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US-20260125682-A1 - CHEMICAL MODIFICATIONS OF SMALL INTERFERING RNA WITH MINIMAL FLUORINE CONTENT

US20260125682A1US 20260125682 A1US20260125682 A1US 20260125682A1US-20260125682-A1

Abstract

The present invention provides oligonucleotides comprising 2′-O-methyl (2′-OMe) and 2′-deoxy-2′-fluoro (2′-F) modifications, compositions thereof, and methods of use for reducing the expression or activity of a gene.

Inventors

  • Weimin Wang
  • Naim Nazef

Assignees

  • NOVO NORDISK A/S

Dates

Publication Date
20260507
Application Date
20250710

Claims (20)

  1. 1 - 30 . (canceled)
  2. 31 . An oligonucleotide comprising: a sense strand comprising 17-36 nucleotides, wherein the sense strand has a first region (R1) and a second region (R2), wherein the second region (R2) of the sense strand comprises a first subregion (S1), a second subregion (S2) and a tetraloop (L) or triloop (triL) that joins the first and second regions, wherein the first and second subregions form a second duplex (D2); an antisense strand comprising 20-22 nucleotides, wherein the antisense strand includes at least 1 single-stranded nucleotide at its 3′-terminus, wherein the sugar moiety of the nucleotide at position 5 of the antisense strand is modified with a 2′-F and the sugar moiety of each of the remaining nucleotides of the antisense strand is modified with a modification selected from 2′-O-propargyl, 2′-O-propylamin, 2′-amino, 2′-ethyl, 2′-aminoethyl (EA), 2′-fluoro (2′-F), 2′-O-methyl (2′-OMe), 2′-O-methoxyethyl (2′-MOE), 2′-O-[2-(methylamino)-2-oxoethyl] (2′-O-NMA), or 2′-deoxy-2′-fluoro-β-d-arabinonucleic acid (2′-FANA), and wherein the sense strand and antisense strand are separate strands; and a first duplex (D1) formed by the first region of the sense strand and the antisense strand, wherein the first duplex has a length of 12-20 base pairs and has 7-10 nucleotides that are modified at the 2′-position of the sugar moiety with 2′-F.
  3. 32 . The oligonucleotide of claim 31 , wherein the sugar moiety at positions 2 and 14 of the antisense strand is modified with 2′-F.
  4. 33 . The oligonucleotide of claim 32 , wherein the sugar moiety at each of up to 3 nucleotides at positions 1, 3, 7, and 10 of the antisense strand is additionally modified with 2′-F.
  5. 34 . The oligonucleotide of claim 31 , wherein the sugar moiety of each of the nucleotides at positions 8-11 of the sense strand is additionally modified with 2′-F.
  6. 35 . The oligonucleotide of claim 31 , wherein the sugar moiety of each of the nucleotides at positions 1-7 and 12-17 or 12-20 of the sense strand are modified with a modification selected from 2′-O-propargyl, 2′-O-propylamin, 2′-amino, 2′-ethyl, 2′-aminoethyl (EA), 2′-O-methyl (2′-OMe), 2′-O-methoxyethyl (2′-MOE), 2′-O-[2-(methylamino)-2-oxoethyl] (2′-O-NMA), or 2′-deoxy-2′-fluoro-β-d-arabinonucleic acid (2′-FANA).
  7. 36 . The oligonucleotide of claim 31 , wherein: the sugar moiety of each of the nucleotides at positions 2, 5, and 14 of the antisense strand is modified with 2′-F; or the sugar moiety of each of the nucleotides at positions 1, 2, 5, and 14 of the antisense strand is modified with 2′-F, or the sugar moiety of each of the nucleotides at positions 1, 2, 3, 5, 7, and 14 of the antisense strand is modified with 2′-F; or the sugar moiety of each of the nucleotides at positions 2, 3, 5, 7, and 14 of the antisense strand is modified with 2′-F; or the sugar moiety of each of the nucleotides at positions 1, 2, 3, 5, 10, and 14 of the antisense strand is modified with 2′-F; or the sugar moiety of each of the nucleotides at positions 2, 3, 5, 10, and 14 of the antisense strand is modified with 2′-F; or the sugar moiety of each of the nucleotides at positions 2, 3, 5, 7, 10, and 14 of the antisense strand is modified with 2′-F; and the sugar moiety of each of the remaining nucleotides of the antisense strand is modified with a modification selected from 2′-O-propargyl, 2′-O-propylamin, 2′-amino, 2′-ethyl, 2′-aminoethyl (EA), 2′-O-methyl (2′-OMe), 2′-O-methoxyethyl (2′-MOE), or 2′-deoxy-2′-fluoro-β-d-arabinonucleic acid (2′-FANA).
  8. 37 . The oligonucleotide of claim 31 , wherein the antisense strand has 3 nucleotides that are modified at the 2′-position of the sugar moiety with 2′-F.
  9. 38 . The oligonucleotide of claim 31 , wherein the second duplex has a length of 1-6 base pairs.
  10. 39 . The oligonucleotide of claim 31 , wherein the second duplex comprises at least one bicyclic nucleotide.
  11. 40 . The oligonucleotide of claim 39 , wherein the second duplex has a length of 1-3 base pairs.
  12. 41 . The oligonucleotide of claim 31 , wherein: the triloop has a nucleotide sequence of GAA or AAA; or the tetraloop is an RNA tetraloop selected from GAAA, UNCG, GNRA, or CUUG or a DNA tetraloop selected from d(GNAB), d(CNNG), or d(TNCG), wherein N is any one of U, A, C, G and R is G or A.
  13. 42 . The oligonucleotide of claim 31 , wherein the sugar moiety of each nucleotide in the second duplex is modified with 2′-O-methyl (2′-OMe).
  14. 43 . The oligonucleotide of claim 31 , wherein 1-3 nucleotides in the triloop or 1-4 nucleotides in the tetraloop are conjugated to a ligand.
  15. 44 . The oligonucleotide of claim 43 , wherein the ligand comprises N-acetylgalactosamine.
  16. 45 . The oligonucleotide of claim 31 , wherein the nucleotide at position 1 of the antisense strand comprises a phosphate mimic.
  17. 46 . The oligonucleotide of claim 31 , wherein the sense strand comprises 36 nucleotides and the antisense strand comprises 22 nucleotides.
  18. 47 . A single-stranded oligonucleotide comprising 20-22 nucleotides, wherein: the sugar moiety of each of the nucleotides at positions 2, 5, and 14 and optionally up to 3 of the nucleotides at positions 1, 3, 7, and 10 of the antisense strand is modified with 2′-F; and the sugar moiety of each of the remaining nucleotides of the antisense strand is modified with a modification selected from 2′-O-propargyl, 2′-O-propylamin, 2′-amino, 2′-ethyl, 2′-aminoethyl (EA), 2′-O-methyl (2′-OMe), 2′-O-methoxyethyl (2′-MOE), 2′-O-[2-(methylamino)-2-oxoethyl] (2′-O-NMA), or 2′-deoxy-2′-fluoro-β-d-arabinonucleic acid (2′-FANA).
  19. 48 . A pharmaceutical composition comprising the oligonucleotide of claim 31 and a pharmaceutically acceptable carrier.
  20. 49 . A method for reducing expression of a target gene in a subject, or treating a disease or disorder in a subject, comprising administering the oligonucleotide of claim 31 to the subject in an amount sufficient to reduce expression of a target gene in the subject.

Description

RELATED APPLICATIONS The present application is a Continuation of U.S. application Ser. No. 17/766,153 filed Apr. 1, 2022, which is a § 371 National Stage of PCT International Application No. PCT/US2020/053999, filed Oct. 2, 2020, which claims priority under 35 U.S.C. § 119(c) from U.S. Provisional Patent Application No. 62/909,278, filed Oct. 2, 2019, each of which is incorporated herein by reference in its entirety. SEQUENCE LISTING The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Jul. 10, 2025, is named 400930-002WO_176023_SL.txt and is 97,813 bytes in size. FIELD OF THE INVENTION The present disclosure relates to oligonucleotides (e.g., RNA interference oligonucleotides) comprising 2′-O-methyl (2′-OMe) and 2′-deoxy-2′-fluoro (2′-F) modifications. BACKGROUND OF THE INVENTION Oligonucleotides for reducing gene expression via RNA interference (RNAi) pathways have been developed. For example, RNAi oligonucleotides have been developed with each strand having sizes of 19-25 nucleotides with at least one 3′ overhang of 1 to 5 nucleotides (see, e.g., U.S. Pat. No. 8,372,968). Longer oligonucleotides have also been developed that are processed by Dicer to generate active RNAi products (see, e.g., U.S. Pat. No. 8,883,996). Further work produced extended double-stranded oligonucleotides where at least one end of at least one strand is extended beyond a duplex targeting region, including structures where one of the strands includes a thermodynamically-stabilizing tetraloop structure (see, e.g., U.S. Pat. Nos. 8,513,207 and 8,927,705, as well as WO2010033225, which are incorporated herein by reference in their entirety). Such structures may include single-stranded extensions (on one or both sides of the molecule) as well as double-stranded extensions. Chemical modification of such RNAi oligonucleotides is essential to fully harness the therapeutic potential of this class of molecules. Various chemical modifications have been developed and applied to RNAi oligonucleotides to improve their pharmacokinetics and pharmacodynamics properties (Deleavey & Damha, CHEM. BIOL., 19:937-954, 2012), and to block innate immune activation (Judge et al., MOL. THER., 13:494-505, 2006). One of the most common chemical modifications is to the 2′-OH of the furanose sugar of the ribonucleotides because of its involvement in the nuclease degradation. Fully chemically modified siRNAs with a combination of 2′-O-methyl (2′-OMe) and 2′-deoxy-2′-fluoro (2′-F) throughout the entire duplex have been reported and have demonstrated excellent stability and RNAi activity (Morrissey et al., HEPATOLOGY, 41:1349-1356, 2005; Allerson et al., J. MED. CHEM., 48:901-904, 2005; Hassler et al., NUCLEIC ACID RES., 46:2185-2196, 2018). More recently, N-acetylgalactosamine (GalNAc) conjugated chemically modified siRNAs have shown effective asialoglycoprotein receptor (ASGPr)-mediated delivery to liver hepatocytes in vivo (Nair et al., J. AM. CHEM. SOC., 136:16958-16961, 2014). Several GalNAc conjugated RNAi platforms including the GalNAc dicer-substrate conjugate (GalXC) platform, have advanced into clinical development for treating a wide range of human diseases. One major concern with using chemically modified nucleoside analogues in the development of oligonucleotide-based therapeutics, including RNAi GalNAc conjugates, is the potential toxicity associated with the modifications. The therapeutic oligonucleotides could slowly degrade in patients, releasing nucleoside analogues that could be potentially phosphorylated and incorporated into cellular DNA or RNA. In the field of antivirus therapeutics, toxicity has emerged during the clinical development of many small molecule nucleotide inhibitors (Feng et al., ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, 60:806-817, 2016). 2′-F modification of fully phosphorothioated antisense oligonucleotide has been reported to cause cellular protein reduction and double-stranded DNA breaks resulting in acute hepatotoxicity in vivo (Shen et al., Nucleic Acid Res., 43:4569-4578, 2015; Shen et al., NUCLEIC ACID RES., 46:2204-2217, 2018). No evidence has been observed so far for such liability of 2′-F modification in the context of RNAi oligonucleotides (Janas et al., NUCLEIC ACID THER., 26:363-371, 2016; Janas et al., NUCLEIC ACID THER., 27:11-22, 2016). Moreover, 2′-F siRNA have been well tolerated in clinical trials. Nonetheless, it is still desirable to minimize the use of unnatural nucleoside analogues such as 2′-F modified nucleosides in therapeutic RNA oligonucleotides. Unlike 2′-deoxy-2′-fluoro RNA, 2′-O-Methyl RNA is a naturally occurring modification of RNA found in tRNA and other small RNAs that arise as a post-transcriptional modification. It is also known that the bulkier 2′-O-Methyl modification confers better metabolic stability as compared to the less bulky 2′-F modification. T