RNA polymerase

Abstract

The invention relates to RNA polymerases, and to variant RNA polymerases. The invention is especially concerned with variant T7 RNA polymerases which generate a modulated amount of double stranded RNA (dsRNA) contamination compared to the parental T7 RNA polymerase during transcription. The invention extends to encoding nucleic acid and protein sequences of such mutant RNA polymerases, and to their use in methods for manufacturing RNA, for example in in vitro transcription (IVT) reactions. The invention extends to RNA manufacturing kits, and to RNA manufactured using such kits, especially for use as vaccine.

Description

[0001] RNA polymerase

[0002] The present invention relates to RNA polymerases, and particularly, although not exclusively, to variant RNA polymerases. The invention is especially concerned with variant T7 RNA polymerases which generate a modulated amount of double stranded RNA (dsRNA) contamination compared to the parental T7 RNA polymerase during transcription. The invention extends to encoding nucleic acid and protein sequences of such mutant RNA polymerases, and to their use in methods for manufacturing RNA, for example in in vitro transcription (IVT) reactions. The invention extends to RNA manufacturing kits, and to RNA manufactured using such kits, especially for use as vaccine.

[0003] Vaccines use the human immune system's ability to react to and remember exposures to pathogen antigens. Thus, vaccines induce a desired immune response that confers protection against infection upon future exposure to a pathogen. Vaccines are both economically and socially important, and in the latest pandemic, especially mRNA vaccines demonstrated their value, both due to extraordinary speedy development as in their superior activity. Simultaneously, the widespread use of the mRNA vaccines against SARS-CoV-2 also demonstrated its shortcomings, including side-effects and / or low vaccination efficiency in specific individuals.

[0004] mRNA vaccines usually comprise a synthetic mRNA molecule encoding the antigen, lipid nanoparticles (LNPs) encapsulating and protecting the mRNA facilitating its delivery into cells, and an adjuvant. Adjuvants are defined as vaccine formulation components added to improve the immune response to the antigen and are a key factor for vaccine efficacy. Traditionally, mineral salts, oil-water emulsions and liposomes have been widely used as adjuvant components. Specifically in mRNA vaccines (e.g., Moderna mRNA-1273 and BioNTech BNT162b2), although the specific impact of each of the components is unknown, ionizable lipids SM-102 and ALC-0315, modified nucleosides pseudouridine, and residual uncapped RNA and dsRNA have been identified as potent immunogenicity factors that serve as built-in and self-adjuvants during vaccination.

[0005] In addition to enhancing the immune response and improving the overall efficacy of vaccines, adjuvants offer other benefits, such as reducing the amount of antigen per vaccine dose and the number of doses required per patient. However, while adjuvants are an essential component of vaccine formulation and efficacy their diversity is scarce and only a few are currently licensed by EMA and FDA for human use. Therefore, the incorporation of novel, safe, efficacious, and scalable supply chain adjuvants isessential to strengthen new-generation vaccines and protect the population from a rapidly spreading pandemic disease agent.

[0006] Although extensively used since the first vaccine formulations, the mechanism of action of adjuvants is not well characterized. However, the current main hypothesis is that adjuvants can elicit a response by activating innate immune cells, which are crucial for initiating and directing adaptive immune responses. The activation of antigen-presenting cells (APC) generates antigen presentation and co-stimulatory signals that can induce the activation of naive T cells, triggering an enhanced adaptive immune response. APCs are equipped with receptors able to detect pathogen-associated molecular patterns (PAMPs).

[0007] Nucleic acids, such as RNA, in the form of double-stranded RNA (dsRNA), are a well-characterized PAMP that can be recognised by MDA-5 and TLR3 receptors. For example, when dsRNA is bound to TLR3, it initiates a signalling cascade that ultimately leads to the activation of genes responsible for type I interferon production, the release of proinflammatory cytokines, the maturation of dendritic cells, the generation of cytotoxic T lymphocyte cells and a Thl-type response. These responses are the hallmark of the effects elicited by adjuvants. Therefore, a careful formulation that includes known or varying amounts of dsRNA in vaccines could generate novel adjuvant vaccine formulations.

[0008] The use of dsRNA as an adjuvant was recently tested in a cancer mRNA vaccine (https: / / doi.org / 10.1073 / pnas.2214320120). The dsRNA was added as a tethered RNA molecule that hybridises with the target RNA forming a dsRNA structure. The efficacy of incorporating exogenous dsRNA into the mRNA vaccine was tested in mouse models of melanoma and lymphoma. The results showed a significantly enhanced cellular immune response, a reduction in tumour size, and an extended lifespan of the mice compared to vaccination without dsRNA as an adjuvant.

[0009] Manufactured RNA is commonly synthesised by in vitro transcription (IVT) by the DNA-dependent RNA polymerases, most commonly the RNA polymerase of bacteriophage T7 (T7RNAP). T7RNAP is a single subunit RNA polymerase of ~100 kDa that catalyses the formation of a phosphodiester bond between ribonucleotides using DNA as a template. In our current understanding, the transcription reaction follows three distinct stages, namely initiation, elongation, and termination, during which T7RNAP adopts two different conformations, i.e. the initiation complex (IC) and elongation complex (EC), as shown in Figure 1. T7RNAP recognises a 23 bp canonicaldouble-stranded DNA (dsDNA) promoter, or (truncated) variants thereof, with high specificity by binding between the nucleotide sequence -17 to -5. Of note, T7RNAP can also bind shorter promoters. The binding of T7RNAP induces the melting of the promoter DNA, primes the addition of the first nucleotide at position +1, most often the GTP nucleotide, and initiates a process known as transcription. Initial transcription consists of very low processivity synthesis of transcripts between 2-9 nt in length, which can be released from the IC in a process called abortive transcription, unless the IC undergoes a transition that forms a stable EC and further elongation of the RNA transcript occurs. The formation of the EC involves a vast conformational change that allows the release of the promoter, the formation of RNA and DNA exit channels, and the increase of stability and processivity of the EC.

[0010] However, under commonly used IVT conditions, T7 RNAP not only catalyses the formation of the target single-strand RNA by elongation of the transcript in direction 5'-3' using DNA as a template, but also the synthesis of spurious products, such as double-stranded RNA and RNA heterogeneous in length at the 3' end. Uncontrolled amounts of contaminating double-stranded RNA (dsRNA) represents a serious limitation in downstream applications, including vaccines, because cellular responses to dsRNA can impair mRNA expression, cause apoptosis, and can induce an undesired immune response, depending on cell type and dose of the RNA. Besides local toxic effects, such responses lead to insufficient expression of the mRNA-encoded antigen, which hampers vaccination efficacy. On the flipside, insufficient adjuvant activity results in insufficient immune system activation to trigger an immune response against the antigen. Thus, there is an urgent need to stringently control the amount of dsRNA byproduct during RNA manufacturing.

[0011] The authors hypothesize that for RNA (e.g. mRNA) vaccines, significant improved vaccination activity and safety profiles can be achieved by stringent control of the ratio of dsRNA:ssRNA, for example by selecting and using different RNA polymerases exhibiting the ability to produce lower or different levels of dsRNA products in an IVT reaction.

[0012] Contaminating dsRNA has been typically controlled using downstream purification strategies, alteration of the IVT conditions, and the use of bacteriophage RNA polymerases homologs of T7 RNAP or T7 RNAP mutants. However, these strategies present problems of costly scalability (purification), insufficient control over the level of dsRNA(all), or / and low yield of the target RNA (RNAP homologues and mutants).Thus, there is a need to provide improved RNA polymerases and / or blends thereof for use in manufacturing RNA, and which result in a controlled and / or predictable amount of dsRNA being produced.

[0013] The amount of dsRNA formed for an RNA sequence is dependent on the primary sequence, the 3' end of the DNA template and the RNA polymerase. Usually, the amount of dsRNA produced by the wildtype RNA polymerase is too high for the intended use, thus requiring a variant RNA polymerase that produces less dsRNA. One particular interesting situation occurs when transcription of an RNA sequence does not produce enough dsRNA for the intended dsRNA-stimulated immune response when the wildtype RNA polymerase is used. In such cases one needs to select a variant RNA polymerase that produces an increased amount of dsRNA to achieve the desired vaccination effect.

[0014] The inventors have carefully investigated the sequence and catalytic activity of T7 RNA polymerase, and developed a series of mutants, based on their research. The catalytic mechanism of T7RNAP involves a two-magnesium-ion-dependent phosphoryl transfer reaction by two aspartic-acid residues (D537 and D812) located in the catalytic centre of the enzyme. In addition to these two aspartic residues, the C-terminal domain of T7RNAP is close to the catalytic centre, which the inventors believe is important for polymerase activity. Limited evidence exists to support this notion, however, modification of the C-terminal carboxy-group negatively affected magnesiumdependent catalysis (Lykke-Andersen J. & Christiansen J. Nucleic Acids Research, 1998;26:5630-5635) and the C-terminus is highly conserved (Mookhtiar KA, et al. Biochemistry, 1991;30:6305-6313). Besides the canonical T7RNAP activity, it has been proposed that T7RNAP can also rebind to its product RNA, producing RNA-templated dsRNA contamination. It can also generate dsRNA contamination via DNA-templated, promoter-less transcription by using the non-template DNA strand.

[0015] Structure-function relationships that provide a mechanistic explanation for dsRNA formation are unknown and thus far the role of the C-terminus in such non-canonical activities has not been characterized. However, previous reports indicated that some phage polymerase homologs to T7RNA polymerases (e.g., at least 20 % identical to T7RNAP wild type) exert lower synthesis of dsRNA contamination. The low sequence homology prevents direct correlation between structure and dsRNA production, especially any correlation of C-terminal amino acids with dsRNA formation is impossible in light of the extensive sequence divergence. Further, such homologues often have other deficiencies, such as lower productivity or undesired reaction conditions, that preclude their use in the manufacturing of synthetic RNA fortherapeutic use. Interestingly, T7RNAP, however, differs from distant homologs in its C-terminal motifs and thus warrants a closer investigation.

[0016] Based on their observations discussed above, the inventors have now hypothesized the importance of a C-terminal motif of T7RNAP, not only for its canonical activity, but also its ability to produce dsRNA. The C-terminal motif of T7RNAP corresponds to D879-F880-A881-F882-A883-COO-. Accordingly, the inventors have developed a series of C-terminal T7RNAP mutants based on this motif, of which several surprisingly result in the production of less dsRNA, with a surprisingly small effect on yield of RNA.

[0017] Thus, in a first aspect of the invention, there is provided a variant RNA polymerase comprising one or more amino acid substitution in its C-terminus compared to the corresponding wild-type C-terminal amino acid sequence, wherein during transcription, the variant RNA polymerase is adapted to produce a modulated amount of double stranded RNA (dsRNA) compared to the corresponding wild-type RNA polymerase.

[0018] The invention is based, at least in part, on the surprising discovery that the C-terminal domain, which, in T7 RNA polymerase, is D879-F880-A881-F882-A883-COO-, is not only important for RNA polymerase activity, but also for dsRNA formation and that the amount of dsRNA produced for a particular ssRNA sequence can be significantly modulated or altered by mutations to the C-terminal domain, and that some phage polymerase mutants with distinct C-terminal motifs exert lower synthesis of dsRNA and some higher. Thus, the variant RNA polymerase of the invention may comprise at least one substitution in the C-terminal motif.

[0019] Modulation of the amount of dsRNA may involve elevating or reducing the dsRNA level that is synthesised. Thus, in one embodiment, the variant RNA polymerase is adapted to produce a higher amount of dsRNA compared to the corresponding wild type RNA polymerase on the same sequence. In another embodiment, however, the variant RNA polymerase is adapted to produce a lower amount of dsRNA compared to the corresponding wild type RNA polymerase on the same sequence.

[0020] As shown herein, variants modified at the C-terminal motif with amino acids distinct from wild-type RNA polymerases produce unpredictable effects. While some substitutions cause enzymatic activity ablation or low RNA yields, some have no detrimental impact on enzymatic activity, and, unexpectedly, some substitutions did not result only in high RNA yields but surprisingly exhibited reduced dsRNA synthesis(e.g. SEQ ID: 100, 197, 216, 124, 172, 76, 201, 220, 262,, 55, 73) or increased dsRNA synthesis (e.g., SEQ ID: 119, 167, 47, 107, 143, 239, 169, 175, 240).

[0021] Accordingly, it will be appreciated, that one or more amino acid substitutions in the C-terminal motif of the RNA polymerase can be associated with a phenotype described herein. However, this effect was unpredictable.

[0022] The variant RNA polymerase may comprise a variant T7, T3, Kll, SP6, KP34, Syn5, or other RNA polymerase. Typically, however, the variant RNA polymerase comprises a variant T7 RNA polymerase.

[0023] The invention provides the wild-type T7 RNA polymerase comprising 883 continuous amino acids. This polypeptide has canonical T7RNAP activity and can result in the synthesis of dsRNA, which may be either RNA- or DNA-dependent dsRNA synthesis. In an embodiment, the amino acid sequence of wild-type RNA polymerase is represented herein, as SEQ ID No: 1, as follows:

[0024] MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHESYEMGEARFRKMFERQLKAGEVADNAAAKPL ITTLLPKMIARINDWFEEVKAKRGKRPTAFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIE DEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEM LIESTGMVSLHRQNAGWGQDSETIELAPEYAEAIATRAGALAGISPMFQPCVVPPKPWTGITGGGYWANGRR PLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPE DIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGND MTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPFCF LAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQA DAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQP AIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTP DGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNFVHSQDGSHLRKTVVWAHEKYG IESFALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAKGNLNLRDIL ESDFAFA

[0025] [SEQ ID No: 1]

[0026] Thus, a wild-type RNA polymerase may comprise an amino acid sequence substantially as set out in SEQ ID No: 1, or a fragment or variant thereof.

[0027] As can be seen in SEQ ID No: 1, the C-terminal amino acid sequence comprises D879-F880-A881-F882-A883-COO-.

[0028] In an embodiment, wild-type RNA polymerase is encoded by a DNA sequence represented herein, as SEQ ID No: 2, as follows: atgaacacgattaacatcgctaagaacgacttctctgacatcgaactggctgctatcccgttcaacactctgg ctgaccattacggtgagcgtttagctcgcgaacagttggcccttgagcatgagtcttacgagatgggtgaagc acgcttccgcaagatgtttgagcgtcaacttaaagctggtgaggttgcggataacgctgccgccaagcctctc atcactaccctactccctaagatgattgcacgcatcaacgactggtttgaggaagtgaaagctaagcgcggca agcgcccgacagccttccagttcctgcaagaaatcaagccggaagccgtagcgtacatcaccattaagaccac tctggcttgcctaaccagtgctgacaatacaaccgttcaggctgtagcaagcgcaatcggtcgggccattgaggacgaggctcgcttcggtcgtatccgtgaccttgaagctaagcacttcaagaaaaacgttgaggaacaactca acaagcgcgtagggcacgtctacaagaaagcatttatgcaagttgtcgaggctgacatgctctctaagggtct actcggtggcgaggcgtggtcttcgtggcataaggaagactctattcatgtaggagtacgctgcatcgagatg ctcattgagtcaaccggaatggttagcttacaccgccaaaatgctggcgtagtaggtcaagactctgagacta tcgaactcgcacctgaatacgctgaggctatcgcaacccgtgcaggtgcgctggctggcatctctccgatgtt ccaaccttgcgtagttcctcctaagccgtggactggcattactggtggtggctattgggctaacggtcgtcgt cctctggcgctggtgcgtactcacagtaagaaagcactgatgcgctacgaagacgtttacatgcctgaggtgt acaaagcgattaacattgcgcaaaacaccgcatggaaaatcaacaagaaagtcctagcggtcgccaacgtaat caccaagtggaagcattgtccggtcgaggacatccctgcgattgagcgtgaagaactcccgatgaaaccggaa gacatcgacatgaatcctgaggctctcaccgcgtggaaacgtgctgccgctgctgtgtaccgcaaggacaagg ctcgcaagtctcgccgtatcagccttgagttcatgcttgagcaagccaataagtttgctaaccataaggccat ctggttcccttacaacatggactggcgcggtcgtgtttacgctgtgtcaatgttcaacccgcaaggtaacgat atgaccaaaggactgcttacgctggcgaaaggtaaaccaatcggtaaggaaggttactactggctgaaaatcc acggtgcaaactgtgcgggtgtcgataaggttccgttccctgagcgcatcaagttcattgaggaaaaccacga gaacatcatggcttgcgctaagtctccactggagaacacttggtgggctgagcaagattctccgttctgcttc cttgcgttctgctttgagtacgctggggtacagcaccacggcctgagctataactgctcccttccgctggcgt ttgacgggtcttgctctggcatccagcacttctccgcgatgctccgagatgaggtaggtggtcgcgcggttaa cttgcttcctagtgaaaccgttcaggacatctacgggattgttgctaagaaagtcaacgagattctacaagca gacgcaatcaatgggaccgataacgaagtagttaccgtgaccgatgagaacactggtgaaatctctgagaaag tcaagctgggcactaaggcactggctggtcaatggctggcttacggtgttactcgcagtgtgactaagcgttc agtcatgacgctggcttacgggtccaaagagttcggcttccgtcaacaagtgctggaagataccattcagcca gctattgattccggcaagggtctgatgttcactcagccgaatcaggctgctggatacatggctaagctgattt gggaatctgtgagcgtgacggtggtagctgcggttgaagcaatgaactggcttaagtctgctgctaagctgct ggctgctgaggtcaaagataagaagactggagagattcttcgcaagcgttgcgctgtgcattgggtaactcct gatggtttccctgtgtggcaggaatacaagaagcctattcagacgcgcttgaacctgatgttcctcggtcagt tccgcttacagcctaccattaacaccaacaaagatagcgagattgatgcacacaaacaggagtctggtatcgc tcctaactttgtacacagccaagacggtagccaccttcgtaagactgtagtgtgggcacacgagaagtacgga atcgaatcttttgcactgattcacgactccttcggtaccattccggctgacgctgcgaacctgttcaaagcag tgcgcgaaactatggttgacacatatgagtcttgtgatgtactggctgatttctacgaccagttcgctgacca gttgcacgagtctcaattggacaaaatgccagcacttccggctaaaggtaacttgaacctccgtgacatctta gagtcggacttcgcgttcgcgtaa

[0029] [SEQ ID No: 2]

[0030] Thus, the wild-type RNA polymerase may be encoded by a nucleotide sequence substantially as set out in SEQ ID No: 2, or a fragment or variant thereof.

[0031] In some embodiments, the variant RNA polymerase has an amino acid sequence comprising at least 80%, 90% or 95% identity to positions 1 to 883 of SEQ ID NO:1. In some embodiments, the variant RNA polymerase comprises an amino acid sequence of at least 96%, 97%, 98% or 99% identity to positions 1 to 883 of SEQ ID NO:1. In other embodiments, the variant RNA polymerase comprises an amino acid sequence of at least 99.1%, 99.2%, 99.3% or 99.4% identity to positions 1 to 883 of SEQ ID NO: 1. It will be appreciated that five mutations (i.e. four C-terminal mutations and one other) represents 0.57% difference to the wild-type sequence.

[0032] The invention provides mutants of T7 RNA polymerase comprising at least 1, 2, 3 or 4 amino acid substitutions corresponding to positions 879, 880, 881, 882, and / or 883 in SEQ ID NO:1 relative to the T7RNAP polymerase. These substitutions generated 240 variants (as shown in Table 1, i.e. the C-terminal motif of each of these variants (VIto V240) is shown herein as SEQ ID No: 11-250) with some unpredicted effects, including improved characteristics, activity ablation, or poor protein expression.

[0033] Thus, in some embodiments, the variant RNA polymerase may comprise a C-terminal motif comprising an amino acid sequence substantially as set out in any one or more of SEQ ID No: 11-250, or a fragment or variant thereof.

[0034] Unexpectedly, and advantageously, some amino acid substitutions generated mutants of T7RNAP that have canonical RNA Polymerase activity but surprisingly resulted in reduced dsRNA contamination (e.g. SEQ ID: 5, 7, 9, 251, 253, 255, 257, 259, 118, 172, 55, 73) or increased dsRNA production (e.g. SEQ ID: 119, 167, 47, 107, 143, 239, 169, 175, 240) on the sequences tested. For polymerase variants exhibiting a desired RNA yield and reduced dsRNA contamination, the 240 variant screen data was further confirmed for 12 variants.

[0035] As described in the Examples, mutants having a modified or substituted F882 showed inferior activity. Thus, typically the variant RNA polymerase comprises an amino acid sequence corresponding to the wild-type sequence at position F882 in SEQ ID No: 1. In other words, in some embodiments, the variant RNA polymerase does not comprise an amino acid substitution at position F882 in SEQ ID No: 1, i.e. the enzyme has a phenylalanine at position 882.

[0036] Therefore, in certain embodiments, the variant RNA polymerase may comprise an amino acid substitution at one or more positions selected from positions D879, F880, A881, and A883 in SEQ ID No: l.

[0037] The variant RNA polymerase may comprise an amino acid substitution at two or more positions selected from positions D879, F880, A881, and A883 in SEQ ID No: l.

[0038] The variant RNA polymerase may comprise an amino acid substitution at three or more positions selected from positions D879, F880, A881, and A883 in SEQ ID No: l.

[0039] The variant RNA polymerase may comprise an amino acid substitution at four or more positions selected from positions D879, F880, A881, and A883 in SEQ ID No: l.In embodiments where the polymerase produces reduced dsRNA levels, the variant RNA polymerase may comprise a C-terminal motif that has a larger volume than the corresponding C-terminus of the wild-type RNA polymerase through substitution of one or more amino acids.

[0040] For reduced dsRNA production, the variant RNA polymerase (T7 RNAP or another RNA polymerase, such as T3, K11, SP6, KP34, Syn5, or any other RNA polymerase) may comprise a C-terminal motif comprising an amino acid sequence, wherein the terminal amino acid is small and more or less polar, wherein the second to last is bulky, aromatic and typically hydrophobic, wherein the third to last amino acid is hydrophobic, and wherein the fourth to last amino acid is bulky and aromatic, and the combination of the four C-terminal amino acids does not correspond to the native C-terminus of the wild-type RNA polymerase.

[0041] However, in embodiments where the polymerase produces increased dsRNA levels, the variant RNA polymerase may comprise a C-terminal motif comprising an amino acid sequence, wherein the terminal amino acid is small and slightly hydrophobic, wherein the second to last is bulky, aromatic and typically hydrophobic, wherein the third to last is small and slightly hydrophobic, and wherein the fourth to last amino acid is bulky and aromatic, and the combination of the four C-terminal amino acids does not correspond to the native C-terminus of the wild-type RNA polymerase.

[0042] Thus, for reduced dsRNA production, the variant RNA polymerase may comprise a C-terminal motif comprising an amino acid sequence wherein the terminal amino acid is selected from C, S, T, A, V or N, wherein the second to last amino acid is selected from F, Y, L, W or H, wherein the third to last amino acid is selected from F, Y, L, I, V, W, A, P, and wherein the fourth to last amino acid is selected from F, Y, H, or W, and the combination of the four C-terminal amino acids does not correspond to the native C-terminus of the wild-type RNA polymerase. The skilled person would readily know how to find the corresponding amino acid positions in any other RNA polymerase, such as T3, Kll, SP6, KP34, or Syn5, for each of the above amino acid residues in T7 RNAP.

[0043] For increased dsRNA production, however, the variant RNA polymerase (T7 RNAP or another RNA polymerase, such as T3, Kll, SP6, KP34, Syn5, or any other RNA polymerase) may comprise a C-terminal motif comprising an aminoacid sequence wherein the terminal amino acid is selected from A, G, or P, typically A, wherein the second to last amino acid is selected from F, Y, L, W or H, typically F, wherein the third to last amino acid is selected from A, G, V, or I, typically A, and wherein the fourth to last amino acid is selected from F, Y, H, or W, typically Y or F, and the combination of the four C-terminal amino acids does not correspond to the native C-terminus of the wild-type RNA polymerase. The skilled person would readily know how to find the corresponding amino acid positions in any other RNA polymerase, such as T3, K11, SP6, KP34, or Syn5, for each of the above amino acid residues in T7 RNAP.

[0044] Hence, in embodiments where the substitution is at position D879, the substitution may be selected from any of the 20 standard amino acids, optionally other than aspartic acid (D), which is the wild-type amino acid at this position. In embodiments where the substitution is at position D879, the substitution may be typically selected from D879T, D879P, D879E, D879V, D879Q, D879S, D879A, and D879I. More typically, however, the D879 substitution comprises a D879V, D879R or D879I substitution for reduction of dsRNA. However, typically the D879 substitution comprises a D879A, D879Q, or D879S for increase of dsRNA.

[0045] In embodiments where the substitution is at position F880, the substitution may be selected from any of the 20 standard amino acids, optionally other than phenylalanine (F), which is the wild-type amino acid at this position. In embodiments where the substitution is at position F880, the substitution may be typically selected from F880K, F880Y, F880H, F880W, and F880A. More typically, however, the F880 substitution comprises a F880Y substitution.

[0046] In embodiments where the substitution is at position A881, the substitution may be selected from any of the 20 standard amino acids, optionally other than alanine (A), which is the wild-type amino acid at this position. In embodiments, where the substitution is at position A881, the substitution may be typically selected from A881F, A881Y, A881L, A881I, A881V, A881W, and A881P. More typically, however, the A881 substitution comprises a A881F for reduction of dsRNA. For increased production of dsRNA, the A881 position is typically unchanged.

[0047] In embodiments where the substitution is at position A882, the substitution may be selected from any of the 20 standard amino acids, optionally other than phenylalanine (F), which is the wild-type amino acid at this position. In embodiments, where the substitution is at position F882, the substitution may be typically selectedfrom F882L, F882Y, F882W and F882H. More typically, however, the F882 substitution comprises a F882L, F882Y, or F882W for reduction of dsRNA. For increased production of dsRNA, the F882 position is typically unchanged.

[0048] In embodiments where the substitution is at position A883, the substitution may be selected from any of the 20 standard amino acids, optionally other than alanine (A), which is the wild-type amino acid at this position. In embodiments, where the substitution is at position A883, the substitution may be typically selected from A883C, A883S, A883T, A883V and A883N. More typically, however, the A883 substitution comprises a A883C or A883S for reduction of dsRNA. For increased production of dsRNA, the A883 position is typically unchanged, or comprises a A883C or A883S substitution.

[0049] The inventors modified wild-type T7 RNA polymerase by substituting amino acid P266L to thereby create a "parental" sequence. Thus, the invention provides a variant T7 RNAP having 883 continuous amino acids with the amino acid substitution P266L. In an embodiment, the amino acid sequence of the parental RNA polymerase is represented herein, as SEQ ID No: 3, as follows:

[0050] MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHESYEMGEARFRKMFERQLKAGEVADNAAAKPL ITTLLPKMIARINDWFEEVKAKRGKRPTAFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIE DEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEM LIESTGMVSLHRQNAGWGQDSETIELAPEYAEAIATRAGALAGISLMFQPCVVPPKPWTGITGGGYWANGRR PLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPE DIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGND MTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPFCF LAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQA DAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQP AIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTP DGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNFVHSQDGSHLRKTVVWAHEKYG IESFALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAKGNLNLRDIL ESDFAFA

[0051] [SEQ ID No: 3]

[0052] Thus, a parental RNA polymerase may comprise an amino acid sequence substantially as set out in SEQ ID No: 3, or a fragment or variant thereof.

[0053] In an embodiment, the parental RNA polymerase is encoded by a DNA sequence represented herein, as SEQ ID No: 4, as follows: atgaacacgattaacatcgctaagaacgacttctctgacatcgaactggctgctatcccgttcaacactctgg ctgaccattacggtgagcgtttagctcgcgaacagttggcccttgagcatgagtcttacgagatgggtgaagc acgcttccgcaagatgtttgagcgtcaacttaaagctggtgaggttgcggataacgctgccgccaagcctctc atcactaccctactccctaagatgattgcacgcatcaacgactggtttgaggaagtgaaagctaagcgcggca agcgcccgacagccttccagttcctgcaagaaatcaagccggaagccgtagcgtacatcaccattaagaccac tctggcttgcctaaccagtgctgacaatacaaccgttcaggctgtagcaagcgcaatcggtcgggccattgag gacgaggctcgcttcggtcgtatccgtgaccttgaagctaagcacttcaagaaaaacgttgaggaacaactca acaagcgcgtagggcacgtctacaagaaagcatttatgcaagttgtcgaggctgacatgctctctaagggtctactcggtggcgaggcgtggtcttcgtggcataaggaagactctattcatgtaggagtacgctgcatcgagatg ctcattgagtcaaccggaatggttagcttacaccgccaaaatgctggcgtagtaggtcaagactctgagacta tcgaactcgcacctgaatacgctgaggctatcgcaacccgtgcaggtgcgctggctggcatctctctgatgtt ccaaccttgcgtagttcctcctaagccgtggactggcattactggtggtggctattgggctaacggtcgtcgt cctctggcgctggtgcgtactcacagtaagaaagcactgatgcgctacgaagacgtttacatgcctgaggtgt acaaagcgattaacattgcgcaaaacaccgcatggaaaatcaacaagaaagtcctagcggtcgccaacgtaat caccaagtggaagcattgtccggtcgaggacatccctgcgattgagcgtgaagaactcccgatgaaaccggaa gacatcgacatgaatcctgaggctctcaccgcgtggaaacgtgctgccgctgctgtgtaccgcaaggacaagg ctcgcaagtctcgccgtatcagccttgagttcatgcttgagcaagccaataagtttgctaaccataaggccat ctggttcccttacaacatggactggcgcggtcgtgtttacgctgtgtcaatgttcaacccgcaaggtaacgat atgaccaaaggactgcttacgctggcgaaaggtaaaccaatcggtaaggaaggttactactggctgaaaatcc acggtgcaaactgtgcgggtgtcgataaggttccgttccctgagcgcatcaagttcattgaggaaaaccacga gaacatcatggcttgcgctaagtctccactggagaacacttggtgggctgagcaagattctccgttctgcttc cttgcgttctgctttgagtacgctggggtacagcaccacggcctgagctataactgctcccttccgctggcgt ttgacgggtcttgctctggcatccagcacttctccgcgatgctccgagatgaggtaggtggtcgcgcggttaa cttgcttcctagtgaaaccgttcaggacatctacgggattgttgctaagaaagtcaacgagattctacaagca gacgcaatcaatgggaccgataacgaagtagttaccgtgaccgatgagaacactggtgaaatctctgagaaag tcaagctgggcactaaggcactggctggtcaatggctggcttacggtgttactcgcagtgtgactaagcgttc agtcatgacgctggcttacgggtccaaagagttcggcttccgtcaacaagtgctggaagataccattcagcca gctattgattccggcaagggtctgatgttcactcagccgaatcaggctgctggatacatggctaagctgattt gggaatctgtgagcgtgacggtggtagctgcggttgaagcaatgaactggcttaagtctgctgctaagctgct ggctgctgaggtcaaagataagaagactggagagattcttcgcaagcgttgcgctgtgcattgggtaactcct gatggtttccctgtgtggcaggaatacaagaagcctattcagacgcgcttgaacctgatgttcctcggtcagt tccgcttacagcctaccattaacaccaacaaagatagcgagattgatgcacacaaacaggagtctggtatcgc tcctaactttgtacacagccaagacggtagccaccttcgtaagactgtagtgtgggcacacgagaagtacgga atcgaatcttttgcactgattcacgactccttcggtaccattccggctgacgctgcgaacctgttcaaagcag tgcgcgaaactatggttgacacatatgagtcttgtgatgtactggctgatttctacgaccagttcgctgacca gttgcacgagtctcaattggacaaaatgccagcacttccggctaaaggtaacttgaacctccgtgacatctta gagtcggacttcgcgttcgcgtaa

[0054] [SEQ ID No: 4]

[0055] Thus, a parental RNA polymerase may be encoded by a nucleotide sequence substantially as set out in SEQ ID No: 4, or a fragment or variant thereof.

[0056] In some embodiments, therefore, the variant RNA polymerase of the invention may comprise an amino acid substitution at position P266 of SEQ ID No: 1. The substitution may be selected from any of the 20 standard amino acids, optionally other than aspartic acid (P), which is the wild-type amino acid at this position.

[0057] Typically, however, the P266 mutations comprises a P266L, P266S, P266Y, P266A, P266G, P266T, P266I, P266V or P266N substitution. More typically, however, the P266 substitution comprises a P266L substitution. For vaccination purposes, it might still be desired to have a substitution of P266, not to have less dsRNA, but to have less abortive transcripts, which contaminate the product if not purified, and lead to higher nucleotide and cap-analogue consumption per amount of full-length RNA product.

[0058] In an embodiment, the variant RNA polymerase may comprise one, two, three, four, five or six amino acid substitutions at positions selected from positions P266, D879, F880, A881, F882, and A883 in SEQ ID No:l. Typically, the variant RNA polymerase may comprise one, two, three, four, or five amino acid substitutions atpositions selected from positions P266, D879, F880, A881, and A883 in SEQ ID No:l.

[0059] Therefore, in one embodiment, the variant RNA polymerase (referred to herein as " V90") may comprise the amino acid substitutions P266L, D879V, F880Y, A881F, and A883C. This embodiment produces lower levels of dsRNA compared to the corresponding wild-type RNA polymerase, and so reduces dsRNA contamination. Thus, the variant RNA polymerase may comprise a C-terminal motif comprising an amino acid sequence substantially as set out as SEQ ID No: 100, or a fragment or variant thereof.

[0060] The full amino acid sequence of this variant is represented herein, as SEQ ID No: 5, as follows:

[0061] MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHESYEMGEARFRKMFERQLKAGEVADNAAAKPL ITTLLPKMIARINDWFEEVKAKRGKRPTAFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIE DEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEM LIESTGMVSLHRQNAGWGQDSETIELAPEYAEAIATRAGALAGISLMFQPCVVPPKPWTGITGGGYWANGRR PLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPE DIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGND MTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPFCF LAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQA DAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQP AIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTP DGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNFVHSQDGSHLRKTVVWAHEKYG IESFALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAKGNLNLRDIL ESVYFFC

[0062] [SEQ ID No: 5]

[0063] Thus, the variant polymerase may comprise an amino acid sequence substantially as set out in SEQ ID No: 5, or a fragment or variant thereof.

[0064] This variant is encoded by a DNA sequence represented herein, as SEQ ID No: 6, as follows: atgaacacgattaacatcgctaagaacgacttctctgacatcgaactggctgctatcccgttcaacactctgg ctgaccattacggtgagcgtttagctcgcgaacagttggcccttgagcatgagtcttacgagatgggtgaagc acgcttccgcaagatgtttgagcgtcaacttaaagctggtgaggttgcggataacgctgccgccaagcctctc atcactaccctactccctaagatgattgcacgcatcaacgactggtttgaggaagtgaaagctaagcgcggca agcgcccgacagccttccagttcctgcaagaaatcaagccggaagccgtagcgtacatcaccattaagaccac tctggcttgcctaaccagtgctgacaatacaaccgttcaggctgtagcaagcgcaatcggtcgggccattgag gacgaggctcgcttcggtcgtatccgtgaccttgaagctaagcacttcaagaaaaacgttgaggaacaactca acaagcgcgtagggcacgtctacaagaaagcatttatgcaagttgtcgaggctgacatgctctctaagggtct actcggtggcgaggcgtggtcttcgtggcataaggaagactctattcatgtaggagtacgctgcatcgagatg ctcattgagtcaaccggaatggttagcttacaccgccaaaatgctggcgtagtaggtcaagactctgagacta tcgaactcgcacctgaatacgctgaggctatcgcaacccgtgcaggtgcgctggctggcatctctctgatgtt ccaaccttgcgtagttcctcctaagccgtggactggcattactggtggtggctattgggctaacggtcgtcgt cctctggcgctggtgcgtactcacagtaagaaagcactgatgcgctacgaagacgtttacatgcctgaggtgt acaaagcgattaacattgcgcaaaacaccgcatggaaaatcaacaagaaagtcctagcggtcgccaacgtaatcaccaagtggaagcattgtccggtcgaggacatccctgcgattgagcgtgaagaactcccgatgaaaccggaa gacatcgacatgaatcctgaggctctcaccgcgtggaaacgtgctgccgctgctgtgtaccgcaaggacaagg ctcgcaagtctcgccgtatcagccttgagttcatgcttgagcaagccaataagtttgctaaccataaggccat ctggttcccttacaacatggactggcgcggtcgtgtttacgctgtgtcaatgttcaacccgcaaggtaacgat atgaccaaaggactgcttacgctggcgaaaggtaaaccaatcggtaaggaaggttactactggctgaaaatcc acggtgcaaactgtgcgggtgtcgataaggttccgttccctgagcgcatcaagttcattgaggaaaaccacga gaacatcatggcttgcgctaagtctccactggagaacacttggtgggctgagcaagattctccgttctgcttc cttgcgttctgctttgagtacgctggggtacagcaccacggcctgagctataactgctcccttccgctggcgt ttgacgggtcttgctctggcatccagcacttctccgcgatgctccgagatgaggtaggtggtcgcgcggttaa cttgcttcctagtgaaaccgttcaggacatctacgggattgttgctaagaaagtcaacgagattctacaagca gacgcaatcaatgggaccgataacgaagtagttaccgtgaccgatgagaacactggtgaaatctctgagaaag tcaagctgggcactaaggcactggctggtcaatggctggcttacggtgttactcgcagtgtgactaagcgttc agtcatgacgctggcttacgggtccaaagagttcggcttccgtcaacaagtgctggaagataccattcagcca gctattgattccggcaagggtctgatgttcactcagccgaatcaggctgctggatacatggctaagctgattt gggaatctgtgagcgtgacggtggtagctgcggttgaagcaatgaactggcttaagtctgctgctaagctgct ggctgctgaggtcaaagataagaagactggagagattcttcgcaagcgttgcgctgtgcattgggtaactcct gatggtttccctgtgtggcaggaatacaagaagcctattcagacgcgcttgaacctgatgttcctcggtcagt tccgcttacagcctaccattaacaccaacaaagatagcgagattgatgcacacaaacaggagtctggtatcgc tcctaactttgtacacagccaagacggtagccaccttcgtaagactgtagtgtgggcacacgagaagtacgga atcgaatcttttgcactgattcacgactccttcggtaccattccggctgacgctgcgaacctgttcaaagcag tgcgcgaaactatggttgacacatatgagtcttgtgatgtactggctgatttctacgaccagttcgctgacca gttgcacgagtctcaattggacaaaatgccagcacttccggctaaaggtaacttgaacctccgtgacatctta gagtcggtgtatttcttctgctaa

[0065] [SEQ ID No: 6]

[0066] Thus, this variant polymerase may be encoded by a nucleotide sequence substantially as set out in SEQ ID No: 6, or a fragment or variant thereof.

[0067] Unexpectedly, this variant exhibits low dsRNA synthesis (e.g. 0.027-0.051 % w / w) and high RNA yield (e.g. 122-199 pg / reaction) for various RNA sequences. As described herein, this variant T7RNAP (e.g., SEQ ID NO: 5) provides better results during IVT reactions offering optimal RNA yields and reduced dsRNA contamination.

[0068] In a second embodiment, the variant RNA polymerase (referred to herein as " V187") may comprise the substitutions P266L, D879I, F880Y, A881F, and A883S. This embodiment produces lower levels of dsRNA compared to the corresponding wild-type RNA polymerase, and so reduces dsRNA contamination. Thus, the variant RNA polymerase may comprise a C-terminal motif comprising an amino acid sequence substantially as set out as SEQ ID No: 197, or a fragment or variant thereof.

[0069] The full amino acid sequence of this variant is represented herein, as SEQ ID No: 7, as follows:

[0070] MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHESYEMGEARFRKMFERQLKAGEVADNAAAKPL ITTLLPKMIARINDWFEEVKAKRGKRPTAFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIE DEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEM LIESTGMVSLHRQNAGWGQDSETIELAPEYAEAIATRAGALAGISLMFQPCVVPPKPWTGITGGGYWANGRR PLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPE DIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGND MTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPFCFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQA DAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQP AIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTP DGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNFVHSQDGSHLRKTVVWAHEKYG IESFALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAKGNLNLRDIL ESIYFFS

[0071] [SEQ ID No: 7]

[0072] Thus, the variant polymerase may comprise an amino acid sequence substantially as set out in SEQ ID No: 7, or a fragment or variant thereof.

[0073] This variant is encoded by a DNA sequence represented herein, as SEQ ID No: 8, as follows: atgaacacgattaacatcgctaagaacgacttctctgacatcgaactggctgctatcccgttcaacactctgg ctgaccattacggtgagcgtttagctcgcgaacagttggcccttgagcatgagtcttacgagatgggtgaagc acgcttccgcaagatgtttgagcgtcaacttaaagctggtgaggttgcggataacgctgccgccaagcctctc atcactaccctactccctaagatgattgcacgcatcaacgactggtttgaggaagtgaaagctaagcgcggca agcgcccgacagccttccagttcctgcaagaaatcaagccggaagccgtagcgtacatcaccattaagaccac tctggcttgcctaaccagtgctgacaatacaaccgttcaggctgtagcaagcgcaatcggtcgggccattgag gacgaggctcgcttcggtcgtatccgtgaccttgaagctaagcacttcaagaaaaacgttgaggaacaactca acaagcgcgtagggcacgtctacaagaaagcatttatgcaagttgtcgaggctgacatgctctctaagggtct actcggtggcgaggcgtggtcttcgtggcataaggaagactctattcatgtaggagtacgctgcatcgagatg ctcattgagtcaaccggaatggttagcttacaccgccaaaatgctggcgtagtaggtcaagactctgagacta tcgaactcgcacctgaatacgctgaggctatcgcaacccgtgcaggtgcgctggctggcatctctctgatgtt ccaaccttgcgtagttcctcctaagccgtggactggcattactggtggtggctattgggctaacggtcgtcgt cctctggcgctggtgcgtactcacagtaagaaagcactgatgcgctacgaagacgtttacatgcctgaggtgt acaaagcgattaacattgcgcaaaacaccgcatggaaaatcaacaagaaagtcctagcggtcgccaacgtaat caccaagtggaagcattgtccggtcgaggacatccctgcgattgagcgtgaagaactcccgatgaaaccggaa gacatcgacatgaatcctgaggctctcaccgcgtggaaacgtgctgccgctgctgtgtaccgcaaggacaagg ctcgcaagtctcgccgtatcagccttgagttcatgcttgagcaagccaataagtttgctaaccataaggccat ctggttcccttacaacatggactggcgcggtcgtgtttacgctgtgtcaatgttcaacccgcaaggtaacgat atgaccaaaggactgcttacgctggcgaaaggtaaaccaatcggtaaggaaggttactactggctgaaaatcc acggtgcaaactgtgcgggtgtcgataaggttccgttccctgagcgcatcaagttcattgaggaaaaccacga gaacatcatggcttgcgctaagtctccactggagaacacttggtgggctgagcaagattctccgttctgcttc cttgcgttctgctttgagtacgctggggtacagcaccacggcctgagctataactgctcccttccgctggcgt ttgacgggtcttgctctggcatccagcacttctccgcgatgctccgagatgaggtaggtggtcgcgcggttaa cttgcttcctagtgaaaccgttcaggacatctacgggattgttgctaagaaagtcaacgagattctacaagca gacgcaatcaatgggaccgataacgaagtagttaccgtgaccgatgagaacactggtgaaatctctgagaaag tcaagctgggcactaaggcactggctggtcaatggctggcttacggtgttactcgcagtgtgactaagcgttc agtcatgacgctggcttacgggtccaaagagttcggcttccgtcaacaagtgctggaagataccattcagcca gctattgattccggcaagggtctgatgttcactcagccgaatcaggctgctggatacatggctaagctgattt gggaatctgtgagcgtgacggtggtagctgcggttgaagcaatgaactggcttaagtctgctgctaagctgct ggctgctgaggtcaaagataagaagactggagagattcttcgcaagcgttgcgctgtgcattgggtaactcct gatggtttccctgtgtggcaggaatacaagaagcctattcagacgcgcttgaacctgatgttcctcggtcagt tccgcttacagcctaccattaacaccaacaaagatagcgagattgatgcacacaaacaggagtctggtatcgc tcctaactttgtacacagccaagacggtagccaccttcgtaagactgtagtgtgggcacacgagaagtacgga atcgaatcttttgcactgattcacgactccttcggtaccattccggctgacgctgcgaacctgttcaaagcag tgcgcgaaactatggttgacacatatgagtcttgtgatgtactggctgatttctacgaccagttcgctgacca gttgcacgagtctcaattggacaaaatgccagcacttccggctaaaggtaacttgaacctccgtgacatctta gagtcgatctatttttttagctaa

[0074] [SEQ ID No: 8]

[0075] Thus, this variant polymerase may be encoded by a nucleotide sequence substantially as set out in SEQ ID No: 8, or a fragment or variant thereof.Unexpectedly, this variant comprises a C-terminal domain with a motif not found in ssRNA polymerase databases. It exhibits low dsRNA synthesis (e.g. 0.033-0.040 % w / w) and high RNA yield (e.g. 167-196 pg / reaction). As described herein, this variant T7RNAP (e.g., SEQ ID NO: 7) provides better results during IVT reactions offering optimal RNA yields and reduced dsRNA contamination.

[0076] In a third embodiment, the variant RNA polymerase (referred to herein as " V206") may comprise the substitutions P266L, D879R, F880Y, and A883C. This embodiment produces lower levels of dsRNA compared to the corresponding wild-type RNA polymerase, and so reduces dsRNA contamination. Thus, the variant RNA polymerase may comprise a C-terminal motif comprising an amino acid sequence substantially as set out as SEQ ID No: 216, or a fragment or variant thereof.

[0077] The full amino acid sequence of this variant is represented herein, as SEQ ID No: 9, as follows:

[0078] MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHESYEMGEARFRKMFERQLKAGEVADNAAAKPL ITTLLPKMIARINDWFEEVKAKRGKRPTAFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIE DEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEM LIESTGMVSLHRQNAGWGQDSETIELAPEYAEAIATRAGALAGISLMFQPCVVPPKPWTGITGGGYWANGRR PLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPE DIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGND MTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPFCF LAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQA DAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQP AIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTP DGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNFVHSQDGSHLRKTVVWAHEKYG IESFALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAKGNLNLRDIL ESRYAFC

[0079] [SEQ ID No: 9] Thus, the variant polymerase may comprise an amino acid sequence substantially as set out in SEQ ID No: 9, or a fragment or variant thereof.

[0080] This variant is encoded by a DNA sequence represented herein, as SEQ ID No: 10, as follows: atgaacacgattaacatcgctaagaacgacttctctgacatcgaactggctgctatcccgttcaacactctgg ctgaccattacggtgagcgtttagctcgcgaacagttggcccttgagcatgagtcttacgagatgggtgaagc acgcttccgcaagatgtttgagcgtcaacttaaagctggtgaggttgcggataacgctgccgccaagcctctc atcactaccctactccctaagatgattgcacgcatcaacgactggtttgaggaagtgaaagctaagcgcggca agcgcccgacagccttccagttcctgcaagaaatcaagccggaagccgtagcgtacatcaccattaagaccac tctggcttgcctaaccagtgctgacaatacaaccgttcaggctgtagcaagcgcaatcggtcgggccattgag gacgaggctcgcttcggtcgtatccgtgaccttgaagctaagcacttcaagaaaaacgttgaggaacaactca acaagcgcgtagggcacgtctacaagaaagcatttatgcaagttgtcgaggctgacatgctctctaagggtct actcggtggcgaggcgtggtcttcgtggcataaggaagactctattcatgtaggagtacgctgcatcgagatg ctcattgagtcaaccggaatggttagcttacaccgccaaaatgctggcgtagtaggtcaagactctgagacta tcgaactcgcacctgaatacgctgaggctatcgcaacccgtgcaggtgcgctggctggcatctctctgatgttccaaccttgcgtagttcctcctaagccgtggactggcattactggtggtggctattgggctaacggtcgtcgt cctctggcgctggtgcgtactcacagtaagaaagcactgatgcgctacgaagacgtttacatgcctgaggtgt acaaagcgattaacattgcgcaaaacaccgcatggaaaatcaacaagaaagtcctagcggtcgccaacgtaat caccaagtggaagcattgtccggtcgaggacatccctgcgattgagcgtgaagaactcccgatgaaaccggaa gacatcgacatgaatcctgaggctctcaccgcgtggaaacgtgctgccgctgctgtgtaccgcaaggacaagg ctcgcaagtctcgccgtatcagccttgagttcatgcttgagcaagccaataagtttgctaaccataaggccat ctggttcccttacaacatggactggcgcggtcgtgtttacgctgtgtcaatgttcaacccgcaaggtaacgat atgaccaaaggactgcttacgctggcgaaaggtaaaccaatcggtaaggaaggttactactggctgaaaatcc acggtgcaaactgtgcgggtgtcgataaggttccgttccctgagcgcatcaagttcattgaggaaaaccacga gaacatcatggcttgcgctaagtctccactggagaacacttggtgggctgagcaagattctccgttctgcttc cttgcgttctgctttgagtacgctggggtacagcaccacggcctgagctataactgctcccttccgctggcgt ttgacgggtcttgctctggcatccagcacttctccgcgatgctccgagatgaggtaggtggtcgcgcggttaa cttgcttcctagtgaaaccgttcaggacatctacgggattgttgctaagaaagtcaacgagattctacaagca gacgcaatcaatgggaccgataacgaagtagttaccgtgaccgatgagaacactggtgaaatctctgagaaag tcaagctgggcactaaggcactggctggtcaatggctggcttacggtgttactcgcagtgtgactaagcgttc agtcatgacgctggcttacgggtccaaagagttcggcttccgtcaacaagtgctggaagataccattcagcca gctattgattccggcaagggtctgatgttcactcagccgaatcaggctgctggatacatggctaagctgattt gggaatctgtgagcgtgacggtggtagctgcggttgaagcaatgaactggcttaagtctgctgctaagctgct ggctgctgaggtcaaagataagaagactggagagattcttcgcaagcgttgcgctgtgcattgggtaactcct gatggtttccctgtgtggcaggaatacaagaagcctattcagacgcgcttgaacctgatgttcctcggtcagt tccgcttacagcctaccattaacaccaacaaagatagcgagattgatgcacacaaacaggagtctggtatcgc tcctaactttgtacacagccaagacggtagccaccttcgtaagactgtagtgtgggcacacgagaagtacgga atcgaatcttttgcactgattcacgactccttcggtaccattccggctgacgctgcgaacctgttcaaagcag tgcgcgaaactatggttgacacatatgagtcttgtgatgtactggctgatttctacgaccagttcgctgacca gttgcacgagtctcaattggacaaaatgccagcacttccggctaaaggtaacttgaacctccgtgacatctta gagtcgcgttatgcgttttgctaa

[0081] [SEQ ID No: 10]

[0082] Thus, this variant polymerase may be encoded by a nucleotide sequence substantially as set out in SEQ ID No: 10, or a fragment or variant thereof.

[0083] Unexpectedly, this variant comprises a C-terminal domain with a motif not found in ssRNA polymerase databases. It exhibits low dsRNA synthesis (e.g. 0.023-0.036 % w / w) and high RNA yield (e.g. 188-206 pg / reaction). As described herein, this variant T7RNAP (e.g., SEQ ID NO: 9) provides better results during IVT reactions offering optimal RNA yields and reduced dsRNA contamination.

[0084] To exclude the possibility that the purification of putative variants of interest failed in the first high-throughput screen, a second was perform for those variants that showed an RNA yield lower than 25 pg, as shown in Figure 9a. For those that improved in yield and dsRNA content, the medium scale validation workflow was repeated and the variants were re-expressed in E. coli, purified and re-profiled by the amount of dsRNA and RNA yield in two independent replicates, as shown in Figure 9c. From this screen, the V114 variant (QYFFC + P266L), the V162 variant (AYFFC + P266L), the V66 variant (EYFFC + P266L), the V191 variant (IYVFS + P266L), and the V210 variant (RYFFC + P266L) were identified as the best performers exhibiting good RNA yields and low dsRNA relative the parental.Therefore, in a fourth embodiment, the variant RNA polymerase (referred to herein as " V114") may comprise the amino acid substitutions P266L, and QYFFC (D879Q, F880Y, A881F, A883C). This embodiment produces lower levels of dsRNA compared to the corresponding wild-type RNA polymerase, and so reduces dsRNA contamination. Thus, the variant RNA polymerase may comprise a C-terminal motif comprising an amino acid sequence substantially as set out as SEQ ID No: 124, or a fragment or variant thereof.

[0085] The full amino acid sequence of this variant is represented herein, as SEQ ID No: 251, as follows:

[0086] MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHESYEMGEARFRKMFERQLKAGEVADNAAAKPL ITTLLPKMIARINDWFEEVKAKRGKRPTAFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIE DEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEM LIESTGMVSLHRQNAGWGQDSETIELAPEYAEAIATRAGALAGISLMFQPCVVPPKPWTGITGGGYWANGRR PLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPE DIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGND MTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPFCF LAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQA DAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQP AIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTP DGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNFVHSQDGSHLRKTVVWAHEKYG IESFALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAKGNLNLRDIL ESQYFFC

[0087] [SEQ ID No: 251]

[0088] Thus, the variant polymerase may comprise an amino acid sequence substantially as set out in SEQ ID No: 251, or a fragment or variant thereof.

[0089] This variant is encoded by a DNA sequence represented herein, as SEQ ID No: 252, as follows: atgaacacgattaacatcgctaagaacgacttctctgacatcgaactggctgctatcccgttcaacactctgg ctgaccattacggtgagcgtttagctcgcgaacagttggcccttgagcatgagtcttacgagatgggtgaagc acgcttccgcaagatgtttgagcgtcaacttaaagctggtgaggttgcggataacgctgccgccaagcctctc atcactaccctactccctaagatgattgcacgcatcaacgactggtttgaggaagtgaaagctaagcgcggca agcgcccgacagccttccagttcctgcaagaaatcaagccggaagccgtagcgtacatcaccattaagaccac tctggcttgcctaaccagtgctgacaatacaaccgttcaggctgtagcaagcgcaatcggtcgggccattgag gacgaggctcgcttcggtcgtatccgtgaccttgaagctaagcacttcaagaaaaacgttgaggaacaactca acaagcgcgtagggcacgtctacaagaaagcatttatgcaagttgtcgaggctgacatgctctctaagggtct actcggtggcgaggcgtggtcttcgtggcataaggaagactctattcatgtaggagtacgctgcatcgagatg ctcattgagtcaaccggaatggttagcttacaccgccaaaatgctggcgtagtaggtcaagactctgagacta tcgaactcgcacctgaatacgctgaggctatcgcaacccgtgcaggtgcgctggctggcatctctctgatgtt ccaaccttgcgtagttcctcctaagccgtggactggcattactggtggtggctattgggctaacggtcgtcgt cctctggcgctggtgcgtactcacagtaagaaagcactgatgcgctacgaagacgtttacatgcctgaggtgt acaaagcgattaacattgcgcaaaacaccgcatggaaaatcaacaagaaagtcctagcggtcgccaacgtaat caccaagtggaagcattgtccggtcgaggacatccctgcgattgagcgtgaagaactcccgatgaaaccggaa gacatcgacatgaatcctgaggctctcaccgcgtggaaacgtgctgccgctgctgtgtaccgcaaggacaagg ctcgcaagtctcgccgtatcagccttgagttcatgcttgagcaagccaataagtttgctaaccataaggccat ctggttcccttacaacatggactggcgcggtcgtgtttacgctgtgtcaatgttcaacccgcaaggtaacgat atgaccaaaggactgcttacgctggcgaaaggtaaaccaatcggtaaggaaggttactactggctgaaaatcc acggtgcaaactgtgcgggtgtcgataaggttccgttccctgagcgcatcaagttcattgaggaaaaccacga gaacatcatggcttgcgctaagtctccactggagaacacttggtgggctgagcaagattctccgttctgcttccttgcgttctgctttgagtacgctggggtacagcaccacggcctgagctataactgctcccttccgctggcgt ttgacgggtcttgctctggcatccagcacttctccgcgatgctccgagatgaggtaggtggtcgcgcggttaa cttgcttcctagtgaaaccgttcaggacatctacgggattgttgctaagaaagtcaacgagattctacaagca gacgcaatcaatgggaccgataacgaagtagttaccgtgaccgatgagaacactggtgaaatctctgagaaag tcaagctgggcactaaggcactggctggtcaatggctggcttacggtgttactcgcagtgtgactaagcgttc agtcatgacgctggcttacgggtccaaagagttcggcttccgtcaacaagtgctggaagataccattcagcca gctattgattccggcaagggtctgatgttcactcagccgaatcaggctgctggatacatggctaagctgattt gggaatctgtgagcgtgacggtggtagctgcggttgaagcaatgaactggcttaagtctgctgctaagctgct ggctgctgaggtcaaagataagaagactggagagattcttcgcaagcgttgcgctgtgcattgggtaactcct gatggtttccctgtgtggcaggaatacaagaagcctattcagacgcgcttgaacctgatgttcctcggtcagt tccgcttacagcctaccattaacaccaacaaagatagcgagattgatgcacacaaacaggagtctggtatcgc tcctaactttgtacacagccaagacggtagccaccttcgtaagactgtagtgtgggcacacgagaagtacgga atcgaatcttttgcactgattcacgactccttcggtaccattccggctgacgctgcgaacctgttcaaagcag tgcgcgaaactatggttgacacatatgagtcttgtgatgtactggctgatttctacgaccagttcgctgacca gttgcacgagtctcaattggacaaaatgccagcacttccggctaaaggtaacttgaacctccgtgacatctta gagtcgcagtattttttttgctaa

[0090] [SEQ ID No: 252]

[0091] Thus, this variant polymerase may be encoded by a nucleotide sequence substantially as set out in SEQ ID No: 252, or a fragment or variant thereof.

[0092] In a fifth embodiment, the variant RNA polymerase (referred to herein as " V162") may comprise the substitutions P266L, and AYFFC (D879A, F880Y, A881F, A883C). This embodiment produces lower levels of dsRNA compared to the corresponding wild-type RNA polymerase, and so reduces dsRNA contamination. Thus, the variant RNA polymerase may comprise a C-terminal motif comprising an amino acid sequence substantially as set out as SEQ ID No: 172, or a fragment or variant thereof.

[0093] The full amino acid sequence of this variant is represented herein, as SEQ ID No: 253, as follows:

[0094] MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHESYEMGEARFRKMFERQLKAGEVADNAAAKPL ITTLLPKMIARINDWFEEVKAKRGKRPTAFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIE DEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEM LIESTGMVSLHRQNAGWGQDSETIELAPEYAEAIATRAGALAGISLMFQPCVVPPKPWTGITGGGYWANGRR PLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPE DIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGND MTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPFCF LAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQA DAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQP AIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTP DGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNFVHSQDGSHLRKTVVWAHEKYG IESFALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAKGNLNLRDIL ESAYFFC

[0095] [SEQ ID No: 253]

[0096] Thus, the variant polymerase may comprise an amino acid sequence substantially as set out in SEQ ID No: 253, or a fragment or variant thereof.

[0097] This variant is encoded by a DNA sequence represented herein, as SEQ ID No: 254, as follows:atgaacacgattaacatcgctaagaacgacttctctgacatcgaactggctgctatcccgttcaacactctgg ctgaccattacggtgagcgtttagctcgcgaacagttggcccttgagcatgagtcttacgagatgggtgaagc acgcttccgcaagatgtttgagcgtcaacttaaagctggtgaggttgcggataacgctgccgccaagcctctc atcactaccctactccctaagatgattgcacgcatcaacgactggtttgaggaagtgaaagctaagcgcggca agcgcccgacagccttccagttcctgcaagaaatcaagccggaagccgtagcgtacatcaccattaagaccac tctggcttgcctaaccagtgctgacaatacaaccgttcaggctgtagcaagcgcaatcggtcgggccattgag gacgaggctcgcttcggtcgtatccgtgaccttgaagctaagcacttcaagaaaaacgttgaggaacaactca acaagcgcgtagggcacgtctacaagaaagcatttatgcaagttgtcgaggctgacatgctctctaagggtct actcggtggcgaggcgtggtcttcgtggcataaggaagactctattcatgtaggagtacgctgcatcgagatg ctcattgagtcaaccggaatggttagcttacaccgccaaaatgctggcgtagtaggtcaagactctgagacta tcgaactcgcacctgaatacgctgaggctatcgcaacccgtgcaggtgcgctggctggcatctctctgatgtt ccaaccttgcgtagttcctcctaagccgtggactggcattactggtggtggctattgggctaacggtcgtcgt cctctggcgctggtgcgtactcacagtaagaaagcactgatgcgctacgaagacgtttacatgcctgaggtgt acaaagcgattaacattgcgcaaaacaccgcatggaaaatcaacaagaaagtcctagcggtcgccaacgtaat caccaagtggaagcattgtccggtcgaggacatccctgcgattgagcgtgaagaactcccgatgaaaccggaa gacatcgacatgaatcctgaggctctcaccgcgtggaaacgtgctgccgctgctgtgtaccgcaaggacaagg ctcgcaagtctcgccgtatcagccttgagttcatgcttgagcaagccaataagtttgctaaccataaggccat ctggttcccttacaacatggactggcgcggtcgtgtttacgctgtgtcaatgttcaacccgcaaggtaacgat atgaccaaaggactgcttacgctggcgaaaggtaaaccaatcggtaaggaaggttactactggctgaaaatcc acggtgcaaactgtgcgggtgtcgataaggttccgttccctgagcgcatcaagttcattgaggaaaaccacga gaacatcatggcttgcgctaagtctccactggagaacacttggtgggctgagcaagattctccgttctgcttc cttgcgttctgctttgagtacgctggggtacagcaccacggcctgagctataactgctcccttccgctggcgt ttgacgggtcttgctctggcatccagcacttctccgcgatgctccgagatgaggtaggtggtcgcgcggttaa cttgcttcctagtgaaaccgttcaggacatctacgggattgttgctaagaaagtcaacgagattctacaagca gacgcaatcaatgggaccgataacgaagtagttaccgtgaccgatgagaacactggtgaaatctctgagaaag tcaagctgggcactaaggcactggctggtcaatggctggcttacggtgttactcgcagtgtgactaagcgttc agtcatgacgctggcttacgggtccaaagagttcggcttccgtcaacaagtgctggaagataccattcagcca gctattgattccggcaagggtctgatgttcactcagccgaatcaggctgctggatacatggctaagctgattt gggaatctgtgagcgtgacggtggtagctgcggttgaagcaatgaactggcttaagtctgctgctaagctgct ggctgctgaggtcaaagataagaagactggagagattcttcgcaagcgttgcgctgtgcattgggtaactcct gatggtttccctgtgtggcaggaatacaagaagcctattcagacgcgcttgaacctgatgttcctcggtcagt tccgcttacagcctaccattaacaccaacaaagatagcgagattgatgcacacaaacaggagtctggtatcgc tcctaactttgtacacagccaagacggtagccaccttcgtaagactgtagtgtgggcacacgagaagtacgga atcgaatcttttgcactgattcacgactccttcggtaccattccggctgacgctgcgaacctgttcaaagcag tgcgcgaaactatggttgacacatatgagtcttgtgatgtactggctgatttctacgaccagttcgctgacca gttgcacgagtctcaattggacaaaatgccagcacttccggctaaaggtaacttgaacctccgtgacatctta gagtcggcgtattttttttgctaa

[0098] [SEQ ID No: 254]

[0099] Thus, this variant polymerase may be encoded by a nucleotide sequence substantially as set out in SEQ ID No: 254, or a fragment or variant thereof.

[0100] In a sixth embodiment, the variant RNA polymerase (referred to herein as " V66") may comprise the substitutions P266L and EYFFC (D879E, F880Y, A881F, A883C). This embodiment produces lower levels of dsRNA compared to the corresponding wild-type RNA polymerase, and so reduces dsRNA contamination. Thus, the variant RNA polymerase may comprise a C-terminal motif comprising an amino acid sequence substantially as set out as SEQ ID No: 76, or a fragment or variant thereof.

[0101] The full amino acid sequence of this variant is represented herein, as SEQ ID No: 255 as follows:MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHESYEMGEARFRKMFERQLKAGEVADNAAAKPL ITTLLPKMIARINDWFEEVKAKRGKRPTAFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIE DEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEM LIESTGMVSLHRQNAGWGQDSETIELAPEYAEAIATRAGALAGISLMFQPCVVPPKPWTGITGGGYWANGRR PLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPE DIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGND MTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPFCF LAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQA DAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQP AIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTP DGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNFVHSQDGSHLRKTVVWAHEKYG IESFALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAKGNLNLRDIL ESEYFFC

[0102] [SEQ ID No: 255]

[0103] Thus, the variant polymerase may comprise an amino acid sequence substantially as set out in SEQ ID No: 255, or a fragment or variant thereof.

[0104] This variant is encoded by a DNA sequence represented herein, as SEQ ID No: 256, as follows: atgaacacgattaacatcgctaagaacgacttctctgacatcgaactggctgctatcccgttcaacactctgg ctgaccattacggtgagcgtttagctcgcgaacagttggcccttgagcatgagtcttacgagatgggtgaagc acgcttccgcaagatgtttgagcgtcaacttaaagctggtgaggttgcggataacgctgccgccaagcctctc atcactaccctactccctaagatgattgcacgcatcaacgactggtttgaggaagtgaaagctaagcgcggca agcgcccgacagccttccagttcctgcaagaaatcaagccggaagccgtagcgtacatcaccattaagaccac tctggcttgcctaaccagtgctgacaatacaaccgttcaggctgtagcaagcgcaatcggtcgggccattgag gacgaggctcgcttcggtcgtatccgtgaccttgaagctaagcacttcaagaaaaacgttgaggaacaactca acaagcgcgtagggcacgtctacaagaaagcatttatgcaagttgtcgaggctgacatgctctctaagggtct actcggtggcgaggcgtggtcttcgtggcataaggaagactctattcatgtaggagtacgctgcatcgagatg ctcattgagtcaaccggaatggttagcttacaccgccaaaatgctggcgtagtaggtcaagactctgagacta tcgaactcgcacctgaatacgctgaggctatcgcaacccgtgcaggtgcgctggctggcatctctctgatgtt ccaaccttgcgtagttcctcctaagccgtggactggcattactggtggtggctattgggctaacggtcgtcgt cctctggcgctggtgcgtactcacagtaagaaagcactgatgcgctacgaagacgtttacatgcctgaggtgt acaaagcgattaacattgcgcaaaacaccgcatggaaaatcaacaagaaagtcctagcggtcgccaacgtaat caccaagtggaagcattgtccggtcgaggacatccctgcgattgagcgtgaagaactcccgatgaaaccggaa gacatcgacatgaatcctgaggctctcaccgcgtggaaacgtgctgccgctgctgtgtaccgcaaggacaagg ctcgcaagtctcgccgtatcagccttgagttcatgcttgagcaagccaataagtttgctaaccataaggccat ctggttcccttacaacatggactggcgcggtcgtgtttacgctgtgtcaatgttcaacccgcaaggtaacgat atgaccaaaggactgcttacgctggcgaaaggtaaaccaatcggtaaggaaggttactactggctgaaaatcc acggtgcaaactgtgcgggtgtcgataaggttccgttccctgagcgcatcaagttcattgaggaaaaccacga gaacatcatggcttgcgctaagtctccactggagaacacttggtgggctgagcaagattctccgttctgcttc cttgcgttctgctttgagtacgctggggtacagcaccacggcctgagctataactgctcccttccgctggcgt ttgacgggtcttgctctggcatccagcacttctccgcgatgctccgagatgaggtaggtggtcgcgcggttaa cttgcttcctagtgaaaccgttcaggacatctacgggattgttgctaagaaagtcaacgagattctacaagca gacgcaatcaatgggaccgataacgaagtagttaccgtgaccgatgagaacactggtgaaatctctgagaaag tcaagctgggcactaaggcactggctggtcaatggctggcttacggtgttactcgcagtgtgactaagcgttc agtcatgacgctggcttacgggtccaaagagttcggcttccgtcaacaagtgctggaagataccattcagcca gctattgattccggcaagggtctgatgttcactcagccgaatcaggctgctggatacatggctaagctgattt gggaatctgtgagcgtgacggtggtagctgcggttgaagcaatgaactggcttaagtctgctgctaagctgct ggctgctgaggtcaaagataagaagactggagagattcttcgcaagcgttgcgctgtgcattgggtaactcct gatggtttccctgtgtggcaggaatacaagaagcctattcagacgcgcttgaacctgatgttcctcggtcagt tccgcttacagcctaccattaacaccaacaaagatagcgagattgatgcacacaaacaggagtctggtatcgc tcctaactttgtacacagccaagacggtagccaccttcgtaagactgtagtgtgggcacacgagaagtacgga atcgaatcttttgcactgattcacgactccttcggtaccattccggctgacgctgcgaacctgttcaaagcag tgcgcgaaactatggttgacacatatgagtcttgtgatgtactggctgatttctacgaccagttcgctgaccagttgcacgagtctcaattggacaaaatgccagcacttccggctaaaggtaacttgaacctccgtgacatctta gagtcggagtattttttttgctaa

[0105] [SEQ ID No: 256]

[0106] Thus, this variant polymerase may be encoded by a nucleotide sequence substantially as set out in SEQ ID No: 256, or a fragment or variant thereof.

[0107] In a seventh embodiment, the variant RNA polymerase (referred to herein as " V191") may comprise the substitutions P266L and IYVFS (D879I, F880Y, A881V, A883S). This embodiment produces lower levels of dsRNA compared to the corresponding wild-type RNA polymerase, and so reduces dsRNA contamination. Thus, the variant RNA polymerase may comprise a C-terminal motif comprising an amino acid sequence substantially as set out as SEQ ID No: 201, or a fragment or variant thereof.

[0108] The full amino acid sequence of this variant is represented herein, as SEQ ID No: 257, as follows:

[0109] MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHESYEMGEARFRKMFERQLKAGEVADNAAAKPL ITTLLPKMIARINDWFEEVKAKRGKRPTAFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIE DEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEM LIESTGMVSLHRQNAGWGQDSETIELAPEYAEAIATRAGALAGISLMFQPCVVPPKPWTGITGGGYWANGRR PLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPE DIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGND MTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPFCF LAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQA DAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQP AIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTP DGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNFVHSQDGSHLRKTVVWAHEKYG IESFALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAKGNLNLRDIL ESIYVFS

[0110] [SEQ ID No: 257]

[0111] Thus, the variant polymerase may comprise an amino acid sequence substantially as set out in SEQ ID No: 257, or a fragment or variant thereof.

[0112] This variant is encoded by a DNA sequence represented herein, as SEQ ID No: 258, as follows: atgaacacgattaacatcgctaagaacgacttctctgacatcgaactggctgctatcccgttcaacactctgg ctgaccattacggtgagcgtttagctcgcgaacagttggcccttgagcatgagtcttacgagatgggtgaagc acgcttccgcaagatgtttgagcgtcaacttaaagctggtgaggttgcggataacgctgccgccaagcctctc atcactaccctactccctaagatgattgcacgcatcaacgactggtttgaggaagtgaaagctaagcgcggca agcgcccgacagccttccagttcctgcaagaaatcaagccggaagccgtagcgtacatcaccattaagaccac tctggcttgcctaaccagtgctgacaatacaaccgttcaggctgtagcaagcgcaatcggtcgggccattgag gacgaggctcgcttcggtcgtatccgtgaccttgaagctaagcacttcaagaaaaacgttgaggaacaactca acaagcgcgtagggcacgtctacaagaaagcatttatgcaagttgtcgaggctgacatgctctctaagggtct actcggtggcgaggcgtggtcttcgtggcataaggaagactctattcatgtaggagtacgctgcatcgagatg ctcattgagtcaaccggaatggttagcttacaccgccaaaatgctggcgtagtaggtcaagactctgagacta tcgaactcgcacctgaatacgctgaggctatcgcaacccgtgcaggtgcgctggctggcatctctctgatgtt ccaaccttgcgtagttcctcctaagccgtggactggcattactggtggtggctattgggctaacggtcgtcgt cctctggcgctggtgcgtactcacagtaagaaagcactgatgcgctacgaagacgtttacatgcctgaggtgtacaaagcgattaacattgcgcaaaacaccgcatggaaaatcaacaagaaagtcctagcggtcgccaacgtaat caccaagtggaagcattgtccggtcgaggacatccctgcgattgagcgtgaagaactcccgatgaaaccggaa gacatcgacatgaatcctgaggctctcaccgcgtggaaacgtgctgccgctgctgtgtaccgcaaggacaagg ctcgcaagtctcgccgtatcagccttgagttcatgcttgagcaagccaataagtttgctaaccataaggccat ctggttcccttacaacatggactggcgcggtcgtgtttacgctgtgtcaatgttcaacccgcaaggtaacgat atgaccaaaggactgcttacgctggcgaaaggtaaaccaatcggtaaggaaggttactactggctgaaaatcc acggtgcaaactgtgcgggtgtcgataaggttccgttccctgagcgcatcaagttcattgaggaaaaccacga gaacatcatggcttgcgctaagtctccactggagaacacttggtgggctgagcaagattctccgttctgcttc cttgcgttctgctttgagtacgctggggtacagcaccacggcctgagctataactgctcccttccgctggcgt ttgacgggtcttgctctggcatccagcacttctccgcgatgctccgagatgaggtaggtggtcgcgcggttaa cttgcttcctagtgaaaccgttcaggacatctacgggattgttgctaagaaagtcaacgagattctacaagca gacgcaatcaatgggaccgataacgaagtagttaccgtgaccgatgagaacactggtgaaatctctgagaaag tcaagctgggcactaaggcactggctggtcaatggctggcttacggtgttactcgcagtgtgactaagcgttc agtcatgacgctggcttacgggtccaaagagttcggcttccgtcaacaagtgctggaagataccattcagcca gctattgattccggcaagggtctgatgttcactcagccgaatcaggctgctggatacatggctaagctgattt gggaatctgtgagcgtgacggtggtagctgcggttgaagcaatgaactggcttaagtctgctgctaagctgct ggctgctgaggtcaaagataagaagactggagagattcttcgcaagcgttgcgctgtgcattgggtaactcct gatggtttccctgtgtggcaggaatacaagaagcctattcagacgcgcttgaacctgatgttcctcggtcagt tccgcttacagcctaccattaacaccaacaaagatagcgagattgatgcacacaaacaggagtctggtatcgc tcctaactttgtacacagccaagacggtagccaccttcgtaagactgtagtgtgggcacacgagaagtacgga atcgaatcttttgcactgattcacgactccttcggtaccattccggctgacgctgcgaacctgttcaaagcag tgcgcgaaactatggttgacacatatgagtcttgtgatgtactggctgatttctacgaccagttcgctgacca gttgcacgagtctcaattggacaaaatgccagcacttccggctaaaggtaacttgaacctccgtgacatctta gagtcgatctatgtttttagctaa

[0113] [SEQ ID No: 258]

[0114] Thus, this variant polymerase may be encoded by a nucleotide sequence substantially as set out in SEQ ID No: 258, or a fragment or variant thereof.

[0115] In an eighth embodiment, the variant RNA polymerase (referred to herein as " V210") may comprise the substitutions P266L and RYFFC (D879R, F880Y, A881F, A883C). This embodiment produces lower levels of dsRNA compared to the corresponding wildtype RNA polymerase, and so reduces dsRNA contamination. Thus, the variant RNA polymerase may comprise a C-terminal motif comprising an amino acid sequence substantially as set out as SEQ ID No: 220, or a fragment or variant thereof.

[0116] The full amino acid sequence of this variant is represented herein, as SEQ ID No: 259, as follows:

[0117] MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHESYEMGEARFRKMFERQLKAGEVADNAAAKPL ITTLLPKMIARINDWFEEVKAKRGKRPTAFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIE DEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEM LIESTGMVSLHRQNAGWGQDSETIELAPEYAEAIATRAGALAGISLMFQPCVVPPKPWTGITGGGYWANGRR PLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPE DIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGND MTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPFCF LAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQA DAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQP AIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTP DGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNFVHSQDGSHLRKTVVWAHEKYG IESFALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAKGNLNLRDIL ESRYFFC

[0118] [SEQ ID No: 259]Thus, the variant polymerase may comprise an amino acid sequence substantially as set out in SEQ ID No: 259, or a fragment or variant thereof.

[0119] This variant is encoded by a DNA sequence represented herein, as SEQ ID No: 260, as follows: atgaacacgattaacatcgctaagaacgacttctctgacatcgaactggctgctatcccgttcaacactctgg ctgaccattacggtgagcgtttagctcgcgaacagttggcccttgagcatgagtcttacgagatgggtgaagc acgcttccgcaagatgtttgagcgtcaacttaaagctggtgaggttgcggataacgctgccgccaagcctctc atcactaccctactccctaagatgattgcacgcatcaacgactggtttgaggaagtgaaagctaagcgcggca agcgcccgacagccttccagttcctgcaagaaatcaagccggaagccgtagcgtacatcaccattaagaccac tctggcttgcctaaccagtgctgacaatacaaccgttcaggctgtagcaagcgcaatcggtcgggccattgag gacgaggctcgcttcggtcgtatccgtgaccttgaagctaagcacttcaagaaaaacgttgaggaacaactca acaagcgcgtagggcacgtctacaagaaagcatttatgcaagttgtcgaggctgacatgctctctaagggtct actcggtggcgaggcgtggtcttcgtggcataaggaagactctattcatgtaggagtacgctgcatcgagatg ctcattgagtcaaccggaatggttagcttacaccgccaaaatgctggcgtagtaggtcaagactctgagacta tcgaactcgcacctgaatacgctgaggctatcgcaacccgtgcaggtgcgctggctggcatctctctgatgtt ccaaccttgcgtagttcctcctaagccgtggactggcattactggtggtggctattgggctaacggtcgtcgt cctctggcgctggtgcgtactcacagtaagaaagcactgatgcgctacgaagacgtttacatgcctgaggtgt acaaagcgattaacattgcgcaaaacaccgcatggaaaatcaacaagaaagtcctagcggtcgccaacgtaat caccaagtggaagcattgtccggtcgaggacatccctgcgattgagcgtgaagaactcccgatgaaaccggaa gacatcgacatgaatcctgaggctctcaccgcgtggaaacgtgctgccgctgctgtgtaccgcaaggacaagg ctcgcaagtctcgccgtatcagccttgagttcatgcttgagcaagccaataagtttgctaaccataaggccat ctggttcccttacaacatggactggcgcggtcgtgtttacgctgtgtcaatgttcaacccgcaaggtaacgat atgaccaaaggactgcttacgctggcgaaaggtaaaccaatcggtaaggaaggttactactggctgaaaatcc acggtgcaaactgtgcgggtgtcgataaggttccgttccctgagcgcatcaagttcattgaggaaaaccacga gaacatcatggcttgcgctaagtctccactggagaacacttggtgggctgagcaagattctccgttctgcttc cttgcgttctgctttgagtacgctggggtacagcaccacggcctgagctataactgctcccttccgctggcgt ttgacgggtcttgctctggcatccagcacttctccgcgatgctccgagatgaggtaggtggtcgcgcggttaa cttgcttcctagtgaaaccgttcaggacatctacgggattgttgctaagaaagtcaacgagattctacaagca gacgcaatcaatgggaccgataacgaagtagttaccgtgaccgatgagaacactggtgaaatctctgagaaag tcaagctgggcactaaggcactggctggtcaatggctggcttacggtgttactcgcagtgtgactaagcgttc agtcatgacgctggcttacgggtccaaagagttcggcttccgtcaacaagtgctggaagataccattcagcca gctattgattccggcaagggtctgatgttcactcagccgaatcaggctgctggatacatggctaagctgattt gggaatctgtgagcgtgacggtggtagctgcggttgaagcaatgaactggcttaagtctgctgctaagctgct ggctgctgaggtcaaagataagaagactggagagattcttcgcaagcgttgcgctgtgcattgggtaactcct gatggtttccctgtgtggcaggaatacaagaagcctattcagacgcgcttgaacctgatgttcctcggtcagt tccgcttacagcctaccattaacaccaacaaagatagcgagattgatgcacacaaacaggagtctggtatcgc tcctaactttgtacacagccaagacggtagccaccttcgtaagactgtagtgtgggcacacgagaagtacgga atcgaatcttttgcactgattcacgactccttcggtaccattccggctgacgctgcgaacctgttcaaagcag tgcgcgaaactatggttgacacatatgagtcttgtgatgtactggctgatttctacgaccagttcgctgacca gttgcacgagtctcaattggacaaaatgccagcacttccggctaaaggtaacttgaacctccgtgacatctta gagtcgcgttattttttttgctaa

[0120] [SEQ ID No: 260]

[0121] Thus, this variant polymerase may be encoded by a nucleotide sequence substantially as set out in SEQ ID No: 260, or a fragment or variant thereof.

[0122] The inventors modified wild-type T7 RNA polymerase by substituting amino acid P266L to thereby create a "parental" sequence. Thus, the invention provides a variant T7 RNAP having 883 continuous amino acids with the amino acid substitution P266L. In an embodiment, the amino acid sequence of the parental RNA polymerase is represented herein, as SEQ ID No: 261, as follows:MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHESYEMGEARFRKMFERQLKAGEVADNAAAKPL ITTLLPKMIARINDWFEEVKAKRGKRPTAFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIE DEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEM LIESTGMVSLHRQNAGWGQDSETIELAPEYAEAIATRAGALAGISLMFQPCVVPPKPWTGITGGGYWANGRR PLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPE DIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGND MTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPFCF LAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQA DAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQP AIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTP DGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNFVHSQDGSHLRKTVVWAHEKYG IESFALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAKGNLNLRDIL ESDFAFA

[0123] [SEQ ID No: 261]

[0124] Thus, a parental RNA polymerase may comprise an amino acid sequence substantially as set out in SEQ ID No: 261, or a fragment or variant thereof.

[0125] In a ninth embodiment, the variant RNA polymerase (referred to herein as " V108") may comprise the substitutions P266L and QFVFN (D879Q, A881V, A883N). This embodiment produces lower levels of dsRNA compared to the corresponding wild-type RNA polymerase, and so reduces dsRNA contamination. Thus, the variant RNA polymerase may comprise a C-terminal motif comprising an amino acid sequence substantially as set out as SEQ ID No: 118, or a fragment or variant thereof.

[0126] The full amino acid sequence of this variant is represented herein, as SEQ ID No: 262, as follows:

[0127] MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHESYEMGEARFRKMFERQLKAGEVADNAAAKPL ITTLLPKMIARINDWFEEVKAKRGKRPTAFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIE DEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEM LIESTGMVSLHRQNAGWGQDSETIELAPEYAEAIATRAGALAGISLMFQPCVVPPKPWTGITGGGYWANGRR PLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPE DIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGND MTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPFCF LAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQA DAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQP AIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTP DGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNFVHSQDGSHLRKTVVWAHEKYG IESFALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAKGNLNLRDIL ESQFVFN

[0128] [SEQ ID No: 262]

[0129] Thus, the variant polymerase may comprise an amino acid sequence substantially as set out in SEQ ID No: 262, or a fragment or variant thereof.

[0130] This variant is encoded by a DNA sequence represented herein, as SEQ ID No: 263 as follows:atgaacacgattaacatcgctaagaacgacttctctgacatcgaactggctgctatcccgttcaacactctgg ctgaccattacggtgagcgtttagctcgcgaacagttggcccttgagcatgagtcttacgagatgggtgaagc acgcttccgcaagatgtttgagcgtcaacttaaagctggtgaggttgcggataacgctgccgccaagcctctc atcactaccctactccctaagatgattgcacgcatcaacgactggtttgaggaagtgaaagctaagcgcggca agcgcccgacagccttccagttcctgcaagaaatcaagccggaagccgtagcgtacatcaccattaagaccac tctggcttgcctaaccagtgctgacaatacaaccgttcaggctgtagcaagcgcaatcggtcgggccattgag gacgaggctcgcttcggtcgtatccgtgaccttgaagctaagcacttcaagaaaaacgttgaggaacaactca acaagcgcgtagggcacgtctacaagaaagcatttatgcaagttgtcgaggctgacatgctctctaagggtct actcggtggcgaggcgtggtcttcgtggcataaggaagactctattcatgtaggagtacgctgcatcgagatg ctcattgagtcaaccggaatggttagcttacaccgccaaaatgctggcgtagtaggtcaagactctgagacta tcgaactcgcacctgaatacgctgaggctatcgcaacccgtgcaggtgcgctggctggcatctctctgatgtt ccaaccttgcgtagttcctcctaagccgtggactggcattactggtggtggctattgggctaacggtcgtcgt cctctggcgctggtgcgtactcacagtaagaaagcactgatgcgctacgaagacgtttacatgcctgaggtgt acaaagcgattaacattgcgcaaaacaccgcatggaaaatcaacaagaaagtcctagcggtcgccaacgtaat caccaagtggaagcattgtccggtcgaggacatccctgcgattgagcgtgaagaactcccgatgaaaccggaa gacatcgacatgaatcctgaggctctcaccgcgtggaaacgtgctgccgctgctgtgtaccgcaaggacaagg ctcgcaagtctcgccgtatcagccttgagttcatgcttgagcaagccaataagtttgctaaccataaggccat ctggttcccttacaacatggactggcgcggtcgtgtttacgctgtgtcaatgttcaacccgcaaggtaacgat atgaccaaaggactgcttacgctggcgaaaggtaaaccaatcggtaaggaaggttactactggctgaaaatcc acggtgcaaactgtgcgggtgtcgataaggttccgttccctgagcgcatcaagttcattgaggaaaaccacga gaacatcatggcttgcgctaagtctccactggagaacacttggtgggctgagcaagattctccgttctgcttc cttgcgttctgctttgagtacgctggggtacagcaccacggcctgagctataactgctcccttccgctggcgt ttgacgggtcttgctctggcatccagcacttctccgcgatgctccgagatgaggtaggtggtcgcgcggttaa cttgcttcctagtgaaaccgttcaggacatctacgggattgttgctaagaaagtcaacgagattctacaagca gacgcaatcaatgggaccgataacgaagtagttaccgtgaccgatgagaacactggtgaaatctctgagaaag tcaagctgggcactaaggcactggctggtcaatggctggcttacggtgttactcgcagtgtgactaagcgttc agtcatgacgctggcttacgggtccaaagagttcggcttccgtcaacaagtgctggaagataccattcagcca gctattgattccggcaagggtctgatgttcactcagccgaatcaggctgctggatacatggctaagctgattt gggaatctgtgagcgtgacggtggtagctgcggttgaagcaatgaactggcttaagtctgctgctaagctgct ggctgctgaggtcaaagataagaagactggagagattcttcgcaagcgttgcgctgtgcattgggtaactcct gatggtttccctgtgtggcaggaatacaagaagcctattcagacgcgcttgaacctgatgttcctcggtcagt tccgcttacagcctaccattaacaccaacaaagatagcgagattgatgcacacaaacaggagtctggtatcgc tcctaactttgtacacagccaagacggtagccaccttcgtaagactgtagtgtgggcacacgagaagtacgga atcgaatcttttgcactgattcacgactccttcggtaccattccggctgacgctgcgaacctgttcaaagcag tgcgcgaaactatggttgacacatatgagtcttgtgatgtactggctgatttctacgaccagttcgctgacca gttgcacgagtctcaattggacaaaatgccagcacttccggctaaaggtaacttgaacctccgtgacatctta gagtcgcagtttgtttttaattaa

[0131] [SEQ ID No: 263]

[0132] Thus, this variant polymerase may be encoded by a nucleotide sequence substantially as set out in SEQ ID No: 263, or a fragment or variant thereof.

[0133] In a tenth embodiment, the variant RNA polymerase (referred to herein as " V162") may comprise the substitutions P266L and AYFFC (D879A, F880Y, A881F, A883C). This embodiment produces lower levels of dsRNA compared to the corresponding wildtype RNA polymerase, and so reduces dsRNA contamination. Thus, the variant RNA polymerase may comprise a C-terminal motif comprising an amino acid sequence substantially as set out as SEQ ID No: 172, or a fragment or variant thereof.

[0134] The full amino acid sequence of this variant is represented herein, as SEQ ID No: 264, as follows:MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHESYEMGEARFRKMFERQLKAGEVADNAAAKPL ITTLLPKMIARINDWFEEVKAKRGKRPTAFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIE DEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEM LIESTGMVSLHRQNAGWGQDSETIELAPEYAEAIATRAGALAGISLMFQPCVVPPKPWTGITGGGYWANGRR PLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPE DIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGND MTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPFCF LAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQA DAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQP AIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTP DGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNFVHSQDGSHLRKTVVWAHEKYG IESFALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAKGNLNLRDIL ESAYFFC

[0135] [SEQ ID No: 264]

[0136] Thus, the variant polymerase may comprise an amino acid sequence substantially as set out in SEQ ID No: 264, or a fragment or variant thereof.

[0137] This variant is encoded by a DNA sequence represented herein, as SEQ ID No: 265, as follows: atgaacacgattaacatcgctaagaacgacttctctgacatcgaactggctgctatcccgttcaacactctgg ctgaccattacggtgagcgtttagctcgcgaacagttggcccttgagcatgagtcttacgagatgggtgaagc acgcttccgcaagatgtttgagcgtcaacttaaagctggtgaggttgcggataacgctgccgccaagcctctc atcactaccctactccctaagatgattgcacgcatcaacgactggtttgaggaagtgaaagctaagcgcggca agcgcccgacagccttccagttcctgcaagaaatcaagccggaagccgtagcgtacatcaccattaagaccac tctggcttgcctaaccagtgctgacaatacaaccgttcaggctgtagcaagcgcaatcggtcgggccattgag gacgaggctcgcttcggtcgtatccgtgaccttgaagctaagcacttcaagaaaaacgttgaggaacaactca acaagcgcgtagggcacgtctacaagaaagcatttatgcaagttgtcgaggctgacatgctctctaagggtct actcggtggcgaggcgtggtcttcgtggcataaggaagactctattcatgtaggagtacgctgcatcgagatg ctcattgagtcaaccggaatggttagcttacaccgccaaaatgctggcgtagtaggtcaagactctgagacta tcgaactcgcacctgaatacgctgaggctatcgcaacccgtgcaggtgcgctggctggcatctctctgatgtt ccaaccttgcgtagttcctcctaagccgtggactggcattactggtggtggctattgggctaacggtcgtcgt cctctggcgctggtgcgtactcacagtaagaaagcactgatgcgctacgaagacgtttacatgcctgaggtgt acaaagcgattaacattgcgcaaaacaccgcatggaaaatcaacaagaaagtcctagcggtcgccaacgtaat caccaagtggaagcattgtccggtcgaggacatccctgcgattgagcgtgaagaactcccgatgaaaccggaa gacatcgacatgaatcctgaggctctcaccgcgtggaaacgtgctgccgctgctgtgtaccgcaaggacaagg ctcgcaagtctcgccgtatcagccttgagttcatgcttgagcaagccaataagtttgctaaccataaggccat ctggttcccttacaacatggactggcgcggtcgtgtttacgctgtgtcaatgttcaacccgcaaggtaacgat atgaccaaaggactgcttacgctggcgaaaggtaaaccaatcggtaaggaaggttactactggctgaaaatcc acggtgcaaactgtgcgggtgtcgataaggttccgttccctgagcgcatcaagttcattgaggaaaaccacga gaacatcatggcttgcgctaagtctccactggagaacacttggtgggctgagcaagattctccgttctgcttc cttgcgttctgctttgagtacgctggggtacagcaccacggcctgagctataactgctcccttccgctggcgt ttgacgggtcttgctctggcatccagcacttctccgcgatgctccgagatgaggtaggtggtcgcgcggttaa cttgcttcctagtgaaaccgttcaggacatctacgggattgttgctaagaaagtcaacgagattctacaagca gacgcaatcaatgggaccgataacgaagtagttaccgtgaccgatgagaacactggtgaaatctctgagaaag tcaagctgggcactaaggcactggctggtcaatggctggcttacggtgttactcgcagtgtgactaagcgttc agtcatgacgctggcttacgggtccaaagagttcggcttccgtcaacaagtgctggaagataccattcagcca gctattgattccggcaagggtctgatgttcactcagccgaatcaggctgctggatacatggctaagctgattt gggaatctgtgagcgtgacggtggtagctgcggttgaagcaatgaactggcttaagtctgctgctaagctgct ggctgctgaggtcaaagataagaagactggagagattcttcgcaagcgttgcgctgtgcattgggtaactcct gatggtttccctgtgtggcaggaatacaagaagcctattcagacgcgcttgaacctgatgttcctcggtcagt tccgcttacagcctaccattaacaccaacaaagatagcgagattgatgcacacaaacaggagtctggtatcgc tcctaactttgtacacagccaagacggtagccaccttcgtaagactgtagtgtgggcacacgagaagtacgga atcgaatcttttgcactgattcacgactccttcggtaccattccggctgacgctgcgaacctgttcaaagcag tgcgcgaaactatggttgacacatatgagtcttgtgatgtactggctgatttctacgaccagttcgctgacca gttgcacgagtctcaattggacaaaatgccagcacttccggctaaaggtaacttgaacctccgtgacatctta gagtcggcgtattttttttgctaa[SEQ ID No: 265]

[0138] Thus, this variant polymerase may be encoded by a nucleotide sequence substantially as set out in SEQ ID No: 265, or a fragment or variant thereof.

[0139] In an eleventh embodiment, the variant RNA polymerase (referred to herein as " V45") may comprise the substitutions P266L and PYVFA (D879P, F880Y, A881V). This embodiment produces lower levels of dsRNA compared to the corresponding wild-type RNA polymerase, and so reduces dsRNA contamination. Thus, the variant RNA polymerase may comprise a C-terminal motif comprising an amino acid sequence substantially as set out as SEQ ID No: 55, or a fragment or variant thereof.

[0140] The full amino acid sequence of this variant is represented herein, as SEQ ID No: 266, as follows:

[0141] MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHESYEMGEARFRKMFERQLKAGEVADNAAAKPL ITTLLPKMIARINDWFEEVKAKRGKRPTAFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIE DEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEM LIESTGMVSLHRQNAGWGQDSETIELAPEYAEAIATRAGALAGISLMFQPCVVPPKPWTGITGGGYWANGRR PLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPE DIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGND MTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPFCF LAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQA DAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQP AIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTP DGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNFVHSQDGSHLRKTVVWAHEKYG IESFALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAKGNLNLRDIL ESPYVFA

[0142] [SEQ ID No: 266]

[0143] Thus, the variant polymerase may comprise an amino acid sequence substantially as set out in SEQ ID No: 266, or a fragment or variant thereof.

[0144] This variant is encoded by a DNA sequence represented herein, as SEQ ID No: 267, as follows: atgaacacgattaacatcgctaagaacgacttctctgacatcgaactggctgctatcccgttcaacactctgg ctgaccattacggtgagcgtttagctcgcgaacagttggcccttgagcatgagtcttacgagatgggtgaagc acgcttccgcaagatgtttgagcgtcaacttaaagctggtgaggttgcggataacgctgccgccaagcctctc atcactaccctactccctaagatgattgcacgcatcaacgactggtttgaggaagtgaaagctaagcgcggca agcgcccgacagccttccagttcctgcaagaaatcaagccggaagccgtagcgtacatcaccattaagaccac tctggcttgcctaaccagtgctgacaatacaaccgttcaggctgtagcaagcgcaatcggtcgggccattgag gacgaggctcgcttcggtcgtatccgtgaccttgaagctaagcacttcaagaaaaacgttgaggaacaactca acaagcgcgtagggcacgtctacaagaaagcatttatgcaagttgtcgaggctgacatgctctctaagggtct actcggtggcgaggcgtggtcttcgtggcataaggaagactctattcatgtaggagtacgctgcatcgagatg ctcattgagtcaaccggaatggttagcttacaccgccaaaatgctggcgtagtaggtcaagactctgagacta tcgaactcgcacctgaatacgctgaggctatcgcaacccgtgcaggtgcgctggctggcatctctctgatgtt ccaaccttgcgtagttcctcctaagccgtggactggcattactggtggtggctattgggctaacggtcgtcgt cctctggcgctggtgcgtactcacagtaagaaagcactgatgcgctacgaagacgtttacatgcctgaggtgt acaaagcgattaacattgcgcaaaacaccgcatggaaaatcaacaagaaagtcctagcggtcgccaacgtaat caccaagtggaagcattgtccggtcgaggacatccctgcgattgagcgtgaagaactcccgatgaaaccggaagacatcgacatgaatcctgaggctctcaccgcgtggaaacgtgctgccgctgctgtgtaccgcaaggacaagg ctcgcaagtctcgccgtatcagccttgagttcatgcttgagcaagccaataagtttgctaaccataaggccat ctggttcccttacaacatggactggcgcggtcgtgtttacgctgtgtcaatgttcaacccgcaaggtaacgat atgaccaaaggactgcttacgctggcgaaaggtaaaccaatcggtaaggaaggttactactggctgaaaatcc acggtgcaaactgtgcgggtgtcgataaggttccgttccctgagcgcatcaagttcattgaggaaaaccacga gaacatcatggcttgcgctaagtctccactggagaacacttggtgggctgagcaagattctccgttctgcttc cttgcgttctgctttgagtacgctggggtacagcaccacggcctgagctataactgctcccttccgctggcgt ttgacgggtcttgctctggcatccagcacttctccgcgatgctccgagatgaggtaggtggtcgcgcggttaa cttgcttcctagtgaaaccgttcaggacatctacgggattgttgctaagaaagtcaacgagattctacaagca gacgcaatcaatgggaccgataacgaagtagttaccgtgaccgatgagaacactggtgaaatctctgagaaag tcaagctgggcactaaggcactggctggtcaatggctggcttacggtgttactcgcagtgtgactaagcgttc agtcatgacgctggcttacgggtccaaagagttcggcttccgtcaacaagtgctggaagataccattcagcca gctattgattccggcaagggtctgatgttcactcagccgaatcaggctgctggatacatggctaagctgattt gggaatctgtgagcgtgacggtggtagctgcggttgaagcaatgaactggcttaagtctgctgctaagctgct ggctgctgaggtcaaagataagaagactggagagattcttcgcaagcgttgcgctgtgcattgggtaactcct gatggtttccctgtgtggcaggaatacaagaagcctattcagacgcgcttgaacctgatgttcctcggtcagt tccgcttacagcctaccattaacaccaacaaagatagcgagattgatgcacacaaacaggagtctggtatcgc tcctaactttgtacacagccaagacggtagccaccttcgtaagactgtagtgtgggcacacgagaagtacgga atcgaatcttttgcactgattcacgactccttcggtaccattccggctgacgctgcgaacctgttcaaagcag tgcgcgaaactatggttgacacatatgagtcttgtgatgtactggctgatttctacgaccagttcgctgacca gttgcacgagtctcaattggacaaaatgccagcacttccggctaaaggtaacttgaacctccgtgacatctta gagtcgccgtatgtttttgcgtaa

[0145] [SEQ ID No: 267]

[0146] Thus, this variant polymerase may be encoded by a nucleotide sequence substantially as set out in SEQ ID No: 267, or a fragment or variant thereof.

[0147] In a twelfth embodiment, the variant RNA polymerase (referred to herein as " V63") may comprise the substitutions P266L and EYAFS (D879E, F880Y, A883S). This embodiment produces lower levels of dsRNA compared to the corresponding wild-type RNA polymerase, and so reduces dsRNA contamination. Thus, the variant RNA polymerase may comprise a C-terminal motif comprising an amino acid sequence substantially as set out as SEQ ID No: 73, or a fragment or variant thereof.

[0148] The full amino acid sequence of this variant is represented herein, as SEQ ID No: 268, as follows:

[0149] MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHESYEMGEARFRKMFERQLKAGEVADNAAAKPL ITTLLPKMIARINDWFEEVKAKRGKRPTAFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIE DEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEM LIESTGMVSLHRQNAGWGQDSETIELAPEYAEAIATRAGALAGISLMFQPCVVPPKPWTGITGGGYWANGRR PLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPE DIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGND MTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPFCF LAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQA DAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQP AIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTP DGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNFVHSQDGSHLRKTVVWAHEKYG IESFALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAKGNLNLRDIL ESEYAFS

[0150] [SEQ ID No: 268]Thus, the variant polymerase may comprise an amino acid sequence substantially as set out in SEQ ID No: 268, or a fragment or variant thereof.

[0151] This variant is encoded by a DNA sequence represented herein, as SEQ ID No: 269, as follows: atgaacacgattaacatcgctaagaacgacttctctgacatcgaactggctgctatcccgttcaacactctgg ctgaccattacggtgagcgtttagctcgcgaacagttggcccttgagcatgagtcttacgagatgggtgaagc acgcttccgcaagatgtttgagcgtcaacttaaagctggtgaggttgcggataacgctgccgccaagcctctc atcactaccctactccctaagatgattgcacgcatcaacgactggtttgaggaagtgaaagctaagcgcggca agcgcccgacagccttccagttcctgcaagaaatcaagccggaagccgtagcgtacatcaccattaagaccac tctggcttgcctaaccagtgctgacaatacaaccgttcaggctgtagcaagcgcaatcggtcgggccattgag gacgaggctcgcttcggtcgtatccgtgaccttgaagctaagcacttcaagaaaaacgttgaggaacaactca acaagcgcgtagggcacgtctacaagaaagcatttatgcaagttgtcgaggctgacatgctctctaagggtct actcggtggcgaggcgtggtcttcgtggcataaggaagactctattcatgtaggagtacgctgcatcgagatg ctcattgagtcaaccggaatggttagcttacaccgccaaaatgctggcgtagtaggtcaagactctgagacta tcgaactcgcacctgaatacgctgaggctatcgcaacccgtgcaggtgcgctggctggcatctctctgatgtt ccaaccttgcgtagttcctcctaagccgtggactggcattactggtggtggctattgggctaacggtcgtcgt cctctggcgctggtgcgtactcacagtaagaaagcactgatgcgctacgaagacgtttacatgcctgaggtgt acaaagcgattaacattgcgcaaaacaccgcatggaaaatcaacaagaaagtcctagcggtcgccaacgtaat caccaagtggaagcattgtccggtcgaggacatccctgcgattgagcgtgaagaactcccgatgaaaccggaa gacatcgacatgaatcctgaggctctcaccgcgtggaaacgtgctgccgctgctgtgtaccgcaaggacaagg ctcgcaagtctcgccgtatcagccttgagttcatgcttgagcaagccaataagtttgctaaccataaggccat ctggttcccttacaacatggactggcgcggtcgtgtttacgctgtgtcaatgttcaacccgcaaggtaacgat atgaccaaaggactgcttacgctggcgaaaggtaaaccaatcggtaaggaaggttactactggctgaaaatcc acggtgcaaactgtgcgggtgtcgataaggttccgttccctgagcgcatcaagttcattgaggaaaaccacga gaacatcatggcttgcgctaagtctccactggagaacacttggtgggctgagcaagattctccgttctgcttc cttgcgttctgctttgagtacgctggggtacagcaccacggcctgagctataactgctcccttccgctggcgt ttgacgggtcttgctctggcatccagcacttctccgcgatgctccgagatgaggtaggtggtcgcgcggttaa cttgcttcctagtgaaaccgttcaggacatctacgggattgttgctaagaaagtcaacgagattctacaagca gacgcaatcaatgggaccgataacgaagtagttaccgtgaccgatgagaacactggtgaaatctctgagaaag tcaagctgggcactaaggcactggctggtcaatggctggcttacggtgttactcgcagtgtgactaagcgttc agtcatgacgctggcttacgggtccaaagagttcggcttccgtcaacaagtgctggaagataccattcagcca gctattgattccggcaagggtctgatgttcactcagccgaatcaggctgctggatacatggctaagctgattt gggaatctgtgagcgtgacggtggtagctgcggttgaagcaatgaactggcttaagtctgctgctaagctgct ggctgctgaggtcaaagataagaagactggagagattcttcgcaagcgttgcgctgtgcattgggtaactcct gatggtttccctgtgtggcaggaatacaagaagcctattcagacgcgcttgaacctgatgttcctcggtcagt tccgcttacagcctaccattaacaccaacaaagatagcgagattgatgcacacaaacaggagtctggtatcgc tcctaactttgtacacagccaagacggtagccaccttcgtaagactgtagtgtgggcacacgagaagtacgga atcgaatcttttgcactgattcacgactccttcggtaccattccggctgacgctgcgaacctgttcaaagcag tgcgcgaaactatggttgacacatatgagtcttgtgatgtactggctgatttctacgaccagttcgctgacca gttgcacgagtctcaattggacaaaatgccagcacttccggctaaaggtaacttgaacctccgtgacatctta gagtcggagtatgcgtttagctaa

[0152] [SEQ ID No: 269]

[0153] Thus, this variant polymerase may be encoded by a nucleotide sequence substantially as set out in SEQ ID No: 269, or a fragment or variant thereof.

[0154] In a thirteenth embodiment, the variant RNA polymerase (referred to herein as "V109") may comprise the substitutions P266L and EYAFA (D879Q, F880Y). This embodiment produces higher levels of dsRNA compared to the corresponding wild-type RNA polymerase, and so increases dsRNA contamination. Thus, the variant RNApolymerase may comprise a C-terminal motif comprising an amino acid sequence substantially as set out as SEQ ID No: 119, or a fragment or variant thereof.

[0155] The full amino acid sequence of this variant is represented herein, as SEQ ID No: 270, as follows:

[0156] MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHESYEMGEARFRKMFERQLKAGEVADNAAAKPL ITTLLPKMIARINDWFEEVKAKRGKRPTAFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIE DEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEM LIESTGMVSLHRQNAGWGQDSETIELAPEYAEAIATRAGALAGISLMFQPCVVPPKPWTGITGGGYWANGRR PLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPE DIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGND MTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPFCF LAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQA DAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQP AIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTP DGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNFVHSQDGSHLRKTVVWAHEKYG IESFALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAKGNLNLRDIL ESQYAFA

[0157] [SEQ ID No: 270]

[0158] Thus, the variant polymerase may comprise an amino acid sequence substantially as set out in SEQ ID No: 270, or a fragment or variant thereof.

[0159] This variant is encoded by a DNA sequence represented herein, as SEQ ID No: 271, as follows: atgaacacgattaacatcgctaagaacgacttctctgacatcgaactggctgctatcccgttcaacactctgg ctgaccattacggtgagcgtttagctcgcgaacagttggcccttgagcatgagtcttacgagatgggtgaagc acgcttccgcaagatgtttgagcgtcaacttaaagctggtgaggttgcggataacgctgccgccaagcctctc atcactaccctactccctaagatgattgcacgcatcaacgactggtttgaggaagtgaaagctaagcgcggca agcgcccgacagccttccagttcctgcaagaaatcaagccggaagccgtagcgtacatcaccattaagaccac tctggcttgcctaaccagtgctgacaatacaaccgttcaggctgtagcaagcgcaatcggtcgggccattgag gacgaggctcgcttcggtcgtatccgtgaccttgaagctaagcacttcaagaaaaacgttgaggaacaactca acaagcgcgtagggcacgtctacaagaaagcatttatgcaagttgtcgaggctgacatgctctctaagggtct actcggtggcgaggcgtggtcttcgtggcataaggaagactctattcatgtaggagtacgctgcatcgagatg ctcattgagtcaaccggaatggttagcttacaccgccaaaatgctggcgtagtaggtcaagactctgagacta tcgaactcgcacctgaatacgctgaggctatcgcaacccgtgcaggtgcgctggctggcatctctctgatgtt ccaaccttgcgtagttcctcctaagccgtggactggcattactggtggtggctattgggctaacggtcgtcgt cctctggcgctggtgcgtactcacagtaagaaagcactgatgcgctacgaagacgtttacatgcctgaggtgt acaaagcgattaacattgcgcaaaacaccgcatggaaaatcaacaagaaagtcctagcggtcgccaacgtaat caccaagtggaagcattgtccggtcgaggacatccctgcgattgagcgtgaagaactcccgatgaaaccggaa gacatcgacatgaatcctgaggctctcaccgcgtggaaacgtgctgccgctgctgtgtaccgcaaggacaagg ctcgcaagtctcgccgtatcagccttgagttcatgcttgagcaagccaataagtttgctaaccataaggccat ctggttcccttacaacatggactggcgcggtcgtgtttacgctgtgtcaatgttcaacccgcaaggtaacgat atgaccaaaggactgcttacgctggcgaaaggtaaaccaatcggtaaggaaggttactactggctgaaaatcc acggtgcaaactgtgcgggtgtcgataaggttccgttccctgagcgcatcaagttcattgaggaaaaccacga gaacatcatggcttgcgctaagtctccactggagaacacttggtgggctgagcaagattctccgttctgcttc cttgcgttctgctttgagtacgctggggtacagcaccacggcctgagctataactgctcccttccgctggcgt ttgacgggtcttgctctggcatccagcacttctccgcgatgctccgagatgaggtaggtggtcgcgcggttaa cttgcttcctagtgaaaccgttcaggacatctacgggattgttgctaagaaagtcaacgagattctacaagca gacgcaatcaatgggaccgataacgaagtagttaccgtgaccgatgagaacactggtgaaatctctgagaaag tcaagctgggcactaaggcactggctggtcaatggctggcttacggtgttactcgcagtgtgactaagcgttc agtcatgacgctggcttacgggtccaaagagttcggcttccgtcaacaagtgctggaagataccattcagcca gctattgattccggcaagggtctgatgttcactcagccgaatcaggctgctggatacatggctaagctgattt gggaatctgtgagcgtgacggtggtagctgcggttgaagcaatgaactggcttaagtctgctgctaagctgctggctgctgaggtcaaagataagaagactggagagattcttcgcaagcgttgcgctgtgcattgggtaactcct gatggtttccctgtgtggcaggaatacaagaagcctattcagacgcgcttgaacctgatgttcctcggtcagt tccgcttacagcctaccattaacaccaacaaagatagcgagattgatgcacacaaacaggagtctggtatcgc tcctaactttgtacacagccaagacggtagccaccttcgtaagactgtagtgtgggcacacgagaagtacgga atcgaatcttttgcactgattcacgactccttcggtaccattccggctgacgctgcgaacctgttcaaagcag tgcgcgaaactatggttgacacatatgagtcttgtgatgtactggctgatttctacgaccagttcgctgacca gttgcacgagtctcaattggacaaaatgccagcacttccggctaaaggtaacttgaacctccgtgacatctta gagtcgcagtatgcgtttgcgtaa

[0160] [SEQ ID No: 271]

[0161] Thus, this variant polymerase may be encoded by a nucleotide sequence substantially as set out in SEQ ID No: 271, or a fragment or variant thereof.

[0162] In a fourteenth embodiment, the variant RNA polymerase (referred to herein as " V157") may comprise the substitutions P266L and AYAFA (D879A, F880Y). This embodiment produces higher levels of dsRNA compared to the corresponding wildtype RNA polymerase, and so increases dsRNA contamination. Thus, the variant RNA polymerase may comprise a C-terminal motif comprising an amino acid sequence substantially as set out as SEQ ID No: 167, or a fragment or variant thereof.

[0163] The full amino acid sequence of this variant is represented herein, as SEQ ID No: 272, as follows:

[0164] MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHESYEMGEARFRKMFERQLKAGEVADNAAAKPL ITTLLPKMIARINDWFEEVKAKRGKRPTAFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIE DEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEM LIESTGMVSLHRQNAGWGQDSETIELAPEYAEAIATRAGALAGISLMFQPCVVPPKPWTGITGGGYWANGRR PLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPE DIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGND MTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPFCF LAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQA DAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQP AIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTP DGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNFVHSQDGSHLRKTVVWAHEKYG IESFALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAKGNLNLRDIL ES AYAFA

[0165] [SEQ ID No: 272]

[0166] Thus, the variant polymerase may comprise an amino acid sequence substantially as set out in SEQ ID No: 272, or a fragment or variant thereof.

[0167] This variant is encoded by a DNA sequence represented herein, as SEQ ID No: 273, as follows: atgaacacgattaacatcgctaagaacgacttctctgacatcgaactggctgctatcccgttcaacactctgg ctgaccattacggtgagcgtttagctcgcgaacagttggcccttgagcatgagtcttacgagatgggtgaagc acgcttccgcaagatgtttgagcgtcaacttaaagctggtgaggttgcggataacgctgccgccaagcctctc atcactaccctactccctaagatgattgcacgcatcaacgactggtttgaggaagtgaaagctaagcgcggca agcgcccgacagccttccagttcctgcaagaaatcaagccggaagccgtagcgtacatcaccattaagaccactctggcttgcctaaccagtgctgacaatacaaccgttcaggctgtagcaagcgcaatcggtcgggccattgag gacgaggctcgcttcggtcgtatccgtgaccttgaagctaagcacttcaagaaaaacgttgaggaacaactca acaagcgcgtagggcacgtctacaagaaagcatttatgcaagttgtcgaggctgacatgctctctaagggtct actcggtggcgaggcgtggtcttcgtggcataaggaagactctattcatgtaggagtacgctgcatcgagatg ctcattgagtcaaccggaatggttagcttacaccgccaaaatgctggcgtagtaggtcaagactctgagacta tcgaactcgcacctgaatacgctgaggctatcgcaacccgtgcaggtgcgctggctggcatctctctgatgtt ccaaccttgcgtagttcctcctaagccgtggactggcattactggtggtggctattgggctaacggtcgtcgt cctctggcgctggtgcgtactcacagtaagaaagcactgatgcgctacgaagacgtttacatgcctgaggtgt acaaagcgattaacattgcgcaaaacaccgcatggaaaatcaacaagaaagtcctagcggtcgccaacgtaat caccaagtggaagcattgtccggtcgaggacatccctgcgattgagcgtgaagaactcccgatgaaaccggaa gacatcgacatgaatcctgaggctctcaccgcgtggaaacgtgctgccgctgctgtgtaccgcaaggacaagg ctcgcaagtctcgccgtatcagccttgagttcatgcttgagcaagccaataagtttgctaaccataaggccat ctggttcccttacaacatggactggcgcggtcgtgtttacgctgtgtcaatgttcaacccgcaaggtaacgat atgaccaaaggactgcttacgctggcgaaaggtaaaccaatcggtaaggaaggttactactggctgaaaatcc acggtgcaaactgtgcgggtgtcgataaggttccgttccctgagcgcatcaagttcattgaggaaaaccacga gaacatcatggcttgcgctaagtctccactggagaacacttggtgggctgagcaagattctccgttctgcttc cttgcgttctgctttgagtacgctggggtacagcaccacggcctgagctataactgctcccttccgctggcgt ttgacgggtcttgctctggcatccagcacttctccgcgatgctccgagatgaggtaggtggtcgcgcggttaa cttgcttcctagtgaaaccgttcaggacatctacgggattgttgctaagaaagtcaacgagattctacaagca gacgcaatcaatgggaccgataacgaagtagttaccgtgaccgatgagaacactggtgaaatctctgagaaag tcaagctgggcactaaggcactggctggtcaatggctggcttacggtgttactcgcagtgtgactaagcgttc agtcatgacgctggcttacgggtccaaagagttcggcttccgtcaacaagtgctggaagataccattcagcca gctattgattccggcaagggtctgatgttcactcagccgaatcaggctgctggatacatggctaagctgattt gggaatctgtgagcgtgacggtggtagctgcggttgaagcaatgaactggcttaagtctgctgctaagctgct ggctgctgaggtcaaagataagaagactggagagattcttcgcaagcgttgcgctgtgcattgggtaactcct gatggtttccctgtgtggcaggaatacaagaagcctattcagacgcgcttgaacctgatgttcctcggtcagt tccgcttacagcctaccattaacaccaacaaagatagcgagattgatgcacacaaacaggagtctggtatcgc tcctaactttgtacacagccaagacggtagccaccttcgtaagactgtagtgtgggcacacgagaagtacgga atcgaatcttttgcactgattcacgactccttcggtaccattccggctgacgctgcgaacctgttcaaagcag tgcgcgaaactatggttgacacatatgagtcttgtgatgtactggctgatttctacgaccagttcgctgacca gttgcacgagtctcaattggacaaaatgccagcacttccggctaaaggtaacttgaacctccgtgacatctta gagtcggcgtatgcgtttgcgtaa

[0168] [SEQ ID No: 273]

[0169] Thus, this variant polymerase may be encoded by a nucleotide sequence substantially as set out in SEQ ID No: 273, or a fragment or variant thereof.

[0170] In a fifteenth embodiment, the variant RNA polymerase (referred to herein as " V37") may comprise the substitutions P266L and PYAFA (D879P, F880Y). This embodiment produces higher levels of dsRNA compared to the corresponding wild-type RNA polymerase, and so increases dsRNA contamination. Thus, the variant RNA polymerase may comprise a C-terminal motif comprising an amino acid sequence substantially as set out as SEQ ID No: 47, or a fragment or variant thereof.

[0171] The full amino acid sequence of this variant is represented herein, as SEQ ID No: 274, as follows:

[0172] MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHESYEMGEARFRKMFERQLKAGEVADNAAAKPL ITTLLPKMIARINDWFEEVKAKRGKRPTAFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIE DEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEM LIESTGMVSLHRQNAGWGQDSETIELAPEYAEAIATRAGALAGISLMFQPCVVPPKPWTGITGGGYWANGRR PLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPE DIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGND MTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPFCFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQA DAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQP AIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTP DGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNFVHSQDGSHLRKTVVWAHEKYG IESFALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAKGNLNLRDIL ESPYAFA

[0173] [SEQ ID No: 274]

[0174] Thus, the variant polymerase may comprise an amino acid sequence substantially as set out in SEQ ID No: 274, or a fragment or variant thereof.

[0175] This variant is encoded by a DNA sequence represented herein, as SEQ ID No: 275, as follows: atgaacacgattaacatcgctaagaacgacttctctgacatcgaactggctgctatcccgttcaacactctgg ctgaccattacggtgagcgtttagctcgcgaacagttggcccttgagcatgagtcttacgagatgggtgaagc acgcttccgcaagatgtttgagcgtcaacttaaagctggtgaggttgcggataacgctgccgccaagcctctc atcactaccctactccctaagatgattgcacgcatcaacgactggtttgaggaagtgaaagctaagcgcggca agcgcccgacagccttccagttcctgcaagaaatcaagccggaagccgtagcgtacatcaccattaagaccac tctggcttgcctaaccagtgctgacaatacaaccgttcaggctgtagcaagcgcaatcggtcgggccattgag gacgaggctcgcttcggtcgtatccgtgaccttgaagctaagcacttcaagaaaaacgttgaggaacaactca acaagcgcgtagggcacgtctacaagaaagcatttatgcaagttgtcgaggctgacatgctctctaagggtct actcggtggcgaggcgtggtcttcgtggcataaggaagactctattcatgtaggagtacgctgcatcgagatg ctcattgagtcaaccggaatggttagcttacaccgccaaaatgctggcgtagtaggtcaagactctgagacta tcgaactcgcacctgaatacgctgaggctatcgcaacccgtgcaggtgcgctggctggcatctctctgatgtt ccaaccttgcgtagttcctcctaagccgtggactggcattactggtggtggctattgggctaacggtcgtcgt cctctggcgctggtgcgtactcacagtaagaaagcactgatgcgctacgaagacgtttacatgcctgaggtgt acaaagcgattaacattgcgcaaaacaccgcatggaaaatcaacaagaaagtcctagcggtcgccaacgtaat caccaagtggaagcattgtccggtcgaggacatccctgcgattgagcgtgaagaactcccgatgaaaccggaa gacatcgacatgaatcctgaggctctcaccgcgtggaaacgtgctgccgctgctgtgtaccgcaaggacaagg ctcgcaagtctcgccgtatcagccttgagttcatgcttgagcaagccaataagtttgctaaccataaggccat ctggttcccttacaacatggactggcgcggtcgtgtttacgctgtgtcaatgttcaacccgcaaggtaacgat atgaccaaaggactgcttacgctggcgaaaggtaaaccaatcggtaaggaaggttactactggctgaaaatcc acggtgcaaactgtgcgggtgtcgataaggttccgttccctgagcgcatcaagttcattgaggaaaaccacga gaacatcatggcttgcgctaagtctccactggagaacacttggtgggctgagcaagattctccgttctgcttc cttgcgttctgctttgagtacgctggggtacagcaccacggcctgagctataactgctcccttccgctggcgt ttgacgggtcttgctctggcatccagcacttctccgcgatgctccgagatgaggtaggtggtcgcgcggttaa cttgcttcctagtgaaaccgttcaggacatctacgggattgttgctaagaaagtcaacgagattctacaagca gacgcaatcaatgggaccgataacgaagtagttaccgtgaccgatgagaacactggtgaaatctctgagaaag tcaagctgggcactaaggcactggctggtcaatggctggcttacggtgttactcgcagtgtgactaagcgttc agtcatgacgctggcttacgggtccaaagagttcggcttccgtcaacaagtgctggaagataccattcagcca gctattgattccggcaagggtctgatgttcactcagccgaatcaggctgctggatacatggctaagctgattt gggaatctgtgagcgtgacggtggtagctgcggttgaagcaatgaactggcttaagtctgctgctaagctgct ggctgctgaggtcaaagataagaagactggagagattcttcgcaagcgttgcgctgtgcattgggtaactcct gatggtttccctgtgtggcaggaatacaagaagcctattcagacgcgcttgaacctgatgttcctcggtcagt tccgcttacagcctaccattaacaccaacaaagatagcgagattgatgcacacaaacaggagtctggtatcgc tcctaactttgtacacagccaagacggtagccaccttcgtaagactgtagtgtgggcacacgagaagtacgga atcgaatcttttgcactgattcacgactccttcggtaccattccggctgacgctgcgaacctgttcaaagcag tgcgcgaaactatggttgacacatatgagtcttgtgatgtactggctgatttctacgaccagttcgctgacca gttgcacgagtctcaattggacaaaatgccagcacttccggctaaaggtaacttgaacctccgtgacatctta gagtcgccgtatgcgtttgcgtaa

[0176] [SEQ ID No: 275]

[0177] Thus, this variant polymerase may be encoded by a nucleotide sequence substantially as set out in SEQ ID No: 275, or a fragment or variant thereof.In a sixteenth embodiment, the variant RNA polymerase (referred to herein as " V109") may comprise the substitutions P266L and QYAFA (D879Q, F880Y). This embodiment produces higher levels of dsRNA compared to the corresponding wildtype RNA polymerase, and so increases dsRNA contamination. Thus, the variant RNA polymerase may comprise a C-terminal motif comprising an amino acid sequence substantially as set out as SEQ ID No: 119, or a fragment or variant thereof.

[0178] The full amino acid sequence of this variant is represented herein, as SEQ ID No: 276, as follows:

[0179] MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHESYEMGEARFRKMFERQLKAGEVADNAAAKPL ITTLLPKMIARINDWFEEVKAKRGKRPTAFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIE DEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEM LIESTGMVSLHRQNAGWGQDSETIELAPEYAEAIATRAGALAGISLMFQPCVVPPKPWTGITGGGYWANGRR PLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPE DIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGND MTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPFCF LAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQA DAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQP AIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTP DGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNFVHSQDGSHLRKTVVWAHEKYG IESFALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAKGNLNLRDIL ESQYAFA

[0180] [SEQ ID No: 276]

[0181] Thus, the variant polymerase may comprise an amino acid sequence substantially as set out in SEQ ID No: 276, or a fragment or variant thereof.

[0182] This variant is encoded by a DNA sequence represented herein, as SEQ ID No: 277, as follows: atgaacacgattaacatcgctaagaacgacttctctgacatcgaactggctgctatcccgttcaacactctgg ctgaccattacggtgagcgtttagctcgcgaacagttggcccttgagcatgagtcttacgagatgggtgaagc acgcttccgcaagatgtttgagcgtcaacttaaagctggtgaggttgcggataacgctgccgccaagcctctc atcactaccctactccctaagatgattgcacgcatcaacgactggtttgaggaagtgaaagctaagcgcggca agcgcccgacagccttccagttcctgcaagaaatcaagccggaagccgtagcgtacatcaccattaagaccac tctggcttgcctaaccagtgctgacaatacaaccgttcaggctgtagcaagcgcaatcggtcgggccattgag gacgaggctcgcttcggtcgtatccgtgaccttgaagctaagcacttcaagaaaaacgttgaggaacaactca acaagcgcgtagggcacgtctacaagaaagcatttatgcaagttgtcgaggctgacatgctctctaagggtct actcggtggcgaggcgtggtcttcgtggcataaggaagactctattcatgtaggagtacgctgcatcgagatg ctcattgagtcaaccggaatggttagcttacaccgccaaaatgctggcgtagtaggtcaagactctgagacta tcgaactcgcacctgaatacgctgaggctatcgcaacccgtgcaggtgcgctggctggcatctctctgatgtt ccaaccttgcgtagttcctcctaagccgtggactggcattactggtggtggctattgggctaacggtcgtcgt cctctggcgctggtgcgtactcacagtaagaaagcactgatgcgctacgaagacgtttacatgcctgaggtgt acaaagcgattaacattgcgcaaaacaccgcatggaaaatcaacaagaaagtcctagcggtcgccaacgtaat caccaagtggaagcattgtccggtcgaggacatccctgcgattgagcgtgaagaactcccgatgaaaccggaa gacatcgacatgaatcctgaggctctcaccgcgtggaaacgtgctgccgctgctgtgtaccgcaaggacaagg ctcgcaagtctcgccgtatcagccttgagttcatgcttgagcaagccaataagtttgctaaccataaggccat ctggttcccttacaacatggactggcgcggtcgtgtttacgctgtgtcaatgttcaacccgcaaggtaacgat atgaccaaaggactgcttacgctggcgaaaggtaaaccaatcggtaaggaaggttactactggctgaaaatcc acggtgcaaactgtgcgggtgtcgataaggttccgttccctgagcgcatcaagttcattgaggaaaaccacga gaacatcatggcttgcgctaagtctccactggagaacacttggtgggctgagcaagattctccgttctgcttc cttgcgttctgctttgagtacgctggggtacagcaccacggcctgagctataactgctcccttccgctggcgt ttgacgggtcttgctctggcatccagcacttctccgcgatgctccgagatgaggtaggtggtcgcgcggttaacttgcttcctagtgaaaccgttcaggacatctacgggattgttgctaagaaagtcaacgagattctacaagca gacgcaatcaatgggaccgataacgaagtagttaccgtgaccgatgagaacactggtgaaatctctgagaaag tcaagctgggcactaaggcactggctggtcaatggctggcttacggtgttactcgcagtgtgactaagcgttc agtcatgacgctggcttacgggtccaaagagttcggcttccgtcaacaagtgctggaagataccattcagcca gctattgattccggcaagggtctgatgttcactcagccgaatcaggctgctggatacatggctaagctgattt gggaatctgtgagcgtgacggtggtagctgcggttgaagcaatgaactggcttaagtctgctgctaagctgct ggctgctgaggtcaaagataagaagactggagagattcttcgcaagcgttgcgctgtgcattgggtaactcct gatggtttccctgtgtggcaggaatacaagaagcctattcagacgcgcttgaacctgatgttcctcggtcagt tccgcttacagcctaccattaacaccaacaaagatagcgagattgatgcacacaaacaggagtctggtatcgc tcctaactttgtacacagccaagacggtagccaccttcgtaagactgtagtgtgggcacacgagaagtacgga atcgaatcttttgcactgattcacgactccttcggtaccattccggctgacgctgcgaacctgttcaaagcag tgcgcgaaactatggttgacacatatgagtcttgtgatgtactggctgatttctacgaccagttcgctgacca gttgcacgagtctcaattggacaaaatgccagcacttccggctaaaggtaacttgaacctccgtgacatctta gagtcgcagtatgcgtttgcgtaa

[0183] [SEQ ID No: 277]

[0184] Thus, this variant polymerase may be encoded by a nucleotide sequence substantially as set out in SEQ ID No: 277, or a fragment or variant thereof.

[0185] In a seventeenth embodiment, the variant RNA polymerase (referred to herein as " V97") may comprise the substitutions P266L and QFAFA (D879Q). This embodiment produces higher levels of dsRNA compared to the corresponding wild-type RNA polymerase, and so increases dsRNA contamination. Thus, the variant RNA polymerase may comprise a C-terminal motif comprising an amino acid sequence substantially as set out as SEQ ID No: 107, or a fragment or variant thereof.

[0186] The full amino acid sequence of this variant is represented herein, as SEQ ID No: 278, as follows:

[0187] MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHESYEMGEARFRKMFERQLKAGEVADNAAAKPL ITTLLPKMIARINDWFEEVKAKRGKRPTAFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIE DEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEM LIESTGMVSLHRQNAGWGQDSETIELAPEYAEAIATRAGALAGISLMFQPCVVPPKPWTGITGGGYWANGRR PLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPE DIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGND MTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPFCF LAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQA DAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQP AIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTP DGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNFVHSQDGSHLRKTVVWAHEKYG IESFALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAKGNLNLRDIL ESQFAFA

[0188] [SEQ ID No: 278]

[0189] Thus, the variant polymerase may comprise an amino acid sequence substantially as set out in SEQ ID No: 278, or a fragment or variant thereof.

[0190] This variant is encoded by a DNA sequence represented herein, as SEQ ID No: 279, as follows:atgaacacgattaacatcgctaagaacgacttctctgacatcgaactggctgctatcccgttcaacactctgg ctgaccattacggtgagcgtttagctcgcgaacagttggcccttgagcatgagtcttacgagatgggtgaagc acgcttccgcaagatgtttgagcgtcaacttaaagctggtgaggttgcggataacgctgccgccaagcctctc atcactaccctactccctaagatgattgcacgcatcaacgactggtttgaggaagtgaaagctaagcgcggca agcgcccgacagccttccagttcctgcaagaaatcaagccggaagccgtagcgtacatcaccattaagaccac tctggcttgcctaaccagtgctgacaatacaaccgttcaggctgtagcaagcgcaatcggtcgggccattgag gacgaggctcgcttcggtcgtatccgtgaccttgaagctaagcacttcaagaaaaacgttgaggaacaactca acaagcgcgtagggcacgtctacaagaaagcatttatgcaagttgtcgaggctgacatgctctctaagggtct actcggtggcgaggcgtggtcttcgtggcataaggaagactctattcatgtaggagtacgctgcatcgagatg ctcattgagtcaaccggaatggttagcttacaccgccaaaatgctggcgtagtaggtcaagactctgagacta tcgaactcgcacctgaatacgctgaggctatcgcaacccgtgcaggtgcgctggctggcatctctctgatgtt ccaaccttgcgtagttcctcctaagccgtggactggcattactggtggtggctattgggctaacggtcgtcgt cctctggcgctggtgcgtactcacagtaagaaagcactgatgcgctacgaagacgtttacatgcctgaggtgt acaaagcgattaacattgcgcaaaacaccgcatggaaaatcaacaagaaagtcctagcggtcgccaacgtaat caccaagtggaagcattgtccggtcgaggacatccctgcgattgagcgtgaagaactcccgatgaaaccggaa gacatcgacatgaatcctgaggctctcaccgcgtggaaacgtgctgccgctgctgtgtaccgcaaggacaagg ctcgcaagtctcgccgtatcagccttgagttcatgcttgagcaagccaataagtttgctaaccataaggccat ctggttcccttacaacatggactggcgcggtcgtgtttacgctgtgtcaatgttcaacccgcaaggtaacgat atgaccaaaggactgcttacgctggcgaaaggtaaaccaatcggtaaggaaggttactactggctgaaaatcc acggtgcaaactgtgcgggtgtcgataaggttccgttccctgagcgcatcaagttcattgaggaaaaccacga gaacatcatggcttgcgctaagtctccactggagaacacttggtgggctgagcaagattctccgttctgcttc cttgcgttctgctttgagtacgctggggtacagcaccacggcctgagctataactgctcccttccgctggcgt ttgacgggtcttgctctggcatccagcacttctccgcgatgctccgagatgaggtaggtggtcgcgcggttaa cttgcttcctagtgaaaccgttcaggacatctacgggattgttgctaagaaagtcaacgagattctacaagca gacgcaatcaatgggaccgataacgaagtagttaccgtgaccgatgagaacactggtgaaatctctgagaaag tcaagctgggcactaaggcactggctggtcaatggctggcttacggtgttactcgcagtgtgactaagcgttc agtcatgacgctggcttacgggtccaaagagttcggcttccgtcaacaagtgctggaagataccattcagcca gctattgattccggcaagggtctgatgttcactcagccgaatcaggctgctggatacatggctaagctgattt gggaatctgtgagcgtgacggtggtagctgcggttgaagcaatgaactggcttaagtctgctgctaagctgct ggctgctgaggtcaaagataagaagactggagagattcttcgcaagcgttgcgctgtgcattgggtaactcct gatggtttccctgtgtggcaggaatacaagaagcctattcagacgcgcttgaacctgatgttcctcggtcagt tccgcttacagcctaccattaacaccaacaaagatagcgagattgatgcacacaaacaggagtctggtatcgc tcctaactttgtacacagccaagacggtagccaccttcgtaagactgtagtgtgggcacacgagaagtacgga atcgaatcttttgcactgattcacgactccttcggtaccattccggctgacgctgcgaacctgttcaaagcag tgcgcgaaactatggttgacacatatgagtcttgtgatgtactggctgatttctacgaccagttcgctgacca gttgcacgagtctcaattggacaaaatgccagcacttccggctaaaggtaacttgaacctccgtgacatctta gagtcgcagtttgcgtttgcgtaa

[0191] [SEQ ID No: 279]

[0192] Thus, this variant polymerase may be encoded by a nucleotide sequence substantially as set out in SEQ ID No: 279, or a fragment or variant thereof.

[0193] In an eighteenth embodiment, the variant RNA polymerase (referred to herein as " V133") may comprise the substitutions P266L and SYAFA (D879S, F880Y). This embodiment produces higher levels of dsRNA compared to the corresponding wildtype RNA polymerase, and so increases dsRNA contamination. Thus, the variant RNA polymerase may comprise a C-terminal motif comprising an amino acid sequence substantially as set out as SEQ ID No: 143, or a fragment or variant thereof.

[0194] The full amino acid sequence of this variant is represented herein, as SEQ ID No: 280, as follows:

[0195] MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHESYEMGEARFRKMFERQLKAGEVADNAAAKPL ITTLLPKMIARINDWFEEVKAKRGKRPTAFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEM LIESTGMVSLHRQNAGWGQDSETIELAPEYAEAIATRAGALAGISLMFQPCVVPPKPWTGITGGGYWANGRR PLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPE DIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGND MTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPFCF LAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQA DAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQP AIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTP DGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNFVHSQDGSHLRKTVVWAHEKYG IESFALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAKGNLNLRDIL ESSYAFA

[0196] [SEQ ID No: 280]

[0197] Thus, the variant polymerase may comprise an amino acid sequence substantially as set out in SEQ ID No: 280, or a fragment or variant thereof.

[0198] This variant is encoded by a DNA sequence represented herein, as SEQ ID No: 281, as follows: atgaacacgattaacatcgctaagaacgacttctctgacatcgaactggctgctatcccgttcaacactctgg ctgaccattacggtgagcgtttagctcgcgaacagttggcccttgagcatgagtcttacgagatgggtgaagc acgcttccgcaagatgtttgagcgtcaacttaaagctggtgaggttgcggataacgctgccgccaagcctctc atcactaccctactccctaagatgattgcacgcatcaacgactggtttgaggaagtgaaagctaagcgcggca agcgcccgacagccttccagttcctgcaagaaatcaagccggaagccgtagcgtacatcaccattaagaccac tctggcttgcctaaccagtgctgacaatacaaccgttcaggctgtagcaagcgcaatcggtcgggccattgag gacgaggctcgcttcggtcgtatccgtgaccttgaagctaagcacttcaagaaaaacgttgaggaacaactca acaagcgcgtagggcacgtctacaagaaagcatttatgcaagttgtcgaggctgacatgctctctaagggtct actcggtggcgaggcgtggtcttcgtggcataaggaagactctattcatgtaggagtacgctgcatcgagatg ctcattgagtcaaccggaatggttagcttacaccgccaaaatgctggcgtagtaggtcaagactctgagacta tcgaactcgcacctgaatacgctgaggctatcgcaacccgtgcaggtgcgctggctggcatctctctgatgtt ccaaccttgcgtagttcctcctaagccgtggactggcattactggtggtggctattgggctaacggtcgtcgt cctctggcgctggtgcgtactcacagtaagaaagcactgatgcgctacgaagacgtttacatgcctgaggtgt acaaagcgattaacattgcgcaaaacaccgcatggaaaatcaacaagaaagtcctagcggtcgccaacgtaat caccaagtggaagcattgtccggtcgaggacatccctgcgattgagcgtgaagaactcccgatgaaaccggaa gacatcgacatgaatcctgaggctctcaccgcgtggaaacgtgctgccgctgctgtgtaccgcaaggacaagg ctcgcaagtctcgccgtatcagccttgagttcatgcttgagcaagccaataagtttgctaaccataaggccat ctggttcccttacaacatggactggcgcggtcgtgtttacgctgtgtcaatgttcaacccgcaaggtaacgat atgaccaaaggactgcttacgctggcgaaaggtaaaccaatcggtaaggaaggttactactggctgaaaatcc acggtgcaaactgtgcgggtgtcgataaggttccgttccctgagcgcatcaagttcattgaggaaaaccacga gaacatcatggcttgcgctaagtctccactggagaacacttggtgggctgagcaagattctccgttctgcttc cttgcgttctgctttgagtacgctggggtacagcaccacggcctgagctataactgctcccttccgctggcgt ttgacgggtcttgctctggcatccagcacttctccgcgatgctccgagatgaggtaggtggtcgcgcggttaa cttgcttcctagtgaaaccgttcaggacatctacgggattgttgctaagaaagtcaacgagattctacaagca gacgcaatcaatgggaccgataacgaagtagttaccgtgaccgatgagaacactggtgaaatctctgagaaag tcaagctgggcactaaggcactggctggtcaatggctggcttacggtgttactcgcagtgtgactaagcgttc agtcatgacgctggcttacgggtccaaagagttcggcttccgtcaacaagtgctggaagataccattcagcca gctattgattccggcaagggtctgatgttcactcagccgaatcaggctgctggatacatggctaagctgattt gggaatctgtgagcgtgacggtggtagctgcggttgaagcaatgaactggcttaagtctgctgctaagctgct ggctgctgaggtcaaagataagaagactggagagattcttcgcaagcgttgcgctgtgcattgggtaactcct gatggtttccctgtgtggcaggaatacaagaagcctattcagacgcgcttgaacctgatgttcctcggtcagt tccgcttacagcctaccattaacaccaacaaagatagcgagattgatgcacacaaacaggagtctggtatcgc tcctaactttgtacacagccaagacggtagccaccttcgtaagactgtagtgtgggcacacgagaagtacgga atcgaatcttttgcactgattcacgactccttcggtaccattccggctgacgctgcgaacctgttcaaagcag tgcgcgaaactatggttgacacatatgagtcttgtgatgtactggctgatttctacgaccagttcgctgacca gttgcacgagtctcaattggacaaaatgccagcacttccggctaaaggtaacttgaacctccgtgacatctta gagtcgagctatgcgtttgcgtaa

[0199] [SEQ ID No: 281]Thus, this variant polymerase may be encoded by a nucleotide sequence substantially as set out in SEQ ID No: 281, or a fragment or variant thereof.

[0200] In a nineteenth embodiment, the variant RNA polymerase (referred to herein as " V229") may comprise the substitutions P266L and DYAFA (F880Y). This embodiment produces higher levels of dsRNA compared to the corresponding wild-type RNA polymerase, and so increases dsRNA contamination. Thus, the variant RNA polymerase may comprise a C-terminal motif comprising an amino acid sequence substantially as set out as SEQ ID No: 239, or a fragment or variant thereof.

[0201] The full amino acid sequence of this variant is represented herein, as SEQ ID No: 282, as follows:

[0202] MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHESYEMGEARFRKMFERQLKAGEVADNAAAKPL ITTLLPKMIARINDWFEEVKAKRGKRPTAFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIE DEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEM LIESTGMVSLHRQNAGWGQDSETIELAPEYAEAIATRAGALAGISLMFQPCVVPPKPWTGITGGGYWANGRR PLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPE DIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGND MTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPFCF LAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQA DAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQP AIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTP DGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNFVHSQDGSHLRKTVVWAHEKYG IESFALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAKGNLNLRDIL ESDYAFA

[0203] [SEQ ID No: 282]

[0204] Thus, the variant polymerase may comprise an amino acid sequence substantially as set out in SEQ ID No: 282, or a fragment or variant thereof.

[0205] This variant is encoded by a DNA sequence represented herein, as SEQ ID No: 283, as follows: atgaacacgattaacatcgctaagaacgacttctctgacatcgaactggctgctatcccgttcaacactctgg ctgaccattacggtgagcgtttagctcgcgaacagttggcccttgagcatgagtcttacgagatgggtgaagc acgcttccgcaagatgtttgagcgtcaacttaaagctggtgaggttgcggataacgctgccgccaagcctctc atcactaccctactccctaagatgattgcacgcatcaacgactggtttgaggaagtgaaagctaagcgcggca agcgcccgacagccttccagttcctgcaagaaatcaagccggaagccgtagcgtacatcaccattaagaccac tctggcttgcctaaccagtgctgacaatacaaccgttcaggctgtagcaagcgcaatcggtcgggccattgag gacgaggctcgcttcggtcgtatccgtgaccttgaagctaagcacttcaagaaaaacgttgaggaacaactca acaagcgcgtagggcacgtctacaagaaagcatttatgcaagttgtcgaggctgacatgctctctaagggtct actcggtggcgaggcgtggtcttcgtggcataaggaagactctattcatgtaggagtacgctgcatcgagatg ctcattgagtcaaccggaatggttagcttacaccgccaaaatgctggcgtagtaggtcaagactctgagacta tcgaactcgcacctgaatacgctgaggctatcgcaacccgtgcaggtgcgctggctggcatctctctgatgtt ccaaccttgcgtagttcctcctaagccgtggactggcattactggtggtggctattgggctaacggtcgtcgt cctctggcgctggtgcgtactcacagtaagaaagcactgatgcgctacgaagacgtttacatgcctgaggtgt acaaagcgattaacattgcgcaaaacaccgcatggaaaatcaacaagaaagtcctagcggtcgccaacgtaat caccaagtggaagcattgtccggtcgaggacatccctgcgattgagcgtgaagaactcccgatgaaaccggaa gacatcgacatgaatcctgaggctctcaccgcgtggaaacgtgctgccgctgctgtgtaccgcaaggacaaggctcgcaagtctcgccgtatcagccttgagttcatgcttgagcaagccaataagtttgctaaccataaggccat ctggttcccttacaacatggactggcgcggtcgtgtttacgctgtgtcaatgttcaacccgcaaggtaacgat atgaccaaaggactgcttacgctggcgaaaggtaaaccaatcggtaaggaaggttactactggctgaaaatcc acggtgcaaactgtgcgggtgtcgataaggttccgttccctgagcgcatcaagttcattgaggaaaaccacga gaacatcatggcttgcgctaagtctccactggagaacacttggtgggctgagcaagattctccgttctgcttc cttgcgttctgctttgagtacgctggggtacagcaccacggcctgagctataactgctcccttccgctggcgt ttgacgggtcttgctctggcatccagcacttctccgcgatgctccgagatgaggtaggtggtcgcgcggttaa cttgcttcctagtgaaaccgttcaggacatctacgggattgttgctaagaaagtcaacgagattctacaagca gacgcaatcaatgggaccgataacgaagtagttaccgtgaccgatgagaacactggtgaaatctctgagaaag tcaagctgggcactaaggcactggctggtcaatggctggcttacggtgttactcgcagtgtgactaagcgttc agtcatgacgctggcttacgggtccaaagagttcggcttccgtcaacaagtgctggaagataccattcagcca gctattgattccggcaagggtctgatgttcactcagccgaatcaggctgctggatacatggctaagctgattt gggaatctgtgagcgtgacggtggtagctgcggttgaagcaatgaactggcttaagtctgctgctaagctgct ggctgctgaggtcaaagataagaagactggagagattcttcgcaagcgttgcgctgtgcattgggtaactcct gatggtttccctgtgtggcaggaatacaagaagcctattcagacgcgcttgaacctgatgttcctcggtcagt tccgcttacagcctaccattaacaccaacaaagatagcgagattgatgcacacaaacaggagtctggtatcgc tcctaactttgtacacagccaagacggtagccaccttcgtaagactgtagtgtgggcacacgagaagtacgga atcgaatcttttgcactgattcacgactccttcggtaccattccggctgacgctgcgaacctgttcaaagcag tgcgcgaaactatggttgacacatatgagtcttgtgatgtactggctgatttctacgaccagttcgctgacca gttgcacgagtctcaattggacaaaatgccagcacttccggctaaaggtaacttgaacctccgtgacatctta gagtcggattatgcgtttgcgtaa

[0206] [SEQ ID No: 283]

[0207] Thus, this variant polymerase may be encoded by a nucleotide sequence substantially as set out in SEQ ID No: 283, or a fragment or variant thereof.

[0208] In a twentieth embodiment, the variant RNA polymerase (referred to herein as " V159") may comprise the substitutions P266L and AYAFS (D879A, F880Y, A883S). This embodiment produces higher levels of dsRNA compared to the corresponding wild-type RNA polymerase, and so increases dsRNA contamination. Thus, the variant RNA polymerase may comprise a C-terminal motif comprising an amino acid sequence substantially as set out as SEQ ID No: 169, or a fragment or variant thereof.

[0209] The full amino acid sequence of this variant is represented herein, as SEQ ID No: 284, as follows:

[0210] MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHESYEMGEARFRKMFERQLKAGEVADNAAAKPL ITTLLPKMIARINDWFEEVKAKRGKRPTAFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIE DEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEM LIESTGMVSLHRQNAGWGQDSETIELAPEYAEAIATRAGALAGISLMFQPCVVPPKPWTGITGGGYWANGRR PLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPE DIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGND MTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPFCF LAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQA DAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQP AIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTP DGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNFVHSQDGSHLRKTVVWAHEKYG IESFALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAKGNLNLRDIL ES AYAFS

[0211] [SEQ ID No: 284]Thus, the variant polymerase may comprise an amino acid sequence substantially as set out in SEQ ID No: 284, or a fragment or variant thereof.

[0212] This variant is encoded by a DNA sequence represented herein, as SEQ ID No: 285, as follows: atgaacacgattaacatcgctaagaacgacttctctgacatcgaactggctgctatcccgttcaacactctgg ctgaccattacggtgagcgtttagctcgcgaacagttggcccttgagcatgagtcttacgagatgggtgaagc acgcttccgcaagatgtttgagcgtcaacttaaagctggtgaggttgcggataacgctgccgccaagcctctc atcactaccctactccctaagatgattgcacgcatcaacgactggtttgaggaagtgaaagctaagcgcggca agcgcccgacagccttccagttcctgcaagaaatcaagccggaagccgtagcgtacatcaccattaagaccac tctggcttgcctaaccagtgctgacaatacaaccgttcaggctgtagcaagcgcaatcggtcgggccattgag gacgaggctcgcttcggtcgtatccgtgaccttgaagctaagcacttcaagaaaaacgttgaggaacaactca acaagcgcgtagggcacgtctacaagaaagcatttatgcaagttgtcgaggctgacatgctctctaagggtct actcggtggcgaggcgtggtcttcgtggcataaggaagactctattcatgtaggagtacgctgcatcgagatg ctcattgagtcaaccggaatggttagcttacaccgccaaaatgctggcgtagtaggtcaagactctgagacta tcgaactcgcacctgaatacgctgaggctatcgcaacccgtgcaggtgcgctggctggcatctctctgatgtt ccaaccttgcgtagttcctcctaagccgtggactggcattactggtggtggctattgggctaacggtcgtcgt cctctggcgctggtgcgtactcacagtaagaaagcactgatgcgctacgaagacgtttacatgcctgaggtgt acaaagcgattaacattgcgcaaaacaccgcatggaaaatcaacaagaaagtcctagcggtcgccaacgtaat caccaagtggaagcattgtccggtcgaggacatccctgcgattgagcgtgaagaactcccgatgaaaccggaa gacatcgacatgaatcctgaggctctcaccgcgtggaaacgtgctgccgctgctgtgtaccgcaaggacaagg ctcgcaagtctcgccgtatcagccttgagttcatgcttgagcaagccaataagtttgctaaccataaggccat ctggttcccttacaacatggactggcgcggtcgtgtttacgctgtgtcaatgttcaacccgcaaggtaacgat atgaccaaaggactgcttacgctggcgaaaggtaaaccaatcggtaaggaaggttactactggctgaaaatcc acggtgcaaactgtgcgggtgtcgataaggttccgttccctgagcgcatcaagttcattgaggaaaaccacga gaacatcatggcttgcgctaagtctccactggagaacacttggtgggctgagcaagattctccgttctgcttc cttgcgttctgctttgagtacgctggggtacagcaccacggcctgagctataactgctcccttccgctggcgt ttgacgggtcttgctctggcatccagcacttctccgcgatgctccgagatgaggtaggtggtcgcgcggttaa cttgcttcctagtgaaaccgttcaggacatctacgggattgttgctaagaaagtcaacgagattctacaagca gacgcaatcaatgggaccgataacgaagtagttaccgtgaccgatgagaacactggtgaaatctctgagaaag tcaagctgggcactaaggcactggctggtcaatggctggcttacggtgttactcgcagtgtgactaagcgttc agtcatgacgctggcttacgggtccaaagagttcggcttccgtcaacaagtgctggaagataccattcagcca gctattgattccggcaagggtctgatgttcactcagccgaatcaggctgctggatacatggctaagctgattt gggaatctgtgagcgtgacggtggtagctgcggttgaagcaatgaactggcttaagtctgctgctaagctgct ggctgctgaggtcaaagataagaagactggagagattcttcgcaagcgttgcgctgtgcattgggtaactcct gatggtttccctgtgtggcaggaatacaagaagcctattcagacgcgcttgaacctgatgttcctcggtcagt tccgcttacagcctaccattaacaccaacaaagatagcgagattgatgcacacaaacaggagtctggtatcgc tcctaactttgtacacagccaagacggtagccaccttcgtaagactgtagtgtgggcacacgagaagtacgga atcgaatcttttgcactgattcacgactccttcggtaccattccggctgacgctgcgaacctgttcaaagcag tgcgcgaaactatggttgacacatatgagtcttgtgatgtactggctgatttctacgaccagttcgctgacca gttgcacgagtctcaattggacaaaatgccagcacttccggctaaaggtaacttgaacctccgtgacatctta gagtcggcgtatgcgtttagctaa

[0213] [SEQ ID No: 285]

[0214] Thus, this variant polymerase may be encoded by a nucleotide sequence substantially as set out in SEQ ID No: 285, or a fragment or variant thereof.

[0215] In a twenty-first embodiment, the variant RNA polymerase (referred to herein as " V165") may comprise the substitutions P266L and AYVFA (D879A, F880Y, A881V). This embodiment produces higher levels of dsRNA compared to the corresponding wild-type RNA polymerase, and so increases dsRNA contamination. Thus, the variantRNA polymerase may comprise a C-terminal motif comprising an amino acid sequence substantially as set out as SEQ ID No: 175, or a fragment or variant thereof.

[0216] The full amino acid sequence of this variant is represented herein, as SEQ ID No: 286, as follows:

[0217] MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHESYEMGEARFRKMFERQLKAGEVADNAAAKPL ITTLLPKMIARINDWFEEVKAKRGKRPTAFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIE DEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEM LIESTGMVSLHRQNAGWGQDSETIELAPEYAEAIATRAGALAGISLMFQPCVVPPKPWTGITGGGYWANGRR PLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPE DIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGND MTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPFCF LAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQA DAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQP AIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTP DGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNFVHSQDGSHLRKTVVWAHEKYG IESFALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAKGNLNLRDIL ESAYVFA

[0218] [SEQ ID No: 286]

[0219] Thus, the variant polymerase may comprise an amino acid sequence substantially as set out in SEQ ID No: 286, or a fragment or variant thereof.

[0220] This variant is encoded by a DNA sequence represented herein, as SEQ ID No: 287, as follows: atgaacacgattaacatcgctaagaacgacttctctgacatcgaactggctgctatcccgttcaacactctgg ctgaccattacggtgagcgtttagctcgcgaacagttggcccttgagcatgagtcttacgagatgggtgaagc acgcttccgcaagatgtttgagcgtcaacttaaagctggtgaggttgcggataacgctgccgccaagcctctc atcactaccctactccctaagatgattgcacgcatcaacgactggtttgaggaagtgaaagctaagcgcggca agcgcccgacagccttccagttcctgcaagaaatcaagccggaagccgtagcgtacatcaccattaagaccac tctggcttgcctaaccagtgctgacaatacaaccgttcaggctgtagcaagcgcaatcggtcgggccattgag gacgaggctcgcttcggtcgtatccgtgaccttgaagctaagcacttcaagaaaaacgttgaggaacaactca acaagcgcgtagggcacgtctacaagaaagcatttatgcaagttgtcgaggctgacatgctctctaagggtct actcggtggcgaggcgtggtcttcgtggcataaggaagactctattcatgtaggagtacgctgcatcgagatg ctcattgagtcaaccggaatggttagcttacaccgccaaaatgctggcgtagtaggtcaagactctgagacta tcgaactcgcacctgaatacgctgaggctatcgcaacccgtgcaggtgcgctggctggcatctctctgatgtt ccaaccttgcgtagttcctcctaagccgtggactggcattactggtggtggctattgggctaacggtcgtcgt cctctggcgctggtgcgtactcacagtaagaaagcactgatgcgctacgaagacgtttacatgcctgaggtgt acaaagcgattaacattgcgcaaaacaccgcatggaaaatcaacaagaaagtcctagcggtcgccaacgtaat caccaagtggaagcattgtccggtcgaggacatccctgcgattgagcgtgaagaactcccgatgaaaccggaa gacatcgacatgaatcctgaggctctcaccgcgtggaaacgtgctgccgctgctgtgtaccgcaaggacaagg ctcgcaagtctcgccgtatcagccttgagttcatgcttgagcaagccaataagtttgctaaccataaggccat ctggttcccttacaacatggactggcgcggtcgtgtttacgctgtgtcaatgttcaacccgcaaggtaacgat atgaccaaaggactgcttacgctggcgaaaggtaaaccaatcggtaaggaaggttactactggctgaaaatcc acggtgcaaactgtgcgggtgtcgataaggttccgttccctgagcgcatcaagttcattgaggaaaaccacga gaacatcatggcttgcgctaagtctccactggagaacacttggtgggctgagcaagattctccgttctgcttc cttgcgttctgctttgagtacgctggggtacagcaccacggcctgagctataactgctcccttccgctggcgt ttgacgggtcttgctctggcatccagcacttctccgcgatgctccgagatgaggtaggtggtcgcgcggttaa cttgcttcctagtgaaaccgttcaggacatctacgggattgttgctaagaaagtcaacgagattctacaagca gacgcaatcaatgggaccgataacgaagtagttaccgtgaccgatgagaacactggtgaaatctctgagaaag tcaagctgggcactaaggcactggctggtcaatggctggcttacggtgttactcgcagtgtgactaagcgttc agtcatgacgctggcttacgggtccaaagagttcggcttccgtcaacaagtgctggaagataccattcagcca gctattgattccggcaagggtctgatgttcactcagccgaatcaggctgctggatacatggctaagctgattt gggaatctgtgagcgtgacggtggtagctgcggttgaagcaatgaactggcttaagtctgctgctaagctgctggctgctgaggtcaaagataagaagactggagagattcttcgcaagcgttgcgctgtgcattgggtaactcct gatggtttccctgtgtggcaggaatacaagaagcctattcagacgcgcttgaacctgatgttcctcggtcagt tccgcttacagcctaccattaacaccaacaaagatagcgagattgatgcacacaaacaggagtctggtatcgc tcctaactttgtacacagccaagacggtagccaccttcgtaagactgtagtgtgggcacacgagaagtacgga atcgaatcttttgcactgattcacgactccttcggtaccattccggctgacgctgcgaacctgttcaaagcag tgcgcgaaactatggttgacacatatgagtcttgtgatgtactggctgatttctacgaccagttcgctgacca gttgcacgagtctcaattggacaaaatgccagcacttccggctaaaggtaacttgaacctccgtgacatctta gagtcggcgtatgtttttgcgtaa

[0221] [SEQ ID No: 287]

[0222] Thus, this variant polymerase may be encoded by a nucleotide sequence substantially as set out in SEQ ID No: 287, or a fragment or variant thereof.

[0223] In a twenty-second embodiment, the variant RNA polymerase (referred to herein as " V230") may comprise the substitutions P266L and DYAFC (F880Y, A883C). This embodiment produces higher levels of dsRNA compared to the corresponding wildtype RNA polymerase, and so increases dsRNA contamination. Thus, the variant RNA polymerase may comprise a C-terminal motif comprising an amino acid sequence substantially as set out as SEQ ID No: 240, or a fragment or variant thereof.

[0224] The full amino acid sequence of this variant is represented herein, as SEQ ID No: 288, as follows:

[0225] MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHESYEMGEARFRKMFERQLKAGEVADNAAAKPL ITTLLPKMIARINDWFEEVKAKRGKRPTAFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIE DEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEM LIESTGMVSLHRQNAGWGQDSETIELAPEYAEAIATRAGALAGISLMFQPCVVPPKPWTGITGGGYWANGRR PLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPE DIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGND MTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPFCF LAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQA DAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQP AIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTP DGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNFVHSQDGSHLRKTVVWAHEKYG IESFALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAKGNLNLRDIL ESDYAFC

[0226] [SEQ ID No: 288]

[0227] Thus, the variant polymerase may comprise an amino acid sequence substantially as set out in SEQ ID No: 288, or a fragment or variant thereof.

[0228] This variant is encoded by a DNA sequence represented herein, as SEQ ID No: 289, as follows: atgaacacgattaacatcgctaagaacgacttctctgacatcgaactggctgctatcccgttcaacactctgg ctgaccattacggtgagcgtttagctcgcgaacagttggcccttgagcatgagtcttacgagatgggtgaagc acgcttccgcaagatgtttgagcgtcaacttaaagctggtgaggttgcggataacgctgccgccaagcctctc atcactaccctactccctaagatgattgcacgcatcaacgactggtttgaggaagtgaaagctaagcgcggca agcgcccgacagccttccagttcctgcaagaaatcaagccggaagccgtagcgtacatcaccattaagaccac tctggcttgcctaaccagtgctgacaatacaaccgttcaggctgtagcaagcgcaatcggtcgggccattgag gacgaggctcgcttcggtcgtatccgtgaccttgaagctaagcacttcaagaaaaacgttgaggaacaactcaacaagcgcgtagggcacgtctacaagaaagcatttatgcaagttgtcgaggctgacatgctctctaagggtct actcggtggcgaggcgtggtcttcgtggcataaggaagactctattcatgtaggagtacgctgcatcgagatg ctcattgagtcaaccggaatggttagcttacaccgccaaaatgctggcgtagtaggtcaagactctgagacta tcgaactcgcacctgaatacgctgaggctatcgcaacccgtgcaggtgcgctggctggcatctctctgatgtt ccaaccttgcgtagttcctcctaagccgtggactggcattactggtggtggctattgggctaacggtcgtcgt cctctggcgctggtgcgtactcacagtaagaaagcactgatgcgctacgaagacgtttacatgcctgaggtgt acaaagcgattaacattgcgcaaaacaccgcatggaaaatcaacaagaaagtcctagcggtcgccaacgtaat caccaagtggaagcattgtccggtcgaggacatccctgcgattgagcgtgaagaactcccgatgaaaccggaa gacatcgacatgaatcctgaggctctcaccgcgtggaaacgtgctgccgctgctgtgtaccgcaaggacaagg ctcgcaagtctcgccgtatcagccttgagttcatgcttgagcaagccaataagtttgctaaccataaggccat ctggttcccttacaacatggactggcgcggtcgtgtttacgctgtgtcaatgttcaacccgcaaggtaacgat atgaccaaaggactgcttacgctggcgaaaggtaaaccaatcggtaaggaaggttactactggctgaaaatcc acggtgcaaactgtgcgggtgtcgataaggttccgttccctgagcgcatcaagttcattgaggaaaaccacga gaacatcatggcttgcgctaagtctccactggagaacacttggtgggctgagcaagattctccgttctgcttc cttgcgttctgctttgagtacgctggggtacagcaccacggcctgagctataactgctcccttccgctggcgt ttgacgggtcttgctctggcatccagcacttctccgcgatgctccgagatgaggtaggtggtcgcgcggttaa cttgcttcctagtgaaaccgttcaggacatctacgggattgttgctaagaaagtcaacgagattctacaagca gacgcaatcaatgggaccgataacgaagtagttaccgtgaccgatgagaacactggtgaaatctctgagaaag tcaagctgggcactaaggcactggctggtcaatggctggcttacggtgttactcgcagtgtgactaagcgttc agtcatgacgctggcttacgggtccaaagagttcggcttccgtcaacaagtgctggaagataccattcagcca gctattgattccggcaagggtctgatgttcactcagccgaatcaggctgctggatacatggctaagctgattt gggaatctgtgagcgtgacggtggtagctgcggttgaagcaatgaactggcttaagtctgctgctaagctgct ggctgctgaggtcaaagataagaagactggagagattcttcgcaagcgttgcgctgtgcattgggtaactcct gatggtttccctgtgtggcaggaatacaagaagcctattcagacgcgcttgaacctgatgttcctcggtcagt tccgcttacagcctaccattaacaccaacaaagatagcgagattgatgcacacaaacaggagtctggtatcgc tcctaactttgtacacagccaagacggtagccaccttcgtaagactgtagtgtgggcacacgagaagtacgga atcgaatcttttgcactgattcacgactccttcggtaccattccggctgacgctgcgaacctgttcaaagcag tgcgcgaaactatggttgacacatatgagtcttgtgatgtactggctgatttctacgaccagttcgctgacca gttgcacgagtctcaattggacaaaatgccagcacttccggctaaaggtaacttgaacctccgtgacatctta gagtcggattatgcgttttgctaa

[0229] [SEQ ID No: 289]

[0230] Thus, this variant polymerase may be encoded by a nucleotide sequence substantially as set out in SEQ ID No: 289, or a fragment or variant thereof.

[0231] In some embodiments, the polypeptides described herein may comprise a purification tag, such as a His-tag, preferably attached to the N-terminal domain.

[0232] The variant RNA polymerase may reduce the amount of dsRNA produced in a transcription reaction by at least 5%, 10% or 20% (w / w) compared to the amount of dsRNA produced by the corresponding wild-type RNA polymerase on the same sequence. The variant RNA polymerase may reduce the amount of dsRNA produced in a transcription reaction by at least 30%, 40% or 50% (w / w) compared to the amount of dsRNA produced by the corresponding wild-type RNA polymerase on the same sequence. In another embodiment, the variant RNA polymerase may reduce the amount of dsRNA produced in a transcription reaction by at least 60%, 70% or 80% (w / w) compared to the amount of dsRNA produced by the corresponding wild-type RNA polymerase on the same sequence. In a typical embodiment, the variant RNA polymerase may reduce the amount of dsRNA produced in a transcription reaction by at least 90%, 95% or 96% (w / w) compared to the amount of dsRNA produced by thecorresponding wild-type RNA polymerase on the same sequence. In a most typical embodiment, the variant RNA polymerase may reduce the amount of dsRNA produced in a transcription reaction by at least 97%, 98% or 99% (w / w) compared to the amount of dsRNA produced by the corresponding wild-type RNA polymerase on the same sequence.

[0233] In a typical embodiment, the variant RNA polymerase may reduce the amount of dsRNA produced in a transcription reaction by at least 20% (w / w) compared to the amount of dsRNA produced by the corresponding wild-type RNA polymerase on the same sequence.

[0234] The variant RNA polymerase may increase the amount of dsRNA produced in a transcription reaction by at least 5%, 10% or 20% (w / w) compared to the amount of dsRNA produced by the corresponding wild-type RNA polymerase on the same sequence. The variant RNA polymerase may increase the amount of dsRNA produced in a transcription reaction by at least 30%, 40% or 50% (w / w) compared to the amount of dsRNA produced by the corresponding wild-type RNA polymerase on the same sequence. In another embodiment, the variant RNA polymerase may increase the amount of dsRNA produced in a transcription reaction by at least 60%, 70% or 80% (w / w) compared to the amount of dsRNA produced by the corresponding wildtype RNA polymerase on the same sequence.

[0235] In a typical embodiment, the variant RNA polymerase may increase the amount of dsRNA produced in a transcription reaction by at least 20% (w / w) compared to the amount of dsRNA produced by the corresponding wild-type RNA polymerase on the same sequence.

[0236] The variant RNA polymerase may yield at least 0.05mg / ml RNA or at least 0.5mg / ml RNA. In an embodiment, the variant RNA polymerase may yield at least lmg / ml RNA, 2mg / ml RNA, 2.5mg / ml RNA, or 3mg / ml RNA. In another embodiment, the variant RNA polymerase may yield at least 4mg / ml RNA, 5mg / ml RNA or 6mg / ml RNA. In yet another embodiment, the variant RNA polymerase may yield at least 7mg / ml RNA, 8mg / ml RNA or 9mg / ml RNA.

[0237] In a second aspect of the invention, there is provided a nucleic acid sequence encoding the variant RNA polymerase according to the first aspect.In some embodiments, the nucleic acid sequence comprises a nucleotide sequence substantially as set out above, comprising or consisting of any one of SEQ ID No: 6, 8, 10, 252, 254, 256, 258, 260, 263, 265, 267, 269, 271, 273, 275, 279, 281, 283, 285, 287, or 289, or a fragment or variant thereof. Especially variants with an altered set of codon usage for one or more amino acids are provided.

[0238] In a third aspect, there is provided an expression cassette comprising the nucleic acid sequence according to the second aspect.

[0239] The nucleic acid sequences of the invention may be harboured in a recombinant vector, for example a recombinant vector for delivery into a host cell of interest to enable production of the variant RNA polymerase.

[0240] Accordingly, in a fourth aspect, there is provided a recombinant vector comprising the expression cassette according to the third aspect.

[0241] The vector of the fourth aspect encoding the variant RNA polymerase may for example be a plasmid, cosmid or phage and / or be a viral vector. Such recombinant vectors are highly useful in the delivery systems of the invention for transforming cells with the nucleotide sequences. The nucleotide sequences may be a DNA sequence, and it is this DNA sequence which encodes the RNA sequence which encodes the RNA polymerase according to the invention.

[0242] Recombinant vectors encoding the RNA polymerase of the invention may also include other functional elements. For example, they may further comprise a variety of other functional elements including a suitable promoter for initiating expression upon introduction of the vector into a suitable host cell. For instance, the vector may be capable of autonomously replicating in the nucleus of the host cell, such as a bacterial cell. In this case, elements which induce or regulate DNA replication may be required in the recombinant vector. Alternatively, the recombinant vector may be designed such that it integrates into the genome of a host cell. In this case, DNA sequences which favour targeted integration (e.g. by homologous recombination) are envisaged. Suitable promoters may include the SV40 promoter, CMV, EFla, PGK, viral long terminal repeats, as well as inducible promoters, such as the Tetracycline inducible system, as examples. The cassette or vector may also comprise a terminator, such as the Beta globin, SV40 polyadenylation sequences or synthetic polyadenylation sequences. The recombinant vector may also comprise a promoter or regulator or enhancer to control expression ofthe nucleic acid as required to produce the variant RNA polymerase. Such promoter is typically not the same or compatible with the RNA polymerase encoded in the vector.

[0243] The vector may also comprise DNA coding for a gene that may be used as a selectable marker in the cloning process, i.e. to enable selection of cells that have been transfected or transformed, and to enable the selection of cells harbouring vectors incorporating heterologous DNA. For example, ampicillin, neomycin, puromycin or chloramphenicol resistance is envisaged. Alternatively, the selectable marker gene may be in a different vector to be used simultaneously with the vector containing the transgene(s). The cassette or vector may also comprise DNA involved with regulating expression of the nucleotide sequence encoding the RNA polymerase, or for targeting the expressed polypeptide to a certain part of the host cell.

[0244] Purified vector may be inserted directly into a host cell by suitable means, e.g. direct endocytotic uptake. The vector may be introduced directly into a host cell (e.g. a eukaryotic or prokaryotic cell) by transfection, infection, electroporation, microinjection, cell fusion, protoplast fusion or ballistic bombardment. Alternatively, vectors of the invention may be introduced directly into a host cell using a particle gun.

[0245] The nucleic acid molecule may (but not necessarily) be one, which becomes incorporated in the DNA of the host cell. Undifferentiated cells may be stably transformed leading to the production of genetically modified daughter cells (in which case regulation of expression in the subject may be required e.g. with specific transcription factors or gene activators). Alternatively, the delivery system may be designed to favour unstable or transient transformation of differentiated cells. When this is the case, regulation of expression may be less important because expression of the DNA molecule will stop when the transformed cells die or stop expressing the protein.

[0246] Alternatively, the delivery system may provide the nucleic acid molecule to the host cell without it being incorporated in a vector. For instance, the nucleic acid molecule may be incorporated within a liposome or virus particle. Alternatively, a "naked" nucleic acid molecule may be inserted into a host cell by a suitable means e.g. direct endocytotic uptake.

[0247] Thus, in a fifth aspect, there is provide a host cell comprising the recombinant vector according to the fourth aspect.It will be appreciated that the variant RNA polymerase described herein may be used to produce RNA.

[0248] Therefore, in a sixth aspect, there is provided use of the variant RNA polymerase according to the first aspect, for preparing an RNA molecule.

[0249] Thus, in a seventh aspect, there is provided a method of preparing an RNA molecule, wherein the method comprises contacting: (i) a template nucleic acid sequence, (ii) the variant RNA polymerase according to the first aspect, and (iii) a plurality of nucleotide triphosphates (NTPs), wherein the variant RNA polymerase transcribes the template nucleic acid sequence to form an RNA molecule.

[0250] In one embodiment, the use of the variant RNA according to the first aspect prevents the need for downstream processing focused at adjusting the dsRNA content. In a typical embodiment, the formed RNA molecule is extracted from the IVT mixture and purified from IVT mixture components, and is substantially ready for use or formulation.

[0251] The RNA molecule prepared by the method may be substantially single-stranded RNA (ssRNA), substantially double stranded RNA (dsRNA) or, typically, a mixture thereof.

[0252] The method may comprise in vivo transcription or in vitro transcription (IVT).

[0253] For some applications, notably using RNA as a vaccine (or for therapeutic uses), it may be desirable to be able to manipulate the ratio of dsRNA to ssRNA that is produced in a transcription reaction.

[0254] Therefore, in an eighth aspect, there is provided a method of modifying the ratio of double stranded RNA (dsRNA) to single stranded RNA (ssRNA) produced in a transcription reaction, wherein the method comprises contacting: (i) a template nucleic acid sequence, (ii) one or more variant RNA polymerase according to the first aspect, and (iii) a plurality of nucleotide triphosphates (NTPs), wherein the variant RNA polymerase transcribes the template nucleic acid sequence to form ssRNA and dsRNA at a desired ratio.

[0255] The ratio of dsRNA to ssRNA may be <0.1:1, or <0.08: 1. Typically, the ratio of dsRNA to ssRNA may be about 0.05:1. More typically, however, the ratio of dsRNA to ssRNA may be <0.05:1, or <0.01:1. These ratios may be for vaccine applications.In some embodiments, the methods may comprise the use of a variant RNA polymerase comprising: (i) an amino acid sequence at least 80%, 90% or 95% identical to positions 1 to 883 of SEQ ID NO:1, and (ii) one or more substitution at a position selected from positions corresponding to positions 266, 879, 880, 881, 882, and 883 of SEQ ID NO:1.

[0256] Typically, the variant RNA polymerase is selected from a polymerase comprises an amino acid sequence comprising any one of SEQ ID No's 5, 7, 9, 251, 253, 255, 257, 259, 262, 264, 266, 268, 270, 272, 274, 278, 280, 282, 284, 286, or 288. or a variant or fragment thereof. Typically, the variant RNA polymerase is selected from a polymerase encoded by a nucleotide sequence comprising any one of SEQ ID No's 6, 8, 10, 252, 254, 256, 258, 260, 263, 265, 267, 269, 271, 273, 275, 279, 281, 283, 285, 287, or 289, or a variant or fragment thereof.

[0257] In some embodiments, the methods may comprise the use of a mixture of variant RNA polymerases to produce a defined amount of dsRNA intermediate between the amount produced by either variant RNA polymerase in a single step, comprising: (i) a template nucleic acid sequence, (ii) a first variant RNA polymerase according to the first aspect, (iii) a second variant RNA polymerase according to the first aspect, and (iv) a plurality of nucleotide triphosphates (NTPs), wherein the variant RNA polymerases transcribe the template nucleic acid sequence to form ssRNA and dsRNA at a desired ratio.

[0258] In such embodiments, the ratio between the first and the second variant RNA polymerase may be adjusted to the relative productivity and the desired amount dsRNA to ssRNA ratio, and may be 1:1, 0.5:1, 0.25:1, or less than 0.25:1.

[0259] The method may comprise testing the first variant RNA polymerase and then measuring RNA (ssRNA and / or dsRNA) yield. The method may comprise testing the second variant RNA polymerase and then measuring RNA yield (ssRNA and / or dsRNA). The operator may determine RNA (ssRNA and / or dsRNA) purity (e.g. using gel / CGE) and dsRNA amount, and then determine the optimum combination of these parameters (i.e. amount of first and second variant RNA polymerase) for the intended RNA product. The method may comprise blending or combining a plurality of variant RNA polymerases (e.g. two, three, four or more variants) to produce the RNA product.Thus, the method may comprise the use of more than one variant RNA polymerase to produce a defined amount of dsRNA intermediate between the amount produced by either variant RNA polymerase by mixing the resultant RNA products in a defined ratio, the method comprising: (i) synthesizing RNA with a first variant RNA polymerase according to the first aspect, and (ii) synthesizing RNA with a second variant RNA polymerase according to the first aspect, and (iii) blending the RNA products in a defined ratio to obtained the desired dsRNA to ssRNA ratio, optionally wherein the dsRNA to ssRNA ratio is about 1:1, 0.5:1, 0.25:1, or less than 0.25:1.

[0260] In some embodiments, a combination of variant RNA polymerases is used, wherein the combination is selected from a group of combinations consisting of: P266L+RYAFC and P266L+QFVFN; P266L+QFVFN and P266L+AYFFC; P266L+AYFFC and P266L+PYVFA; P266L+PYVFA and P266L+EYAFS; P266L+EYAFS and P266L+DFAFA; and P266L+DFAFA and P266L+QYAFA.

[0261] As a result of the amino acid substitutions in the variant RNA polymerase, the methods result in altered dsRNA contamination relative to that which would be produced by the corresponding wild-type RNA polymerase of SEQ ID NO:1 for the same RNA sequence.

[0262] In one embodiment, the methods described herein comprise the use of in vitro transcription (IVT). IVT will be known to the skilled person, and comprises the use of a DNA template, an RNA polymerase, and appropriate amounts of dNTPs for producing a corresponding (encoded) RNA molecule. Thus, the methods may comprise one or more component selected from a group consisting of: DNA template, variant RNA polymerase of the invention, dNTPs, and buffer. Other components that may be necessary or desirable to perform an IVT reaction may be selected from: nucleotides, magnesium ion cofactor, salt, RNAse inhibitor, co-transcriptional capping analogue, pyrophosphatase and spermidine etc.

[0263] The DNA template may comprise an RNA polymerase promoter. The variant RNA polymerase may be variant T7, T3, Kll, SP6, KP34 or Syn5 polymerase. As such the DNA template may comprise a corresponding T7, T3, Kll, SP6, KP34 or Syn5 promoter. The DNA template may comprise 5' and / or 3' UTR. The DNA template may comprise a coding sequence of a target gene. The target gene may encode a therapeutic biomolecule or an immunogen, the latter for use a vaccine.The methods may be performed at a temperature of between about 20°C and 60°C, more typically between 30°C and 40°C, or between about 35°C and 39°C, typically about 37°C.

[0264] The ssRNA may be coding or non-coding. For example, non-coding RNA may be used for RNAi applications. However, typically the ssRNA is coding, and can be used, for example, as an RNA vaccine or RNA therapeutic.

[0265] The ssRNA may be selected from a group consisting of: messenger RNA (mRNA), micro RNA (miRNA); short interfering RNA (siRNA); short hairpin RNA (shRNA); anti-sense RNA; RNA aptamers; self-amplifying RNA (saRNA); interference RNA (RNAi); non-coding RNA; circular RNA; and small RNA. Typically, however, the ssRNA is messenger RNA (mRNA).

[0266] In some embodiments, the methods described herein may be used in a transcription reaction for RNA synthesis involving non-canonical NTPs, such as 2'OMe-UTP, 2'OMe-CTP,2'-Fluoro-dCTP and 2'-Fluoro-dUTP. For example, the variant RNA polymerase (e.g. variant V90) may comprise Y639F and H784A as single mutations in exemplary embodiments, and Y639F / H784A as a double mutant in another exemplary embodiment. Y639F and H784A are known to allow the RNA polymerase to incorporate modified nucleotides substituting the canonical NTP counterpart with 2'OMe-UTP, 2'OMe-CTP,2'-Fluoro-dCTP and 2'-Fluoro-dUTP.

[0267] Thus, any of the variant RNA polymerases described herein may further comprise Y639F and / or H784A.

[0268] The inventors have also developed RNA manufacturing kits, which harness the variant RNA polymerases described herein.

[0269] Thus, in a ninth aspect, there is provided an RNA manufacturing kit comprising one or more variant RNA polymerase according to the first aspect, or the nucleic acid according to the second aspect, and instructions for use.

[0270] In some embodiments, the kit may comprise a plurality of variant RNA polymerases to produce a defined amount of dsRNA intermediate between the amount produced by either variant RNA polymerase. Thus, the kit may comprise a first variant RNA polymerase according to the first aspect and a second variant RNA polymeraseaccording to the first aspect. Such variant RNA polymerases may be provided separately or as a blend.

[0271] An operator may, when using the kit to prepare RNA, use the first variant and then measure RNA (ssRNA and / or dsRNA) yield. The operator may use the second variant and then measure RNA (ssRNA and / or dsRNA) yield. The operator may determine RNA purity (e.g. using gel / CGE) and dsRNA amount, then determine the optimum combination of these parameters (i.e. amount of first and second variant) for the intended RNA product. The kits may comprise a blend or combination of a plurality of variant RNA polymerases (e.g. two, three, four or more variants) to produce the RNA product.

[0272] The instructions may include protocols on how to select the optimal variant RNA polymerase for the intended RNA product characteristics, such as dsRNA content.

[0273] The ratio between the first and the second variant RNA polymerase may be adjusted to the relative productivity and the desired amount dsRNA to ssRNA ratio, and may be 1:1, 0.5:1, 0.25:1, or less than 0.25:1.

[0274] The kit may further comprise one or more component selected from a group consisting of: DNA template, dNTPs, magnesium ion cofactor, salt, RNAse inhibitor, co-transcriptional capping analogue, pyrophosphatase and spermidine etc.

[0275] In a tenth aspect, there is provided an RNA molecule manufactured by the kit of the ninth aspect.

[0276] In an eleventh aspect, there is provided the RNA molecule according to the tenth aspect, for use in stimulating an immune response.

[0277] In a twelfth aspect, there is provided a method of stimulating an immune response in a subject, the method comprising administering, or having administered, to a subject, an immunogenically effective amount of the RNA molecule according to the tenth aspect.

[0278] In a thirteenth aspect, there is provided a vaccine comprising the RNA molecule according to the tenth aspect, and, optionally, an adjuvant (other than dsRNA).It will be appreciated that the invention extends to any nucleic acid or peptide or variant, derivative or analogue thereof, which comprises substantially the amino acid or nucleic acid sequences of any of the sequences referred to herein, including variants or fragments thereof. The terms "substantially the amino acid / nucleotide / peptide sequence", "variant" and "fragment", can be a sequence that has at least 40% sequence identity with the amino acid / nucleotide / peptide sequences of any one of the sequences referred to herein, for example 40% identity with any of the sequence identified herein.

[0279] Amino acid / polynucleotide / polypeptide sequences with a sequence identity which is greater than 65%, or greater than 70%, or greater than 75%, or greater than 80% sequence identity to any of the sequences referred to are also envisaged. In one embodiment, the amino acid / polynucleotide / polypeptide sequence has at least 85% identity with any of the sequences referred to, or at least 90% identity, or at least 92% identity, or at least 95% identity, or at least 97% identity, or at least 98% identity, or at least 99% identity with any of the sequences referred to herein.

[0280] The skilled technician will appreciate how to calculate the percentage identity between two amino acid / polynucleotide / polypeptide sequences. In order to calculate the percentage identity between two amino acid / polynucleotide / polypeptide sequences, an alignment of the two sequences must first be prepared, followed by calculation of the sequence identity value. The percentage identity for two sequences may take different values depending on:- (i) the method used to align the sequences, for example, ClustalW, BLAST, FASTA, Smith-Waterman (implemented in different programs), or structural alignment from 3D comparison; and (ii) the parameters used by the alignment method, for example, local vs global alignment, the pair-score matrix used (e.g. BLOSUM62, PAM250, Gonnet etc.), and gap-penalty, e.g. functional form and constants.

[0281] Having made the alignment, there are many different ways of calculating percentage identity between the two sequences. For example, one may divide the number of identities by: (i) the length of shortest sequence; (ii) the length of alignment; (iii) the mean length of sequence; (iv) the number of non-gap positions; or (v) the number of equivalenced positions excluding overhangs. Furthermore, it will be appreciated that percentage identity is also strongly length dependent. Therefore, the shorter a pair of sequences is, the higher the sequence identity one may expect to occur by chance.Hence, it will be appreciated that the accurate alignment of protein or DNA sequences is a complex process. The popular multiple alignment program ClustalW (Thompson et al., 1994, Nucleic Acids Research, 22, 4673-4680; Thompson et a / ., 1997, Nucleic Acids Research, 24, 4876-4882) is one way for generating multiple alignments of proteins or DNA in accordance with the invention. Suitable parameters for ClustalW may be as follows: For DNA alignments: Gap Open Penalty = 15.0, Gap Extension Penalty = 6.66, and Matrix = Identity. For protein alignments: Gap Open Penalty = 10.0, Gap Extension Penalty = 0.2, and Matrix = Gonnet. For DNA and Protein alignments: ENDGAP = -1, and GAPDIST = 4. Those skilled in the art will be aware that it may be necessary to vary these and other parameters for optimal sequence alignment.

[0282] In one embodiment, calculation of percentage identities between two amino acid / polynucleotide / polypeptide sequences may then be calculated from such an alignment as (N / T)*100, where N is the number of positions at which the sequences share an identical residue, and T is the total number of positions compared including gaps and either including or excluding overhangs. In one embodiment, overhangs are included in the calculation. Hence, one method for calculating percentage identity between two sequences comprises (i) preparing a sequence alignment using the ClustalW program using a suitable set of parameters, for example, as set out above; and (ii) inserting the values of N and T into the following formula:- Sequence Identity = (N / T)*100.

[0283] Alternative methods for identifying similar sequences will be known to those skilled in the art. For example, a substantially similar nucleotide sequence will be encoded by a sequence which hybridizes to DNA sequences or their complements under stringent conditions. By stringent conditions, the inventors mean the nucleotide hybridises to filter-bound DNA or RNA in 3x sodium chloride / sodium citrate (SSC) at approximately 45°C followed by at least one wash in 0.2x SSC / 0.1% SDS at approximately 20-65°C. Alternatively, a substantially similar polypeptide may differ by at least 1, but less than 5, 10, 20, 50 or 100 amino acids from any of the sequences described herein.

[0284] Due to the degeneracy of the genetic code, it is clear that any nucleic acid sequence described herein could be varied or changed without substantially affecting the sequence of the protein encoded thereby, to provide a functional variant thereof. Suitable nucleotide variants are those having a sequence altered by the substitution of different codons that encode the same amino acid within the sequence, thus producing a silent (synonymous) change. Other suitable variants are those having homologousnucleotide sequences but comprising all, or portions of, sequence, which are altered by the substitution of different codons that encode an amino acid with a side chain of similar biophysical properties to the amino acid it substitutes, to produce a conservative change. For example, small non-polar, hydrophobic amino acids include glycine, alanine, leucine, isoleucine, valine, proline, and methionine. Large non-polar, hydrophobic amino acids include phenylalanine, tryptophan and tyrosine. The polar neutral amino acids include serine, threonine, cysteine, asparagine and glutamine. The positively charged (basic) amino acids include lysine, arginine and histidine. The negatively charged (acidic) amino acids include aspartic acid and glutamic acid. It will therefore be appreciated which amino acids may be replaced with an amino acid having similar biophysical properties, and the skilled technician will know the nucleotide sequences encoding these amino acids.

[0285] All of the features described herein (including any accompanying claims, abstracts, and drawings), and / or all of the steps of any method or process so disclosed, may be combined with any of the above aspects in any combination, except combinations where at least some features and / or steps are mutually exclusive.

[0286] For a better understanding of the invention, and to show how embodiments of the same may be carried into effect, reference will now be made, by way of example, to the accompanying Figure, in which:-

[0287] Figure 1a shows the protein structures of the initiation complex (IC) and elongation complex (EC) of T7 RNA polymerase depicting the vast conformational changes observed during RNA transcription. In contrast to the C-terminal domain (CTD), the N-terminal domain (NTD), here represented by cylinders, undergoes a massive structural rearrangement that results in the destruction of the promoter binding domain, the enlargement of the active site and the elongation of the C-helix. Figure lb shows the formation of the RNA pore that allows the accommodation of the nascent RNA transcript during the elongation. Amino acids that form the pore in the EC are represented as a surface. Based on the structure of T7 RNAP during IC (PDB: 2PI4) and EC (PBD: 1H38). Figure 1c shows a schematic representation of the three different stages of transcription during an IVT using a linear DNA template: initiation, elongation and termination. During initiation, the IC binds to the T7 promoter sequence unstably producing short abortive transcripts. The formation of a growing RNA-DNA hybrid promotes the transition from the IC to the EC generating a conformational change that results in a highly processive elongation phase. In the case of an IVT using a linear DNA template, the RNA polymerase reaches the end ofthe DNA template causing a run-off termination of transcription. Also shown are the immunostimulatory dsRNA impurities that are produced during RNA synthesis, including DNA-dependent dsRNAs and RNA-dependent loopback dsRNAs.

[0288] Figure 2 shows the effect of C-terminal substitutions in dsRNA generation by altering the T7 RNA polymerase (T7RNAP) wild-type C-terminal motif (DFAFA (SEQ ID No: 261)) to match those found in RNA polymerases with overall distinct sequences. This was achieved by producing a set of novel T7RNAP variants containing C-terminal motifs of Pseudomonas phage VSW-3 (labelled " TYFFA" (SEQ ID No: 27) - these amino acids at the C-terminus) (VSW-3 has 32.3% overall sequence identity with T7RNAP-P266L-TYFFA)), Klebsiella phage KP34 (labelled " PFFFC" (SEQ ID No: 40) (KP34 has 27.4% overall sequence identity with T7RNAP-P266L-PFFFC)), Ralstonia solanacearum (labelled " PFFFA" (SEQ ID No: 39) (RS has 32.7% overall sequence identity with T7-P266L-PFFFA)), and Pseudomonas phage BIM BV-45 (labelled " VYFFC" (SEQ ID No: 100) (BV-45 has 32.43% overall sequence identity with T7-P266L-VYFFC)). Figure 2a shows the amount of dsRNA produced by wild-type (" DFAFA" (SEQ ID No: 261) at the C-terminus) and the four C-terminal variants, and Figure 2b shows the RNA yield generated by each RNAP. The variant containing the VYFFC (SEQ ID No: 100) C-terminal motif and also a P266L mutation exhibited the greatest dsRNA reduction compared to the wild type, without significantly reducing the RNA yields. This variant was, therefore, selected for further experiments, and is known as " V90" (SEQ ID No: 5).

[0289] Figure 3a shows an embodiment of a molecular model of the C-terminus of T7 RNA polymerase (T7RNAP) wild-type DFAFA (SEQ ID No: 261) (Protein Data Bank (PDB: 1S77). The C-terminal domain positions D879, F880, A881, F882, and A883 and active-site aspartic-acid residues (D537 and D812) are all highlighted. The structure of T7RNAP wild type (PDB: 1S77) served as a template for the modelling of various embodiments of the variant T7RNAP enzyme according to the invention, one embodiment of which is the VYFFC (SEQ ID No: 100) C-terminal variant shown in Figure 2, which is called " V90" (SEQ ID No: 5), and has the following four C-terminal amino acid mutations: D879V, F880Y, A881F, A883C (SEQ ID No: 5) for comparison (see Figure 3b).

[0290] Figure 4 shows a comparison between a commercially available T7RNAP known as H (corresponding to a prior art T7RNAP polymerase mutant commercialised as T7RNAP with allegedly "low" dsRNA production) and different embodiments of the variant V90 T7RNAP enzyme according to the invention, and shows the difference inRNA yields when transcribing different DNA templates denoted 1-10, and the amount of dsRNA that is produced. As can be seen, the variant V90 outperforms the prior art H T7RNAP in yield (187 vs 81 ug) and dsRNA content (0.11 vs 0.19%).

[0291] Figure 5 shows the effect of C-terminal substitutions D879V, F880Y, A881F, A883C with (V90; SEQ ID No: 5) (the VYFFC (SEQ ID No: 100) C-terminal variant) and without substitution P266L on the amounts of dsRNA produced. As can be seen, the C-terminal mutant produces about 0.45% dsRNA compared to only about 0.05% dsRNA produced by the C-terminal variant which has the additional P266L modification.

[0292] Figure 6 shows a 3-dimensional structure of the C-terminal mutant (V90) with the C-terminus being shown as the dark helical coil.

[0293] Figure 7 shows a sequence alignment of the C-terminus of the C-terminal T7RNAP variant V90 (SEQ ID No: 5) (VYFFC; SEQ ID No: 100), variant V187 (SEQ ID No: 7) (IYFFS; SEQ ID No: 197), variant V206 (SEQ ID No: 9) (RYAFC; SEQ ID No: 216), with the corresponding T7RNAP wild type DFAFA(SEQ ID No: 261). Positions 879, 880, 881, 882 and 883 are illustrated with V90 having: D879V, F880Y, A881F, A883C; V187 having: D879I, F880Y, A881F, A883S; and V206 having: D879R, F880Y, A881A, A883C. Position 882 is F for both the wild type and the V90, V187, and V206 variants. The figure also illustrates how position 879 could be varied by using 10 different amino acids, position 880 could be varied using two possible amino acids, position 881 could be varied using three different amino acids and position 883 could be varied using four different amino acids. As such, the total number of variants produced was 10x2x3x4 = 240 variants, as shown in Table 1. Of note, additional mutations could be made in the same domain.

[0294] Figure 8 is a grid showing the sequences of 240 different embodiments of C-terminal mutants, the SEQ ID Nos of each of which are listed in Table 1. The heat map illustrates the propensity of each of the 240 variants to produce dsRNA on a scale of 1.5% dsRNA (poor) to 0.025% dsRNA (good). White coloured variants did not express well, or produced too little RNA to perform well in the screening method.

[0295] Figure 9a shows the results of in vitro transcription for the content of contaminating dsRNA and RNA yield for the data confirmation of all 240 variants prepared, and Figure 9b shows the results of in vitro transcription for the content of contaminating dsRNA and RNA yield for the 12 different variants of interest which were selected. Experiments were performed in triplicates. 11 candidates were initially selected basedon: a yield higher than 55 ug / rxn (X=55), a dsRNA lower than 0.05% dsRNA. The top 3 performers were then selected from this smaller group. V90: VYFFC (SEQ ID No: 100); M187: RYAFC (SEQ ID No: 216); M206: IYFFS (SEQ ID No: 197). Parental: DFAFA (SEQ ID No: 261); Control 1: DYVFC (SEQ ID No: 248); Control 2: QYAFA (SEQ ID No: 119). Figure 9c shows the results for in vitro transcription for the content of contaminating dsRNA and yield for 10 variants of interest that were selected from a second screen. These variants were initially part of a subset that failed to produce acceptable RNA yields in the first high-throughput screen (Figure 9a variants <25 pg). V90: VYFFC (SEQ ID No: 100); V187: RYAFC (SEQ ID No: 216); V206: IYFFS (SEQ ID No: 197). Parental: DFAFA (SEQ ID No: 261); Control 1: DYVFC (SEQ ID No: 248); Control 2: QYAFA (SEQ ID No: 119). V114: QYFFC (SEQ ID No: 124); V162: AYFFC (SEQ ID No: 172); V66: EYFFC (SEQ ID No: 76); V191: IYVFS (SEQ ID No: 201); V210: RYFFC (SEQ ID No: 220).

[0296] Figure 10 shows the results of in vitro transcription for the content of contaminating dsRNA (Figure 10a) and RNA yield (Figure 10b) for wild type (SEQ ID NO:1) and three selected variants V90 (SEQ ID No: 5): VYFFC (SEQ ID No: 100); V187 (SEQ ID No: 7): IYFFS (SEQ ID No: 197); and V206 (SEQ ID No: 9): RYAFC (SEQ ID NO: 216). Experiments were performed in triplicates as part of data confirmation of the first screen (Table 1). T7RNAP wild type DFAFA (SEQ ID No: 261) was the control.

[0297] Figure 11 shows a comparison of enzyme activities between T7RNAP according to the invention (i.e. V90: VYFFC (SEQ ID No: 100); V187: IYFFS (SEQ ID No: 197);

[0298] V206: RYAFC (SEQ ID No: 216)) and various prior art RNA polymerases (i.e.

[0299] Competitors A to H). The integrity of the RNA was analysed by agarose electrophoresis (Figure 11a). Heat map depicting the RNA yield (Figure 11b) and dsRNA content (Figure 11c) produced by three selected variants and various prior art RNA polymerases.

[0300] Figure 12 shows the results of in vitro transcription for the content of dsRNA (top) and RNA yield (bottom) for 7 selected variants of interest transcribing SecNLuc (Figure 12a) and Flue (Figure 12b) DNA templates. Experiments were performed in duplicates. V206: P266L + RYAFC (SEQ ID No:216); V108: P266L + QFVFN (SEQ ID No: 118); V162: P266L + AYFFC (SEQ ID No: 172); V45: P266L + PYVFA (SEQ ID No:55); V63: P266L + EYAFS (SEQ ID No:73); V217: P266L + DFAFA (SEQ ID No:227); V109 P266L + QYAFA (SEQ ID No: 119).

[0301] ExamplesWith reference to Figure 1, besides the canonical T7 RNA polymerase (T7RNAP) activity, T7RNAP also exerts RNA-dependent or DNA-dependent synthesis causing dsRNA formation. Several potential mechanisms have been suggested for this, including non-templated 3' nucleotide additions to the end of the target RNA, strandswitching involving the T7RNAP switching of the DNA template strand to the DNA non-templated strand for 3' nucleotide additions, and RNA primed synthesis. RNA-primed synthesis consists of the synthesis of RNA using RNA as a template. The extension at the 3' end can occur in the same (cis-primed) or two different (trans-primed) RNA molecules, resulting in the production of contaminating dsRNA.

[0302] Although the structure-function relationships encompassing canonical T7RNAP activity are one of the most well-characterized in polymerases, there is no clear mechanistic explanation that links T7RNAP structure or primary amino acid sequence with the synthesis of heterogeneous RNA in length at the 3' end or dsRNA formation. The inventors have therefore investigated the sequence of T7RNAP and produced 240 variants each with a different C-terminal sequence. Each of these variants was examined and scored on the basis of its RNA productivity yield, and also the amount of dsRNA it produces.

[0303] Materials and Methods

[0304] Plasmids, generation of variants and transcription template constructions

[0305] pET28a-T5-His6-RNAPT7-T0ter-Ladqwas constructed in NEB® Stable Competent E. coll (New England Biolabs, Inc) using pET28a (GenScript). A fragment containing an N-terminal hexahistidine tagged T7RNAP (uniprot code P00573) containing the T5 promoter, LacO operator, ribosome binding sequence and lambda TO terminator flanking upstream and downstream respectively was ordered as a gene block (Integrated DNA Technologies) containing Bglll and Xhol restriction sites and cloned it into pET28a. The lad gene present in parental plasmid pET28a was mutated to Laclq(Müller-Hill et al.,1968 Apr; 59(4): 1259–1264.) by site-directed mutagenesis using the Q5 Site-Directed Mutagenesis Kit (New England Biolab, Inc) following manufacturer instructions. T7RNAP variants were generated by site-directed mutagenesis using the Q5 Site-Directed Mutagenesis Kit (New England Biolab, Inc) following manufacturer instructions. All expected sequences were confirmed by Sanger sequencing (Eurofins Genomics). Transcription templates were generated by PCR amplifying a secreted nano Luciferase block (Integrated DNA Technologies) by linker primers containing 3' UTR, T7 promoter, Kozak and 5' UTR polyA sequence upstream and downstream the target sequence, respectively.pET28a-T5-His6-SP6RNAP-T0ter-Ladqis constructed in NEB® Stable Competent E. coli (New England Biolabs, Inc) using pET28a-T5-His6-RNAPT7-T0ter-Ladqas a parental plasmid. The T7RNAP gene is exchanged for SP6RNAP using HiFi assembly with de novo synthesized SP6RNAP gene block (Integrated DNA Technologies) as input according to manufacturer instructions.

[0306] pET28a-T5-His6-T3RNAP-T0ter-Ladqis constructed in NEB® Stable Competent E. coli (New England Biolabs, Inc) using pET28a-T5-His6-RNAPT7-T0ter-Ladqas a parental plasmid. The T7RNAP gene is exchanged forT3RNAP using HiFi assembly with de novo synthesized T3RNAP gene block (Integrated DNA Technologies) as input according to manufacturer instructions.

[0307] pET28a-T5-His6-KllRNAP-T0ter-Ladqis constructed in NEB® Stable Competent E. coli (New England Biolabs, Inc) using pET28a-T5-His6-RNAPT7-T0ter-Ladqas a parental plasmid. The T7RNAP gene is exchanged for K11RNAP (Klepsiella phage Kll) using HiFi assembly with de novo synthesized K11RNAP gene block (Integrated DNA Technologies) as input according to manufacturer instructions.

[0308] SP6RNAP, T3RNAP, and K11RNAP variants are generated by site-directed mutagenesis using the Q5 Site-Directed Mutagenesis kit (New England Biolabs, Inc) following manufacturer instructions. All expected sequences were confirmed by Sanger sequencing (Eurofins Genomics).

[0309] Protein production

[0310] T7RNAP and variants were recombinantly produced in E. coli BL21(DE3) using the pET28a-T5-His6-RNAPT7-T0ter-Ladqvector. In a typical preparation, transformed E. coli BL21(DE3) was grown in ZYM-5052 auto-induction medium as described previously (Studier, 2005, May;41(l):207-34). Bacterial cultures were harvested by centrifugation and either resuspended in lysis buffer (10 mM imidazole, 300 mM NaCI, 1 mM DTT, 50 mM NaxHyPO4, pH 8.0) and sonicated for a medium-scale workflow or resuspended in BugBuster buffer (Novagen) for a high-throughput scale workflow. Cleared lysates were obtained by centrifugation at 15000 x g for 20 min and applied onto PureCube Ni-NTA MagBeads (Cube Biotech). Purification was carried out in batch following the manufacturer's instructions. Protein preparations were dialysed against 2x storage buffer (200 mM NaCI, 20 mM DTT, 2 mM EDTA, 0.2 % Triton X-100, 100 mM Tris-HCI, pH 8.0), diluted at 50% glycerol and stored at -20°C.SP6RNAP, T3RNAP, and K11RNAP variants are recombinantly produced under identical conditions as T7RNAP.

[0311] RNA translation

[0312] For RNA translation experiments in human cells, endotoxins present in proteins were removed from pooled fractions containing T7RNAP or variants using the Pierce™ High-Capacity Endotoxin Removal Resin (THERMO) according to manufacturer recommendations.

[0313] SP6RNAP, T3RNAP, and K11RNAP are similarly treated to remove endotoxins.

[0314] In vitro transcription assays

[0315] Transcription reactions (20 pL) contained 40 mM Tris-HCI pH 8, 35 MgCl₂, 10 mM DTT, 2 mM spermidine, 10 mM of each ribonucleoside triphosphate (40 mM total rNTPs), 0,02 pM of DNA template, 7,5 pM T7 RNAP wt or variant, 40 U RNAse inhibitor and 0,0625U pyrophosphatase. Reactions were incubated at 37°C for 3 h or 4 h. RNA samples were subsequently purified using the RNeasy Mini Kit (QIAGEN). RNA yields were determined by UV absorbance at 260 nm.

[0316] Quantification of dsRNA

[0317] The percentage of dsRNA was calculated by sandwich ELISA assay, using dsRNA-specific mouse monoclonal antibodies J2 and K2 antibodies. 384-well microtiter plates were precoated for 2 h at room temperature (RT) with 0.04 pg / well J2 antibody anti-dsRNA mouse monoclonal antibody (Jena Bioscience) dissolved in PBS (0.15 M NaCI 10 mM K-phosphate buffer, pH 7.2). Plates were then washed once with PBS containing 0.05% Tween-20, blocked with PBS containing 1% BSA, 0.05% Tween-20 for 2 h at RT and washed again once with PBS containing 0.05% Tween-20. Samples, controls and calibration curve (20 pL) were subsequently added and incubated for 2 h at RT. Unbound material was washed thrice with PBS containing 0.05% Tween-20. Bounded antigens were detected by incubation with 5 pg / well of K2 anti-dsRNA mouse monoclonal antibody (Jena Bioscience) dissolved in PBS containing 1% BSA, 0.05% Tween-20 for 1 h at RT. Plates were subsequently washed thrice with PBS containing 0.05% Tween-20 and incubated with a horseradish peroxidase-conjugated 1:1000 diluted goat anti-mouse IgM secondary antibody diluted in PBS containing 1% BSA, 0.05% Tween-20 for 1 h at RT. Before the substrate reaction, plates were washed five times with PBS containing 0.05% Tween-20 exposed to the TMB solution (THERMO) and developed for 10 min at RT. Reactions were stopped in acidic conditions by adding10 pL / well of 2M H₂SO₄. Absorption was measured with a microplate reader at 450 nm.

[0318] Example 1 - Testing the effect of C-terminal substitutions in an RNA Polymerase variant according to the invention

[0319] The effect of C-terminal substitutions in dsRNA generation was first tested by altering the T7 RNAP wild-type C-terminal motif (DFAFA). In this way, a selected set of novel T7RNAP variants was generated containing C-terminal motifs of Pseudomonas phage VSW-3 (TYFFA) (VSW-3 has 32.3% overall seguence identity with T7RNAP-P266L-TYFFA), Klebsiella phage KP34 (PFFFC) (KP34 has 27.4% overall seguence identity with T7RNAP-P266L-PFFFC), Ralstonia solanacearum (PFFFA) (RS has 32.7% overall seguence identity with T7-P266L-PFFFA) and Pseudomonas phage BIM BV-45 (VYFFC) (BV-45 has 32.43% overall seguence identity with T7-P266L-VYFFC).

[0320] Referring to Figure 2a, the variants were then screened by measuring the dsRNA and calculating the RNA yield generated (see Figure 2b) in a standard 20 pL IVT reaction using SecNLuc as a DNA template in three independent replicates. Surprisingly, the variant containing the VYFFC motif and the P266L mutation exhibited the greatest dsRNA reduction compared to the wild type, without significantly reducing the RNA yields. The variant is referred to herein as Variant 90 (V90).

[0321] Figure 3a shows the structure of the C-terminal motif of the DFAFA wild-type T7RNAP and Figure 3b the structure of T7RNAP variant V90, which has the following four C-terminal amino acid mutations: D879V, F880Y, A881F, A883C (SEQ ID No: 5) for comparison.

[0322] Example 2 - Testing the performance of a T7RNAP variant with competitor H using ten different constructs

[0323] The performance of the V90 variant (VYFFC + P266L) was then compared with a competitor H RNAP by measuring the dsRNA and calculating the RNA yield generated in a standard 20 pL in vitro transcription in two independent replicates. The DNA templates for RNA transcription were selected from a range of lengths and types, e.g. plasmid cut with Type II restriction enzymes or synthesized via PCR. Competitor H corresponds to a prior art T7RNAP polymerase mutant commercialised as T7RNAP with low dsRNA production.

[0324] As shown in Figure 4, the experiments revealed that some constructs intrinsically had more propensity to generate dsRNA than others. Overall, the V90 variant (VYFFC +P266L) outperformed competitor H on both average RIMA yield and average % dsRNA content for all 10 constructs, i.e. 0.11% dsRNA (w / w), 187 pg vs 0.19%, 81 pg for dsRNA contamination and yield in V90 vs competitor H, respectively.

[0325] Example 3 - Testing the effect of mutation P266L on the C-terminal motif VYFFC To assess the individual effect of the C-terminal substitution VYFFC in the V90 variant (VYFFC + P266L), a variant containing only the VYFFC (and not P266L) was generated and profiled by analysing the content of dsRNA generated in a standard 20 pL IVT reaction using SecNLuc as DNA template. As shown in Figure 5, the dsRNA percentage produced showed that substitutions at the C-terminal of T7RNAP alone caused a decrease in dsRNA content. However, this effect was increased (i.e. reduced dsRNA production) when the P266L was included in the variant RNAP, i.e. in V90.

[0326] Referring now to Figure 6, there is shown a 3-dimensional structure of the C-terminal mutant (V90) with P266L modification with the C-terminus being shown as the dark helical coil.

[0327] Example 4 - Generation of 240 variants and high-throughput screening

[0328] The surprising discovery that specific substitutions at the C-terminal paired with mutation P266L decreased the generation of dsRNA, gave the framework to set out a wider screen that included the generation of 240 variants (from the 60,000 possible) containing substitutions at the C-terminal motif, as represented in Figure 7 and Table 1.

[0329] Table 1 - The 240 variant RNA polymerases

[0330] Variant Motif: SEQ ID Variant Motif: SEQ ID Variant Motif: SEQ ID No: No: No: No: No: No: 1 TFAFA 11 81 VFVFA 91 161 AYFFA 171 2 TFAFC 12 82 VFVFC 92 162 AYFFC 172 3 TFAFS 13 83 VFVFS 93 163 AYFFS 173 4 TFAFN 14 84 VFVFN 94 164 AYFFN 174 5 TFFFA 15 85 VYAFA 95 165 AYVFA 175 6 TFFFC 16 86 VYAFC 96 166 AYVFC 176 7 TFFFS 17 87 VYAFS 97 167 AYVFS 177 8 TFFFN 18 88 VYAFN 98 168 AYVFN 178 9 TFVFA 19 89 VYFFA 99 169 IFAFA 179 10 TFVFC 20 90 VYFFC 100 170 IFAFC 180 11 TFVFS 21 91 VYFFS 101 171 IFAFS 181 12 TFVFN 22 92 VYFFN 102 172 IFAFN 182

[0331]

[0332] TYAFA 23 93 VYVFA 103 173 IFFFA 183 TYAFC 24 94 VYVFC 104 174 IFFFC 184 TYAFS 25 95 VYVFS 105 175 IFFFS 185 TYAFN 26 96 VYVFN 106 176 IFFFN 186 TYFFA 27 97 QFAFA 107 177 IFVFA 187 TYFFC 28 98 QFAFC 108 178 IFVFC 188 TYFFS 29 99 QFAFS 109 179 IFVFS 189 TYFFN 30 100 QFAFN 110 180 IFVFN 190 TYVFA 31 101 QFFFA 111 181 IYAFA 191 TYVFC 32 102 QFFFC 112 182 IYAFC 192 TYVFS 33 103 QFFFS 113 183 IYAFS 193 TYVFN 34 104 QFFFN 114 184 IYAFN 194 PFAFA 35 105 QFVFA 115 185 IYFFA 195 PFAFC 36 106 QFVFC 116 186 IYFFC 196 PFAFS 37 107 QFVFS 117 187 IYFFS 197 PFAFN 38 108 QFVFN 118 188 IYFFN 198 PFFFA 39 109 QYAFA 119 189 IYVFA 199 PFFFC 40 110 QYAFC 120 190 IYVFC 200 PFFFS 41 111 QYAFS 121 191 IYVFS 201 PFFFN 42 112 QYAFN 122 192 IYVFN 202 PFVFA 43 113 QYFFA 123 193 RFAFA 203 PFVFC 44 114 QYFFC 124 194 RFAFC 204 PFVFS 45 115 QYFFS 125 195 RFAFS 205 PFVFN 46 116 QYFFN 126 196 RFAFN 206 PYAFA 47 117 QYVFA 127 197 RFFFA 207 PYAFC 48 118 QYVFC 128 198 RFFFC 208 PYAFS 49 119 QYVFS 129 199 RFFFS 209 PYAFN 50 120 QYVFN 130 200 RFFFN 210 PYFFA 51 121 SFAFA 131 201 RFVFA 211 PYFFC 52 122 SFAFC 132 202 RFVFC 212 PYFFS 53 123 SFAFS 133 203 RFVFS 213 PYFFN 54 124 SFAFN 134 204 RFVFN 214 PYVFA 55 125 SFFFA 135 205 RYAFA 215 PYVFC 56 126 SFFFC 136 206 RYAFC 216 PYVFS 57 127 SFFFS 137 207 RYAFS 217 PYVFN 58 128 SFFFN 138 208 RYAFN 218 EFAFA 59 129 SFVFA 139 209 RYFFA 219 EFAFC 60 130 SFVFC 140 210 RYFFC 220 EFAFS 61 131 SFVFS 141 211 RYFFS 221 EFAFN 62 132 SFVFN 142 212 RYFFN 222 EFFFA 63 133 SYAFA 143 213 RYVFA 223

[0333]

[0334] 54 EFFFC 64 134 SYAFC 144 214 RYVFC 224 55 EFFFS 65 135 SYAFS 145 215 RYVFS 225 56 EFFFN 66 136 SYAFN 146 216 RYVFN 226 57 EFVFA 67 137 SYFFA 147 217 DFAFA 227 58 EFVFC 68 138 SYFFC 148 218 DFAFC 228 59 EFVFS 69 139 SYFFS 149 219 DFAFS 229 60 EFVFN 70 140 SYFFN 150 220 DFAFN 230 61 EYAFA 71 141 SYVFA 151 221 DFFFA 231 62 EYAFC 72 142 SYVFC 152 222 DFFFC 232 63 EYAFS 73 143 SYVFS 153 223 DFFFS 233 64 EYAFN 74 144 SYVFN 154 224 DFFFN 234 65 EYFFA 75 145 AFAFA 155 225 DFVFA 235 66 EYFFC 76 146 AFAFC 156 226 DFVFC 236 67 EYFFS 77 147 AFAFS 157 227 DFVFS 237 68 EYFFN 78 148 AFAFN 158 228 DFVFN 238 69 EYVFA 79 149 AFFFA 159 229 DYAFA 239 70 EYVFC 80 150 AFFFC 160 230 DYAFC 240 71 EYVFS 81 151 AFFFS 161 231 DYAFS 241 72 EYVFN 82 152 AFFFN 162 232 DYAFN 242 73 VFAFA 83 153 AFVFA 163 233 DYFFA 243 74 VFAFC 84 154 AFVFC 164 234 DYFFC 244 75 VFAFS 85 155 AFVFS 165 235 DYFFS 245 76 VFAFN 86 156 AFVFN 166 236 DYFFN 246 77 VFFFA 87 157 AYAFA 167 237 DYVFA 247 78 VFFFC 88 158 AYAFC 168 238 DYVFC 248 79 VFFFS 89 159 AYAFS 169 239 DYVFS 249 80 VFFFN 90 160 AYAFN 170 240 DYVFN 250

[0335]

[0336] The generation of variants was carried out by selecting a subset of amino acids for each position of interest: 10, 2, 3, and 4 for positions 879, 880, 881, and 883, respectively, resulting in 240 variants (see Figure 7).

[0337] Example 5 - dsRNA and RNA yield profiling for the 240 variants

[0338] The 240 variants were then individually expressed in E. coli at a small scale in a high- throughput screen, purified and profiled by measuring the amount of dsRNA and RNA yield in a standard 20 pL IVT reaction using a plasmid encoding for a luciferase as DNA template. As shown in Figure 8, the majority of the variants were active and displayed varying amounts of dsRNA content and RNA yield (see Figure 8). Variants became candidates of interest if the RNA yield was higher than the screen average (55 pg) and the amount dsRNA was lower than 0.05% (w / w) (see Figure 9a).To validate the selected candidates, the variants were re-expressed in E. coli at a medium scale, purified and re-profiled by the amount of dsRNA and RNA yield in three independent replicates (see Figure 9b). From this screen, the V90 variant (VYFFC + P266L), the V187 variant (IYFFS + P266L) and the V206 variant (RYAFC + P266L) were identified as the best performers exhibiting good RNA yields and low dsRNA relative to wt T7RNAP (see Figure 10).

[0339] To exclude the possibility that the purification of putative variants of interest failed in the first high-throughput screen, a second was perform for those variants that showed an RNA yield lower than 25 pg (Figure 9a). For those that improved in yield and dsRNA content, the medium scale validation workflow was repeated and the variants were re-expressed in E. coli, purified and re-profiled by the amount of dsRNA and RNA yield in two independent replicates (see Figure 9c). From this screen, the V114 variant (QYFFC + P266L), the V162 variant (AYFFC + P266L), the V66 variant (EYFFC + P266L), the V191 variant (IYVFS + P266L), and the V210 variant (RYFFC + P266L) were identified as the best performers exhibiting good RNA yields and low dsRNA relative to the parental.

[0340] Example 6 - Comparison of three selected candidates vs commercial prior art T7RNAP

[0341] The performance of the three selected candidates (V90, V187 and V206) was compared to eight prior art commercially available T7RNAP polymerases, which have been commercialised as RNA polymerase variants with alleged "reduced" dsRNA formation. IVT reactions were carried out according to manufacturers' instructions and the associated RNA integrity, RNA yield and dsRNA content were then analysed. Six different constructs were used as DNA templates, varying in length and DNA source (plasmid or PCR-generated).

[0342] Referring to Figure 11a, the analysis of the RNA integrity by agarose electrophoresis revealed that all candidates (i.e. variants V90, V187 and V206) presented good integrity compared to commercial polymerases. Other than candidate V206 (RYAFC + P266L) in template Fanca, the RNA yields for the candidates were surprisingly superior to those of four out of the eight competitor polymerases tested (competitor A, H, B and D) (see Figure lib). In terms of dsRNA content present in the six RNA constructs, the candidates outperformed six out of the eight competitor polymerases tested, with performance comparable to competitor C (as shown in Figure 11c).Example 7 - transferring variant C-termini according to the invention to homologous RNA polymerases

[0343] To validate the generalizable effect of the C-termini of the invention on homologous RNA polymerase, C-termini from selected T7RNAP variants (V90: P266L + VYFFC, V187: P266L + IYFFS, V206: P266L + RYAFC, V114: P266L + QYFFC, V162: P266L + AYFFC, V66: P266L + EYFFC, V191: P266L + IYVFS, V210: P266L + RYFFC) are cloned via site-directed mutagenesis into SP6, T3 and Kll RNA polymerases. Subseguently, the homologous variant RNA polymerases are expressed as described above and the purified protein is used in IVT reactions, whereafter the RNA is purified and tested for yield and dsRNA. The dsRNA to ssRNA ratio of the variants is compared to the dsRNA to ssRNA ratio of the parental or wild-type homologous RNA polymerase.

[0344] Example 8 - dsRNA and RNA yield profiling for additional variants

[0345] To find RNA polymerase variants with further reduced dsRNA production or higher activity in the context of low dsRNA, novel variants are generated via site-directed mutagenesis similarly to the methods described above. These novel variants have C-termini that are derivatives of the C-termini of the top-performing (i.e., low dsRNA with high RNA yield) T7 RNA polymerases and include the following amino acids at the following locations: the terminal amino acid is selected from C, S, T, A, V or N, wherein the second to last amino acid is selected from F, Y, L, W, or H, wherein the third to last amino acid is selected from F, Y, L, I, V, W, A, P, and wherein the fourth to last amino acid is selected from F, Y, H, or W, and the combination of the four C-terminal amino acids does not correspond to the native C-terminus of the wild-type RNA polymerase. Variants of specific interest are: P266L + VWFFC, P266L + VYWFC, P266L VYFWC, P266L + VYFFT, P266L + VYFLC, P266L + VYFFV, P266L + VWFWC, P266L + VWFLC, P266L + VWFLS, P266L + VLFWC, P266L + VYFLS, P266L + IYFFT, P266L + IWFFC, P266L + IYWFC, P266L + IYFWC, P266L + IYFFT, P266L + IYFLC, P266L + IYFFV, P266L + IWFWC, P266L + IWFLC, P266L + IWFLS, P266L + ILFWC, P266L + IYFLS, P266L + RYALC, P266L + RYFFC, P266L + RYAWC, P266L + RYAFS, P266L + RWAFC, P266L + RWALC, P266 + RWFFC, P266L + RWAWC, P266L + RWAFS, P266L + QYFWC, P266L + QYFFS, P266L + QYFLC, P266L + AYFWC, P266L + AYFLC, P266L + AYFFS, P266L + EYFFS, P266L + EYFWC, P266L + EYFLC, P266L + EYFLT, P266L + IYVFC, P266L + IYVFT, P266L + VWWWC, P266L + VYWWC, P266L + VYWWS, P266L + VYWWT, P266L + VYWFS, P266L + VYWFT, P266L + RYFAS, P266L + VYYYC, P266L + VYYYS, P266L + VYYYT, P266L + RYYYC, P266L + RYYYS, and P266L + RYYYT.To find RNA polymerase variants with further increased dsRNA production or higher activity in the context of high dsRNA, novel variants are generated via site-directed mutagenesis similarly to the methods described above. These novel variants have C-termini that are derivatives of the C-termini of the high dsRNA with high RNA yield variant T7 RNA polymerases and include the following amino acids at the following locations: wherein the terminal amino acid is selected from A, G, or P, typically A, wherein the second to last amino acid is selected from F, Y, L, W or H, typically F, wherein the third to last amino acid is selected from A, G, V, or I, typically A, and wherein the fourth to last amino acid is selected from F, Y, H, or W, typically Y or F, and the combination of the four C-terminal amino acids does not correspond to the native C-terminus of the wild-type RNA polymerase.

[0346] Example 9 - Testing the effect of single mutations Y639F, H784A and double mutant Y639F / H784A in C-terminal mutant V90 in synthetising RNA with non-canonical NTPs To analyse the utilization of non-canonical NTPs in RNA synthesis by a C-terminal mutant, the variant V90 is further mutated to incorporate the prior art modifications Y639F and H784A as single mutations, and Y639F / H784A as a double mutant, via site-directed mutagenesis. After expression, purification and in vitro transcription, the efficiency of the new variants to incorporate modified nucleotides is tested by measuring total RNA yield, substituting the canonical NTP counterpart with 2'OMe-UTP, 2'OMe-CTP,2'-Fluoro-dCTP and 2'-Fluoro-dUTP. The wild-type T7RNAP variants containing the prior art modifications Y639F and H784A as single mutations, and Y639F / H784A as a double mutant, are tested as controls.

[0347] Example 10 - Testing the generation of modulated ratios of dsRNA to ssRNA with selected variants according to the invention

[0348] To validate the ability of variants to generate different ratios of dsRNA to ssRNA during an in vitro transcription reaction, 7 variants were selected from the original 240 screen (See Figure 10a). The selection was based on their ability to produce distinct amounts of dsRNA. The variants were re-expressed in E. coli at a medium scale, purified and re-profiled by the amount of dsRNA and RNA yield using SecNLuc and Flue as DNA templates in two independent replicates, according to methods described above. The variants that were selected to cover a wide dsRNA spectrum were V206: P266L + RYAFC (SEQ ID No:216); V108: P266L + QFVFN (SEQ ID No: 118); V162: P266L + AYFFC (SEQ ID No: 172); V45: P266L + PYVFA (SEQ ID No:55); V63: P266L + EYAFS (SEQ ID No:73); V217: P266L+ DFAFA (SEQ ID No:227); V109: P266L + QYAFA (SEQ ID No:119).Example 11 - Testing the optimal dsRNA content in a mRNA-vaccine

[0349] To verify that an optimized dsRNA content obtained through any of the variant RNA polymerases of the invention provides a superior vaccination, several SARS-CoV-2 Spike protein antigen encoding mRNA, circRNA and saRNA samples are made, each with a different variant RNA polymerase, in order to obtain a different dsRNA content. Mice are vaccinated with the LNP-formulated mRNA, circRNA and saRNA by injection of lpg of mRNA, lpg of circRNA or O.lpg of saRNA intramuscularly, and repeat dosing (booster) after 14 days. Anti-Sars-CoV-2 neutralizing antibody is measured 7 days, 14 days, 28 days, and 90 days after booster. Pro-inflammatory cytokine levels and other inflammatory markers are measured 2, 6, 24 and 72h after injection.

Claims

CLAIMS1. A variant RNA polymerase comprising one or more amino acid substitutions in its C-terminus compared to the corresponding wild-type C-terminal amino acid sequence, wherein during transcription, the variant RNA polymerase is adapted to produce a modulated amount of double stranded RNA (dsRNA) compared to the corresponding wild-type RNA polymerase.

2. The variant RNA polymerase according to claim 1, wherein the variant RNA polymerase is adapted to produce a higher amount of dsRNA compared to the corresponding wild type RNA polymerase on the same sequence.

3. The variant RNA polymerase according to claim 1, wherein the variant RNA polymerase is adapted to produce a lower amount of dsRNA compared to the corresponding wild type RNA polymerase on the same sequence.

4. The variant RNA polymerase according to claim 1, wherein the variant RNA polymerase comprises a variant T7, T3, Kll, SP6, KP34, or Syn5 RNA polymerase, optionally wherein the variant RNA polymerase comprises a variant T7 RNA polymerase.

5. The variant RNA polymerase according to any preceding claim, wherein the wild-type RNA polymerase comprises an amino acid sequence substantially as set out in SEQ ID No: 1, or a fragment or variant thereof; and / or wherein the wild-type RNA polymerase is encoded by a nucleotide sequence substantially as set out in SEQ ID No: 2, or a fragment or variant thereof.

6. The variant RNA polymerase according to any preceding claim, wherein the variant RNA polymerase has an amino acid sequence comprising:(i) at least 80%, 90% or 95% identity to positions 1 to 883 of SEQ ID NO: 1;(ii) at least 96%, 97%, 98% or 99% identity to positions 1 to 883 of SEQ ID NO: 1; and / or(iii) at least 99.1%, 99.2%, 99.3% or 99.4% identity to positions 1 to 883 of SEQ ID NO: 1.

7. The variant RNA polymerase according to any preceding claim, wherein the variant RNA polymerase comprises at least 1, 2, 3 or 4 amino acid substitutionscorresponding to positions 879, 880, 881, 882, and / or 883 in SEQ ID NO: 1 relative to the wild type RNAP polymerase.

8. The variant RNA polymerase according to any preceding claim, wherein the variant RNA polymerase comprises an amino acid sequence corresponding to the wildtype sequence at position F882 in SEQ ID No: 1.

9. The variant RNA polymerase according to any preceding claim, wherein:(i) the variant RNA polymerase comprises an amino acid substitution at one or more position selected from positions D879, F880, A881, F882 and A883 in SEQ ID No: 1;(ii) the variant RNA polymerase comprises an amino acid substitution at two or more positions selected from positions D879, F880, A881, F882 and A883 in SEQ ID No: 1;(iii) the variant RNA polymerase comprises an amino acid substitution at three or more positions selected from positions D879, F880, A881, F882 and A883 in SEQ ID No: 1;(iv) the variant RNA polymerase comprises an amino acid substitution at four or more positions selected from positions D879, F880, A881, F882 and A883 in SEQ ID No: 1.; and / or(v) the variant RNA polymerase comprises an amino acid substitution at five positions selected from positions D879, F880, A881, F882 and A883 in SEQ ID No: 1.

10. The variant RNA polymerase according to any preceding claim, wherein the variant RNA polymerase comprises a C-terminal motif that has a larger volume than the corresponding C-terminus of the wild-type RNA polymerase through substitution of one or more amino acids.

11. The variant RNA polymerase according to any preceding claim, wherein the variant RNA polymerase comprises a C-terminal motif comprising an amino acid sequence, wherein the terminal amino acid is small and more or less polar, wherein the second to last is bulky, aromatic and typically hydrophobic, wherein the third to last amino acid is hydrophobic, and wherein the fourth to last amino acid is bulky and aromatic, and the combination of the four C-terminal amino acids does not correspond to the native C-terminus of the wild-type RNA polymerase.

12. The variant RNA polymerase according to any preceding claim, wherein the variant RNA polymerase comprises a C-terminal motif comprising an amino acid sequence, wherein the terminal amino acid is small and slightly hydrophobic, wherein the second to last is bulky, aromatic and typically hydrophobic, wherein the third to last is small and slightly hydrophobic, and wherein the fourth to last amino acid is bulky and aromatic, and the combination of the four C-terminal amino acids does not correspond to the native C-terminus of the wild-type RNA polymerase.

13. The variant RNA polymerase according to any preceding claim, wherein the variant RNA polymerase comprises a C-terminal motif comprising an amino acid sequence wherein the terminal amino acid is selected from C, S, T, A, V or N, wherein the second to last amino acid is selected from F, Y, L, W or H, wherein the third to last amino acid is selected from F, Y, L, I, V, W, A, P, and wherein the fourth to last amino acid is selected from F, Y, H, or W, and the combination of the four C-terminal amino acids does not correspond to the native C-terminus of the wild-type RNA polymerase.

14. The variant RNA polymerase according to any preceding claim, wherein the variant RNA polymerase comprises a C-terminal motif comprising an amino acid sequence wherein the terminal amino acid is selected from A, G, or P, typically A, wherein the second to last amino acid is selected from F, Y, L, W or H, typically F, wherein the third to last amino acid is selected from A, G, V, or I, typically A, and wherein the fourth to last amino acid is selected from F, Y, H, or W, typically Y or F, and the combination of the four C-terminal amino acids does not correspond to the native C-terminus of the wild-type RNA polymerase.

15. The variant RNA polymerase according to any preceding claim, wherein where the substitution is at position D879, the substitution is selected from D879T, D879P, D879E, D879V, D879Q, D879S, D879A, and D879I.

16. The variant RNA polymerase according to any preceding claim, wherein where the substitution is at position D879, the D879 substitution comprises a D879V, D879Q, D879A, D879R or D879I substitution.

17. The variant RNA polymerase according to any preceding claim, wherein where the substitution is at position F880, the substitution is selected from F880K, F880Y, F880W, F880H and F880A.

18. The variant RNA polymerase according to any preceding claim, wherein where the substitution is at position F880, the F880 substitution comprises a F880Y substitution.

19. The variant RNA polymerase according to any preceding claim, wherein where the substitution is at position A881, the substitution is selected from A881F, A881Y, A881L, A881I, A881W, A881P and A881V.

20. The variant RNA polymerase according to any preceding claim, wherein where the substitution is at position A881, the A881 substitution comprises a A881F substitution.

21. The variant RNA polymerase according to any preceding claim, wherein where the substitution is at position F882, the substitution is selected from F882Y, F882L, F882W, and F882H.

22. The variant RNA polymerase according to any preceding claim, wherein where the substitution is at position F882, the substitution comprises a F882W substitution.

23. The variant RNA polymerase according to any preceding claim, wherein where the substitution is at position A883, the substitution is selected from A883C, A883S, A883T, A883V and A883N.

24. The variant RNA polymerase according to any preceding claim, wherein where the substitution is at position A883, the A883 substitution comprises a A883C or A883S.

25. The variant RNA polymerase according to any preceding claim, wherein the variant RNA polymerase comprises an amino acid substitution at position P266 of SEQ ID No: 1, optionally P266L, P266S, P266Y, P266A, P266G, P266T, P266I, P266V or P266N.

26. The variant RNA polymerase according to any preceding claim, wherein where the substitution is at position P266, the P266 substitution comprises a P266L substitution.

27. The variant RNA polymerase according to any preceding claim, wherein the variant RNA polymerase comprises one, two, three, four, five or six amino acid substitutions at positions selected from positions P266, D879, F880, A881, F882, and A883 in SEQ ID No:1.

28. The variant RNA polymerase according to any preceding claim, wherein the variant RNA polymerase comprises a C-terminal motif comprising an amino acid sequence substantially as set out as SEQ ID No: 100, 197, 216, 118, 124, 172, 55, 73, 76, 119, 201, 220, 167, 47, 107, 143, 239, 169, 175, or 240 or a fragment or variant thereof.

29. The variant RNA polymerase according to any preceding claim, wherein the variant polymerase comprises an amino acid sequence substantially as set out in SEQ ID No: 5, 7 or 9, or a fragment or variant thereof; and / or is encoded by a nucleotide sequence substantially as set out in SEQ ID No: 6, 8, or 10, or a fragment or variant thereof.

30. The variant RNA polymerase according to any preceding claim, wherein the variant polymerase comprises an amino acid sequence substantially as set out in SEQ ID No: 251, 253, 255, 257, 259, 262, 266, 268, 270, 272, 274, 278, 280, 282, 284, 286, or 288. or a fragment or variant thereof; and / or is encoded by a nucleotide sequence substantially as set out in SEQ ID No: 252, 254, 256, 258, 260, 263, 267, 269, 271, 273, 275, 279, 281, 283, 285, 287, or 289, or a fragment or variant thereof.

31. The variant RNA polymerase according to any preceding claim, wherein the variant RNA polymerase reduces the amount of dsRNA produced in a transcription reaction by at least 5%, 10% or 20% (w / w) compared to the amount of dsRNA produced by the corresponding wild-type RNA polymerase on the same sequence; and / or wherein the variant RNA polymerase yields at least 0.05mg / ml RNA or at least 0.5mg / ml RNA.

32. The variant RNA polymerase according to any preceding claim, wherein the variant RNA polymerase increases the amount of dsRNA produced in a transcription reaction by at least 5%, 10% or 20% (w / w) compared to the amount of dsRNA produced by the corresponding wild-type RNA polymerase on the same sequence;and / or wherein the variant RNA polymerase yields at least 0.05mg / ml RNA or at least 0.5mg / ml RNA.

33. The variant RNA polymerase according to any preceding claim, wherein the variant RNA polymerase reduces the amount of dsRNA produced in a transcription reaction by at least 85%, 90% or 95% (w / w) compared to the amount of dsRNA produced by the corresponding wild-type RNA polymerase on the same sequence; and / or wherein the variant RNA polymerase yields at least 2.5mg / ml RNA.

34. A nucleic acid sequence encoding the variant RNA polymerase according to any preceding claim.

35. The nucleic acid according to claim 34, wherein the nucleic acid sequence comprises a nucleotide sequence comprising or consisting of any one of SEQ ID No: 6, 8, 10, 252, 254, 256, 258, 260, 263, 267, 269, 271, 273, 275, 279, 281, 283, 285, 287, or 289. or a fragment or variant thereof.

36. An expression cassette comprising the nucleic acid sequence according to either claim 34 or claim 35.

37. A recombinant vector comprising the expression cassette according to claim 36.

38. A host cell comprising the recombinant vector according to claim 37.

39. Use of the variant RNA polymerase according to any one of claims 1-33, for preparing an RNA molecule.

40. A method of preparing an RNA molecule, wherein the method comprises contacting: (i) a template nucleic acid sequence, (ii) the variant RNA polymerase according to any one of claims 1-33, and (iii) a plurality of nucleotide triphosphates (NTPs), wherein the variant RNA polymerase transcribes the template nucleic acid sequence to form an RNA molecule.

41. A method of modifying the ratio of double stranded RNA (dsRNA) to single stranded RNA (ssRNA) produced in a transcription reaction, wherein the method comprises contacting: (i) a template nucleic acid sequence, (ii) one or more variant RNA polymerase according to any one of claims 1-33, and (iii) a plurality of nucleotidetriphosphates (NTPs), wherein the one or more variant RNA polymerases transcribes the template nucleic acid sequence to form ssRNA and dsRNA at a desired ratio.

42. The method according to claim 41, comprising the use of a mixture of variant RNA polymerases to produce a defined amount of dsRNA intermediate between the amount produced by either variant RNA polymerase in a single step, the method comprising: (i) a first variant RNA polymerase according to any one of claims the first aspect, and (ii) a second variant RNA polymerase according to the first aspect, optionally wherein the ratio between the first and the second variant RNA polymerase is adjusted to the relative productivity and the desired dsRNA to ssRNA ratio, and is 1:1, 0.5:1, 0.25:1, or less than 0.25:1.

43. The method according to claim 41, comprising the use of more than one variant RNA polymerase to produce a defined amount of dsRNA intermediate between the amount produced by either variant RNA polymerase by mixing the resultant RNA products in a defined ratio, the method comprising: (i) synthesizing RNA with a first variant RNA polymerase according to any one of claims 1-33, and (ii) synthesizing RNA with a second variant RNA polymerase according to any one of claims 1-33, and (iii) blending the RNA products in a defined ratio to obtained the desired dsRNA to ssRNA ratio, optionally wherein the dsRNA to ssRNA ratio is 1:1, 0.5: 1, 0.25: 1, or less than 0.25: 1.

44. The method according to claim 41-43, wherein the ratio of dsRNA to ssRNA is <0.05:1, or <0.01:1, optionally for vaccine applications; or the ratio of dsRNA to ssRNA is about 0.005: 1, optionally for therapeutic applications.

45. The method according to claim 41-44, wherein a combination of variant RNA polymerases is used, wherein the combination is selected from a group of combinations consisting of: P266L+RYAFC and P266L+QFVFN; P266L+QFVFN and P266L+AYFFC; P266L+AYFFC and P266L+PYVFA; P266L+PYVFA and P266L+EYAFS; P266L+EYAFS and P266L+DFAFA; and P266L+DFAFA and P266L+QYAFA.

46. The method according to any one of claims 40-45, wherein the ssRNA is selected from a group consisting of: messenger RNA (mRNA), micro RNA (miRNA); short interfering RNA (siRNA); short hairpin RNA (shRNA); anti-sense RNA; RNA aptamers; self-amplifying RNA (saRNA); interference RNA (RNAi); non-coding RNA; circular RNA; and small RNA.

47. An RNA manufacturing kit comprising one or more variant RNA polymerase according to any one of claims 1-33, or the nucleic acid according to either claim 34 or 35, and instructions for use.

48. An RNA molecule manufactured by the kit according to claim 47.

49. The RNA molecule according to claim 48, for use in stimulating an immune response.

50. A vaccine comprising the RNA molecule according to claim 48, and, optionally, an adjuvant.

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