CN117070517A

CN117070517A - siRNA for inhibiting expression of angiotensinogen, conjugate, pharmaceutical composition and application thereof

Info

Publication number: CN117070517A
Application number: CN202310912943.0A
Authority: CN
Inventors: 王书成; 黄河; 王岩; 林国良; 产运霞; 耿玉先; 杨明艳; 汪小君
Original assignee: Beijing Fuyuan Pharmaceutical Co ltd
Current assignee: Beijing Fuyuan Pharmaceutical Co ltd
Priority date: 2022-08-04
Filing date: 2023-07-24
Publication date: 2023-11-17

Abstract

The present application relates to siRNA that inhibit the expression of the Angiotensinogen (AGT) gene, siRNA conjugates, pharmaceutical compositions comprising the same, and uses thereof. Each nucleotide in the siRNA is independently a modified or unmodified nucleotide, the siRNA comprising a sense strand and an antisense strand. The siRNA and the conjugate and the pharmaceutical composition thereof can effectively treat and/or prevent diseases related to the over-expression of AGT genes.

Description

siRNA for inhibiting expression of angiotensinogen, conjugate, pharmaceutical composition and application thereof

Technical Field

The present application relates to siRNA that inhibit the expression of the Angiotensinogen (AGT) gene, siRNA conjugates, pharmaceutical compositions comprising the same, methods of preparation and uses thereof.

Background

Renin (AGT), also known as SERPINA8 or ANHU, is a member of the serine protease inhibitor family and is a component of the renin-angiotensin-aldosterone system (RAAS). It is produced mainly in the liver and released into the circulatory system where renin converts it to angiotensin I. Angiotensin I is then converted to angiotensin II by Angiotensin Converting Enzyme (ACE). Angiotensin II is a peptide hormone that causes vasoconstriction, which in turn can increase blood pressure. Angiotensin II also stimulates secretion of the hormone aldosterone in the adrenal cortex. Aldosterone causes the kidneys to increase reabsorption of sodium and water, resulting in an increase in the volume of fluid in the body, which in turn may increase blood pressure. Excessive stimulation or activity of the RAAS pathway can lead to hypertension.

Hypertension is a major risk factor for a variety of diseases, disorders and conditions such as reduced life expectancy, chronic kidney disease, stroke, myocardial infarction, heart failure, vascular aneurysms (e.g., aortic aneurysms), peripheral arterial disease, cardiac injury (e.g., enlarged or hypertrophic heart), and other cardiovascular-related diseases, disorders and/or conditions. However, current approved therapies for treating hypertension have limitations in that all hypertensive patients fail to achieve adequate blood pressure control. For example, drugs that target portions of the renin-angiotensin-aldosterone system (RAAS) pathway, such as ACE inhibitors and Angiotensin Receptor Blockers (ARBs), have limited ability to inhibit the RAAS pathway (Nobakht et al, nat Rev Nephrol,2011, 7:356-359). In addition, certain antihypertensive drugs (such as ACE inhibitors) are contraindicated in hypertensive patients suffering from renal disease, as they may jeopardize the renal function of the patient. Thus, there is a need to find alternative therapies to inhibit the RAAS pathway and treat hypertension.

At present, no medicine targeting AGT is marketed, and the medicine is still in clinical stage, for example, the antisense nucleic acid medicine ISIS-904 of Ionis is in clinical stage 1 and is used for treating refractory hypertension, so that the development of the medicine targeting AGT has great significance and market prospect.

The present invention aims to provide siRNA, siRNA conjugate and pharmaceutical compositions thereof, which can inhibit the expression of AGT gene in liver, thereby achieving the purpose of disease treatment.

Disclosure of Invention

The present invention provides an siRNA capable of inhibiting AGT gene expression, the siRNA comprising a sense strand and an antisense strand, wherein each nucleotide in the siRNA is independently a modified or unmodified nucleotide, wherein the sense strand comprises nucleotide sequence I, and the antisense strand comprises nucleotide sequence II, which are at least partially reverse complementary to form a double stranded region, wherein the nucleotide sequence I and the nucleotide sequence II are selected from the group consisting of:

(1) The nucleotide sequence I comprises a nucleotide sequence shown as SEQ ID NO. 121, and the nucleotide sequence II comprises a nucleotide sequence shown as SEQ ID NO. 122:

5’-GCUAGUCGCUGCA-3’(SEQ ID NO:121)

5’-UGCAGCGACUAGC-3’(SEQ ID NO:122)，

wherein said nucleotide sequence I is not SEQ ID NO 27 and said nucleotide sequence II is not SEQ ID NO 28;

(2) The nucleotide sequence I comprises a nucleotide sequence shown as SEQ ID NO. 123, and the nucleotide sequence II comprises a nucleotide sequence shown as SEQ ID NO. 124:

5’-CCUUGGAAGGACAAGA-3’(SEQ ID NO:123)

5’-UCUUGUCCUUCCAAGG-3’(SEQ ID NO:124)；

(3) The nucleotide sequence I comprises a nucleotide sequence shown as SEQ ID NO. 125, and the nucleotide sequence II comprises a nucleotide sequence shown as SEQ ID NO. 126:

5’-GUGCUGAACAGCAUUU-3’(SEQ ID NO:125)

5’-AAAUGCUGUUCAGCAC-3’(SEQ ID NO:126)；

(4) The nucleotide sequence I comprises a nucleotide sequence shown as SEQ ID NO. 127, and the nucleotide sequence II comprises a nucleotide sequence shown as SEQ ID NO. 128:

5’-CUACCCAACAGCU-3’(SEQ ID NO:127)

5’-AGCUGUUGGGUAG-3’(SEQ ID NO:128)；

(5) The nucleotide sequence I comprises a nucleotide sequence shown as SEQ ID NO. 1, and the nucleotide sequence II comprises a nucleotide sequence shown as SEQ ID NO. 2;

(6) The nucleotide sequence I comprises a nucleotide sequence shown as SEQ ID NO. 5, and the nucleotide sequence II comprises a nucleotide sequence shown as SEQ ID NO. 6;

(7) The nucleotide sequence I comprises a nucleotide sequence shown as SEQ ID NO. 7, and the nucleotide sequence II comprises a nucleotide sequence shown as SEQ ID NO. 8;

(8) The nucleotide sequence I comprises a nucleotide sequence shown as SEQ ID NO. 53, and the nucleotide sequence II comprises a nucleotide sequence shown as SEQ ID NO. 54;

(9) The nucleotide sequence I comprises a nucleotide sequence shown as SEQ ID NO. 55, and the nucleotide sequence II comprises a nucleotide sequence shown as SEQ ID NO. 56;

(10) The nucleotide sequence I comprises a nucleotide sequence shown as SEQ ID NO. 61, and the nucleotide sequence II comprises a nucleotide sequence shown as SEQ ID NO. 62;

(11) The nucleotide sequence I comprises a nucleotide sequence shown as SEQ ID NO. 63, and the nucleotide sequence II comprises a nucleotide sequence shown as SEQ ID NO. 64;

(12) The nucleotide sequence I comprises a nucleotide sequence shown as SEQ ID NO. 67, and the nucleotide sequence II comprises a nucleotide sequence shown as SEQ ID NO. 68;

(13) The nucleotide sequence I comprises a nucleotide sequence shown as SEQ ID NO. 69, and the nucleotide sequence II comprises a nucleotide sequence shown as SEQ ID NO. 70;

(14) The nucleotide sequence I comprises a nucleotide sequence shown as SEQ ID NO. 119, and the nucleotide sequence II comprises a nucleotide sequence shown as SEQ ID NO. 120;

(15) The nucleotide sequence I comprises a nucleotide sequence shown as SEQ ID NO. 446, and the nucleotide sequence II comprises a nucleotide sequence shown as SEQ ID NO. 447:

5’-UCUGGGUACUA-3’(SEQ ID NO:446)

5’-UAGUACCCAGA-3’(SEQ ID NO:447)；

(16) The nucleotide sequence I comprises a nucleotide sequence shown as SEQ ID NO. 448, and the nucleotide sequence II comprises a nucleotide sequence shown as SEQ ID NO. 449:

5’-CUGCUCCAAUU-3’(SEQ ID NO:448)

5’-AAUUGGAGCAG-3’(SEQ ID NO:449)；

(17) The nucleotide sequence I comprises a nucleotide sequence shown as SEQ ID NO. 450, and the nucleotide sequence II comprises a nucleotide sequence shown as SEQ ID NO. 451:

5’-CUGCAAAACUU-3’(SEQ ID NO:450)

5’-AAGUUUUGCAG-3’(SEQ ID NO:451)；

(18) The nucleotide sequence I comprises a nucleotide sequence shown as SEQ ID NO. 452, and the nucleotide sequence II comprises a nucleotide sequence shown as SEQ ID NO. 453:

5’-CUCUCUAUACCCCU-3’(SEQ ID NO:452)

5’-AGGGGUAUAGAGAG-3’(SEQ ID NO:453)；

(19) The nucleotide sequence I comprises a nucleotide sequence shown as SEQ ID NO. 454, and the nucleotide sequence II comprises a nucleotide sequence shown as SEQ ID NO. 455:

5’-CAAGUGCCCUUCA-3’(SEQ ID NO:454)

5’-UGAAGGGCACUUG-3’(SEQ ID NO:455)；

(20) The nucleotide sequence I comprises a nucleotide sequence shown as SEQ ID NO. 456, and the nucleotide sequence II comprises a nucleotide sequence shown as SEQ ID NO. 457:

5’-ACAGAGUCUACCCAA-3’(SEQ ID NO:456)

5’-UUGGGUAGACUCUGU-3’(SEQ ID NO:457)；

(21) The nucleotide sequence I comprises a nucleotide sequence shown as SEQ ID NO. 458, and the nucleotide sequence II comprises a nucleotide sequence shown as SEQ ID NO. 459:

5’-GUCGACUUUGAGCUGGA-3’(SEQ ID NO:458)

5’-UCCAGCUCAAAGUCGAC-3’(SEQ ID NO:459)；

(22) The nucleotide sequence I comprises a nucleotide sequence shown as SEQ ID NO. 460, and the nucleotide sequence II comprises a nucleotide sequence shown as SEQ ID NO. 461:

5’-GAAAGCAGCCGU-3’(SEQ ID NO:460)

5’-ACGGCUGCUUUC-3’(SEQ ID NO:461)；

(23) The nucleotide sequence I comprises the nucleotide sequence shown as SEQ ID NO. 462, and the nucleotide sequence II comprises the nucleotide sequence shown as SEQ ID NO. 463:

5’-GGCACAAAUGCACCU-3’(SEQ ID NO:462)

5’-AGGUGCAUUUGUGCC-3’(SEQ ID NO:463)；

(24) The nucleotide sequence I comprises a nucleotide sequence shown as SEQ ID NO. 464, and the nucleotide sequence II comprises a nucleotide sequence shown as SEQ ID NO. 465:

5’-GCAGCUGGCAA-3’(SEQ ID NO:464)

5’-UUGCCAGCUGC-3’(SEQ ID NO:465)；

(25) The nucleotide sequence I comprises a nucleotide sequence shown as SEQ ID NO:269, and the nucleotide sequence II comprises a nucleotide sequence shown as SEQ ID NO: 270:

5’-CAAUGCCGGGAAGCCCAAA-3’(SEQ ID NO:269)

5’-UUUGGGCUUCCCGGCAUUG-3’(SEQ ID NO:270)；

(26) The nucleotide sequence I comprises a nucleotide sequence shown as SEQ ID NO. 466, and the nucleotide sequence II comprises a nucleotide sequence shown as SEQ ID NO. 467:

5’-ACCUUCAUACCU-3’(SEQ ID NO:466)

5’-AGGUAUGAAGGU-3’(SEQ ID NO:467)；

(27) The nucleotide sequence I comprises a nucleotide sequence shown as SEQ ID NO. 468, and the nucleotide sequence II comprises a nucleotide sequence shown as SEQ ID NO. 469:

5’-CCAGGAGUUCU-3’(SEQ ID NO:468)

5’-AGAACUCCUGG-3’(SEQ ID NO:469)；

(28) The nucleotide sequence I comprises the nucleotide sequence shown as SEQ ID NO. 470, and the nucleotide sequence II comprises the nucleotide sequence shown as SEQ ID NO. 471:

5’-CCACUGCCCUGCACUUCC-3’(SEQ ID NO:470)

5’-GGAAGUGCAGGGCAGUGG-3’(SEQ ID NO:471)；

(29) The nucleotide sequence I comprises a nucleotide sequence shown as SEQ ID NO:472, and the nucleotide sequence II comprises a nucleotide sequence shown as SEQ ID NO: 473:

5’-AGCUGGUGCUAGUCG-3’(SEQ ID NO:472)

5’-CGACUAGCACCAGCU-3’(SEQ ID NO:473)；

(30) The nucleotide sequence I comprises a nucleotide sequence shown as SEQ ID NO. 474, and the nucleotide sequence II comprises a nucleotide sequence shown as SEQ ID NO. 475:

5’-ACAGCUUAACAA-3’(SEQ ID NO:474)

5’-UUGUUAAGCUGU-3’(SEQ ID NO:475)；

(31) The nucleotide sequence I comprises a nucleotide sequence shown as SEQ ID NO. 476, and the nucleotide sequence II comprises a nucleotide sequence shown as SEQ ID NO. 477:

5’-GCCGUUUCUCCUU-3’(SEQ ID NO:476)

5’-AAGGAGAAACGGC-3’(SEQ ID NO:477)；

(32) The nucleotide sequence I comprises a nucleotide sequence shown as SEQ ID NO. 141, and the nucleotide sequence II comprises a nucleotide sequence shown as SEQ ID NO. 142;

(33) The nucleotide sequence I comprises a nucleotide sequence shown as SEQ ID NO. 145, and the nucleotide sequence II comprises a nucleotide sequence shown as SEQ ID NO. 146;

(34) The nucleotide sequence I comprises a nucleotide sequence shown as SEQ ID NO. 177, and the nucleotide sequence II comprises a nucleotide sequence shown as SEQ ID NO. 178;

(35) The nucleotide sequence I comprises a nucleotide sequence shown as SEQ ID NO. 179, and the nucleotide sequence II comprises a nucleotide sequence shown as SEQ ID NO. 180;

(36) The nucleotide sequence I comprises a nucleotide sequence shown as SEQ ID NO. 181, and the nucleotide sequence II comprises a nucleotide sequence shown as SEQ ID NO. 182;

(37) The nucleotide sequence I comprises a nucleotide sequence shown as SEQ ID NO. 187, and the nucleotide sequence II comprises a nucleotide sequence shown as SEQ ID NO. 188;

(38) The nucleotide sequence I comprises a nucleotide sequence shown as SEQ ID NO. 189, and the nucleotide sequence II comprises a nucleotide sequence shown as SEQ ID NO. 190;

(39) The nucleotide sequence I comprises a nucleotide sequence shown as SEQ ID NO. 191, and the nucleotide sequence II comprises a nucleotide sequence shown as SEQ ID NO. 192;

(40) The nucleotide sequence I comprises a nucleotide sequence shown as SEQ ID NO. 193, and the nucleotide sequence II comprises a nucleotide sequence shown as SEQ ID NO. 194;

(41) The nucleotide sequence I comprises a nucleotide sequence shown as SEQ ID NO. 199, and the nucleotide sequence II comprises a nucleotide sequence shown as SEQ ID NO. 200;

(42) The nucleotide sequence I comprises a nucleotide sequence shown as SEQ ID NO. 201, and the nucleotide sequence II comprises a nucleotide sequence shown as SEQ ID NO. 202;

(43) The nucleotide sequence I comprises a nucleotide sequence shown as SEQ ID NO. 203, and the nucleotide sequence II comprises a nucleotide sequence shown as SEQ ID NO. 204;

(44) The nucleotide sequence I comprises a nucleotide sequence shown as SEQ ID NO. 223, and the nucleotide sequence II comprises a nucleotide sequence shown as SEQ ID NO. 224;

(45) The nucleotide sequence I comprises a nucleotide sequence shown as SEQ ID NO. 243, and the nucleotide sequence II comprises a nucleotide sequence shown as SEQ ID NO. 244;

(46) The nucleotide sequence I comprises a nucleotide sequence shown as SEQ ID NO. 245, and the nucleotide sequence II comprises a nucleotide sequence shown as SEQ ID NO. 246;

(47) The nucleotide sequence I comprises a nucleotide sequence shown as SEQ ID NO. 247, and the nucleotide sequence II comprises a nucleotide sequence shown as SEQ ID NO. 248;

(48) The nucleotide sequence I comprises a nucleotide sequence shown as SEQ ID NO. 281, and the nucleotide sequence II comprises a nucleotide sequence shown as SEQ ID NO. 282.

In one embodiment, the nucleotide sequence I and the nucleotide sequence II are substantially reverse complementary, or fully reverse complementary; by substantially reverse complement is meant that there are no more than 3 base mismatches between the two nucleotide sequences; by substantially reverse complement is meant that there is no more than 1 base mismatch between the two nucleotide sequences; complete reverse complement refers to the absence of mismatches between two nucleotide sequences.

In one embodiment, the sense strand further comprises a nucleotide sequence III, the antisense strand further comprises a nucleotide sequence IV, the nucleotide sequence III and the nucleotide sequence IV are each independently 0-10 nucleotides in length, wherein the nucleotide sequence III is attached at the 5 'end of the nucleotide sequence I, the nucleotide sequence IV is attached at the 3' end of the nucleotide sequence II, the nucleotide sequence III and the nucleotide sequence IV are equal in length and are substantially reverse complementary or fully reverse complementary; by substantially reverse complement is meant that there is no more than 1 base mismatch between the two nucleotide sequences; complete reverse complement refers to the absence of mismatches between two nucleotide sequences; and/or, the nucleotide sequence III is connected at the 3 'end of the nucleotide sequence I, the nucleotide sequence IV is connected at the 5' end of the nucleotide sequence II, and the nucleotide sequence III and the nucleotide sequence IV are equal in length and are substantially reverse complementary or completely reverse complementary; by substantially reverse complement is meant that there is no more than 1 base mismatch between the two nucleotide sequences; complete reverse complement refers to the absence of mismatches between two nucleotide sequences.

In one embodiment, the sense strand further comprises nucleotide sequence V and/or the antisense strand further comprises nucleotide sequence VI, nucleotide sequences V and VI being 0 to 3 nucleotides in length, nucleotide sequence V being linked at the 3 'end of the sense strand constituting the 3' overhang of the sense strand and/or nucleotide sequence VI being linked at the 3 'end of the antisense strand constituting the 3' overhang of the antisense strand. In a preferred embodiment, the nucleotide sequence V or VI is 2 nucleotides in length. In a preferred embodiment, the nucleotide sequence V or VI is two consecutive thymidylate nucleotides or two consecutive uracil ribonucleotides. In a preferred embodiment, the nucleotide sequence V or VI is mismatched or complementary to a nucleotide at the corresponding position of the target mRNA.

In one embodiment, the double stranded region is 15-30 nucleotide pairs in length. In a preferred embodiment, the double stranded region is 17-23 nucleotide pairs in length. In a more preferred embodiment, the double stranded region is 19-21 nucleotide pairs in length.

In one embodiment, the sense strand or the antisense strand has 15-30 nucleotides. In a preferred embodiment, the sense strand or the antisense strand has 19 to 25 nucleotides. In a more preferred embodiment, the sense strand or the antisense strand has 19 to 23 nucleotides.

In one embodiment, at least one nucleotide in the sense strand or the antisense strand is a modified nucleotide and/or at least one phosphate group is a phosphate group having a modification group; preferably, the phosphate group containing a modifying group is a phosphorothioate group formed by substitution of at least one oxygen atom of a phosphodiester bond in the phosphate group with a sulfur atom.

In one embodiment, the siRNA comprises a sense strand that does not comprise a 3' overhang nucleotide.

In one embodiment, the 5 'terminal nucleotide of the sense strand is linked to a 5' phosphate group or a 5 'phosphate derivative group, and/or the 5' terminal nucleotide of the antisense strand is linked to a 5 'phosphate group or a 5' phosphate derivative group.

In one embodiment, the modified nucleotide is selected from the group consisting of a 2 '-fluoro modified nucleotide, a 2' -alkoxy modified nucleotide, a 2 '-substituted alkoxy modified nucleotide, a 2' -alkyl modified nucleotide, a 2 '-substituted alkyl modified nucleotide, a 2' -deoxyribonucleotide, a 2 '-amino modified nucleotide, a 2' -substituted amino modified nucleotide, a nucleotide analog, or a combination of any two or more thereof.

In one embodiment, the modified nucleotide is selected from the group consisting of 2'-F modified nucleotides, 2' -O-CH ₃ Modified nucleotides, 2' -O-CH ₂ -CH ₂ -O-CH ₃ Modified nucleotides, 2' -O-CH ₂ -CH＝CH ₂ Modified nucleotides, 2' -CH ₂ -CH ₂ -CH＝CH ₂ Modified nucleotides, 2' -deoxyribonucleotides, nucleotide analogs, or a combination of any two or more thereof.

In one embodiment, each nucleotide in the sense strand and the antisense strand is independently a 2' -fluoro modified nucleotide or a non-fluoro modified nucleotide. In a preferred embodiment, the 2' -fluoro modified nucleotides are located at positions 7, 9, 10 and 11 of the sense strand in the 5' to 3' direction, the remaining positions being non-fluoro modified nucleotides; the 2' -fluoro modified nucleotide is located at the even position of the antisense strand in the 5' to 3' direction, and the remaining positions are non-fluoro modified nucleotides. In a preferred embodiment, the 2' -fluoro modified nucleotides are located at positions 7, 9, 10 and 11 of the sense strand in the 5' to 3' direction, the remaining positions being non-fluoro modified nucleotides; the 2' -fluoro modified nucleotides are located at positions 2, 6, 14 and 16 of the antisense strand in the 5' to 3' direction, the remaining positions being non-fluoro modified nucleotides. In a preferred embodiment, the 2' -fluoro modified nucleotides are located at positions 7, 9, 10 and 11 of the sense strand in the 5' to 3' direction, the remaining positions being non-fluoro modified nucleotides; the 2' -fluoro modified nucleotides are located at positions 2, 6, 8, 9, 14 and 16 of the antisense strand in the 5' to 3' direction, the remaining positions being non-fluoro modified nucleotides. In a preferred embodiment, the 2' -fluoro modified nucleotides are located at positions 7, 9, 10 and 11 of the sense strand in the 5' to 3' direction, the remaining positions being non-fluoro modified nucleotides; the 2' -fluoro modified nucleotides are located at positions 2, 14 and 16 of the antisense strand in the 5' to 3' direction, with the remaining positions being non-fluoro modified nucleotides. In a preferred embodiment, the 2' -fluoro modified nucleotides are located at positions 9, 10 and 11 of the sense strand in the 5' to 3' direction, the remaining positions being non-fluoro modified nucleotides; the 2' -fluoro modified nucleotide is located at position 14 of the antisense strand in the 5' to 3' direction, the remaining positions being non-fluoro modified nucleotides. In one embodiment, each non-fluoro modified nucleotide is a 2 '-methoxy modified nucleotide, which refers to a nucleotide formed by substitution of the 2' -hydroxy group of the ribosyl group with a methoxy group.

In one embodiment, each non-fluoro modified nucleotide is independently selected from one of a nucleotide or nucleotide analog formed by substitution of the hydroxyl group at the 2' -position of the ribosyl of the nucleotide with a non-fluoro group, the nucleotide analog being selected from one of an iso-nucleotide, LNA, ENA, cET BNA, UNA and GNA.

In one embodiment, each nucleotide in the sense strand and the antisense strand is independently a 2' -fluoro modified nucleotide, a 2' -methoxy modified nucleotide, a 2' -deoxyribonucleotide, a GNA modified nucleotide, or a combination of any two or more thereof. In a preferred embodiment, the 2 '-fluoro modified nucleotides are located at positions 7, 9, 10 and 11 of the sense strand in the 5' to 3 'direction, the remaining positions being 2' -methoxy modified nucleotides; the 2 '-fluoro modified nucleotide is located at the even position of the antisense strand in the 5' to 3 'direction, with the remaining positions being 2' -methoxy modified nucleotides. In a preferred embodiment, the 2 '-fluoro modified nucleotides are located at positions 7, 9, 10 and 11 of the sense strand in the 5' to 3 'direction, the remaining positions being 2' -methoxy modified nucleotides; the 2 '-fluoro modified nucleotides are located at positions 2, 6, 14 and 16 of the antisense strand in the 5' to 3 'direction, with the remaining positions being 2' -methoxy modified nucleotides. In a preferred embodiment, the 2 '-fluoro modified nucleotides are located at positions 7, 9, 10 and 11 of the sense strand in the 5' to 3 'direction, the remaining positions being 2' -methoxy modified nucleotides; the 2 '-fluoro modified nucleotides are located at positions 2, 6, 8, 9, 14 and 16 of the antisense strand in the 5' to 3 'direction, with the remaining positions being 2' -methoxy modified nucleotides. In a preferred embodiment, the 2 '-fluoro modified nucleotides are located at positions 7, 9, 10 and 11 of the sense strand in the 5' to 3 'direction, the remaining positions being 2' -methoxy modified nucleotides; according to the 5 'to 3' direction, 2 '-fluoro modified nucleotides are located at positions 2, 14 and 16 of the antisense strand, GNA modified nucleotides are located at position 6 of the antisense strand, and the remaining positions are 2' -methoxy modified nucleotides. In a preferred embodiment, the 2 '-fluoro modified nucleotides are located at positions 7, 9, 10 and 11 of the sense strand in the 5' to 3 'direction, the remaining positions being 2' -methoxy modified nucleotides; according to the 5 'to 3' direction, 2 '-fluoro modified nucleotides are located at positions 2, 6, 14 and 16 of the antisense strand, GNA modified nucleotides are located at position 7 of the antisense strand, and the remaining positions are 2' -methoxy modified nucleotides. In a preferred embodiment, the 2 '-fluoro modified nucleotides are located at positions 9, 10 and 11 of the sense strand in the 5' to 3 'direction, the remaining positions being 2' -methoxy modified nucleotides; according to the 5 'to 3' direction, the 2 '-fluoro modified nucleotide is located at position 14 of the antisense strand, the deoxyribonucleotide is located at positions 2, 5, 7 and 12 of the antisense strand, and the rest positions are 2' -methoxy modified nucleotides.

In some embodiments, at least one of the following linkages between nucleotides in the siRNA is a phosphorothioate linkage:

a linkage between nucleotide 1 and nucleotide 2 from the 5' end of the sense strand;

a linkage between nucleotide 2 and nucleotide 3 starting at the 5' end of the sense strand;

a linkage between nucleotide 1 and nucleotide 2 from the 3' end of the sense strand;

a linkage between nucleotide 2 and nucleotide 3 starting at the 3' end of the sense strand;

a linkage between nucleotide 1 and nucleotide 2 from the 5' end of the antisense strand;

a linkage between nucleotide 2 and nucleotide 3 from the 5' end of the antisense strand;

a linkage between nucleotide 1 and nucleotide 2 from the 3' end of the antisense strand;

the linkage between nucleotide 2 and nucleotide 3, starting at the 3' end of the antisense strand.

In some embodiments, the siRNA is directed along the 5 'end toward the 3' end, and the sense strand comprises phosphorothioate groups at the positions shown below:

between nucleotide 1 and nucleotide 2 from the 5' end of the sense strand;

Between nucleotide 2 and nucleotide 3 from the 5' end of the sense strand;

between nucleotide 1 and nucleotide 2 from the 3' end of the sense strand;

between nucleotide 2 and nucleotide 3 from the 3' end of the sense strand;

or,

the sense strand comprises phosphorothioate groups at the positions shown below:

between nucleotide 1 and nucleotide 2 from the 5' end of the sense strand;

between nucleotide 2 and nucleotide 3, starting at the 5' end of the sense strand.

In some embodiments, the siRNA is directed along the 5 'end toward the 3' end, and the antisense strand comprises phosphorothioate groups at the positions shown below:

between nucleotide 1 and nucleotide 2 from the 5' end of the antisense strand;

between nucleotide 2 and nucleotide 3 from the 5' end of the antisense strand;

between nucleotide 1 and nucleotide 2 from the 3' end of the antisense strand;

the antisense strand is between nucleotide 2 and nucleotide 3 from the 3' terminus.

In one embodiment, each nucleotide in the sense strand and the antisense strand is independently a 2' -fluoro modified nucleotide, a 2' -methoxy modified nucleotide, a 2' -deoxyribonucleotide, a GNA modified nucleotide, or a combination of any two or more thereof. In a preferred embodiment, the 2' -fluoro modified nucleotides are located at positions 7, 9, 10 and 11 of the sense strand in the 5' to 3' direction, the remaining positions being between the 1 st and 2 nd nucleotides of the 5' end, between the 2 nd and 3 rd nucleotides of the 5' end, between the 1 st and 2 nd nucleotides of the 3' end, and between the 2 nd and 3 rd nucleotides of the 3' end being phosphorothioate linkages; according to the 5 'to 3' direction, the 2 '-fluoro modified nucleotide is located at the even number position of the antisense strand, the rest positions are 2' -methoxy modified nucleotides, the 1 st nucleotide and the 2 nd nucleotide of the 5 'end, the 2 nd nucleotide and the 3 rd nucleotide of the 5' end, the 1 st nucleotide and the 2 nd nucleotide of the 3 'end, and phosphorothioate group connection is formed between the 2 nd nucleotide and the 3 rd nucleotide of the 3' end. In a preferred embodiment, the 2' -fluoro modified nucleotides are located at positions 7, 9, 10 and 11 of the sense strand in the 5' to 3' direction, the remaining positions being 2' -methoxy modified nucleotides, between nucleotide 1 and nucleotide 2 of the 5' end, phosphorothioate linkage between nucleotide 2 and nucleotide 3 of the 5' end, the 3' end being removed; according to the 5 'to 3' direction, the 2 '-fluoro modified nucleotide is located at the even number position of the antisense strand, the rest positions are 2' -methoxy modified nucleotides, the 1 st nucleotide and the 2 nd nucleotide of the 5 'end, the 2 nd nucleotide and the 3 rd nucleotide of the 5' end, the 1 st nucleotide and the 2 nd nucleotide of the 3 'end, and phosphorothioate group connection is formed between the 2 nd nucleotide and the 3 rd nucleotide of the 3' end. In a preferred embodiment, the 2' -fluoro modified nucleotides are located at positions 7, 9, 10 and 11 of the sense strand in the 5' to 3' direction, the remaining positions being 2' -methoxy modified nucleotides, between nucleotide 1 and nucleotide 2 of the 5' end, phosphorothioate linkage between nucleotide 2 and nucleotide 3 of the 5' end, the 3' end being removed; the 2 '-fluoro modified nucleotides are located at positions 2, 6, 14 and 16 of the antisense strand in the 5' to 3 'direction, the remaining positions being 2' -methoxy modified nucleotides, between the 1 st and 2 nd nucleotides at the 5 'end, between the 2 nd and 3 rd nucleotides at the 5' end, between the 1 st and 2 nd nucleotides at the 3 'end, and phosphorothioate linkages between the 2 nd and 3 rd nucleotides at the 3' end. In a preferred embodiment, the 2' -fluoro modified nucleotides are located at positions 7, 9, 10 and 11 of the sense strand in the 5' to 3' direction, the remaining positions being between the 1 st and 2 nd nucleotides of the 5' end, between the 2 nd and 3 rd nucleotides of the 5' end, between the 1 st and 2 nd nucleotides of the 3' end, and between the 2 nd and 3 rd nucleotides of the 3' end being phosphorothioate linkages; the 2 '-fluoro modified nucleotides are located at positions 2, 6, 14 and 16 of the antisense strand in the 5' to 3 'direction, the remaining positions being 2' -methoxy modified nucleotides, between the 1 st and 2 nd nucleotides at the 5 'end, between the 2 nd and 3 rd nucleotides at the 5' end, between the 1 st and 2 nd nucleotides at the 3 'end, and phosphorothioate linkages between the 2 nd and 3 rd nucleotides at the 3' end. In a preferred embodiment, the 2' -fluoro modified nucleotides are located at positions 7, 9, 10 and 11 of the sense strand in the 5' to 3' direction, the remaining positions being between the 1 st and 2 nd nucleotides of the 5' end, between the 2 nd and 3 rd nucleotides of the 5' end, between the 1 st and 2 nd nucleotides of the 3' end, and between the 2 nd and 3 rd nucleotides of the 3' end being phosphorothioate linkages; the 2 '-fluoro modified nucleotides are located at positions 2, 6, 8, 9, 14 and 16 of the antisense strand in the 5' to 3 'direction, with the remaining positions being 2' -methoxy modified nucleotides, between the 1 st and 2 nd nucleotides at the 5 'end, between the 2 nd and 3 rd nucleotides at the 5' end, between the 1 st and 2 nd nucleotides at the 3 'end, and phosphorothioate linkages between the 2 nd and 3 rd nucleotides at the 3' end. In a preferred embodiment, the 2' -fluoro modified nucleotides are located at positions 7, 9, 10 and 11 of the sense strand in the 5' to 3' direction, the remaining positions being between the 1 st and 2 nd nucleotides of the 5' end, between the 2 nd and 3 rd nucleotides of the 5' end, between the 1 st and 2 nd nucleotides of the 3' end, and between the 2 nd and 3 rd nucleotides of the 3' end being phosphorothioate linkages; according to the 5 'to 3' direction, 2 '-fluoro modified nucleotides are located at positions 2, 6, 14 and 16 of the antisense strand, GNA modified nucleotides are located at position 7 of the antisense strand, the rest positions are 2' -methoxy modified nucleotides, between 1 st and 2 nd nucleotides at the 5 'end, between 2 nd and 3 rd nucleotides at the 5' end, between 1 st and 2 nd nucleotides at the 3 'end, and phosphorothioate linkage between 2 nd and 3 rd nucleotides at the 3' end. In a preferred embodiment, the 2' -fluoro modified nucleotides are located at positions 7, 9, 10 and 11 of the sense strand in the 5' to 3' direction, the remaining positions being between the 1 st and 2 nd nucleotides of the 5' end, between the 2 nd and 3 rd nucleotides of the 5' end, between the 1 st and 2 nd nucleotides of the 3' end, and between the 2 nd and 3 rd nucleotides of the 3' end being phosphorothioate linkages; according to the 5 'to 3' direction, 2 '-fluoro modified nucleotides are located at positions 2, 14 and 16 of the antisense strand, GNA modified nucleotides are located at position 6 of the antisense strand, the rest positions are 2' -methoxy modified nucleotides, between 1 st and 2 nd nucleotides at the 5 'end, between 2 nd and 3 rd nucleotides at the 5' end, between 1 st and 2 nd nucleotides at the 3 'end, and phosphorothioate linkage between 2 nd and 3 rd nucleotides at the 3' end. In a preferred embodiment, the 2 '-fluoro modified nucleotides are located at positions 9, 10 and 11 of the sense strand in the 5' to 3 'direction, the remaining positions being 2' -methoxy modified nucleotides, between nucleotide 1 and nucleotide 2 of the 5 'end, between nucleotide 2 and nucleotide 3 of the 5' end, between nucleotide 1 and nucleotide 2 of the 3 'end, between nucleotide 2 and nucleotide 3 of the 3' end being phosphorothioate linkages; according to the 5 'to 3' direction, the 2 '-fluoro modified nucleotide is positioned at the 14 th position of the antisense strand, the deoxyribonucleotide is positioned at the 2 nd, 5, 7 and 12 th positions of the antisense strand, the rest positions are 2' -methoxy modified nucleotides, the 1 st nucleotide and the 2 nd nucleotide of the 5 'terminal, the 2 nd nucleotide and the 3 rd nucleotide of the 5' terminal, the 1 st nucleotide and the 2 nd nucleotide of the 3 'terminal, and the 2 nd nucleotide and the 3 rd nucleotide of the 3' terminal are connected by phosphorothioate groups.

In a specific embodiment, the invention provides an siRNA selected from table 1; preferably, the method comprises the steps of, the siRNA is selected from the group consisting of N-ER-FY0140024, N-ER-FY0140024M1, N-ER-FY0140024MD2, N-ER-FY0140024M1, N-ER-FY0140024MD2, N-ER-FY0140024M2, N-ER-FY0140024, N-ER-FY N-ER-FY0140024, N-ER-FY0140024D2, N-ER-FY0140024M2D2, N-ER-FY0140024M3, N-ER-FY0140024M4, N-ER-FY0140024M5, N-ER-FY0140024M6, N-ER-FY0140024D2, N-ER-FY0140024M2D2, N-ER-FY0140024M 3N-ER-FY 0140024M4, N-ER-FY0140024M5, N-ER-FY0140024D2, N-ER-FY0140024M2D2, N-ER-FY0140024M3, N-ER-FY0140024M4, N-ER-FY0140024M5, N-ER-FY0140024M6, N-ER-FY0140024M2, N-ER-FY0140024M3, N-ER-FY0140024M4, N-ER-FY0140024M5, N-ER-FY0140024M2, N-ER-FY0140024M3, N-ER-FY0140024M 2.

The present invention also provides an siRNA conjugate comprising the siRNA of the present invention and a conjugate group conjugated to the siRNA (as shown in the following formula, a double helix structure represents the siRNA, and the conjugate group is attached to the 3' -end of the sense strand of the siRNA):

x in the above conjugate structure may be selected as O or S, and in one embodiment X is O.

In one embodiment, the conjugate group comprises a pharmaceutically acceptable targeting group and a linker, and the siRNA, the linker and the targeting group are sequentially covalently or non-covalently linked;

preferably, in the siRNA conjugate, the sense strand and the antisense strand of the siRNA are complementary to form a double-stranded region of the siRNA conjugate, and the 3 'end of the sense strand forms a blunt end, the 3' end of the antisense strand having 1-3 protruding nucleotides extending out of the double-stranded region;

or,

in the siRNA conjugate, the sense strand and the antisense strand of the siRNA are complementary to form a double-stranded region of the siRNA conjugate, and the 3 'end of the sense strand forms a blunt end and the 3' end of the antisense strand forms a blunt end.

In one embodiment, the conjugate group is L96 of the formula:

In a specific embodiment, the siRNA conjugate is an siRNA conjugate selected from table 2.

The invention also provides a pharmaceutical composition comprising the siRNA of the invention, or the siRNA conjugate of the invention, and a pharmaceutically acceptable carrier.

The invention also provides a kit comprising the siRNA of the invention, or the siRNA conjugate of the invention, or the pharmaceutical composition of the invention.

The invention also provides the use of the siRNA of the invention, or the siRNA conjugate of the invention, or the pharmaceutical composition of the invention for the preparation of a medicament for inhibiting AGT gene expression.

The invention also provides the use of the siRNA of the invention, or the siRNA conjugate of the invention, or the pharmaceutical composition of the invention for the manufacture of a medicament for the prevention and/or treatment of diseases associated with the overexpression of AGT genes.

In one embodiment, the disease is hypertension, borderline hypertension, primary hypertension, secondary hypertension, isolated systolic or diastolic hypertension, pregnancy-related hypertension, diabetic hypertension, refractory hypertension, paroxysmal hypertension, renal vascular hypertension, hypertension with reduced ejection fraction, gordbla hypertension, low plasma renin activity or plasma renin concentration-related hypertension, ocular hypertension, glaucoma, pulmonary arterial hypertension, portal hypertension, systemic venous hypertension, systolic hypertension, unstable hypertension; hypertensive heart disease, hypertensive kidney disease, atherosclerosis, arteriosclerosis, vascular lesions, diabetic nephropathy, preeclampsia, diabetic retinopathy, chronic heart failure, cardiomyopathy, diabetic cardiomyopathy, glomerulosclerosis, aortic stenosis, aortic aneurysm, ventricular fibrosis, heart failure, myocardial infarction, angina pectoris, stroke, renal disease, renal failure, systemic sclerosis, intrauterine growth retardation (IUGR), fetal growth restriction, obesity, hepatic steatosis/fatty liver, nonalcoholic steatohepatitis (NASH), nonalcoholic fatty liver disease (NAFLD); glucose intolerance, type 2 diabetes (non-insulin dependent diabetes mellitus) and metabolic syndrome.

The application also provides a method of inhibiting AGT gene expression comprising contacting or administering to a subject in need thereof a therapeutically effective amount of an siRNA of the application, or an siRNA conjugate of the application, or a pharmaceutical composition of the application, with cells expressing AGT.

The application also provides a method of treating and/or preventing a disease associated with overexpression of the AGT gene comprising administering to a subject in need thereof a therapeutically effective amount of an siRNA of the application, or an siRNA conjugate of the application, or a pharmaceutical composition of the application.

Advantageous effects

The siRNA, the pharmaceutical composition and the siRNA conjugate provided by the application show excellent AGT gene expression inhibition activity in vitro cell experiments, and have good potential for treating diseases related to AGT gene overexpression. For example, the siRNA and the conjugate thereof disclosed by the application can reduce the expression of AGT mRNA in liver, have low toxic and side effects and good plasma stability, and have good clinical application prospect.

The siRNA provided by the application shows good inhibition effect on AGT genes in human liver cancer cells Huh 7. In some embodiments, the siRNAs provided herein have an inhibition rate of up to 92.63% at a concentration of 0.1nM and an inhibition rate of up to 75.28% at a concentration of 0.01 nM.

In some embodiments, the siRNA provided by the application has higher AGT gene inhibition activity in Huh7 cells, e.g., IC ₅₀ As low as 15.17pM.

In some embodiments, the siRNA conjugates provided herein have high AGT gene inhibition activity in PHH cells, e.g., IC in the case where the siRNA conjugates enter PHH by free uptake ₅₀ As low as 0.559nM; in case the siRNA conjugate enters PHH by transfection, IC ₅₀ As low as 0.004nM.

Detailed Description

Definition of the definition

Throughout the specification, "G" and "G" are used in the art unless otherwise specified "C "," a "," T "and" U "generally represent bases of guanine, cytosine, adenine, thymine, uracil, respectively, but it is also generally known in the art that" G "," C "," a "," T "and" U "each also generally represent nucleotides containing guanine, cytosine, adenine, thymine and uracil, respectively, as bases, which is a common manner in the expression of deoxyribonucleic acid sequences and/or ribonucleic acid sequences, and thus the meaning of" G "," C "," a "," T "," U "in the context of the present disclosure includes the various possible scenarios described above. Lowercase letters a, u, c, g: a nucleotide representing 2' -methoxy modification; af. Gf, cf, uf: a 2' -fluoro modified nucleotide; dA. dG, dC, dU: represents deoxyribonucleotides; the lower case letter s indicates that phosphorothioate linkages are between two nucleotides adjacent to the letter s; p1: indicating that the adjacent nucleotide to the right of P1 is a nucleotide 5' -phosphate; A、U、C、G(underlined + bold + italic): indicating GNA modified nucleotides.

In the above and in the following, the "2 '-fluoro modified nucleotide" refers to a nucleotide in which the hydroxyl group at the 2' -position of the ribosyl group of the nucleotide is substituted with fluorine. "non-fluoro modified nucleotide" refers to a nucleotide or nucleotide analogue in which the hydroxyl group at the 2' -position of the ribosyl of the nucleotide is replaced with a non-fluoro group. In some embodiments, each non-fluoro modified nucleotide is independently selected from one of the nucleotides or nucleotide analogs formed by substitution of the hydroxyl group at the 2' position of the ribosyl of the nucleotide with a non-fluoro group. Nucleotides in which the hydroxyl group at the 2 '-position of the ribosyl group is substituted with a non-fluorine group are well known to those skilled in the art, and may be selected from one of 2' -alkoxy-modified nucleotides, 2 '-substituted alkoxy-modified nucleotides, 2' -alkyl-modified nucleotides, 2 '-substituted alkyl-modified nucleotides, 2' -amino-modified nucleotides, 2 '-substituted amino-modified nucleotides, and 2' -deoxyribonucleotides.

"alkyl" includes straight, branched or cyclic saturated alkyl groups. For example, alkyl groups include, but are not limited to, methyl, ethyl, propyl, and cyclo Propyl, n-butyl, isobutyl, sec-butyl, tert-butyl, cyclobutyl, n-pentyl, cyclohexyl and the like. Exemplary, "C _1-6 "C in" alkyl _1-6 "refers to a group comprising an array of straight, branched, or cyclic forms of 1, 2, 3, 4, 5, or 6 carbon atoms.

"alkoxy" herein refers to an alkyl group attached to the remainder of the molecule through an oxygen atom (-O-alkyl), wherein the alkyl is as defined herein. Non-limiting examples of alkoxy groups include methoxy, ethoxy, trifluoromethoxy, difluoromethoxy, n-propoxy, isopropoxy, n-butoxy, tert-butoxy, n-pentoxy, and the like.

"nucleotide analog" refers to a group that is capable of replacing a nucleotide in a nucleic acid, but that differs in structure from adenine ribonucleotide, guanine ribonucleotide, cytosine ribonucleotide, uracil ribonucleotide, or thymine deoxyribonucleotide. Such as an iso-nucleotide, a bridged nucleotide (bridged nucleic acid, abbreviated as BNA) or an acyclic nucleotide.

BNA refers to a constrained or inaccessible nucleotide. BNA may contain a five-, six-, or seven-membered ring bridging structure with "fixed" C3' -endo-saccharides tucked. The bridge is typically incorporated at the 2'-, 4' -position of the ribose to provide a 2',4' -BNA nucleotide, such as LNA, ENA, cret BNA, etc., where LNA is shown in formula (1), ENA is shown in formula (2), cret BNA is shown in formula (3):

/>

Acyclic nucleotides are a class of nucleotides in which the sugar ring of the nucleotide is opened, such as an Unlocking Nucleic Acid (UNA) or a Glycerolipid Nucleic Acid (GNA), wherein UNA is represented by formula (4), and GNA is represented by formula (5):

in the above formula (4) and formula (5), R is selected from H, OH or alkoxy (O-alkyl).

An isopucleotide refers to a compound in which the position of a base on the ribose ring is changed in a nucleotide, for example, a compound in which the base is shifted from the 1' -position to the 2' -position or the 3' -position of the ribose ring, as shown in formula (6) or (7):

in the compounds of the above formulae (6) to (7), base represents a Base, for example A, U, G, C or T; r is selected from H, OH, F or a non-fluorine group as described above.

In some embodiments, the nucleotide analog is selected from one of an iso-nucleotide, LNA, ENA, cET BNA, UNA, and GNA. In some embodiments, each non-fluoro modified nucleotide is a 2 '-methoxy modified nucleotide, a 2' -deoxyribonucleotide, a GNA modified nucleotide, or a combination of any two or more thereof. In some preferred embodiments, each non-fluoro modified nucleotide is a 2 '-methoxy modified nucleotide, which in the foregoing and hereinafter refers to a nucleotide formed by substitution of the 2' -hydroxy group of the ribosyl group with a methoxy group.

The "2 '-methoxy-modified nucleotide" refers to a nucleotide in which the 2' -hydroxyl group of the ribosyl group is replaced with a methoxy group. The "phosphorothioate group" refers to a phosphorothioate group in which one oxygen atom of a phosphodiester bond in the phosphate group is replaced with a sulfur atom. The "5' -phosphonucleotide" refers to a structure of the formula:

in the context of the present specification, the expressions "complementary" and "reverse complementary" are used interchangeably and have the meaning well known to the person skilled in the art, i.e. in a double stranded nucleic acid molecule the bases of one strand are each paired with a base on the other strand in a complementary manner. In DNA, the purine base adenine (a) is always paired with the pyrimidine base thymine (T) (or uracil (U) in RNA); the purine base guanine (C) is always paired with the pyrimidine base cytosine (G). Each base pair includes a purine and a pyrimidine. When adenine on one strand always pairs with thymine (or uracil) on the other strand, and guanine always pairs with cytosine, the two strands are considered complementary to each other, and the sequence of the strand can be deduced from the sequence of its complementary strand. Accordingly, "mismatch" means in the art that bases at corresponding positions do not exist in complementary pairs in a double-stranded nucleic acid.

In the above and in the following, unless otherwise specified, "substantially reverse complementary" means that there are no more than 3 base mismatches between the two nucleotide sequences involved; "substantially reverse complementary" means that there is no more than 1 base mismatch between two nucleotide sequences; "complete reverse complement" means that there is no base mismatch between the two nucleotide sequences.

In the above and below, the "nucleotide difference" between one nucleotide sequence and another nucleotide sequence means that the base type of the nucleotide at the same position is changed as compared with the former, for example, when one nucleotide base is A in the latter, when the corresponding nucleotide base at the same position in the former is U, C, G or T, it is determined that there is a nucleotide difference between the two nucleotide sequences at the position. In some embodiments, a nucleotide difference is also considered to occur at an original position when the nucleotide is replaced with an abasic nucleotide or its equivalent.

In this context, "overhang" refers to one or more unpaired nucleotides that protrude from the duplex structure of an siRNA when one 3 'end of one strand extends beyond the 5' end of the other strand, or vice versa. By "blunt end" or "blunt end" is meant that there are no unpaired nucleotides at that end of the siRNA, i.e., no nucleotide overhangs. A "blunt-ended" siRNA is one that is double-stranded throughout its length, i.e., has no nucleotide overhangs at either end of the molecule.

In the above and below description of the present application, particularly in describing the preparation method of the siRNA, pharmaceutical composition or siRNA conjugate of the present application, the nucleoside monomer refers to a modified or unmodified nucleoside phosphoramidite monomer used in solid phase phosphoramidite synthesis according to the kind and order of nucleotides in the siRNA or siRNA conjugate to be prepared, unless otherwise specified. Solid phase phosphoramidite synthesis is a method used in RNA synthesis well known to those skilled in the art. Nucleoside monomers useful in the present application are all commercially available.

In the context of the present application, unless otherwise indicated, "conjugated" means that two or more chemical moieties each having a specific function are linked to each other by covalent linkage; accordingly, "conjugate" refers to a compound formed by covalent linkage between the chemical moieties. Further, "siRNA conjugate" means a compound formed by covalently attaching one or more chemical moieties having specific functions to an siRNA. siRNA conjugates are understood to be, depending on the context, the collective term of multiple siRNA conjugates or siRNA conjugates of a certain chemical formula. In the context of the present specification, a "conjugate molecule" is understood to be a specific compound that can be conjugated to an siRNA by reaction, ultimately forming the siRNA conjugate of the application.

Various hydroxyl protecting groups may be used in the present application. In general, the protecting group renders the chemical functional group insensitive to specific reaction conditions and may be appended to and removed from the functional group in the molecule without substantially damaging the remainder of the molecule. In some embodiments, the protecting group is stable under alkaline conditions, but can be removed under acidic conditions. In some embodiments, non-exclusive examples of hydroxyl protecting groups that may be used in the present application include Dimethoxytrityl (DMT), monomethoxytrityl, 9-phenylxanthin-9-yl (Pixyl), and 9- (p-methoxyphenyl) xanthin-9-yl (Mox). In some embodiments, non-exclusive examples of hydroxyl protecting groups that may be used in the present application include Tr (trityl), MMTr (4-methoxytrityl), DMTr (4, 4 '-dimethoxytrityl), and TMTr (4, 4',4 "-trimethoxytrityl).

As used herein, "optional" or "optionally" means that the subsequently described event or circumstance may or may not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not.

The term "subject" as used herein refers to any animal, such as a mammal or a pouched animal. Subjects of the application include, but are not limited to, humans, non-human primates (e.g., rhesus monkeys or other types of macaques), mice, pigs, horses, donkeys, cattle, sheep, rats, rabbits, or any kind of poultry.

As used herein, "treatment" refers to a method of achieving a beneficial or desired result, including but not limited to therapeutic benefit. By "therapeutic benefit" is meant eradication or amelioration of the underlying disorder being treated. In addition, therapeutic benefit is obtained by eradicating or ameliorating one or more of the physiological symptoms associated with the underlying disorder such that an improvement is observed in the subject, although the subject may still be afflicted with the underlying disorder.

As used herein, "preventing" refers to a method of achieving a beneficial or desired result, including but not limited to a prophylactic benefit. To obtain a "prophylactic benefit," the siRNA, siRNA conjugate, or pharmaceutical composition can be administered to a subject at risk of suffering from a particular disease, or to a subject reporting one or more physiological symptoms of the disease, even though a diagnosis of the disease may not have been made.

siRNA

The present application relates to an siRNA capable of inhibiting AGT gene expression. The siRNA of the present application contains a nucleotide group containing a phosphate group, a ribose group, and a base as basic structural units, as is well known to those skilled in the art. Typically, active, i.e., functional, siRNAs are about 12 to 40 nucleotides in length, and in some embodiments about 15 to 30 nucleotides in length.

The siRNA of the application comprises a sense strand and an antisense strand, each nucleotide in the siRNA being independently a modified or unmodified nucleotide, wherein the sense strand comprises a stretch of nucleotide sequence I and the antisense strand comprises a stretch of nucleotide sequence II, the nucleotide sequence I and the nucleotide sequence II being at least partially reverse complementary to form a double-stranded region. In some embodiments, the double stranded region is 15-30 nucleotide pairs in length. In other embodiments, the double stranded region is 17-23 nucleotide pairs in length. In other embodiments, the double stranded region is 19-21 nucleotide pairs in length. In yet other embodiments, the double stranded region is 19 or 21 nucleotide pairs in length.

In some embodiments, the sense strand further comprises a nucleotide sequence III, the antisense strand further comprises a nucleotide sequence IV, the nucleotide sequence III and the nucleotide sequence IV are each independently 0-10 nucleotides in length, the nucleotide sequence III is attached at the 5 'end of nucleotide sequence I, the nucleotide sequence IV is attached at the 3' end of nucleotide sequence II, the nucleotide sequence III and the nucleotide sequence IV are equal in length and are substantially reverse complementary or fully reverse complementary; by substantially reverse complement is meant that there is no more than 1 base mismatch between the two nucleotide sequences; complete reverse complement refers to the absence of mismatches between two nucleotide sequences. In some embodiments, the sense strand further comprises a nucleotide sequence III, the antisense strand further comprises a nucleotide sequence IV, the nucleotide sequence III and the nucleotide sequence IV are each independently 0-10 nucleotides in length, the nucleotide sequence III is attached at the 3 'end of nucleotide sequence I, the nucleotide sequence IV is attached at the 5' end of nucleotide sequence II, the nucleotide sequence III and the nucleotide sequence IV are equal in length and are substantially reverse complementary or fully reverse complementary; by substantially reverse complement is meant that there is no more than 1 base mismatch between the two nucleotide sequences; complete reverse complement refers to the absence of mismatches between two nucleotide sequences. In some embodiments, the sense strand further comprises a nucleotide sequence III, the antisense strand further comprises a nucleotide sequence IV, the nucleotide sequence III and the nucleotide sequence IV are each independently 0-10 nucleotides in length, the nucleotide sequence III is attached at the 5 'end of nucleotide sequence I, the nucleotide sequence IV is attached at the 3' end of nucleotide sequence II, the nucleotide sequence III and the nucleotide sequence IV are equal in length and are substantially reverse complementary or fully reverse complementary; and the nucleotide sequence III is linked at the 3 'end of the nucleotide sequence I, the nucleotide sequence IV is linked at the 5' end of the nucleotide sequence II, the nucleotide sequence III and the nucleotide sequence IV are equal in length and are substantially reverse complementary or completely reverse complementary; by substantially reverse complement is meant that there is no more than 1 base mismatch between the two nucleotide sequences; complete reverse complement refers to the absence of mismatches between two nucleotide sequences.

In some embodiments, the sense strand further comprises nucleotide sequence V and/or the antisense strand further comprises nucleotide sequence VI, nucleotide sequences V and VI being 0 to 3 nucleotides in length, nucleotide sequence V being linked at the 3 'end of the sense strand to form a 3' overhang of the sense strand, and/or nucleotide sequence VI being linked at the 3 'end of the antisense strand to form a 3' overhang of the antisense strand. In some embodiments, the nucleotide sequence V or VI is 2 nucleotides in length. In other embodiments, the nucleotide sequence V or VI is two consecutive thymidines or two consecutive uracils. In other embodiments, the nucleotide sequence V or VI is mismatched or complementary to a nucleotide at a corresponding position in the target mRNA.

The sense and antisense strands provided herein are the same or different in length, and in some embodiments, the sense or antisense strand has 15-30 nucleotides. In other embodiments, the sense strand or the antisense strand has 19 to 25 nucleotides. In other embodiments, the sense strand or the antisense strand has 19 to 23 nucleotides. The length ratio of the sense strand and the antisense strand of the siRNA provided by the application can be 15/15, 16/16, 17/17, 18/18, 19/19, 19/20, 19/21, 19/22, 19/23, 20/19, 20/20, 20/21, 20/22, 20/23, 21/19, 21/20, 21/21, 21/22, 21/23, 22/19, 22/20, 22/21, 22/22, 22/23, 23/19, 23/20, 23/21, 23/22, 23/23, 24/24, 25/25, 26/26, 27/27, 28/28, 29/29, 30/30, 22/24, 22/25, 22/26, 23/24, 23/25, or 23/26, etc. In some embodiments, the siRNA has a length ratio of the sense strand to the antisense strand of 19/19, 21/21, 19/21, 21/23 or 23/23, at which time the siRNA of the present disclosure has better cellular mRNA silencing activity.

It was found that different modification strategies can have distinct effects on the stability, bioactivity, cytotoxicity, etc. of siRNA. For example, various strategies for chemical modification of siRNA were studied in CN102140458B, demonstrating 7 effective modifications, one of which resulted in siRNA that improved blood stability while maintaining substantially equivalent inhibition activity as compared to unmodified siRNA.

The nucleotides in the siRNA of the application are each independently modified or unmodified nucleotides. In some embodiments, each nucleotide in the siRNA of the application is an unmodified nucleotide; in some embodiments, some or all of the nucleotides in the siRNA of the application are modified nucleotides, and such modifications on the nucleotide groups do not result in a significant impairment or loss of the function of the siRNA of the application to inhibit AGT gene expression.

In some embodiments, the siRNA of the application contains at least 1 modified nucleotide. In the context of the present application, the term "modified nucleotide" refers to a nucleotide or nucleotide analogue formed by substitution of the hydroxyl group at the 2' -position of the ribosyl of the nucleotide with other groups, or a nucleotide having a modified base. The modified nucleotide does not result in a significant impairment or loss of function of the siRNA to inhibit gene expression. For example, modified nucleotides disclosed in J.K.Watts, G.F.Deleavey, and M.J.damha, chemically modified siRNA: tools and applications. Drug discovery Today,2008,13 (19-20): 842-55 may be selected.

In some embodiments, at least one nucleotide in the sense strand or the antisense strand of the siRNA provided herein is a modified nucleotide, and/or at least one phosphate group is a phosphate group having a modifying group; in other words, at least a portion of the phosphate groups and/or ribose groups in at least one single-stranded phosphate-sugar backbone in the sense strand and the antisense strand are phosphate groups and/or ribose groups having a modifying group. In some embodiments, the phosphate group containing a modifying group is a phosphorothioate group formed by substitution of at least one oxygen atom of the phosphodiester bond in the phosphate group with a sulfur atom.

In some embodiments, the siRNA comprises a sense strand that does not comprise a 3' overhang nucleotide; that is, the sense strand of the siRNA may have 3' overhang nucleotides that are removed from the sense strand to form blunt ends.

In some embodiments, when there are no protruding nucleotides at the 3 'end of the sense strand after the nucleotide sequence of the sense strand and the nucleotide sequence of the antisense strand are complementary to form a double-stranded region, a nucleotide sequence V is added at the 3' end of the sense strand as the protruding nucleotide. Then, when the nucleotide sequence V is linked to the 3' -end of the sense strand, the nucleotide sequence V is excluded after the chemical modification is completed, and accordingly, the sense strand of the siRNA forms a blunt end.

In some embodiments, when the 3 'end of the sense strand has a protruding nucleotide extending out of the double-stranded region after the nucleotide sequence of the antisense strand is complementary to the nucleotide sequence of the sense strand, the protruding nucleotide at the 3' end of the sense strand is excluded as the nucleotide sequence of the sense strand, and accordingly, the sense strand of the siRNA forms a blunt end.

In some embodiments, the 5' terminal nucleotide of the sense strand is linked to a 5' phosphate group or a 5' phosphate derivative group. In some embodiments, the 5' terminal nucleotide of the antisense strand is linked to a 5' phosphate group or a 5' phosphate derivative group.

Exemplary 5' phosphate groups have the structure:the structure of the 5' phosphate derivative group includes, but is not limited to: />Etc.

The nucleotide at the 5' end of the sense or antisense strand is linked to a 5' phosphate group or 5' phosphate derivative group to form the structure shown below:

wherein Base represents a Base, such as A, U, G, C or T. R 'is hydroxyl or substituted with various groups known to those skilled in the art, for example, 2' -fluoro (2 '-F) modified nucleotide, 2' -alkoxy modified nucleotide, 2 '-substituted alkoxy modified nucleotide, 2' -alkyl modified nucleotide, 2 '-substituted alkyl modified nucleotide, 2' -amino modified nucleotide, 2 '-substituted amino modified nucleotide, 2' -deoxyribonucleotide.

Exemplary modified nucleotides have the structure shown below:

wherein Base represents a Base, such as A, U, G, C or T. The hydroxyl group at the 2' -position of the ribose group is substituted by R. The hydroxyl group at the 2 '-position of these ribose groups may be substituted with various groups known to those skilled in the art, such as, for example, 2' -fluoro (2 '-F) modified nucleotides, 2' -alkoxy modified nucleotides, 2 '-substituted alkoxy modified nucleotides, 2' -alkyl modified nucleotides, 2 '-substituted alkyl modified nucleotides, 2' -amino modified nucleotides, 2 '-substituted amino modified nucleotides, 2' -deoxyribonucleotides.

In some embodiments, the 2 '-alkoxy-modified nucleotide is 2' -methoxy (2 '-OMe,2' -O-CH) ₃ ) Modified nucleotides, and the like.

In some embodiments, the 2' -substituted alkoxy-modified nucleotide is 2' -methoxyethoxy (2 ' -O-CH) ₂ -CH ₂ -O-CH ₃ ) Modified nucleotides, 2' -O-CH ₂ -CH＝CH ₂ Modified nucleotides, and the like.

In some embodiments, the 2 '-substituted alkyl modified nucleotide is 2' -CH ₂ -CH ₂ -CH＝CH ₂ Modified nucleotides, and the like.

In some embodiments, all of the nucleotides in the sense strand and/or the antisense strand are modified nucleotides. In some embodiments, each nucleotide in the sense strand and the antisense strand of the siRNA provided herein is independently a 2' -fluoro modified nucleotide or a non-fluoro modified nucleotide. In some embodiments, each non-fluoro modified nucleotide is a 2 '-methoxy modified nucleotide, a 2' -deoxyribonucleotide, a GNA modified nucleotide, or a combination of any two or more thereof; the 2 '-methoxy modified nucleotide refers to a nucleotide formed by substituting a 2' -hydroxyl group of a ribosyl by methoxy. In some embodiments, the 2' -fluoro modified nucleotides are located at positions 7, 9, 10 and 11 of the sense strand in the 5' to 3' direction, with the remaining positions being non-fluoro modified nucleotides; the 2' -fluoro modified nucleotide is located at the even position of the antisense strand in the 5' to 3' direction, and the remaining positions are non-fluoro modified nucleotides. In some embodiments, the 2' -fluoro modified nucleotides are located at positions 7, 9, 10 and 11 of the sense strand in the 5' to 3' direction, with the remaining positions being non-fluoro modified nucleotides; the 2' -fluoro modified nucleotides are located at positions 2, 6, 14 and 16 of the antisense strand in the 5' to 3' direction, the remaining positions being non-fluoro modified nucleotides. In some embodiments, the 2' -fluoro modified nucleotides are located at positions 7, 9, 10 and 11 of the sense strand in the 5' to 3' direction, with the remaining positions being non-fluoro modified nucleotides; the 2' -fluoro modified nucleotides are located at positions 2, 6, 8, 9, 14 and 16 of the antisense strand in the 5' to 3' direction, the remaining positions being non-fluoro modified nucleotides. In some embodiments, the 2' -fluoro modified nucleotides are located at positions 7, 9, 10 and 11 of the sense strand in the 5' to 3' direction, with the remaining positions being non-fluoro modified nucleotides; the 2' -fluoro modified nucleotides are located at positions 2, 14 and 16 of the antisense strand in the 5' to 3' direction, with the remaining positions being non-fluoro modified nucleotides. In some embodiments, the 2' -fluoro modified nucleotides are located at positions 9, 10 and 11 of the sense strand in the 5' to 3' direction, with the remaining positions being non-fluoro modified nucleotides; the 2' -fluoro modified nucleotide is located at position 14 of the antisense strand in the 5' to 3' direction, the remaining positions being non-fluoro modified nucleotides. In some preferred embodiments, the 2 '-fluoro modified nucleotides are located at positions 7, 9, 10 and 11 of the sense strand in the 5' to 3 'direction, the remaining positions being 2' -methoxy modified nucleotides; the 2 '-fluoro modified nucleotide is located at the even position of the antisense strand in the 5' to 3 'direction, with the remaining positions being 2' -methoxy modified nucleotides. In some preferred embodiments, the 2 '-fluoro modified nucleotides are located at positions 7, 9, 10 and 11 of the sense strand in the 5' to 3 'direction, the remaining positions being 2' -methoxy modified nucleotides; the 2 '-fluoro modified nucleotides are located at positions 2, 6, 14 and 16 of the antisense strand in the 5' to 3 'direction, with the remaining positions being 2' -methoxy modified nucleotides. In some preferred embodiments, the 2 '-fluoro modified nucleotides are located at positions 7, 9, 10 and 11 of the sense strand in the 5' to 3 'direction, the remaining positions being 2' -methoxy modified nucleotides; the 2 '-fluoro modified nucleotides are located at positions 2, 6, 8, 9, 14 and 16 of the antisense strand in the 5' to 3 'direction, with the remaining positions being 2' -methoxy modified nucleotides. In some preferred embodiments, the 2 '-fluoro modified nucleotides are located at positions 7, 9, 10 and 11 of the sense strand in the 5' to 3 'direction, the remaining positions being 2' -methoxy modified nucleotides; according to the 5 'to 3' direction, 2 '-fluoro modified nucleotides are located at positions 2, 14 and 16 of the antisense strand, GNA modified nucleotides are located at position 6 of the antisense strand, and the remaining positions are 2' -methoxy modified nucleotides. In some preferred embodiments, the 2 '-fluoro modified nucleotides are located at positions 7, 9, 10 and 11 of the sense strand in the 5' to 3 'direction, the remaining positions being 2' -methoxy modified nucleotides; according to the 5 'to 3' direction, 2 '-fluoro modified nucleotides are located at positions 2, 6, 14 and 16 of the antisense strand, GNA modified nucleotides are located at position 7 of the antisense strand, and the remaining positions are 2' -methoxy modified nucleotides. In some preferred embodiments, the 2 '-fluoro modified nucleotides are located at positions 9, 10 and 11 of the sense strand in the 5' to 3 'direction, with the remaining positions being 2' -methoxy modified nucleotides; according to the 5 'to 3' direction, the 2 '-fluoro modified nucleotide is located at position 14 of the antisense strand, the deoxyribonucleotide is located at positions 2, 5, 7 and 12 of the antisense strand, and the rest positions are 2' -methoxy modified nucleotides. In some more preferred embodiments, each non-fluoro modified nucleotide is a 2 '-methoxy modified nucleotide, which refers to a nucleotide formed by substitution of the 2' -hydroxy group of the ribosyl group with a methoxy group.

between nucleotide 1 and nucleotide 2 from the 5' end of the sense strand;

Between nucleotide 2 and nucleotide 3 from the 5' end of the sense strand;

between nucleotide 1 and nucleotide 2 from the 3' end of the sense strand;

between nucleotide 2 and nucleotide 3 from the 3' end of the sense strand;

or,

between nucleotide 1 and nucleotide 2 from the 5' end of the sense strand;

between nucleotide 1 and nucleotide 2 from the 5' end of the antisense strand;

between nucleotide 2 and nucleotide 3 from the 5' end of the antisense strand;

between nucleotide 1 and nucleotide 2 from the 3' end of the antisense strand;

siRNA conjugates

The present application relates to an siRNA conjugate comprising the above-described siRNA and a conjugate group conjugated to the siRNA.

In the present application, the sense strand and the antisense strand of the siRNA conjugate form a double-stranded region of the siRNA conjugate, and a blunt end is formed at the 3' -end of the sense strand of the siRNA conjugate. In some embodiments, the 3 'end of the sense strand of the siRNA conjugate forms a blunt end, and the 3' end of the antisense strand of the siRNA conjugate has 1-3 protruding nucleotides extending out of the double-stranded region. In other embodiments, the 3 'end of the sense strand of the siRNA conjugate forms a blunt end and the 3' end of the antisense strand of the siRNA conjugate forms a blunt end.

In some preferred embodiments, the siRNA conjugate is obtained by conjugation of an siRNA to a conjugate group. Wherein the sense strand and the antisense strand of the siRNA are complementary to form a double-stranded region of the siRNA, and the 3 'end of the sense strand of the siRNA forms a blunt end, and the conjugate group is conjugated to the 3' end of the sense strand having the blunt end to form an siRNA conjugate.

In some preferred embodiments, the 3 'end of the sense strand of the siRNA has a protruding nucleotide extending out of the double-stranded region, and the sequence with a 3' blunt end formed after excluding the protruding nucleotide located at the 3 'end in the sense strand is used as the nucleotide sequence for linking the conjugate group, and the conjugate group is linked at the 3' blunt end of the sense strand to form the siRNA conjugate.

In some more preferred embodiments, when there are no protruding nucleotides at the 3 'end of the sense strand after the sense strand is complementary to the nucleotide sequence of the antisense strand to form a double-stranded region, nucleotide sequence V is added at the 3' end of the sense strand as the protruding nucleotide. The 3' -blunt-ended sequence formed after excluding the protruding nucleotide located at the 3' -end in the sense strand is used as a nucleotide sequence for linking the conjugate group, and the conjugate group is linked at the 3' -blunt end of the sense strand to form the siRNA conjugate.

In some more preferred embodiments, when the 3 'end of the sense strand has a protruding nucleotide extending out of the double-stranded region after the nucleotide sequence of the antisense strand is complementary to the nucleotide sequence of the sense strand, the sequence with a 3' blunt end formed after the removal of the protruding nucleotide located at the 3 'end in the sense strand is used as the nucleotide sequence for linking the conjugate group, and the conjugate group is linked at the 3' blunt end of the sense strand to form the siRNA conjugate.

Illustratively, an siRNA of sequence as shown in N-ER-FY0140028M1, having a protruding nucleotide at the 3 'end of the sense strand extending beyond the double-stranded region, excludes the truncated sequence of gscosuagucfcfufufgaaaacuga formed after the protruding-scsa nucleotide at the 3' end in the sense strand as the nucleotide sequence for attachment of the L96 conjugate group, thus forming an siRNA conjugate of the sequence: sense strand

gscosuagucfgcfufufgcaaacuugal 96, antisense strand is

P1usCfsaAfgUfuUfuGfcAfgCfgAfcUfaGfcsAfsc。

In general, the conjugate group comprises at least one pharmaceutically acceptable targeting group, or further comprises a linker (linker), and the siRNA, the linker and the targeting group are sequentially linked. In some embodiments, the targeting group is 1-6. In some embodiments, the targeting group is 2-4. The siRNA molecule may be non-covalently or covalently conjugated to the conjugate group, e.g., may be covalently conjugated to the conjugate group. The conjugation site of the siRNA to the conjugation group may be at the 3' end or the 5' end of the sense strand of the siRNA, or at the 5' end of the antisense strand, or in the internal sequence of the siRNA. In some embodiments, the conjugation site of the siRNA to the conjugation group is at the 3' end of the sense strand of the siRNA.

In some embodiments, the conjugate group may be attached to the phosphate group, the 2' -hydroxyl group, or the base of the nucleotide. In some embodiments, the conjugate group may also be attached to the 3' -hydroxyl group, in which case the nucleotides are linked using a 2' -5' phosphodiester linkage. When a conjugate group is attached to the end of the siRNA strand, the conjugate group is typically attached to the phosphate group of the nucleotide; when a conjugate group is attached to the internal sequence of the siRNA, the conjugate group is typically attached to a ribose sugar ring or base. Various connection means can be referred to as: muthiah Manoharan et al, siRNA conjugates carrying sequentially assembled trivalent N-acetylgalactosamine linked through nucleosides elicit robust gene silencing in vivo in hepatocytocytocytosis, ACS Chemical biology,2015,10 (5): 1181-7.

In some embodiments, the siRNA and the conjugate group may be linked by acid labile, or reducible, chemical bonds that degrade in the acidic environment of the intracellular inclusion bodies, thereby allowing the siRNA to be in a free state. For non-degradable conjugation, the conjugation group can be attached to the sense strand of the siRNA, thereby minimizing the effect of conjugation on siRNA activity.

In some embodiments, the pharmaceutically acceptable targeting group can be a ligand conventionally used in the art of siRNA administration, such as the various ligands described in WO2009082607A2, which is incorporated herein by reference in its entirety.

In some embodiments, the pharmaceutically acceptable targeting group may be selected from one or more of the following ligands formed by the targeting molecule or derivative thereof: lipophilic molecules, such as cholesterol, bile acids, vitamins (e.g. vitamin E), lipid molecules of different chain lengths; polymers, such as polyethylene glycol; polypeptides, such as permeabilizing peptides; an aptamer; an antibody; a quantum dot; sugars, such as lactose, mannose, galactose, N-acetylgalactosamine (GalNAc); folic acid (folate); receptor ligands expressed by hepatic parenchymal cells, such as asialoglycoproteins, asialoglycoresidues, lipoproteins (e.g., high density lipoproteins, low density lipoproteins, etc.), glucagon, neurotransmitters (e.g., epinephrine), growth factors, transferrin, etc.

In some embodiments, each ligand is independently selected from a ligand capable of binding to a cell surface receptor. In some embodiments, at least one ligand is a ligand capable of binding to a hepatocyte surface receptor. In some embodiments, at least one ligand is a ligand capable of binding to a mammalian cell surface receptor. In some embodiments, at least one ligand is a ligand capable of binding to a human hepatocyte surface receptor. In some embodiments, at least one ligand is a ligand capable of binding to liver surface asialoglycoprotein receptor (ASGPR). The class of these ligands is well known to those skilled in the art and generally functions to bind to specific receptors on the surface of target cells, mediating delivery of siRNA linked to the ligand to the target cells.

In some embodiments, the pharmaceutically acceptable targeting group may be any ligand that binds to an asialoglycoprotein receptor (ASGPR) on the surface of mammalian hepatocytes. In some embodiments, each ligand is independently an asialoglycoprotein, such as an asialooomolecular mucin (ASOR) or an Asialofetuin (ASF). In some embodiments, the ligand is a sugar or a derivative of a sugar.

In some embodiments, at least one ligand is a sugar. In some embodiments, each ligand is a sugar. In some embodiments, at least one ligand is a monosaccharide, a polysaccharide, a modified monosaccharide, a modified polysaccharide, or a sugar derivative. In some embodiments, at least one of the ligands may be a monosaccharide, disaccharide, or trisaccharide. In some embodiments, at least one ligand is a modified sugar. In some embodiments, each ligand is a modified sugar. In some embodiments, each ligand is independently selected from a polysaccharide, a modified polysaccharide, a monosaccharide, a modified monosaccharide, a polysaccharide derivative, or a monosaccharide derivative. In some embodiments, each or at least one ligand is selected from the group consisting of: glucose and its derivatives, mannans and its derivatives, galactose and its derivatives, xylose and its derivatives, ribose and its derivatives, fucose and its derivatives, lactose and its derivatives, maltose and its derivatives, arabinose and its derivatives, fructose and its derivatives, and sialic acid.

In some embodiments, each of the ligands may be independently selected from the group consisting of D-mannopyranose, L-mannopyranose, D-arabinose, D-xylose furanose, L-xylose furanose, D-glucose, L-glucose, D-galactose, L-galactose, alpha-D-mannopyranose, beta-D-glucopyranose, alpha-D-glucopyranose, beta-D-glucopyranose, alpha-D-fructofuranose, alpha-D-fructopyranose, alpha-D-galactopyranose, beta-D-galactopyranose, alpha-D-galactofuranose, beta-D-galactosamine, sialic acid, galactosamine, N-acetylgalactosamine, N-trifluoroacetylgalactosamine, N-propionylgalactosamine, N-N-galactosamine, N-isobutyramide, 2-amino-O-3-carboxyethyl-2-deoxy2-D-deoxygalactopyranose, 2-deoxy2-D-deoxygalactopyranose, 4-D-deoxy2-deoxygalactopyranose 2-deoxy-2-sulphonamino-D-glucopyranose, N-glycolyl- α -neuraminic acid, 5-thio- β -D-glucopyranose, 2,3, 4-tri-O-acetyl-1-thio-6-O-trityl- α -D-glucopyranoside methyl ester, 4-thio- β -D-galactopyranose, 3,4,6, 7-tetra-O-acetyl-2-deoxy-1, 5-dithio- α -D-glucoheptopyranoside ethyl ester, 2, 5-anhydro-D-allose nitrile, ribose, D-4-thioribose, L-ribose or L-4-thioribose. Further choices of the ligands can be found in, for example, the description of CN105378082a, incorporated by reference in its entirety.

In some embodiments, the pharmaceutically acceptable targeting group in the siRNA conjugate may be galactose or N-acetylgalactosamine, wherein the galactose or N-acetylgalactosamine molecule may be monovalent, divalent, trivalent, tetravalent. It should be understood that monovalent, divalent, trivalent, tetravalent, as described herein, refer to the molar ratio of siRNA molecules to galactose or N-acetylgalactosamine molecules in the siRNA conjugate after the siRNA molecules form an siRNA conjugate with a conjugate group containing galactose or N-acetylgalactosamine molecules as a targeting group, respectively, being 1:1, 1:2, 1:3, or 1:4. In some embodiments, the pharmaceutically acceptable targeting group is N-acetylgalactosamine. In some embodiments, when the siRNA of the application is conjugated to a conjugate group comprising N-acetylgalactosamine, the N-acetylgalactosamine molecule is trivalent or tetravalent. In some embodiments, the N-acetylgalactosamine molecule is trivalent when the siRNA of the application is conjugated to a conjugate group comprising N-acetylgalactosamine.

The targeting group can be attached to the siRNA molecule via a suitable linker, which can be selected by one skilled in the art depending on the particular type of targeting group. The types of these linkers, targeting groups and the manner of attachment to the siRNA can be found in the disclosure of WO2015006740A2, which is incorporated by reference in its entirety.

Method for synthesizing siRNA

The nucleoside monomers are linked one by one in the 3'-5' direction according to the nucleotide arrangement sequence by a solid-phase phosphoramidite method conventional in the art. Each nucleoside monomer attached includes four steps of deprotection, coupling, capping, oxidation or vulcanization. Wherein, when two nucleotides are connected by phosphate, the connection of the latter nucleoside monomer comprises deprotection, coupling, capping and oxidation. When phosphorothioate is adopted to connect two nucleotides, the following nucleoside monomer is connected, and the four steps of protection, coupling, capping and vulcanization are included.

For example, the synthesis conditions of the siRNA of the present application may be as follows:

the deprotection conditions include: the reaction temperature is 25 ℃, the reaction time is 70 seconds, the deprotection reagent is selected from dichloromethane solution (3%V/V) of dichloroacetic acid, and the molar ratio of the deprotection reagent to the protecting groups on the solid phase carrier is 5:1.

The coupling reaction conditions include: the reaction temperature is 25 ℃, the reaction time is 600 seconds, the coupling reagent is selected from 0.25M acetonitrile solution of 5-ethylthio-1H-tetrazole (ETT), and the molar ratio of the nucleic acid sequence connected on the solid phase carrier to the nucleoside monomer is 1:10.

The capping reaction conditions include: the reaction temperature is 25 ℃, the reaction time is 15 seconds, the capping reagent is selected from mixed solution of CapA (10% acetic anhydride acetonitrile solution) and CapB (10% N-methylimidazole pyridine/acetonitrile solution) with the mol ratio of 1:1, and the mol ratio of the capping reagent to the nucleic acid sequence connected on the solid phase carrier is acetic anhydride: the molar ratio of the N-methylimidazole to the nucleic acid sequences attached to the solid support was 1:1:1.

The oxidation reaction conditions include: the reaction temperature was 25℃and the reaction time was 15 seconds, the oxidizing agent was selected from a 0.05M solution of iodotetrahydrofuran, and the molar ratio of the oxidizing agent to the nucleic acid sequence attached to the solid support in the coupling step was 30:1.

The vulcanization reaction conditions include: the reaction temperature was 25℃and the reaction time was 300 seconds, the sulfiding reagent was selected from the group consisting of hydrogenation Huang Yuansu, and the molar ratio of sulfiding reagent to nucleic acid sequence attached to the solid support in the coupling step was 120:1.

After all nucleoside monomers are connected, the nucleic acid sequences connected on the solid phase carrier are sequentially subjected to cutting, deprotection, purification and desalination to obtain siRNA sense strand and antisense strand, and finally the two strands are subjected to heating annealing to obtain the product.

Methods of cleavage, deprotection, purification, desalting, and annealing are well known in the art. For example, cleavage and deprotection are carried out by contacting the nucleotide sequence to which the solid phase carrier is attached with concentrated ammonia water; purification by chromatography; desalting by reverse phase chromatography; by mixing the sense strand and the antisense strand in equimolar ratio under different strict conditions, the temperature is gradually reduced and cooled.

siRNA conjugate synthesis method

In the first step, DMTR-L96 is reacted with succinic anhydride to give compound L96-A:

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The preparation process comprises the following steps: DMTR-L96, succinic anhydride, 4-dimethylaminopyridine and diisopropylethylamine are added into dichloromethane, stirred and reacted for 24 hours at 25 ℃, then the reaction liquid is washed by 0.5M triethylamine phosphate, the water phase is washed three times by dichloromethane, and the organic phases are combined and evaporated to dryness under reduced pressure to obtain a crude product. Then purifying by column chromatography to obtain the pure L96-A.

Second, L96-A is reacted with NH ₂ SPS reaction gives L96-B:

the preparation process comprises the following steps: L96-A, O-benzotriazol-tetramethyluronium Hexafluorophosphate (HBTU) and Diisopropylethylamine (DIPEA) were mixed and dissolved in acetonitrile, stirred at room temperature for 5 minutes to give a homogeneous solution, and aminomethyl resin (NH) was added ₂ SPS,100-200 meshes) into a reaction liquid, starting a shaking table reaction at 25 ℃, filtering after 18 hours of reaction, and washing a filter cake by dichloromethane and acetonitrile in sequence to obtain the filter cake. Capping the filter cake with CapA/CapB mixed solution to obtain L96-B, namely a solid phase carrier containing conjugate molecules, connecting nucleoside monomers to the conjugate molecules under the coupling reaction, synthesizing siRNA sense strand connected to the conjugate molecules according to the siRNA molecule synthesis method, synthesizing siRNA antisense strand by adopting the siRNA molecule synthesis method, and annealing to obtain the siRNA conjugate.

Pharmaceutical setComposition

The present application provides a pharmaceutical composition comprising the siRNA as described above as an active ingredient and a pharmaceutically acceptable carrier.

The pharmaceutically acceptable carrier may be a carrier conventionally used in the field of siRNA administration, such as, but not limited to, lipid nanoparticles (Lipid Nanoparticle, LNP), magnetic nanoparticles (magnetic nanoparticles, e.g. based on Fe ₃ O ₄ Or Fe (Fe) ₂ O ₃ Carbon nanotubes), mesoporous silica (mesoporous silicon), calcium phosphate nanoparticles (calcium phosphate nanoparticles), polyethylenimine (PEI), polyamidedendrimers (polyamidoamine (PAMAM) dendrimer), polylysine (L-lysine), PLL, chitosan (chitosan), 1, 2-dioleoyl-3-trimethylammoniopropane (1, 2-dioleoyl-3-trimethylonium-propane, DOTAP), poly-D-or L-lactic acid/glycolic acid copolymers (poly (D)&L-lactic/glycolic acid) copolymer, PLGA), poly (aminoethylethylene phosphate) (poly (2-aminoethyl ethylene phosphate), PPEEA) and poly (N, N-dimethylaminoethyl methacrylate) (poly (2-dimethylaminoethyl methacrylate), PDMAEMA) and derivatives thereof.

The content of the siRNA and the pharmaceutically acceptable carrier in the pharmaceutical composition is not particularly required, and can be the conventional content of each component.

In some embodiments, the pharmaceutical composition may further comprise other pharmaceutically acceptable excipients, which may be one or more of various formulations or compounds conventionally employed in the art. For example, the pharmaceutically acceptable additional excipients may include at least one of a pH buffer, a protectant, and an osmolality adjusting agent.

The pH buffer solution can be a tris hydrochloride buffer solution with the pH value of 7.5-8.5 and/or a phosphate buffer solution with the pH value of 5.5-8.5, for example, the pH value of 5.5-8.5.

The protective agent may be at least one of inositol, sorbitol, sucrose, trehalose, mannose, maltose, lactose, and glucose. The protective agent may be present in an amount of 0.01 to 30% by weight, based on the total weight of the pharmaceutical composition.

The osmolality adjusting agent may be sodium chloride and/or potassium chloride. The osmolality adjusting agent is present in an amount such that the osmolality of the pharmaceutical composition is 200-700 milliosmoles per kilogram (mOsm/kg). The amount of osmolality adjusting agent can be readily determined by one skilled in the art based on the desired osmolality.

In some embodiments, the pharmaceutical composition may be a liquid formulation, such as an injection; or freeze-dried powder injection, and is mixed with liquid adjuvant to make into liquid preparation. The liquid formulation may be administered, but is not limited to, for subcutaneous, intramuscular or intravenous injection, and may be administered, but is not limited to, by spraying to the lungs, or by spraying through the lungs to other visceral tissues such as the liver. In some embodiments, the pharmaceutical composition is for intravenous administration.

In some embodiments, the pharmaceutical composition may be in the form of a liposomal formulation. In some embodiments, the pharmaceutically acceptable carrier used in the liposomal formulation comprises an amine-containing transfection compound (which may also be referred to hereinafter as an organic amine), a helper lipid, and/or a pegylated lipid.

The following examples serve to further illustrate the invention without however limiting it in any way.

Examples

Other objects, features and advantages of the present disclosure will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating specific embodiments of the disclosure, are given by way of illustration only, since various changes and modifications within the spirit and scope of the disclosure will become apparent to those skilled in the art from this detailed description.

The experimental techniques and methods used in this example are conventional techniques unless otherwise specified, such as those not specified in the following examples, and are generally performed under conventional conditions such as Sambrook et al, molecular cloning: conditions described in the laboratory Manual (New York: cold Spring Harbor Laboratory Press, 1989) or as recommended by the manufacturer. Materials, reagents and the like used in the examples are all available from a regular commercial source unless otherwise specified.

The cells and reagents used in the following examples are shown in the following table:

EXAMPLE 1 preparation of siRNA

siRNA molecules having the following sequences were synthesized by tenlin biotechnology (shanghai) limited.

TABLE 1siRNA and sequences thereof

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Wherein the capital letters "G", "C", "A", "T" and "U" each generally represent nucleotides containing guanine, cytosine, adenine, thymine and uracil, respectively, as bases; lowercase letters a, u, c, g: a nucleotide representing 2' -methoxy modification; af. Gf, cf, uf: a 2' -fluoro modified nucleotide; dA. dG, dC, dU: represents deoxyribonucleotides; the lower case letter s indicates that phosphorothioate linkages are between two nucleotides adjacent to the letter s; p1: indicating that the adjacent nucleotide to the right of P1 is a nucleotide 5' -phosphate; A、U、C、G(underlined + bold + italic): indicating GNA modified nucleotides.

siRNA conjugates were synthesized by tenlin biotechnology (Shanghai) limited with the following sequences:

table 2siRNA conjugates and sequences thereof:

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wherein, L96 is:

EXAMPLE 2siRNA inhibiting AGT Gene expression

2.1 Experimental materials

Human liver cancer cell Huh7, JCRB cell bank, accession number JCRB0403;

the kit for extracting RNA comprises a kit for extracting RNA,96Kit, QIAGEN-74182;

RNAiMAX transfection reagent, available from Invitrogen under the accession number 13778-150;

opti-medium: serum-reduced medium, available from Gibco under accession number 31985-070;

fastking RT Kit (with gDNase), available from TIANGEN under the accession number KR116-02;

FastStart universal probe master (ROX), available from Roche under the accession number 04914058001;

Target AGT primer and probe set(Thermo,Assay ID:Hs01586213_m1)

Target GAPDH primer and probe set(Thermo,Assay ID:Hs02786624_g1)。

2.2 Experimental methods

2.2.1 Huh7 cells were taken, washed with DPBS, digested with trypsin, adjusted to a cell density of 5.5X105 cells/ml, and seeded into 96-well plates at a density of 20,000 cells per well at 100. Mu.l per well of culture broth. Huh7 cells were placed in 5% CO ₂ Incubate overnight at 37 ℃.

2.2.2 the dry powder of siRNA to be tested was centrifuged at low temperature and high speed and then dissolved in ultra-pure distilled water (ULtraPure Distilled Water) to prepare 100. Mu.M siRNA stock solution.

2.2.3 preparation of siRNA diluent Z at 2nM and siRNA diluent W at 0.2nM

(1) Preparation of 0.1. Mu.M siRNA stock solution X and 0.01. Mu.M siRNA stock solution Y:

a) Taking 2 mu l of 100 mu M siRNA mother liquor prepared in the step 2.2.2, and adding 18 mu l of ultra-pure distilled water to obtain siRNA diluent with the final concentration of 10 mu M;

b) Taking 2 mu l of the 10 mu M siRNA diluent prepared in the step a), and adding 18 mu l of ultra-pure distilled water to obtain the siRNA diluent with the final concentration of 1 mu M;

c) Taking 2 mu l of 1 mu M siRNA dilution liquid prepared in the step b), adding 18 mu l of ultra-pure distilled water, and obtaining siRNA stock solution X with the final concentration of 0.1 mu M;

d) Taking 2 μl of 0.1 μM siRNA stock solution X prepared in step c), adding 18 μl of ultrapure distilled water to obtain 0.01 μM siRNA stock solution Y;

(2) 2. Mu.l each of the above-prepared siRNA stock solution X and siRNA stock solution Y was taken, and 98. Mu.l of Opti-medium was added to each to obtain 2nM siRNA dilution Z and 0.2nM siRNA dilution W, respectively.

2.2.4 transfection of Huh7 cells

(1) Taking outRNAiMAX transfection reagent 3. Mu.l, 97. Mu.l Opti-medium was added to give +.>RNAiMAX transfection reagent dilutions; will->Mixing RNAiMAX transfection reagent diluent with 2nM siRNA diluent Z prepared in step 2.2.3 at a volume ratio of 1:1 to prepare a transfection mixture, standing for 5 minutes, adding 10 μl of the transfection mixture into a 96-well plate to transfect Huh7 cells cultured in step 2.2.1 (final volume 100 μl, concentration of siRNA in the system is 0.1 nM);

(2) Taking outRNAiMAX transfection reagent 3. Mu.l, 97. Mu.l Opti-medium was added to give +.>RNAiMAX transfection reagent dilutions; will->RNAiMAX transfection reagent dilution was mixed with 0.2nM siRNA dilution W prepared in step 2.2.3 at a 1:1 volume ratio to prepare a transfection mixture, which was allowed to stand for 5 minutes, 10. Mu.l of the transfection mixture was added to a 96-well plate to transfect Huh7 cells cultured in step 2.2.1 (final volume 100. Mu.l, concentration of siRNA in the system was 0.01 nM).

Culturing for 24 hours after the transfection; 2 replicates were set for each concentration (0.1 nM and 0.01 nM).

2.2.5 according to96Kit product instructions, total RNA of Huh7 cells obtained in step 2.2.4 was extracted.

2.2.6 reverse transcription of the total RNA extracted to cDNA was performed using a Fastking RT Kit (with gDNase) according to the following procedure:

a) gDNA was removed with gDNase according to the following table;

TABLE 3 Table 3

42 ℃ for 3min; and (4) standing at the temperature of 4 ℃.

b) The reverse transcription procedure was performed as follows

TABLE 4 Table 4

42℃，15min；95℃，3min。

c) The reverse transcription product was stored at-20℃for real-time PCR analysis.

2.2.7 real-time PCR analysis Using FastStart universal probe master (ROX)

a) qPCR reaction mixtures were prepared as shown in the following table, with all reagents placed on ice throughout the run;

TABLE 5

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b) qPCR procedure was performed as follows

95 ℃ for 10 minutes;

95 ℃ for 15 seconds; 60℃for 1 minute (40 cycles).

2.2.8 analysis of results

a) Using Quant Studio 7 software to automatically calculate Ct value by default;

b) The relative expression amount of the gene was calculated using the following formula:

delta ct=ct (AGT gene) -Ct (GAPDH)

ΔΔct=Δct (detection of sample) group) -deltact (Mock group), wherein Mock groups represent groups to which no siRNA was added compared to the test sample groups;

mRNA expression = 2 relative to Mock group ^-ΔΔCt

Inhibition ratio (%) = (relative expression amount of mRNA in Mock group-relative expression amount of mRNA in test sample group)/relative expression amount of mRNA in Mock group×100%

2.3 experimental results

Concentrations of 0.1nM and 0.01nM were chosen for testing.

TABLE 6 inhibition of siRNA of the invention

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As can be seen from Table 6, the siRNA of the present invention significantly inhibited the expression of AGT gene at both 0.1nM and 0.01 nM.

EXAMPLE 3siRNA IC inhibiting AGT Gene expression ₅₀ Measurement

IC for siRNA inhibition of AGT Gene expression in a similar manner to example 2 ₅₀ The assay in which the concentrations of siRNA used for transfection were 10nM, 2.5nM, 0.625nM, 0.156nM, 0.039nM, 0.0098nM, 0.0024nM, 0.0006nM, respectively.

Analysis of results

delta ct=ct (AGT gene) -Ct (GAPDH)

ΔΔΔCt =Δct (detection of sample) group) -delta Ct (Mock group)

mRNA expression = 2 relative to Mock group ^-ΔΔCt 。

Mock group represents: the group to which no siRNA was added was compared with the test sample group.

The log value of siRNA concentration is taken as X axis, the percentage inhibition rate is taken as Y axis, and analysis software Gr is adoptedThe "log (inhibitor) vs. response-variable slope" function of aplPad Prism 8 was used to fit a dose-response curve to derive the IC for each siRNA ₅₀ Values.

The fitting formula is: y=bottom+ (Top-Bottom)/(1+10++LogIC 50-X. Times HillSlope)

Wherein: top represents percent inhibition at the Top plateau, the Top criterion of the curve is typically 80% to 120%; bottom represents the percent inhibition at the Bottom plateau, with Bottom of the curve typically between-20% and 20%; hillSlope represents the slope of the percent inhibition curve.

The experimental results are shown in table 7.

TABLE 7 IC of siRNA ₅₀ (nM)

siRNA ID	IC ₅₀ (pM)
		N-ER-FY0140024M1	30.08
N-ER-FY0140028M1	27.20
		N-ER-FY0140040M1	19.81
N-ER-FY0140045M1	15.17

As can be seen from Table 7, the siRNA of the present invention significantly inhibited AGT gene expression, IC ₅₀ About 30pM or less, and as low as 15.17pM.

EXAMPLE 4 IC of siRNA conjugates to inhibit AGT Gene expression ₅₀ Measurement

4.1 test materials:

human primary hepatocytes PHH cells, supplied by the medicine Mingkang;

PHH medium: invitroGRO CP Meduim serum free BIOVIT, cargo number: s03316;

RNAiMAX transfection reagent, available from Invitrogen, cat: 13778-150;

RNA extraction kit96Kit, available from QIAGEN, cat: QIAGEN-74182;

reverse transcription Kit FastKing RT Kit (With gDNase), available from TianGen, cat: KR116-02;

FastStart Universal Probe master (ROX), available from Roche under the designation: 04914058001;

primers for AGT and GAPDH were provided by Ming Kangde.

4.2 test methods

siRNA conjugates (final siRNA conjugate concentrations of 10nM, 2.5nM, 0.63nM, 0.16nM, 0.04nM, 0.01nM, 0.0024nM and 0.0006nM, compound wells), respectively) were transfected into PHH cells as follows: taking cryopreserved PHH cells, resuscitating, counting, and adjusting cell to 6×10 ⁵ Cell/ml, simultaneous applicationRNAiMax the siRNA conjugates were transferred to cells and seeded into 96-well plates at a density of 54,000 cells per well, with 100. Mu.L of PPH medium added per well. Cells were exposed to 5% CO ₂ Culturing in incubator at 37 ℃. After 48 hours, the medium was removed and the cells were collected for total RNA extraction. Use according to the kit product instructions +. >Total RNA was extracted at 96 Kit.

siRNA conjugates (siRNA conjugates)Final concentrations of the compounds were 500nM, 125nM, 31.25nM, 7.81nM, 1.95nM, 0.49nM, 0.12nM and 0.03nM, respectively, in duplicate) into PHH cells by free uptake as follows: taking cryopreserved PHH cells, resuscitating, counting, and adjusting cell to 6×10 ⁵ Cells/ml, while siRNA conjugate was added, were seeded into 96-well plates at a density of 54,000 cells per well, and 100 μl per well of culture broth. Cells were exposed to 5% CO ₂ Culturing in incubator at 37 ℃. After 48 hours, the medium was removed and the cells were collected for total RNA extraction. Use according to the kit product instructionsTotal RNA was extracted at 96 Kit.

The total RNA extracted was reverse transcribed into cDNA by reverse transcription reaction, and AGT cDNA obtained by reverse transcription was quantitatively amplified by qPCR, using a method similar to that in example 2. GAPDH cDNA will be amplified in parallel as an internal control. The PCR reaction procedure was: 10 minutes at 95℃and then enter a cyclic mode, 95℃for 15 seconds followed by 60℃for 60 seconds for a total of 40 cycles.

Analysis of results:

Delta ct=ct (AGT gene) -Ct (GAPDH)

ΔΔct=Δct (detection of sample) group) -deltact (Mock group), wherein Mock groups represent groups to which no siRNA conjugate was added compared to the test sample groups;

mRNA expression = 2 relative to Mock group ^-ΔΔCt

Inhibition ratio (%) = (relative mRNA expression amount of Mock group-relative mRNA expression amount of test sample group)/relative mRNA expression amount of Mock group×100%.

The log value of siRNA conjugate concentration is taken as X axis, the percent inhibition rate is taken as Y axis, and the "log (inhibitor) vs. response-variable slope" functional module of analytical software GraphPad Prism 8 is adopted to fit the dose-response curve, thereby obtaining the IC of each siRNA conjugate ₅₀ Values.

The experimental results are shown in table 8.

TABLE 8 IC of siRNA conjugates inhibiting AGT Gene expression ₅₀ Value of

As can be seen from Table 8, the siRNA conjugates of the present invention significantly inhibited AGT gene expression, and IC in the case where the siRNA conjugates entered PHH by free uptake ₅₀ As low as 0.559nM; in case the siRNA conjugate enters PHH by transfection, IC ₅₀ As low as 0.004nM.

Example 5 inhibition ratio assay of siRNA and siRNA conjugate to inhibit AGT Gene expression

5.1 test materials:

human primary hepatocytes PHH cells, supplied by the medicine Mingkang;

PHH medium: invitroGRO CP Meduim serum free BIOVIT, cargo number: s03316;

RNAiMAX transfection reagent, available from Invitrogen, cat: 13778-150;

RNA extraction kit96Kit, available from QIAGEN, cat: QIAGEN-74182;

primers for AGT and GAPDH were provided by Ming Kangde.

5.2 test methods

siRNA and siRNA conjugate (final concentrations of siRNA and siRNA conjugate are 5nM and 0.5nM, respectively, countersunk) were transfected into PHH cells by the following procedure: taking cryopreserved PHH cells, resuscitating, counting, and adjusting cell to 6×10 ⁵ Cell/ml, simultaneous applicationRNAiMax was transferred to cells with siRNA and siRNA conjugate, respectively, and inoculated into 96-well plates at a density of 54,000 cells per well, with 100. Mu.L of PPH medium added per well. Cells were exposed to 5% CO ₂ Culturing in incubator at 37 ℃. After 48 hours, the medium was removed and the cells were collected for total RNA extraction. Use according to the kit product instructions +.>Total RNA was extracted at 96 Kit.

siRNA and siRNA conjugate (final concentrations of siRNA and siRNA conjugate are 100nM and 10nM, respectively, in duplicate wells) were entered into PHH cells by free uptake as follows: taking cryopreserved PHH cells, resuscitating, counting, and adjusting cell to 6×10 ⁵ Cells/ml, while siRNA and siRNA conjugate were added separately, and inoculated into 96-well plates at a density of 54,000 cells per well, 100 μl per well of culture broth. Cells were exposed to 5% CO ₂ Culturing in incubator at 37 ℃. After 48 hours, the medium was removed and the cells were collected for total RNA extraction. Use according to the kit product instructionsTotal RNA was extracted at 96 Kit.

Analysis of results:

delta ct=ct (AGT gene) -Ct (GAPDH)

ΔΔct=Δct (detection of sample) group) -deltact (Mock group), wherein Mock represents the group to which no siRNA was added compared to the test sample group;

mRNA expression = 2 relative to Mock group ^-ΔΔCt

TABLE 9 inhibition ratio of siRNA and siRNA conjugate to inhibit AGT Gene expression

As can be seen from table 9, the siRNA and siRNA conjugates of the invention significantly inhibited AGT gene expression.

EXAMPLE 6 inhibition of human AGT (hAGT) Gene expression by siRNA conjugates in humanized mice

C57BL/6-hAGT mice (supplied by Shanghai Nannon model biotechnology Co., ltd.) of 6-8 weeks old were fed to the feeding facility, and after 7 days of adaptive feeding, siRNA conjugates were subcutaneously administered to the mice (6 mice per group) at a single dose of 3mg/kg, respectively. Serum hAGT protein expression levels were measured at 7, 14, 21, 28, 35 and 42 days after administration, thereby obtaining the inhibition rate of hAGT protein expression by the siRNA conjugate.

From the experiment, the siRNA conjugate of the present disclosure has higher inhibition activity on the hAGT gene in vivo, and can reduce the hAGT protein level for a long time, which indicates that the siRNA conjugate of the present invention can better inhibit the generation of hAGT protein.

EXAMPLE 7 plasma kinetics study of siRNA conjugates in CD-1 mice

Animals: CD-1 mice, SPF grade, male, about 30g, purchased from Si Bei Fu (Beijing) Biotechnology Co., ltd.

Dosage and mode of administration: the siRNA conjugates were administered at a dose of 3mg/kg (10 mL/kg), with a single subcutaneous injection following randomization, with 6 mice per group.

Sample collection: samples of whole blood were collected at 10 points at 0.0833, 0.25, 0.5, 1, 2, 4, 8, 24, 36, 48h post-administration. The front 3 of each group is collected for 0.0833, 0.5, 2, 8 and 36 hours, and the rear 3 is collected for 0.25, 1, 4, 24 and 48 hours, and the whole blood is collected and then the blood plasma is centrifugally separated for detection and analysis.

Sample detection and analysis: the concentration of the original drug in plasma samples at each time point was measured by LC-MS/MS method and PK parameters were calculated using WinNonlin software: c (C) _max 、T _max 、AUC、MRT、t _1/2 。

From this experiment, it can be concluded that the siRNA conjugates of the present disclosure have a shorter half-life in plasma and are cleared faster.

EXAMPLE 8 tissue distribution assay of siRNA conjugates in CD-1 mice

Dosage and mode of administration: the siRNA conjugates were administered at a dose of 3mg/kg (10 mL/kg), a single subcutaneous injection following randomization, 3 animals at each time point, and 24 mice total.

Sample collection:

24h after administration: collecting plasma, liver, kidney and spleen; 72h after administration: collecting plasma, liver, kidney and spleen;

168h (1 week) after administration: collecting plasma, liver, kidney, spleen, brain, heart, lung, stomach, small intestine, muscle, testis;

336h (2 weeks) post-dose: collecting plasma, liver, kidney and spleen;

672h (4 weeks) post-dose: collecting plasma, liver, kidney, spleen, brain, heart, lung, stomach, small intestine, muscle, testis; 1008h (6 weeks) after dosing: collecting plasma, liver, kidney and spleen;

1344h (8 weeks) after dosing: collecting plasma, liver, kidney and spleen;

1680h (10 weeks) post-dose: plasma, liver, kidney, spleen, brain, heart, lung, stomach, small intestine, muscle, testis were collected.

Sample detection and analysis: the concentration of the original drug in the plasma and tissue samples at each time point was detected by LC-MS/MS method, and AUC in the plasma and tissue was calculated by trapezoidal area method.

From the experiment, the siRNA conjugate disclosed by the invention is mainly enriched in the liver, has long retention time in tissues and has good stability.

EXAMPLE 9 Single subcutaneous injection of siRNA conjugate C57B/L mice administration MTD assay

C57B/L mice, SPF grade, male, about 25g, purchased from Si Bei Fu (Beijing) Biotechnology Co., ltd. Animals adopt a method of random body weight block according to the body weight of the last 1 day of the adaptation period, and the specific dosage design and grouping are as follows:

Detecting the index:

clinical observation: the administration day is continuously observed for 4 hours, and at least one clinical observation is carried out every day in the recovery period

Weight of: all surviving animals were weighed 2 times per week.

Immunotoxicity: MTD dose animals were alternately bled 1 h.+ -. 2min,4 h.+ -. 5min,8 h.+ -. 10min,24 h.+ -. 20min after D1 dosing, 3 animals per sex/group were harvested at each time point and tested for cytokines (IFN-. Gamma., TNF-. Alpha., IL-2/6/8).

Toxicological kinetics: MTD dose animals are alternately sampled before and 30min 2min,1h 2min,4h 5min,8h 10min and 24h 20min after D1 administration, and blood concentration is detected by collecting 3 animals/sex/animal group at each time point.

Chemistry of blood generation: the animals of the main test group are subjected to R28 sectioning, and the animals of the satellite group are subjected to R7, R14, R21 and R28 sectioning in batches, so as to detect the biochemical property of blood.

Tissue distribution: the main test animals are subjected to R28 sectioning, the satellite animals are subjected to R7, R14, R21 and R28 sectioning in batches, blood and liver are collected, and the tissue drug concentration is detected.

Histopathological examination: animals of the main test group were examined by R28 dissection, and the main organs (heart, liver, spleen, lung, kidney, brain, adrenal gland, thymus, stomach, uterus/testis, ovary/epididymis) and the tissues or organs found abnormal were collected, fixed, and subjected to histopathological examination.

From this experiment, it can be seen that the siRNA conjugates of the present disclosure are less toxic, with an excellent window of drug safety.

Claims

1. An siRNA that inhibits expression of an Angiotensinogen (AGT) gene, said siRNA comprising a sense strand and an antisense strand, wherein each nucleotide in said siRNA is independently a modified or unmodified nucleotide, wherein said sense strand comprises a nucleotide sequence I, and said antisense strand comprises a nucleotide sequence II, said nucleotide sequence I and said nucleotide sequence II being at least partially reverse complementary to form a duplex region, wherein said nucleotide sequence I and nucleotide sequence II are selected from the group consisting of:

5’-GCUAGUCGCUGCA-3’(SEQ ID NO:121)

5’-UGCAGCGACUAGC-3’(SEQ ID NO:122),

5’-CCUUGGAAGGACAAGA-3’(SEQ ID NO:123)

5’-UCUUGUCCUUCCAAGG-3’(SEQ ID NO:124)；

5’-GUGCUGAACAGCAUUU-3’(SEQ ID NO:125)

5’-AAAUGCUGUUCAGCAC-3’(SEQ ID NO:126)；

5’-CUACCCAACAGCU-3’(SEQ ID NO:127)

5’-AGCUGUUGGGUAG-3’(SEQ ID NO:128)；

5’-UCUGGGUACUA-3’(SEQ ID NO:446)

5’-UAGUACCCAGA-3’(SEQ ID NO:447)；

5’-CUGCUCCAAUU-3’(SEQ ID NO:448)

5’-AAUUGGAGCAG-3’(SEQ ID NO:449)；

5’-CUGCAAAACUU-3’(SEQ ID NO:450)

5’-AAGUUUUGCAG-3’(SEQ ID NO:451)；

5’-CUCUCUAUACCCCU-3’(SEQ ID NO:452)

5’-AGGGGUAUAGAGAG-3’(SEQ ID NO:453)；

5’-CAAGUGCCCUUCA-3’(SEQ ID NO:454)

5’-UGAAGGGCACUUG-3’(SEQ ID NO:455)；

5’-ACAGAGUCUACCCAA-3’(SEQ ID NO:456)

5’-UUGGGUAGACUCUGU-3’(SEQ ID NO:457)；

5’-GUCGACUUUGAGCUGGA-3’(SEQ ID NO:458)

5’-UCCAGCUCAAAGUCGAC-3’(SEQ ID NO:459)；

5’-GAAAGCAGCCGU-3’(SEQ ID NO:460)

5’-ACGGCUGCUUUC-3’(SEQ ID NO:461)；

5’-GGCACAAAUGCACCU-3’(SEQ ID NO:462)

5’-AGGUGCAUUUGUGCC-3’(SEQ ID NO:463)；

5’-GCAGCUGGCAA-3’(SEQ ID NO:464)

5’-UUGCCAGCUGC-3’(SEQ ID NO:465)；

5’-CAAUGCCGGGAAGCCCAAA-3’(SEQ ID NO:269)

5’-UUUGGGCUUCCCGGCAUUG-3’(SEQ ID NO:270)；

5’-ACCUUCAUACCU-3’(SEQ ID NO:466)

5’-AGGUAUGAAGGU-3’(SEQ ID NO:467)；

5’-CCAGGAGUUCU-3’(SEQ ID NO:468)

5’-AGAACUCCUGG-3’(SEQ ID NO:469)；

5’-CCACUGCCCUGCACUUCC-3’(SEQ ID NO:470)

5’-GGAAGUGCAGGGCAGUGG-3’(SEQ ID NO:471)；

5’-AGCUGGUGCUAGUCG-3’(SEQ ID NO:472)

5’-CGACUAGCACCAGCU-3’(SEQ ID NO:473)；

5’-ACAGCUUAACAA-3’(SEQ ID NO:474)

5’-UUGUUAAGCUGU-3’(SEQ ID NO:475)；

5’-GCCGUUUCUCCUU-3’(SEQ ID NO:476)

5’-AAGGAGAAACGGC-3’(SEQ ID NO:477)；

2. The siRNA of claim 1, wherein the nucleotide sequence I and the nucleotide sequence II are substantially reverse complementary, substantially reverse complementary or fully reverse complementary; by substantially reverse complement is meant that there are no more than 3 base mismatches between the two nucleotide sequences; by substantially reverse complement is meant that there is no more than 1 base mismatch between the two nucleotide sequences; complete reverse complement refers to the absence of mismatches between two nucleotide sequences.

3. The siRNA of claim 1 or 2, wherein the sense strand further comprises a nucleotide sequence III, the antisense strand further comprises a nucleotide sequence IV, each of the nucleotide sequence III and the nucleotide sequence IV being independently 0-10 nucleotides in length, wherein the nucleotide sequence III is linked at the 5 'end of the nucleotide sequence I, the nucleotide sequence IV is linked at the 3' end of the nucleotide sequence II, the nucleotide sequence III and the nucleotide sequence IV are equal in length and are substantially reverse-complementary or fully reverse-complementary; by substantially reverse complement is meant that there is no more than 1 base mismatch between the two nucleotide sequences; complete reverse complement refers to the absence of mismatches between two nucleotide sequences; and/or, the nucleotide sequence III is connected at the 3 'end of the nucleotide sequence I, the nucleotide sequence IV is connected at the 5' end of the nucleotide sequence II, and the nucleotide sequence III and the nucleotide sequence IV are equal in length and are substantially reverse complementary or completely reverse complementary; by substantially reverse complement is meant that there is no more than 1 base mismatch between the two nucleotide sequences; complete reverse complement refers to the absence of mismatches between two nucleotide sequences.

4. A siRNA according to any one of claims 1-3, said sense strand further comprising nucleotide sequence V and/or said antisense strand further comprising nucleotide sequence VI, nucleotide sequences V and VI being 0 to 3 nucleotides in length, nucleotide sequence V being linked at the 3 'end of said sense strand to form a 3' overhang of the sense strand and/or nucleotide sequence VI being linked at the 3 'end of said antisense strand to form a 3' overhang of the antisense strand; preferably, the nucleotide sequence V or VI is 2 nucleotides in length; more preferably, the nucleotide sequence V or VI is two consecutive thymidines or two consecutive uracils ribonucleotides;

alternatively, the nucleotide sequence V or VI is mismatched or complementary to a nucleotide at the corresponding position of the target mRNA.

5. The siRNA according to any one of claims 1-4, wherein the double stranded region is 15-30 nucleotide pairs in length; preferably, the double-stranded region is 17-23 nucleotide pairs in length; more preferably, the double stranded region is 19-21 nucleotide pairs in length.

6. The siRNA of any one of claims 1-5, wherein the sense strand or the antisense strand has 15-30 nucleotides; preferably, the sense strand or antisense strand has 19-25 nucleotides; more preferably, the sense strand or the antisense strand has 19-23 nucleotides.

7. The siRNA of any one of claims 1-6, wherein at least one nucleotide in the sense strand or the antisense strand is a modified nucleotide and/or at least one phosphate group is a phosphate group having a modification group; preferably, the phosphate group containing a modifying group is a phosphorothioate group formed by substitution of at least one oxygen atom of a phosphodiester bond in the phosphate group with a sulfur atom; and/or, the siRNA comprises a sense strand that does not comprise a 3' overhang nucleotide.

8. The siRNA of any of claims 1-7, wherein the 5 'terminal nucleotide of the sense strand is linked to a 5' phosphate group or a 5 'phosphate derivative group, and/or the 5' terminal nucleotide of the antisense strand is linked to a 5 'phosphate group or a 5' phosphate derivative group.

9. The siRNA of any one of claims 1-8, wherein the modified nucleotide is selected from the group consisting of a 2 '-fluoro modified nucleotide, a 2' -alkoxy modified nucleotide, a 2 '-substituted alkoxy modified nucleotide, a 2' -alkyl modified nucleotide, a 2 '-substituted alkyl modified nucleotide, a 2' -deoxyribonucleotide, a 2 '-amino modified nucleotide, a 2' -substituted amino modified nucleotide, a nucleotide analog, or a combination of any two or more thereof.

10. The siRNA of any of claims 1-9, wherein the modified nucleotide is selected from the group consisting of a 2'-F modified nucleotide, 2' -O-CH ₃ Modified nucleotides, 2' -O-CH ₂ -CH ₂ -O-CH ₃ Modified nucleotides, 2' -O-CH ₂ -CH＝CH ₂ Modified nucleotides, 2' -CH ₂ -CH ₂ -CH＝CH ₂ Modified nucleotides, 2' -deoxyribonucleotides, nucleotide analogs, or a combination of any two or more thereof.

11. The siRNA of any one of claims 1-10, wherein each nucleotide in the sense strand and the antisense strand is independently a 2' -fluoro modified nucleotide or a non-fluoro modified nucleotide;

preferably, the 2' -fluoro modified nucleotides are located at positions 7, 9, 10 and 11 of the sense strand in the 5' to 3' direction, the remaining positions being non-fluoro modified nucleotides; the 2' -fluoro-modified nucleotide is located at the even number position of the antisense strand in the 5' to 3' direction, and the rest positions are non-fluoro-modified nucleotides;

alternatively, the 2' -fluoro modified nucleotides are located at positions 7, 9, 10 and 11 of the sense strand in the 5' to 3' direction, the remaining positions being non-fluoro modified nucleotides; the 2' -fluoro modified nucleotide is located at positions 2, 6, 14 and 16 of the antisense strand according to the 5' to 3' direction, the remaining positions being non-fluoro modified nucleotides;

Alternatively, the 2' -fluoro modified nucleotides are located at positions 7, 9, 10 and 11 of the sense strand in the 5' to 3' direction, the remaining positions being non-fluoro modified nucleotides; the 2' -fluoro modified nucleotides are located at positions 2, 6, 8, 9, 14 and 16 of the antisense strand in the 5' to 3' direction, the remaining positions being non-fluoro modified nucleotides;

alternatively, the 2' -fluoro modified nucleotides are located at positions 7, 9, 10 and 11 of the sense strand in the 5' to 3' direction, the remaining positions being non-fluoro modified nucleotides; the 2' -fluoro modified nucleotide is located at positions 2, 14 and 16 of the antisense strand according to the 5' to 3' direction, and the rest positions are non-fluoro modified nucleotides;

alternatively, the 2' -fluoro modified nucleotides are located at positions 9, 10 and 11 of the sense strand in the 5' to 3' direction, the remaining positions being non-fluoro modified nucleotides; the 2' -fluoro modified nucleotide is located at position 14 of the antisense strand in the 5' to 3' direction, the remaining positions being non-fluoro modified nucleotides;

further preferably, each of the non-fluoro modified nucleotides is a 2 '-methoxy modified nucleotide, which is a nucleotide formed by substituting the 2' -hydroxyl group of the ribosyl group with a methoxy group.

12. The siRNA of claim 11, wherein each non-fluoro modified nucleotide is independently selected from one of a nucleotide or a nucleotide analogue formed by substitution of the hydroxyl group at the 2' -position of the ribosyl of the nucleotide with a non-fluoro group, the nucleotide analogue being selected from one of an iso-nucleotide, LNA, ENA, cET BNA, UNA and GNA.

13. The siRNA of any one of claims 1-12, wherein each nucleotide in the sense strand and the antisense strand is independently a 2' -fluoro modified nucleotide, a 2' -methoxy modified nucleotide, a 2' -deoxyribonucleotide, a GNA modified nucleotide, or a combination of any two or more thereof;

preferably, the 2 '-fluoro modified nucleotides are located at positions 7, 9, 10 and 11 of the sense strand in the 5' to 3 'direction, the remaining positions being 2' -methoxy modified nucleotides; the 2 '-fluoro modified nucleotide is located at the even number position of the antisense strand in the 5' to 3 'direction, and the rest positions are 2' -methoxy modified nucleotides;

alternatively, the 2 '-fluoro modified nucleotides are located at positions 7, 9, 10 and 11 of the sense strand in the 5' to 3 'direction, the remaining positions being 2' -methoxy modified nucleotides; the 2 '-fluoro modified nucleotides are located at positions 2, 6, 14 and 16 of the antisense strand in the 5' to 3 'direction, the remaining positions being 2' -methoxy modified nucleotides;

Alternatively, the 2 '-fluoro modified nucleotides are located at positions 7, 9, 10 and 11 of the sense strand in the 5' to 3 'direction, the remaining positions being 2' -methoxy modified nucleotides; the 2 '-fluoro modified nucleotides are located at positions 2, 6, 8, 9, 14 and 16 of the antisense strand in the 5' to 3 'direction, the remaining positions being 2' -methoxy modified nucleotides;

alternatively, the 2 '-fluoro modified nucleotides are located at positions 7, 9, 10 and 11 of the sense strand in the 5' to 3 'direction, the remaining positions being 2' -methoxy modified nucleotides; in the 5 'to 3' direction, the 2 '-fluoro modified nucleotides are located at positions 2, 14 and 16 of the antisense strand, the GNA modified nucleotide is located at position 6 of the antisense strand, and the remaining positions are 2' -methoxy modified nucleotides;

alternatively, the 2 '-fluoro modified nucleotides are located at positions 7, 9, 10 and 11 of the sense strand in the 5' to 3 'direction, the remaining positions being 2' -methoxy modified nucleotides; according to the 5 'to 3' direction, 2 '-fluoro modified nucleotides are located at positions 2, 6, 14 and 16 of the antisense strand, GNA modified nucleotides are located at position 7 of the antisense strand, and the rest positions are 2' -methoxy modified nucleotides;

Alternatively, the 2 '-fluoro modified nucleotides are located at positions 9, 10 and 11 of the sense strand in the 5' to 3 'direction, the remaining positions being 2' -methoxy modified nucleotides; according to the 5 'to 3' direction, the 2 '-fluoro modified nucleotide is located at position 14 of the antisense strand, the deoxyribonucleotide is located at positions 2, 5, 7 and 12 of the antisense strand, and the rest positions are 2' -methoxy modified nucleotides.

14. The siRNA of any one of claims 1-13, wherein at least one of the linkages between the following nucleotides in the siRNA is a phosphorothioate linkage:

15. The siRNA of any of claims 1-14, wherein said sense strand comprises phosphorothioate groups in the 5 'to 3' direction at positions shown below:

between nucleotide 1 and nucleotide 2 from the 5' end of the sense strand;

between nucleotide 2 and nucleotide 3 from the 5' end of the sense strand;

between nucleotide 1 and nucleotide 2 from the 3' end of the sense strand;

between nucleotide 2 and nucleotide 3 from the 3' end of the sense strand;

or,

between nucleotide 1 and nucleotide 2 from the 5' end of the sense strand;

16. The siRNA of any of claims 1-15, wherein said antisense strand comprises phosphorothioate groups at positions shown below, in the 5 'to 3' direction:

Between nucleotide 1 and nucleotide 2 from the 5' end of the antisense strand;

between nucleotide 2 and nucleotide 3 from the 5' end of the antisense strand;

between nucleotide 1 and nucleotide 2 from the 3' end of the antisense strand;

17. The siRNA of any one of claims 1-16, wherein each nucleotide in the sense strand and the antisense strand is independently a 2' -fluoro modified nucleotide, a 2' -methoxy modified nucleotide, a 2' -deoxyribonucleotide, a GNA modified nucleotide, or a combination of any two or more thereof;

preferably, the 2 '-fluoro modified nucleotides are located at positions 7, 9, 10 and 11 of the sense strand in the 5' to 3 'direction, the remaining positions being 2' -methoxy modified nucleotides, between the 1 st and 2 nd nucleotides of the 5 'end, between the 2 nd and 3 rd nucleotides of the 5' end, between the 1 st and 2 nd nucleotides of the 3 'end, between the 2 nd and 3 rd nucleotides of the 3' end being phosphorothioate linkages; according to the 5' to 3' direction, the 2' -fluoro modified nucleotide is located at the even number position of the antisense strand, the rest positions are 2' -methoxy modified nucleotides, the 1 st nucleotide and the 2 nd nucleotide of the 5' end, the 2 nd nucleotide and the 3 rd nucleotide of the 5' end, the 1 st nucleotide and the 2 nd nucleotide of the 3' end are connected by phosphorothioate groups;

Alternatively, in the 5' to 3' direction, the 2' -fluoro modified nucleotides are located at positions 7, 9, 10 and 11 of the sense strand, the remaining positions being 2' -methoxy modified nucleotides, between nucleotide 1 and nucleotide 2 of the 5' end, between nucleotide 2 and nucleotide 3 of the 5' end being phosphorothioate linkages, the 3' end being removed from the overhang; according to the 5' to 3' direction, the 2' -fluoro modified nucleotide is located at the even number position of the antisense strand, the rest positions are 2' -methoxy modified nucleotides, the 1 st nucleotide and the 2 nd nucleotide of the 5' end, the 2 nd nucleotide and the 3 rd nucleotide of the 5' end, the 1 st nucleotide and the 2 nd nucleotide of the 3' end are connected by phosphorothioate groups;

alternatively, in the 5' to 3' direction, the 2' -fluoro modified nucleotides are located at positions 7, 9, 10 and 11 of the sense strand, the remaining positions being 2' -methoxy modified nucleotides, between nucleotide 1 and nucleotide 2 of the 5' end, between nucleotide 2 and nucleotide 3 of the 5' end being phosphorothioate linkages, the 3' end being removed from the overhang; the 2 '-fluoro modified nucleotides are located at positions 2, 6, 14 and 16 of the antisense strand in the 5' to 3 'direction, the remaining positions being 2' -methoxy modified nucleotides, between the 1 st and 2 nd nucleotides at the 5 'end, between the 2 nd and 3 rd nucleotides at the 5' end, between the 1 st and 2 nd nucleotides at the 3 'end, and between the 2 nd and 3 rd nucleotides at the 3' end being phosphorothioate linkages;

Alternatively, the 2 '-fluoro modified nucleotides are located at positions 7, 9, 10 and 11 of the sense strand in the 5' to 3 'direction, the remaining positions being 2' -methoxy modified nucleotides, between the 1 st and 2 nd nucleotides at the 5 'end, between the 2 nd and 3 rd nucleotides at the 5' end, between the 1 st and 2 nd nucleotides at the 3 'end, and between the 2 nd and 3 rd nucleotides at the 3' end being phosphorothioate linkages; the 2 '-fluoro modified nucleotides are located at positions 2, 6, 14 and 16 of the antisense strand in the 5' to 3 'direction, the remaining positions being 2' -methoxy modified nucleotides, between the 1 st and 2 nd nucleotides at the 5 'end, between the 2 nd and 3 rd nucleotides at the 5' end, between the 1 st and 2 nd nucleotides at the 3 'end, and between the 2 nd and 3 rd nucleotides at the 3' end being phosphorothioate linkages;

alternatively, the 2 '-fluoro modified nucleotides are located at positions 7, 9, 10 and 11 of the sense strand in the 5' to 3 'direction, the remaining positions being 2' -methoxy modified nucleotides, between the 1 st and 2 nd nucleotides at the 5 'end, between the 2 nd and 3 rd nucleotides at the 5' end, between the 1 st and 2 nd nucleotides at the 3 'end, and between the 2 nd and 3 rd nucleotides at the 3' end being phosphorothioate linkages; according to the 5 'to 3' direction, 2 '-fluoro modified nucleotides are positioned at positions 2, 6, 8, 9, 14 and 16 of the antisense strand, the rest positions are 2' -methoxy modified nucleotides, the 1 st nucleotide and the 2 nd nucleotide of the 5 'end, the 2 nd nucleotide and the 3 rd nucleotide of the 5' end, the 1 st nucleotide and the 2 nd nucleotide of the 3 'end and the 2 nd nucleotide and the 3 rd nucleotide of the 3' end are connected by phosphorothioate groups;

Alternatively, the 2 '-fluoro modified nucleotides are located at positions 7, 9, 10 and 11 of the sense strand in the 5' to 3 'direction, the remaining positions being 2' -methoxy modified nucleotides, between the 1 st and 2 nd nucleotides at the 5 'end, between the 2 nd and 3 rd nucleotides at the 5' end, between the 1 st and 2 nd nucleotides at the 3 'end, and between the 2 nd and 3 rd nucleotides at the 3' end being phosphorothioate linkages; according to the 5 'to 3' direction, 2 '-fluoro modified nucleotides are located at positions 2, 6, 14 and 16 of the antisense strand, GNA modified nucleotides are located at position 7 of the antisense strand, the rest positions are 2' -methoxy modified nucleotides, between 1 st and 2 nd nucleotides at the 5 'end, between 2 nd and 3 rd nucleotides at the 5' end, between 1 st and 2 nd nucleotides at the 3 'end, and phosphorothioate linkage between 2 nd and 3 rd nucleotides at the 3' end;

alternatively, the 2 '-fluoro modified nucleotides are located at positions 7, 9, 10 and 11 of the sense strand in the 5' to 3 'direction, the remaining positions being 2' -methoxy modified nucleotides, between the 1 st and 2 nd nucleotides at the 5 'end, between the 2 nd and 3 rd nucleotides at the 5' end, between the 1 st and 2 nd nucleotides at the 3 'end, and between the 2 nd and 3 rd nucleotides at the 3' end being phosphorothioate linkages; according to the 5 'to 3' direction, 2 '-fluoro modified nucleotides are positioned at positions 2, 14 and 16 of the antisense strand, GNA modified nucleotides are positioned at position 6 of the antisense strand, the rest positions are 2' -methoxy modified nucleotides, the 1 st nucleotide and the 2 nd nucleotide of the 5 'terminal, the 2 nd nucleotide and the 3 rd nucleotide of the 5' terminal, the 1 st nucleotide and the 2 nd nucleotide of the 3 'terminal, and phosphorothioate group connection is formed between the 2 nd nucleotide and the 3 rd nucleotide of the 3' terminal;

Alternatively, the 2 '-fluoro modified nucleotides are located at positions 9, 10 and 11 of the sense strand in the 5' to 3 'direction, the remaining positions being 2' -methoxy modified nucleotides, between the 1 st and 2 nd nucleotides at the 5 'end, between the 2 nd and 3 rd nucleotides at the 5' end, between the 1 st and 2 nd nucleotides at the 3 'end, and between the 2 nd and 3 rd nucleotides at the 3' end being phosphorothioate linkages; according to the 5 'to 3' direction, the 2 '-fluoro modified nucleotide is positioned at the 14 th position of the antisense strand, the deoxyribonucleotide is positioned at the 2 nd, 5, 7 and 12 th positions of the antisense strand, the rest positions are 2' -methoxy modified nucleotides, the 1 st nucleotide and the 2 nd nucleotide of the 5 'terminal, the 2 nd nucleotide and the 3 rd nucleotide of the 5' terminal, the 1 st nucleotide and the 2 nd nucleotide of the 3 'terminal, and the 2 nd nucleotide and the 3 rd nucleotide of the 3' terminal are connected by phosphorothioate groups.

18. The siRNA of claim 1, selected from the group consisting of the sirnas of table 1; preferably, the siRNA is selected from the group consisting of N-ER-FY0140024, N-ER-FY0140024M1, N-ER-FY0140024MD2, N-ER-FY0140028, N-ER-FY0140028M1, N-ER-FY0140028MD2, N-ER-FY0140040, N-ER-FY0140040M1, N-ER-FY0140040MD2, N-ER-FY0140045,

N-ER-FY0140045M1、N-ER-FY0140045MD2、N-ER-FY0140032、N-ER-FY014082、N-ER-FY0140024D2、N-ER-FY0140024M2、N-ER-FY0140024M2D2、N-ER-FY0140024M3、N-ER-FY0140024M4、N-ER-FY0140024M5、

N-ER-FY0140024M6、N-ER-FY0140028D2、N-ER-FY0140028M2、

N-ER-FY0140028M2D2、N-ER-FY0140028M3、N-ER-FY0140028M4、N-ER-FY0140028M5、N-ER-FY0140040D2、N-ER-FY0140040M2、

N-ER-FY0140040M2D2、N-ER-FY0140040M3、N-ER-FY0140040M4、N-ER-FY0140040M5、N-ER-FY0140045D2、N-ER-FY0140045M2、

N-ER-FY0140045M2D2、N-ER-FY0140045M3、N-ER-FY0140045M4、N-ER-FY0140045M5、N-ER-FY0140045M6、N-ER-FY0140032M2、

N-ER-FY0140032M3、N-ER-FY0140032M4、N-ER-FY0140032M5、

N-ER-FY014082M2、N-ER-FY014082M3、N-ER-FY014082M4、N-ER-FY014082M5、N-ER-FY014082M2D2。

19. An siRNA conjugate comprising the siRNA of any one of claims 1-18 and a conjugate group conjugated to the siRNA.

20. The siRNA conjugate of claim 19, wherein the conjugate group comprises a pharmaceutically acceptable targeting group and a linker, and the siRNA, the linker and the targeting group are sequentially covalently or non-covalently linked;

or,

21. The siRNA conjugate of claim 20, wherein the conjugate group is L96 of the formula:

22. the siRNA conjugate of any one of claims 19-21, wherein the siRNA conjugate is an siRNA conjugate selected from table 2.

23. A pharmaceutical composition comprising the siRNA of any one of claims 1-18, or the siRNA conjugate of any one of claims 19-22, and a pharmaceutically acceptable carrier.

24. A kit comprising the siRNA of any one of claims 1-18, or the siRNA conjugate of any one of claims 19-22, or the pharmaceutical composition of claim 23.

25. Use of the siRNA of any one of claims 1-18, or the siRNA conjugate of any one of claims 19-22, or the pharmaceutical composition of claim 23, for the preparation of a medicament for inhibiting AGT gene expression.

26. Use of the siRNA of any one of claims 1-18, or the siRNA conjugate of any one of claims 19-22, or the pharmaceutical composition of claim 23, for the manufacture of a medicament for the prevention and/or treatment of diseases associated with the overexpression of AGT genes.

27. The use of claim 26, wherein the disease is hypertension, borderline hypertension, primary hypertension, secondary hypertension, isolated systolic or diastolic hypertension, pregnancy-related hypertension, diabetic hypertension, refractory hypertension, paroxysmal hypertension, renal vascular hypertension, hypertension with reduced ejection fraction, gordbat hypertension, low plasma renin activity or plasma renin concentration-related hypertension, ocular hypertension, glaucoma, pulmonary arterial hypertension, portal hypertension, systemic venous hypertension, systolic hypertension, unstable hypertension; hypertensive heart disease, hypertensive kidney disease, atherosclerosis, arteriosclerosis, vascular lesions, diabetic nephropathy, preeclampsia, diabetic retinopathy, chronic heart failure, cardiomyopathy, diabetic cardiomyopathy, glomerulosclerosis, aortic stenosis, aortic aneurysm, ventricular fibrosis, heart failure, myocardial infarction, angina pectoris, stroke, renal disease, renal failure, systemic sclerosis, intrauterine growth retardation (IUGR), fetal growth restriction, obesity, hepatic steatosis/fatty liver, nonalcoholic steatohepatitis (NASH), nonalcoholic fatty liver disease (NAFLD); glucose intolerance, type 2 diabetes (non-insulin dependent diabetes mellitus) and metabolic syndrome.

28. A method of inhibiting AGT gene expression comprising contacting or administering a therapeutically effective amount of the siRNA of any one of claims 1-18, or the siRNA conjugate of any one of claims 19-22, or the pharmaceutical composition of claim 23, with an AGT expressing cell or to a subject in need thereof.

29. A method of treating and/or preventing a disease associated with overexpression of AGT genes, comprising administering a therapeutically effective amount of the siRNA of any one of claims 1-18, or the siRNA conjugate of any one of claims 19-22, or the pharmaceutical composition of claim 23 to a subject in need thereof.