Sirna for regulating AGT gene expression and use thereof

Abstract

Provided are an siRNA for regulating AGT gene expression and the use thereof. The siRNA is verified in cellular experiments to have a significant inhibitory effect on AGT gene expression, and can be used in the development of a drug for treating and / or preventing diseases related to AGT gene expression.

Description

A siRNA that regulates AGT gene expression and its applications

[0001] This application claims priority to Chinese patent application 2024115457102, filed on November 1, 2024. The entire contents of the aforementioned Chinese patent application are incorporated herein by reference. Technical Field

[0002] This disclosure pertains to the field of small nucleic acid drugs, specifically involving an siRNA that regulates AGT gene expression and its applications. Background Technology

[0003] Nucleic acid drugs, especially oligonucleotide drugs, are widely used due to their simple synthesis and high activity. Oligonucleotide drugs typically include antisense oligonucleotides (ASO), small interfering RNA (siRNA), microRNA (miRNA), and nucleic acid aptamers.

[0004] RNA interference is a natural defense mechanism against foreign genes. siRNA can knock out target genes by recognizing specific sequences and breaking down target mRNA.

[0005] The effective molecule for classical RNA interference consists of a signature 19+2 nucleotide polymer structure, a double helix composed of a 21-nucleotide RNA molecule and a 19-nucleotide corresponding base molecule, including a 2-nucleotide 3' overhang. One strand of the siRNA, the guide strand or antisense strand, is complementary to the target gene's transcript mRNA, while the other strand is designated as the passenger strand or sense strand. The siRNA antisense strand guides the argonin protein to complement the target transcript and becomes part of the RNA-guided silencing complex. The perfect complementarity of the siRNA antisense strand to the target transcript leads to the breakage of the target transcript at the position opposite the 10-11 site of the antisense strand under the catalysis of the argonin protein.

[0006] By chemically modifying the synthesized antisense oligonucleotides, their serum stability can be increased, thereby effectively inhibiting the expression of the target gene.

[0007] Chemical modification of oligonucleotide structures is an effective way to enhance their activity. Chemically modified oligonucleotides can improve their stability against nucleases, their affinity for RNA, and better promote endocytosis and tissue targeting, thereby increasing their activity. All marketed oligonucleotide drugs are chemically modified. Since the approval of the first nucleic acid drug, Fomivirsen (Vitravene), in 1998, the technology for chemical modification of nucleic acid drugs has been continuously upgraded.

[0008] Based on the basic structure of oligonucleotides, namely the bases, sugar rings, phosphate backbones, and ends, chemical modifications can be performed on four parts.

[0009] Base modifications are mainly divided into three forms: purine modification, pyrimidine modification, and base substitution. Purine modifications include modifications with N6-methyladenosine, N1-methyladenosine, and 7-methylguanylic acid; pyrimidine modifications include modifications with 3-methyluridine, 5-methyluridine, 5-methylcytosine, N4-acetylcytidine, pseudouridine, thiouridine, propynourishinidine, and dihydrouridine.

[0010] Sugar ring modification: mainly divided into sugar ring modification and substitution. Sugar ring modification includes 2'-modification, 4'-modification, 5'-modification and isomerization modification, as well as combination modification of these modifications.

[0011] Modification of the phosphate backbone: The hydrolysis of the phosphodiester bonds of oligonucleotides by nucleases in vivo is the main reason for their rapid degradation. Therefore, choosing other suitable analogs to replace the phosphodiester bonds in the backbone can increase its stability. The main modification methods for the phosphate backbone include: thiophosphates, methylphosphates, selenophosphates, methylboryl phosphates, or dithiophosphates; or replacing the phosphate ester groups between nucleosides entirely with groups that do not contain phosphorus atoms, such as replacing P atoms with C, S, and N atoms to form guanidine, S-methylthiourea, etc.

[0012] Terminal modification: Covalently coupling specific groups to the 5' and / or 3' ends of siRNA can enhance cell affinity, tissue selectivity, and cell targeting. Modifying the ends with lipid-soluble small molecules can improve their similarity-to-miscibility with the cell membrane, thereby increasing cellular entry efficiency. The desialylate glycoprotein receptor (ASGPR) is an endocytic receptor specifically expressed by hepatocytes. Utilizing the high-affinity ligand N-acetylgalactosamine (GalNAc) of ASGPR coupled to siRNA can achieve liver-targeted delivery of oligonucleotide drugs.

[0013] Angiotensinogen (AGT) protein is a secreted protein primarily expressed in the liver, but it is also expressed in other tissues such as the brain, gallbladder, heart, and kidneys. AGT protein is cleaved by renin to produce angiotensin I (Ang I), which is then cleaved by angiotensin-converting enzyme (ACE) to produce physiologically active angiotensin II (Ang II). Ang II binds to the angiotensin receptor (ATR), triggering vasoconstriction and raising blood pressure.

[0014] Although a large number of antihypertensive drugs are currently available for treating hypertension, more than two-thirds of patients still struggle to control their blood pressure with a single medication and require two or more drugs. This leads to decreased treatment adherence, increased potential side effects, and compromised treatment efficacy. Therefore, there is a need in this field for alternative and combination therapies for patients with angiotensinogen-related diseases. Summary of the Invention

[0015] To address the problem of poor inhibitory effects of existing siRNAs targeting angiotensinogen (AGT), this disclosure provides an siRNA. After the drug constructed from the siRNA enters the bloodstream, it is endocytosed by hepatocytes and stored in the cellular endosome structure. After the siRNA is released from the endosome or lysosome and enters the cytoplasm, it binds to the RNA-induced silencing complex (RISC) and, mediated by the antisense strand, binds to the mRNA transcribed from the AGT gene, inducing mRNA degradation and thereby inhibiting the translation of AGT protein. As an upstream protein of the renin-angiotensin-aldosterone system (RAAS), inhibiting AGT protein expression fundamentally inhibits the blood pressure-raising effect of the RAAS system, thereby lowering blood pressure. The siRNA provided in this disclosure can significantly reduce AGT gene expression in in vitro cell experiments.

[0016] To address the aforementioned technical problems, this disclosure provides a technical solution: a siRNA (small interfering RNA), wherein the siRNA comprises a sense strand and an antisense strand, and the siRNA comprises any of the following oligonucleotide duplexes:

[0017] 1) The sense chain has a sequence or a fragment thereof as shown in SEQ ID NO:34, or a modified sequence thereof, and the antisense chain has a sequence or a fragment thereof as shown in SEQ ID NO:82, or a modified sequence thereof;

[0018] 2) The sense chain has a sequence or a fragment thereof as shown in SEQ ID NO:39, or a modified sequence thereof, and the antisense chain has a sequence or a fragment thereof as shown in SEQ ID NO:87, or a modified sequence thereof;

[0019] 3) The sense chain has a sequence or a fragment thereof as shown in SEQ ID NO:23, or a modified sequence thereof, and the antisense chain has a sequence or a fragment thereof as shown in SEQ ID NO:71, or a modified sequence thereof;

[0020] 4) The sense chain has a sequence or a fragment thereof as shown in SEQ ID NO:17, or a modified sequence thereof, and the antisense chain has a sequence or a fragment thereof as shown in SEQ ID NO:65, or a modified sequence thereof;

[0021] 5) The sense chain has a sequence or a fragment thereof as shown in SEQ ID NO:35, or a modified sequence thereof, and the antisense chain has a sequence or a fragment thereof as shown in SEQ ID NO:83, or a modified sequence thereof;

[0022] 6) The sense chain has a sequence or a fragment thereof as shown in SEQ ID NO:45, or a modified sequence thereof, and the antisense chain has a sequence or a fragment thereof as shown in SEQ ID NO:93, or a modified sequence thereof;

[0023] 7) The sense chain has a sequence or a fragment thereof as shown in SEQ ID NO:27, or a modified sequence thereof, and the antisense chain has a sequence or a fragment thereof as shown in SEQ ID NO:75, or a modified sequence thereof;

[0024] 8) The sense chain has a sequence or a fragment thereof as shown in SEQ ID NO:30, or a modified sequence thereof, and the antisense chain has a sequence or a fragment thereof as shown in SEQ ID NO:78, or a modified sequence thereof;

[0025] 9) The sense chain has a sequence or a fragment thereof as shown in SEQ ID NO:22, or a modified sequence thereof, and the antisense chain has a sequence or a fragment thereof as shown in SEQ ID NO:70, or a modified sequence thereof;

[0026] The modified sequence contains one or more modified nucleotides.

[0027] In a preferred embodiment of this disclosure, the positive strand is 19-27 nucleotides in length.

[0028] In a preferred embodiment of this disclosure, the antisense strand is 19-27 nucleotides in length.

[0029] In a preferred embodiment of this disclosure, the siRNA comprises any of the following oligonucleotide duplexes:

[0030] 1) The sense chain has a sequence as shown in SEQ ID NO:34 or a modified sequence of SEQ ID NO:34, and the antisense chain has a sequence as shown in SEQ ID NO:82 or a modified sequence of SEQ ID NO:82;

[0031] 2) The sense chain has a sequence as shown in SEQ ID NO:39 or a modified sequence of SEQ ID NO:39, and the antisense chain has a sequence as shown in SEQ ID NO:87 or a modified sequence of SEQ ID NO:87;

[0032] 3) The sense chain has a sequence as shown in SEQ ID NO:23 or a modified sequence of SEQ ID NO:23, and the antisense chain has a sequence as shown in SEQ ID NO:71 or a modified sequence of SEQ ID NO:71;

[0033] 4) The sense chain has a sequence as shown in SEQ ID NO:17 or a modified sequence of SEQ ID NO:17, and the antisense chain has a sequence as shown in SEQ ID NO:65 or a modified sequence of SEQ ID NO:65;

[0034] 5) The sense chain has a sequence as shown in SEQ ID NO:35 or a modified sequence of SEQ ID NO:35, and the antisense chain has a sequence as shown in SEQ ID NO:83 or a modified sequence of SEQ ID NO:83;

[0035] 6) The sense chain has a sequence as shown in SEQ ID NO:45 or a modified sequence of SEQ ID NO:45, and the antisense chain has a sequence as shown in SEQ ID NO:93 or a modified sequence of SEQ ID NO:93;

[0036] 7) The sense chain has a sequence as shown in SEQ ID NO:27 or a modified sequence of SEQ ID NO:27, and the antisense chain has a sequence as shown in SEQ ID NO:75 or a modified sequence of SEQ ID NO:75;

[0037] 8) The sense chain has a sequence as shown in SEQ ID NO:30 or a modified sequence of SEQ ID NO:30, and the antisense chain has a sequence as shown in SEQ ID NO:78 or a modified sequence of SEQ ID NO:78;

[0038] 9) The sense chain has a sequence as shown in SEQ ID NO:22 or a modified sequence of SEQ ID NO:22, and the antisense chain has a sequence as shown in SEQ ID NO:70 or a modified sequence of SEQ ID NO:70;

[0039] The modified sequence contains one or more modified nucleotides.

[0040] In a preferred embodiment of this disclosure, the modified nucleotide includes a 2'-modification of the nucleotide ribose.

[0041] In a preferred embodiment of this disclosure, the 2'-modification is selected from the following modifications: 2'-methoxy, 2'-O-methoxyethyl, 2'-fluoro, 2'-O-benzyl, and 2'-O-methyl-4-pyridine.

[0042] In a preferred embodiment of this disclosure, the 2'-modification is 2'-methoxy and 2'-fluoro.

[0043] In a preferred embodiment of this disclosure, the 2'-modification of each nucleotide ribose is an alternating combination of 2'-methoxy and 2'-fluoro.

[0044] In a preferred embodiment of this disclosure, the nucleotide monomers in the sense strand and the antisense strand are linked by a phosphodiester bond or a thiophosphate diester bond, preferably a 3',5'-phosphodiester bond.

[0045] In a preferred embodiment of this disclosure, the siRNA comprises any of the following oligonucleotide duplexes:

[0046] 1) The positive chain has the sequence shown in SEQ ID NO:34 and the negative chain has the sequence shown in SEQ ID NO:82; or, the positive chain has the sequence shown in SEQ ID NO:130 and the negative chain has the sequence shown in SEQ ID NO:178;

[0047] 2) The sense chain has the sequence shown in SEQ ID NO:39 and the antisense chain has the sequence shown in SEQ ID NO:87; or, the sense chain has the sequence shown in SEQ ID NO:135 and the antisense chain has the sequence shown in SEQ ID NO:183;

[0048] 3) The positive chain has the sequence shown in SEQ ID NO:23 and the negative chain has the sequence shown in SEQ ID NO:71; or, the positive chain has the sequence shown in SEQ ID NO:119 and the negative chain has the sequence shown in SEQ ID NO:167;

[0049] 4) The positive chain has the sequence shown in SEQ ID NO:17 and the negative chain has the sequence shown in SEQ ID NO:65; or, the positive chain has the sequence shown in SEQ ID NO:113 and the negative chain has the sequence shown in SEQ ID NO:161;

[0050] 5) The sense chain has the sequence shown in SEQ ID NO:35 and the antisense chain has the sequence shown in SEQ ID NO:83; or, the sense chain has the sequence shown in SEQ ID NO:131 and the antisense chain has the sequence shown in SEQ ID NO:179;

[0051] 6) The sense chain has the sequence shown in SEQ ID NO:45 and the antisense chain has the sequence shown in SEQ ID NO:93; or, the sense chain has the sequence shown in SEQ ID NO:141 and the antisense chain has the sequence shown in SEQ ID NO:189;

[0052] 7) The positive chain has the sequence shown in SEQ ID NO:27 and the negative chain has the sequence shown in SEQ ID NO:75; or, the positive chain has the sequence shown in SEQ ID NO:123 and the negative chain has the sequence shown in SEQ ID NO:171;

[0053] 8) The sense chain has the sequence shown in SEQ ID NO:30 and the antisense chain has the sequence shown in SEQ ID NO:78; or, the sense chain has the sequence shown in SEQ ID NO:126 and the antisense chain has the sequence shown in SEQ ID NO:174;

[0054] 9) The positive chain has a sequence as shown in SEQ ID NO:22 and the negative chain has a sequence as shown in SEQ ID NO:70; or, the positive chain has a sequence as shown in SEQ ID NO:118 and the negative chain has a sequence as shown in SEQ ID NO:166.

[0055] In a preferred embodiment of this disclosure, at least one nucleotide of the siRNA is conjugated to one or more target ligands.

[0056] In a preferred embodiment of this disclosure, the targeting ligand comprises carbohydrates, amino sugars, cholesterol, peptides, or lipids.

[0057] In a preferred embodiment of this disclosure, the targeting ligand comprises the GalNAc (N-acetylgalactosamine) moiety.

[0058] In a preferred embodiment of this disclosure, the GalNac portion is a monovalent GalNAc portion, a divalent GalNAc portion, a trivalent GalNAc portion, or a tetravalent GalNAc portion.

[0059] To solve the aforementioned technical problems, the present disclosure provides a technical solution as follows: a pharmaceutical composition comprising siRNA as described in the present disclosure and a pharmaceutically acceptable carrier.

[0060] The pharmaceutical compositions described herein can be used to treat diseases or conditions associated with the expression or activity of the AGT gene, such as various forms of hypertension. Such pharmaceutical compositions can be formulated based on delivery models. One example is a composition formulated for systemic administration or targeted delivery to the liver via parenteral delivery, such as intravenous (iv) delivery. Another example is a composition formulated for direct delivery to the brain parenchyma, such as by infusion into the brain, for example, via a continuous pump infusion.

[0061] The pharmaceutical composition may be a solution with or without a buffer solution or a composition containing a pharmaceutically acceptable carrier. Such compositions include, for example, aqueous or crystalline compositions, liposome formulations, micellar formulations, emulsions, and gene therapy carriers.

[0062] In a preferred embodiment of this disclosure, the liposomes (which are pharmaceutically acceptable carriers) in the liposome formulation are LNPs.

[0063] In a preferred embodiment of this disclosure, the pharmaceutical composition further includes one or both of an AGT gene inhibitor and an AGT protein inhibitor, wherein the AGT protein inhibitor includes an antibody targeting the AGT protein, and the AGT gene inhibitor includes one or more of a clipping editor targeting the AGT gene, an antisense oligonucleotide (ASO) targeting the mRNA transcribed from the AGT gene, and a microRNA (miRNA).

[0064] In a preferred embodiment of this disclosure, the pharmaceutical composition further includes a drug for treating hypertension, said drug for treating hypertension including one or more of diuretics, angiotensin-converting enzyme inhibitors, angiotensin II receptor blockers, calcium channel blockers, and beta-blockers.

[0065] In the methods disclosed herein, the siRNA can be administered in a solution. A free siRNA can be administered in a non-buffered solution, such as physiological saline or water. Alternatively, the free siRNA can also be administered in a suitable buffered solution. The buffer solution may include acetate, citrate, alcohol-soluble gluten, carbonate, or phosphate, or any combination thereof. In a preferred embodiment, the buffer solution is phosphate-buffered saline (PBS). The pH and volumetric molar osmotic concentration of the buffer containing the iRNA can be adjusted to suit its administration to the subject.

[0066] In some embodiments, the buffer solution further comprises a reagent for controlling the molar osmotic pressure concentration of the solution, such that the molar osmotic pressure concentration is maintained at a desired value, such as the physiological value in human plasma. Solutes that may be added to the buffer solution to control the molar osmotic pressure concentration include (but are not limited to) proteins, peptides, amino acids, non-metabolitic polymers, vitamins, ions, sugars, metabolites, organic acids, lipids, or salts. In some embodiments, the reagent for controlling the molar osmotic pressure concentration of the solution is a salt. In some embodiments, the reagent for controlling the molar osmotic pressure concentration of the solution is sodium chloride or potassium chloride.

[0067] The pharmaceutical compositions disclosed herein can be administered at a dose sufficient to inhibit the expression of the AGT gene. Typically, a suitable dose of the siRNA disclosed herein is in the range of about 0.001 to about 200.0 mg per kilogram of body weight per day, generally in the range of about 1 to 50 mg per kilogram of body weight per day. The siRNA can be administered, for example, at doses of about 0.01, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3. 5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7. 7, 7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5, 15, 15.5, 16, 16.5, 17, 17.5, 18, 18.5, 19 19.5, 20, 20.5, 21, 21.5, 22, 22.5, 23, 23.5, 24, 24.5, 25, 25.5, 26, 26.5, 27, 27.5, 28, 28.5, 29, 29.5, 30, 31, 32, 33, 34, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or approximately 50 mg / kg.

[0068] The pharmaceutical composition can be administered once daily, or multiple times at different time intervals from 1 to 365 days, or the siRNA can be administered two, three, or more sub-dose at appropriate intervals within a year, or even administered via continuous infusion or delivery using a controlled-release formulation. In this case, the amount of siRNA contained in each sub-dose must be correspondingly less to achieve the total daily dose. Dosage units can also be compounded for delivery over several days, for example using conventional sustained-release formulations that provide sustained siRNA release over a timeframe of several days. Sustained-release formulations are well known in the art and are particularly useful for delivering reagents at specific sites, thus allowing for use with the reagents disclosed herein. In this embodiment, the dosage unit comprises a corresponding plurality of daily doses.

[0069] In other embodiments, a single dose of the siRNA or the pharmaceutical composition can be administered continuously, with subsequent doses given at intervals of no more than 3, 4, or 5 days, or at intervals of no more than 1, 2, 3, or 4 weeks. In some embodiments of this disclosure, a single dose of the pharmaceutical composition is given weekly. In other embodiments of this disclosure, a single dose of the pharmaceutical composition is given every two months.

[0070] Those skilled in the art will understand that certain factors can influence the dosage and timing required to effectively treat a subject, including (but not limited to) the severity of the disease or condition, previous treatments, the subject's overall health and / or age, and other pre-existing conditions. Furthermore, treating a subject with a therapeutically effective dose of the composition may comprise a single treatment or a series of treatments. As described elsewhere herein, the effective dose and in vivo half-life of the individual siRNAs covered by this disclosure can be estimated using conventional methods or based on in vivo testing using suitable animal models.

[0071] Depending on whether local or systemic treatment is desired and depending on the area to be treated, the pharmaceutical compositions of this disclosure can be administered in a variety of ways. Administration can be local (e.g., via a skin patch); pulmonary; such as by inhalation or blowing in a powder or aerosol, including via a nebulizer; intratracheal; intranasal; epidermal; and percutaneous, oral, or parenteral administration. Parenteral administration includes intravenous, intra-arterial, subcutaneous, intraperitoneal, or intramuscular injection or infusion; subdermal, for example, via an implanted device; or intracranial, such as administration within the brain parenchyma, intrasheath, or ventricle.

[0072] The siRNA used in the pharmaceutical compositions, methods, and uses disclosed herein can be formulated for delivery in membrane-bound molecular assemblies, such as liposomes or micelles. As used herein, the term "liposome" refers to a vesicle composed of amphiphilic lipids disposed in at least one bilayer (e.g., one or more bilayers). Liposomes comprise monolayered or multilayered vesicles having a membrane formed from a lipophilic material and an aqueous interior. The aqueous portion contains the siRNA. The lipophilic material separates the aqueous interior from the aqueous exterior, which typically does not contain the siRNA (although in some instances it may include it). Liposomes are useful for the transfer and delivery of active ingredients to sites of action. Because the liposome membrane is structurally similar to a biological membrane, when a liposome is applied to a tissue, the liposome bilayer fuses with the cell membrane bilayer. As the liposome fuses with the cell, the aqueous interior contents, including the siRNA, are delivered into the cell, wherein the siRNA can specifically bind to a target RNA and can mediate RNAi. In some cases, these liposomes are also specifically targeted, for example, to guide the siRNA to a specific cell type.

[0073] Liposomes containing the siRNA can be prepared by various methods. In one example, a lipid component of the liposome is dissolved in a detergent to form micelles. For example, the lipid component can be an amphiphilic cationic lipid or a lipid conjugate. The detergent can have a high critical micelle concentration and can be nonionic. Exemplary detergents include bile salts, CHAPS, octyl glucoside, deoxycholate, and lauroyl sarcosine. The siRNA is then added to micelles containing the lipid component. The cationic groups on the lipid interact with the siRNA and condense around the siRNA to form liposomes. After condensation, the detergent is removed, for example by dialysis, to obtain a liposomal formulation of the siRNA.

[0074] iRNAs, such as the siRNA disclosed herein, can be completely encapsulated in lipid formulations (e.g., LNPs or other nucleic acid-lipid particles).

[0075] As used herein, the term "LNP" refers to a stable nucleic acid-lipid particle. An LNP contains a cationic lipid, a non-cationic lipid, and a lipid that prevents the particle from aggregating (e.g., a PEG-lipid conjugate). LNPs are extremely useful for synthetic applications because they exhibit prolonged cycle life after intravenous (iv) injection and accumulate at distal sites (e.g., at sites physically separate from the administration site).

[0076] In one embodiment, the ratio of lipids to the drug (mass / mass ratio) (e.g., the ratio of lipids to siRNA) will be in the range of about 1:1 to about 50:1, about 1:1 to about 25:1, about 3:1 to about 15:1, about 4:1 to about 10:1, about 5:1 to about 9:1, or about 6:1 to about 9:1.

[0077] In a preferred embodiment of this disclosure, the lipid nanoparticles include cationic lipids, neutral lipids, structural lipids, and polymer-conjugated lipids.

[0078] In a preferred embodiment of this disclosure, the cationic lipid is a compound of formula (I), or its N-oxide, solvate, pharmaceutically acceptable salt, or stereoisomer, wherein G1 is C 1～6 Alkylene; G2 is C 2～8 Alkylene; G3 is C 1～3 Alkylene; L1 is C 6～15 Straight-chain alkyl; L2 is C 12～25 Branched alkyl groups. For example, YK-009 with the structure of formula (II) (see patent CN114044741B).

[0079] In a preferred embodiment of this disclosure, the cationic lipid is a compound of formula (II), or its N-oxide, solvate, pharmaceutically acceptable salt, or stereoisomer, wherein: G1 is C 2～8 Alkylene; G2 is C 2～8 Alkylene; L1 is -C(O)O- or -OC(O)-; L2 is -C(O)O- or -OC(O)-; R1 is C 6～25 Straight-chain or branched alkyl; R2 is C 6～25 Straight-chain or branched alkyl; G3 is HO(CH2)2- or HO(CH2)3-; G4 is HO(CH2)2- or HO(CH2)3-; L is (CH2)2- or -(CH2)3- or -(CH2)4-. For example, YK-401 with formula (II-I) structure, YK-402 with formula (II-II) structure (see patent CN115784921B).

[0080] In a preferred embodiment of this disclosure, the cationic lipid is a compound of formula (III), or its N-oxide, solvate, pharmaceutically acceptable salt, or stereoisomer, wherein: G1 is C 1～6 Alkylene; G2 is C 2～8 Alkylene; R1 is C 6～20 Straight-chain or branched alkyl; R2 is C 12～25Branched alkyl groups; G3 is: HO(CH2)2N(CH3)(CH2)2-, HO(CH2)2N(CH2CH3)(CH2)2-, (HO(CH2)2)2N(CH2)2-, CH3O(CH2)2N(CH3)(CH2)2-, (CH3)2N(CH2)3SC(O)O(CH2)2-, (CH3)2N(CH2)3SC(O)-, CH3NH(CH2)2N(CH3)(CH2)2-, or CH3CH2NH(CH2)2-. For example, YK-201 with formula (III-I) structure, YK-202 with formula (III-II) structure (see patent CN115677518B).

[0081] In a preferred embodiment of this disclosure, the cationic lipid is a compound of formula (IV), or its N-oxide, solvate, pharmaceutically acceptable salt, or stereoisomer, wherein G1 is C 1～8 Alkylene; G2 is C 2～8 Alkylene; R1 is C 6～25 Straight-chain or branched alkyl; R2 is C 12～25 Straight-chain or branched alkyl; G3 is: HO(CH2)2N(R3)CH2CH(OH)CH2-, where R3 is -CH3, -CH2CH3, or -CH2CH2OH. For example, YK-305 with formula (IV-I) and YK-310 with formula (IV-II) (see patent CN115745820B).

[0082] In a preferred embodiment of this disclosure, the cationic lipid is a compound of formula (V), or its N-oxide, solvate, pharmaceutically acceptable salt, or stereoisomer, wherein G 1 and G 2 Each is independently unsubstituted C6-C 10 Alkylene; G 3 For unsubstituted C1-C 12 Alkylene; R 1 and R 2 Each independently is C6-C 24 Alkyl or C6-C 24 alkenyl; R 3 OR 5 N, -C (=O) OR 4 -OC(=O)R 4 or -NR 5 C(=O)R 4 ;R 4 For C1-C 12 hydrocarbon group; and R5 It is an H or C1-C6 hydrocarbon group; for example, ALC0315 with the structure of formula (VI) (see patent CN108368028B).

[0083] In a preferred embodiment of this disclosure, the cationic lipid is a compound of formula (VI), or its N-oxide, solvate, pharmaceutically acceptable salt, or stereoisomer, wherein R4 is selected from -(CH2). n Q and -(CH2) n CHQR; Q is selected from the following groups: -OR, -OH, -O(CH2). n N(R)2, -OC(O)R, -CX3, -CN, -N(R)C(O)R, -N(H)C(O)R, -N(R)S(O)2R, -N(H)S(O)2R, -N(R)C(O)N(R)2, -N(H)C(O)N(R)2, -N(H)C(O)N(H)(R), -N(R)C(S)N(R)2, -N(H)C(S)N(R)2, -N(H)C(S)N(H)(R), -N(R)S(O)2R8 and heterocycles; n is 1, 2 or 3; for example, SM102 of formula (VI-I) (see patent CN110520409A).

[0084] In a preferred embodiment of this disclosure, the cationic lipid is a compound of formula (VII), or its N-oxide, solvate, pharmaceutically acceptable salt, or stereoisomer (see CN102625696B, DLIN-MC3-DMA).

[0085] In a preferred embodiment of this disclosure, the cationic lipids include YK-009, YK-401, YK-305, ALC0315, SM102, and DLIN-MC3-DMA.

[0086] In a preferred embodiment of this disclosure, the molar ratio of the cationic lipid to the neutral lipid is 1:1 to 10:1.

[0087] In a preferred embodiment of this disclosure, the molar ratio of the cationic lipid to the structural lipid is 1:1 to 5:1.

[0088] In a preferred embodiment of this disclosure, the molar ratio of the cationic lipid, the neutral lipid, the structural lipid, and the polymer-conjugated lipid is (25-65):(5-25):(25-70):(0.5-5).

[0089] In a preferred embodiment of this disclosure, the molar ratio of the cationic lipid, the neutral lipid, the structural lipid, and the polymer conjugated lipid is (25-65):(5-25):(25-45):(0.5-5).

[0090] In some more preferred embodiments, the molar ratio of the cationic lipid, the neutral lipid, the structural lipid, and the polymer-conjugated lipid is 50:10:38.5:1.5 or 49:10:39.5:1.5.

[0091] In a preferred embodiment of this disclosure, the neutral lipids include one or more of phosphatidylcholine, phosphatidylethanolamine, sphingomyelin, ceramide, sterols and their derivatives.

[0092] In some more preferred embodiments, the neutral lipid is selected from one or more of the following: 1,2-dilinoleoyl-sn-glycerol-3-phosphate choline (DLPC), 1,2-dimyristoyl-sn-glycerol-3-phosphate choline (DMPC), 1,2-dioleoyl-sn-glycerol-3-phosphate choline (DOPC), 1,2-dipalmitoyl-sn-glycerol-3-phosphate choline (DPPC), 1,2-distearateoyl-sn-glycerol-3-phosphate choline (DSPC), 1,2-diundecanoyl-sn-glycerol-3-phosphate choline (DUPC), 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphate choline (POPC), 1,2-di-O-octadecenyl-sn-glycerol-3-phosphate choline (18:0 Diether 1,2-Dilinoleoyl-sn-glycerol-3-phosphate choline (OChemsPC), 1-hexadecyl-sn-glycerol-3-phosphate choline (C16 Lyso PC), 1,2-dilinoleoyl-sn-glycerol-3-phosphate choline, 1,2-disarachidonicoyl-sn-glycerol-3-phosphate choline, 1,2-bis(docohexanoyl-sn-glycerol-3-phosphate choline), 1,2-dioleoyl-sn-glycerol-3-phosphate ethanolamine (DOPE), 1,2-diphydanyl-sn-glycerol-3-phosphate ethanolamine (ME) 16.0PE), 1,2-distearyl-sn-glycerol-3-phosphate ethanolamine, 1,2-dilinoleoyl-sn-glycerol-3-phosphate ethanolamine, 1,2-dilinolenoyl-sn-glycerol-3-phosphate ethanolamine, 1,2-diarachidonicoyl-sn-glycerol-3-phosphate ethanolamine, 1,2-bis(docosahexaenoicoyl-sn-glycerol-3-phosphate ethanolamine, 1,2-dioleoyl-sn-glycerol-3-phosphate-rac-(1-glycerol) sodium salt (DOPG), dipalmitoylphosphatidylglycerol (DPPG), palmitoyloleoyl 1-Steayl-2-oleoyl-stearoyl-ethanolamine (POPE), 1-stearoyl-2-oleoyl-stearoyl-ethanolamine (DSPE), 1-stearoyl-2-oleoyl-phosphatidylcholine (SOPC), sphingomyelin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyloleoyl-phosphatidylcholine, lysophosphatidylcholine, lysophosphatidylethanolamine (LPE), and mixtures thereof.

[0093] In some more preferred embodiments, the neutral lipid is DOPE and / or DSPC.

[0094] In a preferred embodiment of this disclosure, the structural lipid is selected from one or more of the following: cholesterol, nonsterols, sitosterol, ergosterol, campesterol, stigmasterol, brassosterol, tomatine, ursolic acid, α-tocopherol, and corticosteroids.

[0095] In some more preferred embodiments, the structural lipid is cholesterol.

[0096] In a preferred embodiment of this disclosure, the polymeric conjugated lipid is selected from one or more of the following: PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, and PEG-modified dialkylglycerol.

[0097] In some preferred embodiments, the polymeric conjugated lipid is selected from one or more of the following: distearate phosphatidylethanolamine polyethylene glycol 2000 (DSPE-PEG2000), dimyristoylglycerol-3-methoxy polyethylene glycol 2000 (DMG-PEG2000), and methoxy polyethylene glycol bis(tetradecyl acetamide) (ALC-0159).

[0098] In a specific embodiment of this disclosure, the LNP is RNAiMAX.

[0099] The pharmaceutical compositions described in this disclosure include, but are not limited to, solutions, emulsions, and liposome-containing formulations. These compositions can be derived from a variety of components, including but not limited to preformed liquids, self-emulsifying solids, and self-emulsifying semi-solids. Particularly preferred are formulations targeting the liver when treating liver conditions such as liver cancer.

[0100] The pharmaceutical formulations disclosed herein (which can be conveniently presented in unit dosage forms) can be prepared using conventional techniques well known in the pharmaceutical industry. Such techniques include steps such as combining the active ingredients with the drug carrier or excipient. Generally, these formulations are prepared by uniformly and finely combining the active ingredients with a liquid carrier or a finely dispersed solid carrier, or both, and, if necessary, shaping the product.

[0101] The pharmaceutical compositions described herein can be formulated into any of a number of possible dosage forms, including, but not limited to, tablets, capsules, gel capsules, liquid syrups, soft capsules, suppositories, and enemas. The pharmaceutical compositions described herein can also be formulated as suspensions in aqueous, non-aqueous, or mixed media. Aqueous suspensions may further contain substances that increase the viscosity of the suspension, such substances including, for example, sodium carboxymethyl cellulose, sorbitol, and / or dextran. The suspension may also contain stabilizers.

[0102] Some pharmaceutical compositions disclosed herein also incorporate a carrier compound into the formulation. As used herein, "carrier compound" or "carrier" may refer to a nucleic acid or its analogue that is inert (i.e., not biologically active in itself) but is considered a nucleic acid in vivo, for example by reducing the bioavailability of biologically active nucleic acids by degrading them or promoting their removal from circulation. Co-administration of nucleic acids and carrier compounds (generally in excess of the latter) can result in a significant reduction in the amount of nucleic acid recovered from the liver, kidneys, or other external circulation reservoirs, presumably due to competition for a common receptor between the carrier compound and the nucleic acid. For example, when co-administered with polyinosinic acid, dextran sulfate, polycytidine, or 2,2'-disulfonic acid 4-acetamido-4'-isothiocyanate, the recovery of partially thiophosphorylated dsRNA in liver tissue can be reduced (Miyao et al., DsRNA Research and Development, 1995, 5, 115-121; Takakura et al., DsRNA & Nucleic Acid Drug Development, 1996, 6, 177-183).

[0103] Compared to carrier compounds, a "drug carrier" or "excipient" is a pharmaceutically acceptable solvent, suspending agent, or other pharmaceutically inert medium used to deliver one or more nucleic acids to animals. The excipient can be liquid or solid, and when combined with nucleic acids and other components of a particular pharmaceutical composition, the excipient is selected to provide desired volume, consistency, etc., with reference to the intended manner of administration. Typical drug carriers include, but are not limited to, binders (e.g., pregelatinized corn starch, polyvinylpyrrolidone, or hydroxypropyl methylcellulose); fillers (e.g., lactose and other sugars, microcrystalline cellulose, pectin, gelatin, calcium sulfate, ethyl cellulose, polyacrylate, or dicalcium phosphate); lubricants (e.g., magnesium stearate, talc, silica, colloidal silica, stearic acid, metal stearates, hydrogenated vegetable oils, corn starch, polyethylene glycol, sodium benzoate, sodium acetate, etc.); disintegrants (e.g., starch, sodium starch glycolate, etc.); and wetting agents (e.g., sodium lauryl sulfate, etc.).

[0104] Pharmaceutically acceptable organic or inorganic excipients that are suitable for non-parenteral administration, do not react toxically with nucleic acids, and are suitable for formulating the pharmaceutical compositions described in this disclosure may also be used. Suitable pharmaceutically acceptable carriers include, but are not limited to: water, salt solutions, alcohols, polyethylene glycol, gelatin, lactose, amylose, magnesium stearate, talc, silica, viscous paraffin, hydroxymethyl cellulose, polyvinylpyrrolidone, etc.

[0105] Formulations for topical administration of nucleic acids may include sterile or non-sterile aqueous solutions, non-aqueous solutions in common solvents such as alcohol, or nucleic acid solutions in liquid or solid oil matrices. These solutions may also include buffers, diluents, and other suitable additives. Pharmaceutically acceptable organic or inorganic excipients suitable for non-parenteral administration and not toxic to nucleic acids may be used.

[0106] Suitable pharmaceutically acceptable excipients include, but are not limited to: water, salt solutions, alcohols, polyethylene glycol, gelatin, lactose, amylose, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethyl cellulose, polyvinylpyrrolidone, etc.

[0107] To solve the aforementioned technical problems, the present disclosure provides a technical solution as follows: a kit medicine box, the kit medicine box includes medicine box A, the medicine box A includes one or two of siRNA as described in the present disclosure or a pharmaceutical composition as described in the present disclosure;

[0108] Preferably, the kit further includes a pillbox B, which contains one or both of the following (1) and (2):

[0109] (1) AGT gene inhibitors or AGT protein inhibitors;

[0110] (2) Any one or a combination of two or more of the following groups: hormone preparations, targeted small molecule preparations, proteasome inhibitors, imaging agents, diagnostic agents, chemotherapeutic agents, oncolytic drugs, cytotoxic agents, cytokines, activators of co-stimulatory molecules, inhibitors of inhibitory molecules, and vaccines.

[0111] In a preferred embodiment of this disclosure, the AGT protein inhibitor includes an antibody targeting the AGT protein, and the AGT gene inhibitor includes one or more of a clipping editor targeting the AGT gene, an antisense oligonucleotide (ASO) targeting the mRNA transcribed from the AGT gene, and a microRNA (miRNA).

[0112] To address the aforementioned technical problems, this disclosure provides a technical solution: the use of one or more of the following in the preparation of a drug for the treatment and / or prevention of AGT gene expression-related diseases: siRNA as described in this disclosure, pharmaceutical compositions as described in this disclosure, and kits as described in this disclosure.

[0113] In a preferred embodiment of this disclosure, the AGT gene expression-related diseases include hypertension or diseases related to hypertension.

[0114] In a preferred embodiment of this disclosure, the hypertension-related diseases include borderline hypertension, primary hypertension, secondary hypertension, hypertensive crisis, hypertensive urgency, isolated systolic and diastolic hypertension, pregnancy-related hypertension, diabetic hypertension, refractory hypertension, intractable hypertension, paroxysmal hypertension, renovascular hypertension, Goldblatt hypertension, ocular hypertension, glaucoma, pulmonary hypertension, portal hypertension, systemic venous hypertension, systolic hypertension, and unstable hypertension.

[0115] In a preferred embodiment of this disclosure, the related diseases caused by hypertension include hypertensive heart disease, hypertensive nephropathy, atherosclerosis, arteriosclerosis, vascular disease, diabetic nephropathy, diabetic retinopathy, chronic heart failure, cardiomyopathy, diabetic cardiomyopathy, glomerulosclerosis, aortic coarctation, aortic aneurysm, ventricular fibrosis, and Cushing's syndrome.

[0116] To address the aforementioned technical problems, this disclosure provides a technical solution as follows: siRNA, pharmaceutical composition, or kit as described in this disclosure, used for the treatment and / or prevention of AGT gene expression-related diseases.

[0117] In a preferred embodiment of this disclosure, the AGT gene expression-related diseases include hypertension or diseases related to hypertension.

[0118] In a preferred embodiment of this disclosure, the hypertension-related diseases include borderline hypertension, primary hypertension, secondary hypertension, hypertensive crisis, hypertensive urgency, isolated systolic and diastolic hypertension, pregnancy-related hypertension, diabetic hypertension, refractory hypertension, intractable hypertension, paroxysmal hypertension, renovascular hypertension, Goldblatt hypertension, ocular hypertension, glaucoma, pulmonary hypertension, portal hypertension, systemic venous hypertension, systolic hypertension, and unstable hypertension.

[0119] In a preferred embodiment of this disclosure, the related diseases caused by hypertension include hypertensive heart disease, hypertensive nephropathy, atherosclerosis, arteriosclerosis, vascular disease, diabetic nephropathy, diabetic retinopathy, chronic heart failure, cardiomyopathy, diabetic cardiomyopathy, glomerulosclerosis, aortic coarctation, aortic aneurysm, ventricular fibrosis, and Cushing's syndrome.

[0120] To address the aforementioned technical problems, this disclosure provides a method for treating or preventing AGT gene expression-related diseases, comprising administering to a subject in need a therapeutically effective amount of one or more of the following: siRNA as described in this disclosure, pharmaceutical compositions as described in this disclosure, and a kit as described in this disclosure. This method achieves treatment or prevention by downregulating AGT gene expression.

[0121] In a preferred embodiment of this disclosure, the AGT gene expression-related diseases include hypertension or diseases related to hypertension.

[0122] In a preferred embodiment of this disclosure, the hypertension-related diseases include borderline hypertension, primary hypertension, secondary hypertension, hypertensive crisis, hypertensive urgency, isolated systolic and diastolic hypertension, pregnancy-related hypertension, diabetic hypertension, refractory hypertension, intractable hypertension, paroxysmal hypertension, renovascular hypertension, Goldblatt hypertension, ocular hypertension, glaucoma, pulmonary hypertension, portal hypertension, systemic venous hypertension, systolic hypertension, and unstable hypertension.

[0123] In a preferred embodiment of this disclosure, the related diseases caused by hypertension include hypertensive heart disease, hypertensive nephropathy, atherosclerosis, arteriosclerosis, vascular disease, diabetic nephropathy, diabetic retinopathy, chronic heart failure, cardiomyopathy, diabetic cardiomyopathy, glomerulosclerosis, aortic coarctation, aortic aneurysm, ventricular fibrosis, and Cushing's syndrome.

[0124] The siRNA described herein can be administered to a subject using any administration method known in the art, including (but not limited to) subcutaneous, intravenous, intramuscular, intraocular, intrabronchial, intrapleural, intraperitoneal, intraarterial, lymphatic, cerebrospinal, and any combination thereof. In a preferred embodiment, these agents are administered subcutaneously.

[0125] In another embodiment, the siRNA is administered in combination with another therapeutic agent. The siRNA and the other therapeutic agent may be administered in combination in the same composition, for example, parenterally, or the other therapeutic agent may be administered as part of a separate composition or by another method described herein.

[0126] Other examples of therapeutic agents include those known to treat hypertension or those known to treat cardiovascular and cerebrovascular diseases. For instance, other agents for treating hypertension are selected from angiotensin-converting enzyme inhibitors (e.g., captopril, enalapril, benazepril, perindopril, etc.), angiotensin II receptor antagonists (e.g., losartan, losartan hydrochlorothiazide, valsartan, valsartan hydrochlorothiazide, telmisartan, telmisartan hydrochlorothiazide, olmesartan medoxomil, etc.), and beta-blockers (e.g., propranolol, bisoprolol, metoprolol tartrate, metoprolol succinate, etc.).

[0127] In one embodiment, the siRNA agent is given to the patient, and subsequently, another therapeutic agent is given to the patient (or vice versa). In another embodiment, the iRNA agent and another therapeutic agent are given simultaneously.

[0128] To address the aforementioned technical problems, this disclosure provides a technical solution: a method for reducing AGT gene expression or inhibiting AGT replication, characterized in that the method includes applying one or more of the following to a sample: siRNA as described in this disclosure, a pharmaceutical composition as described in this disclosure, and a kit as described in this disclosure. Preferably, the method is for non-therapeutic purposes.

[0129] In this invention, the amino acid sequence of the targeted AGT is shown in, for example, NCBI Accession No: NM_001384479.1 and NM_001382817.3, and the nucleotide sequence of the gene encoding AGT is shown in, for example, NCBI Accession No: NM_001384479.1 and NM_001382817.3.

[0130] Based on common knowledge in the field, the above-mentioned preferred conditions can be combined arbitrarily to obtain various preferred embodiments of the present invention.

[0131] The reagents and raw materials used in this invention are all commercially available.

[0132] The positive and progressive effects of this invention are as follows:

[0133] The unmodified siRNA provided by this invention has a significant inhibitory effect on the expression of AGT mRNA, for example, the inhibition rate is higher than 50%. After further modification of these siRNAs, some of the modified siRNAs still maintain a high inhibition rate or increase the inhibition rate (for example, the inhibition rate reaches more than 60%). Attached Figure Description

[0134] Figure 1 shows the inhibition rate of AGT by unmodified siRNA, with NC as the control.

[0135] Figure 2 shows the inhibition rate of modified siRNA on AGT, with NC as the control. Detailed Implementation

[0136] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the raw materials used are all commercially available products.

[0137] This disclosure discloses 46 small interfering RNA (siRNA) sequences designed for the AGT mRNA sequence, including 46 sense strands and 46 antisense strands, and these sequences were modified with alternating 2'-F and 2'-Me modifications. Specifically, for the antisense strand of the siRNA sequence, odd-numbered positions (i.e., positions 1, 3, 5, 7...21, 23) were modified with 2'-methoxy, and even-numbered positions (i.e., positions 2, 4, 6, 8...20, 22) were modified with 2'-fluorination. For the sense strand complementary to the antisense strand, at positions on the antisense strand modified with 2'-methoxy, the complementary position on the sense strand was modified with 2'-fluorination; conversely, at positions on the antisense strand modified with 2'-fluorination, the complementary position on the sense strand was modified with 2'-methoxy. The positive control was APC (the Zilebesiran drug sequence from Alnilam), and the negative control was NC (Negative Control), which did not overlap with the AGT mRNA sequence. Unmodified oligonucleotide sequences are shown in Table 1, and oligonucleotides with alternating 2'-F and 2'-Me modifications are shown in Table 2.

[0138] Table 1 Unmodified oligonucleotide sequences

[0139] Table 2 Modified oligonucleotides

[0140] Note: Unmodified sequences are numbered. Alternating modified sequences are prefixed with "A" and followed by "-AL". For example, the unmodified sequence 1583 becomes A1583-AL after alternation modification.

[0141] The nucleotide abbreviations for this article are as follows:

[0142] A = Adenosine-3'-phosphate

[0143] Am = 2'-methoxyadenosine-3'-phosphate

[0144] Ams = 2'-methoxyadenosine-3'-thiophosphate

[0145] Af = 2'-Fluoroadenosine-3'-phosphate

[0146] Afs = 2'-Fluoroadenosine-3'-Thiophosphate

[0147] G = Guanosine-3'-phosphate

[0148] Gm = 2'-methoxyguanosine-3'-phosphate

[0149] Gms = 2'-methoxyguanosine-3'-thiophosphate

[0150] Gf = 2'-Fluoroguanosine-3'-phosphate

[0151] Gfs = 2'-Fluoroguanosine-3'-Thiophosphate

[0152] C = Cytidine-3'-phosphate

[0153] Cm = 2'-methoxycytidine-3'-phosphate

[0154] Cms = 2'-methoxycytidine-3'-thiophosphate

[0155] Cf = 2'-Fluorocytidine-3'-phosphate

[0156] Cfs = 2'-Fluorocytidine-3'-Thiophosphate

[0157] U = uridine-3'-phosphate

[0158] Um = 2'-methoxyuridine-3'-phosphate

[0159] Ums = 2'-methoxyuridine-3'-thiophosphate

[0160] Uf = 2'-fluorouridine-3'-phosphate

[0161] Ufs = 2'-fluorouridine-3'-thiophosphate

[0162] In the following examples of the present invention, the experimental data used for comparison have a p-value < 0.05, indicating that the differences are statistically significant.

[0163] Example 1: Synthesis of Oligonucleotides

[0164] The unmodified oligonucleotide sequences SEQ ID NO.1-48 and 49-96 in Table 1 and the modified oligonucleotide sequences SEQ ID NO.97-144 and 145-192 in Table 2 were synthesized by chemical methods.

[0165] 1. Synthesis of modified oligonucleotides

[0166] 1.1 Synthesis of the modified oligonucleotide sequences in Table 2 (taking A1583-AL as an example)

[0167] The sequence corresponding to the modified oligonucleotide sequence A1583-AL in Table 2 is as follows:

[0168] 5'-Cfs-Ums-Uf-Cm-Uf-Am-Af-Um-Gf-Am-Gf-Um-Cf-Gm-Af-Cm-Uf-Um-Uf-Gm-Af-3' (SEQ ID NO. 130)

[0169] The antisense chain sequence is:

[0170] 5'-Ams-Afs-Am-Gf-Um-Cf-Gm-Af-Cm-Uf-Cm-Af-Um-Uf-Am-Gf-Am-Af-Gms-Afs-Am-3' (SEQ ID NO. 178)

[0171] The positive chain has odd-numbered sites modified with a 2'-fluorine (2'-F) group and even-numbered sites modified with a 2'-methoxy (2'-OMe) group; the negative chain has odd-numbered sites modified with a 2'-methoxy (2'-OMe) group and even-numbered sites modified with a 2'-fluorine (2'-F) group. There are two phosphothioester bonds at the 5' end of the positive chain and two at the 5' / 3' end of the negative chain.

[0172] Instruments and reagents: OligoPilot 400 synthesizer from GE, USA, with cross-linked polystyrene beads as the solid support, model Primer support 5G Unylinker 350 (Cytiva manufacturer).

[0173] Preparation method:

[0174] Prepare the following nucleotide monomer solutions using acetonitrile at a monomer concentration of 0.15 M:

[0175] DMT-A-OMe phosphorus amide monomer (Formula 1), DMT-C-OMe phosphorus amide monomer (Formula 2), DMT-G-OMe phosphorus amide monomer (Formula 3), DMT-U-OMe phosphorus amide monomer (Formula 4), DMT-AF phosphorus amide monomer (Formula 5), ​​DMT-CF phosphorus amide monomer (Formula 6), DMT-GF phosphorus amide monomer (Formula 7), DMT-UF phosphorus amide monomer (Formula 8), and DMT-dT phosphorus amide monomer (Formula 9).

[0176] Prepared using the following steps:

[0177] 1) Deprotection

[0178] The DMT protecting group was removed using a 3% dichloroacetic acid toluene solution as a deprotecting agent, followed by washing with acetonitrile.

[0179] 2) Coupling

[0180] The acetonitrile solutions of each nucleotide monomer were coupled using 0.25 M 5-ethylthiotetrazole as an activator, followed by rinsing with acetonitrile.

[0181] (3) Oxidation / Sulfation

[0182] Oxidation: Oxidation was performed using a 0.05M iodine pyridine / water (90 / 10) solution as the oxidant, followed by rinsing with acetonitrile.

[0183] Vulcanization: Vulcanization is carried out using a 3% pyridine solution of hydroflavin as a sulfiding agent, followed by rinsing with acetonitrile.

[0184] 4) Hydroxyl protection

[0185] Hydroxyl protection was performed using a 10% acetic anhydride tetrahydrofuran solution (CAP A) tetrahydrofuran / pyridine / azirmethylimidazole 74 / 10 / 16 (v / v / v) (CAP B) as the hydroxyl protecting agent, followed by rinsing with acetonitrile.

[0186] Repeat the above operations in a cyclical manner according to the set sequence to obtain a fully protected product.

[0187] 5) Deprotection

[0188] The DMT protecting group of the last nucleotide was removed using a 3% dichloroacetic acid toluene solution as the deprotecting agent, followed by washing with acetonitrile.

[0189] 6) Ammonolysis and purification

[0190] The solid support was transferred to the reactor, concentrated ammonia (25-28%) was added, and the mixture was kept at 60°C for 12 hours for ammonolysis. The system was then cooled to room temperature, and the mixture was transferred to a filter press. The mixture was washed with a mixture of purified water and ethanol, the filtrates were combined, passed through a chromatography column, concentrated, and lyophilized to obtain the product.

[0191] 7) Annealing

[0192] The purified sense and antisense strands were mixed in a 1:1 ratio, heated to 95°C and held for 3 minutes, and then slowly cooled to room temperature to form a double strand.

[0193] 1.2 Synthesis of other modified sequences in Table 2 and positive controls

[0194] Following the above synthesis method, synthesize the other modified oligonucleotides and modified positive control sequences listed in Table 2. For the antisense strand of the siRNA sequence, odd-numbered positions (i.e., positions 1, 3, 5, 7...21, 23) are modified with 2'-methoxy, and even-numbered positions (i.e., positions 2, 4, 6, 8...20, 22) are modified with 2'-fluoride. For the sense strand complementary to the antisense strand, at the positions on the antisense strand where 2'-methoxy is used, the complementary positions on the sense strand are modified with 2'-fluoride; conversely, at the positions on the antisense strand where 2'-fluoride is used, the complementary positions on the sense strand are modified with 2'-methoxy.

[0195] 2. Synthesis of unmodified oligonucleotides

[0196] The sequence of small interfering RNA with sequence number 1583 in Table 1 is as follows:

[0197] Chain of Justice: 5'-CUUCUAAUGAGUCGACUUUGA-3' (SEQ ID NO.34)

[0198] Antonym: 5'-AAAGUCGACUCAUUAGAAGAA-3' (SEQ ID NO.82)

[0199] Instruments and reagents: The Qingke 192P model DNA / RNA automated synthesizer, whose solid support is a universal carrier of cross-linked polystyrene beads, model Primer support 5G Unylinker 350 (Cytiva manufacturer).

[0200] Preparation method:

[0201] The following nucleotide monomer solutions were prepared with acetonitrile at a monomer concentration of 0.15 M: DMT-A-2'-O-TBDMS phosphoramide monomer (Formula 10), DMT-C-2'-O-TBDMS phosphoramide monomer (Formula 11), DMT-G-2'-O-TBDMS phosphoramide monomer (Formula 12), and DMT-U-2'-O-TBDMS phosphoramide monomer (Formula 13).

[0202] Prepared using the following steps:

[0203] A solid support was loaded into the designated position of the synthesizer, and the corresponding fully protected product was obtained after several synthesis cycles. The synthesis cycle included (1) deprotection, (2) coupling, (3) oxidation / sulfidation and (4) hydroxyl protection. The cycle process and the reagents used are described below:

[0204] (1) Deprotection

[0205] The DMT protecting group was removed using a 3% dichloroacetic acid toluene solution as a deprotecting agent, followed by washing with acetonitrile.

[0206] (2) Coupling

[0207] The acetonitrile solutions of each nucleotide monomer were coupled using 0.25 M 5-ethylthiotetrazole as an activator, followed by rinsing with acetonitrile.

[0208] (3) Oxidation / Sulfidation

[0209] Oxidation: Oxidation was performed using a 0.05M iodine pyridine / water (90 / 10) solution as the oxidant, followed by rinsing with acetonitrile.

[0210] Vulcanization: Vulcanization was carried out using a pyridine solution of 3% hydroxanthin as a vulcanizing agent, followed by rinsing with acetonitrile.

[0211] (4) Hydroxyl protection

[0212] Hydroxyl protection was performed using a 10% acetic anhydride tetrahydrofuran solution (CAP A) tetrahydrofuran / pyridine / azirmethylimidazole 74 / 10 / 16 (v / v / v) (CAP B) as the hydroxyl protecting agent, followed by rinsing with acetonitrile.

[0213] Repeat the above operations in a cyclical manner according to the set sequence to obtain a fully protected product.

[0214] (5) Use 3% dichloroacetic acid toluene solution as a deprotection agent to remove the DMT protecting group of the last nucleotide, and then wash with acetonitrile.

[0215] (6) Ammonolysis and purification

[0216] The reacted solid support was transferred to a reactor, and concentrated ammonia (25-28%) was added. After maintaining ammonolysis at 60°C for 12 hours, the system was cooled to room temperature, and the mixture was transferred to a filter press. The mixture was washed with a mixture of purified water and ethanol, and the filtrates were combined, passed through a chromatography column, concentrated, and lyophilized to obtain the 2'-O-TBDMS protected product.

[0217] (7) Remove TBDMS

[0218] DMSO and triethylamine hydrofluoric acid were added to the obtained product, and the reaction was carried out at 60°C for 2 hours. Then, ammonium acetate aqueous solution was added to the reaction solution, and the mixture was shaken to mix. Anhydrous ethanol was added, and the mixture was shaken to mix. Crystallization was then carried out at -20°C for 8-12 hours. After centrifugation, the supernatant was discarded, and the precipitate was washed with anhydrous ethanol to obtain the unmodified single-chain product.

[0219] (8) Annealing

[0220] The obtained unmodified sense and antisense strands were mixed in a 1:1 ratio, heated to 95°C and held for 3 minutes, and then slowly cooled to room temperature to form unmodified double strands.

[0221] 3. Synthesis of other sequences

[0222] Synthesize the other sequences in Table 1 using the method described above.

[0223] Example 2: Inhibitory effect of unmodified oligonucleotides on AGT gene

[0224] The inhibitory activity of the 48 unmodified oligonucleotide sequences synthesized in Example 1 against the AGT gene was tested. The results showed that unmodified sequences 1583, 1812, 1063, and 740 significantly inhibited AGT mRNA expression in HepG2 cells. Sequence 1583 showed the highest inhibition rate at 64.50%, higher than the positive control; sequences 1812, 1063, and 740 also showed inhibition rates above 50%.

[0225] 1. Experimental Materials

[0226] Unmodified oligonucleotides: The unmodified oligonucleotide sequences listed in Table 1 were synthesized in Example 1.

[0227] Cell type: HepG2 cells (Cyagen, H1-1701)

[0228] Positive control: APC (Zilebesiran drug sequence from Alnilam) sense and antisense strands (SEQ ID NO. 47 and 95 in Table 1), synthesized in Example 1.

[0229] Drug solvent: sterile enzyme-free water, Gibco Opti-MEM

[0230] 2. Experimental Methods

[0231] The inhibition of AGT gene mRNA expression in the HepG2 cell line by the sample was detected by qRT-PCR.

[0232] 2.1 Cell Culture

[0233] Take passaged HepG2 cell lines and culture them logarithmically. Culture the logarithmically growing cells in 10% fetal bovine serum DMEM medium (supplemented with 100× penicillin-streptomycin 10 μL / mL, Gibco, 15070063) and place them in a 37℃ cell culture incubator containing 5% CO2. Change the medium once daily. Digest with 0.25% trypsin for passage, centrifuge at 800 rpm for 3 min, discard the supernatant, and add fresh culture medium for further passage.

[0234] 2.2 Cell transfection

[0235] Transfection mixture preparation: Mix Lipofectamine RNAiMAX (Thermo Fisher Scientific) and Opti-MEM at a volume ratio of 2:98 and vortex to mix.

[0236] Transfection reagent preparation: Add 60 μL of Opti-MEM diluted siRNA solution to 60 μL of transfection mixture at a 1:1 volume ratio, vortex to mix, and let stand at room temperature for 15 min to obtain lipid nanoparticles (LNPs). Take 12.5 μL for encapsulation efficiency detection. The encapsulation efficiency meets the transfection requirements.

[0237] Blank control group transfection reagent: Add 60 μL of the prepared transfection mixture to 60 μL of Opti-MEM. Vortex mix and let stand at room temperature for 15 min.

[0238] Add the prepared transfection reagent to each 24-well cell culture plate (100 μL per well) to achieve a final siRNA concentration of 0.07 nM per well. Add 500 μL of cell suspension (containing 1.5 × 10⁻⁶ cells per mL). 5 (1 cell). After mixing using the cross-hatching method, place in a 37°C, 5% CO2 cell culture incubator and incubate for 40 hours.

[0239] 2.3 AGT mRNA detection

[0240] 1) RNA extraction

[0241] a. Aspirate the culture medium from the 24-well plate, add 0.5 mL of 1×PBS to each well to wash the cells, and then aspirate the PBS. Add 0.5 mL of TRIzol reagent to each well, pipette the cells to lyse them thoroughly, and transfer them to a 1.5 mL RNase-free EP tube. Incubate at room temperature for 5 min.

[0242] b. Add 0.1 mL of chloroform to each tube, shake vigorously for 15 seconds, and let stand at room temperature for 5 minutes. Centrifuge at 4°C, 12,000 × g for 15 minutes, and transfer 200 μL of the supernatant to a new EP tube.

[0243] c. Add an equal volume of isopropanol, gently mix the liquid in the tube by inverting it, let it stand at -20℃ for 10 min, centrifuge at 4℃ for 12,000×g for 15 min, and discard the supernatant.

[0244] d. Add 0.5 mL of 75% ethanol, gently wash the RNA precipitate, centrifuge at 12,000 × g for 5 min at 4 °C, and aspirate the supernatant. Repeat the washing once, centrifuge at 12,000 × g for 1 min at 4 °C, and remove any residual ethanol with a micropipette tip.

[0245] e. Allow the residual ethanol to air dry at room temperature for 2-3 minutes, then dissolve it in 40 μL of RNase-free ddH2O.

[0246] 2) RNA concentration detection

[0247] RNA concentration was detected using nanodrop. A 2 μL RNase-free ddH2O solution was used as a blank control, and 2 μL of RNA sample was used for each test. Sample concentrations were recorded.

[0248] 3) AGT mRNA reverse transcription

[0249] The reverse transcription reagent (Takara, RR036A), RNA solution, and water were mixed in a volume ratio of 2:5:3 and reacted in a PCR instrument at 37°C for 15 min, 85°C for 5 s, and finally maintained at 4°C. The cDNA obtained after the reaction was diluted 5-fold with sterile, enzyme-free water.

[0250] 4) Quantitative detection of AGT mRNA

[0251] In a 15 mL centrifuge tube, add qPCR reagent (Takara, RR82WR), upstream primer (GAPDH-F: 5'-AGTATGACAACAGCCTCAAG-3' (SEQ ID NO: 193), AGT-F: 5'-ACTATCTCCCCGGACCATCC-3' (SEQ ID NO: 195)) and downstream primer (GAPDH-R: 5'-TCATGAGTCCTTCCACGATA-3' (SEQ ID NO: 194), AGT-R: 5'-CCTGATGCGGTCATTGCTCA-3' (SEQ ID NO: 196)) at a volume ratio of 5:0.1:0.1 and mix well. Label this solution A.

[0252] Label a 1.5 mL EP tube, add diluted cDNA and water at a volume ratio of 1:3.8 and mix well. Label this solution as solution B.

[0253] Add 5.2 μL of solution A and 4.8 μL of solution B to each well of a 96-well PCR plate. Cover with sealing film, centrifuge at 3000 rpm for 1 min, and then analyze using the PCR instrument.

[0254] *This step is performed on ice, maintaining low temperatures.

[0255] Place the plate into the qPCR instrument and run the following procedure:

[0256] Pre-denaturation: 95℃, 30 seconds;

[0257] Cyclic reaction: 95℃, 5 sec; 60℃, 34 sec; 40 cycles;

[0258] Melting curves: 95℃, 15sec; 60℃, 60sec; 95℃, 15sec.

[0259] The running time is approximately 2 hours. Analyze the experimental results and calculate 2. -ΔΔCt .

[0260] 2.4 Data Processing

[0261] The formula for calculating the AGT mRNA expression rate (%) is as follows:

[0262] Expression rate = (AGT mRNA expression level / AGT mRNA expression level in blank control group) × 100%;

[0263] The formula for calculating the AGT gene expression inhibition rate (%) is as follows:

[0264] AGT gene expression inhibition rate = 1 - expression rate (%).

[0265] 3. Experimental Results

[0266] The specific data on the inhibitory effect of the synthesized unmodified oligonucleotides on AGT gene expression are listed in Table 3 and Figure 1.

[0267] 1) Some unmodified sequences have a significant inhibitory effect on AGT gene expression, including 1583, 1812, 1063 and 740. Among them, the 1583 and 1812 groups have the strongest inhibitory effect, with inhibition rates of 64.50% and 61.04%, respectively.

[0268] Table 3. Inhibition rate of AGT gene expression by unmodified sequences.

[0269] As shown in Table 3, the unmodified sequences 1583, 1812, 1063, and 740 significantly inhibited the expression of AGT mRNA in HepG2 cells. The strongest inhibitory effects were observed in the 1583 and 1812 groups, with inhibition rates of 64.50% and 61.04%, respectively. The inhibition rate of 1583 (64.50%) was higher than that of Yangshen APC (60.65%). The inhibition rates of the other groups were also all above 50%.

[0270] These compounds all exhibit significant inhibitory effects on AGT gene expression, with inhibition rates exceeding 50%.

[0271] 2) Some unmodified sequences have an inhibitory effect on AGT gene expression between 0% and 50%, including double-stranded siRNAs such as 1615, 2034, 1267, 1384, 1221, 904, 1030, 1748, 1155, 760, 1876, 1296, 673, 994, 1642, 1912, 720, 830, 2010, 50, 422, 546, 622, 1323, 1096, 150, 1980, 128, 1494, 1934, 167, etc.

[0272] 4) Some sequences do not inhibit AGT gene expression, including double-stranded siRNAs such as 478, 289, 226, 490, 2059, 262, 1530, 198, 11, 341, and 1461. Among them, the inhibition rate of 1461 is less than -20%. These double-stranded siRNAs do not inhibit AGT gene expression.

[0273] Note: NC inhibited AGT gene expression by 0.73%.

[0274] The unmodified sequences 1583, 1812, 1063, 740, 1615, 2034, 1267, 1384 and 1221 designed in this disclosure can significantly inhibit the expression of the AGT gene (inhibition rate greater than 30%), thereby reducing the concentration of angiotensinogen in the blood, thus lowering blood pressure and achieving the treatment of hypertension.

[0275] Example 3: Effect of alternating modification strategy on oligonucleotide repression of AGT gene

[0276] This disclosure employs an alternating 2′-F and 2′-OMe modification scheme for the unmodified oligonucleotide sequence SEQ ID NO.1-98 designed in this disclosure, and examines the effect of this modification on the activity of the unmodified sequence.

[0277] Activity assay results showed that double-stranded siRNAs modified with alternating 2'-OMe and 2'-F sequences A1583-AL, A1812-AL, and A1063-AL all achieved inhibition rates of over 60% against the AGT gene; A740-AL and A1615-AL also showed high inhibition rates, exceeding 50%.

[0278] Furthermore, it was found that alternating 2'-F and 2'-OMe modifications had different effects on the activity of different sequences. Some sequences showed significantly improved inhibition rates, while others experienced decreased inhibition rates after modification, and still others had virtually no effect on inhibition rates. This indicates that the alternating 2'-OMe and 2'-F modification schemes have inconsistent effects on different sequences, and not all sequences can achieve improved inhibition after the same modification.

[0279] 1. Experimental Materials

[0280] Alternating modified oligonucleotides: The modified oligonucleotides listed in Table 3 are prepared using an alternating modification scheme of 2'-OMe and 2'-F. Specifically, the odd-numbered positions of the sense strand and the even-numbered positions of the antisense strand are modified with 2'-F, and the even-numbered positions of the sense strand and the odd-numbered positions of the antisense strand are modified with 2'-OMe, as synthesized in Example 1.

[0281] Cell type: HepG2 cells (Cyagen, H1-1701)

[0282] Positive control drug: Alternating modified APC, wherein the odd-numbered positions of the sense strand and the even-numbered positions of the antisense strand are both modified with 2'-F, and the even-numbered positions of the sense strand and the odd-numbered positions of the antisense strand are both modified with 2'-OMe, synthesized in Example 1.

[0283] Drug solvent: sterile enzyme-free water, Gibco DMEM.

[0284] 2. Experimental Methods

[0285] The experimental method is the same as in Example 2.

[0286] 3. Experimental Results

[0287] The specific data on the inhibitory effect of alternating modified oligonucleotides on AGT gene expression are shown in Table 4 and Figure 2.

[0288] 1) Five alternating modification sequences, excluding those from *Gynostemma pentaphyllum*, significantly inhibited AGT gene expression. These sequences included A1583-AL, A1812-AL, A1063-AL, and A740-AL. The inhibition rates of A1583-AL, A1812-AL, and A1063-AL all reached 60%. The inhibitory effect was enhanced compared to the unmodified sequences.

[0289] Table 4. Inhibition rate of AGT gene expression by alternating modification sequences.

[0290] As shown in Table 4, the alternating 2'-OMe and 2'-F modification sequences A1583-AL, A1812-AL, A1063-AL, and A740-AL significantly inhibited AGT gene expression, with inhibition rates all exceeding 50%. Among them, the inhibition rates of A1583-AL, A1812-AL, and A1063-AL all reached 60%.

[0291] Therefore, it can be seen that the alternating modification sequences A1583-AL, A1812-AL, A1063-AL and A740-AL can inhibit the expression of the AGT gene, thereby inhibiting the concentration of angiotensinogen in the blood, and can be used to treat hypertension.

[0292] Furthermore, the unmodified sequences corresponding to the aforementioned alternating modified sequences, namely 1583, 1812, 1063, and 740, also significantly inhibited AGT gene expression, with inhibition rates all exceeding 50%. This indicates that the sequences designed in this disclosure, whether unmodified or modified with alternating 2'-OMe and 2'-F, can inhibit AGT gene expression.

[0293] By comparing the inhibition rate with that of the unmodified sequence, it can be seen that alternating modification has different effects on the activity of different sequences, and the inhibition rate increased by modification ranges from 5.01% to 9.76%.

[0294] 2) Some alternating modification sequences have an AGT gene expression inhibition rate between 0% and 50%, including A1615-AL, A2034-AL, A1267-AL, A1384-AL, A1221-AL, A904-AL, A1030-AL, A1748-AL, A1155-AL, A760-AL, A1876-AL, A1296-AL, A673-AL, and A994-AL. Double-stranded siRNAs including L, A1642-AL, A1912-AL, A720-AL, A830-AL, A2010-AL, A50-AL, A422-AL, A546-AL, A622-AL, A1323-AL, A1096-AL, A150-AL, A1980-AL, A128-AL, A1494-AL, A1934AL, and A1675-AL were used. Comparison with unmodified sequences showed that alternating 2'-OMe and 2'-F modifications had different effects on the inhibition rate of different sequences, with alternating modifications increasing the inhibition rate by 2.54%–6.48%.

[0295] 3) Some alternating modification sequences do not inhibit AGT gene expression, including double-stranded siRNAs such as A478-AL, A289-AL, A226-AL, A490-AL, A2059-AL, A262-AL, A1530-AL, A198-AL, A11-AL, A341-AL, and A1461-AL. For example, A1461-AL showed an AGT inhibition rate of -26.60%. Comparison with unmodified sequences shows that alternating 2'-OMe and 2'-F modifications have different effects on the activity of different sequences, with the inhibition rate increasing by -5.99%–2.20% after alternating modification.

[0296] Note: NC inhibited AGT gene expression by -1.5%.

[0297] Therefore, the oligonucleotide sequences designed for AGT mRNA in this disclosure, after alternating modification with 2'-OMe and 2'-F, exhibit significant inhibitory effects on AGT gene expression. These include A1583-AL, A1812-AL, A1063-AL, A740-AL, A1615-AL, A2034-AL, A1267-AL, A1384-AL, and A1030-AL. Among them, A1583-AL, A1812-AL, and A1063-AL show the highest inhibition rates, all reaching 60%, and can be used to develop drugs for treating AGT gene-related diseases.

[0298] Furthermore, the alternating modification of 2'-OMe and 2'-F has inconsistent effects on the different unmodified oligonucleotide sequences designed in this disclosure. Some sequences show increased activity, some show decreased activity, and some show no change in activity. Not all sequences can have their inhibitory effect improved by alternating modification; there will be certain differences between different sequences.

[0299] Example 4: Comparison of suppression rates between the designed sequence of this disclosure and similar sequences disclosed in the prior art.

[0300] Ten sequences with high inhibitory effects on AGT gene expression in this disclosure were compared with similar sequences disclosed in the prior art. The results showed that the unmodified sequence 1812 in this disclosure had no similar disclosed sequences. Sequences 1583, 1063, 740, 1615, 2034, 1267, 1384, 1030, and 994, at the same concentration, showed superior inhibitory effects on AGT mRNA expression in HepG2 cells compared to similar sequences in published patents. For example, the inhibition rate of sequence 1583 in this disclosure was 13.57% higher than that of the similar sequence 1583P; the inhibition rate of sequence 1063 in this disclosure was 28.14% higher than that of the similar sequence 1063P; and the inhibition rate of sequence 740 in this disclosure was 12.51% higher than that of the similar sequence 740P.

[0301] 1. Experimental Materials

[0302] This disclosure includes sequences 1583, 1063, 740, 1615, 2034, 1267, 1384, 1030, and 994, as well as prior art sequences close to these sequences, including sequences 1583P, 1063P, 740P, 1615P, 2034P, 1267P, 1384P, 1030P, and 994P, as detailed in Table 5.

[0303] Table 5 compares the sequences in this disclosure with similar sequences disclosed in the prior art.

[0304] The sequences in Table 5 were synthesized using the same method as in Example 1.

[0305] 2. Experimental Methods

[0306] The inhibition rate of the oligonucleotides listed in Table 5 on the AGT gene was tested, and the specific experimental method was the same as in Example 2.

[0307] 3. Experimental Results

[0308] Table 6 shows the inhibitory activity of sequences similar to those disclosed in this disclosure on AGT gene expression.

[0309] Table 6. Comparison of the activity of the designed sequence in this disclosure with similar sequences disclosed in the prior art.

[0310] As shown in Table 6, the unmodified sequences 1583, 1063, 740, 1615, 2034, 1267, 1384, 1030, and 994 screened in this disclosure exhibit superior inhibitory effects on AGT gene expression compared to similar sequences in previously published patents at the same concentration. For example, the inhibition rate of sequence 1583 disclosed in this disclosure is 64.50%, while the inhibition rate of the similar sequence 1583P disclosed in WO2023014765A1 is only 50.93%, representing an increase of 13.57%. The inhibition rate of sequence 1063 disclosed in this disclosure is 55.34%, which is 28.14% higher than that of the similar sequence 1063P disclosed in CN106574268B. The inhibition rate of sequence 740 disclosed in this disclosure is 50.45%, which is 12.51% higher than that of the similar sequence 740P disclosed in WO2023014765A1.

[0311] Therefore, it can be seen that oligonucleotides with similar sequences do not necessarily have similar inhibition rates of AGT gene expression; rather, they can differ significantly. For example, compared to the previously disclosed sequence 1583P, sequence 1583 differs by one nucleotide at the 3' end of the sense strand and has two fewer nucleotides (AC) at the 5' end of the antisense strand, but its inhibition rate is increased by 13.57%. Similarly, sequence 1063, compared to the previously disclosed sequence 1063P, has the same sense strand but two fewer bases (AA) at the 5' end of the antisense strand, yet its inhibition rate is increased by 28.14%. Furthermore, sequence 740, compared to the previously disclosed sequence 740P, differs by one base at the 3' end of the sense strand and two fewer bases (AC) at the 5' end of the antisense strand, yet its inhibition rate is increased by 12.51%. Although these sequences in this disclosure are only slightly different from those in the prior art, they significantly enhance the inhibition rate of AGT gene expression.

[0312] This disclosure discloses the design of 46 small interfering RNA (siRNA) sequences targeting the AGT mRNA sequence, including 46 sense strands and 46 antisense strands, and these sequences were modified with alternating 2'-OMe and 2'-F modifications. By detecting the inhibitory activity of the unmodified and alternating modified sequences on AGT gene expression, some target sequences with significant inhibitory effects on AGT gene expression were screened.

[0313] 1. Among the 46 unmodified oligonucleotide sequences designed, the inhibitory activities on AGT mRNA expression varied significantly. Some sequences, including 1583, 1812, 1063, and 740, showed significant inhibitory effects on AGT gene expression. Sequence 1583 achieved an inhibition rate of 64.50%, higher than the positive control; 1812 also achieved an inhibition rate of 61.04%, and 1063 and 740 both exceeded 50%. Some sequences exhibited high activity, such as 1615, 2034, and 1267, ranging from 30% to 50%. Some sequences showed low activity, and some had no inhibitory effect.

[0314] Therefore, it can be seen that not all siRNA sequences designed for AGT mRNA sequences have inhibitory activity on AGT gene expression. Different sequences will have very large differences, and it takes a lot of creative work to screen out siRNA sequences that have a significant inhibitory effect on AGT gene expression.

[0315] 2. Forty-six designed siRNA sequences were modified with alternating 2'-F and 2'-Me sequences. Activity assays showed that different sequences, even with the same alternating modification, exhibited significantly different inhibitory activities on AGT gene expression. The alternating modification sequences A1583-AL, A1812-AL, A1063-AL, and A740-AL showed significant inhibitory effects on AGT gene expression, with inhibitory activities exceeding 50%. Other sequences showed activities below 50%.

[0316] Therefore, it can be seen that not all basic sequences have strong inhibitory activity against AGT gene expression after alternating modification with 2'-F and 2'-Me. Different sequences will have very large differences. Screening out siRNA basic sequences that can be enhanced or retained by alternating modification with 2'-F and 2'-Me to significantly inhibit AGT gene expression requires a lot of creative work.

[0317] 3. The 46 designed siRNA sequences, after alternating modification with 2'-F and 2'-Me, had different effects on the activity of different sequences.

[0318] 4. The unmodified sequences 1583, 1063, 740, 1615, 2034, 1267, 1384, 1030, and 994 screened in this disclosure exhibit superior inhibitory effects on AGT gene expression at the same concentration compared to similar sequences in previously published patents. For example, sequence 1583 of this disclosure showed an inhibition rate of 64.50%, while the inhibition rate of the similar sequence 1583P disclosed in WO2023014765A1 was only 50.93%, representing an increase of 13.57%; sequence 1063 of this disclosure showed an inhibition rate of 55.34%, which is 28.14% higher than the similar sequence 1063P disclosed in CN106574268B; and sequence 740 of this disclosure showed an inhibition rate of 50.45%, which is 12.51% higher than the similar sequence 740P disclosed in WO2023014765A1.

[0319] The results show that oligonucleotides with similar sequences do not necessarily have similar inhibition rates of AGT gene expression; rather, they can differ significantly. Although the sequences in this disclosure are only slightly different from those disclosed in the prior art, they significantly enhance the inhibition rate of AGT gene expression.

Claims

1. A siRNA comprising a sense strand and an antisense strand, characterized in that, The siRNA comprises any of the following oligonucleotide duplexes: 1) The sense chain has a sequence or a fragment thereof as shown in SEQ ID NO:34, or a modified sequence thereof, and the antisense chain has a sequence or a fragment thereof as shown in SEQ ID NO:82, or a modified sequence thereof; 2) The sense chain has a sequence or a fragment thereof as shown in SEQ ID NO:39, or a modified sequence thereof, and the antisense chain has a sequence or a fragment thereof as shown in SEQ ID NO:87, or a modified sequence thereof; 3) The sense chain has a sequence or a fragment thereof as shown in SEQ ID NO:23, or a modified sequence thereof, and the antisense chain has a sequence or a fragment thereof as shown in SEQ ID NO:71, or a modified sequence thereof; 4) The sense chain has a sequence or a fragment thereof as shown in SEQ ID NO:17, or a modified sequence thereof, and the antisense chain has a sequence or a fragment thereof as shown in SEQ ID NO:65, or a modified sequence thereof; 5) The sense chain has a sequence or a fragment thereof as shown in SEQ ID NO:35, or a modified sequence thereof, and the antisense chain has a sequence or a fragment thereof as shown in SEQ ID NO:83, or a modified sequence thereof; 6) The sense chain has a sequence or a fragment thereof as shown in SEQ ID NO:45, or a modified sequence thereof, and the antisense chain has a sequence or a fragment thereof as shown in SEQ ID NO:93, or a modified sequence thereof; 7) The sense chain has a sequence or a fragment thereof as shown in SEQ ID NO:27, or a modified sequence thereof, and the antisense chain has a sequence or a fragment thereof as shown in SEQ ID NO:75, or a modified sequence thereof; 8) The sense chain has a sequence or a fragment thereof as shown in SEQ ID NO:30, or a modified sequence thereof, and the antisense chain has a sequence or a fragment thereof as shown in SEQ ID NO:78, or a modified sequence thereof; 9) The sense chain has a sequence or a fragment thereof as shown in SEQ ID NO:22, or a modified sequence thereof, and the antisense chain has a sequence or a fragment thereof as shown in SEQ ID NO:70, or a modified sequence thereof; The modified sequence contains one or more modified nucleotides.

2. The siRNA as described in claim 1, characterized in that, The length of the positive chain is 19-27 nucleotides.

3. The siRNA as described in claim 2, characterized in that, The antisense strand is 19-27 nucleotides in length.

4. The siRNA as described in claim 1, characterized in that, The siRNA comprises any of the following oligonucleotide duplexes: 1) The sense chain has a sequence as shown in SEQ ID NO:34 or a modified sequence of SEQ ID NO:34, and the antisense chain has a sequence as shown in SEQ ID NO:82 or a modified sequence of SEQ ID NO:82; 2) The sense chain has a sequence as shown in SEQ ID NO:39 or a modified sequence of SEQ ID NO:39, and the antisense chain has a sequence as shown in SEQ ID NO:87 or a modified sequence of SEQ ID NO:87; 3) The sense chain has a sequence as shown in SEQ ID NO:23 or a modified sequence of SEQ ID NO:23, and the antisense chain has a sequence as shown in SEQ ID NO:71 or a modified sequence of SEQ ID NO:71; 4) The sense chain has a sequence as shown in SEQ ID NO:17 or a modified sequence of SEQ ID NO:17, and the antisense chain has a sequence as shown in SEQ ID NO:65 or a modified sequence of SEQ ID NO:65; 5) The sense chain has a sequence as shown in SEQ ID NO:35 or a modified sequence of SEQ ID NO:35, and the antisense chain has a sequence as shown in SEQ ID NO:83 or a modified sequence of SEQ ID NO:83; 6) The sense chain has a sequence as shown in SEQ ID NO:45 or a modified sequence of SEQ ID NO:45, and the antisense chain has a sequence as shown in SEQ ID NO:93 or a modified sequence of SEQ ID NO:93; 7) The sense chain has a sequence as shown in SEQ ID NO:27 or a modified sequence of SEQ ID NO:27, and the antisense chain has a sequence as shown in SEQ ID NO:75 or a modified sequence of SEQ ID NO:75; 8) The sense chain has a sequence as shown in SEQ ID NO:30 or a modified sequence of SEQ ID NO:30, and the antisense chain has a sequence as shown in SEQ ID NO:78 or a modified sequence of SEQ ID NO:78; 9) The sense chain has a sequence as shown in SEQ ID NO:22 or a modified sequence of SEQ ID NO:22, and the antisense chain has a sequence as shown in SEQ ID NO:70 or a modified sequence of SEQ ID NO:70; The modified sequence contains one or more modified nucleotides.

5. The siRNA as described in claim 1, characterized in that, The modified nucleotide includes a 2'-modification of the nucleotide ribose; and / or, the nucleotide monomers in the sense strand and the antisense strand are linked by a phosphodiester bond or a thiophosphate diester bond.

6. The siRNA as described in claim 5, characterized in that, The 2'-modification is selected from the following modifications: 2'-methoxy, 2'-O-methoxyethyl, 2'-fluorine, 2'-O-benzyl, and 2'-O-methyl-4-pyridine.

7. The siRNA as described in claim 6, characterized in that, The 2'-modification is 2'-methoxy and 2'-fluoro.

8. The siRNA as described in claim 7, characterized in that, The 2'-modification of the ribose of each nucleotide is an alternating combination of 2'-methoxy and 2'-fluoro; and / or, the nucleotide monomers in the sense strand and the antisense strand are linked by 3',5'-phosphodiester bonds.

9. The siRNA as described in claim 1, characterized in that, The siRNA comprises any of the following oligonucleotide duplexes: 1) The positive chain has the sequence shown in SEQ ID NO:34 and the negative chain has the sequence shown in SEQ ID NO:82; or, the positive chain has the sequence shown in SEQ ID NO:130 and the negative chain has the sequence shown in SEQ ID NO:178; 2) The sense chain has the sequence shown in SEQ ID NO:39 and the antisense chain has the sequence shown in SEQ ID NO:87; or, the sense chain has the sequence shown in SEQ ID NO:135 and the antisense chain has the sequence shown in SEQ ID NO:183; 3) The positive chain has the sequence shown in SEQ ID NO:23 and the negative chain has the sequence shown in SEQ ID NO:71; or, the positive chain has the sequence shown in SEQ ID NO:119 and the negative chain has the sequence shown in SEQ ID NO:167; 4) The positive chain has the sequence shown in SEQ ID NO:17 and the negative chain has the sequence shown in SEQ ID NO:65; or, the positive chain has the sequence shown in SEQ ID NO:113 and the negative chain has the sequence shown in SEQ ID NO:161; 5) The sense chain has the sequence shown in SEQ ID NO:35 and the antisense chain has the sequence shown in SEQ ID NO:83; or, the sense chain has the sequence shown in SEQ ID NO:131 and the antisense chain has the sequence shown in SEQ ID NO:179; 6) The sense chain has the sequence shown in SEQ ID NO:45 and the antisense chain has the sequence shown in SEQ ID NO:93; or, the sense chain has the sequence shown in SEQ ID NO:141 and the antisense chain has the sequence shown in SEQ ID NO:189; 7) The positive chain has the sequence shown in SEQ ID NO:27 and the negative chain has the sequence shown in SEQ ID NO:75; or, the positive chain has the sequence shown in SEQ ID NO:123 and the negative chain has the sequence shown in SEQ ID NO:171; 8) The sense chain has the sequence shown in SEQ ID NO:30 and the antisense chain has the sequence shown in SEQ ID NO:78; or, the sense chain has the sequence shown in SEQ ID NO:126 and the antisense chain has the sequence shown in SEQ ID NO:174; 9) The positive chain has a sequence as shown in SEQ ID NO:22 and the negative chain has a sequence as shown in SEQ ID NO:70; or, the positive chain has a sequence as shown in SEQ ID NO:118 and the negative chain has a sequence as shown in SEQ ID NO:

166.

10. The siRNA according to any one of claims 1-9, characterized in that, At least one nucleotide of the siRNA is conjugated to one or more target ligands.

11. The siRNA as described in claim 10, characterized in that, The targeting ligand comprises carbohydrates, amino sugars, cholesterol, peptides, or lipids.

12. The siRNA as described in claim 11, characterized in that, The targeting ligand contains the GalNAc moiety.

13. The siRNA as described in claim 12, characterized in that, The GalNac portion can be a monovalent GalNAc portion, a divalent GalNAc portion, a trivalent GalNAc portion, or a tetravalent GalNAc portion.

14. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises the siRNA as described in any one of claims 1-13 and a pharmaceutically acceptable carrier.

15. The pharmaceutical composition according to claim 14, characterized in that, The pharmaceutically acceptable carrier is LNP.

16. The pharmaceutical composition of claim 15, characterized in that, The LNP is RNAiMAX contains 2,3-dioleoyloxy-N-[2-(argininosyl)ethyl]-N,N-dimethyl-1-perfluorooctyl trifluoroacetate and dioleoylphosphatidylethanolamine.

17. The pharmaceutical composition of claim 14, characterized in that, The pharmaceutical composition further includes one or both of AGT gene inhibitors and AGT protein inhibitors, wherein the AGT protein inhibitor includes an antibody targeting the AGT protein, and the AGT gene inhibitor includes one or more of a clipping editor targeting the AGT gene, an antisense oligonucleotide targeting the mRNA transcribed from the AGT gene, and microRNA.

18. The pharmaceutical composition of claim 14, characterized in that, The pharmaceutical composition further includes a drug for treating hypertension, which includes one or more of the following: diuretics, angiotensin-converting enzyme inhibitors, angiotensin II receptor blockers, calcium channel blockers, and beta-blockers.

19. A medicine box set, characterized in that, The kit includes a box A, which comprises one or both of the siRNA as described in any one of claims 1-13 or the pharmaceutical composition as described in any one of claims 14-18.

20. The medicine box as described in claim 19, characterized in that, The kit also includes a medicine box B, which contains one or both of the following (1) and (2): (1) AGT gene inhibitors or AGT protein inhibitors; (2) Any one or a combination of two or more of the following groups: hormone preparations, targeted small molecule preparations, proteasome inhibitors, imaging agents, diagnostic agents, chemotherapeutic agents, oncolytic drugs, cytotoxic agents, cytokines, activators of co-stimulatory molecules, inhibitors of inhibitory molecules, and vaccines.

21. The medicine box as described in claim 20, characterized in that, The AGT protein inhibitor includes an antibody that targets the AGT protein, and the AGT gene inhibitor includes one or more of a clipping editor that targets the AGT gene, an antisense oligonucleotide that targets the mRNA transcribed from the AGT gene, and microRNA.

22. The use of one or more of the siRNA as described in any one of claims 1-13, the pharmaceutical composition as described in any one of claims 14-18, and the kit as described in any one of claims 19-21 in the preparation of a treatment for and / or prevention of diseases related to AGT gene expression.

23. The application as described in claim 22, characterized in that, The diseases related to AGT gene expression include hypertension or diseases caused by hypertension.

24. A method for reducing AGT gene expression or inhibiting AGT replication, characterized in that, The method includes administering to a sample one or more of the following: siRNA as described in any one of claims 1-13, a pharmaceutical composition as described in any one of claims 14-18, and a kit as described in any one of claims 19-21.

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