Double-stranded oligonucleotides targeting arg1 and conjugates, pharmaceutical compositions thereof and their use in pulmonary fibrosis

Abstract

This application relates to the field of biotechnology, and particularly to Arg1-targeting double-stranded oligonucleotides and their conjugates, pharmaceutical compositions, and their application in pulmonary fibrosis. The Arg1-targeting double-stranded oligonucleotide comprises a sense strand and an antisense strand, wherein the sense strand and antisense strand are at least partially anticomplementary to form a double-stranded region; the nucleotide sequence of the sense strand comprises a sequence differing from the sequence shown in SEQ ID NO.1 by no more than 3 nucleotides, and the nucleotide sequence of the antisense strand comprises a sequence differing from the sequence shown in SEQ ID NO.2 by no more than 3 nucleotides. Using this Arg1-targeting double-stranded oligonucleotide and its conjugates, or pharmaceutical compositions containing it, to reprogram the nucleic acid of M2 macrophages in pulmonary fibrosis can effectively alter their pro-fibrotic functional state.

Description

Technical Field

[0001] This application relates to the field of biotechnology, and in particular to double-stranded oligonucleotides targeting Arg1 and their conjugates, pharmaceutical compositions and their application in pulmonary fibrosis. Background Technology

[0002] Pulmonary fibrosis is a fatal disease characterized by progressive fibrotic remodeling of the lung interstitium, with idiopathic pulmonary fibrosis (IPF) being a typical example. Existing drugs mainly work by inhibiting fibroblast activation or collagen deposition, but their efficacy is limited and they cannot effectively reverse the disease progression.

[0003] Currently, the main drugs for treating pulmonary fibrosis are pirfenidone and nintedanib, both of which work by inhibiting fibroblast proliferation, reducing extracellular matrix deposition, and blocking fibrosis signaling pathways, thus slowing the decline in lung function to some extent. However, pirfenidone and nintedanib have the following limitations: existing drugs can only slow disease progression but cannot stop or reverse existing pulmonary fibrosis; both pirfenidone and nintedanib have serious side effects. Pirfenidone commonly causes photosensitive rashes, gastrointestinal reactions, and potential hepatotoxicity; nintedanib is most notably associated with diarrhea, often accompanied by decreased appetite, weight loss, and elevated liver enzymes.

[0004] Arg1 (Arginase-1) is a key metabolic enzyme in the urea cycle, catalyzing the hydrolysis of L-arginine to L-ornithine and urea. Ornithine, as a substrate, is converted to proline via OAT (ornithine-pyruvate transaminase), directly supplying fibroblasts for collagen synthesis. Arg1 is a crucial node linking M2 macrophage polarization and metabolic reprogramming in pulmonary fibrosis.

[0005] RNA interference (RNAi) is a highly conserved gene silencing mechanism triggered by double-stranded RNA (dsRNA). It achieves precise regulation of gene expression by specifically degrading target mRNA or inhibiting its translation.

[0006] Therefore, providing a double-stranded oligonucleotide to inhibit Arg1 gene expression is of great significance for developing drugs to alleviate, prevent and / or treat pulmonary fibrosis. Summary of the Invention

[0007] Therefore, it is necessary to provide a double-stranded oligonucleotide targeting Arg1 and its conjugates, a pharmaceutical composition and its application in pulmonary fibrosis.

[0008] In one aspect, a double-stranded oligonucleotide targeting Arg1 is provided, the double-stranded oligonucleotide comprising a sense strand and an antisense strand;

[0009] The nucleotide sequence of the sense strand comprises a sequence that differs from the sequence shown in SEQ ID NO.1 by no more than 3 nucleotides, and the nucleotide sequence of the antisense strand comprises a sequence that differs from the sequence shown in SEQ ID NO.2 by no more than 3 nucleotides.

[0010] Furthermore, the justice chain and the antisense chain are at least partially inversely complementary to form a bichain region.

[0011] In an optional embodiment, the double-stranded oligonucleotide further conforms to at least one of (i) to (vi):

[0012] (i) At least one nucleoside internucleotide bond in the double-stranded oligonucleotide is a modified nucleoside internucleotide bond;

[0013] (ii) The double-stranded oligonucleotide contains at least one modified nucleotide;

[0014] (iii) The nucleotides of the double-stranded oligonucleotides are all unmodified;

[0015] (iv) The double-stranded oligonucleotide contains at least one nucleotide analog;

[0016] (v) The double-stranded oligonucleotide contains at least one 2'-deoxynucleotide; and,

[0017] (vi) The justice chain and the antisense chain each have an independent protruding end.

[0018] In optional embodiments, the modified nucleotides include one or more of the following: 2'-O-methoxyethyl modified nucleotides, 5-methyl modified nucleotides, 2'-O-methyl modified nucleotides, 2-fluorine modified nucleotides, 2'-amino modified nucleotides, 2'-C-alkyl modified nucleotides, 2'-O-alkyl modified nucleotides, bridging nucleic acids, locked nucleic acids, conformationally restricted ethyl modified nucleotides, and base-deficient modified nucleotides.

[0019] In an optional embodiment, the nucleic acid analogue includes at least one of morpholinonucleotide and peptide nucleic acid.

[0020] In an optional implementation, the protrusions of the sense strand and the antisense strand each independently contain 1 to 3 nucleotides.

[0021] In an optional implementation, the protruding ends of the justice chain and the antisense chain are each located independently at the 3' end.

[0022] In an optional embodiment, the nucleotides in the protruding ends of the sense strand and the antisense strand are each independently 2'-deoxynucleotides.

[0023] In an optional embodiment, the double-stranded oligonucleotide is siRNA, the nucleotide sequence of the sense strand is shown in SEQ ID NO.3, and the nucleotide sequence of the antisense strand is shown in SEQ ID NO.4; and the 2'-deoxynucleotides at the 3' end of both the sense strand and the antisense strand are overhangs.

[0024] In a second aspect, a conjugate targeting Arg1 is provided, the conjugate comprising (a) the Arg1-targeting double-stranded oligonucleotide of the first aspect; and (b) one or more targeting delivery ligands linked to (a).

[0025] Thirdly, a pharmaceutical composition is provided, the pharmaceutical composition comprising (I) and (II):

[0026] (I) the double-stranded oligonucleotide targeting Arg1 as described in the first aspect; or the conjugate targeting Arg1 as described in the second aspect; and,

[0027] (II) Delivery carrier.

[0028] In an optional embodiment, the delivery carrier includes at least one of the following: lipid nanoparticles, liposomes, cationic lipids, cationic polymers, metal nanopolymers, nanorods, micelles, microvesicles, cell-penetrating peptides, viral particles, protein coats, and liposomes.

[0029] In an optional implementation, the delivery vector targets M2 macrophages.

[0030] In an optional embodiment, the delivery carrier comprises mannose-modified lipid nanoparticles.

[0031] In an optional embodiment, the lipid component of the lipid nanoparticles includes ionizable lipids, phospholipids, structural lipids, and lipid-polyethylene glycol-mannose conjugates.

[0032] In an optional embodiment, the mannose-modified lipid nanoparticles satisfy one or more of the following conditions:

[0033] The ionizable lipids include at least one of DLin-MC3-DMA, SM-102 and ALC-0315;

[0034] The phospholipids include at least one of DOPE, DSPC, DLPC, DMPC, DOPC, DPPC, DUPC, POPC, DOPG, DPPG, DPPE, DMPE, DSPE, and DOTAP;

[0035] The structural lipids include at least one of cholesterol, coccosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassosterol, and α-tocopherol;

[0036] The lipids in the lipid-polyethylene glycol-mannose conjugate include at least one of DSPE, DMG, ceramide, and DMPE.

[0037] In an optional embodiment, the lipid component includes DLin-MC3-DMA, cholesterol, DOPE, and DSPE-PEG2000-mannose.

[0038] In an optional embodiment, the dosage form of the pharmaceutical composition includes a dosage form suitable for pulmonary administration.

[0039] In optional embodiments, the dosage forms suitable for pulmonary administration include aerosols, dry powder inhalers, liquid formulations for nebulized inhalation, and soft fog inhalers.

[0040] Fourthly, the use of the Arg1-targeting double-stranded oligonucleotide described in the first aspect, the Arg1-targeting conjugate described in the second aspect, or the pharmaceutical composition described in the third aspect in the preparation of products for treating, preventing, or alleviating pulmonary fibrosis is provided.

[0041] The Arg1-targeting double-stranded oligonucleotides provided in this application have shown in both in vitro and in vivo experiments to knock down Arg1 expression, reduce the number of M2 macrophages, and alleviate pulmonary fibrosis in subjects. This application utilizes Arg1-targeting double-stranded oligonucleotides that reprogram the nucleic acid of M2 macrophages in pulmonary fibrosis to regulate the metabolic and functional state of pro-fibrotic M2 macrophages in pulmonary fibrosis. This effectively alters their pro-fibrotic functional state, and by regulating the functional state of M2 macrophages, improves the pulmonary immune microenvironment, transforming them from a pro-fibrotic state to an anti-fibrotic state, thereby effectively reducing the occurrence and development of pulmonary fibrosis. Attached Figure Description

[0042] To more clearly illustrate the technical solutions in the embodiments and examples of this application, and to more completely understand this application and its beneficial effects, the accompanying drawings used in the description of the embodiments or examples will be briefly introduced below. Obviously, the drawings described below are merely some embodiments of this application. Those skilled in the art can obtain other drawings based on these drawings without any creative effort.

[0043] Figure 1The results of flow cytometry in Example 2 are shown in (A) and the statistical analysis of the proportion of mCherry-positive cells in macrophages and other immune cells is shown in (B). The statistical method used was Student's t test, and p < 0.0001.

[0044] Figure 2 The relative expression level of Arg1 mRNA in RAW264.7 cells induced to polarize to the M2 phenotype after in vitro delivery of siArg1 by LNP-Mannose in Example 3 was calculated using One-Way ANOVA. *p < 0.05, **p < 0.01.

[0045] Figure 3 The relative expression level of Arg1 mRNA in BMDM cells induced to polarize to the M2 phenotype after in vitro delivery of siArg1 by LNP-Mannose in Example 4 was statistically analyzed using Student's t-test, with ***p < 0.001.

[0046] Figure 4 The percentage of M2 macrophages in the lungs after LNP-Mannose delivered siArg1 in vivo in Example 4 was calculated using Student's t-test, with *p < 0.05.

[0047] Figure 5 The images show the results of Masson staining of lung tissue after LNP-Mannose delivered siArg1 in vivo in Example 4. The left image shows LNP-Mannose@siCtrl, and the right image shows LNP-Mannose@siArg1. Detailed Implementation

[0048] The present application will be further described in detail below with reference to the accompanying drawings, embodiments, and examples. It should be understood that these embodiments and examples are for illustrative purposes only and are not intended to limit the scope of the present application. The purpose of providing these embodiments and examples is to enable a more thorough and comprehensive understanding of the disclosure of the present application. It should also be understood that the present application can be implemented in many different forms and is not limited to the embodiments and examples described herein. Those skilled in the art can make various modifications or alterations without departing from the spirit of the present application, and the equivalent forms obtained also fall within the protection scope of the present application. For example, features described or illustrated as part of one embodiment can be combined in a suitable manner in another embodiment to produce new embodiments. Furthermore, numerous details are set forth in the following description to provide a fuller understanding of the present application. It should be understood that the present application can be implemented without one or more of these details.

[0049] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used herein in the specification of this application is for descriptive purposes only and is not intended to be limiting of the application.

[0050] Unless otherwise stated or in case of contradiction, the terms or phrases used herein shall have the following meanings:

[0051] The terms “and / or,” “or / and,” and “and / or” as used herein include any one of two or more of the related listed items, as well as any and all combinations of the related listed items. “Any and all combinations” includes any two related listed items, any more related listed items, or a combination of all related listed items. For example, “A and / or B” includes three parallel options: A, B, and “a combination of A and B.”

[0052] In this application, the terms "multiple", "various", "multiple times", "several", "several", etc., unless otherwise specified, refer to a quantity greater than or equal to 2. For example, "one or more" means one or more or more.

[0053] In this application, "optionally", "optional", and "optional" mean that something is optional, that is, it means that it is selected from either "with" or "without".

[0054] In this application, the terms "separately independent", "each...independently selected", "...independently selected", and "...independently selected" are interchangeable and should be interpreted broadly. They mean that in a class of objects, the specific characteristics of each specific object do not affect each other. In this class of objects, the specific characteristics of any two objects can be the same or different.

[0055] In this application, the technical features or solutions described in open-ended language include both closed-ended technical features or solutions consisting of the listed contents and open-ended technical features or solutions that include the listed contents.

[0056] In this application, where the method flow involves multiple steps, unless otherwise explicitly stated herein, there is no strict order restriction on the execution of these steps; they can be executed in any order other than those described. Moreover, any step may include multiple sub-steps or multiple stages, which are not necessarily completed at the same time, but can be executed at different times, and their execution order is not necessarily sequential, but can be performed alternately or simultaneously with other steps or parts of the sub-steps or stages of other steps.

[0057] In this application, the terms "first aspect," "second aspect," "third aspect," "fourth aspect," etc., are used for descriptive purposes only and should not be construed as indicating or implying relative importance or quantity, nor should they be construed as implicitly indicating the importance or quantity of the indicated technical features. Moreover, "first," "second," "third," "fourth," etc., serve only as a non-exhaustive enumeration and should be understood not to constitute a closed limitation on quantity.

[0058] In this application, the term "double-stranded oligonucleotide" refers to a double-stranded structure formed by two oligonucleotides through partial or complete base pairing, wherein the two oligonucleotides include a sense strand and an antisense strand, and the lengths of the sense strand and the antisense strand may be the same or different. Double-stranded oligonucleotides include ribonucleotides, deoxyribonucleotides, or nucleotides containing purine and pyrimidine bases or other natural, chemically or biochemically modified, non-natural or derived nucleotides.

[0059] In this application, 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).

[0060] In this application, the term "nucleic acid molecule" refers to a polymeric form of nucleotides of any length, including ribonucleotides and / or deoxyribonucleotides. Examples of nucleic acid molecules include, but are not limited to, single-stranded, double-stranded, or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or polymers containing purine and pyrimidine bases or other naturally occurring, chemically or biochemically modified, non-natural, or derived nucleotide bases. When a nucleic acid molecule encodes a protein or polypeptide, it may optionally encode the sense or antisense strand. Nucleic acid molecules can be naturally occurring, synthetic, recombinant, or any combination thereof. The terms "nucleic acid molecule," "nucleic acid," and "polynucleotide" are used interchangeably.

[0061] In this application, the overhang of a double-stranded oligonucleotide refers to a single-stranded nucleotide extension formed when the end of one strand of the double-stranded oligonucleotide extends beyond the pairing region of the other complementary strand. The formed single-stranded nucleotide extension may be located at one or both of the sense and antisense strands; the formed single-stranded nucleotide extension may be located at at least one of the 5' end of the sense strand, the 3' end of the sense strand, the 5' end of the antisense strand, and the 3' end of the antisense strand.

[0062] In this application, the term "lipid-polyethylene glycol-mannose conjugate" refers to a three-component chimeric functional molecule, a material composed of lipids, polyethylene glycol (PEG), and mannose sequentially linked by covalent bonds, with PEG serving as a spacer arm, and lipids and mannose located at opposite ends of the PEG. Each PEG may have at least one mannose grafted to one end, for example, but not limited to, at least one, at least two, at least three, or at least four mannose grafts. When at least two mannose grafts are attached to one end of each PEG, the multiple mannose grafts may optionally be linked to different sites on the PEG. In an optional embodiment, the PEG serving as the spacer arm is selected from PEG2000 (polyethylene glycol with a number average molecular weight of 2000 Da).

[0063] In this application, the terms "subject" or "patient" refer to a mammalian subject or patient, and organs, tissues, or cells derived from them. Mammals include, but are not limited to, mice, humans, rats, guinea pigs, rabbits, dogs, cats, hamsters, nude mice, ferrets, pigs, sheep, goats, cattle, horses, donkeys, dogs, cynomolgus monkeys, rhesus monkeys, macaques, baboons, gibbons, golden monkeys, long-tailed macaques, pig-tailed macaques, or chimpanzees. In some embodiments, the subject is a mouse or a human.

[0064] In this application, the terms “treatment,” “relief,” or “improvement” are used interchangeably. These terms refer to methods of achieving beneficial or desired results, including but not limited to treatment benefits. “Treatment benefit” means the eradication or improvement of one or more diseases, conditions, or circumstances associated with the underlying barrier being treated.

[0065] In this application, the terms “prevention” and “avoidance” are used interchangeably to refer to methods for obtaining a beneficial or desired outcome, 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.

[0066] Firstly, some embodiments provide a double-stranded oligonucleotide targeting Arg1. Lung macrophages, particularly M2-type (alternative activation) macrophages, abnormally activate and maintain a pro-fibrotic functional state, playing a crucial role in the occurrence and maintenance of pulmonary fibrosis, as well as driving its development and progression. M2 macrophages promote fibroblast activation, collagen deposition, and tissue remodeling by remodeling the arginine metabolic pathway. Simultaneously, M2 macrophages lack pro-inflammatory and immune clearance functions, and their abnormal accumulation constitutes an important immune basis for pulmonary fibrosis. Arg1 is a key node connecting M2 macrophage polarization and metabolic reprogramming in pulmonary fibrosis. Reducing Arg1 expression levels can transform macrophages from a pro-fibrotic M2 phenotype to an anti-fibrotic functional state, thereby mitigating the occurrence and development of pulmonary fibrosis.

[0067] This Arg1-targeting double-stranded oligonucleotide contains a sense strand and an antisense strand, and the sense strand and antisense strand are at least partially anticomplementary to form a double-stranded region:

[0068] The nucleotide sequence of the sense strand contains a sequence that differs from the sequence shown in SEQ ID NO.1 by no more than 1, 2, or 3 nucleotides; the nucleotide sequence of the antisense strand contains a sequence that differs from the sequence shown in SEQ ID NO.2 by no more than 1, 2, or 3 nucleotides.

[0069] In an optional implementation, the differentially expressed nucleotides in the positive strand from the sequence shown in SEQ ID NO.1 are each independently mutated from one or more sites.

[0070] In an optional implementation, the differentially expressed nucleotides in the antisense strand from the sequence shown in SEQ ID NO.2 are each independently mutated from one or more sites.

[0071] In an optional implementation, the double-stranded oligonucleotide also meets at least one of (i) to (vi):

[0072] (i) At least one nucleoside bond in a double-stranded oligonucleotide is a modified nucleoside bond.

[0073] In an optional embodiment, at least one nucleoside inter-bond is a phosphate thioside inter-bond.

[0074] In an optional embodiment, at least one of the first to third internucleotide bonds at the 5' end of the positive chain is a phosphate thioester internucleotide bond.

[0075] In an optional embodiment, at least one of the first to third nucleoside interbonds at the 3' end of the positive chain is a phosphate thioate nucleoside interbond.

[0076] In an optional embodiment, at least one of the first to third internucleotide bonds at the 5' end of the antisense chain is a phosphate thioester internucleotide bond.

[0077] In an optional embodiment, at least one of the first to third nucleoside interbonds at the 3' end of the antisense chain is a phosphate thioate nucleoside interbond.

[0078] (ii) Double-stranded oligonucleotides contain at least one modified nucleotide.

[0079] In optional embodiments, the modified nucleotides include one or more of the following: 2'-O-methoxyethyl modified nucleotides, 5-methyl modified nucleotides, 2'-O-methyl modified nucleotides, 2-fluorine modified nucleotides, 2'-amino modified nucleotides, 2'-C-alkyl modified nucleotides, 2'-O-alkyl modified nucleotides, bridged nucleic acids (BNA), locked nucleic acids (LNA), conformation-restricted ethyl modified nucleotides (cEt), and base-deficient modified nucleotides.

[0080] (iii) The nucleotides in the double-stranded oligonucleotides are all unmodified.

[0081] (iv) The double-stranded oligonucleotide contains at least one nucleotide analog.

[0082] In an optional implementation, the nucleic acid analogue includes at least one of morpholinonucleotide and peptide nucleic acid.

[0083] (v) The double-stranded oligonucleotide contains at least one 2'-deoxynucleotide.

[0084] And, (vi) the justice chain and the antithesis chain each have independent protruding ends.

[0085] In an alternative implementation, the protruding end of the positive chain contains 1, 2, or 3 nucleotides.

[0086] In an alternative implementation, the overhang of the antisense strand contains 1, 2, or 3 nucleotides.

[0087] In an alternative implementation, the protruding end of the justice chain is located at the 3' end.

[0088] In an optional implementation, the protruding end of the antisense chain is located at the 3' end.

[0089] In an optional implementation, the protruding ends of both the justice chain and the antisense chain are located at the 3' end.

[0090] In an optional implementation, the nucleotide at the overhang of the positive strand is a 2'-deoxynucleotide.

[0091] In an optional implementation, the nucleotide at the overhang of the antisense strand is a 2'-deoxynucleotide.

[0092] In an optional embodiment, the double-stranded oligonucleotide is siRNA, the nucleotide sequence of the sense strand is shown in SEQ ID NO.3, and the nucleotide sequence of the antisense strand is shown in SEQ ID NO.4; and the 2'-deoxynucleotides at the 3' end of both the sense and antisense strands are overhangs.

[0093] In a second aspect, some embodiments provide a conjugate targeting Arg1 comprising (a) a double-stranded oligonucleotide targeting Arg1 as described in the first aspect; and (b) one or more targeted delivery ligands linked to (a).

[0094] In alternative implementations, the targeted delivery ligand may be, for example, but not limited to, a nucleic acid aptamer, a targeted peptide, or a compound drug.

[0095] Thirdly, some embodiments provide a pharmaceutical composition comprising (I) and (II):

[0096] (I) a double-stranded oligonucleotide targeting Arg1 in the first aspect; or a conjugate targeting Arg1 in the second aspect; and,

[0097] (II) Delivery carrier.

[0098] In an optional implementation, the delivery vector targets M2 macrophages. Using a delivery vector targeting M2 macrophages, double-stranded oligonucleotides or their conjugates targeting Arg1 can be precisely delivered to M2 macrophages, achieving efficient and selective action of the active ingredient in lung M2 macrophages. This improves the efficiency of targeted regulation while reducing non-specific effects on other immune cells, thereby obtaining stable and reproducible anti-pulmonary fibrosis effects.

[0099] In optional embodiments, the delivery carrier includes at least one of the following: lipid nanoparticles, liposomes, cationic lipids, cationic polymers, metal nanopolymers, nanorods, micelles, microvesicles, cell-penetrating peptides, viral particles, protein coats, and liposomes.

[0100] In an optional embodiment, the delivery carrier includes mannose-modified lipid nanoparticles (LNP-Mannose). LNP-Mannose achieves specific targeted delivery to M2 macrophages by binding to mannose receptors on the surface of M2 macrophages. Experimental results show that it can effectively target M2 macrophages and facilitates the delivery of Arg1-targeting double-stranded oligonucleotides or their conjugates to target cells.

[0101] In an optional embodiment, the lipid component of the lipid nanoparticles includes ionizable lipids, phospholipids, structural lipids, and lipid-polyethylene glycol-mannose conjugates.

[0102] In an optional embodiment, the molar ratio of ionizable lipids, phospholipids, structural lipids and lipid-polyethylene glycol-mannose conjugates is (30~40):(40~50):(10~20):(1~5).

[0103] In an optional embodiment, the molar ratio of ionizable lipids, phospholipids, structural lipids and lipid-polyethylene glycol-mannose conjugates is (34~36):(46~47):(15~17):(2~3).

[0104] In an optional embodiment, the ionizable lipid includes at least one of DLin-MC3-DMA, SM-102, and ALC-0315.

[0105] In optional embodiments, the phospholipids include DOPE (1,2-dioleoyl-sn-glycerol-3-phosphoethanolamine), DSPC (1,2-distearate-sn-glycerol-3-phosphocholine), DLPC (1,2-dilauroyl-sn-glycerol-3-phosphocholine), DMPC (1,2-dimyristoyl-sn-glycerol-3-phosphocholine), DOPC (1,2-dioleoyl-sn-glycerol-3-phosphocholine), DPPC (1,2-dipalmitoyl-sn-glycerol-3-phosphocholine), DUPC (1,2-dilinoleoyl-sn-glycerol-3-phosphocholine). At least one of the following: choline, POPC (1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphorylcholine), DOPG (1,2-dioleoyl-sn-glycerol-3-phosphorylglycerol), DPPG (1,2-dipalmitoyl-sn-glycerol-3-phosphorylglycerol), DPPE (1,2-dipalmitoyl-sn-glycerol-3-phosphorylethanolamine), DMPE (1,2-dimyristoyl-sn-glycerol-3-phosphorylethanolamine), DSPE (1,2-distearate-sn-glycerol-3-phosphorylethanolamine), and DOTAP (1,2-dioleoyl-3-trimethylammonium propane).

[0106] In an optional embodiment, the structural lipids include at least one of cholesterol, coccosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassosterol, and α-tocopherol.

[0107] In an optional embodiment, the lipid in the lipid-polyethylene glycol-mannose conjugate includes at least one of DSPE (distearate phosphatidylethanolamine), DMG (dimyristoylglycerol), ceramide (Cer), and dimyristoyl phosphatidylethanolamine (DMPE).

[0108] In an optional embodiment, the lipid components include DLin-MC3-DMA, cholesterol, DOPE, and DSPE-PEG2000-mannose.

[0109] In an optional embodiment, the lipid components include DLin-MC3-DMA, cholesterol, DOPE, and DSPE-PEG2000-mannose in a molar ratio of 35:46.5:16:2.5.

[0110] In an optional embodiment, the dosage form of the pharmaceutical composition includes a dosage form suitable for pulmonary administration.

[0111] In optional embodiments, dosage forms suitable for pulmonary administration include aerosols, dry powder inhalers, liquid formulations for nebulized inhalation, and soft fog inhalers.

[0112] In an optional embodiment, the pharmaceutical composition may further include a pharmaceutically acceptable carrier and / or excipient.

[0113] In optional embodiments, the acceptable carrier and pharmaceutically acceptable excipient may be any conventional carrier and / or excipient known in the art. Examples of carriers include, but are not limited to, any physiologically compatible solvent, dispersion medium, coating, antibacterial and antifungal agents, isotonic agents, and delayed absorption agents; examples of excipients include, but are not limited to, fillers, disintegrants, preservatives, solubilizers, and emulsifiers.

[0114] Fourthly, some embodiments provide the use of the Arg1-targeting double-stranded oligonucleotide of the first aspect, the Arg1-targeting conjugate of the second aspect, or the pharmaceutical composition of the third aspect in the preparation of a product for treating, preventing, or alleviating pulmonary fibrosis. The Arg1-targeting conjugate, the Arg1-targeting double-stranded oligonucleotide component in the pharmaceutical composition can serve as an active ingredient for treating, preventing, or alleviating pulmonary fibrosis by inhibiting the expression of the Arg1 gene, thereby treating, preventing, or alleviating the symptoms or signs of pulmonary fibrosis.

[0115] The following are some examples.

[0116] The embodiments of this application will be described in detail below with reference to some examples. It should be understood that these embodiments are only for illustrating this application and are not intended to limit the scope of this application. For experimental methods in the following embodiments where conditions are not specified, please refer to the guidelines given in this application first, or follow experimental manuals or conventional conditions in the art, or follow the conditions recommended by the manufacturer, or refer to experimental methods known in the art.

[0117] In the following examples, the measurement parameters of the raw material components may have slight deviations within the weighing accuracy range unless otherwise specified. Temperature and time parameters are subject to acceptable deviations due to instrument testing accuracy or operational precision.

[0118] Example 1

[0119] This embodiment provides a mannose-modified lipid nanoparticle (LNP-Mannose) comprising the following lipid components: DLin-MC3-DMA, cholesterol, DOPE, and DSPE-PEG2000-mannose; the molar ratio of DLin-MC3-DMA, cholesterol, DOPE, and DSPE-PEG2000-mannose is 35:46.5:16:2.5.

[0120] Lipid nanoparticles loaded with nucleic acid molecules were prepared according to the following method:

[0121] (1) DLin-MC3-DMA, cholesterol, DOPE and DSPE-PEG2000-mannose are mixed in a molar ratio to form an ethanol phase.

[0122] (2) The aqueous phase was prepared using 10 mM citrate buffer (pH 3.0) containing nucleic acid molecules.

[0123] (3) LNPs@nucleic acid molecules were synthesized by rapidly mixing the aqueous phase and the ethanol phase at a volume ratio of 3:1. The resulting LNP-Mannose@nucleic acid molecules were placed at 4°C and dialyzed in 1×PBS buffer for 3 hours using a dialysis device with a molecular weight cutoff of 20,000.

[0124] Example 2

[0125] 1. Mannose-modified lipid nanoparticles (LNP-Mannose@mCherry) loaded with mCherry mRNA were prepared according to the method in Example 1.

[0126] 2. Experimental Methods:

[0127] SPF-grade C57BL / 6 mice (8-10 weeks old) were used as experimental animals. After anesthesia, the mice were slowly injected into their lungs via endotracheal intubation with LNP-Mannose@mCherry lipid nanoparticles containing mannose ligands on their surface. These nanoparticles encapsulated mCherry mRNA. The dosage was 20 μg / mouse (based on mCherry content), and the volume was 40-60 μL. Mice were sacrificed 24 hours after administration, and lung tissue was harvested. The lung tissue was minced and placed in a digestive solution containing collagenase and DNase I, digested at 37°C for 30-60 minutes, and filtered to obtain a single-cell suspension. This suspension was centrifuged, washed, and red blood cells removed before resuspending for later use. The obtained lung tissue single-cell suspension was stained with surface markers, using fluorescently labeled antibodies against mouse CD45 and F4 / 80. After incubation and washing, flow cytometry was performed for detection. For data analysis, cell debris and double cells were first excluded based on forward and side scattering parameters. The staining was performed on CD45... +Further analysis of F4 / 80 expression in cells, including CD45 + F4 / 80 + Cells were defined as lung macrophages, CD45 + F4 / 80 - Cells were defined as other immune cells, and delivery efficiency in different cell populations was assessed using mCherry fluorescence signals.

[0128] 3. Experimental Results: The experimental results are as follows... Figure 1 As shown, the mCherry signal delivered by LNP-Mannose is mainly distributed in the macrophage population. LNP-Mannose can achieve specific delivery in lung macrophages, with a delivery efficiency of over 70% in macrophages, while the delivery rate in other immune cells is approximately 8%.

[0129] Example 3

[0130] 1. This embodiment provides siRNA targeting Arg1, and designs four Arg1-targeting sequences (siArg1-1, siArg1-2, siArg1-3 and siArg1-4), with two 2'-deoxynucleotide overhangs at the 3' ends of the sense and antisense strands. Lipid nanoparticles (LNP-Mannose@siArg1) modified with siArg1-1 to siArg1-4 are prepared according to the method of Example 1.

[0131] 2. RAW264.7 cells (mouse mononuclear macrophage leukemia cells) induced to polarize to the M2 phenotype were selected as test subjects. LNP-Mannose@siArg1 loaded with siArg1-1 to siArg1-4, prepared in the above steps, were added to the culture medium. The final concentration of siArg1 was 50 nM. Cells were collected 48 hours after transfection, and the inhibitory efficiency of siArg1 on Arg1 was detected. The results are as follows. Figure 2 As shown, the results indicate that the knockdown efficiency of siArg1-1 can reach about 70%, which is the best effect, while the knockdown efficiency of siArg1-4 can reach about 50%.

[0132] The nucleotide sequences of siArg1-1 are shown in SEQ ID NO.1 and 2 in Table 1. After adding protruding dTdT ends to the 3' ends of the sense strand and the antisense strand, the siRNAs shown in SEQ ID NO.3 and 4 are obtained.

[0133] Table 1. Nucleotide sequences of siRNA

[0134]

[0135] Example 4

[0136] 1. Prepare lipid nanoparticles loaded with siArg1-1 mannose-modified lipid nanoparticles (LNP-Mannose@siArg1) according to the method of Example 1; prepare lipid nanoparticles loaded with control siRNA (siCtrl) mannose-modified lipid nanoparticles (LNP-Mannose@siCtrl) according to the method of Example 1. The control siRNA is a random sequence with a length of 21 bp.

[0137] 2. In vitro experiments: BMDM (bone marrow-derived macrophages) cells induced to polarize to the M2 phenotype were selected as test subjects. LNP-Mannose@siArg1 and LNP-Mannose@siCtrl, respectively loaded with siArg1-1 and siCtrl prepared in the above steps, were added to the culture medium. The final concentration of siRNA was 50 nM. Cells were collected 48 hours after transfection, and the relative expression level of Arg1 mRNA was detected. The results are as follows: Figure 3 As shown, the results indicate that LNP-Mannose significantly reduced the expression level of Arg1 in M2 macrophages after in vitro delivery of siArg1, suggesting that LNP-Mannose@siArg1 can be effectively taken up by M2 macrophages and significantly inhibit Arg1 expression.

[0138] 3. In vivo experiments: 8-10 week old C57BL / 6 mice were selected as candidates for a pulmonary fibrosis model. After 7 days of bleomycin administration (tracheal administration, dose 2.5 U / kg), LNP-Mannose@siArg1 and LNP-Mannose@siCtrl (final volume 50 μL, siRNA 1 nmol) were delivered to the mouse lung tissue via pulmonary administration. Following administration, relevant detection and analysis were performed on the lung tissue and lung macrophages. Left lung tissue was fixed, paraffin-embedded, and sectioned, and Masson staining was performed to assess the degree of pulmonary fibrosis. The remaining lung tissue was prepared into single-cell suspensions for multicolor flow cytometry analysis.

[0139] Specifically, lung tissue was mechanically minced and enzymatically digested to prepare a single-cell suspension. After filtration through a filter to remove red blood cells, surface marker staining was performed. The antibodies used included fluorescently labeled antibodies against mouse CD45, CD64, MerTK, CD206, and CD86. After antibody incubation, washing, and resuspending according to standard methods, the cells were analyzed by flow cytometry.

[0140] During data analysis, cell debris was first excluded based on forward and side-scattered light parameters, and double-cell pairs were excluded using FSC-A and FSC-H parameters. Cluster analysis was then performed based on these criteria. CD45+ CD64 + MerTK + The cells were defined as a macrophage population. Further subpopulations were established based on CD206 expression, with CD206⁺ macrophages defined as M2-like macrophages. Figure 4 The results showed that LNP-Mannose significantly reduced the proportion of M2 macrophages in the lungs after delivering siArg1 in vivo. Figure 5 The results showed that LNP-Mannose significantly reduced collagen deposition in lung tissue by delivering siArg1 in vivo. Further in vivo experiments indicated that this delivery method could weaken the pro-fibrotic function of M2 macrophages, thereby reducing the degree of lung fibrosis.

[0141] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0142] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims, and the specification and drawings can be used to interpret the content of the claims.

Claims

1. A double-stranded oligonucleotide targeting Arg1, characterized in that, The double-stranded oligonucleotide comprises a sense strand and an antisense strand; The nucleotide sequence of the sense strand comprises a sequence that differs from the sequence shown in SEQ ID NO.1 by no more than 3 nucleotides, and the nucleotide sequence of the antisense strand comprises a sequence that differs from the sequence shown in SEQ ID NO.2 by no more than 3 nucleotides. Furthermore, the justice chain and the antisense chain are at least partially inversely complementary to form a bichain region.

2. The Arg1-targeting double-stranded oligonucleotide according to claim 1, characterized in that, The double-stranded oligonucleotide also meets at least one of (i) to (vi): (i) At least one nucleoside bond in the double-stranded oligonucleotide is a modified nucleoside bond; (ii) The double-stranded oligonucleotide contains at least one modified nucleotide; (iii) The nucleotides of the double-stranded oligonucleotides are all unmodified; (iv) The double-stranded oligonucleotide contains at least one nucleotide analog; (v) The double-stranded oligonucleotide contains at least one 2'-deoxynucleotide; and, (vi) The justice chain and the antisense chain each independently contain protruding ends; Optionally, the modified nucleotides include one or more of the following: 2'-O-methoxyethyl modified nucleotides, 5-methyl modified nucleotides, 2'-O-methyl modified nucleotides, 2-fluorine modified nucleotides, 2'-amino modified nucleotides, 2'-C-alkyl modified nucleotides, 2'-O-alkyl modified nucleotides, bridging nucleic acids, locked nucleic acids, conformation-restricted ethyl modified nucleotides, and base-deficient modified nucleotides. Optionally, the nucleic acid analogue includes at least one of morpholinonucleotide and peptide nucleic acid; Optionally, the overhang of the sense strand and the overhang of the antisense strand each independently contain 1 to 3 nucleotides; Optionally, the protruding ends of the justice chain and the antisense chain are each located independently at the 3' end; Optionally, the nucleotides in the protruding ends of the sense strand and the antisense strand are each independently 2'-deoxynucleotides.

3. The Arg1-targeting double-stranded oligonucleotide according to claim 1 or 2, characterized in that, The double-stranded oligonucleotide is siRNA, the nucleotide sequence of the sense strand is shown in SEQ ID NO.3, and the nucleotide sequence of the antisense strand is shown in SEQ ID NO.4; and the 2'-deoxynucleotides at the 3' end of both the sense strand and the antisense strand are overhangs.

4. A conjugate targeting Arg1, characterized in that, It comprises (a) a double-stranded oligonucleotide targeting Arg1 as described in any one of claims 1 to 3; and (b) one or more targeted delivery ligands linked to (a).

5. A pharmaceutical composition, characterized in that, Includes (Ⅰ) and (Ⅱ): (I) The double-stranded oligonucleotide targeting Arg1 according to any one of claims 1 to 3; or the conjugate targeting Arg1 according to claim 4; and, (II) Delivery carrier.

6. The pharmaceutical composition according to claim 5, characterized in that, The delivery carrier includes at least one of the following: lipid nanoparticles, liposomes, cationic lipids, cationic polymers, metal nanopolymers, nanorods, micelles, microvesicles, cell-penetrating peptides, viral particles, protein coats, and liposomes; Optionally, the delivery vector targets M2 macrophages.

7. The pharmaceutical composition according to claim 6, characterized in that, The delivery carrier comprises mannose-modified lipid nanoparticles; Optionally, the lipid components of the lipid nanoparticles include ionizable lipids, phospholipids, structural lipids, and lipid-polyethylene glycol-mannose conjugates.

8. The mannose-modified lipid nanoparticles according to claim 7, characterized in that, The mannose-modified lipid nanoparticles satisfy one or more of the following conditions: The ionizable lipids include at least one of DLin-MC3-DMA, SM-102 and ALC-0315; The phospholipids include at least one of DOPE, DSPC, DLPC, DMPC, DOPC, DPPC, DUPC, POPC, DOPG, DPPG, DPPE, DMPE, DSPE, and DOTAP; The structural lipids include at least one of cholesterol, coccosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassosterol, and α-tocopherol; The lipids in the lipid-polyethylene glycol-mannose conjugate include at least one of DSPE, DMG, ceramide, and DMPE; Optionally, the lipid components include DLin-MC3-DMA, cholesterol, DOPE, and DSPE-PEG2000-mannose.

9. The pharmaceutical composition according to any one of claims 5 to 8, characterized in that, The dosage forms of the pharmaceutical composition include those suitable for pulmonary administration; Optionally, the dosage forms suitable for pulmonary administration include aerosols, dry powder inhalers, liquid formulations for nebulized inhalation, and soft fog inhalers.

10. The use of the Arg1-targeting double-stranded oligonucleotide of any one of claims 1 to 3, the Arg1-targeting conjugate of claim 4, or the pharmaceutical composition of any one of claims 5 to 9 in the preparation of a product for treating, preventing, or alleviating pulmonary fibrosis.

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