Antisense oligonucleotide for modulating alternative splicing of il1RL2 gene, preparation method therefor and use thereof

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.

Description

Antisense oligonucleotides regulating alternative splicing of the IL1RL2 gene, their preparation and application Technical Field

[0001] 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

[0002] 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.

[0003] 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 activate 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.

[0004] 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.

[0005] 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

[0006] 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.

[0007] 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.

[0008] Specifically, relative to the normal IL1RL2 transcript (NM_003854), the oligonucleotide targets are selected from the following group of regions:

[0009] (1) The exon 4 and intron 3 or their boundary region and the exon 4 and intron 4 or their boundary region;

[0010] (2) The exon 4 and intron 3 or their boundary region;

[0011] (3) The exon 10 and intron 9 or their boundary region and the exon 10 and intron 10 or their boundary region;

[0012] (4) The intron 8 sequence.

[0013] In another preferred embodiment, the oligonucleotide targets a region selected from the following group of regions relative to the normal IL1RL2 transcript (NM_003854):

[0014] (1) The exon 4 and intron 3 or their boundary region and the exon 4 and intron 4 or their boundary region;

[0015] (2) The intron 8 sequence.

[0016] In another preferred embodiment, the oligonucleotide target is selected from the following group of regions:

[0017] (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);

[0018] (2) Located in the c.294-100 to c.467 region of the IL1RL2 transcript (NM_003854);

[0019] (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);

[0020] (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).

[0021] Since ASO targets the pre-mRNA transcribed from the IL1RL2 gene DNA sequence, which contains intron sequences, taking exon 4's CDS as c.294~c.489 as an example, the region targeted by the antisense oligonucleotide corresponds to the sequence of exon 4 and intron 3 or their junction, and the sequence of exon 4 and intron 4 or their junction is: c.294-100~c.344 region or c.439~c.489+100 region. The numbering rule is that upstream introns are marked with "-", and downstream introns are marked with "+".

[0022] In another preferred embodiment, the boundary region refers to a 150nt region including the boundary point; it includes an intronic region of 100nt and an exonic region of 50nt.

[0023] In another preferred embodiment, the boundary points include the boundary points of exon 4 and intron 3, the boundary points of exon 4 and intron 4, the boundary points of exon 10 and intron 9, the boundary points of exon 10 and intron 10, and the sequence of intron 8.

[0024] In another preferred embodiment, the truncated IL1RL2 splice isoform transcript is selected from the group consisting of:

[0025] Z1 transcript, nucleotide sequence as shown in SEQ ID NO.5, contains truncated exon 4;

[0026] Z2 transcript, nucleotide sequence as shown in SEQ ID NO.9, does not contain exon 10;

[0027] The Z3 transcript, with its nucleotide sequence shown in SEQ ID NO.3, does not contain exon 4.

[0028] In another preferred embodiment, the oligonucleotide is an antisense oligonucleotide (ASO).

[0029] In another preferred embodiment, the antisense oligonucleotide is in single-stranded form.

[0030] In another preferred embodiment, the cryptic exon insertion type IL1RL2 splice isoform transcript is selected from the group consisting of:

[0031] The Z4 transcript, with its nucleotide sequence shown in SEQ ID NO.13, retains the hidden exon of intron 8.

[0032] In another preferred embodiment, the antisense oligonucleotide induces an IL1RL2 splice isoform transcript with exon 4 or exon 10 skipping, or containing a hidden exon insertion located in intron 8, compared to the normal IL1RL2 transcript (NM_003854).

[0033] In another preferred embodiment, the exon 4 skipping or the insertion of a hidden exon located in intron 8 both introduce a premature termination codon (PTC), leading to the related transcript entering nonsense degradation (NMD) or the production of truncated IL-36R protein, while simultaneously reducing the expression level of full-length functional IL-36R protein.

[0034] In another preferred embodiment, the protein obtained by translating the truncated or cryptic exon inserted IL1RL2 splice isoform transcript (such as Z1, Z3, Z4) is an extracellular secretory soluble protein.

[0035] In another preferred embodiment, the protein obtained by translating the truncated or cryptic exon inserted IL1RL2 splice isoform transcript is an inhibitory protein that can antagonize IL-36 receptor activation.

[0036] In another preferred embodiment, the nucleotide sequence of the normal IL1RL2 transcript is shown in SEQ ID NO.1.

[0037] In another preferred embodiment, the truncated or cryptic exon insertion IL1RL2 splice isoform transcript encodes a mutant protein selected from the group consisting of:

[0038] The P1 mutant protein has the amino acid sequence shown in SEQ ID NO.6;

[0039] The P2 mutant protein has the amino acid sequence shown in SEQ ID NO.14;

[0040] The P3 mutant protein has the amino acid sequence shown in SEQ ID NO.10;

[0041] The P4 mutant protein has the amino acid sequence shown in SEQ ID NO.4.

[0042] In another preferred embodiment, during the splicing of the IL1RL2 pre-mRNA, the antisense oligonucleotide causes exon 4 to jump, or causes truncated exon 4 to be spliced ​​and integrated, or causes exon 10 to jump, or causes cryptic exon splicing and integration of intron 8, thereby forming a truncated or cryptic exon insertion IL1RL2 splice isoform transcript selected from the group consisting of:

[0043] Z1 transcript, nucleotide sequence as shown in SEQ ID NO.5, contains truncated exon 4;

[0044] Z2 transcript, nucleotide sequence as shown in SEQ ID NO.9, does not contain exon 10;

[0045] The Z3 transcript, with the nucleotide sequence shown in SEQ ID NO.3, does not contain exon 4;

[0046] The Z4 transcript, with its nucleotide sequence shown in SEQ ID NO.13, retains the hidden exon of intron 8.

[0047] In another preferred embodiment, the antisense oligonucleotide is 16-45 nt in length; more preferably 16-30 nt, and even more preferably 18-24 nt.

[0048] In another preferred embodiment, the antisense oligonucleotide specifically binds to the nucleotide sequence corresponding to exon 4 in the IL1RL2 mRNA and has the core sequence shown in SEQ ID NO.15: TATACC (SEQ ID NO.15)

[0049] Each T can be replaced by U independently.

[0050] In another preferred embodiment, "specific binding" means that the antisense oligonucleotide has 100% complementarity with the complementary region of the nucleotide sequence corresponding to exon 4 in the IL1RL2 mRNA (or ≤2 or ≤1 mismatched nucleotides).

[0051] In another preferred embodiment, the antisense oligonucleotide has the structure of formula I: S1-S2-S3 (I)

[0052] in,

[0053] 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').

[0054] S2 is the core sequence TATACC;

[0055] 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').

[0056] Furthermore, each T in the antisense oligonucleotide can be independently replaced by U.

[0057] In another preferred embodiment, L1 is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20.

[0058] In another preferred embodiment, L2 is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15.

[0059] In another preferred embodiment, L1+L2 equals 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, or 39.

[0060] In another preferred embodiment, the antisense oligonucleotide sequence is an oligonucleotide as shown in any of SEQ ID NO.17-32, 65-71.

[0061] In another preferred embodiment, the antisense oligonucleotide structure comprises mA*mU*mU*mC*mU*mU*mU*mU*mU*mU*mU*mA*mC*mC*mU*mU*mA*mA*mC*mC*mA*mC (SEQ ID NO: 17), where * represents phosphate thiocyanate and m represents 2'MOE modification.

[0062] In another preferred embodiment, the antisense oligonucleotide structure comprises mU*mU*mU*mU*mU*mA*mC*mC*mU*mU*mA*mU*mA*mC*mC*mA*mU*mU*mA (SEQ ID NO: 18), where * represents phosphate thioate and m represents 2'MOE modification.

[0063] In another preferred embodiment, the antisense oligonucleotide structure comprises mG*mA*mA*mA*mA*mU*mU*mC*mU*mU*mU*mU*mU*mU*mA*mC*mC*mU*mU*mA*mU*mA*mC*mC (SEQ ID NO: 19), where * represents phosphate thiocyanate and m represents 2'MOE modification.

[0064] In another preferred embodiment, the antisense oligonucleotide structure comprises mC*mC*mU*mU*mA*mU*mA*mC*mC*mA*mC*mU*mU*mU*mA*mU*mU*mG*mG*mA (SEQ ID NO: 20), where * represents phosphate thioate and m represents 2'MOE modification.

[0065] In another preferred embodiment, the antisense oligonucleotide structure comprises mU*mA*mU*mA*mC*mC*mA*mC*mU*mU*mU*mA*mU*mU*mG*mG*mA*mC*mC*mC*mA (SEQ ID NO: 21), where * represents phosphate thiocyanate and m represents 2'MOE modification.

[0066] In another preferred embodiment, the antisense oligonucleotide structure comprises mA*mG*mA*mA*mA*mA*mA*mU*mU*mC*mU*mU*mU*mU*mU*mU*mU*mA*mC*mC*mU*mU*mA*mU*mA*mC*mC (SEQ ID NO: 22), where * represents phosphate thiocyanate and m represents 2'MOE modification.

[0067] In another preferred embodiment, the antisense oligonucleotide structure comprises mA*mA*mG*mA*mA*mA*mA*mA*mU*mU*mC*mU*mU*mU*mU*mU*mU*mA*mC*mC*mU*mU*mA*mU*mA*mC*mC (SEQ ID NO: 23), where * represents phosphate thiocyanate and m represents 2'MOE modification.

[0068] In another preferred embodiment, the antisense oligonucleotide structure comprises mG*mU*mU*mC*mA*mC*mG*mA*mU*mG*mA*mG*mG*mG*mU*mG*mG*mA*mG*mC*mCVmUVmG*mG (SEQ ID NO: 69), where * represents phosphate thiocyanate and m represents 2'MOE modification.

[0069] In another preferred embodiment, the antisense oligonucleotide structure comprises mU*mG*mA*mG*mG*mG*mU*mG*mG*mA*mG*mC*mC*mU*mG*mG*mA*mG*mC*mA*mU*mC*mA (SEQ ID NO: 70), where * represents phosphate thiocyanate and m represents 2'MOE modification.

[0070] In another preferred embodiment, the antisense oligonucleotide structure comprises mG*mG*mU*mG*mG*mA*mG*mC*mC*mU*mG*mG*mA*mG*mC*mA*mA*mU*mC*mA*mG*mU*mU (SEQ ID NO: 71), where * represents phosphate thiocyanate and m represents 2'MOE modification. In another preferred embodiment, the antisense oligonucleotide may be modified or unmodified.

[0071] In another preferred embodiment, one or more or all of the nucleotides in the antisense oligonucleotide are modified.

[0072] In another preferred embodiment, the antisense oligonucleotide comprises a modified polynucleotide backbone.

[0073] In another preferred embodiment, the modified polynucleotide backbone comprises a modified portion of a glycoside that replaces at least one polynucleotide.

[0074] In another preferred embodiment, the modification includes a first-generation modification and a second-generation modification. The first-generation modification includes a phosphate thioester (PS) modification in the phosphate ribose backbone. The second-generation modification includes an alkyl / alkynyl modification at the 2' position, preferably a 2'MOE (methoxyethyl), 2'Ome (methoxy), 2'-LNA (locked nucleic acid), or 2'-cET modification.

[0075] In another preferred embodiment, the phosphate group modified by the first generation is selected from: non-bridging oxygen atoms that replace sulfur atoms, phosphonate groups, thiophosphate groups, phosphate diester groups, morpholine phosphate groups, piperazine phosphate groups, and phosphoroamidate groups.

[0076] In another preferred embodiment, the modified portion is selected from the group consisting of: phosphodiamidomorpholine oligomers (PMO), peptide-coupled phosphodiamidomorpholine oligomers (PPMO), and non-peptide dendritic octaguanidine-labeled morpholine oligomers.

[0077] In another preferred embodiment, the modified polynucleotide backbone may include at least one modified internucleotide linker group.

[0078] In another preferred embodiment, the modified internucleotide linker may comprise a modified phosphate group.

[0079] In another preferred embodiment, the modified phosphate group is selected from: non-bridging oxygen atoms that replace sulfur atoms, phosphonate groups, thiophosphate groups, phosphate diester groups, morpholine phosphate groups, piperazine phosphate groups, and phosphoroamidate groups.

[0080] In another preferred embodiment, the antisense oligonucleotide is chemically linked to one or more conjugates, which enhance the activity or stability of the antisense oligonucleotide, selective cellular uptake, or endosome escape.

[0081] In a second aspect of the invention, a polynucleotide is provided that encodes a variant transcript of IL-36R, wherein, compared to the normal IL1RL2 transcript, the variant transcript is selected from the group consisting of:

[0082] (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;

[0083] (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;

[0084] (3) Transcript Y3, which does not contain the exon 10 sequence and has the nucleotide sequence shown in SEQ ID NO.9;

[0085] (4) Transcript Y4, which does not contain exon 4 sequence and has a nucleotide sequence as shown in SEQ ID NO.3.

[0086] In another preferred embodiment, the polynucleotide is RNA or DNA.

[0087] In a third aspect of the invention, an IL-36R mutant protein is provided, wherein the mutant protein is selected from the group consisting of, compared to the wild-type IL1RL2 protein:

[0088] The P1 mutant protein has the amino acid sequence shown in SEQ ID NO.6;

[0089] The P2 mutant protein has the amino acid sequence shown in SEQ ID NO.14;

[0090] The P3 mutant protein has the amino acid sequence shown in SEQ ID NO.10;

[0091] The P4 mutant protein has the amino acid sequence shown in SEQ ID NO.4.

[0092] In another preferred embodiment, the mutant protein is produced by introducing the antisense oligonucleotide described in the first aspect of the invention into a cell.

[0093] In a fourth aspect of the invention, a pharmaceutical composition is provided comprising the oligonucleotide described in the first aspect of the invention, or a pharmaceutically acceptable salt, hydrate, or solvate thereof, and a pharmaceutically acceptable carrier.

[0094] In another preferred embodiment, the pharmaceutical composition is liquid, solid, or semi-solid.

[0095] In another preferred embodiment, the dosage form of the pharmaceutical composition is an oral dosage form, an injection, or a topical dosage form.

[0096] In another preferred embodiment, the dosage form of the pharmaceutical composition includes transdermal patches, tablets, granules, capsules, oral solutions, or injections.

[0097] In another preferred embodiment, the pharmaceutical composition is a topical pharmaceutical preparation.

[0098] In another preferred embodiment, the carrier is selected from the group consisting of infusion carriers and / or injection carriers. Preferably, the carrier is one or more carriers selected from the group consisting of physiological saline, glucose saline, artificial cerebrospinal fluid, or combinations thereof.

[0099] In a fifth aspect of the invention, there is provided the use of an oligonucleotide as described in the first aspect of the invention, or a pharmaceutical composition as described in the fourth aspect of the invention, in the preparation of a medicament for treating and / or preventing diseases associated with abnormal activation of IL-36R.

[0100] In another preferred embodiment, the IL-36R abnormal activation-related diseases include diseases selected from the group consisting of inflammation, autoimmune diseases, allergic diseases, cancer, or combinations thereof.

[0101] In another preferred embodiment, the autoimmune disease includes psoriasis.

[0102] In another preferred embodiment, the psoriasis includes generalized pustular psoriasis (GPP).

[0103] In another preferred embodiment, the autoimmune disease includes inflammatory bowel disease (IBD).

[0104] In a sixth aspect of the invention, a medicine box is provided, comprising:

[0105] (i) a first container, and a first pharmaceutical composition located in the first container, the first pharmaceutical composition comprising (a1) an oligonucleotide as described in the first aspect of the invention, and (a2) a first pharmaceutically acceptable carrier.

[0106] In another preferred embodiment, the kit may further include (ii) a second container and a detection agent located in the second container, the detection agent being used to detect the presence of the variant transcripts described in the second aspect of the invention, and optionally the wild-type IL-36R transcript, in a sample derived from the subject.

[0107] In another preferred embodiment, the sample includes: blood, skin tissue, drainage and distal lymph node tissue, or a combination thereof.

[0108] In another preferred embodiment, the detection includes qualitative detection and quantitative detection.

[0109] In another preferred embodiment, the medicine box also includes an instruction manual, which states:

[0110] When the detected IL-36R content is more than twice the normal value, it indicates that the subject is suitable for the first pharmaceutical composition in the pharmaceutical product, thereby treating psoriasis.

[0111] In another preferred embodiment, the normal value refers to the value measured in a normal individual.

[0112] In another preferred embodiment, the normal individual refers to an individual who does not have psoriasis.

[0113] In a seventh aspect of the invention, a method for treating and / or preventing diseases associated with abnormal activation of IL-36R is provided, comprising the steps of: administering to a desired subject a safe and effective amount of an oligonucleotide as described in the first aspect of the invention, or a pharmaceutical composition as described in the fourth aspect of the invention.

[0114] In another preferred embodiment, the disease is as defined in the fifth aspect of the invention.

[0115] In another preferred embodiment, the object is a mammal.

[0116] In another preferred embodiment, the mammals include humans or non-human mammals.

[0117] In another preferred embodiment, the non-human mammals include rodents (such as rats and mice) and primates (such as monkeys).

[0118] In an eighth aspect of the invention, a method for inducing the production of IL1RL2 variant mRNA transcripts is provided, the method comprising: delivering to cells an oligonucleotide as described in the first aspect of the invention or a pharmaceutical composition as described in the fourth aspect of the invention, thereby inducing the production of IL1RL2 variant mRNA transcripts.

[0119] In another preferred embodiment, the method is an in vitro method for non-therapeutic purposes.

[0120] In another preferred embodiment, the cells are skin keratinocytes, tumor cells, or immune cells.

[0121] In another preferred embodiment, the tumor is selected from the group consisting of glioma, lung adenocarcinoma, breast cancer, lung squamous cell carcinoma, prostate cancer, gastric cancer, and endometrial cancer.

[0122] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Attached Figure Description

[0123] Figure 1 is a schematic diagram of ASO targeting the IL1RL2 pre-mRNA region. The figure shows the different mRNA splicing induced by ASO targeting different positions on the IL1RL2 pre-mRNA, using the IL1RL2 mRNA (NM_003854) sequence as a nucleotide reference. Specifically, ASO targeting positions ASO-4-xx target exon 4 and intron 3 of IL1RL2 (NM_003854) or their junction, and specifically, the ASO targeting positions ASO-4-xx are from c.294-100 to c.344, or c.439 to c.489+100 of IL1RL2 (NM_003854). The ASO target designated ASO-4part-xx targets exon 4 and intron 3 of IL1RL2 (NM_003854) or their junction region. Specifically, the ASO target location designated ASO-4part-xx is from c.294-100 to c.467 of IL1RL2 (NM_003854). The ASO target designated ASO-10-xx targets exon 10 and intron 9 of IL1RL2 (NM_003854) or their junction region, and exon 10 and intron 10 of IL1RL2 (NM_003854) or their junction region. Specifically, the ASO target location designated ASO-10-xx is from c.1136-100 to c.1186, or c.1247 to c.1297+100. The ASO target sequence of IL1RL2 (NM_003854) with the designation ASO-crpE8-xx is specifically located at positions c.991+2372 to c.991+2522 or c.991+2541 to c.991+2691 of IL1RL2 (NM_003854). The numbering rule is that upstream intron positions are marked with "-", and downstream intron positions are marked with "+".

[0124] Figure 2 shows the IL-36R exon alternative splicing screening results. Figure A represents the RT-PCR gel electrophoresis results for IL-36R exon alternative splicing screening. M represents the protein marker; E1-E3 represent the primers used to screen for alternative splicing, targeting exons 1 and 3 respectively, and used to screen for alternative splicing in exon 2, and so on; CHX represents cyclohexanone; M represents the marker; Figures B-E represent the sequencing alignment results of exon 4, partial sequences of exon 4, exon 6, and exon 10 of IL-36R containing alternative splicing, respectively. The gray areas in Figures B-E are... The bands represent the full-length IL-36R transcript (NM_003854). The red bands in Figure B are the results of sequencing the bands "E2-E4" in Figure A and comparing them with the full-length IL-36R transcript. The red bands in Figure C are the results of sequencing the bands "E3-E5" in Figure A and comparing them with the full-length IL-36R transcript. The red bands in Figure D are the results of sequencing the bands "E5-E7" in Figure A and comparing them with the full-length IL-36R transcript. The red bands in Figure E are the results of sequencing the bands "E9-E11" in Figure A and comparing them with the full-length IL-36R transcript.

[0125] Figure 3. Schematic diagram of IL-36R exon alternative splicing and predicted proteins. The figure shows a schematic diagram of IL-36R exon alternative splicing and predicted proteins, where Q9HB29,2 is the protein encoding recombinant human IL-1RL2 protein; the numbers 1-12 on the mRNA represent exons 1-12 respectively; the yellow area of ​​the Ig domain represents the ligand-binding region; the red area of ​​TM represents the transmembrane region; and the green area of ​​TIR represents the intracellular region.

[0126] Figure 4 shows the RT-PCR gel electrophoresis results for screening IL-36R intron-retaining alternative splicing. M represents the protein marker, and E1-E2 indicate the primers used to screen for intron-retaining alternative splicing, which target exon 1 and exon 2 respectively. These primers are used to screen for intron retention in intron 1, and so on. The results show that partial sequences of intron 1 and intron 8 of IL-36R (named crpE8, whose nucleotide sequence is shown in SEQ ID NO.16) exhibit alternative splicing.

[0127] Figure 5. Schematic diagram of IL-36R intron-retaining alternative splicing and predicted protein. The figure shows a schematic diagram of IL-36R intron-retaining alternative splicing and predicted protein, where the numbers 1-12 on the mRNA represent exons 1-12 respectively; the yellow area of ​​the Ig domain represents the ligand-binding region; the red area of ​​TM represents the transmembrane region; and the green area of ​​TIR represents the intracellular region.

[0128] Figure 6 shows that ASO can regulate the alternative splicing of IL-36R exon 4 at the exogenous level. The figure shows the ASO screening results for IL-36R exon 4 alternative splicing at the exogenous level; where M represents protein marker; "-" is negative control, mini-C2 represents minegene C2; ​​ASO-4-1 to ASO-4-5 represent ASOs numbered 1-5 targeting the 3' splicing signal (3'SS) of exon 4; ASO-4-6 to ASO-4-14 represent ASOs numbered 6-14 targeting the 5' splicing signal (5'SS) of exon 4; hGAPDH is internal control; sIL-36R represents truncated IL-36R transcripts lacking exon 4; the results show that ASOs targeting the 3' splicing signal of exon 4 can effectively regulate the alternative splicing of IL-36R mRNA, which can significantly reduce the production of full-length IL-36R transcripts and increase the production of truncated IL-36R transcripts (sIL-36R).

[0129] Figure 7 shows the screening results of ASOs that can regulate IL-36R exon 4 alternative splicing at the endogenous level in HaCaT cells. ASOs targeting the 3' splicing signal of exon 4 are shown; ASO-4-1, ASO-4-3, ASO-4-15, and ASO-4-16 represent ASOs numbered 1, 3, 15, and 16, respectively, that target the 3' splicing signal of exon 4. Figure A shows the RT-PCR gel electrophoresis results; Figure B shows the statistical results. The results show that ASOs targeting the 3' splicing signal of exon 4 can effectively regulate the alternative splicing of IL-36R mRNA at the endogenous level, that is, they can significantly reduce the full-length IL-36R transcript and increase the production of sIL-36R at the endogenous level in HaCaT cells.

[0130] Figure 8 shows that ASO regulates alternative splicing of IL-36R exon 4 in a dose-dependent manner. This demonstrates that ASO regulates alternative splicing of IL-36R exon 4 in a dose-dependent manner. AB represents the RT-PCR gel electrophoresis images and statistical results of the full-length IL-36R transcript and the truncated IL-36R transcript (sIL-36R) of ASO targeting exon 4 (number 1) at different doses, respectively. CD represents the RT-PCR gel electrophoresis images and statistical results of the full-length IL-36R transcript and the truncated IL-36R transcript (sIL-36R) of ASO targeting exon 4 (number 3) at different doses, respectively.

[0131] Figure 9 shows the changes in p38 MAPK phosphorylation levels in the IL-36R signaling pathway after ASO treatment of HaCaT. The results show that ASOs numbered ASO-4-3, ASO-4-15, and ASO-4-16 significantly inhibited IL-36γ-induced p38 MAPK phosphorylation. Figure A represents the electrophoresis results from Western blotting, where p-p38 represents p38 MAPK phosphorylation; GAPDH represents the internal control; and Figure B shows the statistical results.

[0132] Figure 10 shows the changes in the expression levels of downstream genes in the IL-36R signaling pathway after ASO treatment. Figures A-C show that ASO can inhibit the expression of IL-36γ-induced inflammatory cytokines TNF-α and IL-6, as well as the chemokine CCL20, with ASO-4-3 showing the most significant inhibitory effect.

[0133] Figure 11 shows that ASO targeting mouse IL1RL2 can regulate alternative splicing of IL-36R exon 6 at the endogenous cellular level. The figure shows the screening results of ASOs that can regulate IL-36R exon 6 alternative splicing at the endogenous level in mouse primary skin fibroblasts. mASO-6-1, mASO-6-2, mASO-6-3, and mASO-6-4 represent ASOs numbered 1, 2, 3, and 4, respectively, targeting the 5' splicing signal of mouse IL1RL2 exon 6; mASO-6-5, mASO-6-6, and mASO-6-7 represent ASOs numbered 5, 6, and 7, respectively, targeting the 3' splicing signal of mouse IL1RL2 exon 6. Figure A shows the RT-PCR gel electrophoresis results; Figure B shows the statistical results. The results show that ASOs targeting the 3' splicing signal of exon 6 (mASO-6-5, mASO-6-6, and mASO-6-7) can effectively regulate IL-36R at the endogenous cellular level. Alternative splicing of mRNA can significantly reduce full-length IL-36R transcripts and increase sIL-36R production at the endogenous level in primary mouse skin fibroblasts.

[0134] Figure 12 shows that ASO targeting IL1RL2 in mice significantly improved psoriasis symptoms. The figure illustrates the experimental procedure, daily phenotypic records, and related pathological staining results for ASO intervention in psoriasis-affected mice. Figure A shows the experimental procedure for ASO intervention in mouse psoriasis. Specifically, 62.5 mg of IMQ was applied topically to the back of mice daily. On days 1, 3, and 5, 100 μg (200 μL) of mASO-6-5 was subcutaneously injected. Mice were euthanized on day 7, and back skin was collected for subsequent pathological staining or molecular experiments. Figure B shows the daily phenotypic records of the backs of mice in the blank control group (Blank), the model group (IMQ+PBS), and the drug intervention group (IMQ+ASO). The results showed that the most severe back symptoms were observed in the model group. From day 4 to day 6, the inflammation symptoms on the back of mice in the drug intervention group were significantly improved. The upper part of Figure C shows the back phenotype of mice in the blank control group (Blank), the model group (IMQ+PBS), and the drug intervention group (IMQ+ASO) on day 4. The lower part of Figure C shows the HE staining of the back skin of mice in the blank control group (Blank), the model group (IMQ+PBS), and the drug intervention group (IMQ+ASO) on day 7. The results show that compared with the model group (IMQ+PBS), the epidermal layer of the back skin of mice in the drug intervention group (IMQ+ASO) was significantly thinner. Detailed Implementation

[0135] Through extensive and in-depth research, the inventors unexpectedly discovered an antisense oligonucleotide (ASO) (hereinafter referred to as "the ASO of this invention") that can efficiently inhibit IL1RL2 gene expression. Experiments show that the ASO of this invention can efficiently inhibit IL1RL2 gene expression, thereby significantly inhibiting IL-36R, and further inhibiting the activation of the IL-36R signaling pathway to treat IL-36-related autoimmune diseases, such as psoriasis. Based on the above findings, the inventors completed this invention.

[0136] Specifically, the antisense oligonucleotide therapy provided by this invention can reduce the proportion of mRNA encoding full-length functional IL-36R and increase the expression ratio of mRNA encoding different truncated IL1RL2 proteins by inducing splicing skipping of exon 4 or exon 10 of IL1RL2 gene precursor mRNA, or splicing insertion of cryptic exon sequences of intron 8, thereby achieving activation blockade of the IL-36R signaling pathway. It can be used to treat autoimmune diseases, allergic diseases, neurodegenerative diseases, or cancers associated with abnormal IL-36R activation.

[0137] the term

[0138] As used herein, the term "truncated IL1RL2" includes the extracellular soluble "sIL-36R" protein produced when antisense oligonucleotides cause exon 4 to be cleaved out or a portion of intron 8 to be retained during the splicing of IL1RL2 precursor mRNA, resulting in a truncated splice isoform that does not contain exon 4 or retains a portion of intron 8 (the hidden exon sequence). If antisense oligonucleotides cause exon 10 to be cleaved out, resulting in a truncated splice isoform transcript that does not contain exon 10, then "truncated IL1RL2" is a truncated protein product that lacks the TIR functional domain encoded by exon 10, but still carries a transmembrane segment.

[0139] As used herein, the term “mRNA” refers to the mRNA transcript of a currently known target gene and any other transcript that can be elucidated.

[0140] The term "antisense oligonucleotide" refers to a single-stranded oligonucleotide that has a nucleic acid base sequence that allows hybridization with the corresponding segment of the target nucleic acid.

[0141] As used in this article, the terms "intron 8-part sequence" and "crpE8" are used interchangeably.

[0142] As used in this article, the term "nt" refers to a nucleotide.

[0143] As used in this article, the term "IL-36Ra" refers to an IL-36 receptor antagonist.

[0144] As used in this article, the terms “wild-type IL1RL2 transcript” and “normal IL1RL2 transcript” refer to IL1RL2 (NM_003854).

[0145] As used herein, the term "variant mRNA transcript of IL1RL2" refers to the IL1RL2 (NM_003854) precursor mRNA that, during splicing, isoforms of IL1RL2 are formed by antisense oligonucleotides causing exon 4 or exon 10 to be cleaved or intron 8 to be retained, resulting in either a truncated form that does not contain exon 4 or exon 10 or a form that retains a portion of intron 8, or a truncated form that contains a hidden exon insertion.

[0146] The term "hybridization" refers to the interaction between two or three nucleic acid strands via hydrogen bonds, according to the rules of Watson-Crick DNA complementarity, Hoogstein binding, or other sequence-specific binding known in the art. As used herein, "specific hybridization" is the hybridization of stable duplexes between complementary or substantially complementary polynucleotide strands. For example, a polynucleotide strand with 21 nucleotide units can pair with another polynucleotide strand with 21 nucleotide units, but only 19 bases on each strand are complementary or substantially complementary, resulting in a "duplex" with 19 base pairs. The remaining bases may, for example, be present as 5' and / or 3' overhangs. Furthermore, 100% complementarity is not required within a duplex; substantial complementarity is permitted. Substantial complementarity refers to 75% or higher complementarity. For example, a mismatch in a duplex consisting of 19 base pairs results in 94.7% complementarity, thus making the duplex substantially complementary.

[0147] In this invention, the terms "containing," "comprising," or "including" indicate that various ingredients may be used together in the mixtures or compositions of this invention. Therefore, the terms "consistent with..." and "composed of..." are included in the term "containing."

[0148] As used herein, the term "oligonucleotide" refers to an RNA or DNA molecule that binds to another RNA or DNA (target RNA, DNA), or an oligomeric sequence composed of ribonucleotides and deoxyribonucleotides linked together. For example, if it is an RNA oligonucleotide, it binds to another RNA target through RNA-RNA interactions and alters the activity of the target RNA. Oligonucleotides can upregulate or downregulate the expression and / or function of specific polynucleotides. This definition is intended to include any exogenous RNA or DNA molecule useful from a therapeutic, diagnostic, or other perspective. Such molecules include microRNAs, decoy RNAs, small interfering RNAs (siRNAs), enzyme RNAs, therapeutic editing RNAs, as well as agonist and antagonist RNAs, antisense oligomers, antisense oligonucleotides (ASOs), external guide sequence (EGS) nucleotides, alternative splicers, primers, probes, and other oligomers that hybridize to at least a portion of a target nucleic acid. Similarly, these compounds can be introduced in the form of single-stranded, double-stranded, partially single-stranded, or circular oligomers.

[0149] In this invention, the term "effective amount" refers to the amount of a therapeutic agent that treats, alleviates, or prevents a target disease or condition, or the amount that exhibits a detectable therapeutic or preventative effect. The precise effective amount for a given subject depends on that subject's body size and health status, the nature and severity of the condition, and the choice of the therapeutic agent and / or combination of therapeutic agents administered. Therefore, it is useless to pre-specify an accurate effective amount. However, for a given condition, the effective amount can be determined using routine experiments, and a clinician can judge it accordingly.

[0150] As used herein, the term "pharmaceutically acceptable salt" refers to a salt formed by the compound of the present invention with a base that is suitable for use as a medicine.

[0151] Unless otherwise specified, all compounds mentioned in this invention are intended to include all possible optical isomers, such as compounds with a single chirality, or mixtures of various chiral compounds (i.e., racemates). In all compounds of this invention, each chiral carbon atom may optionally be in the R configuration or the S configuration, or a mixture of the R and S configurations.

[0152] This invention provides methods, compounds, and compositions for regulating IL1RL2 (or IL-36R) expression. In embodiments, the compounds suitable for regulating IL1RL2 (or IL-36R) expression are antisense oligonucleotides.

[0153] In some embodiments, regulation can be performed in cells. In some embodiments, regulation is performed in animals. In some embodiments, the animal is a human. In some embodiments, IL-36R RNA levels are reduced. In some embodiments, IL-36R protein levels and activity are reduced. The reduction occurs in a dose- and time-dependent manner.

[0154] Methods, compounds, and compositions are also provided for the prevention, treatment, and improvement of diseases, conditions, and symptoms. In some embodiments, the IL-36R-related diseases, conditions, and symptoms are inflammation, autoimmune diseases, allergic diseases, cancer, etc. In some embodiments, the autoimmune diseases include psoriasis, etc.

[0155] In some implementations, the treatment method includes administering a pharmaceutical composition containing IL1RL2 antisense oligonucleotides to an individual in need via intravenous injection, subcutaneous injection, or transdermal administration.

[0156] Interleukin-1 receptor-like 2 (IL1RL2)

[0157] As used in this article, the terms “IL1RL2” and “IL-36R” are used interchangeably, and “IL-36 receptor” refers to the gene-encoded protein product.

[0158] IL1RL2 encodes the IL-36 receptor, whose ligand, interleukin-36 (IL-36), is a member of the IL-1 family and plays a crucial role in immune and inflammatory responses. Abnormal activation levels of the interleukin-36 receptor (IL-36R) signaling pathway are associated with the pathogenesis of inflammation, autoimmunity, allergies, and cancer. IL-36R is expressed in various tissues and cells, functioning on macrophages and dendritic cells to induce the production of pro-inflammatory factors, thereby driving innate and adaptive immune responses. Additionally, on T cells, the interleukin-36 receptor is primarily located on naïve CD4+ cells. + IL-36R is expressed on T cells. It can promote naïve CD4+ expression. + IL-36 promotes the proliferation and differentiation of T cells and facilitates the production of inflammatory cytokines. Furthermore, IL-36 can stimulate and activate fibroblasts, epithelial cells, and keratinocytes. This indicates that IL-36R has a broad cellular effect, acting on both immune and non-immune cells.

[0159] IL-36R abnormal activation-related diseases

[0160] Immune homeostasis is crucial for human health. Under normal circumstances, the human immune system is in a balanced state. If the immune system is overactive, it may make mistakes, overreacting to foreign substances and causing allergic reactions to harmless substances, such as food allergies and allergic rhinitis. Internally, it can cause the immune system to mistakenly attack healthy cells, tissues, and organs, leading to autoimmune diseases such as rheumatoid arthritis, systemic lupus erythematosus, inflammatory bowel disease, type 1 diabetes, multiple sclerosis, psoriasis, and over one hundred other autoimmune diseases. Autoimmune diseases can affect any part of the body, weaken bodily functions, and even endanger life.

[0161] The IL-36R signaling pathway consists of three agonists (IL-36α, IL-36β, and IL-36γ) and their IL-36R, as well as two antagonists, IL-36Ra and IL-38. IL-36R has a ligand-binding domain (the immunoglobulin domain) outside the cell membrane and a TIR domain (the domain responsible for intracellular signal transduction) inside the cell membrane. When IL-36α, IL-36β, and IL-36γ bind to IL-36R, they can recruit the co-receptor IL-1RAcP to form a signal transduction complex. By recruiting Myeloid Differentiation Factor 88 (MyD88), they activate IL-1R-associated kinases (IRAKs) and tumor necrosis factor receptor-associated factor 6 (TRAF6), ultimately activating 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. The activity of IL-36 cytokines and the inflammatory response they induce are strictly regulated by some inhibitors, such as the interleukin 36 receptor antagonists IL-36Ra and IL-38. Their binding to IL-36R does not lead to the recruitment of the co-receptor IL-1RAcP, thus exhibiting anti-inflammatory effects.

[0162] The IL-36R signaling pathway plays a crucial role in immune and inflammatory responses. Disruptions in this pathway can lead to autoimmune diseases. Deficiency of interleukin-36 receptor antagonist (DITRA) is a life-threatening monogenic autoinflammatory disease caused by mutations in the IL36RN gene. Patients develop generalized pustular psoriasis (GPP), a skin inflammatory disease caused by IL-36R pathway disruption. It is estimated that 1-2 people per 100,000 in China suffer from GPP. During acute flare-ups, the patient's skin reddens, and numerous sterile pustules erupt across various areas of the body, accompanied by fever, chills, and painful skin lesions. In severe cases, it can lead to organ failure and infectious complications, even death. Studies in GPP patients have shown that it is associated with the overexpression of IL-36 in the skin and the deficiency of the interleukin 36 receptor antagonist IL-36Ra, leading to the overactivation of NF-κB and MAPK-mediated inflammatory signaling. Further research revealed that in the skin of GPP patients, the overactivated IL-36R signaling pathway results in the production of large amounts of pro-inflammatory and chemokines, subsequently leading to the recruitment of immune cells (neutrophils, monocytes, and dendritic cells), resulting in excessive keratinocyte proliferation and pustule formation. Anti-IL-1 treatment has been used clinically to treat DITRA, but the therapeutic effect is poor.

[0163] antisense oligonucleotides

[0164] The antisense oligonucleotides of this invention can be natural nucleotides and / or chemically modified nucleotides. Chemical modification is one of the most effective methods to enhance the efficacy of oligonucleotide drugs. Chemical modification helps antisense oligonucleotides resist degradation by endogenous nucleases, enhances the stability of oligonucleotides in plasma, tissues, and cells, and can also enhance the affinity of oligonucleotides for target nucleic acids, achieving higher antisense activity. Chemical modification includes modification of the polynucleotide backbone, modification of bases, modification of riboglycosyl groups, and the use of nucleic acid analogs.

[0165] As used herein, the term "ASO of the present invention" refers to the oligonucleotide of the first aspect of the present invention.

[0166] As used herein, the terms “antisense oligonucleotide,” “antisense oligonucleotide,” and “antisense nucleotide” are used interchangeably and all refer to the oligonucleotide of the first aspect of this invention.

[0167] In a preferred embodiment, the antisense oligonucleotide target is selected from the following group of regions:

[0168] (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);

[0169] (2) Located in the c.294-100 to c.467 region of the IL1RL2 transcript (NM_003854);

[0170] (3) Located in the c.1136-100 to c.1186 region or c.1247 to c.1247+100 region of the IL1RL2 transcript (NM_003854);

[0171] (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).

[0172] For example, the “c.294-100~c.344 region” refers to the antisense oligonucleotide targeting this region. Specifically, the antisense oligonucleotide is complementary to the nucleotide bases in this region.

[0173] In some embodiments, the modified polynucleotide backbone comprises a modified portion of a glycoside that replaces at least one polynucleotide.

[0174] In some embodiments, the modification includes a first-generation modification and a second-generation modification. The first-generation modification includes thiophosphate (PS) modification in the phosphate ribose backbone. The second-generation modification includes alkyl / alkynyl modification at the 2' position, preferably 2'MOE (methoxyethyl) or 2'Ome (methoxy) modification.

[0175] In some embodiments, the first-generation modified phosphate group is selected from: non-bridged oxygen atoms substituted with sulfur atoms, phosphonate groups, thiophosphate groups, phosphate diester groups, morpholine phosphate groups, piperazine phosphate groups, and phosphoroamidate groups. The modified portion is selected from the group consisting of: phosphodiamidomorpholine oligomers (PMO), peptide-coupled phosphodiamidomorpholine oligomers (PPMO), and non-peptide dendritic octaguanidine-labeled morpholine oligomers.

[0176] In some embodiments, the modified polynucleotide backbone may include at least one modified internucleotide linker group. The modified internucleotide linker group may include a modified phosphate group. The modified phosphate group is selected from: non-bridging oxygen atoms substituted with sulfur atoms, phosphonate groups, thiophosphate groups, phosphodiester groups, morpholinophosphate groups, piperazine phosphate groups, and phosphoroamidate groups.

[0177] In some embodiments, the antisense oligonucleotide is chemically linked to one or more conjugates, which enhance the activity, cellular distribution, and cellular uptake of the antisense oligonucleotide.

[0178] Pharmaceutical Composition

[0179] The present invention also includes pharmaceutical forms, drug conjugates, and pharmaceutical compositions containing antisense oligonucleotides targeting IL1RL2.

[0180] The drug form includes, but is not limited to, pharmaceutically permissible salts, solvates, and prodrugs.

[0181] The drug conjugate is a conjugate containing the above-mentioned antisense oligonucleotide, including but not limited to lipid conjugate, GalNAc conjugate, antibody conjugate, small molecule ligand conjugate, nucleic acid aptamer conjugate, and peptide conjugate.

[0182] The pharmaceutical composition includes pharmaceutically acceptable excipients or carriers.

[0183] The pharmaceutically acceptable carrier can be a carrier commonly used in the field of nucleic acid drugs, including viral carriers (adeno-associated virus, lentivirus, adenovirus, retrovirus, etc.) and non-viral carriers (including but not limited to sugars, polyamines, lipid carriers, nanoparticles, vesicles, exosomes, globular nucleic acids, DNA nanoparticles, etc.).

[0184] The present invention also includes pharmaceutical formulations of antisense oligonucleotides, antisense oligonucleotide drug conjugates, and antisense oligonucleotide drug compositions as described in any of the preceding claims.

[0185] The pharmaceutical preparations may take different forms, including but not limited to transdermal patches, tablets, pills, granules, powders, capsules, injections, lyophilized powder for injection, nasal drops, suppositories, etc.

[0186] Some compounds in this invention may be crystallized or recrystallized with water or various organic solvents, in which case various solvates may be formed. The solvates of this invention include stoichiometric solvates such as hydrates, as well as compounds containing a variable amount of water formed during preparation by low-pressure sublimation drying.

[0187] The present invention also provides a medicine box containing, optionally, the antisense oligonucleotide of the present invention in a container, and packaging inserts, packaging labels, instructions or other labels.

[0188] The kit of the present invention is used to detect whether cancer patients have excessive IL-36R. If so, the antisense oligonucleotide can be administered for treatment.

[0189] use

[0190] The pharmaceutical compositions of the present invention, with antisense oligonucleotides as the main active ingredient, can be used to treat, prevent, and alleviate diseases associated with IL-36R overactivation. According to the prior art, the compounds of the present invention can be used to treat the following diseases: inflammation, autoimmune diseases, allergic diseases, cancer, or combinations thereof.

[0191] Application method

[0192] The antisense oligonucleotides and compositions containing them of the present invention can be administered by any suitable means, such as oral ingestion; transdermal or nasal inhalation; subcutaneous, intravenous, or lumbar puncture injection. The antisense oligonucleotides or compositions containing them of the present invention can be administered in the form of a dosage unit formulation containing a pharmaceutically acceptable, non-toxic carrier or diluent; or in the form of an immediately released or sustained-release formulation.

[0193] The following are suggested effective dosing regimens for administering the antisense oligonucleotides of the present invention to the human body; follow the dosing regimens generally used for other antisense oligonucleotides.

[0194] Based on the information presented in this article, it is useful to treat autoimmune diseases such as psoriasis using pharmaceutically acceptable antisense oligonucleotide formulations.

[0195] The main advantages of this invention include:

[0196] (1) The antisense oligonucleotides provided by the present invention can effectively inhibit the IL1RL2 gene product at the mRNA and protein levels, thereby inhibiting the abnormal activation of the IL-36R signaling pathway to treat autoimmune diseases, such as psoriasis, especially GPP.

[0197] (2) The present invention provides an IL1RL2 antisense oligonucleotide that can promote the expression of truncated IL1RL2 (sIL-36R) by inducing exon 4 skipping or exon 10 skipping of IL1RL2 pre-mRNA or retaining part of the intron 8 sequence, inhibit the IL-36R signaling pathway, and thus treat inflammation, autoimmune diseases, allergic diseases or cancer related to abnormal activation of IL-36R.

[0198] (3) This invention provides the application of reagents that regulate IL1RL2 splicing in the preparation of drugs for treating autoimmune diseases.

[0199] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions as described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or as recommended by the manufacturer. Percentages and parts are by weight unless otherwise stated. Unless otherwise specified, all experimental materials and reagents involved in this invention are commercially available.

[0200] The IL1RL2 related sequences involved in this invention are shown in Table 1.

[0201] Experimental Materials and Methods

[0202] 1. Construction of Minigene plasmid

[0203] To establish a rapid RT-PCR analysis method for alternative splicing events in IL1RL2 exon 4, 1500 bp of the original intron sequences (from introns 3 and 4) were added to both ends of exon 4 based on the full-length human IL-36R transcript. This DNA fragment was then constructed on a minigene backbone plasmid with a CMV promoter, and the plasmid was named minigene C2.

[0204] Table 1 Note: In the table, crpE8 refers to the partial intron 8 sequence that is spliced ​​and integrated into the mRNA.

[0205] 2. Design of antisense oligonucleotides (ASO)

[0206] Antisense oligonucleotide (ASO) technology is a drug development strategy based on chemically modified short oligonucleotides (12-24 nt) that bind to their RNA targets via Watson-Crick base pairing. The ASO sequences were designed according to the ASO design guidelines. ASOs with 2′MOE and PS modifications (2′-O-methoxyethyl sugar with a phosphate thioester backbone) were synthesized by Besun Biotechnology or IDT and purified by HPLC. Detailed ASO sequences are shown in Table 2.

[0207] Table 2 Note: In the table, ASO-4-1 represents the ASO numbered 1 that targets exon 4, and so on; 3'SS represents the 3' end splicing signal; A, U, G, and C represent the unmodified nucleoside moieties; m represents 2'MOE (2'-O-methoxyethyl base); * represents the thiophosphate bond; mASO represents the ASO that targets mouse IL1RL2.

[0208] 3. Cell culture and transfection

[0209] Immortalized human keratinocytes (HaCaT, Cell Bank of Chinese Academy of Sciences, catalog number: SCSP-5091) were cultured in 6-well or 12-well plates (Corning) using DMEM supplemented with 10% (vol / vol) fetal bovine serum (FBS).

[0210] For plasmid transfection, approximately 1.5 μg of the minigene plasmid was transfected into HaCaT cells using Lipofectamine 3000 (Thermo Fisher). For splicing analysis using RT-PCR, cells were treated with MOE-modified ASOs for 48 hours.

[0211] For ASOs modified with 2'MOE (2'-O-methoxyethyl base) and phosphate thioester bonds, cells were transfected with Lipofectamine 3000 (Thermo Fisher) for 48 hours. To suppress nonsense-mediated mRNA degradation (NMD) events, HaCaT cells were incubated with cyclohexanone (CHX, 50 μg / mL) for 4–6 hours before harvest.

[0212] 4. RT-PCR screening for alternative splicing of IL-36R

[0213] Common alternative splicing techniques include exon skipping and intron retention. RT-PCR can be performed by designing upstream and downstream primers for the exon preceding and following the exon to be tested (for example, if the exon to be skipped is exon 2 (E2), then upstream and downstream primers are designed for exon 1 (E1) and exon 3 (E3), respectively; as shown in Figure 2A, "E1-E3" indicates that the exon to be skipped is exon 2 (E2), and similarly, "E3-E5" indicates that the exon to be skipped is exon 4 (E4), and so on). This can detect whether an exon exhibits alternative splicing. If alternative splicing is present in the exon, gel electrophoresis will detect a shorter band than in the normal transcript, and sequencing alignment can then determine whether alternative splicing is present.

[0214] To detect the presence of intron retention due to alternative splicing between adjacent exons, upstream and downstream primers are designed for each exon to perform RT-PCR (e.g., if the retained intron to be tested is intron 1, upstream and downstream primers are designed for exon 1 (E1) and exon 2 (E2), respectively; as shown in Figure 4, "E1-E2" indicates that the retained intron to be tested is intron 1, and similarly, "E8-E9" indicates that the retained intron to be tested is intron 8, and so on). If intron retention is present, a band longer than normal transcripts can be detected by gel electrophoresis, and sequencing alignment can then determine whether intron retention exists. The presence of nonsense-mediated mRNA degradation (NMD) events in RNA can prevent the detection of this portion of RNA. To inhibit NMD-mediated RNA degradation, cyclohexanone (CHX, 50 μg / mL) can be incubated with cultured cells for 4–6 hours before harvesting.

[0215] 5. RT-qPCR

[0216] Following the manufacturer's instructions, total RNA was extracted from HaCaT cells using TRIzol (Invitrogen), and then 1 μg of RNA was reverse transcribed into cDNA using the iScript cDNA Synthesis Kit (Bio-Rad). PCR systems were prepared using a Roche 480SYBR Green I Master (480SYBR Green I Master) with three replicates per sample. RT-qPCR was performed using a Roche 480 Real-Time PCR instrument. GAPDH was used as an internal control. Relevant primers are listed in Table 3.

[0217] Table 3 Note: In the table, IL1RL2_E1~IL1RL2_E12 represent the primer names targeting exons 1 to 12 of the IL1RL2 gene, respectively; mIL1RL2_E5 represents the primer targeting exon 5 of the mouse IL1RL2 gene; and Ms_gapdh represents the primer targeting the mouse GAPDH gene.

[0218] 6. Protein extraction and Western blot analysis

[0219] Cells were collected in RIPA lysis buffer containing protease inhibitors, and the lysis buffer was centrifuged at 12,000g for 15 minutes at 4°C. The supernatant after centrifugation, i.e., the protein extract, was collected, and the protein concentration was measured using a BCA protein quantification kit (Yaxin). The protein sample was then mixed with protein loading buffer in a specific ratio to achieve a uniform protein concentration. The protein was denatured at 85°C for 10 minutes, separated by SDS-PAGE gel electrophoresis, and blotted onto a PVDF membrane. The membrane was blocked with 5% skim milk, and then detected using p-p38 (1:2000, Cell Signaling), p38 (1:2000, Cell Signaling), GAPDH (1:5000, KC-5G5, KANGCHEN), and the corresponding HRP-labeled secondary antibody (1:2000, Cell Signaling). The bands were observed using ECL (TIANGEN) and measured using ImageJ software.

[0220] 7. ASO treatment in psoriatic mice

[0221] The psoriasis mouse model was established using healthy 8-week-old female C57 mice, with the modeling site being the back. The mice were randomly divided into three groups: a blank control group (Blank), a modeling group (IMQ+PBS), and a drug intervention group (IMQ+ASO). In the drug intervention group, 62.5 mg of IMQ was applied topically to the back of the mice daily, and 100 μg (200 μL) of mASO-6-5 was injected subcutaneously on days 1, 3, and 5. In the modeling group, 62.5 mg of IMQ was applied topically to the back of the mice daily, and 200 μL of sterile PBS was injected subcutaneously on days 1, 3, and 5. Symptoms on the back of the mice were recorded daily at the beginning of the experiment. On day 7, the mice were euthanized, and the back skin was harvested for subsequent pathological section staining or molecular experiments.

[0222] 8. Statistical Analysis

[0223] Comparisons among the three groups were performed using one-way ANOVA, followed by Tukey's multiple comparison test. Differences were considered statistically significant at <0.05, expressed as follows: *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001.

[0224] Example 1. Analysis of alternative splicing events in the IL1RL2 gene present in human skin keratinocytes

[0225] To analyze alternative splicing events of the IL1RL2 gene in human keratinocytes, immortalized human keratinocytes (HaCaT) were first cultured and seeded in 6-well plates. When the cells reached 70-80% confluence, they were incubated with cyclohexanone (CHX, 50 μg / mL) for 4-6 hours. Total RNA was then extracted from HaCaT cells using TRIzol (Invitrogen), and 1 μg of RNA was reverse transcribed into cDNA using the iScript cDNA Synthesis Kit (Bio-Rad). For splicing analysis, the transcribed cDNA was used for PCR, employing primer pairs spanning the corresponding exons of the IL1RL2 gene. The primer sequences are listed in Table 3. The experimental procedures were as described above.

[0226] Figure 1 shows the location of the designed ASO target IL1RL2 (NM_003854). Specifically, the ASO targets IL1RL2 (NM_003854) exon 4 and intron 3 or their junction region, and the ASO targets IL1RL2 (NM_003854) exon 4 and intron 4 or their junction region, designated ASO-4-xx. Specifically, the ASO targets designated ASO-4-xx are located at positions c.294-100 to c.344 or c.439 to c.489+100 of IL1RL2 (NM_003854); the ASO targets IL1RL2 (NM_003854) exon 4 and intron 3 or their junction region, designated ASO-4part-xx. Specifically, the ASO targets IL1RL2 (NM_003854) exon 4 and intron 3 or their junction region, designated ASO-4part-xx. The ASO targeting sequence SO-10-xx is IL1RL2 (NM_003854) exon 10 and intron 9 or their junction region, specifically, the ASO targeting position of ASO-10-xx is from c.1136-100 to c.1186 or c.1247 to c.1297+100; the ASO targeting sequence ASO-crpE8-xx is IL1RL2 (NM_003854) intron 8 sequence, specifically, the ASO targeting position of ASO-crpE8-xx is from c.991+2372 to c.991+2522 or c.991+2541 to c.991+2691 of IL1RL2 (NM_003854).

[0227] The experimental results are shown in Figures 2 to 5. The results indicate that alternative splicing exists in exon 4, a partial sequence of exon 4, exon 6, exon 10, intron 1, and a partial sequence of intron 8 (named crpE8) of IL-36R. Among them, exon 4, a partial sequence of exon 4, exon 10, and crpE8 are potential targets for ASO intervention.

[0228] In Figure 2A, "E1-E3" indicates that the primers used to screen for alternative splicing target exon 1 and exon 3, respectively. These primers are used to screen for alternative splicing in exon 2, and so on. Analysis of IL-36R alternative splicing by RT-PCR (Figure 2A) showed that bands "E2-E4", "E3-E5", "E5-E7", and "E9-E11" were shorter than the full-length transcript (NM_003854). Sequencing these bands and comparing them with the full-length IL-36R transcript (NM_003854) confirmed that the bands in "E2-E4" were shorter than those in the full-length IL-36R transcript. The bands in "E3-E5" that are shorter than the full-length IL-36R transcript (NM_003854) have missing parts of exon 4 and the entire exon 4, respectively (Figure 2C). The bands in "E5-E7" and "E9-E11" that are shorter than the full-length IL-36R transcript (NM_003854) have missing the entire exon 6 and exon 10, respectively (Figures 2D-E). This indicates that exon skipping occurs in exon 4, part of exon 4, exon 6, and exon 10 of IL-36R.

[0229] (1) The skipped variable splicing of exon4 in IL-36R was analyzed, and the results are shown in Figure 3:

[0230] If exon4 is missing, i.e. IL-36R lacks exon 4, a stop codon will appear prematurely in exon5, resulting in a protein with only 116 amino acids. This will produce sIL-36R, which contains only the ligand-binding region of IL-36R and lacks the transmembrane and intracellular regions.

[0231] If only a portion of exon 4 is skipped, namely the IL-36R deletion of exon 4, the resulting protein will have only 105 amino acids, and like sIL-36R, it will only contain the ligand-binding region of IL-36R.

[0232] Since the two proteins produced by alternative splicing of exon4 both contain only the ligand-binding region of IL-36R, theoretically both proteins can competitively bind to the IL-36 cytokine, thereby inhibiting the IL-36R signaling pathway. Therefore, exon4 is a target that can be intervened with ASO.

[0233] (2) The variable splicing of exon6 and exon10 skips in IL-36R was analyzed, and the results are shown in Figure 3:

[0234] If exon6 or exon10 is skipped, since both are integer code mutations, neither will produce a stop codon prematurely.

[0235] If exon 6 is skipped, i.e. IL-36R lacks exon 6, the translated protein has 550 amino acids. The ligand-binding region, transmembrane region, and intracellular region of IL-36R are all present. Therefore, exon 6 is not a good target for intervention.

[0236] If exon 10 is skipped, i.e. IL-36R exon 10 is deleted, the translated protein has 521 amino acids. Although its IL-36R ligand binding region, transmembrane region, and intracellular region are all present, the deletion of exon 10 may disrupt the TIR signal transduction function because exon 10 is located in the TIR domain of the intracellular region. Therefore, exon 10 is also a potential target for ASO intervention.

[0237] (3) Analysis of the alternative splicing of intron 1 and intron 8 partial sequences of IL-36R was performed. The results are shown in Figure 5. Introns are retained in parts of intron 1 and intron 8 (named crpE8). Since the translation of IL-36R protein starts from exon 2, the retention of intron 1 is not a good intervention target. The retention of crpE8 will cause premature termination of IL-36R translation. The translated protein has 344 amino acids, with only the ligand-binding region (Figure 5). Therefore, crpE8 is also a potential target for ASO intervention.

[0238] In this invention, the wild-type IL1RL2 transcript is registered under the number NM_003854. In this invention, the alternative splicing transcripts corresponding to partial sequences of exon4, intron1, and crpE8 are not reported for the first time, and all of them can be translated into proteins.

[0239] Example 2. ASO alters IL-36R expression by regulating alternative splicing of exon 4.

[0240] Immortalized human keratinocytes (HaCaT) were cultured and seeded in 6-well plates. When the cells reached approximately 70% confluency, they were simultaneously transfected with 1.5 μg minegene C2 (mini C2) and 100 nM of various alternative saponins (ASOs) targeting the 3' or 5' splicing signals of exon 4. DPBS was used as a negative control. RNA was extracted from the cells after 48 hours and reverse transcribed into cDNA. PCR was used to detect the alternative splicing of exon 4, following the procedures outlined above.

[0241] The experimental results are shown in Figure 6. ASOs targeting the 3' splicing signal of exon 4 (numbered ASO-4-1, ASO-4-2, ASO-4-3, ASO-4-4, and ASO-4-5) effectively regulated alternative splicing of IL-36R mRNA. They significantly reduced the production of full-length IL-36R transcripts or IL-36R transcripts with partial deletion of exon 4, and significantly increased the production of truncated IL-36R (sIL-36R) transcripts with deletion of exon 4. Among these, ASO-4-3 showed the best intervention effect.

[0242] The ASOs targeting the 5' splicing signal of exon 4 (numbered ASO-4-6, ASO-4-7, ASO-4-8, ASO-4-9, ASO-4-10, ASO-4-11, ASO-4-12, ASO-4-13, and ASO-4-14) had no significant effect on the regulation of alternative splicing of IL-36R mRNA.

[0243] Immortalized human keratinocytes (HaCaT) were cultured and seeded in 6-well plates. When the cells reached approximately 70% confluency, they were transfected with 100 nM ASOs (numbered ASO-4-1, ASO-4-3, ASO-4-15, and ASO-4-16) targeting the 3' splicing signal of exon 4. DPBS was used as a negative control. RNA was extracted from the cells after 48 hours and reverse transcribed into cDNA. PCR was used to detect the alternative splicing of exon 4.

[0244] The experimental results are shown in Figure 7. ASO targeting the 3' splicing signal of exon 4 can effectively regulate the alternative splicing of IL-36R mRNA at the endogenous level of cells (Figure 7A). That is, it can significantly reduce the production of full-length IL-36R transcripts at the endogenous level of HaCaT cells and significantly increase the production of truncated IL-36R transcripts (sIL-36R) that lacks exon 4 (Figure 7B).

[0245] Example 3. ASO concentration-dependent regulation of alternative splicing of IL-36R exon 4

[0246] Immortalized human keratinocytes (HaCaT) were cultured and seeded in 6-well plates. When the cells reached approximately 70% confluency, they were transfected with different concentrations of ASO (labeled ASO-4-1 and ASO-4-3): 0 nM, 1 nM, 10 nM, 100 nM, and 300 nM. After 48 hours, RNA was extracted from the cells and reverse transcribed into cDNA. PCR was used to detect alternative splicing of exon 4, and ASO concentration profiles were analyzed.

[0247] The experimental results are shown in Figure 8. ASO regulates alternative splicing of IL-36R exon 4 in a concentration-dependent manner. The full-length IL-36R transcript decreased with increasing concentrations of ASO-4-1 (Figure 8A-B) and ASO-4-3 (Figure 8C-D), respectively; while the sIL-36R transcript lacking exon 4 increased with increasing concentrations of ASO-4-1 (Figure 8A-B) and ASO-4-3 (Figure 8C-D), respectively. Notably, a concentration of ASO-4-3 at 10 nM reduced the full-length IL-36R transcript by approximately 80% (Figure 8D), indicating that ASO-4-3 can regulate IL-36R alternative splicing at extremely low concentrations.

[0248] Example 4. ASO alters the activation level of downstream signaling pathways of IL-36R.

[0249] p38 mitogen-activated protein kinase (p38 MAPK) is a serine / threonine protein kinase that can be activated by various environmental stimuli and inflammatory cytokines. It plays a crucial role in regulating cell growth, responding appropriately to changes in the external environment, and in inflammatory responses. p38 isoforms include p38α, p38β, p38γ, and p38δ, which amplify upstream signals through a conserved three-tiered enzymatic cascade reaction, MAPKKK-MAPKK-MAPK, regulating the expression of genes including, but not limited to, inflammatory factors and chemokines, thereby mediating inflammatory responses. NF-κB, the nuclear factor kappa-light-chain enhancer of activated B cells, is a transcription factor associated with immune responses. It mainly includes NF-κB1 (p50), NF-κB2 (p52), RelA (p65), RelB, and c-Rel, playing a key role in regulating physiological processes such as immune and inflammatory responses. When NF-κB is abnormally activated, it can lead to a variety of diseases, including autoimmune inflammatory diseases, infectious diseases, allergic diseases, and cancer.

[0250] p38 MAPK and NF-κB are downstream molecules of the IL-36R signaling pathway. Studies have shown that when the IL-36R signaling pathway is abnormally activated, p38 MAPK and NF-κB can be activated, i.e., phosphorylated, which leads to the nuclear translocation of AP-1, a downstream molecule of NF-κB and p38 MAPK, promoting the overexpression of inflammatory factors and chemokines, ultimately causing an inflammatory response in the body.

[0251] This embodiment aims to evaluate the effect of ASO on the activation level of the IL-36R signaling pathway by detecting the phosphorylation levels of p38 MAPK and NF-κB after ASO intervention.

[0252] Immortalized human keratinocytes (HaCaT) were cultured and seeded in 12-well plates. When the HaCaT cells reached 70% confluency, they were transfected with ASO (numbered ASO-4-1, ASO-4-3, ASO-4-15, and ASO-4-16) at a concentration of 100 nM for 48 hours. Before protein collection, the cells were stimulated with 100 ng / ml recombinant IL-36γ protein for 30 minutes, and p38MAPK phosphorylation (p-p38) was detected by Western blotting.

[0253] Experimental results, as shown in Figure 9, indicate that IL-36γ can significantly induce p38MAPK phosphorylation, i.e., p-p38 expression. When HaCaT was treated with ASOs (numbered ASO-4-1, ASO-4-3, ASO-4-15, and ASO-4-16), it was observed that ASOs ASO-4-3, ASO-4-15, and ASO-4-16 significantly inhibited the IL-36γ-induced p38 MAPK phosphorylation level (Figures 9A-9B). This demonstrates that ASOs can reduce the activation level of the IL-36R signaling pathway.

[0254] Example 5. ASO alters the expression of downstream genes in the IL-36R signaling pathway

[0255] The IL-36R signaling pathway has three agonists (IL-36α, IL-36β, and IL-36γ) and two antagonists (IL-36Ra and IL-38). Activation of the IL-36R signaling pathway primarily involves the binding of agonists to IL-36R, followed by the recruitment of IL-1RAcP to assemble into a ternary complex. This complex aggregates the intracellular TIR structures of the receptor, activating p38 MAPK and NF-κB signaling, and further promoting the expression of various inflammatory cytokines such as TNF-α, IL-6, IL-17, and IL-1α / β, as well as chemokines CXCL1, CXCL2, CXCL8, and CCL20, thereby activating both the innate and acquired immune systems. IL-36α / β / γ act as amplifiers of skin inflammation, activating keratinocytes and resident immune cells to produce various inflammatory cytokines and chemokines, thereby recruiting more immune cells to the skin and exacerbating the inflammatory response.

[0256] This embodiment aims to evaluate the effect of ASO on the activation level of the IL-36R signaling pathway by detecting changes in the expression levels of downstream genes in the IL-36R signaling pathway after ASO intervention.

[0257] Immortalized human keratinocytes (HaCaT) were cultured and seeded in 6-well plates. When the cells reached 60-70% cell growth, they were transfected with ASO (numbered ASO-4-1, ASO-4-3, ASO-4-15, and ASO-4-16) at a concentration of 100 nM. After 47.5 hours, 100 ng / ml IL-36γ was added to the cells. After 30 minutes, RNA was extracted, and the expression of inflammatory cytokines TNF-α, IL-6, and the chemokine CCL20 was detected by qPCR.

[0258] The experimental results showed that, as shown in Figure 10, ASO could inhibit the expression of IL-36γ-induced inflammatory cytokines TNF-α (Figure 10A), IL-6 (Figure 10B), and chemokine CCL20 (Figure 10C), with ASO-4-3 showing the most significant inhibitory effect.

[0259] Example 6. ASO screening targeting mouse IL1RL2 exon 6

[0260] Testing drugs in animal models remains an indispensable and crucial step in new drug development. It allows for more reliable prediction of the drug's actual response in humans, serving as an important safeguard to ensure drug efficacy and reduce the risks of clinical trials.

[0261] The IL1RL2 gene in humans and mice has low homology, so constructing IL1RL2 humanized mice to test the in vivo efficacy of ASO in treating IL-36R-related autoimmune inflammatory diseases is not a good strategy.

[0262] Since there is currently a lack of animal models for testing ASO treatment targeting the human IL1RL2 gene to treat IL-36R-related autoimmune inflammatory diseases such as psoriasis, this invention will conduct ASO intervention experiments on wild-type mice with psoriasis models. As mentioned earlier, the IL1RL2 gene in humans and mice has poor homology. In order to conduct ASO drug intervention in mice, this invention needs to first screen for ASOs that can regulate mouse IL1RL2 and are similar to the human IL1RL2 gene.

[0263] Through gene structure analysis and experimental testing, the inventors discovered that mice also possess a soluble protein, sIL-36R, which can inhibit the activation of the IL-36R signaling pathway. This protein also contains only the ligand-binding region of IL-36R and can competitively bind to the IL-36 cytokine, thereby inhibiting the IL-36R signaling pathway. However, unlike in humans, the production of sIL-36R in mice is due to the Il1rl2 pre-mRNA skipping exon 6. To conduct ASO intervention experiments in mice, this invention designed seven ASOs targeting mouse Il1rl2 exon 6 and tested and screened them in mouse primary fibroblasts. RT-PCR results (Figures 11A-11B) showed that, compared with the DPBS control group, ASOs targeting the 3' splicing signal of exon 6 (mASO-6-5, mASO-6-6, and mASO-6-7) effectively regulated alternative splicing of exon 6 in mouse IL1rl2, reducing the full-length IL-36R transcript while increasing the production of sIL-36R transcript. Among these, mASO-6-5 showed the best effect, reducing the full-length transcript by approximately 50% at 100 nM. mASO-6-5 will be used in subsequent mouse intervention experiments.

[0264] Example 7. ASO treatment of psoriatic mice

[0265] Psoriasis is a skin disease characterized by the proliferation of epidermal keratinocytes, mediated by the cellular and molecular mechanisms of the innate and adaptive immune systems, and resulting from a combination of genetic, epigenetic, and environmental influences. The most commonly used animal model in preclinical studies is the imiquimod (IMQ)-induced skin lesion and inflammation model, which has a phenotype similar to human psoriasis. IMQ can induce skin lesions in mice, increasing the severity of rashes and erythema, thickening of the epidermis, and histopathological examination revealing dermal infiltration dominated by parakeratosis and inflammatory leukocytes. This ASO intervention mouse model of psoriasis will be established using IMQ.

[0266] The psoriasis mouse model was established using healthy 8-week-old female C57 mice, with the model site being the back. The mice were randomly divided into three groups: a control group, a model group, and a drug intervention group. In the drug intervention group, 62.5 mg of IMQ was applied topically to the back of the mice daily, as shown in Figure 12A. On days 1, 3, and 5, 100 μg (200 μL) of mASO-6-5 was injected subcutaneously. In the model group, 62.5 mg of IMQ was applied topically to the back of the mice daily, and on days 1, 3, and 5, 200 μL of sterile PBS was injected subcutaneously. Symptoms on the back of the mice were recorded daily at the beginning of the experiment. On day 7, the mice were euthanized, and the back skin was harvested for subsequent pathological section staining or molecular experiments.

[0267] The experimental results showed that the mice in the model group exhibited the most pronounced symptoms from day 4 to 6, with obvious erythema and scaling on their skin. Comparing the symptoms on the backs of the mice in the drug intervention group and the model group from day 4 to 6, it was clear that the skin inflammation on the backs of the mice in the drug intervention group was significantly improved (Figure 12B). Further pathological staining results also showed that the epidermal layer of the skin on the back was significantly thinned after mASO-6-5 intervention (Figure 12C). This indicates that mASO-6-5 intervention can improve skin inflammation in mice.

[0268] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. An oligonucleotide, characterized in that, The oligonucleotides interfere with the 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 as described in claim 1, characterized in that, 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+2522 region or c.991+2541~c.991+2691 region of the IL1RL2 transcript (NM_003854).

3. The oligonucleotide as described in claim 1, characterized in that, The truncated IL1RL2 splice isoform transcripts were selected from the following group: Z1 transcript, nucleotide sequence as shown in SEQ ID NO.5, contains truncated exon 4; Z2 transcript, nucleotide sequence as 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, characterized in that, The oligonucleotides mentioned are antisense oligonucleotides (ASO).

5. The oligonucleotide as described in claim 1, characterized in that, The IL1RL2 splice isoform transcripts with cryptic exon insertions were selected from the following group: The Z4 transcript, with its nucleotide sequence shown in SEQ ID NO.13, retains the hidden exon of intron 8.

6. The oligonucleotide as described in claim 1, characterized in that, The truncated or cryptic exon insertion IL1RL2 splice isoform transcript encodes a mutant protein selected from the following group: 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 as described in 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 as described in claim 4, characterized in that, 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 as described in claim 4, characterized in that, The antisense oligonucleotide sequence is any one of the oligonucleotides shown in SEQ ID NO.17-32, 65-71.

10. The oligonucleotide as claimed in claim 4, characterized in that, The antisense oligonucleotide may be modified or unmodified.

11. The oligonucleotide of claim 10, characterized in that, The modification includes first-generation modification and second-generation modification. The first-generation modification includes phosphate thioester modification (PS) in the phosphate ribose backbone. 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 encoding the variant transcript of IL-36R, wherein, compared with the normal IL1RL2 transcript, the variant transcript is selected from the following group: (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 to the wild-type IL1RL2 protein, the mutant protein is selected from the following group: 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 in that, This includes 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, include: (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 in that... The method includes the step of administering a safe and effective amount of the oligonucleotide as described in claim 1, or the pharmaceutical composition as described in claim 14, to the desired recipient.

18. A method for inducing the production of IL1RL2 variant mRNA transcripts, the method comprising: Delivering the oligonucleotide of claim 1 or the pharmaceutical composition of claim 14 to cells induces the production of IL1RL2 variant mRNA transcripts.

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