Modified RNA polymerases

Abstract

This application relates to modified RNA polymerases, compositions or kits comprising the same, and methods of using the same, in particular, in the preparation of ribonucleic acids (RNAs), such as messenger RNAs (mRNAs), in in vitro transcription reactions.

Description

MODIFIED RNA POLYMERASESCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to European application No. 23307423.6 filed 29 December 2023, the entire contents of which are hereby incorporated by reference in their entirety.SUBMISSION OF SEQUENCE LISTING

[0002] The Sequence Listing associated with this application is filed in electronic format as a XML file and hereby incorporated by reference into the specification in its entirety. The name of the XML file containing the Sequence Listing is 01710109-PCT_SL.xlm and the size of the XML file is 95,138 bytes.FIELD

[0003] This application relates to modified RNA polymerases, compositions or kits comprising the same, and methods of using the same, in particular, in the preparation of ribonucleic acids (RNAs), such as messenger RNAs (mRNAs), in in vitro transcription reactions.BACKGROUND

[0004] With the development of in vitro transcription (IVT) methods, synthetic ribonucleic acids (RNAs), such as messenger RNAs (mRNAs), have become an emerging class of gene therapy. IVT is a DNA-templated process that uses a bacteriophage DNA-dependent ribonucleic acid (RNA) polymerase (e.g., SP6, T3, or T7) to synthesize template-directed RNA transcripts. During the IVT reaction, several types of impurities, such as short RNAs or abortive (truncated) transcripts, double-stranded RNAs (dsRNAs), run-on transcripts, polyA tail variants / 3' heterogeneity, and mutated transcripts, are produced at different phases of transcription (FIG. 1). For instance, during the initiation phase, transcription is unstable and RNA polymerase produces many short (truncated) transcripts from about 2-6 nucleotides (nt) in length, a process often referred to as abortive synthesis / initiation or abortive cycling. These short RNAs can interact with RNA polymerase via RNA-templated transcription, producing short dsRNAs. After the synthesis of about eight to twelve nucleotides, the polymerase undergoes a major structural rearrangement and dissociates from the promoter (promoter clearance) to enter into the processive synthesis ofRNA, forming the “elongation complex” until transcription termination. Since the initiation complex is unstable when compared to the elongation complex, abortive RNA transcripts are repeatedly released until the polymerase engages in productive transcription, which produces full- length transcripts. It was reported that both T7 and SP6 RNA polymerases generate abortive RNA transcripts during in vitro synthesis of mRNA (Nam et al., The Journal of Biological Chemistry, 1988, 263(34): 18123-18127; Lee et al., Nucleic Acids Research, 2010,38(18):6045-6053). The full-length transcripts generated by the RNA polymerase can also be used as templates by RNA polymerase to generate long loopback dsRNA species. Often during the elongation phase, RNA polymerase continues to transcribe DNA beyond the point at which termination should be initiated, generating longer than expected RNA transcripts, also referred to as “run-on transcripts”. These run-on transcripts are stochastic, causing 3' heterogeneity which is problematic for downstream applications, such as ligation reactions. All these impurities not only affect protein expression but can also become undesired immunostimulatory byproducts. The presence in a pharmaceutical composition of such impurities generated from IVT methods could impact its safety and efficacy. However, removal of these impurities requires complex purification processes using methods such as reversed-phase high-performance liquid chromatography, which are costly and time consuming.

[0005] Accordingly, there remains a need for an RNA polymerase that is capable of producing full-length RNAs, such as mRNAs, via IVT while generating fewer impurities, such as dsRNAs, abortive RNA transcripts, and / or run-on transcripts.SUMMARY

[0006] Disclosed herein are modified ribonucleic acid (RNA) polymerases that generate fewer impurities, such as double-stranded RNA (dsRNA) and abortive RNA transcripts, when used in in vitro transcription reactions. Accordingly, in one aspect, provided herein is a modified RNA polymerase comprising at least one amino acid substitution relative to a wild-type SP6 RNA polymerase comprising the amino acid sequence of SEQ ID NO: 1, wherein the at least one amino acid substitution is in the N-terminal domain of the modified RNA polymerase or a portion of the C-terminal domain of the modified RNA polymerase that interacts with the N-terminal domain, wherein the modified RNA polymerase produces less double-stranded RNA and / or less abortive RNA transcripts in an in vitro transcription reaction as compared to the wild-type SP6 RNA polymerase, and wherein the modified RNA polymerase comprises an amino acid sequence havingat least about 80% sequence identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the modified RNA polymerase of the present disclosure comprises at least one pair of amino acid substitutions at amino acid positions 35 and 147, 37 and 42, or 131 and 157 as indexed by reference to the wild-type RNA polymerase comprising the amino acid sequence of SEQ ID NO: 1. In some embodiments, the modified RNA polymerase of the present disclosure comprises at least one amino acid substitution at one or more of amino acid positions 19, 20, 36, 41, 128, 154, and 749 as indexed by reference to the wild-type RNA polymerase comprising the amino acid sequence of SEQ ID NO: 1. In some embodiments, the modified RNA polymerase of the present disclosure comprises at least one amino acid substitution at one or more of amino acid positions 19, 20, 41, 128, and 749. In some embodiments, the modified RNA polymerase of the present disclosure further comprises an amino acid substitution at one or more of amino acid positions 19, 20, 36, 41, 128, 154, 458, 464, 538, 566, 749, and 873 as indexed by reference to the wild-type RNA polymerase comprising the amino acid sequence of SEQ ID NO: 1.

[0007] In some embodiments, the at least one pair of amino acid substitutions in the modified RNA polymerase of the present disclosure is at amino acid positions 35 and 147 and / or amino acid positions 37 and 42 as indexed by reference to a wild-type RNA polymerase comprising the amino acid sequence of SEQ ID NO: 1. In some embodiments, the at least one pair of amino acid substitutions is at amino acid positions 35 and 147, and the modified RNA polymerase of the present disclosure further comprises an amino acid substitution at one or more of amino acid positions 19, 20, 36, 41, 128, 154, 458, 464, 538, 566, 749, and 873 as indexed by reference to the wild-type RNA polymerase comprising the amino acid sequence of SEQ ID NO: 1.

[0008] In some embodiments, the modified RNA polymerase of the present disclosure comprises at least two amino acid substitutions at (i) amino acid positions 35 and 147, or (ii) an amino acid position selected from 19, 20, 36, 41, 128, 154, and 749 and an additional amino acid position selected from 19, 20, 36, 41, 128, 154, 464, 538, 458, 464, 538, 566, 749, and 873. In some embodiments, the modified RNA polymerase of the present disclosure comprises amino acid substitutions at amino acid positions 35 and 147, amino acid positions 464 and 749, amino acid positions 538 and 749, amino acid positions 749 and 873, amino acid positions 128 and 873, amino acid positions 128 and 538, or amino acid positions 128 and 749.

[0009] In some embodiments, the modified RNA polymerase of the present disclosure comprises at least three amino acid substitutions at (i) amino acid positions 35 and 147 and aminoacid position selected from 19, 20, 36, 41, 128, 154, 458, 464, 538, 566, 749, and 873; or (ii) an amino acid position selected from 19, 20, 36, 41, 128, 154, and 749 and two additional amino acid positions selected from 19, 20, 36, 41, 128, 154, 464, 538, 458, 464, 538, 566, 749, and 873. In some embodiments, the modified RNA polymerase of the present disclosure comprises amino acid substitutions at amino acid positions 35, 147, and 873, amino acid positions 128, 464, and 749, amino acid positions 35, 147, and 128, amino acid positions 35, 147, and 538, amino acid positions 35, 147, and 464, amino acid positions 35, 147, and 749, amino acid positions 128, 749, and 873, or amino acid positions 35, 147, 464, and 749, e.g. amino acid positions 35, 147, and 873, or amino acid positions 128, 464, and 749.

[0010] In another aspect, provided herein is a modified RNA polymerase comprising at least one amino acid substitution relative to a wild-type SP6 RNA polymerase comprising the amino acid sequence of SEQ ID NO: 1, wherein the at least one amino acid substitution comprises at least one pair of amino acid substitutions at amino acid positions 35 and 147, 37 and 42, or 131 and 157 as indexed by reference to the wild-type RNA polymerase comprising the amino acid sequence of SEQ ID NO: 1, and wherein the modified RNA polymerase comprises an amino acid sequence having at least about 80% sequence identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the modified RNA polymerase of the present disclosure further comprises an amino acid substitution at one or more of amino acid positions 19, 20, 36, 41, 128, 154, 458, 464, 538, 566, 749, and 873 as indexed by reference to the wild-type RNA polymerase comprising the amino acid sequence of SEQ ID NO: 1. In some embodiments, the at least one pair of amino acid substitutions is at amino acid positions 35 and 147 and / or amino acid positions 37 and 42. In some embodiments, the at least one pair of amino acid substitutions is at amino acid positions 35 and 147, and the modified RNA polymerase of the present disclosure further comprises an amino acid substitution at one or more of amino acid positions 19, 20, 41, 128, 458, 464, 538, 566, 749, and 873, e.g., at one or more of amino acid positions 128, 464, 538, 749, and 873, as indexed by reference to the wild-type RNA polymerase comprising the amino acid sequence of SEQ ID NO: 1. In some embodiments, the modified RNA polymerase of the present disclosure comprises amino acid substitutions at amino acid positions 35, 147, and 873, amino acid positions 35, 147, and 128, amino acid positions 35, 147, and 538, amino acid positions 35, 147, and 464, amino acid positions 35, 147, and 749, or amino acid positions 35, 147, 464, and 749, e.g., amino acid positions 35, 147, and 873.

[0011] In yet another aspect, provided herein is a modified RNA polymerase comprising at least one amino acid substitution relative to a wild-type SP6 RNA polymerase comprising the amino acid sequence of SEQ ID NO: 1, wherein the at least one amino acid substitution is at one or more of amino acid positions 19, 20, 36, 41, 128, 154, 458, 464, 538, 566, 749, and 873 as indexed by reference to the wild-type RNA polymerase comprising the amino acid sequence of SEQ ID NO: 1, and wherein the modified RNA polymerase comprises an amino acid sequence having at least about 80% sequence identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the amino acid substitution is at one or more of amino acid positions 19, 20, 41, 128, 458, 464, 538, 566, 749, and 873. In some embodiments, the modified RNA polymerase of the present disclosure comprises at least two amino acid substitutions at amino acid positions selected from 19, 20, 41, 128, 458, 464, 538, 566, 749, and 873. In some embodiments, the modified RNA polymerase of the present disclosure comprises amino acid substitutions at amino acid positions 464 and 749, amino acid positions 538 and 749, amino acid positions 749 and 873, amino acid positions 128 and 873, amino acid positions 128 and 538, or amino acid positions 128 and 749. In some embodiments, the modified RNA polymerase of the present disclosure comprises at least three amino acid substitutions at amino acid positions selected from 19, 20, 41, 128, 458, 464, 538, 566, 749, and 873. In some embodiments, the modified RNA polymerase of the present disclosure comprises amino acid substitutions at amino acid positions 128, 464, and 749 or amino acid positions 128, 749, and 873, e.g. amino acid positions 128, 464, and 749.

[0012] In some embodiments, the amino acid substitution at amino acid position 19 of the modified RNA polymerase of the present disclosure is G19H or G19T. In some embodiments, the amino acid substitution at amino acid position 19 of the modified RNA polymerase of the present disclosure is G19H. In some embodiments, the amino acid substitution at amino acid position 20 of the modified RNA polymerase of the present disclosure is I20E. In some embodiments, the amino acid substitution at amino acid position 36 of the modified RNA polymerase of the present disclosure is E36R. In some embodiments, the amino acid substitution at amino acid position 41 of the modified RNA polymerase of the present disclosure is W41L. In some embodiments, the amino acid substitution at amino acid position 128 of the modified RNA polymerase of the present disclosure is A128K or A128R, optionally A128K. In some embodiments, the amino acid substitution at amino acid position 154 of the modified RNA polymerase of the present disclosure is Al 54V. In some embodiments, the amino acid substitution at amino acid position 458 of themodified RNA polymerase of the present disclosure is N458R or N458K. In some embodiments, the amino acid substitution at amino acid position 464 of the modified RNA polymerase of the present disclosure is K464W. In some embodiments, the amino acid substitution at amino acid position 538 of the modified RNA polymerase of the present disclosure is S538F. In some embodiments, the amino acid substitution at amino acid position 566 of the modified RNA polymerase of the present disclosure is P566W, P566F, or P566Y. In some embodiments, the amino acid substitution at amino acid position 749 of the modified RNA polymerase of the present disclosure is L749F. In some embodiments, the amino acid substitution at amino acid position 873 of the modified RNA polymerase of the present disclosure is F873 Y. In some embodiments, the amino acid substitutions at amino acid positions 35 and 147 of the modified RNA polymerase of the present disclosure are S35E and Y147H, S35L and Y147W, or S35M and Y147W. In some embodiments, the amino acid substitutions at amino acid positions 37 and 42 of the modified RNA polymerase of the present disclosure are S37V and N42L. In some embodiments, the amino acid substitutions at amino acid positions 131 and 157 of the modified RNA polymerase of the present disclosure are Y131F and A157F.

[0013] In some embodiments, the modified RNA polymerase comprises an amino acid sequence having at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the modified RNA polymerase of the present disclosure comprises an amino acid sequence having at least about 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the modified RNA polymerase of the present disclosure comprises an amino acid sequence selected from SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, or SEQ ID NO: 54.

[0014] In a further aspect, provided herein is an artificial nucleic acid encoding any of the modified RNA polymerases of the present disclosure, a vector comprising the artificial nucleicacid, or a host cell comprising such a vector. In some embodiments, provided herein is a composition comprising any of the modified RNA polymerases disclosed herein, an artificial nucleic acid encoding the same, a vector comprising such an artificial nucleic acid, or a host cell comprising the vector.

[0015] In some embodiments, the present disclosure provides a composition or kit comprising any of the modified RNA polymerases disclosed herein and at least one in vitro transcription reaction component, such as nucleoside triphosphates and / or a cap or a cap analog.

[0016] In another aspect, the present disclosure provides a method for producing an RNA in an in vitro transcription reaction, said method comprising contacting a deoxyribonucleic acid (DNA) template with any of the modified RNA polymerases disclosed herein in the presence of nucleoside triphosphates. In some embodiments, the method produces a comparable amount of full-length RNA or more as compared to a control in vitro transcription reaction using an RNA polymerase having the amino acid sequence of SEQ ID NO: 1. In some embodiments, the method produces at least about 10% less double-stranded RNA (dsRNA) as compared to a control in vitro transcription reaction using an RNA polymerase having the amino acid sequence of SEQ ID NO: 1. In some embodiments, the method produces at least about 30% less dsRNA as compared to the control in vitro transcription reaction. In some embodiments, the method produces at least about 50% less dsRNA as compared to the control in vitro transcription reaction. In some embodiments, the method produces at least about 100% less dsRNA as compared to the control in vitro transcription reaction. In some embodiments, the method produces at least about 10% less abortive RNA transcripts as compared to a control in vitro transcription reaction using an RNA polymerase having the amino acid sequence of SEQ ID NO: 1. In some embodiments, the method produces at least about 30% less abortive RNA transcripts as compared to the control in vitro transcription reaction. In some embodiments, the method produces at least about 50% less abortive RNA transcripts as compared to the control in vitro transcription reaction. In some embodiments, the method produces RNA having a yield that is no less than about 35%, e.g. no less than about 50%, of the RNA yield produced in a control in vitro transcription reaction using an RNA polymerase having the amino acid sequence of SEQ ID NO: 1. In some embodiments, the method produces RNA having a yield that is at least about equal to the RNA yield produced in the control in vitro transcription reaction. In some embodiments, the method produces RNA having a yield that is superior to the RNA yield produced in the control in vitro transcription by at least about 10%,20%, 30%, 40%, 50%, or more. In some embodiments, the method of the present disclosure further comprises a step of purifying RNA from the in vitro transcription reaction. In some embodiments, the RNA produced by the method of the present disclosure is messenger RNA (mRNA).BRIEF DESCRIPTION OF THE DRAWINGS

[0017] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate certain embodiments, and together with the written description, serve to explain certain principles of the methods and compositions disclosed herein.

[0018] FIG. 1 depicts impurities produced from the in vitro transcription (IVT) catalyzed by single-subunit RNA polymerases (ssRNAP). NTD: N-terminal domain; CTD: C-terminal domain. Adapted from Fig. 1 of Dousis et al., Nature Biotechnology, 2023, 41 :560-568.

[0019] FIG. 2 depicts a ssRNAP structure in the initiation complex (A) and that in the elongation complex (B). Adapted from Fig. 2 of Yin et al., Science, 2002, 298: 1387-1395.

[0020] FIG. 3 depicts a representative strategy to engineer SP6 RNA polymerase using high throughput screening methods.

[0021] FIG. 4 depicts a graphic showing the production of dsRNA by various modified RNA polymerases selected from the SP6 RNA polymerase mutant library, as described in Example 5.

[0022] FIG. 5 depicts the manufacturability of the selected modified RNA polymerases described in Example 6, as measured by protein yield (mg) of the mutants.

[0023] FIG. 6 depicts a graphic showing that the yield of RNA transcribed in vitro using selected modified RNA polymerases and the wild-type RNA polymerase (WT) had similar profiles.

[0024] FIG. 7 depicts a graphic showing that RNA transcribed in vitro using the modified RNA polymerases had similar integrity profiles to RNA transcribed in vitro using the wild-type RNA polymerase. The molecular weight marker (nt) is shown on the left. Lane 1 : L749F, lane 2: S35E / Y147H, lane 3: W41L, lane 4: G19H, lane 5: 120E, lane 6: S37V / N42L, lane 7: wild-type.

[0025] FIG. 8 depicts a graphic showing the level of dsRNA obtained with the modified RNA polymerases as compared to the level obtained with the wild-type RNA polymerase, expressed as a percentage of the total RNA of the in vitro transcription reaction.

[0026] FIG. 9 depicts a graphic showing the level of abortive RNA transcripts obtained with the modified RNA polymerases as compared to the level of abortive RNA transcripts obtainedwith the wild-type RNA polymerase as determined by LC-MS, expressed as peak intensity per pg RNA.

[0027] FIG. 10 depicts a graphic showing the production of dsRNA by various modified RNA polymerases selected from the SP6 RNA polymerase mutant library, as described in Example 12.

[0028] FIG. 11 depicts a graphic showing integrity of RNA transcribed in vitro using various modified RNA polymerases. The dashed line corresponds to 75% RNA integrity.DETAILED DESCRIPTION

[0029] Reference will now be made in detail to various exemplary embodiments, examples of which are illustrated in the accompanying drawings and discussed in the detailed description that follows. It is to be understood that the following detailed description is provided to give the reader a fuller understanding of certain embodiments, features, and details of aspects of the disclosure, and should not be interpreted as limiting the scope of the disclosure.

[0030] In order for the present disclosure to be more readily understood, certain terms are first defined below. Additional definitions for the following terms and other terms may be set forth through the specification. If a definition of a term set forth below is inconsistent with a definition in an application or patent that is incorporated by reference, the definition set forth in this application should be used to understand the meaning of the term.Definitions

[0031] As used in this specification and the appended claims, the singular forms “a,” “an,” and “the” include plural references unless the context clearly dictates otherwise. Thus, for example, “a ribonucleotide” is understood to represent one or more ribonucleotides. As such, the terms “a,” “an,” “one or more,” and “at least one” can be used interchangeably herein.

[0032] Use of ordinal terms such as “first,” “second,” “third,” etc., in the claims to modify a claim element does not by itself connote any priority, precedence, or order of one claim element over another or the temporal order in which acts of a method are performed, but are used merely as labels to distinguish one claim element having a certain name from another element having a same name (but for use of the ordinal term) to distinguish the claim elements.

[0033] The term “about,” as used herein, refers to an interval of accuracy that a person skilled in the art will understand to still ensure the technical effect of the feature in question. The term indicates a deviation from the indicated numerical value of ±10%. In some embodiments, thedeviation is ±5% of the indicated numerical value. In certain embodiments, the deviation is ±1% of the indicated numerical value. When “about” is present before a series of numbers or a range, it is understood that “about” can modify each of the numbers in the series or range.

[0034] An “amino acid” according to the present disclosure can be any of the twenty naturally occurring (or “standard” amino acids) or variants thereof, such as, for example, D-proline (the D- enantiomer of proline), or any variants that are not naturally found in proteins, such as norleucine. The standard amino acids can be divided into several groups based on their properties. Important factors are charge, hydrophilicity or hydrophobicity, size and functional groups. These properties are important for protein structure and protein-protein interactions. Some amino acids have special properties, such as cysteine that can form covalent disulfide bonds (or disulfide bridges) to other cysteine residues, proline that forms a cycle to the polypeptide backbone, and glycine that is more flexible than other amino acids. Table 1 shows the abbreviations and properties of the standard amino acids.Table 1. Standard amino acids, abbreviations, and properties

[0035] Unless specifically stated or obvious from context, as used herein, the term “or” is understood to be inclusive and covers both “or” and “and.” Furthermore, “and / or” where used herein is to be taken as specific disclosure of each of the two specified features or components with or without the other. Thus, the term “and / or” as used in a phrase such as “A and / or B” herein is intended to include “A and B,” “A or B,” “A” (alone), and “B” (alone). Likewise, the term ’’and / or” as used in a phrase such as “A, B, and / or C” 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).

[0036] As used herein, an “artificial nucleic acid molecule” may typically be understood to be a nucleic acid molecule, e.g., a DNA or an RNA, that does not occur naturally. In other words, an artificial nucleic acid molecule may be understood as a non-natural nucleic acid molecule. Such nucleic acid molecule may be non-natural due to its individual sequence (which does not occur naturally) and / or due to other modifications, e.g., structural modifications of nucleotides that do not occur naturally. An artificial nucleic acid molecule may be a DNA molecule, an RNA molecule, or a hybrid molecule comprising DNA and RNA portions. Typically, artificial nucleic acid molecules may be designed and / or generated by genetic engineering methods to correspond to a desired artificial sequence of nucleotides (heterologous sequence). Further, the term “artificial nucleic acid molecule” is not restricted to mean “one single molecule” but is, typically, understood to comprise an ensemble of identical molecules. Accordingly, it may relate to a plurality of identical molecules contained in an aliquot.

[0037] The phrase “as indexed by reference to” an amino acid sequence, such as the wild-type RNA polymerase comprising the amino acid sequence of SEQ ID NO: 1, as used herein, refers to a normalized biological sequence alignment that allows the comparison of a query sequence (e.g., a modified RNA polymerase sequence to which one or more of the modifications described herein have been or will be applied) to a subject sequence (e.g., a wild-type RNA polymerase polypeptide sequence, such as the SP6 RNA polymerase polypeptide sequence of SEQ ID NO: 1), therebyidentifying amino acid residues in the target sequence that correspond to the same positions in the subject sequence. In general, the target sequence and the query sequence share characteristic portions or features but differ slightly in length and / or sequence identity. For example, the numbering of residues in a specific target sequence or for targeted modification can be identified and described based on the amino acid sequence of a wild-type SP6 RNA polymerase. Sequences are aligned to the full-length protein sequence of the wild-type SP6 RNA polymerase (SEQ ID NO: 1). The N-terminal methionine is residue 1. Accordingly, the phrase “amino acid position x as indexed by reference to the wild-type RNA polymerase comprising the amino acid sequence of SEQ ID NO: 1” is used herein to designate the position / identity of an amino acid residue in a polypeptide of interest (e.g., a modified RNA polymerase) by referring to the corresponding amino acid at position x in the wild-type SP6 RNA polymerase (SEQ ID NO: 1).

[0038] The term “at least,” “less than,” “more than,” or “up to” prior to a number or series of numbers (e.g., “at least two”) is understood to include the number adjacent to the term “at least,” “less than” or “more than,” and all subsequent numbers or integers that could logically be included, as clear from context. When the term “at least,” “less than,” “more than,” or “up to” is present before a series of numbers or a range, it is understood that “at least,” “less than,” “more than,” or “up to” can modify each of the numbers in the series or range.

[0039] As used herein, the term “double-stranded RNA” or “dsRNA” refers to RNA produced during in vitro transcription (IVT) comprising two complementary strands of ribonucleic acids base-paired with each other. During IVT, dsRNA is generated in cis by looping of full-length RNA with internal regions of complementarity. In addition, abortive RNA transcripts are generated during the initiation phase of IVT, and the 3' end of the full-length RNA can prime complementary RNA synthesis from the primary transcripts in trans. Promoter-independent transcription of full-length anti-sense RNA is another mechanism of dsRNA generation.

[0040] 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 an RNA transcript (e.g., by splicing, editing, 5' cap formation, and / or 3' end formation); (3) translation of an RNA into a polypeptide or protein; and / or (4) post- translational modification of a polypeptide or protein. The terms “expression” and “production,” and grammatical equivalents thereof, are used inter-changeably herein.

[0041] Throughout this specification and embodiments, the words “have” and “comprise,” or variations such as “has,” “having,” “comprises,” or “comprising” will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers. It is further understood that wherever embodiments are described herein with the language “comprising” or “having,” or grammatical equivalents thereof, otherwise analogous embodiments described in terms of “consisting of’ and / or “consisting essentially of’ are also provided.

[0042] The term “host” is used herein to refer to a system (e.g., a cell, organism, etc.) in which an RNA or polypeptide of interest is present. In some embodiments, a host is a system that is susceptible to infection with a particular infectious agent. In some embodiments, a host is a system that expresses a particular RNA or polypeptide of interest.

[0043] As used herein, the term “host cell” refers to a cell into which an exogenous nucleic acid (recombinant or otherwise) has been introduced. For example, host cells may be used to produce the modified RNA polymerases described herein by standard recombinant techniques. Persons of skill upon reading this disclosure will understand that such terms refer not only to the particular subject cell, but, to the progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term “host cell: as used herein. In some embodiments, host cells include any prokaryotic and eukaryotic cells suitable for expressing an exogenous DNA (e.g., a recombinant nucleic acid sequence). Exemplary cells include those of prokaryotes and eukaryotes (single-cell or multiple-cell), bacterial cells (e.g., strains of E. coli, Bacillus spp., Streptomyces spp., etc.), mycobacteria cells, fungal cells, yeast cells (e.g., S. cerevisiae, S. pombe, P. pastoris, P. methanolica, etc.), plant cells, insect cells (e.g., SF-9, SF-21, baculovirus-infected insect cells, Trichoplusia ni, etc.), non-human animal cells, human cells, or cell fusions such as, for example, hybridomas or quadromas. In some embodiments, the cell is a human, monkey, ape, hamster, rat, or mouse cell. In some embodiments, the cell is eukaryotic and is selected from the following cells: CHO (e.g., CHO KI, DXB-11 CHO, Veggie-CHO), COS (e.g., COS-7), retinal cell, Vero, CV1, kidney (e.g., HEK293, 293 EBNA, MSR 293, MDCK, HaK, BHK), HeLa, HepG2, WI38, MRC 5, Colo205, HB 8065, HL-60, (e.g., BHK21), Jurkat, Daudi, A431 (epidermal), CV-1, U937, 3T3, L cell, C127 cell, SP2 / 0, NS-0, MMT 060562, Sertoli cell, BRL 3A cell, HT1080 cell, myeloma cell, tumor cell, and a cell linederived from an aforementioned cell. In some embodiments, the cell comprises one or more viral genes, e.g., a retinal cell that expresses a viral gene (e.g., a PER.C6™ cell).

[0044] As used herein, the term “in some embodiments,” “in certain embodiments,” “in other embodiments,” “in some other embodiments,” or the like, refers to embodiments of all aspects of the disclosure, unless the context clearly indicates otherwise.

[0045] As used herein, the term “zzz vitro" refers to events that occur in an artificial environment, e.g., in a test tube or reaction vessel, in cell culture, etc., rather than within a multicellular organism.

[0046] As used herein, the term “zzz vzvo” refers to events that occur within a multi-cellular organism, such as a human or a non-human animal. In the context of cell-based systems, the term may be used to refer to events that occur within a living cell (as opposed to, for example, in vitro systems).

[0047] As used herein, the term “isolated” refers to a substance and / or entity that has been (1) separated from at least some of the components with which it was associated when initially produced (whether in nature and / or in an experimental setting), and / or (2) produced, prepared, and / or manufactured by the hand of man.

[0048] As used herein, the term “messenger RNA” or “mRNA” refers to a polyribonucleotide that encodes at least one polypeptide. The term “mRNA,” as used herein, encompasses both modified and unmodified RNA. mRNA may contain one or more coding and non-coding regions (e.g., a 5' untranslated region and a 3' untranslated region). mRNA can be purified from natural sources, produced using recombinant expression systems and optionally purified, in vitro transcribed, or chemically synthesized. The present disclosure particularly relates to in vitro transcribed mRNA. Where appropriate, for example, in the case of chemically synthesized molecules, mRNA can comprise nucleoside analogues such as analogues having chemically modified bases or sugars, backbone modifications, etc. An mRNA sequence is presented in the 5' to 3' direction unless otherwise indicated. A typical mRNA comprises 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 poly(A) tail.

[0049] The term “mutation” refers to deletion, addition, or substitution of an amino acid residue in the amino acid sequence of a modified protein or polypeptide as compared to the amino acid sequence of a reference protein or polypeptide.

[0050] The term “sequence identity,” as known in the art, is a relationship between two or more polypeptide sequences or two or more polynucleotide sequences, as determined by comparing the sequences. In the art, “identity” also means the degree of sequence relatedness between polypeptide or polynucleotide sequences, as determined by the match between strings of such sequences.

[0051] “Sequence identity” and “sequence similarity” can be readily calculated by known methods, including, but not limited to, those described in Computational Molecular Biology, Lesk, A. M., ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D. W., ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, A. M., and Griffin, H. G., eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991; and Carillo, H., and Lipman, D., Siam J. Applied Math., 48: 1073 (1988). Typical methods to determine identity are designed to give the largest match between the sequences tested. Methods to determine sequence identity and similarity are codified in publicly available computer programs. Typical computer program methods to determine identity and similarity between two sequences include, but are not limited to, the GCG program package (Devereux, J., et al., Nucleic Acids Research 12(1): 387 (1984)), BLASTP, BLASTN, and FASTA (Atschul, S. F. et al., J. Molec. Biol. 215:403-410 (1990). The BLAST X program is publicly available from NCBI and other sources (BLAST Manual, Altschul, S., et al., NCBINLM NIH Bethesda, Md. 20894: Altschul, S., et al., J. Mol. Biol. 215:403-410 (1990). The well-known Needleman-Wunsch algorithm may also be used to determine identity. IgBlast may also be used to determine germline V, D and J gene matches to a query sequence, which is available on the world wide web at ncbi.nlm.nih.gov / igblast / . In some embodiments, the sequence identity is determined using the BLAST X program with the default parameters. Sequence identity is calculated by taking the total number of identical nucleotides or amino acid residues in the alignment, dividing it by the total length of the aligned sequence, and multiplying the result by 100 to obtain a percentage. The reference sequence used for comparisons may be a publicly recognized sequence, such as a wild-type SP6 polymerase sequence, or a specific sequence chosen for its relevance, such as SEQ ID NO: 1. Percent similarity or percent identity as referred to herein is determined after optimal alignment of thesequences to be compared, which may therefore comprise one or more insertions, deletions, truncations and / or substitutions.

[0052] As used herein, the term “sequence-optimized” is used to describe a nucleotide sequence that is modified relative to a naturally occurring or wild-type nucleotide sequence. In the case of a sequence-optimized mRNA, such modifications may include, e.g., codon optimization and / or the use of 5 ' UTRs and / or 3 ' UTRs which are not normally associated with the naturally occurring or wild-type nucleic acid. As used herein, the terms “codon optimization” and “codon-optimized” refer to modifications of the codon composition of a naturally occurring or wild-type nucleic acid encoding a peptide, polypeptide or protein that do not alter its amino acid sequence, thereby improving protein expression of said nucleic acid in a particular expression system. For example, a nucleic acid sequence may be “codon-optimized” for expression in mammalian cells (e.g., CHO cells, human cells, mouse cells etc.), bacterial cells (e.g., E. coif), insect cells, yeast cells or plant cells. In the context of the present disclosure, “codon optimization” may also refer to the process by which one or more optimized nucleotide sequences are arrived at by removing, with filters, less than optimal nucleotide sequences from a list of nucleotide sequences, such as filtering by guanine-cytosine (GC) content, codon adaptation index (CAI), presence of destabilizing nucleic acid sequences or motifs, and / or presence of pause sites and / or terminator signals.

[0053] The terms “short abortive transcript” and “abortive transcript” are used interchangeably herein. They refer to transcripts that are generated during abortive initiation of transcription by an RNA polymerase. Abortive RNA transcripts are commonly observed during an in vitro transcription (IVT) reaction. Abortive RNA transcripts typically comprise less than about 20 nucleotides, including less than about 10 nucleotides. During in vitro synthesis of mRNA, RNA polymerase (RNAP) recognizes its cognate promoter leading to the local melting of a doublestrand DNA template to form the transcriptional “initiation complex.” Transcription during this stage is characterized by the repetitive synthesis and release of two to six nucleotides called “abortive cycling,” which is common to all RNAPs. Even at saturating nucleotide concentrations, abortive RNA transcripts are present in reaction in vitro, although their lengths differ among different RNAPs. After the synthesis of about eight to twelve nucleotides, the polymerase undergoes a major structural rearrangement and dissociates from the promoter (promoter clearance) to enter the processive synthesis of RNA, forming the “elongation complex” untiltranscription termination. Since the initiation complex is unstable, when compared to the elongation complex, abortive RNA transcripts are repeatedly released until the polymerase engages in productive transcription, which produces full-length transcripts.

[0054] As used herein, the terms “SP6 RNA polymerase,” “SP6 polymerase,” and “SP6” are used interchangeably and all refer to a DNA-dependent RNA polymerase obtainable from a Salmonella typhimurium phage, such as an RNA polymerase with the amino acid sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 24.

[0055] As used herein, the term “template DNA” (or “DNA template”) relates to a DNA molecule comprising a nucleic acid sequence encoding an mRNA transcript to be synthesized by in vitro transcription (IVT). The template DNA is used as template for IVT in order to produce the mRNA transcript encoded by the template DNA. The template DNA comprises all elements necessary for IVT, particularly a promoter element for binding of a DNA-dependent RNA polymerase, which is operably linked to the DNA sequence encoding a desired mRNA transcript. Furthermore, the template DNA may comprise primer binding sites 5' and / or 3' of the DNA sequence encoding the mRNA transcript to determine the identity of the DNA sequence encoding the mRNA transcript, e.g., by PCR or DNA sequencing. The “template DNA” in the context of the present disclosure may be a linear or a circular DNA molecule. As used herein, the term “template DNA” may refer to a DNA vector, such as a plasmid DNA, which comprises a nucleic acid sequence encoding the desired mRNA transcript.

[0056] As used herein, the term “truncated transcript” is used to refer to any transcript generated during the elongation phase that is shorter than a full-length RNA (e.g., mRNA) molecule encoded by the DNA template, e.g., as a result of the premature termination of transcription. In some embodiments, a truncated transcript may be less than 90% of the length of the full-length mRNA molecule that is transcribed from the target molecule, e.g., less than 80%, 70%, 60%, 50%, 40%, 30%, 20%, or 10%, including all values and subranges therebetween. The length of an mRNA molecule that encodes a full-length polypeptide or protein is at least 90% of the length of the theoretical transcript length. Indeed, the theoretical transcript length may differ from the measured length using a specific assay. Accordingly, in some embodiments, the term “full-length mRNA” refers to the measured length as characterized when using a specific assay, e.g., gel electrophoresis and detection using UV and UV absorption spectroscopy with separation by capillary gel electrophoresis. In these embodiments, a full-length mRNA that is transcribedfrom a DNA template is at least 95%, e.g., at least 96%, 97%, 98%, or 99%, including all values and subranges therebetween, of a reference mRNA transcript that expressed the full-length polypeptide or protein encoded by the mRNA transcript. In some embodiments, the truncated transcript is less than 100 nucleotides in length, less than 90, less than 80, less than 70, less than 60, less than 50, less than 40, less than 30, less than 20, or less than 10 nucleotides in length. In some embodiments, truncated transcripts are detected or quantified after adding a 5'-cap, and / or a 3 '-poly A tail.

[0057] As used herein, the term “vector” refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a “plasmid,” which refers to a circular double stranded DNA loop into which additional DNA segments may be ligated. Another type of vector is a viral vector, wherein additional DNA segments may be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) can be integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as “expression vectors.”

[0058] The term “wild-type,” as used herein to describe a SP6 RNA polymerase amino acid sequence, refers to the naturally occurring or native amino acid sequence. In some embodiments, the modified SP6 RNA polymerase disclosed herein may include amino acid substitutions, deletion, insertions, or additions relative to the wild-type amino acid sequence in order to render the protein more suitable for use in the methods and compositions of the present disclosure. Without wishing to be bound by any particular theory, the inventors believe that the polymerase function of such a modified enzyme has essentially identical or improved polymerase activity relative to the wild-type or native enzyme.

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

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

[0061] All publications and other reference materials referenced herein are hereby incorporated by reference in their entirety. Although a number of documents are cited herein, this citation does not constitute an admission that any of these documents forms part of the common general knowledge in the art.Modified RNA Polymerase

[0062] Bacteriophage SP6 RNA polymerase is a DNA-dependent RNA polymerase with high sequence specificity for SP6 promoter sequences. Typically, this polymerase catalyzes the 5'— >3 ' in vitro synthesis of RNA on either single-stranded DNA or double-stranded DNA downstream from its promoter and incorporates native ribonucleotides and / or modified ribonucleotides into the polymerized transcript.

[0063] The sequence for bacteriophage SP6 RNA polymerase was initially described in Kotani et al. (Nucleic Acids Res., 1987, 15(6):2653-2664) and is available under GenBank Accession No. Y00105.1 for the nucleotide sequence and P06221.1 for the amino acid sequence. It has the following amino acid sequence:MQDLHAIQLQLEEEMFNGGIRRFEADQQRQIAAGSESDTAWNRRLLSELI APMAEGIQAYKEEYEGKKGRAPRALAFLQCVENEVAAYITMKVVMDML NTDATLQAIAMSVAERIEDQVRFSKLEGHAAKYFEKVKKSLKASRTKSY RHAHNVAVVAEKSVAEKDADFDRWEAWPKETQLQIGTTLLEILEGSVFY NGEPVFMRAMRTYGGKTIYYLQTSESVGQWISAFKEHVAQLSPAYAPCVI PPRPWRTPFNGGFHTEKVASRIRLVKGNREHVRKLTQKQMPKVYKAINALQNTQWQINKDVLAVIEEVIRLDLGYGVPSFKPLIDKENKPANPVPVEFQHLRGRELKEMLSPEQWQQFINWKGECARLYTAETKRGSKSAAVVRMVGQ ARKYSAFESIYFVYAMDSRSRVYVQSSTLSPQSNDLGKALLRFTEGRPVN GVEALKWFCINGANLWGWDKKTFDVRVSNVLDEEFQDMCRDIAADPLT FTQWAKADAPYEFLAWCFEYAQYLDLVDEGRADEFRTHLPVHQDGSCSGIQHYSAMLRDEVGAKAVNLKPSDAPQDIYGAVAQVVIKKNALYMDAD DATTFTSGSVTLSGTELRAMASAWDSIGITRSLTKKPVMTLPYGSTRLTCRESVIDYIVDLEEKEAQKAVAEGRTANKVHPFEDDRQDYLTPGAAYNYMTALIWPSISEVVKAPIVAMKMIRQLARFAAKRNEGLMYTLPTGFILEQKIMA TEMLRVRTCLMGDIKMSLQVETDIVDEAAMMGAAAPNFVHGHDASHLIL TVCELVDKGVTSIAVIHDSFGTHADNTLTLRVALKGQMVAMYIDGNALQ KLLEEHEVRWMVDTGIEVPEQGEFDLNEIMDSEYVFA (SEQ ID NO: 1).

[0064] An alternative sequence for bacteriophage SP6 RNA polymerase is available underGenBank Accession No. AAR90000.1 and has the following amino acid sequence:MQDLHAIQLQLEEEMFNGGIRRFEADQQRQIAAGSESDTAWNRRLLSELIAPMAEGIQAYKEEYEGKKGRAPRALAFLQCVENEVAAYITMKVVMDML NTDATLQAIAMSVAERIEDQVRFSKLEGHAAKYFEKVKKSLKASRTKSY RHAHNVAVVAEKSVAEKDADFDRWEAWPKETQLQIGTTLLEILEGSVFY NGEPVFMRAMRTYGGKTIYYLQTSESVGQWISAFKEHVAQLSPAYAPCVIPPRPWRTPFNGGFHTEKVASRIRLVKGNREHVRKLTQKQMPKVYKAINA LQNTQWQINKDVLAVIEEVIRLDLGYGVPSFKPLIDKENKPANPVPVEFQH LRGRELKEMLSPEQWQQFINWKGECARLYTAETKRGSKSAAVVRMVGQ ARKYSAFESIYFVYAMDSRSRVYVQSSTLSPQSNDLGKALLRFTEGRPVN GVEALKWFCINGANLWGWDKKTFDVRVSNVLDEEFQDMCRDIAADPLT FTQWAKADAPYEFLAWCFEYAQYLDLVDEGRADEFRTHLPVHQDGSCS GIQHYSAMLRDEVGAKAVNLKPSDAPQDIYGAVAQVVIKKNALYMDAD DATTFTSGSVTLSGTELRAMASAWDSIGITRSLTKKPVMTLPYGSTRLTCR ESVIDYIVDLEEKEAQKAVAEGRTANKVHPFEDDRQDYLTPGAAYNYMT ALIWPSISEVVKAPIVAMKMIRQLARFAAKRNEGLMYTLPTGFILEQKIMATEMLRVRTCLMGDIKMSLQVETDIVDEAAMMGAAAPNFVHGHDASHLILTVCELVDKGVTSIAVIHDSFGTHADNTLTLRVALKGQMVAMYIDGNALQ KLLEEHEERWMVDTGIEVPEQGEFDLNEIMDSEYVFA (SEQ ID NO: 24)

[0065] There is currently no known 3-D structure identified for SP6 RNA polymerase. However, based on that of T7 RNA polymerase (e.g., Tahirov et al., Nature, 2002, 420:43-50; Yin et al., Science, 2002, 298: 1387-1395), it is postulated that, similar to T7 RNA polymerase, SP6 RNA polymerase also undergoes initiation and elongation phases during transcription (FIG. 1).

[0066] As described in Yin et al. (Science, 2002, 298: 1387-1395), during the initiation phase, T7 RNA polymerase binds to a specific DNA promoter, opens the duplex at the transcription start site, and initiates RNA synthesis de novo. Transcription during this initiation phase is unstable and characterized by repeated abortive initiation events, leading to the production of short RNA fragments (e.g., 2-6 nucleotides (nt)). After synthesis of 10- to 12-nt-long RNA, the polymerase enters the elongation phase and completes transcription of the mRNA processively without dissociating until termination. The T7 RNA polymerase-DNA complex is substantially more stable in the elongation phase. It is hypothesized that the alpha helix C of the T7 RNA polymerase in the elongation complex becomes larger and thus stabilizes the helical form of the polymerase.

[0067] Without wishing to be bound by any theory, it appears based on the data provided in this application that stabilizing SP6 RNA polymerase in one of the two configurations (i.e., initiation and / or elongation) can improve the outcome of in vitro transcription by, for instance, increasing RNA yield, increasing the production of full-length RNAs, reducing the production of double-stranded RNAs and / or abortive RNA transcripts, and / or improving 3' homogeneity. Thus, the present disclosure is based, in part, on the selection of modified RNA polymerases that are more stable in one of the two configurations as compared to the wild-type SP6 RNA polymerase. In some embodiments, the modified RNA polymerases of the present disclosure are more stable in the initiation phase. In some embodiments, the modified RNA polymerases of the present disclosure are more stable in the elongation phase. The stabilization of the modified RNA polymerases can be predicted based on the free energy of the modified RNA polymerases calculated using any methods known in the art, such as Rosetta energy function (see e.g., Alford et al., J. Chem. Theory Comput., 2017, 13(6):3031-3048), and the ones with the lowest energy forms (i.e., most stable) can be selected.

[0068] Accordingly, provided herein are modified SP6 RNA polymerases in which one or more mutations (e.g., substitution) have been introduced in the amino acid sequence relative to theamino acid sequence of the corresponding wild-type SP6 RNA polymerase. In some embodiments, the modified RNA polymerases disclosed herein possess certain beneficial characteristics, such as producing less dsRNA and / or less abortive RNA transcripts in an in vitro transcription reaction as compared to the corresponding wild-type SP6 RNA polymerase. Also provided are artificial nucleic acid molecules that encode the modified RNA polymerases disclosed herein.

[0069] The modified RNA polymerases of the present disclosure may notably comprise one or more amino acid mutations (e.g., amino acid substitutions) in the N-terminal domain and / or a portion of the C-terminal domain that interacts with the N-terminal domain as compared to the amino acid sequence of the corresponding wild-type SP6 RNA polymerase. While the modified RNA polymerases in this application are described in relation to the wild-type SP6 RNA polymerase having the amino acid sequence of SEQ ID NO: 1, the mutations described in this application can also be introduced into other wild-type SP6 RNA polymerases (e.g., the wild-type SP6 RNA polymerase having the amino acid sequence of SEQ ID NO: 24) or other RNA polymerases that share substantial sequence homology with SP6 RNA polymerase and that undergo initiation and elongation phases during transcription. The sequences of other RNA polymerases can be aligned with the sequence of the SP6 RNA polymerase to identify the location of an amino acid residue in the other RNA polymerase that corresponds to an amino acid residue of interest in a SP6 RNA polymerase.

[0070] The introduced amino acid mutations in the modified RNA polymerases of the disclosure include amino acid substitutions, deletions, or additions. In some embodiments, the one or more amino acid mutations may be independently selected from substitutions, insertions, deletions, and truncations. In some embodiments, the only mutations introduced in the amino acid sequence of the modified RNA polymerase of the disclosure are amino acid substitutions relative to the corresponding wild-type SP6 RNA polymerase and may include conservative and / or nonconservative substitutions.

[0071] Conservative substitutions may be made, for instance, on the basis of similarity in polarity, charge, size, solubility, hydrophobicity, hydrophilicity, and / or the amphipathic nature of the amino acid residues involved. For example, as summarized in Table 1, the 20 naturally occurring amino acids can be grouped into the following six standard amino acid groups: (1) hydrophobic: Met, Ala, Vai, Leu, He; (2) neutral hydrophilic: Cys, Ser, Thr; Asn, Gin; (3) acidic: Asp, Glu; (4) basic: His, Lys, Arg; (5) residues that influence chain orientation: Gly, Pro; and (6)aromatic: Trp, Tyr, Phe. As used herein, “conservative substitutions” are defined as exchanges of an amino acid by another amino acid listed within the same group of the six standard amino acid groups shown above. For example, the exchange of Asp by Glu retains one negative charge in the modified polypeptide. In addition, glycine and proline may be substituted for one another based on their ability to disrupt a-helices. As used herein, “non-conservative substitutions” are defined as exchanges of an amino acid by another amino acid listed in a different group of the six standard amino acid groups shown above.

[0072] In some embodiments, the substitutions may also include non-classical amino acids (e.g. selenocysteine, pyrrolysine, N-formylmethionine P-alanine, GABA and 5-Aminolevulinic acid, 4-aminobenzoic acid (PABA), D-isomers of the common amino acids, 2,4-diaminobutyric acid, a-amino isobutyric acid, 4-aminobutyric acid, Abu, 2-amino butyric acid, y-Abu, s-Ahx, 6- amino hexanoic acid, Aib, 2-amino isobutyric acid, 3-amino propionic acid, ornithine, norleucine, norvaline, hydroxyproline, sarcosme, citrulline, homocitrulline, cysteic acid, t-butylglycine, t- butylalanine, phenylglycine, cyclohexylalanine, P-alanine, fluoro-amino acids, designer amino acids such as P methyl amino acids, C a-methyl amino acids, N a-methyl amino acids, and amino acid analogs in general).

[0073] In some embodiments, amino acid substitutions at a specific amino acid position are chosen based on factors which include, but are not limited to, potential for steric hindrance, charge attraction, charge repulsion, common properties of the amino acid side chain, secondary and / or tertiary structure considerations, and / or frequency of use in respective host cells.

[0074] Accordingly, provided herein is a modified RNA polymerase comprising at least one amino acid substitution relative to a wild-type SP6 RNA polymerase, such as the wild-type SP6 RNA polymerase comprising the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 24, wherein the at least one amino acid substitution is in the N-terminal domain of the modified RNA polymerase or a portion of the C-terminal domain of the modified RNA polymerase that interacts with the N-terminal domain. In another embodiment, provided herein is a modified RNA polymerase comprising at least one amino acid substitution relative to a wild-type SP6 RNA polymerase, such as the wild-type SP6 RNA polymerase comprising the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 24, wherein the at least one amino acid substitution is in the C- terminal domain of the modified RNA polymerase. Advantageously, the modified RNApolymerase produces less double-stranded RNA and / or less abortive RNA transcripts in an in vitro transcription reaction as compared to the corresponding wild type SP6 RNA polymerase.

[0075] In some embodiments, the N-terminal domain of the modified RNA polymerase comprises amino acid residues located in the first half of the modified RNA polymerase. For instance, if the modified RNA polymerase has the full-length of 874 amino acids as the wild-type SP6 RNA polymerase comprising the amino acid sequence of SEQ ID NO: 1, the N-terminal domain of the modified RNA polymerase may comprise amino acid residues located at positions 1 through 437. In some embodiments, the N-terminal domain of the modified RNA polymerase comprises amino acid residues of the modified RNA polymerase between amino acid position 1 and amino acid position 400. In some embodiments, the N-terminal domain of the modified RNA polymerase comprises amino acid residues of the modified RNA polymerase between amino acid position 1 and amino acid position 350. In some embodiments, the N-terminal domain of the modified RNA polymerase comprises amino acid residues of the modified RNA polymerase between amino acid position 1 and amino acid position 300. In some embodiments, the N-terminal domain of the modified RNA polymerase comprises amino acid residues of the modified RNA polymerase between amino acid position 1 and amino acid position 250. In some embodiments, the N-terminal domain of the modified RNA polymerase comprises amino acid residues of the modified RNA polymerase between amino acid position 1 and amino acid position 200. In some embodiments, the N-terminal domain of the modified RNA polymerase comprises amino acid residues of the modified RNA polymerase between amino acid position 1 and amino acid position 160. In some embodiments, the N-terminal domain of the modified RNA polymerase comprises amino acid residues of the modified RNA polymerase between amino acid position 1 and amino acid position 150. In some embodiments, the N-terminal domain of the modified RNA polymerase comprises amino acid residues of the modified RNA polymerase between amino acid position 1 and amino acid position 100. In some embodiments, the N-terminal domain of the modified RNA polymerase comprises amino acid residues of the modified RNA polymerase between amino acid position 1 and amino acid position 50.

[0076] In some embodiments, the portion of the C-terminal domain of the modified RNA polymerase comprises amino acid residues located in the second half of the modified RNA polymerase. For instance, if the modified RNA polymerase has the full-length of 874 amino acids as the wild-type SP6 RNA polymerase comprising the amino acid sequence of SEQ ID NO: 1, theportion of the C-terminal domain of the modified RNA polymerase may comprise amino acid residues located at positions 438 through 874. In some embodiments, the portion of the C-terminal domain of the modified RNA polymerase comprises amino acid residues that form the specificity loop in the modified RNA polymerase. In some embodiments, the portion of the C-terminal domain of the modified RNA polymerase comprises amino acid residues of the modified RNA polymerase after amino acid position 700, including the amino acid residue at position 700 (e.g., amino acid residues 700-874 as indexed by reference to the wild-type SP6 RNA polymerase having the amino acid sequence of SEQ ID NO: 1). In some embodiments, the portion of the C- terminal domain of the modified RNA polymerase comprises amino acid residues of the modified RNA polymerase between amino acid position 700 and amino acid position 800. In some embodiments, the portion of the C-terminal domain of the modified RNA polymerase comprises amino acid residues of the modified RNA polymerase between amino acid position 710 and amino acid position 790. In some embodiments, the portion of the C-terminal domain of the modified RNA polymerase comprises amino acid residues of the modified RNA polymerase between amino acid position 720 and amino acid position 780. In some embodiments, the portion of the C- terminal domain of the modified RNA polymerase comprises amino acid residues of the modified RNA polymerase between amino acid position 730 and amino acid position 770. In some embodiments, the portion of the C-terminal domain of the modified RNA polymerase comprises amino acid residues of the modified RNA polymerase between amino acid position 740 and amino acid position 760. In some embodiments, the portion of the C-terminal domain of the modified RNA polymerase comprises amino acid residues of the modified RNA polymerase between amino acid position 745 and amino acid position 755.

[0077] In some embodiments, the at least one amino acid substitution in the N-terminal domain of the modified RNA polymerase is at one or more of amino acid positions 19, 20, 35, 36, 37, 41, 42, 44, 47, 128, 131, 147, 154, and 157 as indexed by reference to a wild-type RNA polymerase comprising the amino acid sequence of SEQ ID NO: 1. In some embodiments, the at least one amino acid substitution in the portion of the C-terminal domain of the modified RNA polymerase that interacts with the N-terminal domain is at amino acid position 749 as indexed by reference to a wild-type RNA polymerase comprising the amino acid sequence of SEQ ID NO: 1. In some embodiments, the at least one amino acid substitution in the portion of the C-terminal domain is at amino acid position 458, 464, 538, 566, 749, and 873 as indexed by reference to a wild-typeRNA polymerase comprising the amino acid sequence of SEQ ID NO: 1. Said amino acid substitution in the C-terminal domain may notably be in the C-terminal cavity.

[0078] In another aspect, the modified RNA polymerases of the present disclosure comprise at least one amino acid substitution at one or more of amino acid positions 19, 20, 35, 36, 37, 41, 42, 44, 47, 128, 131, 147, 154, 157, 458, 464, 538, 566, 749, and 873 as indexed by reference to a wild-type RNA polymerase comprising the amino acid sequence of SEQ ID NO: 1. In some embodiments, the modified RNA polymerases of the present disclosure comprise at least one amino acid substitution at amino acid position 19 as indexed by reference to a wild-type RNA polymerase comprising the amino acid sequence of SEQ ID NO: 1. In some embodiments, the modified RNA polymerases of the present disclosure comprise at least one amino acid substitution at amino acid position 20 as indexed by reference to a wild-type RNA polymerase comprising the amino acid sequence of SEQ ID NO: 1. In some embodiments, the modified RNA polymerases of the present disclosure comprise at least one amino acid substitution at amino acid position 35 as indexed by reference to a wild-type RNA polymerase comprising the amino acid sequence of SEQ ID NO: 1. In some embodiments, the modified RNA polymerases of the present disclosure comprise at least one amino acid substitution at amino acid position 36 as indexed by reference to a wild-type RNA polymerase comprising the amino acid sequence of SEQ ID NO: 1. In some embodiments, the modified RNA polymerases of the present disclosure comprise at least one amino acid substitution at amino acid position 37 as indexed by reference to a wild-type RNA polymerase comprising the amino acid sequence of SEQ ID NO: 1. In some embodiments, the modified RNA polymerases of the present disclosure comprise at least one amino acid substitution at amino acid position 41 as indexed by reference to a wild-type RNA polymerase comprising the amino acid sequence of SEQ ID NO: 1. In some embodiments, the modified RNA polymerases of the present disclosure comprise at least one amino acid substitution at amino acid position 42 as indexed by reference to a wild-type RNA polymerase comprising the amino acid sequence of SEQ ID NO: 1. In some embodiments, the modified RNA polymerases of the present disclosure comprise at least one amino acid substitution at amino acid position 44 as indexed by reference to a wild-type RNA polymerase comprising the amino acid sequence of SEQ ID NO: 1. In some embodiments, the modified RNA polymerases of the present disclosure comprise at least one amino acid substitution at amino acid position 47 as indexed by reference to a wild-type RNA polymerase comprising the amino acid sequence of SEQ ID NO: 1. In some embodiments, themodified RNA polymerases of the present disclosure comprise at least one amino acid substitution at amino acid position 128 as indexed by reference to a wild-type RNA polymerase comprising the amino acid sequence of SEQ ID NO: 1. In some embodiments, the modified RNA polymerases of the present disclosure comprise at least one amino acid substitution at amino acid position 131 as indexed by reference to a wild-type RNA polymerase comprising the amino acid sequence of SEQ ID NO: 1. In some embodiments, the modified RNA polymerases of the present disclosure comprise at least one amino acid substitution at amino acid position 147 as indexed by reference to a wild-type RNA polymerase comprising the amino acid sequence of SEQ ID NO: 1. In some embodiments, the modified RNA polymerases of the present disclosure comprise at least one amino acid substitution at amino acid position 154 as indexed by reference to a wild-type RNA polymerase comprising the amino acid sequence of SEQ ID NO: 1. In some embodiments, the modified RNA polymerases of the present disclosure comprise at least one amino acid substitution at amino acid position 157 as indexed by reference to a wild-type RNA polymerase comprising the amino acid sequence of SEQ ID NO: 1. In some embodiments, the modified RNA polymerases of the present disclosure comprise at least one amino acid substitution at amino acid position 458 as indexed by reference to a wild-type RNA polymerase comprising the amino acid sequence of SEQ ID NO: 1. In some embodiments, the modified RNA polymerases of the present disclosure comprise at least one amino acid substitution at amino acid position 464 as indexed by reference to a wild-type RNA polymerase comprising the amino acid sequence of SEQ ID NO: 1. In some embodiments, the modified RNA polymerases of the present disclosure comprise at least one amino acid substitution at amino acid position 538 as indexed by reference to a wild-type RNA polymerase comprising the amino acid sequence of SEQ ID NO: 1. In some embodiments, the modified RNA polymerases of the present disclosure comprise at least one amino acid substitution at amino acid position 566 as indexed by reference to a wild-type RNA polymerase comprising the amino acid sequence of SEQ ID NO: 1. In some embodiments, the modified RNA polymerases of the present disclosure comprise at least one amino acid substitution at amino acid position 749 as indexed by reference to a wild-type RNA polymerase comprising the amino acid sequence of SEQ ID NO: 1. In some embodiments, the modified RNA polymerases of the present disclosure comprise at least one amino acid substitution at amino acid position 873 as indexed by reference to a wild-type RNA polymerase comprising the amino acid sequence of SEQ ID NO: 1.

[0079] In some embodiments, the modified RNA polymerases of the present disclosure comprise at least one amino acid substitution at one or more of amino acid positions 19, 20, 36, 41, 128, 154, 458, 464, 538, 566, 749, and 873, e.g. amino acid positions 19, 20, 36, 41, 128, 154, 464, 538, 749, and 873, as indexed by reference to a wild-type RNA polymerase comprising the amino acid sequence of SEQ ID NO: 1. In some embodiments, the modified RNA polymerases of the present disclosure comprise at least one amino acid substitution at one or more of amino acid positions 19, 20, 41, 128, 154, 458, 464, 538, 566 749, and 873, e.g. amino acid positions 19, 20, 41, 128, 154, 464, 538, 749, and 873, as indexed by reference to a wild-type RNA polymerase comprising the amino acid sequence of SEQ ID NO: 1.

[0080] In some embodiments, the modified RNA polymerases of the present disclosure comprise at least two amino acid substitutions at amino acid positions selected from 19, 20, 36, 41, 128, 154, 458, 464, 538, 566, 749, and 873 as indexed by reference to a wild-type RNA polymerase comprising the amino acid sequence of SEQ ID NO: 1, such as at positions 19 and 20, positions 19 and 36, positions 19 and 41, positions 19 and 128, positions 19 and 154, positions 19 and 458, positions 19 and 464, positions 19 and 538, positions 19 and 566, positions 19 and 749, positions 19 and 873, positions 20 and 36, positions 20 and 41, positions 20 and 128, positions 20 and 154, positions 20 and 458, positions 20 and 464, positions 20 and 538, positions 20 and 566, positions 20 and 749, positions 20 and 873, positions 36 and 41, positions 36 and 128, positions 36 and 154, positions 36 and 458, positions 36 and 464, positions 36 and 538, positions 36 and 566, positions 36 and 749, positions 36 and 873, positions 41 and 128, positions 41 and 154, positions 41 and 458, positions 41 and 464, positions 41 and 538, positions 41 and 566, positions 41 and 749, positions 41 and 873, positions 128 and 154, positions 128 and 458, positions 128 and 464, positions 128 and 538, positions 128 and 566, positions 128 and 749, positions 128 and 873, positions 154 and 458, positions 154 and 464, positions 154 and 538, positions 154 and 566, positions 154 and 749, positions 154 and 873, positions 458 and 464, positions 458 and 538, positions 458 and 566, positions 458 and 749, positions 458 and 873, positions 464 and 538, positions 464 and 566, positions 464 and 749, positions 464 and 873, positions 538 and 566, positions 538 and 749, positions 538 and 873, positions 566 and 749, positions 566 and 873, or positions 749 and 873.

[0081] In some embodiments, the modified RNA polymerases of the present disclosure comprise at least two amino acid substitutions at amino acid positions 128 and 538 as indexed byreference to a wild-type RNA polymerase comprising the amino acid sequence of SEQ ID NO: 1. In some embodiments, the modified RNA polymerases of the present disclosure comprise at least two amino acid substitutions at amino acid positions 128 and 749 as indexed by reference to a wild-type RNA polymerase comprising the amino acid sequence of SEQ ID NO: 1. In some embodiments, the modified RNA polymerases of the present disclosure comprise at least two amino acid substitutions at amino acid positions 128 and 873 as indexed by reference to a wildtype RNA polymerase comprising the amino acid sequence of SEQ ID NO: 1. In some embodiments, the modified RNA polymerases of the present disclosure comprise at least two amino acid substitutions at amino acid positions 464 and 749 as indexed by reference to a wildtype RNA polymerase comprising the amino acid sequence of SEQ ID NO: 1. In some embodiments, the modified RNA polymerases of the present disclosure comprise at least two amino acid substitutions at amino acid positions 538 and 749 as indexed by reference to a wildtype RNA polymerase comprising the amino acid sequence of SEQ ID NO: 1. In some embodiments, the modified RNA polymerases of the present disclosure comprise at least two amino acid substitutions at amino acid positions 749 and 873 as indexed by reference to a wildtype RNA polymerase comprising the amino acid sequence of SEQ ID NO: 1.

[0082] In some embodiments, the modified RNA polymerases of the present disclosure comprise at least two amino acid substitutions at amino acid positions 19 and 20 as indexed by reference to a wild-type RNA polymerase comprising the amino acid sequence of SEQ ID NO: 1. In some embodiments, the modified RNA polymerases of the present disclosure comprise at least two amino acid substitutions at amino acid positions 19 and 36 as indexed by reference to a wildtype RNA polymerase comprising the amino acid sequence of SEQ ID NO: 1. In some embodiments, the modified RNA polymerases of the present disclosure comprise at least two amino acid substitutions at amino acid positions 19 and 41 as indexed by reference to a wild-type RNA polymerase comprising the amino acid sequence of SEQ ID NO: 1. In some embodiments, the modified RNA polymerases of the present disclosure comprise at least two amino acid substitutions at amino acid positions 19 and 154 as indexed by reference to a wild-type RNA polymerase comprising the amino acid sequence of SEQ ID NO: 1. In some embodiments, the modified RNA polymerases of the present disclosure comprise at least two amino acid substitutions at amino acid positions 19 and 749 as indexed by reference to a wild-type RNA polymerase comprising the amino acid sequence of SEQ ID NO: 1. In some embodiments, themodified RNA polymerases of the present disclosure comprise at least two amino acid substitutions at amino acid positions 20 and 36 as indexed by reference to a wild-type RNA polymerase comprising the amino acid sequence of SEQ ID NO: 1. In some embodiments, the modified RNA polymerases of the present disclosure comprise at least two amino acid substitutions at amino acid positions 20 and 41 as indexed by reference to a wild-type RNA polymerase comprising the amino acid sequence of SEQ ID NO: 1. In some embodiments, the modified RNA polymerases of the present disclosure comprise at least two amino acid substitutions at amino acid positions 20 and 154 as indexed by reference to a wild-type RNA polymerase comprising the amino acid sequence of SEQ ID NO: 1. In some embodiments, the modified RNA polymerases of the present disclosure comprise at least two amino acid substitutions at amino acid positions 20 and 749 as indexed by reference to a wild-type RNA polymerase comprising the amino acid sequence of SEQ ID NO: 1. In some embodiments, the modified RNA polymerases of the present disclosure comprise at least two amino acid substitutions at amino acid positions 36 and 41 as indexed by reference to a wild-type RNA polymerase comprising the amino acid sequence of SEQ ID NO: 1. In some embodiments, the modified RNA polymerases of the present disclosure comprise at least two amino acid substitutions at amino acid positions 36 and 154 as indexed by reference to a wild-type RNA polymerase comprising the amino acid sequence of SEQ ID NO: 1. In some embodiments, the modified RNA polymerases of the present disclosure comprise at least two amino acid substitutions at amino acid positions 36 and 749 as indexed by reference to a wild-type RNA polymerase comprising the amino acid sequence of SEQ ID NO: 1. In some embodiments, the modified RNA polymerases of the present disclosure comprise at least two amino acid substitutions at amino acid positions 41 and 154 as indexed by reference to a wild-type RNA polymerase comprising the amino acid sequence of SEQ ID NO: 1. In some embodiments, the modified RNA polymerases of the present disclosure comprise at least two amino acid substitutions at amino acid positions 41 and 749 as indexed by reference to a wild-type RNA polymerase comprising the amino acid sequence of SEQ ID NO: 1. In some embodiments, the modified RNA polymerases of the present disclosure comprise at least two amino acid substitutions at amino acid positions 154 and 749 as indexed by reference to a wild-type RNA polymerase comprising the amino acid sequence of SEQ ID NO: 1.

[0083] In some embodiments, the modified RNA polymerases of the present disclosure comprise at least three amino acid substitutions at amino acid positions selected from 19, 20, 36, 41, 128, 154, 458, 464, 538, 566, 749, and 873 as indexed by reference to a wild-type RNA polymerase comprising the amino acid sequence of SEQ ID NO: 1, such as at positions 19, 20, and 36, positions 19, 20, and 41, positions 19, 20, and 128, positions 19, 20, and 154, positions 19, 20, and 458, positions 19, 20, and 464, positions 19, 20, and 538, positions 19, 20, and 566, positions 19, 20, and 749, positions 19, 20, and 873, positions 19, 36, and 41, positions 19, 36, and 128, positions 19, 36, and 154, positions 19, 36, and 458, positions 19, 36, and 464, positions 19, 36, and 538, positions 19, 36, and 566, positions 19, 36, and 749, positions 19, 36, and 873, positions 19, 41, and 128, positions 19, 41, and 154, positions 19, 41, and 458, positions 19, 41, and 464, positions 19, 41, and 538, positions 19, 41, and 566, positions 19, 41, and 749, positions19, 41, and 873, positions 19, 128, and 154, positions 19, 128, and 458, positions 19, 128, and 464, positions 19, 128, and 538, positions 19, 128, and 566, positions 19, 128, and 749, positions 19, 128, and 873, positions 19, 154, and 458, positions 19, 154, and 464, positions 19, 154, and 538, positions 19, 154, and 566, positions 19, 154, and 749, positions 19, 154, and 873, positions 19, 458, and 464, positions 19, 458, and 538, positions 19, 458, and 566, positions 19, 458, and 749, positions 19, 458, and 873, positions 19, 464, and 538, positions 19, 464, and 566, positions 19, 464, and 749, positions 19, 464, and 873, positions 19, 538, and 566, positions 19, 538, and 749, positions 19, 538, and 873, positions 19, 566, and 749, positions 19, 566, and 873. positions 19, 749, and 873, positions 20, 36, and 41, positions 20, 36, and 128, positions 20, 36, and 154, positions 20, 36, and 458, positions 20, 36, and 464, positions 20, 36, and 538, positions 20, 36, and 566, positions 20, 36, and 749, positions 20, 36, and 873, positions 20, 41, and 128, positions20, 41, and 154, positions 20, 41, and 458, positions 20, 41, and 464, positions 20, 41, and 538, positions 20, 41, and 566, positions 20, 41, and 749, positions 20, 41, and 873, positions 20, 128, and 154, positions 20, 128, and 458, positions 20, 128, and 464, positions 20, 128, and 538, positions 20, 128, and 566, positions 20, 128, and 749, positions 20, 128, and 873, positions 20, 154, and 458, positions 20, 154, and 464, positions 20, 154, and 538, positions 20, 154, and 566, positions 20, 154, and 749, positions 20, 154, and 873, positions 20, 458, and 464, positions 20, 458, and 538, positions 20, 458, and 566, positions 20, 458, and 749, positions 20, 458, and 873, positions 20, 464, and 538, positions 20, 464, and 566, positions 20, 464, and 749, positions 20, 464, and 873, positions 20, 538, and 566, positions 20, 538, and 749, positions 20, 538, and 873,positions 20, 566, and 749, positions 20, 566, and 873, positions 20, 749, and 873, positions 36, 41, and 128, positions 36, 41, and 154, positions 36, 41, and 458, positions 36, 41, and 464, positions 36, 41, and 538, positions 36, 41, and 566, positions 36, 41, and 749, positions 36, 41, and 873, positions 36, 128, and 154, positions 36, 128, and 458, positions 36, 128, and 464, positions 36, 128, and 538, positions 36, 128, and 566, positions 36, 128, and 749, positions 36, 128, and 873, positions 36, 154, and 458, positions 36, 154, and 464, positions 36, 154, and 538, positions 36, 154, and 566, positions 36, 154, and 749, positions 36, 154, and 873, positions 36, 458, and 464, positions 36, 458, and 538, positions 36, 458, and 566, positions 36, 458, and 749, positions 36, 458, and 873, positions 36, 464, and 538, positions 36, 464, and 566, positions 36, 464, and 749, positions 36, 464, and 873, positions 36, 538, and 566, positions 36, 538, and 749, positions 36, 538, and 873, positions 36, 566, and 749, positions 36, 566, and 873, positions 36, 749, and 873, positions 41, 128, and 154, positions 41, 128, and 458, positions 41, 128, and 464, positions 41, 128, and 538, positions 41, 128, and 566, positions 41, 128, and 749, positions 41, 128, and 873, positions 41, 154, and 458, positions 41, 154, and 464, positions 41, 154, and 538, positions 41, 154, and 566, positions 41, 154, and 749, positions 41, 154, and 873, positions 41, 458, and 464, positions 41, 458, and 538, positions 41, 458, and 566, positions 41, 458, and 749, positions 41, 458, and 873, positions 41, 464, and 538, positions 41, 464, and 566, positions 41, 464, and 749, positions 41, 464, and 873, positions 41, 538, and 566, positions 41, 538, and 749, positions 41, 538, and 873, positions 41, 566, and 749, positions 41, 566, and 873, positions 41, 749, and 873, positions 128, 154, and 458, positions 128, 154, and 464, positions 128, 154, and 538, positions 128, 154, and 566, positions 128, 154, and 749, positions 128, 154, and 873, positions 128, 458, and 464, positions 128, 458, and 538, positions 128, 458, and 566, positions128, 458, and 749, positions 128, 458, and 873, positions 128, 464, and 538, positions 128, 464, and 566, positions 128, 464, and 749, positions 128, 464, and 873, positions 128, 538, and 566, positions 128, 538, and 749, positions 128, 538, and 873, positions 128, 566, and 749, positions128, 566, and 873, positions 128, 749, and 873, positions 154, 458, and 464, positions 154, 458, and 538, positions 154, 458, and 566, positions 154, 458, and 749, positions 154, 458, and 873, positions 154, 464, and 538, positions 154, 464, and 566, positions 154, 464, and 749, positions 154, 464, and 873, positions 154, 538, and 566, positions 154, 538, and 749, positions 154, 538, and 873, positions 154, 566, and 749, positions 154, 566, and 873, positions 154, 749, and 873, positions 458, 464, and 538, positions 458, 464, and 566, positions 458, 464, and 749, positions458, 464, and 873, positions 458, 538, and 566, positions 458, 538, and 749, positions 458, 538, and 873, positions 458, 566, and 749, positions 458, 566, and 873, positions 458, 749, and 873, positions 464, 538, and 566, positions 464, 538, and 749, positions 464, 538, and 873, positions464, 566, and 749, positions 464, 566, and 873, positions 464, 749, and 873, positions 538, 566, and 749, positions 538, 566, and 873, positions 538, 749, and 873, or positions 566, 749, and 873.

[0084] In some embodiments, the modified RNA polymerases of the present disclosure comprise at least three amino acid substitutions at amino acid positions 19, 20, and 154 as indexed by reference to a wild-type RNA polymerase comprising the amino acid sequence of SEQ ID NO: 1. In some embodiments, the modified RNA polymerases of the present disclosure comprise at least three amino acid substitutions at amino acid positions 19, 20, and 749 as indexed by reference to a wild-type RNA polymerase comprising the amino acid sequence of SEQ ID NO: 1. In some embodiments, the modified RNA polymerases of the present disclosure comprise at least three amino acid substitutions at amino acid positions 19, 36, and 41 as indexed by reference to a wildtype RNA polymerase comprising the amino acid sequence of SEQ ID NO: 1. In some embodiments, the modified RNA polymerases of the present disclosure comprise at least three amino acid substitutions at amino acid positions 19, 36, and 154 as indexed by reference to a wildtype RNA polymerase comprising the amino acid sequence of SEQ ID NO: 1. In some embodiments, the modified RNA polymerases of the present disclosure comprise at least three amino acid substitutions at amino acid positions 19, 36, and 749 as indexed by reference to a wildtype RNA polymerase comprising the amino acid sequence of SEQ ID NO: 1. In some embodiments, the modified RNA polymerases of the present disclosure comprise at least three amino acid substitutions at amino acid positions 19, 41, and 154 as indexed by reference to a wildtype RNA polymerase comprising the amino acid sequence of SEQ ID NO: 1. In some embodiments, the modified RNA polymerases of the present disclosure comprise at least three amino acid substitutions at amino acid positions 19, 41, and 749 as indexed by reference to a wildtype RNA polymerase comprising the amino acid sequence of SEQ ID NO: 1. In some embodiments, the modified RNA polymerases of the present disclosure comprise at least three amino acid substitutions at amino acid positions 19, 154, and 749 as indexed by reference to a wild-type RNA polymerase comprising the amino acid sequence of SEQ ID NO: 1. In some embodiments, the modified RNA polymerases of the present disclosure comprise at least three amino acid substitutions at amino acid positions, 20, 36, and 41 as indexed by reference to a wild-type RNA polymerase comprising the amino acid sequence of SEQ ID NO: 1. In some embodiments, the modified RNA polymerases of the present disclosure comprise at least three amino acid substitutions at amino acid positions, 20, 36, and 154 as indexed by reference to a wildtype RNA polymerase comprising the amino acid sequence of SEQ ID NO: 1. In some embodiments, the modified RNA polymerases of the present disclosure comprise at least three amino acid substitutions at amino acid positions, 20, 36, and 749 as indexed by reference to a wildtype RNA polymerase comprising the amino acid sequence of SEQ ID NO: 1. In some embodiments, the modified RNA polymerases of the present disclosure comprise at least three amino acid substitutions at amino acid positions, 36, 41, and 154 as indexed by reference to a wildtype RNA polymerase comprising the amino acid sequence of SEQ ID NO: 1. In some embodiments, the modified RNA polymerases of the present disclosure comprise at least three amino acid substitutions at amino acid positions, 36, 41, and 749 as indexed by reference to a wildtype RNA polymerase comprising the amino acid sequence of SEQ ID NO: 1. In some embodiments, the modified RNA polymerases of the present disclosure comprise at least three amino acid substitutions at amino acid positions, 36, 154, and 749 as indexed by reference to a wild-type RNA polymerase comprising the amino acid sequence of SEQ ID NO: 1. In some embodiments, the modified RNA polymerases of the present disclosure comprise at least three amino acid substitutions at amino acid positions, 41, 154, and 749 as indexed by reference to a wild-type RNA polymerase comprising the amino acid sequence of SEQ ID NO: 1. In some embodiments, the modified RNA polymerases of the present disclosure comprise at least three amino acid substitutions at amino acid positions 128, 464, and 749 as indexed by reference to a wild-type RNA polymerase comprising the amino acid sequence of SEQ ID NO: 1. In some embodiments, the modified RNA polymerases of the present disclosure comprise at least three amino acid substitutions at amino acid positions 128, 538, and 749 as indexed by reference to a wild-type RNA polymerase comprising the amino acid sequence of SEQ ID NO: 1. In some embodiments, the modified RNA polymerases of the present disclosure comprise at least three amino acid substitutions at amino acid positions 128, 749, and 873 as indexed by reference to a wild-type RNA polymerase comprising the amino acid sequence of SEQ ID NO: 1.

[0085] In some embodiments, the modified RNA polymerases of the present disclosure comprise at least three amino acid substitutions at amino acid positions 128, 464, and 749 as indexed by reference to a wild-type RNA polymerase comprising the amino acid sequence of SEQID NO: 1. In some embodiments, the modified RNA polymerases of the present disclosure comprise at least three amino acid substitutions at amino acid positions 128, 749, and 873 as indexed by reference to a wild-type RNA polymerase comprising the amino acid sequence of SEQ ID NO: 1.

[0086] In some embodiments, the modified RNA polymerases of the present disclosure comprise at least four amino acid substitutions at amino acid positions selected from 19, 20, 36,41, 128, 154, 458, 464, 538, 566, 749, and 873 as indexed by reference to a wild-type RNA polymerase comprising the amino acid sequence of SEQ ID NO: 1. In some embodiments, the modified RNA polymerases of the present disclosure comprise at least five amino acid substitutions at amino acid positions selected from 19, 20, 36, 41, 128, 154, 458, 464, 538, 566, 749, and 873 as indexed by reference to a wild-type RNA polymerase comprising the amino acid sequence of SEQ ID NO: 1. In some embodiments, the modified RNA polymerases of the present disclosure comprise at least six amino acid substitutions at amino acid positions 19, 20, 36, 41, 128, 154, 458, 464, 538, 566, 749, and 873 as indexed by reference to a wild-type RNA polymerase comprising the amino acid sequence of SEQ ID NO: 1.

[0087] In some embodiments, the modified RNA polymerases of the present disclosure comprise at least one pair of amino acid substitutions at amino acid positions 35 and 147, 37 and42, 44 and 47, or 131 and 157 as indexed by reference to a wild-type RNA polymerase comprising the amino acid sequence of SEQ ID NO: 1. In some embodiments, the modified RNA polymerases of the present disclosure comprise at least one pair of amino acid substitutions at amino acid positions 35 and 147 as indexed by reference to a wild-type RNA polymerase comprising the amino acid sequence of SEQ ID NO: 1. In some embodiments, the modified RNA polymerases of the present disclosure comprise at least one pair of amino acid substitutions at amino acid positions 37 and 42 as indexed by reference to a wild-type RNA polymerase comprising the amino acid sequence of SEQ ID NO: 1. In some embodiments, the modified RNA polymerases of the present disclosure comprise at least one pair of amino acid substitutions at amino acid positions 44 and 47 as indexed by reference to a wild-type RNA polymerase comprising the amino acid sequence of SEQ ID NO: 1. In some embodiments, the modified RNA polymerases of the present disclosure comprise at least one pair of amino acid substitutions at amino acid positions 131 and 157 as indexed by reference to a wild-type RNA polymerase comprising the amino acid sequence of SEQ ID NO: 1.

[0088] In some embodiments, the modified RNA polymerases of the present disclosure comprise at least two pairs of amino acid substitutions at amino acid positions 35 and 147, 37 and 42, 44 and 47, or 131 and 157 as indexed by reference to a wild-type RNA polymerase comprising the amino acid sequence of SEQ ID NO: 1. In some embodiments, the modified RNA polymerases of the present disclosure comprise at least two pairs of amino acid substitutions at amino acid positions 35 and 147 and amino acid positions 37 and 42 as indexed by reference to a wild-type RNA polymerase comprising the amino acid sequence of SEQ ID NO: 1. In some embodiments, the modified RNA polymerases of the present disclosure comprise at least two pairs of amino acid substitutions at amino acid positions 35 and 147 and amino acid positions 44 and 47 as indexed by reference to a wild-type RNA polymerase comprising the amino acid sequence of SEQ ID NO: 1. In some embodiments, the modified RNA polymerases of the present disclosure comprise at least two pairs of amino acid substitutions at amino acid positions 35 and 147 and amino acid positions 131 and 157 as indexed by reference to a wild-type RNA polymerase comprising the amino acid sequence of SEQ ID NO: 1. In some embodiments, the modified RNA polymerases of the present disclosure comprise at least two pairs of amino acid substitutions at amino acid positions 37 and 42 and amino acid positions 44 and 47 as indexed by reference to a wild-type RNA polymerase comprising the amino acid sequence of SEQ ID NO: 1. In some embodiments, the modified RNA polymerases of the present disclosure comprise at least two pairs of amino acid substitutions at amino acid positions 37 and 42 and amino acid positions 131 and 157 as indexed by reference to a wild-type RNA polymerase comprising the amino acid sequence of SEQ ID NO: 1. In some embodiments, the modified RNA polymerases of the present disclosure comprise at least two pairs of amino acid substitutions at amino acid positions 44 and 47 and amino acid positions 131 and 157 as indexed by reference to a wild-type RNA polymerase comprising the amino acid sequence of SEQ ID NO: 1.

[0089] In some embodiments, the modified RNA polymerases of the present disclosure comprise at least three pairs of amino acid substitutions at amino acid positions 35 and 147, 37 and 42, 44 and 47, or 131 and 157 as indexed by reference to a wild-type RNA polymerase comprising the amino acid sequence of SEQ ID NO: 1. In some embodiments, the modified RNA polymerases of the present disclosure comprise at least three pairs of amino acid substitutions at amino acid positions 35 and 147, amino acid positions 37 and 42, and amino acid positions 44 and 47 as indexed by reference to a wild-type RNA polymerase comprising the amino acid sequenceof SEQ ID NO: 1. In some embodiments, the modified RNA polymerases of the present disclosure comprise at least three pairs of amino acid substitutions at amino acid positions 35 and 147, amino acid positions 37 and 42, and amino acid positions 131 and 157 as indexed by reference to a wildtype RNA polymerase comprising the amino acid sequence of SEQ ID NO: 1. In some embodiments, the modified RNA polymerases of the present disclosure comprise at least three pairs of amino acid substitutions at amino acid positions 35 and 147, amino acid positions 44 and 47, and amino acid positions 131 and 157 as indexed by reference to a wild-type RNA polymerase comprising the amino acid sequence of SEQ ID NO: 1. In some embodiments, the modified RNA polymerases of the present disclosure comprise at least three pairs of amino acid substitutions at amino acid positions 37 and 42, amino acid positions 44 and 47, and amino acid positions 131 and 157 as indexed by reference to a wild-type RNA polymerase comprising the amino acid sequence of SEQ ID NO: 1. In some embodiments, the modified RNA polymerases of the present disclosure comprise at least four pairs of amino acid substitutions at amino acid positions 35 and 147, 37 and 42, amino acid positions 44 and 47, and amino acid positions 131 and 157 as indexed by reference to a wild-type RNA polymerase comprising the amino acid sequence of SEQ ID NO: 1.

[0090] In some embodiments, the modified RNA polymerases of the present disclosure comprise at least one pair of amino acid substitutions at amino acid positions 35 and 147, 37 and 42, 44 and 47, or 131 and 157 and at least one amino acid substitution at one or more of amino acid positions 19, 20, 36, 41, 128, 154, 458, 464, 538, 566, 749, and 873 as indexed by reference to a wild-type RNA polymerase comprising the amino acid sequence of SEQ ID NO: 1. In some embodiments, the modified RNA polymerases of the present disclosure comprise at least one pair of amino acid substitutions at amino acid positions 35 and 147 and at least one amino acid substitution at one or more of amino acid positions 19, 20, 36, 41, 128, 154, 458, 464, 538, 566, 749, and 873 as indexed by reference to a wild-type RNA polymerase comprising the amino acid sequence of SEQ ID NO: 1. In some embodiments, the modified RNA polymerases of the present disclosure comprise at least one pair of amino acid substitutions at amino acid positions 37 and 42 and at least one amino acid substitution at one or more of amino acid positions 19, 20, 36, 41, 128, 154, 458, 464, 538, 566, 749, and 873 as indexed by reference to a wild-type RNA polymerase comprising the amino acid sequence of SEQ ID NO: 1. In some embodiments, the modified RNA polymerases of the present disclosure comprise at least one pair of amino acid substitutions at amino acid positions 44 and 47 and at least one amino acid substitution at one or more of aminoacid positions 19, 20, 36, 41, 128, 154, 458, 464, 538, 566, 749, and 873 as indexed by reference to a wild-type RNA polymerase comprising the amino acid sequence of SEQ ID NO: 1. In some embodiments, the modified RNA polymerases of the present disclosure comprise at least one pair of amino acid substitutions at amino acid positions 131 and 157 and at least one amino acid substitution at one or more of amino acid positions 19, 20, 36, 41, 128, 154, 458, 464, 538, 566, 749, and 873 as indexed by reference to a wild-type RNA polymerase comprising the amino acid sequence of SEQ ID NO: 1. In some embodiments, the modified RNA polymerase provided herein comprises at least one pair of amino acid substitutions at amino acid positions 35 and 147, and at least one amino acid substitution at one or more of amino acid positions 128, 538, 464, 749, and 873 as indexed by reference to a wild-type RNA polymerase comprising the amino acid sequence of SEQ ID NO: 1. In some embodiments, the modified RNA polymerase provided herein comprises at least one pair of amino acid substitutions at amino acid positions 35 and 147 and two amino acid substitutions at amino acid positions 464 and 749 as indexed by reference to a wildtype RNA polymerase comprising the amino acid sequence of SEQ ID NO: 1.

[0091] In some embodiments, the modified RNA polymerases of the present disclosure comprise at least two pair of amino acid substitutions at amino acid positions 35 and 147, 37 and 42, 44 and 47, or 131 and 157 and at least one amino acid substitution at one or more of amino acid positions 19, 20, 36, 41, 128, 154, 458, 464, 538, 566, 749, and 873 as indexed by reference to a wild-type RNA polymerase comprising the amino acid sequence of SEQ ID NO: 1. In some embodiments, the modified RNA polymerases of the present disclosure comprise at least two pair of amino acid substitutions at amino acid positions 35 and 147 and amino acid positions 37 and 42 and at least one amino acid substitution at one or more of amino acid positions 19, 20, 36, 41, 128, 154, 458, 464, 538, 566, 749, and 873 as indexed by reference to a wild-type RNA polymerase comprising the amino acid sequence of SEQ ID NO: 1. In some embodiments, the modified RNA polymerases of the present disclosure comprise at least two pair of amino acid substitutions at amino acid positions 35 and 147 and amino acid positions 44 and 47 and at least one amino acid substitution at one or more of amino acid positions 19, 20, 36, 41, 128, 154, 458, 464, 538, 566, 749, and 873 as indexed by reference to a wild-type RNA polymerase comprising the amino acid sequence of SEQ ID NO: 1. In some embodiments, the modified RNA polymerases of the present disclosure comprise at least two pair of amino acid substitutions at amino acid positions 35 and 147 and amino acid positions 131 and 157 and at least one amino acid substitution at one or moreof amino acid positions 19, 20, 36, 41, 128, 154, 458, 464, 538, 566, 749, and 873 as indexed by reference to a wild-type RNA polymerase comprising the amino acid sequence of SEQ ID NO: 1. In some embodiments, the modified RNA polymerases of the present disclosure comprise at least two pair of amino acid substitutions at amino acid positions 37 and 42 and amino acid positions 44 and 47 and at least one amino acid substitution at one or more of amino acid positions 19, 20, 36, 41, 128, 154, 458, 464, 538, 566, 749, and 873 as indexed by reference to a wild-type RNA polymerase comprising the amino acid sequence of SEQ ID NO: 1. In some embodiments, the modified RNA polymerases of the present disclosure comprise at least two pair of amino acid substitutions at amino acid positions 37 and 42 and amino acid positions 131 and 157 and at least one amino acid substitution at one or more of amino acid positions 19, 20, 36, 41, 128, 154, 458, 464, 538, 566, 749, and 873 as indexed by reference to a wild-type RNA polymerase comprising the amino acid sequence of SEQ ID NO: 1. In some embodiments, the modified RNA polymerases of the present disclosure comprise at least two pair of amino acid substitutions at amino acid positions 44 and 47 and amino acid positions 131 and 157 and at least one amino acid substitution at one or more of amino acid positions 19, 20, 36, 41, 128, 154, 458, 464, 538, 566, 749, and 873 as indexed by reference to a wild-type RNA polymerase comprising the amino acid sequence of SEQ ID NO: 1.

[0092] In some embodiments, the modified RNA polymerases of the present disclosure comprise at least two pair of amino acid substitutions at amino acid positions 35 and 147, 37 and 42, 44 and 47, or 131 and 157 and at least two amino acid substitutions at amino acid positions selected from 19, 20, 36, 41, 128, 154, 458, 464, 538, 566, 749, and 873 as indexed by reference to a wild-type RNA polymerase comprising the amino acid sequence of SEQ ID NO: 1. In some embodiments, the modified RNA polymerases of the present disclosure comprise at least two pair of amino acid substitutions at amino acid positions 35 and 147 and amino acid positions 37 and 42 and at least two amino acid substitutions at amino acid positions selected from 19, 20, 36, 41, 128, 154, 458, 464, 538, 566, 749, and 873 as indexed by reference to a wild-type RNA polymerase comprising the amino acid sequence of SEQ ID NO: 1. In some embodiments, the modified RNA polymerases of the present disclosure comprise at least two pair of amino acid substitutions at amino acid positions 35 and 147 and amino acid positions 44 and 47 and at least two amino acid substitutions at amino acid positions selected from 19, 20, 36, 41, 128, 154, 458, 464, 538, 566, 749, and 873 as indexed by reference to a wild-type RNA polymerase comprising the amino acidsequence of SEQ ID NO: 1. In some embodiments, the modified RNA polymerases of the present disclosure comprise at least two pair of amino acid substitutions at amino acid positions 35 and 147 and amino acid positions 131 and 157 and at least two amino acid substitutions at amino acid positions selected from 19, 20, 36, 41, 128, 154, 458, 464, 538, 566, 749, and 873 as indexed by reference to a wild-type RNA polymerase comprising the amino acid sequence of SEQ ID NO: 1. In some embodiments, the modified RNA polymerases of the present disclosure comprise at least two pair of amino acid substitutions at amino acid positions 37 and 42 and amino acid positions 44 and 47 and at least two amino acid substitutions at amino acid positions selected from 19, 20, 36, 41, 128, 154, 458, 464, 538, 566, 749, and 873 as indexed by reference to a wild-type RNA polymerase comprising the amino acid sequence of SEQ ID NO: 1. In some embodiments, the modified RNA polymerases of the present disclosure comprise at least two pair of amino acid substitutions at amino acid positions 37 and 42 and amino acid positions 131 and 157 and at least two amino acid substitutions at amino acid positions selected from 19, 20, 36, 41, 128, 154, 458, 464, 538, 566, 749, and 873 as indexed by reference to a wild-type RNA polymerase comprising the amino acid sequence of SEQ ID NO: 1. In some embodiments, the modified RNA polymerases of the present disclosure comprise at least two pair of amino acid substitutions at amino acid positions 44 and 47 and amino acid positions 131 and 157 and at least two amino acid substitutions at amino acid positions selected from 19, 20, 36, 41, 128, 154, 458, 464, 538, 566, 749, and 873 as indexed by reference to a wild-type RNA polymerase comprising the amino acid sequence of SEQ ID NO: 1.

[0093] In some embodiments, the modified RNA polymerases of the present disclosure comprise at least two pair of amino acid substitutions at amino acid positions 35 and 147, 37 and 42, 44 and 47, or 131 and 157 and at least three amino acid substitutions at amino acid positions selected from 19, 20, 36, 41, 128, 154, 458, 464, 538, 566, 749, and 873 as indexed by reference to a wild-type RNA polymerase comprising the amino acid sequence of SEQ ID NO: 1. In some embodiments, the modified RNA polymerases of the present disclosure comprise at least two pair of amino acid substitutions at amino acid positions 35 and 147 and amino acid positions 37 and 42 and at least three amino acid substitutions at amino acid positions selected from 19, 20, 36, 41, 128, 154, 458, 464, 538, 566, 749, and 873 as indexed by reference to a wild-type RNA polymerase comprising the amino acid sequence of SEQ ID NO: 1. In some embodiments, the modified RNA polymerases of the present disclosure comprise at least two pair of amino acid substitutions atamino acid positions 35 and 147 and amino acid positions 44 and 47 and at least three amino acid substitutions at amino acid positions selected from 19, 20, 36, 41, 128, 154, 458, 464, 538, 566, 749, and 873 as indexed by reference to a wild-type RNA polymerase comprising the amino acid sequence of SEQ ID NO: 1. In some embodiments, the modified RNA polymerases of the present disclosure comprise at least two pair of amino acid substitutions at amino acid positions 35 and 147 and amino acid positions 131 and 157 and at least three amino acid substitutions at amino acid positions selected from 19, 20, 36, 41, 128, 154, 458, 464, 538, 566, 749, and 873 as indexed by reference to a wild-type RNA polymerase comprising the amino acid sequence of SEQ ID NO: 1. In some embodiments, the modified RNA polymerases of the present disclosure comprise at least two pair of amino acid substitutions at amino acid positions 37 and 42 and amino acid positions 44 and 47 and at least three amino acid substitutions at amino acid positions selected from 19, 20, 36, 41, 128, 154, 458, 464, 538, 566, 749, and 873 as indexed by reference to a wild-type RNA polymerase comprising the amino acid sequence of SEQ ID NO: 1. In some embodiments, the modified RNA polymerases of the present disclosure comprise at least two pair of amino acid substitutions at amino acid positions 37 and 42 and amino acid positions 131 and 157 and at least three amino acid substitutions at amino acid positions selected from 19, 20, 36, 41, 128, 154, 458, 464, 538, 566, 749, and 873 as indexed by reference to a wild-type RNA polymerase comprising the amino acid sequence of SEQ ID NO: 1. In some embodiments, the modified RNA polymerases of the present disclosure comprise at least two pair of amino acid substitutions at amino acid positions 44 and 47 and amino acid positions 131 and 157 and at least three amino acid substitutions at amino acid positions selected from 19, 20, 36, 41, 128, 154, 458, 464, 538, 566, 749, and 873 as indexed by reference to a wild-type RNA polymerase comprising the amino acid sequence of SEQ ID NO: 1.

[0094] In some embodiments, the amino acid substitution at amino acid position 19 of the modified RNA polymerases according to the present disclosure is G19H or G19T, or a conservative amino acid substitution thereof. In some embodiments, the amino acid substitution at amino acid position 19 is G19H, G19R, or G19K. In some embodiments, the amino acid substitution at amino acid position 19 is G19T, G19P, G19S, G19N, or G19Q. In some embodiments, the amino acid substitution at amino acid position 19 is G19H. In some embodiments, the amino acid substitution at amino acid position 19 is G19T.

[0095] In some embodiments, the amino acid substitution at amino acid position 20 of the modified RNA polymerases according to the present disclosure is I20E or a conservative amino acid substitution thereof. In some embodiments, the amino acid substitution at amino acid position 20 of the modified RNA polymerases according to the present disclosure is I20E or I20D. In some embodiments, the amino acid substitution at amino acid position 20 is I20E.

[0096] In some embodiments, the amino acid substitution at amino acid position 36 of the modified RNA polymerases according to the present disclosure is E36R or a conservative amino acid substitution thereof. In some embodiments, the amino acid substitution at amino acid position 36 is E36R, E36H, or E36K. In some embodiments, the amino acid substitution at amino acid position 36 is E36R.

[0097] In some embodiments, the amino acid substitution at amino acid position 41 of the modified RNA polymerases according to the present disclosure is W41L or a conservative amino acid substitution thereof. In some embodiments, the amino acid substitution at amino acid position 41 is W41L, W41 A, W41 V, W41I, or W41M. In some embodiments, the amino acid substitution at amino acid position 41 is W41L.

[0098] In some embodiments, the amino acid substitution at amino acid position 128 of the modified RNA polymerases according to the present disclosure is A128K or a conservative amino acid substitution thereof. In some embodiments, the amino acid substitution at amino acid position 128 is A128K, A128R, or A128H, e.g., A128K or A128R. In some embodiments, the amino acid substitution at amino acid position 128 is A128K. In some embodiments, the amino acid substitution at amino acid position 128 is A128R.

[0099] In some embodiments, the amino acid substitution at amino acid position 154 of the modified RNA polymerases according to the present disclosure is Al 54V or a conservative amino acid substitution thereof. In some embodiments, the amino acid substitution at amino acid position 154 is Al 54V, Al 541, A154L, or A154M. In some embodiments, the amino acid substitution at amino acid position 154 is Al 54V.

[0100] In some embodiments, the amino acid substitution at amino acid position 458 of the modified RNA polymerases according to the present disclosure is N458R or N458K, or a conservative amino acid substitution thereof. In some embodiments, the amino acid substitution at amino acid position 458 is N458R, N458K, or N458H, e.g., N458R or N458K. In someembodiments, the amino acid substitution at amino acid position 458 is N458R. In some embodiments, the amino acid substitution at amino acid position 458 is N458K.

[0101] In some embodiments, the amino acid substitution at amino acid position 464 of the modified RNA polymerases according to the present disclosure is K464W or a conservative amino acid substitution thereof. In some embodiments, the amino acid substitution at amino acid position 464 is K464W, K464F, or K464Y. In a particular embodiment, the amino acid substitution at amino acid position 464 is K464W.

[0102] In some embodiments, the amino acid substitution at amino acid position 538 of the modified RNA polymerases according to the present disclosure is S538F or a conservative amino acid substitution thereof. In some embodiments, the amino acid substitution at amino acid position 538 is S538F, S538R, or S538W. In a particular embodiment, the amino acid substitution at amino acid position 538 is S538F.

[0103] In some embodiments, the amino acid substitution at amino acid position 566 of the modified RNA polymerases according to the present disclosure is P566W, P566F, or P566Y. In some embodiments, the amino acid substitution at amino acid position 566 is P566W. In some embodiments, the amino acid substitution at amino acid position 566 is P566F. In some embodiments, the amino acid substitution at amino acid position 566 is P566Y.

[0104] In some embodiments, the amino acid substitution at amino acid position 749 of the modified RNA polymerases according to the present disclosure is L749F or a conservative amino acid substitution thereof. In some embodiments, the amino acid substitution at amino acid position 749 is L749F, L749Y, or L749W. In some embodiments, the amino acid substitution at amino acid position 749 is L749F.

[0105] In some embodiments, the amino acid substitution at amino acid position 873 of the modified RNA polymerases according to the present disclosure is F873 Y or a conservative amino acid substitution thereof. In some embodiments, the amino acid substitution at amino acid position 873 is F873 Y or F873W. In a particular embodiment, the amino acid substitution at amino acid position 873 is F873Y.

[0106] In some embodiments, the pair of amino acid substitutions at amino acid positions 35 and 147 of the modified RNA polymerases according to the present disclosure are S35E and Y147H, or a conservative amino acid substitution of S35E and / or Y147H. In some embodiments, the pair of amino acid substitutions at amino acid positions 35 and 147 of the modified RNApolymerases according to the present disclosure are S35E and Y147H, S35E and Y147R, S35E and Y147K, S35D and Y147H, S35D and Y147R, or S35D and Y147K. In some embodiments, the pair of amino acid substitutions at amino acid positions 35 and 147 of the modified RNA polymerases according to the present disclosure are S35E and Y147H.

[0107] In some embodiments, the pair of amino acid substitutions at amino acid positions 35 and 147 of the modified RNA polymerases according to the present disclosure are S35L or S35M, or a conservative amino acid substitution thereof, and Y147W, or a conservative amino acid substitution thereof. In some embodiments, the amino acid substitution at amino acid position 35 is S35L, S35M, S35 A, S35V, or S351. In some embodiments, the amino acid substitution at amino acid position 147 is Y147W or Y 147F. In some embodiments, the pair of amino acid substitutions at amino acid positions 35 and 147 are S35L and Y147H. In some embodiments, the pair of amino acid substitutions at amino acid positions 35 and 147 are S35L and Y147W. In some embodiments, the pair of amino acid substitutions at amino acid positions 35 and 147 are S35M and Y147W.

[0108] In some embodiments, the pair of amino acid substitutions at amino acid positions 37 and 42 of the modified RNA polymerases according to the present disclosure are S37V and N42L, or a conservative amino acid substitution of S37V and / or N42L. In some embodiments, the pair of amino acid substitutions at amino acid positions 37 and 42 of the modified RNA polymerases according to the present disclosure are S37V and N42L, S37V and N42A, S37V and N42V, S37V and N42I, S37V and N42M, S37 A and N42L, S37 A and N42A, S37 A and N42V, S37 A and N42I, S37 A and N42M, S371 and N42L, S371 and N42A, S371 and N42V, S37IV and N42I, , S37IV and N42M, S37L and N42L, S37L and N42A, S37L and N42V, S37L and N42I, S37L and N42M; S37M and N42A; S37M and N42V; S37M and N42I; S37M and N42L; or S37M and N42M. In some embodiments, the pair of amino acid substitutions at amino acid positions 37 and 42 of the modified RNA polymerases according to the present disclosure are S37V and N42L.

[0109] In some embodiments, the pair of amino acid substitutions at amino acid positions 44 and 47 of the modified RNA polymerases according to the present disclosure are R44L and S47V, or a conservative amino acid substitution of R44L and / or S47V. In some embodiments, the pair of amino acid substitutions at amino acid positions 44 and 47 of the modified RNA polymerases according to the present disclosure are R44L and S47V; R44L and S47A; R44L and S47I; R44L and S47L; R44L and S47M; R44A and S47V; R44A and S47A; R44A and S47I; R44A and S47L; R44A and S47M; R44V and S47V; R44V and S47A; R44V and S47I; R44V and S47L; R44V andS47M; R44I and S47V; R44I and S47A; R44I and S47I; R44I and S47L, R44I and S47M; R44M and S47A; R44M and S47I; R44M and S47L; or R44M and S47M. In some embodiments, the pair of amino acid substitutions at amino acid positions 44 and 47 of the modified RNA polymerases according to the present disclosure are R44L and S47V.

[0110] In some embodiments, the pair of amino acid substitutions at amino acid positions 131 and 157 of the modified RNA polymerases according to the present disclosure are Y131F and A157F, or a conservative amino acid substitution of Y131F and / or A157F. In some embodiments, the pair of amino acid substitutions at amino acid positions 131 and 157 of the modified RNA polymerases according to the present disclosure are Y131F and A157F; Y131W and A157F; Y131F and A157Y; Y131F and A157W; Y131W and A157Y; or Y131W and A157W. In some embodiments, the pair of amino acid substitutions at amino acid position 131 and 157 of the modified RNA polymerases according to the present disclosure are Y131F and A157F.[OHl] In some embodiments, provided herein is a modified RNA polymerase comprising amino acid substitutions at amino acid positions 35, 147, and 749 as indexed by reference to a wild-type RNA polymerase comprising the amino acid sequence of SEQ ID NO: 1. In some embodiments, the modified RNA polymerase of the present disclosure comprises amino acid substitutions S35E, Y147H, and L749F, or a conservative amino acid substitution of S35E, Y147H, and / or L749F, as indexed by reference to a wild-type RNA polymerase comprising the amino acid sequence of SEQ ID NO: 1. In some embodiments, the amino acid substitutions at amino acid positions 35, 147, and 749 of the modified RNA polymerases according to the present disclosure are S35E, Y147H, and L749F; S35E, Y147H, and L749Y; S35E, Y147H, and L749W; S35E, Y147R, and L749F; S35E, Y147K, and L749F; S35D, Y147H, and L749F; S35E, Y147R, and L749Y; S35E, Y147R, and L749W; S35E, Y147K, and L749Y; S35E, Y147K, and L749W; S35D, Y147R, and L749F; S35D, Y147K, and L749F; S35D, Y147H, and L749Y; S35D, Y147H, and L749W; S35D, Y147R, and L749Y; S35D, Y147R, and L749W; S35D, Y147K, and L749Y; or S35D, Y147K, and L749W. In some embodiments, the amino acid substitutions at amino acid positions 35, 147, and 749 of the modified RNA polymerases according to the present disclosure are S35E, Y147H, and L749F.

[0112] Also provided herein, in some embodiments, is a modified RNA polymerase comprising amino acid substitutions at amino acid positions 41 and 749 as indexed by reference to a wild-type RNA polymerase comprising the amino acid sequence of SEQ ID NO: 1. In someembodiments, the modified RNA polymerase of the present disclosure comprises amino acid substitutions W41L and L749F, or a conservative amino acid substitution of W41L and / or L749F, as indexed by reference to a wild-type RNA polymerase comprising the amino acid sequence of SEQ ID NO: 1. In some embodiments, the amino acid substitutions at amino acid positions 41 and 749 of the modified RNA polymerases according to the present disclosure are W41L and L749F; W41L and L749Y; W41L and L749W; W41 A and L749F; W41 A and L749Y; W41 A and L749W; W41V and L749F; W41V and L749FY; W41V and L749FW; W41I and L749F; W41I and L749Y; W41I and L749W; W41M and L749F; W41M and L749Y; or W41M and L749W. In some embodiments, the amino acid substitutions at amino acid positions 41 and 749 of the modified RNA polymerases according to the present disclosure are W41L and L749F.

[0113] In some embodiments, provided herein is a modified RNA polymerase comprising amino acid substitutions at amino acid positions 749 and 873 as indexed by reference to a wildtype RNA polymerase comprising the amino acid sequence of SEQ ID NO: 1. In some embodiments, the modified RNA polymerase of the present disclosure comprises amino acid substitutions L749F and F873 Y, or a conservative amino acid substitution of L749F and / or F873 Y, as indexed by reference to a wild-type RNA polymerase comprising the amino acid sequence of SEQ ID NO: 1. In some embodiments, the amino acid substitutions at amino acid positions 749 and 873 of the modified RNA polymerase are L749F and F873 Y, L749F and F873W, L749Y and F873Y, L749Y and F873W, L749W and F873 Y, or L749W and F873W. In some embodiments, the amino acid substitutions at amino acid positions of the modified RNA polymerases according to the present disclosure are L749F and F873 Y.

[0114] In some embodiments, provided herein is a modified RNA polymerase comprising amino acid substitutions at amino acid positions 35, 147, and 873 as indexed by reference to a wild-type RNA polymerase comprising the amino acid sequence of SEQ ID NO: 1. In some embodiments, the modified RNA polymerase of the present disclosure comprises amino acid substitutions S35M, Y147W, and F873Y, or a conservative amino acid substitution of S35M, Y147W, and / or F873Y, as indexed by reference to a wild-type RNA polymerase comprising the amino acid sequence of SEQ ID NO: 1. In some embodiments, the amino acid substitutions at amino acid positions 35, 147, and 873 of the modified RNA polymerases according to the present disclosure are S35L, Y147W, and F873Y; S35L, Y147W, and F873W; S35L, Y147F, and F873Y; S35L, Y147F, and F873W; S35M, Y147W, and F873Y; S35M, Y147W, and F873W; S35M,Y147F, and F873Y; S35M, Y147F, and F873W; S35A, Y147W, and F873Y; S35A, Y147W, and F873W; S35A, Y147F, and F873Y; S35A, Y147F, and F873W; S35V, Y147W, and F873Y; S35V, Y147W, and F873W; S35V, Y147F, and F873 Y; S35V, Y147F, and F873W; S35I, Y147W, and F873Y; S35I, Y147W, and F873W; S35I, Y147F, and F873Y; or S35I, Y147F, and F873W. In some embodiments, the amino acid substitutions at amino acid positions 35, 147, and 873 of the modified RNA polymerases according to the present disclosure are S35M, Y147W, and F873Y. In some embodiments, the amino acid substitutions at amino acid positions 35, 147, and 873 of the modified RNA polymerases according to the present disclosure are S35L, Y147W, and F873Y.

[0115] In some embodiments, provided herein is a modified RNA polymerase comprising amino acid substitutions at amino acid positions 538 and 749 as indexed by reference to a wildtype RNA polymerase comprising the amino acid sequence of SEQ ID NO: 1. In some embodiments, the modified RNA polymerase of the present disclosure comprises amino acid substitutions S538F and L749F, or a conservative amino acid substitution of S538F and / or L749F, as indexed by reference to a wild-type RNA polymerase comprising the amino acid sequence of SEQ ID NO: 1. In some embodiments, the amino acid substitutions at amino acid positions 538 and 749 of the modified RNA polymerases according to the present disclosure are S538F and L749F, S538F and L749Y, S538F and L749W, S538W and L749F, S538W and L749Y, or S538W and L749W. In some embodiments, the amino acid substitutions at amino acid positions 538 and 749 of the modified RNA polymerases according to the present disclosure are S538F and L749F.

[0116] In some embodiments, provided herein is a modified RNA polymerase comprising amino acid substitutions at amino acid positions 464 and 749 as indexed by reference to a wildtype RNA polymerase comprising the amino acid sequence of SEQ ID NO: 1. In some embodiments, the modified RNA polymerase of the present disclosure comprises amino acid substitutions K464W and L749F, or a conservative amino acid substitution of K464W and / or L749F, as indexed by reference to a wild-type RNA polymerase comprising the amino acid sequence of SEQ ID NO: 1. In some embodiments, the amino acid substitutions at amino acid positions 464 and 749 of the modified RNA polymerases according to the present disclosure are K464W and L749F, K464W and L749Y, K464W and L749W, K464F and L749F, K464F and L749Y, K464F and L749W, K464Y and L749F, K464Y and L749Y, or K464Y and L749W. Insome embodiments, the amino acid substitutions at amino acid positions 464 and 749 are K464W and L749F.

[0117] In some embodiments, provided herein is a modified RNA polymerase comprising amino acid substitutions at amino acid positions 128, 464, and 749 as indexed by reference to a wild-type RNA polymerase comprising the amino acid sequence of SEQ ID NO: 1. In some embodiments, the modified RNA polymerase of the present disclosure comprises amino acid substitutions A128K, K464W, and L749F, or a conservative amino acid substitution of A128K, K464W, and / or L749F, as indexed by reference to a wild-type RNA polymerase comprising the amino acid sequence of SEQ ID NO: 1. In some embodiments, the amino acid substitutions at amino acid positions 128, 464, and 749 of the modified RNA polymerases according to the present disclosure are A128K, K464W, and L749F, A128K, K464W, and L749Y, A128K, K464W, and L749W, A128K, K464F, and L749F, A128K, K464F, and L749Y, A128K, K464F, and L749W, A128K, K464Y, and L749F, A128K, K464Y, and L749Y, A128K, K464Y, and L749W, A128R, K464W, and L749F, A128R, K464W, and L749Y, A128R, K464W, and L749W, A128R, K464F, and L749F, A128R, K464F, and L749Y, A128R, K464F, and L749W, A128R, K464Y, and L749F, A128R, K464Y, and L749Y, A128R, K464Y, and L749W, A128H, K464W, and L749F, A128H, K464W, and L749Y, A128H, K464W, and L749W, A128H, K464F, and L749F, A128H, K464F, and L749Y, A128H, K464F, and L749W, A128H, K464Y, and L749F, A128H, K464Y, and L749Y, or A128H, K464Y, and L749W. In some embodiments, the amino acid substitutions at amino acid positions 128, 464, and 749 of the modified RNA polymerases according to the present disclosure are A128K, K464W, and L749F.

[0118] In some embodiments, provided herein is a modified RNA polymerase comprising amino acid substitutions at amino acid positions 128 and 873 as indexed by reference to a wildtype RNA polymerase comprising the amino acid sequence of SEQ ID NO: 1. In some embodiments, the modified RNA polymerase of the present disclosure comprises amino acid substitutions A128K and F873Y, or a conservative amino acid substitution of A128K and / or F873Y, as indexed by reference to a wild-type RNA polymerase comprising the amino acid sequence of SEQ ID NO: 1. In some embodiments, the amino acid substitutions at amino acid positions 128 and 873 of the modified RNA polymerase are A128K and F873Y, A128K and F873W, A128R and F873Y, A128R and F873W, A128H and F873Y, or A128H and F873W. Insome embodiments, the amino acid substitutions at amino acid positions 128 and 873 of the modified RNA polymerases according to the present disclosure are A128K and F873Y.

[0119] In some embodiments, provided herein is a modified RNA polymerase comprising amino acid substitutions at amino acid positions 128 and 538 as indexed by reference to a wildtype RNA polymerase comprising the amino acid sequence of SEQ ID NO: 1. In some embodiments, the modified RNA polymerase of the present disclosure comprises amino acid substitutions A128K and S538F, or a conservative amino acid substitution of A128K and / or S538F, as indexed by reference to a wild-type RNA polymerase comprising the amino acid sequence of SEQ ID NO: 1. In some embodiments, the amino acid substitutions at amino acid positions 128 and 538 of the modified RNA polymerase are A128K and S538F, A128R and S538F, A128H and S538F, or A128K and S538W, A128R and S538W, or A128H and S538W. In some embodiments, the amino acid substitutions at amino acid positions 128 and 538 of the modified RNA polymerase according provided herein are A128K and S538F.

[0120] In some embodiments, provided herein is a modified RNA polymerase comprising amino acid substitutions at amino acid positions 128 and 749 as indexed by reference to a wildtype RNA polymerase comprising the amino acid sequence of SEQ ID NO: 1. In some embodiments, the modified RNA polymerase of the present disclosure comprises amino acid substitutions A128K and L749F, or a conservative amino acid substitution of A128K and / or L749F, as indexed by reference to a wild-type RNA polymerase comprising the amino acid sequence of SEQ ID NO: 1. In some embodiments, the amino acid substitutions at amino acid positions 128 and 749 of the modified RNA polymerase are A128K and L749F, A128K and L749Y, A128R and L749F, A128R and L749Y, A128H and L749F, or A128H and L749Y, A128K and L749W, A128R and L749W, or A128H and L749W. In some embodiments, the amino acid substitutions at amino acid positions 128 and 749 of the modified RNA polymerase provided herein are A128K and L749F.

[0121] In some embodiments, provided herein is a modified RNA polymerase comprising amino acid substitutions at amino acid positions 128, 35, and 147 as indexed by reference to a wild-type RNA polymerase comprising the amino acid sequence of SEQ ID NO: 1. In some embodiments, the modified RNA polymerase provided herein comprises amino acid substitutions A128K, S35L, and Y147W, or a conservative amino acid substitution of A128K, S35L, and / or Y147W as provided herein, as indexed by reference to a wild-type RNA polymerase comprisingthe amino acid sequence of SEQ ID NO: 1. In some embodiments, the conservative amino acid substitution of S35L is S35M. In some embodiments, the amino acid substitutions at amino acid positions 128, 35, and 147 of the modified RNA polymerase provided herein are A128K, S35L, and Y147W. In some embodiments, the amino acid substitutions at amino acid positions 128, 35, and 147 of the modified RNA polymerase provided herein are A128K, S35M, and Y147W.

[0122] In some embodiments, provided herein is a modified RNA polymerase comprising amino acid substitutions at amino acid positions 538, 35 and 147 as indexed by reference to a wildtype RNA polymerase comprising the amino acid sequence of SEQ ID NO: 1. In some embodiments, the modified RNA polymerase provided herein comprises amino acid substitutions S538F, S35L, and Y147W, or a conservative amino acid substitution of S538F, S35L, and / or Y147W as provided herein, as indexed by reference to a wild-type RNA polymerase comprising the amino acid sequence of SEQ ID NO: 1. In some embodiments, the conservative amino acid substitution of S35L is S35M. In some embodiments, the amino acid substitutions at amino acid positions 538, 35, and 147 of the modified RNA polymerase provided herein are S538F, S35L, and Y147W. In some embodiments, the amino acid substitutions at amino acid positions 538, 35, and 147 of the modified RNA polymerase provided herein are S538F, S35M, and Y147W

[0123] In some embodiments, provided herein is a modified RNA polymerase comprising amino acid substitutions at amino acid positions 464, 35, and 147 as indexed by reference to a wild-type RNA polymerase comprising the amino acid sequence of SEQ ID NO: 1. In some embodiments, the modified RNA polymerase of the present disclosure comprises amino acid substitutions K464W, S35L, and Y147W, or a conservative amino acid substitution of K464W, S35L, and / or Y 147W as provided herein, as indexed by reference to a wild-type RNA polymerase comprising the amino acid sequence of SEQ ID NO: 1. In some embodiments, the conservative amino acid substitution of S35L is S35M. In some embodiments, the amino acid substitutions at amino acid positions 464, 35, and 147 of the modified RNA polymerase provided herein are K464W, S35L, and Y147W. In some embodiments, the amino acid substitutions at amino acid positions 464, 35, and 147 of the modified RNA polymerase provided herein are K464W, S35M, and Y147W.

[0124] In some embodiments, provided herein is a modified RNA polymerase comprising amino acid substitutions at amino acid positions 749, 35, and 147 as indexed by reference to a wild-type RNA polymerase comprising the amino acid sequence of SEQ ID NO: 1. In someembodiments, the modified RNA polymerase provided herein comprises amino acid substitutions L749F, S35L, and Y147W, or a conservative amino acid substitution of L749F, S35L, and / or Y147W as provided herein, as indexed by reference to a wild-type RNA polymerase comprising the amino acid sequence of SEQ ID NO: 1.

[0125] In some embodiments, provided herein is a modified RNA polymerase comprising amino acid substitutions at amino acid positions 128, 873, and 749 as indexed by reference to a wild-type RNA polymerase comprising the amino acid sequence of SEQ ID NO: 1. In some embodiments, the modified RNA polymerase provided herein comprises amino acid substitutions A128K, F873Y, and L749F, or a conservative amino acid substitution of A128K, F873Y, and / or L749F, as indexed by reference to a wild-type RNA polymerase comprising the amino acid sequence of SEQ ID NO: 1.

[0126] In some embodiments, provided herein is a modified RNA polymerase comprising amino acid substitutions at amino acid positions 749, 464, 35, and 147 as indexed by reference to a wild-type RNA polymerase comprising the amino acid sequence of SEQ ID NO: 1. In some embodiments, the modified RNA polymerase provided herein comprises amino acid substitutions L749F, K464W, S35L, and Y147W, or a conservative amino acid substitution of L749F, K464W, S35L, and / or Y 147W as provided herein, as indexed by reference to a wild-type RNA polymerase comprising the amino acid sequence of SEQ ID NO: 1.Exemplary Modified RNA Polymerases

[0127] In some embodiments, the modified RNA polymerase of the present disclosure comprises one of the following sequences (amino acid substitution(s): bold and italic).S35E / Y147H variant (SEQ ID NO: 2)MQDLHAIQLQLEEEMFNGGIRRFEADQQRQIAAGEESDTAWNRRLLSELIAPMAEGIQAYKEEYEGKKGRAPRALAFLQCVENEVAAYITMKVVMDML NTDATLQAIAMSVAERIEDQVRFSKLEGHAAKYFEK VKKSLKASRTKSH RHAHNVAVVAEKSVAEKDADFDRWEAWPKETQLQIGTTLLEILEGSVFY NGEPVFMRAMRTYGGKTIYYLQTSESVGQWISAFKEHVAQLSPAYAPCVI PPRPWRTPFNGGFHTEKVASRIRLVKGNREHVRKLTQKQMPKVYKAINA LQNTQWQINKDVLAVIEEVIRLDLGYGVPSFKPLIDKENKPANPVPVEFQH LRGRELKEMLSPEQWQQFINWKGECARLYTAETKRGSKSAAVVRMVGQ ARKYSAFESIYFVYAMDSRSRVYVQSSTLSPQSNDLGKALLRFTEGRPVNGVEALKWFCINGANLWGWDKKTFDVRVSNVLDEEFQDMCRDIAADPLT FTQWAKADAPYEFLAWCFEYAQYLDLVDEGRADEFRTHLPVHQDGSCS GIQHYSAMLRDEVGAKAVNLKPSDAPQDIYGAVAQVVIKKNALYMDAD DATTFTSGSVTLSGTELRAMASAWDSIGITRSLTKKPVMTLPYGSTRLTCR ESVIDYIVDLEEKEAQKAVAEGRTANKVHPFEDDRQDYLTPGAAYNYMT ALIWPSISEVVKAPIVAMKMIRQLARFAAKRNEGLMYTLPTGFILEQKIMA TEMLRVRTCLMGDIKMSLQVETDIVDEAAMMGAAAPNFVHGHDASHLIL TVCELVDKGVTSIAVIHDSFGTHADNTLTLRVALKGQMVAMYIDGNALQ KLLEEHEVRWMVDTGIEVPEQGEFDLNEIMDSEYVFAW41L variant (SEQ ID NO: 3)MQDLHAIQLQLEEEMFNGGIRRFEADQQRQIAAGSESDTALNRRLLSELIA PMAEGIQAYKEEYEGKKGRAPRALAFLQCVENEVAAYITMKVVMDMLN TDATLQAIAMSVAERIEDQVRFSKLEGHAAKYFEKVKKSLKASRTKSYR HAHNVAVVAEKSVAEKDADFDRWEAWPKETQLQIGTTLLEILEGSVFYN GEPVFMRAMRTYGGKTIYYLQTSESVGQWISAFKEHVAQLSPAYAPCVIP PRPWRTPFNGGFHTEKVASRIRLVKGNREHVRKLTQKQMPKVYKAINAL QNTQWQINI<DVLAVIEEVIRLDLGYGVPSFI<PLIDI<ENI<PANPVPVEFQHL RGRELKEMLSPEQWQQFINWKGECARLYTAETKRGSKSAAVVRMVGQA RKYSAFESIYFVYAMDSRSRVYVQSSTLSPQSNDLGKALLRFTEGRPVNG VEALKWFCINGANLWGWDKKTFDVRVSNVLDEEFQDMCRDIAADPLTF TQWAKADAPYEFLAWCFEYAQYLDLVDEGRADEFRTHLPVHQDGSCSGI QHYSAMLRDEVGAKAVNLKPSDAPQDIYGAVAQVVIKKNALYMDADD ATTFTSGSVTLSGTELRAMASAWDSIGITRSLTKKPVMTLPYGSTRLTCRE SVIDYIVDLEEKEAQKAVAEGRTANKVHPFEDDRQDYLTPGAAYNYMTA LIWPSISEVVKAPIVAMKMIRQLARFAAKRNEGLMYTLPTGFILEQKIMAT EMLRVRTCLMGDIKMSLQVETDIVDEAAMMGAAAPNFVHGHDASHLILT VCELVDKGVTSIAVIHDSFGTHADNTLTLRVALKGQMVAMYIDGNALQK LLEEHEVRWMVDTGIEVPEQGEFDLNEIMDSEYVFAG19T variant (SEQ ID NO: 4)MQDLHAIQLQLEEEMFNG7IRRFEADQQRQIAAGSESDTAWNRRLLSELI APMAEGIQAYKEEYEGKKGRAPRALAFLQCVENEVAAYITMKVVMDMLNTDATLQAIAMSVAERIEDQVRFSKLEGHAAKYFEKVKKSLKASRTKSYRHAHNVAVVAEKSVAEKDADFDRWEAWPKETQLQIGTTLLEILEGSVFYNGEPVFMRAMRTYGGKTIYYLQTSESVGQWISAFKEHVAQLSPAYAPCVIPPRPWRTPFNGGFHTEKVASRIRLVKGNREHVRKLTQKQMPKVYKAINALQNTQWQINKDVLAVIEEVIRLDLGYGVPSFKPLIDKENKPANPVPVEFQHLRGRELKEMLSPEQWQQFINWKGECARLYTAETKRGSKSAAVVRMVGQARKYSAFESIYFVYAMDSRSRVYVQSSTLSPQSNDLGKALLRFTEGRPVNGVEALKWFCINGANLWGWDKKTFDVRVSNVLDEEFQDMCRDIAADPLTFTQWAKADAPYEFLAWCFEYAQYLDLVDEGRADEFRTHLPVHQDGSCSGIQHYSAMLRDEVGAKAVNLKPSDAPQDIYGAVAQVVIKKNALYMDAD DATTFTSGSVTLSGTELRAMASAWDSIGITRSLTKKPVMTLPYGSTRLTCR ESVIDYIVDLEEKEAQKAVAEGRTANKVHPFEDDRQDYLTPGAAYNYMT ALIWPSISEVVKAPIVAMKMIRQLARFAAKRNEGLMYTLPTGFILEQKIMATEMLRVRTCLMGDIKMSLQVETDIVDEAAMMGAAAPNFVHGHDASHLILTVCELVDKGVTSIAVIHDSFGTHADNTLTLRVALKGQMVAMYIDGNALQ KLLEEHEVRWMVDTGIEVPEQGEFDLNEIMDSEYVFAG19H variant (SEQ ID NO: 5)MQDLHAIQLQLEEEMFNGHIRRFEADQQRQIAAGSESDTAWNRRLLSELIAPMAEGIQAYKEEYEGKKGRAPRALAFLQCVENEVAAYITMKVVMDML NTDATLQAIAMSVAERIEDQVRFSKLEGHAAKYFEKVKKSLKASRTKSY RHAHNVAVVAEKSVAEKDADFDRWEAWPKETQLQIGTTLLEILEGSVFY NGEPVFMRAMRTYGGKTIYYLQTSESVGQWISAFKEHVAQLSPAYAPCVIPPRPWRTPFNGGFHTEKVASRIRLVKGNREHVRKLTQKQMPKVYKAINALQNTQWQINKDVLAVIEEVIRLDLGYGVPSFKPLIDKENKPANPVPVEFQHLRGRELKEMLSPEQWQQFINWKGECARLYTAETKRGSKSAAVVRMVGQARKYSAFESIYFVYAMDSRSRVYVQSSTLSPQSNDLGKALLRFTEGRPVNGVEALKWFCINGANLWGWDKKTFDVRVSNVLDEEFQDMCRDIAADPLTFTQWAKADAPYEFLAWCFEYAQYLDLVDEGRADEFRTHLPVHQDGSCSGIQHYSAMLRDEVGAKAVNLKPSDAPQDIYGAVAQVVIKKNALYMDAD DATTFTSGSVTLSGTELRAMASAWDSIGITRSLTKKPVMTLPYGSTRLTCR ESVIDYIVDLEEKEAQKAVAEGRTANKVHPFEDDRQDYLTPGAAYNYMTALIWPSISEVVKAPIVAMKMIRQLARFAAKRNEGLMYTLPTGFILEQKIMA TEMLRVRTCLMGDIKMSLQVETDIVDEAAMMGAAAPNFVHGHDASHLIL TVCELVDKGVTSIAVIHDSFGTHADNTLTLRVALKGQMVAMYIDGNALQ K LLEEHE VR WM VDTGIE VPEQGEF DLNE IMD SE YVF AL749F variant (SEQ ID NO: 6)MQDLHAIQLQLEEEMFNGGIRRFEADQQRQIAAGSESDTAWNRRLLSELI APMAEGIQAYKEEYEGKKGRAPRALAFLQCVENEVAAYITMKVVMDML NTDATLQAIAMSVAERIEDQVRFSKLEGHAAKYFEKVKKSLKASRTKSY RHAHNVAVVAEKSVAEKDADFDRWEAWPKETQLQIGTTLLEILEGSVFY NGEPVFMRAMRTYGGKTIYYLQTSESVGQWISAFKEHVAQLSPAYAPCVI PPRPWRTPFNGGFHTEKVASRIRLVKGNREHVRKLTQKQMPKVYKAINALQNTQWQINKDVLAVIEEVIRLDLGYGVPSFKPLIDKENKPANPVPVEFQH LRGRELKEMLSPEQWQQFINWKGECARLYTAETKRGSKSAAVVRMVGQ ARKYSAFESIYFVYAMDSRSRVYVQSSTLSPQSNDLGKALLRFTEGRPVN GVEALKWFCINGANLWGWDKKTFDVRVSNVLDEEFQDMCRDIAADPLT FTQWAKADAPYEFLAWCFEYAQYLDLVDEGRADEFRTHLPVHQDGSCSGIQHYSAMLRDEVGAKAVNLKPSDAPQDIYGAVAQVVIKKNALYMDAD DATTFTSGSVTLSGTELRAMASAWDSIGITRSLTKKPVMTLPYGSTRLTCR ESVIDYIVDLEEKEAQKAVAEGRTANKVHPFEDDRQDYLTPGAAYNYMT ALIWPSISEVVKAPIVAMKMIRQLARFAAKRNEGLMYTLPTGFILEQKIMA TEMLRVRTCFMGDIKMSLQVETDIVDEAAMMGAAAPNFVHGHDASHLILTVCELVDKGVTSIAVIHDSFGTHADNTLTLRVALKGQMVAMYIDGNAL QKLLEEHEVRWMVDTGIEVPEQGEFDLNEIMDSEYVFAE36R variant (SEQ ID NO: 7)MQDLHAIQLQLEEEMFNGGIRRFEADQQRQIAAGS / fSDTAWNRRLLSELI APMAEGIQAYKEEYEGKKGRAPRALAFLQCVENEVAAYITMKVVMDML NTDATLQAIAMSVAERIEDQVRFSKLEGHAAKYFEKVKKSLKASRTKSY RHAHNVAVVAEKSVAEKDADFDRWEAWPKETQLQIGTTLLEILEGSVFY NGEPVFMRAMRTYGGKTIYYLQTSESVGQWISAFKEHVAQLSPAYAPCVI PPRPWRTPFNGGFHTEKVASRIRLVKGNREHVRKLTQKQMPKVYKAINALQNTQWQINKDVLAVIEEVIRLDLGYGVPSFKPLIDKENKPANPVPVEFQHLRGRELKEMLSPEQWQQFINWKGECARLYTAETKRGSKSAAVVRMVGQ ARKYSAFESIYFVYAMDSRSRVYVQSSTLSPQSNDLGKALLRFTEGRPVN GVEALKWFCINGANLWGWDKKTFDVRVSNVLDEEFQDMCRDIAADPLT FTQWAKADAPYEFLAWCFEYAQYLDLVDEGRADEFRTHLPVHQDGSCS GIQHYSAMLRDEVGAKAVNLKPSDAPQDIYGAVAQVVIKKNALYMDAD DATTFTSGSVTLSGTELRAMASAWDSIGITRSLTKKPVMTLPYGSTRLTCR ESVIDYIVDLEEKEAQKAVAEGRTANKVHPFEDDRQDYLTPGAAYNYMT ALIWPSISEVVKAPIVAMKMIRQLARFAAKRNEGLMYTLPTGFILEQKIMA TEMLRVRTCLMGDIKMSLQVETDIVDEAAMMGAAAPNFVHGHDASHLIL TVCELVDKGVTSIAVIHDSFGTHADNTLTLRVALKGQMVAMYIDGNALQ K LLEEHE VR WM VDTGIE VPEQGEF DLNE IMD SE YVF AY131F / A157F variant (SEQ ID NO: 8)MQDLHAIQLQLEEEMFNGGIRRFEADQQRQIAAGSESDTAWNRRLLSELI APMAEGIQAYKEEYEGKKGRAPRALAFLQCVENEVAAYITMKVVMDML NTDATLQAIAMSVAERIEDQVRFSI<LEGHAAI<FFEI<VI<I<SLI<ASRTI<SYR HAHNVAVVFEI<SVAEI<DADFDRWEAWPI<ETQLQIGTTLLEILEGSVFYN GEPVFMRAMRTYGGKTIYYLQTSESVGQWISAFKEHVAQLSPAYAPCVIP PRPWRTPFNGGFHTEKVASRIRLVKGNREHVRKLTQKQMPKVYKAINAL QNTQWQINKDVLAVIEEVIRLDLGYGVPSFKPLIDKENKPANPVPVEFQHL RGRELKEMLSPEQWQQFINWKGECARLYTAETKRGSKSAAVVRMVGQA RKYSAFESIYFVYAMDSRSRVYVQSSTLSPQSNDLGKALLRFTEGRPVNG VEALKWFCINGANLWGWDKKTFDVRVSNVLDEEFQDMCRDIAADPLTF TQWAKADAPYEFLAWCFEYAQYLDLVDEGRADEFRTHLPVHQDGSCSGI QHYSAMLRDEVGAKAVNLKPSDAPQDIYGAVAQVVIKKNALYMDADD ATTFTSGSVTLSGTELRAMASAWDSIGITRSLTKKPVMTLPYGSTRLTCRE SVIDYIVDLEEKEAQKAVAEGRTANKVHPFEDDRQDYLTPGAAYNYMTA LIWPSISEVVKAPIVAMKMIRQLARFAAKRNEGLMYTLPTGFILEQKIMAT EMLRVRTCLMGDIKMSLQVETDIVDEAAMMGAAAPNFVHGHDASHLILT VCELVDKGVTSIAVIHDSFGTHADNTLTLRVALKGQMVAMYIDGNALQK LLEEHE VRWMVDTGIEVPEQGEFDLNEIMDSEYVFAAl 54V variant (SEQ ID NO: 9)MQDLHAIQLQLEEEMFNGGIRRFEADQQRQIAAGSESDTAWNRRLLSELI APMAEGIQAYKEEYEGKKGRAPRALAFLQCVENEVAAYITMKVVMDML NTDATLQAIAMSVAERIEDQVRFSKLEGHAAKYFEKVKKSLKASRTKSY RHAHNVFVVAEKSVAEKDADFDRWEAWPKETQLQIGTTLLEILEGSVFYNGEPVFMRAMRTYGGKTIYYLQTSESVGQWISAFKEHVAQLSPAYAPCVI PPRPWRTPFNGGFHTEKVASRIRLVKGNREHVRKLTQKQMPKVYKAINA LQNTQWQINKDVLAVIEEVIRLDLGYGVPSFKPLIDKENKPANPVPVEFQH LRGRELKEMLSPEQWQQFINWKGECARLYTAETKRGSKSAAVVRMVGQARKYSAFESIYFVYAMDSRSRVYVQSSTLSPQSNDLGKALLRFTEGRPVN GVEALKWFCINGANLWGWDKKTFDVRVSNVLDEEFQDMCRDIAADPLT FTQWAKADAPYEFLAWCFEYAQYLDLVDEGRADEFRTHLPVHQDGSCS GIQHYSAMLRDEVGAKAVNLKPSDAPQDIYGAVAQVVIKKNALYMDADDATTFTSGSVTLSGTELRAMASAWDSIGITRSLTKKPVMTLPYGSTRLTCRESVIDYIVDLEEKEAQKAVAEGRTANKVHPFEDDRQDYLTPGAAYNYMTALIWPSISEVVKAPIVAMKMIRQLARFAAKRNEGLMYTLPTGFILEQKIMATEMLRVRTCLMGDIKMSLQVETDIVDEAAMMGAAAPNFVHGHDASHLILTVCELVDKGVTSIAVIHDSFGTHADNTLTLRVALKGQMVAMYIDGNALQ K LLEEHE VR WM VDTGIE VPEQGEF DLNE IMD SE YVF AI20E variant (SEQ ID NO: 10)MQDLHAIQLQLEEEMFNGGERRFEADQQRQIAAGSESDTAWNRRLLSELI APMAEGIQAYKEEYEGKKGRAPRALAFLQCVENEVAAYITMKVVMDML NTDATLQAIAMSVAERIEDQVRFSKLEGHAAKYFEKVKKSLKASRTKSY RHAHNVAVVAEKSVAEKDADFDRWEAWPKETQLQIGTTLLEILEGSVFYNGEPVFMRAMRTYGGKTIYYLQTSESVGQWISAFKEHVAQLSPAYAPCVI PPRPWRTPFNGGFHTEKVASRIRLVKGNREHVRKLTQKQMPKVYKAINA LQNTQWQINKDVLAVIEEVIRLDLGYGVPSFKPLIDKENKPANPVPVEFQH LRGRELKEMLSPEQWQQFINWKGECARLYTAETKRGSKSAAVVRMVGQARKYSAFESIYFVYAMDSRSRVYVQSSTLSPQSNDLGKALLRFTEGRPVN GVEALKWFCINGANLWGWDKKTFDVRVSNVLDEEFQDMCRDIAADPLT FTQWAKADAPYEFLAWCFEYAQYLDLVDEGRADEFRTHLPVHQDGSCS GIQHYSAMLRDEVGAKAVNLKPSDAPQDIYGAVAQVVIKKNALYMDADDATTFTSGSVTLSGTELRAMASAWDSIGITRSLTKKPVMTLPYGSTRLTCRESVIDYIVDLEEKEAQKAVAEGRTANKVHPFEDDRQDYLTPGAAYNYMTALIWPSISEVVKAPIVAMKMIRQLARFAAKRNEGLMYTLPTGFILEQKIMATEMLRVRTCLMGDIKMSLQVETDIVDEAAMMGAAAPNFVHGHDASHLILTVCELVDKGVTSIAVIHDSFGTHADNTLTLRVALKGQMVAMYIDGNALQ K LLEEHE VR WM VDTGIE VPEQGEF DLNE IMD SE YVF AR44L / S47V variant (SEQ ID NO: 11)MQDLHAIQLQLEEEMFNGGIRRFEADQQRQIAAGSESDTAWNRLLLEELI APMAEGIQAYKEEYEGKKGRAPRALAFLQCVENEVAAYITMKVVMDML NTDATLQAIAMSVAERIEDQVRFSKLEGHAAKYFEKVKKSLKASRTKSY RHAHNVAVVAEKSVAEKDADFDRWEAWPKETQLQIGTTLLEILEGSVFYNGEPVFMRAMRTYGGKTIYYLQTSESVGQWISAFKEHVAQLSPAYAPCVI PPRPWRTPFNGGFHTEKVASRIRLVKGNREHVRKLTQKQMPKVYKAINA LQNTQWQINKDVLAVIEEVIRLDLGYGVPSFKPLIDKENKPANPVPVEFQH LRGRELKEMLSPEQWQQFINWKGECARLYTAETKRGSKSAAVVRMVGQARKYSAFESIYFVYAMDSRSRVYVQSSTLSPQSNDLGKALLRFTEGRPVN GVEALKWFCINGANLWGWDKKTFDVRVSNVLDEEFQDMCRDIAADPLT FTQWAKADAPYEFLAWCFEYAQYLDLVDEGRADEFRTHLPVHQDGSCS GIQHYSAMLRDEVGAKAVNLKPSDAPQDIYGAVAQVVIKKNALYMDADDATTFTSGSVTLSGTELRAMASAWDSIGITRSLTKKPVMTLPYGSTRLTCRESVIDYIVDLEEKEAQKAVAEGRTANKVHPFEDDRQDYLTPGAAYNYMTALIWPSISEVVKAPIVAMKMIRQLARFAAKRNEGLMYTLPTGFILEQKIMATEMLRVRTCLMGDIKMSLQVETDIVDEAAMMGAAAPNFVHGHDASHLILTVCELVDKGVTSIAVIHDSFGTHADNTLTLRVALKGQMVAMYIDGNALQ K LLEEHE VRWM VDTGIE VPEQGEF DLNE IMD SE YVF AS37V / N42L variant (SEQ ID NO: 12)MQDLHAIQLQLEEEMFNGGIRRFEADQQRQIAAGSEEDTAWLRRLLSELI APMAEGIQAYKEEYEGKKGRAPRALAFLQCVENEVAAYITMKVVMDML NTDATLQAIAMSVAERIEDQVRFSKLEGHAAKYFEKVKKSLKASRTKSY RHAHNVAVVAEKSVAEKDADFDRWEAWPKETQLQIGTTLLEILEGSVFYNGEPVFMRAMRTYGGKTIYYLQTSESVGQWISAFKEHVAQLSPAYAPCVIPPRPWRTPFNGGFHTEKVASRIRLVKGNREHVRKLTQKQMPKVYKAINA LQNTQWQINKDVLAVIEEVIRLDLGYGVPSFKPLIDKENKPANPVPVEFQH LRGRELKEMLSPEQWQQFINWKGECARLYTAETKRGSKSAAVVRMVGQ ARKYSAFESIYFVYAMDSRSRVYVQSSTLSPQSNDLGKALLRFTEGRPVN GVEALKWFCINGANLWGWDKKTFDVRVSNVLDEEFQDMCRDIAADPLT FTQWAKADAPYEFLAWCFEYAQYLDLVDEGRADEFRTHLPVHQDGSCS GIQHYSAMLRDEVGAKAVNLKPSDAPQDIYGAVAQVVIKKNALYMDAD DATTFTSGSVTLSGTELRAMASAWDSIGITRSLTKKPVMTLPYGSTRLTCR ESVIDYIVDLEEKEAQKAVAEGRTANKVHPFEDDRQDYLTPGAAYNYMT ALIWPSISEVVKAPIVAMKMIRQLARFAAKRNEGLMYTLPTGFILEQKIMA TEMLRVRTCLMGDIKMSLQVETDIVDEAAMMGAAAPNFVHGHDASHLIL TVCELVDKGVTSIAVIHDSFGTHADNTLTLRVALKGQMVAMYIDGNALQ K LLEEHE VR WM VDTGIE VPEQGEF DLNE IMD SE YVF A

[0128] As can be seen from the sequences of SEQ ID NO: 2-12, the sequences of all the exemplary modified RNA polymerases provided above differ from the wild-type SP6 RNA polymerase of SEQ ID NO: 1 only by the specifically identified amino acid substitution(s) in their respective given variant name (e.g., “S35E / Y147H variant”). For instance, the S35E / Y147H variant of SEQ ID NO: 2 differs from the wild-type SP6 RNA polymerase of SEQ ID NO: 1 only at amino acid positions 35 and 147. By the same logic, accordingly, the respective amino acid sequence of any additional exemplary modified RNA polymerases can be deduced from the sequence of the wild-type SP6 RNA polymerase of SEQ ID NO: 1 based on their respective given variant name. The table below provides a list of additional exemplary modified RNA polymerases with their associated sequence identifiers (i.e., SEQ ID NO) as provided in the accompanied sequence listing which in incorporated by reference herein and forms a part of the instant disclosure.

[0129] Depending on the number of amino acid substitutions introduced into the wild-type SP6 RNA polymerase, the modified RNA polymerases of the present disclosure may have up to about 99.9% sequence identity to the wild-type SP6 RNA polymerase comprising the amino acid sequence of SEQ ID NO: 1. In some embodiments, the modified mRNA polymerase comprises an amino acid sequence having at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the modified RNA polymerases of the present disclosure comprise an amino acid sequence having at least about 90%, such as about 95%, 96%, 97%, 98%, or 99%, sequence identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the modified RNA polymerases of the present disclosure comprise an amino acid sequence selected from SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, or SEQ ID NO: 54.

[0130] Advantageously, the modified RNA polymerases provided herein produce less dsRNA and / or less abortive RNA transcripts in an in vitro transcription reaction as compared to the wild type SP6 RNA polymerase of SEQ ID NO: 1. Also encompassed herein is any other modified RNA polymerase producing less dsRNA and / or less abortive RNA transcripts in an in vitro transcription reaction as compared to the wild type SP6 RNA polymerase of SEQ ID NO: 1.Nucleic Acid Construction and Expression

[0131] The present disclosure further provides artificial nucleic acid molecules encoding the disclosed modified RNA polymerases. The nucleic acids may comprise DNA or RNA and may be wholly or partially synthetic or recombinant. The modified RNA polymerases provided herein can be synthesized as DNA sequences by standard methods known in the art and subsequently cloned and expressed in a recombinant host system using a suitable vector. The modified RNA polymerases provided herein can also be synthesized as RNA, such as messenger RNA (mRNA), sequences. Reference to a nucleotide sequence as set out herein encompasses a DNA molecule with the specified sequence and encompasses an RNA molecule (e.g., mRNA) with the specified sequence in which U is substituted for T, unless context requires otherwise. Other nucleotide derivatives or modified nucleotides can be incorporated into the artificial nucleic acid molecules encoding the disclosed modified RNA polymerases. The synthesized DNA or mRNA sequences encoding the modified RNA polymerases of the disclosure can be codon-optimized so that expression of the encoded protein is improved and optimized for a particular expression system. Any codon optimization algorithms known in the art can be used to generate codon-optimized nucleic acid sequences.

[0132] To express the modified RNA polymerases of the disclosure, suitable recombinant host cells include, but are not limited to, for example, insect cells, mammalian cells, avian cells, bacteria, and yeast cells. Examples of suitable insect cells include, for example, Sf9 cells, Sf21 cells, Tn5 cells, Schneider S2 cells, and High Five cells (a clonal isolate derived from the parental Trichoplusia ni BTI-TN-5B1-4 cell line (Invitrogen)). Examples of suitable mammalian cells include, but are not limited to, for example, Chinese hamster ovary (CHO) cells, human embryonic kidney cells (HEK293 or Expi 293 cells, typically transformed by sheared adenovirus type 5 DNA), NIH-3T3 cells, 293-T cells, Vero cells, and HeLa cells. Suitable avian cells include, but are not limited to, for example, chicken embryonic stem cells (e.g., EBx® cells), chicken embryonic fibroblasts, chicken embryonic germ cells, quail fibroblasts, and duck cells. Suitable insect cell expression systems, such as baculovirus-vectored systems, are known to those of skill in the art and described in, for instance, Summers and Smith, Texas Agricultural Experiment Station Bulletin No. 1555 (1987). Materials and methods for baculovirus / insect cell expression systems are commercially available in kit form from, for example, Invitrogen (San Diego, CA). Avian cell expression systems are also known to those of skill in the art and described in, forexample, U.S. Pat. Nos. 5,340,740; 5,656,479; 5,830,510; 6,114,168; and 6,500,668. Similarly, bacterial (e.g., E. coif) and mammalian cell expression systems are also known in the art and described in, for example, Yeast Genetic Engineering (Barr et al., eds., 1989) Butterworths, London.

[0133] A number of suitable vectors for expression of recombinant proteins in insect or mammalian cells are well-known and conventional in the art. Suitable vectors can contain a number of components, including, but not limited to one or more of the following: an origin of replication; a selectable marker gene; one or more expression control elements, such as a transcriptional control element (e.g., a promoter, an enhancer, a terminator), and / or one or more translation signals; and a signal sequence or leader sequence for targeting to the secretory pathway in a selected host cell (e.g., of mammalian origin or from a heterologous mammalian or nonmammalian species). For example, for expression in insect cells, a suitable baculovirus expression vector, such as pFastBac (Invitrogen), is used to produce recombinant baculovirus particles. The baculovirus particles are amplified and used to infect insect cells to express recombinant protein. For expression in mammalian cells, a vector that will drive expression of the construct in the desired mammalian host cell (e.g., CHO cells) can be used.

[0134] The modified RNA polymerases can be purified using any suitable methods. For example, methods for purifying recombinant polypeptides are known in the art. Suitable methods for purifying desired proteins including precipitation and various types of chromatography, such as hydrophobic interaction, ion exchange, affinity, chelating and size exclusion are well-known in the art. Suitable purification schemes can be created using two or more of these or other suitable methods. If desired, the modified RNA polymerases can include a “tag” that facilitates purification, such as an epitope tag or a histidine (HIS) tag. Such tagged polypeptides can conveniently be purified, for example from conditioned media, by chelating chromatography or affinity chromatography.

[0135] Purified polypeptides can be analyzed by spectroscopic methods known in the art, such as circular dichroism spectroscopy, Fourier-transform infrared spectroscopy, NMR spectroscopy, or X-ray crystallography, to investigate the presence of desired structures like helices and beta sheets.

[0136] Accordingly, in some embodiments, provided herein are artificial nucleic acids encoding any of the modified RNA polymerases described herein. The artificial nucleic acids ofthe disclosure can be in form of a DNA or a RNA, such as a messenger RNA (mRNA). In some embodiments, the artificial nucleic acids of the disclosure are DNA molecules. In some embodiments, the artificial nucleic acids of the disclosure are RNA molecules. In certain embodiments, the artificial nucleic acids of the disclosure are mRNA molecules.

[0137] Also provided, in some embodiments, are vectors comprising the artificial nucleic acids encoding any of the modified RNA polymerases described herein. In certain embodiments, an artificial nucleic acid according to the disclosure is part of a vector, e.g., a plasmid. Such vectors can easily be manipulated by any methods known to the person skilled in the art and can, for instance, be designed for being capable of replication in prokaryotic and / or eukaryotic cells. Thus, further provided herein, in some embodiments, are host cells, including prokaryotic and eukaryotic cells, that comprise such vectors.Compositions and Kits

[0138] In some embodiments, provided herein are compositions comprising any of the modified RNA polymerases disclosed herein, the artificial nucleic acids encoding the same, the vectors comprising such artificial nucleic acids, and / or host cells comprising such artificial nucleic acids or such vectors. In some embodiments, the present disclosure provides a composition suitable for performing in vitro transcription, the composition comprising any of the modified RNA polymerases disclosed herein and at least one in vitro transcription reaction component, such as nucleoside triphosphates, a reaction buffer, and / or a cap or a cap analog. Such compositions, in some embodiments, may be in form of a kit. In some embodiments, the kits of the present disclosure may further comprise instructions on how to perform in vitro transcription.Methods of Use

[0139] Also provided herein are methods for producing an RNA in an in vitro transcription reaction, the method comprising contacting a deoxyribonucleic acid (DNA) template with any of the modified RNA polymerases disclosed herein in the presence of nucleoside triphosphates. In some embodiments, the methods of the disclosure are for producing a mRNA in an in vitro transcription reaction. Advantageously, the use of a modified RNA polymerase provided herein in an IVT reaction produces RNA having one or more improved characteristics as compared to a control reaction. The term “control reaction” as used herein, refers to an IVT reaction that is performed with the RNA polymerase having the amino acid sequence of SEQ ID NO: 1.

[0140] In some embodiments, the methods provided herein produce RNA, such as mRNA, having a yield that is no less than about 35% of the yield produced in a control in vitro transcription reaction using an RNA polymerase having the amino acid sequence of SEQ ID NO: 1. In some embodiments, the methods provided herein produce RNA, such as mRNA, having a yield that is no less than about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of the RNA yield produced in the control in vitro transcription reaction. In some embodiments, the methods provided herein produce RNA, such as mRNA, having a yield that is equal to or superior to the RNA yield produced in the control in vitro transcription reaction. Thus, in some embodiments, the methods provided herein produce RNA, such as mRNA, having a yield that is at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% superior to the RNA yield produced in the control in vitro transcription reaction.

[0141] In some embodiments, the methods of the present disclosure produce a comparable amount of full-length RNA (i.e., + / - about 15%, optionally + / - about 10% or + / - about 5%), such as mRNA, or more as compared to a control in vitro transcription reaction using an RNA polymerase having the amino acid sequence of SEQ ID NO: 1. In some embodiments, the methods of the present disclosure produce up to about 10% more of full-length RNA, such as mRNA, as compared to the control in vitro transcription reaction. In some embodiments, the methods of the present disclosure produce at least about 10% more of full-length RNA, such as mRNA, as compared to the control in vitro transcription reaction. In some embodiments, less than about 25% of the RNA transcripts, such as mRNA transcripts, obtained with the methods provided herein are truncated transcripts. In some embodiments, less than about 20% of the RNA transcripts, such as mRNA transcripts, obtained with the methods provided herein are truncated transcripts.

[0142] In some embodiments, the methods of the present disclosure produce at least about 10% less, at least about 20% less, at least about 30% less, at least about 40% less, at least about 50% less, at least about 60% less, at least about 70% less, at least about 80% less, at least about 90% less, or at least about 100% less dsRNA as compared to a control in vitro transcription reaction using an RNA polymerase having the amino acid sequence of SEQ ID NO: 1 , including any values and subranges between at least about 10% less and at least about 100% less (e.g., at least about 10% less to at least about 70% less). In some embodiments, the methods of the present disclosure produce at least about 10% less dsRNA as compared to a control in vitro transcription reaction using an RNA polymerase having the amino acid sequence of SEQ ID NO: 1. In someembodiments, the methods of the present disclosure produce at least about 20% less dsRNA as compared to the control in vitro transcription reaction. In some embodiments, the methods of the present disclosure produce at least about 30% less dsRNA as compared to the control in vitro transcription reaction. In some embodiments, the methods of the present disclosure produce at least about 40% less dsRNA as compared to the control in vitro transcription reaction. In some embodiments, the methods of the present disclosure produce at least about 50% less dsRNA as compared to the control in vitro transcription reaction. In some embodiments, the methods of the present disclosure produce at least about 60% less dsRNA as compared to the control in vitro transcription reaction. In some embodiments, the methods of the present disclosure produce at least about 70% less dsRNA as compared to the control in vitro transcription reaction. In some embodiments, the methods of the present disclosure produce at least about 80% less dsRNA as compared to the control in vitro transcription reaction. In some embodiments, the methods of the present disclosure produce at least about 90% less dsRNA as compared to the control in vitro transcription reaction. In some embodiments, the methods of the present disclosure produce at least about 100% less dsRNA as compared to the control in vitro transcription reaction.

[0143] In some embodiments, the methods of the present disclosure produce less than about 0.3%, less than 0.25%, less than 0.2%, less than 0.15%, less than 0.10%, less than 0.09%, less than 0.08%, less than 0.07%, less than 0.06%, less than 0.05%, less than 0.04%, less than 0.03%, less than 0.02%, or less than 0.01% dsRNA, including any values and subranges therebetween. In some embodiments, RNA (e.g., mRNA) produced by the methods of the present disclosure is substantially free of dsRNA. The amount of dsRNA produced in an in vitro transcription reaction may be detected using any methods known in the art, such as, for example, by an immunoassay (e.g., ELISA such as a sandwich ELISA, dot blot). In some embodiments, the presence of dsRNA is determined by dot blot using antibody J2. In some embodiments, the presence of dsRNA is determined by ELISA e.g., a sandwich ELISA using antibodies J2 and KI or KI and K2. In some embodiments, the amount of dsRNA produced by the methods provided herein is below the level of quantification, e.g. when determined by ELISA. In some embodiments, the amount of dsRNA is below the limit of detection, e.g. when determined by ELISA. In some embodiments, the RNA obtained by the methods provided herein comprises 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% of dsRNA by weight, e.g., less than 0.5%.

[0144] In some embodiments, the methods of the present disclosure produce at least about 10% less, at least about 20% less, at least about 30% less, at least about 40% less, at least about 50% less, at least about 60% less, at least about 70% less, at least about 80% less, at least about 90% less, or at least about 100% less abortive RNA transcripts as compared to a control in vitro transcription reaction using an RNA polymerase having the amino acid sequence of SEQ ID NO: 1, including any values and subranges between at least about 10% less and at least about 100% less (e.g., at least about 10% less to at least about 70% less). In some embodiments, the methods of the present disclosure produce at least about 10% less abortive RNA transcripts as compared to a control in vitro transcription reaction using an RNA polymerase having the amino acid sequence of SEQ ID NO: 1. In some embodiments, the methods of the present disclosure produce at least about 20% less abortive RNA transcripts as compared to the control in vitro transcription reaction. In some embodiments, the methods of the present disclosure produce at least about 30% less abortive RNA transcripts as compared to the control in vitro transcription reaction. In some embodiments, the methods of the present disclosure produce at least about 40% less abortive RNA transcripts as compared to the control in vitro transcription reaction. In some embodiments, the methods of the present disclosure produce at least about 50% less abortive RNA transcripts as compared to the control in vitro transcription reaction. In some embodiments, the methods of the present disclosure produce at least about 60% less abortive RNA transcripts as compared to the control in vitro transcription reaction. In some embodiments, the methods of the present disclosure produce at least about 70% less abortive RNA transcripts as compared to the control in vitro transcription reaction. In some embodiments, the methods of the present disclosure produce at least about 80% less abortive RNA transcripts as compared to the control in vitro transcription reaction. In some embodiments, the methods of the present disclosure produce at least about 90% less abortive RNA transcripts as compared to the control in vitro transcription reaction. In some embodiments, the methods of the present disclosure produce at least about 100% less abortive RNA transcripts as compared to the control in vitro transcription reaction. The amount of abortive RNA transcripts produced in an in vitro transcription reaction may be detected using any methods known in the art, such as, for example, by liquid chromatography-mass spectrometry (LC-MS).

[0145] In some embodiments, the methods of the present disclosure produce at least about 10% less, at least about 20% less, at least about 30% less, at least about 40% less, at least about 50% less, at least about 60% less, at least about 70% less, at least about 80% less, at least about90% less, or at least about 100% less dsRNA and at least about at least about 10% less, at least about 20% less, at least about 30% less, at least about 40% less, at least about 50% less, at least about 60% less, at least about 70% less, at least about 80% less, at least about 90% less, or at least about 100% less abortive RNA transcripts as compared to a control in vitro transcription reaction using an RNA polymerase having the amino acid sequence of SEQ ID NO: 1, including any values and subranges between at least about 10% less and at least about 100% less (e.g., at least about 10% less to at least about 70% less).

[0146] Full-length, double-stranded, or abortive RNA transcripts may be detected and quantified using any methods available in the art. In some embodiments, the synthesized RNA molecules (e.g., mRNA) are detected using blotting, capillary electrophoresis, chromatography, fluorescence (e.g., immunofluorescence), immunoassay (e.g., ELISA), gel electrophoresis, quantitative hybridization, HPLC, silver stain, spectroscopy, LC-MS, ultraviolet (UV), or UPLC, or a combination thereof. Other detection methods known in the art are included in the present disclosure. In some embodiments, the synthesized RNA molecules (e.g., mRNA) are detected using UV absorption spectroscopy with separation by capillary electrophoresis. In some embodiments, mRNA is first denatured by a Glyoxal dye before gel electrophoresis (“Glyoxal gel electrophoresis”). In some embodiments, synthesized mRNA molecules (e.g., mRNA) are characterized before capping or tailing. In some embodiments, synthesized mRNA molecules (e.g., mRNA) are characterized after capping and tailing.

[0147] In some embodiments, RNA (e.g., mRNA) generated using the modified RNA polymerases and methods disclosed herein comprises less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, less than 1%, less than 0.5%, less than 0.1% impurities other than full-length mRNA, including any values and subranges therebetween. The impurities include IVT contaminants, e.g., proteins, enzymes, free nucleotides, abortive RNA transcripts, dsRNA, and / or run on transcripts.Large-scale mRNA synthesis

[0148] The modified RNA polymerases disclosed herein may be used in the large-scale production of RNA (e.g., mRNA). In some embodiments, the method synthesizes at least 100 mg, 150 mg, 200 mg, 300 mg, 400 mg, 500 mg, 600 mg, 700 mg, 800 mg, 900 mg, 1 g, 5 g, 10 g, 25 g, 50 g, 75 g, 100 g, 250 g, 500 g, 750 g, 1 kg, 5 kg, 10 kg, 50 kg, 100 kg, 1000 kg, or more of RNA (e.g., mRNA) in a single batch, including any values and subranges therebetween. As usedherein, the term “batch” refers to a quantity or amount of RNA (e.g., mRNA) synthesized at one time, e.g., produced according to a single manufacturing setting. A batch may refer to an amount of RNA (e.g., mRNA) synthesized in one reaction that occurs via a single aliquot of enzyme and / or a single aliquot of DNA template for continuous synthesis under one set of conditions. RNA (e.g., mRNA) synthesized at a single batch would not include RNA (e.g., mRNA) synthesized at different times that are combined to achieve the desired amount. Generally, a reaction mixture includes a modified RNA polymerase as disclosed herein, a linear DNA template, and an RNA polymerase reaction buffer (which may include ribonucleotides or may require addition of ribonucleotides).

[0149] In certain embodiments, about 1-100 mg of modified RNA polymerase as disclosed herein is typically used per gram (g) of RNA (e.g., mRNA) produced. In some embodiments, about 1-90 mg, 1-80 mg, 1-60 mg, 1-50 mg, 1-40 mg, 10-100 mg, 10-80 mg, 10-60 mg, or 10-50 mg, including any values and subranges therebetween, of modified RNA polymerase is used per gram of RNA (e.g., mRNA) produced. In some embodiments, about 5-20 mg of modified RNA polymerase is used to produce about 1 gram of RNA (e.g., mRNA). In some embodiments, about 0.5 to 2 grams of modified RNA polymerase is used to produce about 100 grams of RNA (e.g., mRNA). In some embodiments, about 5 to 20 grams of modified RNA polymerase is used to about 1 kilogram of RNA (e.g., mRNA). In some embodiments, at least about 5 mg of modified RNA polymerase is used to produce at least about 1 gram of RNA (e.g., mRNA). In some embodiments, at least about 500 mg of modified RNA polymerase is used to produce at least about 100 grams of RNA (e.g., mRNA). In some embodiments, at least about 5 grams of modified RNA polymerase is used to produce at least about 1 kilogram of RNA (e.g., mRNA). In some embodiments, about 10 mg, 20 mg, 30 mg, 40 mg, 50 mg, 60 mg, 70 mg, 80 mg, 90 mg, or 100 mg, including any values and subranges therebetween, of plasmid DNA is used per gram of RNA (e.g., mRNA) produced. In some embodiments, about 10-30 mg of plasmid DNA is used to produce about 1 gram of RNA (e.g., mRNA). In some embodiments, about 1 to 3 grams of plasmid DNA is used to produce about 100 grams of RNA (e.g., mRNA). In some embodiments, about 10 to 30 grams of plasmid DNA is used to about 1 kilogram of RNA (e.g., mRNA). In some embodiments, at least about 10 mg of plasmid DNA is used to produce at least about 1 gram of RNA (e.g., mRNA). In some embodiments, at least about 1 gram of plasmid DNA is used toproduce at least about 100 grams of RNA (e.g., mRNA). In some embodiments, at least about 10 grams of plasmid DNA is used to produce at least about 1 kilogram of RNA (e.g., mRNA).

[0150] In some embodiments, the concentration of the modified RNA polymerase in the reaction mixture may be from about 1 to 100 nM, 1 to 90 nM, 1 to 80 nM, 1 to 70 nM, 1 to 60 nM, 1 to 50 nM, 1 to 40 nM, 1 to 30 nM, 1 to 20 nM, or about 1 to 10 nM, including any values and subranges therebetween. In certain embodiments, the concentration of the modified RNA polymerase is from about 10 to 50 nM, 20 to 50 nM, or 30 to 50 nM. A concentration of about 100 to about 10000 Units / ml of the modified RNA polymerase may be used, including, for example, concentrations of 100 to 9000 Units / ml, 100 to 8000 Units / ml, 100 to 7000 Units / ml, 100 to 6000 Units / ml, 100 to 5000 Units / ml, 100 to 1000 Units / ml, 200 to 2000 Units / ml, 500 to 1000 Units / ml, 500 to 2000 Units / ml, 500 to 3000 Units / ml, 500 to 4000 Units / ml, 500 to 5000 Units / ml, 500 to 6000 Units / ml, 1000 to 7500 Units / ml, and 2500 to 5000 Units / ml, including any values and subranges therebetween, may be used.

[0151] In some embodiments, the concentration of each ribonucleotide (e.g., ATP, UTP, GTP, and CTP) in a reaction mixture is between about 0.1 mM and about 10 mM, e.g., between about 1 mM and about 10 mM, between about 2 mM and about 10 mM, between about 3 mM and about 10 mM, between about 1 mM and about 8 mM, between about 1 mM and about 6 mM, between about 3 mM and about 10 mM, between about 3 mM and about 8 mM, between about 3 mM and about 6 mM, between about 4 mM and about 5 mM. In some embodiments, each ribonucleotide is at about 5 mM in a reaction mixture. In some embodiments, the total concentration of rNTPs (for example, ATP, GTP, CTP, and UTP combined) used in the reaction is between about 1 mM and about 40 mM. In some embodiments, the total concentration of rNTPs (for example, ATP, GTP, CTP, and UTP combined) used in the reaction is between about 1 mM and about 30 mM, or between about 1 mM and about 28 mM, or between about 1 mM to about 25 mM, or between about 1 mM and about 20 mM, including any values and subranges therebetween. In some embodiments, the total rNTPs concentration is less than about 30 mM. In some embodiments, the total rNTPs concentration is less than about 25 mM. In some embodiments, the total rNTPs concentration is less than about 20 mM. In some embodiments, the total rNTPs concentration is less than about 15 mM. In some embodiments, the total rNTPs concentration is less than about 10 mM.

[0152] The RNA polymerase reaction buffer typically includes a salt / buffering agent, e.g., Tris, HEPES, ammonium sulfate, sodium bicarbonate, sodium citrate, sodium acetate, potassium phosphate sodium phosphate, sodium chloride, or magnesium chloride.

[0153] The pH of the reaction mixture may be between about 6 to 8.5, from about 6.5 to 8.0, from about 7.0 to 7.5, including any values and subranges therebetween, and in some embodiments, the pH is about 7.5.

[0154] Linear or linearized DNA template (e.g., as described above and in an amount / concentration sufficient to provide a desired amount of RNA), the RNA polymerase reaction buffer, and modified RNA polymerase are combined to form the reaction mixture. The reaction mixture is incubated at between about 37°C and about 42°C for about thirty minutes to about six hours, e.g., about sixty to about ninety minutes.

[0155] In some embodiments, about 5 mM NTPs, about 0.05 mg / mL modified RNA polymerase, and about 0.1 mg / ml DNA template in a suitable RNA polymerase reaction buffer (final reaction mixture pH of about 7.5) is incubated at about 37°C to about 42°C for about sixty to about ninety minutes.

[0156] In some embodiments, a reaction mixture contains linearized double stranded DNA template with an SP6 polymerase-specific promoter, modified RNA polymerase, RNase inhibitor, pyrophosphatase, 29 mM NTPs, 10 mM DTT and a reaction buffer (when at lOx is 800 mM HEPES, 20 mM spermidine, 250 mM MgCh, pH 7.7) and quantity sufficient (QS) to a desired reaction volume with RNase-free water; this reaction mixture is then incubated at 37°C for 60 minutes. The polymerase reaction is then quenched by addition of DNase I and a DNase I buffer (when at lOx is 100 mM Tris-HCl, 5 mM MgCh and 25 mM CaCh, pH 7.6) to facilitate digestion of the double-stranded DNA template in preparation for purification.

[0157] In some embodiments, a reaction mixture includes NTPs at a concentration ranging from 1-10 mM, DNA template at a concentration ranging from 0.01-0.5 mg / ml, and SP6 RNA polymerase at a concentration ranging from 0.01-0.1 mg / ml, e.g., the reaction mixture comprises NTPs at a concentration of 5 mM, the DNA template at a concentration of 0.1 mg / ml, and the modified RNA polymerase at a concentration of 0.05 mg / ml.SP6 Promoter

[0158] Any promoter that can be recognized by an SP6 RNA polymerase may be used in the methods disclosed herein. In some embodiments, an SP6 promoter comprises the nucleotidesequence of 5'-ATTTAGGTGACACTATAG-3' (SEQ ID NO: 13). Variants of the SP6 promoter have been discovered and / or created to optimize recognition and / or binding of SP6 to its promoter. Non-limiting variants of the SP6 promoter include but are not limited to the following:5'-ATTTAGGGGACACTATAGAAGAG-3' (SEQ ID NO: 14); 5'-ATTTAGGGGACACTATAGAAGG-3' (SEQ ID NO: 15); 5'-ATTTAGGGGACACTATAGAAGGG-3' (SEQ ID NO: 16); 5'-ATTTAGGTGACACTATAGAA-3' (SEQ ID NO: 17); 5'-ATTTAGGTGACACTATAGAAGA-3' (SEQ ID NO: 18); 5'-ATTTAGGTGACACTATAGAAGAG-3' (SEQ ID NO: 19); 5'-ATTTAGGTGACACTATAGAAGG-3' (SEQ ID NO: 20); 5'-ATTTAGGTGACACTATAGAAGGG-3' (SEQ ID NO: 21); 5'-ATTTAGGTGACACTATAGAAGNG-3' (SEQ ID NO: 22); and 5'-CATACGATTTAGGTGACACTATAG-3' (SEQ ID NO: 23).

[0159] In some embodiments, an SP6 promoter suitable for practicing the methods disclosed herein may have a nucleotide sequence that differs from any of the SP6 promoter sequences described herein by one, two, three, four, five, or six nucleotides. In some embodiments, an SP6 promoter suitable for practicing the methods disclosed herein may have about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more, including any values and subranges therebetween, sequence identity to any one of SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, or SEQ ID NO: 23. In some embodiments, an SP6 promoter suitable for practicing the methods disclosed herein may include one or more additional nucleotides at the 5'- and / or 3 '-end of any of the SP6 promoter sequences described herein.DNA Template

[0160] Various nucleic acid templates may be used in the methods disclosed herein. In some embodiments, DNA templates which are either entirely double-stranded or mostly single-stranded with a double-stranded SP6 promoter sequence can be used.

[0161] Linearized plasmid DNA (linearized via one or more restriction enzymes), linearized genomic DNA fragments (via restriction enzyme and / or physical means), PCR products, and / or synthetic DNA oligonucleotides can be used as templates for in vitro transcription with the modified RNA polymerases disclosed herein, provided that they contain a double-stranded SP6promoter upstream (and in the correct orientation) of the DNA sequence to be transcribed. In some embodiments, the linearized DNA template has a blunt-end.

[0162] In some embodiments, the DNA sequence to be transcribed may be optimized to facilitate more efficient transcription and / or translation. For example, the DNA sequence may be optimized regarding cv.s-regulatory elements (e.g., TATA box, termination signals, and protein binding sites), artificial recombination sites, chi sites, CpG dinucleotide content, negative CpG islands, GC content, polymerase slippage sites, and / or other elements relevant to transcription; the DNA sequence may be optimized regarding cryptic splice sites, mRNA secondary structure, stable free energy of mRNA, repetitive sequences, RNA instability motif, and / or other elements relevant to mRNA processing and stability; the DNA sequence may be optimized regarding codon usage bias, codon adaptability, internal chi sites, ribosomal binding sites (e.g., IRES), premature polyA sites, Shine-Dalgarno (SD) sequences, and / or other elements relevant to translation; and / or the DNA sequence may be optimized regarding codon context, codon-anticodon interaction, translational pause sites, and / or other elements relevant to protein folding. Optimization methods known in the art may be used in the present invention, e.g., GeneOptimizer by ThermoFisher and OptimumGene™, which is described in US 20110081708, the contents of which are incorporated herein by reference in its entirety.

[0163] In some embodiments, the DNA template includes a 5' and / or 3' untranslated region. In some embodiments, a 5' untranslated region includes one or more elements that affect an mRNA’s stability or translation, for example, an iron responsive element. In some embodiments, a 5' untranslated region may be between about 50 and 500 nucleotides in length.

[0164] In some embodiments, a 3' untranslated region includes one or more of a polyadenylation signal, a binding site for proteins that affect an mRNA’s stability of location in a cell, or one or more binding sites for miRNAs. In some embodiments, a 3' untranslated region may be between about 50 and 500 nucleotides in length or longer.

[0165] Exemplary 3' and / or 5' UTR sequences can be derived from mRNA molecules which are stable (e.g., globin, actin, GAPDH, tubulin, histone, and citric acid cycle enzymes) to increase the stability of the sense mRNA molecule. For example, a 5' UTR sequence may include a partial sequence of a CMV immediate-early 1 (IE1) gene, or a fragment thereof to improve the nuclease resistance and / or improve the half-life of the polynucleotide. Also contemplated is the inclusion of a sequence encoding human growth hormone (hGH), or a fragment thereof to the 3' end oruntranslated region of the polynucleotide (e.g., mRNA) to further stabilize the polynucleotide. Generally, these modifications improve the stability and / or pharmacokinetic properties (e.g., halflife) of the polynucleotide relative to their unmodified counterparts, and include, for example modifications made to improve such polynucleotides’ resistance to in vivo nuclease digestion.Nucleotides

[0166] Various naturally-occurring or modified nucleosides may be used to produce RNA (e.g., mRNA) using the modified RNA polymerases disclosed herein. In some embodiments, an RNA (e.g., mRNA) is or comprises natural nucleosides (e.g., adenosine, guanosine, cytidine, uridine); nucleoside analogs (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolo- pyrimidine, 3-methyl adenosine, 5 -methyl cytidine, C5-bromouridine, C5-fluorouridine, C5- iodouridine, C5-propynyl-uridine, C5-propynyl-cytidine, C5-methylcytidine, 7-deazaad enosine, 7-deazaguanosine, 8 -oxoadenosine, 8-oxoguanosine, O(6)-methylguanine, pseudouridine, (e.g., N-l -methyl -pseudouridine), 2-thiouridine, and 2-thiocytidine); chemically modified bases; biologically modified bases (e.g., methylated bases); intercalated bases; modified sugars (e.g., 2’- fluororibose, ribose, 2’ -deoxyribose, arabinose, and hexose); and / or modified phosphate groups (e.g., phosphorothioates and 5'-N-phosphoramidite linkages). In some embodiments, the RNA (e.g., mRNA) molecules comprise one or more phosphorothioate bonds.

[0167] In some embodiments, the RNA (e.g., mRNA) comprises one or more nonstandard nucleotide residues. The nonstandard nucleotide residues may include, e.g., 5-methyl-cytidine (“5mC”), pseudouridine (e.g., a methylpseudouridine, such as N1 -methylpseudouridine) and / or 2- thio-uridine. See, e.g., US Patent Publication US20120195936, U.S. Patent No. 8,278,036 or WO201 1012316 for a discussion of nonstandard residues and their incorporation into mRNA, which are hereby incorporated by reference in their entirety. The presence of nonstandard nucleotide residues may render an mRNA more stable and / or less immunogenic than a control mRNA with the same sequence but containing only standard residues. In further embodiments, the mRNA may comprise one or more nonstandard nucleotide residues chosen from isocytosine, pseudoisocytosine, 5 -bromouracil, 5-propynyluracil, 6-aminopurine, 2-aminopurine, inosine, diaminopurine and 2-chloro-6-aminopurine cytosine, 4'-thiouridine, 5-methylcytosine, 2-thio-l- methyl-l-deaza-pseudouridine, 2-thio-l-methyl-pseudouridine, 2-thio-5-aza-uridine, 2-thio- dihydropseudouridine, 2-thio-dihydrouridine, 2-thiopseudouridine, 4-methoxy-2-thio- pseudouridine, 4-methoxy-pseudouridine, 4-thio-l-methyl-pseudouridine, 4-thio-pseudouridine,5-aza-uridine, dihydropseudouridine, and 5-methoxyuridine, as well as combinations of these modifications and other nucleobase modifications. Some embodiments may further include additional modifications to the furanose ring or nucleobase. Additional modifications may include, for example, sugar modifications or substitutions (e.g., one or more of a 2'-O-alkyl modification, a locked nucleic acid (LNA)). In some embodiments, the RNAs may be complexed or hybridized with additional polynucleotides and / or peptide polynucleotides (PNA). In some embodiments where the sugar modification is a 2'-O-alkyl modification, such modification may include, but are not limited to a 2 '-deoxy-2 '-fluoro modification, a 2'-O-methyl modification, a 2'- O-methoxyethyl modification and a 2'-deoxy modification. In some embodiments, any of these modifications may be present in 0-100% of the nucleotides — for example, more than 0%, 1%, 10%, 25%, 50%, 75%, 85%, 90%, 95%, or 100%, including any values and subranges therebetween, of the constituent nucleotides individually or in combination.Post-synthesis processing

[0168] Typically, a 5' cap and / or a 3' tail may be added to the RNA (e.g., mRNA) after the synthesis. The presence of the 5' cap can provide resistance to nucleases found in most eukaryotic cells. The presence of a 3' tail serves to protect the RNA (e.g., mRNA) from exonuclease degradation.

[0169] A 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; guanosine triphosphate (GTP) is then added to the terminal phosphates via a guanylyl transferase, producing a 5'5'5 triphosphate linkage; and the 7-nitrogen of guanine is then methylated by a methyltransferase. Examples of cap structures include, but are not limited to, m7G(5')ppp (5' (A,G(5')ppp(5')A and G(5')ppp(5')G. Additional cap structures are described in published US Application No. US 2016 / 0032356 and US 2018 / 0125989, which are incorporated herein by reference.

[0170] Typically, a tail structure includes a poly(A) and / or poly(C) tail. A poly-A or poly-C tail on the 3' terminus of mRNA typically includes at least about 50 adenosine or cytosine nucleotides, at least about 100 adenosine or cytosine nucleotides, at least about 150 adenosine or cytosine nucleotides, at least about 200 adenosine or cytosine nucleotides, at least about 250 adenosine or cytosine nucleotides, at least about 300 adenosine or cytosine nucleotides, at least about 350 adenosine or cytosine nucleotides, at least about 400 adenosine or cytosine nucleotides,at least about 450 adenosine or cytosine nucleotides, at least about 500 adenosine or cytosine nucleotides, at least about 550 adenosine or cytosine nucleotides, at least about 600 adenosine or cytosine nucleotides, at least about 650 adenosine or cytosine nucleotides, at least about 700 adenosine or cytosine nucleotides, at least about 750 adenosine or cytosine nucleotides, at least about 800 adenosine or cytosine nucleotides, at least about 850 adenosine or cytosine nucleotides, at least about 900 adenosine or cytosine nucleotides, at least about 950 adenosine or cytosine nucleotides, or at least about 1 kb adenosine or cytosine nucleotides, respectively, including any values and subranges therebetween. In some embodiments, a poly-A or poly-C tail may be about 10 to 800 adenosine or cytosine nucleotides (e.g., about 10 to 200 adenosine or cytosine nucleotides, about 10 to 300 adenosine or cytosine nucleotides, about 10 to 400 adenosine or cytosine nucleotides, about 10 to 500 adenosine or cytosine nucleotides, about 10 to 550 adenosine or cytosine nucleotides, about 10 to 600 adenosine or cytosine nucleotides, about 50 to 600 adenosine or cytosine nucleotides, about 100 to 600 adenosine or cytosine nucleotides, about 150 to 600 adenosine or cytosine nucleotides, about 200 to 600 adenosine or cytosine nucleotides, about 250 to 600 adenosine or cytosine nucleotides, about 300 to 600 adenosine or cytosine nucleotides, about 350 to 600 adenosine or cytosine nucleotides, about 400 to 600 adenosine or cytosine nucleotides, about 450 to 600 adenosine or cytosine nucleotides, about 500 to 600 adenosine or cytosine nucleotides, about 10 to 150 adenosine or cytosine nucleotides, about 10 to 100 adenosine or cytosine nucleotides, about 20 to 70 adenosine or cytosine nucleotides, or about 20 to 60 adenosine or cytosine nucleotides, including any values and subranges therebetween), respectively. In some embodiments, a tail structure includes is a combination of poly(A) and poly(C) tails with various lengths described herein. In some embodiments, a tail structure includes at least about 50%, 55%, 65%, 70%, 75%, 80%, 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, or 99% adenosine nucleotides, including any values and subranges therebetween. In some embodiments, a tail structure includes at least about 50%, 55%, 65%, 70%, 75%, 80%, 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, or 99% cytosine nucleotides, including any values and subranges therebetween.

[0171] The addition of the 5' cap and / or the 3' tail facilitates the detection of abortive RNA transcripts generated during in vitro synthesis because without capping and / or tailing, the size of those prematurely aborted RNA (e.g., mRNA) transcripts can be too small to be detected. Thus, in some embodiments, the 5' cap and / or the 3 ' tail are added to the synthesized RNA (e.g., mRNA)before the RNA (e.g., mRNA) is tested for purity (e.g., the level of abortive RNA transcripts present in the RNA (e.g., mRNA)). In some embodiments, the 5' cap and / or the 3' tail are added to the synthesized RNA (e.g., mRNA) before the RNA (e.g., mRNA) is purified as described herein. In other embodiments, the 5 ' cap and / or the 3 ' tail are added to the synthesized RNA (e.g., mRNA) after the RNA (e.g., mRNA) is purified as described herein.Purification of RNA

[0172] RNA (e.g., mRNA) synthesized using the modified RNA polymerases and methods disclosed herein may be used without further purification. In particular, mRNA synthesized according to the present invention may be used without a step of removing abortive RNA transcripts and / or dsRNA. In some embodiments, the methods of the present disclosure further comprise a step of purifying RNA, such as mRNA, from the in vitro transcription reaction. Any methods known in the art for purifying RNA, such as mRNA, may be used. For example, purification of RNA, such as mRNA, can be performed using centrifugation, filtration and / or chromatographic methods. In some embodiments, the synthesized RNA, such as mRNA, is purified by ethanol precipitation or filtration or chromatography, such as affinity chromatography, or gel purification or any other suitable means. In some embodiments, the RNA, such as mRNA, is purified by HPLC. In some embodiments, the RNA, such as mRNA, is extracted in a standard phenol:chloroform:isoamyl alcohol solution, well known to one of skill in the art. In some embodiments, the RNA, such as mRNA, is purified using Tangential Flow Filtration. Suitable purification methods include those described in US 2016 / 0040154, US 2015 / 0376220, US 2018 / 0251755, and US 2018 / 0251754, all of which are incorporated by reference herein and may be used to practice the methods disclosed herein.

[0173] In some embodiments, the RNA (e.g., mRNA) is purified before capping and tailing. In some embodiments, the RNA (e.g., mRNA) is purified after capping and tailing. In some embodiments, the RNA (e.g., mRNA) is purified both before and after capping and tailing. In some embodiments, the RNA (e.g., mRNA) is purified either before or after or both before and after capping and tailing, by centrifugation. In some embodiments, the RNA (e.g., mRNA) is purified either before or after or both before and after capping and tailing, by filtration. In some embodiments, the RNA (e.g., mRNA) is purified either before or after or both before and after capping and tailing, by Tangential Flow Filtration (TFF). In some embodiments, the RNA (e.g.,mRNA) is purified either before or after or both before and after capping and tailing by chromatography.Protein expression

[0174] mRNA synthesized using the modified RNA polymerases and methods disclosed herein can be used for protein translation. In some embodiments, mRNA synthesized according to the present invention results in comparable or increased protein expression once transfected into cells relative to the protein expression obtained by same amount of mRNA synthesized using a wild type RNA Polymerase comprising the amino acid sequence of SEQ ID NO: 1. In some embodiments, mRNA synthesized using the modified RNA polymerases and methods disclosed herein results in an increase of protein expression of up to about 10%, 15% 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 100% or more, including any values and subranges therebetween, relative to the protein expression obtained by same amount of mRNA synthesized using a wild type RNA polymerase comprising the amino acid sequence of SEQ ID NO: 1.

[0175] Any mRNA may be synthesized using the modified RNA polymerases and methods of the present disclosure. In some embodiments, the synthesized mRNA encodes one or more naturally occurring peptides or proteins. In some embodiments, the synthesized mRNA encodes one or more modified or non-natural peptides or proteins.

[0176] In some embodiments, the synthesized mRNA encodes an intracellular protein. In some embodiments, the synthesized mRNA encodes a cytosolic protein. In some embodiments, the synthesized mRNA encodes a protein associated with the actin cytoskeleton. In some embodiments, the synthesized mRNA encodes a protein associated with the plasma membrane. In some specific embodiments, the synthesized mRNA encodes a transmembrane protein. In some specific embodiments, the synthesized mRNA encodes an ion channel protein. In some embodiments, the synthesized mRNA encodes a perinuclear protein. In some embodiments, the synthesized mRNA encodes a nuclear protein. In some specific embodiments, the synthesized mRNA encodes a transcription factor. In some embodiments, the synthesized mRNA encodes a chaperone protein. In some embodiments, the synthesized mRNA encodes an intracellular enzyme (e.g., mRNA encoding an enzyme associated with urea cycle or lysosomal storage metabolic disorders). In some embodiments, the synthesized mRNA encodes a protein involved in cellular metabolism, DNA repair, transcription and / or translation. In some embodiments, the synthesizedmRNA encodes an extracellular protein. In some embodiments, the synthesized mRNA encodes a protein associated with the extracellular matrix. In some embodiments, the synthesized mRNA encodes a secreted protein. In some embodiments, the synthesized mRNA may be used to express functional proteins or enzymes that are excreted or secreted by one or more target cells into the surrounding extracellular fluid (e.g., mRNA encoding hormones and / or neurotransmitters).

[0177] The modified RNA polymerases and methods disclosed herein can be used for producing a pharmaceutical composition enriched with full-length mRNA molecules encoding a peptide or polypeptide of interest for use in the delivery to or treatment of a subject, e.g., a human subject or a cell of a human subject or a cell that is treated and delivered to a human subject.Representative Embodiments of the Present Disclosure

[0178] Embodiment 1 : A modified ribonucleic acid (RNA) polymerase comprising at least one amino acid substitution, wherein the at least one amino acid substitution is in the N-terminal domain of the modified RNA polymerase or a portion of the C-terminal domain of the modified RNA polymerase that interacts with the N-terminal domain, and wherein the modified RNA polymerase produces less double-stranded RNA and / or less abortive RNA transcripts in an in vitro transcription reaction as compared to the wild type SP6 RNA polymerase, optionally wherein the modified RNA polymerase comprises an amino acid sequence having at least about 80% sequence identity to the amino acid sequence of SEQ ID NO: 1.

[0179] Embodiment 2: The modified RNA polymerase of Embodiment 1, wherein the at least one amino acid substitution comprises at least one pair of amino acid substitutions at amino acid positions 35 and 147, 37 and 42, or 131 and 157 as indexed by reference to the wild-type RNA polymerase comprising the amino acid sequence of SEQ ID NO: 1.

[0180] Embodiment 3: The modified RNA polymerase of Embodiment 1, wherein the at least one amino acid substitution is at one or more of amino acid positions 19, 20, 36, 41, 154, and 749 as indexed by reference to a wild-type RNA polymerase comprising the amino acid sequence of SEQ ID NO: 1.

[0181] Embodiment 4: The modified RNA polymerase of Embodiment 3, wherein the amino acid substitution is at one or more of amino acid positions 19, 20, 41, and 749.

[0182] Embodiment 5: The modified RNA polymerase of Embodiment 2, wherein the at least one amino acid substitution further comprises an amino acid substitution at one or more of aminoacid positions 19, 20, 36, 41, 154, and 749 as indexed by reference to a wild-type RNA polymerase comprising the amino acid sequence of SEQ ID NO: 1.

[0183] Embodiment 6: The modified RNA polymerase of Embodiment 2 or 5, wherein the at least one pair of amino acid substitutions is at amino acid positions 35 and 147 and / or amino acid positions 37 and 42.

[0184] Embodiment 7: The modified RNA polymerase of Embodiment 6, wherein the at least one pair of amino acid substitutions is at amino acid positions 35 and 147 and at amino acid positions 37 and 42, and wherein the at least one amino acid substitution further comprises an amino acid substitution at one or more of amino acid positions 19, 20, 41, and 749 as indexed by reference to a wild-type RNA polymerase comprising the amino acid sequence of SEQ ID NO: 1.

[0185] Embodiment 8: The modified RNA polymerase of any one of Embodiments 1-7, comprising at least two amino acid substitutions at amino acid positions selected from 19, 20, 41, and 749.

[0186] Embodiment 9: The modified RNA polymerase of Embodiment 8, comprising at least three amino acid substitutions at amino acid positions selected from 19, 20, 41, and 749.

[0187] Embodiment 10: The modified RNA polymerase of Embodiment 9, comprising amino acid substitutions at amino acid positions 19, 20, 41, and 749.

[0188] Embodiment 11 : A modified ribonucleic acid (RNA) polymerase comprising at least one amino acid substitution, wherein the at least one amino acid substitution comprises at least one pair of amino acid substitutions at amino acid positions 35 and 147, 37 and 42, or 131 and 157 as indexed by reference to the wild-type RNA polymerase comprising the amino acid sequence of SEQ ID NO: 1.

[0189] Embodiment 12: The modified RNA polymerase of Embodiment 11, wherein the at least one amino acid substitution further comprises an amino acid substitution at one or more of amino acid positions 19, 20, 36, 41, 154, and 749 as indexed by reference to a wild-type RNA polymerase comprising the amino acid sequence of SEQ ID NO: 1.

[0190] Embodiment 13: The modified RNA polymerase of Embodiment 11 or 12, wherein the at least one pair of amino acid substitutions is at amino acid positions 35 and 147 and / or amino acid positions 37 and 42.

[0191] Embodiment 14: The modified RNA polymerase of Embodiment 13, wherein the at least one pair of amino acid substitutions is at amino acid positions 35 and 147 and at amino acidpositions 37 and 42, and wherein the at least one amino acid substitution further comprises an amino acid substitution at one or more of amino acid positions 19, 20, 41, and 749 as indexed by reference to a wild-type RNA polymerase comprising the amino acid sequence of SEQ ID NO: 1.

[0192] Embodiment 15: The modified RNA polymerase of any one of Embodiments 11-14, comprising at least two amino acid substitutions at amino acid positions selected from 19, 20, 41, and 749.

[0193] Embodiment 16: The modified RNA polymerase of Embodiment 15, comprising at least three amino acid substitutions at amino acid positions selected from 19, 20, 41, and 749.

[0194] Embodiment 17: The modified RNA polymerase of Embodiment 16, comprising amino acid substitutions at amino acid positions 19, 20, 41, and 749.

[0195] Embodiment 18: A modified ribonucleic acid (RNA) polymerase comprising at least one amino acid substitution, wherein the at least one amino acid substitution is at one or more of amino acid positions 19, 20, 36, 41, 154, and 749 as indexed by reference to a wild-type RNA polymerase comprising the amino acid sequence of SEQ ID NO: 1.

[0196] Embodiment 19: The modified RNA polymerase of Embodiment 18, wherein the amino acid substitution is at one or more of amino acid positions 19, 20, 41, and 749.

[0197] Embodiment 20: The modified RNA polymerase of Embodiment 18 or 19, comprising at least two amino acid substitutions at amino acid positions selected from 19, 20, 41, and 749.

[0198] Embodiment 21: The modified RNA polymerase of Embodiment 20, comprising at least three amino acid substitutions at amino acid positions selected from 19, 20, 41, and 749.

[0199] Embodiment 22: The modified RNA polymerase of Embodiment 21, comprising amino acid substitutions at amino acid positions 19, 20, 41, and 749.

[0200] Embodiment 23: The modified RNA polymerase of any one of Embodiments 3-22, wherein the amino acid substitution at amino acid position 19 is G19H or G19T.

[0201] Embodiment 24: The modified RNA polymerase of any one of Embodiments 3-23, wherein the amino acid substitution at amino acid position 19 is G19H.

[0202] Embodiment 25: The modified RNA polymerase of any one of Embodiments 3-24, wherein the amino acid substitution at amino acid position 20 is I20E.

[0203] Embodiment 26: The modified RNA polymerase of any one of Embodiments 3-25, wherein the amino acid substitution at amino acid position 41 is W41L.

[0204] Embodiment 27: The modified RNA polymerase of any one of Embodiments 3-26, wherein the amino acid substitution at amino acid position 749 is L749F.

[0205] Embodiment 28: The modified RNA polymerase of any one of Embodiments 2-27, wherein the amino acid substitutions at amino acid positions 35 and 147 are S35E and Y147H.

[0206] Embodiment 29: The modified RNA polymerase of any one of Embodiments 2-28, wherein the amino acid substitutions at amino acid positions 37 and 42 are S37V and N42L.

[0207] Embodiment 30: A modified ribonucleic acid (RNA) polymerase comprising amino acid substitutions at: a) amino acid positions 35, 147, and 749; or b) amino acid positions 41 and 749, as indexed by reference to a wild-type RNA polymerase comprising the amino acid sequence of SEQ ID NO: 1.

[0208] Embodiment 31 : The modified RNA polymerase of Embodiment 30, comprising amino acid substitutions: a) S35E, Y147H, and L749F; or b) W41L and L749F.

[0209] Embodiment 32: The modified RNA polymerase of any one of Embodiments 1-31, comprising an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 1.

[0210] Embodiment 33: The modified RNA polymerase of any one of Embodiments 1-32, wherein the modified RNA polymerases comprises an amino acid sequence selected from SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, or SEQ ID NO: 12.

[0211] Embodiment 34: An artificial nucleic acid encoding the modified RNA polymerase of any one of Embodiments 1-33.

[0212] Embodiment 35: A vector comprising the artificial nucleic acid of Embodiment 34.

[0213] Embodiment 36: A host cell comprising the vector of Embodiment 35.

[0214] Embodiment 37: A composition comprising the modified RNA polymerase of any one of Embodiments 1-33, the artificial nucleic acid of Embodiment 34, the vector of Embodiment 35, or the host cell of Embodiment 36.

[0215] Embodiment 38: A composition or kit comprising the modified RNA polymerase of any one of Embodiments 1-33 and at least one in vitro transcription reaction component, such as nucleoside triphosphates and / or a cap or a cap analog.

[0216] Embodiment 39: A method for producing a ribonucleic acid (RNA) in an in vitro transcription reaction, said method comprising contacting a deoxyribonucleic acid (DNA) template with the modified RNA polymerase of any one of Embodiments 1-33 in the presence of nucleoside triphosphates.

[0217] Embodiment 40: The method of Embodiment 39, wherein the method produces a comparable amount of full-length RNA or more as compared to a control in vitro transcription reaction using an RNA polymerase having the amino acid sequence of SEQ ID NO: 1.

[0218] Embodiment 41 : The method of Embodiment 39 or 40, wherein the method produces at least about 10% less double-stranded RNA (dsRNA) as compared to a control in vitro transcription reaction using an RNA polymerase having the amino acid sequence of SEQ ID NO: 1.

[0219] Embodiment 42: The method of Embodiment 41, wherein the method produces at least about 30% less dsRNA as compared to the control in vitro transcription reaction.

[0220] Embodiment 43 : The method of Embodiment 42, wherein the method produces at least about 50% less dsRNA as compared to the control in vitro transcription reaction.

[0221] Embodiment 44: The method of Embodiment 43, wherein the method produces at least about 100% less dsRNA as compared to the control in vitro transcription reaction.

[0222] Embodiment 45: The method of any one of Embodiments 39-44, wherein the method produces at least about 10% less abortive RNA transcripts as compared to a control in vitro transcription reaction using an RNA polymerase having the amino acid sequence of SEQ ID NO: 1.

[0223] Embodiment 46: The method of Embodiment 45, wherein the method produces at least about 30% less abortive RNA transcripts as compared to the control in vitro transcription reaction.

[0224] Embodiment 47: The method of Embodiment 46, wherein the method produces at least about 50% less abortive RNA transcripts as compared to the control in vitro transcription reaction.

[0225] Embodiment 48: The method of any one of Embodiments 39-47, wherein the method produces RNA having a yield that is no less than about 35%, e.g. no less than about 50%, of theRNA yield produced in a control in vitro transcription reaction using an RNA polymerase having the amino acid sequence of SEQ ID NO: 1.

[0226] Embodiment 49: The method of Embodiment 48, wherein the method produces RNA having a yield that is at least about equal to the RNA yield produced in the control in vitro transcription reaction.

[0227] Embodiment 50: The method of Embodiment 48 or 49, wherein the method produces RNA having a yield that is superior to the RNA yield produced in the control in vitro transcription by at least about 10%, 20%, 30%, 40%, 50%, or more.

[0228] Embodiment 51 : The method of any one of Embodiments 39-50, further comprising a step of purifying RNA from the in vitro transcription reaction.

[0229] Embodiment 52: The method of any one of Embodiments 39-51, wherein the RNA is messenger RNA (mRNA).EXAMPLES

[0230] The following examples are to be considered illustrative and not limiting on the scope of the present disclosure described above.Example 1. Design of Modified SP6 RNA Polymerases and Engineering Strategy

[0231] Modifying the amino acid sequence of an RNA polymerase, such as the SP6 RNA polymerase, may improve its properties, making it more desirable for use in in vitro transcription reactions, such as by increasing transcription efficiency and / or by reducing the amount of impurities (e.g., abortive RNA transcripts and / or dsRNAs) produced. This example illustrates the design of various modified SP6 RNA polymerases with one or more introduced amino acid mutations that contribute to the modified RNA polymerases’ ability to maintain a comparable in vitro transcription yield as the wild-type SP6 RNA polymerase while reducing generation of dsRNAs. As shown in FIG. 1, several types of impurities, such as short RNAs or abortive RNA transcripts, and double-stranded RNAs (dsRNAs), such as short dsRNAs and loopback dsRNAs, are produced at different phases of transcription. Based on the RNA polymerase structure in the initiation complex and in the elongation complex (FIG. 2), a mutagenesis strategy for wild-type SP6 RNA polymerase was developed. The strategy to engineer SP6 RNA polymerase is summarized in FIG. 3. Briefly, a SP6 RNA polymerase mutant library can be generated based on,for example, rational designs or artificial intelligence (Al) assisted designs. This mutant library is then subject to an initial high throughput screening (HTS) using a single assay of a characteristic of interest (here, the level of dsRNA present in the in vitro transcription reaction) followed by validation of potential hits selected from the initial HTS using multiple assays to determine changes in several major characteristics of the RNA that is in vitro transcribed with the RNA polymerase.

[0232] To this end, a SP6 RNA polymerase mutant library consisting of 152 rationally designed mutants was generated based on the wild-type SP6 RNA polymerase having the amino acid sequence of SEQ ID NO: 1 (GenBank Accession No. Y00105.1). The amino acid substitutions evaluated are provided in Table 2 below. The amino acid residue positions listed in Table 2 correspond to those provided in the RNA polymerase of SEQ ID NO: 1.Table 2. Amino acid substitutions in the modified RNA polymerasesExample 2. Expression and Purification of Modified SP6 RNA Polymerases

[0233] Each coding sequence of the modified RNA polymerases designed in Example 1 was cloned into an expression plasmid. A wild-type SP6 RNA polymerase with the amino acid sequence of SEQ ID NO: 1 was also cloned into an expression plasmid and purified as a control.

[0234] An N-terminal His-tag was added to enable affinity purification of the recombinantly produced enzyme, yielding an expression plasmid comprising the His-tagged coding sequence ofthe modified RNA polymerase operatively linked to an IPTG-inducible promoter. The coding sequence encodes a modified RNA polymerase having the amino acid sequence set forth in SEQ ID NO: 1 with the amino acid substitution or combination of amino acid substitutions provided in Table 2

[0235] BL21(DE3) competent Escherichia coli cells (New England Biolab, Ipswich, MA) were transformed with the expression plasmid. Successfully transformed E. coli cells were grown in 96-well plates in 0.6 mL Terrific Broth (TB) auto-induction growth medium supplemented with 100 pg / mL carbenicillin at 37°C overnight with shaking to induce expression of the recombinant protein. Once the cells reached stationary phase, the E. coli cells were harvested by centrifugation and lysed. Lysed cells were clarified by centrifugation followed by filtration through 0.2 pm filters. Insoluble proteins were retained on the filters while the clarified lysates were transferred to King isher™ Flex 2 mL 96-well plates (ThermoFisher Scientific, Waltham, MA) for automated purification of the modified or wild-type RNA polymerases with magnetic beads using the corresponding Ni-IMAC protocol.

[0236] Briefly, lysates were incubated with Pierce™ High Capacity Ni-IMAC Magbeads (ThermoFisher Scientific, Waltham, MA), then beads were washed three times for 10 minutes each in wash buffer (50 mM Tris-HCl pH 8, 1.5 M NaCl, 15 mM imidazole, 5 mM P-mercaptoethanol and 5% glycerol). Modified RNA polymerases were eluted in elution buffer (50 mM Tris-HCl pH 8, 0.3 MNaCl, 0.2 M imidazole, 5 mM P-mercaptoethanol and 5% glycerol) and buffer exchanged to remove salt and imidazole by passing the eluate through 96-well Zeba™ Spin Desalting Plates (ThermoFisher Scientific, Waltham, MA) pre-equilibrated with storage buffer (50 mM Tris-HCl pH 8, 0.3 M NaCl, 5 mM P-mercaptoethanol, 0.1 mM EDTA, 0.1% Tween20, and 5% glycerol). Protein concentration and yield of the modified RNA polymerases or wild-type RNA polymerase was determined by spectrometry at 280 nm using the Lunatic system.Example 3. In vitro Transcription for Preparation of mRNA

[0237] In vitro transcribed mRNA was prepared as described in Example 1 of WO 2021 / 168052, which is incorporated herein by reference. Briefly, an in vitro transcription reaction mixture containing a linearized double-stranded DNA plasmid with an SP6 RNA polymerase-specific promoter, RNA polymerase, RNase inhibitor, pyrophosphatase, NTPs, DTT, and a buffering reagent was prepared with RNase-free water. A linearized template plasmid encoding a mRNAtranscript with a theoretical length of 1941 nucleotides was used. The reaction buffer comprised 25 mM Tris-HCl, 2 mM spermidine, 25 mM MgCh, 0.5 mM NaCl, and pH 7.5. The reaction mixtures were incubated at 37°C for 90 minutes with shaking. In vitro transcribed mRNA was prepared with ATP, CTP, GTP, and N1 -methylpseudouridine to generate in vitro transcribed mRNA comprising a modified ribonucleotide. DNase I was added to stop the reaction, and the reaction mixture was incubated for 15 more minutes at 37°C with shaking. The resulting in vitro transcribed mRNA was purified.

[0238] The affinity-purified modified RNA polymerases or control wild-type polymerase obtained in Example 2 were used for in vitro transcription as described above to assess various parameters, such as mRNA yield, integrity of the in vitro transcribed mRNA, and / or the formation of undesired transcription by-products (e.g., double-stranded RNA and / or abortive RNA transcripts), as described below.Example 4. Protein and RNA Yield of the Modified RNA Polymerases

[0239] Of the 152 modified RNA polymerases expressed recombinantly in E. coli cells and purified, almost half (74) resulted in less than 0.1 mg / mL soluble protein and were not pursued further. The concentration of each of the remaining 77 affinity-purified soluble modified RNA polymerases is provided in Table 3.

[0240] In vitro transcription reactions were performed with the soluble modified RNA polymerases and the control wild-type polymerase as described in Example 3. RNA yield, as determined using the Lunatic system and expressed in ng per pg of RNA polymerase, is provided in Table 3.Table 3. Comparison of RNA polymerase yield and RNA produced by in vitro transcription (IVT) with modified RNA polymerasesWT pol.: wild-type polymerase; NA: not applicable; cone.: concentration.

[0241] Of the 77 modified RNA polymerases, 16 were considered to be enzymatically inactive, as less than 15 pg of RNA was generated in the IVT reaction. As shown in Table 3, 61 modified RNA polymerases were enzymatically active, producing more than 15 pg of RNA in the IVT reaction (Table 3, bold and italic).Example 5. Evaluation of Double-Stranded RNA (dsRNA) Production

[0242] The process of IVT generates dsRNA through base pairing in regions of complementarity within the same strand or on opposite strands yielding dsRNA with 5' or 3' overhangs. Modified RNA polymerases that were soluble and produced acceptable levels of RNA were further evaluated to determine if the level of dsRNA present after IVT was reduced.

[0243] In order to measure the amounts of dsRNA generated during IVT, mRNA synthesis was carried out as described in Example 3, and the dsRNA produced was quantified using a sandwich ELISA. Briefly, 96-well plates were coated with anti-dsRNA monoclonal antibody KI (Cell Signaling Technology, Inc., Danvers, MA) by incubation overnight with a solution of 5 pg / mL KI antibody in PBS at 4°C. The plates were then washed five times with 0.05% Tween 20 in IX PBS (wash buffer) and blocked with a solution of 2% BSA in the wash buffer for one hour at roomtemperature. Purified RNA obtained from the IVT reactions was normalized, added to the wells with dilution buffer (1% BSA in wash buffer), and incubated for at least one hour at room temperature. The plates were then washed five times with wash buffer before adding the anti- dsRNA monoclonal antibody J2 coupled to HRP (Abeam, Inc., Cambridge, United Kingdom) at 1 pg / mL in PBS buffer. The plates were incubated for at least one hour at room temperature and then washed five times. TMB reagent was added to the wells and incubated for 20 minutes at room temperature followed by addition of 2N sulfuric acid to stop the reaction. The absorbance was then measured at 450 nm with a Tecan reader. A calibration curve was generated using a DNA ladder and used to determine the linear range to be used for quantification. The amount of dsRNA in each sample was determined based on the appropriate dilution, with results expressed as a percent ratio of the dsRNA to the total RNA. Two independent replicates were performed. The results are shown in FIG. 4.

[0244] These results demonstrate that performing IVT with certain modified RNA polymerases generated mRNA transcripts comprising reduced levels of dsRNA as compared to the wild-type SP6 RNA polymerase, as determined by ELISA with the J2 antibody. In particular, modified RNA polymerases having any of the following substitutions: S35E / Y147H (considered as a single functional mutation), W41L, G19T, or G19H, showed significantly reduced levels of dsRNA as compared to the wild-type RNA polymerase (p < 0.05, t-test). While not significant, modified RNA polymerases L749F, E36R, Y131F / A157F, I20E, A154V, R44L / S47V, and S37V / N42L showed a tendency for reduced dsRNA levels as compared to the wild-type RNA polymerase.

[0245] This example illustrates that the amount of dsRNA produced by RNA polymerase during an IVT reaction can be reduced by selecting modified RNA polymerases.Example 6. Manufacturability of Selected Modified RNA Polymerases

[0246] The modified RNA polymerases showing reduced dsRNA levels in the IVT reaction of Example 5 were further evaluated. Firstly, enzyme production was scaled-up by about 40-fold to evaluate manufacturability. Briefly, transformed E. coli cells were grown in 25 mL of Terrific Broth (TB) auto-induction growth medium supplemented with 100 pg / mL carbenicillin at 37°C with shaking to induce expression of the recombinant protein. Once the cells reached an ODeoo of about 0.5, the temperature was reduced to 30°C. After overnight incubation, E. coli cells were harvested by centrifugation and lysed. Lysed cells were clarified by centrifugation and thenpurified with nickel affinity chromatography using Pierce™ High Capacity Ni-IMAC Magbeads (ThermoFisher Scientific, Waltham, MA) applied on a 10 mL column with a filter. Beads were washed with 30 mL wash buffer, the modified RNA polymerases were eluted in elution buffer, and then buffer exchanged to remove salt and imidazole by passing the eluate through a 5 mL Zeba™ Spin Desalting Column (ThermoFisher Scientific, Waltham, MA) pre-equilibrated with storage buffer. The concentration of the modified RNA polymerases was determined by spectrometry at 280 nm using the Lunatic system and is shown in FIG. 5 and summarized in Table 4 below.Table 4. Scaled-up RNA polymerase yield for assessing manufacturabilityNA: not applicable

[0247] Protein yield of the modified RNA polymerase having the amino acid substitutions S37V / N42L was nearly 2-fold higher than the wild-type RNA polymerase. In contrast, the yield of the modified RNA polymerase having the amino acid substitution W41L or G19T was about 10-fold or about 2.5-fold lower than the wild-type RNA polymerase, respectively. The remaining modified RNA polymerases showed comparable yield to the wild-type RNA polymerase.Example 7. RNA Yield of Selected Modified RNA Polymerases

[0248] RNA polymerase concentrations were adjusted to 1 mg / mL in storage buffer and in vitro transcribed mRNA was prepared as described in Example 3 in reaction mixture containing 2 pg RNA polymerase. RNA concentration was determined using the Lunatic system.

[0249] As shown in FIG. 6 and in Table 5 below, RNA transcribed in vitro using selected modified RNA polymerases and the wild-type RNA polymerase had similar profiles.Example 8. RNA Integrity Following In Vitro Transcription (IVT) with Selected Modified RNA Polymerases

[0250] Capillary gel electrophoresis was used to evaluate RNA integrity following IVT using an RNA polymerase having one of the following the amino acid substitutions: L749F, S35E / Y147H, W41L, G19H, I20E, and S37V / N42L. Briefly, the standard sensitivity RNA analysis kit (15 nt) was purchased from Agilent (Savage, MD) and used in capillary electrophoresis runs on the Fragment Analyzer instrument with a twelve-capillary array (Agilent, Savage, MD). Upon gel priming, 300 ng of total RNA was mixed with diluent marker at 1 : 11 (RNA:Marker) ratio and 24 mL was loaded per well in a 96-well plate. The molecular weight indicator ladder was prepared by mixing 2 pl of the standard sensitivity RNA ladder with 22 pl diluent marker. Sample injection was at 5.0 kV, 4 seconds, and sample separation at 8.0kV, 40.0 minutes. Electropherogram of each sample was processed through the ProSize 2 software (Advanced Analytical Solutions, Parkersburg, WV), producing tabulated sizes (nt) and abundances of fragments present in the sample.

[0251] As shown in FIG. 7, RNA transcribed in vitro using the modified RNA polymerases had similar integrity profiles to RNA transcribed in vitro using the wild-type RNA polymerase.Example 9. Level of dsRNA Produced by Selected Modified RNA Polymerases

[0252] The level of dsRNA present after IVT was performed according to Example 7 with the modified RNA polymerases produced in Example 6 was evaluated to confirm that dsRNA levels were reduced, as initially observed in the high-throughput screening. The level of dsRNA was quantified by ELISA as described in Example 5.

[0253] The results are shown in FIG. 8. The level of dsRNA obtained with the modified RNA polymerases as compared to the level obtained with the wild-type RNA polymerase, expressed as a percentage, is also provided in Table 5. These results confirm high-throughput screening results. Indeed, dsRNA levels tend to be reduced, with levels comprised between about 37% and about 90% of the amount present when IVT was performed with the wild-type RNA polymerase.Modified RNA polymerases L749F, S35E / Y147H, W41L, G19H, I20E, and S37V / N42L show the greatest reduction in dsRNA (see Table 5).Example 10. Level of Short Abortive RNA Transcripts Produced by Selected RNA Polymerase

[0254] To determine the impact of a given substitution in the amino acid sequence of the RNA polymerase on the initiation phase of transcription, the amount of short abortive RNA transcripts in the IVT reactions described in Example 7 was measured using liquid chromatography-mass spectrometry (LC-MS). 100 pM of a probe oligonucleotide was annealed to the 3' end of the mRNA transcripts (1-2 mg RNA per mL, 21 pL in total) in a thermocycler (Eppendorf, Hamburg, Germany) at 75°C for 10 minutes. The samples were then ramped down to 23°C for 10 minutes and then quickly cooled to 4°C. RNase H nuclease (5000 units / mL, 1 pL, New England BioLabs, Ipswich, MA) and rSAP (1000 units / mL, 4 pL, New England BioLabs, Ipswich, MA) in IX RNase H buffer (New England BioLabs, Ipswich, MA) was added to each sample and incubated in a thermocycler at 37 °C for 40 minutes.

[0255] Ultra-high pressure liquid chromatography (UHPLC) was used for separation of the samples on an Agilent 1290 Infinity II coupled with Agilent InfinityLab Cl 8 2.1 x 100mm, 2.7 pm, 100 A column. The column was heated to 50°C with a flow rate of 500 pL / min. The mobile phase included buffer A (100 mM HFIP 8.6 mM TEA) and buffer B (100% methanol). The gradient started at 1% buffer B and increased to 5% buffer B for the first 3 minutes, followed by a linear ramp to 20% buffer B until 13 minutes. Between 13 and 14 minutes, the percentage of buffer B increased from about 20% to about 50%. At 14 minutes, a 1.5-min rinse at 50% buffer B began, followed by a return to 1% of buffer B at 17 minutes.

[0256] MS spectrum was acquired using Agilent 6545XT AdvanceBio Q-TOF system operating in the negative ion mode. Dual AJS was used as ESI source in full-scan range from 400 to 3000 (m / z). Two spectrum per second were acquired in full-scan MS runs. MS / MS runs were operated in extended dynamic range mode (2 GHz) with a scan range of 100-2500 (m / z). Data was processed by Agilent MassHunter BioConfirm 12.0 and Agilent MassHunter Qualitative Analysis 10.0 software.

[0257] 1 pg of each RNA sample was injected into LC-MS. The results in FIG. 9 correspond to the sum of all peaks between 6.5 and 12 minutes (n=l). Levels of abortive RNA transcriptsobtained with the modified RNA polymerases as compared to the level of abortive RNA transcripts obtained with the wild-type RNA polymerase, expressed as a percentage, are also provided in Table 5

[0258] IVT reactions performed with modified RNA polymerases having the substitutions E36R, Y131F / A157F, A154V, G19H, or S37V / N42L, resulted in amounts of abortive RNA transcripts that were similar to those that were observed when the wild-type RNA polymerase was used. Surprisingly, using modified RNA polymerases having the substitutions S35E / Y147H, W41L, G19T, or I20E, the amounts of abortive RNA transcripts that could be detected in this assay were reduced relative to the wild-type SP6 RNA polymerase. Thus, this example illustrates that the amount of short abortive RNA transcripts produced by SP6 RNA polymerase during an IVT reaction may also be reduced when one or more amino acid substitutions are introduced.Table 5. Summary of characteristics measured for selected modified RNA polymerases, expressed as a percentage of the amount obtained for the wild-type polymerase.Example 11. Design of Modified SP6 RNA Polymerases

[0259] An additional SP6 RNA polymerase mutant library consisting of 83 rationally designed mutants was generated based on the wild-type SP6 RNA polymerase having the amino acidsequence of SEQ ID NO: 1 (GenBank Accession No. Y00105.1) and evaluated as described in Example 1. The amino acid substitutions evaluated are provided in Table 6 below. The amino acid residue positions listed in Table 6 correspond to those provided in the RNA polymerase of SEQ ID NO: 1. Modifications of amino acids S35 and Y147 are considered as a single functional mutation.Table 6. Amino acid substitutions in the modified RNA polymerasesExample 12. Expression and Purification of Additional Modified SP6 RNA Polymerases

[0260] The coding sequence of each of the modified RNA polymerases designed in Example 11 was cloned into an expression plasmid, and the corresponding protein was expressed and purified using the methods provided in Example 2 above. A wild-type SP6 RNA polymerase having the amino acid sequence of SEQ ID NO: 1 and two previously identified SP6 polymerase variants having the substitutions L749F or S35E / Y147H were included as controls.

[0261] The RNA polymerases thus obtained were used for in vitro transcription using the methods provided in Example 3. Various parameters, such as mRNA yield, integrity of the in vitro transcribed mRNA, and / or the formation of undesired transcription by-products (e.g., doublestranded RNA), were assessed using the methods described previously in Examples 5, 7, 8, and 9.Example 13. Protein and RNA Yield of the Modified RNA Polymerases

[0262] Of the 83 modified RNA polymerases expressed recombinantly in E. coli cells and purified, one modified RNA polymerase (having the substitution N559K) resulted in less than 0.1 mg / mL soluble protein and was not pursued further. The amount of total protein produced for each of the remaining 82 affinity-purified soluble modified RNA polymerases in pg and as apercentage of the yield of the wild-type RNA polymerase of SEQ ID NO: 1 is provided in Table 7.

[0263] In vitro transcription reactions were performed with the soluble modified RNA polymerases and the control wild-type polymerase as described previously in Example 3. RNA yield, as determined using the Lunatic system and expressed in pg of RNA product, is provided in Table 7. dsRNA was quantified as described previously in Example 5. The amount of dsRNA produced is expressed as a percentage of the amount of dsRNA produced during IVT with the wild-type SP6 polymerase of SEQ ID NO: 1 in FIG. 10. mRNA integrity was evaluated by capillary gel electrophoresis as provided in Example 8, and results are summarized in FIG. 11.Table 7. Yield of modified RNA polymerases and of RNA produced by IVT with modified RNA polymerases in pg and as compared to wild-type SP6 polymeraseNA: not applicable; WT : wild-type polymerase.

[0264] Of the 82 modified RNA polymerases evaluated here, 17 were considered to be enzymatically inactive, as less than 15 pg of RNA was generated in the IVT reaction (see Table 7). As shown in Table 7, 65 modified RNA polymerases were enzymatically active, producing more than 15 pg of RNA in the IVT reaction (Table 7, bold and italic). Of these, the RNApolymerases having the substitutions: S37I, A128K, A128R, F873Y, F873W, N458R, N458K, S538W, S538R, S538F, K464W, P566W, P566Y, P566F, S35E / Y147R, S35D / Y147R, S35E / Y147K, S35L / Y147W, or S35M / Y147W were identified as having reduced dsRNA production (see FIG. 10), and acceptable RNA yield (i.e., typically no more than about a 2-fold reduction in yield) when compared to that obtained with the wild-type SP6 polymerase. While some of the modified RNA polymerases had slightly lower RNA yield (e.g. A128, S538W), they were nevertheless retained for further evaluation in view of the large reduction in dsRNA production. Integrity of the RNA obtained when using modified RNA polymerase was furthermore superior to 75% for RNA polymerases having the substitutions: S37I, A128K, A128R, F873Y, F873W, N458R, N458K, S538W, S538R, S538F, K464W, P566W, P566Y, P566F, S35L / Y147W, or S35M / Y147W (see FIG. 11). As expected, previously identified modified RNA polymerases having the substitution L749F or S35E / Y147H also showed acceptable RNA yield and integrity and reduced levels of dsRNA production.Example 14. Validation of RNA Yield, RNA Integrity, and dsRNA Level in IVT When Using Selected Modified RNA Polymerases

[0265] RNA yield, RNA integrity, and the level of dsRNA produced during IVT with modified RNA polymerases having the substitution S37I, A128K, F873Y, F873W, N458R, N458K, S538W, S538R, S538F, K464W, P566W, P566F, S35L / Y I47W, or S35M / Y147W were further evaluated to confirm the results obtained in HTS. RNA polymerases having the substitution A128R or P566Y were not evaluated here, as results are expected to be the same as those obtained for A128K and P566W given that R and K as well as W and Y amino acids have similar properties.

[0266] IVT was performed as described in Example 3. RNA concentration and thus yield was determined using the Lunatic system, RNA integrity was evaluated as described in Example 8, and the level of dsRNA was quantified by ELISA as described in Example 5. Results are provided in Table 8 below. RNA yield and dsRNA levels are provided as a percentage of the amount present in the corresponding IVT reaction performed with the wild-type RNA polymerase of SEQ ID NO: 1.Table 8. Evaluation of characteristics of RNA produced by IVT with modified RNA polymerasesWT: wild-type; NA: not applicable

[0267] As shown in Table 8, acceptable RNA yield (i.e. no more than about a 2-fold reduction in yield) was obtained when performing IVT with all modified RNA polymerases. Trends in mRNA integrity were similar to those seen in the high-throughput screening. However, while integrity was superior to 75% in HTS for all selected mutants, RNA integrity was lower than 75% here for RNA polymerase mutants having the substitution S37I, F873W, S538W, or S538R. dsRNA levels were reduced for all modified RNA polymerases, though the level of dsRNA was reduced by only 15.9% for the RNA polymerase having the S538W substitution as compared to the wild-type polymerase of SEQ ID NO: 1. Modified RNA polymerases having the substitutions S37I, F873Y, F873W, S538F, K464W, S35L / Y147W, or S35M / Y147W showed the greatest reduction in dsRNA.

[0268] In view of results of Examples 13 and 14, modified RNA polymerases having the substitutions A128K, A128R, F873Y, N458R, N458K, S538F, K464W, P566W, P566F, P566Y, S35L / Y147W, or S35M / Y147W showed acceptable RNA yield, RNA integrity, and reduction in dsRNA levels.Example 15. Design of a Combinatorial Library of Modified SP6 RNA Polymerases

[0269] Several of the RNA polymerase modifications that were identified above were further tested in combination to determine if RNA characteristics (e.g., yield, integrity, and / or dsRNA levels) could be further improved. Modifications were selected for inclusion in this study were based on results obtained for RNA yield, RNA integrity, and dsRNA levels, as well as location of the substitution within the RNA polymerase. Selected modifications were A128K, F873 Y, S538F, K464W, S35L / Y147W, S35M / Y147W, and L749F.

[0270] The combinations of modifications that were tested are provided in Table 9 below. The amino acid residue positions listed in Table 9 correspond to those provided in the RNA polymerase of SEQ ID NO: 1. Combinations were rationally chosen to combine substitutions that were located in different regions of the RNA polymerase. Modified RNA polymerases having combinations of two or more of the S538F, K464W, and / or F873Y modifications were not expected to show improved properties, given that the selected mutations were all present in the same region of the RNA polymerase (i.e., the C-terminal cavity); they were nevertheless included to validate the approach.Table 9. Amino acid substitutions in the modified RNA polymerases of the combinatorial libraryExample 16. Expression and Purification of Additional Modified SP6 RNA Polymerases

[0271] The coding sequence of each of the modified RNA polymerases designed in Example 15 was cloned into an expression plasmid, and the corresponding protein was expressed and purified using the methods provided in Example 2 above. A wild-type SP6 RNA polymerase comprising the amino acid sequence of SEQ ID NO: 1 was included as a control.

[0272] All modified RNA polymerases designed in Example 15 were soluble and were produced in amounts equal or superior to the level of wild-type polymerase. The amount of modified RNA polymerase that was produced ranged from 101% to 142% of the level of the wild-type polymerase of SEQ ID NO: 1 (data not shown).Example 17. Evaluation of a Combinatorial Library of Modified SP6 RNA Polymerases

[0273] The RNA polymerases obtained from Example 16 were used in in vitro transcription according to the method provided in Example 3. Various parameters, such as RNA yield, integrity of the in vitro transcribed RNA, and / or the formation of undesired transcription by-products (i.e., double-stranded RNA), were assessed using the methods described previously in Examples 5, 7, 8, and 9. Three independent replicates were performed.

[0274] Results are provided in Table 10 below and correspond to the average of the three replicates. RNA yield and dsRNA levels are provided as a percentage of the amount obtained from the IVT reaction performed with the wild-type RNA polymerase of SEQ ID NO: 1 (“% of WT”).Table 10. Evaluation of characteristics of RNA produced by IVT with modified RNA polymerases

[0275] As shown in Table 10, acceptable RNA yield (i.e. no more than about a 2-fold reduction in yield) was obtained when performing IVT with all modified RNA polymerases except those for which an improvement was not expected due to the location of the selected mutations. Indeed, RNA obtained from IVT reactions using modified RNA polymerases having the F873Y / S538F, F873Y / K464W, or S538F / K464W modifications showed RNA yield at only 15-34% of that of a wild-type polymerase. In all cases, dsRNA levels were reduced as compared to the amount present when IVT was performed with the wild-type RNA polymerase. Furthermore, dsRNA levels were reduced as compared to that obtained with modified RNA polymerases having only a single functional mutation, with the exception of the RNA polymerase having the A128K / K464W substitutions, which showed similar levels of dsRNA as for RNA polymerases having either the A128K or K464W substitution alone. In some cases, RNA integrity was also found to be higher when IVT was performed using RNA polymerases in which modifications were combined as compared to that obtained when using modified RNA polymerases having only a single functional mutation, e.g., as was the case for A128K / F873Y or A128K / S538F.

[0276] It was observed that, when performing IVT with an RNA polymerase having the L749F substitution, the RNA yield was similar (see e.g., FIG. 6) or higher (data not shown) to that seen when using the wild-type RNA polymerase. As a tendency for a reduction in RNA yield was observed when performing IVT with an RNA polymerase having the substitutions F873Y, S538F,or K464W (e.g., in the C-terminal cavity), these substitutions were combined with that of L749F to see if yield could be maintained. As shown in Table 10, it was confirmed that the L749F substitution allowed for yield to be restored to a level equal to or higher than that obtained with a wild-type polymerase. Indeed, RNA yield was found to be 137%, 123%, or 115% of that obtained with a wild-type polymerase when the L749F substitution was combined with F873Y, S538F, or K464W, respectively. In contrast, while several modified RNA polymerases previously showed improved characteristics when taken alone, their combination did not necessarily lead to improved RNA characteristics overall. For example, while dsRNA levels were reduced for an RNA polymerase having the modifications A128K / K464W / S35L / Y147W beyond what was seen for each individual functional mutation, RNA yield and integrity also decreased.

[0277] Modified RNA polymerases having the substitutions F873Y / L749F, F873Y / S35M / Y147W, S538F / L749F, K464W / L749F, or A128K / K464W / L749F notably demonstrated low dsRNA along with acceptable RNA integrity and yield.Example 18. Validation of RNA Yield, RNA Integrity, and dsRNA Level in IVT When Using Selected Modified RNA Polymerases

[0278] RNA yield, RNA integrity, and the level of dsRNA produced during IVT with modified RNA polymerases having the F873Y / L749F, F873Y / S35M / Y147W, S538F / L749F, K464W / L749F, or A128K / K464W / L749F were further evaluated to confirm the results obtained in HTS.

[0279] IVT was performed as described in Example 3. RNA concentration and thus yield was determined using the Lunatic system, RNA integrity was evaluated as described in Example 8, and the level of dsRNA was quantified by ELISA as described in Example 5. Two independent replicates were performed. Results are provided in Table 11 below and correspond to the average of the two replicates. RNA yield and dsRNA levels are provided as a percentage as a percentage of the amount obtained from the IVT reaction performed with the wild-type RNA polymerase of SEQ ID NO: 1 (“% of WT”).Table 11. Evaluation of characteristics of RNA produced by IVT with modified RNA polymerases.WT: wild-type polymerase; NA: not applicable

[0280] As shown in Table 11, acceptable RNA yield (i.e. no more than about a 2-fold reduction in yield) was obtained when performing IVT with all modified RNA polymerases. Trends in mRNA integrity were similar to those seen in the high-throughput screening. While integrity was superior to 75% in HTS for all selected mutants, RNA integrity was slightly lower than 75% here for RNA polymerase mutants having the substitutions F873Y / L749F, S538F / L749F, or A128K / K464W / L749F. dsRNA levels were significantly reduced in all cases when compared to the level present when IVT was performed with the wild-type polymerase of SEQ ID NO: 1.

[0281] Taking into account dsRNA, integrity, and yield parameters, in that order, modified RNA polymerases having the substitutions K464W / L749F or F873Y / S35M / Y147W were identified as being of particular interest from among the modified RNA polymerases evaluated in the combinatorial library.Example 19. IVT reaction using modified RNA polymerase on a 2 or 10 mg scale

[0282] IVT reactions were performed using RNA polymerases S35L / Y147W, A128K / K464W / L749F, K464W / L749F, S538F / L749F, F873Y / S35M / Y147W, andF873 Y / L749F at a 2 and 10 mg scale to generate an mRNA having a length of approximately 2000 nucleotides. RNA yield and dsRNA levels are provided as a percentage of the amount obtained from the IVT reaction performed with the wild-type RNA polymerase of SEQ ID NO: 1 (“% of WT”).Table 12. Evaluation of characteristics of RNA produced by IVT with various modified RNA polymerases at a 2 mg scaleWT: wild-type polymerase; NA: not applicable

[0283] RNA yield of modified RNA polymerases was comparable to that of the wild-type polymerase of SEQ ID NO: 1. The RNA polymerase having the modification S35L / Y147W showed the highest yield of all mutants at a level equal or higher to that of the wild-type polymerase at a 2 mg (see Table 12) and also at a 10 mg scale (data not shown). Integrity of RNA obtained with all modified RNA polymerases was found to be superior to 75% (data not shown), and the level of dsRNA was greatly reduced, with a decrease of up to 96% as compared to level obtained when using the wild-type polymerase of SEQ ID NO: 1 (see Table 12), thus confirming the interest of the various modified RNA polymerases identified herein.

[0284] While the foregoing disclosure has been described in some detail by way of illustration and example for purposes of clarity and understanding, it will be clear to one of ordinary skill in the art from a reading of this disclosure that various changes in form and detail can be made without departing from the true scope of the present disclosure and may be practiced within the scope of the appended claims. For example, all constructs, methods, and / or component features, steps, elements, or other aspects thereof can be used in various combinations.

[0285] Claims or descriptions that include “or” between one or more members of a group are considered satisfied if one, more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process unless indicated to the contrary or otherwise evident from the context. The disclosure includes embodiments in which exactly one member of the group is present in, employed in, or otherwise relevant to a given product or process. The disclosure also includes embodiments in which more than one, or the entire group members are present in, employed in, or otherwise relevant to a given product or process. Furthermore, it is to be understood that the disclosure encompasses all variations, combinations, and permutations in which one or more limitations, elements, clauses, descriptive terms, etc., from one or more of the listed claims is introduced into another claim dependent on the same base claim (or, as relevant,any other claim) unless otherwise indicated or unless it would be evident to one of ordinary skill in the art that a contradiction or inconsistency would arise. Where elements are presented as lists, (e.g., in Markush group or similar format) it is to be understood that each subgroup of the elements is also disclosed, and any element(s) can be removed from the group. In general, where embodiments or aspects of the present disclosure, is / are referred to as comprising particular elements, features, etc., certain embodiments or aspects consist, or consist essentially of, such elements, features, etc. For purposes of simplicity those embodiments have not in every case been specifically set forth in so many words herein. It should also be understood that any embodiment or aspect of the present disclosure can be explicitly excluded from the claims, regardless of whether the specific exclusion is recited in the specification.

Claims

CLAIMS1. A modified ribonucleic acid (RNA) polymerase comprising at least one amino acid substitution relative to a wild-type SP6 RNA polymerase comprising the amino acid sequence of SEQ ID NO: 1, wherein the at least one amino acid substitution is in the N-terminal domain of the modified RNA polymerase or a portion of the C-terminal domain of the modified RNA polymerase that interacts with the N-terminal domain, wherein the modified RNA polymerase produces less double-stranded RNA and / or less abortive RNA transcripts in an in vitro transcription reaction as compared to the wild-type SP6 RNA polymerase, and wherein the modified RNA polymerase comprises an amino acid sequence having at least about 80% sequence identity to the amino acid sequence of SEQ ID NO: 1.

2. The modified RNA polymerase of claim 1, wherein the at least one amino acid substitution comprises at least one pair of amino acid substitutions at amino acid positions 35 and 147, 37 and 42, or 131 and 157 as indexed by reference to the wild-type RNA polymerase comprising the amino acid sequence of SEQ ID NO: 1.

3. The modified RNA polymerase of claim 1, wherein the at least one amino acid substitution is at one or more of amino acid positions 19, 20, 36, 41, 128, 154, and 749 as indexed by reference to the wild-type RNA polymerase comprising the amino acid sequence of SEQ ID NO: 1.

4. The modified RNA polymerase of claim 3, wherein the amino acid substitution is at one or more of amino acid positions 19, 20, 41, 128, and 749.

5. The modified RNA polymerase of claim 2, further comprising an amino acid substitution at one or more of amino acid positions 19, 20, 36, 41, 128, 154, 458, 464, 538, 566, 749, and 873 as indexed by reference to the wild-type RNA polymerase comprising the amino acid sequence of SEQ ID NO: 1.

6. The modified RNA polymerase of claim 2 or 5, wherein the at least one pair of amino acid substitutions is at amino acid positions 35 and 147 and / or amino acid positions 37 and 42.

7. The modified RNA polymerase of claim 6, wherein the at least one pair of amino acid substitutions is at amino acid positions 35 and 147, and wherein the at least one amino acid substitution further comprises an amino acid substitution at one or more of amino acid positions 19, 20, 36, 41, 128, 154, 458, 464, 538, 566, 749, and 873 as indexed by reference to the wild-type RNA polymerase comprising the amino acid sequence of SEQ ID NO: 1.

8. The modified RNA polymerase of any one of claims 1-7, comprising at least two amino acid substitutions at (i) amino acid positions 35 and 147, or (ii) an amino acid position selected from 19, 20, 36, 41, 128, 154, and 749 and an additional amino acid position selected from 19, 20, 36, 41, 128, 154, 464, 538, 458, 464, 538, 566, 749, and 873.

9. The modified RNA polymerase of claim 8, comprising amino acid substitutions at amino acid positions 35 and 147, amino acid positions 464 and 749, amino acid positions 538 and 749, amino acid positions 749 and 873, amino acid positions 128 and 873, amino acid positions 128 and 538, or amino acid positions 128 and 749.

10. The modified RNA polymerase of claim 8, comprising at least three amino acid substitutions at: i) amino acid positions 35 and 147 and amino acid position selected from 19, 20, 36, 41, 128, 154, 458, 464, 538, 566, 749, and 873; or ii) an amino acid position selected from 19, 20, 36, 41, 128, 154, and 749 and two additional amino acid positions selected from 19, 20, 36, 41, 128, 154, 464, 538, 458, 464, 538, 566, 749, and 873.

11. The modified RNA polymerase of claim 10, comprising amino acid substitutions at amino acid positions 35, 147, and 873, amino acid positions 128, 464, and 749, amino acid positions 35, 147, and 128, amino acid positions 35, 147, and 538, amino acid positions 35, 147, and 464, amino acid positions 35, 147, and 749, amino acid positions 128, 749, and 873, or amino acid positions 35, 147, 464, and 749, e.g. amino acid positions 35, 147, and 873, or amino acid positions 128, 464, and 749.

12. A modified ribonucleic acid (RNA) polymerase comprising at least one amino acid substitution relative to a wild-type SP6 RNA polymerase comprising the amino acid sequence of SEQ ID NO: 1, wherein the at least one amino acid substitution comprises at least one pair of amino acid substitutions at amino acid positions 35 and 147, 37 and 42, or 131 and 157 as indexed by reference to the wild-type RNA polymerase comprising the amino acid sequence of SEQ ID NO: 1, and wherein the modified RNA polymerase comprises an amino acid sequence having at least about 80% sequence identity to the amino acid sequence of SEQ ID NO: 1.

13. The modified RNApolymerase of claim 12, further comprising an amino acid substitution at one or more of amino acid positions 19, 20, 36, 41, 128, 154, 458, 464, 538, 566, 749, and 873 as indexed by reference to the wild-type RNApolymerase comprising the amino acid sequence of SEQ ID NO: 1.

14. The modified RNA polymerase of claim 12 or 13, wherein the at least one pair of amino acid substitutions is at amino acid positions 35 and 147 and / or amino acid positions 37 and 42.

15. The modified RNA polymerase of claim 14, wherein the at least one pair of amino acid substitutions is at amino acid positions 35 and 147, and wherein the at least one amino acid substitution further comprises an amino acid substitution at one or more of amino acid positions 19, 20, 41, 128, 458, 464, 538, 566, 749, and 873, e.g., at one or more of amino acid positions 128, 464, 538, 749, and 873, as indexed by reference to the wild-type RNApolymerase comprising the amino acid sequence of SEQ ID NO: 1.

16. The modified RNApolymerase of claim 15, comprising amino acid substitutions at amino acid positions 35, 147, and 873, amino acid positions 35, 147, and 128, amino acid positions 35, 147, and 538, amino acid positions 35, 147, and 464, amino acid positions 35, 147, and 749, or amino acid positions 35, 147, 464, and 749, e.g., amino acid positions 35, 147, and 873.

17. A modified ribonucleic acid (RNA) polymerase comprising at least one amino acid substitution relative to a wild-type SP6 RNA polymerase comprising the amino acid sequence ofSEQ ID NO: 1, wherein the at least one amino acid substitution is at one or more of amino acid positions 19, 20, 36, 41, 128, 154, 458, 464, 538, 566, 749, and 873 as indexed by reference to the wild-type RNA polymerase comprising the amino acid sequence of SEQ ID NO: 1, and wherein the modified RNA polymerase comprises an amino acid sequence having at least about 80% sequence identity to the amino acid sequence of SEQ ID NO: 1.

18. The modified RNA polymerase of claim 17, wherein the amino acid substitution is at one or more of amino acid positions 19, 20, 41, 128, 458, 464, 538, 566, 749, and 873.

19. The modified RNA polymerase of claim 17 or 18, comprising at least two amino acid substitutions at amino acid positions selected from 19, 20, 41, 128, 458, 464, 538, 566, 749, and 873.

20. The modified RNA polymerase of claim 19, comprising amino acid substitutions at amino acid positions 464 and 749, amino acid positions 538 and 749, amino acid positions 749 and 873, amino acid positions 128 and 873, amino acid positions 128 and 538, or amino acid positions 128 and 749.

21. The modified RNA polymerase of claim 19, comprising at least three amino acid substitutions at amino acid positions selected from 19, 20, 41, 128, 458, 464, 538, 566, 749, and 873.

22. The modified RNA polymerase of claim 21, comprising amino acid substitutions at amino acid positions 128, 464, and 749 or amino acid positions 128, 749, and 873, e.g. amino acid positions 128, 464, and 749.

23. The modified RNA polymerase of any one of claims 3-22, wherein the amino acid substitution at amino acid position 19 is G19H or G19T.

24. The modified RNA polymerase of claim 23, wherein the amino acid substitution at amino acid position 19 is G19H.I l l25. The modified RNA polymerase of any one of claims 3-24, wherein the amino acid substitution at amino acid position 20 is I20E.

26. The modified RNA polymerase of any one of claims 3-25, wherein the amino acid substitution at amino acid position 36 is E36R.

27. The modified RNA polymerase of any one of claims 3-26, wherein the amino acid substitution at amino acid position 41 is W41L.

28. The modified RNA polymerase of any one of claims 3-27, wherein the amino acid substitution at amino acid position 128 is A128K or A128R, optionally A128K.

29. The modified RNA polymerase of any one of claims 3-28, wherein the amino acid substitution at amino acid position 154 is Al 54V.

30. The modified RNA polymerase of any one of claims 3-29, wherein the amino acid substitution at amino acid position 458 is N458R or N458K.

31. The modified RNA polymerase of any one of claims 3-30, wherein the amino acid substitution at amino acid position 464 is K464W.

32. The modified RNA polymerase of any one of claims 3-31, wherein the amino acid substitution at amino acid position 538 is S538F.

33. The modified RNA polymerase of any one of claims 3-32, wherein the amino acid substitution at amino acid position 566 is P566W, P566F, or P566Y.

34. The modified RNA polymerase of any one of claims 3-33, wherein the amino acid substitution at amino acid position 749 is L749F.

35. The modified RNA polymerase of any one of claims 3-34, wherein the amino acid substitution at amino acid position 873 is F873 Y.

36. The modified RNA polymerase of any one of claims 2-35, wherein the amino acid substitutions at amino acid positions 35 and 147 are S35E and Y147H, S35L and Y147W, or S35M and Y147W.

37. The modified RNA polymerase of any one of claims 2-36, wherein the amino acid substitutions at amino acid positions 37 and 42 are S37V and N42L.

38. The modified RNA polymerase of any one of claims 2-37, wherein the amino acid substitutions at amino acid positions 131 and 157 are Y13 IF and A157F.

39. The modified RNA polymerase of any one of claims 1-38, comprising an amino acid sequence having at least about 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 1.

40. The modified RNA polymerase of any one of claims 1-39, wherein the modified RNA polymerases comprises an amino acid sequence selected from SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, or SEQ ID NO: 54.

41. An artificial nucleic acid encoding the modified RNA polymerase of any one of claims 1- 40.

42. A vector comprising the artificial nucleic acid of claim 41.

43. A host cell comprising the vector of claim 42.

44. A composition comprising the modified RNA polymerase of any one of claims 1-40, the artificial nucleic acid of claim 41, the vector of claim 42, or the host cell of claim 43.

45. A composition or kit comprising the modified RNA polymerase of any one of claims 1-40 and at least one in vitro transcription reaction component, such as nucleoside triphosphates and / or a cap or a cap analog.

46. A method for producing a ribonucleic acid (RNA) in an in vitro transcription reaction, said method comprising contacting a deoxyribonucleic acid (DNA) template with the modified RNA polymerase of any one of claims 1-40 in the presence of nucleoside triphosphates.

47. The method of claim 46, wherein the method produces a comparable amount of full-length RNA or more as compared to a control in vitro transcription reaction using an RNA polymerase having the amino acid sequence of SEQ ID NO: 1.

48. The method of claim 46 or 47, wherein the method produces at least about 10% less doublestranded RNA (dsRNA) as compared to a control in vitro transcription reaction using an RNA polymerase having the amino acid sequence of SEQ ID NO: 1.

49. The method of claim 48, wherein the method produces at least about 30% less dsRNA as compared to the control in vitro transcription reaction.

50. The method of claim 49, wherein the method produces at least about 50% less dsRNA as compared to the control in vitro transcription reaction.

51. The method of claim 50, wherein the method produces at least about 100% less dsRNA as compared to the control in vitro transcription reaction.

52. The method of any one of claims 46-51, wherein the method produces at least about 10% less abortive RNA transcripts as compared to a control in vitro transcription reaction using an RNA polymerase having the amino acid sequence of SEQ ID NO: 1.

53. The method of claim 52, wherein the method produces at least about 30% less abortive RNA transcripts as compared to the control in vitro transcription reaction.

54. The method of claim 53, wherein the method produces at least about 50% less abortive RNA transcripts as compared to the control in vitro transcription reaction.

55. The method of any one of claims 46-54, wherein the method produces RNA having a yield that is no less than about 35%, e.g. no less than about 50%, of the RNA yield produced in a control in vitro transcription reaction using an RNA polymerase having the amino acid sequence of SEQ ID NO: 1.

56. The method of claim 55, wherein the method produces RNA having a yield that is at least about equal to the RNA yield produced in the control in vitro transcription reaction.

57. The method of claim 55 or 56, wherein the method produces RNA having a yield that is superior to the RNA yield produced in the control in vitro transcription by at least about 10%, 20%, 30%, 40%, 50%, or more.

58. The method of any one of claims 47-57, further comprising a step of purifying RNA from the in vitro transcription reaction.

59. The method of any one of claims 47-58, wherein the RNA is messenger RNA (mRNA).

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