RNA molecule

Abstract

The present invention relates to RNA molecules, and to methods of making RNA molecules and to methods of translating RNA molecules into proteins. The present invention relates to an improved method for forming RNA by in vitro transcription, and to the resulting RNA molecules. Furthermore, the present invention relates to a novel method for enhancing the expression and / or translation of RNA, i.e. Protein expression, as well as a method for improving the stability of RNA molecules. The invention also relates to reducing the activation of innate perception, interferon production and / or RNA molecule degradation in a host. The invention also relates to the use of RNA molecules in vaccines and other therapeutic pharmaceutical compositions, as well as their use in immunization and therapy, such as RNAi, gene therapy, gene editing and protein substitution.

Description

Technical Field

[0001] The present invention relates to RNA molecules, and specifically but not exclusively to methods for preparing RNA molecules and methods for translating RNA molecules into proteins. The present invention relates to improved methods for forming RNA by in vitro transcription, and the resulting RNA molecules. In addition, the present invention relates to novel methods for enhancing the expression and / or translation of RNA, i.e., protein expression, and methods for improving the stability of RNA molecules. The present invention also relates to reducing the activation of innate perception, interferon production and / or RNA molecule degradation in the host. The present invention also combines the use of RNA molecules in vaccines and other therapeutic pharmaceutical compositions, as well as their use in immunity and therapy, such as RNAi, gene therapy, gene editing and protein replacement. Background Art

[0002] RNA vaccines and biotherapeutics are now well established and have shown successful utility in the COVID-19 pandemic. Both of the leading COVID-19 candidates, made by Moderna and Pfizer, utilize the introduction of N1-methyl-pseudouridine, a synthetic modified form of the nucleotide triphosphate (NTP) UTP [1]. This serves to reduce the triggering of innate pathways that can limit the expression of encoded proteins, rendering the RNA ineffective for therapeutic or vaccine indications [2, 3]. The exact mechanism of improved expression is not fully understood and may be related to the inhibition of highly immunogenic double-stranded RNA (dsRNA) during in vitro transcription

[11] . However, this approach has little advantage in the case of self-amplifying RNA ( Fig.16 ). In addition, the use of methyl-pseudouridine may not be optimal. In fact, most cellular receptors detect most double-stranded RNA (dsRNA) structures by contacting the ribose backbone with minimal interaction with the base. In this regard, ribose base modifications are predicted to have beneficial effects, for example, 2'-methyl-NTPs, which are involved in innate recognition. However, efficient incorporation of 2'-methyl-NTPs into mRNA or saRNA with sequences exceeding 100 bp has not been achieved previously. In addition, although previous studies have shown that the use of 2'-methyl-NTPs can reduce innate recognition, the natural incorporation of 2'-methyl-NTPs into coding RNAs is generally believed to inhibit or reduce translation

[15] .

[0003] The substitution of ribose 2'-OH with a methyl group is a common, naturally occurring post-transcriptional modification, and 2'-O-methyl (2-OMe) modified RNA has lower immunogenicity and higher stability. 2-OMe modification consists of methylation of the ribose 2'-OH portion, and can therefore occur in all four nucleotides and other atypical nucleotides. 2'-O-methyl modified RNA can be used for short RNA sequences, such as aptamers (less than 20 bp in length), using in vitro transcription methods to maximize the synthesis of modified RNA by RNA polymerases such as T7, T3, SP6, KP34 and Syn5. However, there are currently bottlenecks for the production of longer 2'-O-methyl modified RNAs (>20 bp). For example, strict optimization of the transcription reaction mixture is required to support high and wide-range percentages of nucleotide substitutions of wild-type (i.e., unmodified) NTPs. In addition, even the production of 2'-O-methyl modified RNA aptamers by in vitro transcription is currently inefficient and can be improved. Overcoming these bottlenecks can improve the in vivo application and potential of 2'-methyl RNA intended for use as vaccines or biotherapeutics.

[0004] As mentioned above, ribose 2'-O-methylation is a common type of RNA modification found in eukaryotic mRNA. It is known that ribose 2'-O-methylation of the first and sometimes the second cap-proximal nucleotides (N1 and N2, where N corresponds to any nucleotide) of Cap1-(m7GpppNmN) or Cap2-(m7GpppNmNm) mRNA is used as an important molecular feature for the detection of "self" escape from the immune innate system

[13] . Although this process is common to higher eukaryotes, the role of many internal 2'-O-methylation sites recently discovered in human mRNA coding regions has been less characterized. Recent evidence suggests that the presence of 2'-O-methylated mRNA codons reduces translation efficiency, especially when the modification is present in the second position of the codon

[15] . There are data showing that the presence of 2'-O-methylation in certain mRNAs can selectively reduce translation elongation in vitro and in vivo, which further confirms the above view [16, 17]. Therefore, natural 2'-O-methylation in the mRNA coding region is considered to negatively regulate RNA translation and protein expression

[18] . Therefore, it is necessary to overcome the problems in the art and provide an improved method for preparing modified RNA molecules (e.g., mRNA or self-amplifying RNA) incorporating modified non-natural nucleotide bases. It is also necessary to provide improved methods to enhance RNA translation and protein expression. Summary of the invention

[0005] RNA is composed of the nucleotides ATP, CTP, GTP, and UTP, however, there are a wide range of natural and synthetic modified nucleotides [4]. As described in the Examples, the inventors have demonstrated that incorporation of the modified base 2'-methyl-NTP (2-OMe) surprisingly enhances RNA expression (both self-amplifying RNA (saRNA) and mRNA) in interferon-responsive cells. Replacing ≥75% of GTP with 2'-methyl GTP (Gm) enhanced saRNA expression by 10-fold. Replacing ≥75% of ATP, UTP, or CTP with 2'-methyl modified nucleotides also enhanced saRNA expression in the cell types tested. The inventors believe that combined substitutions may also provide additional advantages. For example, the inventors observed that replacing ≥75% of CTP with 2'-methyl CTP (Cm) enhanced mRNA expression (up to 10-fold; see Fig.33 and 34 ), while substitution of GTP, ATP, or UTP had little effect (see Figures 29 to 32 ). However, combined substitutions of ≥75% or 2'methyl-GTP and -CTP (ie, Gm+Cm) or 2'methyl-GTP, -ATP, -UTP, and -CTP (ie, Gm+Am+Cm+Um) surprisingly enhanced mRNA expression by 100-fold or more (see Figures 23 to 28 , reaching an expression level comparable to that of saRNA (see Fig.21 and 22 ). This was completely unexpected, given the evidence that, in natural contexts, incorporation of 2'-O-methylated NTPs shows a reduction in translation [16-18, 28-29]. Interestingly, these increases in translation were not observed when modified nucleotides were used in which the ribose 2'-OH was replaced by a 2'-fluoro group, suggesting that the effect depends on the use of a larger alkyl group (e.g., methyl) as a replacement for the ribose 2'-OH group (see Fig.18 and 19 ). In addition, in similar model systems, for either mRNA or saRNA, the level of translation enhancement was significantly greater than that seen when using N1-methyl-pseudouridine (see Fig.16 ).

[0006] Thus, the invention described herein overcomes prior art obstacles to producing RNA by replacing 2'-methyl-NTPs and / or other modified NTPs that are not efficiently incorporated by conventional in vitro transcription (IVT) conditions with a substantial proportion of one or more NTPs.

[0007] Therefore, in a first aspect of the present invention, a method for preparing a modified RNA molecule is provided, wherein the method comprises: contacting (i) a template nucleic acid sequence, (ii) an RNA polymerase, and (iii) a plurality of nucleotide triphosphates (NTPs) in the presence of at least 20 mM magnesium ions, wherein one or more of the nucleotide triphosphates (NTPs) are modified nucleotide triphosphates (NTPs), wherein the RNA polymerase transcribes the template nucleic acid sequence to form an RNA molecule comprising at least 20 nucleotides, and wherein at least 25% of the constituent nucleotides in the RNA molecule are modified.

[0008] In a second aspect, there is provided the use of 20 mM magnesium ions in a transcription reaction to prepare a modified RNA molecule comprising at least 20 nucleotides, wherein at least 25% of the constituent nucleotides in the RNA molecule are modified.

[0009] Advantageously, the inventors have designed novel and innovative in vitro transcription reaction conditions for the methods of the present invention. These reaction conditions include higher magnesium ions than previously used (i.e., >20 mM), thus enabling a significant level of replacement of wild-type (i.e., unmodified) nucleotides in the resulting RNA molecules. In the prior art, the use of 2'-methyl-NTPs in short RNA sequences <20 bp (mainly aptamers [5]) has been shown to prevent or reduce the activation of innate immune responses through recognition by membrane and cytoplasmic pattern recognition receptors [6-8]. However, to date, it has not been possible to generate relatively long (>20 bp, 50 bp, or 100 bp, or more) mRNA or saRNA sequences by in vitro transcription in which a significant proportion (i.e., >25%) of single or multiple wild-type unmodified nucleotides are replaced by 2'-methyl-NTPs.

[0010] The in vitro transcription reaction mixture in the method of the present invention requires the use of >20 mM magnesium ions, which is significantly higher than some conventional in vitro transcription methods. The 2'-methyl modified RNA (both mRNA and saRNA) synthesized by the method showed significantly enhanced expression in interferon responsive cells. The inventors believe that the same reaction conditions can therefore facilitate the incorporation of other modified NTPs that are not tolerant to existing IVT conditions.

[0011] Thus, the methods of the present invention advantageously provide improvements in the production of mRNA and saRNA with significant 2'methyl-NTP substitutions (>25%, even up to 100%), and demonstrate that such substitutions can significantly enhance the expression of mRNA and saRNA in interferon-competent cells. This is likely to enhance the in vivo potential of RNA vaccines (e.g., microbial antigens expressed for immunization of infected microorganisms, such as viruses and bacteria, etc., and cancer vaccines expressing oncogenic antigens) and RNA biotherapeutics (e.g., RNAi, protein replacement, gene editing, and gene therapy, etc.), whether delivered as mRNA or saRNA. In addition, the same method is also likely to facilitate the incorporation of other unnatural bases that have not been incorporated into mRNA or saRNA to date [4].

[0012] Preferably, the method comprises the use of at least 30 mM magnesium ions, more preferably at least 35 mM magnesium ions, preferably at least 40 mM magnesium ions, and further preferably at least 50 mM magnesium ions. Preferably, the method comprises the use of at least 60 mM magnesium ions, more preferably at least 70 mM magnesium ions, and further preferably at least 75 mM magnesium ions. Preferably, the method comprises the use of at least 80 mM magnesium ions, more preferably at least 85 mM magnesium ions, more preferably at least 90 mM magnesium ions, and further preferably at least 100 mM magnesium ions.

[0013] Preferably, the method comprises using 50mM to 100mM magnesium ions, more preferably 60mM to 95mM magnesium ions, more preferably 65mM to 90mM magnesium ions, further preferably 70mM to 80mM magnesium ions. Preferably, the method comprises using 71mM to 79mM magnesium ions, more preferably 73mM to 77mM magnesium ions.

[0014] The magnesium ion may be present in the method as Mg 2+ ions. Magnesium ions can be provided in the form of magnesium acetate, magnesium citrate, magnesium sulfate, magnesium gluconate, magnesium lactate, etc. As described in the Examples, the inventors observed that when magnesium ions were provided in the form of magnesium acetate rather than magnesium chloride, RNA expression increased, which was completely unexpected. Therefore, magnesium ions are preferably provided in the form of magnesium acetate. Preferably, magnesium ions are not provided in the form of magnesium chloride.

[0015] The RNA may be single-stranded or double-stranded. Those skilled in the art will appreciate that when the nucleic acid is double-stranded, such as double-stranded RNA, "base length" will refer to the length of base pairs. The RNA may be encoded. For example, encoding RNA may be used for therapeutic applications and vaccine applications.

[0016] The RNA may be non-coding. For example, non-coding RNA may be used in RNAi applications. The RNA may be selected from: messenger RNA (mRNA), microRNA (miRNA); short interfering RNA (siRNA); short hairpin RNA (shRNA); antisense RNA; RNA aptamer; self-amplifying RNA (saRNA); interfering RNA (RNAi); non-coding RNA; circular RNA; and small RNA

[12] .

[0017] Preferably, the RNA is self-amplifying RNA (saRNA) or messenger RNA (mRNA).

[0018] The RNA may be a self-amplifying RNA (saRNA). It will be understood by those skilled in the art that a self-amplifying RNA may contain the basic elements of an mRNA (cap, 5'UTR, 3'UTR, IRES, viral polymerase, and poly(A) tail of variable length), but may be quite long (e.g., 9-12 kb).

[0019] The RNA may be a messenger RNA (mRNA). The mRNA may comprise the basic elements of a cap, a 5'UTR, a 3'UTR, an IRES and a poly(A) tail of variable length.

[0020] The RNA molecule (which may be RNAi, saRNA or mRNA) may be at least 20, 21, 22 or 23 bases in length. The RNA molecule may be at least 24, 25, 26 or 27 bases in length. The RNA molecule may be at least 28, 29, 30 or 31 bases in length. The RNA molecule may be at least 32, 33, 34 or 35 bases in length. The RNA molecule may be at least 36, 37, 38 or 39 bases in length. The RNA molecule may be at least 40, 41, 42, 43, 44 or 45 bases in length. The RNA molecule may be at least 46, 47, 48 or 49 bases in length.

[0021] The RNA molecule (which can be RNAi, saRNA or mRNA) can be at least 50 bases in length, at least 60 bases in length, at least 75 bases in length, at least 100 bases in length, at least 200 bases in length, at least 300 bases in length, at least 400 bases in length, at least 500 bases in length, at least 600 bases in length, at least 700 bases in length, at least 800 bases in length, or at least 900 bases in length.

[0022] The RNA molecule, most preferably a saRNA or mRNA, can be at least 1000 bases in length, at least 2000 bases in length, at least 3000 bases in length, at least 4000 bases in length, at least 5000 bases in length, at least 6000 bases in length, at least 7000 bases in length, at least 8000 bases in length, at least 9000 bases in length, at least 10000 bases in length, at least 11000 bases in length, or at least 12000 bases in length.

[0023] In one embodiment, the RNA molecule is at least 6000 bases in length. In one embodiment, the RNA, most preferably saRNA or mRNA, is at least 6000 bases in length. In a preferred embodiment, the saRNA is at least 6000 bases in length.

[0024] The RNA, most preferably saRNA or mRNA, can be 5000 to 20000 bases in length, 6000 to 15000 bases in length, 7000 to 14000 bases in length, 7500 to 13000 bases in length, 8000 to 12000 bases in length, 8500 to 11000 bases in length, 9000 to 10000 bases in length.

[0025] The method of the invention may be performed in vivo, in vitro or ex vivo. However, it is most preferred that the method is performed in vitro. Preferably, the method comprises in vitro transcription (IVT).

[0026] Preferably, the method comprises the use of an RNA polymerase, which can be selected from: T7; T3; SP6; KP34; Syn5; or other DNA-dependent RNA polymerases; or mutant variants of any of these RNA polymerases. Each of these RNA polymerases is capable of maximizing the synthesis of modified RNA. However, preferably, the method comprises the use of T7 RNA polymerase or a variant thereof. The T7 RNA polymerase variant may include the following mutations (alone or in combination), which are associated with improved processability and / or tolerance of incorporation of modified bases, particularly R425C, K631R, S633P, Y639F, Y639V, S641A, H784A, H784S and / or H784G.

[0027] Preferably, the multiple nucleotide triphosphates (NTPs) are selected from ATP, GTP, CTP and / or UTP. NTP is the building block of RNA. Preferably, the method comprises using each ATP, GTP, CTP and / or UTP in substantially equal proportions. However, in some embodiments, the method may comprise each ATP, GTP, CTP and / or UTP in different proportions, examples of which are given below. This will depend on the sequence of the template nucleic acid and the resulting RNA molecule transcribed thereby.

[0028] Preferably, the method comprises a plurality of nucleotide triphosphates at a concentration of at least 1mM, 2mM, 3mM or 4mM. Preferably, the method comprises a plurality of nucleotide triphosphates at a concentration of at least 5mM, 6mM or 7mM. More preferably, the method comprises a plurality of nucleotide triphosphates at a concentration of at least 8mM, 9mM, 10mM. Suitably, the method comprises a plurality of nucleotide triphosphates at a concentration greater than 10mM. For example, the method comprises a plurality of nucleotide triphosphates at a concentration of at least 12mM, 14mM or 16mM. The method may also comprise a plurality of nucleotide triphosphates at a concentration of at least 18mM, 20mM or 22mM. In other embodiments, the method may comprise a plurality of nucleotide triphosphates at a concentration of at least 25mM, 50mM, 75mM or 100mM.

[0029] The one or more modified NTPs used in the method may be selected from: modified adenosine-5'-triphosphate (ATP); modified cytidine-5'-triphosphate (CTP); modified guanosine-5'-triphosphate (GTP), modified uridine-5'-triphosphate (UTP) and / or modified thymidine-5'-triphosphate (TTP).

[0030] Preferably, the method comprises the use of one or more modified NTPs, including modified ATP, modified CTP, modified GTP, modified UTP and / or modified TTP. Preferably, the method comprises the use of at least one modified NTP selected from the group consisting of modified ATP, modified CTP, modified GTP, modified UTP and / or modified TTP. Preferably, the method comprises the use of at least two modified NTPs selected from the group consisting of modified ATP, modified CTP, modified GTP, modified UTP and / or modified TTP. Preferably, the method comprises the use of at least three modified NTPs selected from the group consisting of modified ATP, modified CTP, modified GTP, modified UTP and / or modified TTP. Preferably, the method comprises the use of at least four modified NTPs selected from the group consisting of modified ATP, modified CTP, modified GTP, modified UTP and / or modified TTP. Preferably, the method comprises the use of at least five modified NTPs selected from the group consisting of modified ATP, modified CTP, modified GTP, modified UTP and / or modified TTP.

[0031] In one embodiment, the one or more modified NTPs may comprise a 2' substituted group, wherein the OH group typically at the 2' position may be substituted with halogen, an optionally substituted aromatic group, NH2, N3, H, an optionally substituted O-alkyl, O-alkenyl or O-alkynyl, or an optionally substituted alkyl, alkenyl or alkynyl, wherein in each case the aromatic group, alkyl, alkenyl or alkynyl is optionally substituted with halogen, oxo, OR, CN, NR2 or SR, wherein R is H or C 1-6 Alkyl, C 2-6 Alkenyl or C 2-6 Alkynyl.

[0032] Alternatively or in addition, one or more modified NTPs may comprise substituted nucleobases. Nucleobases may be substituted with halogen, optionally substituted aromatic groups, NH2, N3, OH, optionally substituted O-alkyl, O-alkenyl or O-alkynyl, or optionally substituted alkyl, alkenyl or alkynyl, wherein in each case, the aromatic group, alkyl, alkenyl or alkynyl is optionally substituted with halogen, oxo, OR, CN, NR2 or SR, wherein R is H or C 1-6 Alkyl, C 2-6 Alkenyl or C 2-6 Alternatively or additionally, the oxo group in the nucleobase may be replaced by a =S group.

[0033] It will be appreciated that when discussing optional substituents and the size of alkyl, alkenyl and / or alkynyl groups, this applies to the optionally substituted alkyl, alkenyl and alkynyl groups themselves as well as the optionally substituted O-alkyl, O-alkenyl and O-alkynyl groups.

[0034] The alkyl, alkenyl or alkynyl group may be an optionally substituted C1-C 20 Alkyl, alkenyl or alkynyl NTP, wherein the alkyl, alkenyl or alkynyl group is optionally substituted with halogen, oxo, OR, CN, NR2 or SR, wherein R is H or C 1-6 Alkyl, C 2-6 Alkenyl or C 2-6 Preferably, the alkyl, alkenyl or alkynyl group may be an optionally substituted C1-C 10 Alkyl, alkenyl or alkynyl NTP, wherein the alkyl, alkenyl or alkynyl group is optionally substituted with halogen, oxo, OR, CN, NR2 or SR, wherein R is H or C 1-6 Alkyl, C 2-6 Alkenyl or C 2-6 Alkynyl.

[0035] In some embodiments, the alkyl, alkenyl or alkynyl group may be an optionally substituted C 1-6 Alkyl, C 2-6 Alkenyl or C 2-6 Alkynyl, wherein the or each alkyl, alkenyl or alkynyl is optionally substituted with halogen, oxo, OR, CN, NR2 or SR, wherein R is H or C 1-6 Alkyl, C 2-6 Alkenyl or C 2-6 In some embodiments, the alkyl, alkenyl or alkynyl group may be an optionally substituted C 1-3 Alkyl, C 2-3 Alkenyl or C 2-3 Alkynyl, wherein the or each alkyl, alkenyl or alkynyl group is substituted with one or more substituents selected from oxo, OH, OMe, NH2 and NRH, wherein R is C 1-6 Alkyl, C 2-6 Alkenyl or C 2-6 More preferably, the alkyl, alkenyl or alkynyl group may be an optionally substituted C 1-3 Alkyl, C 2-3 Alkenyl or C 2-3 Alkynyl wherein the or each alkyl, alkenyl or alkynyl group is unsubstituted.

[0036] Thus, in some embodiments, the alkyl, alkenyl or alkynyl group may be methyl, ethyl, propyl, vinyl, propenyl, ethynyl or propynyl, optionally substituted with one or more substituents selected from oxo, OH, OMe, NH2 and NRH, wherein R is C 1-6 Alkyl, C 2-6 Alkenyl or C 2-6Thus, the alkyl, alkenyl or alkynyl group may be methyl, hydroxymethyl, acetyl, formyl, carbamoylmethyl, methoxycarbonylmethyl, carboxyhydroxymethyl, carboxymethylaminomethyl, methyloxyacetate, or prenylaminomethyl.

[0037] The halogen may be chlorine or bromine. The halogen may be fluorine. Thus, the one or more modified NTPs may comprise a 2'-substituted fluorine NTP.

[0038] The optionally substituted aromatic group may be C6-C 12 Optionally substituted aromatic group. The optionally substituted aromatic group may be an optionally substituted phenyl group.

[0039] Preferably, the one or more modified NTPs comprise a 2'-methyl modified NTP.

[0040] Most preferably, the one or more modified NTPs comprise 2'-O-methyl modified NTPs.

[0041] There are at least 28 identified natural 2'-O-methyl modifications (Nm) of ribonucleosides. Therefore, the one or more modified NTPs can be selected from: 2'-O-methyladenosine (Am); 1,2'-dimethyladenosine (m 1 Am); N6,2'-O-dimethyladenosine (m 6 Am); N 6 ,N 6 ,2-O-trimethyladenosine (m 6,6 Am); 2'-O-methyladenosine (Im); 1,2'-O-dimethyladenosine (m 1 Im); 2'-O-methylguanosine (Gm); 1,2'-O-dimethylguanosine (m 1 Gm); N2,2'-O-dimethylguanosine (m 2 Gm); N2,N2,2'-O-trimethylguanosine (m 2,2 Gm); N2,7,2'-O-trimethylguanosine (m 2,7 Gm); 2'-O-methylcytidine; N 4 ,2'-O-dimethylcytidine (m 4 Cm); N 4 ,N 4 ,2-O-trimethylcytidine (m 4,4 Cm); 5,2'-O-dimethylcytidine (m 5 Cm); N 4 -Acetyl-2'-O-methylcytidine (ac 4 Cm); 2'-O-methyl-5-hydroxymethylcytidine (hm 5 Cm); 5-formyl-2'-O-methylcytosine (f5 Cm); 2'-O-methyluridine (Um); 3,2'-O-dimethyluridine (m 3 Um); 5,2'-O-dimethyluridine (m 5 Um or Tm); 2-thio-2'-O-methyluridine (s 2 Um); 2'-O-methyl-pseudouridine (Ym); 5-carbamoylmethyl-2'-O-methyluridine (ncm 5 Um); 5-methoxycarbonylmethyl-2'-O-methyluridine (mcm 5 Um); 5-(carboxyhydroxymethyl)-2'-O-methyluridine methyl ester (mchm 5 Um); 5-carboxymethylaminomethyl-2'-O-methyluridine (cmbm 5 Um); 2'-O-methyluridine-5-hydroxyacetate (mcmo 5 Um); 5-(isopentenylaminomethyl)-3'O-methyluridine (inm 5 Um); and any other synthetic NTP containing 2'-O-methylated ribose.

[0042] Therefore, preferably, the one or more modified NTPs comprise 2'-O-methyl-modified ATP (2'-O-methyl-ATP; also known as "2-OMe ATP" or "Am"), 2'-O-methyl-modified CTP (2'-O-methyl-CTP; also known as "2-OMeCTP" or "Cm"), 2'-O-methyl-modified GTP (2'-O-methyl-GTP; also known as "2-OMe GTP" or "Gm"), 2'-O-methyl-modified UTP (2'-O-methyl-UTP; also known as "2-OMe UTP" or "Um") and / or 2'-O-methyl-modified TTP (2'-O-methyl-TTP; also known as "2-OMe TTP" or "Tm").

[0043] Preferably, the method comprises the use of one or more modified NTPs, the modified NTP comprising 2'-O-methyl-ATP, 2'-O-methyl-CTP, 2'-O-methyl-GTP, 2'-O-methyl-UTP and / or 2'-O-methyl-TTP. Preferably, the method comprises the use of at least one modified NTP selected from the group consisting of 2'-O-methyl-ATP, 2'-O-methyl-CTP, 2'-O-methyl-GTP, 2'-O-methyl-UTP and 2'-O-methyl-TTP. Preferably, the method comprises the use of at least two modified NTPs selected from the group consisting of 2'-O-methyl-ATP, 2'-O-methyl-CTP, 2'-O-methyl-GTP, 2'-O-methyl-UTP and 2'-O-methyl-TTP. Preferably, the method comprises the use of at least three modified NTPs selected from the group consisting of 2'-O-methyl-ATP, 2'-O-methyl-CTP, 2'-O-methyl-GTP, 2'-O-methyl-UTP and 2'-O-methyl-TTP. Preferably, the method comprises the use of at least four modified NTPs selected from the group consisting of 2'-O-methyl-ATP, 2'-O-methyl-CTP, 2'-O-methyl-GTP, 2'-O-methyl-UTP and 2'-O-methyl-TTP. Preferably, the method comprises the use of at least five modified NTPs selected from the group consisting of 2'-O-methyl-ATP, 2'-O-methyl-CTP, 2'-O-methyl-GTP, 2'-O-methyl-UTP and 2'-O-methyl-TTP.

[0044] However, most preferably, the method comprises the use of at least four modified NTPs selected from the group consisting of 2'-O-methyl-ATP, 2'-O-methyl-CTP, 2'-O-methyl-GTP and 2'-O-methyl-UTP.

[0045] The inventors attempted to achieve maximum incorporation of modified bases into modified RNA molecules. For GTP and ATP, about 75% substitution has been achieved, and for UTP and CTP, about 90% substitution has been achieved. The inventors have observed that for saRNA, modified GTP is most preferably incorporated into modified RNA molecules, and for mRNA, modified GTP and modified CTP are most preferably incorporated.

[0046] Thus, in embodiments where the modified RNA molecule is saRNA, it is preferred that the RNA molecule comprises modified 2'-O-methyl-GTP and unmodified ATP, CTP and / or UTP. In addition, in embodiments where the modified RNA molecule is mRNA, it is preferred that the RNA molecule comprises modified 2'-O-methyl-GTP and modified 2'-O-methyl CTP, as well as unmodified ATP and / or UTP.

[0047] In one embodiment, the one or more modified NTPs may comprise 2'-O-methyl ATP, which is represented herein as formula [I]:

[0048]

[0049] In another embodiment, the one or more modified NTPs may comprise 2'-O-methyl CTP, which is represented herein as formula [II]:

[0050]

[0051] In another embodiment, the one or more modified NTPs may comprise 2'-O-methyl GTP, which is represented herein as formula [III]:

[0052]

[0053] In another embodiment, the one or more modified NTPs may comprise 2'-O-methyl UTP, which is represented herein as formula [IV]:

[0054]

[0055] As described in the Examples, the inventors observed that when one or more modified NTPs comprise modified GTP, saRNA expression levels unexpectedly increased. Therefore, preferably, the one or more modified NTPs comprise modified GTP, more preferably 2'-O-methyl modified GTP. In addition, for mRNA expression levels when the one or more modified NTPs comprise modified CTP, a combination of CTP plus GTP is preferred. Therefore, preferably, for mRNA, the one or more modified NTPs comprise modified CTP plus GTP, more preferably 2'-O-methyl modified CTP plus 2'-O-methyl modified GTP.

[0056] Preferably, one or more NTPs used in the present invention are modified because they are not naturally occurring. Preferably, at least 30%, 35% or 40% of the constituent nucleotides in the resulting RNA molecule are modified, wherein the modified nucleotides may include adenine, cytosine, guanine and / or uracil. More preferably, at least 45%, 50% or 55% of the constituent nucleotides in the resulting RNA molecule are modified, wherein the modified nucleotides may include adenine, cytosine, guanine and / or uracil. Further preferably, at least 60%, 65% or 70%% of the constituent nucleotides in the RNA molecule are modified, wherein the modified nucleotides may include adenine, cytosine, guanine and / or uracil. More preferably, at least 75%, 80% or 85% of the constituent nucleotides in the RNA molecule are modified, wherein the modified nucleotides may include adenine, cytosine, guanine and / or uracil. Most preferably, at least 90%, 95%, 96%, 97%, 98%, 99% or 100% of the constituent nucleotides in the RNA molecule are modified, wherein the modified nucleotides may include adenine, cytosine, guanine and / or uracil.

[0057] As described in the Examples, in embodiments where the RNA molecule produced is mRNA, the inventors unexpectedly observed that replacing ≥75% of CTP enhanced expression by 10-fold. In addition, replacing ≥75% of the wild-type nucleotides with 2'-methyl-CTP plus -GTP or a combination of -GTP, -ATP, -CTP and / or -UTP enhanced mRNA expression by 100-fold, similar to the expression levels shown by saRNA with 75% Gm substitution. This same level of enhancement was also surprisingly observed when ≥75% of the wild-type nucleotides were replaced only by 2'-O-methyl-GTP and 2'-O-methyl-CTP, indicating that replacement of GTP and CTP provided the dominant effect.

[0058] In addition, as described in the Examples, in embodiments where the RNA molecule produced by the method is a saRNA, the inventors found that replacing ≥75% of GTP with 2'-methyl GTP (Gm) in saRNA resulted in surprisingly enhanced expression. In addition, ≥75% Gm modification showed the greatest effect, enhancing expression by 10-fold or more in different cell lines. Substitution with 2'-O-methyl-ATP, UTP or CTP improved expression of saRNA in different cell types, although to a lesser extent than GTP. Further increases in expression were also achieved by combining substitutions of Gm plus Cm or a combination of Gm, Cm, Am and Um. For example, preferably, the method comprises replacing different bases in different proportions in the modified RNA molecule.

[0059] As described herein, for mRNA, the inventors have been able to replace all four bases at a substitution rate of approximately 75%. However, the more complex architecture of saRNA means that achieving such a high ratio of modified bases is more challenging than those observed with mRNA.

[0060] Thus, preferably, the method comprises using a combination of two, three or four different modified NTPs selected from: 2'-O-methyl-ATP, 2'-O-methyl-CTP, 2'-O-methyl-GTP and 2'-O-methyl-UTP. The ratio between the two, three or four different modified NTPs may vary.

[0061] Therefore, the inventors evaluated different combinations of modified bases, as follows:

[0062] For example, for saRNA, the combination of modified bases can be:

[0063] 1.75% Gm;

[0064] 2.75% Gm plus 90% Am and 90% Um;

[0065] 3.75% Gm plus 75% Cm (with or without 90% Am+Um);

[0066] 4.75% Gm plus 50% Cm (with or without 90% Am+Um); or

[0067] 5. 50% Gm plus 50% Cm (with or without 90% Am+Um).

[0068] Preferably, the method comprises using a combination of four different modified NTPs selected from the group consisting of: 2'-O-methyl-ATP, 2'-O-methyl-CTP, 2'-O-methyl-GTP and 2'-O-methyl-UTP. The ratios between the four different modified NTPs may vary.

[0069] Alternatively, for saRNA, the combination of modified bases can be:

[0070] 1. 50%Gm+30%Cm+50%Am+30%Um;

[0071] 2. 50%Gm+90%Cm+50%Am+30%Um;

[0072] 3. 50%Gm+30%Cm+50%Am+90%Um;

[0073] 4. 50%Gm+90%Cm+50%Am+90%Um;

[0074] 5. 25%Gm+30%Cm+25%Am+60%Um;

[0075] 6. 25%Gm+90%Cm+50%Am+60%Um;

[0076] 7. 75%Gm+30%Cm+50%Am+60%Um;

[0077] 8. 75%Gm+60%Cm+50%Am+75%Um;

[0078] 9. 75%Gm+90%Cm+50%Am+60%Um;

[0079] 10. 50%Gm+30%Cm+25%Am+60%Um;

[0080] 11. 50%Gm+90%Cm+25%Am+60%Um;

[0081] 12. 50%Gm+30%Cm+75%Am+60%Um;

[0082] 13. 50%Gm+90%Cm+75%Am+60%Um;

[0083] 14. 25%Gm+60%Cm+50%Am+30%Um;

[0084] 15. 25%Gm+60%Cm+50%Am+90%Um;

[0085] 16. 75%Gm+60%Cm+50%Am+30%Um;

[0086] 17. 75%Gm+60%Cm+50%Am+90%Um;

[0087] 18. 50%Gm+60%Cm+25%Am+30%Um;

[0088] 19. 50%Gm+60%Cm+25%Am+90%Um;

[0089] 20. 50%Gm+60%Cm+75%Am+30%Um;

[0090] 21. 50%Gm+60%Cm+75%Am+90%Um;

[0091] 22. 25%Gm+60%Cm+25%Am+60%Um;

[0092] 23. 75%Gm+60%Cm+25%Am+60%Um;

[0093] 24. 25%Gm+60%Cm+75%Am+60%Um;

[0094] 25. 75%Gm+60%Cm+75%Am+60%Um;

[0095] 26. 50%Gm+60%Cm+50%Am+60%Um;

[0096] 27. 50%Gm+60%Cm+75%Am+75%Um;

[0097] 28. 50%Gm+60%Cm+60%Am+60%Um; or

[0098] 29. 50% Gm + 50% Cm + 50% Am + 50% Um; or any other percentage. For mRNA, the combination of modified bases can be:

[0099] 1.75%Gm+90%Cm (with or without 90%Am+Um);

[0100] 2.75%Gm+75%Cm (with or without 90%Am+Um);

[0101] 3. 50% Gm + 50% Cm (with or without 90% Am + Um); or

[0102] 4.25% Gm + 25% Cm (with or without 90% Am + Um).

[0103] Alternatively, for mRNA, the combination of modified bases can be:

[0104] 1. 50%Gm+30%Cm+50%Am+30%Um;

[0105] 2. 50%Gm+90%Cm+50%Am+30%Um;

[0106] 3. 50%Gm+30%Cm+50%Am+90%Um;

[0107] 4. 50%Gm+90%Cm+50%Am+90%Um;

[0108] 5. 25%Gm+30%Cm+25%Am+60%Um;

[0109] 6. 25%Gm+90%Cm+50%Am+60%Um;

[0110] 7. 75%Gm+30%Cm+50%Am+60%Um;

[0111] 8. 75%Gm+60%Cm+50%Am+75%Um;

[0112] 9. 75%Gm+90%Cm+50%Am+60%Um;

[0113] 10. 50%Gm+30%Cm+25%Am+60%Um;

[0114] 11. 50%Gm+90%Cm+25%Am+60%Um;

[0115] 12. 50%Gm+30%Cm+75%Am+60%Um;

[0116] 13. 50%Gm+90%Cm+75%Am+60%Um;

[0117] 14. 25%Gm+60%Cm+50%Am+30%Um;

[0118] 15. 25%Gm+60%Cm+50%Am+90%Um;

[0119] 16. 75%Gm+60%Cm+50%Am+30%Um;

[0120] 17. 75%Gm+60%Cm+50%Am+90%Um;

[0121] 18. 50%Gm+60%Cm+25%Am+30%Um;

[0122] 19. 50%Gm+60%Cm+25%Am+90%Um;

[0123] 20. 50%Gm+60%Cm+75%Am+30%Um;

[0124] 21. 50%Gm+60%Cm+75%Am+90%Um;

[0125] 22. 25%Gm+60%Cm+25%Am+60%Um;

[0126] 23. 75%Gm+60%Cm+25%Am+60%Um;

[0127] 24. 25%Gm+60%Cm+75%Am+60%Um;

[0128] 25. 75%Gm+60%Cm+75%Am+60%Um;

[0129] 26. 50%Gm+60%Cm+50%Am+60%Um;

[0130] 27. 50%Gm+60%Cm+75%Am+75%Um;

[0131] 28. 50%Gm+60%Cm+60%Am+60%Um; or

[0132] 29. 50%Gm+50%Cm+50%Am+50%Um; or any other percentage.

[0133] In another embodiment, the one or more modified NTPs may comprise 7-deazaguanosine-5-triphosphate (7-deazaGTP), which may be represented herein as formula [V]:

[0134]

[0135] In another embodiment, the one or more modified NTPs may comprise 7-deazaadenosine-5-triphosphate (7-deazaATP), which may be represented herein as formula [VI]:

[0136]

[0137] In another embodiment, the one or more modified NTPs may comprise N1-methyl-pseudouridine, which may be represented herein as formula [VII]:

[0138]

[0139] In some embodiments, the method may include combining one or more 2-O-methyl modified NTPs as defined herein with one or more other modified NTP bases, which may not be 2-O-methyl modified NTPs.

[0140] For example, in embodiments where the RNA molecule is mRNA, one preferred combination may be Gm+Cm, and UTP may be additionally replaced with N'methyl-pseudouridine or fluorinated UTP, etc. Similarly, in embodiments where the RNA molecule is saRNA, one preferred modified NTP is Gm, and UTP may be additionally replaced with N'methyl-pseudouridine or fluorinated UTP.

[0141] Thus, the method may comprise combining:

[0142] (i) one or more modified NTPs comprising a 2' substituted group wherein the OH group, typically at the 2' position, is substituted by halogen, an optionally substituted aromatic group, NH2, N3, H, an optionally substituted O-alkyl, O-alkenyl or O-alkynyl, or an optionally substituted alkyl, alkenyl or alkynyl group, wherein in each case the aromatic group, alkyl, alkenyl or alkynyl group is optionally substituted by halogen, oxo, OR, CN, NR2 or SR, wherein R is H or C 1-6 Alkyl, C 2-6 Alkenyl or C 2-6 Alkynyl; and

[0143] (ii) one or more modified NTPs that are not 2'-O-methyl modified NTPs.

[0144] The halogen may be chlorine, fluorine or bromine. The halogen may be fluorine.

[0145] Preferably, the method comprises combining:

[0146] (i) one or more 2-O-methyl modified NTPs selected from 2'-O-methyl modified ATP (2'-O-methyl-ATP); 2'-O-methyl modified CTP (2'-O-methyl-CTP); 2'-O-methyl modified GTP (2'-O-methyl-GTP); and 2'-O-methyl modified UTP (2'-O-methyl-UTP); and

[0147] (ii) one or more other modified NTP bases that are not 2-O-methyl modified NTPs.

[0148] For example, the one or more other modified NTP bases that are not 2-O-methyl modified NTPs may be N'methyl-pseudouridine, fluorinated UTP, 2'-chloroNTP, 2'-ethyl, 2'-bromoNTP, 2'-aminoNTP, 2'-fluoroNTP or 2'-deoxyNTP, etc. Therefore, preferably, the one or more other modified NTP bases that are not 2-O-methyl modified NTPs are additional 2' modified nucleotides.

[0149] For example, the 2'-chloroNTP can be 2'-chloroATP, 2'-chloroCTP, 2'-chloroGTP, 2'-chloroTTP and / or 2'-chloroUTP.

[0150] For example, the 2'-ethyl NTP can be 2'-ethyl ATP, 2'-ethyl CTP, 2'-ethyl GTP, 2'-ethyl TTP and / or 2'-ethyl UTP.

[0151] For example, the 2'-bromoNTP can be 2'-bromoATP, 2'-bromoCTP, 2'-bromoGTP, 2'-bromoTTP and / or 2'-bromoUTP.

[0152] For example, the 2'-amino NTP can be 2'-amino ATP, 2'-amino CTP, 2'-amino GTP, 2'-amino TTP, and / or 2'-amino UTP.

[0153] For example, the 2'-fluoro NTP can be 2'-fluoro ATP, 2'-fluoro CTP, 2'-fluoro GTP, 2'-fluoro TTP and / or 2'-fluoro UTP.

[0154] The 2'-deoxyNTP may be 2'-deoxyATP, 2'-deoxyCTP, 2'-deoxyGTP, 2'-deoxyTTP and / or 2'-deoxyUTP.

[0155] Preferably, the method comprises combining one or more 2-O-methyl modified NTPs with one or more additional 2' modified nucleotides, which may be selected from the additional 2' modified nucleotides listed in Table 7 below.

[0156] Table 7 - 2-O-methyl modified NTPs and in addition Combination of 2' modified nucleotides

[0157]

[0158]

[0159] Such combinations can be used in any ratio to prepare nucleic acids or modified RNAs of the invention. For example, about 0.1%, 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.9% of one or more 2-O-methyl modified NTPs can be combined with about 99.9%, 99%, 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, 1% or 0.1% of one or more other modified NTP bases (which are not 2-O-methyl modified NTPs), respectively.

[0160] Unless otherwise indicated, the modified nucleotides can completely replace the natural nucleotides of the nucleic acid of the present invention or the modified RNA. As non-limiting examples, the natural nucleotides guanidine, cytosine, adenine, thymine or uracil can be partially replaced by at least one modified nucleotide disclosed herein (e.g., about 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99.9%).

[0161] As a non-limiting example, a 2-O-methyl modified NTP can be partially substituted (e.g., about 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99.9%) with at least one additional 2'-modified nucleotide (e.g., 2'-deoxy GTP, 2'-fluoro GTP). As a non-limiting example, 2-O-methyl modified GTP (Gm) can be partially substituted (e.g., about 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99.9%) with at least one additional 2' modified nucleotide (e.g., 2'-deoxy GTP, 2'-fluoro GTP).

[0162] Thus, as an example, if Gm is used at 90%, it can be used in combination with 2'-deoxyGTP (dGTP) in the following ratios (89:1%, 85:5%, 80:10%, 75:15%, 70:20%, 76:25%, 60:30%, 55:35%, 50:40%, 45:45%, 40:50%, 35:55%, 30:60%, 25:65%, 20:70%, 15:75%, 10:80%, 5:85%).

[0163] In addition, additional 2'-modified NTPs can be used, including 2'-chloro, 2'-ethyl, 2'-bromo, 2'-amino, etc.

[0164] The method may also include the use of DTT. This is believed to enhance RNA yield and RNA polymerase activity.

[0165] The method may also include the use of a pH buffer, such as HEPES, but one skilled in the art will appreciate that other buffers may also be used.

[0166] The method may further comprise the use of a crowding agent, such as poly(ethylene glycol) (PEG) of various molecular weights. For example, the crowding agent may be PEG200 or any additional crowding agent

[32] .

[0167] The method may also include the use of an RNase inhibitor. This prevents RNA degradation if any RNase is present in the reaction.

[0168] The method may further comprise the use of spermidine. This is believed to increase the activity of RNA polymerase, thereby increasing RNA yield.

[0169] The template nucleic acid sequence is preferably transcribed by RNA polymerase to produce a modified RNA molecule. Therefore, preferably, the template nucleic acid sequence comprises DNA. The template nucleic acid can be prepared synthetically, such as dog bone DNA, or prepared by PCR, rolling circle amplification or synthetic amplification. The template nucleic acid can comprise a vector, preferably a plasmid.

[0170] In one embodiment, the template nucleic acid can be transcribed to produce the resulting modified RNA molecule, which can be described as a biotherapeutic RNA molecule, i.e., a molecule used to treat, prevent or improve a patient's disease. The template nucleic acid sequence can encode a therapeutic protein that is derived from an animal or human and treats, prevents or improves a disease in a subject (preferably a human or animal subject). For example, the therapeutic use of the encoded therapeutic protein can include protein replacement, gene editing (e.g., CRISPR-Cas9), gene therapy, or RNAi, etc.

[0171] The biotherapeutic RNA molecule can be selected from: messenger RNA (mRNA), microRNA (miRNA); short interfering RNA (siRNA); short hairpin RNA (shRNA); antisense RNA; RNA aptamer; self-amplifying RNA (saRNA); interfering RNA (RNAi); non-coding RNA; circular RNA; and small RNA.

[0172] In one embodiment, the template nucleic acid can encode proteins and peptides derived from plants. Preferably, the proteins and peptides are plant antigens. The plant antigens can be derived from Ricinus communis.

[0173] In another embodiment, the template nucleic acid encodes an antigen, in which case the resulting modified RNA molecule can be described as a vaccine, i.e., a vaccine for preventing a patient's disease, or a vaccine for therapeutically ameliorating a disease such as cancer. For example, the template nucleic acid sequence can encode an antigen derived from a virus, bacteria, mycoplasma, fungus, animal, plant, algae, parasite or protozoa, or other organism that causes a disease in a subject (preferably a human or animal). Preferably, the template nucleic acid sequence is derived from a virus or bacteria. For example, the template nucleic acid can encode at least a portion of a viral spike protein.

[0174] In another embodiment, the template nucleic acid may encode a tumor immunogen or antigen, or a cancer immunogen or antigen. The tumor immunogen and antigen may be a peptide-containing tumor antigen, such as a polypeptide tumor antigen or a glycoprotein tumor antigen.

[0175] The tumor antigen can be (a) a full-length molecule associated with cancer cells, (b) a homologue and modified forms thereof, including molecules with deleted, added and / or substituted parts, and (c) a fragment thereof.

[0176] Suitable tumor immunogens include class I restricted antigens recognized by CD8+ lymphocytes or class II restricted antigens recognized by CD4+ lymphocytes.

[0177] The tumor antigen may be an antigen associated with a cancer selected from the group consisting of testicular cancer, melanoma, lung cancer, head and neck cancer, NSCLC, breast cancer, gastrointestinal cancer, bladder cancer, colorectal cancer, pancreatic cancer, lymphoma, leukemia, kidney cancer, liver cancer, ovarian cancer, gastric cancer and prostate cancer.

[0178] The tumor antigen may be selected from:

[0179] (a) cancer-testis antigens such as NY-ESO-1, SSX2, SCP1, and RAGE, BAGE, GAGE, and MAGE family polypeptides, e.g., GAGE-1, GAGE-2, MAGE-1, MAGE-2, MAGE-3, MAGE-4, MAGE-5, MAGE-6, and MAGE-12 (which can be used, for example, to address melanoma, lung, head and neck, NSCLC, breast, gastrointestinal, and bladder tumors);

[0180] (b) mutant antigens, e.g., p53 (associated with various solid tumors, e.g., colorectal cancer, lung cancer, head and neck cancer), p21 / Ras (associated with, e.g., melanoma, pancreatic cancer and colorectal cancer), CDK4 (associated with, e.g., melanoma), MUM1 (associated with, e.g., melanoma), caspase-8 (associated with, e.g., head and neck cancer), CIA0205 (associated with, e.g., bladder cancer), HLA-A2-R1701, β-catenin (associated with, e.g., melanoma), TCR (associated with, e.g., T-cell non-Hodgkin's lymphoma), BCR-abl (associated with, e.g., chronic myeloid leukemia), trisaccharide phosphate isomerase, KIA 0205, CDC-27 and LDLR-FUT;

[0181] (c) overexpressed antigens, e.g., galectin 4 (associated with, e.g., colorectal cancer), galectin 9 (associated with, e.g., Hodgkin's disease), proteinase 3 (associated with, e.g., chronic myeloid leukemia), WT1 (associated with, e.g., various leukemias), carbonic anhydrase (associated with, e.g., renal cancer), aldolase A (associated with, e.g., lung cancer), PRAME (associated with, e.g., melanoma), HER-2 / neu (associated with, e.g., breast cancer, colon cancer, lung cancer, and ovarian cancer), alpha-fetoprotein (associated with, e.g., liver cancer), KSA (associated with, e.g., colorectal cancer), gastrin (associated with, e.g., pancreatic cancer and gastric cancer), telomerase catalytic protein, MUC-I (associated with, e.g., breast cancer and ovarian cancer), G-250 (associated with, e.g., renal cell carcinoma), p53 (associated with, e.g., breast cancer, colon cancer), and carcinoembryonic antigen (associated with, e.g., breast cancer, lung cancer, and gastrointestinal cancers such as colorectal cancer);

[0182] (d) shared antigens, e.g., melanin-melanocyte differentiation antigens such as MART-1 / Melan A, gplOO, MClR, melanocyte stimulating hormone receptor, tyrosinase, tyrosinase-related protein-1 / TRP1 and tyrosinase-related protein-2 / TRP2 (associated with, e.g., melanoma);

[0183] (e) prostate-associated antigens, e.g., PAP, PSA, PSMA, PSH-P1, PSM-P1, PSM-P2, which are associated with, e.g., prostate cancer; and / or

[0184] (f) Immunoglobulin idiotype (e.g. associated with myeloma and B-cell lymphoma).

[0185] The template nucleic acid may encode a eukaryotic polypeptide. In one embodiment, the eukaryotic polypeptide is a mammalian polypeptide. The mammalian polypeptide may be selected from: enzymes; enzyme inhibitors; hormones; immune system proteins; receptors; binding proteins; transcription or translation factors; tumor growth expressed proteins; structural proteins and blood proteins.

[0186] The enzyme may be selected from the group consisting of: laccase; gastric lipase; tissue plasminogen activator; streptokinase; cholesterol biosynthetic or degrading sterile enzymes; kinases; phosphodiesterases; methylases; demethylases; dehydrogenases; cellulases; proteases; lipases; phospholipases; aromatases; cytochromes; adenylate or guanylate cyclases and ceramidase.

[0187] The enzyme inhibitor may be a tissue inhibitor of metalloproteinases (TIMP).The hormone may be a growth hormone.

[0188] The immune system protein may be selected from the group consisting of: cytokines; chemokines; lymphokines; erythropoietin; integrins; addressins; selectins; homing receptors; T cell receptors and immunoglobulins.

[0189] The cytokine may be an interleukin, such as IL-2, IL-4 and / or IL-6, colony stimulating factor (CSF), granulocyte colony stimulating factor (G-CSF), granulocyte-macrophage colony stimulating factor (GM-CSF) or tumor necrosis factor (TNF).

[0190] The chemokine may be macrophage inflammatory protein-2 and / or plasminogen activator.

[0191] The lymphokine may be an interferon.

[0192] The immunoglobulin may be a natural, modified or chimeric immunoglobulin or a fragment thereof. Preferably, the immunoglobulin is a chimeric immunoglobulin having dual activities, such as an abzyme or an antibody-toxin chimera.

[0193] The hormone can be selected from: insulin, thyroid hormone, catecholamine, gonadotropin, nutritional hormone, prolactin, oxytocin, dopamine, bovine growth hormone, leptin; growth hormone (e.g., human growth hormone), growth factor (e.g., epidermal growth factor, nerve growth factor, insulin-like growth factor, etc.).

[0194] The receptor may be a steroid hormone receptor or a peptide receptor.Preferably, the receptor is a growth factor receptor.

[0195] The binding protein may be a growth factor binding protein.

[0196] The tumor growth suppressor protein may be a protein that suppresses angiogenesis.

[0197] The structural protein may be selected from the group consisting of: collagen; silk protein; fibrinogen; elastin; tubulin; actin; and myosin.

[0198] The blood protein may be selected from thrombin; serum albumin; factor VII; factor VIII; insulin; factor IX; factor X; tissue-type plasminogen activator; protein C; von Wilebrand factor; antithrombin III; glucocerebrosidase; erythropoietin granulocyte colony stimulating factor (GCSF) or modified factor VIII; and an anticoagulant.

[0199] In a preferred embodiment, the template nucleic acid can encode a cytokine that can regulate lymphatic homeostasis, preferably relates to and preferably induces or enhances the development, initiation, amplification, differentiation and / or survival of T cells. Therefore, preferably, the cytokine is an interleukin. Most preferably IL-2, IL-7, IL-12, IL-15 or IL-21.

[0200] The template nucleic acid may encode a protein capable of enhancing the reprogramming of somatic cells into cells having stem cell characteristics.

[0201] The protein capable of enhancing the reprogramming of somatic cells into cells with stem cell characteristics can be selected from: OCT4, SOX2, NANOG, LIN28, p53, ART-4, BAGE, ss-catenin / m, Bcr-abL CAMEL, CAP-1, CASP-8, CDC27 / m, CD4 / m, CEA, CLAUDIN-12, c-MYC, CT, Cyp-B, DAM, ELF2M, ETV6-AML1, G250, GAGE, GnT-V, GaplOO, HAGE, HER-2 / neu, HPV-E7, HPV-E6, HAST-2 , hTERT (or hTRT), LAGE, LDLR / FUT, MAGE-A, MAGE-B, MAGE-C, MART-1 / Melan-A, MC1R, Myosin / m, MUC1, MUM-1, -2, -3, NA88-A, NF1, NY-ESO-1, NY-BR-1, pl90 Minor BCR-abL (pl90 minor , BCR-abL), Plac-1, Pml / RARa, PRAME, proteinase 3, PSA, PSM, RAGE, RU1 or RU2, SAGE, SART-1 or SART-3, SCGB3A2, SCP1, SCP2, SCP3, SSX, SURVIVIN, TEL / AML1, TPI / m, TRP-1, TRP-2, TRP-2 / INT2, TPTE and WT, preferably WT-1.

[0202] Preferably, MAGE-A is selected from: MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A5, MAGE-A6, MAGE-A7, MAGE-A8, MAGE-A9, MAGE-A10, MAGE-A11 or MAGE-A12

[0203] Preferably, the protein capable of enhancing the reprogramming of somatic cells into cells with stem cell characteristics is OCT4, SOX2, LF4; c-MYC; NANOG; LIN28.

[0204] The template nucleic acid may encode a biomolecule for in vitro cell modification for cell therapy indications. Therefore, preferably, the template nucleic acid may encode a protein selected from immunoglobulins, T cell receptors and NK receptors.

[0205] The template nucleic acid may encode a protein capable of regulating the expression of an endogenous host gene, such as an interfering RNA, such as a small RNA, siRNA or micro RNA.

[0206] In one embodiment, the template nucleic acid may encode an innate inhibitor protein, as described in WO2020 / 254804, PCT / GB2021 / 053362 or PCT / GB2021 / 053361, which counteracts the innate immune response of a subject administered a vaccine comprising the resulting RNA molecule.

[0207] According to a third aspect, there is provided an RNA molecule obtained or obtainable by a method according to the first aspect.

[0208] According to a fourth aspect, there is provided a pharmaceutical composition comprising the RNA molecule according to the third aspect and a pharmaceutically acceptable carrier.

[0209] According to a fifth aspect, there is provided a method of preparing a pharmaceutical composition according to the fourth aspect, the method comprising contacting the RNA molecule according to the third aspect with a pharmaceutically acceptable carrier.

[0210] According to a sixth aspect, there is provided use of the RNA molecule according to the third aspect or the pharmaceutical composition according to the fourth aspect as a medicament.

[0211] According to the seventh aspect, provided is the use of the RNA molecule according to the third aspect or the pharmaceutical composition according to the fourth aspect in treating, preventing or ameliorating a disease (such as cancer) in a subject.

[0212] According to the eighth aspect, a method for treating, preventing or ameliorating a disease (e.g., cancer) in a subject is provided, the method comprising administering or having administered a therapeutically effective amount of an RNA molecule according to the third aspect or a pharmaceutical composition according to the fourth aspect to a subject in need thereof.

[0213] According to the ninth aspect, a vaccine composition is provided, comprising the RNA molecule according to the third aspect or the pharmaceutical composition according to the fourth aspect.

[0214] The vaccine may include a suitable adjuvant. Examples of the adjuvant may include aluminum salts, synthetic forms of DNA, carbohydrates, tablet binders, ion exchange resins, preservatives, polymers, emulsions and / or lipids. Examples of the adjuvant may include monosodium glutamate, sucrose, dextrose, aluminum bovine, human serum albumin, cytosine phosphate guanine, potassium phosphate, povidone C, anhydrous lactose, cellulose, polacrilin potassium, glycerol, asparagine, citric acid, potassium phosphate magnesium sulfate, ammonium ferric citrate, 2-phenoxyethanol, aluminum, β-propiolactone, bovine extract, DOPC, EDTA, formaldehyde, cytosine phosphate guanine (CpG), QS21, saponin, monophosphoryl lipid A (MPLA), squalene, thimerosal, phenol, potassium aluminum sulfate, potassium glutamate, sodium borate, sodium pyrosulfite, urea, PLGA, PVA, PLA, PVP, cyclodextrin-based stabilizers, oil-in-water emulsion adjuvants and / or lipid-based adjuvants.

[0215] According to a tenth aspect, there is provided use of the RNA molecule according to the third aspect, the pharmaceutical composition according to the fourth aspect or the vaccine according to the ninth aspect in stimulating an immune response in a subject.

[0216] Can stimulate immune response to protozoa, bacteria, virus, fungus or cancer.Therefore, described vaccine can be used to express microorganism or pathogenic antigen, to immunize infectious microorganism, such as virus and bacteria etc.However, described vaccine can also be used to express onco-antigen, to prevent cancer.

[0217] According to the eleventh aspect of the present invention, a method for vaccinating a subject is provided, the method comprising administering or having administered a therapeutically effective amount of an RNA molecule according to the third aspect, a pharmaceutical composition according to the fourth aspect, or a vaccine according to the ninth aspect to a subject in need.

[0218] It should be understood that RNA molecules can have significant utility in a wide range of therapeutic applications, such as RNAi, inhibitory RNA, RNA aptamers, etc., where translation of the RNA molecule does not necessarily need to occur. However, applications that require translation of RNA molecules include various vaccines and biotherapeutic methods, such as using mRNA, saRNA, circular RNA, or any RNA sequence that can be translated in vivo and produce a peptide or protein.

[0219] For example, peptides or proteins encoded by RNA molecules can be involved in protein replacement, gene editing, gene therapy, etc.

[0220] It should also be understood that there is an increasing trend towards the use of mRNA and saRNA vaccines in particular, in which the RNA molecule encodes a pathogen, such as a viral coat protein or a region thereof, or an oncogenic antigen, and it is this antigen that is expressed in the immunized host organism and triggers an immune response to the pathogen or tumor.

[0221] As described in the Examples, the inventors not only demonstrated that the method of the first aspect can be used to effectively prepare modified RNA molecules comprising modified NTPs, but also surprisingly demonstrated a significant increase in RNA translation (i.e., protein expression), especially when the modified RNA molecule is mRNA or saRNA. For example, replacing ≥75% of CTP in mRNA enhances expression by up to 10 times, and replacing ≥75% of wild-type nucleotides with 2'-methyl-CTP and -GTP, or -GTP-ATP, -CTP and / or -UTP enhances mRNA expression by up to 100 times, similar to the increase in expression levels demonstrated by saRNA with 75% Gm substitutions. This same level of expression enhancement was also surprisingly observed in mRNA when ≥75% of wild-type nucleotides were replaced only by 2'-O-methyl-GTP and 2'-O-methyl-CTP.

[0222] As described in the Examples, the inventors did not directly measure the amount of the luciferase protein produced, but measured the expression enhancement in terms of luciferase activity. Therefore, luciferase activity can be considered as a substitute for RNA translation, because it should be directly related to the translation amount. In addition, the luciferase data described in the Examples can be used as a substitute for therapeutic agents, basically reflecting the expression of therapeutic proteins. However, as described herein, when using the term "10 times" etc., this means that the luciferase activity actually increased by 10 times. It should be understood that the increase in expression or RNA translation may be more stably related to RNA, so more protein will be produced before degradation. Therefore, translation efficiency (i.e. ribosome speed) may not change, but the total time for ribosomes to make more protein will increase.

[0223] In summary, these data taken together indicate that modified NTPs, particularly 2'-O-methyl modified bases, can be used to increase RNA translation levels for in vitro and in vivo protein production, the latter being particularly advantageous when RNA molecules are used as vaccines or therapeutic biomolecules. Based on the unexpected discovery that the incorporation of 2'-O modified bases enhances, rather than reduces, the expression of naturally incorporated 2'-O methylated bases in coding RNA, the inventors believe that their data also support the following discovery: modified bases can be used to enhance the stability of RNA and reduce the activation of innate perception, interferon production and / or degradation of RNA molecules. In view of the current understanding in the art that the natural incorporation of 2-O-methyl bases actually inhibits RNA translation (i.e., protein production), these effects of 2'-O modified bases on RNA are particularly unexpected.

[0224] Therefore, the inventors believe that any 2'-OH substituted modified NTP can be used to:

[0225] (i) increasing the expression and / or translation of RNA molecules (particularly mRNA or saRNA, and ideally using 2'-O-methyl NTPs);

[0226] (ii) improving the stability of RNA molecules (of any type, regardless of length, and having any of the 2'-OH substitutions described herein); and / or

[0227] (iii) reducing the activation of innate sensing, interferon production and / or degradation of RNA molecules (of any type, regardless of their length, and having any of the 2'-OH substitutions described herein).

[0228] Therefore, according to a twelfth aspect of the present invention, there is provided one or more modified nucleotide triphosphates (NTPs) for:

[0229] (i) enhancing the expression and / or translation of an RNA molecule comprising one or more modified NTPs;

[0230] (ii) enhancing the stability of RNA molecules comprising one or more modified NTPs; and / or

[0231] (iii) reducing the activation of innate sensing, interferon production and / or degradation of RNA molecules comprising one or more modified NTPs,

[0232] wherein the one or more modified NTPs comprise a 2' substituted group, wherein the OH group at the 2' position is typically substituted with halogen, an optionally substituted aromatic group, NH2, N3, H, an optionally substituted O-alkyl, O-alkenyl or O-alkynyl, or an optionally substituted alkyl, alkenyl or alkynyl, wherein in each case the aromatic group, alkyl, alkenyl or alkynyl is optionally substituted with halogen, oxo, OR, CN, NR2 or SR, wherein R is H or C 1-6 Alkyl, C 2-6 Alkenyl or C 2-6 Alkynyl.

[0233] In addition, according to a thirteenth aspect, a method is provided:

[0234] (i) enhancing the expression and / or translation of an RNA molecule comprising one or more modified NTPs;

[0235] (ii) enhancing the stability of RNA molecules comprising one or more modified NTPs; and / or

[0236] (iii) reducing the activation of innate sensing, interferon production and / or degradation of RNA molecules comprising one or more modified NTPs,

[0237] The method comprises incorporating one or more modified nucleotide triphosphates (NTPs) into an RNA molecule, and

[0238] wherein the one or more modified NTPs comprise a 2' substituted group, wherein the OH group at the 2' position is typically substituted with halogen, an optionally substituted aromatic group, NH2, N3, H, an optionally substituted O-alkyl, O-alkenyl or O-alkynyl, or an optionally substituted alkyl, alkenyl or alkynyl, wherein in each case the aromatic group, alkyl, alkenyl or alkynyl is optionally substituted with halogen, oxo, OR, CN, NR2 or SR, wherein R is H or C 1-6 Alkyl, C 2-6 Alkenyl or C 2-6 Alkynyl, and

[0239] Thereby, the expression and / or translation of RNA molecules comprising one or more modified NTPs is enhanced, the stability of RNA molecules comprising one or more modified NTPs is enhanced, and / or the activation of innate sensing, interferon production and / or degradation of RNA molecules comprising one or more modified NTPs is reduced.

[0240] Enhancing the translational expression of modified RNA molecules is an important aspect of the present invention.

[0241] Therefore, according to the fourteenth aspect, a method for enhancing the translation of an RNA molecule is provided, the method comprising translating an RNA molecule comprising one or more 2'-O-methyl modified nucleotide triphosphates (NTPs), wherein the level of translation in the presence of the one or more 2'-O-methyl modified NTPs is greater than the level of translation in the absence of the one or more 2'-O-methyl modified NTPs.

[0242] According to a fifteenth aspect, there is provided a use of one or more 2'-O-methyl modified nucleotide triphosphates (NTPs) in an RNA molecule for enhancing translation of the RNA molecule.

[0243] In a sixteenth aspect, one or more 2'-O-methyl modified nucleotide triphosphates (NTPs) in an RNA molecule are provided for enhancing translation of the RNA molecule.

[0244] The method or use may be performed in vivo, in vitro or ex vivo. However, it is most preferred that the method is performed in vivo.

[0245] In some embodiments, the method of the first aspect can be used to incorporate one or more modified NTPs into RNA molecules. Therefore, the method can include contacting (i) a template nucleic acid sequence, (ii) an RNA polymerase, and (iii) a plurality of nucleotide triphosphates (NTPs) in the presence of at least 20 mM magnesium ions, wherein one or more of the nucleotide triphosphates (NTPs) are modified nucleotide triphosphates (NTPs), wherein the RNA polymerase transcribes the template nucleic acid sequence to form the RNA molecule.

[0246] The one or more modified NTPs may be as defined in relation to the first aspect of the invention. Preferably, the one or more modified NTPs comprise 2'-methyl modified NTPs. Most preferably, the one or more modified NTPs comprise 2'-O-methyl modified NTPs. Thus, the one or more modified NTPs may be selected from: 2'-O-methyladenosine (Am); 1,2'-dimethyladenosine (m 1 Am); N6,2'-O-dimethyladenosine (m 6 Am); N 6 ,N 6 ,2-O-trimethyladenosine (m 6,6 Am); 2'-O-methyladenosine (Im); 1,2'-O-dimethyladenosine (m 1 Im); 2'-O-methylguanosine (Gm); 1,2'-O-dimethylguanosine (m 1 Gm); N2,2'-O-dimethylguanosine (m 2 Gm); N2,N2,2'-O-trimethylguanosine (m2,2 Gm); N2,7,2'-O-trimethylguanosine (m 2,7 Gm); 2'-O-methylcytidine; N 4 ,2'-O-dimethylcytidine (m 4 Cm); N 4 ,N 4 ,2-O-trimethylcytidine (m 4, 4 Cm); 5,2'-O-dimethylcytidine (m 5 Cm); N 4 -Acetyl-2'-O-methylcytidine (ac 4 Cm); 2'-O-methyl-5-hydroxymethylcytidine (hm 5 Cm); 5-formyl-2'-O-methylcytosine (f 5 Cm); 2'-O-methyluridine (Um); 3,2'-O-dimethyluridine (m 3 Um); 5,2'-O-dimethyluridine (m 5 Um or Tm); 2-thio-2'-O-methyluridine (s 2 Um); 2'-O-methyl-pseudouridine (Ym); 5-carbamoylmethyl-2'-O-methyluridine (ncm 5 Um); 5-methoxycarbonylmethyl-2'-O-methyluridine (mcm 5 Um); 5-(carboxyhydroxymethyl)-2'-O-methyluridine methyl ester (mchm 5 Um); 5-carboxymethylaminomethyl-2'-O-methyluridine (cmbm 5 Um); 2'-O-methyluridine-5-hydroxyacetate (mcmo 5 Um); 5-(isopentenylaminomethyl)-3'O-methyluridine (inm 5 Um); and any other synthetic NTP containing 2'-O-methylated ribose.

[0247] Therefore, preferably, the one or more modified NTPs comprise 2'-O-methyl-modified ATP, 2'-O-methyl-modified CTP, 2'-O-methyl-modified GTP, 2'-O-methyl-modified UTP and / or 2'-O-methyl-modified TTP.

[0248] Preferably, the method comprises the use of one or more modified NTPs comprising 2'-O-methyl-ATP, 2'-O-methyl-CTP, 2'-O-methyl-GTP, 2'-O-methyl-UTP and / or 2'-O-methyl-modified TTP. Preferably, the method comprises the use of at least one modified NTP selected from the group consisting of 2'-O-methyl-ATP, 2'-O-methyl-CTP, 2'-O-methyl-GTP, 2'-O-methyl-UTP and 2'-O-methyl-modified TTP. Preferably, the method comprises the use of at least two modified NTPs selected from the group consisting of 2'-O-methyl-ATP, 2'-O-methyl-CTP, 2'-O-methyl-GTP, 2'-O-methyl-UTP and 2'-O-methyl-modified TTP. Preferably, the method comprises the use of at least three modified NTPs selected from the group consisting of 2'-O-methyl-ATP, 2'-O-methyl-CTP, 2'-O-methyl-GTP, 2'-O-methyl-UTP and 2'-O-methyl-modified TTP. Preferably, the method comprises the use of at least four modified NTPs selected from the group consisting of 2'-O-methyl-ATP, 2'-O-methyl-CTP, 2'-O-methyl-GTP, 2'-O-methyl-UTP and 2'-O-methyl-modified TTP. Preferably, the method comprises the use of at least five modified NTPs selected from the group consisting of 2'-O-methyl-ATP, 2'-O-methyl-CTP, 2'-O-methyl-GTP, 2'-O-methyl-UTP and 2'-O-methyl-modified TTP. However, preferably, the method comprises the use of at least four modified NTPs selected from the group consisting of 2'-O-methyl-ATP, 2'-O-methyl-CTP, 2'-O-methyl-GTP and 2'-O-methyl-UTP.

[0249] Preferably, the one or more modified NTPs comprise 2'-O-methyl modified GTP. In embodiments where the modified RNA molecule is saRNA, preferably the RNA molecule comprises modified 2'-O-methyl-GTP and unmodified ATP, CTP and / or UTP. In addition, in embodiments where the modified RNA molecule is mRNA, preferably the RNA molecule comprises modified 2'-O-methyl-GTP and modified 2'-O-methyl CTP, and unmodified ATP and / or UTP.

[0250] The type of RNA molecule can be as described in relation to the first aspect. Thus, the RNA can be single-stranded or double-stranded. The RNA can be coding. For example, coding RNA can be used for therapeutic applications and vaccine applications. The RNA can be non-coding. For example, non-coding RNA can be used for RNAi applications. The RNA can be selected from: messenger RNA (mRNA); self-amplifying RNA (saRNA); microRNA (miRNA); short interfering RNA (siRNA); short hairpin RNA (shRNA); antisense RNA; RNA aptamers; interfering RNA (RNAi); non-coding RNA; circular RNA; and small RNA.

[0251] Advantageously, incorporation of modified NTPs into non-translated RNA molecules (e.g., miRNA, siRNA, shRNA, antisense RNA, RNA aptamers, RNAi, non-coding RNA, circular RNA or small RNA) increases their stability and reduces their detection by innate immune receptors.

[0252] Preferably, the RNA is self-amplifying RNA (saRNA) or messenger RNA (mRNA).

[0253] The length of the RNA molecule may be as described with respect to the first aspect.

[0254] The RNA molecule (which may be RNAi, saRNA or mRNA) may be at least 20, 21, 22 or 23 bases in length. The RNA molecule may be at least 24, 25, 26 or 27 bases in length. The RNA molecule may be at least 28, 29, 30 or 31 bases in length. The RNA molecule may be at least 32, 33, 34 or 35 bases in length. The RNA molecule may be at least 36, 37, 38 or 39 bases in length. The RNA molecule may be at least 40, 41, 42, 43, 44 or 45 bases in length. The RNA molecule may be at least 46, 47, 48 or 49 bases in length.

[0255] The RNA molecule (which can be RNAi, saRNA or mRNA) can be at least 50 bases in length, at least 60 bases in length, at least 75 bases in length, at least 100 bases in length, at least 200 bases in length, at least 300 bases in length, at least 400 bases in length, at least 500 bases in length, at least 600 bases in length, at least 700 bases in length, at least 800 bases in length, or at least 900 bases in length.

[0256] For example, the RNA molecule, most preferably saRNA or mRNA, can be at least 1000 bases in length, at least 2000 bases in length, at least 3000 bases in length, at least 4000 bases in length, at least 5000 bases in length, at least 6000 bases in length, at least 7000 bases in length, at least 8000 bases in length, at least 9000 bases in length, at least 10000 bases in length, at least 11000 bases in length, or at least 12000 bases in length. In one embodiment, the RNA molecule is at least 6000 bases in length. In one embodiment, the RNA, most preferably saRNA or mRNA, is at least 6000 bases in length. In a preferred embodiment, the saRNA is at least 6000 bases in length. The RNA, most preferably saRNA or mRNA, can be 5000 to 20000 bases in length, 6000 to 15000 bases in length, 7000 to 14000 bases in length, 7500 to 13000 bases in length, 8000 to 12000 bases in length, 8500 to 11000 bases in length, 9000 to 10000 bases in length.

[0257] Preferably, one or more NTPs used in the method or use are modified, in that they are not naturally occurring. Preferably, at least 30%, 35% or 40% of the constituent nucleotides in the RNA molecule are modified, wherein the modified nucleotides may include adenine, cytosine, guanine and / or uracil. More preferably, at least 45%, 50% or 55% of the constituent nucleotides in the RNA molecule are modified, wherein the modified nucleotides may include adenine, cytosine, guanine and / or uracil. Further preferably, at least 60%, 65% or 70%% of the constituent nucleotides in the RNA molecule are modified, wherein the modified nucleotides may include adenine, cytosine, guanine and / or uracil. More preferably, at least 75%, 80% or 85% of the constituent nucleotides in the RNA molecule are modified, wherein the modified nucleotides may include adenine, cytosine, guanine and / or uracil. Most preferably, at least 90%, 95%, 96%, 97%, 98%, 99% or 100% of the constituent nucleotides in the RNA molecule are modified, wherein the modified nucleotides may include adenine, cytosine, guanine and / or uracil.

[0258] As described above, the inventors measured the enhancement of RNA translation in terms of luciferase activity, without directly measuring the amount of luciferase protein produced. Therefore, luciferase activity can be considered as a surrogate for RNA translation.

[0259] Suitably, one or more modified NTPs (preferably 2'-O-methyl NTPs) increase the expression and / or translation level of RNA molecules by at least 5 times, 10 times or 2 times compared to the expression and / or translation level that occurs when there is no one or more modified NTPs. More suitably, one or more modified NTPs (preferably 2'-O-methyl NTPs) increase the expression and / or translation level of RNA molecules by at least 5 times, 10 times or 50 times compared to the expression and / or translation level that occurs when there is no one or more modified NTPs. Even more suitably, one or more modified NTPs (preferably 2'-O-methyl NTPs) increase the expression and / or translation level of RNA molecules by at least 75 times, 100 times or 250 times compared to the expression and / or translation level that occurs when there is no one or more modified NTPs. Preferably, the one or more modified NTPs (preferably 2'-O-methyl NTPs) increase the expression and / or translation level of the RNA molecule by at least 500-fold, 750-fold or 1000-fold compared to the expression and / or translation level that occurs in the absence of the one or more modified NTPs. Preferably, the one or more modified NTPs (preferably 2'-O-methyl NTPs) increase the expression and / or translation level of the RNA molecule by at least 2000-fold, 5000-fold or 8000-fold compared to the expression and / or translation level that occurs in the absence of the one or more modified NTPs. Fig.28 As shown, in some embodiments (eg, Gm and Cm of mRNA), some cells surprisingly exhibit 1000 to 8000-fold increased expression.

[0260] The level of expression and / or translation can be measured by the expression of a fluorescent or luminescent protein encoded as a target gene in an RNA molecule (e.g., mRNA or saRNA). For example, a luminescent protein can be a bioluminescent protein, such as a luciferase (e.g., a firefly luciferase protein encoded as a target gene). Therefore, the expression of luciferase can be determined by measuring its enzymatic activity, wherein luciferin is converted into oxyluciferin, and the light emitted can be quantified as relative light units (RLU). The presence of light is proportional to the amount of protein translated. This method has previously been used to assess the effect of pseudouridine incorporation on translation efficiency

[19] .

[0261] Enzymatic incorporation of modified NTPs (preferably 2'-O-methyl NTPs) into RNA has many potential consequences and advantages selected from: altering mRNA stability; preventing innate recognition; reducing RNA degradation; increasing RNA half-life; enhancing recruitment of translation factors and / or ribosomes; and increasing translation.

[0262] Preferably, the method or use enhances protein expression from mRNA, saRNA, circular RNA, or any RNA sequence that can be translated to produce a recombinant protein or peptide.

[0263] In another embodiment, the method or use enhances the stability of RNA (mRNA, miRNA, siRNA, shRNA, antisense RNA, RNA aptamer, self-amplifying RNA (saRNA), interfering RNA (RNAi), non-coding RNA, circular RNA or small RNA). The improved stability provides significant advantages for coding and non-coding biological therapeutic or preventive uses of RNA molecules. Previous studies have shown that the incorporation of 2'-O-methylated NTPs enhances the stability of short RNA oligonucleotides, aptamers and iRNAs [20, 21]. In this regard, 2'-O-methylation increases the resistance of RNA to degradation due to the elimination of hydrogen bonding and nucleophilic properties of 2'-OH. This results in greatly improved stability of 2'-O-methylated RNA against alkaline (even enzymatic) hydrolysis. In addition, 2'-O methylation increases the base pairing strength and stability of alternative RNA conformations

[22] . Although evaluated for short RNAi and aptamers, this has never been done for mRNA or saRNA because there are no methods to incorporate 2'-O-methylated NTPs into RNA sequences larger than >100 bp.

[0264] Incorporation of 2'-O-methylated NTPs into short RNA sequences is achieved by chemical synthesis or in vitro transcription, the latter of which is very inefficient. However, the methods described herein allow the incorporation of modified NTPs (preferably 2'-O-methyl NTPs) of any length (coding or non-coding), thereby improving the stability of the resulting RNA molecules.

[0265] The stability of the RNA molecule can be measured by quantitative PCR, but can also be reflected by the duration of expression, where faster degradation is equivalent to shorter expression. The inventors observed that the duration of expression of saRNA containing 75% 2-O-methyl GTP relative to unmodified GTP was extended to 72 hours (see Figures 9 to 13), and the same was true for mRNA containing 2-O-methyl modified GTP (Gm) plus CTP (Cm) (see Figures 25 to 28 ).

[0266] In another embodiment, the method or use results in a reduction or elimination of innate sensing of RNA by membrane, endosomal and cellular RNA binding proteins that recognize unmodified RNA structures that trigger interferon signaling pathways, and activation of cytoplasmic nucleases (such as RNase L) that degrade RNA

[23] . Reduction of innate sensing of RNA (mRNA, miRNA, siRNA, shRNA, antisense RNA, RNA aptamers, self-amplifying RNA (saRNA), interfering RNA (RNAi), non-coding RNA, circular RNA and small RNA) will provide significant advantages for coding and non-coding biological therapeutic or preventive uses of RNA. Previous studies have shown that the incorporation of 2'-O-methylated NTPs affects innate recognition of short RNA oligonucleotides, aptamers and iRNAs, interferon induction and degradation of related RNAs [24, 25, 26, 27]. However, mRNA or saRNA has not been evaluated as a method for incorporating 2'-O-methylated NTPs in RNA sequences greater than >100 bp. The incorporation of 2'-O-methylated NTPs into short RNA sequences is achieved by chemical synthesis or in vitro transcription, the latter of which is very inefficient. The method presented here allows the incorporation of 2'-O-methylated NTPs of any length to prevent innate RNA recognition, interferon induction, and RNA degradation.

[0267] The activation of innate perception that reduces the degradation of RNA molecules is inferred by improving the expression of modified saRNA and mRNA in interferon competent cells (HeLa and THP-1 cells). Although the incorporation of 2-O-methyl modified bases in HEK293T cells provides some improvements, these cells have impaired innate perception mechanisms, and therefore, any improvement in expression is directly related to the improvement of RNA half-life in the absence of innate recognition. If the expression of unmodified saRNA and mRNA in both HeLa and THP-1 cells is greatly reduced, it reflects the enhancement of degradation caused by the triggering of innate perception mechanisms. Compared with that observed in HEK293T cells, the incorporation of 2'-O-methylated NTPs in saRNA and mRNA provides a more significant increase in expression (fold change) in these two interferon-competent cell types. The induction of type I interferon expression can be measured by ELISA as a surrogate for innate activation.

[0268] It should be understood that the composition, pharmaceutical composition, vaccine or therapeutic RNA molecule can be used in medicine, which can be used as a monotherapy (i.e., the composition is used alone). Alternatively, the composition according to the present invention can be used as an adjuvant or in combination with known therapies for treating, ameliorating or preventing infection or disease.

[0269] The composition of the present invention can be combined in a composition with a variety of different forms, depending on the use mode of the composition. Therefore, for example, the composition can be a powder, tablet, capsule, liquid, ointment, cream, gel, hydrogel, aerosol, spray, micellar solution, transdermal patch, liposome suspension, polymer, emulsion, liposome, lipid nanoparticle, functionalized liposome or lipid nanoparticle (for example, with or encapsulated with peptide, antibody, antibody fragment, polysaccharide, glycoconjugate, DNA or RNA on the surface) form, or any other suitable form that can be applied to humans or animals. The lipid nanoparticle can include one or more components selected from the following: cationic lipids (preferably ionizable); phosphatidylcholine; cholesterol; and polyethylene glycol (PEG)-lipid. It should be understood that the drug carrier according to the present invention should be a drug carrier that gives good tolerance to the subject.

[0270] Drugs including compositions, pharmaceutical compositions or vaccines of the present invention can be used in a variety of ways. For example, oral administration may be desired, in which case the agent may be included in the composition and may, for example, be taken orally in the form of a tablet, capsule or liquid. Compositions including agents and drugs of the present invention may be administered by inhalation (e.g., intranasally). Compositions may also be formulated for topical use. For example, creams or ointments may be applied to the skin. Preparations may be provided in lyophilized form for reconstitution or as solid dosage forms for topical, insertion or injection delivery.

[0271] The compositions, pharmaceutical compositions, vaccines or therapeutic RNA molecules of the present invention may also be incorporated into a slow release or delayed release device. Such a device may, for example, be inserted on or under the skin, and the drug may be released over a period of weeks or even months. The device may be located at least near the treatment site.

[0272] However, in a preferred embodiment, the drug according to the present invention can be administered to a subject by injection into the bloodstream, muscle, skin or directly into the site in need of treatment. The injection can be intravenous (bolus or infusion), subcutaneous (bolus or infusion), intradermal (bolus or infusion), intramuscular (bolus or infusion), intrathecal (bolus or infusion), epidural (bolus or infusion) or intraperitoneal (bolus or infusion).

[0273] It will be appreciated that the amount of a composition, pharmaceutical composition or vaccine or therapeutic RNA molecule required is determined by its biological activity and bioavailability, which in turn depends on the mode of administration, the physiochemical properties of the composition, pharmaceutical composition or vaccine and whether it is used as a monotherapy or in combination therapy.

[0274] The frequency of administration will also be affected by the half-life of the active agent in the subject being treated. The optimal dose for administration can be determined by those skilled in the art and will vary with the composition, pharmaceutical composition or vaccine used, the strength of the pharmaceutical composition, the mode of administration, and the type of treatment. Other factors depending on the specific subject being treated will result in the need to adjust the dose, including subject age, weight, sex, diet, and time of administration.

[0275] The desired dosage may depend on a variety of factors including, but not limited to, the active agent being administered, the disease being treated and / or vaccinated, the subject being treated, and the like.

[0276] Typically, depending on the active agent used, the composition, pharmaceutical composition or vaccine or therapeutic RNA molecule of the present invention may be used at a dose of 0.001 μg / kg body weight to 10 mg / kg body weight, or 0.01 μg / kg body weight to 1 mg / kg body weight. The dose may be understood to be related to the amount of RNA molecule delivered.

[0277] The dosage can be given as a single administration (e.g., a single injection). Alternatively, the composition, pharmaceutical composition, biotherapeutic RNA molecule or vaccine may require more than one administration. As an example, the composition, pharmaceutical composition or vaccine or therapeutic RNA molecule may be administered as an initial primer and subsequent enhancer, or two enhancers (e.g., two or more doses between 0.07 μg and 700 mg, i.e., assuming a body weight of 70 kg) are administered at weekly or monthly intervals, or a biotherapeutic dose is administered repeatedly every day, every week or every month. Alternatively, a sustained-release device can be used to provide the patient with an optimal dose of the composition, pharmaceutical composition or vaccine or therapeutic RNA molecule of the present invention without the need for repeated administration of the dose. The route of administration may involve intravenous, intradermal, subcutaneous, intramuscular, intrathecal, epidural or intraperitoneal injection routes.

[0278] Known procedures, such as those routinely employed by the pharmaceutical industry (e.g., in vivo experiments, clinical trials, etc.), can be used to formulate specific formulations and precise treatment regimens (e.g., dosages and frequency of administration of the agents) of the compositions, pharmaceutical compositions or vaccines or therapeutic RNA molecules according to the present invention.

[0279] "Subject" can be a vertebrate, mammal or livestock. Therefore, the compositions and medicaments according to the present invention can be used to treat any mammal, such as livestock (such as horses), pets, or can be used for other veterinary applications. However, most preferably, the subject is a human.

[0280] A "therapeutically effective amount" of the composition, pharmaceutical composition or vaccine or therapeutic RNA molecule is any amount of the above amount required to produce a therapeutic effect when administered to a subject.

[0281] For example, a therapeutically effective amount of the compositions, pharmaceutical compositions, and vaccines or therapeutic RNA molecules of the invention may contain about 0.0001 mg to about 800 mg of the payload molecule, preferably 0.001 mg to about 650 mg, and preferably about 0.01 mg to about 500 mg of the payload molecule.

[0282] The "pharmaceutically acceptable carrier" referred to herein is any known compound or combination of known compounds known to those skilled in the art that can be used to formulate a pharmaceutical composition.

[0283] In one embodiment, the pharmaceutically acceptable carrier can be solid, and the composition can be in the form of powder or tablet. The solid pharmaceutically acceptable carrier can include one or more substances, which can also be used as flavorings, lubricants, solubilizers, suspending agents, dyes, fillers, glidants, compression aids, inert adhesives, sweeteners, preservatives, dyes, coatings or tablet disintegrating agents. The carrier can also be an encapsulating material. In powder, the carrier is a finely ground solid mixed with a finely ground active agent according to the present invention. In tablets, active agents (for example, compositions of the present invention) can be mixed with a carrier with necessary compression properties in a suitable ratio, and compacted in the desired shape and size. The drug carrier can be a gel, and the composition can be in the form of an emulsifiable paste.

[0284] However, the pharmaceutical carrier can be a liquid, and the pharmaceutical composition is in the form of a solution. Liquid carriers are used to prepare solutions, suspensions, emulsions, syrups, ELISA agents and pressurized compositions. Compositions according to the present invention can be dissolved or suspended in a pharmaceutically acceptable liquid carrier, such as water, an organic solvent, a mixture of the two, or a pharmaceutically acceptable oil or fat. Liquid carriers can contain other suitable pharmaceutical additives, such as solubilizers, emulsifiers, buffers, preservatives, sweeteners, flavoring agents, suspending agents, thickeners, colorants, viscosity modifiers, stabilizers or osmotic regulators. Suitable examples of liquid carriers for oral and parenteral administration include water (partially containing additives as described above, such as cellulose derivatives, preferably sodium carboxymethylcellulose solution), alcohol (including monohydric alcohols and polyols, such as glycols) and derivatives thereof and oils (such as fractionated coconut oil and peanut oil). For parenteral administration, carriers can also be oily esters, such as ethyl oleate and isopropyl myristate. Sterile liquid carriers can be used in sterile liquid form compositions for parenteral administration. The liquid carrier for pressurized compositions can be halogenated hydrocarbon or other pharmaceutically acceptable propellant.

[0285] Liquid pharmaceutical compositions which are sterile solutions or suspensions can be used by, for example, intramuscular, intrathecal, epidural, intraperitoneal, intravenous and subcutaneous injection. The compositions of the present invention can be prepared in any suitable sterile injectable medium.

[0286] The composition and / or pharmaceutical composition of the present invention can be orally administered in the form of a sterile solution or suspension containing other solutes or suspending agents (e.g., saline or glucose sufficient to make the solution isotonic), bile salts, gum arabic, gelatin, sorbitan monooleate, polysorbate 80 (oleate copolymerized with sorbitol and its anhydride and ethylene oxide), etc. The composition and / or pharmaceutical composition of the present invention can also be orally administered in the form of a liquid or solid composition. Compositions suitable for oral administration include solid forms such as pills, capsules, granules, tablets and powders, and liquid forms such as solutions, syrups, ELISA agents and suspensions. Forms that can be used for parenteral administration include sterile solutions, emulsions and suspensions.

[0287] All features described herein (including any accompanying claims, abstract and drawings) and / or all steps of any method or process so disclosed may be combined with any of the above-described aspects in any combination, except combinations where at least some of these features and / or steps are mutually exclusive. BRIEF DESCRIPTION OF THE DRAWINGS

[0288] For a better understanding of the invention, and in order to show how embodiments of the invention may be implemented, reference will now be made, by way of example, to the accompanying drawings, in which:

[0289] Figure 1 It is shown that when magnesium acetate is used, sufficient incorporation of 2' methyl ribose modified NTPs occurs only at magnesium levels >20 nM. A) RNA yield ng / ul, B) RNA product assessed by gel electrophoresis.

[0290] Figure 2 Shows low or no incorporation of 2' methyl ribose modified GTP when magnesium chloride is used. A) RNA yield in ng / ul, B) RNA product assessed by gel electrophoresis.

[0291] Figure 3 This indicates that when magnesium acetate was used instead of manganese acetate, no detectable incorporation of 2'methylribose-modified GTP was observed. A) RNA yield in ng / ul, B) RNA product assessed by gel electrophoresis.

[0292] Figure 4 illustrates the effect of 2'-O-methyl NTP substitution on uncapped saRNA expression of fLuc. Uncapped saRNA encoding fLuc was produced by IVT, in which single nucleotides were replaced by 25%, 50% or 75% 2-O-methyl modified forms. HEK293T cells were transfected with 25ng of saRNA formulated in lipofectamine. A) The expression of luciferase was monitored over 72 hours by measurement of relative light units (see Methods). B) Fold change in expression relative to unmodified saRNA.

[0293] Figure 5 shows the effect of 2'-O-methyl NTP substitution on uncapped saRNA expression of fLuc. Uncapped saRNA encoding fLuc was produced by IVT, in which single nucleotides were replaced by 25%, 50% or 75% 2-O-methyl modified forms. HEK293T cells were transfected with 100ng of saRNA formulated in lipofectamine. A) The expression of luciferase was monitored over 72 hours by measurement of relative light units (see Methods). B) Fold change in expression relative to unmodified saRNA.

[0294] Figure 6 illustrates the effect of 2'-O-methyl NTP substitution on the expression of 5'-capped saRNA of fLuc. 5'-capped saRNA encoding fLuc was produced by IVT, in which a single nucleotide was replaced by 25%, 50% or 75% of 2-O-methyl modified forms. HEK293T cells were transfected with 25ng of saRNA formulated in lipofectamine. A) The expression of luciferase was monitored over 72 hours by measurement of relative light units (see Methods). B) Fold change in expression relative to unmodified saRNA.

[0295] Figure 7 illustrates the effect of 2'-O-methyl NTP substitution on the expression of 5' capped saRNA of fLuc. Capped saRNA encoding fLuc was produced by IVT, in which a single nucleotide was replaced by 25%, 50% or 75% of 2-O-methyl modified forms. HEK293T cells were transfected with 100ng of saRNA formulated in lipofectamine. A) The expression of luciferase was monitored over 72 hours by measurement of relative light units (see methods). B) Fold change in expression relative to unmodified saRNA.

[0296] Figure 8 shows the effect of 2'-O-methyl NTP substitution on uncapped saRNA expression of fLuc in HeLa cells. Uncapped saRNA encoding fLuc was produced by IVT, in which single nucleotides were replaced by 25%, 50% or 75% 2-O-methyl modified forms. HeLa cells were transfected with 25ng of saRNA formulated in lipofectamine. A) The expression of luciferase was monitored over 72 hours by measurement of relative light units (see Methods). B) Fold change in expression relative to unmodified saRNA.

[0297] Figure 9 illustrates the effect of 2'-O-methyl NTP substitution on uncapped saRNA expression of fLuc in HeLa cells. Uncapped saRNA encoding fLuc was produced by IVT in which single nucleotides were substituted with 25%, 50%, or 75% 2-O-methyl modified forms. HeLa cells were transfected with 100 ng of saRNA formulated in lipofectamine. A) Luciferase expression was monitored over 72 hours by measurement of relative light units (see methods). B) Fold change in expression relative to unmodified saRNA.

[0298] Figure 10 illustrates the effect of 2'-O-methyl NTP substitution on 5'-capped saRNA expression of fLuc in HeLa cells. 5'-capped saRNA encoding fLuc was produced by IVT, in which a single nucleotide was replaced by 25%, 50% or 75% of the 2-O-methyl modified form. HeLa cells were transfected with 25ng of saRNA formulated in lipofectamine. A) The expression of luciferase was monitored over 72 hours by measurement of relative light units (see methods). B) Fold change in expression relative to unmodified saRNA.

[0299] Figure 11 illustrates the effect of 2'-O-methyl NTP substitution on 5'-capped saRNA expression of fLuc in HeLa cells. 5'-capped saRNA encoding fLuc was produced by IVT, in which a single nucleotide was replaced by 25%, 50% or 75% of the 2-O-methyl modified form. HeLa cells were transfected with 100ng of saRNA formulated in lipofectamine. A) The expression of luciferase was monitored over 72 hours by measurement of relative light units (see methods). B) Fold change in expression relative to unmodified saRNA.

[0300] Figure 12 shows the effect of 2'-O-methyl NTP substitution on uncapped saRNA expression of fLuc in THP-1 cells. Uncapped saRNA encoding fLuc was produced by IVT, in which a single nucleotide was replaced by 25%, 50% or 75% of 2-O-methyl modified forms. THP-1 cells were transfected with 25ng of uncapped saRNA formulated in lipofectamine. A) The expression of luciferase was monitored over 72 hours by measurement of relative light units (see Methods). B) Fold change in expression relative to unmodified saRNA.

[0301] Figure 13 shows the effect of 2'-O-methyl NTP substitution on uncapped saRNA expression of fLuc in THP-1 cells. Uncapped saRNA encoding fLuc was produced by IVT, in which a single nucleotide was replaced by 25%, 50% or 75% of 2-O-methyl modified forms. THP-1 cells were transfected with 100ng of uncapped saRNA formulated in lipofectamine. A) The expression of luciferase was monitored over 72 hours by measurement of relative light units (see Methods). B) Fold change in expression relative to unmodified saRNA.

[0302] Figure 14 shows the effect of 2'-O-methyl NTP substitution on the expression of 5'-capped saRNA of fLuc in THP-1 cells. 5'-capped saRNA encoding fLuc was produced by IVT, in which a single nucleotide was replaced by 25%, 50% or 75% of 2-O-methyl modified forms. THP-1 cells were transfected with 25ng of uncapped saRNA formulated in lipofectamine. A) The expression of luciferase was monitored over 72 hours by measurement of relative light units (see Methods). B) Fold change in expression relative to unmodified saRNA.

[0303] Figure 15 shows the effect of 2'-O-methyl NTP substitution on the expression of 5'-capped saRNA of fLuc in THP-1 cells. 5'-capped saRNA encoding fLuc was produced by IVT, in which a single nucleotide was replaced by 25%, 50% or 75% of 2-O-methyl modified forms. THP-1 cells were transfected with 25ng of uncapped saRNA formulated in lipofectamine. A) The expression of luciferase was monitored over 72 hours by measurement of relative light units (see Methods). B) Fold change in expression relative to unmodified saRNA.

[0304] Fig.16 Shown is the effect of replacing uncapped and 5'-capped saRNA expression of fLuc with N1 methyl pseudouridine in A) HEK293T cells and B) HeLa cells. Uncapped and 5'-capped saRNA encoding fLuc were produced by IVT, in which the unmodified UTP was replaced by 25%, 50% or 75% N1 methyl pseudouridine. Cells were transfected with 100 ng of uncapped or capped saRNA formulated in lipofectamine and the expression of luciferase was monitored over 72 hours by measurement of relative light units (see Methods).

[0305] Fig.17Shown is the effect of replacing uncapped and 5'-capped saRNA expression of fLuc with 7-deaza-GTP in A) HEK293T cells and B) HeLa cells. Uncapped and 5'-capped saRNA encoding fLuc were produced by IVT, in which unmodified GTP was replaced by 25%, 50% or 75% 7-deaza-GTP. Cells were transfected with 100 ng of uncapped or capped saRNA formulated in lipofectamine and the expression of luciferase was monitored over 72 hours by measurement of relative light units (see Methods).

[0306] Fig.18 The effect of uncapped and 5'-capped saRNA expression of 2'-fluoro substitution fLuc in HEK293T cells is shown. Uncapped and 5'-capped saRNA encoding fLuc were generated by IVT, in which the unmodified bases were replaced by 25%, 50% or 75% 2' fluoro-ATP, CTP or UTP. Cells were transfected with 100ng of uncapped or capped saRNA formulated in lipofectamine, and the expression of luciferase was monitored within 72 hours by measurement of relative light units (see Methods).

[0307] Fig.19 The effect of uncapped and 5'-capped saRNA expression of 2'-fluoro substitution of fLuc in HeLa cells is shown. Uncapped and 5'-capped saRNA encoding fLuc were generated by IVT, in which the unmodified bases were replaced by 25%, 50% or 75% 2' fluoro-ATP, CTP or UTP. Cells were transfected with 100ng of uncapped or capped saRNA formulated in lipofectamine and the expression of luciferase was monitored over 72 hours by measurement of relative light units (see Methods).

[0308] Fig. 20 The effect of 2'-O-methyl NTP substitution on the expression of 5'-capped saRNA and mRNA of fLuc in HEK293T cells is shown. 5'-capped saRNA and mRNA encoding fLuc were generated by IVT. The expression of unmodified saRNA and mRNA was compared with saRNA and mRNA in which 75% of GTP was replaced by 75% of 2'-O-methyl GTP form. In addition, "combined" mRNA was produced by replacing 75% of GTP and ATP and 90% of CTP and UTP with 2'-O-methyl form. HEK293T cells were transfected with 100ng of 5'-capped saRNA and mRNA formulated in lipofectamine. A). The expression of luciferase was monitored over 72 hours by measurement of relative light units. B) Fold change in expression relative to unmodified saRNA.

[0309] Fig.21 The effect of 2'-O-methyl NTP substitution on the expression of 5'-capped saRNA and mRNA of fLuc in HeLa cells is shown. 5'-capped saRNA and mRNA encoding fLuc were generated by IVT. The expression of unmodified saRNA and mRNA was compared with saRNA and mRNA in which 75% of GTP was replaced by 75% of 2'-O-methyl GTP form. In addition, "combined" mRNA was produced by replacing 75% of GTP and ATP and 90% of CTP and UTP with 2'-O-methyl form. Hela cells were transfected with 100ng of 5'-capped saRNA and mRNA formulated in lipofectamine. A). The expression of luciferase was monitored over 72 hours by measurement of relative light units. B) Fold change in expression relative to unmodified saRNA.

[0310] Fig. 22 The effect of 2'-O-methyl NTP substitution on the expression of 5'-capped saRNA and mRNA of fLuc in THP-1 cells is shown. 5'-capped saRNA and mRNA encoding fLuc were generated by IVT. The expression of unmodified saRNA and mRNA was compared with saRNA and mRNA in which 75% of GTP was replaced by 75% of 2'-O-methyl GTP form. In addition, "combined" mRNA was generated by replacing 75% of GTP and ATP and 90% of CTP and UTP with 2'-O-methyl form. THP-1 cells were transfected with 100ng of 5'-capped saRNA and mRNA formulated in lipofectamine. A). The expression of luciferase was monitored over 72 hours by measurement of relative light units. B) Fold change in expression relative to unmodified saRNA.

[0311] Fig.23 The effect of 2'-O-methyl NTP substitution on capped mRNA expression of fLuc in HEK293T cells is shown. Capped mRNA encoding fLuc was produced by IVT in which nucleotides were substituted with ≥75% 2'-O-methyl modified forms. The combined condition was that all four NTPs were substituted. HEK293T cells were transfected with 25ng of capped mRNA formulated in lipofectamine. Luciferase expression was monitored over 72 hours by measurement of relative light units (see methods).

[0312] Fig.24The effect of 2'-O-methyl NTP substitution on capped mRNA expression of fLuc in HEK293T cells is shown. Capped mRNA encoding fLuc was produced by IVT in which nucleotides were substituted with ≥75% 2'-O-methyl modified forms. The combined condition was that all four NTPs were substituted. HEK293T cells were transfected with 25ng of capped mRNA formulated in lipofectamine. The expression of luciferase was monitored over 72 hours by measurement of relative light units and expressed as fold change relative to the expression of unmodified saRNA.

[0313] Fig.25 The effect of 2'-O-methyl NTP substitution on capped mRNA expression of fLuc in HeLa cells is shown. Capped mRNA encoding fLuc was generated by IVT in which nucleotides were substituted with ≥75% 2'-O-methyl modified forms. The combined condition was that all four NTPs were substituted. HeLa cells were transfected with 25 ng of capped mRNA formulated in lipofectamine. Luciferase expression was monitored over 72 hours by measurement of relative light units (see methods).

[0314] Fig.26 The effect of 2'-O-methyl NTP substitution on capped mRNA expression of fLuc in HeLa cells is shown. Capped mRNA encoding fLuc was generated by IVT in which nucleotides were substituted with ≥75% 2'-O-methyl modified forms. The combined condition was that all four NTPs were substituted. HeLa cells were transfected with 25ng of capped mRNA formulated in lipofectamine. Luciferase expression was monitored over 72 hours by measurement of relative light units and expressed as fold change relative to expression of unmodified saRNA.

[0315] Fig. 27 The effect of 2'-O-methyl NTP substitution on capped mRNA expression of fLuc in THP-1 cells is shown. Capped mRNA encoding fLuc was generated by IVT in which nucleotides were substituted with ≥75% 2'-O-methyl modified forms. The combined condition was that all four NTPs were substituted. THP-1 cells were transfected with 25 ng of capped mRNA formulated in lipofectamine. Luciferase expression was monitored over 72 hours by measurement of relative light units (see methods).

[0316] Fig.28The effect of 2'-O-methyl NTP substitution on capped mRNA expression of fLuc in THP-1 cells is shown. Capped mRNA encoding fLuc was generated by IVT in which nucleotides were substituted with ≥75% 2'-O-methyl modified forms. The combined condition was that all four NTPs were substituted. THP-1 cells were transfected with 25ng of capped mRNA formulated in lipofectamine. The expression of luciferase was monitored over 72 hours by measurement of relative light units and expressed as fold change relative to the expression of unmodified saRNA.

[0317] Fig.29 The effect of 2'-O-methyl NTP substitution on capped mRNA expression of fLuc in HEK293T cells is shown. Capped mRNA encoding fLuc was generated by IVT in which individual nucleotides were substituted with ≥75% 2'-O-methyl modified forms. The combined condition was that all four NTPs were substituted. HEK293T cells were transfected with 25 ng of capped mRNA formulated in lipofectamine. Luciferase expression was monitored over 72 hours by measurement of relative light units (see methods).

[0318] Fig.30 The effect of 2'-O-methyl NTP substitution on capped mRNA expression of fLuc in HEK293T cells is shown. Capped mRNA encoding fLuc was generated by IVT in which individual nucleotides were substituted with ≥75% 2'-O-methyl modified forms. The combined condition was that all four NTPs were substituted. HEK293T cells were transfected with 100 ng of capped mRNA formulated in lipofectamine. Luciferase expression was monitored over 72 hours by measurement of relative light units (see Methods).

[0319] Fig.31 The effect of 2'-O-methyl NTP substitution on capped mRNA expression of fLuc in HeLa cells is shown. Capped mRNA encoding fLuc was generated by IVT in which individual nucleotides were substituted with ≥75% 2'-O-methyl modified forms. The combined condition was that all four NTPs were substituted. HeLa cells were transfected with 25 ng of capped mRNA formulated in lipofectamine. Luciferase expression was monitored over 72 hours by measurement of relative light units (see methods).

[0320] Fig.32The effect of 2'-O-methyl NTP substitution on capped mRNA expression of fLuc in HeLa cells is shown. Capped mRNA encoding fLuc was generated by IVT in which individual nucleotides were substituted with ≥75% 2'-O-methyl modified forms. The combined condition was that all four NTPs were substituted. HeLa cells were transfected with 100 ng of capped mRNA formulated in lipofectamine. Luciferase expression was monitored over 72 hours by measurement of relative light units (see Methods).

[0321] Fig.34 The effect of 2'-O-methyl NTP substitution on capped mRNA expression of fLuc in THP-1 cells is shown. Capped mRNA encoding fLuc was produced by IVT in which a single nucleotide was substituted with ≥75% of 2'-O-methyl modified forms. The combined condition was that all four NTPs were substituted. THP-1 cells were transfected with 25ng of capped mRNA formulated in lipofectamine. The expression of luciferase was monitored over 72 hours by measurement of relative light units (see methods) and expressed as a fold change relative to the expression of unmodified saRNA.

[0322] Fig.35 The effect of 2'-O-methyl NTP substitution on capped mRNA expression of fLuc in THP-1 cells is shown. Capped mRNA encoding fLuc was generated by IVT in which individual nucleotides were substituted with ≥75% 2'-O-methyl modified forms. The combined condition was that all four NTPs were substituted. THP-1 cells were transfected with 100 ng of capped mRNA formulated in lipofectamine. Luciferase expression was monitored over 72 hours by measurement of relative light units (see methods).

[0323] Fig.36 Effects of 2'-O-methyl NTP substitutions on capped mRNA expression of fLuc in THP-1 cells are shown. Capped mRNA encoding fLuc was generated by IVT in which individual nucleotides were substituted with ≥75% 2'-O-methyl modified forms. The combined condition was that all four NTPs were substituted. THP-1 cells were transfected with 100 ng of capped mRNA formulated in lipofectamine. Luciferase expression was monitored over 72 hours by measurement of relative light units, shown as fold change relative to unmodified mRNA.

[0324] Fig.37 is a table showing the structures of ATP, CTP, GTP and UTP as unmodified bases, or 2-O-methyl NTP. Also shown are N1-methyl-pseudo-UTP, 7-deaza-GTP, 2'fluoro-NTPs, including 2'fluoro-dTTP.

[0325] Fig.38 is a table showing the structures of ATP, CTP, GTP, and UTP as 2-deoxyNTPs, and also includes dTTP.

[0326] Fig.39 The structures of additional modified NTPs are shown.

[0327] Figure 40 shows a wide range of alternative modified NTPs that provide no benefit or reduce expression when incorporated into saRNA and evaluated for expression of luciferase in A) HEK293T, B) HeLa or C) THP-1 cells. Cells were transfected with 25ng or 100ng of uncapped (pre-capped) or capped saRNA formulated in lipofectamine. Expression of luciferase was monitored over 24 hours by measurement of relative light units, shown as fold change relative to unmodified mRNA.

[0328] Fig.41 The effect of 2'-O-methyl GTP substitution on the expression of capped saRNA of eGFP is shown. IVT uses unmodified NTP to produce capped saRNA encoding eGFP, wherein GTP is replaced by ≥75% 2'-O-methyl modified NTP, and wherein UTP is replaced by 100% N-1-methyl pseudouridine. Unmodified and modified VEEV-eGFP saRNA were transfected three times with 1 μg in 24-well plates of (A) HELA and (B) THP1 cells (error bars represent n=3). Cells were collected and stained after 24 hours of transfection, and eGFP expression was then assessed by flow cytometry. The data show the percentage (%) of cells expressing eGFP (left vertical axis) and medium fluorescence intensity (right vertical axis).

[0329] Fig.42 The effects of single and combined 2'-O-methyl NTP substitutions on capped mRNA expression of fLuc in THP-1 cells are shown. Capped mRNA encoding fLuc was generated by IVT in which nucleotides were substituted with ≥75% 2'-O-methyl GTP, ≥75% 2'-O-methyl ATP, 90% 2'-O-methyl CTP, or 90% 2'-O-methyl UTP, either alone or in different combinations. Substitution of all four NTPs is represented by 2'-O-methyl combinations. THP-1 cells were transfected with 25 ng of capped mRNA formulated in lipofectamine. Luciferase expression was monitored over 48 hours by measurement of relative light units (see Methods).

[0330] Figure 43 shows the effect of combined 2'-O-methyl NTP substitutions on capped mRNA expression of fLuc in HEK293T cells. Conditions 1 to 26 are detailed in Table 6. Capped mRNA encoding fLuc was produced by IVT with nucleotides substituted according to Table 6. HEK293T cells were transfected with 25ng and 100ng of capped mRNA formulated in lipofectamine. A) Expression of luciferase was monitored over 72 hours by measurement of relative light units (see methods). B) Fold change in expression relative to unmodified mRNA.

[0331] Figure 44 shows the effect of combined 2'-O-methyl NTP substitutions on capped mRNA expression of fLuc in HeLa cells. Conditions 1 to 26 are detailed in Table 6. Capped mRNA encoding fLuc was generated by IVT, with nucleotides substituted according to Table 6. HeLa cells were transfected with 25ng and 100ng of capped mRNA formulated in lipofectamine. A) Expression of luciferase was monitored over 72 hours by measurement of relative light units (see Methods). B) Fold change in expression relative to unmodified mRNA.

[0332] Figure 45 shows the effect of combined 2'-O-methyl NTP substitutions on capped mRNA expression of fLuc in THP-1 cells. Conditions 1 to 26 are detailed in Table 6. Capped mRNA encoding fLuc was generated by IVT, with nucleotides substituted according to Table 6. THP-1 cells were transfected with 25ng and 100ng of capped mRNA formulated in lipofectamine. A) Expression of luciferase was monitored over 72 hours by measurement of relative light units (see Methods). B) Fold change in expression relative to unmodified mRNA.

[0333] Figure 46 shows the effect of 2'-O-methyl GTP substitution on the inflammatory properties of saRNA in THP-1 cells. IVT uses unmodified NTP to produce capped saRNA encoding firefly luciferase, wherein GTP is replaced by ≥75% 2'-O-methyl modified NTP, and wherein UTP is replaced by 100% N-1-methyl pseudouridine. Unmodified and modified saRNA were transfected three times with 1 μg in a 24-well plate of THP1 cells (error bars represent n=3). (A) Half of the cell supernatant was collected 24 hours and 48 hours after transfection before performing a luciferase assay to measure antigen expression. (B) The cell supernatant from each time point was then used for MSD assays to determine the different levels of cytokines stimulated by each RNA construct.

[0334] Fig.47The effect of 2'-O-methyl substitution on the expression of capped mRNA of eGFP is shown. Capped mRNA encoding eGFP was generated by IVT using unmodified NTPs, where UTP was replaced by 100% N-1 methyl pseudouridine or where NTP was replaced by 2'-O-methyl modified NTPs (75% Gm, 75% Am, 90% Cm, 90% Um) and labeled as 2'-O-methyl combinations. Unmodified and modified mRNA were transfected three times at 1 μg in 24-well plates of (A) HELA and (B) THP1 cells (error bars represent n=3). Cells were collected and stained 24 hours after transfection, and then eGFP expression was evaluated by flow cytometry. The data show the percentage (%) of cells expressing eGFP (left vertical axis) and medium fluorescence intensity (right vertical axis).

[0335] Figure 48 shows the effect of 2'-O-methyl substitutions on the inflammatory properties of mRNA in THP-1 cells. Capped mRNA encoding firefly luciferase was generated by IVT using unmodified NTPs, where N1-methyl pseudouridine was replaced with UTP or where WT NTPs were replaced with 2'-O-methyl modified NTPs (75% Gm, 75% Am, 90% Cm, 90% Um) and labeled as 2'-O-methyl combinations. Unmodified and modified mRNAs were transfected three times at 1 μg in a 24-well plate of THP1 cells (error bars represent n=3). (A) Half of the cell supernatant was collected 24 hours and 48 hours after transfection before performing a luciferase assay to measure antigen expression. (B) The cell supernatant from each time point was then used for MSD assays to determine the different levels of cytokines stimulated by each RNA construct.

[0336] Figure 49 shows the effect of 2'-O-methyl GTP substitution on in vivo saRNA expression. IVT uses unmodified NTP to produce capped saRNA encoding firefly luciferase, in which GTP is replaced by ≥75% 2'-O-methyl modified NTP. saRNA was formulated in lipid nanoparticles consisting of C12-200 ionizable lipids with DSPC, cholesterol, and DMPE-PEG200. Five female Balb / c mice (n=5 per group) were administered 10 μg LNP-formulated unmodified or modified VEEV-fLucsaRNA IM. (A) Mice were imaged using the IVIS Spectrum in vivo imaging system on the first and fifth days. (B) Luciferase expression was quantified as photons per second using Aura Imaging software. P=0.0005

[0337] Figure 50 shows the effect of 2'-O-methyl GTP substitution on mRNA expression in vivo. Capped mRNA encoding firefly luciferase was generated by IVT using unmodified NTPs, where N1-methyl pseudouridine was replaced with UTP or where WT NTP was replaced with 2'-O-methyl modified NTPs (75% Gm, 75% Am, 90% Cm, 90% Um) and labeled as 2'-O-methyl combinations. mRNA was formulated in lipid nanoparticles composed of C12-200 ionizable lipids with DSPC, cholesterol and DMPE-PEG200. Five female Balb / c mice (n=5 per group) were given 8μg LNP formulated unmodified or modified mRNA IM. (A) Mice were imaged using the IVIS Spectrum in vivo imaging system on days 1 to 3. (B) Luciferase expression was quantified as photons per second using Aura Imaging software. P<0.001.

[0338] Fig.51 2'-O-methyl-GTP modified saRNAs showed enhanced immunogenicity in vivo. VEEV saRNA encoding influenza hemagglutinin protein (VEEV-Ha) downstream of a subgenomic promoter was generated by IVT: unmodified NTPs were used; GTP was replaced by

[0339] ≥75% 2'-O-methyl modified NTP substitution; or wherein UTP is substituted with 100% N-1 methyl pseudouridine. Four groups of female Balb / c mice (n=5 per group) were administered IM with 10 μg of unmodified or modified VEEV-HA saRNA formulated in LNPs, where the mice were bled and immunized at weeks zero and four, and then bled again at week six. (A) Total HA-specific IgG was quantified by ELISA from mouse sera collected at weeks four and six, and (B) neutralizing antibody responses were quantified by neutralization assay with California influenza virus 2009 H1N1 virus. ***Two-way ANOVA-Tukey multiple comparison test P<0.0001. DETAILED DESCRIPTION

[0340] Example

[0341] Materials and methods

[0342] In vitro transcription (IVT)

[0343] The linearized DNA template was used to generate saRNA and mRNA encoding fLuc by in vitro transcription (IVT). The final volume of each IVT reaction was 50uL, and the standardized IVT reaction mixture was as follows:

[0344] Table 1 - Standardized IVT Reaction Mixture

[0345]

[0346]

[0347] Therein, the concentration of magnesium acetate (Mg(OAc)2) was varied, or replaced with magnesium chloride or manganese acetate. The total concentration of each NTP was maintained at 10 nM, however, the relative ratio of unmodified NTP to modified NTP was varied as shown. For example, where 75% GTP was described to be replaced by 2'O-methyl-GTP, this would indicate that the reaction contained 7.5 mM 2'-O-methyl-GTP plus 2.5 mM GTP. The IVT reaction was performed at 37°C for 4 hours. RNA yield was then measured immediately after IVT using the Qubit RNA Broad Range Assay Kit with a Qubit Fluorometer (Thermo Fisher, UK) according to the manufacturer's protocol. RNA was purified using lithium chloride precipitation prior to post-capping. ScriptCap TM Cap 1 end-capping system (CellScript, UK) performs RNA end-capping, and the reaction is incubated at 37 ° C for 2h. After end-capping, RNA is purified again using lithium chloride precipitation, and the final RNA concentration is measured using NanodropOne (Thermo Fisher, UK). In order to evaluate the quality of RNA, the purified RNA and RNA MillenniumMarker Ladder (Thermo Fisher, UK) are mixed with 2x NorthernMax-Gly loading dye (Thermo Fisher, UK) and incubated at 50 ° C for 30min to denature RNA. 1.2% agarose gel with 1x NorthernMax Running Buffer (Thermo Fisher, UK) is prepared. After incubation, the ladder diagram and sample of denaturation are loaded on the gel, and the gel is run at 80V for 45 minutes. The gel is then imaged on GelDoco-It2 (UVP, UK).

[0348] Cells and in vitro transfection

[0349] HEK293T.17 and HeLa cells (ATCC) were cultured in complete Dulbecco's modified Eagle's medium (DMEM) (Gibco, Thermo Fisher, UK) containing 10% fetal bovine serum (FBS), 1% L-glutamine, and 1% penicillin-streptomycin (Thermo Fisher, UK). THP-1 cells (ATCC, USA) were cultured in complete Roswell Park Memorial Institute (RPMI) 1640 (Gibco, Thermo Fisher, UK) containing 10% fetal bovine serum (FBS), 1% L-glutamine, and 1% penicillin-streptomycin (Thermo Fisher, UK). HEK293T.17 and HeLa cells were cultured at 7×10 cells / well 24 hours before transfection. 4 ; THP-1, 1x10 per well 5 The cell density was seeded in a 96-well plate. According to the manufacturer's instructions, transfection of saRNA and mRNA encoding fLuc was performed using Lipofectamine MessengerMAX (Thermo Fisher Scientific, UK). Luminescent signals were measured using a FLUOstar Omeg microplate reader (BMG Labtech, UK).

[0350] Example 1 - Incorporation of modified base 2' methyl-NTPs can be achieved when using magnesium ion concentrations greater than 20 mM (2-OMe-NTP).

[0351] The inventors unexpectedly demonstrated that when using magnesium ion concentrations greater than 20 mM (see Figure 1 ), the incorporation of the modified base 2' methyl-NTP (2-OMe-NTP) can be achieved. In particular, the inventors have observed that for the effective incorporation of 2' methyl-GTP, a magnesium acetate concentration greater than 20 mM is required, and ideally, a concentration of ≤ 75 mM. However, the inventors believe that the maximum concentration of magnesium acetate can even be greater than 80 mM.

[0352] All experiments were performed under conditions with wild-type T7 polymerase, however, the inventors anticipate that the reaction conditions described herein will also favor mutant versions of T7 polymerase, which is more amenable to base substitutions, as well as other RNA polymerases, including T7, T3, and SP6.

[0353] Example 2 - Barely detectable incorporation using magnesium chloride

[0354] Surprisingly, the inventors found that almost no detectable incorporation was observed when magnesium chloride was used, which is most commonly used in in vitro transcription reactions (see Figure 2 ).

[0355] Example 3 - Manganese acetate is ineffective in incorporating 2' methylribose-modified GTP.

[0356] In addition, manganese in the form of manganese acetate was not efficiently incorporated into 2'methyl-ribose-modified GTP ( Figure 3 ).

[0357] Example 4 - Incorporation of 2' methyl-GTP increases cells with impaired innate sensing mechanisms for recognizing RNA Amplitude and expression of uncapped saRNA in (e.g., HEK293T cells).

[0358] As described in the following examples, the inventors evaluated the effects of incorporation of 2'methylribose-modified NTPs on saRNA function in three cell lines: human embryonic kidney 293T (HEK293T) cells have impaired innate sensing mechanisms that can lead to suppressed RNA expression, HeLa cells represent epithelial cells, and THP-1 cells represent myeloid cells.

[0359] To determine whether the potential advantages of incorporating 2'methyl ribose-modified NTPs are affected by 5' capping, the inventors evaluated the effects on uncapped and capped saRNA and mRNA. The saRNA constructs used in these experiments encode the sequence of firefly luciferase (fLuc) downstream of the subgenomic promoter, and the expression of luciferase is detected over time by luciferase activity in cells.

[0360] In transfection experiments performed in HEK293T cells, when 25ug of uncapped saRNA formulated with lipofectamine was used, the inventors observed that the incorporation of 2'methyl-GTP increased the expression of luciferase within 72 hours (Figure 4), with the increase ranging from 2-fold for 25% GM substitution to 6-fold for 75% substitution (Figure 4b). A similar trend was observed when 100ng of uncapped saRNA was used (Figures 5a and b). These data indicate that the incorporation of 2'methyl-GTP (>25%) increases the amplitude and expression of uncapped saRNA in cells with impaired innate sensing mechanisms for recognizing RNA. This suggests that saRNA modified with 2'methyl-GTP can provide enhanced expression and stability even in cells with impaired RNA sensing mechanisms, and that this enhancement is independent of RNA with 5' capping.

[0361] Example 5 - Incorporation of 2' methylribose-modified NTPs enhances the ability of cells with impaired innate RNA sensing (e.g. Figure 3. Amplitude and duration of capped saRNA expression in HEK293T cells.

[0362] The inventors then determined the effect of incorporating 2'methyl ribose-modified NTPs on the function of 5'-capped saRNAs in HEK293T cells. In transfection experiments performed in HEK293T cells, when 25 μg of capped saRNAs prepared with lipofectamine were used, the inventors observed that the expression of unmodified saRNAs increased by one log relative to uncapped RNA (Figure 6a). However, when cells were transfected with 25 ng of saRNA, when GTP was replaced by 2'-O-methyl GTP at a ratio of ≥25%, an increase in expression levels within 72 hours was observed (Figure 6b), and some moderate increases in expression were also observed for 2'-O-methyl ATP, CTP, and UTP replacements. Similar trends were observed using 100 ng saRNA transfection, and the greatest effect of replacing GTP with 2'-O-methyl GTP was observed (Figures 7a and 7b). These data suggest that incorporation of 2'methyl ribose-modified NTPs, particularly Gm, enhances the magnitude and duration of saRNA expression in cells with impaired innate RNA sensing, which can inhibit RNA translation and increase degradation. The inventors anticipate that further gains can be achieved when 2'-O-methyl GTP is used in combination with 2'-O-methyl ATP, CTP, and UTP substitutions.

[0363] For example, for saRNA:

[0364] 1.75% Gm;

[0365] 2.75% Gm plus 90% Am and 90% Um;

[0366] 3.75% Gm plus 75% Cm (with or without 90% Am+Um);

[0367] 4.75% Gm plus 50% Cm (with or without 90% Am+Um);

[0368] 5. 50% Gm plus 50% Cm (with or without 90% Am+Um).

[0369] However, there may be other feasible iteration methods, such as:

[0370] 1. 50%Gm+30%Cm+50%Am+30%Um;

[0371] 2. 50%Gm+90%Cm+50%Am+30%Um;

[0372] 3. 50%Gm+30%Cm+50%Am+90%Um;

[0373] 4. 50%Gm+90%Cm+50%Am+90%Um;

[0374] 5. 25%Gm+30%Cm+25%Am+60%Um;

[0375] 6. 25%Gm+90%Cm+50%Am+60%Um;

[0376] 7. 75%Gm+30%Cm+50%Am+60%Um;

[0377] 8. 75%Gm+60%Cm+50%Am+75%Um;

[0378] 9. 75%Gm+90%Cm+50%Am+60%Um;

[0379] 10. 50%Gm+30%Cm+25%Am+60%Um;

[0380] 11. 50%Gm+90%Cm+25%Am+60%Um;

[0381] 12. 50%Gm+30%Cm+75%Am+60%Um;

[0382] 13. 50%Gm+90%Cm+75%Am+60%Um;

[0383] 14. 25%Gm+60%Cm+50%Am+30%Um;

[0384] 15. 25%Gm+60%Cm+50%Am+90%Um;

[0385] 16. 75%Gm+60%Cm+50%Am+30%Um;

[0386] 17. 75%Gm+60%Cm+50%Am+90%Um;

[0387] 18. 50%Gm+60%Cm+25%Am+30%Um;

[0388] 19. 50%Gm+60%Cm+25%Am+90%Um;

[0389] 20. 50%Gm+60%Cm+75%Am+30%Um;

[0390] 21. 50%Gm+60%Cm+75%Am+90%Um;

[0391] 22. 25%Gm+60%Cm+25%Am+60%Um;

[0392] 23. 75%Gm+60%Cm+25%Am+60%Um;

[0393] 24. 25%Gm+60%Cm+75%Am+60%Um;

[0394] 25. 75%Gm+60%Cm+75%Am+60%Um;

[0395] 26. 50%Gm+60%Cm+50%Am+60%Um;

[0396] 27. 50%Gm+60%Cm+75%Am+75%Um;

[0397] 28. 50%Gm+60%Cm+60%Am+60%Um;

[0398] 29. 50% Gm + 50% Cm + 50% Am + 50% Um; or any other percentage. For mRNA, some combinations may include:

[0399] 1.75%Gm+90%Cm (with or without 90%Am+Um);

[0400] 2.75%Gm+75%Cm (with or without 90%Am+Um);

[0401] 3. 50% Gm + 50% Cm (with or without 90% Am + Um);

[0402] 4.25% Gm + 25% Cm (with or without 90% Am + Um).

[0403] However, as with saRNA, other iterative approaches are possible for mRNA, such as:

[0404] 1. 50%Gm+30%Cm+50%Am+30%Um;

[0405] 2. 50%Gm+90%Cm+50%Am+30%Um;

[0406] 3. 50%Gm+30%Cm+50%Am+90%Um;

[0407] 4. 50%Gm+90%Cm+50%Am+90%Um;

[0408] 5. 25%Gm+30%Cm+25%Am+60%Um;

[0409] 6. 25%Gm+90%Cm+50%Am+60%Um;

[0410] 7. 75%Gm+30%Cm+50%Am+60%Um;

[0411] 8. 75%Gm+60%Cm+50%Am+75%Um;

[0412] 9. 75%Gm+90%Cm+50%Am+60%Um;

[0413] 10. 50%Gm+30%Cm+25%Am+60%Um;

[0414] 11. 50%Gm+90%Cm+25%Am+60%Um;

[0415] 12. 50%Gm+30%Cm+75%Am+60%Um;

[0416] 13. 50%Gm+90%Cm+75%Am+60%Um;

[0417] 14. 25%Gm+60%Cm+50%Am+30%Um;

[0418] 15. 25%Gm+60%Cm+50%Am+90%Um;

[0419] 16. 75%Gm+60%Cm+50%Am+30%Um;

[0420] 17. 75%Gm+60%Cm+50%Am+90%Um;

[0421] 18. 50%Gm+60%Cm+25%Am+30%Um;

[0422] 19. 50%Gm+60%Cm+25%Am+90%Um;

[0423] 20. 50%Gm+60%Cm+75%Am+30%Um;

[0424] 21. 50%Gm+60%Cm+75%Am+90%Um;

[0425] 22. 25%Gm+60%Cm+25%Am+60%Um;

[0426] 23. 75%Gm+60%Cm+25%Am+60%Um;

[0427] 24. 25%Gm+60%Cm+75%Am+60%Um;

[0428] 25. 75%Gm+60%Cm+75%Am+60%Um;

[0429] 26. 50%Gm+60%Cm+50%Am+60%Um;

[0430] 27. 50%Gm+60%Cm+75%Am+75%Um;

[0431] 28. 50%Gm+60%Cm+60%Am+60%Um;

[0432] 29. 50%Gm+50%Cm+50%Am+50%Um; or any other percentage.

[0433] Example 6 - Incorporation of 2' methyl ribose-modified NTPs increases the ability of the cell to recognize RNA with a complete innate sensor The amplitude and expression of uncapped saRNA in cells controlled by GFP (e.g., HeLa cells).

[0434] Next, the inventors evaluated the effect of incorporating 2'methyl ribose-modified NTPs on saRNA function in HeLa cells, which represent epithelial cells. Unlike the HEK293T cells tested in the previous examples, HeLa cells have a complete epithelial innate sensing mechanism that can trigger intracellular signaling pathways in response to RNA, which can reduce RNA expression and increase RNA degradation rate [6-8].

[0435] First, the inventors considered the impact of incorporating 2'-O-methyl modified bases into uncapped saRNA. When 25ng saRNA was used for transfection, the inventors observed that the expression of 2'-O-methyl GTP and the duration of fLuc expression increased within 72 hours (Figure 8a), and when the ratio of 75%-2'-O-methyl GTP to 25% unmodified GTP was used, the increase was 10-fold to 25-fold (Figure 8b). At 48 hours, the expression of 2'-O-methyl ATP and UTP variants increased slightly. However, when 100ng was used for transfection, the inventors observed an increase in expression when 2'-O-methyl GTP was substituted (Figure 9a) at 72 hours, showing a 50-fold increase (Figure 9b). Surprisingly, the inventors observed an increase in expression substituted with 2'-methyl-ATP or CTP at 72 hours, and for 2'-methyl-ATP, it showed a 100-fold increase in expression. These data show that different substitutions may have different effects on expression dynamics.

[0436] Example 7 - Incorporation of 2' methyl ribose-modified NTPs increases the ability of the cell to recognize RNA with a complete innate sensor The amount and expression of capped saRNA in cells controlled by Bcl-2 or 3B (e.g., HeLa cells).

[0437] The inventors then evaluated the effect of incorporating 2'methyl ribose-modified NTPs on capped RNA. Unmodified 5'-capped saRNA (25ng) was expressed 1 log higher in HeLa cells than uncapped saRNA (Figure 10a). Compared with unmodified saRNA, an increase in the expression of saRNA substituted with 2'O-methyl GTP was observed, but the expression change was not obvious compared with uncapped RNA (Figure 10b). Slight but detectable changes were still observed for the substitution of 2'O-methyl ATP, CTP and UTP. When 100ng was transfected, a similar trend was observed (Figure 11a). Here again, 2'O-methyl GTP substitution showed the strongest effect on the amplitude and duration of expression, with a 20-fold increase observed at 48 hours (Figure 11b). Smaller but detectable changes were also seen for 2'-methyl-ATP or CTP substitutions.

[0438] These data suggest that single nucleotides, particularly variants of GTP with 2'O-methyl base modifications, enhance saRNA expression. It is expected that the use of 2'O-methyl-GTP in combination with 2'O-methyl ATP, CTP, and GTP will also result in further increases in expression.

[0439] Example 8 - Incorporation of 2' methyl ribose-modified NTPs increases the sensitivity of the innate sensor for RNA recognition The amplitude and expression of uncapped saRNA in cells controlled by the gene (e.g., THP-1 cells).

[0440] The inventors then evaluated the effect of incorporating 2'methyl-ribose modified NTPs on saRNA function in THP-1 cells (representing myeloid cells, especially monocytes). These cells are particularly resistant to transfection due to sensitive innate sensing mechanisms that can trigger intracellular signaling pathways in response to RNA, which can reduce RNA expression and increase RNA degradation rates.

[0441] First, the inventors considered the effect of incorporating 2'-O-methyl modified bases into uncapped saRNA. When 25ng saRNA was used for transfection, they observed a sharp increase in fLuc expression of 2'-O-methyl GTP (Figure 12a), with a ratio of 75% 2'-O-methyl GTP to 25% unmodified GTP increasing by more than 1000 times at 48 hours (Figure 12b), and expression was detectable at 72 hours, where expression of unmodified saRNA was not observed. For 2'-O-methyl ATP and UTP substitutions, some improvements in expression were observed. When 100ng was used for transfection, the inventors again observed an increase in expression using 2'-O-methyl GTP substitutions at 72 hours (Figure 13a), which showed an 80-fold increase (Figure 13b). The effects of substitutions with 2'-O-methyl ATP, CTP, and UTP on expression were less than 2'-O-methyl GTP.

[0442] Example 9 - Incorporation of 2' methyl ribose-modified NTPs increases the sensitivity of the innate sensor for RNA recognition The amplitude and expression of capped saRNA in cells controlled by the gene expression vector (e.g., THP-1 cells).

[0443] The inventors then evaluated the effect of incorporating 2'methyl-ribose modified NTPs on capped RNA in THP-1 cells. The expression of unmodified 5' capped saRNA (25ng) in THP-1 cells was again one log higher than that of uncapped saRNA (Figure 14a). For 2'O-methyl GTP substitution, an increase in expression relative to unmodified saRNA was observed, with a fold change of 5 to 10 times within 72 hours (Figure 14b), however, this was less obvious than that seen with uncapped RNA. For the substitution of 2'O-methyl ATP, CTP, and UTP, slight but detectable changes were observed. When 100ng was transfected, a similar trend was observed for 2'O-methyl GTP substitution (Figure 15a). Here again, 2'O-methyl GTP substitution showed the strongest effect on the amplitude and duration of expression, with a 40-fold increase observed at 72 hours (Figure 15b). There was almost no observable advantage with substitution of 2'O-methyl ATP, CTP, and UTP.

[0444] These data suggest that replacing GTP with a 2'O-methyl base modification enhances saRNA expression in bone marrow cells. The use of 2'O-methyl-GTP in combination with 2'O-methyl ATP, CTP, and GTP may result in further increases in expression.

[0445] Example 10-N1 methyl-pseudoUTP has no advantage for saRNA expression

[0446] Since N1 methyl-pseudouridine has been previously shown to increase mRNA expression, the inventors determined whether this provided any advantage for saRNA expression. Here, the inventors looked at replacement of UTP with 25% to 100% N1 methyl-pseudouridine. Transfection experiments were performed with 100 ng of uncapped or capped saRNA formulated with lipofectamine. The inventors observed no clear advantage in fLuc expression in HEK293T or HeLa cells (100% and 100% respectively). Fig.16 A and B). Therefore, these data suggest that N1 methyl-pseudoUTP has no advantage over 2'O-methyl GTP for saRNA expression.

[0447] Example 11-2'O-methyl GTP, 7-deaza-GTP substitution increases the amplitude and duration of saRNA expression

[0448] The inventors also evaluated the effect of incorporating 7-deazaguanosine-5-triphosphate (7-deaza-GTP), which has previously been reported to reduce innate recognition of RNA through Toll Like receptor engagement [9]. Here, the inventors observed replacement of GTP with 25% to 100% 7-deaza-GTP. Transfection experiments were performed with 100 ng of uncapped or capped saRNA formulated with lipofectamine. The inventors observed moderately enhanced expression in HEK293T cells at 24 hours, 48 ​​hours, and 72 hours ( Fig.17 A). They also observed enhanced expression in HeLa cells, especially at 72 hours ( Fig.17 B) (2-fold), indicating that 7-deaza-GTP substitution enhances the duration of expression in HeLa cells. These data indicate that, similar to 2'O-methyl GTP, 7-deaza-GTP substitution increases the magnitude and duration of saRNA expression, albeit to a much lesser extent.

[0449] Example 12-2'-Fluoro-modified NTP has no effect on saRNA expression

[0450] As a replacement of the ribose 2'OH with a 2' methyl group modulates saRNA expression, the inventors performed additional experiments to determine the impact of incorporating nucleotides with smaller 2' fluoro groups, which have previously been shown to modulate innate immune activation

[10] . Here, they looked at replacing ATP, CTP, UTP, and TTP with 25% to 100% 2'-fluoro-modified NTPs. Transfection experiments were performed with 100ng of uncapped or capped saRNA formulated with lipofectamine. The inventors observed that in HEK293T cells (Figure 1) with different ratios of 2' fluoro ATP, CTP, TTP, or UTP, the expression of saRNA was significantly increased. Fig.18 ) or HeLa cells ( Fig.19 ), there was no change in the expression of fLuc-capped and uncapped saRNA.

[0451] Example 13 - Data Summary

[0452] Tables 2 and 3 below summarize the effects of base modifications on saRNA expression.

[0453] Table 2 - Effect of base modifications on saRNA expression when using 25ng saRNA with 75% substitution

[0454]

[0455]

[0456] Table 3 - Effect of base modification on saRNA expression when 100 ng saRNA was used

[0457] Base modification HEK293T HeLa THP-1 2'O-methyl-GTP + +++ +++ 2'O-methyl-ATP - +++ + / - 2'O-Methyl-CTP - +++ ++ 2'O-methyl-UTP - ++ + N1 Methyl-pseudoUTP - - ND 7-deaza-GTP + + ND 2'Fluoro-ATP - - ND 2'Fluoro-CTP - - ND 2'Fluoro-TTP - - ND 2'Fluoro-UTP - - ND

[0458] + = 1 to 5 times, ++ = 5 to 10 times, +++ = > 10 times, ND = not determined.

[0459] Example 14 - Substituting 2'O-methyl GTP for GTP enhances mRNA expression

[0460] After determining that 2'O-methyl NTP substitutions enhanced the expression of saRNA, the inventors evaluated its effect on the expression of mRNA, which also encodes firefly luciferase (fLuc). They compared the expression of unmodified mRNA with mRNA in which 75% of GTP was replaced by 2'O-methyl GTP and mRNA in which 75% of GTP and ATP and 90% of CTP and UTP (combined mRNA) were replaced by 2'O-methyl counterparts.

[0461] They evaluated the effect of 2'O-methyl substitution on fLuc expression in HEK293T, HeLa, and THP-1 cells as previously described. Using 5'-capped saRNA and mRNA, the inventors observed that replacing GTP with 2'O-methyl GTP resulted in a modest increase in expression at 24 hours. However, combined substitutions of all four NTPs ( Fig. 20 a) Expression was enhanced 10-fold within 72 hours ( Fig. 20 b) mRNA expression was approximately two-fold lower than that observed with saRNA, which showed a similar pattern of enhanced expression when 75% of GTP was replaced with 2'O-methyl GTP, as described above.

[0462] The inventors then evaluated expression in interferon-competent HeLa cells ( Fig.21 a). When 75% of GTP was replaced with 2'O-methyl GTP, saRNA expression was enhanced by approximately 1 log ( Fig.21 b). Replacing 75% of GTP with 2'O-methyl GTP also had a similar enhancement in mRNA expression. Surprisingly, combined replacement of all NTPs with 2'O-methyl modified NTPs increased mRNA expression to levels comparable to those of saRNA ( Fig.21 a) The same levels observed, with a fold enhancement of ≥100-fold within 72 hours ( Fig.21 b).

[0463] They observed a similar pattern in THP-1 cells, which represent monocytes ( Fig. 22 a). As observed for HeLa cells, saRNA expression was enhanced by approximately 1 log within 72 hours when 75% of GTP was replaced with 2'O-methyl GTP ( Fig. 22b). When evaluating mRNA, expression also enhanced expression 5-fold within 72 hours, however, combined substitution of all NTPs with 2'O-methyl modified NTPs increased mRNA expression to levels comparable to saRNA ( Fig. 22 a) The same levels observed, with a fold enhancement of ≥100-fold within 72 hours ( Fig. 22 b).

[0464] These data surprisingly demonstrate that replacing GTP with 2'O-methyl GTP enhances the expression of both saRNA and mRNA. Further substitution with additional NTPs appears to provide additional benefits for the expression of mRNA, and is expected to be the case for saRNA as well.

[0465] Table 4 - Effects of base modifications on mRNA expression when 100 ng mRNA was used

[0466]

[0467]

[0468] + = 1 to 5 times, ++ = 5 to 10 times, +++ = > 10 times, ND = not determined.

[0469] Example 15 - Replacing GTP and CTP with 2'O-methyl-GTP (Gm) and CTP enhances mRNA expression

[0470] After determining that all four NTP substitutions combined with 2'O-methyl enhanced mRNA expression ( Figure 20 to Figure 22 ), the inventors evaluated the effect of Gm substitution in combination with each of the other three NTPs on expression (fLuc) relative to combined substitutions of all four NTPs (mRNA combination). They compared expression of unmodified mRNA with the following situations: 75% of GTP was replaced by 2'O-methyl GTP alone, 75% of GTP (Gm) was replaced by 75% of ATP (Am), 75% of GTP (Gm) was replaced by 90% of CTP (Cm), 75% of GTP (Gm) was replaced by 90% of UTP (Um), and 75% of GTP, 75% of ATP, 90% of CTP and UTP (combined mRNA) were replaced by 2'O-methyl counterparts.

[0471] They evaluated the effects of 2'O-methyl substitutions on fLuc expression in HEK293T, HeLa, and THP-1 cells as previously described. Using 5'-capped mRNA, the inventors observed that substitution of GTP and CTP with 2'O-methyl GTP and 2'O-methyl CTP provided similar enhancements to those observed when all four NTPs were substituted with 2'O-methyl NTPs. In HEK293T cells, defects in the innate interferon response pathway resulted in a 20-fold enhancement of expression at 24 hours ( Fig.23 and 24).

[0472] The inventors then evaluated expression in interferon-competent HeLa cells ( Fig.25 Surprisingly, by replacing 75% of GTP and 90% of CTP with 2'O-methyl GTP and 2'O-methyl CTP, mRNA expression was enhanced similarly to when all four NTPs were replaced with 2'O-methyl NTPs ( Fig.26 ), with a fold enhancement of ≥70-fold within 72 hours ( Fig.26 ).

[0473] They observed a similar pattern in THP-1 cells, which represent monocytes ( Fig. 27 As observed for HeLa cells, mRNA expression was similarly enhanced by replacing 75% of GTP and 90% of CTP with 2'O-methyl GTP and 2'O-methyl CTP, similar to when all four NTPs were replaced with 2'O-methyl NTPs ( Fig.28 ), with a fold enhancement of ≥1000-fold within 72 hours ( Fig.28 ).

[0474] These data surprisingly demonstrate that replacing GTP with 2'O-methyl GTP, along with replacing CTP with 2'O-methyl GTP and 2'O-methyl CTP, enhances mRNA expression to a similar extent as when all four NTPs are replaced with 2'O-methyl NTPs. Surprisingly, the greatest increase in expression was observed in interferon-competent cells (HeLa and THP-1 cells) relative to HEK293T cells, which have impaired interferon responses and innate signaling pathways.

[0475] Example 16 - Effect of Replacing a Single NTP with 2'O-Methyl-NTP (Nm) on mRNA Expression in HEK293T and HeLa Cells There is little benefit.

[0476] After determining that combined substitution of GTP and CTP with 2'O-methyl-GTP (Gm)_ and -CTP (Cm)_ or substitution of all four NTPs enhanced mRNA expression ( Figure 23-28 ), the inventors evaluated the effect of single NTP substitutions on expression (fLuc). They compared the expression of unmodified mRNA with that of mRNA in which 75% of GTP was replaced by 2'O-methyl GTP (Gm), 75% of ATP was replaced by 2'O-methyl ATP (Am), 90% of CTP was replaced by 2'O-methyl CTP (Cm), or 90% of UTP was replaced by 2'O-methyl UTP (Um).

[0477] They evaluated the effect of 2'O-methyl substitution on fLuc expression in HEK293T and HeLa cells as described previously. Using 5'-capped mRNA, the inventors observed that, as shown by using 25 ng ( Fig.29 ) or 100 ng mRNA ( Fig.30 Substitution of GTP, ATP, CTP, or UTP alone for 2'O-methyl-GTP (Gm), -ATP (Am), -CTP (Cm), or -UTP (Um) had little or no effect on mRNA expression, as determined by measuring fLuc expression in HEK293T cells when 25 ng ( Fig.31 ) or 100 ng of mRNA ( Fig.32 This was also observed in HeLa cells.

[0478] These data surprisingly demonstrate that in both HEK293T and HeLa cells, combined substitutions of GTP with 2'O-methyl GTP and CTP with 2'O-methyl GTP provide a dominant effect on expression enhancement, with substitution of GTP or CTP alone with 2'O-methyl-GTP or -CTP providing little benefit relative to unmodified mRNA.

[0479] Example 17 - Replacing a single CTP with 2'O-methyl-CTP (Cm) enhances mRNA expression in THP-1 cells

[0480] After evaluating the effect of single NTP substitution on mRNA expression in HEK293T cells and HeLa cells, the inventors evaluated the effect of single NTP substitution on expression (fLuc) in THP-1 cells. These cells are mononuclear cells that are highly sensitive to innate activation that triggers robust RNA degradation. They compared the expression of unmodified mRNA with that of mRNA in which 75% of GTP was replaced by 2'O-methyl GTP (Gm), 75% of ATP was replaced by 2'O-methyl ATP (Am), 90% of CTP was replaced by 2'O-methyl CTP (Cm), or 90% of UTP was replaced by 2'O-methyl UTP (Um).

[0481] They evaluated the effect of 2'O-methyl substitution on fLuc expression in THP-1 cells as described previously. Using 5'-capped mRNA, the inventors observed that by using 25 ng ( Fig.33 ) or 100 ng mRNA ( Fig.35), replacing GTP, ATP, CTP, or UTP with 2'O-methyl-GTP (Gm), -ATP (Am), -CTP (Cm), or -UTP (Um) alone had different effects on mRNA expression. Relative to unmodified mRNA, replacing CTP with 2'O-methyl CTP (Cm) provided the greatest effect on expression, showing a 10-fold enhancement with 25 ng mRNA ( Fig.34 ) and a 20-fold enhancement with 100 ng mRNA ( Fig.36 Relative to unmodified mRNA, substitution of 2'O-methyl GTP (Gm) for GTP provided modest enhancement of expression, with a 3-fold enhancement at 25 ng mRNA ( Fig.34 ) and 5-fold enhancement when 100 ng mRNA was used ( Fig.36 Substituting 2'O-methyl ATP (Am) or 2'O-methyl UTP (Um) for ATP or UTP had little effect on expression.

[0482] These data surprisingly demonstrate that while individual substitutions of either CTP or GTP NTP with the 2'O-methylated form enhance expression in THP-1 cells, the combined substitution of GTP with 2'O-methyl GTP and CTP with 2'O-methyl GTP provides a synergistic enhancement of expression that exceeds the additive predicted by the individual substitutions.

[0483] Summarize

[0484] The present inventors have demonstrated that 2'-substituted ribose-modified NTPs, particularly 2'-O-methyl-modified NTPs, can be used to enhance (i) expression and / or translation of RNA molecules comprising one or more modified NTPs; (ii) enhance stability of RNA molecules comprising one or more modified NTPs; and / or (iii) reduce activation of innate sensing, interferon production and / or degradation of RNA molecules comprising one or more modified NTPs.

[0485] Summarize:

[0486] 1. The present invention relates to the use of 2'-modified RNA, in particular mRNA, and saRNA and non-coding RNA, in which a significant percentage of nucleotides (≥25%, up to 100%) are 2'-methyl-NTPs. The mRNA and saRNA sequences are of considerable length (>100 bp), and the wild-type nucleotides are at least partially replaced by 2'-O-methyl-NTPs.

[0487] 2. The modified RNA is synthesized by in vitro transcription, and the reaction mixture optimally includes a high concentration of magnesium ions (ie, >20 mM magnesium ions, ideally 75 nM) and a high concentration of nucleotides (ie, >10 mM nucleotides).

[0488] 3. The present inventors have shown that the presence of magnesium ions in magnesium acetate but not magnesium chloride results in enhanced expression and incorporation of modified NTPs.

[0489] 4. Targeted moiety substitution of wild-type GTP, ATP, CTP and / or UTP with the corresponding 2'-methyl-NTP results in ≤100-fold enhancement in expression and stability of the modified mRNA or saRNA in interferon-responsive-competent cells.

[0490] Example 18 - Substitution of modified NTPs other than 2-O-methyl NTP does not provide any benefit on saRNA expression What are the benefits

[0491] A wider range of modified NTPs were screened for their ability to enhance saRNA expression in human cells in vitro (see Fig.39 ),like Fig.16 , 17 , 18, 19 and 40. These failed to significantly enhance, and in many cases, impaired, fLuc saRNA expression. Notably, many modified NTPs previously shown to enhance mRNA expression, such as pseudouridine, N1-methylpseudouridine, 5-methylcytidine (m5C), N6-methyladenosine (m6A) and 2-thiouridine (s2U), did not provide benefit or reduced expression [30,31].

[0492] These data suggest that saRNA requires specific modifications relative to mRNA to enhance expression, the data are summarized in Table 5 below.

[0493] Table 5 - Effect of base modification on saRNA expression

[0494]

[0495]

[0496] Example 19-2'-O-methyl-GTP enhances the expression of saRNA encoding eGFP

[0497] After determining that replacing GTP with 2'-O-methyl-GTP alone provided a significant effect on fLuc expression, the inventors determined whether the same effect would be observed if the GOI encoded in the saRNA was altered. VEEV saRNAs encoding eGTP downstream of a subgenomic promoter were generated by IVT: using unmodified NTPs; where GTP was replaced with ≥75% 2'-O-methyl-modified NTPs; and where UTP was replaced with 100% N-1 methyl pseudouridine. These constructs were used to transfect HELA and THP1 cells, and after 24 hours, cells were harvested, stained, and assessed for eGFP expression by flow cytometry. In HELA (see Fig.41 A) and THP1 (see Fig.41 B) cells, 2'-O-methyl-GTP-modified VEEV-eGFP saRNA induced a greater number of eGFP-positive cells and increased eGFP median fluorescence intensity (MFI) compared to unmodified and N1-methyl-pseudoUTP-modified VEEV-eGFP. These data indicate that the beneficial effects of 2'-O-methyl-GTP apply to different encoded GOIs. Importantly, N1-methyl pseudoUTP-modified VEEV-eGFP did not produce any eGFP-positive cells in either cell line, in stark contrast to the public findings that the addition of N1-methyl pseudoUTP enhanced the expression of conventional mRNA. These data highlight the surprising observation that saRNA requires different modifications to achieve beneficial modifications relative to mRNA.

[0498] Example 20 - Combined 2'-O-methyl-NTP substitutions enhance expression of mRNA encoding fLuc

[0499] After determining that substitution of GTP with 2'-O-methyl-GTP alone provided a significant effect on fLuc expression, the inventors determined whether further increases in expression could be delivered by combined 2'-O-methyl-NTP substitutions. fLuc mRNA was generated by IVT: using unmodified NTPs; 100% N-1 methyl pseudouridine substitution, and where nucleotides were substituted with ≥75% 2'-O-methyl GTP, ≥75% 2'-O-methyl ATP, 90% 2'-O-methyl CTP, or 90% 2'-O-methyl UTP, either alone or in various combinations. These constructs were used to transfect THP1 cells, and luciferase expression was monitored over 48 hours by measurement of relative light units. Combination substitutions of 2'-O-methyl ATP plus UTP or 2'methyl GTP plus CTP provided enhanced expression relative to substitutions of the single bases alone. However, combined substitutions of all NTPs with 2-O-methyl forms provided the greatest enhancement.

[0500] Example 21 - Combined 2'-O-methyl-NTP substitutions enhance expression of mRNA encoding fLuc

[0501] In determining the 2'-O-methyl-NTP substitution in a fixed ratio (2'-O-methyl combination (see Fig.42 )) provided a significant effect on fLuc expression, the inventors determined how the ratio of 2-O-methyl-NTP substitutions using all four NTPs affected expression. Fluc mRNA was generated by IVT: using unmodified NTPs and a wide range of different 2-O-methyl-NTP substitutions, as detailed in Table 6 below.

[0502] Table 6 - Percent substitution of NTPs for 2-O-methyl modification. Conditions 1 to 26 are evaluated in Figures 43 to 45. estimate

[0503]

[0504] These constructs are used for transfection of HEK293T, HeLa and THP1 cells, and by the measurement of relative light units to monitor the expression of luciferase in 72 hours (referring to Figures 43 to 45). Relative to unmodified mRNA, the combination of all four kinds of 2'-O-methyl NTPs in a wide ratio range replaces enhanced expression. These data show that replacing all four kinds of NTPs with 2-O-methyl NTPs in a wide replacement range is beneficial to improving mRNA expression.

[0505] Example 22 - 2'-O-methyl-GTP-modified saRNA is less inflammatory than unmodified saRNA.

[0506] To determine whether 2'-O-methyl modification can reduce the activation of innate perception of saRNA in host cells, production of interferons and / or degradation, THP1 cells were transfected with unmodified and modified saRNAs and cell supernatants were collected 24 and 48 hours after transfection to characterize the induced cytokine levels, followed by luciferase assays to determine antigen expression levels. THP1 cells were selected for this analysis because they represent monocytes with very robust innate responses. Cytokine levels were measured using the MesoScale Discovery (MSD) assay, where the cytokine panel selected for this experiment included ENA-78, GM-CSF, IFN-β, IL-1α, IL-1β, IP-10, MCP-1, MIP-1α, MIP-1β, MDC, Eotaxin-2, GRO-α, and MCP-3. As previously described, 2'-O-methyl-GTP-modified saRNAs had higher expression of luciferase than unmodified and N1-methyl pseudoUTP-modified saRNAs (see Fig.46A). Evaluation of cytokines and chemokines secreted by THP1 cells transfected with unmodified and modified saRNAs showed that all modified saRNAs showed a significantly reduced inflammatory response compared to unmodified saRNA. Interestingly, this was less pronounced for N1-methyl pseudoUTP-modified saRNA, although N1-methyl pseudoUTP modification failed to show an enhancement of antigen expression (see Fig.46B ). However, 2'-O-methyl-GTP-modified saRNA also showed a reduced inflammatory response compared to unmodified saRNA, although it was not as obvious as N1-methyl pseudoUTP. This suggests that the enhanced antigen expression of saRNA caused by 2'-O-methyl-GTP modification may not only be due to a reduction in the innate response, but also other mechanisms, such as slower degradation.

[0507] Example 23-2'-O-methyl-modification enhances expression of mRNA encoding eGFP

[0508] Having established that 2'-O-methyl-modification provided a significant effect on fLuc expression, the inventors determined whether the same effect would be observed if the encoding GOI was altered. mRNA encoding eGTP was generated by IVT using either unmodified NTPs; in which the UTPs were substituted with 100% N-1 methyl pseudouridine; or in which the NTPs were substituted with 2'-O-methyl-modified NTPs (75% Gm, 75% Am, 90% Cm, 90% Um) and labeled with a 2'-O-methyl combination. These constructs were used to transfect HELA and THP1 cells and after 24 hours, cells were harvested, stained, and assessed for eGFP expression by flow cytometry. In HELA (see Fig.47 A) and THP1 (see Fig.47 B) cells, 2'-O-methyl-modified mRNA induced a greater number of eGFP-positive cells and increased eGFP median fluorescence intensity (MFI) compared to unmodified and N1-methyl pseudoUTP-modified mRNA. These data suggest that the beneficial effects of 2'-O-methyl-modification apply to different encoded GOIs. Importantly, N1-methyl pseudoUTP-modified mRNA did enhance eGFP expression in both cell lines, consistent with published findings for conventional mRNA, although to a lesser extent than 2'O' methyl combination modifications. These data highlight the surprising observation that saRNA requires different modifications to achieve beneficial modifications relative to mRNA (see also Fig.41 and Example 19).

[0509] Example 24 - 2'-O-methyl-modified mRNA is less inflammatory than unmodified mRNA.

[0510] To determine whether 2'-O-methyl modification can reduce the activation of innate sensing, interferon production and / or degradation of mRNA in host cells, THP1 cells were transfected with unmodified and modified mRNA and cell supernatants were collected 24 and 48 hours after transfection to characterize the induced cytokine levels, followed by luciferase assays to determine antigen expression levels. THP1 cells were selected for this analysis because they represent monocytes with very robust innate responses. Cytokine levels were measured using the MesoScale Discovery (MSD) assay, where the cytokine panel selected for this experiment included ENA-78, GM-CSF, IFN-β, IL-1α, IL-1β, IP-10, MCP-1, MIP-1α, MIP-1β, MDC, Eotaxin-2, GRO-α and MCP-3. As previously described, luciferase expression was higher for 2'-O-methyl-modified mRNA compared to unmodified and N1-methyl pseudoUTP-modified mRNA (see Fig.48A ). However, N1-methylpseudouridine-modified mRNA was higher than unmodified mRNA, consistent with previous reports evaluating the mRNA secretion of cytokines and chemokines from THP1 cells transfected with unmodified and modified mRNA, indicating that all modified mRNAs showed a significantly attenuated inflammatory response compared to unmodified mRNA (see Fig.48B Interestingly, it was similar with N1-methyl pseudoUTP and modified mRNA, although the fLuc expression of N1-methyl pseudoUTP modified mRNA was lower than that of 2'-O-methyl modified mRNA (see Fig.48A ). These data suggest that the enhanced antigen expression of mRNA caused by 2'-O-methyl-modification may not only be due to a reduction in the innate response, but also to other mechanisms, such as slower degradation.

[0511] Example 25-2'-O-methyl-GTP enhances saRNA expression in vivo

[0512] Small animal studies were performed to determine the effect of 2'-O-methyl modified saRNA on the duration of in vivo expression. Groups of five female Balb / c mice were injected with 10 μg LNP formulated VEEV-Fluc saRNA, which was produced using unmodified NTPs or 75% 2'-O-methyl-GTP replacement. The PBS group was performed as a comparative control. The LNPs used for these studies were formulated with C12-200 ionizable lipids containing DSPC, cholesterol, and DMPE-PEG200. Mice were imaged using the IVIS Spectrum in vivo imaging system on days 1 and 5 (peak days of saRNA expression) ( Fig.49A). Expression was observed as early as day 1 in the 75% 2'-O-methyl-GTP modified group, which induced higher expression than the unmodified group. Stronger expression levels were also observed on day 5, with a similar trend to that seen on day 1. Total expression was analyzed using Aura Imaging software and quantified as photons / second (p / s) ( Fig.49B ), where VEEV-Fluc modified with 2'-O-methyl-GTP showed significantly higher expression than unmodified GTP on day one, while expression of unmodified GTP gradually decreased over time. These data demonstrate the impact of 2'-O-methyl-GTP modification alone, and as predicted by in vitro studies, further increases in expression are expected when a combination of 2-O-methyl NTPs (Gm+Cm+Am+Um) is used.

[0513] Example 26 - 2'-O-methyl-NTP enhances mRNA expression in vivo

[0514] Small animal studies were performed to determine the effect of 2'-O-methyl modified mRNA on the duration of expression in vivo. Groups of 5 female Balb / c mice were injected with 10 μg of LNP formulated mRNA using unmodified NTPs; wherein UTP was substituted for N1-methyl pseudouridine; or wherein WT NTP was replaced with 2'-O-methyl modified NTPs (75% Gm, 75% Am, 90% Cm, 90% Um) and labeled with 2'-O-methyl combinations (Figure 50). The LNPs used for these studies were formulated with C12-200 ionizable lipids containing DSPC, cholesterol, and DMPE-PEG200. Mice were imaged using the IVIS Spectrum in vivo imaging system on days 1 to 3 ( Fig.50A ). Expression of the 2'-O-methyl-combination modified group was observed as early as day one, with higher induced expression than the unmodified and N1-methylpseudouridine groups. Stronger expression levels were also observed on days two and three, with a similar trend to that seen on day one. Total expression was analyzed using Aura Imaging software and quantified as photons / second (p / s) ( Fig.50B ), where the expression of 2'-O-methyl-combination modified mRNA was significantly higher than that of unmodified mRNA from day 1 to day 3. These data demonstrate the positive effect of 2'-O-methyl-modification on mRNA expression.

[0515] Example 27-2'-O-methyl-GTP enhances the immunogenicity of saRNA in vivo.

[0516] Small animal studies were performed to determine the effect of 2'-O-methyl modified saRNAs on in vivo immunogenicity. VEEV saRNA encoding the influenza hemagglutinin protein (VEEV-Ha) downstream of a subgenomic promoter was generated by IVT: unmodified NTPs were used;

[0517] ≥75% 2'-O-methyl modified NTP substitution; or wherein UTP is substituted with 100% N-1 methyl pseudouridine. At weeks 0 and 4, four groups of 5 female Balb / c mice were immunized intramuscularly with 10 μg LNPs of VEEV-Ha saRNA formulated with: 1) unmodified saRNA, 2) 100% N1-methyl pseudoUTP modified saRNA, 3) 75% 2'-O-methyl-GTP modified saRNA, and 4) PBS ( Fig.51 ). At week 6, mice were bled and euthanized. HA ELISA was performed to assess total HA-specific IgG. The 2'-O-methyl-GTP modified group had significantly higher antibody responses after boosting compared to the unmodified group and the N1-methyl pseudo-UTP modified group ( Fig.51 A). Microneutralization assays were also performed on mouse sera using the California influenza virus 2009 H1N1 strain ( Fig.51 B), the results are consistent with the ELISA data, whereby the 2'-O-methyl-GTP modified group had significantly higher neutralizing antibody responses after boosting compared to all other groups. These data demonstrate the effect of 2'-O-methyl-GTP modification alone on immunogenicity, as predicted by in vitro studies, when 2-O-methyl NTP

[0518] When the combination of (Gm+Cm+Am+Um) is used, additional effects are expected.

[0519] References

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[0552] The following clauses describe embodiments of the present invention:

[0553] 1. A method for preparing a modified RNA molecule, wherein the method comprises: contacting (i) a template nucleic acid sequence, (ii) an RNA polymerase, and (iii) a plurality of nucleotide triphosphates (NTPs) in the presence of at least 20 mM magnesium ions, wherein one or more of the nucleotide triphosphates (NTPs) are modified nucleotide triphosphates (NTPs), wherein the RNA polymerase transcribes the template nucleic acid sequence to form an RNA molecule comprising at least 20 nucleotides, and wherein at least 25% of the constituent nucleotides in the RNA molecule are modified.

[0554] 2. Use of 20 mM magnesium ions in a transcription reaction to prepare a modified RNA molecule comprising at least 20 nucleotides, wherein at least 25% of the constituent nucleotides in the RNA molecule are modified.

[0555] 3. A method or use according to any preceding clause, wherein the method comprises using:

[0556] (i) at least 30 mM magnesium ion, at least 40 mM magnesium ion, at least 50 mM magnesium ion, at least 60 mM magnesium ion, or at least 70 mM magnesium ion; or

[0557] (ii) 50 mM to 100 mM magnesium ion, 60 mM to 95 mM magnesium ion, 65 mM to 90 mM magnesium ion, 70 mM to 80 mM magnesium ion, or 71 mM to 79 mM magnesium ion.

[0558] 4. The method or use according to any preceding clause, wherein the magnesium ion is in the form of Mg 2+ Supplied in ionic form.

[0559] 5. A method or use according to any preceding clause, wherein the magnesium ions are provided in the form of magnesium acetate.

[0560] 6. The method or use according to any preceding clause, wherein the RNA is selected from: messenger RNA (mRNA), micro RNA (miRNA), short interfering RNA (siRNA), short hairpin RNA (shRNA), antisense RNA, RNA aptamer, self-amplifying RNA (saRNA), interfering RNA (RNAi), non-coding RNA, circular RNA, and small RNA.

[0561] 7. The method or use according to any preceding clause, wherein the RNA is self-amplifying RNA (saRNA) or messenger RNA (mRNA).

[0562] 8. A method or use according to any preceding clause, wherein the RNA molecule is:

[0563] (i) at least 20, 21, 22 or 23 bases in length;

[0564] (ii) at least 24, 25, 26 or 27 bases in length;

[0565] (iii) at least 28, 29, 30 or 31 bases in length; or

[0566] (iv) at least 32, 33, 34 or 35 bases in length.

[0567] 9. A method or use according to any preceding clause, wherein the RNA molecule is:

[0568] (i) at least 50 bases in length, at least 60 bases in length, at least 75 bases in length, at least 100 bases in length, at least 200 bases in length, at least 300 bases in length, at least 400 bases in length, at least 500 bases in length, at least 600 bases in length, at least 700 bases in length, at least 800 bases in length, or at least 900 bases in length;

[0569] (ii) at least 1000 bases in length, at least 2000 bases in length, at least 3000 bases in length, at least 4000 bases in length, at least 5000 bases in length, at least 6000 bases in length, at least 7000 bases in length, at least 8000 bases in length, at least 9000 bases in length, at least 10000 bases in length, at least 11000 bases in length, or at least 12000 bases in length.

[0570] 10. A method or use according to any preceding clause, wherein the method comprises using an RNA polymerase selected from the group consisting of: T7; T3; SP6; KP34; Syn5; or other DNA-dependent RNA polymerases; or mutant variants of any of these RNA polymerases.

[0571] 11. A method or use according to any preceding clause, wherein the method comprises using the plurality of nucleotide triphosphates at the following concentrations:

[0572] (i) at least 1 mM, 2 mM, 3 mM or 4 mM;

[0573] (ii) at least 5 mM, 6 mM or 7 mM; or

[0574] (iii) at least 8 mM, 9 mM, 10 mM.

[0575] 12. The method or use according to any preceding clause, wherein the one or more modified NTPs are selected from: modified adenosine-5'-triphosphate (ATP), modified cytidine-5'-triphosphate (CTP), modified guanosine-5'-triphosphate (GTP), and modified uridine-5'-triphosphate (UTP).

[0576] 13. A method or use according to any preceding clause, wherein the method comprises using at least one, two, three, four or five modified NTPs selected from the group consisting of modified ATP, modified CTP, modified GTP, and / or modified UTP.

[0577] 14. A method or use according to any preceding clause, wherein the one or more modified NTPs comprise a 2' substituted group, wherein the OH group, typically at the 2' position, is substituted with halogen, an optionally substituted aromatic group, N3, H, an optionally substituted O-alkyl, O-alkenyl or O-alkynyl, or an optionally substituted alkyl, alkenyl or alkynyl, wherein in each case the aromatic group, alkyl, alkenyl or alkynyl is optionally substituted with halogen, oxo, OR, CN, NR2 or SR, wherein R is H or C 1-6 Alkyl, C 2-6 Alkenyl or C 2-6 Alkynyl.

[0578] 15. A method or use according to any preceding clause, wherein the one or more modified NTPs comprise a substituted nucleobase, wherein the nucleobase is substituted with halogen, an optionally substituted aromatic group, N3, OH, an optionally substituted O-alkyl, O-alkenyl or O-alkynyl, or an optionally substituted alkyl, alkenyl or alkynyl, wherein in each case the aromatic group, alkyl, alkenyl or alkynyl is optionally substituted with halogen, oxo, OR, CN, NR2 or SR, wherein R is H or C 1-6 Alkyl, C 2-6 Alkenyl or C 2-6 Alkynyl, optionally, wherein, alternatively or additionally, the oxo group in the nucleobase is replaced by a =S group.

[0579] 16. The method or use according to any one of clauses 14 to 15, wherein the alkyl, alkenyl or alkynyl group is an optionally substituted C1-C 20 Alkyl, alkenyl or alkynyl NTP, wherein the alkyl, alkenyl or alkynyl is optionally substituted with halogen, oxo, OR, CN, NR2 or SR, wherein R is H or C 1-6 Alkyl, C 2-6 Alkenyl or C 2-6 Alkynyl, preferably wherein the alkyl, alkenyl or alkynyl is an optionally substituted C1-C 10 Alkyl, alkenyl or alkynyl NTP, wherein the alkyl, alkenyl or alkynyl is optionally substituted with halogen, oxo, OR, CN, NR2 or SR, wherein R is H or C 1-6 Alkyl, C 2-6 Alkenyl or C 2-6 Alkynyl.

[0580] 17. The method or use according to any one of clauses 14 to 16, wherein the alkyl, alkenyl or alkynyl group is an optionally substituted C 1-6 Alkyl, C 2-6 Alkenyl or C 2-6 Alkynyl, wherein the alkyl, alkenyl or alkynyl, or each of them is optionally substituted with halogen, oxo, OR, CN, NR2 or SR, wherein R is H or C 1-6 Alkyl, C 2-6 Alkenyl or C 2-6 Alkynyl, preferably wherein the alkyl, alkenyl or alkynyl is an optionally substituted C 1-3 Alkyl, C 2-3 Alkenyl or C 2-3 Alkynyl, wherein the alkyl, alkenyl or alkynyl, or each of them is substituted with one or more substituents selected from oxo, OH, OMe, NH2 and NRH, wherein R is C 1-6 Alkyl, C 2-6 Alkenyl or C 2-6 Alkynyl.

[0581] 18. A method or use according to any preceding clause, wherein the one or more modified NTPs comprise 2'-methyl modified NTPs.

[0582] 19. A method or use according to any preceding clause, wherein the one or more modified NTPs comprise 2'-O-methyl modified NTPs.

[0583] 20. The method or use according to any of the preceding clauses, wherein the one or more modified NTPs are selected from: 2'-O-methyladenosine (Am); 1,2'-dimethyladenosine (m 1 Am); N6,2'-O-dimethyladenosine (m 6 Am); N 6 ,N 6 ,2-O-trimethyladenosine (m 6,6 Am); 2'-O-methyladenosine (Im); 1,2'-O-dimethyladenosine (m 1 Im); 2'-O-methylguanosine (Gm); 1,2'-O-dimethylguanosine (m 1 Gm); N2,2'-O-dimethylguanosine (m 2 Gm); N2,N2,2'-O-trimethylguanosine (m 2,2 Gm); N2,7,2'-O-trimethylguanosine (m 2,7 Gm); 2'-O-methylcytidine; N 4 ,2'-O-dimethylcytidine (m 4 Cm); N 4 ,N 4 ,2-O-trimethylcytidine (m 4,4 Cm); 5,2'-O-dimethylcytidine (m 5 Cm); N 4 -Acetyl-2'-O-methylcytidine (ac 4 Cm); 2'-O-methyl-5-hydroxymethylcytidine (hm 5 Cm); 5-formyl-2'-O-methylcytosine (f 5 Cm); 2'-O-methyluridine (Um); 3,2'-O-dimethyluridine (m 3 Um); 5,2'-O-dimethyluridine (m 5 Um or Tm); 2-thio-2'-O-methyluridine (s 2 Um); 2'-O-methyl-pseudouridine (Ym); 5-carbamoylmethyl-2'-O-methyluridine (ncm 5 Um); 5-methoxycarbonylmethyl-2'-O-methyluridine (mcm 5Um); 5-(carboxyhydroxymethyl)-2'-O-methyluridine methyl ester (mchm 5 Um); 5-carboxymethylaminomethyl-2'-O-methyluridine (cmbm 5 Um); 2'-O-methyluridine-5-hydroxyacetate (mcmo 5 Um); 5-(isopentenylaminomethyl)-3'O-methyluridine (inm 5 Um); and any other synthetic NTP containing 2'-O-methylated ribose.

[0584] 21. A method or use according to any preceding clause, wherein the one or more modified NTPs comprise 2'-O-methyl-modified ATP, 2'-O-methyl-modified CTP, 2'-O-methyl-modified GTP, and / or 2'-O-methyl-modified UTP.

[0585] 22. A method or use according to any preceding clause, wherein the method comprises using at least one, two, three or four modified NTPs selected from the group consisting of 2'-O-methyl-ATP, 2'-O-methyl-CTP, 2'-O-methyl-GTP and 2'-O-methyl-UTP.

[0586] 23. The method or use according to any preceding clause, wherein, when the modified RNA molecule is saRNA, the RNA molecule comprises a modified GTP, preferably a modified 2'-O-methyl-GTP.

[0587] 24. The method or use according to any preceding clause, wherein, when the modified RNA molecule is mRNA, the RNA molecule comprises a modified CTP and a modified GTP, preferably a modified 2'-O-methyl CTP and a modified 2'-O-methyl-GTP.

[0588] 25. The method or use according to any preceding clause, wherein at least 30%, 35% or 40% of the constituent nucleotides in the resulting RNA molecule are modified, wherein the modified nucleotides include adenine, cytosine, guanine and / or uracil; preferably, wherein at least 45%, 50% or 55% of the constituent nucleotides in the resulting RNA molecule are modified, wherein the modified nucleotides include adenine, cytosine, guanine and / or uracil.

[0589] 26. A method or use according to any preceding clause, wherein the method comprises combining one or more 2-O-methyl modified NTPs with one or more other modified NTP bases that are not 2-O-methyl modified NTPs.

[0590] 26. A method or use according to any preceding clause, wherein the method comprises combining:

[0591] (i) one or more modified NTPs comprising a 2' substituted group wherein the OH group, typically at the 2' position, is substituted with halogen, optionally substituted aromatic groups, N3, H, optionally substituted O-alkyl, O-alkenyl or O-alkynyl, or optionally substituted alkyl, alkenyl or alkynyl, wherein in each case the aromatic group, alkyl, alkenyl or alkynyl is optionally substituted with halogen, oxo, OR, CN, NR2 or SR, wherein R is H or C 1-6 Alkyl, C 2-6 Alkenyl or C 2-6 Alkynyl; and

[0592] (ii) one or more modified NTPs that are not 2'-O-methyl modified NTPs.

[0593] 27. A method or use according to any preceding clause, wherein the template nucleic acid sequence comprises DNA, optionally the template nucleic acid comprises a plasmid.

[0594] 28. A method or use according to any preceding clause, wherein the template nucleic acid is transcribed to produce a resulting modified RNA molecule, the modified RNA molecule being selected from: messenger RNA (mRNA), microRNA (miRNA); short interfering RNA (siRNA); short hairpin RNA (shRNA); antisense RNA; RNA aptamer; self-amplifying RNA (saRNA); interfering RNA (RNAi); non-coding RNA; circular RNA; and small RNA.

[0595] 29. A method or use according to any preceding clause, wherein the template nucleic acid sequence encodes an antigen derived from a virus, bacterium, mycoplasma, fungus, animal, plant, algae, parasite or protozoa, or other organism that causes disease in a subject (preferably a human or animal).

[0596] 30. A method or use according to any preceding clause, wherein the template nucleic acid encodes an innate inhibitor protein that counteracts the innate immune response of a subject administered a vaccine comprising an RNA molecule.

[0597] 31. An RNA molecule obtained or obtainable by a method according to any one of clauses 1 to 30.

[0598] 32. A pharmaceutical composition comprising the RNA molecule according to clause 31 and a pharmaceutically acceptable carrier.

[0599] 33. A method of preparing a pharmaceutical composition according to clause 32, the method comprising contacting an RNA molecule according to clause 31 with a pharmaceutically acceptable carrier.

[0600] 34. Use of the RNA molecule according to item 31 or the pharmaceutical composition according to item 32 as a medicament.

[0601] 35. Use of the RNA molecule according to clause 31 or the pharmaceutical composition according to clause 32 for treating, preventing or ameliorating a disease in a subject.

[0602] 36. A vaccine composition comprising the RNA molecule according to clause 31 or the pharmaceutical composition according to clause 32.

[0603] 37. Use of the RNA molecule according to clause 31, the pharmaceutical composition according to clause 32 or the vaccine according to clause 36 for stimulating an immune response in a subject.

[0604] 38. One or more modified nucleotide triphosphates (NTPs) for:

[0605] (i) enhancing the expression and / or translation of an RNA molecule comprising one or more modified NTPs;

[0606] (ii) enhancing the stability of RNA molecules comprising one or more modified NTPs; and / or

[0607] (iii) reducing the activation of innate sensing, interferon production and / or degradation of RNA molecules comprising one or more modified NTPs,

[0608] wherein the one or more modified NTPs comprise a 2' substituted group, wherein the OH group at the 2' position is typically substituted with halogen, an optionally substituted aromatic group, N3, H, an optionally substituted O-alkyl, O-alkenyl or O-alkynyl, or an optionally substituted alkyl, alkenyl or alkynyl, wherein in each case the aromatic group, alkyl, alkenyl or alkynyl is optionally substituted with halogen, oxo, OR, CN, NR2 or SR, wherein R is H or C 1-6 Alkyl, C 2-6 Alkenyl or C 2-6 Alkynyl.

[0609] 39. One or more modified NTPs for the use according to clause 38, wherein the use comprises contacting (i) a template nucleic acid sequence, (ii) an RNA polymerase, and (iii) a plurality of nucleotide triphosphates (NTPs) in the presence of at least 20 mM magnesium ions, wherein one or more of the nucleotide triphosphates (NTPs) are modified nucleotide triphosphates (NTPs), wherein the RNA polymerase transcribes the template nucleic acid sequence to form the RNA molecule.

[0610] 40. One or more modified NTPs for use according to any one of clauses 38 or 39, wherein the one or more modified NTPs are incorporated into the RNA molecule using a method according to any one of clauses 1 to 30.

[0611] 41. One or more modified NTPs for use according to any one of clauses 38 to 40, wherein the one or more modified NTPs are as defined in any one of clauses 1 to 30.

[0612] 42. One or more modified NTPs for use according to any one of clauses 38 to 41, wherein the one or more modified NTPs comprise 2'-O-methyl-modified ATP, 2'-O-methyl-modified CTP, 2'-O-methyl-modified GTP, and / or 2'-O-methyl-modified UTP.

[0613] 43. One or more modified NTPs for use according to any one of clauses 38 to 42, wherein the RNA is selected from: messenger RNA (mRNA); self-amplifying RNA (saRNA); microRNA (miRNA); short interfering RNA (siRNA); short hairpin RNA (shRNA); antisense RNA; RNA aptamer; interfering RNA (RNAi); non-coding RNA; circular RNA; and small RNA.

[0614] 44. One or more modified NTPs for use according to any one of clauses 38 to 43, wherein the RNA molecule is saRNA, optionally wherein the RNA molecule comprises modified 2'-O-methyl-GTP.

[0615] 45. One or more modified NTPs for use according to any one of clauses 38 to 44, wherein the RNA molecule is mRNA, optionally wherein the RNA molecule comprises modified 2'-O-methyl-GTP and modified 2'-O-methyl CTP.

[0616] 46. ​​One or more modified NTPs for use according to any one of clauses 38 to 45, wherein the length of the RNA molecule is as defined in clause 8 or clause 9.

[0617] 47. Use of one or more 2'-O-methyl modified nucleotide triphosphates (NTPs) in an RNA molecule for enhancing translation of said RNA molecule.

Claims

1. One or more modified nucleotide triphosphates (NTPs) for use in: (i) enhancing the expression and / or translation of an RNA molecule comprising said one or more modified NTPs; (ii) enhancing the stability of RNA molecules comprising said one or more modified NTPs; and / or (iii) reducing the activation of innate sensing, interferon production and / or degradation of RNA molecules comprising said one or more modified NTPs, in, The one or more modified NTPs contain a 2' substituted group, wherein the OH group, typically at the 2' position, is replaced by a halogen, an optionally substituted aromatic group, an NH 2 、N 3 , H, optionally substituted O-alkyl, O-alkenyl or O-alkynyl, or optionally substituted alkyl, alkenyl or alkynyl, wherein in each case the aromatic group, alkyl, alkenyl or alkynyl is optionally substituted by halogen, oxo, OR, CN, NR 2 or SR, wherein R is H or C 1-6 Alkyl, C 2-6 Alkenyl or C 2-6 Alkynyl.

2. One or more modified NTPs for use according to claim 1, in, The RNA molecule is selected from: messenger RNA (mRNA), micro RNA (miRNA), short interfering RNA (siRNA), short hairpin RNA (shRNA), antisense RNA, RNA aptamer, self-amplifying RNA (saRNA), interfering RNA (RNAi), non-coding RNA, circular RNA, and small RNA.

3. One or more modified NTPs for use according to claim 1 or 2, in, The RNA molecule is a self-amplifying RNA (saRNA).

4. One or more modified NTPs for use according to claim 1 or 2, in, The RNA molecule is messenger RNA (mRNA).

5. One or more modified NTPs for use according to any one of the preceding claims, in, The one or more modified NTPs are selected from modified adenosine-5'-triphosphate (ATP), modified cytidine-5'-triphosphate (CTP), modified guanosine-5'-triphosphate (GTP), modified uridine-5'-triphosphate (UTP), and modified thymidine-5'-triphosphate (TTP).

6. One or more modified NTPs for use according to any one of the preceding claims, in, The one or more modified NTPs include at least two, three, four or five modified NTPs selected from the group consisting of modified ATP, modified CTP, modified GTP, modified UTP, and / or modified TTP.

7. One or more modified NTPs for use according to any one of the preceding claims, in, The one or more modified NTPs comprise substituted nucleobases, wherein the nucleobases are replaced by halogens, optionally substituted aromatic groups, NH 2 、N 3 , OH, optionally substituted O-alkyl, O-alkenyl or O-alkynyl, or optionally substituted alkyl, alkenyl or alkynyl, wherein in each case the aromatic group, alkyl, alkenyl or alkynyl is optionally substituted by halogen, oxo, OR, CN, NR 2 or SR, wherein R is H or C 1-6 Alkyl, C 2-6 Alkenyl or C 2-6 Alkynyl, optionally, wherein, alternatively or additionally, the oxo group in the nucleobase is replaced by a =S group.

8. One or more modified NTPs for use according to any one of the preceding claims, in, The alkyl, alkenyl or alkynyl group is an optionally substituted C 1 -C 20 Alkyl, alkenyl or alkynyl NTP, wherein the alkyl, alkenyl or alkynyl is optionally substituted by halogen, oxo, OR, CN, NR 2 or SR, wherein R is H or C 1-6 Alkyl, C 2-6 Alkenyl or C 2-6 Alkynyl; preferably wherein the alkyl, alkenyl or alkynyl is an optionally substituted C 1 -C 10 Alkyl, alkenyl or alkynyl NTP, wherein the alkyl, alkenyl or alkynyl is optionally substituted by halogen, oxo, OR, CN, NR 2 or SR, wherein R is H or C 1-6 Alkyl, C 2-6 Alkenyl or C 2-6 Alkynyl.

9. One or more modified NTPs for use according to any one of the preceding claims, in, The alkyl, alkenyl or alkynyl group is an optionally substituted C 1-6 Alkyl, C 2-6 Alkenyl or C 2-6 Alkynyl, wherein the alkyl, alkenyl or alkynyl, or each of them is optionally substituted by halogen, oxo, OR, CN, NR 2 or SR, wherein R is H or C 1-6 Alkyl, C 2-6 Alkenyl or C 2-6 Alkynyl; preferably wherein the alkyl, alkenyl or alkynyl is an optionally substituted C 1-3 Alkyl, C 2-3 Alkenyl or C 2-3 Alkynyl, wherein the alkyl, alkenyl or alkynyl, or each of them is substituted by one or more selected from oxo, OH, OMe, NH 2 and NRH substituents, wherein R is C 1-6 Alkyl, C 2-6 Alkenyl or C 2-6 Alkynyl.

10. One or more modified NTPs for use according to any one of the preceding claims, in, The one or more modified NTPs comprise 2'-methyl modified NTPs.

11. One or more modified NTPs for use according to any one of the preceding claims, in, The one or more modified NTPs comprise 2'-O-methyl modified NTPs.

12. One or more modified NTPs for use according to any one of the preceding claims, in, The one or more modified NTPs are selected from: 2'-O-methyladenosine (Am); 1,2'-dimethyladenosine (m 1 Am); N6,2'-O-dimethyladenosine (m 6 Am); N 6 ,N 6 ,2-O-trimethyladenosine (m 6,6 Am); 2'-O-methyladenosine (Im); 1,2'-O-dimethyladenosine (m 1 Im); 2'-O-methylguanosine (Gm); 1,2'-O-dimethylguanosine (m 1 Gm); N2,2'-O-dimethylguanosine (m 2 Gm); N2,N2,2'-O-trimethylguanosine (m 2,2 Gm); N2,7,2'-O-trimethylguanosine (m 2,7 Gm); 2'-O-methylcytidine; N 4 ,2'-O-dimethylcytidine (m 4 Cm); N 4 ,N 4 ,2-O-trimethylcytidine (m 4,4 Cm); 5,2'-O-dimethylcytidine (m 5 Cm); N 4 -Acetyl-2'-O-methylcytidine (ac 4 Cm); 2'-O-methyl-5-hydroxymethylcytidine (hm 5 Cm); 5-formyl-2'-O-methylcytosine (f 5 Cm); 2'-O-methyluridine (Um); 3,2'-O-dimethyluridine (m 3 Um); 5,2'-O-dimethyluridine (m 5 Um or Tm); 2-thio-2'-O-methyluridine (s 2 Um); 2'-O-methyl-pseudouridine (Ym); 5-carbamoylmethyl-2'-O-methyluridine (ncm 5 Um); 5-methoxycarbonylmethyl-2'-O-methyluridine (mcm 5 Um); 5-(carboxyhydroxymethyl)-2'-O-methyluridine methyl ester (mchm 5 Um); 5-carboxymethylaminomethyl-2'-O-methyluridine (cmbm 5 Um); 2'-O-methyluridine-5-hydroxyacetate (mcmo 5 Um); 5-(isopentenylaminomethyl)-3'O-methyluridine (inm 5 Um); and any other synthetic NTP containing 2'-O-methylated ribose.

13. One or more modified NTPs for use according to any one of the preceding claims, in, The one modified NTP is selected from the group consisting of: 2'-O-methyl-ATP, 2'-O-methyl-CTP, 2'-O-methyl-GTP, and 2'-O-methyl-UTP.

14. One or more modified NTPs for use according to any one of the preceding claims, in, The at least two modified NTPs are selected from the group consisting of: 2'-O-methyl-ATP, 2'-O-methyl-CTP, 2'-O-methyl-GTP, and 2'-O-methyl-UTP.

15. One or more modified NTPs for use according to any one of the preceding claims, in, The at least three modified NTPs are selected from the group consisting of: 2'-O-methyl-ATP, 2'-O-methyl-CTP, 2'-O-methyl-GTP, and 2'-O-methyl-UTP.

16. One or more modified NTPs for use according to any one of the preceding claims, in, The at least four modified NTPs are selected from the group consisting of: 2'-O-methyl-ATP, 2'-O-methyl-CTP, 2'-O-methyl-GTP, and 2'-O-methyl-UTP.

17. One or more modified NTPs for use according to any one of the preceding claims, in, When the RNA molecule is a saRNA, the RNA molecule comprises a modified GTP, preferably a modified 2'-O-methyl-GTP.

18. One or more modified NTPs for use according to any one of the preceding claims, in, When the RNA molecule is mRNA, the RNA molecule comprises a modified CTP and a modified GTP, preferably a modified 2'-O-methyl CTP and a modified 2'-O-methyl-GTP.

19. One or more modified NTPs for use according to any one of the preceding claims, in, At least 30%, 35% or 40% of the constituent nucleotides in the RNA molecule are modified, wherein the modified nucleotides include adenine, cytosine, guanine and / or uracil; preferably, at least 45%, 50% or 55% of the constituent nucleotides in the RNA molecule are modified, wherein the modified nucleotides include adenine, cytosine, guanine and / or uracil.

20. One or more modified NTPs for use according to any one of the preceding claims, in, One or more 2-O-methyl modified NTPs are combined with one or more other modified NTP bases that are not 2-O-methyl modified NTPs.

21. One or more modified NTPs for use according to any one of the preceding claims, in, Combine (i) and (ii) below: (i) the one or more modified NTPs comprising a 2' substituted group, wherein the OH group at the 2' position is usually replaced by a halogen, an optionally substituted aromatic group, an NH 2 、N 3 , H, optionally substituted O-alkyl, O-alkenyl or O-alkynyl, or optionally substituted alkyl, alkenyl or alkynyl, wherein in each case the aromatic group, alkyl, alkenyl or alkynyl is optionally substituted by halogen, oxo, OR, CN, NR 2 or SR, wherein R is H or C 1-6 Alkyl, C 2-6 Alkenyl or C 2-6 Alkynyl; and (ii) one or more modified NTPs that are not 2'-O-methyl modified NTPs.

22. One or more modified NTPs for use according to claim 21, in, Combine (i) and (ii) below: (i) the one or more 2-O-methyl modified NTPs are selected from: 2'-O-methyl modified ATP (2'-O-methyl-ATP), 2'-O-methyl modified CTP (2'-O-methyl-CTP), 2'-O-methyl modified GTP (2'-O-methyl-GTP), and 2'-O-methyl modified UTP (2'-O-methyl-UTP); and (ii) one or more modified NTP bases that are not 2-O-methyl modified NTPs.

23. One or more modified NTPs for use according to claim 21 or 22, in, The one or more modified NTP bases that are not 2-O-methyl modified NTPs are: (i) N'methyl-pseudouridine, fluorinated UTP, 2'-chloroNTP, 2'-ethylNTP, 2'-bromoNTP, 2'-aminoNTP, 2'-fluoroNTP, or 2'-deoxyNTP; or (ii) additional 2' modified nucleotides listed in Table 7.

24. One or more modified NTPs for use according to any one of the preceding claims, in, The RNA molecule is encoded by a template nucleic acid sequence comprising DNA.

25. One or more modified NTPs for use according to claim 24, in, The template nucleic acid sequence encodes an antigen derived from a tumor, virus, bacteria, mycoplasma, fungus, animal, plant, algae, parasite or protozoa, or other organisms that cause disease in a subject (preferably a human or an animal).

26. One or more modified NTPs for use according to claim 24, in, The template nucleic acid encodes a therapeutic protein, which is derived from an animal or a human and treats, prevents or improves a disease in a subject, preferably a human or an animal subject.

27. One or more modified NTPs for use according to any one of claims 22 to 26, in, The template nucleic acid encodes an innate suppressor protein that counteracts the innate immune response of a subject administered a vaccine comprising an RNA molecule.

28. One or more modified NTPs for use according to any one of the preceding claims, in, The use comprises contacting (i) a template nucleic acid sequence, (ii) an RNA polymerase, and (iii) a plurality of nucleotide triphosphates (NTPs) in the presence of at least 20 mM magnesium ions, wherein one or more of the nucleotide triphosphates (NTPs) are modified nucleotide triphosphates (NTPs), wherein the RNA polymerase transcribes the template nucleic acid sequence to form the RNA molecule.

29. A method for preparing a modified RNA molecule, in, The method comprises: contacting (i) a template nucleic acid sequence, (ii) an RNA polymerase, and (iii) a plurality of nucleotide triphosphates (NTPs) in the presence of at least 20 mM magnesium ions, wherein one or more of the nucleotide triphosphates (NTPs) are modified nucleotide triphosphates (NTPs), wherein the RNA polymerase transcribes the template nucleic acid sequence to form an RNA molecule comprising at least 20 nucleotides, and wherein at least 25% of the constituent nucleotides in the RNA molecule are modified.

30. Use of 20 mM magnesium ions in a transcription reaction to prepare a modified RNA molecule comprising at least 20 nucleotides, in, At least 25% of the constituent nucleotides in the RNA molecule are modified.

31. The method or use according to claim 29 or 30, in, The method comprises using: (i) at least 30 mM magnesium ion, at least 40 mM magnesium ion, at least 50 mM magnesium ion, at least 60 mM magnesium ion, or at least 70 mM magnesium ion; or (ii) 50 mM to 100 mM magnesium ion, 60 mM to 95 mM magnesium ion, 65 mM to 90 mM magnesium ion, 70 mM to 80 mM magnesium ion, or 71 mM to 79 mM magnesium ion.

32. The method or use according to any one of claims 29 to 31, in, The magnesium ion is Mg 2+ Preferably, the magnesium ions are provided in the form of magnesium acetate.

33. The method or use according to any one of claims 29 to 32, in, The RNA is as defined in any one of claims 2 to 4.

34. The method or use according to any one of claims 29 to 33, in, The RNA molecule has: (i) at least 20, 21, 22 or 23 bases in length; (ii) at least 24, 25, 26 or 27 bases in length; (iii) at least 28, 29, 30 or 31 bases in length; or (iv) at least 32, 33, 34 or 35 bases in length.

35. The method or use according to any one of claims 29 to 34, in, The RNA molecule has: (i) at least 50 bases in length, at least 60 bases in length, at least 75 bases in length, at least 100 bases in length, at least 200 bases in length, at least 300 bases in length, at least 400 bases in length, at least 500 bases in length, at least 600 bases in length, at least 700 bases in length, at least 800 bases in length, or at least 900 bases in length; (ii) at least 1000 bases in length, at least 2000 bases in length, at least 3000 bases in length, at least 4000 bases in length, at least 5000 bases in length, at least 6000 bases in length, at least 7000 bases in length, at least 8000 bases in length, at least 9000 bases in length, at least 10000 bases in length, at least 11000 bases in length, or at least 12000 bases in length.

36. The method or use according to any one of claims 29 to 35, in, The method comprises using an RNA polymerase selected from: T7; T3; SP6; KP34; Syn5; or other DNA-dependent RNA polymerases; or mutant variants of any of these RNA polymerases.

37. The method or use according to any one of claims 29 to 36, in, The method comprises using the plurality of nucleotide triphosphates at the following concentrations: (i) at least 1 mM, 2 mM, 3 mM or 4 mM; (ii) at least 5 mM, 6 mM or 7 mM; or (iii) at least 8 mM, 9 mM, 10 mM.

38. The method or use according to any one of claims 29 to 37, in, The one or more modified NTPs are as defined in any one of claims 1 to 28.

39. The method or use according to any one of claims 29 to 38, in, The template nucleic acid sequence is as defined in any one of claims 24 to 27.

40. An RNA molecule obtained or obtainable by the method of any one of claims 29 to 39.

41. A pharmaceutical composition comprising the RNA molecule according to claim 40 and a pharmaceutically acceptable carrier.

42. A method for preparing a pharmaceutical composition according to claim 41, the method comprising contacting the RNA molecule according to claim 40 with a pharmaceutically acceptable carrier.

43. Use of the RNA molecule according to claim 40 or the pharmaceutical composition according to claim 41 as a medicament.

44. Use of the RNA molecule according to claim 40 or the pharmaceutical composition according to claim 41 for treating, preventing or ameliorating a disease in a subject.

45. A vaccine composition comprising the RNA molecule according to claim 40 or the pharmaceutical composition according to claim 41.

46. ​​Use of the RNA molecule according to claim 40, the pharmaceutical composition according to claim 41 or the vaccine according to claim 45 for stimulating an immune response in a subject.

47. Use of one or more 2'-O-methyl modified nucleotide triphosphates (NTPs) in an RNA molecule for enhancing translation of said RNA molecule.

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