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WO-2026092294-A1 - ANTISENSE OLIGONUCLEOTIDE FOR MODULATING ALTERNATIVE SPLICING OF IL1RL2 GENE, PREPARATION METHOD THEREFOR AND USE THEREOF

WO2026092294A1WO 2026092294 A1WO2026092294 A1WO 2026092294A1WO-2026092294-A1

Abstract

Provided is an antisense oligonucleotide which interferes with the alternative splicing of an IL1RL2 gene, thereby inducing the production of a truncated IL1RL2 splice isoform transcript. The antisense oligonucleotide can reduce the proportion of mRNA encoding full-length functional IL-36R by means of inducing exon 4 skipping in the pre-mRNA of the IL1RL2 gene, thereby blocking the activation of an IL-36R signaling pathway, and can be used for treating autoimmune diseases, allergic diseases, neurodegenerative diseases or cancers associated with abnormal activation of IL-36R.

Inventors

  • XIONG, Zhiqi
  • CHENG, XUEWEN
  • LI, XUEQUAN
  • LI, KUI

Assignees

  • 中国科学院脑科学与智能技术卓越创新中心
  • 临港国家实验室

Dates

Publication Date
20260507
Application Date
20251023
Priority Date
20241028

Claims (18)

  1. An oligonucleotide, characterized in that the oligonucleotide interferes with alternative splicing of the IL1RL2 gene, thereby inducing the production of truncated or cryptic exon inserted IL1RL2 splice isoform transcripts; Specifically, relative to the normal IL1RL2 transcript (NM_003854), the oligonucleotide targets are selected from the following group of regions: (1) The exon 4 and intron 3 or their boundary region and the exon 4 and intron 4 or their boundary region; (2) The exon 4 and intron 3 or their boundary region; (3) The exon 10 and intron 9 or their boundary region and the exon 10 and intron 10 or their boundary region; (4) The intron 8 sequence.
  2. The oligonucleotide of claim 1, wherein the oligonucleotide targets a region selected from the group consisting of: (1) Located in the c.294-100 to c.344 region or c.439 to c.489+100 region of the IL1RL2 transcript (NM_003854); (2) Located in the c.294-100 to c.467 region of the IL1RL2 transcript (NM_003854); (3) Located in the c.1136-100 to c.1186 region or c.1247 to c.1297+100 region of the IL1RL2 transcript (NM_003854); (4) Located in the c.991+2372～c.991+2522 region or c.991+2541～c.991+2691 region of the IL1RL2 transcript (NM_003854).
  3. The oligonucleotide of claim 1, wherein the truncated IL1RL2 splice isoform transcript is selected from the group consisting of: The Z1 transcript, with the nucleotide sequence shown in SEQ ID NO.5, contains a truncated exon 4; The Z2 transcript, with the nucleotide sequence shown in SEQ ID NO.9, does not contain exon 10; The Z3 transcript, with its nucleotide sequence shown in SEQ ID NO.3, does not contain exon 4.
  4. The oligonucleotide as described in claim 1 is characterized in that the oligonucleotide is an antisense oligonucleotide (ASO).
  5. The oligonucleotide of claim 1, wherein the cryptic exon insertion type IL1RL2 splice isoform transcript is selected from the group consisting of: The Z4 transcript, with its nucleotide sequence shown in SEQ ID NO.13, retains the hidden exon of intron 8.
  6. The oligonucleotide of claim 1, wherein the truncated or cryptic exon insertion IL1RL2 splice isoform transcript encodes a mutant protein selected from the group consisting of: The P1 mutant protein has the amino acid sequence shown in SEQ ID NO. 6; The P2 mutant protein has the amino acid sequence shown in SEQ ID NO.14; The P3 mutant protein has the amino acid sequence shown in SEQ ID NO.10; The P4 mutant protein has the amino acid sequence shown in SEQ ID NO.4.
  7. The oligonucleotide of claim 4, characterized in that the antisense oligonucleotide specifically binds to the nucleotide sequence corresponding to exon 4 in IL1RL2 mRNA and has the core sequence TATACC (SEQ ID NO. 15) shown in SEQ ID NO. 15. Each T in the formula can be replaced by U independently.
  8. The oligonucleotide of claim 4, wherein the antisense oligonucleotide has the structure of formula I: S1-S2-S3(I) in, S1 is a left extension sequence located at the 5' end of the core sequence, and the length L1 of the left extension sequence is 0-20 nt; and when L1≥1, the left extension sequence sequentially includes nucleotides from position 21-L1 to position 20 in 5'-AAGAAAATTCTTTTTTACCT-3' (i.e., when L1＝1, S1 is T; when L1＝2, S1 is CT; ...; when L1＝20, S1 is 5'-AAGAAAATTCTTTTTTACCT-3'). S2 is the core sequence TATACC; S3 is a right extension sequence located at the 3' end of the core sequence, and the length L2 of the right extension sequence is 0-15 nt. When L2 ≥ 1, the right extension sequence includes the nucleotides from position 1 to position L2 of 5'-ACTTTATTGGACCCA-3' (i.e., when L2 = 1, S3 is A; when L2 = 2, S3 is AC; ...; when L2 = 15, S3 is 5'-ACTTTATTGGACCCA-3'). Furthermore, each T in the antisense oligonucleotide can be independently replaced by U.
  9. The oligonucleotide of claim 4, wherein the antisense oligonucleotide sequence is any one of SEQ ID NO. 17-32, 65-71.
  10. The oligonucleotide of claim 4, wherein the antisense oligonucleotide is modified or unmodified.
  11. The oligonucleotide of claim 10 is characterized in that the modification includes a first-generation modification and a second-generation modification, wherein the first-generation modification includes phosphate thioester modification (PS) in the phosphate ribose backbone; and the second-generation modification includes alkyl/alkynyl modification at the 2' position, preferably 2'MOE (methoxyethyl), 2'Ome (methoxy), 2'-LNA (locked nucleic acid), or 2'-cET modification.
  12. A polynucleotide, characterized in that the polynucleotide encodes a variant transcript of IL-36R, wherein, compared with the normal IL1RL2 transcript, the variant transcript is selected from the group consisting of: (1) Transcript Y1, wherein the transcript Y1 is spliced and integrated with a truncated exon 4 sequence and has the nucleotide sequence shown in SEQ ID NO.5; (2) Transcript Y2, which has a hidden exon sequence of intron 8 spliced and integrated, and has a nucleotide sequence as shown in SEQ ID NO.13; (3) Transcript Y3, which does not contain the exon 10 sequence and has the nucleotide sequence shown in SEQ ID NO.9; (4) Transcript Y4, which does not contain exon 4 sequence and has a nucleotide sequence as shown in SEQ ID NO.3.
  13. An IL-36R mutant protein, characterized in that, compared with the wild-type IL1RL2 protein, the mutant protein is selected from the group consisting of: The P1 mutant protein has the amino acid sequence shown in SEQ ID NO. 6; The P2 mutant protein has the amino acid sequence shown in SEQ ID NO.14; The P3 mutant protein has the amino acid sequence shown in SEQ ID NO.10; The P4 mutant protein has the amino acid sequence shown in SEQ ID NO.4.
  14. A pharmaceutical composition characterized by comprising the oligonucleotide of claim 1, or a pharmaceutically acceptable salt, hydrate or solvate thereof, and a pharmaceutically acceptable carrier.
  15. Use of an oligonucleotide as claimed in claim 1, or a pharmaceutical composition as claimed in claim 14, in the preparation of a medicament for treating and/or preventing diseases associated with abnormal activation of IL-36R.
  16. A medicine box, characterized in that it comprises: (i) a first container, and a first pharmaceutical composition located in the first container, the first pharmaceutical composition comprising (a1) an oligonucleotide or oligonucleotide conjugate as claimed in claim 1, and (a2) a first pharmaceutically acceptable carrier.
  17. A method for treating and/or preventing diseases associated with abnormal activation of IL-36R, characterized by the step of administering to a desired subject a safe and effective amount of the oligonucleotide as claimed in claim 1, or the pharmaceutical composition as claimed in claim 14.
  18. A method for inducing the production of IL1RL2 variant mRNA transcripts, the method comprising: delivering to cells an oligonucleotide as described in claim 1 or a pharmaceutical composition as described in claim 14, thereby inducing the production of IL1RL2 variant mRNA transcripts.

Description

Antisense oligonucleotides regulating alternative splicing of the IL1RL2 gene, their preparation and applications Technical Field This invention relates to the field of biotechnology, specifically to antisense oligonucleotides that regulate alternative splicing of the IL1RL2 gene, their preparation methods, and applications. Background Technology Interleukin-36 (IL-36) is a member of the IL-1 family and plays an important role in immune and inflammatory responses. IL1RL2, as the major subunit of the interleukin-36 receptor (IL-36R), activates downstream signaling pathways by forming a heterodimer with the co-receptor IL-1RAcP (IL-1 Receptor Accessory Protein). Abnormal activation levels of the IL-36R signaling pathway are associated with the pathogenesis of inflammation, autoimmunity, allergies, and cancer. IL-36R can be activated by three extracellular ligand agonists (IL-36α, IL-36β, and IL-36γ), and its activation is inhibited by the extracellular secreted antagonists IL-36Ra or IL-38. The extracellular domain of IL-36R is its ligand-binding domain, also known as the immunoglobulin domain. When IL-36R binds to IL-36α, IL-36β, IL-36γ, or IL-36R, it recruits the co-receptor IL-1RAcP to form a signal transduction complex. Both IL-1RL2 and IL-1RAcP have an intracellular TIR (Toll/IL-1 Receptor domain) responsible for intracellular signal transduction. After ligand-induced dimer formation, the TIR domain recruits aptamers, such as MyD88 (Myeloid Differentiation Factor 88), which in turn activates pathways such as IL-1R-associated kinases (IRAKs), tumor necrosis factor receptor-associated factor 6 (TRAF6), or MAPK (P38). Ultimately, this activates transcription factors related to inflammation and immune responses, such as nuclear factor-κB (NF-κB) and activator protein-1 (AP-1), which translocate to the cell nucleus. This leads to the expression of pro-inflammatory factors, chemokines, and secondary mediators of the inflammatory response, thereby exerting a pro-inflammatory effect. Currently, most drugs targeting IL-36R that are under development or on the market are monoclonal antibodies. However, they have certain limitations: First, monoclonal antibodies generally have a short half-life in the body (ranging from one month to a few days). This short half-life leads to frequent dosing, increasing adverse side effects, resulting in high costs for the healthcare system, and reducing patient adherence. Second, most monoclonal antibodies, due to their large size, have limited ability to penetrate and accumulate in tissues; they are confined to the interstitial spaces after injection and cannot deliver proteins in the target tissue to maintain an effective therapeutic concentration. Third, in addition to their immunomodulatory functions, monoclonal antibodies can also induce immune responses in the body, causing other autoimmune inflammations. For example, in a study of 27 patients with multiple sclerosis treated with the monoclonal antibody alemituzumab, nine patients experienced antibody-mediated thyroid autoimmunity after using alemituzumab. Therefore, there is an urgent need in this field to develop a targeted, non-antibody therapy. In particular, there is a pressing need for an antisense oligonucleotide that regulates alternative splicing of the IL1RL2 gene, along with its preparation and application. Summary of the Invention The purpose of this invention is to provide an antisense oligonucleotide that regulates alternative splicing of the IL1RL2 gene, its preparation method, and its application. In a first aspect of the invention, an oligonucleotide is provided that interferes with alternative splicing of the IL1RL2 gene, thereby inducing the production of truncated or cryptic exon inserted IL1RL2 splice isoform transcripts. Specifically, relative to the normal IL1RL2 transcript (NM_003854), the oligonucleotide targets are selected from the following group of regions: (1) The exon 4 and intron 3 or their boundary region and the exon 4 and intron 4 or their boundary region; (2) The exon 4 and intron 3 or their boundary region; (3) The exon 10 and intron 9 or their boundary region and the exon 10 and intron 10 or their boundary region; (4) The intron 8 sequence. In another preferred embodiment, the oligonucleotide targets a region selected from the following group of regions relative to the normal IL1RL2 transcript (NM_003854): (1) The exon 4 and intron 3 or their boundary region and the exon 4 and intron 4 or their boundary region; (2) The intron 8 sequence. In another preferred embodiment, the oligonucleotide target is selected from the following group of regions: (1) Located in the c.294-100 to c.344 region or c.439 to c.489+100 region of the IL1RL2 transcript (NM_003854); (2) Located in the c.294-100 to c.467 region of the IL1RL2 transcript (NM_003854); (3) Located in the c.1136-100 to c.1186 region or c.1247 to c.1297+100 region of the IL1RL2 transcript (NM_003854); (4) Located in the c.991+2372～c.991+252