Modified ribonucleic acids

Abstract

The invention relates to messenger RNAs comprising modified nucleotides, methods of synthesizing such messenger RNAs, and pharmaceutical and vaccine compositions comprising messenger RNAs comprising modified nucleotides for use in treatment.

Description

[0001] MODIFIED RIBONUCLEIC ACIDS

[0002] FIELD OF THE INVENTION

[0003] The invention relates to messenger RNAs comprising modified nucleotides, methods of synthesizing such messenger RNAs, and pharmaceutical and vaccine compositions comprising messenger RNAs comprising modified nucleotides for use in treatment.

[0004] Background of the invention

[0005] Recently, messenger RNA (mRNA) therapeutics have garnered increased interest due to the successful use of in vitro synthesized mRNA vaccines during the COVID-19 pandemic.

[0006] The potential therapeutic advantages of using mRNA instead of viral vectors or plasmids to deliver genetic information to an organism include, for example, improved safety, since RNA does not integrate into the host genome; rapid protein expression, since nuclear import and transcription are not necessary; lack of size / packaging limitations and easy manufacturing, i.e. rapid development and scalability.

[0007] One of the challenges of mRNA technologies is that RNA is highly immunogenic and unstable, which severely limits the scope of application. RNA triggers innate immune pathways in eukaryotic cells, which leads to the production of inflammatory cytokines and suppression of cellular translation.

[0008] Chemical modification of RNA has emerged as a tool to overcome these limitations. Over 120 RNA modifications found in nature have been described (The RNA Modification Database, Rozenski 1999, Nucl Acids Res 27: 196-197). One avenue of modification is incorporation of modified nucleotides in the place of the four common ribonucleotides, adenine (A), guanine (G), cytosine (C) and uracil (U). Common modified bases used are 2-thiouridine (s2U), pseudouridine (i ), N1 -methylpseudouridine (m1 qj), 5-methoxyuridine (5-moll), N6-methyladenosine (m6A), 3-methylcytidine (m3C), 5- methylcytosine (m5C) and N4-acetylcytidine (ac4C). Out of the identified modifications, i and m6A occur most frequently.

[0009] S2U, m6A and 5-moll modifications have been shown to increase RNA stability (Anderson et al. 2011 L. Nucleic Acids Res 39(21):9329-9338, Li et al. 2016, Bioconjugate Chem. 27 (3), 849-853).

[0010] However, foreign mRNA is still rapidly degraded in vivo and additional mRNA modifications that increase expression efficiency of mRNA therapeutics are needed.

[0011] Objectives and Summary of the Invention

[0012] The inventors have surprisingly shown that substitution of ribonucleotides with deoxyribonucleotides in mRNA results in increased mRNA transcription and expression.

[0013] In nature, RNA is composed exclusively of ribonucleotides, and DNA is composed exclusively of deoxyribonucleotide. Modifications of RNA ribonucleotides are known, including analogous nucleotides other than A, C, U and G. However, the inventors have found for the first time that the substitution of even small amounts of one ribonucleotide by its deoxyribonucleotide version can increase transcription of and expression from the resulting mRNA. Substitutions with a modified deoxyribonucleotide further increases this effect.

[0014] Hence, in a first aspect, the invention provides a method of synthesizing an mRNA, comprising the steps of: a) providing a composition comprising ATP, CTP, UTP and GTP ribonucleotides, wherein between 0.1% and 100% of at least one of the ribonucleotides are substituted by a deoxyribonucleotide version of the at least one ribonucleotide; b) adding a nucleic acid template encoding a protein of interest to the composition; and c) contacting the composition with an RNA polymerase under conditions such that the mRNA is synthesized.

[0015] In one embodiment, the mRNA is a self-amplifying RNA (saRNA).

[0016] In one embodiment, the deoxyribonucleotide is a modified or unmodified deoxyribonucleotide.

[0017] The modified deoxyribonucleotide may be the deoxyribonucleotide variant of any naturally occurring modified ribonucleotide according to the RNA Modification Database, described in Cantara et al. 2011 (Nucleic Acids Res . 2011 39 D195-201), which can be accessed at http: / / rna.rega.kuleuven.be / rnamods / , or it may be the deoxyribonucleotide variant of a synthetic modified ribonucleotide.

[0018] In one embodiment, in step a)

[0019] (i) the at least one ribonucleotide that is substituted by a deoxyribonucleotide version is ATP, optionally wherein 0.1% to 100% of ATP is substituted by a nucleotide selected from the group consisting of deoxyadenosine(dA), 2'-lodo-deoxyadenosine, 2'-Bromo- deoxyadenosine, 3'- deoxyadenosine, 7-Deaza-deoxyadenosine, 8-Bromo- deoxyadenosine, 7-Deaza-7-iodo- deoxyadenosine, 2-Hydroxy-deoxyadenosine, 2'- Chloro-deoxyadenosine, 7-Deaza-7-bromo- deoxyadenosine, 2'-Fluoro-deoxyadenosine, Mant-deoxyadenosine, 2'-Mant-3'- deoxyadenosine, 2'NH2- deoxyadenosine, Etheno- deoxyadenosine (E-dA), 5'-(a-seleno)- deoxyadenosine (dAaSe), 5'-(a-thio)- deoxyadenosine (dAaS), N6-(6-Aminohexyl)-deoxyadenosine, N6-methyldeoxyadenosine (m6dA), 2'-0-methyldeoxyadenosine (2'OMedA), N6,2'-0-dimethyldeoxyadenosine (m6dAm), N6,N6,2'-0-trimethyldeoxyadenosine (m62dAm), 8-Oxo-deoxyadenosine, and N6-Methyl-deoxyadenosine; and / or

[0020] (ii) the at least one ribonucleotide that is substituted by a deoxyribonucleotide version is UTP, optionally wherein 0.1% to 100% of UTP is substituted by a nucleotide selected from the group consisting of 2'NH2-deoxyuridine, 3'-Azido-2',3'-dideoxyuridine, Aminoallyl- deoxyuridine, 5-Propargylamino-deoxyuridine, 5-Fluoro-deoxyuridine, 5- Bromo- deoxyuridine, 5-lodo-deoxyuridine, deoxyuridine, 2',3'-Dideoxythymidine, Deoxythymidine, 5'-(a-seleno)-deoxythymidine (dTaSe), 5'-(a-thio)- deoxythymidine(dTaS), 3,2'-0-dimethyldeoxyuridine (m4dll), 2-thiodeoxyuridine (s2dll), 2-Fluoro-dUTP, pseudodeoxyuridine(dMJ), N1 -Methylpseudouridine (m'l d'-P), 2'-O- methyldeoxyuridine (Udm), 4-thiodeoxyuridine (s4dll), 5-methyldeoxyuridine (m5dll), and 5-methoxydeoxyuridine (mo5dll), and / or

[0021] (iii) the at least one ribonucleotide that is substituted by a deoxyribonucleotide version is CTP, optionally wherein 0.1% to 100% of CTP is substituted by a nucleotide selected from the group consisting of deoxycytidine, C8-Alkyne-deoxycytidine, 5-Methyl- deoxycytidine, 2'-Fluoro-deoxycytidine, 5-lodo-deoxycytidine, 5-Bromo- deoxycytidia- seleno)- deoxycytidine (dCaSe), 5'-(a-thio)-deoxycytidine (dCaS), 5-Propargylamino- deoxycytidine, 5-Hydroxymethyl-deoxycytidine, and 2'-0-methyldeoxycytidine (dCm), and / or

[0022] (iv) the at least one ribonucleotide that is substituted by a deoxyribonucleotide version is GTP, optionally wherein 0.1 % to 100% of GTP is substituted by a nucleotide selected from the group consisting of 8-Oxo-deoxyguanosine, 3'-deoxyguanosine, 7-Deaza- deoxyguanosine, 7-Deaza-7-iodo-deoxyguanosine, 6-Thio-deoxyguanosine, 2'-Fluoro- deoxyguanosine, Mant- deoxyguanosine, 2'-Mant-3'-deoxyguanosine, 2'NH2- deoxyguanosine, 5'-(a-seleno)-deoxyguanosine (dCaSe), 5'-(a-thio)-deoxyguanosine (dCaS), 2'-0-methyldeoxyguanosine (dGm), N2,7-dimethyldeoxyguanosine (m2,7dG), 7- methyldeoxguanosine (m7dG), and N2, N2,7-trimethyldeoxyguanosine (m2,7dG).

[0023] In one embodiment, the at least one ribonucleotide that is substituted by a deoxyribonucleotide version is CTP and the deoxyribonucleotide version is modified by a modification is selected from the group consisting of the following naturally or synthetically occurring ribonucleotide or deoxyribonucleotide modifications hm5Cm, s2C, m3C, phm5C, hm5C, f5C, pf5C, ho5C, hm5C, m5C, pC2'3'cp, m4,4C, ac4C, m4C, 2NH2-C, 3’-Azido-2’,3’-ddCTP, C8-Alkyne-C, 5-Propargylamino-C, LCC, CBV, CBR, IC, A5M, CSL, RY, 5CF, 10C, S4C, 73W, CH, N5M, 5IC, M5M, CAR, CFZ, CFL, D2C, 1 mC, m2C, e3C, 3mC, sC, 4mC, 5caC, 5fC, 5hmC, 5gmC and 5mC; or the at least one ribonucleotide that is substituted by a deoxyribonucleotide version is ATP and the deoxyribonucleotide version is modified by a modification is selected from the group consisting of the following naturally or synthetically occurring ribonucleotide or deoxyribonucleotide modifications m1A, msms2i6A, m2,8A, m2A, ms2ct6A, , ms2io6A, ms2hn6A, ms2hn6A, ms2i6A, ms2m6A, ms2t6A, ms2t6A, NADpN, m8A, ct6A, ct6A, ht6A, io6A, pio6A, m62A, ac6A, pac6A, f6A, pf6A, g6A, hm6A, hn6A, m6A, m6t6A, t6A, 2’-Bromo-A, LCA, IG, SRA, AF2, ADS, 2AD, 8AN, PPU, 3DA, AP7, 45A, A9Z, ANZ, A5O, A5L, 5AA, A43, 2HA AD2, 1 mA, 3mA, 4mA, 6mA, 7mA, 9mA, 6hmA, HAP, m2A, AHAP, 6-DMA, ncm6A and sA; or the at least one ribonucleotide that is substituted by a deoxyribonucleotide version is GTP and the deoxyribonucleotide version is modified by a modification is selected from the group consisting of the following naturally or synthetically occurring ribonucleotide or deoxyribonucleotide modifications m1G, preQ1 , preQO, m7G, m2,7G, m2,2,7G, m22G, m2G I, mi l, m1 lm, Im, Q, oQ, dGdAIQ, mdAnQ, preQO, preQ1 , dG+ , 7-Deaza-G, 2’NH2-dG, 7-Deaza-7-iodo-G, 6-thio-dG, dCaSe, dCaS, Fluoro-G, MANT-G, 2-MANT-G, LCG, BGM, 2SG, GRB, GAO, TG, GDO, GMX, XUG, G3A, GS, G38, 2HG, 1 mG, 2mG, 3mG, m6G, 7mG, 8-oxo-G, ADG, 1 ,N(2)-eG, N(2),3-eG, m22G, m2,7dG, m2,2,7dG, 2’NH2-dG and CEG; or the at least one ribonucleotide that is substituted by a deoxyribonucleotide version is UTP and the deoxyribonucleotide version is modified by a modification is selected from the group consisting of the following naturally or synthetically occurring ribonucleotide or deoxyribonucleotide modifications mcmo5U, 2-NH2-dU, 3’-Azido-2’,3’-ddU, Aminoallyl-dU, 5-Propargylamino-dU, 5-Fluoro-dU, 5- dimethyl-2’-dU, 5BU, U37, IU, TLN, BRU, URU, UFB, U5M, UOB, UMO, U5R, B8N, U4M, UAR, UFT, UMS, NYM, MU1 , P1T, M, MU4, putThy, ddT, dTaSe, dTaS, 1mT, 3mT, 04-meT, diHT, 5hmU, 5fU, base J, dhpUra, 5caU, 5-NeOmdU, dU, DHdU, 5-NedU, and dhpdU.

[0024] In one embodiment, the at least one ribonucleotide that is substituted by a deoxyribonucleotide version is CTP and the deoxyribonucleotide version is modified by a modification is selected from the group consisting of the following naturally or synthetically occurring ribonucleotide or deoxyribonucleotide modifications hm5Cm, s2C, m3C, phm5C, hm5C, f5C, pf5C, ho5C, hm5C, m5C, pC2'3'cp, m4,4C, ac4C, m4C, 2NH2-C, 3’-Azido-2’,3’-ddCTP, C8-Alkyne-C, 5-Propargylamino-C, LCC, CBV, CBR, IC, A5M, CSL, RY, 5CF, 10C, S4C, 73W, CH, N5M, 5IC, M5M, CAR, CFZ, CFL, D2C, 1 mC, m2C, e3C, 3mC, sC, 4mC, 5caC, 5fC, 5hmC, 5gmC and 5mC; or the at least one ribonucleotide that is substituted by a deoxyribonucleotide version is ATP and the deoxyribonucleotide version is modified by a modification is selected from the group consisting of the following naturally or synthetically occurring ribonucleotide or deoxyribonucleotide modifications m1A, msms2i6A, m2,8A, m2A, ms2ct6A, , ms2io6A, ms2hn6A, ms2hn6A, ms2i6A, ms2m6A, ms2t6A, ms2t6A, NADpN, m8A, ct6A, ct6A, ht6A, io6A, pio6A, m62A, ac6A, pac6A, f6A, pf6A, g6A, hm6A, hn6A, m6A, m6t6A, t6A, MANT-dATP, 2'-lodo-dATP, 7'-Deaza-dATP, 8'-Bromo-dATP, 7'- Deaza-7'-iodo-dATP, 2'-Hydroxy-dATP, 7'-Deaza-7-bromo-dATP, 2'-Fluoro-dATP, Mant-dATP, 2'NH2-dATP, Etheno-dATP (e-dATP), dATPaSe, dATPaS, 8'-Oxo-dATP, 2’-Bromo-A, LCA, IG, SRA, AF2, ADS, 2AD, 8AN, PPU, 3DA, AP7, 45A, A9Z, ANZ, A5O, A5L, 5AA, A43, 2HA AD2, 1 mA, 3mA, 4mA, 6mA, 7mA, 9mA, 6hmA, HAP, m2A, AHAP, 6-DMA, ncm6A and sA; or the at least one ribonucleotide that is substituted by a deoxyribonucleotide version is GTP and the deoxyribonucleotide version is modified by a modification is selected from the group consisting of the following naturally or synthetically occurring ribonucleotide or deoxyribonucleotide modifications m1G, preQ1 , preQO, m7G, m2,7G, m2,2,7G, m22G, m2G I, mi l, m1 lm, Im, Q, oQ, dGdAIQ, mdAnQ, preQO, preQ1 , dG+ , 7-Deaza-G, 2’NH2-dG, 7-Deaza-7-iodo-G, 6-thio-dG, dCaSe, dCaS, Fluoro-G, MANT-G, 2-MANT-G, LCG, BGM, 2SG, GRB, GAO, TG, GDO, GMX, XUG, G3A, GS, G38, 2HG, 1 mG, 2mG, 3mG, m6G, 7mG, 8-oxo-G, ADG, 1 ,N(2)-eG, N(2),3-eG, m22G, m2,7dG, m2,2,7dG, 2’NH2-dG and CEG; or the at least one ribonucleotide that is substituted by a deoxyribonucleotide version is UTP and the deoxyribonucleotide version is modified by a modification is selected from the group consisting of the following naturally or synthetically occurring ribonucleotide or deoxyribonucleotide modifications mlacpS^P, m1 , ges2U, se2U, s2U, acp3D, acp3 acp3U, mS^P, m3U, s4U, chm5U, mchm5U, inm5s2U, inm5U, nm5ges2U, nm5se2U, nm5s2U, nm5U, nchm5U, ncm5s2U, ncm5U, chm5U, cm5s2U, cmnm5s2U, cmnm5ges2U, cmnm5se2U, cmnm5s2U, cmnm5U, cm5U, cnm5s2U, cnm5U, cnm5U, ho5U, mcm5s2U, mcm5U, mo5U, m5s2U, mnm5ges2U, mnm5se2U, mnm5s2U, mnm5U, m5D, m5U, U, tm5s2U, tm5U, D, cmnm5ges2U, gcmnm5s2U, mnm5ges2U, gmnm5s2U, cmo5U, mcmo5U, 2-NH2-dU, 3’-Azido-2’,3’-ddU, Aminoallyl-dU, 5-Propargylamino-dU, 5-Fluoro-dU, 5- dimethyl-2’-dU, 5BU, U37, IU, TLN, BRU, URU, UFB, U5M, UOB, UMO, U5R, B8N, U4M, UAR, UFT, UMS, NYM, MU1 , P1T, M, MU4, putThy, ddT, dTaSe, dTaS, 1mT, 3mT, 04-meT, diHT, 5hmU, 5fU, base J, dhpUra, 5caU, 5-NeOmdU, dU, DHdU, 5-NedU, and dhpdU.

[0025] In one embodiment, in step a) between 1% to 75% of at least one of the ribonucleotides are substituted by a deoxyribonucleotide version of the at least one ribonucleotide.

[0026] In one embodiment, in step a) the composition comprises ATP, CTP, UTP and GTP and wherein between 1% to 75% of CTP are substituted by a deoxyribonucleotide version of CTP.

[0027] In one embodiment, in step a) the composition comprises ATP, CTP, UTP and GTP and wherein between 1% to 75% of CTP are substituted by a deoxyribonucleotide version of CTP selected from the group consisting of dCTP, 5-Methyl-dCTP, 2'-Fluoro-dCTP, 3'-Azido-2',3'-ddCTP, 7-Deaza-dCTP, C8-Alkyne-dCTP, 5-lodo dCTP, 5-Bromo-dCTP, 2'NH2-dCTP, dCTPaSe, dCTPaS, 5- Propargylamino-dCTP, and 5-Hydroxymethyl-dCTP.

[0028] In one embodiment, the template of step b) is DNA complementary to do coding region of a gene, i.e. complementary to an mRNA. In one embodiment, the template also includes a promoter functionally linked to the coding region.

[0029] In one embodiment, the mRNA is saRNA and the saRNA further encodes Replicase (RNA- dependent RNA Polymerase).

[0030] In one embodiment, RNA Polymerase is added before or during step c).

[0031] In a second aspect, the invention relates to mRNA synthesized by the method of the invention. In a third aspect, the invention relates to an mRNA encoding a protein of interest, wherein at least 0.1% of at least one of the ribonucleotides are substituted by a deoxyribonucleotide version of the at least one ribonucleotide.

[0032] In a further aspect, the invention relates to use of the mRNA of the invention in a method of increasing transcription and / or expression of a protein of interest encoded by the mRNA in vitro compared to an mRNA not comprising deoxyribonucleotides.

[0033] In another aspect, the invention also relates to a pharmaceutical composition comprising the mRNA of the invention and optionally at least one excipient.

[0034] In a further aspect, the invention also relates to a vaccine composition comprising the mRNA of the invention.

[0035] In a further aspect, the invention also relates to the mRNA of the invention, the pharmaceutical composition of the invention or the vaccine composition of the invention for use as a medicament.

[0036] In one embodiment, the invention relates to the mRNA, the pharmaceutical composition or the vaccine composition for use in prevention or treatment of a disease.

[0037] In one embodiment, the prevention or treatment of a disease with the mRNA, the pharmaceutical composition or the vaccine composition for use of the invention comprises increased transcription and / or expression of a protein of interest encoded by the mRNA compared to prevention or treatment with an mRNA not comprising deoxyribonucleotides.

[0038] In one embodiment, the protein of interest encoded by the mRNA is transcribed and expressed in immune cells, preferably in dendritic cells.

[0039] Figure Legends

[0040] Figure 1 : 1% Agarose gel of purified mRNA (120V, 40mA, 40min) M, RNA maker (Thermo Scientific™, SM1821), 25%, 25% 5-Methyl-dCTP substituted CTP; 50%: 50% 5-Methyl-dCTP substituted CTP; 75%: 75% 5-Methyl-dCTP substituted CTP; 100%: 100% 5-Methyl-dCTP substituted CTP.

[0041] Figure 2: Effect of different percentage of nucleotide modifications substitution on Flue expression. Expression levels 24 hours after transfection of 200 ng of RNA encoding Flue reporter into HEK293-T cells with lipofectamine 3000. Fluorescence signal is detected by microplate reader (Omega Fluostar). Signal value minus the background value of the negative control. Error bars represent the standard error of mean (SEM) of n = 4 biological replicates. Cells seeded about 2*10A4 per well. 25% substituted: 25% 5-Methyl-dCTP substitution; 50% substituted: 50% 5-Methyl-dCTP substitution; 75% substituted: 75% 5-Methyl-dCTP substitution; 100% substituted: 75% 5-Methyl- dCTP substitution; WT-Fluc: no modification; RLU / S: Relative light units / second.

[0042] Figure 3: Effect of different deoxyribonucleotide modifications substitution on Flue expression.

[0043] Expression levels 24 hours after transfection of 200 ng of RNA encoding Flue reporter into HEK293-T cells with lipofectamine 3000. Luciferase signal is detected by Pierce™ Firefly Luciferase Glow Assay Kit. Error bars represent the standard error of mean (SEM) of n = 2 biological replicates. Cells seeded about 2x104per well. dATP 25%: 25% dATP substitution; dCTP 25%: 25% dCTP substitution; dGTP 25%: 25% dGTP substitution; dTTP 25%: 25% dTTP substitution; d5mCTP 25%: 25% 5-Methyl-dCTP substitution; m1 qj 100%: 100% (m1 qj) substitution; WT: No modification; qj 100%: 100% (qj) substitution; 5mCTP 25%: 25% 5-Methyl-CTP substitution; dCTP+dTTP (25%): 25% dCTP substitution+ 25% dTTP substitution; d5mCTP +dTTP (25%): 25% 5-Methyl-dCTP substitution+ 25% dTTP substitution; m1 qj 25%: 25% m1 qj substitution; qj 25%: 25% qj substitution; RLU / S: Relative light units / second.

[0044] Figure 4: Effect of different deoxyribonucleotide modifications substitution on Flue expression. Expression levels 24 hours after transfection of 200 ng of RNA encoding Flue reporter into 293T cells or A549 with lipofectamine 3000. Luciferase signal is detected by Pierce™ Firefly Luciferase Glow Assay Kit. Error bars represent the standard error of mean (SEM) of n = 3 biological replicates. 293T Cells seeded about 2x104per well. C1 : 25% C8-Alkyne-dCTP; C2: 25% 5-Methyl-dCTP; C3: 25% 2'- Fluoro-dCTP; C4: 25% 5-lodo-d 5% 5-Bromo-dCTP; C6: 25% 2'NH2-dCTP; C7: 25% 3'- Azido-2',3'-ddCTP; C8: 25% dC 25% dCTPaS; C10: 25% 5-Propargylamino-dCTP; C11 : 25% 5-Hydroxymethyl-dCTP; C1 P; WT: No modification; 25%ip: 25% (qj) substitution; 25% m1 qj: 25% (m1 qj) substitution; 1 % (qj) substitution; 100% m1 qj: 100% (m1 qj) substitution;

[0045] RLU / S: Relative light units / seco

[0046] Figure 5: Effect of different deoxyribonucleotide modifications substitution on Flue expression. Expression levels 24 hours after transfection of 400 ng of RNA encoding Flue reporter into 293T cells with lipofectamine 3000. Luciferase signal is detected by Pierce™ Firefly Luciferase Glow Assay Kit. Error bars represent the standard error of mean (SEM) of n = 3 biological replicates. 293T Cells seeded about 4x104per well. Data are shown as mean ± SEM and analyzed using one-way ANOVA with multiple comparisons tests, ns, not significant, *p < 0.05, **p < 0.01 , ***p < 0.001 , as compared with WT. C2: 25% 5-Methyl-dCTP substituted CTP; C3: 25% 2'-Fluoro-dCTP substituted CTP; C7: 25% 3'- Azido-2',3'-ddCTP substituted CTP; C12: 25% dCTP substituted CTP; WT: No modification, using ATP, CTP, UTP and GTP to synthesize; RLU / S: Relative light units / second.

[0047] Figure 6: Effect of nucleotide modifications on saFLUC expression. Expression levels 48 hours (A) or 96 hours (B) after transfection of 200 ng of RNA encoding a self-amplified luciferase reporter in DC2.4 with lipofectamine 3000. Luciferase signal is detected by Pierce™ Firefly Luciferase Glow Assay Kit. Error bars represent the standard error of mean (SEM) of n = 4 biological replicates. Cells seeded about 2.5x104per well. SaFluc-A: 50% N1-Methyl-pseudourin (Jena Bioscience, NU-890L) substitution; SaFluc-B: 50% 5-Methyl-CTP (Jena Bioscience, NU-1138S) substitution; SaFluc-C: 50% N6-Methyl-ATP (Jena Bioscience, NU-1101 S) substitution; SaFluc-D: 50% 5-Methyl-dCTP substitution(Jena Bioscience, NU-1125S); SaFluc-E: 50% N4-Acetyl-CTP substitution(Jena Bioscience, NU-988S); SaFluc-WT: no modification. Flue: Flue mRNA with 100% N1-Methyl- pseudouridine substitution. PBS: negative control; RLU / 5S: Relative light units / 5 seconds.

[0048] Figure 7: Effect of different deoxyribonucleotide modifications substitution on saFLUC Expression. Expression levels 48 hours after transfection of 200 ng of RNA encoding a self-amplified luciferase reporter into HEK293-T cells with lipofectamine 3000. Luciferase signal is detected by Pierce™ Firefly Luciferase Glow Assay Kit. Error bars represent the standard error of mean (SEM) of n = 5 biological replicates. HEK293TCells seeded about 2.5x104per well. dATP 25%: 25% dATP substitution; dCTP 25%: 25% dCTP substitution; dGTP 25%: 25% dGTP substitution; dTTP 25%: 25% dTTP substitution; d5mCTP 25%: 25% 5-Methyl-dCTP substitution; ml i 100%: 100% (ml i ) substitution; WT: No modification; i 100%: 100% (i ) substitution; 5mCTP 25%: 25% 5-Methyl-CTP substitution; dCTP+dTTP (25%): 25% dCTP substitution+ 25% dTTP substitution; d5mCTP +Dttp (25%): 25% 5-Methyl-dCTP substitution+ 25% dTTP substitution; m1 ip 25%: 25% m1 ip substitution; ip 25%: 25% ip substitution; RLU / S: Relative light units / second.

[0049] Figure 8: Effect of different deoxyribonucleotide modifications substitution on saFluc (self- amplified-Fluc) expression. Expression levels 48 hours after transfection of 200 ng of RNA encoding saFluc reporter into 293T cells with lipofectamine 3000. Luciferase signal is detected by Firefly Lucierase HTS assay (MilliporeSigma, Cat.NO.SCT150). Error bars represent the standard error of mean (SEM) of n = 3 biological replicates. 293T Cells seeded about 4x104per well. Data are shown as mean ± SEM and analyzed using one-way ANOVA with multiple comparisons tests. ****p < 0.001 , as compared with WT. A2: 25% 2'-Bromo-dATP substituted ATP; WT: No modification, using ATP, CTP, UTP and GTP to synthesize; RLU / S: Relative light units / second.

[0050] Figure 9: Effect of different deoxyribonucleotide modifications substitution on saFluc (self- amplified-Fluc) expression. Expression levels 48 hours after transfection of 200 ng of RNA encoding saFluc reporter into 293T cells with lipofectamine 3000. Luciferase signal is detected by Firefly Lucierase HTS assay (MilliporeSigma, Cat.NO.SCT150). Error bars represent the standard error of mean (SEM) of n = 3 biological replicates. 293T Cells seeded about 4x104per well. Data are shown as mean ± SEM and analyzed using one-way ANOVA with multiple comparisons tests. ****p < 0.001 , as compared with WT, 25%ip, 25%m1 ip , 100%ip, 100%m1 ip. G3: 25% 7-Deaza-dGTP substituted GTP; 25%ip: 25%ip substituted UTP, 25%m1 ip: 25% m1 ip substituted UTP, 100%ip: 100%ip substituted UTP, 100%m1 ip: 100% m1 ip substituted UTP, WT: No modification, using ATP, CTP, UTP and GTP to synthesize; RLU / S: Relative light units / second.

[0051] Figure 10: Confirmation of Deoxynucleotides Hybridizing to RNA During In Vitro Transcription. Spectroscopic properties of 25%,50%,75%-Mant-dATP or 25%,50%,75%- Mant-dGTP-incorporated mRNA. The concentration of all RNA candidates is approximately 500 ng / pL, with each sample being repeated three times. The values shown in the figure represent the average of the three repetitions.

[0052] Figure 11 : Effect of different percentage deoxynucleotide substitutions on Flue expression. Firefly luciferase (Flue) expression levels measured 24 hours after transfection of 100 ng RNA encoding the Flue reporter into DC2.4 cells using Lipofectamine™ RNAiMAX. Luciferase activity was quantified using the Pierce™ Firefly Luciferase Glow Assay Kit. X axis indicates % substitution of deoxyribonucleotide for respective ribonucleotide. Error bars indicate the standard error of the mean (SEM) from n = 3 biological replicates. Approximately 4 x 104cells were seeded per well. None: Unmodified RNA (no deoxynucleotide substitutions). Figure 12: Optimal deoxynucleotide substitution percentage for enhancing Flue expression. This figure identifies the most effective percentage of deoxynucleotide substitution that maximizes firefly luciferase (Flue) expression. Firefly luciferase (Flue) expression levels measured 24 hours after transfection of 100 ng LNP-RNA encoding the Flue reporter into DC2.4 cells. Luciferase activity was quantified using the Pierce™ Firefly Luciferase Glow Assay Kit. Error bars indicate the standard error of the mean (SEM) from n = 3 replicates. Approximately 4 x 104cells were seeded per well. X axis indicates % substitution of deoxyribonucleotide for respective ribonucleotide. Mix: 5%-dATP , 5%- dTTP, 25%-dCTP and 25%-dGTP substituted. None: Unmodified RNA (no deoxynucleotide substitutions).

[0053] Figure 13: In vivo evaluations of the translation profiles of optimized deoxynucleotide substitution mRNA. Mice were intramuscularly injected with equal doses (5ug / mouse) of Flue mRNA containing various deoxynucleotide substitution patterns: unmodified (None), 5% dATP, 5% dTTP, 25% dCTP, 25% dGTP substitutions, 25% 5-Methyl-dCTP substitution and Mix(5% dATP, 5% dTTP, 25% dCTP and 25% dGTP mix substitutions). At 6 hours post-injection, in vivo bioluminescence imaging was performed to assess Flue expression. Mice were anesthetized with isoflurane and injected intraperitoneally with D-luciferin (30 mg / mouse). After 5 minutes, the luminescence signal was captured and quantified using the IVIS Lumina Series III imaging system (PerkinElmer). Bioluminescence quantitative analyses of the signal intensities are shown. Data represent mean ± SEM from n = 3 mice per group.

[0054] Figure 14: Optimal deoxynucleotide substitution percentage for enhancing saFluc expression. Self-amplified-Fluc expression levels measured 24 hours after transfection of 100 ng LNP-RNA encoding the Flue reporter into DC2.4 cells or A549 cells. Luciferase activity was quantified using the Pierce™ Firefly Luciferase Glow Assay Kit. Error bars indicate the standard error of the mean (SEM) from n = 3 replicates. Approximately 4 x 104DC2.4 cells or 2.5x 104A549 cells were seeded per well. WT: Unmodified self-amplified-RNA (no deoxynucleotide substitutions). 5%-dATP : 5% dATP substitution, 25%-dCTP : 25% dCTP substitution, 25%-dGTP : 25% dGTP substitution, 5%-dTTP : 5% dTTP substitution , Mix: 5% dATP, 5% dTTP, 25% dCTP and 25% dGTP mix substitutions, 5%- hydroxy-dATP: 5% hydroxy dATP substitution.

[0055] Figure 15: In vivo evaluations of the translation profiles of optimized deoxynucleotide substitution mRNA. Mice were intramuscularly injected with equal doses (5ug / mouse) of saFluc mRNA containing various deoxynucleotide substitution patterns: unmodified (WT), 5% dATP. At 6 hours post-injection, in vivo bioluminescence imaging was performed to assess Flue expression. Mice were anesthetized with isoflurane and injected intraperitoneally with D-luciferin (30 mg / mouse). After 5 minutes, the luminescence signal was captured and quantified using the IVIS Lumina Series III imaging system (PerkinElmer). Bioluminescence quantitative analyses of the signal intensities are shown. Data represent mean ± SEM from n = 3 mice per group.

[0056] Figure 16: Effect of different percentage of 2’-Hydroxy-dATP substitutions on saFluc expression. Self-amplified-Fluc expression levels measured 24 hours after transfection of 100 ng RNA encoding the Flue reporter into DC2.4 cells using Lipofectamine™ RNAiMAX. Luciferase activity was quantified using the Pierce™ Firefly Luciferase Glow Assay Kit. Error bars indicate the standard error of the mean (SEM) from n = 3 replicates. Approximately 4 x 104cells were seeded per well. WT: Unmodified self-amplified-RNA (no deoxynucleotide substitutions). A7-5%: 5% 2’-Hydroxy dATP substitution, A7-10%: 10% 2’-Hydroxy dATP substitution, A7-15%: 15% 2’-Hydroxy dATP substitution, A7-25%: 25% 2’-Hydroxy dATP substitution, A7-50%: 50% 2’-Hydroxy dATP substitution, A7-75%: 75% 2’-Hydroxy dATP substitution.

[0057] DETAILED DESCRIPTION OF THE INVENTION

[0058] Definitions

[0059] Before the invention is described in detail with respect to some of its preferred embodiments, the following general definitions are provided.

[0060] The present invention as illustratively described in the following may suitably be practiced in the absence of any element or elements, limitation or limitations, not specifically disclosed herein.

[0061] The present invention will be described with respect to particular embodiments and with reference to certain figures but the invention is not limited thereto but only by the claims.

[0062] Where the term “comprising” is used in the present description and claims, it does not exclude other elements. For the purposes of the present invention, the term “consisting of” is considered to be a preferred embodiment of the term “comprising of’. If hereinafter a group is defined to comprise at least a certain number of embodiments, this is also to be understood to disclose a group which preferably consists only of these embodiments.

[0063] For the purposes of the present invention, the term “obtained” is considered to be a preferred embodiment of the term “obtainable”. If hereinafter e.g. a compound is defined to be obtainable from a specific source, this is also to be understood to disclose a compound which is obtained from this source.

[0064] Where an indefinite or definite article is used when referring to a singular noun, e.g. “a”, “an” or “the”, this includes a plural of that noun unless something else is specifically stated. The terms “about” or “approximately” in the context of the present invention denote an interval of accuracy that the person skilled in the art will understand to still ensure the technical effect of the feature in question. The term typically indicates deviation from the indicated numerical value of ±10%, and preferably of ±5%.

[0065] Technical terms are used by their common sense. If a specific meaning is conveyed to certain terms, definitions of terms will be given in the following in the context of which the terms are used.

[0066] As described herein "nucleoside" is defined as a compound containing a five-carbon sugar molecule (a pentose or ribose) or derivative thereof, and an organic base, purine or pyrimidine, or a derivative thereof.

[0067] As described herein, "nucleotide" is defined as a nucleoside further comprising a phosphate group. The nucleosides and nucleotides described herein are generally chemically modified on the major groove face. In some embodiments, the major groove chemical modifications can include an amino group, a thiol group, an alkyl group, or a halo group.

[0068] As used herein, the term "alkyl" is meant to refer to a saturated hydrocarbon group which is straight- chained or branched. Example alkyl groups include methyl (Me), ethyl (Et), propyl (e.g., n-propyl and isopropyl), butyl (e.g., n-butyl, isobutyl, t-butyl), pentyl (e.g., n-pentyl, isopentyl, neopentyl), and the like. An alkyl group can contain from 1 to about 20, from 2 to about 20, from 1 to about 12, from 1 to about 8, from 1 to about 6, from 1 to about 4, or from 1 to about 3 carbon atoms.

[0069] As used herein, "halo" or "halogen" includes fluoro, chloro, bromo, and iodo.

[0070] The present disclosure provides for modified nucleosides and nucleotides. The term “nucleotide”, referring to the building blocks of ribonucleic acid (RNA) or deoxyribonucleic acid (DNA), may be used interchangeably to refer to the molecules used in RNA or DNA synthesis (i.e. nucleoside triphosphates, NTP / dNTP), as well as the components of an RNA or DNA polynucleotide (i.e. nucleoside monophosphates).

[0071] When performing a method of mRNA synthesis, nucleotides (ribo- or deoxyribonucleotides) are provided in the reaction mixture (composition) as triphosphates, i.e. NTPs (ATP, GTP, UTP and CTP) or dNTPs (dATP, dGTP, dUTP / dTTP, dCTP). Hence, nucleotides in the composition are referred to as “NTPs” or “dNTPs”. Once synthesized, RNA or DNA is made up of monophsphates, i.e. NMPs / dNMPs. When referring to modified nucleotides, the modification is usually a modification of the base (A, G, U, T, C) and does not affect the phosphate. Hence, before the RNA is synthesized, the modified nucleobase will be provided to the reaction as a triphosphate, for example m5CTP. In the synthesized RNA strand, it will occur as a monophosphate, m5CMP. The modified nucleobase is m5C in either case, therefore, when referring to a nucleotide modification, it is not distinguished between triphosphates and monophosphates throughout the application. When referring to an NTP / dNTP with a modification, this automatically also includes the NMP / dNMP of this modified nucleotide. Modified nucleotides in the monophosphate, diphosphate and triphosphate configuration are comprised in each embodiment.

[0072] A “ribonucleotide” as used herein specifically refers to a nucleotide containing ribose as its pentose component. A “deoxyribonucleotide” as used herein specifically refers to a nucleotide containing deoxyribose as its pentose component. Instead of a hydroxyl group at the second carbon in the ribose ring, deoxyribose carries a hydrogen atom at this position.

[0073] When referring to nucleoside modifications, the terms “nucleoside” and “nucleotide” may sometimes be used interchangeably, as far as the modification does not relate to the phosphate group.

[0074] The term “substituted” as used herein refers to the change of one source of nucleotide to a different variant of this nucleotide. The nucleotides used to synthesize mRNA in an unsubstituted composition are ATP, CTP, UTP and GTP. According to the present invention, instead of providing ATP in the composition, a mixture of 99% ATP and 1% dATP may be provided, this would equate to a 1 % substitution. Accordingly, the composition may comprise a mixture of 75% ATP and 25% dATP, this would equate to a 25% substitution. Furthermore, according to the invention, the composition may comprise a mixture of 75% ATP and 25% modified dATP. As used herein, when referring, for example, to “at least 1 % substitution” or “10% to 50%”, this number always refers to the dNTP variant, and the remaining amount of the nucleotide up to 100% is made up with the unmodified NTP variant (i.e. one of ATP, CTP, UTP and GTP). Preferably, one of ATP, CTP, UTP and GTP is substituted. In some embodiments, two or more of ATP, CTP, UTP and GTP may be substituted. In some embodiments, all of at least one of ATP, CTP, UTP and GTP are substituted, in this case, this ribonucleotide is no longer present. UTP may be substituted by both dUTP and dTTP.

[0075] The term “template” or “nucleic acid template” as used herein refers to a nucleic acid sequence encoding at least one gene of interest. The template according to the invention is a nucleic acid sequence readable by a RNA Polymerase and is transcribed into RNA, preferably mRNA or saRNA. The template comprises a coding sequence, and may optionally further comprise other nucleic acid sequences. Other nucleic acid sequences may include untranslated regions or other non-coding sequences. The template may also include additional genes, including further gene of interest coding regions or other proteins. Other proteins encoded by the template may include enzymes, such as enzymes included in mRNA synthesis. Such enzymes include RNA Polymerase, Replicase, transcription factors, or Capping Enzyme.

[0076] The term “complementary DNA” as used herein refer to DNA that is complementary to an mRNA or saRNA, i.e. it resembles the coding region of a gene without any intronic DNA sequences. The complementary DNA used herein is part of a plasmid, i.e. it is double standed DNA. Usually, the plasmid further comprises a promoter functionally linked to the coding DNA. Preferably, a T7 promoter is linked to the DNA comprising the coding region.

[0077] The term “mRNA” as used herein refers to messenger ribonucleic acid molecules. This may be a polymeric form of nucleotides (polynucleotide) of any length, including deoxyribonucleotides or ribonucleotides, or analogs thereof. An mRNA sequence is typically composed of a specific sequence of four nucleotide bases: adenine (A); cytosine (C); guanine (G); and uracil (U). Thus, the term polynucleotide sequence or nucleic acid sequence is the alphabetical representation of a polynucleotide molecule. The mRNA of the invention may comprise modified nucleotides, such as methylated or capped nucleotides and nucleotide analogs, which may be present in the form of deoxyribonucleotides. If present, modifications to the nucleotide structure may be imparted before or after assembly of the polymer. The term “mRNA” as used herein includes saRNA.

[0078] The term “conditions that allow mRNA synthesis” as used herein refers to conditions under which a nucleic acid template is transcribed into mRNA. Hence, mRNA synthesis means transcription.

[0079] The term “natural” when referring to nucleotide modifications means that the modification has been identified in nature, in any kind of DNA or RNA and in any kind of organisms. The majority of naturally occurring nucleotide modifications occurs in RNAs. Such naturally occurring modifications are published in the RNA Modification Database (described in Cantara et al. 2011 (Nucleic Acids Res . 2011 ;39 D195-201), which can be accessed at http: / / rna.rega.kuleuven.be / rnamods / ). Naturally occurring modifications also include natural non-canonical bases, or “nucleotide analogs”, such as pseudouridine, dihydrouridine, queuosine, wyosine or inosine. Naturally occurring modification in this case refers to modifications that occur naturally in RNAs, i.e. the term refers to modifications that typically involve ribonucleotides. However, when presently referring to modifications, it is also referred to the deoxyribonucleotide variant of any given modified ribonucleotide.

[0080] The term “synthetic” nucleotide modifications refers to modified nucleotides that do not occur in nature. In some embodiments, the nucleotide can be modified on the major groove face and can include replacement of the hydrogen on C-5 of pyrimidines or C-6 of purines with a methyl group or a halo group. As used herein, "halo" or "halogen" includes fluoro, chloro, bromo, and iodo. Synthetic nucleotide modifications are published in the database Modomics, which can be accessed at https: / / iimcb.genesilico.pl / modomics / modifications (Cappannini et al. 2023, Nucleic Acids Research, https: / / doi.Org / 10.1093 / nar / gkad1083).

[0081] The term “self-amplifying RNA” or “saRNA” as used herein refers to a genetically engineered RNA derived from self-replicating single-stranded RNA viruses. Positive-sense alphavirus genomes that have been commonly used for saRNA vaccine design include the Venezuelan equine encephalitis virus (VEE), Sindbis virus (SINV), and Semliki forest virus (SFV). The alphavirus replicase genes encode an RNA-dependent RNA polymerase (RdRP) complex which amplifies synthetic transcripts in situ. The protein of interest, which may be an antigenic or therapeutic sequence, is expressed at high levels as a separate entity and further proteolytic processing of the immunogen is not required. As a result of their self-replicative activity, saRNAs can be delivered at lower concentrations than conventional mRNA to achieve comparable GOI expression. Self-amplifying RNA typically encodes 5' and 3' conserved sequence element (CSE) sequences, the nsP1-4 genes (non-structural protein 1-4), a subgenomic promoter, and the POI. Following in situ translation, the nsP1-4 proteins form an RdRP complex which recognizes flanking CSE sequences and amplifies POI-encoding transcripts. As used herein, the saRNA is encompassed by the term “mRNA”, since it comprises all the features of an mRNA.

[0082] As used herein, “isolated” refers to a nucleic acid molecule or a nucleic acid sequence that has been substantially separated, produced apart from, or purified away from other biological components in the cell or tissue of an organism in which the component occurs, such as other cells, chromosomal and extrachromosomal DNA and RNA, and proteins. Nucleic acids and proteins that have been “isolated” include nucleic acids and proteins purified by standard purification methods. The term also embraces nucleic acids and proteins prepared by recombinant expression in a host cell as well as chemically synthesized nucleic acids and proteins. Isolated proteins or nucleic acids, or cells containing such, in some examples are at least 50% pure, such as at least 75%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 100% pure.

[0083] A "cap" or a "cap nucleotide" means a nucleoside-5 -triphosphate that, under suitable reaction conditions, is used as a substrate by a capping enzyme system and that is thereby joined to the 5 '- end of an uncapped RNA comprising primary RNA transcripts or RNA having a 5'- diphosphate. The nucleotide that is so joined to the RNA is also referred to as a "cap nucleotide" herein. A "cap nucleotide" is a guanine nucleotide that is joined through its 5' end to the 5' end of a primary RNA transcript. The RNA that has the cap nucleotide joined to its 5' end is referred to as "capped RNA" or "capped RNA transcript" or "capped transcript." A common cap nucleoside is 7-methylguanosine or N7 -methylguanosine (sometimes referred to as "standard cap"), which has a structure designated as "m7 G," in which case the capped RNA or "m7 G-capped RNA" has astructure designated as m G(5')ppp(5')Ni(pN)x -OH(3'), or more simply, as m GpppNi(pN)x or m7 G[5']ppp[5']N, wherein m7 G represents the 7-methylguanosine cap nucleoside, ppp represents the triphosphate bridge between the 5' carbons of the cap nucleoside and the first nucleotide of the primary RNA transcript, Ni(pN)x - OH(3') represents the primary RNA transcript, of which Ni is the most 5 '-nucleotide, "p" represents a phosphate group, "G" represents a guanosine nucleoside, "m7 " represents the methyl group on the 7-position of guanine, and "[5']" indicates the position at which the "p" is joined to the ribose of the cap nucleotide and the first nucleoside of the mRNA transcript ("N"). In addition to this "standard cap," a variety of other naturally- occurring and synthetic cap analogs are known in the art. RNA that has any cap nucleotide is referred to as "capped RNA." The capped RNA can be naturally occurring from a biological sample or it can be obtained by in vitro capping of RNA that has a 5' triphosphate group or RNA that has a 5' diphosphate group with a capping enzyme system (e.g., vaccinia capping enzyme system or Saccharomyces cerevisiae capping enzyme system). Alternatively, the capped RNA can be obtained by in vitro transcription (IVT) of a DNA template that contains an RNA polymerase promoter, wherein, in addition to the GTP, the IVT reaction also contains a dinucleotide cap analog (e.g., a m7 GpppG cap analog or an N7 -methyl, 2'-0-methyl-GpppG ARCA cap analog or an N7 -methyl, 3'-0-methyl-GpppG ARCA cap analog) using methods known in the art (e.g., using an AMPLICAP™ T7 capping kit or a MESSAGEMAX™ T7 ARCA-CAPPED MESSAGE Transcription Kit, EPICENTRE, CleanCap® AG reagent (e.g. m7G(5')ppp(5')(2'OMeA)pG) or CleanCAP® AU reagent (e.g. m7G(5')ppp(5')(2'OMeA)pU) or CellScript.

[0084] A “pharmaceutically acceptable carrier” as used herein is essentially non-toxic to recipients at the dosages and concentrations employed and is compatible with other ingredients of the formulation. A pharmaceutically acceptable carrier will not inhibit otherwise adversely affect the function of the agent according to the present invention. Suitable carriers include, but are not limited to water, dextrose, glycerol, saline, ethanol, lipid and any combination thereof. The carrier can contain additional agents such as wetting or emulsifying agents, pH buffering agents, or adjuvants, which enhance the effectiveness of the formulation.

[0085] Suitable carriers include lipid nanoparticle, lipid, cholesterol, peptides, a solvent, aqueous solvent, non-aqueous solvent, polymer, dispersion media, diluent, dispersion, suspension aid, surface active agent, isotonic agent, thickening or emulsifying agent, protein, poloxamine, cell, core-shell nanoparticles, lipoplex, liposome, lipoplex peptide, lipidplex, commonly used inorganic nanoparticles (including gold nanoparticles, iron oxide nanoparticles and mesoporous silica nanoparticles (MSNs)), hyaluronidase, preservative, poloxamer, chitosan, monosaccharides, disaccharides, polysaccharides, cationic lipid (dilinoleylmethyl-4-dimethylaminobutyrate (DLin-MC3-DMA)), phospholipid (1 ,2-distearoyl-sn-glycero-3-phosphocholine (DSPC)), PEG lipid (1 ,2-dimyristoyl-sn- glycerol, methoxypolyethylene glycol and polyethylene glycol-dimyristoyl glycerol (PEG-DMG)), PEG, linear and branched polyethylenimine (PEI), poly(P-amino ester), polyamidoamine (PAMAM) dendrimer, and mixtures thereof. As used herein, the term “excipient” refers to an inert substance which is commonly used as a diluent, vehicle, preservative, binder, or stabilizing agent, and includes, but is not limited to, proteins (e.g., serum albumin, etc.), amino acids (e.g., aspartic acid, glutamic acid, lysine, arginine, glycine, histidine, etc.), fatty acids and phospholipids (e.g., alkyl sulfonates, caprylate, etc.), surfactants (e.g., SDS, polysorbate, nonionic surfactant, etc.), saccharides (e.g., sucrose, maltose, trehalose, etc.) and polyols (e.g., mannitol, sorbitol, etc.).

[0086] The words “treat” or “treating” or “treatment” include preventative treatment, that is, treatment directed to minimizing or partially or completely inhibiting the development of the associated disease, pathological condition, or disorder, such as for example vaccines; curative treatment, that is, cure an disease, pathological condition, or disorder with the goal of a full recovery that includes an acceptable quality of life; palliative treatment, that is, treatment designed for the relief of symptoms rather than the curing of the disease, pathological condition, or disorder; and supportive treatment, that is, treatment employed to supplement another specific therapy directed toward the improvement of the associated disease, pathological condition, or disorder. In an aspect, the terms cover any treatment of a subject, including a mammal (e.g., a human), and includes: (i) preventing the undesired physiological change, disease, pathological condition, or disorder from occurring in a subject that can be predisposed to the disease but has not yet been diagnosed as having it; (ii) inhibiting the physiological change, disease, pathological condition, or disorder, i.e., arresting its development; or (iii) relieving the physiological change, disease, pathological condition, or disorder, i.e., causing regression of the disease.

[0087] For example, treating a disease or disorder can reduce the severity of an established disease, pathological condition, or disorder in a subject by 1 %-100% as compared to a control. In one embodiment, treatment can refer to a 1 %, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% reduction in the severity of a disease, pathological condition, or disorder (such as a genetic disease or disorder). For example, treating a disease, pathological condition, or disorder can reduce one or more symptoms of a disease, pathological condition, or disorder in a subject by 1 %-100% as compared to a control. In one embodiment, treatment can refer to 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% reduction of one or more symptoms of an established a disease, pathological condition, or disorder. It is understood that treatment does not necessarily refer to a cure or complete ablation or eradication of a disease, pathological condition, or disorder. However, treatment can also refer to a cure or complete ablation or eradication of a disease, pathological condition, or disorder.

[0088] An "individual" or "subject" treated in accordance with this invention refers to vertebrates, particularly members of a mammalian species, and includes but is not limited to domestic animals, sports animals, and primates, including humans. In one embodiment, the subject treated in accordance with this invention is a mammal. In one embodiment, the subject treated in accordance with this invention is a human. In another embodiment, the subject treated in accordance with this invention is a nonhuman mammal.

[0089] “Wild-type”, “control” or “reference” gene expression, mRNA or protein levels are determined by a control sample, cell or organisms, or by averaging the expression levels from multiple control samples, cells or organisms. In relation to the present invention, a “control” or “reference” mRNA is an mRNA consisting solely of ribonucleotides, without any deoxyribonucleotide substitutions.

[0090] The term "innate immune response" includes a cellular response to exogenous nucleic acids, including single stranded nucleic acids, generally of viral or bacterial origin, which involves the induction of cytokine expression and release, particularly the interferons, and cell death. Protein synthesis is also reduced during the innate cellular immune response. While it is advantageous to eliminate the innate immune response in a cell, the present disclosure provides substituted mRNAs that substantially reduce the immune response, including interferon signaling, without entirely eliminating such a response. In some embodiments, the immune response is reduced by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, 99.9%, or greater than 99.9% as compared to the immune response induced by a corresponding unsubstituted mRNA. Such a reduction can be measured by expression or activity level of Type 1 interferons or the expression of interferon- regulated genes such as the toll-like receptors (e.g., TLR7 and TLR8). Reduction of innate immune response can also be measured by decreased cell death following one or more administrations of substituted mRNAs to a cell population; e.g., cell death is 10%, 25%, 50%, 75%, 85%, 90%, 95%, or over 95% less than the cell death frequency observed with a corresponding unsubstituted mRNA. Moreover, cell death may affect fewer than 50%, 40%, 30%, 20%, 10%, 5%, 1%, 0.1%, 0.01% or fewer than 0.01% of cells contacted with the substituted mRNAs.

[0091] Nucleic acids for use in accordance with the present disclosure may be prepared according to any available technique including, but not limited to chemical synthesis, enzymatic synthesis, which is generally termed in vitro transcription, enzymatic or chemical cleavage of a longer precursor, etc.

[0092] The modified nucleosides and nucleotides disclosed herein can be prepared from readily available starting materials using the following general methods and procedures. It is understood that where typical or preferred process conditions (i.e., reaction temperatures, times, mole ratios of reactants, solvents, pressures, etc.) are given; other process conditions can also be used unless otherwise stated. Optimum reaction conditions may vary with the particular reactants or solvent used, but such conditions can be determined by one skilled in the art by routine optimization procedures.

[0093] The processes described herein can be monitored according to any suitable method known in the art. For example, product formation can be monitored by spectroscopic means, such as nuclear magnetic resonance spectroscopy (e.g., 1 H or 13 C) infrared spectroscopy, spectrophotometry (e.g., UV-visible), or mass spectrometry, or by chromatography such as high performance liquid chromatography (HPLC) or thin layer chromatography.

[0094] Preparation of modified nucleosides and nucleotides can involve the protection and deprotection of various chemical groups. The need for protection and deprotection, and the selection of appropriate protecting groups can be readily determined by one skilled in the art.

[0095] The reactions of the processes described herein can be carried out in suitable solvents, which can be readily selected by one of skill in the art of organic synthesis. Suitable solvents can be substantially nonreactive with the starting materials (reactants), the intermediates, or products at the temperatures at which the reactions are carried out, i.e., temperatures which can range from the solvent's freezing temperature to the solvent's boiling temperature. A given reaction can be carried out in one solvent or a mixture of more than one solvent. Depending on the particular reaction step, suitable solvents for a particular reaction step can be selected.

[0096] Resolution of racemic mixtures of modified nucleosides and nucleotides can be carried out by any of numerous methods known in the art. An example method includes fractional recrystallization using a "chiral resolving acid" which is an optically active, salt-forming organic acid. Suitable resolving agents for fractional recrystallization methods are, for example, optically active acids, such as the D and L forms of tartaric acid, diacetyltartaric acid, dibenzoyltartaric acid, mandelic acid, malic acid, lactic acid or the various optically active camphorsulfonic acids. Resolution of racemic mixtures can also be carried out by elution on a column packed with an optically active resolving agent (e.g., dinitrobenzoylphenylglycine). Suitable elution solvent composition can be determined by one skilled in the art. Modified nucleic acids need not be uniformly modified along the entire length of the molecule. Different nucleotide modifications and / or backbone structures may exist at various positions in the nucleic acid. One of ordinary skill in the art will appreciate that the nucleotide analogs or other modification(s) may be located at any position(s) of a nucleic acid such that the function of the nucleic acid is not substantially decreased. A modification may also be a 5’ or 3' terminal modification.

[0097] The term “patient,” “target” or “subject” refers to a mammal and includes human and veterinary targets.

[0098] The term “carrier” refers to a diluent, adjuvant, excipient or vehicle with which the compound is administered. Such a pharmaceutical carrier may be a sterile liquid such as water, and oil, including petroleum, animal oil, vegetable oil, or oil of synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, and the like. Water or an aqueous solution, a saline solution, and aqueous dextrose and glycerol solutions are preferably introduced as the carrier, particularly a carrier for injectable solutions. Optionally, the carrier may be a carrier for solid formulations, including one or more selected from a binder (in the case of a compressed pill), a glidant, an encapsulating agent, a flavoring agent, and a coloring agent, but the present invention is not limited thereto.

[0099] Method of mRNA synthesis

[0100] In a first aspect, the invention provides a method of synthesizing an mRNA in vitro, comprising the steps of: a) providing a composition comprising ribonucleotides, wherein between 0.1% to 100% of at least one of the ribonucleotides are substituted by a deoxyribonucleotide version of the at least one ribonucleotide; b) adding a nucleic acid template encoding a protein of interest to the composition; and c) incubating the composition under conditions that allow mRNA synthesis. It is understood throughout the description that the term “mRNA” also includes self-amplifying RNA (saRNA). Hence, in one embodiment, the mRNA is an saRNA.

[0101] In one embodiment, the composition comprises ATP, CTP, UTP and GTP. In one particular embodiment, the four nucleotides are available in equimolar ratio, i.e. 1 :1 :1 :1 . This ratio includes both the ribonucleotide and the substituted deoxyribonucleotide version. Hence, the composition preferably comprises an equimolar ratio of (ATP + dATP and / or modified dATP):(CTP + dCTP and / or modified dCTP):(UTP + dUTP and / or modified dUTP and / or dTTP and / or modified dTTP):(ATP + dGTP and / or modified dGTP).

[0102] In one embodiment, the composition comprises between 2.5mM and 10mM of each one of ATP, CTP, UTP and GTP with a molar ratio of 1 :1 :1 :1 . Preferably, the composition comprises between 5mM and 10mM of each one of ATP, CTP, UTP and GTP with a molar ratio of 1 : 1 : 1 : 1 . More preferably, the composition comprises between 7mM and 8mM of each one of ATP, GTP, UTP and CTP with a molar ratio of 1 : 1 : 1 : 1 .

[0103] In one embodiment, the composition further comprises a reaction buffer suitable for mRNA synthesis. The skilled person is aware of such buffers. In one embodiment, the reaction buffer comprises Tris buffer. In one embodiment, the reaction buffer comprises spermidine. In one embodiment, the reaction buffer comprises magnesium.

[0104] In one embodiment, the composition comprises DTT (Dithiothreitol). In a particular embodiment, the composition comprises between 1 and 10mM DTT, preferably between 4 and 6mM DTT, most preferably 5mM DTT.

[0105] In one embodiment, the method comprises a step of co-transcriptional or post-transcriptional capping.

[0106] In one embodiment, step c) further comprises a step of co-transcriptional capping.

[0107] In one embodiment, the composition comprises a capping agent for co-transcriptional capping of mRNA. In one embodiment, co-transcriptional capping comprises the addition of an anti-reverse cap analog (ARCA) or m7G(5')ppp(5')(2'OMeA)pG. In a preferred embodiment, co-transcriptional capping comprises the addition of m7G(5')ppp(5')(2'OMeA)pG.

[0108] In another embodiment, the method comprises a further step of post-transcriptional capping after initiation of step c) or after completion of step c). In one embodiment, post-transcriptional capping comprises the addition of a capping enzyme and optionally further an mRNA Cap-O- Methyltransferase.

[0109] In one embodiment, the nucleic acid template is a PCR product. In another embodiment, the nucleic acid template is a linearized plasmid. In another embodiment, the nucleic acid template is a synthesized double strand nucleic acid.

[0110] In one embodiment, step c) comprises a step of heating the composition. The exact temperature conditions depend on the activity of the RNA polymerase and can be chosen by the skilled person based on available information, such as the manufacturer’s instructions for commercially available RNA polymerases.

[0111] In one embodiment, step c) comprises a step of heating the composition to a temperature between 30°C and 60°C. Preferably, the composition is heated to a temperature between 30°C and 45°C. Most preferably, composition is heated to a temperature between 35°C and 40°C.

[0112] In another embodiment, step c) comprises a step of intermixing the compositing by continuous shaking. The skilled person is aware that intermixing and heating steps can be performed, for example, using a thermocycler. Alternatively, the heating steps can be performed in a PCR instrument without shaking during the reaction. In this case, intermixing can be achieved by shaking between incubation steps, e.g. by vortexing.

[0113] In another embodiment, step c) comprises incubating the composition for at least 10 minutes. In another embodiment, step c) comprises incubating the composition for at least 30 minutes, at least 60 minutes, at least 90 minutes, at least 120 minutes, or at least 180 minutes. The reaction will eventually slow because of the loss of polymerase activity over time. Hence, an incubation for more than 180 minutes is not necessary.

[0114] In one embodiment, the method further comprises a step of adding DNase after step c), such as DNAse I. In one embodiment, the composition is incubated for an additional 10 to 60 minutes in the presence of DNasel.

[0115] The skilled person is aware that the method can be interrupted and the composition can be stored at between 4°C and -80°C between each step.

[0116] The method may further comprise a step of mRNA purification and / or concentration. In one embodiment, mRNA purification and / or concentration is performed after mRNA transcription. mRNA purification and / or concentration methods are known to the skilled person and may include chromatography methods such as reversed phase, ion exchange (I EX), size exclusion (SEC), hydrophobic interaction (HIC), and affinity chromatography; or may include precipitation by addition of sodium acetate or litihum chloride followed by addition of ethanol or isopropanol.

[0117] Absorbance measurements made on a spectrophotometer can provide an indication of mRNA purity. Hence, in one embodiment, the method further comprises measuring absorbance at 260nm, 280nm and 230nm on a spectrophotometer.

[0118] In one embodiment, the ratio of absorbance at 260nm and 280nm (260 / 280nm) of the mRNA is at least 1 .8. Preferably, the ratio of absorbance at 260nm and 280nm of the mRNA is at least 1 .95. In one particular embodiment, the ratio of absorbance at 260nm and 280nm (260 / 280nm) of the mRNA is between 1 .95 and 2.2.

[0119] In another embodiment, the ratio of absorbance at 260nm and 230nm (260 / 230nm) of the mRNA is at least 2.0. Preferably, the ratio of absorbance at 260nm and 230nm (of the mRNA is at least 2.2. In one particular embodiment, the ratio of absorbance at 260nm and 230nm (260 / 230nm) of the mRNA is between 2.2 and 2.8.

[0120] The method of the invention may also be used for the synthesis of other types of RNA, including untranslated RNA.

[0121] Hence, in one embodiment, the invention also provides a method of synthesizing an RNA in vitro, comprising the steps of: a) providing a composition comprising ribonucleotides, wherein between 0.1% to 100% of at least one of the ribonucleotides are substituted by a deoxyribonucleotide version of the at least one ribonucleotide; b) adding a nucleic acid template to the composition; and c) incubating the composition under conditions that allow RNA synthesis.

[0122] In one embodiment, the RNA is selected from the group consisting of ribosomal RNA (rRNA), transfer RNA (tRNA), circular RNA (circRNA), small nucleolar RNA (snoRNA), micro RNA (miRNA), small interfering RNA (siRNA), small hairpin RNA (shRNA) and piwi-interacting RNA (piRNA). In a preferred embodiment, the RNA is selected from the group consisting of circRNA, miRNA, siRNA, shRNA and piRNA.

[0123] In one embodiment, the RNA is an untranslatable RNA.

[0124] In one embodiment, the mRNA or saRNA is synthesized by in vitro transcription (IVT).

[0125] In one embodiment, the method does not comprise a step of adding a ligase.

[0126] Substitution of ribonucleotides in the composition used in the method of the invention

[0127] In one embodiment, 0.1% to 90% of at least one of the ribonucleotides of the composition are substituted by a deoxyribonucleotide version of the at least one ribonucleotide. In one embodiment, 0.1% to 85%, 0.1 % to 80%, 0.1% to 75%, 0.1% to 70%, 0.1 % to 65%, 0.1% to 60%, 0.1% to 55%,

[0128] 0.1% to 50%, 0.1 % to 45%, 0.1% to 40%, 0.1% to 35%, 0.1 % to 30%, 0.1% to 25%, 0.1% to 20%,

[0129] 0.1% to 15%, 0.1 % to 10%, 0.1% to 5% or 0.1% to 1% of at least one of the ribonucleotides are substituted by a deoxyribonucleotide version of the at least one ribonucleotide.

[0130] In another embodiment, 1% to 100% of at least one of the ribonucleotides of the composition are substituted by a deoxyribonucleotide version of the at least one ribonucleotide. In one embodiment, 1 % to 90%, 1 % to 85%, 1 % to 80%, 1% to 75%, 1% to 70%, 1% to 65%, 1% to 60%, 1% to 55%, 1 % to 50%, 1% to 45%, 1% to 40%, 1% to 35%, 1% to 30%, 1 % to 25%, 1 % to 20%, 1 % to 15%, 1% to 10%, or 1 % to 5% of at least one of the ribonucleotides are substituted by a deoxyribonucleotide version of the at least one ribonucleotide.

[0131] In another embodiment, 5% to 75% of at least one of the ribonucleotides of the composition are substituted by a deoxyribonucleotide version of the at least one ribonucleotide. In one embodiment, 5% to 70%, 5% to 65%, 5% to 60%, 5% to 55%, 5% to 50%, 5% to 45%, 5% to 40%, 5% to 35%, 5% to 30%, 5% to 25%, 5% to 20%, 5% to 15%, or 5% to 10% of at least one of the ribonucleotides are substituted by a deoxyribonucleotide version of the at least one ribonucleotide.

[0132] In another embodiment, 10% to 75% of at least one of the ribonucleotides of the composition are substituted by a deoxyribonucleotide version of the at least one ribonucleotide. In one embodiment, 10% to 70%, 10% to 65%, 10% to 60%, 10% to 55%, 10% to 50%, 10% to 45%, 10% to 40%, 10% to 35%, 10% to 30%, 10% to 25%, 10% to 20%, or 10% to 15%, of at least one of the ribonucleotides are substituted by a deoxyribonucleotide version of the at least one ribonucleotide.

[0133] In another embodiment, 20% to 75% of at least one of the ribonucleotides of the composition are substituted by a deoxyribonucleotide version of the at least one ribonucleotide. In one embodiment, 20% to 70%, 20% to 65%, 20% to 60%, 20% to 55%, 20% to 50%, 20% to 45%, 20% to 40%, 20% to 35%, 20% to 30%, or 20% to 25 of at least one of the ribonucleotides of the composition are substituted by a deoxyribonucleotide version of the at least one ribonucleotide.

[0134] In a preferred embodiment, 1 % to 50% of at least one of the ribonucleotides of the composition are substituted by a deoxyribonucleotide version of the at least one ribonucleotide. In an even more preferred embodiment, 10% to 50% of at least one of the ribonucleotides of the composition are substituted by a deoxyribonucleotide version of the at least one ribonucleotide.

[0135] Hence, in one embodiment, the at least one ribonucleotide that is substituted by a deoxyribonucleotide version is CTP and 1 % to 50% of CTP are substituted by a deoxyribonucleotide version thereof selected from the group consisting of dCTP, 5-Methyl-dCTP, 2'-Fluoro-dCTP, 3'- Azido-2',3'-ddCTP, 7-Deaza-dGTP, C8-Alkyne-dCTP, 5-lodo dCTP, 5-Bromo-dCTP, 2'NH2-dCTP, dCTPaSe, dCTPaS, 5-Propargylamino-dCTP, and 5-Hydroxymethyl-dCTP.

[0136] Hence, in one embodiment, the at least one ribonucleotide that is substituted by a deoxyribonucleotide version is ATP and 1% to 50% of ATP are substituted by a deoxyribonucleotide version thereof selected from the group consisting of dATP and 2'-Bromo-dATP.

[0137] Hence, in one embodiment, the at least one ribonucleotide that is substituted by a deoxyribonucleotide version is UTP and 1 % to 50% of UTP are substituted by dTTP.

[0138] Hence, in one embodiment, the at least one ribonucleotide that is substituted by a deoxyribonucleotide version is GTP and 1% to 50% of GTP are substituted by a deoxyribonucleotide version thereof selected from the group consisting of dGTP and 7-deaza-dGTP.

[0139] For example, 50% substitution of ATP by dATP or modified dATP means that the composition comprises 50% ATP and 50% dATP or modified dATP, CTP, UTP and GTP in a ratio of 1 (ATP+dATP):1 (CTP):1 (UTP):1 (GTP).

[0140] For example, 100% substitution of ATP means that no ATP is comprised in the composition, only dATP or modified dATP. Hence, in one embodiment, the composition comprises three ribonucleotides and one deoxyribonucleotide, wherein the deoxyribonucleotide is not a variant of any of the three ribonucleotides. In one embodiment, at least 0.1% of at least one of the ribonucleotides of the composition are substituted by a deoxyribonucleotide version of the at least one ribonucleotide. In one embodiment, at least 1% of at least one of the ribonucleotides of the composition are substituted by a deoxyribonucleotide version of the at least one ribonucleotide.

[0141] In one embodiment, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% of at least one of the ribonucleotides of the composition are substituted by a deoxyribonucleotide version of the at least one ribonucleotide. In a preferred embodiment, at least 5% of at least one of the ribonucleotides of the composition are substituted by a deoxyribonucleotide version of the at least one ribonucleotide.

[0142] In one embodiment, the at least one ribonucleotide is substituted by the deoxyribonucleotide version and a modified deoxyribonucleotide version. For example, 25% dCTP, 25% m3dCTP and 50% CTP.

[0143] In one embodiment, two ribonucleotides are substituted by a deoxyribonucleotide version. In one embodiment, three ribonucleotides are substituted by a deoxyribonucleotide version. In one embodiment, all four ribonucleotides are substituted by a deoxyribonucleotide version.

[0144] Polymerases

[0145] The method of mRNA synthesis can be performed using any RNA polymerase as long as synthesis of the mRNA from the DNA template that encodes the RNA is specifically and sufficiently initiated from a respective cognate RNA polymerase promoter and full-length mRNA is obtained. In some preferred embodiments, the RNA polymerase is selected from among RNA-dependent RNA Polymerase (RdRP) and DNA-dependent RNA Polymerase (DdRP).

[0146] In another embodiment, the in vitro transcription utilizes any RNA polymerase, or modified DNA polymerase, known in the art. In another embodiment, the in vitro transcription utilizes chemical synthesis.

[0147] Nucleic acid templates comprise a DNA sequence that is complementary to the mRNA to by synthesized. Transcription of DNA templates typically uses a DNA-dependent RNA Polymerase (DdRP). In one embodiment, the DdRP is selected from prokaryotic or eukaryotic RNA Polymerase. In one embodiment, the DdRP is an RNA Polymerase capable of synthesizing mRNA. In one embodiment, the DdRP is selected from the group consisting of T7 RNA polymerase, SP6 RNA polymerase, T3 RNA polymerase and eukaryotic RNA Polymerase II. In a preferred embodiment, the DdRP is selected from the group consisting of T7 RNA polymerase, SP6 RNA polymerase, and T3 RNA polymerase.

[0148] In one embodiment, the RNA Polymerase used in the method of the invention is capable of incorporating deoxyribonucleotides into mRNA. mRNA of the invention In a second aspect, the invention relates to an mRNA molecule synthesized by the method of the invention.

[0149] In another aspect, the invention relates to an mRNA molecule encoding a protein of interest, wherein between 0.1 % to 100% of at least one of the ribonucleotides are substituted by a deoxyribonucleotide version of the at least one ribonucleotide.

[0150] In one embodiment, the mRNA molecule is a purified mRNA molecule.

[0151] In one embodiment, the mRNA is an saRNA.

[0152] In one embodiment, the mRNA is a purified saRNA molecule.

[0153] In one embodiment, 0.1% to 90% of at least one of the ribonucleotides of the mRNA are substituted by a deoxyribonucleotide version of the at least one ribonucleotide. In one embodiment, 0.1% to 85%, 0.1% to 80%, 0.1% to 75%, 0.1 % to 70%, 0.1% to 65%, 0.1 % to 60%, 0.1% to 55%, 0.1% to 50%, 0.1% to 45%, 0.1% to 40%, 0.1 % to 35%, 0.1% to 30%, 0.1 % to 25%, 0.1% to 20%, 0.1% to 15%, 0.1% to 10%, 0.1% to 5% or 0.1% to 1% of at least one of the ribonucleotides are substituted by a deoxyribonucleotide version of the at least one ribonucleotide.

[0154] In another embodiment, 1% to 100% of at least one of the ribonucleotides of the mRNA are substituted by a deoxyribonucleotide version of the at least one ribonucleotide. In one embodiment, 1 % to 90%, 1 % to 85%, 1 % to 80%, 1% to 75%, 1% to 70%, 1% to 65%, 1% to 60%, 1% to 55%, 1 % to 50%, 1% to 45%, 1% to 40%, 1% to 35%, 1% to 30%, 1 % to 25%, 1 % to 20%, 1 % to 15%, 1% to 10%, or 1 % to 5% of at least one of the ribonucleotides are substituted by a deoxyribonucleotide version of the at least one ribonucleotide.

[0155] In another embodiment, 5% to 75% of at least one of the ribonucleotides of the mRNA are substituted by a deoxyribonucleotide version of the at least one ribonucleotide. In one embodiment, 5% to 70%, 5% to 65%, 5% to 60%, 5% to 55%, 5% to 50%, 5% to 45%, 5% to 40%, 5% to 35%, 5% to 30%, 5% to 25%, 5% to 20%, 5% to 15%, or 5% to 10% of at least one of the ribonucleotides are substituted by a deoxyribonucleotide version of the at least one ribonucleotide.

[0156] In another embodiment, 10% to 75% of at least one of the ribonucleotides of the mRNA are substituted by a deoxyribonucleotide version of the at least one ribonucleotide. In one embodiment, 10% to 70%, 10% to 65%, 10% to 60%, 10% to 55%, 10% to 50%, 10% to 45%, 10% to 40%, 10% to 35%, 10% to 30%, 10% to 25%, 10% to 20%, or 10% to 15%, of at least one of the ribonucleotides are substituted by a deoxyribonucleotide version of the at least one ribonucleotide.

[0157] In another embodiment, 20% to 75% of at least one of the ribonucleotides of the mRNA are substituted by a deoxyribonucleotide version of the at least one ribonucleotide. In one embodiment, 20% to 70%, 20% to 65%, 20% to 60%, 20% to 55%, 20% to 50%, 20% to 45%, 20% to 40%, 20% to 35%, 20% to 30%, or 20% to 25 of at least one of the ribonucleotides of the mRNA are substituted by a deoxyribonucleotide version of the at least one ribonucleotide. In a preferred embodiment, 1 % to 50% of at least one of the ribonucleotides of the mRNA are substituted by a deoxyribonucleotide version of the at least one ribonucleotide. In an even more preferred embodiment, 10% to 50% of at least one of the ribonucleotides of the mRNA are substituted by a deoxyribonucleotide version of the at least one ribonucleotide.

[0158] Hence, in one embodiment, the at least one ribonucleotide that is substituted by a deoxyribonucleotide version is CTP and 1 % to 50% of CTP are substituted by a deoxyribonucleotide version thereof selected from the group consisting of dCTP, 5-Methyl-dCTP, 2'-Fluoro-dCTP, 3'- Azido-2',3'-ddCTP, 7-Deaza-dGTP, C8-Alkyne-dCTP, 5-lodo dCTP, 5-Bromo-dCTP, 2'NH2-dCTP, dCTPaSe, dCTPaS, 5-Propargylamino-dCTP, and 5-Hydroxymethyl-dCTP.

[0159] Hence, in one embodiment, the at least one ribonucleotide that is substituted by a deoxyribonucleotide version is ATP and 1% to 50% of ATP are substituted by a deoxyribonucleotide version thereof selected from the group consisting of dATP and 2'-Bromo-dATP.

[0160] Hence, in one embodiment, the at least one ribonucleotide that is substituted by a deoxyribonucleotide version is UTP and 1 % to 50% of UTP are substituted by dTTP.

[0161] Hence, in one embodiment, the at least one ribonucleotide that is substituted by a deoxyribonucleotide version is GTP and 1% to 50% of GTP are substituted by a deoxyribonucleotide version thereof selected from the group consisting of dGTP and 7-deaza-dGTP.

[0162] For example, 100% substitution of ATP means that no ATP is comprised in the mRNA, only dATP and / or modified dATP. Hence, in one embodiment, the mRNA comprises three ribonucleotides and one deoxyribonucleotide, wherein the deoxyribonucleotide is not the deoxy- version of any of the three ribonucleotides.

[0163] In one embodiment, at least 0.1% of at least one of the ribonucleotides of the mRNA are substituted by a deoxyribonucleotide version of the at least one ribonucleotide. In one embodiment, at least 1 % of at least one of the ribonucleotides of the mRNA are substituted by a deoxyribonucleotide version of the at least one ribonucleotide.

[0164] In one embodiment, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% of at least one of the ribonucleotides of the mRNA are substituted by a deoxyribonucleotide version of the at least one ribonucleotide. In a preferred embodiment, at least 5% of at least one of the ribonucleotides of the mRNA are substituted by a deoxyribonucleotide version of the at least one ribonucleotide.

[0165] In one embodiment, the at least one ribonucleotide is substituted by the deoxyribonucleotide version and a modified deoxyribonucleotide version. For example, 25% dCTP, 25% m3dCTP and 50% CTP.

[0166] In one embodiment, two ribonucleotides are substituted by a deoxyribonucleotide version. In one embodiment, three ribonucleotides are substituted by a deoxyribonucleotide version. In one embodiment, all four ribonucleotides are substituted by a deoxyribonucleotide version. Generally, the shortest length of a modified mRNA of the present disclosure can be the length of an mRNA sequence that is sufficient to encode for a dipeptide. In another embodiment, the length of the mRNA sequence is sufficient to encode for a tripeptide. In another embodiment, the length of an mRNA sequence is sufficient to encode for a tetrapeptide. In another embodiment, the length of an mRNA sequence is sufficient to encode for a pentapeptide. In another embodiment, the length of an mRNA sequence is sufficient to encode for a hexapeptide. In another embodiment, the length of an mRNA sequence is sufficient to encode for a heptapeptide. In another embodiment, the length of an mRNA sequence is sufficient to encode for an octapeptide. In another embodiment, the length of an mRNA sequence is sufficient to encode for a nonapeptide. In another embodiment, the length of an mRNA sequence is sufficient to encode for a decapeptide.

[0167] In a further embodiment, the mRNA is greater than 30 nucleotides in length. In another embodiment, the RNA molecule is greater than 35 nucleotides in length. In another embodiment, the length is at least 40 nucleotides. In another embodiment, the length is at least 45 nucleotides. In another embodiment, the length is at least 55 nucleotides. In another embodiment, the length is at least 60 nucleotides. In another embodiment, the length is at least 60 nucleotides. In another embodiment, the length is at least 80 nucleotides. In another embodiment, the length is at least 90 nucleotides. In another embodiment, the length is at least 100 nucleotides. In another embodiment, the length is at least 120 nucleotides. In another embodiment, the length is at least 140 nucleotides. In another embodiment, the length is at least 160 nucleotides. In another embodiment, the length is at least 180 nucleotides. In another embodiment, the length is at least 200 nucleotides. In another embodiment, the length is at least 250 nucleotides. In another embodiment, the length is at least 300 nucleotides. In another embodiment, the length is at least 350 nucleotides. In another embodiment, the length is at least 400 nucleotides. In another embodiment, the length is at least 450 nucleotides. In another embodiment, the length is at least 500 nucleotides. In another embodiment, the length is at least 600 nucleotides. In another embodiment, the length is at least 700 nucleotides. In another embodiment, the length is at least 800 nucleotides. In another embodiment, the length is at least 900 nucleotides. In another embodiment, the length is at least 1000 nucleotides. In another embodiment, the length is at least 1100 nucleotides. In another embodiment, the length is at least 1200 nucleotides. In another embodiment, the length is at least 1300 nucleotides. In another embodiment, the length is at least 1400 nucleotides. In another embodiment, the length is at least 1500 nucleotides. In another embodiment, the length is at least 1600 nucleotides. In another embodiment, the length is at least 1800 nucleotides. In another embodiment, the length is at least 2000 nucleotides. In another embodiment, the length is at least 2500 nucleotides. In another embodiment, the length is at least 3000 nucleotides. In another embodiment, the length is at least 4000 nucleotides. In another embodiment, the length is at least 5000 nucleotides, or greater than 5000 nucleotides.

[0168] In some other embodiments, capped RNA is synthesized co-transcriptionally by using a dinucleotide cap analog in the IVT reaction (e.g., using CleanCap® AG or AU reagent, TriLink Biotechnologies; AMPLICAP™ T7 Kit or a MESSAGE MAX™ T7 ARCA-CAPPED MESSAGE Transcription Kit; EPICENTRE or CellScript, Madison, Wl, USA). If capping is performed co-transcriptionally, preferably the dinucleotide cap analog is an anti-reverse cap analog (ARCA). However, use of a separate IVT reaction, followed by capping with a capping enzyme system, which results in approximately 100% of the RNA being capped, is preferred over co- transcriptional capping, which typically results in only about 80% of the RNA being capped. Thus, in some preferred embodiments, a high percentage of the mRNA molecules used in a method of the present invention are capped (e.g., greater than 80%, greater than 90%, greater than 95%, greater than 98%, greater than 99%, greater than 99.5%, or greater than 99.9% of the population of mRNA molecules are capped).

[0169] In certain embodiments, the mRNA used in the purified RNA preparations is purified to remove substantially, essentially, or virtually all of the contaminants, including substantially, essentially, or virtually all of the RNA contaminants. The present invention is not limited with respect to the purification methods used to purify the mRNA, and the invention includes use of any method that is known in the art or developed in the future in order to purify the mRNA and remove contaminants, including RNA contaminants, that interfere with the intended use of the mRNA. In some embodiments, the mRNA purification is performed using HPLC, but in some other embodiments a gravity flow column is used for the purification. In still another embodiment, the mRNA used in the compositions and methods of the present invention is purified using a process which comprises treating the mRNA with an enzyme that specifically acts (e.g., digests) one or more contaminant RNA or contaminant nucleic acids (e.g., including DNA), but which does not act on (e.g., does not digest) the desired mRNA. For example, in some embodiments, the mRNA used in the compositions and methods of the present invention is purified using a process which comprises treating the mRNA with a ribonuclease III (RNase III) enzyme (e.g., E. coli RNase III) and the mRNA is then purified away from the RNase III digestion products. A ribonuclease III (RNase III) enzyme herein means an enzyme that digests double-stranded RNA greater than about twelve basepairs to shore doublestranded RNA fragments. In some embodiments, the mRNA used in the compositions and methods of the present invention is purified using a process which comprises treating the mRNA with one or more other enzymes that specifically digest one or more contaminant RNAs or contaminant nucleic acids (e.g., including DNA).

[0170] Other components of nucleic acid are optional, and are beneficial in some embodiments. For example, a 5' untranslated region (UTR) and / or a 3'UTR are provided, wherein either or both may independently contain one or more different nucleoside modifications. In such embodiments, nucleoside modifications may also be present in the translatable region. Also provided are nucleic acids containing a Kozak sequence. Additionally, provided are nucleic acids containing one or more intronic nucleotide sequences capable of being excised from the nucleic acid.

[0171] Further, provided are nucleic acids containing an internal ribosome entry site (IRES). An IRES may act as the sole ribosome binding site, or may serve as one of multiple ribosome binding sites of an mRNA. An mRNA containing more than one functional ribosome binding site may encode several peptides or polypeptides that are translated independently by the ribosomes ("multicistronic mRNA"). When nucleic acids are provided with an IRES, further optionally provided is a second translatable region. Examples of IRES sequences that can be used according to the present disclosure include without limitation, those from picomaviruses (e.g. FMDV), pest viruses (CFFV), polio viruses (PV), encephalomyocarditis viruses (ECMV), foot-and-mouth disease viruses (FMDV), hepatitis C viruses (HCV), classical swine fever viruses (CSFV), murine leukemia virus (MLV), simian immune deficiency viruses (SIV) or cricket paralysis viruses (CrPV).

[0172] In some embodiments, the messenger RNA further comprises a poly-A tail. In some embodiments, the messenger RNA further comprises a cap-independent translational enhancer. In some embodiments, the messenger RNA comprises at least about 95% to about 99.9% of all the nucleic acid present in the purified preparation. In some embodiments, the messenger RNA of the invention is significantly less immunogenic than an unmodified preparation of messenger RNA with the same sequence. In some embodiments, the messenger RNA exhibits enhanced ability to be translated by a target cell than an unmodified preparation of messenger RNA with the same sequence. In some embodiments, the messenger RNA exhibits enhanced ability to be translated when delivered to a mammal than an unmodified preparation of messenger RNA with the same sequence.

[0173] In one embodiment, the at least one substituted deoxyribonucleotide version of the at least one ribonucleotide is located in the protein coding sequence of the mRNA or saRNA.

[0174] In one embodiment, the at least one substituted deoxyribonucleotide version of the at least one ribonucleotide is not located in the polyA tail of the mRNA or saRNA.

[0175] Modified Deoxyribonucleotides

[0176] One aspect of the invention is a method of synthesis of an mRNA molecule or an mRNA molecule wherein a percentage of nucleotides of the mRNA are substituted by the deoxyribonucleotide version, and optionally further modified. Hence, in one embodiment, the deoxyribonucleotide is a modified or unmodified deoxyribonucleotide.

[0177] According to the invention, some ribonucleotides that make up the mRNA of the invention are replaced by their deoxyribonucleotide counterpart. In addition to this substitution, the deoxyribonucleotide may also be modified. This can also be the case if the original ribonucleotide was unmodified. Hence, the substitution includes two layers of modification, a change from ribonucleotide to modified ribonucleotide, including naturally occurring modification and synthetic modification; and a change of ribonucleotide to deoxyribonucleotide. The modifications can be done in any order.

[0178] As a basis for the modifications, known mRNA modifications are used, and a deoxy- version of these modified ribonucleotides is prepared. Methods of chemical synthesis to accomplish this are known in the art. The modified deoxyribonucleotides may be synthesized by conversion of a modified ribonucleotide. In one embodiment, ribonucleotide reductase (RNR) mediates the conversion of ribonucleotides to deoxyribonucleotides.

[0179] In an alternative embodiment, the modified deoxyribonucleotides may be synthesized de novo.

[0180] In one embodiment, the nucleotide that is substituted and optionally further modified in an RNA of the methods and compositions of the present invention is uridine (U). In another embodiment, the nucleotide that is substituted and optionally further modified is cytidine (C). In another embodiment, the nucleotide that is substituted and optionally further modified is adenine (A). In another embodiment, the nucleotide that is substituted and optionally further modified is guanine (G).

[0181] The modified deoxyribonucleotide may be the deoxyribonucleotide variant of any naturally occurring modified ribonucleotide, or it may be the deoxyribonucleotide variant of a synthetic modified ribonucleotide, or it may be a naturally occurring or synthetic modified deoxyribonucleotide.

[0182] The modified deoxyribonucleotide may be the deoxyribonucleotide variant of any naturally occurring modified ribonucleotide according to the RNA Modification Database (Cantara et al. 2011 Nucleic Acids Res . 2011 ;39 D195-201), which can be accessed at

[0183] Table 1 depicts a list of naturally occurring modified ribonucleotides in columns 1 (symbol) and 2 (common name).

[0184] In one embodiment, the at least one ribonucleotide is substituted with the deoxy-version of a modified ribonucleotide selected from the databases Modomics (Boccaletto et al. Nucleic Acids Research 2018, 46(D1):D303-D307), NAMDB (The Nucleic Acid Modification Database, http: / / www.namdb.cn) or the RNA Modification Database.

[0185] In one embodiment, the at least one ribonucleotide is substituted with the deoxy-version of a modified ribonucleotide selected from the group consisting of Table 1.

[0186] The modified deoxyribonucleotide may also be a naturally occurring modified deoxyribonucleotide according to Table 2.

[0187] Furthermore, the at least one ribonucleotide may substituted with the deoxy-version of a synthetically modified ribonucleotide or a synthetically modified deoxyribonucleotide selected from the group consisting of Table 3.

[0188] Hence, in one embodiment, the at least one ribonucleotide is substituted with the deoxy-version of a modified ribonucleotide selected from the group consisting of Table 1 or Table 3 or with a modified deoxyribonucleotide selected from the group consisting of Table 2.

[0189] Furthermore, the at least one ribonucleotide may substituted with the deoxy-version of a synthetically modified ribonucleotide or a synthetically modified deoxyribonucleotide selected from the group consisting of Table 3, excluding the last 13 lines of Table 3.

[0190] Hence, in one embodiment, the at least one ribonucleotide is substituted with the deoxy-version of a modified ribonucleotide selected from the group consisting of Table 1 or Table 3, excluding the last 13 lines of Table 3, or with a modified deoxyribonucleotide selected from the group consisting of Table 2.

[0191] Table 1 : Naturally occurring modified ribonucleotides common name and symbol.

[0192]

[0193] Table 2: List of DNA modifications

[0194]

[0195] Table 3: List of synthetic ribonucleotide and deoxyribonucleotide modifications.

[0196]

[0197]

[0198] In another embodiment, the at least one ribonucleotide that is substituted by a deoxyribonucleotide version is CTP and the deoxyribonucleotide version is modified by a modification is selected from the group consisting of the following naturally or synthetically occurring ribonucleotide or deoxyribonucleotide modifications hm5Cm, s2C, m3C, phm5C, hm5C, f5C, pf5C, ho5C, hm5C, m5C, pC2'3'cp, m4,4C, ac4C, m4C, 2NH2-C, 3’-Azido-2’,3’-ddCTP, C8-Alkyne-C, 5-Propargylamino-C, LCC, CBV, CBR, IC, A5M, CSL, RY, 5CF, 10C, S4C, 73W, CH, N5M, 5IC, M5M, CAR, CFZ, CFL, D2C, 1 mC, m2C, e3C, 3mC, sC, 4mC, 5caC, 5fC, 5hmC, 5gmC and 5mC; or wherein the at least one ribonucleotide that is substituted by a deoxyribonucleotide version is ATP and the deoxyribonucleotide version is modified by a modification is selected from the group consisting of the following naturally or synthetically occurring ribonucleotide or deoxyribonucleotide modifications m1A, msms2i6A, m2,8A, m2A, ms2ct6A, , ms2io6A, ms2hn6A, ms2hn6A, ms2i6A, ms2m6A, ms2t6A, ms2t6A, NADpN, m8A, ct6A, ct6A, ht6A, io6A, pio6A, m62A, ac6A, pac6A, f6A, pf6A, g6A, hm6A, hn6A, m6A, m6t6A, t6A, 2’-Bromo-A, LCA, IG, SRA, AF2, ADS, 2AD, 8AN, PPU, 3DA, AP7, 45A, A9Z, ANZ, A5O, A5L, 5AA, A43, 2HA AD2, 1 mA, 3mA, 4mA, 6mA, 7mA, 9mA, 6hmA, HAP, m2A, AHAP, 6-DMA, ncm6A and sA; or wherein the at least one ribonucleotide that is substituted by a deoxyribonucleotide version is GTP and the deoxyribonucleotide version is modified by a modification is selected from the group consisting of the following naturally or synthetically occurring ribonucleotide or deoxyribonucleotide modifications m1 G, preQ1 , preQO, m7G, m2,7G, m2,2,7G, m22G, m2G I, mi l, m1 lm, Im, Q, oQ, dGdAIQ, mdAnQ, preQO, preQ1 , dG+ , 7-Deaza-G, 2’NH2-dG, 7-Deaza-7-iodo-G, 6-thio-dG, dCaSe, dCaS, Fluoro-G, MANT-G, 2-MANT-G, LCG, BGM, 2SG, GRB, GAO, TG, GDO, GMX, XUG, G3A, GS, G38, 2HG, 1 mG, 2mG, 3mG, m6G, 7mG, 8-oxo-G, ADG, 1 ,N(2)-eG, N(2),3-eG, m22G, m2,7dG, m2,2,7dG, 2’NH2-dG and CEG; or wherein the at least one ribonucleotide that is substituted by a deoxyribonucleotide version is UTP and the deoxyribonucleotide version is modified by a modification is selected from the group consisting of the following naturally or synthetically occurring ribonucleotide or deoxyribonucleotide cmo5U, mcmo5U, 2-NH2-dU, 3’-Azido-2’,3’-ddU, Aminoallyl-dU, 5-Propargylamino-dU, 5-Fluoro- dU, 5-dimethyl-2’-dU, 5BU, U37, IU, TLN, BRU, URU, UFB, U5M, UOB, UMO, U5R, B8N, U4M, UAR, UFT, UMS, NYM, MU1 , P1T, M, MU4, putThy, ddT, dTaSe, dTaS, 1 mT, 3mT, 04-meT, diHT, 5hmU, 5fU, base J, dhpUra, 5caU, 5-NeOmdU, dU, DHdU, 5-NedU, and dhpdU.

[0199] In another embodiment, the at least one ribonucleotide that is substituted by a deoxyribonucleotide version is CTP and the deoxyribonucleotide version is modified by a modification is selected from the group consisting of the following naturally or synthetically occurring ribonucleotide or deoxyribonucleotide modifications hm5Cm, s2C, m3C, phm5C, hm5C, f5C, pf5C, ho5C, hm5C, m5C, pC2'3'cp, m4,4C, ac4C, m4C, 2NH2-C, 3’-Azido-2’,3’-ddCTP, C8-Alkyne-C, 5-Propargylamino-C, LCC, CBV, CBR, IC, A5M, CSL, RY, 5CF, 10C, S4C, 73W, CH, N5M, 5IC, M5M, CAR, CFZ, CFL, D2C, 1 mC, m2C, e3C, 3mC, EC, 4mC, 5caC, 5fC, 5hmC, 5gmC and 5mC; or wherein the at least one ribonucleotide that is substituted by a deoxyribonucleotide version is ATP and the deoxyribonucleotide version is modified by a modification is selected from the group consisting of the following naturally or synthetically occurring ribonucleotide or deoxyribonucleotide modifications m1A, msms2i6A, m2,8A, m2A, ms2ct6A, , ms2io6A, ms2hn6A, ms2hn6A, ms2i6A, ms2m6A, ms2t6A, ms2t6A, NADpN, m8A, ct6A, ct6A, ht6A, io6A, pio6A, m62A, ac6A, pac6A, f6A, pf6A, g6A, hm6A, hn6A, m6A, m6t6A, t6A, MANT-dATP, 2'-lodo-dATP, 7'-Deaza-dATP, 8'-Bromo- dATP, 7'-Deaza-7'-iodo-dATP, 2'-Hydroxy-dATP, 7'-Deaza-7-bromo-dATP, 2'-Fluoro-dATP, Mant- dATP, 2'NH2-dATP, Etheno-dATP (e-dATP), dATPaSe, dATPaS, 8'-Oxo-dATP, 2’-Bromo-A, LCA, IG, SRA, AF2, ADS, 2AD, 8AN, PPU, 3DA, AP7, 45A, A9Z, ANZ, A5O, A5L, 5AA, A43, 2HA AD2, 1 mA, 3mA, 4mA, 6mA, 7mA, 9mA, 6hmA, HAP, m2A, AHAP, 6-DMA, ncm6A and sA; or wherein the at least one ribonucleotide that is substituted by a deoxyribonucleotide version is GTP and the deoxyribonucleotide version is modified by a modification is selected from the group consisting of the following naturally or synthetically occurring ribonucleotide or deoxyribonucleotide modifications m1 G, preQ1 , preQO, m7G, m2,7G, m2,2,7G, m22G, m2G I, mi l, m1 lm, Im, Q, oQ, dGdAIQ, mdAnQ, preQO, preQ1 , dG+ , 7-Deaza-G, 2’NH2-dG, 7-Deaza-7-iodo-G, 6-thio-dG, dCaSe, dCaS, Fluoro-G, MANT-G, 2-MANT-G, LCG, BGM, 2SG, GRB, GAO, TG, GDO, GMX, XUG, G3A, GS, G38, 2HG, 1 mG, 2mG, 3mG, m6G, 7mG, 8-oxo-G, ADG, 1 ,N(2)-EG, N(2),3-EG, m22G, m2,7dG, m2,2,7dG, 2’NH2-dG and CEG; or wherein the at least one ribonucleotide that is substituted by a deoxyribonucleotide version is UTP and the deoxyribonucleotide version is modified by a modification is selected from the group consisting of the following naturally or synthetically occurring ribonucleotide or deoxyribonucleotide cmo5U, mcmo5U, 2-NH2-dU, 3’-Azido-2’,3’-ddU, Aminoallyl-dU, 5-Propargylamino-dU, 5-Fluoro- dU, 5-dimethyl-2’-dU, 5BU, U37, IU, TLN, BRU, URU, UFB, U5M, UOB, UMO, U5R, B8N, U4M, UAR, UFT, UMS, NYM, MU1 , P1T, M, MU4, putThy, ddT, dTaSe, dTaS, 1 mT, 3mT, 04-meT, diHT, 5hmU, 5fU, base J, dhpUra, 5caU, 5-NeOmdU, dU, DHdU, 5-NedU, and dhpdU.

[0200] In one embodiment, the at least one ribonucleotide that is substituted by a deoxyribonucleotide version is CTP and the substituted deoxyribonucleotide version is selected from the group consisting of dCTP, 5-Methyl-dCTP, 2'-Fluoro-dCTP, 3'-Azido-2',3'-ddCTP, 7-Deaza-dCTP, C8-Alkyne-dCTP, 5- lodo dCTP, 5-Bromo-dCTP, 2'NH2-dCTP, dCTPaSe, dCTPaS, 5-Propargylamino-dCTP, and 5- Hydroxymethyl-dCTP, 3'-Methyl-dCTP, 2'-O-Methyl-dCTP, 2'-thio-dCTP, N4'-acetyl-dCTP, 5'-formyl- dCTP, 5,2'-O-dimethyl-dCTP, N4-acetyl-2'-O-methyl- dCTP, N4-methyl-dCTP, N4,2'-O-dimethyl- dCTP, 5-hydroxymethyl-dCTP, 5-formyl-2'-0-methyl-dCTP, and N4,N4,2'-O-trimethyl-dCTP; or the at least one ribonucleotide that is substituted by a deoxyribonucleotide version is ATP and the substituted deoxyribonucleotide version is selected from the group consisting of dATP and 2'-Bromo- dATP, T-Methyl-dATP, 2'-Methyl-dATP, N6'-Methyl-dATP, 2'-O-Methyl-dATP, 2'-methylthio-N6- methyl-dATP, N6-isopentenyl-dATP, 2-methylthio-N6-isopentenyl-dATP, N6-(cis- hydroxyisopentenyl)- dATP, N6-glycinylcarbamoyl-dATP, N6-threonylcarbamoyl-dATP, 2-methylthio- N6-threonyl carbamoyl-dATP, N6-methyl-N6-threonylcarbamoyl-dATP, N6- hydroxynorvalylcarbamoyl-dATP, 2-methylthio-N6-hydroxynorvalyl carbamoyl-dATP, N6,N6- dimethyl-dATP, N6,2'-O-dimethyl-dATP, N6,N6,2'-O-trimethyl-dATP, 1 ,2'-O-dimethyl-dATP, N6- acetyl-dATP, 8-methyl-dATP, and cyclic N6-threonylcarbamoyl-dATP; or the at least one ribonucleotide that is substituted by a deoxyribonucleotide version is UTP and the substituted deoxyribonucleotide version is selected from the group consisting of dTTP, dUTP, 2'-O- methyl-dUTP, 3,2'-O-dimethyl-dUTP, 2'NH2-deoxyuridine, 3'-Azido-2',3'-dideoxyuridine, Aminoallyldeoxyuridine, 5-Propargylamino-deoxyuridine, 5-Fluoro-deoxyuridine, 5-Bromo- deoxyuridine, 5- lodo-deoxyuridine, deoxyuridine, 2',3'-Dideoxythymidine, Deoxythymidine, 5'-(a-seleno)- deoxythymidine (dTaSe), 5'-(a-thio)-deoxythymidine(dTaS), 3,2'-0-dimethyldeoxyuridine (m4dll), 2- thiodeoxyuridine (s2dll), 2-Fluoro-dUTP, pseudodeoxyuridine(dMJ), N1 -Methylpseudouridine (m'l d'-P), 2'-0-methyldeoxyuridine (Udm), 4-thiodeoxyuridine (s4dll), 5-methyldeoxyuridine (m5dll), and 5-methoxydeoxyuridine (mo5dll); or the at least one ribonucleotide that is substituted by a deoxyribonucleotide version is GTP and the substituted deoxyribonucleotide version is selected from the group consisting of dGTP, 8-Oxo- deoxyguanosine, 3'-deoxyguanosine, 7-Deaza-deoxyguanosine, 7-Deaza-7-iodo-deoxyguanosine, 6- Thio-deoxyguanosine, 2'-Fluoro-deoxyguanosine, Mant- deoxyguanosine, 2'-Mant-3'- deoxyguanosine, 2'NH2-deoxyguanosine, 5'-(a-seleno)-deoxyguanosine (dCaSe), 5'-(a-thio)- deoxyguanosine (dCaS), 2'-0-methyldeoxyguanosine (dGm), N2,7-dimethyldeoxyguanosine (m2’7dG), 7-methyldeoxguanosine (m7dG), N2, and N2,7-trimethyldeoxyguanosine (m2’2’7dG).

[0201] In one embodiment, the at least one ribonucleotide that is substituted by a deoxyribonucleotide version is CTP and the substituted deoxyribonucleotide version is selected from the group consisting of dCTP, 5-Methyl-dCTP, 2'-Fluoro-dCTP, 3'-Azido-2',3'-ddCTP, 7-Deaza-dGTP, C8-Alkyne-dCTP, 5- lodo dCTP, 5-Bromo-dCTP, 2'NH2-dCTP, dCTPaSe, dCTPaS, 5-Propargylamino-dCTP, and 5- Hydroxymethyl-dCTP; or the at least one ribonucleotide that is substituted by a deoxyribonucleotide version is ATP and the substituted deoxyribonucleotide version is selected from the group consisting of dATP and 2'-Bromo- dATP; or the at least one ribonucleotide that is substituted by a deoxyribonucleotide version is UTP and the substituted deoxyribonucleotide version is dTTP; or the at least one ribonucleotide that is substituted by a deoxyribonucleotide version is GTP and the substituted deoxyribonucleotide version is selected from the group consisting of dGTP and 7-deaza- dGTP. In one embodiment, the at least one ribonucleotide that is substituted by a deoxyribonucleotide version is selected from the group consisting of ATP, CTP and UTP. Preferably, the at least one ribonucleotide that is substituted by a deoxyribonucleotide version is selected from the group consisting of ATP and CTP. Even more preferably, the at least one ribonucleotide that is substituted by a deoxyribonucleotide version is CTP.

[0202] In addition to the substitutions described above, the mRNA may also include further modified nucleotides. In one embodiment, the mRNA may include one or more modified end and / or cap nucleotides selected from the group consisting of 2'3'-cyclic phosphate end, 5' diphosphate end, 5' hydroxyl end, 5' triphosphate end, adenosine-5'-phosphate-2',3'-cyclic phosphate, cytidine-5'- phosphate-2',3'-cyclic phosphate, guanoside-5'-phosphate-2',3'-cyclic phosphate, uridine-5'- monophosphate-2',3'-cyclic phosphate, adenosine pentaphosphate 5' cap (cap Ap5N), adenosine tetraphosphate 5' cap (cap Ap4N), adenosine triphosphate 5' cap (cap A), alphadimethylmonophosphate 5' cap, alpha-methylmonophosphate 5' cap, guanosine triphosphate 5' cap (cap G), N2,7-dimethylguanosine cap (cap DMG), N2,N2,7-trimethylguanosine cap (cap TMG), N6- methyl-adenosine pentaphosphate 5' cap (cap Ap5N), N6-methyl-adenosine tetraphosphate 5' cap (cap Ap4N), N6-methyl-adenosine triphosphate 5' cap (cap A), N7-methyl-guanosine tetraphosphate 5' cap (cap m7Gp4N), and N7-methyl-guanosine cap (cap 0).

[0203] In one embodiment, the mRNA further includes one or more modified nucleotides selected from the group consisting of 5' (3' -dephosphoacetyl-CoA), 5' (3' -dephospho-CoA), 5' (3' -dephosphomalonyl- CoA), and 5' (3' -dephosphosuccinyl-CoA).

[0204] In one embodiment, the mRNA or saRNA comprises one or more modified nucleotides selected from the group consisting of 2'-lodo-dATP; 7'-Deaza-dATP; 8'-Bromo-dATP; 7'-Deaza-7'-iodo-dATP; 2'- Hydroxy-dATP; 2'-Chloro-dATP; 7'-Deaza-7-bromo-dATP; 2'-Fluoro-dATP; Mant-dATP; 2'NH2-dATP; Etheno-dATP (E-dATP); dATPaSe; dATPaS; 8'-Oxo-dATP; N6-Methyl-dATP; 2'-Fluoro-dCTP; 5'- Hydroxymethyl-dCTP and 8'-Oxo-dGTP.In one embodiment, the mRNA or saRNA comprises one or more modified nucleotides selected from the group consisting of 2'-lodo-dATP; 7'-Deaza-dATP; 8'- Bromo-dATP; 7'-Deaza-7'-iodo-dATP; 2'-Hydroxy-dATP; 2'-Chloro-dATP; 7'-Deaza-7-bromo-dATP; 2'-Fluoro-dATP; Mant-dATP; 2'NH2-dATP; Etheno-dATP (E-dATP); dATPaSe; dATPaS; 8'-Oxo- dATP; N6-Methyl-dATP; 2'-Fluoro-dCTP; 5'-Hydroxymethyl-dCTP and 8'-Oxo-dGTP, wherein 25% of one or more of ATP, CTP or GTP are substituted for the one or more modified nucleotide.

[0205] In one embodiment, the mRNA or saRNA comprises at least one modified nucleotide, wherein either

[0206] (i) 25% of ATP is substituted for a modified nucleotide selected from the group consisting of 2'-lodo- dATP; 7'-Deaza-dATP; 8'-Bromo-dATP; 7'-Deaza-7'-iodo-dATP; 2'-Hydroxy-dATP; 2'-Chloro-dATP; 7'-Deaza-7-bromo-dATP; 2'-Fluoro-dATP; Mant-dATP; 2'NH2-dATP; Etheno-dATP (E-dATP); dATPaSe; dATPaS; 8'-Oxo-dATP; and N6-Methyl-dATP; or

[0207] (ii) 25% of CTP is substituted for a modified nucleotide selected from 2'-Fluoro-dCTP and 5'- Hydroxymethyl-dCTP; or

[0208] (iii) 25% of GTP is substituted for 8'-Oxo-dGTP. In a preferred embodiment, the mRNA or saRNA comprises at least one modified nucleotide, wherein 25% of ATP is substituted for a modified nucleotide selected from the group consisting of 2'- lodo-dATP; 7'-Deaza-dATP; 8'-Bromo-dATP; 2'-Hydroxy-dATP; 2'-Chloro-dATP; 7'-Deaza-7-bromo- dATP and Mant-dATP.

[0209] In a preferred embodiment, the mRNA or saRNA comprises one modified nucleotide, wherein 25% of ATP is substituted for a modified nucleotide selected from the group consisting of 2'-lodo-dATP; 7'- Deaza-dATP; 8'-Bromo-dATP; 2'-Hydroxy-dATP; 2'-Chloro-dATP; 7'-Deaza-7-bromo-dATP and Mant-dATP.

[0210] In one embodiment, the mRNA or saRNA consists of ATP, GTP, CTP and TTP, wherein 25% of ATP is substituted for a modified nucleotide selected from the group consisting of 2'-lodo-dATP; 7'-Deaza- dATP; 8'-Bromo-dATP; 2'-Hydroxy-dATP; 2'-Chloro-dATP; 7'-Deaza-7-bromo-dATP and Mant-dATP.

[0211] In one embodiment, the mRNA or saRNA consists of ATP, GTP, CTP and TTP, wherein 25% of ATP in the protein coding region of the RNA is substituted for a modified nucleotide selected from the group consisting of 2'-lodo-dATP; 7'-Deaza-dATP; 8'-Bromo-dATP; 2'-Hydroxy-dATP; 2'-Chloro- dATP; 7'-Deaza-7-bromo-dATP and Mant-dATP.

[0212] In one embodiment, the mRNA or saRNA comprises one or more modified nucleotides selected from the group consisting of deoxyadenosine(dA), 2'-lodo-deoxyadenosine, 2'-Bromo-deoxyadenosine, 3'- deoxyadenosine, 7-Deaza-deoxyadenosine, 8-Bromo-deoxyadenosine, 7-Deaza-7-iodo- deoxyadenosine, 2-Hydroxy-deoxyadenosine, 2'-Chloro-deoxyadenosine, 7-Deaza-7-bromo- deoxyadenosine, 2'-Fluoro-deoxyadenosine, Mant-deoxyadenosine, 2'-Mant-3'- deoxyadenosine, 2'NH2- deoxyadenosine, Etheno-deoxyadenosine (E-dA), 5'-(a-seleno)- deoxyadenosine (dAaSe), 5'- (a-thio)-deoxyadenosine (dAaS), N6-(6-Aminohexyl)-deoxyadenosine, N6-methyldeoxyadenosine (m6dA), 2'-0-methyldeoxyadenosine (2'OMedA), N6,2'-0-dimethyldeoxyadenosine (m6dAm), N6,N6,2'-0-trimethyldeoxyadenosine (m62dAm), 8-Oxo-deoxyadenosine, N6-Methyl- deoxyadenosine, 2'NH2-deoxyuridine, 3'-Azido-2',3'-dideoxyuridine, Aminoallyl- deoxyuridine, 5- Propargylamino-deoxyuridine, 5-Fluoro-deoxyuridine, 5-Bromo- deoxyuridine, 5-lodo-deoxyuridine, deoxyuridine, 2',3'-Dideoxythymidine, Deoxythymidine, 5'-(a-seleno)-deoxythymidine (dTaSe), 5'-(a- thio)-deoxythymidine(dTaS), 3,2'-0-dimethyldeoxyuridine (m4dll), 2-thiodeoxyuridine (s2dll), 2- Fluoro-dUTP, pseudodeoxyuridine(dMJ), N1 -Methylpseudouridine (m'l d'-P), 2'-0-methyldeoxyuridine (Udm), 4-thiodeoxyuridine (s4dll), 5-methyldeoxyuridine (m5dll), 5-methoxydeoxyuridine (mo5dll), deoxycytidine, C8-Alkyne-deoxycytidine, 5-Methyl-deoxycytidine, 2'-Fluoro-deoxycytidine, 5-lodo- deoxycytidine, 5-Bromo- deoxycytidia-seleno)- deoxycytidine (dCaSe), 5'-(a-thio)-deoxycytidine (dCaS), 5-Propargylamino-deoxycytidine, 5-Hydroxymethyl-deoxycytidine, 2'-0-methyldeoxycytidine (dCm), 8-Oxo-deoxyguanosine, 3'-deoxyguanosine, 7-Deaza-deoxyguanosine, 7-Deaza-7-iodo- deoxyguanosine, 6-Thio-deoxyguanosine, 2'-Fluoro-deoxyguanosine, Mant- deoxyguanosine, 2'- Mant-3'-deoxyguanosine, 2'NH2-deoxyguanosine, 5'-(a-seleno)-deoxyguanosine (dCaSe), 5'-(a- thio)-deoxyguanosine (dCaS), 2'-0-methyldeoxyguanosine (dGm), N2,7-dimethyldeoxyguanosine (m2,7dG), 7-methyldeoxguanosine (m7dG), N2, N2,7-trimethyldeoxyguanosine (m2,7dG.

[0213] In one embodiment, the mRNA or saRNA comprises one or more modified nucleotides, wherein 0.1% to 100% of ATP is substituted by a nucleotide selected from the group consisting of deoxyadenosine(dA), 2'-lodo-deoxyadenosine, 2'-Bromo-deoxyadenosine, 3'- deoxyadenosine, 7-Deaza-deoxyadenosine, 8-Bromo-deoxyadenosine, 7-Deaza-7-iodo- deoxyadenosine, 2-Hydroxy-deoxyadenosine, 2'-Chloro-deoxyadenosine, 7-Deaza-7- bromo- deoxyadenosine, 2'-Fluoro-deoxyadenosine, Mant-deoxyadenosine, 2'-Mant-3'- deoxyadenosine, 2'NH2- deoxyadenosine, Etheno-deoxyadenosine (E-dA), 5'-(a-seleno)- deoxyadenosine (dAaSe), 5'-(a-thio)-deoxyadenosine (dAaS), N6-(6-Aminohexyl)- deoxyadenosine, N6-methyldeoxyadenosine (m6dA), 2'-0-methyldeoxyadenosine (2'OMedA), N6,2'-0-dimethyldeoxyadenosine (m6dAm), N6,N6,2'-O- tri methyldeoxyadenosine (m62dAm), 8-Oxo-deoxyadenosine, and N6-Methyl- deoxyadenosine, preferably wherein the substitution of ATP is not in the polyA tail; and / or

[0214] 0.1% to 100% of UTP is substituted by a nucleotide selected from the group consisting of 2'NH2-deoxyuridine, 3'-Azido-2',3'-dideoxyuridine, Aminoallyl- deoxyuridine, 5- Propargylamino-deoxyuridine, 5-Fluoro-deoxyuridine, 5-Bromo- deoxyuridine, 5-lodo- deoxyuridine, deoxyuridine, 2',3'-Dideoxythymidine, Deoxythymidine, 5'-(a-seleno)- deoxythymidine (dTaSe), 5'-(a-thio)-deoxythymidine(dTaS), 3,2'-0-dimethyldeoxyuridine (m4dll), 2-thiodeoxyuridine (s2dll), 2-Fluoro-dUTP, pseudodeoxyuridine(dMJ), N1- Methylpseudouridine (ml d^P), 2'-0-methyldeoxyuridine (Udm), 4-thiodeoxyuridine (s4dll), 5-methyldeoxyuridine (m5dll), and 5-methoxydeoxyuridine (mo5dll), and / or

[0215] 0.1 to 100% of CTP is substituted by a nucleotide selected from the group consisting of deoxycytidine, C8-Alkyne-deoxycytidine, 5-Methyl-deoxycytidine, 2'-Fluoro-deoxycytidine, 5-lodo-deoxycytidine, 5-Bromo- deoxycytidia-seleno)- deoxycytidine (dCaSe), 5'-(a-thio)- deoxycytidine (dCaS), 5-Propargylamino-deoxycytidine, 5-Hydroxymethyl-deoxycytidine, and 2'-0-methyldeoxycytidine (dCm), and / or

[0216] 0.1 to 100% of GTP is substituted by a nucleotide selected from the group consisting of 8- Oxo-deoxyguanosine, 3'-deoxyguanosine, 7-Deaza-deoxyguanosine, 7-Deaza-7-iodo- deoxyguanosine, 6-Thio-deoxyguanosine, 2'-Fluoro-deoxyguanosine, Mant- deoxyguanosine, 2'-Mant-3'-deoxyguanosine, 2'NH2-deoxyguanosine, 5'-(a-seleno)- deoxyguanosine (dCaSe), 5'-(a-thio)-deoxyguanosine (dCaS), 2'-0- methyldeoxyguanosine (dGm), N2,7-dimethyldeoxyguanosine (m2,7dG), 7- methyldeoxguanosine (m7dG), and N2, N2,7-trimethyldeoxyguanosine (m2,7dG).

[0217] In one embodiment, the mRNA or saRNA comprises one or more modified nucleotides, wherein

[0218] (i) 25% to 100% of ATP is substituted by a nucleotide selected from the group consisting of deoxyadenosine(dA), 2'-lodo-deoxyadenosine, 2'-Bromo-deoxyadenosine, 3'- deoxyadenosine, 7-Deaza-deoxyadenosine, 8-Bromo-deoxyadenosine, 7-Deaza-7-iodo- deoxyadenosine, 2-Hydroxy-deoxyadenosine, 2'-Chloro-deoxyadenosine, 7-Deaza-7- bromo- deoxyadenosine, 2'-Fluoro-deoxyadenosine, Mant-deoxyadenosine, 2'-Mant-3'- deoxyadenosine, 2'NH2- deoxyadenosine, Etheno-deoxyadenosine (E-dA), 5'-(a-seleno)- deoxyadenosine (dAaSe), 5'-(a-thio)-deoxyadenosine (dAaS), N6-(6-Aminohexyl)- deoxyadenosine, N6-methyldeoxyadenosine (m6dA), 2'-0-methyldeoxyadenosine (2'OMedA), N6,2'-0-dimethyldeoxyadenosine (m6dAm), N6,N6,2'-O- tri methyldeoxyadenosine (m62dAm), 8-Oxo-deoxyadenosine, and N6-Methyl- deoxyadenosine, preferably wherein the substitution of ATP is not in the polyA tail; and / or 25% to 100% of UTP is substituted by a nucleotide selected from the group consisting of 2'NH2-deoxyuridine, 3'-Azido-2',3'-dideoxyuridine, Aminoallyl- deoxyuridine, 5- Propargylamino-deoxyuridine, 5-Fluoro-deoxyuridine, 5-Bromo- deoxyuridine, 5-lodo- deoxyuridine, deoxyuridine, 2',3'-Dideoxythymidine, Deoxythymidine, 5'-(a-seleno)- deoxythymidine (dTaSe), 5'-(a-thio)-deoxythymidine(dTaS), 3,2'-0-dimethyldeoxyuridine (m4dll), 2-thiodeoxyuridine (s2dll), 2-Fluoro-dUTP, pseudodeoxyuridine(dMJ), N1- Methylpseudouridine (ml d^P), 2'-0-methyldeoxyuridine (Udm), 4-thiodeoxyuridine (s4dll), 5-methyldeoxyuridine (m5dll), and 5-methoxydeoxyuridine (mo5dll), and / or

[0219] 25% to 100% of CTP is substituted by a nucleotide selected from the group consisting of deoxycytidine, C8-Alkyne-deoxycytidine, 5-Methyl-deoxycytidine, 2'-Fluoro-deoxycytidine, 5-lodo-deoxycytidine, 5-Bromo- deoxycytidia-seleno)- deoxycytidine (dCaSe), 5'-(a-thio)- deoxycytidine (dCaS), 5-Propargylamino-deoxycytidine, 5-Hydroxymethyl-deoxycytidine, and 2'-0-methyldeoxycytidine (dCm), and / or

[0220] (iv) 25% to 100% of GTP is substituted by a nucleotide selected from the group consisting of 8-Oxo-deoxyguanosine, 3'-deoxyguanosine, 7-Deaza-deoxyguanosine, 7-Deaza-7-iodo- deoxyguanosine, 6-Thio-deoxyguanosine, 2'-Fluoro-deoxyguanosine, Mant- deoxyguanosine, 2'-Mant-3'-deoxyguanosine, 2'NH2-deoxyguanosine, 5'-(a-seleno)- deoxyguanosine (dCaSe), 5'-(a-thio)-deoxyguanosine (dCaS), 2'-0- methyldeoxyguanosine (dGm), N2,7-dimethyldeoxyguanosine (m2,7dG), 7- methyldeoxguanosine (m7dG), and N2, N2,7-trimethyldeoxyguanosine (m2,7dG). Uses of the mRNA

[0221] In one further aspect, the invention also relates to the use of the mRNA of the invention in a method of increasing transcription and / or expression of a protein of interest encoded by the mRNA in vitro compared to an mRNA not comprising deoxyribonucleotides.

[0222] Self-amplifying RNA (saRNA) technology is particularly advantageous for the development of RNA vaccines. The single-cycle vector system, as described herein, utilizes an alphavirus RNA amplification system, the Venezuelan Equine Encephalitis Virus (VEEV)-based replicon expression vector. This vector expresses the alphavirus nonstructural proteins (nsPs)1-4, which together replicate and transcribe the saRNA resulting in efficient expression of the gene(s) of interest. Due to this self-amplification process, the level and duration of expression of target antigens is higher and longer than that observed with mRNA vaccine platforms. Therefore, prolonged presentation of the antigen to the immune system using this saRNA a platform is expected.

[0223] Hence, in one embodiment, the invention relates to a vaccine composition comprising the saRNA of the invention.

[0224] Self-amplifying RNA (saRNA) molecules comprise a nucleic acid sequence encoding the gene of interest and at least one nucleic acid sequence encoding RNA-dependent RNA Polymerase (RdRP, also called replicase). In one embodiment, the RdRP is a derived from an RNA virus.

[0225] In one embodiment, the RdRP is derived from a virus selected from the group consisting of Coronaviridae, Leviviridae, Cystoviridae, Reoviridae, Totiviridae, Hypoviridae, Partitiviridae, Mononegavirales, Orthmyxoviridae, Bunyvirales, Birnaviridae and Flaviviridae.

[0226] RNA replicase found in positive-strand ssRNA viruses are related to each other, forming three large superfamilies: I. Picorna-, noda-, como-, nepo-, poty-, bymo-, sobemoviruses, and a subset of luteoviruses (beet western yellows virus and potato leafroll virus). II. Carmo-, tombus-, dianthoviruses, another subset of luteoviruses (barley yellow dwarf virus), pestiviruses, hepatitis C virus (HCV), flaviviruses and single-stranded RNA bacteriophages. III. Tobamo-, tobra-, hordei-, tricornaviruses, beet yellows virus, alpha-, rubi-, furoviruses, hepatitis E virus (HEV), potex-, carla-, tymoviruses, and apple chlorotic leaf spot virus.

[0227] Hence, in one embodiment, the RdRP is a RNA replicase derived from a virus selected from the group consisting of picorna-, noda-, como-, nepo-, poty-, bymo-, sobemoviruses, beet western yellows virus and potato leafroll virus, carmo-, tombus-, dianthoviruses, barley yellow dwarf virus, pestiviruses, hepatitis C virus (HCV), flaviviruses, single-stranded RNA bacteriophagestobamo-, tobra-, hordei-, tricornaviruses, beet yellows virus, alpha-, rubi-, furoviruses, hepatitis E virus (HEV), potex-, carla-, tymoviruses, and apple chlorotic leaf spot virus.

[0228] In one embodiment, the RdRP is selected from Bunyavirus RNA replicase and Alphavirus RNA replicase. In a preferred embodiment, the RdRP is an Alphavirus RNA replicase, such as Venezuelan Equine Encephalitis Virus (VEEV) replicase. Although it is possible to use the composition provided in the present invention for the purpose of therapy, the composition may be preferably administered in the form of a pharmaceutical formulation, for example, an admixture with a suitable pharmaceutical excipient, diluent or carrier selected with regard to the intended route of administration and standard pharmaceutical practice. Therefore, according to one aspect of the present invention, there is provided a pharmaceutical composition or formulation including at least one active composition, or a pharmaceutically acceptable derivative thereof, in association with a pharmaceutically acceptable excipient, diluent and / or carrier. The excipient, diluent and / or carrier should be “acceptable” in terms of being compatible with the other ingredients of the formulation and not deleterious to the recipient thereof.

[0229] The composition of the invention may be formulated to be administered in any convenient manner for use in drugs for humans or vertebrates. Therefore, the scope of the present invention includes pharmaceutical compositions including a product of the present invention that is adapted for use in drugs for humans or vertebrates.

[0230] Acceptable excipients, diluents, and carriers for therapeutic use are well known in the field of pharmaceuticals, and the choice of pharmaceutical excipients, diluents, and carriers may be selected with regard to the intended route of administration and standard pharmaceutical practice.

[0231] The dosage of an adjuvant formulation or vaccine composition containing the adjuvant will vary widely, depending upon the nature of the disease, the patient's medical history, the frequency of administration, the administration mode, the clearance of the agents from the host, and the like. The initial dose may be larger, followed by smaller maintenance doses. The dose may be administered as infrequently as monthly or annually to maintain an effective immunological memory.

[0232] Also, the present invention encompasses a pharmaceutical composition and a vaccine. The pharmaceutical composition and vaccine composition of the present invention includes a pharmaceutically acceptable carrier or excipient along with at least one mRNA and one or more adjuvants. Methods of formulating the pharmaceutical composition and vaccine are well known to those having ordinary skill in the art.

[0233] The vaccine compositions of the present invention may include pharmaceutically acceptable diluents, preservatives, solubilizers, emulsifiers, adjuvants and / or carriers. Such compositions have various buffer contents (for example, Tris-HCI, acetate, phosphate), and a pH and ionic strength; and include additives such as surfactants and solubilizers (for example, Tween 80, Polysorbate 80), antioxidants (for example, ascorbic acid, sodium metabisulfite), preservatives (for example, Thimersol, benzyl alcohol), and bulking substances (for example, lactose, mannitol); wherein the materials are incorporated into certain preparations of polymeric compounds such as polylactic acid, polyglycolic acid, and the like or incorporated into liposomes. Hylauronic acid may also be used.

[0234] In one embodiment, the vaccine composition comprises lipid nanoparticle, lipid, cholesterol, peptides, a solvent, aqueous solvent, non-aqueous solvent , polymer, dispersion media, diluent, dispersion, suspension aid, surface active agent, isotonic agent, thickening or emulsifying agent, protein, poloxamine, cell, core-shell nanoparticles, lipoplex, liposome, lipoplex peptide, lipidplex, commonly used inorganic nanoparticles (including gold nanoparticles, iron oxide nanoparticles and mesoporous silica nanoparticles (MSNs)), hyaluronidase, preservative, poloxamer, chitosan, monosaccharides, disaccharides, polysaccharides, cationic lipid (dilinoleylmethyl-4-dimethylaminobutyrate (DLin-MC3- DMA)), phospholipid (1 ,2-distearoyl-sn-glycero-3-phosphocholine (DSPC)), PEG lipid (1 ,2- dimyristoyl-sn-glycerol, methoxypolyethylene glycol and polyethylene glycol-dimyristoyl glycerol (PEG-DMG)), PEG, linear and branched polyethylenimine (PEI), poly(P-amino ester), polyamidoamine (PAMAM) dendrimer, and mixtures thereof.

[0235] Preparations for parenteral administration according to the present invention include a sterile aqueous or non-aqueous solution, a suspension, or an emulsion. Examples of non-aqueous solvents or vehicles include propylene glycol, polyethylene glycol, vegetable oils, such as olive oil and corn oil, gelatin, and injectable organic esters such as ethyl oleate. Such formulations may also contain an adjuvant, a preservative, a wetting agent, an emulsifier, and a dispersing agent. The pharmaceutical compositions may, for example, be sterilized by filtering the compositions through a bacteria- retaining filter, incorporating a sterilizing agent into the compositions, irradiating the compositions, or heating the compositions. These compositions may also be prepared using sterile water or other sterile injectable media, immediately before use thereof.

[0236] In the case of vaccines, it is often observed that a primary challenge with an antigen alone, in the absence of an adjuvant, fails to elicit a humoral or cellular immune response. Therefore, the vaccines of the invention may contain adjuvants including, but not limited to, cholera toxins, fragments and mutants or derivatives having adjuvant properties, E. coli heat-labile enterotoxins, fragments and mutants or derivatives having adjuvant properties, oil-in-water and water-in-oil emulsions, toll-like receptor ligands such as a muramyl dipeptide, E. coli LPS, oligonucleotides containing unmethylated DNA, poly(l:C), lipoteichoic acid, peptidoglycans. Enterotoxins and adjuvants thereof include active derivatives such as cholera toxins, heat-labile E. coli enterotoxins, pertussis toxins, Shiga toxins, and analogues. Other adjuvants such as complete Freund's adjuvants, incomplete Freund's adjuvants, saponin, mineral gels such as aluminum hydroxide, surface active materials such as lysolecithin, pluronic polyols, polyanions, peptides, oil or hydrocarbon emulsions, keyhole limpet hemocyanin, and potentially useful human adjuvants such as N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP), N-acetyl-normuramyl-L-alanyl-D-isoglutamine, N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine- 2-(1 '-2'-dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamine, Bacille Calmette-Guerin (BCG), and Corynebacterium parvum may be used. An adjuvant may serve as a tissue depot that slowly releases the antigens and may also serve as a lymphoid system activator that enhances an immune response in a non-specific manner. When the vaccine is intended for use in human subjects, the adjuvant should be pharmaceutically acceptable.

[0237] Such pharmaceutical compositions and vaccines may be administered orally (in a solid or liquid phase), parenterally (by intramuscular, intraperitoneal, intravenous (IV), or subcutaneous injection), transdermally (either passively or using ionophoresis or electroporation), transmucosally (nasally, vaginally, rectally, or sublingually), or via an inhalation route of administration, or administered using a bioerodible insert, and may be prepared into formulations suitable or each of the routes of administration. In one preferred exemplary embodiment, the compositions or vaccines are administered by means of pulmonary delivery. The compositions or vaccines are delivered to the lungs of a mammal during inhalation, and traverses the epithelial lining of the lungs into the blood stream.

[0238] Nasal delivery or other mucosal delivery of the therapeutic agent is also contemplated. The nasal delivery allows a direct passage of the composition into the blood stream without any necessity for deposition of the product in the lung after the composition is administered to the nose. Formulations for nasal delivery include those with dextran or cyclodextran and saponin as adjuvants.

[0239] The compositions or vaccines of the present invention may be administered in conjunction with one or more additional active ingredients, pharmaceutical compositions, or vaccines. The therapeutic agent of the present invention may be administered to an animal, preferably a mammal, most preferably a human.

[0240] Following the methodologies well-established in the related art, an effective dose and toxicity of the compounds and compositions, which are easily used in in vitro tests, are determined in preclinical studies using a small animal model (for example, mice or rats) in which these drugs may be administered by the same route proposed for the human clinical trials.

[0241] For any vaccine compositions used in the method of the present invention, the therapeutically or prophylactically effective dose may be preferentially estimated from an animal model. A doseresponse curve derived from an animal system is then used to determine testing doses for the initial clinical trials in humans. To determine safety for each of the compositions, the dose and frequency of administration should meet or surpass the requirements anticipated for use in the

[0242] In another embodiment, the present invention provides a method for delivering a recombinant protein to a subject, the method comprising the step of contacting the subject with a purified preparation of an mRNA molecule of the invention, preferably an mRNA molecule synthesized with the method of the invention, thereby delivering a recombinant protein to a subject.

[0243] Further Embodiments

[0244] The invention is also described by the following items:

[0245] 1 . A method of synthesizing an mRNA in vitro, comprising the steps of: a) providing a composition comprising ribonucleotides, wherein between 1 % to 100% of at least one of the ribonucleotides is substituted by a deoxyribonucleotide version of the at least one ribonucleotide; b) adding a nucleic acid template encoding a protein of interest to the composition; and c) incubating the composition under conditions that allow mRNA synthesis.

[0246] 2. The method of item 1 , wherein the mRNA is a self-amplifying RNA (saRNA). 3. The method of item 1 , wherein the deoxyribonucleotide is a modified or unmodified deoxy ribonucleotide.

[0247] 4. The method of any one of the preceding items, wherein in step a) the composition comprises ATP, CTP, UTP and GTP.

[0248] 5. The method of any one of the preceding items, wherein in step a) the composition comprises ATP, CTP, UTP and GTP, and wherein between 1 % to 75% of CTP are substituted by a deoxyribonucleotide version of CTP.

[0249] 6. The method of any one of the preceding items, wherein in step a) the composition comprises ATP, CTP, UTP and GTP and wherein between 1% to 75% of CTP are substituted by a deoxyribonucleotide version of CTP selected from the group consisting of dCTP, 5-Methyl-dCTP, 2'- Fluoro-dCTP, 3'-Azido-2',3'-ddCTP, 7-Deaza-dGTP, C8-Alkyne-dCTP, 5-lodo dCTP, 5-Bromo-dCTP, 2'NH2-dCTP, dCTPaSe, dCTPaS, 5-Propargylamino-dCTP, and 5-Hydroxymethyl-dCTP.

[0250] 7. A method of synthesizing an saRNA in vitro, comprising the steps of: a) providing a composition comprising ATP, CTP, UTP and GTP ribonucleotides, wherein between 0.1% to 100% of at least one of the ribonucleotides is substituted by a deoxyribonucleotide version of the at least one ribonucleotide; b) adding a nucleic acid template encoding a protein of interest to the composition; and c) incubating the composition under conditions that allow saRNA synthesis.

[0251] 8. The method of item 7, wherein the deoxyribonucleotide is a modified or unmodified deoxy ribonucleotide.

[0252] 9. The method of any one of items 7 or 8, wherein in step a) between 1% to 75% of CTP are substituted by a deoxyribonucleotide version of CTP.

[0253] 10. The method of item 9, wherein in step between 1% to 75% of CTP are substituted by a deoxyribonucleotide version of CTP selected from the group consisting of dCTP, 5-Methyl-dCTP, 2'- Fluoro-dCTP, 3'-Azido-2',3'-ddCTP, 7-Deaza-dGTP, C8-Alkyne-dCTP, 5-lodo dCTP, 5-Bromo-dCTP, 2'NH2-dCTP, dCTPaSe, dCTPaS, 5-Propargylamino-dCTP, and 5-Hydroxymethyl-dCTP.

[0254] 11 . An mRNA synthesized by the method of any one of items 1 to 6.

[0255] 12. An saRNA synthesized by the method of any one of items 7 to 10.

[0256] 13. An mRNA encoding a protein of interest, wherein between 0.1% to 100% of at least one of the ribonucleotides are substituted by a deoxyribonucleotide version of the at least one ribonucleotide.

[0257] 14. An saNA encoding a protein of interest, wherein between 0.1 % to 100% of at least one of the ribonucleotides are substituted by a deoxyribonucleotide version of the at least one ribonucleotide.

[0258] 15. The mRNA of any one of items 11 or 13 or the saRNA of any one of items 12 or 14, wherein the deoxyribonucleotide is a modified or unmodified deoxyribonucleotide. 16. The mRNA of any one of items 11 or 13 or the saRNA of any one of items 12 or 14, wherein the at least one ribonucleotide that is substituted by a deoxyribonucleotide version is selected from the group consisting of ATP, CTP and UTP, preferably ATP and CTP, more preferably CTP.

[0259] 17. The mRNA of any one of items 11 or 13 or the saRNA of any one of items 12 or 14, wherein between 1% to 75% of at least one of the ribonucleotides are substituted by a deoxyribonucleotide version of the at least one ribonucleotide.

[0260] 18. The mRNA of any one of items 11 or 13 or the saRNA of any one of items 12 or 14, wherein between 10% to 75% of at least one of the ribonucleotides are substituted by a deoxyribonucleotide version of the at least one ribonucleotide.

[0261] 19. The mRNA of any one of items 11 or 13 or the saRNA of any one of items 12 or 14, wherein between 20% to 75% of at least one of the ribonucleotides are substituted by a deoxyribonucleotide version of the at least one ribonucleotide.

[0262] 20. The mRNA of any one of items 11 or 13 or the saRNA of any one of items 12 or 14, wherein between 25% to 75% of at least one of the ribonucleotides are substituted by a deoxyribonucleotide version of the at least one ribonucleotide.

[0263] 21. The mRNA of any one of items 11 or 13 or the saRNA of any one of items 12 or 14, wherein between 0.1 % to 75% of at least one of the ribonucleotides are substituted by a deoxyribonucleotide version of the at least one ribonucleotide, wherein the at least one ribonucleotide that is substituted by a deoxyribonucleotide version is CTP and the deoxyribonucleotide version is modified by a modification is selected from the group consisting of the following naturally or synthetically occurring ribonucleotide or deoxyribonucleotide modifications hm5Cm, s2C, m3C, phm5C, hm5C, f5C, pf5C, ho5C, hm5C, m5C, pC2'3'cp, m4,4C, ac4C, m4C, 2NH2-C, 3’-Azido-2’,3’-ddCTP, C8-Alkyne-C, 5- Propargylamino-C, LCC, CBV, CBR, IC, A5M, CSL, RY, 5CF, 10C, S4C, 73W, CH, N5M, 5IC, M5M, CAR, CFZ, CFL, D2C, 1 mC, m2C, e3C, 3mC, sC, 4mC, 5caC, 5fC, 5hmC, 5gmC and 5mC; or wherein the at least one ribonucleotide that is substituted by a deoxyribonucleotide version is ATP and the deoxyribonucleotide version is modified by a modification is selected from the group consisting of the following naturally or synthetically occurring ribonucleotide or deoxyribonucleotide modifications m1A, msms2i6A, m2,8A, m2A, ms2ct6A, , ms2io6A, ms2hn6A, ms2hn6A, ms2i6A, ms2m6A, ms2t6A, ms2t6A, NADpN, m8A, ct6A, ct6A, ht6A, io6A, pio6A, m62A, ac6A, pac6A, f6A, pf6A, g6A, hm6A, hn6A, m6A, m6t6A, t6A, 2’-Bromo-A, LCA, IG, SRA, AF2, ADS, 2AD, 8AN, PPU, 3DA, AP7, 45A, A9Z, ANZ, A5O, A5L, 5AA, A43, 2HA AD2, 1 mA, 3mA, 4mA, 6mA, 7mA, 9mA, 6hmA, HAP, m2A, AHAP, 6-DMA, ncm6A and sA; or wherein the at least one ribonucleotide that is substituted by a deoxyribonucleotide version is GTP and the deoxyribonucleotide version is modified by a modification is selected from the group consisting of the following naturally or synthetically occurring ribonucleotide or deoxyribonucleotide modifications m1 G, preQ1 , preQO, m7G, m2,7G, m2,2,7G, m22G, m2G I, mi l, m1 lm, Im, Q, oQ, dGdAIQ, mdAnQ, preQO, preQ1 , dG+ , 7-Deaza-G, 2’NH2-dG, 7-Deaza-7-iodo-G, 6-thio-dG, dCaSe, dCaS, Fluoro-G, MANT-G, 2-MANT-G, LCG, BGM, 2SG, GRB, GAO, TG, GDO, GMX, XUG, G3A, GS, G38, 2HG, 1 mG, 2mG, 3mG, m6G, 7mG, 8-oxo-G, ADG, 1 ,N(2)-eG, N(2),3-eG, m22G, m2,7dG, m2,2,7dG, 2’NH2-dG and CEG; or wherein the at least one ribonucleotide that is substituted by a deoxyribonucleotide version is UTP and the deoxyribonucleotide version is modified by a modification is selected from the group consisting of the following naturally or synthetically occurring ribonucleotide or deoxyribonucleotide cmo5U, mcmo5U, 2-NH2-dU, 3’-Azido-2’,3’-ddU, Aminoallyl-dU, 5-Propargylamino-dU, 5-Fluoro- dU, 5-dimethyl-2’-dU, 5BU, U37, IU, TLN, BRU, URU, UFB, U5M, UOB, UMO, U5R, B8N, U4M, UAR, UFT, UMS, NYM, MU1 , P1T, M, MU4, putThy, ddT, dTaSe, dTaS, 1mT, 3mT, 04-meT, diHT, 5hmU, 5fU, base J, dhpUra, 5caU, 5-NeOmdU, dU, DHdU, 5-NedU, and dhpdU.

[0264] 22. The mRNA of any one of items 11 or 13 or the saRNA of any one of items 12 or 14, wherein between 1% to 75% of at least one of the ribonucleotides are substituted by a deoxyribonucleotide version of the at least one ribonucleotide, wherein the at least one ribonucleotide that is substituted by a deoxyribonucleotide version is CTP and the deoxyribonucleotide version is modified by a modification is selected from the group consisting of the following naturally or synthetically occurring ribonucleotide or deoxyribonucleotide modifications hm5Cm, s2C, m3C, phm5C, hm5C, f5C, pf5C, ho5C, hm5C, m5C, pC2'3'cp, m4,4C, ac4C, m4C, 2NH2-C, 3’-Azido-2’,3’-ddCTP, C8-Alkyne-C, 5- Propargylamino-C, LCC, CBV, CBR, IC, A5M, CSL, RY, 5CF, 10C, S4C, 73W, CH, N5M, 5IC, M5M, CAR, CFZ, CFL, D2C, 1 mC, m2C, e3C, 3mC, sC, 4mC, 5caC, 5fC, 5hmC, 5gmC and 5mC; or wherein the at least one ribonucleotide that is substituted by a deoxyribonucleotide version is ATP and the deoxyribonucleotide version is modified by a modification is selected from the group consisting of the following naturally or synthetically occurring ribonucleotide or deoxyribonucleotide modifications m1A, msms2i6A, m2,8A, m2A, ms2ct6A, , ms2io6A, ms2hn6A, ms2hn6A, ms2i6A, ms2m6A, ms2t6A, ms2t6A, NADpN, m8A, ct6A, ct6A, ht6A, io6A, pio6A, m62A, ac6A, pac6A, f6A, pf6A, g6A, hm6A, hn6A, m6A, m6t6A, t6A, 2’-Bromo-A, LCA, IG, SRA, AF2, ADS, 2AD, 8AN, PPU, 3DA, AP7, 45A, A9Z, ANZ, A5O, A5L, 5AA, A43, 2HA AD2, 1 mA, 3mA, 4mA, 6mA, 7mA, 9mA, 6hmA, HAP, m2A, AHAP, 6-DMA, ncm6A and sA; or wherein the at least one ribonucleotide that is substituted by a deoxyribonucleotide version is GTP and the deoxyribonucleotide version is modified by a modification is selected from the group consisting of the following naturally or synthetically occurring ribonucleotide or deoxyribonucleotide modifications m1 G, preQ1 , preQO, m7G, m2,7G, m2,2,7G, m22G, m2G I, mi l, m1 lm, Im, Q, oQ, dGdAIQ, mdAnQ, preQO, preQ1 , dG+ , 7-Deaza-G, 2’NH2-dG, 7-Deaza-7-iodo-G, 6-thio-dG, dCaSe, dCaS, Fluoro-G, MANT-G, 2-MANT-G, LCG, BGM, 2SG, GRB, GAO, TG, GDO, GMX, XUG, G3A, GS, G38, 2HG, 1 mG, 2mG, 3mG, m6G, 7mG, 8-oxo-G, ADG, 1 ,N(2)-eG, N(2),3-eG, m22G, m2,7dG, m2,2,7dG, 2’NH2-dG and CEG; or wherein the at least one ribonucleotide that is substituted by a deoxyribonucleotide version is UTP and the deoxyribonucleotide version is modified by a modification is selected from the group consisting of the following naturally or synthetically occurring ribonucleotide or deoxyribonucleotide cmo5U, mcmo5U, 2-NH2-dU, 3’-Azido-2’,3’-ddU, Aminoallyl-dU, 5-Propargylamino-dU, 5-Fluoro- dU, 5-dimethyl-2’-dU, 5BU, U37, IU, TLN, BRU, URU, UFB, U5M, UOB, UMO, U5R, B8N, U4M, UAR, UFT, UMS, NYM, MU1 , P1T, M, MU4, putThy, ddT, dTaSe, dTaS, 1mT, 3mT, 04-meT, diHT, 5hmU, 5fU, base J, dhpUra, 5caU, 5-NeOmdU, dU, DHdU, 5-NedU, and dhpdU.

[0265] 23. The mRNA of any one of items 11 or 13 or the saRNA of any one of items 12 or 14, wherein between 10% to 75% of at least one of the ribonucleotides are substituted by a deoxyribonucleotide version of the at least one ribonucleotide, wherein the at least one ribonucleotide that is substituted by a deoxyribonucleotide version is CTP and the deoxyribonucleotide version is modified by a modification is selected from the group consisting of the following naturally or synthetically occurring ribonucleotide or deoxyribonucleotide modifications hm5Cm, s2C, m3C, phm5C, hm5C, f5C, pf5C, ho5C, hm5C, m5C, pC2'3'cp, m4,4C, ac4C, m4C, 2NH2-C, 3’-Azido-2’,3’-ddCTP, C8-Alkyne-C, 5- Propargylamino-C, LCC, CBV, CBR, IC, A5M, CSL, RY, 5CF, 10C, S4C, 73W, CH, N5M, 5IC, M5M, CAR, CFZ, CFL, D2C, 1 mC, m2C, e3C, 3mC, sC, 4mC, 5caC, 5fC, 5hmC, 5gmC and 5mC; or wherein the at least one ribonucleotide that is substituted by a deoxyribonucleotide version is ATP and the deoxyribonucleotide version is modified by a modification is selected from the group consisting of the following naturally or synthetically occurring ribonucleotide or deoxyribonucleotide modifications m1A, msms2i6A, m2,8A, m2A, ms2ct6A, , ms2io6A, ms2hn6A, ms2hn6A, ms2i6A, ms2m6A, ms2t6A, ms2t6A, NADpN, m8A, ct6A, ct6A, ht6A, io6A, pio6A, m62A, ac6A, pac6A, f6A, pf6A, g6A, hm6A, hn6A, m6A, m6t6A, t6A, 2’-Bromo-A, LCA, IG, SRA, AF2, ADS, 2AD, 8AN, PPU, 3DA, AP7, 45A, A9Z, ANZ, A5O, A5L, 5AA, A43, 2HA AD2, 1 mA, 3mA, 4mA, 6mA, 7mA, 9mA, 6hmA, HAP, m2A, AHAP, 6-DMA, ncm6A and sA; or wherein the at least one ribonucleotide that is substituted by a deoxyribonucleotide version is GTP and the deoxyribonucleotide version is modified by a modification is selected from the group consisting of the following naturally or synthetically occurring ribonucleotide or deoxyribonucleotide modifications m1 G, preQ1 , preQO, m7G, m2,7G, m2,2,7G, m22G, m2G I, mi l, m1 lm, Im, Q, oQ, dGdAIQ, mdAnQ, preQO, preQ1 , dG+ , 7-Deaza-G, 2’NH2-dG, 7-Deaza-7-iodo-G, 6-thio-dG, dCaSe, dCaS, Fluoro-G, MANT-G, 2-MANT-G, LCG, BGM, 2SG, GRB, GAO, TG, GDO, GMX, XUG, G3A, GS, G38, 2HG, 1 mG, 2mG, 3mG, m6G, 7mG, 8-oxo-G, ADG, 1 ,N(2)-eG, N(2),3-eG, m22G, m2,7dG, m2,2,7dG, 2’NH2-dG and CEG; or wherein the at least one ribonucleotide that is substituted by a deoxyribonucleotide version is UTP and the deoxyribonucleotide version is modified by a modification is selected from the group consisting of the following naturally or synthetically occurring ribonucleotide or deoxyribonucleotide cmo5U, mcmo5U, 2-NH2-dU, 3’-Azido-2’,3’-ddU, Aminoallyl-dU, 5-Propargylamino-dU, 5-Fluoro- dU, 5-dimethyl-2’-dU, 5BU, U37, IU, TLN, BRU, URU, UFB, U5M, UOB, UMO, U5R, B8N, U4M, UAR, UFT, UMS, NYM, MU1 , P1T, M, MU4, putThy, ddT, dTaSe, dTaS, 1mT, 3mT, 04-meT, diHT, 5hmU, 5fU, base J, dhpUra, 5caU, 5-NeOmdU, dU, DHdU, 5-NedU, and dhpdU.

[0266] 24. The mRNA of any one of items 11 or 13 or the saRNA of any one of items 12 or 14, wherein between 1% to 75% of at least one of the ribonucleotides are substituted by a deoxyribonucleotide version of the at least one ribonucleotide, wherein the at least one ribonucleotide that is substituted by a deoxyribonucleotide version is CTP and the substituted deoxyribonucleotide version is selected from the group consisting of dCTP, 5-Methyl-dCTP, 2'-Fluoro-dCTP, 3'-Azido-2',3'-ddCTP, 7-Deaza- dCTP, C8-Alkyne-dCTP, 5-lodo dCTP, 5-Bromo-dCTP, 2'NH2-dCTP, dCTPaSe, dCTPaS, 5- Propargylamino-dCTP, and 5-Hydroxymethyl-dCTP, 3'-Methyl-dCTP, 2'-O-Methyl-dCTP, 2'-thio- dCTP, N4'-acetyl-dCTP, 5'-formyl-dCTP, 5,2'-O-dimethyl-dCTP, N4-acetyl-2'-O-methyl- dCTP, N4- methyl-dCTP, N4,2'-O-dimethyl- dCTP, 5-hydroxymethyl-dCTP, 5-formyl-2'-0-methyl-dCTP, and N4,N4,2'-O-trimethyl-dCTP; or wherein the at least one ribonucleotide that is substituted by a deoxyribonucleotide version is ATP and the substituted deoxyribonucleotide version is selected from the group consisting of dATP and 2'-Bromo-dATP, 1'-Methyl-dATP, 2'-Methyl-dATP, N6'-Methyl-dATP, 2'-O-Methyl-dATP, 2'- methylthio-N6-methyl-dATP, N6-isopentenyl-dATP, 2-methylthio-N6-isopentenyl-dATP, N6-(cis- hydroxyisopentenyl)- dATP, N6-glycinylcarbamoyl-dATP, N6-threonylcarbamoyl-dATP, 2-methylthio- N6-threonyl carbamoyl-dATP, N6-methyl-N6-threonylcarbamoyl-dATP, N6- hydroxynorvalylcarbamoyl-dATP, 2-methylthio-N6-hydroxynorvalyl carbamoyl-dATP, N6,N6- dimethyl-dATP, N6,2'-O-dimethyl-dATP, N6,N6,2'-O-trimethyl-dATP, 1 ,2'-O-dimethyl-dATP, N6- acetyl-dATP, 8-methyl-dATP, and cyclic N6-threonylcarbamoyl-dATP; or wherein the at least one ribonucleotide that is substituted by a deoxyribonucleotide version is UTP and the substituted deoxyribonucleotide version is selected from the group consisting of dTTP, dUTP, 2'-O-methyl-dUTP, 3,2'-O-dimethyl-dUTP, 2'NH2-deoxyuridine, 3'-Azido-2',3'-dideoxyuridine, Aminoallyl- deoxyuridine, 5-Propargylamino-deoxyuridine, 5-Fluoro-deoxyuridine, 5-Bromo- deoxyuridine, 5-lodo-deoxyuridine, deoxyuridine, 2',3'-Dideoxythymidine, Deoxythymidine, 5'-(a- seleno)-deoxythymidine (dTaSe), 5'-(a-thio)-deoxythymidine(dTaS), 3,2'-0-dimethyldeoxyuridine (m4dU), 2-thiodeoxyuridine (s2dU), 2-Fluoro-dUTP, pseudodeoxyuridine(dlP), N1- Methylpseudouridine (ml d'P), 2'-0-methyldeoxyuridine (Udm), 4-thiodeoxyuridine (s4dU), 5- methyldeoxyuridine (m5dU), and 5-methoxydeoxyuridine (mo5dU); or wherein the at least one ribonucleotide that is substituted by a deoxyribonucleotide version is GTP and the substituted deoxyribonucleotide version is selected from the group consisting of dGTP, 8- Oxo-deoxyguanosine, 3'-deoxyguanosine, 7-Deaza-deoxyguanosine, 7-Deaza-7-iodo- deoxyguanosine, 6-Thio-deoxyguanosine, 2'-Fluoro-deoxyguanosine, Mant- deoxyguanosine, 2'- Mant-3'-deoxyguanosine, 2'NH2-deoxyguanosine, 5'-(a-seleno)-deoxyguanosine (dCaSe), 5'-(a- thio)-deoxyguanosine (dCaS), 2'-0-methyldeoxyguanosine (dGm), N2,7-dimethyldeoxyguanosine (m2’7dG), 7-methyldeoxguanosine (m7dG), N2, and N2,7-trimethyldeoxyguanosine (m2’2’7dG).

[0267] 25. The mRNA of any one of items 11 or 13 or the saRNA of any one of items 12 or 14, wherein between 1% to 75% of at least one of the ribonucleotides are substituted by a deoxyribonucleotide version of the at least one ribonucleotide, wherein the at least one ribonucleotide that is substituted by a deoxyribonucleotide version is CTP and the substituted deoxyribonucleotide version is selected from the group consisting of dCTP, 5-Methyl-dCTP, 2'-Fluoro-dCTP, 3'-Azido-2',3'-ddCTP, 7-Deaza- dGTP, C8-Alkyne-dCTP, 5-lodo dCTP, 5-Bromo-dCTP, 2'NH2-dCTP, dCTPaSe, dCTPaS, 5- Propargylamino-dCTP, and 5-Hydroxymethyl-dCTP; or wherein the at least one ribonucleotide that is substituted by a deoxyribonucleotide version is ATP and the substituted deoxyribonucleotide version is selected from the group consisting of dATP and 2'-Bromo-dATP; or

[0268] Wherein the at least one ribonucleotide that is substituted by a deoxyribonucleotide version is UTP and the substituted deoxyribonucleotide version is dTTP; or

[0269] Wherein the at least one ribonucleotide that is substituted by a deoxyribonucleotide version is GTP and the substituted deoxyribonucleotide version is selected from the group consisting of dGTP and 7- deaza-dGTP.

[0270] 26. Use of the saRNA of any one of items 12 or 14 in a method of increasing transcription and / or expression of a protein of interest encoded by the saRNA in vitro compared to an saRNA not comprising deoxyribonucleotides.

[0271] 27. A pharmaceutical composition comprising the saRNA of any one of items 12 or 14 and optionally at least one excipient.

[0272] 28. A vaccine composition comprising the saRNA of any one of items 12 or 14.

[0273] 29. The saRNA of any one of items 12 or 14 for use as a medicament.

[0274] 30. The mRNA of any one of items 11 or 13 or the saRNA of any one of items 12 or 14, wherein between 0.1 % to 75% of at least one of the ribonucleotides are substituted by a deoxyribonucleotide version of the at least one ribonucleotide, wherein the at least one ribonucleotide that is substituted by a deoxyribonucleotide version is CTP and the deoxyribonucleotide version is modified by a modification is selected from the group consisting of the following naturally or synthetically occurring ribonucleotide or deoxyribonucleotide modifications hm5Cm, s2C, m3C, phm5C, hm5C, f5C, pf5C, ho5C, hm5C, m5C, pC2'3'cp, m4,4C, ac4C, m4C, 2NH2-C, 3’-Azido-2’,3’-ddCTP, C8-Alkyne-C, 5- Propargylamino-C, LCC, CBV, CBR, IC, A5M, CSL, RY, 5CF, 10C, S4C, 73W, CH, N5M, 5IC, M5M, CAR, CFZ, CFL, D2C, 1 mC, m2C, e3C, 3mC, sC, 4mC, 5caC, 5fC, 5hmC, 5gmC and 5mC; or wherein the at least one ribonucleotide that is substituted by a deoxyribonucleotide version is ATP and the deoxyribonucleotide version is modified by a modification is selected from the group consisting of the following naturally or synthetically occurring ribonucleotide or deoxyribonucleotide modifications m1A, msms2i6A, m2,8A, m2A, ms2ct6A, , ms2io6A, ms2hn6A, ms2hn6A, ms2i6A, ms2m6A, ms2t6A, ms2t6A, NADpN, m8A, ct6A, ct6A, ht6A, io6A, pio6A, m62A, ac6A, pac6A, f6A, pf6A, g6A, hm6A, hn6A, m6A, m6t6A, t6A, MANT-dA, 2'-lodo-dA, 7'-Deaza-dA, 8'-Bromo-dA, 7'- Deaza-7'-iodo-dA, 2'-Hydroxy-dA, 7'-Deaza-7-bromo-dA, 2'-Fluoro-dA, Mant-dA, 2'NH2-dA, Etheno- dA (e-dA), dAaSe, dAaS, 8'-Oxo-dA, 2’-Bromo-A, LCA, IG, SRA, AF2, ADS, 2AD, 8AN, PPU, 3DA, AP7, 45A, A9Z, ANZ, A5O, A5L, 5AA, A43, 2HA AD2, 1 mA, 3mA, 4mA, 6mA, 7mA, 9mA, 6hmA, HAP, m2A, AHAP, 6-DMA, ncm6A and EA; or wherein the at least one ribonucleotide that is substituted by a deoxyribonucleotide version is GTP and the deoxyribonucleotide version is modified by a modification is selected from the group consisting of the following naturally or synthetically occurring ribonucleotide or deoxyribonucleotide modifications m1 G, preQ1 , preQO, m7G, m2,7G, m2,2,7G, m22G, m2G I, mi l, m1 lm, Im, Q, oQ, dGdAIQ, mdAnQ, preQO, preQ1 , dG+ , 7-Deaza-G, 2’NH2-dG, 7-Deaza-7-iodo-G, 6-thio-dG, dCaSe, dCaS, Fluoro-G, MANT-G, 2-MANT-G, LCG, BGM, 2SG, GRB, GAO, TG, GDO, GMX, XUG, G3A, GS, G38, 2HG, 1 mG, 2mG, 3mG, m6G, 7mG, 8-oxo-G, ADG, 1 ,N(2)-EG, N(2),3-EG, m22G, m2,7dG, m2,2,7dG, 2’NH2-dG and CEG; or wherein the at least one ribonucleotide that is substituted by a deoxyribonucleotide version is UTP and the deoxyribonucleotide version is modified by a modification is selected from the group consisting of the following naturally or synthetically occurring ribonucleotide or deoxyribonucleotide cmo5U, mcmo5U, 2-NH2-dU, 3’-Azido-2’,3’-ddU, Aminoallyl-dU, 5-Propargylamino-dU, 5-Fluoro- dU, 5-dimethyl-2’-dU, 5BU, U37, IU, TLN, BRU, URU, UFB, U5M, UOB, UMO, U5R, B8N, U4M, UAR, UFT, UMS, NYM, MU1 , P1T, M, MU4, putThy, ddT, dTaSe, dTaS, 1 mT, 3mT, 04-meT, diHT, 5hmU, 5fU, base J, dhpUra, 5caU, 5-NeOmdU, dU, DHdU, 5-NedU, and dhpdU.

[0275] 31. The mRNA of any one of items 11 or 13 or the saRNA of any one of items 12 or 14, wherein between 1% to 75% of at least one of the ribonucleotides are substituted by a deoxyribonucleotide version of the at least one ribonucleotide, wherein the at least one ribonucleotide that is substituted by a deoxyribonucleotide version is CTP and the deoxyribonucleotide version is modified by a modification is selected from the group consisting of the following naturally or synthetically occurring ribonucleotide or deoxyribonucleotide modifications hm5Cm, s2C, m3C, phm5C, hm5C, f5C, pf5C, ho5C, hm5C, m5C, pC2'3'cp, m4,4C, ac4C, m4C, 2NH2-C, 3’-Azido-2’,3’-ddCTP, C8-Alkyne-C, 5- Propargylamino-C, LCC, CBV, CBR, IC, A5M, CSL, RY, 5CF, 10C, S4C, 73W, CH, N5M, 5IC, M5M, CAR, CFZ, CFL, D2C, 1 mC, m2C, e3C, 3mC, sC, 4mC, 5caC, 5fC, 5hmC, 5gmC and 5mC; or wherein the at least one ribonucleotide that is substituted by a deoxyribonucleotide version is ATP and the deoxyribonucleotide version is modified by a modification is selected from the group consisting of the following naturally or synthetically occurring ribonucleotide or deoxyribonucleotide modifications m1A, msms2i6A, m2,8A, m2A, ms2ct6A, , ms2io6A, ms2hn6A, ms2hn6A, ms2i6A, ms2m6A, ms2t6A, ms2t6A, NADpN, m8A, ct6A, ct6A, ht6A, io6A, pio6A, m62A, ac6A, pac6A, f6A, pf6A, g6A, hm6A, hn6A, m6A, m6t6A, t6A, MANT-dA, 2'-lodo-dA, 7'-Deaza-dA, 8'-Bromo-dA, 7'- Deaza-7'-iodo-dA, 2'-Hydroxy-dA, 7'-Deaza-7-bromo-dA, 2'-Fluoro-dA, Mant-dA, 2'NH2-dA, Etheno- dA (e-dA), dAaSe, dAaS, 8'-Oxo-dA, 2’-Bromo-A, LCA, IG, SRA, AF2, ADS, 2AD, 8AN, PPU, 3DA, AP7, 45A, A9Z, ANZ, A5O, A5L, 5AA, A43, 2HA AD2, 1 mA, 3mA, 4mA, 6mA, 7mA, 9mA, 6hmA, HAP, m2A, AHAP, 6-DMA, ncm6A and EA; or wherein the at least one ribonucleotide that is substituted by a deoxyribonucleotide version is GTP and the deoxyribonucleotide version is modified by a modification is selected from the group consisting of the following naturally or synthetically occurring ribonucleotide or deoxyribonucleotide modifications m1 G, preQ1 , preQO, m7G, m2,7G, m2,2,7G, m22G, m2G I, mi l, m1 lm, Im, Q, oQ, dGdAIQ, mdAnQ, preQO, preQ1 , dG+ , 7-Deaza-G, 2’NH2-dG, 7-Deaza-7-iodo-G, 6-thio-dG, dCaSe, dCaS, Fluoro-G, MANT-G, 2-MANT-G, LCG, BGM, 2SG, GRB, GAO, TG, GDO, GMX, XUG, G3A, GS, G38, 2HG, 1 mG, 2mG, 3mG, m6G, 7mG, 8-oxo-G, ADG, 1 ,N(2)-EG, N(2),3-EG, m22G, m2,7dG, m2,2,7dG, 2’NH2-dG and CEG; or wherein the at least one ribonucleotide that is substituted by a deoxyribonucleotide version is UTP and the deoxyribonucleotide version is modified by a modification is selected from the group consisting of the following naturally or synthetically occurring ribonucleotide or deoxyribonucleotide cmo5U, mcmo5U, 2-NH2-dU, 3’-Azido-2’,3’-ddU, Aminoallyl-dU, 5-Propargylamino-dU, 5-Fluoro- dU, 5-dimethyl-2’-dU, 5BU, U37, IU, TLN, BRU, URU, UFB, U5M, UOB, UMO, U5R, B8N, U4M, UAR, UFT, UMS, NYM, MU1 , P1T, M, MU4, putThy, ddT, dTaSe, dTaS, 1 mT, 3mT, 04-meT, diHT, 5hmU, 5fU, base J, dhpUra, 5caU, 5-NeOmdU, dU, DHdU, 5-NedU, and dhpdU.

[0276] 32. The mRNA of any one of items 11 or 13 or the saRNA of any one of items 12 or 14, wherein between 10% to 75% of at least one of the ribonucleotides are substituted by a deoxyribonucleotide version of the at least one ribonucleotide, wherein the at least one ribonucleotide that is substituted by a deoxyribonucleotide version is CTP and the deoxyribonucleotide version is modified by a modification is selected from the group consisting of the following naturally or synthetically occurring ribonucleotide or deoxyribonucleotide modifications hm5Cm, s2C, m3C, phm5C, hm5C, f5C, pf5C, ho5C, hm5C, m5C, pC2'3'cp, m4,4C, ac4C, m4C, 2NH2-C, 3’-Azido-2’,3’-ddCTP, C8-Alkyne-C, 5- Propargylamino-C, LCC, CBV, CBR, IC, A5M, CSL, RY, 5CF, 10C, S4C, 73W, CH, N5M, 5IC, M5M, CAR, CFZ, CFL, D2C, 1 mC, m2C, e3C, 3mC, sC, 4mC, 5caC, 5fC, 5hmC, 5gmC and 5mC; or wherein the at least one ribonucleotide that is substituted by a deoxyribonucleotide version is ATP and the deoxyribonucleotide version is modified by a modification is selected from the group consisting of the following naturally or synthetically occurring ribonucleotide or deoxyribonucleotide modifications m1A, msms2i6A, m2,8A, m2A, ms2ct6A, , ms2io6A, ms2hn6A, ms2hn6A, ms2i6A, ms2m6A, ms2t6A, ms2t6A, NADpN, m8A, ct6A, ct6A, ht6A, io6A, pio6A, m62A, ac6A, pac6A, f6A, pf6A, g6A, hm6A, hn6A, m6A, m6t6A, t6A, MANT-dA, 2'-lodo-dA, 7'-Deaza-dA, 8'-Bromo-dA, 7'- Deaza-7'-iodo-dA, 2'-Hydroxy-dA, 7'-Deaza-7-bromo-dA, 2'-Fluoro-dA, Mant-dA, 2'NH2-dA, Etheno- dA (e-dA), dAaSe, dAaS, 8'-Oxo-dA, 2’-Bromo-A, LCA, IG, SRA, AF2, ADS, 2AD, 8AN, PPU, 3DA, AP7, 45A, A9Z, ANZ, A5O, A5L, 5AA, A43, 2HA AD2, 1 mA, 3mA, 4mA, 6mA, 7mA, 9mA, 6hmA, HAP, m2A, AHAP, 6-DMA, ncm6A and EA; or wherein the at least one ribonucleotide that is substituted by a deoxyribonucleotide version is GTP and the deoxyribonucleotide version is modified by a modification is selected from the group consisting of the following naturally or synthetically occurring ribonucleotide or deoxyribonucleotide modifications m1 G, preQ1 , preQO, m7G, m2,7G, m2,2,7G, m22G, m2G I, mi l, m1 lm, Im, Q, oQ, dGdAIQ, mdAnQ, preQO, preQ1 , dG+ , 7-Deaza-G, 2’NH2-dG, 7-Deaza-7-iodo-G, 6-thio-dG, dCaSe, dCaS, Fluoro-G, MANT-G, 2-MANT-G, LCG, BGM, 2SG, GRB, GAO, TG, GDO, GMX, XUG, G3A, GS, G38, 2HG, 1 mG, 2mG, 3mG, m6G, 7mG, 8-oxo-G, ADG, 1 ,N(2)-EG, N(2),3-EG, m22G, m2,7dG, m2,2,7dG, 2’NH2-dG and CEG; or wherein the at least one ribonucleotide that is substituted by a deoxyribonucleotide version is UTP and the deoxyribonucleotide version is modified by a modification is selected from the group consisting of the following naturally or synthetically occurring ribonucleotide or deoxyribonucleotide cmo5U, mcmo5U, 2-NH2-dU, 3’-Azido-2’,3’-ddU, Aminoallyl-dU, 5-Propargylamino-dU, 5-Fluoro- dU, 5-dimethyl-2’-dU, 5BU, U37, IU, TLN, BRU, URU, UFB, U5M, UOB, UMO, U5R, B8N, U4M, UAR, UFT, UMS, NYM, MU1 , P1T, M, MU4, putThy, ddT, dTaSe, dTaS, 1 mT, 3mT, 04-meT, diHT, 5hmU, 5fU, base J, dhpUra, 5caU, 5-NeOmdU, dU, DHdU, 5-NedU, and dhpdU.

[0277] 33. The mRNA of any one of items 11 or 13 or the saRNA of any one of items 12 or 14, wherein the at least one substituted deoxyribonucleotide version of the at least one ribonucleotide is located in the protein coding sequence of the mRNA or saRNA.

[0278] 34. The mRNA of any one of items 11 or 13 or the saRNA of any one of items 12 or 14, wherein the at least one substituted deoxyribonucleotide version of the at least one ribonucleotide is not located in the polyA tail.

[0279] 35. The mRNA of any one of items 11 or 13 or the saRNA of any one of items 12 or 14, wherein the at least one ribonucleotide that is substituted by a deoxyribonucleotide version is ATP, wherein 0.1 % to 100% of ATP is substituted by a nucleotide selected from the group consisting of deoxyadenosine(dA), 2'-lodo-deoxyadenosine, 2'-Bromo-deoxyadenosine, 3'- deoxyadenosine, 7- Deaza-deoxyadenosine, 8-Bromo-deoxyadenosine, 7-Deaza-7-iodo- deoxyadenosine, 2-Hydroxy- deoxyadenosine, 2'-Chloro-deoxyadenosine, 7-Deaza-7-bromo- deoxyadenosine, 2'-Fluoro- deoxyadenosine, Mant-deoxyadenosine, 2'-Mant-3'- deoxyadenosine, 2'NH2- deoxyadenosine, Etheno-deoxyadenosine (E-dA), 5'-(a-seleno)- deoxyadenosine (dAaSe), 5'-(a-thio)-deoxyadenosine (dAaS), N6-(6-Aminohexyl)-deoxyadenosine, N6-methyldeoxyadenosine (m6dA), 2'-O- methyldeoxyadenosine (2'OMedA), N6,2'-0-dimethyldeoxyadenosine (m6dAm), N6,N6,2'-O- trimethyldeoxyadenosine (m62dAm), 8-Oxo-deoxyadenosine, and N6-Methyl-deoxyadenosine.

[0280] 36. The mRNA of any one of items 11 or 13 or the saRNA of any one of items 12 or 14, wherein the at least one ribonucleotide that is substituted by a deoxyribonucleotide version is UTP, wherein 0.1% to 100% of UTP is substituted by a nucleotide selected from the group consisting of 2'NH2- deoxyuridine, 3'-Azido-2',3'-dideoxyuridine, Aminoallyl- deoxyuridine, 5-Propargylamino- deoxyuridine, 5-Fluoro-deoxyuridine, 5-Bromo- deoxyuridine, 5-lodo-deoxyuridine, deoxyuridine, 2',3'-Dideoxythymidine, Deoxythymidine, 5'-(a-seleno)-deoxythymidine (dTaSe), 5'-(a-thio)- deoxythymidine(dTaS), 3,2'-0-dimethyldeoxyuridine (m4dU), 2-thiodeoxyuridine (s2dU), 2-Fluoro- dUTP, pseudodeoxyuridine(dMJ), N1 -Methylpseudouridine (mld'-P), 2'-0-methyldeoxyuridine (Udm), 4-thiodeoxyuridine (s4dU), 5-methyldeoxyuridine (m5dU), and 5-methoxydeoxyuridine (mo5dU).

[0281] 37. The mRNA of any one of items 11 or 13 or the saRNA of any one of items 12 or 14, wherein the at least one ribonucleotide that is substituted by a deoxyribonucleotide version is CTP, wherein 0.1% to 100% of CTP is substituted by a nucleotide selected from the group consisting of deoxycytidine, C8-Alkyne-deoxycytidine, 5-Methyl-deoxycytidine, 2'-Fluoro-deoxycytidine, 5-lodo-deoxycytidine, 5- Bromo- deoxycytidia-seleno)- deoxycytidine (dCaSe), 5'-(a-thio)-deoxycytidine (dCaS), 5- Propargylamino-deoxycytidine, 5-Hydroxymethyl-deoxycytidine, and 2'-0-methyldeoxycytidine (dCm).

[0282] 38. The mRNA of any one of items 11 or 13 or the saRNA of any one of items 12 or 14, wherein the at least one ribonucleotide that is substituted by a deoxyribonucleotide version is GTP, wherein 0.1% to 100% of GTP is substituted by a nucleotide selected from the group consisting of 8-Oxo- deoxyguanosine, 3'-deoxyguanosine, 7-Deaza-deoxyguanosine, 7-Deaza-7-iodo-deoxyguanosine, 6- Thio-deoxyguanosine, 2'-Fluoro-deoxyguanosine, Mant- deoxyguanosine, 2'-Mant-3'- deoxyguanosine, 2'NH2-deoxyguanosine, 5'-(a-seleno)-deoxyguanosine (dCaSe), 5'-(a-thio)- deoxyguanosine (dCaS), 2'-0-methyldeoxyguanosine (dGm), N2,7-dimethyldeoxyguanosine (m2,7dG), 7-methyldeoxguanosine (m7dG), and N2, N2,7-trimethyldeoxyguanosine (m2,7dG).

[0283] Examples

[0284] Example 1 : mRNA and saRNA transcription plasmid template design and synthesis

[0285] The mRNA transcription template was designed using Firefly luciferase (Flue) reporter gene as the Open Reading Frame (ORF), with 5'UTR, 3'UTR, poly (A) tail, as well as a special cleavage site Bsal after the poly (A) tail, followed by the addition of the T7 promoter sequence before the 5'UTR. The sequence of the plasmid is depicted in SEQ ID NO: 1. The sequences were synthesized by GenScript Company and cloned into pUC57 vector to obtain the plasmid template as well as the recombinant strain.

[0286] The saRNA transcription template was designed following cloning vector pCMV-VEE-GFP, complete sequence (GenBank ID: MH891622.1). This plasmid is depicted in SEQ ID NO: 2. The sequences were synthesized by GenScript Company and cloned into pUC57 vector to obtain the plasmid template as well as the recombinant strain .

[0287] Example 2: Synthesis of Flue mRNA comprising different percentages of 5-Methyl-dCTP

[0288] The transcription reaction using HiScribe® T7 High Yield RNA Synthesis Kit (New ENGLAND Biolabs, E2040S) was prepared according to Table 4. All surfaces and pipettes were treated with RNase Zap to avoid nucleic acid cross-contamination. Template was provided in form of a linearized plasmid, exemplarily expressing firefly luciferase (Flue).

[0289] According to the substitution of either ATP, CTP, UTP and GTP with a modified or unmodified dATP, dCTP, dUTP or dGTP, the other reagents are not changed. For example, for 5-Methyl-dCTP 25% substitution, we use 1.125 pl CTP and 0.375 pl 5-Methyl-dCTP. For 5-Methyl-dCTP 50% substitution, we use 0.75 pl CTP and 0.75 pl 5-Methyl-dCTP. For 5-Methyl-dCTP 75% substitution, we use 0.375 pl CTP and 1 .125 pl 5-Methyl-dCTP, and so on.

[0290] Table 4: Reaction mix for mRNA synthesis

[0291] The reaction was incubated on a heat block at 37°C for 3 hours. 4 pL of DNase I and 2pL of DNase I buffer were added to each reaction and incubated at 37°C for 30 minutes.

[0292] After the end of transcription, the following purification steps were added: Prepare 70% ethanol and chill at -20°C for at least 1 h before use. Begin chilling centrifuge to 4°C. Add more than 10uL 7.5 M LiCI (Thermo Fisher, AM9480) into the samples until it has >2.5 M final concentration. Mix by gentle inversion or swirling. Do not vortex or shake. Chill at -80°C 1 h. entrifuge 10 min under 4°C at 13000 x rpm, the centrifugation step is performed in the precooled centrifuge at 4°C. Discard the supernatant, taking care to retain the pellet and, potentially, any smaller pellet fragments. Wash the pellet with 100 uL cold 70% ethanol. Centrifuge the pellet in 70% ethanol for 5 min at 13,000 x rpm and again using he precooled centrifuge at 4°C. Discard the ethanol supernatant. Use a pipet to remove as much of the residual wash as possible without disturbing the pellet. Repeat steps 14-16 to perform a second wash. Dry the pellet about 5 min. Resuspend the pellet in 60 pl or 30pl DNase / RNase-Free Distilled water.

[0293] Purified mRNA was measured with Nanodrop to determine concentration and puritiy of the RNA, results are shown in Table 5.

[0294] Table 5: The Flue mRNA yield of different percentage 5-Methyl-dCTP substituted CTP

[0295] Table 5 shows that as the concentration of 5-Methyl-dCTP increases, the mRNA product is produced at a lower rate. However, when 5-Methyl-dCTP was added at 100% substitution of CTP, the mRNA product was still synthesized, and the OD280 / OD260 was about 2, indicating very little change in purity.

[0296] The mRNA was visualized using gel electrophoresis, using a 1% agarose gel in MOPS buffer, running at 120V, 40mA for 40min. As can be seen in Figure 1 , all reactions produced RNA bands of the same size, indicating that the same product was produced.

[0297] Figure 2 shows the effect of different percentage of nucleotide modifications substitution on Flue expression.

[0298] Example 3:Effect of different deoxyribonucleotide modifications substitution on Flue expression

[0299] For 25% dATP substitution, the reaction included 5.625 mM ATP and 1.875 mM dATP (Jena Bioscience, NU-1005S) in the final concentration.

[0300] For 25% dCTP substitution, the reaction included 5.625 mM CTP and 1.875 mM dCTP (Jena Bioscience, NU-1005S) in the final concentration.

[0301] For 25% dGTP substitution, the reaction included 5.625 mM CTP and 1.875 mM dCTP (Jena Bioscience, NU-1005S) in the final concentration.

[0302] For 25% dTTP substitution, the reaction included 5.625 mM UTP and 1.875 mM dTTP (Jena Bioscience, NU-1005S) in the final concentration.

[0303] For 25% 5-Methyl-dCTP substitution, the reaction included 5.625 mM CTP and 1.875 mM 5-Methyl- dCTP (Jena Bioscience, NU-1125S) in final concentration.

[0304] For 100% (m1 i ) substitution, the UTP were totally substituted with 7.5mM N1-Methyl-pseudourin (Jena Bioscience, NU-890L).

[0305] For 25% 5-Methyl-CTP substitution, the reaction included 5.625 mM CTP and 1.875 mM 5-Methyl- CTP (Jena Bioscience, NU-1138S) in the final concentration.

[0306] For 100% (i ) substitution, the UTP were totally substituted with 7.5mM Pseudo-UTP (Jena Bioscience, NU-1139S). For 25% dCTP substitution+ 25% dTTP substitution, the reaction included 5.625 mM CTP, 5.625 mM UTP and 1.875 mM dCTP (Jena Bioscience, NU-1005S), 1.875 mM dTTP (Jena Bioscience, NU- 1005S) in the final concentration.

[0307] For 25% 5-Methyl-dCTP substitution+ 25% dTTP substitution, the reaction included 5.625 mM CTP, 5.625 mM UTP and 1.875 mM 5-Methyl-dCTP (Jena Bioscience, NU-1125S), 1.875 mM dTTP (Jena Bioscience, NU-1005S) in final concentration.

[0308] For 25% m1 i substitution, the reaction included 5.625 mM UTP and 1.875 mM N1-Methyl- pseudourin (Jena Bioscience, NU-890L) in final concentration.

[0309] For 25% i substitution, the reaction included 5.625 mM UTP and 1.875 mM Pseudo-UTP (Jena Bioscience, NU-1139S) in the final concentration.

[0310] Figure 3 shows the effect of the above substitutions on Flue expression. The highest amount of expression was reached with 25% 5-Methyl-dCTP (d5mCTP 25%) substituted CTP. 25% 5-Methyl- dCTP performed better than 25% 1 -methylpseudouridine (m1 i ) and 25% pseudouridine (i ), which have been extensively used in mRNA applications, such as the Moderna SARS-CoV-2 vaccine, in which N1-methyl-pseudouridine completely replaced uridine (Corbett et al. 2020, Nature 586:567- 571). Surprisingly, both 25% 5-Methyl-dCTP (C2, Figure 4) and 25% 2'-Fluoro-dCTP (C3, Figure 4) performed similarly or better than 100% 1 -methylpseudouridine (100% ml i , Figure 4.)

[0311] For 25% C8-Alkyne-dCTP substituted, the reaction included 5.625 mM CTP and 1.875 mM C8- Alkyne-dCTP (Jena Bioscience, CLK-T06-S) in the final concentration.

[0312] For 25% 5-Methyl-dCTP substituted, the reaction included 5.625 mM CTP and 1.875 mM 5-Methyl- dCTP (Jena Bioscience, NU-1125S) in the final concentration.

[0313] For 25% 2'-Fluoro-dCTP substituted, the reaction included 5.625 mM CTP and 1.875 mM 2'-Fluoro- dCTP (Jena Bioscience, NU-1214S) in the final concentration.

[0314] For 25% 5-lodo-dCTP substituted, the reaction included 5.625 mM CTP and 1.875 mM 5-lodo-dCTP (Jena Bioscience, NU-128S) in the final concentration.

[0315] For 25% 5-Bromo-dCTP substituted, the reaction included 5.625 mM CTP and 1.875 mM 5-Bromo- dCTP (Jena Bioscience, NU-132S) in the final concentration.

[0316] For 25% 2'NH2-dCTP substituted, the reaction included 5.625 mM CTP and 1.875 mM 2'NH2-dCTP (Jena Bioscience, NU-243S) in the final concentration.

[0317] For 25% 3'-Azido-2',3'-ddCTP substituted, the reaction included 5.625 mM CTP and 1.875 mM 3'- Azido-2',3'-ddCTP (Jena Bioscience, NU-246S) in the final concentration.

[0318] For 25% dCTPaSe substituted, the reaction included 5.625 mM CTP and 1.875 mM dCTPaSe (Jena Bioscience, NU-269S) in the final concentration.

[0319] For 25% dCTPaS substituted, the reaction included 5.625 mM CTP and 1.875 mM dCTPaS (Jena Bioscience, NU-425S) in the final concentration. For 25% 5-Propargylamino-dCTP substituted, the reaction included 5.625 mM CTP and 1.875 mM 5- Propargylamino-dCTP (Jena Bioscience, NU-809S) in the final concentration.

[0320] For 25% 5-Hydroxymethyl-dCTP substituted, the reaction included 5.625 mM CTP and 1.875 mM 5- Hydroxymethyl-dCTP (Jena Bioscience, NU-932S) in the final concentration.

[0321] For 25% dCTP substituted, the reaction included 5.625 mM CTP and 1.875 mM dCTP (Jena Bioscience, NU-1005S) in the final concentration.

[0322] For 25% m1 i substitution, the reaction included 5.625 mM UTP and 1.875 mM N1-Methyl- pseudourin (Jena Bioscience, NU-890L) in final concentration.

[0323] For 25% i substitution, the reaction included 5.625 mM UTP and 1.875 mM Pseudo-UTP (Jena Bioscience, NU-1139S) in the final concentration.

[0324] For 100% (m1 i ) substitution, the UTP were totally substituted with 7.5mM N1-Methyl-pseudourin (Jena Bioscience, NU-890L).

[0325] For 100% (i ) substitution, the UTP were totally substituted with 7.5mM Pseudo-UTP (Jena Bioscience, NU-1139S).

[0326] Figure 4 shows the effect of the modifications above.

[0327] Figure 5 shows the effect of C2: 25% 5-Methyl-dCTP substituted CTP; C3: 25% 2'-Fluoro-dCTP substituted CTP; C7: 25% 3'-Azido-2',3'-ddCTP substituted CTP; C12: 25% dCTP substituted CTP on Flue expression. C2, C3 and C7 performed significantly better than Wildtype, while C12 was comparable with Wildtype.

[0328] Example 4: Effect of different nucleotide modifications and deoxyribonucleotide substitution on saFluc expression

[0329] The SaFluc Plasmids were extracted using Express™ Plasmid Midiprep Kit (ZymoPURE, Cat. No. 4208T) following its instruction and purified with QIAquick PCR Purification Kit (QIAGEN, Cat. No. 28104). After that, following the instruction of the Xba I (New England BioLabs, R0145L) to linearized the plasmid.

[0330] Then following the HiScribe® T7 High Yield RNA Synthesis Kit (New ENGLAND Biolabs, E2040S) instruction with some modify to synthesis the RNA. The specific components are as Table 4, with the lineralized saFLuc plasmid template used instead of the Flue plasmid and also CleanCap Reagent AU (40 mM, Trilink Biotechnologies, N-7114-100) instead of CleanCap Reagent AG.

[0331] According to the different substitution of the NTP, the other compliments are not changed, only the mentioned NTPs were substituted. For example, for 5-Methyl-dCTP 50% substitution, the reaction included 3.75 mM CTP and 3.75 mM 5-Methyl-dCTP (Jena Bioscience, NU-1125S) in final concentration.

[0332] Cell culture, transfection and Firefly Luciferase assay DC2.4 (Sigma-Aldrich, SCC142) were cultured in DMEM (ATCC, 30-2002) with 10% FBS(ATCC, 30- 2020). When confluence reaches 90% the cells were split and seed in a 96 well plate at 2.5*10A4 per well. When the cell confluence reaches in 70%, using Lipofectamine™ 3000 Transfection Reagent (Invitrogen, Cat. No. L3000008) transfected 200 ng RNA per well. After 48 or 96 hours of coincubation, removing the supernant of the cells. And following the instruction of the Pierce™ Firefly Luciferase Glow Assay Kit (Thermo Fisher, 16176) to quantify the expression of firefly luciferase.

[0333] For HEK cell culture, HEK 293T cells (ATCC, CRL-3216™) were cultured in DMEM (ATCC, 30-2002) with 10% FBS (ATCC, 30-2020). When confluence reaches 90% the cells were split and seed in a 96 well plate at 4*10A4 per well in 100 uL DMEM with 2% FBS. When the cell confluence reaches in 70%, using Lipofectamine™ 3000 Transfection Reagent (Invitrogen, Cat. No. L3000008) transfected 200ng or 400ng RNA per well. After 24, 48 or 96 hours, following the instruction of Firefly Luciferase HTS assay (MilliporeSigma, Cat.NO.SCT150) to quantify the expression of firefly luciferase.

[0334] Following the same method as above to quantify the expression of firefly luciferase.

[0335] Substitution of modified deoxyribonucleotides

[0336] For 25% dATP substitution, the reaction included 5.625 mM ATP and 1.875 mM dATP (Jena Bioscience, NU-1005S) in the final concentration.

[0337] For 25% dCTP substitution, the reaction included 5.625 mM CTP and 1.875 mM dCTP (Jena Bioscience, NU-1005S) in the final concentration.

[0338] For 25% dGTP substitution, the reaction included 5.625 mM CTP and 1.875 mM dCTP (Jena Bioscience, NU-1005S) in the final concentration.

[0339] For 25% dTTP substitution, the reaction included 5.625 mM UTP and 1.875 mM dTTP (Jena Bioscience, NU-1005S) in the final concentration.

[0340] For 25% 5-Methyl-dCTP substitution, the reaction included 5.625 mM CTP and 1.875 mM 5-Methyl- dCTP (Jena Bioscience, NU-1125S) in final concentration.

[0341] For 100% (m1 i ) substitution, the UTP were totally substituted with 7.5mM N1-Methyl-pseudourin (Jena Bioscience, NU-890L).

[0342] For 25% 5-Methyl-CTP substitution, the reaction included 5.625 mM CTP and 1.875 mM 5-Methyl- CTP (Jena Bioscience, NU-1138S) in the final concentration.

[0343] For 100% (i ) substitution, the UTP were totally substituted with 7.5mM Pseudo-UTP (Jena Bioscience, NU-1139S).

[0344] For 25% dCTP substitution+ 25% dTTP substitution, the reaction included 5.625 mM CTP, 5.625 mM UTP and 1.875 mM dCTP (Jena Bioscience, NU-1005S), 1.875 mM dTTP (Jena Bioscience, NU- 1005S) in the final concentration. For 25% 5-Methyl-dCTP substitution+ 25% dTTP substitution, the eaction included 5.625 mM CTP, 5.625 mM UTP and 1.875 mM 5-Methyl-dCTP (Jena Bioscience, NU-1125S), 1.875 mM dTTP (Jena Bioscience, NU-1005S) in final concentration.

[0345] For 25% m1 i substitution, the reaction included 5.625 mM UTP and 1.875 mM N1-Methyl- pseudourin (Jena Bioscience, NU-890L) in final concentration.

[0346] For 25% i substitution, the reaction included 5.625 mM UTP and 1.875 mM Pseudo-UTP (Jena Bioscience, NU-1139S) in the final concentration.

[0347] Figure 6 shows the effect of the above nucleotide modifications on saFLUC expression. In line with the results of the Flue mRNA experiments, 50% 5-Methyl-dCTP (“SaFluc-D” in Figure 6) performed best, including better than 100% N1-Methyl-pseudourin (“Flue” in Figure 6).

[0348] Figure 7 shows the effect of different deoxyribonucleotide modifications substitution on saFLUC expression.

[0349] Figure 8 shows the effect of 25% 2'-Bromo-dATP substituted ATP on saFLUC expression.

[0350] Figure 9 shows the effect of further different deoxyribonucleotide modifications substitution on saFLUC expression.

[0351] For 25% 2'-Bromo-dATP substituted ATP, the reaction included 5.625 mM ATP and 1.875 mM 2'- Bromo-dATP (Jena Bioscience, NU-102) in the final concentration.

[0352] For 25% 7-Deaza-dGTP substituted GTP, the reaction included 5.625 mM GTP and 1.875 mM 7- Deaza-dGTP (Jena Bioscience, NU-1179S) in the final concentration.

[0353] For 25% m1 i substitution, the reaction included 5.625 mM UTP and 1.875 mM N1-Methyl- pseudourin (Jena Bioscience, NU-890L) in final concentration.

[0354] For 25% i substitution, the reaction included 5.625 mM UTP and 1.875 mM Pseudo-UTP (Jena Bioscience, NU-1139S) in the final concentration.

[0355] For 100% m1 i substitution, the UTP were totally substituted with 7.5mM N1-Methyl-pseudourin (Jena Bioscience, NU-890L).

[0356] For 100% i substitution, the UTP were totally substituted with 7.5mM Pseudo-UTP (Jena Bioscience, NU-1139S).

[0357] Example 5: Confirmation of Deoxynucleotides Hybridizing to RNA During IN Vitro Transcription

[0358] Different percentage of Mant-dATP and Mant-dGTP modifications substitution FLUC synthesis following the FLUC synthesis Method. According to the different substitution of the NTP, the other compliments are not changed, only the mentioned NTPs were substituted. For example, for 25% Mant-dATP substitution, the reaction included 5.625 mM ATP and 1.875 mM Mant-dATP (Jena Bioscience, NU-203S) in the final concentration. Absorption spectra of samples (500ng / pL in nuclease-free water) were meausing a NanoDrop™ 2000 / 2000c spectrophotometer (Thermo Fisher Scientific, ND2000C). Baseline correction was performed with the corresponding solvent prior to measurements.

[0359] The MANT moiety has specific absorbance characteristics due to its aromatic structure, which can absorb light in the UV-visible range (MANT-G (2'(3')-0-(N-Methylanthraniloyl)-guanosine) or MANT- DATP (2'(3')-0-(N-Methylanthraniloyl)-adenosine). This results in additional absorbance peaks that are not present in unmodified mRNA. MANT typically shows absorbance peaks around 350-400 nm, which are distinct from the typical absorbance of nucleic acids (around 260 nm).

[0360] Figure 10 shows the spectroscopic properties of 25%,50%,75%-Mant-dATP or 25%,50%,75%- Mant-dGTP-incoparated mRNA. The absorbance spectrum shows an additional peak around 350nm, which increases with increasing percentage of MANT availability in the in vitro transcription mixture. This confirms that the modified deoxynucleotides have been incorporated into the RNA during in vitro transcription, and that the incorporation is directly proportional to the percentage of (modified) deoxynucleotides available during the reaction.

[0361] Example 6: Optimizing deoxynucleotide substitution percentage for enhancing Flue expression

[0362] Different percentage deoxynucleotide modifications substitution FLUC synthesis following the FLUC synthesis Method. According to the different substitutions of the NTP, the other compliments are not changed, only the mentioned NTPs were substituted. Then using Lipofectamine RNAiMAX Transfection Reagent (Invitrogen, Cat. No. 13778075) transfected 100 ng RNA per well. After 24 hours of co-incubation, removing the supernant of the cells. And Following the instruction of the Pierce™ Firefly Luciferase Glow Assay Kit (Thermo Fisher, 16176) to quantify the expression of firefly luciferase.

[0363] Figure 11 shows the effect of different percentage deoxynucleotide substitutions on Flue expression.

[0364] Figure 12 identifies the most effective percentage of deoxynucleotide substitution that maximizes firefly luciferase (Flue) expression.

[0365] Firefly luciferase (Flue) expression levels measured 24 hours after transfection of 100 ng LNP-RNA encoding the Flue reporter into DC2.4 cells. Luciferase activity was quantified using the Pierce™ Firefly Luciferase Glow Assay Kit. Error bars indicate the standard error of the mean (SEM) from n = 3 replicates. Approximately 4 x 104cells were seeded per well.

[0366] None: Unmodified RNA (no deoxynucleotide substitutions). Mix: 5%-dATP , 5%-dTTP, 25%-dCTP and 25%-dGTP substituted.

[0367] The data show that all mRNA with deoxyribonucleotide substitutions could be expressed in the cells. Some of the substitutions, such as at least 25% dCTP, and up to 50% dTTP, were effective at increasing expression of Flue compared to unmodified mRNA. A mixture of different substitutions also let to an mRNA that could be readily expressed and showed increased expression compared to WT.

[0368] Example 7 Optimizing deoxynucleotide substitution percentage for enhancing selfamplifying Flue (saFluc) expression saRNA Transcription plasmid template design and synthesis

[0369] The saRNA transcription template was designed following Sequence ID 2 with some modification. The AGG trinucleotide immediately downstream of the T7 promoter (TAATACGACTCACTATA) was deleted. Then the sequences were synthesized by GenScript Company and cloned into pUC57 vector to obtain the plasmid template as well as the recombinant strain .

[0370] SaFluc plasmid template extraction and Linearization:

[0371] The SaFluc Plasmids were extracted using Express™ Plasmid Midiprep Kit (ZymoPURE, Cat. No. 4208T) following its instruction and purified with QIAquick PCR Purification Kit (QIAGEN, Cat. No. 28104). After that, following the instruction of the Xba I (New England BioLabs, R0145L) to linearized the plasmid.

[0372] SaFluc RNA synthesis.

[0373] Then following the HiScribe® T7 High Yield RNA Synthesis Kit (New ENGLAND Biolabs, E2040S) instruction with some modify to synthesis the RNA. The specific components are as table 4.

[0374] According to the different substitution of the NTP, the other compliments are not changed, only the mentioned NTPs were substituted. For example, for 5-Methyl-dCTP 50% substitution, the reaction included 3.75 mM CTP and 3.75 mM 5-Methyl-dCTP (Jena Bioscience, NU-1125S) in final concentration Table 2.

[0375] Then the reaction was incubates at 37°C, 300 rpm in heat block for three hours. 4 pL of DNase I and 2pL of DNase I buffer were added for each reaction (New England BioLabs, B0303), mixed well and incubated at 37°C for 30 minutes. After the end of transcription, the following purification steps were added. Prepared 70% ethanol and chilled at -20°C for at least 1 h before use. Began chilling centrifuge to 4°C. Added more than 10uL 7.5 M LiCI (Thermo Fisher, AM9480) into the samples until it had >2.5 M final concentration. Mixed by gentle inversion or swirling. Did not vortex or shake. Chilled at -80°C 1 h.

[0376] Centrifuged 10 min under 4°C at 13000 x rpm, the centrifugation step was performed in the precooled centrifuge at 4°C. Discarded the supernatant, taking care to retain the pellet and, potentially, any smaller pellet fragments. Washed the pellet with 100 uL cold 70% ethanol. Centrifuged the pellet in 70% ethanol for 5 min at 13,000 x rpm and again using the precooled centrifuge at 4°C. Discarded the ethanol supernatant. Used a pipet to remove as much of the residual wash as possible without disturbing the pellet. Repeat steps 14-16 to perform a second wash. Dried the pellet about 5 min. Resuspended the pellet in 60 pl or 30pl DNase / RNase-Free Distilled water. Then used Nanodrop to determine concentration. 3. Cell culture, transfection and Firefly Luciferase assay

[0377] DC2.4 (Sigma-Aldrich, SCC142) were cultured in DMEM (ATCC, 30-2002) with 10% FBS(ATCC, 30- 2020). When confluence reaches 90% the cells were split and seed in a 96 well plate at 4*1 O*4per well. When the cell confluence reaches in 70%, using Lipofectamine RNAiMAX Transfection Reagent (Invitrogen, Cat. No. 13778075) transfected 100 ng RNA per well. After 24 hours of co-incubation, removing the supernant of the cells. And Following the instruction of the Pierce™ Firefly Luciferase Glow Assay Kit (Thermo Fisher, 16176) to quantify the expression of firefly luciferase.

[0378] Figure 14 shows saFluc expression in cells with dATP, dCTP, dGTP and dTTP and 2’-Hydroxy-dATP dsubstitutions. All substitutions were well expressed. dATP and dGTP substitutions lead to increases in expression compared to unsubstituted WT.

[0379] Figure 16 shows the effect of different percentages of 2’-Hydroxy-dATP substitutions on saFluc expression. In general, all percentages tested, 5% to 75%, were well tolerated. All led to moderate to high increases in saFluc expression compared to wildtype. Smaller amounts of substitution between about 5% and 15% led to the biggest increases in expression.

[0380] Table 1 summarizes further expression data for saFluc mRNA with 25% deoxynucleotide substitutions. The data show that all substitution constructs were expressed at least as well as the WT, while most performed better and lead to increased expression compared to WT. 2'-lodo-dATP, 7'-Deaza-dATP, 8'-Bromo-dATP, 2'-Hydroxy-dATP, 2'-Chloro-dATP, 7'-Deaza-7-bromo-dATP and Mant-dATP lead to increase of expression of at least 3 fold compared to unmodified wildtype saRNA.

[0381] Table 1. Fold-change in expression of self-amplifying Flue (saFluc) mRNA with 25% deoxynucleotide substitutions compared to unmodified controls

[0382] A1 NU-101 2'-lodo-dATP 5.74

[0383] A4 NU-1175S 7'-Deaza-dATP 4.24

[0384] A5 NU-117S 8'-Bromo-dATP 3.18

[0385] A6 NU-1181 S 7'-Deaza-7'-iodo-dATP 1.27

[0386] A7 NU-1209S 2'-Hydroxy-dATP 3.33

[0387] A8 NU-148S 2'-Chloro-dATP 3.20

[0388] A9 NU-149S 7'-Deaza-7-bromo-dATP 3.26

[0389] A10 NU-151S 2'-Fluoro-dATP 2.41

[0390] A11 NU-203S Mant-dATP 3.25

[0391] A13 NU-244S 2'NH2-dATP 1.30 A14 NU-256S Etheno-dATP (E-dATP) 2.42

[0392] A15 NU-267S dATPaSe 2.71

[0393] A16 NU-426S dATPaS 1.28

[0394] A18 NU-889S 8'-Oxo-dATP 2.88

[0395] A19 NU-949S N6-Methyl-dATP 1.19

[0396] C3 NU-1214S 2'-Fluoro-dCTP 1.06

[0397] C11 NU-932S 5'-Hydroxymethyl-dCTP 1.38

[0398] G1 NU-1117S 8'-Oxo-dGTP 1.23

[0399] Expression levels 24 hours after transfection of 100 ng of RNA encoding saFluc reporter into DC2.4 cells with lipofectamine RNAi max. Luciferase signal is detected by Pierce™ Firefly Luciferase Glow Assay Kit. Fold-change were normalized to the unmodified controls WT (sample mean I WT mean). DC2.4 Cells seeded about 4*10A4 per well. Data are shown as mean ± SEM . WT: No modification, using ATP, CTP, UTP and GTP to synthesize.

[0400] Example 7: In vivo evaluations of the translation profiles of deoxynucleotide substitution mRNA.

[0401] Lipid nanoparticle-mRNA preparation (LNP-RNA)

[0402] SM-102 (MCE, 251104), DSPC (MCE, 261435), cholesterol (Sigma-Aldrich, 57-88-5), and DMG- PEG2000 (Avanti, 880151 p-1 g-A-025) were dissolved in ethanol at a molar ratio of 50:10:38.5:1.5. mRNA was diluted in 50 mM citrate buffer (pH 4.0) to a final concentration of 0.17 mg / mL. Lipid and mRNA solutions were mixed at a nitrogen-to-phosphate (N / P) ratio of 6:1 using the NanoAssemblr Ignite microfluidic system (Precision Nanosystems), with a total flow rate of 12 mL / min and an aqueous-to-organic phase flow rate ratio of 3:1. The resulting lipid nanoparticle (LNP)-encapsulated mRNA formulations were dialyzed against phosphate-buffered saline (PBS, pH 7.4) for 24 hours using dialysis tubing (Viskase, USA), and stored at 4°C until further use. Encapsulation efficiency was assessed using the Quant-iT RiboGreen RNA Assay Kit (Invitrogen, USA), and fluorescence was measured with a FLUOstar Omega microplate reader (BMG Labtech).

[0403] For experiments with saRNA, a nitrogen / to / phosphate (N / P) ratio of 8:1 was used.

[0404] Bioluminescence Imaging and Quantification

[0405] To assess in vivo protein expression and tissue distribution of Flue mRNA with varying percentages of deoxynucleotide substitutions, mice were intramuscularly (i.m.) injected with equal doses (5ug / mouse) of unmodified RNA (None) or Flue mRNA containing 5% dATP substitution, 5% dTTP substitution, 25% dCTP substitution, 25% 5-Methyl-dCTP substitution or 25% dGTP substitution. Flue expression was evaluated by bioluminescence imaging at 6 hours post-injection. Mice were anesthetized with isoflurane and administered D-luciferin substrate (30 mg per mouse) via intraperitoneal injection. After 5 minutes, bioluminescent signals were captured and quantified using an in vivo imaging system (IVIS Lumina Series III, PerkinElmer).

[0406] Figure 13 shows the results for Flue mRNA comprising 5% dATP, 5% dTTP, 25% dCTP, 25% dGTP substitutions, 25% 5-Methyl-dCTP substitution and Mix(5% dATP, 5% dTTP, 25% dCTP and 25% dGTP mix substitutions). All substitutions were well tolerated in vivo. The dATP, dGTP, dCTP and dTTP subsitutions all lead to a moderate increase in expression, while the 25% substitution with dm5C or dCTP were especially successful at increasing expression. In Figure 15 shows the results for saFluc mRNA comprissing 5% dATP substitution. This substitution lead to an increase in expression in vivo, which confirms the results of the in vitro experiments.

[0407] Sequences

[0408] SEQ ID NO: 1 - Flue DNA template sequence taatacgactcactataaggggtttcttgctgcagcaacgcgagtgggagcaccaggatctcgggctcggaacgagactgcacggattgtttt aagaaagccaccatggaagacgccaagaatataaagaaaggccctgcacccttctaccccctagaggacggcaccgcaggagagcag ctgcacaaagcaatgaaaaggtatgccctggtgccaggaacgattgccttcactgatgcccacattgaggttgatattacctatgcagagtact tcgagatgtcagtacgcctagccgaggccatgaagagatatggtctgaacacaaaccaccggatcgtcgtctgctcagaaaattctctgcag ttcttcatgcctgtgctgggagcgttattcattggtgtagctgtggcacctgccaatgacatctacaatgagagagaattgctaaacagcatgggc attagtcagcccactgtggttttcgtgtctaaaaaagggcttcagaagatacttaatgtccagaaaaagctaccaataattcaaaagatcatcat tatggactccaagacagactaccaggggttccagtccatgtatacctttgtaaccagccacctcccgccaggcttcaatgaatatgactttgttc cagagtcgtttgatcgggataaaacaattgcgttgatcatgaactcaagtgggtccacgggactccctaagggagtggcccttccacaccgta cagcctgtgtgaggttcagccatgctagagatcccatctttggaaaccagatcatcccagacactgccatactgtctgtagtcccattccatcat ggatttggcatgtttacaacgctgggctacctgatatgcggctttcgagtagtgctcatgtacagatttgaagaagagcttttcctgcgcagtttgc aggattacaaaatccagtctgctctgctggtgcctaccctcttctccttctttgctaaatctacactgattgacaagtacgacttgagcaacctccat gaaattgcttctggtggtgcgccactgagcaaagaagtgggcgaggctgttgcaaagcgcttccacctgcccggcatccgacaaggctacg ggttaactgaaaccacatctgccatattaatcactcctgagggggatgataagcccggtgctgtcgggaaagtggtgccgttttttgaagccaa ggtggtagacctggacacaggcaaaaccttgggagttaatcaacgaggtgaactttgtgtccggggacctatgatcatgagcggatatgtga acaaccctgaagccaccaatgcactgatagacaaggatggctggctgcattccggtgacatcgcctactgggatgaagatgagcacttcttc atcgtggacagactgaagtcactcatcaaatacaagggctatcaggtggccccggcagaattggagtccatccttctgcagcaccccaacat ttttgacgctggggtggctggtttgccagatgacgatgcaggggagctgcctgctgccgtcgttgtcctggagcatggcaagacgatgactgag aaggagattgtggactatgttgccagtcaagtcaccactgcgaagaagctcaggggcggagtggtctttgtggatgaggtgcccaagggcct cacaggaaagctcgatgctaggaaaattcgggagatcctcatcaaggccaaaaaaggggggaagatcgctgtctaataggatcctggag gatttcctcctcttcgagtcgccggtcggttctccgtaaatcgtggcaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaagcatatgactaaa aaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaagagacc

[0409] SEQ ID NO: 2 - SaFluc DNA template sequence TAATACGACTCACTATAAGGataggcggcgcatgagagaagcccagaccaattacctacccaaaatggagaaagttcacgtt gacatcgaggaagacagcccattcctcagagctttgcagcggagcttcccgcagtttgaggtagaagccaagcaggtcactgataatgacc atgctaatgccagagcgttttcgcatctggcttcaaaactgatcgaaacggaggtggacccatccgacacgatccttgacattggaagtgcgc ccgcccgcagaatgtattctaagcacaagtatcattgtatctgtccgatgagatgtgcggaagatccggacagattgtataagtatgcaactaa gctgaagaaaaactgtaaggaaataactgataaggaattggacaagaaaatgaaggagctggccgccgtcatgagcgaccctgacctgg aaactgagactatgtgcctccacgacgacgagtcgtgtcgctacgaagggcaagtcgctgtttaccaggatgtatacgcggttgacggaccg acaagtctctatcaccaagccaataagggagttagagtcgcctactggataggctttgacaccaccccttttatgtttaagaacttggctggagc atatccatcatactctaccaactgggccgacgaaaccgtgttaacggctcgtaacataggcctatgcagctctgacgttatggagcggtcacgt agagggatgtccattcttagaaagaagtatttgaaaccatccaacaatgttctattctctgttggctcgaccatctaccacgagaagagggactt actgaggagctggcacctgccgtctgtatttcacttacgtggcaagcaaaattacacatgtcggtgtgagactatagttagttgcgacgggtacg tcgttaaaagaatagctatcagtccaggcctgtatgggaagccttcaggctatgctgctacgatgcaccgcgagggattcttgtgctgcaaagt gacagacacattgaacggggagagggtctcttttcccgtgtgcacgtatgtgccagctacattgtgtgaccaaatgactggcatactggcaac agatgtcagtgcggacgacgcgcaaaaactgctggttgggctcaaccagcgtatagtcgtcaacggtcgcacccagagaaacaccaatac catgaaaaattaccttttgcccgtagtggcccaggcatttgctaggtgggcaaaggaatataaggaagatcaagaagatgaaaggccacta ggactacgagatagacagttagtcatggggtgttgttgggcttttagaaggcacaagataacatctatttataagcgcccggatacccaaacc atcatcaaagtgaacagcgatttccactcattcgtgctgcccaggataggcagtaacacattggagatcgggctgagaacaagaatcagga aaatgttagaggagcacaaggagccgtcacctctcattaccgccgaggacgtacaagaagctaagtgcgcagccgatgaggctaaggag gtgcgtgaagccgaggagttgcgcgcagctctaccacctttggcagctgatgttgaggagcccactctggaagccgatgtcgacttgatgtta caagaggctggggccggctcagtggagacacctcgtggcttgataaaggttaccagctacgatggcgaggacaagatcggctcttacgctg tgctttctccgcaggctgtactcaagagtgaaaaattatcttgcatccaccctctcgctgaacaagtcatagtgataacacactctggccgaaaa gggcgttatgccgtggaaccataccatggtaaagtagtggtgccagagggacatgcaatacccgtccaggactttcaagctctgagtgaaag tgccaccattgtgtacaacgaacgtgagttcgtaaacaggtacctgcaccatattgccacacatggaggagcgctgaacactgatgaagaat attacaaaactgtcaagcccagcgagcacgacggcgaatacctgtacgacatcgacaggaaacagtgcgtcaagaaagaactagtcact gggctagggctcacaggcgagctggtggatcctcccttccatgaattcgcctacgagagtctgagaacacgaccagccgctccttaccaagt accaaccataggggtgtatggcgtgccaggatcaggcaagtctggcatcattaaaagcgcagtcaccaaaaaagatctagtggtgagcgc caagaaagaaaactgtgcagaaattataagggacgtcaagaaaatgaaagggctggacgtcaatgccagaactgtggactcagtgctctt gaatggatgcaaacaccccgtagagaccctgtatattgacgaagcttttgcttgtcatgcaggtactctcagagcgctcatagccattataaga cctaaaaaggcagtgctctgcggggatcccaaacagtgcggtttttttaacatgatgtgcctgaaagtgcattttaaccacgagatttgcacaca agtcttccacaaaagcatctctcgccgttgcactaaatctgtgacttcggtcgtctcaaccttgttttacgacaaaaaaatgagaacgacgaatc cgaaagagactaagattgtgattgacactaccggcagtaccaaacctaagcaggacgatctcattctcacttgtttcagagggtgggtgaagc agttgcaaatagattacaaaggcaacgaaataatgacggcagctgcctctcaagggctgacccgtaaaggtgtgtatgccgttcggtacaa ggtgaatgaaaatcctctgtacgcacccacctcagaacatgtgaacgtcctactgacccgcacggaggaccgcatcgtgtggaaaacacta gccggcgacccatggataaaaacactgactgccaagtaccctgggaatttcactgccacgatagaggagtggcaagcagagcatgatgc catcatgaggcacatcttggagagaccggaccctaccgacgtcttccagaataaggcaaacgtgtgttgggccaaggctttagtgccggtgc tgaagaccgctggcatagacatgaccactgaacaatggaacactgtggattattttgaaacggacaaagctcactcagcagagatagtattg aaccaactatgcgtgaggttctttggactcgatctggactccggtctattttctgcacccactgttccgttatccattaggaataatcactgggataa ctccccgtcgcctaacatgtacgggctgaataaagaagtggtccgtcagctctctcgcaggtacccacaactgcctcgggcagttgccactgg aagagtctatgacatgaacactggtacactgcgcaattatgatccgcgcataaacctagtacctgtaaacagaagactgcctcatgctttagtc ctccaccataatgaacacccacagagtgacttttcttcattcgtcagcaaattgaagggcagaactgtcctggtggtcggggaaaagttgtccg tcccaggcaaaatggttgactggttgtcagaccggcctgaggctaccttcagagctcggctggatttaggcatcccaggtgatgtgcccaaat atgacataatatttgttaatgtgaggaccccatataaataccatcactatcagcagtgtgaagaccatgccattaagcttagcatgttgaccaag aaagcttgtctgcatctgaatcccggcggaacctgtgtcagcataggttatggttacgctgacagggccagcgaaagcatcattggtgctatag cgcggctgttcaagttttcccgggtatgcaaaccgaaatcctcacttgaagagacggaagttctgtttgtattcattgggtacgatcgcaaggcc cgtacgcacaatccttacaagctttcatcaaccttgaccaacatttatacaggttccagactccacgaagccggatgtgcaccctcatatcatgt ggtgcgaggggatattgccacggccaccgaaggagtgattataaatgctgctaacagcaaaggacaacctggcggaggggtgtgcggag cgctgtataagaaattcccggaaagcttcgatttacagccgatcgaagtaggaaaagcgcgactggtcaaaggtgcagctaaacatatcatt catgccgtaggaccaaacttcaacaaagtttcggaggttgaaggtgacaaacagttggcagaggcttatgagtccatcgctaagattgtcaa cgataacaattacaagtcagtagcgattccactgttgtccaccggcatcttttccgggaacaaagatcgactaacccaatcattgaaccatttgc tgacagctttagacaccactgatgcagatgtagccatatactgcagggacaagaaatgggaaatgactctcaaggaagcagtggctagga gagaagcagtggaggagatatgcatatccgacgactcttcagtgacagaacctgatgcagagctggtgagggtgcatccgaagagttctttg gctggaaggaagggctacagcacaagcgatggcaaaactttctcatatttggaagggaccaagtttcaccaggcggccaaggatatagca gaaattaatgccatgtggcccgttgcaacggaggccaatgagcaggtatgcatgtatatcctcggagaaagcatgagcagtattaggtcgaa atgccccgtcgaagagtcggaagcctccacaccacctagcacgctgccttgcttgtgcatccatgccatgactccagaaagagtacagcgc ctaaaagcctcacgtccagaacaaattactgtgtgctcatcctttccattgccgaagtatagaatcactggtgtgcagaagatccaatgctccca gcctatattgttctcaccgaaagtgcctgcgtatattcatccaaggaagtatctcgtggaaacaccaccggtagacgagactccggagccatc ggcagagaaccaatccacagaggggacacctgaacaaccaccacttataaccgaggatgagaccaggactagaacgcctgagccgat catcatcgaagaggaagaagaggatagcataagtttgctgtcagatggcccgacccaccaggtgctgcaagtcgaggcagacattcacgg gccgccctctgtatctagctcatcctggtccattcctcatgcatccgactttgatgtggacagtttatccatacttgacaccctggagggagctagc gtgaccagcggggcaacgtcagccgagactaactcttacttcgcaaagagtatggagtttctggcgcgaccggtgcctgcgcctcgaacagt attcaggaaccctccacatcccgctccgcgcacaagaacaccgtcacttgcacccagcagggcctgctcgagaaccagcctagtttccacc ccgccaggcgtgaatagggtgatcactagagaggagctcgaggcgcttaccccgtcacgcactcctagcaggtcggtctcgagaaccagc ctggtctccaacccgccaggcgtaaatagggtgattacaagagaggagtttgaggcgttcgtagcacaacaacaatgacggtttgatgcgg gtgcatacatcttttcctccgacaccggtcaagggcatttacaacaaaaatcagtaaggcaaacggtgctatccgaagtggtgttggagagga ccgaattggagatttcgtatgccccgcgcctcgaccaagaaaaagaagaattactacgcaagaaattacagttaaatcccacacctgctaac agaagcagataccagtccaggaaggtggagaacatgaaagccataacagctagacgtattctgcaaggcctagggcattatttgaaggca gaaggaaaagtggagtgctaccgaaccctgcatcctgttcctttgtattcatctagtgtgaaccgtgccttttcaagccccaaggtcgcagtgga agcctgtaacgccatgttgaaagagaactttccgactgtggcttcttactgtattattccagagtacgatgcctatttggacatggttgacggagctt catgctgcttagacactgccagtttttgccctgcaaagctgcgcagctttccaaagaaacactcctatttggaacccacaatacgatcggcagt gccttcagcgatccagaacacgctccagaacgtcctggcagctgccacaaaaagaaattgcaatgtcacgcaaatgagagaattgcccgt attggattcggcggcctttaatgtggaatgcttcaagaaatatgcgtgtaataatgaatattgggaaacgtttaaagaaaaccccatcaggctta ctgaagaaaacgtggtaaattacattaccaaattaaaaggaccaaaagctgctgctctttttgcgaagacacataatttgaatatgttgcagga cataccaatggacaggtttgtaatggacttaaagagagacgtgaaagtgactccaggaacaaaacatactgaagaacggcccaaggtac aggtgatccaggctgccgatccgctagcaacagcgtatctgtgcggaatccaccgagagctggttaggagattaaatgcggtcctgcttccg aacattcatacactgtttgatatgtcggctgaagactttgacgctattatagccgagcacttccagcctggggattgtgttctggaaactgacatcg cgtcgtttgataaaagtgaggacgacgccatggctctgaccgcgttaatgattctggaagacttaggtgtggacgcagagctgttgacgctgat tgaggcggctttcggcgaaatttcatcaatacatttgcccactaaaactaaatttaaattcggagccatgatgaaatctggaatgttcctcacact gtttgtgaacacagtcattaacattgtaatcgcaagcagagtgttgagagaacggctaaccggatcaccatgtgcagcattcattggagatga caatatcgtgaaaggagtcaaatcggacaaattaatggcagacaggtgcgccacctggttgaatatggaagtcaagattatagatgctgtgg tgggcgagaaagcgccttatttctgtggagggtttattttgtgtgactccgtgaccggcacagcgtgccgtgtggcagaccccctaaaaaggct gtttaagcttggcaaacctctggcagcagacgatgaacatgatgatgacaggagaagggcattgcatgaagagtcaacacgctggaaccg agtgggtattctttcagagctgtgcaaggcagtagaatcaaggtatgaaaccgtaggaacttccatcatagttatggccatgactactctagcta gcagtgttaaatcattcagctacctgagaggggcccctataactctctacggctaacctgaatggactacgacatagtctagtccgccaaggat atcATGGAAGATGCCAAAAACATTAAGAAGGGCCCAGCGCCATTCTACCCACTCGAAGACGGGA CCGCCGGCGAGCAGCTGCACAAAGCCATGAAGCGCTACGCCCTGGTGCCCGGCACCATCGCC TTTACCGACGCACATATCGAGGTGGACATTACCTACGCCGAGTACTTCGAGATGAGCGTTCGGC

[0410] TGGCAGAAGCTATGAAGCGCTATGGGCTGAATACAAACCATCGGATCGTGGTGTGCAGCGAGA ATAGCTTGCAGTTCTTCATGCCCGTGTTGGGTGCCCTGTTCATCGGTGTGGCTGTGGCCCCAGC TAACGACATCTACAACGAGCGCGAGCTGCTGAACAGCATGGGCATCAGCCAGCCCACCGTCGT

[0411] ATTCGTGAGCAAGAAAGGGCTGCAAAAGATCCTCAACGTGCAAAAGAAGCTACCGATCATACAA

[0412] AAGATCATCATCATGGATAGCAAGACCGACTACCAGGGCTTCCAAAGCATGTACACCTTCGTGA

[0413] CTTCCCATTTGCCACCCGGCTTCAACGAGTACGACTTCGTGCCCGAGAGCTTCGACCGGGACA

[0414] AAACCATCGCCCTGATCATGAACAGTAGTGGCAGTACCGGATTGCCCAAGGGCGTAGCCCTAC CGCACCGCACCGCTTGTGTCCGATTCAGTCATGCCCGCGACCCCATCTTCGGCAACCAGATCA

[0415] TCCCCGACACCGCTATCCTCAGCGTGGTGCCATTTCACCACGGCTTCGGCATGTTCACCACGCT GGGCTACTTGATCTGCGGCTTTCGGGTCGTGCTCATGTACCGCTTCGAGGAGGAGCTATTCTTG CGCAGCTTGCAAGACTATAAGATTCAATCTGCCCTGCTGGTGCCCACACTATTTAGCTTCTTCGC TAAGAGCACTCTCATCGACAAGTACGACCTAAGCAACTTGCACGAGATCGCCAGCGGCGGGGC

[0416] GCCGCTCAGCAAGGAGGTAGGTGAGGCCGTGGCCAAACGCTTCCACCTACCAGGCATCCGCC AGGGCTACGGCCTGACAGAAACAACCAGCGCCATTCTGATCACCCCCGAAGGGGACGACAAGC

[0417] CTGGCGCAGTAGGCAAGGTGGTGCCCTTCTTCGAGGCTAAGGTGGTGGACTTGGACACCGGTA

[0418] AGACACTGGGTGTGAACCAGCGCGGCGAGCTGTGCGTCCGTGGCCCCATGATCATGAGCGGC

[0419] TACGTTAACAACCCCGAGGCTACAAACGCTCTCATCGACAAGGACGGCTGGCTGCACAGCGGC

[0420] GACATCGCCTACTGGGACGAGGACGAGCACTTCTTCATCGTGGACCGGCTGAAGAGCCTGATC

[0421] AAATACAAGGGCTACCAGGTAGCCCCAGCCGAACTGGAGAGCATCCTGCTGCAACACCCCAAC ATCTTCGACGCCGGGGTCGCCGGCCTGCCCGACGACGATGCCGGCGAGCTGCCCGCCGCAGT CGTCGTGCTGGAACACGGTAAAACCATGACCGAGAAGGAGATCGTGGACTATGTGGCCAGCCA

[0422] GGTTACAACCGCCAAGAAGCTGCGCGGTGGTGTTGTGTTCGTGGACGAGGTGCCTAAAGGACT GACCGGCAAGTTGGACGCCCGCAAGATCCGCGAGATTCTCATTAAGGCCAAGAAGGGCGGCAA GATCGCCGTGTAAgCGGCCGcatacagcagcaattggcaagctgcttacatagaactcgcggcgattggcatgccgccttaaa atttttattttattttttcttttcttttccgaatcggattttgtttttaatatttcaaaaaaaaaaaaaaaaaaaaaaaaaatctaga

Claims

Claims1 . A method of synthesizing an mRNA in vitro, comprising the steps of: a) providing a composition comprising ATP, CTP, UTP and GTP ribonucleotides, wherein between 0.1% to 100% of at least one of the ribonucleotides is substituted by a deoxyribonucleotide version of the at least one ribonucleotide; b) adding a nucleic acid template encoding a protein of interest to the composition; and c) incubating the composition under conditions that allow mRNA synthesis.

2. The method of claim 1 , wherein the deoxyribonucleotide is a modified or unmodified deoxy ribonucleotide.

3. The method of any one of claims 1 or 2, wherein in step a)(v) the at least one ribonucleotide that is substituted by a deoxyribonucleotide version is ATP, optionally wherein 0.1% to 100% of ATP is substituted by a nucleotide selected from the group consisting of deoxyadenosine(dA), 2'-lodo-deoxyadenosine, 2'-Bromo- deoxyadenosine, 3'- deoxyadenosine, 7-Deaza-deoxyadenosine, 8-Bromo- deoxyadenosine, 7-Deaza-7-iodo- deoxyadenosine, 2-Hydroxy-deoxyadenosine, 2'- Chloro-deoxyadenosine, 7-Deaza-7-bromo- deoxyadenosine, 2'-Fluoro-deoxyadenosine, Mant-deoxyadenosine, 2 '-M ant-3'- deoxyadenosine, 2'NH2- deoxyadenosine, Etheno- deoxyadenosine (E-dA), 5'-(a-seleno)- deoxyadenosine (dAaSe), 5'-(a-thio)- deoxyadenosine (dAaS), N6-(6-Aminohexyl)-deoxyadenosine, N6-methyldeoxyadenosine (m6dA), 2'-0-methyldeoxyadenosine (2'OMedA), N6,2'-0-dimethyldeoxyadenosine (m6dAm), N6,N6,2'-0-trimethyldeoxyadenosine (m62dAm), 8-Oxo-deoxyadenosine, and N6-Methyl-deoxyadenosine; and / or(vi) the at least one ribonucleotide that is substituted by a deoxyribonucleotide version is UTP, optionally wherein 0.1% to 100% of UTP is substituted by a nucleotide selected from the group consisting of 2'NH2-deoxyuridine, 3'-Azido-2',3'-dideoxyuridine, Aminoallyl- deoxyuridine, 5-Propargylamino-deoxyuridine, 5-Fluoro-deoxyuridine, 5- Bromo- deoxyuridine, 5-lodo-deoxyuridine, deoxyuridine, 2',3'-Dideoxythymidine, Deoxythymidine, 5'-(a-seleno)-deoxythymidine (dTaSe), 5'-(a-thio)- deoxythymidine(dTaS), 3,2'-0-dimethyldeoxyuridine (m4dU), 2-thiodeoxyuridine (s2dU), 2-Fluoro-dUTP, pseudodeoxyuridine(dMJ), N1 -Methylpseudouridine (ml d'-P), 2'-O- methyldeoxyuridine (Udm), 4-thiodeoxyuridine (s4dU), 5-methyldeoxyuridine (m5dU), and 5-methoxydeoxyuridine (mo5dU), and / or(vii) the at least one ribonucleotide that is substituted by a deoxyribonucleotide version isCTP, optionally wherein 0.1% to 100% of CTP is substituted by a nucleotide selected from the group consisting of deoxycytidine, C8-Alkyne-deoxycytidine, 5-Methyl- deoxycytidine, 2'-Fluoro-deoxycytidine, 5-lodo-deoxycytidine, 5-Bromo- deoxycytidia- seleno)- deoxycytidine (dCaSe), 5'-(a-thio)-deoxycytidine (dCaS), 5-Propargylamino- deoxycytidine, 5-Hydroxymethyl-deoxycytidine, and 2'-0-methyldeoxycytidine (dCm), and / or(viii) the at least one ribonucleotide that is substituted by a deoxyribonucleotide version is GTP, optionally wherein 0.1 % to 100% of GTP is substituted by a nucleotide selected from the group consisting of 8-Oxo-deoxyguanosine, 3'-deoxyguanosine, 7-Deaza- deoxyguanosine, 7-Deaza-7-iodo-deoxyguanosine, 6-Thio-deoxyguanosine, 2'-Fluoro- deoxyguanosine, Mant- deoxyguanosine, 2'-Mant-3'-deoxyguanosine, 2'NH2- deoxyguanosine, 5'-(a-seleno)-deoxyguanosine (dCaSe), 5'-(a-thio)-deoxyguanosine (dCaS), 2'-0-methyldeoxyguanosine (dGm), N2,7-dimethyldeoxyguanosine (m2,7dG), 7- methyldeoxguanosine (m7dG), and N2, N2,7-trimethyldeoxyguanosine (m2,7dG).

4. The method of any one of the preceding claims, wherein in step a) between 1 % to 75% of at least one of the ribonucleotides are substituted by a deoxyribonucleotide version of the at least one ribonucleotide.

5. The method of any one of the preceding claims , wherein the mRNA is self-amplifying RNA (saRNA), non-self-amplifying RNA, circular RNA (circRNA), or an engineered or modified mRNA variant.

6. An mRNA synthesized by the method of any one of claims 1 -6.

7. An mRNA encoding a protein of interest, wherein between 0.1 % to 100% of at least one of the ribonucleotides are substituted by a deoxyribonucleotide version of the at least one ribonucleotide.

8. The mRNA of claim 7, wherein(i) the at least one ribonucleotide that is substituted by a deoxyribonucleotide version is ATP, optionally wherein 0.1% to 100% of ATP is substituted by a nucleotide selected from the group consisting of deoxyadenosine(dA), 2'-lodo-deoxyadenosine, 2'-Bromo- deoxyadenosine, 3'- deoxyadenosine, 7-Deaza-deoxyadenosine, 8-Bromo- deoxyadenosine, 7-Deaza-7-iodo- deoxyadenosine, 2-Hydroxy-deoxyadenosine, 2'- Chloro-deoxyadenosine, 7-Deaza-7-bromo- deoxyadenosine, 2'-Fluoro-deoxyadenosine, Mant-deoxyadenosine, 2 '-M ant-3'- deoxyadenosine, 2'NH2- deoxyadenosine, Etheno- deoxyadenosine (E-dA), 5'-(a-seleno)- deoxyadenosine (dAaSe), 5'-(a-thio)- deoxyadenosine (dAaS), N6-(6-Aminohexyl)-deoxyadenosine, N6-methyldeoxyadenosine(m6dA), 2'-0-methyldeoxyadenosine (2'0MedA), N6,2'-0-dimethyldeoxyadenosine (m6dAm), N6,N6,2'-0-trimethyldeoxyadenosine (m62dAm), 8-Oxo-deoxyadenosine, and N6-Methyl-deoxyadenosine; and / or(ii) the at least one ribonucleotide that is substituted by a deoxyribonucleotide version is UTP, optionally wherein 0.1% to 100% of UTP is substituted by a nucleotide selected from the group consisting of 2'NH2-deoxyuridine, 3'-Azido-2',3'-dideoxyuridine, Aminoallyl- deoxyuridine, 5-Propargylamino-deoxyuridine, 5-Fluoro-deoxyuridine, 5- Bromo- deoxyuridine, 5-lodo-deoxyuridine, deoxyuridine, 2',3'-Dideoxythymidine, Deoxythymidine, 5'-(a-seleno)-deoxythymidine (dTaSe), 5'-(a-thio)- deoxythymidine(dTaS), 3,2'-0-dimethyldeoxyuridine (m4dll), 2-thiodeoxyuridine (s2dll), 2-Fluoro-dUTP, pseudodeoxyuridine(dMJ), N1 -Methylpseudouridine (ml d'-P), 2'-O- methyldeoxyuridine (Udm), 4-thiodeoxyuridine (s4dll), 5-methyldeoxyuridine (m5dll), and 5-methoxydeoxyuridine (mo5dll), and / or(iii) the at least one ribonucleotide that is substituted by a deoxyribonucleotide version is CTP, optionally wherein 0.1% to 100% of CTP is substituted by a nucleotide selected from the group consisting of deoxycytidine, C8-Alkyne-deoxycytidine, 5-Methyl- deoxycytidine, 2'-Fluoro-deoxycytidine, 5-lodo-deoxycytidine, 5-Bromo- deoxycytidia- seleno)- deoxycytidine (dCaSe), 5'-(a-thio)-deoxycytidine (dCaS), 5-Propargylamino- deoxycytidine, 5-Hydroxymethyl-deoxycytidine, and 2'-0-methyldeoxycytidine (dCm), and / or(iv) the at least one ribonucleotide that is substituted by a deoxyribonucleotide version is GTP, optionally wherein 0.1 % to 100% of GTP is substituted by a nucleotide selected from the group consisting of 8-Oxo-deoxyguanosine, 3'-deoxyguanosine, 7-Deaza- deoxyguanosine, 7-Deaza-7-iodo-deoxyguanosine, 6-Thio-deoxyguanosine, 2'-Fluoro- deoxyguanosine, Mant- deoxyguanosine, 2'-Mant-3'-deoxyguanosine, 2'NH2- deoxyguanosine, 5'-(a-seleno)-deoxyguanosine (dCaSe), 5'-(a-thio)-deoxyguanosine (dCaS), 2'-0-methyldeoxyguanosine (dGm), N2,7-dimethyldeoxyguanosine (m2,7dG), 7- methyldeoxguanosine (m7dG), and N2, N2,7-trimethyldeoxyguanosine (m2,7dG).

9. Use of the mRNA of any one of claims 6 to 8 in a method of increasing transcription and / or expression of a protein of interest encoded by the mRNA in vitro compared to an mRNA not comprising deoxyribonucleotides.

10. A pharmaceutical composition comprising the mRNA of any one of claims 6 to 8 and optionally at least one excipient.11 . A vaccine composition comprising the mRNA of any one of claims 6 to 8 and optionally at least one excipient.

12. The mRNA of any one of claims 6 to 8, the pharmaceutical composition of claim 10 or the vaccine composition of claim 11 for use as a medicament.

13. The mRNA of any one of claims 6 to 8, the pharmaceutical composition of claim 10 or the vaccine composition of claim 11 for use in prevention or treatment of a disease.

14. The mRNA, the pharmaceutical composition or the vaccine composition for use according to claim 13, wherein the prevention or treatment results in increased transcription and / or expression of a protein of interest encoded by the mRNA compared to prevention or treatment with an mRNA not comprising deoxyribonucleotides.

15. The mRNA, the pharmaceutical composition or the vaccine composition for use according to claim 14, wherein the protein of interest encoded by the mRNA is transcribed and expressed in immune cells, preferably in dendritic cells.

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