A medicament for treating and preventing metabolic obesity caused by a high-fat diet

Abstract

This invention relates to the field of biomedical technology, specifically to a drug for treating and preventing metabolic obesity caused by a high-fat diet. This invention provides a method to reduce... C2orf74 The use of compositions that reduce gene expression levels in the preparation of medicaments for the prevention and / or treatment of obesity, by utilizing the reduction of gene expression levels. C2orf74 In situ injection of a combination of gene expression levels into the testes of animals can prevent and treat obesity caused by a high-fat diet. Specifically, it can alleviate the obesity phenotype caused by a high-fat diet, including reducing weight gain, reducing fat accumulation, lowering serum triglyceride and cholesterol levels, improving blood glucose regulation and enhancing insulin sensitivity; and reducing the degree of fatty liver degeneration.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to a drug for treating and preventing metabolic obesity caused by a high-fat diet. Background Technology

[0002] Obesity is a metabolic disease characterized by an increase in white adipose tissue and body weight. The incidence of obesity and other associated metabolic disorders is rapidly increasing globally. A high-fat diet is a major cause of lipid metabolism disorders and other metabolic diseases. A long-term high-fat diet leads to excess energy, which is stored as triglycerides in white adipose tissue. White adipose tissue is the primary storage site for lipids. With the onset and progression of obesity, white adipose tissue gradually becomes dysfunctional, its ability to store triglycerides is limited, leading to lipid extravasation and inducing ectopic fat deposition in other tissues and organs, including the liver, thus causing non-alcoholic fatty liver disease. Abnormal blood lipids, including triglycerides and cholesterol, and lipid metabolism disorders can lead to chronic inflammation, induce insulin resistance, and other metabolic syndromes.

[0003] Antisense oligonucleotides (ASOs) are a class of short synthetic oligonucleotides (typically 12-53 nucleotides) whose stability and targeting are enhanced through chemical modification. They can inhibit the expression of target genes by specifically binding to target RNA and mediating its degradation (Keating MF, Drew BG, Calkin AC (2022). Antisense oligonucleotides technologies to combat obesity and fattyliver diseases. Front Physiol. Jan 28;13:839471. doi:10.3389 / fphys.2022.839471).

[0004] Currently, the therapeutic targets actively being developed using the ASO strategy primarily focus on lipid metabolism. These targets aim to reduce lipid anabolism or increase catabolism to decrease lipid accumulation and alleviate obesity phenotypes. However, there is a lack of therapeutic pathways based on other mechanisms. For obesity and metabolic disorders caused by a high-fat diet, simultaneously targeting both lipid metabolism and energy expenditure processes holds promise for more effective treatment and preventative interventions.

[0005] Currently, there is a lack of ASO drugs that can precisely target and treat metabolic obesity caused by a high-fat diet. Summary of the Invention

[0006] This invention aims to at least partially solve one of the technical problems in related technologies. Therefore, one object of this invention is to provide a solution that reduces... C2orf74 The use of compositions that reduce gene expression levels in the preparation of medicaments for the prevention and / or treatment of obesity, the present invention utilizes... C2orf74 In situ injection of a combination of gene expression levels into the testes of animals can prevent and treat obesity caused by a high-fat diet. Specifically, it can alleviate the obesity phenotype caused by a high-fat diet, including reducing weight gain, reducing fat accumulation, lowering serum triglyceride and cholesterol levels, improving blood glucose regulation and enhancing insulin sensitivity; and reducing the degree of fatty liver degeneration.

[0007] The first aspect of the present invention provides a method for reducing C2orf74 Use of the gene expression level composition in the preparation of a medicament for the prevention and / or treatment of obesity.

[0008] According to an embodiment of the present invention, the obesity is obesity induced by a high-fat diet.

[0009] A high-fat diet can induce obesity. The inventors discovered that a high-fat diet can induce... C2orf74 Increased expression levels of the gene, which encodes the C2orf74 protein, specifically localizes within spermatogenic cells in the testes, thereby reducing... C2orf74 The expression level of genes in the testes can significantly alleviate obesity phenotypes, including weight gain, abnormal glucose and lipid metabolism, and liver steatosis.

[0010] According to embodiments of the present invention, the composition for reducing C2orf74 gene expression levels is selected from at least one of the following: Antisense oligonucleotides, siRNAs, miRNAs, specific small molecule inhibitors, specific antibodies, or nanobodies designed targeting the C2orf74 gene.

[0011] According to an embodiment of the present invention, the method for reducing C2orf74 The composition for gene expression level is an antisense oligonucleotide.

[0012] The inventor's ASO-based strategy reduces C2orf74 The gene expression level can be adjusted to treat obesity induced by a high-fat diet and metabolic abnormalities of blood sugar and blood lipids.

[0013] According to an embodiment of the present invention, the nucleic acid sequence of the antisense oligonucleotide is as shown in SEQ ID NO: 1, or has at least 80% identity with SEQ ID NO: 1.

[0014] The inventor designed a line targeting C2orf74The ASO sequence of the mRNA fragment is injected into the seminiferous tubules via the rete testis and ultimately enters the spermatogenic cells. This drug delivery method allows the drug to act in situ in the testes without affecting other organs or tissues. This specific ASO sequence can effectively reduce... C2orf74 Gene expression levels in the testes. In mice obese due to a high-fat diet, ASO administration significantly alleviated the obesity phenotype, including weight gain, abnormal glucose and lipid metabolism, and hepatic steatosis. Simultaneous administration of a high-fat diet and ASO injection to mice significantly prevented the above-mentioned obesity phenotypes induced by a high-fat diet.

[0015] According to embodiments of the present invention, the terminal bases of the antisense oligonucleotide have modifications to enhance sequence stability and / or to increase sequence lipophilicity.

[0016] According to embodiments of the present invention, the modification to enhance sequence stability includes at least one of LNA modification, 2'-OMe modification, 2'-MOE modification, thiophosphate modification, and inverted base modification.

[0017] According to embodiments of the present invention, the modification that increases the lipid solubility of the sequence includes at least one of cholesterol modification, vitamin modification, fatty acid modification, and bile acid modification.

[0018] According to an embodiment of the present invention, one end of the antisense oligonucleotide has an LNA modification and the other end has a cholesterol modification.

[0019] A second aspect of the present invention provides a medicament for the prevention and / or treatment of obesity. According to embodiments of the present invention, the medicament comprises a composition for reducing the expression level of the C2orf74 gene.

[0020] According to an embodiment of the present invention, the obesity is obesity induced by a high-fat diet.

[0021] According to embodiments of the present invention, the composition for reducing C2orf74 gene expression levels is selected from at least one of the following: Antisense oligonucleotides, siRNAs, miRNAs, specific small molecule inhibitors, specific antibodies, or nanobodies designed targeting the C2orf74 gene.

[0022] According to an embodiment of the present invention, the method for reducing C2orf74 The composition for gene expression level is an antisense oligonucleotide.

[0023] According to an embodiment of the present invention, the nucleic acid sequence of the antisense oligonucleotide is as shown in SEQ ID NO: 1, or has at least 80% identity with SEQ ID NO: 1.

[0024] According to embodiments of the present invention, the terminal bases of the antisense oligonucleotide have modifications to enhance sequence stability and / or to increase sequence lipophilicity.

[0025] According to embodiments of the present invention, the modification to enhance sequence stability includes at least one of LNA modification, 2'-OMe modification, 2'-MOE modification, thiophosphate modification, and inverted base modification.

[0026] According to embodiments of the present invention, the modification that increases the lipid solubility of the sequence includes at least one of cholesterol modification, vitamin modification, fatty acid modification, and bile acid modification.

[0027] According to an embodiment of the present invention, one end of the antisense oligonucleotide has an LNA modification and the other end has a cholesterol modification.

[0028] A third aspect of this invention provides an antisense oligonucleotide targeting the C2orf74 gene mRNA. According to embodiments of the invention, the nucleic acid sequence of the antisense oligonucleotide is as shown in SEQ ID NO: 1, or has at least 80% identity with SEQ ID NO: 1.

[0029] According to embodiments of the present invention, the terminal bases of the antisense oligonucleotide have modifications to enhance sequence stability and / or to increase sequence lipophilicity.

[0030] According to embodiments of the present invention, the modification to enhance sequence stability includes at least one of LNA modification, 2'-OMe modification, 2'-MOE modification, thiophosphate modification, and inverted base modification.

[0031] According to embodiments of the present invention, the modification that increases the lipid solubility of the sequence includes at least one of cholesterol modification, vitamin modification, fatty acid modification, and bile acid modification.

[0032] According to an embodiment of the present invention, one end of the antisense oligonucleotide has an LNA modification and the other end has a cholesterol modification.

[0033] A fourth aspect of the present invention provides an expression vector. According to an embodiment of the present invention, the expression vector comprises the coding sequence of the antisense oligonucleotide described in the third aspect.

[0034] A fifth aspect of the present invention provides a recombinant cell or virus. According to embodiments of the present invention, the recombinant cell or virus carries the expression vector described in the fourth aspect.

[0035] Beneficial effects: This invention will target C2orf74An ASO drug containing gene mRNA (nucleic acid sequence as shown in SEQ ID NO: 1), administered orally to the testes, can prevent and treat obesity caused by a high-fat diet. Specifically, it can alleviate the obesity phenotype induced by a high-fat diet, including reducing weight gain, decreasing fat accumulation, lowering serum triglyceride and cholesterol levels, improving glycemic regulation, and enhancing insulin sensitivity; it can also reduce the degree of fatty liver degeneration. Furthermore, this drug can prevent metabolic abnormalities caused by a high-fat diet.

[0036] Additional aspects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. Attached Figure Description

[0037] The above and / or additional aspects and advantages of the present invention will become apparent and readily understood from the description of the embodiments taken in conjunction with the following drawings, in which: Figure 1 The results of trypan blue staining following injection of ASO sequence into the rete testis are shown. Scale bar: 1.5 mm. Figure 2 Targeted mice were shown C2orf74 The ASO sequence knockdown effect was investigated. The expression level of C2orf74 protein in testicular tissue was detected by immunoblotting (A) combined with gray-scale quantitative analysis (B), with β-actin as an internal reference (n=3). The statistical analysis of this figure used an unpaired two-tailed t-test, **p<0.01; the error bars represent the standard error of the mean (SEM). Figure 3 ASO was displayed. C2orf74 The weight gain of mice in the control group and the ASO-Ctrl control group was analyzed. Figure A shows the weekly weight monitoring results (n=10); Figure B shows the fat content detected by time-domain nuclear magnetic resonance (TD-NMR) (n=9). Statistical analysis: Figure A used two-way repeated measures ANOVA combined with Šídák post-hoc test; Figure B used an unpaired two-tailed t-test. *p<0.05; **p<0.01; ***p<0.001; error bars represent the standard error of the mean (SEM). Figure 4 ASO was displayed. C2orf74 The results of blood lipid evaluation in mice in the control group and the ASO-Ctrl control group are shown in Figure A, where A represents the serum triglyceride content (n=10) and B represents the serum cholesterol content (n=10). Statistical analysis: Unpaired two-tailed t-tests were used for both Figure A and Figure B; **p<0.01; error bars represent the standard error of the mean (SEM). Figure 5 ASO was displayed. C2orf74The results of glycemic regulation in mice in the control group and the ASO-Ctrl control group were evaluated. Figure A shows the statistical results of the glucose tolerance test (GTT), with the left graph showing the blood glucose concentration changes at different time points after mice were fasted overnight (12 hours) and injected intraperitoneally with glucose at 2.5 g / kg body weight; the right graph shows the statistical analysis of the area under the curve (AUC) of the corresponding blood glucose level (n=10). Figure B shows the statistical results of the insulin tolerance test (ITT): the left graph shows the blood glucose concentration changes at different time points after mice were fasted for 6 hours and injected intraperitoneally with insulin at 1.6 U / kg body weight; the right graph shows the statistical analysis of the area under the curve (AUC) of the corresponding blood glucose level (n=10). Statistical analysis: The blood glucose change curves in Figures A and B were analyzed using two-way repeated measures ANOVA combined with Šídák post-hoc test; the area under the curve (AUC) of the blood glucose level in Figures A and B were analyzed using an unpaired two-tailed t-test; *p<0.05; **p<0.01; error bars represent the standard error of the mean (SEM). Figure 6 ASO was displayed. C2orf74 The results of fatty liver evaluation in mice of the control group and the ASO-Ctrl control group are shown. A is a representative image of liver hematoxylin-eosin (H&E) staining, scale bar: 50 micrometers; B is a representative image of liver Oil Red O staining, scale bar: 50 micrometers. Figure 7 Targeted mice C2orf74 The ASO sequence knockdown effect was investigated. The expression level of C2orf74 protein in testicular tissue was detected by immunoblotting (A) combined with density quantification analysis (B), with β-actin as an internal reference (n=3). The statistical analysis of this figure used an unpaired two-tailed t-test, **p<0.01; the error bar represents the standard error of the mean (SEM). Figure 8 ASO was displayed. C2orf74 The weight gain of mice in the control group and the ASO-Ctrl control group was analyzed. Figure A shows the weekly weight monitoring results (n=15); Figure B shows the fat mass detected by time-domain nuclear magnetic resonance (TD-NMR) (n=13). Statistical analysis: Figure A used two-way repeated measures ANOVA combined with Šídák post-hoc test; Figure B used an unpaired two-tailed t-test. *p<0.05; **p<0.01; ***p<0.001; error bars represent the standard error of the mean (SEM). Figure 9 ASO was displayed. C2orf74 The results of blood lipid evaluation in mice in the control group and the ASO-Ctrl control group are shown in Figure A, where A represents the serum triglyceride content (n=10) and B represents the serum cholesterol content (n=10). Statistical analysis: Unpaired two-tailed t-tests were used for both Figure A and Figure B; ***p<0.001; error bars represent the standard error of the mean (SEM). Figure 10 ASO was displayed. C2orf74 The results of glycemic regulation in mice in the control group and the ASO-Ctrl control group were evaluated. Figure A shows the statistical results of the glucose tolerance test (GTT). The left graph shows the blood glucose concentration changes at different time points after mice were fasted overnight (12 hours) and injected intraperitoneally with glucose at 2.5 g / kg body weight. The right graph shows the statistical analysis of the area under the curve (AUC) of the corresponding blood glucose level (n=10). Figure B shows the statistical results of the insulin tolerance test (ITT). The left graph shows the blood glucose concentration changes at different time points after mice were fasted for 6 hours and injected intraperitoneally with insulin at 1.6 U / kg body weight. The right graph shows the statistical analysis of the area under the curve (AUC) of the corresponding blood glucose level (n=10). Statistical analysis: The blood glucose change curves in Figures A and B were analyzed using two-way repeated measures ANOVA combined with Šídák post-hoc test; the area under the curve (AUC) of the blood glucose level in Figures A and B were analyzed using an unpaired two-tailed t-test. *p<0.05; **p<0.01; error bars represent the standard error of the mean (SEM). Figure 11 ASO was displayed. C2orf74 The results of fatty liver evaluation in mice of the control group and the ASO-Ctrl control group are shown. In the figure, A is a representative image of liver hematoxylin-eosin (H&E) staining, scale bar: 50 micrometers; B is a representative image of liver Oil Red O staining, scale bar: 50 micrometers. Detailed Implementation

[0038] The embodiments of the present invention are described in detail below. The embodiments described below are exemplary and are only used to explain the present invention, and should not be construed as limiting the present invention.

[0039] It should be noted that the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of indicated technical features. Therefore, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature. Furthermore, in the description of this invention, unless otherwise stated, "a plurality of" means two or more.

[0040] The endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0041] To facilitate understanding of the invention, certain technical and scientific terms are specifically defined below. Unless otherwise expressly defined elsewhere in this document, all other technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art to which this invention pertains.

[0042] In this document, the terms “comprising” or “including” are open-ended expressions, meaning that they include the contents specified in this invention, but do not exclude other aspects.

[0043] In this document, the terms “optionally,” “optionally,” or “optionally” generally refer to an event or condition that may, but may not, occur, and the description includes both cases in which the event or condition occurs and cases in which the event or condition does not occur.

[0044] According to a specific embodiment of the present invention, the present invention provides a method for reducing C2orf74 Use of the gene expression level composition in the preparation of a medicament for the prevention and / or treatment of obesity.

[0045] It should be noted that the obesity mentioned includes any type of obesity caused by any etiology, especially obesity induced by a high-fat diet.

[0046] According to a specific embodiment of the present invention, the composition for reducing the expression level of the C2orf74 gene is selected from at least one of the following: Antisense oligonucleotides, siRNAs, miRNAs, specific small molecule inhibitors, specific antibodies, or nanobodies designed targeting the C2orf74 gene.

[0047] According to a specific embodiment of the present invention, the method for reducing C2orf74 The composition for gene expression level is an antisense oligonucleotide.

[0048] According to a specific embodiment of the present invention, the nucleic acid sequence of the antisense oligonucleotide is as shown in SEQ ID NO: 1, or has at least 80% identity with SEQ ID NO: 1.

[0049] In this paper, “identity” is used to describe nucleic acid sequences relative to a reference sequence, employing the percentage of identical nucleic acids between two nucleic acid sequences determined by conventional methods, for example, see Ausubel et al., eds. (1995), Current Protocols in Molecular Biology, Chapter 19 (Greene Publishing and Wiley-Interscience, New York); and the ALIGN procedure (Dayhoff (1978), Atlas of Protein Sequence and Structure 5: Suppl. 3 (National Biomedical Research Institute)). Foundation, Washington, DC). Numerous algorithms exist for aligning sequences and determining sequence identity, including: the homology alignment algorithm of Needleman et al. (1970) J. Mol. Biol. 48: 443; the local homology algorithm of Smith et al. (1981) Adv. Appl. Math. 2: 482; the similarity search method of Pearson et al. (1988) Proc. Natl. Acad. Sci. 85: 2444; the Smith-Waterman algorithm (Meth. Mol. Biol. 70: 173-187 (1997); and the BLASTP, BLASTN, and BLASTX algorithms (see Altschul et al. (1990) J. Mol. Biol. 215: 403-410). Computer programs utilizing these algorithms are also available, including but not limited to: ALIGN or Megalign (DNASTAR) software, or WU-BLAST-2 (Altschul...). See, Meth.Enzym., 266:460-480 (1996); or GAP, BESTFIT, BLAST Altschul, etc., above, FASTA, and TFASTA, available in Genetics Computing Group (GCG) package, version 8, Madison, Wisconsin, USA; and CLUSTAL in the PC / Gene program provided by Intelligenetics, Mountain View, California.

[0050] In this paper, "at least 80% identity" means at least 80% similarity to the reference sequence, which can be 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% similarity.

[0051] According to a specific embodiment of the present invention, the terminal base of the antisense oligonucleotide has modifications to enhance sequence stability and / or to increase sequence lipophilicity.

[0052] According to a specific embodiment of the present invention, the modification to enhance sequence stability includes, but is not limited to, at least one of LNA modification, 2'-OMe modification, 2'-MOE modification, phosphate thioester modification, and inversion base modification. Preferably, the modification to enhance sequence stability is LNA modification.

[0053] According to a specific embodiment of the present invention, the modification that increases sequence lipid solubility includes, but is not limited to, at least one of cholesterol modification, vitamin modification, fatty acid modification, and bile acid modification. Preferably, the modification that increases sequence lipid solubility is cholesterol modification.

[0054] According to a specific embodiment of the present invention, one end of the antisense oligonucleotide has an LNA modification, and the other end has a cholesterol modification. For example, the 5' end of the antisense oligonucleotide has an LNA modification, and the 3' end has a cholesterol modification.

[0055] According to a specific embodiment of the present invention, the present invention also provides a medicament for preventing and / or treating obesity, said medicament comprising [components for reducing...]. C2orf74 A combination of gene expression levels.

[0056] According to a specific embodiment of the present invention, the obesity is obesity induced by a high-fat diet.

[0057] According to a specific embodiment of the present invention, the composition for reducing the expression level of the C2orf74 gene is selected from at least one of the following: Antisense oligonucleotides, siRNAs, miRNAs, specific small molecule inhibitors, specific antibodies, or nanobodies designed targeting the C2orf74 gene.

[0058] According to a specific embodiment of the present invention, the method for reducing C2orf74 The composition for gene expression level is an antisense oligonucleotide.

[0059] According to a specific embodiment of the present invention, the nucleic acid sequence of the antisense oligonucleotide is as shown in SEQ ID NO: 1, or has at least 80% identity with SEQ ID NO: 1.

[0060] According to a specific embodiment of the present invention, the terminal base of the antisense oligonucleotide has modifications to enhance sequence stability and / or to increase sequence lipophilicity.

[0061] According to a specific embodiment of the present invention, the modifications to enhance sequence stability and the modifications to increase sequence lipophilicity are as described above.

[0062] According to a specific embodiment of the present invention, the terminal base of one end of the antisense oligonucleotide is modified with LNA, and the terminal base of the other end is modified with cholesterol.

[0063] According to a specific embodiment of the present invention, the present invention also provides an antisense oligonucleotide targeting C2orf74 gene mRNA, wherein the nucleic acid sequence of the antisense oligonucleotide is as shown in SEQ ID NO: 1, or has at least 80% identity with SEQ ID NO: 1.

[0064] According to a specific embodiment of the present invention, the terminal base of the antisense oligonucleotide has modifications to enhance sequence stability and / or to increase sequence lipophilicity.

[0065] According to a specific embodiment of the present invention, the modifications to enhance sequence stability and the modifications to increase sequence lipophilicity are as described above.

[0066] According to a specific embodiment of the present invention, the terminal base of one end of the antisense oligonucleotide is modified with LNA, and the terminal base of the other end is modified with cholesterol.

[0067] According to a specific embodiment of the present invention, the present invention provides an expression vector. According to a specific embodiment of the present invention, the expression vector comprises the coding sequence of the antisense oligonucleotide described above.

[0068] According to a specific embodiment of the present invention, the expression vector may be a viral vector or a plasmid vector.

[0069] According to a specific embodiment of the present invention, the present invention also provides a recombinant cell or virus carrying the aforementioned expression vector.

[0070] The present invention will be explained below with reference to embodiments. Those skilled in the art will understand that the following embodiments are for illustrative purposes only and should not be considered as limiting the scope of the invention. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be obtained commercially.

[0071] Example 1: Preparation and Injection of ASO 1. ASO preparation: The ASO sequence of this invention can induce the degradation of the transfer RNA fragment encoded by the C2orf74 gene by complementary binding, thereby reducing its level. The ASO sequence of C2orf74 (ASO- C2orf74 The ASO sequence is 5'-ATCTGAAGATCTGTGGACATGTATG-3' (SEQ ID NO: 1), and the control ASO sequence (ASO-Ctrl) is 5'-ATGAACTTCAGGGTCAGCTTGCCGT-3' (SEQ ID NO: 2). The 5' terminal base of the above ASO sequences was modified with LNA, and the 3' terminal base was modified with cholesterol. LNA modification enhances the stability of the ASO sequence, while cholesterol modification increases its lipid solubility, making it easier to cross the lipid bilayer of cells and reach the intracellular space. The ASO sequences were synthesized by Nanjing GenScript Biotech Co., Ltd. ASO was dissolved in nuclease-free water to prepare solutions with concentrations of 300 μM or 420 μM.

[0072] 2.ASO injection: Eight-week-old male wild-type mice (C57BL6J, purchased from Nanjing Jicui Pharmaceutical) were fed a high-fat diet (60% high-fat feed, purchased from Nantong Trofi, catalog number TP23510) for 10 weeks. The mice's weight increased from an average of 22.71 g to 40.17 g. In contrast, mice fed a normal diet during the same period increased from an average of 23.82 g to 30.31 g, indicating that a high-fat diet can induce an obese phenotype.

[0073] Next, the therapeutic effect of ASO on high-fat diet-induced obesity was evaluated. Mice fed a high-fat diet were injected with ASO at week 10, targeting spermatogenic cells via the testicular rete and seminiferous tubules. The specific procedure was as follows: Mice were first anesthetized and then fixed. The abdomen of the mice was disinfected with 75% alcohol, some hair was removed, a small incision was made, a layer of sterile cloth was placed, and the testicular tissue was gently pulled out of the abdominal cavity and exposed. The efferent ducts were located under a stereomicroscope. The injection needle connected to a pressure infusion pump was slowly and horizontally inserted into the efferent ducts, and the ASO solution was slowly injected using the pressure infusion pump at a rate controlled at 0.15-0.2 μL / min. 10 μL of the targeted ASO solution was injected into each testicular tissue. C2orf74 ASO (ASO-C2orf74) or control ASO (ASO-Ctrl) (both at 300 μM). After injection, leave the needle in place for 5 minutes and slowly withdraw it. Suture the mouse abdomen after injection and apply an appropriate amount of erythromycin ointment to prevent infection. The injection effect was evaluated using pre-mixed trypan blue dye. Figure 1 This indicates that trypan blue adequately fills the seminiferous tubules. A second dose was administered two weeks later, following the same administration procedure. Metabolic parameters were measured two weeks after the last dose. During this period, the mice were fed a consistently high-fat diet.

[0074] Example 2: ASO Knockdown Effect Detection Mice injected with the control ASO sequence (ASO-Ctrl) and the targeted mouse sequence were isolated. C2orf74 ASO sequence (ASO- C2orf74 Mouse testicular tissue was collected and homogenized using 1% Triton X-100 lysis buffer prepared with PBS. The samples were then sonicated and centrifuged at 14,000 rpm for 10 minutes at 4°C. The supernatant was collected for protein concentration determination, followed by Western blotting. A 12% polyacrylamide gel was prepared for electrophoresis of the tissue protein samples: electrophoresis was performed at 80 V for 30 minutes, then increased to 100 V for approximately 90 minutes. Wet transfer was performed at 300 mA for 120 minutes, with the transfer bath kept on ice throughout.

[0075] After transfer, a blocking buffer of 5% skim milk was prepared using TBST solution (20 mM Tris, 150 mM NaCl, 0.1% Tween 20, pH 7.5). The membrane was blocked on a shaker at room temperature for 60 minutes. The primary antibody was then diluted with this blocking buffer, and the membrane was incubated overnight at 4°C. The antibody targeting mouse C2orf74 protein was a rabbit-derived antibody used at a dilution of 1:1,000. The mouse β-actin antibody was purchased from Proteintech (catalog number 66009-1-Ig) and used at a dilution of 1:5,000. The next day, the membrane was washed five times with TBST solution for 5 minutes each time. A secondary antibody carrying HRP of the same species was then diluted with the blocking buffer and incubated at room temperature for 60 minutes. The membrane was washed five times with TBST solution for 5 minutes each time, and the target band was exposed using a developing solution to determine its position and changes.

[0076] The results are as follows Figure 2 As shown, ASO- C2orf74 The protein level of C2orf74 in mouse testicular tissue was significantly reduced by about 70% compared with that in ASO-Ctrl mice, indicating that the ASO sequence can significantly reduce the expression level of C2orf74.

[0077] Example 3: Metabolic phenotypes of ASO-C2orf74 and ASO-Ctrl mice 1. Weight monitoring results: In Example 1, after the first ASO injection, the weight of the mice was monitored, and weekly weight gain data showed that the mice were injected with the targeted drug. C2orf74 Mice with the ASO sequence showed significantly lower body weight gain than control mice. Figure 3 (A). Time-domain nuclear magnetic resonance (TD-NMR) was used to detect fat mass in mice, and it was found that in ASO- C2orf74 The fat levels in the group of mice were significantly lower than those in the control group. Figure 3 (B)

[0078] 2. Detection of triglyceride and cholesterol levels in mouse serum: Serum triglyceride and cholesterol levels are generally positively correlated with obesity levels, and both are core serum biomarkers for lipid metabolism disorders caused by obesity. Triglyceride levels were detected using the Liquid Sample Triglyceride (TG) Enzymatic Assay Kit (catalog number E1003) from Prilely Pharmaceuticals. The specific procedure is as follows: 1) Preparation of standards and working solutions: Dilute 4 mM glycerol standard with distilled water to prepare standard solutions with concentrations of 1000, 500, 250, 125, 62.5, 31.25, and 15.625 μMol / L. At the same time, set up blank control tubes with a concentration of 0. Prepare working solutions by mixing reagents R1:R2 in a ratio of 4:1. 2) Reaction and detection: Add 30 μL of the sample to be tested (standard solution / serum sample) and 170 μL of working solution to each well of the 96-well microplate, and incubate at 37℃ for 15 minutes; read the absorbance value at 550 nm wavelength using the microplate reader. 3) Result calculation: Plot a standard curve based on the absorbance of the standard solution, and calculate the triglyceride level of each serum sample based on the standard curve.

[0079] Cholesterol was analyzed using the Amplex Red kit (Invitrogen, catalog number: A12222). The assay procedure was as follows: 1) Prepare cholesterol standard solutions with concentrations of 0, 0.125, 0.25, 0.5, 1, 2, 4, and 8 mg / mL; 2) Preparation of sample reaction solution: Mix 1 mL of 5× reaction solution (0.5 M potassium dihydrogen phosphate + 0.25 M sodium chloride + 25 mM cholic acid + 0.5% Triton X-100, pH 7.4), 4 mL of deionized water, 75 μL of fluorescent red dye, 50 μL of cholesterol oxidase, 5 μL of cholesterol esterase, and 50 μL of horseradish peroxidase according to the specified ratio, and mix well for later use. 3) Each standard solution and the sample to be tested are set up in 3 replicates. Add 50 μL of substrate and 50 μL of the above mixture to each well, mix well and incubate in a 37°C incubator in the dark for 30 minutes. 4) Read the values ​​using an ELISA reader; 5) Plot a standard curve based on the absorbance of the cholesterol standard solution, and calculate the cholesterol level of each serum sample based on the standard curve.

[0080] The results are as follows Figure 4 As shown in A and B, this indicates that ASO- C2orf74 The levels of triglycerides and cholesterol in the serum of mice were significantly lower than those in ASO-Ctrl mice.

[0081] 3. Glucose tolerance test (GTT) and insulin tolerance test (ITT) Glucose tolerance test (GTT) and intraperitoneal glucose tolerance test (ITT) are core methods for assessing glucose metabolism function and diagnosing insulin resistance. The GTT involves intraperitoneal injection of glucose solution into fasted mice, followed by measurement of blood glucose concentration at different time points, plotting blood glucose curves, and calculating the area under the curve (AUC). Comparison of ASO-Ctrl group mice and ASO- C2orf74 The blood glucose concentration change curves of the group of mice revealed that ASO- C2orf74 The blood glucose concentration in the control group mice decreased more quickly after the initial increase, and the AUC value was significantly lower than that in the control group. This indicates that ASO- C2orf74 The mice in this group had stronger glucose tolerance. Figure 5(A). The ITT experiment involves intraperitoneal injection of insulin into fasting mice, followed by measurement of blood glucose concentration at different time points, plotting blood glucose curves, and calculating the area under the blood glucose curve (AUC). The results show that ASO- C2orf74 The blood glucose concentration in the control group decreased more rapidly, and the AUC value was significantly lower than that in the control group, which means that ASO- C2orf74 The mice in the group had stronger insulin sensitivity. Figure 5 (B)

[0082] 4. Detection of hepatic steatosis: Obesity is the leading risk factor for hepatic steatosis, and the degree of hepatic steatosis can be analyzed by staining liver tissue. Among these methods, hematoxylin-eosin (H&E) staining combined with Oil Red O staining is the best way to visualize hepatic steatosis.

[0083] The steps for hematoxylin-eosin (H&E) staining are as follows: 1) The fixed mouse tissue was dehydrated, cleared, impregnated with paraffin, and embedded in paraffin to make paraffin blocks; it was then cut into 8 mm thick sections using a paraffin microtome and mounted on glass slides. 2) Dewaxing to water: The sections are sequentially immersed in xylene I and II for 10 minutes each, anhydrous ethanol and 95%, 90%, 80% and 70% ethanol for 5 minutes each, then in water for 2-3 minutes, and finally immersed in distilled water for 2 minutes. 3) Staining: Immerse the sections in hematoxylin stain for 3-5 minutes (to achieve a slightly darker stain), then wash with water for 1-2 minutes to remove excess stain; differentiate with 1% HCl-alcohol for 30 seconds (until a pale blue-red color is achieved), then wash with water for 30-60 seconds to return to blue (bright blue / sky blue); immerse in eosin stain for 3-5 minutes, then wash with water for 1 minute to remove excess stain; 4) Dehydration, clearing, and mounting: Sections are sequentially subjected to 80% ethanol, 90% ethanol, anhydrous ethanol I and II, and xylene I and II for 5 minutes each; then mounted with a 2:1 neutral resin-xylene solution and allowed to evaporate in a fume hood for at least 2 hours; 5) Take photos and observe under a microscope.

[0084] The steps for Oil Red O staining are as follows: 1) Mouse tissues were fixed with 4% paraformaldehyde for 24 hours (at 4°C), then transferred to 30% sucrose solution for dehydration for 24 hours, sectioned using a cryostat, and the sections were mounted on glass slides for later use. 2) Preparation of reagents: ① Oil Red stock solution: Dissolve 0.5g of Oil Red powder in 100mL of isopropanol, mix well, filter and store; ② Oil Red working solution: Mix Oil Red stock solution and distilled water in a 3:2 ratio, filter and use. 3) Staining: Wash the sections three times with PBS, stain with oil red in the dark for 15 minutes; then immerse in 60% isopropanol for 3-5 seconds, and rinse with deionized water; then stain with hematoxylin for 3 minutes (to stain the cell nuclei), and wash again with deionized water. 4) After slightly drying the film, add glycerin gelatin sealing tablets and seal it.

[0085] We tested ASO-Ctrl group mice and ASO- C2orf74 Liver tissues from the group of mice were stained with H&E and Oil Red O. The H&E staining results are as follows: Figure 6 As shown in Figure A, ASO- C2orf74 Compared with the ASO-Ctrl group, the number of intracellular vacuoles in the hepatocytes of the mice in the control group was significantly reduced, indicating less fat accumulation in the liver tissue; Oil Red O staining results were as follows. Figure 6 As shown in Figure B, ASO- C2orf74 The lipid droplet content in the livers of the ASO-Ctrl group mice was significantly lower than that of the ASO-Ctrl group mice. These results indicate that ASO- C2orf74 Its use can effectively alleviate hepatic steatosis and improve hepatocyte morphology.

[0086] In summary, the above results indicate that the targeted C2orf74 ASO drugs can effectively treat obesity phenotypes induced by a high-fat diet, including weight gain, glucose and lipid metabolism disorders, and hepatic steatosis.

[0087] Example 4: ASO for the prevention of obesity induced by a high-fat diet Eight-week-old male wild-type mice (C57BL6J, purchased from Nanjing Jicui Pharmaceutical) were fed a high-fat diet (60% high-fat diet, purchased from Nantong Terlofi, catalog number TP23510) and simultaneously treated with ASO using the same injection method as in Example 1, with an injection volume of 6 μL per testis and an ASO concentration of 420 μM. The administration was repeated once at week 10.

[0088] 1. ASO knockdown effect test: Mice injected with the control ASO sequence (ASO-Ctrl) and the targeted mouse sequence were isolated. C2orf74 ASO sequence (ASO- C2orf74 The mouse testicular tissue was subjected to the same methods as in Example 2, including tissue lysis and immunoblotting. Results were as follows: Figure 7 As shown, this indicates that ASO- C2orf74 The protein level of C2orf74 in mouse testicular tissue was significantly reduced by approximately 70% compared to ASO-Ctrl.

[0089] 2. Weight monitoring results: After the first ASO injection, the mice's weight was monitored, and weekly weight gain data showed that the injected targeted mice C2orf74 Mice with the ASO sequence showed significantly lower weight gain than mice with the ASO-Ctrl sequence. Figure 8 (A). Time-domain nuclear magnetic resonance (TD-NMR) was used to detect fat mass in mice, revealing that ASO- C2orf74 The fat level in the group of mice was significantly lower than that in the ASO-Ctrl mice. Figure 8 (B)

[0090] 3. Detection of triglyceride and cholesterol levels in mouse serum: The method for determining triglycerides and cholesterol in mouse serum was the same as in Example 3. The results are as follows: Figure 9 As shown in A and B, this indicates that ASO- C2orf74 The levels of triglycerides and cholesterol in the serum of mice were significantly lower than those in ASO-Ctrl mice.

[0091] 4. Glucose tolerance test (GTT) and insulin tolerance test (ITT): The evaluation methods for GTT and ITT are the same as in Example 3. The GTT results show that ASO- C2orf74 In mice, blood glucose levels rose and then fell back more quickly, and the AUC value was significantly lower than in ASO-Ctrl mice. This suggests that ASO- C2orf74 The mice in this group had stronger glucose tolerance. Figure 10 (A). ITT results indicate that ASO- C2orf74 Mice showed a faster decrease in blood glucose concentration and a significantly lower AUC value compared to the control group, indicating that ASO- C2orf74 The mice in the group had stronger insulin sensitivity. Figure 10 (B)

[0092] 5. Detection of hepatic steatosis: The evaluation method was the same as in Example 3. H&E staining results showed ASO- C2orf74 Compared to ASO-Ctrl mice, the number of intracellular vacuoles in hepatocytes was significantly reduced in these mice, indicating less fat accumulation in the liver tissue. Figure 11 Oil Red O staining results showed ASO- C2orf74 The lipid droplet content in the livers of the group mice was significantly lower than that of the ASO-Ctrl mice ( Figure 11 (B). These results indicate that ASO- C2orf74 Its use can effectively prevent fatty degeneration of the liver and improve the morphology of liver cells.

[0093] In summary, the above results indicate that the targeted C2orf74ASO drugs can effectively prevent obesity phenotypes induced by a high-fat diet, including weight gain, glucose and lipid metabolism disorders, and hepatic steatosis.

[0094] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," "some implementations," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the present invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0095] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.

Claims

1. Use of a composition for reducing C2orf74 the level of gene expression in the manufacture of a medicament for the prevention and / or treatment of obesity.

2. The use according to claim 1, characterized in that, The obesity mentioned is obesity induced by a high-fat diet; Optionally, the composition for reducing C2orf74 gene expression levels is selected from at least one of the following: Antisense oligonucleotides, siRNAs, miRNAs, specific small molecule inhibitors, specific antibodies, or nanobodies designed targeting the C2orf74 gene.

3. The use according to claim 1, characterized in that, The term used to reduce C2orf74 The gene expression level composition is an antisense oligonucleotide; Optionally, the nucleic acid sequence of the antisense oligonucleotide is as shown in SEQ ID NO: 1, or has at least 80% identity with SEQ ID NO: 1; Optionally, the terminal bases of the antisense oligonucleotide have modifications to enhance sequence stability and / or increase sequence lipophilicity; Optionally, the modification to enhance sequence stability includes at least one of LNA modification, 2'-OMe modification, 2'-MOE modification, thiophosphate modification, and inverted base modification; Optionally, the modification that increases the lipid solubility of the sequence includes at least one of cholesterol modification, vitamin modification, fatty acid modification, and bile acid modification; Optionally, one end of the antisense oligonucleotide has an LNA modification and the other end has a cholesterol modification.

4. A drug for preventing and / or treating obesity, characterized in that, This includes compositions for reducing the expression level of the C2orf74 gene.

5. The drug according to claim 4, characterized in that, The obesity mentioned is obesity induced by a high-fat diet; The composition for reducing C2orf74 gene expression levels is selected from at least one of the following: Antisense oligonucleotides, siRNAs, miRNAs, specific small molecule inhibitors, specific antibodies, or nanobodies designed targeting the C2orf74 gene; Optionally, the method for reducing C2orf74 The gene expression level composition is an antisense oligonucleotide; Optionally, the nucleic acid sequence of the antisense oligonucleotide is as shown in SEQ ID NO: 1, or has at least 80% identity with SEQ ID NO: 1; Optionally, the terminal bases of the antisense oligonucleotide have modifications to enhance sequence stability and / or increase sequence lipophilicity; Optionally, the modification to enhance sequence stability includes at least one of LNA modification, 2'-OMe modification, 2'-MOE modification, thiophosphate modification, and inverted base modification; Optionally, the modification that increases the lipid solubility of the sequence includes at least one of cholesterol modification, vitamin modification, fatty acid modification, and bile acid modification; Optionally, one end of the antisense oligonucleotide has an LNA modification and the other end has a cholesterol modification.

6. An antisense oligonucleotide targeting C2orf74 gene mRNA, characterized in that, The nucleic acid sequence of the antisense oligonucleotide is as shown in SEQ ID NO: 1, or has at least 80% identity with SEQ ID NO:

1.

7. The antisense oligonucleotide according to claim 6, characterized in that, The terminal bases of the antisense oligonucleotide have modifications that enhance sequence stability and / or increase sequence lipophilicity; Optionally, the modification to enhance sequence stability includes at least one of LNA modification, 2'-OMe modification, 2'-MOE modification, thiophosphate modification, and inverted base modification; Optionally, the modification that increases the lipid solubility of the sequence includes at least one of cholesterol modification, vitamin modification, fatty acid modification, and bile acid modification; Optionally, one end of the antisense oligonucleotide has an LNA modification and the other end has a cholesterol modification.

8. An expression carrier, characterized in that, The coding sequence comprising the antisense oligonucleotide of claim 6 or 7.

9. A recombinant cell or virus, characterized in that, Carrying the expression vector as described in claim 8.

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