RNA molecule

Abstract

The present invention relates to RNA molecules, and particularly, although not exclusively, to unlocked nucleotides (UNAs) and unlocked nucleic acid (UNA)-RNA molecules per se, to methods for preparing UNA-RNA molecules, and to methods for translating UNA-RNA molecules into protein. The invention extends to improved methods for forming UNA-RNA by in vitro transcription, and to the resultant UNA-RNA molecules. Furthermore, the invention relates to novel methods for enhancing the expression and / or translation of RNA, i.e. protein expression, and to methods for improving the stability of an RNA molecule. The invention also involves reducing the activation of innate sensing, interferon generation and / or degradation of an RNA molecule in a host. The invention also incorporates the use of the UNA-RNA molecules in vaccines and other therapeutic pharmaceutical compositions, and their use in immunisation and therapy, such as RNAi, gene therapy, gene editing and protein replacement. Furthermore, the invention relates to novel methods for enhancing translation of RNA, i.e. protein expression.

Description

[0001] RNA molecule

[0002] The present invention relates to RNA molecules, and particularly, although not exclusively, to unlocked nucleotides (UNAs) and unlocked nucleic acid (UNA)-RNA molecules per se, to methods for preparing UNA-RNA molecules, and to methods for translating UNA-RNA molecules into protein. The invention extends to improved methods for forming UNA-RNA by in vitro transcription, and to the resultant UNA- RNA molecules. Furthermore, the invention relates to novel methods for enhancing the expression and / or translation of RNA, i.e. protein expression, and to methods for improving the stability of an RNA molecule. The invention also involves reducing the activation of innate sensing, interferon generation and / or degradation of an

[0003] RNA molecule in a host. The invention also incorporates the use of the UNA-RNA molecules in vaccines and other therapeutic pharmaceutical compositions, and their use in immunisation and therapy, such as RNAi, gene therapy, gene editing and protein replacement. Furthermore, the invention relates to novel methods for enhancing translation of RNA, i.e. protein expression.

[0004] Unlocked nucleic acid (UNA, or 2', 3'-seco-RNA) is a class of acyclic nucleic acid that lacks C-C bonds between the 2'- and 3'-carbon atoms in the five membered furanose ring [1-6]. This synthetic "sugar backbone mutant" contains all peripheral functional groups of the natural nucleotide except for the disrupted ribose ring (see

[0005] Figure 1). This modification, without the introduction of additional functional groups, makes UNA closely related to natural RNA monomers while simultaneously increasing its flexibility. Importantly, the UNA residues remain able to form Watson- Crick base-pairing with RNA or DNA strands [7], In this respect, UNA is an RNA mimic with high flexibility relative to the parent RNA [1,2],

[0006] Synthesis of UNA phosphoramidites and incorporation into oligonucleotides using standard automated chemical synthesis procedures has been known for over three decades [8-10] and has been applied to the design of siRNA, aptamer, and anti- sense RNA [1,2, 11], The term 'unlocked' is the structural opposite to parent RNA,

[0007] DNA, or 'locked' nucleic acid (LNA) that have intact rigid ribose rings [4] (see Figure 2). Other 2' ribose modifications, including locked and 2'-O-Methyl ribose nucleotides, have long been included in therapeutic oligonucleotides to both reduce hydrolysis and nuclease degradation. UNA oligonucleotides are also highly resistant to nuclease degradation [6, 12] show an extended half-life in serum [6, 13] and reduced toxicity in human cells

[0014] , It is believed that the highly flexible nature of the acyclic UNA backbone prevents the 2'-hydroxy group from attacking the phosphate linkage between RNA nucleotides, which would lead to strand cleavage [15, 16]. The exact mechanism leading to increased nuclease resistance has not been fully characterised, however, UNA is known to destabilise hairpins and reduce duplex stability in double stranded regions

[0017] . It is highly likely that such changes in structure, as well as its flexible nature, reduce recognition by ribonuclease and innate sensing binding proteins associated with innate RNA recognition and RNA degradation [18, 19]. As UNA shows reduced duplex stability, when used for inhibitory oligonucleotides, it is more often used in combination with other 2' modified ribose nucleotides such as 2'F, 2'-O-methyl or locked NTPs [6, 14],

[0008] UNA nucleotides are sometimes used together with other RNA, DNA and 2' ribose modified NTPs (including 2'F, 2'-O-Methyl and LNA) when synthesised as short oligonucleotides as "miximers" or "gapmers" to fine tune anti-sense function [6, 14], Although it has not been previously possible to generate UNA-RNA sequences >25 NTPs, the inventors hypothesised that UNA-NTPs could provide similar properties to longer RNA sequences (such as resistance to nuclease digestion, reduced susceptibility to hydrolysis, and increased serum stability etc.) could they be generated. Critically, unlocked nucleotide triphosphates (UNTPs) have never been assessed for their ability to be incorporated into RNA by in vitro transcription (IVT) using DNA- dependent RNA polymerases encoded by bacteriophages, such as T7, T3, SP6 etc., or to be translated into proteins by cellular ribosomes. This stems from the assumption that the unlocked conformation with a disrupted ring would not represent the required native conformation of RNA to be functional with respect to bacteriophage DNA-dependent RNA polymerase (eg T7, T3, Sp6 etc) or ribosomal translation. In this respect, unlocked nucleic acids are described as synthetic "RNA mimic" or non-natural nucleic acids, also known as xeno nucleic acids (XNAs), representing an artificial chemistry that does not exist in nature

[0020] . Indeed, studies of eukaryotic RNA polymerase, such as RNA polymerase Pol II, indicate that ribose integrity is a prerequisite for DNA transcription

[0021] ,

[0009] This also reflects that, prior to the inventor's discovery described herein, it has only been possible to incorporate one or two UNTPs enzymatically in sequences of up to 25 nucleotides (25nt). A single published study has assessed the ability of six different DNA polymerases from both A- and B-families, including Therminator, KOD, Phusion, Pfu, Klenow, and Taq DNA polymerases to incorporate UNA nucleotides in primer extension assays. At best, these DNA polymerases were able to copy limited stretches of a DNA-template (<25nt) incorporating up to two consecutive UNA-nucleotides in primer extension reactions

[0022] , Thus, while widely used for short antisense RNA (< 100 nucleotides), generated by synthetic processes, to date, it has not been possible to generate sequences enzymatically containing any proportion of unlocked NTPs of sufficient length to encode functional proteins. RNA vaccine and biotherapeutics have recently come of age, showing successful utility in the COVID-19 pandemic. The two lead COVID-19 candidates manufactured by Moderna and Pfizer both utilise the incorporation of Nl-methyl-pseudouridine, a synthetic modified version of the nucleotide triphosphate (NTP) UTP

[0023] . This is used to reduce triggering of innate pathways that can restrict the expression of encoded proteins rendering the RNA ineffective with respect to therapeutic or vaccine indications [24,25]. The exact mechanism for improved expression is not fully understood and may be related to the suppression of highly immunogenic double stranded RNA (dsRNA) during in vitro transcription

[0026] . This approach, however, provides little or no advantage in the context of self-amplifying RNA (saRNA), another modality of RNA constructs used in vaccines. Furthermore, the use of methyl-pseudouridine may not be optimal. Indeed, most cellular receptors detect most double-stranded RNA (dsRNA) structure by contacting the ribose backbone with minimal interaction with the bases. In this respect, ribose base modifications have been predicted to have beneficial effects and UNA with their disrupted ribose ring are highly likely to present a different structural form to that of parent RNA. However, efficient incorporation of UNA-NTPs in mRNA, saRNA, or any sequences over 25nt by an enzymatic process has not previously been achievable. Furthermore, while previous studies have indicated that use of UNA- NTPs can reduce nuclease degradation of short oligonucleotides, no one has previously determined whether the unlocked xeno-nucleic acid can be translated by cellular ribosomes. Indeed, the non-native conformation would suggest that this would be wholly unlikely given that this has not been assessed in the >30 years since their discovery. Currently it has not been possible to generate longer UNA-RNA (>25nt) using an enzymatic process. For instance, strict transcription reaction mixture optimisations are required to support high and wide-ranging percentages of nucleotide substitution of wild-type (i.e. non-modified) NTPs. Furthermore, generation of UNA >25nt has not been successfully achieved for DNA polymerases and has only been achieved for sequences <25nt with replacement of a single or at best two UNTPs in the entire sequence

[0022] , Overcoming these bottlenecks using bacteriophage DNA- dependent RNA polymerases with precise in vitro transcription reaction conditions could greatly improve the in vivo applications and potential of UNA-RNAs intended for use as vaccines or biotherapeutics.

[0010] There is, therefore, a need to overcome the problems in the art, and provide an improved method for preparing a modified RNA molecule (for example, mRNA or self-amplifying RNA) which incorporates modified, non-natural unlocked ribose nucleotides. There is also a need to provide improved methods for enhancing RNA translation and protein expression when using UNA-NTPs. Furthermore, an enzymatic process for generating even short RNA sequences would be advantageous in reducing the cost of production of therapeutic UNAs, such as inhibitory RNA (iRNA, RNAi), and long non-coding RNA, for use in, for example, gene therapy, gene editing, protein replacement.

[0011] RNA is composed by the nucleotides ATP, CTP, GTP and UTP, however, a wide range of natural and synthetic modified nucleotides exist

[0027] , TTP is associated with DNA but has also been used in the generation of unlocked oligonucleotides. As discussed above, however, unlocked nucleic acid does not exist in nature and is an artificial mimic of natural RNA, with a structure that is highly distorted relative to natural RNA [1-6]. To date, it has not been possible to make UNA-RNA of sufficient size to encode proteins and its non-native nature, in particularly the lack of C-C bonds between 2'- and 3'- carbon atoms in the five membered furanose ring and its highly flexible structure, indicate that the ability to engage cellular ribose machinery and generate function proteins would be considered highly unlikely. In this respect, it would be more likely to be similar to eukaryotic RNA polymerases

[0021] and DNA polymerases that are unable to utilise UNTPs

[0022] ,

[0012] As described in the Examples, the inventors have now surprisingly demonstrated that incorporation of the modified UNA-NTPs can be achieved by in vitro transcription, promoted by high levels of magnesium ions, ideally magnesium acetate greater than 20 mM (optimally greater than 40 mM), or magnesium chloride greater than 20 mM (optimally at about 40mM). This opens up the possibility for enzymatic generation of any RNA sequence of any size using unlocked nucleotides. This greatly improves the potential economics of making unlocked RNA sequence of any size, given that enzymatic production of RNA is considered considerably cheaper, and more efficient than current synthetic solid state methods for generating short unlocked RNA sequences used for multiple applications including antisense RNA, RNA aptamers, external guide sequences, ribozymes, short interfering RNA, and, as well as longer RNA greater than 100 nucleotides, such as long non-coding RNA (Lnc-RNA), or encoding RNA: including mRNA; self-amplifying RNA; circular RNA; or any other form of longer RNA. The inventors have also surprisingly shown that UNA-RNA can be translated, generate functional protein, and enhance the expression of RNA (which may be self-amplifying RNA (saRNA) or mRNA), in interferon-responsive cells relative to unmodified RNA. The inventors anticipate that substitution of up to 1-100% of individual NTPs (ATP, CTP, GTP, UTP, TTP) with unlocked NTPs may increase RNA resistance to nuclease and / or hydrolytic degradation and, as a consequence, increase the half-life of RNA and / or its expression.

[0013] The inventors believe that combined UNA-NTP substitutions will have even greater potential to increase resistance to degradation and, as a consequence, enhance the magnitude and / or duration of expression in transduced cells. Combinations could consist of substituting combinations of two, three, four or five types of UNTPs (i.e. ATP, CTP, GTP, UTP and / or TTP).

[0014] The inventor's surprising observation is that unlocked nucleotides can be effectively incorporated into RNA sequences of greater than 100 nucleotides by in vitro transcription, and that these RNA molecules can be translated by ribosomes within living cells, generating functional protein. The inventors have demonstrated that the use of individual and combinations of unlocked nucleotides will increase both the level and duration of protein expression in transduced cells, due to their predicted impact on nuclease resistance. Furthermore, the inventors have shown that different combinations of UNA-NTPs modulate the innate cytokine response to naked unmodified RNA induced when delivered into the cytoplasm of cells due to their different structure and high flexibility relative to parent RNA. This new approach is applicable to all forms of encoding RNA, greater than 25 nucleotides, including messenger RNA (mRNA), self-amplifying RNA (saRNA), circular RNA (cRNA), or to any other form of RNA of a length greater than 25 nucleotides. While the inventors believe that UNTPs on their own have an impact on RNA stability and expression, they believe that they will be most impactful when used in combination with rNTP, dNTPs, 2'-F-NTPs, 2'-O-Methyl NTP and locked NTPs (LNTPs) that have intact furanose rings, as observed for oligonucleotides. Indeed, the inventors anticipate that the combination of highly flexible UNTPs together with ribose intact NTPs is likely to provide alternative RNA structures that are less likely to be recognised by ribose and innate sensing RNA and / or DNA binding proteins. Thus, chimeric DNA / UNA, RNA / UNA, LNA / UNA, 2'F / UNA, 2'-O-Methyl / UNA with alternative structures are preferred.

[0015] While UNAs have traditionally been used with common NTP bases (adenine, cytosine, guanine, uridine and thymidine), the skilled person will appreciate that they could equally be synthesised and used with modified bases known to impact on innate recognition of RNA, such as N-l-methylpseudouridine or 5- methylcytidine, to name just two of a great variety of base modifications that can be generated. Therefore, the invention also provides the use of UNAs containing other base modifications to further augment their functionality.

[0016] Thus, in a first aspect of the invention, there is provided one or more unlocked nucleotide, for use in :

[0017] (i) enhancing the expression and / or translation of an RNA molecule comprising the one or more unlocked nucleotide;

[0018] (ii) enhancing the stability of an RNA molecule comprising the one or more unlocked nucleotide; and / or (iii) reducing the activation of innate sensing, interferon generation and / or degradation of an RNA molecule comprising the one or more unlocked nucleotide.

[0019] In a second aspect, there is provided a method of: (i) enhancing the expression and / or translation of an RNA molecule comprising one or more unlocked nucleotide;

[0020] (ii) enhancing the stability of an RNA molecule comprising one or more unlocked nucleotide; and / or

[0021] (iii) reducing the activation of innate sensing, interferon generation and / or degradation of an RNA molecule comprising one or more unlocked nucleotide, wherein the method comprises incorporating one or more unlocked nucleotide into the RNA molecule, thereby enhancing the expression and / or translation of the RNA molecule comprising the one or more unlocked nucleotide, enhancing the stability of the RNA molecule comprising the one or more locked nucleotide, and / or reducing the activation of innate sensing, interferon generation and / or degradation of the RNA molecule comprising the one or more unlocked nucleotide.

[0022] Enhancing expression of translation of a modified RNA molecule is an important aspect of the invention.

[0023] Accordingly, in a third aspect, there is provided a method of enhancing the translation of an RNA molecule, the method comprising translating an RNA molecule comprising one or more unlocked nucleotide, wherein the level of translation in the presence of the one or more unlocked nucleotide is greater than the level of translation in the absence of the one or more unlocked nucleotide.

[0024] In a fourth aspect, there is provided the use of one or more unlocked nucleotide in an RNA molecule, for enhancing the translation of the RNA molecule. In a fifth aspect, there is provided one or more unlocked nucleotide in an RNA molecule, for use in enhancing the translation of the RNA molecule.

[0025] In another aspect, there is provided use of one or more unlocked nucleotide in an RNA molecule, for: (I) enhancing the expression and / or translation of an RNA molecule comprising the one or more unlocked nucleotide;

[0026] (ii) enhancing the stability of an RNA molecule comprising the one or more unlocked nucleotide; and / or

[0027] (iii) reducing the activation of innate sensing, interferon generation and / or degradation of an RNA molecule comprising the one or more unlocked nucleotide.

[0028] Any of the methods or uses described herein may be carried out in vivo, in vitro or ex vivo. However, most preferably the methods are carried out in vivo.

[0029] As described in the Examples, the inventors have demonstrated that incorporation of unlocked nucleotides (UNTPs, e.g. as shown in Figure 1 and 2) to form UNA as UNA-RNA (either UNA-mRNA or UNA-saRNA) can be achieved by in vitro transcription, promoted by high levels of magnesium ions, ideally magnesium acetate greater than 20 mM, optimally greater than 40 mM, or magnesium chloride greater than 20 mM, optimally greater than 40mM (see Figure 3). The inventors were very surprised to observe that UNA-RNA can be translated, enhance the expression of the RNA and generate functional protein in interferon-responsive cells relative to unmodified RNA, which does not comprise UNAs. Substitution of up to 90% of individual nucleotides (ATP, or UTP) with unlocked nucleotides surprisingly enhances mRNA expression of encoded firefly luciferase (fLuc) (which acts as a marker or surrogate of protein expression) by up to 40-fold in HEK293T cells that have impaired innate immunity sensing (see Figure 4). These data suggest that individual unlocked nucleotides (UNA-NTPs) have the surprising potential to enhance expression in the absence of innate recognition. Substitution of individual nucleotides (NTPs) with unlocked nucleotides (NTPs) enhances fLuc expression in HeLa epithelial cells by up to 200-fold at 48 hours (UNA-ATP) and 160-fold (UNA-UTP) at 48h. Furthermore, combined substitution of both UNA-ATP and UNA-UTP enhanced expression in a similar manner. These data indicate that the incorporation of UNAs enhance both the magnitude and duration of expression in epithelial cells with intact innate sensing pathways (see Figure 5), where the impact hierarchy on expression for the individual NTPs is ATP >UTP.

[0030] Furthermore, substitution of individual nucleotides (NTPs) with unlocked nucleotides (NTPs) enhances fLuc expression in THP-1 monocytic cells by up to 100-fold at 24, 48 and 72 hours (UNA-ATP) and 100-fold (UNA-UTP) at 24h and 200-fold at 48 and

[0031] 72h (UNA-ATP) (Figure 6). Furthermore, combined substitution of both UNA-ATP and UNA-UTP enhanced expression in a similar manner. These data confirm that the incorporation of UNAs enhance both the magnitude and duration of expression in cells with intact innate sensing pathways, in this case demonstrated using a monocytic cell line (see Figure 6). These cells demonstrated the same hierarchy of impact on expression for the individual NTPs where UTP >ATP.

[0032] It was also surprisingly observed that substitution of individual nucleotides (NTPs) with UNAs was able to influence the secretion profile of IP10 (CXCL10) in supernatants harvested from transfected THP-1 cells (see Figure 7). Furthermore, combined substitution of both UNA-ATP and UNA-UTP provided further reduction in IP10 levels. In this respect, IP10 reflects innate response to invading RNA and type I interferon expression [28,29]. These data demonstrate that individual UNTPs impact on innate recognition and associated cytokine induction and that combined UNTPs may provide even greater reduction in innate recognitions. In summary, therefore, these data are highly unexpected given that UNA-RNA has never been considered or assessed for translation and protein production, being a non-native RNA mimic (or xeno-nucleic acid) that cannot be utilized by eukaryotic RNA polymerases and a wide range of DNA polymerases, and has never been assessed for protein translation despite its discovery over three decades ago [21,22],

[0033] Hence, in a sixth aspect of the invention, there is provided a method of preparing an unlocked nucleic acid RNA (UNA-RNA) molecule, wherein the method comprises contacting, in the presence of at least 20mM magnesium ions, (i) a template nucleic acid sequence, (ii) an RNA polymerase, and (ill) a plurality of nucleotide triphosphates (NTPs), one or more of which comprises an unlocked nucleotide, wherein the RNA polymerase transcribes the template nucleic acid sequence to form an UNA-RNA molecule comprising at least 25 nucleotides. In a seventh aspect, there is provided use of at least 20mM magnesium ions in a transcription reaction to prepare an unlocked nucleic acid RNA (UNA-RNA) molecule comprising at least 25 nucleotides.

[0034] Advantageously, the inventors have devised novel and innovative in vitro transcription reaction conditions for use in the methods of the invention. These reaction conditions include higher than previously used magnesium ion concentrations (i.e. >20mM, preferably >40mM, ideally >50mM), and therefore enable significant levels of replacement of wild-type (i.e. unmodified) nucleotides in the resultant UNA-RNA molecule with unlocked nucleotides or UNTPs. To date, it has not been possible to generate RNA sequences by in vitro transcription, of sizable length (>25nt, >50nt, >100nt, > 1000nt, or more) where single or multiple wild-type unmodified nucleotides are replaced by unlocked nucleotides or UNTPs, and where the RNA molecule is expressed and translated into a functional protein. The in vitro transcription reaction mixture in the methods of the invention requires the use of >20mM magnesium ions and is demonstrably higher than some conventional in vitro transcription methods. UNA-RNAs, synthesised from the methods described herein exhibit significantly enhanced expression in interferon responsive cells. This is highly likely to enhance the in vivo potential of RNA vaccines (for example, expressing microbial antigens for immunizing against infectious micro-organisms, such as viruses and bacteria etc., and also cancer vaccines expressing oncogenic antigens) as well as RNA biotherapeutics (for example, RNAi, protein replacement, gene editing and gene therapy etc.) whether delivered as mRNA, saRNA or circular RNA.

[0035] Preferably, the method comprises the use of at least 25mM magnesium ions, preferably at least 30mM magnesium ions, more preferably at least 35mM magnesium ions, preferably at least 40mM magnesium ions, and even more preferably at least 5omM magnesium ions. Preferably, greater than 50mM magnesium ions are used in each embodiment of the the invention. Preferably, the method comprises the use of at least 60mM magnesium ions, more preferably at least 70mM magnesium ions, and even more preferably at least 75mM magnesium ions. Preferably, the method comprises the use of at least 80mM magnesium ions, more preferably at least 85mM magnesium ions, more preferably at least 90mM magnesium ions, and even more preferably at least lOOmM magnesium ions. Preferably, the method comprises the use of less than 120mM magnesium ions, preferably less than lOOmM magnesium ions, more preferably less than 90mM magnesium ions, preferably less than 80mM magnesium ions, and even more preferably less than 75mM magnesium ions.

[0036] Preferably, the method comprises the use of between 50mM and lOOmM magnesium ions, more preferably between 60mM and 95mM magnesium ions, more preferably between 65mM and 90mM magnesium ions, and even more preferably between 70mM and 80mM magnesium ions. Preferably, the method comprises the use of between 71mM and 79mM magnesium ions, more preferably between 73mM and 77mM magnesium ions.

[0037] The magnesium ions may be provided in the method as Mg2+ions. The magnesium ions may be provided as magnesium acetate, magnesium chloride, magnesium citrate, magnesium sulphate, magnesium gluconate, or magnesium lactate, and so on. As described in the Examples, the inventors observed increased RNA expression when the magnesium ions were provided in the form of magnesium acetate, and to a lesser extent, magnesium chloride, and this was totally unexpected. Hence, preferably the magnesium ions are provided as magnesium acetate. Preferably, the magnesium ions are not provided as magnesium chloride.

[0038] As such, in an embodiment in which the method uses magnesium acetate, the method comprises the use of between 30mM and 90mM magnesium acetate, more preferably between 40mM and 80mM magnesium acetate, more preferably between 50mM and 70mM magnesium acetate, and even more preferably between 60mM and 75mM magnesium ions. However, in an embodiment in which the method uses magnesium chloride, the method comprises the use of between 25mM and 55mM magnesium chloride, more preferably between 30mM and 50mM magnesium chloride, more preferably between 35mM and 45mM magnesium chloride, and even more preferably between 38mM and 42mM magnesium chloride.

[0039] The RNA molecule may be single-stranded or double-stranded. The skilled person would appreciate that when the nucleic acid is double-stranded, for example doublestranded RNA, "bases in length" will refer to the length of base pairs. The RNA molecule may be coding. For example, coding RNA may be used for therapeutic applications and vaccine applications.

[0040] The RNA molecule may be non-coding. For example, non-coding RNA may be used for RNAi applications.

[0041] The RNA molecule may be selected from a group consisting of: messenger RNA (mRNA), micro RNA (miRNA); short interfering RNA (siRNA); short hairpin RNA (shRNA); anti-sense RNA; RNA aptamers; self-amplifying RNA (saRNA); interference RNA (RNAi); non-coding RNA; circular RNA; and small RNA.

[0042] Preferably, the RNA molecule is self-amplifying RNA (saRNA) or messenger RNA (mRNA).

[0043] In an embodiment, the RNA molecule may not comprise a cap. However, preferably, the RNA molecule comprises a cap. In some embodiments, the one or more unlocked nucleotide (or UNA) is part of the cap. In other words, preferably the cap comprises the one or more unlocked nucleotide. In other embodiments, however, the one or more unlocked nucleotide (or UNA) is not part of the cap. In other words, preferably the cap does not comprise the one or more unlocked nucleotide. The RNA molecule may be messenger RNA (mRNA). mRNA may contain the basic elements of a cap, 5' UTR, 3'UTR, a Kozak sequence, an IRES, and poly(A) tail of variable length. Examples 1-6 describe mRNA comprising unlocked nucleotides or UNTPs, i.e. UNA-mRNA. The RNA molecule may be self-amplifying RNA (saRNA). The skilled person would appreciate that self-amplifying RNAs may contain the basic elements of mRNA (a cap, 5' UTR, 3'UTR, a Kozak sequence, an IRES, a viral polymerase, and poly(A) tail of variable length), but may be considerably longer (for example 8-12 kb). The RNA molecule may be circular RNA (circRNA). The skilled person would appreciate that circular RNAs are single-stranded, covalently closed RNA molecules that may contain the basic elements of RNA (such a Kozak sequence, and / or an IRES). The RNA molecule (which may be RNAi, saRNA, mRNA or circRNA) may be at least 20,

[0044] 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, at least 40, 41, 42, 43, 44 or 45 bases in length. The RNA molecule may be at least

[0045] 46, 47, 48 or 49 bases in length.

[0046] The RNA molecule (which may be RNAi, saRNA, mRNA or circRNA) may be at least 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.

[0047] The RNA molecule, most preferably saRNA or mRNA, may 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.

[0048] In one embodiment, RNA molecule is at least 6000 bases in length. In one embodiment, the RNA, most preferably saRNA or mRNA, is at least 6000 bases in length. In a preferred embodiment, the saRNA is at least 6000 bases in length.

[0049] The RNA molecule, most preferably saRNA or mRNA, may be between 5000 and

[0050] 20,000 bases in length, between 6000 and 15,000 bases in length, between 7000 and 14,000 bases in length, between 7500 and 13,000 bases in length, between 8000 and

[0051] 12,000 bases in length, between 8500 and 11,000 bases in length, between 9000 and

[0052] 10,000 bases in length.

[0053] The methods of the invention may be carried out in vivo, in vitro or ex vivo. However, most preferably the methods are carried out in vitro. Preferably, the methods comprise in vitro transcription (IVT).

[0054] Preferably, the method comprises the use of an RNA polymerase, which may be selected from a group consisting of: T7; T3; SP6; KP34; Syn5; or other DNA- dependent RNA polymerase; or a mutated variant of any of these RNA polymerases. Each of these RNA polymerases is able to maximise the synthesis of the modified RNA. Preferably, however, the method comprises the use of T7 RNA polymerase or a variant thereof. T7 RNA polymerase variants may include the following mutations (singly or in combination) associated with increased processivity and / or tolerance for incorporation of modified bases, specifically R425C, K631R, S633P, Y639F, Y639V, S641A, H784A, H784S, and / or H784G.

[0055] Preferably, the plurality of nucleotide triphosphates (NTPs) are selected from the group consisting of ATP, GTP, CTP, TTP and / or UTP. NTPs are the building blocks of RNA. Preferably, the method comprises the use of substantially equal proportions of each of ATP, GTP, CTP, TTP and / or UTP. However, in some embodiments, the method may comprise different ratios between each of ATP, GTP, CTP, TTP and / or UTP, examples of which are given below. This will depend on the sequence of the template nucleic acid and resultant RNA molecule to be transcribed therefrom.

[0056] Preferably, the methods comprise the use of the plurality of nucleotide triphosphates at a concentration of at least ImM, 2mM, 3mM or 4mM. Preferably, the methods comprise the use of the plurality of nucleotide triphosphates at a concentration of at least 5mM, 6mM or 7mM. More preferably, the methods comprise the use of the plurality of nucleotide triphosphates at a concentration of at least 8mM, 9mM, lOmM. Suitably, the methods comprise the use of the plurality of nucleotide triphosphates at a concentration of greater than lOmM. For example, the methods may comprise the use of the plurality of nucleotide triphosphates at a concentration of at least 12mM, 14mM, or 16mM. The methods may also comprise the use of the plurality of nucleotide triphosphates at a concentration of at least 18mM, 20mM, or 22mM. In other embodiments, the methods may comprise the use of the plurality of nucleotide triphosphates at a concentration of at least 25mM, 50mM, 75mM or lOOmM.

[0057] It will be appreciated that the one or more unlocked nucleotide may comprise a ribose moiety modified by a lack of C-C bond between 2'- and 3'- carbon atoms in the five membered furanose ring [1-6]. Thus, the one or more unlocked nucleotide preferably comprises the absence of a C-C bond between 2'- and 3'- carbon atoms in the ribose sugar.

[0058] The one or more unlocked nucleotide may be represented herein by formula [I] :

[0059] R1— \nBase wherein Base is a nucleobase;

[0060] R1is a phosphate, a modified phosphate, a phosphate linker, a modified phosphate linker or a 5 to 10 membered heteroarylene, wherein the phosphate linker, the modified phosphate linker or the 5 to 10 membered heteroarylene is a linker between the rest of the unlocked nucleotide of formula [I] and an attachment point to a first adjacent nucleotide in the RNA molecule;

[0061] R3is H or a bond to a second adjacent nucleotide in the RNA molecule; wherein if R1is a phosphate or a modified phosphate then R3is a bond to a second adjacent nucleotide in the RNA molecule;

[0062] R2is H or C1-6 alkyl; and R6is OH, a halogen, an optionally substituted aromatic group, NH2, N3, H, an optionally substituted O-alkyl, O-alkenyl or O-alkynyl group, or an optionally substituted alkyl, alkenyl or alkynyl group, wherein in each instance the aromatic group, alkyl, alkenyl or alkynyl is optionally substituted with halogen, oxo, OR9, CN, NR9R10or SR9; or R2and R6together define a linker;

[0063] R4, R5, R7and R8are independently H or Ci-e alkyl; and

[0064] R9and R10are independently H or Ci-e alkyl, C2-6 alkenyl or C2-6 alkynyl.

[0065] Preferably, the one or more unlocked nucleotide may be represented herein as formula [la] :

[0066] The nucleobase may be a primary nucleobase or a modified nucleobase.

[0067] A primary nucleobase may be a purine or a pyrimidine. A purine may be adenine, guanine or hypoxanthine. A pyrimidine may be cytosine, uracil or thymine.

[0068] The term "modified nucleobase" may be understood by the skilled person. For instance, the modified nucleobase may have the structure of a primary nucleobase with one or more modifications. The one or more modifications may include one or more of: - replacing a hydrogen and / or an NH2 group in the primary nucleobase with an optionally substituted Ci-e alkyl, an optionally substituted C2-6 alkenyl, an optionally substituted C2-6 alkynyl, OR11, NRnR12, COOR11, CONRnR12or a halogen; replacing a double bond with a single bond; - replacing a CH in the structure with an N; replacing an N in the structure with a CR11; replacing an 0 in the structure with an NR11, S, Se or CRnR12; and / or the nucleobase is attached to the rest of the locked nucleotide of formula [I] at a different point to the point where the primary nucleobase would typically be attached; wherein R11and R12are independently H, an optionally substituted Ci-e alkyl, an optionally substituted Ci-e alkenyl, an optionally substituted Ci-e alkynyl or an optionally substituted Ce-12 aryl, or R11and R12together with the atom to which they are bonded form an optionally substituted 3 to 10 membered cycloalkyl or an optionally substituted 3 to 10 membered heterocyclyl.

[0069] An optionally substituted alkyl, alkenyl or alkynyl may be unsubstituted or substituted with one or more of halogen, OR13, COOR13, CONR13R14and NR13R14, wherein R13and R14are H, Ci-e alkyl, C2-6 alkenyl or C2-6 alkynyl.

[0070] An optionally substituted aryl, cycloalkyl or heterocycyl may be unsubstituted or substituted with one or more of halogen, OR13, COOR13, CONR13R14and NR13R14, wherein R13and R14are H, Ci-e alkyl, C2-6 alkenyl or C2-6 alkynyl.

[0071] A halogen may be fluorine, chlorine or bromine.

[0072] An unlocked nucleic acid (UNA) with a modified uracil nucleobase may include pseudouridine (i ), pyridin-4-one ribonucleoside, 5-aza-uridine, 6-aza-uridine, 2- thio-5-aza-uridine, 2-thio-uridine (s2U), 4-thio-uine (s4U), 4-thio-pseudouridine, 2- thio-pseudouridine, 5-hydroxy-uridine (ho5U), 5-aminoallyl- uridine, 5-halo-uridine (e.g., 5-iodo~uridine or 5-bromo-uridine), 3-methyl-uridine (m3U), 5- methoxyuridine (mo^U), uridine 5-oxyacetic acid (cmo^U), uridine 5-oxyacetic acid methyl ester (mcmo^U), 5-carboxymethyl-uridine (cm^U), 1-carboxymethyl- pseudouridine, 5-carboxyhydroxymethyl-uridine (chm^U), 5- carboxyhydroxymethyl-uridine methyl ester (mchm^U), 5-methoxycarbonylmethyl- uridine (mcm^U), 5-methoxycarbonylmethyl-2-thio-uridine (mcm^sU), 5- aminomethyl-2-thio-uridine (nmVU), 5-methylaminomethyl-uridine (mnm^U), 5- methylaminomethyl-2-thio-uridine (mnm5s2U), 5-methylaminomethyl-2-seleno- uridine (mnm5se2U), 5-carbamoylmethyl-uridine (ncm^U), 5- carboxymethylaminomethyl-uridine (cmnm^U), 5- carboxymethylaminomethyl-2- thio-uridine (cmnm^s2U), 5-propynyl-uridine, 1-propynyl- pseudouridine, 5- taurinomethyl-uridine (tm^U), 1-taurinomethyl-pseudouridine, 5-taurinomethyl-2- thio-uridineAmVU), l-taurinomethyl-4-thio-pseudouridine, 5-methyl-uridine (m^u, i.e., having the nucleobase deoxythymine), 1-methyl-pseudouridine (mA), 5- methyl-2-thio-uridine (m^s2U), 1- methyl-4-thio-pseudouridine (m^y) 4-thio-l- methyl-pseudouridine, 3-methyl-pseudouridine (m3y) , 2-thio- 1-methyl- pseudouridine, 1-methyl- 1-deaza-pseudouridine, 2-thio-l -methyl- 1-deaza- pseudouridine, dihydrouridine (D), dihydropseudouridine, 5,6-dihydrouridine, 5- methyl- dihydrouridine (m^D), 2-thio-dihydrouridine, 2-thio-dihydropseudouridine, 2-methoxy-uridine, 2- methoxy-4-thio-uridine, 4-methoxy-pseudouridine, 4- methoxy-2-thio-pseudouridine, Nl-methyl- pseudouridine, 3-(3-amino-3- carboxypropyl)uridine (acp3U), l-methyl-3-(3-amino-3- carboxypropyl)pseudouridine (acp3y), 5-(isopentenylaminomethyl)uridine (inm^U), 5- (isopentenylaminomethyl)-2-thio-uridine (inm^s2U), a-thio-uridine, 1-thio- uridine, deoxythymidine, 5-(2-carbomethoxyvinyl) uridine, and 5-[3-(l-E- propenyla mi no) uridine. In some embodiments, UNA-CTP contains a modified nucleobase. Exemplary base modified UNA-CTPs include 5-aza-cytidine, 6-aza-cytidine, pseudoisocytidine, 3- methyl-cytidine (m3C), N4-acetyl-cytidine (ac4C), 5-formyl-cytidine (f^C), N4- methyl-cytidine (m4C), 5-methyl-cytidine (m^C), 5-halo-cytidine (e.g., 5-iodo- cytidine), 5-hydroxymethyl-cytidine (hm5C), 1-methyl-pseudoisocytidine, pyrrolo- cytidine, pyrrolo- pseudoisocytidine, 2-thio-cytidine (s2C), 2-thio-5-methyl-cytidine, 4-thio-pseudoisocytidine, 4-thio- 1-methyl-pseudoisocytidine, 4-thio-l-methyl-l- deaza-pseudoisocytidine, 1-methyl-l-deaza- pseudoisocytidine, zebularine, 5-aza- zebularine, 5-methyl-zebularine, 5-aza-2-thio-zebularine, 2- thio-zebularine, 2- methoxy-cytidine, 2-methoxy-5-methyl-cytidine, 4-methoxy-pseudoisocytidine, 4- methoxy- 1-methyl-pseudoisocytidine, lysidine (k2C), a-thio-cytidine, 1-thio- cytidine.

[0073] In some embodiments, UNA-ATP contains a modified nucleobase. Exemplary base modified UNA-ATPs include 2-amino-purine, 2, 6-diaminopurine, 2-amino-6-halo- purine (e.g., 2-amino-6-chloro-purine), 6-halo-purine (e.g., 6-chloro-purine), 2- amino-6-methyl-purine, 8-azido-adenosine, 7-deaza-adenine, 7-deaza-8-aza- adenine, 7-deaza-2- amino-purine, 7-deaza-8-aza-2-amino-purine, 7-deaza-2,6- diaminopurine, 7-deaza-8-aza-2,6- diaminopurine, 1-methyl-adenosine ( ^A), 2- methyl-adenine (m2A), N6-methyl-adenosine(m6A), 2- methylthio-N6-methyl- adenosine (ms2m6A), N6-isopentenyl-adenosine (i6A), 2-methylthio-N6- isopentenyl-adenosine (ms2i6A), N6-(cis-hydroxyisopentenyl)adenosine (io6A), 2- methylthio-N6- (cis-hydroxyisopentenyl)adenosine (ms2io6A), N6- glycinylcarbamoyl-adenosine (g6A), N6- threonylcarbamoyl-adenosine (t6A), N6- methyl-N6-threonylcarbamoyl-adenosine (m6t6A), 2- methylthio-N6- threonylcarbamoyl-adenosine (ms2g6A), N6,N6-dimethyl-adenosine (m62A), N6- hydroxynorvalylcarbamoyl-adenosine (hn6A), 2-methylthio-N6- hydroxynorvalylcarbamoyl- adenosine (ms2hn6A), N6-acetyl-adenosine (ac6A), 7- methyl-adenine, 2-methylthio-adenine, 2- methoxy-adenine, a-thio-adenosine, 2- amino-N6-methyl-purine, 1-thio-adenosine, 8-azido- adenosine, and N6-(19-amino- pentaoxanonadecyl)-adenosine.

[0074] An unlocked nucleic acid (UNA) with a modified guanine nucleobase may include inosine (I), 1-methyl-inosine (m1!), wyosine (imG), methylwyosine (mimG), 4- demethyl-wyosine (imG-14), isowyosine (imG2), wybutosine (yW), peroxywybutosine (ozyW), hydroxywybutosine (OHyW), undermodified hydroxybutosine (OHyW*), 7-deaza-guanosine, queuosine (Q), epoxyqueuosine (oQ), galactosyl- queuosine (galQ), mannosyl-queuosine (manQ), 7-cyano-7- deaza-guanosine (preQo), 7- aminomethyl-7-deaza-guanosine (preQi), archaeosine (G+), 7-deaza-8-aza-guanosine, 6-thio- guanosine, 6-thio-7-deaza-guanosine, 6- thio-7-deaza-8-aza-guanosine, 7-methyl-guanosine (m7G), 6-thio-7-methyl- guanosine, 7-methyl-inosine, 6-methoxy-guanosine, 1-methyl-guanosine (mxG) , N2-methyl-guanosine (m2G), N2,N2-dimethyl-guanosine (m2zG), N2,7-dimethyl- guanosine (m27G), N2, N2,7-dimethyl-guanosine (m227G), 8-oxo-guanosine, 7- methyl-8-oxo-guanosine, l-methyl-6- thio-guanosine, N2-methyl-6-thio-guanosine, N2,N2-dimethyl-6-thio-guanosine, a-thio-guanosine, 1-thio-guanosine, 06-methyl- guanosine.

[0075] The nucleobase may be a naturally-occurring or synthetic derivative of a nucleobase, including pyrazolo[3,4-d]pyrimidines, 5-methylcytosine (5-me-C), 5- hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-propynyl uracil and cytosine, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4- thiouracil, 8-halo (e.g., 8-bromo), 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo particularly 5-bromo, 5- trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 8-azaguanine and 8- azaadenine, deazaguanine, 7-deazaguanine, 3-deazaguanine, deazaadenine, 7-deazaadenine, 3- deazaadenine, pyrazolo[3,4- d]pyrimidine, imidazo[l,5-a]l,3,5 triazinones, 9-deazapurines, imidazo[4,5- d]pyrazines, thiazolo[4,5-d]pyrimidines, pyrazin-2-ones, 1,2,4-triazine, pyridazine; and 1,3,5 triazine.

[0076] In some embodiments, the modified nucleobase is 5-methyl-cytosine. In some embodiments, R1is a phosphate. Accordingly, R1may be understood to be

[0077] O ii .

[0078] ■o i o

[0079] O’ In alternative embodiments, R1is a phosphate linker or a modified phosphate linker.

[0080] A phosphate linker may be understood to have the following structure: 0 A modified phosphate or a modified phosphate linker may have the same structure as a phosphate or a phosphate linker, respectively, except one or more of the oxygens may have been replaced with a different substitutent. For instance, one or more of the oxygens in the phosphate or phosphate linker may have been replaced with an NR15, S, Se, BR15, BH3, H, an optionally substituted Ci-e alkyl, an optionally substituted Ci-e alkenyl, an optionally substituted Ci-e alkynyl and / or an optionally substituted Ce-iz aryl and / or the O’ in the phosphate linker may have been replaced with NR15R16, CR15R16, S’, Se’, BH3, H, an optionally substituted Ci-e alkyl, an optionally substituted Ci-e alkenyl, an optionally substituted Ci-e alkynyl or an optionally substituted Ce-iz aryl, wherein R15and R16are independently H, an optionally substituted Ci-e alkyl, an optionally substituted Ci-e alkenyl, an optionally substituted Ci-e alkynyl or an optionally substituted Ce-iz aryl, or R15and R16together with the atom to which they are bonded for an optionally substituted 3 to 10 membered cycloalkyl or an optionally substituted 3 to 10 membered heterocyclyl.

[0081] Examples of modified phosphate groups include, but are not limited to, phosphorothioate, phosphoroselenates, boranophosphates, boranophosphate esters, hydrogen phosphonates, phosphoramidates, phosphorodiamidates, alkyl or aryl phosphonates, and phosphotriesters. Phosphorodithioates have both non- linking oxygens replaced by sulfur. The phosphate linker can also be modified by the replacement of a linking oxygen with nitrogen (bridged phosphoramidates), sulfur (bridged phosphorothioates), and carbon (bridged methylene- phosphonates). The a-thio substituted phosphate moiety is provided to confer stability through the unnatural phosphorothioate backbone linkages. Phosphorothioate DNA and RNA have increased nuclease resistance and subsequently a longer half-life in a cellular environment. Phosphorothioate linked modified nucleic acids or mRNA molecules are expected to also reduce the innate immune response through weaker binding / activation of cellular innate immune molecules.

[0082] In specific embodiments, a modified nucleoside includes an alpha-thio-nucleoside (e.g., 5'- 0-(l-thlophosphate)-adenosine, 5'-0-(l-thiophosphate)-cytidine (a-thio- cytidine), 5'-0-(l-thiophosphate)-guanosine, 5'-0-(l-thiophosphate)-uridine, or 5'-0- (l-thiophosphate)-pseudouridine).

[0083] , H II l-B-P-0

[0084] O wherein indicates the attachment point of the R1group to the rest of the locked nucleotide of formula [I] and indicates the attachment point of the R1group to a first adjacent nucleotide in the RNA molecule.

[0085] In embodiments where one of R1is a 5 to 10 membered heteroarylene, the heteroarylene is preferably a 5 or 6 membered heteroarylene, and more preferably is a 5 membered heteroarylene. The heteroarylene may wherein indicates the attachment point of the R1group to the rest of the locked

[0086] — * nucleotide of formula [I] and indicates the attachment point of the R1group to a first adjacent nucleotide in the RNA molecule.

[0087] O 0

[0088] 5 11 ? 5 11

[0089] -l H ; 5-O-P I --O-f ’ * -i $-O-P I --O -o-p i --o-

[0090] Preferably, R1is 0 or O More preferably, R1is 0

[0091] R2may be H or Ci-e alkyl. More preferably, R2is H or C1-3 alkyl. Even more preferably, R2is H or methyl. In some embodiments, R2is H.

[0092] In some embodiments, R6is OH.

[0093] Alternatively, R6may be an optionally substituted O-alkyl, an optionally substituted O-alkenyl, an optionally substituted O-alkynyl group, an optionally substituted alkyl, an optionally substituted alkenyl or an optionally substituted alkynyl.

[0094] It may be appreciated, when discussing the optional substituents and the size of the alkyl, alkenyl and / or alkynyl, this applies to both the optionally substituted alkyl, alkenyl and alkynyl groups per se as well as the optionally substituted 0- alkyl, O-alkenyl and O-alkynyl groups.

[0095] The alkyl, alkenyl or alkynyl may be an optionally substituted C1-C20 alkyl, an optionally substituted C2-C20 alkenyl or an optionally substituted C2-C20 alkynyl, wherein the alkyl, alkenyl or alkynyl is optionally substituted with halogen, oxo, OR9, CN, NR9R10or SR9. Preferably, the alkyl, alkenyl or alkynyl may be an optionally substituted C1-C10 alkyl, an optionally substituted C2-C10 alkenyl or an optionally substituted C2-C10 alkynyl, wherein the alkyl, alkenyl or alkynyl is optionally substituted with halogen, oxo, OR9, CN, NR9R10or SR9. In some embodiments, the alkyl, alkenyl or alkynyl may be an optionally substituted Ci-e alkyl, an optionally substituted C2-6 alkenyl or an optionally substituted C2-6 alkynyl, wherein the, or each alkyl, alkenyl or alkynyl is optionally substituted with halogen, oxo, OR9, CN, NR9R10or SR9. In some embodiments, the alkyl, alkenyl or alkynyl may be an optionally substituted C1-3 alkyl, an optionally substituted C2-3 alkenyl or an optionally substituted C2-3 alkynyl, wherein the, or each alkyl, alkenyl or alkynyl is unsubstituted or substituted with one or more substituents selected from the group consisting of an oxo, OH, OMe, NH2 and NR9H, wherein R9is Ci-e alkyl, C2-6 alkenyl or C2-6 alkynyl. More preferably, the alkyl, alkenyl or alkynyl may be an optionally substituted C1-3 alkyl, a C2-3 alkenyl or a C2-3 alkynyl, wherein the, or each alkyl, alkenyl or alkynyl is preferably unsubstituted.

[0096] Accordingly, in some embodiments, the 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 an oxo, OH, OMe, NH2 and NR9H, wherein R9is Ci-e alkyl, C2-6 alkenyl or C2-6 alkynyl. Accordingly, the alkyl, alkenyl or alkynyl may be methyl, hydromethyl, acetyl, formyl, carbamoylmethyl, methoxycarbonylmethyl, carboxyhydroxymethyl, carboxymethylaminomethyl, oxyacetic acid methyl ester or isopentenylaminomethyl. Alternatively, R6may be a halogen. The halogen may be a chlorine, or bromine. Preferably the halogen is fluorine. Therefore, the one or more modified nucleotide preferably comprises a 2'-substituted fluoro nucleotide.

[0097] R6may be an optionally substituted aromatic group. The optionally substituted aromatic group may be a C6-C12 optionally substituted aromatic group. The optionally substituted aromatic group may be optionally substituted phenyl.

[0098] Preferably, R6is Me or OMe, more preferably OMe. Alternatively, R2and R6may together define a linker. The linker may be: -L1-X2-X1-

[0099] Wherein L1is a C1-5 alkylene;

[0100] X1is 0, CH2, CO, NR15, Se, S, SO or SO2;

[0101] X2is absent, or is CO, 0, NR17, Se, S, SO or SO2; and Accordingly, the one or more unlocked nucleotide may be represented herein as formula [lb]:

[0102] The one or more unlocked nucleotide may be represented herein as formula [Ibi]:

[0103] L1may be a straight or branched C1-5 alkylene. Preferably, L1is a straight or branched C1-3 alkylene. More preferably, L is -CHz-*, -CH(CH3)-*, -CH2CH2-* or - CH(CH3)CH2-*, where an asterisk indicates a point of attachment to X2in embodiments where it is present and to X1in embodiments where X2is absent. Most preferably, L1is CH2.

[0104] X1may be 0, CH2, NR17, Se or S. In some embodiments, X1is 0.

[0105] X2may be absent. Alternatively, X2may be SO2.

[0106] R4, R5, R7and R8may each independently be H or C1-3 alkyl. Even more preferably, R4, R5, R7and R8are each independently H or methyl. In some embodiments, R4, R5, R7and R8are each H. Accordingly, preferably the use or method comprises using one or more unlocked nucleotide selected from a group consisting of: unlocked adenine; unlocked cytosine; unlocked guanine; unlocked uracil and / or unlocked thymine. Preferably, the use or method comprises using at least two unlocked nucleotides selected from a group consisting of: an unlocked adenine; an unlocked cytosine; an unlocked guanine; an unlocked uracil and / or an unlocked thymine. Preferably, the use or method comprises using at least three unlocked nucleotides selected from a group consisting of: an unlocked adenine; an unlocked cytosine; an unlocked guanine; an unlocked uracil and / or an unlocked thymine. Preferably, the use or method comprises using at least four unlocked nucleotides selected from a group consisting of: an unlocked adenine; an unlocked cytosine; an unlocked guanine; an unlocked uracil and / or an unlocked thymine. Preferably, the use or method comprises using at least five unlocked nucleotides selected from a group consisting of: an unlocked adenine; an unlocked cytosine; an unlocked guanine; an unlocked uracil and / or an unlocked thymine.

[0107] Preferably, one or more of the unlocked nucleotides used in the invention (e.g. in the in vitro transcription reaction or in the resultant UNA-RNA molecule) is unlocked, in that they are not naturally occurring.

[0108] In an embodiment, at least 1%, 2%, 3%, 4% or 5% of the constituent nucleotides in the transcription reaction or in the resultant UNA-RNA molecule may comprise unlocked nucleotides, wherein the unlocked nucleotides may comprise unlocked adenine, unlocked cytosine, unlocked guanine, unlocked thymine and / or unlocked uracil. However, at least 6%, 7%, 8%, 9% or 10% of the constituent nucleotides in the transcription reaction or in the resultant UNA-RNA molecule may comprise unlocked nucleotides, wherein the unlocked nucleotides may comprise unlocked adenine, unlocked cytosine, unlocked guanine, unlocked thymine and / or unlocked uracil. Preferably, at least 15%, 17%, 19%, 20% or 25% of the constituent nucleotides in the transcription reaction or in the resultant UNA-RNA molecule may comprise unlocked nucleotides, wherein the unlocked nucleotides may comprise unlocked adenine, unlocked cytosine, unlocked guanine, unlocked thymine and / or unlocked uracil.

[0109] Preferably, at least 30%, 35% or 40% of the constituent nucleotides in the transcription reaction or in the resultant RNA molecule are unlocked, wherein the unlocked nucleotides may comprise unlocked adenine, unlocked cytosine, unlocked guanine, unlocked thymine and / or unlocked uracil. More preferably, at least 45%, 50% or 55% of the constituent nucleotides in the transcription reaction or in the resultant RNA molecule are unlocked, wherein the unlocked nucleotides may comprise unlocked adenine, unlocked cytosine, unlocked guanine, unlocked thymine and / or unlocked uracil. Even more preferably, at least 60%, 65% or 70% of the constituent nucleotides in the transcription reaction or in the RNA molecule are unlocked, wherein the unlocked nucleotides may comprise unlocked adenine, unlocked cytosine, unlocked guanine, unlocked thymine and / or unlocked uracil. Still more preferably, at least 75%, 80% or 85% of the constituent nucleotides in the transcription reaction or in the RNA molecule are unlocked, wherein the unlocked nucleotides may comprise unlocked adenine, unlocked cytosine, unlocked guanine, unlocked thymine and / or unlocked uracil. Most preferably, at least 90%, 95%, 96%, 97%, 98%, 99% or 100% of the constituent nucleotides in the transcription reaction or in the RNA molecule are unlocked, wherein the unlocked nucleotides may comprise unlocked adenine, unlocked cytosine, unlocked guanine, unlocked thymine and / or unlocked uracil. Preferably, the transcription reaction is an in vitro transcription reaction.

[0110] The data described in the Examples show that combinations of unlocked nucleotides (UNA-NTPs) could provide additional advantages. For example, combined unlocked nucleotide substitutions could potentially be used at lower levels of percentage substitution to enhance expression and additional combinations may be perferable. The inventors anticipate that further levels of enhancement may be seen with substitutions of all four species of UNA-NTPs. It was also observed that substitution of combined unlocked nucleotides had a greater impact than individual unlocked nucleotides with respect to the secretion profile of the cytokine IP 10 (CXCL10), an indicative innate cytokine [28, 29] in supernatants harvested from transfected THP- 1 cells (see Figure 7). Here two, unlocked nucleotide combinations were able to suppress IP-10 induction, where substitution of two unlocked nucleotides was the most effective at suppressing IP-10 responses (see Figure 7).

[0111] As such, these data indicate that combined incorporation unlocked nucleotides, and in particular unlocked uracil (UNA-UTP) with unlocked cytosine (UNA-ATP) reduced IP10 induction greater than individual UNA-NTPs. Accordingly, preferably the method comprises using a combination of two, three or four different unlocked nucleotides selected from a group consisting of: unlocked adenine, unlocked cytosine, unlocked guanine, unlocked thymine, and unlocked uracil. The combinations of two, three, four or five different unlocked nucleotides may be varied in the transcription reaction. Accordingly, the inventors have assessed various different combinations of unlocked nucleotides (UNA-NTPs), as follows: For example, for mRNA these are:

[0112] 1. 90% UNA-ATP+90% UNA-CTP

[0113] 2. 90% UNA-ATP+90% UNA-GTP

[0114] 3. 90% UNA-ATP+90% UNA-UTP

[0115] 4. 90% UNA-ATP+90% UNA-TTP 5. 90% UNA-CTP+90% UNA-GTP

[0116] 6. 90% UNA-CTP+90% UNA-UTP

[0117] 7. 90% UNA-CTP+90% UNA-TTP

[0118] 8. 90% UNA-GTP+90% UNA-UTP

[0119] 9. 90% UNA-GTP+90% UNA-TTP 10. 90% UNA-ATP+90% UNA-CTP+90%UNA-GTP

[0120] 11. 90% UNA-ATP+90% UNA-CTP+90% UNA-TTP

[0121] 12. 90% UNA-ATP+90% UNA-CTP+90% UNA-UTP

[0122] 13. 90% UNA-ATP+90% UNA-GTP+90% UNA-TTP

[0123] 14. 90% UNA-ATP+90% UNA-GTP+90% UNA-UTP 15. 90% UNA-CTP+90% UNA-GTP+90% UNA-TTP

[0124] 16. 90% UNA-CTP+90% UNA-GTP+90% UNA-UTP

[0125] 17. 90% UNA-ATP+90% UNA-CTP+90% UNA-GTP+90% UNA-TTP

[0126] 18. 90% UNA-ATP+90% UNA-CTP+90% UNA-GTP+90% UNA-UTP; or any other percentage ratio .

[0127] For example, for saRNA these are:

[0128] 1. 90% UNA-ATP+90% UNA-CTP

[0129] 2. 90% UNA-ATP+90% UNA-GTP

[0130] 3. 90% UNA-ATP+90% UNA-UTP 4. 90% UNA-ATP+90% UNA-TTP

[0131] 5. 90% UNA-CTP+90% UNA-GTP

[0132] 6. 90% UNA-CTP+90% UNA-UTP

[0133] 7. 90% UNA-CTP+90% UNA-TTP

[0134] 8. 90% UNA-GTP+90% UNA-UTP 9. 90% UNA-GTP+90% UNA-TTP

[0135] 10. 90% UNA-ATP+90% UNA-CTP+90%UNA-GTP

[0136] 11. 90% UNA-ATP+90% UNA-CTP+90% UNA-TTP 12. 90% UNA-ATP+90% UNA-CTP+90% UNA-UTP

[0137] 13. 90% UNA-ATP+90% UNA-GTP+90% UNA-TTP

[0138] 14. 90% UNA-ATP+90% UNA-GTP+90% UNA-UTP

[0139] 15. 90% UNA-CTP+90% UNA-GTP+90% UNA-TTP 16. 90% UNA-CTP+90% UNA-GTP+90% UNA-UTP

[0140] 17. 90% UNA-ATP+90% UNA-CTP+90% UNA-GTP+90% UNA-TTP

[0141] 18. 90% UNA-ATP+90% UNA-CTP+90% UNA-GTP+90% UNA-UTP; or any other percentage ratio. In preferred embodiments, the percentage ratio may be up to 100% substitution.

[0142] The RNA molecule may further comprise one or more modified nucleotide comprising a 2'-substituted group in which the OH group normally at the 2' position is replaced with a halogen (preferably fluorine), an optionally substituted aromatic group, a NH2, a N3, a H, an optionally substituted O-alkyl, O-alkenyl or O-alkynyl group, or an optionally substituted alkyl, alkenyl or alkynyl group, wherein in each instance the aromatic group, alkyl, alkenyl or alkynyl is optionally substituted with halogen, oxo, OR, CN, NR2 or SR, wherein R is H or Ci-e alkyl, C2-6 alkenyl or C2-6 alkynyl.

[0143] Such a combination, in the RNA molecule, of one or more unlocked nucleotide (UNA) and one or more 2'-modified nucleotide can be referred to as a miximer. The 2'-modified nucleotide may be as defined in PCT / GB2023 / 050508, the contents of which are incorporated herein by reference.

[0144] The one or more modified nucleotide may be represented herein by formula [II] : wherein Base is a nucleobase; R17is a phosphate, a modified phosphate, a phosphate linker, a modified phosphate linker or a 5 to 10 membered heteroarylene, wherein the phosphate linker, the modified phosphate linker or the 5 to 10 membered heteroarylene is a linker between the rest of the unlocked nucleotide of formula [I] and an attachment point to a first adjacent nucleotide in the RNA molecule; R18and R21are independently H or Ci-e alkyl;

[0145] R19is H or a bond to a second adjacent nucleotide in the RNA molecule; wherein if R17is a phosphate or a modified phosphate then R19is a bond to a second adjacent nucleotide in the RNA molecule; R20is a halogen, an optionally substituted aromatic group, a NH2, a N3, a H, an optionally substituted O-alkyl, O-alkenyl or O-alkynyl group, or an optionally substituted alkyl, alkenyl or alkynyl group, wherein in each instance the aromatic group, alkyl, alkenyl or alkynyl is optionally substituted with halogen, oxo, OR22, CN, NR22R23or SR22; and R22and R23are independently H or Ci-e alkyl, C2-6 alkenyl or C2-6 alkynyl.

[0146] The one or more modified nucleotide may be represented herein by formula [Ila] : R17-« Q Base

[0147] RIS^L R21R19R20[Ha]

[0148] The nucleobase may be as defined above in relation to formula [I].

[0149] The phosphate, modified phosphate, phosphate linker, modified phosphate linker or 5 to 10 membered heteroarylene may be as defined above in relation to formula [I].

[0150] R18and R21may independently be H or C1-3 alkyl, and more preferably are independently H or methyl. In some embodiments, R8and R21are both H. In one embodiment, R20is an optionally substituted O-alkyl, an optionally substituted O-alkenyl, an optionally substituted O-alkynyl group, an optionally substituted alkyl, an optionally substituted alkenyl or an optionally substituted alkynyl. It may be appreciated, when discussing the optional substituents and the size of the alkyl, alkenyl and / or alkynyl, this applies to both the optionally substituted alkyl, alkenyl and alkynyl groups per se as well as the optionally substituted 0- alkyl, O-alkenyl and O-alkynyl groups. The alkyl, alkenyl or alkynyl may be an optionally substituted C1-C20 alkyl, an optionally substituted C2-C20 alkenyl or an optionally substituted C2-C20 alkenyl alkynyl, wherein the alkyl, alkenyl or alkynyl is optionally substituted with halogen, oxo, OR22, CN, NR22R23or SR22, wherein R22and R23are independently H or Ci-e alkyl, C2-6 alkenyl or C2-6 alkynyl. Preferably, the alkyl, alkenyl or alkynyl may be an optionally substituted C1-C10 alkyl, an optionally substituted C2-C10 alkenyl or an optionally substituted C2-C10 alkynyl, wherein the alkyl, alkenyl or alkynyl is optionally substituted with halogen, oxo, OR22, CN, NR22R23or SR22, wherein R22and R23are H or Ci-e alkyl, C2-6 alkenyl or C2-6 alkynyl.

[0151] In some embodiments, the alkyl, alkenyl or alkynyl may be an optionally substituted Ci-e alkyl, an optionally substituted C2-6 alkenyl or an optionally substituted C2-6 alkynyl, wherein the, or each alkyl, alkenyl or alkynyl is optionally substituted with halogen, oxo, OR22, CN, NR22R23or SR22, wherein R22and R23are H or Ci-e alkyl, C2-6 alkenyl or C2-6 alkynyl. In some embodiments, the alkyl, alkenyl or alkynyl may be an optionally substituted C1-3 alkyl, an optionally substituted C2-3 alkenyl or an optionally substituted C2-3 alkynyl, wherein the, or each alkyl, alkenyl or alkynyl is unsubstituted or substituted with one or more substituents selected from the group consisting of an oxo, OH, OMe, NH2 and NR22H, wherein R22is Ci-e alkyl, C2-6 alkenyl or C2-6 alkynyl. More preferably, the alkyl, alkenyl or alkynyl may be an optionally substituted C1-3 alkyl, an optionally substituted C2-3 alkenyl or an optionally substituted C2-3 alkynyl, wherein the, or each alkyl, alkenyl or alkynyl is preferably unsubstituted. Accordingly, in some embodiments, the 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 an oxo, OH, OMe, NH2 and NR22H, wherein R22is Ci-e alkyl, C2-6 alkenyl or C2-6 alkynyl. Accordingly, the alkyl, alkenyl or alkynyl may be methyl, hydromethyl, acetyl, formyl, carbamoylmethyl, methoxycarbonylmethyl, carboxyhydroxymethyl, carboxymethylaminomethyl, oxyacetic acid methyl ester or isopentenylaminomethyl.

[0152] Preferably, R20is a halogen. The halogen may be a chlorine, or bromine. Preferably the halogen is fluorine. Therefore, the one or more modified nucleotide preferably comprises a 2'-substituted fluoro nucleotide. R20may be an optionally substituted aromatic group. The optionally substituted aromatic group may be a C6-C12 optionally substituted aromatic group. The optionally substituted aromatic group may be optionally substituted phenyl. Preferably, R20is Me or OMe, more preferably OMe.

[0153] Accordingly, preferably the use or method comprises using at least one modified nucleotide selected from a group consisting of: a modified adenine; a modified cytosine; a modified guanine; a modified uracil and / or a modified thymine. Preferably, the use or method comprises using at least two modified nucleotides selected from a group consisting of: a modified adenine; a modified cytosine; a modified guanine; a modified uracil and / or a modified thymine. Preferably, the use or method comprises using at least three modified nucleotides selected from a group consisting of: a modified adenine; a modified cytosine; a modified guanine; a modified uracil and / or a modified thymine. Preferably, the use or method comprises using at least four modified nucleotides selected from a group consisting of: a modified adenine; a modified cytosine; a modified guanine; a modified uracil and / or a modified thymine. Preferably, the use or method comprises using at least five modified nucleotides selected from a group consisting of: a modified adenine; a modified cytosine; a modified guanine; a modified uracil and / or a modified thymine.

[0154] Preferably, the one or more modified nucleotide comprises a 2'-methyl modified nucleotide. Most preferably, the one or more modified nucleotide comprises a 2'-O-methyl modified nucleotide.

[0155] There are at least 28 identified natural 2'-O-methyl modifications (Nm) to ribonucleosides. Accordingly, the one or more modified nucleotide may be selected from a group consisting of: 2'-O-methyladenosine (Am); l,2'-dimethyl-adenosine (m'Am); N6,2'-O-dimethyl-adenosine (m6Am); N6,N6,2-O-trimethyl-adenosine (m6'6Am); 2'-O-methylinosine (Im); l,2'-O-dimethylinosine ( ^Im); 2'-O- methylguanosine (Gm); l,2'-O-dimethylguanosine (m'Gm); N2,2'-O- dimethylguanosine (m2Gm); N2,N2, 2'-O-trimethylguanosine (m2'2Gm); N2,7,2'-O- trimethyl-guanosine (m2'7Gm); 2'-O-methylcytidine; N4,2'-O-di methyl cytidine(m4Cm); N4,N4,2-O-trimethyl-cytidine (m4'4Cm); 5,2'-O-dimethyl-cytidine (m5Cm); N4-acetyl-2'-O-methyl-cytidine (ac4Cm); 2'-O-methyl-5-hydromethyl- cytidine (hm5Cm); 5-formyl-2'-O-methyl-cytidine (f5Cm); 2'-0-methyluridine (Um); 3,2'-O-dimethyluridine (m3llm); 5,2'-O-dimethyluridine (m5Um or Tm); 2-thio-2'-O- methyl-uridine (s2Um); 2'-O-methyl-pseudouridine (Ym); 5-carbamoylmethyl-2'-O- methyluridine (ncm5Um); 5-methoxycarbonylmethyl-2'-O- methyl uridine (mcm5llm); 5-(carboxyhydroxymethyl)-2'-O-methyluridine methyl ester (mchm5Um); 5-carboxymethylaminomethyl-2'-O-methyluridine (cmbm5llm); 2'-O- methyluridine 5-oxyacetic acid methyl ester (mcmo5Um); 5- (isopentenylaminomethyl)-3'O-methyluridine (inm5Um); 2'-O- (trifluoromethyl)adenosine, 2'-O-(trifluoromethyl)guanosine, 2'-O- (trifluoromethyl)cytosine, 2'-O-(trifluoromethyl)uridine; 2'-O- (trifluoromethyl)thymidine; and any other synthetic nucleotide or NTP that contains a 2'-O-methylated ribose.

[0156] Preferably, therefore, the one or more modified nucleotide comprises a 2'-O-methyl modified adenine (2'-O-methyl-Adenine; also known as "2-OMe ATP" or "Am"), a 2'- O-methyl modified cytosine (2'-O-methyl-Cytosine; also known as "2-OMe CTP" or "Cm"), a 2'-O-methyl modified guanine (2'-O-methyl-Guanine; also known as "2- OMe GTP" or"Gm"), a 2'-O-methyl modified uracil (2'-O-methyl-Uracil; also known as "2-OMe UTP" or "Um"), and / or a 2'-O-methyl modified thymine (2'-O-methyl- Thymine; also known as "2-OMe TTP" or "Tm").

[0157] Preferably, the method comprises using one or more modified nucleotide which comprises a 2'-O-methyl-ATP, a 2'-O-methyl-CTP, a 2'-O-methyl-GTP, a 2'-O- methyl-UTP, and / or 2'-O-methyl-TTP. Preferably, the method comprises using at least one modified nucleotide selected from a group consisting of: 2'-O-methyl-ATP, a 2'-O-methyl-CTP, a 2'-O-methyl-GTP, a 2'-O-methyl-UTP, and 2'-O-methyl-TTP. Preferably, the method comprises using at least two modified nucleotides selected from a group consisting of: 2'-O-methyl-ATP, a 2'-O-methyl-CTP, a 2'-O-methyl- GTP, a 2'-O-methyl-UTP, and 2'-O-methyl-TTP. Preferably, the method comprises using at least three modified nucleotides selected from a group consisting of: 2'-O- methyl-ATP, a 2'-O-methyl-CTP, a 2'-O-methyl-GTP, a 2'-O-methyl-UTP, and 2'-O- methyl-TTP. Preferably, the method comprises using at least four modified nucleotides selected from a group consisting of: 2'-O-methyl-ATP, a 2'-O-methyl- CTP, a 2'-O-methyl-GTP, a 2'-O-methyl-UTP, and 2'-O-methyl-TTP. Preferably, the method comprises using at least five modified nucleotides selected from a group consisting of: 2'-O-methyl-ATP, a 2'-O-methyl-CTP, a 2'-O-methyl-GTP, a 2'-O- methyl-UTP, and 2'-O-methyl-TTP. Preferably, the method comprises using at least four modified nucleotides selected from a group consisting of: 2'-O-methyl-ATP, a 2'-O-methyl-CTP, a 2'-O-methyl- GTP, and a 2'-O-methyl-UTP.

[0158] Preferably, however, the method comprises using a modified nucleotide which comprises a 2'-substituted fluoro nucleotide.

[0159] In a preferred embodiment, the use or method of the invention comprises using a combination of:

[0160] (i) one or more unlocked nucleotide represented by formula [I]; and

[0161] (ii) one or more modified nucleotide represented by formula [II].

[0162] Preferably, the use or method of the invention comprises using a combination of: (i) one or more unlocked nucleotide represented by formula [I], wherein R1is , R2is H, R3is a bond, R4is H, R5is H, R6is OH, R7is H, R8is H, and the base is adenine, cytosine, guanine, uracil or thymine; and

[0163] (ii) one or more modified nucleotide represented by formula [II], wherein R17is , R18is hydrogen, R19is a bond, R20is a halogen (preferably fluorine) or OMe, R21is hydrogen, and the base is adenine, cytosine, 5-methyl-cytosine, guanine, uracil or thymine.

[0164] Hence, preferably the RNA molecule comprises one or more unlocked nucleotide represented by formula [I]; and one or more modified nucleotide represented by formula [II].

[0165] The RNA molecule may further comprise one or more locked nucleotide (LNA) comprising a ribose moiety modified by a bridge between the 2 and 4 positions or the 2' and 4' positions in the ribose sugar. Such a combination, in the RNA molecule, of one or more unlocked nucleotide (UNA) and one or more locked modified nucleotides (LNA) can be referred to as a miximer. The locked-modified nucleotide may be as defined in British patent application no. 2310390.6, the contents of which are incorporated herein by reference. The one or more locked nucleotide may be represented herein by formula [III] : wherein Base is a nucleobase;

[0166] L1is a Ci-5 alkylene;

[0167] X3is 0, CH2, CO, NR28, Se, S, SO or S02;

[0168] X4is absent, or is CO, 0, NR28, Se, S, SO or SO2; R24is a phosphate, a modified phosphate, a phosphate linker, a modified phosphate linker or a 5 to 10 membered heteroarylene, wherein the phosphate linker, the modified phosphate linker or the 5 to 10 membered heteroarylene is a linker between the rest of the locked nucleotide of formula [III] and an attachment point to a first adjacent nucleotide in the RNA molecule; R25is H or a bond to a second adjacent nucleotide in the RNA molecule; wherein if

[0169] R24is a phosphate or a modified phosphate then R25is a bond to a second adjacent nucleotide in the RNA molecule;

[0170] R26and R27are independently H or Ci-e alkyl; and

[0171] Preferably, the one or more locked nucleotide may be represented herein as formula [Illa] :

[0172] The nucleobase may be as defined above in relation to formula [I]. The phosphate, modified phosphate, phosphate linker, modified phosphate linker or 5 to 10 membered heteroarylene may be as defined above in relation to formula [I]. L2may be a straight or branched C1-5 alkylene. Preferably, L2is a straight or branched C1-3 alkylene. More preferably, L is -CHz-*, -CH(CH3)-*, -CH2CH2-* or - CH(CH3)CH2-*, where an asterisk indicates a point of attachment to X4in embodiments where it is present and to X3in embodiments where X4is absent. Most preferably, L2is CH2.

[0173] X3may be 0, CH2, NR28, Se or S. In some embodiments, X3is 0.

[0174] X4may be absent. Alternatively, X4may be SO2. R26and R27may independently be H or C1-3 alkyl, and more preferably are independently H or methyl. In some embodiments, R26and R27are both H.

[0175] In a preferred embodiment, the use or method of the invention comprises using a combination of: (i) one or more unlocked nucleotide represented by formula [I]; and

[0176] (II) one or more locked nucleotide represented by formula [III].

[0177] Preferably, the use or method of the invention comprises using a combination of:

[0178] (i) one or more unlocked nucleotide represented by formula [I], wherein R1is , R2is H, R3is a bond, R4is H, R5is H, R6is OH, R7is H, R8is H, and the base is adenine, cytosine, guanine, uracil or thymine; and

[0179] (ii) one or more locked nucleotide represented by formula [III], wherein R24is , R25is a bond, R26is H, R27is H, X3is 0, X4is absent and L2is CH2, and the base is adenine, cytosine, 5-methyl-cytosine, guanine, uracil or thymine.

[0180] In a further preferred embodiment, the use or method of the invention comprises using a combination of: (i) one or more unlocked nucleotide represented by formula [I];

[0181] (II) one or more modified nucleotide represented by formula [II]; and

[0182] (ill) one or more locked nucleotide represented by formula [III]. Preferably, the use or method of the invention comprises using a combination of:

[0183] (i) one or more unlocked nucleotide represented by formula [I], wherein R1is , R2is H, R3is a bond, R4is H, R5is H, R6is OH, R7is H, R8is H, and the base is adenine, cytosine, guanine, uracil or thymine;

[0184] (ii) one or more modified nucleotide represented by formula [II], wherein R17is , R18is hydrogen, R19is a bond, R20is a halogen

[0185] (preferably fluorine) or OMe, R21is hydrogen, and the base is adenine, cytosine, 5-methyl-cytosine, guanine, uracil or thymine; and

[0186] (iii)one or more locked nucleotide represented by formula [III], wherein R24is , R25is a bond, R26is H, R27is H, X3is 0, X4is absent and L2is CH2, and the base is adenine, cytosine, 5-methyl-cytosine, guanine, uracil or thymine.

[0187] Hence, preferably the RNA molecule comprises one or more unlocked nucleotide represented by formula [I]; one or more modified nucleotide represented by formula [II], and one or more locked nucleotide represented by formula [III].

[0188] The method may further comprise use of DTT. This is thought to enhance RNA yield and RNA polymerase activity. The method may further comprise use of a pH buffer, such as HEPES or Tris-HCI, though the skilled person would appreciate that other buffers are available.

[0189] The method may further comprise the use of a crowding agent, for example

[0190] Poly(ethylene glycol) (PEG) of various molecular weights. For example, the crowding agent may be PEG200 or any additional crowding agent

[0030] . The method may further comprise use of an RNase inhibitor. This prevents RNA degradation if any RNase is present in the reaction.

[0191] The method may further comprise use of spermidine. This is thought to improve the activity of the RNA polymerase and therefore the RNA yield.

[0192] The method may further comprise use of a surfactant such as Tween20 or Triton X100. This is thought to improve the activity of the RNA polymerase and therefore the RNA yield

[0031] .

[0193] The method may further comprise use of Betaine. This is thought to improve the activity of the RNA polymerase and therefore the RNA yield

[0031] .

[0194] The template nucleic acid sequence is preferably transcribed by the RNA polymerase to produce the modified RNA molecule. Preferably, therefore, the template nucleic acid sequence comprises DNA. The template nucleic acid may be made synthetically, for example doggybone DNA, or by PCR, rolling circle amplification, or synthetic amplification. The template nucleic acid may comprise a vector, and is preferably a plasmid.

[0195] In one embodiment, the UNA-RNA molecule may be used in the manufacture of recombinant proteins, for example in a cell free system. In a further embodiment the UNA-RNA may be used in the manufacture of cultured meat. In another embodiment the UNA-RNA molecule may be used to differentiate stem cells, preferably ex vivo.

[0196] In one embodiment, the UNA-RNA molecule may also be used in the manufacture of RNAi-based biopesticides against viral and fungal infection / disease / spoilage. In addition, UNA-RNA may be used against pests such as insects (arthropods). Degradation of RNAi in the environment can occur via the actions of ribonucleases, here the use of uNA-RNA with enhanced resistance against ribonucleases is anticipated by the inventors to provide significant advantages for the generation of effective RNAi-based biopesticides. In one embodiment, the template nucleic acid may be transcribed to create the resultant modified RNA molecule, which can be described as being a biotherapeutic RNA molecule, i.e. one which is used therapeutically to treat, prevent or ameliorate a disease in a patient. The template nucleic acid sequence may encode a therapeutic protein, which is derived from an animal or a human, and which treats, prevents or ameliorates disease in a subject, preferably a human or animal subject. For example, therapeutic uses of the encoded therapeutic protein may include protein replacement, gene editing (e.g. CRISPR-Cas9), gene therapy, or RNAi, and so on.

[0197] The biotherapeutic RNA molecule may be selected from a group consisting of: messenger RNA (mRNA), micro RNA (miRNA); short interfering RNA (siRNA); short hairpin RNA (shRNA); anti-sense RNA; RNA aptamer; self-amplifying RNA (saRNA); interference RNA (RNAi); non-coding RNA; circular RNA; and small RNA.

[0198] In one embodiment, the template nucleic acid may encode a protein and peptide derived from a plant. Preferably, the protein and peptide is a plant antigen. The plant antigen may be derived from Ricinus spp., preferably Ricinus communis.

[0199] It will be appreciated that RNAi may be used as an insecticide. Thus, the UNA-RNA molecule may be used as an insecticide. In another embodiment, the template nucleic acid encodes an antigen, in which case the resultant modified RNA molecule can be described as being a vaccine, i.e. one which is used prophylactically to prevent a disease in a patient, or one which is used therapeutically to ameliorate disease, such as cancer. For example, the template nucleic acid sequence may encode an antigen which is derived from a virus, a bacteria, a mycoplasma, a fungus, an animal, a plant, an alga, a parasite, or a protozoan, or other organism which causes a disease in a subject, preferably a human or animal. Preferably, the template nucleic acid sequence is from a virus or a bacterium. For example, the template nucleic acid may encode at least a portion of a viral spike protein.

[0200] In another embodiment, the template nucleic acid may encode a tumour immunogen or antigen, or cancer immunogen or antigen. The tumour immunogen and antigen may be a peptide-containing tumour antigen, such as a polypeptide tumour antigen or glycoprotein tumour antigens.

[0201] The tumour antigen may be (a) a full-length molecule associated with cancer cells, (b) a homolog and modified form of the same, including molecules with deleted, added and / or substituted portions, and (c) a fragment of the same including peptides or strings of peptides.

[0202] Suitable tumour immunogens include: class I-restricted antigens recognized by CD 8+ lymphocytes or class Il-restricted antigens recognized by CD4+ lymphocytes.

[0203] The tumour antigen may be an antigen that is associated with a cancer selected from the group consisting of: a testis cancer, melanoma, lung cancer, head and neck cancer, NSCLC, breast cancer, gastrointestinal cancer, bladder cancer, colorectal cancer, pancreatic cancer, lymphoma, leukaemia, renal cancer, hepatoma, ovarian cancer, gastric cancer and prostate cancer.

[0204] The tumour antigen may be selected from:

[0205] (a) cancer-testis antigens such as NY-ESO-I, SSX2, SCPI as well as RAGE, BAGE, GAGE and MAGE family polypeptides, for example, GAGE-I, GAGE-2, MAGE-I, MAGE- 2, MAGE-3, MAGE-4, MAGE-5, MAGE-6, and MAGE- 12 (which can be used, for example, to address melanoma, lung, head and neck, NSCLC, breast, gastrointestinal, and bladder tumours); (b) mutated antigens, for example, p53 (associated with various solid tumours, e.g., colorectal, lung, head and neck cancer), p21 / Ras (associated with, e.g., melanoma, pancreatic cancer and colorectal cancer), CDK4 (associated with, e.g., melanoma), MUMI (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 nonHodgkins lymphoma), BCR-abl (associated with, e.g., chronic myelogenous leukaemia), triosephosphate isomerase, KIA 0205, CDC-27, and LDLR-FUT;

[0206] (c) over-expressed antigens, for example, Galectin 4 (associated with, e.g., colorectal cancer), Galectin 9 (associated with, e.g., Hodgkin's disease), proteinase 3 (associated with, e.g., chronic myelogenous leukaemia), WT 1 (associated with, e.g., various leukaemias), carbonic anhydrase (associated with, e.g., renal cancer), aldolase A (associated with, e.g., lung cancer), PRAME (associated with, e.g., melanoma), HER-2 / neu (associated with, e.g., breast, colon, lung and ovarian cancer), alpha- fetoprotein (associated with, e.g., hepatoma), KSA (associated with, e.g., colorectal cancer), gastrin (associated with, e.g., pancreatic and gastric cancer), telomerase catalytic protein, MUC-I (associated with, e.g., breast and ovarian cancer), G-250 (associated with, e.g., renal cell carcinoma), p53 (associated with, e.g., breast, colon cancer), and carcinoembryonic antigen (associated with, e.g., breast cancer, lung cancer, and cancers of the gastrointestinal tract such as colorectal cancer); (d) shared antigens, for example, melanoma-melanocyte differentiation antigens such as MART-l / Melan A, gplOO, MOIR, melanocyte-stimulating hormone receptor, tyrosinase, tyrosinase related protein- 1 / TRPI and tyrosinase related protein-2 / TRP2 (associated with, e.g., melanoma); (e) prostate-associated antigens, such as PAP, PSA, PSMA, PSH-PI, PSM-PI, PSM-P2, associated with e.g., prostate cancer; and / or

[0207] (f) immunoglobulin idiotypes (associated with myeloma and B cell lymphomas, for example).

[0208] 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; tumour growth supressing protein; a structural protein and a blood protein.

[0209] The enzyme may be selected from the group consisting of: chymosin; gastric lipase; tissue plasminogen activator; streptokinase; a cholesterol biosynthetic or degradative steriodogenic enzyme; kinases; phosphodiesterases; methylases; de- methylases; dehydrogenases; cellulases; proteases; lipases; phospholipases; aromatases; cytochromes; adenylate or guanylaste cyclases and neuramidases.

[0210] The enzyme inhibitor may be tissue inhibitor of metalloproteinase (TIMP). The hormone may be growth hormone.

[0211] The immune system protein may be selected from the group consisting of: a cytokine; a chemokine; a lymphokine; erythropoietin; an integrin; addressin; selectin; homing receptors; T cell receptors and immunoglobulins.

[0212] The cytokine may be an interleukin, for example IL-2, IL-4 and / or IL-6, colony stimulating factor (CSF), granulocyte colony stimulating factor (G- CSF), granulocyte-macrophage colony stimulating factor (GM-CSF) or tumour necrosis factor (TNF).

[0213] The chemokine may be a macrophage inflammatory protein-2 and / or a plasminogen activator.

[0214] The lymphokine may be an interferon.

[0215] The immunoglobulin may be a natural, modified or chimeric immunoglobulin or a fragment thereof. Preferably, the immunoglobulin is a chimeric immunoglobulin having dual activity such as antibody enzyme or antibody-toxin chimera.

[0216] The hormone may be selected from the group consisting of: insulin, thyroid hormone, catecholamines, gonadotrophines, trophic hormones, prolactin, oxytocin, dopamine, bovine somatotropin, leptins; growth hormones (e.g., human grown hormone), growth factors (e.g., epidermal growth factor, nerve growth factor, insulinlike growth factor and the like).

[0217] The receptor may be a steroid hormone receptor or a peptide receptor. Preferably, the receptor is a growth factor receptor.

[0218] The binding protein may be a growth factor binding protein.

[0219] The tumour growth suppressing protein may be a protein that inhibits angiogenesis.

[0220] The structural protein may be selected from the group consisting of: collagen; fibroin; fibrinogen; elastin; tubulin; actin; and myosin.

[0221] 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 anticoagulants. In one preferred embodiment, the template nucleic acid may encode a cytokine which is capable of regulating lymphoid homeostasis, preferably a cytokine which is involved in and preferably induces or enhances development, priming, expansion, differentiation and / or survival of T cells. Thus, preferably, the cytokine is an interleukin. Most preferably, IL-2, IL-7, IL-12, IL-15, or IL-21.

[0222] The template nucleic acid may encode a protein that is capable of enhancing reprogramming of somatic cells to cells having stem cell characteristics.

[0223] The protein that is capable of enhancing reprogramming of somatic cells to cells having stem cell characteristics may be selected from the group consisting of: OCT4, SOX2, NANOG, LIN28, p53, ART-4, BAGE, ss- catenin / m, Bcr-abL CAMEL, CAP-1, CASP-8, CDC27 / m, CD 4 / 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.

[0224] Preferably, MAGE-A is selected from the group consisting of: MAGE-A 1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A5, MAGE-A6, MAGE- A7, MAGE-A8, MAGE-A9, MAGE-A

[0225] 10, MAGE-A 11, or MAGE-A 12

[0226] Preferably, the protein that is capable of enhancing reprogramming of somatic cells to cells having stem cell characteristics is OCT4, SOX2, LF4; c-MYC; NANOG; LIN28.

[0227] The template nucleic acid may encode a biomolecule that is utilised for the modification of cells ex vivo for cell-therapy indications. Thus, preferably the template nucleic acid may encode a protein selected from the group consisting of an immunoglobulin, a T-cell receptor and NK receptor.

[0228] The template nucleic acid may encode a protein that is capable of regulating expression of endogenous host genes, for example an interfering RNA, such as small RNAs, siRNA or microRNAs.

[0229] In an embodiment, the template nucleic acid may encode an innate inhibitor protein, as described in W02020 / 254804, WO2022 / 129945, WO2022 / 129944 or WO2023 / 111592, which counteracts the innate immune response in a subject administered with a vaccine comprising the resultant RNA molecule.

[0230] In accordance with an eighth aspect, there is provided an RNA molecule obtained or obtainable by the method according to the sixth aspect.

[0231] In a ninth aspect, there is provided an RNA molecule comprising at least 25 nucleotides, wherein one or more of the nucleotides is an unlocked nucleotide. The RNA molecule of the ninth aspect may be defined as in any of the previous aspects. For example, the type and length of the RNA molecule may be as described herein. Also, the type and amount of unlocked nucleotide incorporated in the RNA molecule may be as described herein. In a tenth aspect, there is provided a pharmaceutical composition comprising the RNA molecule according to the eighth aspect or ninth and a pharmaceutically acceptable vehicle.

[0232] In a eleventh aspect, there is provided a method of preparing the pharmaceutical composition according to the tenth aspect, the method comprising contacting the

[0233] RNA molecule according to the eighth or ninth aspect and a pharmaceutically acceptable vehicle.

[0234] In a twelfthaspect, there is provided the RNA molecule according to the eighth or ninth aspect, or the pharmaceutical composition according to the tenth aspect, for use as a medicament.

[0235] In a thirteenth aspect, there is provided the RNA molecule according to the eighth or ninth aspect, or the pharmaceutical composition according to the tenth aspect, for use in treating, preventing or ameliorating a disease in a subject, such as cancer.

[0236] In a fourteenth aspect, there is provided a method of treating, preventing or ameliorating a disease in a subject, such as cancer, the method comprising administering, or having administered, to a subject in need thereof, a therapeutically effective amount of the RNA molecule according to the eighth or ninth aspect, or the pharmaceutical composition according to the tenth aspect. In a fifteenth aspect, there is provided a vaccine composition comprising the RNA molecule according to the eighth or ninth aspect, or the pharmaceutical composition according to the tenth aspect.

[0237] The vaccine may comprise a suitable adjuvant. Examples of adjuvants may include an aluminium salt, a synthetic form of DNA, a carbohydrate, a tablet binder, an ion exchange resin, a preservative, a polymer, an emulsion and / or a lipid. Examples of adjuvants may include monosodium glutamate, sucrose, dextrose, aluminum bovine, human serum albumin, cytosine phosphoguanine, potassium phosphate, plasdone C, anhydrous lactose, cellulose, polacrilin potassium, glycerine, asparagine, citric acid, potassium phosphate magnesium sulfate, iron 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 aluminum sulfate, potassium glutamate, sodium borate, sodium metabisulphite, urea, PLGA, PVA, PLA, PVP, cyclodextrin-based stabilisers, oil in water emulsion adjuvants and / or lipid-based adjuvants. In a sixteenth aspect, there is provided the RNA molecule according to the eighth or ninth aspect, the pharmaceutical composition according to the tenth aspect or the vaccine according to the fifteenth aspect, for use in stimulating an immune response in a subject. The immune response may be stimulated against a protozoa, bacterium, virus, fungus, or cancer. Thus, the vaccine may be used to express a microbial or pathogenic antigen for immunizing against infectious micro-organisms, such as viruses and bacteria etc. However, the vaccine may also be used to express an oncogenic antigen, for preventing cancer.

[0238] In a seventeenth aspect of the invention, there is provided a method of vaccinating a subject, the method comprising administering, or having administered, to a subject in need thereof, a therapeutically effective amount of the RNA molecule according to the eighth or ninth aspect, the pharmaceutical composition according to the tenth aspect or the vaccine according to the fifteenth aspect. It will be appreciated that RNA molecules can have significant utility in a wide range of therapeutic applications, for example RNAi, inhibitory RNA, RNA aptamers, and so on, in which translation of the RNA molecule does not necessarily need to occur. However, applications in which the RNA molecule does need to be translated include various vaccine and biotherapeutic approaches, such as the use of mRNA, saRNA, circular RNA, or any RNA sequence that is able to undergo translation and generate a peptide or protein in vivo.

[0239] For example, the peptide or protein that is encoded by the RNA molecule may be involved in protein replacement, gene editing and gene therapy etc.

[0240] It will also be appreciated that there is a growing trend for the use, in particular, of mRNA and saRNA vaccines, in which the RNA molecule encodes a pathogenic, such as a viral coat protein or region thereof, or an oncogenic antigen, and it is this antigen which is expressed in an immunised host organism and which triggers an immune response to the pathogen or the tumour.

[0241] As described in the Examples, rather than directly measuring the amount of luciferase protein produced, the inventors have instead measured the enhancement of expression in terms of luciferase activity. Luciferase activity can, therefore, be considered as a surrogate of RNA translation, because it should directly relate to the amount of translation. Moreover, the luciferase data described in the examples can be used a surrogate of a therapeutic, essentially mirroring the expression of a therapeutic protein. However, as described herein, when the term "10-fold" or the like is used, it means that there is in fact a 10-fold increase in luciferase activity. It will be appreciated that the increase in expression or RNA translation may be related to the RNA being more stable, and so more protein is made before it is degraded. Accordingly, there may not be a change in translation efficiency (i.e. ribosomal speed), but an increase in the overall time that ribosomes have for making more protein.

[0242] In summary, these data, when taken together, demonstrate that unlocked nucleotides can be used to increase the level of RNA translation for protein production in vitro and in vivo, the latter being especially advantageous when the RNA molecule is used as a vaccine or a therapeutic biomolecule. Based on the unexpected findings that incorporation unlocked nucleotides enhances, rather than reduces, expression, the inventors believe that their data support the finding that modified bases can be used to enhance the stability of RNA, as well as reduce the activation of innate sensing, interferon generation and / or degradation of an RNA molecule. Suitably, the one or more unlocked nucleotide enhances the level of expression and / or translation of the RNA molecule by at least 5-fold, 10-fold or 20-fold increase compared to the level of expression and / or translation that occurs in the absence of the one or more unlocked nucleotide. More suitably, the one or more unlocked nucleotide enhances the level of expression and / or translation of the RNA molecule by at least 30-fold, 40-fold or 50-fold compared to the level of expression and / or translation that occurs in the absence of the one or more unlocked nucleotide. As shown in Figure 4, substitution of up to 90% of individual nucleotides (ATP, or UTP) with unlocked nucleotides surprisingly enhances mRNA expression of encoded firefly luciferase (fLuc) (which acts as a marker or surrogate of protein expression) by up to 40-fold in HEK293T cells that have impaired innate immunity sensing.

[0243] Even more suitably, the one or more unlocked nucleotide enhances the level of expression and / or translation of the RNA molecule by at least 75-fold, 100-fold, 150-fold, 200-fold or 250-fold compared to the level of expression and / or translation that occurs in the absence of the one or more unlocked nucleotide. Substitution of individual nucleotides (NTPs) with unlocked nucleotides (NTPs) enhances fLuc expression in HeLa epithelial cells by up to 200-fold at 48 hours (UNA-ATP) and 160-fold (UNA-UTP) at 48h. As shown in Figure 6, substitution of individual nucleotides (NTPs) with unlocked nucleotides (NTPs) enhances fLuc expression in THP-1 monocytic cells by up to 100-fold at 24, 48 and 72 hours (UNA-ATP) and 100-fold (UNA-UTP) at 24h and 200-fold at 48 and 72h (UNA-ATP).

[0244] Preferably, the one or more unlocked nucleotide enhances the level of expression and / or translation of the RNA molecule by at least 500-fold, 750-fold or 1000-fold compared to the level of expression and / or translation that occurs in the absence of the one or more unlocked nucleotide. Preferably, the one or more unlocked nucleotide enhances the level of expression and / or translation of the RNA molecule by at least 2000-fold, 5000-fold or 8000-fold increase compared to the level of expression and / or translation that occurs in the absence of the one or more unlocked nucleotide. The level of expression and / or translation may be measured by the expression of a fluorescent or luminescent protein encoded as a gene of interest in the RNA molecule (e.g. in either mRNA or saRNA). For example, the luminescent protein may be a bioluminescent protein, such as a luciferase (e.g. firefly luciferase protein encoded as a gene of interest). Thus, expression of luciferase can be determined by measurement of its enzymatic activity, where luciferin is converted to oxyluciferin emitting light that can be quantitated as relative light units (RLUs). The presence of light is proportional to the amount of translated protein. This approach was previously used to assess the impact of pseudouridine incorporation on translational efficiency [24, 25].

[0245] The enzymatic incorporation of unlocked nucleotides in RNA has many potential outcomes and advantages selected from a group consisting of: changing mRNA stability; preventing innate recognition; reducing RNA degradation; increasing RNA half-life; enhancing recruitment of translational factors and / or ribosomes; and increasing translation.

[0246] Preferably, the methods or uses enhance protein expression from mRNA, saRNA, circular RNA or any RNA sequence capable to being translated to generate a recombinant protein or peptide.

[0247] In another embodiment, the methods or uses enhance the stability of RNA (be it mRNA, miRNA; siRNA; shRNA; anti-sense RNA; RNA aptamers; self-amplifying RNA (saRNA); interference RNA (RNAi); non-coding RNA; circular RNA; or small RNA). Improved stability provides a significant advantage to both coding and non-coding biotherapeutic or prophylatic uses of RNA molecules.

[0248] The methods described herein allow the incorporation of unlocked nucleotides of any length (be they coding or non-coding) providing enhanced stability of the resultant RNA molecule.

[0249] Stability of RNA molecules can be measured by quantitative PCR, but is also reflected by the duration of expression, where faster degradation equates to shorter expression.

[0250] In another embodiment, the methods or uses result in reducing or eliminating innate sensing of RNA by membrane, endosomal and cellular RNA binding proteins that recognise unmodified RNA structures that trigger interferon signalling pathways and activation of cytoplasmic nucleases that degrade RNA such as RNase H

[0010] . Reduced innate sensing of RNA (be it mRNA, miRNA; siRNA; shRNA; antisense RNA; RNA aptamers; self-amplifying RNA (saRNA); interference RNA (RNAi); non-coding RNA; circular RNA; and small RNA) would provide significant advantages to both coding and non-coding biotherapeutic or prophylatic uses of RNA. The methods presented here allow incorporation of unlocked nucleotides of any length to prevent innate recognition of RNA, interferon induction and RNA degradation. The data described in the Examples show that incorporation of individual unlocked nucleotides surprisingly increases both the magnitude and expression of capped mRNA in cells that have impaired innate sensing mechanisms for recognizing RNA.

[0251] Reducing the activation of innate sensing, leading to degradation of an RNA molecule is inferred by improved expression of modified RNA in interferon competent cells (HeLa and THP-1 cells). Although the incorporation of unlocked nucleotides provides some improvement in expression in HEK293T cells, these cells have impaired innate sensing mechanisms, therefore, any improvement in expression directly relates to improved RNA half-life in the absence of innate recognition. Expression of unmodified RNA, if greatly diminished in both HeLa and THP-1 cells, reflects enhanced degradation caused by the triggering of innate sensing mechanisms. The incorporation of unlocked nucleotides in saRNA and mRNA provides more pronounced increase of expression in these two interferon- competent cell types (fold change) than that observed in HEK293T cells. Induction of IP10 (CXCL10) expression can be measured by ELISA as a surrogate of innate activation [28, 29]. As shown in Figure 7, it was also surprisingly observed that substitution of individual nucleotides (NTPs) with UNAs was able to influence the secretion profile of IP 10 (CXCL10) in supernatants harvested from transfected THP- 1 cells. Furthermore, combined substitution of both UNA-ATP and UNA-UTP provided further reduction in IP10 levels.

[0252] It will be appreciated that the composition, the pharmaceutical composition, the vaccine or the therapeutic RNA molecule, may be used in a medicament, which may be used as a monotherapy (i.e. use of the composition alone). Alternatively, the composition according to the invention may be used as an adjunct to, or in combination with, known therapies for treating, ameliorating, or preventing an infection or disease. The composition of the invention may be combined in compositions having a number of different forms depending, in particular, on the manner in which the composition is to be used. Thus, for example, the composition may be in the form of a powder, tablet, capsule, liquid, ointment, cream, gel, hydrogel, aerosol, spray, micellar solution, transdermal patch, liposome suspension, polyplex, emulsion, liposome, lipid nanoparticles, functionalised liposomes or lipid nanoparticles (e.g. with peptide, antibody, antibody fragment, glycan, glyco-conjugate, DNA or RNA on the surface or encapsulated) or any other suitable form that may be administered to a person or animal. The lipid nanoparticle may comprise one or more components selected from a group consisting of: a cationic lipid (which is preferably ionisable); phosphatidylcholine; cholesterol; and polyethylene glycol (PEG)-lipid. It will be appreciated that the vehicle of medicaments according to the invention should be one which is well-tolerated by the subject to whom it is given. Medicaments comprising the composition, the pharmaceutical composition or the vaccine of the invention may be used in a number of ways. For instance, oral administration may be required, in which case the agents may be contained within a composition that may, for example, be ingested orally in the form of a tablet, capsule or liquid. Compositions comprising agents and medicaments of the invention may be administered by inhalation (e.g. intranasally). Compositions may also be formulated for topical use. For instance, creams or ointments may be applied to the skin. Formulations may be provided in a lyophilised format for reconstitution or as a solid dosage form for topical, inserted or injected delivery. The composition, the pharmaceutical composition, vaccine or therapeutic RNA molecule of the invention may also be incorporated within a slow- or delayed- release device. Such devices may, for example, be inserted on or under the skin, and the medicament may be released over weeks or even months. The device may be located at least adjacent the treatment site.

[0253] In a preferred embodiment, however, medicaments according to the invention may be administered to a subject by injection into the blood stream, muscle, skin or directly into a site requiring treatment. Injections 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). It will be appreciated that the amount of composition, the pharmaceutical composition or the vaccine or therapeutic RNA molecule that is required is determined by its biological activity and bioavailability, which in turn depends on the mode of administration, the physiochemical properties of the composition, the pharmaceutical composition or the vaccine and whether it is being used as a monotherapy or in a combined therapy.

[0254] The frequency of administration will also be influenced by the half-life of the active agent within the subject being treated. Optimal dosages to be administered may be determined by those skilled in the art, and will vary with the composition, the pharmaceutical composition or the vaccine in use, the strength of the pharmaceutical composition, the mode of administration, and the type of treatment. Additional factors depending on the particular subject being treated will result in a need to adjust dosages, including subject age, weight, gender, diet, and time of administration.

[0255] The required dose may depend upon a number of factors including, but not limited to, the active agent being administered, the disease being treated and / or vaccinated against, the subject being treated, etc.

[0256] Generally, a dose of between 0.001 pg / kg of body weight and 10 mg / kg of body weight, or between 0.01 pg / kg of body weight and 1 mg / kg of body weight, of the composition, the pharmaceutical composition or the vaccine or therapeutic RNA molecule of the invention may be used, depending upon the active agent used. A dose may be understood to relate to the quantity of the RNA molecule which is delivered.

[0257] Doses may be given as a single administration (e.g., a single injection).

[0258] Alternatively, the composition, the pharmaceutical composition, biotherapeutic RNA molecule or the vaccine may require more than one administration. As an example, the composition, the pharmaceutical composition or the vaccine or therapeutic RNA molecule may be administered as an initial primer and a subsequent boost(s), or two boosts administered at between a week or monthly intervals (e.g. two or more doses of between 0.07 pg and 700 mg, i.e., assuming a body weight of 70 kg), or for a biotherapeutic repeat dosing on a daily, weekly or monthly basis.

[0259] Alternatively, a slow-release device may be used to provide optimal doses of the composition, the pharmaceutical composition or the vaccine or therapeutic RNA molecule according to the invention to a patient without the need to administer repeated doses. Routes of administration may incorporate intravenous, intradermal subcutaneous, intramuscular, intrathecal, epidural, or intraperitoneal routes of injection.

[0260] Known procedures, such as those conventionally employed by the pharmaceutical industry (e.g., in vivo experimentation, clinical trials, etc.), may be used to form specific formulations of the composition, the pharmaceutical composition or vaccine or therapeutic RNA molecule according to the invention and precise therapeutic regimes (such as doses of the agents and the frequency of administration).

[0261] A "subject" may be a vertebrate, mammal, or domestic animal. Hence, compositions and medicaments according to the invention may be used to treat any mammal, for example livestock (e.g., a horse), pets, or may be used in other veterinary applications. Most preferably, however, the subject is a human being.

[0262] A "therapeutically effective amount" of the composition, the pharmaceutical composition or the vaccine or therapeutic RNA molecule is any amount which, when administered to a subject, is the amount of the aforementioned that is needed to produce a therapeutic effect.

[0263] For example, a therapeutically effective amount of the composition, the pharmaceutical composition and the vaccine or therapeutic RNA molecule of the invention may comprise from about 0.0001 mg to about 800 mg of the payload molecule, preferably 0.001 mg to about 650 mg, and preferably from about 0.01 mg to about 500 mg of the payload molecule.

[0264] A "pharmaceutically acceptable vehicle" as referred to herein, is any known compound or combination of known compounds that are known to those skilled in the art to be useful in formulating pharmaceutical compositions.

[0265] In one embodiment, the pharmaceutically acceptable vehicle may be a solid, and the composition may be in the form of a powder or tablet. A solid pharmaceutically acceptable vehicle may include one or more substances which may also act as flavouring agents, lubricants, solubilisers, suspending agents, dyes, fillers, glidants, compression aids, inert binders, sweeteners, preservatives, dyes, coatings, or tablet-disintegrating agents. The vehicle may also be an encapsulating material. In powders, the vehicle is a finely divided solid that is in admixture with the finely divided active agents according to the invention. In tablets, the active agent (e.g., composition of the invention) may be mixed with a vehicle having the necessary compression properties in suitable proportions and compacted in the shape and size desired. The pharmaceutical vehicle may be a gel and the composition may be in the form of a cream or the like.

[0266] However, the pharmaceutical vehicle may be a liquid, and the pharmaceutical composition is in the form of a solution. Liquid vehicles are used in preparing solutions, suspensions, emulsions, syrups, elixirs and pressurized compositions. The composition according to the invention may be dissolved or suspended in a pharmaceutically acceptable liquid vehicle such as water, an organic solvent, a mixture of both or pharmaceutically acceptable oils or fats. The liquid vehicle can contain other suitable pharmaceutical additives such as solubilisers, emulsifiers, buffers, preservatives, sweeteners, flavouring agents, suspending agents, thickening agents, colours, viscosity regulators, stabilizers or osmo-regulators. Suitable examples of liquid vehicles for oral and parenteral administration include water (partially containing additives as above, e.g., cellulose derivatives, preferably sodium carboxymethyl cellulose solution), alcohols (including monohydric alcohols and polyhydric alcohols, e.g., glycols) and their derivatives, and oils (e.g., fractionated coconut oil and arachis oil). For parenteral administration, the vehicle can 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 a halogenated hydrocarbon or other pharmaceutically acceptable propellant.

[0267] Liquid pharmaceutical compositions, which are sterile solutions or suspensions, can be utilized by, for example, intramuscular, intrathecal, epidural, intraperitoneal, intravenous and subcutaneous injection. The composition of the invention may be prepared as any appropriate sterile injectable medium.

[0268] The composition and / or the pharmaceutical composition of the invention may be administered orally in the form of a sterile solution or suspension containing other solutes or suspending agents (for example, enough saline or glucose to make the solution isotonic), bile salts, acacia, gelatin, sorbitan monoleate, polysorbate 80 (oleate esters of sorbitol and its anhydrides copolymerized with ethylene oxide) and the like. The composition of the invention and / or the pharmaceutical composition according to the invention can also be administered orally either in liquid or solid composition form. Compositions suitable for oral administration include solid forms, such as pills, capsules, granules, tablets, and powders, and liquid forms, such as solutions, syrups, elixirs, and suspensions. Forms useful for parenteral administration include sterile solutions, emulsions, and suspensions.

[0269] All of the features described herein (including any accompanying claims, abstract and drawings), and / or all of the steps of any method or process so disclosed, may be combined with any of the above aspects in any combination, except combinations where at least some of such features and / or steps are mutually exclusive.

[0270] For a better understanding of the invention, and to show how embodiments of the same may be carried into effect, reference will now be made, by way of example, to the accompanying Figures, in which :-

[0271] Figure 1 shows the structure of an unlocked nucleic acid or nucleotide (UNA) monomer that lacks a C-C bond between the 2'- and 3'-carbon atoms in the five membered furanose ring [2],

[0272] Figure 2 shows the structures of furanose conformation of canonical (DNA, RNA), LNA and UNA nucleotides [2], Figure 3 illustrates that the efficient incorporation of unlocked NTPs occurs at high magnesium levels (75 mM) when using magnesium acetate. A) RNA yield ng / ul, B) RNA product as assessed by gel-electrophoresis.

[0273] Figure 4 illustrates the impact of individual UNTP (ATP, UTP) substitution on mRNA expression of fLuc in HEK293T cells. mRNA encoding fLuc was generated by IVT, where individual nucleotides were substituted with 90% UNA modified versions.

[0274] HEK293T cells were transfected with lOOng of mRNA formulated in Lipofectamine MessengerMax. A). Luciferase expression was monitored over 72h by measurement of relative light units (RLU, see methods). B) Normalized RLU relative to unmodified mRNA at each respective time point. Figure 5 illustrates the impact of individual UNA-NTP (ATP, UTP) and combined (ATP+UTP) substitution on mRNA expression of fLuc in HeLa cells. mRNA encoding fLuc was generated by IVT, where individual nucleotides were substituted with 90% UNA modified versions. HeLa cells were transfected with lOOng of mRNA formulated in Lipofectamine MessengerMax. A). Luciferase expression was monitored over 72h by measurement of relative light units (RLU, see methods). B) Normalized RLU relative to unmodified mRNA at each respective time point.

[0275] Figure 6 illustrates the impact of individual UNA-NTP (ATP, UTP) and combined (ATP+UTP) substitution on mRNA expression of fLuc in THP-1 cells. mRNA encoding fLuc was generated by IVT, where individual nucleotides were substituted with 90% UNA modified versions. THP-1 cells were transfected with lOOng of mRNA formulated in Lipofectamine MessengerMax. A). Luciferase expression was monitored over 72h by measurement of relative light units (RLU, see methods). B) Normalized RLU relative to unmodified mRNA at each respective time point.

[0276] Figure 7 illustrates the impact of individual UNA-NTP (UNA-ATP, and UNA-UTP) and combined (ATP+UTP) substitution on the inflammatory profile of mRNA in THP-1 cells. Capped mRNA encoding firefly luciferase was generated by IVT, using wildtype (WT) NTPs, and where WT NTPs were substituted with 90% UNA-ATP, and / or 90% UNA-UTP. Unmodified and modified mRNA were transfected in 96-well plates of THP1 cells in triplicate with lOOng of mRNA formulated in Lipofectamine MessengerMax (Error bars represent n=3). At 24, 48 and 72h post transfection, supernatants from each condition were collected and then used to run an ELISA assay to determine the different levels of I PIO (CXCL10) stimulated by each RNA construct. A) IP-10 expression in pg / mL. B) Normalized IP-10 relative to unmodified mRNA at each respective time point.

[0277] Examples To date, it has not been possible to make RNA sequences by synthetic chemical approaches of >25 nucleotides (25nt). Furthermore, it has only been possible to incorporate one or two UNTPs enzymatically in sequences of up to 25 nucleotides (25nt) as part of a DNA sequence using a DNA polymerase

[0021] , There has been no previous report of incorporating UNTPs into RNA by in vitro transcription using a bacteriophage RNA-dependent polymerase, such a T7 polymerase, or other similar polymerases (T3, SP6 etc). Therefore, RNA sequences using single unlocked nucleotides, or combinations of unlocked NTPs, has not previously been established. Thus, while widely used for short antisense RNA (< 100 nucleotides), generated by synthetic processes, no-one has ever been able to generate sequences of sufficient length to encode functional proteins. The inventors therefore investigated the effects of the incorporation of unlocked nucleic acids (UNA) into RNA sequences of greater than 25 nucleotides (NTPs), by in vitro transcription. Surprisingly, as discussed in the Examples below, the inventors have now shown that unlocked nucleotides (such as those shown in Figure 1) can be effectively incorporated into RNA sequences of greater than 25 nucleotides by in vitro transcription (Figure 3), and that these can be translated by ribosomes within living cells, generating functional protein (Figures 4-6). Importantly, the inventors have observed that the use of individual and combinations of unlocked nucleotides increases both the level and duration of protein expression in transduced cells. Furthermore, the inventors have demonstrated that individual or combinations of UNTPs can modulate the innate cytokine response to naked unmodified RNA induced when delivered into the cytoplasm of cells. This new approach is likely applicable to all forms of encoding RNA, greater than 100 nucleotides, including messenger RNA, self-amplifying RNA saRNA), circular RNA, or to any other form of RNA of a length greater than 100 nucleotides.

[0278] Accordingly, it is demonstrated for the first time that incorporation of UNA-NTPs can be achieved by in vitro transcription using a bacteriophage DNA dependent RNA polymerase and high concentrations of magnesium (>20mM). The inventors also demonstrate for the first time ever that UNA-RNA can be translated by cellular ribosomes to generate functional proteins and that the incorporation of UNAs into encoding RNA both increases the level and duration of protein expression in transduced cells. Furthermore, they demonstrate for the first time that incorporation of UNAs in RNA sequences greater than 25nt can reduce the inflammatory potential of RNA as evidenced by the reduced expression of IP10 (CXCL10) as secreted cytokine that acts as an indicator of innate inflammatory response to RNA (Figure 7) [28, 29].

[0279] Materials and Methods

[0280] In vitro transcription (IVT) mRNA encoding fLuc was produced by in-vitro transcription (IVT) using a linearised DNA template. The final volume for each IVT reaction was 50uL, and the standardised IVT reaction mix was as follows: Table 1 - Standardised IVT reaction mix 0.5 ua DNA template

[0281] 40 mM DTT

[0282] 40mM HEPES

[0283] 4U RNase Inhibitor per 1 pg DNA

[0284] 100U T7 RNA polymerase per 1 pg DNA 75mM Mg(OAc)2 lOmM Na(OAc)

[0285] 75mM Mg(OAc) lOmM of each GTP, ATP, CTP, UTP

[0286] 0.2mM Spermidine The total concentration of each NTP was kept at lOnM, however, the relative proportion of unmodified to modified NTP was varied as indicated. For example, where it is described that 75% GTP was substituted with UNA-GTP, it indicates that the reaction contained 7.5mM UNA-GTP plus 2.5mM GTP. IVT reactions were performed at 370C for 4 h. RNA yield was then measured right after IVT using the Qubit RNA Broad Range Assay kit with the Qubit Fluorometer (Thermo Fisher, UK) according to the manufacturer's protocol.

[0287] Prior to post capping, RNA was purified using Lithium Chloride precipitation. Post capping of RNA was performed using ScriptCapTM Cap 1 Capping System (CellScript, UK) and reaction was incubated at 37°C for 2h. After post capping, RNA was purified again using lithium chloride precipitation and the final RNA concentration was measured using Nanodrop One (Thermo Scientific, UK). To assess the quality of the RNA, purified RNAs and the 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 with lx NorthernMax Running Buffer (Thermo Fisher, UK) was prepared. After incubation, the denatured ladder and samples were loaded on to the gel and the gel was run at 80 V for 45 min. The gel was then imaged on a GelDoc-It2 (UVP, UK).

[0288] Cell culture and in vitro transfection HEK293T. 17 (ATCC, UK) and HELA (ATCC) cells were cultured in complete Dulbecco's Modified Eagle's Medium (DMEM) (Gibco, UK) containing 10% fetal bovine serum (FBS), 1% L-glutamine (L-glu) and 1% penicillin-streptomycin (Pen- strep) (Thermo Fisher Scientific, UK). THP1 cells (ATCC) were cultured in complete Roswell Park Memorial Institute (RPMI) 1640 medium with 10% FBS, 1 % L-glu and 1% pen-strep. Cells were plated in a 96-well plate 24 h prior to transfection at a density of 7 xlO4cells per well for HEK293T.17 and HeLa cells; and IxlO5cells per well THP-1 cells. Transfection of saRNA and mRNA encoding fLuc was performed using Lipofectamine MessengerMAX (Thermo Fisher, UK) according to the manufacturer's instructions.

[0289] In Vitro Firefly luciferase assay

[0290] At 24, 48 or 72 h post transfection, half of the cell supernatants were discarded from each well and an equal volume of the Bright-GloTM Luciferase (Promega, UK) added to each well then placed in the dark. Cells were incubated with the luciferase reagent for 5 min to ensure complete lysis of the cells. Cells were then pipetted up and down and transferred to a white 96-well Costar® plate (Thermo Fisher Scientific). The luminescence was then measured on the FLUOstar Omega microplate reader (BMG Labtech) using gain 2500, 3000, 3600 and 4095.

[0291] In Vitro IP-10 Profiling

[0292] Cell supernatants were collected at 24 post transfection with RNA and IP 10 levels measured by ELISA (R8iD Systems, SIP100) according to the manufacturer's instructions.

[0293] Example 1 - The incorporation of unlocked-NTPs (UNTPI can be achieved when using magnesium ion concentrations of greater than 20mM

[0294] The inventors have surprisingly demonstrated that incorporation of ribose modified UNTPs can be achieved when using magnesium ion concentrations that are greater than 20mM (see Figure 3). In particular, the inventors have observed that magnesium acetate concentrations of greater than 20mM are required for efficient incorporation of UNA-ATP or UTP, and ideally concentrations of <75mM. However, the inventors believe that the maximum concentration of magnesium acetate could even be greater than 80mM (Figure 3).

[0295] All experiments were performed with wild type T7 polymerase, however, the inventors expect that the reaction conditions described herein would also be advantageous for mutated versions to T7 polymerase shown to be more permissive for NTP substitutions, and also other RNA polymerases including T7, T3 and SP6, and potential mutated variants. As described in the following examples, the inventors then assessed the impact of incorporation of individual UNTPs on the function of mRNA in three cell lines: Human embryonic kidney 293T (HEK293T) cells that have impaired innate sensing mechanisms that may lead to suppressed RNA expression (Example 2), HeLa cells representative of epithelial cells (Example 3), and THP-1 cells representative of myeloid cells (Example 4), the latter two displaying robust innate responses to naked unmodified RNA.

[0296] Example 2 - Incorporation of individual UNTPs increases both the magnitude and expression of capped mRNA in cells that have impaired innate sensing mechanisms for recognizing RNA (e.g. HEK293T cells')

[0297] The mRNA construct used in these experiments encodes the sequence for Firefly luciferase (fLuc) and expression of luciferase is detected by luciferase activity in cells overtime as an indicator of the translation of a functional protein. In transfection experiments performed in HEK293T cells when using lOOug of mRNA formulated with lipofectamine, the inventors observed that incorporation of UNA-

[0298] ATP and UNA-UTP, each increased luciferase expression over 72hs (Figure 4) with a fold increase of ranging from up to 40-fold with 90% substitution at 24h (Figure 4). These data indicate that incorporation of individual UNTPs increases both the magnitude and expression of capped mRNA in cells that have impaired innate sensing mechanisms for recognizing RNA. This suggests that mRNA modified with individual UNTPs may provide enhanced expression and stability even in cells with impaired RNA sensing mechanisms.

[0299] These data indicate that incorporation UNTPs and in particular UNA-ATP and UNA- UTP enhances the magnitude and duration of mRNA expression in cells that have impaired innate RNA sensing that may suppress RNA translation and increase degradation. The inventors anticipate that lower levels of UNTP substitution may be equally effective. For example for mRNA suitable subtitutions range from 1-100%. The inventors anticipate that further gains will be realized when individual UNA- NTPs are used in combination : For example, for mRNA, suitable combinations are: 1. 75% UNA-ATP+90%UNA-5meCTP 2. 90% UNA-ATP+90%UNA-CTP

[0300] 3. 90% UNA-ATP+75% UNA-GTP

[0301] 4. 90% UNA-ATP+90% UNA-TTP

[0302] 5. 90% UNA-ATP+90% UNA-UTP 6. 90% UNA-5meCTP+75% UNA-GTP

[0303] 7. 90% UNA-CTP+75% UNA-GTP

[0304] 8. 90% UNA-5meCTP+90% UNA-TTP

[0305] 9. 90% UNA-CTP+90% UNA-TTP

[0306] 10. 90% UNA-5meCTP+90% UNA-UTP 11. 90% UNA-CTP+90% UNA-UTP

[0307] 12. 75% UNA-GTP+90% UNA-TTP

[0308] 13. 75% UNA-GTP+90% UNA-UTP

[0309] 14. 90% UNA-ATP+90% UNA-5meCTP+75%UNA-GTP

[0310] 15. 90% UNA-ATP+90% UNA-CTP+75%UNA-GTP 16. 75% UNA-ATP+90% UNA-5meCTP+90% UNA-TTP

[0311] 17. 75% UNA-ATP+90% UNA-CTP+90% UNA-TTP

[0312] 18. 75% UNA-ATP+90% UNA-5meCTP+90% UNA-UTP

[0313] 19. 75% UNA-ATP+90% UNA-CTP+90% UNA-UTP

[0314] 20. 75% UNA-ATP+75% UNA-GTP+90% UNA-TTP 21 75% UNA-ATP+75% UNA-GTP+90% UNA-UTP

[0315] 22. 90% UNA-5meCTP+75% UNA-GTP+90% UNA-TTP

[0316] 23. 90% UNA-CTP+75% UNA-GTP+90% UNA-TTP

[0317] 24. 90% UNA-5meCTP+75% UNA-GTP+90% UNA-UTP

[0318] 25. 90% UNA-CTP+75% UNA-GTP+90% UNA-UTP 26. 75% UNA-ATP+90% UNA-5meCTP+75% UNA-GTP+90% UNA-TTP

[0319] 27. 75% UNA-ATP+90% UNA-CTP+75% UNA-GTP+90% UNA-TTP

[0320] 28. 75% UNA-ATP+90% UNA-5meCTP+75% UNA-GTP+90% UNA-UTP

[0321] 29. 75% UNA-ATP+90% UNA-CTP+75% UNA-GTP+90% UNA-UTP, or any other percentage ratios.

[0322] Example 3 - Incorporation of individual UNA-NTPs increases the magnitude and expression of capped mRNA in cells that have intact innate sensing mechanisms for recognizing RNA (e.g. HeLa cells')

[0323] Next, the inventors assessed the impact of incorporation of individual UNTPs (ATP, UTP) and combined (ATP+UTP) on the function of mRNA in HeLa cells representative of epithelial cells. Unlike the HEK293T cells tested in the previous example, HeLa cells have intact epithelial innate sensing mechanisms able to respond to RNA triggering intracellular signaling pathways able to reduce RNA expression and enhance the rate of RNA degradation.

[0324] Substitution of individual NTPs with unlocked NTPs enhances fLuc expression in HeLa epithelial cells by > 100-fold at across 24, 48 and 72 hours for both UNA-UTP and UNA_ATP) and >50-fold with UNA-ATP plus UNA-UTP at 24 and 48h. These data indicate that the incorporation of UNA-NTPs enhance both the magnitude and duration of expression in epithelial cells with intact innate sensing pathways (Figure 5). It is anticipated that the use of different UNA-NTPs in combinations will lead to further gains in expression.

[0325] Example 4 - Incorporation of individual UNA-NTPs increases the magnitude and expression of capped mRNA in cells that have intact innate sensing mechanisms for recognizing RNA (e.o. THP-1 cells') Next, the inventors assessed the impact of incorporation of individual UNTPs (ATP, UTP) and combined (ATP+UTP) on the function of mRNA in THP-1 cells representative of monocytic cells representative of myeloid cells, specifically monocytic cells. These cells are particularly resistant to transfection due to sensitive innate sensing mechanisms able to respond to RNA triggering intracellular signaling pathways able to reduce RNA expression and enhance the rate of RNA degradation.

[0326] Substitution of individual NTPs with unlocked NTPs enhances fLuc expression in THP-1 monocytic cells by up to 100-fold at 24, 48 and 72 hours (UNA-ATP) and 200-fold (UNA-ATP) at 24 and 48h. Combined substitution of UNA-ATP plus UNA- UTP was similar to UNA ATP alone (Figure 6). These data indicate that the incorporation of UNTPs enhance both the magnitude and duration of expression in monocytic cells with intact innate sensing pathways (Figure 6). It is anticipated that the use of different UNA-NTPs in combinations will lead to further gains in expression.

[0327] Example 5 - Summary of the data

[0328] Table 2 below summarises the impact of UNA modifications on mRNA expression.

[0329] Table 2 - The impact of unlocked modifications on mRNA expression when using IQOng mRNA with 90% substitution

[0330] + = 1-10 fold, ++ = 20-40 fold, + + + = > 50 fold

[0331] Example 6 - Incorporation of individual and combined UTPs influences the induction of inflammatory chemokine IP10 (CXCL10) indicative of innate responses to RNA in THP-1 cells.

[0332] To determine whether substitution of individual UNTPs could reduce the activation of innate sensing, interferon generation and / or degradation of mRNA in host cells, THP1 cells were transfected with unmodified vs. mRNA modified with individual UNTPs and the cell supernatant harvested at 24, 48 and 72 h post transfection to characterize the induced cytokine profile. THP1 cells were chosen for this analysis because they represent monocyte cells with very robust innate responses. An Elisa assay was used to measure the levels of IP10 secreted into the supernatant, a marker of the innate inflammatory response to exogenous RNA

[0028] . It was observed that substitution of individual NTPs with UNTPs was able to influence the secretion profile of IP 10 in supernatants harvested from transfected THP-1 cells (Figure 7). These data demonstrate that individual UNTP have potential to impact on innate recognition and associated cytokine induction. Furthermore, combined substitution of both UNA-ATP plus UNA-UTP provided a great reduction in IP10 production suggesting that combinations of UNAs may provide a more profound reduction in innate recognition of RNA.

[0333] Summary

[0334] The inventors show that unlocked NTPs, where the furanose ring lacks C-C bond between 2'- and 3'-carbon atoms, can be used :

[0335] I) to increase to generate UNA-RNA by in vitro transcription using bacteriophage DNA dependent RNA polymerases (eg T7, T3, SP6 etc),

[0336] II) that such UNA-RNA comprising one or more UNTPs will be more resistant to hydrolysis and / or nuclease degradation than unmodified RNA, iii) that changes in the structural presentation of RNA containing one or more

[0337] UNTPs or UNTPs used in combination with other 2' modified ribose NTPs can change the expression profile of RNA encoding protein, in particular mRNA, saRNA or cRNA; and / or iv) RNA containing one or more UNA can reduce the activation of innate sensing, interferon generation and / or degradation of an RNA when used alone or in conjunction with other 2' modified ribose NTPs. The invention involves the use of UNA-RNA, specifically mRNA, saRNA, circular RNA and non-coding RNA, with a significant percentage of nucleotides (>1, up to 100%) being UNA-NTPs. The mRNA and saRNA sequences are of a sizeable length (> lOObp) and wild-type nucleotides are at least partially substituted by UNA-NTPs. The modified RNAs are synthesised via in vitro transcription, and the reaction mixture optimally includes high concentrations of magnesium ions (i.e. >20mM magnesium ions, ideally 75nM magnesium acetate) and high concentrations of nucleotides (i.e. >10mM nucleotides).

[0338] Conclusions The inventors describe herein the invention of generating UNA by in vitro transcription using bacteriophage DNA dependent RNA polymerases (eg T7, T3, SP6 or any bacteriophage polymerase) using optimized reaction buffers that include high levels of magnesium (>20mM, ideally >50mM and most ideally 75mM using magnesium acetate). This invention is novel and non-obvioius, because eukaryotic RNA polymerases

[0021] and wider DNA polymerases

[0022] are unable to incorporate UNTPs into DNA or RNA in sequences over 25nt, and, for sequences <25nt, can only achieve a replacement of one or at best two nucleotides in a short oligonucleotide sequence. References

[0339] [1]. Pasternak A, Wengel J. Unlocked nucleic acid--an RNA modification with broad potential. Org Biomol Chem. 2011;9:3591-3597. doi : 10.1039 / c0ob01085e.

[0340] [2], Campbell MA, Wengel J. Locked vs. unlocked nucleic acids (LNA vs. UNA) : contrasting structures work towards common therapeutic goals. Chem Soc Rev. 2011;40(12) : 5680-5689. doi : 10.1039 / clcsl5048k.

[0341] [3]. Tomaszewska-Antczak A, Guga P, Acyclic Nucleic Acids with Phosphodiester Linkages— Synthesis, Properties and Potential Applications. Appl. Sci. 2021; 11: 12125. doi.org / 10.3390 / appll2412125 [4], Langkjaer N, Pasternak A, Wengel J. UNA (unlocked nucleic acid) : a flexible

[0342] RNA mimic that allows engineering of nucleic acid duplex stability. Bioorg Med Chem. 2009; 17: 5420-5425. doi: 10.1016 / j.bmc.2009.06.045.

[0343] [5]. Werk D, Wengel J, Wengel SL, Grunert HP, Zeichhardt H, Kurreck J. Application of small interfering RNAs modified by unlocked nucleic acid (UNA) to inhibit the heart-pathogenic coxsackievirus B3. FEBS Lett. 2010;584(3): 591-598. doi :

[0344] 10.1016 / j.febslet.2009. 12.007.

[0345] [6]. Laursen MB, Pakula MM, Gao S, Fluiter K, Mook OR, Baas F, Langklaer N, Wengel SL, Wengel J, Kjems J, Bramsen JB. Utilization of unlocked nucleic acid (UNA) to enhance siRNA performance in vitro and in vivo. Mol Biosyst. 2010;6:862- 870. doi : 10.1039 / b918869j.

[0346] [7], Pasternak A, Wengel J. Thermodynamics of RNA duplexes modified with unlocked nucleic acid nucleotides. Nucleic Acids Res. 2010;38(19) :6697-6706. doi : 10.1093 / nar / gkq561.

[0347] [8]. Mikhailov SN, Pfleiderer W. Synthesis of a new class of acyclic 2', 5'- and 3', 5'- oligonucleotide analogs based on 9-[l,5-dihydroxy-4(S)-hydroxymethyl-3-oxapent- 2(R)-yl]-adenine. Tetrahedron Lett., 1985, 26, 2059-2062.

[0348] [9]. Nielsen P, Dreipe LH, Wengel J. Synthesis and evaluation of oligodeoxynucleotides containing acyclic nucleosides: introduction of three novel analogues and a summary. Bioorg Med Chem. 1995;3: 19-28. doi : 10.1016 / 0968- 0896(94)00143-q.

[0349]

[0010] . Mangos MM, Min KL, Viazovkina E, Galarneau EA, Elzagheid MI, Parniak MA, Damha MJ. Efficient RNase H-directed cleavage of RNA promoted by antisense DNA or 2'F-ANA constructs containing acyclic nucleotide inserts. J. Am. Chem. Soc., 2003, 125, 654-661.

[0350]

[0011] . Doessing H, Vester B. Locked and Unlocked Nucleosides in Functional Nucleic Acids Molecules 2011, 16, 4511-4526; doi : 10.3390 / moleculesl6064511

[0351]

[0012] , Itkes AV, Karpeisky M, Kartasheva ON, Mikhailov SN, Moiseyev GP, Pfleiderer W, Charubala R, Yakovlev GI. A route to 2',5'-oligoadenylates with increased stability towards phosphodiesterases. FEBS Lett. 1988, 236, 325-328. DOI: 10.1016 / 0014-5793(88)80048-6

[0352]

[0013] . Bramsen JB, Pakula MM, Hansen TB, Bus C, Langkjaer N, Odadzic D, Smicius R, Wengel SL, Chattopadhyaya J, Engels JW, Herdewijn P, Wengel J, Kjems J. A screen of chemical modifications identifies position-specific modification by UNA to most potently reduce siRNA off-target effects. Nucleic Acids Res. 2010;38: 5761- 5773. doi : 10.1093 / nar / gkq341.

[0353]

[0014] , Bramsen JB, Laursen MB, Nielsen AF, Hansen TB, Bus C, Langkjaer N, Babu BR, Hpjland T, Abramov M, Van Aerschot A, Odadzic D, Smicius R, Haas J, Andree C, Barman J, Wenska M, Srivastava P, Zhou C, Honcharenko D, Hess S, Muller E, Bobkov GV, Mikhailov SN, Fava E, Meyer TF, Chattopadhyaya J, Zerial M, Engels JW, Herdewijn P, Wengel J, Kjems J. A large-scale chemical modification screen identifies design rules to generate siRNAs with high activity, high stability, and low toxicity. Nucleic Acids Res. 2009;37(9):2867-2881. doi : 10.1093 / nar / gkpl06.

[0354]

[0015] . Mikkola S, Ldnnberg T, Ldnnberg H. Phosphodiester models for cleavage of nucleic acids. Beilstein J Org Chem. 2018; 14:803-837. doi : 10.3762 / bjoc. 14.68.

[0355]

[0016] . Zhang K, Hodge J, Chatterjee A, Moon TS, Parker KM. Duplex Structure of Double-Stranded RNA Provides Stability against Hydrolysis Relative to Single- Stranded RNA. Environ Sci Technol. 2021;55:8045-8053. doi : 10.1021 / acs.est. lc01255.

[0356]

[0017] , Kotkowiak W, Kotkowiak M, Kierzek R, Pasternak A. Unlocked nucleic acids: implications of increased conformational flexibility for RNA / DNA triplex formation. Biochem J. 2014;464:203-211. doi : 10.1042 / BJ20141023. PMID: 25226286.

[0357]

[0018] . Goodchild, A., Nopper, N., King, A., Doan, T., Tanudji, M., Arndt, G. M., et al. Sequence determinants of innate immune activation by short interfering RNAs. BMC Immunology, 2009: 10, 40. DOI: 10.1186 / 1471-2172-10-40

[0358] [19.] Hornung, V., Guenthner-Biller, M., Bourquin, C., Ablasser, A., Schlee, M., Uematsu, S., et al. Sequence-specific potent induction of ifn-alpha by short interfering RNA in plasmacytoid dendritic cells through TLR7. Nature Medicine, 2005: 11, 263-270. doi: 10.1038 / nmll91.

[0359]

[0020] , Duffy K, Arangundy-Franklin S, Holliger P. Modified nucleic acids: replication, evolution, and next-generation therapeutics. BMC Biol. 2020; 18: 112. doi : 10.1186 / S12915-020-00803-6.

[0360]

[0021] , Xu L, Plouffe SW, Chong J, Wengel J, Wang D. A chemical perspective on transcriptional fidelity: dominant contributions of sugar integrity revealed by unlocked nucleic acids. Angew Chem Int Ed Engl. 2013;52(47) : 12341-5. doi :

[0361] 10.1002 / anie.201307661.

[0362]

[0022] , Dubois C, Campbell MA, Edwards SL, Wengel J, Veedu RN. Stepping towards highly flexible aptamers: enzymatic recognition studies of unlocked nucleic acid nucleotides. Chem Commun (Camb). 2012;48(44) : 5503-5505. doi : 10.1039 / c2cc31316b.

[0363]

[0023] . Morais P, Adachi H, Yu YT. The Critical Contribution of Pseudouridine to mRNA COVID-19 Vaccines. Front Cell Dev Biol. 2021;9:789427. doi :

[0364] 10.3389 / fcell.2021.789427.

[0024] . Kariko K, Buckstein M, Ni H, Weissman D. Suppression of RNA recognition by Toll-like receptors: the impact of nucleoside modification and the evolutionary origin of RNA. Immunity. 2005;23: 165-75. doi : 10.1016 / j.immuni.2005.06.008.

[0365]

[0025] . Andries O, Me Cafferty S, De Smedt SC, Weiss R, Sanders NN, Kitada T. N(l)-methylpseudouridine-incorporated mRNA outperforms pseudouridine- incorporated mRNA by providing enhanced protein expression and reduced immunogenicity in mammalian cell lines and mice. J Control Release. 2015;217 :337-44. doi: 10.1016 / j.jconrel.2015.08.051.

[0366]

[0026] . Mu X, Greenwald E, Ahmad S, Hur S. An origin of the immunogenicity of in vitro transcribed RNA. Nucleic Acids Res. 2018 Jun l;46(10): 5239-5249.

[0027] , McCown PJ, Ruszkowska A, Kunkier CN, Breger K, Hulewicz JP, Wang MC,

[0367] Springer NA, Brown JA. Naturally occurring modified ribonucleosides. Wiley Interdiscip Rev RNA. 2020; l l:el595. doi : 10.1002 / wrna. l595.

[0368]

[0028] . Elemam NM, Talaat IM, Maghazachi AA. CXCL10 Chemokine: A Critical Player in RNA and DNA Viral Infections. Viruses. 2022; 14:2445. doi: 10.3390 / vl4112445.

[0029] . Buttmann M, Berberich-Siebelt F, Serfling E, Rieckmann P. Interferon-beta is a potent inducer of interferon regulatory factor- 1 / 2-dependent IP-10 / CXCL10 expression in primary human endothelial cells. J Vase Res. 2007;44(l) : 51-60. doi : 10.1159 / 000097977.

[0369]

[0030] . Takahashi S, Matsumoto S, Chilka P, Ghosh S, Okura H, Sugimoto N. Dielectricity of a molecularly crowded solution accelerates NTP misincorporation during RNA-dependent RNA polymerization by T7 RNA polymerase. Sci Rep. 2022; 12: 1149. doi : 10.1038 / s41598-022-05136-8.

[0370]

[0031] . Samnuan K, Blakney AK, McKay PF and Shattock RJ. Design-of-experiments in vitro transcription yield optimization of self-amplifying RNA. FlOOOResearch 2022, 11 :333 (https: / / doi.org / 10.12688 / fl000research.75677. !)

Claims

Claims1. One or more unlocked nucleotide, for use in :(I) enhancing the expression and / or translation of an RNA molecule comprising the one or more unlocked nucleotide;(ii) enhancing the stability of an RNA molecule comprising the one or more unlocked nucleotide; and / or(iii) reducing the activation of innate sensing, interferon generation and / or degradation of an RNA molecule comprising the one or more unlocked nucleotide.

2. One or more unlocked nucleotide, for use according to claim 1, wherein the RNA molecule is coding RNA, optionally for use in therapeutic applications and vaccine applications.

3. One or more unlocked nucleotide, for use according to claim 1, wherein theRNA molecule is non-coding RNA, optionally for use in RNAi applications.

4. One or more unlocked nucleotide, for use according to any preceding claim, wherein the RNA molecule is selected from a group consisting of: messenger RNA (mRNA), micro RNA (miRNA); short interfering RNA (siRNA); short hairpin RNA (shRNA); anti-sense RNA; RNA aptamers; self-amplifying RNA (saRNA); interference RNA (RNAi); non-coding RNA; circular RNA (circRNA); and small RNA.

5. One or more unlocked nucleotide, for use according to any preceding claim, wherein the RNA molecule is self-amplifying RNA (saRNA).

6. One or more unlocked nucleotide, for use according to any preceding claim, wherein the RNA molecule is messenger RNA (mRNA).

7. One or more unlocked nucleotide, for use according to any preceding claim, wherein the RNA molecule comprises a cap, and the cap either does or does not comprise the one or more unlocked nucleotide.

8. One or more unlocked nucleotide, for use according to any preceding claim, wherein the RNA molecule is:(i) at least 20, 21, 22 or 23 bases in length;(ii) at least 28, 29, 30, or 31 bases in length;(iv) at least 36, 37, 38 or 39 bases in length;(v) at least 46, 47, 48 or 49 bases in length;(vi) at least at least 50 bases, at least 60 bases, at least 75 bases, at least 100 bases, at least 200 bases, or at least 300 bases in length; or (vii) at least 400 bases, at least 500 bases, at least 600 bases, at least 700 bases, at least 800 bases, or at least 900 bases in length.

9. One or more unlocked nucleotide, for use according to any preceding claim, wherein the RNA molecule is: (I) at least 1000 bases, at least 2000 bases, at least 3000 bases, at least 4000 bases, or at least 5000 bases in length; or(II), at least 6000 bases, at least 7000 bases, or at least 8000 bases in length.

10. One or more unlocked nucleotide, for use according to any preceding claim, wherein the one or more unlocked nucleotide comprises the absence of a C-C bond between 2'- and 3'- carbon atoms in the ribose sugar.

11. One or more locked nucleotide, for use according to any preceding claim, wherein the one or more unlocked nucleotide is represented herein by formula [I] :wherein Base is a nucleobase;R1is a phosphate, a modified phosphate, a phosphate linker, a modified phosphate linker or a 5 to 10 membered heteroarylene, wherein the phosphate linker, the modified phosphate linker or the 5 to 10 membered heteroarylene is a linker between the rest of the unlocked nucleotide of formula [I] and an attachment point to a first adjacent nucleotide in the RNA molecule;R3is H or a bond to a second adjacent nucleotide in the RNA molecule; wherein if R1is a phosphate or a modified phosphate then R3is a bond to a second adjacent nucleotide in the RNA molecule;R2is H or C1-6 alkyl; and R6is OH, a halogen, an optionally substituted aromatic group, NH2, N3, H, an optionally substituted O-alkyl, O-alkenyl or O-alkynyl group, or an optionally substituted alkyl, alkenyl or alkynyl group, wherein in each instancethe aromatic group, alkyl, alkenyl or alkynyl is optionally substituted with halogen, oxo, OR9, CN, NR9R10or SR9; or R2and R6together define a linker;R4, R5, R7and R8are independently H or Ci-e alkyl; andR9and R10are independently H or Ci-e alkyl, C2-6 alkenyl or C2-6 alkynyl.

12. One or more unlocked nucleotide, for use according to any preceding claim, wherein the one or more locked nucleotide is represented herein as formula [la] :[la].

13. One or more unlocked nucleotide, for use according to any preceding claim, wherein the nucleobase is a primary nucleobase or a modified nucleobase, optionally wherein the primary nucleobase is a purine or a pyrimidine; and / or the modified nucleobase has the structure of a primary nucleobase with one or more modifications, which is selected from : replacing a hydrogen and / or an NH2 group in the primary nucleobase with an optionally substituted Ci-e alkyl, an optionally substituted C2-6 alkenyl, an optionally substituted C2-6 alkynyl, OR11, NRnR12, COOR11, CONRnR12or a halogen; - replacing a double bond with a single bond; replacing a CH in the structure with an N; replacing an N in the structure with a CR11; replacing an 0 in the structure with an NR11, S, Se or CRnR12; and / or the nucleobase is attached to the rest of the locked nucleotide of formula [I] at a different point to the point where the primary nucleobase would typically be attached; wherein R11and R12are independently H, an optionally substituted Ci-e alkyl, an optionally substituted Ci-e alkenyl, an optionally substituted Ci-e alkynyl or an optionally substituted Ce-12 aryl, or R11and R12together with the atom to which they are bonded form an optionally substituted 3 to 10 membered cycloalkyl or an optionally substituted 3 to 10 membered heterocyclyl.

14. One or more unlocked nucleotide, for use according to any preceding claim, whereinbond, R4is H, R5is H, R6is OH, R7is H, R8is H, optionally wherein the base is adenine, cytosine, guanine, uracil or thymine.

15. One or more unlocked nucleotide, for use according to any preceding claim, wherein the use comprises using one or more unlocked nucleotide selected from a group consisting of: unlocked adenine; unlocked cytosine; unlocked guanine; unlocked uracil and unlocked thymine.

16. One or more unlocked nucleotide, for use according to any preceding claim, wherein the RNA molecule further comprises one or more modified nucleotide comprising a 2'-substituted group in which the OH group normally at the 2' position is replaced with a halogen (preferably fluorine), an optionally substituted aromatic group, a NH2, a N3, a H, an optionally substituted O-alkyl, O-alkenyl or O-alkynyl group, or an optionally substituted alkyl, alkenyl or alkynyl group, wherein in each instance the aromatic group, alkyl, alkenyl or alkynyl is optionally substituted with halogen, oxo, OR, CN, NR2 or SR, wherein R is H or Ci-e alkyl, C2-6 alkenyl or C2-6 alkynyl.

17. One or more unlocked nucleotide, for use according to claim 16, wherein the one or more modified nucleotide is represented herein by by formula [II] :R17— vnBase .fl , R19R20[II] wherein Base is a nucleobase;R17is a phosphate, a modified phosphate, a phosphate linker, a modified phosphate linker or a 5 to 10 membered heteroarylene, wherein the phosphate linker, the modified phosphate linker or the 5 to 10 membered heteroarylene is a linker between the rest of the unlocked nucleotide of formula [I] and an attachment point to a first adjacent nucleotide in the RNA molecule;R18and R21are independently H or Ci-e alkyl;R19is H or a bond to a second adjacent nucleotide in the RNA molecule; wherein if R17is a phosphate or a modified phosphate then R19is a bond to a second adjacent nucleotide in the RNA molecule;R20is a halogen, an optionally substituted aromatic group, a NH2, a N3, a H, an optionally substituted O-alkyl, O-alkenyl or O-alkynyl group, or an optionally substituted alkyl, alkenyl or alkynyl group, wherein in each instance the aromatic group, alkyl, alkenyl or alkynyl is optionally substituted with halogen, oxo, OR22, CN, NR22R23or SR22; andR22and R23are independently H or Ci-e alkyl, C2-6 alkenyl or C2-6 alkynyl.

18. One or more unlocked nucleotide, for use according to either claim 16 or 17, wherein the one or more modified nucleotide is represented herein by formula [Ha] :R17-I Q Base19. One or more unlocked nucleotide, for use according to any one of claims 16-18, wherein R9is a halogen, optionally chlorine, or bromine.

20. One or more unlocked nucleotide, for use according to any one of claims 16-19, wherein R9is fluorine.

21. One or more locked nucleotide, for use according to any one of claims 16-18, wherein R9is Me or OMe, preferably OMe.

22. One or more locked nucleotide, for use according to any one of claims 16-21, wherein the use comprises using at least one modified nucleotide selected from a group consisting of: a modified adenine; a modified cytosine; a modified guanine; a modified uracil and / or a modified thymine.

23. One or more locked nucleotide, for use according to any preceding claim, wherein the use comprises using a combination of:(i) one or more locked nucleotide represented by formula [I]; and(II) one or more modified nucleotide represented by formula [II].

24. One or more locked nucleotide, for use according to any preceding claim, wherein the use comprises using a combination of:(i) one or more unlocked nucleotide represented by formula [I], wherein R1is, R2is H, R3is a bond, R4is H, R5is H, R6is OH, R7is H, R8is H, and the base is adenine, cytosine, guanine, uracil or thymine; and one or more modified nucleotide represented by formula [II], wherein R17isO>11s-^-O-P-O-| *0 , R18is hydrogen, R19is a bond, R20is a halogen (preferably fluorine) or OMe, R21is hydrogen, and the base is adenine, cytosine, 5-methyl-cytosine, guanine, uracil or thymine.

25. One or more unlocked nucleotide, for use according to any preceding claim, wherein the RNA molecule further comprises one or more locked nucleotide (LNA) comprising a ribose moiety modified by a bridge between the 2 and 4 positions or the 2' and 4' positions in the ribose sugar.

26. One or more unlocked nucleotide, for use according to claim 25, wherein the one or more locked nucleotide is represented herein by formula [III]:wherein Base is a nucleobase;L1is a C1-5 alkylene;X3is 0, CH2, CO, NR28, Se, S, SO or S02; X4is absent, or is CO, 0, NR28, Se, S, SO or SO2;R24is a phosphate, a modified phosphate, a phosphate linker, a modified phosphate linker or a 5 to 10 membered heteroarylene, wherein the phosphate linker, the modified phosphate linker or the 5 to 10 membered heteroarylene is a linker between the rest of the locked nucleotide of formula [III] and an attachment point to a first adjacent nucleotide in the RNA molecule;R25is H or a bond to a second adjacent nucleotide in the RNA molecule; wherein ifR24is a phosphate or a modified phosphate then R25is a bond to a second adjacent nucleotide in the RNA molecule;R26and R27are independently H or Ci-e alkyl; and27. One or more unlocked nucleotide, for use according to either claim 25 or claim 26, wherein the one or more locked nucleotide is represented herein as formula [Illa] :[Illa],28. One or more unlocked nucleotide, for use according to any one of claims 25-27, wherein the use comprises using a combination of: (i) one or more unlocked nucleotide represented by formula [I]; and(II) one or more locked nucleotide represented by formula [III].

29. One or more unlocked nucleotide, for use according to any one of claims 25-28, wherein the use comprises using a combination of: (i) one or more unlocked nucleotide represented by formula [I], wherein R1is, R2is H, R3is a bond, R4is H, R5is H, R6is OH, R7is H, R8is H, and the base is adenine, cytosine, guanine, uracil or thymine; and(ii) one or more locked nucleotide represented by formula [III], wherein R24is, R25is a bond, R26is H, R27is H, X3is 0, X4is absent and L2is CH2, and the base is adenine, cytosine, 5-methyl-cytosine, guanine, uracil or thymine.

30. One or more unlocked nucleotide, for use according to any preceding claim, wherein the use comprises using a combination of:(i) one or more unlocked nucleotide represented by formula [I];(ii) one or more modified nucleotide represented by formula [II]; and (iii) one or more locked nucleotide represented by formula [III].

31. One or more unlocked nucleotide, for use according to claim 30, wherein the use comprises using a combination of:(i) one or more unlocked nucleotide represented by formula [I], wherein R1is, R2 iSH, R3is a bond, R4is H, R5is H, R6is OH, R7is H, R8is H, and the base is adenine, cytosine, guanine, uracil or thymine;(ii) one or more modified nucleotide represented by formula [II], wherein R17is, R18is hydrogen, R19is a bond, R20is a halogen (preferably fluorine) or OMe, R21is hydrogen, and the base is adenine, cytosine, 5-methyl-cytosine, guanine, uracil or thymine; and(iii)one or more locked nucleotide represented by formula [III], wherein R24is, R25is a bond, R26is H, R27is H, X3is 0, X4is absent and L2is CH2, and the base is adenine, cytosine, 5-methyl-cytosine, guanine, uracil or thymine.

32. A method of preparing an unlocked nucleic acid RNA (UNA-RNA) molecule, wherein the method comprises contacting, in the presence of at least 20mM magnesium ions, (i) a template nucleic acid sequence, (ii) an RNA polymerase, and(iii) a plurality of nucleotide triphosphates (NTPs), one or more of which comprises an unlocked nucleotide, wherein the RNA polymerase transcribes the template nucleic acid sequence to form a UNA-RNA molecule comprising at least 25 nucleotides.

33. Use of >20mM magnesium ions in a transcription reaction to prepare an unlocked nucleic acid RNA (UNA-RNA) molecule comprising at least 25 nucleotides.

34. A method according to claim 32 or a use according to claim 33, wherein the method comprises the use of:(i) at least 25mM magnesium ions, at least 30mM magnesium ions, at least 35mM magnesium ions, at least 40mM magnesium ions, or at least 5omM magnesium ions;(ii) at least 60mM magnesium ions, at least 70mM magnesium ions;(iii) at least 75mM magnesium ions, at least 80mM magnesium ions, at least 85mM magnesium ions, at least 90mM magnesium ions, or at least lOOmM magnesium ions; or (iv) less than 120mM magnesium ions, less than lOOmM magnesium ions, less than 90mM magnesium ions, less than 80mM magnesium ions, or less than 75mM magnesium ions.

35. A method or use according to any one of claims 32-34, wherein the magnesium ions are provided as magnesium acetate, magnesium chloride, magnesium citrate, magnesium sulphate, magnesium gluconate, or magnesium lactate.

36. A method or use according to any one of claims 32-35, wherein the magnesium ions are provided as magnesium acetate.

37. A method or use according to any one of claims 32-36, wherein the method comprises the use of an RNA polymerase, which is selected from a group consisting of: T7; T3; SP6; KP34; Syn5; or other DNA-dependent RNA polymerase; or a mutated variant of any of these RNA polymerases.

38. A method or use according to any one of claims 32-37, wherein the plurality of nucleotide triphosphates (NTPs) are selected from the group consisting of ATP, GTP, CTP, TTP and / or UTP.

39. A method or use according to any one of claims 32-37, wherein the method comprises the use of the plurality of nucleotide triphosphates at a concentration of:(i) at least ImM, 2mM, 3mM or 4mM;(II) at least 5mM, 6mM or 7mM; or (iii) at least 8mM, 9mM, lOmM.

40. A method or use according to any one of claims 32-39, wherein the template nucleic acid sequence comprises DNA, optionally a vector, preferably a plasmid.

41. A method or use according to any one of claims 32-40, wherein the template nucleic acid sequence encodes an antigen which is derived from a tumour, virus, a bacteria, a mycoplasma, a fungus, an animal, a plant, an alga, a parasite, or a protozoan, or other organism which causes a disease in a subject, preferably a human or animal.

42. A method or use according to any one of claims 32-41, wherein the template nucleic acid encodes a therapeutic protein, which is derived from an animal or a human, and which treats, prevents or ameliorates disease in a subject, preferably a human or animal subject.

43. A method or use according to any one of claims 32-42, wherein the template nucleic acid encodes an innate inhibitor protein, which counteracts the innate immune response in a subject administered with a vaccine comprising the RNA molecule.

44. An RNA molecule obtained or obtainable by the method according to any one of claims 32-42.

45. An RNA molecule comprising at least 25 nucleotides, wherein one or more of the nucleotides is an unlocked nucleotide.

46. An RNA molecule according to claim 45, wherein the RNA molecule is as defined in any one of claims 4-9 and / or the locked nucleotide is as defined in any one of claims 10-31.

47. A pharmaceutical composition comprising the RNA molecule according to any one of claims 44-46, and a pharmaceutically acceptable vehicle.

48. A method of preparing the pharmaceutical composition according to claim47. the method comprising contacting the RNA molecule according to any one of claims 44-46 and a pharmaceutically acceptable vehicle.

49. The RNA molecule according to any one of claims 44-46, or the pharmaceutical composition according to claim 47, for use as a medicament.

50. The RNA molecule according to any one of claims 44-46, or the pharmaceutical composition according to claim 47, for use in treating, preventing or ameliorating a disease in a subject.

51. A vaccine composition comprising the RNA molecule according to any one of claims 44-46, or the pharmaceutical composition according to claim 47.

52. The RNA molecule according to any one of claims 44-46, the pharmaceutical composition according to claim 47, or the vaccine according to claim 51, for use in stimulating an immune response in a subject.

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