Ethyl modified RNA caps and methods of use

Abstract

Provided herein are compounds that are cap analogs of a polynucleotide bearing an N-ethylguanosine moiety (e.g., an RNA molecule, such as an mRNA molecule). Capped polynucleotides, such as capped RNA molecules, such as capped mRNA molecules, wherein the 5'end of the RNA molecule comprises a cap analog disclosed herein, pharmaceutical products comprising capped RNA molecules, methods for making capped polynucleotides disclosed herein, and kits for making capped polynucleotides are also provided.

Description

BACKGROUND

[0001] The present disclosure relates generally to RNA cap analogs, polynucleotides (e.g., mRNA) comprising the cap analogs, and methods of making and using the cap analogs.

[0002] Naturally occurring eukaryotic mRNA has a cap structure comprising an N7-methylated guanosine (m7G or 7mG) linked to the first nucleotide of the mRNA via a reverse 5’ to 5’ triphosphate linkage (5’ ppp). The mRNA cap has important roles in cap-dependent initiation of protein synthesis and serves as a protective group from 5’ to 3’ exonuclease cleavage and a unique identifier for recruiting protein factors for precursor mRNA splicing, polyadenylation, and nuclear export. It also acts as an anchor for the recruitment of initiation factors that initiate protein synthesis and 5’ to 3’ circularization of the mRNA during translation.

[0003] Capping can be achieved on synthetic derived mRNA using a variety of strategies, including post-transcriptional enzymatic capping and co-transcriptional capping. The conversion of the 5’- triphosphated end of an mRNA transcript into a capped structure requires several enzymatic steps. The enzymes involved in these steps are referred to as “capping enzymes”. Co-transcriptional capping uses a “capping reagent” that can be incorporated into the growing mRNA strand during the transcription process without the need for additional enzymatic steps. The RNA resulting from these enzymatic steps is referred to as “5’ capped RNA” or simply “capped RNA”.

[0004] The mRNA cap structure is involved in many aspects of mRNA efficacy, including translation efficiency, mRNA stability, and mRNA immunogenicity. However, there is little pharmaceutical chemistry understanding of how these cap structures bind to the relevant enzymes in the capping process (i.e., during mRNA synthesis) or to the translation machinery enzymes such as el F4E.

[0005] There remains a need for synthetic cap analogs that efficiently cap polynucleotides and provide polynucleotides with desirable biological properties, such as capped RNA with desirable translation efficiency. SUMMARY

[0006] Provided herein is a compound of Formula (I) or a stereoisomer, tautomer, or pharmaceutically acceptable salt thereof:

[0007] wherein X is O or S, R 1 is OH or a polynucleotide, and R 2 is H or Me.

[0008] Also provided is an RNA molecule, wherein the 5' end of the RNA molecule comprises a compound, stereoisomer, tautomer, or salt disclosed herein. Also provided is a drug product comprising a capped RNA molecule disclosed herein and one or more pharmaceutically acceptable excipients. Also provided is a kit for capping an RNA molecule, the kit comprising a compound disclosed herein or a stereoisomer, tautomer, or salt thereof and an RNA polymerase. Also provided is a method for making a capped RNA molecule from a polynucleotide template by in vitro transcription, the method comprising: (a) combining more than one nucleotide, a polynucleotide template, and an RNA polymerase to produce a reaction mixture; (b) incubating the reaction mixture; and (c) adding a compound disclosed herein or a stereoisomer, tautomer, or salt thereof to the mixture to produce a capped RNA molecule. BRIEF DESCRIPTION OF DRAWINGS

[0009] Figure 1A "IVT" or in vitro transcription yield of transcription of mRNA comprising RNA cap structure Cap 1, Compound 1, or Compound 2 is shown.

[0010] Figure 1B In vitro expression of mRNA comprising RNA cap structure Cap 1 or RNA cap structure Compound 1 is shown.

[0011] Figure 1C In vivo expression of mRNA comprising RNA cap structure Cap 1 or RNA cap structure Compound 1 is shown.

[0012] Figure 1D Levels of dsRNA produced during IVT reactions with mRNA comprising RNA cap structure Cap 1 or RNA cap structure Compound 1 as determined by sandwich ELISA is shown.

[0013] Figure 2A "IVT" or in vitro transcription yield of transcription of mRNA comprising RNA cap structure Cap 1, Cap 2, Cap 3, Compound 3, or Compound 4 is shown.

[0014] Figure 2B In vitro expression of mRNA comprising RNA cap structure Cap 1, Cap 2, Cap 3, Compound 3, or Compound 4 is shown.

[0015] Figure 2C In vivo expression of mRNA comprising RNA cap structure Cap 1, Cap 2, Cap 3, or Compound 3 is shown.

[0016] Figure 2D Levels of dsRNA produced during IVT reactions with mRNA comprising RNA cap structure Cap 1, Cap 2, Cap 3, or Compound 3 as determined by sandwich ELISA is shown.

[0017] Figure 3A Expression of mRNA without cap structure or mRNA comprising RNA cap structure Cap 1 or Compound 3 is shown.

[0018] Figure 3B Extent of IFNy stimulation of mRNA without cap structure or mRNA comprising RNA cap structure Cap 1 or Compound 3 is shown.

[0019] Figure 3C Extent of IFNa stimulation of mRNA without cap structure or mRNA comprising RNA cap structure Cap 1 or Compound 3 is shown.

[0020] Figure 3D Extent of IP-10 stimulation of mRNA without cap structure or mRNA comprising RNA cap structure Cap 1 or Compound 3 is shown.

[0021] Figure 3E Extent of IL-1a stimulation of mRNA without cap structure or mRNA comprising RNA cap structure Cap 1 or Compound 3 is shown.

[0022] Figure 3F Extent of TNFa stimulation of mRNA without cap structure or mRNA comprising RNA cap structure Cap 1 or Compound 3 is shown. DETAILED DESCRIPTION

[0023] Provided herein are compounds that are cap analogs of polynucleotides (e.g., RNA molecules, such as mRNA molecules). Also provided are capped polynucleotides, e.g., capped RNA molecules, such as capped mRNA molecules, in which the 5’ end of the RNA molecule comprises a cap analog disclosed herein, as well as pharmaceutical products comprising the capped RNA molecules. Also provided are methods for making the capped polynucleotides disclosed herein, as well as kits for making the capped polynucleotides.

[0024] Capped polynucleotides, such as mRNA, can be made by post-transcriptional capping or co-transcriptional capping based on in vitro transcription (IVT) of a DNA template. As used herein, “in vitro transcription” (IVT) refers to a cell-free reaction in which a DNA template is copied by an RNA polymerase to produce an RNA molecule encoded by the DNA template.

[0025] In post-transcriptional capping, RNA from IVT is capped via a series of enzymatic capping reactions. In co-transcriptional capping, a cap analog is added directly to IVT. Synthetic cap analogs have increased capping efficiency, reduced de-capping and potential for reverse capping. RNA capped with these analogs has reduced degradation, increased nuclease resistance, increased translation yield, and reduced immunogenicity.

[0026] Cap analogs and capped polynucleotides Provided herein are compounds that are synthetic cap analogs and methods of making capped polynucleotides, such as mRNA, with cap analogs.

[0027] A cap analog as disclosed herein is a compound of Formula (I) or a stereoisomer, tautomer, or pharmaceutically acceptable salt thereof: , wherein X is O or S, R 1 is OH or a polynucleotide, and R 2 is H or Me. In some cases, the stereochemistry of the phosphorus with the X substitution is (R) (i.e., the compound is an R-P compound of Formula (I)). In some cases, the stereochemistry is (S) (i.e., the compound is an S-P compound of Formula (I)). In some cases, X is O. In some cases, X is S. In some cases, R 1 is OH. In various cases, R 1 is a polynucleotide. In various cases, R 2 is H. In various cases, R 2 is Me.

[0028] Specific compounds of Formula (I) include the compounds listed in Table 1 below.

[0029] Nucleobases, nucleosides, and nucleotides In some cases, a cap analog as described herein is attached to a polynucleotide that comprises at least one non-naturally occurring nucleobase and / or at least one modified nucleobase (i.e., R 1 is a polynucleotide). The term “modified” or “modification” as appropriate refers to structural modifications and / or chemical modifications with respect to A, G, U, or C nucleobases, nucleosides, and / or nucleotides. The nucleotides in the cap analogs of the present disclosure can include non-standard nucleotides, such as non-naturally occurring nucleotides or chemically synthesized nucleotides. The cap analogs of the present disclosure can include any useful modification, such as a modification to the sugar, nucleobase, or internucleoside linkage (e.g., to the linking phosphate, phosphodiester linkage, or phosphodiester backbone). One or more atoms of a pyrimidine nucleobase can be replaced or substituted with an optionally substituted amino group, an optionally substituted thiol, an optionally substituted alkyl group (e.g., methyl or ethyl), or a halogen (e.g., chlorine or fluorine). In certain cases, a modification (e.g., one or more modifications) is present in each of the sugar and internucleoside linkage. A modification according to the present disclosure can be a modification of ribonucleic acid (RNA) to deoxyribonucleic acid (DNA), threose nucleic acid (TNA), glycol nucleic acid (GNA), peptide nucleic acid (PNA), locked nucleic acid (LNA), or a hybrid thereof.

[0030] Different sugar modifications, nucleotide modifications, and / or internucleoside linkages (e.g., backbone structures) can be present in different positions of the cap analog attached to the polynucleotide. For example, for each position, the sugar in the nucleotide can be independently selected from ribose and deoxyribose, and can include modifications such as, but not limited to, 2’-O-alkyl, 2’-O-methoxyethyl, 2’-O-allyl, 2’-O-alkylamine, 2’-fluoro-ribose, 2’-deoxyribose, and locked nucleic acid (LNA). For each position, the base in the nucleotide can be independently selected from adenine, uridine, guanine, or cytidine or an analog of adenine, uridine, guanine, or cytidine, such as a modified adenine, uridine, guanine, or cytidine. Non-limiting examples of adenine, uridine, guanine, and cytidine analogs and modified adenine, uridine, guanine, and cytidine include N6-methyladenine, N1-methyladenine, N6-2’-O-dimethyladenosine, pseudouridine, N1-methylpseudouridine, 5-iodouridine, 4-thiouridine, 2-thiouridine, 5-methyluridine, pseudoisocytosine, 5-methoxy cytosine, 2-thiocytosine, 5-hydroxycytosine, N4-methylcytosine, 5-hydroxymethylcytosine, inosine, N1-methylguanine, 06-methylguanine, 1-methylguanosine, N2-methylguanosine, N2,N2-dimethylguanosine, 2-methylguanosine, N7-methylguanosine, 1-methylguanosine, N2,N7-dimethylguanosine, and isoguanine.

[0031] In some cases, the modification comprises a bicyclic derivative of a nucleotide (LNA, ENA, CLNA, CENA, AENA, etc.), an acyclic nucleotide (UNA, PNA, etc.), or a nucleotide containing a pyranose ring instead of ribose (ANA, HNA).

[0032] In some cases, the modification can be on the backbone. Non-limiting examples include replacing a phospho (PC) group with a phosphorothioate (PS) or boranophosphonate (PB) group, replacing a 3’,5’-phosphodiester linkage with a 2’,5’-phosphodiester linkage, or replacing an ester linkage with an amide linkage, etc.

[0033] In some embodiments, the modification can be on the nucleobase. For example, uridine (U) can be replaced with pseudouridine (ip), 2-thiouridine (s2U), dihydrouridine (D), 5-bromo-U, 5-iodo-U, etc. Purines can be replaced with 2,6-diaminopurine.

[0034] The polynucleotides described herein can include various substitutions and / or insertions from a native polynucleotide or naturally occurring polynucleotide, e.g., in addition to the modifications on the 5’-terminal mRNA cap moiety disclosed herein. As used herein, the term “chemical modification” or “chemically modified” as it pertains to polynucleotides refers to modification of one or more of the position, pattern, percentage, or population of adenosine (A), guanosine (G), uridine (U), thymidine (T), or cytidine (C) ribonucleosides or deoxyribonucleosides and internucleosidic linkages. Generally, these terms are not intended to refer to ribonucleotide modifications in naturally occurring 5’-terminal mRNA cap moieties herein.

[0035] The modifications can be a variety of different modifications. In some embodiments, the region can comprise one, two, or more (optionally different) nucleoside or nucleotide modifications. In some embodiments, the modified polynucleotide introduced into a cell can exhibit reduced degradation in the cell as compared to an unmodified polynucleotide.

[0036] The modifications of the polynucleotides of the present disclosure include, but are not limited to, those listed in detail below. The polynucleotide can comprise naturally occurring, non-naturally occurring modifications, or the polynucleotide can comprise both naturally occurring and non-naturally occurring modifications.

[0037] The polynucleotides of the present disclosure can include any modification, such as a modification to the sugar, nucleobase, or internucleosidic linkage (e.g., to the linking phosphate, phosphodiester linkage, or phosphodiester backbone). One or more atoms of a pyrimidine or purine nucleobase can be replaced or substituted with an optionally substituted amino group, an optionally substituted thiol, an optionally substituted alkyl group (e.g., methyl or ethyl), or a halogen (e.g., chlorine or fluorine).

[0038] In certain embodiments, the modification (e.g., one or more modifications) is present in each of the sugars and internucleosidic linkages. The modifications according to the present disclosure can be modifications that modify ribonucleic acid (RNA) to deoxyribonucleic acid (DNA), threose nucleic acid (TNA), glycol nucleic acid (GNA), peptide nucleic acid (PNA), locked nucleic acid (LNA), or a hybrid thereof. Additional modifications are described herein.

[0039] Non-naturally modified nucleotides can be introduced into a polynucleotide during or after synthesis of the chain to achieve a desired function or property. The modification can be on the internucleotide linkage, purine base, or pyrimidine base, or sugar. The modification can be introduced at the end of the chain or at any other position in the chain with chemical synthesis or with a polymerase. Any region of the polynucleotide can be chemically modified.

[0040] The present disclosure provides polynucleotides comprising unmodified or modified nucleosides and nucleotides, and combinations thereof. As described herein, a "nucleoside" is defined as a moiety comprising a sugar molecule (e.g., a pentose or ribose) or derivative thereof in combination with an organic base (e.g., a purine or pyrimidine) or derivative thereof (also referred to herein as a "nucleobase"). As described herein, a "nucleotide" is defined as a nucleoside comprising a phosphate group. Modified nucleotides can be synthesized by any useful method as described herein (e.g., chemically, enzymatically, or recombinantly to include one or more modified or unnatural nucleosides). A polynucleotide can comprise one or more regions of linked nucleosides. Such regions can have variable backbone linkages. The linkages can be standard phosphodiester linkages, in which case the polynucleotide will comprise nucleotide regions. Any combination of bases / sugars or linkers can be incorporated into the polynucleotides of the present disclosure.

[0041] Modifications (including but not limited to chemical modifications) of polynucleotides (e.g., RNA polynucleotides, such as mRNA polynucleotides) useful in the compositions, methods, and synthetic processes of the disclosure include, but are not limited to, the following: 2-methylthio-N6-(cis-hydroxyisopentenyl)adenosine; 2-methylthio-N6-methyladenosine; 2-methylthio-N6-threonylcarbamoyladenosine; N6-glycylcarbamoyladenosine; N6-isopentenyladenosine; N6-methyladenosine; N6-threonylcarbamoyladenosine; 1,2’-O-dimethyladenosine; 1-methyladenosine; 2’-O-methyladenosine; 2’-O-ribosyladenosine (phosphate); 2-methyladenosine; 2-methylthio-N6-isopentenyladenosine; 2-methylthio-N6-hydroxy-norvalylcarbamoyl adenosine; 2’-O-methyladenosine; 21-O-ribosyladenosine (phosphate); isopentenyladenosine; N6-(cis-hydroxyisopentenyl)adenosine; N6,2’-O-dimethyladenosine; N6,2’-O-dimethyladenosine; N6,N6,2’-O-trimethyladenosine; N6,N6-dimethyladenosine; N6-acetyladenosine; N6-hydroxy-norvalylcarbamoyl adenosine; N6-methyl-N6-threonylcarbamoyl adenosine; 2-methyladenosine; 2-methylthio-N6-isopentenyladenosine; 7-deaza-adenosine; N1-methyl-adenosine; N6,N6(dimethyl)adenine; N6-cis-hydroxy-isopentenyl-adenosine; o-thio-adenosine; 2(amino)adenine; 2(aminopropyl)adenine; 2(methylthio)N6(isopentenyl)adenine; 2-(alkyl)adenine; 2-(aminoalkyl)adenine; 2-(aminopropyl)adenine; 2-(halo)adenine; 2-(halo)adenine; 2-(propyl)adenine; 2’-amino-2’-deoxy-ATP; 2’-azido-2’-deoxy-ATP; 2’-deoxy-2’-a-aminoadenosine TP; 2’-deoxy-2’-a-azidoadenosine TP; 6(alkyl)adenine; 6(methyl)adenine; 6-(alkyl)adenine; 6-(methyl)adenine; 7(deaza)adenine; 8(alkenyl)adenine; 8(alkynyl)adenine; 8(amino)adenine; 8(thioalkyl)adenine; 8-(alkenyl)adenine; 8-(alkyl)adenine; 8-(alkynyl)adenine; 8-(amino)adenine; 8-(halo)adenine; 8-(hydroxyl)adenine; 8-(thioalkyl)adenine; 8-(thiol)adenine; 8-azido-adenosine; azaadenine; deazaadenine; N6(methyl)adenine; N6-(isopentyl)adenine; 7-deaza-8-aza-adenosine; 7-methyladenine; 1-deaza adenosine TP; 2’fluoro-N6-Bz-deoxyadenosine TP; 2’-OMe-2-amino-ATP; 2’O-methyl-N6-Bz-deoxyadenosine TP; 2’-a-ethynyladenosine TP; 2-aminoadenine; 2-amino adenosine TP;2-amino-ATP; 2'-a-trifluoromethyladenosine TP; 2-azidoadenosine TP; 2'-b-ethynyladenosine TP; 2-bromoadenosine TP; 2'-b-trifluoromethyladenosine TP; 2-chloroadenosine TP; 2'-deoxy-2',2'-difluoro adenosine TP; 2'-deoxy-2'-a-mercaptoadenosine TP; 2'-deoxy-2'-a-thiomethoxyadenosine TP; 2'-deoxy-2'-b-aminoadenosine TP; 2'-deoxy-2'-b-azidoadenosine TP; 2'-deoxy-2'-b-bromo adenosine TP; 2'-deoxy-2'-b-chloroadenosine TP; 2'-deoxy-2'-b-fluoroadenosine TP; 2'-deoxy-2'-b-iodoadenosine TP; 2'-deoxy-2'-b-mercaptoadenosine TP; 2'-deoxy-2'-b-thiomethoxyadenosine TP; 2-fluoroadenosine TP; 2-iodoadenosine TP; 2-mercaptoadenosine TP; 2-methoxy-adenine; 2-methylthio-adenine; 2-trifluoromethyladenosine TP; 3-deaza-3-bromo adenosine TP; 3-deaza-3-chloroadenosine TP; 3-deaza-3-fluoroadenosine TP; 3-deaza-3-iodoadenosine TP; 3-deazoadenosine TP; 4'-azidoadenosine TP; 4'-carbocyclic adenosine TP; 4'-ethynyladenosine TP; 5'-high adenosine TP; 8-aza-ATP; 8-bromo-adenosine TP; 8-trifluoromethyladenosine TP; 9-deazoadenosine TP; 2-amino purine; 7-deaza-2,6-diaminopurine; 7-deaza-8-aza-2,6-diaminopurine; 7-deaza-8-aza-2-aminopurine; 2,6-diaminopurine; 7-deaza-8-aza-adenine, 7-deaza-2-aminopurine; 2-thiocytidine; 3-methylcytidine; 5-formylcytidine; 5-hydroxymethylcytidine; 5-methylcytidine; N4-acetylcytidine; 2'-O-methylcytidine; 21-O-methylcytidine; 5,2'-O-dimethylcytidine; 5-formyl-2'-O-methylcytidine; lycomycin; N4,2'-O-dimethylcytidine; N4-acetyl-2'-O-methylcytidine; N4-methylcytidine; N4,N4-dimethyl-2'-OMe-cytidine TP; 4-methylcytidine; 5-azacytidine; pseudoisocytidine; pyrrolocytidine; a-thio-cytidine; 2-(thio)cytosine; 2'-amino-2'-deoxy-CTP; 2'-azido-2'-deoxy-CTP; 2'-deoxy-2'-a-aminocytidine TP; 2'-deoxy-2'-a-azidocytidine TP; 3(deaza)5(nitro)cytosine; 3(methyl)cytosine; 3-(alkyl)cytosine; 3-(deaza)5(nitro)cytosine; 3-(methyl)cytidine; 4,21-O-dimethylcytidine; 5(halo)cytosine; 5(methyl)cytosine; 5(propynyl)cytosine; 5(trifluoromethyl)cytosine; 5-(alkyl)cytosine; 5-(alkynyl)cytosine; 5-(halo)cytosine; 5-(propynyl)cytosine;5-(trifluoromethyl)cytosine; 5-bromo-cytidine; 5-iodo-cytidine; 5-propynyl cytosine; 6-(azo) cytosine; 6-aza-cytidine; aza cytosine; deazacytosine; N4(acetyl) cytosine; 1-methyl-1-deaza-pseudoisocytidine; 1-methyl-pseudoisocytidine; 2-methoxy-5-methyl-cytidine; 2-methoxy-cytidine; 2-thio-5-methyl-cytidine; 4-methoxy-1-methyl-pseudoisocytidine; 4-methoxy-pseudoisocytidine; 4-thio-1-methyl-1-deaza-pseudoisocytidine; 4-thio-1-methyl-pseudoisocytidine; 4-thio-pseudoisocytidine; 5-aza-zebularine; 5-methyl-zebularine; pyrrolo-pseudoisocytidine; Zebularine; (E)-5-(2-bromo-vinyl) cytidine TP; 2,2'-anhydro-cytidine TP hydrochloride; 2'fluoro-N4-Bz-cytidine TP; 2'fluoro-N4-acetyl-cytidine TP; 2'-O-methyl-N4-acetyl-cytidine TP; 2'O-methyl-N4-Bz-cytidine TP; 2'-a-ethynylcytidine TP; 2'-a-trifluoromethylcytidine TP; 2'-b-ethynylcytidine TP; 2'-b-trifluoromethylcytidine TP; 2'-deoxy-2',2'-difluorocytidine TP; 2'-deoxy-2'-a-mercaptocytidine TP; 2'-deoxy-2'-a-thiomethoxycytidine TP; 2'-deoxy-2'-b-aminocytidine TP; 2'-deoxy-2'-b-azidocytidine TP; 2'-deoxy-2'-b-bromocytidine TP; 2'-deoxy-2'-b-chlorocytidine TP; 2'-deoxy-2'-b-fluorocytidine TP; 2'-deoxy-2'-b-iodocytidine TP; 2'-deoxy-2'-b-mercaptocytidine TP; 2'-deoxy-2'-b-thiomethoxycytidine TP; 21-O-methyl-5-(1-propynyl)cytidine TP; 3'-ethynylcytidine TP; 4'-azidocytidine TP; 4'-carbocyclocytidine TP; 4'-ethynylcytidine TP; 5-(1-propynyl)arabinocytidine TP; 5-(2-chloro-phenyl)-2-thiocytidine TP; 5-(4-amino-phenyl)-2-thiocytidine TP; 5-aminoallyl-CTP; 5-cyanocytidine TP; 5-ethynylarabinocytidine TP; 5-ethynylcytidine TP; 5'-homocytidine TP; 5-methoxycytidine TP; 5-trifluoromethyl-cytidine TP; N4-amino-cytidine TP; N4-benzoyl-cytidine TP; pseudoisocytidine; 7-methylguanosine; N2,2'-O-dimethylguanosine; N2-methylguanosine; wyosine; 1,2'-O-dimethylguanosine; 1-methylguanosine; 2'-O-methylguanosine; 2'-O-ribosylguanosine (phosphate); 2'-O-methylguanosine; 2'-O-ribosylguanosine (phosphate); 7-aminomethyl-7-deazaguanosine; 7-cyano-7-deazaguanosine;Archaeosine; methylwyosine; N2,7-dimethylguanosine; N2,N2,2'-O-trimethylguanosine; N2,N2,7-trimethylguanosine; N2,N2-dimethylguanosine; N2,7,2'-O-trimethylguanosine; 6-thio-guanosine; 7-deaza-guanosine; 8-oxo-guanosine; N1-methyl-guanosine; a-thio-guanosine; 2(propyl)guanine; 2-(alkyl)guanine; 2'-amino-2'-deoxy-GTP; 2'-azido-2'-deoxy-GTP; 2'-deoxy-2'-a-aminoguanosine TP; 2'-deoxy-2'-a-azidoguanosine TP; 6(methyl)guanine; 6-(alkyl)guanine; 6-(methyl)guanine; 6-methyl-guanosine; 7(alkyl)guanine; 7(deaza)guanine; 7(methyl)guanine; 7-(alkyl)guanine; 7-(deaza)guanine; 7-(methyl)guanine; 8(alkyl)guanine; 8(alkynyl)guanine; 8(halo)guanine; 8(thioalkyl)guanine; 8-(alkenyl)guanine; 8-(alkyl)guanine; 8-(alkynyl)guanine; 8-(amino)guanine; 8-(halo)guanine; 8-(hydroxy)guanine; 8-(thioalkyl)guanine; 8-(thiol)guanine; azaguanine; deazaguanine; N(methyl)guanine; N-(methyl)guanine; 1-methyl-6-thio-guanosine; 6-methoxy-guanosine; 6-thio-7-deaza-8-aza-guanosine; 6-thio-7-deaza-guanosine; 6-thio-7-methyl-guanosine; 7-deaza-8-aza-guanosine; 7-ethyl-8-oxo-guanosine; N2,N2-dimethyl-6-thio-guanosine; N2-methyl-6-thio-guanosine; 1-Me-GTP; 2'fluoro-N2-isobutyl-guanosine TP; 2'O-methyl-N2-isobutyl-guanosine TP; 2'-a-ethynylguanosine TP; 2'-a-trifluoromethylguanosine TP; 2'-b-ethynylguanosine TP; 2'-b-trifluoromethylguanosine TP; 2'-deoxy-2',2'-difluoroguanosine TP; 2'-deoxy-2'-a-mercapto-guanosine TP; 2'-deoxy-2'-a-thiomethoxy-guanosine TP; 2'-deoxy-2'-b-aminoguanosine TP; 2'-deoxy-2'-b-azidoguanosine TP; 2'-deoxy-2'-b-bromoguanosine TP; 2'-deoxy-2'-b-chloroguanosine TP; 2'-deoxy-2'-b-fluoroguanosine TP; 2'-deoxy-2'-b-iodoguanosine TP; 2'-deoxy-2'-b-mercapto-guanosine TP; 2'-deoxy-2'-b-thiomethoxy-guanosine TP; 4'-azidoguanosine TP; 4'-carbocyclic-guanosine TP; 4'-ethynylguanosine TP; 5'-homoguanosine TP; 8-bromo-guanosine TP; 9-deazaguanosine TP; N2-isobutyl-guanosine TP; 1-methylinosine; inosine; 1,2'-O-dimethylinosine; 2'-O-methylinosine;7-methylguanosine; 2'-O-methylguanosine; epoxyqueuosine; galactosyl-queuosine; mannosylqueuosine; queuosine; allylamino-thymidine; azathiidine; deazathiidine; deoxythymidine; 2'-O-methyluridine; 2-thiouridine; 3-methyluridine; 5-carboxymethyluridine; 5-hydroxyuridine; 5-methyluridine; 5-taurinomethyl-2-thiouridine; 5-taurinomethyluridine; dihydrouridine; pseudouridine; (3-(3-amino-3-carboxypropyl)uridine; 1-methyl-3-(3-amino-5-carboxypropyl)pseudouridine; 1-methylpseudouridine; 1-ethylpseudouridine; 2'-O-methyluridine; 2'-O-methylpseudouridine; 2'-O-methyluridine; 2-thio-2'-O-methyluridine; 3-(3-amino-3-carboxypropyl)uridine; 3,2'-O-dimethyluridine; 3-methyl-pseudouridine TP; 4-thiouridine; 5-(carboxyhydroxymethyl)uridine; 5-(carboxyhydroxymethyl)uridine methyl ester; 5,2'-O-dimethyluridine; 5,6-dihydrouridine; 5-aminomethyl-2-thiouridine; 5-aminocarbonylmethyl-2'-O-methyluridine; 5-aminocarbonylmethyluridine; 5-carboxyhydroxymethyluridine; 5-carboxyhydroxymethyluridine methyl ester; 5-carboxymethylaminomethyl-2'-O-methyluridine; 5-carboxymethylaminomethyl-2-thiouridine; 5-carboxymethylaminomethyl-2-thiouridine; 5-carboxymethylaminomethyluridine; 5-carboxymethylaminomethyluridine; 5-carbonylmethyluridine TP; 5-methoxycarbonylmethyl-2'-O-methyluridine; 5-methoxycarbonylmethyl-2-thiouridine; 5-methoxycarbonylmethyluridine; 5-methyluridine; 5-methoxyuridine; 5-methyl-2-thiouridine; 5-methylaminomethyl-2-selenouridine; 5-methylaminomethyl-2-thiouridine; 5-methylaminomethyluridine; 5-methyldihydrouridine; 5-oxoacetic acid-uridine TP; 5-oxoacetic acid-methyl ester-uridine TP; N1-methyl-pseudouracil; N1-ethyl-pseudouracil; uridine 5-oxoacetic acid; uridine 5-oxoacetic acid methyl ester; 3-(3-amino-3-carboxypropyl)-uridine TP; 5-(isopentenylaminomethyl)-2-thiouridine TP; 5-(isopentenylaminomethyl)-2'-O-methyluridine TP; 5-(isopentenylaminomethyl)uridine TP; 5-propynyluracil; a-thiouridine; 1(aminoalkylaminocarbonylvinyl)-2(thio)-pseudouracil; 1(aminoalkylaminocarbonylvinyl)-2,4(dithio)pseudouracil; 1(aminoalkylaminocarbonylvinyl)-4(thio)pseudouracil; 1(aminoalkylaminocarbonylvinyl)-pseudouracil; 1(aminocarbonylvinyl)-2(thio)pseudouracil; 1(aminocarbonylvinyl)-2,4(dithio)pseudouracil; 1(aminocarbonylvinyl)-4(thio)pseudouracil; 1(aminocarbonylvinyl)-pseudouracil;1-substituted 2-(thio)-pseudouracil; 1-substituted 2,4-(dithio)-pseudouracil; 1-substituted 4(thio)-pseudouracil; 1-substituted pseudouracil; 1-(aminoalkylamino-carbonylvinyl)-2-(thio)-pseudouracil; 1-methyl-3-(3-amino-3-carboxypropyl) pseudouridine TP; 1-methyl-3-(3-amino-3-carboxypropyl) pseudo-UTP; 1-methyl-pseudo-UTP; 1-ethyl-pseudo-UTP; 2(thio) pseudouracil; 2' deoxyuridine; 2'fluorouridine; 2-(thio) uracil; 2,4-(dithio) pseudouracil; 2'methyl-guanosine, 2' amino-guanosine, 2' azido-guanosine, 2' fluoro-guanosine; 2'-amino-2'-deoxy-UTP; 2'-azido-2'-deoxy-UTP; 2'-azido-deoxyuridine TP; 2'-O-methyl pseudouridine; 2' deoxyuridine; 2'fluorouridine; 2'-deoxy-2'-a-aminouridine TP; 2'-deoxy-2'-a-azidouridine TP; 2-methyl pseudouridine; 3(3 amino-3 carboxypropyl) uracil; 4(thio) pseudouracil; 4-(thio) pseudouracil; 4-(thio) uracil; 4-thiouracil; 5(1,3-diazol-1-alkyl) uracil; 5(2-amino propyl) uracil; 5(aminoalkyl) uracil; 5(dimethylaminoalkyl) uracil; 5(guanidinoalkyl) uracil; 5(methoxycarbonylmethyl)-2-(thio) uracil; 5(methoxycarbonyl-methyl) uracil; 5(methyl) 2(thio) uracil; 5(methyl) 2,4(dithio) uracil; 5(methyl) 4(thio) uracil; 5(methylamino methyl)-2(thio) uracil; 5(methylamino methyl)-2,4(dithio) uracil; 5(methylamino methyl)-4(thio) uracil; 5(propynyl) uracil; 5(trifluoromethyl) uracil; 5-(2-amino propyl) uracil; 5-(alkyl)-2-(thio) pseudouracil; 5-(alkyl)-2,4(dithio) pseudouracil; 5-(alkyl)-4(thio) pseudouracil; 5-(alkyl) pseudouracil; 5-(alkyl) uracil; 5-(alkynyl) uracil; 5-(allylamino) uracil; 5-(cyanoalkyl) uracil; 5-(dialkylaminoalkyl) uracil; 5-(dimethylaminoalkyl) uracil; 5-(guanidinoalkyl) uracil; 5-(halo) uracil; 5-(1,3-diazol-1-alkyl) uracil; 5-(methoxy) uracil; 5-(methoxycarbonylmethyl)-2-(thio) uracil; 5-(methoxycarbonyl-methyl) uracil; 5-(methyl) 2(thio) uracil; 5-(methyl) 2,4(dithio) uracil; 5-(methyl) 4(thio) uracil; 5-(methyl)-2-(thio) pseudouracil; 5-(methyl)-2,4(dithio) pseudouracil; 5-(methyl)-4(thio) pseudouracil; 5-(methyl) pseudouracil;5-(methylaminomethyl)-2-(thio)uracil; 5-(methylaminomethyl)-2,4-(dithio)uracil; 5-(methylaminomethyl)-4-(thio)uracil; 5-(propynyl)uracil; 5-(trifluoromethyl)uracil; 5-aminoallyl-uridine; 5-bromo-uridine; 5-iodo-uridine; 5-uracil; 6-(azo)uracil; 6-(azo)uracil; 6-aza-uridine; allylaminouracil; aza-uridine; deazouracil; N3-(methyl)uracil; pseudoUTP-1-2-acetic acid; pseudouracil; 4-thio-pseudoUTP; 1-carboxymethyl-pseudouridine; 1-methyl-1-deazo-pseudouridine; 1-propynyl-uridine; 1-taurate methyl-1- Methyl-uridine; 1-Taurate methyl-4-thiouridine; 1-Taurate methyl-pseudouridine; 2-Methoxy-4-thio-pseudouridine; 2-Thio-1-methyl-1-deazo-pseudouridine; 2-Thio-1-methyl-pseudouridine; 2-Thio-5-aza-uridine; 2-Thio-dihydropseudouridine; 2-Thio-dihydrouridine; 2-Thio-pseudouridine; 4-Methoxy-2-thio-pseudouridine; 4-Methoxy-pseudouridine; 4-Thio-1-methyl-pseudouridine; 4-Thio-pseudouridine; 5-aza-uridine; dihydropseudouridine; (±)1-(2-hydroxypropyl)pseudouridine TP; (2R)-1-(2-hydroxypropyl)pseudouridine TP; (2S)-1-(2-hydroxypropyl)pseudouridine TP; (E)- 5-(2-bromo-vinyl)arabinose-uridine TP; (E)-5-(2-bromo-vinyl)uridine TP; (Z)-5-(2-bromo-vinyl)arabinose-uridine TP; (Z)-5-(2-bromo-vinyl)uridine TP; 1-(2,2,2-trifluoroethyl)-pseudo-UTP; 1-(2,2,3,3,3-pentafluoropropyl)pseudo-uridine TP; 1-(2,2-diethoxyethyl)pseudo-uridine TP; 1-(2,4,6-trimethylbenzyl)pseudo-uridine TP; 1-(2,4,6-trimethyl-benzyl)pseudo-UTP; 1-(2,4,6-trimethyl-phenyl)pseudo-UTP; 1-(2-amino-2-carboxyethyl)pseudo-UTP; 1-(2-amino-ethyl)pseudo-UTP ; 1-(2-hydroxyethyl) pseudouridine TP; 1-(2-methoxyethyl) pseudouridine TP; 1-(3,4-bis-trifluoromethoxybenzyl) pseudouridine TP; 1-(3,4-dimethoxybenzyl) pseudouridine TP; 1-(3-amino-3-carboxypropyl) pseudoUTP; 1-(3-amino-propyl) pseudoUTP; 1-(3-cyclopropyl-prop-2-ynyl) pseudouridine TP; 1-(4-amino-4-carboxybutyl) pseudoUTP; 1-(4-amino-benzyl) pseudoUTP; 1-(4-amino-butyl) pseudoUTP; 1-(4-amino-phenyl) pseudoUTP; 1-(4-azidobenzyl) pseudouridine TP; 1-(4-bromobenzyl) pseudouridine TP; 1-(4-chlorobenzyl) pseudouridine TP;1-(4-Fluorobenzyl) pseudouridine TP; 1-(4-Iodobenzyl) pseudouridine TP; 1-(4- Methanesulfonylbenzyl) pseudouridine TP; 1-(4-Methoxybenzyl) pseudouridine TP; 1-(4- Methoxy-benzyl) pseudo UTP; 1-(4-Methoxy-phenyl) pseudo UTP; 1-(4-Methylbenzyl) pseudouridine TP; 1-(4-Methyl-benzyl) pseudo-UTP; 1-(4-Nitrobenzyl) pseudouridine TP; 1-(4- Nitro-benzyl) pseudo UTP; 1(4-Nitro-phenyl) pseudo UTP; 1-(4-Thiomethoxybenzyl) pseudouridine TP; 1-(4-Trifluoromethoxybenzyl) pseudouridine TP; 1-(4-Trifluoromethylbenzyl) pseudouridine TP; 1-(5-Amino-pentyl) pseudo UTP; 1-(6-Amino-hexyl) pseudo UTP; 1,6-Dimethyl- pseudo UTP; 1-[3-(2-{2-[2-(2-Aminoethoxy)-ethoxy]-ethoxy-ethoxy)-propionyl] pseudouridine TP; 1-13-[2-(2-Aminoethoxy)-ethoxy]-propionyl} pseudouridine TP; 1-Acetyl pseudouridine TP; 1-Alkyl-6-(1-propynyl)-pseudo UTP; 1-Alkyl-6-(2-propynyl)-pseudo UTP; 1-Alkyl-6-allyl- pseudo UTP; 1-Alkyl-6-ethynyl-pseudo UTP; 1-Alkyl-6-homoallyl-pseudo UTP; 1-Alkyl-6- vinyl-pseudo UTP; 1-Allyl pseudouridine TP; 1-Aminomethyl-pseudo UTP; 1-Benzoyl pseudouridine TP; 1-Benzyloxymethyl pseudouridine TP; 1-Benzyl-pseudo UTP; 1-Biotinyl-PEG2- pseudouridine TP; 1-Biotinyl pseudouridine TP; 1-Butyl-pseudo UTP; 1-Cyanomethyl pseudouridine TP; 1-Cyclobutylmethyl-pseudo UTP; 1-Cyclobutyl-pseudo UTP; 1-Cycloheptylmethyl- pseudo UTP; 1-Cycloheptyl-pseudo UTP; 1-Cyclohexylmethyl-pseudo UTP; 1-Cyclohexyl- pseudo UTP; 1-Cyclooctylmethyl-pseudo UTP; 1-Cyclooctyl-pseudo UTP; 1-Cyclopentylmethyl- pseudo UTP; 1-Cyclopentyl-pseudo UTP; 1-Cyclopropylmethyl-pseudo UTP; 1-Cyclopropyl- pseudo UTP; 1-Ethyl-pseudo UTP; 1-Hexyl-pseudo UTP; 1-Homoallyl pseudouridine TP; 1- Hydroxymethyl pseudouridine TP; 1-Isopropyl-pseudo UTP; 1-Me-2-Thio-pseudo UTP; 1-Me-4- Thio-pseudo UTP; 1-Me-α-Thio-pseudo UTP; 1-Methanesulfonylmethyl pseudouridine TP; 1- Methoxymethyl pseudouridine TP; 1-Methyl-6-(2,2,2-trifluoroethyl) pseudo UTP; 1-Methyl-6-(4- morpholino)-pseudo UTP; 1-Methyl-6-(4-thiomorpholino)-pseudo UTP; 1-Methyl-6-(substituted phenyl) pseudo UTP; 1-Methyl-6-amino-pseudo UTP; 1-Methyl-6-azido-pseudo UTP; 1-Methyl-6-bromo-pseudo UTP;1 -methyl-6-butyl-pseudo UTP; 1 -methyl-6-chloro-pseudo UTP; 1 -methyl-6-cyano- pseudo UTP; 1 -methyl-6-dimethylamino-pseudo UTP; 1 -methyl-6-ethoxy-pseudo UTP; 1 -methyl-6-ethylcarboxylate-pseudo UTP; 1 -methyl-6-ethyl-pseudo UTP; 1 -methyl-6-fluoro- pseudo UTP; 1 -methyl-6-formyl-pseudo UTP; 1 -methyl-6-hydroxylamino-pseudo UTP; 1 -methyl-6-hydroxy-pseudo UTP; 1 -methyl-6-iodo-pseudo UTP; 1 -methyl-6- isopropyl-pseudo UTP; 1 -methyl-6-methoxy-pseudo UTP; 1 -methyl-6-methylamino- pseudo UTP; 1 -methyl-6-phenyl-pseudo-UTP; 1 -methyl-6-propyl-pseudo UTP; 1 -methyl-6-tert-butyl-pseudo UTP; 1 -methyl-6-trifluoromethoxy-pseudo UTP; 1 -methyl-6- trifluoromethyl-pseudo UTP; 1 -morpholinomethyl pseudouridine TP; 1 -pentyl-pseudo UTP; 1 -phenyl-pseudo UTP; 1 -pivaloyl pseudouridine TP; 1 -propargyl pseudouridine TP; 1 -propyl-pseudo UTP; 1 -propynyl-pseudouridine; 1 -p-tolyl-pseudo UTP; 1 -tert-butyl-pseudo UTP; 1 -thiomethoxymethyl pseudouridine TP; 1 -thiomorpholinomethyl pseudouridine TP; 1 -trifluoroacetyl pseudouridine TP; 1 -trifluoromethyl-pseudo UTP; 1 -vinyl pseudouridine TP; 2,2'-anhydro-uridine TP; 2'-bromo-deoxyuridine TP; 2'-F-5-methyl-2'-deoxy-UTP; 2'-OMe-5-Me-UTP; 2'-OMe-pseudo UTP; 2'-a-ethynyluridine TP; 2'-a-trifluoromethyluridine TP; 2'-b-ethynyluridine TP; 2'-b-trifluoromethyluridine TP; 2'-deoxy-2',2'-difluorouridine TP; 2'-deoxy-2'-a-mercapto uridine TP; 2'-deoxy-2'-a-thiomethoxyuridine TP; 2'-deoxy-2'-b-aminouridine TP; 2'-deoxy-2'-b-azidouridine TP; 2'-deoxy-2'-b-bromouridine TP; 2'-deoxy-2'-b-chlorouridine TP; 2'-deoxy-2'-b-fluorouridine TP; 2'-deoxy-2'-b-iodouridine TP; 2'-deoxy-2'-b-mercapto uridine TP; 2'-deoxy-2'-b-thiomethoxyuridine TP; 2-methoxy-4-thio-uridine; 2-methoxyuridine; 2'-O-methyl-5-(1-propynyl)uridine TP; 3-alkyl-pseudo UTP; 4'-azidouridine TP; 4'-carbocyclic uridine TP; 4'-ethynyluridine TP; 5-(1-propynyl)arabinouridine TP; 5-(2-furyl)uridine TP; 5-cyano uridine TP; 5-dimethylamino uridine TP; 5'-homouridine TP; 5-iodo-2'-fluoro-deoxyuridine TP; 5-phenylethynyluridine TP;5-trideuteromethyl-6-deuteroxylate uridine TP; 5-trifluoromethyl uridine TP; 5-vinyl arabinosyl uridine TP; 6-(2,2,2-trifluoroethyl)-pseudo UTP; 6-(4-morpholino)-pseudo UTP; 6-(4-thiomorpholino)-pseudo UTP; 6-(substituted phenyl)-pseudo UTP; 6-amino-pseudo UTP; 6-azido-pseudo UTP; 6-bromo-pseudo UTP; 6-butyl-pseudo UTP; 6-chloro-pseudo UTP; 6-cyano-pseudo UTP; 6-dimethylamino-pseudo UTP; 6-ethoxy-pseudo UTP; 6-ethylcarboxylate-pseudo UTP; 6-ethyl-pseudo UTP; 6-fluoro-pseudo UTP; 6-formyl-pseudo UTP; 6-hydroxyamino-pseudo UTP; 6-hydroxy-pseudo UTP; 6-iodo-pseudo UTP; 6-isopropyl-pseudo UTP; 6-methoxy-pseudo UTP; 6-methylamino-pseudo UTP; 6-methyl-pseudo UTP; 6-phenyl-pseudo UTP; 6-phenyl-pseudo UTP; 6-propyl-pseudo UTP; 6-t-butyl-pseudo UTP; 6-trifluoromethoxy-pseudo UTP; 6-trifluoromethyl-pseudo UTP; alpha-thio-pseudo UTP; pseudo uridine 1-(4-methylbenzenesulfonic acid) TP; pseudo uridine 1-(4-methylbenzoic acid) TP; pseudo uridine TP 1-[3-(2-ethoxy)]propionic acid; pseudo uridine TP 1-[3-12-(2-[2-(2-ethoxy)-ethoxy]-ethoxy)-ethoxy]propionic acid; pseudo uridine TP 1-[3-{24242-12(2-ethoxy)-ethoxy-ethoxy]-ethoxy)-ethoxy]propionic acid; pseudo uridine TP 1-[3-12-(2-[2-ethoxy]-ethoxy)-ethoxy]propionic acid; pseudo uridine TP 143-12-(2-ethoxy)-ethoxypropionic acid; pseudo uridine TP 1-methyl phosphonic acid; pseudo uridine TP 1-methyl phosphonic acid diethyl ester; pseudo UTP-N1-3-propionic acid; pseudo UTP-N1-4-butyric acid; pseudo UTP-N1-5-valeric acid; pseudo UTP-N1-6-hexanoic acid; pseudo UTP-N1-7-heptanoic acid; pseudo UTP-N1-methyl-p- benzoic acid; pseudo UTP-N1-p-benzoic acid; Wybutosine; hydroxy Wybutosine; Isowyosine; peroxy Wybutosine; unmodified hydroxy Wybutosine; 4-demethyl Wybutosine; 2,6-(diamino) purine; 1-(aza)-2-(thio)-3-(aza)-phenoxazine-1-yl; 1,3-(diazepino)-2-(oxo)-phenothiazine-1-yl; 1,3-(diazepino)-2-(oxo)-phenoxazine-l-yl; 1,3,5-(triazepino)-2,6-(dioxa)-naphthalene; 2(amino) purine; 2,4,5-(trimethyl) phenyl; 2'methyl-cytidine, 2'amino-cytidine, 2'azido-cytidine, 2'fluoro-cytidine; 21-methyl-adenine, 2'amino-adenine, 2'azido-adenine, 2'fluoro-adenine;2' methyl-uridine, 2' amino-uridine, 2' azido-uridine, 2' fluoro-uridine; 2'-amino-2'-deoxyribose; 2-amino-6-chloro-purine; 2-aza-inosinyl; 2'-azido-2'-deoxyribose; 2' fluoro-2'-deoxyribose; 2'-fluoro modified base; 2'-O-methyl-ribose; 2-oxo-7-amino pyridopyrimidin-3-yl; 2-oxo-pyridopyrimidin-3-yl; 2-pyridinone; 3 nitro-pyrrole; 3-(methyl)-7-(propynyl)isocarbostyrilyl; 3-(methyl)-isocarbostyrilyl; 4-(fluoro)-6-(methyl)benzoimidazole; 4-(methyl)benzoimidazole; 4-(methyl)indolyl; 4,6-(dimethyl)indolyl; 5 nitro-indole; 5-substituted pyrimidine; 5-(methyl)isocarbostyrilyl; 5-nitro-indole; 6-(aza)pyrimidine; 6-(azo)thymine; 6-(methyl)-7-(aza)indolyl; 6-chloro-purine; 6-phenyl-pyrrolo-pyrimidin-2-one-3-yl; 7-(aminoalkylhydroxy)-1-(aza)-2-(thioxo)-3-(aza)-phenothiazin-1-yl; 7-(aminoalkylhydroxy)-1-(aza)-2-(thioxo)-3-(aza)-phenoxazin-1-yl; 7-(aminoalkylhydroxy)-1,3-(diazepino)-2-(oxo)-phenoxazin-1-yl; 7-(aminoalkylhydroxy)-1,3-(diazepino)-2-(oxo)-phenothiazin-1-yl; 7-(aminoalkylhydroxy)-1,3-(diazepino)-2-(oxo)-phenoxazin-1-yl; 7-(aza)indolyl; 7-(guanidinoalkylhydroxy)-1-(aza)-2-(thioxo)-3-(aza)-phenoxazin-1-yl; 7-(guanidinoalkylhydroxy)-1-(aza)-2-(thioxo)-3-(aza)-phenothiazin-1-yl; 7-(guanidinoalkylhydroxy)-1-(aza)-2-(thioxo)-3-(aza)-phenoxazin-1-yl; 7-(guanidinoalkylhydroxy)-1,3-(diazepino)-2-(oxo)-phenoxazin-1-yl; 7-(guanidinoalkyl-hydroxy)-1,3-(diazepino)-2-(oxo)-phenothiazin-1-yl; 7-(guanidinoalkylhydroxy)-1,3-(diazepino)-2-(oxo)-phenoxazin-1-yl; 7-(propynyl)isocarbostyrilyl; 7-(propynyl)isocarbostyrilyl; propynyl-7-(aza)indolyl; 7-deaza-inosinyl; 7-substituted 1-(aza)-2-(thioxo)-3-(aza)-phenoxazin-1-yl; 7-substituted 1,3-(diazepino)-2-(oxo)-phenoxazin-1-yl; 9-(methyl)-imidazopyridinyl; aminoindolyl; anthryl; bis-ortho(aminoalkylhydroxy)-6-phenyl-pyrrolo-pyrimidin-2-one-3-yl; bis-ortho-substituted-6-phenyl-pyrrolo-pyrimidin-2-one-3-yl; difluorotolyl; hypoxanthine; imidazopyridinyl;Inosinyl; Isoquinolinonyl; Isoguanosine; N2-Substituted Purine; N6-Methyl-2- amino-purine; N6-Substituted Purine; N-Alkylated Derivative; Naphthyl; Nitrobenzimidazolyl; Nitroimidazolyl; Nitroindazolyl; Nitropyrazolyl; Ochratoxin; O6-Substituted Purine; O-Alkylated Derivative; Ortho-(aminoalkylhydroxy)-6-phenyl-pyrrolo- pyrimidin-2-one-3-yl; Ortho-Substituted-6-phenyl-pyrrolo-pyrimidin-2-one-3-yl; Oxoformycin TP; Para-(aminoalkylhydroxy)-6-phenyl-pyrrolo-pyrimidin-2-one-3-yl; Para-Substituted-6-phenyl-pyrrolo-pyrimidin-2-one-3-yl; Pentalenyl; Phenanthracenyl; Phenyl; Propynyl-7-(aza)indolyl; Pyrenyl; Pyrido-pyrimidin-3-yl; Pyrido-pyrimidin-3-yl; 2-Oxo-7-amino-pyrido-pyrimidin-3-yl; Pyrrolo-pyrimidin-2-one-3-yl; Pyrrolo-pyrimidinyl; Pyrrolo-pyrazinyl; Stilbenyl; Substituted 1,2,4-Triazole; Tetracenyl; Tubercidin; Xanthine; Xanthosine-5'-TP; 2-Thio-zebularine; 5-Aza-2-thio-zebularine; 7-Deaza-2-amino-purine; Pyrid-4-one ribonucleoside; 2-Amino-nucleoside-TP; Distamycin A TP; Distamycin B TP; Pyrrolosine TP; 2'-OH-Arabinose-adenosine TP; 2'-OH-Arabinose-cytidine TP; 2'-OH-Arabinose-uridine TP; 2'-OH-Arabinose-guanosine TP; 5-(2-Methoxycarbonylvinyl)uridine TP; and N6-(19-Amino-pentaoxanonadecyl)adenosine TP.

[0042] In some embodiments, the polynucleotide (e.g., an RNA polynucleotide, such as an mRNA polynucleotide) comprises a combination of at least two (e.g., 2, 3, 4, or more) of the above-described modified nucleobases.

[0043] In some embodiments, the modified nucleobases in the polynucleotide (e.g., an RNA polynucleotide, such as an mRNA polynucleotide) are selected from the group consisting of: pseudouridine (ip), 2-thiouridine (s2U), 4’-thiouridine, 5-methylcytosine, 2-thio-l-methyl-l-deaza-pseudouridine, 2-thio-l-methyl-pseudouridine, 2-thio-5-aza-uridine, 2-thio-dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-pseudouridine, 4-methoxy-2-thio-pseudouridine, 4-methoxy-pseudouridine, 4-thio-l-methyl-pseudouridine, 4-thio-pseudouridine, 5-aza-uridine, dihydropseudouridine, 5-methyluridine, 5-methoxy-uridine, 21-O-methyluridine, 1-methyl-pseudouridine (m1ip), 1-ethyl-pseudouridine (e1ip), 5-methoxy-uridine (mo5U), 5-methyl-cytidine (m5C), a-thio-guanosine, a-thio-adenosine, 5-cyano uridine, 4’-thiouridine 7-deaza-adenine, 1-methyl-adenosine (mlA), 2-methyl-adenine (m2A), N6-methyl-adenosine (m6A), and 2,6-diaminopurine (I), 1-methyl-inosine (ml I), wyosine (imG), methylwyosine (mimG), 7-deaza-guanosine, 7-cyano-7-deaza-guanosine (preQO), 7-aminomethyl-7-deaza-guanosine (preQ1), 7-methylguanosine (m7G), 1-methylguanosine (ml G), 8-oxo-guanosine, 7-methyl-8-oxo-guanosine, 2,8-dimethyladenosine, 2-ribofuranosylthiouridine, 2-ribosylselenouridine, 3-(3-amino-3-carboxypropyl)-5,6-dihydrouridine, 3-(3-amino-3-carboxypropyl)pseudouridine, 3-methylpseudouridine, 5-(carboxyhydroxymethyl)-2’-O-methyluridinemethyl ester, 5-aminomethyl-2-ribosylthiouridine, 5-aminomethyl-2-ribosylselenouridine, 5-aminomethyluridine, 5-aminocarbonylhydroxymethyluridine, 5-aminocarbonylmethyl-2-ribosylthiouridine, 5-carboxymethyl-2-ribosylthiouridine, 5-carboxymethylaminomethyl-2-ribosylthiouridine, 5-carboxymethylaminomethyl-2-ribosylselenouridine, 5-cyanomethyluridine, 5-hydroxycytidine, 5-methylaminomethyl-2-ribosylthiouridine, 7-aminocarboxypropyl-desmethylwyosine, 7-aminocarboxypropylwyosine, 7-aminocarboxypropylwyosinemethyl ester, 8-methyladenosine, N4,N4-dimethylcytidine, N6-formyladenosine, N6-hydroxymethyladenosine, agmatidine, cyclic N6-serylcarbonyl adenosine, glutamyl-queosine, methylated unmodified hydroxylwybutosine, N4,N4,2'-0-trimethylcytidine, geranylated 5-methylaminomethyl-2-thio-uridine, geranylated 5- carboxymethylaminomethyl-2-thio-uridine, Qbase, preQObase, preQIbase, and combinations of two or more thereof. In some embodiments, the at least one chemically modified nucleoside is selected from the group consisting of pseudouridine, 1 -methyl-pseudouridine, 1 -ethyl- pseudouridine, 5-methylcytosine, 5-methoxyuridine, and combinations thereof. In some embodiments, the polyribonucleotide (e.g., an RNA polyribonucleotide, such as an mRNA polyribonucleotide) comprises at least two (e.g., 2, 3, 4, or more) combinations of the above modified nucleobases. In some embodiments, the polynucleotide (e.g., an RNA polynucleotide, such as an mRNA polynucleotide) comprises at least two (e.g., 2, 3, 4, or more) combinations of the above modified nucleobases.

[0044] In some embodiments, the modified nucleobases in the polynucleotide (e.g., an RNA polynucleotide, such as an mRNA polynucleotide) are selected from the group consisting of 1 -methyl-pseudouridine (m1ip), 1 -ethyl-pseudouridine (e1ip), 5-methoxy-uridine (mo5U), 5-methyl-cytidine (m5C), pseudouridine (ip), a-thio-guanosine, and a-thio-adenosine. In some embodiments, the polyribonucleotide comprises at least two (e.g., 2, 3, 4, or more) combinations of the above modified nucleobases, including but not limited to chemical modifications. In some embodiments, the polynucleotide (e.g., an RNA polynucleotide, such as an mRNA polynucleotide) comprises pseudouridine (ip) and 5-methyl-cytidine (m5C). In some embodiments, the polyribonucleotide (e.g., an RNA, such as an mRNA) comprises 1 -methyl-pseudouridine (m1i). In some embodiments, the polyribonucleotide (e.g., an RNA, such as an mRNA) comprises 1 -ethyl-pseudouridine (e1ip). In some embodiments, the polyribonucleotide (e.g., an RNA, such as an mRNA) comprises 1 -methyl-pseudouridine (m1w) and 5-methyl-cytidine (m5C). In some embodiments, the polyribonucleotide (e.g., an RNA, such as an mRNA) comprises 1 -ethyl-pseudouridine (e1ip) and 5-methyl-cytidine (m5C).

[0045] In some embodiments, the polyribonucleotide (e.g., RNA, such as mRNA) comprises 2-thiouridine (s2U). In some embodiments, the polyribonucleotide (e.g., RNA, such as mRNA) comprises 2-thiouridine and 5-methyl-cytidine (m5C). In some embodiments, the polyribonucleotide (e.g., RNA, such as mRNA) comprises methoxyuridine (mo5U). In some embodiments, the polyribonucleotide (e.g., RNA, such as mRNA) comprises 5-methoxy-uridine (mo5U) and 5-methyl-cytidine (m5C). In some embodiments, the polyribonucleotide (e.g., RNA, such as mRNA) comprises 21-O-methyluridine. In some embodiments, the polyribonucleotide (e.g., RNA, such as mRNA) comprises 21-O-methyluridine and 5-methyl-cytidine (m5C). In some embodiments, the polyribonucleotide (e.g., RNA, such as mRNA) comprises N6-methyl-adenosine (m6A). In some embodiments, the polyribonucleotide (e.g., RNA, such as mRNA) comprises N6-methyl-adenosine (m6A) and 5-methyl-cytidine (m5C).

[0046] In some embodiments, a polynucleotide (e.g., an RNA polynucleotide, such as an mRNA polynucleotide) is uniformly modified (e.g., fully modified, modified throughout the sequence) for a particular modification. For example, a polynucleotide can be uniformly modified with 1-methyl-pseudouridine, meaning that all uridine residues in the mRNA sequence are replaced with 1-methyl-pseudouridine. Similarly, a polynucleotide can be uniformly modified for any type of nucleoside residue present in the sequence by replacement with a modified residue, such as those described above.

[0047] A polynucleotide of the present disclosure can be partially or fully modified along the entire length of the molecule. For example, one or more or all or a given type of nucleotides (e.g., purines or pyrimidines, or any one or more or all of A, G, U, C) can be uniformly modified in a polynucleotide of the present disclosure or in a given predetermined sequence region thereof (e.g., in an mRNA including or not including a poly-A tail). In some embodiments, all nucleotides X in a polynucleotide of the present disclosure (or in a given sequence region thereof) are modified nucleotides, where X can be any one of the nucleotides A, G, U, C, or any one of the combinations A+G, A+U, A+C, G-HU, G-FC, U+C, A+G-HU, A+G-FC, G-HU+C, or A+G+C.

[0048] In some embodiments, the polynucleotide comprises 1% to 100% modified nucleotides (relative to the total nucleotide content, or relative to one or more types of nucleotides, i.e., any one or more of A, G, U, or C), or any intermediate percentage (e.g., 1% to 5%, 1% to 10%, 1% to 20%, 1% to 25%, 1% to 50%, 1% to 60%, 1% to 70%, 1% to 80%, 1% to 90%, 1% to 95%, 10% to 20%, 10% to 25%, 10% to 50%, 10% to 60%, 10% to 70%, 10% to 80%, 10% to 90%, 10% to 95%, 10% to 100%, 20% to 25%, 20% to 50%, 20% to 60%, 20% to 70%, 20% to 80%, 20% to 90%, 20% to 95%, 20% to 100%, 50% to 60%, 50% to 70%, 50% to 80%, 50% to 90%, 50% to 95%, 50% to 100%, 70% to 80%, 70% to 90%, 70% to 95%, 70% to 100%, 80% to 90%, 80% to 95%, 80% to 100%, 90% to 95%, 90% to 100%, and 95% to 100%). It will be understood that any remaining percentage is accounted for by the presence of unmodified A, G, U, or C.

[0049] The polynucleotide can contain a minimum of 1% and a maximum of 100% modified nucleotides, or any intermediate percentage, such as at least 5% modified nucleotides, at least 10% modified nucleotides, at least 25% modified nucleotides, at least 50% modified nucleotides, at least 80% modified nucleotides, or at least 90% modified nucleotides. For example, the polynucleotide can contain modified pyrimidines, such as modified uracils or cytosines. In some embodiments, at least 5%, at least 10%, at least 25%, at least 50%, at least 80%, at least 90%, or 100% of the uracils in the polynucleotide are replaced with modified uracils (e.g., 5-substituted uracils). The modified uracils can be replaced with a compound having a single unique structure, or can be replaced with more than one compound having different structures (e.g., 2, 3, 4, or more unique structures). In some embodiments, at least 5%, at least 10%, at least 25%, at least 50%, at least 80%, at least 90%, or 100% of the cytosines in the polynucleotide are replaced with modified cytosines (e.g., 5-substituted cytosines). The modified cytosines can be replaced with a compound having a single unique structure, or can be replaced with more than one compound having different structures (e.g., 2, 3, 4, or more unique structures).

[0050] Accordingly, in some embodiments, the RNA molecules of the present disclosure comprise a 5' UTR element, an optionally codon-optimized open reading frame, and a 3' UTR element, a poly(A) sequence, and / or a polyadenylation signal, wherein the RNA is not chemically modified.

[0051] In some embodiments, the modified nucleobase is a modified uracil. Exemplary nucleobases and nucleosides having a modified uracil include pseudouridine (ip), pyridin-4-one ribonucleoside, 5-aza-uridine, 6-aza-uridine, 2-thio-5-aza-uridine, 2-thio-uridine (s2U), 4-thio-uridine (s4U), 4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxy-uridine (ho5U), 5-aminoallyl-uridine, 5-halo-uridine (e.g., 5-iodo-uridine or 5-bromo-uridine), 3-methyl-uridine (m3U), 5-methoxy-uridine (mo5U), uridine 5-carboxylic acid (cmo5U), uridine 5-carboxylic acid methyl ester (mcmo5U), 5-carboxymethyl-uridine (cm5U), 1-carboxymethyl-pseudouridine, 5-carboxyhydroxymethyl-uridine (chm5U), 5-carboxyhydroxymethyl-uridine methyl ester (mchm5U), 5-methoxycarbonylmethyl-uridine (mcm5U), 5-methoxycarbonylmethyl-2-thio-uridine (mcm5s2U), 5-aminomethyl-2-thio-uridine (nm5s2U), 5-methylaminomethyl-uridine (mnm5U), 5-methylaminomethyl-2-thio-uridine (mnm5s2U), 5-methylaminomethyl-2-selenouridine (mnm5se2U), 5-carbamoylmethyl-uridine (ncm5U), 5-carboxymethylaminomethyl-uridine (cmnm5U), 5-carboxymethylaminomethyl-2-thio-uridine (cmnm5s2U), 5-propynyl-uridine, 1-propynyl-pseudouridine, 5-taurinomethyl-pseudouridine (im5U), 1-taurinomethyl-pseudouridine, 5-taurinomethyl-2-thio-uridine (Tm5s2U), 1-taurinomethyl-4-thio-pseudouridine, 5-methyl-uridine (m5U, i.e., having the nucleobase deoxythymine), 1-methyl-pseudouridine (m1ip), 1-ethyl-pseudouridine (e1ip), 5-methyl-2-thio-uridine (m5s2U), 1-methyl-4-thio-pseudouridine (m1s4ip), 4-thio-1-methyl-pseudouridine, 3-methyl-pseudouridine (m3ip), 2-thio-1-methyl-pseudouridine, 1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-1-deaza-pseudouridine, dihydrouridine (D), dihydropseudouridine, 5,6-dihydrouridine, 5-methyl-dihydrouridine (m5D), 2-thio-dihydrouridine, 2-thio- dihydrouridine, 2-methoxy-uridine, 2-methoxy-4-thio-uridine, 4-methoxy- pseudouridine, 4-methoxy-2-thio-pseudouridine, N1-methyl-pseudouridine, 3-(3- amino-3-carboxypropyl)uridine (acp3U), 1-methyl-3-(3-amino-3- carboxypropyl)pseudouridine (acp3k), 5-(isopentenylaminomethyl)uridine (inm5U), 5-(isopentenylaminomethyl)-2-thio-uridine (inm5s2U), a-thio-uridine, 2'-O- methyl-uridine (Um), 5,2'-O-dimethyl-uridine (m5Um), 2'-O-methyl-pseudouridine (ipm), 2-thio-2'-O-methyl-uridine (s2Um), 5-methoxycarbonylmethyl-2'-O- methyl-uridine (mcm5Um), 5-carbamoylmethyl-2'-O-methyl-uridine (ncm5Um), 5-carboxymethylaminomethyl-2'-O-methyl-uridine (cmnm5Um), 3,2'-O- dimethyl-uridine (m3Um), and 5-(isopentenylaminomethyl)-2'-O-methyl-uridine (inm5Um), 1-thio-uridine, deoxythymidine, 2'-F-arabinouridine, 2'-F-uridine, 2'-OH- arabinouridine, 5-(2-methoxycarbonylvinyl)uridine, and 5-[3-(1-E- propenylamino)]uridine.

[0052] In some embodiments, the modified nucleobase is a modified cytosine. Exemplary nucleobases and nucleosides having a modified cytosine include 5-aza-cytidine, 6-aza- cytidine, pseudisocytidine, 3-methyl-cytidine (m3C), N4-acetyl-cytidine (ac4C), 5- formyl-cytidine (f5C), N4-methyl-cytidine (m4C), 5-methyl-cytidine (m5C), 5-halo- cytidine (e.g., 5-iodo-cytidine), 5-hydroxymethyl-cytidine (hm5C), 1-methyl-pseudisocytidine, pyrrolo-cytidine, pyrrolo-pseudisocytidine, 2-thio-cytidine (s2C), 2-thio-5- methyl-cytidine, 4-thio-pseudisocytidine, 4-thio-1-methyl-pseudisocytidine, 4-thio-1- methyl-1-deaza-pseudisocytidine, 1-methyl-1-deaza-pseudisocytidine, zebularine, 5- aza-zebularine, 5-methyl-zebularine, 5-aza-2-thio-zebularine, 2-thio-zebularine, 2- methoxy-cytidine, 2-methoxy-5-methyl-cytidine, 4-methoxy-pseudisocytidine, 4- methoxy-1-methyl-pseudisocytidine, lysidine (k2C), o-thio-cytidine, 2’-O-methyl- cytidine (Cm), 5,2’-O-dimethyl-cytidine (m5Cm), N4-acetyl-2’-O-methyl-cytidine (ac4Cm), N4,2’-O-dimethyl-cytidine (m4Cm), 5-formyl-2’-O-methyl-cytidine (f5Cm), N4,N4,2’-O-trimethyl-cytidine (m42Cm), 1-thio-cytidine, 2’-F-arabinocytidine, 2’-F-cytidine, and 2’-OH-arabinocytidine.

[0053] In some embodiments, the modified nucleobase is a modified adenine. Exemplary nucleobases and nucleosides having a modified adenine include 2-amino-purine, 2,6- diaminopurine, 2-amino-6-halo-purine (e.g., 2-amino-6-chloro-purine), 6-halo-purine (e.g., 6-chloro-purine), 2-amino-6-methyl-purine, 8-azido-adenosine, 7-deaza-adenine, 7-deaza-8-aza-adenine, 7-deaza-2-amino-purine, 7-deaza-8-aza-2-diamino-purine, 7-deaza-2,6-diaminopurine, 7-deaza-8-aza-2,6-diaminopurine, 1-methyl-adenosine (mlA), 2-methyl-adenine (m2A), N6-methyl-adenosine (m6A), 2-methylthio-N6-methyl-adenosine (ms2m6A), N6-isopentenyl-adenosine (i6A), 2-methylthio-N6-isopentenyl-adenosine (ms2i6A), N6-(cis-hydroxyisopentenyl)adenosine (io6A), 2-methylthio-N6-(cis- hydroxyisopentenyl)adenosine (ms2io6A), N6-glycinylcarbamoyl-adenosine (g6A), N6- threoninylcarbamoyl-adenosine (t6A), N6-methyl-N6-threoninylcarbamoyl-adenosine (m6t6A), 2-methylthio-N6-threoninylcarbamoyl-adenosine (ms2g6A), N6,N6-dimethyl- adenosine (m62A), N6-hydroxynorvalinylcarbamoyl-adenosine (hn6A), 2-methylthio-N6- hydroxynorvalinylcarbamoyl-adenosine (ms2hn6A), N6-acetyl-adenosine (ac6A), 7- methyl-adenine, 2-methylthio-adenine, 2-methoxy-adenine, o-thio-adenosine, 2'-O- methyl-adenosine (Am), N6,2'-O-dimethyl-adenosine (m6Am), N6,N6,2'-O-trimethyl- adenosine (m62Am), 1,2'-O-dimethyl-adenosine (miAm), 2'-O-ribosyladenosine (phosphate) (Ar(p)), 2-amino-N6-methyl-purine, 1-thio-adenosine, 8-azido-adenosine, 2'-F- arabinosyl-adenosine, 2'-F-adenosine, 2'-OH-arabinosyl-adenosine, and N6-(19-amino- pentadecafluoro-19-yl)-adenosine.

[0054] In some embodiments, the modified nucleobase is a modified guanine. Exemplary nucleobases and nucleosides having modified guanines include inosine (I), 1-methyl-inosine (mil), wyosine (ImG), methylwyosine (mimG), 4-demethyl-wyosine (imG-14), iso-wyosine (imG2), wybutosine (yW), peroxywybutosine (o2yW), hydroxywybutosine (OhyW), unmodified hydroxywybutosine (OhyW*), 7-deaza-guanosine, queuosine (Q), epoxy-queuosine (oQ), galactosyl-queuosine (galQ), mannosyl-queuosine (manQ), 7-cyano-7-deaza-guanosine (preQo), 7-aminomethyl-7-deaza-guanosine (preQi), gualosine (G+), 7-deaza-8-aza-guanosine, 6-thio-guanosine, 6-thio-7-deaza-guanosine, 6-thio-7-deaza-8-aza-guanosine, 7-methyl-guanosine (m7G), 6-thio-7-methyl-guanosine, 7-methyl-inosine, 6-methoxy-guanosine, 1-methyl-guanosine, N2-methyl-guanosine (m2G), N2,N2-dimethyl-guanosine (m22G), N2,7-dimethyl-guanosine (m2’7G), N2,N2,7-dimethyl-guanosine, 8-oxo-guanosine, 7-methyl-8-oxo-guanosine, 1-methyl-6-thio-guanosine, N2-methyl-6-thio-guanosine, N2,N2-dimethyl-6-thio-guanosine, a-thio-guanosine, 2’-O-methyl-guanosine (Gm), N2-methyl-2’-O-methyl-guanosine (m2Gm), N2,N2-dimethyl-2’-O-methyl-guanosine (m22Gm), 1-methyl-2’-O-methyl-guanosine (miGm), N2,7-dimethyl-2’-O-methyl-guanosine (m2’7Gm), 2’-O-methyl-inosine (Im), 1,2’-O-dimethyl-inosine, 2’-O-ribosylguanosine (phosphate) (Gr(p)), 1-thio-guanosine, O6-methyl-guanosine, 2’-F-arabinosyl-guanosine, and 2’-F-guanosine.

[0055] Methods of making cap analogs The cap analogs of the present disclosure can be prepared using general chemical synthesis principles and techniques in a variety of synthetic strategies known in the art. In a reasonable approach, the cap analogs are built from their structural components. These components can be covalently bonded to one another through functional groups, as known in the art, where such functional groups can be present on the components or introduced onto the components using one or more steps. Functional groups that can be used to covalently bond the components together to produce the cap analogs include, but are not limited to, hydroxyl groups, thiol groups, or amino groups. The particular moieties of the different components that are modified to provide the covalent linkages are selected so as to not substantially adversely interfere with other moieties of the components. When necessary and / or desirable, certain moieties on the components can be protected using blocking groups, as known in the art, see, e.g., Green & Wuts, Protective Groups in Organic Synthesis (John Wiley & Sons) (1991).

[0056] It should be appreciated that the particular process conditions used to prepare the cap analogs described herein can be adjusted or selected accordingly to provide the desired physical properties. In some cases, the cap analogs are prepared using a method according to E. Darzynkiewicz et al., "Chemical synthesis and characterization of 7-methylguanosine cap analogues”, Biochem., Vol. 24, pp. 1701-1707 (1985).

[0057] Methods of using the cap analogs to prepare polynucleotides The cap analogs of the present disclosure can be used to co-transcriptionally cap RNA, such as in an in vitro transcription (IVT) reaction.

[0058] In some cases, the method of capping RNA comprises: - combining an NTP, a polynucleotide template, a cap analog, and an RNA polymerase to produce a reaction mixture, and - incubating the reaction mixture under conditions suitable for IVT of the polynucleotide template to produce a capped RNA copy of the polynucleotide template.

[0059] In some cases, the transcription yield from the polynucleotide template to the capped RNA is greater than about 75%, such as about 80%, about 85%, about 90%, or about 95%.

[0060] In some cases, the capping efficiency is at least about 90%, such as about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%. The capped RNA produced by this method can potentially be used without any post-transcriptional enzymatic capping reaction.

[0061] Capping efficiency can be measured using any method known in the art. As non-limiting examples, mass spectrometry (MS), liquid chromatography mass spectrometry (LC / MS), and / or liquid chromatography (such as high performance liquid chromatography (HPLC)) can be used to measure capped and uncapped RNA and calculate capping efficiency. In some other non-limiting examples, a detectable label can be attached to the cap analog to measure capping efficiency.

[0062] The capped RNA produced with this method has higher stability, is more resistant to nucleases, has the same or lower immunogenicity, and / or has the same or higher translation efficiency. In some cases, the half-life of the capped RNA is greater than the half-life of the corresponding natural RNA molecule in a cellular environment.

[0063] As used herein, NTPs (i.e., GTP, CTP, UTP, and ATP) include naturally occurring NTPs or modified NTPs.

[0064] In some cases, the polynucleotide template is a DNA template. The DNA template can comprise any desired sequence encoding a naturally occurring or modified mRNA, tRNA, guide RNA, small inhibitory RNA (siRNA), small activating RNA (saRNA), or microRNA. The DNA can be double-stranded.

[0065] The reaction mixture can be incubated at a temperature between about 30°C to about 60°C, such as about 37°C, between about 30°C to about 40°C, between about 40°C to about 50°C, or between about 50°C to about 60°C. The reaction mixture can be incubated for at least 30 min, such as about 40 min, about 50 min, about 60 min, about 1.5 hours, about 2 hours, about 2.5 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours, about 12 hours, or more.

[0066] The RNA polymerase can be any known RNA polymerase, including natural RNA polymerases and synthetic RNA polymerases. In some cases, the RNA polymerase can be thermostable.

[0067] In some cases, capped RNA can be prepared using solid phase synthesis. Solid phase synthesis is performed on a solid support between filters in a column that allows all reagents and solvents to pass freely through. Coupling agents, protecting groups, and cleavage agents can be selected according to methods known to those of skill in the art.

[0068] According to certain embodiments of the disclosure, cap analogs for use herein include RNA comprising at least one region encoding a peptide (e.g., polypeptide) or protein or functional fragment of the foregoing. As used herein, “functional fragment” refers to a fragment of a peptide (e.g., polypeptide) or protein that retains the ability to induce an immune response. In an embodiment, the encoding RNA is selected from the group consisting of mRNA, viral RNA, retroviral RNA, and self-replicating RNA. In some embodiments, the RNA encodes a viral peptide (e.g., viral polypeptide), viral protein, or functional fragment of the foregoing. In various cases, the RNA encodes a human papillomavirus (HPV) protein, variant thereof, or functional fragment of any of the foregoing. In some cases, the RNA encodes an HPV E6 protein (or variant thereof), an HPV E7 protein (or variant thereof), a combination thereof, or a functional fragment of any of the foregoing. In some cases, the HPV protein is from HPV subtypes HPV 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and / or 68. In various cases, the HPV protein is from HPV subtypes HPV 16 and / or 18. In some cases, the RNA encodes a viral spike protein or functional fragment thereof. In some cases, the RNA encodes a SARS- related coronavirus (e.g., severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome coronavirus (MERS-CoV), human coronavirus 229E (HCoV-229E), human coronavirus 0C43 (HCoV-0C43), human coronavirus HKU1 (HCoV-HKLH), and / or human coronavirus NL63 (HCoV-NL63)). In various embodiments, the RNA encodes a SARS-CoV spike (S) protein, variant thereof, or functional fragment of any of the foregoing. In some cases, the RNA encodes an influenza protein, variant thereof, or functional fragment of any of the foregoing. In various embodiments, the RNA encodes an influenza hemagglutinin (HA) or functional fragment thereof. In some embodiments, the influenza A virus has an HA of a subtype selected from the group consisting of H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14, H15, and H16. In various embodiments, the influenza subtype is an HA strain H1, H2, H3, or H5. In some embodiments, the RNA encodes a combination of the foregoing.

[0069] Non-limiting examples of viruses that cap analogs can encode include: influenza A and influenza B, poliovirus, adenovirus, rabies virus, bovine parainfluenza virus 3, human respiratory syncytial virus, bovine respiratory syncytial virus, canine parainfluenza virus, Newcastle disease virus, herpes simplex virus-1 and herpes simplex virus-2, human papillomavirus, hepatitis A virus, hepatitis B virus, hepatitis C virus and human immunodeficiency virus, cytomegalovirus, varicella-zoster virus, Epstein-Barr virus, Kaposi's sarcoma virus, human herpesvirus-6, human herpesvirus-7, human herpesvirus-8, macaque alphaherpesvirus 1, canine herpesvirus, equine alphaherpesvirus 1, bovine alphaherpesvirus 1, human herpesvirus 2, and herpes simplex virus. Simplex, Gammaherpesvirinae, Avian Alpha Herpesvirus 1, Ebola virus, Marburg virus, Avira, Flavivir, Yellow fever virus, Dengue virus, Japanese encephalitis virus, West Nile virus, Zika virus, Venezuelan equine encephalomyelitis virus, Chikungunya virus, Western equine encephalomyelitis virus, Eastern equine encephalomyelitis virus, Tick-borne encephalitis virus, Kyasanur Forest Disease virus, Alkhurma virus, Omsk Hemorrhagic Fever virus, Hendra virus, Nipah virus, Rubeola virus Virus, Rubella virus, Human parvovirus B19, Smallpox, A virus, Molluscum contagiosum virus, Arenaviridae, Bunyaviridae, Filoviridae, Flaviviruses, Paramyxoviridae, Clonorchiviridae, Flaviviruses, Colorado tick fever virus (coltivirus), Coxsackievirus, Rotavirus, Norovirus, Astrovirus, Adenovirus, Human metapneumovirusMetapneumovirus), rhinovirus, or coronaviruses such as SARS-CoV, SARS-CoV-2, MERS-CoV, HCoV NL63, HKU1, 229E, and OC43, human papillomavirus, Ebola virus, Marburg virus, alphavirus, flavivirus, yellow fever, dengue fever, Japanese encephalitis, West Nile virus, Zika virus, Venezuelan equine encephalomyelitis virus, chikungunya virus, western equine encephalomyelitis virus, eastern equine encephalomyelitis virus, tick-borne encephalitis, Kisana forest disease, Alkhurma disease, Omsk hemorrhagic fever, Hendra virus, Nipah virus, measles virus, rubella virus, human parvovirus B19, human herpesvirus 6, varicella-zoster virus, cytomegalovirus, Epstein-Barr virus, Kaposi's sarcoma virus, human herpesvirus-7, human herpesvirus-8, macaque alpha herpesvirus 1, canine herpesvirus, equine alpha herpesvirus 1, bovine alpha herpesvirus 1 Human herpesvirus 2, herpes simplex virus, gamma herpesvirus subfamily, avian alpha herpesvirus 1, smallpox, alpha virus, molluscum contagiosum virus, hepatitis A virus, hepatitis B virus, hepatitis C, hepatitis D, hepatitis E, polioviruses, arenaviridae, bunyaviridae, filoviridae, flaviviridae, paramyxoviridae or clovenviridae, flaviviruses such as Zika virus, Colorado tick fever virus (coltivirus), Coxsackie virus, rotavirus, norovirus, astrovirus, adenovirus, adenovirus, influenza A virus (influenza) Virus A), human metapneumovirus, rhinovirus, coronavirus, varicella virus, adeno-associated virus, Aichi virus, Australian bat lyssavirus, BK polyomavirus, Banna virus, Barmah forest virus, Bunyamwera virus, La Crosse virus, Bunyavirus snowshoe hare, Cercopithecine herpesvirus, Chandipura virus, Chikungunya virus, Cosavirus A, vaccinia virus, Coxsackie virus, Crimean-Congo hemorrhagic fever virus, dengue virus, Dhori virus, dugbe virus, Duvenhage virus.Human equine encephalitis virus, Ebola virus, Echovirus, encephalomyocarditis virus, European bat rabies virus, GB virus, hepatitis C / G virus, Hantavirus, Hendra virus, hepatitis A virus, hepatitis B virus, hepatitis C virus, hepatitis E virus, hepatitis D virus, horsepox virus, human adenovirus, human astrovirus, human coronavirus, human cytomegalovirus, human enterovirus 68, human enterovirus 70, human papillomavirus 1, human papillomavirus 2, human papillomavirus 16, human papillomavirus 18, human parainfluenza, human parvovirus B19, human respiratory syncytial virus, human rhinovirus, human SARS coronavirus, human spumaretrovirus, human T-lymphotropic virus, human torovirus, influenza A virus. Influenza virus, influenza B virus, influenza C virus, Isfahan virus, JC polyomavirus, Japanese encephalitis virus, Juningsavirus, KI polyomavirus, Kunjin virus, Lagos bat virus, Lake Victoria Marburg virus, Langat virus, Lassa virus, Lordsdale virus, Louping ill virus, lymphocytic choriomeningitis virus, Machupo virus, Mayaro virus, MERS coronavirus, Measles virus, Mengo encephalitis myocarditis virus, Merkel cell polyomavirus, Mokola virus, Molluscum contagiosum virus, Monkeypox virus, Mumps virus, Murray Valley encephalitis virus, New York virus, Nipah virus, Norwalk virus Poliovirus, O'nyong-nyong virus, Orf virus, Oropouche virus, Pichinde virus, poliovirus, Punta toro phlebovirus, Puumala virus, rabies virus, Rift Valley fever virus, Rosavirus A, Ross River virusvirus), Rotavirus A, Rotavirus B, Rotavirus C, Rubella virus, Sagiyama virus, Salivirus A, Sandfly fever Sicilian virus, Sapporo virus, SARS coronavirus 2, Semliki forest virus, Seoul virus, Simian foamy virus, Simian virus 5, Sindbis virus, Southampton virus, St. louis encephalitis virus, Tick-borne powassan virus, Torque teno virus, Toscana virus, Uukuniemi virus, Vaccinia virus, Varicella-zoster virus, Variola virus, Venezuelan equine encephalitis virus, Vesicular stomatitis virus, Western equine encephalitis virus, WU polyomavirus, West Nile virus, Yaba monkey tumor virus, Yaba-like disease virus, Yellow fever virus, Zika virus, Bovine herpesvirus, Pseudorabies virus, Adenoviridae, Bovine adenovirus BAdV-9 = Human adenovirus C, Anelloviridae (proposed family), Torque Teno virus TTV, Bornaviridae, Borna disease virus BDV, Bunyaviridae, Aino virus, Cache valley virus CW, Crimean-Congo hemorrhagic fever virus CCHF, Hantavirus HTNV, Jamestown Canyon virus JCV, LaCrosse virus LACV, Phlebovirus, Rift Valley fever virus RVFV, Caliciviridae, Norovirus, San Miguel sea lion virus SMSV-5SMSV-5), Circoviridae, Bovine circovirus BCV = Porcine circovirus type 2 PCV-2, evolution strain, Coronaviridae, Bovine coronavirus BCoV-1, Bovine torovirus BtoV, Flaviviridae, Bovine viral diarrhea virus BVDV, Japanese encephalitis virus JEV, Kyasanur forest disease virus KFDV, Louping ill virus, Murray Valley encephalitis virus MVE, St. Louis encephalitis virus SLEV, Tick-borne encephalitis virus TBEV, Wesselsbron virus, West Nile virus (including Kunjin), Hepeviridae, Hepatitis E virus HEV, Herpesviridae, Bovine herpes virus BHV-4, Equine herpes virus EHV-1, Infectious bovine rhinotracheitis virus IBR = BHV-1, Pseudorabies virus PRV, Orthomyxoviridae, Douri virus, Influenza A virus, Thogotovirus THOV, Papillomaviridae, Bovine papillomavirus BPV, Paramyxoviridae, Bovine parainfluenza virus BPIV3, Bovine respiratory syncytial virus BRSV, Peste-des-petits ruminants virus PPRV, Rinderpest virus RPV, Parvoviridae, Bovine adeno-associated virus BAAV, Bovine hokovirus BHoV, Picornaviridae, Bovine enterovirus BEV-1, Bovine enterovirus BEV-2, Bovine keratinocyte virus BKV-1 U-1 strain, Encephalomyocarditis virus EMC, Foot-and-mouth disease virus FMDV, Seneca valley virus SW, Polyomaviridae, Bovine polyomavirus BPyV, Poxviridae, Aracatuba virus, Bovine papular stomatitis virus BPSV, Cantagalo virus, Vaccinia virus, Pseudocowpox virus PCPV, Vaccinia virus, Reoviridae, Banna virus BAV, Blue tongue virus BTV, Epizootic hemorrhagic disease virus EHDV, Liaoning virus LNV, Reovirus, Rotavirus, Retroviridae, Bovine foamy virus BFV, Bovine leukemia virus BLV, Rhabdoviridae, Bovine ephemeral fever virus BEFV, Rabies virus, Vesicular stomatitis virus VSV, Togaviridae, Eastern equine encephalitis virus EEEV, Getah virus, Ross River virus RRV, Sindbis virus, Venezuelan equine encephalomyelitis virusvirus, VEE), Anelloviridae (proposed family), Circovirus TTV, Bunyaviridae, Crimean-Congo hemorrhagic fever virus CCHF, Hantaan virus HTNV, Jamestown Canyon virus JCV, La Crosse virus LCV, Caliciviridae, Norovirus, St. Miguel sea lion virus SMSV-5, Sapovirus, Circoviridae, Porcine circovirus PCV-1 and PCV-2, Coronaviridae, Bovine coronavirus BCoV-1, Severe acute respiratory syndrome virus SARS, Infectious gastroenteritis virus TGEV, Filoviridae, Ebola Reston virus, Flaviviridae, Bovine viral diarrhea virus BVDV, Dengue virus, llheus virus, Japanese encephalitis virus JEV, Leaping bug virus, Murray Valley encephalitis virus MVE, Powassan virus, Tick-borne encephalitis virus TBEV, Wesselsbron virus, West Nile virus WNV (including Kunjin), Hepatoviridae, Hepatitis E virus HEV, Herpesviridae, Infectious bovine rhinotracheitis virus IBR = BHV-1, Porcine cytomegalovirus PCMV (B. Potts personal communication), Pseudorabies virus PRV, Orthomyxoviridae, Avian influenza virus (H5N1), Swine influenza virus (H1N1, H1N2), Paramyxoviridae, Bovine parainfluenza virus BPIV3, Menangle virus MENV, Nipah virus NiV, Peste des petits ruminants virus PPRV, Rinderpest virus RPV, Tioman virus TIOV, Parvoviridae, Porcine hokovirus PHoV, Porcine parvovirus PPV, Picornaviridae, Encephalomyocarditis virus EMC, Foot-and-mouth disease virus FMDV, Porcine enterovirus PEV-9, Porcine enterovirus PEV-10, Seneca Valley virus SV, Porcine vesicular disease virus SVDV, Reoviridae, Banna virus BAV, Reovirus, Rotavirus, Retroviridae, Porcine endogenous retrovirus PERV, Rhabdoviridae, Rabies virus, Vesicular stomatitis virus VSV, Togaviridae, Eastern equine encephalitis virus EEEV, Getah virus, Ross River virus RRV, or Venezuelan equine encephalomyelitis VEE.

[0070] In some embodiments, the cap analog encodes an adenovirus, alphavirus, calicivirus (e.g., calicivirus capsid antigen), coronavirus polypeptide, canine distemper virus, Ebola virus polypeptide, enterovirus, flavivirus, hepatitis virus (AE), herpesvirus, infectious peritonitis virus, leukemia virus, Marburg virus, orthomyxovirus, papillomavirus, parainfluenza virus, paramyxovirus, parvovirus, pestivirus, picorna virus (e.g., poliovirus), poxvirus (e.g., vaccinia virus), rabies virus, reovirus, retrovirus, and rotavirus. In certain embodiments, the RNA encodes SARS-CoV-2, HPV (e.g., E6 and / or E7 from HPV16 and / or HPV18), or influenza (e.g., influenza hemagglutinin (HA)).

[0071] In some embodiments, the combination of two or more specific cap analogs as disclosed herein can be particularly useful for therapeutic applications. For example, in some embodiments, one or more cap analogs include a combination of sgRNA (single guide 2 RNA) and mRNA encoding Cas9 as a CRISPR sequence. In still further embodiments, the cap analogs can also be complexed with proteins, such as with a CRISPR / Cas9 ribonucleoprotein complex. In some cases, the cap analogs are complexed with one or more nucleic acids selected from DNA and RNA (e.g., antigenic RNA and adjuvant DNA, such as CpG).

[0072] A pharmaceutically effective amount of a pharmaceutical preparation and / or cap analog (e.g., comprising RNA) can be administered to a cell (such as a mammalian cell) in vitro, ex vivo, or in vivo for therapeutic or diagnostic purposes. The cap analog comprising RNA can be translated into a polypeptide or protein. In some cases, the translation efficiency is greater than about 75%, such as about 80%, about 85%, about 90%, or about 95%.

[0073] The present disclosure also provides kits for preparing capped RNA comprising a cap analog, a RNA polymerase, NTPs, and a DNA template. The kits can also comprise additional enzymes, reaction buffers, and / or instructions for performing the method, such as an incubation temperature.

[0074] Non-limiting examples of reaction buffers include Tris, HEPES, TAPS, MOPS, N-[tris(hydroxymethyl)methyl]glycine, or MES.

[0075] Pharmaceutical products, pharmaceutical preparations, and modes of administration Also provided herein are pharmaceutical products (i.e., pharmaceutical compositions) comprising a cap analog of the disclosure and an effective amount of one or more pharmaceutically acceptable excipients. An “effective amount” includes a “therapeutically effective amount” and a “prophylactically effective amount.” The term “therapeutically effective amount” refers to an amount effective, at dosages and for periods of time necessary, to treat and / or ameliorate a disease or condition in a subject. The term “prophylactically effective amount” refers to an amount effective, at dosages and for periods of time necessary, to prevent and / or substantially reduce the chances of a disease or condition in a subject. As used herein, the terms “patient” and “subject” can be used interchangeably and mean an animal, such as a dog, cat, cow, horse, and sheep (i.e., a non-human animal) and a human. A particular patient or subject is a mammal (e.g., a human). The terms “patient” and “subject” include both males and females. As used herein, the term “excipient” means any pharmaceutically acceptable additive, carrier, diluent, adjuvant, or other ingredient other than the active pharmaceutical ingredient (API) that is appropriately selected with respect to the intended form of administration and is consistent with conventional pharmaceutical practices.

[0076] The cap analogs of the disclosure can be administered to a subject or patient in a therapeutically effective amount. The cap analogs can be administered alone or as part of a pharmaceutically acceptable composition or formulation. Furthermore, the cap analogs can be administered all at once, for example, by bolus injection, multiple administrations, or delivered substantially uniformly over a period of time. It is also noted that the dosage of the cap analogs can vary over time.

[0077] The cap analogs and other pharmaceutically active compounds disclosed herein can be administered to a subject or patient by any appropriate route, if desired, such as orally, rectally, parenterally (e.g., intravenously, intramuscularly, or subcutaneously), intracisternally, intravaginally, intraperitoneally, intravesically, or as a buccal, inhalation, or nasal spray. Administration can be for providing a systemic effect (e.g., enterally or parenterally). All methods available to those skilled in the art for administering pharmaceutically active agents are contemplated.

[0078] Compositions suitable for parenteral injection can include physiologically acceptable, sterile aqueous or nonaqueous solutions, dispersions, suspensions or emulsions, and sterile powders for reconstitution into sterile injectable solutions or dispersions. Examples of suitable aqueous and nonaqueous carriers, diluents, solvents, or vehicles include water, ethanol, polyols (propylene glycol, polyethylene glycol, glycerol, and the like), suitable mixtures thereof, vegetable oils (such as olive oil) and injectable organic esters such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of coating such as lecithin, by the maintenance of required particle size in the case of dispersion and by the use of surfactants.

[0079] These compositions can also contain adjuvants such as preservatives, wetting agents, emulsifying agents, and dispersing agents. Prevention of the growth of microorganisms can be achieved by addition of various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, and the like. It can also be desirable to include isotonic agents, for example, sugars, sodium chloride, and the like. Prolonged absorption of injectable pharmaceutical compositions can be brought about by the use of agents delaying absorption, for example, aluminum monostearate and gelatin.

[0080] Pharmaceutical compositions can be in the form of a sterile injectable aqueous or oleaginous suspension. This suspension can be formulated according to known art using those suitable dispersing or wetting agents and suspending agents which have been mentioned above. The sterile injectable preparation can also be a sterile injectable solution or suspension in a non-toxic parenterally acceptable diluent or solvent, for example, as a solution in 1,3-butanediol. Among the acceptable vehicles and solvents that can be employed are water, Ringer's solution, and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose, any bland fixed oil can be employed including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid find use in the preparation of injectables.

[0081] Compositions for parenteral administration are formulated in sterile medium. Parenteral formulations can be suspensions or solutions containing dissolved cap analogs, depending on the concentration of drug in the media and the agent used. Adjuvants such as local anesthetics, preservatives, and buffering agents can also be added to parenteral compositions.

[0082] When the cap analogs of the present disclosure are used as vaccines, they can comprise one or more immunoadjuvants. As used herein, the term “immunoadjuvant” refers to a compound or mixture of compounds that, when used in conjunction with an immunogen (e.g., a neoantigen), acts to accelerate, prolong, enhance, or modify an immune response. Adjuvants can be non-immunogenic when administered to a host alone, but enhance the immune response of a host to an antigen when administered in conjunction with the antigen. Specifically, the terms “adjuvant” and “immunoadjuvant” are used interchangeably in the present disclosure. Enhancement of the immune response and / or prolongation of the duration of the immune response mediated by an adjuvant can be assessed by any method known in the art, including but not limited to one or more of the following: (i) an increase in the number of antibodies produced in response to immunization with the adjuvant / antigen combination relative to the number of antibodies produced in response to immunization with the antigen alone; (ii) an increase in the number of T cells that recognize the antigen or the adjuvant; and (iii) an increase in the level of one or more cytokines. Adjuvants can be aluminum-based adjuvants, including but not limited to aluminum hydroxide and aluminum phosphate; saponins, such as steroidal saponins and triterpenoid saponins; bacterial flagellin; and some cytokines such as GM-CSF. The choice of adjuvant can depend on the antigen, the vaccine, and the route of administration.

[0083] In some embodiments, adjuvants improve adaptive immune responses to vaccine antigens by modulating innate immunity or facilitating transport and presentation. Adjuvants act directly or indirectly on antigen presenting cells (APCs), including dendritic cells (DCs). Adjuvants can be ligands for toll-like receptors (TLRs) and can directly affect DCs to alter the strength, potency, speed, duration, bias, breadth, and range of adaptive immunity. In other cases, adjuvants can signal through proinflammatory pathways and promote immune cell infiltration, antigen presentation, and effector cell maturation. Such adjuvants include mineral salts, oil emulsions, nanoparticles, and polyelectrolytes, and include colloids and molecular assemblies that exhibit complex, heterogeneous structures. In one example, the composition further comprises pidotimod as an adjuvant. In another example, the composition further comprises CpG as an adjuvant.

[0084] The cap analogs of the present disclosure can be administered to a subject or patient at dosage levels ranging from about 0.1 mg / day to about 3,000 mg / day. For a normal human adult having a body weight of about 70 kg, a dosage in the range of about 0.01 mg to about 100 mg per kilogram of body weight is generally sufficient. The particular dosage and dosage range employed will depend on a number of factors, including the needs of the subject or patient, the severity of the condition or disease being treated, and the pharmacological activity of the compound being administered. Determination of dosage ranges and optimal dosages for a particular subject or patient is within the ordinary skill in the art.

[0085] Methods of use The cap analogs disclosed herein can be delivered to a cell. Accordingly, disclosed herein are methods of delivering a cap analog, such as a nucleic acid (e.g., RNA), to a cell, the method comprising contacting the cell with a cap analog or pharmaceutical composition disclosed herein. In some embodiments, the cell can be contacted in vitro. In some embodiments in which the cell is contacted in vitro, the cell is a HeLa cell. In other embodiments in which the cell is contacted in vivo, the cap analog of the present disclosure is administered to a mammalian subject. The mammalian subject can include, but is not limited to, a human or mouse subject. In yet other embodiments in which the cell is contacted ex vivo, the cell is obtained from a human or mouse subject. In some cases, the cell is a tumor cell. In some cases, the cell is a muscle cell.

[0086] In some embodiments, one or more cap analogs can be delivered for therapeutic use. Non-limiting therapeutic uses include cancer, infectious diseases, autoimmune disorders, and neurological disorders. In certain embodiments, complexes comprising a multi-component delivery system and a polyanionic cargo compound are used as vaccines. Genetic vaccination or administration of nucleic acid molecules (e.g., RNA) to a patient, followed by transcription and / or translation of the encoded genetic information, can be used to treat and / or prevent genetic genetic diseases, but can also be used to treat and / or prevent autoimmune diseases, infectious diseases, cancer or tumor-related diseases, and inflammatory diseases. Genetic vaccination can be used to treat or prevent viral infections, such as those caused by coronaviruses. In the case of coronaviruses, the vaccine target for most of these entities is the spike (S) protein of the coronavirus, a highly glycosylated trimeric class I fusion protein that coats the outside of the virus and is responsible for entry into host cells. The S protein of SARS-CoV-2 shares high structural homology with SARS-CoV-1 and contains several subunits that are critical for viral entry into host cells via the angiotensin-converting enzyme 2 (ACE2) receptor, including the S1 domain, the S2 domain, and the receptor binding domain (RBD). Thus, the S protein and its subunits, as well as accessible peptide sequences within these domains, are attractive vaccine antigen targets. Furthermore, genetic vaccination is particularly useful for the treatment of cancer, as cancer cells express antigens that are not usually recognized and eliminated by the host, as evidenced by the progression of the disease.

[0087] Vaccines. The cap analogs of the present disclosure can also be used as vaccines, where the cap analog is an RNA that can encode an immunogen, antigen, or neoantigen. The immune system of a host provides the means for a rapid and specific protective response to pathogenic microorganisms, and also contributes to the rejection of malignant tumors. The immune response is generally described as including a humoral response, in which antigen-specific antibodies are produced by differentiated B lymphocytes, and a cell-mediated response, in which various types of T lymphocytes eliminate antigens through various mechanisms. For example, CD4 (also known as CD4+) helper T cells, which are capable of recognizing specific antigens, can respond by releasing soluble mediators such as cytokines to recruit additional cells of the immune system to participate in the immune response. CD8 (also known as CD8+) cytotoxic T cells are also capable of recognizing specific antigens and can bind to and destroy or damage cells or particles that carry the antigen. In particular, the cell-mediated immune response, including the cytotoxic T lymphocyte (CTL) response, can be important for eliminating tumor cells and cells infected by microorganisms such as viruses, bacteria, or parasites. It has been found that when one or more of the cap analogs of the present disclosure encode a viral peptide (e.g., a viral polypeptide), a viral protein, or a functional fragment of the foregoing, the cap analog induces an immune response. For example, cap analogs comprising DV-140-F2 or DV-140-F6 / 17 complexed with mRNA encoding HPV E6 / E7 (e.g., from HPV 16 and / or HPV 18) oncogenes, SARS-CoV spike (S) protein, and / or influenza hemagglutinin (HA) elicit strong humoral and cellular immune responses.

[0088] Accordingly, the present disclosure includes a method for inducing an immune response in a subject in need thereof, the method comprising administering to the subject an effective amount of a cap analog of the present disclosure (e.g., formulated into an antigenic composition). Also disclosed herein is a method of treating a viral infection in a subject in need thereof, the method comprising administering to the subject an effective amount of a cap analog of the present disclosure. In some embodiments, the administration is by intramuscular, intratumoral, intravenous, intraperitoneal, or subcutaneous delivery.

[0089] In various embodiments, administering a cap analog of the present disclosure to a subject can result in an increase in the amount of antibodies (e.g., neutralizing antibodies) produced in the subject against a viral antigen relative to the amount of antibodies produced in a subject that is not administered a cap analog of the present disclosure (e.g., formulated into a composition, pharmaceutical formulation, or antigenic composition). In some embodiments, the increase is a 2-fold increase, a 5-fold increase, a 10-fold increase, a 50-fold increase, a 100-fold increase, a 200-fold increase, a 500-fold increase, a 700-fold increase, or a 1000-fold increase.

[0090] The immune response generated by the methods of the disclosure generally includes an antibody response, preferably a neutralizing antibody response, maturation and memory of T cells and B cells, antibody-dependent cell-mediated cytotoxicity (ADCC), antibody cell-mediated phagocytosis (ADCP), complement-dependent cytotoxicity (CDC), and T cell-mediated responses such as CD4+, CD8+. The immune response generated by the cap analog comprising RNA encoding viral antigens as disclosed herein generates an immune response that recognizes and preferably ameliorates and / or neutralizes viral infection as described herein. Methods for assessing antibody responses following administration of an antigen composition (immunization or vaccination) are known in the art and / or described herein. In some embodiments, the immune response includes a T cell-mediated response (e.g., a peptide-specific response, such as a proliferative response or a cytokine response). In some embodiments, the immune response includes both B cell and T cell responses. The antigen composition can be administered in a variety of suitable ways, such as intramuscular injection, intratumoral injection, subcutaneous injection, intradermal administration, and mucosal administration, such as oral or intranasal. Additional modes of administration include, but are not limited to, intravenous, intraperitoneal, intranasal administration, intravaginal, intrarectal, and oral administration. The disclosure also contemplates combinations of different routes of administration in immunized subjects, for example, simultaneous intramuscular and intranasal administration.

[0091] Cancer. Various cancers (e.g., cervical cancer) can be treated with the cap analogs of the disclosure. As used herein, the term “cancer” refers to any of various malignant neoplasms characterized by the proliferation of anaplastic cells that tend to invade surrounding tissues and to be transported to new body sites, and also refers to the pathological condition that is characterized by the growth of such malignant neoplasms. Cancer can be a solid or hematological malignancy and includes, but is not limited to, all types of lymphoma / leukemia, carcinomas, and sarcomas, such as those cancers or tumors found in the anus, bladder, bile duct, bone, brain, breast, cervix, colon / rectum, endometrium, esophagus, eye, gall bladder, head and neck, liver, kidney, larynx, lung, mediastinum (thoracic), mouth, ovary, pancreas, penis, prostate, skin, small intestine, stomach, spinal cord, tailbone, testicle, thyroid, and uterus.

[0092] As non-limiting examples, the cancer that can be treated can be acute granulocytic leukemia, acute lymphocytic leukemia, acute myeloid leukemia, adenocarcinoma, adenosarcoma, adrenal gland cancer, adrenal cortex cancer, anal cancer, anaplastic astrocytoma, angiosarcoma, appendix cancer, astrocytoma, basal cell carcinoma, B-cell lymphoma, bile duct cancer, bladder cancer, bone cancer, bowel cancer, brain cancer, brain stem glioma, brain tumor, breast cancer, carcinoid tumor, cervical cancer, cholangiocarcinoma, chondrosarcoma, chronic lymphocytic leukemia, chronic myeloid leukemia, colon cancer, colorectal cancer, craniopharyngioma, cutaneous lymphoma, cutaneous melanoma, diffuse astrocytoma, ductal carcinoma in situ, endometrial cancer, ependymoma, epithelioid sarcoma, esophageal cancer, Ewing sarcoma, extrahepatic bile duct cancer, eye cancer, fallopian tube cancer, fibrosarcoma, gallbladder cancer, gastric cancer, gastrointestinal cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor, germ cell tumor, general, glioblastoma multiforme, glioma, hairy cell leukemia, head and neck cancer, hemangioendothelioma, Hodgkin lymphoma, Hodgkin disease, Hodgkin lymphoma, hypopharyngeal cancer, invasive ductal carcinoma, invasive lobular carcinoma, inflammatory breast cancer, intestinal cancer, intrahepatic bile duct cancer, invasive I invasive breast cancer, islet cell cancer, jaw cancer, Kaposi sarcoma, kidney cancer, laryngeal cancer, leiomyosarcoma, Leptomeningeal metastases, leukemia, lip cancer, liposarcoma, liver cancer, lobular carcinoma in situ, low-grade astrocytoma, lung cancer, lymph node cancer, lymphoma, male breast cancer, medullary carcinoma, medulloblastoma, melanoma, meningioma, Merkel cell carcinoma, mesenchymal chondrosarcoma, mesenchymal tumor, mesothelioma, metastatic breast cancer, metastatic melanoma, metastatic squamous neck cancer, mixed glioma, mouth cancer, mucinous carcinoma, mucosal melanoma, multiple myeloma, nasal cavity cancer, nasopharyngeal cancer, neck cancer, neuroblastoma, neuroendocrine tumor, non-Hodgkin lymphoma, non-Hodgkin lymphoma, non-small cell lung cancer, oat cell cancer, ocular cancer, ocular melanoma, oligodendroglioma, oral cancer, oral cavity cancer, oropharyngeal cancer, osteogenic sarcoma, osteosarcoma, ovarian cancer, ovarian epithelial cancer, ovarian germ cell tumor, ovarian primary peritoneal cancer, ovarian sex cord stromal tumor, Paget disease, pancreatic cancer, papillary carcinoma, paranasal sinus cancer, parathyroid cancer, pelvic cancer, penile cancer, peripheral nerve cancer, peritoneal cancer, pharyngeal cancer, pheochromocytoma, pilocytic astrocytoma, pineal region tumor, pineoblastoma, pituitary gland cancer, primary central nervous system lymphoma, prostate cancer, rectal cancer, renal cell cancer, renal pelvis cancer, rhabdomyosarcoma, salivary gland cancer, sarcoma,Soft tissue sarcoma, uterine sarcoma, sinus cancer, skin cancer, small cell lung cancer, small intestine cancer, soft tissue sarcoma, spinal cancer, spinal cord cancer, spinal tumor, squamous cell carcinoma, gastric cancer, synovial sarcoma, T-cell lymphoma, testicular cancer, laryngeal cancer, thymoma / thymic carcinoma, thyroid cancer, tongue cancer, tonsil cancer, transitional cell carcinoma, transitional cell carcinoma, triple-negative breast cancer, fallopian tube cancer, tubal cancer, ureteral cancer, ureteral cancer, uterine adenocarcinoma, uterine cancer, uterine sarcoma, vaginal cancer, and vulvar cancer.

[0093] In some embodiments, the cap analogues of this disclosure are used to treat cancers selected from the group consisting of cervical cancer, head and neck cancer, B-cell lymphoma, T-cell lymphoma, prostate cancer, and lung cancer. In some embodiments, the cap analogues can be used to treat cervical cancer.

[0094] Infectious diseases. In some embodiments, the cap analogues of this disclosure are used to treat infectious diseases, such as microbial infections, such as viral infections, bacterial infections, fungal infections, or parasitic infections. Non-limiting examples of infectious diseases include hepatitis (such as HBV or HCV infection), RSV, influenza, adenovirus, rhinovirus, coronavirus, or other viral infections.

[0095] Autoimmune diseases. Various autoimmune diseases and autoimmune-related diseases can be treated with the cap analogs of the disclosure. As used herein, the term "autoimmune disease" refers to a disease in which the body produces antibodies that attack its own tissues. As non-limiting examples, the autoimmune disease can be acute disseminated encephalomyelitis (ADEM), acute necrotizing hemorrhagic leukoencephalitis, Addison's disease, agammaglobulinemia, alopecia areata, amyloidosis, ankylosing spondylitis, anti-GBM / anti-TBM nephritis, antiphospholipid syndrome (APS), autoimmune angioedema, autoimmune aplastic anemia, autoimmune dysautonomia, autoimmune hepatitis, autoimmune hyperlipidemia, autoimmune immunodeficiency, autoimmune inner ear disease (AIED), autoimmune myocarditis, autoimmune oophoritis, autoimmune pancreatitis, autoimmune retinopathy, autoimmune thrombocytopenic purpura (ATP), autoimmune thyroid disease, autoimmune urticarial, axonal & neuronal neuropathies, Balo disease, Behcet's disease, bullous pemphigoid, cardiomyopathy, Castleman disease, celiac disease, Chagas disease, chronic fatigue syndrome**, chronic inflammatory demyelinating polyneuropathy (CIDP), chronic recurrent multifocal osteomyelitis (CRMO), Churg-Strauss syndrome, cicatricial pemphigoid / benign mucosal pemphigoid, Crohn's disease, Cogans syndrome, cold agglutinin disease, congenital heart block, Coxsackie myocarditis, CREST disease, essential mixed cryoglobulinemia, demyelinating neuropathies, dermatitis herpetiformis, dermatomyositis, Devic's disease (neuromyelitis optica), discoid lupus, Dressier's syndrome, endometriosis, eosinophilic esophagitis, eosinophilic fasciitis, erythema nodosum, experimental allergic encephalomyelitis, Evans syndrome, fibromyalgia**, fibrosing alveolitis, giant cell arteritis (temporal arteritis), giant cell myocarditis, glomerulonephritis, Goodpasture's syndrome, granulomatosis with polyangiitis (GPA, formerly known as Wegener's granulomatosis), Graves' disease, Guillain-Barre syndrome, Hashimoto's encephalitis, Hashimoto's thyroiditis, hemolytic anemia, Henoch-Schonlein purpura, hypogammalglobulinemia, idiopathic thrombocytopenic purpura (ITP), inclusion body myositis, inflammatory bowel disease, idiopathic pulmonary fibrosis, idiopathic sprue, interstitial cystitis, juvenile arthritis, Kawasaki's disease, Lambert-Eaton syndrome, lupus, Lyme disease, Marfan syndrome, Meniere's disease, Miller-Fisher syndrome, mixed connective tissue disease, Mooren's ulcer, multiple sclerosis, myasthenia gravis, myositis, neuromyelitis optica, Ord's syndrome, osteoarthritis, paraneoplastic arthropathy, paraplegia, paroxysmal nocturnal hemoglobinuria, pemphigus, pernicious anemia, polyarteritis nodosa, polychondritis, polyglucosuria, polyglandular syndromes, polymyositis, polymyalgia rheumatica, post-myocardial infarction syndrome, primary biliary cirrhosis, primary sclerosing cholangitis, psoriasis, psoriatic arthritis, Raynaud's phenomenon, reactivepurpura), gestational herpes, hypogammaglobulinemia, idiopathic thrombocytopenic purpura (ITP), IgA nephropathy, IgG4-related sclerosing disease, immunomodulatory lipoproteins, inclusion body myositis, interstitial cystitis, juvenile arthritis, juvenile diabetes mellitus (type 1 diabetes), juvenile myositis, Kawasaki syndrome, Lambert-Eaton syndrome, leukocytoclastic vasculitis, lichen planus, lichen sclerosus, ligneous conjunctivitis, linear IgA disease (LAD), lupus (SLE), Lyme disease, chronic Meniere's disease, microscopic polyangiitis, mixed connective tissue disease (MCTD), Mooren's ulcer, Mucha-Habermann disease, multiple sclerosis, myasthenia gravis, myositis, narcolepsy, neuromyelitis optica (Devic's), neutropenia, ocular cicatricial pemphigoid, optic neuritis, palindromic rheumatism, PANDAS (pediatric autoimmune neuropsychiatric disorders associated with streptococcus), paraneoplastic cerebellar degeneration, paroxysmal nocturnal hemoglobinuria (PNH), Parry Romberg syndrome, Parsonnage-Turner syndrome, pars planitis (peripheral uveitis), pemphigus, peripheral neuropathy, perivenous encephalomyelitis, pernicious anemia, POEMS syndrome, polyarteritis nodosa, type I, type II, and type III autoimmune polyglandular syndromes, polymyalgia rheumatica, polymyositis, post myocardial infarction syndrome, postpericardiotomy syndrome, progestin dermatosis, primary biliary cirrhosis, primary sclerosing cholangitis, psoriasis, psoriatic arthritis, idiopathic pulmonary fibrosis, pyoderma gangrenosum, pure red cell anemia, Raynaud's phenomenon, reactive arthritis, reflex sympathetic dystrophy, Reiter's syndrome, relapsing polychondritis, restless leg syndrome, retroperitoneal fibrosis, rheumatic fever, rheumatoid arthritis, sarcoidosis, Schmidt syndrome, scleritis, scleroderma, Sjogren's syndrome, Sperm & testicular autoimmunity, stiff person syndrome, subacute bacterial endocarditis (SBE), Susac's syndrome, sympathetic ophthalmia, Takayasu's arteritis, temporal arteritis / giant cell arteritis, thrombocytopenic purpura (TTP), Tolosa-Hunt syndrome, transverse myelitis, ulcerative colitis, undifferentiated connective tissue disease (UCTD), uveitis, uveomeningitic syndrome, vasculitis, and Vogt-Koyanagi-Harada syndrome.syndrome), transverse myelitis, ulcerative colitis, undifferentiated connective tissue disease (UCTD), uveitis, vasculitis, vesiculobullous dermatosis, vitiligo, and Wegener's granulomatosis (now known as granulomatosis with polyangiitis (GPA)).

[0096] Neurological diseases. Various neurological diseases can be treated with the cap analogs of the disclosure. As non-limiting examples, the neurological disease can be septo-optic dysplasia, acid lipase disease, acid maltase deficiency, acquired epileptic aphasia, acute disseminated encephalomyelitis, attention deficit-hyperactivity disorder (ADHD), Adie’s pupil, Adie’s syndrome, adrenoleukodystrophy, agenesis of the corpus callosum, agnosia, Aicardi Syndrome, Aicardi-Goutieres Syndrome Disorder, AIDS-neurological complications, Alexander disease, Alpers’ disease, alternating hemiplegia, Alzheimer’s disease, amyotrophic lateral sclerosis (ALS), anencephaly, aneurysm, Angelman Syndrome, angiomatosis, anoxia, antiphospholipid syndrome, aphasia, apraxia, arachnoid cyst, arachnoiditis, Arnold-Chiari Malformation, arteriovenous malformation, Asperger Syndrome, ataxia, ataxia telangiectasia, ataxia and cerebellar or spinocerebellar degeneration, atrial fibrillation and stroke, attention deficit-hyperactivity disorder, autism spectrum disorder, autonomic dysfunction, back pain, Barth Syndrome, Batten Disease, Becker’s Myotonia, Behcet’s disease, Bell’s Palsy, benign essential blepharospasm, benign focal muscle atrophy, benign intracranial hypertension, Bernhardt-Roth Syndrome, Binswanger’s Disease, blepharospasm, Bloch-Sulzberger Syndrome, Brachial Plexus Birth Injuries, Brachial Plexus Injuries, Bradbury-Eggleston Syndrome, brain and spinal cord tumors, brain aneurysm, brain injury, Brown-Sequard Syndrome, bulbo-spinal muscular atrophy, cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL), Canavan disease, Carpal Tunnel Syndrome, Cauda Equina Syndrome, Cerebral Palsy, cerebral venous sinus thrombosis, Charcot-Marie-Tooth disease, Chiari Malformation, Chorea, Chronic Inflammatory Demyelinating Polyneuropathy, Chronic Pain, Chronic Regional Pain Syndrome, Cockayne Syndrome, Coffin Lowry Syndrome, Coffin-Siris Syndrome, Congenital Adrenal Hyperplasia, Congenital Cataracts Facial Recognization Syndrome, Congenital Muscular Dystrophy, Cortical Blindness, Cortical Visual Impairment, Craniocerebral Dysraphia, Craniopharyngioma, CREST Syndrome, Crouzon Syndrome, Cushing’s Syndrome, Cystic Fibrosis, Cystinosis, Dandy-Walker Malformation, Dandy-Walker Syndrome, Dandy-Walker Variant, Dementia, Dementia Pugilistica, Dementia with Lewy Bodies, Dercum’s Disease, Devic’s Disease, Diabetes Insipidus, Diabetic Neuropathy, Diffuse Lewy Body Disease, Diffuse Myoclonic Epilepsy, Diffuse Sclerosis, Dravet Syndrome, Duchenne Muscular Dystrophy, Dystonia, Early Infantile Epileptic Encephalopathy, Encephalitis, Encephalopathy, Encephalotrigeminal Angiomatosis, Encephalozelenmalacia, Encephalopathy, Epidermolysis Bullosa, Epilepsy, Epilepsy with Malignant Focal Seizures, Epilepsy with Muscle Cramps and Rigidity, Epilepsy with Myoclonic-Atonic Seizures, Epilepsy with Spasms, Epilepsy with Unverricht’s Myoclonus, Epileptic Encephalopathy, Episodic Familial Infantile Convulsions, Episodic Kinesigenic Hyperekplexia, Episodic Lethal Neonatal Convulsions, Episodic Lethal Neonatal Convulsions, Episodic Kinesigenic Hyperekplexia, Episodic Lethal Neonatal Convulsions,Disease), Carpal Tunnel Syndrome, Causalgia, Cavernomas, Cavernous Angioma, Cavernous Angiomas, Central Cervical Spinal Cord Syndrome, Central Spinal Cord Syndrome, Central Pain Syndrome, Central Pontine Myelinolysis, Head Disorder, Ceramidase Deficiency, Cerebellar Degeneration, Cerebellar Hypoplasia, Cerebral Aneurysm, Cerebral Arteriosclerosis, Cerebral Atrophy, Cerebral Foot Disease, Cerebral Cavernous Angioma, Cerebral Gigantism, Cerebral Hypoxia, Cerebral Palsy, Cerebro-Oculo-Facio-Skeletal Syndrome (COFS), Charcot-Marie-Tooth Disease, Chiari Malformation, Cholesterol Ester Storage Disease, Chorea, Chorea-Red Cell Anemia, Chronic Inflammatory Demyelinating Polyneuropathy (CIDP), Chronic Orthostatic Intolerance, Chronic Pain, Cockayne Syndrome Type II, Coffin Lowry Syndrome, Colpocephaly, Coma, Complex Regional Pain Syndrome, Congenital Bilateral Facial Weakness, Congenital Myasthenia, Congenital Myopathy, Congenital Vascular Cavernous Angioma, Cortical Basal Ganglia Degeneration, Cranial Arteritis, Craniosynostosis, Cree Encephalitis, Creutzfeldt-Jakob Disease, Cumulative Trauma Disorder, Cushing’s Syndrome, Giant Cell Inclusion Disease, Cytomegalovirus Infection, Dancing Eyes-Dancing Feet Syndrome, Dandy-Walker Syndrome, Dawson Disease, DeMorsier’s Syndrome, Dejerine-Klumpke Palsy, Dementia, Dementia - Multi-Infarct, Dementia - Semantic, Dementia Subcortical, Dementia with Lewy Bodies, Dentatorubral Ataxia, Dentatorubral Atrophy, Dermatomyositis, Developmental Apraxia, Devic’s Syndrome,Diabetic neuropathy, diffuse sclerosis, Dravet syndrome, autonomic dysfunction, writing difficulties, reading difficulties, swallowing difficulties, agility disorders, myoclonic cerebellar coordination disorder, progressive cerebellar coordination disorder, dystonia, early infantile epileptic encephalopathy, empty sella syndrome, encephalitis, encephalitis of lethargy, encephalocele, encephalopathy, encephalopathy (familial infantile), trigeminal hemangioma, epilepsy, epileptic hemiplegia, Erb's palsy, Erb-Duchenne and Dejerine-Klumpke palsies, essential tremor, extrapontine myelinolysis, Fabry disease, Fahr syndrome, syncope, familial autonomic dysfunction, familial hemangioma, familial idiopathic basal ganglia calcification, familial periodic paralysis, familial spastic paralysis, Farber's disease Diseases including febrile seizures, fibromuscular dysplasia, Fisher syndrome, infantile relaxation syndrome, foot drop, Friedreich's ataxia, frontotemporal dementia, Gaucher disease, systemic ganglioside deposition syndrome, and Gerstmann's syndrome. Syndrome, Gerstmann-Straussler-Scheinker disease, giant axonal neuropathy, giant cell arteritis, giant cell inclusion body disease, globular leukodystrophy, glossopharyngeal neuralgia, glycogen storage disease, Guillain-Barré syndrome, Hallervorden-Spatz disease, head injury, headache, persistent migraine, unilateral facial muscle spasm, alternating hemiplegia (Hemiplegia Alterans), hereditary neuropathy, hereditary spastic paraplegia, polyneuritis-type hereditary motor ataxia, herpes zoster, otitis herpes zoster, Hirayama syndrome, Holmes-Adie syndrome, holohemorrhagic forebrain malformation, HTLV-1 related myelopathy, Hughes syndrome Syndrome, Huntington's disease, hydrocephalus, hydrocephalus-normal pressure, hydrocephalus, Cushing's syndrome, hypersomnia, hypertonia, hypotonia, hypoxia, immune-mediated encephalomyelitis, inclusion body myositis, pigmentary disorders, infantile hypotonia, infantile axonal dystrophy, infantile phytate storage disease, infantile Refsum diseaseDiseases, infantile spasms, inflammatory myopathy, occipital cleft with exposed brain malformation, enterogenic lipodystrophy, intracranial cysts, intracranial hypertension, Isaacs syndrome, Joubert syndrome, Kearns-Sayre syndrome, Kennedy's disease, Kinsbourne syndrome, Kleine-Levin syndrome, Klippel-Feil syndrome, Klippel-Trenaunay syndrome (KTS), Kliiver-Bucy syndrome, Korsakoff's Amnesic Syndrome, Krabbe disease, Kugelberg-Welander disease, Kuru disease, Lambert-Eaton myasthenia gravis. Myasthenic Syndrome, Landau-Kleffner Syndrome, Lateral Femoral Cutaneous Nerve Entrapment, Lateral Medullary Syndrome, Learning Disabilities, Leigh's Disease, Lennox-Gastaut Syndrome, Lesch-Nyhan Syndrome, Leukodystrophy, Levine-Critchley Syndrome, Lewy Body Dementia, Lipid Storage Disease, Lipoproteinemia, Lissencephaly, Locked-in Syndrome, Luger's Disease Gehrig's disease, lupus-neurological sequelae, Lyme disease-neurological complications, Machado-Joseph disease, macrocephaly, macrocephaly, Melkersson-Rosenthal syndrome, meningitis, meningitis and encephalitis, Menkes disease, paresthesia femoris, metachromatic leukodystrophy, microcephaly, migraine, Miller-Fischer syndromeSyndrome, minor stroke, mitochondrial myopathy, Moebius syndrome, unilateral muscular atrophy, motor neuron disease, Moyamoya disease, mucolipid storage disease, mucopolysaccharidosis, multiple infarct dementia, multifocal motor neuropathy, multiple sclerosis, multiple system atrophy, multiple system atrophy with orthostatic hypotension, muscular dystrophy, myasthenia gravis, myasthenia gravis, myelin-destructive diffuse sclerosis, infantile myoclonic encephalopathy, myoclonic myopathy, myopathy-congenital myopathy, thyrotoxic myopathy. –Thyrotoxic), myotonia, congenital myotonia, narcolepsy, neuroacanthosis, neurodegeneration with iron buildup, neurofibromatosis, neurodegenerative syndrome, neurological complications of AIDS, neurological complications of Lyme disease, neurological consequences of cytomegalovirus infection, neurological manifestations of Pompe disease, neurological sequelae of lupus, neuromyelitis optica, neurogenic myotonia, neuronal ceroid lipofuscin deposition, neuronal migration disorder, hereditary neuropathies, neurosarcoidosis, neurosyphilis, neurotoxicity, neurocavernous nevus, Niemann-Pick disease Diseases, O'Sullivan-McLeod syndrome, occipital neuralgia, Otahara syndrome, olivopontocerebellar atrophy, strabismus-oculoclonus-myoclonus, orthostatic hypotension, overuse syndrome, chronic pain, pantothenic kinase-associated neurodegeneration, paraneoplastic syndrome, paresthesia, Parkinson's disease, paroxysmal choreoathetosis, paroxysmal migraine, Parry-Romberg, Pelizaeus-Merzbacher disease, Pena-Shokeir II syndrome, perineural cysts, periodic paralysis, peripheral neuropathy, periventricular leukomalacia, persistent vegetative state, pervasive developmental disorder, phytate storage disease, Pick's diseaseDiseases, nerve pinching, piriformis syndrome, pituitary adenoma, polymyositis, Pompe disease, porencephaly, post-poliomyelitis syndrome, postherpetic neuralgia, post-infectious encephalomyelitis, orthostatic hypotension, orthostatic orthostatic tachycardia syndrome, orthostatic tachycardia syndrome, primary dentate nucleus atrophy, primary lateral sclerosis, primary progressive aphasia, prion disease, progressive hemifacial atrophy, progressive motor ataxia, progressive multifocal leukoencephalopathy, progressive sclerotic gray matter dystrophy, progressive supranuclear palsy, prosopagnosia, pseudotorch syndrome, pseudotoxoplasmosis syndrome, pseudotumor, psychogenic motor disorders, type I Ramsey-Hunter syndrome, type II Ramsey-Hunter syndrome, Rasmussen encephalitis, reflex sympathetic dystrophy syndrome, Refsum disease, infantile Refsum disease Infantile, repetitive motion disorder, repetitive stress injury, restless legs syndrome, retrovirus-associated myelopathy, Rett syndrome, Reye syndrome, rheumatic encephalitis, Riley-Day syndrome, sacral nerve root cyst, Saint Vitus Dance, salivary gland disorders, Sandhoff disease, Schilder's disease, schizophrenia, Seitelberger disease, seizure disorder, semantic dementia, Septo-Optic dysplasia, severe myoclonic epilepsy in infancy (SMEI), shaken baby syndrome, herpes zoster, Shy-Drager syndrome, Sjögren's syndrome, sleep apnea, sleep apnea-African sleep disorder. Sickness, Sotos syndrome, spasm, spina bifida, spinal cord infarction, spinal cord injury, spinal cord tumor, spinal muscular atrophy, spinocerebellar atrophy, spinocerebellar degeneration, Steele-Richardson-Olszewski syndrome, stiff person syndrome, striatal nigrostriatum degeneration, stroke, Sturge-Weber syndrome, subacute sclerosing panencephalitis, subcortical arteriosclerotic encephalopathy, transient unilateral neuralgia-like headache (SUNT), dysphagia, Sydenham Chorea, syncope, syphilitic myelitis, syringomyelia, syringomyelia, systemic lupus erythematosus, tabes dorsalis, tardive dyskinesia, Tarlov's cyst, Tay-Sachs disease.Disease), temporal arteritis, spinal cord tethering syndrome, Thomsen myotonia, thoracic outlet syndrome, thyrotoxic myopathy, Todd's palsy, Tourette syndrome, transient ischemic attack, transmissible spongiform encephalopathy, transverse myelitis, traumatic brain injury, tremor, trigeminal neuralgia, tropical spastic paraparesis, Troyer syndrome, tuberous sclerosis, vascular erectile tumors, vasculitis syndromes of the central and peripheral nervous system, Von Economo disease, Von Hippel-Lindau disease (VHL), Von Recklinghausen disease, Wallenberg syndrome, Werdnig-Hoffman Disease, Wernicke-Korsakoff Syndrome, West Syndrome, whip-lash injury, Whipple disease, Williams Syndrome, Wilson Disease, Wolman disease, X-linked spinal and bulbar muscular atrophy.

[0097] In jurisdictions that prohibit the patenting of methods performed on the human body, the meaning of "administering" a composition to a human subject or patient shall be limited to prescribing a controlled substance to a human subject or patient who will self-administer by any technique (e.g., oral, inhalation, topical application, injection, insertion, etc.). The broadest reasonable interpretation consistent with the law or regulation defining patentable subject matter is intended. In jurisdictions that do not prohibit the patenting of methods performed on the human body, "administering" of a composition includes both methods performed on the human body and the foregoing activities.

[0098] Examples The presently disclosed subject matter will be better understood by reference to the following examples, which are offered by way of illustration and not by way of limitation. In Table 2, Cap 1, Cap 2, and Cap 3 are comparative compounds to the compounds of the present disclosure.

[0099] Table 1

[0100]

[0101] Table 2

[0102] Materials and Methods In vitro transcription of mRNA comprising cap analogs mRNA was transcribed from DNA templates encoding Lucia using the HiScribe T7 RNA Synthesis Kit (New England Biolabs, Ipswich, MA, USA). N 1 - Reactions were performed with methyl pseudo UTP (TriLink, San Diego, CA, USA) instead of UTP. For 5’ capping of mRNA, cap analog compounds described herein (e.g., Compound 1-Compound 4) or CleanCap (TriLink, San Diego, CA, USA) were added to the IVT reaction as indicated. Reactions were performed at 37 °C for 4 hours.

[0103] Purification of transcripts was performed by TURBO DNase digestion (ThermoFisher Scientific, Waltham, MA, USA) at 37 °C for 30 min followed by RNA Clean XP bead (Beckman Coulter) cleanup. RNA-bound beads were washed 3 times with freshly prepared 70% ethanol, air-dried for 15 minutes, and then eluted in 200 μL of nuclease-free water. Purified mRNA was quantified using a nanodrop (ThermoFisher Scientific, Waltham, MA, USA) and analyzed using a Tapestation (Agilent, Santa Clara, CA, USA).

[0104] In vitro expression of mRNA containing cap analogs Lucia-encoding mRNA containing cap analog compounds described herein (e.g., Compound 1-Compound 4) or CleanCap (TriLink, San Diego, CA, USA) were transfected into HEK 293 cells using Lipofectamine MessengerMAX (ThermoFisher Scientific, Waltham, MA, USA) according to the manufacturer’s protocol.

[0105] Lucia luciferase activity was measured at 2, 4, 6, 24, 48, and 72 hours post transfection. At each time point, 5 pL of media was removed from each well to mix with QUANTI-Luc (InvivoGen, San Diego, CA, USA) and immediately measured using a plate reader (Molecular Devices, San Jose, CA, USA). Following each time point, fresh media containing DMEM with 10% FBS and 50 U / mL penicillin / streptomycin was exchanged in each well. Experiments were performed in 96-well plates in triplicate with a seeding density of 1e4 cells per well and cells were maintained in a 37 °C incubator supplied with 5% CO2.

[0106] In vivo expression of mRNA comprising cap analogs Candidate RNAs were formulated using proprietary lipid-based delivery vehicles (e.g., as described in WO 2020 / 069442 and WO 2023 / 014931) and administered to Balb / c mice at a dose of 15 pg per mouse. In life, serum draws via submandibular vein were performed at selected time points starting 2 hours post-dose. Serum was diluted 5 pL into 50 mL of Quanti-Luc reagent (InvivoGen, San Diego, CA, USA) and immediately measured using a plate reader (Molecular Devices, San Jose, CA, USA).

[0107] Example 1: Synthesis of Compound 1 (COMPD 1) Step-1: Synthesis of N-(9-((2R,3R,4R,5R)-4-hydroxy-5-(hydroxymethyl)-3- methoxytetrahydrofuran-2-yl)-9H-purin-6-yl)benzamide (1)

[0108] To a stirred solution of 2'-OMe-A (50 g, 0.178 mol, 1 eq) in dry pyridine (500 mL) was added TMS-Cl (224 mL, 1.78 mol, 10 eq) drop wise at 0 °C, then stirred at room temperature. After 2 h, distilled benzoyl chloride (30 mL, 0.26 mmol, 1.5 eq) was added to the reaction mixture at 0 °C, then allowed to warm to room temperature (25 °C) over 2 h. The progress of the reaction was monitored by TLC and LCMS. The reaction mixture was cooled to 0 °C, quenched with ice cold water (200 mL), stirred for 30 min, then NH4OH aqueous solution (100 mL) was added, and the mixture was stirred for 30 min and distilled to afford semi pure material. It was further purified by Si02(60-120 mesh) column chromatography by eluting with 0-5% methanol (MeOH) in dichloromethane (DCM) to obtain pure intermediate 1. TLC system: n-Hexane: EtOAc (60:40), Rf: 0.4; MeOH / DCM (10:90), R f Value: 0.4; 1 HNMR (400 MHz, DMSO-d6) δ 8.77 - 8.73 (m, 2H), 8.05 (d, J = 7.6 Hz, 2H), 7.67- 7.64 (m, 1 H), 7.57 - 7.52 (m, 2H), 6.17 (d, J = 5.6 Hz, 1 H), 4.45 -4.39(m, 2H), 4.02 - 4.01 (m, 1 H), 3.72 - 3.69 (m, 1 H), 3.62 - 3.59 (m, 1 H),3.37 (s, 3H);LCMS indicates 73% with 386.3 (M+H) + .

[0109] Step-2: Synthesis of N-(9-((2R,3R,4R,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)- 4-hydroxy-3-methoxytetrahydrofuran-2-yl)-9H-purin-6-yl)benzamide (Int-2)

[0110] To a stirred solution of intermediate 1 (42 g, 109.1 mmol, 1 eq) in dry pyridine (85 mL) was added dry TEA (22.7 mL, 163.6 mmol, 1.5 eq) at 0 °C followed by portion wise addition of DMT-Cl (74 g, 218.2 mmol, 2 eq) and allowed it to continue for 5 h at room temperature. After completion of starting material as per TLC, the reaction mixture was diluted with cold DM water (1 L) and extracted into EtOAC (2 x 350 mL). The combined organic layer was washed with brine solution (300 mL), dried over anhydrous sodium sulphate, filtered and concentrated under reduced pressure to provide semi pure product. It was further purified by Si02(60-120 mesh) column chromatography by eluting with 70-80% EtOAc in hexane to provide Int-2. TLC system: Hexane: EtOAc (70:30), Rf value: 0.5; EtOAc / hexanes (70:30), R f value: 0.5; 1 HNMR (400 MHz, DMSO-d6) δ 11.23 (s, 1 H), 8.69 (s, 1 H), 8.61 (s, 1 H), 8.06 - 8.04 (m, 2H), 7.67 - 7.63 (m, 1 H), 7.57 - 7.54 (m, 2H), 7.37 - 7.35 (m, 2H), 7.28 - 7.18 (m, 7H), 6.86 - 6.83 (m, 4H), 6.19 (d, J = 4.8 Hz, 1 H), 5.35 (d, J = 6.0 Hz, 1 H), 4.56 - 4.53 (m, 1 H), 4.50 - 4.46 (m, 1 H), 4.13 - 4.10 (m, 1 H), 3.72 (s, 6H), 3.40 (s, 3H), 3.27 - 3.25 (m, 2H); LCMS 83%, with m / z: 688.3 (M+H) + .

[0111] Step-3: Synthesis of N-(9-((2R,3R,4R,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-3-methoxy-4-(((2R,3aS,6R,7aS)-3a-methyl-6-(prop-1-en-2-yl)-2-thioxohexahydrobenzo[d][1,3,2]oxathiazepin-2-yl)oxy)tetrahydrofuran-2-yl)-9H-purin-6-yl)benzamide (Int-6)

[0112] At 0 °C, a solution of DBU (4.47 g, 29.0 mmol, 1.2 equivalent) in acetonitrile (5 mL) was added to a stirred solution of Int-2 (10 g, 14.5 mmol, 1 equivalent) and (+)Br-PSI reagent (8.2 g, 18.85 mmol, 1.3 equivalent) in 100 mL of dry-distilled ACN, and stirring was continued for 45 min. After complete consumption of PSI was monitored by TLC, the reaction mixture was distilled to reduce the acetonitrile volume to a maximum of 20 mL. The mixture was purified by silica gel (100-200 mesh) column chromatography with elution of 30% EtOAc in hexane to provide Int-6. TLC system: EtOAc / hexanes (30:70), R f Value: ~0.7; 1 HNMR(400 MHz, DMSO-d6) δ 11.25 (s, 1 H), 8.68 (s, 1 H), 8.60 (s, 1 H), 8.05 (d, J= 7.2 Hz, 2H), 7.67 - 7.63 (m, 1 H), 7.58 - 7.54 (m, 2H), 7.40 - 7.39 (m,2H), 7.28 - 7.19 (m, 7H), 6.86 - 6.83 (m, 4H), 6.13 - 6.12 (m, 1 H), 5.53 -5.49 (m, 1 H), 5.05 - 5.02 (m, 1 H), 4.95 - 4.85 (m, 2H), 4.41 - 4.34 (m, 3H), 3.73 (s, 6H), 3.39 - 3.37 (m, 1H), 3.34 (s, 3H), 2.58 - 2.51 (m, 2H), 2.23 - 2.15 (m, 1H), 1.93 - 1.87 (m, 2H), 1.83 - 1.77 (m, 2H), 1.73 (s, 3H), 1.66 (s, 3H); LCMS 77% has m / z: 933.9 (M+H) + .

[0113] Step-10: Synthesis of N-(9-((2R,3R,4S,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-3,4-dihydroxytetrahydrofuran-2-yl)-6-oxo-6,9-dihydro-1H-purin-2-yl)benzamide (4A)

[0114] To a stirred solution of compound-3A (10 g, 25.83 mmol, 1 eq) in DCM (50 mL, 5 vol) was added AgN03(8.7 g, 51.67 mmol, 2 eq) and DMT-C1 (17.4 g, 51.67 mmol, 2 eq) was added portion wise at 0 °C and then stirred at room temperature for 5 h. After complete consumption of starting material as per TLC, the reaction mixture was diluted with cold water (500 mL) and extracted with DCM (2 x 300 mL), washed with brine solution (250 mL), dried over anhydrous sodium sulphate and concentrated under reduced pressure to provide semi pure compound. Purification was done by normal phase Si02column chromatography with 3-5% MeOH in DCM as eluent to provide compound-4A. TLC system: MeOH / DCM (10:90), R f Value: 0.5; LCMS m / z: 690.0 (M+H) + .

[0115] Synthesis of (2R,3R,4R,5R)-2-(2-benzoylamino-6-oxo-1,6-dihydro-9H-purin-9-yl)-5- ((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)tetrahydrofuran-3,4-diyl bis(2- methylpropanoate) (5A)

[0116] To a stirred solution of compound-4A (15 g, 21.77 mmol, 1 eq) and DMAP (3.9 g, 32.65 mmol, 1.5 eq) in DCM (450 mL, 30 vol) was added isobutyryl chloride (3.4 mL, 32.65 mmol, 1.5 eq) drop wise at 0 °C and then allowed to stir at room temperature for 1 h. After completion of reaction as per TLC, the reaction mixture was cooled to 0 °C, quenched with ice cold water (500 mL) and extracted with DCM (2 x 300 mL), washed with brine solution (250 mL). The organic layer was dried over anhydrous sodium sulphate and concentrated under reduced pressure to provide semi pure compound. Purification was done by normal phase Si02column with 70% EtOAc in hexane as eluent to provide compound-5A. TLC system: MeOH / DCM (05:95), R f Value: 0.4; LCMS m / z: 828.0 (M+H) + .

[0117] Step-12: Synthesis of (2R, 3R, 4R, 5R)-2-(2-benzoylamino-6-oxo-l,6-dihydro-9H-purin-9-yl)-5- (hydroxymethyl)tetrahydrofuran-3,4-diyl bis(2-methylpropanoate) (Nuc-C2)

[0118] To compound-5A (20 g, 71.17 mmol, 1 eq) at 0 °C, a solution of Cl3CCOOH in DCM (5 g / 50 mL) was added by drop wise addition and then it was allowed to stir at room temperature for 2 h. After completion of reaction as per TLC, the reaction mixture was quenched with aqueous NaHC03solution and extracted with DCM (2 x 200 mL), washed with brine solution (350 mL). The organic layer was dried over anhydrous sodium sulphate and concentrated under reduced pressure to afford semi pure compound. Purification was carried out by normal phase Si02column eluting with 5% MeOH in DCM to afford Nuc-C2. TLC system: Methanol:DCM (5:95), Rf: 0.4; MeOH / DCM (05:95), R f Value: 0.4; 1 HNMR (400MHz, DMSO-d6) δ 12.35 (s, 1 H), 11.89 (s, 1 H), 8.34 (s, 1 H), 8.04 - 8.01 (m, 2H), 7.71 - 7.66 (m, 1 H), 7.59 - 7.55 (m, 2H), 6.13 (d, J = 7.2 Hz, 1H), 5.84 - 5.81 (m, 1 H), 5.52 - 5.50 (m, 1 H), 5.39 (t, 1 H), 4.23 - 4.21 (m, 1 H), 4.10 - 4.07 (m, 1 H), 3.72 - 3.68 (m, 2H), 2.65 - 2.61 (m, 1 H), 1.16 (dd, J = 6 Hz, 2.8 Hz, 6H), 1.01 (dd, J = 6.8 Hz, 16 Hz, 6H); LCMS m / z: 526.0 (M+H) + .

[0119] Step-4: Synthesis of (2R, 3R, 4R, 5R)-2-(2-benzamido-6-oxo-l,6-dihydro-9H-purin-9-yl)-5-((((S)-(((2R, 3R, 4R, 5R)-5-(6-benzamido-9H-purin-9-yl)-2-((bis(4- methoxyphenyl)(phenyl)methoxy)methyl)-4-methoxytetrahydrofuran-3-yl)oxy)(hydroxy) thiophosphoryl)oxy)methyl)tetrahydrofuran-3,4-diyl bis(2-methylpropanoate) (22)

[0120] To a stirred solution of Int-6 (9 g, 9.64 mmol, 1 eq) (co-distilled with dry ACN) and Nuc-C2 (7.6 g, 14.46 mmol, 1.5 eq) (co-distilled with dry ACN) in dry distillation ACN (70 mL) was added drop wise a solution of dry distillation DBU (1.45 g, 9.64 mmol, 1 eq) in acetonitrile (15 mL) at 0 °C and stirring was continued for 40 min. After complete consumption of starting material, the reaction mixture was distilled off and then triturated with diethyl ether to afford compound-22. LCMS indicated 34% with m / z: 1292.9 (M+H) + .

[0121] Step-5: Synthesis of (2R, 3R, 4R, 5R)-2-(2-benzamido-6-oxo-l,6-dihydro-9H-purin-9-yl)-5-((((((2R, 3R, 4R, 5R)-5-(6-benzamido-9H-purin-9-yl)-2-(hydroxymethyl)-4- methoxytetrahydrofuran-3-yl)oxy)(hydroxy)phosphoryl)oxy)methyl)tetrahydrofuran-3,4- diyl bis(2-methylpropanoate) (23)

[0122] To a stirred solution of compound-22 (12 g (half pure), 9.28 mmol, 1 eq) in a mixture of ACN:H20 (30 mL, 4:1 ratio) was added selenium dioxide (5.15 g, 46.43 mmol, 5 eq) at 0 °C and the mixture was stirred for 16 h. The progress of the reaction was monitored by LCMS. After completion of the reaction, the reaction mixture was filtered and the mother liquor was distilled off. The crude material was purified by C18 reverse phase column chromatography by eluting with 1 M ammonium bicarbonate buffer and 25-30% ACN. The collected pure fractions were lyophilized to afford compound-23. LCMS 80% with m / z: 974.8 (M+H) + .

[0123] Step-6: Synthesis of (2R,3R,4R,5R)-2-(2-benzoylamino-6-oxo-1,6-dihydro-9H-purin-9-yl)-5-((((((2R,3R,4R,5R)-5-(6-benzoylamino-9H-purin-9-yl)-4-methoxy-2- (phosphonooxy)methyl)tetrahydrofuran-3-yl)oxy)(hydroxy)phosphoryl)oxy)methyl)tetrahydrofuran-3,4-diyl bis(2-methylpropanoate) (24)

[0124] To a stirred solution of compound-23 (2.5 g, 2.56 mmol, 1 eq) in distilled trimethyl phosphate (2.99 mL, 25.66 mmol, 10 eq) was added distilled phosphorous oxychloride (1.9 mL, 20.48 mmol, 8 eq) at 0°C for 4 h after which a clear solution was observed. The progress of the reaction was monitored by LCMS and after complete consumption of the starting material, the reaction mixture was quenched with 1 M TEAB and then lyophilized. The material was purified by C18 reverse phase column chromatography eluting with 1 M aqueous ammonium bicarbonate and 20-25% ACN. The collected pure fractions were lyophilized to afford compound 24 as a triethylammonium salt. LCMS 84% with m / z: 1054.9 (M+H) + .

[0125] Synthesis of 2-amino-9-((2R,3R,4S,5R)-3,4-dihydroxy-5-(((hydroxy(phosphonooxy) phosphoryl)oxy)methyl)tetrahydrofuran-2-yl)-7-ethyl-6-oxo-6,9-dihydro-1H-purin-7-ium (Et7GDP).

[0126]

[0127] To a stirred solution of triethylamine salt of guanosine-5'-diphosphate (7 g, 15.08 mmol, 1 eq) in dry dimethylacetamide (35 mL, 5 vol) was added ethyl iodide (6.5 mL, 79.8 mmol, 5 eq) followed by stirring at room temperature for 7 days. After monitoring the completion of the reaction by LCMS / 1 H NMR, the reaction mixture was added with diethyl ether and the formed solid was filtered, washed with acetone and dried. The obtained crude solid was purified by DEAE Sephadex chromatography by eluting with 12-15% 1 M TEAB aqueous solution and water. The collected desired fractions were lyophilized to obtain semi-pure compound which was further purified twice by preparative HPLC using C18 column to provide Et7GDP. 1HNMR (400 MHz, D20) δ 9.23 (S, 1 H), 6.04 (d, J = 3.2 Hz, 1 H), 4.61 - 4.52 (m, 3H), 4.45 - 4.35 (m, 2H), 4.27 - 4.13 (m, 2H), 1.58 (t, 3H).

[0128] Step-4: Synthesis of 2-amino-9-((2R,3R,4S,5R)-3,4-dihydroxy-5-(((hydroxy((hydroxy(lH- imidazol-l-yl)phosphoryl)oxy)phosphoryl)oxy)methyl)tetrahydrofuran-2-yl)-7-ethyl-6- oxo-6,9-dihydro-lH-purin-7-ium (Et7GDP-lm).

[0129]

[0130] To a solution of Et7GDP (650 mg, 1.35 mmol, 1 eq), TPP (4.17 g, 6.79 mmol, 5 eq), imidazole (920 mg, 13.5 mmol, 10 eq) in a mixture of DMF:DMSO (2 mL:0.3 mL) was added DPS (1.48 g, 6.79 mmol, 5 eq) at 0 °C and then allowed to stir at room temperature for 2 h. After complete consumption of starting material by LCMS, the reaction mixture was cooled to 0 °C and then added cooled sodium perchlorate in acetone. The precipitate formed was collected by centrifuge and washed thoroughly with acetone and then dried. 450 mg of Et7GDP-lm was isolated as off-white solid (yield: 76%); LCMS m / z: 522.0 (M+H) + .

[0131] Step-1: 2-amino-9-((2R,3R,4S,5R)-5-((((((((((2R,3R,4R,5R)-3-(((((2R,3R,4R,5R)-5-(2- benzoylamino-6-oxo-l,6-dihydro-9H-purin-9-yl)-3,4-bis(isobutyryloxy)tetrahydrofuran-2- yl)methoxy)(hydroxy)phosphoryl)oxy)-5-(6-benzoylamino-9H-purin-9-yl)-4- methoxytetrahydrofuran-2-yl)methoxy)(hydroxy)phosphoryl)oxy)(hydroxy)phosphoryl)oxy)(hydroxy)phosphoryl)oxy)methyl)-3,4-dihydroxytetrahydrofuran-2-yl)-7-ethyl-6-oxo- 6,9-dihydro-lH-purin-7-ium (39)

[0132] To a stirred solution of compound-24 (350 mg, 0.332 mmol, 1 eq) (co-distilled with dry toluene) and Et7GDP-lm (435 mg, 0.830 mmol, 2.5 eq) (co-distilled with dry toluene) in dry distillation DMF (10 vol.) was added anhydrous ZnCl2(1.35 g, 9.96 mmol, 30 eq) (dried at 60 °C for 3 h at 0.5 mmHg) at 0 °C and allowed to warm to room temperature (25 °C) and then stirred for 16 h.

[0133] Stage-1 (workup) : The reaction mixture was diluted with cold water (10 mL) and EDTA solid and the obtained clear solution was lyophilized to remove DMF. LCMS indicated compound-39 with 55%, compound-24 with 17%.

[0134] Stage-2 (DEAE Sephadex purification) : The obtained crude compound was purified by DEAE Sephadex eluting with 30-40% ACN and 1 M TEAB aqueous solution. The collected pure fractions were lyophilized to obtain semi-pure compound-39. LCMS indicated compound-39 with 47% of desired m / z.

[0135] Stage-3 (C18 purification) : The above obtained semi-pure compound was further purified by C18 reverse phase preparative HPLC eluting with 48% ACN and 1 M ABC aqueous solution to obtain pure compound-39. LCMS indicated ~80% of desired m / z.

[0136] Step-2: Synthesis of 2-amino-9-((2r,3r,4s,5r)-5-((((((((((2r,3r,4r,5r)-3-(((((2r,3r,4s,5r)-5-(2-amino-6-oxo-l,6-dihydro-9h-purin-9-yl)-3,4- dihydroxytetrahydrofuran-2-yl)methoxy)(hydroxy)phosphoryl)oxy)-5-(6-amino-9h-purin-9-yl)- 4-methoxytetrahydrofuran-2-yl)methoxy)(hydroxy)phosphoryl)oxy)(hydroxy)phosphoryl)oxy)(hydroxy)phosphoryl)oxy)methyl)-3,4-dihydroxytetrahydrofuran-2-yl)-7-ethyl-6-oxo-6,9-dihydro-lh- purin-7-ium (Compd 1).

[0137]

[0138] At 20 °C, 7 M MEOH.NH3(5 vol * 2 times) was added to compound-39 (65 mg, 0.043 mmol, 1 eq) in 25 ml RBF via syringe and stirred at same temperature for 48 h. The progress of reaction was monitored by LCMS and worked up after complete consumption of compound-65.

[0139] Workup The reaction mixture was flushed with nitrogen and then concentrated under reduced pressure to provide semi-pure compound Compd 1.

[0140] DEAE Sephadex purification The obtained semi-pure Compd 1 was further purified by DEAE Sephadex by eluting with 2-3% ACN and 1 M TEAB in water. The collected desired fractions were lyophilized to provide semi-pure Compd 1 as TEA salt.

[0141] Preparative HPLC purification The obtained semi-pure Compd 1 as TEA salt was further purified by preparative HPLC using 1 M ABC in water and 10% ACN: 30% MeOH: 60% water mixture to provide pure Compd 1.

[0142] Example 2: Synthesis of compound 2 (COMPD 2) Step-1: Synthesis of methanesulfonic acid 2-phenoxyethyl ester (A)

[0143] To a stirred solution of SM-1 (30 g, 0.22 mol, 1 eq) in dichloromethane (300 mL, 10 vol) was added triethylamine (41 mL, 0.32 mol, 1.5 eq) and methanesulfonyl chloride (25.1 mL, 0.32 mol, 1.5 eq) at 0 °C and stirred for 5 h. After complete consumption of starting material as per TLC, the reaction mixture was diluted with water and extracted with DCM, then dried over sodium sulfate and concentrated under reduced pressure to provide compound-A; 1 HNMR (400 MHz, CDCl3) δ 7.33 - 7.28 (m, 2H), 7.02 - 6.98 (m, 1 H), 6.91 - 6.88 (m, 2H), 4.59 -4.57 (m, 2H),4.26 - 4.24 (m, 2H), 3.09 (s, 3H).

[0144] Step-4: Synthesis of (2-iodoethoxy)benzene (B)

[0145] To a stirred solution of compound-A (30 g, 0.14 mol, 1 eq) in acetone (300 mL, 10 vol), NaI (31.2 g, 0.21 mol, 1.5 eq) was added and then stirred at reflux temperature for 16 h. After complete consumption of starting material as per TLC, the reaction mixture was diluted with water and extracted with diethyl ether, then dried over sodium sulfate and concentrated under reduced pressure. The obtained crude was purified by silica gel column to afford compound-B; 1 HNMR (400 MHz, CDCl3) δ 7.31 - 7.26(m, 2H), 7.0 - 6.95 (m, 1 H), 6.91 -6.88 (m, 2H), 4.25 (t, 2H), 3.42 (t, 2H).

[0146] Step-5: Synthesis of 2-amino-9-((2R,3R,4S,5R)-3,4-dihydroxy-5-(((hydroxy (phosphonooxy) phosphoryl) oxy) methyl) tetrahydrofuran-2-yl)-6-oxo-7-(2-phenoxyethyl)-6,9-dihydro-1H-purin-7-ium ((CH2CH2-OPh)GDP)

[0147] To a stirred solution of guanosine-5'-diphosphoric acid triethylamine salt (3 g, 6.77 mmol, 1 eq) in dry dimethylacetamide (6 mL, 2 vol), compound-B (3.34 g, 13.54 mmol, 2 eq) was added and then stirred at 70 °C for 16 h. The reaction mixture was diluted with diethyl ether, then the solid formed was washed with acetone and dried. The obtained crude solid was purified by DEAE Sephadex chromatography by eluting with 25% 1 M TEAB in water and water. The collected desired fractions were lyophilized to obtain semi-pure compound which was further purified twice by preparative HPLC using C18 column to afford (CH2CH2-OPh)GDP; LCMS m / z: 564.0 (M) + .

[0148] Step-6: Synthesis of 2-amino-9-((2R,3R,4S,5R)-3,4-dihydroxy-5-(((hydroxy ((hydroxy (1H-imidazol-1-yl) phosphoryl) oxy) phosphoryl) oxy) methyl) tetrahydrofuran-2-yl)-6-oxo-7-(2-phenoxyethyl)-6,9-dihydro-1H-purin-7-ium (CH2CH2-OPh)GDP-lm)

[0149] To a solution of (CH2CH2-OPh)GDP (150 mg, 0.26 mmol, 1 equiv), TPP (423 mg, 1.3 mmol, 5 equiv), imidazole (177 mg, 2.6 mmol, 10 equiv) in a mixture of DMF:DMSO (2 mL:0.3 mL) at 0 °C was added DPS (286 mg, 1.3 mmol, 5 equiv) and allowed to stir at room temperature for 2 h. The reaction mixture was cooled to 0 °C and then cooled sodium perchlorate in acetone was added. The precipitate formed was collected by centrifuge and washed thoroughly with acetone and then dried. The product (CH2CH2-OPh)GDP-lm was isolated; LCMS m / z: 612.0 (M-2H) + .

[0150] 2-Amino-9-((2R,3R,4S,5R)-5-((((((((((2R,3R,4R,5R)-3-(((((2R,3R,4R,5R)-5-(2-benzamido-6-oxo-1,6-dihydro-9H-purin-9-yl)-3,4-bis(isobutyryloxy)tetrahydrofuran-2-yl)methoxy)(hydroxy)phosphoryl)oxy)-5-(6-benzamido-9H-purin-9-yl)-4-methoxytetrahydrofuran-2-yl)methoxy)(hydroxy)phosphoryl)oxy)(hydroxy)phosphoryl)oxy)(hydroxy)phosphoryl)oxy)methyl)-3,4-dihydroxytetrahydrofuran-2-yl)-6-oxo-7-(2-phenoxyethyl)-6,9-dihydro-1H-purin-7-ium (44)

[0151] To a stirred solution of compound-24 (350 mg, 0.332 mmol, 1 equiv) (co-distilled with dry toluene) and (CH2CH2-OPh)GDP-lm (305 mg, 0.498 mmol, 1.5 equiv) (co-distilled with dry toluene) in dry distilled DMF (10 vol) at 0 °C was added anhydrous ZnCl2(0.92 g, 6.64 mmol, 20 equiv) (dried at 60 °C at 0.5 mmHg for 2 h) and allowed to warm to room temperature (25 °C) and then stirred for 48 h. The reaction mixture was diluted with water (10 mL) at 0 °C and then EDTA solid was added and the reaction mixture was kept under lyophilization to remove the DMF solvent. After purification by ion exchange DEAE Sephadex chromatography and preparative HPLC, the pure compound-44 was isolated; LCMS m / z: 1598.0 (M-2H) + .

[0152] Step-2: Synthesis of 2-amino-9-((2R,3R,4S,5R)-5-((((((((((2R,3R,4R,5R)-3-(((((2R,3S,4R,5R)-5-(2-amino-6-oxo-1,6-dihydro-9H-purin-9-yl)-3,4- dihydroxytetrahydrofuran-2-yl)methoxy)(hydroxy)phosphoryl)oxy)-5-(6-amino-9H-purin-9-yl)- 4-methoxytetrahydrofuran-2-yl)(methoxy)(hydroxy)phosphoryl)oxy)(hydroxy)phosphoryl)oxy)( hydroxy)phosphoryl)oxy)methyl)-3,4-dihydroxytetrahydrofuran-2-yl)-6-oxo-7-(2-phenoxyethyl)- 6,9-dihydro-1H-purin-7-ium (Compd 2)

[0153] To compound-44 (48 mg, 0.03 mmol, 1 eq) in 25 mL RBF was added 7 M MeOH.NH3 (5 vol * 2 times) via syringe at 20 °C and stirred at same temperature for 36 h. The progress of reaction was monitored by LCMS and worked up after complete consumption of compound-44. After evaporation of volatiles and trituration with diethyl ether, the obtained crude 39 mg / 41% was purified by DEAE Sephadex chromatography eluted with 1 M ABC / H2O (after confirming stability). The obtained semi-pure Compd 2 was further purified by preparative HPLC using 1 M aqueous ABC and mixture of 10% ACN: 30% MeOH: 60% water to provide pure Compd 2. LCMS and HPLC indicated 98% purity with expected m / z: 1252.8 (M) + .

[0154] Example 3: Synthesis of Cap 3 (Comparative Example) Step-1: Synthesis of N-(9-((2R,3R,4R,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-3- methoxy-4-(((2R,3aS,6R,7aS)-3a-methyl-6-(prop-1-en-2-yl)-2-thioxahexahydrobenzo[d][1,3,2] oxathiolyl-2-yl)oxy)tetrahydrofuran-2-yl)-9H-purin-6-yl)benzamide (Int-6A)

[0155] To a stirred solution of Int-2 (Ref: Clean CAP reagent; 10 g, 14.5 mmol, 1 eq) and (+)-F-PSI reagent (7.1 g, 15.95 mmol, 1.1 eq) in dry ACN (100 mL) was added a solution of DBU (1.4 g, 21.75 mmol, 1.5 eq) in acetonitrile (15 mL) at 0 °C and stirring was continued for 45 min. After complete consumption of (+)-PSI by TLC, the reaction mixture was distilled to reduce the acetonitrile volume to max 20 mL. The mixture was purified by silica gel (100-200 mesh) column chromatography eluting with 30% EtOAc in hexane to afford compound Int-6A. TLC system: 30% MeOH in DCM. EtOAc / hexanes (30:70), R f Value: ~0.7; LCMS m / z: 933.9 (M+H) + .

[0156] Step-2: Synthesis of (2R,3R,4R,5R)-2-(2-benzoylamino-6-oxo-1,6-dihydro-9H-purin-9-yl)-5-((((R)-(((2R,3R,4R,5R)-5-(6-benzoylamino-9H-purin-9-yl)-2-((bis(4- methoxyphenyl)(phenyl)methoxy)methyl)-4-methoxytetrahydrofuran-3-yl)oxy)(hydroxy)thiophosphoryl)oxy)methyl)tetrahydrofuran-3,4-diyl bis(2-methylpropanoate) (45)

[0157] To a stirred solution of Int-6A (10 g, 10.71 mmol, 1 eq) (co-distilled with dry ACN) and Nuc-C2 (7.3 g, 13.92 mmol, 1.3 eq) (co-distilled with dry ACN) in dry ACN (70 mL) was added a solution of dry DBU (2 mL, 16.06 mmol, 1.5 eq) in acetonitrile (15 mL) drop wise at 0 °C and stirring was continued for 1 h. After complete consumption of starting material by TLC, the reaction mixture was distilled to reduce the acetonitrile volume to max 10 mL. The obtained crude solution was purified by C18 / liquid injection reverse phase column chromatography eluting with 1 M aqueous ammonium bicarbonate (ABC) and 40% ACN and the collected pure fractions were lyophilized to afford compound-45. LCMS m / z: 1291.4 (M-2H) + .

[0158] Step-3: Synthesis of (2R,3R,4R,5R)-2-(2-benzamido-6-oxo-l,6-dihydro-9H-purin-9-yl)-5-((((R)-(((2R,3R,4R,5R)-5-(6-benzamido-9H-purin-9-yl)-2-(hydroxymethyl)-4- methoxytetrahydrofuran-3-yl)oxy)(hydroxy)thiophosphoryl)oxy)methyl)tetrahydrofuran-3,4- diyl bis(2-methylpropanoate) (46)

[0159] To a stirred solution of compound-45 (4.1 g, 3.17 mmol, 1 eq) in a mixture of ACN:DCM (20 mL, 1:1 ratio) was added trichloroacetic acid (1.03 g, 6.34 mmol, 2 eq) at 0 °C and stirred for 1 h at room temperature. The progress of the reaction was monitored by LCMS. After completion of starting material, the reaction mixture was distilled under reduced pressure. The obtained semi-pure solid material was purified by C18 reverse phase preparative HPLC using 1 M ammonium bicarbonate buffer solution and 40% ACN. The collected pure fractions were lyophilized to afford compound-46. LCMS m / z: 989.1 (M-H) + .

[0160] Step-4: Synthesis of (2R,3R,4R,5R)-2-(2-benzamido-6-oxo-l,6-dihydro-9H-purin-9-yl)-5-((((R)-(((2R,3R,4R,5R)-5-(6-benzamido-9H-purin-9-yl)-4-methoxy-2-((phosphonooxy)methyl)tetrahydrofuran-3-yl)oxy)(hydroxy)thiophosphoryl)oxy)methyl)tetrahydrofuran-3,4-diyl bis(2-methylpropanoate) (47)

[0161] To a stirred solution of compound-46 (1.8 g, 1.81 mmol, 1 eq) in distilled trimethyl phosphate (1.7 mL, 14.5 mmol, 8 eq) was added distilled phosphorus oxychloride (1.35 mL, 14.5 mmol, 8 eq) at 0 °C and a clear solution was observed after 3 h. The progress of the reaction was monitored by LCMS and after complete consumption of starting material, the reaction mixture was quenched with 1 M triethylammonium bicarbonate (TEAB) and then lyophilized. The obtained semi-pure product was purified by C18 reverse phase preparative HPLC eluting with 1 M aqueous ammonium bicarbonate and 15-20% ACN. The collected pure fractions were lyophilized to afford compound-47. LCMS m / z: 1070.9 (M+H) + .

[0162] Step-5: Synthesis of 2-amino-9-((2R,3R,4S,5R)-5-((((((((((2R,3R,4R,5R)-3-(((R)-(((2R,3R,4R,5R)-5-(2-benzoylamino-6-oxo-l,6-dihydro-9H-purin-9-yl)-3,4- bis(isobutyryloxy)tetrahydrofuran-2-yl)methoxy)(hydroxy)thiophosphoryl)oxy)-5-(6- benzoylamino-9H-purin-9-yl)-4-methoxytetrahydrofuran-2-yl)methoxy)(hydroxy)phosphoryl)oxy)(hydroxy)phosphoryl)oxy)(hydroxy)phosphoryl)oxy)methyl)-3,4-dihydroxytetrahydrofuran-2-yl)-7-methyl-6-oxo- 6,9-dihydro-lH-purin-7-ium (48)

[0163] To a stirred solution of compound-47 (400 mg, 0.37 mmol, 1 eq) (co-distilled with dry toluene) and m7GDP-lm (308 mg, 0.67 mmol, 1.8 eq) (co-distilled with dry toluene) in dry distillation DMF (10 vol) was added anhydrous ZnCl2(1.25 g, 9.25 mmol, 25 eq) (dried at 60 °C for 4 h at 0.5 mmHg) at 0 °C and allowed to warm to room temperature (25 °C) and then stirred for 16 h. After quenching the reaction mixture with chilled water, then added EDTA solid and the obtained clear solution was lyophilized to remove DMF. After two attempts of purification on a DEAE Sephadex column eluted with 1 M TEAB / ACN and lyophilized, 180 mg of compound 48 was obtained as TEA salt. Compound 48 was isolated after purification by C18 reverse phase column chromatography. LCMS m / z: 1507.9 (M-2H) + .

[0164] Step-6: Synthesis of 2-amino-9-((2R,3R,4S,5R)-5-((((((((((2R,3R,4R,5R)-3-(((R)-(((2R,3S,4R,5R)-5-(2-amino-6-oxo-l,6-dihydro-9H-purin-9-yl)-3,4- dihydroxytetrahydrofuran-2-yl)methoxy)(hydroxy)thiophosphoryl)oxy)-5-(6-amino-9H-purin-9-yl)- 4-methoxytetrahydrofuran-2-yl)methoxy)(hydroxy)phosphoryl)oxy)(hydroxy)phosphoryl)oxy)(hydroxy)phosphoryl)oxy)methyl)-3,4-dihydroxytetrahydrofuran-2-yl)-7-ethyl-6-oxo- 6,9-dihydro-lH-purin-7-ium (Cap 3)

[0165] To compound-48 (41 mg, 0.027 mmol, 1 eq) in 25 mL RBF was added 7 M MeOH.NH3(5 vol * 2 times) at 20 °C via syringe and stirred at same temperature for 24 h. The progress of reaction was monitored by LCMS and worked up after complete consumption of compound-48. After evaporation of volatiles, 41.5 mg of crude compound was obtained which was purified by ion exchange DEAE Sephadex and preparative HPLC to afford cap 3. LCMS and HPLC indicated 93% purity with expected m / z: 1162.1 (M) + .

[0166] Example 4: Synthesis of compound 3 (COMPD 3) Step-1: Synthesis of (2R,3R,4R,5R)-2-(2-benzamido-6-oxo-1,6-dihydro-9H-purin-9-yl)-5-((((S)-(((2R,3R,4R,5R)-5-(6-benzamido-9H-purin-9-yl)-2-(hydroxymethyl)-4-methoxytetrahydrofuran-3-yl)oxy)(hydroxy)thiophosphoryl)oxy)methyl)tetrahydrofuran-3,4-diyl bis(2-methylpropanoate) (41)

[0167] To a stirred solution of compound-22 (Ref: Clean CAP reagent; 3.3 g, 2.55 mmol, 1 eq) in a mixture of ACN:DCM (20 mL, 1:1 ratio) was added trichloroacetic acid (0.83 g, 5.10 mmol, 2 eq) at 0 °C and stirred for 1 h at room temperature. The progress of reaction was monitored by LCMS. After complete consumption of starting material, the reaction mixture was distilled under reduced pressure. The obtained crude solid material was purified by C18 reverse phase preparative HPLC using 1 M ammonium bicarbonate buffer solution and 40% ACN. The collected pure fractions were lyophilized to afford compound-41. LCMS m / z: 991.0 (M+H) + .

[0168] Step-2: Synthesis of (2R,3R,4R,5R)-2-(2-benzamido-6-oxo-l,6-dihydro-9H-purin-9-yl)-5-((((S)-(((2R,3R,4R,5R)-5-(6-benzamido-9H-purin-9-yl)-4-methoxy-2- (phosphonooxy)methyl)tetrahydrofuran-3-yl)oxy)(hydroxy)thiophosphoryl)oxy)methyl)tetrahydrofuran-3,4-diyl bis(2-methylpropanoate) (42)

[0169] To a stirred solution of compound-41 (1.6 g, 1.61 mmol, 1 eq) in distilled trimethyl phosphate (1.08 mL, 9.28 mmol, 8 eq) at 0 °C, distilled phosphorus oxychloride (1.5 mL, 16.16 mmol, 10 eq) was added over a period of 3 h after which a clear solution was observed. The progress of the reaction was monitored by LCMS and after complete consumption of the starting material, the reaction mixture was quenched with 1 M triethylammonium bicarbonate (TEAB) and then purified by C18 reverse phase column chromatography eluting with 1 M aqueous ammonium bicarbonate and 15-20% ACN. The collected desired fractions were lyophilized to afford compound-42. LCMS m / z: 1071.0 (M+H) + .

[0170] Step-1: Synthesis of 2-amino-9-((2R,3R,4S,5R)-5-((((((((((2R,3R,4R,5R)-3-(((S)-(((2R,3R,4R,5R)-5-(2-benzamido-6-oxo-l,6-dihydro-9H-purin-9-yl)-3,4-bis(isobutyryloxy)tetrahydrofuran-2-yl)methoxy)(hydroxy)thiophosphoryl)oxy)-5-(6-benzamido-9H-purin-9-yl)-4-methoxytetrahydrofuran-2-yl)methoxy)(hydroxy)phosphoryl)oxy)(hydroxy)phosphoryl)oxy)(hydroxy)phosphoryl)oxy)methyl)-3,4-dihydroxytetrahydrofuran-2-yl)-7-ethyl-6-oxo-6,9-dihydro-lH-purin-7-ium (49)

[0171] To a stirred solution of compound-42 (390 mg, 0.364 mmol, 1 eq) (co-distilled with dry toluene) and Et7GDP-lm (342 mg, 0.656 mmol, 1.8 eq) (co-distilled with dry toluene) in dry distillation DMF (10 vol.) was added anhydrous ZnCl2(1.26 g, 9.1 mmol, 25 eq) (dried at 60 °C for 4 h at 0.5 mmHg) at 0 °C and allowed to warm to room temperature (25 °C) and stirred for 48 h.

[0172] Stage-1 (workup) : The reaction mixture was diluted with cold water (10 mL) and EDTA solid and the obtained clear solution was lyophilized to remove DMF.

[0173] Stage-2 (DEAE Sephadex purification) : The obtained crude compound was purified by DEAE Sephadex eluting with 50% ACN and 1 M TEAB in water. The collected pure fractions were lyophilized to obtain semi-pure compound-49.

[0174] Stage-3 (C18 purification) : The above obtained semi-pure compound was further purified by C18 reverse phase column chromatography eluting with 35% ACN and 1 M ABC in water to provide pure compound-49. LCMS indicated 89% of desired m / z.

[0175] Step-2: Synthesis of 2-amino-9-((2R,3R,4S,5R)-5-((((((((((2R,3R,4R,5R)-3-(((S)-(((2R,3S,4R,5R)-5-(2-amino-6-oxo-l,6-dihydro-9H-purin-9-yl)-3,4- dihydroxytetrahydrofuran-2-yl)methoxy)(hydroxy)thiophosphoryl)oxy)-5-(6-amino-9H-purin-9-yl)- 4-methoxytetrahydrofuran-2-yl)methoxy)(hydroxy)phosphoryl)oxy)(hydroxy)phosphoryl)oxy)(hydroxy) phosphonyl)oxy)methyl)-3,4-dihydroxytetrahydrofuran-2-yl)-7-ethyl-6-oxo-6,9-dihydro-lH-purin-7-ium (Compd 3)

[0176]

[0177] To compound-49 (40 mg, 0.026 mmol, 1 eq) was added 7 M MeOH.NH3 (5 vol * 2 times) at 20 °C and stirred for 36 h at the same temperature. The progress of the reaction was monitored by LCMS and worked up after complete consumption of compound-49.

[0178] Workup The reaction mixture was flushed with nitrogen and then concentrated under reduced pressure to provide semi-pure compound Compd 3.

[0179] DEAE Sephadex purification The obtained semi-pure Compd 3 was further purified by DEAE Sephadex eluting with 2-3% ACN and 1 M TEAB in water. The collected pure fractions were lyophilized to provide Compd 3 as TEA salt.

[0180] Preparative HPLC purification The obtained semi-pure Compd 3 was further purified by preparative HPLC using 1 M ABC in water and 10% ACN: 30% MeOH: 60% water mixture to provide pure Compd 3. LCMS indicated 98% purity with expected m / z: 1176.0 (M) + .

[0181] Example 5: Synthesis of compound 4 (COMPD 4) Step-1: Synthesis of N-(9-((2R,3R,4R,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-3-methoxy-4-(((2R,3aS,6R,7aS)-3a-methyl-6-(prop-1-en-2-yl)-2-thioxahexahydrobenzo[d][1,3,2]oxathia phosphol-2-yl)oxy)tetrahydrofuran-2-yl)-9H-purin-6-yl)benzamide (Int-6A)

[0182] To a stirred solution of Int-2 (Ref: CleanCAP reagent; 10 g, 14.5 mmol, 1 eq) and (+)-F-PSI reagent (7.1 g, 15.95 mmol, 1.1 eq) in dry ACN (100 mL), a solution of DBU (1.4 g, 21.75 mmol, 1.5 eq) in acetonitrile (15 mL) was added and stirring was continued for 45 min. After complete consumption of (+)-PSI was monitored by TLC, the reaction mixture was distilled to reduce the acetonitrile volume to max 20 mL. The mixture was purified by silica gel (100-200 mesh) column chromatography eluting with 30% EtOAc in hexane to provide compound-6A. TLC system: (30:70), Rf value: ~0.7; LCMS m / z: 933.9 (M+H) EtOAc / hexanes (30:70), R f value: ~0.7; LCMS m / z: 933.9 (M+H) + .

[0183] Step-2: Synthesis of (2R,3R,4R,5R)-2-(2-benzamido-6-oxo-l,6-dihydro-9H-purin-9-yl)- 5-((((R)-(((2R,3R,4R,5R)-5-(6-benzamido-9H-purin-9-yl)-2-((bis(4- methoxyphenyl)(phenyl)methoxy)methyl)-4-methoxytetrahydrofuran-3-yl)oxy)(hydroxy) thiophosphoryl)oxy)methyl)tetrahydrofuran-3,4-diyl bis(2-methylpropanoate) (45)

[0184] To a stirred solution of Int-6A (10 g, 10.71 mmol, 1 eq) (co-distilled with dry ACN) and Nuc-C2 (7.3 g, 13.92 mmol, 1.3 eq) (co-distilled with dry ACN) in dry distillation ACN (70 mL) was added drop wise dry distillation DBU (2 mL, 16.06 mmol, 1.5 eq) in acetonitrile (15 mL) at 0°C and stirring was continued for 1 h. After complete consumption of starting material as per TLC, the reaction mixture was distilled to reduce the acetonitrile volume to max 10 mL. The obtained crude solution was purified by C18 / liquid injection reverse phase column chromatography eluted with 1 M aqueous ammonium bicarbonate (ABC) and 40% ACN and the collected pure fractions were lyophilized to afford compound-45. LCMS m / z: 1291.4 (M-2H) + .

[0185] Step-3: Synthesis of (2R,3R,4R,5R)-2-(2-benzamido-6-oxo-l,6-dihydro-9H-purin-9-yl)- 5-((((R)-(((2R,3R,4R,5R)-5-(6-benzamido-9H-purin-9-yl)-2-(hydroxymethyl)-4- methoxytetrahydrofuran-3-yl)oxy)(hydroxy)thiophosphoryl)oxy)methyl)tetrahydrofuran- 3,4-diyl bis(2-methylpropanoate) (46)

[0186] To a stirred solution of compound-45 (4.1 g, 3.17 mmol, 1 eq) in a mixture of ACN:DCM (20 mL, 1:1 ratio) was added trichloroacetic acid (1.03 g, 6.34 mmol, 2 eq) at 0 °C and stirred for 1 h at room temperature. The progress of the reaction was monitored by LCMS. After complete consumption of starting material, the reaction mixture was distilled under reduced pressure. The obtained semi-pure solid material was purified by C18 reverse phase preparative HPLC using 1 M ammonium bicarbonate buffer solution and 40% ACN. The collected pure fractions were lyophilized to afford compound-46. LCMS m / z: 989.1 (M-H) + .

[0187] Step-4: Synthesis of (2R,3R,4R,5R)-2-(2-benzamido-6-oxo-1,6-dihydro-9H-purin-9-yl)-5-((((R)-(((2R,3R,4R,5R)-5-(6-benzamido-9H-purin-9-yl)-4-methoxy-2- (phosphoryloxy)methyl)tetrahydrofuran-3-yl)oxy)(hydroxy)thiophosphoryl)oxy)methyl)tetrahydrofuran-3,4-diyl bis(2-methylpropanoate) (47)

[0188] To a stirred solution of compound-46 (1.8 g, 1.81 mmol, 1 eq) in distilled trimethyl phosphate (1.7 mL, 14.5 mmol, 8 eq) was added distilled phosphoryl chloride (1.35 mL, 14.5 mmol, 8 eq) at 0 °C and a clear solution was observed after 3 h. The progress of the reaction was monitored by LCMS and after complete consumption of starting material, the reaction mixture was quenched with 1 M triethylammonium bicarbonate (TEAB) and then lyophilized. The obtained semi-pure product was purified by C18 reverse phase preparative HPLC eluting with 1 M aqueous ammonium bicarbonate and 15-20% ACN. The collected pure fractions were lyophilized to afford compound-47. LCMS m / z: 1070.9 (M+H) + .

[0189] Step-1: Synthesis of 2-amino-9-((2R,3R,4S,5R)-5-((((((((((2R,3R,4R,5R)-3-(((R)-(((2R,3R,4R,5R)-5-(2-benzamido-6-oxo-1,6-dihydro-9H-purin-9-yl)-3,4-bis(isobutyryloxy)tetrahydrofuran-2-yl)methoxy)(hydroxy)thiophosphoryl)oxy)-5-(6-benzamido-9H-purin-9-yl)-4-methoxytetrahydrofuran-2-yl)methoxy)(hydroxy)phosphoryl)oxy)(hydroxy)phosphoryl)oxy)(hydroxy)phosphoryl)oxy)methyl)-3,4-dihydroxytetrahydrofuran-2-yl)-7-ethyl-6-oxo-6,9-dihydro-1H-purin-7-ium (50)

[0190] To a stirred solution of compound-47 (400 mg, 0.373 mmol, 1 eq) (co-distilled with dry toluene) and Et7GDP-lm (351 mg, 0.672 mmol, 1.8 eq) (co-distilled with dry toluene) in dry distillation DMF (10 vol.) was added anhydrous ZnCl2(1.26 g, 9.35 mmol, 25 eq) (dried at 60 °C for 4 h at 0.5 mmHg) at 0 °C and allowed to warm to room temperature (25 °C) and then stirred for 48 h.

[0191] Stage-1 (workup) : The reaction mixture was diluted with cold water (10 mL) and EDTA solid and the obtained clear solution was lyophilized to remove DMF to provide crude material.

[0192] Stage-2 (DEAE Sephadex purification) : The obtained crude compound was purified by DEAE Sephadex eluting with 60% ACN and 1 M TEAB in water. The collected pure fractions were lyophilized to obtain semi-pure compound-50.

[0193] Stage-3 (C18 purification) : The above obtained semi-pure compound was further purified by C18 reverse phase column chromatography eluting with 55% ACN and 1 M ABC in water to provide pure compound-50. LCMS indicated 92% of desired m / z.

[0194] Step-2: Synthesis of 2-amino-9-((2R,3R,4S,5R)-5-((((((((((2R,3R,4R,5R)-4- (((((2R,3S,4R,5R)-5-(2-amino-6-oxo-l,6-dihydro-9H-purin-9-yl)-3,4- dihydroxytetrahydrofuran-2-yl)methoxy)(hydroxy)phosphoryl)oxy)-5-(6-amino-9H- purin-9-yl)-3-methoxytetrahydrofuran-2-yl)methoxy)(hydroxy)phosphoryl)oxy)(hydroxy)phosphoryl)oxy)(hydroxy)phosphoryl)oxy)methyl)-3,4- dihydroxytetrahydrofuran-2-yl)-7-ethyl-6-oxo-6,9-dihydro-lH-purin-7-ium (Compd 4)

[0195] To compound-50 (41 mg, 0.053 mmol, 1 eq) was added 7 M MeOH.NH3(5 vol * 2 times) at 20 °C and stirred at same temperature for 24 h. The progress of reaction was monitored by LCMS and worked up after complete consumption of compound-50.

[0196] Workup : The reaction mixture was flushed with nitrogen and then concentrated under reduced pressure to provide semi-pure Compd 4.

[0197] DEAE Sephadex purification : The obtained semi-pure Compd 4 was further purified by DEAE Sephadex by eluting with 2-3% ACN and 1 M TEAB in water. The collected pure fractions were lyophilized to provide Compd 4 as TEA salt.

[0198] Preparative HPLC purification : The obtained semi-pure Compd 4 was further purified by preparative HPLC using 1 M ABC in water and 10% ACN: 30% MeOH: 60% water mixture to provide pure Compd 4. LCMS indicated 97% purity with expected m / z: 1157.7 (M) + .

[0199] Example 6: Relative yield, expression and dsRNA of compound 1 Experiments were performed as described above in the Materials and Methods section.

[0200] As shown in Figures 1A-1D , Cap 1, Cap 2 and Cap 3 all have the standard Me7G structure; Compound 1, Compound 3 and Compound 4 have the new Et7G structure; and Compound 2 has an aromatic substitution at this position.

[0201] Compound 1 showed comparable yield and in vivo expression to Cap 1, but produced significantly lower dsRNA levels compared to the Me7G Cap 1 structure.

[0202] Data show that trinucleotides modified with an ethyl group at the 7G position (compounds 1, 3, and 4) were successfully incorporated into IVT, but larger modifications at that position (such as aromatic substitutions) were not incorporated into IVT (see compounds 1 and 2).

[0203] Example 7: Relative yield, expression, and dsRNA of compound 3 The experiment was conducted as described in the Materials and Methods section above.

[0204] like Figures 2A-2D As shown, compared with the Me7G-containing phosphate-thiophosphate-modified caps (cap 2 and cap 3), the Et7G modification on the phosphate-thiophosphate-modified caps (compound 3 and compound 4) showed similar IVT yields, but better expression and lower dsRNA levels.

[0205] Example 8: Evaluation of cap immunogenicity (in vitro) through multiple screening Fresh human dendritic cells (differentiated from PBMCs via GM-CSF+IL-4 stimulation) were seeded at 5e4 cells per well in flat, TC-treated 96-well plates. mRNA was prepared at 2 ng / 1k cells using Lipofectamine MessengerMAX transfection reagent, following the manufacturer's instructions. In short, the mRNA was diluted to 0.02 μg / μl in Opti-MEM serum-depleted medium and mixed with MessengerMax 1:1 to a concentration of 0.01 μg / μl. Two 1:10 dilutions were performed, and 10 μl or 2.5 μl of each dilution was added directly to the resulting doses (2 ng, 0.5 ng, 0.2 ng, 0.05 ng, and 0.02 ng / 1000 DCs) per well. Immunostimulatory RIG-I agonist hairpin RNA (3p-hpRNA, InvivoGen) and double-stranded RNA (5'ppp-dsRNA, InvivoGen) were used as controls and were formulated as described above.

[0206] Stimulated DCs were incubated at 37°C and 5% CO2 for 24 hours. Supernatants were collected and immediately frozen at -80°C. They were subsequently thawed and prepared for Luminex xMAP analysis using the Human Inflammation 20-Plex ProcartaPlex Panel kit (ThermoFisher) according to the manufacturer’s instructions. Data were acquired on a MAGPIX system (Luminex xMAP instrument), and pg / mL values for each analyte were derived from standard curves generated with kit components. Values outside the range of the standard curve were reported as mean fluorescence intensity (MFI).

[0207] As shown in FIG. 3, compound 3 showed lower immunogenicity than cap 1 on key cytokine markers, including IFNy, IFNa, IP-10, IL-1a, and TNFa. Figures 3A-3F As shown in FIG. 3, compound 3 showed lower immunogenicity than cap 1 on key cytokine markers, including IFNy, IFNa, IP-10, IL-1a, and TNFa.

Claims

1. A compound of formula (I) or a stereoisomer, tautomer, or pharmaceutically acceptable salt thereof: in X is O or S; R 1 It is an OH or polynucleotide, and R 2 It is H or Me.

2. The compound, stereoisomer, tautomer, or salt according to claim 1, wherein X is O.

3. The compound, stereoisomer, tautomer, or salt according to claim 1, wherein X is S.

4. The compound, stereoisomer, tautomer, or salt according to any one of claims 1 to 3, wherein R 1 It is OH.

5. The compound, stereoisomer, tautomer, or salt according to any one of claims 1 to 4, wherein R 2 It's H.

6. The compound, stereoisomer, tautomer, or salt according to any one of claims 1 to 4, wherein R 2 It's me.

7. The compounds shown in Table 1 or their stereoisomers, tautomers or pharmaceutically acceptable salts.

8. An RNA molecule, wherein the 5' end of said RNA molecule comprises a compound, stereoisomer, tautomer, or salt according to any one of claims 1 to 7.

9. The RNA molecule according to claim 8, wherein the RNA molecule is mRNA.

10. The RNA molecule of claim 9, wherein the mRNA encodes one or more immunomodulators (e.g., checkpoint inhibitors such as anti-PD-1 antibodies, anti-PDL-1 antibodies, anti-CTLA4, etc., immunosuppressive antagonists, pro-inflammatory agents / cytokines), therapeutic proteins, antibodies, or antigens (e.g., tumor-specific antigens).

11. The RNA molecule according to any one of claims 8 to 10, wherein the RNA molecule has a half-life greater than that of the corresponding native RNA molecule in the cellular environment.

12. The RNA molecule according to any one of claims 8 to 11, compared with an RNA molecule having a 5' end that does not contain a compound, stereoisomer, tautomer or salt according to any one of claims 1 to 7, exhibits reduced formation of double-stranded RNA.

13. A pharmaceutical product comprising an RNA molecule according to any one of claims 8 to 12 and one or more pharmaceutically acceptable excipients.

14. A kit for capping RNA molecules, comprising a compound, stereoisomer, tautomer or salt according to any one of claims 1 to 7, and RNA polymerase.

15. The kit according to claim 14, wherein the RNA molecule is mRNA.

16. A method for preparing capped RNA molecules from a polynucleotide template by in vitro transcription, the method comprising: (a) Combining more than one nucleotide, the polynucleotide template and RNA polymerase to produce a reaction mixture; (b) Incubate the reaction mixture; and (c) Adding the compound, stereoisomer, tautomer or salt according to any one of claims 1 to 7 to the mixture to produce the capped RNA molecule.

17. The method of claim 16, wherein the RNA molecule is mRNA.

18. The method of claim 16 or claim 17, wherein at least a portion of the more than one nucleotide is unmodified.

19. The method according to any one of claims 16 to 18, wherein at least a portion of the more than one nucleotide is modified.

20. The method according to any one of claims 16 to 19, wherein the polynucleotide template is a DNA template.

21. The method according to any one of claims 16 to 20, wherein the capping efficiency is greater than about 95%.

22. The method according to any one of claims 16 to 21, wherein the translation yield is greater than about 75%.

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