Set of antisense oligonucleotides specifically inhibiting thymic stromal lymphopoietin and use thereof

Abstract

A set of antisense oligonucleotides specifically inhibiting thymic stromal lymphopoietin and the use thereof. The antisense oligonucleotide molecules have a length of 16-20 bases. The antisense oligonucleotide molecules have the following use: (1) in the preparation of a preparation for inhibiting the expression level of thymic stromal lymphopoietin; or (2) in the treatment of asthma, atopic dermatitis, chronic obstructive pulmonary disease, sinusitis, and nasal polyps.

Description

A group of antisense oligonucleotides that specifically inhibit thymic stromal lymphopoietin and their applications Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a group of antisense oligonucleotides that specifically inhibit thymic stromal lymphopoietin and their applications. Background Technology

[0002] Asthma is a non-communicable chronic lung disease affecting people of all ages, and is the most common chronic disease in children. It is caused by inflammation and muscle tightness around the airways, leading to difficulty breathing. According to WHO estimates, approximately 262 million people worldwide had asthma in 2019, resulting in 455,000 deaths. The prevalence rate increased by 12.6% compared to 1990. The cost of treating asthma, especially severe asthma, is very high. Between 2013 and 2014, the direct cost of hospitalization for acute asthma attacks in China (per person per attack) reached 11,603 RMB. This demonstrates that, both domestically and internationally, asthma treatment leads to a significant drain on public health resources during diagnosis and treatment, highlighting the continued huge market potential for developing cost-effective treatments and regimens.

[0003] Asthma is classified into three main types: 1. T2 type inflammatory asthma: including eosinophilic (allergic asthma) and non-eosinophilic asthma; 2. Non-T2 type inflammatory asthma; 3. Common type (induced by physical injury). TSLP (Thymic Stromal Lymphopoietin) plays a role in all three types of asthma. TSLP is the newest member of the IL-12 family and is an epithelial cytokine. Its mechanism of action involves activating antigen-presenting cells and playing a crucial role in the maturation of T cell populations. TSLP can act on many immune cells. When TSLP forms a triple complex of TSLP / TSLPR / IL-7Rα, it activates Janus kinase 1 (JAK1) and JAK2, primarily activating STAT5A and STAT5B in downstream signaling pathways, thereby inducing the transcription and expression of inflammatory factors such as IL-13 and IL-4, leading to an inflammatory response.

[0004] Currently, the top five indications for TSLP targeting are asthma, atopic dermatitis, COPD, sinusitis, and nasal polyps. Among these, the drugs targeting TSLP for asthma are all antibody drugs, but their efficacy is limited and they have significant side effects. Nucleic acid drugs, on the other hand, are a novel treatment approach with the advantages of high specificity, significant efficacy, and low toxicity. They have great development potential and are expected to become an effective means of treating asthma.

[0005] Based on this, the present invention is proposed. Summary of the Invention

[0006] This invention first relates to a set of antisense oligonucleotides (ASOs) that specifically inhibit thymic stromal lymphopoietin. The antisense oligonucleotides are 16-20 bases in length, and their target genes are TSLP mRNA and pre-mRNA. The ASOs specifically pair with a specific region of the target gene, and the start site of the specific region is located at:

[0007] TSLPpre-mRNA

[0008] (1)EXON1_INTRON1-2: 1987, 1993;

[0009] (2)INTRON1-2: 2031-2047, 2101-2183, 2216-2247, 2259-2278, 2297, 2336-2727, 2774, 2785;

[0010] (3)EXON2_INTRON2-3:2756-2868;

[0011] (4)INTRON2-3: 2909-2945, 3032-3079, 3096-3175, 3200-3233;

[0012] TSLP mRNA

[0013] (1)5UTR_EXON1: 1~160, 163~199, 231~266, 289~348;

[0014] (2)EXON1 EXON1_2, EXON1_2, EXON1_2EXON2: 328-353;

[0015] (3) EXON2: 356-357, 361, 366-370, 372;

[0016] (4)EXON2 EXON2_3, EXON2_3: 374-381, 383-390;

[0017] (5)EXON3:410-419, 444-462, 494-495, 505;

[0018] (6) EXON3_4: 513-515;

[0019] (7)EXON43UTR:579,608~629,635,673,724,812~861,889~904,987~997,1048, 1099~1154, 1218, 1236~1243, 1391 ~1495, 1552, 1582, 1656~1708, 1740~1901, 1994~2027, 2114~2294, 2364~2380, 2467~2479, 2532~2568, 2590~2593;

[0020] The target gene of the TSLP mRNA is numbered ENST00000344895.4, and its sequence is shown in SEQ ID NO.611; the target gene of the TSLP Pre-mRNA is numbered ENSG00000145777, and its sequence is shown in SEQ ID NO.612.

[0021] Furthermore, the nucleotide sequence of the ASO molecule is shown in SEQ ID NO.1-610.

[0022] The ASO molecule targeting TSLP mRNA is selected from any of the oligonucleotide sequences shown in Table 1 below;

[0023] Table 1. ASO molecules targeting TSLP mRNA

[0024] The ASO molecule targeting TSLP pre-mRNA is selected from any of the oligonucleotide sequences shown in Table 2 below;

[0025] Table 2. ASO molecules targeting TSLP pre-mRNA

[0026] Furthermore, the ASO molecule is a chemically modified version containing chemical modifications;

[0027] Preferably, the modification is: thiolation of the phospholipase bond, methoxyethyl modification at the 2' position (MOE modification), or restriction ethyl-bridged nucleic acid modification at the 2' position (cEt modification);

[0028] More preferably, the modification is: monothiolation of all or part of the phosphate ester bonds of the ASO molecule, and MOE or cEt modification of 3 to 5 bases at the 3' and 5' ends, respectively.

[0029] Furthermore, in the aforementioned ASO molecule:

[0030] (1) All or some of the phospholipid bonds of the nucleotides contain monothioses;

[0031] (2) The 3' end and 5' end each have 3-5 bases modified by MOE or cEt.

[0032] Furthermore, the present invention also relates to the following applications of the aforementioned antisense oligonucleotide molecule (ASO):

[0033] (1) Prepare a formulation that inhibits TSLP expression; or

[0034] (2) To prepare a medicine or pharmaceutical composition for treating diseases related to TSLP; or

[0035] (3) Treatment of TSLP-related diseases; or

[0036] (4) Inhibit the expression of TSLP protein;

[0037] The preferred diseases are asthma, atopic dermatitis, COPD, sinusitis, and nasal polyps.

[0038] Furthermore, the present invention also relates to a medicament or pharmaceutical composition comprising the antisense oligonucleotide molecule (ASO); the medicament is used to treat diseases caused by abnormal expression of the TSLP gene; preferably, the diseases are asthma, atopic dermatitis, COPD, sinusitis, and nasal polyps.

[0039] The drug or pharmaceutical composition comprises a therapeutically effective amount of the antisense oligonucleotide (ASO), and necessary pharmaceutical excipients or delivery carriers.

[0040] The drug or drug composition is an aqueous solvent or an injection; the drug or drug composition is administered via local, intravenous, intramuscular, subcutaneous, or intradermal routes. Detailed Implementation

[0041] Example 1: Design, synthesis and modification of ASO targeting thymic stromal lymphopoietin target genes

[0042] The TSLP mRNA (ENST00000344895.4) from the Ensembl database was selected as the target gene. The sequence of the target gene is shown in SEQ ID NO.611.

[0043] SEQ ID NO.611:

[0044] The TSLP Pre-mRNA (ENSG00000145777) from the Ensembl database was selected as the target gene, and its sequence is shown in SEQ ID NO.612.

[0045] For TSLP mRNA, 442 ASO sequences were designed and selected for synthesis and in vitro efficacy testing. The specific locations and information of these ASOs are shown in Table 3 below. For TSLP pre-mRNA, 168 ASO sequences were designed and selected for synthesis and in vitro efficacy testing. The specific locations and information of these ASOs are shown in Table 4 below.

[0046] Furthermore, chemical modifications were performed on 610 of the aforementioned ASO sequences; specific modifications included: monothiolation of all or part of the phospholipid bonds in the sequence, and MOE modification with 3 to 5 bases at the 3' and 5' ends, respectively.

[0047] Table 3. Specific information on TSLP mRNA ASO molecules

[0048] Table 4. Detailed information on TSLP Pre-mRNA molecules

[0049] Example 2: Testing ASO activity in an in vitro cell model (A549 cells of human non-small cell lung cancer stably transfected with TSLP).

[0050] In this embodiment, the inhibitory effect of the ASO molecules listed in Table 1 on the expression of thymic stromal lymphopoietin (TSLP) in A549 cells stably transfected with TSLP was verified. The specific experimental procedure is as follows:

[0051] (1) Preparation of suspension transfection reagent: Dissolve ASO dry powder in sterile water to a concentration of 10 μM. Dilute 10 μL of ASO stock solution to the required concentration using serum-reduced medium (basalmedia, L530KJ). Dilute Lipofectamine 2000 transfection reagent (Invitrogen, 11668-019) with serum-reduced medium. Mix the transfection reagent dilution and ASO dilution separately to prepare ASO transfection complexes of the preset concentration or concentration gradient. Mix by pipetting and aspirating 3-6 times and let stand at room temperature for 20 min.

[0052] (2) Cell treatment: Cells were plated one day before transfection at a concentration of 5 × 10⁶ cells / mL. 3Cells were seeded into 96-well plates, and 100 μl of F-12K medium containing 10% FBS was added to each well. Before transfection, the confluence of A549 cells should be >70% under a microscope. The old medium was discarded and replaced with 50 μl of F-12K medium containing 10% FBS. The ASO transfection complex prepared in step (1) was added to the 96-well plates and incubated at 37°C in a 5% CO2 incubator. After 5 h, the medium was changed to fresh medium.

[0053] (3) 24 h after transfection, total RNA was extracted from the cells and the expression of TSLP mRNA in the cells was detected by real-time quantitative PCR (Q-RTPCR). The qPCR primers and probes used to amplify the internal reference gene Actin and the target gene TSLP are shown in Table 5-1.

[0054] Table 5-1 qPCR primer and probe sequences

[0055] Relative gene expression was calculated using the 2^-ΔΔCT method (Livak method). The inhibition rate of antisense oligonucleotide mRNA expression was calculated using the following equation: Inhibition rate = (1 - 2^-ΔΔCT) × 100%.

[0056] The experimental groups are as follows:

[0057] Cells treated with modified ASO as indicated by their respective numbers;

[0058] Blank, the blank control group, consists of cells that have not undergone any ASO treatment.

[0059] The efficiency of the ASO sequence described in Table 1 in inhibiting TSLP mRNA and pre-mRNA in A549 cells stably transfected with TSLP is shown in Table 5-2 below.

[0060] The results show that the ASO sequences shown in Tables 1 and 2 have a significant inhibitory effect on the transcription of TSLP mRNA and pre-mRNA.

[0061] Table 5-2 The mRNA inhibition rate (%) of TSLP after treatment of A549 cells with antisense oligonucleotides.

[0062] Finally, it should be noted that the above embodiments are only used to help those skilled in the art understand the essence of the present invention, and are not intended to limit the scope of protection of the present invention.

Claims

1. A group of antisense oligonucleotide molecules (ASOs) that specifically inhibit thymic stromal lymphopoietin, wherein the antisense oligonucleotide molecules are 16-20 bases in length, the target genes are TSLP mRNA and pre-mRNA, and the ASO molecules specifically pair with a specific region of the target gene, wherein the start site of the specific region is located at: The following location of TSLPpre-mRNA: (1)EXON1_INTRON1-2: 1987, 1993; (2)INTRON1-2: 2031-2047, 2101-2183, 2216-2247, 2259-2278, 2297, 2336-2727, 2774, 2785; (3)EXON2_INTRON2-3:2756-2868; (4)INTRON2-3: 2909-2945, 3032-3079, 3096-3175, 3200-3233; The following location of TSLP mRNA: (1)5UTR_EXON1: 1~160, 163~199, 231~266, 289~348; (2)EXON1 EXON1_2, EXON1_2, EXON1_2EXON2: 328-353; (3) EXON2: 356-357, 361, 366-370, 372; (4)EXON2 EXON2_3, EXON2_3: 374-381, 383-390; (5)EXON3:410-419, 444-462, 494-495, 505; (6) EXON3_4: 513-515; (7)EXON43UTR:579,608~629,635,673,724,812~861,889~904,987~997,1048, 1099~1154, 1218, 1236~1243, 1391 ~1495, 1552, 1582, 1656~1708, 1740~1901, 1994~2027, 2114~2294, 2364~2380, 2467~2479, 2532~2568, 2590~2593; The sequence of the TSLP mRNA is shown in SEQ ID NO. 611; The sequence of the TSLP Pre-mRNA is shown in SEQ ID NO.

612.

2. The ASO molecule according to claim 1, characterized in that, The nucleotide sequence of the ASO molecule is shown in any of SEQ ID NO.1-610.

3. The ASO molecule according to claim 1, characterized in that, The ASO molecule is a chemically modified version containing chemical modifications; Preferably, the modification is: thiolation of the phospholipase bond, methoxyethyl modification at the 2' position (MOE modification), or restriction ethyl-bridged nucleic acid modification at the 2' position (cEt modification); More preferably, the modification is: monothiolation of all or part of the phosphate ester bonds of the ASO molecule, and MOE or cEt modification of 3 to 5 bases at the 3' and 5' ends, respectively.

4. The following applications of the antisense oligonucleotide molecule (ASO) according to any one of claims 1-3: (1) Prepare a formulation that inhibits TSLP expression; or (2) To prepare a medicine or pharmaceutical composition for treating diseases related to TSLP; or (3) Treatment of TSLP-related diseases; or (4) Inhibit the expression of TSLP protein; The preferred diseases are asthma, atopic dermatitis, COPD, sinusitis, and nasal polyps.

5. A medicament or pharmaceutical composition comprising any one of the antisense oligonucleotide molecules (ASO) according to claims 1-3, characterized in that, The drug is used to treat diseases caused by abnormal expression of the TSLP gene; preferably, the diseases are asthma, atopic dermatitis, COPD, sinusitis, and nasal polyps.

6. The drug or drug composition according to claim 5, characterized in that, The drug or pharmaceutical composition comprises a therapeutically effective amount of the antisense oligonucleotide (ASO), and necessary pharmaceutical excipients or delivery carriers.

7. The drug or drug composition according to claim 5 or 6, characterized in that, The drug or drug composition is an aqueous solvent or an injection; the drug or drug composition is administered via local, intravenous, intramuscular, subcutaneous, or intradermal routes.

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