SiRNA and application thereof

Abstract

The invention belongs to the technical field of biological medicine, and provides siRNA and application thereof. The siRNA can cause THADA gene silencing and can be used for preparing drugs for preventing or treating lipid metabolism diseases such as hyperlipidemia, fatty liver diseases and atherosclerotic cardiovascular diseases.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a specific small interfering nucleic acid and its applications. Background Technology

[0002] The information disclosed in this background section is intended to enhance understanding of the overall background of the invention and is not necessarily to be construed as an admission or in any way implying that such information constitutes prior art known to those skilled in the art.

[0003] Cardiovascular and metabolic diseases, as major chronic non-communicable diseases worldwide, have become a significant public health problem threatening human life and health. In my country, atherosclerotic cardiovascular diseases (such as ischemic heart disease and stroke) are the leading cause of death, and their disease burden continues to increase, making prevention and control increasingly challenging. This trend is closely related to modern lifestyles and nutrient-rich diets, directly leading to the widespread prevalence of hyperlipidemia, dyslipidemia, and their secondary pathological conditions such as fatty liver disease and atherosclerosis.

[0004] The core manifestation of dyslipidemia is abnormally elevated levels of serum total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), and triglycerides (TG). Over the past three decades, these lipid indicators in the Chinese population have shown a significant upward trend, with a sharp increase in the prevalence of dyslipidemia, resulting in huge medical expenditures and a heavy socioeconomic burden. However, current public awareness, clinical treatment rates, and control rates of dyslipidemia are all extremely low. For high-risk cardiovascular populations, lowering cholesterol and triglyceride levels is the primary goal for effective prevention and control, and the key lies in improving the coverage of lipid-lowering drug therapy and the rate of achieving target lipid levels.

[0005] Current clinical lipid-lowering drugs face fundamental limitations: first-line clinical treatment mainly relies on traditional small-molecule drugs such as statins and fibrates, but monotherapy is difficult to simultaneously control multiple key lipid indicators such as LDL-C and TG to achieve synergistic target levels; moreover, they generally have adverse reactions such as liver function damage, myopathy risk, and high-density lipoprotein cholesterol (HDL-C) abnormalities, resulting in poor patient tolerance and low long-term medication adherence. These shortcomings seriously restrict the effectiveness of prevention and control of dyslipidemia and related cardiovascular and metabolic diseases.

[0006] Faced with this pressing technical bottleneck, there is an urgent need in this field to develop novel lipid-lowering drugs based on gene regulation mechanisms. These drugs should have multi-target synergistic effects, simultaneously regulating core lipid parameters such as LDL-C and TG, significantly reducing the risk of adverse reactions, and ultimately achieving effective prevention and treatment of hyperlipidemia and cardiovascular metabolic diseases. Summary of the Invention

[0007] To address the problems in the prior art, this invention provides a THADA-specific siRNA nucleic acid molecule.

[0008] Another object of the present invention is to provide an application of the above-mentioned siRNA in the preparation of lipid-lowering drugs.

[0009] To achieve the above objectives, the present invention adopts the following technical solution.

[0010] A nucleic acid molecule with a double-stranded structure, comprising an antisense strand and a sense strand, wherein the antisense strand includes a partially complementary binding region to THADA mRNA, the complementary binding region being 15-25 nt, 17-23 nt, or 19-21 nt in length.

[0011] The aforementioned nucleic acid molecules can induce post-transcriptional silencing of the THADA gene, such as RNAi.

[0012] The aforementioned nucleic acid molecules also include their pharmaceutically acceptable salts.

[0013] In some embodiments, the length of the partially complementary binding region of the antisense strand to THADA's mRNA is 19 nt or 21 nt.

[0014] The mRNA is the mature mRNA of the THADA gene or a precursor of the mRNA.

[0015] The THADA gene can be a mouse, monkey, or human THADA gene. In some embodiments, the THADA gene is a monkey or human THADA gene.

[0016] In some embodiments, the initiation sites of the antisense strand complementary binding mRNA are respectively: 135, 137, 139, 144, 189, 246, 269, 275, 277, 315, 337, 342, 360, 499, 515, 533, 534, 536, 537, 547, 560, 582, 583, 584, 593, 630, 631, 676, 696, 702, 717, 721, 722, 728, 734, 735, 738, 741, 769, 846, 848, 85 0, 1156, 1157, 1188, 1189, 1194, 1278, 1344, 1345, 1507, 1508, 1515, 1518, 1523, 1612, 1631, 1738, 1804, 1810, 1811, 1828, 1830, 1838, 1839, 1927, 1935, 1954, 1992, 1993, 2037, 2040, 2045, 2059, 2060, 2088, 2113, 2118, 2123, 2169, 2173 2174, 2175, 2241, 2247, 2263, 2267, 2268, 2289, 2299, 2365, 2368, 2443, 2444, 2445, 2447, 2466, 2469, 2482, 2488, 2549, 2557, 2629, 2637, 2642, 2703, 2717, 2743, 2744, 2745, 2752, 2796, 2872, 2876, 2896, 2917, 2924, 2926, 2961, 2966 3066, 3092, 3112, 3120, 3122, 3162, 3166, 3176, 3207, 3208, 3210, 3224, 3228, 3277, 3279, 3280, 3283, 3287, 3303, 3363, 3403, 3408, 3410, 3483, 3510, 3511, 3575, 3576, 3667, 3668, 3775, 3866, 4023, 4043, 4045, 4069, 4141, 4242, or 4247 bits.

[0017] To achieve higher knockdown efficiency, such as not less than 50%, the initiation sites for the complementary binding mRNA of the antisense strands are 135, 137, 139, 144, 189, 246, 269, 275, 277, 315, 337, 342, 360, 499, 515, 533, 534, 536, 537, 547, 560, 582, 583, 584, 593, 630, 631, 696, 702, 717, 721, 722, 728, 734, 735, 738, and 741. 769, 846, 848, 850, 1156, 1157, 1188, 1189, 1194, 1278, 1344, 1345, 1507, 1508, 1515, 1518, 1523, 1612, 1631, 1738, 1804, 1810, 1811, 1828, 1830, 1838, 1839, 1927, 1935, 1954, 1992, 1993, 2037, 2040, 2045, 2059, 2088, 2113, 21 18, 2123, 2169, 2173, 2174, 2175, 2241, 2247, 2263, 2267, 2268, 2289, 2365, 2368, 2443, 2447, 2466, 2469, 2482, 2488, 2549, 2557, 2629, 2637, 2642, 2703, 2717, 2743, 2744, 2745, 2752, 2796, 2872, 2876, 2896, 2917, 2924, 2926, 2 961, 2966, 3092, 3112, 3120, 3122, 3162, 3166, 3207, 3208, 3210, 3224, 3228, 3279, 3280, 3283, 3287, 3303, 3363, 3403, 3408, 3410, 3483, 3510, 3511, 3575, 3576, 3667, 3668, 3775, 3866, 4023, 4043, 4045, 4069, 4141, 4242, or 4247 bits.

[0018] Furthermore, when the knockdown efficiency is not less than 60%, the initiation sites for the complementary binding of the antisense strand to the mRNA are, respectively, positions 135, 137, 139, 144, 189, 246, 269, 275, 277, 315, 337, 342, 360, 499, 515, 533, 534, 536, 537, 547, 560, 582, 583, 584, 593, 630, 631, 696, 702, 717, 721, 722, 728, and 734. 738, 741, 769, 846, 850, 1156, 1188, 1189, 1194, 1278, 1344, 1345, 1507, 1508, 1515, 1518, 1523, 1612, 1631, 1738, 1804, 1810, 1811, 1828, 1830, 1838, 1839, 1927, 1935, 1954, 1992, 2037, 2045, 2088, 2113, 2118, 21 23, 2169, 2173, 2174, 2175, 2241, 2247, 2263, 2267, 2268, 2289, 2368, 2447, 2466, 2469, 2482, 2488, 2557, 2629, 2637, 2642, 2703, 2717, 2743, 2744, 2745, 2752, 2796, 2872, 2876, 2896, 2917, 2924, 2926, 2961, 3092 3112, 3120, 3122, 3162, 3166, 3207, 3208, 3210, 3224, 3228, 3279, 3280, 3283, 3287, 3303, 3363, 3403, 3408, 3410, 3483, 3510, 3511, 3575, 3576, 3667, 3668, 3775, 3866, 4023, 4043, 4045, 4069, 4141, 4242, or 4247 bits.

[0019] Furthermore, when the knockdown efficiency is not less than 65%, the initiation sites for the antisense strand complementary binding mRNA are respectively: 135, 137, 139, 144, 189, 269, 275, 277, 315, 337, 342, 360, 499, 515, 533, 534, 536, 537, 547, 560, 582, 583, 630, 631, 696, 702, 717, 721, and 72. 2, 728, 734, 738, 769, 846, 850, 1156, 1194, 1278, 1344, 1345, 1508, 1515, 1518, 1523, 1612, 1631, 1804, 1810, 1811, 1828, 1830, 1838, 1839, 1935, 1954, 2037, 2045, 2113, 2123, 2173, 217 4, 2241, 2247, 2263, 2267, 2289, 2368, 2469, 2488, 2557, 2629, 2637, 2642, 2703, 2717, 2743, 2744, 2745, 2752, 2796, 2872, 2876, 2896, 2917, 2924, 2926, 2961, 3092, 3112, 3120, 3122, 31 62, 3166, 3207, 3208, 3210, 3224, 3228, 3279, 3280, 3283, 3287, 3303, 3403, 3408, 3410, 3483, 3510, 3511, 3575, 3576, 3667, 3668, 3775, 3866, 4023, 4043, 4045, 4069, 4141, 4242, or 4247 bits.

[0020] Furthermore, when the knockdown efficiency is not less than 70%, the initiation sites for the complementary binding of the antisense strand to the mRNA are respectively: 135, 137, 139, 144, 189, 275, 277, 337, 342, 360, 499, 515, 533, 534, 536, 537, 547, 560, 582, 583, 630, 631, 696, 702, 717, 722, 728, 734, 738, 769, 846, 850, 1156, 1194, 1278, 1344, 1345, 1515, 1523, 1612, 1631, 1804, 1810, 1811, 1828, 1830, 1838, 1839, 1935, 1954, 2045, 2113, 2123 2173, 2174, 2263, 2267, 2289, 2469, 2637, 2642, 2703, 2717, 2743, 2744, 2745, 2752, 2796, 2876, 2896, 2917, 2924, 2926, 2961, 3092, 3112, 3120, 3122, 3162, 3166 3207, 3210, 3224, 3228, 3279, 3280, 3283, 3303, 3403, 3408, 3410, 3483, 3510, 3511, 3575, 3576, 3667, 3668, 3775, 3866, 4023, 4043, 4045, 4069, 4141, 4242, or 4247 bits.

[0021] Furthermore, when the knockdown efficiency is not less than 75%, the initiation sites for the complementary binding of the antisense strand to the mRNA are respectively: 135, 137, 144, 189, 275, 277, 342, 360, 499, 515, 533, 534, 536, 537, 547, 583, 630, 728, 850, 1156, 1345, 1523, 1810, 1811, 1828, 1830, 1838, 1839, 1935, 2113, 2173, 2174, 2263, 2267, 2469, 2637, 2642, 27... 03, 2717, 2743, 2744, 2745, 2796, 2876, 2896, 2917, 2924, 2926, 2961, 3112, 3120, 3122, 3162, 3166, 3207, 3210, 3224, 3228, 3279, 3280, 3283, 3303, 3408, 3410, 3483, 3510, 3511, 3575, 3576, 3667, 3668, 3775, 3866, 4043, 4045, 4069, 4141, 4242, or 4247.

[0022] Furthermore, when the knockdown efficiency is not less than 80%, the initiation sites for the complementary binding of the antisense strand to the mRNA are respectively: 275, 342, 360, 499, 515, 533, 534, 536, 537, 547, 630, 850, 1156, 1523, 1811, 1828, 1838, 1839, 2113, 2743, 2796, 3 112, 3120, 3122, 3166, 3207, 3210, 3224, 3228, 3280, 3303, 3408, 3410, 3483, 3510, 3511, 3575, 3576, 3667, 3668, 3775, 3866, 4043, 4045, 4069, 4141, 4242, or 4247 bits.

[0023] Furthermore, when the knockdown efficiency is not less than 85%, the start sites of the antisense strand complementary binding mRNA are positions 342, 360, 499, 515, 533, 534, 537, 1839, 2113, 2796, 3122, 3166, 3210, 3303, 3410, 3483, 3510, 3511, 3575, 3576, 3667, 3668, 3775, 3866, 4043, 4045, 4069, 4141, or 4242.

[0024] Furthermore, when the knockdown efficiency is not less than 90%, the start sites for the complementary binding of the antisense strand to the mRNA are positions 342, 360, 499, 534, 3575, 3576, 3667, 3668, 3775, 3866, 4069, or 4242.

[0025] The above mRNA is numbered NM_022065.5 in NCBI.

[0026] The sense chain includes a region that is complementary to the antisense chain; the length of the complementary binding region can be 15-25 bp, 17-23 bp, or 19-21 bp. Specifically, the lengths of the complementary binding regions are 15 bp, 16 bp, 17 bp, 18 bp, 19 bp, 20 bp, 21 bp, 22 bp, 23 bp, 24 bp, and 25 bp.

[0027] In some embodiments, the antisense strand is 21 nt long, the sense strand is 21 nt long, and the complementary binding region of the antisense strand and the sense strand is 19 bp long.

[0028] The antisense strand and / or sense strand described above have protruding nucleotides at one or both ends. The protruding nucleotides are any base nucleotides. In one embodiment, there is one 3' protruding nucleotide; in another embodiment, there are two 3' protruding nucleotides. In one embodiment, the protruding nucleotide is deoxythymidine. The 3' end of the antisense strand and the 5' end of the sense strand may also be covalently linked.

[0029] In some embodiments, the antisense strand continuously comprises any of the nucleotides described in SEQ ID NO: 1-195 and a 3' overhanging nucleotide, and the sense strand correspondingly comprises a complementary nucleotide (SEQ ID NO: 160-318) of any of the nucleotides described in SEQ ID NO: 1-159. In some embodiments, both the antisense strand and the sense strand further comprise a 3' overhanging nucleotide.

[0030] Table 1. Sequences of antisense and sense chains The aforementioned antisense strand and / or sense strand contains at least one modified nucleotide. The modified nucleotide is at least one of phosphate group modification, base modification, and ribose modification. For example, the modified nucleotide is a 5'-thiophosphate modified nucleotide, a 5'-methylphosphonate modified nucleotide, a 5'-vinylphosphonate modified nucleotide, a peptide nucleic acid; pseudouridine acid, 2-thiouridine acid, N1-methylpseudouridine acid, 5-methyluridine acid, 5-methoxyuridine acid, N6-methyladenosine acid, 5-methylcytidine acid, N-ethylpiperidin-6-triazole modified adenosine acid, 5-nitroindole modified nucleotide, 2,4-difluorotoluene modified nucleotide, 6'-phenylpyrrolidinylcytidine acid; 2'-deoxy modified nucleotide, 2'-fluoro modified nucleotide. The nucleotide is selected from at least one of the following: 2'-deoxy-2'-fluorine modified nucleotide, 2'-O-methyl modified nucleotide, 2'-O-methoxyethyl modified nucleotide, 2'-amino modified nucleotide, 2'-O-allyl modified nucleotide, 2'-C-alkyl modified nucleotide, 2'-hydroxy modified nucleotide, nucleotide with 2'-O atom and 4'-C atom cross-linked by methylene or ethylene, unlocked nucleotide, morpholine nucleotide, ethylene glycol nucleotide, tricyclic nucleotide, tetrahydropyran modified nucleotide, 1,5-dehydrated hexadiol modified nucleotide, and fluorescent group modified nucleotide.

[0031] In one embodiment, the modified nucleotide is at least one of 5'-thiophosphate modified nucleotide, 2'-deoxy modified nucleotide, 2'-fluorine modified nucleotide, and 2'-O-methyl modified nucleotide.

[0032] The nucleic acid molecule also contains abase-free nucleotides (AP sites) and / or mismatched base pairs.

[0033] The aforementioned nucleic acid molecules containing a double-stranded structure also include a ligand, which is covalently linked to the nucleic acid molecule. The ligand is selected from N-acetylgalactosamine (GalNAc) and its derivatives or cholesterol and its derivatives, and the ligand can increase the liver targeting of the nucleic acid molecule. The ligand is conjugated to the 3' end, 5' end, or a non-double-stranded region of the nucleic acid molecule. The N-acetylgalactosamine (GalNAc) and its derivatives are conjugated to the 3' end of the sense strand of the nucleic acid molecule. The cholesterol and its derivatives are conjugated to the 5' end of the sense strand of the nucleic acid molecule. In one embodiment, the N-acetylgalactosamine (GalNAc) derivative is: .

[0034] In one embodiment, the connection between the sense strand 3' end of the nucleic acid molecule and the ligand is as follows: Where X is O or S. The nucleic acid molecules of the above conjugated ligands exhibit better liver targeting in experimental monkeys.

[0035] In another embodiment, the cholesterol and its derivatives are conjugated to the 5' end of the sense strand of a nucleic acid molecule.

[0036] The present invention also provides a composition containing the above-mentioned nucleic acid molecule, comprising the above-mentioned nucleic acid molecule with a double-stranded structure and a carrier. The carrier and the nucleic acid molecule are non-covalently linked. The carrier can effectively deliver the nucleic acid molecule and reduce its degradation before reaching the target.

[0037] The vector is selected from any one of lentivirus, adenovirus, adeno-associated virus, lipid nanoparticle (LNP) vector, lipid nanoparticle vector, polyamide-amine dendritic polymer (PAMAM) vector, polyethyleneimine (PEI) vector, albumin vector, zein vector, exovesicles, exosomes, C quantum dots, and Ce quantum dots.

[0038] In some embodiments, the carrier is a lipid nanoparticle carrier. In one embodiment, the composition is prepared based on a Tris-based ionizable lipid disclosed in CN116199646A. In one embodiment, the structure of the Tris-based ionizable lipid is: .

[0039] In another embodiment, the structure of the Tris-based ionizable lipid is as follows: .

[0040] The above-mentioned composition containing nucleic acid molecules and Tris-based ionizable lipid carriers exhibits better stability and duration of action in experimental monkeys.

[0041] The aforementioned nucleic acid molecules or compositions can be used to lower blood cholesterol, low-density lipoprotein cholesterol (LDL-C), and triglycerides in mammals, and to prepare drugs or diagnostic reagents for treating hyperlipidemia, fatty liver disease, atherosclerotic cardiovascular disease, and other cardiovascular and cerebrovascular metabolic diseases.

[0042] The present invention has the following advantages: This invention, through mammalian biological experiments, confirms that inhibiting or disrupting THADA expression in vivo can lower blood cholesterol, low-density lipoprotein cholesterol (LDL-C), and triglyceride levels without affecting high-density lipoprotein cholesterol levels, reduce hepatic triglyceride deposition and hepatic steatosis, and improve atherosclerosis. The effects are clearly defined and without adverse reactions. The siRNA sequence provided by this invention can effectively reduce THADA expression levels in human hepatocytes and can be used to prepare drugs for the prevention and treatment of cardiovascular metabolic diseases such as hyperlipidemia, fatty liver disease, and atherosclerotic cardiovascular disease. This invention provides a novel nucleic acid drug sequence for hyperlipidemia and cardiovascular metabolic diseases, with good practical application value and broad application prospects. Attached Figure Description

[0043] Figure 1 For comparison and Thada Serum total cholesterol (A), serum low-density lipoprotein cholesterol (LDL-C) (B), serum high-density lipoprotein cholesterol (HDL-C) (C), and serum triglyceride (D) levels in liver knockout mice under normal and high-cholesterol diets. Figure 2 For comparison and Thada Oil Red staining morphology of liver tissue in liver knockout mice (A) and liver triglyceride levels (B); Figure 3 For comparison and Thada Oil Red staining of aortic plaques in knockout mice (A) and quantification of plaque area (B); Figure 4 For the development of siRNA in the human hepatocyte cell line HepG2 THADA Knockdown efficiency. Detailed Implementation

[0044] The present invention will be further described below with reference to the embodiments and accompanying drawings, but the present invention is not limited to the following embodiments.

[0045] Example 1: Liver-specific THADA deficiency significantly improved hyperlipidemia and fatty liver disease in mice. 1. Liver-specific Thada Construction of knockout mice Targeting using CRISPR / Cas9 technology Thada A targeting sequence was designed for the exon 8 region of the gene. Two loxp sites were designed and constructed at both ends of the exon 8 region. gRNA, a donor vector containing loxP sites, and Cas9 were co-injected into mouse zygotes to generate conditionally knocked-out progeny of the targeted gene. The mouse genotype was determined by polymerase chain reaction and sequence analysis. Thada -flox mice. Further, the above... Thada -flox mouse and hepatocyte-specific promoters Albumin Driven Cre mice ( Alb -Cre) mating, obtaining offspring through successive generations of breeding. Thada -flox homozygous, simultaneously carrying Alb -Cre mice, that is Thada Hepatocyte-specific knockout (LKO) mice, whose littermates do not carry the virus. Alb -Cre mice were used as controls. The above construction strategy enabled the achievement of THADA-specific deletion and inactivation in mouse hepatocytes.

[0046] 2. Induction of hyperlipidemia The above Thada Liver knockout (LKO) and littermate control (WT) mice were fed a high-fat, high-cholesterol diet (containing 40 kcal% fat and 1.25% cholesterol, Research Diet) for four weeks starting at 8 weeks of age. This high-cholesterol diet resulted in mice exhibiting significant elevated blood lipid levels, hepatic lipid accumulation, and hepatic steatosis, demonstrating metabolic damage phenotypes, and is a commonly used mouse model of hyperlipidemia. Figure 1 As shown in Figure A, compared with mice on a normal diet, mice induced by a high-cholesterol diet showed significantly higher serum total cholesterol levels, confirming the successful induction of the hyperlipidemia model.

[0047] 3. Collection, processing, and testing of mouse blood samples Mice were divided into four groups: control (WT) mice, mice fed a normal diet, and mice fed a normal diet. Thada Liver knockout (LKO) mice on a normal diet, and control (WT) mice on a high-fat, high-cholesterol diet. Thada Liver knockout (LKO) mice were fed a high-fat, high-cholesterol diet. After dietary modeling induction, blood was collected from each group of mice 6 h after fasting. Serum was collected by centrifugation at 3000 rpm for 15 min at 4°C.

[0048] (1) Measurement of total cholesterol levels in mouse serum The total cholesterol levels of the above four groups of mouse serum were determined according to the instructions of the liquid sample total cholesterol enzymatic assay kit.

[0049] Take 190 µL of working solution. Add 10 µL of blank control solution, standard, and mouse serum sample to each working solution, respectively, for a total reaction volume of 200 µL. Incubate at 37℃ for 20 min; the color stabilizes within 60 min after reaction equilibrium. Measure the OD value of each well using a microplate reader. Plot a standard curve and calculate the total cholesterol content in serum by comparing the results with the standard curve.

[0050] The results are as follows Figure 1 As shown in Figure A, under normal dietary conditions, Thada The serum total cholesterol level in liver knockout (LKO) mice was significantly lower than that in the control group. Furthermore, THADA deficiency further reduced the serum total cholesterol level in hyperlipidemic mice after induction with a high-fat, high-cholesterol diet.

[0051] (2) Measurement of serum low-density lipoprotein cholesterol (LDL-C) levels in mice The LDL-C levels in the serum of the four groups of mice were determined according to the instructions of the blood low-density lipoprotein cholesterol enzymatic assay kit.

[0052] LDL cholesterol separation: Take 50 μL of serum, add an equal volume of 50 μL of LDL capture agent, vortex to mix, and incubate at room temperature for 10 min. Centrifuge at 2000 g for 20 min; the precipitate contains LDL cholesterol. Carefully remove the supernatant, centrifuge again at 2000 g for 10 min, and discard the supernatant. Add 100 μL of PBS, vortex to resuspend the precipitate.

[0053] LDL cholesterol assay: Prepare the working solution by adding 190 µL to the wells of the microplate. Add 10 µL of the supernatant prepared after the LDL cholesterol separation step above, and add 10 µL of standard to the standard tubes. The total reaction volume is 200 µL. Incubate at 37℃ for 20 min. Measure the OD value of each tube using a microplate reader. Plot a standard curve and calculate the LDL-C concentration of each group of samples.

[0054] The results are as follows Figure 1 As shown in Figure B, under normal dietary conditions, Thada The serum LDL-C level in liver knockout (LKO) mice was significantly lower than that in the control group. Furthermore, after a high-fat, high-cholesterol diet induced an increase in LDL-C levels in mice, THADA deficiency further significantly reduced serum LDL-C levels in hyperlipidemic mice.

[0055] (3) Measurement of high-density lipoprotein cholesterol (HDL-C) levels in mouse serum The HDL-C levels in the serum of the four groups of mice were measured according to the instructions of the blood high-density lipoprotein cholesterol enzymatic assay kit.

[0056] HDL cholesterol separation: Take 50 μL of serum, add an equal volume of 50 μL of HDL capture agent, vortex to mix, and incubate at room temperature for 10 minutes. Centrifuge at 2000 g for 20 minutes, and the supernatant contains HDL cholesterol.

[0057] HDL cholesterol assay: Prepare the working solution by adding 190 µL to the wells of the microplate. Add 10 µL of the supernatant prepared after the HDL cholesterol separation step above, and add 10 µL of standard to the standard tubes. The total reaction volume is 200 µL. Incubate at 37℃ for 20 min. Measure the OD value of each tube using a microplate reader. Plot a standard curve and calculate the LDL-C concentration of each group of samples.

[0058] The results are as follows Figure 1 As shown in C, regardless of whether one is on a normal diet or a high-cholesterol diet, Thada Compared with control mice, liver knockout (LKO) mice showed no significant change in serum HDL-C, indicating that THADA deficiency does not affect serum HDL-C levels.

[0059] (4) Measurement of serum triglyceride levels in mice: The triglyceride levels in the serum of the four groups of mice were determined according to the instructions of the enzymatic assay kit for liquid sample triglyceride content.

[0060] Dilute the standard accordingly to prepare the working solution. Add 10 μL of the standard or mouse serum sample to a microplate, along with 190 µL of the prepared working solution. Incubate at 37℃ for 15 min. Measure the OD value of each tube using a microplate reader. Plot a standard curve and calculate the triglyceride concentration of each group of samples.

[0061] The results are as follows Figure 1 As shown in D, under normal dietary conditions, Thada The serum triglyceride levels in liver knockout (LKO) mice were significantly lower than those in the control group. Furthermore, after a high-fat, high-cholesterol diet induced a significant increase in serum triglycerides in mice, THADA deficiency further significantly reduced serum triglyceride levels in hyperlipidemic mice.

[0062] 4. Liver histology Oil Red O staining Oil Red O staining (ORO): After dietary induction, equal volumes of liver tissue were collected from mice in each group, embedded in OCT scans, and then frozen sections were prepared. After thawing, the sections were fixed in 4% paraformaldehyde fixative for 20 min. After washing with distilled water, the sections were stained with 60% isopropanol for 2 min, then directly stained in Oil Red O staining solution in the dark for 20 min. After washing with distilled water, the sections were stained with hematoxylin for 2 min, washed with distilled water, and mounted with glycerol gelatin. Images were acquired under a microscope and photographed for analysis.

[0063] 5. Liver triglyceride testing After dietary induction, the mice were fasted for 6 hours before liver tissue samples were collected and flash-frozen in liquid nitrogen. The frozen liver tissue samples were then lysed with lysis buffer, centrifuged at 12000 g for 10 min at 4°C, and the supernatant was collected. The triglyceride content in the supernatant was measured according to the instructions of the enzymatic assay kit for triglyceride content in tissue cells. The absorbance was measured using a microplate reader, and the absolute triglyceride value was calculated using a standard curve. The protein content in the lysate supernatant was measured using the BCA protein quantification kit (purchased from Thermo Fisher Scientific). The triglyceride content of each sample was corrected for protein content.

[0064] The above experimental data are as follows Figure 2 As shown, in control mice with diet-induced fatty liver disease, Oil Red O staining of liver tissue revealed significant lipid accumulation, increased lipid droplets, and vacuolar degeneration, indicating successful induction of the fatty liver disease model. In LKO mice with lost THADA function, the lipid staining area in hepatocytes was significantly reduced, lipid droplet vacuolation was significantly decreased, and no adverse inflammatory changes were observed histologically. Furthermore, triglyceride content assays showed a significant decrease in triglyceride levels and lipid accumulation in the livers of LKO mice.

[0065] The experimental results show that, regardless of whether hyperlipidemia was induced by a normal diet or a high-fat, high-cholesterol diet, liver-specific THADA inactivation significantly reduced serum total cholesterol, low-density lipoprotein cholesterol, and triglyceride levels in mice, without affecting high-density lipoprotein cholesterol levels, demonstrating a significant beneficial effect on improving the lipid profile. These results indicate that specific interference with hepatic THADA expression can be used for the prevention and treatment of lipid metabolism disorders such as hyperlipidemia, hypercholesterolemia, and fatty liver, without significant adverse effects.

[0066] Example 2: THADA deficiency significantly improves the progression of atherosclerotic plaques. 1. Construction of an animal model of atherosclerotic cardiovascular disease right ApoeBackground knockout mice given a high-cholesterol diet can develop hyperlipidemia and atherosclerosis, serving as a commonly used and classic animal model of atherosclerosis. To investigate the therapeutic effect of THADA on atherosclerosis, [the text abruptly ends here]. Thada Heterozygous mice and Apoe Heterozygous mice were mated and bred to obtain Apoe / Thada Double gene knockout (KO) mice, littermates ApoeKO / Thada Wild-type (WT) mice served as controls. Both groups of mice were fed a high-cholesterol diet (containing 40 kcal% fat and 1.25% cholesterol, Research Diet) starting at 8 weeks of age to induce atherosclerosis.

[0067] 2. Aortic dissection and collection in atherosclerotic mice After anesthetizing and fixing the mice in both groups, a systemic perfusion was performed using pre-cooled physiological saline after a cardiac incision. After the blood was drained, the aortic arch and abdominal aorta were separated, and the surrounding fat was removed. The entire aorta was then fixed in 4% paraformaldehyde fixative.

[0068] 3. Assessment of Oil Red O staining plaques in the aorta of atherosclerotic mice The fully fixed aorta was removed from the fixative solution and washed with PBS to remove any remaining fixative. Under a stereomicroscope, the adipose tissue in the adventitia was further removed using fine forceps. The entire aorta was then longitudinally dissected distally along the greater curvature of the aortic arch. The dissected aorta was spread out with the intima facing upwards and fixed to a black dish with a small needle. Prepared Oil Red O staining solution was added for incubation and staining. After discarding the Oil Red stain, the aorta was differentiated with ethanol and washed, followed by microscopic imaging. ImageJ software was used to measure the plaque area and the total intima area. The relative percentage of the plaque was calculated by dividing the total plaque area by the total intima area. This was used to assess the severity of atherosclerotic plaque formation.

[0069] like Figure 3 As shown, the control group Thada Wild-type mice exhibited typical pathological changes of atherosclerosis after induction. Multiple lipid plaques were observed in the aorta stained with Oil Red O, indicating successful atherosclerosis modeling. Compared with the control group, Thada Gene knockout mice showed a significant reduction in Oil Red O staining lipids in the aorta, indicating a marked slowdown in plaque progression. Quantitative analysis of atherosclerotic plaque area showed... Thada The total area of ​​aortic plaques in the gene knockout mice was also significantly reduced compared to the control group.

[0070] The above experimental data fully demonstrate that inhibiting THADA gene expression can effectively alleviate the severity of atherosclerotic plaque lesions. These results provide sufficient in vivo biological evidence for the application of THADA in atherosclerotic cardiovascular diseases.

[0071] Example 3: siRNA in human hepatocyte cell lines THADA Knockdown 1. HepG2 cell line culture and transfection HepG2 cell lines were purchased from the Center for Type Culture Collection, Chinese Academy of Sciences. The cell culture medium was DMEM (Gibco) containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin. All cell lines were stored in a humidified incubator at 37°C and 5% CO2. After digestion with 0.25% trypsin, the cells were cultured at 5.0 × 10⁻⁶ cells / mL. 5 Cells per well were seeded in a 24-well plate.

[0072] Table 2 siRNA sequences The siRNAs listed in Table 2 were artificially synthesized, and then the siRNAs and their control nonsense sequences were transfected into HepG2 cells according to the Lipo3000 transfection reagent instructions.

[0073] 2. RNA extraction and reverse transcription HepG2 cells were collected 24 hours after transfection, and total RNA was extracted using a rapid RNA extraction kit (purchased from Yishan Biotechnology). The concentration and purity of RNA were measured using a NanoDrop One spectrophotometer. Subsequently, following the manufacturer's instructions, 1 μg of RNA was reverse transcribed into 20 μL of cDNA using a PrimeScript RT kit (purchased from Takara Bio).

[0074] 3. Real-time quantitative PCR detection qPCR was performed on a Roche Light Cycler 480 system (Penzberg Roche, Germany). The total reaction volume was 10 μL, containing 1 μL of cDNA, 1 μL of specific primers, 5 μL of SYBR Green Master Mix (purchased from Takara Bio), and 3 μL of ddH2O. Cycling conditions were set as follows: pre-denaturation at 95°C for 10 min, followed by 40 cycles, each consisting of denaturation at 95°C for 10 seconds, annealing at 60°C, and extension for 1 min. Melting profile analysis included one cycle: 95°C for 10 seconds, 60°C for 1 min, and a final step: 95°C for 15 seconds.

[0075] 4. Data Statistical Analysis Export the data to Excel format using CT. control(THADA) -CT β-Actin The results were standardized. To calculate the fold change in relative siRNA silencing efficiency, the data were analyzed using the ΔΔCT method, and the three parallel replicates were analyzed.

[0076] The results are as follows Figure 4 As shown, compared with the control group, siRNA sequences 1-159 were significantly reduced in the human hepatocyte cell line HepG2. THADA The mRNA expression levels of the siRNAs were measured, and the THADA knockdown efficiency of 151 siRNAs reached more than 50%, suggesting that the above siRNA sequences can be effectively used to reduce the expression level of THADA in the liver, so as to improve hyperlipidemia and lipid metabolism disorders.

[0077] The above embodiments, by constructing various in vivo and in vitro models targeting the THADA gene to simulate the pathological processes of hyperlipidemia, fatty liver disease, and atherosclerotic cardiovascular diseases in humans, revealed that inhibiting THADA gene expression can significantly improve lipid metabolism, reduce hyperlipidemia, improve fatty liver disease, and treat cardiovascular metabolic diseases such as atherosclerosis, with no obvious toxic side effects. This indicates that siRNA that inhibits THADA gene expression can be used to prepare novel preventive and therapeutic drugs for the aforementioned lipid metabolism diseases such as hyperlipidemia, fatty liver disease, and atherosclerotic cardiovascular diseases.

[0078] The above description is merely an embodiment of this application and is not intended to limit this application. Various modifications and variations can be made to this application by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principle of this application should be included within the scope of the claims of this application.

Claims

1. Use of a nucleic acid molecule targeting THADA for the treatment of a cardiometabolic disease, characterized in that, The nucleic acid molecule comprises an antisense strand and a sense strand, wherein the antisense strand comprises a complementary binding region to a part of mRNA of THADA; the nucleic acid molecule can cause post-transcriptional silencing of the THADA gene; the nucleic acid molecule also comprises a pharmaceutically acceptable salt thereof.

2. Use according to claim 1, characterized in that, The mRNA is a mature mRNA or a precursor of mRNA of the THADA gene. The cardiovascular metabolic disease comprises hyperlipidemia, dyslipidemia, fatty liver disease, and atherosclerotic cardiovascular disease.

3. Use according to claim 1, characterized in that, The complementary binding region has a length of 15-25 nt.

4. Use according to claim 1, characterized in that, The THADA gene is a THADA gene of a rodent, a primate or a human.

5. The use according to claim 1, characterized in that, The THADA gene is human THADA gene, and the antisense strand is complementary to the start site of mRNA selected from the group consisting of positions 135, 137, 139, 144, 189, 246, 269, 275, 277, 315, 337, 342, 360, 499, 515, 533, 534, 536, 537, 547, 560, 582, 583, 584, 593, 630, 631, 676, 696, 702, 717, 721, 722, 728, 734, 735, 738, 741, 769, 846, 848, 850, 1156, 1157, 1188, 1189, 1194, 1278, 1344, 1345, 1507, 1508, 1515, 1518, 1523, 1612, 1631, 1738, 1804, 1810, 1811, 1828, 1830, 1838, 1839, 1927, 1935, 1954, 1992, 1993, 2037, 2040, 2045, 2059, 2060, 2088, 2113, 2118, 2123, 2169, 2173, 2174, 2175, 2241, 2247, 2263, 2267, 2268, 2289, 2299, 2365, 2368, 2443, 2444, 2445, 2447, 2466, 2469, 2482, 2488, 2549, 2557, 2629, 2637, 2642, 2703, 2717, 2743, 2744, 2745, 2752, 2796, 2872, 2876, 2896, 2917, 2924, 2926, 2961, 2966, 3066, 3092, 3112, 3120, 3122, 3162, 3166, 3176, 3207, 3208, 3210, 3224, 3228, 3277, 3279, 3280, 3283, 3287, 3303, 3363, 3403, 3408, 3410, 3483, 3510, 3511, 3575, 3576, 3667, 3668, 3775, 3866, 4023, 4043, 4045, 4069, 4141, 4242 or 4247; The mRNA is NCBI numbered NM_022065.

5.

6. Use according to claim 1, characterized in that, The sense strand comprises a region of partial complementarity to the antisense strand.

7. Use according to claim 6, characterized in that, The region of partial complementarity is 15-25 bp in length.

8. The use according to claim 1, characterized in that, The antisense strand and / or the sense strand has overhanging nucleotides at one or both ends.

9. Use according to claim 8, characterized in that, The overhanging nucleotides are 1 or 2; the overhanging nucleotides are any base nucleotides.

10. The use according to claim 1, characterized in that, The 3' end of the antisense strand and the 5' end of the sense strand are covalently linked. The 3' end of the antisense strand and the 5' end of the sense strand are covalently linked.

11. Use according to claim 1, characterized in that, the antisense strand comprises, in order, the nucleotides of any one of SEQ ID NOs: 1-159 and 3' terminal overhanging nucleotides, and the sense strand comprises, in order, the nucleotides of any one of SEQ ID NOs: 160-318.

12. Use according to claim 11, characterized in that, the sense strand comprises 3' terminal overhanging nucleotides.

13. Use according to claim 12, characterized in that, the antisense strand comprises, in order, the nucleotides of any one of SEQ ID NOs: 1-159 and 2 deoxythymidine nucleotides at the 3' end, and the sense strand comprises, in order, the nucleotides of any one of SEQ ID NOs: 160-318 and 2 deoxythymidine nucleotides at the 3' end.

14. The use according to claim 1, characterized in that, the nucleic acid molecule further comprises an abasic nucleotide site and / or a mismatch base pair site.

15. The use according to claim 1, characterized in that, the antisense strand and / or the sense strand comprises at least one modified nucleotide.

16. The use according to claim 15, characterized in that, the modified nucleotide is at least one of a phosphate group modification, a base modification, and a ribose modification.

17. Use according to claim 16, characterized in that, the modified nucleotide is at least one of a 5'-phosphorothioate modified nucleotide, a 5'-methylphosphonate modified nucleotide, a 5'-vinylphosphonate modified nucleotide, a peptide nucleic acid; a pseudouridine nucleotide, a 2-thiouridine nucleotide, a N1-methylpseudouridine nucleotide, a 5-methyluridine nucleotide, a 5-methoxyuridine nucleotide, a N6-methyladenosine nucleotide, a 5-methylcytidine nucleotide, a N-ethylpiperidine-6-triazole modified adenosine nucleotide, a 5-nitroindole modified nucleotide, a 2,4-difluorotoluene modified nucleotide, a 6'-phenylpyrrolo-cytidine nucleotide; a 2'-deoxy modified nucleotide, a 2'-fluoro modified nucleotide, a 2'-deoxy-2'-fluoro modified nucleotide, a 2'-O-methyl modified nucleotide, a 2'-O-methoxyethyl modified nucleotide, a 2'-amino modified nucleotide, a 2'-O-allyl modified nucleotide, a 2'-C-alkyl modified nucleotide, a 2'-hydroxyl modified nucleotide, a 2'-O atom and 4'-C atom cross-linked by a methylene or ethylene modified nucleotide, an unlocked nucleotide, a morpholino nucleotide, a glycol nucleotide, a tricyclo nucleotide, a tetrahydropyran modified nucleotide, a 1,5-anhydrohexitol modified nucleotide, and a fluorescent group modified nucleotide.

18. The use according to any one of claims 1 to 17, characterized in that, the nucleic acid molecule further comprises a ligand, the ligand being covalently linked to the nucleic acid molecule.

19. Use according to claim 18, characterized in that, the ligand is conjugated to the 3' end, the 5' end, or a non-double stranded region of the nucleic acid molecule.

20. The use according to claim 18, characterized in that, the ligand is selected from N-acetylgalactosamine and derivatives thereof or cholesterol and derivatives thereof.

21. The use according to claim 20, characterized in that, N-acetylgalactosamine and derivatives thereof are conjugated to the 3' end of the sense strand of the nucleic acid molecule; cholesterol and derivatives thereof are conjugated to the 5' end of the sense strand of the nucleic acid molecule.

22. The use according to claim 20, characterized in that, the derivative of N-acetylgalactosamine is: 。 23. The use according to claim 22, characterized in that, the linkage of the 3' end of the sense strand of the nucleic acid molecule to the ligand is as follows: ； wherein X is O or S.

24. The use according to claim 20, characterized in that, the ligand of the nucleic acid molecule is cholesterol; the ligand is linked to the 5' end of the sense strand.

25. The application according to any one of claims 1-17, characterized in that, the nucleic acid molecule is loaded on a carrier; the carrier and the nucleic acid molecule are linked by a non-covalent linkage.

26. The use according to claim 25, characterized in that, the carrier is selected from any one of a lentivirus, an adenovirus, an adeno-associated virus, a lipid nanoparticle carrier, a lipidoid nanoparticle carrier, a polyamido-amine dendrimer carrier, a polyethyleneimine carrier, an albumin carrier, a zein carrier, an exovesicle, an exosome, a C quantum dot, and a Ce quantum dot.

27. The use according to claim 26, characterized in that, the carrier is a lipid nanoparticle carrier.

28. The use according to claim 27, characterized in that, The lipid nanoparticle carrier is prepared with ionizable lipids based on Tris; the ionizable lipids based on Tris have the following structural formula: ; or, 。

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