ASO inhibitors of the CREPT gene and their inhibitory effect on liver cancer

Abstract

This invention provides an antisense oligonucleotide (ASO) that is complementary to the CREPT gene sequence and effectively inhibits CREPT expression. The antisense oligonucleotide is selected from those having nucleotide sequences as shown in any of SEQ ID Nos. 1-3. This antisense oligonucleotide (ASO) efficiently and specifically targets CREPT, significantly reducing CREPT expression levels in tumor cells, thereby reducing the clonogenic ability of tumor cells and significantly decreasing their invasiveness. This invention also provides extracellular vesicles and pharmaceutical compositions that, when administered to patients, can significantly reduce tumor burden. The antisense oligonucleotide (ASO) of this invention can be used to treat liver cancer.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to ASO inhibitors of the CREPT gene and their inhibitory effect on liver cancer. Background Technology

[0002] CREPT (Cell cycle-related and expression elevated protein in tumor) is a cancer-related gene discovered and cloned by the applicant's laboratory. Compared to adjacent normal tissues and normal tissues, it is highly expressed in various tumor tissues, including liver cancer, pancreatic cancer, glioma, prostate cancer, and colorectal cancer, and is negatively correlated with patient prognosis (Lu et al., 2012). Numerous research groups have reported a consistent phenomenon in different tumors: overexpression of CREPT promotes tumor growth, while inhibition or knockout of CREPT has the opposite effect (Li et al., 2021; Lu et al., 2012). Moreover, CREPT plays an important role in transcriptional regulation, not only interacting with RNA polymerase II (RNAPII) to regulate the expression of target genes such as cyclin D1, but also participating in the regulation of multiple signaling pathways such as Wnt and STAT3, thereby promoting cell growth and tumorigenesis (Lu et al., 2012). Numerous indications suggest that CREPT is a promising new target for cancer treatment.

[0003] The published patent document WO02 / 44321A2 describes in its specification that the nucleoside located at the center of siRNA opposite to the target RNA cleavage site is an important specificity determinant, and even a change in a single nucleoside can reduce RNAi to an undetectable level (see the first paragraph on page 50 of the specification of this patent document).

[0004] However, there are no reports in the current technology of inhibiting CREPT at the mRNA expression level to suppress CREPT-induced tumor formation. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention provides antisense oligonucleotides (ASOs), which are complementary to the CREPT gene sequence and can effectively inhibit CREPT expression; the antisense oligonucleotides are selected from antisense oligonucleotides having nucleotide sequences as shown in any of SEQ ID No. 1 to 3.

[0006] Antisense oligonucleotides (ASOs) are a class of molecular drugs that regulate gene expression at the gene level by specifically binding to target gene DNA or mRNA. Antisense oligonucleotide drugs generally contain 12-28 single-stranded nucleic acids with a specific sequence complementary to the target nucleic acid bases. They can regulate target gene expression by forming an ASO-RNA complex duplex with the target RNA, utilizing site-specific effects. They can also exert their effects by scavenging the RNA strand of the ASO-RNA complex duplex using ribonuclease H. The ASOs used in this invention may contain at least one non-naturally occurring nucleoside.

[0007] In this document, “inhibition of CREPT expression” means that ASO reduces the expression of CREPT gene transcripts and / or CREPT protein in cells or tissues. In some embodiments, the term “inhibition” means complete inhibition (100% inhibition or undetectable level) of CREPT gene transcripts or CREPT protein. In other aspects, the term “inhibition” means inhibition of at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% of the expression of CREPT gene transcripts and / or CREPT protein in cells or tissues.

[0008] CREPT, also known as RPRD1B or C20ORF77, is a gene located on human chromosome 20, discovered in recent years. In humans, the CREPT gene contains five exons, encoding a protein composed of 312 amino acids. CREPT is highly expressed in various tumors, promoting cell proliferation and tumor formation. CREPT has a promoting effect on various malignant tumors, including colorectal cancer, pancreatic cancer, and gastric cancer, and it can participate in the regulation of multiple signaling pathways, such as Wnt, MAPK, STAT3, and NF-κB.

[0009] The antisense oligonucleotide of the present invention may be an antisense oligonucleotide having a homology of ≥75% with any of the nucleotide sequences shown in SEQ ID No. 1 to 3, specifically having a homology of 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%.

[0010] In molecular evolution research, homology generally refers to the degree of similarity between the nucleotide sequences of two nucleic acid molecules or the amino acid sequences of two protein molecules.

[0011] As some embodiments of the present invention, the antisense oligonucleotide further comprises one or more nucleoside analogs.

[0012] "Nucleoside analogs" are variants of natural nucleosides (such as DNA or RNA nucleosides) obtained through modifications to their sugar and / or base moieties. In principle, in the case of oligonucleotides, analogs may only be "silencing" or "equivalent" to the natural nucleoside, meaning they have no functional effect on the way the oligonucleotide acts to inhibit target gene expression. However, such "equivalent" analogs can still be useful if, for example, they are easier or cheaper to manufacture, more stable under storage or manufacturing conditions, or represent a tag or label. However, in some embodiments, nucleoside analogs will have a functional effect on the way ASOs act to inhibit expression: for example, by producing increased binding affinity to the target and / or increased resistance to intracellular nucleases and / or increased ease of transport into the cell. Depending on the natural structure of the nucleotide, chemical modifications can be made to the phosphate backbone, ribose moieties, or bases to improve its stability and reduce activation of the innate immune system.

[0013] In this invention, the ASO may contain more than 1, more than 2, more than 3, more than 4, more than 5, more than 6, more than 7, more than 8, more than 9, more than 10, more than 11, more than 12, more than 13, more than 14, more than 15, more than 16, more than 17, more than 18, or more than 19 nucleoside analogs. In some embodiments, the nucleoside analogs may be the same. In some embodiments, the nucleoside analogs may be different.

[0014] The term "nucleobase" includes purine (e.g., adenine and guanine) and pyrimidine (e.g., uracil, thymine, and cytosine) moieties present in nucleosides and nucleotides that form hydrogen bonds during nucleic acid hybridization. In this document, the term "nucleobase" also encompasses modified nucleobases that may differ from naturally occurring nucleobases but are functional during nucleic acid hybridization. In some embodiments, the nucleobase moieties are modified by modifying or substituting nucleobases.

[0015] In some embodiments, the nucleobase moiety is modified by changing the purine or pyrimidine to a modified purine or pyrimidine, such as a substituted purine or substituted pyrimidine, such as a nucleobase selected from isocytosine, pseudoisocytosine, 5-methylcytosine, 5-thiazono-cytosine, 5-propynyl-cytosine, 5-propynyl-uracil, 5-bromouracil, 5-thiazono-uracil, 2-thiouracil, 2'-thiothymine, inosine, diaminopurine, 6-aminopurine, 2-aminopurine, 2,6-diaminopurine, and 2-chloro-6-aminopurine.

[0016] As some embodiments of the present invention, the antisense oligonucleotide further comprises one or more 5'-methyl-cytosine nucleobases.

[0017] The monomers of ASO described in this invention are coupled together by a linking group. Suitably, each monomer is linked to a 3' adjacent monomer by a linking group. In some embodiments, the continuous nucleotide sequence contains one or more modified internucleotide links. The terms "linking group" or "internucleotide link" are intended to refer to a group capable of covalently coupling two nucleosides together. Non-limiting examples include phosphate groups and thiophosphate groups. In some embodiments, the internucleotide link is modified from its normal phosphodiester to one that is more resistant to nuclease attack, such as a thiophosphate that can be cleaved by RNase H and also allows antisense repression pathways to reduce the expression of target genes.

[0018] As some embodiments of the present invention, the continuous nucleotide sequence of the antisense oligonucleotide includes one or more modified nucleoside-to-nucleotide bonds.

[0019] In some implementations, nucleoside analogs are modified at the 2' position. 2' modification offers significant advantages because conformational maintenance at this site enhances chemical stability, and 2' modification has proven highly suitable for applications, preventing 2'-OH-mediated chain breaks. Furthermore, due to the proximity of the 2' position to the adjacent phosphate group, it provides better protection against nuclease attack. For example, the incorporation of 2'-OMe and 2'-deoxy-2'-fluorine (2'-F) modifications significantly reduces the ability of nucleases to degrade siRNA.

[0020] As some embodiments of the present invention, one or more of the nucleoside analogs include 2'-O-alkyl-RNA; 2'-O-methylRNA (2'-OMe); 2'-alkoxy-RNA; 2'-O-methoxyethyl-RNA (2'-MOE); 2'-amino-DNA; 2'-fluoro-RNA; 2'-fluoro-DNA; Arabica nucleic acid (ANA); 2'-fluoro-ANA; or bicyclic nucleoside analogs.

[0021] The ASO of the present invention may comprise one or more nucleosides having a modified sugar moiety (i.e., a modification of the sugar moiety when compared to the ribosome found in DNA and RNA). Many nucleosides modified with a ribosome have been prepared, primarily with the aim of improving certain properties of the oligonucleotide, such as affinity and / or nuclease resistance. In some embodiments, the modified sugar moiety may be a change in the ribocycle structure. In some embodiments, sugar modification may also be a modification made by changing the substituents on the ribocycle to groups other than hydrogen or by altering the naturally occurring 2'-OH group in the RNA nucleoside.

[0022] As some embodiments of the present invention, one or more of the nucleoside analogs are sugar-modified nucleosides.

[0023] As some embodiments of the present invention, the sugar-modified nucleoside is a 2' sugar-modified nucleoside that enhances affinity.

[0024] Locked nucleosides (LNAs) are modified nucleosides that contain a linker group (called a binucleotide or bridge) between the C2' and C4' ends of the ribose ring (i.e., a 2'-4' bridge), which restricts or locks the conformation of the ribose ring. These nucleosides are also referred to in the literature as bridged nucleic acids or bicyclic nucleic acids (BNAs). When LNAs are incorporated into oligonucleotides of complementary RNA or DNA molecules, the locking of the ribose conformation is associated with enhanced hybridization affinity (double-strand stabilization).

[0025] As some embodiments of the invention, one or more of the nucleoside analogs include LNA. LNA is a 2'-4' biradical-bridged nucleoside.

[0026] As some embodiments of the invention, one or more of the nucleotide analogs are selected from the group consisting of: restricted ethyl nucleoside (cEt), 2',4'-restricted 2'-O-methoxyethyl (cMOE), α-L-LNA, β-D-LNA, 2'-O,4'-C-ethylene-bridged nucleic acid (ENA), amino-LNA, oxy-LNA, thio-LNA, and any combination thereof.

[0027] As some embodiments of the present invention, the one or more modified nucleoside links are thiophosphate links.

[0028] The present invention also provides extracellular vesicles comprising antisense oligonucleotides (ASOs), wherein the antisense oligonucleotides are as described above.

[0029] The term "extracellular vesicle" (EV) refers to a cell-derived vesicle containing a membrane encapsulating its internal space. Extracellular vesicles include all membrane-bound vesicles (e.g., exosomes, nanovesicles) with a diameter smaller than that of the cell from which they originate. In some embodiments, the extracellular vesicles have a diameter ranging from 20 nm to 1000 nm and may contain various macromolecular payloads contained within an internal space (i.e., a lumen), displayed on the outer surface of the extracellular vesicle, and / or transmembrane.

[0030] In some embodiments of the present invention, the extracellular vesicles are exosomes.

[0031] The term "exosome" refers to an extracellular vesicle with a diameter between 20 and 300 nm (e.g., between 40 and 200 nm). Exosomes comprise a membrane enclosing an internal space (i.e., a lumen) and, in some embodiments, can be produced from cells (e.g., production cells) via direct plasma membrane budding or via late endosome fusion with the plasma membrane. In some aspects, exosomes include a scaffold portion. As described below, exosomes can be derived from production cells and isolated from them based on their size, density, biochemical parameters, or combinations thereof. In some embodiments, the extracellular vesicles (e.g., exosomes) disclosed herein are produced by cells expressing one or more transgenic products.

[0032] As some embodiments of the present invention, the extracellular vesicles further include an exogenous targeting portion that causes the extracellular vesicles to target the liver.

[0033] In some embodiments, the EV (e.g., exosome) comprises a targeting moiety, such as an exogenous targeting moiety. In some embodiments, the exogenous targeting moiety comprises a peptide, an antibody or an antigen-binding fragment thereof, a chemical compound, an RNA aptamer, or any combination thereof. In some embodiments, the targeting moiety comprises a microprotein, a designed ankyrin repeat protein (darpin), an anticarrier protein, adnectin, an aptamer, a peptide mimic molecule, a natural ligand of a receptor, a camelid nanobody, or any combination thereof. In some embodiments, the exogenous targeting moiety comprises a full-length antibody, a single-domain antibody, a heavy-chain-only antibody (VHH), a single-chain antibody, a shark heavy-chain-only antibody (VNAR), scFv, Fv, Fab, Fab', F(ab')2, or any combination thereof. In some embodiments, the antibody is a single-chain antibody. In some embodiments, the targeting moiety directs the exosome to the liver.

[0034] As in some embodiments of the present invention, the extracellular vesicle further includes an anchoring portion that anchors the antisense oligonucleotide to the extracellular vesicle.

[0035] Anchoring portions (AMs) can be used to anchor ASO to the extracellular vesicles of the present invention. In some embodiments, anchoring portions (AMs) can be one or more. Anchoring portions can be inserted into the lipid bilayer of extracellular vesicles (e.g., exosomes), thereby allowing exosomes to load ASO. In some embodiments, ASO is directly connected to the anchoring portion or connected via a connector. In some embodiments, the anchoring portion increases the loading of the ASO of the present invention in extracellular vesicles.

[0036] The present invention also provides pharmaceutical compositions comprising: a. the said antisense oligonucleotide or the said extracellular vesicle; and b. a pharmaceutically acceptable diluent, carrier, or salt.

[0037] A pharmaceutical composition refers to a complex of two or more drugs or active molecules that, through synergistic action or effect, aim to treat a disease or improve a health condition. The pharmaceutical composition comprises an EV (e.g., exosome) of the present disclosure with the desired purity and a pharmaceutically acceptable carrier or excipient. The pharmaceutically acceptable excipient or carrier may be determined in part by the specific composition being administered and by the specific method of administering the composition.

[0038] As some embodiments of the present invention, the pharmaceutical composition further includes a second therapeutic agent.

[0039] In some embodiments, the second therapeutic agent and the ASO are co-contained in the extracellular vesicle. In some embodiments, the second therapeutic agent and the ASO are contained in different extracellular vesicles. In some embodiments, the second therapeutic agent and the ASO are not simultaneously contained in the extracellular vesicle. In some embodiments, the second therapeutic agent and the ASO are administered simultaneously. In some embodiments, the second therapeutic agent and the ASO are administered sequentially. In some embodiments, the second therapeutic agent is administered before the ASO. In some embodiments, the second therapeutic agent is administered after the ASO.

[0040] In some embodiments of the present invention, the therapeutic agent is selected from drugs for treating liver cancer.

[0041] As some embodiments of the present invention, the pharmaceutical composition of the present invention may be administered via intravenous injection, intramuscular injection, subcutaneous injection, rectal instillation (rectal administration), eye drops, nasal spray, or oral spray (inhaler), or may be administered topically (surface) or systemically (transdermal). The preferred administration methods are intravenous injection or subcutaneous injection.

[0042] Examples of carriers or diluents include, but are not limited to, water, saline, Ringer's solution, dextran solution, and 5% human serum albumin. The use of such media and compounds for pharmaceutically active substances is well known in the art. Unless any conventional media or compound is incompatible with the extracellular vesicles described herein. The carrier or diluent may be selected depending on the method or route of administration.

[0043] As some embodiments of the present invention, the dosage of the pharmaceutical composition of the present invention is 0.0001~5000 mg, and may be selected from 0.0001, 0.0002, 0.0003, 0.0004, 0.0005, 0.0006, 0.0007, 0.0008, 0.0009, 0.001, 0.002, 0.003, 0.004, 0.005, 0.006, 0.007, 0.008, 0.009, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 120 0, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000, 3100, 3200, 3300, 3400, 3500, 3600, 3700, 3800, 3900, 4000, 4100, 4200, 4300, 4400, 4500, 4600, 4700, 4800, 4900, or 5000 mg.

[0044] As some embodiments of the present invention, the carrier is selected from aqueous carriers, liposomes, polymers, peptides and nanoparticles.

[0045] In some embodiments, pharmaceutically acceptable carriers include one or more aqueous carriers, liposomes, polymers, or peptides. In some embodiments, the aqueous carrier may be, for example, RNase-free water or RNase-free buffer. In some embodiments, the liposomes may be neutral liposomes. In some embodiments, the neutral liposomes specifically target the liver. A method for preparing neutral lipid nanoparticles (LNPs) can be found in the literature: A Virus-Inspired Inhalable Liponanogel Induces Potent Antitumor Immunity and Regression in Metastatic Lung Tumors. Cancer Res. 2024 Jul 15;84(14):2352-2363.

[0046] The present invention also provides a commercial kit comprising the aforementioned antisense oligonucleotide, the aforementioned extracellular vesicles, or the aforementioned pharmaceutical composition, and an instruction manual describing the administration method and dosage.

[0047] As some embodiments of the invention, the commercial pillbox includes one or more containers filled with one or more components of the pharmaceutical composition described herein (such as one or more exosomes provided herein).

[0048] The present invention also provides a diagnostic kit comprising the aforementioned antisense oligonucleotide (ASO), the aforementioned extracellular vesicles, or the aforementioned pharmaceutical composition, and instructions for use.

[0049] As some embodiments of the present invention, the diagnostic kit includes one or more containers filled with one or more components of the pharmaceutical composition described herein (such as one or more exosomes provided herein).

[0050] As some embodiments of the present invention, the diagnostic kit of the present invention may further include instructions for use describing how to apply the antisense oligonucleotide (ASO), the extracellular vesicles, or the pharmaceutical composition to a sample to be tested, thereby determining whether the subject from which the sample originates has cancer.

[0051] The present invention also provides a method for reducing or inhibiting the expression of the CREPT gene in cells in vitro, the method comprising: administering the antisense oligonucleotide (ASO), the extracellular vesicle, or the pharmaceutical composition thereof to cells expressing the CREPT gene, wherein the expression of the CREPT protein in the cells is reduced or inhibited after administration.

[0052] The present invention also provides a method for reducing the proliferation of cancer cell clones and reducing the invasive and metastatic ability of cancer cells in vitro. The method includes: applying the antisense oligonucleotide (ASO), the extracellular vesicles, or the pharmaceutical composition to the cancer cells, wherein the cancer cells, after application, exhibit reduced clonal proliferation, promoted cancer cell apoptosis, and reduced invasive ability.

[0053] The present invention also provides a treatment method for cancer, which includes administering the antisense oligonucleotide, the extracellular vesicle, or the pharmaceutical composition of the present invention to a patient, or administering the drug to the patient according to the instructions for use using the commercial kit described herein.

[0054] The present invention also provides the use of the aforementioned antisense oligonucleotide (ASO), the aforementioned extracellular vesicles, or the aforementioned pharmaceutical composition, wherein the use is in the preparation of a medicament for treating cancer.

[0055] In some embodiments of the present invention, the cancer is selected from liver cancer.

[0056] Liver cancer (HCC), or malignant tumor of the liver, can be divided into two main categories: primary and secondary. Primary malignant liver tumors originate from the epithelial or mesenchymal tissue of the liver; the former is called primary liver cancer, which is a highly prevalent and extremely dangerous malignant tumor. The latter, called sarcoma, is less common compared to primary liver cancer. Secondary or metastatic liver cancer refers to malignant tumors originating from multiple organs throughout the body that invade the liver. It is commonly seen as liver metastasis from malignant tumors of the stomach, bile ducts, pancreas, colorectal region, ovary, uterus, lung, and breast.

[0057] As some embodiments of the present invention, the liver cancer includes primary liver cancer and secondary liver cancer.

[0058] In some embodiments of the invention, the drug is administered to mammals. In some embodiments of the invention, the drug is administered to humans and mice.

[0059] As used herein, “administration” means giving a subject the composition disclosed herein containing EVs (e.g., exosomes) via a pharmaceutically acceptable route. Routes of administration may be intravenous, such as intravenous injection and intravenous infusion. Other routes of administration include, for example, subcutaneous, intramuscular, oral, nasal, and pulmonary administration.

[0060] As described above, the ASO inhibitor of the CREPT gene of the present invention and its tumor-suppressing effect have the following beneficial effects:

[0061] The oligosense nucleotides (ASOs) screened in this invention can efficiently and specifically target CREPT and significantly reduce the expression level of CREPT in liver cancer cells;

[0062] The oligosense nucleotides (ASOs) of the present invention can reduce the clonogenic ability of liver cancer cells and significantly reduce the invasive and migratory abilities of liver cancer cells.

[0063] The ASO targeting CREPT of this invention significantly reduced the tumor burden of liver cancer in mice. Attached Figure Description

[0064] Figure 1 This invention demonstrates the screening of oligosense nucleotides (ASOs) that efficiently and specifically target CREPT, wherein... Figure 1 A protein imprinting assay was used to detect the effect of ASO targeting CREPT on CREPT expression levels in HepG2 hepatocellular carcinoma cells. Figure 1 B protein imprinting assay to detect the effect of ASO targeting CREPT on CREPT expression level in Hep3B liver cancer cells;

[0065] Figure 2 This is to validate the effects of oligosense nucleotides (ASOs) targeting CREPT on the proliferation and invasion of liver cancer cell lines. Figure 2 A protein imprinting assay was used to detect the effect of ASO targeting CREPT on CREPT expression levels in Hep3B and HepG2 liver cancer cells. Figure 2 B. Detection of the effect of ASO targeting CREPT on the clonogenic ability of Hep3B and HepG2 liver cancer cells. Figure 2 C. Detection of the effect of ASO targeting CREPT on the invasion and metastasis of Hep3B and HepG2 liver cancer cells;

[0066] Figure 3 The effect of single-point mutations specifically targeting CREPT oligosense nucleotides (ASOs) is shown. Figure 3 A is a single-point mutation at ASO 23 and 69 in CREPT. Figure 3 B. Detect the effect of ASO single-point mutation in CREPT on the invasive ability of Hep3B liver cancer cells;

[0067] Figure 4 This is shown as an in vivo validation of the effect of the CREPT-targeting oligosense nucleotide ASO on orthotopic liver cancer. Figure 4 A shows the effect of ASO administration time on CREPT expression levels in Hep3B liver cancer cells in a liver cancer model. Figure 4 B shows the effect of ASO targeting CREPT on the in situ tumor weight of Hep3B cells. Figure 4 C shows the effect of ASO targeting CREPT on tumor size. Detailed Implementation

[0068] To make the technical means, creative features, achieved objectives, and effects of this invention readily understandable, the technical solutions in the embodiments of this invention will be clearly and completely described below in conjunction with the embodiments of this invention. Obviously, the described embodiments are merely some embodiments of this invention, and not all embodiments. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0069] [Experimental Materials]

[0070] HepG2 liver cancer cells (Fenghui Biotechnology, WZ010 32)

[0071] Hep3B liver cancer cells (Fenghui Biotechnology, CL0136)

[0072] Mice (Beijing Vital River, Balb / c-null)

[0073] Lipofectamine 2000 transfection reagent (Thermo Fisher Scientific, 2455799)

[0074] Iodine solution (Qingdao Haishi Hainuo Yingnuowei Disinfection Technology Co., Ltd., 0604)

[0075] Meloxicam (Mexican, 150252)

[0076] Stereotactic transducer (Reward, R540)

[0077] Suture needle 5-0 (Ningbo Medical Suture Needle Co., Ltd., 230828)

[0078] microCT (Tsinghua University Instrument Sharing Platform, A18000014)

[0079] The preparation method of neutral lipid nanoparticles (LNPs) can be found in the literature: A Virus-Inspired Inhalable Liponanogel Induces Potent Antitumor Immunity and Regression in Metastatic Lung Tumors. Cancer Res. 2024 Jul 15;84(14):2352-2363.

[0080] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0081] Example 1

[0082] To screen for highly efficient and specific oligosense nucleotides (ASOs) targeting CREPT, we designed 72 ASO sequences from the full-length 5'UTR to 3'UTR of the CREPT transcript in NCBI that could specifically target CREPT RNA sequences. The nucleotide sequences of ASO23, ASO56, and ASO69 are shown in Table 1 as SEQ ID Nos. 1-3, respectively. ASO-Ctrl is a random sequence used as a control group.

[0083] First, we examined whether these ASOs could reduce CREPT expression at the protein level: When the density of HepG2 and Hep3B liver cancer cells was around 40% during their growth phase, different ASO sequences were transfected into the liver cancer cells using Lipofectamine 2000 transfection reagent. The working concentration of the ASO sequences was 50 nM, and the cells were treated for three days. Afterward, protein lysis buffers were extracted for Western blotting experiments.

[0084] The results showed that up to 27-30 ASOs significantly reduced the expression level of CREPT in liver cancer cells. Figure 1 Through preliminary experiments, three oligonucleotides specifically targeting CREPT were identified and screened (Table 1), with sequence numbers SEQ ID No. 1-3. ASO-Ctrl was used as a negative control, with sequence number SEQ ID No. 4.

[0085] Table 1 ASO Sequence and Number

[0086]

[0087] Example 2

[0088] To further illustrate the effectiveness of our screened ASOs, we tested them in hepatocellular carcinoma (HCC) cells. Similarly, when HepG2 and Hep3B HCC cells were in their growth phase (approximately 40% cell density), different ASO sequences were transfected into the cells using Lipofectamine 2000 transfection reagent at a working concentration of 50 nM for three days. Three days later, protein lysis buffers were extracted for Western blotting experiments. We found that the oligosense nucleotides (ASOs) targeting CREPT significantly reduced CREPT expression levels in HepG2 and Hep3B HCC cells. Figure 2 A). Simultaneously, ASO-treated cells were subjected to a cell colony formation assay. The results showed that ASO-targeted interference with CREPT expression significantly affected the in vitro colony formation ability of HepG2 and Hep3B liver cancer cells. Figure 2B). Furthermore, cell-cell experiments on ASO-treated cells revealed that ASO targeting CREPT also led to a decrease in the in vitro invasion and migration abilities of HepG2 and Hep3B liver cancer cells. Figure 2 C). These results demonstrate that our designed oligosense nucleotide ASO targeting CREPT is effective in inhibiting the proliferation, migration, and invasion of different types of hepatocellular carcinoma cells.

[0089] To verify the specificity of the ASOs of the CREPTs we screened, we randomly selected ASOs 23# and 69# for single-point mutation. Figure 3 A), and ASO-treated cells were subjected to cell invasion experiments. The results showed that the ASO NC group significantly reduced the invasive ability of liver cancer cells. When ASO 23# and ASO 69# were mutated at single points, the mutated ASO lost its inhibitory effect on the invasion of liver cancer cells. Figure 3 B). This demonstrates that the ASO sequence is essential for inhibiting CREPT, and the 6th base T of ASO23 and the 12th base A of ASO69 are crucial for inhibiting the invasion of liver cancer cells.

[0090] Example 3

[0091] To investigate the effect of the CREPT-targeting oligosense nucleotide ASO on the proliferation of orthotopic hepatocellular carcinoma cells, we constructed an orthotopic hepatocellular carcinoma model by injecting Hep3B hepatocellular carcinoma cells into the livers of mice. The experimental procedure is as follows:

[0092] First, the mice were anesthetized in a small anesthesia chamber. The fur on the mice's abdomen was shaved with an electric razor, and the abdomen was disinfected with an alcohol swab. The mice were then anesthetized again using a face mask. The anesthesia was maintained throughout the procedure, and the mice's anesthesia status was confirmed by touching them to check for responsiveness. Using sterilized scissors, a 1 cm incision was made in the middle of the mouse's abdomen, sequentially cutting through the skin and peritoneum. Once the liver lobe was exposed, the injection site was determined. An insulin needle containing Hep3B cells was inserted parallel to the liver lobe and slowly injected 60 μL (approximately 1 x 10^6 Hep3B cells). 6 After the injection, press the injection site with a cotton ball for about 1 minute, then slowly rotate the needle out. Next, use a 5-0 suture needle with thread to suture the peritoneum twice, and then suture the skin twice. Disinfect the suture site with iodine, and then inject meloxicam subcutaneously to relieve pain in the mouse. Then transfer the mouse to an electric blanket for about 10 minutes, during which time observe the mouse's heart rate and whether its eyes turn white. After it wakes up, it can be returned to its original cage. Three days later, inject the mouse with meloxicam again to further reduce the pain caused by the surgery.

[0093] Five days later, microCT scans were used to detect the presence of tumors in the liver. Mice with tumors were then grouped for further drug treatment experiments. Nine days after tumor growth, 100 µL (approximately 300 µg) of ASO drug encapsulated in neutral lipid nanoparticles (LNP) was injected once via the tail vein. This was repeated every five days for a total of four injections. Twenty-nine days later, microCT scans were used again to detect tumors in the liver. Subsequently, the mice were dissected, and their livers were examined and photographed to observe the presence and size of tumors. Figure 4 A shows a schematic diagram of the drug delivery strategy for ASO in an orthotopic model of liver cancer. Figure 4 B shows the effect of CREPT's ASO inhibitor on Hep3B tumor formation in the liver. Results showed that, compared to the blank Nacl group and the ASO-Ctrl control group (i.e., the ASO-Ctrl sequence in Table 1), CREPT's ASO inhibitor significantly reduced the tumor burden of mouse liver cancer. Figure 4 BC).

[0094] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make some modifications or alterations to the methods and techniques disclosed above without departing from the scope of the present invention to create equivalent embodiments. Any simple modifications, equivalent changes and alterations made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the scope of the present invention.

Claims

1. An antisense oligonucleotide (ASO), characterized in that, The antisense oligonucleotide is complementary to the CREPT gene sequence and can effectively inhibit the expression of CREPT; the nucleotide sequence of the antisense oligonucleotide is shown in SEQ ID No.

3.

2. The antisense oligonucleotide according to claim 1, characterized in that, The antisense oligonucleotide also contains one or more nucleoside analogues for modification; Alternatively, the antisense oligonucleotide may further contain one or more 5'-methyl-cytosine nucleobase modifications; Alternatively, the continuous nucleotide sequence of the antisense oligonucleotide may contain one or more modified nucleoside-to-nucleotide bonds.

3. The antisense oligonucleotide according to claim 2, characterized in that, One or more of the nucleoside analogs include 2'-O-alkyl-RNA; 2'-O-methylRNA (2'-OMe); 2'-alkoxy-RNA; 2'-O-methoxyethyl-RNA (2'-MOE); 2'-amino-DNA; 2'-fluoro-RNA; 2'-fluoro-DNA; Arabica nucleic acid (ANA); 2'-fluoro-ANA; or bicyclic nucleoside analogs.

4. The antisense oligonucleotide according to claim 2, characterized in that, One or more of the nucleoside analogues are sugar-modified nucleosides.

5. The antisense oligonucleotide according to claim 4, characterized in that, The sugar-modified nucleoside is a 2'-sugar-modified nucleoside that enhances affinity.

6. The antisense oligonucleotide according to claim 2, characterized in that, One or more of the nucleoside analogues include LNA.

7. The antisense oligonucleotide according to claim 2, characterized in that, One or more of the nucleotide analogues are selected from the group consisting of: restricted ethyl nucleoside (cEt), 2',4'-restricted 2'-O-methoxyethyl (cMOE), α-L-LNA, β-D-LNA, 2'-O,4'-C-ethylene-bridged nucleic acid (ENA), amino-LNA, oxy-LNA, thio-LNA, and any combination thereof.

8. The antisense oligonucleotide according to claim 2, characterized in that, The one or more modified nucleoside linkages are thiophosphate linkages.

9. Extracellular vesicles containing antisense oligonucleotides (ASO), characterized in that, The antisense oligonucleotide is the antisense oligonucleotide as described in any one of claims 1 to 3.

10. The extracellular vesicle according to claim 9, characterized in that, The extracellular vesicles are exosomes.

11. The extracellular vesicle according to claim 9, characterized in that, The extracellular vesicles also include an exogenous targeting portion, which enables the extracellular vesicles to target the liver.

12. The extracellular vesicle according to claim 9, characterized in that, The extracellular vesicle also includes an anchoring portion that anchors the antisense oligonucleotide to the extracellular vesicle.

13. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises: a. The antisense oligonucleotide according to any one of claims 1-3, or the extracellular vesicle according to any one of claims 9-12; and b. Pharmaceutically acceptable diluents, carriers, or salts.

14. The pharmaceutical composition according to claim 13, characterized in that, The pharmaceutical composition also includes a second therapeutic agent.

15. The pharmaceutical composition according to claim 14, characterized in that, The second therapeutic agent is selected from drugs used to treat liver cancer.

16. The pharmaceutical composition according to claim 13, characterized in that, The carrier is selected from aqueous carriers, liposomes, polymers, peptides, and nanoparticles.

17. A reagent kit, characterized in that, The kit contains the antisense oligonucleotide (ASO) according to any one of claims 1 to 3, the extracellular vesicles according to any one of claims 9 to 12, or the pharmaceutical composition according to any one of claims 13 to 16, and instructions for use.

18. A method for reducing or inhibiting the expression of the CREPT gene in cells in vitro, characterized in that, The method comprises: administering an antisense oligonucleotide (ASO) according to any one of claims 1 to 3, an extracellular vesicle according to any one of claims 9 to 12, or a pharmaceutical composition according to any one of claims 13 to 16 to cells expressing the CREPT gene, wherein the expression of CREPT protein in the cells is reduced or inhibited after administration.

19. A method for reducing the proliferation of liver cancer cell clones and reducing the invasive and metastatic ability of liver cancer cells in vitro, characterized in that, The method includes: applying the antisense oligonucleotide (ASO) of any one of claims 1 to 3, the extracellular vesicle of any one of claims 9 to 12, or the pharmaceutical composition of any one of claims 13 to 16 to the liver cancer cells, wherein the liver cancer cells reduce clonal proliferation and reduce invasiveness after application.

20. The use of the antisense oligonucleotide (ASO) according to any one of claims 1-3, the extracellular vesicle according to any one of claims 9-12, or the pharmaceutical composition according to any one of claims 13-16, characterized in that, The application is in the preparation of drugs for treating liver cancer.

21. The application according to claim 20, characterized in that, The liver cancer mentioned includes primary liver cancer and secondary liver cancer.

22. The application according to claim 21, characterized in that, The drug is administered to mammals.

23. The application according to claim 22, characterized in that, The drug was administered to humans and mice.

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