-uridine, 5-carboxymethyl-uridine, 5-carboxyhydroxymethyluridine, 5-carboxymethyl-uridine, 5-carboxymethyl-uridine, 5-carboxyhydroxymethyluridine, 5-carboxymethyl-uridine, 5-carboxymethyl-uridine, 5-carboxyhydroxymethyluridine, 5-carboxymethyl-uridine, 5-carboxymethyl-uridine, 5-carboxyhydroxymethyluridine, 5-carboxyhydroxymethyluridine, 5-carboxyhydroxymethyluridine, 5-carboxyhydroxymethyluridine, 5-carboxyhydroxymethyluridine, 5-hydroxymethyl- ... 1-uridine methyl ester, 5-methoxycarbonylmethyluridine, 5-methoxycarbonylmethyl-2-thiouridine, 5-aminomethyl-2-thiouridine, 5-methylaminomethyluridine, 5-methylaminomethyl-2-thiouridine, 5-methylaminomethyl-2-selenouridine, 5-carbamoylmethyluridine, 5-carboxymethylaminomethyluridine, 5-carboxymethylaminomethyl-2-thiouridine, 5-propynyluridine, 1-propynyl-pseudouridine, 5-taurinomethyluridine, 1-taurinomethylpseudouridine, 5-taurinomethyl-2-thiouridine, 1-taurinomethyl-4-thiopseudouridine, 5-methyluridine (m 5U, e.g., having the nucleobase deoxythymine), 1-methyl-pseudouridine, 5-methyl-2-thio-uridine, 1-methyl-4-thio-pseudouridine, 4-thio-1-methyl-pseudouridine, 3-methyl-pseudouridine), 2-thio-1-methyl-pseudouridine, 1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-1-deaza-pseudouridine, dihydrouridine, dihydropseudouridine Uridine, 5,6-dihydrouridine, 5-methyl-dihydrouridine, 2-thio-dihydrouridine, 2-thio-dihydropseudouridine, 2-methoxy-uridine, 2-methoxy-4-thio-uridine, 4-methoxy-pseudouridine, 4-methoxy-2-thio-pseudouridine, N1-methyl-pseudouridine, 3-(3-amino-3-carboxypropyl)uridine, 1-methyl-3-(3-amino-3-carboxypropyl)uridine (Dipropyl)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 and a modified uridine selected from 2'-carboxymethylaminomethyl-2'-O-methyluridine, 3,2'-O-dimethyluridine, 5-(isopentenylaminomethyl)-2'-O-methyluridine, 1-thio-uridine, deoxythymidine, 2'-F-ara-uridine, 2'-F-uridine, 2'-OH-ara-uridine, 5-(2-carbomethoxyvinyl)uridine, and 5-[3-(1-E-propenylamino)uridine].

[0123] 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.

[0124] In some embodiments, the modified ribonucleoside is 5-aza-cytidine, 6-aza-cytidine, pseudoisocytidine, 3-methylcytidine, N 4 -acetylcytidine, 5-formyl-cytidine, N 4 -methylcytidine, 5-methylcytidine, 5-halocytidine (e.g., 5-iodocytidine), 5-hydroxymethylcytidine, 1-methyl-pseudoisocytidine, pyrrolo-cytidine, pyrrolo-pseudoisocytidine, 2-thio-cytidine, 2-thio-5-methylcytidine, 4-thio-pseudoisocytidine, 4-thio-1-methyl-pseudoisocytidine, 4-thio-1-methyl-1-deaza-pseudoisocytidine, 1 -methyl-1-deaza-pseudoisocytidine, zebularine, 5-aza-zebularine, 5-methyl-zebularine, 5-aza-2-thiozebularine, 2-thio-zebularine, 2-methoxycytidine, 2-methoxy-5-methylcytidine, 4-methoxypseudoisocytidine, 4-methoxy-1-methyl-pseudoisocytidine, lysidine, α-thio-cytidine, 2'-O-methylcytidine, 5,2'-O-dimethylcytidine, N 4 -acetyl-2'-O-methylcytidine, N 4 ,2'-O-dimethylcytidine, 5-formyl-2'-O-methylcytidine, N 4 ,N 4, a modified cytidine selected from 2'-O-trimethylcytidine, 1-thio-cytidine, 2'-F-ara-cytidine, 2'-F-cytidine and 2'-OH-ara-cytidine.

[0125] 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 uridine are replaced with modified nucleosides (e.g., N1-methylpseudouridine and 5-methylcytidine).

[0126] In some embodiments, the modified ribonucleoside is a modified purine ribonucleoside. In some embodiments, the modified ribonucleoside is 2-aminopurine, 2,6-diaminopurine, 2-amino-6-halopurine (e.g., 2-amino-6-chloropurine), 6-halopurine (e.g., 6-chloropurine), 2-amino-6-methylpurine, 8-azidoadenosine, 7-deaza-adenine, 7-deaza-8-azaadenine, 7-deaza-2-aminopurine, 7-deaza-8-aza-2-aminopurine, 7-deaza-2,6-diaminopurine, 7-deaza-8-aza-2,6-diaminopurine, 1-methyladenosine, 2-methyladenine, N 6 -Methyladenosine, 2-methylthio-N 6 -methyladenosine, N 6 -Isopentenyl adenosine, 2-methylthio-N 6 -Isopentenyl adenosine, N 6 -(cis-Hydroxyisopentenyl)adenosine, 2-methylthio-N 6 -(cis-hydroxyisopentenyl)adenosine, N 6 -Glycinylcarbamoyl adenosine, N 6 -Threonylcarbamoyladenosine, N 6 -methyl-N 6 -Threonylcarbamoyladenosine, 2-methylthio-N 6-Threonylcarbamoyladenosine, N 6 ,N 6 -Dimethyladenosine, N 6 -Hydroxynorvalylcarbamoyladenosine, 2-methylthio-N 6 -hydroxynorvalylcarbamoyl adenosine, N 6 -acetyladenosine, 7-methyladenine, 2-methylthio-adenine, 2-methoxyadenine, 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-ara-adenosine, 2'-F-adenosine, 2'-OH-ara-adenosine, and N 6 -(19-amino-pentaoxanonadecyl)adenosine.

[0127] In some embodiments, the modified ribonucleoside is inosine, 1-methylinosine, wyosine, methylwyosine, 4-demethylwyosine, isowyosine, wybutosine, peroxywybutosine, hydroxywybutosine, intermediate hydroxywybutosine (undermodified hydroxywybutosine), 7-deaza-guanosine, queuosine, epoxyqueuosine, galactosylqueuosine, mannosylqueuosine, 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, N2 ,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-araguanosine, and 2'-F guanosine.

[0128] In some embodiments, the modified ribonucleoside is a ribonucleoside analog selected from 2-aminoadenosine, inosine, pyrrolo-pyrimidine, 3-methyladenosine, 5-methylcytidine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyl-uridine, C5-propynyl-cytidine, C5-methylcytidine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, O(6)-methylguanine, pseudouridine (e.g., N1-methylpseudouridine), 2-thiouridine, and 2-thiocytidine. See, for example, U.S. Pat. No. 8,278,036 or WO 2011 / 012316 for a description of 5-methylcytidine, pseudouridine and 2-thio-uridine and their incorporation into mRNA.

[0129] In some embodiments, the modified ribonucleoside is selected from pseudouridine, N1-methylpseudouridine, 2-thiouridine, 4'-thiouridine, 5-methylcytidine, 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-methoxyuridine, and 2'-O-methyluridine.

[0130] In some embodiments, the in vitro transcribed mRNA can be RNA in which 25% of the uracil residues are 2-thiouracil and 25% of the cytosine residues are 5-methylcytosine. Teachings for using such modified RNA are disclosed in U.S. Patent Application Publication No. 2012 / 0195936 and International Publication No. WO 2011 / 012316, both of which are incorporated by reference in their entireties. In some embodiments, the in vitro transcribed mRNA can be RNA in which 100% of the uracil residues are N1-methylpseudouracil (sometimes referred to as 1-methylpseudouracil).

[0131] time The length of the IVT reaction can depend on the length of the mRNA transcript. In typical embodiments, the mRNA transcript contains at least 500 ribonucleotides. In some embodiments, the mRNA transcript contains about 500 to about 20,000 ribonucleotides. In some embodiments, the mRNA transcript contains about 700 to about 15,000 ribonucleotides. In some embodiments, the mRNA transcript contains about 800 to about 12,000 ribonucleotides. In some embodiments, the mRNA transcript contains about 1,000 to about 10,000 ribonucleotides. In some embodiments, the mRNA transcript contains about 1,500 to about 7,000 ribonucleotides. In some embodiments, the mRNA transcript contains about 2,000 to about 5,000 ribonucleotides.

[0132] Therefore, the period during which IVT can be performed to synthesize mRNA can vary widely. In some embodiments, IVT is performed for a period of about 30 minutes to about 6 hours. In some embodiments, IVT is performed for a period of about 60 minutes to about 90 hours.

[0133] IVT can be terminated by removing the DNA template, e.g., by adding DNase I and a suitable buffer. For example, the polymerase reaction can be quenched by adding DNase I and DNase I buffer (100 mM Tris-HCl at 10×, 5 mM MgCl2, and 25 mM CaCl2, pH 7.6) to facilitate digestion of the double-stranded DNA template in preparation for purification.

[0134] Large scale synthesis In some embodiments, mRNA is synthesized in batches. In some embodiments, the invention relates to large-scale production of mRNA.

[0135] In some embodiments, a single batch synthesizes about 1 g to about 100 kg of mRNA (e.g., 100 g to 10 kg, or 250 g to 5 kg). In some embodiments, the batch contains at least 1 g of in vitro transcribed mRNA (e.g., 5 g, 10 g, 20 g, 25 g, or 30 g). In other embodiments, the batch contains at least 50 g of in vitro transcribed mRNA (e.g., 75 g, 100 g, 150 g, 200 g, or 250 g).

[0136] In some embodiments, methods herein synthesize at least 500 g, 750 g, 1 kg, 5 kg, 10 kg, 50 kg, 100 kg, 1000 kg, or more of mRNA in a single batch. In some embodiments, 10 kg or more of mRNA is synthesized in a single batch. In some embodiments, 10 kg to 100 kg of mRNA is synthesized in a single batch.

[0137] Exemplary IVT conditions In some embodiments, a suitable reaction mixture comprises a double-stranded DNA template containing a Klebsiella phage KP34 RNA polymerase-specific promoter, Klebsiella phage KP34 RNA polymerase, RNase inhibitor, pyrophosphatase, NTPs, 10 mM DTT, and reaction buffer (25 mM Tris-HCl, 2 mM spermidine, 25 mM MgCl, 0.5 mM NaCl, pH 7.5). In some embodiments, the reaction mixture is incubated at 37° C. for the length of time necessary to complete IVT of the mRNA transcript encoded by the DNA template.

[0138] In some embodiments, the reaction mixture comprises each NTP at a concentration ranging from 1 to 10 mM, a DNA template at a concentration ranging from 0.01 to 0.5 mg / mL, and Klebsiella phage KP34 RNA polymerase at a concentration ranging from 0.01 to 0.1 mg / mL.

[0139] Detection of transcription byproducts During IVT, in addition to the desired mRNA transcript, transcription by-products are formed. The present invention is based, at least in part, on the discovery that Klebsiella phage KP34 RNA polymerase produces very few transcription by-products, such as dsRNA.

[0140] Various methods can be used to characterize in vitro synthesized mRNA transcripts. mRNA transcripts can be detected and quantified using any method available in the art, such as blotting (e.g., dot blot), capillary electrophoresis, chromatography, fluorescence, gel electrophoresis, HPLC, silver staining, spectroscopy, ultraviolet (UV) or UPLC, ultraviolet absorption spectroscopy with separation by capillary electrophoresis, or any combination thereof. In some embodiments, mRNA transcripts are first denatured with glyoxal dye and then analyzed by gel electrophoresis ("glyoxal gel electrophoresis").

[0141] Non-template nucleic acid In some embodiments, the mRNA transcripts produced by IVT using Klebsiella phage KP34 RNA polymerase are substantially free of non-template nucleic acids.

[0142] RNA-dependent 3'-end extension can be measured using an RNA-dependent RNA polymerase (RdRp) assay. A suitable RdRp assay can use an RNA template that can anneal at an internal region of complementarity, thereby looping to form a stretch of double-stranded RNA in cis and self-template to allow 3'-end extension.

[0143] In some embodiments, about 10% or less (eg, about 5% or less, or about 2% or less) by weight of the mRNA transcripts obtained by the methods of the invention comprise non-template nucleic acid.

[0144] double-stranded RNA (dsRNA) In some embodiments, the mRNA transcripts obtained by the methods of the invention are substantially free of dsRNA. In some embodiments, the amount of dsRNA is below the limit of detection. In some embodiments, the presence of dsRNA is determined by dot blot using antibodies J2, K1, or K2 (e.g., J2). In some embodiments, the presence of dsRNA is determined by ELISA, for example, a sandwich ELISA using antibodies J2 and K1, or antibodies K1 and K2. In some embodiments, the amount of dsRNA by weight is below the limit of detection in a 200 ng sample of in vitro synthesized mRNA. In some embodiments, the amount of dsRNA by weight is below the limit of detection in a 200 ng sample of in vitro synthesized mRNA, for example, as measured by dot blot using monoclonal antibody J2.

[0145] In some embodiments, the mRNA transcripts contain less than about 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, 0.05%, or 0.01% by weight, e.g., less than 0.5%, dsRNA. In some embodiments, the amount of dsRNA is determined by ELISA, e.g., sandwich ELISA using antibodies J2 and K1, or antibodies K1 and K2.

[0146] In some embodiments, the amount of dsRNA produced by IVT using Klebsiella phage KP34 RNA polymerase is at least 10-fold less than a corresponding method in which SP6 RNA polymerase is used instead of Klebsiella phage KP34 RNA polymerase. In some embodiments, the amount of dsRNA produced by IVT using Klebsiella phage KP34 RNA polymerase is at least 100-fold less than a corresponding method in which T7 RNA polymerase is used instead of Klebsiella phage KP34 RNA polymerase.

[0147] In some embodiments, the dsRNA produced by IVT using Klebsiella phage KP34 RNA polymerase is less than about 10%, 8%, 5%, 4%, 3%, 2%, or 1% of the dsRNA produced by IVT using SP6 RNA polymerase. In certain embodiments, the dsRNA produced by IVT using Klebsiella phage KP34 RNA polymerase is less than 1% of the dsRNA produced by IVT using SP6 RNA polymerase. In some embodiments, the dsRNA produced by IVT using Klebsiella phage KP34 RNA polymerase is less than 0.5% of the dsRNA produced by IVT using SP6 RNA polymerase.

[0148] In some embodiments, the dsRNA produced by IVT using Klebsiella phage KP34 RNA polymerase is less than 10%, 8%, 5%, 4%, 3%, 2%, or 1% of the dsRNA produced by IVT using T7 RNA polymerase. In certain embodiments, the dsRNA produced by IVT using Klebsiella phage KP34 RNA polymerase is less than 1% of the dsRNA produced by IVT using T7 RNA polymerase. In some embodiments, the dsRNA produced by IVT using Klebsiella phage KP34 RNA polymerase is less than 0.5% of the dsRNA produced by IVT using T7 RNA polymerase.

[0149] Product Integrity The methods of the present invention result in high quality in vitro synthesized mRNA, for example, the present invention provides uniformity / homogeneity of the synthesized mRNA.

[0150] In particular, the compositions of the present invention comprise a plurality of substantially full-length mRNA transcripts. For example, at least 80% of the mRNA transcripts are full-length mRNA molecules. In some embodiments, at least 90% of the mRNA transcripts are full-length mRNA molecules. In some embodiments, at least 95% of the mRNA transcripts are full-length mRNA molecules. Such compositions are said to be "enriched" in full-length mRNA molecules. In some embodiments, the mRNA synthesized according to the present invention is substantially full-length.

[0151] In some embodiments, less than 20% of the mRNA transcripts obtained by the methods of the invention are truncated transcripts. In some embodiments, less than 10% of the mRNA transcripts obtained by the methods of the invention are truncated transcripts. In some embodiments, less than 5% of the mRNA transcripts obtained by the methods of the invention are truncated transcripts.

[0152] Post-Synthesis Processing After IVT, a 5' cap and / or a 3' tail may be added. The presence of a cap is important for providing resistance to nucleases found in most eukaryotic cells. The presence of a "tail" helps protect the mRNA from exonuclease degradation in vivo.

[0153] In some embodiments, the in vitro transcribed mRNA is purified before being capped. In some embodiments, the in vitro transcribed mRNA is purified after being capped. In some embodiments, the in vitro transcribed mRNA is purified before being tailed. In some embodiments, the in vitro transcribed mRNA is purified after tailing. In some embodiments, the in vitro transcribed mRNA is capped before being tailed. In some embodiments, the capped in vitro transcribed mRNA is purified before tailing.

[0154] Optional Tail Addition Step In some embodiments, the 3' tail of the mRNA comprises a poly(A) tail. In some embodiments, the 3' tail of the mRNA comprises a poly(C) tail. In some embodiments, the tail structure comprises at least 50 adenosine or cytosine nucleotides. In typical embodiments, the 3' tail is about 100-500 nucleotides in length. For example, a 3' tail (e.g., a poly(A) tail) of 100-250 nucleotides in length may be particularly useful in therapeutic uses of the mRNA.

[0155] The poly(A) or poly(C) tail at the 3' end of an mRNA typically contains at least 50 adenosine or cytosine nucleotides, at least 100 adenosine or cytosine nucleotides, at least 150 adenosine or cytosine nucleotides, at least 200 adenosine or cytosine nucleotides, at least 250 adenosine or cytosine nucleotides, at least 300 adenosine or cytosine nucleotides, at least 350 adenosine or cytosine nucleotides, at least 400 adenosine or cytosine nucleotides, at least 450 adenosine or cytosine nucleotides, or at least 500 adenosine or cytosine nucleotides, respectively. In some embodiments, the tail structure comprises a combination of poly(A) and poly(C) tails of various lengths as described herein.

[0156] 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 nucleotides, hi 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 nucleotides.

[0157] Optional Capping Step In vitro transcribed mRNAs with a methylated 5' cap structure are efficiently translated in vivo. The IVT process can include a co-transcriptionally added cap analog. Alternatively, the 5' cap structure can be enzymatically added after the IVT reaction is complete. At least 90% of enzymatically capped in vitro transcribed mRNAs can contain the Cap 1 structure.

[0158] Several types of 5' caps are known. The 7-methylguanosine cap (also referred to as "m7G" or "cap0") contains a guanosine linked to the first transcribed nucleotide via a 5'-5'-triphosphate bond. The 5' cap is typically added as follows: first, an RNA terminal phosphatase removes one of the terminal phosphate groups from the 5' nucleotide, leaving two terminal phosphates; then, guanosine triphosphate (GTP) is added to the terminal phosphate by a guanylyltransferase, resulting in a 5'5'5 triphosphate bond; and then, the 7-nitrogen of the guanine is methylated by a methyltransferase. Examples of cap structures include, but are not limited to, mG(5')ppp, (5'(A,G(5')ppp(5')A, and G(5')ppp(5')G. Additional cap structures are described in U.S. Patent Application Publication Nos. 2016 / 0032356 and 2018 / 0125989, which are incorporated herein by reference.

[0159] During co-transcriptional capping, cap analogs are included in the IVT reaction mixture. Cap analogs can be incorporated as the first "base" in the nascent RNA strand. Cap analogs are classified as cap0, cap1, cap2, m 6The cap structure may be Am, or a chemical cap analog. For example, to generate a 5'-guanosine cap structure, the following chemical cap analogs can be used according to the manufacturer's instructions: 3'-O-Me-m7G(5')ppp(5')G (ARCA cap); G(5')ppp(5')A; G(5')ppp(5')G; m7G(5')ppp(5')A; m7G(5')ppp(5')G; m7G(5')ppp(5')(2'OMeA)-pG; m7G(5')ppp(5')(2'OmeA)pU; m7G(5')ppp(5')(2'OmeG)pG (New England BioLabs, Ipswich, MA; TriLink Biotechnologies).

[0160] Vaccinia virus capping enzyme may be used to generate the Cap 0 structure: m7G(5')ppp(5')G. Vaccinia virus capping enzyme and a 2'-O-methyltransferase may both be used to generate the Cap 1 structure: m7G(5')ppp(5')G-2'-O-methyl. The Cap 2 structure may be generated from the Cap 1 structure, followed by 2'-O-methylation of the penultimate 5'-nucleotide using a 2'-O-methyltransferase. The Cap 3 structure may be generated from the Cap 2 structure, followed by 2'-O-methylation of the penultimate 5'-nucleotide using a 2'-O-methyltransferase.

[0161] In some embodiments, the method further comprises a step of capping the in vitro transcribed mRNA. The capping step may include adding a capping enzyme (guanylyltransferase) and guanine. A suitable capping enzyme may be derived from vaccinia virus (vaccinia virus guanylyltransferase). Typically, the capping step also includes adding guanine methyltransferase and 2'-O-methyltransferase. Capping may be performed separately, for example, after IVT. The capping step is generally performed before tailing the in vitro transcribed mRNA.

[0162] In certain embodiments, the in vitro transcribed mRNA may include a 5' cap having the following structure: [ka]

[0163] purification The present inventors have surprisingly discovered that the use of Klebsiella phage KP34 RNA polymerase reduces or eliminates the presence of transcription by-products, particularly dsRNA, in in vitro synthesized mRNA. Thus, the mRNA transcripts produced by the methods of the present invention can be used without the need for additional post-purification steps to remove dsRNA. This greatly simplifies post-synthesis processing of in vitro synthesized mRNA.

[0164] In some embodiments, the methods of the invention further comprise purifying mRNA transcripts obtained from an IVT reaction performed with KP34 RNA polymerase. In some embodiments, mRNA transcripts prepared by the methods of the invention do not require a purification step to remove dsRNA contaminants. In some embodiments, the step of purifying the mRNA transcripts comprises a method other than cellulose chromatography. In some embodiments, the step of purifying the mRNA transcripts comprises a method other than HPLC. In some embodiments, the step of purifying the mRNA transcripts comprises a method other than HPLC using a buffer system comprising triethylammonium acetate and / or acetonitrile. In some embodiments, the step of purifying the mRNA transcripts comprises a method other than anion exchange high performance liquid chromatography.

[0165] In some embodiments, the mRNA transcripts are purified without the use of chaotropic agents. In some embodiments, the mRNA transcripts are purified under non-denaturing conditions. In some embodiments, the mRNA transcripts are purified without the use of lithium chloride, sodium chloride, potassium chloride, guanidinium chloride, guanidinium thiocyanate, guanidinium isothiocyanate, ammonium acetate, and combinations thereof.

[0166] Various methods can be used to purify mRNA. In some embodiments, mRNA is purified by precipitation and centrifugation. In some embodiments, mRNA is purified by filtration, for example, using normal flow filtration (NFF) or tangential flow filtration (TFF).

[0167] Suitable purification methods include those described in U.S. Patent Application Publication Nos. 2016 / 0040154, 2015 / 0376220, 2018 / 0251755, 2018 / 0251754, 2020 / 0095571, 2021 / 0388338, and 2021 / 0002635, all of which are incorporated herein by reference, and WO 2022 / 072836.

[0168] composition In some embodiments, the present invention relates to compositions prepared by the methods of the present invention. In some embodiments, the compositions prepared according to the methods of the present invention comprise mRNA (e.g., for expressing a therapeutic polypeptide or protein) and Klebsiella phage KP34 RNA polymerase, wherein the composition contains less than 1% dsRNA by weight and less than 10% of the mRNA transcripts by weight are abortive transcripts. In certain embodiments, the composition comprises mRNA transcripts that are substantially free of non-template nucleic acids. In some embodiments, the Klebsiella phage KP34 RNA polymerase is removed by NFF or TFF.

[0169] In some embodiments, the invention provides compositions comprising mRNA transcripts (e.g., for expressing a therapeutic polypeptide or protein), wherein the compositions comprise less than 1% dsRNA by weight, wherein less than 10% of the mRNA transcripts by weight are abortive transcripts, and wherein the mRNA transcripts are substantially free of non-template nucleic acid.

[0170] equivalent Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. [Example]

[0171] The following examples are included for illustrative purposes only and are not intended to limit the scope of the invention.

[0172] Example 1. Purification of Klebsiella phage KP34 RNA polymerase This example demonstrates the affinity purification of Klebsiella phage KP34 RNA polymerase.

[0173] The coding sequence for Klebsiella phage KP34 RNA polymerase was cloned into an expression plasmid. An N-terminal His tag was added to allow affinity purification of the recombinantly produced enzyme, resulting in an expression plasmid containing the coding sequence of SEQ ID NO:3 operably linked to an IPTG-inducible promoter. This coding sequence encodes Klebsiella phage KP34 RNA polymerase containing the amino acid sequence set forth in SEQ ID NO:2. BL21 competent E. coli cells (New England Biolabs™) were transformed with the expression plasmid. Successfully transformed E. coli cells were grown in a suitable growth medium containing IPTG to induce expression of the recombinant protein. When the cells reached stationary phase, they were harvested by centrifugation and lysed.

[0174] The cell lysate was loaded onto a Ni-NTA agarose column for immobilized metal affinity chromatography (IMAC). After loading, the column was washed to remove any unbound material. To elute the His-tagged Klebsiella phage KP34 RNA polymerase, an elution buffer containing increasing concentrations of imidazole was added to the column. The crude lysate, the column load material, the flow-through, the wash, and various elution fractions were separated on an SDS-PAGE gel to visualize the enzyme (see Figure 1A). The elution fractions containing Klebsiella phage KP34 RNA polymerase were pooled, and the resulting mixture was subjected to size-exclusion chromatography (SEC) by gel filtration using a Superdex™ 75 column. The gel-filtered composition contained a major peak containing the polymerase (see Figure 1B).

[0175] This example demonstrates that Klebsiella phage KP34 RNA polymerase can be recombinantly expressed in Escherichia coli cells and subsequently purified using affinity chromatography.

[0176] Example 2. Preparation of in vitro transcribed mRNA In vitro transcribed mRNA was prepared as described in Example 1 of International Publication No. 2021 / 168052, incorporated herein by reference. Briefly, for each gram of mRNA to be transcribed, a reaction mixture containing a linearized double-stranded DNA plasmid containing an RNA polymerase-specific promoter, an RNA polymerase (e.g., KP34, SP6, or T7 RNA polymerase as indicated), an RNase inhibitor, pyrophosphatase, NTPs, DTT, and buffering reagents was prepared in RNase-free water. Unless otherwise indicated, the reaction buffer composition for both Klebsiella phage KP34 RNA polymerase and SP6 RNA polymerase was identical (25 mM Tris-HCl, 2 mM spermidine, 25 mM MgCl2, 0.5 mM NaCl, and pH 7.5). The reaction mixture was incubated at 37°C for 60–90 minutes. In vitro transcribed mRNA prepared with ATP, UTP, CTP, and GTP is referred to herein as unmodified (abbreviated as "unmod"). Where indicated, UDP was replaced with N1-methylpseudouridine to prepare in vitro transcribed mRNA containing modified ribonucleotides (abbreviated as "mod"). DNase I was added to stop the reaction, and the reaction mixture was incubated at 37°C for an additional 15 minutes. The resulting in vitro transcribed mRNA was purified.

[0177] Example 3. In vitro transcription The affinity-purified Klebsiella phage KP34 RNA polymerase obtained in Example 1 was used in IVT as described in Example 2 to evaluate various parameters such as mRNA yield, in vitro transcribed mRNA integrity, non-template synthesis, or the formation of undesired transcription by-products (double-stranded RNA).

[0178] For example, to determine yield and product integrity, a linearized template plasmid encoding an mRNA transcript with a theoretical length of 1944 nucleotides was used (also referred to herein as template #1). A short 50-nucleotide template was used to assess the occurrence of non-templated addition during IVT. Where indicated, experiments were performed in parallel to synthesize both unmodified and modified RNAs.

[0179] Example 4. Yield and product integrity This example demonstrates the use of Klebsiella phage KP34 RNA polymerase in place of SP6 RNA polymerase in the synthesis of both unmodified and modified RNA.

[0180] As described in Example 3, a DNA template encoding an mRNA transcript with a theoretical length of 1944 nucleotides was contacted with either SP6 RNA polymerase or Klebsiella phage KP34 RNA polymerase. The coding sequence of the mRNA transcript can be used to express a protein. IVT was performed in the presence of ATP, UTP, CTP, and GTP to generate unmodified mRNA (abbreviated as "unmod"). In addition, IVT was also performed with a reaction mixture containing modified ribonucleotides (containing N1-methylpseudouridine instead of uridine) to synthesize modified mRNA (abbreviated as "mod"). The results of two independent experiments are summarized in Tables 2 and 3.

[0181] The presence of modified ribonucleotides in the reaction mixture did not affect the yield. Notably, the yield achieved with SP6 was, on average, three-fold higher than that achieved with KP34.

[0182] [Table 2]

[0183] [Table 3]

[0184] To determine whether the differences in yield were due to differences in product integrity, the average length of the mRNA transcripts was analyzed by capillary gel electrophoresis. The results of this analysis are summarized in Tables 4 and 5.

[0185] The performance of KP34 was comparable to that of SP6: over 90% of the mRNA was full-length, and the measured length was close to the theoretical value of 1944 nucleotides.

[0186] [Table 4]

[0187] [Table 5]

[0188] To confirm that the non-inferiority in yield was not dependent on the template used, the experiment was repeated using four DNA templates ranging in size from approximately 1100 bp to approximately 4700 bp, in addition to the previously used DNA template #1 (see Example 3). IVT was performed using either SP6 RNA polymerase or KP34 RNA polymerase with reaction mixtures containing N1-methylpseudouridine instead of uridine to synthesize modified mRNA. Table 6 shows that the mRNA yields obtained from the IVT reactions were comparable when using either of these RNA polymerases. Figure 3 graphically displays these data, along with the corresponding standard deviations for each template tested.

[0189] [Table 6]

[0190] The IVT products were subjected to capillary gel electrophoresis to analyze the length of the mRNA transcripts. The integrity of the mRNA obtained with SP6 RNA polymerase or KP34 RNA polymerase was comparable (data not shown).

[0191] This example demonstrates the use of Klebsiella phage KP34 RNA polymerase in place of SP6 RNA polymerase in the synthesis of both unmodified and modified RNA. Approximately 90% or more of the resulting transcripts were full-length.

[0192] Example 5. Non-template transfer This example demonstrates that RNA-dependent 3'-end extension of mRNA transcripts does not occur when Klebsiella phage KP34 RNA polymerase is used in IVT.

[0193] RNA-dependent 3'-end extension was measured using an RNA-dependent RNA polymerase (RdRp) assay. The template was a 50-base-long synthetic RNA (RNA50). This template was allowed to self-anneal. Annealing of the internal region of complementarity resulted in the formation of a double-stranded RNA stretch in cis by forming a loop, allowing self-templated 3'-end extension.

[0194] 0.4 μM of RNA50 template was incubated in the presence of either 0 μM (negative control), 0.1 μM, 0.2 μM, or 0.6 μM T7, SP6, or KP34 RNA polymerase and unmodified ribonucleotides. Each sample also contained pyrophosphatase and RNase inhibitors. Samples were incubated at 37°C for 80 minutes and analyzed by gel electrophoresis on a 15% urea-TBE polyacrylamide gel. RNA was detected using SYBR Gold stain. The results are summarized in Figure 2.

[0195] For both T7 and SP6 RNA polymerases, increasing the polymerase concentration increased the RNA-dependent 3'-end extension of the RNA template. In contrast, no RNA-dependent 3'-end extension was detected during IVT with KP34 RNA polymerase, regardless of the polymerase concentration tested.

[0196] To determine whether the findings observed with short synthetic RNA templates also apply to longer templates, a short 15-base DNA template and a long 1944-base DNA template were amplified in parallel in separate reactions using either SP6 RNA polymerase or KP34 RNA polymerase. Liquid chromatography-mass spectrometry (LC-MS) was used to determine whether non-templated addition of nucleic acids to the 3' end occurred. A 100 μM probe oligonucleotide was annealed to the 3' end of mRNA transcripts (1–2 mg of RNA per mL, 21 μL total) at 75°C for 10 min in a thermocycler (Eppendorf). The sample was then gradually cooled to 23°C over 10 min, followed by rapid cooling to 4°C. RNase H nuclease (5000 units / mL, 1 μL, New England BioLabs) and rSAP (1000 units / mL, 4 μL, New England BioLabs) in 1× RNase H buffer (New England BioLabs) were added to each sample and incubated at 37°C for 40 minutes in a thermocycler.

[0197] Ultra-high pressure liquid chromatography (UHPLC) was used to separate the samples on an Agilent 1290 Infinity II coupled to an Agilent InfinityLab C18 2.1 x 100 mm, 2.7 μm, 100 Å column. The column was heated to 50 °C with a flow rate of 500 μL / min. The mobile phase included Buffer A (100 mM HFIP, 8.6 mM TEA) and Buffer B (100% methanol). The gradient started with 1% Buffer B, increased to 5% Buffer B over the first 3 min, and then linearly increased to 20% Buffer B by 13 min. Between 13 and 14 min, the percentage of Buffer B was increased from 20% to 50%. At 14 min, a 1.5-min rinse with 50% Buffer B was initiated, followed by a return to 1% Buffer B at 17 min.

[0198] In this experiment, when KP34 RNA polymerase was used in the synthesis reaction, no non-templated addition of nucleic acids was observed using short templates. In contrast, SP6 RNA polymerase produced heterogeneous synthesis products. Some of the synthesized RNAs contained additional non-templated nucleic acids at the 3' end. Similarly, when using long templates, the RNAs synthesized by SP6 RNA polymerase were heterogeneous in size, with only 59.3% of the RNAs having the expected 3' end sequence. In contrast, when using KP34 RNA polymerase, the RNAs were uniform in size, with 98% 3' homogeneity.

[0199] This example demonstrates that Klebsiella phage KP34 RNA polymerase does not result in RNA-dependent 3'-end extension of mRNA transcripts when used in IVT, even at high polymerase concentrations. This example also confirms that KP34 RNA polymerase achieves similar 3' homogeneity with long DNA templates. In this regard, KP34 RNA polymerase outperforms both T7 and SP6, making it a highly attractive RNA polymerase for producing high-quality therapeutic RNA.

[0200] Example 6. Double-stranded RNA (dsRNA) This example demonstrates that IVT using Klebsiella phage KP34 RNA polymerase yields undetectable amounts of dsRNA.

[0201] During the IVT process, dsRNA is generated by base pairing of complementary regions within the same strand or opposite strands, resulting in dsRNA with 5' or 3' overhangs. To measure the amount of dsRNA generated during IVT using either KP34 RNA polymerase or SP6 RNA polymerase, mRNA synthesis was performed as described in Example 2. 100 ng, 200 ng, or 400 ng of RNA in a 2 μL sample volume was blotted onto a nitrocellulose membrane. 1 ng, 20 ng, or 40 ng of dsRNA controls were used as references.

[0202] The primary antibody used was anti-dsRNA monoclonal antibody J2. This antibody is typical for detecting dsRNA. This antibody recognizes dsRNA if the helix length is 40 bp or longer. dsRNA recognition is independent of the mRNA sequence and nucleotide composition. The secondary antibody used was anti-mouse IgG HRP. Signals were detected after 1 minute of exposure. The results are shown in Figure 4.

[0203] No dsRNA was detected in either the unmodified or modified RNA transcripts synthesized by Klebsiella phage KP34 RNA polymerase, and the presence of dsRNA was not detected in either the unmodified or modified RNA transcripts synthesized by SP6 RNA polymerase, with a lower signal detected in the modified RNA transcripts.

[0204] These results demonstrate that IVT using Klebsiella phage KP34 RNA polymerase produces mRNA transcripts containing undetectable amounts of dsRNA, as measured by dot blot with the J2 antibody. This example demonstrates that the amount of dsRNA produced by KP34 was many-fold less than the amount of dsRNA produced by SP6 RNA under SP6-optimized IVT conditions.

[0205] Example 7. Improvement of activity and yield This example demonstrates that the activity and yield of Klebsiella phage KP34 RNA polymerase can be increased by removing enzymatically inactive aggregates from affinity-purified enzyme preparations using size-exclusion chromatography (SEC).

[0206] Affinity-purified Klebsiella phage KP34 RNA polymerase was prepared as described in Example 1. The elution fractions containing the enzyme were pooled and concentrated. The resulting concentrated pool was loaded onto a Superdex™ 200 gel filtration column for size-exclusion chromatography using the following buffer: Tris pH 8.0, 200 mM NaCl, 0.1 mM EDTA, 5 mM 2-mercaptoethanol, and 5% glycerol. Under these conditions, two individual peaks could be resolved (Figure 5A). Peak I eluted earlier, indicating that the size of the Klebsiella phage KP34 RNA polymerase in this fraction was larger than that of peak II. The eluates of peak fractions I and II were collected separately. Peak I was estimated to contain approximately 60-70% of the IMAC-purified enzyme.

[0207] The collected peak fractions were separated on an SDS-PAGE gel to visualize Klebsiella phage KP34 RNA polymerase (Figure 5B). SDS-PAGE gel analysis confirmed that peak I and II fractions contained Klebsiella phage KP34 RNA polymerase. The particle size of a representative sample from each peak was measured by dynamic light scattering (DLS). DLS analysis confirmed that peak fraction I contained a majority of particles with a hydrodynamic diameter of approximately 50 nm, corresponding to aggregates of Klebsiella phage KP34 RNA polymerase. In contrast, peak fraction II contained a majority of particles with a hydrodynamic diameter of less than 10 nm.

[0208] To measure the enzymatic activity of the two fractions, an in vitro transcription assay was performed using a short template consisting of 28 nucleotides containing a Klebsiella promoter sequence, using the conditions described in Example 2. This template yielded a 15-nucleotide-long RNA transcript. Peak fraction I contained almost no enzymatic activity. Based on the hydrodynamic diameter, peak fraction I consisted of enzymatically inactive aggregates. In contrast, peak fraction II contained the enzymatically active monomeric form of Klebsiella phage KP34 RNA polymerase.

[0209] The experiment described in Example 4 was repeated using Klebsiella phage KP34 RNA polymerase from peak fraction II. This fraction was estimated to contain less than 5% enzymatically inactive aggregates. Compared to the experiment described in Example 4, there was an approximately two-fold improvement in mRNA yield during in vitro transcription. The monomeric form of Klebsiella phage KP34 RNA polymerase achieved 70% of the mRNA yield produced by SP6 RNA polymerase, as shown in Table 7 below.

[0210] [Table 7]

[0211] The results in Table 7 demonstrate that purification of Klebsiella phage KP34 RNA polymerase to remove enzymatically inactive aggregates results in an enzyme preparation with improved polymerase activity and mRNA yield.

[0212] Example 8. Purification of KP34 polymerase by hydrophobic interaction chromatography Hydrophobic interaction chromatography (HIC) was tested as an alternative purification method to size exclusion chromatography (SEC). Affinity-purified Klebsiella phage KP34 RNA polymerase was prepared as described in Example 1. Several different HIC columns composed of different resins were loaded with Ni 2+ The eluate from the columns was tested by loading them. These were Capto phenyl High Sub, Capto phenyl ImpRes, Capto butyl, Capto butyl ImRes, and Capto octyl, all contained in a high-fluidity agarose matrix. KP34 was successfully purified using HIC. Remarkably, KP34 could be purified using HIC without the need for a gel filtration step.

[0213] Example 9. Sequence optimization improves Klebsiella phage KP34 activity This example demonstrates that mRNA yields from in vitro synthesis reactions utilizing Klebsiella phage KP34 RNA polymerase can be increased by modifying the nucleic acid sequence immediately 3' to the core promoter.

[0214] The present inventors have found that nucleic acid sequences 3' adjacent to the core promoter (SEQ ID NO: 4) can promote KP34 RNA polymerase activity. Table 8 shows the core promoter region (bold) and the nucleic acid sequences 3' adjacent to the core promoter (including a partial sequence of the 5' UTR). Various 3' adjacent sequences were evaluated (underlined). In SEQ ID NO: 22, the core promoter was followed by a stretch of five Gs and one A. In SEQ ID NOs: 23, 24, and 26, the stretch of Gs immediately 3' adjacent to the core promoter was shortened by one, two, or three residues compared to SEQ ID NO: 22. SEQ ID NO: 25 is similar to SEQ ID NO: 24 but also lacks the A 3' adjacent to the stretch of Gs. SEQ ID NO: 27 is identical to SEQ ID NO: 22 except for the replacement of T with A, as indicated.

[0215] [Table 8]

[0216] IVT was performed as described in Example 3 using KP34, whose DNA template contained one of the KP34 promoter sequences listed in Table 8. The IVT reaction conditions were those described in Example 2. KP34 was purified by gel filtration to remove enzymatically inactive aggregates. An IVT reaction performed with SP6 RNA polymerase and a template plasmid containing the SP6 promoter served as a control. Table 9 shows the mRNA yield for each reaction, expressed as a percentage of the yield obtained with SP6.

[0217] [Table 9]

[0218] These results demonstrate that mRNA yields from in vitro synthesis reactions using Klebsiella phage KP34 RNA polymerase can be increased by modifying the nucleic acid sequence 3' adjacent to the core promoter. The mRNA yield from IVT reactions using Klebsiella phage KP34 RNA polymerase can be comparable to that of IVT reactions using SP6 RNA polymerase. Interestingly, Klebsiella phage KP34 RNA polymerase is more versatile than SP6 or T7 RNA polymerase. Unlike SP6 or T7, it can utilize templates with a stretch of four or five Gs in the nucleic acid sequence 3' adjacent to the core promoter.

[0219] Example 10. Modification of promoter-flanking sequences improves RNA yield This example confirms that modifications to the nucleic acid sequence 3' to the core promoter result in increased yields when Klebsiella phage KP34 RNA polymerase is used for in vitro synthesis of mRNA.

[0220] In light of the results summarized in Example 9, additional promoter sequences were tested to determine whether mRNA yields could be further improved. Specifically, variants of the promoters previously tested were generated. The additional promoter sequences are listed in Table 10.

[0221] [Table 10]

[0222] SEQ ID NOS:32-35 are variants of the well-functioning promoter sequences of SEQ ID NOS:23 and 26 shown in Table 8. SEQ ID NOS:32 corresponds to SEQ ID NOS:23 and includes an additional A, as shown in bold and underlined. SEQ ID NOS:33-35 correspond to SEQ ID NOS:26 and includes an additional GA, as shown in bold and underlined. In SEQ ID NOS:34, an additional C is replaced with a G, and in SEQ ID NOS:35, two nucleotides are replaced with two additional Ts. Additional changes are also shown in bold and underlined. The underlining is as shown in Table 8. All of the modifications were within the nucleic acid sequence 3' adjacent to the core promoter (SEQ ID NOS:4, shown in bold in SEQ ID NOS:32-35) and downstream of the transcription start site. SEQ ID NOS:30 and 31 are the T7 and SP6 promoter sequences, respectively (the nucleic acid sequence 3' adjacent to the core promoter, shown in bold, is shown in SEQ ID NOS:28 and 29).

[0223] IVT was performed as described in Example 3 using KP34 DNA templates containing each of the KP34 promoter sequences listed in Tables 8 and 10. To synthesize modified mRNA, IVT was performed with reaction mixtures containing modified ribonucleotides (containing N1-methylpseudouridine instead of uridine). For comparison, previously tested promoter sequences were included. IVT reaction conditions were those described in Example 2.

[0224] The mRNA yields achieved for reactions with each of the promoter sequences tested are summarized in Table 11. The same sample IDs used in Figure 6 are listed in Example 11.

[0225] [Table 11]

[0226] In this experiment, the highest yields were obtained with templates containing the promoter sequences of SEQ ID NOs: 26 and 32. These yields were even higher than those achieved with T7 and SP6 RNA polymerases in this experiment.

[0227] The results in this example clearly demonstrate the effect of modifications made to the nucleic acid sequence 3' to the KP34 core promoter on the mRNA yields achievable with Klebsiella phage KP34 RNA polymerase.

[0228] Example 11. Promoter optimization reduces the amount of short abortive transcripts This example demonstrates that the amount of short abortive transcripts produced by Klebsiella phage KP34 RNA polymerase during an IVT reaction can be reduced by selecting an appropriate promoter sequence.

[0229] To determine the influence of the core promoter and 3'-flanking nucleic acid sequences on transcription initiation, liquid chromatography-mass spectrometry (LC-MS) was used to measure the amount of short abortive transcripts in the IVT reactions described in Example 10. 100 μM probe oligonucleotides were annealed to the 3' ends of mRNA transcripts (1-2 mg of RNA per mL, 21 μL total) at 75°C for 10 minutes in a thermocycler (Eppendorf). The samples were then gradually cooled to 23°C over 10 minutes and then rapidly cooled to 4°C. RNase H nuclease (5000 units / mL, 1 μL, New England BioLabs) and rSAP (1000 units / mL, 4 μL, New England BioLabs) in 1× RNase H buffer (New England BioLabs) were added to each sample and incubated at 37°C for 40 minutes in a thermocycler.

[0230] Ultra-high pressure liquid chromatography (UHPLC) was used to separate the samples on an Agilent 1290 Infinity II coupled to an Agilent InfinityLab C18 2.1 x 100 mm, 2.7 μm, 100 Å column. The column was heated to 50 °C with a flow rate of 500 μL / min. The mobile phase included Buffer A (100 mM HFIP, 8.6 mM TEA) and Buffer B (100% methanol). The gradient started with 1% Buffer B, increased to 5% Buffer B over the first 3 min, and then linearly increased to 20% Buffer B by 13 min. Between 13 and 14 min, the percentage of Buffer B was increased from 20% to 50%. At 14 min, a 1.5-min rinse with 50% Buffer B was initiated, followed by a return to 1% Buffer B at 17 min.

[0231] The results of this experiment are summarized in the bar graph shown in Figure 6, which shows the peak area per μg of sample injected. 1 μg of each sample listed in Table 10 was injected into the LC-MS. The results in Figure 6 represent the sum of all peaks from 6.5 to 12 minutes (n=2).

[0232] IVT reactions performed using DNA templates containing the promoter sequences of SEQ ID NOs: 26 and 35 resulted in amounts of abortive transcripts similar to those observed when DNA templates containing the SP6 or T7 promoters were used with their corresponding RNA polymerases. Surprisingly, the use of either the promoter sequence of SEQ ID NOs: 26 or 35 reduced the amount of abortive transcript that could be detected in this assay by approximately 80% compared to the KP34 promoter sequence of SEQ ID NO: 22. The amount of abortive transcript that could be detected using the promoter sequence of SEQ ID NO: 33 was also reduced.

[0233] This example demonstrates that the amount of short abortive transcripts produced by Klebsiella phage KP34 RNA polymerase during IVT reactions can be reduced by selecting an appropriate promoter sequence. Notably, using a DNA template containing an optimized KP34 promoter sequence resulted in a significant reduction in the amount of short abortive transcripts.

[0234] Example 12. KP34-lysis tag fusion proteins can increase mRNA yield This example demonstrates that fusing Klebsiella phage KP34 RNA polymerase to a lysis tag can further increase mRNA yield.

[0235] To improve the solubility and yield of Klebsiella phage KP34 RNA polymerase during recombinant expression in E. coli, a screen of 22 lysis tags was performed. The tags were fused to the N-terminus of Klebsiella phage KP34 RNA polymerase. The resulting fusion proteins were expressed in E. coli using a 96-well plate format. Each lysis tag was tested in quadruplicate. E. coli cultures were incubated at 30°C for 20 hours.

[0236] At the end of the incubation period, the cells were lysed. The supernatant was clarified by centrifugation and filtered through a 0.2 μm filter. The fusion protein was purified by IMAC as described above. The resulting purified protein was desalted using buffer exchange. The concentration of the purified protein was quantified using absorbance at 280 nm. The amount of protein was normalized before use in the IVT reaction.

[0237] The results of the lysis tag screening are summarized in Figures 6A and 6B. The following lysis tags were tested: APN13, APN36, InfB7, CNP, DxsN, Fh8, GB1, Mocr, Msb, P17, R10, SUMO, SEP, SmbP, Spider, SNUT, T3A, T7A3, T7B, T7C, TalN, and NEXT. Each fusion protein was compared to wild-type Klebsiella phage KP34 RNA polymerase (WT) and SP6, which were expressed and purified using identical conditions. As can be seen in Figure 7A, fusion to InfB7 (322 μg per mL culture), DxsN (280 μg per mL culture), P17 (266 μg per mL culture), SmbP (308 μg per mL culture), or T7A3 (308 μg per mL culture) resulted in the highest total yields compared to WT (224 μg per mL culture).

[0238] The fold increase in solubility compared to WT is shown in Figure 7B. Fusing Klebsiella phage KP34 RNA polymerase to SmbP, T7A3, or DxsN resulted in an approximately 2-fold increase in solubility (1.7-, 1.8-, and 2-fold increases, respectively), while fusion to InfB7 or P17 resulted in an approximately 1.3-fold increase.

[0239] The results of the IVT reactions are summarized in Figure 8. The increased yield and solubility of the fusion proteins was generally reflected in increased mRNA yields. Fusion of Klebsiella phage KP34 RNA polymerase to InfB7, DxsN, P17, SmbP, or T7A3 each tended to result in higher mRNA yields than WT.

Claims

1. 1. A method for producing messenger RNA (mRNA), comprising: a. providing a DNA template comprising a nucleic acid sequence encoding an mRNA transcript for expression of a polypeptide or protein; b. contacting said DNA template with Klebsiella phage KP34 RNA polymerase under conditions suitable for in vitro transcription (IVT) of said mRNA transcript.

2. 10. The method of claim 1, wherein the Klebsiella phage KP34 RNA polymerase provided in step (b) is recombinantly expressed in Escherichia coli cells and purified to remove enzymatically inactive aggregates.

3. 3. The method of claim 2, wherein enzymatically inactive aggregates are removed from an affinity-purified preparation containing the Klebsiella phage KP34 RNA polymerase.

4. 4. The method of claim 3, wherein the enzymatically inactive aggregates are removed by size exclusion chromatography, e.g., gel filtration.

5. 5. The method of any one of claims 1 to 4, wherein the Klebsiella phage KP34 RNA polymerase contains less than 5% enzymatically inactive aggregates.

6. 6. The method of any one of claims 1 to 5, wherein the amino acid sequence of the Klebsiella phage KP34 RNA polymerase is at least 90% identical (e.g., at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical, or identical to) the amino acid sequence of SEQ ID NO:

1.

7. 7. The method of any one of claims 1 to 6, wherein the Klebsiella phage KP34 RNA polymerase is present at a concentration ranging from 0.01 to 0.5 mg / mL.

8. The method of any one of claims 1 to 7, wherein the mRNA transcript comprises at least 500 ribonucleotides.

9. The method according to any one of claims 1 to 8, wherein the amount of double-stranded RNA (dsRNA) contained in the mRNA transcript obtained in step b) is below the detection limit.

10. 10. The method of claim 9, wherein the presence of dsRNA is determined by dot blot using antibody J2.

11. 11. The method of any one of claims 1 to 10, wherein the amount of dsRNA produced in step (b) is at least 10-fold lower compared to a corresponding method in which SP6 RNA polymerase or T7 RNA polymerase is used instead of the Klebsiella phage KP34 RNA polymerase.

12. 12. The method of any one of claims 1 to 11, wherein the mRNA transcripts obtained in step (b) contain less than 1% dsRNA by weight.

13. 13. The method of claim 12, wherein the amount of dsRNA is determined by ELISA using antibodies J2 and K1.

14. 14. The method of any one of claims 1 to 13, wherein no more than 10% by weight of the mRNA transcripts obtained in step (b) comprise non-template nucleic acids.

15. 15. The method of any one of claims 1 to 14, wherein less than 10% by weight of the mRNA transcripts obtained in step (b) are abortive transcripts.

16. 16. The method of claim 15, wherein the abortive transcription product comprises fewer than 20 nucleotides.

17. 17. The method of claim 15 or 16, wherein the abortive transcription product is detectable by gel electrophoresis.

18. 18. The method of any one of claims 1 to 17, comprising synthesizing at least 100 mg of mRNA in a single batch.

19. 19. The method of any one of claims 1 to 18, wherein the DNA template comprises a Klebsiella phage KP34 promoter sequence operably linked to the nucleic acid sequence encoding the mRNA transcript.

20. 20. The method of claim 19, wherein the promoter sequence comprises the nucleic acid sequence set forth in SEQ ID NO:

5.

21. 21. The method of claim 19 or 20, wherein the promoter sequence is optimized to improve the yield of mRNA transcripts.

22. 22. The method of any one of claims 19 to 21, wherein the yield of the mRNA transcript obtained in step b) is equivalent to the yield achieved by a corresponding method using SP6 RNA polymerase or T7 RNA polymerase instead of the Klebsiella phage KP34 RNA polymerase.

23. 23. The method of claim 21 or 22, wherein the promoter sequence comprises the nucleic acid sequence set forth in SEQ ID NO: 7, 8, 9, or 41.

24. 23. The method of claim 21 or 22, wherein the promoter sequence comprises the nucleic acid sequence set forth in SEQ ID NO: 26, 32, or 35, e.g., SEQ ID NO:

26.

25. 25. The method of any one of claims 1 to 24, wherein the DNA template is at a concentration of 0.05 mg / mL to 0.5 mg / mL.

26. The method of any one of claims 1 to 25, wherein the DNA template is linear or linearized.

27. IVT is magnesium chloride (MgCl ２ 27. The method of claim 1, wherein the method is carried out in the presence of

28. The MgCl ２ 28. The method of claim 27, wherein the concentration of is greater than 20 mM.

29. The MgCl ２ 29. The method of claim 28, wherein the concentration of is about 25 mM.

30. 30. The method of any one of claims 1 to 29, wherein the IVT is carried out in the presence of sodium chloride (NaCl).

31. 31. The method of claim 30, wherein the concentration of NaCl is less than 20 mM.

32. 32. The method of claim 31 , wherein the concentration of NaCl is about 0.5 mM.

33. The method of any one of claims 1 to 32, wherein the IVT is carried out in the presence of a buffer.

34. 34. The method of claim 33, wherein the buffering agent is selected from Tris, HEPES, ammonium sulfate, sodium bicarbonate, sodium citrate, sodium acetate, potassium phosphate, and sodium phosphate.

35. 35. The method of claim 34, wherein the buffer is Tris-HCl.

36. 36. The method of claim 35, wherein Tris-HCl is present at a concentration of less than 40 mM.

37. 37. The method of claim 36, wherein Tris-HCl is present at a concentration of about 25 mM.

38. 38. The method of any one of claims 1 to 37, wherein the IVT is performed in the presence of unmodified ribonucleotides.

39. 39. The method of any one of claims 1 to 38, wherein the IVT is performed in the presence of modified ribonucleotides.

40. 40. The method of claim 39, wherein the modified ribonucleotide has a modified nucleoside.

41. 41. The method of claim 40, wherein the modified nucleoside is selected from 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine, 3-methyladenosine, 5-methylcytidine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyl-uridine, C5-propynyl-cytidine, 5-methylcytidine, 5-methoxyuridine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, O(6)-methylguanine, pseudouridine, N1-methylpseudouridine, 2-thiouridine, and 2-thiocytidine.

42. 42. The method of claim 41, wherein the modified nucleoside is pseudouridine, N1-methylpseudouridine, 5-methylcytidine, or 5-methoxyuridine, e.g., the modified nucleoside is N1-methylpseudouridine.

43. 43. The method of any one of claims 1 to 42, wherein the IVT is performed in the presence of ribonucleotides, each ribonucleotide being present at a concentration of 0.1 mM to 10 mM.

44. 44. The method of any one of claims 1 to 43, wherein the IVT is carried out at a pH of 7.0 to 7.

7.

45. 45. The method of claim 44, wherein the pH is about 7.

5.

46. 46. ​​The method of any one of claims 1 to 45, wherein the IVT is performed at a temperature of 37°C to 42°C.

47. 47. The method of claim 46, wherein the temperature is 37°C.

48. 48. The method of any one of claims 1 to 47, wherein the IVT is performed over a period of 30 minutes to 6 hours.

49. 49. The method of any one of claims 1 to 48, wherein the IVT is terminated by the addition of DNase I and DNase I buffer.

50. 50. The method of any one of claims 1 to 49, further comprising purifying the mRNA transcript obtained in step (b) from the KP34 RNA polymerase.

51. 51. The method of claim 50, wherein the step of purifying the mRNA transcripts comprises a method other than (i) cellulose chromatography and / or (ii) high performance chromatography (HPLC) using a buffer system comprising triethylammonium acetate and / or acetonitrile.

52. A composition comprising mRNA transcripts and Klebsiella phage KP34 RNA polymerase, wherein the composition comprises less than 1% by weight of dsRNA and less than 10% by weight of the mRNA transcripts are abortive transcripts.

53. 53. A composition according to claim 52, obtained by a method according to any one of claims 1 to 49.

54. mRNA obtained by the method according to any one of claims 1 to 51.

55. A pharmaceutical composition comprising the mRNA of claim 54.

56. 56. A method of treating or preventing a disease or disorder in a subject, comprising administering to said subject the pharmaceutical composition of claim 55.

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