Double-stranded oligonucleotides targeting ALOX15 mRNA and their applications

Abstract

The application provides a double-stranded oligonucleotide targeting ALOX15 mRNA and an application thereof, and relates to the technical field of biotechnology. A sense strand nucleotide sequence of the double-stranded oligonucleotide comprises a sequence with no more than 8 nucleotides different from the sequence shown in SEQ ID NO. 1, 3, 5, 7 or the like; an antisense strand nucleotide sequence is a sequence with no more than 8 nucleotides different from the sequence shown in SEQ ID NO. 2, 4, 6, 8 or the like; and the sense strand and the antisense strand are at least partially reverse complementary to form a duplex region. The double-stranded oligonucleotide can inhibit ALOX15 mRNA and reduce ALOX15 protein expression in a subject. The double-stranded oligonucleotide can be used for preparing a medicine for treating and / or preventing ALOX15 related diseases (allergic conjunctivitis and dry eye).

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to a double-stranded oligonucleotide targeting ALOX15 mRNA and its applications. Background Technology

[0002] The following statements are provided only as background information in relation to the present invention and do not necessarily constitute prior art.

[0003] Lipoxygenase 15 (ALOX15, also known as 12 / 15-lipoxygenase or 15-LOX-1) is a key member of the lipoxygenase family, widely expressed in various mammalian tissues. Under physiological conditions, ALOX15 participates in cell differentiation, erythrocyte maturation, and membrane remodeling by catalyzing the oxidative metabolism of polyunsaturated fatty acids (such as arachidonic acid and linoleic acid). However, under pathological conditions, abnormal activation of ALOX15 exerts a dual pathogenic effect by regulating inflammation and oxidative stress. On the one hand, it directly acts on cell membrane phospholipids, initiating a lipid peroxidation chain reaction, damaging membrane structure, and inducing ferroptosis; on the other hand, its metabolites (such as 12-HETE and 15-HETE) act as potent pro-inflammatory mediators, chemotactically attracting inflammatory cells and promoting cytokine release, thereby driving a local inflammatory cascade response. Studies have shown that ALOX15-mediated lipid peroxidation is closely related to the pathological processes of various diseases, including atherosclerosis, Alzheimer's disease, Parkinson's disease, tumors, diabetic nephropathy, and ischemia-reperfusion injury.

[0004] Allergic conjunctivitis (AC) is an allergic ocular disease characterized by itchy eyes, tearing, and conjunctival hyperemia. Current clinical treatments mainly include antihistamines, mast cell stabilizers, corticosteroids, and immunosuppressants. However, existing therapies have significant limitations: antihistamines have slow onset and weak efficacy, and may worsen eye discomfort; long-term use of corticosteroids can lead to high intraocular pressure (incidence 10%-40%) and cataracts (incidence 5%-15%). While antibody drugs targeting IgE, IL-5, and IL-4 receptor α can effectively inhibit Th2 immune responses, they are not yet approved for allergic conjunctivitis and face issues such as inconvenient administration, potential immunogenicity, and high cost. Furthermore, existing drugs primarily target allergy symptoms and have limited effectiveness against complications such as corneal ulcers and dry eye, often requiring multiple medications and resulting in poor patient adherence.

[0005] In-depth research shows that the interaction between inflammatory response and oxidative stress is the common pathological basis of allergic conjunctivitis and related dry eye syndrome. After allergens come into contact with the conjunctiva, dendritic cells activate naïve CD4+ T cells to differentiate into Th2 cells, releasing cytokines such as IL-4, IL-5, and IL-13, and inducing B cells to produce IgE. IgE binds to FcεRI receptors on the surface of mast cells, triggering degranulation, releasing histamine and inflammatory mediators such as IL-3, IL-5, and CCL11, and recruiting eosinophils to the site of inflammation. At the same time, the above immune response is accompanied by the production of a large amount of reactive oxygen species (ROS), triggering oxidative stress. On the ocular surface, persistent inflammatory response and oxidative stress lead to tissue damage through a triple mechanism: 1) Inflammatory mediators from mast cells inhibit lacrimal gland function and reduce tear secretion; 2) Meibomian gland dysfunction leads to abnormal lipid secretion, disrupting tear film stability; 3) Long-term oxidative stress induces corneal epithelial cell damage and apoptosis. Tear film disruption and epithelial damage make it easier for allergens to invade tissues, creating a vicious cycle of "allergy-inflammation-oxidative stress-dry eye." Given that current therapies often target single factors and have limited effectiveness in regulating complex immune networks, simultaneously intervening in inflammatory responses and oxidative stress to break this vicious cycle may offer a new strategy for treating allergic conjunctivitis and related dry eye syndromes. However, research on the role of ALOX15 in allergic conjunctivitis and related dry eye syndromes is currently lacking.

[0006] In view of this, the present invention is hereby proposed. Summary of the Invention

[0007] The purpose of this invention is to provide an antisense oligonucleotide that targets ALOX15 mRNA to inhibit or block ALOX15 in subjects, thereby achieving the treatment of allergic conjunctivitis and related dry eye syndrome.

[0008] To solve the above-mentioned technical problems, the present invention adopts the following technical solution: In one aspect, a double-stranded oligonucleotide targeting ALOX15 mRNA is provided, the double-stranded oligonucleotide comprising a sense strand and an antisense strand; The positive strand nucleotide sequence comprises a sequence that differs from the sequence shown in SEQ ID NO. 1, 3, 5 or 7 by no more than 8 nucleotides; The antisense strand nucleotide sequence comprises a sequence that differs from the sequence shown in SEQ ID NO. 2, 4, 6 or 8 by no more than 8 nucleotides; Furthermore, the justice chain and the antisense chain are at least partially complementary in opposite directions, forming a bichain region.

[0009] In a second aspect, a complex of a double-stranded oligonucleotide targeting ALOX15 mRNA is provided, the complex comprising: (a) the double-stranded oligonucleotide targeting ALOX15 mRNA as described in the first aspect; and (b) one or more targeting delivery ligands linked to (a).

[0010] Thirdly, the double-stranded oligonucleotide targeting ALOX15 mRNA as described in the first aspect, or the complex as described in the second aspect, is provided for use in any of the following: (I) Inhibition of subject ALOX15 mRNA and / or reduction of ALOX15 protein expression for non-diagnostic and non-therapeutic purposes.

[0011] (II) Prepare drugs that inhibit ALOX15 mRNA and / or reduce ALOX15 protein expression in subjects.

[0012] (III) Prepare medicines for treating, preventing and / or alleviating pathological conditions or diseases caused by ALOX15 in the subject.

[0013] Fourthly, a pharmaceutical composition is provided, the pharmaceutical composition comprising the double-stranded oligonucleotide targeting ALOX15 mRNA as described in the first aspect, or the complex as described in the second aspect; Optionally, the pharmaceutical composition further includes lipid nanoparticles; the double-stranded oligonucleotide targeting ALOX15 mRNA is loaded onto the lipid nanoparticles; Optionally, the lipid nanoparticles include ROS-responsive ionizable cationic lipids, dioleoylphosphatidylethanolamine, cholesterol, and dimyristoylglycerol-polyethylene glycol.

[0014] Fifthly, a method for inhibiting ALOX15 in a subject for non-diagnostic and therapeutic purposes is provided, the method comprising contacting the subject with a double-stranded oligonucleotide targeting ALOX15 mRNA as described in the first aspect, or a complex as described in the second aspect, or a pharmaceutical composition as described in the fourth aspect.

[0015] Compared with the prior art, the present invention has the following beneficial effects: The double-stranded oligonucleotides targeting ALOX15 mRNA provided by this invention can inhibit ALOX15 mRNA and reduce ALOX15 protein expression in subjects. The double-stranded oligonucleotides targeting ALOX15 mRNA, complexes containing them, and pharmaceutical compositions provided by this invention may help treat and / or prevent ALOX15-related diseases (allergic conjunctivitis and dry eye syndrome). Attached Figure Description

[0016] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0017] Figure 1 Results of ALOX15 screening for allergic conjunctivitis using transcriptome sequencing; A shows the process of establishing a mouse model of allergic conjunctivitis (AC) induced by ragweed pollen (SRW) and ovalbumin (OVA), and a schematic diagram of subsequent clinical symptom analysis and transcriptome sequencing analysis; B shows representative images (left) and corresponding clinical scores (right) of ocular surface redness, tearing, and congestion in mice in the PBS control group and the SRW-induced AC model group. n = 10, one eye was taken from each mouse); C shows representative photographs (left) and corresponding clinical scores (right) of ocular surface redness, tearing, and congestion in mice in the PBS control group and OVA-induced AC model group. n = 10, one eye was taken from each mouse; D is the quantitative analysis of the number of eye scratches in mice in the PBS control group and the SRW model group ( n = 10 mice); E represents the quantitative analysis of the number of eye scratches in mice in the PBS control group and the OVA model group ( n = 10 mice); F is a volcano plot of differentially expressed genes (DEGs) in the conjunctival tissue of mice in the PBS control group and the SRW-induced AC model group. Each RNA-seq sample was prepared by mixing four conjunctival tissues from two mice (each group) n = 2 biological replicates); G represents the KEGG pathway enrichment analysis results of DEGs in conjunctival tissues of the PBS control group and the SRW-induced AC model group; H represents the GO enrichment analysis results of DEGs in conjunctival tissues of the PBS control group and the SRW-induced AC model group; I represents the volcano plot of DEGs in conjunctival tissues of mice in the PBS control group and the OVA-induced AC model group. Each RNA-seq sample was prepared by mixing 4 conjunctival tissues from 2 mice (each group) n= 2 biological replicates); J is the KEGG pathway enrichment analysis result of DEGs in conjunctival tissue of PBS control group and OVA-induced AC model group; K is the GO enrichment analysis result of DEGs in conjunctival tissue of PBS control group and OVA-induced AC model group; L is the heatmap of DEGs related to arachidonic acid (AA) and linoleic acid (LA) metabolic pathways in conjunctival tissue of mice in PBS control group and SRW model group; M is the heatmap of DEGs related to AA and LA metabolic pathways in conjunctival tissue of mice in PBS control group and OVA model group; N is the Venn diagram of upregulated DEGs related to AA and LA metabolic pathways in SRW and OVA-induced AC models; where B, C, D and E data are expressed as mean ± standard deviation, and the two-tailed unpaired Student's t test is used for comparison. p <0.05, p <0.01, p <0.0001; Figure 2 for ALOX15 Expression verification and functional study results of lipoxygenase 15 in allergic conjunctivitis; where A represents the expression of lipoxygenase 15 in conjunctival tissue isolated and subsequently detected by real-time quantitative PCR (RT-qPCR), Western blot (WB), and immunofluorescence (IF). ALOX15 ) Schematic diagram of the experimental procedure for expression; B represents the conjunctival tissue of mice in the PBS control group and the SRW-induced AC model group. ALOX15 relative mRNA expression level (in terms of mRNA relative expression level) Gapdh Quantitative analysis results (as internal reference) n = 6, one eye was taken from each mouse); C represents the Western blot results of ALOX15 and β-actin protein expression in the conjunctival tissue of mice in the PBS control group and the SRW-induced AC model group ( n= 3, one eye per mouse); D shows representative immunofluorescence results of ALOX15 expression (red) in the conjunctival tissue of mice in the PBS control group and SRW-induced AC model group; cell nuclei were stained with DAPI (blue); images are presented at the same magnification; scale bar is 20 μm; E is a schematic diagram of the treatment process for human conjunctival epithelial cells (HconEpic): stimulated with PBS, SRW, or simultaneously transfected with scrambled siRNA or siALOX15 during SRW stimulation, followed by detection of lipid reactive oxygen species (ROS), malondialdehyde (MDA), reduced glutathione (GSH), and oxidized glutathione (GSSG); F shows the Western blot results of ALOX15 and β-actin protein levels in HconEpic cells of the PBS control group and SRW stimulation group; G shows HconEpic cells treated with PBS or SRW, and HconEpic cells simultaneously transfected with scrambled siRNA or siALOX15 during SRW stimulation. ALOX15 Quantitative analysis results of relative mRNA expression levels (using ACTB as an internal reference) n = 5 independent biological experiments); H represents the Western blot results of ALOX15 and β-actin protein levels in HconEpic cells transfected with scrambled siRNA or siALOX15 under SRW stimulation; I represents the representative flow cytometry results (left) and quantitative analysis results of mean fluorescence intensity (MFI) of lipid ROS levels in HconEpic cells transfected with scrambled siRNA or siALOX15-5 under PBS or SRW stimulation and SRW stimulation (right) detected by C11-BODIPY probe. n = 5 independent biological experiments); J represents the quantitative analysis results of the relative MDA levels in HconEpic cells treated with PBS or SRW, and SRW stimulation simultaneously transfected with scrambled siRNA or siALOX15-5. n = 5 independent biological experiments); K represents the quantitative analysis results of GSH levels in HconEpic cells treated with PBS or SRW, and SRW stimulation simultaneously transfected with scrambled siRNA or siALOX15-5. n = 5 independent biological experiments); L represents the quantitative analysis results of GSSG levels in HconEpic cells treated with PBS or SRW, and SRW stimulation simultaneously transfected with scrambled siRNA or siALOX15-5. n= 5 independent biological experiments); M represents the quantitative analysis results of the GSH / GSSG ratio in HconEpic cells treated with PBS or SRW, and simultaneously transfected with scrambled siRNA or siALOX15-5 and stimulated with SRW. n = 5 independent biological experiments); where B, G and IM data are expressed as mean ± standard deviation, and are compared using two-tailed unpaired Student's t test (B) or one-way ANOVA (G and IM); p <0.05, p <0.01, p <0.001, p <0.0001; Figure 3 The images show the construction, characterization, and in vivo retention analysis results of rLNP@siALOX15-5. A is a schematic diagram of the experimental procedure for conjunctival tissue isolation and subsequent reactive oxygen species (ROS) level detection; B is a representative immunofluorescence image of conjunctival tissue stained with the ROS fluorescent probe DCFH-DA (red) in the PBS control group and SRW-induced AC model group mice; cell nuclei were stained with DAPI (blue); images are presented at the same magnification; scale bar is 50 μm; C is a schematic diagram of the construction process and subsequent characterization and evaluation experiments of rLNP@siALOX15-5; D is a representative transmission electron microscope image of rLNP@siALOX15-5 after uranium acetate staining; scale bar is 50 nm; E is the particle size distribution map of rLNP@siALOX15-5 measured by dynamic light scattering; F is the particle size and Zeta potential results of rLNP@siALOX15-5 as shown by nanoparticle tracking analysis. n = 3 independent biological experiments); G is the quantitative analysis result of the encapsulation efficiency of rLNP@siALOX15-5 ( n= 5 independent biological experiments); H is a schematic diagram of the experimental procedure for simulating oxidative stress environment with H2O2 stimulation and subsequent evaluation of the uptake and silencing efficiency of rLNP@siALOX15-5 in HconEpic cells; HconEpic cells were pretreated with hydrogen peroxide (H2O2, 75 µM, 4 h) to simulate oxidative stress, and then incubated with Cy5-siALOX15-5 or rLNP@Cy5-siALOX15-5 for 30 min to assess uptake efficiency, and incubated for 24 h to assess silencing efficiency; I is a representative flow cytometry (left) and MFI quantitative analysis results (right) of Cy5-siALOX15-5 or rLNP@Cy5-siALOX15-5 uptake by HconEpic cells under normal conditions and oxidative stress conditions. n = 3 independent biological experiments); J is siALOX15-5 or rLNP@siALOX15-5 in HconEpic cells. ALOX15 Quantitative analysis results of mRNA (using ACTB as an internal control) silencing efficiency ( n = 3 independent biological experiments); K represents representative in vivo imaging images (left) and quantitative analysis results of relative MFI (right) at different time points after a single topical ophthalmic drop of Cy5-siALOX15-5 or rLNP@Cy5-siALOX15-5 in SRW-induced AC mice; data were normalized at the initial time point (1 minute). n = 3, one eye from each mouse); L represents the representative fluorescence images (left) and quantitative analysis results of MFI (right) of conjunctival tissue at different time points after a single topical eye drop of Cy5-siALOX15-5 or rLNP@Cy5-siALOX15-5 in SRW-induced AC mice. n = 3 (one eye per mouse); images were presented at the same magnification; the scale bar was 20 μm; FG and IL data are expressed as mean ± standard deviation and were compared using one-way ANOVA (I, J and L); p <0.05, p <0.001, p <0.0001; Figure 4This study evaluates the therapeutic efficacy and safety of rLNP@siALOX15-5 in a mouse model of SRW-induced allergic conjunctivitis. Figure A shows a schematic diagram of the administration process for continuous ocular instillation of PBS, siALOX15-5, rLNP@siALOX15-5, or tobramycin-dexamethasone eye drops (TobraDex) into SRW-induced AC mice. Figure B shows representative results of conjunctival hyperemia in each group of mice after continuous ocular administration. Figure C shows the quantitative analysis results of the conjunctival hyperemia area corresponding to Figure B. n = 8, one eye was taken from each mouse; D is the quantitative analysis result of the number of eye-grabbing counts of mice within 30 minutes after the last provocation ( n = 8, one eye was taken from each mouse); E is the quantitative analysis result of mouse tear secretion measured by the phenol red cotton thread method ( n = 8, one eye was taken from each mouse); F represents the quantitative analysis results of immunoglobulin E (IgE) protein levels in mouse conjunctival tissue detected by enzyme-linked immunosorbent assay (ELISA). n = 8, one eye was taken from each mouse); G represents the quantitative analysis results of histamine levels in mouse conjunctival tissue detected by ELISA ( n = 8, one eye was taken from each mouse); H represents the quantitative analysis results of interleukin-4 (IL-4) protein levels in mouse conjunctival tissue detected by ELISA ( n = 8, one eye from each mouse); I represents the quantitative analysis results of interleukin-13 (IL-13) protein levels in mouse conjunctival tissue detected by ELISA ( n = 8, one eye was taken from each mouse); J represents the quantitative analysis results of intraocular pressure (IOP) in mice ( n = 8, one eye was taken from each mouse); K is the quantitative analysis result of malondialdehyde (MDA) level in mouse conjunctival tissue ( n = 8, one eye was taken from each mouse); L is the quantitative analysis result of MDA level in mouse corneal tissue ( n = 8, one eye from each mouse); M represents the representative results of 4-hydroxynonenal (4-HNE) immunohistochemical staining in mouse conjunctival tissue; images are presented at the same magnification; scale bar: top image 100 μm, bottom image 25 μm; N represents the quantitative analysis results of the 4-HNE immunohistochemical staining score corresponding to image M ( n = 8 (one eye was taken from each mouse); where CL and N data are expressed as mean ± standard deviation, and one-way ANOVA was used for comparison. p<0.05, p<0.01, p <0.001, p <0.0001; Figure 5 This study evaluates the therapeutic efficacy and safety of rLNP@siALOX15-5 in an OVA-induced allergic conjunctivitis mouse model. A shows a schematic diagram of the administration process for continuous eye drops of PBS, siALOX15-5, rLNP@siALOX15-5, or TobraDex in OVA-induced AC mice; B shows representative results of conjunctival hyperemia in OVA-induced AC mice after continuous eye drops; and C shows the quantitative analysis results of the conjunctival hyperemia area corresponding to Figure B. n = 7, one eye was taken from each mouse); D is the quantitative analysis result of IL-4 protein level in conjunctival tissue of OVA-induced mice detected by ELISA ( n = 7, one eye was taken from each mouse); E represents the quantitative analysis results of IL-13 protein levels in the conjunctival tissue of OVA-induced mice as detected by ELISA ( n = 7, one eye was taken from each mouse); F represents the quantitative analysis results of OVA-induced IOP in mice ( n = 7 (one eye was taken from each mouse); where CF data are expressed as mean ± standard deviation, and one-way ANOVA was used for comparison; p <0.05, p <0.01, p <0.001; Figure 6 This study evaluates the duration of efficacy of a single dose of rLNP@siALOX15-5 in a mouse model of allergic conjunctivitis. Figure A shows a schematic diagram of the experimental procedure for detecting and analyzing conjunctival hyperemia at different time points after a single eye drop of PBS, siALOX15-5, rLNP@siALOX15-5, or TobraDex in SRW-induced AC mice. Figure B shows representative results of conjunctival hyperemia at different time points after a single dose in SRW-induced AC mice. Figure C shows the quantitative analysis results of the conjunctival hyperemia area corresponding to Figure B. n = 8, one eye was taken from each mouse); D is the number of conjunctival tissue samples taken at different time points after a single dose in SRW-induced AC mice, as detected by RT-qPCR. ALOX15 Quantitative analysis results of relative mRNA expression levels ( n= 8, one eye was taken from each mouse); E represents the concentration of SRW-induced AC mice at different time points after a single dose, as detected by RT-qPCR. Cd4 Quantitative analysis results of relative mRNA expression levels ( n =8, one eye was taken from each mouse); F represents the concentration of SRW-induced AC mice at different time points after a single dose, as detected by RT-qPCR. Il4 Quantitative analysis results of relative mRNA expression levels ( n = 8, one eye was taken from each mouse); G is the concentration of SRW-induced AC mice at different time points after a single dose, as detected by RT-qPCR. Il5 Quantitative analysis results of relative mRNA expression levels ( n =8, one eye was taken from each mouse); H represents the conjunctival tissue of SRW-induced AC mice at different time points after a single dose, as detected by RT-qPCR. Il13 Quantitative analysis results of relative mRNA expression levels ( n = 8, one eye was taken from each mouse); where CH data are expressed as mean ± standard deviation, and one-way ANOVA was used for comparison; p <0.05, p <0.01, p <0.001, p <0.0001; Figure 7 Transcriptomic mechanism analysis of rLNP@siALOX15-5 in the treatment of allergic conjunctivitis; where A is a bar graph of KEGG pathway enrichment analysis of upregulated and downregulated DEGs in conjunctival tissue of mice in the PBS control group and the rLNP@siALOX15-5 treatment group; each RNA-seq sample was prepared by mixing two conjunctival tissues from one mouse (each group) n = 3 mice); B is a bubble chart of GO enrichment analysis of upregulated and downregulated DEGs in the conjunctival tissue of mice in the PBS control group and the rLNP@siALOX15-5 treatment group. Figure 8The graphs are as follows: A shows the expression heatmap of DEGs related to inflammatory signaling pathways in the conjunctival tissues of mice in the PBS, siALOX15-5, rLNP@siALOX15-5, and TobraDex groups; B shows the expression heatmap of DEGs related to ROS generation and antioxidation in the conjunctival tissues of mice in the PBS, siALOX15-5, rLNP@siALOX15-5, and TobraDex groups; C shows the expression heatmap of DEGs related to lipid peroxidation in the conjunctival tissues of mice in the PBS, siALOX15-5, rLNP@siALOX15-5, and TobraDex groups; and D shows the heatmap of the activity of lipid peroxidation, oxidative stress, and inflammatory response-related pathways in the conjunctival tissues of mice in the PBS, siALOX15-5, rLNP@siALOX15-5, and TobraDex groups based on single-sample gene set enrichment analysis (ssGSEA). Figure 9 This study evaluates the therapeutic effect of rLNP@siALOX15-5 on a mouse model of dry eye; where A represents mice with dry eye (DED) in the GSE208297 dataset. ALOX15 Analysis results of relative expression levels ( n = 5 mice); B is a schematic diagram of the establishment of a 0.2% benzalkonium chloride (BAC)-induced dry eye mouse model and the subsequent administration of PBS, siALOX15-5, rLNP@siALOX15-5 or polyvinyl alcohol eye drops (PVA); C is a representative result of corneal fluorescein staining in normal mice and dry eye mice with PBS, siALOX15-5, rLNP@siALOX15-5 or PVA; D is the quantitative analysis result of corneal fluorescein staining score corresponding to Figure C ( n = 7, one eye was taken from each mouse); E is the quantitative analysis result of tear secretion in normal mice and dry eye mice treated with PBS, siALOX15-5, rLNP@siALOX15-5 or PVA by the phenol red cotton thread method. n = 7, one eye per mouse); F represents the representative results of hematoxylin-eosin (H&E) staining of corneal tissue from normal mice and dry-eye mice treated with PBS, siALOX15-5, rLNP@siALOX15-5, or PVA; images are presented at the same magnification; scale bar is 40 μm; G represents the representative results of conjunctival tissue from normal mice and dry-eye mice treated with PBS, siALOX15-5, rLNP@siALOX15-5, or PVA, showing goblet cells; images are presented at the same magnification; scale bar is 100 μm; H represents the quantitative analysis results of the number of conjunctival goblet cells corresponding to Figure G ( n= 7, one eye was taken from each mouse); I represents the quantitative analysis results of MDA levels in conjunctival tissue of normal mice and mice with dry eye syndrome that had received ocular instillation of PBS, siALOX15-5, rLNP@siALOX15-5, or PVA. n = 7, one eye was taken from each mouse); J represents the quantitative analysis results of MDA levels in corneal tissue of normal mice and mice with dry eye syndrome treated with PBS, siALOX15-5, rLNP@siALOX15-5, or PVA. n = 7, one eye per mouse); K represents the representative results of 4-HNE immunohistochemical staining in the conjunctival tissue of normal mice and mice with dry eye syndrome treated with PBS, siALOX15-5, rLNP@siALOX15-5, or PVA; images are presented at the same magnification; scale bar is 50 μm; L represents the representative results of 4-HNE immunohistochemical staining in the corneal tissue of normal mice and mice with dry eye syndrome treated with PBS, siALOX15-5, rLNP@siALOX15-5, or PVA; images are presented at the same magnification; scale bar is 40 μm; where A, D, E, H, I, and J data are expressed as mean ± standard deviation, and comparisons are made using a two-tailed unpaired Student's t test (A) or one-way ANOVA (D, E, H, I, and J); p <0.05, p <0.01, p <0.001, p <0.0001. Detailed Implementation

[0018] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0019] In this document, the terms “comprising” or “including” are open-ended expressions used in this disclosure to mean the phrase “including but not limited to”, and are used interchangeably with it, meaning that they include the contents specified in this disclosure, but do not exclude other contents.

[0020] In this document, the terms “optionally,” “optionally,” or “optionally” generally refer to an event or condition that may or may not occur as described below, and the description includes both cases in which the event or condition occurs and cases in which the event or condition does not occur.

[0021] In this document, “and / or” is used to indicate that one or both of the situations described may occur, for example, A and / or B includes (A and B) and (A or B).

[0022] In this document, unless otherwise stated, any numbering is used to distinguish one entity or behavior from another, and is not required to require or imply any actual relationship, order, or importance between these entities or behaviors, such as numbering i, ii; first, second, etc.

[0023] In this document, the terms “comprising” or “including” mean that the stated elements, integers or steps are included, but do not exclude any other elements, integers or steps.

[0024] In this article, the term "double-stranded oligonucleotide" refers to a double-stranded structure formed by two oligonucleotides through partial or complete base pairing. The two oligonucleotides include a sense strand and an antisense strand, which may or may not be the same length. Double-stranded oligonucleotides include ribonucleotides, deoxyribonucleotides, or nucleotides containing purine and pyrimidine bases or other naturally occurring, chemically or biochemically modified, non-natural, or derived nucleotides.

[0025] In this article, the term "small interfering RNA (siRNA)" refers to a double-stranded RNA of 17 to 25 nucleotides in length, containing a sense strand and an antisense strand. siRNA mediates the targeted cleavage of RNA transcripts via the RISC pathway by forming an RNA-induced silencing complex (RISC).

[0026] In this article, when "connection" refers to the connection of two molecules, the two molecules can be directly connected (e.g., directly connected by chemical bonds) or connected by additional molecules. When two molecules are directly connected, they can be covalently or non-covalently connected. Covalent connection refers to the connection of two molecules through the formation of covalent bonds, which are chemical bonds formed between atoms by sharing electron pairs. Non-covalent connection refers to connection methods other than covalent connection, such as connection through hydrogen bonds, van der Waals forces, electrostatic interactions, hydrophobic interactions, and metal coordination bonds.

[0027] In this document, the terms "subject" or "patient" refer to a mammalian subject or patient, and organs, tissues, or cells derived from them. The mammals include, but are not limited to, humans, rhesus monkeys, rats, mice, guinea pigs, rabbits, dogs, cats, hamsters, nude mice, ferrets, pigs, sheep, goats, cattle, horses, donkeys, dogs, cynomolgus monkeys, macaques, baboons, gibbons, golden monkeys, long-tailed macaques, marmosets, squirrel monkeys, pig-tailed macaques, or chimpanzees. In some embodiments, the subject is a human, a rhesus monkey, or a cynomolgus monkey.

[0028] In this document, the terms “treatment,” “relief,” or “improvement” are used interchangeably. These terms refer to methods of achieving beneficial or desired outcomes, including, but not limited to, treatment benefits. A “treatment benefit” means the eradication or improvement of one or more diseases, conditions, or circumstances associated with the underlying barrier being treated.

[0029] In this article, the terms “prevention” and “avoidance” are used interchangeably to refer to methods for obtaining beneficial or desired outcomes, including but not limited to preventive benefits. To obtain a “preventive benefit,” a drug may be given to a subject at risk of developing a specific disease, or to a subject who reports one or more physiological symptoms of a disease, even if a diagnosis of the disease may not have been made.

[0030] In this document, the term “inhibition” may be used interchangeably with “reduction,” “silencing,” “downregulation,” “blocking,” and other similar terms, and includes any level of inhibition. Inhibition can be assessed by a reduction in the absolute or relative level of one or more of these variables compared to a control level. This control level can be any type of control level used in the art, such as a baseline level before administration or a level determined from an untreated or controlled (e.g., a buffer-only control or an inert agent control) subject, cell, or sample.

[0031] Firstly, a double-stranded oligonucleotide targeting ALOX15 mRNA is provided.

[0032] The double-stranded oligonucleotide comprises a sense strand and an antisense strand; The positive strand nucleotide sequence comprises a sequence that differs from the sequence shown in SEQ ID NO. 1, 3, 5 or 7 by no more than 8 nucleotides; The antisense strand nucleotide sequence comprises a sequence that differs from the sequence shown in SEQ ID NO. 2, 4, 6 or 8 by no more than 8 nucleotides; Furthermore, the justice chain and the antisense chain are at least partially complementary in opposite directions, forming a bichain region.

[0033] In optional embodiments, the sense and antisense nucleotide sequences of the double-stranded oligonucleotide each contain sequences that differ from the sequences shown in SEQ ID NO. 1 and 2 by no more than 8 nucleotides; or, each contain sequences that differ from the sequences shown in SEQ ID NO. 3 and 4 by no more than 8 nucleotides; or, each contain sequences that differ from the sequences shown in SEQ ID NO. 5 and 6 by no more than 8 nucleotides; or, each contain sequences that differ from the sequences shown in SEQ ID NO. 7 and 8 by no more than 8 nucleotides.

[0034] In an optional embodiment, the differential nucleotides in the positive strand that differ from the sequence shown in SEQ ID NO. 1, 3, 5 or 7 originate from mutations at one or more sites; and / or from 1, 2, 3, 4, 5, 6, 7 or 8 nucleotides adjacent to the 5' end and / or 3' end of the fragment containing the ALOX15 mRNA corresponding to the sequence shown in SEQ ID NO. 1, 3, 5 or 7.

[0035] In an optional embodiment, the differentially expressed nucleotides in the antisense strand that are different from the sequence shown in SEQ ID NO. 2, 4, 6 or 8 are mutations at one or more sites; and / or are 1, 2, 3, 4, 5, 6, 7 or 8 nucleotides adjacent to the 5' end and / or 3' end of the fragment containing the ALOX15 mRNA corresponding to the sequence shown in SEQ ID NO. 2, 4, 6 or 8.

[0036] In an optional embodiment, the sense strand nucleotide sequence of the double-stranded oligonucleotide is a sequence containing no more than 1, 2, 3, 4, 5, 6, 7, or 8 nucleotides different from the sequence shown in SEQ ID NO. 7, and the antisense strand nucleotide sequence contains a sequence containing no more than 1, 2, 3, 4, 5, 6, 7, or 8 nucleotides different from the sequence shown in SEQ ID NO. 8. More optionally, the sense strand nucleotide sequence of the double-stranded oligonucleotide is the sequence shown in SEQ ID NO. 7, and the antisense strand nucleotide sequence is the sequence shown in SEQ ID NO. 8.

[0037] In an optional embodiment, at least one nucleoside internucleotide bond of the double-stranded oligonucleotide is a modified nucleoside internucleotide bond.

[0038] In an optional embodiment, at least one nucleoside inter-bond is a phosphate thioside inter-bond.

[0039] In optional embodiments, the first, second, or third internucleotide bond, calculated from the 5' end of the sense strand, is a thiophosphate internucleotide bond; and / or, the first, second, or third internucleotide bond, calculated from the 3' end of the sense strand, is a thiophosphate internucleotide bond; and / or, the first, second, or third internucleotide bond, calculated from the 5' end of the antisense strand, is a thiophosphate internucleotide bond; and / or, the first, second, or third internucleotide bond, calculated from the 3' end of the antisense strand, is a thiophosphate internucleotide bond.

[0040] In an optional embodiment, the double-stranded oligonucleotide contains at least one modified nucleotide.

[0041] In optional embodiments, the modified nucleotide includes one or more of the following: 2'-O-methoxyethyl modified nucleotides, 5'-methyl modified nucleotides, 2'-(S)-restricted ethyl modified nucleotides, 2'-O-methyl modified nucleotides, 2'-deoxy-2'-fluorinated modified nucleotides, 2'-deoxy-modified nucleotides, locked nucleotides, non-locked nucleotides, configuration-restricted nucleotides, restricted ethyl nucleotides, base-free nucleotides, 2'-amino-modified nucleotides, 2'-O-allyl-modified nucleotides, 2'-C-alkyl-modified nucleotides, 2'-hydroxy-modified nucleotides, 2'-O-alkyl-modified nucleotides, morpholinyl nucleotides, bridging nucleic acids, peptide nucleic acids, locked nucleic acids, and non-locked nucleic acids. The modified nucleotide can be one or more modified nucleotides.

[0042] In an optional embodiment, the modified nucleotide includes at least one of 2'-F modified nucleotide, 2'-O-methylated modified nucleotide, and 5'-(E)-VP modified nucleotide.

[0043] In an optional implementation, the first nucleotide at the 5' end of the antisense strand is a nucleotide modified with 5'-(E)-VP.

[0044] In an optional embodiment, the double-stranded oligonucleotide is modified with DV18, wherein the DV18 modification includes: from the 5' to 3' direction, the nucleotides at positions 1-6, 8, and 12-21 of the sense strand are 2'-O-methylated nucleotides, and the nucleotides at positions 7 and 9-11 are 2'-deoxy-2'-fluorinated nucleotides; from the 5' to 3' direction, the nucleotides at positions 1, 3-5, 7, 10-13, 15, and 17-21 of the antisense strand are 2'-O-methylated nucleotides, and the nucleotides at positions 2, 6, 8, 9, 14, and 16 are 2'-deoxy-2'-fluorinated nucleotides; further optionally, the first nucleotide at the 5' end of the antisense strand is a 5'-(E)-VP modified nucleotide.

[0045] In an optional embodiment, the double-stranded oligonucleotide is modified with DV22, wherein the DV22 modification includes: from the 5' to 3' direction, the nucleotides at positions 1-6, 8, and 12-21 of the sense strand are 2'-O-methylated nucleotides, and the nucleotides at positions 7 and 9-12 are 2'-deoxy-2'-fluorinated nucleotides; from the 5' to 3' direction, the nucleotides at positions 1, 3-5, 7-13, 15, and 17-21 of the antisense strand are 2'-O-methylated nucleotides, and the nucleotides at positions 2, 6, 14, and 16 are 2'-deoxy-2'-fluorinated nucleotides; further optionally, the first nucleotide at the 5' end of the antisense strand is a 5'-(E)-VP modified nucleotide.

[0046] In an optional embodiment, the double-stranded oligonucleotide is ESC modified, wherein the ESC modification includes: from the 5' to 3' direction, the nucleotides at positions 2, 4, 6, 8, 12, 14, 16, 18, and 20 of the sense strand are 2'-O-methylated nucleotides, and the nucleotides at positions 1, 3, 5, 7, 9-11, 13, 15, 17, 19, and 21 of the antisense strand are 2'-deoxy-2'-fluorinated nucleotides; from the 5' to 3' direction, the nucleotides at positions 1, 3, 5, 7, 9, 11-13, 15, 17, 19, and 21 of the antisense strand are 2'-O-methylated nucleotides, and the nucleotides at positions 2, 4, 6, 8, 10, 14, 16, 18, and 20 of the antisense strand are 2'-deoxy-2'-fluorinated nucleotides; further optionally, the first nucleotide at the 5' end of the antisense strand is a 5'-(E)-VP modified nucleotide.

[0047] In an optional implementation, the double-stranded oligonucleotide is siRNA.

[0048] In a second aspect, a complex of a double-stranded oligonucleotide targeting ALOX15 mRNA is provided, the complex comprising: (a) the double-stranded oligonucleotide targeting ALOX15 mRNA as described in the first aspect; and (b) one or more targeting delivery ligands linked to (a), the targeting delivery ligands being, for example, but not limited to, nucleic acid aptamers, targeting peptides, or compound drugs.

[0049] Thirdly, a double-stranded oligonucleotide targeting ALOX15 mRNA, or the complex described in the second aspect, is provided for use in any of the following: (I) Inhibition of subject ALOX15 mRNA and / or reduction of ALOX15 protein expression for non-diagnostic and non-therapeutic purposes.

[0050] (II) Prepare drugs that inhibit ALOX15 mRNA and / or reduce ALOX15 protein expression in subjects.

[0051] (III) To prepare medicines for treating, preventing and / or alleviating pathological conditions or diseases caused by ALOX15 in the subject; In an optional implementation, the pathological condition or disease caused by the subject's ALOX15 includes allergic conjunctivitis or dry eye disease.

[0052] Fourthly, a pharmaceutical composition is provided, the pharmaceutical composition comprising the double-stranded oligonucleotide targeting ALOX15 mRNA as described in the first aspect, or the complex as described in the second aspect.

[0053] In an optional embodiment, the pharmaceutical composition further comprises pharmaceutically acceptable optional excipients. The acceptable excipients may be any excipients known in the art and conventionally used. Examples of excipients include, but are not limited to, any physiologically compatible solvents, dispersion media, coatings, antibacterial agents, antifungal agents, pH adjusters, lyophilization protectants, and emulsifiers.

[0054] In optional embodiments, the pharmaceutical composition comprises a double-stranded oligonucleotide for delivering ALOX15 mRNA or a delivery system for delivering the complex. The delivery cells include, but are not limited to, lipid nanoparticles, liposomes, nanoparticles, cationic lipids, cationic polymers, metal nanopolymers, nanorods, micelles, microvesicles, cell-penetrating peptides, viral particles, protein coats, or lipoglobules.

[0055] In an optional embodiment, the lipid nanoparticles comprise ROS-responsive ionizable cationic lipids, dioleoylphosphatidylethanolamine, cholesterol, and dimyristoylglycerol-polyethylene glycol; the molar ratio of the ROS-responsive ionizable cationic lipids, dioleoylphosphatidylethanolamine, cholesterol, and dimyristoylglycerol-polyethylene glycol can be, for example, 50:10:38.5:1.5.

[0056] Fifthly, a method for inhibiting ALOX15 in a subject for non-diagnostic and therapeutic purposes is provided, the method comprising contacting the subject with a double-stranded oligonucleotide targeting ALOX15 mRNA as described in the first aspect, or a complex as described in the second aspect, or a pharmaceutical composition as described in the fourth aspect.

[0057] In an optional implementation, the subjects are selected from humans or mice.

[0058] In an alternative implementation, the subject is a cell, such as a human conjunctival epithelial cell.

[0059] In an optional implementation, the subject is a cell, and the contact includes delivering the double-stranded oligonucleotide targeting ALOX15 mRNA or the complex into the cell using any method known in the art.

[0060] In an optional implementation, the subject is a cell, and the working concentration of the double-stranded oligonucleotide targeting ALOX15 mRNA can be, for example, but not limited to, 0.001 nM, 0.005 nM, 0.014 nM, 0.041 nM, 0.1 nM, 0.3 nM, 0.5 nM, 0.8 nM, 1 nM, 2 nM, 3 nM, 5 nM, 8 nM, 10 nM, 20 nM, 30 nM, 40 nM, 50 nM, 60 nM, 70 nM, 80 nM, or 90 nM.

[0061] In an optional implementation, the subject is an individual organism, and the contact includes administering the drug to the subject via any suitable route known in the art, including but not limited to: oral administration, intravenous administration, intramuscular administration, subcutaneous administration, transdermal administration, airway administration (aerosol), pulmonary administration, nasal administration, ocular administration, or rectal administration.

[0062] The present invention will be further illustrated below with specific embodiments. However, it should be understood that these embodiments are merely for the purpose of more detailed illustration and should not be construed as limiting the present invention in any way.

[0063] Example 1: Screening of ALOX15, a target associated with allergic conjunctivitis In this embodiment, two allergic conjunctivitis models were established using 6-8 week old female BALB / c mice to screen for the ALOX15 target. For the ragweed pollen (SRW, Greer Laboratories, XP56D3A25) induced model: On day 0, 50 μg of SRW pollen and 5 mg of aluminum hydroxide adjuvant (Sigma, A6435) emulsion were injected into the footpads for primary immunization. On day 14, 100 μg of SRW pollen suspension was injected intraperitoneally for booster immunization. From day 28 to 32, 5 μL of SRW suspension was instilled into both eyes daily for challenge. On day 32, 20 minutes after the last challenge, the clinical score was assessed and the number of scratches was counted. On day 33, the mice were sacrificed and samples were collected for RNA-seq. For the ovalbumin (OVA, Sigma, A5503) induced model: On days 0 and 7, subcutaneous injection of an emulsion of OVA (100 μg) and aluminum adjuvant (5 mg) was administered. From days 14 to 21, OVA solution (50 mg / mL, 5 μL) was instilled into both eyes daily for challenge. Clinical scores were assessed 20 minutes after the last challenge on day 21, and the number of scratches was counted. On day 22, animals were sacrificed for RNA-seq. Control groups were treated in parallel with PBS. Figure 1 (A in the middle).

[0064] The results showed that, compared with the PBS control group, mice induced by OVA and SRW exhibited significant symptoms of allergic conjunctivitis, including eyelid edema, conjunctival hyperemia, and a significant increase in eye-scratching, indicating that the model was successfully established. Figure 1 Transcriptome sequencing analysis of conjunctival tissue and KEGG and GO analyses of differentially expressed genes revealed upregulation of arachidonic acid (AA) and linoleic acid (LA) metabolism in both models. GO analysis also indicated upregulation of fatty acid and linoleic acid metabolism, MHCII receptor activity, and extracellular matrix components in SRW-induced allergic conjunctivitis. In OVA-induced allergic conjunctivitis, fatty acid metabolism, arachidonic acid, and linoleic acid metabolic activities were also upregulated. Figure 1 (FK in the model). By further screening for differentially expressed genes in both models, we found... ALOX15 It is the only gene in the AA and LA metabolic pathways that is consistently and stably upregulated in both AC models. Figure 1 LN in (the context of LN).

[0065] Example 2 ALOX15 Expression verification and functional study in allergic conjunctivitis This embodiment will further verify the expression of ALOX15 in conjunctival tissue of allergic conjunctivitis and verify the function of ALOX15 in regulating lipid peroxidation through in vitro cell experiments. Conjunctival tissues were collected from PBS control group and SRW model mice, RNA was extracted, and the expression of ALOX15 in the conjunctival tissues of each group was detected by real-time quantitative PCR (qPCR). ALOX15The mRNA expression level of ALOX15 was measured, and its protein expression and tissue localization were detected by Western blot (WB) or immunofluorescence (IF) staining. Simultaneously, an in vitro cell model was constructed to simulate the in vitro allergic conjunctivitis state. Human conjunctival epithelial cells (HconEpic) were stimulated with SRW at a concentration of 50 μg / mL for 48 hours. Subsequently, the lipid ROS levels of the cells were detected using C11-BODIPY (Invitrogen, D3861). The intracellular contents of MDA, GSH, and GSSG were measured using a kit containing malondialdehyde (MDA, Solarbio Life Sciences, BC0025), reduced glutathione (GSH, Solarbio Life Sciences, BC1175), and oxidized glutathione (GSSG, Solarbio Life Sciences, BC1185), and the GSH / GSSG ratio was calculated. To further validate the function of ALOX15, we designed multiple siRNAs targeting ALOX15 (including siALOX15-1, siALOX15-2, siALOX15-3, siALOX15-4, and siALOX15-5, sequences shown in Table 1), transfected them into HconEpic (40 nM), and detected their knockdown target genes. ALOX15 The efficiency of the study was further examined, and its effect on cellular lipid peroxidation levels was investigated.

[0066] Table 1 siRNA sequence information

[0067] The results are as follows Figure 2 As shown, qPCR results indicated that, compared to the PBS control group, SRW-induced conjunctival tissue in mice... ALOX15 The mRNA expression levels were significantly increased ( Figure 2 The results of Western blot (WB) and IF further confirmed that the protein fluorescence intensity of ALOX15 in the conjunctival tissue of the SRW model group mice was significantly stronger than that in the PBS control group, indicating that ALOX15 was upregulated at the lesion site of allergic conjunctivitis. Figure 2 (D in the text). In vitro experimental results showed that after SRW stimulation for 48 hours, the expression level of ALOX15 in HconEpic cells was significantly higher than that in the PBS control group (D in the text). Figure 2The EF in the SRW stimulation group was significantly lower than that in the PBS control group. Meanwhile, compared with the PBS control group, cells in the SRW stimulation group exhibited significant oxidative stress characteristics: significantly increased lipid ROS levels; significantly increased MDA content (the end product of lipid peroxidation); GSH depletion and GSSG accumulation, leading to a significantly decreased GSH / GSSG ratio. These results indicate that allergens can induce lipid peroxidation in conjunctival epithelial cells by upregulating ALOX15, disrupting cellular redox balance. Transfection of HconEpic with ALOX15-targeting siRNA, qPCR, and Western Blot analysis showed that, compared with the control group, siALOX15-2 to siALOX15-5 levels were all reduced to varying degrees. ALOX15 The mRNA and protein expression levels were measured. Among them, siALOX15-5 showed the most significant inhibitory effect, therefore siALOX15-5 was selected as the preferred sequence for subsequent functional verification experiments. Figure 2 GH (Table 1). Functional experiments showed that knocking down ALOX15 expression with the optimized siALOX15-5 significantly reversed SRW-induced cellular oxidative stress damage. Specifically, compared with the SRW model group, the siALOX15-5 treatment group showed significantly reduced lipid ROS levels, decreased MDA content, increased GSH levels, decreased GSSG levels, and a significantly improved GSH / GSSG ratio. Figure 2 (IM in the middle).

[0068] This embodiment demonstrates that ALOX15 is upregulated in both the lesion site of allergic conjunctivitis and in conjunctival epithelial cells stimulated by allergens, and mediates lipid peroxidation damage. Silencing ALOX15 gene expression with specific siRNA can effectively alleviate this pathological process. This indicates that ALOX15 is a key regulatory target for allergic conjunctivitis, and siRNA targeting ALOX15 has the potential to treat this disease, providing experimental evidence for the subsequent development of nucleic acid-based drug formulations in this invention.

[0069] Example 3: Construction and characterization of ROS-responsive LNP delivery siALOX15-5 (rLNP@siALOX15-5) To achieve in vivo delivery of siALOX15-5 and overcome the ocular drug delivery barrier, this embodiment constructs reactive oxygen species (ROS)-responsive lipid nanoparticles for encapsulating siALOX15-5. Given the elevated ROS levels in the conjunctival tissue of SRW-induced allergic conjunctivitis mice (…),… Figure 3In accordance with section AB, we designed and prepared ROS-responsive LNP@siALOX15-5 (rLNP@siALOX15-5). The preparation was performed using microfluidic technology. The lipid composition included ROS-responsive ionizable cationic lipid (306-TK4-4), dioleoylphosphatidylethanolamine (DOPE), cholesterol, and dimyristoylglycerol-polyethylene glycol (DMG-PEG) in a molar ratio of 50:10:38.5:1.5. The lipids were dissolved in anhydrous ethanol to form a homogeneous organic phase; siALOX15-5 was dissolved in 0.1M sodium citrate buffer (pH 4.5) to prepare an aqueous phase with a concentration of 0.2 mg / mL. The organic and aqueous phases were injected into a microfluidic device at a flow rate ratio of 1:3 for self-assembly, forming rLNP@siALOX15-5. The resulting product was dialyzed with DPBS at 4°C for 4 hours to remove residual ethanol, and then concentrated using an ultrafiltration centrifuge tube with a molecular cutoff of 10 kDa. Figure 3 The prepared nanoparticles were characterized as follows: morphology was observed using transmission electron microscopy (TEM), hydration size and zeta potential were detected using dynamic light scattering (DLS), and encapsulation efficiency was detected using an NGS RNA BR kit (ABP Biosciences, FP009). Simultaneously, the function of rLNP@siALOX15-5 was evaluated in vitro: HconEpic cells were pretreated with hydrogen peroxide (H2O2) to simulate an oxidative stress environment, and the uptake efficiency of rLNP@Cy5-siALOX15-5 by cells was detected by flow cytometry. Figure 3 (H in the original text); the mRNA expression level of ALOX15 after 24 hours of incubation with rLNP@siALOX15-5 was detected by qPCR. Finally, the ocular biodistribution of Cy5-siALOX15-5 and rLNP@Cy5-siALOX15-5 after topical ocular administration was evaluated in a mouse model of allergic conjunctivitis using in vivo imaging and immunofluorescence staining of conjunctival tissue (Cy5 labeling position is at the 5' end of the positive strand).

[0070] Characterization results showed that the prepared rLNP@siALOX15-5 exhibited a spherical structure under transmission electron microscopy. Figure 3 The average hydrated particle size, as determined by dynamic light scattering, is approximately 80 nm (D in the figure). Figure 3 The mean Zeta potential is -6.13 mV (E in the equation). Figure 3 The encapsulation efficiency is approximately 86.5% (F in the text). Figure 3 (G in the text). In vitro functional experiments showed that under H2O2-simulated oxidative stress conditions, HconEpic cells significantly improved the uptake efficiency of rLNP@Cy5-siALOX15-5 (G in the text). Figure 3(I) RT-qPCR results showed that after co-incubation with rLNP@siALOX15-5 for 24 hours, cells ALOX15 The mRNA expression level was significantly downregulated ( Figure 3 J in the middle). Ocular biodistribution studies showed that after topical ocular instillation, the initial residence time of free Cy5-siALOX15-5 and rLNP@Cy5-siALOX15-5 on the ocular surface was similar (J in the middle). Figure 3 The K in the text. However, immunofluorescence analysis of conjunctival tissue showed that free Cy5-siALOX15-5 was detectable 6 hours after administration, and the signal disappeared by 48 hours; while rLNP@Cy5-siALOX15-5 significantly prolonged the retention time of siRNA in conjunctival tissue, and a significant fluorescence signal was still detectable 48 hours after administration. Figure 3 L in the middle.

[0071] This embodiment successfully constructed ROS-responsive lipid nanoparticles rLNP@siALOX15-5, which exhibited uniform particle size, high encapsulation efficiency, and effectively responded to oxidative stress and enhanced cellular uptake in vitro, significantly silencing ALOX15 expression. In vivo distribution experiments confirmed that this delivery system significantly prolonged the retention time of siRNA in conjunctival tissue. These results demonstrate that ROS-responsive LNPs can serve as highly efficient siRNA ocular delivery carriers, laying the foundation for subsequent in vivo pharmacodynamic studies.

[0072] Example 4: Evaluation of the therapeutic effect of rLNP@siALOX15-5 on a mouse model of allergic conjunctivitis To evaluate the in vivo efficacy of rLNP@siALOX15-5, this embodiment conducted a treatment experiment in SRW and OVA-induced allergic conjunctivitis mouse models. The model mice were randomly divided into four groups and administered 5 μL of PBS, siALOX15-5, rLNP@siALOX15-5, or the positive control drug tobramycin-dexamethasone eye drops (TobraDex) once daily for three consecutive weeks. Figure 4 A in Figure 5(A) After drug administration, the degree of conjunctival hyperemia, the number of eye-scratching behaviors, and the amount of tear secretion in mice were observed and recorded. Intraocular pressure (IOP) was measured to evaluate the ocular safety of the drug. After euthanasia, conjunctival and corneal tissues were collected, and the levels of immunoglobulin E (IgE, Solarbio Life Sciences, SEKM-0345), histamine (Mibo, ml103940), interleukin-4 (IL-4, Solarbio Life Sciences, SEKM-0005), and interleukin-13 (IL-13, Solarbio Life Sciences, SEKM-0014) in the tissues were measured. At the same time, the levels of MDA and 4-HNE were measured to assess the level of lipid peroxidation.

[0073] Treatment results showed that free siALOX15-5 had no significant effect on improving conjunctival congestion, while rLNP@siALOX15-5 effectively reduced conjunctival congestion, with efficacy comparable to TobraDex. Figure 4 BC in Figure 5 (BC in the middle). Meanwhile, rLNP@siALOX15-5 and TobraDex both significantly inhibited eye-grabbing behavior in mice ( ). Figure 4 (D in the text), and effectively reduced tear secretion ( Figure 4 The molecular detection results showed that the levels of IgE, histamine, IL-4, and IL-13 in the conjunctival tissue of mice treated with rLNP@siALOX15-5 and TobraDex were significantly reduced. Figure 4 FI in Figure 5 In terms of safety evaluation, long-term local ocular administration of TobraDex led to a significant increase in intraocular pressure in mice, while no significant change in intraocular pressure was observed in the rLNP@siALOX15-5 treatment group, indicating that it has good ocular safety. Figure 4 J in Figure 5 Further analysis of oxidative stress indicators showed that rLNP@siALOX15-5 treatment significantly reduced MDA levels in the conjunctiva and cornea of ​​mice (F). Figure 4 KL in the middle), and reduced the production of 4-HNE in the conjunctival tissue ( Figure 4 (MN in the text).

[0074] This embodiment demonstrates that topical administration of rLNP@siALOX15-5 effectively alleviates clinical symptoms in mice with allergic conjunctivitis, reduces local inflammatory factor levels, and inhibits lipid peroxidation damage, with efficacy comparable to the positive control drug TobraDex. rLNP@siALOX15-5 avoids the intraocular pressure elevation side effect caused by glucocorticoids, exhibiting superior safety. These results indicate that rLNP@siALOX15-5 possesses clinical application potential for the treatment of allergic conjunctivitis.

[0075] Example 5: Evaluation of the duration of efficacy of a single dose of rLNP@siALOX15-5 in a mouse model of allergic conjunctivitis. To evaluate the duration of efficacy of a single dose of rLNP@siALOX15-5, this study conducted experiments in a mouse model of SRW-induced allergic conjunctivitis. The mice were randomly divided into four groups and administered a single eye drop of 5 μL PBS, siALOX15-5, rLNP@siALOX15-5, or TobraDex, respectively. The degree of conjunctival hyperemia was observed and recorded at 6, 24, 48, and 72 hours post-administration. Figure 6 (A) Simultaneously, conjunctival tissue was collected at various time points and detected using RT-qPCR. ALOX15 mRNA expression levels, and CD4 , IL-4 , IL-5 and IL-13 Transcription levels of Th2-type immune-related factors.

[0076] The duration of efficacy observation showed that a single administration of free siALOX15-5 had no significant effect on improving conjunctival congestion. TobraDex showed a slight inhibitory effect 6 hours after administration, which lasted until 24 hours, but had no significant effect between 48 and 72 hours. In contrast, rLNP@siALOX15-5 began to significantly reduce conjunctival congestion 24 hours after administration, and this effect lasted until 48 hours, and essentially disappeared by 72 hours. Figure 6 (BC in the middle). Molecular detection results showed that a single administration of rLNP@siALOX15-5 could effectively inhibit BC in conjunctival tissue. ALOX15 The expression was significantly downregulated. CD4 , IL-4 , IL-5 and IL-13 transcription level ( Figure 6 (DH in the middle).

[0077] This embodiment demonstrates that, compared to the positive control drug TobraDex, rLNP@siALOX15-5 has a slightly later onset of action after a single topical ocular administration, but its therapeutic duration is significantly longer, lasting up to 48 hours post-administration. Simultaneously, a single dose effectively inhibits the expression of the target gene and downstream inflammatory factors. These results indicate that rLNP@siALOX15-5 possesses the advantage of long-lasting action, potentially reducing the frequency of clinical dosing and improving patient compliance.

[0078] Example 6: Transcriptomic mechanism analysis of rLNP@siALOX15-5 in the treatment of allergic conjunctivitis To investigate the molecular mechanism of rLNP@siALOX15-5 in treating allergic conjunctivitis, this study performed transcriptome sequencing analysis on conjunctival tissue samples after repeated drug administration. SRW-induced allergic conjunctivitis mice were repeatedly treated with different treatments (PBS, free siALOX15-5, rLNP@siALOX15-5, or TobraDex), and conjunctival tissue was collected for RNA-seq. Differential gene expression analysis and KEGG and GO enrichment analyses were performed. Subsequently, based on the enrichment results, key genes related to inflammation, oxidative stress, and lipid peroxidation were screened for expression pattern analysis, and single-sample gene set enrichment analysis (ssGSEA) was used to assess changes in the activity of related pathways in each group of samples.

[0079] Transcriptome sequencing results showed that different treatment groups exhibited significantly different transcriptome characteristics. Specifically, the rLNP@siALOX15-5 treatment group showed significant enrichment of downregulated genes in multiple inflammation-related signaling pathways and AA metabolic pathways; while upregulated genes were enriched in the PPAR signaling pathway, AMPK signaling pathway, unsaturated fatty acid biosynthesis, and peroxisome pathway. Figure 7 A in the text). GO analysis further confirmed that the biological processes downregulated by rLNP@siALOX15-5 treatment involve the positive regulation of inflammatory responses, collagen-containing extracellular matrix, and cytokine activity; the upregulated processes involve cellular responses to hydrogen peroxide, peroxisome matrix, and NAD binding (…). Figure 7 Based on pathway enrichment results, the expression changes of key genes related to inflammation and oxidative stress were further analyzed. The results showed that both rLNP@siALOX15-5 and TobraDex could inhibit the expression of inflammation-related genes, while free siALOX15-5 had a weaker effect. Both rLNP@siALOX15-5 and TobraDex treatments downregulated upstream alarmins (…). Il1b, Il33, Tslp, Il36a ), multiple chemokines and their receptors, and inflammatory mediators S100a8 and S100a9 The expression ( Figure 8In addition, both rLNP@siALOX15-5 and TobraDex can inhibit ROS generation-related genes (A). Cybb、 Duox1, Duox2 The expression of ) and upregulation of antioxidant-related genes ( Cat, Gclc, Gpx4, Sod1 The expression of () Figure 8 (B in the text). Notably, rLNP@siALOX15-5 can specifically inhibit the key lipid peroxidase (B). Alox15, Alox12 Alox12e ) and its regulatory enzymes ( Fads1, Cyp4a32, Cyp4f18 The expression of ) was observed, while no effect was observed in TobraDex treatment. Figure 8 (C in the text). Pathway activity analysis based on ssGSEA further confirmed that both rLNP@siALOX15-5 and TobraDex can effectively reduce the activity of nuclear factor κB (NF-κB) and Toll-like receptor (TLR)-related inflammatory signaling pathways, while enhancing the activity of peroxisome and KEAP1-NRF2 antioxidant pathways. Furthermore, rLNP@siALOX15-5 can also significantly reduce the activity of lipid peroxidation-related pathways such as AA and LA metabolism (C in the text). Figure 8 (D in the middle).

[0080] This embodiment reveals through transcriptomic analysis that rLNP@siALOX15-5 exerts its therapeutic effect through multidimensional regulation: on the one hand, it inhibits inflammatory signaling pathways and the expression of inflammatory factors; on the other hand, it activates the antioxidant defense system and has a unique role in inhibiting lipid peroxidation metabolic pathways. Compared with TobraDex, rLNP@siALOX15-5 has the additional advantage of regulating lipid peroxidation on the basis of anti-inflammatory effects, providing a mechanistic explanation for its treatment of allergic conjunctivitis and related oxidative stress damage.

[0081] Example 7: Evaluation of the therapeutic effect of rLNP@siALOX15-5 on a mouse model of dry eye. Given that previous studies have reported dry eye disease in mouse models ALOX15 and MDA levels were all revised upwards ( Figure 9 (A) In this embodiment, the therapeutic potential of rLNP@siALOX15-5 was further evaluated in a mouse model of dry eye induced by 0.2% benzalkonium chloride (BAC, Sigma, 12060). A mouse model of dry eye was established using 6-8 week old C57BL / 6J mice. 5 μL of 0.2% BAC was administered twice daily for 7 days. The mice were then randomly divided into four groups and administered PBS, free siALOX15-5, rLNP@siALOX15-5, or the positive control drug polyvinyl alcohol eye drops (PVA) for two consecutive weeks. Figure 9(B) After drug administration, the degree of corneal epithelial damage was assessed using sodium fluorescein staining, and tear secretion was measured using the phenol red cotton thread method. Corneal and conjunctival tissues were collected, and corneal epithelial thickness was measured using hematoxylin and eosin (H&E) staining, while the number of conjunctival goblet cells was measured using periodic acid-Schiff (PAS) staining. Simultaneously, the levels of MDA and 4-HNE in the tissues were measured to assess lipid peroxidation levels.

[0082] Treatment results showed that, compared with the PBS group and the free siALOX15-5 group, rLNP@siALOX15-5 significantly reduced the corneal fluorescein staining score ( Figure 9 CD), and increases tear secretion, with effects comparable to PVA ( Figure 9 E). H&E staining showed that both rLNP@siALOX15-5 and PVA could effectively restore corneal epithelial thickness, indicating their role in promoting corneal epithelial repair. Figure 9 F in the image). PAS staining further showed that rLNP@siALOX15-5 significantly inhibited the reduction of conjunctival goblet cells, which helps maintain ocular surface barrier function. Figure 9 GH in the middle). Oxidative stress index detection showed that rLNP@siALOX15-5 treatment significantly reduced the levels of MDA and 4-HNE in conjunctival and corneal tissues ( ). Figure 9 (IL in the middle).

[0083] This embodiment confirms that rLNP@siALOX15-5 effectively reduces corneal epithelial damage, promotes tear secretion, restores corneal epithelial thickness, and protects conjunctival goblet cells in a mouse model of dry eye, while inhibiting lipid peroxidation damage. Its efficacy is comparable to that of the positive control drug, polyvinyl alcohol eye drops. These results indicate that rLNP@siALOX15-5 is not only effective for allergic conjunctivitis but also simultaneously relieves dry eye symptoms, demonstrating the potential to treat both comorbidities, providing further experimental evidence for its clinical application.

[0084] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A double-stranded oligonucleotide targeting ALOX15 mRNA, characterized in that, The double-stranded oligonucleotide comprises a sense strand and an antisense strand; The positive strand nucleotide sequence comprises a sequence that differs from the sequence shown in SEQ ID NO. 1, 3, 5 or 7 by no more than 8 nucleotides; The antisense strand nucleotide sequence comprises a sequence that differs from the sequence shown in SEQ ID NO. 2, 4, 6 or 8 by no more than 8 nucleotides; Furthermore, the justice chain and the antisense chain are at least partially complementary in opposite directions, forming a bichain region.

2. The double-stranded oligonucleotide targeting ALOX15 mRNA according to claim 1, characterized in that, The sense strand nucleotide sequence contains a sequence that differs from the sequence shown in SEQ ID NO.7 by no more than 8 nucleotides, and the antisense strand nucleotide sequence contains a sequence that differs from the sequence shown in SEQ ID NO.8 by no more than 8 nucleotides. Optionally, the sense strand nucleotide sequence is the sequence shown in SEQ ID NO.7, and the antisense strand nucleotide sequence is the sequence shown in SEQ ID NO.

8.

3. The double-stranded oligonucleotide targeting ALOX15 mRNA according to claim 1, characterized in that, At least one internucleotide bond is a modified internucleotide bond; Optionally, at least one nucleoside inter-bond is a phosphate thioside inter-bond; Optionally, the first to third internucleotide bonds, counting from the 5' end of the positive chain, are thiophosphate internucleotide bonds; and / or, the first to third internucleotide bonds, counting from the 3' end of the positive chain, are thiophosphate internucleotide bonds; and / or, the first to third internucleotide bonds, counting from the 5' end of the antisense chain, are thiophosphate internucleotide bonds; and / or, the first to third internucleotide bonds, counting from the 3' end of the antisense chain, are thiophosphate internucleotide bonds.

4. The double-stranded oligonucleotide targeting ALOX15 mRNA according to any one of claims 1 to 3, characterized in that, The double-stranded oligonucleotide contains at least one modified nucleotide; Optionally, the modified nucleotides include one or more of the following: 2'-O-methoxyethyl modified nucleotides, 5'-methyl modified nucleotides, 2'-(S)-restricted ethyl modified nucleotides, 2'-O-methyl modified nucleotides, 2'-deoxy-2'-fluorinated modified nucleotides, 2'-deoxy-modified nucleotides, locked nucleotides, non-locked nucleotides, configuration-restricted nucleotides, restricted ethyl nucleotides, base-free nucleotides, 2'-amino-modified nucleotides, 2'-O-allyl-modified nucleotides, 2'-C-alkyl-modified nucleotides, 2'-hydroxy-modified nucleotides, 2'-O-alkyl-modified nucleotides, morpholinyl nucleotides, bridging nucleic acids, peptide nucleic acids, locked nucleic acids, and non-locked nucleic acids. Optionally, the modified nucleotide includes at least one of 2'-F modified nucleotides and 2'-O-methylated modified nucleotides; Optionally, the 5' end of the antisense chain is modified with 5'-(E)-vinyl phosphate.

5. The double-stranded oligonucleotide targeting ALOX15 mRNA according to claim 4, characterized in that, The double-stranded oligonucleotide is modified with DV18, wherein the DV18 modification includes: from the 5' to 3' direction, the nucleotides at positions 1-6, 8, and 12-21 of the sense strand are 2'-O-methylated nucleotides, and the nucleotides at positions 7 and 9-11 are 2'-deoxy-2'-fluorinated nucleotides; from the 5' to 3' direction, the nucleotides at positions 1, 3-5, 7, 10-13, 15, and 17-21 of the antisense strand are 2'-O-methylated nucleotides, and the nucleotides at positions 2, 6, 8, 9, 14, and 16 are 2'-deoxy-2'-fluorinated nucleotides; further optionally, the 5' end of the antisense strand is modified with 5'-(E)-vinyl phosphate; Alternatively, the double-stranded oligonucleotide is modified with DV22, wherein the DV22 modification comprises: from the 5' to 3' direction, the nucleotides at positions 1-6, 8, and 12-21 of the sense strand are 2'-O-methylated nucleotides, and the nucleotides at positions 7 and 9-12 are 2'-deoxy-2'-fluorinated nucleotides; from the 5' to 3' direction, the nucleotides at positions 1, 3-5, 7-13, 15, and 17-21 of the antisense strand are 2'-O-methylated nucleotides, and the nucleotides at positions 2, 6, 14, and 16 are 2'-deoxy-2'-fluorinated nucleotides; further optionally, the 5' end of the antisense strand is modified with 5'-(E)-vinyl phosphate; Alternatively, the double-stranded oligonucleotide is ESC modified, wherein the ESC modification includes: from the 5' to 3' direction, the nucleotides at positions 2, 4, 6, 8, 12, 14, 16, 18, and 20 of the sense strand are 2'-O-methylated nucleotides, and the nucleotides at positions 1, 3, 5, 7, 9-11, 13, 15, 17, 19, and 21 of the antisense strand are 2'-deoxy-2'-fluorinated nucleotides; from the 5' to 3' direction, the nucleotides at positions 1, 3, 5, 7, 9, 11-13, 15, 17, 19, and 21 of the antisense strand are 2'-O-methylated nucleotides, and the nucleotides at positions 2, 4, 6, 8, 10, 14, 16, 18, and 20 of the antisense strand are 2'-deoxy-2'-fluorinated nucleotides; further optionally, the 5' end of the antisense strand is modified with 5'-(E)-vinyl phosphate; Optionally, the double-stranded oligonucleotide is siRNA.

6. A complex of double-stranded oligonucleotides targeting ALOX15 mRNA, characterized in that, The complex contains: (a) the double-stranded oligonucleotide targeting ALOX15 mRNA as described in any one of claims 1 to 5; and (b) one or more targeted delivery ligands linked to (a).

7. The double-stranded oligonucleotide targeting ALOX15 mRNA according to any one of claims 1 to 5, or the complex according to claim 6, used in any one of the following: (I) Inhibition of ALOX15 mRNA and / or reduction of ALOX15 protein expression in subjects for non-diagnostic and non-therapeutic purposes; (II) Prepare drugs that inhibit ALOX15 mRNA and / or reduce ALOX15 protein expression in subjects; (III) Prepare medicines for treating, preventing and / or alleviating pathological conditions or diseases caused by ALOX15 in the subject.

8. The application according to claim 7, characterized in that, The pathological conditions or diseases caused by subject ALOX15 include allergic conjunctivitis or dry eye disease.

9. A pharmaceutical composition, characterized in that, Includes the double-stranded oligonucleotide targeting ALOX15 mRNA as described in any one of claims 1 to 5, or the complex as described in claim 6; Optionally, the pharmaceutical composition further includes lipid nanoparticles; the double-stranded oligonucleotide targeting ALOX15 mRNA is loaded onto the lipid nanoparticles; Optionally, the lipid nanoparticles include ROS-responsive ionizable cationic lipids, dioleoylphosphatidylethanolamine, cholesterol, and dimyristoylglycerol-polyethylene glycol.

10. A method for inhibiting ALOX15 in a subject for non-diagnostic and non-therapeutic purposes, characterized in that, This includes contacting the subject with the double-stranded oligonucleotide targeting ALOX15 mRNA as described in any one of claims 1 to 5, or the complex as described in claim 6, or the pharmaceutical composition as described in claim 9.

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