siRNA targeting AGT gene expression, its conjugate and use

Abstract

The present invention provides siRNA and its conjugate that inhibit AGT gene expression. The siRNA comprises a sense strand and an antisense strand. The siRNA, siRNA conjugate and pharmaceutical composition provided in the present invention have good stability, excellent inhibitory activity against AGT genes, and satisfactory cytotoxic and immunostimulatory activity.

Description

[Technical Field]

[0001] The present invention relates to siRNAs that inhibit the expression of the angiotensinogen (AGT) gene, their conjugates and pharmaceutical compositions, and their use in the prevention and / or treatment of abnormal blood pressure-related diseases. [Background technology]

[0002] Angiotensinogen (AGT) is an important component of the renin-angiotensin system (RAS) in the human body, and the RAS as a whole plays a crucial role in regulating blood pressure in animals. AGT is synthesized and secreted in the liver, and is then converted to angiotensin I by the enzyme renin (REN), which is then converted to angiotensin II by angiotensin-converting enzyme (ACE). Most angiotensin II can bind to type I angiotensin II receptors, resulting in vasoconstriction and increased blood pressure. This molecule also stimulates the production of the hormone aldosterone, which is Na + It induces renal absorption of ions and water, thereby increasing blood volume and further raising blood pressure.

[0003] Hypertension is a serious condition that significantly increases the risk of heart, brain, kidney, and other diseases. According to data published by the World Health Organization, an estimated 1.28 billion adults aged 30-79 worldwide suffer from hypertension. Only 42% of people with hypertension are diagnosed and treated, and only 21% have their blood pressure effectively controlled. Hypertension is a leading cause of premature death worldwide. Furthermore, according to a report in The Lancet (2019;394:1145-58), 2.54 million people died from hypertension in Japan in 2017, making it the leading risk factor for death.

[0004] Currently, first-line drugs clinically used to treat hypertension are divided into five main classes: angiotensinase inhibitors, angiotensin II receptor blockers, β-adrenergic receptor blockers, dihydropyridine calcium channel blockers, and diuretics. These five classes of antihypertensive drugs have completely different mechanisms of action. Depending on the patient's age, the severity of hypertension, and the risk of associated clinical complications, most patients will be treated with one of these drugs. If one method is ineffective, two or even three methods may be used in combination. In high-risk patients, two or even three treatment methods are often used in combination. When blood pressure cannot be effectively controlled by these three methods, it is called refractory hypertension, which is a common and difficult problem in the treatment of hypertension. The development of effective, safe, stable, and long-acting antihypertensive drugs has significant clinical value.

[0005] This application provides an AGT-targeted small interfering RNA (siRNA) formulation that specifically binds to AGT mRNA, disrupts the normal translational template function of AGT mRNA, and thereby interferes with the translation of AGT proteins. This inhibits the RAS pathway at its origin and can be used to treat / prevent diseases associated with the RAS pathway, such as hypertension (including refractory and uncontrolled hypertension).

[0006] Compared to conventional drugs, siRNAs have low stability and are readily degraded by nucleases when administered systemically. Furthermore, there is a need to explore ways to further improve their activity while avoiding off-target effects, immunostimulation, cytotoxicity, and other side effects. Therefore, developing more candidate siRNAs that are stable in the blood, have good in vivo biological activity, have low cytotoxicity, and can effectively inhibit AGT gene expression for extended periods is an urgent challenge to address. At the same time, drug development using these candidate siRNAs to effectively prevent and / or treat hypertension-related diseases by inhibiting AGT gene expression has both the need for clinical research and commercial viability. [Overview of the project]

[0007] The present invention provides an siRNA for inhibiting AGT gene expression, wherein the siRNA comprises a sense strand and an antisense strand, where the antisense strand comprises at least 17 consecutive nucleotides that differ by 4 nucleotides or less from any one of the nucleotide sequences shown in SEQ ID NOs: 102-192, 201-207, 209-220, 222-223, 225-233, 235-250, 252-291, 293-341, and 343-346, the antisense strand is 17-30 nucleotides long, and the sense strand is 17-30 nucleotides long and at least partially complementary to the antisense strand.

[0008] The term "at least partially complementary" means that the two sequences may have a total of 5, 4, 3, or 2 or fewer mismatched base pairs, while retaining the ability to be fully complementary or hybridize under relevant conditions.

[0009] In some embodiments of the present invention, the antisense strand differs from any one of the nucleotide sequences shown in SEQ ID NOs. 102-192, SEQ ID NOs. 201-207, SEQ ID NOs. 209-220, SEQ ID NOs. 222-223, SEQ ID NOs. 225-233, SEQ ID NOs. 235-250, SEQ ID NOs. 252-291, SEQ ID NOs. 293-341, and SEQ ID NOs. 343-346 differs from any one of the nucleotide sequences shown in SEQ ID NOs. 343-346 differs from any one of the nucleotide sequences differs from any one of the nucleotide sequences shown in SEQ ID NOs. 102-192, SEQ ID NOs. 201-207, SEQ ID NOs. 209-220, SEQ ID NOs. 222-223, SEQ ID NOs. 225-233, SEQ ID NOs. 235-250, SEQ ID NOs. 252-291, SEQ ID NOs. 293-341, and SEQ ID NOs. 343-346 differs from any one of the nucleotide sequences differs from any one of the nucleotide sequences shown in SEQ ID NOs. 343-346. In some embodiments of the present invention, the antisense strand differs from any one of the nucleotide sequences shown in SEQ ID NOs: 102-192, 201-207, 209-220, 222-223, 225-233, 235-250, 252-291, 293-341, and 343-346 by only two nucleotides or less. In some embodiments of the present invention, the antisense strand differs from any one of the nucleotide sequences shown in SEQ ID NOs: 102-192, 201-207, 209-220, 222-223, 225-233, 235-250, 252-291, 293-341, and 343-346 by only one nucleotide or less.In some embodiments of the present invention, the antisense chain is one of the nucleotide sequences shown in SEQ ID NOs: 102-192, 201-207, 209-220, 222-223, 225-233, 235-250, 252-291, 293-341, and 343-346.

[0010] In some embodiments of the present invention, the sense strand and the antisense strand have a mismatch of 3 nucleotides or less; in some embodiments, the sense strand and the antisense strand have a mismatch of 2 nucleotides or less; in some embodiments, the sense strand and the antisense strand have a mismatch of 1 nucleotide or less; in some embodiments, the sense strand and the antisense strand are perfectly complementary.

[0011] Preferably, the sense strand and the antisense strand are complementary in at least 15, 16, 17, 18, 19, 20, or 21 nucleotides.

[0012] In some embodiments of the present invention, the antisense strand is 19 to 27 nucleotides long; and the sense strand is 19 to 25 nucleotides long.

[0013] In some embodiments of the present invention, the antisense strand is 19 to 23 nucleotides long; and the sense strand is 19 to 21 nucleotides long.

[0014] In some embodiments of the present invention, the antisense strand is 23 nucleotides long and the sense strand is 21 nucleotides long. In some embodiments of the present invention, the antisense strand is 22 nucleotides long and the sense strand is 20 nucleotides long. In some embodiments of the present invention, the antisense strand is 21 nucleotides long and the sense strand is 21 nucleotides long. In some embodiments of the present invention, the antisense strand is 21 nucleotides long and the sense strand is 19 nucleotides long. In some embodiments of the present invention, the antisense strand is 19 nucleotides long and the sense strand is 19 nucleotides long.

[0015] In some embodiments of the present invention, the siRNA comprises one or more single-stranded nucleotide overhangs, for example, 1, 2, 3, or 4 nucleotide overhangs. In some embodiments of the present invention, the overhangs may be on the sense strand, the antisense strand, or any combination thereof. In some embodiments of the present invention, the overhangs may be located at the 5' end, 3' end, or both ends of the antisense or sense strand of the siRNA.

[0016] In some embodiments of the present invention, the siRNA has a 2-nucleotide overhang at the 3' end of the antisense strand.

[0017] In some embodiments of the present invention, the siRNA has a blunt end. In some embodiments of the present invention, the siRNA has at least one blunt end located at the 5' end of the antisense strand (or the 3' end of the sense strand).

[0018] In some embodiments of the present invention, the siRNA has two blunt ends.

[0019] In some embodiments of the present invention, the siRNA has a nucleotide sequence (5'->3') selected from double strand 1 to double strand 107: [Table 1-1]

Table 1-2

Table 1-3

[0020] In some embodiments of the present invention, the siRNA comprises at least one modified nucleotide.

[0021] In some embodiments of the present invention, all nucleotides in the sense strand and / or the antisense strand of the siRNA are modified nucleotides or nucleotide analogs.

[0022] In some embodiments of the present invention, the modified nucleotide is selected from 2'-methoxy nucleotide, 2'-fluoro nucleotide, 2'-deoxy nucleotide, 2',3'-seco nucleotide analog, 2'-fluoroarabinonucleotide, 2'-methoxyethyl nucleotide, 2'-amino-modified nucleotide, 2'-alkyl-modified nucleotide, 3'-methoxy nucleotide, 2'-allyl-modified nucleotide, nucleotide containing phosphorothioate group, nucleotide containing methylphosphonate group, nucleotide containing 5'-phosphate group, nucleotide containing 5'-phosphate mimic, diol-modified nucleotide, abasic nucleotide, morpholino nucleotide, locked nucleotide (LNA), unlocked nucleotide (UNA), threose nucleotide (TNA), or glycerol nucleotide (GNA), but the present invention is not limited thereto.

[0023] In some embodiments of the present invention, the antisense strand of the siRNA is 23 nucleotides long, with 2'-fluoronucleotides at positions 2, 6, 14, and 16 of the 5' end of the antisense strand, and the remaining positions being 2'-methoxynucleotides; the sense strand of the siRNA is 21 nucleotides long, with 2'-fluoronucleotides at positions 7, 9, 10, and 11 of the 5' end of the sense strand, and the remaining positions being 2'-methoxynucleotides.

[0024] In some embodiments of the present invention, the antisense strand of the siRNA is 23 nucleotides long, with 2'-fluoronucleotides at positions 2, 6, 8, 9, 14, and 16 of the 5' end of the antisense strand, and the remaining positions being 2'-methoxynucleotides; the sense strand of the siRNA is 21 nucleotides long, with 2'-fluoronucleotides at positions 7, 9, 10, and 11 of the 5' end of the sense strand, and the remaining positions being 2'-methoxynucleotides.

[0025] In some embodiments of the present invention, the antisense strand of the siRNA is 23 nucleotides long, with 2'-fluoronucleotides at positions 2, 6, 14, and 16 of the 5' end of the antisense strand, and the remaining positions being 2'-methoxynucleotides; the sense strand of the siRNA is 21 nucleotides long, with 2'-fluoronucleotides at positions 9, 10, and 11 of the 5' end of the sense strand, and the remaining positions being 2'-methoxynucleotides.

[0026] In some embodiments of the present invention, the antisense strand of the siRNA is 23 nucleotides long, with 2'-fluoronucleotides at positions 2, 6, 8, 9, 10, 14, and 16 of the 5' end of the antisense strand, and the remaining positions being 2'-methoxynucleotides; the sense strand of the siRNA is 21 nucleotides long, with 2'-fluoronucleotides at positions 7, 9, 10, and 11 of the 5' end of the sense strand, and the remaining positions being 2'-methoxynucleotides.

[0027] In some embodiments of the present invention, the antisense strand of the siRNA is 23 nucleotides long, with 2'-fluoronucleotides at positions 2, 6, 8, 9, 10, 14, and 16 of the 5' end of the antisense strand, and the remaining positions being 2'-methoxynucleotides. The sense strand of the siRNA is 21 nucleotides long, with 2'-fluoronucleotides at positions 7, 9, 10, 11, and 15 of the 5' end of the sense strand, and the remaining positions being 2'-methoxynucleotides.

[0028] In some embodiments of the present invention, the antisense strand of the siRNA is 23 nucleotides long, with 2'-fluoronucleotides at positions 2, 6, 10, 14, and 16 of the 5' end of the antisense strand, and the remaining positions being 2'-methoxynucleotides; the sense strand of the siRNA is 21 nucleotides long, with 2'-fluoronucleotides at positions 3, 7, 9, 10, and 11 of the 5' end of the sense strand, and the remaining positions being 2'-methoxynucleotides.

[0029] In some embodiments of the present invention, the antisense strand of the siRNA is 23 nucleotides long, with 2'-fluoronucleotides at positions 2, 6, 14, and 16 of the 5' end of the antisense strand, and the remaining positions being 2'-methoxynucleotides; the sense strand of the siRNA is 21 nucleotides long, with 2'-fluoronucleotides at positions 7, 9, 10, and 11 of the 5' end of the sense strand, and the remaining positions being 2'-methoxynucleotides.

[0030] In some embodiments of the present invention, the antisense strand of the siRNA is 23 nucleotides long, with 2, 3, 4, 6, 8, 10, 14, 16, 18, 20, and 22 nucleotides at the 5' end of the antisense strand, and the remaining positions being 2'-methoxynucleotides; the sense strand of the siRNA is 21 nucleotides long, with 2 nucleotides at the 5' end of the sense strand, and the remaining positions being 2'-methoxynucleotides.

[0031] In some embodiments of the present invention, the antisense strand of the siRNA is 23 nucleotides long, with positions 2, 4, 5, 6, 8, 10, 14, 16, 18, 20, and 22 at the 5' end of the antisense strand being 2'-fluoronucleotides, and the remaining positions being 2'-methoxynucleotides; the sense strand of the siRNA is 21 nucleotides long, with position 2 at the 5' end of the sense strand being 2'-fluoronucleotides, and the remaining positions being 2'-methoxynucleotides.

[0032] In some embodiments of the present invention, the antisense strand of the siRNA is 23 nucleotides long, with positions 2, 4, 6, 7, 8, 10, 14, 16, 18, 20, and 22 at the 5' end of the antisense strand being 2'-fluoronucleotides, and the remaining positions being 2'-methoxynucleotides; the sense strand of the siRNA is 21 nucleotides long, with position 2 at the 5' end of the sense strand being 2'-fluoronucleotides, and the remaining positions being 2'-methoxynucleotides.

[0033] In some embodiments of the present invention, the antisense strand of the siRNA is 23 nucleotides long, with 2'-fluoronucleotides at positions 2, 4, 6, 8, 10, 14, 16, 18, and 20 of the 5' end of the antisense strand, and the remaining positions being 2'-methoxynucleotides; the sense strand of the siRNA is 21 nucleotides long, with 2'-fluoronucleotides at positions 1, 3, 5, 7, 9, 10, 11, 13, 15, 17, 19, and 21 of the 5' end of the sense strand, and the remaining positions being 2'-methoxynucleotides.

[0034] In some embodiments of the present invention, the antisense strand of the siRNA is 23 nucleotides long, with 2'-fluoronucleotides at positions 2, 4, 6, 8, 10, 14, 16, 18, 20, and 22 of the 5' end of the antisense strand, and the remaining positions being 2'-methoxynucleotides; the sense strand of the siRNA is 21 nucleotides long, with 2'-fluoronucleotides at positions 1, 3, 5, 7, 9, 10, 11, 13, 15, 17, 19, and 21 of the 5' end of the sense strand, and the remaining positions being 2'-methoxynucleotides.

[0035] In some embodiments of the present invention, the antisense strand of the siRNA is 23 nucleotides long, with 2'-fluoronucleotides at positions 2, 6, 8, 9, 14, and 16 of the 5' end of the antisense strand, glycerol nucleotide (GNA) at position 7, and 2'-methoxynucleotides at the remaining positions. The sense strand of the siRNA is 21 nucleotides long, with 2'-fluoronucleotides at positions 7, 9, 10, and 11 of the 5' end of the sense strand, and 2'-methoxynucleotides at the remaining positions.

[0036] In some embodiments of the present invention, the antisense strand of the siRNA is 23 nucleotides long, with 2'-fluoronucleotides at positions 2, 8, 9, 10, 14, and 16 of the 5' end of the antisense strand, glycerol nucleotide (GNA) at position 6, and 2'-methoxynucleotides at the remaining positions; the sense strand of the siRNA is 21 nucleotides long, with 2'-fluoronucleotides at positions 7, 9, 10, 11, and 15 of the 5' end of the sense strand, and 2'-methoxynucleotides at the remaining positions.

[0037] In some embodiments of the present invention, the antisense strand of the siRNA is 23 nucleotides long, with 2'-fluoronucleotides at positions 2, 8, 9, 10, 14, and 16 of the 5' end of the antisense strand, glycerol nucleotide (GNA) at position 4, and 2'-methoxynucleotides at the remaining positions; the sense strand of the siRNA is 21 nucleotides long, with 2'-fluoronucleotides at positions 7, 9, 10, 11, and 15 of the 5' end of the sense strand, and 2'-methoxynucleotides at the remaining positions.

[0038] In some embodiments of the present invention, the antisense strand of the siRNA is 23 nucleotides long, with 2'-fluoronucleotides at positions 2, 8, 9, 10, 14, and 16 of the 5' end of the antisense strand, glycerol nucleotide (GNA) at position 5, and 2'-methoxynucleotides at the remaining positions; the sense strand of the siRNA is 21 nucleotides long, with 2'-fluoronucleotides at positions 7, 9, 10, 11, and 15 of the 5' end of the sense strand, and 2'-methoxynucleotides at the remaining positions.

[0039] In some embodiments of the present invention, the antisense strand of the siRNA is 23 nucleotides long, with 2'-fluoronucleotides at positions 2, 8, 9, 10, 14, and 16 of the 5' end of the antisense strand, glycerol nucleotide (GNA) at position 7, and 2'-methoxynucleotides at the remaining positions; the sense strand of the siRNA is 21 nucleotides long, with 2'-fluoronucleotides at positions 7, 9, 10, 11, and 15 of the 5' end of the sense strand, and 2'-methoxynucleotides at the remaining positions.

[0040] In some embodiments of the present invention, the antisense strand of the siRNA is 23 nucleotides long, with 2'-fluoronucleotides at positions 2, 8, 9, 10, 14, and 16 of the 5' end of the antisense strand, glycerol nucleotide (GNA) at position 6, and 2'-methoxynucleotides at the remaining positions; the sense strand of the siRNA is 21 nucleotides long, with 2'-fluoronucleotides at positions 9, 10, and 11 of the 5' end of the sense strand, and 2'-methoxynucleotides at the remaining positions.

[0041] In some embodiments of the present invention, the antisense strand of the siRNA is 23 nucleotides long, with 2'-deoxynucleotides at positions 2, 5, 7, and 12 of the 5' end of the antisense strand, 2'-fluoronucleotides at position 14, and 2'-methoxynucleotides at the remaining positions. In some embodiments of the present invention, the sense strand of the siRNA is 21 nucleotides long, with 2'-fluoronucleotides at positions 9, 10, and 11 of the 5' end of the sense strand, and 2'-methoxynucleotides at the remaining positions.

[0042] In some embodiments of the present invention, the antisense strand of the siRNA is 23 nucleotides long, with 2'-deoxynucleotides at positions 2, 5, 7, and 12 of the 5' end of the antisense strand, 2'-fluoronucleotides at positions 6, 8, 9, 10, 14, and 16, and 2'-methoxynucleotides at the remaining positions. In some embodiments of the present invention, the sense strand of the siRNA is 21 nucleotides long, with 2'-fluoronucleotides at positions 7, 9, 10, and 11 of the 5' end of the sense strand, and 2'-methoxynucleotides at the remaining positions.

[0043] In some embodiments of the present invention, the antisense strand of the siRNA is 21 nucleotides long, with 2'-fluoronucleotides at positions 2, 6, 14, and 16 of the 5' end of the antisense strand, and the remaining positions being 2'-methoxynucleotides; the sense strand of the siRNA is 21 nucleotides long, with 2'-fluoronucleotides at positions 5, 7, and 9 of the 5' end of the sense strand, and the remaining positions being 2'-methoxynucleotides.

[0044] In some embodiments of the present invention, the antisense strand of the siRNA is 21 nucleotides long, with 2'-fluoronucleotides at positions 2, 6, 14, and 16 of the 5' end of the antisense strand, and the remaining positions being 2'-methoxynucleotides; the sense strand of the siRNA is 21 nucleotides long, with 2'-fluoronucleotides at positions 7, 9, 10, and 11 of the 5' end of the sense strand, and the remaining positions being 2'-methoxynucleotides.

[0045] In some embodiments of the present invention, the antisense strand of the siRNA is 21 nucleotides long, with 2'-fluoronucleotides at positions 2, 4, 6, 8, 10, 12, 14, 16, 18, and 20 of the 5' end of the antisense strand, and the remaining positions being 2'-methoxynucleotides; the sense strand of the siRNA is 21 nucleotides long, with 2'-fluoronucleotides at positions 9, 10, and 11 of the 5' end of the sense strand, and the remaining positions being 2'-methoxynucleotides.

[0046] In some embodiments of the present invention, the antisense strand of the siRNA is 21 nucleotides long, with 2'-fluoronucleotides at positions 2, 4, 6, 8, 10, 12, 14, 16, 18, and 20 of the 5' end of the antisense strand, and the remaining positions being 2'-methoxynucleotides; the sense strand of the siRNA is 21 nucleotides long, with 2'-fluoronucleotides at positions 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, and 21 of the 5' end of the sense strand, and the remaining positions being 2'-methoxynucleotides.

[0047] In some embodiments of the present invention, the antisense strand of the siRNA is 21 nucleotides long, with 2'-fluoronucleotides at positions 2, 6, 14, and 16 of the 5' end of the antisense strand, and 2'-methoxynucleotides at the remaining positions. The sense strand of the siRNA is 19 nucleotides long, with 2'-fluoronucleotides at positions 7, 8, and 9 of the 5' end of the sense strand, and 2'-methoxynucleotides at the remaining positions.

[0048] In some embodiments of the present invention, the antisense strand of the siRNA is 19 nucleotides long, with 2'-fluoronucleotides at positions 2, 4, 6, 8, 10, 12, 14, 16, and 18 of the 5' end of the antisense strand, and the remaining positions being 2'-methoxynucleotides; the sense strand of the siRNA is 19 nucleotides long, with 2'-fluoronucleotides at positions 7, 8, and 9 of the 5' end of the sense strand, and the remaining positions being 2'-methoxynucleotides.

[0049] In some embodiments of the present invention, the antisense strand of the siRNA is 23 nucleotides long, with the 14th position at the 5' end of the antisense strand being a 2'-fluoronucleotide, the 2nd, 5th, and 7th positions being 2'-deoxynucleotides, the 12th position being a threose nucleotide, and the remaining positions being 2'-methoxynucleotides; the sense strand of the siRNA is 21 nucleotides long, with the 9th, 10th, and 11th positions at the 5' end of the sense strand being 2'-fluoronucleotides, and the remaining positions being 2'-methoxynucleotides.

[0050] In some embodiments of the present invention, the antisense strand of the siRNA is 23 nucleotides long, with 2'-fluoronucleotides at positions 2, 6, 14, and 16 of the 5' end of the antisense strand, and the remaining positions being 2'-methoxynucleotides; the sense strand of the siRNA is 21 nucleotides long, with 2'-fluoronucleotides at positions 9, 10, and 11 of the 5' end of the sense strand, with position 1 being a threose nucleotide, and the remaining positions being 2'-methoxynucleotides.

[0051] In some embodiments of the present invention, the antisense strand of the siRNA is 23 nucleotides long, with 2'-fluoronucleotides at positions 2, 6, 14, and 16 of the 5' end of the antisense strand, threose nucleotide at position 22, and 2'-methoxy nucleotides at the remaining positions; the sense strand of the siRNA is 21 nucleotides long, with 2'-fluoronucleotides at positions 9, 10, and 11 of the 5' end of the sense strand, and 2'-methoxy nucleotides at the remaining positions.

[0052] In some embodiments of the present invention, the antisense strand of the siRNA is 23 nucleotides long, with 2'-fluoronucleotides at positions 2, 6, 14, and 16 of the 5' end of the antisense strand, threose nucleotide at position 23, and 2'-methoxy nucleotides at the remaining positions; the sense strand of the siRNA is 21 nucleotides long, with 2'-fluoronucleotides at positions 9, 10, and 11 of the 5' end of the sense strand, and 2'-methoxy nucleotides at the remaining positions.

[0053] In some embodiments of the present invention, the antisense strand of the siRNA is 22 nucleotides long, with 2'-fluoronucleotides at positions 2, 6, 14, and 16 of the 5' end of the antisense strand, and the remaining positions being 2'-methoxynucleotides; the sense strand of the siRNA is 20 nucleotides long, with 2'-fluoronucleotides at positions 8, 9, and 10 of the 5' end of the sense strand, with 1 being a threose nucleotide, and the remaining positions being 2'-methoxynucleotides.

[0054] In some embodiments of the present invention, the antisense strand of the siRNA is 22 nucleotides long, with 2'-fluoronucleotides at positions 2, 6, 14, and 16 of the 5' end of the antisense strand, threose nucleotide at position 21, and 2'-methoxynucleotides at the remaining positions; the sense strand of the siRNA is 20 nucleotides long, with 2'-fluoronucleotides at positions 8, 9, and 10 of the 5' end of the sense strand, and 2'-methoxynucleotides at the remaining positions.

[0055] In some embodiments of the present invention, the antisense strand of the siRNA is 22 nucleotides long, with 2'-fluoronucleotides at positions 2, 6, 14, and 16 of the 5' end of the antisense strand, threose nucleotide at position 22, and 2'-methoxy nucleotides at the remaining positions; the sense strand of the siRNA is 20 nucleotides long, with 2'-fluoronucleotides at positions 8, 9, and 10 of the 5' end of the sense strand, and 2'-methoxy nucleotides at the remaining positions.

[0056] In some embodiments of the present invention, the antisense strand of the siRNA is 22 nucleotides long, with 2'-fluoronucleotides at positions 2, 6, 14, and 16 of the 5' end of the antisense strand, and the remaining positions being 2'-methoxynucleotides; the sense strand of the siRNA is 20 nucleotides long, with 2'-fluoronucleotides at positions 8, 9, and 10 of the 5' end of the sense strand, and the remaining positions being 2'-methoxynucleotides.

[0057] In some embodiments of the present invention, the antisense strand of the siRNA is 22 nucleotides long, with 2'-fluoronucleotides at positions 2, 6, 14, and 16 of the 5' end of the antisense strand, and the remaining positions being 2'-methoxynucleotides; the sense strand of the siRNA is 20 nucleotides long, with 2'-fluoronucleotides at positions 6, 8, 9, and 10 of the 5' end of the sense strand, and the remaining positions being 2'-methoxynucleotides.

[0058] In some embodiments of the present invention, the antisense strand of the siRNA is 22 nucleotides long, with 2'-fluoronucleotides at positions 2, 6, 14, and 16 of the 5' end of the antisense strand, and the remaining positions being 2'-methoxynucleotides; the sense strand of the siRNA is 20 nucleotides long, with 2'-fluoronucleotides at positions 8, 9, and 10 of the 5' end of the sense strand, 2'-deoxynucleotide at position 6, and the remaining positions being 2'-methoxynucleotides.

[0059] In some embodiments of the present invention, the antisense strand of the siRNA is 22 nucleotides long, with 2'-fluoronucleotides at positions 2, 6, 8, 9, 14, and 16 of the 5' end of the antisense strand, and the remaining positions being 2'-methoxynucleotides; the sense strand of the siRNA is 20 nucleotides long, with 2'-fluoronucleotides at positions 6, 8, 9, and 10 of the 5' end of the sense strand, and the remaining positions being 2'-methoxynucleotides.

[0060] In some embodiments of the present invention, the antisense strand of the siRNA is 22 nucleotides long, with the 14th position at the 5' end of the antisense strand being a 2'-fluoronucleotide, the 2nd, 5th, and 7th positions being 2'-deoxynucleotides, the 12th position being a threose nucleotide, and the remaining positions being 2'-methoxynucleotides; the sense strand of the siRNA is 20 nucleotides long, with the 8th, 9th, and 10th positions at the 5' end of the sense strand being 2'-fluoronucleotides, and the remaining positions being 2'-methoxynucleotides.

[0061] In some embodiments of the Future, the antisense chain of siRNA is 22 nucleotides long, with 3 to 10 positions on the antisense chain (e.g., positions 2, 14, 16; positions 2, 5, 14, 16; positions 2, 4, 6, 14, 16; positions 2, 6, 10, 14, 16; positions 2, 6, 12, 14, 16; positions 2, 5, 10, 14, 16; positions 2, 3, 12, 14, 16; positions 2, 9, 12, 14, 16) ;Positions 2, 6, 8, 9, 14, 16;Positions 2, 3, 5, 12, 14, 16;Positions 2, 8, 9, 12, 14, 16;Positions 2, 7, 9, 12, 14, 16;Positions 2, 4, 6, 8, 10, 14, 16, 18, 20;Positions 2, 4, 5, 6, 8, 10, 12, 14, 16, 18 at the 5' end) are 2'-fluoronucleotides, and the remaining positions are 2'-methoxynucleotides. The sense strand of the siRNA is 20 nucleotides long, with positions 8, 9, and 10 at the 5' end of the sense strand being 2'-fluoronucleotides, one position (preferably position 1 at the 5' end) being a threose nucleotide, one position (preferably position 6 at the 5' end) being a 2'-deoxynucleotide, and the remaining positions being 2'-methoxynucleotide conjugates.

[0062] In some embodiments of the Future, the antisense chain of siRNA is 22 nucleotides long, and the 5' end of the antisense chain is at the following positions: 2, 14, 16; or 2, 5, 14, 16; or 2, 6, 14, 16; or 2, 6, 10, 14, 16; or 2, 6, 12, 14, 16; or 2, 6, 12, 14, 16; or 2, 5, 10, 14, 16; or 2, 3, 12, 14, 16; or 2, 9, 12, 14, 1 Position 6; or positions 2, 6, 8, 9, 14, 16; or positions 2, 3, 5, 12, 14, 16; or positions 2, 8, 9, 12, 14, 16; or positions 2, 7, 9, 12, 14, 16; or positions 2, 4, 6, 8, 10, 14, 16, 18, 20; or positions 2, 4, 5, 6, 8, 10, 12, 14, 16, 18 are 2'-fluoronucleotides, and the remaining positions are 2'-methoxynucleotides; The sense strand of siRNA is 20 nucleotides long, with positions 8, 9, and 10 at the 5' end of the sense strand being 2'-fluoronucleotides, optionally, position 1 at the 5' end being a threose nucleotide, or optionally, position 6 at the 5' end being a 2'-deoxynucleotide, and the remaining positions being 2'-methoxynucleotides.

[0063] In some embodiments of the Future, the antisense chain of siRNA is 22 nucleotides long, and the 5' end of the antisense chain is at the following positions: 2, 14, 16; or 2, 5, 14, 16; or 2, 4, 6, 14, 16; or 2, 6, 10, 14, 16; or 2, 6, 12, 14, 16; or 2, 5, 10, 14, 16; or 2, 3, 12, 14, 16; or 2, 9, 12, 14, 16; or 2, 6, 8, 9, 14, 16; or 2, 3, 5, 12, 1 Positions 4 and 16; or 2, 8, 9, 12, 14, 16; or 2, 7, 9, 12, 14, 16; or 2, 4, 6, 8, 10, 14, 16, 18, 20; or 2, 4, 5, 6, 8, 10, 12, 14, 16, 18, 20 are 2'-fluoronucleotides, and the remaining positions are 2'-methoxynucleotides; the sense strand of the siRNA is 20 nucleotides long, with positions 8, 9 and 10 at the 5' end of the sense strand being 2'-fluoronucleotides, and the remaining positions being 2'-methoxynucleotides.

[0064] In some embodiments of the present invention, the antisense strand of the siRNA is 21 nucleotides long, with 2'-fluoronucleotides at positions 2, 6, 14, and 16 of the 5' end of the antisense strand, and the remaining positions being 2'-methoxynucleotides; the sense strand of the siRNA is 19 nucleotides long, with 2'-fluoronucleotides at positions 7, 8, and 9 of the 5' end of the sense strand, with position 1 being a threose nucleotide, and the remaining positions being 2'-methoxynucleotides.

[0065] In some embodiments of the present invention, the antisense strand of the siRNA is 21 nucleotides long, with 2'-fluoronucleotides at positions 2, 6, 14, and 16 of the 5' end of the antisense strand, threose nucleotide at position 20, and 2'-methoxynucleotides at the remaining positions; the sense strand of the siRNA is 19 nucleotides long, with 2'-fluoronucleotides at positions 7, 8, and 9 of the 5' end of the sense strand, and 2'-methoxynucleotides at the remaining positions.

[0066] In some embodiments of the present invention, the antisense strand of the siRNA is 21 nucleotides long, with 2'-fluoronucleotides at positions 2, 6, 14, and 16 of the 5' end of the antisense strand, threose nucleotide at position 21, and 2'-methoxy nucleotides at the remaining positions; the sense strand of the siRNA is 19 nucleotides long, with 2'-fluoronucleotides at positions 7, 8, and 9 of the 5' end of the sense strand, and 2'-methoxy nucleotides at the remaining positions.

[0067] In some embodiments of the present invention, the antisense strand of the siRNA is 21 nucleotides long, with the 14th position at the 5' end of the antisense strand being a 2'-fluoronucleotide, the 2nd, 5th, and 7th positions being 2'-deoxynucleotides, the 12th position being a threose nucleotide, and the remaining positions being 2'-methoxynucleotides; the sense strand of the siRNA is 19 nucleotides long, with the 7th, 8th, and 9th positions at the 5' end of the sense strand being 2'-fluoronucleotides, and the remaining positions being 2'-methoxynucleotides.

[0068] In some embodiments of the present invention, the antisense strand of siRNA is 22 nucleotides long, and the antisense strand has 3 to 10 positions (e.g., 2, 14, 16; 2, 5, 14, 16; 2, 6, 14, 16; 2, 4, 6, 14, 16; 2, 6, 10, 14, 16; 2, 6, 12th, 14th, 16th; 2nd, 5th, 10th, 14th, 16th; 2nd, 3rd, 12th, 14th, 16th; 2nd, 9th, 12th, 14th, 16th; 2nd, 6th, 8th 2nd, 3rd, 5th, 12th, 14th, 16th; 2nd, 8th, 9th, 12th, 14th, 16th; 2nd, 7th, 9th, 12th, 14th, 16th The sense strand of the siRNA is 20 nucleotides long, with positions 8, 9 and 10 of the 5' end of the sense strand being 2'-fluoronucleotides, optionally one position (preferably position 1 of the 5' end) being a threose nucleotide, optionally one position (preferably position 6 of the 5' end) being a 2'-deoxynucleotide, and the remaining positions being 2'-methoxynucleotide conjugates.

[0069] In some embodiments of the Future, the antisense chain of siRNA is 22 nucleotides long, and the 5' end of the antisense chain is at the following positions: 2, 14, 16; or 2, 5, 14, 16; or 2, 6, 14, 16; or 2, 4, 6, 14, 16; or 2, 6, 10, 14, 16; or 2, 6, 12, 14, 16; or 2, 5, 10, 14, 16 ; or 2nd, 3rd, 12th, 14th, 16th; or 2nd, 9th, 12th, 14th, 16th; or 2nd, 6th, 8th, 9th, 14th, 16th; or 2nd, 3rd, 5th, 12th, 14th, 16th; or 2nd, 8th, 9th, 12th, 14th, 16th; or 2nd, 7th, 9th, 12th, 14th, 16th; or 2nd, 4th, 6th, 8th, 10th, 14th, 16th, 18th, 20th; or 2nd, 4th, 5th, 6th, 8th, 1 Positions 0, 12, 14, 16, and 18 are 2'-fluoronucleotides, and the remaining positions are 2'-methoxynucleotides. The sense strand of the siRNA is 20 nucleotides long, and positions 8, 9, and 10 at the 5' end of the sense strand are 2'-fluoronucleotides. Optionally, position 1 at the 5' end is a threose nucleotide, or optionally, position 6 at the 5' end is a 2'-deoxynucleotide, and the remaining positions are 2'-methoxynucleotides. Preferably, the 2'-deoxynucleotide at position 6 is independently selected from deoxyadenosine monophosphate (dA), deoxyguanosine monophosphate (dG), deoxycytidine monophosphate (dC), deoxythymidine monophosphate (dT), and deoxyuridine monophosphate (dU); more preferably, the 2'-deoxynucleotide at position 6 is deoxythymidine monophosphate (dT).

[0070] In some embodiments of the Future, the antisense chain of siRNA is 22 nucleotides long, and the 5' end of the antisense chain is at the following positions: 2, 14, 16; or 2, 5, 14, 16; or 2, 6, 14, 16; or 2, 4, 6, 14, 16; or 2, 6, 10, 14, 16; or 2, 6, 12, 14, 16; or 2, 5, 10, 14, 16; or 2, 3, 12, 14, 16; or 2, 9, 12, 14, 16; or 2, 6, 8, 9, 14, 16; or 2, 3, 5 , 12th, 14th, 16th; or 2nd, 8th, 9th, 12th, 14th, 16th; or 2nd, 7th, 9th, 12th, 14th, 16th; or 2nd, 4th, 6th, 8th, 10th, 14th, 16th, 18th, 20th; or 2nd, 4th, 5th, 6th, 8th, 10th, 12th, 14th, 16th, 18th are 2'-fluoronucleotides, and the remaining positions are 2'-methoxynucleotides; the sense strand of the siRNA is 20 nucleotides long, and the 8th, 9th, and 10th positions at the 5' end of the sense strand are 2'-fluoronucleotides, and the remaining positions are 2'-methoxynucleotides; or The antisense strand of siRNA is 22 nucleotides long, with positions 2, 6, 14, and 16 at the 5' end of the antisense strand being 2'-fluoronucleotides, and the remaining positions being 2'-methoxynucleotides; the sense strand of siRNA is 20 nucleotides long, with positions 8, 9, and 10 at the 5' end of the sense strand being 2'-fluoronucleotides, position 1 being a threose nucleotide, and the remaining positions being 2'-methoxynucleotides; or The antisense strand of siRNA is 22 nucleotides long, with 2'-fluoronucleotides at positions 2, 6, 14, and 16 of the 5' end of the antisense strand, and 2'-methoxynucleotides at the remaining positions; the sense strand of siRNA is 20 nucleotides long, with 2'-fluoronucleotides at positions 8, 9, and 10 of the 5' end of the sense strand, 2'-deoxynucleotide at position 6, and 2'-methoxynucleotides at the remaining positions; preferably, 2'-deoxynucleotide at position 6. The nucleotide is independently selected from deoxyadenosine monophosphate (dA), deoxyguanosine monophosphate (dG), deoxycytidine monophosphate (dC), deoxythymidine monophosphate (dT), and deoxyuridine monophosphate (dU), more preferably the 2'-deoxynucleotide at position 6 is deoxythymidine monophosphate (dT); more preferably the antisense strand of the siRNA is the nucleotide sequence shown in SEQ ID NO: 105, and the sense strand of the siRNA is the nucleotide sequence shown in SEQ ID NO: 23.

[0071] In some embodiments of the present invention, the antisense strand of the siRNA is 22 nucleotides long, with 2'-fluoronucleotides at positions 2, 6, 14, and 16 of the 5' end of the antisense strand, and the remaining positions being 2'-methoxynucleotides; the sense strand of the siRNA is 20 nucleotides long, with 2'-fluoronucleotides at positions 8, 9, and 10 of the 5' end of the sense strand, and the remaining positions being 2'-methoxynucleotides; Preferably, the antisense strand of the siRNA is the nucleotide sequence shown in SEQ ID NO: 116, and the sense strand of the siRNA is the nucleotide sequence shown in SEQ ID NO: 22.

[0072] In some embodiments of the present invention, the antisense strand of the siRNA is 22 nucleotides long, with the 14th position at the 5' end of the antisense strand being a 2'-fluoronucleotide, the 2nd, 5th, and 7th positions being 2'-deoxynucleotides, the 12th position being a threose nucleotide, and the remaining positions being 2'-methoxynucleotides; the sense strand of the siRNA is 20 nucleotides long, with the 8th, 9th, and 10th positions at the 5' end of the sense strand being 2'-fluoronucleotides, and the remaining positions being 2'-methoxynucleotides; Preferably, the 2'-deoxyribonucleotides at positions 2, 5, and 7 are independently selected from deoxyadenosine monophosphate (dA), deoxyguanosine monophosphate (dG), deoxycytidine monophosphate (dC), deoxythymidine monophosphate (dT), and deoxyuridine monophosphate (dU); more preferably, the 2'-deoxyribonucleotides at positions 2, 5, and 7 are deoxythymidine monophosphate (dT), deoxythymidine monophosphate (dT), and deoxyadenosine monophosphate (dA), respectively; More preferably, the antisense strand of the siRNA is the nucleotide sequence shown in SEQ ID NO: 105, and the sense strand of the siRNA is the nucleotide sequence shown in SEQ ID NO: 23.

[0073] In some embodiments of the present invention, the antisense strand of the siRNA is 21 nucleotides long, with positions 2, 6, 14, and 16 of the 5' end of the antisense strand being 2'-fluoronucleotides and the remaining positions being 2'-methoxynucleotides; the sense strand of the siRNA is 19 nucleotides long, with positions 7, 8, and 9 of the 5' end of the sense strand being 2'-fluoronucleotides, position 1 being a threose nucleotide and the remaining positions being 2'-methoxynucleotides; or The antisense strand of siRNA is 21 nucleotides long, with the 14th position at the 5' end being a 2'-fluoronucleotide, the 2nd, 5th, and 7th positions being 2'-deoxynucleotides, the 12th position being a threose nucleotide, and the remaining positions being 2'-methoxynucleotides. The sense strand of siRNA is 19 nucleotides long, with the 7th, 8th, and 9th positions at the 5' end being 2'-fluoronucleotides, and the remaining positions being 2'-methoxynucleotides. Preferably, the 2'-deoxynucleotides at positions 2, 5, and 7 are independently selected from deoxyadenosine monophosphate (dA), deoxyguanosine monophosphate (dG), deoxycytidine monophosphate (dC), deoxythymidine monophosphate (dT), and deoxyuridine monophosphate (dU); More preferably, the 2'-deoxynucleotides at positions 2, 5, and 7 are deoxythymidine monophosphate (dT), deoxyuridine monophosphate (dU), and deoxyadenosine monophosphate (dA), respectively; More preferably, the antisense strand of the siRNA is the nucleotide sequence shown in SEQ ID NO: 104, and the sense strand of the siRNA is the nucleotide sequence shown in SEQ ID NO: 3.

[0074] In some embodiments of the present invention, the modified nucleotide is a nucleotide in which its phosphate group is modified with a phosphorothioate group. That is, a sulfur atom substitutes for a non-crosslinked oxygen atom in the phosphate diester bond, thereby substituting the phosphate diester bond with a phosphorothioate bond.

[0075] In some embodiments of the present invention, the 5' and 3' ends of the sense chain each independently contain 0, 1, or 2 phosphorothioate groups; and / or, the 5' and 3' ends of the antisense chain each independently contain 1 or 2 phosphorothioate groups.

[0076] In some embodiments of the present invention, at least one bond is a phosphorothioate bond between the nucleotides at positions 1 and 2 of the 5' end of the sense strand, between the nucleotides at positions 2 and 3 of the 5' end of the sense strand, between the nucleotides at positions 1 and 2 of the 3' end of the sense strand, between the nucleotides at positions 2 and 3 of the 3' end of the sense strand, between the nucleotides at positions 1 and 2 of the 3' end of the antisense strand, between the nucleotides at positions 2 and 3 of the 3' end of the antisense strand, between the nucleotides at positions 1 and 2 of the 5' end of the antisense strand, and between the nucleotides at positions 2 and 3 of the 5' end of the antisense strand; preferably, at least four bonds are phosphorothioate bonds; in some embodiments of the present invention, at least six bonds are phosphorothioate bonds; in some embodiments of the present invention, all eight bonds are phosphorothioate bonds.

[0077] In some embodiments of the present invention, the bonds between the nucleotides at positions 1 and 2, and between the nucleotides at positions 2 and 3 of the 5' end of the sense strand, are phosphorothioate bonds.

[0078] In some embodiments of the present invention, the bonds between the nucleotides at positions 1 and 2, and between the nucleotides at positions 2 and 3, at the 5' end of the sense strand are phosphorothioate bonds, and the bonds between the nucleotides at positions 1 and 2, and between the nucleotides at positions 2 and 3, at the 3' end of the sense strand are also phosphorothioate bonds.

[0079] In some embodiments of the present invention, the bonds between the nucleotides at positions 1 and 2, and between the nucleotides at positions 2 and 3 of the 3' end of the antisense chain are phosphorothioate bonds, and the bonds between the nucleotides at positions 1 and 2, and between the nucleotides at positions 2 and 3 of the 5' end are also phosphorothioate bonds.

[0080] In some embodiments of the present invention, the bonds between the nucleotides at positions 1 and 2 of the 5' end of the sense strand, between the nucleotides at positions 2 and 3 of the 5' end of the sense strand, between the nucleotides at positions 1 and 2 of the 3' end of the sense strand, between the nucleotides at positions 2 and 3 of the 3' end of the sense strand, between the nucleotides at positions 1 and 2 of the 3' end of the antisense strand, between the nucleotides at positions 2 and 3 of the 3' end of the antisense strand, between the nucleotides at positions 1 and 2 of the 5' end of the antisense strand, and between the nucleotides at positions 2 and 3 of the 5' end of the antisense strand are all phosphorothioate bonds.

[0081] In some embodiments of the present invention, the sense strand may contain one or more capping residues or portions referred to as “capping residues.” A “capping residue” is a non-nucleotide compound or other portion that can be incorporated into one or more ends of the nucleotide sequence of the siRNA. In some embodiments of the present invention, the capping residues are located at the 5' end, the 3' end, or both the 5' and 3' ends of the sense strand.

[0082] In some embodiments of the present invention, an inverted debase residue (iab or invAb) is added as a capping residue. See F. Czauderna, Nucleic Acids Res., 2003, 31(11), 2705-16. In some embodiments of the present invention, the 5' and / or 3' ends of the sense strand may contain two or more inverted deoxy-basic deoxyribose moieties as capping residues.

[0083] In some embodiments of the present invention, one or more inverted debasing residues (iab or invAb) are added to the 3' end of the sense strand. In some embodiments of the present invention, one or more inverted debasing residues (iab or invAb) are added to the 5' end of the sense strand. In some embodiments of the present invention, one or more inverted debasing residues may be inserted between the delivery carrier portion and the nucleotide sequence of the sense strand of the siRNA. In some embodiments of the present invention, one or more inverted debasing residues are included at or near one or more ends of the sense strand of the siRNA. The inverted debasing residues (iab or invAb) are selected from the following structures: [ka] In the equation, X = O or S.

[0084] In some embodiments of the present invention, one or more inverted debase residues (iab or invAb) are added to the 5' end of the sense strand. In some embodiments of the present invention, one or more inverted debase residues may be inserted between the delivery carrier portion and the nucleotide sequence of the sense strand of the siRNA.

[0085] Inverted debase residues can be linked via phosphate bonds, phosphorothioate bonds, or other nucleoside bonds.

[0086] In some embodiments of the present invention, the first nucleotide at the 5' end of the antisense strand is selected from the following structures: [ka] In the formula, Basse represents a base A, U, G, C, T, or other nucleotide base. In the formula, Basse represents a base A, U, G, C, T, or other nucleotide base.

[0087] In some embodiments of the present invention, the first nucleotide at the 5' end of the antisense strand is an (E)-vinyl phosphate-modified nucleotide.

[0088] In some embodiments of the present invention, the siRNA comprises at least one base-modified nucleotide.

[0089] In some embodiments of the present invention, the base of the base-modified nucleotide is selected from the following structures: [ka] [ka]

[0090] In some embodiments of the present invention, the base-modified nucleotides are located at positions 5, 6, 7, and 8 of the antisense strand of the siRNA.

[0091] In some embodiments of the present invention, the base-modified nucleotide is located in a single-stranded nucleotide overhang within the siRNA.

[0092] Preferably, the antisense strand of the siRNA includes a two-nucleotide overhang, and the base-modified nucleotide is the first nucleotide of the antisense strand overhang of the siRNA.

[0093] Preferably, the antisense strand of the siRNA includes a two-nucleotide overhang, and the base-modified nucleotide is the second nucleotide of the antisense strand overhang of the siRNA.

[0094] The present invention also provides an siRNA conjugate obtained by conjugating the above-mentioned siRNA with a conjugate molecule.

[0095] In the present invention, unless otherwise specified, "conjugation" refers to the covalent linkage between two or more chemical moieties; "conjugate" refers to a compound formed by the covalent linkage of various chemical moieties; and "siRNA conjugate" refers to a compound in which one or more chemical moieties are covalently linked to siRNA. Note that the chemical moieties may be directly bound to siRNA or bound to siRNA via a linker.

[0096] In some embodiments of the present invention, the delivery carrier forms a conjugate molecule and is bound to the siRNA via covalent bonding.

[0097] In some embodiments of the present invention, the delivery carrier forms a conjugate molecule that is independently or simultaneously bound to the 3' or 5' end of the sense strand of the siRNA.

[0098] In some embodiments of the present invention, an siRNA conjugate can be obtained by conjugating the siRNA of the present invention with a pharmaceutically acceptable conjugate molecule. In some embodiments of the present invention, the siRNA is covalently conjugated to the conjugate molecule. To reduce the potential impact of conjugation on siRNA activity, the conjugation site between the siRNA and the conjugate molecule may be located at the 3' or 5' end of the sense strand of the siRNA, or at the 5' end of the antisense strand. In some embodiments, the conjugation site of the siRNA to the conjugate molecule may be located within the internal sequence of the siRNA.

[0099] In the present invention, "delivery carrier" refers to a chemical moiety covalently bound to an siRNA that affects the targeting, activity, intracellular distribution, cellular uptake, or stability of the oligonucleotide. Conjugation of an siRNA to one or more delivery carriers can improve the pharmacological properties of the siRNA. In some embodiments, the delivery carrier moiety modifies or enhances the pharmacokinetic properties of the oligonucleotide by improving its intracellular distribution, bioavailability, metabolism, excretion, permeability, or cellular uptake. Specifically, the delivery carrier can target the oligonucleotide to a specific organ, tissue, or cell type, thereby increasing the efficacy of the oligonucleotide in that organ, tissue, or cell type. At the same time, the delivery carrier moiety can be used to reduce the activity of the oligonucleotide in non-target cell types, tissues, or organs, e.g., off-target activity, or activity in non-target cell types, tissues, or organs.

[0100] Pharmacoherent delivery carriers may be, but are not limited to, one or more of the following delivery carriers or derivatives thereof, conventionally used in the field of siRNA delivery: lipophilic molecules, e.g., cholesterol, bile acids, vitamins (e.g., vitamin E), lipid molecules of different chain lengths; polymers, e.g., polyethylene glycol; polypeptides, e.g., transmembrane peptides; aptamers; antibodies; quantum dots; carbohydrates, e.g., lactose, polylactose, mannose, galactose, N-acetylgalactosamine (GalNAc); folic acid or folate; or receptor ligands expressed by hepatocytes, e.g., asialoclycoproteins, desialylated sugar residues, lipoproteins (e.g., high-density lipoproteins, low-density lipoproteins, etc.), glucagon, neurotransmitters (e.g., adrenaline), growth factors, transferrin, etc.

[0101] In some embodiments of the present invention, the delivery carrier comprises an N-acetylgalactosamine group.

[0102] In some embodiments of the present invention, the delivery carrier is directly bound to the 3' end of the sense strand of the siRNA. In some embodiments of the present invention, the delivery carrier is directly bound to the 5' end of the sense strand of the siRNA. In some embodiments of the present invention, the delivery carrier is bound to the 3' end of the sense strand of the siRNA via an adapter. In some embodiments of the present invention, the delivery carrier is bound to the 5' end of the sense strand of the siRNA via an adapter.

[0103] In some embodiments of the present invention, the delivery carrier comprises N-acetylgalactosamine covalently bound to the 3' end of the sense strand of the siRNA.

[0104] In some embodiments of the present invention, the delivery carrier portion is GalNAc(L96) and has the following structure: [ka]

[0105] In some embodiments of the present invention, GalNAc(L96) is bound to the 3' end of the sense strand of the siRNA.

[0106] In some embodiments of the present invention, GalNAc(L96) is bound to the 5' end of the sense strand of the siRNA.

[0107] In some embodiments of the present invention, GalNAc(L96) is bound to an inverted debase residue (iab or invAb) at the 3' end of the sense strand of the siRNA.

[0108] In some embodiments of the present invention, GalNAc(L96) is bound to an inverted debase residue (iab or invAb) at the 5' end of the sense strand of the siRNA.

[0109] In some embodiments of the present invention, the delivery carrier portion is Ser(GN) and has the following structure: [ka]

[0110] In some embodiments of the present invention, Ser(GN) is bound to the 3' end of the sense strand of the siRNA. In some embodiments of the present invention, Ser(GN) is bound to the 5' end of the sense strand of the siRNA.

[0111] In some embodiments of the present invention, the Ser(GN) moiety is bound to both the 3' and 5' ends of the sense strand of the siRNA.

[0112] In some embodiments of the present invention, the delivery carrier portion is LP-GalNAc (bonded to the 5' end of the sense chain) and has the following structure: [ka]

[0113] In some embodiments of the present invention, the delivery carrier portion is an XY-GalNAc having the following structure (bound to the 3' end of the sense strand). [ka] or [ka]

[0114] In some embodiments of the present invention, the structures of other delivery carrier portions used (bonded to the 5' end of the sense chain) are as follows: [ka] [ka] [ka]

[0115] In some embodiments of the present invention, the structures of other delivery carrier portions used (bonded to the 3' end of the sense chain) are as follows: [ka] [ka]

[0116] Selectively, the delivery carrier moiety is bound to the 3' end of the siRNA sense strand, or the 5' end of the siRNA sense strand, or to the inverted debase residue (iab) at the 3' end of the siRNA sense strand, or to the inverted debase residue (iab) at the 5' end of the siRNA sense strand. Alternatively, one or more delivery carrier moieties are simultaneously bound to the 3' end of the siRNA sense strand, the 5' end of the siRNA sense strand, the inverted debase residue (iab) at the 3' end of the siRNA sense strand, and the inverted debase residue (iab) at the 5' end of the siRNA sense strand. Preferably, the delivery carrier moiety GalNAc(L96) is bound to the 3' end of the siRNA sense strand or to the inverted debase residue (iab) at the 3' end of the siRNA sense strand. The delivery carrier moiety Ser(GN) is bound to both the 3' and 5' ends of the siRNA sense strand. The delivery carrier moiety LP-GalNAc is bound to the 5' end of the siRNA sense strand. The delivery carrier portion XY-GalNAc is bound to the 3' end of the siRNA sense strand. The delivery carrier portion GalNAc(Ser2) is bound to either the 3' or 5' end of the siRNA sense strand, or both. The delivery carrier portion GalNAc(Ser3) is bound to either the 3' or 5' end of the siRNA sense strand. The delivery carrier portion GalNAc(Ser4) is bound to the 3' end of the siRNA sense strand. The delivery carrier portion GalNAc(A1GN) is bound to the 3' end of the siRNA sense strand. The delivery carrier portion GalNAc(A2GN) is bound to the 3' end of the siRNA sense strand. The delivery carrier portion GalNAc(A3GN) is bound to the 5' end of the siRNA sense strand. The delivery carrier portion GalNAc(A4GN) is bound to the 5' end of the siRNA sense strand. The delivery carrier portion GalNAc(A5GN) is bound to the 5' end of the sense strand of the siRNA; the delivery carrier portion GalNAc(NAG25) is bound to the 5' end of the sense strand of the siRNA; and the delivery carrier portion GalNAc(NAG37) is bound to the 5' end of the sense strand of the siRNA.The delivery carrier portions GalNAc(Ser1) and GalNAc(Ser2) are either simultaneously bound to the 3' and 5' ends of the siRNA sense strand, respectively, or simultaneously bound to the 5' and 3' ends of the siRNA sense strand, respectively. Preferably, the delivery carrier portion is selected from GalNAc(L96) bound to the 3' end of the siRNA sense strand, GalNAc(A1GN) bound to the 3' end of the siRNA sense strand, GalNAc(A2GN) bound to the 3' end of the siRNA sense strand, GalNAc(A3GN) bound to the 5' end of the siRNA sense strand, GalNAc(A4GN) bound to the 5' end of the siRNA sense strand, and GalNAc(Ser2) simultaneously bound to both the 3' and 5' ends of the siRNA sense strand.

[0117] The siRNA conjugates listed below are selected from conjugates 1 to 1668 and the positive control PC c. The unmodified nucleotide sequences of the sense and antisense strands of each conjugate correspond to one of the above-listed SEQ ID NOs: 1 to 200 and SEQ ID NOs: 201 to 351 listed herein, respectively. [Table 2-1] [Table 2-2] [Table 2-3] [Table 2-4] [Table 2-5] During the ceremony, C, G, U, A, and T represent the base portion of a nucleotide, including modified and unmodified nucleotides, and I represents the base portion of a modified nucleotide, where the base is [ka] Here, m6A represents the base portion of the modified nucleotide, where the base is [ka] Here, X represents the base portion of the modified nucleotide, where the base is [ka] Here, B represents the base portion of the modified nucleotide, where the base is [ka] That is the case.

[0118] In some embodiments of the present invention, the siRNA conjugate is selected from conjugates 1 to 1668. [Table 3-1] [Table 3-2] [Table 3-3] [Table 3-4] [Table 3-5] [Table 3-6] [Table 3-7] Table 3-8 Table 3-9 Table 3-10 Table 3-11 Table 3-12 Table 3-13 Table 3-14 Table 3-15 Table 3-16 Table 3-17 Table 3-18 Table 3-19 Table 3-20 Table 3-21 Table 3-22 Table 3-23 Table 3-24 Table 3-25 Table 3-26 Table 3-27 Table 3-28 Table 3-29 Table 3-30 Table 3-31 Table 3-32 Table 3-33 Table 3-34 Table 3-35 Table 3-36 Table 3-37 Table 3-38 Table 3-39 Table 3-40 Table 3-41 Table 3-42 Table 3-43 Table 3-44 Table 3-45 Table 3-46 Table 3-47 Table 3-48 Table 3-49 Table 3-50 Table 3-51 Table 3-52 Table 3-53 Table 3-54 Table 3-55 Table 3-56 Table 3-57 Table 3-58 Table 3-59 Table 3-60 Table 3-61 Table 3-62 Table 3-63 Table 3-64 Table 3-65 Table 3-66 Table 3-67 Table 3-68 Table 3-69 Table 3-70 Table 3-71 Table 3-72 Table 3-73 Table 3-74 Table 3-75 Table 3-76 Table 3-77 Table 3-78 Table 3-79 Table 3-80 Table 3-81 Table 3-82 Table 3-83 Table 3-84 Table 3-85 Table 3-86 Table 3-87 Table 3-88 Table 3-89

Table 3-90

[0119] The present invention also provides a pharmaceutical composition comprising any one of the above-mentioned siRNAs and / or any one of the above-mentioned siRNA conjugates and a pharmaceutically acceptable carrier.

[0120] In some embodiments of the present invention, the pharmaceutical composition comprises one siRNA as described in the first aspect. In other embodiments of the present disclosure, the pharmaceutical composition comprises at least two (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 or more, but not limited to) siRNAs as described in the first aspect as active ingredients. Preferably, the at least two siRNAs as described in the first aspect each target different target sequences in the AGT gene, thereby achieving a synergistic effect by acting simultaneously on different target sequences. Here, "different target sequences" means that there is no overlap between the target sequences, or that the number of overlapping consecutive nucleotides between the target sequences is less than 5 (e.g., the number of overlapping consecutive nucleotides is 4, 3, 2, 1 or 0). In this case, the at least two siRNAs as described in the first aspect may be present in any different proportions. Preferably, the at least two siRNAs described in the first embodiment may exist in a molar ratio of 1:100 to 100:1; more preferably, the at least two siRNAs described in the first embodiment may exist in a molar ratio of 1:10 to 10:1, 1:5 to 5:1, or 1:2 to 2:1. In some embodiments of the present invention, the at least two siRNAs described in the first embodiment exist in the same molar ratio.

[0121] The present invention also provides the use of any one of the above siRNAs and / or any one of the above siRNA conjugates and / or the above pharmaceutical composition in the manufacture of a pharmaceutical for treating and / or preventing a pathological condition or disease associated with AGT gene overexpression.

[0122] Furthermore, the pathological condition or disease is a disorder related to abnormal blood pressure.

[0123] The siRNAs, siRNA conjugates, and pharmaceutical compositions provided by the present invention exhibit good stability, excellent inhibitory activity against AGT genes, satisfactory cytotoxicity and immunostimulatory properties, and can significantly reduce blood pressure levels.

[0124] In the present invention, unless otherwise specified, uppercase C, G, U, A, and T represent the base portion of a nucleotide, including modified and unmodified nucleotides; lowercase m indicates that the nucleotide adjacent to the right of identifier m is a 2'-methoxynucleotide; lowercase f indicates that the nucleotide adjacent to the right of identifier f is a 2'-fluoronucleotide; lowercase d indicates that the nucleotide adjacent to the right of identifier d is a 2'-deoxynucleotide; gn indicates that the nucleotide adjacent to the right of identifier gn is a glycerol nucleotide (GNA); tn indicates that the nucleotide adjacent to the right of identifier tn is a threose nucleotide (TNA); *Identifier indicates that the bond between the two nucleotides adjacent to the left and right of identifier * (or between the nucleotide and the delivery carrier portion) is a phosphorothioate bond; eVP indicates that the nucleotide adjacent to its right is an (E)-vinyl phosphate-modified nucleotide, for example, when the base is U, the eVPmU structure is [ka] iab indicates an inverted debase residue; GalNAc(L96) means that the delivery carrier portion GalNAc(L96) is conjugated at this position. Ser(GN) means that the delivery carrier portion Ser(GN) is conjugated at this position.

[0125] In the present invention, unless otherwise specified, the capital letter I indicates the base portion of a base-modified nucleotide, where the base is [ka] m6A represents the base portion of a base-modified nucleotide, where the base is [ka] The capital letter X indicates the base portion of a base-modified nucleotide, where the base is [ka] The capital letter B indicates the base portion of a base-modified nucleotide, where the base is [ka] That is the case.

[0126] In the present invention, unless otherwise specified, the term “complementary” refers to the ability of an oligonucleotide of a first sequence to hybridize with an oligonucleotide of a second sequence under certain conditions to form a double-stranded structure. “At least partially complementary” means that the two sequences may be fully complementary or retain the ability to hybridize under relevant conditions while having a total of 5, 4, 3, or 2 or fewer mismatched base pairs. Furthermore, if two oligonucleotides are designed to form one or more single-stranded overhangs during hybridization, such overhangs should not be considered mismatches for determining complementarity. In the present invention, to satisfy the above hybridization ability requirements, “complementary” sequences may also include, or be entirely formed from, non-Watson-Crick base pairs and / or non-natural and modified nucleotides. Such non-Watson-Crick base pairs include, but are not limited to, G:U swing base pairings or Hoogstein base pairings. Accordingly, in this invention, unless otherwise specified, "mismatch" refers to a situation within an siRNA double-strand molecule where the bases at corresponding positions are not complementaryly paired.

[0127] In this invention, "difference in nucleotide sequence" refers to a change in the type of base at the same or corresponding position in the nucleotide sequence compared to the original nucleotide sequence, unless otherwise specified. For example, if the nucleotide base in the original nucleotide sequence is A, and the nucleotide base at the same or corresponding position is changed to U, C, G, or dT, dC, dG, then a difference in nucleotide sequence is considered to exist at that position. It should be noted that if, compared to the original nucleotide sequence, the nucleotide at the same or corresponding position differs only in the presence or type of modification, then a difference in nucleotide sequence is not considered to exist at that position.

[0128] In the present invention, unless otherwise specified, “pharmaceutically acceptable” means that the carrier, delivery system, diluent, excipient, and / or salts / esters / hydrates formed therefrom are generally chemically or physically compatible with the other components constituting the drug dosage form and physiologically compatible with the receptor.

[0129] In this invention, unless otherwise specified, "inhibition" refers to the downregulation of target gene expression by mRNA degradation via siRNA. "Downregulation" means a decrease of 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% or more, or even 100%, in the absence of siRNA treatment. A 100% decrease in target gene expression level means that no detectable target gene expression exists.

[0130] In the present invention, siRNA may also optionally contain modified nucleotides. These modified nucleotides do not significantly weaken or eliminate the function of siRNA in inhibiting AGT gene expression. Currently, various methods for modifying siRNA exist in the art, including, for example, skeletal modifications (e.g., phosphate group modifications), ribose group modifications, and base modifications (Watts, JK, GFDeleavey, and MJDamha, Chemically modified siRNA: tools and applications. Drug Discov Today, 2008, 13(19-20):842-55).

[0131] Clearly, based on the above description of the present invention and in accordance with common technical knowledge and prior methods in the art, various other modifications, substitutions, or changes can be made without departing from the basic technical concept of the present invention.

[0132] The above-described aspects of the present invention will be further explained by detailed embodiments of the examples. However, this should not be construed as limiting the scope of the above-described subject matter of the present invention to the following embodiments. All techniques implemented based on the above-described aspects of the present invention are included within the scope of the present invention. [Brief explanation of the drawing]

[0133] [Figure 1] A schematic diagram of the synthesis scheme for the conjugate of the present invention is shown. [Figure 2] This shows the process flow for the synthesis of the conjugate of the present invention. [Figure 3] The MS spectrum of test sample conjugate 100 is shown. [Figure 4] The MS spectrum of test sample conjugate 100 is shown. [Figure 5] The 1H-NMR spectrum of test sample conjugate 100 is shown. [Figure 6] The 13C-NMR spectrum of test sample conjugate 100 is shown. [Figure 7]The 31P-NMR spectrum of test sample conjugate 100 is shown. [Figure 8] The 19F-NMR spectrum of test sample conjugate 100 is shown. [Figure 9] The assay for the melting temperature (Tm) of test sample conjugate 100 is shown.

[0134] A specific model for carrying out the present invention Those skilled in the art will know that the siRNAs described in the present invention can be obtained by conventional siRNA preparation methods in the art (e.g., solid-phase synthesis and liquid-phase synthesis), and that custom synthesis services are commercially available for both these solid-phase and liquid-phase methods. Those skilled in the art will also understand that modified nucleotide groups can be introduced into the siRNAs described in the present invention by using nucleotide monomers having the corresponding modifications. Methods for preparing nucleotide monomers having the corresponding modifications are well known to those skilled in the art, and commercially available monomers are also available.

[0135] Example 1: Synthesis of siRNA For the sense and antisense strands of the siRNA sequence of the present invention, as well as the sense and antisense strands of the modified double helix, chain synthesis was initiated using a solid-phase support.

[0136] The preparation process for the conjugate of this application is shown using conjugate 100 as an example.

[0137] A solid support modified with GalNAc(L96) was used for the initiation cycle of sense chain synthesis, and a universal solid support (primer support 5G unilinker 350) was used for the initiation cycle of antisense chain synthesis (Figure 1).

[0138] The structure of the GalNAc(L96) modified solid-phase support (L96-PS) is as follows. [ka]

[0139] The structure of the primer support 5G Unilinker 350 support is as follows: [ka] ● represents a PS (polystyrene) solid support.

[0140] Using the YB-192S synthesizer, a solid-phase synthesis method for phosphoramidite-tryesters was employed. Starting with a solid-phase support, nucleoside monomers were sequentially linked in the 3'-5' direction at a synthesis scale of 0.2 μmol.

[0141] The process flow is shown in Figure 2.

[0142] Process Description: Oligo synthesis was initiated from a PS solid support using a method that constructs oligonucleotide chains on the PS solid support using 3'-O-(2-cyanoethyl)phosphoamidite / 4,4'-dimethoxytrityl (DMT) group protection. Each synthetic cycle involved 5'-hydroxy deblocking, coupling, capping, and oxidation (or thiolation). Each coupling reaction was performed by activating a suitable phosphoramidite monomer and reacting it with the free 5'-hydroxy group of the protected nucleotide or oligonucleotide immobilized on the support. According to the sequence information, the corresponding crude oligonucleotide single-stranded PS-Oligo was obtained by cycle synthesis. The crude oligonucleotide chain was cleaved from the solid support and the associated protecting group was removed. The final product was then obtained by purification by preparative chromatography, desalting by ultrafiltration, annealing, and lyophilization.

[0143] (1) Synthesis procedure The following units were included. 1) Deblocking: In the case of ribonucleotides having a DMT (di-p-methoxytrityl) group at the 5'-OH end, the DMT protecting group was removed from the solid support and the ribonucleotide in the first step of synthesis using trichloroacetic acid (TCA) to expose the 5'-OH of the ribonucleotide for coupling with a new base.

[0144] 2) Coupling: The nucleotide monomer was mixed with an activating reagent and reacted with PS-Oligo on a synthetic column. The activating reagent donated a proton to the nitrogen atom of the diisopropylamino group on the 3'-phosphate, forming a phosphoramidite tetrazole active intermediate. When phosphoramidite tetrazole came into contact with PS-Oligo, a nucleophilic reaction occurred with its 5'-hydroxyl group, resulting in coupling and release of the tetrazole, thereby extending the oligo by one nucleotide.

[0145] 3) Capping: Since the coupling efficiency could not be reached 100%, the 5'-hydroxyl groups of PS-Oligo were capped using a capping reagent to prevent uncoupled PS-Oligo from proceeding to the next coupling step.

[0146] 4) Oxidation or thiolation: After the coupling reaction, the nucleotides were linked to the oligonucleotides on the PS support via a phosphite bond (trivalent phosphorus). This phosphite bond is unstable and susceptible to hydrolysis by acids or bases. The trivalent phosphorus at this site was oxidized to pentavalent phosphorus using an oxidizing reagent. Thilation refers to the formation of a phosphorothioate bond by reacting the trivalent phosphorus in the phosphite bond with a thioting reagent under weakly alkaline conditions. 5) Removal of VP protecting group and ammonolithesis

[0147] The synthesized PS-oligo was transferred from the synthesis column to a centrifuge tube. A deprotection reagent was prepared according to a volume ratio of 3:2:100 = TMS-I:pyridine:DCM, and then added to the centrifuge tube and reacted for 30 minutes. A TEA / acetonitrile = 1:1 solution was prepared, 2-mercaptoethanol (final concentration 2 M) was added and mixed well, and then added to the reaction mixture to stop the reaction. The supernatant was removed, and the PS-oligo was washed twice for 5 minutes each time. After washing, ammonia lysis solution (2-mercaptoethanol (2M) / 28% aqueous ammonia) was added, and ammonia lysis was performed for 10 hours. After ammonia lysis, aqueous ammonia was removed by vacuum centrifugation and the solution was purified.

[0148] Sense and antisense strands of other siRNA sequences of the present invention, as well as sense and antisense strands of modified double helices, were prepared using the same method.

[0149] (2) Purification The diluted sample after ammonolithesis was purified by anion exchange chromatography using high-performance liquid chromatography (HPLC). The chromatography conditions were as follows: Column: PS-15Q10*250mm Flow rate: 4ml / min Detection wavelength: 260nm Ion column preparation and purification gradient program: Mobile phase: A phase: 10mM NaOH solution (pH=10); B phase: 10mM NaOH solution (pH=10) + 2M NaCl [Table 4]

[0150] Next, the prepared samples were subjected to mass spectrometry for confirmation and purity analysis.

[0151] (3) Ultrafiltration After purification, the sample was dissolved in 4 ml of PBS, transferred to a 1 K ultrafiltration tube, and centrifuged at 5000 r / min for 45 minutes. The presence of the sample in the centrifuge tube was detected using Nanodrop. If the presence of the sample was detected at 260 nm, it was confirmed that the ultrafiltration membrane was damaged and the ultrafiltration tube needed to be replaced, and the ultrafiltration was repeated. If the presence of the sample was not detected at 260 nm, 4 ml of RNase water was added and the ultrafiltration was repeated. Ultrafiltration was performed three times, each time for 45 minutes.

[0152] (4) Annealing After ultrafiltration of the sense and antisense strands, the sample was diluted to a concentration of 3 mg / ml. The sense and antisense strands were mixed in a 1:1 molar ratio. A water bath was heated to 90°C, and the mixed sense and antisense strands were placed in the water bath and annealed for 30 minutes. The water bath was then removed, and the mixture was allowed to cool naturally to room temperature for 16 hours. A 10 μL sample was taken for HPLC analysis.

[0153] Typical detection method: LC-MS (Example: Conjugate 100) 1) Equipment model: Waters Xevo G2-XS Qtof 2) Test conditions: Ion source: ESI negative ion source Scanning range: 100~2000 Da Collision energy: 15-25 EV, 25-50 EV 3) The spectra of conjugate 100 are shown in Figures 3 and 4. 4) Spectral analysis:

[0154] When conjugate 100 was detected by denatured IP·RP-LC, the complementary double helix dissociated into single helixes (sense and antisense strands). The sense / antisense precursor ions were then fragmented by tandem mass spectrometry. All detected fragment ions were analyzed and identified using CONFIRM Sequence software. In this experiment, the precursor ions were fragmented at energies of 15–25 eV and 25–50 eV, respectively, resulting in 100% coverage; i.e., the sample sequence matched the theoretical sequence. The confirmed results are shown in the table below.

[0155] Checking the sequence of the sense chain in Conjugate 100 [Table 5]

[0156] Sequence verification of the antisense strand of Conjugate 100 [Table 6]

[0157] Nuclear Magnetic Resonance (NMR) Detection of Conjugate 100 1) Instrument model: Bruker AVANCE III 600M NMR 95.0 mg of the sample was weighed, dissolved in heavy water (D2O), and transferred to an NMR tube for detection. 3) Spectral analysis: 1The 1H-NMR spectrum (Figure 5) showed peaks corresponding to hydrogens of aromatic heterocycles, ribose, methyl groups, amino groups, methylene groups, sugar rings, and alcoholic hydroxyl groups. A series of peaks from 0.95 ppm to 2.5 ppm corresponded to methylene hydrogens not directly bonded to the amide. The peak from 3.17 ppm to 3.20 ppm was attributed to methylene hydrogens adjacent to the amide. The peak from 3.4 ppm to 4.6 ppm belonged to methine hydrogens on the sugar ring, the methyl group at the 2' position, and ether-bonded methylene hydrogens. The peak from 5.1 ppm to 6.5 ppm belonged to methine hydrogen at the 1' position of the sugar ring, aromatic hydrogens of the pyrimidine base, and vinyl hydrogen at the 5' position. The peak from 7.1 ppm to 8.2 ppm belonged to aromatic hydrogens of the purine base.

[0158] 13 The 1C-NMR spectrum (Figure 6) showed peaks corresponding to the carbonyl group, aromatic heterocycle, sugar ring, ether-linked methylene, methylene, and methyl group carbons in the molecule. The peak at 170 ppm–180 ppm was assigned to the carbonyl carbon, the peak at 150 ppm–165 ppm was assigned to the aromatic heterocycle (base) and vinyl group carbons, the peak at 95 ppm–100 ppm was assigned to the methine at the 1' position of ribose, the peak at 70 ppm–95 ppm was assigned to the methylene and methine carbons of ribose, the peak at 55 ppm–70 ppm was assigned to the methoxy and ether-linked methylene carbons, and the peak at 20 ppm–40 ppm was assigned to the methyl carbon at the α position of the acetyl group.

[0159] From these results, 1 H-NMR and 13 1C-NMR revealed aromatic heterocycles (bases), vinyl groups, sugar rings, extracyclic methoxy groups, methylene groups, ether-linked methylene groups, and acetyl groups, demonstrating their consistency with the target molecule's structure.

[0160] 31The P-NMR spectra (Figure 7) showed signals for three types of phosphorus functional groups: thiophosphate diester (P=S) with chemical shifts of 54.5 ppm to 57.5 ppm, phosphate diester (P=O) with chemical shifts of -0.97 ppm to -1.5 ppm, and 5'-vinyl phosphate with a chemical shift of 9.16 ppm. The ratio of P=O bonds to P=S bonds was approximately 5:1, which was consistent with the expected information about the target molecule.

[0161] 19 The 1F-NMR spectrum (Figure 8) shows a peak position between -199 ppm and -201 ppm, corresponding to the 2' position of the fluorine atom in the sugar ring.

[0162] These analyses confirmed that the NMR signal matched that of the target molecule.

[0163] Detection of the melting temperature (Tm) of Conjugate 100: 1) Equipment model: Cary3500 2) Detection conditions: Scanning wavelength: 260nm Temperature scan range: 25°C to 90°C Heating rate: 3°C / min 3) The spectrum of test sample conjugate 100 is shown in Figure 9. 4) Spectral analysis:

[0164] Based on the heating curve of conjugate 100, the software system calculated the Tm at 66.0°C using the Savitzky-Golay algorithm.

[0165] LC-MS detection of synthetic conjugates: [Table 7-1] [Table 7-2] [Table 7-3] Here, PC c is a positive control known to have inhibitory activity against the AGT gene, and its structure was as follows: Sense strand: mG*mU*mCmAmUmCfCmAfCfAfAmUmGmAmGmAmGmUmAmCmA GalNAc (L96) (unmodified nucleotide sequence corresponding to SEQ ID NO: 251); Antisense strand: mU*fG*mUmAmCgnTmCmUmCmAmUmUmGfUmGfGmAmUmGmAmC*mG*mA (unmodified nucleotide sequence corresponding to SEQ ID NO: 348).

[0166] Example 2: In Vitro Activity Assay Cell culture and transfection: Cell culture: Hep3B cells (ATCC) were cultured in MEM complete medium (Gibco, containing 10% FBS) at 37 °C and 5% CO2 until near confluence. Then, the cells were trypsinized and seeded into 12-well plates. Each well contained 2.0×10 5 HepB cells and 1.0 mL of MEM complete medium (Gibco, containing 10% FBS). After culturing at 37 °C and % CO2 for 16 - 24 hours, transfection was performed.

[0167] Cell transfection: 48.5 μL of opti-MEM per well was mixed with 1.5 μL of Lipofectamine RNAiMax (Invitrogen), and then 50 μL of siRNA was added and mixed. The mixture was transferred to a PCR tube and incubated at room temperature for 5 minutes. Finally, this siRNA mixture was added to the cells, and after culturing for 24 hours, RNA was extracted. Single-dose experiments were performed using siRNA duplex concentrations of 10 nM and 0.1 nM, or 0.1 nM and 0.01 nM. IC 50The assay was performed using siRNA double-stranded concentrations of 10 nM, 1.0 nM, 0.1 nM, 0.01 nM, 0.001 nM, 0.0001 nM, and 0.00001 nM. The IC of the compounds was determined using the log(inhibitor)vs.response--variable slope(four parameters) fitting method of GraphPad Prism software. 50 The value was calculated.

[0168] RNA extraction: Using the Total RNA Isolation Kit (Omega, CAT: R6834-02): Cells were harvested, washed with 1% PBS, and then lysed in 400 μL of lysis buffer (containing 2% β-mercaptoethanol). Subsequent steps were carried out according to the kit instructions. Finally, 30 μL of RNase-free water was added, and the cells were incubated for 2 minutes, followed by centrifugation at 14000 g for 2 minutes for RNA recovery.

[0169] cDNA synthesis: cDNA synthesis was performed using the TransGen gDNA Removal and cDNA Synthesis Kit (TransGen Biotechnology Co., Ltd., Beijing, China, Cat# AE311-03). 1 μg of total RNA was added to each sample, and cDNA synthesis was performed using a gradient thermal cycler (LongGene, A600) according to the manufacturer's instructions.

[0170] Real-time fluorescence quantification PCR The synthesized cDNA and master mix (containing primers, qPCR premix, and ultrapure water) were added to a 384-well plate (Bokang Meihua, Cat#PC-0040-9U). The final real-time fluorescence quantitative PCR reaction mixture contained 0.25 μM each of upstream and downstream primers for the target gene (AGT) or internal reference gene (GADPH), and a 1×SYBRGreen premix (Applied Biosystem Cat # A25742).

[0171] Real-time fluorescence PCR was performed using the ΔΔCt method with the ABI QuantStudio® 6 real-time fluorescence PCR system. Three to four independent transfection assays were performed for each duplex, and each transfection was assayed three to four times.

[0172] Human / cynomolgus monkey primary hepatocytes: Free uptake: Cryopreserved primary hepatocytes derived from humans or cynomolgus monkeys are thawed and revived, the number of viable cells is measured, and an appropriate density (6 × 10) is determined. 5 The solution was prepared to the concentration of cells / mL. 10 μL of the 10× compound was added to each well of a collagen-coated cell culture plate. Then, 90 μL of the cell suspension was dispensed into each well of a 96-well collagen-coated plate (5.4 × 10). 4 Cells / well). Cell plates were placed and incubated for 48 hours in a cell culture incubator at 37°C and 5% CO2. Single-dose experiments were performed at siRNA double-strand concentrations of 10 nM and 0.1 nM. IC 50 The assay was performed at siRNA bistrual-strand concentrations of 10 nM, 3.33 nM, 1.11 nM, 0.37 nM, 0.12 nM, 0.041 nM, 0.014 nM, and 0.0046 nM. The IC of the compounds was determined using the log(inhibitor concentration) vs. response-variable gradient (4-parameter) fitting method of GraphPad Prism software. 50 The value was calculated.

[0173] cDNA synthesis: After 48 hours of free uptake, the culture medium was removed and the cells were lysed for RNA extraction. Total RNA was extracted using the RNeasy® 96 kit (QIAGEN-74182) according to the kit instructions. Then, cDNA was synthesized using the FastKing RT Kit (With gDNase) (Tiangen-KR116-02) according to the instructions.

[0174] Real-time fluorescence quantitative PCR: Synthesized cDNA and master mix (containing primers, qPCR premix, and ultrapure water) were added to a 384-well plate. The resulting real-time fluorescence quantitative PCR system contained 0.25 μM and 0.125 μM probes for the upstream and downstream primers of the target gene (AGT) or internal reference gene (GADPH), respectively, and a 1× Universal Probe Master premix (Roche, catalog no. 04914058001).

[0175] Real-time fluorescence PCR was performed using the ΔΔCt method with the ABI QuantStudio® 6 real-time fluorescence PCR system. Three to four independent transfection assays were performed for each duplex, and each transfection was assayed three to four times.

[0176] Tables 1-5 show the results of in vitro activity assays of several siRNA conjugates, with PCc, known to have inhibitory activity against the AGT gene, used as a positive control.

[0177] [Table 8-1] [Table 8-2] [Table 8-3] [Table 8-4]

[0178] [Table 9]

[0179] [Table 10]

[0180] [Table 11]

[0181] [Table 12]

[0182] Example 3: Tritosome Stability The siRNAs to be tested were mixed in rat tritosomes (5 μM) and incubated at 37°C for 24 hours and 48 hours, respectively. After incubation, 1 / 3 of the sample volume of phenol-chloroform (Beyotime, Cat:#p1011) was added. The mixture was vortexed and mixed, then allowed to stand for 5 minutes, and centrifuged at 12,000 rpm for 30 minutes. The upper aqueous phase was collected and transferred to a new centrifuge tube. 1 / 10 volume of 3 M NaAC (Solarbio, Cat:#A1070) was added and mixed, then 2.5 times the volume of pre-cooled anhydrous ethanol and 1 μL of glycogen (Beyotime, Cat:#0812) were added and mixed, allowed to stand at -20°C for 2 minutes, and centrifuged at 12,000 rpm for 30 minutes. The supernatant was discarded, the pellet was air-dried, and dissolved in RNase-free water. The dissolved sample was mixed with formamide loading buffer, and nucleic acid electrophoresis was performed using a 10% PAGE gel. After electrophoresis, the PAGE gel was stained with GelRed (Beyotime, Cat:#D0140) for 30 minutes. Finally, the PAGE gel was imaged using a gel imaging system (Bio-Rad), and grayscale quantitative analysis was performed using Image Lab software. The grayscale quantitative results showed that conjugate 100 exhibited better stability in rat tritosomes compared to the clinical candidate PC c.

[0183] Example 4: Cytotoxic Assay Cell culture: Hep3B cells (ATCC) were cultured in MEM complete medium (Gibco, supplemented with 10% FBS) at 37°C and 5% CO2 until near confluence. The cells were then trypsinized and seeded into 24-well plates, with 0.5 × 10⁶ cells per well. 5 Hep3B cells were added to 0.5 mL of MEM complete medium (Gibco, with 10% FBS), cultured at 37°C and 5% CO2 for 16-24 hours, and then subjected to transfection.

[0184] Cell transfection: 24.25 μL of opt-MEM per well was mixed with 0.75 μL of lipofectamine RNAiMax (Invitrogen), followed by 25 μL of siRNA. This mixture was added to PCR tubes and incubated at room temperature for 5 minutes. Finally, this siRNA mixture was added to cells and cultured for 48 or 96 hours before being subjected to cytotoxicity assays. This experiment was performed at siRNA double-stranded concentrations of 10 nM and 1 nM.

[0185] Cytotoxic assay: Cytotoxicity assays were performed using the CCK-8 assay kit (Beyotime, Cat:#C0040). After 48 / 96 hours of cell transfection, the culture medium was removed, and 500 μL of the corresponding complete culture medium (containing 10% CCK-8) was added to each well. The cells were incubated in the dark at 37°C for 30–60 minutes. The OD values ​​of the samples were measured using a microplate reader (Tecan CAT:#spark 20M) at wavelengths of 450 nm and 620 nm. The assay results showed that the cytotoxicity of conjugates 100, 102, and 106 was similar to that of the clinical PCc molecule; that is, none of them showed significant toxicity.

[0186] Example 5: siRNA immunogenicity assay hPBMC cells (Shanghai Saili Biotechnology Co., Ltd., XFB-HP050A) from three different donors were centrifuged and resuspended in RPMI-1640 cell culture medium (containing 10% FBS and 1% penicillin-streptomycin solution). The mixture was then incubated overnight at 37°C and 5% CO2. The compounds to be tested were diluted to form 20× working solutions. Lipofectamine® 3000 and Opti-Mem were prepared as a mixture in a ratio of 1.5:23.5 and vortexed before use. The prepared 20× working solution and the Lipofectamine® 3000 mixture were mixed in a 1:1 ratio in a 96-well V-bottom plate. After overnight incubation, the medium was discarded and the hPBMC cells were resuspended in RPMI-1640 cell culture medium (containing 10% FBS and 1% penicillin-streptomycin solution). After cell counting, the cells were diluted to the required transfection density and added to a 96-well plate. The final cell count in the cell plate was 2.0 × 10⁶. 5 The cells were divided into wells, with a total volume of 200 μL. The cell plates were then incubated for 24 hours in a 5% CO2, 37°C incubator.

[0187] After transfection of hPBMC cells for 24 hours, the cell supernatant was collected, and the levels of IFN-alpha, IL-6, and TNF-alpha in the hPBMC supernatant were detected using the ProcartaPlex Mix&Match 3-plex Kit. The multiplier of change for each factor in the test compound was calculated. Here, the multiplier of change for the test siRNA, naked siRNA, and polyIC = detected factor concentration / factor concentration in the transfection reagent well; the multiplier of change for GS9688 = detected factor concentration / factor concentration in the DMSO well. The results showed that conjugates 100, 102, 161, 162, and 163 did not show significant immunoactivation in hPBMCs.

[0188] Example 6: Evaluation of Off-Target Potential of siRNA Seed Regions Sequences for detecting on-target activity and seed-region off-target activity were constructed separately for each siRNA sequence. The on-target detection sequence was a sequence that perfectly matched the siRNA being tested, while the seed-region off-target detection sequence was a sequence that perfectly matched the seed region (positions 2-8 at the 5' end) of the antisense strand of the siRNA being tested, with a complete mismatch at all other positions. Five copies of either the on-target or seed-region off-target detection sequence were inserted into each psiCHECK plasmid.

[0189] 293T cells were seeded at 20,000 cells / well in 96-well plates. Lipofectamine 2000 and test siRNA were co-transfected with 25 ng of constructed on-target or off-target psiCHECK plasmids. After 24 hours of incubation at 37°C and 5% CO2, fluorescence values ​​were measured using a Dual-Glo luciferase assay system (Promega, E2920). A control group was prepared by transfecting with psiCHECK plasmids that did not contain siRNA molecules. Each double helix was independently transfected 3–4 times, with transfection concentrations of 50 nM, 10 nM, 2.0 nM, 0.4 nM, 0.08 nM, 0.016 nM, and 0.0032 nM for each siRNA. Target inhibitory effects were determined by measuring sea urchin luciferase levels normalized to constitutively expressed firefly luciferase levels, and IC of on-target activity or seed region off-target activity of siRNA was determined. 50 The off-target risk of the siRNA seed region was calculated as the on-target activity IC5. 50 Seed region off-target active IC 50 The ratio was used for evaluation. The test results showed that the off-target risks attributable to the seed regions of conjugate 100, conjugate 102, and conjugate 161 were lower than those of the clinical molecular PC c.

[0190] Example 7: In vivo activity assay in hAGT mice Male humanized hAGT mice aged 6-8 weeks (provided by Jiangsu Jicui, T054372) were used. Mice were not fasted before grouping and administration. Blood was collected, and serum was separated for hAGT detection by ELISA (abcam#). Mice were randomly divided into four groups (6 mice per group) based on hAGT levels, and this point was designated as day 0. On day 1, PBS, PC c, conjugate 100, conjugate 102, and conjugate 106 were subcutaneously injected at a dose of 1.0 mpk. Blood was collected on days 8, 15, 22, 29, 36, 43, and 50 post-administration, and serum was separated for hAGT detection by ELISA. Serum protein concentrations at each time point were compared to the serum protein concentrations of the PBS group at the corresponding time point. The detection results for different conjugates are shown in Table 6. The candidate compounds showed superior in vitro inhibition of serum hAGT protein in hAGT mice compared to the clinical molecule PC c.

[0191] [Table 13]

[0192] [Table 14]

[0193] Example 8: In vivo activity assay in cynomolgus monkeys In this experiment, twelve male cynomolgus monkeys aged 3–6 years (weighing 2.5–6.0 kg) were selected based on body weight, body temperature, blood pressure, electrocardiogram, serum AGT levels, hematology, and blood biochemistry. These were divided into four groups of three monkeys each. Each animal was subcutaneously injected with a 3.0 mpk test conjugate molecule.

[0194] After a 12-hour fast, blood was collected from cynomolgus monkeys at the same time each week before administration (day 0) and after administration. Whole blood samples were placed in centrifuge tubes without anticoagulants, kept in an icebox for 30 minutes to allow coagulation, and then centrifuged at approximately 1800 × g for 10 minutes at 2–8°C. Serum was separated and stored below -80°C.

[0195] Serum AGT protein levels were measured using the AGT ELISA detection kit (IBL, 27412). The AGT protein content at each detection time point was compared to that in the serum of the same monkeys before administration (day 0) to calculate the inhibition of serum AGT content by the test conjugate at different time points. As shown in Table 8, the 3.0 mpk dose of the candidate compound achieved over 90% inhibition of serum AGT protein in cynomolgus monkeys, showing no significant difference from the results reported for the clinical molecule PCc in the patent (International Publication No. 2019222166A1).

[0196] [Table 15]

Claims

1. A siRNA for inhibiting AGT gene expression, wherein the siRNA comprises a sense strand and an antisense strand, wherein the antisense strand comprises at least 17 consecutive nucleotides that differ by 4 nucleotides or less from any one of the nucleotide sequences shown in SEQ ID NOs: 102 to 192, 201 to 207, 209 to 220, 222 to 223, 225 to 233, 235 to 250, 252 to 291, 293 to 341, and 343 to 346, the antisense strand is 17 to 30 nucleotides long, and the sense strand is 17 to 30 nucleotides long and at least partially complementary to the antisense strand.

2. The siRNA according to claim 1, wherein the antisense strand is 19 to 27 nucleotides long and the sense strand is 19 to 25 nucleotides long.

3. The siRNA according to claim 1, wherein the antisense strand differs from any one of the nucleotide sequences shown in SEQ ID NOs. 102 to 192, SEQ ID NOs. 201 to 207, SEQ ID NOs. 209 to 220, SEQ ID NOs. 222 to 223, SEQ ID NOs. 225 to 233, SEQ ID NOs. 235 to 250, SEQ ID NOs. 252 to 291, SEQ ID NOs. 293 to 341, and SEQ ID NOs. 343 to 346 by only 4 nucleotides or less.

4. The siRNA according to claim 1, wherein the mismatch between the sense strand and the antisense strand is 3 nucleotides or less.

5. The siRNA according to claims 1 to 4, wherein the sequence of the siRNA is selected from double helix 1 to double helix 107.

6. The siRNA according to any one of claims 1 to 5, comprising at least one modified nucleotide.

7. The siRNA according to claim 6, wherein all nucleotides in the sense strand and / or antisense strand of the siRNA are modified nucleotides or nucleotide analogs.

8. The siRNA according to claim 7, wherein the modified nucleotide is selected from the group consisting of 2'-methoxynucleotide, 2'-fluoronucleotide, 2'-deoxynucleotide, 2',3'-seconucleotide analog, 2'-fluoroarabinonucleotide, 2'-methoxyethyl nucleotide, 2'-amino modified nucleotide, 2'-alkyl modified nucleotide, 3'-methoxy nucleotide, 2'-allyl modified nucleotide, phosphorothioate group-containing nucleotide, methylphosphonate group-containing nucleotide, 5'-phosphate group-containing nucleotide, 5'-phosphate mimetic-containing nucleotide, diol modified nucleotide, debasalized nucleotide, morpholino nucleotide, locked nucleotide, unlocked nucleotide, threose nucleotide, and glycerol nucleotide.

9. The siRNA according to any one of claims 1 to 8, wherein the 5' and 3' ends of the sense strand each independently contain zero, one, or two phosphorothioate bonds, and / or the 5' and 3' ends of the antisense strand each independently contain one or two phosphorothioate bonds.

10. The siRNA according to any one of claims 1 to 9, wherein the first nucleotide at the 5' end of the antisense strand is an (E)-vinyl phosphate-modified nucleotide.

11. The siRNA according to any one of claims 1 to 10, comprising at least one base-modified nucleotide.

12. siRNA according to any one of claims 1 to 11, wherein the antisense strand of the siRNA is 22 nucleotides long, and 3 to 10 positions of the antisense strand (for example, positions 2, 14, 16; positions 2, 5, 14, 16; positions 2, 6, 14, 16; positions 2, 4, 6, 14, 16; positions 2, 6, 10, 14, 16; positions 2, 6, 12, 14, 16; positions 2, 5, 10, 14, 16; positions 2, 3, 12, 14, 16; position 2, The positions at positions 9, 12, 14, and 16; positions 2, 6, 8, 9, 14, and 16; positions 2, 3, 5, 12, 14, and 16; positions 2, 8, 9, 12, 14, and 16; positions 2, 7, 9, 12, 14, and 16; positions 2, 4, 6, 8, 10, 14, 16, 18, and 20; positions 2, 4, 5, 6, 8, 10, 12, 14, 16, and 18 at the 5' end are 2'-fluoronucleotides, and the remaining positions are 2'-methoxynucleotides. The sense strand of the siRNA is 20 nucleotides long, and the 8th, 9th, and 10th positions of the 5' end of the sense strand are 2'-fluoronucleotides, optionally one position (preferably position 1 of the 5' end) is a threose nucleotide, optionally one position (preferably position 6 of the 5' end) is a 2'-deoxynucleotide, and the remaining positions are 2'-methoxynucleotide conjugates. Preferably, the antisense strand of the siRNA is 22 nucleotides long, where the positions are: 2, 14, 16; or 2, 5, 14, 16; or 2, 6, 14, 16; or 2, 4, 6, 14, 16; or 2, 6, 10, 14, 16; or 2, 6, 12, 14, 16; or 2, 6, 12, 14, 16; or 2, 5, 10, 14, 16; or 2, 3, 12, 14, 16; or 2, 9, 12, 14, 16; or 2 Positions 1, 6, 8, 9, 14, 16; or 2, 3, 5, 12, 14, 16; or 2, 8, 9, 12, 14, 16; or 2, 7, 9, 12, 14, 16; or 2, 4, 6, 8, 10, 14, 16, 18, 20; or 2, 4, 5, 6, 8, 10, 12, 14, 16, 18 are 2'-fluoronucleotides at the 5' end of the antisense chain, and the remaining positions are 2'-methoxynucleotides. The sense strand of the siRNA is 20 nucleotides long, and the 8th, 9th, and 10th positions of the 5' end of the sense strand are 2'-fluoronucleotides, optionally the 1st position of the 5' end is a threose nucleotide, or optionally the 6th position of the 5' end is a 2'-deoxynucleotide, and the remaining positions are 2'-methoxynucleotides. Preferably, the 2'-deoxynucleotide at position 6 is independently selected from the group consisting of deoxyadenosine monophosphate (dA), deoxyguanosine monophosphate (dG), deoxycytidine monophosphate (dC), deoxythymidine monophosphate (dT), and deoxyuridine monophosphate (dU); More preferably, the 2'-deoxynucleotide at position 6 is deoxythymidine monophosphate (dT), and more preferably, the antisense strand of the siRNA is 22 nucleotides long, where the following positions are present: 2, 14, 16; or 2, 5, 14, 16; or 2, 6, 14, 16; or 2, 4, 6, 14, 16; or 2, 6, 10, 14, 16; or 2, 6, 12, 14, 16; or 2, 5, 10, 14, 16; or 2, 3, 12, 14, 16; or 2, 9, 12, 14, 16; or 2, 6, 8, 9, 14, 16; or 2, 3 1, 5, 12, 14, 16; or 2, 8, 9, 12, 14, 16; or 2, 7, 9, 12, 14, 16; or 2, 4, 6, 8, 10, 14, 16, 18, 20; or 2, 4, 5, 6, 8, 10, 12, 14, 16, 18, 18 are 2'-fluoronucleotides at the 5' end of the antisense strand, and the remaining positions are 2'-methoxynucleotides; or the sense strand of the siRNA is 20 nucleotides long, and the 8, 9 and 10 positions at the 5' end of the sense strand are 2'-fluoronucleotides, and the remaining positions are 2'-methoxynucleotides; or The antisense strand of siRNA is 22 nucleotides long, with 2'-fluoronucleotides at positions 2, 6, 14, and 16 of the 5' end of the antisense strand, and 2'-methoxynucleotides at the remaining positions; the sense strand of siRNA is 20 nucleotides long, with 2'-fluoronucleotides at positions 8, 9, and 10 of the 5' end of the sense strand, 1 being a threose nucleotide, and 2'-methoxynucleotides at the remaining positions, or The antisense strand of the siRNA is 22 nucleotides long, with 2'-fluoronucleotides at positions 2, 6, 14, and 16 of the 5' end of the antisense strand, and 2'-methoxynucleotides at the remaining positions; the sense strand of the siRNA is 20 nucleotides long, with 2'-fluoronucleotides at positions 8, 9, and 10 of the 5' end of the sense strand, 2'-deoxynucleotide at position 6, and 2'-methoxynucleotides at the remaining positions, preferably the 2'-deoxynucleotide at position 6 is independently selected from the group consisting of deoxyadenosine monophosphate (dA), deoxyguanosine monophosphate (dG), deoxycytidine monophosphate (dC), deoxythymidine monophosphate (dT), and deoxyuridine monophosphate (dU); More preferably, the 2'-deoxynucleotide at position 6 is deoxythymidine monophosphate (dT), and more preferably, the antisense strand of the siRNA is the nucleotide sequence shown in SEQ ID NO: 105, and the sense strand of the siRNA is the nucleotide sequence shown in SEQ ID NO:

23.

13. A siRNA according to any one of claims 1 to 11, wherein the antisense strand of the siRNA is 22 nucleotides long, with 2'-fluoronucleotides at positions 2, 6, 14, and 16 of the 5' end of the antisense strand, and the remaining positions being 2'-methoxynucleotides; and the sense strand of the siRNA is 20 nucleotides long, with 2'-fluoronucleotides at positions 8, 9, and 10 of the 5' end of the sense strand, and the remaining positions being 2'-methoxynucleotides; Preferably, the antisense strand of the siRNA is the nucleotide sequence shown in SEQ ID NO: 116, and the sense strand of the siRNA is the nucleotide sequence shown in SEQ ID NO:

22.

14. The siRNA according to any one of claims 1 to 11, The antisense strand of the siRNA is 22 nucleotides long, with the 14th position at the 5' end of the antisense strand being a 2'-fluoronucleotide, the 2nd, 5th, and 7th positions being 2'-deoxynucleotides, the 12th position being a threose nucleotide, and the remaining positions being 2'-methoxynucleotides. The sense strand of the siRNA is 20 nucleotides long, with the 8th, 9th, and 10th positions at the 5' end of the sense strand being 2'-fluoronucleotides, and the remaining positions being 2'-methoxynucleotides. Preferably, the 2'-deoxynucleotides at positions 2, 5, and 7 are independently selected from the group consisting of deoxyadenosine monophosphate (dA), deoxyguanosine monophosphate (dG), deoxycytidine monophosphate (dC), deoxythymidine monophosphate (dT), and deoxyuridine monophosphate (dU), and more preferably, the 2'-deoxynucleotides at positions 2, 5, and 7 are deoxythymidine monophosphate (dT), deoxythymidine monophosphate (dT), and deoxyadenosine monophosphate (dA), respectively. More preferably, the antisense strand of the siRNA is the nucleotide sequence shown in SEQ ID NO: 105, and the sense strand of the siRNA is the nucleotide sequence shown in SEQ ID NO:

23.

15. The siRNA according to any one of claims 1 to 11, The antisense strand of the siRNA is 21 nucleotides long, with 2'-fluoronucleotides at positions 2, 6, 14, and 16 of the 5' end of the antisense strand, and the remaining positions being 2'-methoxynucleotides; or the sense strand of the siRNA is 19 nucleotides long, with 2'-fluoronucleotides at positions 7, 8, and 9 of the 5' end of the sense strand, with threose nucleotide at position 1, and the remaining positions being 2'-methoxynucleotides; or The antisense strand of the siRNA is 21 nucleotides long, with the 14th position at the 5' end of the antisense strand being a 2'-fluoronucleotide, the 2nd, 5th, and 7th positions being 2'-deoxynucleotides, the 12th position being a threose nucleotide, and the remaining positions being 2'-methoxynucleotides. The sense strand of the siRNA is 19 nucleotides long, with the 7th, 8th, and 9th positions at the 5' end of the sense strand being 2'-fluoronucleotides, and the remaining positions being 2'-methoxynucleotides. Preferably, the 2'-deoxynucleotides at positions 2, 5, and 7 are independently selected from the group consisting of deoxyadenosine monophosphate (dA), deoxyguanosine monophosphate (dG), deoxycytidine monophosphate (dC), deoxythymidine monophosphate (dT), and deoxyuridine monophosphate (dU). More preferably, the 2'-deoxynucleotides at positions 2, 5, and 7 are deoxythymidine monophosphate (dT), deoxyuridine monophosphate (dU), and deoxyadenosine monophosphate (dA), respectively. More preferably, the siRNA wherein the antisense strand of the siRNA is the nucleotide sequence shown in SEQ ID NO: 104, and the sense strand of the siRNA is the nucleotide sequence shown in SEQ ID NO:

3.

16. The siRNA according to any one of claims 12 to 15, wherein the 5' and 3' ends of the sense strand each independently contain zero, one, or two phosphorothioate bonds, and / or the 5' and 3' ends of the antisense strand each independently contain one or two phosphorothioate bonds.

17. The siRNA according to any one of claims 12 to 16, wherein the first nucleotide at the 5' end of the antisense strand is an (E)-vinyl phosphate modified nucleotide.

18. The siRNA according to any one of claims 12 to 17, wherein the 3' end and / or 5' end of the sense strand are each bound to an inverted debase residue (iab), preferably the 3' end and 5' end of the sense strand are each bound to an inverted debase residue (iab).

19. An siRNA conjugate obtained by binding the siRNA described in any one of claims 1 to 18 to a conjugate molecule.

20. The siRNA conjugate according to claim 19, wherein the conjugate molecule originates from a delivery carrier portion via covalent bonding and is independently or simultaneously ligated to the 3' or 5' end of the sense strand of the siRNA.

21. The siRNA conjugate according to claim 20, wherein the delivery carrier portion is selected from the group consisting of GalNAc(L96), Ser(GN), GalNAc(Ser1), GalNAc(Ser2), GalNAc(Ser3), GalNAc(Ser4), LP-GalNAc, XY-GalNAc, GalNAc(A1GN), GalNAc(A2GN), GalNAc(A3GN), GalNAc(A4GN), GalNAc(A5GN), GAlNAc(NAG25), and GAlNAc(NAG37), During the ceremony, GalNAc (L96) has the following structure 【Chemistry 1】 、 Ser(GN) has the following structure 【Chemistry 2】 GalNAc(Ser1), GalNAc(Ser2), GalNAc(Ser3), and GalNAc(Ser4) each have the following structures 【Transformation 3】 LP-GalNAc has the following structure 【Chemistry 4】 XY-GalNAc has the following structure 【Transformation 5】 or 【Transformation 6】 GalNAc(A1GN), GalNAc(A2GN), GalNAc(A3GN), GalNAc(A4GN), GalNAc(A5GN), GAlNAc(NAG25), and GAlNAc(NAG37) each have the following structures 【Chemistry 7-1】 【Chemistry 7-2】 Optionally, the delivery carrier portion binds to the 3' end of the siRNA sense strand, or the 5' end of the siRNA sense strand, or the inverted debase residue (iab) at the 3' end of the siRNA sense strand, or the inverted debase residue (iab) at the 5' end of the siRNA sense strand, or one or more of the delivery carrier portions bind to the 3' end of the siRNA sense strand, the 5' end of the siRNA sense strand, the inverted debase residue (iab) at the 3' end of the siRNA sense strand, or the inverted debase residue (i a and b are simultaneously bound, preferably the delivery carrier portion GalNAc (L96) is bound to the 3' end of the sense strand of the siRNA or to the inverted debase residue (iab) of the 3' end of the sense strand of the siRNA, the delivery carrier portion Ser (GN) is simultaneously bound to the 3' and 5' ends of the sense strand of the siRNA, the delivery carrier portion LP-GalNAc is bound to the 5' end of the sense strand of the siRNA, the delivery carrier portion XY-GalNAc is bound to the 3' end of the sense strand of the siRNA, and the delivery carrier portion GalNAc (Ser2) However, the delivery carrier portion GalNAc(Ser3) binds to the 3' end or 5' end of the sense strand of the siRNA, or simultaneously to both the 3' and 5' ends of the sense strand of the siRNA, the delivery carrier portion GalNAc(Ser4) binds to the 3' end of the sense strand of the siRNA, the delivery carrier portion GalNAc(A1GN) binds to the 3' end of the sense strand of the siRNA, and the delivery carrier portion GalNAc(A2G N) binds to the 3' end of the sense strand of the siRNA, the delivery carrier portion GalNAc (A3GN) binds to the 5' end of the sense strand of the siRNA, the delivery carrier portion GalNAc (A4GN) binds to the 5' end of the sense strand of the siRNA, the delivery carrier portion GalNAc (A5GN) binds to the 5' end of the sense strand of the siRNA, the delivery carrier portion GalNAc (NAG25) binds to the 5' end of the sense strand of the siRNA, the delivery carrier portion GalNAc (NAG37) binds to the 5' end of the sense strand of the siRNA,The delivery carrier portions GalNAc(Ser1) and GalNAc(Ser2) are either simultaneously bound to the 3' and 5' ends of the sense strand of the siRNA, respectively, or simultaneously bound to the 5' and 3' ends of the sense strand of the siRNA, Preferably, the delivery carrier portion is an siRNA conjugate selected from the group consisting of GalNAc (L96) bound to the 3' end of the sense strand of the siRNA, GalNAc (A1GN) bound to the 3' end of the sense strand of the siRNA, GalNAc (A2GN) bound to the 3' end of the sense strand of the siRNA, GalNAc (A3GN) bound to the 5' end of the sense strand of the siRNA, GalNAc (A4GN) bound to the 5' end of the sense strand of the siRNA, and GalNAc (Ser2) simultaneously bound to both the 3' and 5' ends of the antisense strand of the siRNA.

22. The siRNA conjugate according to any one of claims 19 to 21, wherein the siRNA conjugate is selected from conjugates 1 to 1613 and conjugates 1617 to 1658.

23. A pharmaceutical composition comprising an siRNA according to any one of claims 1 to 18 and / or an siRNA conjugate according to any one of claims 19 to 22, and a pharmaceutically acceptable carrier.

24. Use of the siRNA according to any one of claims 1 to 18 and / or the siRNA conjugate according to any one of claims 19 to 22 and / or the pharmaceutical composition according to claim 23 in the manufacture of a pharmaceutical for treating and / or preventing a pathological condition or disease associated with AGT gene overexpression.

25. The use according to claim 24, wherein the pathological condition or disease is a disease related to abnormal blood pressure.

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