A siRNA targeting AGT gene expression and its conjugate and use

Abstract

Provided are siRNA and its conjugate for inhibiting AGT gene expression, comprising a sense strand and an antisense strand. The provided siRNA, siRNA conjugate, and pharmaceutical composition have good stability, excellent AGT gene inhibition activity, and satisfactory cytotoxicity and immunostimulation.

Description

Technical Field

[0001] The present invention relates to an siRNA for inhibiting angiotensinogen (AGT) gene expression, a conjugate thereof, a pharmaceutical composition, and use thereof in preventing and / or treating diseases related to abnormal blood pressure. Background Art

[0002] Angiotensinogen (AGT) is an important component of the renin-angiotensin system (RAS) in the human body. The entire RAS plays a key role in regulating blood pressure in animals. AGT is synthesized and secreted in the liver, and then angiotensin I is produced by the enzyme renin (REN), which is then converted into angiotensin II by angiotensin-converting enzyme (ACE). Most angiotensin II can bind to type I angiotensin II receptors, causing vasoconstriction and increasing blood pressure. This molecule also stimulates the production of the hormone aldosterone, which triggers Na + The kidneys absorb ions and water, which leads to an increase in blood volume and further increases in blood pressure.

[0003] Hypertension is a serious condition that significantly increases the risk of heart, brain, and kidney disease, among other illnesses. According to the World Health Organization, an estimated 1.28 billion adults aged 30-79 worldwide suffer from hypertension. Only 42% of hypertensive patients receive diagnosis and treatment, and 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), hypertension caused 2.54 million deaths in my country in 2017, ranking first among all risk factors for death.

[0004] Currently, first-line medications used clinically for the treatment of hypertension are divided into five major categories: angiotensin-converting enzyme inhibitors, angiotensin II receptor blockers, beta-adrenergic receptor blockers, dihydropyridine calcium channel blockers, and diuretics. These five antihypertensive drug classes have distinct mechanisms of action. Depending on the patient's age, severity of hypertension, and risk of related clinical complications, the average patient will receive one of these treatments. If one treatment option is inadequate, a combination of two or even three treatment options may be used. For high-risk patients, a combination of two or even three treatment options may be used. If all three treatment options fail to effectively control blood pressure, the condition is termed refractory hypertension. Refractory hypertension is a common and challenging problem in hypertension treatment. The development of effective, safe, stable, and long-acting antihypertensive drugs holds significant clinical value.

[0005] The present application provides a small interfering RNA (siRNA) preparation targeting AGT, which can specifically bind to AGT mRNA, destroy the normal translation template function of AGT mRNA, thereby preventing it from translating AGT protein, inhibiting the RAS pathway from the source, and is used to treat / prevent related diseases caused by the RAS pathway, such as hypertension, including refractory hypertension and uncontrollable hypertension.

[0006] Compared to traditional drugs, siRNA has poor stability and is susceptible to nuclease degradation when administered systemically. Furthermore, efforts are needed to further enhance its activity while avoiding side effects such as off-target effects, immune stimulation, and cytotoxicity. Therefore, developing more candidate siRNAs that are stable in the blood, exhibit good in vivo bioactivity, have low cytotoxicity, and can effectively inhibit AGT gene expression has become an urgent issue. Furthermore, the development of drugs that can effectively prevent and / or treat hypertension-related diseases using these candidate siRNAs that inhibit AGT gene expression requires clinical research and is commercially viable. Summary of the Invention

[0007] The present invention provides an siRNA for inhibiting AGT gene expression, the siRNA comprising a sense strand and an antisense strand; wherein the antisense strand comprises at least 17 consecutive nucleotides that differ by no more than 4 nucleotides from the nucleotide sequence of any one of SEQ ID NO: 102 to SEQ ID NO: 192, SEQ ID NO: 201 to SEQ ID NO: 207, SEQ ID NO: 209 to SEQ ID NO: 220, SEQ ID NO: 222 to SEQ ID NO: 223, SEQ ID NO: 225 to SEQ ID NO: 233, SEQ ID NO: 235 to SEQ ID NO: 250, SEQ ID NO: 252 to SEQ ID NO: 291, SEQ ID NO: 293 to SEQ ID NO: 341, and SEQ ID NO: 343 to SEQ ID NO: 346, and the antisense strand is 17 to 30 nucleotides in length; and the sense strand is 17 to 30 nucleotides in length and is at least partially complementary to the antisense strand.

[0008] The term "at least partially complementary" means that the two sequences are completely complementary, or have no more than 5, 4, 3 or 2 mismatched base pairs overall, while retaining the ability to hybridize under relevant conditions.

[0009] In some embodiments of the invention, the antisense strand differs from any one of the nucleotide sequences set forth in SEQ ID NO: 102 to SEQ ID NO: 192, SEQ ID NO: 201 to SEQ ID NO: 207, SEQ ID NO: 209 to SEQ ID NO: 220, SEQ ID NO: 222 to SEQ ID NO: 223, SEQ ID NO: 225 to SEQ ID NO: 233, SEQ ID NO: 235 to SEQ ID NO: 250, SEQ ID NO: 252 to SEQ ID NO: 291, SEQ ID NO: 293 to SEQ ID NO: 341, SEQ ID NO: 343 to SEQ ID NO: 346 by no more than 4 nucleotides; in some embodiments of the invention, the antisense strand differs from any one of the nucleotide sequences set forth in SEQ ID NO: 102 to SEQ ID NO: 192, SEQ ID NO: 201 to SEQ ID NO: 207, SEQ ID NO: 209 to SEQ ID NO: 220, SEQ ID NO: 222 to SEQ ID NO: 223, SEQ ID NO: 225 to SEQ ID NO: 233, SEQ ID NO: 235 to SEQ ID NO: 250 NO: 225 to SEQ ID NO: 233, SEQ ID NO: 235 to SEQ ID NO: 250, SEQ ID NO: 252 to SEQ ID NO: 291, SEQ ID NO: 293 to SEQ ID NO: 341, SEQ ID NO: 343 to SEQ ID NO: 346; in some embodiments of the invention, the antisense strand differs from any of the nucleotide sequences shown in SEQ ID NO: 102 to SEQ ID NO: 192, SEQ ID NO: 201 to SEQ ID NO: 207, SEQ ID NO: 209 to SEQ ID NO: 220, SEQ ID NO: 222 to SEQ ID NO: 223, SEQ ID NO: 225 to SEQ ID NO: 233, SEQ ID NO: 235 to SEQ ID NO: 250, SEQ ID NO: 252 to SEQ ID NO: 291, SEQ ID NO: 293 to SEQ ID NO: 341, SEQ ID NO: 343 to SEQ ID NO: Any nucleotide sequence shown in NO:346 differs by no more than 2 nucleotides;In some embodiments of the invention, the antisense strand differs from any of the nucleotide sequences set forth in SEQ ID NO: 102 to SEQ ID NO: 192, SEQ ID NO: 201 to SEQ ID NO: 207, SEQ ID NO: 209 to SEQ ID NO: 220, SEQ ID NO: 222 to SEQ ID NO: 223, SEQ ID NO: 225 to SEQ ID NO: 233, SEQ ID NO: 235 to SEQ ID NO: 250, SEQ ID NO: 252 to SEQ ID NO: 291, SEQ ID NO: 293 to SEQ ID NO: 341, SEQ ID NO: 343 to SEQ ID NO: 346 by no more than 1 nucleotide; in some embodiments of the invention, the antisense strand is SEQ ID NO: 102 to SEQ ID NO: 192, SEQ ID NO: 201 to SEQ ID NO: 207, SEQ ID NO: 209 to SEQ ID NO: 220, SEQ ID NO: 222 to SEQ ID NO: 223, SEQ ID NO: 225 to SEQ ID NO: 233, SEQ ID NO: 235 to SEQ ID NO: 250, Any one of the nucleotide sequences shown in SEQ ID NO: 225 to SEQ ID NO: 233, SEQ ID NO: 235 to SEQ ID NO: 250, SEQ ID NO: 252 to SEQ ID NO: 291, SEQ ID NO: 293 to SEQ ID NO: 341, and SEQ ID NO: 343 to SEQ ID NO: 346.

[0010] In some embodiments of the present invention, the sense strand and the antisense strand have no more than 3 nucleotide mismatches; in some embodiments of the present invention, the sense strand and the antisense strand have no more than 2 nucleotide mismatches; in some embodiments of the present invention, the sense strand and the antisense strand have no more than 1 nucleotide mismatch; in some embodiments of the present invention, the sense strand and the antisense strand are fully complementary.

[0011] Preferably, the sense strand has at least 15, 16, 17, 18, 19, 20 or 21 nucleotides of complementarity with the antisense strand.

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

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

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

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

[0016] In some embodiments of the present invention, the 3' end of the antisense strand of the siRNA has an overhang of 2 nucleotides.

[0017] In some embodiments of the invention, the siRNA has blunt ends. In some embodiments of the 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 invention, the siRNA has two blunt ends.

[0019] In some embodiments of the present invention, the nucleotide sequence (5′→3′) of the siRNA is selected from duplex 1 to duplex 107:

[0020]

[0021]

[0022]

[0023]

[0024]

[0025] In some embodiments of the present invention, the siRNA contains at least one modified nucleotide.

[0026] In some embodiments of the present invention, all nucleotides in the sense strand and / or antisense strand of the siRNA are modified nucleotides or nucleotide analogs.

[0027] In some embodiments of the present invention, the modified nucleotide is selected from 2'-methoxy nucleotides, 2'-fluoro nucleotides, 2'-deoxy nucleotides, 2',3'-split nucleotide analogs, 2'-fluoroarabinonucleotides, 2'-methoxyethyl nucleotides, 2'-amino modified nucleotides, 2'-alkyl modified nucleotides, 3'-methoxy nucleotides, 2'-allyl modified nucleotides, nucleotides containing thiophosphate groups, nucleotides containing methylphosphonate groups, nucleotides containing 5'-phosphate esters, nucleotides containing 5'-phosphate mimetics, diol-modified nucleotides, abasic nucleotides, morpholino nucleotides, locked nucleotides (LNA), unlocked nucleotides (UNA), threose nucleotides (TNA) or glycerol nucleotides (GNA), but the present invention is not limited thereto.

[0028] In some embodiments of the present invention, the antisense strand of the siRNA is 23 nucleotides in length, wherein positions 2, 6, 14, and 16 at the 5' end of the antisense strand are 2'-fluoro nucleotides, and the remaining positions are 2'-methoxy nucleotides; the sense strand of the siRNA is 21 nucleotides in length, wherein positions 7, 9, 10, and 11 at the 5' end of the sense strand are 2'-fluoro nucleotides, and the remaining positions are 2'-methoxy nucleotides.

[0029] In some embodiments of the present invention, the antisense strand of the siRNA is 23 nucleotides in length, wherein positions 2, 6, 8, 9, 14, and 16 at the 5' end of the antisense strand are 2'-fluoro nucleotides, and the remaining positions are 2'-methoxy nucleotides; the sense strand of the siRNA is 21 nucleotides in length, wherein positions 7, 9, 10, and 11 at the 5' end of the sense strand are 2'-fluoro nucleotides, and the remaining positions are 2'-methoxy nucleotides.

[0030] In some embodiments of the present invention, the antisense strand of the siRNA is 23 nucleotides in length, wherein positions 2, 6, 14, and 16 at the 5' end of the antisense strand are 2'-fluoro nucleotides, and the remaining positions are 2'-methoxy nucleotides; the sense strand of the siRNA is 21 nucleotides in length, wherein positions 9, 10, and 11 at the 5' end of the sense strand are 2'-fluoro nucleotides, and the remaining positions are 2'-methoxy nucleotides.

[0031] In some embodiments of the present invention, the antisense strand of the siRNA is 23 nucleotides in length, wherein positions 2, 6, 8, 9, 10, 14, and 16 at the 5' end of the antisense strand are 2'-fluoro nucleotides, and the remaining positions are 2'-methoxy nucleotides; the sense strand of the siRNA is 21 nucleotides in length, wherein positions 7, 9, 10, and 11 at the 5' end of the sense strand are 2'-fluoro nucleotides, and the remaining positions are 2'-methoxy nucleotides.

[0032] In some embodiments of the present invention, the antisense strand of the siRNA is 23 nucleotides in length, wherein positions 2, 6, 8, 9, 10, 14, and 16 at the 5' end of the antisense strand are 2'-fluoro nucleotides, and the remaining positions are 2'-methoxy nucleotides; the sense strand of the siRNA is 21 nucleotides in length, wherein positions 7, 9, 10, 11, and 15 at the 5' end of the sense strand are 2'-fluoro nucleotides, and the remaining positions are 2'-methoxy nucleotides.

[0033] In some embodiments of the present invention, the antisense strand of the siRNA is 23 nucleotides in length, wherein positions 2, 6, 10, 14, and 16 at the 5' end of the antisense strand are 2'-fluoro nucleotides, and the remaining positions are 2'-methoxy nucleotides; the sense strand of the siRNA is 21 nucleotides in length, wherein positions 3, 7, 9, 10, and 11 at the 5' end of the sense strand are 2'-fluoro nucleotides, and the remaining positions are 2'-methoxy nucleotides.

[0034] In some embodiments of the present invention, the antisense strand of the siRNA is 23 nucleotides in length, wherein positions 2, 6, 14, and 16 at the 5' end of the antisense strand are 2'-fluoro nucleotides, and the remaining positions are 2'-methoxy nucleotides; the sense strand of the siRNA is 21 nucleotides in length, wherein positions 7, 9, 10, and 11 at the 5' end of the sense strand are 2'-fluoro nucleotides, and the remaining positions are 2'-methoxy nucleotides.

[0035] In some embodiments of the present invention, the antisense strand of the siRNA is 23 nucleotides in length, wherein positions 2, 3, 4, 6, 8, 10, 14, 16, 18, 20, and 22 at the 5' end of the antisense strand are 2'-fluoro nucleotides, and the remaining positions are 2'-methoxy nucleotides; the sense strand of the siRNA is 21 nucleotides in length, wherein position 2 at the 5' end of the sense strand is a 2'-fluoro nucleotide, and the remaining positions are 2'-methoxy nucleotides.

[0036] In some embodiments of the present invention, the antisense strand of the siRNA is 23 nucleotides in length, wherein positions 2, 4, 5, 6, 8, 10, 14, 16, 18, 20, and 22 at the 5' end of the antisense strand are 2'-fluoro nucleotides, and the remaining positions are 2'-methoxy nucleotides; the sense strand of the siRNA is 21 nucleotides in length, wherein position 2 at the 5' end of the sense strand is a 2'-fluoro nucleotide, and the remaining positions are 2'-methoxy nucleotides.

[0037] In some embodiments of the present invention, the antisense strand of the siRNA is 23 nucleotides in length, wherein positions 2, 4, 6, 7, 8, 10, 14, 16, 18, 20, and 22 at the 5' end of the antisense strand are 2'-fluoro nucleotides, and the remaining positions are 2'-methoxy nucleotides; the sense strand of the siRNA is 21 nucleotides in length, wherein position 2 at the 5' end of the sense strand is a 2'-fluoro nucleotide, and the remaining positions are 2'-methoxy nucleotides.

[0038] In some embodiments of the present invention, the antisense strand of the siRNA is 23 nucleotides in length, wherein positions 2, 4, 6, 8, 10, 14, 16, 18, and 20 at the 5' end of the antisense strand are 2'-fluoro nucleotides, and the remaining positions are 2'-methoxy nucleotides; the sense strand of the siRNA is 21 nucleotides in length, wherein positions 1, 3, 5, 7, 9, 10, 11, 13, 15, 17, 19, and 21 at the 5' end of the sense strand are 2'-fluoro nucleotides, and the remaining positions are 2'-methoxy nucleotides.

[0039] In some embodiments of the present invention, the antisense strand of the siRNA is 23 nucleotides in length, wherein positions 2, 4, 6, 8, 10, 14, 16, 18, 20, and 22 at the 5' end of the antisense strand are 2'-fluoro nucleotides, and the remaining positions are 2'-methoxy nucleotides; the sense strand of the siRNA is 21 nucleotides in length, wherein positions 1, 3, 5, 7, 9, 10, 11, 13, 15, 17, 19, and 21 at the 5' end of the sense strand are 2'-fluoro nucleotides, and the remaining positions are 2'-methoxy nucleotides.

[0040] In some embodiments of the present invention, the antisense strand of the siRNA is 23 nucleotides in length, wherein positions 2, 6, 8, 9, 14, and 16 at the 5' end of the antisense strand are 2'-fluoro nucleotides, position 7 is a glycerol nucleotide (GNA), and the remaining positions are 2'-methoxy nucleotides; the sense strand of the siRNA is 21 nucleotides in length, wherein positions 7, 9, 10, and 11 at the 5' end of the sense strand are 2'-fluoro nucleotides, and the remaining positions are 2'-methoxy nucleotides.

[0041] In some embodiments of the present invention, the antisense strand of the siRNA is 23 nucleotides in length, wherein positions 2, 8, 9, 10, 14, and 16 at the 5' end of the antisense strand are 2'-fluoro nucleotides, position 6 is a glycerol nucleotide (GNA), and the remaining positions are 2'-methoxy nucleotides; the sense strand of the siRNA is 21 nucleotides in length, wherein positions 7, 9, 10, 11, and 15 at the 5' end of the sense strand are 2'-fluoro nucleotides, and the remaining positions are 2'-methoxy nucleotides.

[0042] In some embodiments of the present invention, the antisense strand of the siRNA is 23 nucleotides in length, wherein positions 2, 8, 9, 10, 14, and 16 at the 5' end of the antisense strand are 2'-fluoro nucleotides, position 4 is a glycerol nucleotide (GNA), and the remaining positions are 2'-methoxy nucleotides; the sense strand of the siRNA is 21 nucleotides in length, wherein positions 7, 9, 10, 11, and 15 at the 5' end of the sense strand are 2'-fluoro nucleotides, and the remaining positions are 2'-methoxy nucleotides.

[0043] In some embodiments of the present invention, the antisense strand of the siRNA is 23 nucleotides in length, wherein positions 2, 8, 9, 10, 14, and 16 at the 5' end of the antisense strand are 2'-fluoro nucleotides, position 5 is a glycerol nucleotide (GNA), and the remaining positions are 2'-methoxy nucleotides; the sense strand of the siRNA is 21 nucleotides in length, wherein positions 7, 9, 10, 11, and 15 at the 5' end of the sense strand are 2'-fluoro nucleotides, and the remaining positions are 2'-methoxy nucleotides.

[0044] In some embodiments of the present invention, the antisense strand of the siRNA is 23 nucleotides in length, wherein positions 2, 8, 9, 10, 14, and 16 of the 5' end of the antisense strand are 2'-fluoro nucleotides, position 7 is a glycerol nucleotide (GNA), and the remaining positions are 2'-methoxy nucleotides; the sense strand of the siRNA is 21 nucleotides in length, wherein positions 7, 9, 10, 11, and 15 of the 5' end of the sense strand are 2'-fluoro nucleotides, and the remaining positions are 2'-methoxy nucleotides.

[0045] In some embodiments of the present invention, the antisense strand of the siRNA is 23 nucleotides in length, wherein positions 2, 8, 9, 10, 14, and 16 at the 5' end of the antisense strand are 2'-fluoro nucleotides, position 6 is a glycerol nucleotide (GNA), and the remaining positions are 2'-methoxy nucleotides; the sense strand of the siRNA is 21 nucleotides in length, wherein positions 9, 10, and 11 at the 5' end of the sense strand are 2'-fluoro nucleotides, and the remaining positions are 2'-methoxy nucleotides.

[0046] In some embodiments of the present invention, the antisense strand of the siRNA is 23 nucleotides in length, wherein positions 2, 5, 7, and 12 of the 5' end of the antisense strand are 2'-deoxynucleotides, position 14 is a 2'-fluoro nucleotide, and the remaining positions are 2'-methoxy nucleotides. In some embodiments of the present invention, the sense strand of the siRNA is 21 nucleotides in length, wherein positions 9, 10, and 11 of the 5' end of the sense strand are 2'-fluoro nucleotides, and the remaining positions are 2'-methoxy nucleotides.

[0047] In some embodiments of the present invention, the antisense strand of the siRNA is 23 nucleotides in length, wherein positions 2, 5, 7, and 12 of the 5' end of the antisense strand are 2'-deoxynucleotides, positions 6, 8, 9, 10, 14, and 16 are 2'-fluoro nucleotides, and the remaining positions are 2'-methoxy nucleotides. In some embodiments of the present invention, the sense strand of the siRNA is 21 nucleotides in length, wherein positions 7, 9, 10, and 11 of the 5' end of the sense strand are 2'-fluoro nucleotides, and the remaining positions are 2'-methoxy nucleotides.

[0048] In some embodiments of the present invention, the antisense strand of the siRNA is 21 nucleotides in length, wherein positions 2, 6, 14, and 16 at the 5' end of the antisense strand are 2'-fluoro nucleotides, and the remaining positions are 2'-methoxy nucleotides; the sense strand of the siRNA is 21 nucleotides in length, wherein positions 5, 7, and 9 at the 5' end of the sense strand are 2'-fluoro nucleotides, and the remaining positions are 2'-methoxy nucleotides.

[0049] In some embodiments of the present invention, the antisense strand of the siRNA is 21 nucleotides in length, wherein positions 2, 6, 14, and 16 at the 5' end of the antisense strand are 2'-fluoro nucleotides, and the remaining positions are 2'-methoxy nucleotides; the sense strand of the siRNA is 21 nucleotides in length, wherein positions 7, 9, 10, and 11 at the 5' end of the sense strand are 2'-fluoro nucleotides, and the remaining positions are 2'-methoxy nucleotides.

[0050] In some embodiments of the present invention, the antisense strand of the siRNA is 21 nucleotides in length, wherein positions 2, 4, 6, 8, 10, 12, 14, 16, 18, and 20 at the 5' end of the antisense strand are 2'-fluoro nucleotides, and the remaining positions are 2'-methoxy nucleotides; the sense strand of the siRNA is 21 nucleotides in length, wherein positions 9, 10, and 11 at the 5' end of the sense strand are 2'-fluoro nucleotides, and the remaining positions are 2'-methoxy nucleotides.

[0051] In some embodiments of the present invention, the antisense strand of the siRNA is 21 nucleotides in length, wherein positions 2, 4, 6, 8, 10, 12, 14, 16, 18, and 20 at the 5' end of the antisense strand are 2'-fluoro nucleotides, and the remaining positions are 2'-methoxy nucleotides; the sense strand of the siRNA is 21 nucleotides in length, wherein positions 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, and 21 at the 5' end of the sense strand are 2'-fluoro nucleotides, and the remaining positions are 2'-methoxy nucleotides.

[0052] In some embodiments of the present invention, the antisense strand of the siRNA is 21 nucleotides in length, wherein positions 2, 6, 14, and 16 at the 5' end of the antisense strand are 2'-fluoro nucleotides, and the remaining positions are 2'-methoxy nucleotides; the sense strand of the siRNA is 19 nucleotides in length, wherein positions 7, 8, and 9 at the 5' end of the sense strand are 2'-fluoro nucleotides, and the remaining positions are 2'-methoxy nucleotides.

[0053] In some embodiments of the present invention, the antisense strand of the siRNA is 19 nucleotides in length, wherein positions 2, 4, 6, 8, 10, 12, 14, 16, and 18 at the 5' end of the antisense strand are 2'-fluoro nucleotides, and the remaining positions are 2'-methoxy nucleotides; the sense strand of the siRNA is 19 nucleotides in length, wherein positions 7, 8, and 9 at the 5' end of the sense strand are 2'-fluoro nucleotides, and the remaining positions are 2'-methoxy nucleotides.

[0054] In some embodiments of the present invention, the antisense strand of the siRNA is 23 nucleotides in length, wherein the 14th position at the 5' end of the antisense strand is a 2'-fluoro nucleotide, the 2nd, 5th, and 7th positions are 2'-deoxy nucleotides, the 12th position is a threose nucleotide, and the remaining positions are 2'-methoxy nucleotides; the sense strand of the siRNA is 21 nucleotides in length, wherein the 9th, 10th, and 11th positions at the 5' end of the sense strand are 2'-fluoro nucleotides, and the remaining positions are 2'-methoxy nucleotides.

[0055] In some embodiments of the present invention, the antisense strand of the siRNA is 23 nucleotides in length, wherein the 2nd, 6th, 14th, and 16th positions at the 5' end of the antisense strand are 2'-fluoro nucleotides, and the remaining positions are 2'-methoxy nucleotides; the sense strand of the siRNA is 21 nucleotides in length, wherein the 9th, 10th, and 11th positions at the 5' end of the sense strand are 2'-fluoro nucleotides, the 1st position is a threose nucleotide, and the remaining positions are 2'-methoxy nucleotides.

[0056] In some embodiments of the present invention, the antisense chain of the siRNA is 23 nucleotides in length, wherein the 2nd, 6th, 14th, and 16th positions at the 5' end of the antisense chain are 2'-fluoro nucleotides, the 22nd position is a threose nucleotide, and the remaining positions are 2'-methoxy nucleotides; the sense chain of the siRNA is 21 nucleotides in length, wherein the 9th, 10th, and 11th positions at the 5' end of the sense chain are 2'-fluoro nucleotides, and the remaining positions are 2'-methoxy nucleotides.

[0057] In some embodiments of the present invention, the antisense strand of the siRNA is 23 nucleotides in length, wherein the 2nd, 6th, 14th, and 16th positions at the 5' end of the antisense strand are 2'-fluoro nucleotides, the 23rd position is a threose nucleotide, and the remaining positions are 2'-methoxy nucleotides; the sense strand of the siRNA is 21 nucleotides in length, wherein the 9th, 10th, and 11th positions at the 5' end of the sense strand are 2'-fluoro nucleotides, and the remaining positions are 2'-methoxy nucleotides.

[0058] In some embodiments of the present invention, the antisense strand of the siRNA is 22 nucleotides in length, wherein the 2nd, 6th, 14th, and 16th positions at the 5' end of the antisense strand are 2'-fluoro nucleotides, and the remaining positions are 2'-methoxy nucleotides; the sense strand of the siRNA is 20 nucleotides in length, wherein the 8th, 9th, and 10th positions at the 5' end of the sense strand are 2'-fluoro nucleotides, the 1st position is a threose nucleotide, and the remaining positions are 2'-methoxy nucleotides.

[0059] In some embodiments of the present invention, the antisense strand of the siRNA is 22 nucleotides in length, wherein the 2nd, 6th, 14th, and 16th positions at the 5' end of the antisense strand are 2'-fluoro nucleotides, the 21st position is a threose nucleotide, and the remaining positions are 2'-methoxy nucleotides; the sense strand of the siRNA is 20 nucleotides in length, wherein the 8th, 9th, and 10th positions at the 5' end of the sense strand are 2'-fluoro nucleotides, and the remaining positions are 2'-methoxy nucleotides.

[0060] In some embodiments of the present invention, the antisense strand of the siRNA is 22 nucleotides in length, wherein the 2nd, 6th, 14th, and 16th positions at the 5' end of the antisense strand are 2'-fluoro nucleotides, the 22nd position is a threose nucleotide, and the remaining positions are 2'-methoxy nucleotides; the sense strand of the siRNA is 20 nucleotides in length, wherein the 8th, 9th, and 10th positions at the 5' end of the sense strand are 2'-fluoro nucleotides, and the remaining positions are 2'-methoxy nucleotides.

[0061] In some embodiments of the present invention, the antisense strand of the siRNA is 22 nucleotides in length, wherein the 2nd, 6th, 14th, and 16th positions at the 5' end of the antisense strand are 2'-fluoro nucleotides, and the remaining positions are 2'-methoxy nucleotides; the sense strand of the siRNA is 20 nucleotides in length, wherein the 8th, 9th, and 10th positions at the 5' end of the sense strand are 2'-fluoro nucleotides, and the remaining positions are 2'-methoxy nucleotides.

[0062] In some embodiments of the present invention, the antisense strand of the siRNA is 22 nucleotides in length, wherein the 2nd, 6th, 14th, and 16th positions at the 5' end of the antisense strand are 2'-fluoro nucleotides, and the remaining positions are 2'-methoxy nucleotides; the sense strand of the siRNA is 20 nucleotides in length, wherein the 6th, 8th, 9th, and 10th positions at the 5' end of the sense strand are 2'-fluoro nucleotides, and the remaining positions are 2'-methoxy nucleotides.

[0063] In some embodiments of the present invention, the antisense strand of the siRNA is 22 nucleotides in length, wherein the 2nd, 6th, 14th, and 16th positions at the 5' end of the antisense strand are 2'-fluoro nucleotides, and the remaining positions are 2'-methoxy nucleotides; the sense strand of the siRNA is 20 nucleotides in length, wherein the 8th, 9th, and 10th positions at the 5' end of the sense strand are 2'-fluoro nucleotides, the 6th position is a 2'-deoxy nucleotide, and the remaining positions are 2'-methoxy nucleotides.

[0064] In some embodiments of the present invention, the antisense strand of the siRNA is 22 nucleotides in length, wherein the 2nd, 6th, 8th, 9th, 14th, and 16th positions at the 5' end of the antisense strand are 2'-fluoro nucleotides, and the remaining positions are 2'-methoxy nucleotides; the sense strand of the siRNA is 20 nucleotides in length, wherein the 6th, 8th, 9th, and 10th positions at the 5' end of the sense strand are 2'-fluoro nucleotides, and the remaining positions are 2'-methoxy nucleotides.

[0065] In some embodiments of the present invention, the antisense strand of the siRNA is 22 nucleotides in length, wherein the 14th position at the 5' end of the antisense strand is a 2'-fluoro nucleotide, positions 2, 5, and 7 are 2'-deoxy nucleotides, position 12 is a threose nucleotide, and the remaining positions are 2'-methoxy nucleotides; the sense strand of the siRNA is 20 nucleotides in length, wherein positions 8, 9, and 10 at the 5' end of the sense strand are 2'-fluoro nucleotides, and the remaining positions are 2'-methoxy nucleotides.

[0066] In some embodiments of the present invention, the antisense strand of the siRNA is 22 nucleotides in length, wherein the antisense strand has a nucleotide sequence at positions 3-10 (e.g., positions 2, 14, 16, 2, 5, 14, 16, 2, 4, 6, 14, 16, 2, 6, 10, 14, 16, 2, 6, 12, 14, 16, 2, 5, 10, 14, 16, 2, 3, 12, 14, 16, 2, 9, 10, 14, 16, 2 ... , 12,14,16, 2,6,8,9,14,16, 2,3,5,12,14,16, 2,8,9,12,14,16, 2,7,9,12,14,16, 2,4,6,8,10,14,16,18,20, 2,4,5,6,8,10,12,14,16,18) have 2'-fluoro nucleotides, and the remaining positions are 2'-methoxy nucleotides,

[0067] The sense strand of the siRNA is 20 nucleotides in length, wherein the 8th, 9th and 10th positions at the 5' end of the sense strand are 2'-fluoro nucleotides, optionally a threose nucleotide at one position (preferably the 1st position at the 5' end), optionally a 2'-deoxynucleotide at one position (preferably the 6th position at the 5' end), and the remaining positions are 2'-methoxy nucleotide conjugates.

[0068] In some embodiments of the invention, the antisense strand of the siRNA is 22 nucleotides in length, wherein the antisense strand has 2'-fluoro nucleotides 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, 5, 10, 14, 16, or 2, 3, 12, 14, 16, or 2, 3, 12, 14, 16, or 2, 5, 10, 14, 16, or 2, 3, 12, 14, 16, or 2, 6 ...0, 14, 16, or 2, 6, 12, 14, 16, or 2, or 2,9,12,14,16; or 2,6,8,9,14,16; 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), the remaining positions are 2'-methoxy nucleotides,

[0069] The sense strand of the siRNA is 20 nucleotides in length, wherein the 8th, 9th and 10th positions of the 5' end of the sense strand are 2'-fluoro nucleotides, optionally have a threose nucleotide at the 1st position of the 5' end, or optionally have a 2'-deoxynucleotide at the 6th position of the 5' end, and the remaining positions are 2'-methoxy nucleotides.

[0070] In some embodiments of the invention, the antisense strand of the siRNA is 22 nucleotides in length, wherein the antisense strand has 2'-fluoro nucleotides 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, 6, 8, 9, 14, 16, or 2, 7, 9, 12, 14, 16, or 2, 8, 9 ...6, 12, 14, 16, or 2, 6, 12, 14, 16, or 2, 6, 10, 14, 16, or 2, 6, 12, 14, 16, or 2, , 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), and the remaining positions are 2'-methoxy nucleotides. The sense strand of the siRNA is 20 nucleotides in length, wherein positions 8, 9, and 10 at the 5' end of the sense strand are 2'-fluoro nucleotides, and the remaining positions are 2'-methoxy nucleotides.

[0071] In some embodiments of the present invention, the antisense strand of the siRNA is 21 nucleotides in length, wherein the 2nd, 6th, 14th, and 16th positions at the 5' end of the antisense strand are 2'-fluoro nucleotides, and the remaining positions are 2'-methoxy nucleotides; the sense strand of the siRNA is 19 nucleotides in length, wherein the 7th, 8th, and 9th positions at the 5' end of the sense strand are 2'-fluoro nucleotides, the 1st position is a threose nucleotide, and the remaining positions are 2'-methoxy nucleotides.

[0072] In some embodiments of the present invention, the antisense strand of the siRNA is 21 nucleotides in length, wherein the 2nd, 6th, 14th, and 16th positions at the 5' end of the antisense strand are 2'-fluoro nucleotides, the 20th position is a threose nucleoside, and the remaining positions are 2'-methoxy nucleotides; the sense strand of the siRNA is 19 nucleotides in length, wherein the 7th, 8th, and 9th positions at the 5' end of the sense strand are 2'-fluoro nucleotides, and the remaining positions are 2'-methoxy nucleotides.

[0073] In some embodiments of the present invention, the antisense strand of the siRNA is 21 nucleotides in length, wherein the 2nd, 6th, 14th, and 16th positions at the 5' end of the antisense strand are 2'-fluoro nucleotides, the 21st position is a threose nucleoside, and the remaining positions are 2'-methoxy nucleotides; the sense strand of the siRNA is 19 nucleotides in length, wherein the 7th, 8th, and 9th positions at the 5' end of the sense strand are 2'-fluoro nucleotides, and the remaining positions are 2'-methoxy nucleotides.

[0074] In some embodiments of the present invention, the antisense strand of the siRNA is 21 nucleotides in length, wherein the 14th position at the 5' end of the antisense strand is a 2'-fluoro nucleotide, positions 2, 5, and 7 are 2'-deoxy nucleotides, position 12 is a threose nucleotide, and the remaining positions are 2'-methoxy nucleotides; the sense strand of the siRNA is 19 nucleotides in length, wherein positions 7, 8, and 9 at the 5' end of the sense strand are 2'-fluoro nucleotides, and the remaining positions are 2'-methoxy nucleotides.

[0075] In some embodiments of the invention, the antisense strand of the siRNA is 22 nucleotides in length, wherein the antisense strand has a nucleotide sequence at positions 3-10 (e.g., 2,14,16, 2,5,14,16, 2,6,14,16, 2,4,6,14,16, 2,6,10,14,16, 2,6,12,14,16, 2,5,10,14,16, 2,3,12,14,16) of the 5' end; 2, 9, 12, 14, 16, 2, 6, 8, 9, 14, 16, 2, 3, 5, 12, 14, 16, 2, 8, 9, 12, 14, 16, 2, 7, 9, 12, 14, 16, 2, 4, 6, 8, 10, 14, 16, 18, 20, 2, 4, 5, 6, 8, 10, 12, 14, 16, 18) have 2'-fluoro nucleotides, and the remaining positions have 2'-methoxy nucleotides,

[0076] The sense strand of the siRNA is 20 nucleotides in length, wherein the 8th, 9th and 10th positions at the 5' end of the sense strand are 2'-fluoro nucleotides, optionally a threose nucleotide at one position (preferably the 1st position at the 5' end), optionally a 2'-deoxynucleotide at one position (preferably the 6th position at the 5' end), and the remaining positions are 2'-methoxy nucleotide conjugates.

[0077] In some embodiments of the invention, the antisense strand of the siRNA is 22 nucleotides in length, wherein the antisense strand has 2'-fluoro nucleotides 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, 4, 6, 14, 16, or 2, 6, 10, 14, 16, or 2, 3, 12, 14, 16, or 2, 5, 10, 14, 16, or 2, 3, 12, 14, 16, or 2, 3, 12, 14, 16, or 2, 4, 6, 14, 16, or 2, 6, 10 ... , 14,16; or 2,9,12,14,16; or 2,6,8,9,14,16; 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), the remaining positions are 2'-methoxy nucleotides,

[0078] The sense strand of the siRNA is 20 nucleotides in length, wherein positions 8, 9, and 10 of the 5' end of the sense strand are 2'-fluoro nucleotides, optionally have a threose nucleotide at position 1 of the 5' end, or optionally have a 2'-deoxynucleotide at position 6 of the 5' end, and the remaining positions are 2'-methoxy nucleotides;

[0079] Preferably, the 2'-deoxynucleotide at position 6 is independently selected from: adenine deoxyribonucleotide (dA), guanine deoxyribonucleotide (dG), cytosine deoxyribonucleotide (dC), thymine deoxyribonucleotide (dT), and uracil deoxyribonucleotide (dU); further preferably, the 2'-deoxynucleotide at position 6 is thymine deoxyribonucleotide (dT).

[0080] In some embodiments of the invention, the antisense strand of the siRNA is 22 nucleotides in length, wherein the antisense strand has 2'-fluoro nucleotides 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, 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), and the remaining positions are 2'-methoxy nucleotides, and the sense strand of the siRNA is 20 nucleotides in length, wherein positions 8, 9, 10 at the 5' end of the sense strand are 2'-fluoro nucleotides, and the remaining positions are 2'-methoxy nucleotides; or

[0081] The antisense strand of the siRNA is 22 nucleotides in length, wherein positions 2, 6, 14, and 16 of the 5' end of the antisense strand are 2'-fluoro nucleotides, and the remaining positions are 2'-methoxy nucleotides; the sense strand of the siRNA is 20 nucleotides in length, wherein positions 8, 9, and 10 of the 5' end of the sense strand are 2'-fluoro nucleotides, position 1 is a threose nucleotide, and the remaining positions are 2'-methoxy nucleotides; or

[0082] The antisense strand of the siRNA is 22 nucleotides in length, wherein positions 2, 6, 14, and 16 of the 5' end of the antisense strand are 2'-fluoro nucleotides, and the remaining positions are 2'-methoxy nucleotides; the sense strand of the siRNA is 20 nucleotides in length, wherein positions 8, 9, and 10 of the 5' end of the sense strand are 2'-fluoro nucleotides, position 6 is a 2'-deoxy nucleotide, and the remaining positions are 2'-methoxy nucleotides; preferably, the 2'-deoxy nucleotide at position 6 is independently selected from the group consisting of adenine deoxyribonucleotide (dA), guanine deoxyribonucleotide (dG), cytosine deoxyribonucleotide (dC), thymine deoxyribonucleotide (dT), and uracil deoxyribonucleotide (dU); further preferably, the 2'-deoxy nucleotide at position 6 is thymine deoxyribonucleotide (dT);

[0083] 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.

[0084] In some embodiments of the present invention, the antisense strand of the siRNA is 22 nucleotides in length, wherein positions 2, 6, 14, and 16 of the 5' end of the antisense strand are 2'-fluoro nucleotides, and the remaining positions are 2'-methoxy nucleotides; the sense strand of the siRNA is 20 nucleotides in length, wherein positions 8, 9, and 10 of the 5' end of the sense strand are 2'-fluoro nucleotides, and the remaining positions are 2'-methoxy nucleotides;

[0085] 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.

[0086] In some embodiments of the present invention, the antisense strand of the siRNA is 22 nucleotides in length, wherein the 14th position at the 5' end of the antisense strand is a 2'-fluoro nucleotide, positions 2, 5, and 7 are 2'-deoxy nucleotides, position 12 is a threose nucleotide, and the remaining positions are 2'-methoxy nucleotides; the sense strand of the siRNA is 20 nucleotides in length, wherein positions 8, 9, and 10 at the 5' end of the sense strand are 2'-fluoro nucleotides, and the remaining positions are 2'-methoxy nucleotides;

[0087] Preferably, the 2'-deoxynucleotides at positions 2, 5, and 7 are independently selected from the group consisting of adenine deoxyribonucleotide (dA), guanine deoxyribonucleotide (dG), cytosine deoxyribonucleotide (dC), thymine deoxyribonucleotide (dT), and uracil deoxyribonucleotide (dU);

[0088] Further preferably, the 2'-deoxynucleotides at positions 2, 5, and 7 are respectively: thymidine deoxyribonucleotide (dT), thymidine deoxyribonucleotide (dT), and adenine deoxyribonucleotide (dA);

[0089] 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.

[0090] In some embodiments of the present invention, the antisense strand of the siRNA is 21 nucleotides in length, wherein positions 2, 6, 14, and 16 of the 5' end of the antisense strand are 2'-fluoro nucleotides, and the remaining positions are 2'-methoxy nucleotides; the sense strand of the siRNA is 19 nucleotides in length, wherein positions 7, 8, and 9 of the 5' end of the sense strand are 2'-fluoro nucleotides, position 1 is a threose nucleotide, and the remaining positions are 2'-methoxy nucleotides; or

[0091] The antisense strand of the siRNA is 21 nucleotides in length, wherein the 14th position at the 5' end of the antisense strand is a 2'-fluoro nucleotide, the 2nd, 5th, and 7th positions are 2'-deoxy nucleotides, the 12th position is a threose nucleotide, and the remaining positions are 2'-methoxy nucleotides; the sense strand of the siRNA is 19 nucleotides in length, wherein the 7th, 8th, and 9th positions at the 5' end of the sense strand are 2'-fluoro nucleotides, and the remaining positions are 2'-methoxy nucleotides;

[0092] Preferably, the 2'-deoxynucleotides at positions 2, 5, and 7 are independently selected from the group consisting of adenine deoxyribonucleotide (dA), guanine deoxyribonucleotide (dG), cytosine deoxyribonucleotide (dC), thymine deoxyribonucleotide (dT), and uracil deoxyribonucleotide (dU);

[0093] Further preferably, the 2'-deoxynucleotides at positions 2, 5, and 7 are respectively: thymine deoxyribonucleotide (dT), uracil deoxyribonucleotide (dU), and adenine deoxyribonucleotide (dA);

[0094] 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.

[0095] In some embodiments of the present invention, the modified nucleotide is a nucleotide in which the phosphate group is modified with a phosphorothioate group, that is, a non-bridging oxygen atom in the phosphodiester bond is replaced with a sulfur atom, thereby replacing the phosphodiester bond with a phosphorothioate diester bond.

[0096] In some embodiments of the invention, the 5' and 3' ends of the sense strand independently contain 0, 1 or 2 phosphorothioate linkages; and / or the 5' and 3' ends of the antisense strand independently contain 1 or 2 phosphorothioate linkages.

[0097] In some embodiments of the present invention, at least one of the nucleotides between the 1st and 2nd positions at the 5′ end of the sense chain, between the 2nd and 3rd positions at the 5′ end of the sense chain, between the 1st and 2nd positions at the 3′ end of the sense chain, between the 2nd and 3rd positions at the 3′ end of the sense chain, between the 1st and 2nd positions at the 3′ end of the antisense chain, between the 2nd and 3rd positions at the 3′ end of the antisense chain, between the 1st and 2nd positions at the 5′ end of the antisense chain, and between the 2nd and 3rd positions at the 5′ end of the antisense chain is linked by a thiophosphate group; preferably, at least four of the nucleotides are linked by thiophosphate groups; in some embodiments of the present invention, at least six of the nucleotides are linked by thiophosphate groups; in some embodiments of the present invention, all eight of the nucleotides are linked by thiophosphate groups.

[0098] In some embodiments of the present invention, the nucleotides at positions 1 and 2, and the nucleotides at positions 2 and 3 at the 5' end of the sense strand are linked by phosphorothioate groups.

[0099] In some embodiments of the present invention, the nucleotides at positions 1 and 2 and the nucleotides at positions 2 and 3 of the 5′ end of the sense strand are linked by phosphorothioate groups, and the nucleotides at positions 1 and 2 and the nucleotides at positions 2 and 3 of the 3′ end are linked by phosphorothioate groups.

[0100] In some embodiments of the present invention, the nucleotides at positions 1 and 2, and the nucleotides at positions 2 and 3 at the 3′ end of the antisense strand are linked by phosphorothioate groups, and the nucleotides at positions 1 and 2, and the nucleotides at positions 2 and 3 at the 5′ end are linked by phosphorothioate groups.

[0101] In some embodiments of the present invention, the nucleotides between the 1st and 2nd positions at the 5′ end of the sense chain, the nucleotides between the 2nd and 3rd positions at the 5′ end of the sense chain, the nucleotides between the 1st and 2nd positions at the 3′ end of the sense chain, the nucleotides between the 2nd and 3rd positions at the 3′ end of the sense chain, the nucleotides between the 1st and 2nd positions at the 3′ end of the antisense chain, the nucleotides between the 2nd and 3rd positions at the 3′ end of the antisense chain, the nucleotides between the 1st and 2nd positions at the 5′ end of the antisense chain, and the nucleotides between the 2nd and 3rd positions at the 5′ end of the antisense chain are all linked by phosphorothioate groups.

[0102] In some embodiments of the present invention, the sense strand may include one or more blocking residues or moieties, referred to as "blocking residues." A "blocking residue" is a non-nucleotide compound or other moiety that can be incorporated into one or more ends of the nucleotide sequence of the siRNA. In some embodiments of the present invention, the blocking residue is present at the 5' end, the 3' end, or both the 5' end and the 3' end of the sense strand.

[0103] In some embodiments of the present invention, an inverted abasic 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' end and / or 3' end of the sense strand may contain more than one inverted abasic deoxyribose moiety as a capping residue.

[0104] In some embodiments of the present invention, one or more reverse abasic residues (iab or invAb) are added to the 3 ' end of the sense strand. In some embodiments of the present invention, one or more reverse abasic residues (iab or invAb) are added to the 5 ' end of the sense strand. In some embodiments of the present invention, one or more reverse abasic residues can be inserted between the nucleotide sequence of the delivery vehicle portion and the siRNA sense strand. In some embodiments of the present invention, one or more reverse abasic residues are included near or at one or more ends of the siRNA sense strand. The reverse abasic residues (iab or invAb) are selected from the following structures:

[0105]

[0106] Wherein, X=O or S.

[0107] In some embodiments of the invention, one or more inverted abasic residues (iab or invAb) are added to the 5' end of the sense strand. In some embodiments of the invention, one or more inverted abasic residues may be inserted between the delivery vehicle portion and the nucleotide sequence of the siRNA sense strand.

[0108] Inverted abasic residues can be linked via phosphate, phosphorothioate, or other internucleoside linkages.

[0109] In some embodiments of the present invention, the first nucleotide at the 5' end of the antisense strand is selected from the following structures:

[0110]

[0111] Wherein, Basse is base A, U, G, C, T or other nucleotide bases.

[0112] Wherein, Basse is base A, U, G, C, T or other nucleotide bases.

[0113] In some embodiments of the present invention, the first nucleotide at the 5' end of the antisense strand is a nucleotide modified with (E)-vinyl phosphate.

[0114] In some embodiments of the present invention, the siRNA contains at least one modified nucleotide.

[0115] In some embodiments of the present invention, the base of the base-modified nucleotide is selected from the following structures:

[0116]

[0117] 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.

[0118] In some embodiments of the present invention, the base-modified nucleotides are located at single-stranded nucleotide overhangs in the siRNA.

[0119] Preferably, the siRNA antisense strand contains an overhang of 2 nucleotides, and the base-modified nucleotide is the first nucleotide of the overhang of the siRNA antisense strand.

[0120] Preferably, the siRNA antisense strand contains an overhang of 2 nucleotides, and the base-modified nucleotide is the second nucleotide of the overhang of the siRNA antisense strand.

[0121] The present invention also provides a siRNA conjugate obtained by conjugating the above siRNA with a conjugation molecule.

[0122] In the present invention, unless otherwise specified, "conjugation" refers to the covalent attachment of two or more chemical moieties; "conjugate" refers to a compound formed by covalent attachment of chemical moieties; and "siRNA conjugate" refers to a compound formed by covalent attachment of one or more chemical moieties to an siRNA. It should be noted that each chemical moiety can be attached to the siRNA directly or via a linker.

[0123] In some embodiments of the invention, the delivery vector is linked to the siRNA via a covalent bond to form a conjugate molecule.

[0124] In some embodiments of the present invention, the delivery vector is linked to the 3' end or 5' end of the siRNA sense strand independently or simultaneously through a covalent bond to form a conjugated molecule.

[0125] In some embodiments of the present invention, the siRNA of the present invention can be conjugated with a pharmaceutically acceptable conjugate molecule to obtain an siRNA conjugate. 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 of the siRNA to the conjugate molecule can be at the 3' end or 5' end of the siRNA sense strand, or at the 5' end of the antisense strand. In some embodiments, the conjugation site of the siRNA to the conjugate molecule can also be within the internal sequence of the siRNA.

[0126] In the present invention, "delivery vector" refers to a chemical moiety that is covalently linked to the siRNA and that affects the targeting, activity, cellular distribution, cellular uptake or stability of the oligonucleotide, and the conjugation of siRNA to one or more delivery vectors can improve the pharmacological properties of the siRNA. In certain embodiments, the delivery vector portion is a pharmacokinetic property that modifies or enhances the oligonucleotide by improving the cellular distribution, bioavailability, metabolism, excretion, permeability or cellular uptake of the oligonucleotide. In particular, the delivery vector can target the oligonucleotide to a specific organ, tissue or cell type, thereby enhancing the effectiveness of the oligonucleotide in the organ, tissue or cell type. Simultaneously, the delivery vector portion can be used to reduce the activity of the oligonucleotide in non-target cell types, tissues or organs, for example, off-target activity or activity in non-target cell types, tissues or organs.

[0127] The pharmaceutically acceptable delivery carrier can be a delivery carrier conventionally used in the field of siRNA administration, such as, but not limited to, one or more of the following delivery carriers or their derivatives: lipophilic molecules, such as cholesterol, bile acid, vitamins (such as vitamin E), lipid molecules of different chain lengths; polymers, such as polyethylene glycol; polypeptides, such as membrane-permeable peptides; aptamers; antibodies; quantum dots; carbohydrates, such as lactose, polylactose, mannose, galactose, N-acetylgalactosamine (GalNAc); folic acid (folate); or receptor ligands expressed by hepatocytes, such as asialoglycoproteins, asialoglycosylated residues, lipoproteins (such as high-density lipoproteins, low-density lipoproteins, etc.), glucagon, neurotransmitters (such as epinephrine), growth factors, transferrin, etc.

[0128] In some embodiments of the invention, the delivery vehicle contains an N-acetylgalactosamine group.

[0129] In some embodiments of the present invention, the delivery vector is directly connected to the 3' end of the siRNA sense strand. In some embodiments of the present invention, the delivery vector is directly connected to the 5' end of the siRNA sense strand. In some embodiments of the present invention, the delivery vector is connected to the 3' end of the siRNA sense strand via a joint. In some embodiments of the present invention, the delivery vector is connected to the 5' end of the siRNA sense strand via a joint.

[0130] In some embodiments of the present invention, the delivery vector contains N-acetylgalactosamine, which is covalently linked to the 3' end of the sense strand of the siRNA.

[0131] In some embodiments of the invention, the delivery vector portion is GalNAc(L96) having the following structure:

[0132]

[0133] In some embodiments of the invention, GalNAc(L96) is linked to the 3' end of the sense strand of the siRNA.

[0134] In some embodiments of the invention, GalNAc(L96) is linked to the 5' end of the sense strand of the siRNA.

[0135] In some embodiments of the invention, GalNAc(L96) is linked to the inverted abasic residue (iab or invAb) at the 3' end of the siRNA sense strand.

[0136] In some embodiments of the invention, GalNAc(L96) is linked to the inverted abasic residue (iab or invAb) at the 5' end of the siRNA sense strand.

[0137] In some embodiments of the present invention, the delivery vehicle portion is Ser(GN) having the following structure:

[0138]

[0139] In some embodiments of the invention, Ser(GN) is linked to the 3' end of the sense strand of the siRNA. In some embodiments of the invention, Ser(GN) is linked to the 5' end of the sense strand of the siRNA.

[0140] In some embodiments of the present invention, Ser(GN) is linked to both the 3' end and the 5' end of the sense strand of the siRNA.

[0141] In some embodiments of the present invention, the delivery vector portion is LP-GalNAc (attached to the 5'-end of the sense strand) having the following structure:

[0142]

[0143] In some embodiments of the present invention, the delivery vector portion is XY-GalNAc (attached to the 3' end of the sense strand) having the following structure:

[0144]

[0145] or

[0146]

[0147] In some embodiments of the present invention, the structure of the other delivery vector moiety used (attached to the 5'-end of the sense strand) is as follows:

[0148]

[0149]

[0150]

[0151] In some embodiments of the present invention, the structure of the other delivery vector moiety used (attached to the 3'-end of the sense strand) is as follows:

[0152]

[0153]

[0154] Optionally, the delivery vector portion is linked to the 3' end of the siRNA sense strand, or to the 5' end of the siRNA sense strand, or to the inverted abasic residue (iab) at the 3' end of the siRNA sense strand, or to the inverted abasic residue (iab) at the 5' end of the siRNA sense strand, or one or more of the delivery vector portions are simultaneously linked to the 3' end of the siRNA sense strand, the 5' end of the siRNA sense strand, the inverted abasic residue (iab) at the 3' end of the siRNA sense strand, and the inverted abasic residue (iab) at the 5' end of the siRNA sense strand; preferably, the delivery vector portion The delivery vector portion GalNAc (L96) is linked to the 3' end of the siRNA sense strand or to the inverted abasic residue (iab) at the 3' end of the siRNA sense strand, the delivery vector portion Ser (GN) is simultaneously linked to the 3' end and 5' end of the siRNA sense strand, the delivery vector portion LP-GalNAc is linked to the 5' end of the siRNA sense strand, the delivery vector portion XY-GalNAc is linked to the 3' end of the siRNA sense strand, the delivery vector portion GalNAc (Ser2) is linked to the 3' end of the siRNA sense strand or to the 5' end of the siRNA sense strand or to the siRNA sense strand simultaneously The 3' end and 5' end of the sense strand, the delivery carrier part GalNAc (Ser3) is linked to the 3' end of the siRNA sense strand or to the 5' end of the siRNA sense strand, the delivery carrier part GalNAc (Ser4) is linked to the 3' end of the siRNA sense strand, the delivery carrier part GalNAc (A1GN) is linked to the 3' end of the siRNA sense strand, the delivery carrier part GalNAc (A2GN) is linked to the 3' end of the siRNA sense strand, the delivery carrier part GalNAc (A3GN) is linked to the 5' end of the siRNA sense strand, the delivery carrier part GalNAc (A4GN) Attached to the 5' end of the siRNA sense strand, the delivery carrier part GalNAc (A5GN) is attached to the 5' end of the siRNA sense strand, the delivery carrier part GalNAc (NAG25) is attached to the 5' end of the siRNA sense strand, the delivery carrier part GalNAc (NAG37) is attached to the 5' end of the siRNA sense strand, the delivery carrier part GalNAc (Ser1) and GalNAc (Ser2) are attached to the 3' end of the siRNA sense strand and the 5' end of the siRNA sense strand respectively, or are attached to the 5' end of the siRNA sense strand and the 3' end of the siRNA sense strand respectively at the same time;

[0155] Preferably, the delivery vehicle portion is selected from the following: GalNAc (L96) linked to the 3' end of the siRNA sense strand, GalNAc (AlGN) linked to the 3' end of the siRNA sense strand, GalNAc (A2GN) linked to the 3' end of the siRNA sense strand, GalNAc (A3GN) linked to the 5' end of the siRNA sense strand, GalNAc (A4GN) linked to the 5' end of the siRNA sense strand, and GalNAc (Ser2) linked to both the 3' and 5' ends of the siRNA sense strand.

[0156] The siRNA conjugates listed below are selected from conjugates 1 to 1668 and the positive control PC c, wherein the basic sequences corresponding to the sense chain and antisense chain of each conjugate before modification correspond to one of SEQ ID NO: 1 to SEQ ID NO: 200 listed above and SEQ ID NO: 201 to SEQ ID NO: 351 listed here, respectively.

[0157]

[0158]

[0159]

[0160]

[0161] Among them, C, G, U, A, and T represent the base composition of nucleotides, including modified and unmodified nucleotides; I represents the base composition of base-modified nucleotides, and the base is m6A represents the base composition of base-modified nucleotides, the base is X represents the base composition of the base-modified nucleotide, where B represents the base composition of the base-modified nucleotide, the base is

[0162] In some embodiments of the present invention, the siRNA conjugate is selected from conjugate 1 to conjugate 1668.

[0163]

[0164]

[0165]

[0166]

[0167]

[0168]

[0169]

[0170]

[0171]

[0172]

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[0174]

[0175]

[0176]

[0177]

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[0179]

[0180]

[0181]

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[0183]

[0184]

[0185]

[0186]

[0187]

[0188]

[0189]

[0190]

[0191]

[0192]

[0193]

[0194]

[0195]

[0196]

[0197]

[0198]

[0199]

[0200]

[0201]

[0202]

[0203]

[0204]

[0205]

[0206]

[0207]

[0208]

[0209]

[0210]

[0211]

[0212]

[0213]

[0214]

[0215]

[0216]

[0217]

[0218]

[0219]

[0220]

[0221]

[0222]

[0223]

[0224]

[0225]

[0226]

[0227]

[0228]

[0229]

[0230]

[0231]

[0232]

[0233]

[0234]

[0235]

[0236]

[0237]

[0238]

[0239]

[0240]

[0241]

[0242]

[0243]

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[0347] The present invention also provides a pharmaceutical composition comprising any of the above-mentioned siRNAs and / or any of the above-mentioned siRNA conjugates and a pharmaceutically acceptable carrier.

[0348] In some embodiments of the present invention, the pharmaceutical composition contains one siRNA as described in the first aspect. In other embodiments of the present disclosure, the pharmaceutical composition contains at least two siRNAs as described in the first aspect (for example, but not limited to, two, three, four, five, six, seven, eight, nine, ten or more) as active ingredients. Preferably, the at least two siRNAs as described in the first aspect each target a different target sequence in the AGT gene, thereby being expected to act simultaneously against different target sequences and bring about a synergistic effect. Here, the so-called "different target sequences" refers to the absence of overlap between target sequences, or the number of overlapping consecutive nucleotides between target sequences is less than 5 (for example, 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 can be present in any different ratios. Preferably, the at least two siRNAs as described in the first aspect are present in a molar ratio of 1:100 to 100:1; more preferably, the at least two siRNAs as described in the first aspect are present 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 as described in the first aspect are present in the same molar ratio.

[0349] The present invention also provides use of any of the above siRNAs and / or any of the above siRNA conjugates and / or the above pharmaceutical compositions in the preparation of a medicament for treating and / or preventing pathological conditions or diseases associated with overexpression of the AGT gene.

[0350] Furthermore, the pathological condition or disease is a disease associated with abnormal blood pressure.

[0351] The siRNA, siRNA conjugate and pharmaceutical composition provided by the present invention have good stability, excellent AGT gene inhibition activity, satisfactory cytotoxicity and immunostimulation, and can significantly reduce blood pressure levels.

[0352] In the present invention, unless otherwise specified, capital letters C, G, U, A, and T represent the base composition of nucleotides, including modified and unmodified nucleotides; lowercase letter m indicates that the nucleotide adjacent to the right of the mark m is a 2'-methoxy nucleotide; lowercase letter f indicates that the nucleotide adjacent to the right of the mark f is a 2'-fluoro nucleotide; lowercase letter d indicates that the nucleotide adjacent to the right of the mark d is a 2'-deoxy nucleotide; gn indicates that the nucleotide adjacent to the right of the mark gn is a glycerol nucleotide (GNA); tn indicates that the nucleotide adjacent to the right of the mark tn is a threose nucleotide (TNA); the mark * indicates that the two nucleotides adjacent to the left and right of the mark * (or between the nucleotide and the delivery vector part) are connected by thiophosphate groups; eVP indicates that the nucleotide adjacent to the right is a nucleotide modified with (E)-vinyl phosphate. For example, when the base is U, the structure of eVPmU is iab indicates an inverted abasic residue; GalNAc(L96) indicates that the delivery vehicle moiety GalNAc(L96) is conjugated to this site. Ser(GN) indicates that the delivery vehicle moiety Ser(GN) is conjugated to this site.

[0353] In the present invention, unless otherwise specified, the capital letter I represents the base composition of the base-modified nucleotide. m6A represents the base composition of base-modified nucleotides, the base is The capital letter X represents the base composition of the base-modified nucleotide. The capital letter B represents the base composition of the base-modified nucleotide, the base is

[0354] 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 and form a double-stranded structure. "At least partially complementary" means that the two sequences can be completely complementary, or generally have no more than 5, 4, 3, or 2 mismatched base pairs, while retaining the ability to hybridize under relevant conditions. In addition, when two oligonucleotides are designed to form one or more single-stranded overhangs upon hybridization, such overhangs should not be considered mismatches for the purpose of determining complementarity. In the present invention, when meeting the above hybridization ability requirements, "complementary" sequences may also include or be formed entirely from non-Watson-Crick base pairs and / or base pairs formed from non-natural and modified nucleotides. Such non-Watson-Crick base pairs include, but are not limited to, G:U wobble base pairs or Hoogstein base pairs. Correspondingly, in the present invention, unless otherwise specified, "mismatch" means that the bases at corresponding positions in the siRNA duplex molecule are not paired in a complementary form.

[0355] In the present invention, unless otherwise specified, "a difference in nucleotide sequence" refers to a change in the base type of the nucleotide at the same or corresponding position compared to the original nucleotide sequence. For example, if one 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, etc., a difference in nucleotide sequence is considered to exist at that position. It should be noted that if the nucleotide at the same or corresponding position differs only in the presence or type of modification compared to the original nucleotide sequence, a difference in nucleotide sequence is not considered to exist at that position.

[0356] In the present invention, unless otherwise specified, the term "pharmaceutically acceptable" means that the carrier, vehicle, diluent, excipient and / or the salt / ester / hydrate formed therefrom are generally chemically or physically compatible with other ingredients constituting a pharmaceutical dosage form and physiologically compatible with the receptor.

[0357] In the present invention, unless otherwise specified, the term "inhibit" refers to the down-regulation of target gene expression due to siRNA-mediated degradation of target gene mRNA. The "down-regulation" refers to a decrease in target gene expression by 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%, relative to the absence of siRNA treatment. A 100% decrease in target gene expression means no detectable level of target gene expression.

[0358] In the present invention, the siRNA may further contain modified nucleotides as needed, provided that the modified nucleotides do not significantly weaken or abolish the siRNA's ability to inhibit AGT gene expression. Currently, there are a variety of methods available in the art for modifying siRNA, including backbone modifications (e.g., phosphate group modifications), ribose group modifications, and base modifications (Watts, JK, GF Deleavey, and MJ Damha, Chemically modified siRNA: tools and applications. Drug Discov Today, 2008. 13(19-20): p. 842-55).

[0359] Obviously, based on the above contents of the present invention, according to common technical knowledge and customary means in this field, without departing from the above basic technical ideas of the present invention, other various forms of modifications, replacements or changes can be made.

[0360] The following further describes the above content of the present invention in detail through specific embodiments in the form of examples. However, this should not be construed as limiting the scope of the above subject matter of the present invention to the following examples. All technologies implemented based on the above content of the present invention fall within the scope of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0361] Figure 1 Schematic diagram showing the synthetic route of the conjugates of the present invention.

[0362] Figure 2 Process flow diagram showing the synthesis of the conjugate of the present invention

[0363] Figure 3 、 Figure 4 The MS of the test article conjugate 100 is shown.

[0364] Figure 5 1H-NMR of the test conjugate 100 is shown.

[0365] Figure 6 The 13C-NMR of the test conjugate 100 is shown.

[0366] Figure 7 The 31P-NMR of the test conjugate 100 is shown.

[0367] Figure 8 The 19F-NMR of the test conjugate 100 is shown.

[0368] Figure 9 The melting temperature (Tm) test of the test article conjugate 100 is shown. DETAILED DESCRIPTION

[0369] It is known to those skilled in the art that the siRNA of the present invention can be obtained by conventional siRNA preparation methods in the art (e.g., solid phase synthesis and liquid phase synthesis), wherein both solid phase synthesis and liquid phase synthesis are commercially available. It is also clear to those skilled in the art that modified nucleotide groups can be introduced into the siRNA of the present invention by using nucleotide monomers with corresponding modifications. Methods for preparing nucleotide monomers with corresponding modifications are well known to those skilled in the art, and commercial monomers are also available on the market.

[0370] Example 1: siRNA synthesis

[0371] For the sense and antisense strands of the siRNA sequences of the present invention and the sense and antisense strands of the modified duplexes, strand synthesis is initiated using a solid support.

[0372] Taking conjugate 100 as an example, the preparation process of the conjugate of the present application is described.

[0373] A GalNAc (L96)-modified solid phase support was used as the starting cycle for the positive chain synthesis, and a universal solid phase support (Primer support 5G unylinker 350) was used as the starting cycle for the antisense chain ( Figure 1 ).

[0374] The structure of the GalNAc(L96)-modified solid support (L96-PS) is as follows:

[0375]

[0376] Primer support 5G unylinker 350 carrier structure is as follows:

[0377]

[0378] ●PS stands for polystyrene solid phase carrier

[0379] Using a YB-192S synthesizer and a phosphoramidite triester solid phase synthesis method, starting with a solid phase support, nucleoside monomers were sequentially connected in the 3'-5' direction to perform sequence synthesis on a synthesis scale of 0.2 μmol.

[0380] See the process flow Figure 2 .

[0381] Process description: Oligo synthesis starts with a PS solid phase support and uses a 3′-0-(2-cyanoethyl)phosphoramidite / 4,4′dimethoxytrityl (dimethoxytrityl, DMT) group protection method to assemble oligonucleotide chains on a PS solid phase support. Each synthesis cycle includes 5′-hydroxyl deprotection, coupling, capping and oxidation (thiolation). Each coupling reaction is carried out by activating the appropriate phosphoramidite monomer and reacting it with the free 5′-hydroxyl group of the protected nucleotide or oligonucleotide fixed on the support. According to the sequence information, the corresponding crude oligonucleotide single-strand PS-Oligo is synthesized cyclically, and the crude oligonucleotide single chain is cleaved from the solid phase support and the relevant protecting groups are removed. The final product is then purified by preparative chromatography, desalted by ultrafiltration, annealed, and freeze-dried.

[0382] (1) Synthesis procedure

[0383] Includes the following units:

[0384] 1) Deblocking: The 5'-OH end of the ribonucleotide is protected with a DMT group (di-p-methoxytrityl). In the first step of the synthesis, trichloroacetic acid (TCA) is used to remove the DMT protecting group from the solid phase support and the ribonucleotide, so that the 5'-OH end of the naked ribonucleotide can be coupled with a new base.

[0385] 2) Coupling: Nucleotide monomers are mixed with an activating reagent and react with the PS-Oligo in the synthesis column. The activating reagent donates a proton to the nitrogen atom of the diisopropylamide on the 3'-phosphate, forming a phosphoramidite tetrazole reactive intermediate. Upon contact with the PS-Oligo, the phosphoramidite tetrazole undergoes a nucleophilic reaction with the 5'-hydroxyl group, resulting in coupling and tetrazole removal, extending one nucleotide.

[0386] 3) Capping: Since the coupling efficiency cannot reach 100%, in order to prevent unsuccessfully coupled PS-Oligo from continuing to the next step of coupling, a capping reagent is used to cap the 5'-hydroxyl group of PS-Oligo.

[0387] 4) Oxidation or thiolation: After the coupling reaction, the nucleotide is linked to the oligonucleotide on the PS carrier via a phosphite bond (trivalent phosphorus). This phosphite bond is unstable and easily hydrolyzed by acid and base. The oxidizing agent oxidizes the trivalent phosphorus to pentavalent phosphorus. Thiolation refers to the reaction of the trivalent phosphorus in the phosphite bond with a thiolation agent under weak alkaline conditions to form a phosphorus-sulfur bond.

[0388] 5) VP protecting group removal and aminolysis

[0389] Transfer the synthesized PS-oligo from the synthesis column to a centrifuge tube. Prepare a removal reagent in a volume ratio of 3:2:100 = TMS-I: pyridine: DCM. Add the removal reagent to the centrifuge tube and react for 30 minutes. Prepare a TEA / acetonitrile = 1:1 solution, add 2-mercaptoethanol (final concentration 2M), mix well, and add to the reaction to terminate the reaction. Remove the supernatant and wash the PS-oligo twice for 5 minutes. After washing, add aminolysis solution (2-mercaptoethanol (2M) / 28% ammonia water) and aminolysis for 10 hours. After the aminolysis is completed, vacuum centrifuge to remove the ammonia water and purify.

[0390] The sense strands and antisense strands of other siRNA sequences of the present invention and the sense strands and antisense strands of modified duplexes are prepared using similar methods.

[0391] (2) Purification

[0392] The diluted sample after ammoniolysis was purified by anion column exchange chromatography, and the liquid phase system adopted high performance liquid chromatography. The chromatographic conditions were as follows.

[0393] Chromatographic column: PS-15Q10*250mm

[0394] Flow rate: 4ml / min

[0395] Detection wavelength: 260nm

[0396] Ion column preparation purification gradient program:

[0397] Mobile phase: Phase A: 10mM NaOH solution (PH=10) Phase B: 10mM NaOH solution (PH=10) + 2M NaCl

[0398]

[0399] After preparation, the samples were confirmed by mass spectrometry and purity was confirmed.

[0400] (3) Ultrafiltration

[0401] Dissolve the purified sample in 4 ml of PBS, transfer it to a 1K ultrafiltration tube, and centrifuge it at 5000 rpm for 45 minutes. Then, use a Nanodrop to detect the presence of the sample in the tube. If the sample is detected at a wavelength of 260 nm, the ultrafiltration membrane is damaged and the tube should be replaced and ultrafiltration repeated. If the sample is not detected at a wavelength of 260 nm, add 4 ml of RNase-free water and ultrafiltration again. Ultrafiltration should be repeated three times, each for 45 minutes.

[0402] (4) Annealing

[0403] After ultrafiltration, dilute the sense and antisense strands to 3 mg / ml, then mix them in a 1:1 molar ratio. Heat the mixture in a water bath to 90°C and anneal for 30 minutes. Then, close the water bath and allow the mixture to cool to room temperature for 16 hours. A 10 μL sample was collected for HPLC analysis.

[0404] Representative test method - LC-MS (using conjugate 100 as an example)

[0405] 1) Instrument model: Waters Xevo G2-XS Qtof

[0406] 2) Test conditions

[0407] Ion source: ESI negative ion source

[0408] Scan range: 100~2000Da

[0409] Collision energy: 15-25EV, 25-50EV

[0410] 3) The spectrum of the test conjugate 100 is shown in the attached Figure 3 and Figure 4 shown

[0411] 4) Spectrum analysis

[0412] When the test conjugate 100 was subjected to denaturing IP·RP-LC, the complementary double strands were unwound into single strands (sense and antisense strands). Tandem mass spectrometry then performed gas-phase fragmentation of the positive and antisense strand precursor ions. All detected fragment ions were analyzed and resolved using the CONFIRMSequence software. In this experiment, the precursor ions were fragmented using energies of 15-25 eV and 25-50 eV, respectively. A coverage of 100% was achieved, indicating that the test sample sequence was consistent with the theoretical sequence. The confirmation results are shown in the table below.

[0413] Sequence confirmation of the positive chain of conjugate 100

[0414]

[0415] Sequence confirmation of the antisense strand of conjugate 100

[0416]

[0417] Nuclear magnetic resonance (NMR) testing of conjugate 100

[0418] 1) Instrument model: Bruker AVANCE III 600M NMR

[0419] 2) Detection method: Weigh 95.0 mg of sample and dissolve it in heavy water (D2O), then transfer it to a nuclear magnetic resonance tube for testing.

[0420] 3) Spectrum analysis

[0421] From 1H-NMR ( Figure 5 ) can be used to determine the peak positions of aromatic heterocycles, ribose, methyl, amine, methylene, sugar rings, and alcoholic hydroxyl hydrogens. The peaks from 0.95ppm to 2.5ppm are methylene hydrogens not directly connected to amides, 3.17ppm to 3.20ppm are methylene hydrogens adjacent to amides, 3.4ppm to 4.6ppm are methine hydrogens on sugar rings, 2′-methyl, and ether bond methylene hydrogens, 5.1ppm to 6.5ppm are methine hydrogens at the 1′-position of the sugar ring, pyrimidine base aromatic rings, and 5′-vinyl hydrogens, and 7.1ppm to 8.2ppm are hydrogens on the purine base aromatic rings.

[0422] 13C-NMR( Figure 6The spectrum shows the peak positions of carbonyl groups, aromatic heterocycles, sugar rings, ether linkage methylene groups, methylene groups, and methyl groups within the molecule. The carbonyl carbon is at 170ppm-180ppm, the carbonyl carbon is at 150ppm-165ppm, the carbon atoms corresponding to the aromatic heterocycle (base) and vinyl groups are at 150ppm-165ppm, the 1′-methyl group of ribose is at 95ppm-100ppm, the methylene group and methylene carbon atoms of ribose are at 70ppm-95ppm, the methoxy group and ether linkage methylene carbon atoms are at 55ppm-70ppm, and the α-methyl carbon atoms of the acetyl group are at 20ppm-40ppm.

[0423] The above results show that the aromatic heterocycle (base), vinyl, sugar ring, exocyclic methoxy, methylene, ether bond methylene, and acetyl groups were displayed in 1H-NMR and 13C-NMR, which are consistent with the target molecular structure.

[0424] 31P-NMR( Figure 7 ) The spectrum shows signals of three phosphate functional groups: thiophosphate diester (P=S) with a chemical shift of 54.5ppm to 57.5ppm, phosphate diester (P=O) with a chemical shift of -0.97ppm to -1.5ppm, and 5′-vinyl phosphate with a chemical shift of 9.16ppm. The ratio of P=O bond to P=S bond is approximately 5:1, and the information is consistent with the target molecule.

[0425] From 19F-NMR ( Figure 8 ) The peak position in the spectrum is between -199ppm and -201ppm, which is consistent with the F position at the 2′-position of the sugar ring.

[0426] From the above analysis, it can be seen that the NMR signal is consistent with the target molecule.

[0427] Melting temperature (Tm) test of conjugate 100:

[0428] 1) Instrument model: Cary 3500

[0429] 2) Test conditions

[0430] Scanning wavelength: 260nm

[0431] Temperature scanning range: 25℃~90℃

[0432] Heating rate: 3℃ / min

[0433] 3) The spectrum of the test conjugate 100 is as shown in the attached Figure 9 shown

[0434] 4) Spectrum analysis

[0435] The software system calculated the Tm of the test conjugate to be 66.0°C using the Savitzky-Golay algorithm based on the 100°C temperature increase curve.

[0436] LC-MS detection after conjugate synthesis

[0437]

[0438]

[0439]

[0440]

[0441] Among them, PC c is a positive control, which is known to have an AGT gene inhibition effect. Its structure is as follows:

[0442] Sense strand: mG*mU*mCmAmUmCfCmAfCfAfAmUmGmAmGmAmGmUmAmCmAGalNAc(L96) (the basic sequence before modification corresponds to SEQ ID NO: 251)

[0443] Antisense strand: mU*fG*mUmAmCgnTmCmUmCmAmUmUmGfUmGfGmAmUmGmAmC*mG*mA (the basic sequence before modification corresponds to SEQ ID NO: 348)

[0444] Example 2: In vitro activity detection

[0445] Cell culture and transfection

[0446] Cell culture: Hep3B cells (ATCC) were cultured in MEM complete medium (Gibco, supplemented with 10% FBS) at 37°C in a 5% CO2 environment until nearly confluent. The cells were then trypsinized and plated. 2.0 × 10 5 Hep3B cells and 1.0 mL of MEM complete medium (Gibco, supplemented with 10% FBS) were cultured at 37° C. in a 5% CO 2 environment for 16-24 h before transfection.

[0447] Cell transfection: 1.5 μL of lipofectamine RNAiMax (Invitrogen) was added to 48.5 μL of opti-MEM per well, followed by 50 μL of siRNA. The mixture was added to a PCR tube and incubated at room temperature for 5 minutes. The siRNA mixture was then added to the cells and cultured for 24 hours before RNA extraction. Single-dose experiments were performed with 10 nM and 0.1 nM or 0.1 nM and 0.01 nM siRNA duplex concentrations. IC s0The test experiment was carried out at 10nM, 1.0nM, 0.1nM, 0.01nM, 0.001nM, 0.0001nM and 0.00001nM siRNA duplex concentrations, and the IC50 of the compound was calculated using the log(inhibitor) vs.response--Variable slope(our parameters) fitting method of GraphPad Prism software.

[0448] RNA extraction

[0449] Use the tota1 RNA isolation kit (Omega, cat: R6834-02): collect cells, wash with 1% PBS, then add 400 μL of lysis buffer (containing 2% β-mercaptoethanol) to lyse the cells. Follow the instructions of the RNA isolation kit for subsequent steps. Finally, add 30 μL of RNase-free water, let it stand for 2 minutes, and then centrifuge at 14000g for 2 minutes to collect RNA.

[0450] cDNA synthesis

[0451] cDNA synthesis was performed using the Quanshijin gDNA removal cDNA synthesis kit (Beijing Quanshijin 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.

[0452] Real-time fluorescence quantitative PCR

[0453] The synthesized cDNA and mixed master solution (including primers, qPCR premix and ultrapure water) were added to a 384-well plate (Biocon Biotechnology Cat# PC-0040-9U). The final real-time fluorescence quantitative PCR system contained 0.25 μM of upstream and downstream primers of the target gene (AGT) or internal reference gene (GADPH) and 1× SYBR Green premix (Applied Biosystem Cat# A25742).

[0454] Using the AACt assay in ABIQuantStudio TM Real-time fluorescence PCR was performed in a 6-well real-time fluorescence PCR system. Each duplex was tested for 3-4 independent transfections, and each transfection was performed in triplicate.

[0455] Human / Cynomolgus Monkey Liver Primary Cells

[0456] Free uptake: thaw frozen human or cynomolgus monkey liver primary cells, count live cells, and adjust the density (6×10 5Add 10 μL of 10× compound to the collagen-coated cell culture plate and aliquot 90 μL (5.4×10 4 The cell suspension (1000 cells / well) was transferred to a 96-well collagen-coated plate. The cell plate was incubated in a 37°C, 5% CO2 incubator for 48 hours. Single-dose experiments were performed at 10 nM and 0.1 nM siRNA duplex concentrations. 50 The test experiments were performed at 10 nM, 3.33 nM, 1.11 nM, 0.37 nM, 0.12 nM, 0.041 nM, 0.014 nM and 0.0046 nM siRNA duplex concentrations, and the IC50 of the compounds was calculated using the log (inhibitor) vs. response--Variable slope (four parameters) fitting method of GraphPad Prism software.

[0457] cDNA synthesis: After 48 hours of free uptake, remove the culture medium and lyse the cells for RNA extraction. Use according to the kit instructions. Total RNA was extracted using QIAGEN-74182 RT Kit, and cDNA was synthesized using the FastKing RT Kit (with gDNase) (Tiangen KR116-02) according to the manufacturer's instructions.

[0458] Real-time fluorescence quantitative PCR: The synthesized cDNA and mixed master mix (including primers, qPCR premix, and ultrapure water) were added to a 384-well plate. The final real-time fluorescence quantitative PCR system contained 0.25 μM each of the upstream and downstream primers of the target gene (AGT) or internal reference gene (GADPH), 0.125 μM of the probe, and 1× Universal Probe Master premix (Roche, Cat. No. 04914058001).

[0459] Use the ΔΔCt assay in ABIQuantStudio TM Real-time fluorescence PCR was performed in a 6-well real-time fluorescence PCR system. Each duplex was tested for 3-4 independent transfections, and each transfection was performed in triplicate.

[0460] The in vitro activity test results of some siRNA conjugates are shown in Tables 1 to 5. PC c, which is known to have an AGT gene inhibitory effect, was used as a positive control.

[0461] Table 1. Single-dose Hep3B cell viability

[0462]

[0463]

[0464]

[0465]

[0466] Table 2. Single-dose human primary hepatocyte activity

[0467]

[0468]

[0469] Table 3. Hep3B cell transfection IC 50 Test results

[0470] Conjugate number <![CDATA[IC 50 (pM)]]> Duplex number <![CDATA[IC 50 (pM)]]> Conjugate 68 3.397 Conjugate 71 4.482 Conjugate 100 3.361±2.238 Conjugate 102 3.921 Conjugate 161 1.324 Conjugate 162 2.576 Conjugate 163 1317 PC c 2.645±0.837

[0471] Table 4. IC uptake by PCH cells 50 Test results

[0472] Conjugate number <![CDATA[IC 50 (nM)]]> Duplex number <![CDATA[IC 50 (nM) <!-- 228 -->]]> Conjugate 100 0.286 Conjugate 102 0.311 Conjugate 161 0.133 Conjugate 162 0.056 Conjugate 163 0.192 PC c 0.102

[0473] Table 5. IC uptake by PHH cells 50 Test results

[0474] Conjugate number <![CDATA[IC 50 (nM)]]> Duplex number <![CDATA[IC 50 (nM)]]> Conjugate 100 0.094 Conjugate 102 0.072 Conjugate 161 0.142 Conjugate 162 0.196 Conjugate 163 0.073 PC c 0.173

[0475] Example 3: Tritosome stability

[0476] The test siRNA was placed in rat tritosomes and mixed (5 μM). The mixture was then incubated at 37°C for 24 and 48 hours. After incubation, 1 / 3 of the sample volume of phenol-chloroform (Biyuntian, Cat: #p1011) was added and vortexed. The mixture was allowed to rest for 5 minutes and then centrifuged at 12,000 rpm for 30 minutes. The top aqueous phase was transferred to a new centrifuge tube and 1 / 10 volume of 3M NaAC (solarbio, Cat: #A1070) was added and mixed. Then, 2.5 volumes of pre-chilled anhydrous ethanol and 1 μL of glycogen (Biyuntian, Cat: #0812) were added and mixed. The mixture was allowed to stand at -20°C for 2 minutes. Centrifuged at 12,000 rpm for 30 minutes, the supernatant discarded, and the mixture was air-dried. RNAse-free water was added to dissolve the sample. The dissolved sample was mixed with formamide loading buffer and analyzed by nucleic acid electrophoresis using a 10% polyacrylamide gel. After electrophoresis, the PAGE gel was stained with GelRed (Biyuntian, Cat: #D0140) for 30 minutes. Finally, the gel was imaged using a gel imager (Bio-Rad) and grayscale quantitative analysis was performed using Image Lab software. Grayscale quantitative analysis showed that the rat tritosome stability of conjugate 100 was superior to that of the clinical molecule PC c.

[0477] Example 4: Cytotoxicity test

[0478] Cell culture: Hep3B cells (ATCC) were cultured in MEM complete medium (Gibco, supplemented with 10% FBS) at 37°C in a 5% CO2 environment until nearly confluent. The cells were then trypsinized and plated. 0.5 × 10 cells were added to each well of a 24-well plate. 5 Hep3B cells and 0.5 mL MEM complete medium (Gibco, supplemented with 10% FBS) were cultured at 37° C. in a 5% CO 2 environment for 16-24 h before transfection.

[0479] Cell transfection: 0.75 μL of lipofectamine RNAiMax (Invitrogen) was added to 24.25 μL of opt-MEM per well, followed by 25 μL of siRNA. The mixture was added to a PCR tube and incubated at room temperature for 5 minutes. The siRNA mixture was then added to the cells and cultured for 48 or 96 hours before cytotoxicity testing. This experiment was performed at 10 nM and 1 nM siRNA duplex concentrations.

[0480] Cytotoxicity test:

[0481] Cytotoxicity testing was performed using a CCK-8 kit (Biyuntian, CAT#C0040). 48 / 96 hours after 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 at 37°C in the dark for 30-60 minutes. The OD values of the samples were measured using a microplate reader (Tecan, CAT#spark 20M) at a wavelength of 450 nm compared to a reference wavelength of 620 nm. The results showed that the cytotoxicity of conjugates 100, 102, and 106 was similar to that of clinical PC c molecules, with no significant toxicity.

[0482] Example 5: siRNA immunogenicity test

[0483] hPBMC cells purchased from three different donors (Shanghai Saili Biotechnology Co., Ltd., XFB-HP050A) were centrifuged and resuspended in RPMI-1640 cell culture medium (containing 10% FBS and 1% Penicillin-streptomycin solution). After mixing, they were cultured in a 5% CO2, 37°C incubator overnight. The test compound was diluted to 20× working solution. 3000 and Opti-Mem were prepared into a mixed solution at a ratio of 1.5:23.5 and vortexed before use. 3000 μL of the mixture was mixed at a 1:1 ratio in a 96-well V-bottom plate. Discard the overnight culture medium and resuspend the hPBMC cells in RPMI-1640 cell culture medium (containing 10% FBS and 1% Penicillin-streptomycin solution). After counting the cells, dilute the cells to the desired density for transfection and plate them into a 96-well plate. The final cell count in the plate was 2.0 × 105 cells / well in a total volume of 200 μL. Incubate the plate in a 5% CO2, 37°C incubator for 24 hours.

[0484] 24 hours after hPBMC transfection, cell supernatants were collected and assayed for IFN alpha, IL-6, and TNF alpha using the ProcartaPlex Mix&Match 3-plex Kit. The fold change of each factor was calculated: for test siRNA, nake siRNA, and ployIC, the fold change was calculated as: detected factor concentration / factor concentration in the transfection reagent well; for GS9688, the fold change was calculated as: detected factor concentration / factor concentration in the DMSO well. The results showed that conjugates 100, 102, 161, 162, and 163 did not induce significant immune activation in hPBMCs.

[0485] Example 6: Evaluation of off-target ability of siRNA seed region

[0486] Sequences for detecting on-target activity and off-target activity in the seed region are constructed for each siRNA sequence. On-target detection sequences are sequences that completely match the siRNA being tested. Off-target detection in the seed region is a perfect match with the antisense strand seed region (2-8 at the 5' end) of the siRNA being tested, with no sequence mismatches elsewhere. Five on-target and off-target detection sequences are inserted into each psiCHECK plasmid.

[0487] 293T cells were seeded at 20,000 cells / well in 96-well plates. Test siRNAs were co-transfected with 25 ng of psiCHECK plasmids constructed for either on-target or off-target expression in the seed region using lipofectamine 2000. After 24 hours of incubation at 37°C, 5% CO₂, fluorescence was measured using a Dual-Glo luciferase assay system (Promega, E2920). Three to four independent transfections were performed for each duplex, with psiCHECK plasmid alone transfected without siRNA as a control. The transfection concentrations of each siRNA were 50 nM, 10 nM, 2.0 nM, 0.4 nM, 0.08 nM, 0.016 nM, and 0.0032 nM. Target inhibition was determined by measuring Renilla luciferase activity normalized to constitutively expressed firefly luciferase levels, and the IC₅₀ values of the on-target or off-target activity of the siRNAs were calculated. The off-target risk of the siRNA seed region was assessed by the ratio of off-target activity IC50 of the seed region to on-target activity IC50. The test results showed that the off-target risk of conjugates 100, 102, and 161 due to the seed region was lower than that of the clinical molecule PC c.

[0488] Example 7: In vivo activity detection of hAGT in mice

[0489] 6-8 week old male hAGT humanized mice (provided by Jiangsu Jicui, T054372). The mice were not fasted before group administration, and blood was collected to separate serum for ELISA (abcam#) detection of hAGT. The mice were randomly divided into 4 groups according to hAGT levels, with 6 mice in each group, defined as day 0. On day 1, PBS, PC c, conjugate 100, conjugate 102, and conjugate 106 were subcutaneously injected, respectively, with a drug dose of 1.0 mpk. After administration, blood was collected on day 8, day 15, day 22, day 29, day 36, day 43, and day 50 to separate serum for ELISA detection of hAGT. The serum protein concentration of each group at each time point was compared with the serum protein concentration of the PBS group at that time point. The detection results of different conjugates are shown in Table 6. The candidate compound has a better inhibition on serum hAGT protein in hAGT mice than the clinical molecule PC c.

[0490] Table 6. Changes in hAGT protein expression before and after administration of different siRNA modified duplexes

[0491]

[0492]

[0493] *This value is the relative expression of hAGT protein compared with the PBS control group

[0494] Table 7. Changes in hAGT protein expression before and after administration of different delivery vectors conjugated with modified siRNA duplexes

[0495]

[0496]

[0497] *This value is the relative expression of hAGT protein compared with the PBS control group

[0498] Example 8: In vivo activity detection in common cynomolgus monkeys

[0499] In this study, 12 male cynomolgus macaques aged 3-6 years (weighing 2.5-6.0 kg) were screened based on body weight, temperature, blood pressure, electrocardiogram, serum AGT levels, hematology, and blood biochemistry. The animals were divided into four groups of three macaques each. Each animal received a subcutaneous injection of 3.0 mpk of the test conjugate molecule.

[0500] Blood was collected from cynomolgus monkeys after fasting for 12 hours before administration (day 0) and at fixed times every week after administration. The whole blood samples were placed in a centrifuge tube in an ice box without anticoagulant for 30 minutes, centrifuged at a centrifugal force of approximately 1800×g for 10 minutes at 2-8°C, and the serum was separated and stored below -80°C.

[0501] AGT protein levels in serum were measured using an AGT ELISA kit (IBL, 27412). The AGT protein content at each assay point was compared with the AGT protein content in the serum of the macaque before administration (day 0), and the inhibition of the test conjugate on the AGT content in the macaque serum at different time points was calculated. As shown in Table 8, the candidate compound inhibited the AGT protein content in the macaque serum by more than 90% at a dose of 3.0 mpk, which was not significantly different from the results reported in the clinical molecular PC c patent (WO_2019222166_A1).

[0502] Table 8. Inhibitory effects of different conjugates on serum AGT protein in cynomolgus monkeys after administration

[0503]

Claims

1. siRNA conjugate, characterized in that: The siRNA conjugate is selected from conjugate 100, conjugate 102, conjugate 106, conjugate 162, and conjugate 163: Wherein, C, G, U, A, and T represent the base composition of the nucleotide; m indicates that the nucleotide adjacent to the right of the marker m is a 2'-methoxy nucleotide; f indicates that the nucleotide adjacent to the right of the marker f is a 2'-fluoro nucleotide; d indicates that the nucleotide adjacent to the right of the marker d is a 2'-deoxy nucleotide; the marker * indicates that the two nucleotides adjacent to the left and right of the marker * or the nucleotide and the delivery vector portion are linked by a thiophosphate group; eVP indicates that the nucleotide adjacent to the right is a (E)-vinyl phosphate-modified nucleotide; iab indicates an inverted abasic residue; GalNAc(L96) means that the delivery vector portion GalNAc(L96) is conjugated thereto, and GalNAc(L96) has the following structure:

2. A pharmaceutical composition, characterized in that: The pharmaceutical composition comprises the siRNA conjugate according to claim 1 and a pharmaceutically acceptable carrier.

3. Use of the siRNA conjugate according to claim 1 and / or the pharmaceutical composition according to claim 2 in the preparation of a medicament for treating and / or preventing diseases associated with abnormal blood pressure.

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