RNA molecules

Abstract

The present invention relates to RNA molecules, methods for preparing RNA molecules, and methods for translating RNA molecules into proteins. The present invention extends to improved methods for forming RNA by in vitro transcription, and the resulting RNA molecules. Furthermore, the present invention relates to novel methods for improving RNA expression and / or translation, i.e., protein expression, and methods for improving the stability of RNA molecules. The present invention also includes reducing the activation of innate sensing, interferon production, and / or degradation of RNA molecules in a host. The present invention also incorporates the use of RNA molecules in vaccines and other therapeutic pharmaceutical compositions, and their use in immunization and therapy, such as RNAi, gene therapy, gene editing, and protein replacement.

Description

[Technical field]

[0001] The present invention relates to RNA molecules, and in particular, but not exclusively, to methods for preparing RNA molecules and methods for translating RNA molecules into proteins. The invention extends to improved methods for forming RNA by in vitro transcription and the resulting RNA molecules. Furthermore, the invention relates to novel methods for improving the expression and / or translation of RNA, i.e., protein expression, and methods for improving the stability of RNA molecules. The invention also includes reducing the activation of innate sensing, interferon production, and / or degradation of RNA molecules in a host. The invention also incorporates the use of RNA molecules in vaccines and other therapeutic pharmaceutical compositions, and their use in immunization and therapy, such as RNAi, gene therapy, gene editing, and protein replacement. [Background technology]

[0002] Recently, the era of RNA vaccines and biologics has come and gone, showing successful use in the COVID-19 pandemic. Two COVID-19 lead candidates manufactured by Moderna and Pfizer both utilize the incorporation of N1-methyl-pseudouridine, a synthetic modified version of the nucleotide triphosphate (NTP) UTP [1]. This is used to reduce the induction of innate pathways that may limit the expression of encoded proteins and render RNA ineffective for therapeutic or vaccine applications [2,3]. The exact mechanism of improved expression is not fully understood and may be related to the suppression of highly immunogenic double-stranded RNA (dsRNA) during in vitro transcription

[11] . However, this approach provides little or no benefit for self-amplifying RNA (Figure 16). Furthermore, the use of methyl-pseudouridine may be suboptimal. Indeed, most cellular receptors detect most double-stranded RNA (dsRNA) structures by contacting the ribose backbone with minimal interactions with the bases. In this regard, ribose base modifications are predicted to have beneficial effects, such as the base 2'-methyl-NTP with respect to innate recognition. However, efficient incorporation of 2'-methyl-NTP in mRNA or saRNA of sequences longer than 100 bp has not previously been achievable. Furthermore, previous studies have shown that the use of 2'-methyl-NTP can reduce innate recognition, while the natural incorporation of 2'-methyl-NTP in coding RNA is generally believed to inhibit or reduce translation

[15] . [Prior art documents] [Patent documents]

[0003] [Patent Document 1] WO2020 / 254804 [Patent Document 2] PCT / GB2021 / 053362 [Patent Document 3] PCT / GB2021 / 053361 Summary of the Invention [Problem to be solved by the invention]

[0004] Ribose 2'-OH replacement with a methyl group is a common naturally occurring post-transcriptional modification, and 2'-O-methyl (2-OMe) modified RNAs may be less immunogenic and demonstrate greater stability. 2-OMe modification consists of methylation of the ribose 2'-OH moiety and therefore can occur in all four nucleotides and other non-classical nucleotides. Utilizing in vitro transcription methods that maximize synthesis of modified RNA from RNA polymerases such as T7, T3, SP6, KP34 and Syn5, 2'-O-methyl modified RNAs have been generated for small RNA sequences (less than 20 bp in length), such as aptamers. However, generating longer 2'-O-methyl modified RNAs (greater than 20 bp) is currently problematic. For example, rigorous optimization of the transcription reaction mixture is required to support a high and wide range of percentages of nucleotide substitutions of wild-type (i.e., unmodified) NTPs. Moreover, even the generation of 2'-O-methyl modified RNA aptamers by in vitro transcription is currently inefficient and may leave room for improvement. Overcoming these obstacles may improve the in vivo application and potential of 2'-methyl RNAs intended for use as vaccines or biologics.

[0005] As explained above, ribose 2'-O-methylation is a common type of RNA modification found in eukaryotic mRNAs. Ribose 2'-O-methylation of the first and sometimes second cap-proximal nucleotides (N1 and N2 (N corresponds to any nucleotide)) results in Cap1-(m7GpppNmN) or Cap2-(m7GpppNmNm) mRNAs, which are known to serve as important molecular signatures of "self" to escape detection by the innate immune system

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

[15] . This is further supported by data demonstrating that the presence of 2'-O-methylation in some mRNAs can selectively reduce translation elongation in vitro and in vivo [16, 17]. Natural 2'-O-methylation in mRNA coding regions is therefore understood to negatively regulate RNA translation and protein expression

[18] . Therefore, there is a need in the art to overcome this problem and provide improved methods for preparing modified RNA molecules (e.g., mRNAs or self-amplifying RNAs) that incorporate modified non-natural nucleotide bases. There is also a need to provide improved methods for enhancing RNA translation and protein expression. [Means for solving the problem]

[0006] RNA is composed of the nucleotides ATP, CTP, GTP and UTP, however, a wide range of natural and synthetic modified nucleotides exist [4]. As described in the Examples, the inventors have demonstrated that incorporation of the modified base 2'-methyl-NTP (2-OMe) surprisingly improves RNA expression, both self-amplifying RNA (saRNA) and mRNA, in interferon-responsive cells. Substituting 75% or more of GTP with 2'-methylGTP (Gm) improves saRNA expression by 10-fold. Substituting 75% or more of ATP, UTP or CTP with 2'-methyl modified nucleotides also improved saRNA expression in the cell types tested. The inventors believe that combination substitutions may also provide additional benefits. For example, we observed that replacing 75% or more of CTP with 2'-methyl CTP (Cm) improved mRNA expression (up to 10-fold, see Figures 33 and 34), while replacing GTP, ATP or UTP had little or no effect (see Figures 29-32). However, combined replacement of 75% or more, or 2'-methyl-GTP and -CTP (i.e., Gm+Cm), or 2'-methyl-GTP, -ATP, -UTP and -CTP (i.e., Gm+Am+Cm+Um), surprisingly improved mRNA expression up to or over 100-fold (see Figures 23-28), reaching expression levels comparable to saRNA (see Figures 21 and 22). This was completely unexpected given the evidence that incorporation of 2'-O methylated NTPs in the native environment has been shown to reduce translation [16-18, 28-29]. Interestingly, these increases in translation were not observed when using modified nucleotides in which the ribose 2'-OH was replaced by a 2'-fluoro group, suggesting that this effect was dependent on the use of a bulkier alkyl group (e.g., a methyl group) as a replacement for the ribose 2'-OH group (see Figures 18 and 19). Furthermore, the improvement in translation levels was significantly greater than that seen with N1-methyl-pseudouridine for mRNA or saRNA in similar model systems (see Figure 16).

[0007] Thus, the invention described herein overcomes the hurdles of the prior art in producing RNA by replacing a significant proportion of one or more NTPs with 2'-methyl-NTPs and / or other modified NTPs that are not amenable to efficient incorporation using conventional in vitro transcription (IVT) conditions.

[0008] Thus, in a first aspect of the present invention, there is provided a method of preparing a modified RNA molecule, the method comprising the steps of 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 NTPs are modified nucleotide triphosphates (NTPs), and 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 component nucleotides in the RNA molecule are modified.

[0009] 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 component nucleotides within the RNA molecule are modified.

[0010] Advantageously, the inventors have devised new and innovative in vitro transcription reaction conditions for use in the methods of the invention. These reaction conditions contain higher magnesium ions (i.e., greater than 20 mM) than previously used, and therefore allow for the replacement of significant levels of wild-type (i.e., unmodified) nucleotides in the resulting RNA molecules. The prior art use of 2'-methyl-NTPs in small RNA sequences less than 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, in vitro transcription has not allowed the generation of significant length (20 bp, 50 bp, or 100 bp or more) mRNA or saRNA sequences in which a significant proportion (i.e., greater than 25%) of single or multiple wild-type unmodified nucleotides have been replaced by 2'-methyl-NTPs.

[0011] The in vitro transcription reaction mixture in the method of the present invention requires the use of more than 20 mM magnesium ion, which is obviously higher than some conventional in vitro transcription methods.The 2'-methyl-modified RNAs, both mRNA and saRNA, synthesized from this method exhibit significantly higher expression in interferon-responsive cells.The inventors therefore believe that the same reaction conditions can promote the incorporation of other modified NTPs that were refractory to existing IVT conditions.

[0012] Thus, the method of the present invention advantageously provides improved production of both mRNA and saRNA with significant 2' methyl-NTP substitutions (greater than 25% and even up to 100%), demonstrating that such substitutions can significantly improve the expression of mRNA and saRNA in interferon-competent cells. This is likely to improve the in vivo potential of RNA vaccines (e.g., cancer vaccines expressing microbial antigens to immunize against infectious microorganisms such as viruses and bacteria, and also expressing oncogenic antigens) and RNA biologics (e.g., RNAi, protein replacement therapy, gene editing and gene therapy, etc.), whether delivered as mRNA or saRNA. Furthermore, the same approach is also likely to facilitate the incorporation of other unnatural bases [4] that have not been able to be incorporated into mRNA or saRNA to date.

[0013] For a better understanding of the present invention, and to show how embodiments thereof may be put into effect, reference will now be made, by way of example, to the accompanying drawings in which: [Brief description of the drawings]

[0014] [Figure 1] FIG. 1 shows that when magnesium acetate is used, efficient incorporation of 2′-methyl-ribose modified NTPs occurs only at magnesium levels above 20 nM. A) RNA yield ng / μl, B) RNA product assessed by gel electrophoresis. [Diagram 2] FIG. 1 shows that low or no incorporation of 2′methyl-ribose modified GTP occurs when magnesium chloride is used. A) RNA yield ng / μl, B) RNA product assessed by gel electrophoresis. [Diagram 3] FIG. 1 illustrates that when the use of magnesium acetate is replaced by manganese acetate, no detectable incorporation of 2'methyl-ribose modified GTP is observed. A) RNA yield ng / μl, B) RNA product assessed by gel electrophoresis. [Figure 4]FIG. 4A illustrates the effect of 2'-O-methyl NTP substitution on uncapped saRNA expression of fLuc. Uncapped saRNA encoding fLuc was generated by IVT, where individual nucleotides were replaced by 25, 50 or 75% 2'-O-methyl modified versions. HEK293T cells were transfected with 25 ng saRNA formulated in lipofectamine. Luciferase expression was monitored over 72 hours by measuring relative luminescence (see Methods). FIG. 4B illustrates the effect of 2'-O-methyl NTP substitution on uncapped saRNA expression of fLuc. Uncapped saRNA encoding fLuc was generated by IVT, where individual nucleotides were replaced by 25, 50 or 75% 2'-O-methyl modified versions. HEK293T cells were transfected with 25 ng saRNA formulated in lipofectamine. Fold change in expression relative to unmodified saRNA is shown. [Diagram 5] FIG. 5A shows the effect of 2'-O-methyl NTP substitution on uncapped saRNA expression of fLuc. Uncapped saRNA encoding fLuc was generated by IVT, where individual nucleotides were replaced by 25, 50 or 75% 2'-O-methyl modified versions. HEK293T cells were transfected with 100 ng saRNA formulated in lipofectamine. Luciferase expression was monitored over 72 hours by measuring relative luminescence (see Methods). FIG. 5B shows the effect of 2'-O-methyl NTP substitution on uncapped saRNA expression of fLuc. Uncapped saRNA encoding fLuc was generated by IVT, where individual nucleotides were replaced by 25, 50 or 75% 2'-O-methyl modified versions. HEK293T cells were transfected with 100 ng saRNA formulated in lipofectamine. Fold change in expression relative to unmodified saRNA is shown. [Figure 6]FIG. 6A illustrates the effect of 2'-O-methyl NTP substitution on 5'-capped saRNA expression of fLuc. 5'-capped saRNA encoding fLuc was generated by IVT, where individual nucleotides were replaced by 25, 50 or 75% 2'-O-methyl modified versions. HEK293T cells were transfected with 25 ng of saRNA formulated in lipofectamine. Luciferase expression was monitored over 72 hours by measuring relative luminescence (see Methods). FIG. 6B illustrates the effect of 2'-O-methyl NTP substitution on 5'-capped saRNA expression of fLuc. 5'-capped saRNA encoding fLuc was generated by IVT, where individual nucleotides were replaced by 25, 50 or 75% 2'-O-methyl modified versions. HEK293T cells were transfected with 25 ng of saRNA formulated in lipofectamine. Fold change in expression relative to unmodified saRNA is shown. [Figure 7] FIG. 7A illustrates the effect of 2'-O-methyl NTP substitution on 5' capped saRNA expression of fLuc. Capped saRNA encoding fLuc was generated by IVT, where individual nucleotides were replaced by 25, 50 or 75% 2'-O-methyl modified versions. HEK293T cells were transfected with 100 ng saRNA formulated in lipofectamine. Luciferase expression was monitored over 72 hours by measuring relative luminescence (see Methods). FIG. 7B illustrates the effect of 2'-O-methyl NTP substitution on 5' capped saRNA expression of fLuc. Capped saRNA encoding fLuc was generated by IVT, where individual nucleotides were replaced by 25, 50 or 75% 2'-O-methyl modified versions. HEK293T cells were transfected with 100 ng saRNA formulated in lipofectamine. Fold change in expression relative to unmodified saRNA is shown. [Figure 8]FIG. 8A shows the effect of 2'-O-methyl NTP substitution on uncapped saRNA expression of fLuc in HeLa cells. Uncapped saRNA encoding fLuc was generated by IVT in which individual nucleotides were replaced by 25, 50 or 75% 2'-O-methyl modified versions. HeLa cells were transfected with 25 ng saRNA formulated in lipofectamine. Luciferase expression was monitored over 72 hours by measuring relative luminescence (see Methods). FIG. 8B shows the effect of 2'-O-methyl NTP substitution on uncapped saRNA expression of fLuc in HeLa cells. Uncapped saRNA encoding fLuc was generated by IVT in which individual nucleotides were replaced by 25, 50 or 75% 2'-O-methyl modified versions. HeLa cells were transfected with 25 ng saRNA formulated in lipofectamine. Fold change in expression relative to unmodified saRNA is shown. [Figure 9] FIG. 9A 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, where individual nucleotides were replaced by 25, 50 or 75% 2'-O-methyl modified versions. HeLa cells were transfected with 100 ng saRNA formulated in lipofectamine. Luciferase expression was monitored over 72 hours by measuring relative luminescence (see Methods). FIG. 9B 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, where individual nucleotides were replaced by 25, 50 or 75% 2'-O-methyl modified versions. HeLa cells were transfected with 100 ng saRNA formulated in lipofectamine. Fold change in expression relative to unmodified saRNA is shown. [Figure 10]FIG. 10A 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 generated by IVT, where individual nucleotides were replaced by 25, 50 or 75% 2'-O-methyl modified versions. HeLa cells were transfected with 25 ng of saRNA formulated in lipofectamine. Luciferase expression was monitored over 72 hours by measuring relative luminescence (see Methods). FIG. 10B 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 generated by IVT, where individual nucleotides were replaced by 25, 50 or 75% 2'-O-methyl modified versions. HeLa cells were transfected with 25 ng of saRNA formulated in lipofectamine. The fold change in expression relative to unmodified saRNA is shown. [Figure 11] FIG. 11A 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 generated by IVT, where individual nucleotides were replaced by 25, 50 or 75% 2'-O-methyl modified versions. HeLa cells were transfected with 100 ng of saRNA formulated in lipofectamine. Luciferase expression was monitored over 72 hours by measuring relative luminescence (see Methods). FIG. 11B 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 generated by IVT, where individual nucleotides were replaced by 25, 50 or 75% 2'-O-methyl modified versions. HeLa cells were transfected with 100 ng of saRNA formulated in lipofectamine. The fold change in expression relative to unmodified saRNA is shown. [Figure 12]FIG. 12A shows the effect of 2'-O-methyl NTP substitution on uncapped saRNA expression of fLuc in THP-1 cells. Uncapped saRNA encoding fLuc was generated by IVT, where individual nucleotides were replaced by 25, 50 or 75% 2'-O-methyl modified versions. THP-1 cells were transfected with 25 ng of uncapped saRNA formulated in lipofectamine. Luciferase expression was monitored over 72 hours by measuring relative luminescence (see Methods). FIG. 12B shows the effect of 2'-O-methyl NTP substitution on uncapped saRNA expression of fLuc in THP-1 cells. Uncapped saRNA encoding fLuc was generated by IVT, where individual nucleotides were replaced by 25, 50 or 75% 2'-O-methyl modified versions. THP-1 cells were transfected with 25 ng of uncapped saRNA formulated in lipofectamine. Fold change in expression relative to unmodified saRNA is shown. [Figure 13] Figure 13A shows the effect of 2'-O-methyl NTP substitution on the uncapped saRNA expression of fLuc in THP-1 cells. Uncapped saRNA encoding fLuc was generated by IVT, where individual nucleotides were replaced by 25, 50 or 75% 2'-O-methyl modified versions. THP-1 cells were transfected with 100 ng of uncapped saRNA formulated in lipofectamine. Luciferase expression was monitored over 72 hours by measuring relative luminescence (see Methods). Figure 13B shows the effect of 2'-O-methyl NTP substitution on the uncapped saRNA expression of fLuc in THP-1 cells. Uncapped saRNA encoding fLuc was generated by IVT, where individual nucleotides were replaced by 25, 50 or 75% 2'-O-methyl modified versions. THP-1 cells were transfected with 100 ng of uncapped saRNA formulated in lipofectamine. The fold change in expression relative to unmodified saRNA is shown. [Figure 14]Figure 14A shows the effect of 2'-O-methyl NTP substitution on 5'-capped saRNA expression of fLuc in THP-1 cells. 5'-capped saRNA encoding fLuc was generated by IVT, where individual nucleotides were replaced by 25, 50 or 75% 2'-O-methyl modified versions. THP-1 cells were transfected with 25 ng of uncapped saRNA formulated in lipofectamine. Luciferase expression was monitored over 72 hours by measuring relative luminescence (see Methods). Figure 14B shows the effect of 2'-O-methyl NTP substitution on 5'-capped saRNA expression of fLuc in THP-1 cells. 5'-capped saRNA encoding fLuc was generated by IVT, where individual nucleotides were replaced by 25, 50 or 75% 2'-O-methyl modified versions. THP-1 cells were transfected with 25 ng of uncapped saRNA formulated in lipofectamine. The fold change in expression relative to unmodified saRNA is shown. [Figure 15] Figure 15A shows the effect of 2'-O-methyl NTP substitution on 5'-capped saRNA expression of fLuc in THP-1 cells. 5'-capped saRNA encoding fLuc was generated by IVT, where individual nucleotides were replaced by 25, 50 or 75% 2'-O-methyl modified versions. THP-1 cells were transfected with 25 ng of uncapped saRNA formulated in lipofectamine. Luciferase expression was monitored over 72 hours by measuring relative luminescence (see Methods). Figure 15B shows the effect of 2'-O-methyl NTP substitution on 5'-capped saRNA expression of fLuc in THP-1 cells. 5'-capped saRNA encoding fLuc was generated by IVT, where individual nucleotides were replaced by 25, 50 or 75% 2'-O-methyl modified versions. THP-1 cells were transfected with 25 ng of uncapped saRNA formulated in lipofectamine. The fold change in expression relative to unmodified saRNA is shown. [Figure 16]Figure 1 shows the effect of N1 methyl pseudouridine substitution on uncapped and 5' capped saRNA expression of fLuc in A) HEK293T cells and B) HeLa cells. Uncapped and 5' capped saRNA encoding fLuc were generated by IVT in which 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 luciferase expression was monitored over 72 hours by measuring relative light output (see Methods). [Figure 17] Figure 1 shows the effect of 7-deaza-GTP substitution on uncapped and 5'-capped saRNA expression of fLuc in A) HEK293T cells and B) HeLa cells. Uncapped and 5'-capped saRNA encoding fLuc were generated 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 luciferase expression was monitored over 72 hours by measuring relative light output (see Methods). [Figure 18] Figure 1 shows the effect of 2'-fluoro substitution on uncapped and 5'-capped saRNA expression of fLuc in HEK293T cells. Uncapped and 5'-capped saRNA encoding fLuc were generated by IVT in which unmodified bases were replaced by 25, 50 or 75% 2'fluoro-ATP, CTP or UTP. Cells were transfected with 100 ng of uncapped or capped saRNA formulated in lipofectamine and luciferase expression was monitored over 72 hours by measuring relative light output (see Methods). [Figure 19]Figure 1 shows the effect of 2'-fluoro substitution on uncapped and 5'-capped saRNA expression of fLuc in HeLa cells. Uncapped and 5'-capped saRNA encoding fLuc were generated by IVT in which unmodified bases were replaced by 25, 50 or 75% 2'fluoro-ATP, CTP or UTP. Cells were transfected with 100 ng of uncapped or capped saRNA formulated in lipofectamine and luciferase expression was monitored over 72 hours by measuring relative light output (see Methods). [Figure 20] Figure 1 shows the effect of 2'-O-methyl NTP substitution on 5'-capped saRNA and mRNA expression of fLuc in HEK293T cells. 5'-capped saRNA and mRNA encoding fLuc were generated by IVT. Expression of unmodified saRNA and mRNA was compared to saRNA and mRNA in which 75% of the GTP was replaced by 75% 2'-O-methyl GTP version. In addition, a "combo" mRNA was generated by replacing 75% of the GTP and ATP, and 90% of the CTP and UTP by 2'-O-methyl versions. HEK293T cells were transfected with 100 ng of 5'-capped saRNA and mRNA formulated in lipofectamine. A) Luciferase expression was monitored over 72 hours by measuring relative luminescence. B) Fold change in expression relative to unmodified saRNA is shown. [Figure 21]Figure 1 shows the effect of 2'-O-methyl NTP substitution on 5'-capped saRNA and mRNA expression of fLuc in HeLa cells. 5'-capped saRNA and mRNA encoding fLuc were generated by IVT. Expression of unmodified saRNA and mRNA was compared to saRNA and mRNA in which 75% of the GTP was replaced by 75% 2'-O-methyl GTP version. In addition, a "combo" mRNA was generated by replacing 75% of the GTP and ATP, and 90% of the CTP and UTP by 2'-O-methyl versions. HeLa cells were transfected with 100 ng of 5'-capped saRNA and mRNA formulated in lipofectamine. A) Luciferase expression was monitored over 72 hours by measuring relative luminescence. B) Fold change in expression relative to unmodified saRNA is shown. [Figure 22] Figure 1 shows the effect of 2'-O-methyl NTP substitution on 5'-capped saRNA and mRNA expression of fLuc in THP-1 cells. 5'-capped saRNA and mRNA encoding fLuc were generated by IVT. Expression of unmodified saRNA and mRNA was compared to saRNA and mRNA in which 75% of the GTP was replaced by 75% 2'-O-methyl GTP version. In addition, a "combo" mRNA was generated by replacing 75% of the GTP and ATP, and 90% of the CTP and UTP by 2'-O-methyl versions. THP-1 cells were transfected with 100 ng of 5'-capped saRNA and mRNA formulated in lipofectamine. A) Luciferase expression was monitored over 72 hours by measuring relative luminescence. B) Fold change in expression relative to unmodified saRNA is shown. [Figure 23]Figure 1 shows the effect of 2'-O-methyl NTP substitution on capped mRNA expression of fLuc in HEK293T cells. Capped mRNA encoding fLuc was generated by IVT in which 75% or more of the nucleotides were replaced by 2'-O-methyl modified versions. Combo conditions are conditions in which all four NTPs are replaced. HEK293T cells were transfected with 25ng of capped mRNA formulated in lipofectamine. Luciferase expression was monitored over 72 hours by measuring relative light output (see Methods). [Figure 24] Figure 1 shows the effect of 2'-O-methyl NTP substitution on capped mRNA expression of fLuc in HEK293T cells. Capped mRNA encoding fLuc was generated by IVT in which 75% or more of the nucleotides were replaced by 2'-O-methyl modified versions. Combo conditions are conditions in which all four NTPs are replaced. HEK293T cells were transfected with 25ng of capped mRNA formulated in lipofectamine. Luciferase expression was monitored over 72 hours by measuring relative light output and is expressed as fold change in expression relative to unmodified saRNA. [Diagram 25] Figure 1 shows the effect of 2'-O-methyl NTP substitution on capped mRNA expression of fLuc in HeLa cells. Capped mRNA encoding fLuc was generated by IVT in which 75% or more of the nucleotides were replaced by 2'-O-methyl modified versions. Combo conditions are conditions in which all four NTPs are replaced. HeLa cells were transfected with 25 ng of capped mRNA formulated in lipofectamine. Luciferase expression was monitored over 72 hours by measuring relative light output (see Methods). [Figure 26]Figure 1 shows the effect of 2'-O-methyl NTP substitution on capped mRNA expression of fLuc in HeLa cells. Capped mRNA encoding fLuc was generated by IVT in which 75% or more of the nucleotides were replaced by 2'-O-methyl modified versions. Combo conditions are conditions in which all four NTPs are replaced. HeLa cells were transfected with 25ng of capped mRNA formulated in lipofectamine. Luciferase expression was monitored over 72 hours by measuring relative light output and is expressed as fold change in expression relative to unmodified saRNA. [Figure 27] Figure 1 shows the effect of 2'-O-methyl NTP substitution on capped mRNA expression of fLuc in THP-1 cells. Capped mRNA encoding fLuc was generated by IVT in which 75% or more of the nucleotides were replaced by 2'-O-methyl modified versions. Combo conditions are conditions in which all four NTPs are replaced. THP-1 cells were transfected with 25 ng of capped mRNA formulated in lipofectamine. Luciferase expression was monitored over 72 hours by measuring relative light output (see Methods). [Figure 28] Figure 1 shows the effect of 2'-O-methyl NTP substitution on capped mRNA expression of fLuc in THP-1 cells. Capped mRNA encoding fLuc was generated by IVT, where 75% or more of the nucleotides were replaced by 2'-O-methyl modified versions. Combo conditions are conditions where all four NTPs are replaced. THP-1 cells were transfected with 25ng of capped mRNA formulated in lipofectamine. Luciferase expression was monitored over 72 hours by measuring relative light output and is expressed as fold change in expression relative to unmodified saRNA. [Figure 29]Figure 1 shows the effect of 2'-O-methyl NTP substitution on capped mRNA expression of fLuc in HEK293T cells. Capped mRNA encoding fLuc was generated by IVT in which 75% or more of individual nucleotides were replaced by 2'-O-methyl modified versions. Combo conditions are conditions in which all four NTPs are replaced. HEK293T cells were transfected with 25ng of capped mRNA formulated in lipofectamine. Luciferase expression was monitored over 72 hours by measuring relative light output (see Methods). [Diagram 30] Figure 1 shows the effect of 2'-O-methyl NTP substitution on capped mRNA expression of fLuc in HEK293T cells. Capped mRNA encoding fLuc was generated by IVT in which 75% or more of individual nucleotides were replaced by 2'-O-methyl modified versions. Combo conditions are conditions in which all four NTPs are replaced. HEK293T cells were transfected with 100 ng of capped mRNA formulated in lipofectamine. Luciferase expression was monitored over 72 hours by measuring relative light output (see Methods). [Diagram 31] Figure 1 shows the effect of 2'-O-methyl NTP substitution on capped mRNA expression of fLuc in HeLa cells. Capped mRNA encoding fLuc was generated by IVT in which 75% or more of individual nucleotides were replaced by 2'-O-methyl modified versions. Combo conditions are conditions in which all four NTPs are replaced. HeLa cells were transfected with 25 ng of capped mRNA formulated in lipofectamine. Luciferase expression was monitored over 72 hours by measuring relative light output (see Methods). [Diagram 32]Figure 1 shows the effect of 2'-O-methyl NTP substitution on capped mRNA expression of fLuc in HeLa cells. Capped mRNA encoding fLuc was generated by IVT in which 75% or more of individual nucleotides were replaced by 2'-O-methyl modified versions. Combo conditions are conditions in which all four NTPs are replaced. HeLa cells were transfected with 100 ng of capped mRNA formulated in lipofectamine. Luciferase expression was monitored over 72 hours by measuring relative light output (see Methods). [Diagram 33] Figure 1 shows the effect of 2'-O-methyl NTP substitution on capped mRNA expression of fLuc in THP-1 cells. Capped mRNA encoding fLuc was generated by IVT in which 75% or more of individual nucleotides were replaced by 2'-O-methyl modified versions. Combo conditions are conditions in which all four NTPs are replaced. THP-1 cells were transfected with 25 ng of capped mRNA formulated in lipofectamine. Luciferase expression was monitored over 72 hours by measuring relative light output (see Methods). [Diagram 34] Figure 1 shows the effect of 2'-O-methyl NTP substitution on capped mRNA expression of fLuc in THP-1 cells. Capped mRNA encoding fLuc was generated by IVT, where individual nucleotides were replaced by 75% or more 2'-O-methyl modified versions. Combo conditions are conditions where all four NTPs are replaced. THP-1 cells were transfected with 25ng of capped mRNA formulated in lipofectamine. Luciferase expression was monitored over 72 hours by measuring relative light output (see Methods) and is expressed as fold change in expression relative to unmodified saRNA. [Diagram 35]Figure 1 shows the effect of 2'-O-methyl NTP substitution on capped mRNA expression of fLuc in THP-1 cells. Capped mRNA encoding fLuc was generated by IVT in which 75% or more of individual nucleotides were replaced by 2'-O-methyl modified versions. Combo conditions are conditions in which all four NTPs are replaced. THP-1 cells were transfected with 100 ng of capped mRNA formulated in lipofectamine. Luciferase expression was monitored over 72 hours by measuring relative light output (see Methods). [Diagram 36] Figure 1 shows the effect of 2'-O-methyl NTP substitution on capped mRNA expression of fLuc in THP-1 cells. Capped mRNA encoding fLuc was generated by IVT in which 75% or more of individual nucleotides were replaced by 2'-O-methyl modified versions. Combo conditions are conditions in which all four NTPs are replaced. THP-1 cells were transfected with 100 ng of capped mRNA formulated in lipofectamine. Luciferase expression was monitored over 72 hours by measuring relative light output and is shown as fold change over unmodified mRNA. [Figure 37] 1 is a table showing the structures of ATP, CTP, GTP, and UTP as unmodified bases or 2-O-methyl NTPs. Also shown are 2'fluoro-NTPs, including N1-methyl-pseudo-UTP, 7-deaza-GTP, and 2'fluoro-dTTP. [Figure 38] 1 is a table showing the structures of ATP, CTP, GTP, and UTP as 2-deoxy NTPs, including dTTP. [Figure 39] FIG. 1 shows the structures of alternative modified NTPs. [Figure 40A]Figure 1 shows a wide range of alternative modified NTPs that, when incorporated into saRNA, confer no benefit or reduced expression when evaluated for luciferase expression in HEK293T cells. Cells were transfected with 25 or 100 ng of uncapped (pre-capping) or capped saRNA formulated in lipofectamine. Luciferase expression was monitored over 24 hours by measuring relative luminescence and is shown as fold change over unmodified mRNA. [Figure 40B] Figure 1 shows a wide range of alternative modified NTPs that, when incorporated into saRNA, confer no benefit or reduced expression when evaluated for luciferase expression in HeLa cells. Cells were transfected with 25 or 100 ng of uncapped (pre-capping) or capped saRNA formulated in lipofectamine. Luciferase expression was monitored over 24 hours by measuring relative luminescence and is shown as fold change over unmodified mRNA. [Figure 40C] Figure 1 shows a wide range of alternative modified NTPs that, when incorporated into saRNA, confer no benefit or reduced expression when evaluated for luciferase expression in THP-1 cells. Cells were transfected with 25 or 100 ng of uncapped (pre-capping) or capped saRNA formulated in lipofectamine. Luciferase expression was monitored over 24 hours by measuring relative luminescence and is shown as fold change over unmodified mRNA. [Diagram 41]Figure 1 shows the effect of 2'-O-methyl GTP substitution on capped saRNA expression of eGFP. Capped saRNA encoding eGFP was generated by IVT using unmodified NTPs, GTP was replaced by 75% or more 2'-O-methyl modified NTPs, or UTP was replaced by 100% N-1 methyl pseudouridine. Unmodified and modified VEEV-eGFP saRNAs were transfected in triplicate at 1 μg in 24-well plates of (A) HELA and (B) THP1 cells (error bars represent n=3). 24 hours after transfection, cells were harvested, stained, and then assessed for eGFP expression via flow cytometry. Data show % of cells expressing eGFP (left vertical axis) and median fluorescence intensity (right vertical axis). [Diagram 42] Figure 1 shows the effect of individual and combined 2'-O-methyl NTP substitutions on capped mRNA expression of fLuc in THP-1 cells. Capped mRNA encoding fLuc was generated by IVT in which nucleotides were substituted with 75% or more 2'-O-methyl GTP, 75% or more 2'-O-methyl ATP, 90% 2'-O-methyl CTP or 90% 2'-O-methyl UTP, either alone or in various combinations. Substitution of all four NTPs is indicated as 2'-O-methyl combo. THP-1 cells were transfected with 25 ng of capped mRNA formulated in lipofectamine. Luciferase expression was monitored over 48 hours by measuring relative light output (see Methods). [Figure 43A] Figure 1 shows the effect of combinatorial 2'-O-methyl NTP substitutions on capped mRNA expression of fLuc in HEK293T cells. Conditions 1-26 are detailed in Table 6. Capped mRNA encoding fLuc was generated by IVT, where nucleotides were substituted according to Table 6. HEK293T cells were transfected with 25 ng and 100 ng of capped mRNA formulated in lipofectamine. Luciferase expression was monitored over 72 hours by measuring relative light output (see Methods). [Figure 43B] Figure 1 shows the effect of combinatorial 2'-O-methyl NTP substitutions on capped mRNA expression of fLuc in HEK293T cells. Conditions 1-26 are detailed in Table 6. Capped mRNA encoding fLuc was generated by IVT where nucleotides were substituted according to Table 6. HEK293T cells were transfected with 25ng and 100ng of capped mRNA formulated in lipofectamine. Fold change in expression relative to unmodified mRNA is shown. [Figure 44A] Figure 1 shows the effect of combinatorial 2'-O-methyl NTP substitutions on capped mRNA expression of fLuc in HeLa cells. Conditions 1-26 are detailed in Table 6. Capped mRNA encoding fLuc was generated by IVT where nucleotides were substituted according to Table 6. HeLa cells were transfected with 25 ng and 100 ng of capped mRNA formulated in lipofectamine. Luciferase expression was monitored over 72 hours by measuring relative light output (see Methods). [Figure 44B] Figure 1 shows the effect of combinatorial 2'-O-methyl NTP substitutions on capped mRNA expression of fLuc in HeLa cells. Conditions 1-26 are detailed in Table 6. Capped mRNA encoding fLuc was generated by IVT where nucleotides were substituted according to Table 6. HeLa cells were transfected with 25 ng and 100 ng of capped mRNA formulated in lipofectamine. Fold change in expression relative to unmodified mRNA is shown. [Figure 45A]Figure 1 shows the effect of combinatorial 2'-O-methyl NTP substitutions on capped mRNA expression of fLuc in THP-1 cells. Conditions 1-26 are detailed in Table 6. Capped mRNA encoding fLuc was generated by IVT where nucleotides were substituted according to Table 6. THP-1 cells were transfected with 25 ng and 100 ng of capped mRNA formulated in lipofectamine. Luciferase expression was monitored over 72 hours by measuring relative light output (see Methods). [Figure 45B] Figure 1 shows the effect of combinatorial 2'-O-methyl NTP substitutions on capped mRNA expression of fLuc in THP-1 cells. Conditions 1-26 are detailed in Table 6. Capped mRNA encoding fLuc was generated by IVT where nucleotides were substituted according to Table 6. THP-1 cells were transfected with 25 ng and 100 ng of capped mRNA formulated in lipofectamine. Fold change in expression relative to unmodified mRNA is shown. [Figure 46A] Figure 1 shows the effect of 2'-O-methyl GTP substitution on the inflammatory profile of saRNA in THP-1 cells. Capped saRNA encoding firefly luciferase was generated by IVT using unmodified NTPs, GTP replaced by 75% or more 2'-O-methyl modified NTPs, or UTP replaced by 100% N-1 methyl pseudouridine. Unmodified and modified saRNA were transfected in triplicate at 1 μg in 24-well plates of THP1 cells (error bars represent n=3). At 24 and 48 hours post-transfection, half of the cell supernatant was harvested and then luciferase assay was performed to measure antigen expression. [Figure 46B]Figure 1 shows the effect of 2'-O-methyl GTP substitution on the inflammatory profile of saRNA in THP-1 cells. Capped saRNA encoding firefly luciferase was generated by IVT using unmodified NTPs, GTP replaced by 75% or more 2'-O-methyl modified NTPs, or UTP replaced by 100% N-1 methyl pseudouridine. Unmodified and modified saRNAs were transfected in triplicate at 1 μg in 24-well plates of THP1 cells (error bars represent n=3). MSD assays were then performed using cell supernatants from each time point to determine the various levels of cytokines stimulated by each RNA construct. [Figure 47] Figure 1 shows the effect of 2'-O-methyl substitution on capped mRNA expression of eGFP. Capped mRNA encoding eGFP was generated by IVT using unmodified NTPs, UTP replaced by 100% N-1 methyl pseudouridine, or NTPs replaced by 2'-O-methyl modified NTPs (75% Gm, 75% Am, 90% Cm, 90% Um) and labeled 2'-O-methyl combo. Unmodified and modified mRNAs were transfected in triplicate at 1 μg in 24-well plates of (A) HELA and (B) THP1 cells (error bars represent n=3). 24 hours after transfection, cells were harvested, stained, and then assessed for eGFP expression via flow cytometry. Data show % of cells expressing eGFP (left vertical axis) and median fluorescence intensity (right vertical axis). [Figure 48A]Figure 1 shows the effect of 2'-O-methyl substitution on the inflammatory profile of mRNA in THP-1 cells. Capped mRNA encoding firefly luciferase was generated by IVT using unmodified NTPs, UTP replaced with N1-methyl pseudouridine, or WT NTPs replaced with 2'-O-methyl modified NTPs (75% Gm, 75% Am, 90% Cm, 90% Um) and labeled as 2'-O-methyl combo. Unmodified and modified mRNAs were transfected in triplicate at 1 μg in 24-well plates of THP1 cells (error bars represent n=3). At 24 and 48 hours post-transfection, half of the cell supernatant was harvested and then luciferase assay was performed to measure antigen expression. [Figure 48B] Figure 1 shows the effect of 2'-O-methyl substitution on the inflammatory profile of mRNA in THP-1 cells. Capped mRNA encoding firefly luciferase was generated by IVT using unmodified NTPs, UTP replaced with N1-methyl pseudouridine, or WT NTPs replaced with 2'-O-methyl modified NTPs (75% Gm, 75% Am, 90% Cm, 90% Um) and labeled as 2'-O-methyl combo. Unmodified and modified mRNAs were transfected in triplicate at 1 μg in 24-well plates of THP1 cells (error bars represent n=3). MSD assays were then performed using cell supernatants from each time point to determine the various levels of cytokines stimulated by each RNA construct. [Figure 49]FIG. 49A shows the effect of 2'-O-methyl GTP substitution on saRNA expression in vivo. Capped saRNA encoding firefly luciferase was generated by IVT using unmodified NTPs or with 75% or more of GTP replaced by 2'-O-methyl modified NTPs. saRNA was formulated in lipid nanoparticles consisting of C12-200 ionizable lipids with DSPC, cholesterol and DMPE-PEG200. Groups of 5 female balb / c mice (n=5 per group) were given an IM dose of 10 μg of LNP-formulated unmodified or modified VEEV-fLuc saRNA. Images of mice were acquired on days 1 and 5 using an IVIS Spectrum In Vivo Imaging System. FIG. 49B shows the effect of 2'-O-methyl GTP substitution on saRNA expression in vivo. Capped saRNA encoding firefly luciferase was generated by IVT using either unmodified NTPs or with ≥75% replacement of GTP with 2'-O-methyl modified NTPs. saRNA was formulated in lipid nanoparticles composed of C12-200 ionizable lipids with DSPC, cholesterol, and DMPE-PEG200. Groups of 5 female balb / c mice (n=5 per group) received an IM dose of 10 μg LNP-formulated unmodified or modified VEEV-fLuc saRNA. Luciferase expression was quantified as photons per second using Aura Imaging software. P=0.0005. [Figure 50A]Figure 1 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, UTP replaced with N1-methyl pseudouridine, or WT NTPs replaced with 2'-O-methyl modified NTPs (75% Gm, 75% Am, 90% Cm, 90% Um) and labeled 2'-O-methyl combo. mRNA was formulated in lipid nanoparticles consisting of C12-200 ionizable lipids with DSPC, cholesterol, and DMPE-PEG200. Groups of 5 female balb / c mice (n=5 per group) were given an IM dose of 8 μg LNP-formulated unmodified or modified mRNA. Mice were imaged on days 1-3 using an IVIS Spectrum In Vivo Imaging System. [Figure 50B] Figure 1. Effect of 2'-O-methyl GTP substitution on mRNA expression in vivo. Capped mRNA encoding firefly luciferase was generated by IVT using unmodified NTPs, UTP replaced with N1-methyl pseudouridine, or WT NTPs replaced with 2'-O-methyl modified NTPs (75% Gm, 75% Am, 90% Cm, 90% Um) and labeled 2'-O-methyl combo. mRNA was formulated in lipid nanoparticles consisting of C12-200 ionizable lipids with DSPC, cholesterol, and DMPE-PEG200. Groups of 5 female balb / c mice (n=5 per group) were given an IM dose of 8 μg LNP-formulated unmodified or modified mRNA. Luciferase expression was quantified as photons per second using Aura Imaging software. P<0.001. [Figure 51]2'-O-methyl-GTP modified saRNA shows improved immunogenicity in vivo. VEEV saRNA encoding influenza hemagglutinin protein (VEEV-Ha) downstream of a subgenomic promoter was produced by IVT using unmodified NTPs, or with ≥75% GTP replaced by 2'-O-methyl modified NTPs, or with 100% N-1 methyl pseudouridine replaced by UTP. Four groups of female balb / c mice (n=5 per group) were given a prime-boost regimen with 10 μg IM administration of LNP-formulated unmodified or modified VEEV-HA saRNA, and mice were bled and immunized at weeks 0 and 4, and then bled again at week 6. (A) Total HA-specific IgG was quantified by ELISA from mouse sera collected at weeks 4 and 6, and (B) neutralizing antibody responses were quantified by neutralization assay with influenza A California 2009 H1N1 virus. ****Two-way ANOVA-Tukey's multiple comparison test P<0.0001. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0015] 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, even more 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, even more 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, even more preferably at least 100 mM magnesium ions.

[0016] Preferably, the method comprises the use of 50 mM to 100 mM magnesium ions, more preferably 60 mM to 95 mM magnesium ions, more preferably 65 mM to 90 mM magnesium ions, even more preferably 70 mM to 80 mM magnesium ions. Preferably, the method comprises the use of 71 mM to 79 mM magnesium ions, more preferably 73 mM to 77 mM magnesium ions.

[0017] Magnesium ions are referred to as Mg 2+ Magnesium ions can be provided as magnesium acetate, magnesium citrate, magnesium sulfate, magnesium gluconate, magnesium lactate, etc. As described in the examples, the inventors observed an increase in RNA expression when magnesium ions are provided in the form of magnesium acetate, but not in the form of magnesium chloride, which was completely unexpected. Therefore, preferably, magnesium ions are provided as magnesium acetate. Preferably, magnesium ions are not provided as magnesium chloride.

[0018] The RNA may be single-stranded or double-stranded. Those skilled in the art will understand that when the nucleic acid is double-stranded, e.g., double-stranded RNA, the "length of ~ bases" refers to the length of base pairs. The RNA may be coding RNA. For example, coding RNA may be used for therapeutic and vaccine applications.

[0019] The RNA may be a non-coding RNA. For example, the non-coding RNA may be used for RNAi applications. The RNA may be selected from the group consisting of messenger RNA (mRNA), microRNA (miRNA), small interfering RNA (siRNA), small hairpin RNA (shRNA), antisense RNA, RNA aptamer, self-amplifying RNA (saRNA), interfering RNA (RNAi), non-coding RNA, circular RNA, and small RNA

[12] .

[0020] Preferably, the RNA is a self-amplifying RNA (saRNA) or messenger RNA (mRNA).

[0021] The RNA can be a self-amplifying RNA (saRNA). One skilled in the art will appreciate that a self-amplifying RNA can contain the basic elements of an mRNA (cap, 5'UTR, 3'UTR, IRES, viral polymerase, and a poly(A) tail of variable length), but can be significantly longer (e.g., 9-12 kb).

[0022] The RNA can be messenger RNA (mRNA). The mRNA can contain the basic elements of a cap, a 5'UTR, a 3'UTR, an IRES, and a poly(A) tail of variable length.

[0023] 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.

[0024] The RNA molecule (which may be RNAi, saRNA or mRNA) may 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.

[0025] 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 10,000 bases in length, at least 11,000 bases in length or at least 12000 bases in length.

[0026] In one embodiment, the RNA molecule is at least 6000 bases in length. In one embodiment, the RNA, most preferably the saRNA or mRNA, is at least 6000 bases in length. In a preferred embodiment, the saRNA is at least 6000 bases in length.

[0027] The RNA, most preferably saRNA or mRNA, can be 5,000 to 20,000 bases in length, 6,000 to 15,000 bases in length, 7,000 to 14,000 bases in length, 7,500 to 13,000 bases in length, 8,000 to 12,000 bases in length, 8,500 to 11,000 bases in length, or 9,000 to 10,000 bases in length.

[0028] The method of the invention may be performed in vivo, in vitro or ex vivo. However, most preferably, the method is performed in vitro. Preferably, the method comprises in vitro transcription (IVT).

[0029] Preferably, the method comprises the use of an RNA polymerase that may be 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. Each of these RNA polymerases is capable of maximizing the synthesis of modified RNA. However, preferably, the method comprises the use of a T7 RNA polymerase or a variant thereof. The T7 RNA polymerase variant may comprise the following mutations (alone or in combination), in particular R425C, K631R, S633P, Y639F, Y639V, S641A, H784A, H784S and / or H784G, which are associated with increased processivity and / or tolerance for the incorporation of modified bases.

[0030] Preferably, the plurality of nucleotide triphosphates (NTPs) are selected from the group consisting of ATP, GTP, CTP and / or UTP. NTPs are building blocks of RNA. Preferably, the method comprises the use of substantially equal proportions of each of ATP, GTP, CTP and / or UTP. However, in some embodiments, the method may comprise different ratios between each of ATP, GTP, CTP and / or UTP, examples of which are given below. This depends on the sequence of the template nucleic acid and the resulting RNA molecule transcribed therefrom.

[0031] Preferably, the method comprises the use of multiple nucleotide triphosphates at a concentration of at least 1 mM, 2 mM, 3 mM or 4 mM. Preferably, the method comprises the use of multiple nucleotide triphosphates at a concentration of at least 5 mM, 6 mM or 7 mM. More preferably, the method comprises the use of multiple nucleotide triphosphates at a concentration of at least 8 mM, 9 mM or 10 mM. Suitably, the method comprises the use of multiple nucleotide triphosphates at a concentration of more than 10 mM. For example, the method may comprise the use of multiple nucleotide triphosphates at a concentration of at least 12 mM, 14 mM or 16 mM. Also, the method may comprise the use of multiple nucleotide triphosphates at a concentration of at least 18 mM, 20 mM or 22 mM. In other embodiments, the method may comprise the use of multiple nucleotide triphosphates at a concentration of at least 25 mM, 50 mM, 75 mM or 100 mM.

[0032] The one or more modified NTPs used in the method may be selected from the group consisting of 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).

[0033] Preferably, the method comprises using one or more modified NTPs including modified ATP, modified CTP, modified GTP, modified UTP and / or modified TTP. Preferably, the method comprises using 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 using 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 using 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 using 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 using at least five modified NTPs selected from the group consisting of modified ATP, modified CTP, modified GTP, modified UTP and / or modified TTP.

[0034] In one embodiment, one or more modified NTPs may comprise a 2'-substituted group, where the OH group normally at the 2' position may be replaced by a halogen, an optionally substituted aromatic group, NH, N, H, an optionally substituted O-alkyl, O-alkenyl or O-alkynyl group, or an optionally substituted alkyl, alkenyl or alkynyl group, where in each case the aromatic group, alkyl, alkenyl or alkynyl is optionally substituted by halogen, oxo, OR, CN, NR or SR, where R is H or C. 1~6 Alkyl, C 2~6 Alkenyl or C 2~6 It is alkynyl.

[0035] Alternatively, or in addition, one or more modified NTPs may comprise a substituted nucleobase. The nucleobase may be substituted with a halogen, an optionally substituted aromatic group, NH2, N3, OH, an optionally substituted O-alkyl, O-alkenyl or O-alkynyl group, or an optionally substituted alkyl, alkenyl or alkynyl group, where in each case the aromatic group, alkyl, alkenyl or alkynyl is optionally substituted with a halogen, oxo, OR, CN, NR2 or SR, where R is H or C. 1~6 Alkyl, C 2~6 Alkenyl or C 2~6 Alternatively, or in addition, an oxo group in a nucleobase can be replaced by a =S group.

[0036] When discussing optional substituents and sizes of alkyl, alkenyl and / or alkynyl, it will be understood that this applies to both the optionally substituted alkyl, alkenyl and alkynyl groups themselves, and to the optionally substituted O-alkyl, O-alkenyl and O-alkynyl groups.

[0037] Alkyl, alkenyl or alkynyl is optionally substituted C 1~ C 20 It may be an alkyl, alkenyl or alkynyl NTP, where the alkyl, alkenyl or alkynyl is optionally substituted by halogen, oxo, OR, CN, NR2 or SR, where R is H or C. 1~6 Alkyl, C 2~6 Alkenyl or C 2~6 Preferably, the alkyl, alkenyl or alkynyl is an optionally substituted C1-C 10 It may be an alkyl, alkenyl or alkynyl NTP, where the alkyl, alkenyl or alkynyl is optionally substituted by halogen, oxo, OR, CN, NR2 or SR, where R is H or C. 1~6 Alkyl, C 2~6 Alkenyl or C 2~6 It is alkynyl.

[0038] In some embodiments, the alkyl, alkenyl, or alkynyl is optionally substituted C 1~6 Alkyl, C 2~6 Alkenyl or C 2~6 alkynyl, where the or each alkyl, alkenyl or alkynyl is optionally substituted by halogen, oxo, OR, CN, NR2 or SR, where R is H or C 1~6 Alkyl, C 2~6 Alkenyl or C 2~6 In some embodiments, the alkyl, alkenyl or alkynyl is an optionally substituted C 1~3 Alkyl, C 2~3 Alkenyl or C 2~3 alkynyl, where the or each alkyl, alkenyl or alkynyl is substituted by one or more substituents selected from the group consisting of oxo, OH, OMe, NH and NRH, where R is C 1~6 Alkyl, C 2~6 Alkenyl or C 2~6 More preferably, the alkyl, alkenyl or alkynyl is an optionally substituted C 1~3 Alkyl, C 2~3 Alkenyl or C 2~3 It may also be alkynyl, where the or each alkyl, alkenyl or alkynyl is unsubstituted.

[0039] Thus, in some embodiments, alkyl, alkenyl, or alkynyl may be methyl, ethyl, propyl, ethenyl, propenyl, ethynyl, or propynyl, optionally substituted with one or more substituents selected from the group consisting of oxo, OH, OMe, NH, and NRH, where R is C 1~6 Alkyl, C 2~6 Alkenyl or C 2~6Alkyl, alkenyl or alkynyl may therefore be methyl, hydromethyl, acetyl, formyl, carbamoylmethyl, methoxycarbonylmethyl, carboxyhydroxymethyl, carboxymethylaminomethyl, oxyacetic acid methyl ester or isopentenylaminomethyl.

[0040] The halogen can be chlorine or bromine. The halogen can be fluorine. Thus, the one or more modified NTPs can include a 2'-substituted fluoro NTP.

[0041] The optionally substituted aromatic group is a C-C 12 The optionally substituted aromatic group may be an optionally substituted phenyl.

[0042] Preferably, the one or more modified NTPs include a 2'-methyl modified NTP.

[0043] Most preferably, the one or more modified NTPs comprises a 2'-O-methyl modified NTP.

[0044] At least 28 naturally occurring 2'-O-methyl modifications to ribonucleosides (Nm) have been identified. Thus, one or more modified NTPs may include 2'-O-methyl adenosine (Am), 1,2'-dimethyl-adenosine (m 1 Am), N6,2'-O-dimethyl-adenosine (m 6 Am), N 6 ,N 6 ,2-O-trimethyl-adenosine (m 6,6 Am), 2'-O-methylinosine (Im), 1,2'-O-dimethylinosine (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 2,2Gm), N2,7,2'-O-trimethyl-guanosine (m 2,7 Gm), 2'-O-methylcytidine, N 4 ,2'-O-Dimethylcytidine (m 4 Cm), N 4 ,N 4 ,2-O-trimethyl-cytidine (m 4,4 Cm), 5,2'-O-dimethyl-cytidine (m 5 Cm), N 4 -Acetyl-2'-O-methyl-cytidine (ac 4 Cm), 2'-O-methyl-5-hydromethyl-cytidine (hm 5 Cm), 5-formyl-2'-O-methyl-cytidine (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-methyl-uridine (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-oxyacetic acid methyl ester (mcmo 5 Um), 5-(isopentenylaminomethyl)-3'O-methyluridine (inm 5 Um), and any other synthetic NTP containing 2'-O-methylated ribose.

[0045] Thus, preferably, the one or more modified NTPs include 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-OMe CTP" 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").

[0046] Preferably, the method comprises using one or more modified NTPs including 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 using 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 using 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 using 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 using 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 using 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.

[0047] Most preferably, however, the method comprises the step of using 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.

[0048] The present inventors attempted to achieve maximum incorporation of modified bases in modified RNA molecules. For GTP and ATP, about 75% replacement was achieved, and for UTP and CTP, about 90% replacement was achieved. The present inventors observed that for saRNA, modified GTP was most preferentially incorporated into modified RNA molecules, and for mRNA, modified GTP and modified CTP were most preferentially incorporated.

[0049] Thus, in embodiments where the modified RNA molecule is a saRNA, preferably the RNA molecule comprises modified 2'-O-methyl-GTP, as well as unmodified ATP, CTP and / or UTP. Further, in embodiments where the modified RNA molecule is an mRNA, preferably the RNA molecule comprises modified 2'-O-methyl-GTP and modified 2'-O-methyl CTP, as well as unmodified ATP and / or UTP.

[0050] In one embodiment, the one or more modified NTPs have the formula [I]:

[0051] [ka]

[0052] The present invention may include 2'-O-methyl ATP, which is represented herein as:

[0053] In another embodiment, the one or more modified NTPs have the formula [II]:

[0054] [ka]

[0055] The compound may include 2'-O-methyl-CTP, which is represented herein as:

[0056] In another embodiment, the one or more modified NTPs have the formula [III]:

[0057] [ka]

[0058] The present invention may include 2'-O-methyl-GTP, which is represented herein as:

[0059] In another embodiment, the one or more modified NTPs have the formula [IV]:

[0060] [ka]

[0061] The present invention may include 2'-O-methyl-UTP, which is represented herein as:

[0062] As described in the examples, the present inventors have surprisingly observed an increase in saRNA expression level when one or more modified NTPs include modified GTP.Therefore, preferably, one or more modified NTPs include modified GTP, more preferably 2'-O-methyl modified GTP.Furthermore, for mRNA expression level, when one or more modified NTPs include modified CTP, preferably, it is a combination of CTP and GTP.Therefore, preferably, for mRNA, one or more modified NTPs include modified CTP and GTP, more preferably 2'-O-methyl modified CTP and 2'-O-methyl modified GTP.

[0063] Preferably, one or more of the NTPs used in the present invention are modified in a way that they are not naturally occurring. Preferably, at least 30%, 35% or 40% of the component nucleotides in the resulting RNA molecule are modified, where the modified nucleotides may include adenine, cytosine, guanine and / or uracil. More preferably, at least 45%, 50% or 55% of the component nucleotides in the resulting RNA molecule are modified, where the modified nucleotides may include adenine, cytosine, guanine and / or uracil. Even more preferably, at least 60%, 65% or 70% of the component nucleotides in the RNA molecule are modified, where the modified nucleotides may include adenine, cytosine, guanine and / or uracil. Also more preferably, at least 75%, 80% or 85% of the component nucleotides in the RNA molecule are modified, where 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 component nucleotides in the RNA molecule are modified, where modified nucleotides may include adenine, cytosine, guanine and / or uracil.

[0064] As described in the Examples, in an embodiment in which the RNA molecule produced is mRNA, the inventors surprisingly observed that replacing 75% or more of the CTPs improved expression by up to 10-fold. Furthermore, replacing 75% or more of the wild-type nucleotides with 2'-methyl-CTP and -GTP, or with a combination of -GTP, -ATP, -CTP and / or -UTP improved mRNA expression by up to 100-fold, which was similar to the expression level exhibited by saRNA with 75% Gm replacement. Surprisingly, this same level of improvement was also observed when 75% or more of the wild-type nucleotides were replaced simply with 2'-O-methyl-GTP and 2'-O-methyl-CTP, suggesting that replacing both GTP and CTP gave a dominant effect.

[0065] Furthermore, as described in the Examples, in an embodiment in which the RNA molecule produced by the method is saRNA, the inventors have surprisingly found that replacing 75% or more of GTP in saRNA with 2'-methylGTP (Gm) improves expression. In addition, 75% or more Gm modification showed the greatest effect, improving expression by 10-fold or more in various cell lines. Substitution with 2'-O-methyl-ATP, UTP or CTP improved expression of saRNA in various cell lines, although to a lesser extent than GTP. Further increases in expression are achieved by combination substitution of Gm and Cm, or combination of Gm, Cm, Am and Um. For example, preferably, the method includes replacing various proportions of various bases in the modified RNA molecule.

[0066] As described herein, for mRNA, we have been able to replace all four bases with approximately 75% substitutions. However, the more complex structure of saRNA means that it is more difficult to achieve such a high rate of modified bases compared to the rate seen in mRNA.

[0067] Thus, preferably, the method comprises using a combination of two, three or 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 ratio between the two, three or four different modified NTPs may vary.

[0068] Therefore, we evaluated various combinations of modified bases as follows.

[0069] For example, for saRNA, the modified base combinations are: 1. 75% Gm, 2. 75% Gm and 90% Am and 90% Um, 3. 75% Gm and 75% Cm (with or without 90% Am+Um), 4. 75% Gm and 50% Cm (with or without 90% Am+Um), or 5. 50% Gm and 50% Cm (with or without 90% Am+Um) It could be.

[0070] 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 ratio between the four different modified NTPs may vary.

[0071] Alternatively, for saRNA, the modified base combination is 1. 50% Gm+30% Cm+50% Am+30% Um, 2. 50% Gm + 90% Cm + 50% Am + 30% Um, 3. 50% Gm + 30% Cm + 50% Am + 90% Um, 4. 50%Gm+90%Cm+50%Am+90%Um, 5. 25%Gm+30%Cm+25%Am+60%Um, 6. 25%Gm+90%Cm+50%Am+60%Um, 7. 75% Gm+30% Cm+50% Am+60% Um, 8. 75%Gm+60%Cm+50%Am+75%Um, 9. 75%Gm+90%Cm+50%Am+60%Um, 10.50%Gm+30%Cm+25%Am+60%Um, 11.50%Gm+90%Cm+25%Am+60%Um, 12.50%Gm+30%Cm+75%Am+60%Um, 13.50%Gm+90%Cm+75%Am+60%Um, 14. 25%Gm+60%Cm+50%Am+30%Um, 15. 25%Gm+60%Cm+50%Am+90%Um, 16.75%Gm+60%Cm+50%Am+30%Um, 17.75%Gm+60%Cm+50%Am+90%Um, 18. 50%Gm+60%Cm+25%Am+30%Um, 19.50%Gm+60%Cm+25%Am+90%Um, 20. 50%Gm+60%Cm+75%Am+30%Um, 21. 50%Gm+60%Cm+75%Am+90%Um, 22. 25%Gm+60%Cm+25%Am+60%Um, 23.75%Gm+60%Cm+25%Am+60%Um, 24. 25%Gm+60%Cm+75%Am+60%Um, 25. 75%Gm+60%Cm+75%Am+60%Um, 26. 50%Gm+60%Cm+50%Am+60%Um, 27. 50%Gm+60%Cm+75%Am+75%Um, 28. 50%Gm+60%Cm+60%Am+60%Um, or 29. 50%Gm+50%Cm+50%Am+50%Um, or any other percentage ratio It could be.

[0072] For mRNA, the modified base combinations are: 1. 75% Gm + 90% Cm (with or without 90% Am + Um), 2. 75% Gm + 75% Cm (with or without 90% Am + Um), 3. 50% Gm + 50% Cm (with or without 90% Am + Um), or 4. 25%Gm+25%Cm (with or without 90%Am+Um) It could be.

[0073] Alternatively, for mRNA, the modified base combination is 1. 50% Gm+30% Cm+50% Am+30% Um, 2. 50% Gm + 90% Cm + 50% Am + 30% Um, 3. 50%Gm+30%Cm+50%Am+90%Um、 4. 50%Gm+90%Cm+50%Am+90%Um、 5. 25%Gm+30%Cm+25%Am+60%Um、 6. 25%Gm+90%Cm+50%Am+60%Um、 7. 75%Gm+30%Cm+50%Am+60%Um、 8. 75%Gm+60%Cm+50%Am+75%Um、 9. 75%Gm+90%Cm+50%Am+60%Um、 10. 50%Gm+30%Cm+25%Am+60%Um、 11. 50%Gm+90%Cm+25%Am+60%Um、 12. 50%Gm+30%Cm+75%Am+60%Um、 13. 50%Gm+90%Cm+75%Am+60%Um、 14. 25%Gm+60%Cm+50%Am+30%Um、 15. 25%Gm+60%Cm+50%Am+90%Um、 16. 75%Gm+60%Cm+50%Am+30%Um、 17. 75%Gm+60%Cm+50%Am+90%Um、 18. 50%Gm+60%Cm+25%Am+30%Um、 19. 50%Gm+60%Cm+25%Am+90%Um、 20. 50%Gm+60%Cm+75%Am+30%Um、 21. 50%Gm+60%Cm+75%Am+90%Um、 22. 25%Gm+60%Cm+25%Am+60%Um、 23. 75%Gm+60%Cm+25%Am+60%Um、 24. 25%Gm+60%Cm+75%Am+60%Um、 25. 75%Gm+60%Cm+75%Am+60%Um、 26. 50%Gm+60%Cm+50%Am+60%Um、 27. 50%Gm+60%Cm+75%Am+75%Um, 28. 50%Gm+60%Cm+60%Am+60%Um, or 29. 50%Gm+50%Cm+50%Am+50%Um, or any other percentage ratio It could be.

[0074] In another embodiment, the one or more modified NTPs may include 7-deazaguanosine-5-triphosphate (7-deaza-GTP), which has the formula [V]:

[0075] [ka]

[0076] It may be expressed herein as:

[0077] In another embodiment, the one or more modified NTPs may include 7-deazaadenosine-5-triphosphate (7-deaza-ATP), which has the formula [VI]:

[0078] [ka]

[0079] It may be expressed herein as:

[0080] In another embodiment, the one or more modified NTPs may include N1-methyl-pseudouridine, which has the formula [VII]:

[0081] [ka]

[0082] It may be expressed herein as:

[0083] 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.

[0084] By way of example, in an embodiment in which the RNA molecule is mRNA and one preferred combination may be Gm+Cm, UTP may be further replaced by N'-methyl-pseudouridine or fluorinated UTP, etc. Similarly, in an embodiment in which the RNA molecule is saRNA and one preferred modified NTP is Gm, UTP may be further replaced by N'-methyl-pseudouridine or fluorinated UTP.

[0085] Thus, the method comprises: (i) the OH group, usually at the 2' position, is replaced by a halogen, an optionally substituted aromatic group, NH, N, H, an optionally substituted O-alkyl, O-alkenyl or O-alkynyl group, or an optionally substituted alkyl, alkenyl or alkynyl group, in each case the aromatic group, alkyl, alkenyl or alkynyl is optionally substituted by halogen, oxo, OR, CN, NR or SR, and R is H or C 1~6 Alkyl, C 2~6 Alkenyl or C 2~6 one or more NTPs that contain a 2'-substituted group that is alkynyl; (ii) one or more modified NTPs that are not 2'-O-methyl modified NTPs; The method may include combining the steps of:

[0086] The halogen may be chlorine, fluorine or bromine. The halogen may be fluorine.

[0087] Preferably, the method comprises the steps of: (i) one or more 2-O-methyl modified NTPs selected from the group consisting of 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); (ii) one or more other modified NTP bases that are not 2-O-methyl modified NTPs; The method includes the step of combining the above.

[0088] 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'-chloro NTPs, 2'-ethyl, 2'-bromo NTPs, 2'-amino NTPs, 2'-fluoro NTPs or 2'-deoxy NTPs, etc. Thus, preferably, the one or more other modified NTP bases that are not 2-O-methyl modified NTPs are alternative 2'-modified nucleotides.

[0089] For example, the 2'-chloro NTP can be 2'-chloro ATP, 2'-chloro CTP, 2'-chloro GTP, 2'-chloro TTP and / or 2'-chloro UTP.

[0090] 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.

[0091] For example, the 2'-bromoNTP can be 2'-bromoATP, 2'-bromoCTP, 2'-bromoGTP, 2'-bromoTTP and / or 2'-bromoUTP.

[0092] 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.

[0093] 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.

[0094] The 2'-deoxy NTP can be 2'-deoxy ATP, 2'-deoxy CTP, 2'-deoxy GTP, 2'-deoxy TTP and / or 2'-deoxy UTP.

[0095] Preferably, the method comprises the step of combining one or more 2-O-methyl modified NTPs with one or more alternative 2'-modified nucleotides, which may be selected from the alternative 2'-modified nucleotides listed in Table 7 below.

[0096] [Table 1A]

[0097] [Table 1B]

[0098] Such combinations can be used in any ratio to prepare the nucleic acids or modified RNAs of the present 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%, respectively, of one or more other modified NTP bases that are not 2-O-methyl modified NTPs.

[0099] Unless otherwise indicated, modified nucleotides may be fully substituted for the natural nucleotides of the nucleic acid or modified RNA of the present invention. As a non-limiting example, the natural nucleotides guanidine, cytosine, adenine, thymine or uracil may be partially substituted (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%) with at least one of the modified nucleotides disclosed herein.

[0100] As a non-limiting example, the 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 alternative 2'-modified nucleotide (e.g., 2'-deoxyGTP, 2'-fluoroGTP). 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 alternative 2' modified nucleotide (e.g., 2'-deoxy GTP, 2'-fluoro GTP).

[0101] Thus, as an example, if Gm is used at 90%, it may 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%).

[0102] Additional 2'-modified NTPs may also be used, including 2'-chloro, 2'-ethyl, 2'-bromo, 2'-amino, and the like.

[0103] The method may further include the use of DTT, which is believed to improve RNA yields and RNA polymerase activity.

[0104] The method may further include the use of a pH buffer, such as HEPES, although one of skill in the art will appreciate that other buffers may be used.

[0105] The method may further include 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] .

[0106] The method may further include the use of an RNase inhibitor, which prevents RNA degradation if any RNases are present in the reaction.

[0107] The method may further include the use of spermidine, which is believed to improve the activity of the RNA polymerase and therefore the RNA yield.

[0108] The template nucleic acid sequence is preferably transcribed by an RNA polymerase to generate the modified RNA molecule. Thus, preferably, the template nucleic acid sequence comprises DNA. The template nucleic acid may be produced synthetically (e.g., doggybone DNA) or by PCR, rolling circle amplification, or synthetic amplification. The template nucleic acid may comprise a vector, preferably a plasmid.

[0109] In one embodiment, the template nucleic acid may be transcribed to produce the resulting modified RNA molecule, which may be described as a biologic RNA molecule, i.e., a molecule that is used therapeutically to treat, prevent or ameliorate disease in a patient. The template nucleic acid sequence may be derived from an animal or human and may code for a therapeutic protein that treats, prevents or ameliorate disease in a subject, preferably a human or animal subject. For example, the therapeutic use of the encoded therapeutic protein may include protein replacement therapy, gene editing (e.g., CRISPR-Cas9), gene therapy, or RNAi, etc.

[0110] The biologic RNA molecule may be selected from the group consisting of messenger RNA (mRNA), microRNA (miRNA), small interfering RNA (siRNA), small hairpin RNA (shRNA), antisense RNA, RNA aptamer, self-amplifying RNA (saRNA), interfering RNA (RNAi), non-coding RNA, circular RNA, and small RNA.

[0111] In one embodiment, the template nucleic acid may encode proteins and peptides derived from plants. Preferably, the proteins and peptides are plant antigens. The plant antigens may be derived from castor bean (Ricinus communis).

[0112] In another embodiment, the template nucleic acid encodes an antigen, in which case the resulting modified RNA molecule may be described as a vaccine, i.e., a vaccine used prophylactically to prevent disease in a patient, or a vaccine used therapeutically to ameliorate a disease, such as cancer. For example, the template nucleic acid sequence may encode an antigen derived from a virus, bacterium, mycoplasma, fungus, animal, plant, algae, parasite or protozoan, or other organism that causes disease in a subject, preferably a human or animal. Preferably, the template nucleic acid sequence is of viral or bacterial origin. For example, the template nucleic acid may encode at least a portion of a viral spike protein.

[0113] In another embodiment, the template nucleic acid can encode a tumor immunogen or antigen, or a cancer immunogen or antigen. Tumor immunogens and antigens can be peptide-containing tumor antigens, such as polypeptide tumor antigens or glycoprotein tumor antigens.

[0114] Tumor antigens can be (a) full-length molecules associated with cancer cells, (b) homologs and modified forms of full-length molecules, including molecules with deleted, added and / or substituted portions, and (c) fragments of full-length molecules.

[0115] Suitable tumor immunogens include class I restricted antigens, which are recognized by CD8+ lymphocytes, or class II restricted antigens, which are recognized by CD4+ lymphocytes.

[0116] 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, renal cancer, hepatocellular carcinoma, ovarian cancer, gastric cancer, and prostate cancer.

[0117] Tumor antigens include (a) cancer-testis antigens, such as NY-ESO-I, SSX2, SCP1, and RAGE, BAGE, GAGE ​​and MAGE family polypeptides, such as GAGE-I, GAGE-2, MAGE-I, MAGE-2, MAGE-3, MAGE-4, MAGE-5, MAGE-6 and MAGE-12 (which may be used to address, for example, melanoma, lung tumors, head and neck tumors, NSCLC, breast tumors, gastrointestinal tumors and bladder tumors); (b) mutated antigens, such as p53 (associated with various solid tumors, e.g., colorectal, lung, head and neck cancer), p21 / Ras (associated with, e.g., melanoma, pancreatic 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), CIA 0205 (associated with, e.g., bladder cancer), HLA-A2-R1701, beta-catenin (associated with, e.g., melanoma), TCR (associated with, e.g., T-cell non-Hodgkin's lymphoma), BCR-abl (associated with, e.g., chronic myelogenous leukemia), triosephosphate isomerase, KIA 0205, CDC-27 and LDLR-FUT, (c) overexpressed antigens, e.g., galectin 4 (e.g., associated with colorectal cancer), galectin 9 (e.g., associated with Hodgkin's disease), proteinase 3 (e.g., associated with chronic myeloid leukemia), WT 1 (e.g., associated with various leukemias), carbonic anhydrase (e.g., associated with renal cancer), aldolase A (e.g., associated with lung cancer), PRAME (e.g., associated with melanoma), HER-2 / neu (e.g., associated with breast, colon, lung and ovarian cancer), alpha-fetoprotein (e.g., associated with hepatocellular carcinoma), KSA (e.g., associated with colorectal cancer), gastrin (e.g., associated with pancreatic and gastric cancer), telomerase catalytic protein, MUC-I (e.g., associated with breast and ovarian cancer), G-250 (e.g., associated with renal cell carcinoma), p53 (e.g., associated with breast and colon cancer), and carcinoembryonic antigen (e.g., associated with cancers of the gastrointestinal tract, such as breast, lung and colorectal cancer). (d) shared antigens, e.g., melanoma-melanocyte differentiation antigens, e.g., MART-1 / Melan A, gplOO, MClR, melanocyte stimulating hormone receptor, tyrosinase, tyrosinase-related protein-1 / TRP1, and tyrosinase-related protein-2 / TRP2 (e.g., associated with melanoma); (e) prostate-associated antigens, e.g., PAP, PSA, PSMA, PSH-Pl, PSM-Pl, PSM-P2, and / or (f) immunoglobulin idiotypes (e.g., associated with melanoma and B-cell lymphoma) may be selected from:

[0118] 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 the group consisting of an enzyme, an enzyme inhibitor, a hormone, an immune system protein, a receptor, a binding protein, a transcription or translation factor, a tumor growth suppressor protein, a structural protein, and a blood protein.

[0119] The enzyme may be selected from the group consisting of chymosin, gastric lipase, tissue plasminogen activator, streptokinase, cholesterol biosynthetic or degrading steroidogenic enzymes, kinases, phosphodiesterases, methylases, demethylases, dehydrogenases, cellulases, proteases, lipases, phospholipases, aromatase, cytochromes, adenylate or guanylate cyclases and neuraminidase.

[0120] The enzyme inhibitor may be a tissue inhibitor of metalloproteinase (TIMP).The hormone may be growth hormone.

[0121] 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.

[0122] The cytokine may be an interleukin, such as IL-2, IL-4 and / or IL-6, a colony-stimulating factor (CSF), granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), or tumor necrosis factor (TNF).

[0123] The chemokine may be macrophage inflammatory protein-2 and / or plasminogen activator.

[0124] The lymphokine can be an interferon.

[0125] The immunoglobulin may be a natural, modified or chimeric immunoglobulin, or a fragment thereof. Preferably, the immunoglobulin is a chimeric immunoglobulin with dual activity, such as an antibody enzyme or an antibody-toxin chimera.

[0126] The hormone may be selected from the group consisting of insulin, thyroid hormone, catecholamines, gonadotropins, stimulating hormones, prolactin, oxytocin, dopamine, bovine somatotropin, leptin, growth hormones (e.g., human growth hormone), growth factors (e.g., epidermal growth factor, nerve growth factor, insulin-like growth factor, etc.).

[0127] The receptor may be a steroid hormone receptor or a peptide receptor. Preferably, the receptor is a growth factor receptor.

[0128] The binding protein may be a growth factor binding protein.

[0129] The tumor growth suppressor protein can be a protein that inhibits angiogenesis.

[0130] The structural protein may be selected from the group consisting of collagen, fibroin, fibrinogen, elastin, tubulin, actin and myosin.

[0131] The blood protein may be selected from the group consisting of thrombin, serum albumin, factor VII, factor VIII, insulin, factor IX, factor X, tissue plasminogen activator, protein C, von Willebrand factor, antithrombin III, glucocerebrosidase, erythropoietin, granulocyte colony stimulating factor (GCSF) or modified factor VIII, and an anticoagulant.

[0132] In a preferred embodiment, the template nucleic acid may encode a cytokine capable of regulating lymphatic homeostasis, preferably a cytokine involved in, and preferably inducing or enhancing, the development, priming, expansion, differentiation and / or survival of T cells. Thus, preferably, the cytokine is an interleukin. Most preferably, it is IL-2, IL-7, IL-12, IL-15 or IL-21.

[0133] The template nucleic acid can encode a protein capable of enhancing reprogramming of a somatic cell into a cell with stem cell characteristics.

[0134] Proteins capable of enhancing the reprogramming of somatic cells into cells with stem cell characteristics include 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, 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.

[0135] Preferably, MAGE-A is selected from the group consisting of 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.

[0136] Preferably, the proteins capable of enhancing the reprogramming of somatic cells into cells with stem cell characteristics are OCT4, SOX2, LF4, c-MYC, NANOG, LIN28.

[0137] The template nucleic acid may encode a biomolecule that is utilized to modify cells ex vivo for cell therapy applications. Thus, preferably, the template nucleic acid may encode a protein selected from the group consisting of immunoglobulins, T cell receptors, and NK receptors.

[0138] The template nucleic acid can 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 microRNA.

[0139] In one embodiment, the template nucleic acid may encode an innate inhibitor protein that antagonizes a natural immune response in a subject administered a vaccine containing the resulting RNA molecule, as described in WO2020 / 254804, PCT / GB2021 / 053362, or PCT / GB2021 / 053361.

[0140] According to a third aspect, there is provided an RNA molecule obtained or obtainable by a method according to the first aspect.

[0141] In a fourth aspect, there is provided a pharmaceutical composition comprising an RNA molecule according to the third aspect and a pharma- ceutically acceptable vehicle.

[0142] In a fifth aspect, there is provided a method of preparing a pharmaceutical composition according to the fourth aspect, the method comprising contacting an RNA molecule according to the third aspect with a pharma- ceutically acceptable medium.

[0143] In a sixth aspect, there is provided an RNA molecule according to the third aspect, or a pharmaceutical composition according to the fourth aspect, for use as a medicament.

[0144] In a seventh aspect, there is provided an RNA molecule according to the third aspect, or a pharmaceutical composition according to the fourth aspect, for use in treating, preventing or ameliorating a disease, such as cancer, in a subject.

[0145] In an eighth aspect, there is provided a method of treating, preventing or ameliorating a disease, such as cancer, in a subject, comprising administering, or having administered to a subject in need thereof, a therapeutically effective amount of an RNA molecule according to the third aspect, or a pharmaceutical composition according to the fourth aspect.

[0146] In a ninth aspect, there is provided a vaccine composition comprising an RNA molecule according to the third aspect, or a pharmaceutical composition according to the fourth aspect.

[0147] The vaccine may include a suitable adjuvant. Examples of adjuvants may include aluminum salts, synthetic DNA, carbohydrates, tablet binders, ion exchange resins, preservatives, polymers, emulsions and / or lipids. Examples of adjuvants include monosodium glutamate, sucrose, dextrose, aluminum bovine, human serum albumin, cytosine phosphoguanine, potassium phosphate, plasdone C, anhydrous lactose, cellulose, polacrilin potassium. potassium), glycerin, asparagine, citric acid, potassium phosphate, magnesium sulfate, ferric ammonium citrate, 2-phenoxyethanol, aluminium, beta-propiolactone, bovine extract, DOPC, EDTA, formaldehyde, cytosine phosphoguanine (CpG), QS21, saponin, monophosphoryl lipid A (MPLA), squalene, thimerosal, phenol, potassium aluminium sulfate, potassium glutamate, sodium borate, sodium metabisulfite, urea, PLGA, PVA, PLA, PVP, cyclodextrin based stabilizers, oil in water emulsion adjuvants and / or lipid based adjuvants.

[0148] In a tenth aspect, there is provided an RNA molecule according to the third aspect, a pharmaceutical composition according to the fourth aspect, or a vaccine according to the ninth aspect, for use in stimulating an immune response in a subject.

[0149] The immune response can be stimulated against protozoa, bacteria, viruses, fungi or cancer. Thus, vaccines can be used to express microbial or pathogenic antigens for immunization against infectious microorganisms, such as viruses and bacteria. However, vaccines can also be used to express carcinogenic antigens to prevent cancer.

[0150] In an eleventh aspect of the present invention there is provided a method of vaccinating a subject comprising administering, or having administered to a subject in need thereof, 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.

[0151] It is understood that RNA molecules may have important utility in a wide range of therapeutic applications, such as RNAi, inhibitory RNA, RNA aptamers, etc., in which translation of the RNA molecule does not necessarily have to occur. However, applications in which the RNA molecule needs to be translated include various vaccine and biologic approaches, such as the use of mRNA, saRNA, circular RNA, or any RNA sequence that can be translated in vivo to produce a peptide or protein.

[0152] For example, peptides or proteins encoded by RNA molecules may be involved in protein replacement therapy, gene editing and gene therapy, etc.

[0153] It is also understood that there is an ever-increasing trend in the use, particularly in mRNA and saRNA vaccines, of RNA molecules encoding pathogenic antigens, such as viral coat proteins or regions thereof, or oncogenic antigens, and that it is this antigen that is expressed in an immunized host organism and induces an immune response against the pathogen or tumor.

[0154] 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 containing modified NTPs, but also surprisingly demonstrated a significant increase in RNA translation (i.e., protein expression), especially when the modified RNA molecule is either mRNA or saRNA.For example, replacing 75% or more of the CTP in mRNA improves expression by up to 10-fold, and replacing 75% or more of wild-type nucleotides with 2'-methyl-CTP and -GTP, or with a combination of -GTP, -ATP, -CTP and / or -UTP improves mRNA expression by up to 100-fold, which is similar to the improved expression level also shown by saRNA with 75% Gm replacement.Surprisingly, this same improved expression level was also observed in mRNA when 75% or more of the wild-type nucleotides were simply replaced with 2'-O-methyl-GTP and 2'-O-methyl-CTP.

[0155] As explained in the examples, we did not directly measure the amount of luciferase protein produced, but instead measured the increased expression of luciferase activity. Therefore, luciferase activity can be considered a surrogate for RNA translation, since it should be directly related to the amount of translation. Furthermore, the luciferase data explained in the examples can be used as a surrogate for therapeutic agents, essentially mirroring the expression of therapeutic proteins. However, as explained herein, when "10-fold" or other terms are used, it means that there is actually a 10-fold increase in luciferase activity. It is understood that an increase in expression or RNA translation can be related to the RNA being more stable, and therefore more protein being made before the RNA is degraded. Thus, even if there is no change in translation efficiency (i.e., ribosome speed), there may be an increase in the overall time that the ribosome has to make more protein.

[0156] In summary, these data taken together demonstrate that modified NTPs, particularly 2'-O-methyl modified bases, can be used to increase the level of RNA translation for protein production in vitro and in vivo, and in vivo protein production is particularly advantageous when the RNA molecule is used as a vaccine or therapeutic biomolecule. Based on the unexpected finding that the incorporation of 2'-O modified bases enhances expression rather than reduces it as seen with the natural incorporation of 2'-O methylated bases in coding RNA, the inventors believe that their data also support the finding that modified bases can be used to improve RNA stability and reduce the activation of innate sensing, interferon production, and / or degradation of RNA molecules. These effects of 2'-O modified bases on RNA are particularly unexpected in light of the current understanding in the art that the natural incorporation of 2-O-methyl bases actually inhibits RNA translation, i.e., protein production.

[0157] Therefore, the inventors believe that any 2'-OH substituted modified NTP is (i) improving the expression and / or translation of RNA molecules (particularly mRNA or saRNA and ideally using 2'-O-methyl NTPs); (ii) improve the stability of an RNA molecule (any type of RNA, regardless of its length and with any of the 2'-OH substitutions described herein); and / or (iii) reduce innate sensing activation, interferon production, and / or degradation of RNA molecules (any type of RNA, regardless of its length and with any of the 2'-OH substitutions described herein); I believe it can be used for this purpose.

[0158] Thus, in a twelfth aspect of the present invention, (i) improving expression and / or translation of RNA molecules that contain one or more modified nucleotide triphosphates (NTPs); (ii) improving the stability of an RNA molecule that contains one or more modified NTPs; and / or (iii) reducing the activation of innate sensing, interferon production, and / or degradation of RNA molecules that contain one or more modified NTPs. One or more modified NTPs for use in including 2'-substituted groups, typically in which the OH group at the 2' position is replaced by halogen, an optionally substituted aromatic group, NH, N, H, an optionally substituted O-alkyl, O-alkenyl or O-alkynyl group, or an optionally substituted alkyl, alkenyl or alkynyl group, in each case the aromatic group, alkyl, alkenyl or alkynyl is optionally substituted by halogen, oxo, OR, CN, NR or SR, and R is H or C 1~6 Alkyl, C 2~6 Alkenyl or C 2~6 is alkynyl, Modified NTPs are provided.

[0159] In addition, in the thirteenth aspect, (i) improving expression and / or translation of an RNA molecule that contains one or more modified NTPs; (ii) improving the stability of an RNA molecule that contains one or more modified NTPs; and / or (iii) reducing the activation of innate sensing, interferon production, and / or degradation of an RNA molecule that contains one or more modified NTPs. 1. A method comprising: incorporating one or more modified nucleotide triphosphates (NTPs) into the RNA molecule; One or more of the modified NTPs comprise a 2'-substituted group, typically where the OH group at the 2' position is replaced by a halogen, an optionally substituted aromatic group, NH, N, H, an optionally substituted O-alkyl, O-alkenyl or O-alkynyl group, or an optionally substituted alkyl, alkenyl or alkynyl group, in each case the aromatic group, alkyl, alkenyl or alkynyl is optionally substituted by halogen, oxo, OR, CN, NR or SR, and R is H or C. 1~6 Alkyl, C 2~6 Alkenyl or C 2~6 is alkynyl, This improves expression and / or translation of the RNA molecule comprising one or more modified NTPs, improves stability of the RNA molecule comprising one or more modified NTPs, and / or reduces activation of innate sensing, interferon production, and / or degradation of the RNA molecule comprising one or more modified NTPs. A method is provided.

[0160] Improving the translational expression of modified RNA molecules is an important aspect of the present invention.

[0161] Thus, in a fourteenth aspect, there is provided a method for improving translation of an RNA molecule, 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.

[0162] In a fifteenth aspect, there is provided the use of one or more 2'-O-methyl modified nucleotide triphosphates (NTPs) in an RNA molecule to improve translation of the RNA molecule.

[0163] In a sixteenth aspect, there is provided one or more 2'-O-methyl modified nucleotide triphosphates (NTPs) in an RNA molecule for use in improving translation of the RNA molecule.

[0164] The method or use may be carried out in vivo, in vitro or ex vivo, however most preferably the method is carried out in vivo.

[0165] In some embodiments, one or more modified NTPs may be incorporated into an RNA molecule using the method of the first aspect. Thus, the method may comprise 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, where one or more of the NTPs are modified nucleotide triphosphates (NTPs), and the RNA polymerase transcribes the template nucleic acid sequence to form the RNA molecule.

[0166] The one or more modified NTPs may be as defined with respect to the first aspect of the invention. Preferably, the one or more modified NTPs comprise a 2'-methyl modified NTP. Most preferably, the one or more modified NTPs comprise a 2'-O-methyl modified NTP. Thus, the one or more modified NTPs may comprise 2'-O-methyl adenosine (Am), 1,2'-diethyl-adenosine (m 1 Am), N6,2'-O-dimethyl-adenosine (m 6 Am), N 6 ,N 6 ,2-O-trimethyl-adenosine (m 6,6 Am), 2'-O-methylinosine (Im), 1,2'-O-dimethylinosine (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 2,2Gm), N2,7,2'-O-trimethyl-guanosine (m 2,7 Gm), 2'-O-methylcytidine, N 4 ,2'-O-Dimethylcytidine (m 4 Cm), N 4 ,N 4 ,2-O-trimethyl-cytidine (m 4,4 Cm), 5,2'-O-dimethyl-cytidine (m 5 Cm), N 4 -Acetyl-2'-O-methyl-cytidine (ac 4 Cm), 2'-O-methyl-5-hydromethyl-cytidine (hm 5 Cm), 5-formyl-2'-O-methyl-cytidine (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-methyl-uridine (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-oxyacetic acid methyl ester (mcmo 5 Um), 5-(isopentenylaminomethyl)-3'O-methyluridine (inm 5 Um), and any other synthetic NTP containing 2'-O-methylated ribose.

[0167] 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.

[0168] Preferably, the method comprises using one or more modified NTPs including 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 using 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 using 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 using 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 using 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 using 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. Preferably, however, the method comprises the step of using 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.

[0169] Preferably, the one or more modified NTPs comprise 2'-O-methyl modified GTP. In embodiments where the modified RNA molecule is a saRNA, preferably the RNA molecule comprises modified 2'-O-methyl-GTP, as well as unmodified ATP, CTP and / or UTP. Furthermore, in embodiments where the modified RNA molecule is an mRNA, preferably the RNA molecule comprises modified 2'-O-methyl-GTP and modified 2'-O-methyl CTP, as well as unmodified ATP and / or UTP.

[0170] The type of RNA molecule may be as described for the first aspect. Thus, the RNA may be single-stranded or double-stranded. The RNA may be coding RNA. For example, coding RNA may be used for therapeutic and vaccine applications. The RNA may be non-coding RNA. For example, non-coding RNA may be used for RNAi applications. The RNA may be selected from the group consisting of messenger RNA (mRNA), self-amplifying RNA (saRNA), microRNA (miRNA), small interfering RNA (siRNA), small hairpin RNA (shRNA), antisense RNA, RNA aptamer, interfering RNA (RNAi), non-coding RNA, circular RNA and small RNA.

[0171] Advantageously, incorporation of modified NTPs in a non-translated RNA molecule (e.g., miRNA, siRNA, shRNA, antisense RNA, RNA aptamer, RNAi, non-coding RNA, circular RNA or small RNA) increases the stability of the RNA molecule and reduces detection of the RNA molecule by innate immune receptors.

[0172] Preferably, the RNA is a self-amplifying RNA (saRNA) or messenger RNA (mRNA).

[0173] The length of the RNA molecule may be as described in relation to the first embodiment.

[0174] 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.

[0175] The RNA molecule (which may be RNAi, saRNA or mRNA) may 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.

[0176] For example, the RNA molecule (which may be saRNA or mRNA) may be at least 1000 bases long, at least 2000 bases long, at least 3000 bases long, at least 4000 bases long, at least 5000 bases long, at least 6000 bases long, at least 7000 bases long, at least 8000 bases long, at least 9000 bases long, at least 10,000 bases long, at least 11,000 bases long, or at least 12000 bases long. In one embodiment, the RNA molecule is at least 6000 bases long. In one embodiment, the RNA, most preferably saRNA or mRNA, is at least 6000 bases long. In a preferred embodiment, the saRNA is at least 6000 bases long. The RNA, most preferably saRNA or mRNA, can be 5,000 to 20,000 bases in length, 6,000 to 15,000 bases in length, 7,000 to 14,000 bases in length, 7,500 to 13,000 bases in length, 8,000 to 12,000 bases in length, 8,500 to 11,000 bases in length, or 9,000 to 10,000 bases in length.

[0177] Preferably, one or more of the 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 component nucleotides in the RNA molecule are modified, and the modified nucleotides may include adenine, cytosine, guanine and / or uracil.More preferably, at least 45%, 50% or 55% of the component nucleotides in the RNA molecule are modified, and the modified nucleotides may include adenine, cytosine, guanine and / or uracil.Even more preferably, at least 60%, 65% or 70% of the component nucleotides in the RNA molecule are modified, and the modified nucleotides may include adenine, cytosine, guanine and / or uracil.Even more preferably, at least 75%, 80% or 85% of the component nucleotides in the RNA molecule are modified, and 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 component nucleotides in the RNA molecule are modified, which may include adenine, cytosine, guanine and / or uracil.

[0178] As mentioned above, we measured the enhancement of RNA translation in terms of luciferase activity instead of directly measuring the amount of luciferase protein produced, so luciferase activity can be considered as a surrogate for RNA translation.

[0179] Suitably, the one or more modified NTPs (preferably 2'-O-methyl NTPs) enhance the level of expression and / or translation of the RNA molecule by at least a 5-fold, 10-fold or 2-fold increment, compared to the level of expression and / or translation occurring in the absence of the one or more modified NTPs. More suitably, the one or more modified NTPs (preferably 2'-O-methyl NTPs) enhance the level of expression and / or translation of the RNA molecule by at least a 5-fold, 10-fold or 50-fold increment, compared to the level of expression and / or translation occurring in the absence of the one or more modified NTPs. Even more suitably, the one or more modified NTPs (preferably 2'-O-methyl NTPs) enhance the level of expression and / or translation of the RNA molecule by at least a 75-fold, 100-fold or 250-fold increment, compared to the level of expression and / or translation occurring in the absence of the one or more modified NTPs. Preferably, the one or more modified NTPs (preferably 2'-O-methyl NTPs) enhance the level of expression and / or translation of the RNA molecule by at least 500, 750 or 1000 fold, compared to the level of expression and / or translation occurring in the absence of the one or more modified NTPs. Preferably, the one or more modified NTPs (preferably 2'-O-methyl NTPs) enhance the level of expression and / or translation of the RNA molecule by at least 2000, 5000 or 8000 fold increment, compared to the level of expression and / or translation occurring in the absence of the one or more modified NTPs. As shown in Figure 28, in some embodiments (e.g., Gm and Cm for mRNA), some cells surprisingly exhibit a 1000-8000 fold increase in expression.

[0180] The level of expression and / or translation can be measured by the expression of a fluorescent or luminescent protein encoded as a gene of interest in an RNA molecule (e.g., in either mRNA or saRNA). For example, the luminescent protein can be a bioluminescent protein, such as a luciferase (e.g., a firefly luciferase protein encoded as a gene of interest). Thus, the expression of luciferase can be determined by measuring its enzymatic activity, in which luciferin is converted to oxyluciferin, which emits light that can be quantified as relative light units (RLU). The presence of light is proportional to the amount of protein translated. This technique has previously been used to evaluate the effect of pseudouridine incorporation on translation efficiency

[19] .

[0181] Enzymatic incorporation of modified NTPs (preferably 2'-O-methyl NTPs) in RNA has many potential outcomes and benefits selected from the group consisting of altering mRNA stability, preventing innate recognition, reducing RNA degradation, increasing RNA half-life, improving recruitment of translation factors and / or ribosomes, and increasing translation.

[0182] 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.

[0183] In another embodiment, the method or use improves the stability of RNA, be it mRNA, miRNA, siRNA, shRNA, antisense RNA, RNA aptamer, self-amplifying RNA (saRNA), interfering RNA (RNAi), non-coding RNA, circular RNA or small RNA. Improved stability provides significant benefits for both coding and non-coding biologic therapeutic or prophylactic use of RNA molecules. Previous studies have shown that incorporation of 2'-O-methylated NTPs improves the stability of small RNA oligonucleotides, aptamers and iRNAs [20, 21]. In this regard, 2'-O-methylation increases the resistance of RNA to degradation due to the abolition of hydrogen bonds and the nucleophilic character of 2'-OH. This results in a high increase in the stability of 2'-O-methylated RNA against alkaline (and even enzymatic) hydrolysis. Furthermore, 2'-O methylation increases the base pairing strength and the stability of alternative RNA conformations

[22] . This has been evaluated for small RNAi molecules and aptamers, but not for mRNA or saRNA, as there were no methods available for the incorporation of 2'-O-methylated NTPs in RNA sequences longer than 100 bp.

[0184] The incorporation of 2'-O-methylated NTPs in small RNA sequences has been achieved either by chemical synthesis or by in vitro transcription, the latter of which has been very inefficient. However, the method described herein allows the incorporation of modified NTPs (preferably 2'-O-methyl NTPs) of any length, whether they are coding or non-coding, and provides improved stability of the resulting RNA molecules.

[0185] The stability of the RNA molecules can be measured by quantitative PCR, but is also reflected by the duration of expression, where faster degradation equates to shorter expression. We observe an extended duration of expression to 72 hours for saRNAs containing 75% 2-O-methyl GTP relative to unmodified GTP (see Figures 9-13), as well as an extended duration of expression for mRNAs containing 2-O-methyl modified GTP (Gm) and CTP (Cm) (see Figures 25-28).

[0186] In another embodiment, the method or use results in reducing or eliminating innate sensing of RNA by membrane, endosomal and cellular RNA binding proteins that recognize unmodified RNA structures, where innate sensing triggers the activation of the interferon signaling pathway and cytoplasmic nucleases that degrade RNA, such as RNase L

[23] . Reduction of innate sensing of RNA (be it mRNA, miRNA, siRNA, shRNA, antisense RNA, RNA aptamer, self-amplifying RNA (saRNA), interfering RNA (RNAi), non-coding RNA, circular RNA and small RNA) may provide significant benefits for both coding and non-coding biologic therapeutic or prophylactic use of RNA. Previous studies have shown that incorporation of 2'-O-methylated NTPs affects innate recognition of small RNA oligonucleotides, aptamers and iRNA, interferon induction and associated RNA degradation [24, 25, 26, 27]. However, this method for the incorporation of 2'-O-methylated NTPs in RNA sequences longer than 100bp has not been evaluated for mRNA or saRNA. The incorporation of 2'-O-methylated NTPs in small RNA sequences has been achieved by either chemical synthesis or in vitro transcription, the latter being very inefficient. The method presented herein allows the incorporation of 2'-O-methylated NTPs of any length, preventing the innate recognition of RNA, interferon induction and RNA degradation.

[0187] The reduction in the activation of innate sensing, which leads to the degradation of RNA molecules, is inferred by the improved expression of modified saRNA and mRNA in interferon-competent cells (HeLa and THP-1 cells). Although the incorporation of 2-O-methyl modified bases gives some expression improvement in HEK293T cells, these cells have a defective innate sensing mechanism, and therefore any improvement in expression is directly related to the improvement of RNA half-life in the absence of innate recognition. Whereas the expression of unmodified saRNA and mRNA is greatly reduced in HeLa and THP-1 cells, this reflects the improved degradation caused by the induction of the innate sensing mechanism. The incorporation of 2'-O-methylated NTPs in saRNA and mRNA gives a greater increase in expression (fold change) in these two interferon-competent cell types than observed in HEK293T cells. Induction of type I interferon expression can be measured by ELISA as a surrogate for innate activation.

[0188] It is understood that the compositions, pharmaceutical compositions, vaccines or therapeutic RNA molecules may be used in medicine that may be used as a monotherapy (i.e., use of the composition alone). Alternatively, the compositions according to the invention may be used as an adjunct or in combination with known therapies for treating, ameliorating or preventing infection or disease.

[0189] The compositions of the present invention can be combined in compositions having several different forms, depending in particular on the manner in which the compositions are used. Thus, for example, the compositions can be in the form of powders, tablets, capsules, liquids, ointments, creams, gels, hydrogels, aerosols, sprays, micellar solutions, transdermal patches, liposomal suspensions, polyplexes, emulsions, liposomes, lipid nanoparticles, functionalized liposomes or lipid nanoparticles (e.g., with peptides, antibodies, antibody fragments, glycans, glycoconjugates, DNA or RNA on or encapsulated therein), or any other suitable form that can be administered to humans or animals. The lipid nanoparticles can contain one or more components selected from the group consisting of cationic lipids (preferably ionizable), phosphatidylcholine, cholesterol and polyethylene glycol (PEG)-lipids. It is understood that the medium of the medicament according to the present invention should be a medium that is well tolerated by the subject to which it is given.

[0190] The medicaments, including compositions, pharmaceutical compositions or vaccines of the present invention may be used in several ways. For example, oral administration may be required, in which case the agent may be contained within a composition that can be taken orally, for example in the form of a tablet, capsule or liquid. Compositions containing the agents and medicaments of the present invention may be administered by inhalation (e.g., intranasally). The compositions may also be formulated for topical use. For example, creams or ointments may be applied to the skin. The formulations may be provided in lyophilized form for reconstitution, or as solid dosage forms for topical, insert or injection delivery.

[0191] The compositions, pharmaceutical compositions, vaccines or therapeutic RNA molecules of the present invention may also be incorporated into slow or delayed release devices. Such devices may be inserted, for example, on or under the skin, and the drug may be released over a period of weeks or even months. The devices may be positioned at least adjacent to the treatment site.

[0192] However, in a preferred embodiment, the medicament according to the invention may be administered to a subject by injection into the bloodstream, muscle, skin or directly into the site requiring treatment. Injection may 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).

[0193] It is understood that the amount of a composition, pharmaceutical composition or vaccine, or therapeutic RNA molecule required will be determined by its biological activity and bioavailability, which will depend on the mode of administration of the composition, pharmaceutical composition or vaccine, its physicochemical properties, and whether it is used as a monotherapy or in a combination therapy.

[0194] The frequency of administration is also influenced by the half-life of the active agent in the subject being treated. The optimal dosage to be administered may be determined by one skilled in the art and will vary depending on the composition, pharmaceutical composition or vaccine used, the strength of the pharmaceutical composition, the mode of administration and the type of treatment. Additional factors that depend on the particular subject being treated, including the subject's age, weight, sex, diet and time of administration, will result in the need to adjust the dosage.

[0195] The dosage required may depend on several factors, including, but not limited to, the active agent being administered, the disease being treated and / or vaccinated against, the subject being treated, and the like.

[0196] Generally, depending on the active agent used, a dose of 0.001 μg / kg to 10 mg / kg of body weight, or 0.01 μg / kg to 1 mg / kg of body weight of the composition, pharmaceutical composition or vaccine or therapeutic RNA molecule of the present invention may be used. The dose may be understood to relate to the amount of the RNA molecule to be delivered.

[0197] The dose may be given as a single administration (e.g., a single injection). Alternatively, the composition, pharmaceutical composition, biologic RNA molecule or vaccine may require more than one administration. By way of example, the composition, pharmaceutical composition or vaccine or therapeutic RNA molecule may be administered as an initial primer followed by a boost, or two boosts administered within a week or at monthly intervals (e.g., two or more doses of 0.07 μg to 700 mg, i.e., assuming a body weight of 70 kg), or for daily, weekly or monthly biologic repeat dosing. Alternatively, a slow release device may be used to provide the patient with an optimal dose of the composition, pharmaceutical composition or vaccine or therapeutic RNA molecule according to the invention, without the need to administer repeated doses. The route of administration may incorporate intravenous, intradermal, subcutaneous, intramuscular, intrathecal, epidural or intraperitoneal injection routes.

[0198] Known techniques, e.g. techniques conventionally used by the pharmaceutical industry (e.g. in vivo experiments, clinical trials, etc.), can be used to formulate the specific formulation of the composition, pharmaceutical composition or vaccine or therapeutic RNA molecule according to the invention, as well as the precise treatment regime (e.g. drug dose and frequency of administration).

[0199] A "subject" may be a vertebrate, a mammal, or a livestock animal. Thus, the compositions and medicaments according to the invention may be used to treat any mammal, such as livestock (e.g., horses), pets, or in other veterinary applications. Most preferably, however, the subject is a human.

[0200] A "therapeutically effective amount" of a composition, pharmaceutical composition or vaccine, or a therapeutic RNA molecule is any amount, as referred to above, that is required to produce a therapeutic effect when administered to a subject.

[0201] For example, a therapeutically effective amount of the compositions, pharmaceutical compositions and vaccines or therapeutic RNA molecules of the present invention may contain from about 0.0001 mg to about 800 mg of payload molecule, preferably from 0.001 mg to about 650 mg, preferably from about 0.01 mg to about 500 mg of payload molecule.

[0202] A "pharmaceutically acceptable vehicle" as referred to herein is any known compound, or combination of known compounds, known to those of skill in the art to be useful in formulating a pharmaceutical composition.

[0203] In one embodiment, the pharma- ceutically acceptable vehicle may be a solid, and the composition may be in the form of a powder or tablet. A solid pharma- ceutically acceptable vehicle may contain one or more substances that may also act as flavoring agents, lubricants, solubilizers, suspending agents, dyes, fillers, glidants, compression aids, inert binders, sweeteners, preservatives, dyes, coatings, or tablet disintegrants. The vehicle may also be an encapsulating material. In a powder, the vehicle is a finely divided solid that is present in admixture with the finely divided active agent according to the invention. In a tablet, the active agent (e.g., the composition of the invention) may be mixed in suitable proportions with a vehicle having the necessary compression properties and compressed to the desired shape and size. The pharmaceutical vehicle may be a gel, and the composition may be in the form of a cream or other form.

[0204] However, the pharmaceutical medium may be liquid and the pharmaceutical composition is in the form of a solution. The liquid medium is used in preparing solutions, suspensions, emulsions, syrups, elixirs and pressurized compositions. The composition according to the invention may be dissolved or suspended in a pharma- ceutically acceptable liquid medium, such as water, an organic solvent, a mixture thereof or a pharma- ceutically acceptable oil or fat. The liquid medium may contain other suitable pharmaceutical additives, such as solubilizers, emulsifiers, buffers, preservatives, sweeteners, flavorings, suspending agents, thickeners, colorants, viscosity regulators, stabilizers or osmolality regulators. Suitable examples of liquid media for oral and parenteral administration include water (preferably sodium carboxymethylcellulose solution, partially containing the above-mentioned additives, such as cellulose derivatives), alcohols (including monohydric and polyhydric alcohols, such as glycols) and their derivatives, and oils (such as fractionated coconut oil and peanut oil). For parenteral administration, the medium may also be an oily ester, such as ethyl oleate and isopropyl myristate. Sterile liquid vehicles are useful in sterile liquid form compositions for parenteral administration. The liquid vehicle for pressurized compositions can be halogenated hydrocarbon or other pharma- ceutically acceptable propellants.

[0205] Liquid pharmaceutical compositions that are sterile solutions or suspensions can be utilized 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.

[0206] The composition and / or pharmaceutical composition of the present invention may be orally administered in the form of a sterile solution or suspension containing other solutes or suspending agents (e.g., sufficient salt or glucose to make the solution isotonic), bile salts, acacia, gelatin, sorbitan monooleate, polysorbate 80 (oleic acid esters of sorbitol and its anhydrides copolymerized with ethylene oxide), and the like. The composition and / or pharmaceutical composition of the present invention may also be orally administered in either liquid or solid composition form. Compositions suitable for oral administration include solid forms such as pills, capsules, granules, tablets, and powders, as well as liquid forms such as solutions, syrups, elixirs, and suspensions. Forms useful for parenteral administration include sterile solutions, emulsions, and suspensions.

[0207] All of the features described in this specification (including any accompanying claims, abstract and drawings), and / or all of the steps of any method or process so disclosed, may be combined in any combination with any of the above aspects, except combinations in which at least some of such features and / or steps are mutually exclusive. EXAMPLES

[0208] Materials and Methods In vitro transcription (IVT) saRNA and mRNA encoding fLuc were produced by in-vitro transcription (IVT) using a linearized DNA template. The final volume for each IVT reaction was 50 μL, and the standardized IVT reaction mixture was as follows:

[0209] [Table 2]

[0210] Where indicated, the concentration of magnesium acetate was varied (Mg(OAc)2) or replaced by magnesium chloride or manganese acetate. The total concentration of each NTP was kept at 10 nM, however, the relative percentage of unmodified NTP to modified NTP was varied as indicated. For example, when it is stated that 75% of GTP was replaced by 2'O-methyl-GTP, it indicates that the reaction contained 7.5 mM 2'-O-methyl-GTP and 2.5 mM GTP. IVT reactions were carried out at 37°C for 4 hours. RNA yield was then measured immediately after IVT using Qubit RNA Broad Range Assay kit and Qubit Fluorometer (Thermo Fisher, UK) according to the manufacturer's protocol. Prior to post-transcriptional capping, RNA was purified using lithium chloride precipitation. Post-transcriptional capping of RNA was carried out using ScriptCap™ Cap 1 Capping System (CellScript, UK) and reactions were incubated at 37°C for 2 hours. RNA was purified using post-transcriptional capping, again using lithium chloride precipitation, and the final RNA concentration was measured using a Nanodrop One (Thermo Scientific, UK). To assess RNA quality, purified RNA and RNA Millennium Marker Ladder (Thermo Fisher, UK) were mixed with 2x NorthernMax-Gly Sample Loading Dye (Thermo Fisher, UK) and incubated at 50°C for 30 min to denature the RNA. A 1.2% agarose gel and 1x NorthernMax Running Buffer (Thermo Fisher, UK) were prepared. After incubation, the denatured ladder and samples were loaded onto the gel and run on the gel at 80V for 45 min. Images of the gel were then captured on a GelDoc-It2 (UVP, UK).

[0211] Cells and in vitro transfection HEK293T.17 and HeLa cells (ATCC, USA) were cultured in complete Dulbecco's Modified Eagle 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). Cells were plated in 96-well plates 24 hours prior to transfection at 7 × 10 cells per well for HEK293T.17 and HeLa cells. 4 1 x 10 cells per well for THP-1 cells 5 Cells were seeded at a density of 100x100 / ml. Transfection with saRNA and mRNA encoding fLuc was performed using Lipofectamine MessengerMAX (Thermo Fisher, UK) according to the manufacturer's instructions. Firefly luciferase was measured with the ONE-Glo Luciferase Assay System (Promega, UK) according to the manufacturer's instructions, and luminescence signals were measured using a FLUOstar Omega Plate Reader (BMG Labtech, UK).

[0212] Example 1 Incorporation of the modified base 2' methyl-NTP (2-OMe-NTP) can be achieved when using magnesium ion concentrations above 20 mM The inventors have surprisingly demonstrated that incorporation of modified base 2'-methyl-NTP (2-OMe-NTP) can be achieved when using magnesium ion concentrations above 20 mM (see FIG. 1). In particular, the inventors have observed that magnesium acetate concentrations above 20 mM, and ideally concentrations up to 75 mM, are required for efficient incorporation of 2'-methyl-GTP. However, the inventors believe that the maximum concentration of magnesium acetate may even be above 80 mM.

[0213] All experiments were performed with wild-type T7 polymerase; however, we anticipate that the reaction conditions described herein may also be beneficial for mutant versions of T7 polymerase that have been shown to be more tolerant of base substitutions, as well as other RNA polymerases, including T7, T3, and SP6.

[0214] Example 2 Integration is barely detectable when magnesium chloride is used Surprisingly, we observed that integration was barely detectable when magnesium chloride, the most commonly used agent for in vitro transcription reactions, was used (see FIG. 2).

[0215] Example 3 Manganese acetate was ineffective in incorporation of 2'-methyl-ribose modified GTP Furthermore, replacement of magnesium with a different ion, manganese in the form of manganese acetate, was also ineffective in incorporation of 2'methyl-ribose modified GTP (Figure 3).

[0216] Example 4 Incorporation of 2'-methyl-GTP increases both the absolute level and expression of uncapped saRNA in cells with defective innate sensing mechanisms for RNA recognition (e.g., HEK293T cells). As described in the Examples below, the inventors evaluated the effect of incorporation of 2' methyl-ribose modified NTPs on the function of saRNA in three cell lines (human embryonic kidney 293T (HEK293T) cells, which have a defective innate sensing mechanism that can cause the suppression of RNA expression, HeLa cells, which are representative epithelial cells, and THP-1 cells, which are representative myeloid cells).

[0217] To determine whether the potential benefits of incorporating 2'methyl-ribose modified NTPs are affected by 5' capping, we assessed the effects on both uncapped and capped saRNAs and mRNAs. The saRNA constructs used in these experiments encode a firefly luciferase (fLuc) sequence downstream of a subgenomic promoter, and luciferase expression is detected by luciferase activity in cells over time.

[0218] In transfection experiments performed in HEK293T cells, using 25 μg of uncapped saRNA formulated with Lipofectamine, we observed that incorporation of 2′-methyl-GTP increased luciferase expression over 72 hours ( FIG. 4 ), with the fold increase ranging from 2-fold with 25% GM substitution to 6-fold with 75% substitution ( FIG. 4 b). A similar trend was observed when 100 ng of uncapped saRNA was used ( FIG. 5 a and FIG. 5 b). These data indicate that incorporation of 2′-methyl-GTP (>25%) increases both the absolute level and expression of uncapped saRNA in cells with defective innate sensing mechanisms for recognizing RNA. This suggests that saRNA modified with 2′-methyl-GTP may provide improved expression and stability even in cells with defective RNA sensing mechanisms, and that such improvements are independent of RNA with a 5′ cap.

[0219] Example 5 Incorporation of 2'methyl-ribose modified NTPs improves the absolute and duration of capped saRNA expression in cells with innate RNA sensing deficiencies (e.g., HEK293T cells). We then determined the effect of 2'-methyl-ribose modified NTP incorporation on the function of 5'-capped saRNA in HEK293T cells. In transfection experiments performed in HEK293T cells, when 25 μg of capped saRNA formulated with Lipofectamine was used, we observed that the expression of unmodified saRNA was increased by 1 log compared to uncapped RNA (Figure 6a). Nevertheless, when GTP was replaced by 2'-O-methyl-GTP at a ratio of 25% or more, an increased level of expression was observed over 72 hours when cells were transfected with 25 ng of saRNA (Figure 6b), with some moderate increases in expression also seen for 2'-O-methyl ATP, CTP and UTP replacement. A similar trend was observed when 100 ng saRNA was used for transfection, with the greatest effect observed for the replacement of GTP with 2'-O-methyl-GTP (Figures 7a and 7b). These data indicate that 2'-methyl-ribose modified NTPs, and in particular Gm incorporation, improve the absolute level and duration of saRNA expression in cells with innate RNA sensing deficiencies that can repress RNA translation and increase degradation. We expect that further increases can be realized when 2'-O-methyl-GTP is used in combination with 2'-O-methyl ATP, CTP and UTP substitutions.

[0220] For example, for saRNA these are: 1. 75% Gm 2. 75% Gm and 90% Am and 90% Um 3. 75% Gm and 75% Cm (with or without 90% Am+Um) 4. 75% Gm and 50% Cm (with or without 90% Am+Um) 5. 50% Gm and 50% Cm (with or without 90% Am+Um) It is.

[0221] However, other repeats that may work, e.g. 1. 50% Gm+30% Cm+50% Am+30% Um, 2. 50% Gm + 90% Cm + 50% Am + 30% Um, 3. 50% Gm + 30% Cm + 50% Am + 90% Um, 4. 50%Gm+90%Cm+50%Am+90%Um, 5. 25%Gm+30%Cm+25%Am+60%Um, 6. 25%Gm+90%Cm+50%Am+60%Um, 7. 75% Gm+30% Cm+50% Am+60% Um, 8. 75%Gm+60%Cm+50%Am+75%Um, 9. 75%Gm+90%Cm+50%Am+60%Um, 10.50%Gm+30%Cm+25%Am+60%Um, 11.50%Gm+90%Cm+25%Am+60%Um, 12.50%Gm+30%Cm+75%Am+60%Um, 13.50%Gm+90%Cm+75%Am+60%Um, 14. 25%Gm+60%Cm+50%Am+30%Um, 15. 25%Gm+60%Cm+50%Am+90%Um, 16.75%Gm+60%Cm+50%Am+30%Um, 17.75%Gm+60%Cm+50%Am+90%Um, 18. 50%Gm+60%Cm+25%Am+30%Um, 19.50%Gm+60%Cm+25%Am+90%Um, 20. 50%Gm+60%Cm+75%Am+30%Um, 21. 50%Gm+60%Cm+75%Am+90%Um, 22. 25%Gm+60%Cm+25%Am+60%Um, 23.75%Gm+60%Cm+25%Am+60%Um, 24. 25%Gm+60%Cm+75%Am+60%Um, 25. 75%Gm+60%Cm+75%Am+60%Um, 26. 50%Gm+60%Cm+50%Am+60%Um, 27. 50%Gm+60%Cm+75%Am+75%Um, 28. 50%Gm+60%Cm+60%Am+60%Um, 29. 50%Gm+50%Cm+50%Am+50%Um, or any other percentage ratio But it is possible.

[0222] For mRNA, a specific combination is 1. 75%Gm+90%Cm (with or without 90%Am+Um) 2. 75%Gm+75%Cm (with or without 90%Am+Um) 3. 50%Gm+50%Cm (with or without 90%Am+Um) 4. 25%Gm+25%Cm (with or without 90%Am+Um) may be mentioned.

[0223] However, similar to saRNA, other repeats of mRNA that can act, e.g. 1. 50% Gm+30% Cm+50% Am+30% Um, 2. 50% Gm + 90% Cm + 50% Am + 30% Um, 3. 50% Gm + 30% Cm + 50% Am + 90% Um, 4. 50%Gm+90%Cm+50%Am+90%Um, 5. 25%Gm+30%Cm+25%Am+60%Um, 6. 25%Gm+90%Cm+50%Am+60%Um, 7. 75% Gm+30% Cm+50% Am+60% Um, 8. 75%Gm+60%Cm+50%Am+75%Um, 9. 75%Gm+90%Cm+50%Am+60%Um, 10.50%Gm+30%Cm+25%Am+60%Um, 11.50%Gm+90%Cm+25%Am+60%Um, 12.50%Gm+30%Cm+75%Am+60%Um, 13.50%Gm+90%Cm+75%Am+60%Um, 14. 25%Gm+60%Cm+50%Am+30%Um, 15. 25%Gm+60%Cm+50%Am+90%Um, 16.75%Gm+60%Cm+50%Am+30%Um, 17.75%Gm+60%Cm+50%Am+90%Um, 18. 50%Gm+60%Cm+25%Am+30%Um, 19.50%Gm+60%Cm+25%Am+90%Um, 20. 50%Gm+60%Cm+75%Am+30%Um, 21. 50%Gm+60%Cm+75%Am+90%Um, 22. 25%Gm+60%Cm+25%Am+60%Um, 23.75%Gm+60%Cm+25%Am+60%Um, 24. 25%Gm+60%Cm+75%Am+60%Um, 25. 75%Gm+60%Cm+75%Am+60%Um, 26. 50%Gm+60%Cm+50%Am+60%Um, 27. 50%Gm+60%Cm+75%Am+75%Um, 28. 50%Gm+60%Cm+60%Am+60%Um, 29. 50%Gm+50%Cm+50%Am+50%Um, or any other percentage ratio But it is possible.

[0224] Example 6 Incorporation of 2'methyl-ribose modified NTPs increases the abundance and expression of uncapped saRNA in cells (e.g., HeLa cells) that have an intact innate sensing mechanism for RNA recognition. Next, we evaluated the effect of incorporation of 2'methyl-ribose modified NTPs on the function of saRNA in HeLa cells, a representative epithelial cell. Unlike HEK293T cells tested in the previous example, HeLa cells have an intact epithelial innate sensing machinery capable of responding to RNA-triggered intracellular signaling pathways that can reduce RNA expression and increase the rate of RNA degradation [6-8].

[0225] First, we investigated the impact of incorporating 2'-O-methyl modified bases into uncapped saRNA. When 25 ng of saRNA was used for transfection, we observed an increase in expression and duration of fLuc expression over 72 hours for 2'-O-methyl GTP (Figure 8a), with a 10-25 fold increase when using a ratio of 75% 2'-O-methyl GTP to 25% unmodified GTP (Figure 8b). A moderate increase in expression was observed at 48 hours for both 2'-O-methyl ATP and UTP substitutions. However, when 100 ng was used for transfection, we observed an increase in expression at 72 hours using 2'-O-methyl GTP substitutions (Figure 9a), demonstrating up to a 50-fold increase (Figure 9b). Surprisingly, we observed increased expression at 72 hours with 2'-methyl-ATP or CTP substitution, which demonstrated up to a 100-fold increase in expression for 2'-methyl-ATP. These data indicate that different substitutions can have different effects on the kinetics of expression.

[0226] Example 7 Incorporation of 2'methyl-ribose modified NTPs increases the abundance and expression of capped saRNA in cells with intact innate sensing machinery for RNA recognition (e.g., HeLa cells). We then evaluated the impact of 2'-methyl-ribose modified NTP incorporation on capped RNA. Expression of unmodified 5'-capped saRNA (25 ng) was 1 log higher in HeLa cells than that seen for uncapped saRNA (Figure 10a). Compared to unmodified saRNA, an increase in expression was observed for 2'O-methyl GTP substitution, but the fold change was not greater than that seen for uncapped RNA (Figure 10b). Moderate, but detectable, changes were also observed for substitution with 2'O-methyl ATP, CTP, and UTP. A similar trend was observed when 100 ng was used for transfection (Figure 11a). Again, 2'O-methyl GTP substitution showed the strongest effect on absolute and duration of expression, with a maximum 20-fold increase observed at 48 hours (Figure 11b). Smaller, but detectable changes were also seen for 2'-methyl-ATP or CTP substitution.

[0227] These data indicate that substitution of individual nucleotides, and in particular replacement of GTP with a 2'O-methyl base modification, improves saRNA expression, and the use of 2'O-methyl-GTP in combination with 2'O-methyl ATP, CTP, and GTP is expected to result in further increases in expression.

[0228] Example 8 Incorporation of 2'-methyl-ribose modified NTPs increases the abundance and expression of uncapped saRNA in cells with sensitive innate sensing mechanisms for RNA recognition (e.g., THP-1 cells). We then evaluated the impact of 2'-methyl-ribose modified NTP incorporation on saRNA function in representative myeloid cells, specifically in monocytic THP-1 cells, which are particularly resistant to transfection due to their highly sensitive innate sensing mechanisms capable of responding to RNA-triggered intracellular signaling pathways capable of reducing RNA expression and enhancing the rate of RNA degradation.

[0229] We first investigated the effect of incorporating 2'-O-methyl modified bases into uncapped saRNA. When 25ng of saRNA was used for transfection, they observed a dramatic increase in fLuc expression with 2'-O-methyl GTP (Figure 12a), with a greater than 1000-fold increase at 48 hours when a ratio of 75% 2'-O-methyl GTP to 25% unmodified GTP was used (Figure 12b), and there was detectable expression at 72 hours where none was observed with unmodified saRNA. Some improvement in expression was observed with 2'-O-methyl ATP and UTP substitutions. When 100ng was used for transfection, we again observed an increase in expression at 72 hours when using 2'-O-methyl GTP substitutions (Figure 13a), demonstrating an increase of up to 80-fold (Figure 13b). Substitution with 2'-O-methyl ATP, CTP, and UTP had less effect on expression than 2'-O-methyl GTP.

[0230] Example 9 Incorporation of 2'-methyl-ribose modified NTPs increases the abundance and expression of capped saRNA in cells with sensitive innate sensing mechanisms for RNA recognition (e.g., THP-1 cells). We then evaluated the impact of incorporation of 2'methyl-ribose modified NTPs on capped RNA in THP-1 cells. Expression of unmodified 5' capped saRNA (25 ng) was again 1 log higher than that seen for uncapped saRNA in THP-1 cells (Figure 14a). Compared to unmodified saRNA, increased expression was observed for 2'O-methyl GTP substitution, with fold changes ranging from 5 to 10 fold over 72 hours (Figure 14b), however this was not greater than that seen for uncapped RNA. Moderate, but detectable, changes were observed for substitution with 2'O-methyl ATP, CTP and UTP. A similar trend was observed for 2'O-methyl GTP substitution when 100 ng was used for transfection (Figure 15a). Again, 2'O-methyl GTP substitution showed the strongest effect on absolute and duration of expression, with a maximum 40-fold increase observed at 72 h (Figure 15b). Little discernible benefit was observed for substitutions with 2'O-methyl ATP, CTP, and UTP.

[0231] These data indicate that replacement of GTP with 2'O-methyl base modifications improves saRNA expression in myeloid cells. It is possible that the use of 2'O-methyl-GTP in combination with 2'O-methyl ATP, CTP, and GTP may result in further increases in expression.

[0232] Example 10 N1 methyl-pseudo-UTP does not benefit saRNA expression Since N1-methyl-pseudouridine has previously been shown to increase mRNA expression, we determined whether this confers any benefit on saRNA expression. Here, we examined 25-100% replacement of UTP with N1-methyl-pseudo-UTP. Transfection experiments were performed with 100 ng of uncapped or capped saRNA formulated with Lipofectamine. We did not observe any clear benefit on fLuc expression in either HEK293T or HeLa cells (Figures 16A and 16B, respectively). Therefore, these data suggest that, in contrast to 2'O-methyl GTP, N1-methyl-pseudo-UTP does not confer any benefit on saRNA expression.

[0233] Example 11 2'O-methylGTP, 7-deaza-GTP substitution increases the absolute value and duration of saRNA expression We also evaluated the impact of incorporating 7-deazaguanosine-5-triphosphate (7-deaza-GTP), which has previously been reported to reduce innate recognition of RNA by Toll-like receptor association [9]. Here, we examined 25-100% replacement of GTP with 7-deaza-GTP. Transfection experiments were performed with 100 ng of uncapped or capped saRNA formulated with Lipofectamine. We observed moderate expression enhancement at 24, 48, and 72 hours in HEK293T cells (Figure 17A). They also observed enhanced expression in HeLa cells, especially at 72 hours (Figure 17B) (2-fold), suggesting that 7-deaza-GTP replacement improves the duration of expression in HeLa cells. These data suggest that, similar to 2'O-methylGTP, 7-deaza-GTP substitution increases the absolute level and duration of saRNA expression, although to a lesser extent.

[0234] Example 12 2'-Fluoro modified NTPs have no effect on saRNA expression Since replacement of the ribose 2'OH with a 2'methyl group modulated saRNA expression, we 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 examined replacement of ATP, CTP, UTP, and TTP from 25 to 100% with 2'-fluoro modified NTPs. Transfection experiments were performed with 100 ng of uncapped or capped saRNA formulated with Lipofectamine. We observed no change in the expression of fLuc capped and uncapped saRNA with different percentages of 2'fluoro ATP, CTP, TTP, or UTP in HEK293T cells (Figure 18) or HeLa cells (Figure 19).

[0235] (Example 13) Summarizing the data Tables 2 and 3 below summarize the effects of base modifications on saRNA expression.

[0236] [Table 3]

[0237] [Table 4]

[0238] Example 14 Replacement of GTP with 2'O-methyl GTP improves mRNA expression Having found that 2'O-methyl NTP substitution improves expression of saRNA, the inventors then assessed its effect on the expression of an mRNA that also encodes firefly luciferase (fLuc). They compared the expression of the unmodified mRNA with that of an mRNA in which 75% of the GTPs were replaced by 2'O-methyl GTPs, and with that of an mRNA in which 75% each of the GTPs and ATPs and 90% of the CTPs and UTPs were replaced by their 2'O-methyl counterparts (combo mRNA).

[0239] 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, we observed that replacement of GTP with 2'O-methyl GTP resulted in a modest increase in expression at 24 hours. However, combined replacement of all four NTPs (Figure 20a) improved expression up to 10-fold over 72 hours (Figure 20b). mRNA expression was approximately half that seen with saRNA, which showed a similar pattern of improved expression for 75% replacement of GTP with 2'O-methyl GTP as reported above.

[0240] We then evaluated expression in interferon-competent HeLa cells (Figure 21a). saRNA expression was improved by approximately 1 log with 75% replacement of GTP with 2'O-methyl GTP (Figure 21b). mRNA expression was similarly improved with 75% replacement of GTP with 2'O-methyl GTP. Surprisingly, combinatorial replacement of all 2'O-methyl modified NTPs for NTPs increased mRNA expression to levels comparable to those seen for saRNA (Figure 21a), with a fold increase of up to 100-fold over 72 hours (Figure 21b).

[0241] They observed a similar pattern in THP-1 cells, a representative monocytic cell line (Fig. 22a). Similar to that observed for HeLa cells, saRNA expression was increased by approximately 1 log over 72 hours with replacement of 75% of GTP with 2'O-methyl GTP (Fig. 22b). When mRNA was assessed, expression was also increased 5-fold over 72 hours; however, combinatorial replacement of all 2'O-methyl modified NTPs for NTPs increased mRNA expression to levels comparable to those seen for saRNA (Fig. 22a), with a fold increase of up to 100-fold over 72 hours (Fig. 22b).

[0242] These data surprisingly demonstrate that replacement of GTP with 2'O-methyl GTP enhances expression of both saRNA and mRNA. Further replacement with additional NTPs appears to confer additional benefits to mRNA expression and is expected to do the same for saRNA.

[0243] [Table 5]

[0244] Example 15 Replacement of GTP and CTP with 2'O-methyl-GTP (Gm) and CTP enhances mRNA expression Since the combined 2'O-methyl substitution of all four NTPs was found to improve mRNA expression (Figures 20-22), the inventors then evaluated the effect on expression (fLuc) of Gm substitution in combination with each of the other three NTPs compared to combined substitution of all four NTPs (mRNA combo). They compared the expression of unmodified mRNA with that of mRNA in which 75% of the GTP was replaced by 2'O-methyl GTP alone, 75% substitution of GTP (Gm) and 75% ATP (Am), 75% GTP (Gm) and 90% CTP (Cm), 75% GTP (Gm) and 90% UTP (Um), or 75% of GTP, 75% ATP, CTP, and 90% of UTP (combo mRNA) were replaced by their 2'O-methyl counterparts.

[0245] They evaluated the effect of 2'O-methyl substitution on fLuc expression in HEK293T, HeLa and THP-1 cells as before. Using 5'-capped mRNA, we observed that replacement of GTP and CTP with 2'O-methyl GTP and 2'O-methyl CTP gave an improvement similar to that seen when all four NTPs were replaced with 2'O-methyl NTPs. In HEK293T cells, which are defective in the innate interferon response pathway, this resulted in up to a 20-fold improvement in expression at 24 hours (Figures 23 and 24).

[0246] We then evaluated expression in interferon-competent HeLa cells (Figure 25). Surprisingly, mRNA expression was improved by replacement of 75% and 90% of GTP with 2'O-methyl GTP and 2'O-methyl CTP, as was expression when all four NTPs were replaced with 2'O-methyl NTPs (Figure 26), with a fold increase of up to 70-fold over 72 hours (Figure 26).

[0247] They observed a similar pattern in THP-1 cells, a representative monocytic cell line (Figure 27). Similar to that observed for HeLa cells, mRNA expression was improved by replacement of 75% and 90% of GTP with 2'O-methyl GTP and 2'O-methyl CTP, as was expression when all four NTPs were replaced with 2'O-methyl NTPs (Figure 28), with a fold increase of over 1000-fold over 72 hours (Figure 28).

[0248] These data surprisingly demonstrate that replacement of GTP with 2'O-methyl GTP, along with replacement of CTP with 2'O-methyl GTP and 2'O-methyl CTP, enhances expression of mRNA to a similar extent as expression of mRNA in which all four NTPs were replaced with 2'O-methyl NTPs. Surprisingly, the greatest increase in expression was seen in interferon-competent cells (HeLa and THP-1 cells) compared to HEK293T cells, which have impaired interferon response and innate signaling pathways.

[0249] (Example 16) Substitution of individual NTPs with 2'O-methyl-NTPs (Nm) confers little or no benefit to mRNA expression in HEK293T and HeLa cells. Since the combined replacement of GTP and CTP with 2'O-methyl-GTP (Gm) and -CTP (Cm), or the replacement of all four NTPs, was found to improve mRNA expression (Figures 23-28), the inventors then evaluated the effect of individual NTP replacements 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% replacement of ATP with 2'O-methyl ATP (Am), 90% replacement of CTP with 2'O-methyl CTP (Cm), or 90% replacement of UTP with 2'O-methyl UTP (Um).

[0250] They evaluated the effect of 2'O-methyl substitutions on fLuc expression in HEK293T and HeLa cells as before. Using 5'-capped mRNA, we observed that individual substitutions of GTP, ATP, CTP or UTP with 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 (Figure 29) or 100 ng (Figure 30) of mRNA was used. This was also observed in HeLa cells when 25 ng (Figure 31) or 100 ng (Figure 32) of mRNA was used.

[0251] These data surprisingly demonstrate that the combined replacement of GTP with 2'O-methyl GTP, along with the replacement of CTP with 2'O-methyl GTP, confers a dominant effect on improving expression in both HEK293T and HeLa cells, where the individual replacement of either GTP or CTP with 2'O-methyl-GTP or -CTP confers little or no benefit over unmodified mRNA.

[0252] (Example 17) Replacement of individual CTPs with 2'O-methyl-CTP (Cm) improves mRNA expression in THP-1 cells Having assessed the effect of individual NTP substitutions on mRNA expression in HEK293T and HeLa cells, the inventors then assessed the effect of individual NTP substitutions on expression (fLuc) in THP-1 cells, which are monocytic cells highly susceptible to innate activation that induces robust RNA degradation. They compared the expression of unmodified mRNA with that of mRNA with 75% of GTP replaced by 2'O-methyl GTP (Gm), 75% replacement of ATP with 2'O-methyl ATP (Am), 90% replacement of CTP with 2'O-methyl CTP (Cm), or 90% replacement of UTP with 2'O-methyl UTP (Um).

[0253] They assessed the effect of 2'O-methyl substitution on fLuc expression in THP-1 cells as before. Using 5'-capped mRNA, we observed that individual replacement of GTP, ATP, CTP or UTP with 2'O-methyl-GTP (Gm), -ATP (Am), -CTP (Cm) or -UTP (Um) had differential effects on mRNA expression as determined by measuring fLuc expression in THP-1 cells when using 25 ng (Figure 33) or 100 ng (Figure 35) of mRNA. Replacement of CTP with 2'O-methyl CTP (Cm) had the greatest effect on expression, showing up to a 10-fold increase with 25 ng mRNA (Figure 34) and a 20-fold increase with 100 ng mRNA (Figure 36) compared to unmodified mRNA. Substitution of GTP with 2'O-methyl GTP (Gm) gave a modest increase in expression compared to unmodified mRNA, up to a 3-fold increase with 25 ng mRNA (Figure 34) and a 5-fold increase with 100 ng mRNA (Figure 36). Substitution of ATP or UTP with 2'O-methyl ATP (Am) or 2'O-methyl UTP (Um) had little or no effect on expression.

[0254] These data surprisingly demonstrate that while individual replacement of CTP or GTP NTPs with 2'O-methylated versions improved expression in THP-1 cells, the combined replacement of GTP with 2'O-methyl GTP, along with replacement of CTP with 2'O-methyl GTP, afforded a synergistic improvement in expression beyond the additive improvement predicted from the individual replacements.

[0255] summary The present inventors have demonstrated that 2'-substituted ribose modified NTPs, in particular 2'-O-methyl modified NTPs, can be used to (i) increase expression and / or translation of RNA molecules that comprise one or more modified NTPs, (ii) improve the stability of RNA molecules that comprise one or more modified NTPs, and / or (iii) reduce innate sensing activation, interferon production, and / or degradation of RNA molecules that comprise one or more modified NTPs.

[0256] To summarize: 1. The present invention relates to the use of 2'-modified RNA, in particular mRNA and saRNA as well as non-coding RNA, in which a significant percentage (25% or more, up to 100%) of the nucleotides are 2'-methyl-NTPs. The mRNA and saRNA sequences are sequences of significant length (more than 100 bp) in which wild-type nucleotides are at least partially replaced by 2'-O-methyl-NTPs. 2. The modified RNA is synthesized by in vitro transcription and the reaction mixture optimally contains a high concentration of magnesium ions (i.e., greater than 20 mM magnesium ions, ideally 75 nM) and a high concentration of nucleotides (i.e., greater than 10 mM nucleotides). 3. The inventors have shown that magnesium ions present in magnesium acetate rather than magnesium chloride result in improved expression and incorporation of modified NTPs. 4. Targeted moiety replacement of wild-type GTP, ATP, CTP and / or UTP with their respective 2'-methyl-NTPs resulted in up to 100-fold improvement in expression and stability of modified mRNA or saRNA in interferon-responsive competent cells.

[0257] (Example 18) Substitution of modified NTPs other than 2-O-methyl NTPs fails to confer any benefit on saRNA expression As demonstrated in Figures 16, 17, 18, 19 and 40, a wide range of modified NTPs were screened for their ability to improve saRNA expression in human cells in vitro (see Figure 39). These failed to significantly improve fLuc saRNA expression and in many cases impaired expression. Notably, some modified NTPs previously shown to improve mRNA expression, such as pseudouridine, N1-methylpseudouridine, 5-methylcytidine (m5C), N6-methyladenosine (m6A) and 2-thiouridine (s2U) [30, 31], provided no benefit or reduced expression.

[0258] These data indicate that saRNA requires specific modifications to enhance expression compared to mRNA, and the data are summarized in Table 5 below.

[0259] [Table 6]

[0260] (Example 19) 2'-O-methyl-GTP enhances expression of saRNA encoding eGFP Since replacement of GTP with 2'-O-methyl-GTP alone was found to have a large effect on fLuc expression, we determined whether the same effect would be observed if the GOI encoded in the saRNA was altered. VEEV saRNAs encoding eGFP downstream of a subgenomic promoter were generated by IVT using unmodified NTPs, or in which GTP was replaced by 75% or more of 2'-O-methyl modified NTPs, or in which UTP was replaced by 100% N-1 methyl pseudouridine. These constructs were used to transfect HELA and THP1 cells, and at 24 hours, cells were harvested, stained, and eGFP expression was assessed by flow cytometry. 2'-O-methyl-GTP modified VEEV-eGFP saRNA induces a higher number of eGFP positive cells and an increase in eGFP median fluorescence intensity (MFI) compared to unmodified and N1-methyl pseudo-UTP modified VEEV-eGFP in both HELA (see FIG. 41A) and THP1 (see FIG. 41B) cells. These data indicate that the beneficial effect of 2'-O-methyl-GTP is applicable to various encoded GOIs. Importantly, N1-methyl pseudo-UTP modified VEEV-eGFP did not produce any eGFP positive cells in both cell lines, in contrast to the published findings for conventional mRNAs, where inclusion of N1-methyl pseudo-UTP improves expression. These data highlight the surprising observation that various modifications are required for beneficial modification of saRNAs compared to mRNAs.

[0261] (Example 20) Combinatorial 2'-O-methyl-NTP substitutions improve expression of mRNA encoding fLuc Since substitution of GTP with 2'-O-methyl-GTP alone was found to have a large effect on fLuc expression, we determined whether further increases in expression could be delivered by combined 2'-O-methyl-NTP substitutions. FLuc mRNA was produced by IVT using unmodified NTPs, 100% N-1 methyl pseudouridine substitution, or nucleotides substituted with 75% or more 2'-O-methyl GTP, 75% or more 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 measuring relative light output. Combination substitutions with 2'-O-methyl ATP and UTP or 2'-methyl GTP and CTP improved expression compared to substitution of individual bases alone. However, combination substitutions with all 2-O-methyl versions of NTPs gave the greatest improvement.

[0262] Example 21 Combinatorial 2'-O-methyl-NTP substitutions improve expression of mRNA encoding fLuc Given the dramatic effect of fixed ratio combinatorial substitution of 2'-O-methyl-NTPs (2'-O-methyl combo) (see Figure 42) on fLuc expression, we determined how the ratio of 2-O-methyl-NTP substitutions using all four NTPs affected expression. FLuc mRNA was produced by IVT using unmodified NTPs and a wide range of different 2-O-methyl-NTP substitutions detailed in Table 6 below.

[0263] [Table 7]

[0264] These constructs were used to transfect HEK293T, HeLa and THP1 cells and luciferase expression was monitored over 72 hours by measuring relative luminescence (see Figures 43-45). Combinatorial substitution of all four 2'-O-methyl NTPs over a wide range of ratios improved expression compared to unmodified mRNA. These data indicate that substitution of all four NTPs with 2-O-methyl NTPs over a wide range of substitution ratios is beneficial in improving mRNA expression.

[0265] Example 22 2'-O-methyl-GTP modified saRNA is less inflammatory than unmodified saRNA To determine whether 2'-O-methyl modifications can reduce the activation of innate sensing, interferon production, and / or degradation of saRNA in host cells, THP1 cells were transfected with unmodified or modified saRNA, and cell supernatants were harvested at 24 and 48 hours post-transfection to characterize the induced cytokine profile, followed by luciferase assays to determine the level of antigen expression. THP1 cells were chosen for this analysis because they are monocytic cells with a very robust innate response. Meso Scale Discovery (MSD) assays were used to measure cytokine levels, 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 before, luciferase expression was higher for 2'-O-methyl-GTP modified saRNA compared to both unmodified and N1-methyl pseudo-UTP modified saRNA (see Figure 46A). Evaluation of cytokines and chemokines secreted from THP1 cells transfected with unmodified or modified saRNA showed that all modified saRNAs showed a perceptibly attenuated inflammatory response compared to unmodified saRNA. Interestingly, this was moderately evident for N1-methyl pseudo-UTP modified saRNA, whereas N1-methyl pseudo-UTP modification failed to show an improvement in antigen expression (see Figure 46B). However, 2'-O-methyl-GTP modified saRNA also showed an attenuated inflammatory response compared to unmodified saRNA, but this was not as significantly attenuated as the attenuated inflammatory response by N1-methyl pseudo-UTP. This suggests that the enhanced antigen expression of saRNA induced by 2'-O-methyl-GTP modification may not only be due to a reduction in the innate response, but also due to other mechanisms such as slower degradation.

[0266] Example 23 2'-O-methyl-modifications improve expression of the mRNA encoding eGFP Since 2'-O-methyl-modifications were found to have a large effect on fLuc expression, we determined whether the same effect would be observed if the encoded GOI was changed. eGFP-encoding mRNA was generated by IVT using unmodified NTPs, UTPs replaced by 100% N-1 methyl pseudouridine, or NTPs replaced by 2'-O-methyl modified NTPs (75% Gm, 75% Am, 90% Cm, 90% Um) and labeled with 2'-O-methyl combo. These constructs were used to transfect HELA and THP1 cells, and at 24 hours, cells were harvested, stained, and eGFP expression was assessed by flow cytometry. 2'-O-methyl-modified mRNA induces a higher number of eGFP-positive cells and an increase in eGFP median fluorescence intensity (MFI) compared to unmodified and N1-methyl pseudo-UTP-modified mRNA in both HELA (see FIG. 47A) and THP1 (see FIG. 47B) cells. These data indicate that the beneficial effect of 2'-O-methyl-modification is applicable to various encoded GOIs. Importantly, N1-methyl pseudo-UTP-modified mRNA improved eGFP expression in both cell lines, in agreement with published findings for conventional mRNA, albeit to a lesser extent than 2'O'methyl combo-modification. These data highlight the surprising observation that different modifications are required for beneficial modification of saRNA compared to mRNA (see also FIG. 41 and Example 19).

[0267] (Example 24) 2'-O-methyl-modified mRNA is less inflammatory than unmodified mRNA To determine whether 2'-O-methyl modifications can reduce the activation of innate sensing, interferon production, and / or degradation of mRNA in host cells, THP1 cells were transfected with unmodified or modified mRNA and cell supernatants were harvested at 24 and 48 hours post-transfection to characterize the induced cytokine profile, followed by luciferase assays to determine the level of antigen expression. THP1 cells were chosen for this analysis because they are monocytic cells with a very robust innate response. Meso Scale Discovery (MSD) assays were used to measure cytokine levels, 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 before, luciferase expression was higher for 2'-O-methyl-modified mRNA compared to both unmodified and N1-methylpseudo-UTP modified mRNA (see FIG. 48A). However, N1-methylpseudouridine modified mRNA was higher than unmodified mRNA, consistent with previous reports for mRNA. Evaluation of cytokines and chemokines secreted from THP1 cells transfected with unmodified or modified mRNA showed that all modified mRNAs showed a perceptibly attenuated inflammatory response compared to unmodified mRNA (see FIG. 48B). Interestingly, this was similar for both N1-methylpseudo-UTP and modified mRNA, but fLuc expression was lower for N1-methylpseudo-UTP modified mRNA than for 2'-O-methyl-modified mRNA (see FIG. 48A). These data indicate that the enhanced antigen expression of mRNA induced by 2'-O-methyl-modifications may not only be due to a reduced innate response, but also due to other mechanisms such as slower degradation.

[0268] (Example 25) 2'-O-methyl-GTP enhances saRNA expression in vivo A small animal study was performed to determine the effect of 2'-O-methyl modified saRNA on the duration of expression in vivo. Groups of five female Balb / c mice were injected with 10 μg of LNP-formulated VEEV-Fluc saRNA produced with unmodified NTPs or with 75% 2'-O-methyl-GTP substitution. A PBS group was used as a comparative control. The LNPs used in these studies were formulated with C12-200 ionizable lipids containing DSPC, cholesterol, and DMPE-PEG200. Images of mice were acquired on days 1 and 5 (the day of peak saRNA expression) using an IVIS Spectrum In Vivo Imaging System (Figure 49A). Expression was observed as early as day 1 for the 75% 2'-O-methyl-GTP modified group, inducing higher expression than the unmodified group. Stronger levels of expression were also observed by day 5, with a similar trend as seen on day 1. Using Aura Imaging Software, total expression was analyzed and quantified as photons per second (p / sec) (Figure 49B), and 2'-O-methyl-GTP modified VEEV-Fluc showed significantly higher expression compared to unmodified GTP at day 1, where expression gradually decreased over time. These data demonstrate that the effect of 2'-O-methyl-GTP modification alone, as well as the increase in expression, can be expected when using a combination of 2-O-methyl NTPs (Gm+Cm+Am+Um), as predicted by in vitro studies.

[0269] (Example 26) 2'-O-methyl-NTPs enhance mRNA expression in vivo Small animal studies were performed to determine the effect of 2'-O-methyl modified mRNA on the duration of expression in vivo. Groups of five female Balb / c mice were injected with 10 μg of LNP-formulated mRNA produced using unmodified NTPs, UTP replaced with N1-methylpseudouridine, or WT NTPs replaced with 2'-O-methyl modified NTPs (75% Gm, 75% Am, 90% Cm, 90% Um) and labeled as 2'-O-methyl combo (Figure 50). LNPs used in these studies were formulated using C12-200 ionizable lipids with DSPC, cholesterol, and DMPE-PEG200. Images of mice were acquired on days 1-3 using an IVIS Spectrum In Vivo Imaging System (Figure 50A). Expression was observed as early as day 1 for the 2'-O-methyl-combo modified group, inducing higher expression than both the unmodified and N1-methylpseudouridine groups. Stronger levels of expression were also observed on days 2 and 3, with a similar trend as seen on day 1. Total expression was analyzed and quantified as photons per second (p / sec) using Aura Imaging Software (Figure 50B), and the 2'-O-methyl-combo modified mRNA showed significantly higher expression compared to the unmodified mRNA on days 1-3. These data demonstrate the positive impact of 2'-O-methyl-modification on mRNA expression.

[0270] Example 27 2'-O-methyl-GTP enhances the immunogenicity of saRNA in vivo Small animal studies were performed to determine the effect of 2'-O-methyl modified saRNA on immunogenicity in vivo. VEEV saRNA (VEEV-Ha) encoding influenza hemagglutinin protein downstream of a subgenomic promoter was generated by IVT using unmodified NTPs, with GTP replaced by 75% or more 2'-O-methyl modified NTPs, or with UTP replaced by 100% N-1 methyl pseudouridine. Four groups of five female Balb / c mice were immunized at weeks 0 and 4 with intramuscular injections of 10 μg of 1) unmodified saRNA, 2) 100% N1-methyl pseudo-UTP modified saRNA, 3) 75% 2'-O-methyl-GTP modified saRNA, and 4) LNP-formulated VEEV-Ha saRNA in PBS (Figure 51A). At week 6, mice were bled and euthanized. HA ELISA was performed to evaluate total HA-specific IgG. The 2'-O-methyl-GTP modified group had a significantly higher antibody response after boosting compared to both the unmodified and N1-methyl pseudo-UTP modified groups (Figure 51A). Microneutralization assays were also performed on mouse sera using the influenza A California 2009 H1N1 virus strain (Figure 51B), and the results were consistent with the ELISA data, whereby neutralizing antibody responses were significantly higher in the 2'-O-methyl-GTP modified group after boosting compared to all other groups. These data demonstrate that an additional effect on the immunogenicity of the 2'-O-methyl-GTP modification alone can be expected when using a combination of 2-O-methyl NTPs (Gm+Cm+Am+Um) as predicted by in vitro studies.

[0271] (References) TIFF2025509197000017.tif233166TIFF2025509197000018.tif183166

[0272] The following items describe embodiments of the present invention. 1. A method for preparing a modified RNA molecule, comprising 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 NTPs are modified nucleotide triphosphates (NTPs), and wherein the RNA polymerase transcribes the template nucleic acid sequence to form an RNA molecule comprising at least 20 nucleotides, wherein at least 25% of the component nucleotides in the RNA molecule are modified. 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 component nucleotides in the RNA molecule are modified. 3. The method is (i) at least 30 mM magnesium ions, at least 40 mM magnesium ions, at least 50 mM magnesium ions, at least 60 mM magnesium ions, or at least 70 mM magnesium ions, or (ii) 50 mM to 100 mM magnesium ions, 60 mM to 95 mM magnesium ions, 65 mM to 90 mM magnesium ions, 70 mM to 80 mM magnesium ions, or 71 mM to 79 mM magnesium ions. The method or use according to any preceding item, including the use of 4. Magnesium ions are Mg 2+ 3. The method or use according to any preceding item, wherein the ion is provided as a 5. The method or use of any preceding item, wherein the magnesium ions are provided as magnesium acetate. 6. The method or use according to any preceding item, wherein the RNA is selected from the group consisting of messenger RNA (mRNA), microRNA (miRNA), small interfering RNA (siRNA), small hairpin RNA (shRNA), antisense RNA, RNA aptamer, self-amplifying RNA (saRNA), interfering RNA (RNAi), non-coding RNA, circular RNA, and small RNA. 7. The method or use according to any preceding item, wherein the RNA is self-amplifying RNA (saRNA) or messenger RNA (mRNA). 8. RNA molecules are (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 The method or use according to any preceding item, 9. RNA molecules are (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 10,000 bases in length, at least 11,000 bases in length, or at least 12,000 bases in length The method or use according to any preceding item, 10. The method or use according to any preceding item, wherein the method comprises the use of an RNA polymerase selected from the group consisting of T7, T3, SP6, KP34, Syn5 or other DNA-dependent RNA polymerase, or a mutant variant of any of these RNA polymerases. 11. The method is (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 The method or use of any preceding item, comprising use of a plurality of nucleotide triphosphates at a concentration of 12. The method or use according to any preceding item, wherein the one or more modified NTPs are selected from the group consisting of modified adenosine-5'-triphosphate (ATP), modified cytidine-5'-triphosphate (CTP), modified guanosine-5'-triphosphate (GTP) and modified uridine-5'-triphosphate (UTP). 13. The method or use according to any preceding item, wherein the method comprises a step of using at least 1, 2, 3, 4 or 5 modified NTPs selected from the group consisting of modified ATP, modified CTP, modified GTP and / or modified UTP. 14. One or more modified NTPs comprise a 2'-substituted group, in which the OH group, usually at the 2' position, is replaced by a halogen, an optionally substituted aromatic group, N, H, an optionally substituted O-alkyl, O-alkenyl or O-alkynyl group, or an optionally substituted alkyl, alkenyl or alkynyl group, in each case the aromatic group, alkyl, alkenyl or alkynyl is optionally substituted by halogen, oxo, OR, CN, NR or SR, and R is H or C. 1~6 Alkyl, C 2~6 Alkenyl or C 2~6 The method or use according to any preceding item, wherein R is alkynyl. 15. One or more modified NTPs comprise a substituted nucleobase, wherein the nucleobase is substituted with a halogen, an optionally substituted aromatic group, N, OH, an optionally substituted O-alkyl, O-alkenyl or O-alkynyl group, or an optionally substituted alkyl, alkenyl or alkynyl group, in each case the aromatic group, alkyl, alkenyl or alkynyl is optionally substituted with a halogen, oxo, OR, CN, NR or SR, and R is H or C. 1~6 Alkyl, C 2~6 Alkenyl or C 2~6alkynyl and optionally, or in addition, an oxo group in the nucleobase is replaced by a =S group. 16. An optionally substituted C alkyl, alkenyl or alkynyl group 1~ C 20 an alkyl, alkenyl or alkynyl NTP, where the alkyl, alkenyl or alkynyl is optionally substituted by halogen, oxo, OR, CN, NR2 or SR, and R is H or C 1~6 Alkyl, C 2~6 Alkenyl or C 2~6 Alkynyl, preferably alkyl, alkenyl or alkynyl is an optionally substituted C1-C 10 an alkyl, alkenyl or alkynyl NTP, where the alkyl, alkenyl or alkynyl is optionally substituted by halogen, oxo, OR, CN, NR2 or SR, and R is H or C 1~6 Alkyl, C 2~6 Alkenyl or C 2~6 16. The method or use according to item 14 or 15, wherein the aryl group is alkynyl. 17. An optionally substituted C alkyl, alkenyl or alkynyl group 1~6 Alkyl, C 2~6 Alkenyl or C 2~6 Alkynyl, where the or each alkyl, alkenyl or alkynyl is optionally substituted by halogen, oxo, OR, CN, NR2 or SR, and R is H or C 1~6 Alkyl, C 2~6 Alkenyl or C 2~6 alkynyl, preferably alkyl, alkenyl or alkynyl is optionally substituted C 1~3 Alkyl, C 2~3 Alkenyl or C 2~3 alkynyl, where the or each alkyl, alkenyl or alkynyl is substituted by one or more substituents selected from the group consisting of oxo, OH, OMe, NH and NRH; R is C 1~6 Alkyl, C 2~6 Alkenyl or C 2~617. The method or use according to any one of items 14 to 16, wherein R is alkynyl. 18. The method or use according to any preceding item, wherein the one or more modified NTPs comprise a 2'-methyl modified NTP. 19. The method or use of any preceding item, wherein the one or more modified NTPs include 2'-O-methyl modified NTPs. 20. The one or more modified NTPs are 2'-O-methyl adenosine (Am), 1,2'-dimethyl-adenosine (m 1 Am), N6,2'-O-dimethyl-adenosine (m 6 Am), N 6 ,N 6 ,2-O-trimethyl-adenosine (m 6,6 Am), 2'-O-methylinosine (Im), 1,2'-O-dimethylinosine (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-trimethyl-guanosine (m 2,7 Gm), 2'-O-methylcytidine, N 4 ,2'-O-Dimethylcytidine (m 4 Cm), N 4 ,N 4 ,2-O-trimethyl-cytidine (m 4,4 Cm), 5,2'-O-dimethyl-cytidine (m 5 Cm), N 4 -Acetyl-2'-O-methyl-cytidine (ac 4 Cm), 2'-O-methyl-5-hydromethyl-cytidine (hm 5 Cm), 5-formyl-2'-O-methyl-cytidine (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-methyl-uridine (s 2Um), 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-oxyacetic acid methyl ester (mcmo 5 Um), 5-(isopentenylaminomethyl)-3'O-methyluridine (inm 5 The method or use according to any preceding item, wherein the NTP is selected from the group consisting of NTPs containing 2'-O-methylated ribose, Um, and any other synthetic NTP containing 2'-O-methylated ribose. 21. The method or use according to any preceding item, 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. 22. The method or use according to any preceding item, wherein the method comprises a step of 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. 23. The method or use according to any preceding item, wherein when the modified RNA molecule is saRNA, the RNA molecule comprises modified GTP, preferably modified 2'-O-methyl-GTP. 24. The method or use according to any preceding item, wherein when the modified RNA molecule is mRNA, the RNA molecule comprises modified CTP and modified GTP, preferably modified 2'-O-methyl-CTP and modified 2'-O-methyl-GTP. 25. A method or use according to any preceding item, wherein at least 30%, 35% or 40% of the component nucleotides in the resulting RNA molecule are modified, and the modified nucleotides comprise adenine, cytosine, guanine and / or uracil, preferably at least 45%, 50% or 55% of the component nucleotides in the resulting RNA molecule are modified, and the modified nucleotides comprise adenine, cytosine, guanine and / or uracil. 26. A method or use according to any preceding item, wherein the method comprises a step of 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. 26. The method is (i) the OH group, usually at the 2' position, is replaced by halogen, an optionally substituted aromatic group, N, H, an optionally substituted O-alkyl, O-alkenyl or O-alkynyl group, or an optionally substituted alkyl, alkenyl or alkynyl group, in each case the aromatic group, alkyl, alkenyl or alkynyl is optionally substituted by halogen, oxo, OR, CN, NR or SR, and R is H or C 1~6 Alkyl, C 2~6 Alkenyl or C 2~6 one or more NTPs that contain a 2'-substituted group that is alkynyl; (ii) one or more modified NTPs that are not 2'-O-methyl modified NTPs; The method or use according to any preceding item, comprising combining 27. The method or use of any preceding item, wherein the template nucleic acid sequence comprises DNA, and optionally, the template nucleic acid comprises a plasmid. 28. The method or use according to any preceding item, wherein a template nucleic acid is transcribed to generate a resulting modified RNA molecule selected from the group consisting of messenger RNA (mRNA), microRNA (miRNA), small interfering RNA (siRNA), small hairpin RNA (shRNA), antisense RNA, RNA aptamer, self-amplifying RNA (saRNA), interfering RNA (RNAi), non-coding RNA, circular RNA, and small RNA. 29. The method or use of any preceding item, wherein the template nucleic acid sequence encodes an antigen derived from a virus, bacterium, mycoplasma, fungus, animal, plant, algae, parasite or protozoan, or other organism that causes disease in a subject, preferably a human or animal. 30. A method or use according to any preceding item, wherein the template nucleic acid encodes an innate inhibitor protein that counteracts a natural immune response in a subject administered a vaccine containing the resulting RNA molecule. 31. An RNA molecule obtained or obtainable by the method according to any one of items 1 to 30. 32. A pharmaceutical composition comprising the RNA molecule according to item 31 and a pharma- ceutically acceptable vehicle. 33. A method for preparing the pharmaceutical composition according to item 32, comprising contacting the RNA molecule according to item 31 with a pharma- ceutically acceptable medium. 34. The RNA molecule according to item 31 or the pharmaceutical composition according to item 32 for use as a medicament. 35. The RNA molecule according to item 31 or the pharmaceutical composition according to item 32 for use in treating, preventing or ameliorating a disease in a subject. 36. A vaccine composition comprising the RNA molecule according to item 31 or the pharmaceutical composition according to item 32. 37. The RNA molecule according to item 31, the pharmaceutical composition according to item 32, or the vaccine according to item 36, for use in stimulating an immune response in a subject. 38. (i) Improving expression and / or translation of an RNA molecule that contains one or more modified NTPs; (ii) improving the stability of an RNA molecule that contains one or more modified NTPs; and / or (iii) reducing the activation of innate sensing, interferon production, and / or degradation of RNA molecules that contain one or more modified NTPs. One or more modified nucleotide triphosphates (NTPs) for use in including 2'-substituted groups, typically in which the OH group at the 2' position is replaced by halogen, an optionally substituted aromatic group, N, H, an optionally substituted O-alkyl, O-alkenyl or O-alkynyl group, or an optionally substituted alkyl, alkenyl or alkynyl group, in each case the aromatic group, alkyl, alkenyl or alkynyl is optionally substituted by halogen, oxo, OR, CN, NR or SR, and R is H or C 1~6 Alkyl, C 2~6 Alkenyl or C 2~6 is alkynyl, One or more modified NTPs. 39. One or more modified nucleotide triphosphates (NTPs) for use according to item 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 NTPs are modified nucleotide triphosphates (NTPs), and wherein the RNA polymerase transcribes the template nucleic acid sequence to form an RNA molecule. 40. One or more modified NTPs for use according to item 38 or 39, wherein the one or more modified NTPs are incorporated into an RNA molecule using a method according to any one of items 1 to 30. 41. One or more modified NTPs for use according to any one of items 38 to 40, wherein the one or more modified NTPs are as defined in any one of items 1 to 30. 42. The one or more modified NTPs for use according to any one of items 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. 43. The one or more modified NTPs for use according to any one of items 38 to 42, wherein the RNA is selected from the group consisting of messenger RNA (mRNA), self-amplifying RNA (saRNA), microRNA (miRNA), small interfering RNA (siRNA), small hairpin RNA (shRNA), antisense RNA, RNA aptamer, interfering RNA (RNAi), non-coding RNA, circular RNA and small RNA. 44. The one or more modified NTPs for use according to any one of items 38 to 43, wherein the RNA molecule is a saRNA, and optionally the RNA molecule comprises a modified 2'-O-methyl-GTP. 45. The one or more modified NTPs for use according to any one of items 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. 46. ​​One or more modified NTPs for use according to any one of items 38 to 45, wherein the length of the RNA molecule is as defined in item 8 or item 9. 47. One or more 2'-O-methyl modified nucleotide triphosphates (NTPs) in an RNA molecule for use in improving translation of the RNA molecule.

Claims

1. (i) improving the expression and / or translation of RNA molecules containing one or more modified nucleotide triphosphates (NTPs); (ii) improving the stability of RNA molecules that contain one or more modified NTPs; and / or (iii) reducing innate sensing activation, interferon production, and / or degradation of RNA molecules containing one or more modified NTPs. One or more modified NTPs for use in containing 2'-substituted groups, where the OH group at the 2' position is usually replaced by 2'-O-methyl; One or more modified NTPs.

2. 2. The one or more modified NTPs for use according to claim 1, wherein the RNA molecule is selected from the group consisting of messenger RNA (mRNA), microRNA (miRNA), small interfering RNA (siRNA), small hairpin RNA (shRNA), antisense RNA, RNA aptamer, self-amplifying RNA (saRNA), interfering RNA (RNAi), non-coding RNA, circular RNA and small RNA.

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

4. 3. The one or more modified NTPs for use according to claim 1 or 2, wherein the RNA molecule is a messenger RNA (mRNA).

5. (i) the one or more modified NTPs are selected from the group consisting of 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); (ii) the one or more modified NTPs comprise 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; (iii) The one or more modified NTPs are 2'-O-methyladenosine (Am), N6,2'-O-dimethyl-adenosine (m 6 Am), N 6 ,N 6 ,2-O-trimethyl-adenosine (m 6,6 Am), 2'-O-methylinosine (Im), 1,2'-O-dimethylinosine (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-trimethyl-guanosine (m 2,7 Gm), 2'-O-methylcytidine, N 4 ,2'-O-dimethylcytidine (m 4 Cm), N 4 ,N 4 ,2-O-trimethyl-cytidine (m 4,4 Cm), 5,2'-O-dimethyl-cytidine (m 5 Cm), N 4 -acetyl-2'-O-methyl-cytidine (ac 4 Cm), 2'-O-methyl-5-hydromethyl-cytidine (hm 5 Cm), 5-formyl-2'-O-methyl-cytidine (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-methyl-uridine (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-oxyacetic acid methyl ester (mcmo 5 Um), and any other synthetic NTP containing 2'-O-methylated ribose; (iv) 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; (v) 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; (vi) 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; (vii) 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; (viii) when the RNA molecule is saRNA, the RNA molecule comprises a modified GTP, preferably a modified 2'-O-methyl-GTP; (ix) if the RNA molecule is mRNA, the RNA molecule comprises modified 2'-O-methyl-CTP and modified 2'-O-methyl-GTP; (x) at least 30%, 35%, or 40% of the component nucleotides in the RNA molecule are modified, and the modified nucleotides comprise adenine, cytosine, guanine, and / or uracil, preferably at least 45%, 50%, or 55% of the component nucleotides in the RNA molecule are modified, and the modified nucleotides comprise adenine, cytosine, guanine, and / or uracil; (xi) 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; (xii) the one or more 2-O-methyl modified NTPs are selected from the group consisting of 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 one or more other modified NTP bases that are not 2-O-methyl modified NTPs; In combination, optionally one or more other modified NTP bases that are not 2-O-methyl modified NTPs, (i) N'methyl-pseudouridine, fluorinated UTP, 2'-chloro NTP, 2'-ethyl NTP, 2'-bromo NTP, 2'-amino NTP, 2'-fluoro NTP or 2'-deoxy NTP, or (ii) alternative 2'-modified nucleotides listed in Table 7 and / or (xiii) the RNA molecule is encoded by a template nucleic acid sequence comprising DNA, and optionally the template nucleic acid sequence encodes an antigen derived from a tumor, a virus, a bacterium, a mycoplasma, a fungus, an animal, a plant, an algae, a parasite or a protozoan, or other organism that causes disease in a subject, preferably a human or an animal; or 3. The one or more modified NTPs for use according to claim 1 or 2, wherein the template nucleic acid is derived from an animal or a human and encodes a therapeutic protein that treats, prevents or ameliorates a disease in a subject, preferably a human or an animal, and optionally the template nucleic acid encodes an innate inhibitor protein that counteracts the innate immune response in a subject administered a vaccine comprising the RNA molecule.

6. 3. The one or more modified NTPs for use according to claim 1 or 2, 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 NTPs are modified nucleotide triphosphates (NTPs) comprising a 2'-substituted group in which the OH group normally at the 2' position is replaced with 2'-O-methyl, and wherein the RNA polymerase transcribes the template nucleic acid sequence to form an RNA molecule.

7. An RNA molecule comprising at least 100 nucleotides in which one or more of the nucleotides contains a 2'-substituted group, usually an OH group at the 2' position, replaced by a 2'-O-methyl.

8. i) the RNA is as defined in claim 2; and / or (ii) the RNA molecule is (i) 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; or (ii) at least 1,000 bases in length, at least 2,000 bases in length, at least 3,000 bases in length, at least 4,000 bases in length, at least 5,000 bases in length, at least 6,000 bases in length, at least 7,000 bases in length, at least 8,000 bases in length, at least 9,000 bases in length, at least 10,000 bases in length, at least 11,000 bases in length, or at least 12,000 bases in length The RNA molecule of claim 7,

9. A pharmaceutical composition comprising the RNA molecule of claim 7 and a pharmaceutically acceptable vehicle.

10. 10. A method for preparing the pharmaceutical composition of claim 9, comprising contacting the RNA molecule of claim 7 or 8 with a pharmaceutically acceptable medium.

11. 10. An RNA molecule according to claim 7 or 8, or a pharmaceutical composition according to claim 9, for use as a medicament.

12. 10. The RNA molecule of claim 7 or 8, or the pharmaceutical composition of claim 9, for use in treating, preventing or ameliorating a disease in a subject.

13. A vaccine composition comprising an RNA molecule according to claim 7 or 8, or a pharmaceutical composition according to claim 9.

14. 10. The RNA molecule of claim 7 or 8, or the pharmaceutical composition of claim 9, for use in stimulating an immune response in a subject.

15. One or more 2'-O-methyl modified nucleotide triphosphates (NTPs) in an RNA molecule for use in improving translation of the RNA molecule.

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