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CN-122003503-A - RAGE-targeting antisense oligonucleotides

CN122003503ACN 122003503 ACN122003503 ACN 122003503ACN-122003503-A

Abstract

The present invention relates to efficient splice switching antisense oligonucleotides that modulate the alternative splicing of pre-mRNA encoding late glycosylated end product Receptors (RAGE) or portions thereof, and methods of using said antisense oligonucleotides for treating RAGE-associated disorders. In one aspect, the invention provides an Antisense Oligonucleotide (AON) consisting of nucleotide sequence TGCCGCCTTTGCCACAAG (5 '-3'; SEQ ID NO: 1) or GCCGCCTTTGCCACAAGA (5 '-3'; SEQ ID NO: 2), preferably wherein the antisense oligonucleotide comprises at least one modified nucleotide.

Inventors

  • C j wright
  • THOMAS MYRON C.

Assignees

  • 瑞奇生物技术私人有限公司

Dates

Publication Date
20260508
Application Date
20241018
Priority Date
20231020

Claims (20)

  1. 1. An Antisense Oligonucleotide (AON), the antisense oligonucleotide consists of the following nucleotide sequence: TGCCGCCTTTGCCACAAG (5 '-3'; SEQ ID NO: 1), or GCCGCCTTTGCCACAAGA(5'-3';SEQ ID NO: 2)。
  2. 2. The antisense oligonucleotide of claim 1, wherein the antisense oligonucleotide comprises at least one modified nucleotide.
  3. 3. The antisense oligonucleotide of claim 2, wherein the modified nucleotide has a modified internucleotide linkage.
  4. 4. The antisense oligonucleotide of claim 3, wherein the modified internucleotide linkage has a phosphorothioate linkage.
  5. 5. The antisense oligonucleotide of any one of claims 2-4, wherein the modified nucleotide has a modified sugar moiety or glycomimetic.
  6. 6. The antisense oligonucleotide of claim 5 wherein the modified sugar moiety or glycomimetic is selected from the group consisting of morpholino, 2 'O-methyl (2' -OMe) and 2 '-O-methoxyethyl (2' -MOE), 2',4' constrained 2 '-O-methoxyethyl (cMOE), 2',4 'constrained 2' -O-ethyl (cEt), and Locked Nucleic Acid (LNA), preferably the modified sugar moiety is a 2 '-O-methoxyethyl (2' MOE) sugar moiety.
  7. 7. The antisense oligonucleotide of any one of claims 2-6, wherein the modified nucleotide has a modified nucleobase.
  8. 8. The antisense oligonucleotide of claim 7, wherein the modified nucleobase is a C5-methylated cytosine.
  9. 9. The antisense oligonucleotide of any one of claims 1-8, wherein the 5 'end of the antisense oligonucleotide has a 5' hydroxyl group.
  10. 10. The antisense oligonucleotide of any one of claims 1-5 and 7-9, wherein the antisense oligonucleotide is RNA or each nucleotide in the antisense oligonucleotide is a ribonucleotide.
  11. 11. The antisense oligonucleotide according to any one of claims 1 to 10, wherein one or more of thymine (T) as shown in SEQ ID No.1 or 2 is replaced by uracil (U) and/or pseudouracil (ψ).
  12. 12. The antisense oligonucleotide of claim 11, wherein one, two or three of the three consecutive T's shown in SEQ ID No. 1 or 2 are replaced by pseudouracil (ψ).
  13. 13. The antisense oligonucleotide according to any one of claims 1 to 10, wherein said T is C5-methyl-U.
  14. 14. The antisense oligonucleotide of any one of claims 1-13, wherein each nucleotide of the antisense oligonucleotide is modified and comprises: Phosphorothioate linkages, and -2 '-O-methoxyethyl (2' -MOE) sugar moiety.
  15. 15. The antisense oligonucleotide of any one of claims 1-13, wherein each nucleotide of the antisense oligonucleotide is modified and comprises: Phosphorothioate linkages, and -A 2 '-O-methoxyethyl (2' -MOE) sugar moiety, And wherein each cytosine in the antisense oligonucleotide is C5-methylated.
  16. 16. The antisense oligonucleotide of any one of claims 1-13, wherein each nucleotide of the antisense oligonucleotide is modified and comprises: Phosphorothioate linkages, and -A 2 '-O-methoxyethyl (2' -MOE) sugar moiety, And wherein each cytosine in the antisense oligonucleotide is a C5-methylated uracil and each T is a C5-methylated uracil.
  17. 17. The antisense oligonucleotide of any one of claims 1-16, wherein the antisense oligonucleotide consists of one of the following structures: -continuous process , Wherein x 1 is S and x 2 is O, or x 1 is O and x 2 is S; and MOE is 2-methoxyethyl.
  18. 18. A conjugate comprising the antisense oligonucleotide of claims 1-17 conjugated to a molecule.
  19. 19. The conjugate of claim 18, wherein the molecule is a cell-penetrating peptide (CPP).
  20. 20. The conjugate of claim 18, wherein the conjugate is in Vivo Morpholino Oligomer (VMO) or Peptide Phosphodiamide Morpholino Oligomer (PPMO).

Description

RAGE-targeting antisense oligonucleotides Technical Field The present invention relates to splice switching antisense oligonucleotides that modulate the alternative splicing of pre-mRNA encoding late glycosylated end product Receptors (RAGE) or portions thereof, and methods of using said antisense oligonucleotides for treating RAGE-associated disorders. RELATED APPLICATIONS The present application claims the benefit of priority from australian provisional application number 2023903354 filed 10/20 in 2023, the entire disclosure of which is incorporated herein by reference. Background The late glycosylation end product Receptor (RAGE) is a multivalent type I transmembrane glycoprotein belonging to the immunoglobulin (Ig) superfamily. The human RAGE (agent) gene is located within the major histocompatibility complex class III region on chromosome 6. It comprises 11 exons and 10 introns and a 5' flanking region that regulates its transcription. The transcribed RAGE mRNA was about 1.4 kb with a short 3' UTR. 50 The kDa-55 kDa glycosylated RAGE protein is constitutively expressed in a limited range of cells (e.g., vascular endothelium, type I lung cells, leukocytes), although RAGE expression can be induced in most cell types and tissues following injury, stress, hypoxia or inflammation, providing a channel for pro-inflammatory and pro-proliferative signaling. Thus, RAGE expression is upregulated in inflammatory and metabolic disorders including, but not limited to, neurodegenerative diseases, cancer, cardiovascular diseases, diabetes, autoimmune and ischemic injury, where RAGE is also associated with development and progression. Human RAGE consists of an immunoglobulin-like ectodomain, a single transmembrane domain, and a short (42 amino acids) cytoplasmic tail. The extracellular domain of RAGE (also known as the extracellular domain) comprises three immunoglobulin-like regions, an N-terminal V-domain, followed by two C-domains (known as C and C' or alternatively C1 and C2). Binding of advanced glycation end products (AGEs) and non-AGE ligands to the extracellular domain of RAGE activates intracellular signaling cascades associated with inflammation, injury, and cell proliferation and differentiation. RAGE activation also triggers a positive feedback loop in which RAGE ligand-receptor interactions increase RAGE expression via nfkb activation, thereby enhancing subsequent RAGE-induced cellular activation. Indeed, the inventors know that the only means to strongly down-regulate RAGE expression is to reduce RAGE activation. This is in contrast to other receptors in which increased levels of ligand reduce receptor expression. In humans, the cytoplasmic tail of RAGE is 43 amino acids long (residues 362-404). The cytoplasmic tail contains motifs critical for RAGE-dependent cell activation, but not ligand binding. The cytoplasmic tail of RAGE can be transactivated upon activation of a co-localized G-protein coupled receptor by its cognate ligand without the need for the AGE of a non-AGE ligand to bind to the extracellular domain of RAGE (also known as ligand independent activation of RAGE), thereby activating the same pathway (Pickering et al, J CLIN INVEST 2019; 129: 406-421). The RAGE ligand independent activation of certain co-localized activated G protein-coupled receptors on the cytoplasmic tail of RAGE appears to be an important pathway for RAGE activation in vivo. In both ligand-dependent and ligand-independent activation of RAGE, intracellular signaling is mediated by the cytoplasmic domain of RAGE, which interacts with a range of signaling partners. Alternative splicing of RAGE is also important for modulating RAGE activity by producing RAGE isoforms that have altered ability to be activated by ligand-dependent and ligand-independent signaling pathways. Alternative splicing of RAGE is altered in disease states including malignancy, diabetes and alzheimer's disease. Alternative splicing appears to be important for RAGE regulation/deregulation, and splice modified antisense oligonucleotides have been described that can promote alternative splicing. However, there remains a need for new and/or improved antisense oligonucleotides that more effectively promote alternative splicing, thereby promoting the production of endogenous soluble RAGE and reducing membrane-bound RAGE. The reference to any prior art in the specification is not intended to be an acknowledgement or suggestion that this prior art forms part of the common general knowledge in any jurisdiction or that this prior art could reasonably be expected to be understood by a person skilled in the art, considered relevant and/or combined with other prior art. Disclosure of Invention The present invention is based on the design and generation of antisense oligonucleotides that exhibit high potency to promote alternative splicing of RAGE precursor mRNA (e.g., comprising exon 9b and/or excluding (e.g., skipping) exon 10), such that the generation of endogenous s