Modified ribonucleic acid and uses thereof

Abstract

Disclosed herein are modified ribonucleotides comprising nucleosides comprising N4-acetylcytidine and / or 5-hydroxymethyluridine, and polyribonucleotides comprising the modified ribonucleotides. Also provided herein are compositions comprising the polyribonucleotides of the disclosure, as well as methods of making and using the compositions.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 63 / 185,925, filed May 7, 2021, and U.S. Provisional Patent Application No. 63 / 273,031, filed October 28, 2021, the entire contents of each of which are incorporated by reference herein. [Background technology]

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

[0003] The present disclosure identifies certain challenges associated with the use of RNA as a therapeutic. For example, in some embodiments, the present disclosure identifies certain problems that may be encountered with immunogenicity resulting from the administration of RNA therapeutics, which may hinder efforts to use high doses or repeated administration of RNA therapeutics. The immunogenicity resulting from the administration of RNA therapeutics in response to the RNA molecule itself should be contrasted with the immunogenicity caused, for example, by the polypeptide encoded by the RNA molecule, which may be desirable as a result of, for example, an RNA vaccine. The present disclosure also identifies challenges associated with payload expression that may be encountered with the administration of RNA therapeutics. For example, currently used RNA therapeutics do not usually provide high and / or sustained expression of the payload encoded by the RNA.

[0004] In particular, the present disclosure provides techniques for reducing immunogenicity and / or increasing payload expression of RNA therapeutics, for example, by providing polyribonucleotides comprising modified ribonucleotides as disclosed herein. In some embodiments, the modified ribonucleotides comprise ribonucleotides comprising N4-acetylcytidine and / or ribonucleotides comprising 5-hydroxymethyluridine. Without wishing to be bound by theory, the present disclosure proposes that polyribonucleotides comprising N4-acetylcytidine and / or 5-hydroxymethyluridine, when administered to a cell, tissue or subject, can achieve reduced immunogenicity through reduced activation of the innate immune response. In some embodiments, the reduced activation of the innate immune response by the polyribonucleotide comprising N4-acetylcytidine and / or 5-hydroxymethyluridine, or the composition comprising the same, 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), allows, for example, repeated administration of the polyribonucleotide or the composition comprising the same, e.g., at least two administrations. In some embodiments, the polyribonucleotide comprising N4-acetylcytidine and / or 5-hydroxymethyluridine, or the composition comprising the same, can be administered at a higher dose than a reference ribonucleotide that (i) has fewer acetyl groups on the nucleobase (e.g., the nucleobase does not contain an acetyl group) and / or (ii) has fewer hydroxymethyl groups (e.g., does not contain a hydroxymethyl group).

[0005] The present disclosure reports for the first time the insight that N4-acetylcytidine and 5-hydroxymethyluridine residues can synergistically interact in polyribonucleotides to reduce immunogenicity, increase cell viability, and / or increase protein or polypeptide expression when administered to a cell, tissue, or subject. In particular, the present disclosure provides the insight that polyribonucleotides containing both N4-acetylcytidine and 5-hydroxymethyluridine residues have higher payload expression and reduced immunogenicity compared to polyribonucleotides containing only either modification. For example, in some embodiments, polyribonucleotides containing 5-hydroxymethyluridine residues increase payload expression. As another example, polyribonucleotides containing N4-acetylcytidine have reduced immunogenicity. As shown in Example 3 herein, the combination of polyribonucleotides containing both N4-acetylcytidine and 5-hydroxymethyluridine residues enhances payload expression and significantly reduces immunogenicity.

[0006] The present disclosure further provides the insight that polyribonucleotides containing both N4-acetylcytidine and 5-hydroxymethyluridine residues can inhibit the sensing of uncapped RNA, an insight that is particularly useful when using RNA in applications involving repeated administration and / or high-dose regimens, including gene therapy and enzyme replacement, where innate immune sensing of RNA remains a significant barrier.

[0007] Among other things, the present disclosure provides techniques for enhancing expression from RNA therapeutics by providing polyribonucleotides that include modified ribonucleotides, e.g., ribonucleotides that include uridine nucleosides that include one or more modifications and / or ribonucleotides that include cytidine nucleosides that include one or more modifications. In some embodiments, the modified ribonucleotides include ribonucleotides that include N4-acetylcytidine and / or ribonucleotides that include 5-hydroxymethyluridine. In some embodiments, the polyribonucleotides described herein encode a payload, e.g., as described herein. Without wishing to be bound by theory, the present disclosure proposes that when polyribonucleotides that include N4-acetylcytidine and / or 5-hydroxymethyluridine are administered to a cell, tissue, or subject, high levels of payload expression can be achieved.

[0008] The techniques provided herein for reducing the immunogenicity and / or enhancing payload expression of RNA therapeutics are particularly useful for the delivery of therapeutics such as antibody therapeutics, immunomodulatory therapies, gene therapies and / or other therapies (e.g., as described herein) where the stability and / or shelf life of the therapeutic formulation is important for therapeutic efficacy. For example, antibody therapeutic formulations (e.g., antibody therapeutics including antibodies, antibody fragments or alternative antibody formats) are generally time and / or temperature sensitive and may not be very stable over long periods of time. This results in degradation products from the antibody therapeutic that may be toxic and / or ineffective. When administered to a subject, degradation of the antibody therapeutic formulation may result in reduced expression of the product, reduced efficacy, and even increased immunogenicity (e.g., from degradation products or degradation of other formulation components). Such antibody therapeutics or immunomodulatory therapies have a short half-life when delivered to a subject and are rapidly cleared from the body, thus limiting their therapeutic window. Moreover, such antibody therapies, immunomodulatory therapies, gene therapies and / or other therapies (e.g., as described herein) are often formulated in a way that makes repeated delivery impractical and / or undesirable (e.g., due to inconvenience, inability to administer repeatedly to a subject, or other related factors).

[0009] In contrast, administration of the polyribonucleotides disclosed herein can provide sustained, continuous, and / or high expression of a therapeutic agent, and can further allow for repeated administration of a therapeutic agent (e.g., antibody therapy, immunomodulatory therapy, gene therapy, and / or other therapy (e.g., as described herein)). The polyribonucleotides disclosed herein are not subject to the challenges of other therapeutic agents (e.g., antibody therapeutic agents), such as degradation products in therapeutic formulations and / or inability to be administered repeatedly, because the therapeutic agent is produced, e.g., in the cell or subject. This provides a particular advantage to the use of the polyribonucleotides disclosed herein when administering a therapeutic agent, at least because the polyribonucleotides deliver the therapeutic agent in the form of RNA, which is converted to the corresponding therapeutic agent in vivo.

[0010] The technology disclosed herein relates, inter alia, to the structure, e.g., modification, of polyribonucleotides, and the advantages of such modifications, e.g., reduced immunogenicity and / or increased payload expression. The advantages associated with the polyribonucleotides disclosed herein are not limited by payload. Instead, the polyribonucleotides disclosed herein can be used with any payload or multiple payloads to provide, for example, the advantages of reduced immunogenicity and / or increased payload expression.

[0011] Thus, the present disclosure further provides the recognition that polyribonucleotides containing N4-acetylcytidine residues and / or 5-hydroxymethyluridine residues can function well in a number of therapeutic compositions, including gene therapy, antibody therapy, immunomodulatory therapy, and vaccines.

[0012] The present disclosure further provides the insight that inclusion of N4-acetylcytidine and / or 5-hydroxymethyluridine in polyribonucleotides can inhibit recognition of uncapped RNA (e.g., mRNA) by a subject's immune system, e.g., by RIG-I sensing.

[0013] Also provided herein are compositions comprising N4-acetylcytidine and / or 5-hydroxymethyluridine, as well as methods of making and using the same.

[0014] The present disclosure provides modified ribonucleotides comprising a nucleoside comprising an acetyl group, wherein the nucleoside is N4-acetylcytidine and the modified ribonucleotide has (a) a 5' monophosphate, (b) a 5' diphosphate; or (c) a 5' triphosphate.

[0015] In some embodiments, the modified ribonucleotide comprises N4-acetylcytidine and has the following structure: [ka]

[0016] Also provided herein are polyribonucleotides comprising one or more modified ribonucleotides disclosed herein, e.g., polyribonucleotides comprising nucleosides comprising acetyl groups, wherein the nucleoside is N4-acetylcytidine.

[0017] In some embodiments, the polyribonucleotide comprises cytidine residues, and at least 5% of the cytidine residues in the polyribonucleotide comprise N4-acetylcytidine.

[0018] In some embodiments, the polyribonucleotide comprises cytidine residues, and less than 100% of the cytidine residues in the polyribonucleotide comprise N4-acetylcytidine.

[0019] 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.

[0020] In some embodiments, the polyribonucleotide comprises cytidine residues, and about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 99% of the cytidine residues in the polyribonucleotide comprise N4-acetylcytidine.

[0021] In some embodiments, the polyribonucleotide comprises cytidine residues, and at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95%, or at least 99% of the cytidine residues in the polyribonucleotide comprise N4-acetylcytidine.

[0022] In some embodiments, the polyribonucleotide comprises cytidine residues, and greater than 60% of the cytidine residues in the polyribonucleotide comprise N4-acetylcytidine.

[0023] In some embodiments, the polyribonucleotide comprises cytidine residues, and about 60-100%, about 65%-100%, about 70%-100%, about 75%-100%, about 80%-100%, about 85%-100%, about 90%-100%, about 95%-100%, about 60%-95%, about 60%-90%, about 60%-85%, about 60%-80%, about 60%-75%, about 60%-70%, or about 60%-65% of the cytidine residues in the polyribonucleotide comprise N4-acetylcytidine.

[0024] In some embodiments, the polyribonucleotide comprising N4-acetylcytidine further comprises one or more modified ribonucleotides other than N4-acetylcytidine. In some embodiments, the one or more modified ribonucleotides comprise a nucleoside selected from adenosine, guanosine, cytidine or uridine, or a combination thereof. In some embodiments, the one or more modified ribonucleotides comprise a hydroxymethyl group. In some embodiments, the nucleoside of the one or more modified ribonucleotides is 5-hydroxymethyluridine. In some embodiments, the polyribonucleotide comprising 5-hydroxymethyluridine has (a) a 5' monophosphate; (b) a 5' diphosphate; or (c) a 5' triphosphate.

[0025] In some embodiments, the polyribonucleotide comprising N4-acetylcytidine further comprises uridine, wherein about 5% to 100% of the uridine is substituted with 5-hydroxymethyluridine.

[0026] The present disclosure provides modified ribonucleotides that include a nucleoside that includes a hydroxymethyl group, where the nucleoside is 5-hydroxymethyluridine and the modified ribonucleotide has (a) a 5' phosphate; (b) a 5' diphosphate; or (c) a 5' triphosphate. [ka]

[0027] Also provided herein are polyribonucleotides that contain one or more modified ribonucleotides disclosed herein, e.g., polyribonucleotides that contain nucleosides that contain hydroxymethyl groups, where the nucleoside is 5-hydroxymethyluridine.

[0028] In some embodiments, the polyribonucleotide comprises uridine residues, and at least 5% of the cytidine residues in the polyribonucleotide comprise 5-hydroxymethyluridine.

[0029] In some embodiments, the polyribonucleotide comprises uridine residues, and less than 100% of the uridine residues in the polyribonucleotide comprise 5-hydroxymethyluridine.

[0030] 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.

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

[0032] In some embodiments, the polyribonucleotide comprises uridine residues, and at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95%, or at least 99% of the uridine residues in the polyribonucleotide comprise 5-hydroxymethyluridine.

[0033] In some embodiments, the polyribonucleotide comprises uridine residues, and greater than 60% of the uridine residues in the polyribonucleotide comprise 5-hydroxymethyluridine.

[0034] In some embodiments, the polyribonucleotide comprises uridine residues, and about 60-100%, about 65%-100%, about 70%-100%, about 75%-100%, about 80%-100%, about 85%-100%, about 90%-100%, about 95%-100%, about 60%-95%, about 60%-90%, about 60%-85%, about 60%-80%, about 60%-75%, about 60%-70%, or about 60%-65% of the uridine residues in the polyribonucleotide comprise 5-hydroxymethyluridine.

[0035] In some embodiments, the polyribonucleotide comprising 5-hydroxymethyluridine further comprises one or more additional modified ribonucleotides other than 5-hydroxymethyluridine. In some embodiments, the one or more additional modified ribonucleotides comprise a nucleoside selected from adenosine, guanosine, cytidine or uridine, or a combination thereof. In some embodiments, the one or more additional modified ribonucleotides comprise an acetyl group. In some embodiments, the nucleoside of the one or more additional modified ribonucleotides is N4-acetylcytidine.

[0036] In some embodiments, the polyribonucleotide comprising 5-hydroxymethyluridine further comprises cytidine, wherein between about 5% and 100% of the cytidines are substituted with N4-acetylcytidine.

[0037] The present disclosure provides polyribonucleotides comprising one or more modified ribonucleotides, the one or more modified ribonucleotides comprising one or both of the following:

[0038] (i) 5-hydroxymethyluridine, and (a) the 5' monophosphate; (b) the 5' diphosphate; or (c) the 5' triphosphate, and the following structure: [ka]

[0039] (ii) N-acetylcytidine, and (a) a 5' monophosphate; (b) a 5' diphosphate; or (c) a 5' triphosphate, and the following structure: [ka]

[0040] In some embodiments of a polynucleotide comprising cytidine and uridine residues, (a) at least 5% of the cytidine residues in the polyribonucleotide comprise N4-acetylcytidine; and / or (b) at least 5% of the uridine residues in the polyribonucleotide comprise 5-hydroxymethyluridine.

[0041] In some embodiments of a polynucleotide comprising cytidine and uridine residues, (a) less than 100% of the cytidine residues in the polyribonucleotide comprise N4-acetylcytidine; and / or (b) less than 100% of the uridine residues in the polyribonucleotide comprise 5-hydroxymethyluridine.

[0042] In some embodiments of a polynucleotide comprising cytidine residues and uridine residues: (a) 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% ~25%, about 5%-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% is N and / or (b) 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.

[0043] In some embodiments of a polynucleotide comprising cytidine and uridine residues: (a) about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 99% of the cytidine residues in the polyribonucleotide are and / or (b) about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 99% of the uridine residues in the polyribonucleotide contain 5-hydroxymethyluridine.

[0044] In some embodiments of a polynucleotide comprising cytidine and uridine residues: (a) 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 are N4-acetylcytidine; and / or (b) 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 contain 5-hydroxymethyluridine.

[0045] In some embodiments of a polynucleotide comprising cytidine and uridine residues, (a) greater than 60% of the cytidine residues in the polyribonucleotide comprise N4-acetylcytidine; and / or (b) greater than 60% of the uridine residues in the polyribonucleotide comprise 5-hydroxymethyluridine.

[0046] In some embodiments of the polynucleotide comprising cytidine residues and uridine residues: (a) about 60-100%, about 65%-100%, about 70%-100%, about 75%-100%, about 80%-100%, about 85%-100%, about 90%-100%, about 95%-100%, about 60%-95%, about 60%-90%, about 60%-85%, about 60%-80%, about 60%-75%, about 60%-70%, or about 60%-65% of the cytidine residues in the polyribonucleotide and / or (b) about 60 to 100%, about 65% to 100%, about 70% to 100%, about 75% to 100%, about 80% to 100%, about 85% to 100%, about 90% to 100%, about 95% to 100%, about 60% to 95%, about 60% to 90%, about 60% to 85%, about 60% to 80%, about 60% to 75%, about 60% to 70%, or about 60% to 65% of the uridine residues in the polyribonucleotide contain 5-hydroxymethyluridine.

[0047] In some embodiments of a polynucleotide comprising cytidine and uridine residues, (a) about 60-100% of the cytidine residues in the polyribonucleotide comprise N4-acetylcytidine; and / or (b) about 60-100% of the uridine residues in the polyribonucleotide comprise 5-hydroxymethyluridine.

[0048] In some embodiments of a polynucleotide comprising cytidine and uridine residues, (a) at least about 60% of the cytidine residues in the polyribonucleotide comprise N4-acetylcytidine; and / or (b) between about 60% and 100% of the uridine residues in the polyribonucleotide comprise 5-hydroxymethyluridine.

[0049] In some embodiments of a polynucleotide comprising cytidine and uridine residues, (a) at least about 70% of the cytidine residues in the polyribonucleotide comprise N4-acetylcytidine; and / or (b) about 60-100% of the uridine residues in the polyribonucleotide comprise 5-hydroxymethyluridine.

[0050] In some embodiments of a polynucleotide comprising cytidine and uridine residues, (a) at least about 75% of the cytidine residues in the polyribonucleotide comprise N4-acetylcytidine; and / or (b) about 60-100% of the uridine residues in the polyribonucleotide comprise 5-hydroxymethyluridine.

[0051] In some embodiments of a polynucleotide comprising cytidine and uridine residues, (a) at least about 80% of the cytidine residues in the polyribonucleotide comprise N4-acetylcytidine; and / or (b) about 60-100% of the uridine residues in the polyribonucleotide comprise 5-hydroxymethyluridine.

[0052] In some embodiments of a polynucleotide comprising cytidine and uridine residues, (a) at least about 85% of the cytidine residues in the polyribonucleotide comprise N4-acetylcytidine; and / or (b) about 60-100% of the uridine residues in the polyribonucleotide comprise 5-hydroxymethyluridine.

[0053] In some embodiments of a polynucleotide comprising cytidine and uridine residues, (a) at least about 90% of the cytidine residues in the polyribonucleotide comprise N4-acetylcytidine; and / or (b) about 60-100% of the uridine residues in the polyribonucleotide comprise 5-hydroxymethyluridine.

[0054] In some embodiments of a polynucleotide comprising cytidine and uridine residues, (a) at least about 95% of the cytidine residues in the polyribonucleotide comprise N4-acetylcytidine; and / or (b) about 60-100% of the uridine residues in the polyribonucleotide comprise 5-hydroxymethyluridine.

[0055] In some embodiments of a polynucleotide comprising cytidine and uridine residues, (a) at least about 99% of the cytidine residues in the polyribonucleotide comprise N4-acetylcytidine; and / or (b) about 60-100% of the uridine residues in the polyribonucleotide comprise 5-hydroxymethyluridine.

[0056] In some embodiments of a polynucleotide comprising cytidine and uridine residues, (a) about 100% of the cytidine residues in the polyribonucleotide comprise N4-acetylcytidine; and / or (b) about 60-100% of the uridine residues in the polyribonucleotide comprise 5-hydroxymethyluridine.

[0057] In some embodiments of a polynucleotide comprising cytidine and uridine residues, (a) about 60-100% of the cytidine residues in the polyribonucleotide comprise N4-acetylcytidine; and / or (b) at least about 60% of the uridine residues in the polyribonucleotide comprise 5-hydroxymethyluridine.

[0058] In some embodiments of a polynucleotide comprising cytidine and uridine residues, (a) about 60-100% of the cytidine residues in the polyribonucleotide comprise N4-acetylcytidine; and / or (b) at least about 70% of the uridine residues in the polyribonucleotide comprise 5-hydroxymethyluridine.

[0059] In some embodiments of a polynucleotide comprising cytidine and uridine residues, (a) about 60-100% of the cytidine residues in the polyribonucleotide comprise N4-acetylcytidine; and / or (b) at least about 75% of the uridine residues in the polyribonucleotide comprise 5-hydroxymethyluridine.

[0060] In some embodiments of a polynucleotide comprising cytidine and uridine residues, (a) about 60-100% of the cytidine residues in the polyribonucleotide comprise N4-acetylcytidine; and / or (b) at least about 80% of the uridine residues in the polyribonucleotide comprise 5-hydroxymethyluridine.

[0061] In some embodiments of a polynucleotide comprising cytidine and uridine residues, (a) about 60-100% of the cytidine residues in the polyribonucleotide comprise N4-acetylcytidine; and / or (b) at least about 85% of the uridine residues in the polyribonucleotide comprise 5-hydroxymethyluridine.

[0062] In some embodiments of a polynucleotide comprising cytidine and uridine residues, (a) about 60-100% of the cytidine residues in the polyribonucleotide comprise N4-acetylcytidine; and / or (b) at least about 90% of the uridine residues in the polyribonucleotide comprise 5-hydroxymethyluridine.

[0063] In some embodiments of a polynucleotide comprising cytidine and uridine residues, (a) about 60-100% of the cytidine residues in the polyribonucleotide comprise N4-acetylcytidine; and / or (b) at least about 95% of the uridine residues in the polyribonucleotide comprise 5-hydroxymethyluridine.

[0064] In some embodiments of a polynucleotide comprising cytidine and uridine residues, (a) about 60-100% of the cytidine residues in the polyribonucleotide comprise N4-acetylcytidine; and / or (b) at least about 99% of the uridine residues in the polyribonucleotide comprise 5-hydroxymethyluridine.

[0065] In some embodiments of a polynucleotide comprising cytidine and uridine residues, (a) about 60-100% of the cytidine residues in the polyribonucleotide comprise N4-acetylcytidine; and / or (b) about 100% of the uridine residues in the polyribonucleotide comprise 5-hydroxymethyluridine.

[0066] In some embodiments of a polynucleotide comprising cytidine and uridine residues, (a) at least about 60% of the cytidine residues in the polyribonucleotide comprise N4-acetylcytidine; and / or (b) at least about 60% of the uridine residues in the polyribonucleotide comprise 5-hydroxymethyluridine.

[0067] In some embodiments of a polynucleotide comprising cytidine and uridine residues, (a) at least about 70% of the cytidine residues in the polyribonucleotide comprise N4-acetylcytidine; and / or (b) at least about 70% of the uridine residues in the polyribonucleotide comprise 5-hydroxymethyluridine.

[0068] In some embodiments of a polynucleotide comprising cytidine and uridine residues, (a) at least about 75% of the cytidine residues in the polyribonucleotide comprise N4-acetylcytidine; and / or (b) at least about 75% of the uridine residues in the polyribonucleotide comprise 5-hydroxymethyluridine.

[0069] In some embodiments of a polynucleotide comprising cytidine and uridine residues, (a) at least about 80% of the cytidine residues in the polyribonucleotide comprise N4-acetylcytidine; and / or (b) at least about 80% of the uridine residues in the polyribonucleotide comprise 5-hydroxymethyluridine.

[0070] In some embodiments of a polynucleotide comprising cytidine and uridine residues, (a) at least about 85% of the cytidine residues in the polyribonucleotide comprise N4-acetylcytidine; and / or (b) at least about 85% of the uridine residues in the polyribonucleotide comprise 5-hydroxymethyluridine.

[0071] In some embodiments of a polynucleotide comprising cytidine and uridine residues, (a) at least about 90% of the cytidine residues in the polyribonucleotide comprise N4-acetylcytidine; and / or (b) at least about 90% of the uridine residues in the polyribonucleotide comprise 5-hydroxymethyluridine.

[0072] In some embodiments of a polynucleotide comprising cytidine and uridine residues, (a) at least about 95% of the cytidine residues in the polyribonucleotide comprise N4-acetylcytidine; and / or (b) at least about 95% of the uridine residues in the polyribonucleotide comprise 5-hydroxymethyluridine.

[0073] In some embodiments of a polynucleotide comprising cytidine and uridine residues, (a) at least about 99% of the cytidine residues in the polyribonucleotide comprise N4-acetylcytidine; and / or (b) at least about 99% of the uridine residues in the polyribonucleotide comprise 5-hydroxymethyluridine.

[0074] In some embodiments of a polynucleotide comprising cytidine and uridine residues, (a) about 100% of the cytidine residues in the polyribonucleotide comprise N4-acetylcytidine; and / or (b) about 100% of the uridine residues in the polyribonucleotide comprise 5-hydroxymethyluridine.

[0075] In some embodiments, the polyribonucleotides disclosed herein are characterized by observed reduced immunogenicity when assessed in cells, tissues or organisms to which the polyribonucleotide has been administered, as compared to an appropriate reference comparator.

[0076] In some embodiments, the reference comparator includes an otherwise similar cell, tissue, or organism that has been administered a comparable polyribonucleotide that (i) contains fewer acetyl groups on the nucleobases than the polyribonucleotide in the composition (e.g., does not contain any acetyl groups on the nucleobases); and / or (ii) contains fewer hydroxymethyl groups than the polyribonucleotide in the composition (e.g., does not contain any hydroxymethyl groups). In some embodiments, the comparable polynucleotide contains less (e.g., does not contain) N4-acetylcytidine and / or less (e.g., does not contain) 5-hydroxymethyluridine than the polyribonucleotide in the composition disclosed herein.

[0077] In some embodiments, the reduced immunogenicity comprises reduced activation of innate immune response-induced toxicity. In some embodiments, the reduced activation of the immune response comprises reduced activation of NF-kb or the NF-kb pathway; IRF or the IRF pathway; and / or other inflammatory cytokines in a cell, tissue, or organism. In some embodiments, the reduced activation of the immune response comprises reduced detection of uncapped RNA by a molecular sensor, e.g., RIG-I. In some embodiments, the uncapped RNA comprises RNA that does not have a cap structure, e.g., as described herein. In some embodiments, the uncapped RNA comprises RNA that has a 5' phosphate group and / or a 5' hydroxyl group.

[0078] In some embodiments, the polyribonucleotides disclosed herein are characterized by observing increased expression of the payload when assessed in a cell, tissue, or organism to which the polyribonucleotide has been administered, as compared to a suitable reference comparator. In some embodiments, the reference comparator comprises an otherwise similar cell, tissue, or organism to which a comparable polyribonucleotide has been administered that contains (i) low N4-acetylcytidine nucleosides (e.g., does not contain any N4-acetylcytidine nucleosides); and / or (ii) low 5-hydroxymethyluridine nucleosides (e.g., does not contain any 5-hydroxymethyluridine nucleosides).

[0079] In some embodiments, the increase in expression of the payload is about 1.2-fold, about 1.5-fold, about 2-fold, about 4-fold, about 5-fold, about 10-fold, or about 20-fold compared to a reference standard.

[0080] In some embodiments, the increase in expression of the payload is about 1.2-fold to about 20-fold, about 1.5-fold to about 20-fold, about 2-fold to about 20-fold, about 4-fold to about 20-fold, about 5-fold to about 20-fold, about 10-fold to about 20-fold, about 1.2-fold to about 10-fold, about 1.2-fold to about 5-fold, about 1.2-fold to about 4-fold, about 1.2-fold to about 2-fold, or about 1.2-fold to about 1.5-fold.

[0081] In some embodiments, the payload is or includes a polypeptide encoded by a polyribonucleotide comprising one or more modified ribonucleotides, e.g., as described herein.

[0082] In some embodiments, the payload is or comprises a polyribonucleotide located in a polyribonucleotide that comprises one or more modified ribonucleotides, e.g., as described herein.

[0083] In some embodiments, the reduced immunogenicity allows for repeated administration of polyribonucleotide, for example, two or more administrations. In some embodiments, the repeated administration comprises 2, 3, 4, 5, 6, 7, 8, 9, or 10 doses of polyribonucleotide. In some embodiments, the repeated administration comprises the same dose of polyribonucleotide compared to the previous dose. In some embodiments, the repeated administration comprises a different dose of polyribonucleotide compared to the previous dose.

[0084] In some embodiments, the reduced immunogenicity allows for the administration of higher doses of polyribonucleotides relative to a suitable reference standard. In some embodiments, the reference standard comprises a comparable polyribonucleotide that comprises (i) fewer acetyl groups on the nucleobases (e.g., does not contain any acetyl groups on the nucleobases); and / or (ii) fewer hydroxymethyl groups (e.g., does not contain any hydroxymethyl groups) than the polyribonucleotides in the compositions disclosed herein. In some embodiments, the comparable polynucleotide comprises less (e.g., does not contain) N4-acetylcytidine and / or less (e.g., does not contain) 5-hydroxymethyluridine than the polyribonucleotides in the compositions disclosed herein. In some embodiments, the polyribonucleotides disclosed herein are characterized by an increase in cell viability observed when assessed in cells, tissues, or organisms to which the polyribonucleotides are administered, compared to the suitable reference standard. In some embodiments, the reference standard is the cell viability of an otherwise similar cell, tissue, or organism administered a comparable polyribonucleotide that contains (i) fewer acetyl groups on the nucleobases (e.g., does not contain any acetyl groups on the nucleobases); and / or (ii) fewer hydroxymethyl groups (e.g., does not contain any hydroxymethyl groups) than the polyribonucleotide in the compositions disclosed herein. In some embodiments, the comparable polynucleotide contains less (e.g., does not contain) N4-acetylcytidine and / or less (e.g., does not contain) 5-hydroxymethyluridine than the polyribonucleotide in the compositions disclosed herein. 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.

[0085] In some embodiments, the polyribonucleotides disclosed herein are or comprise RNA oligos.

[0086] In some embodiments, the polyribonucleotides disclosed herein are or comprise messenger RNA (mRNA).

[0087] In some embodiments, the polyribonucleotide disclosed herein is or comprises a gRNA.

[0088] In some embodiments, the polyribonucleotides disclosed herein are or comprise inhibitory RNA.

[0089] In some embodiments, the polyribonucleotide disclosed herein is or comprises an miRNA or miRNA.

[0090] In some embodiments, the polyribonucleotides disclosed herein are or comprise antisense oligonucleotides.

[0091] The present disclosure provides a composition comprising one or more polyribonucleotides disclosed herein, for example, one or more modified ribonucleotides disclosed herein.In some embodiments, modified ribonucleotide comprises a nucleoside comprising an acetyl group, and the nucleoside is N4-acetylcytidine.In some embodiments, modified ribonucleotide comprises a nucleoside comprising a hydroxymethyl group, and the nucleoside is 5-hydroxymethyluridine.

[0092] In some embodiments, the composition is a pharmaceutical composition. In some embodiments, the pharmaceutical composition is or comprises an immunogenic composition. In some embodiments, the pharmaceutical composition is or comprises an immunotherapy. In some embodiments, the pharmaceutical composition is or comprises an immunomodulatory therapy. In some embodiments, the pharmaceutical composition is or comprises an immunomodulatory therapy. In some embodiments, the pharmaceutical composition is or comprises a vaccine. In some embodiments, the pharmaceutical composition is or comprises a gene therapy. In some embodiments, the pharmaceutical composition is or comprises a chemotherapy. In some embodiments, the pharmaceutical composition is or comprises a protein replacement therapy. In some embodiments, the pharmaceutical composition is or comprises an immunotherapy. In some embodiments, the pharmaceutical composition is or comprises a cell engineering therapy.

[0093] In some embodiments, the composition comprises double-stranded RNA.

[0094] Also provided herein are methods that include administering to a cell, tissue, or subject one or more of the polyribonucleotides disclosed herein or a composition comprising one or more thereof.

[0095] In some embodiments, the method further comprises determining 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.

[0096] In some embodiments, the cell, tissue or subject to which the polyribonucleotide or composition comprising the same is administered shows improved cell viability compared to the reference cell viability. In some embodiments, the reference cell viability is the cell viability of a cell, tissue or subject to which a comparable polyribonucleotide or composition comprising the same is administered, which comprises (i) fewer acetyl groups (e.g., no acetyl groups on the nucleobases) than the polyribonucleotide in the composition disclosed herein; and / or (ii) fewer hydroxymethyl groups (e.g., no hydroxymethyl groups). In some embodiments, the comparable polynucleotide comprises fewer (e.g., no) N4-acetylcytidine and / or fewer (e.g., no) 5-hydroxymethyluridine than the polyribonucleotide in the composition disclosed herein.

[0097] In some embodiments, the cells, tissues, or subjects to which the polyribonucleotide or a composition comprising the same has been administered exhibit a reduced immune response to the polyribonucleotide. In some embodiments, the methods disclosed herein further comprise determining the immune system response of the cells, tissues, or subjects to which the polyribonucleotide or a composition comprising the same has been administered.

[0098] In some embodiments, the immune response comprises a response of the innate immune system, including innate immune system-induced toxicity. In some embodiments, determining the response of the innate immune system comprises determining the levels of NF-κB, IRF, and / or other inflammatory cytokines in the cell, tissue, or subject. In some embodiments, determining the response of the innate immune system comprises determining the level of detection of uncapped RNA by a molecular sensor, e.g., RIG-I.

[0099] In some embodiments, the cells, tissues, or subjects to which the polyribonucleotide or a composition comprising the same is administered show high expression of the payload. In some embodiments, the methods disclosed herein further comprise determining the expression of the payload in the cells, tissues, or subjects to which the polyribonucleotide or a composition comprising the same is administered. In some embodiments, the payload is or comprises a polypeptide encoded by a polyribonucleotide comprising one or more modified ribonucleotides. In some embodiments, the payload is or comprises a polyribonucleotide located in a polyribonucleotide comprising one or more modified ribonucleotides. In some embodiments, determining the expression of the payload comprises determining the expression of an RNA, a polypeptide, or both. In some embodiments, the increase in expression of the payload is about 1.2-fold, about 1.5-fold, about 2-fold, about 4-fold, about 5-fold, about 10-fold, or about 20-fold compared to the reference.

[0100] In some embodiments, the cell, tissue, or subject to which the polyribonucleotide or composition comprising the same is administered shows a reduced innate immune system response compared to the reference. In some embodiments, the reference is the innate immune system response of a cell, tissue, or subject to which a comparable polyribonucleotide or composition comprising the same is administered that contains (i) fewer acetyl groups on the nucleobases (e.g., no acetyl groups on the nucleobases); and / or (ii) fewer hydroxymethyl groups (e.g., no hydroxymethyl groups) than the polyribonucleotide in the composition disclosed herein. In some embodiments, the comparable polynucleotide contains less (e.g., no) N4-acetylcytidine and / or less (e.g., no) 5-hydroxymethyluridine than the polyribonucleotide in the composition disclosed herein. In some embodiments, the method disclosed herein further comprises determining the efficacy of the polyribonucleotide or composition comprising the same in the cell, tissue, or subject to which the polyribonucleotide or composition comprising the same is administered. In some embodiments, determining the efficacy comprises determining an antibody response or a cellular response in the cell, tissue, or subject.

[0101] In some embodiments, the cell, tissue, or subject to which the polyribonucleotide or composition comprising the same is administered shows a higher antibody response or cellular response compared to the reference. In some embodiments, the reference is the antibody response or cellular response of the cell, tissue, or subject to which a comparable polyribonucleotide or composition comprising the same is administered that comprises (i) fewer acetyl groups (e.g., no acetyl groups on the nucleobases) than the polyribonucleotide in the composition disclosed herein; and / or (ii) fewer hydroxymethyl groups (e.g., no hydroxymethyl groups). In some embodiments, the comparable polynucleotide comprises fewer (e.g., no) N4-acetylcytidines and / or fewer (e.g., no) 5-hydroxymethyluridines than the polyribonucleotide in the composition disclosed herein.

[0102] In some embodiments, the method comprises administering the polyribonucleotide or a composition comprising the same to a cell, tissue or subject at least two times. In some embodiments, the method comprises administering the polyribonucleotide or a composition comprising the same to a cell, tissue or subject at least two times, 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, the method comprises administering the polyribonucleotide or a composition comprising the same to a cell, tissue or subject at least two times. In some embodiments, the method comprises administering the polyribonucleotide or a composition comprising the same to a cell, tissue or subject at least three times. In some embodiments, the method comprises administering the polyribonucleotide or a composition comprising the same to a cell, tissue or subject at least four times. In some embodiments, the method comprises administering the polyribonucleotide or a composition comprising the same to a cell, tissue or subject at least five times. In some embodiments, the method comprises administering the polyribonucleotide or a composition comprising the same to a cell, tissue or subject at least six times. In some embodiments, the method comprises administering the polyribonucleotide or a composition comprising the same to the cell, tissue or subject at least 7 times. In some embodiments, the method comprises administering the polyribonucleotide or a composition comprising the same to the cell, tissue or subject at least 8 times. In some embodiments, the method comprises administering the polyribonucleotide or a composition comprising the same to the cell, tissue or subject at least 9 times. In some embodiments, the method comprises administering the polyribonucleotide or a composition comprising the same to the cell, tissue or subject at least 10 times.

[0103] In some embodiments, at least two administrations of a polyribonucleotide or a composition comprising same to a cell, tissue or subject does not result in a decrease in the efficacy of the polyribonucleotide or a composition comprising same compared to administration of a single dose of the polyribonucleotide or a composition comprising same.

[0104] In some embodiments, the method comprises administering a polyribonucleotide or a composition comprising the same to a cell, tissue or subject at a higher dose than a suitable reference comparator. In some embodiments, the reference comparator comprises a comparable polyribonucleotide that comprises (i) fewer acetyl groups on the nucleobases (e.g., no acetyl groups on the nucleobases); and / or (ii) fewer hydroxymethyl groups (e.g., no hydroxymethyl groups) than the polyribonucleotide in the composition disclosed herein. In some embodiments, the comparable polynucleotide comprises less (e.g., no) N4-acetylcytidine and / or less (e.g., no) 5-hydroxymethyluridine than the polyribonucleotide in the composition disclosed herein. In some embodiments of any of the methods disclosed 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.

[0105] In some embodiments, the method is a method of stimulating an immune response.

[0106] In some embodiments, the method is a method of vaccination.

[0107] In some embodiments, the method is a method of antibody therapy.

[0108] In some embodiments, the method is a method of immunomodulatory therapy.

[0109] In some embodiments, the method is a method of gene therapy.

[0110] In some embodiments, the methods include delivery of one or more components of a gene therapy, such as a gRNA.

[0111] In some embodiments, the method is a cell therapy engineering method.

[0112] In some embodiments, the method is an immunotherapy method, hi some embodiments, the immunotherapy method comprises delivery of an immune modulating therapy and / or an immune checkpoint therapy.

[0113] 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.

[0114] In some embodiments, the method is a method of chemotherapy.

[0115] Also provided herein are methods of vaccination comprising administering to a cell, tissue or subject one or more of the polyribonucleotides disclosed herein or compositions comprising same.

[0116] 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.

[0117] Provided herein are methods of providing antibody therapy comprising administering one or more of the polyribonucleotides disclosed herein or compositions comprising same to a cell, tissue or subject. In some embodiments, the antibody therapy comprises an antibody, a fragment, variant, or 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 bicomponent 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.

[0118] Provided herein is a method of providing an immunomodulatory therapy comprising administering one or more of the polyribonucleotides disclosed herein or compositions comprising the same to a cell, tissue or subject. In some embodiments, the immunomodulatory therapy comprises a cytokine or variant or fragment thereof, a chemokine or variant or fragment thereof, a T cell modulator, a NK cell modulator, a B cell modulator, a myeloid cell modulator, a modulator of other immune cells, or a 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.

[0119] 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.

[0120] 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.

[0121] 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.

[0122] The present disclosure provides a method for obtaining a lower level of immunogenicity in a subject receiving a polyribonucleotide comprising a modified ribonucleotide or a composition comprising the same, compared to a subject receiving a comparable unmodified polyribonucleotide. In some embodiments, the method comprises administering to the subject a polyribonucleotide comprising a modified ribonucleotide or a composition comprising the same.

[0123] 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 5' hydroxyl group at the 5' end of the polyribonucleotide.

[0124] 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.

[0125] In some embodiments, the subject receiving the polyribonucleotide comprising the modified ribonucleotide, or the composition comprising same, and the subject receiving the comparable unmodified polyribonucleotide are the same subject.

[0126] In some embodiments, the subject receiving the polyribonucleotide comprising the modified ribonucleotide, or the composition comprising same, and the subject receiving the comparable unmodified polyribonucleotide are different subjects.

[0127] Provided herein is a method for preparing an 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 the RNA composition.

[0128] Disclosed herein are cells comprising the polyribonucleotides disclosed herein or compositions comprising same.

[0129] Also disclosed herein is the use of the modified ribonucleotides disclosed herein in the production of polyribonucleotides.

[0130] 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.

[0131] 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.

[0132] 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.

[0133] 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.

[0134] 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.

[0135] 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.

[0136] 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.

[0137] 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.

[0138] 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.

[0139] 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.

[0140] Provided herein are compositions comprising the polyribonucleotides disclosed herein for use as vaccines.

[0141] The present disclosure provides compositions comprising the polyribonucleotides disclosed herein for use as immunotherapeutics.

[0142] Provided herein are compositions comprising the polyribonucleotides disclosed herein for use as antibody therapeutics.

[0143] Provided herein are compositions comprising the polyribonucleotides disclosed herein for use as immunomodulatory therapies.

[0144] Provided herein are compositions comprising the polyribonucleotides disclosed herein for use as gene therapy agents.

[0145] The present disclosure provides compositions comprising the polyribonucleotides disclosed herein for use as protein replacement therapy.

[0146] Provided herein are compositions comprising the polyribonucleotides disclosed herein for use as cell engineering therapies.

[0147] The present disclosure provides compositions comprising the polyribonucleotides disclosed herein for use as chemotherapy.

[0148] In some embodiments of any of the uses or methods provided herein, the polyribonucleotide or a composition comprising same is administered to a cell, tissue or subject.

[0149] 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]

[0150] [Figure 1] 1 is a graph showing the viability of A549 cells following transfection with RNA synthesized using the indicated percentage of N4-acetylcytidine in place of unmodified (eg, natural) cytidine.

[0151] [Diagram 2] FIG. 1 is a graph showing activation of an NF-κb reporter by RNA synthesized with the indicated percentages of N4-acetylcytidine in place of unmodified cytidine.

[0152] [Diagram 3] 1 is a graph showing activation of an IRF reporter by RNA synthesized with the indicated percentages of N4-acetylcytidine in place of unmodified cytidine.

[0153] [Figure 4] 1 is a graph showing expression of the luciferase gene by RNA synthesized using the indicated percentages of N4-acetylcytidine in place of unmodified cytidine.

[0154] [Diagram 5] FIG. 1 is a graph showing luciferase gene expression normalized to cell viability by RNA synthesized using the indicated percentages of N4-acetylcytidine in place of unmodified cytidine.

[0155] [Figure 6]FIG. 1 is a graph showing the time course of luciferase expression following repeated administration of luciferase RNA with the indicated chemical modifications and synthesized at the indicated IVT synthesis temperatures in BALb / c mice. Mice were administered three doses of LNP-formulated RNA. Each dose was separated by 72 hours, and mice were imaged for luciferase expression at 6, 27, and 51 hours after administration of each dose. The x-axis provides time point information. For example, 1.6, 1.27, and 1.51 indicate 6, 27, and 51 hours after administration of the first dose. The same is true for time points after the second dose (see time points 2.6, 2.27, and 2.51) and the third dose (see time points 3.6, 3.27, and 3.51).

[0156] [Figure 7] 1 shows the differences in serum cytokines in BALB / c mice administered various RNAs.

[0157] [Figure 8] FIG. 1 is a graph showing luciferase expression in BALB / c mice administered 1 microgram of luc2 RNA with the indicated % substitutions of N4-acetylcytidine or unmodified luc2 RNA.

[0158] [Figure 9] Graph showing IgG antibody titers against SARS-CoV-2 RNA vaccine candidates in mice administered the indicated nucleotide compositions.

[0159] [Figure 10] The figure shows the expression of a luciferase reporter gene by repeated administration of 100% Ac4C / 100% 5hmU modified RNA at 72 hour intervals.

[0160] [Figure 11] The structure of 5-hydroxymethyluridine triphosphate (5hmU) is shown.

[0161] [Figure 12]Cell viability with RNA synthesized using the indicated percentage of 5-hydroxymethyluridine in place of natural uridine is shown.

[0162] [Figure 13] Activation of the IRF reporter by RNA synthesized with the indicated percentage of 5-hydroxymethyluridine in place of natural uridine is shown.

[0163] [Figure 14] Shown is the activity of the NF-κB reporter with RNA synthesized using the indicated percentage of 5-hydroxymethyluridine in place of natural uridine.

[0164] [Figure 15] Expression of the luciferase gene by RNA synthesized using the indicated percentage of 5-hydroxymethyluridine in place of natural uridine is shown.

[0165] [Figure 16] Luciferase gene expression normalized to cell viability with RNA synthesized using the indicated percentage of 5-hydroxymethyluridine in place of natural uridine is shown.

[0166] [Figure 17] Shown is cell viability with RNA synthesized using the indicated percentage of 5-hydroxymethyluridine instead of natural uridine in combination with 100% Ac4C instead of cytidine at a dose of 50 ng.

[0167] [Figure 18] Activation of the IRF reporter by RNA synthesized using the indicated percentage of 5-hydroxymethyluridine instead of natural uridine in combination with 100% Ac4C instead of cytidine at a dose of 50 ng is shown.

[0168] [Figure 19]Activation of an NF-κB reporter by RNA synthesized using the indicated percentage of 5-hydroxymethyluridine instead of natural uridine in combination with 100% Ac4C instead of cytidine at a dose of 50 ng is shown.

[0169] [Figure 20] Luciferase gene expression normalized to untreated is shown with RNA synthesized using the indicated percentage of 5-hydroxymethyluridine instead of natural uridine in combination with 100% Ac4C instead of cytidine at a dose of 50 ng.

[0170] [Figure 21] Luciferase gene expression normalized to cell viability is shown with RNA synthesized using the indicated percentage of 5-hydroxymethyluridine instead of natural uridine in combination with 100% Ac4C instead of cytidine at a dose of 50 ng.

[0171] [Figure 22] Cell viability is shown with RNA synthesized using the indicated percentage of 5-hydroxymethyluridine instead of natural uridine in combination with 100% Ac4C instead of cytidine at a dose of 200 ng.

[0172] [Figure 23] Activation of the IRF reporter by RNA synthesized using the indicated percentage of 5-hydroxymethyluridine instead of natural uridine in combination with 100% Ac4C instead of cytidine at a dose of 200 ng is shown.

[0173] [Figure 24] Activation of an NF-κB reporter by RNA synthesized using the indicated percentage of 5-hydroxymethyluridine instead of natural uridine in combination with 100% Ac4C instead of cytidine at a dose of 200 ng is shown.

[0174] [Diagram 25]Luciferase gene expression normalized to untreated is shown with RNA synthesized using the indicated percentage of 5-hydroxymethyluridine instead of natural uridine in combination with 100% Ac4C instead of cytidine at a dose of 200 ng.

[0175] [Figure 26] Shown is luciferase gene expression normalized to cell viability with RNA synthesized using the indicated percentage of 5-hydroxymethyluridine instead of natural uridine in combination with 100% Ac4C instead of cytidine at a dose of 200 ng.

[0176] [Figure 27] Cell viability with RNA synthesized using the indicated percentage of N4-acetylcytidine instead of natural cytidine in combination with 100% 5Hmu instead of uridine at a dose of 50 ng is shown.

[0177] [Figure 28] Activation of the IRF reporter by RNA synthesized using the indicated percentages of N4-acetylcytidine in place of natural cytidine in combination with 100% 5Hmu in place of uridine at a dose of 50 ng is shown.

[0178] [Figure 29] Shown is activation of an NF-κB reporter by RNA synthesized using the indicated percentages of N4-acetylcytidine instead of natural cytidine in combination with 100% 5hmu instead of uridine at a dose of 50 ng.

[0179] [Diagram 30] Luciferase gene expression normalized to untreated is shown with RNA synthesized using the indicated percentages of N4-acetylcytidine instead of natural cytidine in combination with 100% 5hmu instead of uridine at a dose of 50 ng.

[0180] [Diagram 31]Luciferase gene expression normalized to cell viability is shown with RNA synthesized using the indicated percentages of N4-acetylcytidine in place of natural cytidine in combination with 100% 5hmu in place of uridine at a dose of 50 ng.

[0181] [Diagram 32] Cell viability with RNA synthesized using the indicated percentage of N4-acetylcytidine instead of natural cytidine in combination with 100% 5Hmu instead of uridine at a dose of 200 ng is shown.

[0182] [Diagram 33] Activation of the IRF reporter by RNA synthesized using the indicated percentages of N4-acetylcytidine in place of natural cytidine in combination with 100% 5Hmu in place of uridine at a dose of 200 ng is shown.

[0183] [Diagram 34] Shown is activation of an NF-κB reporter by RNA synthesized using the indicated percentages of N4-acetylcytidine instead of natural cytidine in combination with 100% 5Hmu instead of uridine at a dose of 200 ng.

[0184] [Diagram 35] Luciferase gene expression normalized to untreated is shown with RNA synthesized using the indicated percentages of N4-acetylcytidine instead of natural cytidine in combination with 100% 5hmu instead of uridine at a dose of 50 ng.

[0185] [Diagram 36] Luciferase gene expression normalized to cell viability is shown with RNA synthesized using the indicated percentages of N4-acetylcytidine in place of natural cytidine in combination with 100% 5hmu in place of uridine at a dose of 200 ng.

[0186] [Figure 37]Cell viability is shown with RNA synthesized using the indicated percentage of 5-hydroxymethyluridine instead of natural uridine in combination with 100% Ac4C instead of cytidine at a dose of 200 ng without CIAP treatment.

[0187] [Figure 38] Activation of the IRF reporter by RNA synthesized using the indicated percentage of 5-hydroxymethyluridine instead of natural uridine in combination with 100% Ac4C instead of cytidine at a dose of 200 ng without CIAP treatment is shown.

[0188] [Figure 39] Activation of the NF-κB reporter by RNA synthesized using the indicated percentage of 5-hydroxymethyluridine instead of natural uridine in combination with 100% Ac4C instead of cytidine at a dose of 200 ng without CIAP treatment is shown.

[0189] [Diagram 40] Luciferase gene expression normalized to untreated is shown with RNA synthesized using the indicated percentage of 5-hydroxymethyluridine instead of natural uridine in combination with 100% Ac4C instead of cytidine at a dose of 200 ng without CIAP treatment.

[0190] [Diagram 41] Luciferase gene expression normalized to cell viability is shown with RNA synthesized using the indicated percentage of 5-hydroxymethyluridine instead of natural uridine in combination with 100% Ac4C instead of cytidine at a dose of 200 ng without CIAP treatment.

[0191] [Diagram 42] Cell viability is shown with RNA synthesized using the indicated percentages of N4-acetylcytidine instead of natural cytidine in combination with 100% 5Hmu instead of uridine at a dose of 200 ng without CIAP treatment.

[0192] [Diagram 43] Activation of the IRF reporter by RNA synthesized using the indicated percentages of N4-acetylcytidine instead of natural cytidine in combination with 100% 5Hmu instead of uridine at a dose of 200 ng without CIAP treatment is shown.

[0193] [Diagram 44] Shown is activation of an NF-κB reporter by RNA synthesized using the indicated percentages of N4-acetylcytidine instead of natural cytidine in combination with 100% 5Hmu instead of uridine at a dose of 200 ng without CIAP treatment.

[0194] [Diagram 45] Luciferase gene expression normalized to untreated is shown with RNA synthesized using the indicated percentages of N4-acetylcytidine instead of natural cytidine in combination with 100% 5hmu instead of uridine at a dose of 200 ng without CIAP treatment.

[0195] [Figure 46] Luciferase gene expression normalized to cell viability is shown with RNA synthesized using the indicated percentages of N4-acetylcytidine instead of natural cytidine in combination with 100% 5Hmu instead of uridine at a dose of 200 ng without CIAP treatment.

[0196] [Figure 47] Luciferase reporter gene expression following repeated administration of 9 μg of 100% Ac4C / 100% 5hmU modified RNA versus 100% N1-methylpseudouridine modified RNA at 72 hour intervals is shown.

[0197] [Figure 48] Shown is the systemic cytokine response to 100% Ac4C / 100% 5hmU Luc2 RNA 9ug versus 100% N1-methylpseudouridine Luc2 RNA.

[0198] [Figure 49] IgG antibody titers in response to vaccination with the indicated RNA encoding SARS-CoV-2 vaccine candidates. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0199] Specific Definitions 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 the referenced value. In general, a person skilled in the art familiar with the context will understand the appropriate degree of variation 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 are within 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 referenced value.

[0200] Antibody: As used herein, the term "antibody" refers to a polypeptide that contains elements of a canonical immunoglobulin sequence sufficient to confer specific binding to a particular target antigen. As known in the art, an intact antibody, as produced in nature, is a tetrameric chemical entity of approximately 150 kD, composed of two identical heavy chain polypeptides (about 50 kD each) and two identical light chain polypeptides (about 25 kD each) associated with each other, commonly referred to as a "Y-shaped" structure. Each heavy chain is composed of at least four domains, each about 110 amino acids long: an amino-terminal variable (VH) domain (located at the tip of the Y structure) followed by three constant domains: CH1, CH2, and a carboxy-terminal CH3 (located at the base of the Y stem). A short region known as the "switch" connects the heavy chain variable and constant regions. A "hinge" connects the CH2 and CH3 domains to the rest of the antibody. Two disulfide bonds in this hinge region connect the two heavy chain polypeptides to each other in an intact antibody. Each light chain is composed of two domains: an amino-terminal variable (VL) domain followed by a carboxy-terminal constant (CL) domain, which are separated from each other by another "switch". An intact antibody tetramer is composed of two heavy-light chain dimers in which the heavy and light chains are linked to each other by one disulfide bond; two other disulfide bonds connect the heavy chain hinge regions to each other, thus connecting the dimers to form a tetramer. In addition, naturally produced antibodies are usually glycosylated in the CH2 domain. Each domain in a natural antibody has a structure characterized by an "immunoglobulin fold" formed from two beta sheets (e.g., three-, four-, or five-stranded sheets) packed against each other in a compressed antiparallel beta barrel. Each variable domain contains three hypervariable loops known as "complementarity determining regions" (CDR1, CDR2 and CDR3), and four somewhat invariant "framework" regions (FR1, FR2, FR3 and FR4).When a natural antibody folds, the CDR loop regions of both the heavy and light chains join in three-dimensional space such that the FR regions form beta sheets to provide the structural framework of the domain, creating a single hypervariable antigen-binding site located at the tip of a Y-structure. The Fc region of a naturally occurring antibody binds to elements of the complement system and also to receptors on effector cells, such as effector cells that mediate cytotoxicity. As is known in the art, the affinity and / or other binding properties of the Fc region for the Fc receptor can be modulated through glycosylation or other modifications. In some embodiments, the antibodies produced and / or utilized in accordance with the present invention comprise a glycosylated Fc domain, including Fc domains with such glycosylation that have been modified or engineered. In some embodiments, the antibodies produced and / or utilized in accordance with the present disclosure comprise one or more modifications on the Fc domain, such as effector null mutations, such as LALA, LAGA, FEGG, AAGG, or AAGA mutations. For purposes of this disclosure, in certain embodiments, any polypeptide or complex of polypeptides that includes a sufficient immunoglobulin domain sequence as found in a natural antibody may be referred to and / or used as an "antibody," regardless of whether such polypeptide is naturally generated (e.g., generated by an organism in response to an antigen) or generated by recombinant engineering, chemical synthesis, or other artificial systems or methodologies. In some embodiments, an antibody is polyclonal; in some embodiments, an antibody is monoclonal. In some embodiments, an antibody has constant region sequences characteristic of a dog antibody, a feline antibody, a mouse antibody, a rabbit antibody, a primate antibody, or a human antibody. In some embodiments, the antibody sequence elements are human, humanized, primatized, chimeric, etc., as known in the art. Furthermore, the term "antibody" as used herein can refer to any of the constructs or formats known or developed in the art for utilizing the structural and functional features of antibodies in alternative presentations, in appropriate embodiments (unless otherwise stated or clear from the context).For example, in some embodiments, the antibody utilized in accordance with the present invention is in a format selected from, but not limited to, an intact IgA, IgG, IgE or IgM antibody; a bi- or multispecific antibody (e.g., Zybodies®, etc.); an antibody fragment such as a Fab fragment, a Fab' fragment, a F(ab')2 fragment, a Fd' fragment, a Fd fragment, and isolated CDRs thereof; a single chain Fv; a polypeptide-Fc fusion; a single domain antibody, an alternative scaffold or an antibody mimic (e.g., anticalin, FN3 monobody, DARPin, affibody, affilin, affimer, affitin, alphabody, avimer, fynomer, Im7, VLR, VNAR, trimab, crossmab, trident); a nanobody, a binanobody, a F(ab')2, a Fab', a di-sdFv, a single domain antibody, a trifunctional antibody, a diabody, and a minibody. In some embodiments, the relevant formats may be or may include Adnectins®; Affibodies®; Affilins®; Anticalins®; Avimers®; BiTEs®; cameloid antibodies; Centyrins®; Ankyrin repeat proteins or DARPINs®; Dual Affinity Retargeting (DART) agents; Fynomers®; Shark single domain antibodies such as IgNAR; Immunomobilizing Monoclonal T Cell Receptors against Cancer (ImmTAC); KALBITOR®; Microproteins; Nanobodies®; Minibodies; Masked antibodies (e.g., Probodies®, etc.); Small Modular ImmunoPharmaceuticals (SMIPs™); Single chain or tandem diabodies (TandAbs®); TCR-like antibodies; Trans-bodies®; TrimerX®; VHHs. In some embodiments, the antibody may lack covalent modifications (eg, glycan attachment) that it would have if produced in nature.In some embodiments, the antibodies may contain covalent modifications (e.g., the attachment of a glycan, a payload (e.g., a detectable moiety, a therapeutic moiety, a catalytic moiety, etc.), or other pendant groups (e.g., polyethylene glycol, etc.).

[0201] 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 be aware of the various routes that may be utilized for administration to a subject, e.g., a human, under appropriate circumstances. For example, in some embodiments, administration may be ocular, oral, parenteral, topical, and the like. In some particular embodiments, administration may be bronchial (e.g., by bronchial instillation), buccal, transdermal (e.g., may be or may include one or more of topical, intradermal, interdermal, transdermal, and the like), 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, and the like. In some embodiments, administration may include only a single administration. In some embodiments, administration may include the application of a certain number of doses. In some embodiments, administration may include intermittent (e.g., multiple doses spaced apart in time) 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.

[0202] Antigen: As used herein, the term "antigen" refers to a chemical entity 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 but may or may not induce a specific physiological response in an organism. In general, an antigen may be or include any chemical entity, such as, for example, a small molecule, a nucleic acid, a polypeptide, a carbohydrate, a lipid, a polymer (in some embodiments other than a biopolymer [e.g., other than a nucleic acid or amino acid polymer]), and the like. 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 materials, such as cell extracts or other relatively crude preparations of antigen-containing sources). 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.

[0203] Delivery / Contacting: 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 can be cultured in vitro or ex vivo, or can be present in 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 can vary depending on the 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) can 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) can 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 in a subject by administering the fusion polynucleotide (e.g., as described herein) or the fusion polypeptide (e.g., as described herein) to the subject.

[0204] 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.

[0205] Fragment: A "fragment" of a substance or entity as described herein comprises a discrete portion of the 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 comprises a characteristic structural element or portion 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 in 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.

[0206] 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 bonds 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 relative to that of the natural residue (eg, 2'-fluororibose, ribose, 2'-deoxyribose, arabinose, and hexose).In some embodiments, a nucleic acid has a nucleotide sequence that encodes a functional gene product, such as an RNA or a polypeptide. In some embodiments, a nucleic acid has a nucleotide sequence that includes one or more introns. In some embodiments, a 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, 4000 , 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, the particular number of nucleotides refers to, for example, the number of nucleotides on one strand of the fusion polynucleotide.

[0207] Polypeptide: As used herein, the term "polypeptide" generally has its art-recognized meaning of a polymer of at least three or more amino acids. Those skilled in the art will appreciate that the term "polypeptide" is intended to be general enough 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.

[0208] 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 can include a backbone structure as described in the definition of "nucleic acid / oligonucleotide" above. A polyribonucleotide can be a regulatory RNA (e.g., siRNA, microRNA, etc.), or a messenger RNA (mRNA) oligonucleotide. In some embodiments, a polyribonucleotide is an mRNA oligonucleotide, the polyribonucleotide typically includes a poly(A) region at its 3' end. In some embodiments, a polyribonucleotide is an mRNA oligonucleotide, the polyribonucleotide typically includes a cap structure at its 5' end, e.g., as recognized in the art, for recognizing and binding to ribosomes to initiate translation of the mRNA. In some embodiments, a polyribonucleotide includes an RNA oligonucleotide. When the number of ribonucleotides is used, for example, as an indicator of the size of a polynucleotide, the particular number of nucleotides refers, for example, to the number of ribonucleotides on a single strand.

[0209] Subject: As used herein, the term "subject" refers to an organism, typically a mammal (e.g., a human). In some embodiments, the subject is suffering from 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, the subject is one who possesses one or more characteristics characteristic of a susceptibility to 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 who has been administered a diagnosis and / or therapy.

[0210] Variant: As used herein, the term "variant" refers to an entity that exhibits sufficient structural identity with a reference entity, but that differs structurally from the reference entity in the presence or level of one or more chemical moieties relative to the reference entity. In many embodiments, a variant also differs functionally from the 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 in addition, 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 relative to a reference polypeptide.

[0211] 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 various general and more specific references cited and discussed throughout this specification. See, for example, 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.

[0212] Detailed Description of Specific Embodiments The recent introduction of RNA-based vaccines against SARS-CoV-2 has brought to the forefront the capabilities of a therapeutic platform that has been in development for quite some time. Although RNA therapeutics have real potential for application in various indications, their widespread application beyond vaccines is still limited by side effects caused by unwanted activation of the body's innate immune response. Over the course of human evolution, the human body has developed mechanisms to fight a wide variety of pathogens. The introduction of foreign RNA into human cells can trigger a number of innate immune sensors that recognize the RNA as non-self. Cytoplasmic sensors such as MDA5, PKR, and OAS can all recognize dsRNA contaminants generated during RNA synthesis or functional secondary structures inherent in the RNA transcript itself. RIG-I is an example of a major sensor of uncapped RNA, while TLR3, 7, and 8 are examples of common sensors of "non-self" RNA. These sensors can promote antiviral responses that lead to the inhibition of protein translation. Coupled with translation inhibition, an excessive inflammatory response commonly occurs and can manifest as severe side effects in patients. Effective RNA therapeutics need to deliver high levels of a protein of interest while being well tolerated by patients receiving the treatment, both of which can be achieved by bypassing the body's innate immune sensors.

[0213] One potential technique to address this challenge is to use chemically modified nucleotides that attenuate the excessive innate immune response to RNA sufficiently so that side effects are tolerable and efficacy is not significantly reduced. Thus, the present disclosure provides techniques for reducing the immunogenicity of RNA therapeutics by providing polyribonucleotides that include modified ribonucleotides. In some embodiments, the modified ribonucleotides include ribonucleotides that include N4-acetylcytidine and / or ribonucleotides that include 5-hydroxymethyluridine. As discussed herein, the reduced immunogenicity is due to administration of the RNA therapeutic in response to the RNA molecule itself and should be contrasted with immunogenicity caused, for example, by a polypeptide encoded by the RNA molecule, which may be desirable as a result of, for example, an RNA vaccine. Specifically disclosed herein is the novel discovery that in vitro transcribed RNA containing N4-acetylcytidine instead of unmodified cytidine and / or 5-hydroxymethyluridine instead of unmodified uridine can improve the efficacy of RNA therapeutics by significantly reducing undesired innate immune responses.

[0214] Previous studies have found that N4-acetylcytidine can increase the translation of RNA transcripts containing N4-acetylcytidine as a result of improved interaction with tRNAs that recognize codons containing cytidine at the wobble position (Arango, et al. (2018), Cell, 175(7):1872-1886). This study did not address the effect of N4-acetylcytidine on the immunogenicity of RNA transcripts containing N4-acetylcytidine or the therapeutic efficacy of RNA compositions containing N4-acetylcytidine. In fact, Arango et al. did not find any difference in the phosphorylation of eIF2a, which may be an indirect indicator of innate immune responses, between RNAs with and without N4-acetylcytidine.

[0215] The present disclosure demonstrates for the first time the surprising discovery that RNA (e.g., mRNA) incorporating N4-acetylcytidine results in a significant improvement in cell viability and a reduction in RNA-induced toxicity in vivo. In particular, disclosed herein is the discovery that the degree of improvement in cell viability is influenced by the proportion of cytidine nucleotides substituted by N4-acetylcytidine. In some embodiments, a reduction in polypeptide expression, e.g., reporter protein expression, is observed in polyribonucleotides in which all cytidines are N4-acetylcytidine. In some embodiments, the preferred ratio of modified nucleotides to unmodified nucleotides is likely to depend on the particular codon composition of the nucleotide sequence in question and the therapeutic application for which it is used.

[0216] The present disclosure also recognizes that polyribonucleotides comprising N4-acetylcytidine and / or 5-hydroxymethyluridine mediate evasion of the innate immune system, improve the viability of cells into which the polyribonucleotide is introduced, and / or increase expression of a payload in cells into which the polyribonucleotide is introduced. In some embodiments, evasion of the innate immune system comprises a reduction in activation of NF-κb or the NF-κb pathway; IRF or the IRF pathway; and / or other inflammatory cytokines in a cell, tissue, or organism into which the polyribonucleotide is introduced. In some embodiments, evasion of the innate immune system comprises a reduction in detection of uncapped RNA in a cell, tissue, or organism into which the polyribonucleotide is introduced.

[0217] Among other things, disclosed herein is the discovery that the degree of innate immune evasion and the magnitude of increased payload expression are influenced by the percentage of cytidine nucleotides in a polyribonucleotide that are substituted with N4-acetylcytidine and / or the percentage of uridine nucleotides that are substituted with 5-hydroxymethyluridine. In some embodiments, replacing the majority (e.g., about 75% or more) of the cytidines in a polyribonucleotide with N4-acetylcytidine provides enhanced innate immune evasion (e.g., complete innate immune evasion) and / or increased payload expression. In some embodiments, replacing the majority (e.g., about 75% or more) of the uridines in a polyribonucleotide with 5-hydroxymethyluridine provides enhanced innate immune evasion (e.g., complete innate immune evasion) and / or increased payload expression. In some embodiments, substitution of a majority (e.g., about 75% or more) of the cytidines in a polyribonucleotide with N4-acetylcytidine and substitution of a majority (e.g., about 75% or more) of the uridines in a polyribonucleotide with 5-hydroxymethyluridine provides enhanced innate immune evasion (e.g., complete innate immune evasion) and / or increased payload expression.

[0218] The insights and discoveries provided in this disclosure further allow for tuning (e.g., the ability to make incremental changes) of payload expression and / or immunogenicity from polyribonucleotides comprising one or more modified ribonucleotides, for example, as disclosed herein. For example, a particular level of expression of a payload from a polyribonucleotide can be achieved by using one or more modified ribonucleotides described herein based on the desired application of the polyribonucleotide comprising one or more modified ribonucleotides. Furthermore, a particular level of immunogenicity associated with a polyribonucleotide can be achieved by using one or more modified ribonucleotides described herein based on the desired application of the polyribonucleotide comprising one or more modified ribonucleotides.

[0219] The present disclosure also provides insight that immunogenicity from polyribonucleotides may be reduced, for example, by changing the percentage of cytidine nucleosides substituted with N4-acetylcytidine in polyribonucleotides. For example, polyribonucleotides that do not have N4-acetylcytidine in place of cytidine can be associated with a certain level of immunogenicity. Polyribonucleotides with, for example, 25% N4-acetylcytidine in place of cytidine can provide reduced immunogenicity compared to the level observed from polyribonucleotides that do not have N4-acetylcytidine in place of cytidine. Figures 2 and 3 herein provide exemplary reductions in immunogenicity by polyribonucleotides with more than 25% cytidine nucleosides substituted with N4-acetylcytidine. Polyribonucleotides with, for example, 50% N4-acetylcytidine in place of cytidine can provide even greater reductions in immunogenicity compared to the level observed from polyribonucleotides that do not have N4-acetylcytidine in place of cytidine.

[0220] The present disclosure also provides insight that payload expression may be increased, for example, by modifying the percentage of uridine nucleosides substituted with 5-hydroxymethyluridine in a polyribonucleotide. For example, a polyribonucleotide that does not have 5-hydroxymethyluridine in place of a uridine residue can provide a certain level of payload expression. A polyribonucleotide that has, for example, 25% 5-hydroxymethyluridine in place of a uridine can provide increased expression compared to the level observed from a polyribonucleotide that does not have 5-hydroxymethyluridine in place of a uridine. A polyribonucleotide that has, for example, 50% 5-hydroxymethyluridine in place of a uridine can provide an even greater increase in expression compared to the level observed from a polyribonucleotide that does not have 5-hydroxymethyluridine in place of a uridine. Figure 16 herein provides an exemplary increase in payload expression by a polyribonucleotide in which more than 50% of the uridines are substituted with 5-hydroxymethyluridine.

[0221] In some embodiments, the payload is or comprises a polypeptide encoded by a polyribonucleotide comprising modified ribonucleotides.

[0222] In some embodiments, the payload is or comprises RNA located in a polyribonucleotide that comprises modified ribonucleotides.

[0223] In some embodiments, the use of polyribonucleotides containing N4-acetylcytidine and / or 5-hydroxymethyluridine allows for improved efficacy of RNA therapeutics containing the polyribonucleotides and / or improved tolerability in subjects administered the polyribonucleotides.

[0224] In some embodiments, polyribonucleotides containing N4-acetylcytidine and / or 5-hydroxymethyluridine allow for repeated administration without loss of payload expression and / or therapeutic efficacy.

[0225] In some embodiments, polyribonucleotides containing N4-acetylcytidine and / or 5-hydroxymethyluridine allow for administration of high doses of polyribonucleotides without reducing payload expression and / or increasing immunogenicity. In some embodiments, the high doses of polyribonucleotides disclosed herein refer to doses of RNA therapeutics currently used in patients, e.g., RNA therapeutics approved by the FDA or used in clinical trials. For example, Damase TR et al., (2021) Front.Bioeng.Biotechnol., https: / / doi.org / 10.3389 / fbioe.2021.628137, incorporated herein by reference in its entirety, provides RNA therapeutics currently approved by the FDA or in clinical trials (see Table 1 therein). For the RNA therapeutics discussed in Damase 2021, one of skill in the art will understand that the approved doses of any of the RNA therapeutics can be obtained from the FDA-approved package for the agent or from the clinical trial website accessible at https: / / clinicaltrials.gov / . For example, the approved dose of eteplirsen is 30 mg / kg; the approved dose of patisiran is 0.3 mg / kg for patients weighing less than 100 kg and 30 mg for patients weighing 100 kg or more; and the approved dose of the COVID-19 vaccine mRNA-1273 (Moderna) is 100 micrograms.

[0226] In some embodiments, polyribonucleotides comprising N4-acetylcytidine and / or 5-hydroxymethyluridine allow for administration (e.g., repeated administration) of the polyribonucleotide about every hour, about every 2 hours, about every 3 hours, about every 4 hours, about every 5 hours, about every 6 hours, about every 8 hours, about every 10 hours, about every 12 hours, about every 24 hours, about every 36 hours, about every 48 hours, or about every 72 hours. In some embodiments, the polyribonucleotide is administered about every 1-72 hours, about every 2-72 hours, about every 3-72 hours, about every 4-72 hours, about every 5-72 hours, about every 6-72 hours, about every 8-72 hours, about every 10-72 hours, about every 12-72 hours, about every 24-72 hours, about every 36-72 hours, about every 48-72 hours, about every 1-48 hours, about every 1-36 hours, about every 1-24 hours, about every 1-12 hours, about every 1-10 hours, about every 1-8 hours, about every 1-6 hours, about every 1-5 hours, about every 1-4 hours, about every 1-3 hours, or about every 1-2 hours. In some embodiments, polyribonucleotides comprising N4-acetylcytidine and / or 5-hydroxymethyluridine allow for hourly, daily, weekly, monthly, or yearly administration (e.g., repeated administration) of polyribonucleotides.

[0227] Acetylated Nucleotides In particular, provided herein is a polyribonucleotide comprising one or more modified ribonucleotides comprising a nucleoside comprising an acetyl group. In some embodiments, the nucleoside of the modified ribonucleotide is N4-acetylcytidine, and the modified ribonucleotide has a 5' monophosphate, a 5' diphosphate, or a 5' triphosphate.

[0228] In some embodiments, the nucleoside of the modified ribonucleotide is N4-acetylcytidine and the modified ribonucleotide has the structure: [ka]

[0229] In some embodiments, the polyribonucleotide disclosed herein comprises cytidine residues. In some embodiments, at least 5% of the cytidine residues in the polyribonucleotide comprise N4-acetylcytidine. In some embodiments, less than 100% of the cytidine residues in the polyribonucleotide comprise N4-acetylcytidine.

[0230] In some embodiments, the polyribonucleotides disclosed herein comprise cytidine residues, wherein at least 5% of the cytidine residues in the polyribonucleotide comprise N4-acetylcytidine.

[0231] In some embodiments, the polyribonucleotides disclosed herein comprise cytidine residues, wherein at least 10% of the cytidine residues in the polyribonucleotide comprise N4-acetylcytidine.

[0232] In some embodiments, the polyribonucleotides disclosed herein comprise cytidine residues, wherein at least 15% of the cytidine residues in the polyribonucleotide comprise N4-acetylcytidine.

[0233] In some embodiments, the polyribonucleotides disclosed herein comprise cytidine residues, wherein at least 20% of the cytidine residues in the polyribonucleotide comprise N4-acetylcytidine.

[0234] In some embodiments, the polyribonucleotides disclosed herein comprise cytidine residues, wherein at least 25% of the cytidine residues in the polyribonucleotide comprise N4-acetylcytidine.

[0235] In some embodiments, the polyribonucleotides disclosed herein comprise cytidine residues, wherein at least 30% of the cytidine residues in the polyribonucleotide comprise N4-acetylcytidine.

[0236] In some embodiments, the polyribonucleotides disclosed herein comprise cytidine residues, wherein at least 35% of the cytidine residues in the polyribonucleotide comprise N4-acetylcytidine.

[0237] In some embodiments, the polyribonucleotides disclosed herein comprise cytidine residues, wherein at least 40% of the cytidine residues in the polyribonucleotide comprise N4-acetylcytidine.

[0238] In some embodiments, the polyribonucleotides disclosed herein comprise cytidine residues, wherein at least 45% of the cytidine residues in the polyribonucleotide comprise N4-acetylcytidine.

[0239] In some embodiments, the polyribonucleotides disclosed herein comprise cytidine residues, wherein at least 50% of the cytidine residues in the polyribonucleotide comprise N4-acetylcytidine.

[0240] In some embodiments, the polyribonucleotides disclosed herein comprise cytidine residues, wherein at least 55% of the cytidine residues in the polyribonucleotide comprise N4-acetylcytidine.

[0241] In some embodiments, the polyribonucleotides disclosed herein comprise cytidine residues, wherein at least 60% of the cytidine residues in the polyribonucleotide comprise N4-acetylcytidine.

[0242] In some embodiments, the polyribonucleotides disclosed herein comprise cytidine residues, wherein at least 65% of the cytidine residues in the polyribonucleotide comprise N4-acetylcytidine.

[0243] In some embodiments, the polyribonucleotides disclosed herein comprise cytidine residues, wherein at least 70% of the cytidine residues in the polyribonucleotide comprise N4-acetylcytidine.

[0244] In some embodiments, the polyribonucleotides disclosed herein comprise cytidine residues, wherein at least 75% of the cytidine residues in the polyribonucleotide comprise N4-acetylcytidine.

[0245] In some embodiments, the polyribonucleotides disclosed herein comprise cytidine residues, wherein at least 80% of the cytidine residues in the polyribonucleotide comprise N4-acetylcytidine.

[0246] In some embodiments, the polyribonucleotides disclosed herein comprise cytidine residues, wherein at least 85% of the cytidine residues in the polyribonucleotide comprise N4-acetylcytidine.

[0247] In some embodiments, the polyribonucleotides disclosed herein comprise cytidine residues, wherein at least 90% of the cytidine residues in the polyribonucleotide comprise N4-acetylcytidine.

[0248] In some embodiments, the polyribonucleotides disclosed herein comprise cytidine residues, wherein at least 95% of the cytidine residues in the polyribonucleotide comprise N4-acetylcytidine.

[0249] In some embodiments, the polyribonucleotides disclosed herein comprise 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 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 N4-acetylcytidine.

[0250] In some embodiments, the polyribonucleotides disclosed herein comprise cytidine residues, and more than about 60% of the cytidine residues in the polyribonucleotide comprise N4-acetylcytidine.

[0251] In some embodiments, the polyribonucleotides disclosed herein comprise cytidine residues, and greater than about 65% of the cytidine residues in the polyribonucleotide comprise N4-acetylcytidine.

[0252] In some embodiments, the polyribonucleotides disclosed herein comprise cytidine residues, and more than about 70% of the cytidine residues in the polyribonucleotide comprise N4-acetylcytidine.

[0253] In some embodiments, the polyribonucleotides disclosed herein comprise cytidine residues, and more than about 75% of the cytidine residues in the polyribonucleotide comprise N4-acetylcytidine.

[0254] In some embodiments, the polyribonucleotides disclosed herein comprise cytidine residues, and greater than about 80% of the cytidine residues in the polyribonucleotide comprise N4-acetylcytidine.

[0255] In some embodiments, the polyribonucleotides disclosed herein comprise cytidine residues, and greater than about 85% of the cytidine residues in the polyribonucleotide comprise N4-acetylcytidine.

[0256] In some embodiments, the polyribonucleotides disclosed herein comprise cytidine residues, and greater than about 90% of the cytidine residues in the polyribonucleotide comprise N4-acetylcytidine.

[0257] In some embodiments, the polyribonucleotides disclosed herein comprise cytidine residues, and greater than about 95% of the cytidine residues in the polyribonucleotide comprise N4-acetylcytidine.

[0258] In some embodiments, the polyribonucleotides disclosed herein comprise cytidine residues, and greater than about 99% of the cytidine residues in the polyribonucleotide comprise N4-acetylcytidine.

[0259] In some embodiments, the polyribonucleotides disclosed herein comprise cytidine residues, wherein about 5% of the cytidine residues in the polyribonucleotide comprise N4-acetylcytidine.

[0260] In some embodiments, the polyribonucleotides disclosed herein comprise cytidine residues, wherein about 10% of the cytidine residues in the polyribonucleotide comprise N4-acetylcytidine.

[0261] In some embodiments, the polyribonucleotides disclosed herein comprise cytidine residues, wherein about 15% of the cytidine residues in the polyribonucleotide comprise N4-acetylcytidine.

[0262] In some embodiments, the polyribonucleotides disclosed herein comprise cytidine residues, wherein about 20% of the cytidine residues in the polyribonucleotide comprise N4-acetylcytidine.

[0263] In some embodiments, the polyribonucleotides disclosed herein comprise cytidine residues, wherein about 25% of the cytidine residues in the polyribonucleotide comprise N4-acetylcytidine.

[0264] In some embodiments, the polyribonucleotides disclosed herein comprise cytidine residues, wherein about 30% of the cytidine residues in the polyribonucleotide comprise N4-acetylcytidine.

[0265] In some embodiments, the polyribonucleotides disclosed herein comprise cytidine residues, wherein about 35% of the cytidine residues in the polyribonucleotide comprise N4-acetylcytidine.

[0266] In some embodiments, the polyribonucleotides disclosed herein comprise cytidine residues, wherein about 40% of the cytidine residues in the polyribonucleotide comprise N4-acetylcytidine.

[0267] In some embodiments, the polyribonucleotides disclosed herein comprise cytidine residues, wherein about 45% of the cytidine residues in the polyribonucleotide comprise N4-acetylcytidine.

[0268] In some embodiments, the polyribonucleotides disclosed herein comprise cytidine residues, wherein about 50% of the cytidine residues in the polyribonucleotide comprise N4-acetylcytidine.

[0269] In some embodiments, the polyribonucleotides disclosed herein comprise cytidine residues, wherein about 55% of the cytidine residues in the polyribonucleotide comprise N4-acetylcytidine.

[0270] In some embodiments, the polyribonucleotides disclosed herein comprise cytidine residues, wherein about 60% of the cytidine residues in the polyribonucleotide comprise N4-acetylcytidine.

[0271] In some embodiments, the polyribonucleotides disclosed herein comprise cytidine residues, wherein about 65% of the cytidine residues in the polyribonucleotide comprise N4-acetylcytidine.

[0272] In some embodiments, the polyribonucleotides disclosed herein comprise cytidine residues, wherein about 75% of the cytidine residues in the polyribonucleotide comprise N4-acetylcytidine.

[0273] In some embodiments, the polyribonucleotides disclosed herein comprise cytidine residues, wherein about 80% of the cytidine residues in the polyribonucleotide comprise N4-acetylcytidine.

[0274] In some embodiments, the polyribonucleotides disclosed herein comprise cytidine residues, wherein about 85% of the cytidine residues in the polyribonucleotide comprise N4-acetylcytidine.

[0275] In some embodiments, the polyribonucleotides disclosed herein comprise cytidine residues, wherein about 90% of the cytidine residues in the polyribonucleotide comprise N4-acetylcytidine.

[0276] In some embodiments, the polyribonucleotides disclosed herein comprise cytidine residues, wherein about 95% of the cytidine residues in the polyribonucleotide comprise N4-acetylcytidine.

[0277] In some embodiments, the polyribonucleotides disclosed herein comprise cytidine residues, wherein about 99% of the cytidine residues in the polyribonucleotide comprise N4-acetylcytidine.

[0278] In some embodiments, the polyribonucleotides disclosed herein comprise cytidine residues, wherein 100% of the cytidine residues in the polyribonucleotide comprise N4-acetylcytidine.

[0279] In some embodiments, a polyribonucleotide disclosed herein (e.g., a polyribonucleotide comprising cytidine residues, wherein about 5-100% of the cytidine residues comprise N4-acetylcytidine) comprises one or more additional modified ribonucleotides. In some embodiments, the one or more additional modified ribonucleotides comprise a nucleoside selected from adenosine, guanosine, cytidine or uridine, or a combination thereof. In some embodiments, the one or more additional modified ribonucleotides comprise a 5-hydroxymethyl group. In some embodiments, the one or more additional modified ribonucleotides comprise 5-hydroxymethyluridine. In some embodiments, 5% to 100% of the uridine residues in a polyribonucleotide comprising uridine are 5-hydroxymethyluridine.

[0280] In some embodiments, the polyribonucleotide can have a length of at least 5 nucleotides or more. In some embodiments, the polyribonucleotide can 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.

[0281] 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 It can have a length of about 85 nucleotides, about 5 nucleotides to about 80 nucleotides, about 5 nucleotides to about 75 nucleotides, about 5 nucleotides to about 70 nucleotides, about 5 nucleotides to about 65 nucleotides, about 5 nucleotides to about 60 nucleotides, about 5 nucleotides to about 55 nucleotides, about 5 nucleotides to about 50 nucleotides, about 5 nucleotides to about 45 nucleotides, about 5 nucleotides to about 40 nucleotides, about 5 nucleotides to about 5 nucleotides 35 nucleotides, about 5 nucleotides to about 30 nucleotides, about 5 nucleotides to about 25 nucleotides, about 5 nucleotides to about 20 nucleotides, about 5 nucleotides to about 15 nucleotides, or about 5 nucleotides to about 10 nucleotides.

[0282] In some embodiments, the polyribonucleotide is from about 5 nucleotides to about 200,000 nucleotides, from about 10 nucleotides to about 200,000 nucleotides, from 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, from about 1000 nucleotides to about It can have a length of 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.

[0283] 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.

[0284] 5-Hydroxymethyl modified nucleotides In particular, provided herein are polyribonucleotides that include one or more modified ribonucleotides that include a nucleoside that includes a 5-hydroxymethyl group. In some embodiments, the nucleoside of the modified ribonucleotide is 5-hydroxymethyluridine, and the modified ribonucleotide has a 5' monophosphate, a 5' diphosphate, or a 5' triphosphate.

[0285] In some embodiments, the nucleoside of the modified ribonucleotide is 5-hydroxymethyluridine, and the modified ribonucleotide has the structure: [ka]

[0286] In some embodiments, the polyribonucleotides disclosed herein comprise uridine residues. In some embodiments, at least 5% of the uridine residues in the polyribonucleotide comprise 5-hydroxymethyluridine. In some embodiments, less than 100% of the uridine residues in the polyribonucleotide comprise 5-hydroxymethyluridine.

[0287] In some embodiments, the polyribonucleotides disclosed herein comprise uridine residues, wherein at least 5% of the uridine residues in the polyribonucleotide comprise 5-hydroxymethyluridine.

[0288] In some embodiments, the polyribonucleotides disclosed herein comprise uridine residues, wherein at least 10% of the uridine residues in the polyribonucleotide comprise 5-hydroxymethyluridine.

[0289] In some embodiments, the polyribonucleotides disclosed herein comprise uridine residues, wherein at least 15% of the uridine residues in the polyribonucleotide comprise 5-hydroxymethyluridine.

[0290] In some embodiments, the polyribonucleotides disclosed herein comprise uridine residues, wherein at least 20% of the uridine residues in the polyribonucleotide comprise 5-hydroxymethyluridine.

[0291] In some embodiments, the polyribonucleotides disclosed herein comprise uridine residues, wherein at least 25% of the uridine residues in the polyribonucleotide comprise 5-hydroxymethyluridine.

[0292] In some embodiments, the polyribonucleotides disclosed herein comprise uridine residues, wherein at least 30% of the uridine residues in the polyribonucleotide comprise 5-hydroxymethyluridine.

[0293] In some embodiments, the polyribonucleotides disclosed herein comprise uridine residues, wherein at least 35% of the uridine residues in the polyribonucleotide comprise 5-hydroxymethyluridine.

[0294] In some embodiments, the polyribonucleotides disclosed herein comprise uridine residues, wherein at least 40% of the uridine residues in the polyribonucleotide comprise 5-hydroxymethyluridine.

[0295] In some embodiments, the polyribonucleotides disclosed herein comprise uridine residues, wherein at least 45% of the uridine residues in the polyribonucleotide comprise 5-hydroxymethyluridine.

[0296] In some embodiments, the polyribonucleotides disclosed herein comprise uridine residues, wherein at least 50% of the uridine residues in the polyribonucleotide comprise 5-hydroxymethyluridine.

[0297] In some embodiments, the polyribonucleotides disclosed herein comprise uridine residues, wherein at least 55% of the uridine residues in the polyribonucleotide comprise 5-hydroxymethyluridine.

[0298] In some embodiments, the polyribonucleotides disclosed herein comprise uridine residues, wherein at least 60% of the uridine residues in the polyribonucleotide comprise 5-hydroxymethyluridine.

[0299] In some embodiments, the polyribonucleotides disclosed herein comprise uridine residues, wherein at least 65% of the uridine residues in the polyribonucleotide comprise 5-hydroxymethyluridine.

[0300] In some embodiments, the polyribonucleotides disclosed herein comprise uridine residues, wherein at least 70% of the uridine residues in the polyribonucleotide comprise 5-hydroxymethyluridine.

[0301] In some embodiments, the polyribonucleotides disclosed herein comprise uridine residues, wherein at least 75% of the uridine residues in the polyribonucleotide comprise 5-hydroxymethyluridine.

[0302] In some embodiments, the polyribonucleotides disclosed herein comprise uridine residues, wherein at least 80% of the uridine residues in the polyribonucleotide comprise 5-hydroxymethyluridine.

[0303] In some embodiments, the polyribonucleotides disclosed herein comprise uridine residues, wherein at least 85% of the uridine residues in the polyribonucleotide comprise 5-hydroxymethyluridine.

[0304] In some embodiments, the polyribonucleotides disclosed herein comprise uridine residues, wherein at least 90% of the uridine residues in the polyribonucleotide comprise 5-hydroxymethyluridine.

[0305] In some embodiments, the polyribonucleotides disclosed herein comprise uridine residues, wherein at least 95% of the uridine residues in the polyribonucleotide comprise 5-hydroxymethyluridine.

[0306] In some embodiments, the polyribonucleotides disclosed herein comprise uridine residues, wherein at least 99% of the uridine residues in the polyribonucleotide comprise 5-hydroxymethyluridine.

[0307] In some embodiments, the polyribonucleotides disclosed herein contain uridine 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 99% of the uridine residues in the polyribonucleotide. ~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% contains 5-hydroxymethyluridine.

[0308] In some embodiments, the polyribonucleotides disclosed herein comprise uridine residues, and greater than 60% of the uridine residues in the polyribonucleotide comprise 5-hydroxymethyluridine.

[0309] In some embodiments, the polyribonucleotides disclosed herein comprise uridine residues, and greater than 65% of the uridine residues in the polyribonucleotide comprise 5-hydroxymethyluridine.

[0310] In some embodiments, the polyribonucleotides disclosed herein comprise uridine residues, and greater than 70% of the uridine residues in the polyribonucleotide comprise 5-hydroxymethyluridine.

[0311] In some embodiments, the polyribonucleotides disclosed herein comprise uridine residues, and greater than 75% of the uridine residues in the polyribonucleotide comprise 5-hydroxymethyluridine.

[0312] In some embodiments, the polyribonucleotides disclosed herein comprise uridine residues, and greater than 80% of the uridine residues in the polyribonucleotide comprise 5-hydroxymethyluridine.

[0313] In some embodiments, the polyribonucleotides disclosed herein comprise uridine residues, and greater than 85% of the uridine residues in the polyribonucleotide comprise 5-hydroxymethyluridine.

[0314] In some embodiments, the polyribonucleotides disclosed herein comprise uridine residues, and greater than 90% of the uridine residues in the polyribonucleotide comprise 5-hydroxymethyluridine.

[0315] In some embodiments, the polyribonucleotides disclosed herein comprise uridine residues, and greater than 95% of the uridine residues in the polyribonucleotide comprise 5-hydroxymethyluridine.

[0316] In some embodiments, the polyribonucleotides disclosed herein comprise uridine residues, and greater than 99% of the uridine residues in the polyribonucleotide comprise 5-hydroxymethyluridine.

[0317] In some embodiments, the polyribonucleotides disclosed herein comprise uridine residues, wherein about 5% of the uridine residues in the polyribonucleotide comprise 5-hydroxymethyluridine.

[0318] In some embodiments, the polyribonucleotides disclosed herein comprise uridine residues, wherein about 10% of the uridine residues in the polyribonucleotide comprise 5-hydroxymethyluridine.

[0319] In some embodiments, the polyribonucleotides disclosed herein comprise uridine residues, wherein about 15% of the uridine residues in the polyribonucleotide comprise 5-hydroxymethyluridine.

[0320] In some embodiments, the polyribonucleotides disclosed herein comprise uridine residues, wherein about 20% of the uridine residues in the polyribonucleotide comprise 5-hydroxymethyluridine.

[0321] In some embodiments, the polyribonucleotides disclosed herein comprise uridine residues, wherein about 25% of the uridine residues in the polyribonucleotide comprise 5-hydroxymethyluridine.

[0322] In some embodiments, the polyribonucleotides disclosed herein comprise uridine residues, wherein about 30% of the uridine residues in the polyribonucleotide comprise 5-hydroxymethyluridine.

[0323] In some embodiments, the polyribonucleotides disclosed herein comprise uridine residues, wherein about 35% of the uridine residues in the polyribonucleotide comprise 5-hydroxymethyluridine.

[0324] In some embodiments, the polyribonucleotides disclosed herein comprise uridine residues, wherein about 40% of the uridine residues in the polyribonucleotide comprise 5-hydroxymethyluridine.

[0325] In some embodiments, the polyribonucleotides disclosed herein comprise uridine residues, wherein about 45% of the uridine residues in the polyribonucleotide comprise 5-hydroxymethyluridine.

[0326] In some embodiments, the polyribonucleotides disclosed herein comprise uridine residues, wherein about 50% of the uridine residues in the polyribonucleotide comprise 5-hydroxymethyluridine.

[0327] In some embodiments, the polyribonucleotides disclosed herein comprise uridine residues, wherein about 55% of the uridine residues in the polyribonucleotide comprise 5-hydroxymethyluridine.

[0328] In some embodiments, the polyribonucleotides disclosed herein comprise uridine residues, wherein about 60% of the uridine residues in the polyribonucleotide comprise 5-hydroxymethyluridine.

[0329] In some embodiments, the polyribonucleotides disclosed herein comprise uridine residues, wherein about 65% of the uridine residues in the polyribonucleotide comprise 5-hydroxymethyluridine.

[0330] In some embodiments, the polyribonucleotides disclosed herein comprise uridine residues, wherein about 75% of the uridine residues in the polyribonucleotide comprise 5-hydroxymethyluridine.

[0331] In some embodiments, the polyribonucleotides disclosed herein comprise uridine residues, wherein about 80% of the uridine residues in the polyribonucleotide comprise 5-hydroxymethyluridine.

[0332] In some embodiments, the polyribonucleotides disclosed herein comprise uridine residues, wherein about 85% of the uridine residues in the polyribonucleotide comprise 5-hydroxymethyluridine.

[0333] In some embodiments, the polyribonucleotides disclosed herein comprise uridine residues, wherein about 90% of the uridine residues in the polyribonucleotide comprise 5-hydroxymethyluridine.

[0334] In some embodiments, the polyribonucleotides disclosed herein comprise uridine residues, wherein about 95% of the uridine residues in the polyribonucleotide comprise 5-hydroxymethyluridine.

[0335] In some embodiments, the polyribonucleotides disclosed herein comprise uridine residues, wherein about 99% of the uridine residues in the polyribonucleotide comprise 5-hydroxymethyluridine.

[0336] In some embodiments, the polyribonucleotides disclosed herein comprise uridine residues, wherein 100% of the uridine residues in the polyribonucleotide comprise 5-hydroxymethyluridine.

[0337] In some embodiments, a polyribonucleotide disclosed herein (e.g., a polyribonucleotide comprising uridine residues in which about 5-100% of the uridine residues comprise 5-hydroxymethyluridine) comprises one or more additional modified ribonucleotides other than 5-hydroxymethyluridine. In some embodiments, the one or more additional modified ribonucleotides comprise a nucleoside selected from adenosine, guanosine, cytidine or uridine, or a combination thereof. In some embodiments, the one or more additional modified ribonucleotides comprise an acetyl group. In some embodiments, the one or more additional modified ribonucleotides comprise N4-acetylcytidine. In some embodiments, 5% to 100% of the cytidine residues in a polyribonucleotide comprising cytidine are N4-acetylcytidine.

[0338] In some embodiments, the polyribonucleotide can have a length of at least 5 nucleotides or more. In some embodiments, the polyribonucleotide can 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.

[0339] 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 It can have a length of about 85 nucleotides, about 5 nucleotides to about 80 nucleotides, about 5 nucleotides to about 75 nucleotides, about 5 nucleotides to about 70 nucleotides, about 5 nucleotides to about 65 nucleotides, about 5 nucleotides to about 60 nucleotides, about 5 nucleotides to about 55 nucleotides, about 5 nucleotides to about 50 nucleotides, about 5 nucleotides to about 45 nucleotides, about 5 nucleotides to about 40 nucleotides, about 5 nucleotides to about 5 nucleotides 35 nucleotides, about 5 nucleotides to about 30 nucleotides, about 5 nucleotides to about 25 nucleotides, about 5 nucleotides to about 20 nucleotides, about 5 nucleotides to about 15 nucleotides, or about 5 nucleotides to about 10 nucleotides.

[0340] In some embodiments, the polyribonucleotide is from about 5 nucleotides to about 200,000 nucleotides, from about 10 nucleotides to about 200,000 nucleotides, from 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, from about 1000 nucleotides to about It can have a length of 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.

[0341] 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.

[0342] composition In particular, the present disclosure provides compositions. The compositions disclosed herein include one or more polyribonucleotides that include one or more modified ribonucleotides that include a base that includes an acetyl group and / or a 5-hydroxymethyl group. In some embodiments, the nucleoside of the modified ribonucleotide is N4-acetylcytidine. In some embodiments, the nucleoside of the modified ribonucleotide is 5-hydroxymethyluridine.

[0343] In some embodiments, a composition comprising one or more modified ribonucleotides, eg, one or more polyribonucleotides comprising N4-acetylcytidine and / or 5-hydroxymethyluridine, is a pharmaceutical composition.

[0344] In some embodiments, a composition comprising one or more modified ribonucleotides, e.g., one or more polyribonucleotides comprising N4-acetylcytidine and / or 5-hydroxymethyluridine, is or comprises an immunogenic composition. An immunogenic composition is a composition that induces an immune response. In some embodiments, an immunogenic composition comprising one or more polyribonucleotides does not itself induce an immune response, but rather, the one or more polyribonucleotides encode, for example, one or more polypeptides that induce an immune response.

[0345] In some embodiments, the composition comprising one or more modified ribonucleotides, eg, one or more polyribonucleotides comprising N4-acetylcytidine and / or 5-hydroxymethyluridine, is a vaccine.

[0346] In some embodiments, the composition comprising one or more modified ribonucleotides, eg, one or more polyribonucleotides comprising N4-acetylcytidine and / or 5-hydroxymethyluridine, is or comprises an antibody therapy.

[0347] In some embodiments, a composition comprising one or more modified ribonucleotides, eg, one or more polyribonucleotides comprising N4-acetylcytidine and / or 5-hydroxymethyluridine, is or comprises an immunomodulatory therapy.

[0348] In some embodiments, a composition comprising one or more modified ribonucleotides, eg, one or more polyribonucleotides comprising N4-acetylcytidine and / or 5-hydroxymethyluridine, is or comprises a gene therapy.

[0349] In some embodiments, the composition comprising one or more modified ribonucleotides, eg, one or more polyribonucleotides comprising N4-acetylcytidine and / or 5-hydroxymethyluridine, is or comprises a chemotherapy.

[0350] In some embodiments, a composition comprising one or more modified ribonucleotides, eg, one or more polyribonucleotides comprising N4-acetylcytidine and / or 5-hydroxymethyluridine, is or comprises a protein replacement therapy.

[0351] In some embodiments, a composition comprising one or more modified ribonucleotides, eg, one or more polyribonucleotides comprising N4-acetylcytidine and / or 5-hydroxymethyluridine, is or comprises an immunotherapy.

[0352] In some embodiments, a composition comprising one or more modified ribonucleotides, eg, one or more polyribonucleotides comprising N4-acetylcytidine and / or 5-hydroxymethyluridine, is or comprises a cell engineering therapy.

[0353] In some embodiments, a composition comprising one or more polyribonucleotides comprising one or more modified ribonucleotides, e.g., N4-acetylcytidine and / or 5-hydroxymethyluridine, comprises double-stranded RNA. In some embodiments, a composition comprising one or more polyribonucleotides comprising one or more modified ribonucleotides, e.g., N4-acetylcytidine and / or 5-hydroxymethyluridine, does not comprise double-stranded RNA.

[0354] In some embodiments, compositions comprising the polyribonucleotides disclosed herein are characterized by observed reduced immunogenicity when assessed in cells, tissues, or organisms administered the polyribonucleotides, as compared to a suitable reference comparator. In some embodiments, the reference comparator comprises an otherwise similar cell, tissue, or organism administered a composition comprising a comparable polyribonucleotide that comprises fewer acetyl groups and / or fewer 5-hydroxymethyl groups on the nucleobases. In some embodiments, the comparable polyribonucleotide is a polyribonucleotide that comprises (i) fewer N4-acetylcytidine nucleosides (e.g., no N4-acetylcytidine nucleosides); and / or (ii) fewer 5-hydroxymethyluridine nucleosides (e.g., no 5-hydroxymethyluridine nucleosides) compared to the polyribonucleotides disclosed herein. In some embodiments, reduced immunogenicity comprises reduced activation of innate immune response-induced toxicity. In some embodiments, the reduced activation of the immune response comprises reduced activation of NFkb or the NFkb pathway; IRF or the IRF pathway; and / or other inflammatory cytokines in a cell, tissue, or organism. In some embodiments, the reduced activation of the immune response comprises reduced detection of uncapped RNA by a molecular sensor, e.g., RIG-I.

[0355] In some embodiments, the reduced immunogenicity allows for repeated administration of a composition comprising a polyribonucleotide disclosed herein to a cell, tissue, or subject, for example, at least two administrations. 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 a dose of the composition that is the same as that of a previous administration of the composition. In some embodiments, the repeated administration comprises administration of a dose of the composition that is different from that of a previous administration of the composition.

[0356] In some embodiments, the repeated administration of the compositions 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.

[0357] 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 as compared to a first dose of a composition comprising a polyribonucleotide disclosed herein.

[0358] 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 comprising fewer acetyl groups on the nucleobases than the polyribonucleotides in the composition. In some embodiments, the reference standard comprises a comparable polyribonucleotide comprising fewer acetyl groups and / or fewer 5-hydroxymethyl groups on the nucleobases. In some embodiments, the comparable polyribonucleotide is a polyribonucleotide comprising (i) fewer N4-acetylcytidine nucleosides (e.g., no N4-acetylcytidine nucleosides); and / or (ii) fewer 5-hydroxymethyluridine nucleosides (e.g., no 5-hydroxymethyluridine nucleosides) compared to the polyribonucleotides disclosed herein.

[0359] In some embodiments, compositions comprising the polyribonucleotides disclosed herein are characterized by observing higher cell viability when assessed in cells, tissues, or organisms administered the polyribonucleotides, as compared to a suitable reference comparator. In some embodiments, the reference comparator is the cell viability of cells, tissues, or organisms administered a comparable polyribonucleotide that contains fewer acetyl groups and / or fewer 5-hydroxymethyl groups on the nucleobases. In some embodiments, the comparable polyribonucleotide is a polyribonucleotide that contains (i) fewer N4-acetylcytidine nucleosides (e.g., no N4-acetylcytidine nucleosides); and / or (ii) fewer 5-hydroxymethyluridine nucleosides (e.g., no 5-hydroxymethyluridine nucleosides) compared to the polyribonucleotides disclosed herein.

[0360] 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.

[0361] In some embodiments, cell viability is a measure of the number of cells in a cell, tissue or subject that are alive at one or more time points.

[0362] In some embodiments, the compositions disclosed herein are or comprise in vitro transcribed polyribonucleotides comprising modified ribonucleotides disclosed herein.

[0363] In some embodiments, a composition disclosed herein is or comprises an expression vector comprising one or more polynucleotides disclosed herein.

[0364] In some embodiments, the compositions disclosed herein comprise a polyribonucleotide comprising one or more modified ribonucleotides disclosed herein.

[0365] 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, where a first polyribonucleotide comprises a first modified ribonucleotide and a second polyribonucleotide comprises 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.

[0366] 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.

[0367] 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.

[0368] 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.

[0369] Pharmaceutical Compositions In some embodiments, the composition comprising one or more modified ribonucleotides, such as one or more polyribonucleotides comprising N4-acetylcytidine and / or 5-hydroxymethyluridine, 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.

[0370] In some embodiments, the pharmaceutical composition may include a pharma- ceutically acceptable carrier or excipient, which as used herein includes 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, etc., suitable 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 formulating 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, or others, dextrose, fatty acid esters, etc., and combinations thereof.

[0371] If desired, the pharmaceutical composition can be mixed with auxiliary agents (e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, coloring agents, flavoring agents, and / or aromatic agents) that do not adversely react with the active compounds or interfere with their activity. In certain embodiments, water-soluble carriers suitable for intravenous administration are used. In some embodiments, the pharmaceutical composition can be sterile.

[0372] Suitable pharmaceutical compositions can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents, if desired.The pharmaceutical composition can be a liquid solution, suspension, or emulsion.

[0373] The pharmaceutical composition may be formulated according to routine procedures as a pharmaceutical composition adapted for administration to humans. The formulation of the pharmaceutical composition must be suitable for the mode of administration. For example, in some embodiments, a composition for intravenous administration is usually a solution in a 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 in unit dosage form, for example as a dry lyophilized powder or water-free concentrate in an air-sealed container such as an ampoule or sachet indicating the quantity of active agent, or are mixed together. When the pharmaceutical composition is administered by infusion, the pharmaceutical composition may be dispensed in an infusion bottle containing sterile pharmaceutical grade water, saline or dextrose / water. When the pharmaceutical composition is administered by injection, an ampoule of sterile water for injection or saline may be provided so that the ingredients may be mixed prior to administration.

[0374] Although 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 of skill in the art that such compositions are generally suitable for administration to any type of animal or in vitro or ex vivo cell. Modifications of pharmaceutical compositions suitable for administration to humans to make them suitable for administration to a variety of animals or in vitro or ex vivo cells are well understood and can be designed and / or implemented by those of skill in the art, e.g., veterinary pharmacologists, with no more than routine experimentation, if any.

[0375] 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 bringing into association 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 multiple dosage unit.

[0376] The pharmaceutical compositions of 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.

[0377] RNA preparations In particular, provided herein are compositions comprising polyribonucleotides comprising N4-acetylcytidine and / or 5-hydroxymethyluridine, and formulations thereof. In some embodiments, the compositions comprising polyribonucleotides disclosed herein are formulated in lipid nanoparticle (LNP) formulations.

[0378] In some embodiments, the polyribonucleotides disclosed herein encode polypeptides. In some embodiments, the polyribonucleotides disclosed herein are messenger RNA or comprise messenger RNA. In some embodiments, the compositions comprising polyribonucleotides comprising messenger RNA are formulated in lipid nanoparticle (LNP) formulations.

[0379] In some embodiments, the polyribonucleotide disclosed herein is or comprises a gRNA. In some embodiments, a composition comprising a polyribonucleotide comprising a gRNA is formulated in a lipid nanoparticle (LNP) formulation.

[0380] In some embodiments, the polyribonucleotides disclosed herein are or comprise an inhibitory RNA. In some embodiments, compositions comprising polyribonucleotides comprising an inhibitory RNA are formulated in lipid nanoparticle (LNP) formulations.

[0381] In some embodiments, the polyribonucleotide disclosed herein is or comprises a miRNA or siRNA. In some embodiments, the composition comprising a polyribonucleotide comprising a miRNA or siRNA is formulated in a lipid nanoparticle (LNP) formulation.

[0382] In some embodiments, the polyribonucleotides disclosed herein are or comprise antisense oligonucleotides. In some embodiments, compositions comprising polyribonucleotides, including antisense oligonucleotides, are formulated in lipid nanoparticle (LNP) formulations.

[0383] In some embodiments, the present disclosure provides LNP formulations comprising the polyribonucleotides disclosed herein for use in pharmaceutical compositions, eg, immunogenic compositions.

[0384] Methods of using the compositions disclosed herein The present disclosure provides, inter alia, methods of using the polyribonucleotides disclosed herein or compositions comprising same.

[0385] 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.

[0386] In some embodiments, provided herein is a method of vaccination comprising administering to a cell, tissue, or subject a polyribonucleotide disclosed herein, or a composition comprising a polyribonucleotide disclosed herein.

[0387] In some embodiments, provided herein is a method of antibody 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 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 bicomponent 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.

[0388] In some embodiments, disclosed herein is an immunomodulatory 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 immunomodulatory therapy comprises a cytokine or a variant or fragment thereof, a chemokine or a variant or fragment thereof, a T cell modulator, a NK cell modulator, a B cell modulator, a myeloid cell modulator, a modulator of other immune cells, or a 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 (CAR) therapy.

[0389] 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, e.g., a guide RNA and / or a Cas polypeptide.

[0390] In some embodiments, provided herein is a method 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.

[0391] 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.

[0392] 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, In some embodiments, the methods of immunotherapy comprise delivery of an immune modulatory therapy and / or an immune checkpoint therapy.

[0393] In some embodiments, disclosed herein are methods of protein replacement 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 protein replacement therapy comprises delivery of enzyme replacement therapy.

[0394] In some embodiments, provided herein is a method of chemotherapy comprising administering to a cell, tissue, or subject a polyribonucleotide disclosed herein, or a composition comprising a polyribonucleotide disclosed herein.

[0395] In some embodiments, the methods or uses disclosed herein include determining 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.

[0396] In some embodiments, a cell, tissue, or subject administered with a polyribonucleotide or a composition comprising the same exhibits improved cell viability compared to a reference cell viability. In some embodiments, the reference cell viability is the cell viability of a cell, tissue, or organism administered with a comparable polyribonucleotide or a composition comprising the same that comprises fewer acetyl groups and / or fewer 5-hydroxymethyl groups on the nucleobases. In some embodiments, the comparable polyribonucleotide comprises (i) fewer N4-acetylcytidine nucleosides (e.g., no N4-acetylcytidine nucleosides); and / or (ii) fewer 5-hydroxymethyluridine nucleosides (e.g., no 5-hydroxymethyluridine nucleosides) compared to the polyribonucleotides disclosed herein.

[0397] In some embodiments, the methods or uses disclosed herein further comprise determining the immune system response of a cell, tissue or subject to which the polyribonucleotide or composition comprising the same has been administered. In some embodiments, the immune response comprises an innate immune system response, including 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 a combination thereof, in the cell, tissue or subject. In some embodiments, determining the innate immune system response comprises determining the level of detection of uncapped RNA in the cell, tissue or subject.

[0398] In some embodiments, the cells, tissues, or subjects to which the polyribonucleotide or composition comprising the same is administered show a reduced innate immune system response compared to a reference. In some embodiments, the reference is the innate immune system response of the cells, tissues, or subjects to which a comparable polyribonucleotide or composition comprising the same is administered that contains fewer acetyl groups and / or fewer 5-hydroxymethyl groups on the nucleobases. In some embodiments, the comparable polyribonucleotide is a polyribonucleotide that contains (i) fewer N4-acetylcytidine nucleosides (e.g., no N4-acetylcytidine nucleosides); and / or (ii) fewer 5-hydroxymethyluridine nucleosides (e.g., no 5-hydroxymethyluridine nucleosides) compared to the polyribonucleotides disclosed herein.

[0399] 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.

[0400] In some embodiments, determining the effectiveness includes determining an antibody response or cellular response in a cell, tissue, or subject. In some embodiments, the cell, tissue, or subject to which the polyribonucleotide or composition comprising the same is administered shows a higher antibody response or cellular response compared to a reference. In some embodiments, the reference is the antibody response or cellular response of a cell, tissue, or subject to which a comparable polyribonucleotide or composition comprising the same is administered that contains (i) less N4-acetylcytidine nucleosides (e.g., no N4-acetylcytidine nucleosides); and / or (ii) less 5-hydroxymethyluridine nucleosides (e.g., no 5-hydroxymethyluridine nucleosides) compared to the polyribonucleotides disclosed herein.

[0401] 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.

[0402] In some embodiments, the methods or uses disclosed herein comprise administering multiple doses of polyribonucleotide or a composition comprising the same to a cell, tissue or subject.In some embodiments, the second or subsequent doses of polyribonucleotide or a composition comprising the same have substantially similar efficacy in a cell, tissue or subject compared to administering the first dose of a composition comprising polyribonucleotide.

[0403] 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 comprising fewer acetyl groups and / or fewer 5-hydroxymethyl groups on the nucleobases. In some embodiments, the comparable polyribonucleotide comprises (i) fewer N4-acetylcytidine nucleosides (e.g., no N4-acetylcytidine nucleosides); and / or (ii) fewer 5-hydroxymethyluridine nucleosides (e.g., no 5-hydroxymethyluridine nucleosides) than the polyribonucleotides disclosed herein.

[0404] 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.

[0405] In some embodiments of any of the methods or uses disclosed herein, the cell is a mammalian cell.

[0406] In some embodiments of any of the methods or uses disclosed herein, the tissue is mammalian tissue.

[0407] In some embodiments of any of the methods disclosed herein, the subject is a mammal. In some embodiments, the mammal is a human.

[0408] kit Another aspect of the present disclosure further provides a pharmaceutical pack or kit. In some embodiments, the kit can include a polyribonucleotide or composition as described herein. In some embodiments, the kit can be used in any applicable method, such as the methods described herein. EXAMPLES

[0409] Example 1: Reducing immunogenicity and improving efficacy of RNA containing N4-acetylcytidine This example demonstrates that the use of RNA containing N4-acetylcytidine (Ac4C) instead of natural cytidine can reduce undesirable immunogenicity associated with in vitro transcribed RNA.

[0410] method

[0411] Generation of IVT templates: For experiments using Luc2 RNA, the luc2 gene encoding an optimized version of firefly luciferase was amplified from pGL4.10[luc2] (Promega). Amplifications were performed in 20 μL reactions consisting of 0.25 μM of each primer Luc2_fwd and Luc2_rev, 1× Herculase II buffer, 25 mM of each dNTP, 30 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. PCR products were purified using SpriSelect beads (Beckman Coulter) at a 0.8-fold ratio relative to the PCR reaction volume and eluted in 45 μL of nuclease-free water. 42.5 μL of the eluted product was subjected to treatment with 125 U of Dpn1 enzyme (New England Biolabs) in a 50 μL reaction to digest the template plasmid. The digested product was purified using SpriSelect beads (Beckman Coulter) at a 0.65x ratio to digest reaction volume and eluted in 40 μL of nuclease-free water. This digested primary PCR product was then amplified at 50°C in a 20 μL reaction consisting of 0.25 μM of each primer T7-AGG_fwd and 120 pA_rev, 1x Herculase II buffer, 25 mM of each dNTP, 10 ng of Luc2 primary amplicon, and 0.4 μL of Herculase II enzyme. This secondary PCR product was cleaned up using SpriSelect beads (BeckmanCoulter) at a 0.8x ratio to PCR reaction volume and eluted in 10 mM Tris-HCl pH 8.5.

[0412] The sequences of the primers used were as follows: Luc2_fwd: CTTGTTCTTT TTGCAGAAGC TCAGAATAAA CGCTCAACTT TGGCCACCat ggaagatgcc aaaaacatta agaagggc (SEQ ID NO: 1)

[0413] Luc2_rev AGAATGTGAA GAAACTTTCT TTTTATTAGG AGCAGATACG AATGGCTACA TTTTGGGGGA CAACATTTTG TAAAGTGTAA GTTGGTATTA TGTAGCTTAG AGACTCCATT CGGGTGTTCT TGAGGCTGGT CTATCATTAc acggcgatct tgccgcc (SEQ ID NO: 2)

[0414] T7-AGG_fwd gaattTAATA CGACTCACTA TAAGGcttgt tctttttgca gaagc (SEQ ID NO: 3)

[0415] 120pA_rev TTTTTTTTTT TTTTTTTTTT TTTTTTTTTT TTTTTTTTTT TTTTTTTTTT TTTTTTTTTT TTTTTTTTTT TTTTTTTTTT TTTTTTTTTT TTTTTTTTTT TTTTTTTTTT TTTTTTTTTT agaatgtgaa gaaactttct ttttattag (SEQ ID NO: 4)

[0416] For experiments investigating RNA vaccine candidates, DNA sequences encoding the designed candidates were ordered as gblocks (IDT). Gblocks were resuspended at a concentration of 20ng / μL in 10mM Tris-HCl pH 8.5. T7 templates were generated by PCR amplification at 50°C in a 20μL reaction consisting of 0.25μM of each primer T7-AGG_fwd and 120pA_rev, 1× Herculase II buffer, 25mM of each dNTP, 10ng of gblock, and 0.4μL of Herculase II enzyme. The PCR products were cleaned up using SpriSelect beads (BeckmanCoulter) at a 0.8x ratio to the PCR reaction volume and eluted in 10mM Tris-HCl pH 8.5.

[0417] The sequences of the primers used were as follows:

[0418] T7-AGG_fwd gaattTAATA CGACTCACTA TAAGGcttgt tctttttgca gaagc (SEQ ID NO: 3)

[0419] 120pA_rev TTTTTTTTTT TTTTTTTTTT TTTTTTTTTT TTTTTTTTTT TTTTTTTTTT TTTTTTTTTT TTTTTTTTTT TTTTTTTTTT TTTTTTTTTT TTTTTTTTTT TTTTTTTTTT TTTTTTTTTT agaatgtgaa gaaactttct ttttattag (SEQ ID NO: 4)

[0420] In vitro transcription (IVT) of Luc2 RNA for A549 assay: Luc2 RNA was synthesized in a 20 μL IVT reaction consisting of 200 ng Luc2T7 template, 20 mM MgCl2, 7.5 mM each NTP, 7.5 mM CleanCapAG (TriLink), 1× HiScribe transcription buffer, and 2 uL HiScribe polymerase mix (NEB) and incubated for 1 h at 37° C. To examine the effect of a gradient of increasing substitution percentages with N4-acetylcytidine, the corresponding percentages of CTP were replaced with Ac4CTP (Jena BioScience) in the IVT mixture.

[0421] All IVT products were cleaned up using Monarch 500ug RNA Cleanup Kit (NEB) and eluted in 88μL nuclease free water. The eluted products were then digested in a 100μL reaction consisting of 1× DNase I buffer and 10U DNase I (RNase free) (New England Biolabs) at 37°C for 10 minutes to degrade the DNA template. The DNase I treated samples were cleaned up using Monarch 500ug RNA Cleanup Kit (NEB) and eluted in 88μL nuclease free water.

[0422] The DNase I-treated products, containing the Cap1 structure added co-transcriptionally, were treated with 1x CutSmart buffer and 25 U of Quick CIP (NEB) 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.

[0423] The Quick CIP treated RNA was cleaned up using Monarch 500ug RNA Cleanup Kit (NEB) and eluted in 1mM sodium citrate, pH 6.5.

[0424] RNA quantification: RNA concentrations were measured using a NanoDrop OneC spectrophotometer (Thermo Scientific).

[0425] Culture method of A549 cells: 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.

[0426] One day prior to transfection, cells were seeded in 96-well plates at 2,000 cells / well. Lipofectamine MessengerMAX transfection reagent (ThermoFisher) was used to transfect 50ng of each RNA using a 1:1.5μg:uL ratio of RNA:MessengerMAX. Transfections were performed in triplicate.

[0427] Viability and luciferase expression were measured using the ONE-Glo+Tox luciferase reporter and cell viability assay (Promega). NF-κB activation was measured via the SEAP reporter gene using QUANTI-Blue detection reagent (InvivoGen) as described by the manufacturer. IRF pathway activation was measured via Lucia luciferase gene activity using QUANTI-Luc detection reagent (InvivoGen) as described by the manufacturer.

[0428] In vitro transcription (IVT) of Luc2 RNA for in vivo experiments: Luc2 RNA was synthesized in a 20 μL IVT reaction consisting of 200 ng Luc2T7 template, 20 mM MgCl2, 7.5 mM each NTP, 7.5 mM CleanCapAG (TriLink), 1× HiScribe transcription buffer, and 2 uL HiScribe polymerase mix (NEB) and incubated for 1 h at 37° C. For RNA conditions using chemically modified nucleotides, UTP was replaced with N1-methyl pseudo-UTP (TriLink) or cytidine was replaced with N4-acetyl-CTP (Jena BioScience) at the indicated ratios in the IVT mix.

[0429] All IVT products were cleaned up using Monarch 500μg RNA Cleanup Kit (NEB) and eluted in 88μL nuclease free water. The eluted products were then digested in a 100μL reaction consisting of 1× DNase I buffer and 10U DNase I (RNase free) (New England Biolabs) at 37°C for 10 minutes to degrade the DNA template. The DNase I treated samples were cleaned up using Monarch 500ug RNA Cleanup Kit (NEB) and eluted in 88μL nuclease free water.

[0430] The DNase I-treated products, which contained the Cap1 structure added co-transcriptionally, were treated with 1x CutSmart buffer and 25 U of Quick CIP (NEB) for 5 min at 37°C as a polishing step to remove the rarely immunogenic 5' triphosphate from RNA transcripts that did not incorporate Clean Cap AG.

[0431] The Quick CIP treated RNA was cleaned up using Monarch 500ug RNA Cleanup Kit (NEB) and eluted in 1mM sodium citrate, pH 6.5.

[0432] In vitro transcription (IVT) of RNA vaccine candidates: RNA vaccine candidates were synthesized in a 20 μL IVT reaction consisting of 200 ng Luc2T7 template, 20 mM MgCl2, 7.5 mM each NTP, 7.5 mM CleanCapAG (TriLink), 1× HiScribe transcription buffer, and 2 uL HiScribe polymerase mix (NEB) and incubated for 1 h at 37° C. For RNA conditions using chemically modified nucleotides, UTP was replaced with N1-methyl pseudo-UTP (TriLink) or cytidine was replaced with N4-acetyl-CTP (Jena BioScience) at the indicated ratios in the IVT mix.

[0433] All IVT products were cleaned up using Monarch 500μg RNA Cleanup Kit (NEB) and eluted in 88μL nuclease free water. The eluted products were then digested in a 100μL reaction consisting of 1× DNase I buffer and 10U DNase I (RNase free) (New England Biolabs) at 37°C for 10 minutes to degrade the DNA template. The DNase I treated samples were cleaned up using Monarch 500ug RNA Cleanup Kit (NEB) and eluted in 88μL nuclease free water.

[0434] The DNase I-treated products, which contained the Cap1 structure added co-transcriptionally, were treated with 1x CutSmart buffer and 25 U of Quick CIP (NEB) for 5 min at 37°C as a polishing step to remove the rarely immunogenic 5' triphosphate from RNA transcripts that did not incorporate Clean Cap AG.

[0435] The Quick CIP treated RNA was cleaned up using Monarch 500ug RNA Cleanup Kit (NEB) and eluted in 1mM sodium citrate, pH 6.5.

[0436] Formulations for in vivo RNA experiments: Formulations of RNA in lipid nanoparticles (RNA-LNPs) were prepared using a microfluidic mixer (Precision Nanosystems, Vancouver, BC). Briefly, using the manufacturer's recommended formulation parameters, the GenVoy-ILM lipid mixture (Precision Nanosystems NWW0042) was diluted to 12.5 mM in absolute ethanol and combined with an aqueous solution of RNA (0.14 mg / mL) in PNI buffer (Precision Nanosystems NWW0043). The formulation was immediately diluted 30:1 with phosphate buffered saline (Gibco 10010023), concentrated using an Amicon centrifugal filter (MilliporeSigma UFc901008), and adjusted to the appropriate final volume with PBS. Formulations were stored at 4 °C for up to 8 days prior to in vivo administration.

[0437] Repeated-dose RNA administration study in mice: Animal studies were performed according to 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=4 per condition) were acclimated for 3 days before the start of the study. During the course of the repeated-dose Luc2 study, animals received three RNA administrations at 72-h intervals, and animals were imaged by whole-body bioluminescence imaging 6, 27, and 51 h after each RNA administration. All RNA injections consisted of 200uL of RNA-LNP formulation (1ug Luc2 RNA dose per animal) delivered via tail vein injection. 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 via 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, 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 ventral side up in the imaging chamber and maintained under 3% isoflurane anesthesia throughout imaging. Images were acquired using field of view D, with 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 to recover. Mice were euthanized 72 h after the third RNA administration, at which point blood was collected via an intracardiac stick. Serum was separated from blood by centrifugation at 1200×g for 10 min at 4° C. in MiniCollect serum separator tubes (Greiner Bio-One 450472).Fresh serum aliquots were stored at 4°C for <24 h and then shipped on ice to IDEXX BioAnalytics (North Grafton, MA) for Mouse Liver Panel testing (code 60405). Serum aliquots frozen at -80°C were shipped on dry ice to IDEXX BioAnalytics (Columbia, MO) for Mouse Cytokine 25-plex Panel testing (code 62579).

[0438] Vaccine immunogenicity screening in mice: All 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=4 per condition) were allowed to acclimate for 3 days before the start of the study. On day 1, mice were injected with 50uL of RNA-LNP formulation into the right quadriceps (10ug RNA dose per animal). On day 4, mice were injected with 50uL of the same RNA-LNP formulation used for the first dose into the left quadriceps (again 10ug dose per animal). Mice were euthanized on day 11, at which point blood was collected via an intracardiac stick. Serum was separated from blood by centrifugation at 1200×g for 10 min at 4° C. in MiniCollect serum separator tubes (Greiner Bio-One 450472). Fresh serum was stored at 4° C. and used for evaluation of immunogenicity by ELISA, and the remainder was aliquoted and frozen at −80° C. An aliquot of serum frozen at −80° C. was shipped on dry ice to IDEXX BioAnalytics (Columbia, MO) for Mouse Cytokine 25-plex Panel testing (code 62579).

[0439] result:

[0440] The work described in this Example demonstrates that the use of N4-acetylcytidine (Ac4C) in place of natural cytidine can reduce the undesirable immunogenicity associated with in vitro transcribed RNA. Table 1 shows that the nucleotide was readily accepted by T7 RNA polymerase and incorporated into in vitro transcribed RNA. As shown herein, the reduced immunogenicity and improved cell viability from the Ac4C substitution enhanced the therapeutic efficacy of the RNA product.

[0441] [Table 1]

[0442] A significant improvement in cell viability was evident from the data presented in Figure 1. This trend in improved viability followed a similar trend in the reduction of both immunogenic markers, NF-κB and IRF, as shown in Figures 2 and 3. In summary, increasing the substitution percentage of Ac4C reduced the toxicity of the exogenous RNA, which in some embodiments led to improved patient tolerability of RNA therapeutics incorporating Ac4C.

[0443] In contrast to the data presented in Arango, et al., Figures 4 and 5 show that reporter gene expression was indeed reduced when cytidines were completely replaced with Ac4C. In determining the ideal percentage of Ac4C to use, a trade-off between reduced immunogenicity and increased protein expression can be accounted for. The ideal percentage of expression will likely depend, for example, on the codon composition of the RNA in question. This phenomenon of the ideal replacement percentage of Ac4C depending on the application was evident from the in vivo experiments described herein.

[0444] RNA fully modified with Ac4C was well suited for repeated dosing of therapeutic proteins, as complete substitution of Ac4C significantly reduced immunogenicity. As shown in Figure 6, RNA fully modified with Ac4C (100%Ac4c 37C) indeed outperformed the state-of-the-art N1-methylpseudouridine (100%mPseudo 37C) at the first and second doses (compare data 6, 27, and 51 hours after the first and second doses of 100%Ac4c 37C and 100%mPseudo 37C). This is likely due to the reduction in the local inflammatory response by Ac4C, which is not achieved by N1-methylpseudouridine, allowing for even higher protein expression. By the third dose, the efficacy of Ac4C and N1-methylpseudouridine appeared to be comparable. This could have been due to enhanced systemic inflammation reaching a threshold with both nucleotides. Figure 7 shows the systemic cytokines that were up- or down-regulated in response to each of the RNA conditions. It is noteworthy that none of the cytokines examined in the IDEXX BioAnalytics Mouse Cytokine 25-plex Panel test were significantly affected in response to RNA with 100% Ac4C to cytidine. However, N1-methylpseudouridine did show an increase in IP-10 expression. Thus, the convergence in protein expression at the third dose may have been due to some systemic cytokines not examined within the panel. Nonetheless, the data demonstrated the ability of N4-acetylcytidine to prevent undesired innate immune responses and improve expression of proteins of interest compared to N1-methylpseudouridine.

[0445] Comparison of the data in FIG. 6 with that in FIG. 8 shows that different advantages were achieved using different percentages of Ac4C. The highest absolute expression of the Luc2 reporter was achieved with 75% substitution, while full substitution allowed for improvements in repeated dosing. In addition, FIG. 9 shows that peak IgG antibody titers from the SARS-CoV-2 RNA vaccine candidates examined were achieved with 75% substitution. In some embodiments, this percentage of substitution that appears to be best for both absolute protein expression and antibody titers may be coincidental, as the even higher antibody titers with 75% substitution may be due to, for example, a combination of high antigen expression and a slightly immunostimulatory adjuvant effect from the RNA. This data showed that the efficacy of the RNA was clearly improved by the use of RNA containing Ac4C. Based on these findings, the percentage of Ac4C may be maximized for a particular application by optimizing the percentage of Ac4C substitution.

[0446] Example 2: Increased expression of RNA containing 5-hydroxymethyluridine This example demonstrates that the use of RNA containing 5-hydroxymethyluridine (5hmU) instead of natural uridine can reduce the undesirable immunogenicity associated with in vitro transcribed RNA. The method used in this example is similar to that described in Example 3 below.

[0447] To identify other modified ribonucleotides (other than N4-acetylcytidine shown in Example 1) with improved properties such as reduced immunogenicity and increased expression, an extensive screen of chemically modified nucleotides was conducted. Based on this screen, one of the potential candidates that emerged was 5-hydroxymethyluridine (5hmU), shown in FIG.

[0448] Messenger RNA (RNA) with 0% to 100% substitution of uridine with 5-hydroxymethyluridine was synthesized and the effect of the RNA on cell viability and immunogenicity was examined. As shown in Figure 12, RNA in which all uridines were substituted with 5hmU showed little effect on cell viability. Figures 13 and 14 demonstrate that RNA in which all uridines were substituted with 5hmU had little or no effect on immunogenicity (see Figure 13 for IRF reporter activation and Figure 14 for NF-κB reporter activity).

[0449] We also examined reporter gene expression using RNA with uridines substituted with 5hmU (0% to 100%). As shown in Figures 15 and 16, RNAs with a high percentage of uridines substituted with 5hmU (e.g., greater than about 75%) showed a greater than 5-fold increase in reporter gene expression compared to RNAs with a low percentage of uridines substituted with 5hmU (less than about 75%).

[0450] This data indicates that RNA containing 5-hydroxymethyluridine has desirable properties and can be used for therapeutic purposes.

[0451] Example 3: Inhibition of innate immune sensing and improved expression of RNA containing 5-hydroxymethyluridine and N4-acetylcytidine This example shows that using RNA containing both 5-hydroxymethyluridine (5hmU) instead of uridine and N4-acetylcytidine instead of cytidine can reduce undesirable immunogenicity associated with in vitro transcribed RNA, reduce detection of uncapped RNA that is a by-product of the in vitro RNA transcription reaction, and / or increase expression of the RNA or a polypeptide encoded by the RNA. This example further demonstrates the in vivo expression profile of RNA containing 5-hydroxymethyluridine and N4-acetylcytidine. As described in more detail below, the dually modified RNA allows for repeated administration with similar payload expression at each dose.

[0452] method

[0453] Generation of IVT template: The luc2 gene encoding an optimized version 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, 30 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. PCR products were purified using SpriSelect beads (Beckman Coulter) at a 0.8-fold ratio relative to the PCR reaction volume and eluted in 45 μL of nuclease-free water. 42.5 μL of the eluted product was subjected to treatment with 125 U of Dpn1 enzyme (New England Biolabs) in a 50 μL reaction to digest the template plasmid. The digestion products were purified using SpriSelect beads (Beckman Coulter) at a 0.65x ratio to the digestion reaction volume and eluted in 40 μL of nuclease-free water. This digested primary PCR product was then amplified at 50°C in a 20 μL reaction consisting of 0.25 μM of each primer T7-AGG_fwd and 120 pA_rev, 1× Herculase II buffer, 25 mM of each dNTP, 10 ng of Luc2 primary amplicon, and 0.4 μL of Herculase II enzyme. This secondary PCR product was cleaned up using SpriSelect beads (Beckman Coulter) at a 0.8x ratio to the PCR reaction volume and eluted in 10 mM Tris-HCl pH 8.5.

[0454] The sequences of the primers used were as follows:

[0455] Luc2_fwd: CTTGTTCTTTTTGCAGAAGCTCAGAATAAACGCTCAACTTTGGCCACCatggaagatgccaaaaacattaagaagggc (SEQ ID NO: 1)

[0456] Luc2_rev: AGAATGTGAA GAAACTTTCT TTTTATTAGG AGCAGATACG AATGGCTACA TTTTGGGGGA CAACATTTTG TAAAGTGTAA GTTGGTATTA TGTAGCTTAG AGACTCCATT CGGGTGTTCT TGAGGCTGGT CTATCATTAc acggcgatct tgccgcc(SEQ ID NO: 2)

[0457] T7-AGG_fwd: gaattTAATA CGACTCACTA TAAGGcttgt tctttttgca gaagc(SEQ ID NO: 3)

[0458] 120pA_rev: TTTTTTTTTT TTTTTTTTTT TTTTTTTTTT TTTTTTTTTT TTTTTTTTTT TTTTTTTTTT TTTTTTTTTT TTTTTTTTTT TTTTTTTTTT TTTTTTTTTT TTTTTTTTTT TTTTTTTTTT agaatgtgaa gaaactttct ttttattag(SEQ ID NO: 4)

[0459] In vitro transcription (IVT) of Luc2 RNA: Luc2 RNA was synthesized in a 20 μL IVT reaction consisting of 200 ng Luc2T7 template, 20 mM MgCl2, 7.5 mM each NTP, 7.5 mM CleanCap AG (TriLink), 1× HiScribe transcription buffer, and 2 uL HiScribe polymerase mix (NEB) and incubated at 37°C for 1 h. To investigate the synergistic effect of N4-acetylcytidine or 5-hydroxymethyluridine, the corresponding proportions of CTP or UTP were replaced with Ac4CTP (Jena BioScience) or 5hmUTP (TriLink) in the IVT mixture. In the gradient of increasing Ac4CTP, the proportion of 5hmU was kept constant at 100%. In the gradient of increasing 5hmU, the proportion of Ac4C was kept constant at 100%. RNA using natural nucleotides was made in parallel to serve as a control representing a highly immunogenic RNA.

[0460] All IVT products were cleaned up using Monarch 500μg RNA Cleanup Kit (NEB) and eluted in 88μL nuclease free water. The eluted products were then digested in a 100μL reaction consisting of 1× DNase I buffer and 10U DNase I (RNase free) (New England Biolabs) at 37°C for 10 minutes to degrade the DNA template. The DNase I treated samples were cleaned up using Monarch 500ug RNA Cleanup Kit (NEB) and eluted in 88μL nuclease free water.

[0461] The DNase I-treated products, which contained the co-transcriptionally added Cap1 structure, were treated with 1× DNase I buffer (NEB) and 100 U of 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 step because Promega's CIAP in NEB's DNase I buffer is more effective at polishing RNA 5' ends than the CIAP enzyme in its own buffer.

[0462] CIAP-treated RNA was cleaned up using the Monarch 500ug RNA Cleanup Kit (NEB) and eluted in nuclease-free H2O.

[0463] RNA quantification: RNA concentrations were measured using a NanoDrop OneC spectrophotometer (Thermo Scientific).

[0464] Culture method of A549 cells: 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.

[0465] One day prior to transfection, cells were seeded in 96-well plates at 2,000 cells / well. Lipofectamine MessengerMAX transfection reagent (ThermoFisher) was used to transfect 50ng of each RNA using a 1:1.5μg:uL ratio of RNA:MessengerMAX. Transfections were performed in triplicate.

[0466] Viability and luciferase expression were measured using the ONE-Glo+Tox luciferase reporter and cell viability assay (Promega). NF-κB activation was measured via the SEAP reporter gene using QUANTI-Blue detection reagent (InvivoGen) as described by the manufacturer. IRF pathway activation was measured via Lucia luciferase gene activity using QUANTI-Luc detection reagent (InvivoGen) as described by the manufacturer.

[0467] RNA-LNP formulations: Formulations of RNA in lipid nanoparticles (RNA-LNPs) were prepared using an Ignite microfluidic mixer (Precision Nanosystems, Vancouver, BC). Briefly, using the manufacturer's recommended formulation parameters, the GenVoy-ILM lipid mixture (Precision Nanosystems NWW0042) was diluted to 12.5 mM in absolute ethanol and combined with an aqueous solution of RNA (0.14 mg / mL) in PNI buffer (Precision Nanosystems NWW0043). The formulation was immediately diluted 30:1 with phosphate buffered saline (Gibco 10010023), concentrated using an Amicon centrifugal filter (MilliporeSigma UFc901008), and adjusted to the estimated final volume with PBS. The RNA was then analyzed using a Stunner UV-VIS / DLS instrument (Unchained The formulations were characterized using a ELISA kit (DeepLab) and further diluted with PBS as necessary to achieve the correct payload concentration (ug / mL). Formulations were stored at 4°C until in vivo administration.

[0468] Single-dose and repeated-dose Luc2 RNA 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 acclimated for at least 2 days before the start of the study. For the single-dose study (Figure 8), animals received one RNA-LNP administration via IV. During the course of the repeated-dose study, animals received three RNA administrations via IV, spaced 72 hours apart. In both cases, mice were imaged by whole-body bioluminescence imaging at three time points after each RNA administration (approximately 6 hours, approximately 24 hours, and approximately 48 hours after administration). All RNA injections consisted of 200uL of RNA-LNP formulation (Luc2 RNA doses of 1-9ug per animal) delivered via tail vein injection. For whole-body bioluminescence imaging, animals were injected with 200uL of D-luciferin K+ salt (PerkinElmer 122799) diluted to 15mg / mL in PBS via intraperitoneal (IP) injection 10min prior to imaging time point. Mice were placed under 3% isoflurane anesthesia in an induction chamber 3min 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 ventral side up in the imaging chamber and maintained at 3% isoflurane anesthesia throughout imaging. Images were acquired using field of view D and exposure was continued until 30,000 photons were collected or 1min had elapsed, whichever occurred first. After imaging, animals were returned to their home cages for recovery.

[0469] Blood collection and analysis: Mice were euthanized 72 hours after the third RNA administration, at which time blood was collected via an intracardiac stick. Alternatively, mice in a second set of cages (in addition to the IVIS imaged mice) were euthanized 6 hours after RNA administration (a single dose of 9ug, a single dose of 1ug, or three doses of 1ug at 72 hour intervals), at which time blood was collected via an intracardiac stick (Figure 49). Serum was separated from blood by centrifugation at 1200xg for 10 minutes at 4°C in MiniCollect serum separator tubes (Greiner Bio-One 450472). Aliquots of serum were frozen at -80°C and shipped on dry ice to IDEXX BioAnalytics (Columbia, MO facility; test code 62579) for mouse cytokine 25plex panel testing.

[0470] Immunogenicity Screening of RNA Vaccines in Mice: All 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=4 per condition) were allowed to acclimate for at least 2 days before the start of the study. On day 0, mice were injected with 50uL of a priming formulation of RNA-LNP in the right quadriceps (10ug RNA dose per animal). On day 7 (Figure 9) or day 21 (Figure 50), mice were injected with 50uL of a boosting formulation of the same RNA-LNP formulation used for the priming dose in the left quadriceps (again 10ug dose per animal). Mice were euthanized on day 14 (Figure 9) or day 35 (Figure 50), at which time blood was collected via intracardiac stick. Serum was separated from blood by centrifugation at 1200 x g for 10 minutes at 4°C in MiniCollect serum separator tubes (Greiner Bio-One 450472). Fresh serum was stored at 4°C and used for evaluation of immunogenicity by ELISA.

[0471] Serum antibody titers: Total IgG antigen-specific antibodies were measured by enzyme-linked immunosorbent assay (ELISA). Briefly, sera were serially diluted 1:10. Plates (Thermo Scientific Nunc 442404) were coated with 2ug / mL SARS-CoV-2 spike protein (Sino Biological 40589-V08B1) in PBS overnight at 4°C. Plates were washed three times with 0.1% Tween 20 in PBS and then blocked with SuperBlock PBS blocking buffer (Thermo Scientific 37515) for 1 hour at room temperature. Plates were then incubated with diluted serum for 2 hours at room temperature and then washed three times with 0.1% Tween 20 in PBS. Plates were then incubated with goat anti-mouse IgG HRP-conjugated secondary antibody (Millipore Sigma AP127P) for 1 hour at room temperature and then washed three times with 0.1% Tween 20 in PBS. Finally, the plates were developed with SigmaFast OPD reagent (P(187)) for 10 min at room temperature, stopped with 3N HCL and the absorbance was read at 490 nm on a Promega Discovery plate reader.

[0472] result:

[0473] Given the effect of N4-acetylcytidine (Ac4C) in reducing immunogenicity and improving cell viability, RNAs with gradients of either Ac4C or 5-hydromethyluridine (5hmU) were examined in the context of full nucleotide substitution without a gradient to determine whether there was a synergistic effect between the two modified nucleotides. It was hypothesized that the several-fold increase in expression from 100% 5hmU (as shown in Example 2 and data therein) may compensate for the decrease in protein expression observed with 100% ac4C (see Example 1 and data therein), while maintaining the low immunogenicity advantage provided by 100% ac4C substitution.

[0474] Figures 17-21 show the effect of increasing the 5hmU ratio at an RNA dose of 50 ng, while Figures 22-26 use the same samples but at a higher dose of 200 ng. In these samples, the natural cytidines were fully replaced with ac4C, except for the unmodified control. The effect on cell viability of RNA with fully replaced ac4C with varying degrees of 5hmU replacement is shown in Figure 17 (50 ng) and Figure 22 (200 ng). Inhibition of IRF activation by RNA with fully replaced ac4C with varying degrees of 5hmU replacement is shown in Figure 18 (50 ng) and Figure 23 (200 ng). Inhibition of NF-κB activation by RNA with fully replaced ac4C with varying degrees of 5hmU replacement is shown in Figure 19 (50 ng) and Figure 24 (200 ng). Reporter gene expression by RNA with fully substituted ac4C with varying degrees of 5hmU substitution is shown in Figures 20-21 (50 ng) and Figures 25-26 (200 ng). As shown in Figures 20-21 and Figures 25-26, high luciferase expression was observed in RNA with fully substituted ac4C with varying degrees of 5hmU substitution.

[0475] Figures 27-36 show data from follow-up experiments examining RNA samples at doses of 50 ng (Figures 27-31) and 200 ng (Figures 32-36) in which all uridines were replaced with 5hmU and the substitution rate of ac4C was varied.

[0476] The effect of RNA with fully substituted 5hmU with varying degrees of ac4C substitution on cell viability is shown in Figure 27 (50 ng) and Figure 32 (200 ng). Inhibition of IRF activation by RNA with fully substituted 5hmU with varying degrees of ac4C substitution is shown in Figure 28 (50 ng) and Figure 33 (200 ng). Inhibition of NF-κB activation by RNA with fully substituted 5hmU with varying degrees of ac4C substitution is shown in Figure 29 (50 ng) and Figure 34 (200 ng). Expression of reporter genes by RNA with fully substituted 5hmU with varying degrees of ac4C substitution is shown in Figures 30-31 (50 ng) and Figures 35-36 (200 ng). As shown in Figures 30-31 and Figures 35-36, high luciferase expression was observed with RNA with fully substituted 5hmU with varying degrees of ac4C substitution.

[0477] In both 200ng data sets, the IRF activation signal of unmodified RNA was lower than that seen with 50ng transfections. Since this coincided with the low luciferase expression signal, one possible explanation for this observation is that unmodified RNA may further activate PKR, leading to a global inhibition of translation. The NF-κB signal elicited by unmodified RNA was nearly indistinguishable from that seen in untreated cells.

[0478] Taken together, the data elucidate the role of each individual chemically modified nucleotide on the RNA. All samples with uridines fully substituted with 5hmU had reduced NF-κB, which may suggest that 5hmU plays a role in reducing TLR signaling. Conversely, all samples with cytidines fully substituted with Ac4C had lower IRF, which may suggest that Ac4C plays a role in reducing signaling through cytoplasmic innate immune sensors. Complete innate immune evasion was only seen when both chemically modified nucleotides were used at 100% substitution, regardless of dose. One observation from Figures 18 and 19 is that the use of both chemically modified nucleotides at a dose of 50 ng resulted in a decoupling of NF-κB and IRF activation. Without wishing to be bound by any particular theory, the relatively high NF-κB activation and low IRF activation may suggest a shift to protein-driven NF-κB activation, as NF-κB activation appears to begin to correlate with higher luciferase expression. The data show that there is a strong synergy between the two modified nucleotides (Ac4C and 5-hmU) and this observed synergy is demonstrated, for example, by the fact that dually modified RNAs significantly outperform RNAs bearing each of the individual modifications alone.

[0479] This disclosure is the first to report the combination of Ac4C and 5-hmU modified nucleotides and their associated beneficial effects. While the combined use of both modified U and C nucleotides has been described previously (see U.S. Pat. No. 8,278,036), the currently disclosed combination of ac4C and 5hmU is crucially different from the previously described combination of pseudouridine (Ψ) and 5-methylcytosine (m5c). Moreover, recent studies suggest that m5C may actually impair the positive effects of N1-methylpseudouridine (Svitkin, et al., NAR, 2017). Rather, our combination represents a clear improvement over the state of the art in chemically modified RNA, as we see a direct synergistic effect between our two modifications.

[0480] Also provided herein are data demonstrating that the ac4C / 5hmU combination directly inhibits the recognition of uncapped RNA. This is, to the best of our knowledge, the first demonstration of this effect with a chemically modified RNA combination. The IVT workflow used in this example utilizes co-transcriptional capping with CleanCap AG. The main advantage of this method is that it produces RNA with a high capping efficiency that alleviates downstream processing by avoiding enzymatic capping using the vaccinia capping system. Unfortunately, co-transcriptional capping also produces uncapped by-products because the RNA polymerase initiates with a standard nucleotide rather than a cap analog. The uncapped fraction is estimated to account for approximately 4-5% of the product. To address this challenge and reduce the immunogenicity associated with the sensing of uncapped RNA by RIG-I, it is typical to treat the RNA with a phosphatase enzyme that cleaves off the immunogenic 5' triphosphate while leaving the RNA cap intact. Previously published studies have suggested that in addition to 5' triphosphate, another ligand for RIG-I is polyU / UC within the RNA sequence (Schnell, Loo, Marcotrigiano & Gale, PLoS Pathogen, 2012). Therefore, experiments were performed to analyze whether RNA dually modified with ac4C and 5hmU reduces RIG-I activation upon introduction of samples containing residual uncapped RNA.

[0481] In these experiments, RNA was transfected at a dose of 200 ng prior to phosphatase treatment of the RNA used in Figures 16-36. Figures 37-46 show that unmodified, non-phosphatase-treated RNA performed substantially worse than unmodified, phosphatase-treated RNA. Complete replacement of each chemically modified nucleotide individually with the unmodified nucleotide nearly restored cell viability and expression compared to phosphatase-untreated RNA, but still resulted in high immunogenicity as indicated by IRF and NF-κB reporters. However, phosphatase-untreated Ac4C / 5hmU doubly modified RNA produced both high viability and expression with little natural immunogenicity above background.

[0482] Figure 37 shows that cell viability for phosphatase-untreated ac4C / 5hmU dual modified RNA was highest when the RNA had 100% ac4C for cytidines and greater than about 75% 5-hydroxymethyluridine for uridines. The same RNAs as in Figure 37 were examined for IRF activation (Figure 38), NF-κB activation (Figure 39) and reporter expression (Figures 40-41). Figure 38 shows that there is no activation of IRFs by phosphatase-untreated ac4C / 5hmU dual modified RNA when the RNA has 100% ac4C for cytidines and greater than about 75% 5-hydroxymethyluridine for uridines. Figure 39 shows that NF-κB activation was reduced by about 40% when the RNA had 100% ac4C for cytidine and greater than about 75% 5-hydroxymethyluridine for uridine (compared to unmodified RNA), and NF-κB activation was reduced by about 20% when the RNA had 100% ac4C for cytidine and greater than about 75% 5-hydroxymethyluridine for uridine (compared to unmodified RNA). Figures 40-41 show high reporter expression when the RNA had 100% ac4C for cytidine and 0-100% 5-hydroxymethyluridine for uridine.

[0483] Figure 42 shows that cell viability for phosphatase-untreated ac4C / 5hmU dual modified RNA was highest when the RNA had 100% 5-hydroxymethyluridine to uridine and greater than about 50% ac4C to cytidine. The same RNAs as in Figure 41 were examined for IRF activation (Figure 43), NF-κB activation (Figure 44), and reporter expression (Figures 45-46). Figure 43 shows significantly reduced IRF activation by phosphatase-untreated ac4C / 5hmU dual modified RNA (relative to unmodified RNA) when the RNA had 100% 5-hydroxymethyluridine to uridine and greater than about 50% ac4C to cytidine. No IRF activation was observed when the RNA had 100% 5-hydroxymethyluridine to uridine and 100% ac4C to cytidine. Figure 44 shows reduced NF-κB activation when the RNA has 100% 5-hydroxymethyluridine to uridine and approximately 50-75% ac4C to cytidine. Figures 45-46 show high reporter expression when the RNA has 100% 5-hydroxymethyluridine to uridine and 0-100% ac4C to cytidine.

[0484] A number of experiments were performed to investigate the effects of the novel chemically modified RNAs disclosed herein in in vivo models. It was observed that 100% Ac4C / 100% 5hmU RNA was superior to state-of-the-art RNAs using N1-methylpseudouridine. Figure 10 shows that repeated administration of dual modified RNAs over a therapeutically relevant dose range at a relatively short interval of 72 hours resulted in comparable protein expression at each dose. Previously, repeated administration at such short intervals resulted in a gradual decrease in expression with each repeated administration due to systemic immune stimulation with each administration. This has prevented RNAs from being used for frequent administration in therapeutic applications. The present application is the first to show that RNAs containing the modifications disclosed herein, e.g., Ac4C and / or 5hmU, do not have this limitation and can be used for repeated administration, e.g., frequent repeated administration, in therapeutic applications.

[0485] FIG. 47 shows that modified RNAs provided comparable reporter protein expression at repeated doses over 72 hours compared to state-of-the-art N1-methylpseudouridine modified RNAs at a high mouse dose of 9ug. Because mammalian immune systems have evolved inflammatory pathways to respond to foreign nucleic acids and halt pathogenic replication, it is reasonable to assume that the use of modified RNAs disclosed herein provides best-in-class reductions in RNA immunogenicity, resulting in comparable protein expression with each repeated dose. This point is illustrated in FIG. 48, where a high dose of 9ug N1-methylpseudouridine RNA produced higher levels of key systemic inflammatory markers compared to 9ug modified RNAs disclosed herein. This data also suggests that in some embodiments, the use of modified RNAs disclosed herein allows for the delivery of higher doses of RNA with better patient tolerance due to reduced inflammatory responses.

[0486] Figure 49 shows that vaccination with 100% Ac4C / 100% 5hmU RNA encoding a SARS-CoV-2 vaccine candidate resulted in higher IgG antibody titers than unmodified RNA or RNA with 100% Ac4C only. Without being bound to any particular theory, this may be a function of higher expression and availability of the antigen to the immune system, allowing more immune cells to recognize the antigen and contribute to higher antibody titers.

[0487] These data show that complete substitution of natural C and U nucleosides with Ac4C and 5hmU inhibits innate immune sensing and increases expression of RNA-encoded proteins. This combination of Ac4C and 5hmU modified nucleotides is the first nucleotide combination shown to directly inhibit sensing of uncapped RNA. Improved results were obtained when a large proportion of both nucleotides were substituted (e.g., about 75% or more substitutions at both nucleotides). In some embodiments, the percentage of substitution at each nucleotide is 100%, rather than an undetermined percentage less than 100%.

[0488] When using RNA in applications involving repeated administration and / or high-dose regimens, including gene therapy and enzyme replacement, the innate immune detection of RNA remains a major barrier.The data described herein has great implications for expanding the usefulness of RNA as a therapeutic.The data suggest that innate immune evasion can be achieved by polyribonucleotides containing ac4C and 5hmU.

[0489] Exemplary embodiments Embodiment 1. A modified ribonucleotide comprising a nucleoside comprising an acetyl group, wherein the nucleoside is N4-acetylcytidine, the modified ribonucleotide having (a) a 5' monophosphate, (b) a 5' diphosphate; or (c) a 5' triphosphate, and having the structure: [ka]

[0490] Embodiment 2. A polyribonucleotide comprising one or more modified ribonucleotides as described in embodiment 1.

[0491] Embodiment 3. The polyribonucleotide of embodiment 2, wherein the polyribonucleotide comprises cytidine residues, and at least 5% of the cytidine residues in the polyribonucleotide comprise N4-acetylcytidine.

[0492] Embodiment 4. The polyribonucleotide of embodiment 3, wherein the polyribonucleotide comprises cytidine residues, and less than 100 of the cytidine residues in the polyribonucleotide comprise N4-acetylcytidine.

[0493] Embodiment 5. The polyribonucleotide contains 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 ... 5. The polyribonucleotide according to any one of embodiments 2 to 4, 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 N4-acetylcytidine.

[0494] Embodiment 6. The polyribonucleotide of any one of embodiments 2 to 4, wherein the polyribonucleotide comprises cytidine residues, and wherein 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.

[0495] Embodiment 7. The polyribonucleotide of any one of embodiments 2 to 6, wherein the polyribonucleotide further comprises one or more modified ribonucleotides other than N4-acetylcytidine.

[0496] Embodiment 8. The polyribonucleotide of embodiment 7, wherein the one or more modified ribonucleotides comprise a nucleoside selected from adenosine, guanosine, cytidine or uridine, or a combination thereof.

[0497] Embodiment 9. The polyribonucleotide of embodiment 7 or 8, wherein the one or more modified ribonucleotides comprises a hydroxymethyl group.

[0498] Embodiment 10. The polyribonucleotide of embodiment 9, wherein the nucleoside of the one or more modified ribonucleotides is 5-hydroxymethyluridine, and the modified ribonucleotide has (a) a 5' monophosphate, (b) a 5' diphosphate; or (c) a 5' triphosphate, and the structure: [ka]

[0499] Embodiment 11. The polyribonucleotide of any one of embodiments 7 to 10, wherein the polyribonucleotide comprises uridine residues, and at least about 5% of the uridines in the polyribonucleotide comprise 5-hydroxymethyluridine.

[0500] Embodiment 12. The polyribonucleotide of any one of embodiments 7 to 10, wherein the polyribonucleotide comprises uridine residues, and at least less than 100% of the uridines in the polyribonucleotide comprise 5-hydroxymethyluridine.

[0501] Embodiment 13. The polyribonucleotide comprises a uridine residue, and the uridines 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 The polyribonucleotide according to any one of embodiments 7 to 12, wherein 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.

[0502] Embodiment 14. The polyribonucleotide of any one of embodiments 7 to 13, wherein the polyribonucleotide comprises uridine residues, and wherein 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 uridines in the polyribonucleotide comprise 5-hydroxymethyluridine.

[0503] Embodiment 15. A polyribonucleotide according to any one of embodiments 2 to 14, characterized in that reduced immunogenicity is observed when assessed in a cell, tissue or organism to which the polyribonucleotide has been administered, compared to an appropriate reference standard.

[0504] Embodiment 16. The polyribonucleotide of embodiment 15, wherein the reference comparison standard comprises an otherwise similar cell, tissue or organism that has been administered a comparable polyribonucleotide that comprises fewer acetyl groups on the nucleobases than the polyribonucleotide in the composition (e.g., no acetyl groups on the nucleobases).

[0505] Embodiment 17. The polyribonucleotide of embodiment 15 or 16, wherein the reduced immunogenicity comprises a reduced activation of innate immune response-induced toxicity.

[0506] Embodiment 18. The polyribonucleotide of embodiment 17, wherein the reduced activation of the immune response comprises a reduced activation of NFκb, IRF pathways, and / or other cytokines resulting from inflammation in a cell, tissue, or organism.

[0507] Embodiment 19. The polyribonucleotide of any one of embodiments 15 to 18, wherein the reduced immunogenicity allows for repeated administration of the polyribonucleotide.

[0508] Embodiment 20. The polyribonucleotide of embodiment 19, wherein the reduced immunogenicity allows for the administration of higher doses of said polyribonucleotide relative to an appropriate reference standard.

[0509] Embodiment 21. The polyribonucleotide of embodiment 20, wherein the reference comparison standard comprises a comparable polyribonucleotide comprising fewer acetyl groups on the nucleobases (e.g., no acetyl groups on the nucleobases).

[0510] Embodiment 22. A polyribonucleotide described in any one of embodiments 2 to 21, characterized in that when evaluated in a cell, tissue or organism to which the polyribonucleotide has been administered, an increase in cell viability is observed compared to an appropriate reference comparison standard.

[0511] Embodiment 23. The polyribonucleotide of embodiment 22, wherein the reference comparison standard is the cell viability of a cell, tissue or organism administered a comparable polyribonucleotide comprising fewer acetyl groups on the nucleobases (e.g., no acetyl groups on the nucleobases).

[0512] Embodiment 24. The polyribonucleotide of embodiment 22 or 23, wherein cell viability is a measure of the length of time that one or more cells of said cell, tissue or subject are alive.

[0513] Embodiment 25. The polyribonucleotide of embodiment 23 or 24, wherein cell viability is a measure of the number of cells in said cell, tissue or subject that are alive at one or more time points.

[0514] Embodiment 26 The polyribonucleotide of any one of embodiments 2 to 25, wherein the polyribonucleotide is or comprises messenger RNA (mRNA).

[0515] Embodiment 27. A polyribonucleotide according to any one of embodiments 2 to 25, wherein the polyribonucleotide is or comprises an RNA oligo.

[0516] Embodiment 28. A polyribonucleotide according to any one of embodiments 2 to 25, wherein the polyribonucleotide is or comprises a gRNA.

[0517] Embodiment 29. The polyribonucleotide of any one of embodiments 2 to 25, wherein the polyribonucleotide is or comprises an inhibitory RNA.

[0518] Embodiment 30. The polyribonucleotide of embodiment 29, wherein the polyribonucleotide is or comprises a miRNA or siRNA.

[0519] Embodiment 31. The polyribonucleotide of any one of embodiments 2 to 25, wherein the polyribonucleotide is or comprises an antisense oligonucleotide.

[0520] Embodiment 32. A composition comprising one or more polyribonucleotides according to any one of embodiments 2 to 31.

[0521] Embodiment 33. The composition of embodiment 32, wherein the composition is a pharmaceutical composition.

[0522] Embodiment 34. The composition of embodiment 33, wherein the pharmaceutical composition is or comprises an immunogenic composition.

[0523] Embodiment 35. The method of embodiment 33, wherein the pharmaceutical composition is or comprises a vaccine.

[0524] Embodiment 36 The composition of embodiment 33, wherein the pharmaceutical composition is or comprises a gene therapy.

[0525] Embodiment 37. The composition of embodiment 33, wherein the pharmaceutical composition is or comprises a chemotherapy.

[0526] Embodiment 38 The composition of embodiment 33, wherein the pharmaceutical composition is or comprises a protein replacement therapy.

[0527] Embodiment 39. The composition of embodiment 33, wherein the pharmaceutical composition is or comprises an immunotherapy, antibody therapy and / or immunomodulatory therapy.

[0528] Embodiment 40. The composition of embodiment 33, wherein the pharmaceutical composition is or comprises a cell-engineered therapy.

[0529] Embodiment 41. The composition of any one of embodiments 32 to 30, wherein the composition comprises double-stranded RNA.

[0530] Embodiment 42. A method of administration, comprising administering to a cell, tissue or subject one or more polyribonucleotides according to any one of embodiments 2 to 31, or a composition according to any one of embodiments 32 to 41.

[0531] Embodiment 43 The method of embodiment 42, further comprising determining cell viability of the cell, tissue or subject.

[0532] Embodiment 44 The method of embodiment 43, wherein cell viability is a measure of the length of time one or more cells of said cell, tissue or subject are alive.

[0533] Embodiment 45 The method of embodiment 44, wherein cell viability is a measure of the number of cells in said cell, tissue or subject that are alive at one or more time points.

[0534] Embodiment 46. The method according to any one of embodiments 43 to 45, wherein the cell, tissue or subject to which the polyribonucleotide or a composition comprising the polyribonucleotide has been administered exhibits improved cell viability compared to a reference cell viability.

[0535] Embodiment 47. The method of embodiment 46, wherein the reference cell viability is the cell viability of the cell, tissue or subject that has been administered a comparable polyribonucleotide or a composition comprising a comparable polyribonucleotide that comprises fewer acetyl groups on the nucleobases (e.g., no acetyl groups on the nucleobases).

[0536] Embodiment 48. The method of any one of embodiments 43 to 47, further comprising determining the response of the immune system of the cell, tissue or subject to which the polyribonucleotide or a composition comprising the polyribonucleotide has been administered.

[0537] Embodiment 49. The method of embodiment 48, wherein the immune response comprises a response of the innate immune system, including innate immune system-induced toxicity.

[0538] Embodiment 50. The method of embodiment 49, wherein assessing the response of the innate immune system comprises determining levels of NF-κB, IRF, and / or other inflammatory cytokines in the cell, tissue, or subject.

[0539] Embodiment 51. The method according to any one of embodiments 48 to 50, wherein the cell, tissue or subject to which the polyribonucleotide or a composition comprising the polyribonucleotide has been administered exhibits a reduced response of the innate immune system compared to a reference.

[0540] Embodiment 52. The method of embodiment 51, wherein the reference is a response of the innate immune system of a cell, tissue or subject that has been administered a comparable polyribonucleotide or a composition comprising a comparable polyribonucleotide that comprises fewer acetyl groups on the nucleobases (e.g., no acetyl groups on the nucleobases).

[0541] Embodiment 53. The method of any one of embodiments 42 to 52, further comprising determining the effectiveness of the polyribonucleotide or the composition comprising the polyribonucleotide in the cell, tissue or subject to which the polyribonucleotide or the composition comprising the polyribonucleotide has been administered.

[0542] Embodiment 54 The method of embodiment 53, wherein determining the effectiveness comprises determining an antibody response or a cellular response in the cell, tissue or subject.

[0543] Embodiment 55. The method of embodiment 54, wherein the cell, tissue or subject to which the polyribonucleotide or a composition comprising the polyribonucleotide has been administered exhibits an increased antibody or cellular response compared to a reference.

[0544] Embodiment 56. The method of embodiment 55, wherein the reference is an antibody response or a cellular response of the cell, tissue or subject that has been administered a comparable polyribonucleotide or a composition comprising a comparable polyribonucleotide that comprises fewer acetyl groups on the nucleobases (e.g., no acetyl groups on the nucleobases).

[0545] Embodiment 57. The method of any one of embodiments 42 to 56, wherein the method comprises administering the polyribonucleotide or a composition comprising the polyribonucleotide to the cell, tissue or subject at least twice.

[0546] Embodiment 58. The method of any one of embodiments 42 to 57, wherein the method comprises administering the polyribonucleotide or a composition comprising the polyribonucleotide to the cell, tissue or subject 2, 3, 4, 5, 6, 7, 8, 9, or 10 times.

[0547] Embodiment 59. The method of embodiment 57 or 58, wherein at least two administrations of the polyribonucleotide or the composition comprising the polyribonucleotide to the cell, tissue or subject does not reduce the effectiveness of the polyribonucleotide or the composition comprising the polyribonucleotide compared to administration of a single dose of the polyribonucleotide or the composition comprising the polyribonucleotide.

[0548] Embodiment 60. The method of any one of embodiments 42 to 59, wherein the method comprises administering to the cell, tissue or subject the polyribonucleotide or a composition comprising the polyribonucleotide at a dose that is higher than a suitable reference standard.

[0549] Embodiment 61. The method of embodiment 60, wherein the reference comparison standard comprises a comparable polyribonucleotide comprising fewer acetyl groups on the nucleobases (e.g., no acetyl groups on the nucleobases).

[0550] Embodiment 62. The method of any one of embodiments 42 to 61, wherein the cell is a mammalian cell, the tissue is a mammalian tissue, or the subject is a mammal.

[0551] Embodiment 63 The method of embodiment 62, wherein the mammal is a human.

[0552] Embodiment 64. The method of any one of embodiments 42 to 63, wherein the method is a method of stimulating an immune response.

[0553] Embodiment 65. The method according to any one of embodiments 42 to 64, wherein said method is a vaccination method.

[0554] Embodiment 66 The method of any one of embodiments 42 to 64, wherein the method is a gene therapy method.

[0555] Embodiment 67. The method of embodiment 66, wherein the gene therapy method comprises delivery of one or more components of gene therapy, such as gRNA.

[0556] Embodiment 68. The method of any one of embodiments 42 to 64, wherein the method is a cell therapy engineering method.

[0557] Embodiment 69. The method according to any one of embodiments 42 to 64, wherein said method is an immunotherapy method or an antibody therapy method.

[0558] Embodiment 70. The method of embodiment 69, wherein the immunotherapy method comprises delivery of an immune modulating therapy and / or an immune checkpoint therapy.

[0559] Embodiment 71 The method of any one of embodiments 42 to 63, wherein the method is a method of protein replacement therapy.

[0560] Embodiment 72 The method of embodiment 71, wherein said method of protein replacement therapy comprises delivery of enzyme replacement therapy.

[0561] Embodiment 73. The method of any one of embodiments 42 to 64, wherein the method is a chemotherapy method.

[0562] Embodiment 74. A method of vaccination, comprising administering to a cell, tissue or subject one or more polyribonucleotides described in any one of embodiments 2 to 31, or a composition described in any one of embodiments 32 to 41.

[0563] Embodiment 75. A method of immunotherapy comprising administering to a cell, tissue or subject one or more polyribonucleotides according to any one of embodiments 2 to 31, or a composition according to any one of embodiments 32 to 41.

[0564] Embodiment 76. A method of gene therapy comprising administering to a cell, tissue or subject one or more polyribonucleotides described in any one of embodiments 2 to 31, or a composition described in any one of embodiments 32 to 41.

[0565] Embodiment 77. A method for protein replacement therapy, comprising administering to a cell, tissue or subject one or more polyribonucleotides described in any one of embodiments 2 to 31, or a composition described in any one of embodiments 32 to 41.

[0566] Embodiment 78. A method of cell engineering therapy, comprising administering to a cell, tissue or subject one or more polyribonucleotides described in any one of embodiments 2 to 31, or a composition described in any one of embodiments 32 to 41.

[0567] Embodiment 79. A method for producing an RNA composition, comprising introducing at least one modified ribonucleotide according to embodiment 1 into a polyribonucleotide.

[0568] Embodiment 80 The method of embodiment 79, wherein said method does not include removing double-stranded RNA from said RNA composition.

[0569] Embodiment 81. A cell comprising a polyribonucleotide according to any one of embodiments 2 to 31, or a composition according to any one of embodiments 32 to 41.

[0570] Embodiment 82. Use of a modified ribonucleotide according to embodiment 1 in the manufacture of polyribonucleotides.

[0571] Embodiment 83. Use of a polyribonucleotide according to any one of embodiments 2 to 31, or a composition according to any one of embodiments 32 to 41, for stimulating an immune response.

[0572] Embodiment 84. Use of a polyribonucleotide according to any one of embodiments 2 to 31, or a composition according to any one of embodiments 32 to 41, as a vaccine.

[0573] Embodiment 85. Use of a polyribonucleotide according to any one of embodiments 2 to 31, or a composition according to any one of embodiments 32 to 41, as immunotherapy, for example as antibody therapy, immunomodulatory therapy and / or immune checkpoint therapy.

[0574] Embodiment 86. Use of a polyribonucleotide according to any one of embodiments 2 to 31 or a composition according to any one of embodiments 32 to 41 as a gene therapy.

[0575] Embodiment 87. Use of a polyribonucleotide according to any one of embodiments 2 to 31, or a composition according to any one of embodiments 32 to 41, as a protein replacement therapy.

[0576] Embodiment 88. Use of a polyribonucleotide according to any one of embodiments 2 to 31, or a composition according to any one of embodiments 32 to 41, as a cell engineering therapy.

[0577] Embodiment 89. Use of a polyribonucleotide according to any one of embodiments 2 to 31, or a composition according to any one of embodiments 32 to 41, as a chemotherapy.

[0578] Embodiment 90. The use according to any one of embodiments 82 to 89, wherein the polyribonucleotide or a composition comprising the polyribonucleotide is administered to a cell, tissue or subject.

[0579] Embodiment 91. The use of embodiment 90, wherein the cell is a mammalian cell, the tissue is a mammalian tissue, or the subject is a mammal.

[0580] Embodiment 92. The use according to embodiment 91, wherein the mammal is a human.

[0581] Embodiment 93. A modified ribonucleotide comprising a nucleoside comprising a hydroxymethyl group, wherein the nucleoside is 5-hydroxymethyluridine, the modified ribonucleotide having (a) a 5' monophosphate, (b) a 5' diphosphate; or (c) a 5' triphosphate, and having the structure: [ka]

[0582] Embodiment 94. A polyribonucleotide comprising one or more modified ribonucleotides according to embodiment 93.

[0583] Embodiment 95. The polyribonucleotide of embodiment 94, wherein the polyribonucleotide comprises uridine residues, and at least about 5% of the uridine residues in the polyribonucleotide comprise 5-hydroxymethyluridine.

[0584] Embodiment 96 The polyribonucleotide of embodiment 94, wherein the polyribonucleotide comprises uridine residues, and wherein less than 100% of the uridine residues in the polyribonucleotide comprise 5-hydroxymethyluridine.

[0585] Embodiment 97. 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%, The polyribonucleotide according to any one of embodiments 94 to 96, 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.

[0586] Embodiment 98. The polyribonucleotide of any one of embodiments 94 to 96, wherein the polyribonucleotide comprises uridine residues, and wherein 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.

[0587] Embodiment 99. The polyribonucleotide of any one of embodiments 94 to 96, wherein the polyribonucleotide comprises uridine residues, and greater than 60% of the uridine residues in the polyribonucleotide comprise 5-hydroxymethyluridine.

[0588] Embodiment 100. The polyribonucleotide according to embodiment 99, wherein about 60 to 100%, about 65% to 100%, about 70% to 100%, about 75% to 100%, about 80% to 100%, about 85% to 100%, about 90% to 100%, about 95% to 100%, about 60% to 95%, about 60% to 90%, about 60% to 85%, about 60% to 80%, about 60% to 75%, about 60% to 70%, or about 60% to 65% of the uridine residues in the polyribonucleotide contain 5-hydroxymethyluridine.

[0589] Embodiment 101. The polyribonucleotide of embodiment 99 or 100, wherein about 75% of the uridine residues in the polyribonucleotide comprise 5-hydroxymethyluridine.

[0590] Embodiment 102. The polyribonucleotide of embodiment 99 or 100, wherein 100% of the uridine residues in the polyribonucleotide comprise 5-hydroxymethyluridine.

[0591] Embodiment 103. The polyribonucleotide of any one of embodiments 94 to 102, wherein the polyribonucleotide further comprises one or more modified ribonucleotides other than 5-hydroxymethyluridine.

[0592] Embodiment 104. The polyribonucleotide of embodiment 103, wherein the one or more modified ribonucleotides comprise a nucleoside selected from adenosine, guanosine, cytidine or uridine, or a combination thereof.

[0593] Embodiment 105. The polyribonucleotide of embodiment 103 or 104, wherein the one or more modified ribonucleotides comprises an acetyl group.

[0594] Embodiment 106. A polyribonucleotide according to any one of embodiments 103 to 105, wherein the nucleoside of the one or more modified ribonucleotides is N4-acetylcytidine, and the modified ribonucleotide has (a) a 5' monophosphate, (b) a 5' diphosphate; or (c) a 5' triphosphate, and has the following structure: [ka]

[0595] Embodiment 107. The polyribonucleotide of embodiment 106, wherein the polyribonucleotide comprises cytidine residues, and at least 5% of the cytidine residues in the polyribonucleotide comprise N4-acetylcytidine.

[0596] Embodiment 108. The polyribonucleotide of embodiment 106, wherein the polyribonucleotide comprises cytidine residues, and wherein less than 100% of the cytidine residues in the polyribonucleotide comprise N4-acetylcytidine.

[0597] Embodiment 109. 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% of the cytidine residues in the polyribonucleotide. , 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.

[0598] Embodiment 110. The polyribonucleotide of any one of embodiments 106 to 108, wherein the polyribonucleotide comprises cytidine residues, and wherein 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.

[0599] Embodiment 111. The polyribonucleotide of embodiment 106, wherein the polyribonucleotide comprises cytidine residues, and more than 60% of the cytidine residues in the polyribonucleotide comprise N4-acetylcytidine.

[0600] Embodiment 112. The polyribonucleotide according to embodiment 111, wherein about 60 to 100%, about 65% to 100%, about 70% to 100%, about 75% to 100%, about 80% to 100%, about 85% to 100%, about 90% to 100%, about 95% to 100%, about 60% to 95%, about 60% to 90%, about 60% to 85%, about 60% to 80%, about 60% to 75%, about 60% to 70%, or about 60% to 65% of the cytidine residues in the polyribonucleotide contain N4-acetylcytidine.

[0601] Embodiment 113. The polyribonucleotide of embodiment 111 or 112, wherein at least about 75% of the cytidine residues in the polyribonucleotide comprise N4-acetylcytidine.

[0602] Embodiment 114. The polyribonucleotide of embodiment 111 or 112, wherein 100% of the cytidine residues in the polyribonucleotide comprise N4-acetylcytidine.

[0603] Embodiment 115. A polyribonucleotide comprising one or more modified ribonucleotides, wherein the one or more modified ribonucleotides comprise one or both of the following:

[0604] (i) 5-hydroxymethyluridine, and the modified ribonucleotide has (a) a 5' monophosphate; (b) a 5' diphosphate; or (c) a 5' triphosphate, and has the structure: [ka] and

[0605] (ii) N-acetylcytidine, and the modified ribonucleotide has (a) a 5' monophosphate; (b) a 5' diphosphate; or (c) a 5' triphosphate, and has the structure: [ka]

[0606] Embodiment 116. The polyribonucleotide of embodiment 115, wherein: (a) the polyribonucleotide comprises cytidine residues, and at least 5% of the cytidine residues in the polyribonucleotide comprise N4-acetylcytidine; and / or (b) the polyribonucleotide comprises uridine residues, and at least 5% of the uridine residues in the polyribonucleotide comprise 5-hydroxymethyluridine.

[0607] Embodiment 117. The polyribonucleotide of embodiment 115, wherein: (a) the polyribonucleotide comprises cytidine residues, and less than 100% of the cytidine residues in the polyribonucleotide comprise N4-acetylcytidine; and / or (b) the polyribonucleotide comprises uridine residues, and less than 100% of the uridine residues in the polyribonucleotide comprise 5-hydroxymethyluridine.

[0608] Embodiment 118. (a) 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 20%, about 5% to 25%, about 5% to 30%, about 5% to 35%, about 5% to 30%, about 5% to 25%, about 5% to 30 ... 5%, 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% and / or (b) the polyribonucleotide contains uridine residues, and the uridine residues in the polyribonucleotide account for 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%, The polyribonucleotide according to any one of embodiments 115 to 117, wherein 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 5-hydroxymethyluridine.

[0609] Embodiment 119. (a) 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; and / or (b) the polyribonucleotide comprises N4-acetylcytidine. 119. The polyribonucleotide of any one of embodiments 115-118, wherein the ribonucleotide 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.

[0610] Embodiment 120. The polyribonucleotide of embodiment 115, wherein: (a) the polyribonucleotide comprises cytidine residues, and more than 60% of the cytidine residues in the polyribonucleotide comprise N4-acetylcytidine; and / or (b) the polyribonucleotide comprises uridine residues, and more than 60% of the uridine residues in the polyribonucleotide comprise 5-hydroxymethyluridine.

[0611] Embodiment 121. (a) about 60-100%, about 65%-100%, about 70%-100%, about 75%-100%, about 80%-100%, about 85%-100%, about 90%-100%, about 95%-100%, about 60%-95%, about 60%-90%, about 60%-85%, about 60%-80%, about 60%-75%, about 60%-70%, about 60%-65% of the cytidine residues in the polyribonucleotide contain N4-acetylcytidine; and / or (b) the polyribonucleotide comprises N4-acetylcytidine; 121. The polyribonucleotide according to embodiment 115 or 120, wherein about 60 to 100%, about 65% to 100%, about 70% to 100%, about 75% to 100%, about 80% to 100%, about 85% to 100%, about 90% to 100%, about 95% to 100%, about 60% to 95%, about 60% to 90%, about 60% to 85%, about 60% to 80%, about 60% to 75%, about 60% to 70%, or about 60% to 65% of the uridine residues in the polyribonucleotide comprise 5-hydroxymethyluridine.

[0612] Embodiment 122. The polyribonucleotide of embodiment 115 or 120, wherein (a) at least about 75% of the cytidine residues in the polyribonucleotide comprise N4-acetylcytidine; and / or (b) at least about 75% of the uridine residues in the polyribonucleotide comprise 5-hydroxymethyluridine.

[0613] Embodiment 123. The polyribonucleotide of embodiment 115 or 120, wherein (a) 100% of the cytidine residues in the polyribonucleotide comprise N4-acetylcytidine; and / or (b) 100% of the uridine residues in the polyribonucleotide comprise 5-hydroxymethyluridine.

[0614] Embodiment 124. A polyribonucleotide described in any one of embodiments 94 to 123, characterized in that reduced immunogenicity is observed when assessed in a cell, tissue or organism to which the polyribonucleotide has been administered, compared to an appropriate reference standard.

[0615] Embodiment 125. The polyribonucleotide of embodiment 124, wherein the reference comparison standard comprises an otherwise similar cell, tissue or organism that has been administered a comparable polyribonucleotide that comprises: (i) fewer N4-acetylcytidine nucleosides (e.g., no N4-acetylcytidine nucleosides); and / or (ii) fewer 5-hydroxymethyluridine nucleosides (e.g., no 5-hydroxymethyluridine nucleosides).

[0616] Embodiment 126. The polyribonucleotide of embodiment 124 or 125, wherein the reduced immunogenicity comprises a reduced activation of innate immune response-induced toxicity.

[0617] Embodiment 127. The polyribonucleotide of embodiment 126, wherein the reduced activation of the immune response comprises a reduced activation of the NFκb, IRF pathways, and / or other cytokines resulting from inflammation in said cell, tissue, or organism.

[0618] Embodiment 128. The polyribonucleotide of embodiment 126 or 127, wherein the reduced activation of the immune response comprises reduced detection of uncapped RNA by a molecular sensor.

[0619] Embodiment 129. The polyribonucleotide of embodiment 128, wherein the molecular sensor is or comprises RIG-I.

[0620] Embodiment 130. The polyribonucleotide of any one of embodiments 124 to 129, wherein the reduced immunogenicity allows repeated administration of said polyribonucleotide.

[0621] Embodiment 131. The polyribonucleotide according to any one of embodiments 124 to 130, wherein the reduced immunogenicity allows for the administration of higher doses of said polyribonucleotide compared to a suitable reference standard.

[0622] Embodiment 132. The polyribonucleotide of embodiment 131, wherein the reference comparison standard comprises a comparable polyribonucleotide that comprises: (i) fewer N4-acetylcytidine nucleosides (e.g., no N4-acetylcytidine nucleosides); and / or (ii) fewer 5-hydroxymethyluridine nucleosides (e.g., no 5-hydroxymethyluridine nucleosides).

[0623] Embodiment 133. A polyribonucleotide described in any one of embodiments 94 to 132, characterized in that, when evaluated in a cell, tissue or organism to which the polyribonucleotide has been administered, an increase in cell viability is observed compared to an appropriate reference comparison standard.

[0624] Embodiment 134. The polyribonucleotide of embodiment 133, wherein the reference comparison standard is the cell viability of a cell, tissue or organism administered a comparable polyribonucleotide that contains (i) less N4-acetylcytidine nucleosides (e.g., no N4-acetylcytidine nucleosides); and / or (ii) less 5-hydroxymethyluridine nucleosides (e.g., no 5-hydroxymethyluridine nucleosides).

[0625] Embodiment 135. The polyribonucleotide of embodiment 133 or 134, wherein cell viability is a measure of the length of time one or more cells of said cell, tissue or subject are alive.

[0626] Embodiment 136. The polyribonucleotide of any one of embodiments 133 to 135, wherein cell viability is a measure of the number of cells in said cell, tissue or subject that are alive at one or more time points.

[0627] Embodiment 137. A polyribonucleotide described in any one of embodiments 94 to 136, characterized in that, when evaluated in a cell, tissue or organism to which the polyribonucleotide has been administered, elevated expression of the payload is observed compared to an appropriate reference standard.

[0628] Embodiment 138. The polyribonucleotide of embodiment 137, wherein the reference comparison standard comprises an otherwise similar cell, tissue or organism that has been administered a comparable polyribonucleotide that contains (i) fewer N4-acetylcytidine nucleosides (e.g., no N4-acetylcytidine nucleosides); and / or (ii) fewer 5-hydroxymethyluridine nucleosides (e.g., no 5-hydroxymethyluridine nucleosides).

[0629] Embodiment 139. The polyribonucleotide of embodiment 137 or 138, wherein said increase in expression of said payload is about 1.2-fold, about 1.5-fold, about 2-fold, about 4-fold, about 5-fold, about 10-fold, or about 20-fold compared to a reference standard.

[0630] Embodiment 140. The polyribonucleotide of any one of embodiments 137 to 139, wherein the payload is or comprises a polypeptide encoded by the polyribonucleotide comprising one or more modified ribonucleotides.

[0631] Embodiment 141. A polyribonucleotide according to any one of embodiments 137 to 140, wherein the payload is or comprises a polyribonucleotide located in the polyribonucleotide comprising one or more modified ribonucleotides.

[0632] Embodiment 142. The polyribonucleotide of any one of embodiments 2 to 31 or 94 to 141, wherein the polyribonucleotide is or comprises an RNA oligo.

[0633] Embodiment 143. The polyribonucleotide of any one of embodiments 2 to 31 or 94 to 141, wherein the polyribonucleotide is or comprises messenger RNA (mRNA).

[0634] Embodiment 144. The polyribonucleotide of any one of embodiments 2 to 31 or 94 to 141, wherein the polyribonucleotide is or comprises a gRNA.

[0635] Embodiment 145. The polyribonucleotide of any one of embodiments 2 to 31 or 94 to 141, wherein the polyribonucleotide is or comprises an inhibitory RNA.

[0636] Embodiment 146. A polyribonucleotide according to any one of embodiments 2 to 31 or 94 to 141, wherein the polyribonucleotide is or comprises a miRNA or siRNA.

[0637] Embodiment 147. The polyribonucleotide of any one of embodiments 2 to 31 or 94 to 141, wherein the polyribonucleotide is or comprises an antisense oligonucleotide.

[0638] Embodiment 148. A composition comprising one or more polyribonucleotides according to any one of embodiments 94 to 147.

[0639] Embodiment 149. The composition of embodiment 148, wherein the composition is a pharmaceutical composition.

[0640] Embodiment 150. The composition of embodiment 149, wherein the pharmaceutical composition is or comprises an immunogenic composition.

[0641] Embodiment 151. The composition of embodiment 149, wherein the pharmaceutical composition is or comprises a vaccine.

[0642] Embodiment 152. The composition of embodiment 149, wherein the pharmaceutical composition is or comprises a gene therapy.

[0643] Embodiment 153. The composition of embodiment 149, wherein the pharmaceutical composition is or comprises a chemotherapy.

[0644] Embodiment 154. The composition of embodiment 149, wherein the pharmaceutical composition is or comprises a protein replacement therapy.

[0645] Embodiment 155. The composition of embodiment 149, wherein the pharmaceutical composition is or comprises an immunotherapy, antibody therapy and / or immunomodulatory therapy.

[0646] Embodiment 156. The composition of embodiment 149, wherein the pharmaceutical composition is or comprises a cell engineering therapy.

[0647] Embodiment 157. The composition of any one of embodiments 149 to 156, wherein the composition comprises double-stranded RNA.

[0648] Embodiment 158. A method comprising administering to a cell, tissue or subject one or more polyribonucleotides according to any one of embodiments 94 to 147, or a composition according to any one of embodiments 148 to 157.

[0649] Embodiment 159. The method of embodiment 158, further comprising determining cell viability of the cell, tissue or subject.

[0650] Embodiment 160. The method of embodiment 158, wherein cell viability is a measure of the length of time that one or more cells of said cell, tissue or subject are alive.

[0651] Embodiment 161 The method of embodiment 158, wherein cell viability is a measure of the number of cells in said cell, tissue or subject that are alive at one or more time points.

[0652] Embodiment 162. The method according to any one of embodiments 158 to 161, wherein the cell, tissue or subject to which the polyribonucleotide or a composition comprising the polyribonucleotide has been administered exhibits improved cell viability compared to a reference cell viability.

[0653] Embodiment 163. The method of embodiment 162, wherein the reference comparison standard is cell viability of a cell, tissue or subject administered a comparable polyribonucleotide that contains (i) less N4-acetylcytidine nucleosides (e.g., no N4-acetylcytidine nucleosides); and / or (ii) less 5-hydroxymethyluridine nucleosides (e.g., no 5-hydroxymethyluridine nucleosides).

[0654] Embodiment 164. The method of any one of embodiments 158 to 163, further comprising determining the response of the immune system of the cell, tissue or subject to which the polyribonucleotide or a composition comprising the polyribonucleotide has been administered.

[0655] Embodiment 165. The method of embodiment 164, wherein the immune response comprises a response of the innate immune system, including innate immune system-induced toxicity.

[0656] Embodiment 166. The method of embodiment 165, wherein assessing the response of the innate immune system comprises determining levels of NF-κB, IRF, and / or other inflammatory cytokines in the cell, tissue, or subject.

[0657] Embodiment 167. The method of embodiment 166, wherein determining the response of the innate immune system comprises determining the level of detection of uncapped RNA by a molecular sensor.

[0658] Embodiment 168. The method of embodiment 167, wherein the molecular sensor is or comprises RIG-I.

[0659] Embodiment 169. The method according to any one of embodiments 164 to 168, wherein the cell, tissue or subject to which the polyribonucleotide or a composition comprising the polyribonucleotide has been administered exhibits a reduced response of the innate immune system compared to a reference.

[0660] Embodiment 170. The method of any one of embodiments 158 to 169, wherein the reference is a response of the innate immune system of a cell, tissue or subject that has been administered a comparable polyribonucleotide or a composition comprising the comparable polyribonucleotide that contains (i) fewer N4-acetylcytidine nucleosides (e.g., no N4-acetylcytidine nucleosides); and / or (ii) fewer 5-hydroxymethyluridine nucleosides (e.g., no 5-hydroxymethyluridine nucleosides).

[0661] Embodiment 171. The method of any one of embodiments 32 to 78 or 158 to 170, further comprising determining expression of the payload in the cell, tissue or subject to which the polyribonucleotide or a composition comprising the polyribonucleotide has been administered.

[0662] Embodiment 172. The method of embodiment 171, wherein the payload is or comprises a polypeptide encoded by the polyribonucleotide comprising one or more modified ribonucleotides.

[0663] Embodiment 173. The method of embodiment 171, wherein the payload is or comprises a polyribonucleotide located in the polyribonucleotide comprising one or more modified ribonucleotides.

[0664] Embodiment 174. The method of any one of embodiments 171 to 173, wherein determining the expression of the payload comprises determining the expression of an RNA, or a polypeptide, or both.

[0665] Embodiment 175. The method according to any one of embodiments 171 to 174, wherein the cell, tissue or subject to which the polyribonucleotide or a composition comprising the polyribonucleotide has been administered exhibits elevated expression of the payload compared to a reference.

[0666] Embodiment 176. The method of any one of embodiments 171 to 175, wherein the reference is expression of a payload in a cell, tissue or subject that has been administered a comparable polyribonucleotide or a composition comprising the comparable polyribonucleotide, which comprises (i) fewer N4-acetylcytidine nucleosides (e.g., no N4-acetylcytidine nucleosides); and / or (ii) fewer 5-hydroxymethyluridine nucleosides (e.g., no 5-hydroxymethyluridine nucleosides).

[0667] Embodiment 177. The method of any one of embodiments 171 to 176, wherein the increased expression of the payload is about 1.2-fold, about 1.5-fold, about 2-fold, about 4-fold, about 5-fold, about 10-fold, or about 20-fold compared to the reference.

[0668] Embodiment 178. The method of any one of embodiments 158 to 177, wherein the method comprises administering the polyribonucleotide or a composition comprising the polyribonucleotide to the cell, tissue or subject at least twice.

[0669] Embodiment 179. The method of any one of embodiments 158 to 178, wherein the method comprises administering the polyribonucleotide or a composition comprising the polyribonucleotide to the cell, tissue or subject 2, 3, 4, 5, 6, 7, 8, 9, or 10 times.

[0670] Embodiment 180. The method of embodiment 178 or 179, wherein at least two administrations of the polyribonucleotide or the composition comprising the polyribonucleotide to the cell, tissue or subject does not reduce the effectiveness of the polyribonucleotide or the composition comprising the polyribonucleotide compared to administration of a single dose of the polyribonucleotide or the composition comprising the polyribonucleotide.

[0671] Embodiment 181. The method of any one of embodiments 158 to 180, wherein the method comprises administering to the cell, tissue or subject the polyribonucleotide or a composition comprising the polyribonucleotide at a dose that is higher than a suitable reference standard.

[0672] Embodiment 182. The method of embodiment 181, wherein the reference comparison standard comprises a comparable polyribonucleotide that contains (i) fewer N4-acetylcytidine nucleosides (e.g., no N4-acetylcytidine nucleosides); and / or (ii) fewer 5-hydroxymethyluridine nucleosides (e.g., no 5-hydroxymethyluridine nucleosides).

[0673] Embodiment 183. The method of any one of embodiments 158 to 182, wherein the cell is a mammalian cell, the tissue is a mammalian tissue, or the subject is a mammal.

[0674] Embodiment 184. The method of embodiment 182, wherein the mammal is a human.

[0675] Embodiment 185. The method of any one of embodiments 158 to 184, wherein the method is a method of stimulating an immune response.

[0676] Embodiment 186. The method according to any one of embodiments 158 to 185, wherein said method is a vaccination method.

[0677] Embodiment 187. The method of any one of embodiments 158 to 185, wherein the method is a gene therapy method.

[0678] Embodiment 188. The method of embodiment 187, wherein the gene therapy method comprises delivery of one or more components of gene therapy, such as gRNA.

[0679] Embodiment 189. The method of any one of embodiments 158 to 185, wherein the method is a cell therapy engineering method.

[0680] Embodiment 190. The method according to any one of embodiments 158 to 185, wherein said method is an immunotherapy method or an antibody therapy method.

[0681] Embodiment 191. The method of embodiment 190, wherein the immunotherapy method comprises delivery of an immune modulating therapy and / or an immune checkpoint therapy.

[0682] Embodiment 192. The method of any one of embodiments 158 to 184, wherein the method is a method of protein replacement therapy.

[0683] Embodiment 193. The method of embodiment 192, wherein said method of protein replacement therapy comprises delivery of enzyme replacement therapy.

[0684] Embodiment 194. The method of any one of embodiments 158 to 185, wherein the method is a chemotherapy method.

[0685] Embodiment 195. A method of vaccination, comprising administering to a cell, a tissue or a subject one or more polyribonucleotides described in any one of embodiments 94 to 147 or a composition described in any one of embodiments 148 to 157.

[0686] Embodiment 196. A method of immunotherapy comprising administering to a cell, tissue or subject one or more polyribonucleotides described in any one of embodiments 94 to 147 or a composition described in any one of embodiments 148 to 157.

[0687] Embodiment 197. A method of gene therapy comprising administering to a cell, tissue or subject one or more polyribonucleotides described in any one of embodiments 94 to 147 or a composition described in any one of embodiments 148 to 157.

[0688] Embodiment 198. A method for protein replacement therapy, comprising administering to a cell, tissue or subject one or more polyribonucleotides described in any one of embodiments 94 to 147, or a composition described in any one of embodiments 148 to 157.

[0689] Embodiment 199. A method of cell engineering therapy, comprising administering to a cell, tissue or subject one or more polyribonucleotides described in any one of embodiments 94 to 147, or a composition described in any one of embodiments 148 to 157.

[0690] Embodiment 200. A method for obtaining a lower level of immunogenicity in a subject receiving a polyribonucleotide described in any one of embodiments 2 to 31 or 94 to 147, or a composition described in any one of embodiments 32 to 41 or 148 to 157, compared to a subject receiving a comparable unmodified polyribonucleotide, the method comprising administering to the subject the polyribonucleotide or a composition comprising the polyribonucleotide.

[0691] Embodiment 201. The method according to embodiment 200, wherein the polyribonucleotide according to any one of embodiments 2 to 31 or 94 to 147, or the composition according to any one of embodiments 32 to 41 or 148 to 157, does not contain a 5' cap, e.g., a 5'-5' triphosphate-linked guanosine.

[0692] Embodiment 202. The method according to embodiment 200 or 201, wherein the polyribonucleotide according to any one of embodiments 2 to 31 or 94 to 147, or the composition according to any one of embodiments 32 to 41 or 148 to 157, comprises a 5' phosphate group and / or a 5' hydroxyl group at the 5' end of the polyribonucleotide.

[0693] Embodiment 203. The subject receiving the polyribonucleotide according to any one of embodiments 2 to 31 or 94 to 147, or the composition according to any one of embodiments 32 to 41 or 148 to 157, and the subject receiving a comparable unmodified polyribonucleotide are the same subject. The method according to any one of embodiments 200 to 202.

[0694] Embodiment 204. The subject receiving the polyribonucleotide according to any one of embodiments 2 to 31 or 94 to 147, or the composition according to any one of embodiments 32 to 41 or 148 to 157, and the subject receiving the comparable unmodified polyribonucleotide are different subjects. The method according to any one of embodiments 200 to 202.

[0695] Embodiment 205. A method for producing an RNA composition, comprising introducing at least one modified ribonucleotide according to embodiment 93 or 115 into a polyribonucleotide.

[0696] Embodiment 206. The method of embodiment 205, wherein the polyribonucleotide further comprises one or more modified ribonucleotides other than 5-hydroxymethyluridine as described in any one of embodiments 103 to 106.

[0697] Embodiment 207 The method of embodiment 205 or 206, wherein the method does not include removing double-stranded RNA from the RNA composition.

[0698] Embodiment 208. A cell comprising a polyribonucleotide according to any one of embodiments 94 to 147, or a composition according to any one of embodiments 148 to 157.

[0699] Embodiment 209. Use of a modified ribonucleotide according to embodiment 93 or 115 in the manufacture of a polyribonucleotide.

[0700] Embodiment 210. The use according to embodiment 209, wherein the polyribonucleotide further comprises one or more modified ribonucleotides other than 5-hydroxymethyluridine.

[0701] Embodiment 211. Use of a polyribonucleotide according to any one of embodiments 94 to 147, or a composition according to any one of embodiments 148 to 157, for stimulating an immune response.

[0702] Embodiment 212. Use of a polyribonucleotide according to any one of embodiments 94 to 147, or a composition according to any one of embodiments 148 to 157, as a vaccine.

[0703] Embodiment 213. Use of a polyribonucleotide according to any one of embodiments 94 to 147, or a composition according to any one of embodiments 148 to 157, as immunotherapy, for example as antibody therapy, immunomodulatory therapy and / or immune checkpoint therapy.

[0704] Embodiment 214. Use of a polyribonucleotide according to any one of embodiments 94 to 147 or a composition according to any one of embodiments 148 to 157 as a gene therapy.

[0705] Embodiment 215. Use of a polyribonucleotide according to any one of embodiments 94 to 147 or a composition according to any one of embodiments 148 to 157 as a protein replacement therapy.

[0706] Embodiment 216. Use of a polyribonucleotide according to any one of embodiments 94 to 147 or a composition according to any one of embodiments 148 to 157 as a cell engineering therapy.

[0707] Embodiment 217. Use of a polyribonucleotide according to any one of embodiments 94 to 147 or a composition according to any one of embodiments 148 to 157 as a chemotherapy.

[0708] Embodiment 218. The use according to any one of embodiments 200 to 217, wherein the polyribonucleotide or a composition comprising the polyribonucleotide is administered to a cell, tissue or subject.

[0709] Embodiment 219. The use of embodiment 218, wherein the cell is a mammalian cell, the tissue is a mammalian tissue, or the subject is a mammal.

[0710] Embodiment 220. The use of embodiment 219, wherein the mammal is a human.

[0711] Equivalent Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Unless otherwise expressly stated or unless a contradiction or inconsistency would be apparent to one of ordinary skill in the art, the invention should be understood to encompass all variations, combinations, and permutations of one or more limitations, elements, clauses, descriptive terms, etc., from one or more of the recited claims that are introduced into another claim (or any other related claim) that depends from the same base claim. Furthermore, it should also be understood that any embodiment or aspect of the invention may be expressly excluded from the scope of the claims, regardless of whether a specific exclusion is described herein. The scope of the invention is not intended to be limited to the detailed description of the invention set forth above, but rather is as set forth in the following claims.

Claims

1. A modified ribonucleotide comprising a nucleoside containing an acetyl group, said nucleoside being N4-acetylcytidine, said modified ribonucleotide comprising (a) a 5′ monophosphate, (b) a 5' diphosphate, or (c) the modified ribonucleotide having a 5' triphosphate.

2. A polyribonucleotide comprising one or more modified ribonucleotides according to claim 1.

3. the polyribonucleotide comprises a cytidine nucleoside; and (i) at least 5% of the cytidine nucleosides in the polyribonucleotide comprise N4-acetylcytidine; or (ii) the polyribonucleotide of claim 2, wherein less than 100% of the cytidine nucleosides in the polyribonucleotide comprise N4-acetylcytidine.

4. The polyribonucleotide of claim 2 or 3, further comprising one or more modified ribonucleotides other than N4-acetylcytidine.

5. 5. The polyribonucleotide of claim 4, wherein said nucleoside of said one or more modified ribonucleotides is 5-hydroxymethyluridine and said modified ribonucleotide has a 5' monophosphate.

6. The polyribonucleotide of claim 2 or 3, characterized in that reduced immunogenicity is observed when assessed in a cell, tissue or organism to which the polyribonucleotide has been administered, compared to a suitable reference comparison standard, optionally comprising an otherwise similar cell, tissue or organism to which the reference comparison standard has been administered a comparable polyribonucleotide that contains fewer acetyl groups on the nucleobases (e.g., does not contain any acetyl groups on the nucleobases).

7. The polyribonucleotide of claim 6 , wherein the reduced immunogenicity comprises reduced activation of the innate immune response.

8. (i) the reduction in activation of the innate immune response includes a reduction in activation of NFκb, IRF pathways, and / or other cytokines resulting from inflammation in the cell, tissue, or organism; (ii) reduced immunogenicity allows repeated administration of said polyribonucleotide; and / or (iii) the reduced immunogenicity allows for the administration of higher doses of the polyribonucleotide as compared to the reference comparison standard.

9. The polyribonucleotide of claim 2 or 3, characterized in that when assessed in a cell, tissue or organism to which the polyribonucleotide has been administered, an increase in cell viability is observed compared to a suitable reference comparison standard, optionally wherein the reference comparison standard is the cell viability of a cell, tissue or organism to which a comparable polyribonucleotide has been administered that contains fewer acetyl groups on the nucleobases (e.g., does not contain any acetyl groups on the nucleobases).

10. 10. The polyribonucleotide of claim 9, wherein cell viability is or comprises: (i) a measure of the length of time that one or more cells of the cell, tissue or subject are alive; and / or (ii) a measure of the number of cells of the cell, tissue or subject that are alive at one or more points in time.

11. A modified ribonucleotide comprising a nucleoside containing a hydroxymethyl group, said nucleoside being 5-hydroxymethyluridine, said modified ribonucleotide comprising (a) a 5′ monophosphate, (b) a 5' diphosphate, or (c) the modified ribonucleotide having a 5' triphosphate.

12. A polyribonucleotide comprising one or more modified ribonucleotides according to claim 11.

13. the polyribonucleotide comprises a uridine nucleoside; (i) at least 5% of the uridine nucleosides in the polyribonucleotide comprise 5-hydroxymethyluridine; (ii) less than 100% of the uridine nucleosides in the polyribonucleotide comprise 5-hydroxymethyluridine; or (iii) the polyribonucleotide of claim 12, wherein greater than 60% of the uridine nucleosides in the polyribonucleotide comprise 5-hydroxymethyluridine.

14. The polyribonucleotide of claim 12 or 13, further comprising one or more modified ribonucleotides other than 5-hydroxymethyluridine.

15. 15. The polyribonucleotide of claim 14, wherein said nucleoside of said one or more modified ribonucleotides is N4-acetylcytidine and said modified ribonucleotide has a 5' monophosphate.

16. 1. A polyribonucleotide comprising one or more modified ribonucleotides, said one or more modified ribonucleotides comprising: (i) 5-hydroxymethyluridine, wherein the modified ribonucleotide has a 5' monophosphate; and (ii) N4-acetylcytidine, wherein the modified ribonucleotide has a 5' monophosphate. The polyribonucleotide comprising either or both of the following:

17. the polyribonucleotide comprises a cytidine nucleoside; and (a) at least 5% of the cytidine nucleosides in said polyribonucleotide comprise N4-acetylcytidine, and / or said polyribonucleotide comprises uridine nucleosides, wherein at least 5% of the uridine nucleosides in said polyribonucleotide comprise 5-hydroxymethyluridine; (b) less than 100% of the cytidine nucleosides in the polyribonucleotide comprise N4-acetylcytidine, and / or the polyribonucleotide comprises uridine nucleosides, and less than 100% of the uridine nucleosides in the polyribonucleotide comprise 5-hydroxymethyluridine; or 17. The polyribonucleotide of claim 16, wherein: (c) greater than 60% of the cytidine nucleosides in the polyribonucleotide comprise N4-acetylcytidine; and / or (b) the polyribonucleotide comprises uridine nucleosides, and greater than 60% of the uridine nucleosides in the polyribonucleotide comprise 5-hydroxymethyluridine.

18. a reduction in immunogenicity is observed when assessed in a cell, tissue or organism to which the polyribonucleotide has been administered, compared to an appropriate reference standard; Optionally, the reference comparison standard comprises an otherwise similar cell, tissue or organism that has been administered a comparable polyribonucleotide that contains (i) fewer N4-acetylcytidine nucleosides (e.g., no N4-acetylcytidine nucleosides), and / or (ii) fewer 5-hydroxymethyluridine nucleosides (e.g., no 5-hydroxymethyluridine nucleosides).

19. The polyribonucleotide of claim 18, wherein the reduced immunogenicity comprises reduced activation of the innate immune response.

20. A decrease in activation of the innate immune response (i) a reduction in the activation of NFκb, IRF pathways, and / or other cytokines of inflammation in said cell, tissue, or organism; and / or 20. The polyribonucleotide of claim 19, wherein (ii) the reduced activation of the immune response comprises reduced detection of uncapped RNA by a molecular sensor, and optionally, the molecular sensor is or comprises RIG-I.

21. The polyribonucleotide of claim 18, wherein the reduced immunogenicity allows for (i) repeated administration of the polyribonucleotide and / or (ii) administration of higher doses of the polyribonucleotide compared to the reference comparison standard.

22. When assessed in a cell, tissue or organism to which the polyribonucleotide has been administered, an increase in cell viability is observed compared to an appropriate reference standard; Optionally, the reference comparison standard is cell viability of a cell, tissue or organism that has been administered a comparable polyribonucleotide that contains (i) fewer N4-acetylcytidine nucleosides (e.g., no N4-acetylcytidine nucleosides), and / or (ii) fewer 5-hydroxymethyluridine nucleosides (e.g., no 5-hydroxymethyluridine nucleosides).

23. (i) cell viability is a measure of the length of time that one or more cells of said cell, tissue or subject are alive; or (ii) cell viability is a measure of the number of cells in the cell, tissue or subject that are alive at one or more time points.

24. When evaluated in a cell, tissue or organism to which the polyribonucleotide has been administered, increased expression of the payload is observed compared to an appropriate reference standard; Optionally, the reference comparison standard comprises an otherwise similar cell, tissue or organism that has been administered a comparable polyribonucleotide that contains (i) fewer N4-acetylcytidine nucleosides (e.g., no N4-acetylcytidine nucleosides), and / or (ii) fewer 5-hydroxymethyluridine nucleosides (e.g., no 5-hydroxymethyluridine nucleosides).

25. 25. The polyribonucleotide of claim 24, wherein the increase in expression of the payload is about 1.2-fold, about 1.5-fold, about 2-fold, about 4-fold, about 5-fold, about 10-fold, or about 20-fold compared to the reference standard.

26. The payload is (i) a polypeptide encoded by the polyribonucleotide; or (ii) polyribonucleotides 25. The polyribonucleotide of claim 24, which is or comprises:

27. 17. The polyribonucleotide of any one of claims 2, 12 or 16, wherein the polyribonucleotide is or comprises an RNA oligo, messenger RNA (mRNA), gRNA, inhibitory RNA, miRNA or siRNA, an antisense oligonucleotide, or a combination thereof.

28. 20. A composition comprising one or more polyribonucleotides according to any one of claims 2, 12 or 16.

29. 30. The composition of claim 28, wherein the composition is a pharmaceutical composition.

30. 30. The composition of claim 29, wherein the pharmaceutical composition is or comprises an immunogenic composition, a vaccine, a gene therapy, a chemotherapy, a protein replacement therapy, an immunotherapy, an antibody therapy, an immunomodulatory therapy, a cell engineering therapy, or a combination thereof.

31. A cell comprising a polyribonucleotide according to any one of claims 2, 12 or 16.

32. Use of a modified ribonucleotide according to claim 1 or 11 in the manufacture of an RNA composition.

33. Use of a composition comprising a polyribonucleotide according to any one of claims 2, 12 or 16 in the preparation of a drug for delivering a polyribonucleotide to a cell, tissue or subject.

34. The use of claim 2, (i) a method of stimulating an immune response; (ii) methods of antibody therapy; (iii) methods of immunomodulation; (iv) methods of vaccination; (v) gene therapy methods; (vi) cell therapy engineering methods; (vii) immunotherapeutic methods; (viii) methods of protein replacement therapy; (ix) a chemotherapy method, or (x) Any combination of (i) to (ix) The use according to claim 33, 35. A composition for use in delivering a polyribonucleotide to a cell, tissue or subject, the composition comprising a polyribonucleotide described in any one of claims 2, 12 or 16.

36. The use of claim 2 (i) a method of stimulating an immune response; (ii) methods of antibody therapy; (iii) methods of immunomodulation; (iv) methods of vaccination; (v) gene therapy methods; (vi) cell therapy engineering methods; (vii) immunotherapeutic methods; (viii) methods of protein replacement therapy; (ix) a chemotherapy method, or (x) Any combination of (i) to (ix) 36. The composition for use according to claim 35,

37. The composition for use described in claim 35, wherein delivery comprises administration of the polyribonucleotide to the cell, tissue or subject.

38. The method of claim 37, wherein cell viability of the cell, tissue, or subject is determined, and the cell, tissue, or subject to which the polyribonucleotide or a composition comprising the polyribonucleotide has been administered exhibits improved cell viability compared to a reference cell viability; The reference cell viability is (i) fewer N4-acetylcytidine nucleosides (e.g., no N4-acetylcytidine nucleosides), and / or (ii) fewer 5-hydroxymethyluridine nucleosides (e.g., no 5-hydroxymethyluridine nucleosides); or a composition comprising the comparable polyribonucleotide, or the cell viability of a cell, tissue or subject administered the comparable polyribonucleotide.

36. A composition for use according to claim 35.

39. The method of claim 39, further comprising determining a response of the immune system of the cell, tissue or subject to which the polyribonucleotide or a composition comprising the polyribonucleotide has been administered, the cell, tissue or subject to which the polyribonucleotide or a composition comprising the polyribonucleotide has been administered exhibits a reduced response of the innate immune system compared to a reference; The reference (i) fewer N4-acetylcytidine nucleosides (e.g., no N4-acetylcytidine nucleosides), and / or (ii) fewer 5-hydroxymethyluridine nucleosides (e.g., no 5-hydroxymethyluridine nucleosides); a response of the innate immune system of a cell, tissue or subject administered a comparable polyribonucleotide comprising the same or a composition comprising the comparable polyribonucleotide; 36. A composition for use according to claim 35. (i) the efficacy of the polyribonucleotide or the composition comprising the polyribonucleotide is determined in the cell, tissue or subject to which the polyribonucleotide or the composition comprising the polyribonucleotide has been administered, the efficacy being determined by determining an antibody response or a cellular response in the cell, tissue or subject; and / or (ii) determining expression of the payload in the cell, tissue, or subject to which the polyribonucleotide or a composition comprising the polyribonucleotide has been administered; The payload is (a) a polypeptide encoded by the polyribonucleotide; or (b) polyribonucleotides is or contains 36. A composition for use according to claim 35. (i) the cell is a mammalian cell. (ii) the tissue is mammalian tissue, or (iii) the subject is a mammal; 36. A composition for use according to claim 35.

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