ASO for prevention and treatment of cancer metastasis and use thereof

Abstract

Provided are an antisense oligonucleotide (ASO) for prevention and treatment of cancer metastasis and a use thereof. The present disclosure specifically relates to an ASO or a pharmaceutically acceptable salt thereof, wherein the ASO specifically targets lncRNA GMAN, and the nucleotide sequence of the lncRNA GMAN is as shown in SEQ ID NO: 1. The provided ASO sequence targeting lncRNA GMAN can significantly inhibit invasion and metastasis of gastric cancer cells, and has huge potential to treat cancer and inhibit cancer metastasis.

Description

ASO for preventing and treating cancer metastasis and its use

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This disclosure claims priority to Chinese Patent Application No. 202410143725X filed on February 1, 2024, and the entire text of the above-mentioned Chinese Patent Application is incorporated herein by reference. Technical Field

[0003] The present disclosure belongs to the field of biomedicine and relates to an antisense oligonucleotide (ASO) for preventing and treating cancer metastasis and its use. Background Art

[0004] In 2020, gastric cancer ranked third in both new morbidity and mortality among malignant tumors. It is a common and serious disease in my country, accounting for 40% of new cases worldwide, severely impacting the health of the Chinese population. Early-stage gastric cancer is rare in my country, and most cases are already in the advanced stage and have metastasized by the time they are discovered. Consequently, the prognosis is poor, with a five-year survival rate of less than 10%, making it a leading cause of death among gastric cancer patients. Therefore, identifying treatment options for gastric cancer metastasis is crucial for the treatment of patients with advanced gastric cancer.

[0005] Nucleic acid therapy is becoming one of the emerging therapies, and has made significant progress in the treatment of metabolic diseases or genetic diseases. Small nucleic acid drugs mainly include antisense oligonucleotides (ASOs), small interfering RNA (siRNA), microRNA (miRNA), etc. ASOs are single-stranded nucleic acid polymers with a length of usually 15-30 nucleotides. ASOs can regulate gene expression in different ways: ASOs bind to target mRNA to form a steric hindrance effect, thereby preventing RNA-binding protein complexes from binding to mRNA and performing translation functions; ASOs bind to target RNA through complementary base pairing and then recruit nucleases to degrade the target RNA; ASOs bind to the splicing site of pre-mRNA, resulting in abnormal translation of target exons.

[0006] ASO drugs have the characteristics of high specificity, easy design, and rich target areas. Currently, nine ASO drugs have been approved for the treatment of rare diseases, such as hypercholesterolemia, spinal muscular atrophy, Duchenne muscular dystrophy, transthyretin amyloidosis polyneuropathy, and chylomicronemia syndrome. However, the development of small nucleic acid drugs still faces many difficulties. ASO drugs are easily degraded after entering the body and cannot reach target cells to function. They also have low cellular uptake efficiency, low affinity for binding to target molecules, and drug toxicity, among other factors that limit the development and application of ASO drugs.

[0007] The applicant has screened a long non-coding lncRNA GMAN that is closely related to gastric cancer metastasis and patient prognosis. lncRNA GMAN promotes the distal metastasis of gastric cancer cells by promoting the translation of Ephrin A1. Therefore, targeting lncRNA GMAN has potential value in treating gastric cancer metastasis. At present, research reports on lncRNA are generally still in the laboratory stage, and there are no clear reports on using lncRNA as a drug target for clinical trials or preclinical studies. At present, there are few ASO drugs on the market. The 10 ASO drugs on the market are approved for the treatment of rare diseases (one of which has been withdrawn from the market), and there are no ASO drugs for tumor treatment. Therefore, the new small nucleic acid ASO drug designed for lncRNA GMAN provides a new and effective disease treatment approach for the diagnosis and treatment of gastric cancer. Summary of the Invention

[0008] In order to solve the problems existing in the prior art, the present disclosure aims to provide an antisense oligonucleotide (ASO) for preventing and treating cancer metastasis and its use.

[0009] In one aspect, the present disclosure provides an antisense oligonucleotide or a pharmaceutically acceptable salt thereof, wherein the antisense oligonucleotide specifically targets lncRNA GMAN, and the nucleotide sequence of lncRNA GMAN is as SEQ ID NO: 1.

[0010] In another aspect, the present disclosure provides a conjugate comprising the aforementioned antisense oligonucleotide or a fragment thereof, and optionally a pharmaceutically acceptable carrier;

[0011] Preferably, the carrier is selected from any one of galactosamine GalNac modification, lipid nanoparticles, adeno-associated virus vectors, adenovirus vectors, albumin nanoparticles or exosomes;

[0012] Preferably, the carrier is a lipid nanoparticle.

[0013] In another aspect, the present disclosure provides a pharmaceutical composition comprising the aforementioned antisense oligonucleotide or a fragment thereof or the aforementioned conjugate, and at least another pharmaceutical component.

[0014] In another aspect, the present disclosure provides a reagent or kit comprising the aforementioned antisense oligonucleotide or a fragment thereof;

[0015] Preferably, the dosage form of the agent includes an injection;

[0016] Preferably, the injection includes aseptic or sterilized solution, water injection, oil injection or powder injection.

[0017] In another aspect, the present disclosure provides a use of the aforementioned antisense oligonucleotide or a fragment thereof, the aforementioned pharmaceutical composition, the aforementioned reagent or the aforementioned kit in the preparation of a drug for preventing or treating cancer metastasis;

[0018] Preferably, the cancer is selected from one or more of nasal cavity and paranasal sinus malignancies, nasopharyngeal cancer, oral cancer, laryngeal cancer, salivary gland tumors, intracranial tumors, thyroid cancer, tongue cancer, lung cancer, esophageal cancer, cardia cancer, breast cancer, mediastinal tumors, gastric cancer, colorectal cancer, rectal cancer, liver cancer, pancreatic cancer and periampullary cancer, small intestinal malignancies, kidney cancer, prostate cancer, bladder cancer, cervical cancer, ovarian cancer, skin malignant melanoma or lymphoma.

[0019] The beneficial effects of this application are at least as follows:

[0020] The disclosed ASO sequence targeting lncRNA GMAN can significantly inhibit the invasion and metastasis of gastric cancer cells, showing great potential for treating gastric cancer metastasis and filling the current gap in small nucleic acid-based therapeutics for tumor metastasis. Furthermore, the combination of ASO and chemotherapy drugs can significantly inhibit the invasive ability of gastric cancer cells, with greater efficacy than chemotherapy drugs alone, demonstrating its potential for treating cancer and inhibiting cancer metastasis. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 shows the sequence information of ASOs after different types of modifications, taking GMAN3 as an example.

[0022] FIG2 is a graph showing the effect of ASO sequences GMAN 1 / 2 / 3 / 4 / 5 / 6 on the expression level of Ephrin A1 protein in gastric cancer cells detected by western blot.

[0023] FIG3 is a protein immunoblot assay showing the effect of phosphorothioate-modified ASO sequences PS-GMAN 1 / 3 / 4 on the expression level of Ephrin A1 protein in gastric cancer cells.

[0024] FIG4 is a graph showing the effect of 2'OMe-GMAN3 on the expression level of Ephrin A1 protein in gastric cancer cells detected by western blot.

[0025] Figure 5 shows the experimental results of PS-GMAN1 / 3 / 4 on gastric cancer cell lines BGC-823, MKN45 and PDX cells, *, P<0.05; **, P<0.01; ***, P<0.001.

[0026] FIG6 shows the experimental results of PS-GMAN1 / 3 / 4 on the lung cancer cell line A549, ***, P<0.001.

[0027] FIG7 shows the experimental results of 2'-OMe GMAN sequences 1 / 3 / 4 on gastric cancer cell line MKN45 and PDX cells, **, P<0.01.

[0028] FIG8 is a safety test of ASO in mice at a dose of 3.75 mg / kg.

[0029] FIG9 shows the therapeutic effect of a high-dose (2.5 mg / kg) ASO sequence 2'-OMe GMAN 3 on liver metastasis of gastric cancer cells as detected by BLI; *, P<0.05.

[0030] Figure 10 shows the therapeutic effect of a low-dose (0.5 mg / kg) ASO sequence 2'-OMe GMAN 3 on gastric cancer cell liver metastasis as assessed by BLI; *, P < 0.05; **, P < 0.01. The bar in the left image is 10 mm, and the bar in the middle image is 200 μm.

[0031] Figure 11 shows the effects of ASO combined with chemotherapy drugs on the invasion and metastasis of gastric cancer cells. *, P < 0.05; **, P < 0.01.

[0032] FIG12 shows the effect of GMAN3 truncated or extended sequences on Ephrin A1 protein expression in gastric cancer PDX cells.

[0033] FIG13 shows the effects of GMAN3 truncated or extended sequences on the invasion and metastasis abilities of gastric cancer cell line MKN45 or PDX cells, **, P<0.01. DETAILED DESCRIPTION

[0034] I. Definition

[0035] In this disclosure, unless otherwise indicated, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, terms and laboratory procedures related to protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, and chemistry used herein are those commonly used in the respective fields. To facilitate a better understanding of this disclosure, definitions and explanations of relevant terms are provided below.

[0036] The terms "including" and "having" in this disclosure, as well as any variations thereof, are intended to cover non-exclusive inclusions. For example, a process, method, apparatus, product or device comprising a series of steps is not limited to the listed steps or modules, but may optionally include steps that are not listed, or may optionally include other steps that are inherent to these processes, methods, products or devices. The "plurality" mentioned in this disclosure refers to two or more. "And / or" describes the association relationship of associated objects, indicating that three relationships may exist. For example, A and / or B may represent three situations: A exists alone, A and B exist at the same time, and B exists alone. The character " / " generally indicates that the objects associated before and after are in an "or" relationship.

[0037] As used herein, the term "or combinations thereof" refers to all permutations and combinations of the terms listed before the term. For example, "A, B, C, or combinations thereof" is intended to include at least one of: A, B, C, AB, AC, BC, or ABC, and also BA, CA, CB, ACB, CBA, BCA, BAC, or CAB if order is important in the particular context. Continuing with this example, combinations containing repetitions of one or more terms or items, such as BB, AAA, AAB, BBC, AAABCCCC, CBBAAA, CABABB, etc., are expressly included. The skilled artisan will understand that, unless otherwise apparent from the context, there is generally no limit on the number of terms or items in any combination.

[0038] With respect to polypeptide sequences, the phrase "substantially identical" is understood to mean exhibiting at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference polypeptide sequence. With respect to nucleic acid sequences, the term is understood to mean nucleotide sequences that exhibit at least greater than 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference nucleic acid sequence.

[0039] In the description herein, references to “some embodiments,” “some implementation schemes,” or “some implementation plans” describe a subset of all possible embodiments, but it will be understood that “some embodiments” may be the same subset or different subsets of all possible embodiments and may be combined with each other without conflict.

[0040] As used herein, the term "lncRNA" refers to long noncoding RNA, a class of long noncoding RNAs greater than 200 nt in length, which are an important component of the noncoding genome. lncRNA is involved in a variety of biological processes, including DNA methylation, histone modification, RNA post-transcriptional regulation, and protein translation regulation, and is involved in the regulation of various physiological and pathological processes. In some embodiments of the present disclosure, the lncRNA is lncRNA GMAN, whose nucleotide sequence is shown in SEQ ID NO: 1. In some embodiments, lncRNA GMAN promotes the distal metastasis of gastric cancer cells by promoting the translation of Ephrin A1.

[0041] As used herein, the term "antisense oligonucleotide" (ASO) is defined as an oligonucleotide that can regulate the expression of a target gene by hybridizing with a target nucleic acid, particularly with a continuous sequence on the target nucleic acid. Antisense oligonucleotides are not substantially double-stranded, and therefore are not siRNA or shRNA. In certain embodiments, antisense oligonucleotides disclosed herein are single-stranded. It should be understood that the single-stranded oligonucleotides disclosed herein can form a hairpin structure or an intermolecular duplex structure (a duplex between two molecules of the same oligonucleotide), as long as the internal or intermediate self-complementarity degree is less than 50% in the total length of the oligonucleotide. Antisense oligonucleotides disclosed herein are modified oligonucleotides. As used herein, the term "antisense oligonucleotide" can refer to the complete sequence of the antisense oligonucleotide, or in some embodiments, refers to its continuous nucleotide sequence. In some embodiments, the length of the ASO of the present disclosure is 15-30nt. In some embodiments, the length of the ASO of the present disclosure is 15-25nt.

[0042] Chemical modifications of ASOs, such as phosphorothioate DNA, PMO, and peptide nucleic acids, improve nuclease resistance and enhance cellular uptake. 2'-OMe, 2'-MOE, and locked nucleic acids are often used in combination to improve stability and enhance target binding, and are generally less toxic than unmodified ASOs. tcDNA, a conformationally constrained DNA analog, can enhance potency and tissue uptake after systemic administration. The choice of delivery vehicle is crucial for the biodistribution of small nucleic acid therapeutics and their protection against nucleases and the immune system. Currently, the majority of approved or late-stage clinical nucleic acid therapeutics utilize delivery systems that include N-acetylated galactosamine (GalNac) conjugation, lipid nanoparticles (LNPs), adeno-associated viral vectors (AAV), and adenoviral vectors. Albumin nanoparticles and exosomes also offer potential delivery vehicles. GalNac conjugation, in which GalNac is covalently conjugated to the 3' end of nucleic acids in a trivalent manner, is a common delivery method for currently marketed siRNA therapeutics, promoting target organ accumulation and cellular uptake. LNP is a lipid nanoparticle loaded with small nucleic acid drugs. It provides a protected compartment for nucleic acid drugs, isolating them from nuclease activity and immune components. It is usually composed of five components: small nucleic acid drugs, ionizable cationic lipids, phospholipids, cholesterol and polyethylene glycol (PEG) lipids. The different lipid components that make up LNP play an important role in maintaining the neutral charge on the LNP surface, promoting tissue cell uptake, and endosome escape. LNP-loaded siRNA drugs have been approved for marketing. Engineered viral vectors are also effective DNA delivery vectors. They can actively deliver their payloads to the cell nucleus. Some drugs based on recombinant viral vector-mediated transgene delivery have been approved by the FDA and EMA. In addition to retinal diseases, AAV vector-mediated in vivo therapies are also used in late-stage clinical evaluation for the treatment of liver and central nervous system-related diseases.

[0043] As used herein, the term "siRNA" refers to small interfering ribonucleic acid (siRNA) RNAi agents. siRNA is a type of double-stranded RNA molecule and is referred to as short interfering RNA or silencing RNA in the art. siRNA typically comprises a sense strand (also referred to as a follower strand) and an antisense strand (also referred to as a guide strand), wherein the length of each strand is 17-30 nucleotides, typically 19-25 nucleosides in length, wherein the antisense strand is complementary to the target nucleic acid (suitably mature mRNA sequence) (such as complete complementarity), and the sense strand is complementary to the antisense strand so that the sense strand and the antisense strand form a duplex or duplex region. The siRNA chain can form a blunt-ended duplex, or advantageously the sense strand and the antisense strand 3' end can form a 3' single-stranded overhang of, for example, 1, 2, or 3 nucleosides. In some embodiments, both the sense strand and the antisense strand have 2nt 3' single-stranded overhangs. Therefore, the length of the duplex region can be, for example, 17-25 nucleotides, such as 21-23 nucleotides in length.

[0044] As used herein, the term "Transwell" is a laboratory product used for cell culture and studying biological processes such as cell migration and invasion. It consists of a transparent polycarbonate or polyester membrane with pores between the top and bottom layers, allowing cells to pass through.

[0045] As used herein, the term "tc-DNA" refers to a class of constrained DNA analogs in which each nucleotide is modified by the introduction of a cyclopropane ring to restrict the conformational flexibility of the backbone and optimize the geometry of the backbone torsion angle γ. tc-DNA, which contains high adenine and thymine bases, forms exceptionally stable AT base pairs with complementary RNA.

[0046] As used herein, the term "nucleoside" refers to a compound comprising a core base and a sugar covalently attached to the core base. Further, the term "nucleoside" is intended to include naturally occurring or modified nucleosides or nucleoside mimetics that can be incorporated into oligomers using natural or chemical oligomer synthesis methods. Generally and preferably, the term "nucleoside" used herein refers to naturally occurring nucleosides, modified nucleosides or nucleoside mimetics. The term "modified nucleoside" is intended to include modifications to the sugar and / or core base of a nucleoside as known to those skilled in the art and described herein. The term "nucleoside mimetics" is intended to include those structures for replacing sugars and core bases. Examples of nucleoside mimetics include nucleosides in which the core base is replaced by a phenoxazine moiety (e.g., 9-(2-aminoethoxy)-1,3-diazaphenoxazine-2-one group) and the sugar moiety is replaced by a cyclohexenyl or bicyclohexyl moiety. The term "nucleoside" also includes combinations of modifications, such as more than one nucleobase modification, more than one sugar modification, or at least one nucleobase and at least one sugar modification.

[0047] The sugar of the nucleoside includes, but is not limited to, a monocyclic, bicyclic or tricyclic ring system, preferably a tricyclic or bicyclic ring system or a monocyclic ribose or deoxyribose. Modifications of the sugar further include, but are not limited to, modified stereochemical configurations, at least one substitution of a group, or at least one deletion of a group. Modified sugars are generally and preferably modified forms of the ribosyl moiety (i.e., furanosyl moiety) naturally present in RNA and DNA, such as bicyclic sugars, tetrahydropyranoses, 2'-modified sugars, 3'-modified sugars, 4'-modified sugars, 5'-modified sugars, or 4'-substituted sugars. Examples of suitable sugar modifications are known to those of skill in the art and include, but are not limited to, 2', 3' and / or 4' substituted nucleosides (e.g., 4'-S-modified nucleosides); 2'-O-modified RNA nucleotide residues, such as 2'-O-alkyl or 2'-O-(substituted)alkyl, for example, 2'-O-methyl, 2'-O-(2-cyanoethyl), 2'-O-(2-methoxy)ethyl (2'-MOE), 2'-O-(2-thiomethyl)ethyl; 2'-O-(haloalkoxy)methyl, for example, 2'-O-(2-chloroethoxy)methyl (MCEM), 2'-O-( 2'-O-alkoxycarbonyl, for example 2'-O-[2-(methoxycarbonyl)ethyl] (MOCE), 2'-O-[2-(N-methylcarbamoyl)ethyl] (MCE), 2'-O-[2-(N,N-dimethylcarbamoyl)ethyl] (DMCE), in particular 2'-O-methyl modification or 2'-O-methoxyethyl (2'-O-MOE); or other modified sugar moieties, such as morpholino (PMO), cationic morpholino (PMOPlus) or modified morpholino groups, such as PMO-X. The term "PMO-X" refers to a modified morpholino group comprising at least one 3' or 5' terminal modification, such as a 3'-fluorescent label, a 3' quencher (e.g., 3'-carboxyfluorescein, 3'-Gene Tools Blue, 3'-lissamine, 3'-dabcyl), a 3'-affinity tag and a functional group for chemical attachment (e.g., 3'-biotin, 3'-primary amine, 3'-disulfide amide, 3'-pyridyl disulfide), a 5'-terminal modification (5'-primary amine, 5'-dabcyl), 3'-azide, 3'-alkyne, 5'-azide, 5'-alkyne, or as disclosed in WO2011 / 150408 and US2012 / 0065169.

[0048] A "bicyclic sugar moiety" comprises two interconnected ring systems, such as a bicyclic nucleoside, wherein the sugar moiety has a 2'-O-CH(alkyl)-4' or 2'-O-CH2-4' group, locked nucleic acid (LNA), xylose-LNA, α-L-LNA, β-D-LNA, cEt (2'-O,4'-C constrained ethyl) LNA, cMOEt (2'-O,4'-C constrained methoxyethyl) LNA, ethylene-bridged nucleic acid (ENA), hexitol nucleic acid (HNA), fluorinated HNA (F-HNA), pyranosyl-RNA (p-RNA) or 3'-deoxypyranosyl-DNA (p-DNA).

[0049] As used herein, the term "oligonucleotide" refers to a compound comprising at least two nucleosides that are separately connected to each other by an internucleoside linking group. Therefore, the term "oligonucleotide" as used herein includes and generally and preferably refers to an oligomeric compound comprising at least two nucleosides connected by an internucleoside linking group, wherein the at least two nucleosides are independently selected from naturally occurring nucleosides, modified nucleosides or nucleoside mimics. Therefore, the term "oligonucleotide" as used herein includes and generally and preferably refers to an oligomeric compound comprising at least two nucleosides connected by an internucleoside linking group, wherein the at least two nucleosides are independently selected from naturally occurring nucleosides, modified nucleosides or nucleoside mimics.

[0050] As used herein, the term "pharmaceutical composition" refers to a mixture or solution comprising a therapeutically effective amount of an active pharmaceutical ingredient together with a pharmaceutically acceptable excipient for administration to a mammal (eg, a human) in need thereof.

[0051] As used herein, the term "pharmaceutically acceptable salt" refers to salts derived from a variety of organic and inorganic counterions known in the art. Pharmaceutically acceptable acid addition salts can be formed with inorganic and organic acids. Inorganic acids from which salts can be derived include, for example, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, and phosphoric acid. Organic acids from which salts can be derived include, for example, acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, and salicylic acid. Pharmaceutically acceptable base addition salts can be formed with inorganic and organic bases. Inorganic bases from which salts can be derived include, for example, sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, and aluminum. Organic bases from which salts can be derived include, for example, primary, secondary, and tertiary amines, substituted amines (including naturally occurring substituted amines), cyclic amines, and basic ion exchange resins. Specific examples include isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine and ethanolamine. In some embodiments, the pharmaceutically acceptable base addition salt is selected from ammonium, potassium, sodium, calcium and magnesium salts, preferably the pharmaceutically acceptable salt is a sodium salt.

[0052] As used herein, the term "LNP" or "lipid nanoparticle" is a vesicle comprising a lipid layer that encapsulates a pharmaceutically active molecule (e.g., a nucleic acid molecule, e.g., an iRNA or a plasmid from which the iRNA is transcribed). LNPs are described, for example, in U.S. Patent Nos. 6,858,225, 6,815,432, 8,158,601, and 8,058,069, the entire contents of which are incorporated herein by reference.

[0053] As used herein, "subject" refers to any animal, preferably a mammal, more preferably a human, who is to undergo or has undergone treatment according to the methods of the embodiments of the present application. The term "mammal" as used herein encompasses any mammal. Examples of mammals include, but are not limited to, cattle, horses, sheep, pigs, cats, dogs, mice, rats, rabbits, guinea pigs, non-human primates (NHPs) such as monkeys or apes, humans, etc., more preferably humans.

[0054] The term "therapeutically effective amount" refers to an amount of a compound or molecule of the present invention that, when administered to a subject, (i) treats or prevents a particular disease, condition, or disorder, (ii) reduces, ameliorates, or eliminates one or more symptoms of a particular disease, condition, or disorder, or (iii) prevents or delays the onset of one or more symptoms of a particular disease, condition, or disorder as described herein. The therapeutically effective amount depends on the compound, the disease state being treated, the severity of the disease being treated, the age and relative health of the subject, the route and form of administration, the judgment of the attending medical or veterinary doctor, and other factors.

[0055] II. Specific Implementation Methods

[0056] In one aspect, the present disclosure provides an antisense oligonucleotide or a pharmaceutically acceptable salt thereof, wherein the antisense oligonucleotide specifically targets lncRNA GMAN, and the nucleotide sequence of lncRNA GMAN is as SEQ ID NO: 1.

[0057] In some embodiments, the antisense oligonucleotide is single-stranded, 15-30 nt.

[0058] In some embodiments, the antisense oligonucleotide is single-stranded, 15-25 nt.

[0059] In some embodiments, the antisense oligonucleotide comprises a nucleotide sequence selected from any one of SEQ ID NOs: 2-432. In some embodiments, the antisense oligonucleotide comprises a nucleotide sequence selected from any one of SEQ ID NOs: 2-7. In some embodiments, the nucleotide sequence of the antisense oligonucleotide is as shown in SEQ ID NO: 4.

[0060] In some embodiments, the antisense oligonucleotide comprises modified nucleotides. In some embodiments, at least part of the nucleotides of the antisense oligonucleotide are modified nucleotides. In some embodiments, each nucleotide of the antisense oligonucleotide is a modified nucleotide. In some embodiments, the modified nucleotide is selected from 2'-methoxy modified nucleotides, 2'-fluoro modified nucleotides, 2'-deoxy nucleotides, inosine or inosine nucleotides, 5'-phosphate nucleotides, 2',3'-seco nucleotide mimetics, locked nucleotides (LNA), unlocked nucleic acid nucleotides (UNA), glycol nucleic acid nucleotides (GNA), bicyclic nucleic acids (BNA), 2'-F-arabino nucleotides, 2'-methoxyethyl nucleotides, abasic nucleotides, ribitol, inverted nucleotides, inverted abasic nucleotides (Invab), inverted 2'-OMe nucleotides, inverted 2'-deoxy nucleotides, 2'-amino modified nucleotides, 2'-alkyl modified nucleotides, morpholino nucleotides, 3'-OMe nucleotides, phosphate group modified nucleotides, terminal nucleotides linked to a cholesterol derivative or dodecanoic acid bisdecylamide group, 2'-amino modified nucleotides, phosphoramidates, non-natural base containing nucleotides, or 5'-phosphate mimetic modified nucleotides. In some embodiments, the 5'-phosphate mimetic is selected from 5'-oxymethylphosphonate, 5'-vinylphosphonate, a 5'-vinyl phosphate analog, 5'vinylphosphonate-2'-N-acetyl, or 5'-malonylphosphonate.

[0061] In some embodiments, the antisense oligonucleotide comprises at least one internucleotide bond. In some embodiments, the internucleotide bond is a phosphorothioate bond. In some embodiments, the nucleotide sequence of the antisense oligonucleotide comprises a sequence selected from any one of SEQ ID NOs: 433-436 or a fragment thereof. In some embodiments, the nucleotide sequence of the antisense oligonucleotide is as shown in any one of SEQ ID NOs: 433-436.

[0062] In some embodiments, the antisense oligonucleotide comprises a phosphorothioate bond between any two nucleotides. In some embodiments, the antisense oligonucleotide further comprises at least one 2'-methoxy modified nucleotide. In some embodiments, the antisense oligonucleotide further comprises at least one 2'-methoxy modified nucleotide. In some embodiments, the antisense oligonucleotide is selected from the nucleotide sequence shown in any one of SEQ ID NOs: 437-440. In some embodiments, the nucleotide sequence of the antisense oligonucleotide is as shown in SEQ ID NO: 439.

[0063] In some embodiments, the antisense oligonucleotide comprises multiple modifications, and the modified nucleotide sequence comprises a sequence according to any one of SEQ ID NOs: 441-458 or a fragment thereof.

[0064] In another aspect, the present disclosure provides a conjugate comprising the aforementioned antisense oligonucleotide or a fragment thereof, and optionally a pharmaceutically acceptable carrier.

[0065] In some embodiments, the carrier is selected from any one of galactosamine GalNac modification, lipid nanoparticles, adeno-associated virus vectors, adenovirus vectors, albumin nanoparticles, or exosomes.

[0066] In some embodiments, the carrier is modified with galactosamine GalNac, wherein the GalNac is trivalently covalently coupled to the 3' end of the antisense oligonucleotide.

[0067] In some embodiments, the carrier is a lipid nanoparticle.

[0068] In another aspect, the present disclosure provides a pharmaceutical composition comprising the aforementioned antisense oligonucleotide or a fragment thereof or the aforementioned conjugate, and at least another pharmaceutical composition.

[0069] In some embodiments, the pharmaceutical composition contains at least one other pharmaceutical component. In some embodiments, the pharmaceutical component is selected from trastuzumab, ramucirumab, apatinib, lapatinib, pertuzumab, everolimus, bevacizumab, olaparib, TDM-1, nimotuzumab, cetuximab, panitumumab, erlotinib, icotinib, gefitinib, afatinib, sorafenib, sunitinib, regorafenib, crizotinib, onartuzumab, ponatinib, dovitinib, fluorouracil (5-FU), capecitabine, S-1, oxaliplatin, epirubicin, capecitabine, paclitaxel, docetaxel, cisplatin, carboplatin, irinotecan, pembrolizumab, nebulizumab, atezolizumab, avelumab or duivalumab.

[0070] In another aspect, the present disclosure provides a reagent or a kit comprising the aforementioned antisense oligonucleotide or a fragment thereof, the aforementioned conjugate, or the aforementioned pharmaceutical composition.

[0071] In some embodiments, the dosage form of the agent includes an injection; in some embodiments, the injection includes a sterile or sterilized solution, water injection, oil injection or powder injection.

[0072] On the other hand, the present disclosure provides a use of the aforementioned antisense oligonucleotide or fragment thereof, the aforementioned pharmaceutical composition, the aforementioned reagent or kit in the preparation of a drug for preventing or treating cancer or inhibiting cancer metastasis.

[0073] In some embodiments, the cancer is selected from one or more of nasal cavity and paranasal sinus malignancies, nasopharyngeal cancer, oral cancer, laryngeal cancer, salivary gland tumors, intracranial tumors, thyroid cancer, tongue cancer, lung cancer, esophageal cancer, cardia cancer, breast cancer, mediastinal tumors, stomach cancer, colorectal cancer, rectal cancer, liver cancer, pancreatic cancer and periampullary cancer, small intestinal malignancies, kidney cancer, prostate cancer, bladder cancer, cervical cancer, ovarian cancer, skin malignant melanoma, and lymphoma.

[0074] In another aspect, the present disclosure provides a method for preventing or treating cancer metastasis, comprising administering the aforementioned antisense oligonucleotide or fragment thereof, the aforementioned pharmaceutical composition, the aforementioned reagent or kit of parts of the present disclosure to a subject.

[0075] In the preferred embodiment of the present disclosure, the mode of administration for preventing or treating cancer metastasis comprises any suitable method known in the art, including but not limited to, oral, oral, sublingual, eyeball, local, parenteral, rectal, intrathecal, intracytoplasmic reticulum groove, groin, intravesical, local (such as powder, ointment or drops), or nasal route. But, for many therapeutic uses, preferred route of administration / mode is parenteral administration (such as intravenous injection, subcutaneous injection, intraperitoneal injection, intramuscular injection). Technicians should understand that route of administration and / or mode will change according to intended purpose. In a preferred embodiment, antisense oligonucleotide of the present invention, pharmaceutical composition are given by intravenous infusion or injection.

[0076] The present disclosure is described in more detail below with reference to specific examples, which are, however, for illustrative purposes only and do not limit the present disclosure. The reagents and biological materials described in the following examples, unless otherwise specified, can be obtained from commercial sources.

[0077] Example 1: Design and preparation of ASO

[0078] The inventors have identified a long noncoding RNA (lncRNA) called GMAN, which is closely associated with gastric cancer metastasis and patient prognosis. GMAN promotes distal metastasis of gastric cancer cells by promoting the translation of Ephrin A1. The nucleotide sequence of lncRNA GMAN is shown in SEQ ID NO:1. Based on this sequence, the inventors designed an ASO targeting this lncRNA and commissioned Shanghai Gene to synthesize it. The ASO was designed as a single strand of 15-30 nt. Figure 1 shows a partial ASO sequence, and the detailed ASO sequence information can be found in SEQ ID NOs. 1-432.

[0079] Table 1 ASO naked sequences

[0080] Example 2: Thio-modification of ASO

[0081] For example, some nucleotides at the 3' and 5' ends of the naked nucleic acid sequences of GMAN 1 / 2 / 3 / 4 were thiolated, and the sequence information after modification is shown in Table 2 below.

[0082] Table 2 ASO modified sequences Note: * represents phosphorothioate modification.

[0083] Example 3: 2' methoxy modification of ASO

[0084] Based on the thiolated sequences of Example 2, the inventors further performed full thiolation modification on the nucleic acid backbone of GMAN 1 / 2 / 3 / 4, and simultaneously methoxylated the 2' position of the ribose sugars at each of the five bases at both ends of the sequence before synthesizing them. For example, the modified sequence information is shown in Table 3 below.

[0085] Table 3 ASO modified sequences Note: * represents phosphorothioate modification; m represents 2'OMe modification; non-underlined letters represent deoxyribonucleotides; underlined letters represent ribonucleotides.

[0086] Example 4: ASO GMAN3 sequence can be modified in different ways

[0087] In order to further explore the effects of other modification methods on the ASO sequence, the inventors chose to perform different types of modifications on the ASO GMAN 3 sequence, such as full sulfo modification, 2'OMe modification, full sulfo combination 2'OMe modification, 2'-MOE modification (2'-methoxyethyl modification), full sulfo combination 2'-MOE modification, 2'-F modification (2'-fluoro modification), full sulfo combination 2'-F modification, LNA modification (locked nucleic acid modification), full sulfo combination LNA modification, 2'-cEt modification (2'-constrained ethyl), full sulfo combination 2'-cEt modification, tcDNA modification (three-ring DNA modification), full sulfo combination tcDNA modification, PMO modification (phosphodiamidate morpholino oligonucleotide modification) and PNA modification (peptide nucleic acid modification). For example, the sequence after some modifications can be seen in Figure 1.

[0088] Example 5: Both naked and modified ASO sequences can significantly inhibit the expression of Ephrin A1 in cells

[0089] Since lncRNA GMAN is located in the Ephrin A1 gene cluster and promotes metastasis through Ephrin A1, the inventors tested the expression of Ephrin A1 to reflect the knockdown efficiency of lncRNA GMAN and its ability to promote metastasis. The specific experimental steps are as follows:

[0090] 1. Cell Transfection with ASO

[0091] Gastric cancer cell lines BGC-823 and MKN45 in the logarithmic growth phase were plated in six-well plates and transfected when the cell density reached about 50%. 1 mL of 1640 medium containing 10% fetal bovine serum was replaced 1 h before transfection. The transfection system was then prepared according to the instructions of the transfection reagent. 100 pmol ASO (GMAN1 / 2 / 3 / 4 group, PS-GMAN1 / 3 / 4 group, 2'OMe-GMAN1 / 3 group) was diluted with 200 μl of 1× buffer, and a blank control was set up. After mixing, 8 μL Genmute (purchased from SignaGen, USA) was added, mixed again, and incubated for 15 min. Then, the cells were added to the cell culture medium and shaken. The culture medium was replaced after 24 h.

[0092] 2. Western Blot Detection of Ephrin A1 Expression in Gastric Cancer Cells

[0093] Total protein was extracted using RIPA lysis buffer (purchased from Shanghai Biotech) and the procedure was performed according to the instructions of the reagent. 6 ) was added with 200 μl of RIPA lysis buffer containing 1× PMSF (purchased from Shanghai Bio-Tech). The cells were scraped off with a cell scraper and transferred to a 1.5 mL EP tube. The cells were placed on ice and disrupted using a sonicator. The cell lysate was then centrifuged at 12,000 g at 4°C for 10 minutes to remove unlysed cell debris. The supernatant was aspirated and transferred to a new 1.5 mL EP tube. Loading buffer was added, mixed thoroughly, and incubated in a 100°C water bath for 10 minutes. The resulting lysate can be stored at -20°C until needed.

[0094] (1) Preparation of SDS-PAGE separation gel and stacking gel:

[0095] The 10% separation gel formula (10 mL) is shown in Table 4 below:

[0096] Table 4 Separation gel formula

[0097] The formula of 5% stacking gel (4 mL) is shown in Table 5 below:

[0098] Table 5 Concentrating gel formula

[0099] (2) Western Blot experiment

[0100] The markers and processed protein samples were added to the sample wells in the corresponding order. The voltage was adjusted to 60V for electrophoresis for 1 hour, then the voltage was adjusted to 100V until the end of the electrophoresis. Before transfer, the PVDF membrane was cut into 8 cm long and 6 cm wide pieces and activated with 100% methanol for 15 seconds. The transfer voltage was 100V for 90 minutes. After transfer, the PVDF membrane was blocked in 5% skim milk at room temperature for 1 hour. Then, the membrane was incubated with Ephrin A1 antibody (Wuhan Abotek, A5341) at 4°C overnight. After the primary antibody incubation, the membrane was washed three times with 1× TBST at room temperature for 10 minutes each. Then, a secondary antibody (Wuhan Abotek, AS014) of appropriate dilution was added and incubated for 1 hour. The PVDF membrane was then placed in a Bio-Rad instrument and incubated with a colorimetric solution (purchased from Hangzhou Fude) for 30 seconds before colorimetric scanning.

[0101] 3. Experimental Results

[0102] The WB results are shown in Figures 2-4. The results show that both the naked and modified ASO sequences targeting lncRNA GMAN disclosed in this application can significantly inhibit the protein expression level of Ephrin A1 in gastric cancer cells, indicating that both the naked and modified ASO sequences can inhibit the expression of Ephrin A1 by downregulating the level of lncRNA GMAN. Similarly, the naked ASO sequences and their modified sequences disclosed in the Examples of this application, such as SEQ ID NOs. 3 and 6-432, also exhibit the ability to inhibit the expression of Ephrin A1.

[0103] Example 6: ASO significantly inhibits cancer cell invasion

[0104] Because lncRNA GMAN can significantly promote the invasion and metastasis of gastric cancer cells, targeting and downregulating lncRNA GMAN expression may be a potential strategy for treating gastric cancer metastasis. To further test the effectiveness of ASOs in inhibiting tumor metastasis in other cancer types, the inventors transfected ASOs into gastric cancer cell lines (BGC-823 and MKN45), gastric cancer PDX cells, and lung cancer cell line A549, and examined their effects on the invasiveness of different tumor cells. For example, modified GMAN1, GMAN3, and GMAN4 were used in this example.

[0105] 1. Cell Transfection with ASO

[0106] Gastric cancer cell lines BGC-823, MKN45, and PDX cells in the logarithmic growth phase, and lung cancer cell line A549 were plated in six-well plates. When the cell density reached about 50%, transfection was performed. 1 mL of fresh culture medium was replaced 1 h before transfection. The transfection system was then prepared according to the instructions of the transfection reagent. 100 pmol ASO (PS-GMAN1 / 3 / 4 group, 2'OMe-GMAN1 / 3 group) was diluted with 200 μL of 1× buffer, and a blank control was set up. After mixing, 8 μL of Genmute (purchased from SignaGen, USA) was added. After mixing again, the cells were incubated for 15 min, then added to the cell culture medium, shaken, and the culture medium was replaced after 24 h.

[0107] 2. Cell Invasion Assay

[0108] 50 μL of 0.5% gelatin (purchased from Shanghai Sangon, China) was placed in an 8 μm Transwell (24-well, purchased from Corning, USA) in advance and incubated in a cell culture incubator at 37°C for 2 h. The transfected cells were digested and counted, and the cells were resuspended in medium containing 1% FBS. 1×10 5 To a 200μL cell suspension, add 750μL of 10% FBS-containing 1640 medium to the lower chamber and incubate in an incubator for the appropriate time. After the migration period, fix the transwell with 4% PFA for 10 minutes. Stain with 0.1% crystal violet for 15 minutes. Gently wash off excess crystal violet with PBS. Carefully wipe away cells from the upper layer of the transwell using a cotton swab. Observe cells that have invaded the lower layer of the transwell under a microscope, photograph them, and perform statistical analysis.

[0109] 3. Experimental Results

[0110] By transfecting the small nucleic acid drug ASO PS-GMAN1, PS-GMAN3, and PS-GMAN4 sequences into gastric cancer cell lines (BGC-823, MKN45, and PDX cells), it was demonstrated that ASO can significantly inhibit the invasion of gastric cancer cells, see Figure 5. In the lung cancer cell line A549, both PS-GMAN3 used alone and ASO PS-GMAN1 / 3 / 4 used in combination can significantly inhibit the invasion of cancer cells, see Figure 6. In addition, methoxy-modified small nucleic acid drugs (2'-OMe GMAN sequence 1, 2'-OMe GMAN sequence 3, 2'-OMe GMAN sequence 4, whose nucleotide sequences are shown in SEQ ID NO.806, 808, and 809, respectively) also showed a good effect of inhibiting the invasion of gastric cancer cells (Figure 7). Similarly, the naked ASO sequences and their modified sequences shown in SEQ ID NO.3, 6-432 disclosed in the examples of this application also showed a good ability to inhibit the spread and invasion of tumor cells. In summary, both the naked sequence and modified sequence of the small nucleic acid drug ASO prepared in the present application can show good effects in inhibiting the invasion and metastasis of cancer cells, and can also exert the ability to inhibit the invasion and metastasis of cancer cells in other cancer types besides gastric cancer.

[0111] Example 7: Safe Dosage of LNP-ASO in Mice

[0112] Before the in vivo pharmacodynamic study, in order to explore the safe dose for use in mice, the inventors further studied the dosage of LNP-ASO. For example, in this example, the 2'-OMe modified sequence of GMAN3 (SEQ ID NO. 439) was used for safety verification. The specific experimental process is as follows:

[0113] 1. LNP-ASO Preparation

[0114] D-Lin-MC3-DMA (purchased from MedChemExpress, USA), cholesterol (purchased from MilliporeSigma, USA), DSPC (purchased from MedChemExpress, USA), and DMG-PEG2000 (purchased from MedChemExpress, USA) were dissolved in ethanol at a molar ratio of 50:38.5:10:1.5. The ASO GMAN3 was dissolved in citrate buffer (25 mM, pH 4.0). The lipid solution and ASO GMAN3 were then mixed using a microfluidic mixing device at a volume ratio of 1:6 at a flow rate of 114 μL / min to prepare a LNP-ASO solution. The prepared GMAN3-LNP was then dialyzed to PBS and purified by centrifugal ultrafiltration (MWCO 10 kDa, Amicon, purchased from MilliporeSigma, USA) to remove free ASO and concentrate. The concentrated and purified GMAN3-LNP preparation was used for subsequent in vivo experiments.

[0115] 2. Safety Study of LNP-ASO in Mice

[0116] First, healthy NSG mice were randomly divided into two groups, with 5 mice in each group. The drug was injected intraperitoneally for 3 consecutive days, and starting from the 6th day, it was injected again for 3 days. The experimental group was injected with lipid nanoparticles encapsulated with the ASO GMAN3 sequence (3.75mg / kg), and the control group was injected with PBS. Body weight was measured once a week. After the experiment, blood samples were collected from the mice for analysis of liver and kidney function. At the same time, samples of the heart, liver, spleen, lung, and kidney organs of the mice were collected and stained with HE to detect whether the functions of the main organs of the mice were abnormal.

[0117] 3. Experimental Results

[0118] The results of the mouse safety experiment are shown in Figure 8. The results showed that compared with the PBS group, the weight of the mice in the LNP-GMAN3 group did not decrease significantly, and no obvious damage was found in the major organs (heart, liver, spleen, lung, and kidney) of the mice treated with LNP-GMAN3. At the same time, further evaluation of systemic toxicity and liver and kidney function analysis showed that compared with PBS, the serum levels of aspartate aminotransferase (AST), alkaline phosphatase (ALP), creatinine (CR), and uric acid (UA) in the LNP-GMAN3-treated mice were not significantly increased. These results confirm the safety of the ASO drugs disclosed in this application for in vivo use.

[0119] Example 8: ASO inhibits gastric cancer cell metastasis in a mouse model

[0120] To test the anti-tumor efficacy of the small nucleic acid drug ASO GMAN sequence targeting the lncRNA GMAN, the inventors exemplified the ASO GMAN sequence shown in SEQ ID NO:439 for LNP encapsulation and injected 2.5 mg / kg ASO 2'-OMe-GMAN 3 into the tail vein of model mice. To further verify the efficacy of the ASO and explore the lowest dose, the model mice were treated with 1 / 5 of the previously administered dose. The specific experimental procedures are as follows:

[0121] 1. NSG mouse liver metastasis experiment

[0122] The corresponding ASO-LNP preparation was prepared according to the method in Example 7. NSG mice were anesthetized and then underwent spleen injection surgery. Gastric cancer PDX cells stably expressing luciferase gene (1×10 6 ) was injected into 10 NSG mice through the spleen to establish a liver metastasis model of gastric cancer cells. Before treatment, the mice were randomly divided into 2 groups, with 5 mice in each group. On the second day after the completion of the operation, drug injection was started through the tail vein, and the injection was continued for 3 consecutive days, and the fourth drug injection was performed on the 6th day. The experimental group was injected with lipid nanoparticles (Lipid Nanoparticle, LNP) coated with 2'-OMe-GMAN 3 (2.5 mg / kg), and the control group was injected with LNP that did not contain the ASO sequence targeting lncRNA GMAN. On the 7th day, in vivo bioluminescence imaging (BLI) was performed using the Xenogen IVIS200 imaging system to observe the metastasis of gastric cancer cells and perform statistical analysis.

[0123] The inventors also delivered a smaller dose of 2'-OMe-GMAN 3-encapsulated LNPs (0.5 mg / kg) to several additional model mice to verify the therapeutic efficacy of the ASO at this concentration. Similarly, drug injections were administered via the tail vein starting on the second day after surgery for three consecutive days, followed by another three-day infusion starting on the sixth day. Bioluminescence imaging was performed twice during treatment to observe gastric cancer cell metastasis, and the results were statistically analyzed.

[0124] 2. Experimental Results

[0125] The experimental results after ASO administration in the high-dose group are shown in Figure 9, and the experimental results after ASO administration in the low-dose group are shown in Figure 10. Both BLI results showed that gastric cancer PDX cells exhibited severe liver metastasis in mice treated with the control group, while mice treated with the small nucleic acid drug targeting lncRNA GMAN were able to significantly inhibit the metastasis of gastric cancer PDX cells, even at a low dose. Liver histological analysis also confirmed that liver metastasis was significantly suppressed in mice treated with the small nucleic acid drug targeting lncRNA GMAN, compared with mice in the control group, with a significant reduction in liver metastatic lesions (Figure 4.3).

[0126] Similarly, the naked ASO sequences and modified sequences thereof as shown in SEQ ID NO. 3, 6-432 disclosed in the examples of the present application also exhibit the ability to inhibit the metastasis of gastric cancer cells.

[0127] In summary, in vivo treatment data demonstrate that targeting lncRNA GMAN is effective in treating gastric cancer metastasis. The ASO small nucleic acid drug disclosed in this application has enormous potential for clinical treatment, filling the current gap in small nucleic acid drug treatments for tumor metastasis. Given that lncRNA GMAN is significantly overexpressed in metastatic tumor tissues and that ASO drugs have strong targeting specificity, which can reduce off-target effects, the drugs developed using the naked and modified ASO sequences targeting lncRNA GMAN disclosed in this application have potential value in treating tumor metastasis.

[0128] Example 9: Combination of ASO and chemotherapy drugs significantly inhibits gastric cancer cell invasion

[0129] Clinically, chemotherapy drugs can effectively kill tumor cells and shrink primary tumor tissue. However, prior art shows that chemotherapy drugs have a weak therapeutic effect on metastatic tumor lesions and may stimulate tumor cells to further metastasize. Considering the good effect of the ASO drug provided by the present disclosure in inhibiting tumor metastasis, it is highly complementary to chemotherapy drugs and may have a better therapeutic effect when used in combination with chemotherapy drugs. Therefore, in this example, the ASO drug is combined with chemotherapy drugs to detect the effect on the invasion and metastasis of gastric cancer cells. Exemplarily, the ASO drug used in this example is 2'-OMe-GMAN 3 (SEQ ID NO.439).

[0130] 1. Cell Transfection with ASO

[0131] Gastric cancer PDX cells in the logarithmic growth phase were plated in a six-well plate. Transfection was performed when the cell density reached about 50%. 1 mL of fresh culture medium was replaced 1 h before transfection. The transfection system (ASO: 2'OMe-NC, 2'OMe-GMAN3) was prepared according to the instructions of the transfection reagent. After incubation for 15 min, the cells were added to the cell culture medium and shaken. The culture medium was replaced after 24 h.

[0132] 2. Treatment of cells with chemotherapy drugs

[0133] 10 μM oxaliplatin (Oxaliplatin, purchased from MedChemExpress, USA) was added to the transfected cells, and subsequent invasion experiments were performed after treatment for 48 hours.

[0134] 3. Cell Invasion Assay

[0135] 50 μL of 0.5% gelatin (purchased from Shanghai Biotechnology Co., Ltd., China) was spread in advance in an 8 μm Transwell (24-well, purchased from Corning, USA) and incubated in a cell culture incubator at 37°C for 2 h. 5 Each cell was resuspended in 200 μL of 1% FBS-containing medium and added to the transwell chamber. Subsequently, 750 μL of 10% FBS-containing 1640 medium was added to the lower chamber and incubated in an incubator for the appropriate time. After the migration period, the transwell was fixed with 4% PFA for 10 minutes. The cells were then stained with 0.1% crystal violet for 15 minutes. Excess crystal violet was gently washed off with PBS. Cells in the upper layer of the transwell chamber were carefully wiped away with a cotton swab. Cells invading the lower layer of the transwell chamber were observed under a microscope, photographed, and analyzed.

[0136] 4. Experimental Results

[0137] The experimental results are shown in Figure 11. As shown in Figure 11, the combination of the chemotherapy drug oxaliplatin and 2'-OMe-GMAN 3 can significantly inhibit the invasive ability of gastric cancer cells, and the effect is better than that of the chemotherapy drug alone. Similarly, the naked ASO sequences and their modified sequences as shown in SEQ ID NOs. 3, 6-432 disclosed in the Examples of this application, combined with chemotherapy drugs, also significantly inhibited the invasion of gastric cancer cells.

[0138] In summary, the ASO disclosed in this application can significantly inhibit gastric cancer cell invasion, and this anti-tumor invasion and metastasis effect can be enhanced when combined with chemotherapy drugs. The combined use of ASO drugs and chemotherapy drugs has the potential to better treat cancer and inhibit cancer metastasis.

[0139] Example 10: Truncated or extended ASO sequences significantly inhibited Ephrin A1 protein expression and invasion and metastasis abilities of gastric cancer cells

[0140] As mentioned above, the ASO small nucleic acid drugs targeting lncRNA GMAN disclosed in this application can significantly inhibit the in vitro invasive ability of gastric cancer cells and can effectively treat the metastasis of gastric cancer cells in vivo. In order to further verify whether the truncated or extended sequences derived from GMAN1-GMAN6 also have the aforementioned properties, in this example, the inventors randomly screened four truncated or extended sequences of GMAN3 (SEQ ID NO: 4): 15nt, 17nt, 25nt, and 30nt, and tested their effects on Ephrin A1 protein expression and invasive and metastatic ability of gastric cancer cells. The truncated or extended sequence information is shown in Table 6, and the modified sequence information is shown in Table 7.

[0141] Table 6 Truncated or extended sequences of GMAN3

[0142] Table 7 Truncated or extended modified sequences of GMAN3 Note: * represents phosphorothioate modification; m represents 2'OMe modification; non-underlined letters represent deoxyribonucleic acid; underlined letters represent ribonucleotides.

[0143] The specific experimental process is as follows:

[0144] 1. Cell Transfection with ASO

[0145] Gastric cancer cell line MKN45 and gastric cancer PDX cells in the logarithmic growth phase were plated in six-well plates. Transfection was performed when the cell density reached about 50%. 1 mL of fresh culture medium was replaced 1 h before transfection. The transfection system (ASO: 2'OMe-Ctrl, 2'OMe-GMAN3-15nt, 2'OMe-GMAN3-17nt, 2'OMe-GMAN3-25nt, 2'OMe-GMAN3-30nt, 2'OMe-GMAN3-19nt) was prepared according to the instructions of the transfection reagent. After incubation for 15 min, the cells were added to the cell culture medium, shaken, and the culture medium was replaced after 24 h.

[0146] 2. Western Blot Detection of Ephrin A1 Expression in Gastric Cancer Cells

[0147] The total protein of the transfected cells was extracted. The total protein of the cells was extracted using RIPA lysis buffer (purchased from Shanghai Biyuntian) and the operation was carried out according to the instructions. Gastric cancer cells (about 1×10 6) was added with 200 μl of RIPA lysis buffer containing 1× PMSF (purchased from Shanghai Bio-Tech). The cells were scraped off with a cell scraper and transferred to a 1.5 mL EP tube. The cells were disrupted using a sonicator at low temperature. The cell lysate was then centrifuged at 12,000 g at 4°C for 10 minutes. The supernatant was transferred to a new 1.5 mL EP tube, and loading buffer was added. The mixture was mixed and incubated in a 100°C water bath for 10 minutes. The resulting lysate was then analyzed for protein expression by Western blotting.

[0148] 3. Gastric Cancer Cell Invasion Assay

[0149] Transfected cells were further tested for invasion. For the invasion assay, transfected cells were plated onto Transwell chambers pretreated with 0.5% gelatin. 750 μL of 10% FBS-containing 1640 medium was added to the lower chamber and the cells were incubated in an incubator for the appropriate time. After the migration period, the transwells were fixed with 4% PFA for 10 minutes. The cells were then stained with 0.1% crystal violet for 15 minutes. Excess crystal violet was gently washed off with PBS. Cells in the upper layer of the Transwell chamber were carefully wiped away with a cotton swab. Cells invading the lower layer of the Transwell chamber were observed under a microscope, photographed, and analyzed statistically.

[0150] 4. Experimental Results

[0151] By transfecting 2'OMe-15nt / 17nt / 25nt / 30nt / 19nt ASO sequences into the gastric cancer cell line MKN45 and gastric cancer PDX cells, the inventors found that truncated or extended ASO sequences could still inhibit Ephrin A1 protein expression (see Figure 12). In addition, both truncated and extended ASO sequences were able to significantly inhibit the invasive ability of gastric cancer cells (see Figure 13). Therefore, truncated or extended ASO sequences derived from GMAN1-GMAN6, namely the naked ASO sequences shown in SEQ ID NOs. 3, 6-432 and their modified sequences disclosed in the Examples of this application, have the potential to treat gastric cancer metastasis.

[0152] The technical features of the above-mentioned embodiments can be combined arbitrarily. In order to make the description concise, not all possible combinations of the technical features in the above-mentioned 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.

[0153] The above-described embodiments merely represent several implementation methods of the present disclosure. While the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that a person of ordinary skill in the art could make various modifications and improvements without departing from the spirit of the present disclosure, all of which fall within the scope of protection of the present disclosure. Therefore, the scope of protection of the present patent shall be determined by the appended claims.

[0154] Table 8 ASO naked sequences

Claims

1. An antisense oligonucleotide or a pharmaceutically acceptable salt thereof, wherein: The antisense oligonucleotide specifically targets lncRNA GMAN, and the nucleotide sequence of lncRNA GMAN is as SEQ ID NO:

1.

2. The antisense oligonucleotide or a pharmaceutically acceptable salt thereof according to claim 1, wherein The antisense oligonucleotide comprises a nucleotide sequence selected from any one of SEQ ID NOs: 2-801; Preferably, the antisense oligonucleotide is selected from the nucleotide sequence shown in any one of SEQ ID NOs: 2-432; Preferably, the antisense oligonucleotide is selected from the nucleotide sequence shown in any one of SEQ ID NOs: 2-7; Preferably, the nucleotide sequence of the antisense oligonucleotide is shown in SEQ ID NO:

4.

3. The antisense oligonucleotide or a pharmaceutically acceptable salt thereof according to any one of claims 1 or 2, wherein The antisense oligonucleotide comprises modified nucleotides; Preferably, at least part of the nucleotides of the antisense oligonucleic acid are modified nucleotides; Preferably, each nucleotide of the antisense oligonucleotide is a modified nucleotide; Preferably, the modified nucleotide is selected from 2'-methoxy modified nucleotides, 2'-fluoro modified nucleotides, 2'-deoxy nucleotides, inosine or inosine nucleotides, 5'-phosphate nucleotides, 2',3'-seco nucleotide mimetics, locked nucleotides (LNA), unlocked nucleic acid nucleotides (UNA), glycol nucleic acid nucleotides (GNA), bicyclic nucleic acids (BNA), 2'-F-arabino nucleotides, 2'-methoxyethyl nucleotides, abasic nucleotides, ribitol, inverted nucleotides, inverted abasic nucleotides (Invab), inverted 2'-OMe nucleotides, inverted 2'-deoxy nucleotides, 2'-amino modified nucleotides, 2'-alkyl modified nucleotides, morpholino nucleotides, 3'-OMe nucleotides, phosphate group modified nucleotides, terminal nucleotides linked to a cholesterol derivative or a dodecanoic acid bisdecylamide group, 2'-amino modified nucleotides, phosphoramidates, non-natural bases containing nucleotides or 5'-phosphate mimetic modified nucleotides; Preferably, the 5'-phosphate mimetic is selected from 5'-oxymethylphosphonate, 5'-vinylphosphonate, a 5'-vinylphosphonate analogue, 5'vinylphosphonate-2'-N-acetyl or 5'-malonylphosphonate.

4. The antisense oligonucleotide or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 3, wherein The antisense oligonucleotide comprises at least one internucleotide linkage; Preferably, the internucleotide bond is a phosphorothioate bond; Preferably, the nucleotide sequence of the antisense oligonucleotide comprises a sequence selected from any one of SEQ ID NOs: 433-436 or a fragment thereof; Preferably, the nucleotide sequence of the antisense oligonucleotide is shown in any one of SEQ ID NOs: 433-436.

5. The antisense oligonucleotide or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 4, wherein: Any two nucleotides in the antisense oligonucleotide contain a phosphorothioate bond; Preferably, the antisense oligonucleotide further comprises at least one 2'-methoxy modified nucleotide; Preferably, any two nucleotides in the antisense nucleotide contain a phosphorothioate bond, and the nucleotides at positions 1 to 5 from the 5' end and the 3' end are 2'-methoxy-modified nucleotides; Preferably, the antisense oligonucleotide is selected from the nucleotide sequence shown in any one of SEQ ID NOs: 437-440; Preferably, the nucleotide sequence of the antisense oligonucleotide is shown as SEQ ID NO:

439.

6. A conjugate comprising the antisense oligonucleotide or a fragment thereof according to any one of claims 1 to 5, and optionally a pharmaceutically acceptable carrier; Preferably, the carrier is selected from any one of galactosamine GalNac modification, lipid nanoparticles, adeno-associated virus vectors, adenovirus vectors, albumin nanoparticles or exosomes; Preferably, the carrier is a lipid nanoparticle.

7. A pharmaceutical composition comprising the antisense oligonucleotide or a fragment thereof according to any one of claims 1 to 5 or the conjugate according to claim 6, and at least another pharmaceutical component.

8. The pharmaceutical composition according to claim 7, wherein The pharmaceutical component is selected from trastuzumab, ramucirumab, apatinib, lapatinib, pertuzumab, everolimus, bevacizumab, olaparib, TDM-1, nimotuzumab, cetuximab, panitumumab, erlotinib, icotinib, gefitinib, afatinib, sorafenib, sunitinib, regorafenib, crizotinib, onartuzumab, ponatinib, dovitinib, fluorouracil (5-FU), capecitabine, S-1, oxaliplatin, epirubicin, capecitabine, paclitaxel, docetaxel, cisplatin, carboplatin, irinotecan, pembrolizumab, nebulizumab, atezolizumab, avelumab or duivalumab.

9. A reagent or kit comprising the antisense oligonucleotide or fragment thereof according to any one of claims 1 to 5, the conjugate according to claim 6, or the pharmaceutical composition according to claims 7 to 8; Preferably, the dosage form of the agent includes an injection; Preferably, the injection includes aseptic or sterilized solution, water injection, oil injection or powder injection.

10. Use of the antisense oligonucleotide or fragment thereof according to any one of claims 1 to 5, the conjugate according to claim 6, the pharmaceutical composition according to any one of claims 7 to 8, or the reagent or kit according to claim 9 in the preparation of a drug for preventing or treating cancer or inhibiting cancer metastasis; Preferably, the cancer is selected from one or more of nasal cavity and paranasal sinus malignancies, nasopharyngeal cancer, oral cancer, laryngeal cancer, salivary gland tumors, intracranial tumors, thyroid cancer, tongue cancer, lung cancer, esophageal cancer, cardia cancer, breast cancer, mediastinal tumors, gastric cancer, colorectal cancer, rectal cancer, liver cancer, pancreatic cancer and periampullary cancer, small intestinal malignancies, kidney cancer, prostate cancer, bladder cancer, cervical cancer, ovarian cancer, skin malignant melanoma or lymphoma.

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