Dsrna for simultaneously inhibiting expression of PCSK9 and AGT target genes and use thereof

Abstract

Provided are a double-stranded ribonucleic acid (dsRNA) agent that is capable of simultaneously reducing the expression of PCSK9 and AGT target genes and is used for PCSK9 and AGT-related diseases and conditions, and a use thereof.

Description

A dsRNA that simultaneously inhibits the expression of PCSK9 and AGT target genes and its applications

[0001] Cross-references to related applications

[0002] This application claims priority to Chinese patent application No. 2024116584266, filed on November 20, 2024, the entire contents of which are incorporated herein by reference. Technical Field

[0003] This disclosure belongs to the field of biochemistry and provides a double-stranded RNA (dsRNA) agent that simultaneously inhibits the expression of PCSK9 and AGT target genes and is used to treat diseases and conditions related to PCSK9 and AGT gene expression. Background Technology

[0004] Cardiovascular disease (CVD) is the leading chronic non-communicable disease threatening human health worldwide. It is projected that by 2050, the number of CVD patients globally will rise to 1.14 billion, potentially leading to 35.6 million deaths (European Journal of Preventive Cardiology (2024) 00, 1–15). CVD, primarily atherosclerotic cardiovascular disease (ASCVD) (such as ischemic heart disease and ischemic stroke), is the leading cause of death among urban and rural residents in my country, accounting for over 40% of all deaths.

[0005] Epidemiological, genetic, and clinical intervention studies have provided ample evidence that low-density lipoprotein cholesterol (LDL-C) is a key risk factor for ASCVD, and most national or regional lipid management guidelines recommend LDL-C as the primary target for lipid-lowering therapy. Statins are currently the first-line drugs for treating elevated LDL-C levels, but they have several shortcomings in clinical application: some patients experience adverse reactions, LDL-C reduction is not fully achieved, and Lp(a) levels may increase. PCSK9 inhibitors reduce LDL-C by approximately 50%–70% (Chinese Lipid Management Guidelines, 2023), with both monoclonal antibodies and siRNAs already approved for marketing. In particular, PCSK9 monoclonal antibodies are recommended as a preferred drug for controlling LDL-C in guidelines from multiple countries.

[0006] A close relationship exists between blood pressure levels and the risk of morbidity and mortality from cardiovascular disease (CVD). A global observational analysis of 61 populations (1 million individuals, aged 40-89 years) (Lancet. 2002 Dec 14; 360(9349):1903-13) found a continuous, independent, and direct positive correlation between blood pressure and the risk of stroke, coronary events, and cardiovascular disease mortality. For every 20 mmHg increase in systolic blood pressure or every 10 mmHg increase in diastolic blood pressure, the risk of CVD doubled. In China, approximately two-thirds of patients with hyperlipidemia also have hypertension, and 27.9% of patients with hypertension also have hyperlipidemia. Clinical statistical analysis shows that simultaneously lowering blood pressure and LDL-C is more effective than single-factor reduction in reducing cardiovascular events (N Engl J Med 2016; 374:2032-43).

[0007] Angiotensinogen (AGT) is a crucial component of the renin-angiotensin system (RAS) in the human body, and the entire RAS plays a key role in the regulation of blood pressure in animals. AGT is synthesized and secreted in the liver, where it is converted into angiotensin I by the enzyme renin (REN), and then into angiotensin II by angiotensin-converting enzyme (ACE). Most angiotensin II binds to type I angiotensin II receptors, leading to vasoconstriction and increased blood pressure. This molecule also stimulates the production of the hormone alpha-1, which triggers renal reabsorption of Na+ ions and water, resulting in increased blood volume and further elevated blood pressure. Existing clinical data indicate that reducing hepatic AGT secretion levels using double-stranded RNA (dsRNA) agents can effectively control blood pressure.

[0008] This application provides a double-stranded ribonucleic acid (dsRNA) agent that simultaneously targets PCSK9 and AGT target genes. It can specifically bind to PCSK9 and AGT mRNA, disrupt the normal translation template function of PCSK9 and AGT mRNA, thereby preventing the translation of PCSK9 and AGT proteins, inhibiting the related protein pathways at the source, and can be used to treat / prevent related diseases caused by the related protein pathways.

[0009] In summary, PCSK9 and AGT are associated with lipid metabolism disorders and cardiovascular diseases. However, traditional drugs and single-target siRNA drugs suffer from high dosing frequency and low patient compliance. Therefore, developing more candidate double-stranded RNAs (dsRNAs) that are stable in the blood, possess good biological activity, have low cytotoxicity, and can effectively inhibit the expression of dual-target genes (PCSK9 and AGT) is an urgent problem to be solved. Furthermore, developing drugs using these candidate dsRNAs that inhibit the expression of dual-target genes to effectively prevent and / or treat hyperlipidemia-related diseases and hypertension-related diseases is both necessary for clinical research and a realistic possibility for commercialization. Summary of the Invention

[0010] This disclosure provides a double-stranded ribonucleic acid (dsRNA) agent that simultaneously inhibits the expression of PCSK9 and AGT target genes, and has the following characteristics:

[0011] a. A first nucleic acid single strand and a second nucleic acid single strand that are partially or completely complementary; the two nucleic acid single strands are capable of forming a stable double-stranded structure; the first nucleic acid single strand and the second nucleic acid single strand are 30-80 nt in length, each containing a region that is at least partially complementary to the PCSK9 mRNA or AGT mRNA sequence, and each containing at least two unmodified RNA nucleotides or DNA nucleotides.

[0012] b. Each nucleic acid single strand is conjugated with at least one carrier group containing an ASGPR ligand, wherein the carrier group of the ASGPR ligand is conjugated to each nucleic acid single strand; each nucleic acid single strand is represented by formula (I):

[0013] Each single strand of nucleic acid is represented by equation (I):

[0014] In this context, each N is an independent unmodified or chemically modified nucleotide; each L is an independent carrier group, either present or absent; (N) represents a series of unmodified nucleotides; and the sum of X1, X2, X3, X4 and X5 is an integer from 30 to 80.

[0015] In some preferred embodiments of this disclosure, at least one of the first and second nucleic acid single strands has a structure as shown in formula (II):

[0016] 5'N X2 —(N) X3 —N X4 —L 3' (II).

[0017] In some preferred embodiments of this disclosure, both the first and second nucleic acid single strands have the structure shown in formula (II).

[0018] In some preferred embodiments of this disclosure, the first or second nucleic acid single strand has a structure as shown in formula (III):

[0019] 5'N X2 —(N) X3 —N X4 3' (III).

[0020] In some preferred embodiments of this disclosure, the first nucleic acid single strand has a structure as shown in Formula (II); and the second nucleic acid single strand has a structure as shown in Formula (III).

[0021] In some preferred embodiments of this disclosure, the first nucleic acid single strand has a structure as shown in Formula (III); and the second nucleic acid single strand has a structure as shown in Formula (II).

[0022] In some specific embodiments of this disclosure, the dsRNA can form a stable Bubble structure at a position of 15-30 nt at the 5' end of nucleic acid single strand 1 or nucleic acid single strand 2; the Bubble structure contains at least two unmodified RNA nucleotides or DNA nucleotides; the Bubble structure can be recognized and cleaved by nucleases in vitro or in vivo, cleaving into two independent siRNA conjugates, and the two cleaved siRNA conjugates can independently inhibit the target mRNAs of PCSK9 and AGT, respectively, through the RNAi-induced silencing complex process.

[0023] Furthermore, the nucleic acid conjugate can form 0, 1, or more Bubble structures at the 15-30 nt position of the 5' end of nucleic acid single strand 1 or nucleic acid single strand 2.

[0024] In some embodiments of this disclosure, X1 in the nucleic acid conjugate is an integer from 14 to 29; the nucleic acid conjugate can form 0, 1, or more Bubble structures at the (N) position.

[0025] In some embodiments of this disclosure, the region at least partially complementary to the mRNA sequence of the target gene is located at the (N) position or on the 5′ side of the Bubble structure.

[0026] Furthermore, the position of (N) or the Bubble structure contains at least two unmodified RNA or DNA nucleotides.

[0027] In some preferred embodiments of this disclosure, the position of (N) or the Bubble structure comprises unmodified RNA nucleotides (rN) or DNA nucleotides (dN).

[0028] In some embodiments of this disclosure, the carrier group containing the ASGPR ligand has, but is not limited to, the following structural fragments:

[0029] In some preferred embodiments of this disclosure, the carrier group containing the ASGPR ligand is GalNAc (L96) or Ser (GN), or GalNAc (Ser1), GalNAc (Ser2), GalNAc (Ser3), GalNAc (Ser4), or LP-GalNAc, XY-GalNAc, GalNAc (A1GN), GalNAc (A1dGN), GalNAc (A3GN), GalNAc (A3dGN), GalNAc (A5GN), GAlNAc (NAG25), GAlNAc (NAG37).

[0030] GalNAc(L96) has the following structure:

[0031] Ser(GN) can have the following structure:

[0032] GalNAc(Ser1), GalNAc(Ser2), GalNAc(Ser3), and GalNAc(Ser4) have the following structures:

[0033] LP-GalNAc has the following structure:

[0034] XY-GalNAc has the following structure:

[0035] GalNAc(A1GN), GalNAc(A1dGN), GalNAc(A3GN), GalNAc(A3dGN), GalNAc(A5GN), GAlNAc(NAG25), and GAlNAc(NAG37) have the following structures:

[0036] In some specific embodiments of this disclosure, the two single strands of the dsRNA agent contain at least one modified nucleotide.

[0037] In some specific embodiments of this disclosure, at least 90% of the nucleotides in the two single strands of the dsRNA agent are modified nucleotides.

[0038] In some specific embodiments of this disclosure, the modified nucleotides include: threonine, 2'-O-methyl nucleotide, 2'-fluoronucleotide, 2'-deoxynucleotide, 2'3'-seco nucleotide mimic, locked nucleotide, unlocked nucleic acid nucleotide (UNA), glycol nucleic acid nucleotide (GNA), 2'-F-arabinonucleotide, 2'-methoxyethyl nucleotide, baseless nucleotide, ribitol, reverse nucleotide, reverse baseless nucleotide, reverse 2'-OMe nucleotide, reverse 2'-deoxynucleotide, 2'-amino-modified nucleotide, 2'-alkyl-modified nucleotide, morpholinonucleotide, and 3'-OMe nucleotide, 2'-O-C22H45 nucleotide, nucleotide containing a 5'-thiophosphate group, 2'-amino-modified nucleotide, aminophosphate, or nucleotide containing a non-natural base.

[0039] In some specific embodiments of this disclosure, each single strand of the dsRNA agent contains at least one phosphate thioester nucleoside linker.

[0040] In some specific embodiments of this disclosure, the two single strands of the dsRNA agent have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 thiophosphate groups linked at the 5' end, 3' end, or in the middle.

[0041] In some specific embodiments of this disclosure, the dsRNA agent contains a phosphate ester or phosphate ester mimicry-modified nucleotide at the 5' end of each single strand.

[0042] In some specific embodiments of this disclosure, the phosphate mimic at the 5' end of each single strand of the dsRNA agent is a nucleotide modified with 5'-vinyl phosphate; further, the phosphate mimic is a nucleotide modified with 5'-(E)-vinyl phosphate.

[0043] In some specific embodiments of this disclosure, the phosphate ester, phosphate analog, or 5'-vinyl phosphate modified nucleotide has the following structure:

[0044] Wherein, Base is a base A, U, G, C, T or other modified bases.

[0045] In some specific embodiments of this disclosure, the dsRNA agent has the nucleic acid conjugate structure shown in Table 1.

[0046] In some specific embodiments of this disclosure, the dsRNA agent is selected from nucleic acid conjugates 001 to 138 in Table 2.

[0047] In some preferred embodiments of this disclosure, the dsRNA agent is a nucleic acid conjugate 045, 133, 134, 135, 136, 137, or 138. In another aspect, this disclosure also provides a pharmaceutical composition for inhibiting the expression of PCSK9 and AGT target genes, the pharmaceutical composition comprising the aforementioned dsRNA agent and a pharmaceutically acceptable carrier.

[0048] In another aspect, this disclosure also provides the use of the aforementioned dsRNA agents or pharmaceutical compositions for the treatment and / or prevention of pathological conditions or diseases associated with PCSK9 and / or AGT gene expression.

[0049] In another aspect, this disclosure also provides the use of the aforementioned dsRNA agents or pharmaceutical compositions in the preparation of medicaments for treating pathological conditions or diseases associated with PCSK9 and / or AGT expression.

[0050] In another aspect, this disclosure also provides a dsRNA agent or pharmaceutical composition for treating pathological conditions or diseases associated with PCSK9 and / or AGT expression.

[0051] In another aspect, this disclosure also provides a method for treating pathological conditions or diseases related to PCSK9 and / or AGT expression. The method includes administering the aforementioned dsRNA agent or pharmaceutical composition to a subject in need.

[0052] Further, the disease or condition is one or more of the following: hyperlipidemia, nonfamilial hypercholesterolemia, polygenic hypercholesterolemia, familial hypercholesterolemia, homozygous familial hypercholesterolemia and heterozygous familial hypercholesterolemia, coronary heart disease, myocardial infarction, stroke and atherosclerosis, borderline hypertension, essential hypertension, secondary hypertension, isolated systolic or diastolic hypertension, pregnancy-related hypertension, diabetic hypertension, refractory hypertension, intractable hypertension, paroxysmal hypertension, renovascular hypertension, Goldblatt hypertension, hypertension related to low plasma renin activity or plasma renin concentration, ocular hypertension, glaucoma, pulmonary arterial hypertension. Hypertension, portal hypertension, systemic venous hypertension, systolic hypertension, unstable hypertension, hypertensive heart disease, hypertensive nephropathy, atherosclerosis, arteriosclerosis, vascular disease, diabetic nephropathy, diabetic retinopathy, chronic heart failure, cardiomyopathy, diabetic cardiomyopathy, nocturnal hypertension, glomerulosclerosis, aortic coarctation, aortic aneurysm, ventricular fibrosis, heart failure, myocardial infarction, angina pectoris, stroke, kidney disease, kidney failure, systemic sclerosis, intrauterine growth retardation, fetal growth restriction, obesity, hepatic steatosis / fatty liver, non-alcoholic steatohepatitis, non-alcoholic fatty liver disease, glucose intolerance, type 2 diabetes, and metabolic syndrome, etc.

[0053] In some embodiments of this disclosure, the dsRNA agent or pharmaceutical composition is administered by injection or subcutaneous administration.

[0054] In some preferred embodiments of this disclosure, the dsRNA agent or pharmaceutical composition is administered via subcutaneous injection.

[0055] In some embodiments of this disclosure, the dsRNA agent or pharmaceutical composition is administered together with excipients, including buffered or non-buffered solutions.

[0056] In some preferred embodiments of this disclosure, the non-buffered solution is saline or water, or the buffered solution comprises acetate, citrate, alcohol-soluble gluten, carbonate, or phosphate or phosphate-buffered saline, or any combination thereof.

[0057] In some preferred embodiments of this disclosure, the object of need is a person.

[0058] In some preferred embodiments of this disclosure, the dsRNA agent or pharmaceutical composition is administered at a dose of 1-100 mg / kg.

[0059] Sequence Description

[0060] In some embodiments of this disclosure, the dsRNA agent that simultaneously inhibits the expression of PCSK9 and AGT genes is composed of complementary pairings of nucleic acid single strand 1 and nucleic acid single strand 2, wherein the nucleotide sequence of the first or second nucleic acid single strand is selected from the unmodified nucleotide sequences shown in Table 1a.

[0061] Table 1a. Unmodified nucleotide sequences

[0062] In some embodiments of this disclosure, the first or second nucleic acid single strand comprises a nucleotide sequence as shown in any of SEQ ID NOs:1000-1034.

[0063] In some embodiments of this disclosure, the nucleotide sequence of the first or second nucleic acid single strand is selected from the following combinations:

[0064] (1) SEQ ID NO: 1000 and 1001;

[0065] (2) SEQ ID NO: 1002 and 1001;

[0066] (3) SEQ ID NO: 1003 and 1004;

[0067] (4) SEQ ID NO: 1003 and 1005;

[0068] (5) SEQ ID NO: 1006 and 1005;

[0069] (6) SEQ ID NO: 1000 and 1007;

[0070] (7) SEQ ID NO: 1008 and 1009;

[0071] (8) SEQ ID NO: 1010 and 1005;

[0072] (9) SEQ ID NO: 1011 and 1012;

[0073] (10) SEQ ID NO: 1013 and 1012;

[0074] (11) SEQ ID NO: 1014 and 1015;

[0075] (12) SEQ ID NO: 1014 and 1016;

[0076] (13) SEQ ID NO: 1017 and 1016;

[0077] (14) SEQ ID NO: 1011 and 1018;

[0078] (15) SEQ ID NO: 1019 and 1020;

[0079] (16) SEQ ID NO: 1021 and 1016;

[0080] (17) SEQ ID NO: 1022 and 1023;

[0081] (18) SEQ ID NO: 1024 and 1023;

[0082] (19) SEQ ID NO: 1025 and 1026;

[0083] (20)SEQ ID NO: 1025 and 1027;

[0084] (21) SEQ ID NO: 1028 and 1027;

[0085] (22) SEQ ID NO: 1022 and 1029;

[0086] (23) SEQ ID NO: 1030 and 1031;

[0087] (24) SEQ ID NO: 1032 and 1027;

[0088] (25)SEQ ID NO:1033 and 1034.

[0089] In some embodiments of this disclosure, the first or second nucleic acid single strand may be chemically modified as follows:

[0090] Among them, B1, B2, B3, B4, B5, and B6 are each independently nucleotides modified by 2'-OMe, 2'-F, LNA, and 2'-OMOE, respectively;

[0091] T1, T2, T3, and T4 are each independently nucleotides modified with 2'-F, 2'-OMe, or DNA.

[0092] Z1, Z2, and Z3 are each independently nucleotides of 2'-F, 2'-OMe, 2'-DNA, and RNA (2'-OH modified);

[0093] q 1 ~q 13 Each is an integer between 0 and 10, independent of the others.

[0094] This disclosed nucleic acid double-stranded conjugate incorporates at least two specific nucleotide bases (unmodified DNA or RNA) into each nucleic acid single strand to form a stable Bubble structure. This Bubble structure can be specifically recognized by endonucleases and cleaved in vivo into two independent siRNA conjugate sites, respectively inhibiting two target genes through an RNAi-induced silencing complex process. At the sites specifically recognized and cleaved by the endonucleases, this nucleic acid double-stranded conjugate can form zero, one, or more Bubble structures.

[0095] In some preferred embodiments of this disclosure, the nucleotide sequence of the Bubble structure is selected from dArU, dTrCdT, or dAdTrUdT.

[0096] In some embodiments of this disclosure, the nucleotide sequence of the Bubble structure on the first nucleic acid single strand is dTrCdT, and the nucleotide sequence of the Bubble structure on the second nucleic acid single strand is dTrCdT.

[0097] In some embodiments of this disclosure, the nucleotide sequence of the Bubble structure on the first nucleic acid single strand is dArU, and the nucleotide sequence of the Bubble structure on the second nucleic acid single strand is dTrCdT.

[0098] In some embodiments of this disclosure, the nucleotide sequence of the Bubble structure on the first nucleic acid single strand is dArU, and the nucleotide sequence of the Bubble structure on the second nucleic acid single strand is dAdTrUdT.

[0099] In some embodiments of this disclosure, the first nucleic acid single strand comprises the modified nucleotide sequence of nucleic acid single strand 1 of the modified double strands 1 to 138 in Table 1b.

[0100] In some embodiments of this disclosure, the second nucleic acid single strand comprises the modified nucleotide sequence of the nucleic acid single strand 2 of the modified double strands 1 to 138 in Table 1b.

[0101] In some embodiments of this disclosure, the first or second nucleic acid single strand further comprises a 5′-(E)-vinyl phosphate modified nucleotide at its 5′ end.

[0102] In some preferred embodiments of this disclosure, the first nucleic acid single strand comprises the modified nucleotide sequence of nucleic acid single strand 1 of the modified double strands 045, 133, 134, 135, 136, 137, and 138 in Table 1b.

[0103] In some embodiments of this disclosure, the second nucleic acid single strand comprises the modified nucleotide sequence of nucleic acid single strand 2 of the modified double strands 045, 133, 134, 135, 136, 137, and 138 in Table 1b.

[0104] In some embodiments of this disclosure, the nucleotide sequence combination of the first or second nucleic acid single strand is selected from:

[0105] (1) Nucleotide sequences of single strand 1 and single strand 2 of nucleic acid modified by double strand 045 in Table 1b;

[0106] (2) Nucleotide sequences of single strand 1 and single strand 2 of nucleic acid modified by double strand 133 in Table 1b;

[0107] (3) Nucleotide sequences of single strand 1 and single strand 2 of nucleic acid that modify double strand 134 in Table 1b;

[0108] (4) Nucleotide sequences of single strand 1 and single strand 2 of nucleic acid modified by double strand 135 in Table 1b;

[0109] (5) Nucleotide sequences of single strand 1 and single strand 2 of nucleic acid modified by double strand 136 in Table 1b;

[0110] (6) Nucleotide sequences of single strand 1 and single strand 2 of nucleic acid modified by double strand 137 in Table 1b;

[0111] (7) The nucleotide sequences of single strand 1 and single strand 2 of the nucleic acid modified by double strand 138 in Table 1b.

[0112] In some embodiments of this disclosure, the modified duplexes of the dsRNA agent that simultaneously inhibits PCSK9 and AGT gene expression are shown in Table 1b:

[0113] Table 1b. Modified duplexes with PCSK9 and AGT target mRNA inhibitory effects

[0114] In some specific embodiments of this disclosure, the dsRNA agent that simultaneously inhibits the expression of PCSK9 and AGT genes has the molecular structure shown in Table 2, wherein each double-stranded ribonucleic acid is composed of complementary pairings of nucleic acid single strand 1 and nucleic acid single strand 2:

[0115] The modification schemes used in Tables 1b and 2 are as follows:

[0116] In this context, uppercase letters A, C, G, U, I, T, and 5mC represent adenosine-3'-phosphate, cytidine-3'-phosphate, guanosine-3'-phosphate, uridine-3'-phosphate, inosine-3'-phosphate, 5-methyluracil-3'-phosphate, and 5-methylcytosine-3'-phosphate, respectively; lowercase letter m indicates that the nucleotide adjacent to its right is 2'-methoxy modified; lowercase letter f indicates that the nucleotide adjacent to its right is 2'-fluoro modified; lowercase letter d represents that the nucleotide adjacent to its right is DNA; lowercase letter r represents that the nucleotide adjacent to its right is 2'-hydroxy; the two nucleotides adjacent to each other by an asterisk (*) are linked by a thiophosphate group; lowercase letter gn indicates that the nucleotide adjacent to its right is glycerol nucleotide (GNA); lowercase letter moe indicates that the nucleotide adjacent to its right is 2'-O-MOE modified; and eVP represents 5'-(E)-vinyl phosphate modification.

[0117] Table 2. Double-stranded ribonucleic acid conjugates with PCSK9 and AGT target mRNA inhibitory effects

[0118] The disclosed dsRNA exhibits good stability, excellent dual-target gene inhibitory activity, low off-target risk, and satisfactory cytotoxicity and immunogenicity in the process of inhibiting PCSK9 and AGT target genes.

[0119] Regarding the definition of terms used in this disclosure: unless otherwise stated, the initial definitions provided herein for groups or terms apply to the group or term throughout the specification; for terms not specifically defined herein, the meanings that a person skilled in the art would give them should be given in light of the disclosure and context.

[0120] In this disclosure, all scopes disclosed should be considered as disclosures of all sub-scopes and all point values ​​within the scope. For example, the disclosure of 1-100 should be considered as disclosure of the scopes 1-20, 20-30, etc., as well as the point values ​​20, 30, 40, 50, 60, 70, 80, 90, and 100.

[0121] In this disclosure, "dsRNA" refers to a double-stranded ribonucleic acid molecule having a double helix structure comprising two antiparallel and substantially complementary nucleic acid strands (nucleic acid single strand 1 and nucleic acid single strand 2, each single strand having a length of 30-80 nucleotides). This dsRNA can be recognized and cleaved into siRNA by nucleases in vitro or in vivo. The cleaved siRNA induces the degradation of target RNA (e.g., mRNA) through a post-transcriptional gene silencing mechanism known herein as RNA interference or RNAi.

[0122] In this disclosure, "nucleic acid drug" refers to an RNA or RNA-like (e.g., chemically modified RNA) oligonucleotide molecule that can reduce or inhibit the translation of messenger RNA (mRNA) in a sequence-specific manner.

[0123] Nucleic acid molecules can exert their effects through RNA interference mechanisms (e.g., by interacting with the mRNA interference pathway mechanism (RNA-induced silencing complex RISC) in mammalian cells) or any other mechanism or pathway. While the term "nucleic acid drug" as used herein is considered to primarily exert its effects through RNA interference mechanisms, the nucleic acid drug is not limited to or restricted to any particular pathway or mechanism of action. Types of nucleic acid drug molecules include, but are not limited to, single-stranded antisense oligonucleotides, short interfering RNA (siRNA), double-stranded RNA (dsRNA), microRNA (miRNA), short hairpin RNA (shRNA), and dicer enzyme substrates. The nucleic acid drugs described in this disclosure consist of oligonucleotide chains having at least a partial complementarity to the mRNA that serves as the target. In some embodiments, the nucleic acid drug or conjugate described in this disclosure is double-stranded and consists of a single nucleic acid strand 1 and a single nucleic acid strand 2 that is at least partially complementary to the single nucleic acid strand 1.

[0124] The terms “silence,” “reduction,” “inhibition,” “downregulation,” or “knockdown” refer to a reduction or decrease in the expression level of a given gene when it is administered directly into cells, tissues, organs, or animals treated with the nucleic acid drug molecules described in this disclosure, compared to administration into cells, tissues, organs, or animals that have not been so treated.

[0125] "Inhibition of PCSK9 expression" and "Inhibition of AGT expression" include inhibition of the PCSK9 and AGT genes at any level, such as at least partially stopping the expression of the PCSK9 and AGT genes, for example, inhibiting at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%.

[0126] The term "sequence" or "nucleotide sequence" refers to the order or sequence of nucleobases or nucleotides, expressed alphabetically using standard nucleotide nomenclature.

[0127] The term "complementarity," used to describe the relationship between a first nucleotide sequence (e.g., the sense strand of a nucleic acid drug or target mRNA) and a second nucleotide sequence (e.g., a single-stranded antisense oligonucleotide or a double-stranded antisense strand of a nucleic acid drug), refers to the ability of an oligonucleotide or oligonucleotide containing the first nucleotide sequence to hybridize with an oligonucleotide or oligonucleotide containing the second nucleotide sequence under certain conditions (under mammalian physiological conditions or similar in vitro conditions) to form base pairs and a double-stranded or double-helical structure. A complementary sequence contains either a Watson-Crick base pair or a non-Watson-Crick base pair and contains native or modified nucleotides or nucleotide analogs to a degree sufficient to satisfy the hybridization requirements described above. For example, for the purpose of determining identity or complementarity, monomers mA and fA are complementary to U (or T) and equivalent to A.

[0128] In this disclosure, "complete complementarity" means that in the hybridized nucleobase or nucleotide sequence pair, all (100%) bases of the sequential sequence of the first oligonucleotide will hybridize with the same number of bases of the sequential sequence of the second oligonucleotide. The sequential sequence may comprise all or part of the first or second nucleotide sequence.

[0129] In this disclosure, "partial complementarity" means that in the hybridized nucleobase or nucleotide sequence pair, at least 70% (but not all) of the bases in the sequential sequence of the first oligonucleotide will hybridize with the same number of bases in the sequential sequence of the second oligonucleotide. The sequential sequence may comprise all or part of the first or second nucleotide sequence.

[0130] In this disclosure, the terms “complementary,” “fully complementary,” and “partially complementary” are used to refer to the nucleobase or nucleotide matching between nucleic acid single strand 1 and nucleic acid single strand 2 of the dsRNA agent, or between the antisense strand of the RNAi agent and the sequence of the target mRNA.

[0131] In this disclosure, "unmodified RNA" is ribonucleotide (2'-hydroxynucleotide); "unmodified DNA" is deoxyribonucleotide (2'-deoxynucleotide).

[0132] In this disclosure, "Bubble structure" refers to the phenomenon where, during the annealing and pairing process of two single-stranded nucleic acids, due to incomplete matching or mismatch of bases, the double-stranded nucleic acid cannot form a standard double-stranded RNA structure, and instead forms a raised portion, i.e. a loop-like or bubble-like region, in the incompletely matched or mismatched region (unpaired region). Detailed Implementation

[0133] Those skilled in the art will recognize that the double-stranded ribonucleic acid described herein can be obtained using conventional dsRNA preparation methods (e.g., solid-phase synthesis and liquid-phase synthesis), both of which are commercially available custom services. Those skilled in the art will also understand that modified nucleotide groups can be introduced into the double-stranded ribonucleic acid described herein using appropriately modified nucleotide monomers. Methods for preparing appropriately modified nucleotide monomers are well known to those skilled in the art, and commercially available monomers are also available.

[0134] Example 1: Synthesis of double-stranded ribonucleic acid (dsRNA)

[0135] For the single-stranded RNA (dsRNA) of this disclosure, single-stranded RNA 1 and single-stranded RNA 2, CPG is used as a solid-phase carrier; CPG modified with GalNAc(L96) is used as the starting cycle for the synthesis of single-stranded RNA 1 and single-stranded RNA 2.

[0136] Using a YB-192S synthesizer, a solid-phase synthesis of phosphoramidite was performed, starting with a solid support and sequentially linking nucleoside monomers in the 3'-5' direction to achieve a synthesis scale of 0.2 μmol.

[0137] The phosphorus amide monomer is linked through a continuous cycle of four chemical reactions: deprotection, coupling, oxidation / sulfidation, and capping. The phosphorus amide monomer is prepared as a 0.05 M acetonitrile solution, with 0.3 M BTT in acetonitrile as the activator, a 3% trichloroacetic acid / dichloromethane solution as the deprotecting agent, a 0.05 M iodine / pyridine / tetrahydrofuran / aqueous solution (v / v / v = 2 / 1 / 7) as the oxidizing agent, acetic anhydride / acetonitrile solution as capping agent A (v / v = 2 / 8), pyridine / N-methylimidazolium / tetrahydrofuran solution as capping agent B (v / v / v = 10 / 16 / 74) as the capping agent, and a 0.05 M DDTT solution of pyridine / acetonitrile as the thiochemical agent (v / v = 4 / 6).

[0138] After solid-phase synthesis, the support was transferred to a 2 mL centrifuge tube, and 0.8 mL of concentrated ammonia was added. The mixture was then sealed and reacted at 55 °C for 16 h. After cooling to room temperature, the solution was transferred to a 2 mL centrifuge tube and concentrated to dryness. 0.2 mL of anhydrous DMSO was added to dissolve the solution, followed by 0.25 mL of triethylamine trihydrofluoride. The reaction was carried out at 65 °C for 2 h. After the reaction was complete, the mixture was cooled to room temperature, and the crude sequence was obtained by ethanol precipitation.

[0139] The crude product was purified by reversed-phase HPLC, and the collected fraction was lyophilized. Ethanol precipitation was performed by adding 0.3 mL of 1M sodium acetate solution and 0.9 mL of ethanol to replace the sequence with sodium salt. Then, desalting was carried out using a 3KD ultrafiltration tube to remove excess free salt.

[0140] Nucleic acid single strand 1 and nucleic acid single strand 2 were prepared into an aqueous solution of a certain concentration. Nucleic acid single strand 1 and nucleic acid single strand 2 were mixed at a molar ratio of 1:1.05, incubated at 95°C for 5 minutes, and then naturally cooled to room temperature. The target product (double-stranded ribonucleic acid) was obtained by freeze drying. The compounds are shown in Table 2.

[0141] Example 2: In vitro activity assay - free uptake by human primary hepatocytes

[0142] After resuscitation, human primary hepatocytes were diluted with culture medium to a density of 600,000 cells / mL. Different concentrations of conjugates were added to 96-well collagen plates at 10 μL / well, followed by 90 μL / well of human primary hepatocytes (54,000 cells / well). A PBS control group was also included. The plates were incubated at 37°C with 5% CO2 for 48 hours. After 48 hours, the culture medium was removed, and cells were collected for RNA extraction. Total RNA was extracted using the RNeasy 96 Kit (QIAGEN-74182) according to the kit instructions. cDNA was then synthesized using the HiScript III RT SuperMix for qPCR (+gDNAwiper) (Vazyme-R323-01) according to the kit instructions. Real-time PCR was performed using the ΔΔCt assay in an Applied Biosystems-QuantStudio 7 Flex real-time PCR system (Table).

[0143] Table 3. Results of free uptake assay in human primary hepatocytes

[0144] Example 3: RNAi Off-Target Analysis - Human Primary Hepatocytes' Free RNA Take-Up (Seq)

[0145] After resuscitation, human primary hepatocytes were diluted with culture medium to a density of 670,000 cells / mL. Different concentrations of conjugates were added at 50 μL / well to 24-well collagen plates, followed by 450 μL / well of human primary hepatocytes. A PBS control group was also included. The plates were then incubated at 37°C with 5% CO2 for 48 hours. After 48 hours, the culture medium was removed, and the cells were collected for RNA extraction. Total RNA was extracted using the RNeasy 96 Kit (QIAGEN-74182) according to the kit instructions. 1 μg of extracted total RNA was collected and processed using the Ribo-off rRNA Depletion Kit (Human / Mouse / Rat) (Vazyme N406-02), VAHTS Universal V6 RNA-seq Library Prep Kit for Illumina (Vazyme NR604-02), and VAHTS RNA Multiplex Oligos Set1-Set2 for Illumina (Vazyme N323 / N324) kits for library construction. All samples were sequenced using a next-generation sequencer (NovaSeq6000, Illumina), and the sequencing results were compared with those of blank samples to identify genes significantly downregulated after RNAi treatment (log2FoldChange ≤ -1, p-adjust < 0.05). Subsequently, cDNA was synthesized using the HiScript III RT SuperMix for qPCR (+gDNA wiper) (Vazyme-R323-01) according to the manufacturer's instructions. The significantly downregulated gene was validated by real-time fluorescence PCR using the ΔΔCt assay in the Applied Biosystems-QuantStudio 7Flex real-time fluorescence PCR system.

[0146] Sequencing analysis of RNA samples from human primary hepatocytes treated with conjugate 133, combined with qPCR off-target gene validation, revealed that only the mRNA levels of PCSK9 and AGT target genes were significantly reduced. This indicates that conjugate 133 has no significant off-target effects in human primary hepatocytes, and its inhibition of PCSK9 and AGT target genes is highly specific.

[0147] Example 4: In vivo activity assay of double-stranded ribonucleic acid (dsRNA)

[0148] This experiment used male humanized mice with the hPCSK9 and hAGT genes. Mice were placed in an acclimatization facility for at least 3 days before drug administration, and blood was collected to separate serum. Mice were then grouped according to body weight, with 3-5 mice in each group. A single subcutaneous injection was administered, designated as D0. Blood was collected from all mice on D7, D14, D21, and D28 after drug administration, and serum was separated. The levels of hPCSK9 or hAGT protein in the separated mouse serum were detected by ELISA.

[0149] Table 3. Results of conjugate 133 in single-transfer mice: Inhibition of PCSK9 protein

[0150] Table 4. Results of conjugate 133 in single-transfer mice: Inhibition of AGT protein Note: A+: Indicates inhibition rate ≥ 90%; A: Indicates 90% > inhibition rate ≥ 80%; B: Indicates 80% > inhibition rate ≥ 70%; C: Indicates 70% > inhibition rate ≥ 60%; D: Indicates 60% > inhibition rate ≥ 50%; ND: Indicates no inhibition rate;

[0151] Example 5: In vivo activity assay of double-stranded ribonucleic acid

[0152] Healthy male cynomolgus macaques were selected during their environmental adaptation period based on body weight, hematology, and blood biochemistry levels. They were randomly assigned to groups of three. Serum samples were collected on days -14, -7, and 1 (before drug administration). On day 0 of the study, cynomolgus macaques (referred to herein as "cynomolgus macaques") were subcutaneously injected with isotonic saline (vehicle control) or 6 mg / kg (mpk) of the delivery vehicle of this disclosure, prepared in isotonic saline and containing a nucleic acid conjugate as described herein. Serum was then collected on days 7, 14, 21, 28, 35, 42, and 56. The protein expression levels of PCSK9 and AGT in the cynomolgus macaque serum were measured.

[0153] Table 5. Results of conjugate 133 in cynomolgus monkeys: inhibition of PCSK9 protein.

[0154] Table 6. Results of conjugate 133 in cynomolgus monkeys: Inhibition of AGT protein Note: A+: Indicates inhibition rate ≥ 90%; A: Indicates 90% > inhibition rate ≥ 80%; B: Indicates 80% > inhibition rate ≥ 70%; C: Indicates 70% > inhibition rate ≥ 60%; D: Indicates 60% > inhibition rate ≥ 50%; ND: Indicates no inhibition rate;

Claims

1. A dsRNA agent that simultaneously inhibits the expression of PCSK9 and AGT genes, comprising: a. A first nucleic acid single strand and a second nucleic acid single strand that are partially or completely complementary; the two nucleic acid single strands are capable of forming a stable double-stranded structure; the first nucleic acid single strand and the second nucleic acid single strand are 30-80 nt in length, each containing a region that is at least partially complementary to the PCSK9 mRNA or AGT mRNA sequence, and each containing at least two unmodified RNA nucleotides and / or DNA nucleotides. b. Each nucleic acid single strand is conjugated with at least one carrier group containing an ASGPR ligand, wherein, The carrier group of the ASGPR ligand is conjugated to each nucleic acid single strand; each nucleic acid single strand is represented by formula (I): In this context, each N is an independent unmodified or chemically modified nucleotide; each L is an independent carrier group, either present or absent; (N) represents a series of unmodified nucleotides; and the sum of X1, X2, X3, X4 and X5 is an integer from 30 to 80.

2. The dsRNA agent according to claim 1, wherein, X1 and / or X5 are 0 on the first and / or second nucleic acid single strands; Preferably, at least one of the first and second nucleic acid single strands has a structure as shown in formula (II): 5' N X2 —(N) X3 —N X4 —L 3' (II); and / or The first or second nucleic acid single strand has a structure as shown in formula (III): 5′ N X2 —(N) X3 —N X4 3' (III)。 3. The dsRNA agent according to claim 1 or 2, wherein, The sum of X1 and X2 is an integer between 14 and 29; the dsRNA agent can form 0, 1 or more Bubble structures at position (N); the region that is at least partially complementary to the PCSK9 mRNA or AGT mRNA sequence is located on the 5′ side of position (N).

4. The dsRNA agent according to any one of claims 1-3, characterized in that: The first or second single-stranded nucleic acid contains a nucleotide sequence as shown in SEQ ID NOs:1000-1034.

5. The dsRNA agent according to any one of claims 1-4, characterized in that: The nucleotide sequences of the first and second nucleic acid single strands are selected from the following combinations: (1) SEQ ID NO: 1000 and 1001; (2) SEQ ID NO: 1002 and 1001; (3) SEQ ID NO: 1003 and 1004; (4) SEQ ID NO: 1003 and 1005; (5) SEQ ID NO: 1006 and 1005; (6) SEQ ID NO: 1000 and 1007; (7) SEQ ID NO: 1008 and 1009; (8) SEQ ID NO: 1010 and 1005; (9) SEQ ID NO: 1011 and 1012; (10) SEQ ID NO: 1013 and 1012; (11) SEQ ID NO: 1014 and 1015; (12) SEQ ID NO: 1014 and 1016; (13) SEQ ID NO: 1017 and 1016; (14) SEQ ID NO: 1011 and 1018; (15) SEQ ID NO: 1019 and 1020; (16) SEQ ID NO: 1021 and 1016; (17) SEQ ID NO: 1022 and 1023; (18) SEQ ID NO: 1024 and 1023; (19) SEQ ID NO: 1025 and 1026; (20)SEQ ID NO: 1025 and 1027; (21) SEQ ID NO: 1028 and 1027; (22) SEQ ID NO: 1022 and 1029; (23) SEQ ID NO: 1030 and 1031; (24) SEQ ID NO: 1032 and 1027; (25)SEQ ID NO:1033 and 1034.

6. The dsRNA agent according to any one of claims 1-5, characterized in that: The first or second single-stranded nucleic acid of the dsRNA agent contains at least one modified nucleotide.

7. The dsRNA agent according to any one of claims 1-6, characterized in that: At least 90% of the nucleotides in the first or second single-stranded nucleic acid of the dsRNA agent are modified nucleotides.

8. The dsRNA agent according to any one of claims 1-7, characterized in that: The modified nucleotides include threonine, 2'-O-methylnucleotide, 2'-fluoronucleotide, 2'-deoxynucleotide, 2'3'-seco nucleotide mimic, locked nucleotide, unlocked nucleonucleotide (UNA), glycol nucleonucleotide (GNA), 2'-F-arabinonucleotide, 2'-methoxyethyl nucleotide, abase-free nucleotide, ribitol, reverse nucleotide, reverse abase-free nucleotide, reverse 2'-OMe nucleotide, reverse 2'-deoxynucleotide, 2'-amino-modified nucleotide, 2'-alkyl-modified nucleotide, morpholinonucleotide, and 3'-OMe nucleotide, 2'-OC 22 H 45 Nucleotides, nucleotides containing a 5'-thiophosphate group, nucleotides modified with a 2'-amino group, aminophosphates, or nucleotides containing a non-natural base, or one or more of these.

9. The dsRNA agent according to any one of claims 1-8, characterized in that: Each single strand of the double-stranded ribonucleic acid has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 thiophosphate groups linked between the 5' end, 3' end, or intermediate nucleotides.

10. The dsRNA agent according to any one of claims 1-9, characterized in that: Each nucleic acid single strand contains a phosphate ester or phosphate ester mimic at its 5' end; preferably, the phosphate mimic is 5'-vinyl phosphate; more preferably, the phosphate mimic is 5'-(E)-vinyl phosphate.

11. The dsRNA agent according to any one of claims 1-10, characterized in that: The first nucleic acid single strand contains the modified nucleotide sequence of nucleic acid single strand 1 of modified double strands 1 to 138 in Table 1b; And / or the second nucleic acid single strand contains the modified nucleotide sequence of the nucleic acid single strand 2 of the modified double strands 1 to 138 in Table 1b; And / or the first or second nucleic acid single strand further comprises a 5′-(E)-vinyl phosphate modified nucleotide at its 5′ end; Preferably, the first nucleic acid single strand comprises the modified nucleotide sequence of nucleic acid single strand 1 of the modified double strands 045, 133, 134, 135, 136, 137, and 138 in Table 1b; Preferably, the second nucleic acid single strand comprises the modified nucleotide sequence of nucleic acid single strand 2 of the modified double strands 045, 133, 134, 135, 136, 137, and 138 in Table 1b.

12. The dsRNA agent according to any one of claims 1-11, characterized in that: The modified nucleotide sequence combination of the first or second single-stranded nucleic acid is selected from the modified nucleotide sequence combinations of the double strands 045, 133, 134, 135, 136, 137, and 138 in Table 1b; Preferably, the nucleotide sequence combination of the first or second nucleic acid single strand is selected from: (1) Nucleotide sequences of single strand 1 and single strand 2 of nucleic acid modified by double strand 045 in Table 1b; (2) Nucleotide sequences of single strand 1 and single strand 2 of nucleic acid modified by double strand 133 in Table 1b; (3) Nucleotide sequences of single strand 1 and single strand 2 of nucleic acid that modify double strand 134 in Table 1b; (4) Nucleotide sequences of single strand 1 and single strand 2 of nucleic acid modified by double strand 135 in Table 1b; (5) Nucleotide sequences of single strand 1 and single strand 2 of nucleic acid modified by double strand 136 in Table 1b; (6) Nucleotide sequences of single strand 1 and single strand 2 of nucleic acid modified by double strand 137 in Table 1b; (7) The nucleotide sequences of single strand 1 and single strand 2 of the nucleic acid modified by double strand 138 in Table 1b.

13. The dsRNA agent according to any one of claims 1-12, characterized in that: The dsRNA agent has liver-targeting properties; Preferably, the carrier group containing the ASGPR ligand is GalNAc (L96) or Ser (GN), or GalNAc (Ser1), GalNAc (Ser2), GalNAc (Ser3), GalNAc (Ser4), or LP-GalNAc, XY-GalNAc, GalNAc (A1GN), GalNAc (A1dGN), GalNAc (A3GN), GalNAc (A3dGN), GalNAc (A5GN), GAlNAc (NAG25), GAlNAc (NAG37). GalNAc(L96) has the following structure: Ser(GN) has the following structure: GalNAc(Ser1), GalNAc(Ser2), GalNAc(Ser3), and GalNAc(Ser4) have the following structures: LP-GalNAc has the following structure: XY-GalNAc has the following structure: GalNAc(A1GN), GalNAc(A1dGN), GalNAc(A3GN), GalNAc(A3dGN), GalNAc(A5GN), GAlNAc(NAG25), and GAlNAc(NAG37) have the following structures:

14. The dsRNA agent according to claims 1-13, characterized in that: The dsRNA agent has liver targeting; the carrier group containing the ASGPR ligand has the following structure:

15. The dsRNA agent according to claims 1-14, characterized in that: The dsRNA agent is selected from nucleic acid conjugates 001 to 138 in Table 2; preferably, the dsRNA agent is nucleic acid conjugate 045 or nucleic acid conjugate 133.

16. A pharmaceutical composition for inhibiting the expression of PCSK9 and AGT target genes, said pharmaceutical composition comprising a dsRNA agent according to any one of claims 1-15 and a pharmaceutically acceptable carrier.

17. The pharmaceutical composition according to claim 16, characterized in that, The pharmaceutical composition is an injectable formulation or a subcutaneous formulation; preferably, the pharmaceutical composition is a subcutaneous injection formulation.

18. The pharmaceutical composition according to claim 17, characterized in that, The pharmaceutical composition further comprises excipients, including buffer solutions or non-buffer solutions; Preferably, the buffer solution comprises acetate, citrate, alcohol-soluble gluten, carbonate or phosphate or phosphate-buffered saline or any combination thereof, or the non-buffered solution is saline or water.

19. The use of the dsRNA agent according to any one of claims 1-15 or the pharmaceutical composition according to any one of claims 16-18 for the treatment and / or prevention of pathological conditions or diseases associated with PCSK9 and / or AGT gene expression; preferably, the pathological condition or disease is hyperlipidemia, nonfamilial hypercholesterolemia, polygenic hypercholesterolemia, familial hypercholesterolemia, homozygous familial hypercholesterolemia and heterozygous familial hypercholesterolemia, coronary heart disease, myocardial infarction, stroke and atherosclerosis, borderline hypertension, essential hypertension, secondary hypertension, isolated systolic or diastolic hypertension, pregnancy-related hypertension, diabetic hypertension, refractory hypertension, intractable hypertension, paroxysmal hypertension, renovascular hypertension, Goldblatt. Hypertension, hypertension with low plasma renin activity or plasma renin concentration, ocular hypertension, glaucoma, pulmonary hypertension, portal hypertension, systemic venous hypertension, systolic hypertension, unstable hypertension, hypertensive heart disease, hypertensive nephropathy, atherosclerosis, arteriosclerosis, vascular disease, diabetic nephropathy, diabetic retinopathy, chronic heart failure, cardiomyopathy, diabetic cardiomyopathy, nocturnal hypertension, glomerulosclerosis, aortic coarctation, aortic aneurysm, ventricular fibrosis, heart failure, myocardial infarction, angina pectoris, stroke, kidney disease, kidney failure, systemic sclerosis, intrauterine growth retardation, fetal growth restriction, obesity, hepatic steatosis / fatty liver, non-alcoholic steatohepatitis, non-alcoholic fatty liver disease, glucose intolerance, type 2 diabetes, and metabolic syndrome, etc.

20. Use of the dsRNA agent according to any one of claims 1-15 or the pharmaceutical composition according to any one of claims 16-18 in the preparation of a medicament for treating pathological conditions or diseases related to PCSK9 and / or AGT expression; preferably, the pathological condition or disease is hyperlipidemia, nonfamilial hypercholesterolemia, polygenic hypercholesterolemia, familial hypercholesterolemia, homozygous familial hypercholesterolemia and heterozygous familial hypercholesterolemia, coronary heart disease, myocardial infarction, stroke and atherosclerosis, borderline hypertension, essential hypertension, secondary hypertension, isolated systolic or diastolic hypertension, pregnancy-related hypertension, diabetic hypertension, refractory hypertension, intractable hypertension, paroxysmal hypertension, renovascular hypertension, Goldblatt syndrome. Hypertension, hypertension with low plasma renin activity or plasma renin concentration, ocular hypertension, glaucoma, pulmonary hypertension, portal hypertension, systemic venous hypertension, systolic hypertension, unstable hypertension, hypertensive heart disease, hypertensive nephropathy, atherosclerosis, arteriosclerosis, vascular disease, diabetic nephropathy, diabetic retinopathy, chronic heart failure, cardiomyopathy, diabetic cardiomyopathy, nocturnal hypertension, glomerulosclerosis, aortic coarctation, aortic aneurysm, ventricular fibrosis, heart failure, myocardial infarction, angina pectoris, stroke, kidney disease, kidney failure, systemic sclerosis, intrauterine growth retardation, fetal growth restriction, obesity, hepatic steatosis / fatty liver, non-alcoholic steatohepatitis, non-alcoholic fatty liver disease, impaired glucose tolerance, type 2 diabetes, and metabolic syndrome, etc.

21. A dsRNA agent or pharmaceutical composition for treating pathological conditions or diseases related to PCSK9 and / or AGT expression; wherein, The dsRNA agent is defined as described in any one of claims 1-15, and the pharmaceutical composition is defined as described in any one of claims 16-18; preferably, the pathological condition or disease is hyperlipidemia, nonfamilial hypercholesterolemia, polygenic hypercholesterolemia, familial hypercholesterolemia, homozygous familial hypercholesterolemia and heterozygous familial hypercholesterolemia, coronary heart disease, myocardial infarction, stroke and atherosclerosis, borderline hypertension, essential hypertension, secondary hypertension, isolated systolic or diastolic hypertension, pregnancy-related hypertension, diabetic hypertension, refractory hypertension, intractable hypertension, paroxysmal hypertension, renovascular hypertension, Goldblatt hypertension, low plasma renin activity or plasma renin concentration. Related conditions include hypertension, ocular hypertension, glaucoma, pulmonary hypertension, portal hypertension, systemic venous hypertension, systolic hypertension, unstable hypertension, hypertensive heart disease, hypertensive nephropathy, atherosclerosis, arteriosclerosis, vascular disease, diabetic nephropathy, diabetic retinopathy, chronic heart failure, cardiomyopathy, diabetic cardiomyopathy, nocturnal hypertension, glomerulosclerosis, aortic coarctation, aortic aneurysm, ventricular fibrosis, heart failure, myocardial infarction, angina pectoris, stroke, kidney disease, kidney failure, systemic sclerosis, intrauterine growth retardation, fetal growth restriction, obesity, hepatic steatosis / fatty liver, non-alcoholic steatohepatitis, non-alcoholic fatty liver disease, glucose intolerance, type 2 diabetes, and metabolic syndrome.

22. A method for treating pathological conditions or diseases related to PCSK9 and / or AGT expression, wherein, The method includes administering to a subject in need the dsRNA agent according to any one of claims 1-15 or the pharmaceutical composition according to any one of claims 16-18; preferably, the pathological condition or disease is hyperlipidemia, nonfamilial hypercholesterolemia, polygenic hypercholesterolemia, familial hypercholesterolemia, homozygous familial hypercholesterolemia and heterozygous familial hypercholesterolemia, coronary heart disease, myocardial infarction, stroke and atherosclerosis, borderline hypertension, essential hypertension, secondary hypertension, isolated systolic or diastolic hypertension, pregnancy-related hypertension, diabetic hypertension, refractory hypertension, intractable hypertension, paroxysmal hypertension, renovascular hypertension, Goldblatt hypertension, low plasma renin activity or Hypertension related to plasma renin concentration, ocular hypertension, glaucoma, pulmonary hypertension, portal hypertension, systemic venous hypertension, systolic hypertension, unstable hypertension, hypertensive heart disease, hypertensive nephropathy, atherosclerosis, arteriosclerosis, vascular disease, diabetic nephropathy, diabetic retinopathy, chronic heart failure, cardiomyopathy, diabetic cardiomyopathy, nocturnal hypertension, glomerulosclerosis, aortic coarctation, aortic aneurysm, ventricular fibrosis, heart failure, myocardial infarction, angina pectoris, stroke, kidney disease, kidney failure, systemic sclerosis, intrauterine growth retardation, fetal growth restriction, obesity, hepatic steatosis / fatty liver, non-alcoholic steatohepatitis, non-alcoholic fatty liver disease, glucose intolerance, type 2 diabetes, and metabolic syndrome, etc.

23. The method of claim 22, wherein the dsRNA agent or pharmaceutical composition is administered by injection or subcutaneous administration; preferably, the dsRNA agent or pharmaceutical composition is administered by subcutaneous injection.

24. The method of claim 22, wherein the dsRNA agent or pharmaceutical composition is administered together with an excipient, the excipient comprising a buffered solution or a non-buffered solution; Preferably, the non-buffered solution is saline or water, or the buffered solution includes acetate, citrate, alcohol-soluble gluten, carbonate, or phosphate or phosphate-buffered saline or any combination thereof.

25. The method of claim 22, wherein the object of need is a person.

26. The method of claim 22, wherein the dsRNA agent or pharmaceutical composition is administered at a dose of 1-100 mg / kg.

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