US-12618064-B2 - Acetylated ribonucleic acids and uses thereof

Abstract

Disclosed herein is a modified ribonucleotide comprising a nucleoside comprising 2′-O-acetylated ribose, and polyribonucleotides comprising the same. Also provided herein are compositions comprising a polyribonucleotide of the present disclosure and methods of making and using the same.

Inventors

• Nikhil Dhar
• Kyle Backman
• Dario de Jesus Davila Pasillas
• Justin Sean Huang
• Nikolai Eroshenko

Assignees

• HELIX NANOTECHNOLOGIES INC

Dates

Publication Date

20260505

Application Date

20241219

Claims (20)

1. 1 . An in vitro transcription mixture comprising: (a) a DNA template, (b) at least one RNA polymerase, and (c) a plurality of ribonucleotides comprising one or more modified ribonucleotides, wherein the one or more modified ribonucleotides comprises: (i) a 5′-triphosphate, and (ii) a nucleoside comprising a 2′-O-acetylated ribose and a nucleobase, wherein the nucleobase is an adenine or a modified version thereof, a guanine or a modified version thereof, a cytosine or a modified version thereof, or a uracil or a modified version thereof.
2. 2 . The in vitro transcription mixture of claim 1 , wherein the mixture further comprises one or more polyribonucleotides, whereby the one or more polyribonucleotides were produced by incubating components (a), (b) and (c) in the mixture.
3. 3 . The in vitro transcription mixture of claim 2 , wherein the one or more polyribonucleotides comprises ribose moieties that are 2′-O-acetylated.
4. 4 . The in vitro transcription mixture of claim 3 , wherein the one or more polyribonucleotides has at least 5% ribose moieties that are 2′-O-acetylated.
5. 5 . The in vitro transcription mixture of claim 3 , wherein all of the ribose moieties in the one or more polyribonucleotides are 2′-O-acetylated.
6. 6 . The in vitro transcription mixture of claim 2 , wherein the one or more polyribonucleotides comprises one or more ribonucleotides that do not comprise a 2′-O-acetylated ribose.
7. 7 . The in vitro transcription mixture of claim 1 , wherein the RNA polymerase is or comprises: a bacteriophage RNA polymerase, a mitochondrial RNA polymerase, a eukaryotic RNA polymerase, a bacterial RNA polymerase, or any combination thereof.
8. 8 . The in vitro transcription mixture of claim 1 , wherein the RNA polymerase is or comprises: a T7 RNA polymerase, a T3 RNA polymerase, a SP6 RNA polymerase, a N4 virion RNA polymerase, a viral RNA polymerase, or a variant of any of the foregoing.
9. 9 . The in vitro transcription mixture of claim 2 , wherein the one or more polyribonucleotides comprises a coding region.
10. 10 . The in vitro transcription mixture of claim 9 , wherein the one or more polyribonucleotides encodes one or more target polypeptides.
11. 11 . The in vitro transcription mixture of claim 2 , wherein the one or more polyribonucleotides comprises a guide RNA, a short hairpin RNA, an siRNA, a microRNA, a long non-coding RNA, a circular RNA, or any combination thereof.
12. 12 . The in vitro transcription mixture of claim 1 , wherein the one or more modified ribonucleotides comprise one or more subsets of modified ribonucleotides, wherein each subset of modified ribonucleotides comprises a nucleobase that is N4-acetyl-cytidine (ac4C), 5-hydroxymethyluridine, N1-methylpseudouridine, pyridin-4-one ribonucleoside, 5-aza-uridine, 6-aza-uridine, 2-thio-5-aza-uridine, 2-thio-uridine (s2U), 5-methyl cytidine (m5C), 5-aza-cytidine, 6-aza-cytidine, pseudoisocytidine, 3-methyl-cytidine (m3C), 5-formyl-cytidine (f5C), N4-methyl-cytidine (m4C), 2-amino-purine, 2, 6-diaminopurine, 2-amino-6-halo-purine, 6-halo-purine, inosine (I), 1-methyl-inosine (m1 I), wyosine (imG), or methylwyosine (mimG).
13. 13 . The in vitro transcription mixture of claim 12 , wherein the one or more subsets of modified ribonucleotides comprise: (i) a subset of modified ribonucleotides comprising a nucleobase that is N4-acetyl-cytidine (ac4C), (ii) a subset of modified ribonucleotides comprising a nucleobase that is 5-hydroxymethyluridine, (iii) a subset of modified ribonucleotides comprising a nucleobase that is N1-methylpseudouridine, or (iv) any combination thereof.
14. 14 . The in vitro transcription mixture of claim 2 , further comprising a 5′ cap.
15. 15 . The in vitro transcription mixture of claim 2 , wherein the one or more polyribonucleotides further comprise a 5′ cap.
16. 16 . The in vitro transcription mixture of claim 14 , wherein the 5′ cap does not comprise a 2′-O-acetylated ribose.
17. 17 . The in vitro transcription mixture of claim 14 , wherein the 5′ cap comprises a 2′-O-acetylated ribose.
18. 18 . An in vitro transcription mixture comprising: (a) a DNA template, (b) at least one RNA polymerase, and (c) a plurality of ribonucleotides comprising one or more modified ribonucleotides, wherein the one or more modified ribonucleotides comprises: (i) a 5′-triphosphate, and (ii) a nucleoside comprising a 2′-O-acetylated ribose and a nucleobase, wherein the nucleobase is N4-acetylcytosine.
19. 19 . The in vitro transcription mixture of claim 18 , wherein the mixture further comprises one or more polyribonucleotides, wherein the one or more polyribonucleotides were produced by incubating components (a), (b) and (c) in the mixture, wherein the one or more polyribonucleotides comprise ribonucleotides comprising cytidine, and wherein at least about 5% of the ribonucleotides comprising cytidine nucleosides comprise an N4-acetylcytosine nucleobase.
20. 20 . The in vitro transcription mixture of claim 18 , wherein the one or more modified ribonucleotides further comprises: (i) a 5′-triphosphate, and (ii) a nucleoside comprising a 2′-O-acetylated ribose and a nucleobase, wherein the nucleobase is 5-hydroxymethyluracil.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS This application is a Divisional of U.S. application Ser. No. 18/143,533 filed on May 4, 2023, which claims priority to U.S. Provisional Patent Application 63/338,429 filed on May 4, 2022, the entire contents of each of which is hereby incorporated by reference. SEQUENCE LISTING The instant application contains a Sequence Listing which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. Said XML copy, created on Jul. 5, 2023 is named 2012611-0057_SL.xml and is 4,908 bytes in size. BACKGROUND RNA therapeutics is a new and emerging field. SUMMARY The present disclosure identifies certain challenges with the production of RNA for use in applications such as therapeutic applications, and challenges with the use of RNAs as therapeutics. For example, in some embodiments, the present disclosure identifies certain problems that can be encountered with using currently available RNAs for therapeutics due to, e.g., the short-lived activity and/or instability of RNAs. In some embodiments, instability of an RNA can be traced, e.g., to the reactivity of the hydroxyl group on carbon 2 of the ribose of a ribonucleotide. Among other things, the present disclosure provides technologies related to 2′-O-acetylated nucleotides in which a hydroxyl group on carbon 2 of a ribose is acetylated (2′-O-acetylated ribose). Without wishing to be bound by theory, in some embodiments, a ribonucleotide having a 2′-O-acetylated ribose can maintain crucial hydrogen bonding interactions necessary for transcription, translation, and duplex formation, while simultaneously having reduced reactivity that would otherwise promote RNA autohydrolysis and nuclease degradation. In some embodiments, a ribonucleotide having a 2′-O-acetylated ribose is more stable than a comparable RNA having fewer 2′-O-acetyl groups on a ribose. Among the technologies provided by this disclosure are technologies for using 2′-O-acetylated nucleotide triphosphates as a reagent for in vitro transcription of polyribonucleotides with RNA polymerase. In some embodiments, RNA polymerase can use 2′-O-acetylated nucleotide triphosphates to produce 2′-O-acetylated polyribonucleotides which can be translated by the intracellular machinery of a cell. In some embodiments, 2′-O-acetylated polyribonucleotides can be used for a variety of applications, including therapeutic applications, research applications, diagnostic applications, agricultural applications, and any other suitable applications. The present disclosure also provides technologies for reducing immunogenicity of RNA therapeutics by providing a polyribonucleotide comprising a modified ribonucleotide, e.g., a 2′-O-acetylated ribose. Without wishing to be bound by theory, the present disclosure proposes that a polyribonucleotide comprising a 2′-O-acetylated ribose can achieve reduced immunogenicity when administered in a cell, tissue or subject by reduced activation of an innate immune response. In some embodiments, reduced activation of an innate immune response, e.g., reduced activation of NF-kb or an NF-kb pathway, IRF or an IRF pathway, and/or other inflammatory cytokines; or reduced detection of uncapped RNA by a molecular sensor (e.g., RIG-I), with a polyribonucleotide comprising a 2′-O-acetylated ribose or a composition comprising the same allows for repeated dosing of, e.g., at least two doses of, said polyribonucleotide or a composition comprising the same. In some embodiments, a polyribonucleotide comprising a 2′-O-acetylated ribose, or a composition comprising the same can be administered at a higher dose compared to a reference polyribonucleotide that includes fewer 2′-O-acetyl groups on a ribose. Also provided herein are technologies for increasing expression from RNA therapeutics by providing a polyribonucleotide comprising a modified ribonucleotide, e.g., a ribonucleotide comprising a 2′-O-acetylated ribose. Without wishing to be bound by theory, the present disclosure proposes that a polyribonucleotide comprising 2′-O-acetylated ribose can achieve increased levels of protein or polypeptide expression when administered in a cell, tissue or subject, as compared to administration of a comparable polyribonucleotide having fewer 2′-O-acetyl groups on a ribose. Further provided herein are technologies for increasing persistence of RNA therapeutics by providing a polyribonucleotide comprising a modified ribonucleotide, e.g., a ribonucleotide comprising a 2′-O-acetylated ribose. Without wishing to be bound by theory, the present disclosure proposes that a polyribonucleotide comprising 2′-O-acetylated ribose can achieve increased persistence when administered in a cell, tissue or subject, as compared to administration of a comparable polyribonucleotide having fewer 2′-O-acetyl groups on a ribose. In some embodiments, increased persistence is a result of increased resistance to one or more nucleases. In some embodiments, increa