CN118339294A

CN118339294A - DsRNA, preparation method and application thereof

Info

Publication number: CN118339294A
Application number: CN202280080991.1A
Authority: CN
Inventors: 张瑱; 邓永岩; 李云飞; 林晓燕; 侯哲; 张建羽; 耿俊; 黄龙飞; 周雅琴; 吕珍珍
Original assignee: Shanghai Tuojie Biomedical Technology Co ltd
Current assignee: Shanghai Tuojie Biomedical Technology Co ltd
Priority date: 2021-12-16
Filing date: 2022-12-16
Publication date: 2024-07-12
Also published as: WO2023109932A1; TW202334424A

Abstract

Provides dsRNA, a preparation method and application thereof. Also provided are pharmaceutical compositions, cells or kits comprising the dsrnas. The dsRNA can interfere with the expression of the APOC3 gene, and prevent and/or treat related diseases.

Description

DsRNA, preparation method and application thereof

The present disclosure claims priority to chinese patent application 202111542323.X, 12/16/2021, and to chinese patent application 202210059877.2, 19/2022, 01/20, the present disclosure incorporates the entirety of the above-mentioned chinese patent applications.

Technical Field

The present disclosure relates to a dsRNA that can be targeted for delivery into a cell, exerting the effects of RNA interference. The disclosure also relates to methods of making and using the dsrnas.

Technical Field

RNA interference (RNAi) is an effective way to silence gene expression. It is counted that, of the disease-associated proteins in humans, approximately more than 80% of proteins cannot be targeted by currently conventional small molecule drugs and biomacromolecule preparations, which are non-patentable proteins. By utilizing the RNA interference technology, proper siRNA can be designed according to mRNA encoding the proteins, target mRNA is specifically targeted and degraded, and thus, the generation of related proteins is inhibited. Therefore, siRNA has very important drug development prospect. However, to achieve the therapeutic objective RNA interference effect in vivo, it is necessary to deliver siRNA molecules to specific cells in vivo.

The targeting ligand is adopted to conjugate siRNA, and the targeting ligand and the receptor molecular structure on the surface of the cell membrane are utilized, so that endocytosis enters the cell, and the targeting ligand is an effective drug delivery mode. For example, asialoglycoprotein receptor (ASGPR) is a receptor specifically expressed by liver cells, and has the characteristics of high abundance on the surface of liver cells and rapid intracellular and extracellular conversion. Mono-and polysaccharide molecules such as galactose, galactosamine, N-acetylgalactosamine and the like have high affinity for ASGPR. Literature reports (10.16476/j. Pibb.2015.0028) that the use of a cluster of galactosamine molecules (GalNAc) can effectively deliver RNA to hepatocytes, and that GalNAc molecules designed as a trivalent or tetravalent cluster of molecules can significantly enhance the ability of monovalent or divalent GalNAc molecules to target hepatocytes.

Different molecular cluster structures, and different modes of attachment to RNA, can significantly affect siRNA activity in vivo, with higher activity meaning better therapeutic effect, or lower doses, which also means lower toxic response with equivalent efficacy. Therefore, the reasonable design of the covalent connection mode of the targeting ligand and the siRNA is of great importance.

APOC3 is synthesized mainly in the liver and plays an important role in the production, metabolism and clearance from plasma of triglyceride-rich lipoproteins. Expression of APOC3 in the liver may promote secretion of Very Low Density Lipoprotein (VLDL) rich in triglycerides. In addition, APOC3 can further increase serum triglyceride levels by inhibiting the catabolism of triglyceride-rich lipoproteins by inhibiting the activity of lipoprotein lipase and liver lipase. Furthermore, APOC3 can also inhibit liver clearance of triglyceride-rich lipoproteins and their residual particles by interfering with the binding of triglyceride-rich lipoproteins to liver receptors.

Elevated ApoC3 levels are associated with elevated triglyceride levels and diseases such as cardiovascular disease, metabolic syndrome, obesity and diabetes. In recent years APOC3 has become a promising target for the treatment of diseases associated with hypertriglyceridemia. Elevated serum triglyceride levels are identified as an independent risk factor for cardiovascular disease and as a contributor to the progression of atherosclerosis. Individuals with severe hypertriglyceridemia also have a high risk of developing recurrent pancreatitis.

Disclosure of Invention

In a first aspect, the present disclosure provides a double-stranded ribonucleic acid (dsRNA) comprising an siRNA comprising a sense strand and an antisense strand, and one or more ligands conjugated thereto, said antisense strand comprising a chemical modification represented by formula (I), a tautomer thereof, or a pharmaceutically acceptable salt thereof, at least one nucleotide position from position 2 to position 8 of the 5' end thereof:

Wherein: y is selected from O, NH and S;

Each X is independently selected from CR ₄(R ₄')、S、NR ₅ and NH-CO, wherein R ₄、R ₄'、R ₅ is each independently H or C ₁-C ₆ alkyl;

j ₂ is H or C ₁-C ₆ alkyl;

n=0, 1 or 2; m=0, 1 or 2; s=0 or 1;

r ₃ is selected from H, OH, halogen, NH ₂、C ₁-C ₆ alkyl, C ₁-C ₆ alkoxy, C ₂-C ₆ alkenyl, C ₂-C ₆ alkynyl, S-CH ₃、NCH ₃(CH ₃)、OCH ₂CH ₂OCH ₃, -O-alkylamino, and (CH ₂) _pR ₆; wherein R ₆ is selected from OH, halogen, methoxy, ethoxy, N ₃、C ₂-C ₆ alkenyl, and C ₂-C ₆ alkynyl, p=1, 2, or 3;

q ₁ is Q ₂ is R ₂; or Q ₁ is R ₂,Q ₂ is

Wherein:

R ₁ is selected from H, C ₁-C ₆ alkyl, C ₁-C ₆ alkoxy, C ₂-C ₆ alkenyl, C ₂-C ₆ alkynyl and (CH ₂) _qR ₇; wherein R ₇ is selected from OH, halogen, methoxy, ethoxy, N ₃、C ₂-C ₆ alkenyl and C ₂-C ₆ alkynyl, q=1, 2 or 3;

J ₁ is H or C ₁-C ₆ alkyl;

R ₂ is selected from H, OH, halogen, NH ₂、C ₁-C ₆ alkyl, C ₁-C ₆ alkoxy, C ₂-C ₆ alkenyl, C ₂-C ₆ alkynyl, S-CH ₃、NCH ₃(CH ₃)、OCH ₂CH ₂OCH ₃, -O-alkylamino, and (CH ₂) _rR ₈; wherein R ₈ is selected from OH, halogen, methoxy, ethoxy, N ₃、C ₂-C ₆ alkenyl, and C ₂-C ₆ alkynyl, R = 1,2, or 3;

Optionally, R ₁ and R ₂ are directly linked to form a ring;

B is a base;

The chemical modification shown in the formula (I), the tautomer or the pharmaceutically acceptable salt modification thereof is not

The ligand is a compound shown as a formula (II) or pharmaceutically acceptable salt thereof,

Wherein L ₁ is a C ₁-C ₃₀ alkyl chain, or a C ₁-C ₃₀ alkyl chain comprising a break with one or more oxygen, sulfur, nitrogen atoms, or c=o;

R ₁₁ and R ₁₂ are independently a bond, NR ₁₆, c=o or-OC (=o) -;

q ₃ is

Is a single bond or a double bond, and whenR ₁₃ is independently CR ₁₇R ₁₈、NR ₁₆, O or S when a single bond is presentWhen a double bond, R ₁₃ is independently CR ₁₉ or N;

R ₁₄ is independently CR ₁₉ or N;

Ring a is cycloalkyl, heterocycloalkyl, aryl, or heteroaryl, with or without ring a present, and R ₁₅ is independently CR ₁₉ or N when ring a is not present, and R ₁₅ is independently CR ₁₇R ₁₈、NR ₁₆ or O when ring a is not present;

R ₁₆ and R ₁₉ are independently hydrogen, deuterium, alkyl, alkoxy, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, SR ', S (=o) R', S (=o) ₂R'、S(＝O) ₂ NR '(R "), C (=o) R', C (=o) OR 'OR C (=o) NR' (R"), said alkyl, alkoxy, cycloalkyl, heterocycloalkyl, aryl OR heteroaryl being optionally substituted with one OR more groups selected from halogen, hydroxy, oxo, nitro, cyano, C _1-6 alkyl, C _1-6 alkoxy, C _3-7 cycloalkyl, 3-12 membered heterocycloalkyl, 6-12 membered aryl, 5-12 membered heteroaryl, SR ', S (=o) R', S (=o) ₂R'、S(＝O) ₂ NR '(R "), C (=o) R', C (=o) OR 'and C (=o) NR' (R").

R ₁₇ and R ₁₈ are independently hydrogen, deuterium, alkyl, alkoxy, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, SR ', S (=o) R', S (=o) ₂R'、S(＝O) ₂ NR '(R "), C (=o) R', C (=o) OR 'OR C (=o) NR' (R"), said alkyl, alkoxy, cycloalkyl, heterocycloalkyl, aryl OR heteroaryl being optionally substituted with one OR more groups selected from halogen, hydroxy, oxo, nitro, cyano, C _1-6 alkyl, C _1-6 alkoxy, C _3-7 cycloalkyl, 3-12 membered heterocycloalkyl, 6-12 membered aryl, 5-12 membered heteroaryl, SR ', S (=o) R', S (=o) ₂R'、S(＝O) ₂ NR '(R "), C (=o) R', C (=o) OR 'and C (=o) NR' (R").

R' and R "are independently hydrogen, deuterium, hydroxy, alkyl, alkoxy, cycloalkyl, heterocycloalkyl, aryl or heteroaryl, said alkyl, alkoxy, cycloalkyl, heterocycloalkyl, aryl or heteroaryl optionally substituted with one or more substituents selected from halogen, hydroxy, oxo, nitro and cyano;

m1, n1, p1 and q1 are independently 0, 1, 2,3 or 4;

B1 is

R _b1、R _b2、R _b3、R _b4、R _b5、R _b6 and R _b7 are independently-C (=o) -, -NHC (=o) -, -C (=o) O-, -C (=o) - (CH ₂) _z8 -O-, or-NHC (=o) - (CH ₂) _z9 -O-;

z1, z2, z3, z4, z5, z6, z7, z8 and z9 are independently integers from 0 to 10;

L ₂ is a C ₁-C ₃₀ alkyl chain, or a C ₁-C ₃₀ alkyl chain comprising a break with one or more oxygen, sulfur, nitrogen atoms, or c=o;

r1 is an integer of 1 to 10.

In some embodiments (certain groups or features are defined below, undefined groups or features are described in any of the other embodiments, hereinafter referred to as "in some embodiments"), when X is nh—co, R ₁ is not H.

In some embodiments, the chemical modification shown in formula (I), a tautomer thereof, or a pharmaceutically acceptable salt thereof is replaced with a 2' -methoxy modification.

In some embodiments, at least one nucleotide of the antisense strand at positions 2-8 of its 5 'end is a 2' -methoxy modified nucleotide.

In some embodiments, the chemical modification of formula (I) is selected from the group consisting of chemical modifications of formula (I-1):

Wherein: y is selected from O, NH and S;

Each J ₁、J ₂ is independently H or C ₁-C ₆ alkyl;

n=0, 1 or 2; m=0, 1 or 2; s=0 or 1;

Optionally, R ₁ and R ₂ are directly linked to form a ring;

B is as defined in formula (I).

In some embodiments, B is selected from the group consisting of a purine base, a pyrimidine base, an indole, a 5-nitroindole, and a 3-nitropyrrole.

In some embodiments, B is selected from adenine, guanine, isoguanine, hypoxanthine, xanthine, C2 modified purine, N8 modified purine, 2, 6-diaminopurine, 6-dimethylaminopurine, 2-aminopurine, N6-alkyladenine, O6-alkylguanine, 7-deazapurine, cytosine, 5-methylcytosine, isocytosine, pseudocytosine, uracil, pseudouracil, 2-thiouridine, 4-thiouridine, C5 modified pyrimidine, thymine, indole, 5-nitroindole, and 3-nitropyrrole.

In some embodiments, B is selected from adenine, guanine, 2, 6-diaminopurine, 6-dimethylaminopurine, 2-aminopurine, cytosine, uracil, thymine, indole, 5-nitroindole, and 3-nitropyrrole.

In some embodiments, B is the same base as when the nucleotide at that position of the antisense strand is not modified.

In some embodiments, the chemical modification of formula (I) is selected from

Wherein Y is selected from O, NH and S;

n=0, 1 or 2; m=0, 1 or 2; s=0 or 1;

Each J ₁、J ₂ is independently H or C ₁-C ₆ alkyl;

R ₂ is selected from H, C ₁-C ₆ alkyl, C ₁-C ₆ alkoxy, S-CH ₃、NCH ₃(CH ₃)、OCH ₂CH ₂OCH ₃, -O-alkylamino, and (CH ₂) _rR ₈; wherein R ₈ is selected from OH, halogen, methoxy, ethoxy, N ₃、C ₂-C ₆ alkenyl, and C ₂-C ₆ alkynyl; R = 1,2, or 3;

Optionally, R ₁ and R ₂ are directly linked to form a ring;

B is as defined in formula (I).

In some embodiments, each X is independently selected from CR ₄(R ₄')、S、NR ₅ and NH-CO, wherein R ₄、R ₄'、R ₅ is each independently H or C ₁-C ₃ alkyl;

n=0, 1 or 2; m=0, 1 or 2; s=0 or 1;

Each J ₁、J ₂ is independently H or C ₁-C ₃ alkyl;

r ₃ is selected from H, OH, halogen, NH ₂、C ₁-C ₃ alkyl, C ₁-C ₃ alkoxy, C ₂-C ₄ alkenyl, C ₂-C ₄ alkynyl, S-CH ₃、NCH ₃(CH ₃)、OCH ₂CH ₂OCH ₃, -O-alkylamino, and (CH ₂) _pR ₆; wherein R ₆ is selected from OH, halogen, methoxy, ethoxy, N ₃、C ₂-C ₆ alkenyl, and C ₂-C ₆ alkynyl, p=1, 2, or 3;

R ₁ is selected from H, C ₁-C ₃ alkyl, C ₁-C ₃ alkoxy, C ₂-C ₄ alkenyl, C ₂-C ₄ alkynyl and (CH ₂) _qR ₇; wherein R ₇ is selected from OH, halogen, methoxy, ethoxy, N ₃、C ₂-C ₄ alkenyl and C ₂-C ₄ alkynyl, q=1, 2 or 3;

r ₂ is selected from H, OH, halogen, NH ₂、C ₁-C ₃ alkyl, C ₁-C ₃ alkoxy, C ₂-C ₄ alkenyl, C ₂-C ₄ alkynyl, S-CH ₃、NCH ₃(CH ₃)、OCH ₂CH ₂OCH ₃, -O-alkylamino, and (CH ₂) _rR ₈; wherein R ₈ is selected from OH, halogen, methoxy, ethoxy, N ₃、C ₂-C ₄ alkenyl, and C ₂-C ₄ alkynyl, R = 1,2, or 3;

Optionally, R ₁ and R ₂ are directly linked to form a ring;

B is as defined in formula (I).

In some embodiments, each X is independently selected from CR ₄(R ₄')、S、NR ₅ and NH-CO, wherein R ₄、R ₄'、R ₅ is each independently H, methyl, ethyl, n-propyl, or isopropyl;

n=0, 1 or 2; m=0, 1 or 2; s=0 or 1;

Each J ₁、J ₂ is independently H or methyl;

R ₃ is selected from H, OH, F, cl, NH ₂, methyl, ethyl, N-propyl, isopropyl, methoxy, ethoxy, N-propoxy, isopropoxy, vinyl, allyl, ethynyl, propargyl, S-CH ₃、NCH ₃(CH ₃)、OCH ₂CH ₂OCH ₃, -O-methylamino, -O-ethylamino, and (CH ₂) _pR ₆; wherein R ₆ is selected from OH, F, cl, methoxy, ethoxy, N ₃, vinyl, allyl, ethynyl, and propargyl, p = 1 or 2;

R ₁ is selected from H, methyl, ethyl, N-propyl, isopropyl, methoxy, ethoxy, N-propoxy, isopropoxy, vinyl, allyl, ethynyl, propargyl, and (CH ₂) _qR ₇; wherein R ₇ is selected from OH, F, cl, methoxy, ethoxy, N ₃, vinyl, allyl, ethynyl, and propargyl, q=1 or 2;

R ₂ is selected from H, OH, F, cl, NH ₂, methyl, ethyl, N-propyl, isopropyl, methoxy, ethoxy, N-propoxy, isopropoxy, vinyl, allyl, ethynyl, propargyl, S-CH ₃、NCH ₃(CH ₃)、OCH ₂CH ₂OCH ₃, -O-methylamino, -O-ethylamino, and (CH ₂) _rR ₈; wherein R ₈ is selected from OH, F, cl, methoxy, ethoxy, N ₃, vinyl, allyl, ethynyl, and propargyl, R = 1 or 2;

Optionally, R ₁ and R ₂ are directly linked to form a ring;

B is as defined in formula (I).

n=0, 1 or 2; m=0, 1 or 2; s=0 or 1;

Each J ₁、J ₂ is independently H or methyl;

Optionally, R ₁ and R ₂ are directly linked to form a ring;

B is as defined in formula (I).

In some embodiments, Y is O or NH; each X is independently selected from NH-CO, CH ₂, and NH;

n=0 or 1; m=0 or 1; s=0 or 1;

Each J ₁、J ₂ is independently H;

R ₁ is selected from H, methyl and CH ₂ OH;

R ₂ is selected from H, OH, NH ₂, methyl and CH ₂ OH;

R ₃ is selected from H, OH, NH ₂, methyl and CH ₂ OH;

Optionally, R ₁ and R ₂ are directly linked to form a ring;

B is as defined in formula (I).

n=0 or 1; m=0 or 1; s=0 or 1;

Each J ₁、J ₂ is independently H;

R ₁ is selected from H, methyl and CH ₂ OH;

r ₂ is selected from H, methyl and CH ₂ OH;

R ₃ is selected from H, OH, NH ₂, methyl and CH ₂ OH;

Optionally, R ₁ and R ₂ are directly linked to form a ring;

B is as defined in formula (I).

In some embodiments, Y is O or NH;

each X is independently selected from CR ₄(R ₄')、NR ₅ and NH-CO, R ₄、R ₄'、R ₅ is each independently H or C ₁-C ₆ alkyl;

j ₂ is H or C ₁-C ₆ alkyl;

n=0 or 1; m=0 or 1; s=0 or 1;

R ₃ is selected from H, OH, NH ₂、C ₁-C ₆ alkyl, C ₁-C ₆ alkoxy and (CH ₂) _pR ₆;R ₆ is selected from OH, methoxy and ethoxy, p=1, 2 or 3;

q ₁ is Q ₂ is R ₂; or Q ₁ is R ₂,Q ₂ is

R ₁ is selected from H, OH, C ₁-C ₆ alkyl, C ₁-C ₆ alkoxy and (CH ₂) _qR ₇;R ₇ is selected from OH, methoxy and ethoxy, q=1, 2 or 3;

J ₁ is H or C ₁-C ₆ alkyl;

R ₂ is selected from H, OH, C ₁-C ₆ alkyl, C ₁-C ₆ alkoxy and (CH ₂) _rR ₈;R ₈ is selected from OH, methoxy and ethoxy, r=1, 2 or 3;

Optionally, R ₁ and R ₂ are directly linked to form a 3-6 membered ring;

B is a base;

In some embodiments, X is independently selected from CR ₄(R ₄') and NH-CO.

In some embodiments, X is independently selected from CR ₄(R ₄').

In some embodiments, R ₃ is selected from H, C ₁-C ₆ alkyl and (CH ₂) _pR ₆).

In some embodiments, R ₃ is selected from H and C ₁-C ₆ alkyl.

In some embodiments, R ₁ is selected from H, C ₁-C ₆ alkyl and (CH ₂) _qR ₇).

In some embodiments, R ₁ is selected from H and C ₁-C ₆ alkyl.

In some embodiments, R ₂ is selected from H, OH, C ₁-C ₆ alkyl, and (CH ₂) _rR ₈).

In some embodiments, R ₂ is selected from H, C ₁-C ₆ alkyl and (CH ₂) _rR ₈).

In some embodiments, Y is O;

Each X is independently selected from CR ₄(R ₄ ') and NH-CO, R ₄ and R ₄' are each independently H or C ₁-C ₆ alkyl;

j ₂ is H or C ₁-C ₆ alkyl;

R ₃ is selected from H, C ₁-C ₆ alkyl and (CH ₂) _pR ₆;R ₆ is selected from OH, p=1, 2 or 3;

q ₁ is Q ₂ is R ₂; or Q ₁ is R ₂,Q ₂ is

R ₁ is selected from H, C ₁-C ₆ alkyl and (CH ₂) _qR ₇;R ₇ is selected from OH, q=1, 2 or 3;

J ₁ is H or C ₁-C ₆ alkyl;

R ₂ is selected from H, OH, C ₁-C ₆ alkyl and (CH ₂) _rR ₈;R ₈ is selected from OH, r=1, 2 or 3;

Optionally, R ₁ and R ₂ are directly linked to form a 5-6 membered ring;

B is a base.

In some embodiments, Y is O;

Each X is independently selected from CR ₄(R ₄'),R ₄ and R ₄' is each independently H or C ₁-C ₆ alkyl;

J ₂ is H;

R ₃ is selected from H and C ₁-C ₆ alkyl;

q ₁ is Q ₂ is R ₂; or Q ₁ is R ₂,Q ₂ is

R ₁ is selected from H and C ₁-C ₆ alkyl;

J ₁ is H or C ₁-C ₆ alkyl;

r ₂ is selected from H, C ₁-C ₆ alkyl and (CH ₂) _rR ₈;R ₈ is selected from OH, r=1, 2 or 3;

Optionally, R ₁ and R ₂ are directly linked to form a 5-6 membered ring;

B is a base.

In some embodiments, Y is O.

In some embodiments, X is independently selected from CR ₄(R ₄')、NR ₅ and NH-CO, and R ₄、R ₄'、R ₅ is each independently H, methyl, ethyl, n-propyl, or isopropyl. In some embodiments, X is independently selected from NH-CO, CH ₂, and NH. In some embodiments, X is independently selected from NH-CO and CH ₂. In some embodiments, X is CH ₂.

In some embodiments, J ₂ is H or methyl. In some embodiments, J ₂ is H.

In some embodiments, R ₃ is selected from H, OH, NH ₂, methyl, ethyl, n-propyl, isopropyl, methoxy, ethoxy, n-propoxy, isopropoxy, and (CH ₂) _pR ₆,R ₆ is selected from OH, methoxy, and ethoxy, p=1 or 2. In some embodiments, R ₃ is selected from H, methyl, ethyl, n-propyl, isopropyl, and (CH ₂) _pR ₆,R ₆ is selected from OH, p=1 or 2. In some embodiments, R ₃ is selected from H and methyl.

In some embodiments, R ₁ is selected from H, OH, methyl, ethyl, n-propyl, isopropyl, methoxy, ethoxy, n-propoxy, isopropoxy, and (CH ₂) _qR ₇,R ₇ is selected from OH, q=1 or 2. In some embodiments, R ₁ is selected from H, methyl, ethyl, n-propyl, isopropyl, and (CH ₂) _qR ₇,R ₇ is selected from OH, q=1 or 2. In some embodiments, R ₁ is selected from H and methyl.

In some embodiments, R ₂ is selected from H, OH, methyl, ethyl, n-propyl, isopropyl, methoxy, ethoxy, n-propoxy, isopropoxy, and (CH ₂) _rR ₈,R ₈ is selected from OH, r=1 or 2. In some embodiments, R ₂ is selected from H, OH, methyl, ethyl, n-propyl, isopropyl, and (CH ₂) _rR ₈,R ₈ is selected from OH, r=1 or 2. In some embodiments, R ₂ is selected from H, methyl, and CH ₂ OH.

In some embodiments, R ₁ and R ₂ are directly linked to form a 5-6 membered ring. In some embodiments, R ₁ and R ₂ are directly linked to form a 3-6 membered cycloalkyl. In some embodiments, R ₁ and R ₂ are directly linked to form cyclopentyl or cyclohexyl.

In some embodiments, the chemical modification of formula (I) is selected from any one of the following structures:

Wherein: b is selected from purine bases, pyrimidine bases, indoles, 5-nitroindoles and 3-nitropyrroles.

In some embodiments, the nucleotide comprising the chemical modification of formula (I), a tautomer thereof, or a pharmaceutically acceptable salt thereof is selected from the group consisting of nucleotides comprising the chemical modification of formula (I'), a tautomer thereof, or a pharmaceutically acceptable salt thereof,

Wherein: y is selected from O, NH and S;

j ₂ is H or C ₁-C ₆ alkyl;

n=0, 1 or 2; m=0, 1 or 2; s=0 or 1;

q _1' is Q _2' is R ₂; or Q _1' is R ₂,Q _2' is

Wherein:

J ₁ is H or C ₁-C ₆ alkyl;

Optionally, R ₁ and R ₂ are directly linked to form a ring;

B is a base;

M is O or S;

The chemical modification represented by the formula (I'), a tautomer thereof or a pharmaceutically acceptable salt thereof is not

In some embodiments, when X is nh—co, R ₁ is not H.

In some embodiments, the chemical modification of formula (I ') is selected from the group consisting of chemical modifications of formula (I' -1):

Wherein: y is selected from O, NH and S;

Each J ₁、J ₂ is independently H or C ₁-C ₆ alkyl;

n=0, 1 or 2; m=0, 1 or 2; s=0 or 1;

M is O or S;

Optionally, R ₁ and R ₂ are directly linked to form a ring;

B is as defined in formula (I').

In some embodiments, the chemical modification of formula (I ') is selected from the group consisting of chemical modifications of formula (I' -2):

wherein Y is selected from O, NH and S;

n=0, 1 or 2; m=0, 1 or 2; s=0 or 1;

Each J ₁、J ₂ is independently H or C ₁-C ₆ alkyl;

Optionally, R ₁ and R ₂ are directly linked to form a ring;

M is O or S;

B is as defined in formula (I').

n=0, 1 or 2; m=0, 1 or 2; s=0 or 1;

Each J ₁、J ₂ is independently H or C ₁-C ₃ alkyl;

Optionally, R ₁ and R ₂ are directly linked to form a ring;

B is as defined in formula (I').

n=0, 1 or 2; m=0, 1 or 2; s=0 or 1;

Each J ₁、J ₂ is independently H or methyl;

Optionally, R ₁ and R ₂ are directly linked to form a ring;

B is as defined in formula (I').

In some embodiments, B is selected from adenine, guanine, isoguanine, hypoxanthine, xanthine, C2 modified purine, N8 modified purine, 2, 6-diaminopurine, 6-dimethylaminopurine, 2-aminopurinin, N6-alkyladenine, O6-alkylguanine, 7-deazapurine, cytosine, 5-methylcytosine, isocytosine, pseudocytosine, uracil, pseudouracil, 2-thiouridine, 4-thiouridine, C5 modified pyrimidine, thymine, indole, 5-nitroindole, and 3-nitropyrrole.

n=0, 1 or 2; m=0, 1 or 2; s=0 or 1;

Each J ₁、J ₂ is independently H or methyl;

R ₁ is selected from H, methyl, ethyl, N-propyl, isopropyl, methoxy, ethoxy, N-propoxy, isopropoxy, vinyl, allyl, ethynyl, propargyl, and (CH ₂) _qR ₇; wherein R ₇ is selected from OH, F, cl, methoxy, ethoxy, N ₃, vinyl, allyl, ethynyl, and propargyl, q=1 or 2;0)

Optionally, R ₁ and R ₂ are directly linked to form a ring;

B is as defined in formula (I').

n=0 or 1; m=0 or 1; s=0 or 1;

Each J ₁、J ₂ is independently H;

R ₁ is selected from H, methyl and CH ₂ OH;

R ₂ is selected from H, OH, NH ₂, methyl and CH ₂ OH;

R ₃ is selected from H, OH, NH ₂, methyl and CH ₂ OH;

Optionally, R ₁ and R ₂ are directly linked to form a ring;

B is as defined in formula (I').

n=0 or 1; m=0 or 1; s=0 or 1;

Each J ₁、J ₂ is independently H;

R ₁ is selected from H, methyl and CH ₂ OH;

r ₂ is selected from H, methyl and CH ₂ OH;

R ₃ is selected from H, OH, NH ₂, methyl and CH ₂ OH;

Optionally, R ₁ and R ₂ are directly linked to form a ring;

B is as defined in formula (I').

In some embodiments, Y is O or NH;

j ₂ is H or C ₁-C ₆ alkyl;

n=0 or 1; m=0 or 1; s=0 or 1;

q _1' is Q _2' is R ₂; or Q _1' is R ₂,Q _2' is

J ₁ is H or C ₁-C ₆ alkyl;

Optionally, R ₁ and R ₂ are directly linked to form a 3-6 membered ring;

M is O or S;

B is a base;

In some embodiments, X is independently selected from CR ₄(R ₄') and NH-CO.

In some embodiments, X is independently selected from CR ₄(R ₄').

In some embodiments, R ₃ is selected from H and C ₁-C ₆ alkyl.

In some embodiments, R ₁ is selected from H and C ₁-C ₆ alkyl.

In some embodiments, Y is O;

j ₂ is H or C ₁-C ₆ alkyl;

q _1' is Q _2' is R ₂; or Q _1' is R ₂,Q _2' is

J ₁ is H or C ₁-C ₆ alkyl;

Optionally, R ₁ and R ₂ are directly linked to form a 5-6 membered ring;

M is O or S;

B is a base.

In some embodiments, Y is O;

J ₂ is H;

R ₃ is selected from H and C ₁-C ₆ alkyl;

q _1' is Q _2' is R ₂; or Q _1' is R ₂,Q _2' is

R ₁ is selected from H and C ₁-C ₆ alkyl;

J ₁ is H or C ₁-C ₆ alkyl;

Optionally, R ₁ and R ₂ are directly linked to form a 5-6 membered ring;

M is O or S;

B is a base.

In some embodiments, Y is O.

In some embodiments, J ₂ is H or methyl. In some embodiments, J ₂ is H.

In some embodiments, the chemical modification represented by formula (I') is selected from any one of the following structures:

wherein: m is O or S;

b is selected from purine bases, pyrimidine bases, indoles, 5-nitroindoles, and 3-nitropyrroles.

wherein: m is O or S;

And those in which adenine in their structure is replaced with guanine, cytosine, uracil or thymine.

In some embodiments, L ₁ is a C ₁-C ₃₀ alkyl chain, or a C ₁-C ₃₀ alkyl chain comprising a break with one or more oxygen, sulfur, nitrogen atoms, or c=o;

R ₁₁ and R ₁₂ are independently a bond, NR ₁₆ or c=o;

q ₃ is

R ₁₃ is CR ₁₇R ₁₈、NR ₁₆, O or S;

R ₁₄ is CR ₁₉;

R ₁₅ is independently CR ₁₇R ₁₈、NR ₁₆ or O;

R ₁₆ to R ₁₉ are independently hydrogen, deuterium or alkyl;

m1, p1 and q1 are independently 0, 1,2, 3 or 4;

B1 is

R _b5、R _b6 and R _b7 are independently-C (=o) -, -NHC (=o) -, -C (=o) O-, -C (=o) - (CH ₂) _z8 -O-, or-NHC (=o) - (CH ₂) _z9 -O-;

z5, z6, z7, z8 and z9 are independently integers from 0 to 10;

r1 is an integer of 1 to 10.

In some embodiments, L ₁ is- (CH ₂) _j11-C(＝O)-(CH ₂) _j12 -;

R ₁₁ and R ₁₂ are independently a bond, NR ₁₆ or c=o;

R ₁₆ is hydrogen or C _1-6 alkyl;

q ₃ is

R ₁₃ is CR ₁₇R ₁₈ or O;

R ₁₄ is CR ₁₉;

R ₁₅ is independently CR ₁₇R ₁₈ or O;

R ₁₇ to R ₁₉ are independently hydrogen or alkyl;

m1, p1 and q1 are independently 0 or 1;

B1 is

R _b5、R _b6 and R _b7 are independently-C (=O) - (CH ₂) _z8 -O-or-NHC (=O) - (CH ₂) _z9 -O-;

z8 and z9 are independently integers from 0 to 10;

L ₂ is- (CH ₂) _j15-(OCH ₂CH ₂) _1-4-(CH ₂) _j16) -or

J15 and j16 are independently integers from 0 to 4;

r1 is 3, 4, 5 or 6.

In some embodiments, L ₁ can be L ₃ or L ₃-R ₁₁₀-R ₁₁₁-L ₃, where L ₃ is independently a C ₁-C ₁₂ alkyl chain, - (CH ₂) _j11-C(＝O)-(CH ₂) _j12 -or -(CH ₂) _j13-(CH ₂CH ₂O) _1-4-(CH ₂) _j14-,R ₁₁₀) and R ₁₁₁ is independently a bond, -NR ₁₁₂ -, -C (=O) -or-OC (=O) -, R ₁₁₂ is hydrogen or C ₁-C ₁₂ alkyl, j11, j12, j13 and j14 are independently integers from 0 to 10, in some embodiments, j11, j12, j13 and j14 are independently integers from 0 to 2 or 4 to 10, in some embodiments, j11, j12, j13 and j14 are independently 0, 1, 2, 6, 7, 8, 9 or 10.

In some embodiments, L ₁ may be- (CH ₂) _j11-C(＝O)-(CH ₂) _j12 -, with j11 and j12 being as defined in any of the previous schemes.

In some embodiments, L ₁ may beThe definition of j12 is the same as any one of the previous schemes, wherein the a1 end is connected with B1, and the B1 end is connected with R ₁₁.

In some embodiments, L ₁ may be Wherein, the a1 end is connected with B ₁, and the B1 end is connected with R ₁₁.

In some embodiments, R ₁₁ can be a bond and R ₁₂ can be c=o.

In some embodiments, R ₁₁ can be a bond and R ₁₂ can be NR ₁₆,R ₁₆ as defined in any of the previous schemes.

In some embodiments, R ₁₁ can be a bond and R ₁₂ can be-OC (=o) -.

In some embodiments, R ₁₁ can be NR ₁₆ and R ₁₂ can be c=o, and R ₁₆ is as defined in any of the previous schemes.

In some embodiments, R ₁ can be NR ₁₆ and R ₁₂ can be-OC (=o) -, and R ₁₆ is as defined in any of the previous embodiments.

In some embodiments, R ₁₂ can be NR ₁₆ and R ₁₁ can be c=o, and R ₁₆ is as defined in any of the previous schemes.

In some embodiments, R ₁₂ can be NR ₁₆ and R ₁₁ can be-OC (=o) -, and R ₁₆ is as defined in any of the previous embodiments.

In some embodiments, R ₁₁ can be NH and R ₁₂ can be c=o.

In some embodiments, R ₁₂ can be NH and R ₁₁ can be c=o.

In some embodiments, R ₁₆ can be hydrogen or C _1-6 alkyl.

In some embodiments, R ₁₆ can be hydrogen, methyl, ethyl, propyl, or isopropyl.

In some embodiments, R ₁₆ can be hydrogen.

In some embodiments, R ₁₇ and R ₁₈ may be hydrogen.

In some embodiments, R ₁₉ can be hydrogen.

In some embodiments, ring a, when present, may be a C _6-12 aryl group.

In some embodiments, ring a may be phenyl.

In some embodiments, m1 may be 0 or 1.

In some embodiments, m1 may be 3.

In some embodiments, n1 may be 0 or 1.

In some embodiments, p1 and q1 are independently 0 or 1.

In some embodiments, p1=1 and q1=1.

In some embodiments, p1=1 and q1=0.

In some embodiments, p1=0 and q1=1.

In some embodiments, p1=0 and q1=0.

In some embodiments, z1, z2, z3, z4, z5, z6, z7, z8, and z9 may independently be integers from 0 to 4. In some embodiments, z1, z2, z3, z4, z5, z6, z7, z8, and z9 can independently be 0, 1, or 2.

In some embodiments, B ₁ may beR _b1、R _b2、R _b3 and R _b4 are independently-C (=O) -or-NHC (=O) -, the N atom is attached to L ₁, and z1, z2, z3 and z4 are as defined in any of the previous schemes.

In some embodiments, B ₁ may beR _b1、R _b2、R _b3 and R _b4 are independently-C (=O) -or-NHC (=O) -, the N atom is attached to L ₁, R _b1、R _b3 and R _b4 are the same, and z1, z2, z3 and z4 are as defined in any of the previous schemes.

In some embodiments, B ₁ may be

In some embodiments, B ₁ may beR _b5、R _b6 and R _b7 are independently-C (=O) - (CH ₂) _z8 -O-or-NHC (=O) - (CH ₂) _z9 -O-, the N atom is attached to L ₁ and z5, z6, z7, z8 and z9 are as defined in any of the previous schemes.

In some embodiments, B ₁ may beR _b5、R _b6 and R _b7 are independently-C (=O) - (CH ₂) _z8 -O-or-NHC (=O) - (CH ₂) _z9 -O-, the N atom is attached to L ₁, R _b5、R _b6 and R _b7 are the same, and z5, z6, z7, z8 and z9 are as defined in any of the previous schemes.

In some embodiments, B ₁ may be

In some embodiments, L ₂ can be L ₄ or L ₄-R ₁₃-R ₁₄-L ₄, wherein L ₄ is independently a C ₁-C ₁₂ alkyl chain or -(CH ₂) _j15-(OCH ₂CH ₂) _1-4-(CH ₂) _j16-,R ₁₁₃ and R ₁₁₄ are independently a bond, -NR ₁₁₅ -, -C (=o) -or-OC (=o) -, R ₁₁₅ is independently hydrogen or C ₁-C ₁₂ alkyl, and j15 and j16 are independently integers from 0 to 10. In some embodiments, j15 and j16 are independently integers from 0 to 6. In some embodiments, j15 and j16 are independently 0, 1,2,3, or 4.

In some embodiments, L ₂ may be- (CH ₂) _j15-(OCH ₂CH ₂) _1-4-(CH ₂) _j16 -, with j15 and j16 being as defined in any of the preceding schemes.

In some embodiments, L ₂ may beIn some embodiments, L ₂ may beWherein the left side is connected with an O atom, and the right side is connected with B ₁.

In some embodiments, L ₂ can be a C ₁-C ₁₂ alkyl chain.

In some embodiments, L ₂ may be

In some embodiments, L ₂ may beIn some embodiments, L ₂ may beIn some embodiments, L ₂ may beIn some embodiments, L ₂ may beWherein, the a3 end is connected with the O atom, and the B3 end is connected with the B ₁.

In some embodiments, L ₂ may beWherein, the a3 end is connected with the O atom, and the B3 end is connected with the B ₁.

In some embodiments, r1 may be 3, 4, 5, or 6. In some embodiments, r1 may be 3.

In some embodiments, Q ₃ may beIn some embodiments, Q ₃ may beWherein R ₁₃、R ₁₄、R ₁₅ and n1 are as defined in any one of the preceding schemes.

In some embodiments of the present invention, in some embodiments,Can beWherein R ₁₃、R ₁₄、R ₁₅, p1 and q1 are as defined in any one of the preceding schemes.

In some embodiments of the present invention, in some embodiments,Can be Wherein R ₁₃、R ₁₄、R ₁₅, p1 and q1 are as defined in any one of the preceding schemes.

In some embodiments of the present invention, in some embodiments,Can beIn some embodiments of the present invention, in some embodiments,Can beIn some embodiments of the present invention, in some embodiments,Can beThe definition of p1 and q1 is as in any one of the previous schemes.

In some embodiments of the present invention, in some embodiments,Can be In some embodiments of the present invention, in some embodiments,Can be

In some embodiments of the present invention, in some embodiments,Can be The definition of p1 and q1 is as in any one of the previous schemes.

In some embodiments of the present invention, in some embodiments,Can beWherein R ₁₃、R ₁₄, n1, p1 and q1 are as defined in any one of the preceding schemes.

In some embodiments of the present invention, in some embodiments,Can beIn some embodiments of the present invention, in some embodiments,Can beN1, p1 and q1 are as defined in any one of the preceding schemes.

In some embodiments of the present invention, in some embodiments,Can beN1, p1 and q1 are as defined in any one of the preceding schemes.

In some embodiments, the ligand may be any one of the following structures or a pharmaceutically acceptable salt thereof,

In some embodiments, the ligand may be of the structure or a pharmaceutically acceptable salt thereof,

In some embodiments, the chemical modification of formula (I) is B is selected from guanine, adenine, cytosine and uracil; and the ligand is any one of the following structures or pharmaceutically acceptable salts thereof,

In some embodiments, the chemical modification of formula (I) is B is selected from guanine, adenine, cytosine and uracil, and the ligand is any one of the following structures or pharmaceutically acceptable salts thereof,

In some embodiments, the chemical modification of formula (I) is B is selected from guanine, adenine, cytosine and uracil; and the ligand is of the following structure or pharmaceutically acceptable salt thereof,

In some embodiments, the N-acetyl-galactosamine moiety in the above ligand may be replaced with N-trifluoroacetyl galactosamine, N-propionyl galactosamine, N-butyryl galactosamine, or N-isobutyryl galactosamine.

In some embodiments, the siRNA and the ligand are covalently or non-covalently linked.

In some embodiments, the 3 'and/or 5' end of the sense strand may be conjugated to the ligand.

In some embodiments, the 3' end of the sense strand may be conjugated to the ligand.

In some embodiments, the ligand is linked to the siRNA terminus via a phosphate group or a phosphorothioate group.

In some embodiments, the ligand is linked to the siRNA terminal via a phosphodiester group or a phosphorothioate diester group.

In some embodiments, the ligand is linked to the siRNA end via a phosphodiester group.

In some embodiments, the ligand is indirectly linked to the siRNA end via a phosphate group or a phosphorothioate group.

In some embodiments, the ligand is directly linked to the siRNA end via a phosphate group or a phosphorothioate group.

In some embodiments, the ligand is directly linked to the 3' end of the sense strand of the siRNA through a phosphate group or a phosphorothioate group.

In some embodiments, the phosphate group is a phosphate monoester group or a phosphate diester group. In some embodiments, the phosphate group is a phosphodiester group.

In some embodiments, the phosphorothioate group is a phosphorothioate monoester group or a phosphorothioate diester group. In some embodiments, the phosphorothioate group is a phosphorothioate diester group.

In some embodiments, to facilitate entry of the siRNA into the cell, a lipophilic group such as cholesterol may be introduced at the end of the sense strand of the siRNA, including covalent bond with small interfering nucleic acids, such as end-introduced cholesterol, lipoproteins, vitamin E, etc., to facilitate interaction with mRNA within the cell through the cell membrane comprised of the lipid bilayer. Meanwhile, siRNA can also be modified by non-covalent bonds, such as through hydrophobic bonds or ionic bonds to phospholipid molecules, polypeptides, cationic polymers and the like, so as to increase stability and biological activity.

In some embodiments, a nucleotide comprising a chemical modification of formula (I), a tautomer thereof, or a pharmaceutically acceptable salt thereof is located at position 5, 6, or 7 of the 5' end of the antisense strand.

In some embodiments, a nucleotide comprising a chemical modification of formula (I), a tautomer thereof, or a pharmaceutically acceptable salt thereof is located at position 7 at the 5' end of the antisense strand.

In some embodiments, the chemical modification of formula (I), tautomer thereof, or pharmaceutically acceptable salt thereof modifies at position 5 of its 5' end, and B is selected from adenine, guanine, 2, 6-diaminopurine, 6-dimethylaminopurine, 2-aminopurine, cytosine, uracil, thymine, indole, 5-nitroindole, and 3-nitropyrrole.

In some embodiments, when the chemical modification of formula (I), a tautomer thereof, or a pharmaceutically acceptable salt thereof is modified at position 6 of its 5' end, B is selected from adenine, guanine, 2, 6-diaminopurine, 6-dimethylaminopurine, 2-aminopurine, cytosine, uracil, thymine, indole, 5-nitroindole, and 3-nitropyrrole.

In some embodiments, when the chemical modification of formula (I), a tautomer thereof, or a pharmaceutically acceptable salt thereof is modified at position 7 of its 5' end, B is selected from adenine, guanine, 2, 6-diaminopurine, 6-dimethylaminopurine, 2-aminopurine, cytosine, uracil, thymine, indole, 5-nitroindole, and 3-nitropyrrole.

In some embodiments, when the chemical modification of formula (I), a tautomer thereof, or a pharmaceutically acceptable salt thereof is modified at position 8 of its 5' end, B is selected from adenine, guanine, 2, 6-diaminopurine, 6-dimethylaminopurine, 2-aminopurine, cytosine, uracil, thymine, indole, 5-nitroindole, and 3-nitropyrrole.

In some embodiments, B is the same base as the antisense strand when the 5 th nucleotide at its 5' end is not modified.

In some embodiments, B is the same base as the antisense strand when the nucleotide at 6 of its 5' end is not modified.

In some embodiments, B is the same base as the antisense strand when the nucleotide at position 7 of its 5' end is not modified.

In some embodiments, B is the same base as the antisense strand when the 8 th nucleotide at its 5' end is not modified.

In some embodiments, at least one additional nucleotide in the sense strand and/or the antisense strand is a modified nucleotide selected from the group consisting of: 2' -methoxy-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' -fluoro-modified nucleotide, 2' -deoxynucleotide, 2' -deoxy-2 ' -fluoro-modified nucleotide, 3' -deoxy-thymine nucleotide, isonucleotide, LNA, ENA, cET, UNA, GNA; in some embodiments, the modified nucleotides are independently selected from the group consisting of: 2 '-methoxy-modified nucleotide or 2' -fluoro-modified nucleotide.

In some embodiments, the sense strand contains three consecutive nucleotides with the same modification. In some embodiments, the three nucleotides with the same modification are 2' -fluoro modified nucleotides.

In some embodiments, the nucleotides at positions 2, 4, 6, 10, 12, 14, 16 and 18 of the antisense strand are each independently a 2' -fluoro modified nucleotide in a 5' to 3' orientation.

In some embodiments, the antisense strand is at least partially reverse-complementary to a target sequence to mediate RNA interference; in some embodiments, no more than 5, no more than 4, no more than 3, no more than 2, no more than 1 mismatches exist between the antisense strand and the target sequence; in some embodiments, the antisense strand is fully reverse-complementary to the target sequence.

In some embodiments, the sense strand is at least partially reverse-complementary to the antisense strand to form a double-stranded region; in some embodiments, there are no more than 5, no more than 4, no more than 3, no more than 2, no more than 1 mismatches between the sense strand and the antisense strand; in some embodiments, the sense strand is fully reverse-complementary to the antisense strand.

In some embodiments, the sense strand and the antisense strand each independently have 16 to 35, 16 to 34, 17 to 33, 18 to 32, 18 to 31, 18 to 30, 18 to 29, 18 to 28, 18 to 27, 18 to 26, 18 to 25, 18 to 24, 18 to 23, 19 to 25, 19 to 24, or 19 to 23 nucleotides (e.g., 19, 20, 21, 22, 23 nucleotides).

In some embodiments, the sense strand and the antisense strand are the same or different in length, the sense strand is 19-23 nucleotides in length, and the antisense strand is 19-26 nucleotides in length. Thus, the length ratio of the sense strand to the antisense strand in the dsrnas provided by the present disclosure may be 19/20、19/21、19/22、19/23、19/24、 19/25、19/26、20/20、20/21、20/22、20/23、20/24、20/25、20/26、21/20、21/21、21/22、21/23、21/24、21/25、21/26、22/20、22/21、22/22、22/23、22/24、22/25、22/26、23/20、23/21、23/22、23/23、23/24、23/25 or 23/26. In some embodiments, the ratio of the length of the sense strand to the antisense strand is 19/21, 21/23, or 23/25. In some embodiments, the ratio of the length of the sense strand to the antisense strand is 19/21.

In some embodiments, the siRNA comprises one or two blunt ends.

In some embodiments, the siRNA comprises an overhang of 1 to 4 unpaired nucleotides, e.g., 1, 2, 3, 4.

In some embodiments, the siRNA comprises an overhang at the 3' end of the antisense strand.

In some embodiments, the sense strand comprises a nucleotide sequence (5 '-3') as shown in the following formula:

N _aN _aN _aN _aXN _aN _bN _bN _bN _aN _aN _aN _aN _aN _aN _aN _aN _aN _a

Wherein each X is independently N _a or N _b;N _a is a2 '-methoxy modified nucleotide and N _b is a 2' -fluoro modified nucleotide.

In some embodiments, the sense strand comprises a nucleotide sequence represented by the formula:

5'-N _aN _aN _aN _aN _aN _aN _bN _bN _bN _aN _aN _aN _aN _aN _aN _aN _aN _aN _a-3'; Or alternatively, the first and second heat exchangers may be,

5'-N _aN _aN _aN _aN _bN _aN _bN _bN _bN _aN _aN _aN _aN _aN _aN _aN _aN _aN _a-3';

Wherein N _a is a2 '-methoxy modified nucleotide and N _b is a 2' -fluoro modified nucleotide.

In some embodiments, the antisense strand comprises a nucleotide sequence represented by the formula:

5'-N _a'N _b'N _a'N _b'N _a'N _b'W'N _a'N _a'N _b'N _a'N _b'N _a'N _b'N _a'N _b'N _a'N _b'N _a'N _a'N _a'-3';

Wherein N _a 'is a 2' -methoxy modified nucleotide and N _b 'is a 2' -fluoro modified nucleotide; w 'represents a 2' -methoxy modified nucleotide or a nucleotide comprising a chemical modification represented by formula (I), a tautomer thereof, or a pharmaceutically acceptable salt thereof.

In some specific embodiments, W represents a 2' -methoxy modified nucleotide.

In some specific embodiments, W represents a nucleotide comprising a chemical modification represented by formula (I), a tautomer thereof, or a pharmaceutically acceptable salt thereof.

In some specific embodiments, the chemical modification represented by formula (I) is selected from:

wherein: b is selected from guanine, adenine, cytosine and uracil; in some specific embodiments, B is the same base as the antisense strand when the nucleotide at position 7 of its 5' end is not modified.

Wherein: m is O or S; wherein: b is selected from guanine, adenine, cytosine or uracil; in some specific embodiments, B is the same base as the antisense strand when the nucleotide at position 7 of its 5' end is not modified.

In some specific embodiments, M is S. In some specific embodiments, M is O.

In some embodiments, at least one phosphate group in the sense strand and/or the antisense strand is a phosphate group having a modification group that allows for increased stability of the siRNA in a biological sample or environment; in some embodiments, the phosphate group having a modifying group is a phosphorothioate group. In some embodiments, the phosphate group having a modifying group is a phosphorothioate diester group.

In some embodiments, the phosphorothioate diester group is present in at least one of the following positions:

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

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

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

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

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

The 3' end of the antisense strand is between nucleotide 2 and nucleotide 3.

In some embodiments, the sense strand and/or antisense strand includes a plurality of phosphorothioate diester groups therein, the phosphorothioate diester groups being present in:

Between nucleotide 1 and nucleotide 2 of the 5' end of the sense strand; and, a step of, in the first embodiment,

Between nucleotide 2 and nucleotide 3 of the 5' end of the sense strand; and, a step of, in the first embodiment,

The 5' end of the antisense strand is between nucleotide 1 and nucleotide 2; and, a step of, in the first embodiment,

The 5' end of the antisense strand is between nucleotide 2 and nucleotide 3; and, a step of, in the first embodiment,

The 3' end of the antisense strand is between nucleotide 1 and nucleotide 2; and, a step of, in the first embodiment,

The 3' end of the antisense strand is between nucleotide 2 and nucleotide 3.

5'-NMSNMSNMNMNFNMNFNFNFNMNMNMNMNMNMNMNMNMNM-3', or,

5’-NmsNmsNmNmNmNmNfNfNfNmNmNmNmNmNmNmNmNmNm-3’，

Where Nm represents any 2 '-methoxy modified nucleotide, e.g. 2' -methoxy modified C, G, U, A; nf represents any 2 '-fluoro modified nucleotide, for example, 2' -fluoro modified C, G, U, A;

Lower case letter s in the middle means that there is a phosphorothioate diester linkage between two nucleotides adjacent to the letter s; the lower case letter s indicates that the one nucleotide adjacent to the left of the letter s is terminated with a phosphorothioate diester group when the 3' end is first.

In some embodiments, the antisense strand comprises a nucleotide sequence represented by the formula: 5'-Nm' sNf 'sNm' Nf 'Nm Nf' W 'Nm' Nm 'Nf' Nm 'Nf' Nm 'Nf' Nm 'sNm' sNm '-3';

where Nm ' represents any 2' -methoxy modified nucleotide, e.g. 2' -methoxy modified C, G, U, A; nf ' represents any 2' -fluoro modified nucleotide, for example, 2' -fluoro modified C, G, U, A;

Lower case letter s in the middle indicates that a phosphorothioate diester linkage is between two nucleotides adjacent to the left and right of the letter s, and lower case letter s in the first of the 3' ends indicates that one nucleotide adjacent to the left of the letter s is terminated with a phosphorothioate diester;

w 'represents a 2' -methoxy modified nucleotide or a nucleotide comprising a chemical modification represented by formula (I), a tautomer thereof, or a pharmaceutically acceptable salt thereof.

In some embodiments, W represents a 2' -methoxy modified nucleotide.

In some embodiments, the chemical modification represented by formula (I) is selected from:

Wherein: b is selected from guanine, adenine, cytosine and uracil; in some embodiments, B is the same base as the antisense strand when the nucleotide at position 7 of its 5' end is not modified.

In some embodiments, M is S. In some specific embodiments, M is O.

In some embodiments, the siRNA is an siRNA targeting an apolipoprotein C3 (APOC 3) gene.

In some embodiments, the sense strand of the siRNA comprises at least 15 consecutive nucleotides that differ by NO more than 3 nucleotides from the nucleotide sequence of any one of SEQ ID NOs.1 to SEQ ID NOs.4, and/or,

The antisense strand comprises at least 19 consecutive nucleotides differing by NO more than 3 nucleotides from the nucleotide sequence of either SEQ ID NO. 5 or SEQ ID NO. 6.

In some embodiments, the sense strand of the siRNA comprises any one of SEQ ID NO:1 to SEQ ID NO:4, and/or the antisense strand comprises any one of SEQ ID NO:5 or SEQ ID NO: 6.

In some embodiments, the siRNA is any of the following:

The nucleotide sequence of the sense strand comprises SEQ ID NO. 1, and the nucleotide sequence of the antisense strand comprises SEQ ID NO. 5;

The nucleotide sequence of the sense strand comprises SEQ ID NO. 2 and the nucleotide sequence of the antisense strand comprises SEQ ID NO. 5;

The nucleotide sequence of the sense strand comprises SEQ ID NO. 3 and the nucleotide sequence of the antisense strand comprises SEQ ID NO. 6;

The nucleotide sequence of the sense strand comprises SEQ ID NO. 4 and the nucleotide sequence of the antisense strand comprises SEQ ID NO. 6.

In some embodiments, the siRNA is any of the following:

the nucleotide sequence of the sense strand is selected from SEQ ID NO. 1, and the nucleotide sequence of the antisense strand is selected from SEQ ID NO. 5;

The nucleotide sequence of the sense strand is selected from SEQ ID NO. 2, and the nucleotide sequence of the antisense strand is selected from SEQ ID NO. 5;

The nucleotide sequence of the sense strand is selected from SEQ ID NO. 3, and the nucleotide sequence of the antisense strand is selected from SEQ ID NO. 6;

The nucleotide sequence of the sense strand is selected from SEQ ID NO. 4, and the nucleotide sequence of the antisense strand is selected from SEQ ID NO. 6.

In the present disclosure, according to the 5'-3' direction,

SEQ ID NO. 1 is UAUUCUCAGUGCUCUCCUA;

SEQ ID NO. 2 is UAUUCUCAGUGCUCUCCUG;

SEQ ID NO. 3 is GCACCGUUAAGGACAAGUC;

SEQ ID NO. 4 is GCACCGUUAAGGACAAGUU;

SEQ ID NO. 5 is UAGGAGAGCACUGAGAAUACU;

SEQ ID NO. 6 is AACUUGUCCUUAACGGUGCUC.

In some embodiments, the siRNA is any of the following:

The nucleotide sequence of the sense strand comprises 5'-UAUUCUCAGUGCUCUCCUZ _b1 -3' (SEQ ID NO: 24) and the nucleotide sequence of the antisense strand comprises 5'-UAGGAGAGCACUGAGAAUACU-3' (SEQ ID NO: 5);

Or the nucleotide sequence of the sense strand comprises 5'-GCACCGUUAAGGACAAGUZ _b2 -3' (SEQ ID NO: 52) and the nucleotide sequence of the antisense strand comprises 5'-AACUUGUCCUUAACGGUGCUC-3' (SEQ ID NO: 6);

Wherein Z _b1 is A or G; z _b2 is C or U.

In some embodiments, the dsRNA may be any one of the following structures or a pharmaceutically acceptable salt thereof:

Wherein Z is siRNA, 3' end of sense strand of the siRNA is directly connected with ligand through phosphodiester group, definition of the siRNA is as described in any scheme of the disclosure.

In some embodiments, the dsRNA may be any one of the following structures or a pharmaceutically acceptable salt thereof,

Wherein Z is siRNA, 3' end of sense strand of the siRNA is directly connected with ligand through phosphodiester group, definition of the siRNA is as in any one of the previous schemes.

In some embodiments, the dsRNA may be of the structure or a pharmaceutically acceptable salt thereof,

In some embodiments, the nucleotide sequence of the sense strand of the dsRNA of the present disclosure comprises any one of SEQ ID NO 7 to SEQ ID NO 14, and/or,

The nucleotide sequence of the antisense strand of the dsRNA of the present disclosure comprises any one of SEQ ID NO. 17 or SEQ ID NO. 18;

In some embodiments, the dsRNA is any one of the following:

the sense strand comprises the nucleotide sequence shown in SEQ ID NO. 7, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO. 17;

the sense strand comprises the nucleotide sequence shown in SEQ ID NO. 9, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO. 17;

the sense strand comprises the nucleotide sequence shown as SEQ ID NO. 11, and the antisense strand comprises the nucleotide sequence shown as SEQ ID NO. 18;

the sense strand comprises the nucleotide sequence shown as SEQ ID NO. 13, and the antisense strand comprises the nucleotide sequence shown as SEQ ID NO. 18;

The sense strand comprises the nucleotide sequence shown in SEQ ID NO. 8, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO. 17;

the sense strand comprises the nucleotide sequence shown in SEQ ID NO. 10, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO. 17;

The sense strand comprises the nucleotide sequence shown in SEQ ID NO. 12, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO. 18;

The sense strand comprises the nucleotide sequence shown as SEQ ID NO. 14 and the antisense strand comprises the nucleotide sequence shown as SEQ ID NO. 18.

In some embodiments, the dsRNA is any one of the following:

The sense strand is selected from SEQ ID NO. 7, and the antisense strand is selected from SEQ ID NO. 17;

The sense strand is selected from SEQ ID NO. 9, and the antisense strand is selected from SEQ ID NO. 17;

The sense strand is selected from SEQ ID NO. 11, and the antisense strand is selected from SEQ ID NO. 18;

the sense strand is selected from SEQ ID NO. 13, and the antisense strand is selected from SEQ ID NO. 18;

The sense strand is selected from SEQ ID NO. 8, and the antisense strand is selected from SEQ ID NO. 17;

the sense strand is selected from SEQ ID NO. 10, and the antisense strand is selected from SEQ ID NO. 17;

the sense strand is selected from SEQ ID NO. 12, and the antisense strand is selected from SEQ ID NO. 18;

The sense strand is selected from SEQ ID NO. 14 and the antisense strand is selected from SEQ ID NO. 18.

In some embodiments, the dsRNA is any one of the following:

Comprising SEQ ID NO. 7 and comprising SEQ ID NO. 17;

Comprising SEQ ID NO 9 and comprising SEQ ID NO 17;

Comprising SEQ ID NO. 11 and comprising SEQ ID NO. 18;

comprising SEQ ID NO. 13 and comprising SEQ ID NO. 18;

comprising SEQ ID NO 8 and comprising SEQ ID NO 17;

Comprising SEQ ID NO 10 and comprising SEQ ID NO 17;

Comprising SEQ ID NO. 12 and comprising SEQ ID NO. 18; or (b)

Comprises SEQ ID NO. 14, SEQ ID NO. 18

In some embodiments, the dsRNA is any one of the following:

SEQ ID NO. 7 and SEQ ID NO. 17;

SEQ ID NO. 9 and SEQ ID NO. 17;

SEQ ID NO. 11 and SEQ ID NO. 18;

SEQ ID NO. 13 and SEQ ID NO. 18;

SEQ ID NO. 8 and SEQ ID NO. 17;

SEQ ID NO. 10 and SEQ ID NO. 17;

SEQ ID NO. 12 and SEQ ID NO. 18; or (b)

SEQ ID NO. 14 and SEQ ID NO. 18.

In the present disclosure, according to the 5'-3' direction,

SEQ ID NO. 7 is

UmsAmsUmUmCfUmCfAfGfUmGmCmUmCmUmCmCmUmAm-NAG0052’；

SEQ ID NO. 8 is

UmsAmsUmUmCmUmCfAfGfUmGmCmUmCmUmCmCmUmAm-NAG0052’；

SEQ ID NO. 9 is

UmsAmsUmUmCfUmCfAfGfUmGmCmUmCmUmCmCmUmGm-NAG0052’；

SEQ ID NO. 10 is

UmsAmsUmUmCmUmCfAfGfUmGmCmUmCmUmCmCmUmGm-NAG0052’；

SEQ ID NO. 11 is

GmsCmsAmCmCfGmUfUfAfAmGmGmAmCmAmAmGmUmCm-NAG0052’；

SEQ ID NO. 12 is

GmsCmsAmCmCmGmUfUfAfAmGmGmAmCmAmAmGmUmCm-NAG0052’；

SEQ ID NO. 13 is

GmsCmsAmCmCmGmUfUfAfAmGmGmAmCmAmAmGmUmUm-NAG0052’；

SEQ ID NO. 14 is

GmsCmsAmCmCfGmUfUfAfAmGmGmAmCmAmAmGmUmUm-NAG0052’；

SEQ ID NO. 17 is

UmsAfsGmGfAmGf(-)hmpNA(A)GmCmAfCmUfGmAfGmAfAmUfAmsCmsUm；

SEQ ID NO. 18 is

AmsAfsCmUfUmGf(-)hmpNA(U)CmCmUfUmAfAmCfGmGfUmGfCmsUmsCm。

Wherein af=adenine 2'-F ribonucleoside (adenine 2' -F ribonucleoside); cf=cytosine 2'-F ribonucleoside (cytosine 2' -F ribonucleoside); uf=uracil 2'-F ribonucleoside (uracil 2' -F ribonucleoside); gf=guanine 2'-F ribonucleoside (guanine 2' -F ribonucleoside); am = adenine 2'-OMe ribonucleoside (adenine 2' -OMe ribonucleoside); cm = cytosine 2'-OMe ribonucleoside (cytosine 2' -OMe ribonucleoside); gm=guanine 2'-OMe ribonucleoside (guanine 2' -OMe ribonucleoside); um = uracil 2'-OMe ribonucleoside (uracil 2' -OMe ribonucleoside);

s represents a phosphorothioate diester linkage between two nucleotides adjacent to the letter s;

NAG0052' represents

(-) HmpNA (A) represents(-) HmpNA (U) represents

In some embodiments, the dsRNA is selected from TRD007972, TRD007996, TRD007997, TRD008081, TRD007972-1, TRD007996-1, TRD007997-1, or TRD008081-1.

In some embodiments, the dsRNA is selected from the following structures or pharmaceutically acceptable salts thereof (sequences, in order of appearance, SEQ ID NO 8 and SEQ ID NO 17 respectively):

wherein af=adenine 2'-F ribonucleoside (adenine 2' -F ribonucleoside); cf=cytosine 2'-F ribonucleoside (cytosine 2' -F ribonucleoside); uf=uracil 2'-F ribonucleoside (uracil 2' -F ribonucleoside); am = adenine 2'-OMe ribonucleoside (adenine 2' -OMe ribonucleoside); cm = cytosine 2'-OMe ribonucleoside (cytosine 2' -OMe ribonucleoside); gf=guanine 2'-F ribonucleoside (guanine 2' -F ribonucleoside); gm=guanine 2'-OMe ribonucleoside (guanine 2' -OMe ribonucleoside); um=uracil 2'-OMe ribonucleoside (uracil 2' -OMe ribonucleoside). Represents a phosphorothioate diester group, and,Represents a group of a phosphoric acid diester,

NAG0052' represents

(-) HmpNA (A) represents

In some embodiments, the pharmaceutically acceptable salt may be a salt conventional in the art, including but not limited to: sodium salt, potassium salt, ammonium salt, amine salt, and the like.

In some embodiments, the dsRNA is TRD007972-1, which is of the structure (in order of appearance, SEQ ID NO 8 and SEQ ID NO 17 respectively):

Wherein af=adenine 2'-F ribonucleoside (adenine 2' -F ribonucleoside); cf=cytosine 2'-F ribonucleoside (cytosine 2' -F ribonucleoside); uf=uracil 2'-F ribonucleoside (uracil 2' -F ribonucleoside); am = adenine 2'-OMe ribonucleoside (adenine 2' -OMe ribonucleoside); cm = cytosine 2'-OMe ribonucleoside (cytosine 2' -OMe ribonucleoside); gf=guanine 2'-F ribonucleoside (guanine 2' -F ribonucleoside); gm=guanine 2'-OMe ribonucleoside (guanine 2' -OMe ribonucleoside); um = uracil 2'-OMe ribonucleoside (uracil 2' -OMe ribonucleoside); im = hypoxanthine 2'-OMe ribonucleosides (Inosine 2' -OMe ribonucleoside).

Represents a phosphorothioate diester group, and,Represents a group of a phosphoric acid diester,

NAG0052' represents

(-) HmpNA (A) represents

The dsRNA described in the present disclosure is selected from synthetic sources or prepared in vitro.

In another aspect, the present disclosure provides a compound of synthetic origin or a compound prepared in vitro, selected from the dsRNA of the present disclosure. In another aspect, the present disclosure provides a pharmaceutical composition comprising the dsRNA described above.

In some embodiments, the pharmaceutical composition further comprises one or more pharmaceutically acceptable excipients. Various drug delivery systems are known and can be used in the dsRNA or pharmaceutical compositions of the present disclosure, e.g., encapsulated in liposomes, microparticles, microcapsules, recombinant cells capable of expressing the compound, receptor-mediated endocytosis, construction of nucleic acids as part of a retrovirus or other vector.

In some embodiments, the dsRNA or pharmaceutical compositions of the present disclosure are administered in a conventional manner, either topically (e.g., directly injected or implanted) or systemically, or by oral, rectal, or parenteral routes including, but not limited to, subcutaneous injection, intravenous injection, intramuscular injection, intraperitoneal injection, transdermal administration, inhalation administration (e.g., aerosol), mucosal administration (e.g., sublingual, intranasal), intracranial administration, and the like.

In some embodiments, the dsRNA or pharmaceutical compositions provided by the present disclosure can be administered by injection, e.g., intravenous, intramuscular, intradermal, subcutaneous, intraduodenal, or intraperitoneal injection.

In some embodiments, the dsRNA or pharmaceutical compositions provided by the present disclosure can be packaged in a kit.

In another aspect, the present disclosure provides use of the dsRNA described above or the pharmaceutical composition described above in the preparation of a medicament.

In some embodiments, the effective amount or effective dose of the dsRNA or pharmaceutical composition is about 0.001mg/kg body weight to about 200mg/kg body weight, about 0.01mg/kg body weight to about 100mg/kg body weight, or about 0.5mg/kg body weight to about 50mg/kg body weight.

In some embodiments, the medicament may be used to prevent and/or treat diseases associated with APOC3 gene expression. In some embodiments, the disease is selected from the group consisting of hypertriglyceridemia, obesity, hyperlipidemia, lipid and/or cholesterol metabolic abnormalities, atherosclerosis, cardiovascular disease, coronary artery disease, hypertriglyceridemia-induced pancreatitis, metabolic syndrome, type II diabetes, familial chylomicronemia syndrome, or familial partial lipid malnutrition.

In some embodiments, the medicament may be used to prevent and/or treat diseases mediated by elevated triglyceride levels or elevated cholesterol levels. In some embodiments, the disease is selected from the group consisting of hypertriglyceridemia, obesity, hyperlipidemia, lipid and/or cholesterol metabolic abnormalities, atherosclerosis, cardiovascular disease, coronary artery disease, hypertriglyceridemia-induced pancreatitis, metabolic syndrome, type II diabetes, familial chylomicronemia syndrome, or familial partial lipid malnutrition.

In another aspect, the present disclosure provides the use of a dsRNA as described above or a pharmaceutical composition as described above in the manufacture of a medicament for preventing and/or treating a disease in a subject.

In some embodiments, the disease may be a disease associated with APOC3 gene expression. In some embodiments, the disease is selected from the group consisting of hypertriglyceridemia, obesity, hyperlipidemia, lipid and/or cholesterol metabolic abnormalities, atherosclerosis, cardiovascular disease, coronary artery disease, hypertriglyceridemia-induced pancreatitis, metabolic syndrome, type II diabetes, familial chylomicronemia syndrome, or familial partial lipid malnutrition.

In some embodiments, the disease may be a disease mediated by elevated triglyceride levels or elevated cholesterol levels. In some embodiments, the disease is selected from the group consisting of hypertriglyceridemia, obesity, hyperlipidemia, lipid and/or cholesterol metabolic abnormalities, atherosclerosis, cardiovascular disease, coronary artery disease, hypertriglyceridemia-induced pancreatitis, metabolic syndrome, type II diabetes, familial chylomicronemia syndrome, or familial partial lipid malnutrition.

In another aspect, the present disclosure provides a method of preventing and/or treating a disease comprising administering to a subject an effective amount or effective dose of the dsRNA described above or the pharmaceutical composition described above.

In another aspect, the present disclosure provides a method of reducing the level of low density lipoprotein in a subject comprising administering to the subject an effective amount or effective dose of the dsRNA described above or the pharmaceutical composition described above.

In another aspect, the present disclosure provides a method for silencing an APOC3 gene or mRNA thereof in a cell in vivo or in vitro comprising the step of introducing into the cell the dsRNA described above or the pharmaceutical composition described above.

In another aspect, the present disclosure provides a method of inhibiting expression of an APOC3 gene or mRNA thereof comprising administering to a subject an effective amount or dose of the dsRNA described above or the pharmaceutical composition described above.

The dsRNA or pharmaceutical composition of the present disclosure can reduce the expression level of a target gene or mRNA thereof in a cell, cell population, tissue, or subject, or the like, comprising: administering to a subject a therapeutically effective amount of a dsRNA or pharmaceutical composition described herein, thereby inhibiting expression of a target gene or mRNA thereof in the subject.

In some embodiments, the subject has been previously identified as having a pathological upregulation of a target gene or mRNA thereof in a targeted cell, cell population, tissue, or subject.

A subject as described in this disclosure refers to a subject diagnosed with (or suspected of having, or susceptible to) a disease or condition that would benefit from a reduction or inhibition of target mRNA expression.

In another aspect, the present disclosure provides a method of delivering an oligonucleotide to the liver comprising administering to a subject an effective amount or effective dose of the dsRNA described above or the pharmaceutical composition described above.

In another aspect, the present disclosure provides an RNAi (RNA interference) agent comprising the dsRNA described above or the pharmaceutical composition described above.

In another aspect, the present disclosure also provides a cell comprising the dsRNA described above or the pharmaceutical composition described above.

In another aspect, the disclosure also provides a kit comprising the dsRNA described above or the pharmaceutical composition described above.

In the present disclosure, the above dsRNA or pharmaceutical composition, when contacted with a cell expressing a target gene, is prepared from, for example: other methods such as PCR or branched DNA (bDNA) based methods, or protein based methods such as immunofluorescence assays, e.g., western Blot or flow cytometry assays, the dsRNA or pharmaceutical composition described above will inhibit expression of the target gene by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%.

In the present disclosure, the above dsRNA or pharmaceutical composition, when contacted with a cell expressing a target gene, is prepared from, for example: the percentage of residual expression of target gene mRNA by the above dsRNA or pharmaceutical compositions is no more than 99%, no more than 95%, no more than 90%, no more than 85%, no more than 80%, no more than 75%, no more than 70%, no more than 65%, no more than 60%, no more than 55%, no more than 50%, no more than 45%, no more than 40%, no more than 35%, no more than 30%, no more than 25%, no more than 20%, no more than 15%, or no more than 10% as determined by other methods such as PCR or branched DNA (bDNA) based methods, or protein based methods such as immunofluorescence assays, e.g., western Blot or flow cytometry.

In the present disclosure, the above dsRNA or pharmaceutical composition, when contacted with a cell expressing a target gene, is prepared from, for example: psiCHECK activity screening and luciferase reporter gene assays, other methods such as PCR or branched DNA (bDNA) based methods, or protein based methods such as immunofluorescence assays, e.g., western Blot, or flow cytometry assays, dsRNA reduces off-target activity by at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70% or at least 75% while maintaining target activity.

In the present disclosure, the above dsRNA or pharmaceutical composition, when contacted with a cell expressing a target gene, is prepared from, for example: psiCHECK activity screening and luciferase reporter gene assays, other methods such as PCR or branched DNA (bDNA) based methods, or protein based methods such as immunofluorescence assays, e.g., western Blot, or flow cytometry assays, dsRNA reduces off-target activity by at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70% or at least 75% while target activity is reduced by at most 19%, at most 15%, at most 10%, at most 5% or more than 1%.

In the present disclosure, the above dsRNA or pharmaceutical composition, when contacted with a cell expressing a target gene, is prepared from, for example: psiCHECK activity screening and luciferase reporter assay, other methods such as PCR or branched DNA (bDNA) based methods, or protein based methods such as immunofluorescence assays, e.g., western Blot, or flow cytometry assays, dsRNA reduces off-target activity by at least 20%, at least 25%, at least 45%, at least 30%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, or at least 80% while increasing target activity by at least 1%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 70%, at least 75%, or at least 80%, as determined by Western Blot, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, or at least 75%.

The present disclosure also provides a method of making a dsRNA or pharmaceutical composition comprising: the ligand, siRNA, dsRNA or pharmaceutical composition described in the present disclosure is synthesized.

Interpretation of the terms

For easier understanding of the present disclosure, some technical and scientific terms are specifically defined below. Unless defined otherwise herein, all other technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

The compounds of the present disclosure may exist in particular geometric or stereoisomeric forms. The present disclosure contemplates all such compounds, including cis and trans isomers, (-) -and (+) -pairs of enantiomers, (R) -and (S) -enantiomers, diastereomers, (D) -isomers, (L) -isomers, and racemic mixtures and other mixtures thereof, such as enantiomerically or diastereomerically enriched mixtures, all of which are within the scope of the disclosure. Additional asymmetric carbon atoms may be present in substituents such as alkyl groups. All such isomers and mixtures thereof are included within the scope of the present disclosure. The asymmetric carbon atom containing compounds of the present disclosure may be isolated in optically active pure or racemic forms. Optically pure forms can be resolved from the racemic mixture or synthesized by using chiral starting materials or chiral reagents.

Optically active (R) -and (S) -isomers and D and L isomers can be prepared by chiral synthesis or chiral reagents or other conventional techniques. If one enantiomer of a compound of the present disclosure is desired, it may be prepared by asymmetric synthesis or derivatization with chiral auxiliary wherein the resulting diastereomeric mixture is separated and the auxiliary group cleaved to provide the pure desired enantiomer. Or when the molecule contains a basic functional group (e.g., amino) or an acidic functional group (e.g., carboxyl), with a suitable optically active acid or base to form a diastereomeric salt, followed by diastereomeric resolution by conventional methods well known in the art, and recovery of the pure enantiomer. Furthermore, separation of enantiomers and diastereomers is typically accomplished by the use of chromatography employing a chiral stationary phase, optionally in combination with chemical derivatization (e.g., carbamate formation from amine).

In the chemical structure of the compounds of the present disclosure, the bondIndicating unspecified configuration, i.e. bonds if chiral isomers are present in the chemical structureMay beOr (b)Or at the same time containAndTwo configurations. In the chemical structure of the compounds of the present disclosure, the bondNot specifying configuration, i.e. keysThe configuration of (a) may be E-type or Z-type, or both E and Z configurations may be included.

In the chemical structural formula of the present disclosure,One or more of any group may be attached according to the scope of the invention described herein; asterisks indicate chiral centers.

The compounds and intermediates of the present disclosure may also exist in different tautomeric forms without specifying the configuration, and all such forms are included within the scope of the present disclosure. The term "tautomer" or "tautomeric form" refers to structural isomers of different energies that can interconvert via a low energy barrier. For example, proton tautomers (also known as proton transfer tautomers) include tautomers via proton transfer, such as keto-enol and imine-enamine, lactam-lactam isomerization. Examples of lactam-lactam balances are between a and B as shown below.

All compounds in the present disclosure may be drawn as form a or form B. All tautomeric forms are within the scope of the invention. The naming of the compounds does not exclude any tautomers.

The present disclosure also includes some isotopically-labeled compounds of the present disclosure which are identical to those recited herein, but for the replacement of one or more atoms by an atom having an atomic weight or mass number different from the atomic weight or mass number usually found in nature. Examples of isotopes that can be incorporated into compounds of the present disclosure include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine, iodine, and chlorine, such as ²H、 ³H、 ¹¹C、 ¹³C、 ¹⁴C、 ¹³N、 ¹⁵N、 ¹⁵O、 ¹⁷O、 ¹⁸O、 ³¹P、 ³²P、 ³⁵S、 ¹⁸F、 ¹²³I、 ¹²⁵I and ³⁶ Cl, respectively, and the like.

Unless otherwise indicated, when a position is specifically designated as deuterium (D), that position is understood to be deuterium (i.e., at least 10% deuterium incorporation) having an abundance that is at least 1000 times greater than the natural abundance of deuterium (which is 0.015%). The natural abundance of a compound in an example can be at least 1000 times greater than the abundance of deuterium, at least 2000 times greater than the abundance of deuterium, at least 3000 times greater than the abundance of deuterium, at least 4000 times greater than the abundance of deuterium, at least 5000 times greater than the abundance of deuterium, at least 6000 times greater than the abundance of deuterium, or higher than the abundance of deuterium. The present disclosure also includes various deuterated forms of compounds of formula (I), formula (I'), formula (II). Each available hydrogen atom attached to a carbon atom may be independently replaced with a deuterium atom. Those skilled in the art are able to synthesize deuterated forms of the compounds of formula (I), formula (I'), formula (II) with reference to the relevant literature. Commercially available deuterated starting materials may be used in the preparation of the deuterated forms of the compounds of formula (I), formula (I'), formula (II), or they may be synthesized using conventional techniques with deuterated reagents including, but not limited to, deuterated borane, tridentate borane tetrahydrofuran solution, deuterated lithium aluminum hydride, deuterated iodoethane, deuterated iodomethane, and the like.

Unless otherwise indicated, "optionally," "optional," or "optionally" means that the subsequently described event or circumstance may but need not occur, and that the description includes instances where the event or circumstance occurs or does not. For example, "optionally, R ₁ and R ₂ are directly linked in a ring" means that R ₁ and R ₂ are directly linked in a ring may occur, but need not be, and the description includes the case where R ₁ and R ₂ are directly linked in a ring and the case where R ₁ and R ₂ are not.

The terms "about" and "approximately" refer to values that are within acceptable tolerances for the particular values being determined by one of ordinary skill in the art, which values depend in part on how the measurement or determination is made (i.e., the limits of the measurement system). For example, "about" may mean within 1 or exceeding a standard deviation of 1. Or "about" or "substantially comprising" may mean a range of up to 20%, for example, between 1% and 15%, between 1% and 10%, between 1% and 5%, between 0.5% and 1%, in this disclosure, each instance preceded by the term "about" by the numerical value or numerical range also includes the given number of embodiments. Unless otherwise indicated, when a particular value is found in the present disclosure and claims, the meaning of "about" or "consisting essentially of" should be assumed to be within the acceptable error range for that particular value.

In this disclosure, the term "comprising" may be replaced by "consisting of … …".

Unless otherwise indicated, the "compounds", "chemical modifications", "ligands", "dsRNA", "nucleic acids" and "RNAi" of the present disclosure may each independently exist in the form of salts, mixed salts or non-salts (e.g., free acids or free bases). When present in salt or mixed salt form, it may be a pharmaceutically acceptable salt.

The "pharmaceutically acceptable salt" may be selected from inorganic salts or organic salts, and may also include pharmaceutically acceptable acid addition salts and pharmaceutically acceptable base addition salts.

By "pharmaceutically acceptable acid addition salt" is meant a salt with an inorganic or organic acid that retains the biological effectiveness of the free base without other side effects. Inorganic acid salts include, but are not limited to, hydrochloride, hydrobromide, sulfate, nitrate, phosphate, and the like; organic acid salts include, but are not limited to, formate, acetate, 2-dichloroacetate, trifluoroacetate, propionate, hexanoate, octanoate, decanoate, undecylenate, glycolate, gluconate, lactate, sebacate, adipate, glutarate, malonate, oxalate, maleate, succinate, fumarate, tartrate, citrate, palmitate, stearate, oleate, cinnamate, laurate, malate, glutamate, pyroglutamate, aspartate, benzoate, methanesulfonate, benzenesulfonate, p-toluenesulfonate, alginate, ascorbate, salicylate, 4-aminosalicylate, naphthalenedisulfonate, and the like. These salts can be prepared by methods known in the art.

By "pharmaceutically acceptable base addition salt" is meant a salt formed with an inorganic or organic base that is capable of maintaining the bioavailability of the free acid without other side effects. Salts derived from inorganic bases include, but are not limited to, sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, aluminum, and the like. Preferred inorganic salts are ammonium, sodium, potassium, calcium and magnesium salts, preferably sodium salts. Salts derived from organic bases include, but are not limited to, the following: primary, secondary and tertiary amines, substituted amines including natural substituted amines, cyclic amines and basic ion exchange resins such as ammonia, isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, ethanolamine, diethanolamine, triethanolamine, dimethylethanolamine, 2-dimethylaminoethanol, 2-diethylaminoethanol, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, choline, betaine, ethylenediamine, glucosamine, methylglucamine, theobromine, purine, piperazine, piperidine, N-ethylpiperidine, polyamine resins and the like. Preferred organic bases include isopropylamine, diethylamine, ethanolamine, trimethylamine, dicyclohexylamine, choline, and caffeine. These salts can be prepared by methods known in the art.

"Alkyl" refers to saturated aliphatic hydrocarbon groups, such as straight and branched chain groups (C ₁-C ₃₀ alkyl) comprising 1 to 30 carbon atoms, and further such as alkyl groups containing 1 to 6 carbon atoms (C ₁-C ₆ alkyl), and further such as alkyl groups containing 1 to 3 carbon atoms (C ₁-C ₃ alkyl). Non-limiting examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, 1-dimethylpropyl, 1, 2-dimethylpropyl, 2-dimethylpropyl, and various branched isomers thereof, and the like.

The term "alkenyl" refers to a hydrocarbon group containing at least one double bond. Non-limiting examples of alkenyl groups include, but are not limited to: vinyl, 1-propenyl, 2-propenyl, 1-butenyl or 2-butenyl and various branched isomers thereof.

The term "alkynyl" refers to a hydrocarbon group containing at least one triple bond. Non-limiting examples of alkynyl groups include, but are not limited to: ethynyl, 1-propynyl, 2-propynyl, 1-butynyl or 2-butynyl and various branched isomers thereof.

The term "alkoxy" refers to-O- (alkyl) wherein alkyl is as defined above. Non-limiting examples of alkoxy groups include: methoxy, ethoxy, propoxy, butoxy.

"Cycloalkyl" refers to a saturated or partially unsaturated monocyclic or polycyclic cyclic hydrocarbon substituent, the cycloalkyl ring containing from 3 to 20 carbon atoms, preferably from 3 to 6 carbon atoms, more preferably 5-6 carbon atoms. Non-limiting examples of monocyclic cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cyclohexadienyl, and the like; polycyclic cycloalkyl groups include spiro, fused and bridged cycloalkyl groups.

"Heterocyclyl" refers to a saturated or partially unsaturated monocyclic or polycyclic cyclic hydrocarbon substituent comprising 3 to 20 ring atoms, wherein one or more ring atoms are heteroatoms selected from nitrogen, oxygen, or S (O) _m (where m is an integer from 0 to 2), but excluding the ring portion of-O-O-, -O-S-, or-S-S-, and the remaining ring atoms are carbon. Preferably containing 3 to 12 ring atoms, of which 1 to 4 are heteroatoms; more preferably from 3 to 7 ring atoms. Non-limiting examples of "heterocycloalkyl" include:

Etc.

The heterocycloalkyl ring may be fused to an aryl or heteroaryl ring, wherein the ring attached to the parent structure is a heterocycloalkyl group, non-limiting examples of which include:

etc.

"Aryl" refers to a 6 to 14 membered all-carbon monocyclic or fused polycyclic (i.e., rings sharing adjacent pairs of carbon atoms) group having a conjugated pi-electron system, preferably 6 to 12 membered, such as phenyl and naphthyl. The aryl ring may be fused to a heteroaryl, heterocycloalkyl, or cycloalkyl ring, wherein the ring attached to the parent structure is an aryl ring, non-limiting examples of which include:

"heteroaryl" refers to a heteroaromatic system containing from 1 to 4 heteroatoms, from 5 to 14 ring atoms, wherein the heteroatoms are selected from oxygen, sulfur, and nitrogen. Heteroaryl is preferably 6 to 12 membered, more preferably 5 or 6 membered. For example. Non-limiting examples of which include: imidazolyl, furyl, thienyl, thiazolyl, pyrazolyl, oxazolyl (oxazolyl), isoxazolyl (isoxazolyl), pyrrolyl, tetrazolyl, pyridyl, pyrimidinyl, thiadiazole, pyrazinyl, triazolyl, indazolyl, benzimidazolyl, Etc.

The heteroaryl ring may be fused to an aryl, heterocycloalkyl, or cycloalkyl ring, wherein the ring attached to the parent structure is a heteroaryl ring, non-limiting examples of which include:

the term "hydroxy" refers to an-OH group.

The term "halogen" refers to fluorine, chlorine, bromine or iodine.

The term "cyano" refers to-CN.

The term "amino" refers to-NH ₂.

The term "nitro" refers to-NO ₂.

The term "oxo" refers to an =o substituent.

In the present disclosure, a "phosphate group" may be a phosphomonoester group, a phosphodiester group, or a phosphotriester group, preferably a phosphodiester group; the term "phosphate group" in the term "phosphorothioate group" is also intended to have the same meaning.

In the present disclosure, phosphorothioate diester refers to a modified phosphodiester group having a non-bridging oxygen atom replaced with a sulfur atom, which may be used(M is an S atom) are used interchangeably.

"Substituted" means that one or more hydrogen atoms, preferably up to 5, more preferably 1 to 3 hydrogen atoms in the group are independently substituted with a corresponding number of substituents. When the substituent is ketone or oxo (i.e., =o), then two (2) hydrogens on the atom are replaced.

In the context of the present disclosure, a groupIn (a) and (b)The moiety may be replaced by any group capable of achieving ligation to an adjacent nucleotide.

The term "linked," when referring to a connection between two molecules, refers to the connection of the two molecules by covalent bonds or the association of the two molecules via non-covalent bonds (e.g., hydrogen or ionic bonds), including direct connection, indirect connection.

The term "directly linked" means that a first compound or group is linked to a second compound or group without any intervening atoms or groups of atoms.

The term "indirectly attached" refers to a first compound or group being attached to a second compound or group through an intervening group, compound or molecule (e.g., a linking group).

"Pharmaceutical composition" means a mixture comprising one or more of the compounds described herein or a physiologically acceptable salt or prodrug thereof, and other chemical components, such as physiologically acceptable carriers and excipients. The purpose of the pharmaceutical composition is to promote the administration to organisms, facilitate the absorption of active ingredients and thus exert biological activity.

"Pharmaceutically acceptable excipients" include, but are not limited to, any auxiliary, carrier, glidant, sweetener, diluent, preservative, dye/colorant, flavoring agent, surfactant, wetting agent, dispersing agent, suspending agent, stabilizing agent, isotonic agent, buffer, solvent, or emulsifying agent that has been approved by the U.S. Food and Drug Administration (FDA) for use in humans or livestock animals.

As used herein, the term "inhibit" may be used interchangeably with "reduce," "silence," "down-regulate," "repress," and other similar terms, and includes any level of inhibition. Inhibition may be assessed by a decrease in absolute or relative levels of one or more of these variables as compared to control levels. The control level may be any type of control level used in the art, such as a pre-dosing baseline level or a level determined from a similar untreated or control (e.g., buffer-only control or inert control) -treated subject, cell, or sample. For example, the extent of inhibition of expression of a target gene by an siRNA can be characterized by a residual amount of mRNA, e.g., a residual amount of mRNA of not greater than 99%, not greater than 95%, not greater than 90%, not greater than 85%, not greater than 80%, not greater than 75%, not greater than 70%, not greater than 65%, not greater than 60%, not greater than 55%, not greater than 50%, not greater than 45%, not greater than 40%, not greater than 35%, not greater than 30%, not greater than 25%, not greater than 20%, not greater than 15%, or not greater than 10%. The inhibition rate of target gene expression can be usedLuciferase ASSAY SYSTEM detects, respectively reads a Firefly (Firefly) chemiluminescence value and a Renilla (Renilla) chemiluminescence value, and calculates a relative value ratio=ren/Fir, and an inhibition Ratio (%) =1- (ratio+siRNA/Ratioreporter only) ×100%; in the present disclosure, the remaining mRNA expression amount ratio (or remaining activity%) =100% -inhibition (%).

An "effective amount" or "effective dose" comprises an amount sufficient to ameliorate or prevent a symptom or condition of a medical condition. An effective amount is also meant to be an amount sufficient to permit or facilitate diagnosis. The effective amount for a particular patient or veterinary subject may vary depending on the following factors: such as the condition to be treated, the general health of the patient, the route of administration and the dosage and severity of the side effects. An effective amount may be the maximum dose or regimen that avoids significant side effects or toxic effects.

As used herein, "subject," "patient," "subject," or "individual" are used interchangeably and include a human or non-human animal, such as a mammal, e.g., a human or monkey.

As used herein, the sense strand (also known as the SS, or sense strand) refers to a strand comprising a sequence that is identical or substantially identical to a target mRNA sequence; the antisense strand (also known AS the AS or AS strand) refers to a strand having a sequence complementary to the target mRNA sequence.

In this disclosure, the "5 'region" of the sense strand or antisense strand, i.e., "5' end", is used interchangeably. For example, the nucleotides at positions 2 to 8 of the 5 '-region of the antisense strand may be replaced with the nucleotides at positions 2 to 8 of the 5' -end of the antisense strand. Similarly, the "3' region", "3' end" and "3' end" of the sense strand or antisense strand may be used interchangeably.

In the context of describing the sense strand of the siRNA described herein, the term "at least 15 consecutive nucleotides of the nucleotide sequence of any one of SEQ ID nos. 1 to 4 differ by NO more than 3 nucleotide sequences" is intended to mean that the sense strand of the siRNA described herein comprises at least 15 consecutive nucleotides as the sense strand of any one of SEQ ID nos. 1 to 4, or at least 15 consecutive nucleotides of the sense strand of any one of SEQ ID nos. 1 to 4 differ by NO more than 3 nucleotide sequences, optionally by NO more than 2 nucleotide sequences, optionally by 1 nucleotide sequence. Optionally, the siRNA sense strand described herein comprises at least 16 consecutive nucleotides of any one of SEQ ID NO. 1 to SEQ ID NO. 4, or differs by NO more than 3 nucleotide sequences, optionally by NO more than 2 nucleotide sequences, optionally by 1 nucleotide sequence, from at least 16 consecutive nucleotides of any one of SEQ ID NO. 1 to SEQ ID NO. 4;

In the context of describing the antisense strand of an siRNA described herein, the term "at least 15 consecutive nucleotides differing by NO more than 3 nucleotide sequences from either of the antisense strands of SEQ ID NO. 5 or SEQ ID NO. 6" is intended to mean at least 15 consecutive nucleotides of either of the antisense strands of SEQ ID NO. 5 or SEQ ID NO. 6 described herein, or at least 15 consecutive nucleotides differing by NO more than 3 nucleotide sequences from either of the antisense strands of SEQ ID NO. 5 or SEQ ID NO. 6, optionally, by NO more than 2 nucleotide sequences, optionally, by 1 nucleotide sequence.

In the context of the present disclosure, "G", "C", "a", "T" and "U" represent nucleotides, respectively, comprising bases of guanine, cytosine, adenine, thymidine and uracil, respectively, unless otherwise specified. The lower case letter d indicates that the adjacent nucleotide to the right of the letter d is a deoxyribonucleotide; the lower case letter m indicates that the adjacent nucleotide to the left of the letter m is a methoxy modified nucleotide; the lower case letter f indicates that the adjacent nucleotide to the left of the letter f is a fluoro-modified nucleotide; the lower case letter s indicates that there is a phosphorothioate diester linkage between two nucleotides adjacent to and about the letter s.

As used in this disclosure, the term "2 '-fluoro (2' -F) 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, and "non-fluoro modified nucleotide" refers to a nucleotide or nucleotide analogue in which the hydroxyl group at the 2' -position of the ribosyl group of the nucleotide is substituted with a non-fluorine group.

As used in this disclosure, the term "2' -methoxy (2 ' -OMe) -modified nucleotide" refers to a nucleotide formed by substitution of the 2' -hydroxy group of the ribosyl group with a methoxy group.

In the context of the present disclosure, the presence of a "nucleotide difference" in one nucleotide sequence from another means that the base type of the nucleotide at the same position is changed in the former as compared with the latter, for example, in the case where one nucleotide base is A, 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 that 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.

The term "dsRNA" refers to a double-stranded RNA molecule capable of RNA interference, comprising a sense strand and an antisense strand.

As used herein, the terms "complementary" or "reverse complementary" are used interchangeably and have the meaning well known to those skilled in the art that in a double stranded nucleic acid molecule, the bases of one strand pair with the bases on the other strand in a complementary manner. In DNA, the purine base adenine is always paired with the pyrimidine base thymine (or uracil in RNA); the purine base guanine always pairs with the pyrimidine base cytosine. 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.

The term "chemical modification" or "modification" includes all changes in the nucleotide through chemical means, such as the addition or removal of chemical moieties, or substitution of one chemical moiety for another.

The term "base" encompasses any known DNA and RNA base, base analogue, such as purine or pyrimidine, which also includes the natural compounds adenine, thymine, guanine, cytosine, uracil, inosine and natural analogues. The base analogue may also be a universal base.

The terms "blunt end" or "blunt end" are used interchangeably to refer to the absence of unpaired nucleotides or nucleotide analogs, i.e., no nucleotide overhang, at a given end of the siRNA. In most cases, an siRNA that is blunt-ended at both ends will be double-stranded throughout its length.

The siRNA provided by the present disclosure can be obtained by methods of preparation conventional in the art (e.g., methods of solid phase synthesis and liquid phase synthesis). Among them, solid-phase synthesis already has commercial subscription services. Methods of preparing nucleoside monomers having corresponding modifications and methods of introducing modified nucleotide groups into siRNA can also be known to those of skill in the art by introducing modified nucleotide groups into siRNA described in the present disclosure using nucleoside monomers having corresponding modifications.

Drawings

FIG. 1 shows the expression level of mRNA in TTR at 7 days after administration of TRD002218 and TRD 007205.

FIG. 2 shows the expression levels of mRNA in TTR at day 28 after administration of TRD002218 and TRD 007205.

Detailed Description

The present disclosure is further described below in connection with the examples, which are not intended to limit the scope of the present disclosure. Experimental methods for which specific conditions are not noted in the examples of the present disclosure are generally performed under conventional conditions or under conditions suggested by the manufacturer of the raw materials or goods. Reagents of particular origin are not noted, but are available from any supplier of molecular biological reagents in quality/purity for molecular biological applications.

Example 1: preparation of chemical modifications

1.1 Synthesis of Compounds 1-1a and 1-1b

Compound 1 (500 mg,3.42 mmol) and triethylamine (Et ₃ N,692mg,6.84mmol,0.95 mL) were dissolved in dichloromethane (DCM, 10 mL), a solution of 4-toluenesulfonyl chloride (TsCl, 719 mg,3.76 mmol) in dichloromethane (10 mL) was added dropwise under ice, after the addition was completed, the reaction was stirred at room temperature overnight, after the reaction was completed, quenched with water, the aqueous phase was extracted three times with dichloromethane (15 mL), the combined organic phases were washed with saturated aqueous sodium bicarbonate (10 mL) first, then with saturated brine (20 mL), and then the solvent was evaporated under reduced pressure to give crude 2 (820 mg, 80%) which was used directly in the next reaction. MS m/z: c ₁₄H ₂₁O ₅S,[M+H] ⁺ theory: 301.10 actual measurement: 301.2.

Compound 3 (239 mg,1.22 mmol) was dissolved in dimethylformamide (DMF, 10 mL), naH (60% in mineral oil, 93mg,2.33 mmol) was added under ice-bath, stirring was carried out for 30min under this reaction, then compound 2 (350 mg,1.16 mmol) was added dropwise, the reaction was stirred at 60℃for 5H after the addition was completed, after the reaction was completed, water was added to quench the reaction, the aqueous phase was extracted three times with ethyl acetate (15 mL), the combined organic phases were washed three times with water (10 mL) first, then with saturated brine (10 mL), followed by evaporation of the solvent under reduced pressure, and reversed phase preparative HPLC (C ¹⁸, condition: 5-50% (A: H ₂O,B:CH ₃ CN), flow rate: 70 mL/min) was carried out to give 220mg of compound 4 after lyophilization. MS m/z: c ₁₉H ₂₁N ₅O ₃Na,[M+Na] ⁺ theory: 390.16, actual measurement: 390.3.

Compound 4 (1.50 g,4.08 mmol) was dissolved in a mixture of 20mL of acetic acid and water (4:1) at room temperature, stirred at 60℃for 30min, after the reaction was completed, the solvent was evaporated under reduced pressure, and 1.10g of Compound 5 was obtained by lyophilization through reverse phase preparative HPLC (C ¹⁸, condition: 5-25% (A: H ₂O,B:CH ₃ CN), flow rate: 70 mL/min). MS m/z: c ₁₆H ₁₈N ₅O ₃,[M+H] ⁺ theory: 328.13, actual measurement: 328.4.

Compound 5 (1.00 g,3.05 mmol) was dissolved in pyridine (Py, 10 mL), a solution of 4,4' -dimethoxytrityl chloride (DMTrCl, 1.50g,4.58 mmol) in pyridine (5 mL) was added dropwise in ice, the reaction was stirred at room temperature overnight after the addition, quenched with water after the reaction was completed, the solvent was evaporated under reduced pressure, and 1.00g of Compound 6 was obtained after lyophilization by reverse phase preparative HPLC (C ¹⁸, condition: 5-80% (A: H ₂O,B:CH ₃ CN), flow rate: 70 mL/min). MS m/z: c ₃₇H ₃₆N ₅O ₅,[M-H] ⁺ theory: 630.26, actual measurement: 630.5. racemate compound 6 was subjected to chiral column (DaicelIE 250.6mm, 5 μm, A: n-hexane, B: ethanol) gives 410mg 6A (-) and 435mg 6B (+).

Compound 6A (-) (200 mg,0.32 mmol), tetrazole (11 mg,0.16 mmol), N-methylimidazole (5 mg,0.06 mmol), 3A molecular sieves (500 mg) were dissolved in 10mL of acetonitrile, compound 7 (144 mg,0.48 mmol) was added at room temperature, and stirred at room temperature overnight. After the reaction was completed, the molecular sieve was filtered off, dichloromethane (30 mL) was added, and the mixture was washed three times with saturated aqueous sodium bicarbonate (10 mL) and then with saturated brine (20 mL), and the filtrate was dried by spin-drying and subjected to reverse phase preparative HPLC (C ¹⁸, condition: 5-100% (A: water, B: CH ₃ CN), flow rate: 70 mL/min) to obtain 200mg of compound 1-1a after lyophilization. MS m/z: c ₄₀H ₃₉N ₆O ₇ P, [ M-diisopropyl+OH ] ⁺ theory: 747.26, actual measurement ：747.6.1H NMR(400MHz,Acetonitrile-d ₃)δ7.56,7.54(2s,1H),7.36-7.27(m,2H),7.24-7.21(m,7H),6.83-6.80(m,4H),4.12-4.10(m,2H),3.75-3.68(m,10H),3.20-2.80(m,2H),2.68-2.54(m,4H),1.22-1.04(m,18H).

Compound 6B (+) (200 mg,0.32 mmol), tetrazole (11 mg,0.16 mmol), N-methylimidazole (5 mg,0.06 mmol), 3A molecular sieves (500 mg) were dissolved in 10mL of acetonitrile, compound 7 (144 mg,0.48 mmol) was added at room temperature, and stirred at room temperature overnight. After the reaction was completed, the molecular sieve was filtered off, dichloromethane (30 mL) was added, and the mixture was washed three times with saturated aqueous sodium bicarbonate (10 mL) and then with saturated brine (20 mL), and the filtrate was dried by spin-drying and subjected to reverse phase preparative HPLC (C ¹⁸, conditions: 5-100% (A: water, B: CH ₃ CN), flow rate: 70 mL/min) to obtain 200mg of compound 1-1B after lyophilization. MS m/z: c ₄₀H ₃₉N6O ₇ P, [ M-diisopropyl+OH ] ⁺ theory: 747.26, actual measurement: 747.5.

1.2 Synthesis of Compounds 1-6a

Compound 1 (10 g,68.404 mmol), compound 2 (15 g,62.186 mmol) and triphenylphosphine (32.62 g,124.371 mmol) were dissolved in anhydrous THF (30 mL) and DIAD (24.650 mL,124.371 mmol) was slowly added dropwise at 0deg.C. The reaction mixture was reacted at 25℃for 12 hours. LCMS showed the reaction was complete. The reaction was extracted with ethyl acetate (200 mL) and water (200 mL), the organic phase was dried and the filtrate was concentrated, and the resulting residue was purified by forward column (DCM/meoh=10/1) to give the desired product 3 (20 g).

Compound 3 (20 g,28.585 mmol) was dissolved in acetic acid (24 mL,426.016 mmol) and H ₂ O (12 mL) and stirred at 60℃for 1 hour. The reaction mixture was then dried by spin-drying, and THF (12 mL) and H ₂ O (12 mL) were added thereto, followed by stirring at 80℃for 7 hours. LCMS showed the reaction was complete. The reaction was extracted with ethyl acetate (200 mL) and water (100 mL), and the aqueous phase was added with sodium carbonate solids until a significant amount of the solids had precipitated in the aqueous phase. The solid was filtered, washed with water, and the filter cake was pulled up with an oil pump to give the objective compound 5 (9 g).

Compound 5 (6.8 g,18.581 mmol) was dissolved in pyridine (80 mL) under nitrogen, TMSCL (14.250 mL,111.489 mmol) was slowly added at 0deg.C and stirred for 2h. Then Isobutyryl chloride (2.044 mL,19.511 mmol) was added at 0deg.C and stirred at 25deg.C for 1h. LCMS showed the reaction was complete. Extraction with dichloromethane (200 mL) and water (200 mL) and drying of the organic phase followed by stirring and purification on a forward column (DCM: meoh=10:1) peak at 4.8%) afforded compound 6 (12 g) as a yellow oil.

Compound 6 (5.5 g, 12.390 mmol) was dissolved in pyridine (30 mL) under nitrogen, MOLECULAR SIEVE 4A 1/16 (7 g, 12.390 mmol) was added, then DMTrCl (5.04 g, 14.87mmol) solid was added in portions at 0deg.C and reacted for 2h at 25deg.C. TLC (PE: etoac=1:1, rf=0.69) showed the reaction was complete. The reaction mixture was treated with TJN200879,879-040-P1 in combination. The reaction was extracted with ethyl acetate (200 mL) and water (200 mL), and the organic phase was dried and spun-dried and purified on a forward column (PE: etOAc, peak 84%) to give compound 7 (12 g) as a yellow oil.

Compound 7 (12 g,15.389 mmol) was dissolved in EtOAc (140 mL) and wet Pd/C on carbon (7 g,15.389 mmol) was added and the reaction was allowed to react for 2 hours under hydrogen (15 Psi) at 25 ℃. TLC (PE: etoac=0:1, rf=0.09) showed the reaction was complete. The reaction mixture was filtered, and the cake was washed three times with ethyl acetate (30 mL), and the filtrate was collected. After spin-drying the filtrate was added 50mL of dichloromethane and 2mL of triethylamine and purified by forward column (DCM: meoh=10:1, peak at 0.5%) to give 9g (yellow foamy solid) the resulting racemic compound SFC was resolved to give the product title compound 7A (-) (3.9 g) and the title compound 7B (+) (3.8 g).

Compound 7A (-) (3.30 g,5.40 mmol), tetrazole (190 mg,2.70 mmol), 1-methylimidazole (90 mg,1.10 mmol), 3A molecular sieves (500 mg) were dissolved in 30mL of acetonitrile, compound 8 (2.50 g,8.10 mmol) was added at room temperature, and stirred at room temperature for 2h. After completion of the reaction, the molecular sieve was filtered off, DCM (150 mL) was added, washed with saturated aqueous sodium bicarbonate (30 mL x 3), then with saturated brine (30 mL), the filtrate was dried by spinning and lyophilized to give 1-6a (2.9 g, 66%) by reverse phase preparative HPLC (C18, condition:5-100% (a: water, B: CH3 CN), flow rate: 70 mL/min). MS m/z: c43H55N7O7P [ m+h ] +, theory: 812.38, actual measurement ：812.5.1H NMR(400MHz,Acetonitrile-d3)δ7.56,7.54(2s,1H),7.36-7.27(m,2H),7.24-7.21(m,7H),6.83-6.80(m,4H),4.12-4.10(m,2H),3.75-3.68(m,10H),3.20-2.80(m,2H),2.68-2.54(m,4H),1.22-1.04(m,18H).

1.3 Synthesis of Compounds 1-7a

Compound 1 (5 g,23.1272 mmol), compound 2 (6.76 g,46.254 mmol) and triphenylphosphine (7.28 g,27.753 mmol) were dissolved in 30mL dioxane under nitrogen and DEAD (5.502 mL,27.753 mmol) was slowly added dropwise at 0deg.C. After the completion of the dropwise addition, the reaction was slowly warmed to 25℃and continued for 1 hour. To the reaction mixture was added 100mL of H ₂ O and 100mL of EtOAc, and the organic phase was combined, dried, filtered, concentrated, and purified by column chromatography (PE: etoac=1:1) to give the desired product (4 g).

Compound 3 (3.3 g) was dissolved in HOAc (16 mL) and H ₂ O (4 mL) and heated in an oil bath at 60℃for 0.5H. The residue obtained by spin-drying the reaction mixture was purified by forward column (PE: etoac=0:1 passage through a column) to give the desired product 4 (3 g).

Compound 4 (3 g,8.873 mmol) was dissolved in 5mL of pyridine and a solution of DMTrCl (3.91 g,11.535 mmol) in 10mL of pyridine was slowly added dropwise under nitrogen at 0deg.C. After the completion of the dropwise addition, the reaction was warmed to 25℃and continued for 1 hour. To the reaction mixture was added 50mL of water and 100mL of ethyl acetate for extraction. The aqueous phase was extracted three more times with 100mL ethyl acetate and the organic phases were combined, dried, filtered and concentrated and purified on a forward column (with PE: etoac=2:1). Target product 5 (4 g) was obtained.

Compound 5 (4 g,5.769 mmol) was dissolved in methanol (10 mL), saturated NH3 methanol solution (40 mL) was added, and the mixture was reacted at 0℃for 6h. The reaction solution was spin-dried and purified by a forward column (PE: etoac=0:1) to give 2.4g of the racemic compound, which was resolved by SFC to give the desired product 6A (750 mg,100% purity) and the desired product 6B (400 mg,99.16% purity).

Compound 6A (-) (700 mg,1.40 mmol), tetrazole (50 mg,0.70 mmol), 1-methylimidazole (23 mg,0.28 mmol), 3A molecular sieves (500 mg) were dissolved in 10mL of acetonitrile, compound 7 (630 mg,2.10 mmol) was added at room temperature, and stirred at room temperature for 2h. After completion of the reaction, the molecular sieve was filtered off, DCM (50 mL) was added, washed with saturated aqueous sodium bicarbonate (10 mL x 3), then with saturated brine (20 mL), the filtrate was dried by spinning and lyophilized to give 1-7a (700 mg, 72%) by reverse phase preparative HPLC (C18, condition:5-100% (a: water B: CH ₃ CN), flow rate: 70 mL/min). MS m/z: C38H47N4O7PNa [ m+na ] +, theory: 725.32, actual measurement: 725.5.

1.4 Synthesis of Compounds 1-8a

Compound 1 (8.5 g,76.508 mmol), compound 2 (30.64 g,91.809 mmol) was dissolved in DMF (150 mL), CS2CO3 (29.91 g,91.809 mmol) was added and reacted under nitrogen for 12h at 90 ℃. LCMS detects completion of the reaction. The reaction was filtered, oil pump dried, and purified by column chromatography (80 g, dcm/meoh=10/1 to 5/1) to give the desired product 3 (13.5 g,80% purity).

Compound 3 (10.5 g,35.105 mmol) was dissolved in pyridine (65 mL) and CH ₃ CN (65 mL), bzCl (4.894 mL,42.126 mmol) was added dropwise to the solution and reacted at 25℃for 2h. LCMS detected completion of most starting material reaction, quench with H ₂ O (100 mL), extract with EtOAc (100 mL X3), dry spin-dry, purify by column separation (combination TJN 200872-101) (80 g, pe/etoac=10/1-0/1, dcm/meoh=10/1) to afford the desired product 4 (14 g,90% purity).

Compound 4 (14 g,36.694 mmol) was dissolved in HOAc (56 mL,314.796 mmol) and H ₂ O (14 mL) and reacted at 60℃for 2H, LCMS indicated the reaction was complete. Oil pump concentration and forward column separation (40 g, dcm/meoh=1/0 to 5/1) gave the desired product 5 (8.4 g,90% purity &2.4g,80% purity).

Compound 5 (7.4 g,21.957 mmol), DMAP (0.54 g, 4.3991 mmol), MOLECULAR SIEVE 4A (11.1 g,2.967 mmol) was dissolved in pyridine (60 mL), stirred for 10min under ice, then DMTrCl (8.93 g,26.348 mmol) was added and the reaction stirred for 1.8h. LCMS detects about 19% starting material remaining, about 60% target MS. The fractions were combined (TJN 200872-105& 106) and purified together. H ₂ O (50 mL) was added to the reaction solution, extracted with DCM (50 mL X3), dried, spin-dried, and column separated (120 g, PE/(EA: DCM: TEA=1:1:0.05) =1/0-0/1 to DCM/MeOH=10/1) to give the target compound 6 (11 g,89% purity, TJN200872-105&106& 107), and the starting material (3.0 g,70% purity) was recovered.

Compound 6 (15 g,22.041 mmol) was isolated by SFC (DAICEL CHIRALPAK AD (250 mm. Times.50 mm,10 um); 0.1% NH ₃H ₂ O EtOH, B:45% -45%;200 ml/min) to give target product 6A (5.33 g,94.29% purity), target product 6B (6.14 g,97.91% purity), and compound 6 was recovered at 1.0g.

Compound 6B (-) (5.4 g,8.92 mmol), tetrazole (312 mg,4.46 mmol), 1-methylimidazole (146 mg,1.78 mmol), 3A molecular sieves (500 mg) were dissolved in 40mL of acetonitrile, compound 7 (4 g,13.4 mmol) was added at room temperature, and stirred at room temperature for 2h. After completion of the reaction, the molecular sieve was filtered off, DCM (200 mL) was added, washed with saturated aqueous sodium bicarbonate (30 mL x 3), then with saturated brine (50 mL), the filtrate was dried by spinning and subjected to reverse phase preparative HPLC (C18, condition:5-100% (a: water, B: CH ₃ CN), flow rate: 70 mL/min) to give 1-8a (5.8 g, 80%) after lyophilization. MS m/z: c45H51N5O7P, [ m+h ] +, theory: 804.36, actual measurement: 804.4.

Example 2: synthesis of dsRNA

The synthesis of dsRNA is not different from the common phosphoramidite solid-phase synthesis method, and when the nucleotide modified at the 5' 7 th position of AS chain is synthesized, the synthesized phosphoramidite monomer is used for replacing the parent sequence original nucleotide. The synthesis process is briefly described as follows: nucleoside phosphoramidite monomers were individually linked according to the synthetic procedure starting with the Universal CPG vector on a Dr.Oligo48 synthesizer (Biolytic). In addition to the above described nucleoside phosphoramidite monomers at position 7 of the AS-strand 5', the remaining nucleoside monomer starting materials, 2' -F RNA, 2' -O-methyl RNA, and other nucleoside phosphoramidite monomers, were purchased from Shanghai megadimension or Stuzhou Ji Ma. 5-ethylthio-1H-tetrazole (ETT) was used as the activator (0.6M acetonitrile solution), 0.22M PADS was dissolved in a 1:1 volume ratio of acetonitrile and trimethylpyridine (Ke Lema, suzhou) solution as the sulfiding agent, and iodopyridine/water solution (Ke Lema) as the oxidizing agent.

After the solid phase synthesis was completed, the oligoribonucleotides were cleaved from the solid support and immersed in a 3:1 solution of 28% ammonia and ethanol at 50℃for 16 hours. The supernatant was then transferred to another centrifuge tube, concentrated to dryness, purified using C18 reverse chromatography, mobile phase 0.1M TEAA and acetonitrile, and DMTr removed using 3% trifluoroacetic acid solution. The target oligonucleotides were collected, lyophilized, and identified as target products by LC-MS, and then quantified by UV (260 nm).

The obtained single-stranded oligonucleotide is annealed according to complementary pairing according to the equimolar ratio, and finally the obtained dsRNA is dissolved in 1 XPBS and adjusted to the concentration required by the experiment for standby.

Example 3: psiCHECK activity screening assay

DsRNA samples were synthesized as described above, and plasmids were derived from the biological engineering (Shanghai) Co., ltd. The psiCHECK experimental consumables are shown in table 1.

TABLE 1 psicheck assay consumables and reagents

The experimental steps are as follows: cell plating, cell transfection, wherein specific formulation amounts of the transfection complexes are shown in table 2.

TABLE 2 amount of transfection complex required per well of 96 well plates

	Dosage/pore	Opti-MEM
Plasmid mixture	0.05μL	10μL
Lipofectamine 2000	0.2μL	10μL

Note that: lipo:0.2 μl/well; plasmid: 0.05 μl/well; opti-MEM: 10. Mu.L/well.

According to Table 3, the working fluid was diluted to different concentrations according to different experimental requirements and prepared as it was. After 24h of transfection, the method is carried out according toThe test protocol of the Luciferase ASSAY SYSTEM test kit was followed. Calculating a relative value ratio=ren/Fir (Renilla/firefly Ratio); calculating inhibition Ratio 1- (ratio+dsrna/Ratioreporter only) ×100% = inhibition Ratio (%); in the present disclosure, residual activity% (also referred to as mRNA residual expression amount% or mRNA residual expression ratio) =100% -inhibition (%).

TABLE 3 Multi-concentration Point dsRNA dilution protocol

Final concentration (nM)	Adding water and dsRNA
/	/
40	4μL dsRNA(20μM)+96μL H ₂O
13.33333333	30μL dsRNA+60μL H ₂O
4.444444444	30μL dsRNA+60μL H ₂O
1.481481481	30μL dsRNA+60μL H ₂O
0.49382716	30μL dsRNA+60μL H ₂O
0.164609053	30μL dsRNA+60μL H ₂O
0.054869684	30μL dsRNA+60μL H ₂O
0.018289895	30μL dsRNA+60μL H ₂O
0.006096632	30μL dsRNA+60μL H ₂O
0.002032211	30μL dsRNA+60μL H ₂O
0.000677404	30μL dsRNA+60μL H ₂O

Example 4: characterization of different chemical modifications

Wherein: we define hmpNA the nucleotide synthesized from 2-hydroxymethyl-1, 3-propanediol as starting material;

(+) hmpNA (a) is obtained by solid phase synthesis of nucleoside phosphoramidite monomer 1-1b of section 1.1 of example, absolute configuration is (S) -hmpNA (a);

(-) hmpNA (a) is obtained by solid phase synthesis of nucleoside phosphoramidite monomer 1-1a of section 1.1 of example, absolute configuration (R) -hmpNA (a);

Similarly, the base species of substitution hmpNA were obtained by solid phase synthesis to obtain the following structures and confirm the absolute configuration:

(+) hmpNA (G), absolute configuration (S) -hmpNA (G);

(-) hmpNA (G), absolute configuration (R) -hmpNA (G);

(+) hmpNA (C), absolute configuration (S) -hmpNA (C);

(-) hmpNA (C), absolute configuration (R) -hmpNA (C);

(+) hmpNA (U), absolute configuration (R) -hmpNA (U);

(-) hmpNA (U), absolute configuration (S) -hmpNA (U).

The absolute configuration of (S) -hmpNA (G), (R) -hmpNA (G), (S) -hmpNA (C), (R) -hmpNA (C), (S) -hmpNA (U) and (R) -hmpNA (U) is confirmed by X-Ray diffraction of the intermediate or derivative thereof.

The structure of the intermediate or derivative is as follows:

TJ-NA067, the detection crystal is colorless block (0.30X0.10X0.04 mm 3), belonging to monoclinic system P21 space group. Unit cell parameters α＝90°,β＝118.015(4)°,γ＝90°, Z=4. The density dc=1.389 g/cm3, the electron number F (000) =504.0 in the unit cell, the linear absorption coefficient μ (Cu kα) =0.840 mm-1 in the unit cell, and the diffraction experiment temperature t=150.00 (11) K were calculated.

6A (+) (0.30X0.20X0.10mm3) of colorless block-shaped detection crystal belongs to monoclinic system P21 space group. Unit cell parametersα＝90°,β＝113.876(3)°,γ＝90°, Z=2. The density dc=0.999 g/cm3, the electron number F (000) = 1318.0 in the unit cell, the linear absorption coefficient μ (Cu kα) =0.570 mm-1 of the unit cell, and the diffraction experiment temperature t=100.01 (18) K were calculated.

TJ-NA048, the detection crystal is colorless needle-shaped (0.30X0.04 X0.04 mm 3), belonging to the monoclinic system P1 space group. Unit cell parametersα＝85.007(4)°,β＝88.052(4)°,γ＝70.532(4)°, Z=2. The density dc=1.366 g/cm3, the electron number F (000) =620.0 in the unit cell, the linear absorption coefficient μ (Cu kα) =0.856 mm-1 in the unit cell, and the diffraction experiment temperature t=150.00 (13) K were calculated.

TJ-NA092, the detection crystal is colorless prism (0.30X0.10X0.10mm3), belonging to the triclinic system P1 space group. Unit cell parametersα＝93.146(2)°,β＝101.266(2)°,γ＝96.134(2)°, Z=2. The density dc=1.412 g/cm3, the electron number F (000) =228.0 in the unit cell, the linear absorption coefficient μ (Cu kα) =0.945 mm-1 of the unit cell, and the diffraction experiment temperature t=100.00 (10) K were calculated.

Example 5: sequence dependent experiments with dsrnas comprising different chemical modifications

Abasic modifications are known to have sequence dependencies, and thus the inventors tested the experimental compounds of the present disclosure over a number of different sequences. dsRNA targeting mRNA of different genes (ANGPTL 3, HBV-S, HBV-X) (the sequences are shown in Table 4) was used, the 7 th site of the 5' end of AS strand was modified with the compounds (+) hmpNA (A), (-) hmpNA (A) of example 1 and GNA (A) AS a control (the sequences are shown in Table 5), and then the targeting activity and the off-targeting activity were compared with the parent sequence.

TABLE 4 dsRNA sequences targeting different genes

In the above table, the capital letter G, A, C, U represents a nucleotide containing guanine, adenine, cytosine, and uracil, the lowercase letter m represents a2 '-methoxy modification, the lowercase letter f represents a 2' -fluoro modification, and the lowercase letter s represents a phosphorothioate diester linkage between two nucleotides adjacent to the letter s; the following is the same.

TABLE 5 dsRNA sequences comprising chemical modifications targeting different genes

As a result of the target activity experiment, see Table 6, GNA (A) showed a clear sequence dependence, and the difference of the target activities of different sequences was clear. The experimental compounds of the present disclosure do not show significant sequence dependence and are more universally applicable.

As can be seen by reference to table 7, the experimental compounds of the present disclosure significantly reduced the off-target activity of dsRNA relative to the parent sequence.

TABLE 6 results of on-target Activity of dsRNA against different target sequences

TABLE 7 off-target Activity results of dsRNA against different target sequences

Example 6: preparation of NAG0052, L96

Compounds NAG0024 and NAG0026 were purchased from Tianjin Ming Kangde New drug development Co. Unless otherwise indicated, the reagents used in the examples below are all commercially available.

Compound 3

(3) Synthesis of Compound NAG0052

Starting material compound 1 was purchased from Jiangsu-times pharmaceutical technology Co., ltd. The synthetic route for compound NAG0052 is shown below:

The synthesis and identification of the specific intermediates and end products involved in the above schemes are as follows:

Compound 2

To a solution of Compound 1 (12.3 mL,101 mmol) in THF (300 mL) was added NaH (12.2 g,304mmol, 60% purity) in portions at 0deg.C under nitrogen. The mixture was stirred at 20℃for 1 hour and then cooled to 0℃again, then benzyl bromide (36.3 mL,304 mmol) was added dropwise to the system, and stirred at 20℃for 12 hours. The reaction was quenched with H ₂ O (100 mL) and extracted with EtOAc (200 mL. Times.2). The combined organic phases were washed with saturated brine (100 mL), dried over Na ₂SO ₄, filtered, and the residue obtained by concentration was separated by silica gel column chromatography to give the title compound 2 (20.0 g,51.8mmol, 51% yield).

LCMS:t _R＝2.615 and 2.820min in 30-90AB_7min_220&254_Shimadzu.lcm(Xtimate C18,3um,2.1*30mm),MS(ESI)m/z＝351.2[M+Na] ⁺.

¹H NMR:(400MHz,CDCl ₃)δppm 7.35-7.12(m,10H),5.06-4.95(m,1H),4.51-4.39(m,4H),4.24-3.87(m,2H),3.50-3.40(m,2H),3.38-3.20(m,3H),2.20-1.91(m,2H).

Compounds 3 and 4

To a solution of compound 2 (13.0 g,33.6 mmol) in DCM (300 mL) at 20deg.C under nitrogen is added TMSCN (13.5 mL,101 mmol) in one portion followed by dropwise addition of a solution of TMSOTF (9.14 mL,50.5 mmol) in DCM (30 mL). The reaction solution was stirred at 20℃for 15 hours. After the reaction was completed, the system was quenched with saturated aqueous NaHCO ₃ (80 mL) and extracted with DCM (150 mL x 2), the combined organic phases were washed with saturated brine (80 mL), dried over Na ₂SO ₄, filtered and concentrated to give the title compound 3 (3.30 g,9.18mmol, 27% yield) as a pale yellow oil, compound 4 (8.50 g,9.18mmol, 70% yield) after separation by silica gel column chromatography.

Compound 3

¹H NMR:(400MHz,CDCl ₃)δppm 7.42-7.29(m,10H),4.81(t,J＝7.8Hz,1H),4.65-4.49(m,4H),4.30-4.21(m,2H),3.65-3.57(m,1H),3.57-3.49(m,1H),2.49-2.40(m,2H).

Compound 4

¹H NMR:(400MHz,CDCl ₃)δppm 7.42-7.26(m,10H),4.93-4.87(m,1H),4.65-4.48(m,4H),4.43-4.38(m,1H),4.21-4.17(m,1H),3.79-3.70(m,1H),3.54(d,J＝4.0Hz,1H),2.45-2.37(m,2H).

Compound 5

A solution of Compound 4 (3.00 g,9.28 mmol) in THF (15 mL) was added dropwise to a solution of LiAlH ₄ (0.79 g,20.9 mmol) in THF (15 mL) at 0deg.C under nitrogen, and the system was reacted at 0deg.C for 1 hour after the dropwise addition. TLC (PE: etoac=3:1) monitored complete disappearance of starting material. Sodium sulfate decahydrate was slowly added to the reaction solution until bubbling did not occur. The reaction mixture was then filtered, and the cake was washed three times with methylene chloride (60 mL), and the filtrate was collected and dried to give the objective compound 5 (3.00 g, yield 90%).

¹H NMR:(400MHz,DMSO-d ₆)δppm 7.40-7.14(m,10H),4.54-4.38(m,4H),4.06-3.99(m,2H),3.91(q,J＝6.4Hz,1H),3.48-3.37(m,2H),2.67-2.52(m,2H),2.21-2.18(m,1H),1.77-1.73(m,1H).

Compound 6

Compound 5 (3.00 g,8.25 mmol) was dissolved in DCM (30 mL) under nitrogen and TEA (3.44 mL,24.7 mmol) and CbzCl (1.76 mL,12.4 mmol) were added and reacted at 20℃for 2h. LCMS showed the reaction was complete. The reaction mixture was extracted with dichloromethane (30 mL) and water (60 mL). The organic phase was washed three times with water (60 ml x 3), dried over anhydrous sodium sulfate, and concentrated to dryness on a forward column (PE: etoac=1:1) to give the title compound 6 (2.5 g, 90% yield).

LCMS:t _R＝0.810min in 5-95AB_1min,MS(ESI)m/z＝462.2[M+H] ⁺。

¹H NMR:(400MHz,CDCl ₃)δppm 7.39-7.29(m,15H),5.35(s,1H),5.15-5.01(m,2H),4.72(d,J＝6.0Hz,1H),4.54-4.40(m,3H),4.26(s,1H),4.23-4.18(m,1H),4.11-4.04(m,1H),3.54-3.41(m,3H),3.37-3.25(m,1H),2.34-2.23(m,1H),1.85-1.79(m,1H).

Compound 7

Compound 6 (2.00 g,3.90 mmol) was dissolved in DCM (5 mL) under nitrogen and BCl ₃ in THF (1M, 27.3 mL) was added at-78deg.C and reacted for 1 hour. TLC (DCM: meoh=10:1) monitored complete disappearance of starting material. The reaction was quenched by the addition of methanol (20 mL) at-78 ℃, concentrated and purified by forward column (DCM: meoh=10:1) to give the title compound 7 (2.00 g, 60% yield).

¹H NMR:(400MHz,CD ₃OD)δppm 7.41-7.23(m,5H),5.08(s,2H),4.25-4.07(m,2H),3.85-3.75(m,1H),3.63-3.56(m,1H),3.54-3.48(m,1H),3.30-3.27(m,2H),2.34-2.21(m,1H),1.71-1.64(m,1H).

Compound 8

Compound 7 (0.50 g,1.78 mmol) was dissolved in pyridine (5 mL) under nitrogen, 4A molecular sieve (500 mg) and DMTrCl (0.66 mL,2.13 mmol) were added at 0deg.C, and then warmed to 20deg.C for 1.5 hours. TLC (PE: etoac=2:1) monitored complete disappearance of starting material. The reaction solution was extracted with ethyl acetate (60 mL) and water (60 mL), and the organic phase was washed three times with water (60 ml×3), dried over anhydrous sodium sulfate, concentrated, and purified by a forward column (PE: etoac=1:1) to give the objective compound 8 (800 mg, yield 90%).

¹H NMR:(400MHz,CDCl ₃)δppm 7.44(d,J＝7.6Hz,2H),7.37-7.23(m,11H),7.22-7.15(m,1H),6.84(d,J＝8.8Hz,4H),5.09(s,2H),4.31-4.17(m,2H),4.02-3.91(m,1H),3.84-3.73(m,6H),3.33(s,1H),3.28(s,1H),3.19-3.01(m,2H),2.34-2.25(m,1H),1.70-1.62(m,1H).

Compound 9

Compound 8 (800 mg,1.234 mmol) was dissolved in EtOAc (5 mL) and Pd/C10% (800 mg,7.517 mmol) was added and the reaction was carried out under H ₂ conditions (15 Psi) at 20℃for 1 hour. LCMS showed the reaction was complete. The reaction solution was filtered, and the cake was washed three times with dichloromethane (100 mL) and methanol (100 mL), concentrated, and separated by reverse phase column to give compound 9 (300 mg, 54%).

LCMS:t _R＝2.586min in 10-80CD_3min MS(ESI)m/z＝450.2[M+H] ⁺。

Compound 11

Compound 10 (435 mg,1.780 mmol) was dissolved in DCM (10 mL), DIEA (0.4471 mL,2.67 mmol) and HATU (677 mg,1.78 mmol) were added and then Compound 9 (400 mg,0.890 mmol) was added and reacted at 20℃for 1 hour. TLC (DCM: meoh=10:1) monitored the reaction was complete. The reaction was extracted with dichloromethane (60 mL) and water (60 mL), the organic phase was washed three times with water (60 mL x 3), dried over anhydrous sodium sulfate, concentrated and purified on a forward column (PE: etoac=0:1 column, product peak at 100%) to give the title compound 11 (600 mg, yield 90%).

LCMS:t _R＝2.745min in 30-90CD_3min,MS(ESI)m/z＝698.4[M+Na] ⁺。

¹H NMR:(400MHz,CD ₃OD)δppm 7.46-7.38(m,2H),7.35-7.24(m,6H),7.22-7.16(m,1H),6.90-6.78(m,4H),4.29-4.21(m,2H),4.02-3.95(m,1H),3.77(s,6H),3.66-3.62(m,3H),3.41(s,1H),3.18-3.04(m,2H),2.36-2.17(m,5H),1.71-1.50(m,5H),1.39-1.25(m,14H).

Compound 12

Compound 11 (600 mg,0.799 mmol) was dissolved in THF (3 mL) and H ₂ O (1 mL), liOH.H2 ₂ O (134 mg,3.20 mmol) was added and reacted at 20℃for 12 hours. TLC (DCM: meoh=10:1) showed the reaction was complete. The reaction solution was dried by spin-drying, dissolved in water (5 mL) and methanol (5 mL), and purified by reverse column (H ₂O:CH ₃ cn=1:1, peak around 35%) to give the objective compound 12 (460 mg, yield 100%, lithium salt).

LCMS:t _R＝1.346min in 10-80CD_3min,MS(ESI)m/z＝684.3[M+Na] ⁺。

HPLC:t _R＝1.879min in 10-80CD_6min。

¹H NMR:(400MHz,CD ₃OD)δppm 7.47-7.39(m,2H),7.35-7.24(m,6H),7.22-7.15(m,1H),6.91-6.79(m,4H),4.31-4.18(m,2H),4.02-3.95(m,1H),3.78(s,6H),3.44-3.33(m,2H),3.18-3.04(m,2H),2.35-2.27(m,1H),2.24-2.10(m,4H), 1.70-1.51(m,5H),1.31-1.23(m,12H).

Compound 13

Compound NAG0024 (271mg, 0.151 mmol) was dissolved in anhydrous THF (2 mL) and anhydrous DMF (4 mL) at room temperature under nitrogen, 3A molecular sieve was added followed by compound 12 (100 mg,0.151 mmol), HOBt (25 mg,0.181 mmol), DCC (38 mg,0.181 mmol) and DIEA (39 mg,0.302 mmol) in sequence. The reaction mixture was reacted at 45℃for 16 hours. After the reaction was complete, LC-MS was quenched with water and filtered. After concentration of the filtrate, purification by C18 reverse phase column (H ₂ O/MeCN) afforded compound 13 (210 mg, 57% yield).

Compound NAG0052

Compound 13 (230 mg,0.094 mmol) was dissolved in pyridine (5 mL), molecular sieves were added, DMAP (12 mg,0.283 mmol) was added, and succinic anhydride (28 mg,0.283 mmol) was added at room temperature. The mixture was stirred at 50℃for 16 hours under nitrogen. LCMS detected complete reaction and filtered spin-dry. After purification on a C18 reverse phase column, purification by preparative HPLC gave the title compound NAG0052 (123 mg,0.048mmol, 51% yield).

MS (ESI) m/z=2535.3 [ m-1] ^- theory: 2536.2.

¹H NMR(400MHz,Acetonitrile-d ₃)δ7.48-7.43(m,2H),7.37-7.12(m,11H),7.00-6.85(m,10H),6.66(s,1H),5.31(dd,J＝3.4,1.1Hz,3H),5.20-5.13(m,1H),5.05(dd,J＝11.3,3.4Hz,3H),4.56(d,J＝8.5Hz,3H),4.30(dd,J＝7.7,5.3Hz,1H),4.18-3.93(m,14H),3.79(s,10H),3.65(q,J＝4.7,3.6Hz,13H),3.56-3.07(m,24H),2.56(s,6H),2.37(t,J＝5.8Hz,10H),2.17(t,J＝7.5Hz,9H),2.02-1.96(m,20H),1.88(s,8H),1.82-1.73(m,2H),1.60(dt,J＝15.0,7.3Hz,16H),1.27(s,13H).

(4) Synthesis of L96

L96 of the above formula was prepared according to the method described in patent application WO2014025805A 1.

Synthesis of example 7 dsRNA

1. Homemade resin with carrier

Compound NAG0052 (157 mg,0.062 mmol) containing carboxylic acid groups was dissolved in anhydrous DMF (3 mL), and after complete dissolution of the substrate, anhydrous acetonitrile (4 mL), DIEA (0.03 mL,0.154mmol,2.5 eq) and HBTU (35 mg,0.093mmol,1.5 eq) were added sequentially. After the reaction mixture was mixed uniformly, a large Kong An methyl resin (476 mg, blank load 0.41mmol/g, target load 0.1 mmol/g) was added. The reaction solution was placed on a shaking table (temperature: 25 ℃ C., rotation speed: 200 rpm) and shaken overnight. The reaction was filtered and the filter cake was washed with DCM, anhydrous acetonitrile, respectively, and the solids were collected and dried overnight in vacuo.

The above solid was dispersed in anhydrous acetonitrile (5 mL), pyridine (0.18 mL), DMAP (3 mg), NMI (0.12 mL) and CapB1 (2.68 mL) were added sequentially. The reaction solution was placed on a shaking table (temperature: 25 ℃ C., rotation speed: 200 rpm) and shaken for 2 hours. The reaction solution was filtered, and the filter cake was washed with anhydrous acetonitrile, and the solid was collected and dried under vacuum overnight to give a supported resin. The loading was determined to be 0.1mmol/g.

2. For NAG0052 which has been attached to the resin, nucleoside monomers were attached one by one in the 3'-5' direction in the nucleotide arrangement order, using the resin as a starting point. Each nucleoside monomer attached includes four steps of deprotection, coupling, capping, oxidation or vulcanization. The operation is conventional in the art.

The compound NAG0052 is connected to the sequence through solid phase synthesis, and after aminolysis, a part of functional groups of NAG0052 structure are removed to become NAG0052'.

The resulting dsRNA had sense and antisense strands shown in tables 8 and 9.

TABLE 8 list of dsRNAs

DsRNA numbering	Sense strand numbering	Antisense strand numbering
TRD002218	TJR4373-SS	TJR0414-AS
TRD007205	TJR013485S	TJR0414-AS

TABLE 9 nucleic acid sequences of sense and antisense strands

The structure of the above ligand is as follows:

wherein TRD002218 is used as a reference positive compound, and Z represents siRNA.

Example 8 dsRNA inhibition of target Gene mRNA expression in vivo

The experiment examines the inhibition efficiency of dsRNA conjugated with different structures to the mRNA expression quantity of a target gene in vivo

Male 6-8 week old C57BL/6 mice were randomly grouped, 6 in each group, 3 at each time point, and TRD007205, reference positive TRD002218, and PBS were administered to each group of mice, respectively.

All animals calculated the dose in total on a per-body basis, and were given in a single dose by subcutaneous injection, with dsRNA (in terms of siRNA) doses of 1mg/kg and 5mL/kg of dose volume. Mice were sacrificed 7 days and 28 days after dosing, livers were collected and saved with RNA later (SIGMA ALDRICH Co.); subsequently, liver tissues are homogenized by a tissue homogenizer, and total RNA of the liver tissues is extracted by using a tissue RNA extraction kit (known medical science and technology, FG 0412) according to the operation steps marked by the operation instructions. The total RNA is reversely transcribed into cDNA and the expression quantity of TTR mRNA in liver tissue is detected by adopting a real-time fluorescence quantitative PCR method. In the fluorescent quantitative PCR method, the mRNA expression levels of TTR and GAPDH are detected using Taqman probe primers for TTR and GAPDH, respectively, using glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene as an internal reference gene.

Table 10. Grouping information of experimental compounds in mice:

Table 11. Sequences of detection primers are as follows:

TTR mRNA expression amount was calculated according to the following equation:

TTR mRNA expression level= [ test set TTR mRNA expression level/test set GAPDH MRNA expression level)/(control set TTR mRNA expression level/control set GAPDH MRNA expression level) ]x100%.

The inhibition efficiency of dsRNA conjugated with different structures of the present disclosure on the target gene mRNA expression level in vivo 7 days and 28 days after administration is shown in fig. 1 and 2, respectively. From the results shown in fig. 1, TRD007205 had a good effect on inhibition of TTR mRNA expression at 7 days after administration. As can be seen from FIG. 2, the TRD007205 had better effect of inhibiting the expression level of mRNA of the target gene than TRD002218 after 28 days of administration.

Example 9 Synthesis of dsRNA

1. Homemade resin with carrier

The specific operation was the same as in example 7.

2. Using a resin with NAG0052 as a starting material, nucleoside monomers were linked one by one in the nucleotide arrangement order from the 3'-5' direction. Each nucleoside monomer attached includes four steps of deprotection, coupling, capping, oxidation or vulcanization. Reference is made specifically to the synthetic method of example 2.

The dsRNA produced had sense and antisense strands shown in table 12 and table 13. The corresponding naked sequences are shown in table 14.

TABLE 12 list of dsRNAs

TABLE 13 sense and antisense strands of dsRNA

TABLE 14 naked sequences corresponding to the nucleic acid sequences of the sense and antisense strands of dsRNA

Wherein, (-) hmpNA (a), (-) hmpNA (G), (-) hmpNA (C), (-) hmpNA (U) are structured as described in example 4.

NAG0052' has the structure:

The structure of A (GNA) is:

The structure of L10 is:

Example 10 dsRNA on-target Activity against different targets

In vitro molecular level simulation of dsRNA was screened for target activity in HEK293A cells using 9 concentration gradients.

The human APOC3 gene is used to construct the target sequence corresponding to dsRNA, and the target sequence is inserted into psiCHECK-2 plasmid. The plasmid contains a Renilla luciferase gene and a firefly luciferase gene. As a Dual reporter system, the target sequence of dsRNA was inserted into the 3' UTR region of Renilla luciferase gene, and the activity of dsRNA on the target sequence was reflected by detection of Renilla luciferase expression after firefly luciferase calibration, using Dual-Luciferase Reporter ASSAY SYSTEM (Promega, E2940).

HEK293A cells were cultured in DMEM high sugar medium containing 10% fetal bovine serum at 37 ℃ under 5% co ₂. 24h before transfection, HEK293A cells were seeded in 96-well plates at a density of 8X 10 ³ cells per well and 100. Mu.L of medium per well.

According to the instructions, the cells were co-transfected with dsRNA and the corresponding plasmid using Lipofectamine2000 (ThermoFisher, 11668019), using 0.2. Mu.LL per well of Lipofectamine 2000. Plasmid transfection was 20ng per well. For the plasmid in the target sequence, the dsRNA was set to 9 concentration points in total, with the highest concentration point at a final concentration of 20nM, 3-fold gradient dilution, 20nM,6.6667nM,2.2222nM,0.7407nM,0.2469nM,0.0823nM,0.0274nM,0.0091nM,0.0030nM. 24h after transfection, the target level was detected using Dual-Luciferase Reporter ASSAY SYSTEM (Promega, E2940). The on-target activity of the detected sequences is shown in Table 15. The results indicate that the TRD007972, TRD007996, TRD007997, TRD007972-1, TRD007996-1, TRD007997-1 compounds have high levels of on-target inhibitory activity against the APOC3 gene in the psiCHECK system.

TABLE 15 screening results of dsRNA psi-CHECK for target Activity

Example 11 dsRNA inhibitory Activity against human APOC3 in human primary hepatocytes (PHH) -7 concentration points

7 Concentration gradients were used in human primary hepatocytes (PHH) for screening dsRNA for human primary hepatocytes (PHH) activity. The final transfection concentration for each dsRNA sample was 20nM, 5-fold gradient dilution and 7 concentration points.

PHH was frozen in liquid nitrogen 24h before transfection, and human primary hepatocytes (PHH) were resuscitated and plated in 96-well plates at a plating density of 3X 10 ⁴ cells per well, 80. Mu.L of medium per well.

Referring to the product instruction manual, lipofectamine RNAi MAX (thermo fisher, 13778150) was used to transfect dsRNA, with a gradient final concentration of 10nM,2nM,0.4nM,0.08nM,0.016 nM,0.0032nM and 0.00064nM for dsRNA transfection. After 24 hours of treatment, total cellular RNA extraction, RNA reverse transcription experiments (Takara, 6210B) and quantitative real-time PCR detection (thermo fisher, 4444557) were performed using a high throughput cellular RNA extraction kit, the mRNA levels of human APOC3 were determined and corrected based on GAPDH reference gene levels.

In the quantitative real-time PCR detection, a probe Q-PCR detection experiment was used, and the primer information is shown in Table 16.

TABLE 16 Taqman primer information Table

Primer name	SEQ ID NO	Primer sequences
hAPOC3-PF	46	TGCCTCCCTTCTCAGCTTCA
hAPOC3-PR	47	GGGAACTGAAGCCATCGGTC
hAPOC3-P	48	ATGAAGCACGCCACCAAGACCGCCA
hGAPDH-PF1-MGB	49	GACCCCTTCATTGACCTCAACTAC
hGAPDH-PR1-MGB	50	TTGACGGTGCCATGGAATTT
hGAPDH-P1-MGB	51	TTACATGTTCCAATATGATTCC

The result analysis method comprises the following steps:

After the Q-PCR detection experiment is finished, corresponding Ct values are obtained according to the threshold value automatically set by the system, and the expression of a certain gene can be relatively quantified through Ct value comparison: comparing Ct refers to calculating the difference in gene expression by the difference between the Ct values of the reference genes, also referred to as 2 ^-△△Ct, ΔΔct= [ (Ct experimental group gene of interest-Ct experimental group reference) - (Ct control group gene of interest-Ct control group reference) ]. Inhibition ratio (%) = (residual amount of 1-target gene expression) ×100%.

Results are expressed as the remaining percentage of human APOC3mRNA expression relative to dsRNA treated cells. The results of IC ₅₀ for inhibition are shown in Table 17.

The results showed that TRD007972, TRD007996, TRD007997, TRD007972-1, TRD007996-1, TRD007997-1 had high levels of on-target inhibitory activity against the APOC3 gene in PHH cells.

TABLE 17 Multi-dose inhibitory Activity of dsRNA in PHH cells

EXAMPLE 12 psiCHECK antisense strand (AS strand) off-target level verification

In vitro molecular level simulated off-target level screening was performed on dsRNA using 9 concentration gradients in HEK-293A cells. The results indicate that the dsrnas of the present disclosure have high activity while also having low off-targeting. Experimental procedure for psiCHECK activity screening assay reference example 11.

Constructing off-target sequence corresponding to dsRNA sequence and inserting into psiCHECK-2 plasmid. The plasmid contains a Renilla luciferase gene and a firefly luciferase gene. As a Dual reporter system, the target sequence of dsRNA was inserted into the 3' UTR region of Renilla luciferase gene, and the activity of dsRNA on the target sequence was reflected by detection of Renilla luciferase expression after firefly luciferase calibration, using Dual-Luciferase Reporter ASSAY SYSTEM (Promega, E2940).

The GSSM target plasmid construction rule corresponding to the dsRNA is as follows:

Aiming at the antisense strand, constructing a targeting plasmid GSSM which is completely complementary with 1-8 positions of the 5' end of the antisense strand and is completely mismatched with bases at other positions, wherein the base mismatch correspondence rules are that A and C are matched and G and T are matched.

The results are shown in Table 18.

The results indicate that TRD007972-1 does not have the risk of seed region off-target.

TABLE 18 screening results for off-target Activity of the AS-chain seed region psiCHECK (GSSM)

Claims

A double-stranded ribonucleic acid (dsRNA), wherein the double-stranded ribonucleic acid comprises an siRNA comprising a sense strand and an antisense strand, and one or more ligands conjugated thereto, the antisense strand comprising a chemical modification represented by formula (I), a tautomer thereof, or a pharmaceutically acceptable salt thereof, at least one nucleotide position from position 2 to position 8 of the 5' end thereof:

The chemical modification shown in the formula (I) is selected from any one of the following structures:

B is each independently a base at the position corresponding to the 2 nd to 8 th positions of the 5' end of the antisense strand;

the ligand has the following structure or a pharmaceutically acceptable salt thereof:

The siRNA is an siRNA targeting an apolipoprotein C3 (APOC 3) gene.
The dsRNA of claim 1, wherein the sense strand of the siRNA comprises at least 15 consecutive nucleotides differing by NO more than 3 nucleotides from the nucleotide sequence of any one of SEQ ID NOs 1 to 4, and/or,

The antisense strand comprises at least 19 consecutive nucleotides differing by NO more than 3 nucleotides from the nucleotide sequence of either of SEQ ID NO. 5 or SEQ ID NO. 6;

Preferably, the sense strand of the siRNA comprises the nucleotide sequence shown in any one of SEQ ID NO. 1 to SEQ ID NO. 4, and/or,

The antisense strand comprises the nucleotide sequence shown in any one of SEQ ID NO. 5 or SEQ ID NO. 6;

More preferably, the process is carried out,

The sense strand of the siRNA comprises a nucleotide sequence shown as SEQ ID NO. 1, and the antisense strand comprises a nucleotide sequence shown as SEQ ID NO. 5;

The sense strand comprises the nucleotide sequence shown in SEQ ID NO. 2, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO. 5;

The sense strand comprises the nucleotide sequence shown in SEQ ID NO. 3, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO. 6; or (b)

The sense strand comprises the nucleotide sequence shown in SEQ ID NO. 4 and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO. 6.
The dsRNA of claim 1 or 2, wherein the 3' end of the sense strand is conjugated to the ligand.
The dsRNA of any one of claims 1-3, wherein said ligand is linked to the 3' end of said siRNA sense strand by a phosphate group or a phosphorothioate group; preferably through a phosphodiester group or a phosphorothioate diester group, more preferably through a phosphodiester group.
The dsRNA of any one of claims 1-4, wherein the chemically modified, tautomer thereof or pharmaceutically acceptable salt thereof modified nucleotide represented by formula (I) is located at position 5, 6 or 7, preferably at position 7, of the 5' end of the antisense strand.
The dsRNA of any of claim 1-5, wherein,

At least one additional nucleotide in the sense strand and/or the antisense strand is a modified nucleotide.
The dsRNA of any of claim 1-6, wherein,

The sense strand comprises or consists of a nucleotide sequence represented by the formula:

5'-N _aN _aN _aN _aN _aN _aN _bN _bN _bN _aN _aN _aN _aN _aN _aN _aN _aN _aN _a-3'; Or alternatively, the first and second heat exchangers may be,

5'-N _aN _aN _aN _aN _bN _aN _bN _bN _bN _aN _aN _aN _aN _aN _aN _aN _aN _aN _a-3';

Wherein N _a is a2 '-methoxy modified nucleotide and N _b is a 2' -fluoro modified nucleotide.
The dsRNA of any one of claims 1-7, wherein said antisense strand comprises or consists of a nucleotide sequence represented by the formula:

5'-N _a'N _b'N _a'N _b'N _a'N _b'W'N _a'N _a'N _b'N _a'N _b'N _a'N _b'N _a'N _b'N _a'N _b'N _a'N _a'N _a'-3';

Wherein,

N _a 'is a 2' -methoxy modified nucleotide and N _b 'is a 2' -fluoro modified nucleotide;

w' represents a nucleotide comprising a chemical modification selected from the group consisting of the following structures:

Wherein: b corresponds to the base at position 7 of the 5' -end of the antisense strand.
The dsRNA of any one of claims 1-8, wherein at least one phosphate group in the sense strand and/or antisense strand is a phosphate group having a modifying group; preferably, the phosphate group having a modifying group is a phosphorothioate diester group.
The dsRNA of claim 9, wherein said phosphorothioate diester group is present in at least one of the following positions:

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

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

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

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

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

The 3' end of the antisense strand is between nucleotide 2 and nucleotide 3;

Preferably, the method comprises the steps of,

The sense strand and/or antisense strand includes a plurality of phosphorothioate diester groups therein, the phosphorothioate diester groups being present in:

Between nucleotide 1 and nucleotide 2 of the 5' end of the sense strand; and, a step of, in the first embodiment,

Between nucleotide 2 and nucleotide 3 of the 5' end of the sense strand; and, a step of, in the first embodiment,

The 5' end of the antisense strand is between nucleotide 1 and nucleotide 2; and, a step of, in the first embodiment,

The 5' end of the antisense strand is between nucleotide 2 and nucleotide 3; and, a step of, in the first embodiment,

The 3' end of the antisense strand is between nucleotide 1 and nucleotide 2; and, a step of, in the first embodiment,

The 3' end of the antisense strand is between nucleotide 2 and nucleotide 3.
Wherein, the dsRNA comprises a double-stranded wire,

The dsRNA comprises a sense strand shown in SEQ ID NO. 8 and an antisense strand shown in SEQ ID NO. 17; or alternatively, the first and second heat exchangers may be,

Comprises a sense strand shown in SEQ ID NO. 7 and an antisense strand shown in SEQ ID NO. 17; or alternatively, the first and second heat exchangers may be,

Comprises a sense strand shown in SEQ ID NO. 9 and an antisense strand shown in SEQ ID NO. 17; or alternatively, the first and second heat exchangers may be,

Comprises a sense strand shown in SEQ ID NO. 11 and an antisense strand shown in SEQ ID NO. 18; or alternatively, the first and second heat exchangers may be,

Comprises a sense strand shown in SEQ ID NO. 13 and an antisense strand shown in SEQ ID NO. 18; or alternatively, the first and second heat exchangers may be,

Comprises a sense strand shown in SEQ ID NO. 10 and an antisense strand shown in SEQ ID NO. 17; or alternatively, the first and second heat exchangers may be,

Comprises a sense strand shown in SEQ ID NO. 12 and an antisense strand shown in SEQ ID NO. 18; or alternatively, the first and second heat exchangers may be,

Comprising a sense strand shown as SEQ ID NO. 14 and an antisense strand shown as SEQ ID NO. 18.
The dsRNA of any one of claims 1-11, wherein said dsRNA is selected from the following structures or pharmaceutically acceptable salts thereof:

Wherein Af = adenine 2' -F ribonucleoside; cf=cytosine 2' -F ribonucleoside; uf = uracil 2' -F ribonucleoside; am = adenine 2' -OMe ribonucleoside; cm = cytosine 2' -OMe ribonucleoside; gf = guanine 2' -F ribonucleoside; gm=guanine 2' -OMe ribonucleosides; um = uracil 2' -OMe ribonucleoside. Represents a phosphorothioate diester group, and,Represents a group of a phosphoric acid diester,

NAG0052' represents

(-) HmpNA (A) represents
A pharmaceutical composition comprising the dsRNA of any one of claims 1-12; preferably, the pharmaceutical composition further comprises one or more pharmaceutically acceptable excipients.
Use of the dsRNA of any one of claims 1-12 or the pharmaceutical composition of claim 13 in the manufacture of a medicament;

The medicament is for reducing the level of low density lipoprotein in a subject, or for preventing and/or treating a disease mediated by elevated triglyceride levels or elevated cholesterol levels; preferably, the disorder mediated by elevated triglyceride levels or elevated cholesterol levels is selected from the group consisting of hypertriglyceridemia, obesity, hyperlipidemia, lipid and/or cholesterol metabolic abnormalities, atherosclerosis, cardiovascular disease, coronary artery disease, hypertriglyceridemia-induced pancreatitis, metabolic syndrome, type II diabetes, familial chylomicronemia syndrome, or familial partial lipid malnutrition.
A method of inhibiting expression of an APOC3 gene or mRNA thereof comprising administering to a subject an effective amount or effective dose of the dsRNA of any one of claims 1-12 or the pharmaceutical composition of claim 13.
A method of delivering an oligonucleotide to the liver comprising administering to a subject an effective amount or effective dose of the dsRNA of any one of claims 1-12 or the pharmaceutical composition of claim 13.
A cell comprising the dsRNA of any one of claims 1-12.
A vector comprising the dsRNA of any one of claims 1-12.
A kit comprising the dsRNA of any one of claims 1-12 or the pharmaceutical composition of any one of claims 13.
A method of making a dsRNA or pharmaceutical composition comprising: synthesizing a ligand, siRNA, dsRNA or pharmaceutical composition according to any one of claims 1-12 or claim 13.