Rnai reagent having specific modification combination and use thereof

Abstract

An RNAi reagent for inhibiting the expression of a target gene, said reagent using a specific combination of modified nucleotides. A composition comprising the RNAi reagent and a method for using the RNAi reagent to inhibit the expression of a target gene.

Description

RNAi reagents with specific modification combinations and their uses Technical Field

[0001] This invention belongs to the field of biomedicine and relates to an RNAi reagent that inhibits the expression of target genes, which uses a specific combination of modified nucleotides to exert its effect. Background Technology

[0002] Oligonucleotides are polymers of nucleotides. As nucleic acid inhibitor molecules, oligonucleotides can regulate intracellular mRNA levels and have shown early promise in the treatment of genetic diseases, metabolic diseases, cancer, and viral infections. Nucleic acid inhibitor molecules can regulate mRNA expression through a different set of mechanisms, including RNA interference (RNAi).

[0003] RNAi is a conserved pathway found in most eukaryotes, in which a double-stranded RNA molecule (dsRNA) inhibits the expression of a target gene with a complementary sequence to the dsRNA. In a typical RNAi pathway, the longer dsRNA is cleaved by a nuclease (Dicer) into a shorter RNA duplex called small interfering RNA (“siRNA”). siRNA has been shown to associate with the nuclease, trans-activating response RNA-binding protein (TRBP), and Argonaute 2 (“Ago2”) to form a complex, sometimes referred to as the RNA-induced silencing complex (“RISC”). Ago2 is a nuclease that uses the antisense strand (also known as the guide strand) of the siRNA to guide the sequence-specific cleavage of the target mRNA.

[0004] Various double-stranded RNAi inhibitor molecular structures have been developed over the years. For example, early work on RNAi inhibitor molecules focused on double-stranded nucleic acid molecules mimicking natural siRNA, where each strand has 19-25 nucleotides and includes at least one 3' overhang with 1 to 5 nucleotides (see, for example, U.S. Patent No. 8,372,968). Subsequently, longer double-stranded RNAi inhibitor molecules were developed, which were cleaved in vivo by endonucleases into active RNAi inhibitor molecules (see, for example, U.S. Patent No. 8,883,996). Subsequent work developed extended double-stranded nucleic acid inhibitor molecules in which at least one end of at least one strand extends beyond the double-stranded target region of the molecule, one of the strands comprising a thermodynamically stable tetracyclic structure (see, for example, U.S. Patent Nos. 8,513,207, 8,927,705, WO 2010 / 033225, and WO 2016 / 100401). These structures include single-strand extensions (on one or both sides of the molecule) and double-strand extensions.

[0005] Compared with traditional drug therapies such as small molecule drugs and protein drugs, nucleic acid drugs have advantages such as simple design, short development cycle, high success rate, strong specificity, and the ability to fundamentally regulate pathogenic genes, providing solutions for the treatment of many intractable diseases. Currently, the main problems with nucleic acid drugs are poor in vivo stability, easily degraded by nucleases or cleared by the kidneys; secondly, nucleic acid drugs carry a negative charge, making it difficult to cross barriers or membranes to reach cells and exert their effects; finally, nucleic acid drugs have weak targeting in vivo, failing to precisely target specific cells or tissues, potentially leading to off-target effects, side effects, and damage to the body. Drugs entering cells also face the risk of lysosomal hydrolysis and non-specific protein adsorption during circulation, rendering them ineffective. Chemically modifying the bases, phosphate groups, and ribose in the basic nucleotide units of nucleic acid drugs can alter their physicochemical properties, making them more stable and enhancing their resistance to degradation by endogenous endonucleases and exonucleases, thereby improving delivery efficiency.

[0006] Based on the location of the chemical modification, the chemical modification methods applied in RNAi molecules are divided into phosphate ester modification, base modification, and ribose modification. When chemical modifications are applied to dsRNA design, they constitute the structural motif of dsRNA according to specific rules, including the modification method and structural design. Early siRNAs typically used partial modifications with 2'-OMe, 2'-F, or PS. Current siRNA modifications cover almost the entire length and are developing towards diversification and refinement. Alnylam has developed various siRNA structural motifs, listed from earliest to latest development time, including: STC (Standard template chemistry), ESC (Enhanced stabilization chemistry), Advanced ESC, and ESC+. Currently, all three Alnylam siRNA drugs approved use the ESC modification method. ESC further adds four additional PS modifications to the 5'-terminus of the antisense strand and the 3'-terminus of the sense strand, and reduces the number of 2'-F substitutions, based on STC. These changes significantly enhance the potency and duration of siRNA. Advanced ESC technology aims to further reduce the number of 2'-F substitutions and optimize their position. ESC+ introduces GNA into the seed region of the antisense strand, which can significantly mitigate off-target effects caused by miRNA-like recognition (i.e., partial matching between the seed region and non-target mRNA) and reduce hepatotoxicity. (See, for example, Hu, B., Zhong, L., Weng, Y. et al. Therapeutic siRNA: state of the art. Sig Transduct Target Ther 5, 101 (2020).) Summary of the Invention

[0007] In a first aspect, the present invention provides an RNAi reagent for inhibiting the expression of a target gene, comprising a sense strand and an antisense strand, each strand having 17 to 30 nucleotides, wherein each nucleotide is independently a modified or unmodified nucleotide; wherein the antisense strand is complementary to at least a portion of the mRNA of the target gene, and the sense strand and the antisense strand are at least partially complementary to form a double-stranded region.

[0008] The 5th and 7th positions of the 5' end of the antisense strand contain 2'-deoxynucleotides; the sense strand position corresponding to the 12th position of the 5' end of the antisense strand contains a nucleotide that is not modified by 2'-F.

[0009] In some embodiments of the invention, positions 2, 9, 12, 14, 16, 18, 20 or 22 at the 5' end of the antisense strand further comprise 2'-deoxynucleotides.

[0010] In some embodiments of the present invention, the second position at the 5' end of the antisense strand contains a 2'-F modified nucleotide or a 2'-deoxynucleotide.

[0011] In some embodiments of the present invention, the sense position corresponding to the 12th position at the 5' end of the antisense strand contains a 2'-methoxy modified nucleotide or a 2'-deoxy nucleotide.

[0012] In some embodiments of the present invention, the 15th position of the 5' end of the antisense strand contains a heat-stabilized modified nucleotide.

[0013] In some embodiments of the present invention, the thermally stabilized modified nucleotide refers to the double-stranded oligonucleotide whose thermal dissociation temperature Tm increases after introduction; preferably, the thermal dissociation temperature Tm increases by at least 0.05°C; more preferably, the thermal dissociation temperature Tm increases by 0.5-4°C.

[0014] In some specific embodiments of the present invention, the heat-stabilized modified nucleotide is selected from nucleotides modified with 2'-O-methoxyethyl, nucleotides modified with 2'-O-allyl, nucleotides modified with 2'-C-allyl, nucleotides modified with 2'-O-2-N-methylamino-2-oxomethyleneethyl, nucleotides modified with 2'-O-2-N,N-dimethylaminoethyl, nucleotides modified with 2'-O-3-aminopropyl, nucleotides modified with 2'-O-2,4-dinitrophenyl, nucleotides modified with 2'-O-(N-methylacetamido)-modified, nucleotides modified with 2',4'-disubstituted, locked nucleotides (LNA), bicyclic nucleotides (BNA), or restricted ethyl-bridged nucleotides (cEt); preferably, the heat-stabilized modified nucleotide is selected from nucleotides modified with 2'-O-methoxyethyl.

[0015] In some embodiments of the present invention, the 14th position of the 5' end of the antisense strand contains a nucleotide modified with 2'-F.

[0016] In some embodiments of the present invention, the sense strand position corresponding to the 11th and / or 13th position at the 5' end of the antisense strand contains a nucleotide modified with 2'-F.

[0017] In some embodiments of the present invention, the 12th and / or 16th position of the 5' end of the antisense strand contains a nucleotide modified with 2'-F.

[0018] In some embodiments of the present invention, the sense strand position corresponding to the 6th position at the 5' end of the antisense strand contains a nucleotide modified with 2'-F.

[0019] In some embodiments of the present invention, the sense strand position corresponding to the 9th position at the 5' end of the antisense strand contains a nucleotide modified with 2'-F.

[0020] In some embodiments of the present invention, the sense strand position corresponding to the 12th position at the 5' end of the antisense strand contains a 2'-deoxynucleotide.

[0021] In some embodiments of the present invention, the sense strand position corresponding to the 6th position at the 5' end of the antisense strand contains a 2'-deoxynucleotide.

[0022] In some embodiments of the present invention, the sense strand position corresponding to the 15th position at the 5' end of the antisense strand contains a 2'-deoxynucleotide.

[0023] In some embodiments of the present invention, each strand of the sense and antisense strands of the RNAi reagent has 17 to 25 nucleotides; preferably, each strand has 19 to 23 nucleotides.

[0024] In some embodiments of the invention, each strand of the sense and antisense strands of the RNAi reagent has 19, 20, 21, 22, or 23 nucleotides.

[0025] In some embodiments of the present invention, the sense strand of the RNAi reagent has two fewer nucleotides than the antisense strand, and the ratio of the number of nucleotides in the sense strand to the antisense strand is 19 / 21, 20 / 22, or 21 / 23.

[0026] In some embodiments of the present invention, the RNAi reagent comprises one or more single-stranded nucleotide overhangs; preferably, the overhangs are located at the 3' end of the antisense strand of the RNAi reagent.

[0027] In some embodiments of the present invention, the RNAi reagent has blunt ends; preferably, the RNAi reagent has at least one blunt end located at the 5' end of the antisense strand.

[0028] In some embodiments of the invention, the 12th position of the 3' end of the sense strand contains a nucleotide that is not modified by 2'-F.

[0029] In some embodiments of the present invention, the 12th position of the 3' end of the sense strand comprises a 2'-methoxy modified nucleotide or a 2'-deoxy nucleotide.

[0030] In some embodiments of the present invention, the 11th and / or 13th position of the 3' end of the sense strand contains a nucleotide modified with 2'-F.

[0031] In some embodiments of the present invention, the 6th position of the 3' end of the sense strand contains a nucleotide modified with 2'-F.

[0032] In some embodiments of the present invention, the 9th position of the 3' end of the sense strand contains a nucleotide modified with 2'-F.

[0033] In some embodiments of the present invention, the 12th position of the 3' end of the sense strand contains a 2'-deoxynucleotide.

[0034] In some embodiments of the invention, the 6th, 7th, 9th, 10th, 14th, 17th and / or 19th positions of the 3' end of the sense strand contain 2'-deoxynucleotides.

[0035] In some embodiments of the present invention, the 15th position of the 3' end of the sense strand contains a 2'-deoxynucleotide.

[0036] In some embodiments of the present invention, the remaining positions in the sense or antisense strand of the RNAi reagent are modified nucleotides; preferably, the modified nucleotides are selected from 2'-methoxy-modified nucleotides, 2'-F-modified nucleotides, 2'-deoxynucleotides, 2'-O-methoxyethyl-modified nucleotides, threonucleotides (TNA), ethylene glycol nucleotides (GNA), locked nucleotides (LNA), open cyclic nucleotides (UNA), bicyclic nucleotides (BNA), 2',5'-linked nucleotides, 2'-amino-modified nucleotides, 2'-alkyl-modified nucleotides, 2'-allyl-modified nucleotides, debased nucleotides, morpholinonucleotides, and phosphate ester-modified nucleotides.

[0037] In some embodiments of the present invention, in the RNAi reagent, three or all of the positions 2, 12, 14, and 16 at the 5' end of the antisense strand contain 2'-F modified nucleotides, the 5th and 7th positions contain 2'-deoxynucleotides, and the 15th position contains a 2'-O-methoxyethyl modified nucleotide; the 9th, 11th, and 13th positions at the 3' end of the sense strand contain 2'-F modified nucleotides.

[0038] In a further preferred embodiment, the 2nd, 9th, 12th, 14th, or 16th position of the 5' end of the antisense strand contains a 2'-deoxynucleotide.

[0039] In a further preferred embodiment, the sixth position of the 3' end of the sense strand contains a nucleotide modified with 2'-F.

[0040] In some embodiments of the present invention, in the RNAi reagent, the 2nd, 12th, 14th, and 16th positions of the 5' end of the antisense strand contain 2'-F modified nucleotides, the 5th and 7th positions contain 2'-deoxynucleotides, and the 15th position contains a 2'-O-methoxyethyl modified nucleotide; the 9th, 11th, and 13th positions of the 3' end of the sense strand contain 2'-F modified nucleotides, and the 18th, 20th, or 22nd positions of the 5' end of the antisense strand also contain 2'-deoxynucleotides.

[0041] In some embodiments of the present invention, in the RNAi reagent, the antisense strand contains 2'-F modified nucleotides at positions 2, 12, 14, and 16 of its 5' end, 2'-deoxynucleotides at positions 5 and 7, and 2'-O-methoxyethyl modified nucleotides at position 15; the sense strand contains 2'-F modified nucleotides at positions 9, 11, and 13 of its 3' end, and 2'-deoxynucleotides at positions 6, 7, 10, 12, 14, 15, 17, or 19 of its 3' end, or 2'-deoxynucleotides at positions 11 and 13 of its 3' end, and 2'-deoxynucleotides at position 9 of its 3' end.

[0042] In some specific embodiments of the present invention, the RNAi reagent has a combination of modifications selected from any of the following:

[0043] 1) The antisense strand contains 2'-F modified nucleotides at positions 2, 12, 14, and 16 at the 5' end, 2'-deoxynucleotides at positions 5 and 7, and 2'-O-methoxyethyl modified nucleotides at position 15; the sense strand contains 2'-F modified nucleotides at positions 9, 11, and 13 at the 3' end.

[0044] 2) The antisense strand contains 2'-F modified nucleotides at positions 2, 14, and 16 of the 5' end; 2'-deoxynucleotides at positions 5, 7, and 12; and 2'-O-methoxyethyl modified nucleotides at position 15. The sense strand contains 2'-F modified nucleotides at positions 9, 11, and 13 of the 3' end.

[0045] 3) The antisense strand contains 2'-F modified nucleotides at positions 2, 12, and 14 at the 5' end; 2'-deoxynucleotides at positions 5, 7, and 16; and 2'-O-methoxyethyl modified nucleotides at position 15. The sense strand contains 2'-F modified nucleotides at positions 6, 9, 11, and 13 at the 3' end.

[0046] 4) The antisense strand contains 2'-F modified nucleotides at positions 12, 14, and 16 of the 5' end; 2'-deoxynucleotides at positions 2, 5, and 7; and 2'-O-methoxyethyl modified nucleotides at position 15. The sense strand contains 2'-F modified nucleotides at positions 9, 11, and 13 of the 3' end.

[0047] 5) The antisense strand contains 2'-F modified nucleotides at positions 12, 14, and 16 of the 5' end; 2'-deoxynucleotides at positions 2, 5, and 7; and 2'-O-methoxyethyl modified nucleotides at position 15. The sense strand contains 2'-F modified nucleotides at positions 6, 9, 11, and 13 of the 3' end.

[0048] 6) The antisense strand contains 2'-F modified nucleotides at positions 2, 14, and 16 of the 5' end; 2'-deoxynucleotides at positions 5, 7, and 9; and 2'-O-methoxyethyl modified nucleotides at position 15. The sense strand contains 2'-F modified nucleotides at positions 9, 11, and 13 of the 3' end.

[0049] 7) The antisense strand contains 2'-F modified nucleotides at positions 2, 12, and 16 of the 5' end; 2'-deoxynucleotides at positions 5, 7, and 14; and 2'-O-methoxyethyl modified nucleotides at position 15; the sense strand contains 2'-F modified nucleotides at positions 9, 11, and 13 of the 3' end.

[0050] 8) The antisense strand contains 2'-F modified nucleotides at positions 2, 12, 14, and 16 at the 5' end, 2'-deoxynucleotides at positions 5 and 7, and 2'-O-methoxyethyl modified nucleotides at position 15; the sense strand contains 2'-F modified nucleotides at positions 9, 11, and 13 at the 3' end, and 2'-deoxynucleotides at position 15.

[0051] 9) The antisense strand contains 2'-F modified nucleotides at positions 2, 12, 14, and 16 at the 5' end; 2'-deoxynucleotides at positions 5, 7, and 18; and 2'-O-methoxyethyl modified nucleotides at position 15. The sense strand contains 2'-F modified nucleotides at positions 9, 11, and 13 at the 3' end.

[0052] 10) The antisense strand contains 2'-F modified nucleotides at positions 2, 12, 14, and 16 at the 5' end; 2'-deoxynucleotides at positions 5, 7, and 20; and 2'-O-methoxyethyl modified nucleotides at position 15. The sense strand contains 2'-F modified nucleotides at positions 9, 11, and 13 at the 3' end.

[0053] 11) The antisense strand contains 2'-F modified nucleotides at positions 2, 12, 14, and 16 at the 5' end; 2'-deoxynucleotides at positions 5, 7, and 22; and 2'-O-methoxyethyl modified nucleotides at position 15. The sense strand contains 2'-F modified nucleotides at positions 9, 11, and 13 at the 3' end.

[0054] 12) The antisense strand contains 2'-F modified nucleotides at positions 2, 12, 14, and 16 at the 5' end, 2'-deoxynucleotides at positions 5 and 7, and 2'-O-methoxyethyl modified nucleotides at position 15; the sense strand contains 2'-F modified nucleotides at positions 6, 9, 11, and 13 at the 3' end.

[0055] 13) The antisense strand contains 2'-F modified nucleotides at positions 2, 12, 14, and 16 at the 5' end, 2'-deoxynucleotides at positions 5 and 7, and 2'-O-methoxyethyl modified nucleotides at position 15; the sense strand contains 2'-F modified nucleotides at positions 9, 11, and 13 at the 3' end, and 2'-deoxynucleotides at position 6.

[0056] 14) The antisense strand contains 2'-F modified nucleotides at positions 2, 12, 14, and 16 at the 5' end, 2'-deoxynucleotides at positions 5 and 7, and 2'-O-methoxyethyl modified nucleotides at position 15; the sense strand contains 2'-F modified nucleotides at positions 9, 11, and 13 at the 3' end, and 2'-deoxynucleotides at position 19.

[0057] 15) The antisense strand contains 2'-F modified nucleotides at positions 2, 12, 14, and 16 at the 5' end, 2'-deoxynucleotides at positions 5 and 7, and 2'-O-methoxyethyl modified nucleotides at position 15; the sense strand contains 2'-F modified nucleotides at positions 9, 11, and 13 at the 3' end, and 2'-deoxynucleotides at position 14.

[0058] 16) The antisense strand contains 2'-F modified nucleotides at positions 2, 12, 14, and 16 at the 5' end, 2'-deoxynucleotides at positions 5 and 7, and 2'-O-methoxyethyl modified nucleotides at position 15; the sense strand contains 2'-F modified nucleotides at positions 9, 11, and 13 at the 3' end, and 2'-deoxynucleotides at position 17.

[0059] 17) The antisense strand contains 2'-F modified nucleotides at positions 2, 12, 14, and 16 at the 5' end, 2'-deoxynucleotides at positions 5 and 7, and 2'-O-methoxyethyl modified nucleotides at position 15; the sense strand contains 2'-F modified nucleotides at positions 11 and 13 at the 3' end, and 2'-deoxynucleotides at position 9.

[0060] 18) The antisense strand contains 2'-F modified nucleotides at positions 2, 12, 14, and 16 at the 5' end, 2'-deoxynucleotides at positions 5 and 7, and 2'-O-methoxyethyl modified nucleotides at position 15; the sense strand contains 2'-F modified nucleotides at positions 9, 11, and 13 at the 3' end, and 2'-deoxynucleotides at position 12.

[0061] 19) The antisense strand contains 2'-F modified nucleotides at positions 2, 12, 14, and 16 at the 5' end, 2'-deoxynucleotides at positions 5 and 7, and 2'-O-methoxyethyl modified nucleotides at position 15; the sense strand contains 2'-F modified nucleotides at positions 9, 11, and 13 at the 3' end, and 2'-deoxynucleotides at position 7.

[0062] 20) The antisense strand contains 2'-F modified nucleotides at positions 2, 12, 14, and 16 at the 5' end, 2'-deoxynucleotides at positions 5 and 7, and 2'-O-methoxyethyl modified nucleotides at position 15; the sense strand contains 2'-F modified nucleotides at positions 9, 11, and 13 at the 3' end, and 2'-deoxynucleotides at position 10.

[0063] Preferably, the remaining positions in the sense or antisense strand of the RNAi reagent are nucleotides modified with 2'-methoxy groups.

[0064] On the other hand, the present invention provides an RNAi reagent for inhibiting the expression of a target gene, comprising a sense strand and an antisense strand, each strand having 17 to 30 nucleotides, wherein each nucleotide is independently a modified or unmodified nucleotide; wherein the antisense strand is complementary to at least a portion of the mRNA of the target gene, and the sense strand and the antisense strand are at least partially complementary to form a double-stranded region.

[0065] The antisense chain includes the sequence shown in formula IA;

[0066] 3'-(N) n NNN E NN N'N NNNN d NN d NNN F N-5' (Form IA)

[0067] in,

[0068] n represents an integer from 0 to 8;

[0069] N F Nucleotides representing 2'-F modifications;

[0070] Each N d They represent 2'-deoxynucleotides;

[0071] Each N, N', or N E Each can be used independently to represent a modified or unmodified nucleotide;

[0072] The nucleotide corresponding to N' in the sense strand is not a nucleotide modified by 2'-F.

[0073] In some embodiments of the present invention, the nucleotide corresponding to N' in the sense strand is selected from nucleotides containing 2'-methoxy modification or 2'-deoxynucleotides.

[0074] In some embodiments of the present invention, N E The nucleotides are selected from those containing heat-stabilized modifications; the heat-stabilized modifications refer to the double-stranded oligonucleotides whose thermal dissociation temperature Tm increases after introduction; preferably, the thermal dissociation temperature Tm increases by at least 0.05°C; more preferably, the thermal dissociation temperature Tm increases by 0.5-4°C.

[0075] In some specific embodiments of the present invention, the heat-stabilized modified nucleotide is selected from nucleotides modified with 2'-O-methoxyethyl, nucleotides modified with 2'-O-allyl, nucleotides modified with 2'-C-allyl, nucleotides modified with 2'-O-2-N-methylamino-2-oxomethyleneethyl, nucleotides modified with 2'-O-2-N,N-dimethylaminoethyl, nucleotides modified with 2'-O-3-aminopropyl, nucleotides modified with 2'-O-2,4-dinitrophenyl, nucleotides modified with 2'-O-(N-methylacetamido)-modified, nucleotides modified with 2',4'-disubstituted, locked nucleotides (LNA), bicyclic nucleotides (BNA), or restricted ethyl-bridged nucleotides (cEt); preferably, the heat-stabilized modified nucleotide is selected from nucleotides modified with 2'-O-methoxyethyl.

[0076] In some embodiments of the present invention, the antisense chain includes the sequence shown by formula IAa;

[0077] 3'-(N) n N a17 N a16 N E N F N a13 N'N a11 Na10 N a9 N a8 N d N a6 N d N a4 N a3 N F N a1 -5' (Formula IAa)

[0078] in,

[0079] n represents an integer from 0 to 8;

[0080] Each N F These represent nucleotides modified by 2'-F;

[0081] Each N d They represent 2'-deoxynucleotides;

[0082] Each N and N' independently represents a modified or unmodified nucleotide;

[0083] N a1 N a3 N a4 N a6 N a8 N a9 N a10 N a11 N a13 N a16 N a17 Each can be used independently to represent a modified or unmodified nucleotide;

[0084] N E This represents a nucleotide containing a heat-stabilizing modification; preferably, N E This represents a nucleotide modified with 2'-O-methoxyethyl.

[0085] In some embodiments of the present invention, the sense chain includes the sequence shown in Formula II;

[0086] 5'-(N) m NNNNNN T NNNNNNNNNN N-3' (Equation II)

[0087] in,

[0088] m represents an integer from 0 to 8;

[0089] N T This represents a nucleotide that is not modified by 2'-F;

[0090] Each N independently represents a modified or unmodified nucleotide.

[0091] In some embodiments of the present invention, N T This represents a nucleotide or 2'-deoxynucleotide containing a 2'-methoxy group.

[0092] In some embodiments of the present invention, the sense chain includes the sequence shown in Form IIA;

[0093] 5'-(N) m N s17 N s16 N s15 N s14 N F N T N F N s10 N s9 N s8 N s7 N s6 N s5 N s4 N s3 N s2 N s1 -3' (Formula IIA)

[0094] in,

[0095] m represents an integer from 0 to 8;

[0096] N T This represents a nucleotide containing a 2'-methoxy group or a 2'-deoxynucleotide.

[0097] Each N F These represent nucleotides modified by 2'-F;

[0098] Each N independently represents a modified or unmodified nucleotide;

[0099] N s1 N s2 N s3 N s4 N s5 N s6 N s7 N s8 N s9 N s10 N s14 N s15 N s16 N s17 Each can be used to represent a modified or unmodified nucleotide.

[0100] In some embodiments of the present invention, the sense chain includes the sequence represented by formula IIAa;

[0101] 5'-(N) m N s17 N s16 N s15 N s14 N F N T N F N s10 N F N s8 N s7 N s6 N s5 N s4 N s3 N s2 N s1 -3' (Formula IIAa)

[0102] in,

[0103] m represents an integer from 0 to 8;

[0104] N T This represents a nucleotide containing a 2'-methoxy group or a 2'-deoxynucleotide.

[0105] Each N F These represent nucleotides modified by 2'-F;

[0106] Each N independently represents a modified or unmodified nucleotide;

[0107] N s1 N s2 N s3 N s4 N s5 N s6 N s7 N s8 N s10 N s14 N s15 N s16 N s17 Each can be used to represent a modified or unmodified nucleotide.

[0108] In some embodiments of the present invention, the sense chain includes the sequence represented by formula IIAb;

[0109] 5'-(N) m N s17 N s16 N s15 N s14 N F Nd N F N s10 N s9 N s8 N s7 N s6 N s5 N s4 N s3 N s2 N s1 -3' (Formula IIAb)

[0110] in,

[0111] m represents an integer from 0 to 8;

[0112] Each N F These represent nucleotides modified by 2'-F;

[0113] N d Represents 2'-deoxynucleotide;

[0114] Each N independently represents a modified or unmodified nucleotide;

[0115] N s1 N s2 N s3 N s4 N s5 N s6 N s7 N s8 N s9 N s10 N s14 N s15 N s16 N s17 Each can be used to represent a modified or unmodified nucleotide.

[0116] In some embodiments of the present invention, the sense chain includes the sequence represented by IIAc;

[0117] 5'-(N) m N s17 N s16 N s15 N s14 N F N T N F N s10 N s9 N s8 N s7 N F N s5 N s4 N s3 N s2N s1 -3' (Form IIAc)

[0118] in,

[0119] m represents an integer from 0 to 8;

[0120] N T This represents a nucleotide containing a 2'-methoxy group or a 2'-deoxynucleotide.

[0121] Each N F These represent nucleotides modified by 2'-F;

[0122] Each N independently represents a modified or unmodified nucleotide;

[0123] N s1 N s2 N s3 N s4 N s5 N s7 N s8 N s9 N s10 N s14 N s15 N s16 N s17 Each can be used to represent a modified or unmodified nucleotide.

[0124] In some embodiments of the present invention, the sense chain includes the sequence represented by IIAd;

[0125] 5'-(N) m N s17 N s16 N d N s14 N F N T N F N s10 N s9 N s8 N s7 N s6 N s5 N s4 N s3 N s2 N s1 -3' (Formula IIAd)

[0126] in,

[0127] m represents an integer from 0 to 8;

[0128] N T This represents a nucleotide containing a 2'-methoxy group or a 2'-deoxynucleotide.

[0129] Each N F These represent nucleotides modified by 2'-F;

[0130] N d Represents 2'-deoxynucleotide;

[0131] Each N independently represents a modified or unmodified nucleotide;

[0132] N s1 N s2 N s3 N s4 N s5 N s6 N s7 N s8 N s9 N s10 N s14 N s16 N s17 Each can be used to represent a modified or unmodified nucleotide.

[0133] In some embodiments of the present invention, the nucleotide modified in the RNAi reagent is selected from 2'-methoxy-modified nucleotides, 2'-F-modified nucleotides, 2'-deoxynucleotides, 2'-O-methoxyethyl-modified nucleotides, threonucleotides (TNA), ethylene glycol nucleotides (GNA), locked nucleotides (LNA), open-ring nucleotides (UNA), bicyclic nucleotides (BNA), 2',5'-linked nucleotides, 2'-amino-modified nucleotides, 2'-alkyl-modified nucleotides, 2'-allyl-modified nucleotides, debased nucleotides, morpholinonucleotides, and nucleotides modified with phosphate ester groups.

[0134] In some specific embodiments of the present invention, the RNAi reagent comprises an antisense strand having any of the following modifications:

[0135] 1)3'-(N) n N a17 N F N MOE N F N a13 N F N a11 N a10 N a9 N a8 N d N a6 N d N a4 N a3 N F N a1 -5';

[0136] 2)3’-(N) n N a17 N F N MOE N F N a13 N d N a11 N a10 N a9 N a8 N d N a6 N d N a4 N a3 N F N a1 -5’;

[0137] 3)3’-(N) n N a17 N d N MOE N F N a13 N F N a11 N a10 N a9 N a8 N d N a6 N d N a4 N a3 N F N a1 -5’;

[0138] 4)3’-(N) n N a17 N F N MOE N F N a13 N F N a11 N a10 N a9 N a8 N d N a6 N d N a4 N a3 N d N a1 -5’;

[0139] 5)3’-(N) n N a17 N F NMOE N F N a13 N a12 N a11 N a10 N d N a8 N d N a6 N d N a4 N a3 N F N a1 -5’;

[0140] 6)3’-(N) n N a17 N F N MOE N d N a13 N F N a11 N a10 N a9 N a8 N d N a6 N d N a4 N a3 N F N a1 -5’;

[0141] 7)3’-N a23 N a22 N a21 N a20 N a19 N d N a17 N F N MOE N F N a13 N F N a11 N a10 N a9 N a8 N d N a6 N d N a4 N a3 N F N a1 -5’;

[0142] 8)3’-N a23 N a22 N a21 N d Na19 N a18 N a17 N F N MOE N F N a13 N F N a11 N a10 N a9 N a8 N d N a6 N d N a4 N a3 N F N a1 -5';

[0143] 9)3'-N a23 N d N a21 N a20 N a19 N a18 N a17 N F N MOE N F N a13 N F N a11 N a10 N a9 N a8 N d N a6 N d N a4 N a3 N F N a1 -5';

[0144] in,

[0145] n represents an integer from 0 to 8; preferably, n is 2, 3, 4, 5, 6 or 7;

[0146] Each N F These represent nucleotides modified by 2'-F;

[0147] Each N d They represent 2'-deoxynucleotides;

[0148] N MOE Represents a nucleotide modified with 2'-O-methoxyethyl;

[0149] Each N independently represents a modified or unmodified nucleotide;

[0150] N a1N a3 N a4 N a6 N a8 N a9 N a10 N a11 N a12 N a13 N a17 N a18 N a19 N a20 N a21 N a22 N a23 Each can independently represent a modified or unmodified nucleotide; preferably, N a1 N a3 N a4 N a6 N a8 N a9 N a10 N a11 N a12 N a13 N a17 N a18 N a19 N a20 N a21 N a22 N a23 All of them are nucleotides containing 2'-methoxy groups.

[0151] In some specific embodiments of the present invention, the RNAi reagent comprises a sense strand having any of the following modifications:

[0152] 1)5'-(N) m N s17 N s16 N s15 N s14 N F N T N F N s10 N F N s8 N s7 N s6 N s5 N s4 N s3 N s2 N s1 -3';

[0153] 2)5'-(N) m N s17 N s16 N s15 Ns14 N F N d N F N s10 N F N s8 N s7 N s6 N s5 N s4 N s3 N s2 N s1 -3’;

[0154] 3)5’-(N) m N s17 N s16 N s15 N s14 N F N T N F N s10 N F N s8 N s7 N F N s5 N s4 N s3 N s2 N s1 -3’;

[0155] 4)5’-(N) m N s17 N s16 N d N s14 N F N T N F N s10 N F N s8 N s7 N s6 N s5 N s4 N s3 N s2 N s1 -3’;

[0156] 5)5’-(N) m N s17 N s16 N s15 N s14 N F N T N F N s10 NF N s8 N s7 N d N s5 N s4 N s3 N s2 N s1 -3’;

[0157] 6)5’-N s21 N s20 N d N s18 N s17 N s16 N s15 N s14 N F N T N F N s10 N F N s8 N s7 N s6 N s5 N s4 N s3 N s2 N s1 -3’;

[0158] 7)5’-(N) m N s17 N s16 N s15 N d N F N T N F N s10 N F N s8 N s7 N s6 N s5 N s4 N s3 N s2 N s1 -3’;

[0159] 8)5’-(N) m N d N s16 N s15 N s14 N F N T N F N s10 N F N s8 N s7N s6 N s5 N s4 N s3 N s2 N s1 -3’;

[0160] 9)5’-(N) m N s17 N s16 N s15 N s14 N F N T N F N s10 N d N s8 N s7 N s6 N s5 N s4 N s3 N s2 N s1 -3’;

[0161] 10)5’-(N) m N s17 N s16 N s15 N s14 N F N T N F N s10 N F N s8 N d N s6 N s5 N s4 N s3 N s2 N s1 -3’;

[0162] 11)5’-(N) m N s17 N s16 N s15 N s14 N F N T N F N d N F N s8 N s7 N s6 N s5 N s4 N s3 N s2 Ns1 -3';

[0163] in,

[0164] m represents an integer from 0 to 8; preferably, n is 2, 3, 4, 5, 6 or 7;

[0165] Each N F These represent nucleotides modified by 2'-F;

[0166] Each N d They represent 2'-deoxynucleotides;

[0167] N T This represents a nucleotide containing a 2'-methoxy group.

[0168] Each N independently represents a modified or unmodified nucleotide;

[0169] N s1 N s2 N s3 N s4 N s5 N s6 N s7 N s8 N s10 N s14 N s15 N s16 N s17 N s18 N s20 N s21 Each can independently represent a modified or unmodified nucleotide; preferably, N s1 N s2 N s3 N s4 N s5 N s6 N s7 N s8 N s10 N s14 N s15 N s16 N s17 N s18 N s20 N s21 All of them are nucleotides containing 2'-methoxy groups.

[0170] In some embodiments of the present invention, the RNAi reagent comprises at least one modified phosphate ester nucleoside linker; preferably, the modified phosphate ester nucleoside linker is selected from thiophosphate nucleoside links, dithiophosphate nucleoside links, and nucleotides with methylphosphonate groups.

[0171] In some embodiments of the present invention, the 5' and 3' ends of the sense strand of the RNA reagent independently contain 0, 1, 2, 3, 4, or 5 phosphate-thioester nucleoside links, respectively; and / or the 5' and 3' ends of the antisense strand of the RNA reagent independently contain 0, 1, 2, 3, 4, or 5 phosphate-thioester nucleoside links, respectively.

[0172] In some embodiments of the present invention, the RNAi reagent further comprises one or more ligand groups or linker groups.

[0173] In some embodiments of the present invention, the ligand group or linker group is conjugated to the sense or antisense strand of the RNAi reagent; preferably, the ligand group or linker group is conjugated to the 3' end of the sense strand, and / or the ligand group or linker group is conjugated to the 5' end of the sense strand, and / or the ligand group or linker group is conjugated to the 3' end of the antisense strand.

[0174] The present invention also provides a composition comprising any of the above-described RNAi reagents and a pharmaceutically acceptable carrier.

[0175] The present invention also provides a method for inhibiting the expression of a target gene in a cell, the method comprising: delivering any of the above-described RNAi reagents or compositions to a subject, such that the RNAi reagent is delivered to a specific target of the subject.

[0176] The present invention also provides a method for treating a disease, the method comprising: delivering any of the above-described RNAi reagents or compositions to a subject, such that the RNAi reagent is delivered to a specific target of the subject, the disease being selected from viral diseases, neuromuscular diseases, bacterial infections, inflammatory and immune diseases, metabolic diseases, liver diseases, kidney diseases, cardiovascular diseases, ophthalmic diseases, lung diseases, and rare diseases.

[0177] The present invention also provides the use of any of the above-described RNAi reagents or compositions in the preparation of medicaments for viral diseases, neuromuscular diseases, bacterial infections, inflammatory and immune diseases, metabolic diseases, liver diseases, kidney diseases, cardiovascular diseases, ophthalmic diseases, lung diseases, and rare diseases.

[0178] The present invention also provides any of the above-mentioned RNAi reagents or compositions for use in inhibiting the expression of target genes in cells or for treating viral diseases, neuromuscular diseases, bacterial infections, inflammatory and immune diseases, metabolic diseases, liver diseases, kidney diseases, cardiovascular diseases, eye diseases, lung diseases and rare diseases.

[0179] The present invention provides one or more RNAi reagents with specific modification combinations, which can effectively reduce the expression of target genes in tissue cells.

[0180] The RNAi reagents with one or more specific modification combinations provided by this invention have a certain enhancement effect on activity compared with existing general-purpose RNAi reagents.

[0181] The present invention provides one or more RNAi reagents with specific modification combinations, especially when applied to siRNA, which have good knockdown effects on different target genes (TTR, AGT, C5, ANPTL3, MUC5AC, INHBE, TMPRSS6, HBV, CFB, APOC3, C3, etc.).

[0182] The RNAi reagents with specific modification combinations provided by this invention are applicable to siRNA sequences of different lengths (sense / antisense strand lengths of 19 / 19, 19 / 21, 21 / 21, 21 / 23, 20 / 22, etc.) and exhibit high activity when applied to siRNA. Attached Figure Description

[0183] Figure 1 shows the serum HBsAg expression results in transgenic mice in Example 6;

[0184] Figure 2 shows the results of TTR expression in mouse serum in Example 7;

[0185] Figure 3 shows the results of CFB mRNA expression in mouse liver in Example 8;

[0186] Figure 4 shows the results of hAPOC3 downregulation in mouse serum in Example 9;

[0187] Figure 5 shows the relative expression results of mouse liver C3 mRNA in Example 10. Detailed Implementation

[0188] Invention Details

[0189] In a first aspect, the present invention provides an RNAi reagent for inhibiting the expression of a target gene. The “RNAi” or “RNAi reagent” according to the present invention is an active molecule containing RNA (or a derivative thereof), wherein the active agent is capable of mediating the targeted cleavage of messenger RNA (mRNA) via the RNA-induced silencing complex (RISC) pathway.

[0190] Intrinsic RNAi (RNA interference) mechanisms in organisms typically involve a series of processes, including: Dicer processing long dsRNA into short 19-21 base pairs (bp) siRNA; siRNA binding to Ago protein to form an RNA-induced silencing complex (RISC); the AGO protein cleaving the sense strand of the siRNA and releasing it; subsequently, the mature RISC bound to the antisense strand cleaves the mRNA that is anticomplementary to the antisense strand through a sequence complementation mechanism. Based on this RNA interference mechanism, various artificial RNAi molecules with different structures have been developed. These structures can enter the RNAi pathway at different stages to achieve sequence-specific cleavage of target gene transcripts. See, for example, Molecules 2019, 24, 2211; doi:10.3390 / molecules24122211 (this literature is hereby incorporated herein by reference in its entirety for the purposes of this invention). Artificial RNAi molecules with such structures include, for example, siRNA molecules having a double-stranded region and one or two overhangs, long siRNA molecules that can serve as substrates for the Dicer enzyme, short hairpin RNA (shRNA) that can be processed by Dicer to produce siRNA structures, and long single-stranded siRNA molecules containing only an antisense strand. It is understood that these molecular forms all fall within the scope of the RNAi activators of the present invention. However, in some aspects, preferably, the RNAi activator according to the present invention refers to an siRNA molecule comprising an oligonucleotide double strand, i.e., a sense strand (sense oligonucleotide) and an antisense strand (antisense oligonucleotide), wherein the sense strand is complementary to at least a portion of the sequence of the antisense strand (e.g., at least 15, 16, 17, 18, or 19 consecutive nucleotides) to form an oligonucleotide double-stranded region. For example, siRNA molecules having a 19-21 nt double-stranded region and a 2-nt 3' overhang, or siRNA molecules containing a 20-22 nt antisense strand and a 15-16 nt short sense strand, thus having an atypical long overhang, are all within the scope of the present invention. Furthermore, molecules in which the sense and antisense strands of the disclosed RNAi molecule are covalently linked together by a single nucleotide strand or other linkage (e.g., the shRNA molecule described below) are also considered in this invention.

[0191] In this invention, the RNAi-related term "antisense strand" refers to an oligonucleotide chain in which the RNAi activator contains a region complementary to a consecutive nucleotide of the target sequence. In this invention, the RNAi-related term "sense strand" refers to an oligonucleotide chain in which the RNAi activator contains a region complementary to at least a portion of the antisense strand to form a double-stranded region.

[0192] For the purpose of inhibiting target mRNA expression, as those skilled in the art know, the oligonucleotide used as the sense strand does not participate in direct complementary binding to the target gene, and does not need to have perfectly complementary base pairing with the antisense oligonucleotide in the duplex region. Therefore, in some aspects, the sense strand according to the invention may include at least one or more of the following properties: substantially complementary to the consecutive nucleotides of the antisense strand in the hybridization duplex region, for example, at least 70%, at least 80%, at least 90%, or 100% complementary. Similarly, for the purpose of inhibiting target mRNA expression, as those skilled in the art know, the antisense strand serving as a guide RNAi for specific binding to the target mRNA may also contain sequences that are not 100% complementary to the consecutive nucleotide regions of the target mRNA; for example, the complementarity may be at least 80%, at least 90%, or 95% complementary; however, in some cases, 100% complementarity is more preferred. According to the purpose of the invention, in some aspects, when considering the complementarity of the sequence motif of the antisense strand with the consecutive nucleotides of the target gene sequence, the presence of insertions and deletions is preferably not permitted. With regard to the sense and antisense strands of the present invention, in some aspects, when the complementary region is not perfectly complementary to the continuous nucleotide region, the mismatch may be located inside or at the end of the region, for example, a mismatch of 3, 2 or 1 nucleotides at the 5' and / or 3' ends.

[0193] In some embodiments, the present invention provides an RNAi reagent whose antisense strand comprises a sequence motif complementary to at least 17, at least 18, at least 19, at least 20, or all of the consecutive nucleotides in a target gene sequence. In some embodiments, the complementarity refers to at least 80% complementarity, i.e., nucleotide mismatches at no more than 20% of the positions in the consecutive nucleotide regions of the target gene sequence. In other embodiments, the complementarity refers to at least 85%, 90%, or 95% complementarity. In still other embodiments, the complementarity refers to 100% complementarity.

[0194] In some embodiments, the present invention also provides a sense strand complementary to at least a portion of the sequence in the antisense strand according to the invention, and a double-stranded RNAi reagent comprising such an antisense strand and a sense strand. In some embodiments, the at least portion of the sequence in the antisense strand has, for example, at least 17, 18, 19, 20, 21, 22, 23 or more nucleotides. In other embodiments, the complementarity refers to at least 80%, 85%, 90%, 95% complementarity, or 100% complementarity. In some embodiments, the length of the sense strand is, for example, about 17, 18, 19, 20, 21, 22, 23, 24, 25 or more nucleotides.

[0195] In this invention, when double-stranded RNAi reagents or siRNAs are involved, the double-stranded region formed by the hybridization of the sense and antisense strands can be of any length that allows for specific degradation of the target mRNA via the RISC pathway. In some embodiments, this length is 17 to 30 base pairs (“bp”), or can be any length within this range, such as 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 bp, and any subranges thereof, including but not limited to 17-30 bp, 17-26 bp, 17-23 bp, 17-22 bp, 17-21 bp, 17-20 bp, 17-19 bp, 17-18 bp, 18-30 bp, 18-26 bp, 18-23 bp, 18- 22bp, 18-21bp, 18-20bp, 19-30bp, 19-26bp, 19-23bp, 19-22bp, 19-21bp, 19-20bp, 19bp, 20-30bp, 20-26bp, 20-25bp, 20-24bp, 20-23bp, 20-22bp, 20-21bp, 20bp, 21-30bp, 21-26bp, 21-25bp, 21-24bp, 21-23bp, 21-22bp, 21bp, 22bp, or 23bp. In some embodiments, the double-stranded RNAi reagent and siRNA according to the present invention have a double-stranded region of about 19 to about 30bp. In some embodiments, the double-stranded RNAi reagent and siRNA according to the present invention have a double-stranded region of about 19 to about 27bp. In some embodiments, the double-stranded RNAi reagent and siRNA according to the present invention have a double-stranded region of about 19 to about 25 bp. In some embodiments, the double-stranded RNAi reagent and siRNA according to the present invention have a double-stranded region of about 19 to about 23 bp. In some embodiments, the double-stranded RNAi reagent and siRNA according to the present invention have a double-stranded region of about 19 to about 21 bp. In some embodiments, the double-stranded RNAi reagent and siRNA according to the present invention have a double-stranded region of about 21 bp.

[0196] In some embodiments, the double-stranded RNAi reagent or siRNA according to the invention may have one or two overhangs. When the 3' end of one strand of the double-stranded oligonucleotide constituting the double-stranded RNAi reagent or siRNA extends beyond the 5' end of the other strand, or when the 5' end of one strand extends beyond the 3' end of the other strand, unpaired nucleotides form overhangs. The length of the overhang can be at least one nucleotide; optionally, the overhang can contain at least two, three, four, five, or more nucleotides. The overhang can contain modified or unmodified nucleotides, or be composed of modified or unmodified nucleotides. The overhang can be located on the sense strand, the antisense strand, or any combination thereof. Furthermore, the overhang can be located at the 5' end and / or the 3' end of the antisense strand or the sense strand. In some preferred embodiments, the RNAi reagent according to the invention comprises a double-stranded structure with overhangs consisting of an antisense strand and a sense strand, and preferably, the overhang is a single 3' overhang consisting of one, two, three, or four nucleotides from the terminal 3' end of the antisense strand. More preferably, the overhang is a single 3' overhang composed of the last 2 nucleotides of the antisense strand 3'.

[0197] The RNAi reagent according to the invention can also have zero protrusions. In the case of zero protrusions, all ends of the RNAi reagent are blunt ends, and such molecules lack 3' or 5' single-stranded nucleotide protrusions.

[0198] Unless otherwise specified, the nucleosides in the RNAi reagents of this invention may be unmodified (i.e., comprising naturally occurring RNA nucleosides) or (and preferably) modified, as long as they retain the desired functional activity (i.e., capable of forming the desired double-stranded structure and allowing or mediating specific degradation of the target RNA via the RISC pathway). Such RNA modifications may occur at the base moiety, sugar moiety, and / or phosphate ester linkage of the nucleotide.

[0199] In some aspects, the present invention covers specific modifications of the described RNAi reagents. Examples of modifications that may be mentioned include, for example: 2'-methoxy-modified nucleotides, 2'-F-modified nucleotides, 2'-deoxynucleotides, 2'-O-methoxyethylnucleotide modifications, threononucleotide (TNA) modifications, glycol nucleotide (GNA) modifications, locked nucleotide (LNA) modifications, open-ring nucleotide (UNA) modifications, bicyclic nucleotide (BNA) modifications, 2',5'-linked nucleotides, conformation-restricted nucleotide modifications, 2'-amino-modified nucleotides, 2'-alkyl-modified nucleotides, 2'-allyl-modified nucleotides, and debased nucleosides. Acid modification, 2'-O-alkylnucleotide, morpholinonucleotide, aminophosphamide nucleotide modification, nucleotide modification with non-natural bases, tetrahydropyranonucleotide modification, 1,5-dehydrated hexadiol nucleotide modification, cyclohexenyl nucleotide modification, nucleotide modification containing thiophosphate groups, nucleotide modification containing methylphosphate groups, nucleotide modification containing 2'-phosphates, nucleotide modification containing 5'-vinylphosphates, thermostable nucleotide modification, thermostable nucleotide modification, and 2-O-(N-methylacetamide) nucleotide modification; and combinations thereof.

[0200] In some embodiments, the RNAi reagent according to the present invention may have one or more modifications inside the nucleic acid molecule or at one or both ends. Examples of nucleoside base modifications, ribose moiety modifications, and phosphate backbone modifications that can be used in the RNAi reagent of the present invention are described below.

[0201] Nucleoside base modification

[0202] “G,” “C,” “A,” “T,” and “U” typically represent nucleotides containing guanine, cytosine, adenine, thymine nucleotides, and uracil as bases, respectively. However, those skilled in the art will recognize that guanine, cytosine, adenine, and uracil can be replaced by other parts without substantially altering the base-pairing properties of the oligonucleotide containing the nucleotide with such a substitution. Examples of such nucleoside base modifications that can be used to generate RNAi activators include the substitution of nucleotides containing uracil, guanine, or adenine with nucleotides containing, for example, inosine; and the replacement of adenine and cytosine in oligonucleotides with guanine and uracil, respectively, to form a GU Wobble base pair with the target mRNA. In addition, other examples of modified nucleoside bases that can be used to generate RNAi activators include, but are not limited to: 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xantine, 4-acetylcytosine, 5-(carboxyhydroxymethyl)uracil, 5-carboxymethylaminomethyl-2-thiouracil, 5-carboxymethylaminomethyluracil, dihydrouracil, β-D-galactosylqueosine, inosine, N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7 -Methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, β-D-mannosyl queosine, 5'-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N6-isopentenyladenine, uracil-5-hydroxyacetic acid (v), wybutoxosine, pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-hydroxyacetic acid methyl ester, uracil-5-hydroxyacetic acid (v), 5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl)uracil, (acp3)w, and 2,6-diaminopurine. These modified nucleoside bases are all within the scope of this invention.

[0203] Sugar modification

[0204] Compared to the ribose moiety present in natural RNA, the oligonucleotides of the RNAi reagent of the present invention can contain one or more nucleosides with modified sugar moieties (i.e., sugar-modified nucleotides). Numerous ribose-modified nucleotides have been developed, primarily with the aim of improving certain properties of oligonucleotides, such as affinity and / or nuclease resistance, and off-target effects. Sugar modification can include modifications by replacing the naturally occurring 2'-OH group on the ribose ring of the RNA nucleoside with other groups. Furthermore, substituents can be introduced, for example, at the 2', 3', 4', or 5' positions of the sugar ring. The following are some specific examples of 2'-modified nucleotides:

[0205] Nucleotide analogues

[0206] In some embodiments, the RNAi reagent of the present invention may comprise one or more nucleotide analogs. Available nucleotide analogs include, for example, nucleotide analogs formed by replacing the ribocycle structure with one of the following: a hexose ring (HNA), a threonose ring (TNA), ethylene glycol (GNA, an acyclic nucleotide analog in which the pentose sugar of the nucleotide is replaced by propylene glycol), a locked nucleic acid structure (LNA, a bicyclic ring with a double-base bridge between the C2 and C4 carbons on the ribocycle), or a non-locked nucleic acid structure (UNA, a ribocycle lacking a bond between the C2 and C3 carbons). Other examples of available nucleotide analogs include, for example, bicyclic hexosennucleotides or tricyclic nucleic acids; and peptide nucleic acids (PNA) or morpholinonucleotides. Other examples include the modified nucleotides described in WO2025067240A1.

[0207] Here are some specific examples of nucleotide analogs:

[0208] Phosphate modification

[0209] Various phosphate ester modifications for use in RNAi reagents are known in the art. Such modified internucleotide linkages include, for example, thiophosphates, chiral thiophosphates, dithiophosphates, triphosphates, aminoalkyl phosphates, methyl and other alkylphosphonates (including 3'-alkylphosphonates and chiral phosphonates), phosphonites, and aminophosphates (including 3'-aminoaminophosphates and aminoalkylaminophosphates). Furthermore, modified internucleotide linkages that do not contain phosphorus atoms are also within the scope of this invention. In some embodiments, thiophosphate internucleotide linkages are introduced into the nucleosides of the oligonucleotides of the RNAi reagent of this invention. This modification can enhance the nuclease stability of said oligonucleotides.

[0210] In some embodiments, the RNAi reagent according to the invention may optionally also include chemical modifications at the 5' and / or 3' ends, i.e., non-nucleotide or nucleoside chemical portions linked to the ends of the oligonucleotide chains (sense and / or antisense strands) of the RNAi. Examples of chemical portions linked to the 5' ends of the oligonucleotide chains may include, but are not limited to, 5'-terminal phosphate modifications, preferably wherein said modifications are selected from: 5'-(E)-vinylphosphonate (5'-(E)-VP), 5'-methylphosphonate (5'-MP), (S)-5'-C-methyl analogs, and 5'-thiophosphate (5'-PS).

[0211] In some embodiments, the present invention provides an RNAi reagent for inhibiting the expression of a target gene, comprising a sense strand and an antisense strand, each strand having 17 to 30 nucleotides, wherein each nucleotide is independently a modified or unmodified nucleotide; wherein the antisense strand is complementary to at least a portion of the mRNA of the target gene, and the sense strand and the antisense strand are at least partially complementary to form a double-stranded region.

[0212] The 5th and 7th positions of the 5' end of the antisense strand contain 2'-deoxynucleotides; the sense strand position corresponding to the 12th position of the 5' end of the antisense strand contains a nucleotide that is not modified by 2'-F.

[0213] In this invention, the sense strand position corresponding to a certain position of the antisense strand refers to the position of the sense strand in the double-stranded region that corresponds to a certain position of the antisense strand when the sense strand and the antisense strand are at least partially complementary to form a double-stranded region. For example, when the sense strand and the antisense strand completely complement each other to form a double-stranded region, if the 5' end of the antisense strand (i.e., the 3' end of the sense strand) has a blunt end, the 12th position of the 5' end of the antisense strand corresponds to the 12th position of the 3' end of the sense strand; or, for another example, if the 3' end of the sense strand has two nucleotide overhangs, the 12th position of the 5' end of the antisense strand corresponds to the 14th position of the 3' end of the sense strand.

[0214] The nucleotide not modified by 2'-F can be an unmodified nucleotide or other modified nucleotides not modified by 2'-F. In some embodiments of the present invention, the nucleotide not modified by 2'-F can be a nucleotide containing 2'-methoxy or 2'-deoxynucleotide; that is, the sense position corresponding to the 12th position at the 5' end of the antisense strand contains a nucleotide containing 2'-methoxy or 2'-deoxynucleotide.

[0215] In some embodiments of the invention, positions 2, 9, 12, 14, 16, 18, 20 or 22 at the 5' end of the antisense strand further comprise 2'-deoxynucleotides.

[0216] In some embodiments of the present invention, the second position at the 5' end of the antisense strand contains a 2'-F modified nucleotide or a 2'-deoxynucleotide.

[0217] In some embodiments of the present invention, the 15th position of the 5' end of the antisense strand contains a heat-stabilized modified nucleotide;

[0218] In some embodiments of the present invention, the thermally stabilized modified nucleotide refers to the double-stranded oligonucleotide whose thermal dissociation temperature Tm increases after introduction; in some embodiments of the present invention, the thermal dissociation temperature Tm increases by at least 0.05°C; in some embodiments of the present invention, the thermal dissociation temperature Tm increases by 0.1-6°C; in some embodiments of the present invention, the thermal dissociation temperature Tm increases by 0.5-4°C.

[0219] In some specific embodiments of the present invention, the heat-stabilized modified nucleotide is selected from nucleotides modified with 2'-O-methoxyethyl, nucleotides modified with 2'-O-allyl, nucleotides modified with 2'-C-allyl, nucleotides modified with 2'-O-2-N-methylamino-2-oxomethyleneethyl, nucleotides modified with 2'-O-2-N,N-dimethylaminoethyl, nucleotides modified with 2'-O-3-aminopropyl, nucleotides modified with 2'-O-2,4-dinitrophenyl, nucleotides modified with 2'-O-(N-methylacetamido)-modified, nucleotides modified with 2',4'-disubstituted, locked nucleotides (LNA), bicyclic nucleotides (BNA), or restricted ethyl-bridged nucleotides (cEt); preferably, the heat-stabilized modified nucleotide is selected from nucleotides modified with 2'-O-methoxyethyl.

[0220] In some embodiments of the present invention, the 14th position of the 5' end of the antisense strand contains a nucleotide modified with 2'-F.

[0221] In some embodiments of the present invention, the sense strand position corresponding to the 11th and / or 13th position at the 5' end of the antisense strand contains a nucleotide modified with 2'-F.

[0222] In some embodiments of the present invention, the 12th and / or 16th position of the 5' end of the antisense strand contains a nucleotide modified with 2'-F.

[0223] In some embodiments of the present invention, the sense strand position corresponding to the 6th position at the 5' end of the antisense strand contains a nucleotide modified with 2'-F.

[0224] In some embodiments of the present invention, the sense strand position corresponding to the 9th position at the 5' end of the antisense strand contains a nucleotide modified with 2'-F.

[0225] In some embodiments of the present invention, the sense strand position corresponding to the 12th position at the 5' end of the antisense strand contains a 2'-deoxynucleotide.

[0226] In some embodiments of the present invention, the sense strand position corresponding to the 6th position at the 5' end of the antisense strand contains a 2'-deoxynucleotide.

[0227] In some embodiments of the present invention, the sense strand position corresponding to the 15th position at the 5' end of the antisense strand contains a 2'-deoxynucleotide.

[0228] In some embodiments of the present invention, each strand of the sense and antisense strands of the RNAi reagent has 17 to 25 nucleotides; preferably, each strand has 19 to 23 nucleotides.

[0229] In some embodiments of the present invention, the sense strand of the RNAi reagent has 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides; in some embodiments of the present invention, the antisense strand of the RNAi reagent has 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides.

[0230] In some embodiments of the present invention, the sense strand of the RNAi reagent has 19 nucleotides and the antisense strand has 19 nucleotides; or the sense strand of the RNAi reagent has 19 nucleotides and the antisense strand has 21 nucleotides; the sense strand of the RNAi reagent has 20 nucleotides and the antisense strand has 20 nucleotides; the sense strand of the RNAi reagent has 20 nucleotides and the antisense strand has 22 nucleotides; the sense strand of the RNAi reagent has 21 nucleotides and the antisense strand has 21 nucleotides; or the sense strand of the RNAi reagent has 21 nucleotides and the antisense strand has 23 nucleotides.

[0231] In some embodiments of the present invention, the RNAi reagent comprises one or more single-stranded nucleotide overhangs; the overhangs can be the result of one strand being longer than another, or the result of two strands of equal length being interleaved; preferably, the overhangs are located at the 3' end of the antisense strand of the RNAi reagent. The overhangs can be 1-5 nucleotides long, 2-5 nucleotides long, 1-4 nucleotides long, 2-4 nucleotides long, 1-3 nucleotides long, 2-3 nucleotides long, or 1-2 nucleotides long.

[0232] In some embodiments of the present invention, the RNAi reagent has blunt ends; preferably, the RNAi reagent has at least one blunt end located at the 5' end of the antisense strand (i.e., the 3' end of the sense strand).

[0233] In some embodiments of the present invention, when the 5' end of the antisense strand (i.e. the 3' end of the sense strand) is blunt, the 12th position of the 3' end of the sense strand contains a nucleotide that is not modified by 2'-F.

[0234] In some embodiments of the present invention, when the 5' end of the antisense strand (i.e. the 3' end of the sense strand) is blunt, the 12th position of the 3' end of the sense strand contains a 2'-methoxy modified nucleotide or a 2'-deoxy nucleotide.

[0235] In some embodiments of the present invention, when the 5' end of the antisense strand (i.e. the 3' end of the sense strand) is blunt, the 11th and / or 13th positions of the 3' end of the sense strand contain a nucleotide modified with 2'-F.

[0236] In some embodiments of the present invention, when the 5' end of the antisense strand (i.e. the 3' end of the sense strand) is blunt, the 6th position of the 3' end of the sense strand contains a nucleotide modified with 2'-F.

[0237] In some embodiments of the present invention, when the 5' end of the antisense strand (i.e. the 3' end of the sense strand) is blunt, the 9th position of the 3' end of the sense strand contains a nucleotide modified with 2'-F.

[0238] In some embodiments of the present invention, when the 5' end of the antisense strand (i.e. the 3' end of the sense strand) is flat-ended, the 12th position of the 3' end of the sense strand contains a 2'-deoxynucleotide.

[0239] In some embodiments of the present invention, when the antisense strand 5' end (i.e. the sense strand 3' end) is flat-ended, the 6th, 7th, 9th, 10th, 14th, 17th and / or 19th positions of the sense strand 3' end contain 2'-deoxynucleotides.

[0240] In some embodiments of the present invention, when the antisense strand 5' end (i.e. the sense strand 3' end) is flat-ended, the 15th position of the sense strand 3' end contains a 2'-deoxynucleotide.

[0241] In some embodiments of the present invention, the remaining positions in the sense or antisense strand of the RNAi reagent are modified nucleotides; preferably, the modified nucleotides are selected from 2'-methoxy-modified nucleotides, 2'-F-modified nucleotides, 2'-deoxynucleotides, 2'-O-methoxyethyl-modified nucleotides, threonucleotides (TNA), ethylene glycol nucleotides (GNA), locked nucleotides (LNA), open-ring nucleotides (UNA), bicyclic nucleotides (BNA), 2',5'-linked nucleotides, 2'-amino-modified nucleotides, 2'-alkyl-modified nucleotides, 2'-allyl-modified nucleotides, debased nucleotides, morpholinonucleotides, and phosphate ester-modified nucleotides, but the present invention is not limited thereto. In some embodiments of the present invention, the modified nucleotides at the remaining positions in the sense or antisense strand of the RNAi reagent are the modified nucleotides described in WO2025067240A1. In some embodiments of the present invention, the nucleotide at the 5' end of the sense strand of the RNAi reagent is the modified nucleotide described in WO2025067240A1.

[0242] In some specific embodiments of the present invention, the RNAi reagent has a combination of modifications selected from any of the following:

[0243] 1) The antisense strand contains 2'-F modified nucleotides at positions 2, 12, 14, and 16 at the 5' end, 2'-deoxynucleotides at positions 5 and 7, and 2'-O-methoxyethyl modified nucleotides at position 15; the sense strand contains 2'-F modified nucleotides at positions 9, 11, and 13 at the 3' end.

[0244] 2) The antisense strand contains 2'-F modified nucleotides at positions 2, 14, and 16 of the 5' end; 2'-deoxynucleotides at positions 5, 7, and 12; and 2'-O-methoxyethyl modified nucleotides at position 15. The sense strand contains 2'-F modified nucleotides at positions 9, 11, and 13 of the 3' end.

[0245] 3) The antisense strand contains 2'-F modified nucleotides at positions 2, 12, and 14 at the 5' end; 2'-deoxynucleotides at positions 5, 7, and 16; and 2'-O-methoxyethyl modified nucleotides at position 15. The sense strand contains 2'-F modified nucleotides at positions 6, 9, 11, and 13 at the 3' end.

[0246] 4) The antisense strand contains 2'-F modified nucleotides at positions 12, 14, and 16 of the 5' end; 2'-deoxynucleotides at positions 2, 5, and 7; and 2'-O-methoxyethyl modified nucleotides at position 15. The sense strand contains 2'-F modified nucleotides at positions 9, 11, and 13 of the 3' end.

[0247] 5) The antisense strand contains 2'-F modified nucleotides at positions 12, 14, and 16 of the 5' end; 2'-deoxynucleotides at positions 2, 5, and 7; and 2'-O-methoxyethyl modified nucleotides at position 15. The sense strand contains 2'-F modified nucleotides at positions 6, 9, 11, and 13 of the 3' end.

[0248] 6) The antisense strand contains 2'-F modified nucleotides at positions 2, 14, and 16 of the 5' end; 2'-deoxynucleotides at positions 5, 7, and 9; and 2'-O-methoxyethyl modified nucleotides at position 15. The sense strand contains 2'-F modified nucleotides at positions 9, 11, and 13 of the 3' end.

[0249] 7) The antisense strand contains 2'-F modified nucleotides at positions 2, 12, and 16 of the 5' end; 2'-deoxynucleotides at positions 5, 7, and 14; and 2'-O-methoxyethyl modified nucleotides at position 15; the sense strand contains 2'-F modified nucleotides at positions 9, 11, and 13 of the 3' end.

[0250] 8) The antisense strand contains 2'-F modified nucleotides at positions 2, 12, 14, and 16 at the 5' end, 2'-deoxynucleotides at positions 5 and 7, and 2'-O-methoxyethyl modified nucleotides at position 15; the sense strand contains 2'-F modified nucleotides at positions 9, 11, and 13 at the 3' end, and 2'-deoxynucleotides at position 15.

[0251] 9) The antisense strand contains 2'-F modified nucleotides at positions 2, 12, 14, and 16 at the 5' end; 2'-deoxynucleotides at positions 5, 7, and 18; and 2'-O-methoxyethyl modified nucleotides at position 15. The sense strand contains 2'-F modified nucleotides at positions 9, 11, and 13 at the 3' end.

[0252] 10) The antisense strand contains 2'-F modified nucleotides at positions 2, 12, 14, and 16 at the 5' end; 2'-deoxynucleotides at positions 5, 7, and 20; and 2'-O-methoxyethyl modified nucleotides at position 15. The sense strand contains 2'-F modified nucleotides at positions 9, 11, and 13 at the 3' end.

[0253] 11) The antisense strand contains 2'-F modified nucleotides at positions 2, 12, 14, and 16 at the 5' end; 2'-deoxynucleotides at positions 5, 7, and 22; and 2'-O-methoxyethyl modified nucleotides at position 15. The sense strand contains 2'-F modified nucleotides at positions 9, 11, and 13 at the 3' end.

[0254] 12) The antisense strand contains 2'-F modified nucleotides at positions 2, 12, 14, and 16 at the 5' end, 2'-deoxynucleotides at positions 5 and 7, and 2'-O-methoxyethyl modified nucleotides at position 15; the sense strand contains 2'-F modified nucleotides at positions 6, 9, 11, and 13 at the 3' end.

[0255] 13) The antisense strand contains 2'-F modified nucleotides at positions 2, 12, 14, and 16 at the 5' end, 2'-deoxynucleotides at positions 5 and 7, and 2'-O-methoxyethyl modified nucleotides at position 15; the sense strand contains 2'-F modified nucleotides at positions 9, 11, and 13 at the 3' end, and 2'-deoxynucleotides at position 6.

[0256] 14) The antisense strand contains 2'-F modified nucleotides at positions 2, 12, 14, and 16 at the 5' end, 2'-deoxynucleotides at positions 5 and 7, and 2'-O-methoxyethyl modified nucleotides at position 15; the sense strand contains 2'-F modified nucleotides at positions 9, 11, and 13 at the 3' end, and 2'-deoxynucleotides at position 19.

[0257] 15) The antisense strand contains 2'-F modified nucleotides at positions 2, 12, 14, and 16 at the 5' end, 2'-deoxynucleotides at positions 5 and 7, and 2'-O-methoxyethyl modified nucleotides at position 15; the sense strand contains 2'-F modified nucleotides at positions 9, 11, and 13 at the 3' end, and 2'-deoxynucleotides at position 14.

[0258] 16) The antisense strand contains 2'-F modified nucleotides at positions 2, 12, 14, and 16 at the 5' end, 2'-deoxynucleotides at positions 5 and 7, and 2'-O-methoxyethyl modified nucleotides at position 15; the sense strand contains 2'-F modified nucleotides at positions 9, 11, and 13 at the 3' end, and 2'-deoxynucleotides at position 17.

[0259] 17) The antisense strand contains 2'-F modified nucleotides at positions 2, 12, 14, and 16 at the 5' end, 2'-deoxynucleotides at positions 5 and 7, and 2'-O-methoxyethyl modified nucleotides at position 15; the sense strand contains 2'-F modified nucleotides at positions 11 and 13 at the 3' end, and 2'-deoxynucleotides at position 9.

[0260] 18) The antisense strand contains 2'-F modified nucleotides at positions 2, 12, 14, and 16 at the 5' end, 2'-deoxynucleotides at positions 5 and 7, and 2'-O-methoxyethyl modified nucleotides at position 15; the sense strand contains 2'-F modified nucleotides at positions 9, 11, and 13 at the 3' end, and 2'-deoxynucleotides at position 12.

[0261] 19) The antisense strand contains 2'-F modified nucleotides at positions 2, 12, 14, and 16 at the 5' end, 2'-deoxynucleotides at positions 5 and 7, and 2'-O-methoxyethyl modified nucleotides at position 15; the sense strand contains 2'-F modified nucleotides at positions 9, 11, and 13 at the 3' end, and 2'-deoxynucleotides at position 7.

[0262] 20) The antisense strand contains 2'-F modified nucleotides at positions 2, 12, 14, and 16 at the 5' end, 2'-deoxynucleotides at positions 5 and 7, and 2'-O-methoxyethyl modified nucleotides at position 15; the sense strand contains 2'-F modified nucleotides at positions 9, 11, and 13 at the 3' end, and 2'-deoxynucleotides at position 10.

[0263] Preferably, the remaining positions in the sense or antisense strand of the RNAi reagent are nucleotides modified with 2'-methoxy groups.

[0264] In other embodiments of the present invention, an RNAi reagent for inhibiting the expression of a target gene is provided, comprising a sense strand and an antisense strand, each strand having 17 to 30 nucleotides, wherein each nucleotide is independently a modified or unmodified nucleotide; wherein the antisense strand is complementary to at least a portion of the mRNA of the target gene, and the sense strand and the antisense strand are at least partially complementary to form a double-stranded region.

[0265] The antisense chain includes the sequence shown in formula IA;

[0266] 3'-(N) n NNN E NN N'N NNNN d NN d NNN F N-5' (Form IA)

[0267] in,

[0268] n represents an integer from 0 to 8; preferably, n represents 2, 3, 4, 5, or 6.

[0269] N F Nucleotides representing 2'-F modifications;

[0270] Each Nd They represent 2'-deoxynucleotides;

[0271] Each N, N', or N E Each can be used independently to represent a modified or unmodified nucleotide;

[0272] The nucleotide corresponding to N' in the sense strand is not a nucleotide modified by 2'-F.

[0273] The nucleotide not modified by 2'-F can be an unmodified nucleotide or other modified nucleotides not modified by 2'-F. In some embodiments of the present invention, the nucleotide not modified by 2'-F can be a nucleotide containing 2'-methoxy modification or a 2'-deoxynucleotide; that is, the nucleotide corresponding to N' in the sense strand is selected from nucleotides containing 2'-methoxy modification or 2'-deoxynucleotides.

[0274] In some embodiments of the present invention, N E The nucleotides are selected from those containing heat-stabilized modifications; the heat-stabilized modifications refer to the increase in the thermal dissociation temperature Tm of the double-stranded oligonucleotide after introduction; in some embodiments of the present invention, the thermal dissociation temperature Tm increases by at least 0.05°C; in some embodiments of the present invention, the thermal dissociation temperature Tm increases by 0.1-6°C; in some embodiments of the present invention, the thermal dissociation temperature Tm increases by 0.5-4°C.

[0275] In some specific embodiments of the present invention, the heat-stabilized modified nucleotide is selected from nucleotides modified with 2'-O-methoxyethyl, nucleotides modified with 2'-O-allyl, nucleotides modified with 2'-C-allyl, nucleotides modified with 2'-O-2-N-methylamino-2-oxomethyleneethyl, nucleotides modified with 2'-O-2-N,N-dimethylaminoethyl, nucleotides modified with 2'-O-3-aminopropyl, nucleotides modified with 2'-O-2,4-dinitrophenyl, nucleotides modified with 2'-O-(N-methylacetamido)-modified, nucleotides modified with 2',4'-disubstituted nucleotides, locked nucleotides (LNA), bicyclic nucleotides (BNA), or restricted ethyl-bridged nucleotides (cEt); preferably, the heat-stabilized modified nucleotide is selected from nucleotides modified with 2'-O-methoxyethyl.

[0276] In some embodiments of the present invention, the antisense chain includes the sequence shown by formula IAa;

[0277] 3'-(N) n N a17 N a16 N E N F N a13 N'N a11 Na10 N a9 N a8 N d N a6 N d N a4 N a3 N F N a1 -5' (Formula IAa)

[0278] in,

[0279] n represents an integer from 0 to 8;

[0280] Each N F These represent nucleotides modified by 2'-F;

[0281] Each N d They represent 2'-deoxynucleotides;

[0282] Each N and N' independently represents a modified or unmodified nucleotide;

[0283] N a1 N a3 N a4 N a6 N a8 N a9 N a10 N a11 N a13 N a16 N a17 Each can be used independently to represent a modified or unmodified nucleotide;

[0284] N E This represents a nucleotide containing a heat-stabilizing modification; preferably, N E This represents a nucleotide modified with 2'-O-methoxyethyl.

[0285] On the other hand, in some embodiments of the present invention, the sense chain includes the sequence shown in Formula II;

[0286] 5'-(N) m NNNNNN T NNNNNNNNNN N-3' (Equation II)

[0287] in,

[0288] m represents an integer from 0 to 8; preferably, m represents 2, 3, 4, 5, or 6.

[0289] N T This represents a nucleotide that is not modified by 2'-F;

[0290] Each N independently represents a modified or unmodified nucleotide.

[0291] The non-2'-F modified nucleotide can be an unmodified nucleotide or other modified nucleotides that are not 2'-F modified. In some embodiments of the present invention, the non-2'-F modified nucleotide can be a nucleotide containing 2'-methoxy modification or a 2'-deoxynucleotide; that is, N... T This represents a nucleotide or 2'-deoxynucleotide containing a 2'-methoxy group.

[0292] In some embodiments of the present invention, the sense chain includes the sequence shown in Form IIA;

[0293] 5'-(N) m N s17 N s16 N s15 N s14 N F N T N F N s10 N s9 N s8 N s7 N s6 N s5 N s4 N s3 N s2 N s1 -3' (Formula IIA)

[0294] in,

[0295] m represents an integer from 0 to 8;

[0296] N T This represents a nucleotide containing a 2'-methoxy group or a 2'-deoxynucleotide.

[0297] Each N F These represent nucleotides modified by 2'-F;

[0298] Each N independently represents a modified or unmodified nucleotide;

[0299] N s1 N s2 N s3 N s4 N s5 N s6 N s7 N s8 N s9 N s10 N s14 N s15N s16 N s17 Each can be used to represent a modified or unmodified nucleotide.

[0300] In some embodiments of the present invention, the sense chain includes the sequence represented by formula IIAa;

[0301] 5'-(N) m N s17 N s16 N s15 N s14 N F N T N F N s10 N F N s8 N s7 N s6 N s5 N s4 N s3 N s2 N s1 -3' (Formula IIAa)

[0302] in,

[0303] m represents an integer from 0 to 8;

[0304] N T This represents a nucleotide containing a 2'-methoxy group or a 2'-deoxynucleotide.

[0305] Each N F These represent nucleotides modified by 2'-F;

[0306] Each N independently represents a modified or unmodified nucleotide;

[0307] N s1 N s2 N s3 N s4 N s5 N s6 N s7 N s8 N s10 N s14 N s15 N s16 N s17 Each can be used to represent a modified or unmodified nucleotide.

[0308] In some embodiments of the present invention, the sense chain includes the sequence represented by formula IIAb;

[0309] 5'-(N) m N s17N s16 N s15 N s14 N F N d N F N s10 N s9 N s8 N s7 N s6 N s5 N s4 N s3 N s2 N s1 -3' (Formula IIAb)

[0310] in,

[0311] m represents an integer from 0 to 8;

[0312] Each N F These represent nucleotides modified by 2'-F;

[0313] N d Represents 2'-deoxynucleotide;

[0314] Each N independently represents a modified or unmodified nucleotide;

[0315] N s1 N s2 N s3 N s4 N s5 N s6 N s7 N s8 N s9 N s10 N s14 N s15 N s16 N s17 Each can be used to represent a modified or unmodified nucleotide.

[0316] In some embodiments of the present invention, the sense chain includes the sequence represented by IIAc;

[0317] 5'-(N) m N s17 N s16 N s15 N s14 N F N T N F N s10 N s9 N s8 N s7 NF N s5 N s4 N s3 N s2 N s1 -3' (Form IIAc)

[0318] in,

[0319] m represents an integer from 0 to 8;

[0320] N T This represents a nucleotide containing a 2'-methoxy group or a 2'-deoxynucleotide.

[0321] Each N F These represent nucleotides modified by 2'-F;

[0322] Each N independently represents a modified or unmodified nucleotide;

[0323] N s1 N s2 N s3 N s4 N s5 N s7 N s8 N s9 N s10 N s14 N s15 N s16 N s17 Each can be used to represent a modified or unmodified nucleotide.

[0324] In some embodiments of the present invention, the sense chain includes the sequence represented by IIAd;

[0325] 5'-(N) m N s17 N s16 N d N s14 N F N T N F N s10 N s9 N s8 N s7 N s6 N s5 N s4 N s3 N s2 N s1 -3' (Formula IIAd)

[0326] in,

[0327] m represents an integer from 0 to 8;

[0328] N T This represents a nucleotide containing a 2'-methoxy group or a 2'-deoxynucleotide.

[0329] Each N F These represent nucleotides modified by 2'-F;

[0330] N d Represents 2'-deoxynucleotide;

[0331] Each N independently represents a modified or unmodified nucleotide;

[0332] N s1 N s2 N s3 N s4 N s5 N s6 N s7 N s8 N s9 N s10 N s14 N s16 N s17 Each can be used to represent a modified or unmodified nucleotide.

[0333] In some embodiments of the present invention, the nucleotides modified in the RNAi reagent are selected from 2'-methoxy-modified nucleotides, 2'-F-modified nucleotides, 2'-deoxynucleotides, 2'-O-methoxyethyl-modified nucleotides, threonucleotides (TNA), ethylene glycol nucleotides (GNA), locked nucleotides (LNA), open-ring nucleotides (UNA), bicyclic nucleotides (BNA), 2',5'-linked nucleotides, 2'-amino-modified nucleotides, 2'-alkyl-modified nucleotides, 2'-allyl-modified nucleotides, debased nucleotides, morpholinonucleotides, and phosphate ester-modified nucleotides, but the present invention is not limited thereto.

[0334] In some specific embodiments of the present invention, the RNAi reagent comprises an antisense strand having any of the following modifications:

[0335] 1)3'-(N) n N a17 N F N MOE N F N a13 N F N a11 N a10 N a9 N a8 N d Na6 N d N a4 N a3 N F N a1 -5’;

[0336] 2)3’-(N) n N a17 N F N MOE N F N a13 N d N a11 N a10 N a9 N a8 N d N a6 N d N a4 N a3 N F N a1 -5’;

[0337] 3)3’-(N) n N a17 N d N MOE N F N a13 N F N a11 N a10 N a9 N a8 N d N a6 N d N a4 N a3 N F N a1 -5’;

[0338] 4)3’-(N) n N a17 N F N MOE N F N a13 N F N a11 N a10 N a9 N a8 N d N a6 N d N a4 N a3 N d Na1 -5’;

[0339] 5)3’-(N) n N a17 N F N MOE N F N a13 N a12 N a11 N a10 N d N a8 N d N a6 N d N a4 N a3 N F N a1 -5’;

[0340] 6)3’-(N) n N a17 N F N MOE N d N a13 N F N a11 N a10 N a9 N a8 N d N a6 N d N a4 N a3 N F N a1 -5’;

[0341] 7)3’-N a23 N a22 N a21 N a20 N a19 N d N a17 N F N MOE N F N a13 N F N a11 N a10 N a9 N a8 N d N a6 N d N a4 N a3 N F N a1-5';

[0342] 8)3'-N a23 N a22 N a21 N d N a19 N a18 N a17 N F N MOE N F N a13 N F N a11 N a10 N a9 N a8 N d N a6 N d N a4 N a3 N F N a1 -5';

[0343] 9)3'-N a23 N d N a21 N a20 N a19 N a18 N a17 N F N MOE N F N a13 N F N a11 N a10 N a9 N a8 N d N a6 N d N a4 N a3 N F N a1 -5';

[0344] in,

[0345] n represents an integer from 0 to 8; preferably, n is 2, 3, 4, 5, 6 or 7;

[0346] Each N F These represent nucleotides modified by 2'-F;

[0347] Each N d They represent 2'-deoxynucleotides;

[0348] N MOERepresents a nucleotide modified with 2'-O-methoxyethyl;

[0349] Each N independently represents a modified or unmodified nucleotide;

[0350] N a1 N a3 N a4 N a6 N a8 N a9 N a10 N a11 N a12 N a13 N a17 N a18 N a19 N a20 N a21 N a22 N a23 Each can independently represent a modified or unmodified nucleotide; preferably, N a1 N a3 N a4 N a6 N a8 N a9 N a10 N a11 N a12 N a13 N a17 N a18 N a19 N a20 N a21 N a22 N a23 All of them are nucleotides containing 2'-methoxy groups.

[0351] In some more specific embodiments of the invention, the RNAi reagent comprises an antisense strand having any of the following modifications:

[0352] 1)3'-(N M )6N M N F N MOE N F N M N F N M N M N M N M N d N M N d N M N M N F NM -5';

[0353] 2)3'-(N M )6N M N F N MOE N F N M N M N M N M N M N M N d N M N d N M N M N F N M -5';

[0354] 3)3'-(N M )4N M N F N MOE N F N M N F N M N M N M N M N d N M N d N M N M N F N M -5';

[0355] 4)3'-(N M )4N M N F N MOE N F N M N M N M N M N M N M N d N M N d N M N M N F N M -5';

[0356] 5)3'-(N M )6N M NF N MOE N F N M N d N M N M N M N M N d N M N d N M N M N F N M -5';

[0357] 6)3'-(N M )6N M N d N MOE N F N M N F N M N M N M N M N d N M N d N M N M N F N M -5';

[0358] 7)3'-(N M )6N M N F N MOE N F N M N F N M N M N M N M N d N M N d N M N M N d N M -5';

[0359] 8)3'-(N M )6N M N F N MOE N F N M N M NM N M N d N M N d N M N d N M N M N F N M -5';

[0360] 9)3'-(N M )6N M N F N MOE N d N M N F N M N M N M N M N d N M N d N M N M N F N M -5';

[0361] 10)3'-(N M )4N M N d N M N F N MOE N F N M N F N M N M N M N M N d N M N d N M N M N F N M -5';

[0362] 11)3'-(N M )2N M N d N M N M N M N F N MOE N F N M NF N M N M N M N M N d N M N d N M N M N F N M -5';

[0363] 12)3'-N M N d N M N M N M N M N M N F N MOE N F N M N F N M N M N M N M N d N M N d N M N M N F N M -5';

[0364] in,

[0365] Each N F These represent nucleotides modified by 2'-F;

[0366] Each N d They represent 2'-deoxynucleotides;

[0367] N MOE Represents a nucleotide modified with 2'-O-methoxyethyl;

[0368] Each N M These represent nucleotides modified with 2'-methoxy groups.

[0369] In some specific embodiments of the present invention, the RNAi reagent comprises a sense strand having any of the following modifications:

[0370] 1)5'-(N) m N s17 N s16 N s15 N s14 NF N T N F N s10 N F N s8 N s7 N s6 N s5 N s4 N s3 N s2 N s1 -3’;

[0371] 2)5’-(N) m N s17 N s16 N s15 N s14 N F N d N F N s10 N F N s8 N s7 N s6 N s5 N s4 N s3 N s2 N s1 -3’;

[0372] 3)5’-(N) m N s17 N s16 N s15 N s14 N F N T N F N s10 N F N s8 N s7 N F N s5 N s4 N s3 N s2 N s1 -3’;

[0373] 4)5’-(N) m N s17 N s16 N d N s14 N F N T N F N s10 N F Ns8 N s7 N s6 N s5 N s4 N s3 N s2 N s1 -3’;

[0374] 5)5’-(N) m N s17 N s16 N s15 N s14 N F N T N F N s10 N F N s8 N s7 N d N s5 N s4 N s3 N s2 N s1 -3’;

[0375] 6)5’-N s21 N s20 N d N s18 N s17 N s16 N s15 N s14 N F N T N F N s10 N F N s8 N s7 N s6 N s5 N s4 N s3 N s2 N s1 -3’;

[0376] 7)5’-(N) m N s17 N s16 N s15 N d N F N T N F N s10 N F N s8 N s7 N s6N s5 N s4 N s3 N s2 N s1 -3’;

[0377] 8)5’-(N) m N d N s16 N s15 N s14 N F N T N F N s10 N F N s8 N s7 N s6 N s5 N s4 N s3 N s2 N s1 -3’;

[0378] 9)5’-(N) m N s17 N s16 N s15 N s14 N F N T N F N s10 N d N s8 N s7 N s6 N s5 N s4 N s3 N s2 N s1 -3’;

[0379] 10)5’-(N) m N s17 N s16 N s15 N s14 N F N T N F N s10 N F N s8 N d N s6 N s5 N s4 N s3 N s2 N s1 -3’;

[0380] 11)5'-(N) m N s17 N s16 N s15 N s14 N F N T N F N d N F N s8 N s7 N s6 N s5 N s4 N s3 N s2 N s1 -3';

[0381] in,

[0382] m represents an integer from 0 to 8; preferably, n is 2, 3, 4, 5, 6 or 7;

[0383] Each N F These represent nucleotides modified by 2'-F;

[0384] Each N d They represent 2'-deoxynucleotides;

[0385] N T This represents a nucleotide containing a 2'-methoxy group.

[0386] Each N independently represents a modified or unmodified nucleotide;

[0387] N s1 N s2 N s3 N s4 N s5 N s6 N s7 N s8 N s10 N s14 N s15 N s16 N s17 N s18 N s20 N s21 Each can independently represent a modified or unmodified nucleotide; preferably, N s1 N s2 N s3 N s4 N s5 N s6 N s7 Ns8 N s10 N s14 N s15 N s16 N s17 N s18 N s20 N s21 All of them are nucleotides containing 2'-methoxy groups.

[0388] In some more specific embodiments of the invention, the RNAi reagent comprises a sense strand having any of the following modifications:

[0389] 1)5'-(N M )4N M N M N M N M N F N T N F N M N F N M N M N M N M N M N M N M N M -3';

[0390] 2)5'-(N M )2N M N M N M N M N F N T N F N M N F N M N M N M N M N M N M N M N M -3';

[0391] 3)5'-(N M )4N M N M N M N M N F N T N F N M NF N M N M N F N M N M N M N M N M -3';

[0392] 4)5'-(N M )4N M N M N d N M N F N T N F N M N F N M N M N M N M N M N M N M N M -3';

[0393] 5)5'-(N M )4N M N M N M N M N F N T N F N M N F N M N M N d N M N M N M N M N M -3';

[0394] 6)5'-(N M )2N d N M N M N M N M N M N F N T N F N M N F N M N M NM N M N M N M N M N M -3';

[0395] 7)5'-(N M )4N M N M N M N d N F N T N F N M N F N M N M N M N M N M N M N M N M -3';

[0396] 8)5'-(N M )4N d N M N M N M N F N T N F N M N F N M N M N M N M N M N M N M N M -3';

[0397] 9)5'-(N M )4N M N M N M N M N F N T N F N M N d N M N M N M N M N M N M N M NM -3';

[0398] 10) 5'-(N M )4N M N M N M N M N F N d N F N M N F N M N M N M N M N M N M N M N M -3';

[0399] 11) 5'-(N M )4N M N M N M N M N F N T N F N M N F N M N d N M N M N M N M N M N M -3';

[0400] 12) 5'-(N M )4N M N M N M N M N F N T N F N d N F N M N M N M N<​​​​​​​​​​​​​​​F These represent nucleotides modified by 2'-F;

[0403] Each N d They represent 2'-deoxynucleotides;

[0404] N T This represents a nucleotide containing a 2'-methoxy group.

[0405] Each N M These represent nucleotides modified with 2'-methoxy groups.

[0406] In some more specific embodiments of the invention, the RNAi reagent comprises a combination of antisense and sense strands having any of the modifications selected from Table A below. In the table below, numbers 1 to 23, representing nucleotide positions, indicate the position of the nucleotide in the antisense or sense strand, counting from 5' to 3'. For example, in modification template RM11, the sense strand, counting from 5' to 3', has nucleotides at positions 9, 11, and 13 modified with f, and the other nucleotides modified with m; the antisense strand, counting from 5' to 3', has nucleotides at positions 2, 12, 14, and 16 modified with f, positions 5 and 7 being 2'-deoxynucleotides, position 15 being a 2'-O-methoxyethyl modified nucleoside, and the other nucleotides modified with m.

[0407] Table A: Modification Template

[0408] In some embodiments of the present invention, the RNAi reagent comprises at least one modified phosphate ester nucleoside linker; preferably, the modified phosphate ester nucleoside linker is selected from thiophosphate nucleoside links, dithiophosphate nucleoside links, and nucleotides with methylphosphonate groups.

[0409] In some embodiments of the present invention, the 5' and 3' ends of the sense strand of the RNA reagent independently contain 0, 1, 2, 3, 4, or 5 phosphate-thioester nucleoside links, respectively; and / or the 5' and 3' ends of the antisense strand of the RNA reagent independently contain 0, 1, 2, 3, 4, or 5 phosphate-thioester nucleoside links, respectively.

[0410] In some embodiments of the present invention, at least one of the following connections is a phosphate thiophosphate nucleoside link: between the nucleotides at positions 1 and 2 of the 5' end of the sense strand; between the nucleotides at positions 2 and 3 of the 5' end of the sense strand; between the nucleotides at positions 1 and 2 of the 3' end of the sense strand; between the nucleotides at positions 2 and 3 of the 3' end of the antisense strand; between the nucleotides at positions 2 and 3 of the 3' end of the antisense strand; between the nucleotides at positions 1 and 2 of the 5' end of the antisense strand; and between the nucleotides at positions 2 and 3 of the 5' end of the antisense strand. In some embodiments of the present invention, at least four connections are phosphate thiophosphate nucleoside links. In some embodiments of the present invention, at least six connections are phosphate thiophosphate nucleoside links. In some embodiments of the present invention, all eight connections are phosphate thiophosphate nucleoside links.

[0411] In some embodiments of the present invention, the nucleotides at positions 1 and 2, and at positions 2 and 3, of the sense strand are linked by phosphate thioester nucleosides.

[0412] In some embodiments of the present invention, the nucleotides at positions 1 and 2, and positions 2 and 3 at the 5' end of the sense strand are linked by phosphate thioester nucleosides, and the nucleotides at positions 1 and 2, and positions 2 and 3 at the 3' end are linked by phosphate thioester nucleosides.

[0413] In some embodiments of the present invention, the nucleotides at positions 1 and 2 at the 3' end of the antisense strand are linked by phosphate thioester nucleosides, and the nucleotides at positions 1 and 2 at the 5' end are linked by phosphate thioester nucleosides.

[0414] In some embodiments of the present invention, the nucleotides at positions 1 and 2 of the 5' end of the sense strand, the nucleotides at positions 2 and 3 of the 5' end of the sense strand, the nucleotides at positions 1 and 2 of the 3' end of the sense strand, the nucleotides at positions 2 and 3 of the 3' end of the sense strand, the nucleotides at positions 1 and 2 of the 3' end of the antisense strand, the nucleotides at positions 2 and 3 of the 3' end of the antisense strand, the nucleotides at positions 1 and 2 of the 5' end of the antisense strand, and the nucleotides at positions 2 and 3 of the 5' end of the antisense strand are all linked by phosphate thioester nucleosides.

[0415] On the other hand, in some embodiments of the present invention, the RNAi reagent further comprises one or more ligand groups or linker groups.

[0416] This invention provides conjugate compounds formed by covalently linking an oligonucleotide double strand of RNAi according to the invention to a non-nucleotide portion (conjugate portion). In this document, such conjugates are also referred to as "RNAi conjugates" or "siRNA conjugates".

[0417] The conjugation of the oligonucleotide duplex of the RNAi reagent of the present invention to one or more non-nucleotide moieties can, for example, improve the pharmacological properties of the RNAi of the present invention by affecting the activity, cellular distribution, cellular uptake, or stability of the oligonucleotide. In some embodiments, the conjugated moiety modulates or enhances the pharmacokinetic properties of the RNAi of the present invention by improving the cellular distribution, bioavailability, metabolism, excretion, permeability, and / or cellular uptake of the oligonucleotide. In particular, the conjugation can direct the oligonucleotide to a specific organ, tissue, or cell type and thus enhance the effectiveness of the RNAi of the present invention in such organ, tissue, or cell type. Simultaneously, the conjugation can reduce the activity of the RNAi of the present invention in non-target cell types, tissues, or organs (e.g., off-target activity or activity in non-target cell types, tissues, or organs).

[0418] In some embodiments, the conjugate portion used in the RNAi activator of the present invention may be selected from: antibodies, peptides, peptide mimics, aptamers, small chemical compounds, lipids, cell-penetrating peptide polymers, or nanoparticle conjugates.

[0419] In other embodiments, the conjugation portion of the RNAi activator of the present invention may be selected from sugars, cell surface receptor ligands, drugs, hormones, lipophilic substances, polymers, proteins, peptides, toxins (e.g., bacterial toxins), vitamins, viral proteins (e.g., capsids), or combinations thereof. In some embodiments, for example, the conjugation portion may include a lipid portion, such as cholesterol, bile acids, aliphatic chains, etc.

[0420] In some embodiments, the conjugation moiety is or comprises a sugar moiety. Sugar conjugation moieties include, but are not limited to, galactose, lactose, N-acetylgalactosamine, mannose, and mannose-6-phosphate. Sugar conjugations can be used to enhance delivery or activity in a range of tissues such as the liver and / or muscle. In other embodiments, preferably, the conjugation moiety of the RNAi activator of the present invention is a compound moiety capable of binding to the desialylate glycoprotein receptor (ASGPR). For example, monovalent, divalent, and trivalent N-acetylgalactosamine and its derivatives conjugation moieties are all suitable for binding to ASGPR and are therefore suitable for use in the present invention.

[0421] In some embodiments, the conjugate moiety is attached to the 5' and / or 3' terminal nucleotide of the sense strand and optionally the 5' and / or 3' terminal nucleotide of the antisense strand of the RNAi reagent of the present invention, optionally via a thiophosphate group or a phosphate group. In some cases, the conjugate moiety may also be conjugated to the internal sequence of the RNAi oligonucleotide. In some embodiments, the conjugate moiety may be attached to a phosphate group, a 2′-hydroxyl group, or a base of the nucleotide. When the conjugate moiety is attached to the end of an RNAi (such as siRNA) oligonucleotide chain, the conjugate moiety is typically attached to a phosphate group of the nucleotide; when the conjugate moiety is attached to the internal sequence of an RNAi (such as siRNA) oligonucleotide, the conjugate moiety is typically attached to a sugar ring of the ribose or a base.

[0422] The conjugate portion can be directly linked to the oligonucleotide duplex of the RNAi of the present invention or linked via a linker portion (e.g., a adapter). In some embodiments of the present invention, the RNAi conjugate of the present invention may optionally include a linker region located between the oligonucleotide duplex of the RNAi and the conjugate portion.

[0423] In some embodiments of the present invention, the ligand group or linker group is conjugated to the sense or antisense strand of the RNAi reagent; preferably, the ligand group or linker group is conjugated to the 3' end of the sense strand, and / or the ligand group or linker group is conjugated to the 5' end of the sense strand, and / or the ligand group or linker group is conjugated to the 3' end of the antisense strand.

[0424] In some embodiments of the present invention, the present invention provides RNAi conjugates comprising a conjugate moiety linked to the RNAi double-stranded oligonucleotide of the present invention, wherein the conjugate moiety comprises a trivalent GalNAc ligand having the following structure:

[0425] In some embodiments of the present invention, the present invention provides RNAi conjugates comprising a conjugate moiety linked to an RNAi oligonucleotide of the present invention, wherein the conjugate moiety has an L96 structure as shown below:

[0426] The wavy line on the right indicates a connection to the RNAi oligonucleotide chain, preferably via a phosphodiester bond or a thiophosphate diester bond.

[0427] The present invention also provides a composition comprising any of the above-described RNAi reagents and a pharmaceutically acceptable carrier.

[0428] In this invention, a "pharmaceutical composition" comprises a pharmaceutically effective amount of one or more RNAi reagents, a pharmaceutically acceptable carrier, and optionally other therapeutic agents that act synergistically with the RNAi reagents. As used herein, "pharmaceuticalally effective amount," "therapeutically effective amount," or simply "effective amount" refers to an amount of RNAi active agent that effectively produces the desired pharmacological, therapeutic, or preventative outcome. For example, a given clinical treatment is considered effective if a measurable parameter associated with a disease or condition is reduced by at least 10%, and the therapeutically effective amount of the drug used to treat said disease or condition is the amount necessary to produce a reduction of at least 10% in said parameter. In other embodiments, a given clinical treatment is considered effective when a measurable parameter associated with a disease or condition exhibits a reduction of at least 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, or 95%, and the therapeutically effective amount of the drug used to treat said disease or condition is the amount necessary to produce a reduction of at least 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, or 95%, respectively. The term "pharmaceutically acceptable carrier" refers to a carrier for administering the therapeutically active agent. Such carriers include, but are not limited to, saline, buffered saline, glucose, water, glycerol, ethanol, and combinations thereof.

[0429] In some embodiments, the present invention provides pharmaceutical compositions comprising the RNAi reagent or RNAi conjugate of the present disclosure as an active ingredient and pharmaceutically acceptable diluents, carriers, and / or excipients (e.g., PBS buffer, physiological saline, water). The purpose of the pharmaceutical compositions is to facilitate administration to a living organism, thereby promoting the absorption of the active ingredient and the exertion of its biological activity. Pharmaceutically acceptable diluents, carriers, and / or excipients used in the present invention include any suitable pharmaceutically acceptable diluents, carriers, and / or excipients known in the art.

[0430] In some embodiments, the RNAi reagent or RNAi conjugate according to the invention may be present in a non-buffered solution, preferably saline or water. In other embodiments, the RNAi reagent or RNAi conjugate according to the invention may be present in a buffered solution, preferably comprising acetate, citrate, alcohol-soluble gluten, carbonate, or phosphate, or any combination thereof; more preferably, the buffered solution is phosphate-buffered saline (PBS). In some embodiments, the RNAi reagent or RNAi conjugate according to the invention is formulated as a subcutaneous formulation. In other embodiments, the RNAi reagent or RNAi conjugate according to the invention is formulated as an intravenous formulation.

[0431] The present invention also provides a method for inhibiting the expression of a target gene in a cell, the method comprising: delivering any of the above-described RNAi reagents to a subject, such that the RNAi reagent is delivered to a specific target of the subject.

[0432] Pharmaceutical compositions comprising the RNAi reagent or RNAi conjugate of the present invention can be administered via a variety of suitable routes of administration, including but not limited to buccal, inhalation (including blowing or deep inhalation), nasal, oral, parenteral, implantation, injection, or infusion (via epidural, intra-articular, intra-articular, intracapsular, intracardiac, intracranial, intradermal, intramuscular, intraorbital, intraperitoneal, intraspinal, intrasternal, intrasheath, intravenous, subarachnoid, subcapsular, subcutaneous, subepidermal, transendothelial, transtracheal, transvascular, rectal, sublingual, local, and / or vaginal routes). Administration can be performed by injection, infusion, skin patch, or any other method known in the art. Formulations for administration can be powdered, atomized, aerosolized, granulated, or suitably prepared for delivery.

[0433] The present invention also provides the use of any of the above-mentioned RNAi reagents in the preparation of medicaments for viral diseases, neuromuscular diseases, bacterial infections, inflammatory and immune diseases, metabolic diseases, liver diseases, kidney diseases, cardiovascular diseases, ophthalmic diseases, lung diseases, and rare diseases.

[0434] In this invention, when the terms "comprising" or "including" are used, unless otherwise specified, they also cover situations where the elements, integers, or steps mentioned are constituted. For example, when referring to an oligonucleotide that "comprising" a specific sequence, it is also intended to cover oligonucleotides that consist of that specific sequence.

[0435] In this invention, unless otherwise specified, the term "complementary" refers to the ability of an oligonucleotide of a first sequence to hybridize with an oligonucleotide of a second sequence under certain conditions and form a double-stranded structure. "At least partially complementary" means that the two sequences can be completely complementary, or have no more than 5, 4, 3, or 2 mismatched base pairs in total, while retaining the ability to hybridize under the relevant conditions. Furthermore, where the two oligonucleotides are designed to form one or more single-stranded overhangs upon hybridization, such overhangs should not be considered mismatches for determining complementarity. In this invention, to satisfy the above hybridization ability requirements, the "complementary" sequence may also include or consist entirely of base pairs formed from non-Watson-Crick base pairs and / or from non-natural and modified nucleotides. Such non-Watson-Crick base pairs include, but are not limited to, G:U swing base pairs or Hoogstein base pairs. Correspondingly, in this invention, unless otherwise specified, "mismatch" means that in the siRNA double-stranded molecule, the bases at corresponding positions are not paired in a complementary manner.

[0436] In this invention, for the purposes of this invention, the expression "complementarity" or "complementarity" associated with siRNA is preferably not less than 70%, that is, at least 70% of the base positions in the double-stranded region formed by complementary hybridization are complementary, i.e., the number of mismatched positions in the continuous nucleotide sequence forming the double-stranded region is less than 30%. For example, for a 21-base-pair double-stranded region, not less than 70% complementarity means that the double-stranded region forms no more than 6, 5, 4, 3, 2, 1, or 0 mismatched base pairs during hybridization. Preferably, the presence of insertions and deletions is not allowed when calculating the complementarity % of the continuous nucleotide sequence in the double-stranded region. It should be understood that when the two oligonucleotides of the siRNA are designed to form one or more single-stranded overhangs during hybridization, such overhangs will not be considered mismatches when determining complementarity. For example, for the purposes described herein, an siRNA comprising a 21-nucleotide positive oligonucleotide chain and a 23-nucleotide negative oligonucleotide chain can still be considered “perfectly complementary” if the longer negative oligonucleotide contains a 21-nucleotide sequence that is perfectly complementary to the shorter positive oligonucleotide.

[0437] In this invention, the term "protruding end" is used to describe an unpaired nucleotide located at the 3' or 5' end of the double-stranded region of a double-stranded oligonucleotide.

[0438] In this invention, "G", "C", "A", "T", and "U" typically represent nucleotides containing guanine, cytosine, adenine, thymine nucleotides, and uracil as bases, respectively. However, those skilled in the art will know that guanine, cytosine, adenine, and uracil can be replaced by other parts without substantially altering the base-pairing properties of the oligonucleotide containing the nucleotide with such replacement parts. Examples of nucleoside base modifications that can be used to generate RNAi reagents include the substitution of nucleotides containing uracil, guanine, or adenine with nucleotides containing, for example, inosine; and the replacement of adenine and cytosine in oligonucleotides with guanine and uracil, respectively, to form a GU Wobble base pair with the target mRNA.

[0439] In this invention, "nucleotide" refers to the structural unit of oligonucleotides and polynucleotides, and for the purposes of this invention, includes naturally occurring nucleotides and modified nucleotides. In nature, RNA nucleotides comprise a sugar moiety (ribose), a nucleobase moiety, and a phosphate ester group. Herein, a modified nucleotide refers to a nucleotide that, corresponding to a natural RNA nucleotide, has modifications in its sugar moiety and / or nucleobase moiety and / or phosphate ester group.

[0440] In this invention, unless otherwise stated, the term "conjugation" refers to the covalent connection between two or more chemical moieties, each having a specific function; correspondingly, "conjugate" refers to a compound formed by covalently connecting these chemical moieties. Accordingly, in this document, siRNA conjugates refer to compounds formed by covalently linking one or more chemical moieties having a specific function to the oligonucleotide chain of siRNA. In this document, the specific chemical moieties covalently linked to the oligonucleotide chain of siRNA, or the specific compounds that can conjugate such specific chemical moieties to RNAi via a reaction, are also referred to as "conjugate moieties." In some embodiments of the invention, the conjugate moieties are non-nucleoside or non-nucleotide chemical moieties, but this does not preclude the possibility that the conjugate moieties are linked to oligonucleotides via nucleosides or nucleotides or their analogues or derivatives.

[0441] In this invention, "nucleotide" refers to the structural unit of oligonucleotides and polynucleotides, and for the purposes of this invention, includes naturally occurring nucleotides and modified nucleotides. In nature, RNA nucleotides comprise a sugar moiety (ribose), a nucleobase moiety, and a phosphate ester group. In this document, a modified nucleotide refers to a nucleotide that, corresponding to a natural RNA nucleotide, has modifications in its sugar moiety and / or nucleobase moiety.

[0442] In this invention, the term "modified nucleoside" or "nucleoside modification" refers to a modified nucleoside formed by introducing one or more sugar moieties and / or one or more (nuclear)base moieties, compared to the corresponding RNA nucleoside. Therefore, the term "modified nucleoside" may also be used interchangeably with the term "modified nucleotide." In some preferred embodiments according to the invention, the modified nucleoside comprises a modified sugar moieties.

[0443] Obviously, based on the above description of the present invention, and according to common technical knowledge and conventional methods in the field, various other modifications, substitutions or alterations can be made without departing from the basic technical concept of the present invention.

[0444] Detailed Implementation Plan

[0445] The following detailed embodiments further illustrate the above-described content of the present invention. However, this should not be construed as limiting the scope of the present invention to the following examples. All technologies implemented based on the above-described content of the present invention fall within the scope of the present invention.

[0446] Those skilled in the art will recognize that the siRNA described in this invention can be obtained using conventional siRNA preparation methods (e.g., solid-phase synthesis and liquid-phase synthesis), both of which are commercially available custom-made services. Those skilled in the art will also understand that modified nucleotide groups can be introduced into the siRNA described in this invention using appropriately modified nucleotide monomers. Methods for preparing appropriately modified nucleotide monomers are well known to those skilled in the art, and commercially available monomers are also available.

[0447] Example 1: Preparation of Standard Modified siRNA

[0448] The siRNA was synthesized using a phosphorus amide solid-phase synthesis method. When synthesizing nucleotides modified at various positions in the SS and AS chains, the synthesized phosphorus amide monomers were used to replace the original nucleotides of the parent sequence.

[0449] The synthesis process is briefly described as follows: Using an LK-48E synthesizer (Lingkun), starting with a Universal CPG carrier, nucleoside phosphoramide monomers were linked one by one according to the synthesis program. In addition to DT3-A, U, G, and C described above, the remaining nucleoside monomer raw materials, such as 2'-F RNA and 2'-O-methyl RNA, were purchased from Shanghai Zhaowei. 5'-Ethylthio-1H-tetrazole (ETT) was used as the activator (0.6M acetonitrile solution), 0.22M PADS dissolved in a 1:1 volume ratio of trimethylpyridine (Suzhou Kelama) solution was used as the sulfidation agent, and iodopyridine / water solution (Kelama) was used as the oxidizing agent.

[0450] After solid-phase synthesis, the oligonucleotides were cleaved from the solid support and soaked in a 3:1 solution of 28% ammonia and ethanol at 50°C for 16 hours. After centrifugation, the supernatant was transferred to another centrifuge tube, concentrated, and evaporated to dryness. Purification was then performed using C18 reversed-phase chromatography with 0.1M TEAA and acetonitrile as the mobile phase, and DMTr was removed using 3% trifluoroacetic acid solution. The target oligonucleotides were collected, lyophilized, identified as the target product by LCMS, and quantified by UV (260 nm) spectroscopy.

[0451] The obtained single-stranded oligonucleotides were annealed according to complementary pairing in equimolar ratios, and the resulting double-stranded siRNA was dissolved in 1xPBS and adjusted to the required concentration for the experiment.

[0452] The sense and antisense strands contained in the modified siRNAs are shown in Table 1. Each siRNA was analyzed by LC-MS to confirm identity, quantified by UV (260 nm), and its purity was determined by HPLC analysis.

[0453] Table 1. Exemplary sequences of parental siRNA molecules.

[0454] Table 2. Example sequences of modified siRNA molecules

[0455] Table 3

[0456] Example 2: Detection of the free uptake activity of conjugates in primary hepatocytes

[0457] On day 0, cryopreserved primary cynomogus hepatocytes (PCH) were thawed and adjusted to a cell density of 2 × 10⁵ cells / well. 190 μl of each cell was then seeded into 96-well plates. Maintenance medium (Miaoshun (Shanghai) Biotechnology Co., Ltd., catalog number: HEP064) was used. Simultaneously, 10 μl of the test GalNAc-siRNA was added to each well. Control wells (PBS negative control group) without GalNAc-siRNA were also included. Three test concentrations were prepared for each sample, and three replicates were prepared for each concentration. After 48 hours of culture, cells were collected for RNA extraction. RT-qPCR was used to detect the mRNA expression levels of the target genes (e.g., AGT, primers: CCTTCCACCTCGTCATCC (SEQ ID NO:189), ACGTCTTGGCCTGTATCG (SEQ ID NO:190)) in the samples, while the mRNA expression level of the internal reference gene GAPDH was also detected (primers: TTTGGTATCGTGGAAGGA (SEQ ID NO:191), AGGCAGGGATGATGTTCT (SEQ ID NO:192)). Intracellular RNA was extracted using the VAMNE Magnetic Cell / Tissue TotalRNA Kit (96 Prepackaged) (vazyme, RMA102-C1-P21*96T) according to the manufacturer's instructions. III. The 1st Strand cDNA Synthesis Kit (vazyme, R312-02) was used to reverse transcribe the DNA into cDNA, which was then quantified by qPCR.

[0458] The relative expression level of AGT mRNA is calculated using the following formula: Fold changes (target gene) = 2 - ((Ct target gene - Ct reference gene) experimental group - (Ct target gene - Ct reference gene) control group); relative mRNA inhibition rate (%) = (1 - relative expression level of AGT mRNA in the test group / relative expression level of AGT mRNA in the negative control group) × 100%.

[0459] The results are shown in Tables 4 and 5.

[0460] Table 4. In vitro activity of primary cynomolgus monkey hepatocytes

[0461] Table 5. In vitro activity of primary cynomolgus monkey hepatocytes

[0462] Example 3: Preparation of modified siRNA

[0463] Following the preparation method described in Example 1, the modified siRNAs shown in Table 6 and Table 7, and the modified siRNAs with delivery ligands shown in Table 7-1 were prepared.

[0464] Table 6. Modified siRNA sequences

[0465] Table 7. Modified siRNA sequences

[0466] Table 7-1 Modified siRNA sequences

[0467] Example 4: In vitro testing of modified siRNA

[0468] This study evaluated the effects of different siRNA motifs related to MUC5AC, INHBE, and TMPRSS6 on gene silencing efficiency under different modified templates in A549, Huh7, and Hep3B cell lines. A total of 3 × 6 × 7 different siRNA nucleic acid motif molecules involving 3 different gene targets were analyzed, as detailed in Table 8 below. Each siRNA compound was evaluated at two concentrations: 1 nM and 0.5 nM. Cells were seeded in 96-well plates at a density of 20,000 cells / well. SiRNA compounds were transfected using Lipofectamine RNAiMAX transfection reagent at concentrations of 1 nM and 0.5 nM, with three replicates for each concentration. A mock control group was also included. 24 hours post-transfection, mRNA was extracted for qPCR detection. The primers for MUC5AC are 5'-CAGACCCAGACCACCTTCAC-3' (SEQ ID NO:593) and 5'-GAGGAGGTGCTCTGAGTTGG-3' (SEQ ID NO:594), respectively; the primers for INHBE are 5'-CTGGATGGGTTGCACCTGAC-3' (SEQ ID NO:595) and 5'-TAGGCTGAAGTGGAGTCTGTG-3' (SEQ ID NO:596), respectively; the primers for TMPRSS6 are 5'-CGGGAATCTAGTGCCTTCCG-3' (SEQ ID NO:597) and 5'-GAGGGGTCCCTCCCCAAA-3' (SEQ ID NO:598), respectively; and the primers for the corresponding reference housekeeping gene B2M are 5'-AGATGAGTATGCCTGCCGTG-3' (SEQ ID NO:594). NO:599) and 5'-TTCAAACCTCCATGATGCTGC-3' (SEQ ID NO:600). The relative inhibition rate of each compound was calculated using the formula Fold changes(target gene) = 2 - ((Ct target gene - Ct reference gene) experimental group - (Ct target gene - Ct reference gene) control group). The inhibition rate of the compound formed by the RM11 modified motif was used as the baseline to evaluate the effect of different modified siRNAs on the mRNA inhibition of MUC5AC, INHBE, and TMPRSS6. A value >100 indicated that the activity of the modified siRNA was superior to the baseline data. Specific results are shown in Table 9 below.

[0469] Table 8. siRNA motifs

[0470] Table 9. In vitro test data

[0471] Example 5: In vitro testing of modified siRNA

[0472] This embodiment uses the HepG2 cell line to evaluate the effect of RM11-modified template-introduced deoxygenated or fluorinated modification on the inhibitory effect of CFB mRNA via siRNA transfection. A total of three different siRNA nucleic acid motifs were analyzed, and their sequences are shown in Table 10 below. Each siRNA compound used in the evaluation was processed at three concentrations: 5 nM, 0.5 nM, and 0.05 nM. Cells were seeded in 96-well plates at a density of 20,000 cells / well. Transfection with the siRNA compounds was performed using Lipofectamine RNAiMAX transfection reagent at concentrations of 5 nM, 0.5 nM, and 0.05 nM, with three replicates for each concentration. A mock control group was also included. 24 hours after transfection, mRNA was extracted for qPCR detection. The primers and probes for CFB were from Thermo, cat#.4351368, and the reference housekeeping gene was Thermo, cat#.4326317E. The relative inhibition rate of each compound was calculated using the formula Fold changes(target gene) = 2 - ((Ct target gene - Ct reference gene) experimental group - (Ct target gene - Ct reference gene) control group). The inhibition rate of compounds formed from RM11-modified motifs was used as a baseline to evaluate the effect of introducing RM11-modified templates into DNA or f-modification on the inhibitory effect on CFB mRNA. A value >100 indicated that the introduced modification had better activity than the baseline data. Specific results are shown in Table 11 below.

[0473] The results showed that, among the three different motifs, RM11-modified templates exhibited good DNA compatibility at positions SS7, AS18, AS20, AS22, SS16, SS3, SS8, SS5, SS10, SS15, SS12, and SS13, with good f-compatibility at position SS16. Modifications at these positions did not significantly affect the original RM11 modification activity; in fact, they could even enhance the activity of certain compounds at certain concentrations and with different compounds.

[0474] Table 10. siRNA motifs

[0475] Table 11. In vitro test data

[0476] Example 6: Transgenic Mouse Animal Experiment

[0477] This embodiment uses a transgenic HBV mouse model to evaluate the in vivo efficacy of HBV siRNA molecules with different modifications. The transgenic mouse strain was C57B / 6N-Tg(1.28HBV) / Vst, purchased from Beijing Vitonda Biotechnology Co., Ltd. Mice were grouped according to serum HBsAg levels and body weight, ensuring no statistically significant differences in serum HBsAg and body weight. Mice with lower HBsAg levels and lighter body weight were removed from the experiment. Each group consisted of 4 males, and the drug was administered at a dose of 3 mg / kg via a single subcutaneous dose. The saline group served as the negative control (NC). Blood samples were collected from the orbital venous plexus of mice before administration (day 0) and after administration (days 7, 14, 21, 28, and 35). The collected blood samples were incubated at room temperature for 3 minutes, centrifuged at 5 rpm for 1 minute, and the supernatant was used for HBsAg detection. The reagent used for detection was a Hepatitis B virus surface antigen assay kit (Mike), and the detection method was performed according to the corresponding instructions. The serum HBsAg expression level after drug administration was compared with the corresponding Log10 value before self-administration. The reduction effect compared to the pre-administration level was used to evaluate the efficacy of the conjugate. The corresponding efficacy results are shown in Figure 1. Analysis of the HBsAg knockdown effect on days 7, 14, 21, 28, and 35 after drug administration revealed that the knockdown effect of CM20-149 was superior to that of CM20-148 and CM20-327.

[0478] Example 7: Mouse Animal Experiment

[0479] This study used a wild-type C57BL / 6 mouse model to evaluate the in vivo efficacy of TTR siRNA molecules with different modifications. On day 0 before administration, mice were grouped according to serum TTR protein levels and body weight, ensuring no statistically significant difference in serum TTR protein levels and body weight. Mice with lower TTR levels and lighter body weight were removed from the experiment. Each group consisted of four males, and the dosage was 0.5 mg / kg, administered subcutaneously as a single dose. The saline group served as the negative control (NC). Blood samples were collected from the orbital venous plexus of mice before administration (day 0) and after administration (days 7, 14, 21, 28, 35, 42, and 49). The collected blood samples were incubated at room temperature for 3 minutes, centrifuged at 5 rpm for 1 minute, and the supernatant was used for TTR detection. The reagent used was the mouse TTR ELISA kit Abcam ab282297, and the detection method was performed according to the corresponding instructions. Serum TTR expression levels after administration were compared with serum TTR expression levels before administration. The serum TTR downregulation results at the corresponding time points are shown in Figure 2. On day 49 after drug administration, CM20-158 and CM20-151 showed better TTR knockdown effects than CM20-150 and CM20-328.

[0480] Example 8: Mouse Animal Experiment

[0481] This study used a wild-type C57BL / 6 mouse model to evaluate the in vivo efficacy of CFB siRNA molecules with different modifications. On day 0 before administration, mice were grouped according to body weight, ensuring no statistically significant difference in weight; mice with abnormal weight were removed from the experiment. The saline group consisted of 2 males, while the other experimental groups each consisted of 5 males. The dosage was 10 mg / kg, administered subcutaneously as a single dose. The saline group, containing the drug solvent, served as the negative control (NC). After drug administration (day 56), mice were euthanized and their livers were dissected. The relative expression of CFB mRNA in the liver was detected using qPCR. The forward and reverse primers for the CFB target gene were 5'-AGCCTTCCTGCCAAGATTCC-3' (SEQ ID NO: 643) and 5'-CCCGAGGGGTCTAGGACAAT-3' (644), respectively. The reference housekeeping gene was RPL32, with forward and reverse primers of 5'-CTGCCATCTGTTTTACGGCA-3' (SEQ ID NO: 645) and 5'-ATCAGGATCTGGCCCTTGAAC-3' (SEQ ID NO: 646), respectively. The relative expression value of CFB mRNA was calculated using the formula: Fold changes(target gene) = 2 - ((Ct target gene - Ct reference gene) experimental group - (Ct target gene - Ct reference gene) control group). The corresponding efficacy results are shown in Figure 3. On day 56 after administration, CM20-153 showed significantly better inhibitory effects on CFB mRNA in mouse liver than CM20-329 and CM20-152.

[0482] Example 9: Mouse Animal Experiment

[0483] This embodiment uses a humanized hAPOC3 transgenic mouse model from Jicui Biotechnology to evaluate the in vivo efficacy of APOC3 siRNA molecules with different modifications. On day 0 before drug administration, mice were grouped according to serum hAPOC3 protein levels and body weight, ensuring no statistically significant differences in serum hAPOC3 protein levels and body weight. Mice with abnormal hAPOC3 levels or body weight were removed from the experiment. Each group consisted of 5 males, and the drug was administered at a dose of 1 mg / kg via a single subcutaneous dose. The saline group served as the negative control (NC). Blood samples were collected from the orbital venous plexus of mice before drug administration (day 0) and after drug administration (days 7, 14, and 21). The collected blood samples were incubated at room temperature for 3 minutes, centrifuged at 5 rpm for 1 minute, and the supernatant was used for hAPOC3 detection. The reagent used was the Human APOC3 ELISA kit Thermo EHAPOC3, and the detection method was performed according to the corresponding instructions. Serum hAPOC3 expression levels after drug administration were compared with serum hAPOC3 expression levels before drug administration. The serum hAPOC3 downregulation results at the corresponding time points are shown in Figure 4. On days 7, 14, and 21 after drug administration, CM20-193, CM20-154, CM20-155, CM20-156, and CM20-157 showed comparable APOC3 knockdown effects.

[0484] Example 10: Mouse Animal Experiment

[0485] This embodiment uses a wild-type C57BL / 6 mouse model to evaluate the in vivo efficacy of C3 siRNA molecules with different modifications. On day 0 before administration, mice were grouped according to body weight, ensuring no statistically significant difference in weight; mice with abnormal weight were removed from the experiment. Each group consisted of 4 males, and the dosage was 3 mg / kg, administered subcutaneously as a single dose. The saline group served as the negative control (NC). After administration (day 14), the mice were euthanized and their livers were dissected. The relative expression of C3 mRNA in the liver was detected using qPCR. The forward and reverse primers for the C3 target genes were 5'-CTGGAGAGCGAAGAGACCAT-3' (SEQ ID NO: 647) and 5'-TTCCTTACTGGCTGGAATCTTGA-3' (SEQ ID NO: 648), respectively. The reference gene was RPL32, with forward and reverse primers of 5'-CTGCCATCTGTTTTACGGCA-3' and 5'-ATCAGGATCTGGCCCTTGAAC-3', respectively. The relative expression value of C3 mRNA was calculated using the formula: Fold changes (target gene) = 2 - ((Ct target gene - Ct reference gene) experimental group - (Ct target gene - Ct reference gene) control group). The corresponding efficacy results are shown in Figure 5. On day 14 after administration, CM20-159, CM20-160, and CM20-161 all showed significant inhibitory effects on C5 mRNA in mouse liver.

Claims

1. An RNAi reagent for inhibiting the expression of a target gene, comprising a sense strand and an antisense strand, each strand having 17 to 30 nucleotides, wherein each nucleotide is independently a modified or unmodified nucleotide; wherein the antisense strand is complementary to at least a portion of the mRNA of the target gene, and the sense strand and the antisense strand are at least partially complementary to form a double-stranded region. The 5th and 7th positions of the 5' end of the antisense strand contain 2'-deoxynucleotides; optionally, the 2nd, 9th, 12th, 14th, 16th, 18th, 20th or 22nd positions of the 5' end of the antisense strand further contain 2'-deoxynucleotides. The sense position corresponding to the 12th position at the 5' end of the antisense strand contains a nucleotide that is not modified by 2'-F.

2. The RNAi reagent according to claim 1, characterized in that: The second position at the 5' end of the antisense strand contains a nucleotide modified with 2'-F or a 2'-deoxynucleotide.

3. The RNAi reagent according to any one of claims 1 to 2, characterized in that: The sense strand position corresponding to the 12th position at the 5' end of the antisense strand contains a 2'-methoxy modified nucleotide or a 2'-deoxy nucleotide.

4. The RNAi reagent according to any one of claims 1 to 3, characterized in that: The 15th position of the 5' end of the antisense strand contains a nucleotide with a heat-stabilized modification.

5. The RNAi reagent according to claim 4, characterized in that: The thermally stabilized nucleotide refers to the double-stranded oligonucleotide whose thermal dissociation temperature Tm increases after introduction; preferably, the thermal dissociation temperature Tm increases by at least 0.05°C; more preferably, the thermal dissociation temperature Tm increases by 0.5-4°C.

6. The RNAi reagent according to claim 5, characterized in that: The heat-stabilized modified nucleotide is selected from nucleotides modified with 2'-O-methoxyethyl, nucleotides modified with 2'-O-allyl, nucleotides modified with 2'-C-allyl, nucleotides modified with 2'-O-2-N-methylamino-2-oxomethyleneethyl, nucleotides modified with 2'-O-2-N,N-dimethylaminoethyl, nucleotides modified with 2'-O-3-aminopropyl, nucleotides modified with 2'-O-2,4-dinitrophenyl, nucleotides modified with 2'-O-(N-methylacetamido)-modified, 2',4'-disubstituted nucleotides, locked nucleotides (LNA), bicyclic nucleotides (BNA), or restricted ethyl-bridged nucleotides (cEt); preferably, the heat-stabilized modified nucleotide is selected from nucleotides modified with 2'-O-methoxyethyl.

7. The RNAi reagent according to any one of claims 1 to 6, characterized in that: The 14th position of the 5' end of the antisense strand contains a nucleotide modified with 2'-F.

8. The RNAi reagent according to any one of claims 1 to 7, characterized in that: The sense strand position corresponding to the 11th and / or 13th position at the 5' end of the antisense strand contains a nucleotide modified with 2'-F.

9. The RNAi reagent according to any one of claims 1 to 8, characterized in that: The 12th and / or 16th position of the 5' end of the antisense strand contains a nucleotide modified with 2'-F.

10. The RNAi reagent according to any one of claims 1 to 8, characterized in that: The sense strand position corresponding to the 6th position at the 5' end of the antisense strand contains a nucleotide modified with 2'-F.

11. The RNAi reagent according to any one of claims 1 to 8, characterized in that: The sense strand position corresponding to the 9th position at the 5' end of the antisense strand contains a nucleotide modified with 2'-F.

12. The RNAi reagent according to any one of claims 1 to 8, characterized in that: The sense strand position corresponding to the 12th position at the 5' end of the antisense strand contains a 2'-deoxynucleotide.

13. The RNAi reagent according to any one of claims 1 to 8, characterized in that: The sense strand position corresponding to the 6th position at the 5' end of the antisense strand contains a 2'-deoxynucleotide.

14. The RNAi reagent according to any one of claims 1 to 8, characterized in that: The sense strand position corresponding to the 15th position at the 5' end of the antisense strand contains a 2'-deoxynucleotide.

15. The RNAi reagent according to any one of claims 1 to 14, characterized in that: Each strand of the sense and antisense strands of the RNAi reagent has 17 to 25 nucleotides; preferably, each strand has 19 to 23 nucleotides; or, the sense strand of the RNAi reagent has two fewer nucleotides than the antisense strand, and the ratio of the number of nucleotides in the sense and antisense strands is 19 / 21, 20 / 22, or 21 / 23.

16. The RNAi reagent according to any one of claims 1 to 15, characterized in that: The RNAi reagent comprises one or more single-stranded nucleotide overhangs; preferably, the overhangs are located at the 3' end of the antisense strand of the RNAi reagent.

17. The RNAi reagent according to any one of claims 1 to 16, characterized in that: The RNAi reagent has blunt ends; preferably, the RNAi reagent has at least one blunt end located at the 5' end of the antisense strand.

18. The RNAi reagent according to claim 17, characterized in that: The 12th position of the 3' end of the sense strand contains a nucleotide that is not modified by 2'-F.

19. The RNAi reagent according to claim 17, characterized in that: The 12th position of the 3' end of the sense strand contains a 2'-methoxy modified nucleotide or a 2'-deoxy nucleotide.

20. The RNAi reagent according to claim 17, characterized in that: The 11th and / or 13th position of the 3' end of the sense strand contains a nucleotide modified with 2'-F.

21. The RNAi reagent according to claim 17, characterized in that: The sixth position at the 3' end of the sense strand contains a nucleotide modified with 2'-F.

22. The RNAi reagent according to claim 17, characterized in that: The 9th position of the 3' end of the sense strand contains a nucleotide modified with 2'-F.

23. The RNAi reagent according to claim 17, characterized in that: The 12th position of the 3' end of the sense strand contains a 2'-deoxynucleotide.

24. The RNAi reagent according to claim 17, characterized in that: The 6th, 7th, 9th, 10th, 14th, 17th and / or 19th positions of the 3' end of the sense strand contain 2'-deoxynucleotides.

25. The RNAi reagent according to claim 17, characterized in that: The 15th position of the 3' end of the sense strand contains a 2'-deoxynucleotide.

26. The RNAi reagent according to any one of claims 1-25, characterized in that: The remaining positions in the sense or antisense strand of the RNAi reagent are modified nucleotides; preferably, the modified nucleotides are selected from 2'-methoxy modified nucleotides, 2'-F modified nucleotides, 2'-deoxynucleotides, 2'-O-methoxyethyl modified nucleotides, threonucleotides (TNA), ethylene glycol nucleotides (GNA), locked nucleotides (LNA), open cyclic nucleotides (UNA), bicyclic nucleotides (BNA), 2',5'-linked nucleotides, 2'-amino modified nucleotides, 2'-alkyl modified nucleotides, 2'-allyl modified nucleotides, debased nucleotides, morpholinonucleotides, and nucleotides modified with phosphate ester groups.

27. The RNAi reagent according to claim 26, characterized in that: In the RNAi reagent, three or all of the positions 2, 12, 14, and 16 at the 5' end of the antisense strand contain nucleotides modified with 2'-F, positions 5 and 7 contain 2'-deoxynucleotides, and position 15 contains a nucleotide modified with 2'-O-methoxyethyl; positions 9, 11, and 13 at the 3' end of the sense strand contain nucleotides modified with 2'-F.

28. The RNAi reagent according to claim 27, characterized in that: The 2nd, 9th, 12th, 14th, or 16th position of the 5' end of the antisense strand further comprises a 2'-deoxynucleotide.

29. The RNAi reagent according to claim 27, characterized in that: The sixth position at the 3' end of the sense strand further comprises a nucleotide modified with 2'-F.

30. The RNAi reagent according to claim 26, characterized in that: In the RNAi reagent, the antisense strand contains 2'-F modified nucleotides at positions 2, 12, 14, and 16 of its 5' end, 2'-deoxynucleotides at positions 5 and 7, and 2'-O-methoxyethyl modified nucleotides at position 15; the sense strand contains 2'-F modified nucleotides at positions 9, 11, and 13 of its 3' end, and the antisense strand also contains 2'-deoxynucleotides at positions 18, 20, or 22 of its 5' end.

31. The RNAi reagent according to claim 26, characterized in that: In the RNAi reagent, the antisense strand contains 2'-F modified nucleotides at positions 2, 12, 14, and 16 of its 5' end, 2'-deoxynucleotides at positions 5 and 7, and 2'-O-methoxyethyl modified nucleotides at position 15; the sense strand contains 2'-F modified nucleotides at positions 9, 11, and 13 of its 3' end, and 2'-deoxynucleotides at positions 6, 7, 10, 12, 14, 15, 17, or 19 of its 3' end, or 2'-deoxynucleotides at positions 11 and 13 of its 3' end, and 2'-deoxynucleotides at position 9 of its 3' end.

32. The RNAi reagent according to any one of claims 1-31, characterized in that: The RNAi reagent has a combination of modifications selected from any of the following: 1) The antisense strand contains 2'-F modified nucleotides at positions 2, 12, 14, and 16 at the 5' end, 2'-deoxynucleotides at positions 5 and 7, and 2'-O-methoxyethyl modified nucleotides at position 15; the sense strand contains 2'-F modified nucleotides at positions 9, 11, and 13 at the 3' end. 2) The antisense strand contains 2'-F modified nucleotides at positions 2, 14, and 16 of the 5' end; 2'-deoxynucleotides at positions 5, 7, and 12; and 2'-O-methoxyethyl modified nucleotides at position 15. The sense strand contains 2'-F modified nucleotides at positions 9, 11, and 13 of the 3' end. 3) The antisense strand contains 2'-F modified nucleotides at positions 2, 12, and 14 at the 5' end; 2'-deoxynucleotides at positions 5, 7, and 16; and 2'-O-methoxyethyl modified nucleotides at position 15. The sense strand contains 2'-F modified nucleotides at positions 6, 9, 11, and 13 at the 3' end. 4) The antisense strand contains 2'-F modified nucleotides at positions 12, 14, and 16 of the 5' end; 2'-deoxynucleotides at positions 2, 5, and 7; and 2'-O-methoxyethyl modified nucleotides at position 15. The sense strand contains 2'-F modified nucleotides at positions 9, 11, and 13 of the 3' end. 5) The antisense strand contains 2'-F modified nucleotides at positions 12, 14, and 16 of the 5' end; 2'-deoxynucleotides at positions 2, 5, and 7; and 2'-O-methoxyethyl modified nucleotides at position 15. The sense strand contains 2'-F modified nucleotides at positions 6, 9, 11, and 13 of the 3' end. 6) The antisense strand contains 2'-F modified nucleotides at positions 2, 14, and 16 of the 5' end; 2'-deoxynucleotides at positions 5, 7, and 9; and 2'-O-methoxyethyl modified nucleotides at position 15. The sense strand contains 2'-F modified nucleotides at positions 9, 11, and 13 of the 3' end. 7) The antisense strand contains 2'-F modified nucleotides at positions 2, 12, and 16 of the 5' end; 2'-deoxynucleotides at positions 5, 7, and 14; and 2'-O-methoxyethyl modified nucleotides at position 15; the sense strand contains 2'-F modified nucleotides at positions 9, 11, and 13 of the 3' end. 8) The antisense strand contains 2'-F modified nucleotides at positions 2, 12, 14, and 16 at the 5' end, 2'-deoxynucleotides at positions 5 and 7, and 2'-O-methoxyethyl modified nucleotides at position 15; the sense strand contains 2'-F modified nucleotides at positions 9, 11, and 13 at the 3' end, and 2'-deoxynucleotides at position 15. 9) The antisense strand contains 2'-F modified nucleotides at positions 2, 12, 14, and 16 at the 5' end; 2'-deoxynucleotides at positions 5, 7, and 18; and 2'-O-methoxyethyl modified nucleotides at position 15. The sense strand contains 2'-F modified nucleotides at positions 9, 11, and 13 at the 3' end. 10) The antisense strand contains 2'-F modified nucleotides at positions 2, 12, 14, and 16 at the 5' end; 2'-deoxynucleotides at positions 5, 7, and 20; and 2'-O-methoxyethyl modified nucleotides at position 15. The sense strand contains 2'-F modified nucleotides at positions 9, 11, and 13 at the 3' end. 11) The antisense strand contains 2'-F modified nucleotides at positions 2, 12, 14, and 16 at the 5' end; 2'-deoxynucleotides at positions 5, 7, and 22; and 2'-O-methoxyethyl modified nucleotides at position 15. The sense strand contains 2'-F modified nucleotides at positions 9, 11, and 13 at the 3' end. 12) The antisense strand contains 2'-F modified nucleotides at positions 2, 12, 14, and 16 at the 5' end, 2'-deoxynucleotides at positions 5 and 7, and 2'-O-methoxyethyl modified nucleotides at position 15; the sense strand contains 2'-F modified nucleotides at positions 6, 9, 11, and 13 at the 3' end. 13) The antisense strand contains 2'-F modified nucleotides at positions 2, 12, 14, and 16 at the 5' end, 2'-deoxynucleotides at positions 5 and 7, and 2'-O-methoxyethyl modified nucleotides at position 15; the sense strand contains 2'-F modified nucleotides at positions 9, 11, and 13 at the 3' end, and 2'-deoxynucleotides at position 6. 14) The antisense strand contains 2'-F modified nucleotides at positions 2, 12, 14, and 16 at the 5' end, 2'-deoxynucleotides at positions 5 and 7, and 2'-O-methoxyethyl modified nucleotides at position 15; the sense strand contains 2'-F modified nucleotides at positions 9, 11, and 13 at the 3' end, and 2'-deoxynucleotides at position 19. 15) The antisense strand contains 2'-F modified nucleotides at positions 2, 12, 14, and 16 at the 5' end, 2'-deoxynucleotides at positions 5 and 7, and 2'-O-methoxyethyl modified nucleotides at position 15; the sense strand contains 2'-F modified nucleotides at positions 9, 11, and 13 at the 3' end, and 2'-deoxynucleotides at position 14. 16) The antisense strand contains 2'-F modified nucleotides at positions 2, 12, 14, and 16 at the 5' end, 2'-deoxynucleotides at positions 5 and 7, and 2'-O-methoxyethyl modified nucleotides at position 15; the sense strand contains 2'-F modified nucleotides at positions 9, 11, and 13 at the 3' end, and 2'-deoxynucleotides at position 17. 17) The antisense strand contains 2'-F modified nucleotides at positions 2, 12, 14, and 16 at the 5' end, 2'-deoxynucleotides at positions 5 and 7, and 2'-O-methoxyethyl modified nucleotides at position 15; the sense strand contains 2'-F modified nucleotides at positions 11 and 13 at the 3' end, and 2'-deoxynucleotides at position 9. 18) The antisense strand contains 2'-F modified nucleotides at positions 2, 12, 14, and 16 at the 5' end, 2'-deoxynucleotides at positions 5 and 7, and 2'-O-methoxyethyl modified nucleotides at position 15; the sense strand contains 2'-F modified nucleotides at positions 9, 11, and 13 at the 3' end, and 2'-deoxynucleotides at position 12. 19) The antisense strand contains 2'-F modified nucleotides at positions 2, 12, 14, and 16 at the 5' end, 2'-deoxynucleotides at positions 5 and 7, and 2'-O-methoxyethyl modified nucleotides at position 15; the sense strand contains 2'-F modified nucleotides at positions 9, 11, and 13 at the 3' end, and 2'-deoxynucleotides at position 7. 20) The antisense strand contains 2'-F modified nucleotides at positions 2, 12, 14, and 16 at the 5' end, 2'-deoxynucleotides at positions 5 and 7, and 2'-O-methoxyethyl modified nucleotides at position 15; the sense strand contains 2'-F modified nucleotides at positions 9, 11, and 13 at the 3' end, and 2'-deoxynucleotides at position 10.

33. The RNAi reagent according to claim 32, characterized in that: The remaining positions in the sense or antisense strand of the RNAi reagent are nucleotides modified with 2'-methoxy groups.

34. An RNAi reagent for inhibiting the expression of a target gene, comprising a sense strand and an antisense strand, each strand having 17 to 30 nucleotides, wherein each nucleotide is independently a modified or unmodified nucleotide; wherein the antisense strand is complementary to at least a portion of the mRNA of the target gene, and the sense strand and the antisense strand are at least partially complementary to form a double-stranded region. The antisense chain includes the sequence shown in formula IA; 3’-(N) n NNN E NNN’NNNNN d NN d NNN F N-5’ (Formula IA) in, n represents an integer from 0 to 8; N F Nucleotides representing 2'-F modifications; Each N d They represent 2'-deoxynucleotides; Each N, N', or N E Each can be used independently to represent a modified or unmodified nucleotide; The nucleotide corresponding to N' in the sense strand is not a nucleotide modified by 2'-F.

35. The RNAi reagent according to claim 34, characterized in that: The nucleotide corresponding to N' in the sense strand is selected from nucleotides containing 2'-methoxy modification or 2'-deoxynucleotides.

36. The RNAi reagent according to any one of claims 34-35, characterized in that: N E The nucleotides are selected from those containing heat-stabilized modifications; the heat-stabilized modifications refer to the double-stranded oligonucleotides whose thermal dissociation temperature Tm increases after introduction; preferably, the thermal dissociation temperature Tm increases by at least 0.05°C; more preferably, the thermal dissociation temperature Tm increases by 0.5-4°C.

37. The RNAi reagent according to claim 36, characterized in that: The heat-stabilized modified nucleotide is selected from nucleotides modified with 2'-O-methoxyethyl, nucleotides modified with 2'-O-allyl, nucleotides modified with 2'-C-allyl, nucleotides modified with 2'-O-2-N-methylamino-2-oxomethyleneethyl, nucleotides modified with 2'-O-2-N,N-dimethylaminoethyl, nucleotides modified with 2'-O-3-aminopropyl, nucleotides modified with 2'-O-2,4-dinitrophenyl, nucleotides modified with 2'-O-(N-methylacetamido)-modified, 2',4'-disubstituted nucleotides, locked nucleotides (LNA), bicyclic nucleotides (BNA), or restricted ethyl-bridged nucleotides (cEt); preferably, the heat-stabilized modified nucleotide is selected from nucleotides modified with 2'-O-methoxyethyl.

38. The RNAi reagent according to claim 34, characterized in that: The antisense chain includes the sequence shown in formula IAa; 3’-(N) n N a17 N a16 N E N F N a13 N’N a11 N a10 N a9 N a8 N d N a6 N d N a4 N a3 N F N a1 -5’ (Formula IAa) in, n represents an integer from 0 to 8; Each N F These represent nucleotides modified by 2'-F; Each N d They represent 2'-deoxynucleotides; Each N and N' independently represents a modified or unmodified nucleotide; N a1 N a3 N a4 N a6 N a8 N a9 N a10 N a11 N a13 N a16 N a17 Each can be used independently to represent a modified or unmodified nucleotide; N E This represents a nucleotide containing a heat-stabilizing modification; preferably, N E This represents a nucleotide modified with 2'-O-methoxyethyl.

39. The RNAi reagent according to any one of claims 34-38, characterized in that: The meaningful chain includes the sequence shown in Formula II; 5'-(N) m NNNNNN T NNNNNNNNNNN-3' (Formula II) in, m represents an integer from 0 to 8; N T This represents a nucleotide that is not modified by 2'-F; Each N independently represents a modified or unmodified nucleotide.

40. The RNAi reagent according to claim 39, characterized in that: N T This represents a nucleotide or 2'-deoxynucleotide containing a 2'-methoxy group.

41. The RNAi reagent according to any one of claims 39-40, characterized in that: The meaningful chain includes the sequence shown in Form IIA; 5′-(N) m N s17 N s16 N s15 N s14 N F N T N F N s10 N s9 N s8 N s7 N s6 N s5 N s4 N s3 N s2 N s1 -3' (IIA) in, m represents an integer from 0 to 8; N T This represents a nucleotide containing a 2'-methoxy group or a 2'-deoxynucleotide. Each N F These represent nucleotides modified by 2'-F; Each N independently represents a modified or unmodified nucleotide; N s1 N s2 N s3 N s4 N s5 N s6 N s7 N s8 N s9 N s10 N s14 N s15 N s16 N s17 Each can be used to represent a modified or unmodified nucleotide.

42. The RNAi reagent according to any one of claims 39-41, characterized in that: The meaningful chain includes the sequence shown in formula IIAa; 5’-(N) m N s17 N s16 N s15 N s14 N F N T N F N s10 N F N s8 N s7 N s6 N s5 N s4 N s3 N s2 N s1 -3’ (Formula IIAa) in, m represents an integer from 0 to 8; N T This represents a nucleotide containing a 2'-methoxy group or a 2'-deoxynucleotide. Each N F These represent nucleotides modified by 2'-F; Each N independently represents a modified or unmodified nucleotide; N s1 N s2 N s3 N s4 N s5 N s6 N s7 N s8 N s10 N s14 N s15 N s16 N s17 Each can be used to represent a modified or unmodified nucleotide.

43. The RNAi reagent according to any one of claims 39-41, characterized in that: The meaningful chain includes the sequence shown by formula IIAb; 5’-(N) m N s17 N s16 N s15 N s14 N F N d N F N s10 N s9 N s8 N s7 N s6 N s5 N s4 N s3 N s2 N s1 ​ in, m represents an integer from 0 to 8; Each N F These represent nucleotides modified by 2'-F; N d Represents 2'-deoxynucleotide; Each N independently represents a modified or unmodified nucleotide; N s1 N s2 N s3 N s4 N s5 N s6 N s7 N s8 N s9 N s10 N s14 N s15 N s16 N s17 Each can be used to represent a modified or unmodified nucleotide.

44. The RNAi reagent according to any one of claims 39-41, characterized in that: The meaningful chain includes the sequence shown by IIAc; 5’-(N) m N s17 N s16 N s15 N s14 N F N T N F N s10 N s9 N s8 N s7 N F N s5 N s4 N s3 N s2 N s1 -3’ (Formula IIAc) in, m represents an integer from 0 to 8; N T This represents a nucleotide containing a 2'-methoxy group or a 2'-deoxynucleotide. Each N F These represent nucleotides modified by 2'-F; Each N independently represents a modified or unmodified nucleotide; N s1 N s2 N s3 N s4 N s5 N s7 N s8 N s9 N s10 N s14 N s15 N s16 N s17 Each can be used to represent a modified or unmodified nucleotide.

45. The RNAi reagent according to any one of claims 39-41, characterized in that: The meaningful chain includes the sequence shown by IIAd; 5’-(N) m N s17 N s16 N d N s14 N F N T N F N s10 N s9 N s8 N s7 N s6 N s5 N s4 N s3 N s2 N s1 -3’ (Formula IIAd) in, m represents an integer from 0 to 8; N T This represents a nucleotide containing a 2'-methoxy group or a 2'-deoxynucleotide. Each N F These represent nucleotides modified by 2'-F; N d Represents 2'-deoxynucleotide; Each N independently represents a modified or unmodified nucleotide; N s1 N s2 N s3 N s4 N s5 N s6 N s7 N s8 N s9 N s10 N s14 N s16 N s17 Each can be used to represent a modified or unmodified nucleotide.

46. ​​The RNAi reagent according to any one of claims 34-45, characterized in that: The nucleotides modified in the RNAi reagent are selected from 2'-methoxy-modified nucleotides, 2'-F-modified nucleotides, 2'-deoxynucleotides, 2'-O-methoxyethyl-modified nucleotides, threonucleotides (TNA), ethylene glycol nucleotides (GNA), locked nucleotides (LNA), open-ring nucleotides (UNA), bicyclic nucleotides (BNA), 2',5'-linked nucleotides, 2'-amino-modified nucleotides, 2'-alkyl-modified nucleotides, 2'-allyl-modified nucleotides, debased nucleotides, morpholinonucleotides, and nucleotides modified with phosphate ester groups.

47. The RNAi reagent according to any one of claims 34-46, characterized in that: The RNAi reagent contains an antisense strand having any of the following modifications: 1)3’-(N) n N a17 N F N MOE N F N a13 N F N a11 N a10 N a9 N a8 N d N a6 N d N a4 N a3 N F N a1 -5’; 2)3’-(N) n N a17 N F N MOE N F N a13 N d N a11 N a10 N a9 N a8 N d N a6 N d N a4 N a3 N F N a1 -5’; 3)3’-(N) n N a17 N d N MOE N F N a13 N F N a11 N a10 N a9 N a8 N d N a6 N d N a4 N a3 N F N a1 -5’; 4)3’-(N) n N a17 N F N MOE N F N a13 N F N a11 N a10 N a9 N a8 N d N a6 N d N a4 N a3 N d N a1 -5’; 5)3’-(N) n N a17 N F N MOE N F N a13 N a12 N a11 N a10 N d N a8 N d N a6 N d N a4 N a3 N F N a1 -5’; 6)3’-(N) n N a17 N F N MOE N d N a13 N F N a11 N a10 N a9 N a8 N d N a6 N d N a4 N a3 N F N a1 -5’; 7)3’-N a23 N a22 N a21 N a20 N a19 N d N a17 N F N MOE N F N a13 N F N a11 N a10 N a9 N a8 N d N a6 N d N a4 N a3 N F N a1 -5’; 8)3’-N a23 N a22 N a21 N d N a19 N a18 N a17 N F N MOE N F N a13 N F N a11 N a10 N a9 N a8 N d N a6 N d N a4 N a3 N F N a1 -5’; 9)3’-N a23 N d N a21 N a20 N a19 N a18 N a17 N F N MOE N F N a13 N F N a11 N a10 N a9 N a8 N d N a6 N d N a4 N a3 N F N a1 -5’; in, n represents an integer from 0 to 8; preferably, n is 2, 3, 4, 5, 6 or 7; Each N F These represent nucleotides modified by 2'-F; Each N d They represent 2'-deoxynucleotides; N MOE Represents a nucleotide modified with 2'-O-methoxyethyl; Each N independently represents a modified or unmodified nucleotide; N a1 N a3 N a4 N a6 N a8 N a9 N a10 N a11 N a12 N a13 N a17 N a18 N a19 N a20 N a21 N a22 N a23 Each can independently represent a modified or unmodified nucleotide; preferably, N a1 N a3 N a4 N a6 N a8 N a9 N a10 N a11 N a12 N a13 N a17 N a18 N a19 N a20 N a21 N a22 N a23 All of them are nucleotides containing 2'-methoxy groups.

48. The RNAi reagent according to any one of claims 34-47, characterized in that: The RNAi reagent contains a sense strand having any of the following modifications: 1)5’-(N) m N s17 N s16 N s15 N s14 N F N T N F N s10 N F N s8 N s7 N s6 N s5 N s4 N s3 N s2 N s1 -3’; 2)5’-(N) m N s17 N s16 N s15 N s14 N F N d N F N s10 N F N s8 N s7 N s6 N s5 N s4 N s3 N s2 N s1 -3’; 3)5’-(N) m N s17 N s16 N s15 N s14 N F N T N F N s10 N F N s8 N s7 N F N s5 N s4 N s3 N s2 N s1 -3’; 4)5’-(N) m N s17 N s16 N d N s14 N F N T N F N s10 N F N s8 N s7 N s6 N s5 N s4 N s3 N s2 N s1 -3’; 5)5’-(N) m N s17 N s16 N s15 N s14 N F N T N F N s10 N F N s8 N s7 N d N s5 N s4 N s3 N s2 N s1 -3’; 6)5’-N s21 N s20 N d N s18 N s17 N s16 N s15 N s14 N F N T N F N s10 N F N s8 N s7 N s6 N s5 N s4 N s3 N s2 N s1 -3’; 7)5’-(N) m N s17 N s16 N s15 N d N F N T N F N s10 N F N s8 N s7 N s6 N s5 N s4 N s3 N s2 N s1 -3’; 8)5’-(N) m N d N s16 N s15 N s14 N F N T N F N s10 N F N s8 N s7 N s6 N s5 N s4 N s3 N s2 N s1 -3’; 9)5’-(N) m N s17 N s16 N s15 N s14 N F N T N F N s10 N d N s8 N s7 N s6 N s5 N s4 N s3 N s2 N s1 -3’; 10)5’-(N) m N s17 N s16 N s15 N s14 N F N T N F N s10 N F N s8 N d N s6 N s5 N s4 N s3 N s2 N s1 -3’; 11)5’-(N) m N s17 N s16 N s15 N s14 N F N T N F N d N F N s8 N s7 N s6 N s5 N s4 N s3 N s2 N s1 -3’; in, m represents an integer from 0 to 8; preferably, n is 2, 3, 4, 5, 6 or 7; Each N F These represent nucleotides modified by 2'-F; Each N d They represent 2'-deoxynucleotides; N T This represents a nucleotide containing a 2'-methoxy group. Each N independently represents a modified or unmodified nucleotide; N s1 N s2 N s3 N s4 N s5 N s6 N s7 N s8 N s10 N s14 N s15 N s16 N s17 N s18 N s20 N s21 Each can independently represent a modified or unmodified nucleotide; preferably, N s1 N s2 N s3 N s4 N s5 N s6 N s7 N s8 N s10 N s14 N s15 N s16 N s17 N s18 N s20 N s21 All of them are nucleotides containing 2'-methoxy groups.

49. The RNAi reagent according to any one of claims 1-48, characterized in that: The RNAi reagent comprises at least one modified phosphate ester nucleoside linker; preferably, the modified phosphate ester nucleoside linker is selected from thiophosphate nucleoside links, dithiophosphate nucleoside links, and nucleotides with methylphosphonate groups.

50. The RNAi reagent according to claim 49, characterized in that: The sense strand of the RNA reagent contains 0, 1, 2, 3, 4, or 5 phosphate-thioester nucleoside links independently at its 5' and 3' ends, respectively; and / or the antisense strand of the RNA reagent contains 0, 1, 2, 3, 4, or 5 phosphate-thioester nucleoside links independently at its 5' and 3' ends, respectively.

51. The RNAi reagent according to any one of claims 1-50, characterized in that: The RNAi reagent also contains one or more ligand groups or linker groups.

52. The RNAi reagent according to claim 51, characterized in that: The ligand group or linker group is conjugated to the sense or antisense strand of the RNAi reagent; preferably, the ligand group or linker group is conjugated to the 3' end of the sense strand, and / or the ligand group or linker group is conjugated to the 5' end of the sense strand, and / or the ligand group or linker group is conjugated to the 3' end of the antisense strand.

53. A composition comprising the RNAi reagent of any one of claims 1-52, and a pharmaceutically acceptable carrier.

54. A method for inhibiting the expression of a target gene in a cell, the method comprising: delivering an RNAi reagent according to any one of claims 1-52 or a composition according to claim 53 to a subject, such that the RNAi reagent is delivered to a specific target of the subject.

55. Use of the RNAi reagent of any one of claims 1-52 or the composition of claim 53 in the preparation of medicaments for viral diseases, neuromuscular diseases, bacterial infections, inflammatory and immune diseases, metabolic diseases, liver diseases, kidney diseases, cardiovascular diseases, ophthalmic diseases, lung diseases, and rare diseases.

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