SiRNA combination for inhibiting expression of LOC157273 and application thereof

Abstract

The invention relates to the technical field of biological medicine, and provides a siRNA combination for inhibiting expression of LOC157273 and application of the siRNA combination, and the siRNA combination comprises one or more of 63 siRNA1, siRNA2, siRNA3, siRNA4, siRNA25, siRNA45, siRNA48 and the like. The siRNA composition provided by the invention can be used for targeted therapy of metabolic diseases related to the gene by inhibiting expression of the LOC157273 gene, and intervention on abnormal glucose metabolism, lipid metabolism and protein metabolism is realized.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to an siRNA combination for inhibiting LOC157273 expression and an application thereof. Background Art

[0002] With the change of lifestyle and the intensification of population aging, metabolic diseases such as type 2 diabetes (T2D) and overweight and dyslipidemia have become increasingly serious public health issues. T2D patients are often accompanied by overweight, abnormal low-density lipoprotein (LDL) and total cholesterol (TC) levels, forming a vicious cycle and significantly increasing the risk of cardiovascular disease. Although existing drugs have significant effects on single metabolic indicators (such as blood sugar and weight) in the treatment of metabolic syndrome, their limitations are reflected in the lack of coordinated regulation of multi-system metabolic abnormalities and pathological mechanisms. At the same time, clinical treatment mostly relies on hypoglycemic and lipid-lowering drugs and weight loss interventions for patients, but there are long-term problems such as diminished efficacy and many adverse reactions, making it difficult to achieve comprehensive improvement of metabolic indicators and cure of the disease.

[0003] To address this issue, Huayuan Biopharmaceuticals has applied for siRNA that inhibits LOC157273 gene expression in patent 202210990726.9. This can silence the expression of LOC157273, thereby inhibiting the protein PP1R3B to regulate glycogen synthesis and lower blood glucose levels in the peripheral circulation. However, the drug in this application mainly focuses on the glycogen metabolism pathway and lacks synergistic intervention effects on co-morbid mechanisms such as dyslipidemia, hepatic steatosis, and chronic inflammation. If we want to achieve the joint prevention of multiple diseases, we still need to rely on a combination drug strategy.

[0004] However, the application of innovative targeted drugs in vivo faces significant challenges: the actual effects vary significantly from expectations, making effective translation from the laboratory to the clinic difficult. Furthermore, for the in vivo delivery of oligonucleotide therapeutics, nucleic acids, and double-stranded siRNA, current receptor-specific ligand-bound iRNA duplexes and their preparation methods are insufficient in terms of effectiveness and practicality. Therefore, there is an urgent need to develop new, more targeted, and highly effective long-acting therapeutics. Summary of the Invention

[0005] The present invention aims to provide an siRNA combination for inhibiting LOC157273 expression and its use. The siRNA combination contains at least one duplex region, which can inhibit expression of the target gene LOC157273 in cells. This siRNA combination focuses on innovative drug combinations, breaking through the limitations of traditional treatment methods and the bottleneck of single-target optimization.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0007] The present invention provides an siRNA combination for inhibiting the expression of LOC157273, wherein the siRNA combination comprises siRNA1, siRNA2, siRNA3, siRNA4, siRNA5, siRNA6, siRNA7, siRNA8, siRNA9, siRNA10, siRNA11, siRNA12, siRNA13, siRNA14, siRNA15, siRNA16, siRNA17, siRNA18, siRNA19, siRNA20, siRNA21, siRNA22, siRNA23, siRNA24, siRNA25, siRNA26, siRNA27, siRNA28, siRNA29, and siRNA30. , one or more of siRNA31, siRNA32, siRNA33, siRNA34, siRNA35, siRNA36, siRNA37, siRNA38, siRNA39, siRNA40, siRNA41, siRNA42, siRNA43, siRNA44, siRNA45, siRNA46, siRNA47, siRNA48, siRNA49, siRNA50, siRNA51, siRNA52, siRNA53, siRNA54, siRNA55, siRNA56, siRNA57, siRNA58, siRNA59, siRNA60, siRNA61, siRNA62, and siRNA63.

[0008] Preferably, the siRNA combination comprises one or more of siRNA25, siRNA45, and siRNA48.

[0009] Preferably, the mass ratio of siRNA25, siRNA45 and siRNA48 in the siRNA combination is 1-3:1-3:1-3, and more preferably 1:1:1.

[0010] Preferably, the siRNA1 comprises the sense strand shown in SEQ ID No.72 and the antisense strand shown in SEQ ID No.73; the siRNA2 comprises the sense strand shown in SEQ ID No.74 and the antisense strand shown in SEQ ID No.75; the siRNA3 comprises the sense strand shown in SEQ ID No.76 and the antisense strand shown in SEQ ID No.77; the siRNA4 comprises the sense strand shown in SEQ ID No.78 and the antisense strand shown in SEQ ID No.79; the siRNA5 comprises the sense strand shown in SEQ ID No.80 and the antisense strand shown in SEQ ID No.81; the siRNA6 comprises the sense strand shown in SEQ ID No.82 and the antisense strand shown in SEQ ID No.83; the siRNA7 comprises the sense strand shown in SEQ ID No.84 and the antisense strand shown in SEQ ID No.85; the siRNA8 comprises the sense strand shown in SEQ ID No.86 and the antisense strand shown in SEQ ID No.87; the siRNA9 comprises the sense strand shown in SEQ ID No.88 and the antisense strand shown in SEQ ID No.89. No. 89; the siRNA 10 includes the sense strand shown in SEQ ID No. 90 and the antisense strand shown in SEQ ID No. 91; the siRNA 11 includes the sense strand shown in SEQ ID No. 92 and the antisense strand shown in SEQ ID No. 93; the siRNA 12 includes the sense strand shown in SEQ ID No. 94 and the antisense strand shown in SEQ ID No. 95; the siRNA 13 includes the sense strand shown in SEQ ID No. 96 and the antisense strand shown in SEQ ID No. 97; the siRNA 14 includes the sense strand shown in SEQ ID No. 98 and the antisense strand shown in SEQ ID No. 99; the siRNA 15 includes the sense strand shown in SEQ ID No. 100 and the antisense strand shown in SEQ ID No. 101; the siRNA 16 includes the sense strand shown in SEQ ID No. 102 and the antisense strand shown in SEQ ID No. 103; the siRNA 17 includes the sense strand shown in SEQ ID No. 104 and the antisense strand shown in SEQ ID No. No.105; the siRNA18 comprises the sense strand as shown in SEQ ID No.106 and the antisense strand as shown in SEQ ID No.107; the siRNA19 comprises the sense strand as shown in SEQ ID No.108 and the antisense strand as shown in SEQ ID No.109; the siRNA20 comprises the sense strand as shown in SEQ ID No.110 and the antisense strand as shown in SEQ ID No.111; the siRNA 21 includes the sense strand as shown in SEQ ID No.112 and the antisense strand as shown in SEQ ID No.113; the siRNA 22 includes the sense strand as shown in SEQ ID No.114 and the antisense strand as shown in SEQ ID No.115; the siRNA 23 includes the sense strand as shown in SEQ ID No.116 and the antisense strand as shown in SEQ ID No.117; the siRNA 24 includes the sense strand as shown in SEQ ID No.118 and the antisense strand as shown in SEQ ID No.119; the siRNA 25 includes the sense strand as shown in SEQ ID No.120 and the antisense strand as shown in SEQ ID No.121; the siRNA 26 includes the sense strand as shown in SEQ ID No.122 and the antisense strand as shown in SEQ ID No.123; the siRNA 27 includes the sense strand as shown in SEQ ID No.124 and the antisense strand as shown in SEQ ID No.125; the siRNA 28 includes the sense strand as shown in SEQ ID No.129 and the antisense strand as shown in SEQ ID No.130; 126 and the antisense strand as shown in SEQ ID No. 127; the siRNA29 includes the sense strand as shown in SEQ ID No. 128 and the antisense strand as shown in SEQ ID No. 129; the siRNA30 includes the sense strand as shown in SEQ ID No. 130 and the antisense strand as shown in SEQ ID No. 131; the siRNA31 includes the sense strand as shown in SEQ ID No. 132 and the antisense strand as shown in SEQ ID No. 133; the siRNA32 includes the sense strand as shown in SEQ ID No. 134 and the antisense strand as shown in SEQ ID No. 135; the siRNA33 includes the sense strand as shown in SEQ ID No. 136 and the antisense strand as shown in SEQ ID No. 137; the siRNA34 includes the sense strand as shown in SEQ ID No. 138 and the antisense strand as shown in SEQ ID No. 139; the siRNA35 includes the sense strand as shown in SEQ ID No. 140 and the antisense strand as shown in SEQ ID No.141; the siRNA36 includes the sense strand shown in SEQ ID No.142 and the antisense strand shown in SEQ ID No.143; the siRNA37 includes the sense strand shown in SEQ ID No.144 and the antisense strand shown in SEQ ID No.145; the siRNA38 includes the sense strand shown in SEQ ID No.146 and the antisense strand shown in SEQ ID No.147; the siRNA39 includes the sense strand shown in SEQ ID No.148 and the antisense strand shown in SEQ ID No.149; the siRNA40 includes the sense strand shown in SEQ ID No.150 and the antisense strand shown in SEQ ID No.151; the siRNA41 includes the sense strand shown in SEQ ID No.152 and the antisense strand shown in SEQ ID No.153; the siRNA42 includes the sense strand shown in SEQ ID No.154 and the antisense strand shown in SEQ ID No.155; the siRNA43 includes the sense strand shown in SEQ ID No.156 and the antisense strand shown in SEQ ID No.157; the siRNA44 includes the sense strand shown in SEQ ID No.158 and the antisense strand shown in SEQ ID No.159; the siRNA45 includes the sense strand shown in SEQ ID No.160 and the antisense strand shown in SEQ ID No.161; the siRNA46 includes the sense strand shown in SEQ ID No.162 and the antisense strand shown in SEQ ID No. 163; the siRNA47 includes the sense strand shown in SEQ ID No.164 and the antisense strand shown in SEQ ID No.165; the siRNA48 includes the sense strand shown in SEQ ID No.166 and the antisense strand shown in SEQ ID No.167; the siRNA49 includes the sense strand shown in SEQ ID No.168 and the antisense strand shown in SEQ ID No.169; the siRNA50 includes the sense strand shown in SEQ ID No.170 and the antisense strand shown in SEQ ID No.171; the siRNA51 includes the sense strand shown in SEQ ID No.172 and the antisense strand shown in SEQ ID No.173; the siRNA52 includes the sense strand shown in SEQ ID No.174 and the antisense strand shown in SEQ ID No.175; the siRNA53 includes the sense strand shown in SEQ ID No.176 and the antisense strand shown in SEQ ID No.177; the siRNA54 includes the sense strand shown in SEQ ID No.176 and the antisense strand shown in SEQ ID No.177 No.178 and the antisense strand as shown in SEQ ID No.179; the siRNA55 includes the sense strand as shown in SEQ ID No.180 and the antisense strand as shown in SEQ ID No.181; the siRNA56 includes the sense strand as shown in SEQ ID No.182 and the antisense strand as shown in SEQ ID No.183; the siRNA57 includes the sense strand as shown in SEQ ID No.184 and the antisense strand shown in SEQ ID No. 185; the siRNA 58 comprises the sense strand shown in SEQ ID No. 186 and the antisense strand shown in SEQ ID No. 187; the siRNA 59 comprises the sense strand shown in SEQ ID No. 188 and the antisense strand shown in SEQ ID No. 189; the siRNA 60 comprises the sense strand shown in SEQ ID No. 190 and the antisense strand shown in SEQ ID No. 191; the siRNA 61 comprises the sense strand shown in SEQ ID No. 192 and the antisense strand shown in SEQ ID No. 193; the siRNA 62 comprises the sense strand shown in SEQ ID No. 194 and the antisense strand shown in SEQ ID No. 195; and the siRNA 63 comprises the sense strand shown in SEQ ID No. 196 and the antisense strand shown in SEQ ID No. 197.

[0011] Preferably, the 3' ends of the sense strand and the antisense strand are both overhanged with dTdT.

[0012] The present invention also provides a chemical modification method of the siRNA combination, which includes: changing non-linking phosphate oxygen, replacing non-linking phosphate oxygen, changing linking phosphate oxygen, replacing linking phosphate oxygen, replacing phosphate part with "dephosphorylated" linker, modifying naturally occurring bases, replacing naturally occurring bases, replacing ribose phosphate backbone and modifying ribose phosphate backbone one or more.

[0013] Preferably, the chemical modification method comprises, in the direction from the 5' end to the 3' end:

[0014] The nucleotides at positions 2 and 14 of the sense strand are modified with one or two of 2'-fluoro modification, 4'-thio modification, 2'-fluoroarabinose nucleic acid, and acyclic nucleotide analogs, and the antisense strand is modified with 2'-methoxyethoxy;

[0015] or

[0016] The sense strand is modified with 2'-methoxyethoxy, and the nucleotides at positions 2 and 14 of the antisense strand are modified with one or two of 2'-fluoro modification, 4'-thio modification, 2'-fluoroarabinose nucleic acid, and acyclic nucleotide analogs.

[0017] The present invention also provides a siRNA combination modified by a chemical modification method.

[0018] The present invention also provides a drug conjugate comprising a ligand and the siRNA combination.

[0019] Preferably, the ligand is connected to the 3' end of the sense strand of the siRNA modification via a linker group, and the ligand is GalNAc.

[0020] The present invention also provides use of the siRNA combination, the siRNA modification combination, or the drug conjugate in preparing a drug for inhibiting the expression of DC624545.

[0021] The LOC157273 gene can regulate the expression of PPP1R3B, participate in the conversion and regulation of blood sugar and blood lipid cholesterol, and play a role in patients with type 2 diabetes. Among them, the protein encoded by PPP1R3B is a protein phosphatase target subunit that can promote liver glycogen synthesis. When LOC157273 negatively regulates PPP1R3B, it will hinder the conversion of glucose into glycogen in the body, thereby inhibiting glucose synthesis. PCSK9 binds to the low-density lipoprotein receptor (LDL-R) on the surface of hepatocytes, promoting the endocytosis and degradation of LDL-R, leading to increased levels of low-density lipoprotein cholesterol (LDL-C) in the blood, thereby increasing the risk of atherosclerosis and cardiovascular disease.

[0022] The drug combination must contain at least one duplex region, the first strand of which must be at least partially complementary to the RNA transcribed from the target gene and contain modified or unmodified nucleotides at multiple sites to assist in nucleic acid processing by the RNA-induced silencing complex (RISC).

[0023] The innovative nucleic acid structure involved in the present invention is a key tool for accurately inhibiting the expression of target genes in cells. The nucleic acid has at least one duplex region, which is composed of a partial fragment of the first chain and a partially complementary fragment of the second chain. Among them, the first chain is complementary to the partial fragment of RNA transcribed from the target gene, and the first chain is distributed with modified or unmodified nucleotides at multiple positions. The special arrangement of these nucleotides can effectively promote the processing of nucleic acids by the RNA-induced silencing complex (RISC).

[0024] The siRNA targeting the LOC157273 gene target has a GC content greater than 30% and less than 50%, a Tm of 21.5° C., avoids continuous GC, has an AT content of more than 4, and has a silencing efficiency greater than 80%.

[0025] The beneficial effects of the present invention are:

[0026] (1) The present invention provides a siRNA combination that can target and treat metabolic diseases related to the LOC157273 gene by inhibiting its expression. Existing drugs mostly focus on a single metabolic pathway and lack synergistic intervention for co-morbid mechanisms such as dyslipidemia, hepatic steatosis, and chronic inflammation. In addition, innovative targeted drugs face significant bottlenecks in terms of in vivo delivery efficiency, efficacy, and clinical transformation. The siRNA combination designed based on the technical solution of the present invention contains at least one siRNA molecule, which can intervene in abnormal glucose metabolism, lipid metabolism, and protein metabolism by regulating the LOC157273 target.

[0027] (2) The siRNA combination of this application is applicable to a variety of diseases related to metabolic disorders: ① Lipid metabolism disorders, such as hyperlipidemia, hypercholesterolemia, and hypertriglyceridemia, accompanied by abnormal indicators such as increased serum free fatty acids and increased small, dense low-density lipoprotein cholesterol (sdLDL-C); ② Glucose metabolism disorders, including prodromal symptoms such as diabetes (type 1 and / or type 2), insulin resistance, obesity, and glucose intolerance; ③ Cardiovascular complications, such as atherosclerosis and hypertension caused by abnormal lipid metabolism. In addition, it is also applicable to metabolic syndrome and its related diseases, including visceral obesity, hyperinsulinemia, elevated serum C-reactive protein (CRP), and other disorders of glucose and lipid metabolism and abnormal chronic inflammatory indicators.

[0028] (3) By targeting the LOC157273 gene, this invention provides a multi-mechanism synergistic intervention strategy for metabolic diseases and comorbidities, potentially overcoming the limitations of existing single-pathway drugs in treating comorbidities. Its scope of application encompasses not only the specific diseases mentioned above, but also all pathological conditions associated with LOC157273 gene expression or activity, providing a new direction for the development of more targeted and effective long-term therapeutic drugs.

[0029] The present invention uses siRNA silencing to lower blood lipids, which has the following advantages compared to existing technologies: the combination of specific antisense and sense chain modifications even brings unexpected advantages, some of which are manifested in enhancing the ability to inhibit target gene expression, enhancing serum stability and / or increasing target specificity. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 Comparison of the glucose-lowering efficiency of patented siRNA and siRNA combination (the patented application described in this application is 202210990726.9);

[0031] Figure 2 Comparison of weight loss efficiency of patented siRNA and siRNA combination;

[0032] Figure 3Comparison of the LDL-lowering efficiency of patented siRNA and siRNA combination;

[0033] Figure 4 Comparison of CHO reduction efficiency of patented siRNA and siRNA combination;

[0034] Figure 5 Comparison of TG reduction efficiency of patented siRNA and siRNA combination;

[0035] Figure 6 PAS staining of sections from mice in the control group;

[0036] Figure 7 PAS staining of sections of mice in the experimental group after administration of siRNA No. 12;

[0037] Figure 8 PAS staining of sections after administration of siRNA 2+4 to mice in the experimental group;

[0038] Figure 9 PAS staining of sections of mice in the experimental group after administration of siRNA 2+3+4;

[0039] Figure 10 PAS staining of sections of mice in the experimental group after administration of siRNA 2+3+4+8. DETAILED DESCRIPTION

[0040] The technical solutions provided by the present invention are described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0041] Example 1

[0042] The innovative nucleic acid structure involved in the present invention is a core tool for precisely inhibiting the expression of target genes in cells. The nucleic acid contains at least one duplex region composed of partially complementary segments of the first and second strands, wherein the first strand is complementary to a portion of the RNA transcribed from the target gene and contains modified or unmodified nucleotides at multiple sites. This can effectively promote the processing of nucleic acids by the RNA-induced silencing complex (RISC), thereby achieving specific inhibition of target gene expression.

[0043] Gene expression level: The siRNA targeting the target site must meet the following standards: GC content must be between 30% and 50%, Tm = 21.5°C, avoid continuous GC, AT content of more than 4, and silencing efficiency greater than 80%.

[0044] Gene expression levels after siRNA administration and the effects of glycogen synthesis and related genes were measured in HepG2 cells to comprehensively evaluate the potential of siRNA as a therapeutic agent for metabolic syndrome-related diseases and to select target siRNAs. Experimental methods: 150,000 cells were seeded per 6-well plate. After 24 hours of culture, cells were transfected with GalNAc and lysed 48 hours later. Total RNA was extracted, and gene expression was measured using the internationally accepted two-step Taqman qRT-PCR technique. The primers used for each related gene and LOC157273 are shown in Table 1. Changes in glycogen synthesis and related genes were also measured simultaneously to comprehensively evaluate the potential of siRNA as a therapeutic agent for metabolic syndrome-related diseases.

[0045] Before cell lysis and administration, the glycogen content in the cell culture supernatant was measured using the Glycogen Assay Kit (Fluorometric) produced by Cell Biolabs. The assay principle is as follows: First, glycogen is broken down into glucose monomers by the action of amyloglucosidase; then, the glucose monomers are oxidized by glucose oxidase to produce D-gluconic acid and hydrogen peroxide. Finally, the hydrogen peroxide reacts specifically with the colorimetric probe in the assay kit. The absorbance is measured spectrophotometrically at a wavelength of 550 nm. The glycogen content in the unknown sample is then determined based on a pre-prepared glycogen standard curve.

[0046] Table 1

[0047]

[0048] Example 2

[0049] siRNA synthesis: After obtaining the gene sequence of LOC157273 from the Genebank database, the R language was used to screen for qualified 21bp siRNAs. siRNAs with a GC content of 50% were further selected. The maximum Tm of the seed double-strand stability was set to 21.51°C to optimize the selection of qualified siRNAs. Simultaneously, the siRNA sequence was chemically synthesized, and two overhanging dTdT residues were added to the 3' end to enhance the siRNA's stability.

[0050] In terms of nucleotide modification details, each group of siRNA uses GalNAc as a delivery system, and there are special requirements for the 2nd and 14th nucleotides at the 5' end of the first chain: the 2nd and 14th nucleotides at the 5' end of the first chain can be modified with one or two selected from 2'F, 4'-S, 2'-FANA and UNA modifications; while the 2nd and 14th nucleotides at the 5' end of the second chain can be modified with 2'O-MOE.

[0051] Experiments were conducted in HepG2 cells to detect the silencing effect of single siRNA on LOC157273 at the cellular level and the detection of cellular glycogen synthesis and related genes. The target combination siRNA was further optimized. The detailed sequences of the siRNA results obtained by cell experiment screening are shown in Table 2.

[0052] Table 2

[0053]

[0054]

[0055]

[0056]

[0057]

[0058]

[0059]

[0060] Example 3 Data Preprocessing

[0061] The silencing efficiency of siRNA was calculated based on the expression level of LOC157273. The calculation formula is as follows:

[0062] Silencing efficiency = 1-expression rate Formula 1

[0063] In this study, each siRNA had multiple evaluation indicators, including target gene silencing efficiency (range, 60-95%), glycogen uptake increase ratio (10-40%), PCSK9 reduction rate (20-80%), and PPP1R3B expression enhancement rate (some exceeding 200%). The value ranges and dimensions of these multiple evaluation indicators vary significantly. Without normalization, the maximum value indicator (such as PPP1R3B) will dominate the principal component analysis or comprehensive score, thereby masking the contribution of other key effect indicators.

[0064] To eliminate the aforementioned dimensionality effects, we used Z-score standardization to normalize each column of variables. This involves subtracting the mean from each value and then dividing it by the standard deviation. This method not only preserves the relative distribution characteristics of the variables but also makes different indicators comparable in distance calculation and variance interpretation. It is particularly suitable for analytical methods such as PCA and multi-indicator comprehensive scoring.

[0065] Weighted calculation:

[0066] After obtaining the standardized data matrix, we added a certain weight to each indicator and calculated the score of each siRNA, see Table 3.

[0067] Table 3

[0068] index Weight LOC157273 silencing efficiency 0.4 Glycogen uptake growth rate 0.1 PPP1R3B growth rate 0.4 PCSK9 reduction rate 0.1

[0069] Calculate the final score of each siRNA and select the top three. The formula is as follows:

[0070]

[0071] To comprehensively evaluate the overall performance of each siRNA sequence across multiple functional indicators, this technique used principal component analysis (PCA) to reduce the dimensionality of the standardized experimental data. The experimental results are shown in Table 4. PCA can transform the original multidimensional data into several uncorrelated principal components, thereby revealing the main variation trends and potential structural characteristics among the data.

[0072] This method enables a holistic, visual analysis of siRNA response patterns, effectively identifying candidate sequences that consistently excel across multiple metrics, such as target gene silencing efficiency, glycogen uptake capacity, and targeted gene expression regulation. Furthermore, combined with clustering trends, it can aid in the screening of highly effective siRNA sequences with stable functionality and synergistic mechanisms of action for subsequent experimental validation and drug development.

[0073] Based on the comprehensive weighted scores and PCA clustering results, siRNAs with high scores and tight clustering in the principal component space are selected as highly effective and functionally stable candidate molecules for subsequent in vitro and in vivo validation and drug development processes.

[0074] This process ensures the scientific integration and rational screening of complex multi-index data, and improves the screening efficiency and accuracy of candidate siRNAs.

[0075] Table 4

[0076]

[0077]

[0078]

[0079]

[0080] Example 4

[0081] At the cellular level, siRNAs were combined into combination drugs in the form of two-by-two, three-by-three, and four-by-four combinations. The silencing efficiency levels of various combinations were verified using HepG2 cells. Tables 5, 6, and 7 screened some of the combinations for display.

[0082] Table 5 siRNA pairwise combinations

[0083]

[0084]

[0085] Table 6 siRNA triple combinations

[0086]

[0087]

[0088]

[0089] Table 7 siRNA 4-4 combinations

[0090]

[0091]

[0092] Example 5

[0093] Combining the experimental results of individual siRNAs, a data model was constructed using data analysis methods. Three siRNA sequences were selected as target drugs for the next step of the experiment: siRNA25, siRNA45, and siRNA48. siRNA25, siRNA45, and siRNA48 were used together to prepare a combined siRNA agent that inhibited LOC157273 expression. The mass ratio of siRNA25, siRNA45, and siRNA48 in the combined siRNA agent was 1:1:1.

[0094] This experiment used GalNAc triplets to link siRNA. In terms of nucleotide modification details, there are special requirements for the 2nd and 14th nucleotides at the 5' end of the first chain: nucleotides 2 and 14 at the 5' end of the first chain are modified with 2'F; while the 2nd and 14th nucleotides at the 5' end of the second chain are modified with 2'O-MOE.

[0095] The specific sequences of siRNA25, siRNA45, and siRNA48 as well as their modified sequences are shown in Table 8 below.

[0096] Table 8

[0097]

[0098]

[0099] At the same time, a patented siRNA was used as a control test drug. The specific sequence of the control test drug siRNA and the modified sequence are shown in Table 9 below.

[0100] Table 9

[0101]

[0102] Each experimental and control group received a single subcutaneous injection of 4 mg / kg of the drug into cynomolgus macaques with T2D and obesity (these macaques were induced on a high-density lipoprotein (HFD) without any other medications). The day of administration was designated Day 0. Daily monitoring was performed for 24 consecutive days after administration to comprehensively evaluate the therapeutic efficacy of the double-stranded RNA agent by measuring glucose, body weight, low-density lipoprotein (LDL), triglycerides, and total cholesterol. A before-after control was used as the control.

[0103] The specific testing process is as follows:

[0104] ① Sample processing: After blood collection, immediately inject the blood into the EDTA anticoagulant tube (purple cap) and mix it by inverting 8 times to prevent coagulation.

[0105] ② After blood collection, inject it into a non-anticoagulant tube (red cap) or a heparin anticoagulant tube (green cap, suitable for emergency biochemistry), let it stand at room temperature for 30 minutes to allow the blood to coagulate, and centrifuge (3000 rpm, 10 minutes) to separate the serum / plasma.

[0106] ③ The relevant indicators were tested by automatic blood tester. The test results of each indicator are shown in Figures 1 to 5 .

[0107] Conclusion: This study transfected GalNAc-siRNA into HepG2 cells, extracted nucleic acids, and measured expression levels using qPCR. Three siRNAs with the highest silencing efficacy from LOC157273 (siRNA48, siRNA45, and siRNA25) were selected and tested as a combination. The results showed that a single LOC157273 inhibitor had only modest therapeutic effects on various indicators, but some indicators were unable to be suppressed to normal or remained within normal ranges. The combination formulation, however, demonstrated long-term and stable inhibitory effects on all indicators simultaneously.

[0108] Example 6 Construction of transgenic mice expressing the non-coding gene LOC157273

[0109] 1. Animal selection: C57BL / 6J mice (6-8 weeks old, used as fertilized egg donors and surrogate mothers, ICR male mice (vasectomized, used to prepare pseudopregnant mothers)

[0110] 2. Expression Vector Construction

[0111] 2.1 Target gene cloning

[0112] 2.1.1 Obtain the LOC157273 gene sequence from NCBI (accession number: NR_040039.1);

[0113] 2.1.2 Introduce appropriate restriction sites (including EcoRI site);

[0114] 2.1.3 PCR amplification using mouse genomic DNA as template

[0115] PCR reaction system: 10 μL 5× Master Mix, 4 μL dNTP Mix (2.5 mM each), 2 μL upstream primer (10 μM), 2 μL downstream primer (10 μM), 1 μL template DNA, 0.5 μL Phusion enzyme, and ddH₂O to 50 μL. PCR reaction conditions: initial denaturation at 98°C for 30 seconds, denaturation at 98°C for 10 seconds, annealing at 55-65°C for 30 seconds, extension at 72°C for 30 seconds (30 cycles), and final extension at 72°C for 5 minutes.

[0116] 2.2 Vector construction

[0117] 2.2.1 Double-digest the PCR product and expression vector (e.g., pCAGGS) with BamHI and EcoRI.

[0118] 2.2.2 Enzyme digestion reaction system (50 μL): DNA 20 μL, 10× Buffer 5 μL, BamHI 2 μL, EcoRI 2 μL, add ddH2O to 50 μL, and incubate at 37°C for 2 h.

[0119] 2.2.3 The digestion products were separated by agarose gel electrophoresis and recovered.

[0120] 2.2.4 Ligation reaction system (20 μL): 1 μL vector DNA, 3 μL insert fragment, 2 μL 10× T4 ligase buffer, 1 μL T4 DNA ligase, make up to 20 μL with ddH2O, incubate overnight at 16°C, transform DH5α competent cells, plate on LB plates containing ampicillin, pick single colonies, extract plasmids for enzyme digestion and sequencing verification.

[0121] 2.3 Pronuclear microinjection: 6-8 week old C57BL / 6J female mice were selected and intraperitoneally injected with 5 IU of PMSG. 48 hours later, 5 IU of hCG was injected intraperitoneally. Immediately after hCG injection, the mice were housed with C57BL / 6J male mice.

[0122] 2.4 Fertilized egg collection: Check the vaginal plug the next morning after cage closure. If there is a vaginal plug, it is considered a successful pregnancy.

[0123] Female mice were sacrificed by cervical dislocation, and the oviducts were removed and placed in M2 medium containing hyaluronidase to disperse the cumulus cells. Normally morphologically fertilized eggs were selected and washed three times in M2 medium. The fertilized eggs were then transferred to KSOM medium and cultured in a 37°C, 5% CO2 incubator until ready for use.

[0124] 2.5 DNA solution preparation: Extract the recombinant plasmid using an endotoxin-free plasmid extraction kit, linearize the plasmid with BamHI and EcoRI, extract with phenol-chloroform, and purify the DNA by ethanol precipitation. Dissolve the DNA in microinjection buffer and adjust the concentration to 2 ng / μL. Centrifuge at 12,000 rpm for 15 minutes and collect the supernatant for injection.

[0125] 2.6 Microinjection operation: Add a drop of M2 culture medium on the microinjection slide, cover with mineral oil, move the fertilized egg into the drop, adjust the micromanipulator, fix the fertilized egg with the egg holding needle, insert the injection needle into the male pronucleus, slowly inject the DNA solution, and observe the expansion of the pronucleus. After injection, the fertilized egg is washed with KSOM culture medium and placed in the incubator to recover for 1 hour.

[0126] 2.7 Embryo transfer: 6-8 week old ICR female mice were placed in the same cage with vasectomized male mice. The vaginal plug was checked the next morning. The female mice with vaginal plug were designated as pseudo-pregnant mice (gestational day 0.5).

[0127] 2.8 Embryo transplantation: Anesthetize the pseudopregnant female mouse with isoflurane, shave and disinfect the back, make a 1 cm skin incision in the midline of the back, separate the muscle layer, find the ovaries and fallopian tubes, fix them with forceps, and use a transplant cannula to transfer 20-30 injected fertilized eggs into the ampulla of the fallopian tube. Suture the muscles and skin, and give analgesics after surgery.

[0128] 2.9 Identification of offspring mice

[0129] 2.9.1 Genomic DNA extraction: Cut the tip of the mouse tail (about 0.5 cm), add 200 μL of lysis buffer (50 mM Tris-HCl pH 8.0, 100 mM EDTA, 100 mM NaCl, 1% SDS) and 2 μL of proteinase K (20 mg / mL), incubate at 55°C overnight, and the next day add 200 μL of isopropanol to precipitate the DNA, wash with 70% ethanol, dry, and dissolve in 50 μL of TE buffer.

[0130] 2.9.2 PCR Identification

[0131] PCR reaction system (25 μL): 2× Taq MasterMix 12.5 μL, upstream primer (10 μM) 1 μL, downstream primer (10 μM) 1 μL, template DNA 2 μL, ddH2O to 25 μL

[0132] PCR reaction conditions: pre-denaturation at 94°C for 5 minutes, denaturation at 94°C for 30 seconds, annealing at 55°C for 30 seconds, extension at 72°C for 30 seconds (35 cycles), and final extension at 72°C for 5 minutes.

[0133] 3. Take the successfully established transgenic mice with the non-coding gene LOC157273 and divide them into groups for injection:

[0134] From single siRNA to multiple siRNA combinations, the siRNA and siRNA combination with the highest cell silencing efficiency were selected for each combination and injected into transgenic mice. The siRNA numbers corresponding to the specific groups are shown in Table 10. The mice were subcutaneously injected according to the groups in Table 10. After 7 days of administration, the liver tissues of the mice were sliced ​​and PAS stained. The experimental results of each group are shown in Table 10. Figures 6-10 shown.

[0135] Table 10

[0136]

[0137]

[0138] Conclusion: With the increase of silencing efficiency of the target gene (LOC157273), the expression of downstream PPP1R3B increased, and glycogen synthesis increased. Among them, the glycogen synthesis of the combination (2+3+4+8) increased by more than double compared with the glycogen synthesis content of the control group mice after administration.

[0139] As can be seen from the above examples, the present invention provides a siRNA combination for inhibiting LOC157273 expression and its use. The siRNA combination comprises one or more of 63 genes, including siRNA1, siRNA2, siRNA3, siRNA4, siRNA25, siRNA45, and siRNA48. By inhibiting LOC157273 gene expression, the siRNA combination provided by the present invention can target and treat metabolic diseases associated with this gene, thereby intervening in abnormal glucose, lipid, and protein metabolism.

[0140] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. An siRNA combination for inhibiting LOC157273 expression, characterized in that: The siRNA combination includes siRNA1, siRNA2, siRNA3, siRNA4, siRNA5, siRNA6, siRNA7, siRNA8, siRNA9, siRNA10, siRNA11, siRNA12, siRNA13, siRNA14, siRNA15, siRNA1 6. siRNA17, siRNA18, siRNA19, siRNA20, siRNA21, siRNA22, siRNA23, siRNA24, siRNA25, siRNA26, siRNA27, siRNA28, siRNA29, siRNA30, siRNA31, siRNA3 2. One or more of siRNA33, siRNA34, siRNA35, siRNA36, siRNA37, siRNA38, siRNA39, siRNA40, siRNA41, siRNA42, siRNA43, siRNA44, siRNA45, siRNA46, siRNA47, siRNA48, siRNA49, siRNA50, siRNA51, siRNA52, siRNA53, siRNA54, siRNA55, siRNA56, siRNA57, siRNA58, siRNA59, siRNA60, siRNA61, siRNA62, and siRNA63.

2. The siRNA combination according to claim 1, characterized in that The siRNA combination comprises one or more of siRNA25, siRNA45, and siRNA48.

3. The siRNA combination according to claim 1, characterized in that The siRNA1 includes a sense strand as shown in SEQ ID No. 72 and an antisense strand as shown in SEQ ID No. 73; The siRNA2 includes a sense strand as shown in SEQ ID No. 74 and an antisense strand as shown in SEQ ID No. 75; The siRNA3 includes a sense strand as shown in SEQ ID No. 76 and an antisense strand as shown in SEQ ID No. 77; The siRNA4 includes a sense strand as shown in SEQ ID No. 78 and an antisense strand as shown in SEQ ID No. 79; The siRNA5 includes a sense strand as shown in SEQ ID No.80 and an antisense strand as shown in SEQ ID No.81; The siRNA6 includes a sense strand as shown in SEQ ID No.82 and an antisense strand as shown in SEQ ID No.83; The siRNA7 includes a sense strand as shown in SEQ ID No.84 and an antisense strand as shown in SEQ ID No.85; The siRNA8 includes a sense strand as shown in SEQ ID No.86 and an antisense strand as shown in SEQ ID No.87; The siRNA9 includes a sense strand as shown in SEQ ID No.88 and an antisense strand as shown in SEQ ID No.89; The siRNA 10 includes a sense strand as shown in SEQ ID No. 90 and an antisense strand as shown in SEQ ID No. 91; The siRNA11 includes a sense strand as shown in SEQ ID No.92 and an antisense strand as shown in SEQ ID No.93; The siRNA12 includes a sense strand as shown in SEQ ID No.94 and an antisense strand as shown in SEQ ID No.95; The siRNA13 includes a sense strand as shown in SEQ ID No.96 and an antisense strand as shown in SEQ ID No.97; The siRNA14 includes a sense strand as shown in SEQ ID No.98 and an antisense strand as shown in SEQ ID No.99; The siRNA15 includes a sense strand as shown in SEQ ID No.100 and an antisense strand as shown in SEQ ID No.101; The siRNA16 includes a sense strand as shown in SEQ ID No. 102 and an antisense strand as shown in SEQ ID No. 103; The siRNA17 includes a sense strand as shown in SEQ ID No. 104 and an antisense strand as shown in SEQ ID No. 105; The siRNA18 includes a sense strand as shown in SEQ ID No. 106 and an antisense strand as shown in SEQ ID No. 107; The siRNA19 includes a sense strand as shown in SEQ ID No. 108 and an antisense strand as shown in SEQ ID No. 109; The siRNA 20 includes a sense strand as shown in SEQ ID No. 110 and an antisense strand as shown in SEQ ID No. 111; The siRNA21 includes a sense strand as shown in SEQ ID No.112 and an antisense strand as shown in SEQ ID No.113; The siRNA22 includes a sense strand as shown in SEQ ID No.114 and an antisense strand as shown in SEQ ID No.115; The siRNA23 includes a sense strand as shown in SEQ ID No.116 and an antisense strand as shown in SEQ ID No.117; The siRNA24 includes a sense strand as shown in SEQ ID No.118 and an antisense strand as shown in SEQ ID No.119; The siRNA25 includes a sense strand as shown in SEQ ID No.120 and an antisense strand as shown in SEQ ID No.121; The siRNA26 includes a sense strand as shown in SEQ ID No. 122 and an antisense strand as shown in SEQ ID No. 123; The siRNA27 includes a sense strand as shown in SEQ ID No. 124 and an antisense strand as shown in SEQ ID No. 125; The siRNA28 includes a sense strand as shown in SEQ ID No. 126 and an antisense strand as shown in SEQ ID No. 127; The siRNA29 includes a sense strand as shown in SEQ ID No.128 and an antisense strand as shown in SEQ ID No.129; The siRNA30 includes a sense strand as shown in SEQ ID No.130 and an antisense strand as shown in SEQ ID No.131; The siRNA31 includes a sense strand as shown in SEQ ID No.132 and an antisense strand as shown in SEQ ID No.133; The siRNA32 includes a sense strand as shown in SEQ ID No. 134 and an antisense strand as shown in SEQ ID No. 135; The siRNA33 includes a sense strand as shown in SEQ ID No. 136 and an antisense strand as shown in SEQ ID No. 137; The siRNA34 includes a sense strand as shown in SEQ ID No. 138 and an antisense strand as shown in SEQ ID No. 139; The siRNA35 includes a sense strand as shown in SEQ ID No. 140 and an antisense strand as shown in SEQ ID No. 141; The siRNA36 includes a sense strand as shown in SEQ ID No. 142 and an antisense strand as shown in SEQ ID No. 143; The siRNA37 includes a sense strand as shown in SEQ ID No. 144 and an antisense strand as shown in SEQ ID No. 145; The siRNA38 includes a sense strand as shown in SEQ ID No. 146 and an antisense strand as shown in SEQ ID No. 147; The siRNA39 includes a sense strand as shown in SEQ ID No. 148 and an antisense strand as shown in SEQ ID No. 149; The siRNA40 includes a sense strand as shown in SEQ ID No. 150 and an antisense strand as shown in SEQ ID No. 151; The siRNA41 includes a sense strand as shown in SEQ ID No. 152 and an antisense strand as shown in SEQ ID No. 153; The siRNA42 includes a sense strand as shown in SEQ ID No. 154 and an antisense strand as shown in SEQ ID No. 155; The siRNA43 includes a sense strand as shown in SEQ ID No. 156 and an antisense strand as shown in SEQ ID No. 157; The siRNA44 includes a sense strand as shown in SEQ ID No. 158 and an antisense strand as shown in SEQ ID No. 159; The siRNA45 includes a sense strand as shown in SEQ ID No. 160 and an antisense strand as shown in SEQ ID No. 161; The siRNA46 includes a sense strand as shown in SEQ ID No. 162 and an antisense strand as shown in SEQ ID No. 163; The siRNA47 includes a sense strand as shown in SEQ ID No. 164 and an antisense strand as shown in SEQ ID No. 165; The siRNA48 includes a sense strand as shown in SEQ ID No. 166 and an antisense strand as shown in SEQ ID No. 167; The siRNA49 includes a sense strand as shown in SEQ ID No. 168 and an antisense strand as shown in SEQ ID No. 169; The siRNA50 includes a sense strand as shown in SEQ ID No. 170 and an antisense strand as shown in SEQ ID No. 171; The siRNA51 includes a sense strand as shown in SEQ ID No. 172 and an antisense strand as shown in SEQ ID No. 173; The siRNA52 includes a sense strand as shown in SEQ ID No. 174 and an antisense strand as shown in SEQ ID No. 175; The siRNA53 includes a sense strand as shown in SEQ ID No. 176 and an antisense strand as shown in SEQ ID No. 177; The siRNA54 includes a sense strand as shown in SEQ ID No. 178 and an antisense strand as shown in SEQ ID No. 179; The siRNA55 includes a sense strand as shown in SEQ ID No. 180 and an antisense strand as shown in SEQ ID No. 181; The siRNA56 includes a sense strand as shown in SEQ ID No. 182 and an antisense strand as shown in SEQ ID No. 183; The siRNA57 includes a sense strand as shown in SEQ ID No. 184 and an antisense strand as shown in SEQ ID No. 185; The siRNA58 includes a sense strand as shown in SEQ ID No. 186 and an antisense strand as shown in SEQ ID No. 187; The siRNA59 includes a sense strand as shown in SEQ ID No. 188 and an antisense strand as shown in SEQ ID No. 189; The siRNA60 includes a sense strand as shown in SEQ ID No. 190 and an antisense strand as shown in SEQ ID No. 191; The siRNA61 includes a sense strand as shown in SEQ ID No. 192 and an antisense strand as shown in SEQ ID No. 193; The siRNA62 includes a sense strand as shown in SEQ ID No. 194 and an antisense strand as shown in SEQ ID No. 195; The siRNA63 includes a sense strand as shown in SEQ ID No.196 and an antisense strand as shown in SEQ ID No.

197.

4. The siRNA combination according to claim 3, characterized in that The 3' ends of both the sense and antisense strands have dTdT overhangs.

5. A method for chemically modifying the siRNA combination according to any one of claims 1 to 4, characterized in that: The chemical modification methods include: changing non-linking phosphate oxygens, replacing non-linking phosphate oxygens, changing linking phosphate oxygens, replacing linking phosphate oxygens, replacing phosphate moieties with "dephosphorylated" linkers, modifying naturally occurring bases, replacing naturally occurring bases, replacing the ribose phosphate backbone, and modifying the ribose phosphate backbone.

6. The chemical modification method according to claim 5, characterized in that The chemical modification method includes, from the 5' end to the 3' end: The nucleotides at positions 2 and 14 of the sense strand are modified with one or two of 2'-fluoro modification, 4'-thio modification, 2'-fluoroarabinose nucleic acid, and acyclic nucleotide analogs, and the antisense strand is modified with 2'-methoxyethoxy; or The sense strand is modified with 2'-methoxyethoxy, and the nucleotides at positions 2 and 14 of the antisense strand are modified with one or two of 2'-fluoro modification, 4'-thio modification, 2'-fluoroarabinose nucleic acid, and acyclic nucleotide analogs.

7. The siRNA combination modified by the chemical modification method of claim 5 or 6.

8. A drug conjugate, characterized in that Comprising a ligand and the siRNA combination according to claim 7.

9. The drug conjugate according to claim 8, characterized in that The ligand is connected to the 3' end of the sense strand of the siRNA modification through a linking group, and the ligand is GalNAc.

10. Use of the siRNA combination according to any one of claims 1 to 4, the modified siRNA combination according to claim 7, or the drug conjugate according to any one of claims 8 to 9 in the preparation of a drug for inhibiting the expression of DC624545.

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