RNA polymerase variants

Abstract

The RNA polymerase variant can realize efficient transcription of RNA. In some embodiments, the RNA polymerase variants are capable of achieving RNA transcription with high capping efficiency and / or low levels of double-stranded RNA contamination.

Description

[0001] Related Applications

[0002] This application claims the benefit of U.S. Provisional Application No. 63 / 331,145, filed on April 14, 2022, under 35 U.S.C. §119(e), the contents of which are incorporated herein by reference in their entirety.

[0003] References to Electronic Sequence Listings

[0004] The contents of the electronic Sequence Listing (M137870217WO00-SEQ-HJD.xml; size: 31,370 bytes; and creation date: April 12, 2023) are incorporated herein by reference in their entirety. Background Art

[0005] The emergence of therapeutic agents based on ribonucleic acid (RNA) requires polymerases that produce RNA and produce few byproducts with abnormal activity. Transcripts produced by in vitro transcription using bacteriophage T7 RNA polymerase show immunostimulatory activity that is usually unexpected and uncontrollable. This immunostimulatory activity of T7 transcripts is caused by its abnormal activity of initiating transcription from the promoter-free deoxyribonucleic acid (DNA) end. This activity leads to the production of antisense RNA that is completely complementary to the expected sense RNA product, and thus produces long double-stranded RNA (dsRNA) that can strongly stimulate unexpected immune responses. In addition, in the presence of cap analogs, bacteriophage T7 RNA polymerase produces T7 transcripts with low 5' end capping efficiency, partly because the polymerase has a low binding affinity to the cap analogs. Summary of the invention

[0006] Some aspects include T7 RNA polymerase variants and in vitro transcription methods using these variants, which have been shown to reduce dsRNA contaminants and / or increase co-transcriptional 5' end capping efficiency relative to a control (e.g., a wild-type T7 RNA polymerase comprising the amino acid sequence of SEQ ID NO: 1).

[0007] Some aspects provide a ribonucleic acid (RNA) polymerase variant, the RNA polymerase variant comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 2-9, wherein the amino acid sequence comprises an amino acid substitution at position D351 and at least two additional amino acid substitutions relative to the RNA polymerase comprising the amino acid sequence of SEQ ID NO: 1.

[0008] Some aspects provide an RNA polymerase variant comprising an amino acid sequence comprising at least one, at least two, at least three, or at least four amino acid substitutions relative to a wild-type T7 RNA polymerase (e.g., a wild-type T7 RNA polymerase comprising the amino acid sequence of SEQ ID NO: 1).

[0009] Some aspects provide an RNA polymerase variant comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, or 100% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 2-9.

[0010] Some aspects provide an RNA polymerase variant, comprising: an amino acid sequence, relative to a wild-type T7 RNA polymerase comprising the amino acid sequence of SEQ ID NO: 1, comprising (i) an amino acid substitution at position E350, (ii) an amino acid substitution at D351, and (iii) an amino acid substitution at position K387, position N437, or at position K387 and position N437.

[0011] In some embodiments, the amino acid sequence of the variant comprises an amino acid substitution at position K387.

[0012] In some embodiments, the amino acid sequence of the variant comprises an amino acid substitution at position N437.

[0013] In some embodiments, the amino acid sequence of the variant comprises an amino acid substitution at position K387 and at position N437.

[0014] In some embodiments, the amino acid substitution at position K387 is a polar neutral amino acid.

[0015] In some embodiments, the polar neutral amino acid is selected from asparagine (N), cysteine ​​(C), glutamine (Q), methionine (M), serine (S), and threonine (T).

[0016] In some embodiments, the polar neutral amino acid is asparagine (K387N).

[0017] In some embodiments, the polar neutral amino acid is cysteine ​​(K387C).

[0018] In some embodiments, the polar neutral amino acid is glutamine (K387Q).

[0019] In some embodiments, the polar neutral amino acid is methionine (K387M).

[0020] In some embodiments, the polar neutral amino acid is serine (K387S).

[0021] In some embodiments, the polar neutral amino acid is threonine (K387T).

[0022] In some embodiments, the amino acid substitution at position N437 is an aromatic amino acid.

[0023] In some embodiments, the aromatic amino acid is selected from tryptophan (W), tyrosine (Y), and phenylalanine (F).

[0024] In some embodiments, the aromatic amino acid is tryptophan (N437W).

[0025] In some embodiments, the aromatic amino acid is tyrosine (N437Y).

[0026] In some embodiments, the aromatic amino acid is phenylalanine (N437F).

[0027] Other aspects provide an RNA polymerase variant comprising an amino acid sequence, relative to a wild-type T7 RNA polymerase comprising the amino acid sequence of SEQ ID NO 1, the amino acid sequence comprising (i) an amino acid substitution at position E350, (ii) an amino acid substitution at D351, and (iii) an amino acid substitution at position D653.

[0028] In some embodiments, the amino acid substitution at position D653 is an aromatic amino acid.

[0029] In some embodiments, the aromatic amino acid is selected from tryptophan (W), tyrosine (Y), and phenylalanine (F).

[0030] In some embodiments, the aromatic amino acid is tryptophan (D653W).

[0031] In some embodiments, the aromatic amino acid is tyrosine (D653Y).

[0032] In some embodiments, the aromatic amino acid is phenylalanine (D653F).

[0033] In some embodiments, the amino acid substitution at position E350 is an aromatic amino acid.

[0034] In some embodiments, the aromatic amino acid is selected from tryptophan (W), tyrosine (Y), and phenylalanine (F).

[0035] In some embodiments, the aromatic amino acid is tryptophan (E350W).

[0036] In some embodiments, the aromatic amino acid is tyrosine (E350Y).

[0037] In some embodiments, the aromatic amino acid is phenylalanine (E350F).

[0038] In some embodiments, the amino acid substitution at position D351 is a non-polar aliphatic amino acid.

[0039] In some embodiments, the non-polar aliphatic amino acid is selected from alanine (A), glycine (G), isoleucine (I), leucine (L), proline (P), and valine (V).

[0040] In some embodiments, the non-polar aliphatic amino acid is alanine (D351A).

[0041] In some embodiments, the non-polar aliphatic amino acid is glycine (D351G).

[0042] In some embodiments, the non-polar aliphatic amino acid is isoleucine (D351I).

[0043] In some embodiments, the non-polar aliphatic amino acid is leucine (D351L).

[0044] In some embodiments, the non-polar aliphatic amino acid is proline (D351P).

[0045] In some embodiments, the non-polar aliphatic amino acid is valine (D351V).

[0046] Other aspects provide an RNA polymerase variant, comprising: an amino acid sequence having at least 70% identity with the amino acid sequence of SEQ ID NO: 1, wherein, relative to the wild-type T7 RNA polymerase comprising the amino acid sequence of SEQ ID NO: 1, the amino acid sequence of the variant comprises (i) an amino acid substitution at position E350, (ii) an amino acid substitution at D351, and (iii) amino acid substitutions at position K387, position N437, or at position K387 and position N437.

[0047] In some embodiments, the amino acid sequence is at least 75%, at least 80%, at least 85%, at least 95%, or at least 98% identical to the amino acid sequence of SEQ ID NO:1.

[0048] In some embodiments, the amino acid sequence of the variant comprises an amino acid substitution at position K387.

[0049] In some embodiments, the amino acid sequence of the variant comprises an amino acid substitution at position N437.

[0050] In some embodiments, the amino acid sequence of the variant comprises an amino acid substitution at position K387 and at position N437.

[0051] In some embodiments, the amino acid substitution at position K387 is a polar neutral amino acid.

[0052] In some embodiments, the polar neutral amino acid is selected from asparagine (K387N), cysteine ​​(K387C), glutamine (K387Q), methionine (K387M), serine (K387S), and threonine (K387T).

[0053] In some embodiments, the amino acid substitution at position N437 is an aromatic amino acid.

[0054] In some embodiments, the aromatic amino acid is selected from tryptophan (N437W), tyrosine (N437Y), and phenylalanine (N437F).

[0055] Other aspects provide an RNA polymerase variant, comprising: an amino acid sequence having at least 70% identity with the amino acid sequence of SEQ ID NO: 1, wherein relative to the wild-type T7 RNA polymerase comprising the amino acid sequence of SEQ ID NO: 1, the amino acid sequence of the variant comprises (i) an amino acid substitution at position E350, (ii) an amino acid substitution at D351, and (iii) an amino acid substitution at position D653.

[0056] In some embodiments, the amino acid sequence is at least 75%, at least 80%, at least 85%, at least 95%, or at least 98% identical to the amino acid sequence of SEQ ID NO:1.

[0057] In some embodiments, the amino acid substitution at position D653 is an aromatic amino acid.

[0058] In some embodiments, the aromatic amino acid is selected from tryptophan (D653W), tyrosine (D653Y), and phenylalanine (D653F).

[0059] In some embodiments, the amino acid substitution at position E350 is an aromatic amino acid.

[0060] In some embodiments, the aromatic amino acid is selected from tryptophan (E350W), tyrosine (E350Y), and phenylalanine (E350F).

[0061] In some embodiments, the amino acid substitution at position D351 is a non-polar aliphatic amino acid.

[0062] In some embodiments, the non-polar aliphatic amino acid is selected from alanine (D351A), glycine (D351G), isoleucine (D351I), leucine (D351L), proline (D351P), and valine (D351V).

[0063] Some aspects provide an RNA polymerase variant comprising the amino acid sequence of SEQ ID NO: 2, wherein X 1 is an aromatic amino acid, optionally selected from W, Y, and F; X 2 is selected from non-polar aliphatic amino acids, optionally selected from A, G, I, L, P and V; X 3 is a polar neutral amino acid, optionally selected from N, C, Q, M, S and T; and X 4 is an aromatic amino acid, optionally selected from W, Y and F. In some embodiments, the RNA polymerase variant comprises the amino acid sequence of SEQ ID NO:6.

[0064] Some aspects provide an RNA polymerase variant comprising the amino acid sequence of SEQ ID NO: 3, wherein X 1 is an aromatic amino acid, optionally selected from W, Y, and F; X 2 is selected from non-polar aliphatic amino acids, optionally selected from A, G, I, L, P and V; and X 4 is an aromatic amino acid, optionally selected from W, Y and F. In some embodiments, the RNA polymerase variant comprises the amino acid sequence of SEQ ID NO:7.

[0065] Some aspects provide an RNA polymerase variant comprising the amino acid sequence of SEQ ID NO: 4, wherein X 1 is an aromatic amino acid, optionally selected from W, Y, and F; X 2 is selected from non-polar aliphatic amino acids, optionally selected from A, G, I, L, P and V; and X 3 is a polar neutral amino acid, optionally selected from N, C, Q, M, S and T. In some embodiments, the RNA polymerase variant comprises the amino acid sequence of SEQ ID NO:8.

[0066] Some aspects provide an RNA polymerase variant comprising the amino acid sequence of SEQ ID NO: 5, wherein X 1 is an aromatic amino acid, optionally selected from W, Y, and F; X 2 is selected from non-polar aliphatic amino acids, optionally selected from A, G, I, L, P and V; and X 5is an aromatic amino acid, optionally selected from W, Y and F. In some embodiments, the RNA polymerase variant comprises the amino acid sequence of SEQ ID NO:9.

[0067] Some aspects provide a method comprising: producing messenger RNA (mRNA) in an in vitro transcription reaction comprising DNA, nucleoside triphosphates, the RNA polymerase variant of any of the preceding paragraphs, and optionally a cap analog.

[0068] In some embodiments, the reaction includes a cap analog.

[0069] In some embodiments, the cap analog is a dinucleotide cap analog, a trinucleotide cap analog, or a tetranucleotide cap analog. In some embodiments, the cap analog is a tetranucleotide cap analog.

[0070] In some embodiments, the cap analog is a trinucleotide cap analog comprising a GAG sequence. In some embodiments, the GAG ​​cap analog comprises a compound selected from the group consisting of:

[0071]

[0072] In some embodiments, the tetranucleotide cap analog comprises a GGAG sequence. In some embodiments, the tetranucleotide cap analog comprises a compound selected from the group consisting of:

[0073]

[0074]

[0075] In some embodiments, the DNA includes a 2'-deoxythymidine residue or a 2'-deoxycytidine residue at position +1.

[0076] Some aspects include a composition or kit comprising the RNA polymerase variant of any of the preceding paragraphs and an in vitro transcription (IVT) reagent selected from the group consisting of DNA, nucleoside triphosphates, and a cap analog.

[0077] Some aspects include a nucleic acid encoding the RNA polymerase variant of any of the preceding paragraphs. BRIEF DESCRIPTION OF THE DRAWINGS

[0078] Figures 1A-1D A graph depicting the functional properties of transcribed RNA products produced by in vitro transcription (IVT) reactions involving exemplary RNA polymerase variants is shown. After oligo dT purification, the yield of transcribed RNA products was analyzed ( Figure 1A ), percentage of capped RNA ( Figure 1B), the percentage of RNA tailing according to the Tris RP (reverse phase) method (i.e., the percentage of RNA containing a poly A tail) ( Figure 1C ) and the amount of dsRNA ( Figure 1D ).

[0079] Figures 2A-2C A graph depicting the functional properties of transcribed RNA products produced by in vitro transcription (IVT) reactions involving exemplary RNA polymerase variants in the presence of different levels of GGAG cap analogs is shown. After oligo dT purification, the transcribed RNA products were analyzed for the percentage of capped RNA ( Figure 2A ), yield( Figure 2B ) and the percentage of RNA tailing according to the Tris RP (reverse phase) method (i.e., the percentage of RNA containing a poly A tail) ( Figure 2C ). DETAILED DESCRIPTION

[0080] RNA polymerase (e.g., DNA-dependent RNA polymerase) is an enzyme that catalyzes the sequential addition of ribonucleotides to the 3' end of a growing RNA chain (RNA transcription in the 5'→3' direction), wherein nucleoside triphosphates (NTPs) serve as substrates for the enzyme and the sequence of nucleotides is specified by a DNA template. Transcription depends on the complementary pairing of bases. The two chains of the double helix are partially separated, and one of the separated chains serves as a template (DNA template). RNA polymerase then catalyzes the alignment of free nucleotides on the DNA template by the complementary bases in the template. Therefore, if RNA polymerase catalyzes the sequential addition of ribonucleotides to the 3' end of a growing RNA chain, RNA polymerase is considered to have RNA polymerase activity.

[0081] DNA-directed RNA polymerases are able to initiate RNA synthesis in the absence of a primer; the first catalytic phase of initiation is called de novo RNA synthesis. De novo synthesis is a unique phase in the transcription cycle in which the RNA polymerase binds two nucleotides rather than a nascent RNA polymer and a single nucleotide. For bacteriophage T7 RNA polymerase, transcription clearly prefers to start with GTP at the +1 and +2 positions. The position at which the initiating nucleotide binds to the RNA polymerase is different from that described for the elongation complex (Kennedy WP et al. J Mol Biol. 2007; 370(2): 256-68). The selection bias in favor of GTP as the initiating nucleotide is achieved by shape complementarity, extensive protein side chains, and strong base stacking interactions with the guanine moiety in the enzyme active site. Therefore, the initiating GTP provides the greatest stabilizing force for the open promoter conformation (Kennedy et al. 2007). In some embodiments, the RNA polymerase variant comprises one or more amino acid substitutions at one or more binding site residues for de novo RNA synthesis, without being bound by theory, which alters the RNA polymerase affinity for the cap analog of the in vitro transcription reaction, for example, resulting in improved capping efficiency at low cap analog concentrations.

[0082] Therefore, in some aspects, RNA polymerase variants include RNA polymerases comprising two or more amino acid substitutions at binding site residues for de novo RNA synthesis. RNA polymerase variants are enzymes having RNA polymerase activity and at least one substitution and / or modification relative to a corresponding wild-type RNA polymerase. In some embodiments, relative to a wild-type RNA polymerase, amino acid substitutions are selected from positions 350, 351, 387, 437, and 653, wherein the wild-type RNA polymerase comprises the amino acid sequence of SEQ ID NO:1.

[0083] Structural studies of T7 RNA polymerase show that the conformation of the N-terminal domain changes substantially between the initiation phase and the extension phase of transcription. The N-terminal domain contains a C-helical subdomain and a promoter binding domain, which includes two segments separated by a subdomain H. The promoter binding domain and the bound promoter rotate approximately 45 degrees during the synthesis of the 8-nt RNA transcript, thereby allowing the promoter to maintain contact when the active site expands to accommodate the growing heteroduplex. The C-helical subdomain moves moderately to its extended conformation, while subdomain H remains in its initiation phase position-rather than its extension phase position at a distance of more than 70 angstroms. Structural comparison of the initiation and extension complexes of T7 RNA polymerase reveals extensive conformational changes within the N-terminal 267 residues (N-terminal domain) and very few changes in the rest of the RNA polymerase. The rigid body rotation of the promoter binding domain and the refolding of the N-terminal C-helix (residues 28-71) and H (residues 151-190) subdomains are responsible for eliminating the promoter binding site, expanding the active site and generating an exit tunnel for the RNA transcript. In particular, the residues E42-G47 of T7 RNA polymerase (present as a β-loop structure in the initiation complex) adopt an α-helical structure in the extension complex. The structural changes in the N-terminal domain explain the increase in the stability and processivity of the extension complex (see, e.g., Durniak, KJ et al., Science 322 (5901): 553–557, 2008, which is incorporated herein by reference). T7 RNA polymerase also includes 'N helix' (residues 374-409), which functions to deflect the direction of the 5' end of the RNA transcript when the RNA transcript is separated from the template and affects the stability and processivity of the extension complex (e.g., by the interaction between residues 385-395 and the ribose backbone). The 'O helix' (residues 627-640) of RNA polymerase functions to stabilize incoming NTPs during insertion and prevent backtracking during synthesis of the RNA transcript. Finally, the 'Y helix' (residues 644-661) functions to stabilize the template base at the n+1 position of the growing RNA transcript.

[0084] In some aspects, it is an RNA polymerase variant (e.g., T7 RNA polymerase variant) that promotes conformational changes from RNA polymerase initiation complexes to RNA polymerase extension complexes. In some embodiments, relative to wild-type RNA polymerase, RNA polymerase variants include at least one, at least two, at least three, or at least four amino acid modifications, which cause at least one three-dimensional ring structure of RNA polymerase variants to undergo conformational changes to helical structures when RNA polymerase variants are transformed from initiation complexes to extension complexes. Therefore, in some embodiments, relative to wild-type amino acids, at least one amino acid modification has a high helical tendency.

[0085] In addition, in some aspects, it is an RNA polymerase variant (e.g., T7 RNA polymerase variant) that increases the stability and continuous synthesis ability of the extension complex relative to wild-type T7 RNA polymerase, prevents backtracking and stabilizes the incoming NTP and template. In some embodiments, relative to wild-type RNA polymerase, the RNA polymerase variant comprises at least one, at least two, at least three, or at least four amino acid modifications that increase the stability and continuous synthesis ability of the extension complex, prevents backtracking and stabilizes the incoming NTP and template. In some embodiments, relative to wild-type RNA polymerase, the RNA polymerase variant comprises at least one, at least two, at least three, or at least four amino acid modifications in 'N helix' (residues 374-409) (e.g., to increase the stability and continuous synthesis ability of the extension complex). In some embodiments, relative to wild-type RNA polymerase, the RNA polymerase variant comprises at least one, at least two, at least three, or at least four amino acid modifications in 'O helix' (residues 627-640) (e.g., to stabilize the incoming NTP during insertion and prevent backtracking). In some embodiments, the RNA polymerase variant comprises at least one, at least two, at least three, or at least four amino acid modifications in the 'Y helix' (residues 644-661) relative to wild-type RNA polymerase (eg, to stabilize growing RNA transcripts).

[0086] Therefore, some aspects provide RNA polymerase variants comprising multiple amino acid substitutions and / or modifications relative to wild-type RNA polymerase. In some embodiments, the RNA polymerase variant comprises an amino acid sequence comprising (a) an amino acid substitution at a binding site residue for de novo RNA synthesis, and (b) an amino acid substitution that promotes conformational changes from an RNA polymerase initiation complex to an RNA polymerase extension complex.

[0087] In some embodiments, the use of RNA polymerase variants in an in vitro transcription reaction increases transcription efficiency relative to a control RNA polymerase. For example, the use of RNA polymerase variants can increase transcription efficiency (e.g., RNA yield and / or transcription rate) by at least 20%. In some embodiments, the use of RNA polymerase variants increases transcription efficiency (e.g., RNA yield and / or transcription rate) by at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or at least 10%. In some embodiments, the use of RNA polymerase variants increases transcription efficiency by 20-100%, 20-90%, 20-80%, 20-70%, 20-60%, 20-50%, 30-100%, 30-90%, 30-80%, 30-70%, 30-60%, 30-50%, 40-100%, 40-90%, 40-80%, 40-70%, 40-60%, 40-50%, 50-100%, 50-90%, 50-80%, 50-70% or 50-60%. In some embodiments, the use of RNA polymerase variants increases total RNA productivity by at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or at least 10%. In some embodiments, the use of RNA polymerase variants increases total RNA yield by 20-100%, 20-90%, 20-80%, 20-70%, 20-60%, 20-50%, 30-100%, 30-90%, 30-80%, 30-70%, 30-60%, 30-50%, 40-100%, 40-90%, 40-80%, 40-70%, 40-60%, 40-50%, 50-100%, 50-90%, 50-80%, 50-70% or 50-60%. In some embodiments, the use of RNA polymerase variants increases the transcription rate by at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or at least 10%. In some embodiments, the use of RNA polymerase variants increases the transcription rate by 20-100%, 20-90%, 20-80%, 20-70%, 20-60%, 20-50%, 30-100%, 30-90%, 30-80%, 30-70%, 30-60%, 30-50%, 40-100%, 40-90%, 40-80%, 40-70%, 40-60%, 40-50%, 50-100%, 50-90%, 50-80%, 50-70% or 50-60%. In some embodiments, the control RNA polymerase is a wild-type RNA polymerase comprising the amino acid sequence of SEQ ID NO: 1 ("wild-type T7 RNA polymerase").

[0088] Surprisingly, the RNA polymerase variants enable the use of much lower concentrations (amounts) of cap analogs in in vitro transcription reactions, producing equivalent amounts of capped RNA as those produced using wild-type T7 RNA polymerase. See e.g. Figures 1A-2C And Examples 1-2. In some embodiments, when half the concentration of the cap analog is used in the in vitro transcription reaction, the use of the RNA polymerase variant in the in vitro transcription reaction increases the yield of the capped RNA. In some embodiments, when only 25%, 50% or 75% of the cap analog is used in the in vitro transcription reaction, the use of the RNA polymerase variant in the in vitro transcription reaction increases the yield of the capped RNA. For example, when only 25%, 50% or 75% of the cap analog is used in the in vitro transcription reaction, the use of the RNA polymerase variant can increase the yield of the capped RNA by at least 20%. In some embodiments, when only 25%, 50% or 75% of the cap analog is used in the in vitro transcription reaction, the use of the RNA polymerase variant increases the yield of the capped RNA by at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or at least 100%. In some embodiments, the use of the RNA polymerase variant increases the yield of capped RNA by 20-100%, 20-90%, 20-80%, 20-70%, 20-60%, 20-50%, 30-100%, 30-90%, 30-80%, 30-70%, 30-60%, 30-50%, 40-100%, 40-90%, 40-80%, 40-70%, 40-60%, 40-50%, 50-100%, 50-90%, 50-80%, 50-70%, or 50-60% when only 25%, 50% or 75% concentration of the cap analog is used in the in vitro transcription reaction. In some embodiments, the control RNA polymerase is wild-type T7 RNA polymerase.

[0089] In some embodiments, the use of RNA polymerase variants increases the total yield of capped RNA by at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 10%. In some embodiments, the use of RNA polymerase variants increases the total yield of capped RNA by 20-100%, 20-90%, 20-80%, 20-70%, 20-60%, 20-50%, 30-100%, 30-90%, 30-80%, 30-70%, 30-60%, 30-50%, 40-100%, 40-90%, 40-80%, 40-70%, 40-60%, 40-50%, 50-100%, 50-90%, 50-80%, 50-70%, or 50-60%.

[0090] In some embodiments, the use of RNA polymerase variants in in vitro transcription reactions increases the co-transcriptional capping efficiency. For example, the use of RNA polymerase variants can increase the co-transcriptional capping efficiency (e.g., the percentage of transcripts containing cap analogs) by at least 20%. In some embodiments, the use of RNA polymerase variants increases the co-transcriptional capping efficiency (e.g., the percentage of transcripts containing cap analogs) by at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 100%. In some embodiments, the use of RNA polymerase variants increases the co-transcriptional capping efficiency by 20-100%, 20-90%, 20-80%, 20-70%, 20-60%, 20-50%, 30-100%, 30-90%, 30-80%, 30-70%, 30-60%, 30-50%, 40-100%, 40-90%, 40-80%, 40-70%, 40-60%, 40-50%, 50-100%, 50-90%, 50-80%, 50-70%, or 50-60%. In some embodiments, the control RNA polymerase is wild-type T7 RNA polymerase.

[0091] In some embodiments, at least 50% of the mRNA comprises a functional cap analog. For example, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 95%, or 100% of the mRNA may comprise a cap analog. In some embodiments, 50%-100%, 50%-90%, 50%-80%, or 50%-70% of the mRNA comprises a cap analog.

[0092] In some embodiments, the use of RNA polymerase variants in an in vitro transcription reaction improves the 3' homogeneity of RNA at half the concentration of cap analogs used in the in vitro transcription reaction. For example, the use of RNA polymerase variants can improve the 3' homogeneity of RNA by at least 20% when only 25%, 50% or 75% concentration of cap analogs are used in the in vitro transcription reaction. In some embodiments, the use of RNA polymerase variants improves the 3' homogeneity by at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or at least 100% when only 25%, 50% or 75% concentration of cap analogs are used in the in vitro transcription reaction. In some embodiments, the use of the RNA polymerase variant increases 3' homogeneity by 20-100%, 20-90%, 20-80%, 20-70%, 20-60%, 20-50%, 30-100%, 30-90%, 30-80%, 30-70%, 30-60%, 30-50%, 40-100%, 40-90%, 40-80%, 40-70%, 40-60%, 40-50%, 50-100%, 50-90%, 50-80%, 50-70%, or 50-60% when only 25%, 50% or 75% concentration of the cap analog is used in the in vitro transcription reaction. In some embodiments, the control RNA polymerase is wild-type T7 RNA polymerase.

[0093] In some embodiments, at least 50% of the mRNAs produced in the in vitro transcription reaction that contain the RNA polymerase variant exhibit 3' homogeneity. For example, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 95%, or 100% of the mRNAs exhibit 3' homogeneity. In some embodiments, 50%-100%, 50%-90%, 50%-80%, or 50%-70% of the mRNAs exhibit 3' homogeneity.

[0094] In some embodiments, the mRNA comprising the RNA polymerase variant produced in the in vitro transcription reaction has a 3' homogeneity greater than a threshold value. In some embodiments, the threshold value is 50% or at least 50%. For example, the threshold value can be 55%, 60%, 65%, 70%, 75%, 80%, 85% or 90%.

[0095] In some embodiments, the use of RNA polymerase variants in in vitro transcription reactions improves transcription fidelity (e.g., mutation rate). For example, the use of RNA polymerase variants can improve transcription fidelity by at least 20%. In some embodiments, the use of RNA polymerase variants improves transcription fidelity by at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 100%. In some embodiments, the use of RNA polymerase variants improves transcription fidelity by 20-100%, 20-90%, 20-80%, 20-70%, 20-60%, 20-50%, 30-100%, 30-90%, 30-80%, 30-70%, 30-60%, 30-50%, 40-100%, 40-90%, 40-80%, 40-70%, 40-60%, 40-50%, 50-100%, 50-90%, 50-80%, 50-70% or 50-60%. RNA polymerase variants that improve transcription fidelity will produce RNA transcripts (e.g., mRNA transcripts) with a lower mutation rate or total mutation count than a control RNA polymerase. In some embodiments, the control RNA polymerase is a wild-type T7 RNA polymerase.

[0096] In some embodiments, the mRNA produced using the RNA polymerase variant has less than 1 mutation per 100 nucleotides relative to the DNA template. For example, the mRNA produced can have less than 1 mutation per 200, 300, 400, 500, 600, 700, 800, 900 or 1000 nucleotides relative to the DNA template.

[0097] In some embodiments, the use of RNA polymerase variants in the in vitro transcription reaction reduces the amount of double-stranded RNA (dsRNA) contamination in the in vitro transcription reaction. For example, the use of RNA polymerase variants can reduce the amount of dsRNA contamination in the in vitro transcription reaction by at least 20%. In some embodiments, the use of RNA polymerase variants reduces the amount of dsRNA contamination in the in vitro transcription reaction by at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or at least 100%. In some embodiments, the use of RNA polymerase variants reduces the amount of dsRNA contamination in in vitro transcription reactions by 20-100%, 20-90%, 20-80%, 20-70%, 20-60%, 20-50%, 30-100%, 30-90%, 30-80%, 30-70%, 30-60%, 30-50%, 40-100%, 40-90%, 40-80%, 40-70%, 40-60%, 40-50%, 50-100%, 50-90%, 50-80%, 50-70% or 50-60%. In some embodiments, the control RNA polymerase is wild-type T7 RNA polymerase.

[0098] In some embodiments, the concentration of dsRNA contamination is less than 10 ng / 25 μg of mRNA product. In some embodiments, the concentration of dsRNA contamination is less than 5 ng / 25 μg of mRNA product. For example, the concentration of dsRNA contamination can be less than 4 ng / 25 μg of mRNA product, less than 3 ng / 25 μg of mRNA product, less than 2 ng / 25 μg of mRNA product, or less than 1 ng / 25 μg of mRNA product. In some embodiments, the concentration of dsRNA contamination is 0.5-1, 0.5-2, 0.5-3, 0-.4 or 0.5-5 ng / 25 μg of mRNA product.

[0099] In some embodiments, the mRNA comprising the RNA polymerase variant produced in the in vitro transcription reaction has a dsRNA below a threshold amount. In some embodiments, the threshold is 10 ng. In some embodiments, the threshold is 5 ng. In some embodiments, the threshold is 4 ng, 3 ng, 2 ng or 1 ng.

[0100] Amino acid substitutions and modifications

[0101] Relative to wild-type (WT) RNA polymerase, RNA polymerase variants include at least one amino acid substitution, preferably at least two amino acid substitutions. For example, with respect to the WT T7 RNA polymerase having the amino acid sequence of SEQ ID NO: 1, glutamic acid (E) at position 350 is considered to be a "wild-type amino acid", while the substitution of glutamic acid for tryptophan at position 350 is considered to be an "amino acid substitution". In some embodiments, relative to a WT RNA polymerase (e.g., a WT T7 RNA polymerase having the amino acid sequence of SEQ ID NO: 1), an RNA polymerase variant is a T7 RNA polymerase variant comprising at least one (one or more) amino acid substitutions.

[0102] In some embodiments, RNA T7 polymerase variants include at least two amino acid substitutions. In some embodiments, RNA T7 polymerase variants include at least three amino acid substitutions. In some embodiments, RNA T7 polymerase variants include at least four amino acid substitutions. In some embodiments, RNA T7 polymerase variants include at least five amino acid substitutions.

[0103] In some embodiments, RNA polymerase variants include amino acid sequences, the amino acid sequence includes (at least one) amino acid modification, which causes the RNA polymerase variant's ring structure to undergo conformational changes to helical structures when the RNA polymerase variant changes from the initiation complex to the extension complex. In some embodiments, the amino acid substitution is a high-propensity amino acid substitution. Examples of high-propensity helical amino acids include alanine, isoleucine, leucine, arginine, methionine, lysine, glutamine and / or glutamic acid.

[0104] In some embodiments, RNA polymerase variants include amino acid sequences, the amino acid sequences include (at least one) amino acid substitution to introduce polar neutral amino acids. In some embodiments, polar neutral amino acids are selected from asparagine (N), cysteine ​​(C), glutamine (Q), methionine (M), serine (S) and threonine (T). In some embodiments, RNA polymerase variants include amino acid sequences, the amino acid sequences include (at least one) amino acid substitution to introduce aromatic amino acids. In some embodiments, aromatic amino acids are selected from tryptophan (W), tyrosine (Y) and phenylalanine (F). In some embodiments, RNA polymerase variants include amino acid sequences, the amino acid sequences include (at least one) amino acid substitution to introduce non-polar aliphatic amino acids. In some embodiments, non-polar aliphatic amino acids are selected from alanine (A), glycine (G), isoleucine (I), leucine (L), proline (P) and valine (V). In some embodiments, RNA polymerase variants include amino acid sequences, the amino acid sequences include (at least one) amino acid substitution to introduce positively charged amino acids. In some embodiments, the positively charged amino acid is selected from lysine (K), arginine (R) and histidine (H). In some embodiments, the RNA polymerase variant comprises an amino acid sequence comprising (at least one) amino acid substitution to introduce a negatively charged amino acid. In some embodiments, the negatively charged amino acid is selected from aspartic acid (D) and glutamic acid (E).

[0105] In some embodiments, RNA polymerase variants include amino acid sequences, and the amino acid sequence is included in (at least one) amino acid modification at the position of non-conservative amino acid residues.Conservative amino acid residues are amino acids or amino acid types (e.g., single amino acids such as Gly or Ser, or amino acid groups sharing similar properties such as amino acids with acidic functional groups) that are usually shared between multiple homologous sequences of the same protein. Conservative amino acid residues can be identified using sequence alignments of homologous amino acid sequences.Sequence alignments of approximately 1000 RNA polymerase sequences obtained using basic local alignments (Basic Local Alignment) searches allow determination of 240 positions of SEQ ID NO:1 that are most likely to be conserved in RNA polymerase sequences. The 240 positions of SEQ ID NO: 1 that are most likely conserved among RNA polymerase sequences are at positions 5-6, 39, 269-277, 279, 281-282, 323-333, 411-448, 454-470, 472-474, 497-516, 532-560, 562-573, 626-646, 691, 693-702, 724-738, 775-794, 805-820, 828-833, 865-867, and 877-879. Thus, in some embodiments, the RNA polymerase variants include RNA polymerases comprising (at least one) amino acid modification at a position other than one of positions 5-6, 39, 269-277, 279, 281-282, 323-333, 411-448, 454-470, 472-474, 497-516, 532-560, 562-573, 626-646, 691, 693-702, 724-738, 775-794, 805-820, 828-833, 865-867, and 877-879 of SEQ ID NO: 1. In some embodiments, the RNA polymerase variant can further comprise any number of amino acid modifications at any number of positions other than one of positions 5-6, 39, 269-277, 279, 281-282, 323-333, 411-448, 454-470, 472-474, 497-516, 532-560, 562-573, 626-646, 691, 693-702, 724-738, 775-794, 805-820, 828-833, 865-867, and 877-879 of SEQ ID NO: 1.In some embodiments, the RNA polymerase variant comprising the amino acid sequence of any one of SEQ ID NOs: 2-9 may further comprise (at least one) additional amino acid modification at a position other than one of positions 5-6, 39, 269-277, 279, 281-282, 323-333, 411-448, 454-470, 472-474, 497-516, 532-560, 562-573, 626-646, 691, 693-702, 724-738, 775-794, 805-820, 828-833, 865-867, and 877-879. Conversely, non-conserved amino acid positions are most likely to undergo modification or mutation. Thus, in some embodiments, RNA polymerase variants include RNA polymerases comprising (at least one) amino acid modification at positions 1-4, 7-38, 40-268, 278, 280, 283-322, 334-410, 449-453, 471, 475-496, 517-531, 561, 574-625, 647-690, 692, 703-723, 739-774, 795-804, 821-827, 834-864, 868-876, and 880-883. In some embodiments, the RNA polymerase variant comprising the amino acid sequence of any one of SEQ ID NOs: 2-9 may further comprise (at least one) additional amino acid modification at positions 1-4, 7-38, 40-268, 278, 280, 283-322, 334-410, 449-453, 471, 475-496, 517-531, 561, 574-625, 647-690, 692, 703-723, 739-774, 795-804, 821-827, 834-864, 868-876, and 880-883.

[0106] In some embodiments, the RNA polymerase variants comprising the amino acid sequence of any one of SEQ ID NO:2-9 may further include (at least one) amino acid modification at any amino acid position that does not destroy the secondary or tertiary structure of the RNA polymerase protein. In some embodiments, the RNA polymerase variants comprising the amino acid sequence of any one of SEQ ID NO:2-9 may further include (at least one) amino acid modification at any amino acid position that does not destroy the ability of the RNA polymerase protein to fold. In some embodiments, the RNA polymerase variants comprising the amino acid sequence of any one of SEQ ID NO:2-9 may further include (at least one) amino acid modification at any amino acid position that does not destroy the ability of the RNA polymerase protein to bind nucleic acid (e.g., DNA).

[0107] In some embodiments, the RNA polymerase variant comprises an amino acid sequence comprising an amino acid substitution at position 437 (e.g., N437Y), an amino acid substitution at position 387 (e.g., K387S), an amino acid substitution at position 350 (e.g., E350W), and an amino acid substitution at position 351 (e.g., D351V) relative to a wild-type RNA polymerase, wherein the wild-type RNA polymerase comprises the amino acid sequence of SEQ ID NO: 1. In some embodiments, the RNA polymerase variant comprises N437Y, K387S, E350W, and D351V substitutions relative to a wild-type RNA polymerase, wherein the wild-type RNA polymerase comprises the amino acid sequence of SEQ ID NO: 1.

[0108] In some embodiments, the RNA polymerase variant comprises an amino acid sequence comprising an amino acid substitution at position 437 (e.g., N437Y), an amino acid substitution at position 350 (e.g., E350W), and an amino acid substitution at position 351 (e.g., D351V) relative to a wild-type RNA polymerase, wherein the wild-type RNA polymerase comprises the amino acid sequence of SEQ ID NO: 1. In some embodiments, the RNA polymerase variant comprises N437Y, E350W, and D351V substitutions relative to a wild-type RNA polymerase, wherein the wild-type RNA polymerase comprises the amino acid sequence of SEQ ID NO: 1.

[0109] In some embodiments, the RNA polymerase variant comprises an amino acid sequence relative to a wild-type RNA polymerase, the amino acid sequence comprising an amino acid substitution at position 387 (e.g., K387S), an amino acid substitution at position 350 (e.g., E350W), and an amino acid substitution at position 351 (e.g., D351V), wherein the wild-type RNA polymerase comprises the amino acid sequence of SEQ ID NO: 1. In some embodiments, the RNA polymerase variant comprises K387S, E350W, and D351V substitutions relative to a wild-type RNA polymerase, wherein the wild-type RNA polymerase comprises the amino acid sequence of SEQ ID NO: 1.

[0110] In some embodiments, the RNA polymerase variant comprises an amino acid sequence comprising an amino acid substitution at position 653 (e.g., D653W), an amino acid substitution at position 350 (e.g., E350W), and an amino acid substitution at position 351 (e.g., D351V) relative to a wild-type RNA polymerase, wherein the wild-type RNA polymerase comprises the amino acid sequence of SEQ ID NO: 1. In some embodiments, the RNA polymerase variant comprises D653W, E350W, and D351V substitutions relative to a wild-type RNA polymerase, wherein the wild-type RNA polymerase comprises the amino acid sequence of SEQ ID NO: 1.

[0111] In some embodiments, the amino acid substitution at position K387 is a polar neutral amino acid. In some embodiments, the polar neutral amino acid is selected from asparagine (N), cysteine ​​(C), glutamine (Q), methionine (M), serine (S) and threonine (T). Therefore, in some embodiments, the amino acid substitution at position K387 is K387N, K387C, K387Q, K387M, K387S or K387T.

[0112] In some embodiments, the amino acid substitution at position N437 is an aromatic amino acid. In some embodiments, the aromatic amino acid is selected from tryptophan (W), tyrosine (Y) and phenylalanine (F). Therefore, in some embodiments, the amino acid substitution at position N437 is N437W, N437Y or N437F.

[0113] In some embodiments, the amino acid substitution at position D653 is an aromatic amino acid. In some embodiments, the aromatic amino acid is selected from tryptophan (W), tyrosine (Y) and phenylalanine (F). Therefore, in some embodiments, the amino acid substitution at position D653 is D653W, D653Y or D653F.

[0114] In some embodiments, the amino acid substitution at position E350 is an aromatic amino acid. In some embodiments, the aromatic amino acid is selected from tryptophan (W), tyrosine (Y) and phenylalanine (F). Therefore, in some embodiments, the amino acid substitution at position E350 is E350W, E350Y or E350F.

[0115] In some embodiments, the amino acid substitution at position D351 is a non-polar aliphatic amino acid. In some embodiments, the non-polar aliphatic amino acid is selected from alanine (A), glycine (G), isoleucine (I), leucine (L), proline (P) and valine (V). Therefore, in some embodiments, the amino acid substitution at position D351 is D351A, D351G, D351I, D351L, D351P or D351V.

[0116] In some embodiments, relative to the wild-type RNA polymerase, the RNA polymerase variant comprises an amino acid sequence comprising an amino acid substitution at position R379 (e.g., R379A), wherein the wild-type RNA polymerase comprises the amino acid sequence of SEQ ID NO: 1. In some embodiments, the amino acid substitution at position R379 is a non-polar aliphatic amino acid; a polar neutral amino acid; an aromatic amino acid or a charged amino acid. In some embodiments, the non-polar aliphatic amino acid is selected from alanine (A), glycine (G), isoleucine (I), leucine (L), proline (P) and valine (V). In some embodiments, the aromatic amino acid is selected from tryptophan (W), tyrosine (Y) and phenylalanine (F). In some embodiments, the charged amino acid is a positively charged amino acid (e.g., lysine (K) or histidine (H)) or a negatively charged amino acid (e.g., glutamic acid (E) or aspartic acid (D)). In some embodiments, the amino acid substitution at position R379 is R379A, R379K, R379E, or R379W.

[0117] In some embodiments, relative to the wild-type RNA polymerase, the RNA polymerase variant comprises an amino acid sequence comprising an amino acid substitution at position Y385 (e.g., Y385A), wherein the wild-type RNA polymerase comprises the amino acid sequence of SEQ ID NO: 1. In some embodiments, the amino acid substitution at position Y385 is a non-polar aliphatic amino acid; a polar neutral amino acid; an aromatic amino acid or a charged amino acid. In some embodiments, the non-polar aliphatic amino acid is selected from alanine (A), glycine (G), isoleucine (I), leucine (L), proline (P) and valine (V). In some embodiments, the aromatic amino acid is selected from tryptophan (W) and phenylalanine (F). In some embodiments, the charged amino acid is a positively charged amino acid (e.g., lysine (K), histidine (H) or arginine (R)) or a negatively charged amino acid (e.g., glutamic acid (E) or aspartic acid (D)). In some embodiments, the amino acid substitution at position Y385 is Y385A, Y385K, Y385W, or Y385V.

[0118] In some embodiments, relative to the wild-type RNA polymerase, the RNA polymerase variant comprises an amino acid sequence comprising an amino acid substitution at position R386 (e.g., R386A), wherein the wild-type RNA polymerase comprises the amino acid sequence of SEQ ID NO: 1. In some embodiments, the amino acid substitution at position R386 is a non-polar aliphatic amino acid; a polar neutral amino acid; an aromatic amino acid or a charged amino acid. In some embodiments, the non-polar aliphatic amino acid is selected from alanine (A), glycine (G), isoleucine (I), leucine (L), proline (P) and valine (V). In some embodiments, the charged amino acid is a positively charged amino acid (e.g., lysine (K) or histidine (H)) or a negatively charged amino acid (e.g., glutamic acid (E) or aspartic acid (D)). In some embodiments, the aromatic amino acid is selected from tryptophan (W), tyrosine (Y) and phenylalanine (F). In some embodiments, the amino acid substitution at position R386 is R386A, R386K or R386Y.

[0119] In some embodiments, relative to wild-type RNA polymerase, RNA polymerase variants include an amino acid sequence comprising an amino acid substitution (e.g., D388A) at position D388, wherein the wild-type RNA polymerase comprises the amino acid sequence of SEQ ID NO: 1. In some embodiments, the amino acid substitution at position D388 is a non-polar aliphatic amino acid; a polar neutral amino acid; an aromatic amino acid or a charged amino acid. In some embodiments, the non-polar aliphatic amino acid is selected from alanine (A), glycine (G), isoleucine (I), leucine (L), proline (P) and valine (V). In some embodiments, the polar neutral amino acid is selected from asparagine (N), cysteine ​​(C), glutamine (Q), methionine (M), serine (S) and threonine (T). In some embodiments, aromatic amino acids are selected from tryptophan (W), tyrosine (Y) and phenylalanine (F). In some embodiments, the amino acid substitution at position D388 is D388A, D388N or D388Y.

[0120] In some embodiments, relative to the wild-type RNA polymerase, the RNA polymerase variant comprises an amino acid sequence comprising an amino acid substitution at position K389 (e.g., K389A), wherein the wild-type RNA polymerase comprises the amino acid sequence of SEQ ID NO: 1. In some embodiments, the amino acid substitution at position K389 is a non-polar aliphatic amino acid; a polar neutral amino acid; an aromatic amino acid or a charged amino acid. In some embodiments, the non-polar aliphatic amino acid is selected from alanine (A), glycine (G), isoleucine (I), leucine (L), proline (P) and valine (V). In some embodiments, the polar neutral amino acid is selected from asparagine (N), cysteine ​​(C), glutamine (Q), methionine (M), serine (S) and threonine (T). In some embodiments, the aromatic amino acid is selected from tryptophan (W), tyrosine (Y) and phenylalanine (F). In some embodiments, the amino acid substitution at position K389 is K389A, K389S or K389Y.

[0121] In some embodiments, the RNA polymerase variant comprises an amino acid sequence relative to a wild-type RNA polymerase, the amino acid sequence comprising an amino acid substitution at position R391 (e.g., R391A), wherein the wild-type RNA polymerase comprises the amino acid sequence of SEQ ID NO: 1. In some embodiments, the amino acid substitution at position R391 is a non-polar aliphatic amino acid; a polar neutral amino acid; an aromatic amino acid or a charged amino acid. In some embodiments, the non-polar aliphatic amino acid is selected from alanine (A), glycine (G), isoleucine (I), leucine (L), proline (P) and valine (V). In some embodiments, the amino acid substitution at position R391 is R391A.

[0122] In some embodiments, relative to the wild-type RNA polymerase, the RNA polymerase variant comprises an amino acid sequence comprising an amino acid substitution at position R394 (e.g., R394A), wherein the wild-type RNA polymerase comprises the amino acid sequence of SEQ ID NO: 1. In some embodiments, the amino acid substitution at position R394 is a non-polar aliphatic amino acid; a polar neutral amino acid; an aromatic amino acid or a charged amino acid. In some embodiments, the non-polar aliphatic amino acid is selected from alanine (A), glycine (G), isoleucine (I), leucine (L), proline (P) and valine (V). In some embodiments, the polar neutral amino acid is selected from asparagine (N), cysteine ​​(C), glutamine (Q), methionine (M), serine (S) and threonine (T). In some embodiments, the aromatic amino acid is selected from tryptophan (W), tyrosine (Y) and phenylalanine (F). In some embodiments, the amino acid substitution at position R394 is R394A, R394Q or R394Y.

[0123] In some embodiments, the RNA polymerase variant comprises an amino acid sequence relative to a wild-type RNA polymerase, the amino acid sequence comprising an amino acid substitution at position R395 (e.g., R395A), wherein the wild-type RNA polymerase comprises the amino acid sequence of SEQ ID NO: 1. In some embodiments, the amino acid substitution at position R395 is a non-polar aliphatic amino acid; a polar neutral amino acid; an aromatic amino acid or a charged amino acid. In some embodiments, the non-polar aliphatic amino acid is selected from alanine (A), glycine (G), isoleucine (I), leucine (L), proline (P) and valine (V). In some embodiments, the amino acid substitution at position R395 is R395A.

[0124] In some embodiments, relative to the wild-type RNA polymerase, the RNA polymerase variant comprises an amino acid sequence comprising an amino acid substitution at position D471 (e.g., D471A), wherein the wild-type RNA polymerase comprises the amino acid sequence of SEQ ID NO: 1. In some embodiments, the amino acid substitution at position D471 is a non-polar aliphatic amino acid; a polar neutral amino acid; an aromatic amino acid or a charged amino acid. In some embodiments, the non-polar aliphatic amino acid is selected from alanine (A), glycine (G), isoleucine (I), leucine (L), proline (P) and valine (V). In some embodiments, the charged amino acid is a positively charged amino acid (e.g., lysine (K), histidine (H) or arginine (R)) or a negatively charged amino acid (e.g., glutamic acid). In some embodiments, the aromatic amino acid is selected from tryptophan (W), tyrosine (Y) and phenylalanine (F). In some embodiments, the amino acid substitution at position D471 is D471A, D471E or D471Y.

[0125] In some embodiments, the RNA polymerase variant comprises an amino acid sequence relative to a wild-type RNA polymerase, the amino acid sequence comprising an amino acid substitution at position R627 (e.g., R627A), wherein the wild-type RNA polymerase comprises the amino acid sequence of SEQ ID NO: 1. In some embodiments, the amino acid substitution at position R627 is a non-polar aliphatic amino acid; a polar neutral amino acid; an aromatic amino acid or a charged amino acid. In some embodiments, the non-polar aliphatic amino acid is selected from alanine (A), glycine (G), isoleucine (I), leucine (L), proline (P) and valine (V). In some embodiments, the amino acid substitution at position R627 is R627A.

[0126] In some embodiments, the RNA polymerase variant comprises an amino acid sequence relative to a wild-type RNA polymerase, the amino acid sequence comprising an amino acid substitution at position R631 (e.g., R631A), wherein the wild-type RNA polymerase comprises the amino acid sequence of SEQ ID NO: 1. In some embodiments, the amino acid substitution at position R631 is a non-polar aliphatic amino acid; a polar neutral amino acid; an aromatic amino acid or a charged amino acid. In some embodiments, the non-polar aliphatic amino acid is selected from alanine (A), glycine (G), isoleucine (I), leucine (L), proline (P) and valine (V). In some embodiments, the amino acid substitution at position R631 is R631A.

[0127] In some embodiments, relative to the wild-type RNA polymerase, the RNA polymerase variant comprises an amino acid sequence comprising an amino acid substitution at position R632 (e.g., R632A), wherein the wild-type RNA polymerase comprises the amino acid sequence of SEQ ID NO: 1. In some embodiments, the amino acid substitution at position R632 is a non-polar aliphatic amino acid; a polar neutral amino acid; an aromatic amino acid or a charged amino acid. In some embodiments, the non-polar aliphatic amino acid is selected from alanine (A), glycine (G), isoleucine (I), leucine (L), proline (P) and valine (V). In some embodiments, the aromatic amino acid is selected from tryptophan (W), tyrosine (Y) and phenylalanine (F). In some embodiments, the polar neutral amino acid is selected from asparagine (N), cysteine ​​(C), glutamine (Q), methionine (M), serine (S) and threonine (T). In some embodiments, the amino acid substitution at position R632 is R632D, R632A, R632Q or R632Y.

[0128] In some embodiments, the RNA polymerase variant comprises an amino acid sequence relative to a wild-type RNA polymerase, the amino acid sequence comprising an amino acid substitution at position G640 (e.g., G640A), wherein the wild-type RNA polymerase comprises the amino acid sequence of SEQ ID NO: 1. In some embodiments, the amino acid substitution at position G640 is a non-polar aliphatic amino acid; a polar neutral amino acid; an aromatic amino acid or a charged amino acid. In some embodiments, the non-polar aliphatic amino acid is selected from alanine (A), glycine (G), isoleucine (I), leucine (L), proline (P) and valine (V). In some embodiments, the amino acid substitution at position G640 is G640A.

[0129] In some embodiments, the RNA polymerase variant comprises an amino acid sequence relative to a wild-type RNA polymerase, the amino acid sequence comprising an amino acid substitution at position G645 (e.g., G645A), wherein the wild-type RNA polymerase comprises the amino acid sequence of SEQ ID NO: 1. In some embodiments, the amino acid substitution at position G645 is a non-polar aliphatic amino acid; a polar neutral amino acid; an aromatic amino acid or a charged amino acid. In some embodiments, the non-polar aliphatic amino acid is selected from alanine (A), glycine (G), isoleucine (I), leucine (L), proline (P) and valine (V). In some embodiments, the amino acid substitution at position G645 is G645A.

[0130] In some embodiments, the RNA polymerase variant comprises an amino acid sequence relative to a wild-type RNA polymerase, the amino acid sequence comprising an amino acid substitution at position Q648 (e.g., Q648A), wherein the wild-type RNA polymerase comprises the amino acid sequence of SEQ ID NO: 1. In some embodiments, the amino acid substitution at position Q648 is a non-polar aliphatic amino acid; a polar neutral amino acid; an aromatic amino acid or a charged amino acid. In some embodiments, the non-polar aliphatic amino acid is selected from alanine (A), glycine (G), isoleucine (I), leucine (L), proline (P) and valine (V). In some embodiments, the aromatic amino acid is selected from tryptophan (W), tyrosine (Y) and phenylalanine (F). In some embodiments, the charged amino acid is a positively charged amino acid (e.g., lysine (K), histidine (H) or arginine (R)) or a negatively charged amino acid (e.g., glutamic acid (E) or aspartic acid (D)). In some embodiments, the amino acid substitution at position Q648 is Q648A, Q648R, Q648D, Q648E, or Q648Y.

[0131] In some embodiments, the RNA polymerase variant comprises an amino acid sequence relative to a wild-type RNA polymerase, the amino acid sequence comprising an amino acid substitution at position Q649 (e.g., Q649A), wherein the wild-type RNA polymerase comprises the amino acid sequence of SEQ ID NO: 1. In some embodiments, the amino acid substitution at position Q649 is a non-polar aliphatic amino acid; a polar neutral amino acid; an aromatic amino acid or a charged amino acid. In some embodiments, the non-polar aliphatic amino acid is selected from alanine (A), glycine (G), isoleucine (I), leucine (L), proline (P) and valine (V). In some embodiments, the amino acid substitution at position Q649 is Q649A.

[0132] In some embodiments, the RNA polymerase variant comprises an amino acid sequence relative to a wild-type RNA polymerase, the amino acid sequence comprising an amino acid substitution at position E652 (e.g., E652A), wherein the wild-type RNA polymerase comprises the amino acid sequence of SEQ ID NO: 1. In some embodiments, the amino acid substitution at position E652 is a non-polar aliphatic amino acid; a polar neutral amino acid; an aromatic amino acid or a charged amino acid. In some embodiments, the non-polar aliphatic amino acid is selected from alanine (A), glycine (G), isoleucine (I), leucine (L), proline (P) and valine (V). In some embodiments, the aromatic amino acid is selected from tryptophan (W), tyrosine (Y) and phenylalanine (F). In some embodiments, the charged amino acid is a positively charged amino acid (e.g., lysine (K), histidine (H) or arginine (R)). In some embodiments, the amino acid substitution at position E652 is E652R, E652A or E652Y.

[0133] In some embodiments, relative to the wild-type RNA polymerase, the RNA polymerase variant comprises an amino acid sequence comprising an amino acid substitution at position D653 (e.g., D653A), wherein the wild-type RNA polymerase comprises the amino acid sequence of SEQ ID NO: 1. In some embodiments, the amino acid substitution at position D653 is a non-polar aliphatic amino acid; a polar neutral amino acid; an aromatic amino acid or a charged amino acid. In some embodiments, the non-polar aliphatic amino acid is selected from alanine (A), glycine (G), isoleucine (I), leucine (L), proline (P) and valine (V). In some embodiments, the aromatic amino acid is selected from tryptophan (W), tyrosine (Y) and phenylalanine (F). In some embodiments, the charged amino acid is a positively charged amino acid (e.g., lysine (K), histidine (H) or arginine (R)). In some embodiments, the amino acid substitution at position D653 is D653K, D653A or D653Y.

[0134] In some embodiments, the RNA polymerase variant comprises an amino acid sequence relative to a wild-type RNA polymerase, the amino acid sequence comprising an amino acid substitution at position Q656 (e.g., Q656A), wherein the wild-type RNA polymerase comprises the amino acid sequence of SEQ ID NO: 1. In some embodiments, the amino acid substitution at position Q656 is a non-polar aliphatic amino acid; a polar neutral amino acid; an aromatic amino acid or a charged amino acid. In some embodiments, the non-polar aliphatic amino acid is selected from alanine (A), glycine (G), isoleucine (I), leucine (L), proline (P) and valine (V). In some embodiments, the aromatic amino acid is selected from tryptophan (W), tyrosine (Y) and phenylalanine (F). In some embodiments, the charged amino acid is a positively charged amino acid (e.g., lysine (K), histidine (H) or arginine (R)) or a negatively charged amino acid (e.g., glutamic acid (E) or aspartic acid (D)). In some embodiments, the amino acid substitution at position Q656 is Q656K, Q656A, Q656E, or Q656Y.

[0135] In some embodiments, the RNA polymerase variant comprises an amino acid sequence relative to a wild-type RNA polymerase, the amino acid sequence comprising an amino acid substitution at position P657 (e.g., P657A), wherein the wild-type RNA polymerase comprises the amino acid sequence of SEQ ID NO: 1. In some embodiments, the amino acid substitution at position P657 is a non-polar aliphatic amino acid; a polar neutral amino acid; an aromatic amino acid or a charged amino acid. In some embodiments, the non-polar aliphatic amino acid is selected from alanine (A), glycine (G), isoleucine (I), leucine (L), proline (P) and valine (V). In some embodiments, the aromatic amino acid is selected from tryptophan (W), tyrosine (Y) and phenylalanine (F). In some embodiments, the charged amino acid is a positively charged amino acid (e.g., lysine (K), histidine (H) or arginine (R)) or a negatively charged amino acid (e.g., glutamic acid (E) or aspartic acid (D)). In some embodiments, the amino acid substitution at position P657 is P657G, P657A, P657E, or P657Y.

[0136] Table 1. RNA polymerase variants

[0137]

[0138]

[0139]

[0140]

[0141]

[0142] In some embodiments, the RNA polymerase variant further comprises one or more purification tags. For example, the RNA polymerase variant may comprise a histidine purification tag (e.g., an amino acid sequence of -HHHHHH- (SEQ ID NO: 14)) or any other amino acid sequence that can be used for purification. A histidine purification tag or a similar charged amino acid sequence can bind Ni 2+ Resin. In some embodiments, the histidine purification tag comprises the amino acid sequence of -HHHHHHV- (SEQ ID NO: 15). In some embodiments, the purification tag is an N-terminal purification tag covalently attached to the N-terminus of the RNA polymerase variant. In some embodiments, the purification tag is a C-terminal purification tag covalently attached to the C-terminus of the RNA polymerase variant. In some embodiments, the protein purification tag is a FLAG tag (e.g., -DYKDDDK- amino acid sequence (SEQ ID NO: 16)) or a hemagglutinin tag. In some embodiments, the RNA polymerase variant comprising an N-terminal His tag comprises any one of SEQ ID NO: 10-13.

[0143] Table 2. RNA polymerase variants containing an N-terminal His tag

[0144]

[0145]

[0146]

[0147] In some embodiments, the RNA polymerase variant has 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% identity with an RNA polymerase comprising the amino acid sequence of any one of SEQ ID NOs: 2-13. In some embodiments, the RNA polymerase variant may share at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95% identity with an RNA polymerase comprising the amino acid sequence of SEQ ID NO: 1.

[0148] The term "identity" refers to the relationship between the sequences of two or more polypeptides (e.g., enzymes) or polynucleotides (nucleic acids), as determined by comparing sequences. Identity also refers to the degree of sequence relatedness between sequences, as determined by the number of matches between strings of two or more amino acid residues or nucleic acid residues. Identity measures the percentage of identity matches between the smaller sequences in two or more sequences with gap comparisons (if any) processed by a specific mathematical model or computer program (e.g., "algorithm"). The identity of related proteins or nucleic acids can be easily calculated by known methods. When applied to polypeptide or polynucleotide sequences, "percent (%) identity" is defined as the percentage of residues (amino acid residues or nucleic acid residues) in a candidate amino acid or nucleic acid sequence that are identical to the residues in the amino acid sequence or nucleic acid sequence of the second sequence after aligning the sequences and introducing gaps (if necessary) to achieve maximum percentage identity. Methods and computer programs for comparison are well known in the art. It is understood that identity depends on the calculation of percentage identity, but its value may vary due to gaps and penalties introduced in the calculation. Typically, variants of a particular polynucleotide or polypeptide (e.g., an 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 a particular reference polynucleotide or polypeptide, as determined by sequence alignment programs and parameters as described herein and known to those skilled in the art. Such tools for alignment include those of the BLAST suite (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 popular local alignment technique is based on the Smith-Waterman algorithm (Smith, TF and Waterman, MS (1981) "Identification of common molecular subsequences." J. Mol. Biol. 147: 195-197).A general global alignment technique 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 Best Global Sequence Alignment Algorithm (FOGSAA) has been developed, which is said to produce global alignments of nucleotide and protein sequences faster than other best global alignment methods, including the Needleman-Wunsch algorithm.

[0149] RNA capping

[0150] The multivalent RNA composition may include one or more mRNAs having an open reading frame encoding a protein or peptide. Each of these mRNAs may have a 5' cap. The 5' cap may be added during a co-IVT reaction (e.g., transcription co-capping) or after an IVT reaction.

[0151] Some aspects also include a polynucleotide comprising both a 5' cap and a polynucleotide (eg, a polynucleotide comprising a nucleotide sequence encoding a polypeptide to be expressed).

[0152] The 5' cap structure of native mRNA participates in nuclear export, increases mRNA stability and binds mRNA cap-binding proteins (CBPs), such as eIF4E, which are responsible for mRNA stability and translation competence in cells to form mature circular mRNA species through the association of CBP with poly(A) binding proteins. The cap further facilitates the removal of 5' proximal introns during mRNA splicing.

[0153] Endogenous mRNA molecules can be capped at the 5' end, thereby generating a 5'-ppp-5'-triphosphate bond between the terminal guanosine cap residue of the mRNA molecule and the sense nucleotide transcribed at the 5'-end. This 5'-guanylate cap can then be methylated to produce an N7-methyl-guanylate residue. The ribose of the terminal and / or pre-terminal transcribed nucleotides at the 5' end of the mRNA can also be optionally 2'-O-methylated. 5'-decapping can target nucleic acid molecules, such as mRNA molecules, for degradation by hydrolysis and cleavage of the guanylate cap structure.

[0154] In some embodiments, a polynucleotide (eg, a polynucleotide comprising a nucleotide sequence encoding a polypeptide) is incorporated into a cap moiety.

[0155] In some embodiments, the polynucleotide comprises a non-hydrolyzable cap structure that prevents decapping and thus increases the half-life of the mRNA. Because the hydrolysis of the cap structure requires the cleavage of the 5'-ppp-5' phosphodiester bond, modified nucleotides can be used during the capping reaction. For example, the vaccinia capping enzyme from New England Biolabs (Ipswich, MA) can be used with α-thio-guanosine nucleotides according to the manufacturer's instructions to produce thiophosphate bonds in the 5'-ppp-5' cap. Additional modified guanosine nucleotides such as α-methyl-phosphonic acid and seleno-phosphate nucleotides can be used.

[0156] Additional modifications include, but are not limited to, 2'-O-methylation of the ribose of the 5'-end and / or the 5'-end pre-nucleotide of the polynucleotide (as mentioned above) on the 2'-hydroxyl of the sugar ring. A plurality of different 5'-cap structures can be used to produce the 5'-cap of nucleic acid molecules, such as polynucleotides used as mRNA molecules. Cap analogs, also referred to herein as synthetic cap analogs, chemical caps, chemical cap analogs or structural or functional cap analogs, are different from natural (i.e., endogenous, wild-type or physiological) 5'-caps in their chemical structure while retaining the cap function. Cap analogs can be chemically (i.e., non-enzymatically) or enzymatically synthesized and / or attached to polynucleotides.

[0157] For example, the anti-reverse cap analog (ARCA) cap contains two guanines linked by a 5'-5'-triphosphate group, one of which contains an N7 methyl group and a 3'-O-methyl group (i.e., N7,3'-O-dimethyl-guanosine-5'-triphosphate-5'-guanosine (m 7 G-3'mppp-G; which can be equivalently named 3'O-Me-m 7 G(5')ppp(5')G). The 3'-O atom of the other unmodified guanine becomes attached to the 5'-terminal nucleotide of the capping polynucleotide. The N7- and 3'-O-methylated guanines provide the terminal portion of the capping polynucleotide.

[0158] Another exemplary cap is mCAP, which is similar to ARCA but has a 2'-O-methyl group on guanosine (ie, N7, 2'-O-dimethyl-guanosine-5'-triphosphate-5'-guanosine, m 7 Gm-ppp-G).

[0159] Another exemplary cap is m 7 G-ppp-Gm-AG (ie, N7, guanosine-5'-triphosphate-2'-O-dimethyl-guanosine-adenosine-guanosine).

[0160] In some embodiments, the cap is a dinucleotide cap analog. As a non-limiting example, the dinucleotide cap analog can be modified with a boranophosphate group or a selenophosphate group at different phosphate positions, such as the dinucleotide cap analogs described in U.S. Pat. No. US 8,519,110, the contents of which are incorporated herein by reference in their entirety.

[0161] In another embodiment, the cap is a cap analog that is a N7-(4-chlorophenoxyethyl) substituted dinucleotide form of a cap analog known in the art and / or described herein. Non-limiting examples of N7-(4-chlorophenoxyethyl) substituted dinucleotide forms of cap analogs include N7-(4-chlorophenoxyethyl)-G(5')ppp(5')G and N7-(4-chlorophenoxyethyl)-m 3 ' -O G(5')ppp(5')G cap analogs (see, e.g., various cap analogs and methods of synthesizing cap analogs described in Kore et al. Bioorganic & Medicinal Chemistry 2013 21:4570-4574; the contents of which are incorporated herein by reference in their entirety). In another embodiment, the cap analog is a 4-chloro / bromophenoxyethyl analog.

[0162] The polynucleotides may also be enzymatically capped after manufacture (whether by IVT or chemical synthesis) to produce a more authentic 5'-cap structure. As used herein, the phrase "more authentic" refers to a feature that closely reflects or mimics an endogenous or wild-type feature in structure or function. That is, a "more authentic" feature is more representative of endogenous, wild-type, natural or physiological cellular function and / or structure than a synthetic feature or analog of the prior art, or is superior in one or more aspects to a corresponding endogenous, wild-type, natural or physiological feature. Non-limiting examples of more authentic 5' cap structures are those cap structures that, among other things, have enhanced cap-binding protein binding, increased half-life, reduced sensitivity to 5' endonucleases and / or reduced 5' decapping compared to synthetic 5' cap structures (or wild-type, natural or physiological 5' cap structures) known in the art. For example, a recombinant vaccinia virus capping enzyme and a recombinant 2'-O-methyltransferase can generate a canonical 5'-5'-triphosphate bond between the 5'-terminal nucleotide of a polynucleotide and a guanine cap nucleotide, wherein the cap guanine contains N7 methylation and the 5'-terminal nucleotide of the mRNA contains a 2'-O-methyl group. This structure is referred to as a cap 1 structure. Compared to other 5' cap analog structures known in the art, for example, this cap results in higher translational capacity and cellular stability and reduced activation of cellular proinflammatory cytokines. Cap structures include, but are not limited to, 7mG(5')ppp(5')N,pN2p (cap 0), 7mG(5')ppp(5')NlmpNp (cap 1), and 7mG(5')-ppp(5')NlmpN2mp (cap 2).

[0163] As a non-limiting example, post-manufacturing capping of chimeric polynucleotides may be more efficient because nearly 100% of the chimeric polynucleotides may be capped, as compared to approximately 80% when the cap analog is attached to the chimeric polynucleotide during an in vitro transcription reaction.

[0164] In some embodiments, the 5' terminal cap may include an endogenous cap or a cap analog. In some embodiments, the 5' terminal cap may include a guanine analog. Available guanine analogs include, but are not limited to, inosine, N1-methyl-guanosine, 2'fluoro-guanosine, 7-deaza-guanosine, 8-oxo-guanosine, 2-amino-guanosine, LNA-guanosine, and 2-azido-guanosine.

[0165] Exemplary caps are also described, including those that can be used in co-transcriptional capping methods for ribonucleic acid (RNA) synthesis using an RNA polymerase, such as a wild-type RNA polymerase or a variant thereof, such as those described. In one embodiment, the cap can be added when the RNA is produced in a "one-pot" reaction, without the need for a separate capping reaction. Thus, in some embodiments, the method includes reacting a polynucleotide template with an RNA polymerase variant, a nucleoside triphosphate, and a cap analog under in vitro transcription reaction conditions to produce an RNA transcript.

[0166] In some embodiments, the cap analog binds to a polynucleotide template comprising a promoter region comprising a transcription start site having a first nucleotide at nucleotide position +1, a second nucleotide at nucleotide position +2, and a third nucleotide at nucleotide position +3. In some embodiments, the cap analog hybridizes to the polynucleotide template at least at polynucleotide position +1, such as at positions +1 and +2, or at positions +1, +2, and +3.

[0167] For example, the cap analog can be 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. As used herein, the term "cap" comprises an inverted G nucleotide and may comprise additional nucleotides 3' of the inverted G, for example, 1, 2 or more nucleotides 3' of the inverted G and 5' of the 5' UTR.

[0168] Exemplary caps include the sequence or The underlined italic G is an inverted G.

[0169] In some embodiments, the nucleotide cap (eg, a trinucleotide cap or a tetranucleotide cap) comprises formula (I):

[0170] or a stereoisomer, tautomer or salt thereof, wherein

[0171]

[0172] Ring B1 is modified or unmodified guanine;

[0173] Ring B2 and Ring B3 are each independently a nucleobase or a modified nucleobase;

[0174] X2 is O, S(O) p NR 24 or CR 25 R 26 , where p is 0, 1, or 2;

[0175] Y0 is O or CR6R7;

[0176] Y1 is O, S(O) n , CR6R7 or NR8, wherein n is 0, 1 or 2;

[0177] Each --- is a single bond or does not exist, wherein when each --- is a single bond, Yi is O, S(O) n , CR6R7 or NR8; and when each --- is absent, Y1 is absent;

[0178] Y2 is (OP(O)R4) m , where m is 0, 1 or 2, or -O-(CR 40 R 41 )u-Q0-(CR 42 R 43 )v-, where Q0 is a bond, O, S(O) r NR 44 or CR 45 R 46 , r is 0, 1 or 2, and each of u and v is independently 1, 2, 3 or 4;

[0179] Each R2 and R2' is independently halo, LNA or OR3;

[0180] each R3 is independently H, C1-C6 alkyl, C2-C6 alkenyl or C2-C6 alkynyl, and when R3 is C1-C6 alkyl, C2-C6 alkenyl or C2-C6 alkynyl, it is optionally substituted with one or more of: halo, OH and C1-C6 alkoxy optionally substituted with one or more OH or OC(O)-C1-C6 alkyl;

[0181] Each R4 and R4' is independently H, halo, C1-C6 alkyl, OH, SH, SeH or BH3 - ;

[0182] Each of R6, R7 and R8 is independently -Q1-T1, wherein Q1 is a bond or a C1-C3 alkyl linker optionally substituted with one or more of halo, cyano, OH and C1-C6 alkoxy, and T1 is H, halo, OH, COOH, cyano or R s1 , where R s1 It is C1-C3 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C1-C6 alkoxy, C(O)O-C1-C6 alkyl, C3-C8 cycloalkyl, C6-C 10 Aryl, NR 31 R 32 ,(NR 31 R32 R 33 ) + , 4- to 12-membered heterocycloalkyl or 5- or 6-membered heteroaryl, and R s1 Optionally substituted with one or more substituents selected from the group consisting of halo, OH, oxo, C1-C6 alkyl, COOH, C(O)O-C1-C6 alkyl, cyano, C1-C6 alkoxy, NR 31 R 32 ,(NR 31 R 32 R 33 ) + , C3-C8 cycloalkyl, C6-C 10 aryl, 4- to 12-membered heterocycloalkyl, and 5- or 6-membered heteroaryl;

[0183] R 10 , R 11 , R 12 , R 13 , R 14 and R 15 Each of the above is independently -Q2-T2, wherein Q2 is a bond or a C1-C3 alkyl linker optionally substituted with one or more of halo, cyano, OH, and C1-C6 alkoxy, and T2 is H, halo, OH, NH2, cyano, NO2, N3, R s2 OR s2 , where R s2 It is C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C8 cycloalkyl, C6-C 10 Aryl, NHC(O)-C1-C6 alkyl, NR 31 R 32 ,(NR 31 R 32 R 33 ) + , 4- to 12-membered heterocycloalkyl or 5- or 6-membered heteroaryl, and R s2 Optionally substituted with one or more substituents selected from the group consisting of halo, OH, oxo, C1-C6 alkyl, COOH, C(O)O-C1-C6 alkyl, cyano, C1-C6 alkoxy, NR 31 R 32 ,(NR 31 R 32 R 33 ) + , C3-C8 cycloalkyl, C6-C 10 aryl, 4- to 12-membered heterocycloalkyl, and 5- or 6-membered heteroaryl; or alternatively, R 12 With R 14 Together is oxo, or R 13With R 15 Together is oxygen,

[0184] R 20 , R 21 , R 22 and R 23 Each of the above is independently -Q3-T3, wherein Q3 is a bond or a C1-C3 alkyl linker optionally substituted with one or more of halo, cyano, OH, and C1-C6 alkoxy, and T3 is H, halo, OH, NH2, cyano, NO2, N3, R S3 OR S3 , where R S3 It is C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C8 cycloalkyl, C6-C 10 Rs3 is optionally substituted by one or more substituents selected from the group consisting of halo, OH, oxo, C1-C6 alkyl, COOH, C(O)O-C1-C6 alkyl, cyano, C1-C6 alkoxy, amino, mono-C1-C6 alkylamino, di-C1-C6 alkylamino, C3-C8 cycloalkyl, C6-C 10 aryl, 4- to 12-membered heterocycloalkyl, and 5- or 6-membered heteroaryl; R 24 , R 25 and R 26 Each of is independently H or C1-C6 alkyl;

[0185] R 27 and R 28 Each of which is independently H or OR 29 ; or R 27 With R 28 Together we form OR 30 -O; each R 29 are independently H, C1-C6 alkyl, C2-C6 alkenyl or C2-C6 alkynyl, and when R 29 is C1-C6 alkyl, C2-C6 alkenyl or C2-C6 alkynyl, optionally substituted by one or more of the following: halo, OH and C1-C6 alkoxy optionally substituted by one or more OH or OC(O)-C1-C6 alkyl;

[0186] R 30 is a C1-C6 alkylene group optionally substituted by one or more of a halogen group, an OH group, and a C1-C6 alkoxy group;

[0187] R 31 , R 32 and R 33Each of the following is independently H, C1-C6 alkyl, C3-C8 cycloalkyl, C6-C 10 Aryl, 4- to 12-membered heterocycloalkyl, or 5- or 6-membered heteroaryl;

[0188] R 40 , R 41 , R 42 and R 43 Each of which is independently H, halo, OH, cyano, N3, OP(O)R 47 R 48 or optionally one or more OP(O)R 47 R 48 Substituted C1-C6 alkyl, or one R 41 and an R 43 Together with the carbon atom to which they are attached and Q0, they form C4-C 10 Cycloalkyl, 4-membered to 14-membered heterocycloalkyl, C6-C 10 aryl or 5- to 14-membered heteroaryl, and each of the cycloalkyl, heterocycloalkyl, phenyl or 5- to 6-membered heteroaryl is optionally substituted with one or more of: OH, halo, cyano, N3, oxo, OP(O)R 47 R 48 , C1-C6 alkyl, C1-C6 haloalkyl, COOH, C(O)O-C1-C6 alkyl, C1-C6 alkoxy, C1-C6 haloalkoxy, amino, mono-C1-C6 alkylamino, and di-C1-C6 alkylamino;

[0189] R 44 is H, C1-C6 alkyl or amine protecting group;

[0190] R 45 and R 46 Each of which is independently H, OP(O)R 47 R 48 or optionally one or more OP(O)R 47 R 48 Substituted C1-C6 alkyl, and

[0191] R 47 and R 48 Each of which is independently H, halo, C1-C6 alkyl, OH, SH, SeH or BH3 - .

[0192] It should be understood that the cap analog may include any of the cap analogs described in International Publication No. WO 2017 / 066797, published on April 20, 2017, which is incorporated herein by reference in its entirety.

[0193] In some embodiments, the B2 middle position can be a non-ribose molecule, such as arabinose.

[0194] In some embodiments, R2 is ethyl based.

[0195] Thus, in some embodiments, the trinucleotide cap comprises the following structure:

[0196]

[0197] In other embodiments, the trinucleotide cap comprises the following structure:

[0198] In other embodiments, the trinucleotide cap comprises the following structure:

[0199] In other embodiments, the trinucleotide cap comprises the following structure:

[0200]

[0201] Thus, in some embodiments, the tetranucleotide cap comprises the following structure:

[0202] In other embodiments, the tetranucleotide cap comprises the following structure:

[0203] In other embodiments, the tetranucleotide cap comprises the following structure:

[0204] In other embodiments, the tetranucleotide cap comprises the following structure:

[0205]

[0206] In some embodiments, R is alkyl (e.g., C1-C6 alkyl). In some embodiments, R is methyl (e.g., C1 alkyl). In some embodiments, R is ethyl (e.g., C2 alkyl). In some embodiments, R is hydrogen.

[0207] 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. 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.

[0208] In some embodiments, the trinucleotide cap comprises a sequence selected from the group consisting of: 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.

[0209] In some embodiments, the trinucleotide cap comprises m 7 GpppApA. In some embodiments, the trinucleotide cap comprises m 7 GpppApC. In some embodiments, the trinucleotide cap comprises m 7 GpppApG. In some embodiments, the trinucleotide cap comprises m 7In some embodiments, the trinucleotide cap comprises m 7 GpppCpA. In some embodiments, the trinucleotide cap comprises m 7 GpppCpC. In some embodiments, the trinucleotide cap comprises m 7 GpppCpG. In some embodiments, the trinucleotide cap comprises m 7 In some embodiments, the trinucleotide cap comprises m 7 GpppGpA. In some embodiments, the trinucleotide cap comprises m 7 GpppGpC. In some embodiments, the trinucleotide cap comprises m 7 GpppGpG. In some embodiments, the trinucleotide cap comprises m 7 In some embodiments, the trinucleotide cap comprises m 7 GpppUpA. In some embodiments, the trinucleotide cap comprises m 7 GpppUpC. In some embodiments, the trinucleotide cap comprises m 7 GpppUpG. In some embodiments, the trinucleotide cap comprises m 7 GpppUpU.

[0210] In some embodiments, the trinucleotide cap comprises a sequence selected from the group consisting of: 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、m7 G 3'OMe pppUpC、m 7 G 3'OMe pppUpG and m 7 G 3'OMe pppUpU.

[0211] In some embodiments, the trinucleotide cap comprises m 7 G 3'OMe pppApA. In some embodiments, the trinucleotide cap comprises m 7 G 3'OMe pppApC. In some embodiments, the trinucleotide cap comprises m 7 G 3'OMe pppApG. In some embodiments, the trinucleotide cap comprises m 7 G 3'OMe pppApU. In some embodiments, the trinucleotide cap comprises m 7 G 3'OMe pppCpA. In some embodiments, the trinucleotide cap comprises m 7 G 3'OMe pppCpC. In some embodiments, the trinucleotide cap comprises m 7 G 3'OMe pppCpG. In some embodiments, the trinucleotide cap comprises m 7 G 3'OMe In some embodiments, the trinucleotide cap comprises m 7 G 3'OMe pppGpA. In some embodiments, the trinucleotide cap comprises m 7 G 3'OMe pppGpC. In some embodiments, the trinucleotide cap comprises m 7 G 3'OMe pppGpG. In some embodiments, the trinucleotide cap comprises m 7 G 3' OMe pppGpU. In some embodiments, the trinucleotide cap comprises m 7 G 3'OMe pppUpA. In some embodiments, the trinucleotide cap comprises m 7 G 3'OMe pppUpC. In some embodiments, the trinucleotide cap comprises m 7 G 3'OMe pppUpG. In some embodiments, the trinucleotide cap comprises m 7 G 3'OMe pppUpU.

[0212] In other embodiments, the trinucleotide cap comprises a sequence selected from the group consisting of: 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 U 2'OMe pA、m 7 G 3'OMe U 2'OMe pC、m 7 G 3' OMe U 2'OMe pG and m 7 G 3'OMe U 2'OMe pU.

[0213] In some embodiments, the trinucleotide cap comprises m 7 G 3'OMe pppA 2'OMe pA. In some embodiments, the trinucleotide cap comprises m 7 G 3'OMe pppA 2'OMeIn some embodiments, the trinucleotide cap comprises m 7 G 3'OMe pppA 2'OMe pG. 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 pA. 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 pG. 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 pA. 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 pG. 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 U 2'OMe pA. In some embodiments, the trinucleotide cap comprises m 7 G 3'OMe U 2'OMe In some embodiments, the trinucleotide cap comprises m 7 G 3'OMe U 2'OMe pG. In some embodiments, the trinucleotide cap comprises m 7 G 3'OMe U 2'OMe pU.

[0214] In other embodiments, the trinucleotide cap comprises a sequence selected from the group consisting of: m 7 GpppA2'OMe pA、m 7 GpppA 2'OMe pC、m 7 GpppA 2'OMe pG、m 7 GpppA 2'OMe pU、m 7 Gppp 2'OMe pA、m 7 Gppp 2'OMe pC、m 7 Gppp 2' OMe pG、m 7 Gppp 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.

[0215] In some embodiments, the trinucleotide cap comprises m 7 GpppA 2'OMe pA. In some embodiments, the trinucleotide cap comprises m 7 GpppA 2'OMe In some embodiments, the trinucleotide cap comprises m 7 GpppA 2'OMe pG. In some embodiments, the trinucleotide cap comprises m 7 GpppA 2'OMe In some embodiments, the trinucleotide cap comprises m 7 Gppp 2'OMe pA. In some embodiments, the trinucleotide cap comprises m 7 Gppp 2'OMe In some embodiments, the trinucleotide cap comprises m 7 Gppp 2'OMe pG. In some embodiments, the trinucleotide cap comprises m 7 Gppp 2'OMeIn some embodiments, the trinucleotide cap comprises m 7 GpppG 2'OMe pA. In some embodiments, the trinucleotide cap comprises m 7 GpppG 2'OMe In some embodiments, the trinucleotide cap comprises m 7 GpppG 2'OMe pG. In some embodiments, the trinucleotide cap comprises m 7 GpppG 2' OMe In some embodiments, the trinucleotide cap comprises m 7 GpppU 2'OMe pA. In some embodiments, the trinucleotide cap comprises m 7 GpppU 2'OMe In some embodiments, the trinucleotide cap comprises m 7 GpppU 2'OMe pG. In some embodiments, the trinucleotide cap comprises m 7 GpppU 2'OMe pU.

[0216] In some embodiments, the trinucleotide cap comprises m 7 Gpppm 6 A 2'Ome pG. In some embodiments, the trinucleotide cap comprises m 7 Gpppe 6 A 2'Ome pG.

[0217] 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.

[0218] In some embodiments, the trinucleotide cap comprises any of the following structures:

[0219]

[0220] In some embodiments, the cap analog comprises a tetranucleotide cap. In some embodiments, the cap analog comprises GGAG.

[0221] In some embodiments, the tetranucleotide cap comprises any of the following structures:

[0222]

[0223] In some embodiments, the tetranucleotide cap comprises trinucleotides as shown above. In some embodiments, the tetranucleotide cap comprises m7 GpppN1N2N3, wherein N1, N2 and N3 are optional (i.e., may not be present or one or more may be present) and are independently natural, modified or non-natural nucleoside bases. In some embodiments, m7 G is further methylated, for example, at the 3' position. In some embodiments, m7 G comprises an O-methyl group at the 3' position. In some embodiments, N1, N2 and N3, if present, are optionally independently adenine, uracil, guanidine, thymine or cytosine. In some embodiments, one or more (or all) of N1, N2 and N3, if present, are methylated, for example, at the 2' position. In some embodiments, one or more (or all) of N1, N2 and N3, if present, have an O-methyl group at the 2' position.

[0224] In some embodiments, the tetranucleotide cap comprises the following structure:

[0225]

[0226] Wherein B1, B2 and B3 are independently natural, modified or non-natural nucleoside bases; and R1, R2, R3 and R4 are independently OH or O-methyl. In some embodiments, R3 is O-methyl and R4 is OH. In some embodiments, R3 and R4 are O-methyl. In some embodiments, R4 is O-methyl. In some embodiments, R1 is OH, R2 is OH, R3 is O-methyl, and R4 is OH. In some embodiments, R1 is OH, R2 is OH, R3 is O-methyl, and R4 is O-methyl. In some embodiments, at least one of R1 and R2 is O-methyl, R3 is O-methyl, and R4 is OH. In some embodiments, at least one of R1 and R2 is O-methyl, R3 is O-methyl, and R4 is O-methyl.

[0227] In some embodiments, B1, B3 and B3 are natural nucleoside bases. In some embodiments, at least one of B1, B2 and B3 is a modified or non-natural base. In some embodiments, at least one of B1, B2 and B3 is N6-methyladenine. In some embodiments, B1 is adenine, cytosine, thymine or uracil. In some embodiments, B1 is adenine, B2 is uracil, and B3 is adenine. In some embodiments, R1 and R2 are OH, R3 and R4 are O-methyl, B1 is adenine, B2 is uracil, and B3 is adenine.

[0228] In some embodiments, the tetranucleotide cap comprises a sequence selected from the group consisting of GAAA, GACA, GAGA, GAUA, GCAA, GCCA, GCGA, GCUA, GGAA, GGCA, GGGA, GGUA, GUCA, and GUUA. In some embodiments, the tetranucleotide cap comprises a sequence selected from the group consisting of GAAG, GACG, GAGG, GAUG, GCAG, GCCG, GCGG, GCUG, GGAG, GGCG, GGGG, GGUG, GUCG, GUGG, and GUUG. In some embodiments, the tetranucleotide cap comprises a sequence selected from the group consisting of GAAU, GACU, GAGU, GAUU, GCAU, GCCU, GCGU, GCUU, GGAU, GGCU, GGGU, GGUU, GUAU, GUCU, GUGU, and GUUU. In some embodiments, the tetranucleotide cap comprises a sequence selected from the group consisting of GAAC, GACC, GAGC, GAUC, GCAC, GCCC, GCGC, GCUC, GGAC, GGCC, GGGC, GGUC, GUAC, GUCC, GUGC, and GUUC.

[0229] In some embodiments, the tetranucleotide cap comprises a sequence selected from the group consisting of: m 7 G 3'OMe pppApApN、m 7 G 3'OMe pppApCpN、m 7 G 3'OMe pppApGpN、m 7 G 3'OMe pppApUpN、m 7 G 3'OMe pppCpApN、m 7 G 3'OMe pppCpCpN、m 7 G 3'OMe pppCpGpN、m 7 G 3'OMe pppCpUpN、m 7 G 3'OMe pppGpApN、m 7 G 3'OMe pppGpCpN、m 7 G 3'OMe pppGpGpN、m 7 G 3'OMe pppGpUpN、m 7 G 3'OMe pppUpApN、m 7 G 3'OMe pppUpCpN、m 7 G3'OMe pppUpGpN and m 7 G 3'OMe pppUpUpN, wherein N is a natural, modified or non-natural nucleobase.

[0230] In other embodiments, the tetranucleotide cap comprises a sequence selected from the group consisting of: m 7 G 3'OMe pppA 2'OMe pApN、m 7 G 3'OMe pppA 2'OMe pCpN、m 7 G 3'OMe pppA 2'OMe pGpN、m 7 G 3'OMe pppA 2'OMe pUpN、m 7 G 3'OMe pppC 2'OMe pApN、m 7 G 3'OMe pppC 2'OMe pCpN、m 7 G 3'OMe pppC 2'OMe pGpN、m 7 G 3'OMe pppC 2'OMe pUpN、m 7 G 3'OMe pppG 2'OMe pApN、m 7 G 3'OMe pppG 2'OMe pCpN、m 7 G 3'OMe pppG 2'OMe pGpN、m 7 G 3'OMe pppG 2'OMe pUpN、m 7 G 3'OMe U 2'OMe pApN、m 7 G 3'OMe U 2'OMe pCpN、m 7 G 3'OMe U 2'OMe pGpN and m 7 G 3'OMe U 2'OMe pUpN, wherein N is a natural, modified or non-natural nucleobase.

[0231] In other embodiments, the tetranucleotide cap comprises a sequence selected from the group consisting of: m 7 GpppA 2'OMe pApN、m 7 GpppA 2'OMe pCpN、m 7 GpppA 2'OMe pGpN、m 7 GpppA 2'OMe pUpN、m 7 Gppp 2'OMe pApN、m 7 Gppp 2'OMe pCpN、m 7 Gppp 2'OMe pGpN、m 7 Gppp 2'OMe pUpN、m 7 GpppG 2'OMe pApN、m 7 GpppG 2'OMe pCpN、m 7 GpppG 2'OMe pGpN、m 7 GpppG 2'OMe pUpN、m 7 GpppU 2'OMe pApN、m 7 GpppU 2'OMe pCpN、m 7 GpppU 2'OMe pGpN and m 7 GpppU 2'OMe pUpN, wherein N is a natural, modified or non-natural nucleobase.

[0232] In other embodiments, the tetranucleotide cap comprises a sequence selected from the group consisting of: m 7 G 3'OMe pppA 2'OMe pA 2' OMe pN、m 7 G 3'OMe pppA 2'OMe pC 2'OMe pN、m 7 G 3'OMe pppA 2'OMe pG 2'OMe pN、m 7 G 3'OMe pppA 2'OMe pU 2'OMe pN、m 7 G 3' OMe pppC2'OMe pA 2'OMe pN、m 7 G 3'OMe pppC 2'OMe pC 2'OMe pN、m 7 G 3'OMe pppC 2'OMe pG 2'OMe pN、m 7 G 3'OMe pppC 2' OMe pU 2'OMe pN、m 7 G 3'OMe pppG 2'OMe pA 2'OMe pN、m 7 G 3'OMe pppG 2'OMe pC 2'OMe pN、m 7 G 3'OMe pppG 2'OMe pG 2'OMe pN、m 7 G 3'OMe pppG 2'OMe pU 2'OMe pN、m 7 G 3'OMe U 2'OMe pA 2'OMe pN、m 7 G 3'OMe U 2'OMe pC 2'OMe pN、m 7 G 3' OMe U 2'OMe pG 2'OMe pN and m 7 G 3'OMe U 2'OMe pU 2'OMe pN, wherein N is a natural, modified or non-natural nucleobase.

[0233] In other embodiments, the tetranucleotide cap comprises a sequence selected from the group consisting of: m 7 GpppA 2'OMe pA 2'OMe pN、m 7 GpppA 2'OMe pC 2'OMe pN、m 7 GpppA 2'OMe pG 2'OMe pN、m 7 GpppA 2'OMepU 2'OMe pN、m 7 Gppp 2'OMe pA 2'OMe pN、m 7 Gppp 2'OMe pC 2'OMe pN、m 7 Gppp 2'OMe pG 2'OMe pN、m 7 Gppp 2'OMe pU 2'OMe pN、m 7 GpppG 2'OMe pA 2'OMe pN、m 7 GpppG 2'OMe pC 2'OMe pN、m 7 GpppG 2'OMe pG 2'OMe pN、m 7 GpppG 2'OMe pU 2'OMe pN、m 7 GpppU 2'OMe pA 2'OMe pN、m 7 GpppU 2'OMe pC 2'OMe pN、m 7 GpppU 2'OMe pG 2'OMe pN and m 7 GpppU 2'OMe pU 2'OMe pN, wherein N is a natural, modified or non-natural nucleobase.

[0234] In some embodiments, the tetranucleotide cap comprises GGAG. In some embodiments, the tetranucleotide cap comprises the following structure:

[0235]

[0236] The capping efficiency of a post-transcriptional or co-transcriptional capping reaction may vary. The term "capping efficiency" may refer to the amount (e.g., expressed as a percentage) of mRNA that contains a cap structure relative to the total mRNA in a mixture (e.g., a post-translational capping reaction or a co-transcriptional calling reaction). In some embodiments, the capping efficiency of the capping reaction is at least 60%, 70%, 80%, 90%, 95%, 99%, or 99.9% (e.g., at least 60%, 70%, 80%, 90%, 95%, 99%, or 99.9% of the input mRNA contains a cap after the capping reaction). In some embodiments, a multivalent co-IVT reaction does not affect the capping efficiency of the mRNA produced by the IVT reaction.

[0237] In vitro transcription method

[0238] Some aspects relate to a method of producing (e.g., synthesizing) an RNA transcript (e.g., an mRNA transcript), the method comprising contacting a DNA template (e.g., a first input DNA and a second input DNA) with an RNA polymerase (e.g., T7 RNA polymerase, a T7 RNA polymerase variant, etc.) under conditions that result in production of the RNA transcript.

[0239] Some aspects relate to a method of performing an IVT reaction, the method comprising contacting a DNA template with an RNA polymerase (eg, T7 RNA polymerase, such as a T7 RNA polymerase variant) in the presence of nucleoside triphosphates and a buffer under conditions that result in production of an RNA transcript.

[0240] Other aspects provide a co-transcriptional capping method comprising 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.

[0241] In some embodiments, a co-transcriptional capping method for RNA synthesis comprises reacting a polynucleotide template with: (a) a T7 RNA polymerase variant comprising at least one amino acid substitution relative to a wild-type RNA polymerase (e.g., a T7 polymerase variant comprising amino acid substitutions at positions 437, 387, 350, and 351 relative to SEQ ID NO: 1), (b) a nucleoside triphosphate, and (c) a cap analog (e.g., comprising the sequence GpppA 2'Ome pG) to produce an RNA transcript in which the polynucleotide template comprises a 2'-deoxythymidine residue at template position +1.

[0242] IVT conditions generally require a purified linear DNA template containing a promoter, nucleoside triphosphates, a buffer system comprising 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 can be performed by incubating the DNA template with an RNA polymerase and nucleoside triphosphates, including GTP, ATP, CTP, and UTP (or nucleotide analogs), in a transcription buffer. This reaction produces an RNA transcript having a 5' terminal guanosine triphosphate.

[0243] "Percent identity," "sequence identity," "% identity," or "% sequence identity" (as they are used interchangeably herein) of two sequences (e.g., nucleic acids or amino acids) refers to a quantitative measure of the similarity between two sequences (e.g., nucleic acids or amino acids). Percent identity can be determined using the algorithm of Karlin and Altschul, Proc. Natl. Acad. Sci. USA 87:2264-68, 1990, as modified by Karlin and Altschul, Proc. Natl. Acad. Sci. USA 90:5873-77, 1993. Such an algorithm is incorporated into the NBLAST and XBLAST programs (version 2.0) of Altschul et al., J. Mol. Biol. 215:403-10, 1990. BLAST protein searches can be performed with the XBLAST program with score = 50 and wordlength = 3 to obtain amino acid sequences homologous to a protein molecule of interest. In the case of a gap between two sequences, Gapped BLAST can be used as described in Altschul et al., Nucleic Acids Res. 25(17):3389-3402, 1997. When using BLAST and Gapped BLAST programs, the default parameters of the corresponding programs (e.g., XBLAST and NBLAST) can be used. Unless otherwise stated, when specifying a percentage identity or a range thereof (e.g., at least, greater than, etc.), the endpoints should be inclusive and the range (e.g., at least 70% identity) should include all ranges within the cited range.

[0244] Input deoxyribonucleic acid (DNA) serves as a nucleic acid template for RNA polymerase. The DNA template may include a polynucleotide encoding a polypeptide of interest (e.g., an antigenic polypeptide). In some embodiments, the DNA template includes an RNA polymerase promoter (e.g., a T7 RNA polymerase promoter), which is located at the 5' of the polynucleotide encoding the polypeptide of interest and is operably connected to the polynucleotide. The DNA template may also include a nucleotide sequence encoding a polyadenylated (poly A) tail at the 3' end of the gene of interest. In some embodiments, the input DNA includes plasmid DNA (pDNA). The term "plasmid DNA" or "pDNA" may refer to an extrachromosomal DNA molecule that is physically separated from chromosomal DNA in a cell and can be replicated independently. In some embodiments, plasmid DNA is isolated from a cell (e.g., as a plasmid DNA preparation). In some embodiments, plasmid DNA includes a replication origin, which may contain one or more heterologous nucleic acids, such as nucleic acids encoding therapeutic proteins that can be used as RNA polymerase templates. Plasmid DNA may be circularized or linear (e.g., plasmid DNA that has been linearized by restriction enzyme digestion).

[0245] In some embodiments, each input DNA (e.g., population of input DNA molecules) in a co-IVT reaction is obtained from a different source (e.g., synthesized separately in different cells or cell populations). In some embodiments, each input DNA (e.g., population of input DNA) is obtained from a different bacterial cell or bacterial cell population. For example, in a co-IVT reaction with three populations of input DNA, a first input DNA is produced in bacterial cell population A, a second input DNA is produced in bacterial cell population B, and a third input DNA is produced in bacterial population C, wherein each of A, B, and C is not the same bacterial culture (e.g., co-cultured in the same container or plate). In another example, two input DNAs obtained from different sources are i) chemically synthesized in separate synthesis reactions, or ii) produced by separate amplifications (e.g., polymerase chain reactions (PCR reactions)). Methods for obtaining input DNA populations (e.g., plasmid DNA) are known, such as described by Sambrook, Joseph. Molecular Cloning: a Laboratory Manual. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press, 2001.

[0246] Some aspects include normalizing the amount of DNA used in the multivalent co-IVT reaction. In some embodiments, the normalization is based on the molar mass of the input DNA. In some embodiments, the normalization is based on the degradation rate of the input DNA. In some embodiments, the normalization is based on the degradation rate of the resulting mRNA (e.g., measured based on the poly A variants present in the reaction mixture, or T7 polymerase ineffective transcripts or truncated transcripts). In some embodiments, the normalization is based on the nucleotide content of the input DNA (e.g., the amount of A, G, C, U, or any combination thereof). In some embodiments, the normalization is based on the purity of the input DNA. In some embodiments, the normalization is based on the poly A tailing efficiency of the input DNA. In some embodiments, the normalization is based on the length of the input DNA.

[0247] In some embodiments, normalization is based on the lowest level present in the input DNA (e.g., lowest molar mass, degradation rate (e.g., degradation rate of the input DNA and / or output RNA), nucleotide content, purity, and / or poly A tailing efficiency). In some embodiments, normalization is based on the highest level present in the input DNA (e.g., highest molar mass, degradation rate (e.g., degradation rate of the input DNA and / or output RNA), nucleotide content, purity, and / or poly A tailing efficiency). In some embodiments, normalization is based on the RNA production rate of the input DNA (e.g., the highest RNA production rate of the input DNA or the lowest RNA production rate of the input DNA in the reaction mixture).

[0248] Some aspects relate to IVT methods in which the amount of input DNA (e.g., a first DNA or a second DNA) is adjusted or normalized to improve the production of a multivalent RNA composition having a predefined ratio of mRNA components. The present disclosure is based in part on the discovery that certain factors that affect the purity of a multivalent RNA composition, such as large differences in size between input DNAs during IVT (e.g., differences of more than 100, 200, 500, 1000 or more nucleotides in length) and / or the poly A-tailing efficiency of a given DNA, can be addressed by normalizing the amount of input DNA based on one or more of these factors prior to IVT. For example, in some embodiments, the amount of two input DNAs is calculated based on the desired molar ratio of the first RNA to the second RNA transcribed from the input DNA. In some embodiments, the calculation includes determining a plasmid mass ratio based on the desired molar ratio of the input DNA. In some embodiments, the amount of input DNA is normalized based on the highest poly A-tailing efficiency of the input DNA during IVT.

[0249] The amount of input DNA (e.g., a population of input DNA molecules) used in an IVT reaction can vary, depending on the number of different RNA molecules desired to be included in the multivalent RNA composition. In some embodiments, the IVT reaction mixture comprises 2 or more different input DNAs, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 or more different input DNAs. In some embodiments, the IVT reaction comprises more than 10 different input DNAs. The term "different input DNA" encompasses input DNAs encoding different RNAs, e.g., different RNAs having: i) different lengths (whether or not the RNAs are identical over the entire length of the shorter of the two lengths), ii) different nucleotide sequences, iii) different chemical modification patterns, or iv) any combination of the foregoing.

[0250] The concentration of each of the population of DNA molecules can also vary. In some embodiments, the concentration of each population of DNA molecules in the IVT reaction is in the range of about 0.005 mg / mL to about 0.5 mg / ml. In some embodiments, the concentration of each population of DNA molecules in the IVT reaction is in the range of about 0.02 mg / ml to about 0.05 mg / ml, 0.02 to about 0.15 mg / ml, about 0.05 mg / ml to about 0.20 mg / ml, about 0.175 to about 0.3 mg / ml, about 0.2 mg / ml to about 0.5 mg / ml, about 0.3 mg / ml to about 0.6 mg / ml, about 0.5 mg / ml to about 0.75 mg / ml, about 0.5 ... In the range of about 1.0 mg / ml, about 0.75 mg / ml to about 0.9 mg / ml, about 0.75 mg / ml to about 1.5 mg / ml, about 0.8 mg / ml to about 1.2 mg / ml, about 1.0 mg / ml to about 1.5 mg / ml, about 1.0 mg / ml to about 2.5 mg / ml, about 1.5 mg / ml to about 3.0 mg / ml, about 2.0 mg / ml to about 4.0 mg / ml or about 2.5 mg / ml to about 5.0 mg / ml.

[0251] In some embodiments, the input DNA added to the IVT reaction is a predefined ratio of DNA, which may include ratios between 2, 3, 4, 5, 6, 7, 8, 9, 10 or more different input DNAs (e.g., depending on the number of different RNAs in the composition). In some embodiments, the predefined ratio of input DNA includes ratios between more than 10 input DNAs. The term "predefined ratio of input DNA" may refer to the desired final ratio of DNA molecules in the IVT reaction. The desired final ratio of the input DNA may depend on the final peptide or polypeptide product encoded by the RNA encoded by the input DNA. In some embodiments, the input DNA may have a desired ratio, which may include 2 to 8 input DNAs (e.g., a:b, a:b:c, a:b:c:d, a:b:c:d:e, a:b:c:d:e:f, a:b:c:d:e:f:g, a:b:c:d:e:f:g:h, etc., where each of ah is a number between 1 and 10). In some embodiments, the predefined input DNA ratio is different from the predefined mRNA ratio.

[0252] The size of two or more input DNAs (e.g., DNA from two or more different input DNA populations) can vary. In some embodiments, the input DNA comprises from about 15 to about 8,000 base pairs (e.g., 15 to 50, 15 to 100, 15 to 200, 15 to 300, 15 to 400, 15 to 500, 15 to 600, 15 to 700, 15 to 800, 15 to 900, 15 to 1000, 15 to 1200, 15 to 1400, 15 to 1500, 15 to 1800, 15 to 2000, 15 to 2500, 15 to 3000, 50 to 4000, 50 to 5000, 50 to 6000, 50 to 7000, 50 to 8000, 50 to 9000, 50 to 1000, 50 to 1200, 50 to 1400, 50 to 1500, 50 to 1800 100, 50 to 200, 50 to 300, 50 to 400, 50 to 500, 50 to 600, 50 to 700, 50 to 800, 50 to 900, 50 to 1000, 50 to 1200, 50 to 1400, 50 to 1500, 50 to 1800, 50 to 2000, 50 to 2500, 50 to 3000, 100 to 200, 100 to 300, 100 to 400, 100 to 500, 100 to 600, 100 to 700 , 100 to 800, 100 to 900, 100 to 1000, 100 to 1200, 100 to 1400, 100 to 1500, 100 to 1800, 100 to 2000, 100 to 2500, 100 to 3000, 200 to 300, 200 to 400, 200 to 500, 200 to 600, 200 to 700, 200 to 800, 200 to 900, 200 to 1000, 200 to 1500, 200 to 300 or 0, 500 to 1000, 500 to 1500, 500 to 2000, 500 to 2500, 500 to 3000, 1000 to 1500, 1000 to 2000, 1000 to 2500, 1000 to 3000, 1500 to 3000, 2500 to 3000, 2000 to 3000, 2500 to 4000, 3000 to 5000, 3500 to 6500, 5000 to 7500, or 6500 to 8000 base pairs.

[0253] The mass of each population of input DNA molecules in an IVT reaction can vary. In some embodiments, the mass of each population of input DNA varies based on the total volume of the IVT reaction mixture. In some embodiments, the mass of each population of each input DNA molecule in the IVT mixture individually varies from about 0.5% to about 99.9% of the total input DNA present in the IVT reaction mixture. In some embodiments, the molar ratio of each population of input DNA molecules in an IVT reaction can vary.

[0254] In some embodiments, two or more input DNA molecules used in an IVT reaction have different lengths (e.g., contain different numbers of nucleotides). In some embodiments, the difference in length between two or more (e.g., 3, 4, 5, 6, 7, 8, 9, 10, or more) different input DNA molecules in an IVT reaction mixture is greater than 70 base pairs, 80 base pairs, 90 base pairs, or 100 base pairs (e.g., two input DNAs in the composition are not within 70, 80, 90, or 100 base pairs of each other in length). In some embodiments, the difference in length between two or more (e.g., 3, 4, 5, 6, 7, 8, 9, 10, or more) different input DNA molecules is more than 100 base pairs, such as 500 base pairs, 1000 base pairs, 1500 base pairs, 2000 base pairs, 3000 base pairs, 4000 base pairs, 5000 base pairs, 6000 base pairs, 7000 base pairs, 8000 base pairs, or more.

[0255] In some embodiments, two or more input DNA molecules used in the IVT reaction encode mRNA molecules of different lengths (e.g., comprising different numbers of nucleotides). In some embodiments, the difference in length between two or more mRNA molecules encoded by different input DNA molecules in the IVT reaction mixture is greater than 70 nucleotides, 80 nucleotides, 90 nucleotides, or 100 nucleotides (e.g., two input DNAs in the composition encode mRNA molecules that are not within 70, 80, 90, or 100 nucleotides of each other in length). In some embodiments, the difference in length between two or more mRNA molecules encoded by different input DNA molecules is more than 100 nucleotides, such as 500 nucleotides, 1000 nucleotides, 1500 nucleotides, 2000 nucleotides, 3000 nucleotides, 4000 nucleotides, or more.

[0256] In some embodiments, the multivalent IVT includes co-transcription of at least 2 different input DNAs (e.g., at least 2 of DNA A, B, C, D, E, F, F, H, I, J, etc.) at a ratio of A:B:C:D:E:F:G:H:I:J, wherein if DNA A is normalized to 1, one or more of DNAs B, C, D, E, F, G, H, I, J, etc. may each independently be present in an amount (e.g., concentration) of 0.01 to 100 times the amount (e.g., concentration) of A, such as 0.05 to 20 times the amount of A, 0.1 to 10 times the amount of A, 0.2 to 5 times the amount of A, 0.3 to 3 times the amount of A, 0.5 to 2 times the amount of A, 0.75 to 1.4 times the amount of A, 0.8 to 1.25 times the amount of A, or 0.9 to 1.15 times the amount of A. One or more of DNA B, C, D, E, F, G, H, I or J may also be absent.

[0257] In some embodiments, a multivalent RNA composition is produced by combining RNA transcripts (e.g., mRNA) from separate sources. In some embodiments, a multivalent RNA composition is produced by separately transcribing two or more DNA templates in separate IVT reactions and combining the transcribed RNAs. In some embodiments, RNA transcripts are produced by IVT and then added to one or more other RNAs. The RNAs can be combined in any desired amount to produce a multivalent RNA composition comprising two or more RNAs in a specific ratio.

[0258] In some embodiments, the RNA transcript is a product of an IVT reaction. In some embodiments, the RNA transcript is a messenger RNA (mRNA) comprising a nucleotide sequence encoding a polypeptide of interest (e.g., a therapeutic protein or therapeutic peptide) attached to a poly A tail. In some embodiments, the mRNA is a modified mRNA (mmRNA) comprising at least one modified nucleotide.

[0259] Nucleoside triphosphates (NTPs) may comprise 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 comprise unmodified ATP. In some embodiments, the NTPs of the IVT reaction comprise modified ATP. In some embodiments, the NTPs of the IVT reaction comprise unmodified UTP. In some embodiments, the NTPs of the IVT reaction comprise modified UTP. In some embodiments, the NTPs of the IVT reaction comprise unmodified GTP. In some embodiments, the NTPs of the IVT reaction comprise modified GTP. In some embodiments, the NTPs of the IVT reaction comprise unmodified CTP. In some embodiments, the NTPs of the IVT reaction comprise modified CTP.

[0260] The composition of the NTPs in the IVT reaction can also vary. In some embodiments, each NTP in the IVT reaction is present in equimolar amounts. In some embodiments, each NTP in the IVT reaction is present in non-equimolar amounts. For example, ATP in excess of GTP, CTP, and UTP can be used. As a non-limiting example, an IVT reaction can include 7.5 millimoles of GTP, 7.5 millimoles of CTP, 7.5 millimoles of UTP, and 3.75 millimoles of ATP. In some embodiments, the molar ratio of G:C:U:A is 2:1:0.5:1. In some embodiments, the molar ratio of G:C:U:A is 1:1:0.7:1. In some embodiments, the molar ratio of G:C:A:U is 1:1:1:1. The same IVT reaction can include 3.75 millimoles of a cap analog (e.g., a trinucleotide cap or a tetranucleotide 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. In some embodiments, the amount of NTPs in a co-IVT reaction is calculated empirically. For example, the consumption rate of each NTP in an IVT reaction can be empirically determined for each individual input DNA, and then a balanced ratio of NTPs based on those individual NTP consumption rates can be added to a co-IVT comprising multiple input DNAs.

[0261] In some embodiments, the IVT reaction mixture further comprises a cap analog. The concentrations of nucleoside triphosphates and cap analogs present in the IVT reaction may vary. In some embodiments, the NTPs and cap analogs are present in the reaction at equimolar concentrations. In some embodiments, the molar ratio of the cap analog (e.g., a trinucleotide cap or a tetranucleotide cap) to the nucleoside triphosphate in the reaction is greater than 1:1. For example, the molar ratio of the cap analog to the 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 the cap analog (e.g., a trinucleotide cap or a tetranucleotide cap) to the nucleoside triphosphate in the reaction is less than 1:1. For example, the molar ratio of cap analog (e.g., trinucleotide cap or tetranucleotide 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.

[0262] In some embodiments, the RNA transcript (e.g., mRNA transcript) comprises a modified nucleobase selected from the group consisting of pseudouridine (ψ), 1-methylpseudouridine (m 1 ψ), 5-methoxyuridine (mo 5 U), 5-methylcytidine (m 5 C), α-thio-guanosine and α-thio-adenosine. In some embodiments, the RNA transcript (eg, mRNA transcript) includes a combination of at least two (eg, 2, 3, 4 or more) of the aforementioned modified nucleobases.

[0263] In some embodiments, the RNA transcript (e.g., mRNA transcript) includes pseudouridine (ψ). In some embodiments, the RNA transcript (e.g., mRNA transcript) includes 1-methylpseudouridine (m 1 In some embodiments, the RNA transcript (e.g., mRNA transcript) includes 5-methoxyuridine (m 5 In some embodiments, the RNA transcript (e.g., mRNA transcript) includes 5-methylcytidine (m 5 C). In some embodiments, the RNA transcript (eg, mRNA transcript) includes α-thio-guanosine. In some embodiments, the RNA transcript (eg, mRNA transcript) includes α-thio-adenosine.

[0264] In some embodiments, polynucleotides (e.g., RNA polynucleotides, such as mRNA polynucleotides) are uniformly modified (e.g., fully modified, modified throughout the entire sequence) for a specific modification. For example, polynucleotides can be uniformly modified with 1-methylpseudouridine (m 1 ψ) modification, meaning that all uridine residues in the mRNA sequence are replaced by 1-methylpseudouridine (m 1 ψ) replacement.Similarly, polynucleotides can be uniformly modified for any type of nucleoside residues present in the sequence by replacing with modified residues (such as any one of those residues set forth above).Alternately, polynucleotides (e.g., RNA polynucleotides, such as mRNA polynucleotides) may not be uniformly modified (e.g., partially modified, a part of the sequence is modified).Every possibility represents a separate embodiment.

[0265] The buffer system of the IVT reaction mixture can vary. In some embodiments, the buffer system contains tris. For example, the concentration of tris used in the IVT reaction can be 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.

[0266] In some embodiments, the buffer system contains dithiothreitol (DTT). For example, the concentration of DTT used in the IVT reaction can be 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.

[0267] In some embodiments, the buffer system contains magnesium. In some embodiments, the NTPs present in the IVT reaction are combined with magnesium ions (Mg 2+ For example, the molar ratio of NTP to magnesium ion can be 1:0.25, 1:0.5, 1:1, 1:2, 1:3, 1:4 or 1:5.

[0268] In some embodiments, the NTPs present in the IVT reaction plus a cap analog (e.g., a trinucleotide cap such as GAG) are combined with a magnesium ion (Mg 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.

[0269] In some embodiments, the buffer system contains Tris-HCl, spermidine (e.g., at a concentration of 1-30 mM), X-100 (polyethylene glycol p-(1,1,3,3-tetramethylbutyl)-phenyl ether) and / or polyethylene glycol (PEG).

[0270] In some embodiments, the IVT method further comprises a step of separating (e.g., purifying) the in vitro transcription product (e.g., mRNA) from other reaction components. In some embodiments, the separation comprises chromatography of the IVT reaction mixture. In some embodiments, the chromatography comprises size-based (e.g., length-based) chromatography. In some embodiments, the chromatography comprises oligo dT chromatography.

[0271] The addition of nucleoside triphosphates (NTPs) to the 3' end of the growing RNA chain is catalyzed by a polymerase, such as T7 RNA polymerase, e.g., a T7 RNA polymerase variant (e.g., an RNA polymerase comprising D653W / E350W / D351V substitutions). 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.

[0272] Surprisingly, T7 RNA polymerase variants (e.g., RNA polymerases comprising D653W / E350W / D351V substitutions) and cap analogs (e.g., GpppA 2'Ome pG), for example, results in the production of RNA transcripts wherein greater than 80% of the RNA transcripts produced comprise a functional cap. In some embodiments, greater than 85% of the RNA transcripts produced comprise a functional cap. In some embodiments, greater than 90% of the RNA transcripts produced comprise a functional cap. In some embodiments, greater than 95% of the RNA transcripts produced comprise a functional cap. In some embodiments, greater than 96% of the RNA transcripts produced comprise a functional cap. In some embodiments, greater than 97% of the RNA transcripts produced comprise a functional cap. In some embodiments, greater than 98% of the RNA transcripts produced comprise a functional cap. In some embodiments, greater than 99% of the RNA transcripts produced comprise a functional cap.

[0273] It was also surprising to find that the use of a polynucleotide template comprising a 2'-deoxythymidine residue or a 2'-deoxycytidine residue at template position +1 resulted in the production of RNA transcripts, wherein greater than 80% (e.g., greater than 85%, greater than 90%, or greater than 95%) of the RNA transcripts produced comprise a functional cap. Thus, in some embodiments, the polynucleotide (e.g., DNA) template used, e.g., in an IVT reaction, comprises a 2'-deoxythymidine residue at template position +1. In other embodiments, the polynucleotide (e.g., DNA) template used, e.g., in an IVT reaction, comprises a 2'-deoxycytidine residue at template position +1.

[0274] application

[0275] RNA transcripts produced using RNA polymerase variants may include mRNA (including modified mRNA and / or unmodified RNA), lncRNA, self-replicating RNA, circular RNA, CRISPR guide RNA, etc. In an embodiment, the RNA is an RNA (e.g., mRNA or self-replicating RNA) encoding a polypeptide (e.g., a therapeutic polypeptide). Therefore, RNA transcripts produced using RNA polymerase variants can be used in countless applications.

[0276] For example, RNA transcripts can be used to produce polypeptides of interest, such as therapeutic proteins, vaccine antigens, and the like. In some embodiments, RNA transcripts are therapeutic RNAs. Therapeutic mRNAs are mRNAs encoding therapeutic proteins (the term 'protein' encompasses peptides). Therapeutic proteins mediate various effects in host cells or subjects to treat diseases or improve the signs and symptoms of diseases. For example, therapeutic proteins can replace defective or abnormal proteins, enhance the function of endogenous proteins, provide cells with new functions (e.g., inhibit or activate endogenous cell activity, or act as a delivery agent for another therapeutic compound (e.g., antibody-drug conjugates). Therapeutic mRNAs can be used for the treatment of the following diseases and conditions: bacterial infections, viral infections, parasitic infections, cell proliferation disorders, genetic disorders, and autoimmune disorders. Other diseases and conditions are covered herein.

[0277] The RNA transcripts produced using RNA polymerase variants can encode one or more biologics. Biologics are polypeptide-based molecules that can be used to treat, cure, alleviate, prevent or diagnose serious or life-threatening diseases or medical conditions. Biologics include, but are not limited to, allergy extracts (e.g., for allergy injections and testing), blood components, gene therapy products, human tissue or cell products for transplantation, vaccines, monoclonal antibodies, cytokines, growth factors, enzymes, thrombolytics, and immunomodulators, etc.

[0278] One or more biologics currently on sale or in development may be encoded by RNA produced by RNA polymerase variants. Although not wishing to be bound by theory, it is believed that incorporating the encoding polynucleotides of known biologics into RNA will result in improved therapeutic efficacy, at least in part due to the specificity, purity and / or selectivity of the construct design.

[0279] The RNA transcripts produced using RNA polymerase variants can encode one or more antibodies. The term "antibody" includes monoclonal antibodies (including full-length antibodies with immunoglobulin Fc regions), antibody compositions with multiple epitope specificities, multispecific antibodies (e.g., bispecific antibodies, double antibodies, and single-chain molecules) and antibody fragments. The term "immunoglobulin" (Ig) is used interchangeably with "antibody" in this article. Monoclonal antibodies are antibodies obtained from a population of substantially homogeneous antibodies, that is, the single antibodies comprising the population are identical, except for possible naturally occurring mutations and / or post-translational modifications (e.g., isomerization, amidation) that may exist in trace amounts. Monoclonal antibodies have a high degree of specificity (for a single antigenic site).

[0280] Monoclonal antibodies specifically include chimeric antibodies (immunoglobulins), and fragments of such antibodies, in which a portion of the heavy chain and / or light chain is identical or homologous to a corresponding sequence in an antibody derived from a particular species or belonging to a particular antibody class or subclass, and the remainder of the chain is identical or homologous to a corresponding sequence in an antibody derived from another species or belonging to another antibody class or subclass, as long as it exhibits the desired biological activity. Chimeric antibodies include, but are not limited to, "primatized" antibodies, which contain variable domain antigen-binding sequences derived from non-human primates (e.g., Old World Monkey, ape, etc.) and human constant region sequences.

[0281] RNA transcripts produced using RNA polymerase variants can encode one or more vaccine antigens. Vaccine antigens are biological preparations that improve immunity to specific diseases or infectious agents. One or more vaccine antigens currently on sale or in development can be encoded by RNA. Vaccine antigens encoded in RNA can be used to treat conditions or diseases in many therapeutic areas, such as but not limited to cancer, allergies and infectious diseases. In some embodiments, cancer vaccines can be personalized cancer vaccines in the form of concatemers or single RNAs encoding peptide epitopes or combinations thereof.

[0282] RNA transcripts produced using RNA polymerase variants 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 variety of animals, such as, but not limited to, microorganisms, invertebrates, plants, amphibians, birds, fish, and mammals.

[0283] In some embodiments, RNA transcripts are used as radiolabeled RNA probes. In some embodiments, RNA transcripts are used for non-isotopic RNA labeling. In some embodiments, RNA transcripts are used as guide RNA (gRNA) for gene targeting. In some embodiments, RNA transcripts (e.g., mRNA) are used for in vitro translation and microinjection. In some embodiments, RNA transcripts are used for RNA structure, processing and catalysis research. In some embodiments, RNA transcripts are used for RNA amplification. In some embodiments, RNA transcripts are used as antisense RNA for gene expression experiments.

[0284] Wild-type T7 RNA polymerase

[0285] MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHESYEMGEARFRKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTAFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAKHFKKNVEEQLNKRVGHV YKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIATRAGALAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPFCFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNFVHSQDGSHLRKTVVWAHEKYGIESFALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAKGNLNLRDILESDFAFA(SEQ ID NO:1)

[0286] Additional embodiments

[0287] The additional embodiments are covered by the numbered paragraphs 1 - 71 below:

[0288] 1. A ribonucleic acid (RNA) polymerase variant, comprising: an amino acid sequence, relative to a wild-type T7 RNA polymerase comprising the amino acid sequence of SEQ ID NO: 1, comprising (i) an amino acid substitution at position E350, (ii) an amino acid substitution at position D351, and (iii) an amino acid substitution at position K387, position N437, or at position K387 and position N437.

[0289] 2. The RNA polymerase variant of paragraph 1, wherein the amino acid sequence of the variant comprises an amino acid substitution at position K387.

[0290] 3. The RNA polymerase variant of paragraph 1, wherein the amino acid sequence of the variant comprises an amino acid substitution at position N437.

[0291] 4. The RNA polymerase variant of paragraph 1, wherein the amino acid sequence of the variant comprises amino acid substitutions at position K387 and position N437.

[0292] 5. The RNA polymerase variant of any one of paragraphs 1-4, wherein the amino acid substitution at position K387 is a polar neutral amino acid.

[0293] 6. The RNA polymerase variant of paragraph 5, wherein the polar neutral amino acid is selected from asparagine (N), cysteine ​​(C), glutamine (Q), methionine (M), serine (S) and threonine (T).

[0294] 7. The RNA polymerase variant of paragraph 6, wherein the polar neutral amino acid is asparagine (K387N).

[0295] 8. The RNA polymerase variant of paragraph 6, wherein the polar neutral amino acid is cysteine ​​(K387C).

[0296] 9. The RNA polymerase variant of paragraph 6, wherein the polar neutral amino acid is glutamine (K387Q).

[0297] 10. The RNA polymerase variant of paragraph 6, wherein the polar neutral amino acid is methionine (K387M).

[0298] 11. The RNA polymerase variant of paragraph 6, wherein the polar neutral amino acid is serine (K387S).

[0299] 12. The RNA polymerase variant of paragraph 6, wherein the polar neutral amino acid is threonine (K387T).

[0300] 13. The RNA polymerase variant of any of paragraphs 1-12, wherein the amino acid substitution at position N437 is an aromatic amino acid.

[0301] 14. The RNA polymerase variant of paragraph 13, wherein the aromatic amino acid is selected from tryptophan (W), tyrosine (Y) and phenylalanine (F).

[0302] 15. The RNA polymerase variant of paragraph 14, wherein the aromatic amino acid is tryptophan (N437W).

[0303] 16. The RNA polymerase variant of paragraph 14, wherein the aromatic amino acid is tyrosine (N437Y).

[0304] 17. The RNA polymerase variant of paragraph 14, wherein the aromatic amino acid is phenylalanine (N437F).

[0305] 18. A ribonucleic acid (RNA) polymerase variant, comprising an amino acid sequence, relative to a wild-type T7 RNA polymerase comprising the amino acid sequence of SEQ ID NO 1, comprising (i) an amino acid substitution at position E350, (ii) an amino acid substitution at D351, and (iii) an amino acid substitution at position D653.

[0306] 19. The RNA polymerase variant of paragraph 18, wherein the amino acid substitution at position D653 is an aromatic amino acid.

[0307] 20. The RNA polymerase variant of paragraph 19, wherein the aromatic amino acid is selected from tryptophan (W), tyrosine (Y) and phenylalanine (F).

[0308] 21. The RNA polymerase variant of paragraph 20, wherein the aromatic amino acid is tryptophan (D653W).

[0309] 22. The RNA polymerase variant of paragraph 20, wherein the aromatic amino acid is tyrosine (D653Y).

[0310] 23. The RNA polymerase variant of paragraph 20, wherein the aromatic amino acid is phenylalanine (D653F).

[0311] 24. The RNA polymerase variant of any of paragraphs 1-23, wherein the amino acid substitution at position E350 is an aromatic amino acid.

[0312] 25. The RNA polymerase variant of paragraph 24, wherein the aromatic amino acid is selected from tryptophan (W), tyrosine (Y) and phenylalanine (F).

[0313] 26. The RNA polymerase variant of paragraph 25, wherein the aromatic amino acid is tryptophan (E350W).

[0314] 27. The RNA polymerase variant of paragraph 25, wherein the aromatic amino acid is tyrosine (E350Y).

[0315] 28. The RNA polymerase variant of paragraph 25, wherein the aromatic amino acid is phenylalanine (E350F).

[0316] 29. The RNA polymerase variant of paragraphs 1-28, wherein the amino acid substitution at position D351 is a non-polar aliphatic amino acid.

[0317] 30. The RNA polymerase variant of paragraph 29, wherein the non-polar aliphatic amino acid is selected from alanine (A), glycine (G), isoleucine (I), leucine (L), proline (P) and valine (V).

[0318] 31. The RNA polymerase variant of paragraph 30, wherein the non-polar aliphatic amino acid is alanine (D351A).

[0319] 32. The RNA polymerase variant of paragraph 30, wherein the non-polar aliphatic amino acid is glycine (D351G).

[0320] 33. The RNA polymerase variant of paragraph 30, wherein the non-polar aliphatic amino acid is isoleucine (D351I).

[0321] 34. The RNA polymerase variant of paragraph 30, wherein the non-polar aliphatic amino acid is leucine (D351L).

[0322] 35. The RNA polymerase variant of paragraph 30, wherein the non-polar aliphatic amino acid is proline (D351P).

[0323] 36. The RNA polymerase variant of paragraph 30, wherein the non-polar aliphatic amino acid is valine (D351V).

[0324] 37. An RNA polymerase variant, comprising: an amino acid sequence having at least 70% identity with the amino acid sequence of SEQ ID NO: 1, wherein relative to the wild-type T7 RNA polymerase comprising the amino acid sequence of SEQ ID NO: 1, the amino acid sequence of the variant comprises (i) an amino acid substitution at position E350, (ii) an amino acid substitution at D351, and (iii) amino acid substitutions at position K387, position N437, or position K387 and position N437.

[0325] 38. The RNA polymerase variant of paragraph 37, wherein the amino acid sequence is at least 75%, at least 80%, at least 85%, at least 95% or at least 98% identical to the amino acid sequence of SEQ ID NO: 1.

[0326] 39. The RNA polymerase variant of paragraph 37 or 38, wherein the amino acid sequence of the variant comprises an amino acid substitution at position K387.

[0327] 40. The RNA polymerase variant of paragraph 37 or 38, wherein the amino acid sequence of the variant comprises an amino acid substitution at position N437.

[0328] 41. The RNA polymerase variant of paragraph 37 or 38, wherein the amino acid sequence of the variant comprises an amino acid substitution at position K387 and at position N437.

[0329] 42. The RNA polymerase variant of any of paragraphs 37-41, wherein the amino acid substitution at position K387 is a polar neutral amino acid.

[0330] 43. The RNA polymerase variant of paragraph 42, wherein the polar neutral amino acid is selected from asparagine (K387N), cysteine ​​(K387C), glutamine (K387Q), methionine (K387M), serine (K387S) and threonine (K387T).

[0331] 44. The RNA polymerase variant of any of paragraphs 37-42, wherein the amino acid substitution at position N437 is an aromatic amino acid.

[0332] 45. The RNA polymerase variant of paragraph 44, wherein the aromatic amino acid is selected from tryptophan (N437W), tyrosine (N437Y) and phenylalanine (N437F).

[0333] 46. ​​An RNA polymerase variant, comprising: an amino acid sequence having at least 70% identity with the amino acid sequence of SEQ ID NO: 1, wherein relative to the wild-type T7 RNA polymerase comprising the amino acid sequence of SEQ ID NO: 1, the amino acid sequence of the variant comprises (i) an amino acid substitution at position E350, (ii) an amino acid substitution at D351, and (iii) an amino acid substitution at position D653.

[0334] 47. The RNA polymerase variant of paragraph 46, wherein the amino acid sequence is at least 75%, at least 80%, at least 85%, at least 95% or at least 98% identical to the amino acid sequence of SEQ ID NO: 1.

[0335] 48. The RNA polymerase variant of paragraph 46 or 47, wherein the amino acid substitution at position D653 is an aromatic amino acid.

[0336] 49. The RNA polymerase variant of paragraph 48, wherein the aromatic amino acid is selected from tryptophan (D653W), tyrosine (D653Y) and phenylalanine (D653F).

[0337] 50. The RNA polymerase variant of any of paragraphs 37-49, wherein the amino acid substitution at position E350 is an aromatic amino acid.

[0338] 51. The RNA polymerase variant of paragraph 50, wherein the aromatic amino acid is selected from tryptophan (E350W), tyrosine (E350Y) and phenylalanine (E350F).

[0339] 52. The RNA polymerase variant of any of paragraphs 37-51, wherein the amino acid substitution at position D351 is a non-polar aliphatic amino acid.

[0340] 53. The RNA polymerase variant of paragraph 52, wherein the non-polar aliphatic amino acid is selected from alanine (D351A), glycine (D351G), isoleucine (D351I), leucine (D351L), proline (D351P) and valine (D351V).

[0341] 54. A ribonucleic acid (RNA) polymerase variant, the RNA polymerase variant comprising the amino acid sequence of SEQ ID NO: 2, wherein X 1 is an aromatic amino acid, optionally selected from W, Y, and F; X 2 is selected from non-polar aliphatic amino acids, optionally selected from A, G, I, L, P and V; X 3is a polar neutral amino acid, optionally selected from N, C, Q, M, S and T; and X 4 is an aromatic amino acid, optionally selected from W, Y and F.

[0342] 55. A ribonucleic acid (RNA) polymerase variant, comprising the amino acid sequence of SEQ ID NO: 6.

[0343] 56. A ribonucleic acid (RNA) polymerase variant, the RNA polymerase variant comprising the amino acid sequence of SEQ ID NO: 3, wherein X 1 is an aromatic amino acid, optionally selected from W, Y, and F; X 2 is selected from non-polar aliphatic amino acids, optionally selected from A, G, I, L, P and V; and X 4 is an aromatic amino acid, optionally selected from W, Y and F.

[0344] 57. A ribonucleic acid (RNA) polymerase variant, comprising the amino acid sequence of SEQ ID NO:7.

[0345] 58. A ribonucleic acid (RNA) polymerase variant, the RNA polymerase variant comprising the amino acid sequence of SEQ ID NO: 4, wherein X 1 is an aromatic amino acid, optionally selected from W, Y, and F; X 2 is selected from non-polar aliphatic amino acids, optionally selected from A, G, I, L, P and V; and X 3 is a polar neutral amino acid, optionally selected from N, C, Q, M, S and T.

[0346] 59. A ribonucleic acid (RNA) polymerase variant, comprising the amino acid sequence of SEQ ID NO: 8.

[0347] 60. A ribonucleic acid (RNA) polymerase variant, the RNA polymerase variant comprising the amino acid sequence of SEQ ID NO: 5, wherein X 1 is an aromatic amino acid, optionally selected from W, Y, and F; X 2 is selected from non-polar aliphatic amino acids, optionally selected from A, G, I, L, P and V; and X 5 is an aromatic amino acid, optionally selected from W, Y and F.

[0348] 61. A ribonucleic acid (RNA) polymerase variant, comprising the amino acid sequence of SEQ ID NO:9.

[0349] 62. A method comprising: producing messenger RNA (mRNA) in an in vitro transcription reaction comprising DNA, nucleoside triphosphates, the RNA polymerase variant of any of paragraphs 1-53, and optionally a cap analog.

[0350] 63. The method of paragraph 62, wherein the reaction includes a cap analog.

[0351] 64. The method of paragraph 62 or 63, wherein the cap analog is a dinucleotide cap analog, a trinucleotide cap analog, or a tetranucleotide cap analog.

[0352] 65. The method of paragraph 64, wherein the cap analog is a trinucleotide cap analog comprising a GAG sequence.

[0353] 66. The method of paragraph 65, wherein the GAG ​​cap analog comprises a compound selected from the group consisting of:

[0354]

[0355] 67. The method of paragraph 64, wherein the tetranucleotide cap analog comprises a GGAG sequence.

[0356] 68. The method of paragraph 67, wherein the tetranucleotide cap analog comprises a compound selected from the group consisting of:

[0357]

[0358]

[0359] 69. The method of any of paragraphs 62-68, wherein the DNA comprises a 2'-deoxythymidine residue or a 2'-deoxycytidine residue at position +1.

[0360] 70. A composition or kit comprising the RNA polymerase variant of any of paragraphs 1-61 and an in vitro transcription (IVT) reagent selected from the group consisting of DNA, nucleoside triphosphates, and a cap analog.

[0361] 71. A nucleic acid encoding the RNA polymerase variant of any of paragraphs 1-61.

[0362] Example

[0363] Example 1. IVT reactions using RNA polymerase variants

[0364] In vitro transcription (IVT) reactions were performed using DNA templates, GGAG cap analogs, and selected individual RNA polymerase variants as provided in Table 1. Specifically, RNA polymerase variants comprising N437Y, K387S, E350W, and D351V substitutions (SEQ ID NO: 6; "Variant A"); N437Y, E350W, and D351V substitutions (SEQ ID NO: 7; "Variant B"); K387S, E350W, and D351V substitutions (SEQ ID NO: 8; "Variant C"); and D653W, E350W, and D351V substitutions (SEQ ID NO: 9; "Variant D") were tested in this example. Reactions were also performed using a control T7 RNA polymerase (SEQ ID NO: 1). Following the IVT reaction, the transcribed RNA product from each reaction was characterized to address the quality of the RNA product, including total RNA yield, capping efficiency (percent of total RNA containing the GGAG cap), dsRNA contamination, and tail purity.

[0365] After oligo dT purification, the total yield of total RNA was measured by UV absorption. The total RNA product was analyzed by LC-MS to determine the capping efficiency (i.e., the percentage of transcribed RNA containing a GGAG cap). In this example, a standard ELISA was used after the IVT reaction to assess dsRNA contaminants (e.g., dsRNA longer than 40 nucleotide base pairs). The Tris RP (reverse phase) method was used to assess the percentage of tailed RNA (i.e., the percentage of transcribed RNA containing a poly A tail).

[0366] Each RNA polymerase variant tested produced RNA with at least 80% capped RNA (percentage of total RNA containing a GGAG cap) and at least about 80% tailed RNA (i.e., the percentage of transcribed RNA containing a poly A tail) in an IVT reaction. RNA polymerase variants comprising N437Y, E350W, and D351V substitutions (SEQ ID NO: 7); and K387S, E350W, and D351V substitutions (SEQ ID NO: 8) produced less than 0.007% dsRNA (weight: weight). In addition, the total RNA yield of each RNA polymerase variant tested (greater than 8 mg / mL) was comparable to that of the control T7 RNA polymerase.

[0367] In each test characteristic, each tested RNA polymerase variant performs comparable or better than the control T7 RNA polymerase. Specifically, relative to the control T7 RNA polymerase, N437Y+K387S+E350W+D351V provides RNA with higher capping efficiency (about 85% capped RNA), similar yield and similar tailing purity. Relative to the control T7 RNA polymerase, N437Y+E350W+D351V provides RNA with higher capping efficiency (about 80% capped RNA), similar yield, similar tailing purity and similar dsRNA pollution. Relative to the control T7 RNA polymerase, K387S+E350W+D351V provides RNA with higher capping efficiency (about 83% capped RNA), similar yield, higher tailing purity (about 85% tailing RNA) and less dsRNA pollution (0.00327dsRNA weight: weight). Relative to the control T7 RNA polymerase, D653W+E350W+D351V provided RNA with higher capping efficiency (approximately 95% capped RNA).

[0368] Data for each RNA polymerase tested are provided in Tables 3 and Figures 1A-1D middle.

[0369] Table 3. Properties of RNA produced by RNA polymerase used in Example 1

[0370]

[0371] Example 2. RNA polymerase variants produce RNA products with high levels of capping efficiency at low concentrations of GGAG cap analogs

[0372] In vitro transcription reaction was performed using DNA template, equimolar NTP, variable amount of GGAG tetranucleotide cap analog (0.25mM, 0.5mM, 0.75mM, 1mM, 1.25mM, 1.5mM, 3mM) and T7 RNA polymerase. In this example, RNA polymerase variants comprising N437Y, K387S, E350W and D351V substitutions (SEQ ID NO: 6); N437Y, E350W and D351V substitutions (SEQ ID NO: 7); K387S, E350W and D351V substitutions (SEQ ID NO: 8); and D653W, E350W and D351V substitutions (SEQ ID NO: 9) were tested. Control T7 RNA polymerase was also used for reaction.

[0373] Following the IVT reaction, the mRNA product was oligo-dT purified and then analyzed by LC-MS to determine the % capped RNA (i.e., the percentage of transcribed RNA that contained a cap), by HPLC to determine the RNA yield of the reaction, and by the Tris RP (reverse phase) method to determine the percentage of tailed RNA.

[0374] In the presence of the GGAG cap analog, each of the tested RNA polymerase variants produced RNA with a higher percentage of capped RNA than the control polymerase variant, regardless of the concentration of the GGAG analog ( Figure 2A ). Even at the lowest tested concentration of GGAG cap analog (0.25 mM), all tested variants produced at least 50% capped RNA, much higher than the approximately 25% capped RNA produced by the control polymerase variant. At 1.5 mM GGAG cap analog, all tested variants produced approximately 80-95% capped RNA.

[0375] Each tested variant produced comparable yields relative to the control polymerase variant ( Figure 2B ) and percentage of tailed RNA ( Figure 2C )'s RNA.

[0376] These data demonstrate that each RNA polymerase variant tested (i.e., N437Y+K387S+E350W+D351V; N437Y+E350W+D351V; K387S+E350W+D351V; and D653W+E350W+D351V) is able to produce RNA with higher capping efficiency than the control T7 RNA polymerase without giving up any yield or tailing content.

[0377] Equivalents and scope

[0378] All references, patents, and patent applications disclosed herein are incorporated by reference, in some cases in their entirety, for the subject matter for which they are cited.

[0379] The indefinite articles "a" and "an" as used in the specification and claims, unless expressly indicated to the contrary, should be understood to mean "at least one".

[0380] It should also be understood that in any method claimed herein that includes more than one step or action, the order of the steps or actions of the method is not necessarily limited to the order in which the steps or actions of the method are recited unless explicitly stated to the contrary.

[0381] In the claims and the foregoing specification, all transitional phrases such as "comprising," "including," "carrying," "having," "containing," "involving," "having," "consisting of," etc. shall be understood as open-ended, i.e., meaning including but not limited to. Only the transitional phrases "consisting of" and "consisting essentially of" shall be closed or semi-closed transitional phrases, respectively, as described in Section 2111.03 of the U.S. Patent Office Manual of Patent Examining Procedures.

Claims

1. A ribonucleic acid (RNA) polymerase variant, comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 2-9, wherein the amino acid sequence comprises an amino acid substitution at position D351 and at least two additional amino acid substitutions relative to the RNA polymerase comprising the amino acid sequence of SEQ ID NO:

1.

2. The RNA polymerase variant of claim 1, comprising an amino acid sequence having at least 95% sequence identity with the amino acid sequence of any one of SEQ ID NOs: 2-9.

3. The RNA polymerase variant of claim 1 or 2, comprising an amino acid sequence comprising the amino acid sequence of any one of SEQ ID NOs: 2-9.

4. The RNA polymerase variant of any one of claims 1 to 3, comprising at least one, at least two, at least three or at least four amino acid substitutions relative to the wild-type T7 RNA polymerase comprising the amino acid sequence of SEQ ID NO:

1.

5. A ribonucleic acid (RNA) polymerase variant, comprising the amino acid sequence of SEQ ID NO:

6.

6. A ribonucleic acid (RNA) polymerase variant, comprising the amino acid sequence of SEQ ID NO:

7.

7. A ribonucleic acid (RNA) polymerase variant, comprising the amino acid sequence of SEQ ID NO:

8.

8. A ribonucleic acid (RNA) polymerase variant, comprising the amino acid sequence of SEQ ID NO:

9.

9. A method comprising: Messenger RNA (mRNA) is produced in an in vitro transcription reaction comprising DNA, nucleoside triphosphates, and the RNA polymerase variant of any one of claims 1-8.

10. The method of claim 9, wherein the reaction further comprises a cap analog.

11. The method of claim 10, wherein the cap analog is a dinucleotide cap analog, a trinucleotide cap analog, or a tetranucleotide cap analog.

12. The method of claim 11, wherein the cap analog is a trinucleotide cap analog comprising a GAG sequence.

13. The method of claim 11, wherein the cap analog comprises a compound selected from the group consisting of:

14. The method of claim 11, wherein the tetranucleotide cap analog comprises a GGAG sequence.

15. The method of claim 11, wherein the tetranucleotide cap analog comprises a compound selected from the group consisting of:

16. The method of any one of claims 9-15, wherein the DNA comprises a 2'-deoxythymidine residue or a 2'-deoxycytidine residue at position +1.

17. A composition or kit comprising the RNA polymerase variant of any one of claims 1 to 9 and an in vitro transcription (IVT) reagent selected from the group consisting of DNA, nucleoside triphosphates and cap analogs.

18. A nucleic acid encoding the RNA polymerase variant according to any one of claims 1 to 9.

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