CN-121986164-A - RNA editing oligonucleotides and uses thereof

Abstract

The disclosure features compositions and methods useful for treating a disorder in a subject in need thereof, to which deamination of adenosine in RNA produces a therapeutic outcome.

Inventors

• STEVE FAULHABER
• C. BROWN
• MOYER TOM
• M.R. PTA
• M. Masayevsky

Assignees

• 科罗生物公司

Dates

Publication Date

20260505

Application Date

20240715

Priority Date

20230713

Claims (20)

1. 1. An oligonucleotide comprising the structure: [A m ]-X 1 -X 2 -X 3 -[B n ] Wherein the method comprises the steps of M+n is 24 to 50, n is at least 4, and m is at least 20; -X 1 -X 2 -X 3 -is the central triplet of the oligonucleotide; x 1 is position-1 of the oligonucleotide, X 2 is position 0 of the oligonucleotide, and X 3 is position +1 of the oligonucleotide; [A] m is a first domain located at positions- (m+1) to-2 of the oligonucleotide; [B] n is a second domain located at positions +2 to + (n+1) of the oligonucleotide; each a and B is a nucleotide comprising a nucleobase, a sugar ("a/B sugar") and an internucleotide linkage; Each of X 1 、X 2 and X 3 comprises a nucleobase, a sugar ("X sugar") and an internucleotide linkage; The a/B sugar and the X 3 sugar are selected from the group consisting of 2 '-methoxy-ribose, 2' -MOE-ribose, 2 '-deoxy-2' -fluoro-ribose, 2 '-fluoro-arabinose, 2-methoxy-arabinose, 2' deoxyribose, and Locked Nucleic Acid (LNA); The X 1 sugar is 2' -deoxidized-2 ' -fluoro ribose or 2' -deoxidized ribose; The X 2 sugar is selected from the group consisting of 2 '-methoxy-ribose, 2' -MOE-ribose, 2 '-deoxy-2' -fluoro ribose, 2 '-fluoro-arabinose, 2-methoxy-arabinose, 2' -deoxyribose, locked Nucleic Acid (LNA), and β -homodna sugar; The a/B sugar and the X sugar together are 10-70% 2 '-deoxy-2' -fluoro ribose; the internucleotide linkages of the oligonucleotide are 30% -100% phosphorothioate and phosphoroamidate linkages, and 3 to 20 internucleotide linkages are phosphorophosphoramidate linkages; (i) between the nucleotide at position- (m+1) and the nucleotide at position- (m) (5 'end), (ii) between the nucleotide at position+ (n) and the nucleotide at position+ (n+1) (3' end), or (iii) the internucleotide linkage at each of the 5 'end and the 3' end of the oligonucleotide is a phosphoramidate linkage, and The internucleotide linkages between the nucleotide at position- (m) and the nucleotide at position- (m-1) and the internucleotide linkages between the nucleotide at position + (n-1) and the nucleotide at position + (n) are independently phosphorothioate linkages or phosphoroamidate linkages.
2. 2. The oligonucleotide of claim 1, wherein the X 2 nucleobase is cytosine or isodU.
3. 3. The oligonucleotide of claim 1 or claim 2, wherein the X 3 nucleobase is guanosine, hypoxanthine, or 7-deazaguanine.
4. 4. The oligonucleotide of any one of claims 1 to 3, wherein no more than four consecutive a/B saccharides are 2 '-deoxy-2' -fluoro ribose.
5. 5. The oligonucleotide of any one of claims 1 to 4, wherein the a/B sugar and the X sugar together are 20-50% 2 '-deoxy-2' -fluoro ribose.
6. 6. The oligonucleotide of any one of claims 1 to 5, wherein the a/B sugar is selected from the group consisting of 2' -methoxy-ribose, 2' -MOE-ribose, 2' -deoxy-2 ' -fluoro ribose, and 2' deoxyribose.
7. 7. The oligonucleotide of any one of claims 1 to 6, wherein the X 2 nucleobase is a cytosine.
8. 8. The oligonucleotide of any one of claims 1 to 7, wherein the X 2 sugar is a β -homodna sugar.
9. 9. The oligonucleotide of any one of claims 1 to 7, wherein the X 2 sugar is 2' -deoxyribose.
10. 10. The oligonucleotide of any one of claims 1 to 9, wherein the X 1 sugar is 2 '-deoxy-2' -fluoro ribose.
11. 11. The oligonucleotide of any one of claims 1 to 9, wherein the X 1 sugar is 2' -deoxyribose.
12. 12. The oligonucleotide of any one of claims 1 to 11, wherein the X 3 nucleobase is hypoxanthine.
13. 13. The oligonucleotide of any one of claims 1 to 12, wherein the internucleotide linkage between X 1 and X 2 is a phosphorothioate linkage or a phosphodiester linkage.
14. 14. The oligonucleotide of claim 13, wherein the internucleotide linkage between X 1 and X 2 is phosphorothioate.
15. 15. The oligonucleotide of any one of claims 1 to 14, wherein the internucleotide linkage between X 2 and X 3 is a phosphorothioate linkage or a phosphodiester linkage.
16. 16. The oligonucleotide of claim 15, wherein the internucleotide linkage between X 2 and X 3 is phosphorothioate.
17. 17. The oligonucleotide of any one of claims 1 to 16, wherein the internucleotide linkage between the nucleotide at position-2 and X 1 is a phosphorothioate linkage or a phosphodiester linkage.
18. 18. The oligonucleotide of claim 17, wherein the internucleotide linkage between the nucleotide at position-2 and X 1 is phosphorothioate.
19. 19. The oligonucleotide of any one of claims 1 to 18, wherein the internucleotide linkage between the nucleotide at position-9 and the nucleotide at position-8 is a phosphoramidate.
20. 20. The oligonucleotide of any one of claims 1 to 19, wherein the internucleotide linkage between the nucleotide at position-11 and the nucleotide at position-10 is a phosphoramidate.

Description

RNA editing oligonucleotides and uses thereof Incorporation of electronically submitted materials by reference As an independent part of this disclosure, the present application contains a sequence listing (file name: 50004_seqliping. Txt; size: 1,229,802 bytes; created on: 2024, 7, 11) in computer-readable form, which is incorporated by reference in its entirety. Background Adenosine Deaminase (ADAR) acting on RNA is an enzyme that binds to double-stranded RNA (dsRNA) and converts adenosine to inosine by deamination. In RNA, inosine acts similarly to guanosine for translation and replication. Thus, the conversion of adenosine to inosine in mRNA may cause a codon change that may alter the encoded protein and its function. There are three types of ADAR proteins known to be expressed in humans, namely ADAR1, ADAR2 and ADAR3.ADAR1 and ADAR2 are expressed systemically, whereas ADAR3 is expressed only in the brain. ADAR1 and ADAR2 have catalytic activity, while ADAR3 is considered inactive. Synthetic single stranded oligonucleotides have been shown to be able to utilize ADAR proteins to edit target RNAs by deaminating specific adenosines in the target RNAs. The oligonucleotide is complementary to the target RNA except for at least one mismatch relative to the adenosine to be deaminated. However, the previously disclosed methods have not been shown to have the selectivity and/or stability required to achieve their use as therapies. Thus, there is a need for new oligonucleotides that are capable of utilizing ADAR proteins in a therapeutically effective manner to selectively edit target RNAs. Disclosure of Invention The present disclosure provides oligonucleotides, compositions, and methods for deaminating adenosine in a target RNA (e.g., adenosine that can be deaminated to produce a therapeutic result), for example, in a subject in need thereof. In some embodiments, the target RNA is mRNA. Adenosine Deaminase (ADAR) acting on RNA is an editing enzyme that recognizes certain structural motifs of double-stranded RNA (dsRNA) and edits adenosine to inosine, thereby allowing recoding of amino acid codons, which may lead to changes in the encoded protein and its function. Nucleobases surrounding an editing site, particularly the nucleobase immediately 5 'and the nucleobase immediately 3' of the editing site, referred to as triplets together with the editing site, play an important role in deamination of adenosine. The preference of U at the 5 'position and G at the 3' position relative to the editing site is revealed by analysis of yeast RNAs efficiently edited by overexpressed human ADAR2 and ADAR 1. See Wang et al, (2018) Biochemistry 57:1640-1651, eifler et al, (2013) Biochemistry 52:7857-7869 and Eggington et al, (2011) natural communication (Nat. Commun.) 319:1-9. Regardless of the adjacent bases, recruiting ADAR to a specific site of a selected transcript and deaminating adenosine holds great promise for the treatment of disease. Based on structural and modeling studies of the editing site of dsRNA/ADAR complexes, several structural features have been identified that can be incorporated into guide oligonucleotides, whose characteristics can increase the recruitment of ADAR and can increase the editing efficiency of target RNAs. Novel oligonucleotides with chemical modifications are shown, such as α -homodna capable of recruiting ADAR proteins and deaminating adenosines in target RNAs with different peripheral base compositions. In addition, structure-activity relationship (SAR) studies revealed that 2 '-O-methyl (2' -OMe) modifications to ribose of some, but not all, nucleosides in the guide oligonucleotide are compatible with efficient ADAR recruitment and editing, in addition to triplet modifications. In one aspect, described herein is an oligonucleotide comprising the structure: [Am]-X1-X2-X3-[Bn] Wherein the method comprises the steps of M+n is 24 to 50, n is at least 4, and m is at least 20; -X 1-X2-X3 -is the central triplet of the oligonucleotide; x 1 is position-1 of the oligonucleotide, X 2 is position 0 of the oligonucleotide, and X 3 is position +1 of the oligonucleotide; [A] m is a first domain located at positions- (m+1) to-2 of the oligonucleotide; [B] n is a second domain located at positions +2 to + (n+1) of the oligonucleotide; each a and B is a nucleotide comprising a nucleobase, a sugar ("a/B sugar") and an internucleotide linkage; Each of X 1、X2 and X 3 comprises a nucleobase, a sugar ("X sugar") and an internucleotide linkage; The a/B sugar and the X 3 sugar are selected from the group consisting of 2 '-methoxy-ribose, 2' -MOE-ribose, 2 '-deoxy-2' -fluoro-ribose, 2 '-fluoro-arabinose, 2-methoxy-arabinose, 2' deoxyribose, and Locked Nucleic Acid (LNA); The X 1 sugar is 2' -deoxidized-2 ' -fluoro ribose or 2' -deoxidized ribose; The X 2 sugar is selected from the group consisting of 2 '-methoxy-ribose, 2' -MOE-ribose, 2 '-deoxy-2' -fluoro ribose, 2 '-fluoro-arabinose, 2-methoxy-arabinose, 2' -de