Double-stranded ribonucleic acids targeting jak1 gene and uses thereof

Abstract

The present application belongs to the field of biological medicine, and particularly relates to double-stranded ribonucleic acid targeting JAK1 gene and application thereof. Specifically, the present application provides a novel dsRNA targeting JAK1 gene, wherein the dsRNA comprises a sense strand and an antisense strand forming a double-stranded region, and the sense strand and the antisense strand are complementary or substantially complementary. The present application also provides a conjugate comprising the dsRNA, a delivery preparation, a cell and a pharmaceutical composition, and use thereof in reducing or inhibiting JAK1 gene expression and / or activity, preventing and / or treating a disease or disorder mediated by JAK1 gene and / or preparing a personal care product.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to a double-stranded ribonucleic acid (dsRNA) targeting the JAK1 gene, conjugates, delivery formulations, cell and pharmaceutical compositions, and their use in reducing or inhibiting JAK1 gene expression and / or activity, preventing and / or treating JAK1 gene-mediated diseases or conditions and / or preparing personal care products. Background Technology

[0002] Janus kinase 1 (JAK1) is a member of the JAK kinase family, which includes JAK1, JAK2, JAK3, and TYK2. JAKs are non-receptor tyrosine kinases located on the inner side of the cell membrane, primarily acting as messengers of cytokine signals. In terms of biological function, when extracellular cytokines bind to their corresponding receptors, they activate receptor-coupled JAK1. Activated JAK1 phosphorylates the receptor, providing a docking site for downstream signal transducer and activator of transcription (STAT) proteins. Phosphorylated STAT proteins form dimers that enter the nucleus and directly bind to the DNA sequence of specific genes, thereby regulating gene transcription and influencing cell fate, such as triggering inflammatory responses, promoting cell proliferation, or differentiation. JAK1-mediated signals are very broad, including important pro-inflammatory signals such as type I and type II interferons and gp130 receptor-dependent cytokines, and their function is tissue-specific.

[0003] Abnormal activation of JAK1 is a core factor leading to various autoimmune diseases. In atopic dermatitis, the overactive Th2 immune response produces large amounts of cytokines such as IL-4, IL-13, and IL-31, which are transmitted through JAK1, driving itching, impaired skin barrier function, and inflammation. In alopecia areata, perifollicular immune cells attack hair follicles by releasing cytokines such as interferon-γ; the JAK-STAT pathway is the core signaling pathway for these attacks.

[0004] Given the crucial role of JAK1 in disease, the development of highly selective JAK1 inhibitors has become a hot topic in drug research and development, with a trend towards shifting from early broad-spectrum JAK1 inhibitors to highly selective ones. Several selective JAK1 inhibitors have already been approved for marketing, including abuxitinib and utpatinib for the treatment of moderate to severe atopic dermatitis, and amaxitinib, originally developed in China, which was approved in March 2025 for the treatment of ankylosing spondylitis. Regarding investigational drugs, Jiangsu Weikai'er's VC005 tablets, as a "second-generation" highly selective JAK1 inhibitor, retains potent inhibition of JAK1 while further reducing inhibitory activity against JAK2, and has initiated Phase III clinical trials for ankylosing spondylitis and moderate to severe atopic dermatitis.

[0005] JAK1, a core protein in the JAK-STAT signaling pathway, plays a crucial role in the pathogenesis of autoimmune diseases. Drug development is moving towards higher selectivity and better safety, and small nucleic acids, due to their inherent characteristics, possess higher specificity and better safety, making them one of the best choices for novel JAK1-targeting drugs. Although intervention strategies targeting JAK1 (such as small molecule inhibitors and peptide mimics) have been explored, most of these methods are still in the preclinical research stage and face challenges such as difficulty in targeting the protein-protein interaction interface and potential off-target effects. Against this backdrop, utilizing gene silencing technology, especially small interfering RNA (siRNA) technology, to directly target and degrade JAK1 mRNA, thereby efficiently and specifically reducing its protein level at the source of gene expression, provides a novel and more advantageous direction for developing innovative therapies for autoimmune diseases.

[0006] Therefore, there is an urgent need to develop siRNA drugs that target JAK1 with high specificity, excellent efficacy, and high safety. Summary of the Invention

[0007] The purpose of this application is to provide a novel double-stranded RNA (dsRNA) targeting the JAK1 gene and its use in reducing or inhibiting JAK1 gene expression and / or activity, preventing and / or treating JAK1 gene-mediated diseases or conditions, and / or preparing personal care products.

[0008] In a first aspect, the present invention provides a double-stranded ribonucleic acid (dsRNA) targeting the JAK1 gene, comprising a sense strand and an antisense strand, wherein the nucleotide sequence of the sense strand comprises at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, or at least 21 consecutive nucleotides selected from any of the sequences shown in SEQ ID NO: 1-461, and the nucleotide difference does not exceed 0, 1, 2, or 3; and / or wherein the nucleotide sequence of the antisense strand comprises at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, or at least 22 consecutive nucleotides selected from any of the sequences shown in SEQ ID NO: 462-922, and the nucleotide difference does not exceed 0, 1, 2, or 3.

[0009] In a second aspect, the present invention provides a double-stranded ribonucleic acid (dsRNA) targeting the JAK1 gene, comprising a sense strand and an antisense strand, wherein the nucleotide sequence of the sense strand comprises at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, or at least 21 consecutive nucleotides selected from any of the sequences shown in SEQ ID NO: 923-1383, 1885, 1886, and 1887, and the nucleotide difference does not exceed 0, 1, 2, or 3; and / or wherein the nucleotide sequence of the antisense strand comprises at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, or at least 22 consecutive nucleotides selected from any of the sequences shown in SEQ ID NO: 1384-1844, and the nucleotide difference does not exceed 0, 1, 2, or 3.

[0010] In a third aspect, the present invention provides a double-stranded ribonucleic acid (dsRNA) targeting the JAK1 gene, comprising a sense strand and an antisense strand, wherein at least one nucleotide in the sense strand and the antisense strand is a modified nucleotide.

[0011] In a fourth aspect, the present invention provides a coupling of formula (I), R1—B—R2 Formula (I), Wherein R1 is a targeting ligand, delivery enhancer, or hydrophobic modifier, B is an optional adapter, and R2 is selected from the dsRNA described in this invention.

[0012] In a fifth aspect, the present invention provides a delivery formulation comprising dsRNA as described in the present invention and a pharmaceutically acceptable carrier.

[0013] In a sixth aspect, the present invention provides a cell comprising dsRNA according to the present invention.

[0014] In a seventh aspect, the present invention provides a pharmaceutical composition comprising dsRNA according to the present invention, a conjugate according to the present invention, a delivery formulation according to the present invention, or a cell according to the present invention, and a pharmaceutically acceptable excipient.

[0015] In an eighth aspect, the present invention provides the use of dsRNA, conjugates, delivery formulations, cells, or pharmaceutical compositions according to the present invention in the preparation of medicaments for the prevention and / or treatment of JAK1 gene-mediated diseases or conditions.

[0016] In a ninth aspect, the present invention provides the use of the dsRNA, conjugate, delivery formulation, cell, or pharmaceutical composition according to the present invention in the preparation of a medicament for reducing or inhibiting JAK1 gene expression and / or activity.

[0017] In a tenth aspect, the present invention provides a method for reducing or inhibiting JAK1 gene expression and / or activity, the method comprising the step of contacting a dsRNA according to the present invention, a conjugate according to the present invention, a delivery formulation according to the present invention, a cell according to the present invention, or a pharmaceutical composition according to the present invention with a cell.

[0018] In an eleventh aspect, the present invention provides the use of dsRNA, conjugates, delivery formulations, cells, or pharmaceutical compositions according to the present invention in the preparation of personal care products, wherein the personal care products include skin care products, hair care products, or body hygiene products.

[0019] Compared with the prior art, the present invention has at least the following beneficial effects: First, the novel dsRNA obtained through synthesis and screening in this invention exhibits excellent inhibitory activity at different concentrations, both in its naked and modified sequences, and demonstrates superior activity levels in various cell lines derived from humans, mice, rats, and pigs. The novel dsRNA provided by this invention exhibits excellent overall inhibitory activity at different concentrations and in multiple cell lines, demonstrating outstanding potential for broad-spectrum cross-species applications. Furthermore, the optimal three sequences of this invention show significantly better overall activity than known sequences in the prior art (such as the multiple positive control sequences in this invention) across multiple concentrations and cell lines, indicating a very broad application prospect.

[0020] Second, the naked and modified sequences of the novel dsRNA of this invention still exhibit strong knockout efficiency even at extremely low usage levels.

[0021] Third, the dsRNA of the present invention has the advantages of high gene silencing activity, low immunogenicity, high stability and low off-target risk, making it suitable for highly efficient gene silencing therapy.

[0022] Fourth, the dsRNA and its conjugates, delivery formulations, or cell or pharmaceutical compositions containing the dsRNA of the present invention can significantly inhibit JAK1 expression in skin tissue, thereby providing an effective treatment for skin diseases such as atopic dermatitis, vitiligo, or alopecia areata.

[0023] Fifth, the dsRNA of the present invention can also be coupled with a targeted delivery system. In particular, after coupling with a targeted ligand, such as lipids or lipid-based molecules, it can mimic the properties of natural lipids, efficiently mediate the entry of dsRNA into cells, better enhance the targeting ability to target organs such as skin, and can maintain its existence in the target organ for a longer period of time, effectively prolonging the half-life. Attached Figure Description

[0024] Figure 1 The results show the IC50 of the candidate modified sequence on the JAK1 gene determined using human cell lines. 50 result.

[0025] Figure 2 The results show the IC50 of the candidate modified sequence on the JAK1 gene, determined using mouse cell lines. 50 result.

[0026] Figures 3-6 The results show the effects of candidate naked sequences on JAK1 gene expression as determined using HepG2, N2A, BRL, and PK15 cells.

[0027] Figure 7 This demonstrates the effect of the candidate sequence of the present invention on the JAK1 gene in mouse skin. Detailed Implementation

[0028] In a first aspect, the present invention provides a double-stranded ribonucleic acid (dsRNA) targeting the JAK1 gene, comprising a sense strand and an antisense strand, wherein the nucleotide sequence of the sense strand comprises at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, or at least 21 consecutive nucleotides selected from any of the sequences shown in SEQ ID NO: 1-461, and the nucleotide difference does not exceed 0, 1, 2, or 3; and / or wherein the nucleotide sequence of the antisense strand comprises at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, or at least 22 consecutive nucleotides selected from any of the sequences shown in SEQ ID NO: 462-922, and the nucleotide difference does not exceed 0, 1, 2, or 3.

[0029] In some embodiments, the nucleotide sequence of the sense strand comprises at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, or at least 21 consecutive nucleotides selected from any of the sequences shown in SEQ ID NO: 379, 381, and 461, with no more than 0, 1, 2, or 3 nucleotide differences, and / or the nucleotide sequence of the antisense strand comprises at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, or at least 22 consecutive nucleotides selected from any of the sequences shown in SEQ ID NO: 840, 842, and 922, with no more than 0, 1, 2, or 3 nucleotide differences.

[0030] In some implementations, the combination of the sense and antisense strands of the dsRNA is selected from the combinations listed in Table 1.

[0031] In some embodiments, the dsRNA comprises a sense strand selected from SEQ ID NO: 379, 381 and 461 and an antisense strand selected from SEQ ID NO: 840, 842 and 922.

[0032] In some embodiments, the length of the sense strand does not exceed 26 nucleotides; and / or the length of the antisense strand does not exceed 28 nucleotides.

[0033] In some embodiments, the length of the sense strand does not exceed 24 nucleotides; and / or the length of the antisense strand does not exceed 26 nucleotides.

[0034] In some embodiments, the length of the sense strand does not exceed 22 nucleotides; and / or the length of the antisense strand does not exceed 24 nucleotides.

[0035] In some embodiments, the dsRNA comprises a sense strand and an antisense strand selected from the following: a) The sense strand, whose nucleotide sequence comprises at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, or at least 21 consecutive nucleotides of SEQ ID NO: 379, with no more than 0, 1, 2, or 3 nucleotide differences; and the antisense strand, whose nucleotide sequence comprises at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, or at least 22 consecutive nucleotides of SEQ ID NO: 840, with no more than 0, 1, 2, or 3 nucleotide differences. b) The sense strand, whose nucleotide sequence comprises at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, or at least 21 consecutive nucleotides of SEQ ID NO: 381, with no more than 0, 1, 2, or 3 nucleotide differences; and the antisense strand, whose nucleotide sequence comprises at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, or at least 22 consecutive nucleotides of SEQ ID NO: 842, with no more than 0, 1, 2, or 3 nucleotide differences. c) The sense strand, whose nucleotide sequence comprises at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, or at least 21 consecutive nucleotides of SEQ ID NO: 461, with no more than 0, 1, 2, or 3 nucleotide differences; and the antisense strand, whose nucleotide sequence comprises at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, or at least 22 consecutive nucleotides of SEQ ID NO: 922, with no more than 0, 1, 2, or 3 nucleotide differences. Preferably, the length of the sense strand does not exceed 26 nucleotides; and / or the length of the antisense strand does not exceed 28 nucleotides; More preferably, the length of the sense strand does not exceed 24 nucleotides; and / or the length of the antisense strand does not exceed 26 nucleotides; More preferably, the length of the sense strand does not exceed 22 nucleotides; and / or the length of the antisense strand does not exceed 24 nucleotides.

[0036] In some embodiments, the dsRNA comprises or consists of a sense strand and an antisense strand selected from the following: a) The sense strand with nucleotide sequence as shown in SEQ ID NO: 379 and the antisense strand with nucleotide sequence as shown in SEQ ID NO: 840; b) The sense strand with nucleotide sequence as shown in SEQ ID NO: 381 and the antisense strand with nucleotide sequence as shown in SEQ ID NO: 842; c) The sense strand with nucleotide sequence as shown in SEQ ID NO: 461 and the antisense strand with nucleotide sequence as shown in SEQ ID NO: 922.

[0037] In a second aspect, the present invention provides a double-stranded ribonucleic acid (dsRNA) targeting the JAK1 gene, comprising a sense strand and an antisense strand, wherein the nucleotide sequence of the sense strand comprises at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, or at least 21 consecutive nucleotides selected from any of the sequences shown in SEQ ID NO: 923-1383, 1885, 1886, and 1887, and the nucleotide difference does not exceed 0, 1, 2, or 3; and / or wherein the nucleotide sequence of the antisense strand comprises at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, or at least 22 consecutive nucleotides selected from any of the sequences shown in SEQ ID NO: 1384-1844, and the nucleotide difference does not exceed 0, 1, 2, or 3.

[0038] In some embodiments, the combination of the modified sense strand and the modified antisense strand of the dsRNA is selected from the combinations listed in Table 2.

[0039] In some embodiments, the nucleotide sequence of the sense strand comprises at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, or at least 21 consecutive nucleotides selected from any of the sequences shown in SEQ ID NO: 1301, 1303, 1383, 1885, 1886, and 1887, with no more than 0, 1, 2, or 3 nucleotide differences; and / or the nucleotide sequence of the antisense strand comprises at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, or at least 22 consecutive nucleotides selected from any of the sequences shown in SEQ ID NO: 1762, 1764, and 1844, with no more than 0, 1, 2, or 3 nucleotide differences.

[0040] In some embodiments, the dsRNA comprises a sense strand selected from SEQ ID NO: 1301, 1303, 1383, 1885, 1886, and 1887 and an antisense strand selected from SEQ ID NO: 1762, 1764, and 1844. Preferably, the length of the sense strand does not exceed 26 nucleotides; and / or the length of the antisense strand does not exceed 28 nucleotides; More preferably, the length of the sense strand does not exceed 24 nucleotides; and / or the length of the antisense strand does not exceed 26 nucleotides; More preferably, the length of the sense strand does not exceed 22 nucleotides; and / or the length of the antisense strand does not exceed 24 nucleotides.

[0041] In some embodiments, the dsRNA comprises a sense strand and an antisense strand selected from the following: a) The sense strand, whose nucleotide sequence comprises at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, or at least 21 consecutive nucleotides of SEQ ID NO: 1301, with no more than 0, 1, 2, or 3 nucleotide differences; and the antisense strand, whose nucleotide sequence comprises at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, or at least 22 consecutive nucleotides of SEQ ID NO: 1762, with no more than 0, 1, 2, or 3 nucleotide differences. b) The sense strand, whose nucleotide sequence comprises at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, or at least 21 consecutive nucleotides of SEQ ID NO: 1303, with no more than 0, 1, 2, or 3 nucleotide differences; and the antisense strand, whose nucleotide sequence comprises at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, or at least 22 consecutive nucleotides of SEQ ID NO: 1764, with no more than 0, 1, 2, or 3 nucleotide differences. c) The sense strand, whose nucleotide sequence comprises at least 15, 16, 17, 18, 19, 20, or 21 consecutive nucleotides of SEQ ID NO: 1383, with no more than 0, 1, 2, or 3 nucleotide differences; and the antisense strand, whose nucleotide sequence comprises at least 15, 16, 17, 18, 19, 20, or 21 consecutive nucleotides of SEQ ID NO: 1844, or at least 22 consecutive nucleotides, with no more than 0, 1, 2, or 3 nucleotide differences. d) The sense strand, whose nucleotide sequence comprises at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, or at least 21 consecutive nucleotides of SEQ ID NO: 1885, with no more than 0, 1, 2, or 3 nucleotide differences; and the antisense strand, whose nucleotide sequence comprises at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, or at least 22 consecutive nucleotides of SEQ ID NO: 1762, with no more than 0, 1, 2, or 3 nucleotide differences. e) The sense strand, whose nucleotide sequence comprises at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, or at least 21 consecutive nucleotides of SEQ ID NO: 1886, with no more than 0, 1, 2, or 3 nucleotide differences; and the antisense strand, whose nucleotide sequence comprises at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, or at least 22 consecutive nucleotides of SEQ ID NO: 1764, with no more than 0, 1, 2, or 3 nucleotide differences. f) The sense strand, whose nucleotide sequence comprises at least 15, 16, 17, 18, 19, 20, or 21 consecutive nucleotides of SEQ ID NO: 1887, with no more than 0, 1, 2, or 3 nucleotide differences; and the antisense strand, whose nucleotide sequence comprises at least 15, 16, 17, 18, 19, 20, or 21 consecutive nucleotides of SEQ ID NO: 1844, or at least 22 consecutive nucleotides, with no more than 0, 1, 2, or 3 nucleotide differences. Preferably, the length of the sense strand does not exceed 26 nucleotides; and / or the length of the antisense strand does not exceed 28 nucleotides; More preferably, the length of the sense strand does not exceed 24 nucleotides; and / or the length of the antisense strand does not exceed 26 nucleotides; More preferably, the length of the sense strand does not exceed 22 nucleotides; and / or the length of the antisense strand does not exceed 24 nucleotides.

[0042] In some embodiments, the dsRNA comprises or consists of a sense strand and an antisense strand selected from the following: a) The sense strand with nucleotide sequence as shown in SEQ ID NO: 1301 and the antisense strand with nucleotide sequence as shown in SEQ ID NO: 1762; b) The sense strand with nucleotide sequence as shown in SEQ ID NO: 1303 and the antisense strand with nucleotide sequence as shown in SEQ ID NO: 1764; c) The sense strand with nucleotide sequence as shown in SEQ ID NO: 1383 and the antisense strand with nucleotide sequence as shown in SEQ ID NO: 1844; d) The sense strand with the nucleotide sequence shown in SEQ ID NO: 1885 and the antisense strand with the nucleotide sequence shown in SEQ ID NO: 1762; e) The sense strand with the nucleotide sequence shown in SEQ ID NO: 1886 and the antisense strand with the nucleotide sequence shown in SEQ ID NO: 1764; f) The sense strand with nucleotide sequence as shown in SEQ ID NO: 1887 and the antisense strand with nucleotide sequence as shown in SEQ ID NO: 1844.

[0043] In a third aspect, the present invention provides a double-stranded ribonucleic acid (dsRNA) targeting the JAK1 gene, comprising a sense strand and an antisense strand, wherein at least one nucleotide in the sense strand and the antisense strand is a modified nucleotide.

[0044] In some embodiments, the modified nucleotides are each independently selected from one or more of the following: 2'-halogenated nucleotides, 2'-deoxy-modified nucleotides, 2'-alkyl-modified nucleotides, 2'-substituted alkyl-modified nucleotides, 2'-alkoxy-modified nucleotides, 2'-substituted alkoxy-modified nucleotides, 2'-amino-modified nucleotides, 2'-substituted amino-modified nucleotides, thiophosphate nucleosides or nucleotide analogs, 2'-fluoro-modified nucleotides, 2'-methoxy-modified nucleotides, 2'-O-CH2-CH2-O-CH3-modified nucleotides, 2'-O-CH2-CH=CH2-modified nucleotides, 2'-CH2-CH2-CH=CH2-modified nucleotides, and thiophosphate-modified nucleotides.

[0045] In some implementations, the justice chain and antisense chain include modifications selected from any of the following groups: a) The sense strand contains the following modification: nucleotides with 2'-fluorination at positions 6, 8, 9, 10 and 11 starting from the 5' end; and / or the antisense strand contains the following modification: nucleotides with 2'-fluorination at positions 2, 3, 5, 7, 10, 14 and 16 starting from the 5' end; b) The sense strand contains the following modification: nucleotides with 2'-fluorination at positions 6, 8, 9 and 10 starting from the 5' end; and / or the antisense strand contains the following modification: nucleotides with 2'-fluorination at positions 2, 3, 5, 7, 10, 14 and 16 starting from the 5' end; c) The sense strand contains the following modification: nucleotides with 2'-fluorination at positions 4, 6, 8, 10 and 14 starting from the 5' end; and / or the antisense strand contains the following modification: nucleotides with 2'-fluorination at positions 2, 5, 6, 14 and 16 starting from the 5' end; d) The sense strand contains the following modification: nucleotides with 2'-fluorination at positions 4, 6, 8, 9, 10 and 14 starting from the 5' end; and / or the antisense strand contains the following modification: nucleotides with 2'-fluorination at positions 2, 5, 6, 14 and 16 starting from the 5' end; e) The sense strand contains the following modification: nucleotides with 2'-fluorination at positions 6, 8, 9 and 10 starting from the 5' end; and / or the antisense strand contains the following modification: nucleotides with 2'-fluorination at positions 2, 3, 5, 6, 7, 10, 14 and 16 starting from the 5' end; f) The sense strand contains the following modification: nucleotides with 2'-fluorination at positions 4, 6, 8, 9, 10 and 14 starting from the 5' end; and / or the antisense strand contains the following modification: nucleotides with 2'-fluorination at positions 2, 3, 5, 6, 10, 14 and 16 starting from the 5' end; g) The sense strand contains the following modification: nucleotides with 2'-fluorination at positions 6, 8, 9 and 10 starting from the 5' end; and / or the antisense strand contains the following modification: nucleotides with 2'-fluorination at positions 2, 5, 6, 14 and 16 starting from the 5' end.

[0046] In some embodiments, the sense strand comprises a nucleotide with a 2'-fluorinated modification at positions 6, 8, 9, 10, and 11 starting from the 5' end; and the antisense strand comprises a nucleotide with a 2'-fluorinated modification at positions 2, 3, 5, 7, 10, 14, and 16 starting from the 5' end.

[0047] In some embodiments, the sense strand comprises a nucleotide with a 2'-fluorinated modification at positions 6, 8, 9, and 10 starting from the 5' end; and the antisense strand comprises a nucleotide with a 2'-fluorinated modification at positions 2, 3, 5, 7, 10, 14, and 16 starting from the 5' end.

[0048] In some implementations, the chain of justice further includes the following modifications: a) Nucleotides modified with phosphate thioester at positions 1 and 2 from the 5' end and positions 2 and 3 from the 3' end; and / or b) The remaining sites on the positive strand are nucleotides modified with 2'-methoxy groups.

[0049] In some embodiments, the antisense chain further includes modifications selected from: a) wherein the antisense strand is a phosphate-thiophosphate modified nucleotide at positions 1 and 2 from the 5' end and positions 2 and 3 from the 3' end; and / or b) The remaining sites of the antisense strand are nucleotides modified with 2'-methoxy groups.

[0050] In some embodiments, the positive strand comprises the following modifications: a) nucleotides with a 2'-fluorinated modification at positions 6, 8, 9, 10, and 11 starting from the 5' end; b) nucleotides with a phosphate thioester modification at positions 1 and 2 starting from the 5' end and positions 2 and 3 starting from the 3' end; and c) nucleotides with a 2'-methoxy modification at the remaining positions; and The antisense strand comprises the following modifications: a) nucleotides with 2'-fluorination at positions 2, 3, 5, 7, 10, 14 and 16 from the 5' end; b) nucleotides with phosphate thioester modification at positions 1 and 2 from the 5' end and positions 2 and 3 from the 3' end; and c) nucleotides with 2'-methoxy modification at the remaining positions.

[0051] In some embodiments, the positive strand comprises the following modifications: a) nucleotides with a 2'-fluorinated modification at positions 6, 8, 9, and 10 starting from the 5' end; b) nucleotides with a phosphate thioester modification at positions 1 and 2 starting from the 5' end and positions 2 and 3 starting from the 3' end; and c) nucleotides with a 2'-methoxy modification at the remaining positions; and The antisense strand comprises the following modifications: a) nucleotides with 2'-fluorination at positions 2, 3, 5, 7, 10, 14 and 16 from the 5' end; b) nucleotides with phosphate thioester modification at positions 1 and 2 from the 5' end and positions 2 and 3 from the 3' end; and c) nucleotides with 2'-methoxy modification at the remaining positions.

[0052] In some embodiments, the nucleotide sequence of the sense strand comprises at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, or at least 21 consecutive nucleotides selected from any of the sequences shown in SEQ ID NO: 1-461, with no more than 0, 1, 2, or 3 nucleotide differences, and / or the nucleotide sequence of the antisense strand comprises at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, or at least 22 consecutive nucleotides selected from any of the sequences shown in SEQ ID NO: 462-922, with no more than 0, 1, 2, or 3 nucleotide differences.

[0053] In a fourth aspect, the present invention provides a coupling of formula (I), R1—B—R2 Formula (I), Wherein R1 is a targeting ligand, delivery enhancer, or hydrophobic modifier, B is an optional adapter, and R2 is selected from dsRNA according to the present invention.

[0054] In some embodiments, the R1 is selected from: cholesterol, fatty acids, cyclic RGD peptides, N-acetylgalactosamine (GalNAc), folic acid (FA), steroids, lithocholic acid (LCA), open-ring steroids, lipids, gangliosides and nucleoside analogs, endocannabinoids, vitamins, eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), and docosuccinic acid (DCA).

[0055] In some embodiments, the R1 is docosuccinic acid (DCA).

[0056] Preferably, wherein R1 is .

[0057] In some embodiments, the connector is selected from one of the following groups: -NH-(CH2) n -C(CH2OH)(CH3)- , -NH(CH2) n CO- , -NH(CH2) n NH- , -O-CO-(CH2) n -CO- , -O-CO-(CH2CH2O) n CH2CH2CO- , -NH-(CH2CH2O) n -CH2CH2CO- , -NH-(CH2CH2O) n -CH2CH2NH- , -CO-CH(NH2)-CH2-imidazole- , -CO-CH2-NH-CH2-CO-CH(NH2)-CH(CH3)- , -CO-CH(NH2)-CH(CH3)-CH2CH2- , -CO-CH(NH2)-CH2C(CH3)2- , -CO-CH(NH2)-CH2CH2-S-CH2- , -CO-CH(NH2)-CH2-phenyl- , -CO-CH(NH2)-CH2-p-methoxyphenyl- , -CO-CH(NH2)-CH2-β-indole- , -NH-(CH2) n -triazole- , -NH-(CH2CH2O) n -triazole- , -CO-(CH2) n -triazole- , -CO-(CH2CH2O) n -triazole- , -NH-(CH2) n -S-(succinimide-3-yl-N)- , -NH-(CH2CH2O) n -S-(succinimide-3-yl-N)- , -CO-(CH2) n -S-(succinimide-3-yl-N)- , -CO-(CH2CH2O) n -S-(succinimide-3-yl-N)- , -NH-(CH2) n -(succinimide-3-yl-N)-S- , -NH-(CH2CH2O) n -(succinimide-3-yl-N)-S- , -CO-(CH2) n -(succinimide-3-yl-N)-S- , -CO-(CH2CH2O) n -(succinimide-3-yl-N)-S- , -CH(NH2)-CH2-imidazole- , -CH2-NH-CH2-CO-CH(NH2)-CH(CH3)- , -CH(NH2)-CH(CH3)-CH2CH2- , -CH(NH2)-CH2C(CH3)2- , -CH(NH2)-CH2CH2-S-CH2- , -CH(NH2)-CH2-phenyl- , -CH(NH2)-CH2-p-methoxyphenyl- , -CH(NH2)-CH2-β-indole- , , , , , , , , and , in This indicates the connection end with the target ligand. This indicates the linker end with the dsRNA, further, This indicates coupling with the 3' end of the positive strand of the dsRNA; Where n is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15.

[0058] In some implementations, the connector is or ,in This indicates the connection end with the target ligand. This indicates the linker end with the dsRNA, further, This indicates coupling with the 3' end of the positive strand of the dsRNA.

[0059] In some embodiments, the 3' end of the sense chain is connected to the targeting ligand, delivery enhancer or hydrophobic modifier, or the connector; and / or the 5' end of the antisense chain comprises a vinyl phosphate ester.

[0060] In some embodiments, the coupling of the present invention has the structure shown in formula (II): Equation (II).

[0061] In a fifth aspect, the present invention provides a delivery formulation comprising dsRNA as described in the present invention and a pharmaceutically acceptable carrier.

[0062] In some embodiments, the vector is selected from one or more of the following: lentiviral vectors, retroviral vectors, adenovirus vectors, adeno-associated virus vectors, poxvirus vectors, herpesvirus vectors, liposomes, lipid nanoparticles, polymers, peptides, antibodies, aptamers, N-acetylgalactosamine (GalNAc), lipid polymers, polymer nanoparticles, inorganic nanoparticles, cationic nanoemulsions, exosomes, and biological microvesicles.

[0063] In a sixth aspect, the present invention provides a cell comprising dsRNA according to the present invention.

[0064] In a seventh aspect, the present invention provides a pharmaceutical composition comprising dsRNA according to the present invention, a conjugate according to the present invention, a delivery formulation according to the present invention, or a cell according to the present invention, and a pharmaceutically acceptable excipient.

[0065] In an eighth aspect, the present invention provides the use of dsRNA, conjugates, delivery formulations, cells, or pharmaceutical compositions according to the present invention in the preparation of medicaments for the prevention and / or treatment of JAK1 gene-mediated diseases or conditions.

[0066] In some implementations, the diseases or conditions mediated by the JAK1 gene include one or more of the following: cancer, psoriasis, rheumatoid arthritis, inflammatory bowel disease, asthma, systemic lupus erythematosus, multiple sclerosis, graft-versus-host disease, transplant rejection, atopic dermatitis, alopecia areata, vitiligo, and ankylosing spondylitis.

[0067] In a ninth aspect, the present invention provides the use of the dsRNA, conjugate, delivery formulation, cell, or pharmaceutical composition according to the present invention in the preparation of a medicament for reducing or inhibiting JAK1 gene expression and / or activity.

[0068] In a tenth aspect, the present invention provides a method for reducing or inhibiting JAK1 gene expression and / or activity, the method comprising the step of contacting a dsRNA according to the present invention, a conjugate according to the present invention, a delivery formulation according to the present invention, a cell according to the present invention, or a pharmaceutical composition according to the present invention with a cell.

[0069] In an eleventh aspect, the present invention provides the use of dsRNA, conjugates, delivery formulations, cells, or pharmaceutical compositions according to the present invention in the preparation of personal care products, wherein the personal care products include skin care products, hair care products, or body hygiene products.

[0070] In some implementations, the personal care product is used to prevent or improve hair loss, promote hair growth, prevent or improve skin depigmentation, promote skin pigmentation recovery, relieve itching, repair the skin barrier, and / or reduce skin inflammation.

[0071] In some implementations, the personal care product is used to prevent or improve skin symptoms associated with diseases or conditions caused by abnormally elevated JAK1 gene expression.

[0072] definition Unless specifically defined, the nomenclature used in the analytical chemistry, synthetic organic chemistry, and medical and medicinal chemistry described herein, as well as the procedures and techniques used in these fields, are well-known and commonly used in the art. Standard techniques may be used for chemical synthesis and analysis. Where permitted, all patents, applications, published applications and other publications mentioned herein, GENBANK accession numbers and related sequence information available from databases such as the National Center for Biotechnology Information (NCBI), and other data relating to the literature discussed herein are incorporated herein by reference, both in their entirety and by reference. For the purposes of this invention, the terminology used herein is explained below, but the invention is not limited by these explanations.

[0073] Throughout this specification, unless the context otherwise requires, the terms “comprise,” “comprises,” and “comprising” shall be understood to mean including the steps or elements or groups of steps or elements described herein, but not excluding any other steps or elements or groups of steps or elements.

[0074] "Composed of..." means, but is not limited to, anything that follows the phrase "composed of...". Therefore, the phrase "composed of..." indicates that the listed elements are necessary or mandatory, and that no other elements may exist. "Substantially composed of..." means including any elements listed following the phrase "substantially composed of...", and is limited to other elements that do not interfere with or contribute to the activities or actions specified in the public content of the listed elements.

[0075] Throughout this specification, references to "an embodiment," "an embodiment," "a particular embodiment," "a related embodiment," "a certain embodiment," "another embodiment," or "a further embodiment," or combinations thereof, mean that a particular feature, structure, or characteristic described in connection with said embodiment is included in at least one embodiment of the invention. Therefore, the appearance of the foregoing phrases in various places throughout this specification does not necessarily refer to the same embodiment. Furthermore, a particular feature, structure, or characteristic may be combined in one or more embodiments in any suitable manner.

[0076] As used in this article, the term "PCR amplification" refers to the use of polymerase chain reaction (PCR) to amplify a DNA sequence to obtain more copies of the DNA sequence.

[0077] As used herein, "effective amount" in the context of modulating activity or treating or preventing symptoms means, in the form of a single dose or a portion of a series of doses, the amount of active ingredient that effectively modulates, treats, prevents, or improves the effect of said activity, or treats or prevents or improves said symptoms, administered to a subject requiring said modulation, treatment, or prevention. Effective amount will vary depending on factors such as the health and physical condition of the subject being treated, the taxonomic group of the subject being treated, the formulation of the composition, the assessment of the medical condition, and other relevant factors.

[0078] As used in this article, "therapeutic effective dose" means the amount of medicine that provides therapeutic benefit to an individual.

[0079] As used in this article, “inhibit expression or activity” means to reduce or block expression or activity and does not necessarily indicate complete elimination of expression or activity.

[0080] As used herein, "pharmaceutically acceptable carrier" means a medium or diluent that does not interfere with the structure of the oligonucleotide. Some of these carriers enable pharmaceutical compositions to be formulated, for example, into tablets, pills, sugar-coated pills, capsules, liquids, gels, syrups, pastes, suspensions, and lozenges for oral administration to a subject.

[0081] As used herein, "pharmaceutical composition" means a mixture of substances suitable for administration to a subject. For example, a pharmaceutical composition may comprise an antisense oligonucleotide and a sterile aqueous solution. In some embodiments, the pharmaceutical composition exhibits activity in a free uptake assay of certain cell lines.

[0082] As used in this article, "subject" means a human or non-human animal selected for treatment or therapy.

[0083] As used in this article, "target" refers to the protein that needs to be regulated.

[0084] As used in this article, "target gene" refers to the gene that encodes the target.

[0085] As used in this article, “targeting” refers to the process of designing and selecting antisense compounds that will specifically hybridize with the target nucleic acid and induce the desired effect.

[0086] As used in this article, “target nucleic acid,” “target RNA,” “target RNA transcript,” and “nucleic acid target” all refer to nucleic acids that can be targeted by antisense compounds.

[0087] As used in this article, “treatment” means the application of a composition to alter or improve a disease or symptom.

[0088] As used herein, the terms “oligonucleotide,” “polynucleotide,” or “oligomery” are used interchangeably and refer to polymers of nucleotides, including but not limited to single-stranded, double-stranded, or partially double-stranded nucleic acid molecules of DNA, RNA, or DNA / RNA hybrids, oligonucleotide chains containing regular and irregular alternating deoxyribosyl and ribosyl moieties, and modified and naturally or unnaturally existing frameworks for such oligonucleotides.

[0089] As used herein, a "nucleoside" is a compound composed of a purine or pyrimidine base and ribose or deoxyribose; a "ribonucleotide," also called a "nucleotide," is a compound composed of a purine or pyrimidine base, ribose or deoxyribose, and a phosphate group; and an "oligonucleotide" is a nucleic acid molecule (RNA or DNA) having a length of, for example, less than 100, 200, 300, or 400 nucleotides. Oligonucleotides may contain ribonucleotides, deoxyribonucleotides, and / or modified nucleotides, including, for example, modified ribonucleotides. Oligonucleotides may be single-stranded or double-stranded. Oligonucleotides may or may not have a double-stranded region. As a set of non-limiting examples, oligonucleotides may be, but are not limited to, small interfering RNA (siRNA), microRNA (miRNA), short hairpin RNA (shRNA), cleatase substrate interfering RNA (dsiRNA), antisense oligonucleotides, short siRNA, or single-stranded siRNA.

[0090] As used herein, "deoxyribonucleotide" means a nucleotide having a hydrogen atom at the 2' position of the sugar portion of the nucleotide. Deoxyribonucleotides can be modified by any of a variety of substituents.

[0091] As used herein, the term "complementary" refers to a structural relationship between nucleotides (e.g., on opposite nucleic acids or on opposite regions of a single nucleic acid chain) that allows nucleotides to form base pairs with each other. For example, a purine nucleotide complementary to a pyrimidine nucleotide of an opposite nucleic acid can pair up with each other by forming hydrogen bonds.

[0092] As used herein, "base complementarity" refers to the ability of an antisense oligonucleotide's nucleic acid bases to accurately pair (i.e., hybridize) with corresponding nucleic acid bases in a target nucleic acid, mediated by Watson-Crick, Hoogsteen, or anti-Hoogsteen hydrogen bonds between the corresponding nucleic acid bases. For example, in DNA, adenine (A) is complementary to thymine (T). For example, in RNA, adenine (A) is complementary to uracil (U). In some embodiments, complementary nucleic acid bases refer to nucleic acid bases in the antisense compound that can pair with nucleic acid bases in its target nucleic acid. For example, if a nucleic acid base at a certain position in the antisense compound can form a hydrogen bond with a nucleic acid base at a certain position in the target nucleic acid, then the hydrogen bond position between the oligonucleotide and the target nucleic acid at that nucleic acid base pair is considered complementary.

[0093] As used in this article, "complementarity" refers to the ability of the nucleic acid bases of the first nucleic acid and the second nucleic acid to pair.

[0094] As used herein, "fully complementary" or "100% complementary" means that all nucleic acid bases of the first nucleic acid have complementary nucleic acid bases in the second nucleic acid. In some implementations, the first nucleic acid is an antisense compound and the target nucleic acid is the second nucleic acid.

[0095] As used herein, the term "complementary region" refers to a region on the antisense strand that is completely or substantially complementary to the target mRNA sequence. In cases where the complementary region is not completely complementary to the target sequence, mismatches can be located in the interior or terminal regions of the molecule. Typically, the most tolerant mismatches are located in the terminal regions, for example, within 5, 4, 3, 2, or 1 nucleotides at the 5' and / or 3' ends. The antisense strand portion most sensitive to mismatches is called the "seed region." For example, in a siRNA containing a 19-nt strand, the 19th position (from 5' to 3') can tolerate some mismatches. The terms "complementary," "completely complementary," and "substantially complementary" can be used relative to the base pairing between the sense and antisense strands of the siRNA, or between the antisense strand of the siRNA reagent and the target sequence.

[0096] As used herein, the term "difference" refers to a "nucleotide difference" between two nucleotide sequences. This means that the type of bases at the same position of the nucleotides has changed compared to the former. For example, if a nucleotide base in the latter is A, and the corresponding nucleotide base at the same position in the former is U, C, G, or T, then a nucleotide difference at that position is considered to exist between the two nucleotide sequences. In some embodiments, replacing the nucleotide at the original position with a baseless nucleotide or a nucleotide analog can also be considered a nucleotide difference at that position.

[0097] As used in this article, the term "mismatch" refers to a double-stranded nucleic acid where the bases at corresponding positions are not paired in a complementary manner.

[0098] As used herein, the term "siRNA" is a class of double-stranded RNA molecules that can mediate the silencing of a target RNA (e.g., mRNA, such as transcripts of genes encoding proteins) that is complementary to it. siRNA is typically double-stranded, consisting of an antisense strand complementary to the target RNA and a sense strand complementary to that antisense strand. For convenience, such mRNA is also referred to herein as the mRNA to be silenced. Such genes are also referred to as target genes. Typically, the RNA to be silenced is an endogenous gene or a pathogen gene. In addition, RNAs other than mRNA (e.g., tRNA) and viral RNA can also be targeted.

[0099] As used herein, the term "dsRNA" refers to a ribonucleic acid molecular complex with a double-stranded structure, comprising two antiparallel and substantially complementary nucleic acid strands. These two strands have "sense" and "antisense" directions relative to the target RNA (such as the JAK1 gene). In some embodiments of the invention, double-stranded RNA (dsRNA) mediates the degradation of the target RNA (such as mRNA) through a post-transcriptional gene silencing mechanism of RNA interference.

[0100] Generally, most nucleotides in each strand of a dsRNA molecule are ribonucleotides, but as described in detail herein, each or both strands may also contain one or more non-ribonucleotides, such as deoxyribonucleotides or modified nucleotides. Additionally, as used herein, “iRNA” may contain chemically modified ribonucleotides; iRNA may contain substantial modifications at multiple nucleotide sites. As used herein, the term “modified nucleotide” refers to a nucleotide that independently has a modified sugar moiety, a modified internucleotide bond, or a modified nucleobase, or any combination thereof. Therefore, the term modified nucleotide encompasses substitution, addition, or removal of, for example, functional groups or atoms, of nucleoside bonds, sugar moieties, or nucleobases. Modifications to pharmaceuticals suitable for use in this invention include all types of modifications disclosed herein or known in the art. For the purposes of this specification and claims, any such modifications are covered by “iRNA” or “RNAi pharmaceutical” as used in siRNA-type molecules.

[0101] As used in this article, the term "antisense strand" refers to a strand of dsRNA that contains regions that are fully or substantially complementary to the target sequence.

[0102] As used herein, the term "sense chain" refers to another chain of dsRNA that includes regions substantially complementary to the region referred to herein as the antisense chain.

[0103] As used in this article, “continuous nucleic acid bases” or “continuous nucleotides” means nucleic acid bases that are adjacent to each other.

[0104] As used in this article, a "base" is the basic building block for the synthesis of nucleosides, nucleotides, and nucleic acids. Its constituent elements include nitrogen, hence it is also called a "nitrogenous base." Unless otherwise specified, the capital letters A, U, T, G, and C in this article represent the base composition of nucleotides, namely adenine, uracil, thymine, guanine, and cytosine, respectively.

[0105] As used in this article, “unmodified nucleic acid bases” means purine bases adenine (A) and guanine (G), and pyrimidine bases thymine (T), cytosine (C), and uracil (U).

[0106] As used in this article, "modified nucleic acid base" means any nucleic acid base other than adenine, cytosine, guanine, thymine, or uracil.

[0107] As used in this article, "modified nucleoside" means a nucleoside that independently has a modified sugar moiety and / or a modified nucleic acid base.

[0108] As used in this article, "modified nucleotide" means a nucleotide that independently has a modified sugar moiety, a modified nucleoside bond, or a modified nucleic acid base.

[0109] As used herein, “modified oligonucleotide” means an oligonucleotide containing at least one modified nucleoside bond, a modified sugar, and / or a modified nucleic acid base.

[0110] As used in this article, “2’-O-methoxyethyl” (also known as 2’-MOE and 2’-OCH2CH2-OCH3) refers to the O-methoxyethyl modification at the 2’ position of the furanose ring.

[0111] As used herein, “2'-O-methoxyethyl modified nucleosides / nucleotides”, “methoxyethyl modified nucleosides / nucleotides”, “2'-MOE nucleosides / nucleotides” or “2'-OME nucleosides / nucleotides” are used interchangeably and are intended to include 2'-MOE modified nucleosides / nucleotides.

[0112] As used in this article, "docosalicylic acid" or "DCA" can also be called docosalic acid, docosahexalic acid, behenic acid, oleic acid, succinic acid, etc. It is a carboxylic acid and a saturated fatty acid with the molecular formula C. 21 H 43 COOH, structural formula as shown below: .

[0113] As used herein, the term “connector” or “linking group” means an organic part that connects two parts of a compound, such as covalently attaching two parts of a compound, or two different compounds.

[0114] Example For the purpose of clarity and concise description, features are described using only a portion of some of the same or separate embodiments herein; however, it should be understood that the scope of this disclosure may include some embodiments having combinations of all or some of the described features. The invention is described in more detail below with reference to specific embodiments; however, these embodiments are for illustrative purposes only and are not intended to limit the invention.

[0115] In this article, unless otherwise specified, uppercase letters C, G, U, A, and T represent the base composition of nucleotides; lowercase letter m indicates that the nucleotide adjacent to the left of the letter m is a nucleotide modified with 2'-methoxy; lowercase letter f indicates that the nucleotide adjacent to the left of the letter f is a nucleotide modified with 2'-fluoride; and lowercase letter s indicates that the nucleotide adjacent to the left of the letter s is a nucleotide modified with phosphate thioester.

[0116] Example 1: Design and synthesis of naked dsRNA sequences, modified sequences, and positive control sequences For the mRNA coding region of the JAK1 gene, following the principles of 21-23 bp length, GC content of 36%-52%, and thermodynamic asymmetry, candidate sequences were designed using methods commonly used in the art, and dsRNA was synthesized using conventional methods in the art. After purification, screening, identification, and functional verification, the naked dsRNA candidate sequences of the present invention were obtained. The specific sequence information is shown in Table 1 below.

[0117] Table 1. Information on candidate naked sequences for dsRNA

[0118] Then, the naked dsRNA candidate sequences in Table 1 were further modified using modification methods known in the art, and the modified dsRNA candidate sequences are shown in Table 2 below.

[0119] Table 2. Information on candidate dsRNA modification sequences

[0120] In addition, the dsRNA used as a positive control in this application was synthesized using conventional methods in the art, and its sequence is shown in Table 3 below.

[0121] Table 3. dsRNA sequence information used as positive controls

[0122] Positive control sources: PC1: CN117642508A; PC2 and PC8: Tang Q, et al., Rational design of a JAK1-selective siRNA inhibitor for the modulation of autoimmunity in the skin. Nat Commun 2023 Nov 4;14(1):7099. doi: 10.1038 / s41467-023-42714-4. PMID: 37925520; PMCID: PMC10625637; PC3- PC4: WO2024256707A1; PC5- PC7: CN121046378 A.

[0123] Example 2: Testing the knockout activity of candidate modified sequences on the JAK1 gene in human cell lines 1. JAK1 siRNA transfection of HepG2 cells HepG2 cells were cultured in DMEM medium containing 10% fetal bovine serum at 37°C with 5% CO2. When the cells were in the logarithmic growth phase and in good condition (70% confluence), they were seeded and transfected. Cell density was adjusted to 8 x 102 cells per well. 4 Cells were seeded into 24-well plates; transfection complexes were prepared using OMEM and RNAimax with the dsRNA synthesized in Example 1, with final transfection concentrations of 3 nM, 0.3 nM, and 0.03 nM, respectively. The transfection complexes were added to the 24-well plates, shaken well, and incubated in a 5% CO2, 37°C incubator for 48 h; in addition to the experimental groups, the following control groups were also set up: the NC group was a negative control (without related siRNA), and the RNAimax group was a transfection reagent control (without dsRNA).

[0124] 2. Real-time quantitative PCR analysis Cells were lysed 48 hours after transfection, and total RNA was extracted using a column extraction kit (Novizan). Real-time quantitative PCR was performed using a QuantStudio 5 PCR instrument (Thermo) with GAPDH gene as an internal control and TaqMan probe assay reagent (Novizan) as the standard. The primers used are listed in Table 4 below. Table 4

[0125] 3. Data Analysis After the PCR reaction, relative quantification was performed using the 2^-ΔΔCt(Livak) method with the reference gene as the standard. Screening was conducted by qPCR, and the sequences were ranked based on their activity levels at 0.03 nM. The results are shown in Table 5. As can be seen from the table, all the dsRNA candidate modification sequences designed in this invention have knockout effects on the JAK1 gene. Furthermore, the top 20 candidate modification sequences with the highest activity levels at 0.03 nM were selected for further screening and analysis, including further analysis of their activity levels at 0.3 nM and 3 nM. Finally, 12 candidate modification sequences exhibiting excellent activity levels at all tested concentrations (0.03 nM, 0.3 nM, and 3 nM) were selected for subsequent example studies.

[0126] Table 5

[0127] Example 3: Determining the IC50 of candidate modified sequences on the JAK1 gene using human cell lines 50 Twelve candidate modification sequences that exhibited excellent activity at all tested concentrations in Example 2 were selected and transfected into HepG2 cells using these 12 candidate modification sequences. The final transfection concentrations were 10, 2.5, 0.625, 0.1562, 0.03906, 0.009765, 0.002441, and 0.0006103 nM, respectively. Real-time quantitative PCR analysis was performed after transfection, repeated twice and the results were recorded. The IC50 of the 12 candidate modification sequences was also measured. 50 Values. The results are shown in Tables 6-7 and 7-8. Figure 1 As shown in Table 6, the data from two repeated experiments are recorded.

[0128] The experimental data from this embodiment show that the 12 selected candidate modification sequences still exhibit good activity at concentrations as low as 0.0006103 nM. Furthermore, all 12 candidate modification sequences have low IC50 values. 50 The value indicates that all 12 candidate modification sequences of the present invention have excellent inhibitory activity.

[0129] Table 6

[0130] Table 7

[0131] Example 4: Testing the knockout activity of candidate modified sequences on the JAK1 gene in mouse cell lines Through homology comparison, eight sequences with the highest mouse homology (ZYJ049-M1, ZYJ093-M1, ZYJ175-M1, ZYJ179-M1, ZYJ260-M1, ZYJ379-M1, ZYJ381-M1, and ZYJ461-M1) were selected from the 12 candidate modified sequences with high activity levels verified in Example 3 for activity level detection in the mouse cell line of this example. The remaining four sequences (e.g., ZYJ181-M1), although also exhibiting excellent activity in Example 3, were excluded from this example due to their low mouse homology.

[0132] 1. JAK1 siRNA transfection of N2A cells N2A cells were cultured in MEM medium containing 10% fetal bovine serum at 37°C with 5% CO2. When the cells were in the logarithmic growth phase and in good condition (70% confluence), they were seeded and transfected. Cell density was adjusted to 1 x 102 cells per well. 5 Cells were seeded into 24-well plates. Transfection complexes were prepared using OMEM and RNAimax with 8 groups of dsRNA candidate modifications to achieve final transfection concentrations of 3 nM, 0.3 nM, and 0.03 nM, respectively. The transfection complexes were added to the 24-well plates, shaken well, and incubated in a 5% CO2, 37°C incubator for 48 h. In addition to the experimental groups, the following control groups were set up: the NC group was a negative control (without siRNA), and the RNAimax group was a transfection reagent control (without siRNA).

[0133] 2. Real-time quantitative PCR analysis Cells were lysed 48 hours after transfection, and total RNA was extracted using a column extraction kit (Novizan). Real-time quantitative PCR was performed using a QuantStudio 5 PCR instrument (Thermo) with GAPDH gene as an internal control and TaqMan probe assay reagent (Novizan) as the standard. The primers used are listed in Table 8 below. Table 8

[0134] 3. Data Analysis After the PCR reaction, relative quantification was performed using the 2^-ΔΔCt(Livak) method with the reference gene as the standard. The qPCR screening results are shown in Table 9. The data in the table below show that the eight selected candidate modification sequences exhibited good knockout activity against the JAK1 gene in mouse cell lines, achieving good knockout effects even at concentrations as low as 0.03 nM.

[0135] Specifically, ZYJ379-M1, ZYJ381-M1, ZYJ179-M1, and ZYJ461-M1 exhibited relatively high activity at a concentration of 0.03 nM, with values ​​of 0.778, 0.852, 0.878, and 0.878, respectively. However, the activity of ZYJ179-M1 at 0.3 nM and 3 nM was significantly lower than that of ZYJ379-M1, ZYJ461-M1, and ZYJ381-M1. Therefore, the inventors selected ZYJ379-M1, ZYJ381-M1, and ZYJ461-M1, which demonstrated excellent activity at all three concentrations (0.03 nM, 0.3 nM, and 3 nM), for further research.

[0136] Table 9

[0137] Example 5: Determining the IC50 of candidate modified sequences on the JAK1 gene using mouse cell lines 50 N2A cells were transfected with three candidate modification sequences, ZYJ379-M1, ZYJ381-M1, and ZYJ461-M1, which exhibited excellent activity at all tested concentrations as described in Example 4. The final transfection concentrations were 10 nM, 2.5 nM, 0.625 nM, 0.1562 nM, 0.03906 nM, 0.009765 nM, 0.002441 nM, and 0.0006103 nM, respectively. Real-time quantitative PCR analysis was performed after transfection, repeated twice and the results were recorded. The IC50 of the three candidate modification sequences was also measured. 50 Values. Results are shown in Tables 10 and 11. Figure 2 As shown in Table 10, the data from two repeated experiments are recorded.

[0138] The data results from this embodiment show that the three candidate modification sequences ZYJ379-M1, ZYJ381-M1, and ZYJ461-M1 all exhibited excellent inhibitory activity in mouse cells, with ZYJ381-M1 showing the highest IC50 value. 50 The lowest (0.2545 nM) concentration showed the strongest inhibitory activity; ZYJ379-M1 was the second highest (IC50). 50 (0.2715 nM); ZYJ461-M1 IC 50 Relatively high (0.3883 nM).

[0139] Table 10

[0140] Table 11

[0141] Example 6: Determining the effect of naked sequences on JAK1 gene expression in multiple genera cell lines In this embodiment, 40 candidate modified sequences with the highest activity at each measured concentration in Example 2 were selected. Cell lines HepG2, N2A, BRL, and PK15 derived from humans, mice, rats, and pigs were used to detect the inhibitory activity of the naked sequences of these 40 candidate modified sequences in multiple cell lines.

[0142] 1. JAK1 siRNA transfection of cells HepG2, N2A, BRL, and PK15 cells were cultured in complete culture medium at 37°C with 5% CO2. Cells were seeded and transfected when they were in the logarithmic growth phase and in good condition (70% confluence). Cell density was adjusted to 8 x 10 cells per well. 4 Cells were seeded into 24-well plates; transfection complexes were prepared using OMEM and RNAimax with naked dsRNA candidate sequences to achieve final transfection concentrations of 3 nM, 0.3 nM, and 0.03 nM, respectively. The transfection complexes were added to the 24-well plates, shaken well, and incubated in a 5% CO2, 37°C incubator for 48 h. In addition to the experimental groups, the following control groups were also set up: the NC group was a negative control (without siRNA), and the RNAimax group was a transfection reagent control (without siRNA).

[0143] 2. Real-time quantitative PCR analysis: Cells were lysed 48 h after transfection, and total RNA was extracted using a column extraction kit (Novizan). Using the GAPDH gene as an internal control, real-time quantitative PCR was performed using a QuantStudio 5 real-time PCR system (Thermo) with TaqMan probe assay reagents (Novizan). Primers used are shown in Table 12 below. Table 12

[0144] 3. Data Analysis After the PCR reaction, relative quantification was performed using the 2^-ΔΔCt(Livak) method with the reference gene as the standard. The results of qPCR screening are shown in Tables 13A-13D. As can be seen from the data in the table below, the 40 naked sequences determined in this example all exhibited certain inhibitory activity in various cell lines, demonstrating broad-spectrum and efficient JAK1 knockout. Comprehensive analysis of the inhibitory activity data of the 40 naked sequences at different concentrations and in various cell lines revealed that ZYJ379, ZYJ381, and ZYJ461 exhibited the best overall activity levels in all tested cell lines, demonstrating excellent broad-spectrum application potential.

[0145] Specifically, taking the activity at 0.03 nM as an example, in the human HepG2 cell line, the activity levels of ZYJ379, ZYJ381, and ZYJ461 ranked 2nd, 13th, and 11th, respectively. In the mouse N2A cell line, the activity levels of ZYJ379, ZYJ381, and ZYJ461 ranked 4th, 6th, and 3rd, respectively. In the porcine kidney cell line PK15, the activity levels of ZYJ379, ZYJ381, and ZYJ461 ranked 2nd, 6th, and 7th, respectively. In the rat hepatocyte cell line BRL, the activity levels of ZYJ379, ZYJ381, and ZYJ461 ranked 22nd, 4th, and 1st, respectively. Moreover, ZYJ379, ZYJ381, and ZYJ461 also exhibited high-ranking and excellent activities at both 0.3 nM and 3 nM. Taking into account the activity levels across different cell lines and at different concentrations, ZYJ379, ZYJ381, and ZYJ461 were the three sequences with the best performance among these 40 naked sequences.

[0146] Table 13A: Activity in the human cell line HepG2

[0147] Table 13B: Activity in mouse cell line N2A

[0148] Table 13C: Activity in porcine kidney cell line PK15

[0149] Table 13D: Activity in the rat hepatocyte line BRL

[0150] Example 7: Determining the effect of candidate naked sequences on JAK1 gene expression in multiple genera cell lines 1. JAK1 siRNA transfection of cells HepG2, N2A, BRL, and PK15 cells were cultured in complete culture medium at 37°C with 5% CO2. Cells were seeded and transfected when they were in the logarithmic growth phase and in good condition (70% confluence). Cell density was adjusted to 8 x 10 cells per well. 4Cells were seeded into 24-well plates. Transfection complexes were prepared using OMEM and RNAimax with naked dsRNA candidate sequences to achieve final transfection concentrations of 3 nM, 0.3 nM, 0.03 nM, 10, 2.5, 0.625, 0.03906, 0.009765, 0.002441, 0.0006103, and 0.000152588 nM, respectively. The transfection complexes were added to the 24-well plates, mixed well, and incubated in a 5% CO2, 37°C incubator for 48 h. In addition to the experimental groups, the following control groups were set up: the NC group was a negative control (without siRNA), and the RNAimax group was a transfection reagent control (without siRNA).

[0151] 2. Real-time quantitative PCR analysis Cells were lysed 48 h after transfection, and total RNA was extracted using a column extraction kit (Novizan). Real-time quantitative PCR was performed using a QuantStudio 5 PCR instrument (Thermo) with the GAPDH gene as an internal control and TaqMan probe assay kit (Novizan). Primers used were as shown in Table 12 of Example 6.

[0152] 3. Data Analysis After the PCR reaction, relative quantification was performed using the 2^-ΔΔCt(Livak) method, with the reference gene as the standard. The results of qPCR screening are shown in Tables 14-19. Figure 3-6 As shown in Table 15, the data from two repeated experiments are recorded. The results of this embodiment show that ZYJ379, ZYJ381, and ZYJ461 can significantly inhibit JAK1 gene expression in all four test cell lines, and the combined inhibitory activity of the three in various concentrations and cell lines is significantly better than that of the positive controls.

[0153] The data in the table below show that, at a concentration of 0.03 nM, ZYJ379, ZYJ381, and ZYJ461 exhibited higher activity in the BRL cell line than all positive controls; their activity in HepG2 cells was higher than all positive controls except PC6, but the activity of ZYJ379, ZYJ381, and ZYJ461 at 0.3 nM was higher than that of PC6; their activity in the N2A cell line was higher than that of all positive controls except PC1; and their activity in the PK15 cell line was higher than that of all positive controls except PC4.

[0154] Furthermore, ZYJ379, ZYJ381, and ZYJ461 showed IC50 values ​​in four test cell lines. 50The values ​​were all low, demonstrating stable and excellent cross-species inhibitory activity, and significantly superior to most positive controls, validating its broad-spectrum and highly effective characteristics. For example, in the HepG2 cell line, ZYJ379, ZYJ381, and ZYJ461 all showed activity, with ZYJ381 exhibiting the highest activity (its IC50 value was [missing value]). 50 The value was 0.06111, while the IC50 values ​​of the positive controls PC1-PC7 were... 50 The values ​​ranged from 0.08 to 0.22, indicating that the activities of PC1-PC7 were approximately 1-3 times lower than those of ZYJ379, ZYJ381, and ZYJ461. In the PK15 and N2A cell lines, some positive controls (such as PC2, PC5, and PC6) exhibited extremely poor activity, while ZYJ379, ZYJ381, and ZYJ461 all showed relatively high activity, superior to most positive controls.

[0155] Therefore, it can be seen that the three candidate naked sequences ZYJ379, ZYJ381, and ZYJ461 of the present invention all exhibit excellent and broad-spectrum JAK1 gene repressive activity in HepG2, N2A, BRL, and PK15 cell lines, that is, they all show significantly better activity than the positive control in four different species of cell lines: human, mouse, rat, and pig.

[0156] Table 14. Activity of naked sequences and positive controls in various cell lines

[0157] Table 15

[0158] Table 16. IC50 measured in the HepG2 cell line 50

[0159] Table 17. IC50 measured in the PK15 cell line 50

[0160] Table 18. IC50 measured in the N2A cell line 50

[0161] Table 19. IC50 measured in BRL cell lines 50

[0162] Example 8: Effect of the candidate sequence of the present invention on the JAK1 gene in mouse skin Female C57BL / 6 mice aged 6-8 weeks (Beijing Vital River Laboratory Animal Technology Co., Ltd.) were used in the experiment. Mice were randomly divided into groups of 10 mice each based on body weight. Each mouse received a single subcutaneous injection of 0.15 mg of the test sample (ZYJ379-M6, ZYJ381-M6, ZYJ461-M6, PC8, NC, with sequences shown in Table 20 and structures shown in structural formula (II) below) into the back. Skin tissue from the injection site was collected on day 3 after hair removal. RNA was extracted from the tissue using the Trizol method and detected by qPCR. Results are as follows: Figure 7 As shown, the relative mRNA level of PC8 in the control group was approximately 0.7, while the relative mRNA levels of ZYJ379-M6, ZYJ381-M6, and ZYJ461-M6 of the present invention were all below 0.4, significantly lower than that of the control group. In other words, compared to the control group of the prior art, ZYJ379-M6, ZYJ381-M6, and ZYJ461-M6 of the present invention exhibit significantly higher inhibitory effects on JAK1 mRNA levels in mouse skin tissue.

[0163] Equation (II) Table 20 .

[0164] Having fully described the invention, those skilled in the art will understand that the invention can be carried out in a wide and equivalent range of conditions, formulations and other parameters without affecting the scope of any embodiments provided herein.

[0165] All patents, patent applications and publications cited in this invention are incorporated herein by reference in their entirety.

Claims

1. A double-stranded ribonucleic acid (dsRNA) targeting the JAK1 gene, wherein the dsRNA comprises a sense strand and an antisense strand selected from any one of the following groups: a) The sense strand, whose nucleotide sequence is SEQ ID NO: 379, and the antisense strand, whose nucleotide sequence is SEQ ID NO: 840; b) The sense strand, with the nucleotide sequence SEQ ID NO: 381, and the antisense strand, with the nucleotide sequence SEQ ID NO: 842; c) a sense strand having a nucleotide sequence comprising SEQ ID NO: 461, and an antisense strand having a nucleotide sequence comprising SEQ ID NO: 922, wherein the sense strand is no more than 22 nucleotides in length and the antisense strand is no more than 24 nucleotides in length.

2. A double-stranded ribonucleic acid (dsRNA) targeting the JAK1 gene, wherein the dsRNA comprises a sense strand and an antisense strand selected from any one of the following groups: a) The sense strand, whose nucleotide sequence is SEQ ID NO: 1301, and the antisense strand, whose nucleotide sequence is SEQ ID NO: 1762; b) The sense strand, with the nucleotide sequence SEQ ID NO: 1303, and the antisense strand, with the nucleotide sequence SEQ ID NO: 1764; c) a sense strand having a nucleotide sequence comprising SEQ ID NO: 1383, and an antisense strand having a nucleotide sequence comprising SEQ ID NO: 1844, wherein the sense strand is no more than 22 nucleotides in length; and the antisense strand is no more than 24 nucleotides in length; d) The sense strand, with the nucleotide sequence SEQ ID NO: 1885, and the antisense strand, with the nucleotide sequence SEQ ID NO: 1762; e) The sense strand, with the nucleotide sequence SEQ ID NO: 1886, and the antisense strand, with the nucleotide sequence SEQ ID NO: 1764; f) A positive strand having a nucleotide sequence comprising SEQ ID NO: 1887, and an antisense strand having a nucleotide sequence comprising SEQ ID NO: 1844, wherein the positive strand is no more than 22 nucleotides in length and the antisense strand is no more than 24 nucleotides in length.

3. A coupling of formula (I), R1—B—R2 Formula (I), R1 can be a targeting ligand, delivery enhancer, or hydrophobic modifier. B is an arbitrarily existing connector, and R2 is a dsRNA selected from any one of claims 1-2.

4. The conjugate according to claim 3, wherein R1 is selected from: cholesterol, fatty acids, cyclic RGD peptides, N-acetylgalactosamine (GalNAc), folic acid (FA), lithocholic acid (LCA), open-ring steroids, gangliosides, endocannabinoids, eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), and docosuccinic acid (DCA).

5. The coupling compound according to claim 3 or 4, wherein R1 is DCA (dodecanoic acid).

6. The coupling according to claim 3, wherein the connector is selected from one of the group consisting of: -NH-(CH2) n -C(CH2OH)(CH3)- , -NH(CH2) n CO- , -NH(CH2) n NH- , -O-CO-(CH2) n -CO- , -O-CO-(CH2CH2O) n CH2CH2CO- , -NH-(CH2CH2O) n -CH2CH2CO- , -NH-(CH2CH2O) n -CH2CH2NH- , -CO-CH(NH2)-CH2-imidazole- , -CO-CH2-NH-CH2-CO-CH(NH2)-CH(CH3)- , -CO-CH(NH2)-CH(CH3)-CH2CH2- , -CO-CH(NH2)-CH2C(CH3)2- , -CO-CH(NH2)-CH2CH2-S-CH2- , -CO-CH(NH2)-CH2-phenyl- , -CO-CH(NH2)-CH2-p-methoxyphenyl- , -CO-CH(NH2)-CH2-β-indole- , -NH-(CH2) n -triazole- , -NH-(CH2CH2O) n -triazole- , -CO-(CH2) n -triazole- , -CO-(CH2CH2O) n -triazole- , -NH-(CH2) n -S-(succinimide-3-yl-N)- , -NH-(CH2CH2O) n -S-(succinimide-3-yl-N)- , -CO-(CH2) n -S-(succinimide-3-yl-N)- , -CO-(CH2CH2O) n -S-(succinimide-3-yl-N)- , -NH-(CH2) n -(succinimide-3-yl-N)-S- , -NH-(CH2CH2O) n -(succinimide-3-yl-N)-S- , -CO-(CH2) n -(succinimide-3-yl-N)-S- , -CO-(CH2CH2O) n -(succinimide-3-yl-N)-S- , -CH(NH2)-CH2-imidazole- , -CH2-NH-CH2-CO-CH(NH2)-CH(CH3)- , -CH(NH2)-CH(CH3)-CH2CH2- , -CH(NH2)-CH2C(CH3)2- , -CH(NH2)-CH2CH2-S-CH2- , -CH(NH2)-CH2-phenyl- , -CH(NH2)-CH2-p-methoxyphenyl- , -CH(NH2)-CH2-β-indole- , , , , , , , , and , in This indicates the connection end with the target ligand. This indicates the linker end of the dsRNA; Where n is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15.

7. The coupling according to claim 3, wherein the connector is or ,in This indicates the connection end with the target ligand. This indicates the linker end to the dsRNA.

8. The conjugate of claim 3, wherein the 3' end of the sense chain is connected to the targeting ligand, delivery enhancer or hydrophobic modifier, or the connector; and / or wherein the 5' end of the antisense chain comprises a vinyl phosphate ester.

9. A delivery formulation comprising dsRNA according to any one of claims 1-2 and a pharmaceutically acceptable carrier.

10. The delivery formulation according to claim 9, wherein the carrier is selected from one or more of the following: lentiviral vectors, retroviral vectors, adenoviral vectors, poxvirus vectors, herpesvirus vectors, liposomes, lipid nanoparticles, peptides, antibodies, aptamers, N-acetylgalactosamine (GalNAc), lipid polymers, polymer nanoparticles, inorganic nanoparticles, cationic nanoemulsions, and exosomes.

11. A recombinant cell comprising dsRNA according to any one of claims 1-2.

12. A pharmaceutical composition comprising dsRNA according to any one of claims 1-2, a conjugate according to any one of claims 3-8, a delivery formulation according to claim 9 or 10 or a cell according to claim 11, and a pharmaceutically acceptable excipient.

13. Use of the dsRNA according to any one of claims 1-2, the conjugate according to any one of claims 3-8, the delivery formulation according to claim 9 or 10, the cell according to claim 11, or the pharmaceutical composition according to claim 12 in the preparation of a medicament for the prevention and / or treatment of JAK1 gene-mediated diseases or conditions, wherein the JAK1 gene-mediated diseases or conditions are one or more of the following: psoriasis, atopic dermatitis, alopecia areata, vitiligo.

14. Use of the dsRNA according to any one of claims 1-2, the conjugate according to any one of claims 3-9, the delivery formulation according to claim 9 or 10, the cell according to claim 11, or the pharmaceutical composition according to claim 12 in the preparation of personal care products, wherein the personal care products are skin care products or hair care products.

15. The use according to claim 14, wherein the personal care product is used to prevent or improve hair loss, promote hair growth, prevent or improve skin depigmentation, promote skin pigmentation recovery, relieve itching, repair the skin barrier, and / or reduce skin inflammation.

16. The use according to claim 15, wherein the personal care product is used to prevent or improve skin symptoms associated with diseases or conditions caused by abnormally elevated JAK1 gene expression.

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