RNA polymerase variants

Abstract

To provide, in some aspects, RNA polymerase variants comprising at least one amino acid substitution, relative to the wild-type RNA polymerase, that causes at least one loop structure of the RNA polymerase variant to undergo a conformational change to a helix structure as the RNA polymerase variant transitions from an initiation complex to an elongation complex.SOLUTION: The present disclosure provides, in some aspects, variant RNA polymerases, the use of which increases the transcription efficiency while reducing the number of double-stranded RNA contaminates and run-on transcripts produced during an in vitro transcription reaction. In some embodiments, the RNA polymerase variants (e.g., T7 RNA polymerase variants) include mutations of residues that have a high-helix propensity (e.g., alanine) or that cause the backbone flexibility to specifically match that of an elongation complex. Also described are an amino acid substitution in the region of a C-linker of T7 RNA polymerase and a C-helix including S43A and G47A, and an addition of a C-terminal glycine residue. A co-transcriptional method using a cap analog is also described.SELECTED DRAWING: None

Description

[Technical field]

[0001] Related Applications This application claims the benefit under 35 U.S.C. §119(e) of U.S. Provisional Application No. 62 / 547,677, filed August 18, 2017, U.S. Provisional Application No. 62 / 628,484, filed February 9, 2018, U.S. Provisional Application No. 62 / 638,684, filed March 5, 2018, and U.S. Provisional Application No. 62 / 677,527, filed May 29, 2018, each of which is incorporated herein by reference in its entirety. [Background technology]

[0002] In vitro transcription (IVT) uses bacteriophage DNA-dependent ribonucleic acid (RNA) polymerases (e.g., SP6, T3, and T7) to synthesize mRNA transcripts against a template. Problems in an IVT reaction can result in complete failure (e.g., no transcripts produced) or transcripts that are incorrectly sized (e.g., shorter or longer than expected). Specific problems associated with IVT reactions include, for example, abortive (truncated) transcripts, run-on transcripts, polyA tail variants / 3' heterogeneity, mutated transcripts, and / or double-stranded contaminants generated during the reaction.

[0003] RNA polymerases exhibit three phases of transcription: initiation, elongation, and termination. In the initiation phase, RNA polymerase binds to a specific promoter DNA sequence, opening the DNA duplex and delivering the template strand to the active site. For example, T7 RNA polymerase forms a structure called the initiation complex, which contains a six-helix bundle subdomain (promoter-binding domain) that interacts with the promoter and initiates DNA duplex melting. While bound to the promoter, the polymerase generates many short (truncated) transcripts, 2-12 nucleotides (nt) in length, a process often referred to as abortive synthesis / initiation. Truncated RNA transcripts cannot be converted into full-length transcripts by RNA polymerase and become by-products that accumulate during transcription. After transition to the elongation phase and release of the promoter, the polymerase works on the DNA template to generate full-length RNA transcripts.

[0004] During the elongation phase, RNA polymerase often continues to transcribe DNA beyond the position where termination should have begun, generating longer than expected RNA transcripts ("run-on transcripts"). T7 RNA polymerase, for example, adds nucleotides to the end of a transcript before it "falls off" from the template. Studies suggest that over 70% of transcripts generated in vitro by T7 RNA polymerase may be run-on transcripts. In some cases, these aberrant RNA products are twice as long as the encoded sequence. Because run-on transcription is stochastic, there is often a lot of 3' heterogeneity in the products in a given IVT reaction. This 3' heterogeneity is problematic for downstream applications such as ligation reactions that rely on RNA transcripts of defined length and / or nucleotide composition. Summary of the Invention

[0005] During transcription initiation, RNA polymerase balances two opposing steps. The polymerase must first bind to the promoter tightly enough to allow dissociation of the two DNA strands (one of which is the template strand) and initiate transcription. The polymerase must then release the promoter and enter a highly processive elongation step. Competition between these two steps results in the generation of abortive transcripts. The polymerase repeatedly attempts to remove the promoter but is unable to overcome the transition barrier, releasing a short (abortive) RNA product. Conversely, polymerases often do not "run off" the DNA template, generating a population of RNA transcripts with 3' heterogeneity. Provided herein are variant RNA polymerases, run-on transcripts, double-stranded contaminants, or any combination thereof, that increase transcription efficiency and 3' homogeneity generated in in vitro transcription (IVT) reactions, for example.

[0006] During the transition from initiation to elongation, RNA polymerase undergoes a conformational change that requires extensive rearrangements of the amino-terminal domain (N-terminal domain) (see, e.g., Bandwar, RP et al. Journal of Biological Chemistry 282, 22879-22886 (2009); Guillerez, J. et al. Proc National Acad Sci 102, 5958-5963 (2005); Durniak, K. et al. Science (New York, NY) 322, 553 (2008); and Tahirov, TH et al. Nature 420, 43-50 (2002), each of which is incorporated herein by reference). Within this N-terminal domain are the "C helix" (e.g., amino acids 28-71 of T7 RNA polymerase) and the "C linker" (e.g., amino acids 258-266 of T7 RNA polymerase), each of which contains a subregion (amino acids 42-47 and 257-262, respectively) that undergoes a conformational change from a loop to a helix structure in which the promoter binding site is abandoned, the active site is expanded, and an exit tunnel for the RNA transcript is created as the RNA polymerase transitions from the initiation complex to the elongation complex (see, e.g., FIG. 10). Mutations to the C helix subregion and / or the linker region of the C linker may drive the conformational equilibrium for the elongation complex by increasing the thermodynamic stability of the elongation complex relative to the initiation complex. Without being bound by theory, it is believed that mutations in select regions, such as the C-helix structure and / or the linker region of the C-linker, can alter how the exit tunnel of the polymerase interacts with the transcript during elongation, significantly reducing transcription errors (e.g., run-on transcripts). Thus, the variant polymerases of the present disclosure include (at least one) mutation in the C-helix and / or C-linker to drive conformational equilibrium for the elongation complex.

[0007] As provided herein, other regions of the N-terminal domain, including loop structures that transition to helical structures as the polymerase proceeds from initiation to elongation, can be mutated (referred to as substitutions, point mutations). Non-limiting examples of such loop-to-helix regions include regions spanning amino acids 55-73, 164-169, or 176-187 of T7 RNA polymerase (e.g., SEQ ID NO:1), or regions of other single-subunit RNA polymerases that are homologous (e.g., at least 80%, at least 90%, at least 95%, or at least 98% identical) to the aforementioned regions (see, e.g., Cermakian, N. et al. J Mol Evol 45, 671-681 (1997)). Non-limiting examples of other single-subunit RNA polymerases include T3 RNA polymerase, K11 RNA polymerase, and SP6 RNA polymerase.

[0008] In some embodiments, the RNA polymerase variant (e.g., a T7 RNA polymerase variant) contains mutations to residues that have high helical propensity (e.g., alanine) or that specifically match backbone flexibility to that of the elongation complex.

[0009] Further provided herein are RNA polymerase variants that include at least one additional amino acid at the C-terminus of the polymerase. For example, T7 RNA polymerase variants can include at least one additional amino acid, such as an additional glycine (G), at the C-terminus of the polymerase. Surprisingly, the C-terminal "foot" region (e.g., "FAFA" at positions 880-883 of wild-type T7 RNAP) can include at least one additional amino acid, such as an additional glycine (G) at the C-terminus of the polymerase. (SEQ ID NO:172) A population of RNA transcripts generated using T7 RNA polymerase modified to include an additional G at the C-terminal glycine (i.e., the region containing the amino acid ...FAFAG) shows less 3' heterogeneity. For example, as shown in Figure 23, (SEQ ID NO:329)T7 RNA polymerase variants, including those with the 3' end variant, generate a population of RNA transcripts in which at least 85% of the transcripts are homogeneous at their 3' ends. This data is particularly unexpected given that previous studies have shown that C-terminal additions / insertions abolish T7 polymerase function (Gardner LP, et al. Biochemistry 36, 2908-2918 (1997) and Gross L, et al. Journal of Molecular Biology 228, 488-505 (1992)).

[0010] Also provided herein are methods for co-transcriptionally capping ssRNA (e.g., mRNA) with cap analogs (e.g., trinucleotides) in in vitro transcription assays using the T7 RNA polymerase variants described herein (e.g., T7 RNA polymerase variant G47A or G47A* C-terminal variant). Efficient co-transcriptional capping typically involves a double-stranded DNA (dsDNA) template that initiates transcription using 5′ATP and equimolar concentrations of NTPs and trinucleotides. Under these conditions, T7 RNA polymerase has significantly reduced initiation activity with 5′ATP. Unexpectedly, the data provided herein demonstrate that GAG trinucleotides (e.g., m 7 GpppA 2′OMe In the presence of pG), for example, the limited initiation activity of T7 RNA polymerase with 5' ATP is driven by trinucleotides rather than 5' ATP, resulting in the co-transcriptional production of capped RNA. Surprisingly, in some embodiments, greater than 90% of the generated RNA comprises single-stranded full-length transcripts, at least 90% of the generated RNA contains a functional cap, and the generated RNA does not exhibit a substantial cytokine response after IVT without purification.

[0011] Thus, some aspects of the present disclosure provide an RNA polymerase variant comprising at least one amino acid substitution that causes at least one loop structure of the RNA polymerase variant to undergo a conformational change to a helical structure as the RNA polymerase variant transitions from an initiation complex to an elongation complex, as compared to a wild-type RNA polymerase. In some embodiments, the at least one amino acid substitution is estimated to cause a larger negative change in folding free energy in the elongation complex than in the initiation complex. In some embodiments, the at least one amino acid substitution has a higher helical propensity compared to the wild-type amino acid. In some embodiments, the RNA polymerase variant comprises (at least one) additional amino acid residue at the C-terminus. For example, the RNA polymerase variant (e.g., T7 RNA polymerase variant) may comprise a glycine (G) at the C-terminus.

[0012] In some embodiments, the RNA polymerase is T7 RNA polymerase. In some embodiments, the RNA polymerase is T3 RNA polymerase. In some embodiments, the RNA polymerase is SP6 RNA polymerase. In some embodiments, the RNA polymerase is K11 RNA polymerase.

[0013] In some embodiments, at least one loop structure is in a C-helix structure. In some embodiments, at least one loop structure is in a C-linker structure. In some embodiments, at least one loop structure is present at amino acids 55-73, 164-169, or 176-187 of T7 RNA polymerase, or a region within an RNA polymerase that is homologous to T7 RNA polymerase.

[0014] In some embodiments, the at least one amino acid substitution is at least one high helix propensity amino acid substitution. For example, the at least one high helix propensity amino acid substitution may be selected from alanine, isoleucine, leucine, arginine, methionine, lysine, glutamine, and / or glutamic acid. In some embodiments, the at least one high helix propensity amino acid substitution is alanine. In some embodiments, the at least one high helix propensity amino acid substitution is isoleucine. In some embodiments, the at least one high helix propensity amino acid substitution is leucine. In some embodiments, the at least one high helix propensity amino acid substitution is arginine. In some embodiments, the at least one high helix propensity amino acid substitution is methionine. In some embodiments, the at least one high helix propensity amino acid substitution is lysine. In some embodiments, the at least one high helix propensity amino acid substitution is glutamine. In some embodiments, the at least one high helix propensity amino acid substitution is glutamic acid.

[0015] In some embodiments, a T7 RNA polymerase is modified to include at least one amino acid substitution of a high helix-prone amino acid at at least one position selected from E42 (e.g., E42R), S43 (e.g., S43A), Y44 (e.g., Y44A), E45 (e.g., E45R / L), M46 (e.g., M46A), G47 (e.g., G47A), A255 (e.g., A255K / Q / Y / I), R257 (e.g., R257A), A258 (e.g., A258R / E / L), G259 (e.g., G259A), A260 (e.g., A260R / E / L), L261 (e.g., L261A), and A262 (e.g., A262R / E / L). A T7 RNA polymerase, in some embodiments, can further comprise one or more additional amino acid substitutions (in addition to at least one high-helix-propensity amino acid substitution). Thus, the present disclosure encompasses further modifications of existing (e.g., currently available and / or commercially available) T7 RNA polymerase variants with one or more high-helix-propensity amino acid substitutions provided herein.

[0016] In some embodiments, the T7 RNA polymerase comprises the amino acid sequence of SEQ ID NO:1 modified to include at least one amino acid substitution of a high helix-propensity amino acid at a position selected from E42 (e.g., E42R), S43 (e.g., S43A), Y44 (e.g., Y44A), E45 (e.g., E45R / L), M46 (e.g., M46A), and G47 (e.g., G47A). In some embodiments, the at least one amino acid substitution comprises S43A. In some embodiments, the at least one amino acid substitution comprises G47A.

[0017] In some embodiments, the T7 RNA polymerase comprises the amino acid sequence of SEQ ID NO:99, SEQ ID NO:100, SEQ ID NO:294, SEQ ID NO:295, or SEQ ID NO:296 modified to include at least one amino acid substitution of a high helix-propensity amino acid at a position selected from E42 (e.g., E42R), S43 (e.g., S43A), Y44 (e.g., Y44A), E45 (e.g., E45R / L), M46 (e.g., M46A), and G47 (e.g., G47A). In some embodiments, the at least one amino acid substitution comprises S43A. In some embodiments, the at least one amino acid substitution comprises G47A.

[0018] In some embodiments, the T7 RNA polymerase comprises the amino acid sequence of SEQ ID NO:1 modified to include at least one amino acid substitution of a high helix-propensity amino acid at a position selected from A255 (e.g., A255K / Q / Y / I), R257 (e.g., R257A), A258 (e.g., A258R / E / L), G259 (e.g., G259A), A260 (e.g., A260R / E / L), L261 (e.g., L261A), and A262 (e.g., A262R / E / L). In some embodiments, the at least one amino acid substitution comprises R257A. In some embodiments, the at least one amino acid substitution comprises G259A.

[0019] In some embodiments, the T7 RNA polymerase comprises the amino acid sequence of SEQ ID NO:99, SEQ ID NO:100, SEQ ID NO:294, SEQ ID NO:295, or SEQ ID NO:296 modified to include at least one amino acid substitution of a high helix-propensity amino acid at a position selected from A255 (e.g., A255K / Q / Y / I), R257 (e.g., R257A), A258 (e.g., A258R / E / L), G259 (e.g., G259A), A260 (e.g., A260R / E / L), L261 (e.g., L261A), and A262 (e.g., A262R / E / L). In some embodiments, the at least one amino acid substitution comprises R257A. In some embodiments, the at least one amino acid substitution comprises G259A.

[0020] In some embodiments, also provided herein is a T7 RNA polymerase comprising the amino acid sequence of SEQ ID NO:1 modified to include an amino acid substitution of a high helical propensity amino acid (e.g., alanine, isoleucine, leucine, arginine, methionine, lysine, glutamine, and / or glutamic acid) at position G47, S43, R257, or G259. In some embodiments, further provided is a T7 RNA polymerase comprising the amino acid sequence of SEQ ID NO:99, SEQ ID NO:100, SEQ ID NO:294, SEQ ID NO:295, or SEQ ID NO:296 modified to include an amino acid substitution of a high helical propensity amino acid (e.g., alanine, isoleucine, leucine, arginine, methionine, lysine, glutamine, and / or glutamic acid) at position G47, S43, R257, or G259.

[0021] In some embodiments, a T7 RNA polymerase of this disclosure comprises the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5. In some embodiments, a T7 RNA polymerase of this disclosure comprises the amino acid sequence of SEQ ID NO: 107 or 108, SEQ ID NO: 109 or 110, SEQ ID NO: 111 or 112, or SEQ ID NO: 113 or 114.

[0022] In some embodiments, the T7 RNA polymerase comprises at least one additional C-terminal amino acid. In some embodiments, the T7 RNA polymerase comprises at least two additional C-terminal amino acids.

[0023] In some embodiments, the at least two additional C-terminal amino acids comprise the same type of amino acid (e.g., all Gly, all Ala). In some embodiments, the at least two additional C-terminal amino acids comprise at least two different types of amino acids (e.g., GlyAla, AlaGly).

[0024] In some embodiments, the T7 RNA polymerase comprises at least three additional C-terminal amino acids, in some embodiments, the at least three additional C-terminal amino acids comprise at least two or at least three of the same or different types of amino acids (e.g., GlyGlyGly, AlaAlaAla).

[0025] In some embodiments, the T7 RNA polymerase comprises between 1 and 10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) additional C-terminal amino acids. In some embodiments, the T7 RNA polymerase comprises between 1 and 5 additional C-terminal amino acids.

[0026] In some embodiments, the T7 RNA polymerase is FAFAX n (SEQ ID NO:171) In some embodiments, X is glycine (G). In some embodiments, n is 1, 2, 3, 4, or 5. In some embodiments, n is greater than 1, and thus the C-terminal motif is, for example, FAFXX, FAFAXXX. (SEQ ID NO:319) , FAFAXXXX (SEQ ID NO:320) , or FAFAXXXXX (SEQ ID NO:321) It should be understood that in embodiments where, for example, X' can be the same amino acid or a different amino acid. For example, the C-terminal motif is FAFAGG (SEQ ID NO:322) Or FAFAGGG (SEQ ID NO:323) or the C-terminal motif may be FAFAGA (SEQ ID NO:324) , FAFAGC (SEQ ID NO:325) , FAFAGAA (SEQ ID NO:326) , FAFAGAG (SEQ ID NO:327) , FAFAGAC (SEQ ID NO:328) etc. Other combinations of C-terminal amino acids may be used.

[0027] In some embodiments, the T7 RNA polymerase is FAFAG (SEQ ID NO:329) The C-terminus contains the motif.

[0028] In some embodiments, the T7 RNA polymerase is XAFAX n Motif, FXFAX n Motif, FAXAX n Motif, or FAFXX n The present disclosure includes a variety of C-terminal, F-terminal, and C-terminal motifs, where each X is any amino acid and n is any integer greater than zero. 880 A 881 F 882 A 883 (SEQ ID NO:172) motif, where one or more of amino acids at positions 880, 881, 882, or 883 (e.g., as compared to wild-type T7 RNAP, e.g., SEQ ID NO: 1) are replaced with an additional C-terminal amino acid (X n ), has been modified to contain at least one amino acid substitution, with or without

[0029] In some aspects, the present disclosure provides an RNA polymerase that comprises at least one additional C-terminal amino acid compared to a corresponding wild-type RNA polymerase. In some embodiments, the RNA polymerase is selected from T7 RNA polymerase, T3 RNA polymerase, and SP6 RNA polymerase.

[0030] In some embodiments, the RNA polymerase further comprises at least one additional amino acid substitution, hi some embodiments, the RNA polymerase further comprises an amino acid substitution corresponding to an amino acid substitution in SEQ ID NO:1 selected from G47A, S43A, R257A, and G259A.

[0031] In some embodiments, the RNA polymerase is a T7 RNA polymerase comprising an amino acid sequence at least 90%, at least 95%, at least 98%, or at least 99% identical to SEQ ID NO:1, modified to include an amino acid at position 43±1 (e.g., 42, 43, or 44), 47±1 (e.g., 46, 47, or 48), 257±1 (e.g., 256, 257, or 258), and / or 259±1 (e.g., 258, 259, or 260), optionally where the amino acid substitution is an alanine (A). In some embodiments, the RNA polymerase is a T3 RNA polymerase comprising an amino acid sequence at least 90%, at least 95%, or at least 98% identical to SEQ ID NO:6, modified to include amino acid substitutions at positions corresponding to positions 43±1, 47±1, 257±1, and / or 259±1 (based on sequence or structural alignment) of wild-type T7 RNA polymerase, optionally where the amino acid substitution is alanine (A). In some embodiments, the RNA polymerase is a SP6 RNA polymerase comprising an amino acid sequence at least 90%, at least 95%, at least 98%, or at least 99% identical to SEQ ID NO:7, modified to include amino acid substitutions at positions corresponding to positions 43±1, 47±1, 257±1, and / or 259±1 (based on sequence or structural alignment) of wild-type T7 RNA polymerase, optionally where the amino acid substitution is alanine (A).

[0032] Some embodiments of the present disclosure provide a T7 RNA polymerase comprising an amino acid sequence at least 90%, at least 95%, at least 98%, or at least 99% identical to SEQ ID NO:1, modified to include an amino acid substitution at position 43, 47, 257, and / or 259, optionally wherein the amino acid substitution is an alanine (A).

[0033] Another aspect of the disclosure provides a T3 RNA polymerase comprising an amino acid sequence at least 90%, at least 95%, or at least 98% identical to SEQ ID NO:6, modified to include amino acid substitutions at positions corresponding to positions 43, 47, 257, and / or 259 of wild-type T7 RNA polymerase, optionally wherein the amino acid substitution is alanine (A). Thus, in some embodiments, a T3 RNA polymerase comprises an amino acid sequence at least 90%, at least 95%, or at least 98% identical to SEQ ID NO:6, modified to include amino acid substitutions at positions 44, 48, 258, and / or 260, optionally wherein the amino acid substitution is alanine (A).

[0034] Yet other aspects of the disclosure provide an SP6 RNA polymerase comprising an amino acid sequence at least 90%, at least 95%, or at least 98% identical to SEQ ID NO:7, modified to include amino acid substitutions at positions corresponding to positions 43, 47, 257, and / or 259 of wild-type T7 RNA polymerase, optionally wherein the amino acid substitution is alanine (A). Thus, in some embodiments, an SP6 TNA polymerase comprises an amino acid sequence at least 90%, at least 95%, or at least 98% identical to SEQ ID NO:7, modified to include amino acid substitutions at positions 15, 19, 230, and / or 232, optionally wherein the amino acid substitution is alanine (A).

[0035] The present disclosure also provides a method of producing RNA comprising contacting a DNA template with an RNA polymerase variant described herein under conditions that result in the production of an RNA transcript (eg, IVT conditions).

[0036] The present disclosure further provides a method of performing an IVT reaction, comprising contacting a DNA template with an RNA polymerase variant provided herein in the presence of nucleoside triphosphates (NTPs) and a buffer under conditions that result in the production of an RNA transcript.

[0037] In some embodiments, the generated RNA transcripts, when delivered to cells, optionally in unpurified form, stimulate at least 50% (e.g., at least 60%, at least 70%, at least 80%, at least 95%, or at least 98%) less cytokine response compared to RNA generated using a wild-type RNA polymerase under the same IVT conditions.

[0038] In some embodiments, the concentration of dsRNA transcripts produced by IVT is at least 50% (e.g., at least 60%, at least 70%, at least 80%, at least 95%, or at least 98%) lower compared to dsRNA transcripts produced using a wild-type polymerase.

[0039] In some embodiments, less than 20% (eg, less than 15%, less than 10%, less than 5%) of the RNA transcripts produced exhibit 3' heterogeneity.

[0040] In some embodiments, less than 50% (eg, less than 40%, less than 30%, less than 20%, less than 10%) of the RNA transcripts produced are truncated RNA transcripts.

[0041] In some embodiments, less than 50% (eg, less than 40%, less than 30%, less than 20%, less than 10%) of the RNA transcripts produced are run-on RNA transcripts.

[0042] In some embodiments, the amount of full-length RNA transcript produced is at least 15-fold greater than the amount of the DNA template.

[0043] In some embodiments, the ratio of truncated RNA transcripts to full-length RNA transcripts produced is less than 1:1.

[0044] In some embodiments, the RNA transcripts produced have less than 1 mutation per 100 nucleotides compared to the DNA template.

[0045] The disclosure provides, in some aspects, nucleic acids encoding RNA polymerase variants, and in some embodiments, vectors (e.g., plasmids) and / or host cells (e.g., mammalian cells, e.g., human cells) comprising the nucleic acids.

[0046] Also provided are RNA transcripts produced by the methods of the present disclosure. In some embodiments, the RNA transcripts (e.g., mRNA) are formulated in lipid nanoparticles. The lipid nanoparticles may include, for example, a molar ratio of 20-60% ionizable amino lipid, 5-25% non-cationic lipid, 25-55% sterol, and 0.5-15% PEG-modified lipid. See, for example, WO2017 / 070624, published April 27, 2017, which is incorporated herein by reference.

[0047] Other compositions and kits that include the RNA polymerase variants are encompassed herein.

[0048] Also provided herein is a co-transcriptional capping method for ribonucleic acid (RNA) synthesis, comprising reacting a polynucleotide template with a T7 RNA polymerase variant, a nucleoside triphosphate, and a cap analog under in vitro transcription reaction conditions to produce an RNA transcript.

[0049] In some embodiments, greater than 80%, greater than 85%, or greater than 90% of the RNA transcripts produced contain a functional cap, in some embodiments, greater than 95% of the RNA transcripts produced contain a functional cap.

[0050] In some embodiments, the nucleoside triphosphates comprise unmodified or modified ATP, modified or unmodified UTP, modified or unmodified GTP, and / or modified or unmodified CTP.

[0051] In some embodiments, the T7 polymerase variant comprises the amino acid sequence of SEQ ID NO:1 (or an amino acid sequence having 90% to 99%, e.g., at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, identity to SEQ ID NO:1), modified to include at least one amino acid substitution of a high propensity amino acid at a position selected from E42, S43, Y44, E45, M46, G47, A255, R257, and G259. In some embodiments, the T7 polymerase variant comprises the amino acid sequence of SEQ ID NO:1 (or an amino acid sequence having 90% to 99%, e.g., at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, identity to SEQ ID NO:1), modified to include an amino acid substitution of G47A. In some embodiments, the T7 polymerase variant comprises the amino acid sequence of SEQ ID NO:1 (or an amino acid sequence having 90% to 99%, e.g., at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, identity to SEQ ID NO:1), modified to include the S43A amino acid substitution.

[0052] In some embodiments, the T7 polymerase variant comprises the amino acid sequence of SEQ ID NO: 99 (or an amino acid sequence having 90% to 99%, e.g., at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, identity to SEQ ID NO: 99), modified to include at least 99 amino acid substitutions of high propensity amino acids at positions selected from E42, S43, Y44, E45, M46, G47, A255, R257, and G259. In some embodiments, the T7 polymerase variant comprises the amino acid sequence of SEQ ID NO: 99 (or an amino acid sequence having 90% to 99%, e.g., at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, identity to SEQ ID NO: 99), modified to include an amino acid substitution of G47A. In some embodiments, the T7 polymerase variant comprises the amino acid sequence of SEQ ID NO: 99 (or an amino acid sequence having 90% to 99%, e.g., at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, identity to SEQ ID NO: 99), modified to include an S43A amino acid substitution.

[0053] In some embodiments, the T7 polymerase variant comprises the amino acid sequence of SEQ ID NO: 100 (or an amino acid sequence having 90% to 99%, e.g., at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, identity to SEQ ID NO: 100), modified to include at least 100 amino acid substitutions of high propensity amino acids at positions selected from E42, S43, Y44, E45, M46, G47, A255, R257, and G259. In some embodiments, the T7 polymerase variant comprises the amino acid sequence of SEQ ID NO: 100 (or an amino acid sequence having 90% to 99%, e.g., at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, identity to SEQ ID NO: 100), modified to include an amino acid substitution of G47A. In some embodiments, the T7 polymerase variant comprises the amino acid sequence of SEQ ID NO:100 (or an amino acid sequence having 90% to 99%, e.g., at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, identity to SEQ ID NO:100), modified to include an S43A amino acid substitution.

[0054] In some embodiments, the nucleoside triphosphate and the cap analog are present in equimolar concentrations in the reaction. In some embodiments, the molar ratio of the cap analog to the nucleoside triphosphate in the reaction is greater than 1:1. In some embodiments, the molar ratio of the cap analog to the nucleoside triphosphate in the reaction is less than 1:1.

[0055] In some embodiments, the cap analog is a dinucleotide cap, a trinucleotide cap, or a tetranucleotide cap, hi some embodiments, the cap analog is a trinucleotide cap.

[0056] In some embodiments, the trinucleotide cap comprises a sequence selected from the following sequences: GAA, GAC, GAG, GAU, GCA, GCC, GCG, GCU, GGA, GGC, GGG, GGU, GUA, GUC, GUG, and GUU.

[0057] In some embodiments, the trinucleotide cap has the following sequence: 7 GppApA, m 7 GpppApC, m 7 GppApG, m 7 GppApU,m 7 GppCpA, m 7 GppCpC, m 7 GppCpG, m 7 GppCpU,m 7 GppGpA, m 7 GppGpC, m 7 GppGpG, m 7 GppGpU,m 7 GpppUpA,m 7 GpppUpC,m 7 GpppUpG, and m 7 GpppUpU.

[0058] In some embodiments, the trinucleotide cap has the following sequence: 7 G 3′OMe pppApA, m 7 G 3′OMe pppApC, m 7 G 3′OMe pppApG, m 7 G 3′OMe pppApU,m 7 G 3′OMe pppCpA, m 7 G 3′OMe pppCpC, m 7 G 3′OMe pppCpG,m 7 G 3′OMe pppCpU,m 7 G 3′OMe pppGpA,m 7 G 3′OMe pppGpC, m 7 G 3′OMepppGpG, m 7 G 3′OMe pppGpU,m 7 G 3′OMe pppUpA,m 7 G 3′OMe pppUpC,m 7 G 3′OMe pppUpG, and m 7 G 3′OMe pppUpU.

[0059] In some embodiments, the trinucleotide cap has the following sequence: 7 G 3′OMe pppA 2′OMe pA, m 7 G 3′OMe pppA 2′OMe pC, m 7 G 3′OMe pppA 2′OMe pG,m 7 G 3′OMe pppA 2′OMe pU,m 7 G 3′OMe pppC 2′OMe pA, m 7 G 3′OMe pppC 2′OMe pC, m 7 G 3′OMe pppC 2′OMe pG,m 7 G 3′OMe pppC 2′OMe pU,m 7 G 3′OMe pppG 2′OMe pA, m 7 G 3′OMe pppG 2′OMe pC, m 7 G 3′OMe pppG 2′OMe pG,m 7 G 3′OMe pppG 2′OMe pU,m 7 G 3′OMe pppU 2′OMe pA, m 7 G 3′OMe pppU 2′OMe pC, m 7 G 3′OMe pppU 2′OMe pG, and m 7G 3′OMe pppU 2′OMe pU.

[0060] In some embodiments, the trinucleotide cap has the following sequence: 7 GpppA 2′OMe pA, m 7 GpppA 2′OMe pC, m 7 GpppA 2′OMe pG,m 7 GpppA 2′OMe pU,m 7 GpppC 2′OMe pA, m 7 GpppC 2′OMe pC, m 7 GpppC 2′OMe pG,m 7 GpppC 2′OMe pU,m 7 GpppG 2′OMe pA, m 7 GpppG 2′OMe pC, m 7 GpppG 2′OMe pG,m 7 GpppG 2′OMe pU,m 7 GpppU 2′OMe pA, m 7 GpppU 2′OMe pC, m 7 GpppU 2′OMe pG, and m 7 GpppU 2′OMe pU.

[0061] In some embodiments, the trinucleotide cap comprises a sequence selected from the following sequences: GAG, GCG, GUG, and GGG. In some embodiments, the trinucleotide cap comprises the sequence GAG. In some embodiments, the trinucleotide cap comprises the sequence 7 GpppA 2′Ome Contains pG.

[0062] In some embodiments, the polynucleotide template comprises a 2'-deoxythymidine residue at template position +1. In some embodiments, the polynucleotide template comprises a 2'-deoxycytidine residue at template position +1. In some embodiments, the polynucleotide template comprises a 2'-deoxyadenosine residue at template position +1. In some embodiments, the polynucleotide template comprises a 2'-deoxyguanosine residue at template position +1.

[0063] A co-transcriptional capping method for RNA synthesis, comprising: coupling a polynucleotide template to (a) a T7 RNA polymerase variant, the T7 RNA polymerase variant comprising at least one amino acid substitution, as compared to a wild-type RNA polymerase, that causes at least one loop structure of the RNA polymerase variant to undergo a conformational change to a helical structure as the RNA polymerase variant transitions from an initiation complex to an elongation complex; (b) a nucleoside triphosphate; and (c) a GpppA sequence, under in vitro transcription reaction conditions to produce an RNA transcript. 2′Ome Also provided herein are methods comprising reacting a polynucleotide template with a trinucleotide cap that contains pG, wherein the polynucleotide template contains a 2'-deoxythymidine residue at template position +1.

[0064] In some embodiments, the generated RNA transcripts, when delivered to cells, optionally in an unpurified form, do not stimulate a detectable cytokine response.

[0065] Further provided herein is a composition comprising in vitro transcribed (IVT) RNA and a pharma- ceutically acceptable excipient, the composition being substantially free of cytokine-induced RNA contaminants without post-IVT purification.

[0066] The composition, in some embodiments, comprises an IVT RNA and a pharma- ceutically acceptable excipient, wherein the composition has less than 5% uncapped RNA species.

[0067] In some embodiments, more than 80%, more than 85%, or more than 90% of the IVT RNA comprises a functional cap, in some embodiments, more than 95% of the IVT RNA comprises a functional cap.

[0068] In some embodiments, the IVT RNA is not chemically modified, hi other embodiments, the IVT RNA is chemically modified.

[0069] In some embodiments, greater than 95% of the IVT RNA comprises a single-stranded full-length transcript.

[0070] In some embodiments, the RNA is prepared by subjecting a polynucleotide template to in vitro transcription reaction conditions to generate an RNA transcript, comprising: (a) a T7 RNA polymerase variant, the T7 RNA polymerase variant comprising at least one amino acid substitution, relative to a wild-type RNA polymerase, that causes at least one loop structure of the RNA polymerase variant to undergo a conformational change to a helical structure as the RNA polymerase variant transitions from an initiation complex to an elongation complex; (b) a nucleoside triphosphate; and (c) a sequence GpppA 2′Ome The polynucleotide template contains a 2'-deoxythymidine residue at template position +1, produced by a process that includes reacting with a pG-containing trinucleotide cap.

[0071] In some aspects, the disclosure provides a T7 RNAP variant comprising an amino acid sequence of SEQ ID NO:294-313, where x is any amino acid and n is any integer, e.g., 1-5 (e.g., 1, 2, 3, 4, or 5).

[0072] In some aspects, the present disclosure provides a method of performing an IVT reaction, comprising contacting a DNA template with an RNA polymerase variant of the present disclosure in the presence of nucleoside triphosphates and a buffer under conditions that result in the production of RNA transcripts. In some embodiments, the generated RNA stimulates at least 50% lower cytokine response compared to dsRNA transcripts generated using wild type when delivered to cells, optionally in unpurified form. In some embodiments, less than 30% of the RNA transcripts generated by the IVT reaction exhibit 3' heterogeneity. [Brief description of the drawings]

[0073] [Figure 1] HPLC chromatograms at 260 nm of human erythropoietin (hEPO) mRNA produced using wild-type (WT) T7 polymerase or T7 polymerase variants G47A* (with a C-terminal G) and S43A* (with a C-terminal G) are shown. [Diagram 2] The left panel shows a graph depicting IFNβ responses in BJ fibroblasts transfected with unmodified hEPO RNA transcripts generated using WT T7 RNA polymerase with or without reverse phase (RP) purification or one of the T7 RNA polymerase variants S43A* (with a C-terminal G) or G47A* (with a C-terminal G). The right panel shows a graph depicting hEPO expression in the transfected cells. [Diagram 3]The top panel shows a graph depicting IFNβ responses in BJ fibroblasts transfected with chemically modified hEPO RNA transcripts (N1-methylpseudouracil (m1ψ) modified) produced using WT T7 RNA polymerase with or without reverse phase (RP) purification or one of the T7 RNA polymerase variants S43A* (with a C-terminal G) or G47A* (with a C-terminal G). The bottom panel shows a graph depicting IFNβ responses in cells transfected with chemically modified hEPO RNA transcripts (5-methoxy-uridine (mo5U) modified) produced using WT T7 RNA polymerase with or without reverse phase (RP) purification or one of the T7 RNA polymerase variants S43A* (with a C-terminal G) or G47A* (with a C-terminal G). [Figure 4] The top and bottom panels show graphs depicting hEPO expression in cells transfected with the hEPO RNA transcripts used in Figure 3. N1-methylpseudouracil (m1ψ)-modified hEPO expression is shown in the top graph, and 5-methoxy-uridine (mo5U)-modified hEPO expression is shown in the bottom graph. [Diagram 5] 1 shows a graph depicting IP10 responses in monocyte-derived macrophages transfected with chemically modified hEPO RNA transcripts produced using WT T7 RNA polymerase or T7 RNA polymerase variants S43A* (with a C-terminal G) or G47A* (with a C-terminal G). [Figure 6]The top panel shows a graph depicting IP10 responses in monocyte-derived macrophages transfected with chemically modified hEPO RNA transcripts (N1-methylpseudouracil (m1ψ)) generated using WT T7 RNA polymerase or T7 RNA polymerase variants S43A* (with a C-terminal G) or G47A* (with a C-terminal G). The bottom panel shows a graph depicting IP10 responses in monocyte-derived macrophages transfected with chemically modified hEPO RNA transcripts (5-methoxy-uridine (mo5U)) generated using WT T7 RNA polymerase or T7 RNA polymerase variants S43A* (with a C-terminal G) or G47A* (with a C-terminal G). [Figure 7] Graphs are shown showing the concentration of contaminating double-stranded (ds) RNA detected using dsRNA ELISA using 1 μg of chemically unmodified hEPO RNA transcript (left) or 5 μg of chemically unmodified hEPO RNA transcript produced by IVT reactions using WT T7 RNA polymerase or T7 RNA polymerase variants S43A* (with a C-terminal G) or G47A* (with a C-terminal G). [Figure 8] FIG. 1 shows a graph depicting the concentration of contaminating dsRNA detected using dsRNA ELISA with chemically modified hEPO RNA transcripts (N1-methylpseudouracil (m1ψ) modified, top; 5-methoxy-uridine (mo5U) modified, bottom). [Figure 9A] 1 shows mass chromatogram results from RNase T1 tail digestion of hEPO RNA transcripts generated using WT T7 RNA polymerase or T7 RNA polymerase variants S43A* (with a C-terminal G) or G47A* (with a C-terminal G). LCMS analysis results are shown. [Figure 9B]9A shows mass chromatogram results from RNase T1 tail digestion of hEPO RNA transcripts generated using WT T7 RNA polymerase or T7 RNA polymerase variants S43A* (with a C-terminal G) or G47A* (with a C-terminal G). Quantification of the 3' end population distribution of FIG. 9A is shown. [Figure 10] A schematic showing the C-helix and C-linker loop structures changing conformation to C-helix and C-loop helix as T7 RNA polymerase transitions from the initiation complex to the elongation complex. Generated from PDB crystal structures 1MSW and 1QLN and rendered in the Molecular Operating Environment [Chemical Computing Group ULC]. [Figure 11A] A schematic diagram is shown of the ligation of the "leftmer" RNA transcript generated by IVT to a "rightmer" fluorescently labeled polyA signal using a DNA splint in the presence of ligase. Even a single nucleotide overhang at the ligation site effectively abolishes ligation. [Figure 11B] 1 is a PAGE-D gel showing ligation efficiency of lightmers generated with WT T7 RNA polymerase and T7 RNA polymerase variants G47A* (with a C-terminal G) or S43A* (with a C-terminal G). [Figure 12] Shown is a radioactive gel in which 32P-GTP was used to label the abortive transcript or 32P-CTP to label the reverse complement. IVT was performed using short model transcripts. The data demonstrates that the T7 RNA polymerase variants S43A* (with a C-terminal G) and G47A* (with a C-terminal G) reduce reverse complement formation. [Figure 13] FIG. 1 is a schematic diagram of a conventional method for producing capped mRNA by in vitro transcription. [Figure 14A]Graph showing the results of a co-transcriptional capping assay using either wild-type T7 RNA polymerase or a T7 RNA polymerase variant (G47A*) of the present disclosure. Examples of dinucleotide caps (vaccinia cap1) or trinucleotide caps (GAG or GmAG) are shown. The co-transcriptional capping assay was used in a trinucleotide cap assay. [Figure 14B] 1 is a graph showing the results of a co-transcriptional capping assay using either wild-type T7 RNA polymerase or a T7 RNA polymerase variant of the disclosure (G47A*). 1 shows the mRNA yield of the co-transcriptional capping assay. [Figure 14C] 1 is a graph showing the results of a co-transcriptional capping assay using either wild-type T7 RNA polymerase or a T7 RNA polymerase variant (G47A*) of the present disclosure, demonstrating high integrity of mRNA produced in the co-transcriptional capping assay. [Figure 14D] 1 is a graph showing the results of a co-transcriptional capping assay using either wild-type T7 RNA polymerase or a T7 RNA polymerase variant (G47A*) of the present disclosure, which shows that capped mRNAs generated by WT T7 RNA polymerase induced cytokine production in BJ fibroblasts, whereas capped mRNAs generated by the T7 RNA polymerase variant G47A* (with a C-terminal G) surprisingly did not induce cytokine production. [Figure 14E]1 is a graph showing the results of a co-transcriptional capping assay using either wild-type T7 RNA polymerase or the T7 RNA polymerase variant (G47A*) of the present disclosure. Capped mRNA produced by WT T7 RNA polymerase did not express the encoded protein (hEPO) in BJ fibroblasts, while capped mRNA produced by T7 RNA polymerase variant G47A* (with C-terminal G) resulted in hEPO expression levels comparable to the control. The control is mRNA produced by WT T7 RNA polymerase and capped with vaccinia cap 1. [Figure 15] Figure 1 shows the results of LC-mass spectrometry of mRNAs generated in a co-transcriptional capping assay. The results unexpectedly show that the T7 RNA polymerase variant G47A* (with a C-terminal G) generated cleaner mRNAs than WT T7 RNA polymerase. The rate at which trinucleotides were incorporated appeared to be comparable for both enzymes. [Figure 16A] FIG. 13 is a graph comparing the cytokine responses of mRNA generated in a co-transcriptional capping assay and capped with a GAG trinucleotide or mRNA capped in a standard capping assay using vaccinia cap1. [Figure 16B] 1 is a graph comparing expression of mRNA generated in a co-transcriptional capping assay and capped with a GAG trinucleotide or mRNA capped in a standard capping assay using vaccinia cap1. [Figure 17] FIG. 1 is a schematic diagram showing the co-transcriptional capping assay described herein using T7 RNA polymerase variant G47A* and the trinucleotide cap m7GpppA2′OmepG. [Figure 18A] 1 shows liquid chromatography mass spectrometry (LCMS) results comparing the capping efficiency of mRNA beginning with 5′ ATP or 5′ GTP. Analysis of intact mRNA is shown. [Figure 18B]Liquid chromatography mass spectrometry (LCMS) results showing the comparison of capping efficiency of mRNA beginning with 5'ATP or 5'GTP. Analysis of the 5' end of mRNA truncated by RNase H is shown. [Figure 19A] 1 is a graph showing analysis of 1-methylpseudouridine chemically modified mRNAs generated from PCR fragment templates of three model constructs (hEPO, luciferase, and eGFP), which are shown to have the same sequence as analyzed by RNaseT1 fingerprinting assay. [Figure 19B] 1 is a graph showing analysis of mRNA chemically modified with 1-methylpseudouridine generated from PCR fragment templates of three model constructs (hEPO, luciferase, and eGFP), demonstrating that the mRNA has a high degree of integrity. [Figure 19C] 1 is a graph showing analysis of 1-methylpseudouridine chemically modified mRNA generated from PCR fragment templates of three model constructs (hEPO, luciferase, and eGFP), showing that the mRNA did not induce a cytokine response in BJ fibroblasts. [Figure 20A] Graph showing analysis of mRNA chemically modified with 1-methylpseudouridine generated from plasmid templates of three model constructs (hEPO, Luc, and eGFP).NTP consumption in a co-transcriptional capping assay using hEPO plasmid as template is shown. [Figure 20B] Graph showing analysis of mRNA chemically modified with 1-methylpseudouridine generated from plasmid templates of three model constructs (hEPO, Luc, and eGFP).NTP consumption in a co-transcriptional capping assay using eGFP plasmid as template is shown. [Figure 20C] 1 is a graph showing analysis of mRNA chemically modified with 1-methylpseudouridine generated from plasmid templates of three model constructs (hEPO, Luc, and eGFP), demonstrating high integrity of the mRNA products. [Figure 20D] 1 is a graph showing analysis of mRNA chemically modified with 1-methylpseudouridine produced from plasmid templates of three model constructs (hEPO, Luc, and eGFP).Cytokine response of mRNA products is shown. [Figure 20E] 1 is a graph showing analysis of mRNA chemically modified with 1-methylpseudouridine generated from plasmid templates of three model constructs (hEPO, Luc, and eGFP).Figure 2 shows expression of mRNA encoding hEPO in BJ fibroblasts. [Figure 20F] Graph showing analysis of mRNA chemically modified with 1-methylpseudouridine generated from plasmid templates of three model constructs (hEPO, Luc, and eGFP).Expression of mRNA encoding luciferase (Luc) in BJ fibroblasts. [Figure 20G] Graph showing analysis of mRNA chemically modified with 1-methylpseudouridine generated from plasmid templates of three model constructs (hEPO, Luc, and eGFP).Expression of mRNA encoding eGFP in BJ fibroblasts. [Figure 21] 1 is a graph showing that greater than 85% (e.g., about 90%) of mRNA transcripts produced using the T7 RNAP variant G47A*, which contains a C-terminal glycine (G), have a hydroxyl group at the 3′ end. [Figure 22] Figure 1 shows unmodified hEPO mRNA production from IVT reactions using WT or G47A T7 RNAP variants, some of which contain additional amino acids at the C-terminus (e.g., one or two glycines, or two alanines). [Figure 23] 1 shows 1-methyl-pseudouridine-modified hEPO mRNA production from IVT reactions containing the indicated WT, G47A, or S43A / G47A T7 RNAP variants, some of which have additional amino acids at the C-terminus (e.g., one or two glycines, or two alanines). [Figure 24A]FIG. 1 shows the results of RNAse T1 tail digests of hEPO mRNA transcripts produced using the indicated WT, G47A, or S43A / G47A T7 RNAP variants, some of which have additional amino acids (e.g., one or two glycines, or two alanines) at the C-terminus. FIG. 1 shows the results of LCMS analysis of hEPO mRNA produced from IVT reactions using WT T7 RNAP with equimolar concentrations of NTPs (WT EQ), WT T7 RNAP with excess GTP and ATP (WT alpha), or G47A* T7 RNAP with an additional C-terminal glycine and equimolar concentrations of NTPs (G47A* EQ). [Figure 24B] 24A shows the results of RNAse T1 tail digests of hEPO mRNA transcripts generated using the indicated WT, G47A, or S43A / G47A T7 RNAP variants, some of which have additional amino acids (e.g., one or two glycines, or two alanines) at the C-terminus. Quantification of the 3'-end population distribution is shown in FIG. 24B. [Figure 24C] 1 shows the results of RNAse T1 tail digests of hEPO mRNA transcripts produced using the indicated WT, G47A, or S43A / G47A T7 RNAP variants, some of which have additional amino acids (e.g., one or two glycines, or two alanines) at the C-terminus. The percentage of homogenous 3'-end populations produced in IVT reactions using unmodified mRNA is shown. [Figure 24D] 1 shows the results of RNAse T1 tail digests of hEPO mRNA transcripts produced using the indicated WT, G47A, or S43A / G47A T7 RNAP variants, some of which have additional amino acids (e.g., one or two glycines, or two alanines) at the C-terminus. The percentage of homogenous 3'-end populations produced in IVT reactions using 1-methyl-pseudouridine modified mRNA is shown. [Figure 25A]FIG. 1 shows a graph comparing the cytokine response of BJ fibroblasts to unmodified mRNA generated from IVT reactions containing the indicated WT, G47A, or S43A / G47A T7 RNAP variants, some of which have substitutions at the C-terminus. [Figure 25B] Graphs comparing the cytokine responses of BJ fibroblasts to 1-methyl-pseudouridine modified mRNAs generated from IVT reactions containing the indicated WT, G47A, or S43A / G47A T7 RNAP variants, some of which have substitutions at the C-terminus. [Figure 26A] Radiolabeled mRNA generated from IVT reactions containing the indicated WT or G47A T7 RNAP variants, some of which have additional amino acids at the C-terminus. IVT mRNA products are separated by size on a denaturing polyacrylamide gel. Either unmodified or 1-methyl-pseudouridine-modified mRNA was radiolabeled with 32P-CTP. [Figure 26B] Radiolabeled mRNA generated from IVT reactions containing the indicated WT or G47A T7 RNAP variants, some of which have additional amino acids at the C-terminus. IVT mRNA products are separated by size on a denaturing polyacrylamide gel. Reverse complement mRNA is radiolabeled with 32P-CTP. [Figure 27A] 1 shows a graph of data demonstrating that trinucleotide-capped G47A* mRNA encoding firefly luciferase (ffLuc) induces similar IP-10 serum cytokine levels in vivo as mRNA controls on day 1. [Figure 27B] 1 shows a graph of data demonstrating that trinucleotide-capped G47A* mRNA encoding firefly luciferase (ffLuc) induces similar IP-10 serum cytokine levels in vivo as mRNA controls at day 29. [Figure 28A]1 shows a graph of data demonstrating that trinucleotide-capped G47A* mRNA encoding ffLuc induces similar in vitro baseline cytokine levels in BJ fibroblasts (BJF) as mRNA controls. [Figure 28B] 1 shows a graph of data demonstrating that trinucleotide-capped G47A* mRNA encoding ffLuc induces similar baseline cytokine levels in vitro as mRNA controls in monocyte-derived macrophages (MDMs). [Figure 29] 13 shows a graph of data demonstrating that trinucleotide-capped G47A* mRNA encoding ffLuc maintains high expression in vivo following six weekly administrations. [Diagram 30] 1 shows a graph of data demonstrating that trinucleotide-capped G47A* mRNA encoding ffLuc induces low anti-PEG IgM levels after six weekly doses. [Diagram 31] 1 shows a graph of data demonstrating that trinucleotide-capped G47A* mRNA encoding ffLuc shows similar low B cell activation as the mRNA control. [Figure 32A] 1 shows a graph of data demonstrating that trinucleotide-capped G47A* mRNA encoding human erythropoietin (hEPO) induces similar IP-10 serum cytokine levels in vivo on day 1 as mRNA controls. [Figure 32B] 1 shows a graph of data demonstrating that trinucleotide-capped G47A* mRNA encoding human erythropoietin (hEPO) induces similar IP-10 serum cytokine levels in vivo as mRNA controls at day 22. [Figure 33A] 1 shows a graph of data demonstrating that trinucleotide-capped G47A* mRNA encoding hEPO induces similar baseline cytokine levels in vitro in BJ fibroblasts (BJF) as mRNA controls. [Figure 33B]1 shows a graph of data showing that trinucleotide-capped G47A* mRNA encoding hEPO induces similar in vitro baseline cytokine levels in monocyte-derived macrophages (MDMs) as mRNA controls. [Diagram 34] 1 shows a graph of data demonstrating that trinucleotide-capped G47A* mRNA encoding hEPO maintains high expression in vivo following six weekly doses. [Diagram 35] 1 shows a graph of data demonstrating that trinucleotide-capped G47A* mRNA encoding hEPO induces low anti-PEG IgM levels after six weekly doses. [Diagram 36] 1 shows a graph of data demonstrating that trinucleotide-capped G47A* mRNA encoding hEPO shows reduced B cell activation similar to the mRNA control. [Figure 37A] Figure 2 shows that the G47A* T7 RNA polymerase variant does not affect indel frequency. [Figure 37B] Figure 2 shows that the G47A* T7 RNA polymerase variant does not affect point mutation frequency. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0074] The present disclosure provides RNA polymerase (RNAP) variants that increase transcription efficiency and 3' homogeneity while reducing run-on transcripts and / or double-stranded contaminants generated, for example, during in vitro transcription (IVT) reactions. In some embodiments, these RNAP variants, which contain single amino acid substitutions, facilitate the RNAP conformational transition from the initiation complex to the elongation complex, thereby alleviating many of the problems associated with the transcription initiation step.

[0075] Unexpected experimental results are provided herein showing that modification(s) of the N-terminal C-helix and / or C-linker structure(s) of DNA-dependent RNA polymerase (e.g., T7 RNA polymerase) drives the conformational equilibrium of the polymerase into an elongation complex, facilitating the release of the DNA template promoter and the initiation of a highly processive elongation phase. Surprisingly, the use of the polymerase variants provided herein in IVT reactions reduces the 3' heterogeneity between the generated transcripts and also reduces (or eliminates) the generation of double-stranded contaminants. Furthermore, the results show that the purity and expression levels of the transcripts generated using the polymerase variants are comparable to those of the transcripts generated using the wild-type polymerase.

[0076] Thus, the use of the RNA polymerase variants of the present disclosure reduces many of the problems associated with IVT reaction products. Wild-type T7 RNA polymerase (RNAP) is commonly used in both industry and academia, and some of its activities significantly impair the purity of the resulting RNA transcripts. In particular, the use of this wild-type T7 RNAP enzyme results in the non-templated addition of nucleotides to the 3' end of the RNA transcript. For example, wild-type T7 RNAP inserts at least one, and often more than one, non-templated nucleotide at the 3' end that appears to be largely unfavorable to the identity of the nucleobase. Surprisingly, the T7 RNAP variants provided herein reduce the occurrence of 3' heterogeneity. In some embodiments, less than 30% of the RNA transcripts generated by IVT using the RNA polymerase variants of the present disclosure exhibit 3' heterogeneity. In some embodiments, less than 20% (e.g., less than 15%, 10%, or 5%) of the RNA transcripts generated by IVT using the RNA polymerase variants of the present disclosure exhibit 3' heterogeneity. In some embodiments, between 1-20%, 1-15%, 1-10%, or 1-5% of the RNA transcripts produced by IVT exhibit 3' heterogeneity. In some embodiments, less than 1% of the RNA transcripts produced by IVT using an RNA polymerase variant of the disclosure exhibit 3' heterogeneity.

[0077] Thus, the use of the RNA polymerase variants of the present disclosure, for example in an IVT reaction, results in the generation of "more homogeneous" RNA transcripts (a population of RNA transcripts with reduced heterogeneity / increased homogeneity at the 3' end). In some embodiments, at least 70% of the RNA transcripts generated using the RNA polymerase variants of the present disclosure exhibit 3' homogeneity. In some embodiments, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% of the RNA transcripts generated using the RNA polymerase variants of the present disclosure exhibit 3' homogeneity. In some embodiments, at least 90% of the RNA transcripts generated using the RNA polymerase variants of the present disclosure exhibit 3' homogeneity. In some embodiments, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the RNA transcripts generated using the RNA polymerase variants of the present disclosure exhibit 3' homogeneity.

[0078] Also surprising herein was the finding that RNA transcripts produced using T7 RNA polymerase variants with amino acid substitutions (e.g., S43A and / or G47A of SEQ ID NO:1) or C-terminal additions (e.g., WT, S43A*, and / or G47A* with one or more amino acid additions) stimulate a lower cytokine response compared to RNA produced using a wild-type RNA polymerase, optionally when delivered to cells in unpurified form (e.g., not purified by reverse phase chromatography). In some embodiments, there is no detectable cytokine response from cells receiving RNA transcripts produced using the T7 RNA mutant polymerases of the present disclosure. In some embodiments, RNA produced using an RNA polymerase variant (e.g., a T7 RNAP S43A* variant and / or a T7 RNAP G47A* variant) stimulates at least 50% (e.g., 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%) less cytokine response compared to RNA produced using a wild-type RNA polymerase. In some embodiments, RNA produced using an RNA polymerase variant (e.g., T7 RNAP S43A* variant and / or T7 RNAP G47A* variant) stimulates a 50-60%, 50-70%, 50-80%, 50-90%, 50-100%, 60-70%, 60-80%, 60-90%, 60-100%, 70-80%, 70-90%, 70-100%, 80-90%, 80-100%, or 90-100% lower cytokine response compared to RNA produced using a wild-type RNA polymerase. In some embodiments, RNA produced using an RNA polymerase variant (e.g., T7 RNAP S43A* variant and / or T7 RNAP G47A* variant) stimulates at least a 2-fold, 3-fold, 4-fold, or 5-fold lower cytokine response compared to RNA produced using a wild-type RNA polymerase. In some embodiments, the cells used to test cytokine responses are human fibroblasts (eg, BJ (ATCC® CRL-2522™) cells).In some embodiments, the cells used to test cytokine responses are monocyte-derived macrophages (MDMs).

[0079] In some embodiments, the concentration of dsRNA transcripts generated using an RNA polymerase variant is at least 50% (e.g., 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%) lower than dsRNA transcripts generated using a wild-type polymerase. In some embodiments, the concentration of dsRNA transcripts generated is 50-60%, 50-70%, 50-80%, 50-90%, 50-100%, 60-70%, 60-80%, 60-90%, 60-100%, 70-80%, 70-90%, 70-100%, 80-90%, 80-100%, or 90-100% lower than dsRNA transcripts generated using a wild-type polymerase. In some embodiments, the concentration of dsRNA transcripts produced is at least 2-fold, 3-fold, 4-fold, or 5-fold lower compared to dsRNA transcripts produced using a wild-type polymerase.

[0080] Use of the disclosed RNA polymerase variants unexpectedly resulted in the production of double-stranded contaminants and fewer run-on transcripts.

[0081] In some embodiments, less than 50% (e.g., less than 45%, less than 40%, less than 35%, less than 30%, less than 25%, less than 20%, less than 15%, less than 10%, less than 5%, or less than 1%) of the RNA transcripts generated using the RNA polymerase variant, e.g., by IVT, are double-stranded contaminants. In some embodiments, 1-50%, 1-40%, 1-30%, 1-20%, 1-10%, 1-5%, 5-50%, 5-40%, 5-30%, 5-20%, 5-10%, 10-50%, 10-40%, 10-30%, or 10-20% of the generated RNA transcripts are double-stranded contaminants.

[0082] In some embodiments, less than 50% (e.g., less than 45%, less than 40%, less than 35%, less than 30%, less than 25%, less than 20%, less than 15%, less than 10%, less than 5%, or less than 1%) of the RNA transcripts generated using the RNA polymerase variant are run-on RNA transcripts. In some embodiments, 1-50%, 1-40%, 1-30%, 1-20%, 1-10%, 1-5%, 5-50%, 5-40%, 5-30%, 5-20%, 5-10%, 10-50%, 10-40%, 10-30%, or 10-20% of the generated RNA transcripts are run-on RNA transcripts.

[0083] In some embodiments, the amount of full-length RNA transcripts generated using the RNA polymerase variant is at least 15 times greater than the amount of the DNA template. For example, the amount of full-length RNA transcripts generated can be at least 20 times, 30 times, 40 times, 45 times, 50 times, 60 times, 70 times, 80 times, 90 times, or 100 times greater than the amount of the DNA template. In some embodiments, the amount of full-length RNA transcripts generated can be 15-100 times, 15-90 times, 15-80 times, 15-70 times, 15-60 times, 15-50 times, 15-40 times, 15-30 times, 15-20 times, 20-100 times, 20-90 times, 20-80 times, 20-70 times, 20-60 times, 20-50 times, 20-40 times, or 20-30 times greater than the amount of the DNA template. In some embodiments, the amount of full-length RNA transcript produced is 2-fold, 3-fold, 4-fold, or 5-fold greater than the amount of DNA template.

[0084] In some embodiments, the ratio of double-stranded contaminants:full-length RNA transcripts generated using an RNA polymerase variant is less than 1: 1. For example, the ratio of double-stranded contaminants:full-length RNA transcripts generated is 0.9: 1, 0.8: 1, 0.7: 1, 0.6: 1, 0.5: 1, 0.4: 1, 0.3: 1, 0.2: 1, or 0.1: 1.

[0085] In some embodiments, the RNA transcripts generated using the RNA polymerase variant have less than 1 mutation per 100 nucleotides compared to the DNA template. For example, the generated RNA transcripts may have less than 1 mutation per 200, 300, 400, 500, 600, 700, 800, 900, or 1000 nucleotides compared to the DNA template.

[0086] RNA polymerase RNA polymerase (DNA-dependent RNA polymerase) is an enzyme that catalyzes the sequential addition of ribonucleotides to the 3' end of a growing RNA strand (transcription of RNA in the 5' → 3' direction), with nucleoside triphosphates (NTPs) acting as substrates for the enzyme, and the sequence of nucleotides specified by a DNA template. Transcription depends on the complementary pairing of bases. The two strands of the double helix are separated locally, and one of the separated strands serves as a template (DNA template). The RNA polymerase then catalyzes the alignment of free nucleotides on the DNA template with the complementary bases in the template. Thus, an RNA polymerase is considered to have RNA polymerase activity if the polymerase catalyzes the sequential addition of ribonucleotides to the 3' end of a growing RNA strand.

[0087] T7 RNA polymerase (T7 RNAP) is a 99 kDa DNA-dependent RNA polymerase encoded by the genome of bacteriophage T7 and is highly specific for the T7 phage promoter. Structural studies of T7 RNAP have shown that the conformation of the N-terminal domain changes significantly between the initiation and elongation phases of transcription. The N-terminal domain contains a C-helix subdomain and a promoter-binding domain (comprising two segments separated by subdomain H). The promoter-binding domain and bound promoter rotate approximately 45 degrees during synthesis of the 8 nt RNA transcript, allowing the active site to expand to accommodate the growing heteroduplex while maintaining promoter contact. The C-helix subdomain moves slowly towards its elongation conformation, whereas subdomain H remains in its initiation phase position, but not in its elongation phase position, more than 70 Å away. Comparison of the structures of T7 RNAP initiation and elongation complexes revealed major conformational changes within the N-terminal 267 residues (the N-terminal domain) and minor changes in the remainder of the RNAP. Rigid body rotations of the promoter binding domain and refolding of the terminal C-helix (residues 28-71) and H (residues 151-190) subdomains are responsible for abolishing the promoter binding site, expanding the active site, and creating an exit tunnel for the RNA transcript. The structural changes within the N-terminal domain are the main contributor to the increased stability and processivity of the elongation complex (see, e.g., Durniak, KJ et al., Science 322(5901): 553-557, 2008, incorporated herein by reference).

[0088] In some embodiments, provided herein is an RNA polymerase variant (e.g., a T7 RNAP variant) that promotes a conformational change from an RNAP initiation complex to an RNAP elongation complex. An RNA polymerase variant is an enzyme that has RNA polymerase activity and at least one substitution compared to the corresponding wild-type RNA polymerase. As indicated above, an RNA polymerase is considered to have RNA polymerase activity if the polymerase catalyzes the successive addition of ribonucleotides to the 3' end of a growing RNA strand. For example, an enzyme that comprises the amino acid sequence of SEQ ID NO: 1 with an amino acid substitution at position S43 (e.g., S43A) or G47 (e.g., G47A) and maintains RNA polymerase activity is considered to be a T7 RNAP variant of wild-type T7 RNAP (SEQ ID NO: 1).

[0089] In some embodiments, the RNA polymerase variant comprises at least one amino acid substitution that causes at least one three-dimensional loop structure of the RNA polymerase variant to undergo a conformational change to a helical structure as the RNA polymerase variant transitions from an initiation complex to an elongation complex, as compared to a wild-type RNA polymerase. Thus, in some embodiments, at least one amino acid modification has a high helical propensity compared to the wild-type amino acid.

[0090] Examples of loop structures include, but are not limited to, amino acids (aa) 42 to 47 in the C helix structure of the T7 RNA polymerase initiation complex (IC) conformation (e.g., aa 28 to 71 in SEQ ID NO: 1) and amino acids 257 to 262 in the C linker structure (e.g., amino acids 258 to 266 in SEQ ID NO: 1).

[0091] Also provided herein are RNA polymerase variants (e.g., T7 RNAP variants) that include at least one additional amino acid at the C-terminus. The at least one additional amino acid, in some embodiments, is selected from alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine. In some embodiments, the at least one additional amino acid is a polar amino acid. In some embodiments, the at least one additional amino acid is a non-polar amino acid. In some embodiments, the at least one additional amino acid is glycine. In some embodiments, the at least one additional amino acid is alanine. In some embodiments, the at least one additional amino acid is serine.

[0092] In some embodiments, the C-terminus of the RNA polymerase contains the following consensus sequence: FAFAX n (Sequence number 171 ), where x is any amino acid, and n is any integer, such as 1 to 5 (e.g., 1, 2, 3, 4, or 5). In some embodiments, the C-terminus of the RNA polymerase comprises the following consensus sequence: FAFAG n (Sequence number 330 ), where n is any integer, for example, 1 to 5. In some embodiments, the C-terminus of the RNA polymerase comprises the following consensus sequence: FAFAA n ( SEQ ID NO:331 ), where n is any integer, for example, 1 to 5. In some embodiments, the C-terminus of the RNA polymerase comprises the following consensus sequence: FAFAR n (Sequence number 332 ), where n is any integer, for example, 1 to 5. In some embodiments, the C-terminus of the RNA polymerase comprises the following consensus sequence: FAFAN n (Sequence number 333), where n is any integer, for example, 1 to 5. In some embodiments, the C-terminus of the RNA polymerase comprises the following consensus sequence: FAFAD n (Sequence number 334 ), where n is any integer, for example, 1 to 5. In some embodiments, the C-terminus of the RNA polymerase comprises the following consensus sequence: FAFAC n (Sequence number 335 ), where n is any integer, for example, 1 to 5. In some embodiments, the C-terminus of the RNA polymerase comprises the following consensus sequence: FAFAE n (Sequence number 336 ), where n is any integer, for example, 1 to 5. In some embodiments, the C-terminus of the RNA polymerase comprises the following consensus sequence: FAFAQ n (SEQ ID NO: 302), where n is any integer, for example, 1 to 5. In some embodiments, the C-terminus of the RNA polymerase comprises the following consensus sequence: FAFAH n (SEQ ID NO: 303), where n is any integer, for example, 1 to 5. In some embodiments, the C-terminus of the RNA polymerase comprises the following consensus sequence: FAFAI n (SEQ ID NO: 304), where n is any integer, for example, 1 to 5. In some embodiments, the C-terminus of the RNA polymerase comprises the following consensus sequence: FAFAL n (SEQ ID NO: 305), where n is any integer, for example, 1 to 5. In some embodiments, the C-terminus of the RNA polymerase comprises the following consensus sequence: FAFAK n (SEQ ID NO: 306), where n is any integer, for example, 1 to 5. In some embodiments, the C-terminus of the RNA polymerase comprises the following consensus sequence: FAFAM n (SEQ ID NO: 307), where n is any integer, for example, 1 to 5. In some embodiments, the C-terminus of the RNA polymerase comprises the following consensus sequence: FAFAF n (SEQ ID NO: 308), where n is any integer, for example, 1 to 5. In some embodiments, the C-terminus of the RNA polymerase comprises the following consensus sequence: FAFAP n(SEQ ID NO: 309), where n is any integer, for example, 1 to 5. In some embodiments, the C-terminus of the RNA polymerase comprises the following consensus sequence: FAFAS n (SEQ ID NO: 310), where n is any integer, for example, 1 to 5. In some embodiments, the C-terminus of the RNA polymerase comprises the following consensus sequence: FAFAT n (SEQ ID NO: 311), where n is any integer, for example, 1 to 5. In some embodiments, the C-terminus of the RNA polymerase comprises the following consensus sequence: FAFAW n (SEQ ID NO: 312), where n is any integer, for example, 1 to 5. In some embodiments, the C-terminus of the RNA polymerase comprises the following consensus sequence: FAFAY n (SEQ ID NO: 313), where n is any integer, for example, 1 to 5. In some embodiments, the C-terminus of the RNA polymerase comprises the following consensus sequence: FAFAV n (SEQ ID NO: 314), where n is any integer, for example, 1 to 5.

[0093] In some embodiments, the C-terminal motif (FAFA (SEQ ID NO:172) or FAX n (SEQ ID NO:171) ) contains an amino acid substitution at one or more of positions 880, 881, 882, or 883 compared to wild-type T7 RNAP (e.g., SEQ ID NO: 1). Thus, the present disclosure provides a variety of C-terminal F 880 A 881 F 882 A 883 (SEQ ID NO:172) motif, and one or more of amino acids at positions 880, 881, 882, or 883 have an additional C-terminal amino acid (X n ) has been modified to include at least one amino acid substitution.

[0094] Amino acid substitutions The RNA polymerase variant of the present disclosure comprises at least one amino acid substitution compared to WT RNA polymerase.For example, with respect to WT T7 RNA polymerase having the amino acid sequence of SEQ ID NO: 1, serine at position 43 is considered as "wild type amino acid", and substitution of serine at position 43 with alanine is considered as "amino acid substitution" with high helical propensity.

[0095] The average globular protein contains 30% α-helices, the most common type of secondary structure. Some amino acids occur more frequently in α-helices than others, this tendency being known as helical propensity. See, e.g., Pace, NC and Scholtz, JM Biophysical Journal, 75:422-427 (1998). In some embodiments, at least one amino acid substitution has a high helical propensity compared to the wild-type amino acid. In general, high helical propensity amino acid substitutions are selected to thermodynamically bias the population of polymerase conformers towards the elongation complex. Relative ΔΔGs (free energies) are calculated for the substitutions in the IC and elongation complex (EC) structures using publicly available software (e.g., Rosetta from the University of Washington and Schrödinger's Maestro). Substitutions are then selected based on the calculations and additional knowledge including, for example, amino acid helical propensities and / or polypeptide backbone phi-psi compatibility.

[0096] In some embodiments, the RNA polymerase variant is a T7 RNA polymerase variant that includes at least one (one or more) amino acid substitution compared to WT T7 RNA polymerase (e.g., WT T7 RNA polymerase having the amino acid sequence of SEQ ID NO: 1). In some embodiments, the amino acid substitution is a high helical propensity amino acid substitution. Examples of high helical propensity amino acids include alanine, isoleucine, leucine, arginine, methionine, lysine, glutamine, and / or glutamic acid.

[0097] In some embodiments, the RNA polymerase variant is a T7 RNA polymerase variant that includes at least one additional amino acid at the C-terminus (e.g., SEQ ID NO:99, SEQ ID NO:100, SEQ ID NO:294, SEQ ID NO:295, or SEQ ID NO:296, or an amino acid sequence having 90%-99%, e.g., at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, identity to SEQ ID NO:99, SEQ ID NO:100, SEQ ID NO:294, SEQ ID NO:295, or SEQ ID NO:296). In some embodiments, the RNA polymerase variant is a T7 RNA polymerase variant that includes at least one additional amino acid and an amino acid substitution at the C-terminus compared to WT T7 RNA polymerase. In some embodiments, the amino acid substitution is a high helix-propensity amino acid substitution.

[0098] Provided herein are two approaches that can be used to identify high or higher helical propensity amino acid substitutions that thermodynamically bias / favor the conformational equilibrium towards EC. In both approaches, care is taken to avoid amino acid substitutions at any positions directly involved in promoter binding or catalysis (based on structural review, literature search). In method A, specialized software is used to identify mutations in EC and IC (ΔΔG mut ) to calculate the change in the free energy of protein folding. All possible amino acid substitutions at the desired sequence positions are evaluated by this method. The favorable amino acid substitutions (i.e., those that favor EC over IC) are calculated based on the ΔΔG mut Difference

number

[0099] In some embodiments, the amino acid substitution is a high helix propensity amino acid (e.g., alanine, isoleucine, leucine, arginine, methionine, lysine, glutamine, and / or glutamic acid) substitution at any one of amino acid positions 42-47 (E42, S43, Y44, E45, M46, and / or G47) of SEQ ID NO: 1. In some embodiments, the amino acid substitution is a high helix propensity amino acid (e.g., alanine, isoleucine, leucine, arginine, methionine, lysine, glutamine, and / or glutamic acid) substitution at any one of amino acid positions 42-47 (E42, S43, Y44, E45, M46, and / or G47) of an amino acid sequence having 90-99%, e.g., at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, identity to SEQ ID NO: 1. In some embodiments, the amino acid substitution is a high helix propensity amino acid substitution at amino acid position 42 of SEQ ID NO:1. In some embodiments, the amino acid substitution is a high helix propensity amino acid substitution at amino acid position 43 of SEQ ID NO:1. In some embodiments, the amino acid substitution is a high helix propensity amino acid substitution at amino acid position 44 of SEQ ID NO:1. In some embodiments, the amino acid substitution is a high helix propensity amino acid substitution at amino acid position 45 of SEQ ID NO:1. In some embodiments, the amino acid substitution is a high helix propensity amino acid substitution at amino acid position 46 of SEQ ID NO:1. In some embodiments, the amino acid substitution is a high helix propensity amino acid substitution at amino acid position 47 of SEQ ID NO:1.

[0100] In some embodiments, the amino acid substitution is a high helical propensity amino acid (e.g., alanine, isoleucine, leucine, arginine, methionine, lysine, glutamine, and / or glutamic acid) substitution at any one of amino acid positions 42-47 (E42, S43, Y44, E45, M46, and / or G47) of SEQ ID NO: 99, 100, 294, 295, or 296. The amino acid substitution is a high helix propensity amino acid (e.g., alanine, isoleucine, leucine, arginine, methionine, lysine, glutamine, and / or glutamic acid) substitution at any one of amino acid positions 42-47 (E42, S43, Y44, E45, M46, and / or G47) of an amino acid sequence having 90-99%, e.g., at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, identity to SEQ ID NO: 99, 100, 294, 295, or 296. In some embodiments, the amino acid substitution is a high helix propensity amino acid substitution at amino acid position 42 of SEQ ID NO: 99, 100, 294, 295, or 296. In some embodiments, the amino acid substitution is a high helix propensity amino acid substitution at amino acid position 43 of SEQ ID NO: 99, 100, 294, 295, or 296. In some embodiments, the amino acid substitution is a high helix propensity amino acid substitution at amino acid position 44 of SEQ ID NO: 99, 100, 294, 295, or 296. In some embodiments, the amino acid substitution is a high helix propensity amino acid substitution at amino acid position 45 of SEQ ID NO: 99, 100, 294, 295, or 296. In some embodiments, the amino acid substitution is a high helix propensity amino acid substitution at amino acid position 46 of SEQ ID NO: 99, 100, 294, 295, or 296. In some embodiments, the amino acid substitution is a high helix propensity amino acid substitution at amino acid position 47 of SEQ ID NO: 99, 100, 294, 295, or 296.

[0101] In some embodiments, the amino acid substitution is an alanine at any one of amino acid positions 42-47 of SEQ ID NO:1. In some embodiments, the amino acid substitution is an alanine at any one of amino acid positions 42-47 of an amino acid sequence having 90-99%, e.g., at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, identity to SEQ ID NO:1. Thus, in some embodiments, the amino acid substitution is an alanine at position 42 of SEQ ID NO:1. In some embodiments, the amino acid substitution is an alanine at position 43 of SEQ ID NO:1. In some embodiments, the amino acid substitution is an alanine at position 44 of SEQ ID NO:1. In some embodiments, the amino acid substitution is an alanine at position 45 of SEQ ID NO:1. In some embodiments, the amino acid substitution is an alanine at position 46 of SEQ ID NO:1. In some embodiments, the amino acid substitution is an alanine at position 47 of SEQ ID NO:1.

[0102] In some embodiments, the amino acid substitution is an alanine at any one of amino acid positions 42-47 of SEQ ID NO: 99, 100, 294, 295, or 296. In some embodiments, the amino acid substitution is an alanine at any one of amino acid positions 42-47 of an amino acid sequence having 90-99%, e.g., at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, identity to SEQ ID NO: 99, 100, 294, 295, or 296. Thus, in some embodiments, the amino acid substitution is an alanine at position 42 of SEQ ID NO: 99, 100, 294, 295, or 296. In some embodiments, the amino acid substitution is an alanine at position 43 of SEQ ID NO: 99, 100, 294, 295, or 296. In some embodiments, the amino acid substitution is an alanine at position 44 of SEQ ID NO: 99, 100, 294, 295, or 296. In some embodiments, the amino acid substitution is an alanine at position 45 of SEQ ID NO: 99, 100, 294, 295, or 296. In some embodiments, the amino acid substitution is an alanine at position 46 of SEQ ID NO: 99, 100, 294, 295, or 296. In some embodiments, the amino acid substitution is an alanine at position 47 of SEQ ID NO: 99, 100, 294, 295, or 296.

[0103] In some embodiments, the amino acid substitution is isoleucine at any one of amino acid positions 42-47 of SEQ ID NO:1. In some embodiments, the amino acid substitution is isoleucine at any one of amino acid positions 42-47 of an amino acid sequence having 90-99%, e.g., at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, identity to SEQ ID NO:1. Thus, in some embodiments, the amino acid substitution is isoleucine at position 42 of SEQ ID NO:1. In some embodiments, the amino acid substitution is isoleucine at position 43 of SEQ ID NO:1. In some embodiments, the amino acid substitution is isoleucine at position 44 of SEQ ID NO:1. In some embodiments, the amino acid substitution is isoleucine at position 45 of SEQ ID NO:1. In some embodiments, the amino acid substitution is isoleucine at position 46 of SEQ ID NO:1. In some embodiments, the amino acid substitution is isoleucine at position 47 of SEQ ID NO:1.

[0104] In some embodiments, the amino acid substitution is an isoleucine at any one of amino acid positions 42-47 of SEQ ID NO: 99, 100, 294, 295, or 296. In some embodiments, the amino acid substitution is an isoleucine at any one of amino acid positions 42-47 of an amino acid sequence having 90-99%, e.g., at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, identity to SEQ ID NO: 99, 100, 294, 295, or 296. Thus, in some embodiments, the amino acid substitution is an isoleucine at position 42 of SEQ ID NO: 99, 100, 294, 295, or 296. In some embodiments, the amino acid substitution is an isoleucine at position 43 of SEQ ID NO: 99, 100, 294, 295, or 296. In some embodiments, the amino acid substitution is an isoleucine at position 44 of SEQ ID NO: 99, 100, 294, 295, or 296. In some embodiments, the amino acid substitution is an isoleucine at position 45 of SEQ ID NO: 99, 100, 294, 295, or 296. In some embodiments, the amino acid substitution is an isoleucine at position 46 of SEQ ID NO: 99, 100, 294, 295, or 296. In some embodiments, the amino acid substitution is an isoleucine at position 47 of SEQ ID NO: 99, 100, 294, 295, or 296.

[0105] In some embodiments, the amino acid substitution is a leucine at any one of amino acid positions 42-47 of SEQ ID NO:1. In some embodiments, the amino acid substitution is a leucine at any one of amino acid positions 42-47 of an amino acid sequence having 90-99%, e.g., at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, identity to SEQ ID NO:1. Thus, in some embodiments, the amino acid substitution is a leucine at position 42 of SEQ ID NO:1. In some embodiments, the amino acid substitution is a leucine at position 43 of SEQ ID NO:1. In some embodiments, the amino acid substitution is a leucine at position 44 of SEQ ID NO:1. In some embodiments, the amino acid substitution is a leucine at position 45 of SEQ ID NO:1. In some embodiments, the amino acid substitution is a leucine at position 46 of SEQ ID NO:1. In some embodiments, the amino acid substitution is a leucine at position 47 of SEQ ID NO:1.

[0106] In some embodiments, the amino acid substitution is a leucine at any one of amino acid positions 42-47 of SEQ ID NO: 99, 100, 294, 295, or 296. In some embodiments, the amino acid substitution is a leucine at any one of amino acid positions 42-47 of an amino acid sequence having 90-99%, e.g., at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, identity to SEQ ID NO: 99, 100, 294, 295, or 296. Thus, in some embodiments, the amino acid substitution is a leucine at position 42 of SEQ ID NO: 99, 100, 294, 295, or 296. In some embodiments, the amino acid substitution is a leucine at position 43 of SEQ ID NO: 99, 100, 294, 295, or 296. In some embodiments, the amino acid substitution is a leucine at position 44 of SEQ ID NO: 99, 100, 294, 295, or 296. In some embodiments, the amino acid substitution is a leucine at position 45 of SEQ ID NO: 99, 100, 294, 295, or 296. In some embodiments, the amino acid substitution is a leucine at position 46 of SEQ ID NO: 99, 100, 294, 295, or 296. In some embodiments, the amino acid substitution is a leucine at position 47 of SEQ ID NO: 99, 100, 294, 295, or 296.

[0107] In some embodiments, the amino acid substitution is an arginine at any one of amino acid positions 42-47 of SEQ ID NO:1. In some embodiments, the amino acid substitution is an arginine at any one of amino acid positions 42-47 of an amino acid sequence having 90-99%, e.g., at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, identity to SEQ ID NO:1. Thus, in some embodiments, the amino acid substitution is an arginine at position 42 of SEQ ID NO:1. In some embodiments, the amino acid substitution is an arginine at position 43 of SEQ ID NO:1. In some embodiments, the amino acid substitution is an arginine at position 44 of SEQ ID NO:1. In some embodiments, the amino acid substitution is an arginine at position 45 of SEQ ID NO:1. In some embodiments, the amino acid substitution is an arginine at position 46 of SEQ ID NO:1. In some embodiments, the amino acid substitution is an arginine at position 47 of SEQ ID NO:1.

[0108] In some embodiments, the amino acid substitution is an arginine at any one of amino acid positions 42-47 of SEQ ID NO: 99, 100, 294, 295, or 296. In some embodiments, the amino acid substitution is an arginine at any one of amino acid positions 42-47 of an amino acid sequence having 90-99%, e.g., at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, identity to SEQ ID NO: 99, 100, 294, 295, or 296. Thus, in some embodiments, the amino acid substitution is an arginine at position 42 of SEQ ID NO: 99, 100, 294, 295, or 296. In some embodiments, the amino acid substitution is an arginine at position 43 of SEQ ID NO: 99, 100, 294, 295, or 296. In some embodiments, the amino acid substitution is an arginine at position 44 of SEQ ID NO: 99, 100, 294, 295, or 296. In some embodiments, the amino acid substitution is an arginine at position 45 of SEQ ID NO: 99, 100, 294, 295, or 296. In some embodiments, the amino acid substitution is an arginine at position 46 of SEQ ID NO: 99, 100, 294, 295, or 296. In some embodiments, the amino acid substitution is an arginine at position 47 of SEQ ID NO: 99, 100, 294, 295, or 296.

[0109] In some embodiments, the amino acid substitution is a methionine at any one of amino acid positions 42-47 of SEQ ID NO:1. In some embodiments, the amino acid substitution is a methionine at any one of amino acid positions 42-47 of an amino acid sequence having 90-99%, e.g., at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, identity to SEQ ID NO:1. Thus, in some embodiments, the amino acid substitution is a methionine at position 42 of SEQ ID NO:1. In some embodiments, the amino acid substitution is a methionine at position 43 of SEQ ID NO:1. In some embodiments, the amino acid substitution is a methionine at position 44 of SEQ ID NO:1. In some embodiments, the amino acid substitution is a methionine at position 45 of SEQ ID NO:1. In some embodiments, the amino acid substitution is a methionine at position 46 of SEQ ID NO:1. In some embodiments, the amino acid substitution is a methionine at position 47 of SEQ ID NO:1.

[0110] In some embodiments, the amino acid substitution is a methionine at any one of amino acid positions 42-47 of SEQ ID NO: 99, 100, 294, 295, or 296. In some embodiments, the amino acid substitution is a methionine at any one of amino acid positions 42-47 of an amino acid sequence having 90-99%, e.g., at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, identity to SEQ ID NO: 99, 100, 294, 295, or 296. Thus, in some embodiments, the amino acid substitution is a methionine at position 42 of SEQ ID NO: 99, 100, 294, 295, or 296. In some embodiments, the amino acid substitution is a methionine at position 43 of SEQ ID NO: 99, 100, 294, 295, or 296. In some embodiments, the amino acid substitution is a methionine at position 44 of SEQ ID NO: 99, 100, 294, 295, or 296. In some embodiments, the amino acid substitution is a methionine at position 45 of SEQ ID NO: 99, 100, 294, 295, or 296. In some embodiments, the amino acid substitution is a methionine at position 46 of SEQ ID NO: 99, 100, 294, 295, or 296. In some embodiments, the amino acid substitution is a methionine at position 47 of SEQ ID NO: 99, 100, 294, 295, or 296.

[0111] In some embodiments, the amino acid substitution is a lysine at any one of amino acid positions 42-47 of SEQ ID NO:1. In some embodiments, the amino acid substitution is a lysine at any one of amino acid positions 42-47 of an amino acid sequence having 90-99%, e.g., at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, identity to SEQ ID NO:1. Thus, in some embodiments, the amino acid substitution is a lysine at position 42 of SEQ ID NO:1. In some embodiments, the amino acid substitution is a lysine at position 43 of SEQ ID NO:1. In some embodiments, the amino acid substitution is a lysine at position 44 of SEQ ID NO:1. In some embodiments, the amino acid substitution is a lysine at position 45 of SEQ ID NO:1. In some embodiments, the amino acid substitution is a lysine at position 46 of SEQ ID NO:1. In some embodiments, the amino acid substitution is a lysine at position 47 of SEQ ID NO:1.

[0112] In some embodiments, the amino acid substitution is a lysine at any one of amino acid positions 42-47 of SEQ ID NO: 99, 100, 294, 295, or 296. In some embodiments, the amino acid substitution is a lysine at any one of amino acid positions 42-47 of an amino acid sequence having 90-99%, e.g., at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, identity to SEQ ID NO: 99, 100, 294, 295, or 296. Thus, in some embodiments, the amino acid substitution is a lysine at position 42 of SEQ ID NO: 99, 100, 294, 295, or 296. In some embodiments, the amino acid substitution is a lysine at position 43 of SEQ ID NO: 99, 100, 294, 295, or 296. In some embodiments, the amino acid substitution is a lysine at position 44 of SEQ ID NO: 99, 100, 294, 295, or 296. In some embodiments, the amino acid substitution is a lysine at position 45 of SEQ ID NO: 99, 100, 294, 295, or 296. In some embodiments, the amino acid substitution is a lysine at position 46 of SEQ ID NO: 99, 100, 294, 295, or 296. In some embodiments, the amino acid substitution is a lysine at position 47 of SEQ ID NO: 99, 100, 294, 295, or 296.

[0113] In some embodiments, the amino acid substitution is a glutamine at any one of amino acid positions 42-47 of SEQ ID NO:1. In some embodiments, the amino acid substitution is a glutamine at any one of amino acid positions 42-47 of an amino acid sequence having 90-99%, e.g., at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, identity to SEQ ID NO:1. Thus, in some embodiments, the amino acid substitution is a glutamine at position 42 of SEQ ID NO:1. In some embodiments, the amino acid substitution is a glutamine at position 43 of SEQ ID NO:1. In some embodiments, the amino acid substitution is a glutamine at position 44 of SEQ ID NO:1. In some embodiments, the amino acid substitution is a glutamine at position 45 of SEQ ID NO:1. In some embodiments, the amino acid substitution is a glutamine at position 46 of SEQ ID NO:1. In some embodiments, the amino acid substitution is a glutamine at position 47 of SEQ ID NO:1.

[0114] In some embodiments, the amino acid substitution is a glutamic acid at any one of amino acid positions 42-47 of SEQ ID NO: 99, 100, 294, 295, or 296. In some embodiments, the amino acid substitution is a glutamine at any one of amino acid positions 42-47 of an amino acid sequence having 90-99%, e.g., at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, identity to SEQ ID NO: 99, 100, 294, 295, or 296. Thus, in some embodiments, the amino acid substitution is a glutamic acid at position 42 of SEQ ID NO: 99, 100, 294, 295, or 296. In some embodiments, the amino acid substitution is a glutamic acid at position 43 of SEQ ID NO: 99, 100, 294, 295, or 296. In some embodiments, the amino acid substitution is a glutamic acid at position 44 of SEQ ID NO: 99, 100, 294, 295, or 296. In some embodiments, the amino acid substitution is a glutamic acid at position 45 of SEQ ID NO: 99, 100, 294, 295, or 296. In some embodiments, the amino acid substitution is a glutamic acid at position 46 of SEQ ID NO: 99, 100, 294, 295, or 296. In some embodiments, the amino acid substitution is a glutamic acid at position 47 of SEQ ID NO: 99, 100, 294, 295, or 296.

[0115] In some embodiments, the amino acid substitution is a glutamic acid at any one of amino acid positions 42-47 of SEQ ID NO:1. In some embodiments, the amino acid substitution is a glutamic acid at any one of amino acid positions 42-47 of an amino acid sequence having 90-99%, e.g., at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, identity to SEQ ID NO:1. Thus, in some embodiments, the amino acid substitution is a glutamic acid at position 42 of SEQ ID NO:1. In some embodiments, the amino acid substitution is a glutamic acid at position 43 of SEQ ID NO:1. In some embodiments, the amino acid substitution is a glutamic acid at position 44 of SEQ ID NO:1. In some embodiments, the amino acid substitution is a glutamic acid at position 45 of SEQ ID NO:1. In some embodiments, the amino acid substitution is a glutamic acid at position 46 of SEQ ID NO:1. In some embodiments, the amino acid substitution is a glutamic acid at position 47 of SEQ ID NO:1.

[0116] In some embodiments, the amino acid substitution is a glutamic acid at any one of amino acid positions 42-47 of SEQ ID NO: 99, 100, 294, 295, or 296. In some embodiments, the amino acid substitution is a glutamic acid at any one of amino acid positions 42-47 of an amino acid sequence having 90-99%, e.g., at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, identity to SEQ ID NO: 99, 100, 294, 295, or 296. Thus, in some embodiments, the amino acid substitution is a glutamic acid at position 42 of SEQ ID NO: 99, 100, 294, 295, or 296. In some embodiments, the amino acid substitution is a glutamic acid at position 43 of SEQ ID NO: 99, 100, 294, 295, or 296. In some embodiments, the amino acid substitution is a glutamic acid at position 44 of SEQ ID NO: 99, 100, 294, 295, or 296. In some embodiments, the amino acid substitution is a glutamic acid at position 45 of SEQ ID NO: 99, 100, 294, 295, or 296. In some embodiments, the amino acid substitution is a glutamic acid at position 46 of SEQ ID NO: 99, 100, 294, 295, or 296. In some embodiments, the amino acid substitution is a glutamic acid at position 47 of SEQ ID NO: 99, 100, 294, 295, or 296.

[0117] In some embodiments, the amino acid substitution is a high helix-propensity amino acid (e.g., alanine, isoleucine, leucine, arginine, methionine, lysine, glutamine, and / or glutamic acid) substitution at any one of amino acid positions 257-262 (R257, A258, G259, A260, L261, and / or A262) of SEQ ID NO:1. In some embodiments, the amino acid substitution is a high helix propensity amino acid (e.g., alanine, isoleucine, leucine, arginine, methionine, lysine, glutamine, and / or glutamic acid) substitution at any one of amino acid positions 257-262 (R257, A258, G259, A260, L261, and / or A262) of an amino acid sequence having 90-99%, e.g., at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, identity to SEQ ID NO:1. In some embodiments, the amino acid substitution is a high helix propensity amino acid substitution at amino acid position 257 of SEQ ID NO:1. In some embodiments, the amino acid substitution is a high helix propensity amino acid substitution at amino acid position 258 of SEQ ID NO:1. In some embodiments, the amino acid substitution is a high helix propensity amino acid substitution at amino acid position 259 of SEQ ID NO:1. In some embodiments, the amino acid substitution is a high helix-propensity amino acid substitution at amino acid position 260 of SEQ ID NO:1. In some embodiments, the amino acid substitution is a high helix-propensity amino acid substitution at amino acid position 261 of SEQ ID NO:1. In some embodiments, the amino acid substitution is a high helix-propensity amino acid substitution at amino acid position 262 of SEQ ID NO:1.

[0118] The amino acid substitution is a high helix-propensity amino acid (e.g., alanine, isoleucine, leucine, arginine, methionine, lysine, glutamine, and / or glutamic acid) substitution at any one of amino acid positions 257 to 262 (R257, A258, G259, A260, L261, and / or A262) of SEQ ID NO: 99, 100, 294, 295, or 296. In some embodiments, the amino acid substitution is a high helix propensity amino acid (e.g., alanine, isoleucine, leucine, arginine, methionine, lysine, glutamine, and / or glutamic acid) substitution at any one of amino acid positions 257-262 (R257, A258, G259, A260, L261, and / or A262) of an amino acid sequence having 90-99%, e.g., at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, identity to SEQ ID NO: 99, 100, 294, 295, or 296. In some embodiments, the amino acid substitution is a high helix propensity amino acid substitution at amino acid position 257 of SEQ ID NO: 99, 100, 294, 295, or 296. In some embodiments, the amino acid substitution is a high helix propensity amino acid substitution at amino acid position 258 of SEQ ID NO: 99, 100, 294, 295, or 296. In some embodiments, the amino acid substitution is a high helix propensity amino acid substitution at amino acid position 259 of SEQ ID NO: 99, 100, 294, 295, or 296. In some embodiments, the amino acid substitution is a high helix propensity amino acid substitution at amino acid position 260 of SEQ ID NO: 99, 100, 294, 295, or 296. In some embodiments, the amino acid substitution is a high helix propensity amino acid substitution at amino acid position 261 of SEQ ID NO: 99, 100, 294, 295, or 296. In some embodiments, the amino acid substitution is a high helix propensity amino acid substitution at amino acid position 262 of SEQ ID NO: 99, 100, 294, 295, or 296.

[0119] In some embodiments, the amino acid substitution is an alanine at any one of amino acid positions 257-262 of SEQ ID NO:1. In some embodiments, the amino acid substitution is an alanine at any one of amino acid positions 257-262 of an amino acid sequence having 90-99%, e.g., at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, identity to SEQ ID NO:1. Thus, in some embodiments, the amino acid substitution is an alanine at position 257 of SEQ ID NO:1. In some embodiments, the amino acid substitution is an alanine at position 258 of SEQ ID NO:1. In some embodiments, the amino acid substitution is an alanine at position 259 of SEQ ID NO:1. In some embodiments, the amino acid substitution is an alanine at position 260 of SEQ ID NO:1. In some embodiments, the amino acid substitution is an alanine at position 261 of SEQ ID NO:1. In some embodiments, the amino acid substitution is an alanine at position 262 of SEQ ID NO:1.

[0120] In some embodiments, the amino acid substitution is an alanine at any one of amino acid positions 257-262 of SEQ ID NO: 99, 100, 294, 295, or 296. In some embodiments, the amino acid substitution is an alanine at any one of amino acid positions 257-262 of an amino acid sequence having 90-99%, e.g., at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, identity to SEQ ID NO: 99, 100, 294, 295, or 296. Thus, in some embodiments, the amino acid substitution is an alanine at position 257 of SEQ ID NO: 99, 100, 294, 295, or 296. In some embodiments, the amino acid substitution is an alanine at position 258 of SEQ ID NO: 99, 100, 294, 295, or 296. In some embodiments, the amino acid substitution is an alanine at position 259 of SEQ ID NO: 99, 100, 294, 295, or 296. In some embodiments, the amino acid substitution is an alanine at position 260 of SEQ ID NO: 99, 100, 294, 295, or 296. In some embodiments, the amino acid substitution is an alanine at position 261 of SEQ ID NO: 99, 100, 294, 295, or 296. In some embodiments, the amino acid substitution is an alanine at position 262 of SEQ ID NO: 99, 100, 294, 295, or 296.

[0121] In some embodiments, the amino acid substitution is isoleucine at any one of amino acid positions 257-262 of SEQ ID NO:1. In some embodiments, the amino acid substitution is isoleucine at any one of amino acid positions 257-262 of an amino acid sequence having 90-99%, e.g., at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, identity to SEQ ID NO:1. Thus, in some embodiments, the amino acid substitution is isoleucine at position 257 of SEQ ID NO:1. In some embodiments, the amino acid substitution is isoleucine at position 258 of SEQ ID NO:1. In some embodiments, the amino acid substitution is isoleucine at position 259 of SEQ ID NO:1. In some embodiments, the amino acid substitution is isoleucine at position 260 of SEQ ID NO:1. In some embodiments, the amino acid substitution is isoleucine at position 261 of SEQ ID NO:1. In some embodiments, the amino acid substitution is isoleucine at position 262 of SEQ ID NO:1.

[0122] In some embodiments, the amino acid substitution is an isoleucine at any one of amino acid positions 257-262 of SEQ ID NO: 99, 100, 294, 295, or 296. In some embodiments, the amino acid substitution is an isoleucine at any one of amino acid positions 257-262 of an amino acid sequence having 90-99%, e.g., at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, identity to SEQ ID NO: 99, 100, 294, 295, or 296. Thus, in some embodiments, the amino acid substitution is an isoleucine at position 257 of SEQ ID NO: 99, 100, 294, 295, or 296. In some embodiments, the amino acid substitution is an isoleucine at position 258 of SEQ ID NO: 99, 100, 294, 295, or 296. In some embodiments, the amino acid substitution is an isoleucine at position 259 of SEQ ID NO: 99, 100, 294, 295, or 296. In some embodiments, the amino acid substitution is an isoleucine at position 260 of SEQ ID NO: 99, 100, 294, 295, or 296. In some embodiments, the amino acid substitution is an isoleucine at position 261 of SEQ ID NO: 99, 100, 294, 295, or 296. In some embodiments, the amino acid substitution is an isoleucine at position 262 of SEQ ID NO: 99, 100, 294, 295, or 296.

[0123] In some embodiments, the amino acid substitution is a leucine at any one of amino acid positions 257-262 of SEQ ID NO:1. The amino acid substitution is a leucine at any one of amino acid positions 257-262 of an amino acid sequence having 90-99%, e.g., at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, identity to SEQ ID NO:1. Thus, in some embodiments, the amino acid substitution is a leucine at position 257 of SEQ ID NO:1. In some embodiments, the amino acid substitution is a leucine at position 258 of SEQ ID NO:1. In some embodiments, the amino acid substitution is a leucine at position 259 of SEQ ID NO:1. In some embodiments, the amino acid substitution is a leucine at position 260 of SEQ ID NO:1. In some embodiments, the amino acid substitution is a leucine at position 261 of SEQ ID NO:1. In some embodiments, the amino acid substitution is a leucine at position 262 of SEQ ID NO:1.

[0124] In some embodiments, the amino acid substitution is a leucine at any one of amino acid positions 257-262 of SEQ ID NO: 99, 100, 294, 295, or 296. In some embodiments, the amino acid substitution is an isoleucine at any one of amino acid positions 257-262 of an amino acid sequence having 90-99%, e.g., at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, identity to SEQ ID NO: 99, 100, 294, 295, or 296. Thus, in some embodiments, the amino acid substitution is a leucine at position 257 of SEQ ID NO: 99, 100, 294, 295, or 296. In some embodiments, the amino acid substitution is a leucine at position 258 of SEQ ID NO: 99, 100, 294, 295, or 296. In some embodiments, the amino acid substitution is a leucine at position 259 of SEQ ID NO: 99, 100, 294, 295, or 296. In some embodiments, the amino acid substitution is a leucine at position 260 of SEQ ID NO: 99, 100, 294, 295, or 296. In some embodiments, the amino acid substitution is a leucine at position 261 of SEQ ID NO: 99, 100, 294, 295, or 296. In some embodiments, the amino acid substitution is a leucine at position 262 of SEQ ID NO: 99, 100, 294, 295, or 296.

[0125] In some embodiments, the amino acid substitution is an arginine at any one of amino acid positions 257-262 of SEQ ID NO:1. In some embodiments, the amino acid substitution is an arginine at any one of amino acid positions 257-262 of an amino acid sequence having 90-99%, e.g., at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, identity to SEQ ID NO:1. Thus, in some embodiments, the amino acid substitution is an arginine at position 257 of SEQ ID NO:1. In some embodiments, the amino acid substitution is an arginine at position 258 of SEQ ID NO:1. In some embodiments, the amino acid substitution is an arginine at position 259 of SEQ ID NO:1. In some embodiments, the amino acid substitution is an arginine at position 260 of SEQ ID NO:1. In some embodiments, the amino acid substitution is an arginine at position 261 of SEQ ID NO:1. In some embodiments, the amino acid substitution is an arginine at position 262 of SEQ ID NO:1.

[0126] In some embodiments, the amino acid substitution is an arginine at any one of amino acid positions 257-262 of SEQ ID NO: 99, 100, 294, 295, or 296. In some embodiments, the amino acid substitution is an arginine at any one of amino acid positions 257-262 of an amino acid sequence having 90-99%, e.g., at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, identity to SEQ ID NO: 99, 100, 294, 295, or 296. Thus, in some embodiments, the amino acid substitution is an arginine at position 257 of SEQ ID NO: 99, 100, 294, 295, or 296. In some embodiments, the amino acid substitution is an arginine at position 258 of SEQ ID NO: 99, 100, 294, 295, or 296. In some embodiments, the amino acid substitution is an arginine at position 259 of SEQ ID NO: 99, 100, 294, 295, or 296. In some embodiments, the amino acid substitution is an arginine at position 260 of SEQ ID NO: 99, 100, 294, 295, or 296. In some embodiments, the amino acid substitution is an arginine at position 261 of SEQ ID NO: 99, 100, 294, 295, or 296. In some embodiments, the amino acid substitution is an arginine at position 262 of SEQ ID NO: 99, 100, 294, 295, or 296.

[0127] In some embodiments, the amino acid substitution is a methionine at any one of amino acid positions 257-262 of SEQ ID NO:1. In some embodiments, the amino acid substitution is a methionine at any one of amino acid positions 257-262 of an amino acid sequence having 90-99%, e.g., at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, identity to SEQ ID NO:1. Thus, in some embodiments, the amino acid substitution is a methionine at position 257 of SEQ ID NO:1. In some embodiments, the amino acid substitution is a methionine at position 258 of SEQ ID NO:1. In some embodiments, the amino acid substitution is a methionine at position 259 of SEQ ID NO:1. In some embodiments, the amino acid substitution is a methionine at position 260 of SEQ ID NO:1. In some embodiments, the amino acid substitution is a methionine at position 261 of SEQ ID NO:1. In some embodiments, the amino acid substitution is a methionine at position 262 of SEQ ID NO:1.

[0128] In some embodiments, the amino acid substitution is a methionine at any one of amino acid positions 257-262 of SEQ ID NO: 99, 100, 294, 295, or 296. In some embodiments, the amino acid substitution is a methionine at any one of amino acid positions 257-262 of an amino acid sequence having 90-99%, e.g., at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, identity to SEQ ID NO: 99, 100, 294, 295, or 296. Thus, in some embodiments, the amino acid substitution is a methionine at position 257 of SEQ ID NO: 99, 100, 294, 295, or 296. In some embodiments, the amino acid substitution is a methionine at position 258 of SEQ ID NO: 99, 100, 294, 295, or 296. In some embodiments, the amino acid substitution is a methionine at position 259 of SEQ ID NO: 99, 100, 294, 295, or 296. In some embodiments, the amino acid substitution is a methionine at position 260 of SEQ ID NO: 99, 100, 294, 295, or 296. In some embodiments, the amino acid substitution is a methionine at position 261 of SEQ ID NO: 99, 100, 294, 295, or 296. In some embodiments, the amino acid substitution is a methionine at position 262 of SEQ ID NO: 99, 100, 294, 295, or 296.

[0129] In some embodiments, the amino acid substitution is a lysine at any one of amino acid positions 257-262 of SEQ ID NO:1. In some embodiments, the amino acid substitution is a lysine at any one of amino acid positions 257-262 of an amino acid sequence having 90-99%, e.g., at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, identity to SEQ ID NO:1. Thus, in some embodiments, the amino acid substitution is a lysine at position 257 of SEQ ID NO:1. In some embodiments, the amino acid substitution is a lysine at position 258 of SEQ ID NO:1. In some embodiments, the amino acid substitution is a lysine at position 259 of SEQ ID NO:1. In some embodiments, the amino acid substitution is a lysine at position 260 of SEQ ID NO:1. In some embodiments, the amino acid substitution is a lysine at position 261 of SEQ ID NO:1. In some embodiments, the amino acid substitution is a lysine at position 262 of SEQ ID NO:1.

[0130] In some embodiments, the amino acid substitution is a lysine at any one of amino acid positions 257-262 of SEQ ID NO: 99, 100, 294, 295, or 296. In some embodiments, the amino acid substitution is a lysine at any one of amino acid positions 257-262 of an amino acid sequence having 90-99%, e.g., at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, identity to SEQ ID NO: 99, 100, 294, 295, or 296. Thus, in some embodiments, the amino acid substitution is a lysine at position 257 of SEQ ID NO: 99, 100, 294, 295, or 296. Thus, in some embodiments, the amino acid substitution is a lysine at position 258 of SEQ ID NO: 99, 100, 294, 295, or 296. Thus, in some embodiments, the amino acid substitution is a lysine at position 259 of SEQ ID NO: 99, 100, 294, 295, or 296. Thus, in some embodiments, the amino acid substitution is a lysine at position 260 of SEQ ID NO: 99, 100, 294, 295, or 296. Thus, in some embodiments, the amino acid substitution is a lysine at position 261 of SEQ ID NO: 99, 100, 294, 295, or 296. Thus, in some embodiments, the amino acid substitution is a lysine at position 262 of SEQ ID NO: 99, 100, 294, 295, or 296.

[0131] In some embodiments, the amino acid substitution is a glutamine at any one of amino acid positions 257-262 of SEQ ID NO:1. In some embodiments, the amino acid substitution is a glutamine at any one of amino acid positions 257-262 of an amino acid sequence having 90-99%, e.g., at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, identity to SEQ ID NO:1. Thus, in some embodiments, the amino acid substitution is a glutamine at position 257 of SEQ ID NO:1. In some embodiments, the amino acid substitution is a glutamine at position 258 of SEQ ID NO:1. In some embodiments, the amino acid substitution is a glutamine at position 259 of SEQ ID NO:1. In some embodiments, the amino acid substitution is a glutamine at position 260 of SEQ ID NO:1. In some embodiments, the amino acid substitution is a glutamine at position 261 of SEQ ID NO:1. In some embodiments, the amino acid substitution is a glutamine at position 262 of SEQ ID NO:1.

[0132] In some embodiments, the amino acid substitution is a glutamine at any one of amino acid positions 257-262 of SEQ ID NO: 99, 100, 294, 295, or 296. In some embodiments, the amino acid substitution is a glutamine at any one of amino acid positions 257-262 of an amino acid sequence having 90-99%, e.g., at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, identity to SEQ ID NO: 99, 100, 294, 295, or 296. Thus, in some embodiments, the amino acid substitution is a glutamine at position 257 of SEQ ID NO: 99, 100, 294, 295, or 296. In some embodiments, the amino acid substitution is a glutamine at position 258 of SEQ ID NO: 99, 100, 294, 295, or 296. In some embodiments, the amino acid substitution is a glutamine at position 259 of SEQ ID NO: 99, 100, 294, 295, or 296. In some embodiments, the amino acid substitution is a glutamine at position 260 of SEQ ID NO: 99, 100, 294, 295, or 296. In some embodiments, the amino acid substitution is a glutamine at position 261 of SEQ ID NO: 99, 100, 294, 295, or 296. In some embodiments, the amino acid substitution is a glutamine at position 262 of SEQ ID NO: 99, 100, 294, 295, or 296.

[0133] In some embodiments, the amino acid substitution is a glutamic acid at any one of amino acid positions 257-262 of SEQ ID NO:1. In some embodiments, the amino acid substitution is a glutamic acid at any one of amino acid positions 257-262 of an amino acid sequence having 90-99%, e.g., at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, identity to SEQ ID NO:1. Thus, in some embodiments, the amino acid substitution is a glutamic acid at position 257 of SEQ ID NO:1. In some embodiments, the amino acid substitution is a glutamic acid at position 258 of SEQ ID NO:1. In some embodiments, the amino acid substitution is a glutamic acid at position 259 of SEQ ID NO:1. In some embodiments, the amino acid substitution is a glutamic acid at position 260 of SEQ ID NO:1. In some embodiments, the amino acid substitution is a glutamic acid at position 261 of SEQ ID NO:1. In some embodiments, the amino acid substitution is a glutamic acid at position 262 of SEQ ID NO:1.

[0134] In some embodiments, the amino acid substitution is a glutamic acid at any one of amino acid positions 257-262 of SEQ ID NO: 99, 100, 294, 295, or 296. In some embodiments, the amino acid substitution is a glutamic acid at any one of amino acid positions 257-262 of an amino acid sequence having 90-99%, e.g., at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, identity to SEQ ID NO: 99, 100, 294, 295, or 296. Thus, in some embodiments, the amino acid substitution is a glutamic acid at position 257 of SEQ ID NO: 99, 100, 294, 295, or 296. In some embodiments, the amino acid substitution is a glutamic acid at position 258 of SEQ ID NO: 99, 100, 294, 295, or 296. In some embodiments, the amino acid substitution is a glutamic acid at position 259 of SEQ ID NO: 99, 100, 294, 295, or 296. In some embodiments, the amino acid substitution is a glutamic acid at position 260 of SEQ ID NO: 99, 100, 294, 295, or 296. In some embodiments, the amino acid substitution is a glutamic acid at position 261 of SEQ ID NO: 99, 100, 294, 295, or 296. In some embodiments, the amino acid substitution is a glutamic acid at position 262 of SEQ ID NO: 99, 100, 294, 295, or 296.

[0135] In some embodiments, the T7 RNA polymerase variant comprises the amino acid sequence of SEQ ID NO:1 modified to include amino acid substitutions of high helix-propensity amino acids at positions G47, S43, R257, and / or G259. In some embodiments, the T7 RNA polymerase variant comprises an amino acid sequence having 90-99%, e.g., at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, identity to SEQ ID NO:1 modified to include amino acid substitutions of high helix-propensity amino acids at positions G47, S43, R257, and / or G259.

[0136] In some embodiments, the T7 RNA polymerase variant comprises the amino acid sequence of SEQ ID NO: 99, 100, 294, 295, or 296 modified to include amino acid substitutions of high helix propensity amino acids at positions G47, S43, R257, and / or G259. In some embodiments, the T7 RNA polymerase variant comprises an amino acid sequence having 90-99%, e.g., at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, identity to SEQ ID NO: 99, 100, 294, 295, or 296 modified to include amino acid substitutions of high helix propensity amino acids at positions G47, S43, R257, and / or G259.

[0137] In some embodiments, the T7 RNA polymerase variant comprises the amino acid sequence of SEQ ID NO: 2. In some embodiments, the T7 RNA polymerase variant comprises the amino acid sequence of SEQ ID NO: 3. In some embodiments, the T7 RNA polymerase variant comprises the amino acid sequence of SEQ ID NO: 4. In some embodiments, the T7 RNA polymerase variant comprises the amino acid sequence of SEQ ID NO: 5.

[0138] In some embodiments, the T7 RNA polymerase variant comprises the amino acid sequence of SEQ ID NO: 107 or 108. In some aspects, the T7 RNA polymerase variant comprises the amino acid sequence of SEQ ID NO: 109 or 110. In some aspects, the T7 RNA polymerase variant comprises the amino acid sequence of SEQ ID NO: 111 or 112. In some aspects, the T7 RNA polymerase variant comprises the amino acid sequence of SEQ ID NO: 113 or 114.

[0139] Also provided herein are RNA polymerase variants having at least two (two or more) substitutions. In some embodiments, the RNA polymerase comprises at least two high helix-propensity amino acid (e.g., alanine, isoleucine, leucine, arginine, methionine, lysine, glutamine, and / or glutamic acid) substitutions at any one of amino acid positions 42-47 (E42, S43, Y44, E45, M46, and / or G47) and / or amino acid positions 257-262 (R257, A258, G259, A260, L261, and / or A262) of SEQ ID NO:1 or an amino acid sequence having 90-99%, e.g., at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, identity to SEQ ID NO:1.

[0140] For example, the RNA polymerase variant may include amino acid substitutions (e.g., alanine, isoleucine, leucine, arginine, methionine, lysine, glutamine, and / or glutamic acid) at E42 and S43, E42 and Y44, E42 and E45, E42 and M46, E42 and G47, S43 and Y44, S43 and E45, S43 and M46, S43 and G47, Y44 and E45, Y44 and M46, Y44 and G47, E45 and M46, E45 and G47, or M46 and G47 of SEQ ID NO:1 or an amino acid sequence having 90-99%, e.g., at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO:1.

[0141] In some embodiments, the RNA polymerase variant is SEQ ID NO:1 or an amino acid sequence having 90-99%, e.g., at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, identity to SEQ ID NO:1. 8 and G259, A258 and A260, A258 and L261, A258 and A262, G259 and A260, G259 and L261, G259 and A262, A260 and L261, A260 and A262, or L261 and A262 may include amino acid substitutions (e.g., alanine, isoleucine, leucine, arginine, methionine, lysine, glutamine, and / or glutamic acid) at

[0142] In some embodiments, the RNA polymerase variant is selected from the group consisting of SEQ ID NO:1 or amino acid sequences having 90-99%, e.g., at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, identity to SEQ ID NO:1, including E42 and R257, E42 and A258, E42 and G259, E42 and A260, E42 and L261, E42 and A262, S43 and R257, S43 and A258, S43 and G259, S43 and A260, S43 and L261, S43 and A262, Y44 and R257, Y44 and A258, Y44 and G259, Y44 and and A262, E45 and R257, E45 and A258, E45 and G259, E45 and A260, E45 and L261, E45 and A262, M46 and R257, M46 and A258, M46 and G259, M46 and A260, M46 and L261, M46 and A262, G47 and R257, G47 and A258, G47 and G259, G47 and A260, G47 and L261, or G47 and A262 (e.g., alanine, isoleucine, leucine, arginine, methionine, lysine, glutamine, and / or glutamic acid).

[0143] In some embodiments, the RNA polymerase comprises amino acid substitutions at S43A and G47A of SEQ ID NO:1 or an amino acid sequence having 90 to 99%, e.g., at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO:1. In some embodiments, the RNA polymerase comprises amino acid substitutions at S43A and R257A of SEQ ID NO:1 or an amino acid sequence having 90 to 99%, e.g., at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO:1. In some embodiments, the RNA polymerase comprises amino acid substitutions at S43A and G259A of SEQ ID NO:1 or an amino acid sequence having 90 to 99%, e.g., at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO:1. In some embodiments, the RNA polymerase comprises amino acid substitutions at G47A and R257A in SEQ ID NO:1 or an amino acid sequence having 90 to 99%, e.g., at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO:1. In some embodiments, the RNA polymerase comprises amino acid substitutions at G47A and R257A in SEQ ID NO:1 or an amino acid sequence having 90 to 99%, e.g., at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO:1. In some embodiments, the RNA polymerase comprises amino acid substitutions at R257A and G259A in SEQ ID NO:1 or an amino acid sequence having 90 to 99%, e.g., at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO:1.In some embodiments, the RNA polymerase includes amino acid substitutions at G47A and G259A in SEQ ID NO:1 or an amino acid sequence having 90-99%, e.g., at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, identity to SEQ ID NO:1.

[0144] In some embodiments, the RNA polymerase variant comprises at least three (or at least four, or at least five) highly helical propensity amino acid (e.g., alanine, isoleucine, leucine, arginine, methionine, lysine, glutamine, and / or glutamic acid) substitutions at any one of amino acid positions 42-47 (E42, S43, Y44, E45, M46, and / or G47) and / or amino acid positions 257-262 (R257, A258, G259, A260, L261, and / or A262) of SEQ ID NO:1 or an amino acid sequence having 90-99%, e.g., at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, identity to SEQ ID NO:1.

[0145] In some embodiments, the RNA polymerase variants are E42R, S43A, S43E, S43L, S43R, E45R, E45L, M46A, G47A, G47E, G47L, G47R, N165W, E167M, E167N, E168I, E168T, E168V, A181F, A181W, G184M, E187F, A255Q, A255K, A255I, A255Y, R257A, R257E, R257L, R257W, G259A, G259B, G259C, G259D, G259E, G259F, G259G, G259H, G259I ... 1 or an amino acid sequence having 90 to 99%, for example, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, identity to SEQ ID NO:1, selected from the group consisting of SEQ ID NO:1, 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, and SEQ ID NO:1.

[0146] In some embodiments, the RNA polymerase comprises at least two high helix-prone amino acid (e.g., alanine, isoleucine, leucine, arginine, methionine, lysine, glutamine, and / or glutamic acid) substitutions at any one of amino acid positions 42-47 (E42, S43, Y44, E45, M46, and / or G47) and / or amino acid positions 257-262 (R257, A258, G259, A260, L261, and / or A262) of SEQ ID NO: 99, 100, 294, 295, or 296 or an amino acid sequence having 90-99%, e.g., at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, identity to SEQ ID NO: 99, 100, 294, 295, or 296.

[0147] For example, the RNA polymerase variant can be any of the amino acid positions E42 and S43, E42 and Y44, E42 and E50, E42 and S51, E42 and Y52, E42 and E60, E42 and S61, E42 and S62, E42 and S63, E42 and Y44, E42 and E64, E42 and S65, E42 and S66, E42 and S67, E42 and S68, E42 and S69, E42 and S61, E42 and S62, E42 and S63, E42 and S64, E42 and S65, E42 and S66, E42 and S67, E42 and S68, E42 and S69 ... In some embodiments, the polypeptide may include amino acid substitutions (e.g., alanine, isoleucine, leucine, arginine, methionine, lysine, glutamine, and / or glutamic acid) at: S43 and Y44, S43 and E45, S43 and M46, S43 and G47, Y44 and E45, Y44 and M46, Y44 and G47, E45 and M46, E45 and G47, or M46 and G47.

[0148] In some embodiments, the RNA polymerase variant is selected from the group consisting of SEQ ID NO: 99, 100, 294, 295, or 296, or an amino acid sequence having 90-99%, e.g., at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, identity to SEQ ID NO: 99, 100, 294, 295, or 296. Amino acid substitutions at 257 and L261, R257 and A262, A258 and G259, A258 and A260, A258 and L261, A258 and A262, G259 and A260, G259 and L261, G259 and A262, A260 and L261, A260 and A262, or L261 and A262 (e.g., alanine, isoleucine, leucine, arginine, methionine, lysine, glutamine, and / or glutamic acid).

[0149] In some embodiments, the RNA polymerase variant is selected from the group consisting of SEQ ID NO: 99, 100, 294, 295, or 296, or amino acid positions E42 and R257, E42 and A258, E42 and G259, E42 and A260, E42 and L261, E42 and A262, S43 and R257, S43 and A258, S43 and G259, S43 and A260, S43 and L261, S43 and A262, Y44 and R257, Y45 and R257, Y46 and R258, Y47 and R259, Y48 and R259, Y49 and R259, Y50 and R257, Y51 and R257, Y52 and R258, Y53 and R259, Y54 and R258, Y55 and R259, Y56 and R259, Y57 and R257, Y58 and R258, Y59 and R259, Y60 and R261, Y60 and R261, Y60 and R262, Y44 and R257, Y59 and R258, Y59 and R258, Y60 ... 44 and A258, Y44 and G259, Y44 and A260, Y44 and L261, Y44 and A262, E45 and R257, E45 and A258, E45 and G259, E45 and A260, E45 and L261, E45 and A262, M46 and R257, M46 and A258, M46 and G259, M46 and A260, M4 and L261, M46 and A262, G47 and R257, G47 and A258, G47 and G259, G47 and A260, G47 and L261, or G47 and A262 amino acid substitutions (e.g., alanine, isoleucine, leucine, arginine, methionine, lysine, glutamine, and / or glutamic acid).

[0150] In some embodiments, the RNA polymerase comprises the amino acid substitutions S43A and G47A of SEQ ID NO: 99, 100, 294, 295, or 296 or an amino acid sequence having 90-99%, e.g., at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, identity to SEQ ID NO: 99, 100, 294, 295, or 296. In some embodiments, the RNA polymerase comprises the amino acid substitutions S43A and R257A of SEQ ID NO: 99, 100, 294, 295, or 296 or an amino acid sequence having 90-99%, e.g., at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, identity to SEQ ID NO: 99, 100, 294, 295, or 296. In some embodiments, the RNA polymerase comprises amino acid substitutions S43A and G259A in SEQ ID NO: 99, 100, 294, 295, or 296 or an amino acid sequence having 90-99%, e.g., at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, identity to SEQ ID NO: 99, 100, 294, 295, or 296. In some embodiments, the RNA polymerase comprises amino acid substitutions G47A and R257A in SEQ ID NO: 99, 100, 294, 295, or 296 or an amino acid sequence having 90-99%, e.g., at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, identity to SEQ ID NO: 99, 100, 294, 295, or 296. In some embodiments, the RNA polymerase includes the amino acid substitutions G47A and R257A in SEQ ID NO: 99, 100, 294, 295, or 296 or an amino acid sequence having 90-99%, e.g., at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, identity to SEQ ID NO: 99, 100, 294, 295, or 296.In some embodiments, the RNA polymerase comprises amino acid substitutions R257A and G259A in SEQ ID NO: 99, 100, 294, 295, or 296 or an amino acid sequence having 90-99%, e.g., at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, identity to SEQ ID NO: 99, 100, 294, 295, or 296. In some embodiments, the RNA polymerase comprises amino acid substitutions G47A and G259A in SEQ ID NO: 99, 100, 294, 295, or 296 or an amino acid sequence having 90-99%, e.g., at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, identity to SEQ ID NO: 99, 100, 294, 295, or 296.

[0151] In some embodiments, the RNA polymerase variant comprises at least three (or at least four, or at least five) highly helical propensity amino acid (e.g., alanine, isoleucine, leucine, arginine, methionine, lysine, glutamine, and / or glutamic acid) substitutions at any one of amino acid positions 42-47 (E42, S43, Y44, E45, M46, and / or G47) and / or amino acid positions 257-262 (R257, A258, G259, A260, L261, and / or A262) of SEQ ID NO: 99, 100, 294, 295, or 296 or an amino acid sequence having 90-99%, e.g., at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, identity to SEQ ID NO: 99, 100, 294, 295, or 296.

[0152] In some embodiments, the RNA polymerase variant is selected from E42R, S43A, S43E, S43L, S43R, E45R, E45L, M46A, G47A, G47E, G47L, G47R, N165W, E167M, E167N, E168I, E168T, E168V, A181F, A181W, G184M, E187F, R257A, R257E, R257L, R257W, G259A, G259E, G259L, G259R, A260W, and A260R. 99, 100, 294, 295, or 296, or an amino acid sequence having 90 to 99%, e.g., at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, identity to SEQ ID NO: 99, 100, 294, 295, or 296, or at least one (or at least two, or at least three, or at least four, or at least five) amino acid substitution(s).

[0153] In some embodiments, the RNA polymerase variants are E42R, S43A, S43E, S43L, S43R, E45R, E45L, M46A, G47A, G47E, G47L, G47R, N165W, E167M, E167N, E168I, E168T, E168V, A181F, A181W, G184M, E187F, R257A, R257E, R257L, R257W, G259A, G259E, G259L, G259R ... At least one (or at least two, or at least three, or at least four, or at least five) amino acid substitution(s) selected from 59R, A260W, and A260R, and further comprising at least one (or at least two, or at least three, or at least four, or at least five) other amino acid substitutions (e.g., amino acid substitutions not provided herein).

[0154] Thus, the present disclosure relates to: E42R, S43A, S43E, S43L, S43R, E45R, E45L, M46A, G47A, G47E, G47L, G47R, N165W, E167M, E167N, E168I, E168T, E168V, A181F, A181W, G184M, E187F, A255K, A255Q, A255Y, A255I, R257A, R257E, R257L, R257W, G259A, G259E, G259L, G259 The present invention encompasses RNA polymerase variants that contain at least one (or at least two, or at least three, or at least four, or at least five) amino acid substitution(s) selected from A260R, A260W, and A260R, and have at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO:1.

[0155] The term "identity" refers to the relationship between two or more polypeptide (e.g., enzyme) or polynucleotide (nucleic acid) sequences, as determined by comparing the sequences. Identity also refers to the degree of sequence relatedness between or among sequences, as determined by the number of matches between strings of two or more amino acid or nucleic acid residues. Identity measures the percent identity match between two or more sequences, with gap alignment (if any) specified by a particular mathematical model or computer program (e.g., "algorithm"). The identity of related proteins or nucleic acids can be readily calculated by known methods. "Percent (%) identity" as applied to a polypeptide or polynucleotide sequence is defined as the percentage of residues (amino acid or nucleic acid residues) in a candidate amino acid or nucleic acid sequence that are identical to the residues in the amino acid or nucleic acid sequence of a second sequence after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent identity. Methods and computer programs for alignment are well known in the art. It is understood that identity depends on the calculation of percent identity, but may vary in value depending on gaps and penalties introduced in the calculation. In general, variants of a particular polynucleotide or polypeptide (e.g., antigen) have at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, but less than 100% sequence identity to that of a particular reference polynucleotide or polypeptide as determined by sequence alignment programs and parameters described herein and known to those of skill in the art. Such alignment tools include the BLAST suite of tools (Stephen F. Altschul, et al (1997), "Gapped BLAST and PSI-BLAST: a new generation of protein database search programs", Nucleic Acids Res. 25:3389-3402).Another common local alignment method is based on the Smith-Waterman algorithm (Smith, TF & Waterman, MS (1981) "Identification of common molecular subsequences." J. Mol. Biol. 147:195-197). A common global alignment method based on dynamic programming is the Needleman-Wunsch algorithm (Needleman, SB & Wunsch, CD (1970) "A general method applicable to the search for similarities in the amino acid sequences of two proteins." J. Mol. Biol. 48:443-453). Recently, the Fast Optimal Global Sequence Alignment Algorithm (FOGSAA) has been developed, which is said to generate global alignments of nucleotide and protein sequences faster than other optimal global alignment methods, including the Needleman-Wunsch algorithm.

[0156] Trinucleotide Cap Also provided herein is a co-transcriptional capping method for ribonucleic acid (RNA) synthesis. That is, the RNA is produced in a "one-pot" reaction and does not require a separate capping reaction. Thus, the method, in some embodiments, comprises reacting a polynucleotide template with a T7 RNA polymerase variant, nucleoside triphosphates, and a cap analog under in vitro transcription reaction conditions to produce an RNA transcript.

[0157] The cap analog can be, for example, a dinucleotide cap, a trinucleotide cap, or a tetranucleotide cap. In some embodiments, the cap analog is a dinucleotide cap. In some embodiments, the cap analog is a trinucleotide cap. In some embodiments, the cap analog is a tetranucleotide cap.

[0158] The trinucleotide cap, in some embodiments, is a compound of formula (I) [ka] or a stereoisomer, tautomer, or salt thereof, wherein [ka] Ring B 1 is a modified or unmodified guanine, Ring B 2 and Ring B 3 are each independently a nucleobase or a modified nucleobase; X 2 is O, S(O) p , N.R. 24 , or CR 25 R 26 where p is 0, 1, or 2; Y 0 is O or CR 6 R 7 and Y1 is O, S(O) n , C.R. 6 R 7 , or N.R. 8 where n is 0, 1, or 2; Each - is a single bond or is absent. When each - is a single bond, Yi is O, S(O) n , C.R. 6 R 7 , or N.R. 8 and for each --- absent, Y 1 is void, Y 2 is (OP(O)R4 ) m (m is 0, 1, or 2) or -O-(CR 40 R 41 )uQ 0 -(CR 42 R 43 )v-, where Q 0 is a bond, O, S(O) r , N.R. 44 , or CR 45 R 46 wherein r is 0, 1, or 2; and each of u and v is independently 1, 2, 3, or 4; Each R 2 and R 2 ´ are independently halo, LNA, or OR 3 and Each R 3 are independently H, C 1 -C 6 Alkyl, C 2 -C 6 Alkenyl, or C 2 -C 6 alkynyl, R 3 is C 1 -C 6 Alkyl, C 2 -C 6 Alkenyl, or C 2 -C 6 When alkynyl, it is halo, OH, or one or more of OH or OC(O)-C 1 -C 6 C optionally substituted with alkyl 1 -C 6 Optionally substituted with one or more of alkoxyl, Each R 4 and R 4 ´ is independently H, halo, C 1 -C 6 Alkyl, OH, SH, SeH, or BH 3 - and R 6 , R 7 , and R 8 Each of the following is independently -Q 1 -T 1 where Q1 is a bond, or halo, cyano, OH, and C 1 -C 6 C optionally substituted with one or more of alkoxy 1 -C 3 is an alkyl linker, T 1 is H, halo, OH, COOH, cyano, or R s1 where R s1 is C 1 -C 3 Alkyl, C 2 -C 6 Alkenyl, C 2 -C 6 Alkynyl, C 1 -C 6 Alkoxyl, C(O)OC 1 -C 6 Alkyl, C 3 -C 8 Cycloalkyl, C 6 -C 10 Aryl, NR 31 R 32 , (NR 31 R 32 R 33 ) + , 4-12 membered heterocycloalkyl, or 5- or 6-membered heteroaryl; R s1 is halo, OH, oxo, C 1 -C 6 Alkyl, COOH, C(O)OC 1 -C 6 Alkyl, Cyano, C 1 -C 6 Alkoxyl, NR 31 R 32 , (NR 31 R 32 R 33 ) + , C 3 -C 8 Cycloalkyl, C 6 -C 10 optionally substituted with one or more substituents selected from the group consisting of aryl, 4- to 12-membered heterocycloalkyl, and 5- or 6-membered heteroaryl; R 10 , R 11 , R12 , R 13 R 14 , and R 15 Each of the following is independently -Q 2 -T 2 where Q 2 is a bond, or halo, cyano, OH, and C 1 -C 6 C optionally substituted with one or more of alkoxy 1 -C 3 is an alkyl linker, T 2 H, halo, OH, NH 2 , Cyano, NO 2 , N 3 , R s2 OR s2 where R s2 is C 1 -C 6 Alkyl, C 2 -C 6 Alkenyl, C 2 -C 6 Alkynyl, C 3 -C 8 Cycloalkyl, C 6 -C 10 Aryl, NHC(O)-C 1 -C 6 Alkyl, NR 31 R 32 , (NR 31 R 32 R 33 ) + , 4-12 membered heterocycloalkyl, or 5- or 6-membered heteroaryl; R s2 is halo, OH, oxo, C 1 -C 6 Alkyl, COOH, C(O)OC 1 -C 6 Alkyl, Cyano, C 1 -C 6 Alkoxyl, NR 31 R 32 , (NR 31 R 32 R 33 ) + , C 3 -C 8 Cycloalkyl, C 6 -C10 Optionally substituted with one or more substituents selected from the group consisting of aryl, 4- to 12-membered heterocycloalkyl, and 5- or 6-membered heteroaryl, or R 12 is R 14 and together with oxo, or R 13 is R 15 Together with oxo, R 20 , R 21 , R 22 , and R 23 Each of the following is independently -Q 3 -T 3 where Q 3 is a bond, or halo, cyano, OH, and C 1 -C 6 C optionally substituted with one or more of alkoxy 1 -C 3 is an alkyl linker, T 3 H, halo, OH, NH 2 , Cyano, NO 2 , N 3 , R S3 OR S3 where R S3 is C 1 -C 6 Alkyl, C 2 -C 6 Alkenyl, C 2 -C 6 Alkynyl, C 3 -C 8 Cycloalkyl, C 6 -C 10 Aryl, NHC(O)-C 1 -C 6 Alkyl, mono-C 1 -C 6 Alkylamino, Di-C 1 -C 6 alkylamino, 4-12 membered heterocycloalkyl, or 5- or 6-membered heteroaryl; Rs 3 is halo, OH, oxo, C 1 -C 6 Alkyl, COOH, C(O)OC 1 -C 6 Alkyl, Cyano, C1 -C 6 Alkoxy, Amino, Mono-C 1 -C 6 Alkylamino, Di-C 1 -C 6 Alkylamino, C 3 -C 8 Cycloalkyl, C 6 -C 10 optionally substituted with one or more substituents selected from the group consisting of aryl, 4- to 12-membered heterocycloalkyl, and 5- or 6-membered heteroaryl; R 24 , R 25 , and R 26 each independently is H or C 1 -C 6 is alkyl, R 27 and R 28 each independently is H or OR 29 or R 27 and R 28 Together, OR 30 -O, and each R 29 are independently H, C 1 -C 6 Alkyl, C 2 -C 6 Alkenyl, or C 2 -C 6 alkynyl, R 29 , is C 1 -C 6 Alkyl, C 2 -C 6 Alkenyl, or C 2 -C 6 When alkynyl, it is halo, OH, or one or more of OH or OC(O)-C 1 -C 6 C optionally substituted with alkyl 1 -C 6 Optionally substituted with one or more of alkoxyl, R 30 are halo, OH, and C 1 -C 6 C optionally substituted with one or more of alkoxyl 1-C 6 is alkylene, R 31 , R 32 , and R 33 each independently represents H, C 1 -C 6 Alkyl, C 3 -C 8 Cycloalkyl, C 6 -C 10 aryl, 4- to 12-membered heterocycloalkyl, or 5- or 6-membered heteroaryl; R 40 , R 41 , R 42 , and R 43 each independently represents H, halo, OH, cyano, N 3 ,OP(O)R 47 R 48 , or one or more OP(O)R 47 R 48 C optionally replaced with 1 -C 6 alkyl or one R 41 and one R 43 are the carbon atoms to which they are attached and Q 0 Together with C 4 -C 10 Cycloalkyl, 4-14 membered heterocycloalkyl, C 6 -C 10 aryl, or 5-14 membered heteroaryl, each of which is selected from the group consisting of cycloalkyl, heterocycloalkyl, phenyl, and 5-6 membered heteroaryl, and each of which is selected from the group consisting of OH, halo, cyano, N 3 , OXO, OP(O)R 47 R 48 , C 1 -C 6 Alkyl, C 1 -C 6 Haloalkyl, COOH, C(O)OC 1 -C 6 Alkyl, C 1 -C 6 Alkoxyl, C 1 -C 6 Haloalkoxyl, Amino, Mono-C 1 -C 6Alkylamino and di-C 1 -C 6 optionally substituted with one or more of alkylamino; R 44 , H, C 1 -C 6 an alkyl or amine protecting group, R 45 and R 46 each of which is independently H, OP(O)R 47 R 48 , or one or more OP(O)R 47 R 48 C optionally replaced with 1 -C 6 is alkyl, R 47 and R 48 each independently represents H, halo, C 1 -C 6 Alkyl, OH, SH, SeH, or BH 3 - It is.

[0159] It is understood that the cap analogs provided herein may include any of the cap analogs described in International Publication WO2017 / 066797, published April 20, 2017, the entirety of which is incorporated herein by reference.

[0160] In some embodiments, B 2 The intermediate position can be a non-ribose molecule, such as arabinose.

[0161] In some embodiments, R 2 is an ethyl type.

[0162] Thus, in some embodiments, the trinucleotide cap comprises the following structure: [ka]

[0163] In other embodiments, the trinucleotide cap comprises the following structure: [ka]

[0164] In yet other embodiments, the trinucleotide cap comprises the following structure: [ka]

[0165] In yet other embodiments, the trinucleotide cap comprises the following structure: [ka]

[0166] The trinucleotide cap, in some embodiments, comprises a sequence selected from the following sequences: GAA, GAC, GAG, GAU, GCA, GCC, GCG, GCU, GGA, GGC, GGG, GGU, GUA, GUC, GUG, and GUU. In some embodiments, the trinucleotide cap comprises GAA. In some embodiments, the trinucleotide cap comprises GAC. In some embodiments, the trinucleotide cap comprises GAG. In some embodiments, the trinucleotide cap comprises GAU. In some embodiments, the trinucleotide cap comprises GCA. In some embodiments, the trinucleotide cap comprises GCC. In some embodiments, the trinucleotide cap comprises GCG. In some embodiments, the trinucleotide cap comprises GCU. In some embodiments, the trinucleotide cap comprises GGA. In some embodiments, the trinucleotide cap comprises GGC. In some embodiments, the trinucleotide cap comprises GGG. In some embodiments, the trinucleotide cap comprises GGU. In some embodiments, the trinucleotide cap comprises GUA. In some embodiments, the trinucleotide cap comprises GUC. In some embodiments, the trinucleotide cap comprises GUG. In some embodiments, the trinucleotide cap comprises GUU.

[0167] In some embodiments, the trinucleotide cap has the following sequence: 7 GppApA, m 7 GpppApC, m 7 GppApG, m 7 GppApU,m 7 GppCpA, m 7 GppCpC, m 7 GppCpG, m 7 GppCpU,m 7 GppGpA, m 7 GppGpC, m 7 GppGpG, m 7 GppGpU,m 7 GpppUpA,m7 GpppUpC,m 7 GpppUpG, and m 7 GpppUpU.

[0168] In some embodiments, the trinucleotide cap is 7 In some embodiments, the trinucleotide cap comprises m 7 In some embodiments, the trinucleotide cap comprises m 7 In some embodiments, the trinucleotide cap comprises m 7 In some embodiments, the trinucleotide cap comprises m 7 In some embodiments, the trinucleotide cap comprises m 7 In some embodiments, the trinucleotide cap comprises m 7 In some embodiments, the trinucleotide cap comprises m 7 In some embodiments, the trinucleotide cap comprises m 7 In some embodiments, the trinucleotide cap comprises GpppGpA. 7 In some embodiments, the trinucleotide cap comprises GpppGpC. 7 In some embodiments, the trinucleotide cap comprises m 7 In some embodiments, the trinucleotide cap comprises m 7 In some embodiments, the trinucleotide cap comprises m 7 In some embodiments, the trinucleotide cap comprises m 7 In some embodiments, the trinucleotide cap comprises m 7 Includes GpppUpU.

[0169] The trinucleotide cap, in some embodiments, has the following sequence: 7 G 3′OMe pppApA, m 7 G 3′OMe pppApC, m 7 G 3′OMe pppApG, m 7 G 3′OMe pppApU,m 7 G 3′OMe pppCpA, m 7 G 3′OMe pppCpC, m 7 G 3′OMe pppCpG,m 7 G 3′OMe pppCpU,m 7 G 3′OMe pppGpA,m 7 G 3′OMe pppGpC, m 7 G 3′OMe pppGpG, m 7 G 3′OMe pppGpU,m 7 G 3′OMe pppUpA,m 7 G 3′OMe pppUpC,m 7 G 3′OMe pppUpG, and m 7 G 3′OMe pppUpU.

[0170] In some embodiments, the trinucleotide cap is 7 G 3′OMe In some embodiments, the trinucleotide cap comprises m 7 G 3′OMe In some embodiments, the trinucleotide cap comprises pppApC. 7 G 3′OMe In some embodiments, the trinucleotide cap comprises pppApG. 7 G 3′OMe In some embodiments, the trinucleotide cap comprises pppApU. 7 G 3′OMe In some embodiments, the trinucleotide cap comprises pppCpA.7 G 3′OMe In some embodiments, the trinucleotide cap comprises m 7 G 3′OMe In some embodiments, the trinucleotide cap comprises m 7 G 3′OMe In some embodiments, the trinucleotide cap comprises pppCpU. 7 G 3′OMe In some embodiments, the trinucleotide cap comprises pppGpA. 7 G 3′OMe In some embodiments, the trinucleotide cap comprises pppGpC. 7 G 3′OMe In some embodiments, the trinucleotide cap comprises pppGpG. 7 G 3′OMe In some embodiments, the trinucleotide cap comprises pppGpU. 7 G 3′OMe In some embodiments, the trinucleotide cap comprises pppUpA. 7 G 3′OMe In some embodiments, the trinucleotide cap comprises pppUpC. 7 G 3′OMe In some embodiments, the trinucleotide cap comprises pppUpG. 7 G 3′OMe Includes pppUpU.

[0171] The trinucleotide cap, in some embodiments, has the following sequence: 7 G 3′OMe pppA 2′OMe pA, m 7 G 3′OMe pppA 2′OMe pC, m 7 G 3′OMe pppA 2′OMe pG,m 7 G 3′OMe pppA 2′OMe pU,m 7 G 3′OMe pppC 2′OMe pA, m 7 G3′OMe pppC 2′OMe pC, m 7 G 3′OMe pppC 2′OMe pG,m 7 G 3′OMe pppC 2′OMe pU,m 7 G 3′OMe pppG 2′OMe pA, m 7 G 3′OMe pppG 2′OMe pC, m 7 G 3′OMe pppG 2′OMe pG,m 7 G 3′OMe pppG 2′OMe pU,m 7 G 3′OMe pppU 2′OMe pA, m 7 G 3′OMe pppU 2′OMe pC, m 7 G 3′OMe pppU 2′OMe pG, and m 7 G 3′OMe pppU 2′OMe pU.

[0172] In some embodiments, the trinucleotide cap is 7 G 3′OMe pppA 2′OMe In some embodiments, the trinucleotide cap comprises m 7 G 3′OMe pppA 2′OMe In some embodiments, the trinucleotide cap comprises m 7 G 3′OMe pppA 2′OMe In some embodiments, the trinucleotide cap comprises m 7 G 3′OMe pppA 2′OMe In some embodiments, the trinucleotide cap comprises m 7 G 3′OMe pppC 2′OMe In some embodiments, the trinucleotide cap comprises m 7 G 3′OMepppC 2′OMe In some embodiments, the trinucleotide cap comprises m 7 G 3′OMe pppC 2′OMe In some embodiments, the trinucleotide cap comprises m 7 G 3′OMe pppC 2′OMe In some embodiments, the trinucleotide cap comprises m 7 G 3′OMe pppG 2′OMe In some embodiments, the trinucleotide cap comprises m 7 G 3′OMe pppG 2′OMe In some embodiments, the trinucleotide cap comprises m 7 G 3′OMe pppG 2′OMe In some embodiments, the trinucleotide cap comprises m 7 G 3′OMe pppG 2′OMe In some embodiments, the trinucleotide cap comprises m 7 G 3′OMe pppU 2′OMe In some embodiments, the trinucleotide cap comprises m 7 G 3′OMe pppU 2′OMe In some embodiments, the trinucleotide cap comprises m 7 G 3′OMe pppU 2′OMe In some embodiments, the trinucleotide cap comprises m 7 G 3′OMe pppU 2′OMe Contains pU.

[0173] The trinucleotide cap may further comprise, in some embodiments, the following sequence: 7 GpppA 2′OMe pA, m 7 GpppA 2′OMe pC, m 7 GpppA 2′OMe pG,m 7 GpppA2′OMe pU,m 7 GpppC 2′OMe pA, m 7 GpppC 2′OMe pC, m 7 GpppC 2′OMe pG,m 7 GpppC 2′OMe pU,m 7 GpppG 2′OMe pA, m 7 GpppG 2′OMe pC, m 7 GpppG 2′OMe pG,m 7 GpppG 2′OMe pU,m 7 GpppU 2′OMe pA, m 7 GpppU 2′OMe pC, m 7 GpppU 2′OMe pG, and m 7 GpppU 2′OMe pU.

[0174] In some embodiments, the trinucleotide cap is 7 GpppA 2′OMe In some embodiments, the trinucleotide cap comprises m 7 GpppA 2′OMe In some embodiments, the trinucleotide cap comprises m 7 GpppA 2′OMe In some embodiments, the trinucleotide cap comprises m 7 GpppA 2′OMe In some embodiments, the trinucleotide cap comprises m 7 GpppC 2′OMe In some embodiments, the trinucleotide cap comprises m 7 GpppC 2′OMe In some embodiments, the trinucleotide cap comprises m 7 GpppC 2′OMe In some embodiments, the trinucleotide cap comprises m 7 GpppC 2′OMeIn some embodiments, the trinucleotide cap comprises m 7 GpppG 2′OMe In some embodiments, the trinucleotide cap comprises m 7 GpppG 2′OMe In some embodiments, the trinucleotide cap comprises m 7 GpppG 2′OMe In some embodiments, the trinucleotide cap comprises m 7 GpppG 2′OMe In some embodiments, the trinucleotide cap comprises m 7 GpppU 2′OMe In some embodiments, the trinucleotide cap comprises m 7 GpppU 2′OMe In some embodiments, the trinucleotide cap comprises m 7 GpppU 2′OMe In some embodiments, the trinucleotide cap comprises m 7 GpppU 2′OMe Contains pU.

[0175] In some embodiments, the trinucleotide cap comprises GAG. In some embodiments, the trinucleotide cap comprises GCG. In some embodiments, the trinucleotide cap comprises GUG. In some embodiments, the trinucleotide cap comprises GGG.

[0176] In vitro transcription method Some embodiments of the present disclosure provide methods of producing (synthesizing) an RNA transcript (e.g., an mRNA transcript) comprising contacting a DNA template with an RNA polymerase (e.g., a T7 RNA polymerase, such as a T7 RNA polymerase variant) under conditions that result in the production of the RNA transcript.

[0177] In some embodiments, the method comprises contacting a DNA template with a T7 RNA polymerase (e.g., SEQ ID NO: 1, 99, or 100) variant having an S43A substitution (e.g., an S43A T7 RNAP variant or an S43A* T7 RNAP variant) under conditions that result in the production of an RNA transcript. In other embodiments, the method comprises contacting a DNA template with a T7 RNA polymerase (e.g., SEQ ID NO: 1, 99, or 100) variant having a G47A substitution (e.g., a G47A T7 RNAP variant or a G47A* T7 RNAP variant) under conditions that result in the production of an RNA transcript. In yet other embodiments, the method comprises contacting a DNA template with a T7 RNA polymerase (e.g., SEQ ID NO: 1, 99, or 100) variant having an R257A substitution (e.g., an R257A T7 RNAP variant or an R257A* T7 RNAP variant) under conditions that result in the production of an RNA transcript. In yet other embodiments, the method includes contacting a DNA template with a T7 RNA polymerase (e.g., SEQ ID NO: 1, 99, or 100) variant having a G259A substitution (e.g., a G259A T7 RNAP variant or a G259A* T7 RNAP variant) under conditions that result in the production of an RNA transcript.

[0178] In some embodiments, the method includes contacting the DNA template with a T7 RNA polymerase variant that includes (at least one) additional C-terminal amino acid (e.g., Gly, Ala, GlyGly, AlaAla, GlyAla, or AlaGly).

[0179] In some embodiments, the method further comprises: reacting the DNA template with one or more of the following amino acids: E42R, S43A, S43E, S43L, S43R, E45R, E45L, M46A, G47A, G47E, G47L, G47R, N165W, E167M, E167N, E168I, E168T, E168V, A181F, A181W, G184M, E187F, A255Q, A255K, A255I, A The method comprises contacting the T7 RNA polymerase with a T7 RNA polymerase variant comprising a 255Y, R257A, R257E, R257L, R257W, G259A, G259E, G259L, G259R, A260W, or A260R substitution, or any combination of two or more of the foregoing substitutions and optionally an additional C-terminal amino acid (e.g., Gly, Ala, GlyGly, AlaAla, GlyAla, or AlaGly).

[0180] In some aspects, the disclosure provides methods of performing an IVT reaction, comprising contacting a DNA template with an RNA polymerase (e.g., a T7 RNA polymerase, such as a T7 RNA polymerase variant) in the presence of nucleoside triphosphates and a buffer under conditions that result in the production of an RNA transcript.

[0181] Another aspect of the disclosure provides a co-transcriptional capping method that includes reacting a polynucleotide template with a T7 RNA polymerase variant, nucleoside triphosphates, and a cap analog under conditions to produce an in vitro transcript, to produce an RNA transcript.

[0182] In some embodiments, the co-transcriptional capping method for RNA synthesis comprises coupling a polynucleotide template to (a) a T7 RNA polymerase variant, the T7 RNA polymerase variant comprising at least one amino acid substitution, as compared to a wild-type RNA polymerase, that causes at least one loop structure of the RNA polymerase variant to undergo a conformational change to a helical structure as the RNA polymerase variant transitions from an initiation complex to an elongation complex, under in vitro transcription reaction conditions to produce an RNA transcript, (b) a nucleoside triphosphate, and (c) a nucleotide sequence selected from the group consisting of GpppA, Gppp ...2′Ome The polynucleotide template comprises reacting with a trinucleotide cap containing pG, wherein the polynucleotide template contains a 2'-deoxythymidine residue at template position +1.

[0183] IVT conditions typically require a purified linear DNA template with a promoter, nucleoside triphosphates, a buffer system containing dithiothreitol (DTT) and magnesium ions, and an RNA polymerase. The exact conditions used in the transcription reaction depend on the amount of RNA required for a particular application. A typical IVT reaction is performed by incubating a DNA template with RNA polymerase and nucleoside triphosphates containing GTP, ATP, CTP, UTP (or nucleotide analogs) in a transcription buffer. RNA transcripts with 5'-terminal guanosine triphosphate are generated from this reaction.

[0184] Deoxyribonucleic acid (DNA) is simply a nucleic acid template for RNA polymerase. The DNA template may include a polynucleotide that encodes a polypeptide of interest (e.g., an antigenic polypeptide). The DNA template, in some embodiments, includes an RNA polymerase promoter (e.g., a T7 RNA polymerase promoter) located 5' from the polynucleotide that encodes the polypeptide of interest and operably linked to the polynucleotide. The DNA template may also include a nucleotide sequence that encodes a polyadenylation (polyA) tail located at the 3' end of the gene of interest.

[0185] Polypeptides of interest include, but are not limited to, biologics, antibodies, antigens (vaccines), and therapeutic proteins. The term "protein" includes peptides.

[0186] The RNA transcript, in some embodiments, is the product of an IVT reaction. The RNA transcript, in some embodiments, is a messenger RNA (mRNA) that includes a nucleotide sequence encoding a polypeptide of interest linked to a polyA tail. In some embodiments, the mRNA is a modified mRNA (mmRNA) that includes at least one modified nucleotide.

[0187] A nucleotide comprises a nitrogenous base, a pentose sugar (ribose or deoxyribose), and at least one phosphate group. Nucleotides include nucleoside monophosphates, nucleoside diphosphates, and nucleoside triphosphates. A nucleoside monophosphate (NMP) comprises a nucleobase and one phosphate bound to a ribose. A nucleoside diphosphate (NDP) comprises a nucleobase and two phosphates bound to a ribose. A nucleoside triphosphate (NTP) comprises a nucleobase and three phosphates bound to a ribose. A nucleotide analog is a compound that has the general structure of a nucleotide or is structurally similar to a nucleotide. Nucleotide analogs include, for example, nucleobase analogs, sugar analogs, and / or analogs of the phosphate group(s) of a nucleotide.

[0188] A nucleoside contains a nitrogenous base and a pentose sugar. Thus, a nucleoside and a phosphate group form a nucleotide. A nucleoside analog is a compound that has the general structure of a nucleoside or is structurally similar to a nucleoside. For example, a nucleoside analog includes an analog of the nucleic acid base and / or an analog of the sugar of the nucleoside.

[0189] The term "nucleotide" is understood to include naturally occurring, synthetic, and modified nucleotides, unless otherwise specified. Examples of naturally occurring nucleotides provided herein for use in generating RNA, e.g., in IVT reactions, include adenosine triphosphate (ATP), guanosine triphosphate (GTP), cytidine triphosphate (CTP), uridine triphosphate (UTP), and 5-methyluridine triphosphate (MTP).5 In some embodiments, adenosine diphosphate (ADP), guanosine diphosphate (GDP), cytidine diphosphate (CDP), and / or uridine diphosphate (UDP) are used.

[0190] Examples of nucleotide analogs include antiviral nucleotide analogs, phosphate analogs (soluble or immobilized, hydrolyzable or non-hydrolyzable), dinucleotides, trinucleotides, tetranucleotides, such as cap analogs, or precursors / substrates for enzymatic capping (vaccinia or ligase), nucleotides labeled with functional groups to facilitate ligation / conjugation of the cap or 5' portion (IRES), 5'PO to facilitate ligation of the cap or 5' portion, 4 Examples of antiviral nucleotide / nucleoside analogs include, but are not limited to, nucleotides labeled with , or nucleotides labeled with functional groups / protecting groups that can be chemically or enzymatically cleaved. Examples of antiviral nucleotide / nucleoside analogs include, but are not limited to, ganciclovir, entecavir, telbivudine, vidarabine, and cidofovir.

[0191] Modified nucleotides may include modified nucleobases. For example, an RNA transcript (e.g., an mRNA transcript) of the present disclosure may include modified nucleobases selected from pseudouridine (ψ), 1-methylpseudouridine (m1ψ), 1-ethylpseudouridine, 2-thiouridine, 4'-thiouridine, 2-thio-1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-pseudouridine, 2-thio-5-aza-uridine, 2-thio-dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-pseudouridine, 4-methoxy-2-thio-pseudouridine, 4-methoxy-pseudouridine, 4-thio-1-methyl-pseudouridine, 4-thio-pseudouridine, 5-aza-uridine, dihydropseudouridine, 5-methyluridine, 5-methoxyuridine (mo5U), and 2'-O-methyluridine. In some embodiments, an RNA transcript (eg, an mRNA transcript) includes a combination of at least two (eg, 2, 3, 4 or more) of the foregoing modified nucleobases.

[0192] Nucleoside triphosphates (NTPs) provided herein can include unmodified or modified ATP, modified or unmodified UTP, modified or unmodified GTP, and / or modified or unmodified CTP. In some embodiments, the NTPs of the IVT reaction include unmodified ATP. In some embodiments, the NTPs of the IVT reaction include modified ATP. In some embodiments, the NTPs of the IVT reaction include unmodified UTP. In some embodiments, the NTPs of the IVT reaction include modified UTP. In some embodiments, the NTPs of the IVT reaction include unmodified GTP. In some embodiments, the NTPs of the IVT reaction include modified GTP. In some embodiments, the NTPs of the IVT reaction include unmodified CTP. In some embodiments, the NTPs of the IVT reaction include modified CTP.

[0193] The concentrations of nucleoside triphosphate and cap analog present in the IVT reaction can vary. In some embodiments, the NTP and cap analog are present in equimolar concentrations in the reaction. In some embodiments, the molar ratio of cap analog (e.g., trinucleotide cap) to nucleoside triphosphate in the reaction is greater than 1:1. For example, the molar ratio of cap analog to nucleoside triphosphate in the reaction can be 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 15:1, 20:1, 25:1, 50:1, or 100:1. In some embodiments, the molar ratio of cap analog (e.g., trinucleotide cap) to nucleoside triphosphate in the reaction is less than 1:1. For example, the molar ratio of cap analog (e.g., trinucleotide cap) to nucleoside triphosphate in the reaction can be 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:15, 1:20, 1:25, 1:50, or 1:100.

[0194] The composition of NTPs in the IVT reaction may also vary. For example, ATP may be used in excess of GTP, CTP, and UTP. As a non-limiting example, an IVT reaction may include 7.5 millimoles of GTP, 7.5 millimoles of CTP, 7.5 millimoles of UTP, and 3.75 millimoles of ATP. The same IVT reaction may include 3.75 millimoles of a cap analog (e.g., a trinucleotide cap). In some embodiments, the molar ratio of G:C:U:A:cap is 1:1:1:0.5:0.5. In some embodiments, the molar ratio of G:C:U:A:cap is 1:1:0.5:1:0.5. In some embodiments, the molar ratio of G:C:U:A:cap is 1:0.5:1:1:0.5. In some embodiments, the molar ratio of G:C:U:A:cap is 0.5:1:1:1:0.5.

[0195] In some embodiments, the RNA transcript (e.g., the mRNA transcript) may contain pseudouridine (ψ), 1-methylpseudouridine (m 1 ψ), 5-methoxyuridine (mo5 U), 5-methylcytidine (m 5 C), α-thioguanosine, and α-thioadenosine. In some embodiments, an RNA transcript (e.g., an mRNA transcript) includes a combination of at least two (e.g., 2, 3, 4 or more) of the foregoing modified nucleobases.

[0196] In some embodiments, the RNA transcript (e.g., the mRNA transcript) includes pseudouridine (ψ). In some embodiments, the RNA transcript (e.g., the mRNA transcript) includes 1-methylpseudouridine (m 1 In some embodiments, the RNA transcript (e.g., the mRNA transcript) includes 5-methoxyuridine (mo 5 In some embodiments, the RNA transcript (e.g., the mRNA transcript) includes 5-methylcytidine (mU). 5 In some embodiments, the RNA transcript (e.g., the mRNA transcript) includes α-thio-guanosine. In some embodiments, the RNA transcript (e.g., the mRNA transcript) includes α-thio-adenosine.

[0197] In some embodiments, a polynucleotide (e.g., an RNA polynucleotide, such as an mRNA polynucleotide) is uniformly modified (e.g., completely modified, modified throughout the sequence) for a particular modification. For example, a polynucleotide is modified with 1-methylpseudouridine (mPs) or ... 1 ψ), which means that all uridine residues in the mRNA sequence are modified with 1-methylpseudouridine (m 1ψ). Similarly, a polynucleotide can be uniformly modified for any type of nucleoside residue present in the sequence by substitution with a modified residue such as any of those described above. Alternatively, a polynucleotide (e.g., an RNA polynucleotide such as an mRNA polynucleotide) can be non-uniformly modified (e.g., partially modified, where part of the sequence is modified). Each option represents a separate embodiment of the present invention.

[0198] In some embodiments, the buffer system comprises tris. For example, the concentration of tris used in the IVT reaction is at least 10 mM, at least 20 mM, at least 30 mM, at least 40 mM, at least 50 mM, at least 60 mM, at least 70 mM, at least 80 mM, at least 90 mM, at least 100 mM, or at least 110 mM phosphate. In some embodiments, the concentration of phosphate is 20-60 mM or 10-100 mM.

[0199] In some embodiments, the buffer system includes dithiothreitol (DTT). The concentration of DTT used in the IVT reaction can be, for example, at least 1 mM, at least 5 mM, or at least 50 mM. In some embodiments, the concentration of DTT used in the IVT reaction is 1-50 mM or 5-50 mM. In some embodiments, the concentration of DTT used in the IVT reaction is 5 mM.

[0200] In some embodiments, the buffer system comprises magnesium. In some embodiments, the NTPs and magnesium ions (Mg 2+ ; e.g. MgCl 2 The molar ratio of NTP to magnesium ion is 1:1 to 1:5. For example, the molar ratio of NTP to magnesium ion can be 1:1, 1:2, 1:3, 1:4, or 1:5.

[0201] In some embodiments, the NTP and cap analog (e.g., a trinucleotide cap such as GAG) present in the IVT reaction and magnesium ion (Mg 2+ ; e.g. MgCl 2 For example, the molar ratio of NTP+trinucleotide cap (e.g., GAG) to magnesium ion can be 1:1, 1:2, 1:3, 1:4, or 1:5.

[0202] In some embodiments, the buffer system comprises Tris-HCl, spermidine (e.g., at a concentration of 1-30 mM), TRITON® X-100 (polyethylene glycol p-(1,1,3,3-tetramethylbutyl)-phenyl ether), and / or polyethylene glycol (PEG).

[0203] The addition of nucleoside triphosphates (NTPs) to the 3' end of a growing RNA strand is catalyzed by a polymerase, such as T7 RNA polymerase, e.g., any one or more of the T7 RNA polymerase variants of the present disclosure (e.g., S43A and / or G47A). In some embodiments, the RNA polymerase (e.g., a T7 RNA polymerase variant) is present in the reaction (e.g., an IVT reaction) at a concentration of 0.01 mg / ml to 1 mg / ml. For example, the RNA polymerase can be present in the reaction at a concentration of 0.01 mg / mL, 0.05 mg / ml, 0.1 mg / ml, 0.5 mg / ml, or 1.0 mg / ml.

[0204] Surprisingly, for example, in in vitro transcription reactions, the T7 RNAP variants provided herein (e.g., E42, S43, Y44, E45, M46, G47, A255, R257, or G259, e.g., S43A or G47A) can be used in combination with cap analogs (e.g., GpppA 2′OmepG) are used in combination to generate RNA transcripts, where greater than 80% of the generated RNA transcripts include a functional cap. In some embodiments, greater than 85% of the generated RNA transcripts include a functional cap. In some embodiments, greater than 90% of the generated RNA transcripts include a functional cap. In some embodiments, greater than 95% of the generated RNA transcripts include a functional cap. In some embodiments, greater than 96% of the generated RNA transcripts include a functional cap. In some embodiments, greater than 97% of the generated RNA transcripts include a functional cap. In some embodiments, greater than 98% of the generated RNA transcripts include a functional cap. In some embodiments, greater than 99% of the generated RNA transcripts include a functional cap.

[0205] It was also surprising to find that using a polynucleotide template that includes a 2'-deoxythymidine or 2'-deoxycytidine residue at template position +1 produces RNA transcripts, with more than 80% (e.g., more than 85%, more than 90%, or more than 95%) of the generated RNA transcripts containing a functional cap. Thus, in some embodiments, for example, a polynucleotide (e.g., DNA) template used in an IVT reaction includes a 2'-deoxythymidine residue at template position +1. In other embodiments, for example, a polynucleotide (e.g., DNA) template used in an IVT reaction includes a 2'-deoxycytidine residue at template position +1.

[0206] Purpose The RNA transcripts generated according to the present disclosure include mRNA (including modified mRNA and / or unmodified RNA), lncRNA, self-replicating RNA, circular RNA, CRISPR guide RNA, etc. In embodiments, the RNA is an RNA (e.g., an mRNA or a self-replicating RNA) that encodes a polypeptide (e.g., a therapeutic polypeptide). Thus, the RNA transcripts generated using the RNA polymerase variants of the present disclosure can be used in numerous applications.

[0207] For example, the RNA transcripts can be used to generate a polypeptide of interest, such as a therapeutic protein, a vaccine antigen, and the like. In some embodiments, the RNA transcript is a therapeutic RNA. A therapeutic mRNA is an mRNA that encodes a therapeutic protein (the term "protein" includes peptides). Therapeutic proteins mediate various effects in a host cell or subject to treat a disease or ameliorate signs and symptoms of a disease. For example, therapeutic proteins replace missing or abnormal proteins, enhance the function of an endogenous protein, provide a novel function to a cell (e.g., inhibit or activate an endogenous cellular activity, or act as a delivery agent for another therapeutic compound, such as an antibody-antibody conjugate). Therapeutic mRNAs can be useful in treating the following diseases and conditions: bacterial infections, viral infections, parasitic infections, cell proliferation disorders, genetic disorders, and autoimmune disorders. Other diseases and conditions are encompassed herein.

[0208] The protein of interest encoded by the mRNA provided herein can be essentially any protein. In some embodiments, the therapeutic protein is a cytokine, growth factor, antibody, or fusion protein. Non-limiting examples of therapeutic proteins include blood factors (such as Factor VIII and Factor VII), complement factors, low density lipoprotein receptor (LDLR), and MUT1. Non-limiting examples of cytokines include interleukins, interferons, chemokines, lymphokines, and the like. Non-limiting examples of growth factors include erythropoietin, EGF, PDGF, FGF, TGF, IGF, TNF, CSF, MCSF, GMCSF, and the like. Non-limiting examples of antibodies include adalimumab, infliximab, rituximab, ipilimumab, tocilizumab, canakinumab, itolizumab, tralokinumab. Non-limiting examples of fusion proteins include, for example, etanercept, abatacept, and belatacept.

[0209] In some embodiments, the protein of interest is human erythropoietin, LDLR (used to inhibit cholesterol), or MUT1 (used to treat methylmalonic acidemia (MMA). In other embodiments, the protein of interest encoded by the mRNA is a therapeutic antibody, including but not limited to those antibodies described above.

[0210] The RNA transcripts generated using the RNA polymerase variants disclosed herein may code for one or more biologics. Biologics are polypeptide-based molecules that can be used to treat, cure, mitigate, prevent, or diagnose serious or life-threatening diseases or conditions. Biologies include, but are not limited to, allergen extracts (e.g., for allergy shots and tests), blood components, gene therapy products, human tissue or cell products used for transplantation, vaccines, monoclonal antibodies, cytokines, growth factors, enzymes, thrombolytic agents, and immunomodulators, among others.

[0211] One or more biologics currently on the market or in development may be encoded by the RNA of the present invention. Without wishing to be bound by theory, it is believed that incorporating the encoding polynucleotides of known biologics into the RNA of the present disclosure may result in improved therapeutic efficacy, due at least in part to the specificity, purity, and / or selectivity of the construct design.

[0212] The RNA transcripts generated using the RNA polymerase variants disclosed herein may encode one or more antibodies. The term "antibody" includes monoclonal antibodies (including full-length antibodies with immunoglobulin Fc regions), antibody compositions with polyepitopic specificity, multispecific antibodies (e.g., bispecific antibodies, diabodies, and single-chain molecules), and antibody fragments. The term "immunoglobulin" (Ig) is used interchangeably with "antibody" herein. A monoclonal antibody is an antibody obtained from a population of substantially homogeneous antibodies. That is, the individual antibodies that make up the population are identical except for possible naturally occurring mutations and / or post-translational modifications (e.g., isomerization, amidation) that may be present in minor amounts. Monoclonal antibodies are highly specific and directed against a single antigenic site.

[0213] Monoclonal antibodies specifically include chimeric antibodies (immunoglobulins) in which a portion of the heavy and / or light chain is identical or homologous to corresponding sequences of antibodies derived from a particular species or belonging to a particular antibody class or subclass, and the remainder of the chain(s) is identical or homologous to corresponding sequences of antibodies derived from another antibody or belonging to another antibody class or subclass, and fragments of such antibodies so long as they exhibit the desired biological activity. Chimeric antibodies include, but are not limited to, "primatized" antibodies that contain variable domain antigen-binding sequences derived from a non-human primate (e.g., Old World monkeys, apes, etc.) and human constant region sequences.

[0214] Antibodies encoded by the RNA of the present disclosure may be utilized to treat conditions or diseases in many therapeutic areas, such as hematology, cardiovascular, CNS, poisoning (including antitoxins), dermatology, endocrinology, gastrointestinal, medical imaging, musculoskeletal, oncology, immunology, respiratory, sensory, and anti-infective.

[0215] The RNA transcripts produced using the RNA polymerase variants disclosed herein can code for one or more vaccine antigens. Vaccine antigens are biological preparations that improve immunity against a particular disease or infectious agent. One or more vaccine antigens currently on the market or under development can be encoded by the RNA of the present disclosure. RNA-encoded vaccine antigens can be utilized to treat conditions or diseases in many therapeutic areas, such as cancer, allergy, infectious diseases, etc. In some embodiments, the cancer vaccine can be a personalized cancer vaccine in the form of individual RNAs encoding concatemers or peptide epitopes or combinations thereof.

[0216] The RNA transcripts generated using the RNA polymerase variants disclosed herein can be designed to encode one or more antimicrobial peptides (AMPs) or antiviral peptides (AVPs). AMPs and AVPs have been isolated and described from a wide range of animals, including, but not limited to, microorganisms, invertebrates, plants, amphibians, birds, fish, and mammals. Antimicrobial polypeptides can block cell fusion and / or viral entry by one or more enveloped viruses (e.g., HIV, HCV). For example, antimicrobial polypeptides can comprise or consist of a synthetic peptide corresponding to a region, e.g., a contiguous sequence of at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, or 60 amino acids of a viral envelope protein, e.g., HIV-1 gpl20 or gp41 transmembrane subunit. The amino acid and nucleotide sequences of HIV-1 gp120 or gp41 are described, for example, in Kuiken et al., (2008). "HIV Sequence Compendium", Los Alamos National Laboratory.

[0217] In some embodiments, the RNA transcripts are used as radiolabeled RNA probes. In some embodiments, the RNA transcripts are used for nonisotopic RNA labeling. In some embodiments, the RNA transcripts are used as guide RNAs (gRNAs) for gene targeting. In some embodiments, the RNA transcripts (e.g., mRNAs) are used for in vitro translation and microinjection. In some embodiments, the RNA transcripts are used for RNA structure, processing, and catalysis studies. In some embodiments, the RNA transcripts are used for RNA amplification. In some embodiments, the RNA transcripts are used as antisense RNAs for gene expression experiments. Other uses are encompassed by the present disclosure.

[0218] composition The T7 RNAP variants of the present disclosure, in some embodiments, when used in combination with cap analogs, such as trinucleotide caps, in an IVT reaction, produce RNA that does not induce a detectable cytokine response, even without post-IVT purification. Thus, in some embodiments, provided herein are compositions comprising an IVT RNA and a pharma- ceutically acceptable excipient, the compositions being substantially free (e.g., free or containing less than 10%, less than 1%, less than 0.1%, or less than 0.01%) of cytokine-inducing RNA contaminants, without post-IVT purification.

[0219] As described elsewhere herein, the disclosed T7 RNAP variants and methods of the disclosure generate RNA transcripts, at least 80% (e.g., 80%-90%, 80-95%, 90-95%, 80-99%, 90-99%, or 90-100%) of the transcripts contain a functional cap. Accordingly, compositions comprising an IVT RNA and a pharma- ceutically acceptable excipient are also provided herein, wherein the composition comprises less than 20% (e.g., 5-15%, 5-10%, 1-15%, or 1-10%) uncapped RNA species. In some embodiments, the composition comprises less than 15% (e.g., 5-10%, 1-10%, or 1-5%) uncapped RNA species. In some embodiments, the composition comprises less than 10% uncapped RNA species. In some embodiments, the composition comprises less than 5% uncapped RNA species.

[0220] In some embodiments, greater than 80% of the IVT RNA comprises a functional cap. In some embodiments, greater than 85% of the IVT RNA comprises a functional cap. In some embodiments, greater than 90% of the IVT RNA comprises a functional cap. In some embodiments, greater than 95% of the IVT RNA comprises a functional cap.

[0221] In some embodiments, the IVT RNA is not chemically modified, while in other embodiments, the IVT RNA is chemically modified.

[0222] In some embodiments, unexpectedly, more than 80% of the IVT RNA comprises single-stranded full-length transcripts. For example, more than 85% of the IVT RNA may comprise single-stranded full-length transcripts. In some embodiments, more than 90% of the IVT RNA comprises single-stranded full-length transcripts, and more than 95% of the IVT RNA comprises single-stranded full-length transcripts.

[0223] In some embodiments, the RNA is prepared by subjecting a polynucleotide template to in vitro transcription reaction conditions to generate an RNA transcript, comprising: (a) a T7 RNA polymerase variant, the T7 RNA polymerase variant comprising at least one amino acid substitution, relative to a wild-type RNA polymerase, that causes at least one loop structure of the RNA polymerase variant to undergo a conformational change to a helical structure as the RNA polymerase variant transitions from an initiation complex to an elongation complex; (b) a nucleoside triphosphate; and (c) a sequence GpppA 2′Ome The polynucleotide template is generated by a method comprising reacting with a trinucleotide cap containing pG, the polynucleotide template containing a 2'-deoxythymidine residue at template position +1.

[0224] kit Also provided herein is a kit, such as an in vitro transcription kit, that includes the RNA polymerase variant of the present disclosure.The kit may include any one or more (at least one) IVT components and any one or more (at least one) RNA polymerase variant described herein.For example, the kit may include a buffer system, NTP, and a T7 RNA polymerase variant having the amino acid sequence of SEQ ID NO: 1, 99, or 100, with at least one (or at least two, or at least three) amino acid substitutions at positions E42, S43, Y44, E45, M46, G47, A255, R257, A258, G259, A260, L261, and / or A262, and optionally at least one amino acid addition at the C-terminus.

[0225] Additional Embodiments Additional embodiments of the present disclosure include those in the following numbered paragraphs: Paragraph 1. A ribonucleic acid (RNA) polymerase variant, comprising at least one amino acid substitution, compared to a wild-type RNA polymerase, that causes at least one loop structure of the RNA polymerase variant to undergo a conformational change to a helical structure as the RNA polymerase variant transitions from an initiation complex to an elongation complex.

[0226] Paragraph 2. The RNA polymerase variant of paragraph 1, wherein the at least one amino acid substitution has an increased helical propensity compared to a wild-type amino acid.

[0227] Paragraph 3. The RNA polymerase variant described in paragraph 1 or 2, wherein the RNA polymerase is T7 RNA polymerase.

[0228] Paragraph 4. An RNA polymerase variant described in any one of paragraphs 1 to 3, wherein the at least one loop structure is in a C-helix structure.

[0229] Paragraph 5. An RNA polymerase variant described in any one of paragraphs 1 to 4, wherein the at least one loop structure is in a C linker structure.

[0230] Paragraph 6. An RNA polymerase variant according to any one of paragraphs 1 to 4, wherein the at least one amino acid substitution is at least one high-helix-propensity amino acid substitution.

[0231] Paragraph 7. The RNA polymerase variant of Paragraph 6, wherein the at least one high-helix-prone acid substitution is selected from alanine, isoleucine, leucine, methionine, lysine, glutamine, and / or glutamic acid.

[0232] Paragraph 8. The RNA polymerase variant of Paragraph 7, wherein the at least one high-helix-prone amino acid substitution is an alanine.

[0233] Paragraph 9. The RNA polymerase variant of any one of Paragraphs 4 to 8, wherein the T7 RNA polymerase comprises an amino acid sequence identified by SEQ ID NO:1, SEQ ID NO:99, or SEQ ID NO:100, modified to include at least one amino acid substitution of a high helix-propensity amino acid at a position selected from E42, S43, Y44, E45, M46, G47, R257, and G259.

[0234] Paragraph 10. The RNA polymerase variant of Paragraph 9, wherein the at least one amino acid substitution comprises S43A.

[0235] Paragraph 11. The RNA polymerase variant of Paragraph 9, wherein the at least one amino acid substitution comprises G47A.

[0236] Paragraph 12. The RNA polymerase variant of any one of Paragraphs 5 to 8, wherein the T7 RNA polymerase comprises an amino acid sequence identified by SEQ ID NO:1, SEQ ID NO:99, or SEQ ID NO:100, modified to include at least one amino acid substitution of a high helix-propensity amino acid at a position selected from R257, A258, G259, A260, L261, and A262.

[0237] Paragraph 13. The RNA polymerase variant according to Paragraph 12, wherein the at least one amino acid substitution comprises R257A.

[0238] Paragraph 14. The RNA polymerase variant of Paragraph 12, wherein the at least one amino acid substitution comprises G259A.

[0239] Paragraph 15. A T7 ribonucleic acid (RNA) polymerase comprising an amino acid sequence identified by SEQ ID NO:1, SEQ ID NO:99, or SEQ ID NO:100, modified to include an amino acid substitution of a high helical propensity amino acid at positions G47, S43, R257, or G259.

[0240] Paragraph 16. The T7 RNA polymerase of Paragraph 15, wherein the high helix-prone amino acids are selected from alanine, isoleucine, leucine, methionine, lysine, glutamine, and / or glutamic acid.

[0241] 17. The T7 RNA polymerase of paragraph 16, wherein the high helix-prone amino acid is alanine.

[0242] Paragraph 18. A T7 RNA polymerase comprising an amino acid sequence identified by SEQ ID NO:2, SEQ ID NO:107, or SEQ ID NO:108.

[0243] Paragraph 19. A T7 RNA polymerase comprising an amino acid sequence identified by SEQ ID NO:3, SEQ ID NO:109, or SEQ ID NO:110.

[0244] Paragraph 20. A T7 RNA polymerase comprising an amino acid sequence identified by SEQ ID NO:4, SEQ ID NO:111, or SEQ ID NO:112.

[0245] Paragraph 21. A T7 RNA polymerase comprising an amino acid sequence identified by SEQ ID NO:5, SEQ ID NO:113, or SEQ ID NO:114.

[0246] Paragraph 22. A method for producing ribonucleic acid (RNA), comprising contacting a DNA template with an RNA polymerase according to any one of Paragraphs 1 to 21 under conditions that result in the production of an RNA transcript.

[0247] Paragraph 23. A method for performing an in vitro transcription (IVT) reaction, comprising contacting a DNA template with an RNA polymerase according to any one of Paragraphs 1 to 21 in the presence of nucleoside triphosphates and a buffer under conditions resulting in the production of an RNA transcript.

[0248] Paragraph 24. The method of Paragraph 23, wherein the generated RNA transcripts, when delivered to a cell, optionally in unpurified form, stimulate at least a 50% lower cytokine response compared to RNA generated using a wild-type RNA polymerase.

[0249] Paragraph 25. The method of any one of paragraphs 23 to 24, wherein the concentration of the double-stranded RNA transcripts produced by IVT is at least 50% lower compared to dsRNA transcripts produced using a wild-type polymerase.

[0250] Paragraph 26. The method of any one of Paragraphs 23 to 25, wherein less than 20% of the generated RNA transcripts exhibit 3' heterogeneity.

[0251] Paragraph 27. The method of any one of Paragraphs 23 to 26, wherein less than 50% of the produced RNA transcripts are truncated RNA transcripts.

[0252] Paragraph 28. The method of any one of Paragraphs 23 to 28, wherein less than 50% of the produced RNA transcripts are run-on RNA transcripts.

[0253] Paragraph 29. The method of any one of Paragraphs 23 to 28, wherein the amount of full-length RNA transcript produced is at least 15-fold greater than the amount of DNA template.

[0254] Paragraph 30. The method of any one of Paragraphs 23 to 29, wherein the ratio of truncated RNA transcripts:full-length RNA transcripts produced is less than 1:1.

[0255] Paragraph 31. The method of any one of Paragraphs 23 to 30, wherein the produced RNA transcripts have less than 1 mutation per 100 nucleotides compared to the DNA template.

[0256] Paragraph 32. A nucleic acid encoding an RNA polymerase according to any one of Paragraphs 1 to 21.

[0257] Paragraph 33. A vector comprising the nucleic acid of Paragraph 33.

[0258] Paragraph 34. A host cell comprising a nucleic acid according to paragraph 33 or a vector according to paragraph 34.

[0259] Paragraph 35. A kit comprising an RNA polymerase according to any one of Paragraphs 1 to 21.

[0260] Paragraph 36. A composition comprising an RNA polymerase according to any one of Paragraphs 1 to 21.

[0261] Paragraph 37. Ribonucleic acid (RNA) produced by a method according to any one of paragraphs 22 to 31.

[0262] Paragraph 38. The RNA according to Paragraph 37 formulated in a lipid nanoparticle.

[0263] Paragraph 39. The RNA of Paragraph 38, wherein the lipid nanoparticles comprise a molar ratio of 20-60% ionizable amino lipid, 5-25% non-cationic lipid, 25-55% sterol, and 0.5-15% PEG-modified lipid.

[0264] Paragraph 40. A co-transcriptional capping method for ribonucleic acid (RNA) synthesis, comprising reacting a polynucleotide template with a T7 RNA polymerase variant, a nucleoside triphosphate, and a cap analog under in vitro transcription reaction conditions to produce an RNA transcript.

[0265] Paragraph 41. The method of Paragraph 40, wherein more than 80%, more than 85%, or more than 90% of the generated RNA transcripts comprise a functional cap.

[0266] Paragraph 42. The method of Paragraph 41, wherein greater than 95% of the produced RNA transcripts comprise a functional cap.

[0267] Paragraph 43. The method of any one of Paragraphs 40 to 42, wherein the nucleoside triphosphates comprise unmodified or modified ATP, modified or unmodified UTP, modified or unmodified GTP, and / or modified or unmodified CTP.

[0268] Paragraph 44. The method according to any one of Paragraphs 40 to 43, wherein the T7 RNA polymerase variant is a T7 polymerase variant according to any one of Paragraphs 1 to 21.

[0269] Paragraph 45. The method of Paragraph 44, wherein said T7 polymerase variant comprises an amino acid sequence identified by SEQ ID NO:1, SEQ ID NO:99, or SEQ ID NO:100 modified to include at least one amino acid substitution of a high propensity amino acid at a position selected from E42, S43, Y44, E45, M46, G47, R257, and G259.

[0270] Paragraph 46. The method of any one of Paragraphs 40 to 45, wherein the T7 polymerase variant comprises an amino acid sequence identified by SEQ ID NO:1, SEQ ID NO:99, or SEQ ID NO:100 modified to include an amino acid substitution at position G47A.

[0271] Paragraph 47. The method of any one of Paragraphs 40 to 45, wherein the T7 polymerase variant comprises an amino acid sequence identified by SEQ ID NO:1, SEQ ID NO:99, or SEQ ID NO:100 modified to include an S43A amino acid substitution.

[0272] Paragraph 48. The method of any one of Paragraphs 40 to 47, wherein the nucleoside triphosphate and cap analog are present in equimolar concentrations in the reaction.

[0273] Paragraph 49. The method of any one of Paragraphs 40 to 47, wherein the molar ratio of cap analog to nucleoside triphosphate in the reaction is greater than 1:1.

[0274] 50. The method of any one of paragraphs 40 to 47, wherein the molar ratio of cap analog to nucleoside triphosphate in the reaction is less than 1:1.

[0275] Paragraph 51. The method of any one of Paragraphs 40 to 50, wherein the cap analog is a dinucleotide cap, a trinucleotide cap, or a tetranucleotide cap.

[0276] Paragraph 52. The method of any one of Paragraphs 40 to 50, wherein the cap analog is a trinucleotide cap.

[0277] Paragraph 53. The method of Paragraph 51, wherein the trinucleotide cap comprises a sequence selected from the following sequences: GAA, GAC, GAG, GAU, GCA, GCC, GCG, GCU, GGA, GGC, GGG, GGU, GUA, GUC, GUG, and GUU.

[0278] Paragraph 54. The trinucleotide cap has the following sequence: 7 GppApA, m 7 GpppApC, m 7 GppApG, m 7 GppApU,m 7 GppCpA, m 7 GppCpC, m 7 GppCpG, m 7 GppCpU,m 7 GppGpA, m 7 GppGpC, m 7 GppGpG, m 7 GppGpU,m 7 GpppUpA,m 7 GpppUpC,m 7 GpppUpG, and m 7 54. The method of paragraph 53, comprising a sequence selected from GpppUpU.

[0279] Paragraph 55. The trinucleotide cap has the following sequence: 7 G 3′OMe pppApA, m 7 G3′OMe pppApC, m 7 G 3′OMe pppApG, m 7 G 3′OMe pppApU,m 7 G 3′OMe pppCpA, m 7 G 3′OMe pppCpC, m 7 G 3′OMe pppCpG,m 7 G 3′OMe pppCpU,m 7 G 3′OMe pppGpA,m 7 G 3′OMe pppGpC, m 7 G 3′OMe pppGpG, m 7 G 3′OMe pppGpU,m 7 G 3′OMe pppUpA,m 7 G 3′OMe pppUpC,m 7 G 3′OMe pppUpG, and m 7 G 3′OMe 54. The method of paragraph 53, comprising a sequence selected from pppUpU.

[0280] Paragraph 56. The trinucleotide cap has the following sequence: 7 G 3′OMe pppA 2′OMe pA, m 7 G 3′OMe pppA 2′OMe pC, m 7 G 3′OMe pppA 2′OMe pG,m 7 G 3′OMe pppA 2′OMe pU,m 7 G 3′OMe pppC 2′OMe pA, m 7 G 3′OMe pppC 2′OMe pC, m 7 G 3′OMe pppC 2′OMe pG,m 7 G 3′OMe pppC 2′OMe pU,m 7 G3′OMe pppG 2′OMe pA, m 7 G 3′OMe pppG 2′OMe pC, m 7 G 3′OMe pppG 2′OMe pG,m 7 G 3′OMe pppG 2′OMe pU,m 7 G 3′OMe pppU 2′OMe pA, m 7 G 3′OMe pppU 2′OMe pC, m 7 G 3′OMe pppU 2′OMe pG, and m 7 G 3′OMe pppU 2′OMe 54. The method of paragraph 53, comprising a sequence selected from pU.

[0281] Paragraph 57. The trinucleotide cap has the following sequence: 7 GpppA 2′OMe pA, m 7 GpppA 2′OMe pC, m 7 GpppA 2′OMe pG,m 7 GpppA 2′OMe pU,m 7 GpppC 2′OMe pA, m 7 GpppC 2′OMe pC, m 7 GpppC 2′OMe pG,m 7 GpppC 2′OMe pU,m 7 GpppG 2′OMe pA, m 7 GpppG 2′OMe pC, m 7 GpppG 2′OMe pG,m 7 GpppG 2′OMe pU,m 7 GpppU 2′OMe pA, m 7 GpppU 2′OMe pC, m 7 GpppU 2′OMe pG, and m 7GpppU 2′OMe 54. The method of paragraph 53, comprising a sequence selected from pU.

[0282] Paragraph 58. The method of any one of Paragraphs 53 to 57, wherein the trinucleotide cap comprises a sequence selected from the following sequences: GAG, GCG, GUG, and GGG.

[0283] Paragraph 59. The method of Paragraph 58, wherein the trinucleotide cap comprises the sequence GAG.

[0284] Paragraph 60. The trinucleotide cap, 2′Ome 60. The method of paragraph 59, comprising pG.

[0285] Paragraph 61. The method of any one of Paragraphs 40 to 60, wherein the polynucleotide template comprises a 2'-deoxythymidine residue at template position +1.

[0286] Paragraph 62. The method of any one of Paragraphs 40 to 60, wherein the polynucleotide template comprises a 2'-deoxycytidine residue at template position +1.

[0287] Paragraph 63. A co-transcriptional capping method for RNA synthesis, comprising: coupling a polynucleotide template to (a) a T7 RNA polymerase variant, the T7 RNA polymerase variant comprising at least one amino acid substitution, as compared to a wild-type RNA polymerase, that causes at least one loop structure of the RNA polymerase variant to undergo a conformational change to a helical structure as the RNA polymerase variant transitions from an initiation complex to an elongation complex; (b) a nucleoside triphosphate; and (c) a nucleotide sequence of the sequence GpppA, ... 2′Ome reacting the polynucleotide template with a trinucleotide cap containing pG, wherein the polynucleotide template contains a 2'-deoxythymidine residue at template position +1.

[0288] Paragraph 64. The method of any one of Paragraphs 40 to 63, wherein the produced RNA transcripts, when delivered to a cell, optionally in unpurified form, do not stimulate a detectable cytokine response.

[0289] Paragraph 65. A composition comprising in vitro transcribed (IVT) RNA and a pharma- ceutically acceptable excipient, said composition being substantially free of cytokine-induced RNA contaminants without post-IVT purification.

[0290] Paragraph 66. A composition comprising an in vitro transcribed (IVT) RNA and a pharma- ceutically acceptable excipient, the composition having less than 5% uncapped RNA species.

[0291] Paragraph 67. The composition of Paragraph 65 or 66, wherein more than 80%, 85%, or 90% of said IVT RNA comprises a functional cap.

[0292] Paragraph 68. The composition of Paragraph 67, wherein more than 95% of said IVT RNA comprises a functional cap.

[0293] Paragraph 69. The composition according to any one of Paragraphs 65 to 68, wherein said IVT RNA is not chemically modified.

[0294] Paragraph 70. The composition of any one of paragraphs 65 to 68, wherein the IVT RNA is chemically modified.

[0295] Paragraph 71. The composition of any one of Paragraphs 65 to 70, wherein greater than 95% of the IVT RNA comprises a single-stranded full-length transcript.

[0296] Paragraph 72. The RNA, Under in vitro transcription reaction conditions to produce an RNA transcript, a polynucleotide template is transduced with (a) a T7 RNA polymerase variant, the T7 RNA polymerase variant comprising at least one amino acid substitution, as compared to a wild-type RNA polymerase, that causes at least one loop structure of the RNA polymerase variant to undergo a conformational change to a helical structure as the RNA polymerase variant transitions from an initiation complex to an elongation complex; (b) a nucleoside triphosphate; and (c) a GpppA sequence. 2′Ome 72. The composition of any one of claims 65-71, produced by a process comprising reacting with a trinucleotide cap comprising pG, wherein the polynucleotide template comprises a 2'-deoxythymidine residue at template position +1.

[0297] Paragraph 73. A T7 RNA polymerase comprising at least one additional C-terminal amino acid compared to wild-type T7 RNA polymerase.

[0298] Paragraph 74. The T7 RNA polymerase of Paragraph 73, comprising at least two additional C-terminal amino acids.

[0299] Paragraph 75. The T7 RNA polymerase of Paragraph 74, wherein the at least two additional C-terminal amino acids comprise amino acids of the same type or at least two different types.

[0300] Paragraph 76. A T7 RNA polymerase according to any one of Paragraphs 73 to 75, comprising 1 to 10 additional C-terminal amino acids.

[0301] 77. The T7 RNA polymerase of Paragraph 76, comprising 1 to 5 additional C-terminal amino acids.

[0302] Paragraph 78. The T7 RNA polymerase is FAFAX n (SEQ ID NO:171)78. The T7 RNA polymerase of any one of paragraphs 73 to 77, comprising a C-terminus that comprises the motif, wherein X is any amino acid and n is any integer greater than zero.

[0303] If paragraph 79.X is G or A, then, optionally, X n 79. The T7 RNA polymerase of paragraph 78, wherein is GG or AA.

[0304] 80. The T7 RNA polymerase according to paragraph 78 or 79, wherein paragraph 80.n is 1, 2, 3, 4 or 5.

[0305] Paragraph 81.FAFAG (SEQ ID NO:329) T7 RNA polymerase, containing a C-terminal motif.

[0306] Paragraph 82. XAFAX n Motif, FAXAX n Motif, FAXAX n Motif, or FAFXX n a C-terminus containing a motif, wherein each X is any amino acid and n is any integer greater than zero, T7 RNA polymerase.

[0307] Paragraph 83. The T7 RNA polymerase according to any one of Paragraphs 73 to 82, wherein said T7 RNA polymerase comprises at least one substitution and optionally at least one additional substitution at a position corresponding to position S43, G47, R257, or G259 of wild-type T7 RNA polymerase.

[0308] Paragraph 84. The T7 RNA polymerase of Paragraph 83, wherein the wild-type T7 RNA polymerase comprises the amino acid sequence identified by SEQ ID NO:1.

[0309] Paragraph 85. A T7 RNA polymerase comprising the amino acid sequence of SEQ ID NO:99 modified to include at least one substitution selected from G47, S43, R257, and G259, and optionally at least one additional amino acid substitution.

[0310] Paragraph 86. A T7 RNA polymerase comprising the amino acid sequence of any one of SEQ ID NOs: 100, 294, 296, or 296 modified to include at least one substitution selected from G47, S43, R257, and G259, and optionally at least one additional amino acid substitution.

[0311] Paragraph 87. The T7 RNA polymerase according to any one of Paragraphs 83 to 86, comprising at least one substitution selected from S43A, G47A, R257A, and G259A.

[0312] Paragraph 88. A T7 RNA polymerase comprising the amino acid sequence of any one of SEQ ID NOs: 294-313, wherein x is any amino acid and n is any integer, e.g., 1-5 (e.g., 1, 2, 3, 4, or 5), and optionally, the T7 RNA polymerase further comprises at least one additional amino acid substitution.

[0313] Paragraph 89. A method of conducting an in vitro transcription (IVT) reaction, comprising contacting a DNA template with the RNA polymerase of any of Paragraphs 73 to 88 in the presence of nucleoside triphosphates and a buffer under conditions that result in the production of an RNA transcript.

[0314] Paragraph 90. The method of Paragraph 89, wherein the produced RNA, when delivered to a cell, stimulates at least a 50% lower cytokine response as compared to a dsRNA transcript produced using WT T7 RNAP.

[0315] 91. The method of paragraph 90, wherein the RNA is delivered to the cell in an unpurified form.

[0316] Paragraph 92. The method of any one of Paragraphs 89 to 92, wherein less than 30% of the generated RNA transcripts exhibit 3' heterogeneity.

[0317] Paragraph 93. A nucleic acid encoding an RNA polymerase according to any one of Paragraphs 73 to 92.

[0318] Paragraph 94. A vector comprising the nucleic acid of Paragraph 93.

[0319] Paragraph 95. A host cell comprising a nucleic acid according to Paragraph 93 or a vector according to Paragraph 94.

[0320] Paragraph 96. A kit comprising an RNA polymerase according to any one of Paragraphs 73 to 92.

[0321] Paragraph 97. A composition comprising an RNA polymerase according to any one of Paragraphs 73 to 92.

[0322] Paragraph 98. Ribonucleic acid (RNA) produced by a method according to any one of Paragraphs 89 to 92.

[0323] Paragraph 99. The RNA according to Paragraph 98 formulated in a lipid nanoparticle.

[0324] Paragraph 100. The RNA of Paragraph 98, wherein the lipid nanoparticles comprise a molar ratio of 20-60% ionizable amino lipid, 5-25% non-cationic lipid, 25-55% sterol, and 0.5-15% PEG-modified lipid.

[0325] Paragraph 101. An RNA polymerase comprising at least one additional C-terminal amino acid compared to a corresponding wild-type RNA polymerase, optionally wherein the at least one additional C-terminal amino acid comprises glycine (G) and / or alanine (A).

[0326] Paragraph 102. The RNA polymerase according to Paragraph 101, wherein the RNA polymerase is selected from T7 RNA polymerase, T3 RNA polymerase, and SP6 RNA polymerase.

[0327] Paragraph 103. The RNA polymerase of Paragraph 101 or 102, wherein the RNA polymerase further comprises at least one additional amino acid substitution, optionally an amino acid substitution corresponding to an amino acid substitution in SEQ ID NO:1 selected from S43A, G47A, R257A, and G259A.

[0328] Paragraph 104. The RNA polymerase of any one of Paragraphs 101 to 104, wherein the RNA polymerase is a T7 RNA polymerase comprising an amino acid sequence at least 90%, at least 95%, or at least 98% identical to SEQ ID NO:1 modified to include amino acid substitutions at positions 43, 47, 257, and / or 259, and optionally, the amino acid substitutions are alanine (A).

[0329] Paragraph 105. The RNA polymerase of any one of Paragraphs 101 to 104, wherein the RNA polymerase is a T3 RNA polymerase comprising an amino acid sequence at least 90%, at least 95%, or at least 98% identical to SEQ ID NO: 6 modified to include amino acid substitutions at positions 44, 48, 258, and / or 260, and optionally, the amino acid substitutions are alanine (A).

[0330] Paragraph 106. The RNA polymerase of any one of Paragraphs 101 to 104, wherein the RNA polymerase is an SP6 RNA polymerase comprising an amino acid sequence at least 90%, at least 95%, or at least 98% identical to SEQ ID NO:7 modified to include amino acid substitutions at positions 15, 19, 230, and / or 232, and optionally, the amino acid substitutions are alanine (A).

[0331] Paragraph 107. A T7 RNA polymerase comprising an amino acid sequence at least 90%, at least 95%, or at least 98% identical to SEQ ID NO:1 modified to include amino acid substitutions at positions 43, 47, 257, and / or 259, optionally wherein the amino acid substitution is an alanine (A).

[0332] Paragraph 108. A T3 RNA polymerase comprising an amino acid sequence at least 90%, at least 95%, or at least 98% identical to SEQ ID NO:6 modified to include amino acid substitutions at positions 44, 48, 258, and / or 260, optionally wherein the amino acid substitution is an alanine (A).

[0333] Paragraph 109. An SP6 RNA polymerase comprising an amino acid sequence at least 90%, at least 95%, or at least 98% identical to SEQ ID NO:7 modified to include amino acid substitutions at positions 15, 19, 230, and / or 232, optionally wherein the amino acid substitution is an alanine (A). [Table 1-1] [Table 1-2] [Table 2-1] [Table 2-2] [Table 2-3] [Table 2-4] EXAMPLES

[0334] Example 1. Generation of variant T7 RNA polymerase C-helix and C-linker T7 RNA polymerase variants were generated with the substitutions shown in Tables 3 and 4. [Table 3-1] [Table 3-2] [Table 3-3]

Table 3-4

Table 3-5

Table 3-6

Table 3-7

Table 3-8

Table 3-9

Table 3-10

Table 3-11

Table 3-12

Table 3-13

Table 3-14

Table 3-15

Table 3-16

Table 3-17

Table 3-18

Table 3-19

Table 3-20

Table 3-21

Table 3-22

Table 3-23

Table 3-24

Table 3-25

Table 3-26

Table 3-27

Table 3-28

Table 3-29

Table 3-30

Table 3-31

Table 3-32

Table 3-33

Table 3-34

Table 3-35

Table 3-36

Table 3-37

Table 3-38

Table 3-39

Table 3-40

Table 3-41

Table 3-42

Table 3-43

Table 3-44

Table 3-45

Table 3-46

Table 3-47

Table 3-48

Table 3-49

Table 3-50

Table 3-51

Table 3-52

Table 3-53

Table 3-54

Table 3-55

Table 3-56

Table 3-57

Table 3-58

Table 3-59

Table 3-60

Table 3-61

Table 3-62

Table 3-63

Table 3-64

Table 3-65

Table 3-66

Table 3-67

Table 3-68

Table 3-69

Table 3-70

Table 3-71

Table 3-72

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[0335] Example 2. The purity of hEPO mRNA produced using T7 RNA polymerase variants is comparable to that of hEPO mRNA produced using wild-type T7 RNA polymerase. In vitro transcription reactions were performed using hEPO DNA template and (1) wild-type (WT) T7 RNA polymerase (WT #1 and WT #2), (2) G47A C-terminal G T7 RNA polymerase variant ("G47A*") (SEQ ID NO: 110), and (3) S43A C-terminal G T7 RNA polymerase variant ("S43A*") (SEQ ID NO: 108). DBAA (dibutylammonium acetate) HPLC analysis showed that the purity of all hEPO transcripts was similar (Figure 1).

[0336] Example 3. hEPO mRNA produced using T7 RNA polymerase variants does not elicit a cellular cytokine response. BJ fibroblasts were transfected with hEPO mRNA produced by the IVT reaction described in Example 2. The mRNA was either unpurified or purified by reverse phase (RP) chromatography. As shown in Figure 2 (left graph), unpurified mRNA produced using S43A* or G47A* T7 RNA polymerase variants was as "cold" (did not elicit a cytokine response) as purified mRNA produced using WT T7 polymerase. Approximately equivalent mRNA expression levels are shown in Figure 2 (right graph). The mRNA used in these experiments did not contain any nucleotide modifications.

[0337] m 1ψ nucleotide modification (Figure 3, top graph) or mo 5 The same cell transfection assay was repeated using hEPO mRNA containing either the G47A* or S43A* U nucleotide modification (Figure 3, bottom graph). 1 BJ fibroblasts transfected with ψ hEPO mRNA were incubated with unpurified mEPO mRNA generated using WT T7 RNA polymerase. 1 ψ hEPO mRNA transfected cells showed a lower IFNβ response than did cells transfected with hEPO mRNA. Approximately equivalent mRNA expression levels are shown in FIG.

[0338] Monocyte-derived macrophages (MDMs) were then used to further test cytokine responses. The ratio of IP10 to hypoxanthine-guanine phosphoribosyltransferase (HPRT) was measured in MDMs transfected with hEPO mRNA produced under the conditions described in Example 2. Since MDMs are highly sensitive to unmodified chemistry, no dramatic differences between samples were seen using unmodified hEPO mRNA. However, unpurified hEPO mRNA produced using G47A* and S43A* T7 RNA polymerase variants showed no significant differences between samples. 1 MDMs transfected with ψ hEPO mRNA were transfected with purified m 1 showed a lower IP10 response than MDMs transfected with ψ hEPO mRNA (Fig. 5 , top graph).

[0339] Example 4. T7 RNA polymerase variants produce mRNA associated with less contaminating dsRNA than WT T7 RNA polymerase. Using a standard dsRNA ELISA, unmodified hEPO mRNA (Figure 7) or 1 ψ nucleotide modification (Figure 8, upper graph) or mo 5We assessed dsRNA contamination (e.g., longer than 40 nucleotide base pairs) in IVT reactions used to generate hEPO mRNA containing either U nucleotide modifications (Figure 8, bottom graph). The dsRNA assay showed that the S43A* and G47A* T7 RNA polymerase variants generated less dsRNA than the WT T7 RNA polymerase (Figures 7 and 8).

[0340] Example 5. T7 RNA polymerase variants reduce mRNA 3' heterogeneity. The 3' heterogeneity of transcripts can be measured using RNAse T1 digestion. RNAse T1 specifically cleaves mRNA after a G nucleotide. Endonucleotide cleavage results in a 5' hydroxide (OH) and a 3' monophosphate (mP) "scar". Thus, RNAse T1 digestion can be used to distinguish between transcripts with and without untemplated additions at the 3' end. In this example, the hEPO mRNA produced in Example 2 was modified to include a polyA tail and an XbaI restriction site (e.g., A n The hEPO mRNA produced using WT T7 RNA polymerase, S43A* T7 RNA polymerase variant, or G47A* T7 RNA polymerase variant was digested with RNAse T1 and analyzed by LCMS to generate oligo fingerprints. The homogenous 3′ end (5′OH / 3′OH) was found to peak at 32780 Da, while the “scar” (5′OH / 3′mP) peaked at 32860 Da (FIG. 9A). The “scar” indicates that the transcript had an untemplated addition at the carboxy terminus. The hEPO mRNA produced using T7 RNA polymerase variant, S43A*, or G47A*, had a higher 3′ end population distribution, indicating that they have more homogenous 3′ ends and improved 3′ end heterogeneity (FIG. 9B).

[0341] Example 6. T7 RNA polymerase variants that generate "more homogeneous" 3' ends An assay was used to examine the extent of untemplated addition produced by the T7 RNAP variants, G47A* and S43A*. In this assay, the enzyme polymerase was used to generate a ligation leftmer (see, for example, FIG. 11A). The leftmer was then annealed to a perfectly complementary DNA splint immediately adjacent to the 5′-monophosphorylated lightmer RNA. The DNA splint is typically 40 nt long, and the lightmer is typically modified with a fluorophore at the 5′ end. DNA ligase I was then added under catalytic conditions, and ligation was allowed to proceed for the desired reaction time before the reaction was quenched with EDTA. The mixture was then denatured in 4M urea at 95° C. for 5 min before being loaded onto a denaturing polyacrylamide gel (PAGE-D) (FIG. 11B). The percentage of acrylamide in the gel depends on the expected length of the construct, but is typically 6% acrylamide for leftomer lengths >50 nt and 20% acrylamide for leftomers approaching typical mRNA lengths. The 6% acrylamide gel was run at a constant 180V for 25 minutes, and the 20% acrylamide gel was run at a constant 180V for 120 minutes. The gel was then imaged on a Typhoon or similar scanner using excitation and emission wavelengths appropriate for the fluorophores present in the lightmer and the newly ligated product. The lightmer and ligated product will migrate differently on the gel based on size, and the intensity of the two bands will accurately correspond to the efficiency of the ligation reaction. The greater the extent of untemplated nucleotide addition during the enzymatic synthesis of the leftmer, the lower the expected ligation yield.

[0342] Ligation yields were calculated as the ratio of bands corresponding to unligated rightmer and ligated full-length products. Reactions were terminated early to exaggerate differences in reaction yields. Based on these results, T7 RNAP variant G47A* incorporated the fewest untemplated nucleotides at the 3´ end of leftmer RNAs.

[0343] Example 7. Generation of capped mRNA in a single in vitro transcription reaction In standard in vitro transcription (IVT) assays, T7 RNA polymerase preferably initiates transcription with 5′GTP. Single-stranded RNA (ssRNA) molecules generated by such standard IVT assays require a separate enzymatic capping step (Figure 13). For example, Cap1 RNA (7mGpppN2′-Om-RNA) can be generated in a capping assay using vaccinia capping and 2′-O-methyltransferase (2′OMTase). Cap1 is typically used for efficient protein translation and mRNA stability in cells. However, some mRNA sequences are difficult to cap, and capping / methylation enzymes are very expensive.

[0344] The present disclosure provides, in some embodiments, a method for co-transcriptionally capping ssRNA (e.g., mRNA) with trinucleotides in an in vitro transcription assay using a T7 RNA polymerase variant (e.g., T7 RNA polymerase variant G47A*) described herein. Efficient co-transcriptional capping typically involves a double-stranded DNA (dsDNA) template initiating transcription with 5′ ATP and equimolar concentrations of NTPs and trinucleotides. Typically, T7 RNA polymerase has significantly reduced initiation activity with 5′ ATP. However, XAG (X is any nucleotide, e.g., 5′ m In the presence of a trinucleotide (Gppp), the limited initiation activity of T7 RNA polymerase using 5' ATP allows initiation to be driven by the trinucleotide but not the 5' ATP, resulting in the cotranscriptional production of capped mRNA.

[0345] Exemplary commercially available di- and trinucleotide caps are shown in FIG. 14A. Vaccinia Cap 1 is a typical di-nucleotide cap and cannot be added co-transcriptionally. Another di-nucleotide cap that can be added co-transcriptionally is Anti-Reverse Cap Analog (ARCA), which is also commercially available (e.g., from Thermo Fisher, catalog number: AM8045). ARCA cleaves the 3'OH group (m 7 G) is -OCH 3 ARCA is a modified cap analogue in which ARCA is replaced by ARCA. ARCA is used as a control in some of the cotranscriptional capping assays described in this study.

[0346] In the co-transcriptional capping assay, a human EPO (hEPO) DNA template was used. 5 mM NTPs with or without 5 mM trinucleotides were also present in the assay mixture ( m GppAG, m The unmodified mRNA products were analyzed by RNase H cap assay or LCMS. As shown in Table 5, wild-type (WT) T7 RNA polymerase and T7 RNA polymerase variant G47A* were able to generate fully capped mRNA with equally high efficiency. RNA yields were comparable among different IVT reactions (Figure 14B), and mRNA products with high integrity were generated (Figure 14C). [Table 5]

[0347] The immune stimulatory activity of the mRNA products was evaluated by assessing their ability to induce cytokine (IFNβ) production in BJ fibroblasts. Interestingly, WT T7 RNA polymerase produced unmodified mRNA that induced a high cytokine response, whereas T7 RNA polymerase variant G47A* produced unmodified mRNA that did not stimulate cytokine production in BJ fibroblasts (Figure 14D), minimizing the need for further purification of RNA after IVT / capping assay. hEPO expression from the mRNA products was also evaluated. hEPO expression was observed from mRNA produced by WT T7 RNA polymerase, whereas mRNA produced by T7 RNA polymerase variant G47A* resulted in hEPO expression comparable to 1-methyl-pseudouridine modified mRNA capped with vaccinia cap 1 (Figure 14E). LCMS experiments also show that the T7 RNA polymerase variant G47A* produced more homogeneous RNA than WT T7 RNA polymerase, although trinucleotide incorporation efficiency appeared to be comparable for the two enzymes (Figure 15).

[0348] In a different experiment, the capping efficiency of T7 RNA polymerase variant G47A* was compared to that achieved in a standard capping assay using vaccinia cap 1. In this experiment, the reaction mixture contained a hEPO dsDNA template containing a deoxythymidine at the +1 position on the template strand (also named "Astart" for one nucleotide of the first template), T7 RNA polymerase variant G47A*, and equimolar (7.5 mM) NTPs and GAG trinucleotides. The resulting mRNA products were purified using oligo-dT and analyzed for capping efficiency, in vitro cytokine response, and in vitro expression. The results show that T7 RNA polymerase variant G47A* produced capped mRNA with a high efficiency comparable to the standard capping process (Table 6), the mRNA did not induce a cytokine response in BJ fibroblasts (Figure 16A), and the mRNA led to high hEPO expression in BJ fibroblasts (Figure 16B). [Table 6]

[0349] In summary, it has been shown herein that the T7 RNA polymerase variant G47A* can efficiently initiate transcription at 3′ dTTP of the template strand in the presence of XAG trinucleotides (e.g., GAG or GmAG). Provided that the trinucleotides are equimolar in concentration as NTPs in the assay, they can be added co-transcriptionally to generate fully capped mRNAs that do not stimulate an immune response, resulting in high protein expression (Figure 17).

[0350] Example 8. Comparison of transcription initiation with 5'GTP (Gstart) or 5'ATP (Astart) Using the co-transcriptional capping assay described in Example 7, we compared capping efficiency to model constructs in which transcription initiation requires 5'-GTP (Gstart) or 5'-ATP (Astart). Model constructs encoding 5'UTR and 47-mer RNA oligonucleotides were used as templates for IVT. Total RNA products were assessed by mass spectrometry, and the results show that Astart constructs incorporate GAG ​​or GmAG trinucleotides more efficiently compared to Gstart constructs (Figure 18A). Although the data is not shown here, it was also observed that Cstart and Ustart constructs generated small amounts of capped products.

[0351] Additionally, the RNA products were subjected to RNase H cleavage and the cleaved 5' ends of the RNA products were analyzed using mass spectrometry to determine the presence of a cap, with similar results obtained as for the total RNA product (Figure 18B).

[0352] Trinucleotide titration experiments were also performed using the model constructs used in Figures 18A and 18B. In the co-transcriptional capping assay, 5 mM NTPs (A, U, G, C modified in equimolar ratio) were used, while the concentration of trinucleotides (GAG or GmAG) was varied to evaluate the capping efficiency at different molar ratios of NTPs and trinucleotides. The crude RNA products were then analyzed by LC-MS. The results show that the capping efficiency is highest when NTPs and trinucleotides are in equimolar ratio for both GAG and GmAG (Table 7). [Table 7]

[0353] Example 9. Capping of chemically modified mRNA with 1-methyl-pseudouridine Co-transcriptional capping of mRNA chemically modified with 1-methyl-pseudouridine was also evaluated. Three model constructs were used: hEPO, Luc (encoding luciferase), and eGFP. All IVT templates were Astart templates. Templates could be PCR fragments or plasmids. Capping assay reaction mixtures included template, T7 RNA polymerase variant G47A*, 7.5 mM NTPs, and 7.5 mM trinucleotides (GAG or GmAG). UTP in the NTPs was replaced with 1-methyl-pseudouridine to generate chemically modified mRNA. Vaccinia Cap 1 was used as a production control in standard capping assays. The mRNA products were analyzed using RNase H cap assays, T1 fingerprinting, and fragment analyzers to determine their integrity. The ability of the mRNA products to induce cytokine responses and express the encoded proteins in BJ fibroblasts was also tested.

[0354] As shown in Table 8, the 1-methyl-pseudouridine chemically modified mRNAs of all three model constructs generated from PCR fragments were efficiently capped, with capping efficiencies comparable to the Vaccinia Cap1 control. Chemistry A indicates the use of unmodified uridine triphosphate nucleotides, chemistry B indicates the use of 1-methyl-pseudouridine triphosphate nucleotides. Chemistry C indicates the use of 5-methyl-cytidine triphosphate and 1-methyl-pseudouridine triphosphate nucleotides. Similar results were obtained for the 1-methyl-pseudouridine chemically modified mRNAs generated from plasmid templates (Table 9). Furthermore, RNase T1 fingerprinting analysis showed that the mRNAs generated from the model constructs using PCR fragment templates were of the correct sequence (Figure 19A). The mRNAs generated from either the PCR fragment templates (Figure 19B) or the plasmid templates (Figure 20C) also showed a high degree of integrity. Capped mRNAs generated from PCR fragment templates as well as plasmid templates did not induce any cytokine expression in BJ fibroblasts (Figure 19C, Figure 20D). Furthermore, mRNAs generated from plasmid templates led to expression of the encoded proteins in BJ fibroblasts (Figure 20E-G). Expression levels of GAG-capped mRNAs were slightly higher, comparable to, or slightly lower than expression levels from vaccinia cap1 control mRNA. [Table 8] [Table 9]

[0355] NTP consumption in co-transcriptional capping assays was also evaluated for reactions using plasmid templates for model constructs hEPO (Figure 20A) and eGFP (Figure 20B). Results show that GAG and GmAG were barely consumed over the duration of the assay (2 hours). This is consistent with the fact that the concentration of trinucleotides in the assay was extremely over-concentrated. It may be possible to recover the trinucleotides after the assay is completed.

[0356] Example 10. Substitutions at the C-terminus of T7 RNA polymerase that affect RNA yield G47A* polymerase contains an extra glycine (a "foot glycine") at the C-terminus of the translated protein. The presence of a foot glycine was confirmed by trypsin digestion and mass spectrometry of purified G47A* protein (data not shown). The role of the C-terminal foot region of G47A* was examined in more detail due to its proximity to the active site of G47A*.

[0357] In vitro transcription (IVT) reactions were performed using hEPO DNA template and (1) wild-type (WT) T7 RNA polymerase (WT), (2) WT T7 RNA polymerase with a foot glycine (WT+G), (3) WT T7 RNA polymerase with two foot glycines (WT+GG), (4) wild-type RNA polymerase with two foot alanines (WT+AA), (5) G47A T7 RNA polymerase variant, (6) G47A* T7 RNA polymerase variant with a foot glycine (G47A+G), (7) G47A T7 RNA polymerase variant with two foot glycines (G47A+GG), (7) G47A T7 RNA polymerase variant with two foot alanines (G47A+AA), (9) S43A / G47A T7 IVT reactions were performed using an RNA polymerase variant (S43A / G47A), and an S43A / G47A T7 RNA polymerase variant with (10) foot glycine (S43A / G47A+G). The IVT reaction mixture contained hEPO DNA template, one of the T7 RNA polymerase variants listed above, and NTPs. IVT reactions with unmodified chemistry (FIG. 22) were performed with standard NTPs (ATP, CTP, GTP, UTP), while IVT reactions with pseudouridine-modified (m1ψ) chemistry (FIG. 23) contained 1-methylpseudouridine instead of UTP to generate chemically modified mRNA. RNA yields were measured by UV absorption.

[0358] hEPO mRNA yield decreased with the presence of a footglycine in either WT or G47A* variant T7 RNA polymerase (Figures 22 and 23). The mRNA yield decreased less with G47A* than with WT T7 RNA polymerase, but continued to decrease with either two footglycine or two footalanine residues.

[0359] Example 11.3´-Effect of T7 RNA polymerase foot glycine on mRNA heterogeneity As in Example 5, hEPO mRNA produced using WT or G47A* T7 RNAP with foot glycines was digested with RNAse T1 and analyzed by LCMS to generate oligo fingerprints. WT T7 RNAP IVT reactions were performed with either equimolar concentrations of NTPs (WT EQ) or molar excess of GTP and ATP (WT alpha(A)). LCMS analysis revealed that homogenous 3′ ends (5′OH / 3′OH) peaked at 32780 Da and the “scar” (5′OH / 3′mP) peaked at 32860 Da (FIG. 24A). The “scar” indicates that the transcript had an untemplated addition at the 3′ end. hEPO mRNA produced using equimolar concentrations of WT T7 RNAP and NTPs (WT EQ) reduced the 3′ end population distribution and showed more run-on products with reduced 3′ homogeneity and untemplated additions (FIG. 24B). However, hEPO mRNAs produced using G47A* T7 RNAP with a foot glycine had a higher 3′-end population distribution, indicating that they had more homogenous 3′-ends and improved 3′-end heterogeneity (Figure 24B).

[0360] To directly test the effect of the T7 RNAP foot region on hEPO 3´ mRNA uniformity, IVT reactions were performed with hEPO DNA template and (1) wild-type T7 RNAP (WT), (2) WT T7 RNAP with a foot glycine (WT+G), (3) WT T7 RNAP with two foot glycines (WT+GG), (4) wild-type T7 RNAP with two foot alanines (WT+AA), (5) G47A T7 RNAP variant, (6) G47A* T7 RNAP variant with a foot glycine (G47A+G), (7) G47A T7 RNAP variant with two foot glycines (G47A+GG), (8) G47A T7 RNAP variant with two foot alanines (G47A+AA), (9) S43A / G47A T7 RNAP variant with a foot alanine (G47A+AA), (10) S43A / G47A T7 RNAP variant with a foot alanine (G47A+AA), (11) S43A / G47A T7 RNAP variant with a foot alanine (G47A+AA), (12) S43A / G47A T7 RNAP variant with a foot alanine (G47A+AA), (13) S43A / G47A T7 RNAP variant with a foot alanine (G47A+AA), (14) S43A / G47A T7 RNAP variant with a foot alanine (G47A+AA), (15) S43A / G47A T7 RNAP variant with a foot alanine (G47A+AA), (16) S43A / G47A T7 RNAP variant with a foot alanine (G47A+AA), (17) S43A / G47A T7 RNAP variant with a foot alanine (G47A+AA), (18) S43A / G47A T7 RNAP variant with a foot a (10) T7 RNAP variant (S43A / G47A), and (11) S43A / G47A T7 RNAP variant with a foot glycine (S43A / G47A+G). The IVT reaction mixture contained hEPO DNA template, one of the T7 RNAP variants listed above, and equimolar NTPs. IVT reactions were performed that produced either unmodified mRNA (Figure 24C) or pseudouridine-modified mRNA (Figure 24D). hEPO mRNA was digested with RNase T1 as in Example 5 and above. The presence of a foot glycine increased the uniformity of the hEPO mRNA 3' end by 60% (Figures 24C-D). Two foot glycines or two alanines in T7 RNAP also improved the uniformity of the 3' end by 60-70% over WT or G47A T7 RNAP (Figures 24C and 24D). These results indicate that the presence of a single foot glycine in T7 RNAP can increase the 3'-end uniformity observed in Figure 24B.

[0361] Example 12. Effect of T7 RNAP foot glycine on immune stimulation The role of T7 RNAP footglycine in stimulating immune responses to hEPO mRNA products was evaluated by assessing their ability to induce cytokine (IFNβ) production in BJ fibroblasts, as in Example 7. Interestingly, WT T7 RNAP-produced unmodified and pseudouridine-modified hEPO mRNA induced high cytokine responses, whereas WT T7 RNAP with footglycine-produced unmodified and pseudouridine-modified hEPO mRNA stimulated less cytokine production in BJ fibroblasts (FIG. 25). hEPO mRNA produced by the G47A* variant T7 RNAP with footglycine failed to stimulate cytokine production in BJ fibroblasts, indicating an additive effect between footglycine and the G47A T7 RNA polymerase variant in failing to induce an immune response (FIG. 25).

[0362] Example 13. Effect of T7 RNA polymerase foot glycine on mRNA production An assay was developed to examine the effect of the footglycine residue on hEPO mRNA species produced by WT and G47A variant T7 RNAPs. The T7 RNAPs tested were: (1) WT+G (footglycine), (2) G47A variant, or (3) G47A variant+G (G47A* variant). IVT reactions consisted of one of the listed T7 RNAPs, shORFan template, NTPs (either unmodified or modified UTP), and 32 P-CTP was included. Reactions were terminated with EDTA after the desired length of time. The reaction mixture was then denatured in 4M urea at 95° C. for 5 minutes before being loaded onto a denaturing polyacrylamide gel (20% acrylamide) (FIG. 26A). The gel was run at 20 watts for 30 minutes and then at 40 watts for 2 hours. Images were transferred to a phosphorimager screen before imaging using a Typhoon scanner (1 hour exposure time). 32The use of P-CTP labels the full-length product (shown as shORFan mRNA), the run-on transcription product (upper box) (Figure 26A), and the reverse complement mRNA product (Figure 26A, lower box). The results are also summarized in Table 10 below. These data show that T7 RNAP with a footglycine is associated with less contaminating dsRNA than either WT or G47A T7 RNAP (Figure 26A (lower box) and Figure 26B). Furthermore, footglycine is associated with reduced run-on transcription (Figure 26A (upper box), Figure 26B) compared to WT or G47A variant T7 RNAP. [Table 10]

[0363] Example 14. Evaluation of repeated administration of trinucleotide-capped G47A* mRNA encoding firefly luciferase In vitro transcribed mRNA is typically purified, for example, by reverse phase chromatography (RP) to remove cytokine-induced impurities (dsRNA) and process impurities (e.g., protein / DNA) and improve total RNA purity. However, this purification process often results in significant loss of mRNA product. Eliminating RP reduces turnaround time, eliminates operational and operational complexity at scale, and saves costs. Furthermore, eliminating RP increases mRNA expression levels because the high temperatures used during RP can hydrolyze a fraction of the mRNA. Therefore, an mRNA product and purification strategy was developed to eliminate the need for RP. The mRNA produced by the method provided in this example is referred to as "trinucleotide-capped G47A* mRNA" because it was found to be capped with the trinucleotide GpppA in an in vitro transcription assay using a G47A* T7 RNAP variant (with a G47A substitution and an additional C-terminal G). 2′Ome This is an mRNA that is cotranscriptionally capped with pG.

[0364] In this example, a single 0.5 mpk dose of mRNA encoding firefly luciferase (ffLuc) was formulated in MC3 lipid nanoparticles and administered weekly to C57Bl / 6 mice (n=5) for 6 weeks (days 1, 8, 15, 22, 29, and 36). The following mRNAs were administered: (1) unmodified mRNA produced using wild-type T7 polymerase in the presence of equimolar concentrations of NTPs and purified by oligo dT purification (Chemical A Process 1 dT), (2) pseudouridine-modified mRNA produced using wild-type T7 polymerase in the presence of excess concentrations of GTP / ATP and purified by reverse phase chromatography (Chemical B Process 2 RP), (3) pseudouridine-modified mRNA produced using wild-type T7 polymerase in the presence of excess concentrations of GTP / ATP and purified by oligo dT purification (Chemical B Process 2 dT), (4) pseudouridine-modified mRNA produced using wild-type T7 polymerase in the presence of equimolar concentrations of NTPs and purified by oligo dT purification (Chemical B Process 1 dT), (5) pseudouridine-modified trinucleotide-capped G47A* mRNA (trinucleotide capped and produced using the G47A* T7 RNAP variant) purified by reverse phase chromatography (Chemical B Process 3 RP), (6) pseudouridine modified trinucleotide capped G47A* mRNA purified by oligo dT purification (Chemistry B Process 3 dT), and (7) uncapped pseudouridine modified mRNA produced using the G47A* T7 RNAP variant and purified by oligo dT purification (Chemistry B Process 4 dT). Six hours after each dose, the following were assessed: serum cytokine levels, mRNA expression, and anti-PEG IgM levels. B cell activation was assessed six hours after the 36th dose. Chemistry A indicates the use of unmodified uridine triphosphate nucleotides and chemistry B indicates the use of 1-methyl-pseudouridine triphosphate nucleotides.Process 1 refers to equimolar NTPs in the IVT and vaccinia cap 1, Process 2 refers to IVT with 4:2:1:1 GTP:ATP:CTP:UTP (see WO2018 / 053209 A1 published March 22, 2018, which is incorporated by reference in its entirety) and vaccinia cap 1, Process 3 refers to equimolar NTPs and GAGs in the IVT, and Process 4 refers to equimolar NTPs in the IVT and no cap.

[0365] Results from serum cytokine assessment (IP-10) in mice on days 1 and 29 are presented in Figures 27A and 27B and show that trinucleotide-capped G47A* mRNA induced serum cytokine levels in vivo that were similar to the alpha mRNA control. Results were similar for in vitro experiments in which mRNA was delivered to BJ human fibroblasts (Figure 28A) and monocyte-derived macrophages (MDM) (Figure 28B).

[0366] mRNA expression studies (Figure 29) showed that trinucleotide-capped G47A* mRNA remained highly expressed in vivo after six biweekly doses.

[0367] Anti-PEG IgM assessment (Figure 30) showed that N1U trinucleotide capped G47A* mRNA produced low anti-PEG IgM levels after six biweekly doses.

[0368] B cell activation assays (FIG. 31) showed that activation of splenic B cells transfected with trinucleotide-capped G47A* mRNA was low and similar to the alpha mRNA control.

[0369] Taken together, these results indicate that trinucleotide-capped G47A* mRNA encoding ffLuc that was not RP-purified does not induce cytokine responses above baseline, maintains low anti-PEG IgM levels in vivo, and exhibits low B cell activation.

[0370] Example 15. Evaluation of repeated administration of trinucleotide-capped G47A* mRNA encoding human erythropoietin In this example, a single 0.5 mpk dose of mRNA encoding human erythropoietin (hEPO) was formulated in MC3 lipid nanoparticles and administered weekly to C57Bl / 6 mice (n=5) for 6 weeks (days 1, 8, 15, 22, 29, and 36). The following mRNAs were administered: (1) unmodified mRNA produced using wild-type T7 polymerase in the presence of equimolar concentrations of NTPs and purified by oligo dT purification (Chemical A Process 1 dT), (2) pseudouridine-modified mRNA produced using wild-type T7 polymerase in the presence of excess concentrations of GTP / ATP and purified by reverse phase chromatography (Chemical B Process 2 RP), (3) pseudouridine-modified mRNA produced using wild-type T7 polymerase in the presence of excess concentrations of GTP / ATP and purified by oligo dT purification (Chemical B Process 2 dT), (4) pseudouridine-modified mRNA produced using wild-type T7 polymerase in the presence of equimolar concentrations of NTPs and purified by oligo dT purification (Chemical B Process 1 dT), (5) pseudouridine-modified trinucleotide-capped G47A* mRNA (trinucleotide capped and produced using the G47A* T7 RNAP variant) purified by reverse phase chromatography (Chemical B Process 3 RP), (6) pseudouridine modified trinucleotide capped G47A* mRNA purified by oligo dT purification (Chemistry B Process 3 dT), and (7) uncapped pseudouridine modified mRNA produced using the G47A* T7 RNAP variant and purified by oligo dT purification (Chemistry B Process 4 dT). Six hours after each dose, the following were assessed: serum cytokine levels, mRNA expression, and anti-PEG IgM levels. B cell activation was assessed six hours after the 36th dose. Chemistry A indicates the use of unmodified uridine triphosphate nucleotides and chemistry B indicates the use of 1-methyl-pseudouridine triphosphate nucleotides.Process 1 refers to equimolar NTPs in the IVT and vaccinia cap 1, process 2 refers to IVT with 4:2:1:1 GTP:ATP:CTP:UTP and vaccinia cap 1, process 3 refers to equimolar NTPs and GAG in the IVT, and process 4 refers to equimolar NTPs in the IVT and no cap.

[0371] Results from serum cytokine assessment (IP-10) in mice on days 1 and 22 are presented in Figures 32A and 32B and show that trinucleotide-capped G47A* mRNA induced serum cytokine levels in vivo that were similar to the alpha mRNA control. Results were similar for in vitro experiments in which mRNA was delivered to BJ human fibroblasts (Figure 33A) and monocyte-derived macrophages (MDM) (Figure 33B).

[0372] mRNA expression studies (Figure 34) showed that trinucleotide-capped G47A* mRNA remained highly expressed in vivo after six biweekly doses.

[0373] Anti-PEG IgM assessment (Figure 35) showed that trinucleotide capped G47A* mRNA produced low anti-PEG IgM levels after six biweekly doses.

[0374] B cell activation assays (FIG. 36) showed that activation of splenic B cells transfected with trinucleotide-capped G47A* mRNA was low and similar to the alpha mRNA control.

[0375] Taken together, these results indicate that trinucleotide-capped G47A* mRNA encoding non-RP-purified hEPO does not induce cytokine responses above baseline, maintains low anti-PEG IgM levels in vivo, and exhibits low B cell activation.

[0376] Example 16. Assessment of indel and point mutation frequencies mRNA was prepared with either WT or G47A* enzyme using process 1 (equimolar NTPs) or process 2 (4:2:1:1 GTP:ATP:CTP:UTP). For next generation sequencing (NGS), mRNA was converted to cDNA by reverse transcriptase and adapters were ligated to prepare sequencing libraries. NGS data was compared to parental sequences and any indels observed were tabulated. Indel frequencies were comparable for WT and G47A* (Figure 37A). Similarly, NGS data was analyzed for any point mutations. Point mutation frequencies were comparable for WT and G47A* (Figure 37B).

[0377] All references, patents, and patent applications disclosed herein are incorporated by reference with respect to the subject matter for which each is indicated, which in some cases may include the entire document.

[0378] As used herein in the specification and the claims, the indefinite articles "a" and "an" should be understood to mean "at least one," unless clearly stated to the contrary.

[0379] Unless clearly stated to the contrary, in any method claimed herein that includes more than one step or act, the order of the method steps or acts is not necessarily limited to the order in which the method steps or acts are recited.

[0380] In the claims, as well as in the above specification, all transitional phrases such as "comprising," "including," "carrying," "having," "containing," "involving," "holding," "composed of," and the like, are to be understood to mean open-ended, i.e., including but not limited to. Only the transitional phrases "consisting of" and "consisting essentially of" are intended to be closed or semi-closed transitional phrases, respectively, as set forth in the United States Patent Office Manual of Patent Examining Procedures, Section 2111.03.

Claims

1. A ribonucleic acid (RNA) polymerase variant, comprising an amino acid substitution, compared to a wild-type RNA polymerase, that causes a loop structure of the RNA polymerase variant to undergo a conformational change to a helical structure as the RNA polymerase variant transitions from an initiation complex to an elongation complex.

2. The RNA polymerase variant of claim 1 , wherein the amino acid substitution has a high helical propensity compared to the wild-type amino acid.

3. The RNA polymerase variant of claim 1 or 2, wherein the RNA polymerase is T7 RNA polymerase.

4. 、 The RNA polymerase variant according to any one of claims 1 to 3, wherein the loop structure is in a C-helix structure.

5. The RNA polymerase variant according to any one of claims 1 to 4, wherein the loop structure is in a C linker structure.

6. The RNA polymerase variant according to any one of claims 1 to 4, wherein the amino acid substitution is a high helix-propensity amino acid substitution.

7. 7. The RNA polymerase variant of claim 6, wherein the high helix-prone acid substitutions are selected from alanine, isoleucine, leucine, methionine, lysine, glutamine, and glutamic acid.

8. The RNA polymerase variant of claim 7, wherein the high helix-prone amino acid substitution is alanine.

9. The RNA polymerase variant of any one of claims 1 to 8, wherein the T7 RNA polymerase variant comprises an amino acid sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO:

1.

10. 10. The RNA polymerase variant of claim 9, wherein the T7 RNA polymerase comprises an amino acid sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO:1 modified to include amino acid substitutions of high helix-propensity amino acids at positions selected from E42, S43, Y44, E45, M46, G47, A255, R257, A258, G259, A260, L261, and A262.

11. The RNA polymerase variant of claim 10 , wherein the amino acid substitution comprises S43A.

12. The RNA polymerase variant of claim 10 , wherein the amino acid substitution comprises G47A.

13. The RNA polymerase variant of any one of claims 1 to 12, further comprising an additional C-terminal amino acid.

14. The RNA polymerase variant of claim 13, wherein the additional C-terminal amino acid comprises glycine (G).

15. The RNA polymerase variant of claim 11, 13, or 14, wherein the RNA polymerase variant comprises the amino acid sequence of SEQ ID NO:

108.

16. The RNA polymerase variant of any one of claims 12 to 14, wherein the RNA polymerase variant comprises the amino acid sequence of SEQ ID NO:

110.

17. A ribonucleic acid (RNA) polymerase variant that contains additional C-terminal amino acids compared to a wild-type RNA polymerase.

18. The RNA polymerase variant of claim 17, wherein the RNA polymerase variant is a T7 RNA polymerase variant.

19. The RNA polymerase variant of claim 18, wherein the RNA polymerase variant has at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to wild-type T7 RNA polymerase.

20. The RNA polymerase variant of claim 17 or 18, wherein the RNA polymerase variant comprises an amino acid substitution compared to wild-type T7 RNA polymerase.

21. 21. The RNA polymerase variant of any one of claims 17 to 20, comprising two additional C-terminal amino acids.

22. 22. The RNA polymerase variant of claim 21, wherein the two additional C-terminal amino acids comprise the same type of amino acid or two different types of amino acids.

23. 23. The RNA polymerase variant of any one of claims 17 to 22, comprising 1 to 5 or 1 to 10 additional C-terminal amino acids.

24. FAX ｎ (SEQ ID NO:171) A RNA polymerase variant comprising a C-terminus comprising the motif, wherein X is any amino acid and n is any integer greater than zero.

25. 25. The RNA polymerase variant of claim 24, wherein X is G or A.

26. 26. The RNA polymerase variant of claim 24 or 25, wherein n is any integer greater than zero, optionally wherein n is 1, 2, 3, 4, or 5.

27. A RNA polymerase variant, optionally a T7 RNA polymerase variant, comprising a C-terminus containing a FAFAG (SEQ ID NO: 329) motif.

28. XAFAX ｎ Motif, FXFAX ｎ Motif, FAXAX ｎ Motif or FAFXX ｎ wherein each X is any amino acid and n is any integer greater than zero.

29. The RNA polymerase variant of any one of claims 17 to 28, further comprising an amino acid substitution.

30. 30. The RNA polymerase variant of claim 29, wherein the amino acid substitutions are high helix-prone amino acids at positions selected from E42, S43, Y44, E45, M46, G47, R257, A258, G259, A260, L261, and A262 of the wild-type RNA polymerase, and optionally wherein the wild-type RNA polymerase comprises the amino acid sequence of SEQ ID NO:1 or an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO:

1.

31. The RNA polymerase variant of claim 30, wherein the high helix-prone amino acid is alanine.

32. The RNA polymerase variant of claim 31 , wherein the amino acid substitution is S43A or G47A.

33. A method for producing ribonucleic acid (RNA) comprising contacting a DNA template with an RNA polymerase according to any one of claims 1 to 32 under conditions which result in the production of an RNA transcript.

34. 33. A method of performing an in vitro transcription (IVT) reaction comprising contacting a DNA template with an RNA polymerase according to any one of claims 1 to 32 in the presence of nucleoside triphosphates and a buffer under conditions that result in the production of an RNA transcript.

35. 35. The method of claim 33 or 34, wherein the generated RNA transcripts, when delivered to a cell, optionally in unpurified form, stimulate at least a 50% lower cytokine response compared to RNA generated using a wild-type RNA polymerase.

36. 36. The method of any one of claims 33 to 35, wherein the concentration of double-stranded RNA (dsRNA) transcripts produced is at least 50% lower compared to dsRNA transcripts produced using a wild-type polymerase.

37. 37. The method of any one of claims 33 to 36, wherein less than 20% of the RNA transcripts produced exhibit 3' heterogeneity.

38. 38. The method of any one of claims 33 to 37, wherein less than 50% of the RNA transcripts produced are double-stranded contaminants.

39. 39. The method of any one of claims 33 to 38, wherein less than 50% of the RNA transcripts produced are run-on RNA transcripts.

40. 40. The method of any one of claims 33 to 39, wherein the amount of full-length RNA transcript produced is at least 15-fold greater than the amount of said DNA template.

41. 41. The method of any one of claims 33 to 40, wherein the ratio of double-stranded contaminants:full-length RNA transcripts produced is less than 1:

1.

42. 42. The method of any one of claims 33 to 41, wherein the produced RNA transcripts have less than 1 mutation per 100 nucleotides compared to the DNA template.

43. A nucleic acid encoding the RNA polymerase variant according to any one of claims 1 to 32.

44. A vector comprising the nucleic acid of claim 43.

45. 45. A host cell comprising the nucleic acid of claim 43 or the vector of claim 44.

46. A composition or kit comprising an RNA polymerase variant according to any one of claims 1 to 32 and optionally in vitro transcription (IVT) reagents.

47. 43. Ribonucleic acid (RNA), optionally messenger RNA (mRNA), produced by the method of any one of claims 33 to 42.

48. 48. The RNA of claim 47 formulated in a lipid nanoparticle, optionally wherein the lipid nanoparticle comprises a molar ratio of 20-60% ionizable amino lipid, 5-25% non-cationic lipid, 25-55% sterol, and 0.5-15% PEG-modified lipid.

49. A co-transcriptional capping method for ribonucleic acid (RNA) synthesis, comprising reacting a polynucleotide template with an RNA polymerase variant, optionally a T7 RNA polymerase variant, nucleoside triphosphates, and a cap analog under in vitro transcription reaction conditions to produce an RNA transcript.

50. 50. The method of claim 49, wherein greater than 80%, greater than 85%, greater than 90%, or greater than 95% of the RNA transcripts produced contain a functional cap.

51. 51. The method of claim 49 or 50, wherein the nucleoside triphosphates comprise unmodified or modified ATP, modified or unmodified UTP, modified or unmodified GTP, and / or modified or unmodified CTP.

52. The method according to any one of claims 49 to 51, wherein the RNA polymerase variant is an RNA polymerase variant according to any one of claims 1 to 32.

53. 53. The method of any one of claims 49 to 52, wherein the nucleoside triphosphate and cap analog are present in equimolar concentrations in the reaction.

54. 54. The method of any one of claims 49-53, wherein the molar ratio of cap analog to nucleoside triphosphate in the reaction is greater than or equal to 1:1, and optionally the nucleoside triphosphate comprises GTP and / or ATP.

55. 55. The method of any one of claims 49 to 54, wherein the cap analog is a dinucleotide cap, a trinucleotide cap, or a tetranucleotide cap.

56. 56. The method of claim 55, wherein the cap comprises a sequence selected from the following sequences: GAA, GAC, GAG, GAU, GCA, GCC, GCG, GCU, GGA, GGC, GGG, GGU, GUA, GUC, GUG, and GUU.

57. The trinucleotide cap has the following sequence: (a) m ７ GpppApA,m ７ GpppApC, m ７ GpppApG, m ７ GpppApU,m ７ GpppCpA,m ７ GpppCpC, m ７ GpppCpG, m ７ GpppCpU,m ７ GpppGpA,m ７ GpppGpC, m ７ GpppGpG,m ７ GpppGpU,m ７ GpppUpA,m ７ GpppUpC, m ７ GpppUpG, and m ７ GpppUpU; (b) m ７ G ３′ＯＭｅ pppApA, m ７ G ３′ＯＭｅ pppApC, m ７ G ３′ＯＭｅ pppApG, m ７ G ３′ＯＭｅ pppApU, m ７ G ３′ＯＭｅ pppCpA, m ７ G ３′ＯＭｅ pppCpC, m ７ G ３′ＯＭｅ pppCpG, m ７ G ３′ＯＭｅ pppCpU, m ７ G ３′ＯＭｅ pppGpA, m ７ G ３′ＯＭｅ pppGpC, m ７ G ３′ＯＭｅ pppGpG, m ７ G ３′ＯＭｅ pppGpU, m ７ G ３′ＯＭｅ pppUpA, m ７ G ３′ＯＭｅ pppUpC, m ７ G ３′ＯＭｅ pppUpG, and m ７ G ３′ＯＭｅ pppUpU; (c) m ７ G ３′ＯＭｅ pppA ２′ＯＭｅ pA, m ７ G ３′ＯＭｅ pppA ２′ＯＭｅ pC, m ７ G ３′ＯＭｅ pppA ２′ＯＭｅ pG, m ７ G ３′ＯＭｅ pppA ２′ＯＭｅ pU, m ７ G ３′ＯＭｅ pppC ２′ＯＭｅ pA, m ７ G ３′ＯＭｅ pppC ２′ＯＭｅ pC, m ７ G ３′ＯＭｅ pppC ２′ＯＭｅ pG, m ７ G ３′ＯＭｅ pppC ２′ＯＭｅ pU, m ７ G ３′ＯＭｅ pppG ２′ＯＭｅ pA, m ７ G ３′ＯＭｅ pppG ２′ＯＭｅ pC, m ７ G ３′ＯＭｅ pppG ２′ＯＭｅ pG, m ７ G ３′ＯＭｅ pppG ２′ＯＭｅ pU, m ７ G ３′ＯＭｅ pppU ２′ＯＭｅ pA, m ７ G ３′ＯＭｅ pppU ２′ＯＭｅ pC, m ７ G ３′ＯＭｅ pppU ２′ＯＭｅ pG, and m ７ G ３′ＯＭｅ pppU ２′ＯＭｅ pU; or (d) m ７ GpppA ２′ＯＭｅ pA,m ７ GpppA ２′ＯＭｅ pC, m ７ GpppA ２′ＯＭｅ pG,m ７ GpppA ２′ＯＭｅ pU,m ７ GpppC ２′ＯＭｅ pA,m ７ GpppC ２′ＯＭｅ pC, m ７ GpppC ２′ＯＭｅ pG,m ７ GpppC ２′ＯＭｅ pU,m ７ GpppG ２′ＯＭｅ pA,m ７ GpppG ２′ＯＭｅ pC, m ７ GpppG ２′ＯＭｅ pG,m ７ GpppG ２′ＯＭｅ pU,m ７ GpppU ２′ＯＭｅ pA,m ７ GpppU ２′ＯＭｅ pC, m ７ GpppU ２′ＯＭｅ pG, and m ７ GpppU ２′ＯＭｅ 57. The method of claim 56, comprising a sequence selected from pU.

58. 58. The method of claim 56 or 57, wherein the trinucleotide cap comprises a sequence selected from the following sequences: GAG, GCG, GUG, and GGG.

59. 59. The method of claim 58, wherein the trinucleotide cap comprises the sequence GAG.

60. The trinucleotide cap is GpppA ２′Ｏｍｅ 60. The method of claim 59, comprising pG.

61. 61. The method of any one of claims 49 to 60, wherein the polynucleotide template comprises a 2'-deoxythymidine residue at template position +1.

62. 61. The method of any one of claims 49 to 60, wherein the polynucleotide template comprises a 2'-deoxycytidine residue at template position +1.

63. A co-transcriptional capping method for RNA synthesis, comprising: coupling a polynucleotide template to (a) a T7 RNA polymerase variant, the T7 RNA polymerase variant comprising at least one amino acid substitution, as compared to a wild-type RNA polymerase, that causes at least one loop structure of the RNA polymerase variant to undergo a conformational change to a helical structure as the RNA polymerase variant transitions from an initiation complex to an elongation complex; (b) a nucleoside triphosphate; and (c) a nucleotide sequence of the sequence GpppA; ２′Ｏｍｅ reacting the polynucleotide template with a trinucleotide cap containing pG, wherein the polynucleotide template contains a 2'-deoxythymidine residue at template position +1.

64. 64. The method of any one of claims 49-63, wherein the produced RNA transcripts, when delivered to a cell, optionally in unpurified form, do not stimulate a detectable cytokine response or do not stimulate a cytokine response above a baseline control.

65. A composition comprising in vitro transcribed (IVT) RNA and a pharma- ceutically acceptable excipient, the composition being substantially free of cytokine-induced RNA contaminants without post-IVT purification.

66. A composition comprising an in vitro transcribed (IVT) RNA and a pharma- ceutically acceptable excipient, the composition comprising less than 5% uncapped RNA species.

67. 67. The composition of claims 65 or 66, wherein more than 80%, more than 85%, more than 90%, or more than 95% of the IVT RNA comprises a functional cap.

68. 68. The composition of any one of claims 65-67, wherein the IVT RNA is not chemically modified.

69. 68. The composition of any one of claims 65-67, wherein the IVT RNA is chemically modified.

70. 70. The composition of any one of claims 65-69, wherein greater than 95% of the IVT RNA comprises a single-stranded full-length transcript.

71. The RNA is Under in vitro transcription reaction conditions to produce an RNA transcript, a polynucleotide template is transfected with (a) a T7 RNA polymerase variant, the T7 RNA polymerase variant comprising at least one amino acid substitution, as compared to a wild-type RNA polymerase, that causes at least one loop structure of the RNA polymerase variant to undergo a conformational change to a helical structure as the RNA polymerase variant transitions from an initiation complex to an elongation complex; (b) a nucleoside triphosphate; and (c) a nucleic acid sequence comprising the sequence GpppA. ２′Ｏｍｅ 71. The composition of any one of claims 65-70, wherein the polynucleotide template comprises a 2'-deoxythymidine residue at template position +1, produced by a process comprising reacting with a trinucleotide cap comprising pG.

72. 1. A T7 ribonucleic acid (RNA) polymerase having at least 90% identity to a wild-type T7 RNA polymerase, comprising an amino acid sequence modified to include (a) an alanine at an amino acid position corresponding to amino acid 47 of SEQ ID NO:1, and (b) an additional C-terminal glycine.

73. 73. The T7 RNA polymerase of claim 72, further comprising a trinucleotide cap.

74. The trinucleotide cap is GpppA ２′Ｏｍｅ 74. The T7 RNA polymerase of claim 73, which is pG.

75. 1. A T7 ribonucleic acid (RNA) polymerase having at least 90% identity to a wild-type T7 RNA polymerase, comprising an amino acid sequence modified to include (a) an alanine at an amino acid position corresponding to amino acid 43 of SEQ ID NO:1, and (b) an additional C-terminal glycine.

76. 76. The T7 RNA polymerase of claim 75, further comprising a trinucleotide cap.

77. The trinucleotide cap is GpppA ２′Ｏｍｅ 77. The T7 RNA polymerase of claim 76, which is pG.

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