Group of antisense oligonucleotides targeting JAK1 and use thereof

Abstract

A group of antisense oligonucleotides targeting JAK1 and a use thereof. Each antisense oligonucleotide has a length of 16-20 bases. The antisense oligonucleotides have the following uses: (1) preparing a formulation for inhibiting the expression level of JAK1; (2) preparing a drug for treating atopic dermatitis (AD) related to JAK1; (3) treating AD related to JAK1; or (4) inhibiting the expression of JAK1 protein.

Description

A group of antisense oligonucleotides targeting JAK1 and their applications Technical Field

[0001] This invention belongs to the field of biotechnology, specifically, it relates to a group of antisense oligonucleotides targeting JAK1 and their applications. Background Technology

[0002] Atopic dermatitis (AD) is a chronic, relapsing, inflammatory skin disease. Patients often have co-existing atopic conditions such as allergic rhinitis and asthma, thus it is considered a systemic disease. Over the past 30 years, the prevalence of AD has been increasing globally, reaching 10%–20% in children in developed countries, ranking first among non-fatal skin disease burdens. Although the exact pathogenesis of AD is unclear, immune abnormalities, skin barrier dysfunction, and skin microbiota dysbiosis are generally considered important factors. Currently, medications used to treat atopic dermatitis include corticosteroids, TCIs, antihistamines, immunosuppressants, JAK (Janus-activated kinase) inhibitors, and biologics. Among these, JAK inhibitors are recommended as systemic treatment candidates in the 2023 Chinese AD clinical pathway expert consensus.

[0003] The pathogenesis of Alzheimer's disease (AD) involves soluble mediators binding to specific transmembrane receptors, subsequently initiating intracellular signaling via the JAK signaling transduction and STAT pathways. AD has a complex pathogenesis, with the JAK and STAT pathways playing a central role in regulating multiple immune axes involved in the pathogenesis of AD, inducing the expression of various cytokines, particularly Th2 cytokines, including interleukin (IL)-4, IL-5, IL-13, IL-31, and thymic stromal lymphopoietin (TSLP), leading to chronic inflammation and pruritus symptoms in AD. Topical application of JAK inhibitors alleviates Th2-mediated skin reactions and pruritus, and repairs skin barrier dysfunction, making them suitable for the treatment of non-atopic dermatitis. Currently, indications for targeting JAK1 include alopecia areata, atopic dermatitis, vitiligo, and psoriasis. Among these, drugs targeting JAK1 for atopic dermatitis are all small molecule drugs with limited efficacy and significant side effects.

[0004] Nucleic acid drugs are a novel treatment approach with advantages such as high specificity, significant efficacy, and low toxicity. They have great development potential and are expected to become an effective means of treating atopic dermatitis.

[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) targeting the JAK1 (Janus-activated kinase 1) gene. The antisense oligonucleotides are 16-20 bases in length and specifically pair with a specific region of the target gene. The start site of the specific region of the target gene that specifically pairs with the ASO is located at the following position in the pre-mRNA of JAK1:

[0007] (1)EXON1_INTRON1-2:202-208

[0008] (2)INTRON1-2:1335,7218-7219,18148-18152,18154,19773,26394,40970-40981,40983,40985,44033,441 65-44168, 45377-45378, 45409-45411, 55391, 58240, 61342-61345, 74503-74505, 74636-74639, 74687-74689

[0009] (3)INTRON2-3:82483

[0010] (4)INTRON2-3_EXON3:83071

[0011] (5)INTRON3-4:86737-86741, 86856-86858

[0012] (6)INTRON4-5:89170-89176

[0013] (7)INTRON5-6:95754

[0014] (8)INTRON6-7:98439

[0015] (9)INTRON6-7_EXON7:99334-99339

[0016] (10)INTRON7-8_EXON8:101567

[0017] (11)INTRON8-9:104269

[0018] (12)INTRON11-12:112469-112474

[0019] (13)INTRON14-15_EXON15:120907-120909

[0020] (14)INTRON23-24_EXON24:131040-131043

[0021] (15)INTRON3-4_EXON4 INTRON4-5:89435-89436, 87398-87399

[0022] Alternatively, the start site of a specific region of the target gene that is specifically paired with the aforementioned ASO is located at the following position on the JAK1 mRNA:

[0023] (1)5UTR EXON1 EXON1_2: 202

[0024] (2)EXON2_3: 289, 292, 295-297

[0025] (3)EXON3: 305-306, 309, 315, 319-320, 342

[0026] (4) EXON4 EXON4_5: 602

[0027] (5) EXON5: 680, 754

[0028] (6) EXON5_6: 775, 777

[0029] (7) EXON6: 889-890

[0030] (8) EXON6_7: 934-941

[0031] (9) EXON6_7 EXON7: 942-947

[0032] (10)EXON7: 945-949, 957-958, 977-978, 980, 1041-1042, 1044, 1046, 1087, 1108-1109, 1141, 1145

[0033] (11)EXON8: 1307-1310, 1315, 1320, 1338, 1370-1372, 1375, 1377, 1414, 1416, 1421, 1423-1424, 1430-1431, 1433-1435

[0034] (12) EXON9: 1499, 1526

[0035] (13)EXON10: 1672-1678, 1680-1681, 1692, 1694-1695, 1699

[0036] (14)EXON11: 1773, 1776, 1802, 1880

[0037] (15)EXON13: 2083-2084, 2140, 2142-2144

[0038] (16) EXON14_15 EXON15: 2282

[0039] (17)EXON15: 2318-2319, 2321-2327, 2374-2375

[0040] (18)EXON17: 2612-2615, 2617, 2621-2640, 2642, 2645

[0041] (19)EXON18: 2770-2771, 2773, 2777, 2804, 2806

[0042] (20)EXON20: 2951, 2956, 3028-3035, 3037-3038, 3041, 3098, 3104-3105, 3108

[0043] (21)EXON21: 3166, 3229, 3230-3231

[0044] (22) EXON21_22: 3250-3257

[0045] (23)EXON22: 3274-3290, 3310-3311, 3362, 3366-3377, 3380

[0046] (24)EXON23_24EXON24: 3555, 3558-3559

[0047] (25)EXON24: 2556, 3558, 3560, 3562-3563, 3580-3581

[0048] (26)EXON25: 3683-3689, 3720-3721, 3723

[0049] (27)EXON253UTR: 4804-4810, 5040-5043, 5046-5049

[0050] The target gene of JAK1 mRNA is numbered ENST00000342505.5, and its sequence is shown in SEQ ID NO.429.

[0051] The target gene of the JAK1 Pre-mRNA is numbered ENSG00000162434, and its sequence is shown in SEQ ID NO.430.

[0052] Furthermore, the nucleotide sequence of the ASO molecule is as shown in any of SEQ ID NO.1-428.

[0053] Furthermore, the ASO molecule is a chemically modified version containing chemical modifications;

[0054] 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);

[0055] 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.

[0056] Furthermore, in the aforementioned ASO molecule:

[0057] (1) All or some of the phospholipid bonds of the nucleotides contain monothioses;

[0058] (2) The 3' end and 5' end each have 3-5 bases modified by MOE or cEt.

[0059] More preferably, in the ASO molecule:

[0060] (1) All or some of the phospholipid bonds of the nucleotides contain monothioses;

[0061] (2) ASO with a length of 20nt has 5 bases at the 3' end and 5' end respectively modified with MOE, and ASO with a length of 16nt has 3 bases at the 3' end and 5' end respectively modified with cEt.

[0062] Furthermore, the present invention also relates to the following applications of the aforementioned antisense oligonucleotide molecule (ASO):

[0063] (1) Prepare a formulation that inhibits JAK1 expression; or

[0064] (2) To prepare a drug or drug composition for treating JAK1-associated atopic dermatitis (AD); or

[0065] (3) Treatment of JAK1-associated atopic dermatitis (AD); or

[0066] (4) Inhibit the expression of JAK1 protein.

[0067] Furthermore, the present invention also relates to a medicament or pharmaceutical composition comprising the antisense oligonucleotide molecule (ASO); the medicament is used to treat a disease caused by abnormal expression of the JAK1 gene; preferably, the disease is atopic dermatitis (AD).

[0068] The drug or pharmaceutical composition comprises a therapeutically effective amount of the antisense oligonucleotide (ASO), and necessary pharmaceutical excipients or delivery carriers.

[0069] 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

[0070] Example 1: Design, synthesis and modification of ASO targeting non-receptor tyrosine protein kinase 1 target genes

[0071] The JAK1 mRNA (ENST00000342505.5) from the Ensembl database was selected as the target gene. The sequence of the target gene is shown in SEQ ID NO.429.

[0072] SEQ ID NO.429:

[0073] The JAK1 pre-mRNA (ENSG00000162434) from the Ensembl database was selected as the target gene, and its sequence is shown in SEQ ID NO.430.

[0074] For JAK1 mRNA, 306 ASO molecules were designed and selected (Table 1) for synthesis and in vitro efficacy testing. The specific locations and information of these ASOs are shown in Table 3 below. For JAK1 pre-mRNA, 122 ASO molecules were designed and selected (Table 2) for synthesis and in vitro efficacy testing. The specific locations and information of these ASOs are shown in Table 4 below.

[0075] Furthermore, 428 of the aforementioned ASO sequences were chemically modified, specifically as follows:

[0076] (1) Monothiolation of phospholipase bonds in all or part of the nucleotides in the sequence; and

[0077] (2) ASO with a length of 20nt has 5 bases at the 3' end and 5' end respectively modified with MOE; ASO with a length of 16nt has 3 bases at the 3' end and 5' end respectively modified with cEt.

[0078] Table 1: ASO targeting JAK1 mRNA

[0079] Table 2. ASO targeting JAK1 pre-mRNA

[0080] Table 3. Detailed information on JAK1 mRNA ASO molecules

[0081] Table 4. Detailed information on the JAK1 Pre-mRNA molecule

[0082] Example 2: Testing ASO activity using an in vitro cell model (HaCaT human immortalized epidermal cells)

[0083] In this embodiment, the inhibitory effect of the ASO molecules listed in Table 1 on the expression of non-receptor tyrosine protein kinase 1 (JAK1) in HaCaT cells was verified. The specific experimental procedure is as follows:

[0084] (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.

[0085] (2) Cell treatment: Cells were plated one day before transfection at a concentration of 5 × 10⁶ cells / mL. 3 Cells were seeded into 96-well plates, and 100 μl of DMEM medium containing 10% FBS was added to each well. Before transfection, the HaCaT cell confluence rate should be >60% as observed under a microscope. The old medium was discarded and replaced with 50 μl of DMEM 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 hours, the medium was changed to fresh medium.

[0086] (3) 24 h after transfection, total RNA was extracted from the cells and the expression of JAK1 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 JAK1 are shown in Table 5-1.

[0087] Table 5-1 qPCR primer and probe sequences

[0088] 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%.

[0089] The experimental groups are as follows:

[0090] Cells treated with modified ASO as indicated by their respective numbers;

[0091] Blank, the blank control group, consists of cells that have not undergone any ASO treatment.

[0092] The efficiency of the ASO sequence described in Table 1 in inhibiting JAK1 mRNA in HaCaT cells is shown in Table 5-2 below.

[0093] The results show that the ASO sequence shown in Table 1 has a significant inhibitory effect on the transcription of JAK1 mRNA.

[0094] Table 5-2 The inhibition rate (%) of JAK1 mRNA in HaCaT cells after treatment with antisense oligonucleotides.

[0095] The efficiency of the ASO sequence described in Table 2 in inhibiting JAK1 Pre-mRNA in HaCaT cells is shown in Table 5-3 below.

[0096] The results show that the ASO sequence shown in Table 2 has a significant inhibitory effect on the transcription of JAK1 Pre-mRNA.

[0097] Table 5-3 shows the efficiency of the ASO sequence described in Table 2 in inhibiting JAK1 Pre-mRNA in HaCaT cells.

[0098] 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 oligonucleotides (ASO molecules) targeting the JAK1 (Janus-activated kinase 1) gene, characterized in that, The antisense oligonucleotide molecule is 16-20 bases in length and specifically pairs with a specific region of the target gene. The start site of the specific region of the target gene that specifically pairs with the ASO molecule is located on the mRNA and pre-mRNA of JAK1. Specifically, it is located at the following position in the pre-mRNA of JAK1: (1)EXON1_INTRON1-2:202-208 (2)INTRON1-2:1335,7218-7219,18148-18152,18154,19773,26394,40970-40981,40983,40985,44033,441 65-44168, 45377-45378, 45409-45411, 55391, 58240, 61342-61345, 74503-74505, 74636-74639, 74687-74689 (3)INTRON2-3:82483 (4)INTRON2-3_EXON3:83071 (5)INTRON3-4:86737-86741, 86856-86858 (6)INTRON4-5:89170-89176 (7)INTRON5-6:95754 (8)INTRON6-7:98439 (9)INTRON6-7_EXON7:99334-99339 (10)INTRON7-8_EXON8:101567 (11)INTRON8-9:104269 (12)INTRON11-12:112469-112474 (13)INTRON14-15_EXON15:120907-120909 (14)INTRON23-24_EXON24:131040-131043 (15)INTRON3-4_EXON4 INTRON4-5:89435-89436, 87398-87399 Alternatively, it may be located at the following position on the JAK1 mRNA: (1) 5UTREXON1 EXON1_2: 202 (2)EXON2_3: 289, 292, 295-297 (3)EXON3: 305-306, 309, 315, 319-320, 342 (4) EXON4 EXON4_5: 602 (5) EXON5: 680, 754 (6) EXON5_6: 775, 777 (7) EXON6: 889-890 (8) EXON6_7: 934-941 (9) EXON6_7 EXON7: 942-947 (10)EXON7: 945-949, 957-958, 977-978, 980, 1041-1042, 1044, 1046, 1087, 1108-1109, 1141, 1145 (11)EXON8: 1307-1310, 1315, 1320, 1338, 1370-1372, 1375, 1377, 1414, 1416, 1421, 1423-1424, 1430-1431, 1433-1435 (12) EXON9: 1499, 1526 (13)EXON10: 1672-1678, 1680-1681, 1692, 1694-1695, 1699 (14)EXON11: 1773, 1776, 1802, 1880 (15)EXON13: 2083-2084, 2140, 2142-2144 (16) EXON14_15 EXON15: 2282 (17)EXON15: 2318-2319, 2321-2327, 2374-2375 (18)EXON17: 2612-2615, 2617, 2621-2640, 2642, 2645 (19)EXON18: 2770-2771, 2773, 2777, 2804, 2806 (20)EXON20: 2951, 2956, 3028-3035, 3037-3038, 3041, 3098, 3104-3105, 3108 (21)EXON21: 3166, 3229, 3230-3231 (22) EXON21_22: 3250-3257 (23)EXON22: 3274-3290, 3310-3311, 3362, 3366-3377, 3380 (24)EXON23_24EXON24: 3555, 3558-3559 (25)EXON24: 2556, 3558, 3560, 3562-3563, 3580-3581 (26)EXON25: 3683-3689, 3720-3721, 3723 (27)EXON253UTR: 4804-4810, 5040-5043, 5046-5049 The sequence of the JAK1 mRNA is shown in SEQ ID NO.

429. The sequence of the JAK1 Pre-mRNA is shown in SEQ ID NO.

430.

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.1-428.

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

4. The ASO molecule according to claim 3, characterized in that, The chemical modification is as follows: (1) All or some of the phospholipid bonds of the nucleotides contain monothioses; (2) The 3' end and 5' end each have 3-5 bases modified by MOE or cEt.

5. The ASO molecule according to claim 4, characterized in that, The chemical modification is as follows: (1) All or some of the phospholipid bonds of the nucleotides contain monothioses; (2) ASO with a length of 20nt has 5 bases at the 3' end and 5' end respectively modified with MOE, and ASO with a length of 16nt has 3 bases at the 3' end and 5' end respectively modified with cEt.

6. The following applications of the antisense oligonucleotide molecule according to any one of claims 1-5: (1) Prepare a formulation that inhibits JAK1 expression; or (2) To prepare a drug or drug composition for treating JAK1-associated atopic dermatitis (AD); or (3) Treatment of JAK1-associated atopic dermatitis (AD); or (4) Inhibit the expression of JAK1 protein.

7. A pharmaceutical product or pharmaceutical composition comprising the antisense oligonucleotide molecule (ASO) according to any one of claims 1-5, characterized in that, The drug or drug composition is used to treat diseases caused by abnormal expression of the JAK1 gene; preferably, the disease is atopic dermatitis (AD); The drug or pharmaceutical composition comprises: a therapeutically effective amount of the antisense oligonucleotide (ASO), and necessary pharmaceutical excipients or delivery carriers.

8. The drug or drug composition according to claim 7, 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.

Images