Search

US-20260125681-A1 - DOUBLE-STRANDED RNAi AGENTS FOR TARGETING AND REGULATING HBV GENE EXPRESSION, AND A USE THEREOF

US20260125681A1US 20260125681 A1US20260125681 A1US 20260125681A1US-20260125681-A1

Abstract

The present disclosure provides a double-stranded RNAi agent for targeting and regulating HBV gene expression and a use thereof. The double-stranded RNAi agent comprises an antisense strand and a sense strand complementary to the antisense strand forming a duplex region. The nucleotide sequence of the antisense strand is as shown in SEQ ID NO: 8, 11, or 13, or the nucleotide sequence of the antisense strand is a modified sequence of the sequence shown in SEQ ID NO: 8, 11, or 13. Results from cell and animal experiments demonstrate that the double-stranded RNAi agent provided in the present disclosure can significantly reduce the expression of one or more HBV genes, block the viral life cycle, and can be used to develop drugs for treating diseases related to HBV gene expression.

Inventors

  • Zeao HUANG
  • Gengshen Song
  • Shuo Yang
  • Yang Yang
  • Zhikang TIAN
  • Yucheng Wu
  • Kai XIONG
  • Zhongcai Gao

Assignees

  • HANGZHOU TIANLONG PHARMACEUTICAL CO., LTD.

Dates

Publication Date
20260507
Application Date
20250708
Priority Date
20240708

Claims (20)

  1. 1 . A double-stranded RNAi agent, wherein the double-stranded RNAi agent comprises an antisense strand and a sense strand complementary to the antisense strand forming a double-stranded region, wherein a nucleotide sequence of the antisense strand is as shown in SEQ ID NO: 8, 11, or 13, or a nucleotide sequence of the antisense strand is a modified sequence of a sequence shown in SEQ ID NO: 8, 11, or 13.
  2. 2 . The double-stranded RNAi agent according to claim 1 , wherein the double-stranded RNAi agent comprises an oligonucleotide duplex formed by pairing of the sense strand and the antisense strand; wherein the sense strand has a sequence as shown in SEQ ID NO: 1, 4, or 6, or a fragment thereof, or a modified sequence of the sequence or fragment thereof, preferably, wherein the sense strand and the antisense strand comprise at least one modified nucleotide, preferably wherein at least one of the modified nucleotides is selected from one or more of the group consisting of: deoxy-nucleotide, 3′-terminal deoxy-thymidine nucleotide, 2′-O-methyl-modified nucleotide, 2′-fluoro-modified nucleotide, 2′-deoxy-modified nucleotide, locked nucleotide, unlocked nucleotide, conformationally restricted nucleotide, constrained ethyl nucleotide, abasic nucleotide, 2-amino-modified nucleotide, 2′-O-allyl-modified nucleotide, 2′-C-alkyl-modified nucleotide, 2′-hydroxy-modified nucleotide, 2′-O-methoxyethyl-modified nucleotide, 2′-O-alkyl-modified nucleotide, morpholino nucleotide, phosphoramidate, nucleotide containing unnatural bases, tetrahydropyran-modified nucleotide, 1,5-anhydrohexitol-modified nucleotide, cyclohexenyl-modified nucleotide, nucleotide containing phosphorothioate groups, nucleotide containing methylphosphonate groups, nucleotide containing 5′-phosphate, and nucleotide containing 5′-phosphate mimetics.
  3. 3 . The double-stranded RNAi agent according to claim 1 , wherein the double-stranded RNAi agent has the function of inhibiting HBV gene expression.
  4. 4 . The double-stranded RNAi agent according to claim 1 , wherein at least one strand of the double-stranded RNAi agent comprises a 3′ overhang of at least 2 nucleotides, or wherein the duplex region of the double-stranded RNAi agent is 20 nucleotide pairs in length, or wherein the sense strand of the double-stranded RNAi agent has 20 nucleotides, and the antisense strand has 22 nucleotides, or wherein all modifications on the nucleotides of the sense and antisense strands are chemical modifications at the 2′ position of a nucleotide ribose, preferably wherein the chemical modifications at the 2′ position of the nucleotide ribose are selected from one or more of the group consisting of: 2′-methoxy, 2′-O-methoxyethyl, 2′-fluoro, 2′-benzyloxy, 2′-methylcarbonylamino, and 2′-pyridylmethoxy, preferably wherein the chemical modification at the 2′ position of the nucleotide ribose is 2′-methoxy or 2′-fluoro.
  5. 5 . The double-stranded RNAi agent according to claim 1 , wherein the nucleotides are linked by 3′,5′-phosphodiester bonds, preferably wherein the 3′,5′-phosphodiester bond comprises a phosphorothioate modification.
  6. 6 . The double-stranded RNAi agent according to claim 1 , wherein a 5′ carbon atom of the 5′ terminal nucleotide glycoside of the antisense strand is phosphorylated, preferably wherein the phosphorylated 5′ phosphorylated group includes one or more selected from 5′-vinylphosphonate group, 5′-methylphosphonate group, 5′-C-methylphosphate group, 5′-phosphorothioate group, and 5′-phosphate group, with the structure being R is hydrogen, hydroxyl, amino, C 1-4 alkyl, aryl, C 1-4 alkoxy, C 1-4 alkylcarbonylamino, or halogen; the base is selected from any one of adenine, guanine, cytosine, thymine, and uracil.
  7. 7 . The double-stranded RNAi agent according to claim 1 , wherein terminal nucleotides of the sequence are linked by a 3′,5′-phosphodiester bond containing a phosphorothioate modification, forming a chirally pure 3′,5′-phosphorothioate bond, preferably wherein a 5′ end of the sense strand and the antisense strand comprises 1 to 3 phosphorothioate linkages, and a 3′ end of the antisense strand comprises 1 to 3 phosphorothioate linkages.
  8. 8 . The double-stranded RNAi agent according to claim 1 , wherein the antisense strand adopts one of the modification patterns outlined in the table below: Antisense strand (AS) 5′-3′ No. 1 2 3 4 5 6 7 8 9 10 11 A 2′-OMe + PS + EVP 2′-F + PS 2′-OMe 2′-F 2′-F 2′-F 2′-OMe 2′-OMe 2′-OMe 2′-OMe 2′-OMe B 2′-OMe + PS + EVP 2′-F + PS 2′-F 2′-F 2′-OMe 2′-F 2′-OMe 2′-OMe 2′-OMe 2′-OMe 2′-OMe C 2′-OMe + PS + EVP 2′-F + PS 2′-OMe 2′-OMe 2′-F 2′-F 2′-OMe 2′-OMe 2′-OMe 2′-OMe 2′-OMe D 2′-OMe + PS + EVP 2′-F + PS 2′-F 2′-OMe 2′-OMe 2′-F 2′-OMe 2′-OMe 2′-OMe 2′-OMe 2′-OMe E 2′-OMe + PS + EVP 2′-F + PS 2′-OMe 2′-OMe 2′-OMe 2′-F 2′-OMe 2′-OMe 2′-OMe 2′-OMe 2′-OMe F 2′-OMe + PS + EVP 2′-F + PS 2′-F 2′-OMe 2′-F 2′-F 2′-OMe 2′-OMe 2′-OMe 2′-OMe 2′-OMe Antisense strand (AS) 5′-3′ No. 12 13 14 15 16 17 18 19 20 21 22 A 2′-OMe 2′-OMe 2′-F 2′-OMe 2′-F 2′-OMe 2′-OMe 2′-OMe 2′-OMe + PS 2′-OMe + PS 2′-OMe B 2′-OMe 2′-OMe 2′-F 2′-OMe 2′-F 2′-OMe 2′-OMe 2′-OMe 2′-OMe + PS 2′-OMe + PS 2′-OMe C 2′-OMe 2′-OMe 2′-F 2′-OMe 2′-F 2′-OMe 2′-OMe 2′-OMe 2′-OMe + PS 2′-OMe + PS 2′-OMe D 2′-OMe 2′-OMe 2′-F 2′-OMe 2′-F 2′-OMe 2′-OMe 2′-OMe 2′-OMe + PS 2′-OMe + PS 2′-OMe E 2′-OMe 2′-OMe 2′-F 2′-OMe 2′-F 2′-OMe 2′-OMe 2′-OMe 2′-OMe + PS 2′-OMe + PS 2′-OMe F 2′-OMe 2′-OMe 2′-F 2′-OMe 2′-F 2′-OMe 2′-OMe 2′-OMe 2′-OMe + PS 2′-OMe + PS 2′-OMe ; or Antisense strand (AS) 5′-3′ No. 1 2 3 4 5 6 7 8 9 10 11 12 A 2′-OMe + PS + EVP 2′-F + PS 2′-OMe 2′-F 2′-F 2′-F 2′-OMe 2′-OMe 2′-OMe 2′-OMe 2′-OMe 2′-OMe B 2′-OMe + PS + EVP 2′-F + PS 2′-F 2′-F 2′-OMe 2′-F 2′-OMe 2′-OMe 2′-OMe 2′-OMe 2′-OMe 2′-OMe C 2′-OMe + PS + EVP 2′-F + PS 2′-OMe 2′-OMe 2′-F 2′-F 2′-OMe 2′-OMe 2′-OMe 2′-OMe 2′-OMe 2′-OMe D 2′-OMe + PS + EVP 2′-F + PS 2′-F 2′-OMe 2′-OMe 2′-F 2′-OMe 2′-OMe 2′-OMe 2′-OMe 2′-OMe 2′-OMe E 2′-OMe + PS + EVP 2′-F + PS 2′-OMe 2′-OMe 2′-OMe 2′-F 2′-OMe 2′-OMe 2′-OMe 2′-OMe 2′-OMe 2′-OMe F 2′-OMe + PS + EVP 2′-F + PS 2′-F 2′-OMe 2′-F 2′-F 2′-OMe 2′-OMe 2′-OMe 2′-OMe 2′-OMe 2′-OMe Antisense strand (AS) 5′-3′ No. 13 14 15 16 17 18 19 20 21 22 23 A 2′-OMe 2′-F 2′-OMe 2′-F 2′-OMe 2′-OMe 2′-OMe 2′-OMe 2′-OMe + PS 2′-OMe + PS 2′-OMe B 2′-OMe 2′-F 2′-OMe 2′-F 2′-OMe 2′-OMe 2′-OMe 2′-OMe 2′-OMe + PS 2′-OMe + PS 2′-OMe C 2′-OMe 2′-F 2′-OMe 2′-F 2′-OMe 2′-OMe 2′-OMe 2′-OMe 2′-OMe + PS 2′-OMe + PS 2′-OMe D 2′-OMe 2′-F 2′-OMe 2′-F 2′-OMe 2′-OMe 2′-OMe 2′-OMe 2′-OMe + PS 2′-OMe + PS 2′-OMe E 2′-OMe 2′-F 2′-OMe 2′-F 2′-OMe 2′-OMe 2′-OMe 2′-OMe 2′-OMe + PS 2′-OMe + PS 2′-OMe F 2′-OMe 2′-F 2′-OMe 2′-F 2′-OMe 2′-OMe 2′-OMe 2′-OMe 2′-OMe + PS 2′-OMe + PS 2′-OMe and/or the sense strand adopts one of the modification patterns outlined in the table below: Sense strand (SS) 5′-3′ No. 1 2 3 4 5 6 7 8 9 10 a 2′-OMe + PS 2′-OMe + PS 2′-OMe 2′-OMe 2′-OMe 2′-F 2′-OMe 2′-F 2′-F 2′-F b 2′-OMe + PS 2′-OMe + PS 2′-OMe 2′-OMe 2′-OMe 2′-F 2′-OMe 2′-F 2′-OMe 2′-F Sense strand (SS) 5′-3′ No. 11 12 13 14 15 16 17 18 19 20 a 2′-OMe 2′-OMe 2′-OMe 2′-OMe 2′-OMe 2′-F 2′-OMe 2′-OMe 2′-OMe 2′-OMe b 2′-OMe 2′-OMe 2′-OMe 2′-OMe 2′-OMe 2′-F 2′-OMe 2′-OMe 2′-OMe 2′-OMe or Sense strand (SS) 5′-3′ No. 1 2 3 4 5 6 7 8 9 10 11 a 2′-OMe + PS 2′-OMe + PS 2′-OMe 2′-OMe 2′-OMe 2′-OMe 2′-F 2′-OMe 2′-F 2′-F 2′-F b 2′-OMe + PS 2′-OMe + PS 2′-OMe 2′-OMe 2′-OMe 2′-OMe 2′-F 2′-OMe 2′-F 2′-OMe 2′-F Sense strand (SS) 5′-3′ No. 12 13 14 15 16 17 18 19 20 21 a 2′-OMe 2′-OMe 2′-OMe 2′-OMe 2′-OMe 2′-F 2′-OMe 2′-OMe 2′-OMe 2′-OMe b 2′-OMe 2′-OMe 2′-OMe 2′-OMe 2′-OMe 2′-F 2′-OMe 2′-OMe 2′-OMe 2′-OMe wherein 2′-OMe is 2′-methoxy; 2′-F is 2′-fluoro; PS is phosphorothioate backbone; EVP is 5′-(E)-vinylphosphonate, preferably wherein the modification patterns of the double-stranded RNAi agent are as follows: the antisense strand is modified by pattern A and the sense strand modified by pattern a; the antisense strand is modified by pattern A and the sense strand modified by pattern b; the antisense strand is modified by pattern B and the sense strand modified by pattern a; the antisense strand is modified by pattern B and the sense strand modified by pattern b; the antisense strand is modified by pattern C and the sense strand modified by pattern b; the antisense strand is modified by pattern D and the sense strand modified by pattern b; the antisense strand is modified by pattern E and the sense strand modified by pattern a; or the antisense strand is modified by pattern F and the sense strand modified by pattern b.
  9. 9 . The double-stranded RNAi agent according to claim 1 , wherein the antisense strand is modified with modifying groups from the second to the eighth positions starting from the 5′ end, where the modifying groups are selected from one or more of UNA, GNA, and DNA, and the structures of UNA and GNA are the base is selected from any one of adenine, guanine, cytosine, thymine, and uracil.
  10. 10 . The double-stranded RNAi agent according to claim 1 , wherein the double-stranded RNAi agent comprises an oligonucleotide duplex selected from any one of the following sense and antisense strand pairs: (1) the sense strand has the sequence as shown in SEQ ID NO: 81, and the antisense strand has the sequence as shown in SEQ ID NO: 179, 180, 181, 182, or 184; (2) the sense strand has the sequence as shown in SEQ ID NO: 82, and the antisense strand has the sequence as shown in SEQ ID NO: 179, 180, or 183; (3) the sense strand has the sequence as shown in SEQ ID NO: 83, and the antisense strand has the sequence as shown in SEQ ID NO: 185 or 186; (4) the sense strand has the sequence as shown in SEQ ID NO: 84, and the antisense strand has the sequence as shown in SEQ ID NO: 186; (5) the sense strand has the sequence as shown in SEQ ID NO: 85, and the antisense strand has the sequence as shown in SEQ ID NO: 186; (6) the sense strand has the sequence as shown in SEQ ID NO: 96, and the antisense strand has the sequence as shown in SEQ ID NO: 201, 202, 203, or 204; (7) the sense strand has the sequence as shown in SEQ ID NO: 97, and the antisense strand has the sequence as shown in SEQ ID NO: 201 or 202; (8) the sense strand has the sequence as shown in SEQ ID NO: 98, and the antisense strand has the sequence as shown in SEQ ID NO: 205 or 206; (9) the sense strand has the sequence as shown in SEQ ID NO: 99, and the antisense strand has the sequence as shown in SEQ ID NO: 206; (10) the sense strand has the sequence as shown in SEQ ID NO: 100, and the antisense strand has the sequence as shown in SEQ ID NO: 206; (11) the sense strand has the sequence as shown in SEQ ID NO: 106, and the antisense strand has the sequence as shown in SEQ ID NO: 213, 214, 215, or 216; (12) the sense strand has the sequence as shown in SEQ ID NO: 107, and the antisense strand has the sequence as shown in SEQ ID NO: 214 or 213; (13) the sense strand has the sequence as shown in SEQ ID NO: 108, and the antisense strand has the sequence as shown in SEQ ID NO: 217 or 218; (14) the sense strand has the sequence as shown in SEQ ID NO: 109, and the antisense strand has the sequence as shown in SEQ ID NO: 218; (15) the sense strand has the sequence as shown in SEQ ID NO: 110, and the antisense strand has the sequence as shown in SEQ ID NO: 218; (16) the sense strand has the sequence as shown in SEQ ID NO: 306, and the antisense strand has the sequence as shown in SEQ ID NO: 321.
  11. 11 . A conjugate, wherein the conjugate comprises the double-stranded RNAi agent according to claim 1 and a ligand conjugated to the double-stranded RNAi agent, preferably wherein the ligand is conjugated to a 3′-end or a 5′-end of an oligonucleotide sense strand, or wherein the ligand is one or more GalNAc derivatives attached via a bivalent or trivalent branched linker, or a GalNAc derivative attached via a monovalent linker, or wherein the ligand is: wherein X is hydrogen, a hydroxyl protecting group, or H, and the hydroxyl protecting group includes acetyl, benzoyl, or isobutyryl; Y is an amine protecting group or H, and the amine protecting group is formyl, acetyl, propionyl, n-butyryl, or isobutyryl; n is an integer between 0 and 20; q, r, and s are independently integers between 1 and 7, or wherein the ligand is:
  12. 12 . The conjugate according to claim 11 , wherein the ligand is: wherein X is oxygen, nitrogen, or sulfur; Y is an alkyl or aromatic group; R 1 is oxygen or sulfur; R 2 is hydrogen, an amino group, a C 1-4 alkyl group, an aromatic group, a C 1-4 alkoxy group, or a halogen; A is —(CH 2 ) a —, —(CH 2 CH 2 O) b —, —((CH 2 ) c NHCO) d —, or —((CH 2 ) c CONH) d —, where a is an integer from 1 to 15, b is an integer from 1 to 7, c is an integer from 1 to 7, and d is an integer from 1 to 5; B is —(CH 2 ) e —, where e is an integer from 0 to 7; L is —CONH— or —NHCO—; X 1 is —(CH 2 ) f — or —(CH 2 CH 2 O)CH 2 —, where f is an integer from 1 to 5; X 2 is —(CH 2 ) g —, where g is an integer from 1 to 6; Y 1 is 0 or 1; Y 2 is 0, 1, or 2; Y 3 is 1, 2, or 3; m is an integer from 0 to 4; n is an integer from 0 to 4, preferably wherein the ligand is G4, G5, G6, or G7: preferably wherein the conjugate is represented by the following structures:
  13. 13 . The conjugate according to claim 11 , wherein the ligand is: wherein X is oxygen, nitrogen, or sulfur; Y is an alkyl or aromatic group; R 1 is oxygen or sulfur; R 2 is hydrogen, an amino group, a C 1-4 alkyl group, an aromatic group, a C 1-4 alkoxy group, or a halogen; A is —(CH 2 ) a —, —(CH 2 CH 2 O) b —, —((CH 2 ) c NHCO) d —, or —((CH 2 ) c CONH) d —, where a is an integer from 1 to 15, b is an integer from 1 to 7, c is an integer from 1 to 7, and d is an integer from 1 to 5; B is —(CH 2 ) e —, where e is an integer from 0 to 7; L is —CONH— or —NHCO—; X 1 is —(CH 2 ) f — or —(CH 2 CH 2 O) f CH 2 —, where f is an integer from 1 to 5; X 2 is —(CH 2 ) g —, where g is an integer from 1 to 6; Y 1 is 0 or 1; Y 2 is 0, 1, or 2; Y 3 is 1, 2, or 3; m is an integer from 0 to 4; n is an integer from 0 to 4, preferably wherein the ligand is G101, G102, G103, G105, or G106: preferably wherein the conjugate is represented by the following structures:
  14. 14 . The conjugate according to claim 11 , wherein the conjugate comprises an oligonucleotide duplex selected from any one of the following sense and antisense strand pairs: (1) the sense strand has the sequence as shown in SEQ ID NO: 326, 327 or 328, and the antisense strand has the sequence as shown in SEQ ID NO: 179; (2) the sense strand has the sequence as shown in SEQ ID NO: 329, 330 or 331, and the antisense strand has the sequence as shown in SEQ ID NO: 181; (3) the sense strand has the sequence as shown in SEQ ID NO: 338 or 341, and the antisense strand has the sequence as shown in SEQ ID NO: 201; (4) the sense strand has the sequence as shown in SEQ ID NO: 350, 351 or 352, and the antisense strand has the sequence as shown in SEQ ID NO: 321.
  15. 15 . The conjugate according to claim 11 , wherein the conjugate has the function of inhibiting HBV gene expression.
  16. 16 . A pharmaceutical composition comprising the double-stranded RNAi agent according to claim 1 , along with a pharmaceutically acceptable carrier.
  17. 17 . The pharmaceutical composition according to claim 16 , wherein the double-stranded RNAi agent or the conjugate is administered in a non-buffered solution, preferably wherein the non-buffered solution is saline or water, or wherein the double-stranded RNAi agent or the conjugate is administered with a buffered solution, preferably wherein the buffered solution comprises acetate, citrate, prolamine, carbonate, or phosphate, or any combination thereof, preferably wherein the buffered solution is phosphate-buffered solution, or wherein the double-stranded RNAi agent or the conjugate is formulated into a lipid formulation for delivery within membranous molecular assemblies, preferably wherein a mass-to-mass ratio of lipid to the double-stranded RNAi agent or the conjugate is 1:1 to 50:1, 1:1 to 25:1, 3:1 to 15:1, 4:1 to 10:1, 5:1 to 9:1, or 6:1 to 9:1, preferably wherein the lipid formulation is a nucleic acid-lipid particle, preferably wherein the lipid formulation is a lipid nanoparticle.
  18. 18 . The pharmaceutical composition according to claim 17 , wherein the lipid nanoparticle comprises a cationic lipid, a neutral lipid, a structural lipid, and a polymer-conjugated lipid.
  19. 19 . The pharmaceutical composition according to claim 18 , wherein the cationic lipid is a compound of formula (I), or a N-oxide, solvate, pharmaceutically acceptable salt, or stereoisomer thereof, wherein G 1 is a C 1-6 alkylene group; G 2 is a C 2-s alkylene group; G 3 is a C 1-3 alkylene group; L 1 is a C 6-15 linear alkyl group; L 2 is a C 12-25 branched alkyl group, preferably wherein the cationic lipid is YK-009 of formula (I-I):
  20. 20 . The pharmaceutical composition according to claim 18 , wherein the cationic lipid is a compound of formula (II), or a N-oxide, solvate, pharmaceutically acceptable salt, or stereoisomer thereof, wherein G 1 is a C 2-8 alkylene group; G 2 is a C 2-8 alkylene group; L 1 is —C(O)O— or —OC(O)—; L 2 is —C(O)O— or —OC(O)—; R 1 is a C 6-25 linear or branched alkyl group; R 2 is a C 6-25 linear or branched alkyl group; G 3 is HO(CH 2 ) 2 — or HO(CH 2 ) 3 —; G 4 is HO(CH 2 ) 2 — or HO(CH 2 ) 3 —; L is —(CH 2 ) 2 —, —(CH 2 ) 3 —, or —(CH 2 ) 4 —, preferably wherein the cationic lipid is YK-402 of formula (II-II):

Description

SEQUENCE LISTING The instant application contains a Sequence Listing which has been submitted electronically in XML file format and is hereby incorporated by reference in its entirety. Said XML copy, created on Jun. 30, 2025, is named S3920.10196US01_Sequence_Listing and is 3,579,904 bytes in size. TECHNICAL FIELD The present disclosure relates to the field of nucleic acid modification technology, and specifically, to a double-stranded RNAi agent for targeting and regulating HBV gene expression, and a use thereof. BACKGROUND Nucleic acid drugs, particularly oligonucleotide drugs, have been widely used due to their simple synthesis and high activity. Oligonucleotide drugs typically include antisense oligonucleotides (ASOs), small interfering RNAs (siRNAs), microRNAs (miRNAs), and nucleic acid aptamers, among others. Oligonucleotides are short DNA or RNA molecules or oligomers that can easily bind to their complementary oligonucleotides, DNA, or RNA in a sequence-specific manner to form duplexes or, less commonly, hybrids. This fundamental property enables oligonucleotides to have broad applications in gene detection, research, and medicine. In nature, oligonucleotides are often small RNA molecules that play a role in the regulation of gene expression or are intermediates derived from the degradation of larger nucleic acid molecules. RNA interference (RNAi) is a natural defense mechanism against exogenous genes. siRNA can downregulate target genes by recognizing specific sequences and degrading the target mRNA. An effective molecule of a classical RNAi consists of a characteristic 19+2 nucleotide polymer structure (a double-helix structure composed of a 21-nucleotide RNA molecule paired with a 19-nucleotide molecule of complementary bases, comprising a 3′ end overhang of two nucleotides). One strand (the guide strand or antisense strand) of siRNA is complementary to the target gene's transcript mRNA, while the other strand is designated as the passenger strand (or sense strand). The siRNA (antisense strand) guides the Argonaute protein (AGO2) to complementary to the target transcript and becomes part of the RNA-induced silencing complex (RISC). The complete complementarity between the siRNA (antisense strand) and the target leads to the cleavage of the target transcript at positions 10-11 corresponding to the guide strand (antisense strand), catalyzed by the AGO2 protein. siRNA possesses inherent advantages compared to small molecules and antibody drugs, as siRNA executes its function through Watson-Crick base pairing with mRNA, whereas small molecules and monoclonal antibody drugs require recognition of the complex three-dimensional structure of specific proteins. Therefore, many diseases cannot be treated with small molecules or monoclonal antibodies because their target molecules are highly active and lack identifiable molecular structures with sufficient affinity and binding specificity. The mechanism of action of siRNA drugs enables them to regulate the expression of target proteins at the genetic level, offering superior target specificity compared to small molecules or antibody drugs. The principle of base complementarity also allows siRNA to have a broader therapeutic range, simpler design, and shorter development cycles. In natural oligonucleotides, nucleotides are linked by phosphodiester bonds, making them highly susceptible to nucleases under physiological conditions. As a result, natural, unmodified oligonucleotide drugs are rapidly degraded by nucleases in vivo, exhibit low activity, and have poor druggability. Chemical modification of oligonucleotide structures is an effective approach to enhance their activity. Such modifications can improve their stability against nucleases, increase their affinity for RNA, and promote cellular uptake and tissue targeting, thereby effectively regulating the expression of target genes. Based on the basic structure of oligonucleotides—comprising the nucleobase, sugar ring, phosphate backbone, and terminal—chemical modifications can be performed in four parts: 1) Nucleobase modifications, which primarily include purine modifications, pyrimidine modifications, and nucleobase replacements. Purine modifications include N6-methyladenosine, Ni-methyladenosine, and 7-methylguanosine modifications. Pyrimidine modifications include 3-methyluridine, 5-methyluridine, 5-methylcytidine, N4-acetylcytidine, pseudouridine, thiouridine, propynyluridine, and dihydrouridine modifications, among others.2) Sugar ring modifications, which mainly include modifications and replacements of the sugar moiety. Sugar ring modifications include 2′-, 4′-, and 5′-modifications, isomer modifications, and combinations thereof. Among siRNA modifications, the most common 2-modifications are 2′-OMe (2′-methoxy) and 2′-F (2′-fluoro). Compared to natural siRNA, siRNA modified with both 2′-OMe and 2′-F exhibits higher Tm values, stronger serum stability, and improved activity.3) Phosphate backbone mo