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CN-122003499-A - Antisense oligonucleotides and medical uses thereof

CN122003499ACN 122003499 ACN122003499 ACN 122003499ACN-122003499-A

Abstract

The present invention provides antisense oligonucleotides having a 2 '-O-methoxyethyl sugar modification at each position and at least one chiral internucleotide phosphorothioate linkage with Sp configuration at the 3' -terminus. The invention also relates to the use of such antisense oligonucleotides in medicine. The invention also provides compositions, including pharmaceutical compositions, comprising such antisense oligonucleotides. In addition, a method of manufacture is provided.

Inventors

  • JONATHAN HALL
  • Anastasia Ashkinazar
  • Eva Maria manz

Assignees

  • 苏黎世联邦理工学院

Dates

Publication Date
20260508
Application Date
20231019

Claims (15)

  1. 1. An antisense oligonucleotide comprising SEQ ID NO. 1 or a pharmaceutically acceptable salt thereof, Wherein the antisense oligonucleotide has a2 '-O-methoxyethyl sugar modification on each of its nucleotides and a chiral internucleotide phosphorothioate linkage of Sp configuration at the 3' -terminus.
  2. 2. The antisense oligonucleotide or pharmaceutically acceptable salt thereof according to claim 1, wherein the antisense oligonucleotide contains only phosphorothioate linkages.
  3. 3. The antisense oligonucleotide or pharmaceutically acceptable salt thereof according to claim 1 or 2, wherein the antisense oligonucleotide further has at least one or at least two chiral internucleotide phosphorothioate linkages having an Sp configuration that is continuous with the chiral internucleotide phosphorothioate linkages at the 3' -terminus.
  4. 4. The antisense oligonucleotide or a pharmaceutically acceptable salt thereof according to claim 1, Wherein all cytosine residues are 5-methylcytosine and Wherein the antisense oligonucleotide comprises: A chiral internucleotide phosphorothioate linkage having the Sp configuration at the 3' -terminus, while all other internucleotide linkages are achiral-controlled phosphorothioate linkages, or Two consecutive chiral internucleotide phosphorothioate linkages having Sp configuration at the 3' -terminus, while all other internucleotide linkages are achiral controlled phosphorothioate linkages, or Three consecutive chiral internucleotide phosphorothioate linkages having Sp configuration at the 3' -terminus, while all other internucleotide linkages are achiral controlled phosphorothioate linkages, or One chiral internucleotide phosphorothioate linkage with the Sp configuration at the 3' -terminus, while all other internucleotide linkages are chiral control phosphorothioate linkages with the Rp configuration.
  5. 5. A composition comprising a plurality of antisense oligonucleotides according to any one of claims 1 to 4 and/or pharmaceutically acceptable salts thereof.
  6. 6. A pharmaceutical composition comprising a pharmaceutically acceptable carrier or diluent, and The antisense oligonucleotide and/or pharmaceutically acceptable salt thereof according to any one of claims 1 to 4, or The plurality of antisense oligonucleotides and/or pharmaceutically acceptable salts thereof of claim 5.
  7. 7. A kit comprising a pharmaceutically acceptable carrier or diluent and one of the following in lyophilized form: the antisense oligonucleotide or pharmaceutically acceptable salt thereof according to any one of claims 1 to 4, or The composition of claim 5.
  8. 8. The antisense oligonucleotide or pharmaceutically acceptable salt thereof according to any one of claims 1 to 4, or The composition according to claim 5, or The pharmaceutical composition according to claim 6, or The kit according to claim 7, Is used as a medicament.
  9. 9. The antisense oligonucleotide or pharmaceutically acceptable salt thereof according to any one of claims 1 to 4, or The composition according to claim 5, or The pharmaceutical composition according to claim 6, or The kit according to claim 7, Can be used for treating spinal muscular atrophy.
  10. 10. A method of promoting the inclusion of exon 7 in an SMN2 transcript in a cell, tissue or organ comprising contacting the cell, tissue or organ with the antisense oligonucleotide of any one of claims 1 to 4 or a pharmaceutically acceptable salt thereof or the composition of claim 5, or the pharmaceutical composition of claim 6 in vitro.
  11. 11. A method of preparing an antisense oligonucleotide according to any one of claims 1 to 4, comprising the steps of: a) Providing a functionalized solid support represented by formula I, I is a kind of , Wherein L is a linker, preferably selected from: And ; SS stands for solid support; Ar 1 is phenyl optionally substituted with halo, C 1 -C 6 alkyl, C 3 -C 6 cycloalkyl or C 1 -C 4 alkoxy; x is selected from sulfur and oxygen; R 1 represents a hydrogen or hydroxy protecting group selected from the group consisting of trityl, 4-monomethoxytrityl, 4 '-dimethoxytrityl, 4' '-trimethoxytrityl, 9-phenylton-9-yl and 9- (p-methoxyphenyl) ton-9-yl, preferably selected from the group consisting of 4,4' -dimethoxytrityl and 4-Shan Jialiu-yl trityl; R 2 is selected from methyl and phenyl; R 3 is selected from hydrogen, acetyl and trifluoroacetyl; b) The following series of reactions were carried out: reacting a functionalized solid support of formula I with a nucleoside building block of formula (IIa-T), (IIb-T) or (IIc-T) to obtain a support-bound 3-mer oligonucleotide, and Reacting the support-bound 3-mer oligonucleotide with a nucleoside building block of formula (IIa-C), (IIb-C) or (IIc-C) to obtain a support-bound 4-mer oligonucleotide, followed by Reacting the support-bound 4-mer with a nucleoside building block of formula (IIa-G), (IIb-G) or (IIc-G) to obtain a support-bound 5-mer oligonucleotide, and Reacting the support-bound 5-mer with a nucleoside building block of formula (IIa-T), (IIb-T) or (IIc-T) to obtain a support-bound 6-mer oligonucleotide, and Reacting the support-bound 6-mer with a nucleoside building block of formula (IIa-A), (IIb-A) or (IIc-A) to obtain a support-bound 7-mer oligonucleotide, and Reacting the support-bound 7-mer with a nucleoside building block of formula (IIa-A), (IIb-A) or (IIc-A) to obtain a support-bound 8-mer oligonucleotide, and Reacting the support-bound 8-mer with a nucleoside building block of formula (IIa-T), (IIb-T) or (IIc-T) to obtain a support-bound 9-mer oligonucleotide, and Reacting the support-bound 9-mer with a nucleoside building block of formula (IIa-A), (IIb-A) or (IIc-A) to obtain a support-bound 10-mer oligonucleotide, and Reacting the support-bound 10-mer with a nucleoside building block of formula (IIa-C), (IIb-C) or (IIc-C) to obtain a support-bound 11-mer oligonucleotide, and Reacting the support-bound 11-mer with a nucleoside building block of formula (IIa-T), (IIb-T) or (IIc-T) to obtain a support-bound 12-mer oligonucleotide, and Reacting the support-bound 12-mer with a nucleoside building block of formula (IIa-T), (IIb-T) or (IIc-T) to obtain a support-bound 13-mer oligonucleotide, and Reacting the support-bound 13-mer with a nucleoside building block of formula (IIa-T), (IIb-T) or (IIc-T) to obtain a support-bound 14-mer oligonucleotide, and Reacting the support-bound 14-mer with a nucleoside building block of formula (IIa-C), (IIb-C) or (IIc-C) to obtain a support-bound 15-mer oligonucleotide, and Reacting the support-bound 15-mer with a nucleoside building block of formula (IIa-A), (IIb-A) or (IIc-A) to obtain a support-bound 16-mer oligonucleotide, and Reacting the support-bound 16-mer with a nucleoside building block of formula (IIa-C), (IIb-C) or (IIc-C) to obtain a support-bound 17-mer oligonucleotide, and Reacting the support-bound 17-mer with a nucleoside building block of formula (IIa-T), (IIb-T) or (IIc-T) to obtain a support-bound 18-mer oligonucleotide comprising SEQ ID NO. 1, Wherein the reaction comprises the steps of: b-1) the deprotection of the substrate, B-2) the coupling of the two components, B-3) end capping, and B-4) oxidizing or vulcanizing the mixture, B-1), b-2), b-3), b-4), b-1), b-2), b-4), b-3), b-2), b-3), b-4), b-1), or b-2), b-4), b-3), b-1). C) Optionally cleaving R 1 from the support-bound oligonucleotide, and D) Cleaving the support-bound 18-mer oligonucleotide from the solid support to obtain an antisense oligonucleotide, Wherein the nucleic acid construct units of the formulae (IIa-G), (IIa-T), (IIa-C), (IIa-A), (IIb-G), (IIb-T), (IIb-C), (IIb-A), (IIc-G), (IIc-T), (IIc-C) and (IIc-A) are respectively represented by the following formulae: formula (IIa-G) formula (IIa-T) formula (IIa-C) formula (IIa-A) Formula (IIb-G) formula (IIb-T) formula (IIb-C) formula (IIb-A) Formula (IIc-G) formula (IIc-T) formula (IIc-C) formula (IIc-A) Wherein Ar 2 is phenyl optionally substituted with halogen, C 1 -C 6 alkyl, C 3 -C 6 cycloalkyl or C 1 -C 4 alkoxy, and R 4 represents a protecting group for a hydroxyl group selected from the group consisting of trityl, 4-monomethoxytrityl, 4 '-dimethoxytrityl, 4' '-trimethoxytrityl, 9-phenylton-9-yl and 9- (p-methoxyphenyl) ton-9-yl, preferably selected from the group consisting of 4,4' -dimethoxytrityl and 4-Shan Jialiu-yltrityl.
  12. 12. The method of claim 11, wherein step a) comprises A-1) providing a solid support represented by formula III Formula III , Wherein L is selected from: And ; SS stands for solid support SS; R 1 represents a hydrogen or hydroxy protecting group selected from the group consisting of trityl, 4-monomethoxytrityl, 4 '-dimethoxytrityl, 4' '-trimethoxytrityl, 9-phenylton-9-yl and 9- (p-methoxyphenyl) ton-9-yl, preferably selected from the group consisting of 4,4' -dimethoxytrityl and 4-Shan Jialiu-yl trityl; R 2 is selected from methyl and phenyl, then A-2) reacting the nucleoside building blocks of formula (IIa-G) with a solid support of formula III, thereby obtaining a nucleotide bound to the support, and then Reacting the nucleoside building blocks of formula (IIb-G) with a nucleotide bound to a support, thereby obtaining a support-bound 2-mer oligonucleotide.
  13. 13. An antisense oligonucleotide represented by SEQ ID No. 1 obtainable by the method according to claim 11 or 12.
  14. 14. The antisense oligonucleotide of claim 13, wherein at least 95% of the antisense oligonucleotide remains full length after incubation in mouse liver lysate for at least 5 days as measured by LC-MS.
  15. 15. The antisense oligonucleotide of claim 13 or 14, wherein the full length antisense oligonucleotide is increased by at least 5 percent compared to sodium norcinacate after incubation in mouse liver lysate for at least 5 days as measured by LC-MS.

Description

Antisense oligonucleotides and medical uses thereof Technical Field The present invention relates to the field of antisense oligonucleotides, in particular splice switching oligonucleotides (SPLICE SWITCHING oligonucleosides), and their use in medicine, in particular in the treatment of spinal muscular atrophy. Background Antisense oligonucleotide: Antisense oligonucleotides (ASOs) are relatively short single-stranded nucleic acids, typically between 10 and 30 nucleotides in length, that bind to complementary messenger RNAs (mRNA, including pre-mRNA). In contrast to many traditional drugs, which typically inhibit protein function by binding to a specific pocket of an enzyme, ASO alters protein biosynthesis as the protein is formed. General classification of ASOs can be made according to their respective mechanisms of action, in particular according to whether they function by a so-called "target occupancy" mechanism or by an "occupancy-mediated degradation" mechanism. ASOs that act by "occupancy-mediated degradation" include in particular RNase H-dependent ASOs, which are commonly used to down-regulate pathogenic proteins by enzymatic degradation of the respective mRNA. An example of such an RNase H-dependent ASO is miphene (mipomersen), which binds to ApoB-100 mRNA and induces its degradation by RNase H, thereby inhibiting translation of the ApoB-100 protein. In contrast, ASOs that act primarily through a so-called "target-occupying" mechanism do not typically induce enzymatic RNA degradation. A notable example of a so-called "target-occupying" ASO is a Splice Switching Oligonucleotide (SSO) that binds to certain regions of the pre-mRNA involved in splicing, such as splice sites or regulatory elements, in order to regulate splicing in the event that normal splicing is disrupted. In particular, SSO can interfere with endogenous RNA splicing mechanisms by sterically blocking the accessibility of certain splice sites and/or regulatory elements. The ASO field, and particularly the SSO field, has recently attracted attention due to the progress of treatment of neurological diseases. For example, sodium norcinacalcet (nusinersen), sold under the name Spinraza, was the first drug to be used in the treatment of spinal muscular atrophy (SMA, a rare neuromuscular disease). Treatment of spinal muscular atrophy: SMA is a rare neuromuscular disease caused by homozygous deletion of the SMN1 gene encoding the motor neuron Survival (SMN) protein. In humans, SMN proteins are produced additionally from the SMN2 gene. However, the production of SMN protein from the SMN2 gene is limited. Typically, SMN2 protein expression is inhibited by a splice enhancer sequence, resulting in the exclusion of exon 7, and thus an unstable truncated SMN protein. Sodium norcinal is an ASO that binds to and alters splicing of SMN2 pre-mRNA, thereby including exon 7 and restoring loss of SMN protein. In addition, SMA can be treated by a recently developed gene therapy called zoqinma (zolgensma). However, the use of zoqinma is limited due to the high costs associated with such gene therapy methods. Chemical oligonucleotide synthesis: General methods of chemical oligonucleotide synthesis are known in the art and generally involve the stepwise addition of nucleoside building blocks to the 5' end of a growing oligonucleotide strand bound to a solid support. Each addition is commonly referred to as a synthesis cycle and typically includes four chemical reactions: deprotection, removal of the protecting group of the 5 '-hydroxy group (e.g., 4' -dimethoxytrityl) of the solid support-attached oligonucleotide or the hydroxy protecting group present on the solid support in the first synthesis cycle (i.e., when no nucleoside is attached to the solid support, the universal solid support). Coupling Once the hydroxyl protecting group is removed, the free 5' -OH of the solid support-attached oligonucleotide or the free hydroxyl of the solid support can react with the next nucleoside, which is typically added as a monomeric nucleoside building block, such as a Phosphoramidite (PA) building block or an oxazaphospholane (oxazaphospholidine, OAP) building block. The coupling reaction results in the formation of phosphite triester linkages. Blocking unreacted 5' -hydroxyl or free hydroxyl groups of the solid support) is blocked, for example, using acetic anhydride in the presence of a base. If not blocked, these hydroxyl groups will react in the next cycle, resulting in an oligonucleotide comprising internal deletions. Oxidation or sulfidation the phosphite triesters formed during the coupling reaction are relatively unstable under the conditions of oligonucleotide synthesis. Thus, it must be converted to a more stable phosphorus species, particularly to a phosphodiester bond, typically by oxidation using aqueous iodine or to a phosphorothioate bond, typically by sulfidation using commercially available sulfiding reagents, before the next synthesis cycle begins. So