WO2024097739A2

WO2024097739A2 - Engineered vaccinia capping enzyme variants

Info

Publication number: WO2024097739A2
Application number: PCT/US2023/078328
Authority: WO
Inventors: Marina Mikhailovna BAKHTINA; Margie Tabuga BORRA-GARSKE; Jason FELL; Stephan JENNE; Kalyanaraman KRISHNAMOORTHY; Nikki D. KRUSE; Melissa Ann MAYO; Jovana Nazor; Aksiniya Lyubenova PETKOVA; Marcus ROHOVIE
Original assignee: Codexis, Inc.
Priority date: 2022-11-03
Filing date: 2023-11-01
Publication date: 2024-05-10

Abstract

The present invention provides engineered vaccina capping enzyme and vaccinia virus capping enzyme polypeptides and compositions thereof, as well as polynucleotides encoding the engineered vaccinia capping enzyme and vaccinia virus capping enzyme subunit polypeptides. The disclosure also provides methods for use of the engineered vaccina capping enzymes and vaccinia virus capping enzyme subunits, as well as compositions thereof for diagnostic, molecular biological tools, and other purposes.

Description

ENGINEERED VACCINIA CAPPING ENZYME VARIANTS

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Patent Application Serial No. 63/382,135, filed November 3, 2022, which is hereby incorporated by reference herein in its entirety.

REFERENCE TO SEQUENCE LISTING, TABLE OR COMPUTER PROGRAM

[0002] The Sequence Listing concurrently submitted herewith as file name CX9-237WOl_ST26.xml is herein incorporated by reference. The electronic copy of the Sequence Listing was created on October 31, 2023, with a file size of 2,192,915 bytes.

FIELD OF THE INVENTION

[0003] The present invention provides engineered vaccina capping enzyme and vaccinia virus capping enzyme polypeptides and compositions thereof, as well as polynucleotides encoding the engineered vaccinia capping enzyme and vaccinia virus capping enzyme subunit polypeptides. The disclosure also provides methods for use of the engineered vaccina capping enzymes and vaccinia virus capping enzyme subunits, as well as compositions thereof for diagnostic, molecular biological tools, and other purposes.

BACKGROUND

[0004] Eukaryotic and prokaryotic cells contain multiple types of RNA, at least some of which can be characterized by the different chemical constituents present at their 5’ ends. The eukaryotic mRNA 5’ cap structure is necessary for pre-mRNA processing, as well as mRNA export, translation initiation, and stability. Thus, the 5’ guanine-N7-methyl cap structure is a central feature of eukaryotic mRNA (See e.g., Fuchs et al., RNA 22: 1-13 [2016]).

SUMMARY OF THE INVENTION

[0005] The present invention provides engineered vaccina capping enzyme and vaccinia virus capping enzyme polypeptides and compositions thereof, as well as polynucleotides encoding the engineered vaccinia capping enzyme and vaccinia virus capping enzyme subunit polypeptides. The disclosure also provides methods for use of the engineered vaccina capping enzymes and vaccinia virus capping enzyme subunits, as well as compositions thereof for diagnostic, molecular biological tools, and other purposes.

[0006] The present invention provides engineered vaccinia virus capping enzymes, vaccinia virus capping enzyme subunits, and/or functional fragments thereof, comprising a polynucleotide sequence having at least 75% or more sequence identity to a reference sequence comprising SEQ ID NO: 1 or to a reference sequence of SEQ ID NO: 1, wherein the polynucleotide sequence comprises one or more substitutions relative to the reference sequence comprising SEQ ID NO: 1, or to the reference sequence of SEQ ID NO: 1.

[0007] The present invention further provides engineered vaccina virus capping enzymes and/or vaccinia virus capping enzyme subunits comprising polypeptide sequences having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence comprising residues 12 to 855 of SEQ ID NO: 3, or to the reference sequence of SEQ ID NO: 3.

[0008] In some embodiments, the engineered vaccinia virus capping enzyme and/or vaccinia virus capping enzyme subunit comprises a polypeptide sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence comprising residues 12 to 855 of SEQ ID NO: 14, 105, 166, 242, 288, 318, 371, and/or 373, or to a reference sequence of SEQ ID NO: 14, 105, 166, 242, 288, 318, 371, and/or 373.

[0009] In some embodiments, the present invention provides engineered vaccinia virus capping enzymes and/or vaccinia virus capping enzyme subunits comprising one or more substitutions or substitution sets at amino acid positions selected from 209/212/259/431, 221/304/347/533/550/745/792, 304/347/350/459/787/792, and 831, and any combinations thereof, wherein the amino acid positions are numbered with reference to SEQ ID NO: 3. In some further embodiments, the present invention provides engineered vaccinia virus capping enzymes and/or vaccinia virus capping enzyme subunits comprising one or more substitutions or substitution sets at amino acid positions selected from 42, 44, 97, 184, 210, 211, 332, 355, 368, 433, 450, 542, 546, 654/656, 679, 680, and/or 702, and any combinations thereof, wherein the amino acid positions are numbered with reference to SEQ ID NO: 3.

[0010] In some additional embodiments, the present invention provides engineered vaccinia virus capping enzymes and/or vaccinia virus capping enzyme subunits comprising one or more substitutions or substitution sets at amino acid positions selected from 209/212/221/431, 209/212/221/745/831, 209/212/533/831, 212/221/431/533, 212/221/431/533/550/745, 212/221/745/831, 221/259/431/533/745, 221/259/431/550, 221/259/550/831, 221/431/550/745, 221/550, 259/533/550/831, 259/533/745, 259/745/831, 259/831, 431, 533, 533/550/745/831, 533/745, and/or 550/745/831, and any combinations thereof, wherein the amino acid positions are numbered with reference to SEQ ID NO: 14.

[0011] In some further embodiments, the present invention provides engineered vaccinia virus capping enzymes and/or vaccinia virus capping enzyme subunits comprising one or more substitutions or substitution sets at amino acid positions selected from 184/355/368/542/546/654/680, and/or 225, and any combinations thereof, wherein the amino acid positions are numbered with reference to SEQ ID NO: 105. [0012] In still some further embodiments, the present invention provides engineered vaccinia virus capping enzymes and/or vaccinia virus capping enzyme subunits comprising one or more substitutions or substitution sets at amino acid positions selected from 45, 97, 143, 209, 304, 330, 343, 596, 598, 651, and/or 818, and any combinations thereof, wherein the amino acid positions are numbered with reference to SEQ ID NO: 166.

[0013] In some additional embodiments, the present invention provides engineered vaccinia virus capping enzymes and/or vaccinia virus capping enzyme subunits comprising one or more substitutions or substitution sets at amino acid positions selected from 45/97/143/209/304/818, 45/97/143/209/304/818/820, 45/97/143/209/818, 45/97/143/209/818/855, 45/97/143/304/446/732/818/855, 45/97/143/304/818, 45/97/143/304/818/820/855, 45/97/209/446/818/820, 45/97/446/818, 45/143, 45/143/209/304/818/820/855, 45/143/209/818, 45/143/304, 45/143/818, 45/143/818/820/855, and/or 45/143/818/855, and any combinations thereof, wherein the amino acid positions are numbered with reference to SEQ ID NO: 242.

[0014] In some further embodiments, the present invention provides engineered vaccinia virus capping enzymes and/or vaccinia virus capping enzyme subunits comprising one or more substitutions or substitution sets at amino acid positions selected from 43, 421, 458, 458/562, 474, 552, 594, 601, and/or 815, and any combinations thereof, wherein the amino acid positions are numbered with reference to SEQ ID NO: 288.

[0015] In some additional embodiments, the present invention provides engineered vaccinia virus capping enzymes and/or vaccinia virus capping enzyme subunits comprising one or more substitutions or substitution sets at amino acid positions selected from 239, 239/251, 239/251/258, 239/258, 251, 258, and/or 474/552/594/601, and any combinations thereof, wherein the amino acid positions are numbered with reference to SEQ ID NO: 318.

[0016] In some further embodiments, the present invention provides engineered vaccinia virus capping enzymes and/or vaccinia virus capping enzyme subunits comprising one or more substitutions or substitution sets at amino acid positions selected from 81/184, 209/355/542, 211, 228, 236, 249, 362, 431, 446, 474, 596, 602, 642, 654, 657, 679, 732, 818, and/or 820, and any combinations thereof, wherein the amino acid positions are numbered with reference to SEQ ID NO: 371.

[0017] In still some additional embodiments, the present invention provides engineered vaccinia virus capping enzymes and/or vaccinia virus capping enzyme subunits comprising one or more substitutions or substitution sets at amino acid positions selected from 43/97/209/304/548/551/601, 43/209/304/548/551, 43/209/355/542/548/551, 43/542/601, 97/304/542, 97/548, 209/332/355/542, 209/355/542, and/or 542/548/551/601, and any combinations thereof, wherein the amino acid positions are numbered with reference to SEQ ID NO: 373.

[0018] In some additional embodiments, the present invention provides engineered vaccinia virus capping enzymes and/or vaccinia virus capping enzyme subunits comprising a polypeptide sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence comprising residues 12 to 855 of SEQ ID NO: 3, 14, 105, 166, 242, 288, 318, 371, and/or 373, or to a reference sequence of SEQ ID NO: 3, 14, 105, 166, 242, 288, 318, 371, and/or 373, with the proviso that the polypeptide sequence does not include the sequence comprising residues 12 to 855 of SEQ ID NO:3.

[0019] In some further embodiments, the engineered vaccinia virus capping enzyme and/or vaccinia virus capping enzyme subunit polypeptide sequence comprises one or more amino acid substitutions relative to the reference sequence comprising residues 12 to 855 of SEQ ID NO: 14, or to the reference sequence of SEQ ID NO: 14, with the proviso that the polypeptide sequence does not include the sequence comprising residues 12 to 855 of SEQ ID NO: 3.

[0020] In some additional embodiments, the engineered vaccinia virus capping enzyme and/or vaccinia virus capping enzyme subunit polypeptide sequence comprises one or more amino acid substitutions relative to the reference sequence comprising residues 12 to 855 of SEQ ID NO: 105, or to the reference sequence of SEQ ID NO: 105, with the proviso that the polypeptide sequence does not include the sequence comprising residues 12 to 855 of SEQ ID NO: 3.

[0021] In some further embodiments, the engineered vaccinia virus capping enzyme and/or vaccinia virus capping enzyme subunit polypeptide sequence comprises one or more amino acid substitutions relative to the reference sequence comprising residues 12 to 855 of SEQ ID NO: 166, or to the reference sequence of SEQ ID NO: 166, with the proviso that the polypeptide sequence does not include the sequence comprising residues 12 to 855 of SEQ ID NO: 3.

[0022] In some additional embodiments, the vaccinia virus capping enzyme and/or vaccinia virus capping enzyme subunit polypeptide sequence comprises one or more amino acid substitutions relative to the reference sequence comprising residues 12 to 855 of SEQ ID NO: 242, or to the reference sequence of SEQ ID NO: 242, with the proviso that the polypeptide sequence does not include the sequence comprising residues 12 to 855 of SEQ ID NO: 3.

[0023] In some further embodiments, the engineered vaccinia virus capping enzyme and/or vaccinia virus capping enzyme subunit polypeptide sequence comprises one or more amino acid substitutions relative to the reference sequence comprising residues 12 to 855 of SEQ ID NO: 288, or to the reference sequence of SEQ ID NO: 288, with the proviso that the polypeptide sequence does not include the sequence comprising residues 12 to 855 of SEQ ID NO: 3.

[0024] In some additional embodiments, the engineered vaccinia virus capping enzyme and/or vaccinia virus capping enzyme subunit polypeptide sequence comprises one or more amino acid substitutions relative to the reference sequence comprising residues 12 to 855 of SEQ ID NO: 318, or to the reference sequence of SEQ ID NO: 318, with the proviso that the polypeptide sequence does not include the sequence comprising residues 12 to 855 of SEQ ID NO: 3.

[0025] In still some further embodiments, the engineered vaccinia virus capping enzyme and/or vaccinia virus capping enzyme subunit polypeptide sequence comprises one or more amino acid substitutions relative to the reference sequence comprising residues 12 to 855 of SEQ ID NO: 371, or to the reference sequence of SEQ ID NO: 371, with the proviso that the polypeptide sequence does not include the sequence comprising residues 12 to 855 of SEQ ID NO: 3.

[0026] In some additional embodiments, the engineered vaccinia virus capping enzyme and/or vaccinia virus capping enzyme subunit polypeptide sequence comprises one or more amino acid substitutions relative to the reference sequence comprising residues 12 to 855 of SEQ ID NO: 373, or to the reference sequence of SEQ ID NO: 373, with the proviso that the polypeptide sequence does not include the sequence comprising residues 12 to 855 of SEQ ID NO: 3.

[0027] The present invention also provides engineered vaccinia virus capping enzymes and/or vaccinia virus capping enzyme subunits comprising a polypeptide sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence comprising SEQ ID NO: 5, 107, and/or 168, or to a reference sequence of SEQ ID NO: 5, 107, and/or 168.

[0028] The present invention further provides engineered vaccinia virus capping enzymes and/or vaccinia virus capping enzyme subunits comprising a substitution at amino acid position 208, wherein the amino acid positions are numbered with reference to SEQ ID NO: 5. In some additional embodiments, the present invention provides engineered vaccinia virus capping enzymes and/or vaccinia virus capping enzyme subunits comprising one or more substitutions or substitution sets at amino acid positions selected from 78, 78/208/225, 78/208/225/274, 208, and/or 225, and any combinations thereof, wherein the amino acid positions are numbered with reference to SEQ ID NO: 5.

[0029] The present invention also provides engineered vaccinia virus capping enzymes and/or vaccinia virus capping enzyme subunits comprising a substitution at amino acid position 225, wherein the amino acid positions are numbered with reference to SEQ ID NO: 107.

[0030] The present invention further provides engineered vaccinia virus capping enzymes and/or vaccinia virus capping enzyme subunits comprising one or more substitutions at amino acid positions 239 and/or 258, and any combinations thereof, wherein the amino acid positions are numbered with reference to SEQ ID NO: 168. In some embodiments, the present invention provides engineered vaccinia virus capping enzymes and/or vaccinia virus capping enzyme subunits comprising one or more substitutions or substitution sets at amino acid positions selected from 239, 239/251, 239/251/258, 239/258, 251, and/or 258, and any combinations thereof, wherein the amino acid positions are numbered with reference to SEQ ID NO: 168.

[0031] The present invention also provides engineered vaccinia virus capping enzymes and/or vaccinia virus capping enzyme subunits, wherein the vaccinia virus capping enzymes and vaccinia virus capping enzyme subunits comprise a polypeptide sequence comprising a substitution or substitution set provided in Table 6.2, 6.3, 7.1, 8.1, 9.2, 10.2, 11.1, 12.1, 13.1, and/or 13.2, wherein the substitution or substitution set is relative to the reference sequence of SEQ ID NO: 2, 3, 5, 14, 105, 107, 166, 168, 242, 288, 318, 371, and/or 373. In some embodiments, the engineered vaccinia virus capping enzyme and/or vaccinia virus capping enzyme subunit comprises a polypeptide sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a sequence comprising an engineered vaccinia virus capping enzyme and/or vaccinia virus capping enzyme subunit set forth in Table 6.2, 6.3, 7.1, 8.1, 9.2, 10.2, 11.1, 12.1, 13.1, and/or 13.2.

[0032] The present invention also provides engineered vaccinia virus capping enzyme and/or vaccinia virus capping enzyme subunit polypeptide sequences comprising a sequence comprising residues 12 to 855 of SEQ ID NO: 3, 14, 105, 166, 242, 288, 318, 371, and/or 373.

[0033] The present invention also provides engineered vaccinia virus capping enzyme and/or vaccinia virus capping enzyme subunit polypeptide sequences comprising SEQ ID NO: 5, 105, and/or 168.

[0034] The present invention also provides engineered vaccinia virus capping enzymes and/or vaccinia virus capping enzyme subunits that have capping activity.

[0035] The present invention also provides engineered vaccinia virus capping enzymes and/or vaccinia virus capping enzyme subunits having at least one improved property as compared to the vaccinia virus capping enzyme subunits set forth in SEQ ID NO: 3 and/or 5. In some embodiments, the present invention also provides engineered vaccinia virus capping enzymes and/or vaccinia virus capping enzyme subunits having at least one improved property, as compared to the vaccinia virus capping enzyme subunits of SEQ ID NO: 3 and/or 5, wherein the improved property is selected from increased activity, increased stability, increased soluble expression, increased thermostability, increased capping, increased thermotolerance, increase resistance to inhibitors, and increased resistance to protease.

[0036] In some additional embodiments, the present invention provides purified engineered vaccinia virus capping enzymes and/or vaccinia virus capping enzyme subunits.

[0037] The present invention also provides engineered polynucleotides encoding engineered vaccinia virus capping enzymes and/or vaccinia virus capping enzyme subunits. In some embodiments, the present invention provides engineered polynucleotides comprising at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference polynucleotide sequence of nucleotide residues 34 to 2529 of SEQ ID NO: 2, or to a reference polynucleotide sequence of SEQ ID NO: 2 encoding an engineered vaccinia virus capping enzyme, vaccinia virus capping enzyme subunit, or a functional fragment thereof, wherein the encoded vaccinia virus capping enzyme and/or vaccinia virus capping enzyme subunit polypeptide comprises one or more substitutions at one or more amino acid positions relative to the reference sequence comprising residues 12 to 855 of SEQ ID NO: 3, or to a reference sequence of SEQ ID NO: 3.

[0038] The present invention further provides engineered polynucleotide sequences encoding an engineered vaccinia virus capping enzyme and/or vaccinia virus capping enzyme subunit comprising polypeptide sequences having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence comprising residues 12 to 855 of SEQ ID NO: 3, 14, 105, 166, 242, 288, 318, 371, and/or 373, or to a reference sequence of SEQ ID NO: 3, 14, 105, 166, 242, 288, 318, 371, and/or 373.

[0039] The present invention further provides engineered polynucleotide sequences comprising nucleotide residues 34 to 2529 of SEQ ID NO: 2, 13, 104, 165, 241, 287, 317, 370, and/or 372, or comprises the polynucleotide sequence of SEQ ID NO: 2, 13, 104, 165, 241, 287, 317, 370, and/or 372.

[0040] The present invention further provides engineered polynucleotide sequences encoding vaccinia virus capping enzymes and/or vaccinia virus capping enzyme subunits, wherein the polynucleotide sequences are codon-optimized.

[0041] The present invention also provides expression vectors comprising at least one engineered polynucleotide sequence provided herein. The present invention further provides host cells transformed with at least one polynucleotide provided herein or at least one expression vector provided herein.

[0042] The present invention also provides methods of producing an engineered vaccinia virus capping enzyme and/or an engineered vaccinia virus capping enzyme subunit polypeptide in a host cell comprising culturing a host cell provided herein under suitable culture conditions, such that at least one engineered vaccinia virus capping enzyme and/or vaccinia virus capping enzyme subunit is produced. In some embodiments of the methods, the vaccinia virus capping enzyme subunit is selected from DI and/or D 12. In some additional embodiments, the methods further comprise recovering at least one engineered vaccinia virus capping enzyme and/or vaccinia virus capping enzyme subunit from the culture and/or host cells. In some further embodiments, the methods further comprise a step of purifying the at least one engineered vaccinia virus capping enzyme and/or vaccinia virus capping enzyme subunit. In some embodiments, the vaccinia virus capping enzyme subunit purified is DI and/or D 12.

[0043] The present invention also provides compositions comprising at least one engineered vaccinia virus capping enzyme and/or vaccinia virus capping enzyme subunit. In some embodiments, the composition comprises vaccinia virus capping enzyme subunit DI and/or D 12. In some additional embodiments, the composition comprises at least one buffer. In some further embodiments, the composition comprises one or more substrates. In some additional embodiments, the composition comprises one or more methyl donors.

[0044] The present invention also provides methods of capping RNA, comprising providing uncapped RNA, a methyl donor, and comprising contacting the uncapped RNA with the engineered vaccinia virus capping enzyme subunits provided herein, under conditions suitable for capping the uncapped RNA to produce capped RNA. [0045] The present invention also provides methods of incorporating labeled GTP in RNA containing 5’ terminal triphosphates, comprising contacting labeled GTP and RNA components with the engineered vaccinia virus capping enzyme subunits provided herein, under conditions suitable for incorporating labeled GTP to produce labeled RNA.

[0046] The present invention also provides kits comprising at least one engineered vaccinia virus capping enzyme and/or vaccinia virus capping enzyme subunit provided herein. In some embodiments, the kits further comprise at least one buffer.

DESCRIPTION OF THE FIGURES

[0047] Figure 1 provides a schematic showing the vaccinia virus capping enzyme reaction.

DESCRIPTION OF THE INVENTION

[0048] The present invention provides engineered vaccina capping enzyme and vaccinia virus capping enzyme polypeptides and compositions thereof, as well as polynucleotides encoding the engineered vaccinia capping enzyme and vaccinia virus capping enzyme subunit polypeptides. The disclosure also provides methods for use of the engineered vaccina capping enzymes and vaccinia virus capping enzyme subunits, as well as compositions thereof for diagnostic, molecular biological tools, and other purposes.

[0049] In the basic cap structure of eukaryotes, (m⁷G or cap-0), a 5’ N7-methylguanosine is attached to the mRNA by an unusual 5 ’-5’ triphosphate linkage. Capping enzymes are brought to the RNA polymerase II via the carboxy-terminal domain (CTD) (See, Cho et al., Genes Dev., 11 :3319-3326 [1997], These enzymes act co-transcriptionally when the transcript reaches a length of about 20-30 nucleotides (See, Salditt-Georgieff et al., Cell, 19: 69-78 [1980]). There are three successive enzymatic steps that are necessary for formation of cap-0 (See, Figure 1). These steps involve RNA triphosphatase (TPase), RNA guanylyltransferase (GTase), and RNA guanine -7-methyltransferase activities (N7MTase). The RNA triphosphatase removes the 5’ gamma-phosphate from the growing pre-mRNA strand, in order to produce an mRNA containing a 5’ diphosphate. Next, the RNA guanylyltransferase transfers guanine monophosphate from at guanosine triphosphate donor to the 5 ’ end of the mRNA being processed, in order to produce an unmethylated cap structure. Finally, the guanine-7-methyltransferase methylates the N7 position of the guanine base by using S-adenosyl methionine (SAM) as a methyl donor. While the most predominant RNA cap in yeast is the cap-0 structure, in higher eukaryotes the RNA can be additionally methylated at the nucleotides following the cap.

[0050] Viruses that replicate in the cytoplasm of eukaryotes rely on their own genes encoding enzymes that provide a cap structure for viral transcripts. Thus, viruses either carry genes that encode the enzymes responsible for capping or encode a cap-snatching machinery that transfers caps from cellular mRNAs (See e.g., Reguera et al., Curr. Opin. Struct. Biol., 36: 75-84 [2016]). The vaccinia virus RNA capping enzyme (VCE) is typically a heterodimer consisting of a 97 kDa subunit encoded by the vaccinia virus DIR gene, and a 33 kDa subunit encoded by the vaccinia virus D12L gene. VCE also contains a 2’-O-methyltransferase enzyme (VP39). This enzyme converts the cap-0 structure into a cap-1 structure (See e.g., Barbosa and Moss J. Biol. Chem., 253: 7692-7697 [1978]; Schnierle et al., Proc. Natl. Acad. Sc., 89: 2897-2901 [2014]; and Hodel et al., Mol. Cell., 1:443-447 [1998]).

[0051] In spite of the essential role of the cap structure, detailed knowledge regarding the interactions of the 5’ end of mRNAs and adapter proteins and decapping enzymes is rather limited. This is largely due to the challenges associated with the in vitro production of large quantities of pure and homogenous capped RNA. The present invention provides compositions and methods suitable for the production of such pure and homogenous capped RNA in quantities that are suitable for diagnostic, therapeutic, vaccine, and research purposes. It is not intended that the present invention be limited to any particular use. In addition to providing such capped RNA, the VCE variant enzymes provided herein exhibit improved properties, including capping efficiency, soluble expression, and thermotolerance, as compared to wild-type VCE.

Abbreviations and Definitions

[0052] Unless defined otherwise, all technical and scientific terms used herein generally have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Generally, the nomenclature used herein and the laboratory procedures of cell culture, molecular genetics, microbiology, organic chemistry, analytical chemistry and nucleic acid chemistry described below are those well-known and commonly employed in the art. Such techniques are well- known and described in numerous texts and reference works well known to those of skill in the art. Standard techniques, or modifications thereof, are used for chemical syntheses and chemical analyses. [0053] Although any suitable methods and materials similar or equivalent to those described herein find use in the practice of the present invention, some methods and materials are described herein. It is to be understood that this invention is not limited to the particular methodology, protocols, and reagents described, as these may vary, depending upon the context they are used by those of skill in the art. Accordingly, the terms defined immediately below are more fully described by reference to the application as a whole. All patents, patent applications, articles and publications mentioned herein, both supra and infra, are hereby expressly incorporated herein by reference.

[0054] As used herein, the singular "a", "an," and "the" include the plural references, unless the context clearly indicates otherwise.

[0055] As used herein, the term “comprising” and its cognates are used in their inclusive sense (i.e., equivalent to the term “including” and its corresponding cognates). [0056] It is to be further understood that where description of embodiments use the term “comprising” and its cognates, the embodiments can also be described using language “consisting essentially of’ or “consisting of.”

[0057] Numeric ranges are inclusive of the numbers defining the range. Thus, every numerical range disclosed herein is intended to encompass every narrower numerical range that falls within such broader numerical range, as if such narrower numerical ranges were all expressly written herein. It is also intended that every maximum (or minimum) numerical limitation disclosed herein includes every lower (or higher) numerical limitation, as if such lower (or higher) numerical limitations were expressly written herein.

[0058] As used herein, the term “about” means an acceptable error for a particular value. In some instances “about” means within 0.05%, 0.5%, 1.0%, or 2.0%, of a given value range. In some instances, “about” means within 1, 2, 3, or 4 standard deviations of a given value.

[0059] Furthermore, the headings provided herein are not limitations of the various aspects or embodiments of the invention which can be had by reference to the application as a whole. Accordingly, the terms defined immediately below are more fully defined by reference to the application as a whole. Nonetheless, in order to facilitate understanding of the invention, a number of terms are defined below.

[0060] Unless otherwise indicated, nucleic acids are written left to right in 5' to 3' orientation; amino acid sequences are written left to right in amino to carboxy orientation, respectively.

[0061] As used herein, the “EC” number refers to the Enzyme Nomenclature of the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology (NC-IUBMB). The IUBMB biochemical classification is a numerical classification system for enzymes based on the chemical reactions they catalyze.

[0062] As used herein, “ATCC” refers to the American Type Culture Collection whose biorepository collection includes genes and strains.

[0063] As used herein, “NCBI” refers to National Center for Biological Information and the sequence databases provided therein.

[0064] As used herein, the term “DNA” refers to deoxyribonucleic acid.

[0065] As used herein, the term “RNA” refers to ribonucleic acid. As used herein “mRNA” refers to messenger RNA, while “rRNA” refers to ribosomal RNA, “tRNA” refers to transfer RNA, and “miRNA” refers to micro RNA.

[0066] As used herein, the terms “fusion protein,” and “chimeric protein” and “chimera” refer to hybrid proteins created through the joining of two or more genes that originally encoded separate proteins. In some embodiments, fusion proteins are created by recombinant technology (e.g., molecular biology techniques known in the art).

[0067] As used herein, “VCE” refers to vaccinia virus capping enzyme. In some embodiments, the term refers to a “vaccinia virus capping enzyme subunit,” such as subunits DI and D12, taken alone or in combination. As used herein, the term refers to the enzymes used by vaccinia virus to co-opt host cell machinery to produce viral proteins. The VCE forms a cap-0 structure (m⁷Gppp5’N) at the 5’ end of uncapped RNA molecules through the activities of the DI and D12 subunits. In infected cells, capping viral transcripts allows the transcription by the host cells. In the presence of the VCE enzymes of the present invention, transcripts may be rapidly capped in vitro, in the presence of reaction buffer, GTP (guanosine triphosphate), and a methyl donor (e.g., SAM). Capping by the VCE subunits of the present invention may be nearly 100% efficient and all capped structures added in the proper orientation (e.g., compared to co-transcriptional addition of some cap analogs). In the presence of labeled GTP, the 5’ terminal triphosphate of transcripts may be labeled. In some embodiments, the transcripts are RNA that contain the 5 'terminal triphosphate which will become labeled in the presence of labeled GTP via the VCE enzyme.

[0068] As used herein, the term “cap” refers to natural caps (e.g., ⁷mG), as well as a compound of the general formula R3p₃Nl-p-N(_X), where R3 is guanine, adenine, cytosine, uridine, or analogs thereof (e.g., N⁷ -methylguanosine; m⁷G), p₃ is a triphosphate linkage, N 1 and N_x are ribonucleosides, x is 0-8, and p is, independently for each position, a phosphate group, a phosphothioate, phosphorodithoate, alkylphosphanoate, arylphosphonoate, or an N-phosphoramidate linkage. In some embodiments, cap analogs are added at the 5’ end of an RNA transcript in the process referred to as “post-transcriptional capping,” to yield a 5’ capped RNA.

[0069] As used herein, the terms “DI” and “D 1 subunit” refer to the 97 kDa vaccinia virus capping enzyme subunit encoded by the vaccinia virus DIR gene (See, GeneID:3707562, UniProtKB ID: YP 232988.1). DI is a catalytic subunit with RNA triphosphatase, RNA guanylyltransferase, and RNA N⁷-guanine methyltransferase enzymatic activities (See, Cong and Shuman, J. Biol. Chem., 268: 7256-7260 [1993], Niles and Christen, J. Biol. Chem., 268:24986-24899 [1993], Higman and Niles, J. Biol. Chem., 269: 14982-14987 [1994], Mao and Shuman, J. Biol. Chem., 269: 24472-24479 [1994], Shuman and Moss, Meth. Enzymol., 181: 170-180 [1990]; and Gong and Shuman Virol., 309: 125-134 [2003]). In some embodiments, the DI subunit comprises an amino acid sequence at least about 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80%, 79%, 78%, 77%, 76%, 75%, 74%, 73%, 72%, 71%, or 70% sequence identity to SEQ ID NO: 3. In some additional embodiments, the DI subunit comprises a histidine tag (e.g., at the N- terminus). Unless otherwise indicated, the term refers to the whole subunit. In some embodiments, the DI subunit comprises at least one modified amino acid (e.g., hydroxylated, phosphorylated, myristoylated, palmitoylated, isoprenylated, sulfated, ubiquitinated, glycosylated (e.g., N-linked or O- linked), lipoylated, acetylated, alkylated (e.g., methylated), biotinylated, amidated, oxidized (e.g., by cysteines forming disulfide bonds or by reduction). It is not intended that the term be limited to any specific modifications to DI.

[0070] As used herein, the terms “D12” and “D12 subunit” refer to the 33 kDa subunit encoded by the vaccinia virus D12L gene (See, GenelD: 3707515; UniProtKB ID: YP 232999.1). The D12 subunit is regulatory, having no known enzymatic activity. However, the D12 subunit significantly stimulates the RNA N7-guanine methyltransferase activity of the DI subunit (See, Higman et al., J. Biol. Chem., 267: 16430-16437 [1992], Higman et al., J. Biol. Chem., 269: 14974-14981 [1994], and Mao and Shuman, supra). In some embodiments, the D 12 subunit comprises an amino acid sequence at least about 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80%, 79%, 78%, 77%, 76%, 75%, 74%, 73%, 72%, 71%, or 70% sequence identity to SEQ ID NO: 5. In some additional embodiments, the D12 subunit comprises a histidine tag (e.g., at the N-terminus). Unless otherwise indicated, the term refers to the whole subunit. In some embodiments, the D12 subunit comprises at least one modified amino acid (e.g., hydroxylated, phosphorylated, myristoylated, palmitoylated, isoprenylated, sulfated, ubiquitinated, glycosylated (e.g., N-linked or O-linked), lipoylated, acetylated, alkylated (e.g., methylated), biotinylated, amidated, oxidized (e.g., by cysteines forming disulfide bonds or by reduction). It is not intended that the term be limited to any specific modifications to D12.

[0071] As used herein, the term “vaccinia virus capping enzyme activity,” “synthetic activity,” and “capping activity” are used interchangeably herein, and refer to the ability of an enzyme to produce capO on RNA transcripts.

[0072] As used herein, the term “polymerase” refers to a class of enzymes that polymerize nucleoside triphosphates. Polymerases use a template nucleic acid strand to synthesize a complementary nucleic acid strand. The template strand and synthesized nucleic acid strand can independently be either DNA or RNA. Polymerases known in the art include but are not limited to DNA polymerases (e.g., E. coli DNA poll, T. aquaticus DNA polymerase (Taq), DNA-dependent RNA polymerases, and reverse transcriptases). As used herein, the polymerase is a polypeptide or protein containing sufficient amino acids to carry out a desired enzymatic function of the polymerase. In some embodiments, the polymerase does not contain all of the amino acids found in the native enzyme, but only those which are sufficient to allow the polymerase to carry out a desired catalytic activity, including but not limited to 5 ’-3’ polymerization, 5 ’-3’ exonuclease, and 3 ’-5’ exonuclease activities.

[0073] As used herein, the terms “duplex” and “ds” refer to a double -stranded nucleic acid (e.g., DNA) molecule comprised of two single -stranded polynucleotides that are complementary in their sequence (A pairs to T, C pairs to G), arranged in an antiparallel 5 ’ to 3 ’ orientation, and held together by hydrogen bonds between the nucleobases (i.e., adenine [A], guanine [G], cytosine [C], and thymine [T]).

[0074] As used here, the terms “protein,” “polypeptide,” and “peptide” are used interchangeably to denote a polymer of at least two amino acids covalently linked by an amide bond, regardless of length or post-translational modification (e.g., glycosylation or phosphorylation).

[0075] As used herein, the term “amino acids” are referred to herein by either their commonly known three-letter symbols or by the one-letter symbols recommended by IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise, may be referred to by their commonly accepted single letter codes. The abbreviations used for the genetically encoded amino acids are conventional and are as follows: alanine (Ala or A), arginine (Arg or R), asparagine (Asn or N), aspartate (Asp or D), cysteine (Cys or C), glutamate (Glu or E), glutamine (Gin or Q), histidine (His or H), isoleucine (He or I), leucine (Leu or L), lysine (Lys or K), methionine (Met or M), phenylalanine (Phe or F), proline (Pro or P), serine (Ser or S), threonine (Thr or T), tryptophan (Trp or W), tyrosine (Tyr or Y), and valine (Vai or V). When the three-letter abbreviations are used, unless specifically preceded by an “L” or a “D” or clear from the context in which the abbreviation is used, the amino acid may be in either the L- or D-configuration about a-carbon (C_a). For example, whereas “Ala” designates alanine without specifying the configuration about the a-carbon, “D-Ala” and “L-Ala” designate D-alanine and L-alanine, respectively. When the one-letter abbreviations are used, upper case letters designate amino acids in the L-configuration about the a-carbon and lower case letters designate amino acids in the D-configuration about the a-carbon. For example, “A” designates L-alanine and “a” designates D- alanine. When polypeptide sequences are presented as a string of one-letter or three-letter abbreviations (or mixtures thereof), the sequences are presented in the amino (N) to carboxy (C) direction in accordance with common convention.

[0076] The abbreviations used for the genetically encoding nucleosides are conventional and are as follows: adenosine (A); guanosine (G); cytidine (C); thymidine (T); and uridine (U). Unless specifically delineated, the abbreviated nucleosides may be either ribonucleosides or 2’- deoxyribonucleosides. The nucleosides may be specified as being either ribonucleosides or 2’- deoxyribonucleosides on an individual basis or on an aggregate basis. When nucleic acid sequences are presented as a string of one-letter abbreviations, the sequences are presented in the 5’ to 3’ direction in accordance with common convention, and the phosphates are not indicated.

[0077] As used herein, the terms “engineered,” “recombinant,” “non-naturally occurring,” and “variant,” when used with reference to a cell, a polynucleotide or a polypeptide refers to a material or a material corresponding to the natural or native form of the material that has been modified in a manner that would not otherwise exist in nature or is identical thereto but produced or derived from synthetic materials and/or by manipulation using recombinant techniques.

[0078] As used herein, “wild-type” and “naturally-occurring” refer to the form found in nature. For example, a wild-type polypeptide or polynucleotide sequence is a sequence present in an organism that can be isolated from a source in nature and which has not been intentionally modified by human manipulation.

[0079] As used herein, “coding sequence” refers to that part of a nucleic acid (e.g., a gene) that encodes an amino acid sequence of a protein.

[0080] As used herein, the term “percent (%) sequence identity” refers to comparisons among polynucleotides and polypeptides, and are determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide or polypeptide sequence in the comparison window may comprise additions or deletions (i. e. , gaps) as compared to the reference sequence for optimal alignment of the two sequences. The percentage may be calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity. Alternatively, the percentage may be calculated by determining the number of positions at which either the identical nucleic acid base or amino acid residue occurs in both sequences or a nucleic acid base or amino acid residue is aligned with a gap to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity. Those of skill in the art appreciate that there are many established algorithms available to align two sequences. Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith and Waterman (Smith and Waterman, Adv. Appl. Math., 1981, 2:482), by the homology alignment algorithm of Needleman and Wunsch (Needleman and Wunsch, J. Mol. Biol., 1970, 48:443), by the search for similarity method of Pearson and Lipman (Pearson and Lipman, Proc. Natl. Acad. Sci. USA, 1988, 85:2444), by computerized implementations of these algorithms (e.g., GAP, BESTFIT, FASTA, and TFASTA in the GCG Wisconsin Software Package), or by visual inspection, as known in the art. Examples of algorithms that are suitable for determining percent sequence identity and sequence similarity include, but are not limited to the BLAST and BLAST 2.0 algorithms (See e.g., Altschul et al., J. Mol. Biol., 1990, 215: 403-410; and Altschul et al., Nucleic Acids Res., 1977, 3389-3402). Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information website. This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length “W” in the query sequence, which either match or satisfy some positive-valued threshold score “T,” when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold (See, Altschul et al, supra). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are then extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters “M” (reward score for a pair of matching residues; always >0) and “N” (penalty score for mismatching residues; always <0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity “X” from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a wordlength (W) of 11, an expectation (E) of 10, M=5, N=-4, and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a wordlength (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (See e.g., Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA, 1989, 89: 10915). Exemplary determination of sequence alignment and % sequence identity can employ the BESTFIT or GAP programs in the GCG Wisconsin Software package (Accelrys, Madison WI), using default parameters provided.

[0081] As used herein, “reference sequence” refers to a defined sequence used as a basis for a sequence comparison. A reference sequence may be a subset of a larger sequence, for example, a segment of a full-length gene or polypeptide sequence. Generally, a reference sequence is at least 20 nucleotide or amino acid residues in length, at least 25 residues in length, at least 50 residues in length, at least 100 residues in length or the full length of the nucleic acid or polypeptide. Since two polynucleotides or polypeptides may each (1) comprise a sequence (i.e., a portion of the complete sequence) that is similar between the two sequences, and (2) may further comprise a sequence that is divergent between the two sequences, sequence comparisons between two (or more) polynucleotides or polypeptide are typically performed by comparing sequences of the two polynucleotides or polypeptides over a “comparison window” to identify and compare local regions of sequence similarity. In some embodiments, a “reference sequence” can be based on a primary amino acid sequence, where the reference sequence is a sequence that can have one or more changes in the primary sequence. For instance, the phrase “a reference sequence based on SEQ ID NO: 2, having a valine at the residue corresponding to X200” (or “a reference sequence based on SEQ ID NO: 2, having a valine at the residue corresponding to position 712”) refers to a reference sequence in which the corresponding residue at position X200 in SEQ ID NO: 2 (e.g., an alanine), has been changed to valine.

[0082] As used herein, “comparison window” refers to a conceptual segment of at least about 20 contiguous nucleotide positions or amino acids residues wherein a sequence may be compared to a reference sequence of at least 20 contiguous nucleotides or amino acids and wherein the portion of the sequence in the comparison window may comprise additions or deletions (i.e., gaps) of 20 percent or less as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The comparison window can be longer than 20 contiguous residues, and includes, optionally 30, 40, 50, 100, or longer windows.

[0083] As used herein, “corresponding to”, “reference to,” and “relative to” when used in the context of the numbering of a given amino acid or polynucleotide sequence refer to the numbering of the residues of a specified reference sequence when the given amino acid or polynucleotide sequence is compared to the reference sequence. In other words, the residue number or residue position of a given polymer is designated with respect to the reference sequence rather than by the actual numerical position of the residue within the given amino acid or polynucleotide sequence. For example, a given amino acid sequence, such as that of an engineered vaccinia virus capping enzyme, can be aligned to a reference sequence by introducing gaps to optimize residue matches between the two sequences. In these cases, although the gaps are present, the numbering of the residue in the given amino acid or polynucleotide sequence is made with respect to the reference sequence to which it has been aligned. In some embodiments, the sequence is tagged (e.g., with a histidine tag).

[0084] As used herein, “mutation” refers to the alteration of a nucleic acid sequence. In some embodiments, mutations result in changes to the encoded polypeptide sequence (i.e., as compared to the original sequence without the mutation). In some embodiments, the mutation comprises a substitution, such that a different amino acid is produced (e.g., substitution of an aspartic acid with tryptophan). In some alternative embodiments, the mutation comprises an addition, such that an amino acid is added to the original polypeptide sequence. In some further embodiments, the mutation comprises a deletion, such that an amino acid is deleted from the original polypeptide sequence. Any number of mutations may be present in a given sequence.

[0085] As used herein, “amino acid difference” and “residue difference” refer to a difference in the amino acid residue at a position of a polypeptide sequence relative to the amino acid residue at a corresponding position in a reference sequence. The positions of amino acid differences generally are referred to herein as “Xn,” where n refers to the corresponding position in the reference sequence upon which the residue difference is based. For example, a “residue difference at position X209 as compared to SEQ ID NO: 3” (or a “residue difference at position 209 as compared to SEQ ID NO: 3”) refers to a difference of the amino acid residue at the polypeptide position corresponding to position 209 of SEQ ID NO: 3. Thus, if the reference polypeptide of SEQ ID NO: 3 has an arginine at position 209, then a “residue difference at position X209 as compared to SEQ ID NO: 3” refers to an amino acid substitution of any residue other than arginine at the position of the polypeptide corresponding to position 209 of SEQ ID NO: 3. In most instances herein, the specific amino acid residue difference at a position is indicated as “XnY” where “Xn” specified the corresponding residue and position of the reference polypeptide (as described above), and “Y” is the single letter identifier of the amino acid found in the engineered polypeptide (i.e., the different residue than in the reference polypeptide). In some instances (e.g., in the Tables in the Examples), the present invention also provides specific amino acid differences denoted by the conventional notation “AnB”, where A is the single letter identifier of the residue in the reference sequence, “n” is the number of the residue position in the reference sequence, and B is the single letter identifier of the residue substitution in the sequence of the engineered polypeptide. In some instances, a polypeptide of the present invention can include one or more amino acid residue differences relative to a reference sequence, which is indicated by a list of the specified positions where residue differences are present relative to the reference sequence. In some embodiments, where more than one amino acid can be used in a specific residue position of a polypeptide, the various amino acid residues that can be used are separated by a

(e.g., X421E/X421G, X421E/G, or M421E/G). The present invention includes engineered polypeptide sequences comprising one or more amino acid differences that include either/or both conservative and non-conservative amino acid substitutions, as well as insertions and deletions of amino acids in the sequence.

[0086] As used herein, the terms “amino acid substitution set” and “substitution set” refers to a group of amino acid substitutions within a polypeptide sequence. In some embodiments, substitution sets comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more amino acid substitutions. In some embodiments, a substitution set refers to the set of amino acid substitutions that is present in any of the variant vaccinia virus capping enzyme polypeptides listed in any of the Tables in the Examples. In these substitution sets, the individual substitutions are separated by a semicolon

e.g., N81D/C184L) or slash

e.g., N81D/C184L). In some embodiments, the “substitution” comprises the deletion of an amino acid.

[0087] As used herein, “conservative amino acid substitution” refers to a substitution of a residue with a different residue having a similar side chain, and thus typically involves substitution of the amino acid in the polypeptide with amino acids within the same or similar defined class of amino acids. By way of example and not limitation, an amino acid with an aliphatic side chain may be substituted with another aliphatic amino acid (e.g., alanine, valine, leucine, and isoleucine); an amino acid with hydroxyl side chain is substituted with another amino acid with a hydroxyl side chain (e.g., serine and threonine); an amino acids having aromatic side chains is substituted with another amino acid having an aromatic side chain (e.g., phenylalanine, tyrosine, tryptophan, and histidine); an amino acid with a basic side chain is substituted with another amino acid with a basis side chain (e.g., lysine and arginine); an amino acid with an acidic side chain is substituted with another amino acid with an acidic side chain (e.g., aspartic acid or glutamic acid); and a hydrophobic or hydrophilic amino acid is replaced with another hydrophobic or hydrophilic amino acid, respectively.

[0088] As used herein, “non-conservative substitution” refers to substitution of an amino acid in the polypeptide with an amino acid with significantly differing side chain properties. Non-conservative substitutions may use amino acids between, rather than within, the defined groups and affect: (a) the structure of the peptide backbone in the area of the substitution (e.g., proline for glycine); (b) the charge or hydrophobicity; and/or (c) the bulk of the side chain. By way of example and not limitation, exemplary non-conservative substitutions include an acidic amino acid substituted with a basic or aliphatic amino acid; an aromatic amino acid substituted with a small amino acid; and a hydrophilic amino acid substituted with a hydrophobic amino acid.

[0089] As used herein, “deletion” refers to modification to the polypeptide by removal of one or more amino acids from the reference polypeptide. Deletions can comprise removal of 1 or more amino acids, 2 or more amino acids, 5 or more amino acids, 10 or more amino acids, 15 or more amino acids, or 20 or more amino acids, up to 10% of the total number of amino acids, or up to 20% of the total number of amino acids making up the reference enzyme while retaining enzymatic activity and/or retaining the improved properties of an engineered polymerase enzyme. Deletions can be directed to the internal portions and/or terminal portions of the polypeptide. In various embodiments, the deletion can comprise a continuous segment or can be discontinuous. Deletions are indicated by and may be present in substitution sets.

[0090] As used herein, “insertion” refers to modification to the polypeptide by addition of one or more amino acids from the reference polypeptide. Insertions can be in the internal portions of the polypeptide, or to the carboxy or amino terminus. Insertions as used herein include fusion proteins as is known in the art. The insertion can be a contiguous segment of amino acids or separated by one or more of the amino acids in the naturally occurring polypeptide.

[0091] As used herein, “functional fragment” and “biologically active fragment” are used interchangeably herein, to refer to a polypeptide that has an amino-terminal and/or carboxy-terminal deletion(s) and/or internal deletions, but where the remaining amino acid sequence is identical to the corresponding positions in the sequence to which it is being compared (e.g., a full length engineered vaccinia virus capping enzyme of the present invention) and that retains substantially all of the activity of the full-length polypeptide.

[0092] As used herein, “isolated polypeptide” refers to a polypeptide which is substantially separated from other contaminants that naturally accompany it (e.g., protein, lipids, and polynucleotides). The term embraces polypeptides which have been removed or purified from their naturally-occurring environment or expression system (e.g., host cell or in vitro synthesis). The recombinant vaccinia virus capping enzyme polypeptides may be present within a cell, present in the cellular medium, or prepared in various forms, such as lysates or isolated preparations. As such, in some embodiments, the recombinant vaccinia virus capping enzyme polypeptides provided herein are isolated polypeptides. [0093] As used herein, “substantially pure polypeptide” refers to a composition in which the polypeptide species is the predominant species present (i.e., on a molar or weight basis it is more abundant than any other individual macromolecular species in the composition), and is generally a substantially purified composition when the object species comprises at least about 50 percent of the macromolecular species present by mole or % weight. Generally, a substantially pure vaccinia virus capping enzyme composition will comprise about 60% or more, about 70% or more, about 80% or more, about 90% or more, about 95% or more, and about 98% or more of all macromolecular species by mole or % weight present in the composition. In some embodiments, the object species is purified to essential homogeneity (i.e., contaminant species cannot be detected in the composition by conventional detection methods) wherein the composition consists essentially of a single macromolecular species. Solvent species, small molecules (<500 Daltons), and elemental ion species are not considered macromolecular species. In some embodiments, the isolated recombinant vaccinia virus capping enzyme polypeptides are substantially pure polypeptide compositions.

[0094] As used herein, “isolated nucleotide” refers to a nucleotide which is substantially separated from other contaminants that naturally accompany it (e.g., protein, lipids, and polynucleotides). The term embraces polynucleotides which have been removed or purified from their naturally-occurring environment or expression system (e.g., host cell or in vitro synthesis). The recombinant vaccinia virus capping enzyme polynucleotides may be present within a cell, present in the cellular medium, or prepared in various forms, such as lysates or isolated preparations. As such, in some embodiments, the recombinant vaccinia virus capping enzyme polynucleotides provided herein are isolated polynucleotides.

[0095] As used herein, “substantially pure polynucleotide” refers to a composition in which the polynucleotide species is the predominant species present (i.e., on a molar or weight basis it is more abundant than any other individual macromolecular species in the composition), and is generally a substantially purified composition when the object species comprises at least about 50 percent of the macromolecular species present by mole or % weight. Generally, a substantially pure vaccinia virus capping enzyme composition will comprise about 40% or more, 50% or more 60% or more, about 70% or more, about 80% or more, about 90% or more, about 95% or more, and about 98% or more of all macromolecular species by mole or % weight present in the composition. In some embodiments, the object species is purified to essential homogeneity (i.e., contaminant species cannot be detected in the composition by conventional detection methods) wherein the composition consists essentially of a single macromolecular species. Solvent species, small molecules (<500 Daltons), and elemental ion species are not considered macromolecular species. In some embodiments, the isolated recombinant vaccinia virus capping enzyme polynucleotides are substantially pure polypeptide compositions.

[0096] As used herein, “improved enzyme property” refers to an engineered vaccinia virus capping enzyme polypeptide that exhibits an improvement in any enzyme property as compared to a reference Vaccinia virus capping enzyme polypeptide, such as a wild-type vaccinia virus capping enzyme polypeptide (e.g., the wild-type vaccinia virus capping enzyme polypeptide sequence of residues 12 to 855 of SEQ ID NO: 3 or SEQ ID NO: 5) or another engineered vaccinia virus capping enzyme polypeptide. Improved properties include but are not limited to such properties as increased production of capped RNA product, soluble expression, and thermotolerance.

[0097] As used herein, “increased enzymatic activity” and “enhanced catalytic activity” refer to an improved property of the engineered vaccinia virus capping enzyme polypeptides, which can be represented by an increase in specific activity (e.g., product produced/time/weight protein) and/or an increase in percent conversion of the substrate to the product (e.g., percent conversion of starting amount of substrate to product in a specified time period using a specified amount of vaccinia virus capping enzyme) as compared to the reference vaccinia virus capping enzyme (e.g., wild-type vaccinia virus capping enzyme and/or another engineered vaccinia virus capping enzyme).

Exemplary methods to determine enzyme activity are provided in the Examples. Any property relating to enzyme activity may be affected, including the classical enzyme properties of K_m, V_max or kcat, changes of which can lead to increased enzymatic activity. Improvements in enzyme activity can be from about 1.1 fold the enzymatic activity of the corresponding wild-type enzyme, to as much as 2- fold, 5 -fold, 10-fold, 20-fold, 25 -fold, 50-fold, 75 -fold, 100-fold, 150-fold, 200-fold or more enzymatic activity than the naturally occurring vaccinia virus capping enzyme or another engineered vaccinia virus capping enzyme from which the vaccinia virus capping enzyme polypeptides were derived.

[0098] As used herein, the phrases “reducing sensitivity to proteolysis” and “reducing proteolytic sensitivity” are used interchangeably herein mean that an engineered vaccinia virus capping enzyme polypeptide according to the invention will have a higher enzyme activity compared to a reference vaccinia virus capping enzyme in a standard assay (e.g., as disclosed in the Examples) after treatment with one or more proteases.

[0099] As used herein, “conversion” refers to the enzymatic conversion (or biotransformation) of substrate(s) to the corresponding product(s). “Percent conversion” refers to the percent of the substrate that is converted to the product within a period of time under specified conditions. Thus, the “enzymatic activity” or “activity” of a vaccinia virus capping enzyme polypeptide can be expressed as “percent conversion” of the substrate to the product in a specific period of time. In some embodiments, the substrate is uncapped RNA and the product is capped RNA.

[0100] As used herein, “hybridization stringency” relates to hybridization conditions, such as washing conditions, in the hybridization of nucleic acids. Generally, hybridization reactions are performed under conditions of lower stringency, followed by washes of varying but higher stringency. The term “moderately stringent hybridization” refers to conditions that permit target-DNA to bind a complementary nucleic acid that has about 60% identity, preferably about 75% identity, about 85% identity to the target DNA, with greater than about 90% identity to target-polynucleotide. Exemplary moderately stringent conditions are conditions equivalent to hybridization in 50% formamide, 5x Denhart's solution, 5xSSPE, 0.2% SDS at 42°C, followed by washing in 0.2xSSPE, 0.2% SDS, at 42°C. “High stringency hybridization” refers generally to conditions that are about 10°C or less from the thermal melting temperature T_m as determined under the solution condition for a defined polynucleotide sequence. In some embodiments, a high stringency condition refers to conditions that permit hybridization of only those nucleic acid sequences that form stable hybrids in 0.018M NaCl at 65°C (i.e., if a hybrid is not stable in 0.018M NaCl at 65°C, it will not be stable under high stringency conditions, as contemplated herein). High stringency conditions can be provided, for example, by hybridization in conditions equivalent to 50% formamide, 5x Denhart's solution, 5xSSPE, 0.2% SDS at 42°C, followed by washing in O.l xSSPE, and 0.1% SDS at 65°C. Another high stringency condition comprises hybridizing in conditions equivalent to hybridizing in 5X SSC containing 0. 1% (w:v) SDS at 65°C and washing in O. lx SSC containing 0.1% SDS at 65°C. Other high stringency hybridization conditions, as well as moderately stringent conditions, are described in the references cited above.

[0101] As used herein, “codon optimized” refers to changes in the codons of the polynucleotide encoding a protein to those preferentially used in a particular organism such that the encoded protein is more efficiently expressed in that organism. Although the genetic code is degenerate, in that most amino acids are represented by several codons, called “synonyms” or “synonymous” codons, it is well known that codon usage by particular organisms is nonrandom and biased towards particular codon triplets. This codon usage bias may be higher in reference to a given gene, genes of common function or ancestral origin, highly expressed proteins versus low copy number proteins, and the aggregate protein coding regions of an organism's genome. In some embodiments, the polynucleotides encoding the vaccinia virus capping enzyme enzymes are codon optimized for optimal production from the host organism selected for expression.

[0102] As used herein, “control sequence” refers herein to include all components that are necessary or advantageous for the expression of a polynucleotide and/or polypeptide of the present invention. Each control sequence may be native or foreign to the nucleic acid sequence encoding the polypeptide. Such control sequences include, but are not limited to, leaders, polyadenylation sequences, propeptide sequences, promoter sequences, signal peptide sequences, initiation sequences, and transcription terminators. At a minimum, the control sequences include a promoter, and transcriptional and translational stop signals. In some embodiments, the control sequences are provided with linkers for the purpose of introducing specific restriction sites facilitating ligation of the control sequences with the coding region of the nucleic acid sequence encoding a polypeptide.

[0103] As used herein, “operably linked” refers to a configuration in which a control sequence is appropriately placed (i.e., in a functional relationship) at a position relative to a polynucleotide of interest such that the control sequence directs or regulates the expression of the polynucleotide encoding a polypeptide of interest.

[0104] As used herein, “promoter sequence” refers to a nucleic acid sequence that is recognized by a host cell for expression of a polynucleotide of interest, such as a coding sequence. The promoter sequence contains transcriptional control sequences that mediate the expression of a polynucleotide of interest. The promoter may be any nucleic acid sequence which shows transcriptional activity in the host cell of choice including mutant, truncated, and hybrid promoters, and may be obtained from genes encoding extracellular or intracellular polypeptides either homologous or heterologous to the host cell.

[0105] As used herein, “suitable reaction conditions” or “suitable conditions” refers to those conditions in the enzymatic conversion reaction solution (e.g., ranges of enzyme loading, substrate loading, temperature, pH, buffers, co-solvents, etc.) under which a vaccinia vims capping enzyme polypeptide of the present invention is capable of converting a substrate to the desired product compound. Exemplary “suitable reaction conditions” are provided herein (See, the Examples).

[0106] As used herein, “loading”, such as in “compound loading” or “enzyme loading” refers to the concentration or amount of a component in a reaction mixture at the start of the reaction. “Substrate” in the context of an enzymatic conversion reaction process refers to the compound or molecule acted on by the vaccinia vims capping enzyme polypeptide. [0107] As used herein, “product” in the context of an enzymatic conversion process refers to the compound or molecule resulting from the action of the vaccinia virus capping enzyme polypeptide on the substrate. In some embodiments, the substrate is uncapped RNA and the product is capped RNA. [0108] As used herein, “culturing” refers to the growing of a population of microbial cells under suitable conditions using any suitable medium (e.g., liquid, gel, or solid).

[0109] Recombinant polypeptides (e.g., vaccinia virus capping enzyme variants) can be produced using any suitable methods known the art. For example, there is a wide variety of different mutagenesis techniques well known to those skilled in the art. In addition, mutagenesis kits are also available from many commercial molecular biology suppliers. Methods are available to make specific substitutions at defined amino acids (site-directed), specific or random mutations in a localized region of the gene (regio-specific), or random mutagenesis over the entire gene (e.g., saturation mutagenesis). Numerous suitable methods are known to those in the art to generate enzyme variants, including but not limited to site-directed mutagenesis of single-stranded DNA or double-stranded DNA using PCR, cassette mutagenesis, gene synthesis, error-prone PCR, shuffling, and chemical saturation mutagenesis, or any other suitable method known in the art. Non-limiting examples of methods used for DNA and protein engineering are provided in the following patents: US Pat. No. 6,117,679; US Pat. No. 6,420,175; US Pat. No. 6,376,246; US Pat. No. 6,586,182; US Pat. No. 7,747,391; US Pat. No. 7,747,393; US Pat. No. 7,783,428; and US Pat. No. 8,383,346. After the variants are produced, they can be screened for any desired property (e.g., high or increased activity, or low or reduced activity, increased thermal activity, increased thermal stability, and/or acidic pH stability, etc.). In some embodiments, “recombinant vaccinia virus capping enzyme polypeptides” (also referred to herein as “engineered vaccinia virus capping enzyme polypeptides,” “engineered vaccinia virus capping enzymes,” “variant vaccinia virus capping enzyme enzymes,” and “vaccinia virus capping enzyme variants”) find use.

[0110] As used herein, a "vector" is a DNA construct for introducing a DNA sequence into a cell. In some embodiments, the vector is an expression vector that is operably linked to a suitable control sequence capable of effecting the expression in a suitable host of the polypeptide encoded in the DNA sequence. In some embodiments, an "expression vector" has a promoter sequence operably linked to the DNA sequence (e.g., transgene) to drive expression in a host cell, and in some embodiments, also comprises a transcription terminator sequence.

[0111] As used herein, the term "expression" includes any step involved in the production of the polypeptide including, but not limited to, transcription, post-transcriptional modification, translation, and post-translational modification. In some embodiments, the term also encompasses secretion of the polypeptide from a cell.

[0112] As used herein, the term “produces” refers to the production of proteins and/or other compounds by cells. It is intended that the term encompass any step involved in the production of polypeptides including, but not limited to, transcription, post-transcriptional modification, translation, and post-translational modification. In some embodiments, the term also encompasses secretion of the polypeptide from a cell.

[0113] As used herein, an amino acid or nucleotide sequence (e.g., a promoter sequence, signal peptide, terminator sequence, etc.) is "heterologous" to another sequence with which it is operably linked if the two sequences are not associated in nature.

[0114] As used herein, the terms “host cell” and “host strain” refer to suitable hosts for expression vectors comprising DNA provided herein (e.g., a polynucleotide sequences encoding at least one Vaccinia virus capping enzyme variant). In some embodiments, the host cells are prokaryotic or eukaryotic cells that have been transformed or transfected with vectors constructed using recombinant DNA techniques as known in the art.

[0115] As used herein, the term “analogue” means a polypeptide having more than 70 % sequence identity but less than 100% sequence identity (e.g., more than 75%, 78%, 80%, 83%, 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity) with a reference polypeptide. In some embodiments, analogues include non-naturally occurring amino acid residues including, but not limited, to homoarginine, ornithine and norvaline, as well as naturally occurring amino acids. In some embodiments, analogues also include one or more D-amino acid residues and non-peptide linkages between two or more amino acid residues.

[0116] As used herein, the term “effective amount” means an amount sufficient to produce the desired result. One of general skill in the art may determine what the effective amount by using routine experimentation.

[0117] The terms “isolated” and “purified” are used to refer to a molecule (e.g., an isolated nucleic acid, polypeptide, etc.) or other component that is removed from at least one other component with which it is naturally associated. The term “purified” does not require absolute purity, rather it is intended as a relative definition.

[0118] As used herein, “cell-free DNA” refers to DNA circulating freely in the bloodstream and is not contained by or associated with cells. In some embodiments, cell-free DNA comprises DNA originally derived and released from normal somatic or germ line cells, cancer cells, fetal cells, microbial cells, or viruses.

[0119] As used herein, “amplification” refers to nucleic acid replication. In some embodiments, the term refers to replication of specific template nucleic acid.

[0120] As used herein, “polymerase chain reaction” and “PCR” refer to the methods described in US Pat Nos. 4,683,195 and 4,6884,202, hereby incorporated by reference. These methods find use in increasing the concentration of a segment of a target sequence or an entire target sequence in a mixture or purified DNA, without cloning or purification being required. The sequence of denaturation, annealing and extension constitute a “cycle.” The steps of denaturing, primer annealing, and polymerase extension can be repeated many times (i.e., multiple cycles are used), to obtain a high concentration of amplified DNA. The process is well-known in the art and numerous variations have been developed over the years since the method was first described. With PCR, it is possible to amplify a single copy of a specific target sequence to a level that is detectable by several different methodologies, including but not limited to hybridization with a labeled probe, incorporation of biotinylated primers followed by avidin-enzyme conjugate detection, incorporation of ³²P-labeled deoxyribonucleotide triphosphates (e.g., dCTP or dATP) into the amplified segment, etc. In addition to genomic DNA, any oligonucleotide sequence amenable to amplification can be copies using PCR with an appropriate set of primers. PCR products can also serve as templates for amplification.

[0121] As used herein, “target” when used in reference to a method employing a DNA polymerase, refers to the region of nucleic acid for preparation of a complementary DNA. The “target” is sorted out from other nucleic acids present in the methods using a DNA polymerase. In some embodiments, a “segment” is a region of nucleic acid within the target sequence.

[0122] As used herein, “target DNA” when used in context of methods utilizing DNA polymerase refers to the DNA, all or a portion thereof, that is the object for preparation of a complementary DNA copy. The target DNA can be the whole of the DNA sequence or a portion thereof, such as a segment of the DNA sequence.

[0123] As used herein, “target RNA” refers to the RNA, all or a portion thereof, that is the object for preparation of a complementary DNA copy. The target RNA can be the whole of the RNA sequence or a portion thereof, such as a segment of the RNA sequence.

[0124] As used herein, “sample template” refers to nucleic acid originating from a sample which is analyzed for the presence of target nucleic acid. In contrast, “background template” refers to nucleic acid other than sample template that may or may not be present within a sample. Background template may be inadvertently included in the sample, it may result from carryover, or may be due to the presence of nucleic acid contaminants from which the target nucleic acid is purified. For example, in some embodiments, nucleic acids from organisms other than those to be detected may be present as background in a test sample. However, it is not intended that the present invention be limited to any specific nucleic acid samples or templates.

[0125] As used herein, “amplifiable nucleic acid” is used in reference to nucleic acids which may be amplified by any amplification method, including but not limited to PCR. In most embodiments, amplifiable nucleic acids comprise sample templates.

[0126] As used herein, “PCR product”, “PCR fragment,” and “amplification product” refer to the resultant compounds obtained after two or more cycles of PCR amplification (or other amplification method, as indicated by the context), typically comprising the steps of denaturation, annealing, and extension. The terms encompass the situation wherein there has been amplification of one or more segments of one or more target sequences.

[0127] As used herein, “amplification reagents” and “PCR reagents” refer to those reagents (e.g., deoxyribonucleotide triphosphates, buffer, etc.), needed for amplification except for the primers, nucleic acid template, and the amplification enzyme. Typically, amplification reagents, along with other reaction components are placed and contained in a reaction vessel (e.g., test tube, microwell, etc.). It is not intended that the present invention be limited to any specific amplification reagents, as any suitable reagents find use in the present invention.

[0128] As used herein, “primer” refers to an oligonucleotide (i.e., a sequence of nucleotides), whether occurring naturally or produced synthetically, recombinantly, or by amplification, which is capable of acting as a point of initiation of nucleic acid synthesis, when placed under conditions in which synthesis of a primer extension product that is complementary to a nucleic acid strand is induced (i.e., in the presence of nucleotides and an inducing agent such as a DNA polymerase, and at a suitable temperature and pH). In most embodiments, primers a single -stranded, but in some embodiments, they are double -stranded. In some embodiments, the primers are of sufficient length to prime the synthesis of extension products in the presence of DNA polymerase. The exact primer length depends upon many factors, as known to those skilled in the art.

[0129] As used herein, “probe” refers to an oligonucleotide (i.e., a sequence of nucleotides), whether occurring naturally or produced synthetically, recombinantly, or by amplification, which is capable of hybridizing to another oligonucleotide of interest. Probes find use in the detection, identification, and/or isolation of particular gene sequences of interest. In some embodiments, probes are labeled with a “reporter molecule” (also referred to as a “label”) that aids in the detection of the probe in a suitable detection system (e.g., fluorescent, radioactive, luminescent, enzymatic, and other systems). It is not intended that the present invention be limited to any particular detection system or label. Primers, deoxyribonucleotides, and deoxyribonucleosides may contain labels. Indeed, it is not intended that the labeled composition of the present invention be limited to any particular component. Illustrative labels include, but are not limited to ³²P, ³⁵S, and fluorescent molecules (e.g., fluorescent dyes, including but not limited to green fluorescent protein).

[0130] The term “subject” encompasses mammals such as humans, non-human primates, livestock, companion animals, and laboratory animals (e.g., rodents and lagamorphs). It is intended that the term encompass females as well as males.

[0131] As used herein, the term “patient” means any subject that is being assessed for, treated for, or is experiencing disease.

[0132] As used herein, the term “sample” refers to a material or substance for reaction with a vaccinia virus capping enzyme. In some embodiments, the sample is a “biological sample,” which refers to sample of biological tissue or fluid. Such samples are typically from humans, but include tissues isolated from non-human primates, or rodents (e.g., mice, and rats), and includes sections of tissues such as biopsy and autopsy samples, frozen sections taken for histological purposes, blood, plasma, serum, sputum, stool, tears, mucus, hair, skin, etc. A "biological sample" also refers to a cell or population of cells or a quantity of tissue or fluid from organisms. In some embodiments, the biological sample has been removed from an animal, but the term "biological sample" can also refer to cells or tissue analyzed in vivo (i.e., without removal from the animal). Typically, a "biological sample" will contain cells from the animal or of organisms, but the term can also refer to noncellular biological material, such as noncellular fractions of blood, saliva, or urine. Numerous types of biological samples can be used with the enzymes, compositions, and method in the present invention, including, but not limited to, a tissue biopsy, a blood sample, a buccal scrape, a saliva sample, or a nipple discharge. As used herein, a "tissue biopsy" refers to an amount of tissue removed from an animal, preferably a human, for diagnostic analysis. In a patient with cancer, tissue may be removed from a tumor, allowing the analysis of cells within the tumor. "Tissue biopsy" can refer to any type of biopsy, such as needle biopsy, fine needle biopsy, surgical biopsy, etc.

Engineered Vaccinia Virus Capping Enzyme Polypeptides

[0133] In some embodiments herein, when a particular vaccinia virus capping enzyme variant (i.e., an engineered vaccinia virus capping enzyme polypeptide) is referred to by reference to modification of particular amino acid residues in the sequence of a wild-type vaccinia virus capping enzyme or reference vaccinia virus capping enzyme polypeptide, it is to be understood that variants of another vaccinia virus capping enzyme modified in the equivalent position(s) (as determined from the optional amino acid sequence alignment between the respective amino acid sequences) are encompassed herein. For example, for a substitution at specified amino acid position(s) numbered in reference to SEQ ID: 3, an equivalent amino acid position(s) can be readily ascertained for another reference sequence, such as a reference sequence comprising residues 12 to 855 of SEQ ID NO: 3, 14, 105, 166, 242, 288, 318, 371, or 373, or such a reference sequence as SEQ ID NO: 3, 14, 105, 166, 242, 288, 318, 371, or 373.

[0134] In some embodiments, the engineered vaccinia virus capping enzyme polypeptide variants of the present invention are useful in performing capping reactions. In some embodiments, the engineered vaccinia virus capping enzyme variants of the present invention find use in diagnostics, vaccines, therapeutics, and research applications. These engineered vaccinia virus capping enzyme variants can be used in solution, as well as in immobilized embodiments. In some embodiments, the engineered vaccinia virus capping enzyme can be prepared and used as non-fusion polypeptides or as fusion polypeptides.

[0135] In some embodiments, an engineered vaccinia virus capping enzyme or a functional fragment thereof of the present invention comprises a polypeptide comprising at least 70%, at least 75%, or at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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% sequence identity to a reference sequence comprising residues 12 to 855 of SEQ ID NO: 3, wherein the engineered vaccinia virus capping enzyme comprises one or more substitutions relative to the reference sequence comprising residues 12 to 855 of SEQ ID NO:3. In some embodiments, an engineered vaccinia virus capping enzyme or a functional fragment thereof of the present invention comprises a polypeptide comprising at least about 70%, at least about 75%, or at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to a reference sequence comprising residues 12 to 855 of SEQ ID NO: 3, wherein the engineered vaccinia virus capping enzyme comprises one or more substitutions relative to the reference sequence comprising residues 12 to 855 of SEQ ID NO:3.

[0136] In some embodiments, an engineered vaccinia virus capping enzyme or a functional fragment thereof of the present invention comprises a polypeptide comprising at least 70%, at least 75%, or at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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% sequence identity to a reference sequence of SEQ ID NO: 3, wherein the recombinant vaccinia virus capping enzyme comprises one or more substitutions relative to the reference sequence of SEQ ID NO: 3.

[0137] In some additional embodiments, the present invention provides an engineered vaccinia virus capping enzyme or a functional fragment thereof, comprising a polypeptide sequence having at least 75% or more sequence identity to a reference sequence comprising residues 12 to 855 of SEQ ID NO: 178, or to a reference sequence of SEQ ID NO: 3, wherein the polypeptide sequence comprises one or more substitutions relative to the reference sequence comprising residues 12 to 855 of SEQ ID NO: 178, or to the reference sequence of SEQ ID NO: 3.

[0138] In some embodiments, the engineered vaccinia virus capping enzyme comprises a polypeptide sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence comprising residues 12 to 855 of SEQ ID NO: 3 or to the reference sequence of SEQ ID NO: 3.

[0139] In some embodiments, the engineered vaccinia virus capping enzyme or a functional fragment thereof, comprises a polypeptide sequence having at least 80% or more sequence identity to reference sequence comprising residues 12 to 855 of SEQ ID NO: 14, wherein the polypeptide sequence comprises one or more substitutions relative to the reference sequence comprising residues 12 to 855 of SEQ ID NO: 14.

[0140] In some embodiments, the engineered vaccinia virus capping enzyme or a functional fragment thereof comprises a polypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence comprising residues 12 to 855 of SEQ ID NO: 14, wherein the polypeptide sequence comprises one or more substitutions relative to the reference sequence comprising residues 12 to 855 of SEQ ID NO: 14.

[0141] In some embodiments, the reference sequence comprises the sequence of SEQ ID NO: 14. In some embodiments, the engineered vaccinia virus capping enzyme or a functional fragment thereof, comprises a polypeptide sequence having at least 80% or more sequence identity to a reference sequence of SEQ ID NO: 14, wherein the polypeptide sequence comprises one or more substitutions relative to SEQ ID NO: 14.

[0142] In some additional embodiments, the present invention provides an engineered vaccinia virus capping enzyme or a functional fragment thereof, comprising a polypeptide sequence having at least 75% or more sequence identity to a reference sequence comprising residues 12 to 855 of SEQ ID NO: 178, or to a reference sequence of SEQ ID NO: 14, wherein the polypeptide sequence comprises one or more substitutions relative to the reference sequence comprising residues 12 to 855 of SEQ ID NO: 178, or to the reference sequence of SEQ ID NO: 14.

[0143] In some embodiments, the engineered vaccinia virus capping enzyme comprises a polypeptide sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence comprising residues 12 to 855 of SEQ ID NO: 14 or to the reference sequence of SEQ ID NO: 14.

[0144] In some embodiments, the engineered vaccinia virus capping enzyme comprises a polypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence of SEQ ID NO: 105, wherein the polypeptide sequence comprises one or more substitutions relative to the reference sequence of SEQ ID NO: 105. In some embodiments, the engineered vaccinia virus capping enzyme comprises a polypeptide sequence having at least about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more sequence identity to the reference sequence of SEQ ID NO: 105, wherein the polypeptide sequence comprises one or more substitutions relative to the reference sequence of SEQ ID NO: 105.

[0145] In some embodiments, the reference sequence comprises the sequence of SEQ ID NO: 105. In some embodiments, the engineered vaccinia virus capping enzyme or a functional fragment thereof, comprises a polypeptide sequence having at least 80% or more sequence identity to a reference sequence of SEQ ID NO: 105, wherein the polypeptide sequence comprises one or more substitutions relative to SEQ ID NO: 105.

[0146] In some additional embodiments, the present invention provides an engineered vaccinia virus capping enzyme or a functional fragment thereof, comprising a polypeptide sequence having at least 75% or more sequence identity to a reference sequence comprising residues 12 to 855 of SEQ ID NO: 178, or to a reference sequence of SEQ ID NO: 105, wherein the polypeptide sequence comprises one or more substitutions relative to the reference sequence comprising residues 12 to 855 of SEQ ID NO: 178, or to the reference sequence of SEQ ID NO: 105.

[0147] In some embodiments, the engineered vaccinia virus capping enzyme comprises a polypeptide sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence comprising residues 12 to 855 of SEQ ID NO: 105 or to the reference sequence of SEQ ID NO: 105. [0148] In some embodiments, the engineered vaccinia vims capping enzyme comprises a polypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence of SEQ ID NO: 166, wherein the polypeptide sequence comprises one or more substitutions relative to the reference sequence of SEQ ID NO: 166. In some embodiments, the engineered vaccinia vims capping enzyme comprises a polypeptide sequence having at least about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more sequence identity to the reference sequence of SEQ ID NO: 166, wherein the polypeptide sequence comprises one or more substitutions relative to the reference sequence of SEQ ID NO: 166.

[0149] In some embodiments, the reference sequence comprises the sequence of SEQ ID NO: 166. In some embodiments, the engineered vaccinia vims capping enzyme or a functional fragment thereof, comprises a polypeptide sequence having at least 80% or more sequence identity to a reference sequence of SEQ ID NO: 166, wherein the polypeptide sequence comprises one or more substitutions relative to SEQ ID NO: 166.

[0150] In some additional embodiments, the present invention provides an engineered vaccinia vims capping enzyme or a functional fragment thereof, comprising a polypeptide sequence having at least 75% or more sequence identity to a reference sequence comprising residues 12 to 855 of SEQ ID NO: 178, or to a reference sequence of SEQ ID NO: 166, wherein the polypeptide sequence comprises one or more substitutions relative to the reference sequence comprising residues 12 to 855 of SEQ ID NO: 178, or to the reference sequence of SEQ ID NO: 166.

[0151] In some embodiments, the engineered vaccinia vims capping enzyme comprises a polypeptide sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence comprising residues 12 to 855 of SEQ ID NO: 166 or to the reference sequence of SEQ ID NO: 166.

[0152] In some embodiments, the engineered vaccinia vims capping enzyme comprises a polypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence of SEQ ID NO: 242, wherein the polypeptide sequence comprises one or more substitutions relative to the reference sequence of SEQ ID NO: 242. In some embodiments, the engineered vaccinia vims capping enzyme comprises a polypeptide sequence having at least about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more sequence identity to the reference sequence of SEQ ID NO: 242, wherein the polypeptide sequence comprises one or more substitutions relative to the reference sequence of SEQ ID NO: 242.

[0153] In some embodiments, the reference sequence comprises the sequence of SEQ ID NO: 242. In some embodiments, the engineered vaccinia vims capping enzyme or a functional fragment thereof, comprises a polypeptide sequence having at least 80% or more sequence identity to a reference sequence of SEQ ID NO: 242, wherein the polypeptide sequence comprises one or more substitutions relative to SEQ ID NO: 242.

[0154] In some additional embodiments, the present invention provides an engineered vaccinia virus capping enzyme or a functional fragment thereof, comprising a polypeptide sequence having at least 75% or more sequence identity to a reference sequence comprising residues 12 to 855 of SEQ ID NO: 178, or to a reference sequence of SEQ ID NO: 242, wherein the polypeptide sequence comprises one or more substitutions relative to the reference sequence comprising residues 12 to 855 of SEQ ID NO: 178, or to the reference sequence of SEQ ID NO: 242.

[0155] In some embodiments, the engineered vaccinia virus capping enzyme comprises a polypeptide sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence comprising residues 12 to 855 of SEQ ID NO: 242 or to the reference sequence of SEQ ID NO: 242.

[0156] In some embodiments, the engineered vaccinia virus capping enzyme comprises a polypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence of SEQ ID NO: 288, wherein the polypeptide sequence comprises one or more substitutions relative to the reference sequence of SEQ ID NO: 288. In some embodiments, the engineered vaccinia virus capping enzyme comprises a polypeptide sequence having at least about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more sequence identity to the reference sequence of SEQ ID NO: 288, wherein the polypeptide sequence comprises one or more substitutions relative to the reference sequence of SEQ ID NO: 288.

[0157] In some embodiments, the reference sequence comprises the sequence of SEQ ID NO: 288. In some embodiments, the engineered vaccinia virus capping enzyme or a functional fragment thereof, comprises a polypeptide sequence having at least 80% or more sequence identity to a reference sequence of SEQ ID NO: 288, wherein the polypeptide sequence comprises one or more substitutions relative to SEQ ID NO: 288.

[0158] In some additional embodiments, the present invention provides an engineered vaccinia virus capping enzyme or a functional fragment thereof, comprising a polypeptide sequence having at least 75% or more sequence identity to a reference sequence comprising residues 12 to 855 of SEQ ID NO: 288, or to a reference sequence of SEQ ID NO: 288, wherein the polypeptide sequence comprises one or more substitutions relative to the reference sequence comprising residues 12 to 855 of SEQ ID NO: 178, or to the reference sequence of SEQ ID NO: 288.

[0159] In some embodiments, the engineered vaccinia virus capping enzyme comprises a polypeptide sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence comprising residues 12 to 855 of SEQ ID NO: 288 or to the reference sequence of SEQ ID NO: 288.

[0160] In some embodiments, the engineered vaccinia virus capping enzyme comprises a polypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence of SEQ ID NO: 318. wherein the polypeptide sequence comprises one or more substitutions relative to the reference sequence of SEQ ID NO: 318. In some embodiments, the engineered vaccinia virus capping enzyme comprises a polypeptide sequence having at least about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more sequence identity to the reference sequence of SEQ ID NO: 318, wherein the polypeptide sequence comprises one or more substitutions relative to the reference sequence of SEQ ID NO: 318.

[0161] In some embodiments, the reference sequence comprises the sequence of SEQ ID NO: 318. In some embodiments, the engineered vaccinia virus capping enzyme or a functional fragment thereof, comprises a polypeptide sequence having at least 80% or more sequence identity to a reference sequence of SEQ ID NO: 318, wherein the polypeptide sequence comprises one or more substitutions relative to SEQ ID NO: 318.

[0162] In some additional embodiments, the present invention provides an engineered vaccinia virus capping enzyme or a functional fragment thereof, comprising a polypeptide sequence having at least 75% or more sequence identity to a reference sequence comprising residues 12 to 855 of SEQ ID NO: 318, or to a reference sequence of SEQ ID NO: 318, wherein the polypeptide sequence comprises one or more substitutions relative to the reference sequence comprising residues 12 to 855 of SEQ ID NO: 178, orto the reference sequence of SEQ ID NO: 318.

[0163] In some embodiments, the engineered vaccinia virus capping enzyme comprises a polypeptide sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence comprising residues 12 to 855 of SEQ ID NO: 318 or to the reference sequence of SEQ ID NO: 318.

[0164] In some embodiments, the engineered vaccinia virus capping enzyme comprises a polypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence of SEQ ID NO: 371, wherein the polypeptide sequence comprises one or more substitutions relative to the reference sequence of SEQ ID NO: 371. In some embodiments, the engineered vaccinia virus capping enzyme comprises a polypeptide sequence having at least about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more sequence identity to the reference sequence of SEQ ID NO: 371, wherein the polypeptide sequence comprises one or more substitutions relative to the reference sequence of SEQ ID NO: 371. [0165] In some embodiments, the reference sequence comprises the sequence of SEQ ID NO: 371. In some embodiments, the engineered vaccinia virus capping enzyme or a functional fragment thereof, comprises a polypeptide sequence having at least 80% or more sequence identity to a reference sequence of SEQ ID NO: 371, wherein the polypeptide sequence comprises one or more substitutions relative to SEQ ID NO: 371.

[0166] In some additional embodiments, the present invention provides an engineered vaccinia virus capping enzyme or a functional fragment thereof, comprising a polypeptide sequence having at least 75% or more sequence identity to a reference sequence comprising residues 12 to 855 of SEQ ID NO: 371, or to a reference sequence of SEQ ID NO: 371, wherein the polypeptide sequence comprises one or more substitutions relative to the reference sequence comprising residues 12 to 855 of SEQ ID NO: 371, orto the reference sequence of SEQ ID NO: 371.

[0167] In some embodiments, the engineered vaccinia virus capping enzyme comprises a polypeptide sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence comprising residues 12 to 855 of SEQ ID NO: 371 or to the reference sequence of SEQ ID NO: 371.

[0168] In some embodiments, the engineered vaccinia virus capping enzyme comprises a polypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence of SEQ ID NO: 373, wherein the polypeptide sequence comprises one or more substitutions relative to the reference sequence of SEQ ID NO: 373. In some embodiments, the engineered vaccinia virus capping enzyme comprises a polypeptide sequence having at least about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more sequence identity to the reference sequence of SEQ ID NO: 373, wherein the polypeptide sequence comprises one or more substitutions relative to the reference sequence of SEQ ID NO: 373.

[0169] In some embodiments, the reference sequence comprises the sequence of SEQ ID NO: 373. In some embodiments, the engineered vaccinia virus capping enzyme or a functional fragment thereof, comprises a polypeptide sequence having at least 80% or more sequence identity to a reference sequence of SEQ ID NO: 373, wherein the polypeptide sequence comprises one or more substitutions relative to SEQ ID NO: 373.

[0170] In some additional embodiments, the present invention provides an engineered vaccinia virus capping enzyme or a functional fragment thereof, comprising a polypeptide sequence having at least 75% or more sequence identity to a reference sequence comprising residues 12 to 855 of SEQ ID NO: 373, or to a reference sequence of SEQ ID NO: 373, wherein the polypeptide sequence comprises one or more substitutions relative to the reference sequence comprising residues 12 to 855 of SEQ ID NO: 373, or to the reference sequence of SEQ ID NO: 373. [0171] In some embodiments, the engineered vaccinia vims capping enzyme comprises a polypeptide sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence comprising residues 12 to 855 of SEQ ID NO: 373 or to the reference sequence of SEQ ID NO: 373.

[0172] In some embodiments, the engineered vaccinia vims capping enzyme comprises at least one substitutions or substitution sets at positions selected from 209/212/259/431, 221/304/347/533/550/745/792, 304/347/350/459/787/792, and 831, or combinations thereof, wherein the amino acid positions are relative to the reference sequence of SEQ ID NO: 3. In some embodiments, the engineered vaccinia vims capping enzyme comprises at least one substitutions or substitution sets selected from 209T/212K/259P/431N, 221N/304K/347E/533K/550K/745E/792E, 304K/347E/350K/459E/787S/792E, and 83 IE, wherein the amino acid positions are relative to the reference sequence of SEQ ID NO: 3. In some embodiments, the engineered vaccinia vims capping enzyme comprises at least one substitutions or substitution sets selected from

R209T/E212K/V259P/S43 IN, K221N/I304K/S347E/S533K/H550K/S745E/V792E, I304K/S347E/V350K/N459E/N787S/V792E, and G83 IE, wherein the amino acid positions are relative to the reference sequence of SEQ ID NO: 3.

[0173] In some embodiments, the engineered vaccinia vims capping enzyme comprises at least one substitutions or substitution sets at positions selected from 42, 44, 97, 184, 210, 211, 332, 355, 368, 433, 450, 542, 546, 654/656, 679, 680, and 702, or combinations thereof, wherein the amino acid positions are relative to the reference sequence of SEQ ID NO: 3. In some embodiments, the engineered vaccinia vims capping enzyme comprises at least one substitutions or substitution sets selected from 42P, 44S, 97R, 184C, 184Q, 210F, 21 IK, 332A, 355Q, 355W, 368R, 433G, 450T, 542T, 542V, 546T, 654M/656D, 679C, 679E, 679G, 679Q, 680E, and 702G, wherein the amino acid positions are relative to the reference sequence of SEQ ID NO: 3. In some embodiments, the engineered vaccinia vims capping enzyme comprises at least one substitutions or substitution sets selected from A42P, E44S, H97R, K184C, K184Q, D210F, N21 IK, V332A, R355Q, R355W, T368R, I433G, Y450T, E542T, E542V, S546T, K654M/Y656D, Y679C, Y679E, Y679G, Y679Q, F680E, and A702G, wherein the amino acid positions are relative to the reference sequence of SEQ ID NO: 3. [0174] In some embodiments, the engineered vaccinia vims capping enzyme comprises at least one substitutions or substitution sets at positions selected from 209/212/221/431, 209/212/221/745/831, 209/212/533/831, 212/221/431/533, 212/221/431/533/550/745, 212/221/745/831, 221/259/431/533/745, 221/259/431/550, 221/259/550/831, 221/431/550/745, 221/550, 259/533/550/831, 259/533/745, 259/745/831, 259/831, 431, 533, 533/550/745/831, 533/745, and 550/745/831, or combinations thereof, wherein the amino acid positions are relative to the reference sequence of SEQ ID NO: 14. In some embodiments, the engineered vaccinia vims capping enzyme comprises at least one substitutions or substitution sets selected from 20917212K/221N/43 IN, 209T/212K/221N/745E/831E, 209T/212K/533K/831E, 212K/221N/431N/533K, 212K/221N/431N/533K/550K/745E, 212K/221N/745E/831E, 221N/259P/431N/533K/745E, 221N/259P/431N/550K, 221N/259P/550K/831E, 221N/431N/550K/745E, 221N/550K, 259P/533K/550K/831E, 259P/533K/745E, 259P/745E/831E, 259P/831E, 43 IN, 533K, 533K/550K/745E/831E, 533K/745E, and 550K/745E/831E, wherein the amino acid positions are relative to the reference sequence of SEQ ID NO: 14. In some embodiments, the engineered vaccinia virus capping enzyme comprises at least one substitutions or substitution sets selected from R209T/E212K/K221N/S431N, R209T/E212K/K221N/S745E/G831E, R209T/E212K/S533K/G831E, E212K/K221N/S431N/S533K, E212K/K221N/S431N/S533K/H550K/S745E, E212K/K221N/S745E/G83 IE, K221N/V259P/S431N/S533K/S745E, K221N/V259P/S431N/H550K, K221N/V259P/H550K/G831E, K221N/S431N/H550K/S745E, K221N/H550K, V259P/S533K/H550K/G831E, V259P/S533K/S745E, V259P/S745E/G831E, V259P/G831E, S431N, S533K, S533K/H550K/S745E/G831E, S533K/S745E, and H550K/S745E/G831E, wherein the amino acid positions are relative to the reference sequence of SEQ ID NO: 14.

[0175] In some embodiments, the engineered vaccinia virus capping enzyme comprises at least one substitutions or substitution sets at positions selected from 184/355/368/542/546/654/680, and 225, or combinations thereof, wherein the amino acid positions are relative to the reference sequence of SEQ ID NO: 105. In some embodiments, the engineered vaccinia virus capping enzyme comprises at least one substitutions or substitution sets selected from 184C/355W/368R/542T/546T/654M/680E, and 225A, wherein the amino acid positions are relative to the reference sequence of SEQ ID NO: 105. In some embodiments, the engineered vaccinia virus capping enzyme comprises at least one substitutions or substitution sets selected from K184C/R355W/T368R/E542T/S546T/K654M/F680E, T225A, wherein the amino acid positions are relative to the reference sequence of SEQ ID NO: 105. [0176] In some embodiments, the engineered vaccinia virus capping enzyme comprises at least one substitutions or substitution sets at positions selected from 45, 97, 143, 209, 304, 330, 343, 596, 598, 651 , and 818, or combinations thereof, wherein the amino acid positions are relative to the reference sequence of SEQ ID NO: 166. In some embodiments, the engineered vaccinia virus capping enzyme comprises at least one substitutions or substitution sets selected from 45C, 97T, 143D, 143K, 209G, 304V, 330F, 343L, 596V, 598C, 5981, 651R, and 818W, wherein the amino acid positions are relative to the reference sequence of SEQ ID NO: 166. In some embodiments, the engineered vaccinia virus capping enzyme comprises at least one substitutions or substitution sets selected from I45C, H97T, L143D, L143K, R209G, K304V, E330F, K343L, F596V, D598C, D598I, I651R, and D818W, wherein the amino acid positions are relative to the reference sequence of SEQ ID NO: 166.

[0177] In some embodiments, the engineered vaccinia virus capping enzyme comprises at least one substitutions or substitution sets at positions selected from 45/97/143/209/304/818, 45/97/143/209/304/818/820, 45/97/143/209/818, 45/97/143/209/818/855, 45/97/143/304/446/732/818/855, 45/97/143/304/818, 45/97/143/304/818/820/855, 45/97/209/446/818/820, 45/97/446/818, 45/143, 45/143/209/304/818/820/855, 45/143/209/818, 45/143/304, 45/143/818, 45/143/818/820/855, and 45/143/818/855, or combinations thereof, wherein the amino acid positions are relative to the reference sequence of SEQ ID NO: 242. In some embodiments, the engineered vaccinia virus capping enzyme comprises at least one substitutions or substitution sets selected from 45C/97R/143D/209G/304V/818W,

45 C/97R/143D/304V/446K/732V/818W/855 Q, 45 C/97R/209G/446K/818W/820Q, 45C/97T/143D/209G/304V/818W/820Q, 45C/97T/143D/209G/818W, 45C/97T/143D/209G/818W/855Q, 45C/97T/143K/304V/818W, 45C/97T/143K/304V/818W/820Q/855Q, 45C/97T/446K/818W, 45C/143D/209G/304V/818W/820Q/855Q, 45C/143D/209G/818W, 45C/143D/304V, 45C/143D/818K/855Q, 45C/143D/818W/820Q/855Q, 45C/143K, and 45C/143K/818W, wherein the amino acid positions are relative to the reference sequence of SEQ ID NO: 242. In some embodiments, the engineered vaccinia virus capping enzyme comprises at least one substitutions or substitution sets selected from I45C/H97R/L143D/R209G/K304V/D818W,

145 C/H97R/L 143D/K304V/F446K/T732V/D818W/R855 Q, I45C/H97R/R209G/F446K/D818W/P820Q, I45C/H97T/L143D/R209G/K304V/D818W/P820Q, I45C/H97T/L143D/R209G/D818W, I45C/H97T/L143D/R209G/D818W/R855Q, I45C/H97T/L143K/K304V/D818W, I45C/H97T/L143K/K304V/D818W/P820Q/R855Q, I45C/H97T/F446K/D818W, I45C/L143D/R209G/K304V/D818W/P820Q/R855Q, I45C/L143D/R209G/D818W, I45C/L143D/K304V, I45C/L143D/D818K/R855Q,

I45C/L143D/D818W/P820Q/R855Q, I45C/L143K, and I45C/L143K/D818W, wherein the amino acid positions are relative to the reference sequence of SEQ ID NO: 242.

[0178] In some embodiments, the engineered vaccinia virus capping enzyme comprises at least one substitutions or substitution sets at positions selected from 43, 421, 458, 458/562, 474, 552, 594, 601, and 815, or combinations thereof, wherein the amino acid positions are relative to the reference sequence of SEQ ID NO: 288. In some embodiments, the engineered vaccinia virus capping enzyme comprises at least one substitutions or substitution sets selected from 43N, 421E, 421G, 458C, 458D, 458G, 458G/562S, 474G, 552G, 594R, 601R, 601S, and 815R.

[0179] , wherein the amino acid positions are relative to the reference sequence of SEQ ID NO: 288. In some embodiments, the engineered vaccinia virus capping enzyme comprises at least one substitutions or substitution sets selected from Y43N, M421E, M421G, Y458C, Y458D, Y458G, Y458G/P562S, N474G, Y552G, K594R, N601R, N601S, and T815R, wherein the amino acid positions are relative to the reference sequence of SEQ ID NO: 288.

[0180] In some embodiments, the engineered vaccinia virus capping enzyme comprises at least one substitutions or substitution sets at positions selected from 239, 239/251, 239/251/258, 239/258, 251, 258, and 474/552/594/601, or combinations thereof, wherein the amino acid positions are relative to the reference sequence of SEQ ID NO: 318, In some embodiments, the engineered vaccinia virus capping enzyme comprises at least one substitutions or substitution sets selected from 239R, 239R/251T, 239R/251T/258V, 239R/258V, 25 IT, 258V, and 474G/552G/594R/601R, wherein the amino acid positions are relative to the reference sequence of SEQ ID NO: 318, In some embodiments, the engineered vaccinia virus capping enzyme comprises at least one substitutions or substitution sets selected from T239R, T239R/R251T, T239R/R251T/Y258V, T239R/Y258V, R251T, Y258V, N474G/Y552G/K594R/N601R, wherein the amino acid positions are relative to the reference sequence of SEQ ID NO: 318,

[0181] In some embodiments, the engineered vaccinia virus capping enzyme comprises at least one substitutions or substitution sets at positions selected from 81/184, 209/355/542, 211, 228, 236, 249, 362, 431, 446, 474, 596, 602, 642, 654, 657, 679, 732, 818, and 820, or combinations thereof, wherein the amino acid positions are relative to the reference sequence of SEQ ID NO: 371. In some embodiments, the engineered vaccinia virus capping enzyme comprises at least one substitutions or substitution sets selected from 81D/184L, 209G/355R/542G, 21 IQ, 228S, 236G, 249R, 362T, 431E, 446V, 474L, 596G, 6021, 642D, 654R, 657G, 679E, 732E, 818K, 820F, and 820Y , wherein the amino acid positions are relative to the reference sequence of SEQ ID NO: 371. In some embodiments, the engineered vaccinia virus capping enzyme comprises at least one substitutions or substitution sets selected from N81D/C184L, R209G/W355R/T542G, N211Q, R228S, E236G, K249R, K362T, S431E, F446V, G474L, F596G, K602I, E642D, M654R, K657G, Y679E, T732E, W818K, Q820F, and Q820Y, wherein the amino acid positions are relative to the reference sequence of SEQ ID NO: 371.

[0182] In some embodiments, the engineered vaccinia virus capping enzyme comprises at least one substitutions or substitution sets at positions selected from 43/97/209/304/548/551/601, 43/209/304/548/551, 43/209/355/542/548/551, 43/542/601, 97/304/542, 97/548, 209/332/355/542, 209/355/542, and 542/548/551/601, or combinations thereof, wherein the amino acid positions are relative to the reference sequence of SEQ ID NO: 373. In some embodiments, the engineered vaccinia virus capping enzyme comprises at least one substitutions or substitution sets selected from 43N/97T/209G/304V/548G/551G/601R, 43N/209G/304V/548G/551G, 43N/209G/355R/542G/548G/551G, 43N/542G/601R, 97T/304V/542G, 97T/548G, 209G/332G/355R/542G, 209G/355R/542G, and 542G/548G/551G/601R, wherein the amino acid positions are relative to the reference sequence of SEQ ID NO: 373. In some embodiments, the engineered vaccinia virus capping enzyme comprises at least one substitutions or substitution sets selected from Y43N/H97T/R209G/K304V/V548G/Q551G/N601R, Y43N/R209G/K304V/V548G/Q551G, Y43N/R209G/W355R/T542G/V548G/Q551G, Y43N/T542G/N601R, H97T/K304V/T542G, H97T/V548G, R209G/V332G/W355R/T542G, R209G/W355R/T542G, and T542G/V548G/Q551G/N601R, wherein the amino acid positions are relative to the reference sequence of SEQ ID NO: 373.

[0183] In some embodiments, the engineered vaccinia virus capping enzyme comprises a polypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence of SEQ ID NO: 5, wherein the polypeptide sequence comprises one or more substitutions relative to the reference sequence of SEQ ID NO: 5. In some embodiments, the engineered vaccinia virus capping enzyme comprises a polypeptide sequence having at least about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more sequence identity to the reference sequence of SEQ ID NO: 5, wherein the polypeptide sequence comprises one or more substitutions relative to the reference sequence of SEQ ID NO: 5.

[0184] In some embodiments, the reference sequence comprises the sequence of SEQ ID NO: 5. In some embodiments, the engineered vaccinia virus capping enzyme or a functional fragment thereof, comprises a polypeptide sequence having at least 80% or more sequence identity to a reference sequence of SEQ ID NO: 5, wherein the polypeptide sequence comprises one or more substitutions relative to SEQ ID NO: 5.

[0185] In some embodiments, the engineered vaccinia virus capping enzyme comprises a polypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence of SEQ ID NO: 107, wherein the polypeptide sequence comprises one or more substitutions relative to the reference sequence of SEQ ID NO: 107. In some embodiments, the engineered vaccinia virus capping enzyme comprises a polypeptide sequence having at least about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more sequence identity to the reference sequence of SEQ ID NO: 107, wherein the polypeptide sequence comprises one or more substitutions relative to the reference sequence of SEQ ID NO: 107.

[0186] In some embodiments, the reference sequence comprises the sequence of SEQ ID NO: 107. In some embodiments, the engineered vaccinia virus capping enzyme or a functional fragment thereof, comprises a polypeptide sequence having at least 80% or more sequence identity to a reference sequence of SEQ ID NO: 107, wherein the polypeptide sequence comprises one or more substitutions relative to SEQ ID NO: 107.

[0187] In some embodiments, the engineered vaccinia virus capping enzyme comprises a polypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence of SEQ ID NO: 168, wherein the polypeptide sequence comprises one or more substitutions relative to the reference sequence of SEQ ID NO: 168. In some embodiments, the engineered vaccinia virus capping enzyme comprises a polypeptide sequence having at least about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more sequence identity to the reference sequence of SEQ ID NO: 168, wherein the polypeptide sequence comprises one or more substitutions relative to the reference sequence of SEQ ID NO: 168.

[0188] In some embodiments, the reference sequence comprises the sequence of SEQ ID NO: 168. In some embodiments, the engineered vaccinia virus capping enzyme or a functional fragment thereof, comprises a polypeptide sequence having at least 80% or more sequence identity to a reference sequence of SEQ ID NO: 168, wherein the polypeptide sequence comprises one or more substitutions relative to SEQ ID NO: 168.

[0189] In some embodiments, the engineered vaccinia virus capping enzyme comprises a substitution at position 208, or combinations thereof, wherein the amino acid positions are relative to the reference sequence of SEQ ID NO: 5. In some embodiments, the engineered vaccinia virus capping enzyme comprises the substitution 208R, wherein the amino acid positions are relative to the reference sequence of SEQ ID NO: 5. In some embodiments, the engineered vaccinia virus capping enzyme comprises the substitution L208R, wherein the amino acid positions are relative to the reference sequence of SEQ ID NO: 5.

[0190] In some embodiments, the engineered vaccinia virus capping enzyme comprises at least one substitutions or substitution sets at positions selected from 8, 78/208/225, 78/208/225/274, 208, and 225, or combinations thereof, wherein the amino acid positions are relative to the reference sequence of SEQ ID NO: 5. In some embodiments, the engineered vaccinia virus capping enzyme comprises at least one substitutions or substitution sets selected from 78L, 78L/208R/225T, 78L/208W/225T/274P, 208W, and 225T, wherein the amino acid positions are relative to the reference sequence of SEQ ID NO: 5. In some embodiments, the engineered vaccinia virus capping enzyme comprises at least one substitutions or substitution sets selected from V78L, V78L/L208R/V225T, V78L/L208W/V225T/I274P, L208W, and V225T, wherein the amino acid positions are relative to the reference sequence of SEQ ID NO: 5.

[0191] In some embodiments, the engineered vaccinia virus capping enzyme comprises a substitution at position 225, or combinations thereof, wherein the amino acid positions are relative to the reference sequence of SEQ ID NO: 107. In some embodiments, the engineered vaccinia virus capping enzyme comprises the substitution 225A, wherein the amino acid positions are relative to the reference sequence of SEQ ID NO: 107. In some embodiments, the engineered vaccinia virus capping enzyme comprises the substitution T225A, wherein the amino acid positions are relative to the reference sequence of SEQ ID NO: 107.

[0192] In some embodiments, the engineered vaccinia virus capping enzyme comprises at least one substitutions or substitution sets at positions selected from 239 and 258, or combinations thereof, wherein the amino acid positions are relative to the reference sequence of SEQ ID NO: 168. In some embodiments, the engineered vaccinia virus capping enzyme comprises at least one substitutions or substitution sets selected from 239R and 258V, wherein the amino acid positions are relative to the reference sequence of SEQ ID NO: 168. In some embodiments, the engineered vaccinia virus capping enzyme comprises at least one substitutions or substitution sets selected from T239R and Y258V, wherein the amino acid positions are relative to the reference sequence of SEQ ID NO: 168. [0193] In some embodiments, the engineered vaccinia virus capping enzyme comprises at least one substitutions or substitution sets at positions selected from 239, 239/251, 239/251/258, 239/258, 251, and 258, or combinations thereof, wherein the amino acid positions are relative to the reference sequence of SEQ ID NO: 168. In some embodiments, the engineered vaccinia virus capping enzyme comprises at least one substitutions or substitution sets selected from 239R, 239R/251T, 239R/251T/258V, 239R/258V, 25 IT, and 258V, wherein the amino acid positions are relative to the reference sequence of SEQ ID NO: 168. In some embodiments, the engineered vaccinia virus capping enzyme comprises at least one substitutions or substitution sets selected from T239R, T239R/R25 IT, T239R/R251T/Y258V, T239R/Y258V, R25 IT, and Y258V, wherein the amino acid positions are relative to the reference sequence of SEQ ID NO: 168.

[0194] The present invention further provides engineered vaccinia virus capping enzymes comprising polypeptide sequences having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence comprising residues 12 to 855 of SEQ ID NO: 3, 14, 105, 166, 242, 288, 318, 371, 373, or to a reference sequence of SEQ ID NO: 3, 14, 105, 166, 242, 288, 318, 371, and/or 373.

[0195] In some embodiments, the engineered vaccinia virus capping enzyme comprises a polypeptide sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence comprising residues 12 to 855 of SEQ ID NO: 3, 14, 105, 166, 242, 288, 318, 371, and/or 373, or to a reference sequence of SEQ ID NO: 3, 14, 105, 166, 242, 288, 318, 371, and/or 373, with the proviso that the polypeptide sequence does not include the sequence comprising residues 12 to 855 of SEQ ID NO:3 14, 105, 166, 242, 288, 318, 371, and/or 373.

[0196] The present invention further provides engineered vaccinia virus capping enzymes comprising polypeptide sequences having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence comprising residues 12 to 855 of SEQ ID NO: 3 or to a reference sequence SEQ ID NO: 3.

[0197] In some embodiments, the engineered vaccinia virus capping enzyme comprises a polypeptide sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence comprising residues 12 to 855 of SEQ ID NO: 3, or to a reference sequence SEQ ID NO: 3, with the proviso that the polypeptide sequence does not include the sequence comprising residues 12 to 855 of SEQ ID NO: 3.

[0198] The present invention further provides engineered vaccinia virus capping enzymes comprising polypeptide sequences having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence comprising residues 12 to 855 of SEQ ID NO: 14, or to a reference sequence SEQ ID NO: 14. [0199] In some embodiments, the engineered vaccinia virus capping enzyme comprises a polypeptide sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence comprising residues 12 to 855 of SEQ ID NO: 14, or to a reference sequence SEQ ID NO: 14, with the proviso that the polypeptide sequence does not include the sequence comprising residues 12 to 855 of SEQ ID NO: 14.

[0200] The present invention further provides engineered vaccinia virus capping enzymes comprising polypeptide sequences having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence comprising residues 12 to 855 of SEQ ID NO: 105, or to a reference sequence SEQ ID NO: 105. [0201] In some embodiments, the engineered vaccinia virus capping enzyme comprises a polypeptide sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence comprising residues 12 to 855 of SEQ ID NO: 105, or to a reference sequence SEQ ID NO: 105, with the proviso that the polypeptide sequence does not include the sequence comprising residues 12 to 855 of SEQ ID NO: 105.

[0202] The present invention further provides engineered vaccinia virus capping enzymes comprising polypeptide sequences having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence comprising residues 12 to 855 of SEQ ID NO: 166, or to a reference sequence SEQ ID NO: 166. [0203] In some embodiments, the engineered vaccinia virus capping enzyme comprises a polypeptide sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence comprising residues 12 to 855 of SEQ ID NO: 166, or to a reference sequence SEQ ID NO: 166, with the proviso that the polypeptide sequence does not include the sequence comprising residues 12 to 855 of SEQ ID NO: 166.

[0204] The present invention further provides engineered vaccinia virus capping enzymes comprising polypeptide sequences having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence comprising residues 12 to 855 of SEQ ID NO: 242, or to a reference sequence SEQ ID NO: 242.

[0205] In some embodiments, the engineered vaccinia virus capping enzyme comprises a polypeptide sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence comprising residues 12 to 855 of SEQ ID NO: 242, or to a reference sequence SEQ ID NO: 242, with the proviso that the polypeptide sequence does not include the sequence comprising residues 12 to 855 of SEQ ID NO: 242.

[0206] The present invention further provides engineered vaccinia virus capping enzymes comprising polypeptide sequences having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence comprising residues 12 to 855 of SEQ ID NO: 288, or to a reference sequence SEQ ID NO: 288.

[0207] In some embodiments, the engineered vaccinia virus capping enzyme comprises a polypeptide sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence comprising residues 12 to 855 of SEQ ID NO: 288, or to a reference sequence SEQ ID NO: 288, with the proviso that the polypeptide sequence does not include the sequence comprising residues 12 to 855 of SEQ ID NO: 288.

[0208] The present invention further provides engineered vaccinia virus capping enzymes comprising polypeptide sequences having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence comprising residues 12 to 855 of SEQ ID NO: 318, or to a reference sequence SEQ ID NO: 318. [0209] In some embodiments, the engineered vaccinia virus capping enzyme comprises a polypeptide sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence comprising residues 12 to 855 of SEQ ID NO: 318, or to a reference sequence SEQ ID NO: 318, with the proviso that the polypeptide sequence does not include the sequence comprising residues 12 to 855 of SEQ ID NO: 318.

[0210] The present invention further provides engineered vaccinia virus capping enzymes comprising polypeptide sequences having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence comprising residues 12 to 855 of SEQ ID NO: 371, or to a reference sequence SEQ ID NO: 371.

[0211] In some embodiments, the engineered vaccinia virus capping enzyme comprises a polypeptide sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence comprising residues 12 to 855 of SEQ ID NO: 371, or to a reference sequence SEQ ID NO: 371, with the proviso that the polypeptide sequence does not include the sequence comprising residues 12 to 855 of SEQ ID NO: 371.

[0212] The present invention further provides engineered vaccinia virus capping enzymes comprising polypeptide sequences having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence comprising residues 12 to 855 of SEQ ID NO: 373, or to a reference sequence SEQ ID NO: 373. [0213] In some embodiments, the engineered vaccinia vims capping enzyme comprises a polypeptide sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence comprising residues 12 to 855 of SEQ ID NO: 373, or to a reference sequence SEQ ID NO: 373, with the proviso that the polypeptide sequence does not include the sequence comprising residues 12 to 855 of SEQ ID NO: 373.

[0214] The present invention further provides engineered vaccinia vims capping enzymes comprising polypeptide sequences having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence comprising SEQ ID NO: 5, 107, or 168, or to a reference sequence of SEQ ID NO: 5, 107, or 168. [0215] In some embodiments, the engineered vaccinia vims capping enzyme comprises a polypeptide sequence comprising a substitution or substitution set provided in Table 6.2, 6.3, 7.1, 8.1,

9.2, 10.2, 11.1, 12.1, 13.1, and/or 13.2, wherein the substitution or substitution set is relative to the reference sequence of SEQ ID NO: 3, 14, 105, 166, 242, 288, 318, 371, 373 or SEQ ID NO: 5, 107, or 168.

[0216] In some embodiments, the engineered vaccinia vims capping enzyme comprises a polypeptide sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a sequence comprising residues 12 to 855 of an engineered vaccinia vims capping DI enzyme variant set forth in Table 6.2, 6.3, 7.1, 8.1,

9.2, 10.2, 11.1, 12.1, 13.1, and/or 13.2.

[0217] In some embodiments, the engineered vaccinia vims capping enzyme comprises a polypeptide sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a sequence comprising an engineered vaccinia vims capping D12 enzyme variant set forth in Table 7.1, 8.1, 11.1, and/or 12.1.

[0218] In some embodiments, the engineered vaccinia vims capping enzyme comprises a polypeptide sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a sequence of an engineered vaccinia vims capping enzyme variant set forth in Table 6.2, 6.3, 7.1, 8.1, 9.2, 10.2, 11.1, 12.1, 13.1, and/or

13.2. In some embodiments, the engineered vaccinia vims capping enzyme comprises a polypeptide sequence comprising at least one substitution provided in Table 6.2, 6.3, 7.1, 8.1, 9.2, 10.2, 11.1, 12.1, 13.1, and/or 13.2, wherein the substitution is relative to the reference sequence comprising residues 12 to 855 of SEQ ID NO: 3, 14, 105, 166, 242, 288, 318, 371, and/or 373, or to the reference sequence of SEQ ID NO: 3, 14, 105, 166, 242, 288, 318, 371, and/or 373. In some embodiments, the engineered vaccinia vims capping enzyme comprises a polypeptide sequence comprising at least one substitution provided in Table 6.2, 6.3, 7.1, 8.1, 9.2, 10.2, 11.1, 12.1, 13.1, and/or 13.2, wherein the substitution is relative to the reference sequence comprising SEQ ID NO: 5, 107, and/or 168, or to the reference sequence of SEQ ID NO: 5, 107, and/or 168. [0219] In some embodiments, an engineered vaccinia vims capping enzyme comprises a polypeptide sequence comprising at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence comprising residues 12 to 855 of a corresponding SEQ ID NO. set forth in Table 6.2, 6.3, 7.1, 8.1, 9.2, 10.2, 11.1, 12.1, 13.1, and/or 13.2 or to a reference sequence of a corresponding SEQ ID NO. set forth in Table 6.2, 6.3, 7.1,

8.1, 9.2, 10.2, 11.1, 12.1, 13.1, and/or 13.2. In some embodiments, the engineered vaccinia vims DI capping enzyme comprises a polypeptide sequence comprising residues 12 to 855 of a corresponding SEQ ID NO. set forth in Table 6.2, 6.3, 7.1, 8.1, 9.2, 10.2, 11.1, 12.1, 13.1, and/or 13.2, or a polypeptide sequence comprising a corresponding SEQ ID NO. set forth in Table 6.2, 6.3, 7.1, 8.1,

9.2, 10.2, 11.1, 12.1, 13.1, and/or 13.2. In some embodiments, the engineered vaccinia vims D12 capping enzyme comprises a polypeptide sequence comprising a corresponding SEQ ID NO. set forth in Table 6.2, 7.1, 8.1, 11.1, and/or 12.1, or a polypeptide sequence comprising a corresponding SEQ ID NO. set forth in Table 6.2, 7.1, 8.1, 11.1, and/or 12.1.

[0220] In some embodiments, the engineered vaccinia vims capping enzyme comprises a polypeptide sequence comprising a sequence selected from SEQ ID NOS: 6-617.

[0221] In some embodiments, the engineered vaccinia vims capping enzyme comprises a polypeptide sequence comprising a sequence selected from SEQ ID NOS: 6-617, wherein the polypeptide optionally has 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10 substitutions in the polypeptide sequence.

[0222] In some embodiments, the engineered vaccinia vims capping enzyme comprises a polypeptide comprising 1, 2, 3, 4, up to 5 substitutions in the polypeptide sequence. In some embodiments, the engineered vaccinia vims capping enzyme comprises substitutions that are conservative substitutions. In some embodiments, the engineered vaccinia vims capping enzyme polypeptide has 1, 2, 3, 4, or up to 5 substitutions in the polypeptide sequence. In some embodiments, the engineered vaccinia vims capping enzyme polypeptide has 1, 2, 3, or 4 substitutions in the polypeptide sequence. In some embodiments, the substitutions comprise non-conservative substitutions. In some embodiments, the substitutions comprise conservative substitutions. In some embodiments, the substitutions comprise non-conservative of conservative substitutions. In some embodiments, guidance on non-conservative and conservative substitutions are provided by the variants disclosed herein. In some embodiments, the foregoing substitutions can be in addition to any of the above described embodiments.

[0223] In some embodiments, the engineered vaccinia vims capping enzyme comprises a fusion protein.

[0224] It will be apparent that the description herein, including the Examples, provide Tables showing sequence stmctural information correlating specific amino acid sequence features with the functional activity of the engineered vaccinia vims capping enzyme polypeptides. This structurefunction correlation information is provided in the form of specific amino acid residue differences relative to the reference polypeptides of SEQ ID NO: 3, 5, 14, 105, 107, 166, 168, 242, 288, 318, 371, and/or 373, as well as associated experimentally determined activity data for the exemplary engineered vaccinia virus capping enzyme polypeptides. Such information provides guidance and information on substitutions implemented in preparing engineered vaccinia virus capping enzyme variants.

[0225] In some embodiments, the engineered vaccinia virus capping enzyme of the present invention has capping enzyme activity. In some embodiments, the engineered vaccinia virus capping enzyme has at least one improved property as compared to the vaccinia virus capping enzyme comprising residues 12 to 855 of SEQ ID NO: 3 and/or compared to the vaccinia virus capping enzyme of SEQ ID NO: 3. In some embodiments, the engineered vaccinia virus capping enzyme has one or more of improved property selected from increased production of capped RNA product, soluble expression, and thermotolerance.

[0226] In some embodiments, the engineered vaccinia virus capping enzyme of the present invention has capping enzyme activity. In some embodiments, the engineered vaccinia virus capping enzyme has at least one improved property as compared to the vaccinia virus capping enzyme comprising SEQ ID NO: 5 and/or compared to the vaccinia virus capping enzyme of SEQ ID NO: 5. In some embodiments, the engineered vaccinia virus capping enzyme has one or more of improved property selected from increased production of capped RNA product, soluble expression, and thermotolerance. [0227] In some embodiments, the engineered vaccinia virus capping enzyme polypeptides of the present invention are produced by cultivating a host cell, such as a microorganism, comprising at least one polynucleotide sequence encoding at least one engineered vaccinia virus capping enzyme polypeptide under conditions which are conducive for producing the vaccinia virus capping enzyme polypeptide. In some embodiments, the engineered vaccinia virus capping enzyme polypeptide is subsequently recovered from the resulting culture medium and/or cells.

[0228] In some embodiments, the engineered vaccinia virus capping enzyme polypeptide described herein is an isolated composition. In some embodiments, the engineered vaccinia virus capping enzyme polypeptide is a purified composition, as further discussed herein.

[0229] In some embodiments, the present invention further provides functional fragments or biologically active fragments of engineered vaccinia virus capping enzyme polypeptides described herein. Thus, for each and every embodiment herein of an engineered vaccinia virus capping enzyme, a functional fragment or biologically active fragment of the engineered vaccinia virus capping enzyme is provided herewith. In some embodiments, a functional fragment or biologically active fragments of an engineered vaccinia virus capping enzyme comprises at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% of the activity of the vaccinia virus capping enzyme polypeptide from which it was derived (i.e., the parent vaccinia virus capping enzyme). In some embodiments, functional fragments or biologically active fragments comprise at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% of the parent sequence of the vaccinia virus capping enzyme. In some embodiments the functional fragment will be truncated by less than 5, less than 10, less than 15, less than 10, less than 25, less than 30, less than 35, less than 40, less than 45, and less than 50 amino acids.

[0230] In some embodiments, a functional fragment of an engineered vaccinia virus capping enzyme herein comprises at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the parent sequence of the engineered vaccinia virus capping enzyme. In some embodiments the functional fragment will be truncated by less than 5, less than 10, less than 15, less than 10, less than 25, less than 30, less than 35, less than 40, less than 45, less than 50, less than 55, less than 60, less than 65, or less than 70 amino acids.

Polynucleotides Encoding Engineered Polypeptides., Expression Vectors and Host Cells

[0231] In another aspect, the present invention provides polynucleotides encoding the engineered vaccinia virus capping enzyme polypeptides described herein. In some embodiments, the polynucleotides are operatively linked to one or more heterologous regulatory sequences that control gene expression to create a vaccinia virus capping enzyme capable of expressing the polypeptide. In some embodiments, expression constructs containing at least one heterologous polynucleotide encoding the engineered vaccinia virus capping enzyme polypeptide (s) is introduced into appropriate host cells to express the corresponding vaccinia virus capping enzyme polypeptide(s).

[0232] As will be apparent to the skilled artisan, availability of a protein sequence and the knowledge of the codons corresponding to the various amino acids provide a description of all the polynucleotides capable of encoding the subject polypeptides. The degeneracy of the genetic code, where the same amino acids are encoded by alternative or synonymous codons, allows an extremely large number of nucleic acids to be made, all of which encode an engineered vaccinia virus capping enzyme polypeptide of the present invention. Thus, the present invention provides methods and compositions for the production of each and every possible variation of engineered vaccinia virus capping enzyme polynucleotides that could be made that encode the engineered vaccinia virus capping enzyme polypeptides described herein by selecting combinations based on the possible codon choices, and all such variations are to be considered specifically disclosed for any polypeptide described herein, including the amino acid sequences presented in the Examples (e.g., in Table 6.2, 6.3, 7.1, 8.1, 9.2, 10.2, 11.1, 12.1, 13.1, and/or 13.2) and in the Sequence Listing.

[0233] In some embodiments, the codons are preferably optimized for utilization by the chosen host cell for protein production. For example, preferred codons used in bacteria are typically used for expression in bacteria, and preferred codons used in mammalian cells are typically used for expression in mammalian cells. Consequently, codon optimized polynucleotides encoding the engineered vaccinia virus capping enzyme polypeptides contain preferred codons at about 40%, 50%, 60%, 70%, 80%, 90%, or greater than 90% of the codon positions in the full length coding region. [0234] In some embodiments, the codons are preferably optimized for utilization by the chosen host cell for protein production. For example, preferred codons used in bacteria are typically used for expression in bacteria. Consequently, codon optimized polynucleotides encoding the engineered vaccinia virus capping enzyme polypeptides contain preferred codons at about 40%, 50%, 60%, 70%, 80%, 90%, or greater than 90% of the codon positions in the full length coding region.

[0235] In some embodiments, the polynucleotide encodes an engineered vaccinia virus capping enzyme polypeptide having vaccinia virus capping enzyme activity, wherein the polypeptide comprises an amino acid sequence having at least at least 70%, at least 75%, or at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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% sequence identity to a reference sequence comprising residues 12 to 855 of SEQ ID NO: 3, or to a reference sequence of SEQ ID NO: 3, wherein the polypeptide sequence comprises one or more substitutions relative to the reference sequence of SEQ ID NO:3, as described herein.

[0236] In some embodiments, the polynucleotide encodes an engineered vaccinia virus capping enzyme polypeptide having vaccinia virus capping enzyme activity, wherein the polypeptide comprises an amino acid sequence having at least at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to a reference sequence comprising residues 12 to 855 of SEQ ID NO: 3, or to a reference sequence of SEQ ID NO: 3, wherein the polypeptide sequence comprises one or more substitutions relative to the reference sequence of SEQ ID NO:3, as described herein.

[0237] In some embodiments, the polynucleotide encodes an engineered vaccinia virus capping enzyme polypeptide having vaccinia virus capping enzyme activity, wherein the polypeptide comprises an amino acid sequence having at least at least 70%, at least 75%, or at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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% sequence identity to a reference sequence comprising residues 12 to 855 of SEQ ID NO: 14, or to a reference sequence of SEQ ID NO: 14, wherein the polypeptide sequence comprises one or more substitutions relative to the reference sequence of SEQ ID NO: 14, as described herein.

[0238] In some embodiments, the polynucleotide encodes an engineered vaccinia virus capping enzyme polypeptide having vaccinia virus capping enzyme activity, wherein the polypeptide comprises an amino acid sequence having at least at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to a reference sequence comprising residues 12 to 855 of SEQ ID NO: 14, or to a reference sequence of SEQ ID NO: 14, wherein the polypeptide sequence comprises one or more substitutions relative to the reference sequence of SEQ ID NO: 14, as described herein.

[0239] In some embodiments, the polynucleotide encodes an engineered vaccinia virus capping enzyme polypeptide having vaccinia virus capping enzyme activity, wherein the polypeptide comprises an amino acid sequence having at least at least 70%, at least 75%, or at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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% sequence identity to a reference sequence comprising residues 12 to 855 of SEQ ID NO: 105, or to a reference sequence of SEQ ID NO: 105, wherein the polypeptide sequence comprises one or more substitutions relative to the reference sequence of SEQ ID NO: 105, as described herein.

[0240] In some embodiments, the polynucleotide encodes an engineered vaccinia virus capping enzyme polypeptide having vaccinia virus capping enzyme activity, wherein the polypeptide comprises an amino acid sequence having at least at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to a reference sequence comprising residues 12 to 855 of SEQ ID NO: 105, or to a reference sequence of SEQ ID NO: 105, wherein the polypeptide sequence comprises one or more substitutions relative to the reference sequence of SEQ ID NO: 105, as described herein.

[0241] In some embodiments, the polynucleotide encodes an engineered vaccinia virus capping enzyme polypeptide having vaccinia virus capping enzyme activity, wherein the polypeptide comprises an amino acid sequence having at least at least 70%, at least 75%, or at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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% sequence identity to a reference sequence comprising residues 12 to 855 of SEQ ID NO: 166, or to a reference sequence of SEQ ID NO: 166, wherein the polypeptide sequence comprises one or more substitutions relative to the reference sequence of SEQ ID NO: 166, as described herein.

[0242] In some embodiments, the polynucleotide encodes an engineered vaccinia virus capping enzyme polypeptide having vaccinia virus capping enzyme activity, wherein the polypeptide comprises an amino acid sequence having at least at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to a reference sequence comprising residues 12 to 855 of SEQ ID NO: 166, or to a reference sequence of SEQ ID NO: 166, wherein the polypeptide sequence comprises one or more substitutions relative to the reference sequence of SEQ ID NO: 166, as described herein. [0243] In some embodiments, the polynucleotide encodes an engineered vaccinia vims capping enzyme polypeptide having vaccinia vims capping enzyme activity, wherein the polypeptide comprises an amino acid sequence having at least at least 70%, at least 75%, or at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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% sequence identity to a reference sequence comprising residues 12 to 855 of SEQ ID NO: 242, or to a reference sequence of SEQ ID NO: 242, wherein the polypeptide sequence comprises one or more substitutions relative to the reference sequence of SEQ ID NO:242, as described herein.

[0244] In some embodiments, the polynucleotide encodes an engineered vaccinia vims capping enzyme polypeptide having vaccinia vims capping enzyme activity, wherein the polypeptide comprises an amino acid sequence having at least at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to a reference sequence comprising residues 12 to 855 of SEQ ID NO:

242, or to a reference sequence of SEQ ID NO: 242, wherein the polypeptide sequence comprises one or more substitutions relative to the reference sequence of SEQ ID NO:242, as described herein.

[0245] In some embodiments, the polynucleotide encodes an engineered vaccinia vims capping enzyme polypeptide having vaccinia vims capping enzyme activity, wherein the polypeptide comprises an amino acid sequence having at least at least 70%, at least 75%, or at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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% sequence identity to a reference sequence comprising residues 12 to 855 of SEQ ID NO: 288, or to a reference sequence of SEQ ID NO: 288, wherein the polypeptide sequence comprises one or more substitutions relative to the reference sequence of SEQ ID NO:288, as described herein.

[0246] In some embodiments, the polynucleotide encodes an engineered vaccinia vims capping enzyme polypeptide having vaccinia vims capping enzyme activity, wherein the polypeptide comprises an amino acid sequence having at least at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to a reference sequence comprising residues 12 to 855 of SEQ ID NO:

288, or to a reference sequence of SEQ ID NO: 288, wherein the polypeptide sequence comprises one or more substitutions relative to the reference sequence of SEQ ID NO:288, as described herein.

[0247] In some embodiments, the polynucleotide encodes an engineered vaccinia vims capping enzyme polypeptide having vaccinia vims capping enzyme activity, wherein the polypeptide comprises an amino acid sequence having at least at least 70%, at least 75%, or at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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% sequence identity to a reference sequence comprising residues 12 to 855 of SEQ ID NO: 318, or to a reference sequence of SEQ ID NO: 318, wherein the polypeptide sequence comprises one or more substitutions relative to the reference sequence of SEQ ID NO:318, as described herein.

[0248] In some embodiments, the polynucleotide encodes an engineered vaccinia virus capping enzyme polypeptide having vaccinia virus capping enzyme activity, wherein the polypeptide comprises an amino acid sequence having at least at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to a reference sequence comprising residues 12 to 855 of SEQ ID NO:

318, or to a reference sequence of SEQ ID NO: 318, wherein the polypeptide sequence comprises one or more substitutions relative to the reference sequence of SEQ ID NO:318, as described herein.

[0249] In some embodiments, the polynucleotide encodes an engineered vaccinia virus capping enzyme polypeptide having vaccinia virus capping enzyme activity, wherein the polypeptide comprises an amino acid sequence having at least at least 70%, at least 75%, or at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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% sequence identity to a reference sequence comprising residues 12 to 855 of SEQ ID NO: 371, or to a reference sequence of SEQ ID NO: 371, wherein the polypeptide sequence comprises one or more substitutions relative to the reference sequence of SEQ ID NO:371, as described herein.

[0250] In some embodiments, the polynucleotide encodes an engineered vaccinia virus capping enzyme polypeptide having vaccinia virus capping enzyme activity, wherein the polypeptide comprises an amino acid sequence having at least at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to a reference sequence comprising residues 12 to 855 of SEQ ID NO:

371, or to a reference sequence of SEQ ID NO: 371, wherein the polypeptide sequence comprises one or more substitutions relative to the reference sequence of SEQ ID NO:371, as described herein.

[0251] In some embodiments, the polynucleotide encodes an engineered vaccinia virus capping enzyme polypeptide having vaccinia virus capping enzyme activity, wherein the polypeptide comprises an amino acid sequence having at least at least 70%, at least 75%, or at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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% sequence identity to a reference sequence comprising residues 12 to 855 of SEQ ID NO: 373, or to a reference sequence of SEQ ID NO: 373, wherein the polypeptide sequence comprises one or more substitutions relative to the reference sequence of SEQ ID NO:373, as described herein.

[0252] In some embodiments, the polynucleotide encodes an engineered vaccinia virus capping enzyme polypeptide having vaccinia virus capping enzyme activity, wherein the polypeptide comprises an amino acid sequence having at least at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to a reference sequence comprising residues 12 to 855 of SEQ ID NO: 373, or to a reference sequence of SEQ ID NO: 373, wherein the polypeptide sequence comprises one or more substitutions relative to the reference sequence of SEQ ID NO:373, as described herein.

[0253] In some embodiments, the present invention provides an engineered polynucleotide encoding the engineered vaccinia virus capping enzyme comprises at least one substitutions or substitution sets at positions selected from 209/212/259/431, 221/304/347/533/550/745/792, 304/347/350/459/787/792, and 831, or combinations thereof, wherein the amino acid positions are relative to the reference sequence of SEQ ID NO: 3. In some embodiments, the engineered polynucleotide encodes an engineered vaccinia virus capping enzyme comprising at least one substitutions or substitution sets selected from 209T/212K/259P/431N, 221N/304K/347E/533K/550K/745E/792E, 304K/347E/350K/459E/787S/792E, and 83 IE, wherein the amino acid positions are relative to the reference sequence of SEQ ID NO: 3. In some embodiments, the engineered polynucleotide encodes an engineered vaccinia virus capping enzyme comprising at least one substitutions or substitution sets selected from R209T/E212K/V259P/S431N, K221N/I304K/S347E/S533K/H550K/S745E/V792E, I304K/S347E/V350K/N459E/N787S/V792E, and G83 IE, wherein the amino acid positions are relative to the reference sequence of SEQ ID NO: 3. [0254] In some embodiments, the engineered polynucleotide encodes an engineered vaccinia virus capping enzyme comprising at least one substitutions or substitution sets at positions selected from 42, 44, 97, 184, 210, 211, 332, 355, 368, 433, 450, 542, 546, 654/656, 679, 680, and 702, or combinations thereof, wherein the amino acid positions are relative to the reference sequence of SEQ ID NO: 3. In some embodiments, the engineered polynucleotide encodes an engineered vaccinia virus capping enzyme comprising at least one substitutions or substitution sets selected from 42P, 44S, 97R, 184C, 184Q, 210F, 21 IK, 332A, 355Q, 355W, 368R, 433G, 450T, 542T, 542V, 546T, 654M/656D, 679C, 679E, 679G, 679Q, 680E, and 702G, wherein the amino acid positions are relative to the reference sequence of SEQ ID NO: 3. In some embodiments, the engineered polynucleotide encodes an engineered vaccinia virus capping enzyme comprising at least one substitutions or substitution sets selected from A42P, E44S, H97R, K184C, K184Q, D210F, N21 IK, V332A, R355Q, R355W, T368R, I433G, Y450T, E542T, E542V, S546T, K654M/Y656D, Y679C, Y679E, Y679G, Y679Q, F680E, and A702G, wherein the amino acid positions are relative to the reference sequence of SEQ ID NO: 3.

[0255] In some embodiments, the engineered polynucleotide encodes an engineered vaccinia virus capping enzyme comprising at least one substitutions or substitution sets at positions selected from 209/212/221/431, 209/212/221/745/831, 209/212/533/831, 212/221/431/533, 212/221/431/533/550/745, 212/221/745/831, 221/259/431/533/745, 221/259/431/550, 221/259/550/831, 221/431/550/745, 221/550, 259/533/550/831, 259/533/745, 259/745/831, 259/831, 431, 533, 533/550/745/831, 533/745, and 550/745/831, or combinations thereof, wherein the amino acid positions are relative to the reference sequence of SEQ ID NO: 14. In some embodiments, the engineered polynucleotide encodes an engineered vaccinia virus capping enzyme comprising at least one substitutions or substitution sets selected from 209T/212K/221N/431N, 209T/212K/221N/745E/831E, 209T/212K/533K/831E, 212K/221N/431N/533K, 212K/221N/431N/533K/550K/745E, 212K/221N/745E/831E, 221N/259P/431N/533K/745E, 221N/259P/431N/550K, 221N/259P/550K/831E, 221N/431N/550K/745E, 221N/550K, 259P/533K/550K/831E, 259P/533K/745E, 259P/745E/831E, 259P/831E, 43 IN, 533K, 533K/550K/745E/831E, 533K/745E, and 550K/745E/831E, wherein the amino acid positions are relative to the reference sequence of SEQ ID NO: 14. In some embodiments, the engineered polynucleotide encodes an engineered vaccinia virus capping enzyme comprising at least one substitutions or substitution sets selected from R209T/E212K/K221N/S431N, R209T/E212K/K221N/S745E/G831E, R209T/E212K/S533K/G831E, E212K/K221N/S431N/S533K, E212K/K221N/S431N/S533K/H550K/S745E, E212K/K221N/S745E/G831E,

K221N/V259P/S431N/S533K/S745E, K221N/V259P/S431N/H550K, K221N/V259P/H550K/G83 IE, K221N/S431N/H550K/S745E, K221N/H550K, V259P/S533K/H550K/G831E, V259P/S533K/S745E, V259P/S745E/G831E, V259P/G831E, S431N, S533K, S533K/H550K/S745E/G831E, S533K/S745E, and H550K/S745E/G831E, wherein the amino acid positions are relative to the reference sequence of SEQ ID NO: 14.

[0256] In some embodiments, the engineered polynucleotide encodes an engineered vaccinia virus capping enzyme comprising at least one substitutions or substitution sets at positions selected from 184/355/368/542/546/654/680, and 225, or combinations thereof, wherein the amino acid positions are relative to the reference sequence of SEQ ID NO: 105. In some embodiments, the engineered polynucleotide encodes an engineered vaccinia virus capping enzyme comprising at least one substitutions or substitution sets selected from 184C/355W/368R/542T/546T/654M/680E, and 225A, wherein the amino acid positions are relative to the reference sequence of SEQ ID NO: 105. In some embodiments, the engineered polynucleotide encodes an engineered vaccinia virus capping enzyme comprising at least one substitutions or substitution sets selected from K184C/R355W/T368R/E542T/S546T/K654M/F680E, T225A, wherein the amino acid positions are relative to the reference sequence of SEQ ID NO: 105. [0257] In some embodiments, the engineered polynucleotide encodes an engineered vaccinia virus capping enzyme comprising at least one substitutions or substitution sets at positions selected from 45, 97, 143, 209, 304, 330, 343, 596, 598, 651, and 818, or combinations thereof, wherein the amino acid positions are relative to the reference sequence of SEQ ID NO: 166. In some embodiments, the engineered polynucleotide encodes an engineered vaccinia virus capping enzyme comprising at least one substitutions or substitution sets selected from 45C, 97T, 143D, 143K, 209G, 304V, 330F, 343L, 596V, 598C, 5981, 651R, and 818W, wherein the amino acid positions are relative to the reference sequence of SEQ ID NO: 166. In some embodiments, the engineered polynucleotide encodes an engineered vaccinia virus capping enzyme comprising at least one substitutions or substitution sets selected from I45C, H97T, L143D, L143K, R209G, K304V, E330F, K343L, F596V, D598C, D598I, 1651R, and D818W, wherein the amino acid positions are relative to the reference sequence of SEQ ID NO: 166.

[0258] In some embodiments, the engineered polynucleotide encodes an engineered vaccinia virus capping enzyme comprising at least one substitutions or substitution sets at positions selected from 45/97/143/209/304/818, 45/97/143/209/304/818/820, 45/97/143/209/818, 45/97/143/209/818/855, 45/97/143/304/446/732/818/855, 45/97/143/304/818, 45/97/143/304/818/820/855, 45/97/209/446/818/820, 45/97/446/818, 45/143, 45/143/209/304/818/820/855, 45/143/209/818, 45/143/304, 45/143/818, 45/143/818/820/855, and 45/143/818/855, or combinations thereof, wherein the amino acid positions are relative to the reference sequence of SEQ ID NO: 242. In some embodiments, the engineered polynucleotide encodes an engineered vaccinia virus capping enzyme comprising at least one substitutions or substitution sets selected from

45 C/97R/143D/209G/304 V/818W, 45 C/97R/ 143D/304V/446K/732V/818W/855 Q, 45C/97R/209G/446K/818W/820Q, 45C/97T/143D/209G/304V/818W/820Q, 45C/97T/143D/209G/818W, 45C/97T/143D/209G/818W/855Q, 45C/97T/143K/304V/818W, 45C/97T/143K/304V/818W/820Q/855Q, 45C/97T/446K/818W, 45C/143D/209G/304V/818W/820Q/855Q, 45C/143D/209G/818W, 45C/143D/304V, 45C/143D/818K/855Q, 45C/143D/818W/820Q/855Q, 45C/143K, and 45C/143K/818W, wherein the amino acid positions are relative to the reference sequence of SEQ ID NO: 242. In some embodiments, the engineered polynucleotide encodes an engineered vaccinia virus capping enzyme comprising at least one substitutions or substitution sets selected from I45C/H97R/L143D/R209G/K304V/D818W,

145 C/H97R/L 143D/K304V/F446K/T732V/D818W/R855 Q, I45C/H97R/R209G/F446K/D818W/P820Q, I45C/H97T/L143D/R209G/K304V/D818W/P820Q, I45C/H97T/L143D/R209G/D818W, I45C/H97T/L143D/R209G/D818W/R855Q, I45C/H97T/L143K/K304V/D818W, I45C/H97T/L143K/K304V/D818W/P820Q/R855Q, I45C/H97T/F446K/D818W, I45C/L143D/R209G/K304V/D818W/P820Q/R855Q, I45C/L143D/R209G/D818W, I45C/L143D/K304V, I45C/L143D/D818K/R855Q, I45C/L143D/D818W/P820Q/R855Q, I45C/L143K, and I45C/L143K/D818W, wherein the amino acid positions are relative to the reference sequence of SEQ ID NO: 242.

[0259] In some embodiments, the engineered polynucleotide encodes an engineered vaccinia virus capping enzyme comprising at least one substitutions or substitution sets at positions selected from 43, 421, 458, 458/562, 474, 552, 594, 601, and 815, or combinations thereof, wherein the amino acid positions are relative to the reference sequence of SEQ ID NO: 288. In some embodiments, the engineered polynucleotide encodes an engineered vaccinia virus capping enzyme comprising at least one substitutions or substitution sets selected from 43N, 421E, 421G, 458C, 458D, 458G, 458G/562S, 474G, 552G, 594R, 601R, 601S, and 815R, wherein the amino acid positions are relative to the reference sequence of SEQ ID NO: 288. In some embodiments, the engineered polynucleotide encodes an engineered vaccinia virus capping enzyme comprising at least one substitutions or substitution sets selected from Y43N, M421E, M421G, Y458C, Y458D, Y458G, Y458G/P562S, N474G, Y552G, K594R, N601R, N601S, and T815R, wherein the amino acid positions are relative to the reference sequence of SEQ ID NO: 288.

[0260] In some embodiments, the engineered polynucleotide encodes an engineered vaccinia virus capping enzyme comprising at least one substitutions or substitution sets at positions selected from 239, 239/251, 239/251/258, 239/258, 251, 258, and 474/552/594/601, or combinations thereof, wherein the amino acid positions are relative to the reference sequence of SEQ ID NO: 318, In some embodiments, the engineered polynucleotide encodes an engineered vaccinia virus capping enzyme comprising at least one substitutions or substitution sets selected from 239R, 239R/251T, 239R/251T/258V, 239R/258V, 25 IT, 258V, and 474G/552G/594R/601R, wherein the amino acid positions are relative to the reference sequence of SEQ ID NO: 318, In some embodiments, the engineered polynucleotide encodes an engineered vaccinia virus capping enzyme comprising at least one substitutions or substitution sets selected from T239R, T239R/R251T, T239R/R251T/Y258V, T239R/Y258V, R25 IT, Y258V, N474G/Y552G/K594R/N601R, wherein the amino acid positions are relative to the reference sequence of SEQ ID NO: 318,

[0261] In some embodiments, the engineered polynucleotide encodes an engineered vaccinia virus capping enzyme comprising at least one substitutions or substitution sets at positions selected from 81/184, 209/355/542, 211, 228, 236, 249, 362, 431, 446, 474, 596, 602, 642, 654, 657, 679, 732, 818, and 820, or combinations thereof, wherein the amino acid positions are relative to the reference sequence of SEQ ID NO: 371. In some embodiments, the engineered polynucleotide encodes an engineered vaccinia virus capping enzyme comprising at least one substitutions or substitution sets selected from 81D/184L, 209G/355R/542G, 211Q, 228S, 236G, 249R, 362T, 431E, 446V, 474L, 596G, 6021, 642D, 654R, 657G, 679E, 732E, 818K, 820F, and 820Y, wherein the amino acid positions are relative to the reference sequence of SEQ ID NO: 371. In some embodiments, the engineered polynucleotide encodes an engineered vaccinia virus capping enzyme comprising at least one substitutions or substitution sets selected from N81D/C184L, R209G/W355R/T542G, N21 IQ, R228S, E236G, K249R, K362T, S431E, F446V, G474L, F596G, K602I, E642D, M654R, K657G, Y679E, T732E, W818K, Q820F, and Q820Y, wherein the amino acid positions are relative to the reference sequence of SEQ ID NO: 371.

[0262] In some embodiments, the engineered polynucleotide encodes an engineered vaccinia virus capping enzyme comprising at least one substitutions or substitution sets at positions selected from 43/97/209/304/548/551/601, 43/209/304/548/551, 43/209/355/542/548/551, 43/542/601, 97/304/542, 97/548, 209/332/355/542, 209/355/542, and 542/548/551/601, or combinations thereof, wherein the amino acid positions are relative to the reference sequence of SEQ ID NO: 373. In some embodiments, the engineered polynucleotide encodes an engineered vaccinia virus capping enzyme comprising at least one substitutions or substitution sets selected from 43N/97T/209G/304V/548G/551G/601R, 43N/209G/304V/548G/551G, 43N/209G/355R/542G/548G/551G, 43N/542G/601R, 97T/304V/542G, 97T/548G, 209G/332G/355R/542G, 209G/355R/542G, and 542G/548G/551G/601R, wherein the amino acid positions are relative to the reference sequence of SEQ ID NO: 373. In some embodiments, the engineered polynucleotide encodes an engineered vaccinia virus capping enzyme comprising at least one substitutions or substitution sets selected from

Y43N/H97T/R209G/K304V/V548G/Q551G/N60IR, Y43N/R209G/K304V/V548G/Q551G, Y43N/R209G/W355R/T542G/V548G/Q551G, Y43N/T542G/N601R, H97T/K304V/T542G, H97T/V548G, R209G/V332G/W355R/T542G, R209G/W355R/T542G, and

T542G/V548G/Q551G/N601R, wherein the amino acid positions are relative to the reference sequence of SEQ ID NO: 373.

[0263] In some embodiments, the engineered polynucleotide encodes an engineered vaccinia virus capping enzyme polypeptide having vaccinia virus capping enzyme activity, wherein the polypeptide comprises an amino acid sequence having at least at least 70%, at least 75%, or at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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% sequence identity to a reference sequence comprising SEQ ID NO: 5, or to a reference sequence of SEQ ID NO: 5, wherein the polypeptide sequence comprises one or more substitutions relative to the reference sequence of SEQ ID NO: 5, as described herein.

[0264] In some embodiments, the engineered polynucleotide encodes an engineered vaccinia virus capping enzyme polypeptide having vaccinia virus capping enzyme activity, wherein the polypeptide comprises an amino acid sequence having at least at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to a reference sequence comprising SEQ ID NO: 5, or to a reference sequence of SEQ ID NO: 5, wherein the polypeptide sequence comprises one or more substitutions relative to the reference sequence of SEQ ID NO: 5, as described herein.

[0265] In some embodiments, the engineered polynucleotide encodes an engineered vaccinia virus capping enzyme polypeptide having vaccinia virus capping enzyme activity, wherein the polypeptide comprises an amino acid sequence having at least at least 70%, at least 75%, or at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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% sequence identity to a reference sequence comprising SEQ ID NO: 107, or to a reference sequence of SEQ ID NO: 107, wherein the polypeptide sequence comprises one or more substitutions relative to the reference sequence of SEQ ID NO: 107, as described herein.

[0266] In some embodiments, the engineered polynucleotide encodes an engineered vaccinia virus capping enzyme polypeptide having vaccinia virus capping enzyme activity, wherein the polypeptide comprises an amino acid sequence having at least at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to a reference sequence comprising SEQ ID NO: 107, or to a reference sequence of SEQ ID NO: 107, wherein the polypeptide sequence comprises one or more substitutions relative to the reference sequence of SEQ ID NO: 107, as described herein.

[0267] In some embodiments, the engineered polynucleotide encodes an engineered vaccinia virus capping enzyme polypeptide having vaccinia virus capping enzyme activity, wherein the polypeptide comprises an amino acid sequence having at least at least 70%, at least 75%, or at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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% sequence identity to a reference sequence comprising SEQ ID NO: 168, or to a reference sequence of SEQ ID NO: 168, wherein the polypeptide sequence comprises one or more substitutions relative to the reference sequence of SEQ ID NO: 168, as described herein.

[0268] In some embodiments, the engineered polynucleotide encodes an engineered vaccinia virus capping enzyme polypeptide having vaccinia virus capping enzyme activity, wherein the polypeptide comprises an amino acid sequence having at least at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to a reference sequence comprising SEQ ID NO: 168, or to a reference sequence of SEQ ID NO: 168, wherein the polypeptide sequence comprises one or more substitutions relative to the reference sequence of SEQ ID NO: 168, as described herein. [0269] In some embodiments, the engineered polynucleotide encodes an engineered vaccinia virus capping enzyme comprising a substitution at position 208, wherein the amino acid positions are relative to the reference sequence of SEQ ID NO: 5. In some embodiments, the engineered polynucleotide encodes an engineered vaccinia virus capping enzyme comprising the substitution 208R, wherein the amino acid positions are relative to the reference sequence of SEQ ID NO: 5. In some embodiments, the engineered polynucleotide encodes an engineered vaccinia virus capping enzyme comprising the substitution L208R, wherein the amino acid positions are relative to the reference sequence of SEQ ID NO: 5.

[0270] In some embodiments, the engineered polynucleotide encodes an engineered vaccinia virus capping enzyme comprising at least one substitutions or substitution sets at positions selected from 78, 78/208/225, 78/208/225/274, 208, and 225, or combinations thereof, wherein the amino acid positions are relative to the reference sequence of SEQ ID NO: 5. In some embodiments, the engineered polynucleotide encodes an engineered vaccinia virus capping enzyme comprising at least one substitutions or substitution sets selected from 78L, 78L/208R/225T, 78L/208W/225T/274P, 208W, and 225T, wherein the amino acid positions are relative to the reference sequence of SEQ ID NO: 5. In some embodiments, the engineered polynucleotide encodes an engineered vaccinia virus capping enzyme comprising at least one substitutions or substitution sets selected from V78L, V78L/L208R/V225T, V78L/L208W/V225T/I274P, L208W, and V225T, wherein the amino acid positions are relative to the reference sequence of SEQ ID NO: 5.

[0271] In some embodiments, the engineered polynucleotide encodes an engineered vaccinia virus capping enzyme comprising a substitution at position 225, wherein the amino acid positions are relative to the reference sequence of SEQ ID NO: 107. In some embodiments, the engineered polynucleotide encodes an engineered vaccinia virus capping enzyme comprising the substitution 225A, wherein the amino acid positions are relative to the reference sequence of SEQ ID NO: 108. In some embodiments, the engineered polynucleotide encodes an engineered vaccinia virus capping enzyme comprising the substitution T225A, wherein the amino acid positions are relative to the reference sequence of SEQ ID NO: 107.

[0272] In some embodiments, the engineered polynucleotide encodes an engineered vaccinia virus capping enzyme comprising at least one substitutions or substitution sets at positions selected from 239 and 258, or combinations thereof, wherein the amino acid positions are relative to the reference sequence of SEQ ID NO: 168. In some embodiments, the engineered polynucleotide encodes an engineered vaccinia virus capping enzyme comprising at least one substitutions or substitution sets selected from 239R, and 258V, wherein the amino acid positions are relative to the reference sequence of SEQ ID NO: 168. In some embodiments, the engineered polynucleotide encodes an engineered vaccinia virus capping enzyme comprising at least one substitutions or substitution sets selected from T239R and Y258V, wherein the amino acid positions are relative to the reference sequence of SEQ ID NO: 168. [0273] In some embodiments, the engineered polynucleotide encodes an engineered vaccinia virus capping enzyme comprising at least one substitutions or substitution sets at positions selected from 239, 239/251, 239/251/258, 239/258, 251, and 258, or combinations thereof, wherein the amino acid positions are relative to the reference sequence of SEQ ID NO: 168. In some embodiments, the engineered polynucleotide encodes an engineered vaccinia virus capping enzyme comprising at least one substitutions or substitution sets selected from 239R, 239R/251T, 239R/251T/258V, 239R/258V, 25 IT, and 258V, wherein the amino acid positions are relative to the reference sequence of SEQ ID NO: 168. In some embodiments, the engineered polynucleotide encodes an engineered vaccinia virus capping enzyme comprising at least one substitutions or substitution sets selected from T239R, T239R/R251T, T239R/R251T/Y258V, T239R/Y258V, R251T, and Y258V, wherein the amino acid positions are relative to the reference sequence of SEQ ID NO: 168.

[0274] In some additional embodiments, the present invention provides an engineered polynucleotide encoding a vaccinia virus capping enzyme or a functional fragment thereof, comprising a polypeptide sequence having at least 75% or more sequence identity to a reference sequence comprising residues 12 to 855 of SEQ ID NO: 3, 14, 105, 166, 242, 288, 318, 371 and/or 373, orto a reference sequence of SEQ ID NO: 3, 14, 105, 166, 242, 288, 318, 371 and/or 373, wherein the polypeptide sequence comprises one or more substitutions relative to the reference sequence comprising residues 12 to 855 of SEQ ID NO: 3, 14, 105, 166, 242, 288, 318, 371 and/or 373, or to the reference sequence of SEQ ID NO: 3, 14, 105, 166, 242, 288, 318, 371 and/or 373.

[0275] In some embodiments, the engineered polypeptide encodes an engineered vaccinia virus capping enzyme comprising a polypeptide sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence comprising residues 12 to 855 of SEQ ID NO: 3, 14, 105, 166, 242, 288, 318, 371 and/or 373 or to the reference sequence of SEQ ID NO: 3, 14, 105, 166, 242, 288, 318, 371 and/or 373.

[0276] In some additional embodiments, the present invention provides an engineered polynucleotide encoding a vaccinia virus capping enzyme or a functional fragment thereof, comprising a polypeptide sequence having at least 75% or more sequence identity to a reference sequence comprising SEQ ID NO: 5, 107, and/or 168, or to a reference sequence of SEQ ID NO: 5, 107, and/or 168, wherein the polypeptide sequence comprises one or more substitutions relative to the reference sequence comprising SEQ ID NO: 5, 107, and/or 168, orto the reference sequence of SEQ ID NO: 5, 107, and/or 168.

[0277] In some embodiments, the engineered polypeptide encodes an engineered vaccinia virus capping enzyme comprising a polypeptide sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence comprising SEQ ID NO: 5, 107, and/or 168, or to a reference sequence of SEQ ID NO: 5, 107, and/or 168, wherein the polypeptide sequence comprises one or more substitutions relative to the reference sequence comprising SEQ ID NO: 5, 107, and/or 168, or to the reference sequence of SEQ ID NO: 5, 107, and/or 168.

[0278] In some embodiments, the polynucleotide encodes an engineered vaccinia virus capping enzyme comprising a polypeptide sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence comprising residues 12 to 855 of SEQ ID NO: 3, 14, 105, 166, 242, 288, 318, 371 and/or 373, wherein the polypeptide sequence optionally comprises one or more amino acid substitutions relative to SEQ ID NO: 3, 14, 105, 166, 242, 288, 318, 371 and/or 373, with the proviso that the polypeptide sequence does not comprise the sequence comprising residues 12 to 855 of SEQ ID NO: 3, 14, 105, 166, 242, 288, 318, 371 and/or 373.

[0279] In some embodiments, the polynucleotide encodes an engineered vaccinia virus capping enzyme comprising a polypeptide sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence comprising residues 12 to 855 of SEQ ID NO: 3, wherein the polypeptide sequence optionally comprises one or more amino acid substitutions relative to SEQ ID NO: 3, with the proviso that the polypeptide sequence does not comprise the sequence comprising residues 12 to 855 of SEQ ID NO: 3.

[0280] In some embodiments, the polynucleotide encodes an engineered vaccinia virus capping enzyme comprising a polypeptide sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence comprising residues 12 to 855 of SEQ ID NO: 14, wherein the polypeptide sequence optionally comprises one or more amino acid substitutions relative to SEQ ID NO: 14, with the proviso that the polypeptide sequence does not comprise the sequence comprising residues 12 to 855 of SEQ ID NO: 14.

[0281] In some embodiments, the polynucleotide encodes an engineered vaccinia virus capping enzyme comprising a polypeptide sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence comprising residues 12 to 855 of SEQ ID NO: 105, wherein the polypeptide sequence optionally comprises one or more amino acid substitutions relative to SEQ ID NO: 105, with the proviso that the polypeptide sequence does not comprise the sequence comprising residues 12 to 855 of SEQ ID NO: 105.

[0282] In some embodiments, the polynucleotide encodes an engineered vaccinia virus capping enzyme comprising a polypeptide sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence comprising residues 12 to 855 of SEQ ID NO: 166, wherein the polypeptide sequence optionally comprises one or more amino acid substitutions relative to SEQ ID NO: 166, with the proviso that the polypeptide sequence does not comprise the sequence comprising residues 12 to 855 of SEQ ID NO: 166.

[0283] In some embodiments, the polynucleotide encodes an engineered vaccinia virus capping enzyme comprising a polypeptide sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence comprising residues 12 to 855 of SEQ ID NO: 242, wherein the polypeptide sequence optionally comprises one or more amino acid substitutions relative to SEQ ID NO: 242, with the proviso that the polypeptide sequence does not comprise the sequence comprising residues 12 to 855 of SEQ ID NO: 242.

[0284] In some embodiments, the polynucleotide encodes an engineered vaccinia virus capping enzyme comprising a polypeptide sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence comprising residues 12 to 855 of SEQ ID NO: 288, wherein the polypeptide sequence optionally comprises one or more amino acid substitutions relative to SEQ ID NO: 288, with the proviso that the polypeptide sequence does not comprise the sequence comprising residues 12 to 855 of SEQ ID NO: 288.

[0285] In some embodiments, the polynucleotide encodes an engineered vaccinia virus capping enzyme comprising a polypeptide sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence comprising residues 12 to 855 of SEQ ID NO: 318, wherein the polypeptide sequence optionally comprises one or more amino acid substitutions relative to SEQ ID NO: 318, with the proviso that the polypeptide sequence does not comprise the sequence comprising residues 12 to 855 of SEQ ID NO: 318.

[0286] In some embodiments, the polynucleotide encodes an engineered vaccinia virus capping enzyme comprising a polypeptide sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence comprising residues 12 to 855 of SEQ ID NO: 371, wherein the polypeptide sequence optionally comprises one or more amino acid substitutions relative to SEQ ID NO: 371, with the proviso that the polypeptide sequence does not comprise the sequence comprising residues 12 to 855 of SEQ ID NO: 371.

[0287] In some embodiments, the polynucleotide encodes an engineered vaccinia virus capping enzyme comprising a polypeptide sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence comprising residues 12 to 855 of SEQ ID NO: 373, wherein the polypeptide sequence optionally comprises one or more amino acid substitutions relative to SEQ ID NO: 373, with the proviso that the polypeptide sequence does not comprise the sequence comprising residues 12 to 855 of SEQ ID NO: 373. [0288] In some embodiments, the polynucleotide encodes an engineered vaccinia vims capping enzyme comprising a polypeptide sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence comprising SEQ ID NO: 5, wherein the polypeptide sequence optionally comprises one or more amino acid substitutions relative to SEQ ID NO: 5.

[0289] In some embodiments, the polynucleotide encodes an engineered vaccinia vims capping enzyme comprising a polypeptide sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence comprising SEQ ID NO: 107, wherein the polypeptide sequence optionally comprises one or more amino acid substitutions relative to SEQ ID NO: 107.

[0290] In some embodiments, the polynucleotide encodes an engineered vaccinia vims capping enzyme comprising a polypeptide sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence comprising SEQ ID NO: 168, wherein the polypeptide sequence optionally comprises one or more amino acid substitutions relative to SEQ ID NO: 168.

[0291] In some embodiments, for each of the foregoing embodiments, the specific amino acid substitutions described herein for the substitution or substitution set can be used for the encoded engineered vaccinia vims capping enzyme polypeptide.

[0292] In some embodiments, the polynucleotide encodes an engineered vaccinia vims capping enzyme comprising a polypeptide sequence comprising at least one substitution provided in Table 6.2, 6.3, 7.1, 8.1, 9.2, 10.2, 11.1, 12.1, 13.1, and/or 13.2, wherein the substitution is relative to SEQ ID NO: 2, 3, 14, 105, 166, 242, 288, 318, 371, and/or 373. In some embodiments, the polynucleotide encodes an engineered vaccinia vims capping enzyme comprising a polypeptide sequence comprising at least one substitution provided in Table 6.2, 7.1, 8.1, 9.2, 11.1, and/or 12.1, wherein the substitution is relative to SEQ ID NO: 5, 107, and/or 168.

[0293] In some embodiments, the polynucleotide encodes an engineered vaccinia vims capping enzyme comprising at least one substitution or substitution set provided in Table 6.2, 6.3, 7.1, 8.1, 9.2, 10.2, 11.1, 12.1, 13.1, and/or 13.2, wherein the substitution or substitution set is relative to SEQ ID NO: 2, 3, 14, 105, 166, 242, 288, 318, 371, 373. In some embodiments, the polynucleotide encodes an engineered vaccinia vims capping enzyme comprising a polypeptide sequence comprising at least one substitution provided in Table 6.2, 7.1, 8.1, 9.2, 11.1, and/or 12.1, wherein the substitution is relative to SEQ ID NO: 5, 107, and/or 168.

[0294] In some embodiments, the polynucleotide encodes an engineered DI vaccinia vims capping enzyme comprising a polypeptide sequence comprising at least 75%, 80%, 85%, 86%, 887%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a sequence comprising residues 12 to 855 of a corresponding SEQ ID NO. set forth in Table 6.2, 6.3, 7.1, 8.1, 9.2, 10.2, 11.1, 12.1, 13.1, and/or 13.2, corresponding to a DI variant. In some embodiments, the polynucleotide encodes an engineered D 1 vaccinia virus capping enzyme comprising a polypeptide sequence comprising at least 75%, 80%, 85%, 86%, 887%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a sequence of a corresponding SEQ ID NO. set forth in Table 6.2, 6.3, 7.1, 8.1, 9.2, 10.2, 11.1, 12.1, 13.1, and/or 13.2, corresponding to a D 1 variant. In some embodiments, the encoded engineered D 1 vaccinia virus capping enzyme comprises a polypeptide sequence comprising residues 12 to 855 of a corresponding SEQ ID NO. set forth in Table 6.2, 6.3, 7.1, 8.1, 9.2, 10.2, 11.1, 12.1, 13.1, and/or 13.2, corresponding to a D 12 variant. In some embodiments, the encoded engineered DI vaccinia virus capping enzyme comprises a polypeptide sequence corresponding to a corresponding SEQ ID NO. set forth in Table 6.2, 6.3, 7.1, 8.1, 9.2, 10.2, 11.1, 12.1, 13.1, and/or 13.2, corresponding to a Dl variant.

[0295] In some embodiments, the polynucleotide encodes an engineered D12 vaccinia virus capping enzyme comprising a polypeptide sequence comprising at least 75%, 80%, 85%, 86%, 887%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a sequence comprising residues 12 to 855 of a corresponding SEQ ID NO. set forth in Table 6.2, 7.1,

8.1, 11.1, and/or 12.1, corresponding to a D 12 variant. In some embodiments, the polynucleotide encodes an engineered D12 vaccinia virus capping enzyme comprising a polypeptide sequence comprising at least 75%, 80%, 85%, 86%, 887%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a sequence of a corresponding SEQ ID NO. set forth in Table 6.2, 7.1, 8.1, 11.1, and/or 12.1, corresponding to a D12 variant. In some embodiments, the encoded engineered vaccinia virus capping enzyme comprises a polypeptide sequence comprising residues 12 to 855 of a corresponding SEQ ID NO. set forth in Table 6.2, 7.1, 8.1, 11.1, and/or 12.1, corresponding to a D 12 variant. In some embodiments, the encoded engineered D12 vaccinia virus capping enzyme comprises a polypeptide sequence corresponding to a SEQ ID NO. set forth in Table

6.2, 7.1, 8.1, 11.1, and/or 12.1, corresponding to a D12 variant.

[0296] In some embodiments, the polynucleotide encodes an engineered vaccinia virus capping enzyme sequence comprising residues 12 to 855 of a corresponding sequence of SEQ ID NO: 6-617, or a fragment thereof, wherein the polypeptide sequence has optionally 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10 substitutions in the polypeptide sequence, as compared with SEQ ID NO: 3.

[0297] In some embodiments, the polynucleotide encodes an engineered vaccinia virus capping enzyme sequence comprising a corresponding sequence among SEQ ID NO: 6-617, or a fragment thereof, wherein the polypeptide sequence has optionally 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10 substitutions in the polypeptide sequence, as compared with SEQ ID NO: 5. In some embodiments, the encoded engineered vaccinia virus capping enzyme polypeptide includes 1, 2, 3, 4, up to 5 substitutions in the polypeptide sequence. In some embodiments, the engineered vaccinia virus capping enzyme polypeptide includes 1, 2, 3, or 4 substitutions in the polypeptide sequence. In some embodiments, the substitutions comprise non-conservative of conservative substitutions. In some embodiments, the substitutions comprise conservative substitutions. In some embodiments, the substitutions comprise non-conservative substitutions. In some embodiments, guidance on nonconservative and conservative substitutions are provided by the variants disclosed herein.

[0298] In some embodiments, the polynucleotide encodes an engineered vaccinia virus capping enzyme polypeptide comprising a polypeptide sequence comprising residues 12 to 855 of SEQ ID NO: 3, 14, 105, 166, 242, 288, 318, 371, or 373, or encodes a polypeptide sequence of SEQ ID NO: 3, 14, 105, 166, 242, 288, 318, 371, or 373, wherein the polypeptide sequence has optionally 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10 substitutions in the polypeptide sequence. In some embodiments, the encoded vaccinia virus capping enzyme includes 1, 2, 3, 4, up to 5 substitutions in the polypeptide sequence. In some embodiments, the encoded vaccinia virus capping enzyme includes 1, 2, 3, or 4 substitutions in the polypeptide sequence.

[0299] In some embodiments, the polynucleotide encodes an engineered vaccinia virus capping enzyme polypeptide comprising a polypeptide sequence comprising SEQ ID NO: 5, 107, or 168, or encodes a polypeptide sequence of SEQ ID NO: 5, 107, or 168, wherein the polypeptide sequence has optionally 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10 substitutions in the polypeptide sequence. In some embodiments, the encoded vaccinia virus capping enzyme includes 1, 2, 3, 4, up to 5 substitutions in the polypeptide sequence. In some embodiments, the encoded vaccinia virus capping enzyme includes 1, 2, 3, or 4 substitutions in the polypeptide sequence.

[0300] In some embodiments, the polynucleotide comprises a polynucleotide sequence comprising at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference polynucleotide sequence comprising SEQ ID NO: 1, or to a reference polynucleotide sequence of SEQ ID NO: 1, encoding an engineered vaccinia virus capping enzyme or a functional fragment thereof.

[0301] In some embodiments, the polynucleotide comprises a polynucleotide sequence comprising at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference polynucleotide sequence comprising nucleotide residues 34 to 2565 of SEQ ID NO: 2, or to a reference polynucleotide sequence of SEQ ID NO: 2, encoding an engineered vaccinia virus capping enzyme or a functional fragment thereof, wherein the engineered polypeptide comprises one or more substitutions at one or more amino acid positions relative to the reference sequence comprising residues 12 to 855 of SEQ ID NO:3, or to the reference sequence of SEQ ID NO: 3.

[0302] In some embodiments, the polynucleotide comprises a polynucleotide sequence comprising at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference polynucleotide sequence comprising nucleotide residues 34 to 2565 of SEQ ID NO: 13, or to a reference polynucleotide sequence of SEQ ID NO: 13, encoding an engineered vaccinia virus capping enzyme or a functional fragment thereof, wherein the engineered polypeptide comprises one or more substitutions at one or more amino acid positions relative to the reference sequence comprising residues 12 to 855 of SEQ ID NO: 14, or to the reference sequence of SEQ ID NO: 14. [0303] In some embodiments, the polynucleotide comprises a polynucleotide sequence comprising at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference polynucleotide sequence comprising nucleotide residues 34 to 2565 of SEQ ID NO: 104, or to a reference polynucleotide sequence of SEQ ID NO: 104, encoding an engineered vaccinia virus capping enzyme or a functional fragment thereof, wherein the engineered polypeptide comprises one or more substitutions at one or more amino acid positions relative to the reference sequence comprising residues 12 to 855 of SEQ ID NO: 105, or to the reference sequence of SEQ ID NO: 105.

[0304] In some embodiments, the polynucleotide comprises a polynucleotide sequence comprising at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference polynucleotide sequence comprising nucleotide residues 34 to 2565 of SEQ ID NO: 165, or to a reference polynucleotide sequence of SEQ ID NO: 165, encoding an engineered vaccinia virus capping enzyme or a functional fragment thereof, wherein the engineered polypeptide comprises one or more substitutions at one or more amino acid positions relative to the reference sequence comprising residues 12 to 855 of SEQ ID NO: 166, or to the reference sequence of SEQ ID NO: 166.

[0305] In some embodiments, the polynucleotide comprises a polynucleotide sequence comprising at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference polynucleotide sequence comprising nucleotide residues 34 to 2565 of SEQ ID NO: 241, or to a reference polynucleotide sequence of SEQ ID NO: 241, encoding an engineered vaccinia virus capping enzyme or a functional fragment thereof, wherein the engineered polypeptide comprises one or more substitutions at one or more amino acid positions relative to the reference sequence comprising residues 12 to 855 of SEQ ID NO:242, or to the reference sequence of SEQ ID NO: 242.

[0306] In some embodiments, the polynucleotide comprises a polynucleotide sequence comprising at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference polynucleotide sequence comprising nucleotide residues 34 to 2565 of SEQ ID NO: 287, or to a reference polynucleotide sequence of SEQ ID NO: 287, encoding an engineered vaccinia virus capping enzyme or a functional fragment thereof, wherein the engineered polypeptide comprises one or more substitutions at one or more amino acid positions relative to the reference sequence comprising residues 12 to 855 of SEQ ID NO:288, or to the reference sequence of SEQ ID NO: 288.

[0307] In some embodiments, the polynucleotide comprises a polynucleotide sequence comprising at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference polynucleotide sequence comprising nucleotide residues 34 to 2565 of SEQ ID NO: 317, or to a reference polynucleotide sequence of SEQ ID NO: 317, encoding an engineered vaccinia virus capping enzyme or a functional fragment thereof, wherein the engineered polypeptide comprises one or more substitutions at one or more amino acid positions relative to the reference sequence comprising residues 12 to 855 of SEQ ID NO:318, or to the reference sequence of SEQ ID NO: 318.

[0308] In some embodiments, the polynucleotide comprises a polynucleotide sequence comprising at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference polynucleotide sequence comprising nucleotide residues 34 to 2565 of SEQ ID NO: 370, or to a reference polynucleotide sequence of SEQ ID NO: 370, encoding an engineered vaccinia virus capping enzyme or a functional fragment thereof, wherein the engineered polypeptide comprises one or more substitutions at one or more amino acid positions relative to the reference sequence comprising residues 12 to 855 of SEQ ID NO:371, or to the reference sequence of SEQ ID NO: 371.

[0309] In some embodiments, the polynucleotide comprises a polynucleotide sequence comprising at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference polynucleotide sequence comprising nucleotide residues 34 to 2565 of SEQ ID NO: 372, or to a reference polynucleotide sequence of SEQ ID NO: 372, encoding an engineered vaccinia virus capping enzyme or a functional fragment thereof, wherein the engineered polypeptide comprises one or more substitutions at one or more amino acid positions relative to the reference sequence comprising residues 12 to 855 of SEQ ID NO:373, or to the reference sequence of SEQ ID NO: 373.

[0310] In some embodiments, the polynucleotide comprises a polynucleotide sequence comprising at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference polynucleotide sequence comprising SEQ ID NO: 4, or to a reference polynucleotide sequence of SEQ ID NO: 4, encoding an engineered vaccinia virus capping enzyme or a functional fragment thereof, wherein the engineered polypeptide comprises one or more substitutions at one or more amino acid positions relative to the reference sequence comprising SEQ ID NO:5, or to the reference sequence of SEQ ID NO: 5.

[0311] In some embodiments, the polynucleotide comprises a polynucleotide sequence comprising at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference polynucleotide sequence comprising SEQ ID NO: 106, or to a reference polynucleotide sequence of SEQ ID NO: 106, encoding an engineered vaccinia virus capping enzyme or a functional fragment thereof, wherein the engineered polypeptide comprises one or more substitutions at one or more amino acid positions relative to the reference sequence comprising SEQ ID NO: 107, or to the reference sequence of SEQ ID NO: 107.

[0312] In some embodiments, the polynucleotide comprises a polynucleotide sequence comprising at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference polynucleotide sequence comprising SEQ ID NO: 167, or to a reference polynucleotide sequence of SEQ ID NO: 167, encoding an engineered vaccinia virus capping enzyme or a functional fragment thereof, wherein the engineered polypeptide comprises one or more substitutions at one or more amino acid positions relative to the reference sequence comprising SEQ ID NO: 168, or to the reference sequence of SEQ ID NO: 168.

[0313] In some embodiments, the polynucleotide encoding an engineered DI vaccinia virus capping enzyme comprises a polynucleotide sequence comprising at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a polynucleotide sequence of nucleotide residues 34 to 2529 of a corresponding sequence provided in SEQ ID NOS: 6-617, wherein the polynucleotide encodes an engineered DI vaccinia virus capping enzyme, as described herein. In some embodiments, the polynucleotide encoding an engineered DI vaccinia virus capping enzyme comprises a polynucleotide sequence of nucleotide residues 34 to 2529 of a sequence provided in SEQ ID NOS: 6-617.

[0314] In some embodiments, the polynucleotide encoding an engineered D12 vaccinia virus capping enzyme comprises a polynucleotide sequence comprising at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a polynucleotide sequence corresponding to a sequence included within SEQ ID NO: 6-617, wherein the polynucleotide encodes an engineered D12 vaccinia virus capping enzyme, as described herein.

[0315] In some embodiments, the polynucleotide encoding an engineered D12 vaccinia virus capping enzyme comprises a polynucleotide sequence provided in SEQ ID NOS: 6-617.

[0316] In some embodiments, the polynucleotide encoding an engineered vaccinia virus capping enzyme comprises a polynucleotide sequence comprising at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a sequence provided in SEQ ID NOS: 6-617, wherein the polynucleotide encodes an engineered vaccinia virus capping enzyme, as described herein.

[0317] In some embodiments, the polynucleotide encoding an engineered vaccinia virus capping enzyme comprises a polynucleotide sequence comprising at least about 75%, about 80%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or more sequence identity to a sequence provided in SEQ ID NOS: 6-617, wherein the polynucleotide encodes an engineered vaccinia virus capping enzyme, as described herein.

[0318] In some embodiments, the polynucleotide encoding an engineered vaccinia virus capping enzyme comprises polypeptide sequences included within SEQ ID NOS: 6-617.

[0319] In some embodiments, an isolated polynucleotide encoding any of the engineered vaccinia virus capping enzyme polypeptides herein is manipulated in a variety of ways to facilitate expression of the vaccinia virus capping enzyme polypeptide. In some embodiments, the polynucleotides encoding the vaccinia virus capping enzyme polypeptides comprise expression vectors where one or more control sequences is present to regulate the expression of the vaccinia virus capping enzyme polynucleotides and/or polypeptides. Manipulation of the isolated polynucleotide prior to its insertion into a vector may be desirable or necessary depending on the expression vector utilized. Techniques for modifying polynucleotides and nucleic acid sequences utilizing recombinant DNA methods are well known in the art. In some embodiments, the control sequences include among others, promoters, leader sequences, polyadenylation sequences, propeptide sequences, signal peptide sequences, and transcription terminators. In some embodiments, suitable promoters are selected based on the host cells selection. For bacterial host cells, suitable promoters for directing transcription of the nucleic acid constructs of the present invention, include, but are not limited to promoters obtained from the E. coli lac operon, Streptomyces coelicolor agarase gene (dagA), Bacillus subtilis levansucrase gene (sacB), Bacillus licheniformis alpha-amylase gene (amyL), Bacillus stearothermophilus maltogenic amylase gene (amyM), Bacillus amyloliquefaciens alpha-amylase gene (amyQ), Bacillus licheniformis penicillinase gene (penP), Bacillus subtilis xylA and xylB genes, and prokaryotic beta-lactamase gene (See e.g., Villa-Kamaroff et al., Proc. Natl Acad. Sci. USA, 1978, 75:3727-3731), as well as the tac promoter (See e.g., DeBoer et al., Proc. Natl Acad. Sci. USA, 1983, 80:21-25). Exemplary promoters for filamentous fungal host cells, include, but are not limited to promoters obtained from the genes for Aspergillus oryzae TAKA amylase, Rhizomucor miehei aspartic proteinase, Aspergillus niger neutral alpha-amylase, Aspergillus niger acid stable alpha-amylase, Aspergillus niger or Aspergillus awamori glucoamylase (glaA), Rhizomucor miehei lipase, Aspergillus oryzae alkaline protease, Aspergillus oryzae triose phosphate isomerase, Aspergillus nidulans acetamidase, and Fusarium oxysporum trypsin-like protease (See e.g., WO 96/00787), as well as the NA2-tpi promoter (a hybrid of the promoters from the genes for Aspergillus niger neutral alpha-amylase and Aspergillus oryzae triose phosphate isomerase), and mutant, truncated, and hybrid promoters thereof. Exemplary yeast cell promoters include, but are not limited to the genes for Saccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae galactokinase (GALI), Saccharomyces cerevisiae alcohol dehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH2/GAP), and Saccharomyces cerevisiae 3 -phosphoglycerate kinase. Other useful promoters for yeast host cells are known in the art (See e.g., Romanos et al., Yeast, 1992, 8:423-488).

[0320] In some embodiments, the control sequence is also a suitable transcription terminator sequence (i.e., a sequence recognized by a host cell to terminate transcription). In some embodiments, the terminator sequence is operably linked to the 3' terminus of the nucleic acid sequence encoding the vaccinia virus capping enzyme polypeptide. Any suitable terminator which is functional in the host cell of choice finds use in the present invention. Exemplary transcription terminators for filamentous fungal host cells can be obtained from the genes for Aspergillus oryzae TAKA amylase, Aspergillus niger glucoamylase, Aspergillus nidulans anthranilate synthase, Aspergillus niger alpha-glucosidase, and Fusarium oxysporum trypsin-like protease. Exemplary terminators for yeast host cells can be obtained from the genes for Saccharomyces cerevisiae enolase, Saccharomyces cerevisiae cytochrome C (CYC1), and Saccharomyces cerevisiae glyceraldehyde-3- phosphate dehydrogenase. Other useful terminators for yeast host cells are known in the art (See e.g., Romanos et al., supra).

[0321] In some embodiments, the control sequence is also a suitable leader sequence (i.e., a nontranslated region of an mRNA that is important for translation by the host cell). In some embodiments, the leader sequence is operably linked to the 5' terminus of the nucleic acid sequence encoding the vaccinia virus capping enzyme polypeptide. Any suitable leader sequence that is functional in the host cell of choice find use in the present invention. Exemplary leaders for filamentous fungal host cells are obtained from the genes for Aspergillus oryzae TAKA amylase, and Aspergillus nidulans triose phosphate isomerase. Suitable leaders for yeast host cells are obtained from the genes for Saccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae 3- phosphoglycerate kinase, Saccharomyces cerevisiae alpha-factor, and Saccharomyces cerevisiae alcohol dehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH2/GAP).

[0322] In some embodiments, the control sequence is also a polyadenylation sequence (i.e., a sequence operably linked to the 3' terminus of the nucleic acid sequence and which, when transcribed, is recognized by the host cell as a signal to add polyadenosine residues to transcribed mRNA). Any suitable polyadenylation sequence which is functional in the host cell of choice finds use in the present invention. Exemplary polyadenylation sequences for filamentous fungal host cells include, but are not limited to the genes for Aspergillus oryzae TAKA amylase, Aspergillus niger glucoamylase, Aspergillus nidulans anthranilate synthase, Fusarium oxysporum trypsin-like protease, and Aspergillus niger alpha-glucosidase. Useful polyadenylation sequences for yeast host cells are known (See e.g., Guo and Sherman, Mol. Cell. Biol., 1995, 15:5983-5990).

[0323] In some embodiments, the control sequence is also a signal peptide (i.e., a coding region that codes for an amino acid sequence linked to the amino terminus of a polypeptide and directs the encoded polypeptide into the cell's secretory pathway). In some embodiments, the 5' end of the coding sequence of the nucleic acid sequence inherently contains a signal peptide coding region naturally linked in translation reading frame with the segment of the coding region that encodes the secreted polypeptide. Alternatively, in some embodiments, the 5' end of the coding sequence contains a signal peptide coding region that is foreign to the coding sequence. Any suitable signal peptide coding region which directs the expressed polypeptide into the secretory pathway of a host cell of choice finds use for expression of the engineered polypeptide(s). Effective signal peptide coding regions for bacterial host cells are the signal peptide coding regions include, but are not limited to those obtained from the genes for Bacillus NC1B 11837 maltogenic amylase, Bacillus stearothermophilus alpha-amylase, Bacillus licheniformis subtilisin, Bacillus licheniformis betalactamase, Bacillus stearothermophilus neutral proteases (nprT, nprS, nprM), and Bacillus suhtilis prsA. Further signal peptides are known in the art (See e.g., Simonen and Palva, Microbiol. Rev., 1993, 57: 109-137). In some embodiments, effective signal peptide coding regions for filamentous fungal host cells include, but are not limited to the signal peptide coding regions obtained from the genes for Aspergillus oryzae TAKA amylase, Aspergillus niger neutral amylase, Aspergillus niger glucoamylase, Rhizomucor miehei aspartic proteinase, Humicola insolens cellulase, and Humicola lanuginosa lipase. Useful signal peptides for yeast host cells include, but are not limited to those from the genes for Saccharomyces cerevisiae alpha-factor and Saccharomyces cerevisiae invertase.

[0324] In some embodiments, the control sequence is also a propeptide coding region that codes for an amino acid sequence positioned at the amino terminus of a polypeptide. The resultant polypeptide is referred to as a “proenzyme,” “propolypeptide,” or “zymogen.” A propolypeptide can be converted to a mature active polypeptide by catalytic or autocatalytic cleavage of the propeptide from the propolypeptide. The propeptide coding region may be obtained from any suitable source, including, but not limited to the genes for Bacillus subtilis alkaline protease (aprE), Bacillus subtilis neutral protease (nprT), Saccharomyces cerevisiae alpha-factor, Rhizomucor miehei aspartic proteinase, and Myceliophthora thermophila lactase (See e.g., WO 95/33836). Where both signal peptide and propeptide regions are present at the amino terminus of a polypeptide, the propeptide region is positioned next to the amino terminus of a polypeptide and the signal peptide region is positioned next to the amino terminus of the propeptide region.

[0325] In some embodiments, regulatory sequences are also utilized. These sequences facilitate the regulation of the expression of the polypeptide relative to the growth of the host cell. Examples of regulatory systems are those that cause the expression of the gene to be turned on or off in response to a chemical or physical stimulus, including the presence of a regulatory compound. In prokaryotic host cells, suitable regulatory sequences include, but are not limited to the lac, tac, and trp operator systems. In yeast host cells, suitable regulatory systems include, but are not limited to the ADH2 system or GALI system. In filamentous fungi, suitable regulatory sequences include, but are not limited to the TAKA alpha-amylase promoter, Aspergillus niger glucoamylase promoter, and Aspergillus oryzae glucoamylase promoter.

[0326] In another aspect, the present invention provides a recombinant expression vector comprising a polynucleotide encoding an engineered vaccinia virus capping enzyme polypeptide, and one or more expression regulating regions such as a promoter and a terminator, a replication origin, etc., depending on the type of hosts into which they are to be introduced. In some embodiments, the various nucleic acid and control sequences described herein are joined together (i.e., operably linked) to produce recombinant expression vectors which include one or more convenient restriction sites to allow for insertion or substitution of the nucleic acid sequence encoding the vaccinia virus capping enzyme polypeptide at such sites. Alternatively, in some embodiments, the nucleic acid sequence of the present invention is expressed by inserting the nucleic acid sequence or a nucleic acid construct comprising the sequence into an appropriate vector for expression. In some embodiments involving the creation of the expression vector, the coding sequence is located in the vector so that the coding sequence is operably linked with the appropriate control sequences for expression. [0327] The recombinant expression vector may be any suitable vector (e.g., a plasmid or virus), that can be conveniently subjected to recombinant DNA procedures and bring about the expression of the vaccinia virus capping enzyme polynucleotide sequence. The choice of the vector typically depends on the compatibility of the vector with the host cell into which the vector is to be introduced. The vectors may be linear or closed circular plasmids.

[0328] In some embodiments, the expression vector is an autonomously replicating vector (i.e., a vector that exists as an extra-chromosomal entity, the replication of which is independent of chromosomal replication, such as a plasmid, an extra-chromosomal element, a minichromosome, or an artificial chromosome). The vector may contain any means for assuring self-replication. In some alternative embodiments, the vector is one in which, when introduced into the host cell, it is integrated into the genome and replicated together with the chromosome(s) into which it has been integrated. Furthermore, in some embodiments, a single vector or plasmid, or two or more vectors or plasmids which together contain the total DNA to be introduced into the genome of the host cell, and/or a transposon is utilized.

[0329] In some embodiments, the expression vector contains one or more selectable markers, which permit easy selection of transformed cells. A “selectable marker” is a gene, the product of which provides for biocide or viral resistance, resistance to heavy metals, prototrophy to auxotrophs, and the like. Examples of bacterial selectable markers include, but are not limited to the dal genes from Bacillus subtilis or Bacillus licheniformis, or markers, which confer antibiotic resistance such as ampicillin, kanamycin, chloramphenicol or tetracycline resistance. Suitable markers for yeast host cells include, but are not limited to ADE2, HIS3, LEU2, LYS2, MET3, TRP1, and URA3. Selectable markers for use in fdamentous fungal host cells include, but are not limited to, amdS (acetamidase; e.g., from A. nidulans or A. orzyae), argB (ornithine carbamoyltransferases), bar (phosphinothricin acetyltransferase; e.g., from .S', hygroscopicus), hph (hygromycin phosphotransferase), niaD (nitrate reductase), pyrG (orotidine-5 '-phosphate decarboxylase; e.g., from A. nidulans or A. orzyae), sC (sulfate adenyltransferase), and trpC (anthranilate synthase), as well as equivalents thereof. In another aspect, the present invention provides a host cell comprising at least one polynucleotide encoding at least one engineered Vaccinia virus capping enzyme polypeptide of the present invention, the polynucleotide(s) being operatively linked to one or more control sequences for expression of the engineered vaccinia virus capping enzyme enzyme(s) in the host cell. Host cells suitable for use in expressing the polypeptides encoded by the expression vectors of the present invention are well known in the art and include but are not limited to, bacterial cells, such as E. coli, Vibrio fluvialis, Streptomyces and Salmonella typhimurium cells; fungal cells, such as yeast cells (e.g., Saccharomyces cerevisiae or Pichia pastoris (ATCC Accession No. 201178)); insect cells such as Drosophila S2 and Spodoptera Sf9 cells; animal cells such as CHO, COS, BHK, 293, and Bowes melanoma cells; and plant cells. Exemplary host cells also include various Escherichia coli strains (e.g., W3110 (AfhuA) and BL21). [0330] Accordingly, in another aspect, the present invention provides methods of producing the engineered vaccinia virus capping enzyme polypeptides, where the methods comprise culturing a host cell capable of expressing a polynucleotide encoding the engineered vaccinia virus capping enzyme polypeptide under conditions suitable for expression of the polypeptide. In some embodiments, the methods further comprise the step(s) of isolating and/or purifying the vaccinia virus capping enzyme polypeptides, as described herein.

[0331] Appropriate culture media and growth conditions for host cells are well known in the art. It is contemplated that any suitable method for introducing polynucleotides for expression of the vaccinia virus capping enzyme polypeptides into cells will find use in the present invention. Suitable techniques include, but are not limited to electroporation, biolistic particle bombardment, liposome mediated transfection, calcium chloride transfection, and protoplast fusion.

[0332] Engineered vaccinia virus capping enzyme polypeptides with the properties disclosed herein can be obtained by subjecting the polynucleotide encoding the naturally occurring or engineered vaccinia virus capping enzyme polypeptide to any suitable mutagenesis and/or directed evolution methods known in the art, and/or as described herein. An exemplary directed evolution technique is mutagenesis and/or DNA shuffling (See e.g., Stemmer, Proc. Natl. Acad. Sci. USA, 1994, 91: 10747- 10751; WO 95/22625; WO 97/0078; WO 97/35966; WO 98/27230; WO 00/42651; WO 01/75767 and U.S. Pat. 6,537,746). Other directed evolution procedures that can be used include, among others, staggered extension process (StEP), in vitro recombination (See e.g., Zhao et al., Nat. Biotechnol., 1998, 16:258-261), mutagenic PCR (See e.g., Caldwell et al., PCR Methods Appl., 1994, 3 : S 136- SI40), and cassette mutagenesis (See e.g., Black et al., Proc. Natl. Acad. Sci. USA, 1996, 93:3525- 3529).

[0333] Mutagenesis and directed evolution methods can be readily applied to vaccinia virus capping enzyme -encoding polynucleotides to generate variant libraries that can be expressed, screened, and assayed. Any suitable mutagenesis and directed evolution methods find use in the present invention and are well known in the art (See e.g., US Patent Nos. 5,605,793, 5,811,238, 5,830,721, 5,834,252, 5,837,458, 5,928,905, 6,096,548, 6,117,679, 6,132,970, 6,165,793, 6,180,406, 6,251,674, 6,265,201,

6.277.638, 6,287,861, 6,287,862, 6,291,242, 6,297,053, 6,303,344, 6,309,883, 6,319,713, 6,319,714, 6,323,030, 6,326,204, 6,335,160, 6,335,198, 6,344,356, 6,352,859, 6,355,484, 6,358,740, 6,358,742, 6,365,377, 6,365,408, 6,368,861, 6,372,497, 6,337,186, 6,376,246, 6,379,964, 6,387,702, 6,391,552, 6,391,640, 6,395,547, 6,406,855, 6,406,910, 6,413,745, 6,413,774, 6,420,175, 6,423,542, 6,426,224, 6,436,675, 6,444,468, 6,455,253, 6,479,652, 6,482,647, 6,483,011, 6,484,105, 6,489,146, 6,500,617,

6.500.639, 6,506,602, 6,506,603, 6,518,065, 6,519,065, 6,521,453, 6,528,311, 6,537,746, 6,573,098, 6,576,467, 6,579,678, 6,586,182, 6,602,986, 6,605,430, 6,613,514, 6,653,072, 6,686,515, 6,703,240, 6,716,631, 6,825,001, 6,902,922, 6,917,882, 6,946,296, 6,961,664, 6,995,017, 7,024,312, 7,058,515, 7,105,297, 7,148,054, 7,220,566, 7,288,375, 7,384,387, 7,421,347, 7,430,477, 7,462,469, 7,534,564, 7,620,500, 7,620,502, 7,629,170, 7,702,464, 7,747,391, 7,747,393, 7,751,986, 7,776,598, 7,783,428, 7,795,030, 7,853,410, 7,868,138, 7,783,428, 7,873,477, 7,873,499, 7,904,249, 7,957,912, 7,981,614, 8,014,961, 8,029,988, 8,048,674, 8,058,001, 8,076,138, 8,108,150, 8,170,806, 8,224,580, 8,377,681, 8,383,346, 8,457,903, 8,504,498, 8,589,085, 8,762,066, 8,768,871, 9,593,326, 9,665,694, 9,684,771, and all related PCT and non-US counterparts; Ling et al., Anal. Biochem., 1997, 254(2): 157-78; Dale et al., Meth. Mol. Biol., 1996, 57:369-74; Smith, Ann. Rev. Genet., 1985, 19:423-462; Botstein et al., Science, 1985, 229: 1193-1201; Carter, Biochem. J., 1986, 237: 1-7; Kramer et al., Cell, 1984, 38:879- 887; Wells et al., Gene, 1985, 34:315-323; Minshull et al., Curr. Op. Chem. Biol., 1999, 3:284-290; Christians et al., Nat. Biotechnol., 1999, 17:259-264; Crameri et al., Nature, 1998, 391:288-291; Crameri, et al., Nat. Biotechnol., 1997, 15:436-438; Zhang et al., Proc. Nat. Acad. Sci. U.S.A., 1997, 94:4504-4509; Crameri et al., Nat. Biotechnol., 1996, 14:315-319; Stemmer, Nature, 1994, 370:389- 391; Stemmer, Proc. Nat. Acad. Sci. USA, 1994, 91: 10747-10751; EP 3 049 973; WO 95/22625; WO 97/0078; WO 97/35966; WO 98/27230; WO 00/42651; WO 01/75767; WO 2009/152336; and WO 2015/048573, all of which are incorporated herein by reference).

[0334] In some embodiments, the enzyme clones obtained following mutagenesis treatment are screened by subjecting the enzyme preparations to a defined temperature (or other assay conditions) and measuring the amount of enzyme activity remaining after heat treatments or other suitable assay conditions. Clones containing a polynucleotide encoding a vaccinia virus capping enzyme polypeptide are then isolated from the gene, sequenced to identify the nucleotide sequence changes (if any), and used to express the enzyme in a host cell. Measuring enzyme activity from the expression libraries can be performed using any suitable method known in the art (e.g., standard biochemistry techniques, such as HPEC analysis).

[0335] For engineered polypeptides of known sequence, the polynucleotides encoding the enzyme can be prepared by standard solid-phase methods, according to known synthetic methods. In some embodiments, fragments of up to about 100 bases can be individually synthesized, then joined (e.g., by enzymatic or chemical ligation methods, or polymerase mediated methods) to form any desired continuous sequence. For example, polynucleotides and oligonucleotides disclosed herein can be prepared by chemical synthesis using the classical phosphoramidite method (See e.g., Beaucage et al., Tet. Eett., 1981, 22: 1859-69; and Matthes et al., EMBO J., 1984, 3:801-05), as it is typically practiced in automated synthetic methods. According to the phosphoramidite method, oligonucleotides are synthesized (e.g., in an automatic DNA synthesizer, purified, annealed, ligated and cloned in appropriate vectors).

[0336] Accordingly, in some embodiments, a method for preparing the engineered vaccinia virus capping enzyme polypeptide can comprise: (a) synthesizing a polynucleotide encoding a polypeptide comprising an amino acid sequence selected from the amino acid sequence of any variant as described herein, and (b) expressing the vaccinia virus capping enzyme polypeptide encoded by the polynucleotide. In some embodiments of the method, the amino acid sequence encoded by the polynucleotide can optionally have one or several (e.g., up to 3, 4, 5, or up to 10) amino acid residue deletions, insertions and/or substitutions. In some embodiments, the amino acid sequence has optionally 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 1-15, 1-20, 1-21, 1-22, 1-23, 1-24, 1-25, 1-30, 1- 35, 1-40, 1-45, or 1-50 amino acid residue deletions, insertions and/or substitutions. In some embodiments, the amino acid sequence has optionally 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 30, 35, 40, 45, or 50 amino acid residue deletions, insertions and/or substitutions. In some embodiments, the amino acid sequence has optionally 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 18, 20, 21, 22, 23, 24, or 25 amino acid residue deletions, insertions and/or substitutions. In some embodiments, the substitutions are conservative or non-conservative substitutions.

[0337] The expressed engineered vaccinia virus capping enzyme polypeptide can be evaluated for any desired improved property or a combination of properties (e.g., activity, capping efficiency, stability, thermostability, soluble expression, etc.) using any suitable assay known in the art, including but not limited to the assays and conditions described herein.

[0338] In some embodiments, any of the engineered vaccinia virus capping enzyme polypeptides expressed in a host cell are recovered from the cells and/or the culture medium using any one or more of the well-known techniques for protein purification, including, among others, lysozyme treatment, sonication, filtration, salting-out, ultra-centrifugation, and chromatography.

[0339] Chromatographic techniques for isolation of the vaccinia virus capping enzyme polypeptides include, among others, reverse phase chromatography, high-performance liquid chromatography, ionexchange chromatography, hydrophobic -interaction chromatography, size-exclusion chromatography, gel electrophoresis, and affinity chromatography. Conditions for purifying a particular enzyme depends, in part, on factors such as net charge, hydrophobicity, hydrophilicity, molecular weight, molecular shape, etc., and will be apparent to those having skill in the art. In some embodiments, affinity techniques may be used to isolate the improved vaccinia virus capping enzyme enzymes. For affinity chromatography purification, any antibody that specifically binds a vaccinia virus capping enzyme polypeptide of interest may find use. For the production of antibodies, various host animals, including but not limited to rabbits, mice, rats, etc., are immunized by injection with a vaccinia virus capping enzyme polypeptide, or a fragment thereof. In some embodiments, the vaccinia virus capping enzyme polypeptide or fragment is attached to a suitable carrier, such as BSA, by means of a side chain functional group or linkers attached to a side chain functional group.

[0340] In some embodiments, the engineered vaccinia virus capping enzyme polypeptide is produced in a host cell by a method comprising culturing a host cell (e.g., an E. coli strain) comprising a polynucleotide sequence encoding an engineered vaccinia virus capping enzyme polypeptide as described herein under conditions conducive to the production of the engineered vaccinia virus capping enzyme polypeptide and recovering the engineered vaccinia virus capping enzyme polypeptide from the cells and/or culture medium. In some embodiments, the host cell produces more than one engineered vaccinia virus capping enzyme polypeptide. [0341] In some embodiments, the present invention provides a method of producing an engineered vaccinia virus capping enzyme polypeptide comprising culturing a recombinant cell comprising a polynucleotide sequence encoding an engineered vaccinia virus capping enzyme polypeptide as described herein, under suitable culture conditions to allow the production of the engineered vaccinia virus capping enzyme polypeptide and optionally recovering the engineered vaccinia virus capping enzyme polypeptide from the culture and/or cultured cells. In some embodiments, the host cell produces more than one engineered vaccinia virus capping enzyme polypeptide. In some embodiments, the host cell is a bacterial cell.

[0342] In some embodiments, the isolated or purified engineered vaccinia virus capping enzyme polypeptides are combined with other ingredients and compounds to provide compositions and formulations comprising the engineered vaccinia virus capping enzyme polypeptide as appropriate for different applications and uses (e.g., diagnostic methods and compositions). In some embodiments, a composition comprises at least one engineered vaccinia virus capping enzyme of the present invention. In some embodiments, the composition further comprises a buffer. In some embodiments, the composition further comprises a substrate, such as nucleotide substrates (e.g., NTPs, NTP analogs, and/or modified NTPs), RNA substrate length, RNA substrate structure, and/or at least methyl donor.

Uses of Engineered Vaccinia Virus Capping Enzyme Polypeptides and Kits

[0343] In another aspect, the present invention provides uses of the engineered vaccinia virus capping enzymes for diagnostic and molecular biological uses, as well as therapeutic and vaccine uses. In addition, the present invention finds use in research uses including, but not limited to labeling of RNA containing 5’ terminal triphosphates.

[0344] The foregoing and other aspects of the invention may be better understood in connection with the following non-limiting examples.

EXPERIMENTAL

[0345] The following Examples, including experiments and results achieved, are provided for illustrative purposes only and are not to be construed as limiting the present invention. Thus, the present invention is described in further detail in the following Examples, which are not in any way intended to limit the scope of the invention as claimed.

[0346] In the experimental disclosure below, the following abbreviations apply: ppm (parts per million); M (molar); mM (millimolar), uM and pM (micromolar); nM (nanomolar); mol (moles); gm and g (gram); mg (milligrams); ug and pg (micrograms); L and 1 (liter); ml and mb (milliliter); cm (centimeters); mm (millimeters); um and pm (micrometers); sec. (seconds); min(s) (minute(s)); h(s) and hr(s) (hour(s)); Q (ohm); pf (microfarad); U (units); MW (molecular weight); rpm (rotations per minute); ref (relative centrifugal force); psi and PSI (pounds per square inch); °C (degrees Centigrade); RT and rt (room temperature); NGS (next-generation sequencing); ds (double stranded); ss (single stranded); CDS (coding sequence); DNA (deoxyribonucleic acid); RNA (ribonucleic acid); dNTPs (deoxynucleoside triphosphates); ATP (adenosine triphosphate); GTP (guanosine triphosphate); dTTP (thymidine triphosphate or deoxythymidine triphosphate); CTP (cytidine triphosphate); UTP (uridine triphosphate); gDNA (human genomic DNA); E. coli W3110 (commonly used laboratory E. coli strain, available from the Coli Genetic Stock Center [CGSC], New Haven, CT); HTP (high throughput); SFP (shake flask powder); HPLC (high pressure liquid chromatography); LC-MS (liquid chromatography - mass spectrometry); ddH2O (double distilled water); PBS (phosphate buffered saline); S-adenosyl methionine (SAM); SDS (sodium dodecyl sulfate); BSA (bovine serum albumin); DTT (dithiothreitol); EDTA (ethylenediaminetetraacetic acid); CAM (chloramphenicol); CAT (chloramphenicol acetyltransferase); LB (Luria-Bertani broth); TB (Terrific Broth); GITC (guanidinium isothiocyanate); IPTG (isopropyl [3-D-l- thiogalactopyranoside); FIOPC (fold improvements over positive control); IVT (in vitro transcription); PCR (polymerase chain reaction); SPRI (solid phase reversible immobilization); Sigma-Aldrich (Sigma-Aldrich, St. Louis, MO); Biotium (Biotium, Inc., Fremont, CA); Eppendorf (Eppendorf North America, Enfield, CT); Microfluidics (Microfluidics International, Inc., Westwood, MA); NEB (New England Biolabs, Inc., Ipswich, MA); Cytiva (Cytiva, Inc., Marlborough, MA); BioRad (Bio-Rad Laboratories, Inc., Hercules, CA); IDT (Integrated DNA Technologies, Coralville, IA); Twist (Twist Bioscience, South San Francisco, CA); Codexis (Codexis, Inc., Redwood City, CA); Promega (Promega, Inc., Madison, WI); Perkin Elmer (Perkin Elmer, Inc, Waltham, MA); Harvard Apparatus (Harvard Apparatus, Holliston, MA); Millipore (Millipore, Corp., Billerica MA); Covaris (Covaris, Inc., Woburn, MA); MagBio (MagBio Genomics, Inc., Gaithersburg, MD); Qiagen (Qiagen Inc., Germantown, MD); Illumina (Illumina, Inc., San Diego, CA); BD Biosciences (BD Biosciences, San Jose, CA); Difco (Difco Laboratories, BD Diagnostic Systems, Detroit, MI); Kuhner (Adolf Kuhner, AG, Basel, Switzerland); Zymo (Zymo Research, Irvine, CA); Agilent (Agilent Technologies, Inc., Santa Clara, CA); ThermoFisher (part of Thermo Fisher Scientific, Waltham, MA); GE Healthcare (GE Healthcare Bio-Sciences, Piscataway, NJ); and Bio-Rad (BioRad Laboratories, Hercules, CA).

EXAMPLE 1

Expression Vectors

[0347] To produce VCE, genes for the two subunits were cloned in either of two different expression vectors. For Examples 6-8, the genes were cloned in pCKl 10900 vector (See, US Pat. Nos. 7,629,157 and 9,714,437, both of which are hereby incorporated by reference) which are operatively linked to the lac promoter under control of the lacl repressor. The expression vector also contains the P15a origin of replication and the chloramphenicol resistance gene. For Examples 9-13, the genes were cloned in pJVl 10900 vector (See, US Pat. No. 10184117). The resulting plasmids were transformed into E. coli W3110, using standard methods known in the art. The transformants were isolated by subjecting the cells to chloramphenicol selection, as known in the art (See e.g., US Pat. No. 8,383,346 and WO 2010/144103).

EXAMPLE 2

E. coli High-Throughput Expression of Recombinant VCE Genes

[0348] In a 96-well format, single colonies were picked and grown in 190 pL LB containing 1% glucose and 30 pg/mL chloramphenicol (CAM), at 30 °C, 200 rpm, and 85% relative humidity. Following overnight growth, 20 pL of the grown cultures were transferred into a deep-well plate containing 380 pL of TB with 30 pg/mL CAM. The cultures were grown at 30 °C, 250 rpm, with 85% relative humidity for approximately 2.25 hours. When the optical density (OD₆oo) of the cultures reached 0.4 - 0.8, expression of the VCE gene was induced by addition of IPTG to a final concentration of 1 mM. Following induction, growth was continued for 18-20 hours at 30 °C, 250 rpm with 85% relative humidity. Cells were harvested by centrifugation at 4000 rpm at 4 °C for 10 - 20 minutes and the media discarded. The cell pellets were stored at -80 °C until ready for use.

EXAMPLE 3

E. coli Shake Flask Expression and Purification of Recombinant VCE Genes [0349] Shake-flask procedures find use in the generation of engineered VCE polypeptide shake flask powders, which are useful for secondary screening assays and/or use in the biocatalytic processes described herein. Shake flask powder (SFP) preparation of enzymes provides a more purified preparation (e.g., up to 30% of total protein) of the engineered enzyme, as compared to the cell lysate used in HTP assays and also allows for the use of more concentrated enzyme solutions. To start the cultures, a single colony of E. coli, transformed with a plasmid encoding an engineered polypeptide of interest, was inoculated into 6 mL LB with 30 pg/mL CAM and 1% glucose. The culture was grown overnight (at least 16 hours) in an incubator at 30 °C, with shaking at 250 rpm. Following the overnight growth, 5 mL of the culture was inoculated into 250 mL of TB with 30 pg/mL CAM, in a IL shake flask. The 250 mL culture was grown at 30 °C at 250 rpm, for 2-3 hours until ODeoo reached 0.4 - 0.8. Expression of the VCE gene was induced by addition of IPTG to a final concentration of 1 mM. Growth was continued for an additional 18-20 hours at 30 °C and 250 rpm. Cells were harvested by transferring the culture into a pre-weighed centrifuge bottle, then centrifuged at 7,000 rpm for 10 minutes at 4 °C. The supernatant was discarded. The remaining cell pellet was weighed. In some embodiments, the cells are stored at -80 °C until ready to use.

[0350] For lysis, the cell pellet was resuspended in 12 mL of cold 50 mM Tris-HCl, pH 8.0, 0.1% Triton X-100, 300 mM NaCl, 10 % glycerol, 1 mM DTT. The cells were lysed using a 110L MICROFLUID IZER® processor system (Microfluidics). Cell debris was removed by centrifugation at 10,000 rpm for 60 minutes at 4 °C. The clarified lysate was collected and purified using HisPur Ni- NTA spin columns (ThermoFisher), following ThermoFisher’s protocol. The columns were equilibrated with 50 mM Tris-HCl, pH 8.0, 0.1% Triton X-100, 300 mM NaCl, 10 % glycerol, 1 mM DTT, with 10 mM imidazole. VCE was eluted using 50 mM Tris-HCl, pH 8.0, 0.1% Triton X-100, 300 mM NaCl, 10 % glycerol, 1 mM DTT, 250 mM imidazole. Eluted VCE was desalted using PD- 10 column (Cytiva), using Cytiva’s recommendation, into 4 mL of 40 mM Tris pH 8.0, 0.1% Triton X-100, 200 mM NaCl, 2 mM DTT, 0.2 mM EDTA. Then, 4 mL of 80% glycerol was added prior to storage at -20 °C.

EXAMPLE 4

Production of RNA Substrate Used in Examples 6 - 8

[0351] For Examples 6 - 8, GlmS-16a mRNA was used as a substrate to evaluate VCE activity. An engineered transcription DNA template GlmS-16A (SEQ ID NO: 618 ) includes a T7 RNAP promoter sequence coupled to the coding sequence for the Bacillus anthracis GlmS-16A riboswitch (SEQ ID NO: 619). Upon induction with glucoseamine-6-phosphate, the GlmS riboswitch self-cleaves and releases a 16-mer RNA (SEQ ID NO: 620). SEQ ID: 618 was expressed in TOPIO cells, following Qiagen’s protocol. The DNA was purified from the cell pellet using a Qiagen Maxiprep Plasmid Kit (Qiagen), following the manufacturer’s protocol. Dried purified DNA was resuspended in nuclease- free water and linearized using /Gm HI (NEB), according to the NEB’s instructions, followed by heat inactivation at 80 °C. SEQ ID NO: 619 was then generated via in vitro transcription (IVT), in which T7 RNA polymerase was incubated with 0.05 mg/mL purified DNA template, 0.002 U/uL pyrophosphatase (NEB), 6 mM ATP, 6 mM GTP, 6 mM UTP, 6 mM CTP, 30 mM MgCh, 10 mM DTT, 1 U/pL RNasin inhibitor, in 50 mM Tris HC1 pH 7.9, for 2 hours, at 37 °C. The RNA was subsequently purified using one of two methods described below.

[0352] Zymo column purification or sodium acetate precipitation was used to clean up the mRNA substrate prior to use in a VCE enzymatic activity assay. IVT-generated mRNA was purified using a Zymo clean and concentrator kit (Zymoresearch), according to the Zymoresearch’s protocol. Alternately, the IVT reaction was treated with 0.3 M sodium acetate followed by 0.7 volumes of isopropanol and allowed to precipitate at -20 °C for at least 2 hours. After centrifugation and supernatant removal, the pellet was resuspended in nuclease free water at 40 °C with shaking and then concentrated and buffer exchanged into nuclease-free water using centrifugal concentrators to reach a final concentration > 0.7 g/L. EXAMPLE 5

Production of RNA Substrate Used in Examples 9 - 13

[0353] The RNA15 substrate (Table 5-1), used for Examples 9 - 13, was prepared by IVT reaction. DNA oligos 1 and 2 (Table 5-1) were annealed to make a DNA template for HICAP™ T7 RNA polymerase (Codexis). The IVT reactions were carried out using 1 pM DNA, 6 mM GTP, 6 mM CTP, 6 mM ATP, 2 mM UTP, 0. 125 pg/pL HICAP™ RNA polymerase, 1 U/pL RNase inhibitor (NEB), 1 U/pL pyrophosphatase (NEB), 30 mM Tris-HCl pH 8.0, 26.9 mM MgCh, 3 mM DTT, for 4 hours, at 30 °C. The reaction was quenched by addition of 1/10 volume of 0.5M EDTA, and the resulting RNA15 was purified using a MONARCH® RNA Cleanup Kit (500 pg) (NEB), following NEB’s protocol. RNA concentration was determined using Qubit™ microRNA assay (ThermoFisher), following manufacturer’s protocol.

[0354] Fluorescently labeled AlexaFluor488-RNA15 substrate (Table 5-1) was prepared similarly to non-labeled RNA 15 with small changes in the IVT reaction composition. Reactions were carried out using GTP, CTP and ATP, at2.5 mM (each), 50 pM ChromaTide™ AlexaFluor488-UTP (ThermoFisher), and 0.025 pg/pL HICAP™ RNA polymerase. The RNA concentration was measured using absorption at 492 nM and using the extinction coefficient 62000 M^-1cm^-1.

EXAMPLE 6

Evolution and Screening of Engineered Polypeptides Derived from SEQ ID NO:1 for Improved RNA Capping Activity

[0355] The engineered polynucleotide (SEQ ID NO: 1), encoding the polypeptide having VCE activity of SEQ ID NOS: 3 and 5, was used to generate the further engineered polypeptides of Table 6-2. These polypeptides displayed improved VCE activity (e.g., % conversion of capO RNA product), as compared to the starting polypeptide. Directed evolution began with the polynucleotide set forth in SEQ ID NO: 1. Engineered polypeptides were then selected as starting “backbone” gene sequences. Libraries of engineered polypeptides were generated using various well-known techniques (e.g., saturation mutagenesis, recombination of previously identified beneficial amino acid differences). Recombinant genes were expressed in high-throughput, as described in Example 2, and screened using HTP assay and analysis methods that measured the polypeptides’ ability to generate capO RNA product.

[0356] The polypeptide encoded by each gene was produced in HTP, as described in Example 2. The cell pellets were lysed by addition of 300 pL of Tris-HCl pH 8 buffer, containing 1 g/L lysozyme, 0.5 g/L Polymyxin B sulfate (PMBS), 0.1% Triton X-100, 0.3 M NaCl, 10% glycerol, and DNasel (NEB) into each well. The plates were sealed and shaken at room temperature for 2 hours. Following lysis, the plates were centrifuged at 4000 rpm for 20 minutes at 4 °C to pellet the cell debris. The clarified lysates were purified using His-PUR plates (ThermoFisher), following ThermoFisher Scientific’s protocol. The plates were washed with 400 pL of Wash buffer (0.05 M Tris-HCl, pH 8, with 0.1% Triton X-100, 0.3 M NaCl, 10% glycerol, 0.001 M DTT, and 0.01 M imidazole) prior to application of the lysate. Clarified lysate was applied to the column, followed by wash with 2 x 400 pL Wash buffer. The VCE variant was eluted using 70 pL elution buffer, containing 0.05 M Tris- HCl, pH 8, with 0.1% Triton X-100, 0.3 M NaCl, 10% glycerol, 0.001 M DTT, and 0.25 M imidazole. After His-PUR purification, samples were desalted using ZEBA™ spin desalting plates, 7K MWCO (ThermoFisher), following ThermoFisher’s protocol. The enzyme was eluted in 0.04 M Tris-HCl, pH 8, with 0.1% Triton X-100, 0.2 M NaCl, 10% glycerol, 0.002 M DTT, and 0.0002 M EDTA. Samples were then diluted 1 : 1 with 90% glycerol and stored at -20 °C until use.

[0357] Purified VCE samples were evaluated in BioRad hardshell plates, in 20.3 pL total volume. For each reaction, 1 pL of the lysate was incubated with 0.45 g/L GlmS-16A RNA, purified using Zymo column as described in Example 4, 0.98 mM MgCU, 0.98 mM DTT, 0.1 mM S-adenosyl methionine (SAM), 0.5 mM GTP, 0.49 U/pL RNAse inhibitor (NEB), 6.5 mM glucosamine 6 phosphate, 4.9 mM NaCl, in 49 mM Tris HC1 at pH 8.0. Ribozyme cleavage and enzyme activity both occurred in parallel at 37 °C for 2 hours. The reaction was quenched by addition of 1.2 mM ethylenediaminetetraacetic acid (EDTA). Samples were diluted two-fold and analyzed by LC-MS, as described in Table 6.1.

[0358] Purified VCE activity was also evaluated using the riboswitch mRNA substrate purified using sodium acetate precipitation, as described in Example 4. Purified VCE samples were diluted 30x in 20 mM Tris HC1, pH 8.0. Reactions were set up with 5 pL diluted VCE enzyme, 0.44 g/L GlmS-16A RNA substrate, 0.97 mM MgCl₂, 0.97 mM DTT, 0.1 mM SAM, 0.5 mM GTP, 0.48 U/pL RNAse inhibitor (NEB), 6.5 mM glucosamine 6 phosphate, 48 mM Tris HC1 at pH 8.0, 4.8 mM NaCl, in 20.7 pL total volume. Samples were incubated at 37 °C for 1 hour. The reaction was quenched by addition of 0.84 mM EDTA, followed by heat treatment at 75 °C for 15 min. Subsequently, 2.9 mM glucosamine 6 phosphate was added to the reaction to activate the GlmS ribozyme. Ribozyme cleavage was done at 37 °C for 60 min. Samples were analyzed by LC-MS, as described in Table 6.1 and results shown on Table 6-3.

EXAMPLE 7

Evolution and Screening of Engineered Polypeptides Derived from SEQ ID: 12

[0359] The engineered polynucleotide (SEQ ID NO: 12) encoding the polypeptide with VCE activity of SEQ ID NO: 14 and 5 was used to generate the engineered polypeptides of Table 7-1. These polypeptides displayed improved VCE activity under the desired conditions (e.g., the production of capO RNA product) as compared to the starting polypeptide. Directed evolution began with the polynucleotide set forth in SEQ ID NO: 12. Libraries of engineered polypeptides were generated using various well-known techniques (e.g., saturation mutagenesis, recombination of previously identified beneficial amino acid differences). Recombinant genes were expressed in high-throughput, as described in Example 2, and screened using the HTP assay below and the analytical method described

[0360] The cell pellets were lysed, similar to Example 6, by addition of 300 pL of Tris-HCl pH 8 buffer, containing 1 g/L lysozyme, 0.5 g/L PMBS, 0.1% Triton X- 100, 0.3 M NaCl, 10% glycerol, and DNasel into each well. The plates were sealed and shaken at room temperature for 2 hours. Following lysis, the plates were centrifuged at 4000 rpm for 20 minutes at 4 °C to pellet the cell debris. The clarified lysates were purified using His-PUR plates. The plates were washed with 400 pL of wash buffer (0.05 M Tris-HCl, pH 8, with 0. 1% Triton X-100, 0.3 M NaCl, 10% glycerol, 0.001 M DTT, and 0.01 M imidazole) prior to application of the lysate. Clarified lysates were applied to the plate, followed by wash with 2 x 400 pL wash buffer. VCE variants were eluted using 70 pL elution buffer, containing 0.05 M Tris-HCl, pH 8, with 0.1% Triton X-100, 0.3 M NaCl, 10% glycerol, 0.001 M DTT, and 0.25 M imidazole. After His-PUR purification, samples were desalted using ZEBA™ spin desalting plates, 7K MWCO. The enzymes were eluted in 0.04 M Tris-HCl, pH 8, with 0.1% Triton X-100, 0.2 M NaCl, 10% glycerol, 0.002 M DTT, and 0.0002 M EDTA. Samples were then diluted 1 : 1 with 90% glycerol at stored at -20 °C until use.

[0361] Evaluation was done using GlmS-16A RNA substrate prepared using sodium acetate precipitation, as described in Example 4. Activity of the purified enzymes was evaluated using a similar assay as described in Example 6. Purified lysates were diluted 30x in 20 mM Tris HC1, pH 8.0. Reactions were carried out using 5 pL enzyme, 0.44 g/L GlmS-16A RNA substrate, 0.97 mM MgCl₂, 0.97 mM DTT, 0.1 mM SAM, 0.5 mM GTP, 0.48 U/pL RNAse inhibitor, 6.5 mM glucosamine 6 phosphate, 48 mM Tris HC1 at pH 8.0, 4.8 mM NaCl, in 20.7 pL total volume/well. Samples were incubated at 37 °C for 1 hour. The reaction was quenched by addition of 0.84 mM EDTA, followed by heat treatment at 75 °C for 15 min. Subsequently, 2.9 mM glucosamine 6 phosphate was added to the reaction to activate the GlmS ribozyme. Ribocleavage was run at 37 °C for 60 min. Samples were analyzed by LC-MS, as described in Table 6.1.

EXAMPLE 8

Evolution and Screening of Engineered Polypeptides Derived from SEQ ID NO: 103 for Improved RNA Capping Activity

[0362] The engineered polynucleotide (SEQ ID NO: 103) encoding the polypeptide with VCE activity of SEQ ID NO: 105 and 107 was used to generate the engineered polypeptides of Table 8-1. These polypeptides displayed improved VCE activity under the desired conditions (e.g., the production of capO RNA product) as compared to the starting polypeptide. Directed evolution began with the polynucleotide set forth in SEQ ID NO: 103. Libraries of engineered polypeptides were generated using various well-known techniques (e.g., saturation mutagenesis, recombination of previously identified beneficial amino acid differences). Recombinant genes were expressed in high- throughput, as described in Example 2, and screened using the HTP assay, described below, and the analytical method described in Table 6-1.

[0363] Similar to Example 6, the cell pellets were lysed by addition of 300 pL of Tris-HCl pH 8 buffer, containing 1 g/L lysozyme, 0.5 g/L PMBS, 0.1% Triton X- 100, 0.3 M NaCl, 10% glycerol, and DNasel into each well. The plates were sealed and shaken at room temperature for 2 hours. Following lysis, the plates were centrifuged at 4000 rpm for 20 minutes at 4 °C to pellet the cell debris. The clarified lysates were purified using His-PUR plates. The plates were washed with 400 pL of wash buffer (0.05 M Tris-HCl, pH 8, with 0. 1% Triton X-100, 0.3 M NaCl, 10% glycerol, 0.001 M DTT, and 0.01 M imidazole) prior to application of the lysate. Clarified lysates were applied to the plate, followed by wash with 2 x 400 pL wash buffer. The VCE variants were eluted using 70 pL elution buffer, containing 0.05 M Tris-HCl, pH 8, with 0.1% Triton X-100, 0.3 M NaCl, 10% glycerol, 0.001 M DTT, and 0.25 M imidazole. After His-PUR purification, samples were desalted using ZEBA™ spin desalting plates, 7K MWCO. The enzymes were eluted in 0.04 M Tris-HCl, pH 8, with 0.1% Triton X-100, 0.2 M NaCl, 10% glycerol, 0.002 M DTT, and 0.0002 M EDTA. Samples were then diluted 1 : 1 with 90% glycerol at stored at -20 °C until use.

[0364] Evaluation was done using GlmS-16A RNA substrate prepared using sodium acetate precipitation, as described in Example 4. Purified enzymes were diluted 120x in 20 mM Tris HC1, pH 8.0. Reactions were set up using with 5 pL enzyme, 0.44 g/L GlmS-16A RNA substrate, 0.97 mM MgC12, 0.97 mM DTT, 0.1 mM SAM, 0.5 mM GTP, 0.48 U/pL RNAse inhibitor, 48 mM Tris HC1 at pH 8.0, 4.8 mM NaCl, in 20.7 pL total volume/well. Samples were incubated at 37 °C for 1 hour. Subsequently, 4.3 mM glucosamine 6 phosphate was added in order to activate the GlmS ribozyme ribocleavage, which was done at 37 °C for 15 min. The reaction was quenched by addition of 0.66 mM EDTA and heat treatment at 75 °C for 15 min. Samples were analyzed by LC-MS, as described in Table 6.1.

EXAMPLE 9

Evolution and Screening of Engineered Polypeptides Derived from SEQ ID NO: 164 for Improved RNA Capping Activity

[0365] The engineered polynucleotide (SEQ ID NO: 164) encoding the polypeptide with VCE activity of SEQ ID NO: 166 and 168 was used to generate the engineered polypeptides of Table 9-2. These polypeptides displayed improved VCE activity under the desired conditions (e.g., the production of capO RNA product) as compared to the starting polypeptide. Directed evolution began with the polynucleotide set forth in SEQ ID NO: 164. Libraries of engineered polypeptides were generated using various well-known techniques (e.g., saturation mutagenesis, recombination of previously identified beneficial amino acid differences). Recombinant genes were expressed in high- throughput, as described in Example 2, and screened using the HTP assay below and the analytical method described in Table 9-1

[0366] High throughput cell pellets were lysed by addition of 400 pL lysis buffer/well, containing 50 mM Tris-HCl, pH 8.0, 0. 1% Triton X-100, 0.3 M NaCl, 10% glycerol, 1 mM DTT, 10 mM imidazole, 0.5 g/L PMBS, 1 g/L lysozyme, 0.5 U/mL DNase I (NEB), one protease inhibitor tablet (Roche)/100 mL lysis buffer. Plates were sealed and shaken for 2 hours at room temperature. After 2 hours, the plates were centrifuged at 4000 rpm in 4 °C for 10-15 minutes. The clarified lysate was purified using HisPur™ Ni-NTA plates (ThermoFisher), using the ThermoFisher’s protocol. The plates were preequilibrated with 50 mM Tris-HCl, pH 8.0, 0.1% Triton X-100, 0.3 M NaCl, 10% glycerol, 1 mM DTT, 10 mM imidazole. The clarified lysate was applied to the HisPur Ni-NTA plates, 200 pL/well. After the binding step, the resin was washed 2 times, each wash with 600 pL of 50 mM Tris-HCl, pH 8.0, 0. 1% Triton X-100, 0.3 M NaCl, 10% glycerol, 1 mM DTT, 10 mM imidazole. The enzyme was eluted with 70 pL of 50 mM Tris-HCl, pH 8.0, 0.1% Triton X-100, 0.3 M NaCl, 10% glycerol, 1 mM DTT, 150 mM imidazole. The eluted enzyme was then mixed with 80% (v/v) glycerol to a final glycerol concentration of 40%.

[0367] Enzyme assays were carried out in a 96-well BioRad hardshell plates, in 20 pL total volume/well. The reactions were carried out using 1 % (v/v) HTP lysate, 20 pM RNA15 (described in Example 5), 0.1 mM SAM, 0.5 mM GTP, 0.5 U/mol RNase inhibitor (NEB), 50 mM Tris-HCl, pH 8.0, 5 mM NaCl, 1 mM MgCU, 1 mM DTT. Reactions were incubated at 37 °C for 1 hour. Samples were quenched by addition of 40 pL of 5 mM EDTA, made in RNAse-free water (ThermoFisher). VCE activity (i.e., the production of capO RNA product) was evaluated using the following LC-MS method in Table 9.1.

EXAMPLE 10

Evolution and Screening of Engineered Polypeptides Derived from SEQ ID NO:240 for Improved RNA Capping Activity

[0368] The engineered polynucleotide (SEQ ID NO: 240) encoding the polypeptide with VCE activity of SEQ ID NO: 242 and 168 was used to generate the engineered polypeptides of Table 10-2. These polypeptides displayed improved VCE activity under the desired conditions (e.g., the production of capO RNA) as compared to the starting polypeptide. Directed evolution began with the polynucleotide set forth in SEQ ID NO: 240. Libraries of engineered polypeptides were generated using various well-known techniques (e.g., saturation mutagenesis, recombination of previously identified beneficial amino acid differences). Recombinant genes were expressed in high-throughput, as described in Example 2, and screened using the HTP assay, described below, and the analytical method described in Table 10-1.

[0369] High throughput pellets were resuspended at room-temperature for 45 mins in 400 pL lysis reagent/well, containing of 50 mM Tris-HCl, pH 8.0, 0.1% Triton X-100, 0.3 M NaCl, 10% glycerol, 1 mM DTT, 10 mM imidazole, 0.5 g/L PMBS, 1 g/L lysozyme, 0.5 U/mL DNasel (NEB), one protease inhibitor tablet (NEB)/ 100 mL lysis buffer. Then, 170 pL of resuspended cells was transferred to 96-well BioRad hardshell plates, sealed and incubated for 30 min at 43.5 °C. After 30 minutes, the plates were centrifuged at 4000 rpm, at 4 °C, for 5 minutes. The clarified lysates were used in the assay.

[0370] Enzyme assay was carried out in a 96-well BioRad hardshell plates, in 20 pL total volume/well. The reactions were carried out using 0.05 % (v/v) HTP lysate, 5-10 pM RNA15 (described in Example 5), 0.1 pM AF488-RNA15 (described in Example 5), 0.1 mM SAM, 0.5 mM GTP, 0.5 U/mol RNase inhibitor (NEB), 50 mM Tris-HCl, pH 8.0, 5 mM NaCl, 1 mM MgCh, 1 mM DTT. Reactions were incubated at 37 °C for 1 hour. Samples were quenched by addition of 20 pL of 5 mM EDTA. Quenched samples were diluted 10-fold in RNase-free water and then 10-fold in Hi -Di formamide (Applied Biosystems), containing Alexa-Fluor633 DNA ladder, which was custom-made for Codexis (sequences are provided in Table 10-1). VCE activity was evaluated using the following capillary electrophoresis method set forth in Table 10.1.

EXAMPLE 11

Evolution and Screening of Engineered Polypeptides Derived SEQ ID NO:286 for Improved RNA Capping Activity

[0371] The engineered polynucleotide (SEQ ID NO: 286), encoding the polypeptide with VCE activity of SEQ ID NO: 288 and 168, was used to generate the engineered polypeptides of Table 11-1. These polypeptides displayed improved VCE activity under the desired conditions (e.g., the production of CapO RNA) as compared to the starting polypeptide. Directed evolution began with the polynucleotide set forth in SEQ ID NO: 286. Libraries of engineered polypeptides were generated using various well-known techniques (e.g., saturation mutagenesis, recombination of previously identified beneficial amino acid differences). Recombinant genes were expressed in high-throughput, as described in Example 2, and screened using the HTP assay below and the analytical method described in Table 10-1.

[0372] Similar to Example 10, high throughput pellets were resuspended at room -temperature for 45 mins in 400 pL lysis reagent/well, containing of 50 mM Tris-HCl, pH 8.0, 0.1% Triton X-100, 0.3 M NaCl, 10% glycerol, 1 mM DTT, 10 mM imidazole, 0.5 g/L PMBS, 1 g/L lysozyme, 0.5 U/mL DNase I, one protease inhibitor tablet/100 mb lysis buffer. Then, 170 pL of resuspended cells was transferred to 96-well BioRad hardshell plates, sealed and incubated for 30 min at 48 °C. Plates were sealed and shaken for 30 minutes at room temperature. After 30 minutes, the plates were centrifuged at 4000 rpm, in 4 °C, for 5 minutes. The clarified lysate was used in the assay. The enzyme assay was carried out in a 96-well BioRad hardshell plates, in 20 pL total volume/well. The reactions were carried out using 0.073 % (v/v) HTP lysate, 5 pM RNA15 (described in Example 5), 0.1 pM AF488- RNA15 (described in Example 5), 0.1 mM SAM, 0.5 mM GTP, 0.5 U/mol RNase inhibitor, 50 mM Tris-HCl, pH 8.0, 5 mM NaCl, 1 mM MgC12, 1 mM DTT. Reactions were incubated at 37 °C for 1 hour. Samples were quenched by addition of 20 pL of 5 mM EDTA. Quenched samples were diluted 10-fold in RNase-free water and then 10-fold in Hi -Di formamide, containing the custom-made AlexaFlour633 DNA ladder (see Table 10-1). VCE activity was evaluated using the capillary electrophoresis method set forth in Table 10.1.

EXAMPLE 12

Evolution and Screening of Engineered Polypeptides Derived from SEQ ID NO:316 for Improved RNA Capping Activity

[0373] The engineered polynucleotide (SEQ ID NO: 316), encoding the polypeptide with VCE activity of SEQ ID NO: 318 and 168, was used to generate the engineered polypeptides of Table 12-1. These polypeptides displayed improved VCE activity under the desired conditions (e.g., the production of CapO RNA) as compared to the starting polypeptide. Directed evolution began with the polynucleotide set forth in SEQ ID NO: 316. Libraries of engineered polypeptides were generated using various well-known techniques (e.g., saturation mutagenesis, recombination of previously identified beneficial amino acid differences). Recombinant genes were expressed in high-throughput, as described in Example 2, and screened using the HTP assay below and the analytical method described in Table 10-1.

[0374] Similar to Example 10, high throughput pellets were resuspended at room -temperature for 30 mins in 400 pL lysis reagent/well, containing of 50 mM Tris-HCl, pH 8.0, 0.1% Triton X-100, 0.3 M NaCl, 10% glycerol, 1 mM DTT, 10 mM imidazole, 0.5 g/L PMBS, 1 g/L lysozyme, 0.5 U/mL DNase I, one protease inhibitor tablet/ 100 mb lysis buffer. Then 170 pL of resuspended samples were transferred to 96-well PCR plates, sealed and incubated for 30 min at 50 °C. After 30 minutes, the plates were centrifuged at 4000 rpm, at 4 °C, for 5 minutes. The clarified lysate was used in the assay. The enzyme assay was carried out in a 96-well PCR plates, in 20 pL total volume/well. The reactions were carried out using 0.025 % (v/v) HTP lysate, 5 pM RNA15 (described in Example 5), 0.075 pM AF488 -RNA 15 (de scribed in Example 5), 0.1 mM SAM, 0.5 mM GTP, 0.5 U/mol RNase inhibitor, 50 mM Tris-HCl, pH 8.0, 5 mM NaCl, 1 mM MgCh, 1 mM DTT. Reactions were incubated at 37 °C for 1 hour. Then, 10 pL of the samples were quenched by addition of 90 pL of 0.25 mM EDTA. Then, 2 microL of the quenched samples were diluted further 10-fold in 18 pL of Hi-Di formamide, containing the custom-made AlexaFluor633 DNA ladder (see Table 10-1). VCE activity was evaluated using the capillary electrophoresis method set forth in Table 10.1.

EXAMPLE 13

Evolution and Screening of Engineered Polypeptides Derived from SEQ ID NO:371 for Improved RNA Capping Activity

[0375] The engineered polynucleotide (SEQ ID NO: 371), encoding the polypeptide with VCE activity of SEQ ID NO: 373 and 375, was used to generate the engineered polypeptides of Table 13-1. These polypeptides displayed improved VCE activity under the desired conditions (e.g., the production of capO RNA product) as compared to the starting polypeptide. Directed evolution began with the polynucleotide set forth in SEQ ID NO: 371. Libraries of engineered polypeptides were generated using various well-known techniques (e.g., saturation mutagenesis, recombination of previously identified beneficial amino acid differences). Recombinant genes were expressed in high- throughput, as described in Example 2, and screened using the HTP assay below and the analytical method described in Table 10-1.

[0376] Similar to Example 10, high throughput pellets were resuspended at room -temperature for 30 mins in 400 pL lysis reagent/well, containing of 50 mM Tris-HCl, pH 8.0, 0.1% Triton X-100, 0.3 M NaCl, 10% glycerol, 1 mM DTT, 10 mM imidazole, 0.5 g/L PMBS, 1 g/L lysozyme, 0.5 U/mL DNase I, one protease inhibitor tablet/ 100 mb lysis buffer. After 30 mins, 170 pL of the resuspended samples were transferred to BioRad hardshell 96 we 11 -plates, sealed and shaken for 30 minutes at 45 °C. The plates were centrifuged at 4000 rpm at 4 °C for 10-15 minutes. The clarified lysate was used in the assay. The enzyme assay was carried out in a 96-well PCR plates, in 20 pL total volume/well. The reactions were carried out using 0.01 % (v/v) HTP lysate, 5 pM RNA15 (described in Example 5), 0.075 pM AF488-RNA15 (described in Example 5), 0.1 mM SAM, 0.5 mM GTP, 0.5 U/mol RNase inhibitor, 50 mM Tris-HCl, pH 8.0, 5 mM NaCl, 1 mM MgC12, 1 mM DTT. Reactions were incubated at 37 °C for 1 hour. Then, 10 pL of the samples were quenched by addition of 90 pL of 0.25 mM EDTA. Then, 2 uL of the quenched samples were diluted further 10-fold in 18 pL of Hi-Di formamide, containing the custom-made AlexaFluor633 DNA ladder (see Table 10-1) VCE activity was evaluated using the capillary electrophoresis method set forth in Table 10.1.

[0377] In a separate evaluation, a set of high throughput pellets were resuspended at room temperature for 30 mins in 400 pL lysis reagent/well, containing of 50 mM Tris-HCl, pH 8.0, 0.1% Triton X-100, 0.3 M NaCl, 10% glycerol, 1 mM DTT, 10 mM imidazole, 0.5 g/L PMBS, 1 g/L lysozyme, 0.5 U/mL DNase I, one protease inhibitor tablet 100 mb lysis buffer. After 30 mins, 170 pL of the resuspended samples were transferred to BioRad hardshell 96 well-plate, sealed and shaken for 30 minutes at 52 °C. The plates were centrifuged at 4000 rpm at 4 °C for 10-15 minutes. The clarified lysate was used in the assay. The enzyme assay was carried out in a 96-well PCR plates, in 20 pL total volume/well. The reactions were carried out using 0.01 % (v/v) HTP lysate, 5 pM RNA15 (described in Example 5), 0.075 pM AF488-RNA15 (described in Example 5), 0.1 mM SAM, 0.5 mM GTP, 0.5 U/mol RNase inhibitor, 50 mM Tris-HCl, pH 8.0, 5 mM NaCl, 1 mM MgC12, 1 mM DTT. Reactions were incubated at 37 °C for 1 hour. Then, 10 pL of the samples were quenched by addition of 90 pL of 0.25 mM EDTA. Then, 2 uL of the quenched samples were diluted further 10- fold in 18 pL of Hi-Di formamide, containing the custom-made AlexaFluor633 DNA ladder (see Table 10-1). VCE activity was evaluated using the capillary electrophoresis method set forth in Table 10.1.

[0378] While the invention has been described with reference to the specific embodiments, various changes can be made and equivalents can be substituted to adapt to a particular situation, material, composition of matter, process, process step or steps, thereby achieving benefits of the invention without departing from the scope of what is claimed.

[0379] For all purposes in the United States of America, each and every publication and patent document cited in this disclosure is incorporated herein by reference as if each such publication or document was specifically and individually indicated to be incorporated herein by reference. Citation of publications and patent documents is not intended as an indication that any such document is pertinent prior art, nor does it constitute an admission as to its contents or date.

Claims

CLAIMS WHAT IS CLAIMED IS:

1. An engineered vaccinia virus capping enzyme, vaccinia virus capping enzyme subunit, or a functional fragment thereof, comprising a polynucleotide sequence having at least 75% or more sequence identity to a reference sequence comprising SEQ ID NO: 1 or to a reference sequence of SEQ ID NO: 1, wherein the polynucleotide sequence comprises one or more substitutions relative to the reference sequence comprising SEQ ID NO: 1, or to the reference sequence of SEQ ID NO: 1.

2. An engineered vaccina virus capping enzyme or vaccinia virus capping enzyme subunit comprising a polypeptide sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence comprising residues 12 to 855 of SEQ ID NO: 3, or to the reference sequence of SEQ ID NO: 3.

3. The engineered vaccinia virus capping enzyme or vaccinia virus capping enzyme subunit of Claim 2, comprising a polypeptide sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence comprising residues 12 to 855 of SEQ ID NO: 14, 105, 166, 242, 288, 318, 371, and/or 373, or to a reference sequence of SEQ ID NO: 14, 105, 166, 242, 288, 318, 371, and/or 373.

4. The engineered vaccinia virus capping enzyme or vaccinia virus capping enzyme subunit of Claim 3, comprising one or more substitutions or substitution sets at amino acid positions selected from 209/212/259/431, 221/304/347/533/550/745/792, 304/347/350/459/787/792, and/or 831, and any combinations thereof, wherein the amino acid positions are numbered with reference to SEQ ID NO: 3.

5. The engineered vaccinia virus capping enzyme or vaccinia virus capping enzyme subunit of Claim 3, comprising one or more substitutions or substitution sets at amino acid positions selected from 42, 44, 97, 184, 210, 211, 332, 355, 368, 433, 450, 542, 546, 654/656, 679, 680, and/or 702, and any combinations thereof, wherein the amino acid positions are numbered with reference to SEQ ID NO: 3.

6. The engineered vaccinia virus capping enzyme or vaccinia virus capping enzyme subunit of Claim 3, comprising one or more substitutions or substitution sets at amino acid positions selected from 209/212/221/431, 209/212/221/745/831, 209/212/533/831, 212/221/431/533, 212/221/431/533/550/745, 212/221/745/831, 221/259/431/533/745, 221/259/431/550, 221/259/550/831, 221/431/550/745, 221/550, 259/533/550/831, 259/533/745, 259/745/831, 259/831, 431, 533, 533/550/745/831, 533/745, and/or 550/745/831, and any combinations thereof, wherein the amino acid positions are numbered with reference to SEQ ID NO: 14.

7. The engineered vaccinia virus capping enzyme or vaccinia virus capping enzyme subunit of Claim 3, comprising one or more substitutions or substitution sets at amino acid positions selected from 184/355/368/542/546/654/680, and/or 225, and any combinations thereof, wherein the amino acid positions are numbered with reference to SEQ ID NO: 105.

8. The engineered vaccinia virus capping enzyme or vaccinia virus capping enzyme subunit of Claim 3, comprising one or more substitutions or substitution sets at amino acid positions selected from 45, 97, 143, 209, 304, 330, 343, 596, 598, 651, and/or 818, and any combinations thereof, wherein the amino acid positions are numbered with reference to SEQ ID NO: 166.

9. The engineered vaccinia virus capping enzyme or vaccinia virus capping enzyme subunit of Claim 3, comprising one or more substitutions or substitution sets at amino acid positions selected from 45/97/143/209/304/818, 45/97/143/209/304/818/820, 45/97/143/209/818, 45/97/143/209/818/855, 45/97/143/304/446/732/818/855, 45/97/143/304/818, 45/97/143/304/818/820/855, 45/97/209/446/818/820, 45/97/446/818, 45/143, 45/143/209/304/818/820/855, 45/143/209/818, 45/143/304, 45/143/818, 45/143/818/820/855, and/or 45/143/818/855, and any combinations thereof, wherein the amino acid positions are numbered with reference to SEQ ID NO: 242.

10. The engineered vaccinia virus capping enzyme or vaccinia virus capping enzyme subunit of Claim 3, comprising one or more substitutions or substitution sets at amino acid positions selected from 43, 421, 458, 458/562, 474, 552, 594, 601, and/or 815, and any combinations thereof, wherein the amino acid positions are numbered with reference to SEQ ID NO: 288.

11. The engineered vaccinia virus capping enzyme or vaccinia virus capping enzyme subunit of Claim 3, comprising one or more substitutions or substitution sets at amino acid positions selected from 239, 239/251, 239/251/258, 239/258, 251, 258, and/or 474/552/594/601, and any combinations thereof, wherein the amino acid positions are numbered with reference to SEQ ID NO: 318.

12. The engineered vaccinia virus capping enzyme or vaccinia virus capping enzyme subunit of Claim 3, comprising one or more substitutions or substitution sets at amino acid positions selected from 81/184, 209/355/542, 211, 228, 236, 249, 362, 431, 446, 474, 596, 602, 642, 654, 657, 679, 732, 818, and/or 820, and any combinations thereof, wherein the amino acid positions are numbered with reference to SEQ ID NO: 371.

13. The engineered vaccinia virus capping enzyme or vaccinia virus capping enzyme subunit of Claim 3, comprising one or more substitutions or substitution sets at amino acid positions selected from 43/97/209/304/548/551/601, 43/209/304/548/551, 43/209/355/542/548/551, 43/542/601, 97/304/542, 97/548, 209/332/355/542, 209/355/542, and/or 542/548/551/601, and any combinations thereof, wherein the amino acid positions are numbered with reference to SEQ ID NO: 373.

14. The engineered vaccinia virus capping enzyme or vaccinia virus capping enzyme subunit of any of Claims 1-13, comprising a polypeptide sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence comprising residues 12 to 855 of SEQ ID NO: 3, 14, 105, 166, 242, 288, 318, 371, and/or 373, or to a reference sequence of SEQ ID NO: 3, 14, 105, 166, 242, 288, 318, 371, and/or 373, with the proviso that the polypeptide sequence does not include the sequence comprising residues 12 to 855 of SEQ ID NO:3.

15. The engineered vaccinia virus capping enzyme or vaccinia virus capping enzyme subunit of Claim 14, wherein the polypeptide sequence comprises one or more amino acid substitutions relative to the reference sequence comprising residues 12 to 855 of SEQ ID NO: 14, or to the reference sequence of SEQ ID NO: 14, with the proviso that the polypeptide sequence does not include the sequence comprising residues 12 to 855 of SEQ ID NO: 3.

16. The engineered vaccinia virus capping enzyme or vaccinia virus capping enzyme subunit of Claim 14, wherein the polypeptide sequence comprises one or more amino acid substitutions relative to the reference sequence comprising residues 12 to 855 of SEQ ID NO: 105, or to the reference sequence of SEQ ID NO: 105, with the proviso that the polypeptide sequence does not include the sequence comprising residues 12 to 855 of SEQ ID NO: 3.

17. The engineered vaccinia virus capping enzyme or vaccinia virus capping enzyme subunit of Claim 14, wherein the polypeptide sequence comprises one or more amino acid substitutions relative to the reference sequence comprising residues 12 to 855 of SEQ ID NO: 166, or to the reference sequence of SEQ ID NO: 166, with the proviso that the polypeptide sequence does not include the sequence comprising residues 12 to 855 of SEQ ID NO: 3.

18. The vaccinia virus capping enzyme or vaccinia virus capping enzyme subunit of Claim 14, wherein the polypeptide sequence comprises one or more amino acid substitutions relative to the reference sequence comprising residues 12 to 855 of SEQ ID NO: 242, or to the reference sequence of SEQ ID NO: 242, with the proviso that the polypeptide sequence does not include the sequence comprising residues 12 to 855 of SEQ ID NO: 3.

19. The engineered vaccinia virus capping enzyme or vaccinia virus capping enzyme subunit of Claim 14, wherein the polypeptide sequence comprises one or more amino acid substitutions relative to the reference sequence comprising residues 12 to 855 of SEQ ID NO: 288, or to the reference sequence of SEQ ID NO: 288, with the proviso that the polypeptide sequence does not include the sequence comprising residues 12 to 855 of SEQ ID NO: 3.

20. The engineered vaccinia virus capping enzyme or vaccinia virus capping enzyme subunit of Claim 14, wherein the polypeptide sequence comprises one or more amino acid substitutions relative to the reference sequence comprising residues 12 to 855 of SEQ ID NO: 318, or to the reference sequence of SEQ ID NO: 318, with the proviso that the polypeptide sequence does not include the sequence comprising residues 12 to 855 of SEQ ID NO: 3.

21. The engineered vaccinia virus capping enzyme or vaccinia virus capping enzyme subunit of Claim 14, wherein the polypeptide sequence comprises one or more amino acid substitutions relative to the reference sequence comprising residues 12 to 855 of SEQ ID NO: 371, or to the reference sequence of SEQ ID NO: 371, with the proviso that the polypeptide sequence does not include the sequence comprising residues 12 to 855 of SEQ ID NO: 3.

22. The engineered vaccinia virus capping enzyme or vaccinia virus capping enzyme subunit of Claim 14, wherein the polypeptide sequence comprises one or more amino acid substitutions relative to the reference sequence comprising residues 12 to 855 of SEQ ID NO: 373, or to the reference sequence of SEQ ID NO: 373, with the proviso that the polypeptide sequence does not include the sequence comprising residues 12 to 855 of SEQ ID NO: 3.

23. An engineered vaccinia virus capping enzyme or vaccinia virus capping enzyme subunit comprising a polypeptide sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence comprising SEQ ID NO: 5, 107, and/or 168, or to a reference sequence of SEQ ID NO: 5, 107, and/or 168.

24. The engineered vaccinia virus capping enzyme or vaccinia virus capping enzyme subunit of Claim 23, comprising a substitution at amino acid position 208, wherein the amino acid positions are numbered with reference to SEQ ID NO: 5.

25. The engineered vaccinia virus capping enzyme or vaccinia virus capping enzyme subunit of Claim 23, comprising one or more substitutions or substitution sets at amino acid positions selected from 78, 78/208/225, 78/208/225/274, 208, and/or 225, and any combinations thereof, wherein the amino acid positions are numbered with reference to SEQ ID NO: 5.

26. The engineered vaccinia virus capping enzyme or vaccinia virus capping enzyme subunit of Claim 23, comprising a substitution at amino acid position 225, wherein the amino acid positions are numbered with reference to SEQ ID NO: 107.

27. The engineered vaccinia virus capping enzyme or vaccinia virus capping enzyme subunit of Claim 23, comprising one or more substitutions at amino acid positions 239 and/or 258, and any combinations thereof, wherein the amino acid positions are numbered with reference to SEQ ID NO: 168.

28. The engineered vaccinia virus capping enzyme or vaccinia virus capping enzyme subunit of Claim 23, comprising one or more substitutions or substitution sets at amino acid positions selected from 239, 239/251, 239/251/258, 239/258, 251, and/or 258, and any combinations thereof, wherein the amino acid positions are numbered with reference to SEQ ID NO: 168.

29. The engineered vaccinia virus capping enzyme or vaccinia virus capping enzyme subunit of any of Claims 1-28, wherein the vaccinia virus capping enzyme comprises a polypeptide sequence comprising a substitution or substitution set provided in Table 6.2, 6.3, 7.1, 8.1, 9.2, 10.2,

11.1, 12.1, 13.1, and/or 13.2, wherein the substitution or substitution set is relative to the reference sequence of SEQ ID NO: 2, 3, 5, 14, 105, 107, 166, 168, 242, 288, 318, 371, and/or 373.

30. The engineered vaccinia virus capping enzyme or vaccinia virus capping enzyme subunit of any of Claims 1-29, wherein the engineered vaccinia virus capping enzyme comprises a polypeptide sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a sequence comprising an engineered vaccinia virus capping enzyme set forth in Table 6.2, 6.3, 7.1, 8.1, 9.2, 10.2, 11.1, 12.1, 13.1, and/or

13.2.

31. The engineered vaccinia virus capping enzyme or vaccinia virus capping enzyme subunit of Claim 3, wherein the polypeptide sequence comprises a sequence comprising residues 12 to 855 of SEQ ID NO: 3, 14, 105, 166, 242, 288, 318, 371, and/or 373.

32. The engineered vaccinia virus capping enzyme or vaccinia virus capping enzyme subunit of Claim 23, wherein the polypeptide sequence comprises SEQ ID NO: 5, 105, and/or 168.

33. The engineered vaccinia virus capping enzyme or vaccinia virus capping enzyme subunit of any one of Claims 0-32, wherein the engineered vaccinia virus capping enzyme has capping activity.

34. The vaccinia virus capping enzyme or vaccinia virus capping enzyme subunit of any one of Claims 0-33, having at least one improved property as compared to the vaccinia virus capping enzyme of SEQ ID NO: 3 and/or 5.

35. The engineered vaccinia virus capping enzyme or vaccinia virus capping enzyme subunit of Claim 34, having at least one improved property, as compared to the vaccinia virus capping enzyme of SEQ ID NO: 3 and/or 5, wherein the improved property is selected from increased activity, increased stability, increased soluble expression, increased thermostability, increased capping, increased thermotolerance, increase resistance to inhibitors, and increased resistance to protease.

36. The engineered vaccinia virus capping enzyme or vaccinia virus capping enzyme subunit of any one of Claims 0-35, wherein the vaccinia virus capping enzyme is purified.

37. An engineered polynucleotide encoding an engineered vaccinia virus capping enzyme or vaccinia virus capping enzyme subunit of any one of Claims 1-36.

38. A engineered polynucleotide comprising at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference polynucleotide sequence of nucleotide residues 34 to 2529 of SEQ ID NO: 2, or to a reference polynucleotide sequence of SEQ ID NO: 2 encoding an engineered vaccinia virus capping enzyme, vaccinia virus capping enzyme subunit, or a functional fragment thereof, wherein the encoded vaccinia virus capping enzyme polypeptide comprises one or more substitutions at one or more amino acid positions relative to the reference sequence comprising residues 12 to 855 of SEQ ID NO: 3, or to a reference sequence of SEQ ID NO: 3.

39. The engineered polynucleotide of Claim 37 or 38, wherein the engineered polynucleotide sequence encodes an engineered vaccinia virus capping enzyme or vaccinia virus capping enzyme subunit comprising a polypeptide sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence comprising residues 12 to 855 of SEQ ID NO: 3, 14, 105, 166, 242, 288, 318, 371, and/or 373, orto a reference sequence of SEQ ID NO: 3, 14, 105, 166, 242, 288, 318, 371, and/or 373.

40. The engineered polynucleotide of Claim 39, wherein the engineered polynucleotide sequence comprises nucleotide residues 34 to 2529 of SEQ ID NO: 2, 13, 104, 165, 241, 287, 317, 370, and/or 372, or comprises the polynucleotide sequence of SEQ ID NO: 2, 13, 104, 165, 241, 287, 317, 370, and/or 372.

41. The engineered polynucleotide sequence of any one of Claims 37-40, wherein the engineered polynucleotide sequence is codon-optimized.

42. An expression vector comprising at least one engineered polynucleotide sequence of any one of Claims 37-41.

43. A host cell transformed with an engineered polynucleotide of any one of Claims 37- 41, or an expression vector of Claim 42.

44. A method of producing an engineered vaccinia virus capping enzyme or an engineered vaccinia virus capping enzyme subunit polypeptide in a host cell comprising culturing a host cell of Claim 43, under suitable culture conditions, such that at least one engineered vaccinia virus capping enzyme or vaccina virus capping enzyme subunit is produced.

45. The method of Claim 44, wherein said vaccinia virus capping enzyme subunit is selected from DI and D12.

46. The method of Claim 44 or 45, further comprising recovering at least one engineered vaccinia virus capping enzyme or vaccinia virus capping enzyme subunit from the culture and/or host cells.

47. The method of Claim 46, further comprising a step of purifying the at least one engineered vaccinia virus capping enzyme or vaccinia virus capping enzyme subunit.

-HO-

48. A composition comprising at least one engineered vaccinia virus capping enzyme or vaccinia virus capping enzyme subunit of any one of Claims 1-36.

49. The composition of Claim 48, comprising at least one buffer.

50. The composition of Claim 48 or 29, further comprising one or more substrates.

51. The composition of any one of Claims 49-50, further comprising one or more methyl donors.

52. A method of capping RNA, comprising providing uncapped RNA, a methyl donor, and comprising contacting the uncapped RNA with the engineered vaccinia virus capping enzyme subunits provided in any one of Claims 1-36, under conditions suitable for capping the uncapped RNA to produce capped RNA.

53. A method of incorporating labeled GTP in RNA containing 5’ terminal triphosphates, comprising contacting labeled GTP and RNA components with the engineered vaccinia virus capping enzyme subunits provided in any of Claims 1-36, under conditions suitable for incorporating the labeled GTP to produce labeled RNA.

54. A kit comprising at least one engineered vaccinia virus capping enzyme of any one of Claims 1-36.

55. The kit of Claim 54, further comprising at least one buffer.