RNAi agent and composition for inhibiting AGT gene expression and application of RNAi agent and composition

Abstract

The invention relates to an RNAi agent for inhibiting AGT gene expression, a composition and application of the RNAi agent and the composition, and belongs to the technical field of biological medicine. The RNAi agent is composed of a positive-sense strand and an antisense strand, the positive-sense strand and the antisense strand are complementary in base pairing, the positive-sense strand contains nucleotide sequences which are shown as SEQ ID NO: 1-3 and have no more than three nucleotide differences, and the antisense strand contains nucleotide sequences which are shown as SEQ ID NO: 22-24 and have no more than three nucleotide differences; the nucleotides of the positive-sense strand and the antisense strand are partially or completely modified nucleotides. The synthesized siRNA has a remarkable effect on inhibiting AGT gene expression of Hep 3B cells, and the inhibition rate exceeds 60%; the inhibition rate of the modified siRNA on AGT gene expression of Hep 3B cells reaches 80% or above, and the modified siRNA can be used for preparing drugs for preventing, treating and relieving diseases and symptoms related to AGT.

Description

Technical Field

[0001] The present invention relates to an RNAi agent and a composition for inhibiting AGT gene expression and uses thereof, belonging to the technical field of biomedicine. Background Art

[0002] Angiotensinogen AGT, also known as SerpinA8, generates angiotensin I (ANGI) under the action of renin enzyme, and ANGI generates angiotensin II (ANGII) through angiotensin converting enzyme. ANGII causes arterial contraction and increases blood pressure, and promotes the secretion of aldosterone in the adrenal cortex. Aldosterone participates in Na + / K + Excretion causes an increase in sodium ions and water retention, which increases blood volume and thus causes an increase in blood pressure.

[0003] Hypertension is one of the main causes of cardiovascular diseases, causing stroke, myocardial infarction, atherosclerosis, renal failure, etc. Dietary control and drug therapy are the main means to control blood pressure and prevent related complications. Current antihypertensive drugs include calcium channel blockers, angiotensin converting enzyme inhibitors, angiotensin receptor antagonists, thiazide diuretics and beta-blockers, but some patients still fail to effectively control their blood pressure. Therefore, there is still a clinical need for effective antihypertensive drugs to achieve effective control of patients' blood pressure. Summary of the invention

[0004] The purpose of the present invention is to provide a new technical solution to improve or solve the technical problems existing in the prior art as described above.

[0005] The technical solution provided by the present invention is as follows: an RNAi agent for inhibiting the expression of the AGT gene, the RNAi agent consisting of a sense chain and an antisense chain with complementary base pairing, the sense chain containing a nucleotide sequence with no more than 3 nucleotide differences shown in SEQ ID NO:1-3; the antisense chain containing a nucleotide sequence with no more than 3 nucleotide differences shown in SEQ ID NO:22-24.

[0006] Based on the above technical solution, the present invention can also be improved as follows.

[0007] Further, the sense strand comprises a 3' overhang of 0, 1, or 2 nucleotides, and the antisense strand comprises a 3' overhang of 0, 1, or 2 nucleotides.

[0008] Furthermore, the lengths of the sense strand and the antisense strand are 19-25 nucleotides; the sense strand and the antisense strand are at least 100% complementary, 90% complementary, or 80% complementary.

[0009] Further, part or all of the nucleotides of the sense strand and the antisense strand are modified nucleotides, and the modified nucleotides are independently selected from 2'-methoxy modified nucleotides, 2'-fluoro modified nucleotides, 2'-aminoethyl modified nucleotides, 2'-O-methoxyethyl modified nucleotides, thiophosphate modified nucleotides and vinyl phosphate modified nucleotides.

[0010] Furthermore, the phosphorothioate-modified nucleotide is located at the end of the sense strand, and the sense strand contains at least 1, 2 or 3 phosphorothioate-modified nucleotides; the phosphorothioate-modified nucleotide is located at the end of the antisense strand, and the antisense strand contains at least 1, 2 or 3 phosphorothioate-modified nucleotides; the vinyl phosphate-modified nucleotide is located at the first position of the antisense strand.

[0011] Furthermore, the sense strand contains a modified nucleotide sequence with no more than 3 nucleotide differences as shown in SEQ ID NOs: 43-55; and the antisense strand contains a modified nucleotide sequence with no more than 3 nucleotide differences as shown in SEQ ID NOs: 56-68.

[0012] Furthermore, the sense strand and the antisense strand are connected with ligands, and the ligands are conjugated to the sense strand and the antisense strand. Preferably, the ligands are conjugated to the 3' end and / or the 5' end of the sense strand, and more preferably, the ligands are conjugated to the 3' end of the sense strand.

[0013] Furthermore, the ligand is an asialoglycoprotein receptor ligand, and the ligand is galactose or N-acetylgalactosamine, preferably trivalent or tetravalent N-acetylgalactosamine.

[0014] Furthermore, the sense strand contains a modified nucleotide sequence with no more than 3 nucleotide differences as shown in SEQ ID NOs: 69-77; and the antisense strand contains a modified nucleotide sequence with no more than 3 nucleotide differences as shown in SEQ ID NOs: 78-86.

[0015] Furthermore, a composition for inhibiting the expression of the AGT gene comprises the RNAi agent for inhibiting the expression of the AGT gene and a pharmaceutically acceptable carrier, and also includes but is not limited to a pharmaceutically acceptable excipient, diluent, buffer and / or stabilizer.

[0016] Furthermore, the pharmaceutically acceptable carrier includes ionizable lipids and cationic lipids; the pharmaceutically acceptable carrier also includes pegylated lipids and auxiliary lipids.

[0017] Furthermore, the RNAi agent or the composition for inhibiting AGT gene expression is used in the preparation of drugs for preventing and / or treating and / or alleviating diseases related to inhibiting AGT gene expression.

[0018] Compared with the prior art, the technical solution provided by the present invention has the following beneficial effects:

[0019] The synthesized siRNA of the present invention has a significant effect on the inhibition of AGT gene expression in Hep 3B cells, with an inhibition rate of more than 60%; the modified siRNA can achieve an inhibition rate of more than 80% on AGT gene expression, and can be used for the preparation of drugs for preventing, treating and alleviating AGT-related diseases and symptoms. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 is the mass spectrum of the H03 compound of the present invention;

[0021] Figure 2 It is the mass spectrum of the H05 compound of the present invention. DETAILED DESCRIPTION

[0022] The principles and features of the present invention are described below in conjunction with examples. The examples are only used to explain the present invention and are not used to limit the scope of the present invention.

[0023] Example 1: Synthesis of Compound H03

[0024] 1) Synthesis of Compound 2

[0025]

[0026] Compound 1 (50.0 g, 128.4 mmol) was dissolved in 300 mL of anhydrous 1,2-dichloroethane, and trimethylsilyl trifluoromethanesulfonate (TMSOTf, 34.3 g, 154.3 mmol) was added in an ice-water bath and nitrogen protection, and the reaction was continued overnight at room temperature. 500 mL of saturated sodium bicarbonate aqueous solution was added to the reaction solution, stirred evenly, and the organic phase was separated. The aqueous phase was extracted 3 times with 200 mL of dichloromethane, and the organic phases were combined, and washed once with 200 mL of saturated sodium bicarbonate solution and 200 mL of saturated sodium chloride in turn, and the organic phase was separated, dried over anhydrous sodium sulfate, and the solvent was evaporated under reduced pressure to obtain 38.3 g of a light yellow viscous syrupy crude product. The product was directly put into the next step without purification.

[0027] 2) Synthesis of compound 3

[0028]

[0029] Compound 2 (38.0 g, 115.4 mmol) was dissolved in 200 mL of anhydrous 1,2-dichloroethane, 5-hexene-1-ol (12.3 g, 122.8 mmol) was added, and the mixture was stirred at room temperature for 30 min. Then, trimethylsilyl trifluoromethanesulfonate (TMSOTf, 12.3 g, 55.4 mmol) was added under ice bath and nitrogen protection, and the mixture was stirred at room temperature overnight. The reaction solution was diluted with 200 mL of dichloromethane, and then 400 mL of saturated sodium bicarbonate aqueous solution was added and stirred for 10 min to wash, and the organic phase was separated; the aqueous phase was extracted with 200 mL of dichloromethane, and the organic phase was washed once with 200 mL of saturated sodium bicarbonate aqueous solution and 200 mL of saturated sodium chloride in sequence, and the separated organic phase was dried with anhydrous sodium sulfate, and the solvent was evaporated under reduced pressure to obtain 39.6 g of a yellow syrupy product.

[0030] MS m / z[M+H] + (ESI):430.05.

[0031] 3) Synthesis of compound 4

[0032]

[0033] Compound 3 (39.5 g, 92.0 mmol) was dissolved in a mixed solvent of 200 mL of dichloromethane and 200 mL of acetonitrile, and 270 mL of deionized water and sodium periodate (59.0 g, 275.8 mmol) were added respectively, stirred for 10 min under an ice-water bath, and ruthenium trichloride (600 mg, 2.9 mmol) was added, and the reaction was allowed to react overnight at room temperature. The reaction solution was stirred with 800 mL of deionized water, and a saturated aqueous sodium bicarbonate solution was added to adjust the pH to about 7.5, and the phases were separated. The aqueous phase was extracted 3 times with 500 mL of dichloromethane, and the organic phase was discarded. The aqueous phase was adjusted to a pH of about 3.0 with solid citric acid, extracted 3 times with 500 mL of dichloromethane, and the organic phases were combined, dried over anhydrous sodium sulfate, and the solvent was evaporated under reduced pressure to obtain 9.2 g of the product.

[0034] MS m / z[M+H] + (ESI):448.57.

[0035] 4) Synthesis of Compound 5

[0036]

[0037] Compound 4 (3.2 g, 7.1 mmol) was dissolved in 32 mL of dichloromethane, and diisopropylethylamine (2.8 g, 21.7 mmol) and 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU, 3.3 g, 8.7 mmol) were added, and the mixture was stirred and reacted overnight at room temperature. The reaction solution was added with 100 mL of saturated sodium bicarbonate aqueous solution and stirred for 10 min for washing, and the organic phase was separated, and the aqueous phase was extracted with 100 mL of dichloromethane, and the organic phases were combined and washed with 100 mL of saturated sodium bicarbonate aqueous solution and 100 mL of saturated sodium chloride, respectively, and the phases were separated, and the organic phase was dried with anhydrous sodium sulfate, and the product was separated with an eluent (dichloromethane: methanol = 2% to 20%, V: V) silica gel column, and the solvent was spin-dried to obtain 3.0 g of a yellow syrupy product.

[0038] MS m / z[M+H] + (ESI):604.48.

[0039] 5) Synthesis of Compound 6

[0040]

[0041] Compound 5 (40.0 g, 66.3 mmol) was dissolved in 160 mL of dichloromethane, and 80 mL of dioxane hydrochloride solution (4 M) was added in batches, and the mixture was stirred at room temperature for 4 h. The solvent was evaporated under reduced pressure to obtain 42.0 g of a foamy light orange solid.

[0042] 6) Synthesis of Compound 8

[0043]

[0044] Compound 7 (2.0 g, 16.6 mmol) was dissolved in 50 mL of dichloromethane, and 4,4'-dimethoxytriphenylmethane (DMTrCl, 6.8 g, 20.1 mmol) and triethylamine (4.0 g, 39.5 mmol) were added, and the mixture was stirred and reacted overnight at room temperature. The reaction solution was washed twice with 50 mL of saturated sodium chloride water, and the phases were separated; the organic phase was dried over anhydrous sodium sulfate and dried under reduced pressure to obtain 9.0 g of a dark green viscous crude product.

[0045] MS m / z[M+H] + (ESI):423.23.

[0046] 7) Synthesis of Compound 9

[0047]

[0048] Compound 8 (3.0 g, 7.1 mmol) was dissolved in 30 mL of tetrahydrofuran and lithium hydroxide monohydrate (LiOH .H2O) aqueous solution (0.9 g dissolved in 9 mL water), stirred and reacted overnight at room temperature. The reaction solution was allowed to stand for phase separation, the organic phase was washed twice with 30 mL saturated sodium chloride, dried over anhydrous sodium sulfate, and dried under reduced pressure. The crude product was washed with 80 mL methyl tert-butyl ether, filtered, and the filter cake was dried to obtain 1.5 g of light yellow solid powder.

[0049] 8) Synthesis of Compound 10

[0050]

[0051] Compound 9 (1.0 g, 2.4 mmol) was dissolved in 10 mL of dichloromethane, tert-butyl 4-aminobutyrate hydrochloride (0.47 g, 2.4 mmol), Carter condensation agent (BOP, 1.28 g, 2.9 mmol), triethylamine (0.61 g, 6.0 mmol) were added, and the mixture was stirred and reacted overnight at room temperature. The reaction solution was washed 3 times with 10 mL of saturated sodium bicarbonate aqueous solution and once with 10 mL of saturated sodium chloride, and the phases were separated. The organic phase was dried over anhydrous sodium sulfate and spin-dried to obtain 1.3 g of a light orange-yellow semisolid.

[0052] MS m / z[M+H] + (ESI):550.38.

[0053] 9) Synthesis of Compound 11

[0054]

[0055] Compound 10 (1.2 g, 2.2 mmol) was dissolved in 18 mL of tetrahydrofuran, 6 mL of methanol was added, and lithium hydroxide monohydrate (LiOH . H2O) aqueous solution (0.83g is dissolved in 12mL water), stirring reaction is spent overnight at room temperature. The reaction solution is spin-dried under reduced pressure, and 100mL saturated sodium chloride, 50mL tetrahydrofuran are added, and phase separation is performed. The aqueous phase is extracted 2 times with 50mL tetrahydrofuran again, and the organic phase is combined, and washed 1 time with 50mL saturated sodium chloride, and phase separation is performed. The organic phase is dried over anhydrous sodium sulfate, and is spin-dried to obtain 1.1g of transparent viscous material.

[0056] 10) Synthesis of Compound 13

[0057]

[0058] Compound 12 (10.0 g, 23.5 mmol) was dissolved in 100 mL of dichloromethane, and L-glutamic acid di-tert-butyl ester hydrochloride (7.0 g, 23.5 mmol), diisopropylethylamine (10.6 g, 81.8 mmol), 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU, 9.8 g, 25.8 mmol) were added, and the mixture was stirred and reacted overnight at room temperature. The reaction solution was washed 3 times with 100 mL of saturated sodium bicarbonate aqueous solution, and once with 100 mL of saturated sodium chloride, and the phases were separated. The organic phase was dried with anhydrous sodium sulfate and spin-dried to obtain 21 g of a viscous substance.

[0059] MS m / z[M+H] + (ESI):667.79.

[0060] 11) Synthesis of Compound 14

[0061]

[0062] Compound 13 (19.0 g, 28.5 mmol) was dissolved in 500 mL of dichloromethane, and 95 mL of trifluoroacetic acid was added, and the mixture was stirred at room temperature for 4 h. The reaction solution was dried under reduced pressure, and 200 mL of dichloromethane was added to slurry the mixture twice, and the mixture was filtered. The filter cake was dried at 45°C to obtain 11.7 g of a light purple solid.

[0063] MS m / z[M+H] + (ESI):499.56.

[0064] 12) Synthesis of Compound 15

[0065]

[0066] Compound 14 (3.5 g, 7.0 mmol) was dissolved in 120 mL of N, N-dimethylformamide, and compound 6 (12.5 g, 23.2 mmol), diisopropylethylamine (9.5 g, 73.7 mmol), 2-(7-azabenzotriazole)-N, N, N', N'-tetramethyluronium hexafluorophosphate (HATU, 9.6 g, 25.3 mmol) were added, and the reaction was stirred at room temperature overnight. 800 mL of saturated sodium chloride was added to the reaction solution, and then 200 mL of dichloromethane was added to extract twice, and the organic phases were combined. The organic phase was washed once with 200 mL of saturated sodium chloride, separated, and dried over anhydrous sodium sulfate, dried under reduced pressure, added 50 mL of dichloromethane, crystallized with 50 mL of methyl tert-butyl ether, filtered, and the filter cake was redissolved with methanol and evaporated under reduced pressure to obtain 15.1 g of yellow-white solid.

[0067] MS m / z[M+H] + (ESI):1955.17.

[0068] 13) Synthesis of Compound 16

[0069]

[0070] Compound 15 (10.0 g, 5.1 mmol) was dissolved in 100 mL of N,N-dimethylformamide and 40 mL of tetrahydrofuran, and 100 mL of diisopropylethylamine was added, and the mixture was stirred and reacted overnight in an oil bath at 55°C. The reaction solution was dried under reduced pressure to form an oily substance, and ethyl acetate was stirred and refluxed. After standing, the supernatant was poured out and dried under reduced pressure to obtain 4.0 g of a yellow-brown foam solid.

[0071] MS m / z[M+H] + (ESI):1732.92.

[0072] 14) Synthesis of Compound 17

[0073]

[0074] Compound 16 (2.0 g, 1.15 mmol) was dissolved in 20 mL of dichloromethane, and compound 11 (0.58 g, 1.15 mmol), diisopropylethylamine (0.37 g, 2.89 mmol), 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU, 0.48 g, 1.27 mmol) were added, and the reaction was stirred overnight at room temperature. 200 mL of sodium chloride solution was added to the reaction solution, and 200 mL of dichloromethane was extracted, and the phases were separated, and the organic phase was dried under reduced pressure. 200 mL of ethyl acetate was added, stirred and crystallized, filtered, and the filter cake was redissolved with methanol and dried under reduced pressure to obtain 1.3 g of brown foamy solid.

[0075] MS m / z[M+H] + (ESI):2209.31.

[0076] 15) Synthesis of H03

[0077]

[0078] Compound 17 (1.2 g, 0.54 mmol) was dissolved in 12 mL of dichloromethane, and succinic anhydride (544 mg, 5.43 mmol), triethylamine (825 mg, 8.15 mmol), and 4-dimethylaminopyridine (DMAP, 664 mg, 5.43 mmol) were added, and the reaction was stirred at room temperature overnight. The reaction solution was diluted with 200 mL of dichloromethane, washed once with 200 mL of sodium chloride solution, washed twice with 200 mL of saturated ammonium chloride aqueous solution, and washed twice with 200 mL of NaCl solution. After phase separation, the organic phase was dried over anhydrous sodium sulfate and dried under reduced pressure to obtain 840 mg of a yellow-brown solid crude product. The preparative liquid phase was further separated and purified, model Gilson GX281, chromatographic column: watersX-bridge C18, 19*250 mm, 10 μm. Mobile phase A: ammonium bicarbonate aqueous solution, pH = 8-9; mobile phase B: acetonitrile. The flow rate was 20 ml / min, the monitoring wavelength was 214 nm, and gradient elution was performed. After freeze-drying, a light yellow solid powder was obtained.

[0079] The mass spectrum of compound H03 is shown in Figure 1 As shown, the molecular weight measured by high-resolution mass spectrometry is HRMS m / z[M+Na]-(ESI):2331.00, which is consistent with the theoretical molecular weight.

[0080] Example 2: Synthesis of Compound H05

[0081] 1) Synthesis of Compound 20

[0082]

[0083] Compound 19 (100 g, 651 mmol) was dissolved in 1.3 L acetonitrile, and then triethylamine (161.4 g, 1595 mmol) was added at room temperature. Ethyl trifluoroacetate was then added in a 25°C water bath and reacted at room temperature for 24 h. The reaction solution was filtered, the filtrate was concentrated, washed with 1 L ethyl acetate, and the insoluble matter was removed by filtration. The filtrate was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to constant weight to obtain 157 g of a brown oily crude product. The product was used directly in subsequent reactions without further purification.

[0084] 2) Synthesis of Compound 21

[0085]

[0086] Compound 20 (651 mmol) was dissolved in 1.6 L of dichloromethane, triethylamine (98.8 g, 976 mmol) was added, and nitrogen was purged. DMTrCl (4.269 g, 12.6 mmol) was added to the reaction under nitrogen protection, and the reaction was carried out at room temperature for 4 h. 1.5 L of saturated sodium chloride was added to the reaction solution, and the layers were stirred and separated. The aqueous phase was extracted twice with 600 mL of dichloromethane, and the organic phases were combined and concentrated under reduced pressure to constant weight to obtain 384 g of a crude brown oil. The product was used directly in subsequent reactions without further purification.

[0087] 3) Synthesis of Compound 22

[0088]

[0089] Compound 21 (651 mmol) was dissolved in 1.8 L methanol, and then potassium hydroxide aqueous solution (73.06 g, 1302 mmol dissolved in 430 mL deionized water) was added to the reaction, and the reaction was carried out at room temperature for 70 min. The reaction solution was concentrated to remove the solvent, and 400 mL deionized water was added to wash twice. The sticky solid was dissolved in ethyl acetate and washed with saturated sodium chloride. About 300 g of syrup was obtained by spin drying.

[0090] 4) Synthesis of Compound 23

[0091]

[0092] Monomethyl dodecanedioate (7.4 g, 30.1 mmol) was placed in a 500 mL three-necked flask, compound 22 (12.7 g, 30.1 mmol) was added, BOP (16.0 g, 36.2 mmol) was added, and 74 mL of dichloromethane was added to stir and dissolve. The reaction flask was placed in a normal temperature water bath, triethylamine (7.6 g, 75.4 mmol) was slowly added under stirring, and stirred at room temperature overnight. The reaction solution was washed 3 times with 80 mL of saturated sodium bicarbonate aqueous solution, 1 time with 100 mL of deionized water, dried over anhydrous sodium sulfate, and dried under reduced pressure to obtain 29.3 g of an orange-yellow oil.

[0093] 5) Synthesis of Compound 24

[0094]

[0095] Compound 23 (29.3 g, 45.4 mmol) was placed in a 1L single-mouth bottle, 300 mL of tetrahydrofuran was added to dissolve, and an aqueous solution of lithium hydroxide monohydrate (LiOH·H2O) (17.2 g, 408.3 mmol dissolved in 170 mL of deionized water) was added under stirring, and stirred at room temperature overnight. The reaction solution was allowed to stand for phase separation, and the upper organic phase was washed twice with 170 mL of saturated sodium chloride, dried, and dried under reduced pressure. 260 mL of dichloromethane was added to dissolve, 520 mL of methyl tert-butyl ether was added dropwise, and the precipitated solid was filtered and dried to obtain 18.8 g of white powder solid.

[0096] 6) Synthesis of Compound 25

[0097]

[0098] Compound 16 (2.0 g, 1.15 mmol) was dissolved in 20 mL of N, N-dimethylformamide, and compound 24 (729 mg, 1.15 mmol), diisopropylethylamine (0.37 g, 2.89 mmol), 2-(7-azabenzotriazole)-N, N, N', N'-tetramethyluronium hexafluorophosphate (HATU, 0.48 g, 1.27 mmol) were added, and the reaction was stirred at room temperature overnight. 300 mL of saturated sodium chloride was added to the reaction solution, and 200 mL of dichloromethane was used for extraction 3 times. The organic phases were combined, washed once with 200 mL of saturated sodium chloride, separated, and the organic phase was dried and dried under reduced pressure. 200 mL of ethyl acetate was added for washing, filtered, and the filter cake was redissolved in methanol and dried under reduced pressure to obtain 2.1 g of brown solid.

[0099] 7) Synthesis of H05

[0100]

[0101] Compound 25 (2.0 g, 0.85 mmol) was dissolved in 40 mL of dichloromethane, and succinic anhydride (853 mg, 8.52 mmol), triethylamine (1.3 g, 12.78 mmol) and 4-dimethylaminopyridine (1.0 g, 8.52 mmol) were added. The mixture was stirred at room temperature overnight.

[0102] The reaction solution was diluted with 200 mL of dichloromethane, and 20 mL of methanol was added to increase the solubility of the product. Wash once with 200 mL of sodium chloride, twice with 200 mL of saturated ammonium chloride aqueous solution, and twice with 200 mL of sodium chloride. Phase separation, drying of the organic phase, spin drying, and recrystallization of the crude product with a mixed solution of methanol: dichloromethane: ethyl acetate (100:10:200, V:V:V) were obtained, and filtration was performed. The crude product was further separated and purified by preparative liquid phase, model Gilson GX281, chromatographic column: waters X-bridge C18, 19*250mm, 10μm. Mobile phase A: ammonium bicarbonate aqueous solution, pH=8-9; mobile phase B: acetonitrile. Flow rate 20 ml / min, monitoring wavelength 214 nm, gradient elution, and freeze-dried to obtain a light yellow solid powder.

[0103] The mass spectrum of compound H05 is shown in Figure 2 As shown, the molecular weight measured by high-resolution mass spectrometry is HRMS m / z[M+Na]-(ESI):2469.10, which is consistent with the theoretical molecular weight.

[0104] Synthesis of solid supports H03-CPG and H05-CPG for compounds H03 and H05

[0105] Compound H03 (46 mg, 20 μmol) was added to 10 mL of N, N-dimethylformamide, and then benzotriazole-N, N, N', N'-tetramethyluronium hexafluorophosphate (HBTU, 19 mg, 50 μmol), N, N-diisopropylethylamine (DIEA, 16.2 mg, 125 μmol), and 0.4 g of amino-modified solid phase carrier (CPG-LCAA-NH2) were added, and the mixture was shaken at 25°C for 24 hours. After the reaction was completed, the mixture was washed with acetonitrile and dichloromethane in turn. 20% acetic anhydride / 80% acetonitrile was added, and the mixture was shaken at 25°C for 24 hours. After the reaction was completed, the mixture was washed with acetonitrile and dichloromethane in turn to obtain the solid phase carrier target product H03-CPG. Similarly, the synthesis of the solid phase carrier H05-CPG of compound H05 was synthesized according to the synthesis method of the solid phase carrier H03-CPG of compound H03 mentioned above.

[0106] siRNA synthesis

[0107] Each RNA single strand was synthesized using phosphoramidite solid phase, starting with Universal CPG vector, and using a DNA synthesizer to sequentially connect nucleoside phosphoramidite monomers according to the synthesis program. The connection of each nucleoside monomer includes four steps of deprotection, coupling, oxidation, and capping. After coupling, the thiophosphate backbone was introduced using a 0.1M pyridine solution of N,N-dimethyl-N'-(3-thio-3H-1,2,4-dithiazole-5-yl)formamidine. After the synthesis was completed, the nucleotide chain connected to the solid phase carrier was reacted in 28% ammonia water at 60°C for 5 hours, the oligonucleotide chain was cut from the solid phase carrier, and the protecting group was removed. The 2'-O-TBDMS protecting group was removed with methylamine and triethylamine trihydrofluoric acid. The crude product was purified by gradient elution with sodium bromide solution to obtain RNA single strands. The purified RNA single strands were desalted and freeze-dried, and the purity was detected by ion pair reverse phase chromatography, and the product molecular weight was analyzed by LC-MS. The sense strand and the antisense strand were dissolved in deionized water, mixed in an equimolar ratio, annealed to form double-stranded siRNA, and adjusted to the concentration required for the experiment, and stored at 2-8°C for later use.

[0108] The information of the synthesized siRNA sequence is shown in Table 1; Table 2 is the synthesized modified siRNA; Table 3 is the siRNA-I-1 conjugate. Among them, A, U, G, C represent adenine, uracil, guanine, and cytosine nucleotides; m represents a 2'-Ome modified nucleotide; f represents a 2'-F modified nucleotide; s represents a thiophosphate; vp represents a vinyl phosphate modified nucleotide; H05 represents a galactosamine carrier. The synthesized siRNA sequence is shown in Table 1:

[0109] Table 1 Synthesized siRNA sequences

[0110]

[0111] The modified siRNA sequences are shown in Table 2:

[0112] Table 2 Modified siRNA sequences

[0113]

[0114]

[0115]

[0116] Synthesis of H05-siRNA conjugates

[0117] The sense chain of GalNAc-siRNA containing H05 was synthesized and purified starting from the H05-CPG vector, and the antisense chain was synthesized and purified starting from the universal CPG vector. The synthesized sense chain and antisense chain were tested for purity by ion pair reverse phase chromatography, and the product molecular weight was analyzed by LC-MS. After mixing in an equimolar ratio, annealing was performed to form double-stranded siRNA, and the concentration required for the experiment was adjusted and stored at 2-8°C for future use. The sequence of the GalNAc-siRNA conjugate is shown in Table 3.

[0118] Table 3 GalNAc-siRNA conjugate sequences

[0119]

[0120]

[0121] Inhibitory effect of siRNA on AGT gene expression in Hep 3B cells

[0122] The human liver cancer cell line Hep 3B was used to investigate the inhibitory effect of the siRNA shown in Table 2 on AGT gene expression.

[0123] Hep 3B cells were cultured at a rate of 1×10 4 Inoculate in 96-well cell culture plates, add DMEM (purchased from Gibco, C11995500BU) complete medium containing 10% fetal bovine serum (purchased from Gibco, 12483020), and place in a cell culture incubator at 37°C and 5% CO2. Hep 3B cells grow to about 80-90% density, use Lipofectamin2000 as a transfection reagent, and dilute Lipofectamin2000 and siRNA stock solution with opUi-MEM (purchased from Gibco, 31985070) according to the instructions, mix the diluted siRNA and diluted Lipofectamin2000 at a volume ratio of 1:1, incubate at room temperature for 30 minutes, add to the cell culture plate, and add DMEM medium containing 10% fetal bovine serum to make the siRNA concentration 25nM, and continue to culture in a cell culture incubator at 37°C and 5% CO2 for 24 hours.

[0124] The total RNA of each group was extracted using a magnetic bead method cell total RNA extraction kit (purchased from TIANGEN, DP761) according to the instructions. cDNA was obtained by reverse transcription using the method described in III 1st Strand cDNA Synthesis SuperMix (purchased from Yisheng Biotechnology Co., Ltd., 11141ES60). qPCR was performed using the Universal TaqMan multiplex qPCR master mix (UDG plus) kit (purchased from Yisheng Biotechnology Co., Ltd., 11212ES08), and the results were calculated using the ΔΔCt method to calculate the AGT mRNA expression in each group. The primer sequences used are shown in Table 4. The results of siRNA inhibition of Hep 3B cell AGT are shown in Table 5.

[0125] Table 4 Primer sequences

[0126]

[0127]

[0128] Table 5 Inhibition results of siRNA on AGT in Hep 3B cells

[0129] serial number Inhibition rate (%) SD HL1001 89.73 2.32 HL1002 84.32 3.51 HL1003 76.91 2.95 HL1004 85.83 2.40 HL1005 88.54 2.53 HL1006 89.08 0.60 HL1007 70.30 3.98 HL1008 70.61 4.65 HL1009 88.92 1.29 HL1010 72.16 10.18 HL1011 68.63 6.24 HL1012 80.28 9.66 HL1013 79.76 2.64 HL1014 81.10 0.66 HL1015 82.19 3.00 HL1016 70.11 2.59 HL1017 88.42 1.36 HL1018 76.01 2.56 HL1019 75.98 2.01 HL1020 82.68 2.40 HL1021 72.15 2.84 HL1022 69.70 8.94

[0130] As shown in Table 5, each group had a significant effect on the inhibition of AGT gene expression in Hep 3B cells, with the inhibition rate exceeding 60%.

[0131] Inhibitory effect of modified siRNA on AGT gene expression in Hep 3B cells

[0132] The human liver cancer cell line Hep 3B was used to investigate the inhibitory effect of the modified siRNA on the expression of the AGT gene. The results of the inhibition of the modified siRNA on the expression of the AGT gene in Hep 3B cells are shown in Table 6.

[0133] Table 6 Inhibition results of siRNA on AGT in Hep 3B cells

[0134] serial number Inhibition rate (%) SD HL2001 87.87 1.60 HL2002 83.94 3.56 HL2003 86.00 6.09 HL2004 85.61 3.08 HL2005 84.77 2.40 HL2006 87.35 2.90 HL2007 85.57 2.72 HL2008 80.80 2.95 HL2009 86.94 2.67 HL2010 88.04 2.04 HL2011 89.81 2.33 HL2012 85.41 3.70 HL2013 86.40 2.25

[0135] As shown in Table 6, each group has a significant effect on inhibiting the AGT gene expression of Hep 3B cells, and the inhibition rate can reach more than 80%.

[0136] Therefore, the modified siRNA of the present invention has a significant effect on inhibiting AGT gene expression, with an inhibition rate of more than 80%, and can be used for the preparation of drugs for preventing, treating and alleviating diseases and symptoms related to AGT gene expression.

[0137] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. An RNAi agent for inhibiting AGT gene expression, characterized in that: The RNAi agent consists of a sense strand and an antisense strand with complementary base pairing, wherein the sense strand contains a nucleotide sequence with no more than 3 nucleotide differences as shown in SEQ ID NOs: 1-3; and the antisense strand contains a nucleotide sequence with no more than 3 nucleotide differences as shown in SEQ ID NOs: 22-24.

2. The RNAi agent for inhibiting AGT gene expression according to claim 1, characterized in that: The lengths of the sense strand and the antisense strand are 19-25 nucleotides; the sense strand and the antisense strand are at least 100% complementary, 90% complementary, or 80% complementary.

3. The RNAi agent for inhibiting AGT gene expression according to claim 1, characterized in that: The sense strand comprises a 3' overhang of 0, 1 or 2 nucleotides and the antisense strand comprises a 3' overhang of 0, 1 or 2 nucleotides.

4. The RNAi agent for inhibiting AGT gene expression according to claim 1, characterized in that: Part or all of the nucleotides of the sense strand and the antisense strand are modified nucleotides, and the modified nucleotides are selected from any one of 2'-methoxy modified nucleotides, 2'-fluoro modified nucleotides, 2'-aminoethyl modified nucleotides, 2'-O-methoxyethyl modified nucleotides, thiophosphate modified nucleotides and vinyl phosphate modified nucleotides.

5. The RNAi agent for inhibiting AGT gene expression according to claim 4, characterized in that: The phosphorothioate-modified nucleotide is located at the end of the sense strand, and the sense strand contains at least 1, 2 or 3 phosphorothioate-modified nucleotides; the phosphorothioate-modified nucleotide is located at the end of the antisense strand, and the antisense strand contains at least 1, 2 or 3 phosphorothioate-modified nucleotides; the vinyl phosphate-modified nucleotide is located at the first position of the antisense strand.

6. The RNAi agent for inhibiting AGT gene expression according to claim 4, characterized in that: The sense strand contains a modified nucleotide sequence with no more than 3 nucleotide differences shown in SEQ ID NOs: 43-55; the antisense strand contains a modified nucleotide sequence with no more than 3 nucleotide differences shown in SEQ ID NOs: 56-68.

7. The RNAi agent for inhibiting AGT gene expression according to claim 1, characterized in that: The sense strand and the antisense strand are connected with a ligand, and the ligand is conjugated to the sense strand and the antisense strand; the ligand is an asialoglycoprotein receptor.

8. The RNAi agent for inhibiting AGT gene expression according to claim 7, characterized in that: The sense strand contains a modified nucleotide sequence with no more than 3 nucleotide differences shown in SEQ ID NOs: 69-77; the antisense strand contains a modified nucleotide sequence with no more than 3 nucleotide differences shown in SEQ ID NOs: 78-86.

9. A composition for inhibiting AGT gene expression, characterized in that: The invention comprises the RNAi agent according to any one of claims 1 to 8 and a pharmaceutically acceptable carrier.

10. Use of the RNAi agent for inhibiting AGT gene expression as claimed in any one of claims 1 to 8 or the composition as claimed in claim 9 in the preparation of a drug for preventing and / or treating and / or alleviating diseases related to inhibition of AGT gene expression.

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