Acetylated ribonucleic acid and uses thereof

Abstract

Disclosed herein are modified ribonucleotides comprising nucleosides comprising 2'-O-acetylated ribose, and polyribonucleotides comprising the same. Also provided herein are compositions comprising the polyribonucleotides of the disclosure, as well as methods for making and using the same. The disclosure identifies certain challenges associated with making RNA for use in applications, such as therapeutic applications, and challenges associated with using RNA as a therapeutic agent. For example, in some embodiments, the disclosure identifies certain problems that may be encountered when using currently available RNA as a therapeutic agent, for example, due to the temporal activity and / or instability of RNA.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 63 / 338,429, filed May 4, 2022, the entire contents of which are incorporated herein by reference. [Background technology]

[0002] RNA therapeutics is a new and emerging field. Summary of the Invention [Means for solving the problem]

[0003] The present disclosure identifies certain challenges in the generation of RNA for use in applications, such as therapeutic applications, and in the use of RNA as a therapeutic agent. For example, in some embodiments, the present disclosure identifies certain problems that may be encountered in using currently available RNA as a therapeutic agent, for example, due to the temporal activity and / or instability of RNA. In some embodiments, the instability of RNA may be due, for example, to the reactivity of the hydroxyl group on the 2-carbon of the ribose of ribonucleotides.

[0004] In particular, the present disclosure provides technology related to 2-O-acetylated nucleotides, in which the hydroxyl group on the 2-carbon of ribose is acetylated (2-O-acetylated ribose). Without wishing to be bound by theory, in some embodiments, ribonucleotides with 2-O-acetylated ribose can maintain the crucial hydrogen bonding interactions required for transcription, translation, and duplex formation, while at the same time reducing reactivity that would otherwise promote autohydrolysis and nuclease degradation of RNA. In some embodiments, ribonucleotides with 2-O-acetylated ribose are more stable than comparable RNAs with fewer 2-O-acetyl groups on the ribose.

[0005] Among the techniques provided by the present disclosure are techniques for using 2-O-acetylated nucleotide triphosphates as reagents for in vitro transcription of polyribonucleotides by RNA polymerase. In some embodiments, the RNA polymerase can use the 2-O-acetylated nucleotide triphosphates to generate 2-O-acetylated polyribonucleotides that can be translated by the intracellular machinery of a cell. In some embodiments, the 2-O-acetylated polyribonucleotides can be used for a variety of applications, including therapeutic applications, research applications, diagnostic applications, agricultural applications, and any other suitable applications.

[0006] The present disclosure also provides techniques for reducing the immunogenicity of RNA therapeutics by providing modified ribonucleotides, e.g., polyribonucleotides comprising 2-O-acetylated ribose. Without wishing to be bound by theory, the present disclosure proposes that when polyribonucleotides comprising 2-O-acetylated ribose are administered to a cell, tissue, or subject, reduced immunogenicity may be achieved by reduced activation of innate immune response. In some embodiments, reduced activation of innate immune response, e.g., reduced activation of NF-kb or NF-kb pathway, IRF or IRF pathway, and / or other inflammatory cytokines, or reduced detection of uncapped RNA by molecular sensors (e.g., RIG-I), by polyribonucleotides comprising 2-O-acetylated ribose or compositions comprising same, allows, for example, repeated administration of the polyribonucleotide or compositions comprising same, e.g., at least two administrations. In some embodiments, a polyribonucleotide comprising 2-O-acetylated ribose, or a composition comprising same, may be administered at a higher dose compared to a reference polyribonucleotide that comprises fewer 2'-O-acetyl groups on the ribose.

[0007] Also provided herein is a technique for increasing expression from RNA therapeutics by providing polyribonucleotides that include modified ribonucleotides, such as ribonucleotides that include 2-O-acetylated ribose.Without wishing to be bound by theory, the present disclosure proposes that polyribonucleotides that include 2-O-acetylated ribose can achieve increased levels of protein or polypeptide expression when administered to cells, tissues or subjects, when compared to administration of a comparable polyribonucleotide that has fewer 2'-O-acetyl groups on ribose.

[0008] Further provided herein is a technique for increasing the persistence of RNA therapeutics by providing a polyribonucleotide comprising modified ribonucleotides, e.g., ribonucleotides comprising 2-O-acetylated ribose. Without wishing to be bound by theory, the present disclosure proposes that a polyribonucleotide comprising 2-O-acetylated ribose may achieve increased persistence when administered to a cell, tissue, or subject, when compared to administration of a comparable polyribonucleotide having fewer 2'-O-acetyl groups on the ribose. In some embodiments, the increased persistence is the result of increased resistance to one or more nucleases. In some embodiments, the increased persistence may also allow for reduced dosing frequency and / or reduced doses of modified ribonucleotides, e.g., polyribonucleotides comprising ribonucleotides comprising 2-O-acetylated ribose.

[0009] Also provided herein are compositions comprising polyribonucleotides containing 2-O-acetylated ribose, as well as methods of making and using the same.

[0010] The present disclosure provides modified ribonucleotides comprising a nucleoside, the nucleoside comprising a ribose moiety comprising an acetyl group, the ribose being 2'-O-acetylated. In some embodiments, the modified ribonucleotide comprises the following: [ka] The structure is

[0011] (a) wherein X is a 5' monophosphate, a 5' diphosphate, or a 5' triphosphate;

[0012] (b) R is a nucleobase selected from adenine or a modified form thereof, guanine or a modified form thereof, cytosine or a modified form thereof, or uracil or a modified form thereof.

[0013] In some embodiments, the modified ribonucleotide comprises a 5' triphosphate and the following: [ka] The structure is

[0014] wherein R is a nucleobase selected from adenine or a modified form thereof, guanine or a modified form thereof, cytosine or a modified form thereof, or uracil or a modified form thereof.

[0015] In some embodiments, the nucleobase is adenine. In some embodiments, the polyribonucleotide comprising adenine has a 5' triphosphate and the following: [ka] It has the structure:

[0016] In some embodiments, the nucleobase is guanine. In some embodiments, the polyribonucleotide comprising guanine has a 5' triphosphate and the following: [ka] It has the structure:

[0017] In some embodiments, the nucleobase is cytosine. In some embodiments, the polyribonucleotide comprising cytosine has a 5' triphosphate and the following: [ka] It has the structure:

[0018] In some embodiments, the nucleobase is uracil. In some embodiments, the polyribonucleotide comprises a 5' triphosphate and the following: [ka] It has the structure:

[0019] Also provided herein is a polyribonucleotide that comprises one or more modified ribonucleotides, such as ribonucleotides that comprise 2-O-acetylated ribose. In some embodiments, the polyribonucleotide comprises a plurality of ribonucleotides selected from adenine, guanine, cytosine, or uracil, or any combination thereof.

[0020] In some embodiments, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least about 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% of the ribose moieties in the polyribonucleotide are acetylated (2'-O-acetylated).

[0021] In some embodiments, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% of the ribose moieties in the polyribonucleotide are acetylated (2'-O-acetylated).

[0022] In some embodiments, at least about 95% of the ribose moieties are 2'-O-acetylated.

[0023] In some embodiments, 100% of the ribose moieties in the polyribonucleotide are acetylated (2'-O-acetylated).

[0024] In some embodiments, the polyribonucleotide comprises a cap structure. In some embodiments, the cap structure does not comprise 2'-O-acetylated ribose. In some embodiments, the cap structure comprises 2'-O-acetylated ribose.

[0025] In some embodiments, the polyribonucleotides comprising 2-O-acetylated ribose (e.g., comprising an adenine with 2-O-acetylated ribose, a guanine with 2-O-acetylated ribose, a cytosine with 2-O-acetylated ribose, and / or a uracil with 2-O-acetylated ribose) further comprise modifications including a modified backbone, a modified nucleobase, or any combination thereof.

[0026] In some embodiments, polyribonucleotides that comprise 2-O-acetylated ribose (e.g., adenine with 2-O-acetylated ribose, guanine with 2-O-acetylated ribose, cytosine with 2-O-acetylated ribose, and / or uracil with 2-O-acetylated ribose) further comprise a modified backbone.

[0027] In some embodiments, the polyribonucleotide comprising 2-O-acetylated ribose (e.g., comprising adenine comprising 2-O-acetylated ribose, guanine comprising 2-O-acetylated ribose, cytosine comprising 2-O-acetylated ribose, and / or uracil comprising 2-O-acetylated ribose) further comprises a modified nucleobase. In some embodiments, the nucleobase comprising a modification is selected from adenine, guanine, cytosine, or uracil. In some embodiments, the nucleobase comprising a modification is an adenine. In some embodiments, the nucleobase comprising a modification is a cytosine. In some embodiments, the nucleobase comprising a modification is a guanine. In some embodiments, the nucleobase comprising a modification is uracil.

[0028] In some embodiments, the nucleobase modification comprises a modification known in the art or as disclosed herein. In some embodiments, the nucleobase modification comprises N4-acetyl-cytidine (ac4C), 5-hydroxymethyluridine (5-hmU), N1-methylpseudouridine, pseudouridine, pyridin-4-one ribonucleoside, 5-aza-uridine, 6-aza-uridine, 2-thio-5-aza-uridine, 2-thio-uridine (s2U), 5-methylcytidine (m5C), 5-aza-cytidine, 6-aza-cytidine, 5-methyl ... cytidine, pseudoisocytidine, 3-methyl-cytidine (m3C), 5-formyl-cytidine (f5C), N4-methyl-cytidine (m4C), 2-amino-purine, 2,6-diaminopurine, 2-amino-6-halo-purine, 6-halo-purine, inosine (I), 1-methyl-inosine (m1I), wyosine (imG), methyl wyosine (mimG), or any combination thereof.

[0029] In some embodiments, the polyribonucleotide comprising 2-O-acetylated ribose (e.g., comprising an adenine comprising 2-O-acetylated ribose, a guanine comprising 2-O-acetylated ribose, a cytosine comprising 2-O-acetylated ribose, and / or a uracil comprising 2-O-acetylated ribose) further comprises a nucleoside comprising an acetyl group, wherein the nucleoside is N4-acetylcytidine, and the modified ribonucleotide is any of the following: [ka] It has the structure:

[0030] In some embodiments, the polyribonucleotide comprises cytidine residues, and the cytidine residues in the polyribonucleotide are about 5% to 99%, about 5% to 95%, about 5% to 90%, about 5% to 85%, about 5% to 80%, about 5% to 75%, about 5% to 70%, about 5% to 65%, about 5% to 60%, about 5% to 55%, about 5% to 50%, about 5% to 45%, about 5% to 40%, about 5% to 35%, about 5% to 30%, about 5% to 25%, about 5% to 20%. , about 5% to 15%, about 5% to 10%, about 10% to 99%, about 15% to 99%, about 20% to 99%, about 25% to 99%, about 30% to 99%, about 35% to 99%, about 40% to 99%, about 45% to 99%, about 50% to 99%, about 55% to 99%, about 60% to 99%, about 65% to 99%, about 70% to 99%, about 80% to 99%, about 85% to 99%, about 90% to 99%, or about 95% to 99% contain N4-acetylcytidine.

[0031] In some embodiments, the polyribonucleotide comprises cytidine residues, and at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% of the cytidine residues in the polyribonucleotide comprise N4-acetylcytidine.

[0032] In some embodiments, the polyribonucleotide comprising 2-O-acetylated ribose (e.g., adenine comprising 2-O-acetylated ribose, guanine comprising 2-O-acetylated ribose, cytosine comprising 2-O-acetylated ribose, and / or uracil comprising 2-O-acetylated ribose) further comprises one or more modified ribonucleotides comprising a hydroxymethyl group. In some embodiments, the one or more modified ribonucleotides comprise a nucleoside selected from adenosine, guanosine, cytidine, or uridine, or any combination thereof. In some embodiments, the nucleoside of the one or more modified ribonucleotides is 5-hydroxymethyluridine, and the modified ribonucleotide comprises one of the following: [ka] It has the structure:

[0033] In some embodiments, the polyribonucleotide comprises uridine residues, and the uridine residues in the polyribonucleotide are about 5% to 99%, about 5% to 95%, about 5% to 90%, about 5% to 85%, about 5% to 80%, about 5% to 75%, about 5% to 70%, about 5% to 65%, about 5% to 60%, about 5% to 55%, about 5% to 50%, about 5% to 45%, about 5% to 40%, about 5% to 35%, about 5% to 30%, about 5% to 25%, about 5% to 20%, about 5% to 15%, about 5% to 10%, about 10% to 99%, about 15% to 99%, about 20% to 99%, about 25% to 99%, about 30% to 99%, about 35% to 99%, about 40% to 99%, about 45% to 99%, about 50% to 99%, about 55% to 99%, about 60% to 99%, about 65% to 99%, about 70% to 99%, about 80% to 99%, about 85% to 99%, about 90% to 99%, or about 95% to 99% contains 5-hydroxymethyluridine.

[0034] In some embodiments, the polyribonucleotide comprises uridine residues, and at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% of the uridine residues in the polyribonucleotide comprise 5-hydroxymethyluridine.

[0035] The present disclosure also provides a method for producing polyribonucleotides comprising 2-O-acetylated ribose. In some embodiments, disclosed herein is a method for producing polyribonucleotides comprising incubating an in vitro transcription mixture, the in vitro transcription mixture comprising (i) a DNA template, (ii) at least one RNA polymerase or a variant or fragment thereof, and (iii) a plurality of ribonucleotides comprising at least one modified ribonucleotide comprising 2'-O-acetylated ribose, thereby producing polyribonucleotides comprising 2'-O-acetylated ribose.

[0036] Also disclosed herein is an in vitro transcription mixture useful for generating polyribonucleotides comprising 2'-O-acetylated ribose. In some embodiments, disclosed herein is an in vitro transcription mixture comprising (i) a DNA template, (ii) at least one RNA polymerase or a variant or fragment thereof, and (iii) a plurality of ribonucleotides comprising at least one modified ribonucleotide comprising 2'-O-acetylated ribose.

[0037] In some embodiments, the method or in vitro transcription mixture generates a plurality of polyribonucleotides. In some embodiments, each polyribonucleotide in the plurality of polyribonucleotides comprises a 2'-O-acetylated ribose.

[0038] In some embodiments, the polyribonucleotides produced using the methods or in vitro transcription mixtures disclosed herein further comprise one or more modifications, for example, one or more base modifications.

[0039] In some embodiments, the RNA polymerase is selected from bacteriophage RNA polymerase, mitochondrial RNA polymerase, eukaryotic RNA polymerase, bacterial RNA polymerase, or any combination thereof. In some embodiments, the RNA polymerase comprises T7 RNA polymerase, T3 RNA polymerase, SP6 RNA polymerase, viral RNA polymerase, N4 virion RNA polymerase, or a variant of any of the foregoing.

[0040] In some embodiments, the polyribonucleotides generated by the methods disclosed herein or used in the in vitro transcription mixture include polyribonucleotides that include a coding region.

[0041] In some embodiments, the polyribonucleotides generated by the methods disclosed herein or used in the in vitro transcription mixture comprise polyribonucleotides that do not include coding regions.

[0042] In some embodiments, the polyribonucleotides generated by the methods disclosed herein or using the in vitro transcription mixtures include guide RNA, short hairpin RNA, siRNA, microRNA, long non-coding RNA, or messenger RNA (mRNA), circular RNA, or any combination thereof.

[0043] In some embodiments, the polyribonucleotides generated by the methods disclosed herein or using the in vitro transcription mixture encode a payload. In some embodiments, the payload comprises one or more target polypeptides.

[0044] In some embodiments of the methods of making ribonucleotides disclosed herein, the incubating step is carried out at a temperature of at least 37°C.

[0045] In some embodiments of the methods of making ribonucleotides disclosed herein, the incubation step is performed at a temperature of about 45°C, 46°C, 47°C, 48°C, 49°C, 50°C, 51°C, 52°C, 53°C, 54°C, 55°C, or greater.

[0046] In some embodiments of the methods of making ribonucleotides disclosed herein, the incubation step is carried out for at least 1 hour, at least 2 hours, at least 3 hours, at least 4 hours or more.

[0047] Further provided herein are compositions comprising polyribonucleotides disclosed herein, e.g., polyribonucleotides made according to the methods disclosed herein. In some embodiments, the compositions are or comprise pharmaceutical compositions. In some embodiments, the compositions are pharmaceutical compositions. In some embodiments, the pharmaceutical compositions are or comprise immunogenic compositions. In some embodiments, the pharmaceutical compositions are or comprise antibody therapy. In some embodiments, the pharmaceutical compositions are or comprise immunomodulatory therapy. In some embodiments, the pharmaceutical compositions are or comprise vaccines. In some embodiments, the pharmaceutical compositions are or comprise gene therapy. In some embodiments, the pharmaceutical compositions are or comprise chemotherapy. In some embodiments, the pharmaceutical compositions are or comprise protein replacement therapy. In some embodiments, the pharmaceutical compositions are or comprise immunotherapy. In some embodiments, the pharmaceutical compositions are or comprise cell engineering therapy.

[0048] In some embodiments, the composition comprises double-stranded RNA.

[0049] Also provided herein is a method comprising administering to a cell, tissue, or subject a polyribonucleotide disclosed herein, or a composition comprising the same. In some embodiments, the method further comprises determining cell viability of the cell, tissue, or subject.

[0050] In some embodiments, cell viability is a measure of the length of time that one or more cells of a cell, tissue or subject are alive. In some embodiments, cell viability is a measure of the number of cells of a cell, tissue or subject that are alive at one or more time points. In some embodiments, a cell, tissue or subject that is administered with polyribonucleotide or a composition comprising the same shows improved cell viability when compared to a reference cell viability. In some embodiments, the reference cell viability is the cell viability of a cell, tissue or subject that is administered with a comparable polyribonucleotide or a composition comprising the same that contains less 2'-O-acetylated ribose (e.g., does not contain any 2'-O-acetylated ribose).

[0051] In some embodiments, the method further comprises determining an immune system response of a cell, tissue or subject to which the polyribonucleotide or a composition comprising the same has been administered. In some embodiments, the immune response comprises an innate immune system response comprising an innate immune system-induced toxicity. In some embodiments, determining the innate immune system response comprises determining the levels of NF-κB, IRF, and / or other inflammatory cytokines in the cell, tissue or subject. In some embodiments, the cell, tissue or subject to which the polyribonucleotide or a composition comprising the same has been administered exhibits a reduced innate immune system response when compared to a reference. In some embodiments, the reference is the innate immune system response of a cell, tissue or subject to which an equivalent polyribonucleotide or a composition comprising the same has been administered that comprises less 2′-O-acetylated ribose (e.g., no 2′-O-acetylated ribose).

[0052] In some embodiments, the administration method disclosed herein further comprises determining the effectiveness of the polyribonucleotide or the composition comprising the same in a cell, tissue or subject to which the polyribonucleotide or the composition comprising the same is administered. In some embodiments, determining the effectiveness comprises determining an antibody response or a cellular response in the cell, tissue or subject. In some embodiments, the cell, tissue or subject to which the polyribonucleotide or the composition comprising the same is administered shows an increase in the antibody response or the cellular response when compared to a reference. In some embodiments, the reference is the antibody response or the cellular response of a cell, tissue or subject to which an equivalent polyribonucleotide or a composition comprising the same is administered that contains less 2'-O-acetylated ribose (e.g., does not contain any 2'-O-acetylated ribose).

[0053] In some embodiments, the method comprises administering polyribonucleotide or a composition comprising thereof to a cell, tissue or subject at least twice. In some embodiments, the method comprises administering polyribonucleotide or a composition comprising thereof to a cell, tissue or subject at least twice, at least three times, at least four times, at least five times, at least six times, at least seven times, at least eight times, at least nine times or at least ten times. In some embodiments, administering polyribonucleotide or a composition comprising thereof to a cell, tissue or subject at least twice does not result in a decrease in the efficacy of polyribonucleotide or a composition comprising thereof compared to administering one dose of polyribonucleotide or a composition comprising thereof.

[0054] In some embodiments, the method comprises administering to a cell, tissue or subject a polyribonucleotide or a composition comprising the same at a higher dose than a suitable reference standard. In some embodiments, the reference standard comprises a comparable polyribonucleotide that comprises less 2'-O-acetylated ribose (e.g., no 2'-O-acetylated ribose). In some embodiments, the cell is a mammalian cell, the tissue is a mammalian tissue, or the subject is a mammal.

[0055] In some embodiments, the polyribonucleotides disclosed herein are or comprise RNA oligos.

[0056] In some embodiments, the polyribonucleotide disclosed herein comprises a coding region. In some embodiments, the coding region encodes a gene product. In some embodiments, the gene product is or comprises a polypeptide. In some embodiments, the gene product is or comprises a transcript.

[0057] In some embodiments, the polyribonucleotides disclosed herein do not include coding regions.

[0058] In some embodiments, the polyribonucleotides disclosed herein are or comprise messenger RNA (mRNA).

[0059] In some embodiments, the polyribonucleotide disclosed herein is or comprises a gRNA.

[0060] In some embodiments, the polyribonucleotides disclosed herein are or comprise inhibitory RNA.

[0061] In some embodiments, the polyribonucleotide disclosed herein is or comprises an miRNA or siRNA.

[0062] In some embodiments, the polyribonucleotides disclosed herein are or comprise long non-coding RNA.

[0063] In some embodiments, the polyribonucleotides disclosed herein are or comprise antisense oligonucleotides.

[0064] In some embodiments, the method is a method of stimulating an immune response.

[0065] In some embodiments, the method is a method of vaccination.

[0066] In some embodiments, the method is a method of antibody therapy.

[0067] In some embodiments, the method is a method of immunomodulatory therapy.

[0068] In some embodiments, the method is a method of gene therapy.

[0069] In some embodiments, the methods include delivery of one or more components of a gene therapy, such as a gRNA.

[0070] In some embodiments, the method is a cell therapy engineering method.

[0071] In some embodiments, the method is a method of immunotherapy, hi some embodiments, the method of immunotherapy comprises delivery of an immune modulating therapy and / or an immune checkpoint therapy.

[0072] In some embodiments, the method is a method of protein replacement therapy. In some embodiments, the method of protein replacement therapy comprises the delivery of enzyme replacement therapy.

[0073] In some embodiments, the method is a method of chemotherapy.

[0074] Also provided herein are methods of vaccination comprising administering to a cell, tissue or subject one or more of the polyribonucleotides disclosed herein or a composition comprising same.

[0075] Provided herein are methods of immunotherapy comprising administering to a cell, tissue or subject one or more of the polyribonucleotides disclosed herein or compositions comprising same.

[0076] Provided herein is a method of providing antibody therapy comprising administering one or more of the polyribonucleotides disclosed herein or a composition comprising the same to a cell, tissue or subject. In some embodiments, the antibody therapy comprises an antibody, a fragment, a variant, or a fusion thereof. In some embodiments, the antibody therapy comprises a fragment (e.g., scFv, Fab or other fragment) comprising an antigen recognition domain, or an intact antibody, or a polypeptide comprising an antigen binding specificity fused to an Fc. In some embodiments, the antibody therapy comprises a bispecific antibody, a multispecific antibody, a heterodimeric antibody, a Crossmab, a DVD-Ig, a two-in-one IgG, an IgG-sc-FV, a scFv-scFv, a BiTE, a DART, a diabody, a Fab-scFv fusion, a Fab-Fab fusion, a tandem antibody, or any other antibody format recognized in the art.

[0077] Provided herein is a method for providing an immunomodulatory therapy, comprising administering one or more polyribonucleotides disclosed herein or compositions comprising the same to a cell, tissue or subject. In some embodiments, the immunomodulatory therapy comprises a cytokine or a variant or fragment thereof, a chemokine or a variant or fragment thereof, a T cell regulator, a NK cell regulator, a B cell regulator, a myeloid cell regulator, any other immune cell regulator, or any combination thereof. In some embodiments, the immunomodulatory therapy comprises a chimeric antigen receptor (CAR) therapy. In some embodiments, the immunomodulatory therapy comprises an engineered T cell receptor (TCR) therapy.

[0078] Provided herein are methods of gene therapy comprising administering to a cell, tissue or subject one or more of the polyribonucleotides disclosed herein or compositions comprising same.

[0079] The present disclosure provides methods of protein replacement therapy comprising administering to a cell, tissue or subject one or more of the polyribonucleotides disclosed herein or compositions comprising same.

[0080] Also disclosed herein are methods of cell engineering therapy comprising administering to a cell, tissue or subject one or more of the polyribonucleotides disclosed herein or compositions comprising same.

[0081] The present disclosure provides a method for obtaining a lower level of immunogenicity in a subject receiving a polyribonucleotide comprising modified ribonucleotides or a composition comprising the same, when compared to a subject receiving an equivalent unmodified polyribonucleotide. In some embodiments, the method comprises administering to a subject a polyribonucleotide comprising modified ribonucleotides or a composition comprising the same.

[0082] In some embodiments of any of the methods, uses, or compositions disclosed herein, the polyribonucleotide comprising the modified ribonucleotide does not include a 5' cap, e.g., a 5'-5' triphosphate linked guanosine. In some embodiments, the polyribonucleotide comprising the modified ribonucleotide comprises a 5' phosphate group and / or a hydroxyl group at the 5' end of the polyribonucleotide.

[0083] In some embodiments of any of the methods, uses, or compositions disclosed herein, the polyribonucleotide comprising modified ribonucleotides comprises a 5' cap, for example a 5'-5' triphosphate linked guanosine.

[0084] In some embodiments, the subject receiving a polyribonucleotide comprising modified ribonucleotides or a composition comprising same, and the subject receiving an equivalent unmodified polyribonucleotide are the same subject.

[0085] In some embodiments, the subject receiving a polyribonucleotide comprising a modified ribonucleotide or a composition comprising same, and the subject receiving an equivalent unmodified polyribonucleotide are different subjects.

[0086] Provided herein is a method for producing RNA composition, comprising introducing at least one modified ribonucleotide disclosed herein into polyribonucleotide.In some embodiments, the method does not include removing double-stranded RNA from RNA composition.

[0087] Disclosed herein are cells comprising the polyribonucleotides disclosed herein or compositions comprising same.

[0088] Also disclosed herein is the use of the modified ribonucleotides disclosed herein in making polyribonucleotides.

[0089] The present disclosure provides the use of a polyribonucleotide disclosed herein, or a composition comprising same, in the preparation of a medicament for stimulating an immune response.

[0090] Provided herein is the use of a polyribonucleotide disclosed herein, or a composition comprising same, in the preparation of a medicament for use as a vaccine.

[0091] The present disclosure provides for the use of a polyribonucleotide disclosed herein, or a composition comprising same, in the preparation of a medicament for use as an immunotherapy.

[0092] Provided herein is the use of a polyribonucleotide disclosed herein, or a composition comprising same, in the preparation of a medicament for use as an antibody therapy.

[0093] Provided herein is the use of a polyribonucleotide disclosed herein, or a composition comprising same, in the preparation of a medicament for use as an immunomodulatory therapy.

[0094] Provided herein is the use of a polyribonucleotide disclosed herein, or a composition comprising same, in the preparation of a medicament for use as a gene therapy.

[0095] The present disclosure provides for the use of a polyribonucleotide disclosed herein, or a composition comprising same, in the preparation of a medicament for use as a protein replacement therapy.

[0096] Provided herein is the use of a polyribonucleotide disclosed herein, or a composition comprising same, in the preparation of a medicament for use as a cell engineering therapy.

[0097] The present disclosure provides for the use of a polyribonucleotide disclosed herein, or a composition comprising same, in the preparation of a medicament for use as a chemotherapy.

[0098] The present disclosure provides the use of a polyribonucleotide disclosed herein, or a composition comprising same, in the preparation of a medicament for stimulating an immune response.

[0099] Provided herein are compositions comprising the polyribonucleotides disclosed herein for use as vaccines.

[0100] The present disclosure provides compositions comprising the polyribonucleotides disclosed herein for use as immunotherapeutics.

[0101] Provided herein are compositions comprising the polyribonucleotides disclosed herein for use as antibody therapy.

[0102] Provided herein are compositions comprising the polyribonucleotides disclosed herein for use as immunomodulatory therapies.

[0103] Provided herein are compositions comprising the polyribonucleotides disclosed herein for use as gene therapy.

[0104] The present disclosure provides compositions comprising the polyribonucleotides disclosed herein for use as protein replacement therapy.

[0105] Provided herein are compositions comprising the polyribonucleotides disclosed herein for use as cell engineering therapies.

[0106] The present disclosure provides compositions comprising the polyribonucleotides disclosed herein for use as chemotherapy.

[0107] In some embodiments of any of the uses or methods provided herein, a polyribonucleotide or a composition comprising same is administered to a cell, tissue, or subject.

[0108] In some embodiments of any of the uses or methods provided herein, the cell is a mammalian cell, the tissue is a mammalian tissue, or the subject is a mammal. In some embodiments, the mammal is a human. [Brief description of the drawings]

[0109] [Figure 1A] 1 provides structures of natural base 2-O-acetyl ribonucleotide analogs. [Figure 1B] 1 provides structures of natural base 2-O-acetyl ribonucleotide analogs. [Figure 1C] 1 provides structures of natural base 2-O-acetyl ribonucleotide analogs. [Figure 1D] 1 provides structures of natural base 2-O-acetyl ribonucleotide analogs.

[0110] [Diagram 2] FIG. 1 is a graph showing luciferase gene expression normalized to cell viability by dose of RNA synthesized with the indicated nucleotide composition.

[0111] [Diagram 3] 1 is a graph showing the viability of A549 cells after transfection with each dose of RNA synthesized with the indicated nucleotide composition.

[0112] [Figure 4] 1 is a graph showing IRF reporter activation by dose of RNA synthesized with the indicated nucleotide composition.

[0113] [Diagram 5] 5 is a graph showing NF-KB reporter activation by dose of RNA synthesized with the indicated nucleotide composition.

[0114] [Figure 6] FIG. 1 is a graph showing 1H NMR (300 MHz, DMSO-d6) of silyl-protected cytidine 2.

[0115] [Figure 7] Mass spectrum of silyl-protected cytidine 2 (ESI+, 100% CH3OH, TOF).

[0116] [Figure 8] FIG. 1 is an HPLC chromatogram of silyl-protected cytidine 2 (CHOH).

[0117] [Figure 9] 1 is a graph showing 1H NMR (300 MHz, DMSO-d6) of diacetylated cytidine 3.

[0118] [Figure 10] Mass spectrum (ESI+, 100% CH3OH, TOF) of diacetylated cytidine 3, retention time (4.534).

[0119] [Figure 11] FIG. 1 is an HPLC chromatogram (CHOH) of diacetylated cytidine 3 (an unstable compound on LC-MS).

[0120] [Figure 12] FIG. 1 is a graph showing 1H NMR (300 MHz, DMSO-d6) of 2'-O-acetylated silyl protected cytidine 4.

[0121] [Figure 13] Mass spectrum of 2'-O-acetylated silyl-protected cytidine 4 (ESI+, 100% CH3OH, TOF).

[0122] [Figure 14] FIG. 1 is an HPLC chromatogram (CHOH) of 2′-O-acetylated silyl-protected cytidine 4, an LC-MS unstable compound.

[0123] [Figure 15-1] FIG. 1 is a graph showing 1H NMR (300 MHz, DMSO-d6) of N,O-diacetyl-cytidine 6 (mixture of 2'-OAc and 3'-OAc positional isomers). [Figure 15-2] FIG. 1 is a graph showing 1H NMR (300 MHz, DMSO-d6) of N,O-diacetyl-cytidine 6 (mixture of 2'-OAc and 3'-OAc positional isomers).

[0124] [Figure 16] Mass spectrum (ESI+, 100% CH3OH, TOF) of N,O-diacetyl-cytidine 6 (mixture of 2'-OAc and 3'-OAc positional isomers).

[0125] [Figure 17] FIG. 1 is an HPLC chromatogram (CHOH) of N,O-diacetyl-cytidine 6 (a mixture of 2'-OAc and 3'-OAc positional isomers).

[0126] [Figure 18-1] FIG. 1 is a graph showing 1H NMR (300 MHz, DMSO-d6) of 2'-O-monoacetyl-cytidine 7 (mixture of 2'-OAc and 3'OAc). [Figure 18-2] FIG. 1 is a graph showing 1H NMR (300 MHz, DMSO-d6) of 2'-O-monoacetyl-cytidine 7 (mixture of 2'-OAc and 3'OAc).

[0127] [Figure 19] Mass spectrum of 2'-O-acetyl-cytidine 7 (ESI+, 100% CH3OH, TOF).

[0128] [Figure 20] 1 is a graph showing the HPLC chromatogram of 2'-O-acetyl-cytidine 7 (CHOH).

[0129] [Figure 21] FIG. 1 is a graph showing the H NMR (300 MHz, DO) of N,O-diacetyl-cytidine triphosphate sodium salt 9 (mixture of 2′-OAc and 3′-OAc).

[0130] [Figure 22] FIG. 31P NMR (121 MHz, DO) of N,O-diacetyl-cytidine triphosphate sodium salt 9 (mixture of 2′-OAc and 3′-OAc positional isomers).

[0131] [Diagram 23] FIG. 1 shows the H NMR (300 MHz, DO) of 2-O-acetyl-cytidine triphosphate sodium salt 11 (mixture of 2′-OAc and 3′-OAc positional isomers).

[0132] [Figure 24] FIG. 31P NMR (121 MHz, DO) of 2-O-acetylcytidine triphosphate sodium salt 11 (mixture of 2'-OAc and 3'-OAc positional isomers).

[0133] [Diagram 25] FIG. 1 is a graph showing 1H NMR (300 MHz, DMSO-d6) of silyl-protected uridine 13.

[0134] [Figure 26] Mass spectrum of silyl-protected uridine 13 (ESI+, 100% CH3OH, TOF).

[0135] [Figure 27] HPLC chromatogram of silyl-protected uridine 13 (CHOH).

[0136] [Figure 28] FIG. 1 is a graph showing the 1H NMR (300 MHz, DO) of 2′-O-acetyl-silyl protected uridine 14.

[0137] [Figure 29] Mass spectrum of 2'-O-acetyl-silyl protected uridine 14 (ESI+, 100% CH3OH, TOF).

[0138] [Diagram 30] HPLC chromatogram of 2'-O-acetyl-silyl protected uridine 14.

[0139] [Diagram 31] FIG. 1 shows the H NMR (300 MHz, DMSO-d6) of 2′-O-acetyl-uridine 15 (mixture of 2′-OAc and 3′-OAc positional isomers).

[0140] [Diagram 32] Mass spectrum (ESI+, 100% CH3OH, TOF) of 2'-O-acetyl-uridine 15 (mixture of 2'-OAc and 3'-OAc positional isomers).

[0141] [Diagram 33] FIG. 1 is an HPLC chromatogram (CHOH) of 2′-O-acetyl-uridine 15 (a mixture of 2′-OAc and 3′-OAc positional isomers).

[0142] [Diagram 34] FIG. 1 shows the H NMR (300 MHz, DO) of 2′-O-acetyl-uridine triphosphate 17 (mixture of 2′OAc and 3′-OAc positional isomers).

[0143] [Diagram 35]FIG. 31P NMR (121 MHz, DO) of 2'-O-acetyl-uridine triphosphate 17 (mixture of 2'OAc and 3'-OAc positional isomers).

[0144] [Diagram 36] FIG. 1 shows the 1H NMR (300 MHz, DMSO-d6) of silyl-protected adenosine 19.

[0145] [Figure 37] Mass spectrum of silyl-protected adenosine 19 (ESI+, 100% CH3OH, TOF).

[0146] [Figure 38] FIG. 1 is an HPLC chromatogram of silyl-protected adenosine 19 (CHOH).

[0147] [Figure 39] FIG. 1 shows the 1H NMR (300 MHz, DMSO-d6) of 2'-O-acetylated silyl protected adenosine 20.

[0148] [Diagram 40] Mass spectrum of 2'-O-acetylated silyl protected adenosine 20 (ESI+, 100% CHOH, TOF).

[0149] [Diagram 41] FIG. 1 is an HPLC chromatogram of 2′-O-acetylated silyl protected adenosine 20 (CHOH).

[0150] [Diagram 42] FIG. 1 is a graph showing 1H NMR (300 MHz, DMSO-d6) of 2'-OAc Adenosine 21 (min).

[0151] [Diagram 43] Mass spectrum (ESI+, 100% CH3OH, TOF) of 2'-OAc adenosine 21 (minor).

[0152] [Diagram 44] HPLC chromatogram (CHOH) of 2'-OAc Adenosine 21 (minor).

[0153] [Diagram 45] FIG. 1 is a graph showing 1H NMR (300 MHz, DMSO-d6) of 3'-OAc Adenosine 22 (main).

[0154] [Diagram 46] Mass spectrum (ESI+, 100% CH3OH, TOF) of 3'-OAc adenosine 22 (major).

[0155] [Figure 47] HPLC chromatogram (CHOH) of 3'-OAc adenosine 22 (major).

[0156] [Figure 48] FIG. 1 is a graph showing 1H NMR (300 MHz, DO) of 2'-O-acetyl adenosine triphosphate sodium salt 24.

[0157] [Figure 49] FIG. 1 is a graph showing the 31P NMR (121 MHz, DO) of 2′-O-acetyladenosine triphosphate sodium salt 24.

[0158] [Figure 50] Mass spectrum of 2'-O-acetyl adenosine triphosphate sodium salt 24 (ESI-, 100% CHOH, TOF).

[0159] [Figure 51] FIG. 1 shows the 1H NMR (300 MHz, DMSO-d6) of silyl-protected guanosine 26.

[0160] [Figure 52] Mass spectrum of silyl-protected guanosine 26 (ESI+, 100% CH3OH, TOF).

[0161] [Figure 53] HPLC chromatogram of silyl-protected guanosine 26 (CHOH).

[0162] [Figure 54] FIG. 1 shows the 1H NMR (300 MHz, DMSO-d6) of 2'-O-acetyl-silyl protected guanosine 27.

[0163] [Figure 55] Mass spectrum of 2'-O-acetyl-silyl protected guanosine 27 (ESI+, 100% CH3OH, TOF).

[0164] [Figure 56] FIG. 1 is an HPLC chromatogram of 2′-O-acetyl-silyl protected guanosine 27 (CHOH).

[0165] [Figure 57] 1H NMR (300 MHz, DMSO-d6) of 2'-O-acetylguanosine 28.

[0166] [Figure 58] Mass spectrum of 2'-O-acetylguanosine 28 (ESI+, 100% CH3OH, TOF).

[0167] [Figure 59] HPLC chromatogram of 2'-O-acetylguanosine 28 (CHOH).

[0168] [Figure 60] 1 is a graph showing 1H NMR (300 MHz, DO) of 2'-O-acetyl-guanosine triphosphate 30.

[0169] [Figure 61] 31P NMR (121 MHz, DO) of 2'-O-acetyl-guanosine triphosphate 30.

[0170] [Figure 62] (A-B) are graphs showing in vivo luciferase gene expression from LNP-delivered 2-O acetylated RNA or LNP-delivered N1-methylpseudouridine modified RNA. Luciferase gene expression was measured in all animals 6, 24, and 48 hours after administration of LNP. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0171] A specific definition About or Approximately: As used herein, the terms "about" and "approximately" when used herein with respect to a value refer to a similar value in the context of a reference value. In general, those skilled in the art who are familiar with the context will understand the associated degree of dispersion encompassed by "about" or "approximately" in that context. For example, in some embodiments, the term "about" or "approximately" may encompass a range of values ​​that is 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less of the reference value.

[0172] Administering: As used herein, the term "administering" or "administration" generally refers to administering a composition to a subject to achieve delivery of an agent that is or is contained in the composition. Those skilled in the art will recognize the various routes that may be utilized for administration to a subject, e.g., a human, in the appropriate circumstances. For example, in some embodiments, administration may be ocular, oral, parenteral, topical, etc. In some specific embodiments, administration may be bronchial (e.g., by bronchial instillation), buccal, transdermal (e.g., may be or may include one or more of topical skin, intradermal, interdermal, transdermal, etc.), enteral, intraarterial, intradermal, intragastric, intramedullary, intramuscular, intranasal, intraperitoneal, intrathecal, intravenous, intraventricular, intraspecific organ (e.g., intrahepatic), mucosal, nasal, oral, rectal, subcutaneous, sublingual, topical, tracheal (e.g., by intratracheal instillation), vaginal, vitreous, etc. In some embodiments, administration may include only a single dose. In some embodiments, administration may include the application of a fixed number of doses. In some embodiments, administration may include intermittent (e.g., multiple doses spaced apart in time) dosing and / or periodic (e.g., individual doses spaced apart over a period of time) dosing. In some embodiments, administration may include continuous dosing (e.g., perfusion) for at least a selected period of time.

[0173] Antigen: The term "antigen" as used herein refers to a chemical that elicits an immune response and / or (ii) binds to a T cell receptor (e.g., when presented by an MHC molecule) or an antibody. In some embodiments, an antigen elicits a humoral response (e.g., including production of antigen-specific antibodies). In some embodiments, an antigen elicits a cellular response (e.g., engagement of T cells whose receptors specifically interact with the antigen). In some embodiments, an antigen comprises at least one epitope of a target protein. In some embodiments, an epitope may be a linear epitope. In some embodiments, an epitope may be a conformational epitope. In some embodiments, an antigen binds to an antibody and may or may not elicit a specific physiological response in an organism. In general, an antigen may be or include any chemical, such as a small molecule, a nucleic acid, a polypeptide, a carbohydrate, a lipid, a polymer (in some embodiments, other than a biological polymer [e.g., other than a nucleic acid or amino acid polymer]), etc. In some embodiments, an antigen is or includes a polypeptide. In some embodiments, an antigen is or includes a glycan. Those skilled in the art will appreciate that, in general, antigens may be provided in isolated or pure form, or alternatively, may be provided in crude form (e.g., together with other substances, such as in an extract (e.g., a cellular extract) or other relatively crude preparation of an antigen-containing source). In some embodiments, antigens utilized in accordance with the present invention are provided in crude form. In some embodiments, the antigen is a recombinant antigen.

[0174] Delivery / Contact: As used interchangeably herein, the terms "delivery," "delivering," or "contacting" refer to the introduction of a fusion polynucleotide (e.g., as described herein) or a fusion polypeptide (e.g., as described herein) into a target cell. The target cell may be cultured in vivo or ex vivo, or may be present within a subject (in vivo). The method of introducing a fusion polynucleotide (e.g., as described herein) or a fusion polypeptide (e.g., as described herein) into a target cell may vary by in vitro, ex vivo, or in vivo application. In some embodiments, a fusion polynucleotide (e.g., as described herein) or a fusion polypeptide (e.g., as described herein) may be introduced into a target cell in cell culture by in vitro transfection. In some embodiments, a fusion polynucleotide (e.g., as described herein) or a fusion polypeptide (e.g., as described herein) may be introduced into a target cell via a delivery vehicle (e.g., complexed with a nanoparticle, a liposome, and / or a cell-penetrating agent). In some embodiments, a fusion polynucleotide (e.g., as described herein) or a fusion polypeptide (e.g., as described herein) can be introduced into a target cell within a subject by administering the fusion polynucleotide (e.g., as described herein) or the fusion polypeptide (e.g., as described herein) to the subject.

[0175] Expression: As used herein, the term "expression" of a nucleic acid sequence refers to the production of any gene product from the nucleic acid sequence. In some embodiments, the gene product can be a transcript. In some embodiments, the gene product can be a polypeptide. In some embodiments, expression of a nucleic acid sequence involves one or more of: (1) production of an RNA template from a DNA sequence (e.g., by transcription); (2) processing of the RNA transcript (e.g., by splicing, editing, etc.); (3) translation of the RNA into a polypeptide or protein; and / or (4) post-translational modification of the polypeptide or protein.

[0176] Functional: As used herein, the term "functional" is used to refer to forms or fragments of an entity that exhibit particular properties and / or activities.

[0177] Fragment: A "fragment" of a substance or entity as described herein comprises a discrete portion of a whole, but has a structure that lacks one or more portions found in the whole. In some embodiments, a fragment consists of such a discrete portion. In some embodiments, a fragment consists of or includes characteristic structural elements or portions found in the whole. In some embodiments, a fragment comprises a polynucleotide fragment. In some embodiments, a fragment comprises a polypeptide fragment. In some embodiments, a polynucleotide or polypeptide fragment comprises or consists of at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500 or more monomeric units (e.g., residues) found throughout the entire polynucleotide or polypeptide. In some embodiments, a polynucleotide or polypeptide fragment comprises or consists of at least about 5%, 10%, 15%, 20%, 25%, 30%, 25%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more of the monomeric units (e.g., residues) found in the entire polynucleotide or entire polypeptide. The entire polypeptide or entire polynucleotide may in some embodiments be referred to as the "parent" of the polynucleotide or polypeptide fragment.

[0178] Gene product or expression product: As used herein, the term "gene product" or "expression product" generally refers to the RNA transcribed from a gene (before and / or after processing) or the polypeptide (before and / or after modification) encoded by the RNA transcribed from a gene.

[0179] Nucleic Acid / Oligonucleotide / Polynucleotide: As used herein, the terms "nucleic acid" and "polynucleotide" and "oligonucleotide" are used interchangeably and refer to a polymer of three or more nucleotides. In some embodiments, a nucleic acid comprises DNA. In some embodiments, a nucleic acid comprises RNA. In some embodiments, a nucleic acid comprises messenger RNA (mRNA). In some embodiments, a nucleic acid is single-stranded. In some embodiments, a nucleic acid is double-stranded. In some embodiments, a nucleic acid comprises both single-stranded and double-stranded portions. In some embodiments, a nucleic acid comprises a backbone comprising one or more phosphodiester bonds. In some embodiments, a nucleic acid comprises a backbone comprising both phosphodiester and non-phosphodiester bonds. For example, in some embodiments, a nucleic acid may comprise a backbone comprising one or more phosphorothioate or 5'-N-phosphoramidite bonds and / or one or more peptide bonds, e.g., as in "peptide nucleic acids." In some embodiments, a nucleic acid includes one or more, or all, naturally occurring residues (e.g., adenine, cytosine, deoxyadenosine, deoxycytidine, deoxyguanosine, deoxythymidine, guanine, thymine, uracil). In some embodiments, a nucleic acid includes one or more, or all, non-naturally occurring residues. In some embodiments, the non-natural residue comprises a nucleoside analog (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine, 3-methyladenosine, 5-methylcytidine, C-5 propynyl-cytidine, C-5 propynyl-uridine, 2-aminoadenosine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyl-uridine, C5-propynyl-cytidine, C5-methylcytidine, 2-aminoadenosine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, 6-O-methylguanine, 2-thiocytidine, methylated bases, intercalating bases, and combinations thereof). In some embodiments, the non-natural residue comprises one or more modified sugars (eg, 2'-fluororibose, ribose, 2'-deoxyribose, arabinose, and hexose) when compared to that of the natural residue.In some embodiments, the nucleic acid has a nucleotide sequence that encodes a functional gene product, such as an RNA or a polypeptide. In some embodiments, the nucleic acid has a nucleotide sequence that includes one or more introns. In some embodiments, the nucleic acid may be prepared by isolation from a natural source, enzymatic synthesis (e.g., by polymerization based on a complementary template, e.g., in vivo or in vitro polymerization), replication in a recombinant cell or system, or chemical synthesis. In some embodiments, the nucleic acid is at least 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 20, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4 In some embodiments, the nucleic acid sequence is greater than or equal to 1,000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500, 10,000, 10,500, 11,000, 11,500, 12,000, 12,500, 13,000, 13,500, 14,000, 14,500, 15,000, 15,500, 16,000, 16,500, 17,000, 17,500, 18,000, 18,500, 19,000, 19,500, or 20,000 or more residues or nucleotides in length. When the number of nucleotides is used as an indication of the size of, for example, a fusion polynucleotide, a particular number of nucleotides refers, for example, to the number of nucleotides on one strand of the fusion polynucleotide.

[0180] Polypeptide: The term "polypeptide" as used herein generally has its art-recognized meaning of a polymer of at least three or more amino acids. Those skilled in the art will understand that the term "polypeptide" is intended to encompass not only polypeptides having the complete sequences listed herein, but also polypeptides that represent functional, biologically active, or characteristic fragments, portions, or domains of such complete polypeptides (e.g., fragments, portions, or domains that retain at least one activity). Polypeptides may contain L-amino acids, D-amino acids, or both, and may contain any of a variety of amino acid modifications or analogs known in the art. Useful modifications include, for example, terminal acetylation, amidation, methylation, and the like. In some embodiments, polypeptides may include natural amino acids, unnatural amino acids, synthetic amino acids, and combinations thereof.

[0181] Polyribonucleotide: As used herein, the term "polyribonucleotide" refers to a polymer of three or more ribonucleotides. In some embodiments, a polyribonucleotide is single-stranded. In some embodiments, a polyribonucleotide is double-stranded. In some embodiments, a polyribonucleotide includes both single-stranded and double-stranded portions. In some embodiments, a polyribonucleotide may include a backbone structure as described in the definition of "nucleic acid / oligonucleotide" above. A polyribonucleotide may include a coding region that encodes a gene product, or a polyribonucleotide may not include a coding region. Additionally, a polyribonucleotide may be a regulatory RNA (e.g., siRNA, microRNA, etc.), or a messenger RNA (mRNA) oligonucleotide. In some embodiments, where the polyribonucleotide is a polyribonucleotide that includes a coding region, e.g., an mRNA oligonucleotide, the polyribonucleotide typically includes a poly(A) region at its 3' end. In some embodiments, where the polyribonucleotide is a polyribonucleotide that includes a coding region, e.g., an mRNA oligonucleotide, the polyribonucleotide typically includes a cap structure at its 5' end, e.g., recognized in the art, for recognizing the polyribonucleotide, e.g., an mRNA, and binding it to a ribosome to initiate translation. In some embodiments, when the polyribonucleotide does not include a coding region, the polyribonucleotide may include a long non-coding RNA (lncRNA), a microRNA, an siRNA, a piRNA, a snoRNA, a snRNA, an exRNA, a scaRNA, a rRNA, a tRNA, or a combination thereof. In some embodiments, the polyribonucleotide includes an RNA oligonucleotide. When the number of ribonucleotides is used, e.g., as an indicator of the size of a polynucleotide, a certain number of nucleotides refers, for example, to the number of ribonucleotides on a single strand.

[0182] Subject: As used herein, the term "subject" refers to an organism, typically a mammal (e.g., a human). In some embodiments, the subject is afflicted with a disease, disorder, or condition. In some embodiments, the subject is susceptible to a disease, disorder, or condition. In some embodiments, the subject exhibits one or more symptoms or characteristics of a disease, disorder, or condition. In some embodiments, the subject does not exhibit any symptoms or characteristics of a disease, disorder, or condition. In some embodiments, a subject is one who has one or more characteristics that characterize a susceptibility or risk for a disease, disorder, or condition. In some embodiments, the subject is a patient. In some embodiments, the subject is an individual to whom and / or to whom diagnosis and / or therapy is administered.

[0183] Variant: As used herein, the term "variant" refers to an entity that exhibits sufficient structural identity with a reference entity, but that is structurally distinct from the reference entity in the presence or level of one or more chemical moieties when compared to the reference entity. In many embodiments, a variant also differs functionally from its reference entity. In general, whether a particular entity is properly considered to be a "variant" of a reference entity is based on the degree of structural identity with the reference entity. For example, a variant polypeptide may differ from a reference polypeptide as a result of one or more differences in amino acid sequence and / or one or more differences in chemical moieties (e.g., carbohydrates, lipids, etc.) covalently attached to the polypeptide backbone. Alternatively or additionally, in some embodiments, a variant polypeptide does not share at least one characteristic sequence element with a reference polypeptide. In some embodiments, a reference polypeptide has one or more biological activities. In some embodiments, a variant polypeptide shares one or more of the biological activities of a reference polypeptide. In some embodiments, a variant polypeptide lacks one or more of the biological activities of a reference polypeptide. In some embodiments, a variant polypeptide exhibits a reduced level of one or more biological activities when compared to a reference polypeptide.

[0184] Standard techniques may be used for recombinant DNA, oligonucleotide synthesis, e.g., RNA synthesis, as well as tissue culture and transformation (e.g., electroporation, lipofection). Enzymatic reactions and purification techniques may be performed according to manufacturer's specifications or as commonly accomplished in the art or as described herein. The techniques and procedures described above may generally be performed according to conventional methods well known in the art and as described in the various general and more specific references cited and described throughout the specification. See, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual (2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989)), which is incorporated herein by reference for any purpose.

[0185] DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS RNA, such as messenger RNA (mRNA), has enormous potential to provide life-changing therapies for diseases previously deemed untreatable. However, in the overall landscape of therapeutic modalities, RNA technology is still in the early stages of its development. Although much progress has been made in the art to bring the technology to its current form, further improvements to the platform still need to be made to truly realize its full potential.

[0186] One of the unsolved problems is that RNA is an inherently unstable molecule. Some improvement in this regard has been seen as a result of the proposed mechanism of increased base stacking and hydrogen bonding by using pseudouridine derivatives instead of uridine (Hudson, Bloomingdale, & Znosko, RNA 2013). However, in its current commercial form, its stability and activity are still very transient (Anand & Stahel, Patient Saf Surg 2021). In fact, the transient nature of RNA is mainly considered as a platform advantage, contributing to its safety as a therapeutic modality. However, the other side of this perceived advantage is that in order to use RNA for indications other than vaccination, it is important to extend the half-life of therapeutic RNA at least somewhat beyond its current half-life. This is because indications such as cancer therapy and enzyme replacement require the expression of the therapeutic protein of interest for a period of time, for example, for optimal benefit to the patient. A therapeutic that loses activity within a few hours, and therefore its benefit to the patient, would require frequent dosing and formulation costs. This is likely to be intolerable and unsustainable for the average patient, and may limit the use of the therapy. In some embodiments, extending the half-life of RNA does not mean making the RNA permanently active. Rather, it is to increase the half-life of RNA so that the required dosing frequency is more tolerable. It is also important to note that the optimal RNA half-life for a given indication may be different from another indication, but the underlying theme is that some degree of extension is required.

[0187] Without wishing to be bound by theory, in some embodiments, the instability of RNA, even in base-modified RNA, may be due to the reactivity of, for example, the hydroxyl group on the 2-carbon of the ribose of ribonucleotides. When RNA is exposed to conditions of high pH, ​​high temperature, divalent cations, or some combination thereof, oxygen atoms can launch a nucleophilic attack on the phosphate backbone of the RNA strand. This is the same hydroxyl group targeted by the ubiquitous RNase enzyme, which rapidly degrades any RNA (Brown & Pasloske, Methods in Enzymology 2001). The lack of this hydroxyl group is what gives DNA its extraordinary stability compared to RNA. Therefore, reducing the reactivity of this hydroxyl group should improve the stability of RNA. This phenomenon is observed when 2-O-methylnucleoside triphosphate (NTP) is used with RNA, which increases the thermal stability of the RNA duplex and provides resistance to nucleases (Assi, et al., NAR 2020). However, the problem with using 2-O-methyl and other currently available 2-O modified NTPs is that they can only be incorporated into short RNA oligonucleotides by chemical synthesis, because only error-prone RNA polymerases can use 2-O-methyl NTPs as substrates due to the loss of critical hydrogen bonding interactions (Meyer, et al., NAR 2015). The size limitations of RNAs made by chemical synthesis using 2-O-methyl NTPs also mean that gene-length RNAs are currently not possible with this method. When considering RNA therapeutics, these problems are completely superseded by the fact that 2-O-methyl modifications throughout the RNA transcript inhibit its translation by steric hindrance of critical ribosomal RNA residues and codon-anticodon interactions (Choi, et al., Nat Struct Mol Biol 2018).

[0188] Given the knowledge in the art, it was hypothesized that an ideal 2-O-modification for RNA applications would be a functional group that could maintain the crucial hydrogen bonding interactions necessary for transcription, translation, and duplex formation while at the same time reducing the reactivity that would otherwise promote RNA autohydrolysis and nuclease degradation. Based on biochemistry principles, it was further hypothesized that 2-O-acetylated NTPs (FIGS. 1A-1D) could provide such a profile. Thus, the present disclosure provides technology related to 2-O-acetylated nucleotides in which the hydroxyl group on the 2-carbon of ribose is acetylated (2-O-acetylated ribose). The technology provided in the present disclosure includes modified ribonucleotides comprising 2-O-acetylated ribose, polyribonucleotides comprising modified ribonucleotides comprising 2-O-acetylated ribose, compositions comprising such modified ribonucleotides and / or polyribonucleotides, as well as methods of making such polyribonucleotides and methods of using such polyribonucleotides for a variety of applications.

[0189] Tyrosine (Tyr) 639 of T7 RNA polymerase appears to be the key identifiable amino acid residue that determines whether T7 RNA polymerase can use nucleotides as substrates (Makinen, et al., Nature Communications 2021). It was hypothesized that the carbonyl oxygen of the acetyl group could function as a hydrogen bond acceptor and maintain a critical interaction with Tyr639 to allow the use of 2-O-acetyl nucleotides by wild-type bacteriophage RNA polymerases such as T7 RNA polymerase. This is in contrast to 2-O-methyl NTPs, in which the methyl group prevents hydrogen bonding between the 2' oxygen and Tyr639, thus preventing the use of 2-O-methyl NTPs for in vitro transcription.

[0190] In a previous study, RNA chemically acetylated posttranscriptionally with acetic anhydride was found to be translationally active in a wheat germ cell-free translation system (Ovodov & Alakhov, FEBS 1990). The authors of this study were unable to confirm specific acetylation of 2-OH, and only claimed that up to 75% of the 2-OH groups in the RNA were acetylated. Although Ovodov and Alakhov also used a cell-free translation system, their study did not report whether chemically acetylated RNA could be introduced into living cells and / or translated by the intracellular ribosomal machinery. Thus, the utility of Ovodov and Alakhov's chemically acetylated RNA was limited to artificial cell-free translation systems.

[0191] Among other things disclosed herein is the novel discovery that 2-O-acetylated nucleotide triphosphates can be accepted by RNA polymerase as substrates for in vitro transcription of RNA. The present disclosure also provides the insight that RNA polymerase can use 2-O-acetylated nucleotide triphosphates to generate fully acetylated RNA. In some embodiments, the RNA produced by the methods disclosed herein is 100% 2-O-acetylated, except for cap structures that do not contain 2-O-acetylated ribose, for example, by co-transcriptional capping. In some embodiments, the RNA produced by the methods disclosed herein is 100% 2-O-acetylated, including cap structures that contain 2-O-acetylation. The present discovery of novel modified nucleotides that can be used to generate RNA using in vitro transcription reactions allows the use of 2-O-acetylated ribose in the manufacture of RNA for a variety of applications, including therapeutic applications, research applications, diagnostic applications, agricultural applications, and any other suitable applications.

[0192] Moreover, the present disclosure also demonstrates for the first time the surprising finding that RNA containing 2-O-acetylated ribose can be translated by the intracellular ribosomal machinery of a cell. Figures 2-5 herein provide exemplary polypeptide expression from 2-O-acetylated RNA. Also provided herein is the finding that the yield of 2-O-acetylated RNA is comparable to RNA without this modification. Table 1 provides exemplary yields that can be obtained with 2-O-acetylated RNA.

[0193] Further, the present disclosure provides the discovery that polyribonucleotides comprising 2-O-acetylated ribose, such as those produced in an in vitro transcription reaction, are functional and have desirable properties, such as reduced immunogenicity, avoidance of innate immune system activation, improved viability of cells into which the polyribonucleotide is introduced, increased expression of payloads in cells into which the polyribonucleotide is introduced, and / or increased persistence of the polyribonucleotide. In some embodiments, evasion of the innate immune system comprises reduced activation of NFkb or the NFkb pathway, IRF or the IRF pathway, and / or other inflammatory cytokines in cells, tissues, or organisms into which the polyribonucleotide is introduced. In some embodiments, evasion of the innate immune system comprises reduced detection of uncapped RNA in cells, tissues, or organisms into which the polyribonucleotide is introduced. In some embodiments, in vitro transcribed RNA containing 2-O-acetylated ribose may improve the efficacy of RNA therapeutics by reducing undesired innate immune responses. In some embodiments, the reduction of undesired innate immune responses by RNA containing 2-O-acetylated ribose may be further enhanced (e.g., undesired innate immune responses may be further reduced) by including one or more base modifications in the RNA. For example, Figures 4 and 5 show the reduction of innate immune activation by polyribonucleotides that include 2-O-acetylated ribose in combination with an exemplary modified cytosine base (N4-acetyl-cytidine, Ac4C). This data supports the use of polyribonucleotides that include 2-O-acetylated ribose to reduce innate immune activation while increasing payload expression and / or persistence.

[0194] Also provided herein is the finding that the expression level of RNA can be regulated by including 2-O-acetylated ribose in RNA.For example, Figure 2 herein provides an exemplary increase in payload expression with polyribonucleotide that includes 2-O-acetylated ribose when compared with polyribonucleotide that does not have 2-O-acetylated ribose.

[0195] Further provided herein is the finding that polyribonucleotides with 2-O acetylated ribose can increase the persistence of polyribonucleotides. For example, Figures 62A-62B herein provide exemplary data showing equivalent gene expression from LNP-delivered 2-O acetylated polyribonucleotides compared to LNP-delivered N1-methylpseudouridine-modified polyribonucleotides. This observation, given the increased immunogenicity seen with 2-O ribose acetylated polyribonucleotides without any additional modifications (Example 1, Figures 4 and 5), suggests that polyribonucleotides with 2-O acetylated ribose have increased persistence, likely due to increased resistance to nuclease degradation.

[0196] Thus, the present disclosure provides the finding that the use of polyribonucleotides comprising 2-O acetylated ribose alone or in combination with one or more base modifications can be useful for applications (e.g., therapeutic applications) in which increased persistence (e.g., increased nuclease resistance) of polyribonucleotides is desired.Without wishing to be bound by any particular theory, polyribonucleotides with increased persistence can be achieved by combining reduced immunogenicity through base modifications (e.g., Ac4C, 5hmU, N1-methylpseudouridine, etc.) with nuclease resistance through ribosome-compatible backbone modifications (e.g., 2-O ribose acetylation as described herein).

[0197] In some embodiments, the provided methods may be useful for enhancing the persistence or uptake of polyribonucleotides comprising 2-O acetylated ribose in target cells. In some embodiments, the persistence or uptake of polyribonucleotides comprising 2-O acetylated ribose in target cells is enhanced by at least 30% or more when compared to the persistence or uptake of other similar polyribonucleotides that do not have 2-O acetylated ribose or that have less 2-O acetylated ribose. In some embodiments, the persistence or uptake of polyribonucleotides comprising 2-O acetylated ribose in target cells is enhanced by at least 30% or more when compared to the persistence or uptake of other similar polyribonucleotides that do not have 2-O acetylated ribose or that have less 2-O acetylated ribose.

[0198] As shown in Figures 62A-62B, the present disclosure further provides that 2-O-acetylated polyribonucleotides can be delivered in vivo by LNPs. Thus, the present disclosure provides the discovery that 2-O-acetylated polyribonucleotides can be delivered in a delivery vehicle, such as LNPs, to deliver polyribonucleotides to a cell, tissue, or subject, to treat a disease or disorder, and / or to ameliorate the symptoms of a disease or disorder.

[0199] In some embodiments, the payload is or comprises a polypeptide encoded by a polyribonucleotide comprising modified ribonucleotides.

[0200] In some embodiments, the payload is or comprises RNA located within a polyribonucleotide that comprises modified ribonucleotides.

[0201] In some embodiments, the use of polyribonucleotides containing 2-O-acetylated ribose allows for improved efficacy and / or improved tolerability in subjects administered the RNA therapeutics containing same.

[0202] 2'-O-acetylated nucleotides In particular, provided herein is a polyribonucleotide comprising one or more modified ribonucleotides comprising a ribose moiety comprising an acetyl group instead of a hydroxyl group. In some embodiments, the modified ribonucleotide comprises a 2'-O-acetylated ribose. In some embodiments, the modified ribonucleotide comprising a 2'-O-acetylated ribose comprises a 5' monophosphate, a 5' diphosphate or a 5' triphosphate. In some embodiments, the modified ribonucleotide comprising a 2'-O-acetylated ribose comprises a nucleobase selected from adenine or a modified form thereof, guanine or a modified form thereof, cytosine or a modified form thereof, or uracil or a modified form thereof.

[0203] In some embodiments, the modified ribonucleotide is: [ka] The structure is

[0204] (a) wherein X is a 5' monophosphate, a 5' diphosphate, or a 5' triphosphate;

[0205] (b) R is a nucleobase selected from adenine or a modified form thereof, guanine or a modified form thereof, cytosine or a modified form thereof, or uracil or a modified form thereof.

[0206] In some embodiments, the modified ribonucleotide comprises a 2'-O-acetylated ribose, a 5' triphosphate and the following: [ka] The structure is

[0207] wherein R is a nucleobase selected from adenine (e.g., unmodified or modified adenine), guanine (e.g., unmodified or modified guanine), cytosine (e.g., unmodified or modified cytosine), or uracil (e.g., unmodified or modified uracil).

[0208] In some embodiments, the modified ribonucleotide comprising a 2'-O-acetylated ribose comprises an adenine and a 5' monophosphate, a 5' diphosphate, or a 5' triphosphate. [ka] It has the structure:

[0209] In some embodiments, the modified ribonucleotide comprising a 2'-O-acetylated ribose comprises guanine and a 5' monophosphate, a 5' diphosphate, or a 5' triphosphate. [ka] It has the structure:

[0210] In some embodiments, the modified ribonucleotide comprising a 2'-O-acetylated ribose comprises a cytosine and a 5' monophosphate, a 5' diphosphate, or a 5' triphosphate. [ka] It has the structure:

[0211] In some embodiments, the modified ribonucleotide comprising a 2'-O-acetylated ribose comprises uracil and a 5' monophosphate, a 5' diphosphate, or a 5' triphosphate. [ka] It has the structure:

[0212] In some embodiments, the modified ribonucleotide comprising a 2'-O-acetylated ribose comprises an N4-acetylcytidine and a 5' monophosphate, a 5' diphosphate, or a 5' triphosphate. [ka] It has the structure:

[0213] In some embodiments, the modified ribonucleotide comprising a 2'-O-acetylated ribose comprises 5-hydroxymethyluridine and a 5' monophosphate, a 5' diphosphate, or a 5' triphosphate. [ka] It has the structure:

[0214] In some embodiments, the modified ribonucleotide comprising a 2'-O-acetylated ribose comprises N1-methylpseudouridine and a 5' monophosphate, a 5' diphosphate, or a 5' triphosphate. [ka] It has the structure:

[0215] Also provided herein is a polyribonucleotide comprising one or more modified ribonucleotides, e.g., nucleotides comprising 2-O-acetylated ribose. In some embodiments, the nucleotide comprises modified nucleotides, e.g., as described herein, or unmodified nucleotides.

[0216] In some embodiments, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least about 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% of the ribose moieties in the polyribonucleotide are acetylated (2'-O-acetylated).

[0217] In some embodiments, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% of the ribose moieties in the polyribonucleotide are acetylated (2'-O-acetylated).

[0218] In some embodiments, 100% of the ribose moieties in the polyribonucleotide are acetylated (2'-O-acetylated).

[0219] In some embodiments, the ribose moiety in the polyribonucleotide is about 5% to 99%, about 5% to 95%, about 5% to 90%, about 5% to 85%, about 5% to 80%, about 5% to 75%, about 5% to 70%, about 5% to 65%, about 5% to 60%, about 5% to 55%, about 5% to 50%, about 5% to 45%, about 5% to 40%, about 5% to 35%, about 5% to 30%, about 5% to 25%, about 5% to 20%, about 5% to 15%, about 5% to 10%, about 5% to 15% or about 5% to 10%. %, about 10% to 99%, about 15% to 99%, about 20% to 99%, about 25% to 99%, about 30% to 99%, about 35% to 99%, about 40% to 99%, about 45% to 99%, about 50% to 99%, about 55% to 99%, about 60% to 99%, about 65% to 99%, about 70% to 99%, about 80% to 99%, about 85% to 99%, about 90% to 99%, or about 95% to 99% are acetylated (2'-O-acetylated).

[0220] In some embodiments, the ribose moiety in the polyribonucleotide is about 76% to about 100%, about 77% to about 100%, about 78% to about 100%, about 79% to about 100%, about 80% to about 100%, about 81% to about 100%, about 82% to about 100%, about 83% to about 100%, about 84% to about 100%, about 85% to about 100%, about 86% to about 100%, about 87% to about 100%, or about 88% to about 100%. 00%, about 88% to about 100%, about 89% to about 100%, about 90% to about 100%, about 91% to about 100%, about 92% to about 100%, about 93% to about 100%, about 94% to about 100%, about 95% to about 100%, about 96% to about 100%, about 97% to about 100%, about 98% to about 100%, and about 99% to about 100% are acetylated (2'-O-acetylated).

[0221] In some embodiments, the ribose moiety in the polyribonucleotide is about 5% to 99%, about 5% to 95%, about 5% to 90%, about 5% to 85%, about 5% to 80%, about 5% to 75%, about 5% to 70%, about 5% to 65%, about 5% to 60%, about 5% to 55%, about 5% to 50%, about 5% to 45%, about 5% to 40%, about 5% to 35%, about 5% to 30%, about 5% to 25%, about 5% to 20%, about 5% to 15%, about 5% to 10%, about 5% to 15% or about 5% to 10%. %, about 10% to 99%, about 15% to 99%, about 20% to 99%, about 25% to 99%, about 30% to 99%, about 35% to 99%, about 40% to 99%, about 45% to 99%, about 50% to 99%, about 55% to 99%, about 60% to 99%, about 65% to 99%, about 70% to 99%, about 80% to 99%, about 85% to 99%, about 90% to 99%, or about 95% to 99% are acetylated (2'-O-acetylated).

[0222] In some embodiments, about 76% to about 99%, about 76% to about 98%, about 76% to about 97%, about 76% to about 96%, about 76% to about 95%, about 76% to about 94%, about 76% to about 93%, about 76% to about 92%, about 76% to about 91%, about 76% to about 90%, about 76% to about 89%, about 76% to about 88%, about 76% to about 87%, about 76% to about 86%, about 76% to about 85%, about 76% to about 84%, about 76% to about 83%, about 76% to about 82%, about 76% to about 81%, about 76% to about 80%, about 76% to about 79%, about 76% to about 78%, and about 76% to about 77% are acetylated (2'-O-acetylated).

[0223] In some embodiments, the polyribonucleotide comprises a cap structure, and the cap structure does not comprise a 2'-O-acetylated ribose.

[0224] In some embodiments, the polyribonucleotide is fully 2-O-acetylated (100% of the ribose moieties are 2-O-acetylated) except for the cap structure. In some embodiments, the cap structure of the polyribonucleotide comprises a hydroxyl group on the 2-carbon of the ribose.

[0225] In some embodiments, the polyribonucleotide comprises a cap structure that is less than 100% 2-O-acetylated (less than 100% of the ribose moieties are 2-O-acetylated) and does not contain any 2'-O-acetylated ribose. In some embodiments, the cap structure of the polyribonucleotide comprises a hydroxyl group on the 2-carbon of ribose.

[0226] In some embodiments, the polyribonucleotide comprises a cap structure that is at least 5% 2-O-acetylated (at least 5% of the ribose moieties are 2-O-acetylated) and does not contain 2'-O-acetylated ribose, hi some embodiments, the cap structure of the polyribonucleotide comprises a hydroxyl group on the 2-carbon of ribose.

[0227] In some embodiments, the polyribonucleotide comprises a cap structure, and the cap structure comprises a 2'-O-acetylated ribose.

[0228] In some embodiments, the polyribonucleotide is fully 2-O-acetylated (100% of the ribose moieties are 2-O-acetylated) and contains a 2-O-acetylated cap structure.

[0229] In some embodiments, the polyribonucleotides are less than 100% 2-O-acetylated (less than 100% of the ribose moieties are 2-O-acetylated) and include a 2-O-acetylated cap structure.

[0230] In some embodiments, the polyribonucleotide is at least 5% 2-O-acetylated (at least 5% of the ribose moieties are 2-O-acetylated) and comprises a 2-O-acetylated cap structure.

[0231] In some embodiments, the polyribonucleotide may have a length of at least 5 nucleotides or more. In some embodiments, the polyribonucleotide may have a length of at least 5 nucleotides, at least 10 nucleotides, at least 15 nucleotides, at least 20 nucleotides, at least 25 nucleotides, at least 30 nucleotides, at least 35 nucleotides, at least 40 nucleotides, at least 45 nucleotides, at least 50 nucleotides, at least 55 nucleotides, at least 60 nucleotides, at least 65 nucleotides, at least 70 nucleotides, at least 75 nucleotides, at least 80 nucleotides, at least 85 nucleotides, at least 90 nucleotides, at least 95 nucleotides, at least 100 nucleotides, at least 200 nucleotides, at least 300 nucleotides, at least 400 nucleotides, at least 500 nucleotides, at least 1000 nucleotides, at least 2000 nucleotides, at least 5000 nucleotides or more.

[0232] In some embodiments, the polyribonucleotide may be from about 5 nucleotides to about 200,000 nucleotides, from about 5 nucleotides to about 150,000 nucleotides, from about 5 nucleotides to about 100,000 nucleotides, from about 5 nucleotides to about 50,000 nucleotides, from about 5 nucleotides to about 10,000 nucleotides, from about 5 nucleotides to about 5000 nucleotides, from about 5 nucleotides to about 1000 nucleotides, from about 5 nucleotides to about 500 nucleotides, from about 5 nucleotides to about 400 nucleotides, from about 5 nucleotides to about 300 nucleotides, from about 5 nucleotides to about 200 nucleotides, from about 5 nucleotides to about 100 nucleotides, from about 5 nucleotides to about 90 nucleotides, from about 5 nucleotides to about 5 The length of the nucleic acid may be from about 5 nucleotides to about 85 nucleotides, from about 5 nucleotides to about 80 nucleotides, from about 5 nucleotides to about 75 nucleotides, from about 5 nucleotides to about 70 nucleotides, from about 5 nucleotides to about 65 nucleotides, from about 5 nucleotides to about 60 nucleotides, from about 5 nucleotides to about 55 nucleotides, from about 5 nucleotides to about 50 nucleotides, from about 5 nucleotides to about 45 nucleotides, from about 5 nucleotides to about 40 nucleotides, from about 5 nucleotides to about 35 nucleotides, from about 5 nucleotides to about 30 nucleotides, from about 5 nucleotides to about 25 nucleotides, from about 5 nucleotides to about 20 nucleotides, from about 5 nucleotides to about 15 nucleotides, or from about 5 nucleotides to about 10 nucleotides.

[0233] In some embodiments, the polyribonucleotide may be from about 5 nucleotides to about 200,000 nucleotides, from about 10 nucleotides to about 200,000 nucleotides, from about 15 nucleotides to about 200,000 nucleotides, from about 20 nucleotides to about 200,000 nucleotides, from about 30 nucleotides to about 200,000 nucleotides, from about 40 nucleotides to about 200,000 nucleotides, from about 50 nucleotides to about 200,000 nucleotides, from about 100 nucleotides to about 200,000 nucleotides, from about 200 nucleotides to about 200,000 nucleotides, from about 300 nucleotides to about 200,000 nucleotides, from about 400 nucleotides to about 200,000 nucleotides, from about 500 nucleotides to about 200,000 nucleotides, The nucleic acid sequence may have a length of about 1 to about 200,000 nucleotides, about 2000 nucleotides to about 200,000 nucleotides, about 3000 nucleotides to about 200,000 nucleotides, about 4000 nucleotides to about 200,000 nucleotides, about 5000 nucleotides to about 200,000 nucleotides, about 10,000 nucleotides to about 200,000 nucleotides, about 20,000 nucleotides to about 200,000 nucleotides, about 30,000 nucleotides to about 200,000 nucleotides, about 40,000 nucleotides to about 200,000 nucleotides, about 50,000 nucleotides to about 200,000 nucleotides, about 100,000 nucleotides to about 200,000 nucleotides, or about 150,000 nucleotides to about 200,000 nucleotides.

[0234] In some embodiments, a polyribonucleotide can have a length of up to 200,000 nucleotides, up to 150,000 nucleotides, up to 100,000 nucleotides, or up to 50,000 nucleotides.

[0235] Additional nucleotide modifications In some embodiments, the polyribonucleotide comprising a 2'-O-acetylated ribose further comprises a modification comprising a modified backbone, a modified nucleobase, or any combination thereof.

[0236] In some embodiments, the polyribonucleotide comprising a 2'-O-acetylated ribose further comprises a modified backbone.

[0237] In some embodiments, the polyribonucleotide comprising 2'-O-acetylated ribose further comprises modified nucleobases. The modifications on the nucleobases include modifications at one or more of adenine, cytosine, uracil, or guanine.

[0238] In some embodiments, the polyribonucleotide comprising a 2'-O-acetylated ribose further comprises a modified adenine.In some embodiments, the modified adenine is 2-amino-purine, 2,6-diaminopurine, 2-amino-6-halo-purine (e.g., 2-amino-6-chloro-purine), 6-halo-purine (e.g., 6-chloro-purine), 2-amino-6-methyl-purine, 8-azido-adenosine, 7-deaza-adenine, 7-deaza-8-aza-adenine, 7-deaza-2-amino-purine, 7-deaza-8-aza-2-amino-purine, 7-deaza-2,6-diaminopurine, 7-deaza-8-aza-2,6-diaminopurine, 1-Methyl-adenosine (m1A), 2-Methyl-adenine (m2A), N6-Methyl-adenosine (m6A), 2-Methylthio-N6-methyl-adenosine (ms2m6A), N6-Isopentenyl-adenosine (i6A), 2-Methylthio-N6-isopentenyl-adenosine (ms2i6A), N6-(cis-hydroxyisopentenyl)adenosine (io6A), 2-Methylthio-N6-(cis-hydroxyisopentenyl)adenosine (ms2io6A), N6-Glycinylcarbamoyl-adenosine (g6A) , N6-threonylcarbamoyl-adenosine (t6A), N6-methyl-N6-threonylcarbamoyl-adenosine (m6t6A), 2-methylthio-N6-threonylcarbamoyl-adenosine (ms2g6A), N6,N6-dimethyl-adenosine (m\A), N6-hydroxynorvalylcarbamoyl-adenosine (hn6A), 2-methylthio-N6-hydroxynorvalylcarbamoyl-adenosine (ms2hn6A), N6-acetyl-adenosine (ac6A), 7-methyl-adenine, 2-methylthio- adenine, 2-methoxy-adenine, a-thio-adenosine, 2'-O-methyl-adenosine (Am), N6,2'-O-dimethyl-adenosine (m6Am), N6,N6,2'-O-trimethyl-adenosine (m62Am), 1,2'-O-dimethyl-adenosine (m1Am), 2'-O-ribosyladenosine (phosphate) (Ar(p)), 2-amino-N6-methyl-purine, 1-thio-adenosine, 8-azidoadenosine, 2'-F-ara-adenosine, 2'-F-adenosine, 2'-OH-ara-adenosine, N6-(l 9-amino-pentaoxanonadecyl)-adenosine, or any combination thereof.

[0239] In some embodiments, a polyribonucleotide comprising 2'-O-acetylated ribose and modified adenines further comprises one or more modified nucleobases other than modified adenines, eg, as described herein.

[0240] In some embodiments, the polyribonucleotide comprising 2'-O-acetylated ribose further comprises a modified cytosine. In some embodiments, the modified cytosine is selected from the group consisting of 5-aza-cytidine, 6-aza-cytidine, pseudoisocytidine, 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, and the like. 5-methyl-1-deaza-pseudoisocytidine, 1-methyl-1-deaza-pseudoisocytidine, zebularine, 5-methyl-1-deaza-pseudoisocytidine, 1 ... The-Zebularine, 5-Methyl-Zebularine, 5-Aza-2-Thio-Zebularine, 2-Thio-Zebularine, 2-Methoxy-Cytidine, 2-Methoxy-5-Methyl-Cytidine, 4-Methoxy-Pseudoisocytidine, 4-Methoxy-1-Methyl-Pseudoisocytidine, Lysidine (k2C), a-Thio-Cytidine, 2'-O-Methyl-Cytidine (Cm), 5,2'-O-Dimethyl-Cytidine (m 5Cm), N4-acetyl-2'-O-methyl-cytidine (ac4Cm), N4,2'-O-dimethylcytidine (m4Cm), 5-formyl-2'-O-methyl-cytidine (f5Cm), N4,N4,2'-O-trimethyl-cytidine (m\Cm), 1-thio-cytidine, 2'-F-ara-cytidine, 2'-F-cytidine, 2'-OH-ara-cytidine, or any combination thereof.

[0241] In some embodiments, the polyribonucleotides comprising 2'-O-acetylated ribose comprise modified cytosines, including N4-acetyl-cytidine (ac4C).

[0242] In some embodiments, a polyribonucleotide comprising 2'-O-acetylated ribose and modified cytosine further comprises one or more modified nucleobases other than modified cytosine, eg, as described herein.

[0243] In some embodiments, a polyribonucleotide comprising a 2'-O-acetylated ribose and a modified cytosine comprising Ac4C further comprises one or more modified nucleobases other than a modified cytosine comprising Ac4C, e.g., as described herein.

[0244] In some embodiments, the polyribonucleotide comprising 2'-O-acetylated ribose further comprises a modified uracil. In some embodiments, the modified uracil is selected from the group consisting of 5-hydroxymethyluridine, pseudouridine, 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, 4-thio-uridine (s4U), 5-amino-uridine, 5-halo-uridine (e.g., 5-iodo-uridine or 5-bromo-uridine), 5-methyl-uridine, 5-amino ... Lysine (m3U), 5-methoxy-uridine (mo5U), uridine 5-oxyacetic acid (cmo5U), uridine 5-oxyacetic 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 (mcm5U) s2U), 5-aminomethyl-2-thio-uridine (nm5s2U), 5-methylaminomethyl-uridine (nmm5U), 5-methylaminomethyl-2-thio-uridine (nmm5s2U), 5-methylaminomethyl-2-seleno-uridine (nmm5se2U), 5-carbamoylmethyl-uridine (ncm5U), 5-carboxymethylaminomethyl-uridine (cmnm5U), 5-carboxymethylaminomethyl-2-thio-uridine (cnmm5s2U), 5-propynyl-uridine, 1-propynyl pseudo-uridine Uridine, 5-taurinomethyl-uridine (TITI5U), 1-taurinomethyl-pseudouridine, 5-taurinomethyl-2-thio-uridine (TITI5s2U), l-taurinomethyl-4-thio-pseudouridine, 5-methyl-uridine (m5U, i.e., with the nucleobase deoxythymine), 5-methyl-2-thio-uridine (m5s2U), l-methyl-4-thio-pseudouridine, 4-thio-1-methyl-pseudouridine, 3-methyl-pseudouridine, 2-thio-1-methyl-pseudouridine,l-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-l-deaza-pseudouridine, dihydrouridine (D), dihydropseudouridine, 5,6-dihydrouridine, 5-methyldihydrouridine (m5D), 2-thio-dihydrouridine, 2-thio-dihydropseudouridine, 2-methoxy-uridine, 2-methoxy-4-thio-uridine, 4-methoxy-pseudouridine, 4-methoxy-2-thio-pseudouridine, N1-methylpseudouridine, 3-(3-amino-3-carboxypropyl)uridine (acp3U), l-methyl-3-(3-amino-3-carboxypropyl)pseudouridine, 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, 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 (cnmm5Um). m), 3,2'-O-dimethyluridine (m3Um), and 5-(isopentenylaminomethyl)-2'-O-methyl-uridine (inm5Um), 1-thio-uridine, deoxythymidine, 2'-F-ara-uridine, 2'-F-uridine, 2'-OH-ara-uridine, 5-(2-carbomethoxyvinyl)uridine, 5-[3-(1-E-propenylamino)uridine, or any combination thereof.

[0245] In some embodiments, the polyribonucleotide comprising 2'-O-acetylated ribose further comprises modified uracils, including 5-hydroxymethyluridine.

[0246] In some embodiments, the polyribonucleotide comprising 2'-O-acetylated ribose further comprises modified uracils, including N1-methylpseudouridine.

[0247] In some embodiments, a polyribonucleotide comprising 2'-O-acetylated ribose and modified uracil further comprises one or more modified nucleobases other than modified uracil, eg, as described herein.

[0248] In some embodiments, a polyribonucleotide comprising 2'-O-acetylated ribose and a modified uracil, including 5-hydroxymethyluridine, further comprises one or more modified nucleobases other than a modified uracil, including 5-hydroxymethyluridine, e.g., as described herein.

[0249] In some embodiments, a polyribonucleotide comprising 2'-O-acetylated ribose and a modified uracil, including N1-methylpseudouridine, further comprises one or more modified nucleobases other than a modified uracil, including N1-methylpseudouridine, e.g., as described herein.

[0250] In some embodiments, the polyribonucleotide comprising 2'-O-acetylated ribose further comprises a modified guanine. In some embodiments, the modified guanine is inosine (I), 1-methyl-inosine (m1I), wyosine (imG), methylwyosine (mimG), 4-demethyl-wyosine (imG-14), isowyosine (imG2), wyosine (yW), peroxywyosine (o2yW), hydroxywyosine (OHyW), undermodified hydroxywyosine (OHyW), or undermodified hydroxywyosine (OHyW). *), 7-deaza-guanosine, queuosine (Q), epoxyqueuosine (oQ), galactosylqueuosine, mannosylqueuosine (manQ), 7-cyano-7-deaza-guanosine (preQ0), 7-aminomethyl-7-deaza-guanosine (preQ1), archaeosine (G+), 7-deaza-8-aza-guanosine, 6-thioguanosine, 6-thio-7-deaza-guanosine Anosine, 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 (m1G), N2-methyl-guanosine (m2G), N2,N2-dimethyl-guanosine (m\G), N2,7-dimethyl-guanosine (m27G), N2,N2,7-dimethyl-guanosine (m2'2 '7G), 8-oxo-guanosine, 7-methyl-8-oxo-guanosine, l-methyl-6-thio-guanosine, N2-methyl-6-thio-guanosine, N2,N2-dimethyl-6-thio-guanosine, a-thio-guanosine, 2'-O-methyl-guanosine (Gm), N2-methyl-2'-O-methyl-guanosine (m2Gm), N2,N2-dimethyl-2'-O-methyl-guanosine (m22Gm), l-methyl -2'-O-methyl-guanosine (m1Gm), N2,7-dimethyl-2'-O-methylguanosine (m2'7Gm), 2'-O-methyl-inosine (Im), 1,2'-O-dimethyl-inosine (m1Im), 2'-O-ribosylguanosine (phosphate) (Gr(p)), 1-thio-guanosine, O6-methyl-guanosine, 2'-F-ara-guanosine, 2'-F-guanosine, or any combination thereof.

[0251] In some embodiments, a polyribonucleotide comprising a 2'-O-acetylated ribose and a modified guanine further comprises one or more modified nucleobases other than modified guanine, eg, as described herein.

[0252] In some embodiments, the polyribonucleotides comprising 2'-O-acetylated ribose further comprise a purine analog and / or a pyrimidine analog. In some embodiments, the polyribonucleotides comprising 2'-O-acetylated ribose comprise nucleobases, including naturally occurring and synthetic derivatives of bases, such as pyrazolo[3,4-d]pyrimidine, 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-propynyluracil and cytosine, 6-azouracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo (e.g., 8-bromo), 8-amino, These include 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo, especially 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 8-azaguanine and 8-azaadenine, deazaguanine, 7-deazaguanine, 3-deazaguanine, deazaadenine, 7-deazaadenine, 3-deazaadenine, pyrazolo[3,4-d]pyrimidines, imidazo[1,5-a]1,3,5 triazinones, 9-deazapurines, imidazo[4,5-d]pyrazines, thiazolo[4,5-d]pyrimidines, pyrazin-2-ones, 1,2,4-triazines, pyridazines, 1,3,5 triazines, or any combination thereof.

[0253] Exemplary nucleotide modifications, including nucleobase modifications, include those provided in International Patent Application WO2013 / 052523, filed October 3, 2012, the entire contents of which are incorporated herein by reference.

[0254] N4-acetylcytidine modified nucleotides In some embodiments, the polyribonucleotide comprising 2'-O-acetylated ribose further comprises a base comprising an acetyl group, the nucleoside is N4-acetylcytidine, and the modified ribonucleotide is: [ka] It has the structure:

[0255] In some embodiments, the polyribonucleotides disclosed herein comprise cytidine residues. In some embodiments, at least 1% of the cytidine residues comprise 2-O acetylated ribose. In some embodiments, no more than 90% of the cytidine residues comprise 2-O acetylated ribose.

[0256] In some embodiments, the polyribonucleotide disclosed herein comprises cytidine residues, and at least 5% of the cytidine residues comprise N4-acetylcytidine. In some embodiments, at least 1% of the N4-acetylcytidine residues comprise 2-O acetylated ribose. In some embodiments, 90% or less of the N4-acetylcytidine residues comprise 2-O acetylated ribose.

[0257] In some embodiments, the polyribonucleotide disclosed herein comprises cytidine residues, and less than 100% of the cytidine residues comprise N4-acetylcytidine. In some embodiments, at least 1% of the N4-acetylcytidine residues comprise 2-O acetylated ribose. In some embodiments, 90% or less of the N4-acetylcytidine residues comprise 2-O acetylated ribose.

[0258] In some embodiments, the polyribonucleotide disclosed herein comprises cytidine residues, and at least 5% of the cytidine residues in the polyribonucleotide comprise N4-acetylcytidine. In some embodiments, the polyribonucleotide disclosed herein comprises cytidine residues, and at least 10% of the cytidine residues in the polyribonucleotide comprise N4-acetylcytidine. In some embodiments, the polyribonucleotide disclosed herein comprises cytidine residues, and at least 15% of the cytidine residues in the polyribonucleotide comprise N4-acetylcytidine. In some embodiments, the polyribonucleotide disclosed herein comprises cytidine residues, and at least 20% of the cytidine residues in the polyribonucleotide comprise N4-acetylcytidine. In some embodiments, the polyribonucleotide disclosed herein comprises cytidine residues, and at least 25% of the cytidine residues in the polyribonucleotide comprise N4-acetylcytidine. In some embodiments, the polyribonucleotide disclosed herein comprises cytidine residues, and at least 30% of the cytidine residues in the polyribonucleotide comprise N4-acetylcytidine. In some embodiments, the polyribonucleotide disclosed herein comprises cytidine residues, and at least 35% of the cytidine residues in the polyribonucleotide comprise N4-acetylcytidine. In some embodiments, the polyribonucleotide disclosed herein comprises cytidine residues, and at least 40% of the cytidine residues in the polyribonucleotide comprise N4-acetylcytidine. In some embodiments, the polyribonucleotide disclosed herein comprises cytidine residues, and at least 45% of the cytidine residues in the polyribonucleotide comprise N4-acetylcytidine. In some embodiments, the polyribonucleotide disclosed herein comprises cytidine residues, and at least 50% of the cytidine residues in the polyribonucleotide comprise N4-acetylcytidine. In some embodiments, the polyribonucleotides disclosed herein comprise cytidine residues, wherein at least 55% of the cytidine residues in the polyribonucleotide comprise N4-acetylcytidine.In some embodiments, the polyribonucleotide disclosed herein comprises cytidine residues, and at least 60% of the cytidine residues in the polyribonucleotide comprise N4-acetylcytidine. In some embodiments, the polyribonucleotide disclosed herein comprises cytidine residues, and at least 65% of the cytidine residues in the polyribonucleotide comprise N4-acetylcytidine. In some embodiments, the polyribonucleotide disclosed herein comprises cytidine residues, and at least 70% of the cytidine residues in the polyribonucleotide comprise N4-acetylcytidine. In some embodiments, the polyribonucleotide disclosed herein comprises cytidine residues, and at least 75% of the cytidine residues in the polyribonucleotide comprise N4-acetylcytidine. In some embodiments, the polyribonucleotide disclosed herein comprises cytidine residues, and at least 80% of the cytidine residues in the polyribonucleotide comprise N4-acetylcytidine. In some embodiments, the polyribonucleotide disclosed herein comprises cytidine residues, and at least 85% of the cytidine residues in the polyribonucleotide comprise N4-acetylcytidine. In some embodiments, the polyribonucleotide disclosed herein comprises cytidine residues, and at least 90% of the cytidine residues in the polyribonucleotide comprise N4-acetylcytidine. In some embodiments, the polyribonucleotide disclosed herein comprises cytidine residues, and at least 95% of the cytidine residues in the polyribonucleotide comprise N4-acetylcytidine. In some embodiments, at least 1% of the N4-acetylcytidine residues comprise 2-O acetylated ribose. In some embodiments, 90% or less of the N4-acetylcytidine residues comprise 2-O acetylated ribose.

[0259] In some embodiments, the polyribonucleotide disclosed herein comprises cytidine residues. In some embodiments, about 5% to 99%, about 5% to 95%, about 5% to 90%, about 5% to 85%, about 5% to 80%, about 5% to 75%, about 5% to 70%, about 5% to 65%, about 5% to 60%, about 5% to 55%, about 5% to 50%, about 5% to 45%, about 5% to 40%, about 5% to 35%, about 5% to 30%, about 5% to 25%, about 5% to 20%, about 5% to 15%, about 5% to 5% of the cytidine residues in the polyribonucleotide. % to 10%, about 10% to 99%, about 15% to 99%, about 20% to 99%, about 25% to 99%, about 30% to 99%, about 35% to 99%, about 40% to 99%, about 45% to 99%, about 50% to 99%, about 55% to 99%, about 60% to 99%, about 65% to 99%, about 70% to 99%, about 80% to 99%, about 85% to 99%, about 90% to 99%, or about 95% to 99% of the N4-acetylcytidine residues comprise N4-acetylcytidine. In some embodiments, at least 1% of the N4-acetylcytidine residues comprise 2-O acetylated ribose. In some embodiments, 90% or less of the N4-acetylcytidine residues comprise 2-O acetylated ribose.

[0260] In some embodiments, the polyribonucleotide disclosed herein comprises cytidine residues, and more than about 60% of the cytidine residues in the polyribonucleotide comprise N4-acetylcytidine. In some embodiments, the polyribonucleotide disclosed herein comprises cytidine residues, and more than about 65% of the cytidine residues in the polyribonucleotide comprise N4-acetylcytidine. In some embodiments, the polyribonucleotide disclosed herein comprises cytidine residues, and more than about 70% of the cytidine residues in the polyribonucleotide comprise N4-acetylcytidine. In some embodiments, the polyribonucleotide disclosed herein comprises cytidine residues, and more than about 75% of the cytidine residues in the polyribonucleotide comprise N4-acetylcytidine. In some embodiments, the polyribonucleotide disclosed herein comprises cytidine residues, and more than about 80% of the cytidine residues in the polyribonucleotide comprise N4-acetylcytidine. In some embodiments, the polyribonucleotide disclosed herein comprises cytidine residues, and more than about 85% of the cytidine residues in the polyribonucleotide comprise N4-acetylcytidine. In some embodiments, the polyribonucleotide disclosed herein comprises cytidine residues, and more than about 90% of the cytidine residues in the polyribonucleotide comprise N4-acetylcytidine. In some embodiments, the polyribonucleotide disclosed herein comprises cytidine residues, and more than about 95% of the cytidine residues in the polyribonucleotide comprise N4-acetylcytidine. In some embodiments, the polyribonucleotide disclosed herein comprises cytidine residues, and more than about 99% of the cytidine residues in the polyribonucleotide comprise N4-acetylcytidine. In some embodiments, the polyribonucleotide disclosed herein comprises cytidine residues, and more than about 5% of the cytidine residues in the polyribonucleotide comprise N4-acetylcytidine. In some embodiments, at least 1% of the N4-acetylcytidine residues comprise 2-O acetylated ribose. In some embodiments, up to 90% of the N4-acetylcytidine residues contain a 2-O acetylated ribose.

[0261] In some embodiments, the polyribonucleotide disclosed herein comprises cytidine residues, and about 10% of the cytidine residues in the polyribonucleotide comprise N4-acetylcytidine. In some embodiments, the polyribonucleotide disclosed herein comprises cytidine residues, and about 15% of the cytidine residues in the polyribonucleotide comprise N4-acetylcytidine. In some embodiments, the polyribonucleotide disclosed herein comprises cytidine residues, and about 20% of the cytidine residues in the polyribonucleotide comprise N4-acetylcytidine. In some embodiments, the polyribonucleotide disclosed herein comprises cytidine residues, and about 25% of the cytidine residues in the polyribonucleotide comprise N4-acetylcytidine. In some embodiments, the polyribonucleotide disclosed herein comprises cytidine residues, and about 30% of the cytidine residues in the polyribonucleotide comprise N4-acetylcytidine. In some embodiments, the polyribonucleotide disclosed herein comprises cytidine residues, and about 35% of the cytidine residues in the polyribonucleotide comprise N4-acetylcytidine. In some embodiments, the polyribonucleotide disclosed herein comprises cytidine residues, and about 40% of the cytidine residues in the polyribonucleotide comprise N4-acetylcytidine. In some embodiments, the polyribonucleotide disclosed herein comprises cytidine residues, and about 45% of the cytidine residues in the polyribonucleotide comprise N4-acetylcytidine. In some embodiments, the polyribonucleotide disclosed herein comprises cytidine residues, and about 50% of the cytidine residues in the polyribonucleotide comprise N4-acetylcytidine. In some embodiments, the polyribonucleotide disclosed herein comprises cytidine residues, and about 55% of the cytidine residues in the polyribonucleotide comprise N4-acetylcytidine. In some embodiments, the polyribonucleotides disclosed herein comprise cytidine residues, and about 60% of the cytidine residues in the polyribonucleotide comprise N4-acetylcytidine.In some embodiments, the polyribonucleotides disclosed herein comprise cytidine residues, and about 65% of the cytidine residues in the polyribonucleotide comprise N4-acetylcytidine.In some embodiments, the polyribonucleotide disclosed herein comprises cytidine residues, and about 75% of the cytidine residues in the polyribonucleotide comprise N4-acetylcytidine. In some embodiments, the polyribonucleotide disclosed herein comprises cytidine residues, and about 80% of the cytidine residues in the polyribonucleotide comprise N4-acetylcytidine. In some embodiments, the polyribonucleotide disclosed herein comprises cytidine residues, and about 85% of the cytidine residues in the polyribonucleotide comprise N4-acetylcytidine. In some embodiments, the polyribonucleotide disclosed herein comprises cytidine residues, and about 90% of the cytidine residues in the polyribonucleotide comprise N4-acetylcytidine. In some embodiments, the polyribonucleotide disclosed herein comprises cytidine residues, and about 95% of the cytidine residues in the polyribonucleotide comprise N4-acetylcytidine. In some embodiments, the polyribonucleotide disclosed herein comprises cytidine residues, and about 99% of the cytidine residues in the polyribonucleotide comprise N4-acetylcytidine. In some embodiments, the polyribonucleotide disclosed herein comprises cytidine residues, and 100% of the cytidine residues in the polyribonucleotide comprise N4-acetylcytidine. In some embodiments, at least 1% of the N4-acetylcytidine residues comprise 2-O acetylated ribose. In some embodiments, 90% or less of the N4-acetylcytidine residues comprise 2-O acetylated ribose.

[0262] In some embodiments, a polyribonucleotide in which at least 5% of the cytidine residues are N4-acetylcytidine comprises at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least about 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% 2'-O-acetylated ribose. In some embodiments, at least 1% of the N4-acetylcytidine residues comprise 2-O acetylated ribose. In some embodiments, 90% or less of the N4-acetylcytidine residues comprise 2-O acetylated ribose.

[0263] In some embodiments, a polyribonucleotide in which at least 5% of the cytidine residues are N4-acetylcytidine comprises at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% 2'-O-acetylated ribose. In some embodiments, at least 1% of the N4-acetylcytidine residues comprise 2-O acetylated ribose. In some embodiments, 90% or less of the N4-acetylcytidine residues comprise 2-O acetylated ribose.

[0264] In some embodiments, the polyribonucleotide in which at least 5% of the cytidine residues are N4-acetylcytidine contains 100% 2'-O-acetylated ribose.

[0265] In some embodiments, the polyribonucleotide in which at least 5% of the cytidine residues are N4-acetylcytidine is about 5% to 99%, about 5% to 95%, about 5% to 90%, about 5% to 85%, about 5% to 80%, about 5% to 75%, about 5% to 70%, about 5% to 65%, about 5% to 60%, about 5% to 55%, about 5% to 50%, about 5% to 45%, about 5% to 40%, about 5% to 35%, about 5% to 30%, about 5% to 25%, about 5% to 20%, About 5% to 15%, about 5% to 10%, about 10% to 99%, about 15% to 99%, about 20% to 99%, about 25% to 99%, about 30% to 99%, about 35% to 99%, about 40% to 99%, about 45% to 99%, about 50% to 99%, about 55% to 99%, about 60% to 99%, about 65% to 99%, about 70% to 99%, about 80% to 99%, about 85% to 99%, about 90% to 99%, or about 95% to 99% 2'-O-acetylated ribose. In some embodiments, at least 1% of the N4-acetylcytidine residues contain 2-O acetylated ribose. In some embodiments, 90% or less of the N4-acetylcytidine residues contain 2-O acetylated ribose.

[0266] In some embodiments, a polyribonucleotide in which at least 5% of the cytidine residues are N4-acetylcytidine is about 76% to about 100%, about 77% to about 100%, about 78% to about 100%, about 79% to about 100%, about 80% to about 100%, about 81% to about 100%, about 82% to about 100%, about 83% to about 100%, about 84% to about 100%, about 85% to about 100%, about 86% to about 100%, about 87% to about 100%, about 88% to about 100%, about 89% to about 100%, about 90% to about 100%, about 91% to about 100%, about 92% to about 100%, about 93% to about 100%, about 94% to about 100%, about 95% to about 100%, about 96% to about 100%, about 97% to about 100%, about 98% to about 100%, about 99% to about 100%, about 10 ... About 100%, about 87% to about 100%, about 88% to about 100%, about 89% to about 100%, about 90% to about 100%, about 91% to about 100%, about 92% to about 100%, about 93% to about 100%, about 94% to about 100%, about 95% to about 100%, about 96% to about 100%, about 97% to about 100%, about 98% to about 100%, about 99% to about 100% 2'-O-acetylated ribose. In some embodiments, at least 1% of the N4-acetylcytidine residues contain 2-O acetylated ribose. In some embodiments, 90% or less of the N4-acetylcytidine residues contain 2-O acetylated ribose.

[0267] In some embodiments, a polyribonucleotide comprising cytidine residues including 2-O acetylated ribose and N4-acetylcytidine (e.g., a portion of N4-acetylcytidine having 2-O ribose acetylation) further comprises one or more modified nucleotides.

[0268] 5-Hydroxymethyl modified nucleotides In some embodiments, the polyribonucleotide comprising 2'-O-acetylated ribose further comprises a base comprising a hydroxymethyl group, the nucleoside is 5-hydroxymethyluridine, and the modified ribonucleotide is [ka] It has the structure:

[0269] In some embodiments, the polyribonucleotides disclosed herein comprise uridine residues. In some embodiments, at least 1% of the uridine residues comprise 2-O acetylated ribose. In some embodiments, no more than 90% of the uridine residues comprise 2-O acetylated ribose.

[0270] In some embodiments, the polyribonucleotides disclosed herein comprise uridine, and at least 5% of the uridine residues comprise 5-hydroxymethyluridine. In some embodiments, at least 1% of the 5-hydroxymethyluridine residues comprise 2-O acetylated ribose. In some embodiments, no more than 90% of the 5-hydroxymethyluridine residues comprise 2-O acetylated ribose.

[0271] In some embodiments, the polyribonucleotides disclosed herein comprise uridine, and less than 100% of the uridine residues comprise 5-hydroxymethyluridine. In some embodiments, at least 1% of the 5-hydroxymethyluridine residues comprise 2-O acetylated ribose. In some embodiments, 90% or less of the 5-hydroxymethyluridine residues comprise 2-O acetylated ribose.

[0272] In some embodiments, the polyribonucleotides disclosed herein comprise uridine residues, and at least 5% of the uridine residues in the polyribonucleotide comprise 5-hydroxymethyluridine. In some embodiments, the polyribonucleotides disclosed herein comprise uridine residues, and at least 10% of the uridine residues in the polyribonucleotide comprise 5-hydroxymethyluridine. In some embodiments, the polyribonucleotides disclosed herein comprise uridine residues, and at least 15% of the uridine residues in the polyribonucleotide comprise 5-hydroxymethyluridine. In some embodiments, the polyribonucleotides disclosed herein comprise uridine residues, and at least 20% of the uridine residues in the polyribonucleotide comprise 5-hydroxymethyluridine. In some embodiments, the polyribonucleotides disclosed herein comprise uridine residues, and at least 25% of the uridine residues in the polyribonucleotide comprise 5-hydroxymethyluridine. In some embodiments, the polyribonucleotides disclosed herein comprise uridine residues, and at least 30% of the uridine residues in the polyribonucleotide comprise 5-hydroxymethyluridine. In some embodiments, the polyribonucleotides disclosed herein comprise uridine residues, and at least 35% of the uridine residues in the polyribonucleotide comprise 5-hydroxymethyluridine. In some embodiments, the polyribonucleotides disclosed herein comprise uridine residues, and at least 40% of the uridine residues in the polyribonucleotide comprise 5-hydroxymethyluridine. In some embodiments, the polyribonucleotides disclosed herein comprise uridine residues, and at least 45% of the uridine residues in the polyribonucleotide comprise 5-hydroxymethyluridine. In some embodiments, the polyribonucleotides disclosed herein comprise uridine residues, and at least 50% of the uridine residues in the polyribonucleotide comprise 5-hydroxymethyluridine. In some embodiments, the polyribonucleotides disclosed herein comprise uridine residues, and at least 55% of the uridine residues in the polyribonucleotide comprise 5-hydroxymethyluridine.In some embodiments, the polyribonucleotides disclosed herein comprise uridine residues, and at least 60% of the uridine residues in the polyribonucleotide comprise 5-hydroxymethyluridine. In some embodiments, the polyribonucleotides disclosed herein comprise uridine residues, and at least 65% of the uridine residues in the polyribonucleotide comprise 5-hydroxymethyluridine. In some embodiments, the polyribonucleotides disclosed herein comprise uridine residues, and at least 70% of the uridine residues in the polyribonucleotide comprise 5-hydroxymethyluridine. In some embodiments, the polyribonucleotides disclosed herein comprise uridine residues, and at least 75% of the uridine residues in the polyribonucleotide comprise 5-hydroxymethyluridine. In some embodiments, the polyribonucleotides disclosed herein comprise uridine residues, and at least 80% of the uridine residues in the polyribonucleotide comprise 5-hydroxymethyluridine. In some embodiments, the polyribonucleotides disclosed herein comprise uridine residues, and at least 85% of the uridine residues in the polyribonucleotide comprise 5-hydroxymethyluridine. In some embodiments, the polyribonucleotides disclosed herein comprise uridine residues, and at least 90% of the uridine residues in the polyribonucleotide comprise 5-hydroxymethyluridine. In some embodiments, the polyribonucleotides disclosed herein comprise uridine residues, and at least 95% of the uridine residues in the polyribonucleotide comprise 5-hydroxymethyluridine. In some embodiments, the polyribonucleotides disclosed herein comprise uridine residues, and at least 99% of the uridine residues in the polyribonucleotide comprise 5-hydroxymethyluridine. In some embodiments, at least 1% of the 5-hydroxymethyluridine residues comprise 2-O acetylated ribose. In some embodiments, 90% or less of the 5-hydroxymethyluridine residues comprise 2-O acetylated ribose.

[0273] In some embodiments, the polyribonucleotide disclosed herein contains uridine residues. In some embodiments, the uridine residues in the polyribonucleotide are about 5% to 99%, about 5% to 95%, about 5% to 90%, about 5% to 85%, about 5% to 80%, about 5% to 75%, about 5% to 70%, about 5% to 65%, about 5% to 60%, about 5% to 55%, about 5% to 50%, about 5% to 45%, about 5% to 40%, about 5% to 35%, about 5% to 30%, about 5% to 25%, about 5% to 20%, about 5% to 15%, about 5% to 20%, about 5% to 3 ... 10%, about 10%-99%, about 15%-99%, about 20%-99%, about 25%-99%, about 30%-99%, about 35%-99%, about 40%-99%, about 45%-99%, about 50%-99%, about 55%-99%, about 60%-99%, about 65%-99%, about 70%-99%, about 80%-99%, about 85%-99%, about 90%-99%, or about 95%-99% comprise 5-hydroxymethyluridine. In some embodiments, at least 1% of the 5-hydroxymethyluridine residues comprise 2-O acetylated ribose. In some embodiments, 90% or less of the 5-hydroxymethyluridine residues comprise 2-O acetylated ribose.

[0274] In some embodiments, the polyribonucleotides disclosed herein comprise uridine residues, and more than 60% of the uridine residues in the polyribonucleotide comprise 5-hydroxymethyluridine. In some embodiments, the polyribonucleotides disclosed herein comprise uridine residues, and more than 65% of the uridine residues in the polyribonucleotide comprise 5-hydroxymethyluridine. In some embodiments, the polyribonucleotides disclosed herein comprise uridine residues, and more than 70% of the uridine residues in the polyribonucleotide comprise 5-hydroxymethyluridine. In some embodiments, the polyribonucleotides disclosed herein comprise uridine residues, and more than 75% of the uridine residues in the polyribonucleotide comprise 5-hydroxymethyluridine. In some embodiments, the polyribonucleotides disclosed herein comprise uridine residues, and more than 80% of the uridine residues in the polyribonucleotide comprise 5-hydroxymethyluridine. In some embodiments, the polyribonucleotides disclosed herein comprise uridine residues, and more than 85% of the uridine residues in the polyribonucleotide comprise 5-hydroxymethyluridine. In some embodiments, the polyribonucleotides disclosed herein comprise uridine residues, and more than 90% of the uridine residues in the polyribonucleotide comprise 5-hydroxymethyluridine. In some embodiments, the polyribonucleotides disclosed herein comprise uridine residues, and more than 95% of the uridine residues in the polyribonucleotide comprise 5-hydroxymethyluridine. In some embodiments, the polyribonucleotides disclosed herein comprise uridine residues, and more than 99% of the uridine residues in the polyribonucleotide comprise 5-hydroxymethyluridine. In some embodiments, at least 1% of the 5-hydroxymethyluridine residues comprise 2-O acetylated ribose. In some embodiments, 90% or less of the 5-hydroxymethyluridine residues comprise 2-O acetylated ribose.

[0275] In some embodiments, the polyribonucleotides disclosed herein comprise uridine residues, and about 5% of the uridine residues in the polyribonucleotide comprise 5-hydroxymethyluridine. In some embodiments, the polyribonucleotides disclosed herein comprise uridine residues, and about 10% of the uridine residues in the polyribonucleotide comprise 5-hydroxymethyluridine. In some embodiments, the polyribonucleotides disclosed herein comprise uridine residues, and about 15% of the uridine residues in the polyribonucleotide comprise 5-hydroxymethyluridine. In some embodiments, the polyribonucleotides disclosed herein comprise uridine residues, and about 20% of the uridine residues in the polyribonucleotide comprise 5-hydroxymethyluridine. In some embodiments, the polyribonucleotides disclosed herein comprise uridine residues, and about 25% of the uridine residues in the polyribonucleotide comprise 5-hydroxymethyluridine. In some embodiments, the polyribonucleotides disclosed herein comprise uridine residues, and about 30% of the uridine residues in the polyribonucleotide comprise 5-hydroxymethyluridine. In some embodiments, the polyribonucleotides disclosed herein comprise uridine residues, and about 35% of the uridine residues in the polyribonucleotide comprise 5-hydroxymethyluridine. In some embodiments, the polyribonucleotides disclosed herein comprise uridine residues, and about 40% of the uridine residues in the polyribonucleotide comprise 5-hydroxymethyluridine. In some embodiments, the polyribonucleotides disclosed herein comprise uridine residues, and about 45% of the uridine residues in the polyribonucleotide comprise 5-hydroxymethyluridine. In some embodiments, the polyribonucleotides disclosed herein comprise uridine residues, and about 50% of the uridine residues in the polyribonucleotide comprise 5-hydroxymethyluridine. In some embodiments, the polyribonucleotides disclosed herein comprise uridine residues, and about 55% of the uridine residues in the polyribonucleotide comprise 5-hydroxymethyluridine.In some embodiments, the polyribonucleotides disclosed herein comprise uridine residues, and about 60% of the uridine residues in the polyribonucleotide comprise 5-hydroxymethyluridine. In some embodiments, the polyribonucleotides disclosed herein comprise uridine residues, and about 65% of the uridine residues in the polyribonucleotide comprise 5-hydroxymethyluridine. In some embodiments, the polyribonucleotides disclosed herein comprise uridine residues, and about 75% of the uridine residues in the polyribonucleotide comprise 5-hydroxymethyluridine. In some embodiments, the polyribonucleotides disclosed herein comprise uridine residues, and about 80% of the uridine residues in the polyribonucleotide comprise 5-hydroxymethyluridine. In some embodiments, the polyribonucleotides disclosed herein comprise uridine residues, and about 85% of the uridine residues in the polyribonucleotide comprise 5-hydroxymethyluridine. In some embodiments, the polyribonucleotides disclosed herein comprise uridine residues, and about 90% of the uridine residues in the polyribonucleotide comprise 5-hydroxymethyluridine. In some embodiments, the polyribonucleotides disclosed herein comprise uridine residues, and about 95% of the uridine residues in the polyribonucleotide comprise 5-hydroxymethyluridine. In some embodiments, the polyribonucleotides disclosed herein comprise uridine residues, and about 99% of the uridine residues in the polyribonucleotide comprise 5-hydroxymethyluridine. In some embodiments, at least 1% of the 5-hydroxymethyluridine residues comprise 2-O acetylated ribose. In some embodiments, no more than 90% of the 5-hydroxymethyluridine residues comprise 2-O acetylated ribose.

[0276] In some embodiments, the polyribonucleotides disclosed herein comprise uridine residues, and 100% of the uridine residues in the polyribonucleotide comprise 5-hydroxymethyluridine. In some embodiments, at least 1% of the 5-hydroxymethyluridine residues comprise 2-O acetylated ribose. In some embodiments, 90% or less of the 5-hydroxymethyluridine residues comprise 2-O acetylated ribose.

[0277] In some embodiments, a polyribonucleotide in which at least 5% of the uridine residues are 5-hydroxymethyluridine comprises at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least about 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% 2'-O-acetylated ribose. In some embodiments, at least 1% of the 5-hydroxymethyluridine residues comprise 2-O acetylated ribose. In some embodiments, 90% or less of the 5-hydroxymethyluridine residues comprise 2-O acetylated ribose.

[0278] In some embodiments, a polyribonucleotide in which at least 5% of the uridine residues are 5-hydroxymethyluridine comprises at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% 2'-O-acetylated ribose. In some embodiments, at least 1% of the 5-hydroxymethyluridine residues comprise 2-O acetylated ribose. In some embodiments, 90% or less of the 5-hydroxymethyluridine residues comprise 2-O acetylated ribose.

[0279] In some embodiments, the polyribonucleotide in which at least 5% of the uridine residues are 5-hydroxymethyluridine contains 100% 2'-O-acetylated ribose.

[0280] In some embodiments, a polyribonucleotide in which at least 5% of the uridine residues are 5-hydroxymethyluridine is about 5% to 99%, about 5% to 95%, about 5% to 90%, about 5% to 85%, about 5% to 80%, about 5% to 75%, about 5% to 70%, about 5% to 65%, about 5% to 60%, about 5% to 55%, about 5% to 50%, about 5% to 45%, about 5% to 40%, about 5% to 35%, about 5% to 30%, about 5% to 25%, about 5% to 20%. , about 5%-15%, about 5%-10%, about 10%-99%, about 15%-99%, about 20%-99%, about 25%-99%, about 30%-99%, about 35%-99%, about 40%-99%, about 45%-99%, about 50%-99%, about 55%-99%, about 60%-99%, about 65%-99%, about 70%-99%, about 80%-99%, about 85%-99%, about 90%-99%, or about 95%-99% 2'-O-acetylated ribose. In some embodiments, at least 1% of the 5-hydroxymethyluridine residues comprise 2-O acetylated ribose. In some embodiments, 90% or less of the 5-hydroxymethyluridine residues comprise 2-O acetylated ribose.

[0281] In some embodiments, a polyribonucleotide in which at least 5% of the uridine residues are 5-hydroxymethyluridine is about 76% to about 100%, about 77% to about 100%, about 78% to about 100%, about 79% to about 100%, about 80% to about 100%, about 81% to about 100%, about 82% to about 100%, about 83% to about 100%, about 84% to about 100%, about 85% to about 100%, about 86% to about 100%, about 87% to about 100%, about 88% to about 100%, about 89% to about 100%, about 90% to about 100%, about 91% to about 100%, about 92% to about 100%, about 93% to about 100%, about 94% to about 100%, about 95% to about 100%, about 96% to about 100%, about 97% to about 100%, about 98% to about 100%, about 99% to about 100%, about 10 ... % to about 100%, about 87% to about 100%, about 88% to about 100%, about 89% to about 100%, about 90% to about 100%, about 91% to about 100%, about 92% to about 100%, about 93% to about 100%, about 94% to about 100%, about 95% to about 100%, about 96% to about 100%, about 97% to about 100%, about 98% to about 100%, or about 99% to about 100% 2'-O-acetylated ribose. In some embodiments, at least 1% of the 5-hydroxymethyluridine residues comprise 2-O-acetylated ribose. In some embodiments, 90% or less of the 5-hydroxymethyluridine residues comprise 2-O-acetylated ribose.

[0282] In some embodiments, a polyribonucleotide comprising uridine residues including 2-O acetylated ribose and 5-hydroxymethyluridine (e.g., a portion of 5-hydroxymethyluridine having 2-O ribose acetylation) further comprises one or more modified nucleotides.

[0283] N1-Methylpseudouridine-modified nucleotides In some embodiments, the polyribonucleotide comprising 2'-O-acetylated ribose further comprises a base comprising a methyl group, the nucleoside is N1-methylpseudouridine (m1Ψ), and the modified ribonucleotide is: [ka] It has the structure:

[0284] In some embodiments, the polyribonucleotides comprising 2'-O-acetylated ribose comprise uridine residues, and at least 5% of the uridine residues comprise N1-methylpseudouridine. In some embodiments, at least 1% of the N1-methylpseudouridine residues comprise 2-O acetylated ribose. In some embodiments, no more than 90% of the N1-methylpseudouridine residues comprise 2-O acetylated ribose.

[0285] In some embodiments, the polyribonucleotide comprising 2'-O-acetylated ribose comprises uridine residues, and less than 100% of the uridine residues in the polyribonucleotide comprise N1-methylpseudouridine. In some embodiments, at least 1% of the N1-methylpseudouridine residues comprise 2-O acetylated ribose. In some embodiments, 90% or less of the N1-methylpseudouridine residues comprise 2-O acetylated ribose.

[0286] In some embodiments, the polyribonucleotides comprising 2'-O-acetylated ribose comprise uridine residues, where at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 99% of the uridine residues in the polyribonucleotide comprise N1-methylpseudouridine. In some embodiments, the polyribonucleotides comprising 2'-O-acetylated ribose comprise uridine residues, where at least 10% of the uridine residues in the polyribonucleotide comprise N1-methylpseudouridine.

[0287] In some embodiments, the polyribonucleotide containing 2'-O-acetylated ribose contains uridine residues, and the uridine residues are about 5% to 99%, about 5% to 95%, about 5% to 90%, about 5% to 85%, about 5% to 80%, about 5% to 75%, about 5% to 70%, about 5% to 65%, about 5% to 60%, about 5% to 55%, about 5% to 50%, about 5% to 45%, about 5% to 40%, about 5% to 35%, about 5% to 30%, about 5% to 25%, about 5% to 20%, ... , about 5% to 15%, about 5% to 10%, about 10% to 99%, about 15% to 99%, about 20% to 99%, about 25% to 99%, about 30% to 99%, about 35% to 99%, about 40% to 99%, about 45% to 99%, about 50% to 99%, about 55% to 99%, about 60% to 99%, about 65% to 99%, about 70% to 99%, about 80% to 99%, about 85% to 99%, about 90% to 99%, or about 95% to 99% comprises N1-methylpseudouridine. In some embodiments, the polyribonucleotide comprising 2'-O-acetylated ribose comprises uridine residues, and at least 10% of the uridine residues in the polyribonucleotide comprise N1-methylpseudouridine.

[0288] In some embodiments, the polyribonucleotides comprising 2'-O-acetylated ribose comprise uridine residues, where more than 60%, no more than 70%, no more than 80%, no more than 90%, no more than 95%, or no more than 99% of the uridine residues comprise N1-methylpseudouridine. In some embodiments, the polyribonucleotides comprising 2'-O-acetylated ribose comprise uridine residues, where at least 10% of the uridine residues in the polyribonucleotide comprise N1-methylpseudouridine.

[0289] In some embodiments, the polyribonucleotides comprising 2'-O-acetylated ribose comprise uridine residues, and about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, or about 99% of the uridine residues in the polyribonucleotide comprise N1-methylpseudouridine. In some embodiments, the polyribonucleotides comprising 2'-O-acetylated ribose comprise uridine residues, and at least 10% of the uridine residues in the polyribonucleotide comprise N1-methylpseudouridine.

[0290] In some embodiments, the polyribonucleotides comprising 2'-O-acetylated ribose comprise uridine residues, and 100% of the uridine residues in the polyribonucleotide comprise N1-methylpseudouridine. In some embodiments, the polyribonucleotides comprising 2'-O-acetylated ribose comprise uridine residues, and at least 10% of the uridine residues in the polyribonucleotide comprise N1-methylpseudouridine.

[0291] In some embodiments, a polyribonucleotide in which at least 5% of the uridine residues are N1-methylpseudouridine comprises at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least about 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% 2'-O-acetylated ribose. In some embodiments, a polyribonucleotide comprising 2'-O-acetylated ribose comprises uridine residues, and at least 10% of the uridine residues in the polyribonucleotide comprise N1-methylpseudouridine.

[0292] In some embodiments, a polyribonucleotide in which at least 5% of the uridine residues are N1-methylpseudouridine comprises at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% 2'-O-acetylated ribose. In some embodiments, a polyribonucleotide comprising 2'-O-acetylated ribose comprises uridine residues, and at least 10% of the uridine residues in the polyribonucleotide comprise N1-methylpseudouridine.

[0293] In some embodiments, a polyribonucleotide in which at least 5% of the uridine residues are N1-methylpseudouridine comprises 100% 2'-O-acetylated ribose. In some embodiments, a polyribonucleotide that comprises 2'-O-acetylated ribose comprises uridine residues, and at least 10% of the uridine residues in the polyribonucleotide comprise N1-methylpseudouridine.

[0294] In some embodiments, a polyribonucleotide in which at least 5% of the uridine residues are N1-methylpseudouridine is about 5% to 99%, about 5% to 95%, about 5% to 90%, about 5% to 85%, about 5% to 80%, about 5% to 75%, about 5% to 70%, about 5% to 65%, about 5% to 60%, about 5% to 55%, about 5% to 50%, about 5% to 45%, about 5% to 40%, about 5% to 35%, about 5% to 30%, about 5% to 25%, about 5% to 20%. , about 5%-15%, about 5%-10%, about 10%-99%, about 15%-99%, about 20%-99%, about 25%-99%, about 30%-99%, about 35%-99%, about 40%-99%, about 45%-99%, about 50%-99%, about 55%-99%, about 60%-99%, about 65%-99%, about 70%-99%, about 80%-99%, about 85%-99%, about 90%-99%, or about 95%-99% 2'-O-acetylated ribose. In some embodiments, the polyribonucleotide comprising 2'-O-acetylated ribose comprises uridine residues, and at least 10% of the uridine residues in the polyribonucleotide comprise N1-methylpseudouridine.

[0295] In some embodiments, the polyribonucleotide in which at least 5% of the uridine residues are N1-methylpseudouridine is about 76% to about 100%, about 77% to about 100%, about 78% to about 100%, about 79% to about 100%, about 80% to about 100%, about 81% to about 100%, about 82% to about 100%, about 83% to about 100%, about 84% to about 100%, about 85% to about 100%, about 86% to about 100%, about 87% to about 100%, about 88% to about 100%, about 89% to about 100%, about 90% to about 100%, about 91% to about 100%, about 92% to about 100%, about 93% to about 100%, about 94% to about 100%, about 95% to about 100%, about 96% to about 100%, about 97% to about 100%, about 98% to about 100%, about 99% to about 100%, about 10 ... % to about 100%, about 87% to about 100%, about 88% to about 100%, about 89% to about 100%, about 90% to about 100%, about 91% to about 100%, about 92% to about 100%, about 93% to about 100%, about 94% to about 100%, about 95% to about 100%, about 96% to about 100%, about 97% to about 100%, about 98% to about 100%, or about 99% to about 100% 2'-O-acetylated ribose. In some embodiments, the polyribonucleotide comprising 2'-O-acetylated ribose comprises uridine residues, and at least 10% of the uridine residues in the polyribonucleotide comprise N1-methylpseudouridine.

[0296] In some embodiments, a polyribonucleotide comprising uridine residues including 2-O acetylated ribose and N1-methylpseudouridine (e.g., a portion of N1-methylpseudouridine having 2-O ribose acetylation) further comprises one or more modified nucleotides.

[0297] composition Among other things, the present disclosure provides compositions. The compositions disclosed herein include one or more polyribonucleotides that include one or more modified ribonucleotides that include 2'-O-acetylated ribose.

[0298] In some embodiments, a composition comprising one or more polyribonucleotides comprising 2'-O-acetylated ribose is or comprises a pharmaceutical composition.

[0299] In some embodiments, the composition comprising one or more polyribonucleotides comprising 2'-O-acetylated ribose is or comprises an immunogenic composition.An immunogenic composition is a composition that induces an immune response.In some embodiments, the immunogenic composition comprising one or more polyribonucleotides does not induce an immune response by itself, but rather, one or more polyribonucleotides encode, for example, one or more polypeptides that induce an immune response.

[0300] In some embodiments, the composition comprising one or more polyribonucleotides comprising 2'-O-acetylated ribose is or comprises a vaccine.

[0301] In some embodiments, the composition comprising one or more polyribonucleotides comprising 2'-O-acetylated ribose is or comprises a gene therapy.

[0302] In some embodiments, the composition comprising one or more polyribonucleotides comprising 2'-O-acetylated ribose is or comprises a chemotherapeutic agent.

[0303] In some embodiments, the composition comprising one or more polyribonucleotides comprising 2'-O-acetylated ribose is or comprises a protein replacement therapy.

[0304] In some embodiments, the composition comprising one or more polyribonucleotides comprising 2'-O-acetylated ribose is or comprises an immunotherapy.

[0305] In some embodiments, a composition comprising one or more polyribonucleotides comprising 2'-O-acetylated ribose is or comprises a cell engineering therapy.

[0306] In some embodiments, the composition comprising one or more polyribonucleotides comprising 2'-O-acetylated ribose comprises double-stranded RNA.

[0307] In some embodiments, a composition comprising one or more polyribonucleotides comprising 2'-O-acetylated ribose does not include double-stranded RNA.

[0308] In some embodiments, compositions comprising polyribonucleotides disclosed herein are characterized by observed reduced immunogenicity when assessed in cells, tissues, or organisms administered the polyribonucleotides, as compared to an appropriate reference comparator. In some embodiments, the reference comparator comprises other similar cells, tissues, or organisms administered compositions comprising equivalent polyribonucleotides that contain less 2'-O-acetylated ribose than the polyribonucleotides in the composition (e.g., no 2'-O-acetylated ribose). In some embodiments, reduced immunogenicity comprises reduced activation of innate immune response-induced toxicity. In some embodiments, reduced activation of immune response comprises reduced activation of NFkb or NFkb pathway, IRF or IRF pathway, and / or other inflammatory cytokines in cells, tissues, or organisms. In some embodiments, reduced activation of immune response comprises reduced detection of uncapped RNA by molecular sensors, e.g., RIG-I.

[0309] In some embodiments, the reduced immunogenicity allows for repeated administration, for example at least two administrations, of a composition comprising a polyribonucleotide disclosed herein to a cell, tissue or subject. In some embodiments, the repeated administration comprises at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine or at least ten doses of a composition comprising a polyribonucleotide disclosed herein. In some embodiments, the repeated administration comprises administration of the same dose of the composition compared to a previously administered dose of the composition. In some embodiments, the repeated administration comprises administration of a different dose of the composition compared to a previously administered dose of the composition.

[0310] In some embodiments, the repeated administration of a composition disclosed herein comprises administering a first dose at a first time point, followed by a subsequent dose at a second time point, in some embodiments, the first time point is about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 1 week, about 2 weeks, about 3 weeks, about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, or about 12 months prior to the second time point.

[0311] In some embodiments, a second or subsequent dose of a composition comprising a polyribonucleotide disclosed herein has substantially similar efficacy in a cell, tissue, or subject compared to the first dose of a composition comprising a polyribonucleotide disclosed herein.

[0312] In some embodiments, the reduced immunogenicity allows for the administration of higher doses of compositions comprising the polyribonucleotides disclosed herein relative to an appropriate reference standard. In some embodiments, the reference standard comprises a comparable polyribonucleotide that comprises less 2'-O-acetylated ribose (e.g., no 2'-O-acetylated ribose) compared to the polyribonucleotides disclosed herein.

[0313] In some embodiments, compositions comprising the polyribonucleotides disclosed herein are characterized by an increase in cell viability observed when assessed in a cell, tissue or organism to which the polyribonucleotides are administered, compared to a suitable reference comparison standard. In some embodiments, the reference comparison standard is the cell viability of a cell, tissue or organism to which a comparable polyribonucleotide is administered that contains less 2'-O-acetylated ribose (e.g., no 2'-O-acetylated ribose) compared to the polyribonucleotides disclosed herein.

[0314] In some embodiments, cell viability is a measure of the length of time that one or more cells of a cell, tissue, or subject remain viable.

[0315] In some embodiments, cell viability is a measure of the number of cells in a cell, tissue, or subject that are viable at one or more points in time.

[0316] In some embodiments, the compositions disclosed herein are or comprise in vitro transcribed polyribonucleotides that comprise modified ribonucleotides disclosed herein.

[0317] In some embodiments, a composition disclosed herein is or comprises an expression vector that includes one or more polynucleotides disclosed herein.

[0318] In some embodiments, the compositions disclosed herein comprise a polyribonucleotide comprising one or more modified ribonucleotides disclosed herein.

[0319] In some embodiments, the compositions disclosed herein comprise a plurality of polyribonucleotides, each comprising one or more modified ribonucleotides disclosed herein. In some embodiments, the compositions comprise a plurality of ribonucleotides, the first polyribonucleotide comprising a first modified ribonucleotide and the second polyribonucleotide comprising a second modified ribonucleotide. In some embodiments, the first modified ribonucleotide and the second modified ribonucleotide are the same modified ribonucleotide. In some embodiments, the first modified ribonucleotide and the second modified ribonucleotide are different modified ribonucleotides. In some embodiments, the first polyribonucleotide and / or the second polyribonucleotide further comprises one or more modified ribonucleotides.

[0320] In some embodiments, the compositions disclosed herein are administered at a dose of about 5 ng to about 1000 ng, about 5 ng to about 900 ng, about 5 ng to about 800 ng, about 5 ng to about 700 ng, about 5 ng to about 600 ng, about 5 ng to about 500 ng, about 5 ng to about 400 ng, about 5 ng to about 300 ng, about 5 ng to about 200 ng, about 5 ng to about 100 ng, about 5 ng to about 90 ng, about 5 ng to about 80 ng, about 5 ng to about 70 ng, about 5 ng to about 60 ng, about 5 ng to about 50 ng, about 5 ng to about 40 ng, about 5 ng to about 30 ng, about 5 ng to about 20 ng, or about 5 ng to about 10 ng. In some embodiments, the compositions disclosed herein are administered at a dose of about 10 ng to about 1000 ng, about 20 ng to about 1000 ng, about 30 ng to about 1000 ng, about 40 ng to about 1000 ng, about 50 ng to about 1000 ng, about 60 ng to about 1000 ng, about 70 ng to about 1000 ng, about 80 ng to about 1000 ng, about 90 ng to about 1000 ng, about 100 ng to about 1000 ng, about 200 ng to about 1000 ng, about 300 ng to about 1000 ng, about 40 ng to about 1000 ng, about 50 ng to about 1000 ng, about 60 ng to about 1000 ng, about 700 ng to about 1000 ng, about 800 ng to about 1000 ng, or about 900 ng to about 1000 ng.

[0321] In some embodiments, the compositions disclosed herein are administered at a dose of about 5 ng, about 10 ng, about 20 ng, about 30 ng, about 40 ng, about 50 ng, about 60 ng, about 70 ng, about 80 ng, about 90 ng, about 100 ng, 150 ng, about 200 ng, about 250 ng, about 300 ng, about 350 ng, about 400 ng, about 450 ng, about 500 ng, about 550 ng, about 600 ng, about 650 ng, about 700 ng, about 750 ng, about 800 ng, about 850 ng, about 900 ng, about 950 ng, or about 1000 ng.

[0322] In some embodiments, the compositions disclosed herein are administered at a dose of at least 5 ng, at least 10 ng, at least 20 ng, at least 30 ng, at least 40 ng, at least 50 ng, at least 60 ng, at least 70 ng, at least 80 ng, at least 90 ng, at least 100 ng, at least 150 ng, at least 200 ng, at least 250 ng, at least 300 ng, at least 350 ng, at least 400 ng, at least 450 ng, at least 500 ng, at least 550 ng, at least 600 ng, at least 650 ng, at least 700 ng, at least 750 ng, at least 800 ng, at least 850 ng, at least 900 ng, at least 950 ng, or at least 1000 ng.

[0323] Pharmaceutical Compositions In some embodiments, the composition comprising one or more polyribonucleotides comprising 2'-O-acetylated ribose is a pharmaceutical composition. In some embodiments, the pharmaceutical composition further comprises a pharma- ceutically acceptable excipient. The pharmaceutical composition of the present disclosure may comprise a polypeptide disclosed herein, a polynucleotide disclosed herein, or an expression vector comprising a polynucleotide disclosed herein.

[0324] In some embodiments, pharmaceutical compositions may include pharma- ceutically acceptable carriers or excipients, as used herein, including any and all solvents, dispersion media, diluents, or other liquid vehicles, dispersing or suspending aids, surface active agents, isotonicity agents, thickening or emulsifying agents, preservatives, solid binders, lubricants, and the like, as appropriate for the particular dosage form desired. Remington's The Science and Practice of Pharmacy, 21st Edition, AR Gennaro (Lippincott, Williams & Wilkins, Baltimore, MD, 2006; incorporated herein by reference) discloses various excipients used in the formulation of pharmaceutical compositions and known techniques for their preparation. Suitable pharma-ceutically acceptable carriers include, but are not limited to, water, salt solutions (e.g., NaCl), saline, buffered saline, glycerol, sugars, such as mannitol, sucrose, and the like, dextrose, fatty acid esters, and the like, and combinations thereof.

[0325] The pharmaceutical composition may be mixed with auxiliary substances (e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, colorants, flavorings, and / or aromatic substances, etc.) as necessary, which do not adversely react with the active compound or interfere with its activity. In certain embodiments, a water-soluble carrier suitable for intravenous administration is used. In some embodiments, the pharmaceutical composition may be sterilized.

[0326] Suitable pharmaceutical compositions, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents.The pharmaceutical composition can be a liquid solution, suspension, or emulsion.

[0327] The pharmaceutical composition can be formulated according to the usual procedures as a pharmaceutical composition suitable for administration to humans. The formulation of the pharmaceutical composition must suit the method of administration. For example, in some embodiments, a composition for intravenous administration is usually a solution in sterile isotonic aqueous buffer. If necessary, the composition may also include a solubilizing agent and a local anesthetic to ease pain at the site of the injection. Generally, the ingredients are supplied separately or mixed in unit dosage form, for example as a lyophilized powder or water-free concentrate in a hermetically sealed container, such as an ampule or sachette indicating the quantity of active agent. If the pharmaceutical composition is to be administered by injection, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water, saline, or dextrose / water. If the pharmaceutical composition is to be administered by injection, an ampule of sterile water for injection or saline can be provided so that the ingredients can be mixed prior to administration.

[0328] While the description of pharmaceutical compositions provided herein is primarily directed to pharmaceutical compositions suitable for ethical administration to humans, it will be understood by those skilled in the art that such compositions are generally suitable for administration to any type of animal or cell, in vitro or ex vivo. Modifications of pharmaceutical compositions suitable for administration to humans to provide compositions suitable for administration to a variety of animals or cells, in vitro or ex vivo, are well understood and an ordinarily skilled professional, e.g., a veterinary pharmacologist, can design and / or perform such modifications with only routine experimentation, if any.

[0329] The formulations of the pharmaceutical compositions described herein may be prepared by any method known or hereafter developed in the art of pharmacology. In general, such methods of preparation include the step of combining the active ingredient with a diluent or other excipient and / or one or more other accessory ingredients, and then, if necessary and / or desirable, shaping and / or packaging the product into the desired single- or multi-dosage unit.

[0330] Pharmaceutical compositions according to the present disclosure may be prepared, packaged, and / or sold in bulk, as a single unit dose, and / or as a plurality of single unit doses. As used herein, a "unit dose" is a discrete amount of a pharmaceutical composition described herein.

[0331] Method for preparing polyribonucleotides containing 2'-O-acetylated ribose The present disclosure provides, inter alia, a method for producing polyribonucleotides comprising 2-O-acetylated ribose. In some embodiments, disclosed herein is a method for producing polyribonucleotides, comprising incubating an in vitro transcription mixture, the in vitro transcription mixture comprising (i) a DNA template, (ii) at least one RNA polymerase or a variant or fragment thereof, and (iii) a plurality of ribonucleotides, the plurality of ribonucleotides comprising at least one modified ribonucleotide comprising 2'-O-acetylated ribose, thereby producing polyribonucleotides comprising 2'-O-acetylated ribose.

[0332] Also disclosed herein is an in vitro transcription mixture useful for generating polyribonucleotides comprising 2'-O-acetylated ribose. In some embodiments, disclosed herein is an in vitro transcription mixture comprising (i) a DNA template, (ii) at least one RNA polymerase or a variant or fragment thereof, and (iii) a plurality of ribonucleotides comprising at least one modified ribonucleotide comprising 2'-O-acetylated ribose.

[0333] In some embodiments, the method or in vitro transcription mixture generates a plurality of polyribonucleotides. In some embodiments, each polyribonucleotide in the plurality of polyribonucleotides comprises a 2'-O-acetylated ribose.

[0334] In some embodiments, the RNA polymerase is selected from bacteriophage RNA polymerase, mitochondrial RNA polymerase, eukaryotic RNA polymerase, bacterial RNA polymerase, or any combination thereof. In some embodiments, the RNA polymerase comprises T7 RNA polymerase, T3 RNA polymerase, SP6 RNA polymerase, viral RNA polymerase, N4 virion RNA polymerase, or a variant of any of the foregoing.

[0335] In some embodiments, the polyribonucleotides produced by the methods disclosed herein or using the in vitro transcription mixtures include a coding region, which in some embodiments encodes a gene product, e.g., as described herein.

[0336] In some embodiments, the polyribonucleotides generated by the methods disclosed herein or using the in vitro transcription mixture do not include coding regions. In some embodiments, the polyribonucleotides that do not include coding regions may also be referred to as non-coding RNA. Exemplary non-coding RNAs include, but are not limited to, long non-coding RNAs (lncRNAs), microRNAs, siRNAs, piRNAs, snoRNAs, snRNAs, exRNAs, scaRNAs, rRNAs, and tRNAs.

[0337] In some embodiments, the polyribonucleotides generated by the methods disclosed herein or using the in vitro transcription mixtures include guide RNA, short hairpin RNA, siRNA, microRNA, long non-coding RNA, circular RNA, or messenger RNA (mRNA), or any combination thereof.

[0338] In some embodiments, the polyribonucleotides generated by the methods disclosed herein or using the in vitro transcription mixture encode a payload. In some embodiments, the payload comprises one or more target polypeptides.

[0339] In some embodiments of the methods of making ribonucleotides disclosed herein, the incubating step is carried out at a temperature of at least 37°C.

[0340] In some embodiments of the methods of making ribonucleotides disclosed herein, the incubation step is performed at a temperature of about 45°C, 46°C, 47°C, 48°C, 49°C, 50°C, 51°C, 52°C, 53°C, 54°C, 55°C, or greater.

[0341] In some embodiments of the methods of making ribonucleotides disclosed herein, the incubation step is carried out for at least 1 hour, at least 2 hours, at least 3 hours, at least 4 hours or more.

[0342] RNA preparations In particular, provided herein are compositions comprising polyribonucleotides comprising 2'-O-acetylated ribose, and formulations thereof. In some embodiments, the compositions comprising polyribonucleotides disclosed herein are formulated into lipid nanoparticle (LNP) formulations.

[0343] In some embodiments, the polyribonucleotide disclosed herein encodes a polypeptide.In some embodiments, the polyribonucleotide disclosed herein is or comprises messenger RNA.In some embodiments, the composition comprising the polyribonucleotide comprising messenger RNA is formulated into lipid nanoparticle (LNP) formulation.

[0344] In some embodiments, the polyribonucleotide disclosed herein is or comprises a gRNA. In some embodiments, the composition comprising the polyribonucleotide comprising the gRNA is formulated into a lipid nanoparticle (LNP) formulation.

[0345] In some embodiments, the polyribonucleotide disclosed herein is or comprises an inhibitory RNA. In some embodiments, the composition comprising the polyribonucleotide comprising the inhibitory RNA is formulated into a lipid nanoparticle (LNP) formulation.

[0346] In some embodiments, the polyribonucleotide disclosed herein is or comprises miRNA or siRNA. In some embodiments, the composition comprising the polyribonucleotide comprising miRNA or siRNA is formulated into a lipid nanoparticle (LNP) formulation.

[0347] In some embodiments, the polyribonucleotide disclosed herein is or comprises an antisense oligonucleotide. In some embodiments, the composition comprising the polyribonucleotide comprising the antisense oligonucleotide is formulated into a lipid nanoparticle (LNP) formulation.

[0348] In some embodiments, the present disclosure provides LNP formulations comprising the polyribonucleotides disclosed herein for use in pharmaceutical compositions, such as immunogenic compositions.

[0349] Methods of using the compositions disclosed herein The present disclosure provides, inter alia, methods of using the polyribonucleotides disclosed herein, or compositions comprising same.

[0350] In some embodiments, provided herein are methods of administering a polyribonucleotide disclosed herein, or a composition comprising a polyribonucleotide disclosed herein, to a cell, tissue, or subject.

[0351] In some embodiments, provided herein are methods of vaccination comprising administering to a cell, tissue, or subject a polyribonucleotide disclosed herein or a composition comprising a polyribonucleotide disclosed herein.

[0352] In some embodiments, disclosed herein are methods of gene therapy comprising administering to a cell, tissue, or subject a polyribonucleotide disclosed herein or a composition comprising a polyribonucleotide disclosed herein. In some embodiments, the gene therapy method comprises delivery of one or more components of gene therapy, such as a guide RNA and / or a Cas polypeptide.

[0353] In some embodiments, provided herein are methods of stimulating an immune response comprising administering to a cell, tissue, or subject a polyribonucleotide disclosed herein, or a composition comprising a polyribonucleotide disclosed herein.

[0354] In some embodiments, also provided herein are methods of engineering cell therapy comprising administering to a cell, tissue, or subject a polyribonucleotide disclosed herein or a composition comprising a polyribonucleotide disclosed herein.

[0355] In some embodiments, provided herein are methods of immunotherapy comprising administering to a cell, tissue, or subject a polyribonucleotide disclosed herein or a composition comprising a polyribonucleotide disclosed herein, hi some embodiments, the methods of immunotherapy comprise delivery of antibody therapy and / or immune checkpoint therapy.

[0356] In some embodiments, disclosed herein are methods of protein replacement therapy comprising administering to a cell, tissue or subject a polyribonucleotide as disclosed herein or a composition comprising a polyribonucleotide as disclosed herein. In some embodiments, the protein replacement therapy comprises delivery of enzyme replacement therapy.

[0357] In some embodiments, provided herein are methods of chemotherapy comprising administering to a cell, tissue, or subject a polyribonucleotide disclosed herein or a composition comprising a polyribonucleotide disclosed herein.

[0358] In some embodiments, the method or use disclosed herein comprises determining the cell viability of a cell, tissue or subject.In some embodiments, cell viability is a measure of the length of time that one or more cells of a cell, tissue or subject are alive.In some embodiments, cell viability is a measure of the number of cells of a cell, tissue or subject that are alive at one or more time points.

[0359] In some embodiments, the cell, tissue or subject administered with polyribonucleotide or a composition comprising the same shows improved cell viability when compared to a reference cell viability. In some embodiments, the reference cell viability is the cell viability of a cell, tissue or subject administered with a comparable polyribonucleotide or a composition comprising the same that contains less 2'-O-acetylated ribose (e.g., does not contain any 2'-O-acetylated ribose) compared to the polyribonucleotide disclosed herein.

[0360] In some embodiments, the method or use disclosed herein further comprises determining an immune system response of a cell, tissue or subject to which a polyribonucleotide or a composition comprising the same has been administered. In some embodiments, the immune response comprises an innate immune system response including an innate immune system-induced toxicity. In some embodiments, determining the innate immune system response comprises determining the level and / or activation of NF-κB or the NF-κB pathway, IRF or the IRF pathway, or inflammatory cytokines, or any combination thereof, in the cell, tissue or subject. In some embodiments, determining the innate immune system response comprises determining the detection level of uncapped RNA in the cell, tissue or subject.

[0361] In some embodiments, the cells, tissues, or subjects to which the polyribonucleotide or composition comprising the same is administered exhibit a reduced innate immune system response when compared to a reference. In some embodiments, the reference is the innate immune system response of the cells, tissues, or subjects to which the polyribonucleotide or composition comprising the same is administered that contains less 2'-O-acetylated ribose (e.g., does not contain any 2'-O-acetylated ribose) compared to the polyribonucleotides disclosed herein.

[0362] In some embodiments, the methods or uses disclosed herein further comprise determining the effectiveness of the polyribonucleotide or a composition comprising same in a cell, tissue or subject to which the polyribonucleotide or a composition comprising same has been administered.

[0363] In some embodiments, determining the effectiveness comprises determining the antibody response or cellular response in a cell, tissue or subject. In some embodiments, the cell, tissue or subject to which the polyribonucleotide or the composition comprising it is administered shows an increase in antibody response or cellular response when compared to a reference. In some embodiments, the reference is the antibody response or cellular response of a cell, tissue or subject to which the equivalent polyribonucleotide or the composition comprising it is administered, which comprises less 2'-O-acetylated ribose (e.g., does not comprise any 2'-O-acetylated ribose) compared to the polyribonucleotide disclosed herein.

[0364] In some embodiments, the methods or uses disclosed herein comprise administering a polyribonucleotide or a composition comprising the same to a cell, tissue or subject at least twice. In some embodiments, the methods disclosed herein comprise administering a polyribonucleotide or a composition comprising the same to a cell, tissue or subject at least twice, at least three times, at least four times, at least five times, at least six times, at least seven times, at least eight times, at least nine times, or at least ten times.

[0365] In some embodiments, the method or use disclosed herein comprises administering multiple doses of polyribonucleotide or a composition comprising it to a cell, tissue or subject.In some embodiments, the second or subsequent doses of polyribonucleotide or a composition comprising it have substantially similar efficacy in a cell, tissue or subject compared to the administration of the first dose of the composition comprising polyribonucleotide.

[0366] In some embodiments of any of the methods or uses disclosed herein, the polyribonucleotide or the composition comprising it is administered to a cell, tissue or subject at a higher dose than a suitable reference standard. In some embodiments, the reference standard comprises a comparable polyribonucleotide that comprises less 2'-O-acetylated ribose (e.g., no 2'-O-acetylated ribose) compared to the polyribonucleotide disclosed herein.

[0367] In some embodiments of any of the methods or uses disclosed herein, the composition is administered via any one of the following routes of administration: intramuscular, intravenous, subcutaneous, intrathecal, intradermal, ocular, intranasal, sublingual, or oral.

[0368] In some embodiments of any of the methods or uses disclosed herein, the cell is a mammalian cell.

[0369] In some embodiments of any of the methods or uses disclosed herein, the tissue is mammalian tissue.

[0370] In some embodiments of any of the methods or uses disclosed herein, the subject is a mammal. In some embodiments, the mammal is a human.

[0371] kit Another aspect of the present disclosure further provides a pharmaceutical pack or kit. In some embodiments, the kit may include a polyribonucleotide or composition as described herein. In some embodiments, the kit may be used in any applicable method, such as the methods described herein. EXAMPLES

[0372] Example 1: Use of 2-O-acetylated NTPs by T7 RNA polymerase as substrates for in vitro transcription This example demonstrates that wild-type T7 RNA polymerase can use 2'-O-acetylated nucleotide triphosphates as substrates for the in vitro transcription of RNA, and that the resulting RNA is translationally active.

[0373] method:

[0374] Example 3 discloses the methods used to generate the 2'-O-acetylated nucleotide triphosphates used in this example and disclosed herein.

[0375] In vitro transcription (IVT) of Luc2 RNA for the A549 assay

[0376] Luc2 RNA was synthesized in a 20 μL IVT reaction consisting of 200 ng Luc2 T7 template, 20 mM MgCl2, 7.5 mM each of either native or 2-O-acetyl NTPs, 7.5 mM CleanCap AG (TriLink), 1x HiScribe transcription buffer, and 2 uL HiScribe polymerase mix (NEB) and incubated at 37°C for 4 hours.

[0377] All IVT products were cleaned up using Monarch (500 μg) RNA Clean Up kit (NEB) and eluted in 88 μL nuclease-free water. The eluted products were then digested in a 100 μL reaction consisting of 1× DNase I buffer and 10 U DNase I (RNase-free) (New England Biolabs) at 37° C. for 5 minutes to degrade the DNA template. The DNase I-treated samples were cleaned up using Monarch (500 ug) RNA Clean Up kit (New England Biolabs) and eluted in 88 μL nuclease-free water.

[0378] The DNAse I-treated products, bearing the co-transcriptionally added Cap1 structure, were treated with 1x DNase I buffer (NEB) and 100 U of Calf Intestinal Alkaline Phosphotase (CIAP) (Promega) for 5 min at 37°C as a polishing step to remove rare immunogenic 5' triphosphates from RNA transcripts that did not incorporate CleanCap AG. DNase I buffer was used for this enzyme step because we found that the CIAP enzyme works well in DNAse I buffer for this RNA polishing step (unpublished data).

[0379] CIAP-treated RNA was cleaned up using the Monarch (500ug) RNA Clean Up kit (NEB) and eluted in 100uL of nuclease-free water.

[0380] RNA quantification: RNA concentrations were determined using a NanoDrop OneC spectrophotometer (Thermo Scientific).

[0381] A549 cell culture method: A549-Dual (InvivoGen) were cultured in high glucose GlutaMAX Dulbecco's modified Eagle's medium supplemented with 10% heat-inactivated fetal bovine serum, 100 units / mL penicillin, 100 μg / mL streptomycin, 10 μg / mL blasticidin, and 100 μg / mL zeocin, and maintained at 37°C and 5% CO2.

[0382] One day prior to transfection, cells were seeded in 96-well plates at 2,000 cells / well. 50 or 100 ng of each RNA was transfected using Lipofectamine MessengerMAX Transfection Reagent (ThermoFisher) using a 1:1.5 μg:uL ratio of RNA:MessengerMAX. Transfections were performed in triplicate.

[0383] Viability and luciferase expression were determined using the ONE-Glo+Tox Luciferase Reporter and Cell Viability Assay (Promega). NF-κB activation was measured via the SEAP reporter gene using the QUANTI-Blue detection reagent (InvivoGen) as described by the manufacturer. IRF pathway activation was measured via the activity of the Lucia luciferase gene using the QUANTI-Luc detection reagent (InvivoGen) as described by the manufacturer.

[0384] result:

[0385] 2-O-acetylated RNA reduces immunogenicity and improves cell viability. As shown in Table 1, T7 RNA polymerase used 2-O-acetyl analogs to generate significant amounts of fully 2-O-acetylated full-length RNA, although the yields were reduced compared to natural nucleotides.

[0386] [Table 1]

[0387] Not only were the fully 2-O-acetylated full-length RNAs translationally active, but they were also superior to unmodified RNAs in terms of reporter gene expression, cell viability, and immunogenicity. Figure 2 shows that fully 2-O-acetylated RNAs can be introduced into eukaryotic cells and translated by the eukaryotic ribosomal machinery to be expressed better than unmodified RNAs. Without wishing to be bound by any particular theory, we expect that this observation is due to the improved stability and / or half-life of 2-O-acetylated RNAs.

[0388] The results of the experiment further demonstrated that 2-O-acetylated RNA is less immunogenic than its unmodified counterpart. Figure 3 shows the improvement in cell viability with 2-O-acetylated RNA compared to unmodified RNA. This trend for improved viability was paralleled by a reduction in both immunogenicity markers NF-Kb (Figure 5) and IRF (Figure 4).

[0389] In assays testing the immunogenicity of 2-O-acetylated RNA (see Figures 4-5), increased immunogenicity was observed at 100 ng of 2-O-acetylated RNA. Without wishing to be bound by any particular theory, it was hypothesized that this increased immunogenicity at higher doses of 2-O-acetylated RNA could be due to novel RNA structures detected by cytoplasmic RNA sensors, increased protein load on the cells due to extended RNA half-life, or a combination of such factors. To test this hypothesis, a 2-O-acetyl analog of the base-modified nucleotide N4-acetylcytidine, which previously showed reduced natural immunogenicity, was tested with 2-O-acetylated RNA (see Figures 4 and 5, last bar on the right labeled "50 ng 100% 2-O-acetylated Ac4C Luc2"). In some embodiments, this data provides support for using 2-O-acetyl nucleotide triphosphates that are also base-modified to further improve RNA performance when using higher doses of 2-O-acetyl RNA.

[0390] Characterization of 2-O-acetylation of nucleotides: NMR data was obtained on purified ribose acetylated NTPs to confirm complete 2-O-acetylation of the nucleotides used. The data show that the purified ribose acetylated NTPs consisted exclusively of a mixture of 2-O-acetyl and 3-O-acetyl NTPs (see Example 3 and Figures 6-61). This mixture was likely due to spontaneous switching of the acetyl group position when present in solution as triphosphates. However, this spontaneous switching and the resulting mixture of structures does not indicate that the RNA used in this example was not fully 2-O-acetylated, since RNA transcription requires the 3'OH of ribose available for chain elongation. Therefore, any RNA products generated when 2-O-acetylated NTPs are used for transcription should have only 2-O modifications, since the 3-O-acetyl group will function as a transcription terminator.

[0391] Conclusion: Taken together, this example and the data herein demonstrate for the first time that 2'-O-acetylated RNA can be transcribed by wild-type T7 RNA polymerase and that the resulting RNA performs better than unmodified RNA. These findings highlight the discovery of a novel class of nucleotide triphosphates that can be readily used for in vitro transcription of RNA for a variety of applications, including as therapeutic agents in medical applications.

[0392] Example 2: In vivo gene expression from LNP-delivered 2-O-ribose acetylated RNA is equivalent to LNP-delivered N1-methylpseudouridine modified RNA This example describes in vivo gene expression from animals administered LNP formulations containing 2-O ribose acetylated RNA or N1-methylpseudouridine modified RNA.

[0393] Methods: Generation of Luc2 IVT template: The luc2 gene, encoding an optimized form of firefly luciferase, was amplified from pGL4.10[luc2] (Promega). Amplification was performed in a 20 μL reaction consisting of 0.25 μM of each primer Luc2_fwd and Luc2_rev, 1× Herculase II buffer, 25 mM of each dNTP, 15 ng of pGL4.10[luc2] plasmid (Promega), 0.25 M betaine, and 0.4 μL of Herculase II enzyme at an annealing temperature of 70° C. The PCR product was purified using a DNA Clean&Concentrator-25 Kit (Zymo Research) according to the manufacturer's protocol and eluted in 45 μL of nuclease-free water. 42.5uL of the eluted product was subjected to treatment with 125U of Dpn1 enzyme (New England Biolabs) in a 50μL reaction to digest the template plasmid. The digested product was purified again using the DNA Clean&Concentrator-25 Kit (Zymo Research) according to the manufacturer's protocol and eluted in 40μL of nuclease-free water. The digested primary PCR product was then amplified at 50°C in a 20μL reaction consisting of 0.25μM of each primer T7-AGG_fwd and 120pA_rev, 1x Herculase II buffer, 25mM of each dNTP, 15ng of Luc2 primary amplification product, and 0.4μL of Herculase II enzyme. The secondary PCR product was cleaned up using the DNA Clean&Concentrator-25 Kit (Zymo Research) according to the manufacturer's protocol and eluted in nuclease-free water.

[0394] The primer sequences used were as follows:

[0395] Luc2_fwd:

[0396] CTTGTTCTTT TTGCAGAAGC TCAGAATAAA CGCTCAACTT TGGCCACCat ggaagatgcc aaaaacatta agaagggc (SEQ ID NO: 1)

[0397] Luc2_rev

[0398] AGAATGTGAA GAAACTTTCT TTTTATTAGG AGCAGATACG AATGGCTACA TTTTGGGGGA CAACATTTTG TAAAGTGTAA GTTGGTATTA TGTAGCTTAG AGACTCCATT CGGGTGTTCT TGAGGCTGGT CTATCATTAc acggcgatct tgccgcc (SEQ ID NO: 2)

[0399] T7-AGG_fwd:

[0400] gaattTAATA CGACTCACTA TAAGGcttgt tctttttgca gaagc (SEQ ID NO: 3)

[0401] 120pA_rev

[0402] TTTTTTTTTT TTTTTTTTTT TTTTTTTTTT TTTTTTTTTT TTTTTTTTTT TTTTTTTTTT TTTTTTTTTT TTTTTTTTTT TTTTTTTTTT TTTTTTTTTT TTTTTTTTTT TTTTTTTTTT agaatgtgaa gaaactttct ttttattag (SEQ ID NO: 4)

[0403] In vitro transcription (IVT) of Luc2 RNA for A549 assay: Luc2 RNA was synthesized in a 20 μL IVT reaction consisting of 200 ng Luc2 T7 template, 20 mM MgCl2, 7.5 mM each of either native NTPs or 2-O-acetyl NTPs, 7.5 mM CleanCap AG (TriLink), 1x HiScribe transcription buffer, and 2 uL HiScribe polymerase mix (NEB) and incubated at 37°C for 4 hours.

[0404] All IVT products were cleaned up using Monarch (500 μg) RNA Clean Up kit (NEB) and eluted in 88 μL nuclease-free water. The eluted products were then digested in a 100 μL reaction consisting of 1× DNase I buffer and 10 U DNase I (RNase-free) (New England Biolabs) at 37° C. for 5 minutes to degrade the DNA template. The DNase I-treated samples were cleaned up using Monarch (500 ug) RNA Clean Up kit (New England Biolabs) and eluted in 88 μL nuclease-free water.

[0405] The DNAse I-treated products, bearing the co-transcriptionally added Cap1 structure, were treated with 1x DNase I buffer (NEB) and 100 U of Calf Intestinal Alkaline Phosphotase (CIAP) (Promega) for 5 min at 37°C as a polishing step to remove rare immunogenic 5' triphosphates from RNA transcripts that did not incorporate CleanCap AG. DNase I buffer was used for this enzyme step because we found that the CIAP enzyme works well in DNAse I buffer for this RNA polishing step (unpublished data).

[0406] CIAP-treated RNA was cleaned up using the Monarch (500ug) RNA Clean Up kit (NEB) and eluted in 100uL of nuclease-free water.

[0407] RNA quantification: RNA concentrations were determined using a NanoDrop OneC spectrophotometer (Thermo Scientific).

[0408] A549 cell culture method: A549-Dual (InvivoGen) were cultured in high glucose GlutaMAX Dulbecco's modified Eagle's medium supplemented with 10% heat-inactivated fetal bovine serum, 100 units / mL penicillin, 100 μg / mL streptomycin, 10 μg / mL blasticidin, and 100 μg / mL zeocin, and maintained at 37°C and 5% CO2.

[0409] One day prior to transfection, cells were seeded in 96-well plates at 2,000 cells / well. 50 or 100 ng of each RNA was transfected using Lipofectamine MessengerMAX Transfection Reagent (ThermoFisher) using a 1:1.5 μg:uL ratio of RNA:MessengerMAX. Transfections were performed in triplicate.

[0410] Viability and luciferase expression were determined using the ONE-Glo+Tox Luciferase Reporter and Cell Viability Assay (Promega). NF-κB activation was measured via the SEAP reporter gene using the QUANTI-Blue detection reagent (InvivoGen) as described by the manufacturer. IRF pathway activation was measured via the activity of the Lucia luciferase gene using the QUANTI-Luc detection reagent (InvivoGen) as described by the manufacturer.

[0411] In vitro transcription (IVT) of Luc2 RNA for in vivo testing: For 2-O-acetylated Luc2 mRNA, mRNA was synthesized in a 20 μL IVT reaction consisting of 200 ng Luc2 T7 template, 20 mM MgCl2, 7.5 mM of each 2-O-acetyl NTP, 7.5 mM CleanCap AG (TriLink), 1x HiScribe transcription buffer, and 2 uL HiScribe polymerase mix (NEB) and incubated at 37°C for 4 hours.

[0412] For N1-methylpseudouridine modified Luc2 mRNA, mRNA was synthesized in a 20 μL IVT reaction consisting of 200 ng Luc2 T7 template, 20 mM MgCl2, 7.5 mM of each NTP with N1-methylpseudouridine triphosphate fully substituted for uridine triphosphate, 7.5 mM CleanCap AG (TriLink), 1x HiScribe transcription buffer, and 2 uL HiScribe polymerase mix (NEB) and incubated at 37°C for 4 hours.

[0413] All IVT products were cleaned up using Monarch (500 μg) RNA Clean Up kit (NEB) and eluted in 88 μL nuclease-free water. The eluted products were then digested in a 100 μL reaction consisting of 1× DNase I buffer and 10 U DNase I (RNase-free) (New England Biolabs) at 37° C. for 5 minutes to degrade the DNA template. The DNase I-treated samples were cleaned up using Monarch (500 ug) RNA Clean Up kit (New England Biolabs) and eluted in 88 μL nuclease-free water.

[0414] The DNAse I-treated products, bearing the co-transcriptionally added Cap1 structure, were treated with 1x DNase I buffer (NEB) and 100 U of Calf Intestinal Alkaline Phosphotase (CIAP) (Promega) for 5 min at 37°C as a polishing step to remove rare immunogenic 5' triphosphates from RNA transcripts that did not incorporate CleanCap AG. DNase I buffer was used for this enzyme step because we found that the CIAP enzyme works well in DNAse I buffer for this RNA polishing step (unpublished data).

[0415] The IVT product was cleaned up using the Monarch (500 μg) RNA Clean Up kit (NEB) and eluted in 100 μL of 1 mM sodium citrate (pH 6.5) (ThermoFisher). The IVT product was cleaned up using the Monarch (500 μg) RNA Clean Up kit (NEB) and eluted in 100 μL of 1 mM sodium citrate (pH 6.5) (ThermoFisher).

[0416] RNA quantification: RNA concentrations were determined using a NanoDrop OneC spectrophotometer (Thermo Scientific).

[0417] mRNA-LNP Formulations. Formulations of mRNA in lipid nanoparticles (mRNA-LNP) were prepared using an Ignite microfluidic mixer (Precision Nanosystems, Vancouver, BC). Briefly, using the manufacturer's recommended formulation parameters, the GenVoy-ILM lipid mixture (PrecisionNanosystems NWW0042) was diluted to 12.5 mM in absolute ethanol and combined with an aqueous mRNA solution (0.14 mg / mL) in PNI buffer (Precision Nanosystems NWW0043). The formulation was immediately diluted 30:1 in phosphate buffered saline (Gibco 10010023), concentrated using an Amicon centrifugal filter (MilliporeSigma UFc901008), and adjusted to the estimated final volume with PBS. The formulation was then characterized on a Stunner UV-VIS / DLS instrument (Unchained Labs) and then further diluted with PBS as necessary to achieve the correct payload concentration (ug / mL). The formulations were stored at 4° C. until in vivo administration.

[0418] mRNA administration studies in mice. Animal studies were performed according to the guidelines set forth by Charles River Accelerator Development Lab (CRADL, Cambridge, MA, USA) and approved by the CRADL Institutional Animal Care and Use Committee (IACUC). Female BALB / C mice (7-9 weeks old) were purchased from Charles River Laboratories (Wilmington, MA, USA) and housed at CRADL. Mice (n=5 per condition) were allowed to acclimate for at least 2 days before the start of the study. For single-dose studies (Figure 6), animals received a single IM administration of mRNA-LNP. Mice were imaged by whole-body bioluminescence imaging at three time points after mRNA injection (approximately 6 hours, approximately 24 hours, and approximately 48 hours after administration). All mRNA injections consisted of 50 uL of mRNA-LNP formulation (1 ug Luc2 mRNA dose per animal) delivered via IM injection in the left flank. For whole-body bioluminescence imaging, animals were injected with 200 uL of D-luciferin K+ salt (PerkinElmer 122799) diluted to 15 mg / mL in PBS by intraperitoneal (IP) injection 10 min prior to imaging time point. Mice were placed under 3% isoflurane anesthesia in an induction chamber 3 min prior to imaging and then moved to an isoflurane delivery nose cone (IVIS-Spectrum Model 124262, Perkin Elmer, Waltham, MA) in the imaging chamber immediately prior to imaging. Mice were positioned left flank up in the imaging chamber and maintained at 3% isoflurane throughout imaging. Images were acquired using field of view D and exposure continued until 30,000 photons were collected or 1 min had elapsed, whichever occurred first. After imaging, animals were returned to their home cages for recovery.

[0419] Results: Figures 62A and 62B show that reporter gene expression from fully 2-O-acetylated LNP-delivered RNA is comparable to that from LNP-delivered N1-methylpseudouridine-modified RNA, a commonly used modification for RNA therapeutics. Reporter gene expression levels were comparable for both RNAs at 6, 24, and 48 hours after administration. This data suggests that 2-O-acetylated RNA has similar in vivo persistence compared to that of N1-methylpseudouridine-modified RNA.

[0420] This result was unexpected for several reasons, one of which is the in vitro data showing that IRF and NK-kB signaling persists with 2-O-acetylated RNA that does not have base modifications (Example 1 and Figures 4-5). Because IRF and NF-kB activity can result in upregulation of nucleases from an antiviral response, and because the levels of reporter expression seen with 2-O acetylated RNA are similar to N1-methylpseudouridine modified RNA, without wishing to be bound by any particular theory, the data suggests that the use of 2-O acetylated NTPs for in vitro transcription may provide RNA with increased persistence, e.g., increased resistance to nuclease degradation.

[0421] In some embodiments, the use of 2-O-acetylated derivatives of known base-modified NTPs for in vitro transcription can provide RNAs with increased persistence compared to commonly used N1-methylpseudouridine-modified mRNAs. Without wishing to be bound by any particular theory, polyribonucleotides with increased persistence can be achieved by combining reduced immunogenicity through base modifications (e.g., Ac4C, 5hmU, N1-methylpseudouridine, etc.) with nuclease resistance through ribosome-compatible backbone modifications (e.g., 2-O ribose acetylation as described herein).

[0422] Example 3: Synthesis of 2-O-acetylated NTPs This example describes exemplary methods for the synthesis of 2-O-acetylated NTPs as used in Examples 1 and 2 and disclosed herein. Figures 6-61 provide exemplary structures made using the synthetic methods disclosed below.

[0423] Solvents and reagents were purchased from Sigma-Aldrich, VWR, or Fisher Scientific and used without further purification. Substrates were purchased from Combi-blocks and used without further purification. Reactions were monitored either by thin layer chromatography (TLC) or by analytical liquid chromatography mass spectrometry (LC-MS) using a Waters Acquity Ultra Performance LC system and a Synapt high definition mass spectrometer. 1H NMR and 31P NMR spectra were recorded on a Varian Unity INOVA spectrometer (300 MHz). All chemical shifts are reported in ppm and coupling constants J are reported in Hertz (Hz). NMR solvent peaks were referenced as follows: (1H NMR) CDCl3: 7.27 ppm, DMSO-d6: 2.50 ppm, DO: 4.65 ppm. Compounds were purified by flash column chromatography on a Teledyne ISCO Combi-Flash system using normal-phase silica gel (SiliCycle Inc.) or reversed-phase (Teledyne Gold-C18 or C18Aq) prepacked columns, and an AKTA avant chromatography system on a HiTrap™ DEAE FF prepacked column. Compound purity was determined by analytical HPLC (Waters Acquity Ultra Performance) using an Acquity UPLC CSH C18 1.7 μm (50 mm×2.1 mm) column and a flow rate of 0.3 mL / min. Gradient conditions: Solvent A (0.05% formic acid in water) and solvent B (0.05% formic acid in acetonitrile): 0–0.1 min 95% A, 0.1–4.0 min 5–95% B (linear gradient), 4.0–5.0 min 95% B, UV detection at 254 nm and 220 nm, and a Waters Spherisorb, SAX column 80 Å, 5 μm 4.6 mm × 250 mm.

[0424] Synthesis of TIPD silyl-protected cytidine (2) [ka] Scheme 1. Synthesis of silyl-protected cytidine 2

[0425] Cytidine 1 (3.0 g, 12.34 mmol) was dissolved in pyridine and coevaporated (3×30 mL) to a white suspension. The coevaporated substrate 1 was dissolved in pyridine (40 mL) and 1,3-dichloro-1,1,3,3-tetraisopropyldisiloxane (TIPDSCl2) (4.3428 mL, 13.575 mmol) and 4-dimethylaminopyridine (DMAP) (0.7539 g, 6.171 mmol) were added. The reaction was stirred at room temperature for approximately 24 hours and monitored by TLC. Once no starting material was detected, the mixture was diluted with CHCl (25 mL). The resulting solution was transferred to a separatory funnel and washed with water (1×25 mL). The aqueous layer was then extracted with additional CHCl (2×25 mL). The organic layers were combined, washed with brine and dried over MgSO4 to give crude silyl protected 2 as a white solid which was purified by silica gel flash chromatography (CH2Cl2 / CH3OH, modified with Et3N 5%) to give 2 (3.91 g, 65%). TLC (CH2Cl2:CH3OH=95 / 5, v / v): Rf=0.75. 1H NMR (300 MHz, DMSO-d6, δ): 7.69 (d, 3J = 7.4 Hz, 1H), 7.15 (d, 3J = 13.0 Hz, 2H), 5.63 (t, 3J = 5.9 Calculated value for m / z [C21H40N3O6Si2]+ [M+H]+ 486.2450; Actual value 486.2000.

[0426] Synthesis of diacetylated silyl-protected cytidine 3 [ka] Scheme 2. Synthesis of diacetylated silyl-protected cytidine 3

[0427] In an oven-dried RBF (25 mL), cytidine 2 (0.2 g, 0.412 mmol) was dissolved in anhydrous CH3CN (10 mL) to form a white emulsion. 4-Dimethylaminopyridine (DMAP) (0.2518 g, 2.061 mmol) was added, followed by acetyl chloride (AcCl) (29.3 μl, 4.122 mmol). The solution dissolved and the reaction was stirred at room temperature for 10 min under argon. Upon completion of the reaction, the reaction mixture was concentrated and the crude material was dissolved in dichloromethane CHCl (25 mL). The resulting solution was transferred to a separatory funnel and washed with water (1×25 mL). The aqueous layer was then extracted with additional CHCl (2×25 mL). The organic layers were combined, washed with brine, and dried over magnesium sulfate MgSO4 to give crude silyl-protected 3 as a white solid. The residue was purified by flash column chromatography (ethyl acetate / methanol 9:1) to give compound 3 as the main fraction in 82% yield and minor compound 4 in 18% yield. TLC (ethyl acetate:CH3OH=9 / 1, v / v): Rf=0.90, 1 H NMR (300 MHz, DMSO-d6, δ):10.94 (s, 1H), 8.01 (d, 3 J = 7.6 Hz, 1H), 7.18 (d, 3 J = 7.5 Hz, 1H), 5.68 (s, 1H), 5.44 (d, 3 MS (ESI) + , 100% CH3OH, TOF):m / z [C 25 H 44 N3O8Si2] + [M+H] + Calculated value 570.2661; measured value 570.2802.

[0428] Synthesis of mono-2'O-acetylated silyl-protected cytidine 4 [ka] Scheme 3. Synthesis of mono-2'O-acetylated silyl-protected cytidine 4

[0429] Alternatively, compound 4 is synthesized as follows: Cytidine 2 (0.2 g, 0.412 mmol) was dissolved in anhydrous CH3CN (10 mL) to form a white emulsion. 4-Dimethylaminopyridine (DMAP) (0.2518 g, 2.061 mmol) was added, followed by acetyl chloride (AcCl) (29.3 μl, 4.122 mmol). The solution dissolved and the reaction was stirred at room temperature for 3 hours under argon. Once no starting material was observed, the reaction mixture was concentrated and the crude material was dissolved in dichloromethane CHCl (25 mL). The resulting solution was transferred to a separatory funnel and washed with water (1×25 mL). The aqueous layer was then extracted with additional CHCl (2×25 mL). The organic layers were combined, washed with brine, and dried over MgSO4 to give the crude silyl protection as a white solid. The residue was purified by flash column chromatography (ethyl acetate / methanol 9:1) to give compound 4 (63% yield) as the main fraction, as well as minor compounds 3 (13%) and 5 (18% yield). TLC (ethyl acetate:CH3OH=9 / 1, v / v): Rf=0.54, 1 H NMR (300 MHz, DMSO-d6, δ):10.86 (s, 1H), 8.09 (d, 3 J = 7.3 Hz, 1H), 7.16 (d, 3 J = 7.3 Hz, 1H), 5.74-5.71 (m, 1H), 5.55-5.53 (m, 1H), 4.20-4.13 (m, 1H), 4.02-3.94 (m, 3H), 3.94-3.86 (m, 1H), 2.05 (s, 3H), 1.02-0.91 (m, 31H). MS (ESI + , 100% CH3OH, TOF):m / z [C 23 H 42 N3O7Si2] + [M+H] + Calculated value 528.2556; measured value 528.2685.

[0430] Synthesis of N-Ac,2'-OAc cytidine 6 [ka] Scheme 4. Synthesis of N,O-diacetylated cytidine 6 (forming a mixture of 2'-OAc and 3'-OAc regioisomers)

[0431] Cytidine 3 (1.2 g, 2.11 mmol) was dissolved in anhydrous methanol (21 mL) in an oven-dried RBF (25 mL). Acetic acid (0.277 mL, 4.85 mmol) and ammonium fluoride NHF (0.172 g, 4.64 mmol) were added to the solution. The reaction mixture was stirred at room temperature for 48 hours. When no starting material was observed, the reaction mixture was concentrated and the crude material was dry loaded onto silica. The compound was purified on flash column chromatography (ethyl acetate / methanol 9:1) to give compound 6 as a white solid (0.352 g, 51%). 1 H NMR (300 MHz, DMSO-d6, δ): (mixture of 2'-OAc and 3'-OAc regioisomers) 10.91 (s, 1.2H), 8.41-8.32 (m, 1.4H), 7.20-7.14 (m, 1.4H), 5.91 (d, 3 J = 3.8 Hz, 0.4H), 5.82 (d, 3 J = 4.8 Hz, 1H), 5.13 (t, 3 J = 4.5 Hz, 0.4H ), 4.98 (t, 3 J = 5.0 Hz, 1H), 4.26 (t, 3 J = 4.9 Hz, 1H), 4.18 (t, 3 J = 5.6 Hz, 0.4H ), 4.09 (dt, 3 J = 4.8, 4 MS (ESI + , 100% CH3OH, TOF):m / z [C 13 H 18N3O7] + [M+H] + Calculated value 328.1139; measured value 328.2317.

[0432] Synthesis of monoacetylcytidine 7 [ka] Scheme 5. Synthesis of monoacetylcytidine 7

[0433] In an oven-dried RBF (25 mL), cytidine analog 4 (0.61 g, 1.16 mmol) was dissolved in anhydrous methanol (12 mL). Acetic acid (0.153 mL, 2.68 mmol) and ammonium fluoride NHF (0.095 g, 2.56 mmol) were added to the solution. The reaction was stirred at room temperature for 48 hours. Once no more starting material was observed, the reaction mixture was concentrated and the crude material was dry loaded onto silica. The compound was purified on flash column chromatography (ethyl acetate / methanol 9:1) to give compound 7 as a white solid (0.216 g, 46%). 1 H NMR (300 MHz, DMSO-d6, δ): (mixture of 2'-OAc and 3'-OAc regioisomers) 7.81 (t, 3 J = 8.0 Hz, 1.8H (signal overlap), 7.26-7.11 (m, 4H), 5.95 (d, 3 J = 5.1 Hz, 0.3H ), 5.81 (d, 3 J = 5.9 Hz, 1H), 5.72 (m, 2.3H), 5.61 (d, 3 J = 5.7 Hz, 1H), 5.05-4.98 (m, 2H), 4.21 (m, 1.3H), 4.04-3.97 (m, 2H), 3.91 (d, 3 J = 3.1 Hz, 1H), 3.84-3.79 (m, 1H), 3.65-3.50 (m, 4H), 2.06( s, 3H), 2.03 (s, 1H).MS (ESI + , 100% CH3OH, TOF):m / z [C 11 H 16N3O6] + [M+H] + Calculated value 286.1034; measured value 286.1571.

[0434] Triphosphorylation of cytidine derivatives [ka] Scheme 6. Triphosphorylation and salt exchange to form diacetylated cytidine triphosphate sodium salt 9.

[0435] The method was adapted from the literature (see Kore, AR; et. al. Curr. Protoc. Nucleic Acid Chem. 2012, Unit 13.10). In an oven-dried vial, tributylammonium pyrophosphate (0.143 g, 0.26 mmol) was dissolved in anhydrous acetonitrile (1.1 mL). Tributylamine (0.44 mL, 1.83 mmol) was added and the mixture was kept at -20 °C. In a separate oven-dried, argon-filled round-bottom flask equipped with a stir bar, cytidine derivative 6 (0.10 g, 0.31 mmol) was added and dissolved in trimethyl phosphate (1.4 mL). The reaction mixture was evacuated and backfilled with argon three times and stirred at room temperature for 10 min. The RBF / vial was cooled in an ice bath (5 °C-0 °C) and stirred for 30 min. The first portion of phosphorus oxychloride (20 μl, 0.21 mmol) was added and stirred for 10 min. A second portion of phosphorus oxychloride (20 μl, 0.21 mmol) was added and the reaction was stirred for 35 min. The pyrophosphate solution was then added dropwise to the mixture and the flask was cooled in an ice / NaCl bath (-5°C to -10°C) and stirred for 15 min. The septum was removed and water (7.3 mL) was added dropwise to the mixture. The solution was transferred to a separatory funnel and the aqueous layer was extracted with CHCl (3×2 mL) and the organic layer was discarded. The pH of the aqueous layer was adjusted to 6.5 using ammonium hydroxide and stored at 4°C overnight. The pH of the solution was rechecked and readjusted if necessary. The compound was purified on an AKTA purification system. The column was connected to an AKTA purification apparatus equipped with a programmable gradient pump system and UV detector and washed with water, 2.0 M NaCl, followed by water. The product solution was loaded onto the column at a flow rate of 75 ml / min and UV detection was performed at 254 nm. The column was eluted with a linear gradient of 0-1 M triethylammonium bicarbonate (TEAB) buffer. Appropriate fractions were analyzed by analytical HPLC using mobile phase A and mobile phase B. 10 μl fractions were injected through a 5 μM Hypersil SAX column (4.6 mm×25 cm) at a flow rate of 1.0 mL / min using the following gradient conditions: 0% to 50% mobile phase B (over 5 min) 50% to 75% mobile phase B (over 5 min) 75% to 100% mobile phase B (over 2 min) 100% mobile phase B (2 min) 100% to 0% mobile phase B (over 2 min).

[0436] The appropriate fractions containing phosphoric acid were combined and concentrated (35°C-38°C water bath and 1-10 mm vacuum). The resulting residue was coevaporated with water (3 x 2 ml) and dried under vacuum overnight. The resulting triethylamine salt of cytidine triphosphate was dissolved in water (2.6 mL).

[0437] Sodium perchlorate (0.422 g, 3.46 mmol) was dissolved in acetone (7.6 mL) in a centrifuge vial and the CTP solution was added dropwise to the sodium perchlorate / acetone solution over 5 min. The resulting mixture was centrifuged at 704×g and 4° C. for 10 min and the supernatant was discarded. The resulting pellet was dried under vacuum for 1 h, dissolved in water (2.6 mL), dissolved in the sodium perchlorate / acetone solution, and the centrifugation process was repeated. The resulting solid was dried under high vacuum at ambient temperature overnight and weighed to give the sodium salt of CTP9 as a white solid (0.027 g, 14%). 1 H NMR (300 MHz, DO, δ): (mixture of regioisomers) 8.23 ​​(d, 3 J = 7.4 Hz, 1H), 7.77 (d, 3 J = 7.6 Hz, 1H), 7.20 (d, 3 J = 7.7 Hz, 1H), 5.97-5.94 (m, 1H), 5.83-5.80 (m, 2H), 4.23-4.10 (m, 11H), 2.07-2.03 (m, 6H). 31 P NMR (121 MHz, D2O, δ):-5.49 (d, 3 J = 16.1 Hz, 1P), -10.68 (dd, 3 J = 15.7, 4 J =7.4 Hz, 1P), -19.13- -19.62 (m, 1P).

[0438] Triphosphorylation of 2'-O-acetyl-cytidine [ka] Scheme 7. Triphosphorylation and salt exchange to form 2'-O-acetylcytidine triphosphate sodium salt 11 (mixture of 2'-OAc and 3'-OAc regioisomers).

[0439] General procedure for triphosphorylation Error! Bookmark not defined. Performed as described above with the scale stated. In an oven-dried vial, dissolve tributylammonium pyrophosphate (0.164 g, 0.30 mmol) in anhydrous acetonitrile (1.3 mL) and tributylamine (0.50 mL, 2.10 mmol) and store the mixture at -20 °C. In an oven-dried, argon-filled round-bottom flask or vial equipped with a stir bar, add the cytidine derivative (0.10 g, 0.35 mmol) and dissolve in trimethyl phosphate (1.60 mL). Evacuate the reaction mixture and backfill with argon three times. Stir the reaction mixture at room temperature for 10 min. Cool the RBF / vial in an ice bath (5 °C to 0 °C) and stir for 30 min. Add the first portion of phosphorus oxychloride (22.9 μl, 0.25 mmol) and stir for 10 min. A second portion of phosphorus oxychloride (22.9 μl, 0.25 mmol) is added to the vial and stirred for 35 min. Pyrophosphate solution is added to the mixture. The flask is cooled in an ice / NaCl bath (-5°C to -10°C) and stirred for 15 min. The septum is removed and water (7.3 mL) is added dropwise to the mixture. The solution is transferred to a separatory funnel and the aqueous layer is extracted with CHCl (3×2 mL) and the organic layer is discarded. The pH of the aqueous layer is adjusted to 6.5 using ammonium hydroxide. Purification and salt exchange were carried out as described in the procedure above to give 11 as a white solid (0.025 g, 14%). 1 H NMR (300 MHz, D2O, δ):7.82 (d, 3 J = 7.56 Hz, 1H), 5.97 (d, 3 J = 7.7 Hz, 1H), 5.84 (d, 3 J = 4.4 Hz, 1H), 4.28-4.24 (m, 1H), 4.16 (t,3 J = 4.75 Hz, 1H), 4.11-4.05 (m, 4H), 2.06-2.02 (m, 3H). 31 P NMR (121 MHz;D2O, δ): -7.55- -7.86 (m, 1P), -11.30 (d, 3 J = 19.1 Hz, 1P), -22.36 (m, 1P).

[0440] TIPDSi-protection of uridine [ka] Scheme 8. Synthesis of silyl-protected uridine 13

[0441] Uridine 12 (3.0 g, 12.30 mmol) was dissolved in pyridine and coevaporated (3×30 mL) to a white suspension. The coevaporated substrate 12 was dissolved in pyridine (40 mL) and TIPDSCl2 (4.260 mL, 13.52 mmol) and DMAP (0.7508 g, 6.14 mmol) were added. The reaction was stirred at room temperature for about 24 h and monitored by TLC. Once no starting material was detected, the mixture was diluted with CHCl (25 mL). The resulting solution was transferred to a separatory funnel and washed with water (1×25 mL). The aqueous layer was then extracted with additional CHCl (2×25 mL). The organic layers were combined, washed with brine, and dried over MgSO4 to give crude silyl-protected 13 as a white solid, which was purified by silica gel flash chromatography (CHCl / CHOH, modified with 5% EtN). Yield (3.67 g, 61%). Compound 13 was stored at -20°C until use. TLC (CH2Cl2:CH3OH=95 / 5, v / v): Rf=0.75, 1 H NMR (300 MHz, DMSO-d6, δ):11.36 (s, 1H), 8.54 (dd, 3 J = 5.8, 4 J =1.7 Hz, 1H), 7.66 (d, 3 J = 8.1 Hz, 1H), 5.60 (d, 3J = 4.4 Hz, 1H), 4.12-4.07 (m, 4H), 3.96-3.85 (m, 3H), 1.01 (d, 3 J = 3.9 Hz, 36H). MS (ESI + , 100% CH3OH, TOF):m / z [C 21 H 39 N2O7Si2] + [M+H] + Calculated value 487.2290; measured value 487.3135.

[0442] Acetylation of silyl-protected uridine [ka] Scheme 9. Synthesis of 2'-O-acetylsilyl protected uridine 14

[0443] In an oven-dried RBF (25 mL), uridine 13 (2.0 g, 4.11 mmol) was dissolved in anhydrous CH3CN (40 mL) to form a white emulsion. DMAP (1.16 g, 9.46 mmol) was added, followed by acetyl chloride AcCl (0.67 mL, 9.46 mmol). The solution dissolved and the reaction was stirred at room temperature for 35 min under argon. Once the reaction was complete (no starting material detected), the reaction mixture was concentrated and the crude material was dissolved in dichloromethane CHCl (25 mL). The resulting solution was transferred to a separatory funnel and washed with water (1×25 mL). The aqueous layer was then extracted with additional CHCl (2×25 mL). The organic layers were combined, washed with brine, and dried over magnesium sulfate MgSO4 to give crude silyl-protected 14 as a white solid. The residue was purified by flash column chromatography (ethyl acetate / methanol 9:1) to give 14 as the major compound in a yield of 1.88 g (87%). TLC (ethyl acetate:CH2Cl2=1 / 1, v / v): Rf=0.75, 1 H NMR (300 MHz, DMSO-d6, δ):11.40 (s, 1H), 7.64 (d, 3 J = 8.1 Hz, 1H), 5.63 (d, 3J = 1.4 Hz, 1H), 5.56 (dd, 3 J = 8.0, 4 J = 2.0 Hz, 1H), 5.49 (dd, 3 J = 5.7, 4 J = 1.3 Hz, 1H), 4.53 (dd, 3 J = 8.4, 4 J = 5.8 Hz, 1H), 4.04 (m, 1H), 3.91 (m, 1H), 3.81 (dt, 3 J = 7.8, 4 J = 3.7 Hz, 1H), 2.05 (s, 3H), 1.02-0.93 (m, 28H). MS (ESI + , 100% CH3OH, TOF):m / z [C 23 H 40 N2NaO8Si2] + [M+Na] + Calculated value: 551.2215; measured value: 551.2078

[0444] Silyl deprotection of acetylated uridine [ka] Scheme 10. Synthesis of 2'O-acetyluridine 15

[0445] Compound 14 (1.2 g, 2.27 mmol) was dissolved in anhydrous methanol (22 mL) in an oven-dried RBF (25 mL). Acetic acid (0.299 mL, 5.22 mmol) was added to the solution followed by NHF (0.185 g, 5.00 mmol). The reaction mixture was stirred at room temperature for 48 hours. Once no starting material was observed, the reaction mixture was concentrated and the crude material was dry loaded onto silica. The compound was purified on flash column chromatography (ethyl acetate / methanol 9:1) to give compound 15 as a white solid (0.222 g, 34%) (mixture of 2'-OAc and 3'-OAc regioisomers). 1 H NMR (300 MHz, DMSO-d6, δ): 11.37 (s, 1.4H), 7.87 (d, 3J = 12.2, d, 3 J = 8.1 Hz, (two overlapping doublets) 1.6H), 5.96 (d, 3 J = 5.9 Hz, 0.6H), 5.79 (d, 3 J = 6.8 Hz, 1H), 5.74-5.64 (m, 2.6H), 5.51 (d, 3 J = 5.4 Hz, 0.6H), 5.27 (t, 3 J = 5.0 Hz, 1H), 5.20 (t, 3 J = 4.8 Hz, 0.6H), 5.11-5.06 (m, 1.6H), 4.22 (dq, 3 J = 11.5, 4 MS (ESI) + , 100% CH3OH, TOF):m / z [C 11 H 14 N2NaO7] + [M+Na] + Calculated value: 309.0693; measured value: 309.2410

[0446] Triphosphorylation of uridine derivatives [ka] Scheme 11. Triphosphorylation and salt exchange, synthesis of 2'-O-acetyluridine triphosphate sodium salt 17

[0447] This method was based on the literature. Error!Bookmark not defined. In an oven-dried vial, tributylammonium pyrophosphate (0.114 g, 0.21 mmol) was dissolved in anhydrous CH3CN (0.90 mL). Tributylamine (0.35 mL, 1.47 mmol) was added and the mixture was stored at -20 °C. In a separate oven-dried argon-filled round-bottom flask or vial equipped with a stir bar, uridine derivative 15 (0.07 g, 0.25 mmol) and proton sponge (0.052 g, 0.25 mmol) were added and dissolved in trimethyl phosphate (0.84 mL). The reaction mixture was evacuated and backfilled with argon three times. The reaction mixture was stirred at room temperature for 10 min, then the RBF / vial was cooled in an ice / NaCl bath (-5 °C to -10 °C) and stirred for 30 min. The first portion of phosphorus oxychloride (13.5 μl, 0.14 mmol) was added and stirred for 5 min. A second portion of phosphorus oxychloride (9.3 μl, 0.10 mmol) was added and stirred for 10 min. Cold pyrophosphoric acid solution was added and stirred for 10 min. The septum was removed and water (7.3 mL) was added dropwise to the mixture. The solution was transferred to a separatory funnel and the aqueous layer was extracted with CHCl (3×2 mL) and the organic layer was discarded. The pH of the aqueous layer was adjusted to 6.5 using ammonium hydroxide. Purification and salt exchange were carried out as described in the general procedure above to give 17 as a white solid (4.2 mg, 3%). 1 H NMR (300 MHz, D2O, δ):7.81 (d, 3 J = 8.4 Hz, 2H), 5.83-5.78 (m, 3H), 4.30-4.21 (m, 3H), 4.08 (m, 4H), 3.50 (d, 3 J = 11.4 Hz, 1H), 2.05 (s, 3H). 31 P NMR (121 MHz, D2O, δ): -6.18 (m, 1P), -11.17 (m, 1P), -21.68 (s, 1P).

[0448] TIPDSi-Protection of Adenosine [ka] Scheme 12. Synthesis of silyl-protected adenosine 19

[0449] In a 100 mL round bottom flask, adenosine 18 (0.3 g, 1.12 mmol) was dissolved in pyridine and coevaporated (3×5 mL) to a white suspension. The coevaporated substrate 18 was dissolved in pyridine (4 mL) and TIPDSCl2 (0.40 mL, 1.25 mmol) and DMAP (0.0686 g, 0.561 mmol) were added. The reaction was stirred at room temperature for about 23 h and monitored by TLC. Once no starting material was detected, the mixture was diluted with CHCl (25 mL). The resulting solution was transferred to a separatory funnel and washed with water (1×25 mL). The aqueous layer was then extracted with additional CHCl (2×15 mL). The organic layers were combined, washed with brine, and dried over MgSO4 to give crude silyl-protected 19 as a white solid, which was purified by silica gel flash chromatography (CH2Cl2 / CH3OH, 5%, 0.05% Et3N). Yield (0.885 g, 76%). Compound 19 was stored at -20 °C until use. 1 H NMR (300 MHz, DMSO-d6, δ):8.19 (s, 1H), 8.05 (s, 1H), 5.85 (s, 1H), 5.63 (d, 3 J = 4.6 Hz, 1H), 4.79-4.75 (m, 1H), 4.49 (t, 3 J = 4.7 Hz, 1H), 4.06-3.88 (m, 3H), 1.02 (s, 28H). MS (ESI + , 100% CH3OH, TOF):m / z [C 22 H 40 N5O5Si2] + [M+H] + Calculated value 510.2563; measured value 510.3010.

[0450] Acetylation of silyl-protected adenosine [ka] Scheme 13. Synthesis of 2'-OAc-adenosine 20

[0451] In an oven-dried RBF (25 mL), adenosine 19 (5.83 g, 11.44 mmol) was dissolved in anhydrous CH3CN (100 mL) to form a white emulsion. 4-Dimethylaminopyridine DMAP (3.214 g, 26.31 mmol) was added, followed by acetyl chloride AcCl (1.3 mL, 18.30 mmol). The solution dissolved and the reaction was stirred at room temperature under argon for 1.5 h. Upon reaction completion, the reaction mixture was concentrated and the crude material was dissolved in dichloromethane CHCl (25 mL). The resulting solution was transferred to a separatory funnel and washed with water (1×25 mL). The aqueous layer was then extracted with additional CHCl (2×25 mL). The organic layers were combined, washed with brine (1×25 mL) and dried over magnesium sulfate MgSO4 to give crude silyl protected 20 as a white solid. The residue was purified by flash column chromatography (CH2Cl2 / CH3OH, modified with 5% Et3N) to give compound 20 as the major fraction in yield (5.567 g, 88%). TLC (CH2Cl2 / CH3OH, modified with 5% Et3N, v / v): 1 H NMR (300 MHz, DMSO-d6, δ): 8.23 ​​(s, 1H), 8.02 (s, 1H), 7.39 (s, 2H), 6.07 (s, 1H), 5.86 (dd, 3 J = 5.8, 4 J =1.0 Hz, 1H), 5.24 (dd, 3 J = 8.4, 4 J =5.8 Hz, 1H), 4.01-3.87 (m, 3H), 2.08 (s, 3H), 1.04-0.93 (m, 36H). + , 100% CH3OH, TOF):m / z [C 24 H 42 N5O6Si2] + [M+H] + Calculated value 552.2668; measured value 552.2981.

[0452] Silyl deprotection of acetylated adenosine [ka] Scheme 14. Synthesis of 2'-OAc adenosine 21

[0453] Adenosine derivative 20 (0.50 g, 0.906 mmol) was dissolved in anhydrous methanol (10 mL) in an oven-dried RBF (50 mL). Acetic acid (1.2 mL, 2.08 mmol) was added to the solution followed by ammonium fluoride NHF (0.074 g, 1.99 mmol). The reaction was stirred at 70 °C for 6 h. Once no starting material was observed, the reaction mixture was concentrated and the crude material was concentrated and redissolved in water (1 x 15 mL), filtered through a celite plug and the plug was rinsed with water (3 x 15 mL). The compounds were purified by aqueous reverse-phase flash chromatography (HO / CHCN 7:3) to give 21 (minor) as a white solid (0.0258 g, 9%, mixture of 2'-OAc and 3'-OAc isomers) and 22 (major) as a white solid (0.064 g, 23%, mixture of 2'-OAc and 3'-OAc isomers).

[0454] (minor peak on reversed phase chromatogram): 1 H NMR (300 MHz, DMSO-d6, δ): (mixture of 2'-OAc and 3'-OAc regioisomers); 8.36 (s, 1H), 8.12 (s, 1H), 7.39 (s, 2H), 6.13 (d, 3 J = 6.4 Hz, 1H), 5.61 (m, 2H), 5.48 (dd, 3 J = 7.0, 4 J = 4.5 Hz, 1H), 4.95 (m, 0.04H, corresponding to 3'-OAc regioisomer) 4.44-4.41 (m, 1H), 3.98 (m, 1H), 3.70-3.65 (m, 1H), 3.58-3.52 (m, 1H), 2.10 (s, 0.1H), 2.00 (s, 3H).MS (ESI + , 100% CH3OH, TOF):m / z [C 12 H 16 N5O5] + [M+H] +Calculated value 310.1146; measured value 310.1014.

[0455] (Main peak on reversed phase chromatogram): 1 H NMR (300 MHz, DMSO-d6, δ): (mixture of 2'-OAc and 3'-OAc isomers): 8.36 (s, 1H), 8.12 (s, 1H), 7.41 (s, 2H), 6.145(d, 3 J = 6.47, 0.04H corresponding to the 2'-OAc isomer), 5.87 (d, 3 J = 7.2 Hz, 1H), 5.80 (d, 3 J = 5.7 Hz, 1H), 5.67 (dd, 3 J = 7.5, 4 J = 4.5 Hz, 1H), 5.26 (dd, 3 J = 5.2, 4 J =1.9 Hz, 1H), 4.86 (q, 3 J = 5.9 Hz, 1H), 4.12 (d, 3 J = 2.1 Hz, 1H), 3.67 (dt, 3 J = 12.2, 4 J = 4.0 Hz, 1H), 3.57 (ddd, 3 J = 11.8, 4 J = 7.9, 4 J =3.6 Hz, 1H), 2.10 (s, 3H). MS (ESI + , 100% CH3OH, TOF):m / z [C 12 H 16 N5O5] + [M+H] + Calculated value 310.1146; measured value 310.1075.

[0456] Triphosphorylation of adenosine derivatives [ka] Scheme 15. Synthesis of 2'-O-acetyladenosine triphosphate sodium salt 24 via triphosphorylation and salt exchange protocol.

[0457] This method was adapted from the literature. Error!Bookmark not defined. In an oven-dried, argon-filled vial, dissolve tributylammonium pyrophosphate (0.109 g, 0.199 mmol) in anhydrous acetonitrile (0.85 mL) and tributylamine (0.33 mL, 1.391 mmol) and store the mixture at -20 °C. In an oven-dried, argon-filled round-bottom flask or vial equipped with a stir bar, add adenosine derivative mixture 21 / 22 (2'-OAc / 3'-OAc isomers) (0.0725 g, 0.234 mmol) and tributylamine (0.11 mL, 0.467 mmol) and dissolve in trimethyl phosphate (1 mL). Evacuate the reaction mixture and backfill with argon three times. Stir the reaction mixture at room temperature for 10 min. Cool the RBF / vial in an ice / NaCl bath (-5 °C to -10 °C) and stir for 30 min. Add the first portion of phosphorus oxychloride (26 μl, 0.278 mmol) and stir for 3 min. Add the second portion of phosphorus oxychloride (13 μl, 0.139 mmol) to the vial and stir for 5 min. Add the pyrophosphate solution and stir for 10 min. The reaction was monitored on an HPLC-SAX column. Remove the septum and add water (5 mL) dropwise to the mixture. Transfer the solution to a separatory funnel, extract the aqueous layer with CHCl (3 × 2 mL) and discard the organic layer. Adjust the pH of the aqueous layer to 6.5 using ammonium. Purification was performed on an AKTA column and the appropriate fractions containing phosphoric acid were combined and concentrated in a water bath (35 °C-38 °C and vacuum 1-10 mm). Coevaporate the resulting residue with water (3 × 2 ml) and dry it under vacuum overnight. Dissolve the resulting triethylamine salt of cytidine triphosphate in water (2.6 mL).

[0458] Sodium perchlorate (0.062 g, 0.508 mmol) was dissolved in acetone (2.5 mL) in a 15 mL centrifuge vial and the ATP solution was added dropwise to the sodium perchlorate / acetone solution over 5 min. The resulting mixture was centrifuged at 704×g and 4° C. for 10 min and the supernatant was discarded. The resulting pellet was dried under vacuum for 1 h, dissolved in water (2.6 mL), dissolved in the sodium perchlorate / acetone solution, and the centrifugation process was repeated. The resulting solid was dried under high vacuum at ambient temperature overnight and weighed to give the sodium salt of ATP24 as a white solid (30.8 mg, 21%). 1 H NMR (300 MHz, D2O, δ):8.34 (d, 3 J = 10.3 Hz, 2H), 8.27 (s, 1H), 8.03 (d, 3 J = 2.3 Hz, 2H), 8.00 (s, 1H), 6.07 (dd, 3 J = 4.1, 4 J = 1.8 Hz, 1H), 5.96 (d, 3 J = 7.6 Hz, 1H), 5.92-5.90 (m, 1H), 5.37-5.33 (m, 1H), 5.30 (dd, 3 J = 5.4, 4 J = 1.7 Hz, 2H), 4.84-4.79 (m, 2H), 4.58 (dd, 3 J = 3.5, 4 J =1.6 Hz, 2H), 4.38-4.33 (m, 2H), 4.18-4.16 (m, 2H), 4.12-4.08 (m, 3H), 4.08-4.03 (m, 2H), 4.01-3.95 (m, 2H), 2.00 (d, 3 J = 5.3 Hz, 15H), 1.94 (s, 3H). 31 P NMR (121 MHz, D2O, δ):-8.68- -9.23 (m, 1P), -11.41 (d, 3 J = 19.5 Hz, 1P), -22.73 (t, 3 J = 19.6 Hz, 1P). MS (ESI -, 100% CH3OH, TOF):m / z [C 12 H 17 N5O 14 P3] - [MH] - Calculated value 547.9990; measured value 547.7838.

[0459] TIPDSi-Protection of Guanosine [ka] Scheme 16. Synthesis of silyl-protected guanosine 26

[0460] In a 100 mL round bottom flask, guanosine 25 (1.5 g, 5.30 mmol) was dissolved in pyridine and coevaporated (3×15 mL) to a white suspension. The coevaporated substrate 25 was dissolved in pyridine (19 mL) and TIPDSCl2 (1.86 mL, 5.83 mmol) and DMAP (0.323 g, 2.65 mmol) were added. The reaction was stirred at room temperature for about 23 h and monitored by TLC. Once no starting material was detected, the mixture was diluted with CHCl (25 mL). Addition of water results in precipitation. The mixture was filtered over a short plug of celite to give crude silyl protected 26 as a white solid in yield (1.3916 g, 50%). Compound 26 was stored at −20° C. until use. 1 H NMR (300 MHz, DMSO-d6, δ) 10.64 (s, 1H), 7.75 (s, 1H), 6.49 (s, 2H), 5.65 (s, 1H), 5.59 (s, 1H), 4.33 (dd, 3 J = 8.0, 3 J =5.1 Hz, 1H), 4.24-4.23 (m, 1H), 4.06 (dd, 3 J = 12.6, 3 J = 2.9 Hz, 1H), 3.97 (dt, 3 J = 7.9, 4 J =2.8 Hz, 1H), 3.90 (m, 1H), 1.02-0.98 (m, 28H). MS (ESI +, 100% CH3OH, TOF):m / z [C 22 H 40 N5O6Si2] + [M+H] + Calculated value: 526.2512; measured value: 526.3105

[0461] Acetylation of silyl-protected guanosine [ka] Scheme 17. Synthesis of 2'-O-acetyl-silyl protected guanosine 27

[0462] In an oven-dried RBF (25 mL), guanosine 26 (1.084 g, 2.062 mmol) was dissolved in anhydrous CH3CN (20 mL) to form a white emulsion. 4-Dimethylaminopyridine DMAP (0.58 g, 4.742 mmol) was added, followed by acetyl chloride (AcCl) (0.23 mL, 3.299 mmol). The solution dissolved and the reaction was stirred at room temperature under argon for 30 min. Upon completion of the reaction (monitored by TLC), the reaction mixture was concentrated and the crude material was dissolved in CHCl (25 mL). The resulting solution was transferred to a separatory funnel and washed with water (2×25 mL). The aqueous layer was then extracted with additional CHCl (2×25 mL). The organic layers were combined, washed with brine, and dried over MgSO4 to give crude silyl-protected 27 as a white solid. The residue was purified by flash column chromatography (CH2Cl2 / CH3OH, modified with 5% Et3N) to give a white solid (0.9295 g, 79%). 1 H NMR (300 MHz, DMSO-d6, δ):10.68 (s, 1H), 7.86 (s, 1H), 6.41 (s, 2H), 5.85 (s, 1H), 5.73 (s, 0.3H), 5.57 (d, 3 MS (ESI +, 100% CH3OH, TOF):m / z [C 24 H 42 N5O7Si2] + [M+H] + Calculated value: 568.2617; measured value: 568.3389

[0463] Silyl deprotection of acetylated guanosine [ka] Scheme 18. Synthesis of 2'-OAc-guanosine 28

[0464] In an oven-dried RBF (50 mL), guanosine analog 27 (0.7239 g, 1.275 mmol) was dissolved in anhydrous methanol (13 mL). Acetic acid (170 μL, 2.932 mmol) was added to the solution followed by ammonium fluoride NHF (0.1039 g, 2.805 mmol). The reaction was stirred at room temperature for 72 h. When no starting material was observed on TLC, the reaction mixture was concentrated and the crude material was concentrated and redissolved in water (1×25 mL), filtered through a celite plug and the plug was rinsed with water (3×25 mL). The compound was purified on aqueous reverse-phase flash chromatography (H2O / CH3CN 7:3) to give the major compound 28 as a white solid (0.011 g, 23%) and the minor compound 29 as a white solid (0.0047 g, 4%). 1 H NMR (300 MHz, DMSO-d6, δ):10.66 (s, 1H), 7.94 (s, 1H), 6.49-6.45 (m, 3H), 5.77-5.75 (m, 1H), 5.66 (d, 3 J = 7.2 Hz, 1H), 5.26-5.21 (m, 1H), 5.20 (dt, 3 J = 5.4, 4 J = 2.7 Hz, 1H), 4.69-4.62 (m, 1H), 4.36 (dt, 3 J = 3.2, 4J =1.5 Hz, ), 4.03-4.00 (m, 1H), 3.62-3.51 (m, 3H), 2.08 (s, 3H), 2.01 (s, 0.05H). + , 100% CH3OH, TOF):m / z [C 12 H 16 N5O5] + [M+H] + Calculated value: 326.1095; measured value: 326.1147

[0465] Triphosphorylation of guanosine derivatives [ka] Scheme 19. Triphosphorylation and salt exchange, synthesis of 2'-OAc guanosine triphosphate sodium salt 30

[0466] This method was adapted from the literature. Error!Bookmark not defined. In an oven-dried vial, dissolve tributylammonium pyrophosphate (0.136 g, 0.249 mmol) in anhydrous acetonitrile (1.3 mL) and tributylamine (0.41 mL, 1.740 mmol) and store the mixture at -20 °C. In an oven-dried, argon-filled round-bottom flask or vial equipped with a stir bar, add the guanosine derivative (0.0954 g, 0.293 mmol) and tributylamine (0.21 mL, 0.87 mmol) and dissolve in trimethyl phosphate (1 mL). Evacuate the reaction mixture and backfill with argon three times. Stir the reaction mixture at room temperature for 10 min. Cool the RBF / vial in an ice / NaCl bath (-5 °C to -10 °C) and stir for 30 min. Add the first portion of phosphorus oxychloride (35 μl, 0.373 mmol) and stir for 3 min. A second portion of phosphorus oxychloride (23 μl, 0.249 mmol) is added to the vial and stirred for 5 min. Pyrophosphate solution is added and stirred for 10 min. The reaction was monitored on an HPLC-SAX column. The septum was removed and water (5 mL) was added dropwise to the mixture. The solution was transferred to a separatory funnel, the aqueous layer was extracted with CHCl (3×2 mL) and the organic layer was discarded. The pH of the aqueous layer was adjusted to 6.5 using ammonium hydroxide. Purification was performed on an AKTA column and the appropriate fractions containing phosphoric acid were combined and concentrated in a water bath (35°C-38°C and 1-10 mm vacuum). The resulting residue was coevaporated with water (3×2 ml) and dried under vacuum overnight. The obtained triethylamine salt of guanosine triphosphate was dissolved in water (2.6 mL).

[0467] Sodium perchlorate (0.0954 g, 0.781 mmol) was dissolved in acetone (3 mL) in a 15 mL centrifuge vial and the GTP solution was added dropwise to the sodium perchlorate / acetone solution over 5 min. The resulting mixture was centrifuged at 704×g and 4° C. for 10 min and the supernatant was discarded. The resulting pellet was dried under vacuum for 1 h, dissolved in water (2.6 mL), dissolved in the sodium perchlorate / acetone solution, and the centrifugation process was repeated. The resulting solid was dried under high vacuum at ambient temperature overnight and weighed to give the sodium salt of GTP30 as a white solid (6.4 mg, 4%). 1 H NMR (300 MHz, D2O, δ):8.11 (s, 0.3H), 8.08 (s, 2H), 7.98 (s, 1H), 6.02 (d, 3 J = 3.6 Hz, 1H), 5.87 (dd, 3 J = 10.2, 3 J =7.2 Hz, 2H), 5.57 (d, 3 J = 4.7 Hz, 1H), 5.46 (dd, 3 J = 5.3, 3 J = 0.6 Hz, 1H), 5.04 (t, 3 J = 6.6 Hz, 1H), 4.59-4.56 (m, 1H), 4.45 (s, 1H), 4.27-4.12 (m, 7H), 2.19 (s, 1H), 2.16 (s, 3H), 2.11 (s, 1H). 31 P NMR (121 MHz, D2O, δ): -6.27- -6.62 (m, 1P), -11.21 (dt, 3 J = 19.7, 3 J = 7.7 Hz, 1P), -22.25 (m, 1P).

[0468] Exemplary embodiments Embodiment 1. A modified ribonucleotide comprising a nucleoside, wherein the nucleoside comprises a ribose moiety comprising an acetyl group, wherein the ribose is 2'-O-acetylated, and wherein the modified ribonucleotide is selected from the group consisting of: [ka] The structure is

[0469] (a) wherein X is a 5' monophosphate, a 5' diphosphate, or a 5' triphosphate;

[0470] (b) The modified ribonucleotide, wherein R is a nucleobase selected from adenine or a modified form thereof, guanine or a modified form thereof, cytosine or a modified form thereof, or uracil or a modified form thereof.

[0471] Embodiment 2. The modified ribonucleotide of embodiment 1, wherein the nucleobase is adenine.

[0472] Embodiment 3. The modified ribonucleotide comprises a 5' triphosphate and the following: [ka] 3. The modified ribonucleotide of embodiment 2, having the structure:

[0473] Embodiment 4. The modified ribonucleotide of embodiment 1, wherein the nucleobase is guanine.

[0474] Embodiment 5. The modified ribonucleotide comprises a 5' triphosphate and the following: [ka] 5. The modified ribonucleotide of embodiment 4, having the structure:

[0475] Embodiment 6 The modified ribonucleotide of embodiment 1, wherein the nucleobase is a cytosine.

[0476] Embodiment 7. The modified ribonucleotide comprises a 5' triphosphate and the following: [ka] 7. The modified ribonucleotide of embodiment 6, having the structure:

[0477] Embodiment 8. The modified ribonucleotide of embodiment 1, wherein the nucleobase is N4-acetylcytidine.

[0478] Embodiment 9. The modified ribonucleotide comprises a 5' triphosphate and the following: [ka] 9. The modified ribonucleotide of embodiment 8, having the structure:

[0479] Embodiment 10. The modified ribonucleotide of embodiment 1, wherein the nucleobase is uracil.

[0480] Embodiment 11. The modified ribonucleotide comprises a 5' triphosphate and the following: [ka] 11. The modified ribonucleotide of embodiment 10, having the structure:

[0481] Embodiment 12. The modified ribonucleotide of embodiment 1, wherein the nucleobase is 5-hydroxymethyluridine.

[0482] Embodiment 13. The modified ribonucleotide comprises a 5' triphosphate and the following: [ka] 13. The modified ribonucleotide of embodiment 12, having the structure:

[0483] Embodiment 14 The modified ribonucleotide of embodiment 1, wherein the nucleobase is N1-methylpseudouridine.

[0484] Embodiment 15. The modified ribonucleotide comprises a 5' triphosphate and the following: [ka] 15. The modified ribonucleotide of embodiment 14, having the structure:

[0485] Embodiment 16 A polyribonucleotide comprising one or more modified ribonucleotides according to any one of the preceding embodiments.

[0486] Embodiment 17. The polyribonucleotide of embodiment 16, wherein at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least about 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% of the ribose moieties are acetylated (2'-O-acetylated).

[0487] Embodiment 18. The polyribonucleotide of embodiment 16, wherein at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% of the ribose moieties are acetylated (2'-O-acetylated).

[0488] Embodiment 19. The polyribonucleotide of any one of embodiments 16 to 18, wherein 100% of the ribose moieties are acetylated (2'-O-acetylated).

[0489] Embodiment 20. About 5% to 99%, about 5% to 95%, about 5% to 90%, about 5% to 85%, about 5% to 80%, about 5% to 75%, about 5% to 70%, about 5% to 65%, about 5% to 60%, about 5% to 55%, about 5% to 50%, about 5% to 45%, about 5% to 40%, about 5% to 35%, about 5% to 30%, about 5% to 25%, about 5% to 20%, about 5% to 15%, about 5% to 10%, about 10% to 99%, about 15% to 99% 20% to 99%, about 25% to 99%, about 30% to 99%, about 35% to 99%, about 40% to 99%, about 45% to 99%, about 50% to 99%, about 55% to 99%, about 60% to 99%, about 65% to 99%, about 70% to 99%, about 80% to 99%, about 85% to 99%, about 90% to 99%, or about 95% to 99% is acetylated (2'-O-acetylated).

[0490] Embodiment 21. The polyribonucleotide of embodiment 20, wherein at least 95% of the ribose moieties are 2'-O-acetylated.

[0491] Embodiment 22. A polyribonucleotide according to any one of embodiments 16 to 21, wherein the polyribonucleotide comprises a cap structure, and the cap structure does not comprise 2'-O-acetylated ribose.

[0492] Embodiment 23. A polyribonucleotide according to any one of embodiments 16 to 21, wherein the polyribonucleotide comprises a cap structure, and the cap structure comprises a 2'-O-acetylated ribose.

[0493] Embodiment 24. The polyribonucleotide of any one of embodiments 16 to 22, wherein the polyribonucleotide further comprises a modification comprising a modified backbone, a modified nucleobase, or any combination thereof.

[0494] Embodiment 25. The polyribonucleotide of embodiment 24, wherein the polyribonucleotide comprises a modified nucleobase.

[0495] Embodiment 26. The polyribonucleotide of embodiment 25, wherein the nucleobase containing a modification is selected from adenine, guanine, cytosine, or uracil.

[0496] Embodiment 27. The polyribonucleotide of embodiment 26, wherein the nucleobase is adenine.

[0497] Embodiment 28. The polyribonucleotide of embodiment 26, wherein the nucleobase is guanine.

[0498] Embodiment 29. The polyribonucleotide of embodiment 26, wherein the nucleobase is cytosine.

[0499] Embodiment 30. The polyribonucleotide of embodiment 26, wherein the nucleobase is uracil.

[0500] Embodiment 31. The modification is N4-acetyl-cytidine (ac4C), 5-hydroxymethyluridine, N1-pseudomethyluridine, pseudouridine, pyridin-4-one ribonucleoside, 5-aza-uridine, 6-aza-uridine, 2-thio-5-aza-uridine, 2-thio-uridine (s2U), 5-methylcytidine (m5C), 5-aza-cytidine, 6-aza-cytidine, pseudoisocytidine, 3-methyl-cytidine, 17. The polyribonucleotide of any one of embodiments 14 to 16, comprising cytidine (m3C), 5-formyl-cytidine (f5C), N4-methyl-cytidine (m4C), 2-amino-purine, 2,6-diaminopurine, 2-amino-6-halo-purine, 6-halo-purine, inosine (I), 1-methyl-inosine (m1I), wyosine (imG), methyl wyosine (mimG), or a combination thereof.

[0501] Embodiment 32. The polyribonucleotide of any one of embodiments 16 to 31, wherein the ribonucleotide comprises a nucleoside that includes an acetyl group, and the nucleoside is N4-acetylcytidine.

[0502] Embodiment 33. The ribonucleotide comprises a nucleoside comprising an acetyl group, the nucleoside being N4-acetylcytidine, and the modified ribonucleotide is: [ka] The polyribonucleotide according to any one of embodiments 16 to 31, having the structure:

[0503] Embodiment 34. The polyribonucleotide comprises cytidine residues, and about 5% to 99%, about 5% to 95%, about 5% to 90%, about 5% to 85%, about 5% to 80%, about 5% to 75%, about 5% to 70%, about 5% to 65%, about 5% to 60%, about 5% to 55%, about 5% to 50%, about 5% to 45%, about 5% to 40%, about 5% to 35%, about 5% to 30%, about 5% to 25%, about 5% to 20%, about 5% to 15%, about 5% to 10%, about 5% to 25%, about 5% to 3 ... The polyribonucleotide according to embodiment 32 or 33, wherein 0%, about 10% to 99%, about 15% to 99%, about 20% to 99%, about 25% to 99%, about 30% to 99%, about 35% to 99%, about 40% to 99%, about 45% to 99%, about 50% to 99%, about 55% to 99%, about 60% to 99%, about 65% to 99%, about 70% to 99%, about 80% to 99%, about 85% to 99%, about 90% to 99%, or about 95% to 99% comprises N4-acetylcytidine.

[0504] Embodiment 35. The polyribonucleotide of embodiment 32 or 33, wherein the polyribonucleotide comprises cytidine residues, and wherein at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% of the cytidine residues in the polyribonucleotide comprise N4-acetylcytidine.

[0505] Embodiment 36. A polyribonucleotide according to any one of embodiments 33 to 35, wherein at least 1% of the N4-acetylcytidine residues contain 2-O acetylated ribose.

[0506] Embodiment 37. A polyribonucleotide according to embodiment 33 or 34, wherein no more than 90% of the N4-acetylcytidine residues contain 2-O acetylated ribose.

[0507] Embodiment 38. The polyribonucleotide according to any one of embodiments 33 to 35, wherein the polyribonucleotide further comprises, in addition to N-acetylcytidine, one or more additional ribonucleotides, such as modified ribonucleotides.

[0508] Embodiment 39. The polyribonucleotide of embodiment 38, wherein the one or more ribonucleotides (e.g., modified ribonucleotides) comprise a nucleoside selected from adenosine, guanosine, cytidine, or uridine, or a combination thereof.

[0509] Embodiment 40. The polyribonucleotide of any one of embodiments 16 to 39, wherein the ribonucleotide comprises a nucleoside that includes a hydroxymethyl group, and the nucleoside is 5-hydroxymethyluridine.

[0510] Embodiment 41. The ribonucleotide comprises a nucleoside comprising a hydroxymethyl group, wherein the nucleoside is 5-hydroxymethyluridine, and the modified ribonucleotide is selected from the group consisting of: [ka] The polyribonucleotide according to any one of embodiments 16 to 39, having the structure:

[0511] Embodiment 42. The polyribonucleotide contains uridine residues, and about 5% to 99%, about 5% to 95%, about 5% to 90%, about 5% to 85%, about 5% to 80%, about 5% to 75%, about 5% to 70%, about 5% to 65%, about 5% to 60%, about 5% to 55%, about 5% to 50%, about 5% to 45%, about 5% to 40%, about 5% to 35%, about 5% to 30%, about 5% to 25%, about 5% to 20%, about 5% to 15%, about 5% to 1 ... 42. The polyribonucleotide according to embodiment 40 or 41, wherein about 10% to 99%, about 15% to 99%, about 20% to 99%, about 25% to 99%, about 30% to 99%, about 35% to 99%, about 40% to 99%, about 45% to 99%, about 50% to 99%, about 55% to 99%, about 60% to 99%, about 65% to 99%, about 70% to 99%, about 80% to 99%, about 85% to 99%, about 90% to 99%, or about 95% to 99% comprises 5-hydroxymethyluridine.

[0512] Embodiment 43. The polyribonucleotide of embodiment 40 or 41, wherein the polyribonucleotide comprises uridine residues, and wherein at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% of the uridine residues in the polyribonucleotide comprise 5-hydroxymethyluridine.

[0513] Embodiment 44. A polyribonucleotide according to any one of embodiments 41 to 43, wherein at least 1% of the 5-hydroxymethyluridine residues contain 2-O acetylated ribose.

[0514] Embodiment 45. The polyribonucleotide of any one of embodiments 41 to 43, wherein no more than 90% of the 5-hydroxymethyluridine residues contain 2-O acetylated ribose.

[0515] Embodiment 46. The polyribonucleotide of any one of embodiments 39 to 44, wherein the polyribonucleotide further comprises, in addition to 5-hydroxymethyluridine, one or more additional ribonucleotides, such as modified ribonucleotides.

[0516] Embodiment 47. The polyribonucleotide of embodiment 46, wherein the one or more ribonucleotides (e.g., modified ribonucleotides) comprise a nucleoside selected from adenosine, guanosine, cytidine, or uridine, or a combination thereof.

[0517] Embodiment 48. The polyribonucleotide of any one of embodiments 16 to 46, wherein the ribonucleotide comprises a nucleoside that includes N1-methylpseudouridine.

[0518] Embodiment 49. The ribonucleotide comprises a nucleoside comprising N1-methylpseudouridine, and the modified ribonucleotide is: [ka] The polyribonucleotide according to any one of embodiments 16 to 46, having the structure:

[0519] Embodiment 50. The polyribonucleotide comprises uridine residues, and about 5% to 99%, about 5% to 95%, about 5% to 90%, about 5% to 85%, about 5% to 80%, about 5% to 75%, about 5% to 70%, about 5% to 65%, about 5% to 60%, about 5% to 55%, about 5% to 50%, about 5% to 45%, about 5% to 40%, about 5% to 35%, about 5% to 30%, about 5% to 25%, about 5% to 20%, about 5% to 15%, about 5% to 1 ... 50% to 99%, about 55% to 99%, about 60% to 99%, about 65% to 99%, about 70% to 99%, about 80% to 99%, about 85% to 99%, about 90% to 99%, or about 95% to 99% of the polyribonucleotide according to embodiment 48 or 49 comprises N1-methylpseudouridine.

[0520] Embodiment 51. The polyribonucleotide of embodiment 48 or 49, wherein the polyribonucleotide comprises uridine residues, and wherein at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% of the uridine residues in the polyribonucleotide comprise N1-methylpseudouridine.

[0521] Embodiment 52. A polyribonucleotide according to any one of claims 48 to 51, wherein at least 1% of the N1-methylpseudouridine residues contain 2-O acetylated ribose.

[0522] Embodiment 53. A polyribonucleotide according to any one of claims 48 to 51, wherein no more than 90% of the N1-methylpseudouridine residues contain 2-O acetylated ribose.

[0523] Embodiment 54. The polyribonucleotide of any one of claims 48 to 53, wherein the polyribonucleotide further comprises, in addition to N1-methylpseudouridine, one or more additional ribonucleotides, such as modified ribonucleotides.

[0524] Embodiment 55. The polyribonucleotide of embodiment 54, wherein the one or more ribonucleotides (e.g., modified ribonucleotides) comprise a nucleoside selected from adenosine, guanosine, cytidine, or uridine, or a combination thereof.

[0525] Embodiment 56. A polyribonucleotide described in any one of claims 16 to 55, wherein when the polyribonucleotide contains one or more modified ribonucleotides of a particular type that includes 2-O acetylated ribose, at least 1% of the modified ribonucleotides of the particular type have 2-O acetylated ribose.

[0526] Embodiment 57. A polyribonucleotide described in any one of claims 16 to 55, wherein when the polyribonucleotide contains one or more modified ribonucleotides of a particular type that includes 2-O acetylated ribose, no more than 90% of the modified ribonucleotides of the particular type have 2-O acetylated ribose.

[0527] Embodiment 58. The polyribonucleotide of embodiment 56 or 57, wherein the polyribonucleotide further comprises (i) one or more ribonucleotides, and / or (ii) one or more modified ribonucleotides.

[0528] Embodiment 59. A polyribonucleotide according to any one of embodiments 16 to 58, characterized in that reduced immunogenicity is observed when assessed in a cell, tissue or organism to which the polyribonucleotide has been administered, as compared to an appropriate reference standard.

[0529] Embodiment 60. The polyribonucleotide of embodiment 59, wherein the reference comparison standard comprises other similar cells, tissues or organisms administered an equivalent polyribonucleotide that contains less 2'-O-acetylated ribose than the polyribonucleotide in the composition (e.g., no 2'-O-acetylated ribose at all).

[0530] Embodiment 61 The polyribonucleotide of embodiment 59, wherein the reduced immunogenicity comprises reduced activation of innate immune response-induced toxicity.

[0531] Embodiment 62. The polyribonucleotide of embodiment 61, wherein the reduced activation of the immune response comprises a reduced activation of the NFkb pathway, the IRF pathway, and / or other cytokines resulting from inflammation in said cell, said tissue or said organism.

[0532] Embodiment 63. The polyribonucleotide of any one of embodiments 60 to 62, wherein the reduced immunogenicity allows repeated administration of the polyribonucleotide.

[0533] Embodiment 64. A polyribonucleotide according to any one of embodiments 60 to 63, wherein the reduced immunogenicity allows for the administration of higher doses of said polyribonucleotide relative to an appropriate reference standard.

[0534] Embodiment 65. The polyribonucleotide of embodiment 64, wherein the reference comparison standard comprises an equivalent polyribonucleotide that contains less 2'-O-acetylated ribose than the polyribonucleotide in the composition (e.g., no 2'-O-acetylated ribose at all).

[0535] Embodiment 66. A polyribonucleotide according to any one of the preceding embodiments, characterized in that, when assessed in a cell, tissue or organism to which said polyribonucleotide has been administered, an increase in cell viability is observed compared to a suitable reference comparison standard.

[0536] Embodiment 67. The polyribonucleotide of embodiment 66, wherein the reference comparison standard is the cell viability of a cell, tissue or organism administered an equivalent polyribonucleotide containing less 2'-O-acetylated ribose (e.g., no 2'-O-acetylated ribose).

[0537] Embodiment 68. The polyribonucleotide of embodiment 66 or 67, wherein cell viability is a measure of the length of time that one or more cells of said cell, said tissue or subject are alive.

[0538] Embodiment 69. The polyribonucleotide of embodiment 66 or 67, wherein cell viability is a measure of the number of cells of said cell, said tissue or subject that are viable at one or more time points.

[0539] Embodiment 70. A polyribonucleotide according to any one of the preceding embodiments, characterized in that an increase in the persistence of the polyribonucleotide is observed when assessed in a cell, tissue or organism to which the polyribonucleotide has been administered, compared to a suitable reference comparator.

[0540] Embodiment 71. The polyribonucleotide of embodiment 70, wherein the reference comparison standard is the persistence of an equivalent polyribonucleotide that contains less 2'-O-acetylated ribose (e.g., no 2'-O-acetylated ribose at all).

[0541] Embodiment 72. The polyribonucleotide of embodiment 70 or 71, wherein the persistence of the polyribonucleotide is increased by at least 15%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%.

[0542] Embodiment 73. The polyribonucleotide of any one of embodiments 70 to 72, wherein the increased persistence is the result of increased resistance to one or more nucleases.

[0543] Embodiment 74. The polyribonucleotide of embodiment 73, wherein the one or more nucleases comprise an endonuclease.

[0544] Embodiment 75. The polyribonucleotide of embodiment 73, wherein the one or more nucleases comprise an exonuclease.

[0545] Embodiment 76. A polyribonucleotide according to any one of embodiments 70 to 75, wherein the increased persistence allows the polyribonucleotide to be administered in a lesser amount and / or more frequently compared to an otherwise similar polyribonucleotide that contains less 2'-O-acetylated ribose (e.g., no 2'-O-acetylated ribose at all).

[0546] Embodiment 77. A polyribonucleotide according to any one of the preceding embodiments, characterized in that, when assessed in a cell, tissue or organism to which the polyribonucleotide has been administered, an increase in expression and / or activity of a gene product encoded by the polyribonucleotide is observed compared to a suitable reference standard.

[0547] Embodiment 78. The polyribonucleotide of embodiment 77, wherein the reference comparison standard is the expression and / or activity of a gene product in a cell, tissue or organism administered an equivalent polyribonucleotide containing less 2'-O-acetylated ribose (e.g., no 2'-O-acetylated ribose at all).

[0548] Embodiment 79. The polyribonucleotide of embodiment 78, wherein the expression and / or activity of a gene product is increased by at least 15%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%.

[0549] Embodiment 80. The polyribonucleotide of any one of embodiments 16 to 79, wherein the polyribonucleotide comprises a coding region.

[0550] Embodiment 81. The polyribonucleotide of embodiment 79, wherein the coding region encodes a gene product, e.g., a payload.

[0551] Embodiment 82: The polyribonucleotide of embodiment 81, wherein said gene product is or comprises a polypeptide.

[0552] Embodiment 83: The polyribonucleotide of embodiment 81, wherein said gene product is or comprises a transcript.

[0553] Embodiment 84. The polyribonucleotide of any one of embodiments 16 to 79, wherein the polyribonucleotide is a messenger RNA (mRNA).

[0554] Embodiment 85. The polyribonucleotide of any one of embodiments 16 to 79, wherein the polyribonucleotide is a circRNA.

[0555] Embodiment 86. The polyribonucleotide of any one of embodiments 16 to 79, wherein the polyribonucleotide is a gRNA.

[0556] Embodiment 87. The polyribonucleotide of any one of embodiments 16 to 79, wherein the polyribonucleotide is an inhibitory RNA.

[0557] Embodiment 88. The polyribonucleotide of any one of embodiments 16 to 79, wherein the polyribonucleotide is an miRNA or siRNA.

[0558] Embodiment 89. The polyribonucleotide of any one of embodiments 16 to 79, wherein the polyribonucleotide is an antisense oligonucleotide.

[0559] Embodiment 90. The polyribonucleotide of any one of embodiments 16 to 79, wherein the polyribonucleotide is a long non-coding RNA.

[0560] Embodiment 91. A method for producing an RNA composition, comprising introducing one or more modified ribonucleotides according to any one of embodiments 1 to 15 into a polyribonucleotide.

[0561] Embodiment 92 The method of embodiment 91, wherein the method does not include removing double-stranded RNA from the RNA composition.

[0562] Embodiment 93 The method according to embodiment 91 or 92, wherein the method is an in vitro transcription reaction method.

[0563] Embodiment 94. The method of any one of embodiments 91 to 94, wherein the method further comprises introducing one or more unmodified ribonucleotides.

[0564] Embodiment 95. A method for making polyribonucleotides, comprising incubating an in vitro transcription mixture comprising a DNA template, at least one RNA polymerase or a variant or fragment thereof, and a plurality of ribonucleotides comprising at least one modified ribonucleotide comprising 2'-O-acetylated ribose, thereby generating polyribonucleotides comprising 2'-O-acetylated ribose.

[0565] Embodiment 96 The method of embodiment 95, wherein the method produces a plurality of polyribonucleotides.

[0566] Embodiment 97. An in vitro transcription mixture comprising: (i) a DNA template; (ii) at least one RNA polymerase or a variant or fragment thereof; and (iii) a plurality of ribonucleotides comprising at least one modified ribonucleotide comprising a 2'-O-acetylated ribose.

[0567] Embodiment 98. The in vitro transcription mixture of embodiment 97, wherein the mixture produces polyribonucleotides comprising 2'-O-acetylated ribose.

[0568] Embodiment 99. The in vitro transcription mixture of embodiment 49, wherein the mixture produces a plurality of polyribonucleotides.

[0569] Embodiment 100. The method of embodiment 96, or the mixture of embodiments 97 to 99, wherein each polyribonucleotide in the plurality of polyribonucleotides comprises 2'-O-acetylated ribose.

[0570] Embodiment 101. The method or mixture of embodiment 100, wherein each polyribonucleotide in the plurality has at least 5% 2'-O-acetylated ribose.

[0571] Embodiment 102. The method or mixture of embodiment 100, wherein each polyribonucleotide in the plurality has 90% or less 2'-O-acetylated ribose.

[0572] Embodiment 103. The method or mixture of embodiment 100, wherein each polyribonucleotide in the plurality has 100% 2'-O-acetylated ribose.

[0573] Embodiment 104. The method according to embodiment 95 or the mixture according to embodiment 97, wherein the RNA polymerase is selected from a bacteriophage RNA polymerase, a mitochondrial RNA polymerase, a eukaryotic RNA polymerase, a bacterial RNA polymerase, or a combination thereof.

[0574] Embodiment 105. The method according to embodiment 104 or the mixture according to embodiment 104, wherein the RNA polymerase comprises T7 RNA polymerase, T3 RNA polymerase, SP6 RNA polymerase, N4 virion RNA polymerase, viral RNA polymerase, or a variant of any of the foregoing.

[0575] Embodiment 106. The method of embodiment 95 or the mixture of embodiment 97, wherein the polyribonucleotide comprises a coding region.

[0576] Embodiment 107. The method of embodiment 95 or the mixture of embodiment 97, wherein the polyribonucleotide comprises a guide RNA, a short hairpin RNA, an siRNA, a microRNA, a long non-coding RNA, a circular RNA, or a messenger RNA (mRNA), or a combination thereof.

[0577] Embodiment 108. The method according to embodiment 106 or 107, or the mixture according to embodiment 106 or 107, wherein said polyribonucleotides encode one or more target polypeptides.

[0578] Embodiment 109. A composition comprising one or more polyribonucleotides according to any one of embodiments 16 to 90.

[0579] Embodiment 110. A composition comprising one or more polyribonucleotides produced according to the method of any one of embodiments 91 to 96 or 100 to 108.

[0580] Embodiment 111. A composition comprising one or more polyribonucleotides produced using an in vitro transcription reaction mixture according to any one of embodiments 97 to 108.

[0581] Embodiment 112. The composition of any one of embodiments 109 to 111, wherein the composition is a pharmaceutical composition.

[0582] Embodiment 113. The composition of embodiment 112, wherein the pharmaceutical composition is or comprises an immunogenic composition.

[0583] Embodiment 114. The composition of embodiment 112, wherein the pharmaceutical composition is or comprises a vaccine.

[0584] Embodiment 115. The composition of embodiment 112, wherein the pharmaceutical composition is or comprises a gene therapy.

[0585] Embodiment 116. The composition of embodiment 112, wherein the pharmaceutical composition is or comprises a chemotherapy.

[0586] Embodiment 117. The composition of embodiment 112, wherein the pharmaceutical composition is or comprises a protein replacement therapy.

[0587] Embodiment 118. The composition of embodiment 112, wherein the pharmaceutical composition is or comprises an immunotherapy.

[0588] Embodiment 119. The composition of embodiment 112, wherein the pharmaceutical composition is or comprises a cell engineering therapy.

[0589] Embodiment 120. The composition of embodiment 112, wherein the composition comprises double-stranded RNA.

[0590] Embodiment 121. A method comprising administering to a cell, tissue or subject one or more polyribonucleotides described in any one of embodiments 16 to 90, or a composition described in any one of embodiments 109 to 120.

[0591] Embodiment 122 The method of embodiment 121, further comprising determining cell viability of said cell, said tissue or said subject.

[0592] Embodiment 123 The method of embodiment 122, wherein cell viability is a measure of the length of time that one or more cells of said cell, said tissue or said subject are alive.

[0593] Embodiment 124. The method of embodiment 122, wherein cell viability is a measure of the number of cells of said cell, said tissue or said subject that are viable at one or more time points.

[0594] Embodiment 125. The method according to any one of embodiments 121 to 124, wherein the cell, the tissue or the subject to which the polyribonucleotide or a composition comprising same has been administered exhibits improved cell viability when compared to a reference cell viability.

[0595] Embodiment 126. The method of embodiment 125, wherein the reference cell viability is the cell viability of a cell, tissue or subject administered an equivalent polyribonucleotide or a composition comprising the same that contains less 2'-O-acetylated ribose (e.g., no 2'-O-acetylated ribose at all).

[0596] Embodiment 127. The method of any one of embodiments 121 to 126, further comprising determining the immune system response of the cell, the tissue or the subject to which the polyribonucleotide or a composition comprising same has been administered.

[0597] Embodiment 128. The method of embodiment 127, wherein the immune response comprises an innate immune system response comprising an innate immune system-induced toxicity.

[0598] Embodiment 129. The method of embodiment 128, wherein determining the innate immune system response comprises determining levels of NF-κB, IRF, and / or other inflammatory cytokines in the cell, tissue, or subject.

[0599] Embodiment 130. The method according to any one of embodiments 127 to 129, wherein the cell, the tissue or the subject to which the polyribonucleotide or a composition comprising same has been administered exhibits a reduced innate immune system response compared to a reference.

[0600] Embodiment 131. The method of embodiment 130, wherein the reference is the innate immune system response of a cell, tissue or subject administered an equivalent polyribonucleotide containing less 2'-O-acetylated ribose (e.g., no 2'-O-acetylated ribose) or a composition containing the same.

[0601] Embodiment 132. The method of any one of embodiments 121 to 131, further comprising determining the effectiveness of the polyribonucleotide or a composition comprising same in the cell, tissue or subject to which the polyribonucleotide or a composition comprising same has been administered.

[0602] Embodiment 133. The method of embodiment 132, wherein determining the effectiveness comprises determining an antibody response or a cellular response in the cell, the tissue or the subject.

[0603] Embodiment 134. The method of embodiment 132 or 133, wherein the cell, the tissue or the subject to which the polyribonucleotide or a composition comprising same has been administered exhibits an increased antibody or cellular response when compared to a reference.

[0604] Embodiment 135. The method of embodiment 134, wherein the reference is an antibody response or a cellular response of a cell, tissue or subject that has been administered an equivalent polyribonucleotide containing less 2'-O-acetylated ribose (e.g., no 2'-O-acetylated ribose at all) or a composition containing the same.

[0605] Embodiment 136. The method of any one of embodiments 121 to 135, further comprising determining the persistence of polyribonucleotides in said cell, said tissue or said subject as compared to a reference.

[0606] Embodiment 137. The method of embodiment 136, wherein the reference is the persistence of an equivalent polyribonucleotide or composition comprising the same that contains less 2'-O-acetylated ribose (e.g., no 2'-O-acetylated ribose) administered to a cell, tissue or subject.

[0607] Embodiment 138 The method of embodiment 136 or 137, wherein administration of the polyribonucleotide results in an increased persistence of the polyribonucleotide when compared to a reference.

[0608] Embodiment 139. The method of embodiment 138, wherein the persistence of the polyribonucleotide is increased by at least 15%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%.

[0609] Embodiment 140. The method of any one of embodiments 121 to 139, further comprising determining the expression and / or activity of a gene product encoded by the polyribonucleotide in the cell, tissue or subject compared to a reference.

[0610] Embodiment 141. The method of embodiment 140, wherein the reference is the expression and / or activity of an equivalent polyribonucleotide or composition comprising the same that contains less 2'-O-acetylated ribose (e.g., no 2'-O-acetylated ribose) that has been administered to a cell, tissue or subject.

[0611] Embodiment 142 The method of embodiment 141 or 142, wherein administration of the polyribonucleotide results in increased expression and / or activity of the polyribonucleotide when compared to a reference.

[0612] Embodiment 143. The method of embodiment 142, wherein the expression and / or activity of the gene product is increased by at least 15%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%.

[0613] Embodiment 144. The method according to any one of embodiments 121 to 143, wherein the method comprises administering the polyribonucleotide or a composition comprising same to the cell, the tissue or the subject at least twice.

[0614] Embodiment 145. The method according to any one of embodiments 121 to 144, wherein the method comprises administering the polyribonucleotide or a composition comprising same to the cell, tissue or subject at least 2 times, at least 3 times, at least 4 times, at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times or at least 10 times.

[0615] Embodiment 146. The method of embodiment 144 or 145, wherein at least two administrations of the polyribonucleotide or a composition comprising same to the cell, tissue or subject does not result in a decrease in the effectiveness of the polyribonucleotide or a composition comprising same compared to administration of one dose of the polyribonucleotide or a composition comprising same.

[0616] Embodiment 147. The method according to any one of embodiments 121 to 146, wherein the method comprises administering to the cell, the tissue or the subject the polyribonucleotide or a composition comprising same at a higher dose compared to a suitable reference standard.

[0617] Embodiment 148. The method of embodiment 147, wherein the reference comparison standard comprises an equivalent polyribonucleotide that contains less 2'-O-acetylated ribose (e.g., no 2'-O-acetylated ribose at all).

[0618] Embodiment 149. The method of any one of embodiments 121 to 148, wherein the cell is a mammalian cell, the tissue is a mammalian tissue, or the subject is a mammal.

[0619] Embodiment 150. The method of embodiment 149, wherein the mammal is a human.

[0620] Embodiment 151. The method of any one of embodiments 121 to 150, wherein the method is a method of stimulating an immune response.

[0621] Embodiment 152. The method according to any one of embodiments 121 to 150, wherein the method is a vaccination method.

[0622] Embodiment 153. The method of any one of embodiments 121 to 150, wherein the method is a method of gene therapy.

[0623] Embodiment 154. The method of embodiment 153, wherein the gene therapy method comprises delivery of one or more components of gene therapy, such as gRNA.

[0624] Embodiment 155. The method of any one of embodiments 121 to 150, wherein the method is a cell therapy engineering method.

[0625] Embodiment 156. The method of any one of embodiments 121 to 150, wherein the method is an immunotherapy method.

[0626] Embodiment 157. The method of embodiment 156, wherein the immunotherapy method comprises delivery of antibody therapy and / or immune checkpoint therapy.

[0627] Embodiment 158. The method of any one of embodiments 121 to 150, wherein the method is a method of protein replacement therapy.

[0628] Embodiment 159. The method of embodiment 158, wherein the protein replacement therapy method comprises delivery of enzyme replacement therapy.

[0629] Embodiment 160. The method of any one of embodiments 121 to 150, wherein the method is a chemotherapy method.

[0630] Embodiment 161. A method of vaccination comprising administering to a cell, tissue or subject one or more polyribonucleotides described in any one of embodiments 16 to 90, or a composition described in any one of embodiments 109 to 120.

[0631] Embodiment 162. A method of immunotherapy comprising administering to a cell, tissue or subject one or more polyribonucleotides described in any one of embodiments 16 to 90 or a composition described in any one of embodiments 109 to 120.

[0632] Embodiment 163. A method of gene therapy comprising administering to a cell, tissue or subject one or more polyribonucleotides described in any one of embodiments 16 to 90 or a composition described in any one of embodiments 109 to 120.

[0633] Embodiment 164. A method for protein replacement therapy comprising administering to a cell, tissue or subject one or more polyribonucleotides described in any one of embodiments 16 to 90, or a composition described in any one of embodiments 109 to 120.

[0634] Embodiment 165. A method of cell engineering therapy comprising administering to a cell, tissue or subject one or more polyribonucleotides described in any one of embodiments 16 to 90, or a composition described in any one of embodiments 109 to 120.

[0635] Embodiment 166. A method for increasing the persistence of polyribonucleotides in a cell, tissue or subject, comprising administering to the cell, tissue or subject one or more polyribonucleotides described in any one of embodiments 16 to 90, or a composition described in any one of embodiments 109 to 120.

[0636] Embodiment 167. A method for increasing the expression and / or activity of a gene product encoded by a polyribonucleotide in a cell, tissue or subject, the method comprising administering to the cell, tissue or subject one or more polyribonucleotides described in any one of embodiments 16 to 90, or a composition described in any one of embodiments 109 to 120.

[0637] Embodiment 168. A cell comprising a polyribonucleotide according to any one of embodiments 116 to 90, or a composition according to any one of embodiments 109 to 120.

[0638] Embodiment 169. Use of a modified ribonucleotide according to embodiment 1 in the production of a polyribonucleotide.

[0639] Embodiment 170. Use of a polyribonucleotide according to any one of embodiments 16 to 90 or a composition according to any one of embodiments 109 to 120 for stimulating an immune response.

[0640] Embodiment 171. Use of a polyribonucleotide according to any one of embodiments 16 to 90 or a composition according to any one of embodiments 109 to 120 as a vaccine.

[0641] Embodiment 172. Use of a polyribonucleotide according to any one of embodiments 16 to 90 or a composition according to any one of embodiments 109 to 120 as an immunotherapy.

[0642] Embodiment 173. Use of a polyribonucleotide according to any one of embodiments 16 to 90 or a composition according to any one of embodiments 109 to 120 as gene therapy.

[0643] Embodiment 174. Use of a polyribonucleotide according to any one of embodiments 16 to 90 or a composition according to any one of embodiments 109 to 120 as a protein replacement therapy.

[0644] Embodiment 175. Use of a polyribonucleotide according to any one of embodiments 116 to 90 or a composition according to any one of embodiments 109 to 120 as a cell engineering therapy.

[0645] Embodiment 176. Use of a polyribonucleotide according to any one of embodiments 16 to 90 or a composition according to any one of embodiments 109 to 120 as chemotherapy.

[0646] Embodiment 177. The use according to any one of embodiments 169 to 176, wherein the polyribonucleotide or a composition comprising it is administered to a cell, tissue or subject.

[0647] Embodiment 178. The use of embodiment 177, wherein the cell is a mammalian cell, the tissue is a mammalian tissue, or the subject is a mammal.

[0648] Embodiment 179. The use according to embodiment 178, wherein the mammal is a human.

[0649] Doctrine of Equivalents Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, numerous equivalents to the specific embodiments of the invention described herein. It should be understood that the invention encompasses all variations, combinations, and permutations of one or more limitations, elements, clauses, descriptive terms, etc. from one or more of the enumerated claims that are introduced into another claim that is dependent on the same base claim (or any other related claim), unless otherwise specified or unless a contradiction or inconsistency would be obvious to one of ordinary skill in the art. It should also be understood that any embodiment or aspect of the invention may be expressly excluded from the claims, regardless of whether a specific exclusion is described in the specification. The scope of the invention is not limited to the above description, but is instead set forth in the following claims.

Claims

1. A polyribonucleotide comprising one or more modified ribonucleotides, wherein the one or more modified ribonucleotides (i) 5'-monophosphate, and (ii) Nucleosides containing 2'-O-acetylated ribose and nucleic acid bases A polyribonucleotide comprising the above, wherein the nucleic acid base is adenine or a modified form thereof, guanine or a modified form thereof, cytosine or a modified form thereof, or uracil or a modified form thereof.

2. (i) At least 80% of the ribose portion of the polyribonucleotide is 2'-O-acetylated, and / or (ii) The polyribonucleotide includes a cap structure, The polyribonucleotide according to claim 1.

3. The polyribonucleotide according to claim 1 or 2, wherein the polyribonucleotide further comprises one or more ribonucleotides that do not contain 2'-O-acetylated ribose.

4. When evaluated in cells, tissues, or organisms to which the aforementioned polyribonucleotides have been administered, (i) A decrease in immunogenicity is observed compared to an appropriate reference standard; (ii) An increase in the persistence of the polyribonucleotide is observed in relation to an appropriate reference comparison criterion; (iii) An increase in the expression and / or activity of the gene product encoded by the polyribonucleotide is observed in relation to an appropriate reference comparison criterion; or (iv) Any combination of (i) to (iii) A polyribonucleotide according to any one of claims 1 to 3, characterized by the above.

5. (i) The polyribonucleotide includes a coding region; (ii) The polyribonucleotide includes RNA oligonucleotides, messenger RNA, gRNA, inhibitory RNA, miRNA, siRNA, antisense oligonucleotides, long non-coding RNA, or circular RNA; or (iii) The gene product comprises (a) a polypeptide encoded by the polyribonucleotide containing one or more modified ribonucleotides, or (b) a polyribonucleotide located within the polyribonucleotide containing one or more modified ribonucleotides. The polyribonucleotide according to claim 3.

6. A polyribonucleotide according to any one of claims 1 to 3, further comprising N4-acetylcytidine (ac4C), 5-hydroxymethyluridine, N1-methylpseudolidine, pyridine-4-onyribonucleoside, 5-azauridine, 6-azauridine, 2-thio-5-azauridine, 2-thiouridine (s2U), 5-methylcytidine (m5C), 5-azauridine, 6-azauridine, pseudoisocytidine, 3-methylcytidine (m3C), 5-formylcytidine (f5C), N4-methylcytidine (m4C), 2-aminopurine, 2,6-diaminopurine, 2-amino-6-halopurine, 6-halopurine, inosine (I), 1-methylinosine (m1I), waiosin (imG), methylwaiosin (mimG), or any combination thereof.

7. A cell containing polyribonucleotides according to any one of claims 1 to 3.

8. A composition comprising a polyribonucleotide comprising one or more modified ribonucleotides, wherein the one or more modified ribonucleotides (i) 5'-monophosphate, and (ii) Nucleosides containing 2'-O-acetylated ribose and nucleic acid bases A composition comprising the above, wherein the nucleic acid base is adenine or a modified form thereof, guanine or a modified form thereof, cytosine or a modified form thereof, or uracil or a modified form thereof.

9. The composition according to claim 8, wherein at least 80% of the ribose portion of the polyribonucleotide is 2'-O-acetylated, and / or the polyribonucleotide includes a cap structure.

10. The composition according to claim 9, wherein the composition is a pharmaceutical composition and comprises an immunogenic composition, a vaccine, gene therapy, chemotherapy, protein replacement therapy, immunotherapy, antibody therapy, immunomodulatory therapy, cell engineering therapy, or any combination thereof.

11. A composition comprising the polyribonucleotide according to any one of claims 1 to 3, for use in delivering the polyribonucleotide to a cell, tissue or subject.

12. The use according to claim 11, wherein the cells are mammalian cells, the tissue is mammalian tissue, or the subject is a mammal.

13. The aforementioned use (i) Methods to stimulate an immune response, (ii) Methods of antibody therapy, (iii) an immunomodulatory method; (IV) Vaccination Method, (v) Methods of gene therapy, (vi) Engineering methods for cell therapy, (vii) Methods of immunotherapy, (viiii) Methods of protein replacement therapy, (ix) Method of chemotherapy, or Any combination of (x)(i) to (ix) The use according to claim 11, which is for the purpose of.

14. (i) the cell viability of the cells, tissue or target, (ii) The immune system response of the cell, tissue or target, (iii) The efficacy of the polyribonucleotide in the cells, tissues or subjects, (iv) The persistence of the polyribonucleotide in the cell, tissue or subject, (v) Expression and / or activity of the gene product encoded by the polyribonucleotide in the cell, tissue or subject, or (vi) Any combination of (i) to (v) The use according to claim 11, which includes determining the

15. Use of a composition comprising the polyribonucleotide according to claim 1 or 2 in the preparation of a pharmaceutical for delivering the polyribonucleotide to cells, tissues or subjects.

16. The use described above is (i) Methods to stimulate an immune response, (ii) Methods of antibody therapy, (iii) an immunomodulatory method; (IV) Vaccination Method, (v) Methods of gene therapy, (vi) Engineering methods for cell therapy, (vii) Methods of immunotherapy, (viiii) Methods of protein replacement therapy, (ix) Method of chemotherapy, or Any combination of (x)(i) to (ix) The use described in claim 14 is for the purpose of the use described in claim 14.

17. An in vitro transfer mixture, (a) DNA template and (b) at least one RNA polymerase, (c) Multiple ribonucleotides containing one or more modified ribonucleotides The modifications include, and the one or more modified ribonucleotides (i) 5'-triphosphate, and (ii) Nucleosides containing 2'-O-acetylated ribose and nucleic acid bases An in vitro transfer mixture containing the above.

18. The in vitro transcription mixture according to claim 17, wherein the nucleic acid base is adenine or a modified form thereof, guanine or a modified form thereof, cytosine or a modified form thereof, or uracil or a modified form thereof.

19. The one or more modified ribonucleotides comprises one or more subsets of modified ribonucleotides, each subset of modified ribonucleotides being N4-acetylcytidine (ac4C), 5-hydroxymethyluridine, N1-methylpsuduridine, pyridine-4-one ribonucleoside, 5-azauridine, 6-azauridine, 2-thio-5-azauridine, 2-thiouridine (s2U), 5-methylcytidine (m5C), 5-azauridine, 6-azauridine The in vitro transcription mixture according to claim 17 or 18, comprising a nucleic acid base which is din, pseudoisocytidine, 3-methylcytidine (m3C), 5-formylcytidine (f5C), N4-methylcytidine (m4C), 2-aminopurine, 2,6-diaminopurine, 2-amino-6-halopurine, 6-halopurine, inosine (I), 1-methylinosine (m1I), yosin (imG), or methylyosin (mimG), or any combination thereof.

20. The in vitro transcription mixture according to claim 16, wherein the mixture further comprises one or more polyribonucleotides, the one or more polyribonucleotides being produced by incubating components (a), (b), and (c) in the mixture.

21. The in vitro transcription mixture according to claim 16, wherein one or more polyribonucleotides have a ribose moiety that is at least 5% 2'-O-acetylated.

22. The in vitro transcription mixture according to claim 20, comprising one or more polyribonucleotides that do not contain 2'-O-acetylated ribose.

23. The in vitro transcription mixture according to claim 16, wherein the RNA polymerase is bacteriophage RNA polymerase, mitochondrial RNA polymerase, eukaryotic RNA polymerase, bacterial RNA polymerase, or any combination thereof.

24. The in vitro transcription mixture according to claim 16, wherein the RNA polymerase is T7 RNA polymerase, T3 RNA polymerase, SP6 RNA polymerase, N4 virion RNA polymerase, viral RNA polymerase, or any of the above variants, or comprises the same.

25. The in vitro transcription mixture according to claim 20, wherein one or more polyribonucleotides include a coding region.

26. The in vitro transcription mixture according to claim 21, wherein the one or more polyribonucleotides comprises a guide RNA, a short hairpin RNA, a siRNA, a microRNA, a long non-coding RNA, a circular RNA, or any combination thereof.

27. ​​The in vitro transcription mixture according to claim 20, wherein one or more polyribonucleotides encode one or more target polypeptides.

28. The in vitro transfer mixture according to claim 21, further comprising a 5' cap.

29. The in vitro transcription mixture according to claim 22, further comprising one or more polyribonucleotides with a 5' cap.

30. A polyribonucleotide prepared according to the in vitro transcription mixture described in Claim 23.

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