Modified double-stranded ribonucleic acid and use thereof

Abstract

Disclosed in the present invention are a dsRNA capable of enhancing the effect of inhibiting target gene expression, a conjugate thereof, and a use thereof in the preparation of a medicament for treating related diseases.

Description

Modified double-stranded RNA and its applications

[0001] Cross-references

[0002] This application claims priority to Chinese invention patent application No. 202411179535.X, filed August 26, 2024; Chinese invention patent application No. 202510040868.2, filed January 9, 2025; Chinese invention patent application No. 202510045726.5, filed January 10, 2025; and Chinese invention patent application No. 202511029130.2, filed July 24, 2025, the entire contents of which are incorporated herein by reference. Technical Field

[0003] This invention relates to a class of dsRNAs that can enhance the effect of inhibiting target gene expression, their conjugates, and their application in the preparation of drugs for treating related diseases. Background Technology

[0004] RNA interference (RNAi) is a highly conserved evolutionary phenomenon characterized by the efficient and specific degradation of homologous mRNA induced by double-stranded RNA (dsRNA). RNA molecules inhibit gene expression by disrupting specific mRNAs. Because RNAi technology can specifically knock out or shut down the expression of specific genes, it has rapidly become one of the most popular research tools in gene function research and gene therapy, and is widely used to explore gene function and treat various diseases.

[0005] Lp(a) is a heterogeneous low-density lipoprotein particle composed of low-density lipoprotein-like particles (LDL-like particles) and apolipoprotein a (Apo(a)). LDL-like particles contain apolipoprotein B (ApoB-100), which is linked to apolipoprotein a via disulfide bonds. High Lp(a) levels are an independent risk factor for cardiovascular disease. The gene for apolipoprotein a, a core component of Lp(a), is LPA. LPA is primarily expressed in the liver, and LPA inhibitors can be used to treat diseases associated with high LPA levels, including cardiovascular disease and other related conditions.

[0006] Given the proven advantages of RNAi, such as long-lasting effects and low toxicity, the application of siRNA or dsRNA in treating related diseases by targeting LPA to downregulate its mRNA level and thereby inhibiting its expression is highly anticipated. Summary of the Invention

[0007] In a first aspect, this application provides a dsRNA comprising a sense strand and an antisense strand, wherein the length of the sense strand and the antisense strand are independently 17 to 35 nucleotides, wherein:

[0008] Each nucleotide may be independently and optionally modified.

[0009] The sense strand comprises a first strand, and the antisense strand comprises a second strand. The first and second strands are anti-complementary. Starting from the 5' end of the first strand, positions 9, 11, and 13 of the first strand are nucleotides containing a 2'-F modification, and optionally position 15 of the first strand is a nucleotide containing a 2'-F modification.

[0010] Starting from the 5' end of the second strand, position 14 of the second strand is a nucleotide containing a 2'-F modification.

[0011] In some embodiments of the first aspect, this application provides a dsRNA comprising a sense strand and an antisense strand, the sense strand and the antisense strand being independently 17 to 35 nucleotides in length, the nucleotides being independently and optionally modified, wherein the sense strand comprises a first strand, the antisense strand comprises a second strand, the first strand and the second strand are anticomplementary, and starting from the 5' end of the first strand, positions 9, 11, 13 and 15 of the first strand are nucleotides containing 2'-F modification, and starting from the 5' end of the second strand, position 14 of the second strand is a nucleotide containing 2'-F modification.

[0012] In some embodiments of the first aspect, this application provides a dsRNA comprising a sense strand and an antisense strand, wherein the length of the sense strand and the antisense strand are independently 17 to 35 nucleotides, wherein:

[0013] Each nucleotide may be independently and optionally modified.

[0014] The sense strand comprises a first strand, and the antisense strand comprises a second strand. The first and second strands are anti-complementary. Starting from the 5' end of the first strand, position 9 of the first strand is a nucleotide containing a 2'-F modification, and optionally position 11 of the first strand is a nucleotide containing a 2'-F modification.

[0015] Starting from the 5' end of the second strand, positions 2 and 14 of the second strand are nucleotides containing 2'-F modification, and positions 5, 7, and 12 of the second strand are nucleotides containing 2'-deoxy modification.

[0016] In some embodiments of the first aspect of this application, the second position of the second strand is a nucleotide containing a 2'-deoxy modification or a nucleotide containing a 2'-F modification, and the 5th, 7th and 12th positions of the second strand are nucleotides containing a 2'-deoxy modification.

[0017] In some embodiments of the first aspect of this application, positions 2 and 16 of the second strand are nucleotides containing 2'-F modifications.

[0018] In some embodiments of the first aspect of this application, positions 2 and 16 of the second strand are nucleotides containing 2'-F modification, and the antisense strand further includes a 3' extension and a 5' extension. The 3' extension is connected to the 3' end of the second strand and has a length of 0-5 nucleotides, and the 5' extension is connected to the 5' end of the second strand and has a length of at least 3 nucleotides. The dsRNA can be cleaved between the 3' end nucleotide of the 5' extension and the 5' end nucleotide of the second strand, and the resulting cleavage product containing the first and second strands can silence gene expression through RNA interference.

[0019] In some embodiments of the first aspect of this application, the 6th position of the second strand is a nucleotide containing a 2'-F modification.

[0020] In some embodiments of the first aspect of this application, the second strand has a nucleotide at position 2 containing a 2'-deoxy modified nucleotide or a nucleotide containing a 2'-F modified nucleotide, and positions 5, 7, and 12 of the second strand have nucleotides containing a 2'-deoxy modified nucleotide. The antisense strand also contains a 3' extension and a 5' extension. The 3' extension is connected to the 3' end of the second strand and has a length of 0-5 nucleotides. The 5' extension is connected to the 5' end of the second strand and has a length of at least 3 nucleotides. The dsRNA can be cleaved between the 3' end nucleotide of the 5' extension and the 5' end nucleotide of the second strand. The resulting cleavage product containing the first and second strands can silence gene expression through RNA interference.

[0021] Secondly, this application provides a dsRNA comprising a sense strand and an antisense strand, wherein the length of the sense strand and the antisense strand are independently 17-35 nucleotides, wherein:

[0022] Each nucleotide may be independently and optionally modified.

[0023] The sense strand comprises a first strand, and the antisense strand comprises a second strand. The first and second strands are inversely complementary. Starting from the 5' end of the first strand, positions 9, 10, and 11 or positions 9, 11, and 13 of the first strand are nucleotides containing a 2'-F modification. Starting from the 5' end of the second strand, position 2 of the second strand is a nucleotide containing a 2'-deoxy modification or a nucleotide containing a 2'-F modification, positions 5, 7, and 12 of the second strand are nucleotides containing a 2'-deoxy modification, and position 14 of the second strand is a nucleotide containing a 2'-F modification.

[0024] In some embodiments of the second aspect, this application provides a dsRNA comprising a sense strand and an antisense strand, the length of which is independently 17 to 35 nucleotides, and the nucleotides are independently and optionally modified. The sense strand comprises a first strand, and the antisense strand comprises a second strand. The first and second strands are anticomplementary. Starting from the 5' end of the first strand, positions 9 and 11 of the first strand are nucleotides containing 2'-F modification, and positions 10 or 13 of the first strand are nucleotides containing 2'-F modification. Starting from the 5' end of the second strand, position 2 of the second strand is a nucleotide containing 2'-deoxy modification or a nucleotide containing 2'-F modification, positions 5, 7, and 12 of the second strand are nucleotides containing 2'-deoxy modification, and position 14 of the second strand is a nucleotide containing 2'-F modification.

[0025] In some embodiments of the second aspect of this application, starting from the 5' end of the first strand, positions 9, 10, and 11 of the first strand are nucleotides containing 2'-F modification; starting from the 5' end of the second strand, position 2 of the second strand is a nucleotide containing 2'-deoxy modification; the antisense strand further includes a 3' extension and a 5' extension; the 3' extension is connected to the 3' end of the second strand and has a length of 0-5 nucleotides; the 5' extension is connected to the 5' end of the second strand and has a length of at least 3 nucleotides; the dsRNA can be cleaved between the 3' end nucleotide of the 5' extension and the 5' end nucleotide of the second strand; the resulting cleavage product containing the first and second strands can silence gene expression through RNA interference.

[0026] NA interference is used to silence gene expression.

[0027] Thirdly, this application provides a dsRNA comprising a sense strand and an antisense strand, wherein the length of the sense strand and the antisense strand are independently 17-35 nucleotides, wherein:

[0028] Each nucleotide may be independently and optionally modified.

[0029] The sense strand comprises a first strand, and the antisense strand comprises a second strand. The first and second strands are inversely complementary. Starting from the 5' end of the first strand, positions 9, 10, and 11 or positions 9, 11, and 13 of the first strand are nucleotides containing a 2'-F modification. And starting from the 5' end of the second strand, positions 2, 14, and 16 of the second strand are nucleotides containing a 2'-F modification.

[0030] In some embodiments of the third aspect, this application provides a dsRNA comprising a sense strand and an antisense strand, the length of which is independently 17 to 35 nucleotides, and the nucleotides are independently and optionally modified. The sense strand comprises a first strand, and the antisense strand comprises a second strand. The first and second strands are anticomplementary. Starting from the 5' end of the first strand, positions 9 and 11 of the first strand are nucleotides containing 2'-F modification, and positions 10 or 13 of the first strand are nucleotides containing 2'-F modification. Starting from the 5' end of the second strand, positions 2, 14, and 16 of the second strand are nucleotides containing 2'-F modification.

[0031] In some embodiments of the third aspect of this application, the antisense strand further comprises a 3' extension and a 5' extension, wherein the 3' extension is connected to the 3' end of the second strand and has a length of 0-5 nucleotides, and the 5' extension is connected to the 5' end of the second strand and has a length of at least 3 nucleotides. The dsRNA can be cleaved between the 3' end nucleotide of the 5' extension and the 5' end nucleotide of the second strand, and the resulting cleavage product containing the first and second strands can silence gene expression through RNA interference.

[0032] In some embodiments of the third aspect of this application, the 10th or 13th position of the first strand is a nucleotide containing a 2'-F modification and the 6th position of the second strand is a nucleotide containing a 2'-F modification.

[0033] In some embodiments of the third aspect of this application, the 13th position of the first strand is a nucleotide containing a 2'-F modification and the 7th position of the second strand is a nucleotide containing a 2'-F modification.

[0034] In some embodiments of the third aspect of this application, positions 8 and 9 of the second strand are nucleotides containing 2'-F modification.

[0035] In some embodiments of the third aspect of this application, the 7th position of the first strand is a nucleotide containing a 2'-F modification.

[0036] Fourthly, this application provides a dsRNA comprising a sense strand and an antisense strand, wherein the length of the sense strand and the antisense strand are independently 17-35 nucleotides, wherein:

[0037] Each nucleotide may be independently and optionally modified.

[0038] The sense strand comprises a first strand, and the antisense strand comprises a second strand. The first and second strands are anti-complementary. Starting from the 5' end of the first strand, positions 9, 10, and 11 of the first strand are nucleotides containing a 2'-F modification.

[0039] Starting from the 5' end of the second strand, positions 2, 5, 7, and 12 of the second strand are nucleotides containing 2'-deoxy modification, and position 14 of the second strand is a nucleotide containing 2'-F modification.

[0040] In some embodiments of the fourth aspect, this application provides a dsRNA comprising a sense strand and an antisense strand, the sense strand and the antisense strand each being independently 17 to 35 nucleotides in length, the nucleotides being independently and optionally modified, wherein the sense strand comprises a first strand, the antisense strand comprises a second strand, the first strand and the second strand are anticomplementary, and counting from the 5' end of the first strand, positions 9, 10, and 11 of the first strand are nucleotides containing 2'-F modification; counting from the 5' end of the second strand, positions 2, 5, 7, and 12 of the second strand are nucleotides containing 2'-F modification. - A deoxy-modified nucleotide, wherein position 14 of the second strand is a nucleotide containing a 2'-F modification, the antisense strand further includes a 3' extension and a 5' extension, the 3' extension being attached to the 3' end of the second strand and having a length of 0-5 nucleotides, the 5' extension being attached to the 5' end of the second strand and having a length of at least 3 nucleotides, the dsRNA being cleaved between the 3' end nucleotide of the 5' extension and the 5' end nucleotide of the second strand, the resulting cleavage product containing the first and second strands being able to silence gene expression through RNA interference.

[0041] Fifthly, this application provides a dsRNA comprising a sense strand and an antisense strand, wherein the length of the sense strand and the antisense strand are independently 17 to 35 nucleotides, wherein:

[0042] Each nucleotide may be independently and optionally modified.

[0043] The sense strand comprises a first strand, and the antisense strand comprises a second strand. The first and second strands are anti-complementary. Starting from the 5' end of the first strand, positions 9, 10, and 12 of the first strand are nucleotides containing a 2'-F modification.

[0044] Starting from the 5' end of the second strand, positions 2, 6, 14, and 16 of the second strand are nucleotides containing a 2'-F modification.

[0045] In some embodiments of the fifth aspect, this application provides a dsRNA comprising a sense strand and an antisense strand, the sense strand and the antisense strand being independently 17 to 35 nucleotides in length, the nucleotides being independently and optionally modified, wherein the sense strand comprises a first strand, the antisense strand comprises a second strand, the first strand and the second strand are anticomplementary, and starting from the 5' end of the first strand, positions 9, 10, and 12 of the first strand are nucleotides containing 2'-F modification; starting from the 5' end of the second strand, positions 2, 6, 14, and 16 of the second strand are nucleotides containing 2'-F modification; the antisense strand further comprises a 3' extension and a 5' extension, the 3' extension being connected to the 3' end of the second strand and having a length of 0-5 nucleotides, the 5' extension being connected to the 5' end of the second strand and having a length of at least 3 nucleotides; the dsRNA is cleavable between the 3' end nucleotide of the 5' extension and the 5' end nucleotide of the second strand, and the resulting cleavage product comprising the first strand and the second strand can silence gene expression through RNA interference.

[0046] In some embodiments of the first to fifth aspects of this application, starting from the 3' end of the aforementioned 5' extension, the two nucleotides of the aforementioned 5' extension are selected from optionally modified natural analogs of G, A, G, non-natural analogs of G, natural analogs of A, and non-natural analogs of A.

[0047] In some embodiments of the first to fifth aspects of this application, the two nucleotides of the 5' extension, starting from the 3' end of the 5' extension, are selected from optionally modified G, natural analogs of G, and non-natural analogs of G.

[0048] In some embodiments of the first to fifth aspects of this application, the 5' nucleotide of the second chain is selected from natural analogs of A, U, I, U, non-natural analogs of U, natural analogs of A, non-natural analogs of A, natural analogs of I, and non-natural analogs of I.

[0049] Sixthly, this application provides a dsRNA comprising a sense strand and an antisense strand, wherein the length of the sense strand and the antisense strand are independently 17 to 35 nucleotides, wherein:

[0050] The justice chain comprises a first chain, and the antisense chain comprises a second chain; the first chain and the second chain are opposite and complementary.

[0051] The antisense chain also includes a 3' extension and a 5' extension.

[0052] The 3' extension is connected to the 3' end of the second strand and has a length of 0-5 nucleotides.

[0053] The 5' extension is connected to the 5' end of the second strand and is at least 3 nucleotides in length.

[0054] The dsRNA can be cleaved between the 3' end nucleotide of the 5' extension and the 5' end nucleotide of the second strand to obtain cleaved dsRNA.

[0055] The cleaved dsRNA contains a first strand and a second strand and is capable of silencing the expression of target genes through RNA interference.

[0056] In some embodiments of the first to sixth aspects of this application, a blocking group is attached to the 5' nucleotide of the 5' extension; further, the blocking group is M06 or InvAB.

[0057] In some embodiments of the first to sixth aspects of this application, starting from the 3' end of the aforementioned 5' extension, positions 1 and 2 of the aforementioned 5' extension are nucleotides containing 2'-F modifications.

[0058] In some embodiments of the first to sixth aspects of this application, the aforementioned blocking group is connected to the 5' end nucleotide residue of the aforementioned 5' extension via a thiophosphate bond, and the nucleotide residues at positions 1 and 2 of the aforementioned second chain are connected via a thiophosphate bond, starting from the 5' end of the aforementioned second chain.

[0059] In some embodiments of the first to sixth aspects of this application, starting from the 3' end of the antisense strand, the nucleotide residues at positions 1 and 2 of the antisense strand are linked by a phosphate thioester bond, and the nucleotide residues at positions 2 and 3 of the antisense strand are linked by a phosphate thioester bond.

[0060] In some embodiments of the first to sixth aspects of this application, starting from the 3' end of the aforementioned positive chain, the nucleotide residues at positions 1 and 2 of the aforementioned positive chain are linked by a phosphate thioester bond, and the nucleotide residues at positions 2 and 3 of the aforementioned positive chain are linked by a phosphate thioester bond.

[0061] In a seventh aspect, this application provides a dsRNA conjugate comprising some of the schemes and delivery systems described in the first to sixth aspects above, wherein the delivery system includes delivery systems targeting the liver and targeting extrahepatic tissues.

[0062] In some embodiments of the seventh aspect of this application, the aforementioned delivery system includes a ligand delivery system targeting ASGPR, a ligand delivery system targeting transferrin receptors, a ligand delivery system targeting Apelin receptors, a ligand delivery system targeting CD206, a ligand delivery system targeting integrin receptors, or a ligand delivery system targeting low-density lipoprotein-related proteins.

[0063] In some embodiments of the seventh aspect of this application, the aforementioned dsRNA conjugate is wherein the 3' end nucleotide of the positive strand is linked to a delivery system.

[0064] In some embodiments of the seventh aspect of this application, the aforementioned delivery system is L96.

[0065] In some embodiments of the seventh aspect of this application, the residues of the 3' end nucleotide of the aforementioned positive chain are linked to the L96 residue by a phosphate ester bond.

[0066] Eighthly, this application provides a dsRNA conjugate comprising some of the schemes and delivery systems described in the first to seventh aspects, wherein the 5' end nucleotide residue of the positive strand is linked to the inverted abasic nucleotide residue via a phosphate thioester bond, the inverted abasic nucleotide is linked to the delivery system, and starting from the 3' end of the positive strand, the 1st and 2nd nucleotide residues of the positive strand are linked by a phosphate thioester bond.

[0067] In some embodiments of the eighth aspect of this application, the aforementioned delivery system includes a ligand delivery system targeting ASGPR, a ligand delivery system targeting transferrin receptors, a ligand delivery system targeting Apelin receptors, a ligand delivery system targeting CD206, a ligand delivery system targeting integrin receptors, or a ligand delivery system targeting low-density lipoprotein-related proteins.

[0068] In some embodiments of the eighth aspect of this application, the aforementioned delivery system is NAG37.

[0069] In some embodiments of the eighth aspect of this application, the aforementioned inverted non-basic nucleotide residue is linked to the NAG37 residue via a thiophosphate bond.

[0070] In some embodiments of aspects one through eight of this application, the above modifications are independently selected from one, two, or three combinations of the following: 2'-OMe modification, 2'-F modification, 2'-deoxygenation modification, VP modification, 5'-MP modification, PS modification, PS2 modification, MP modification, MOP modification, invAb modification, and invAB modification.

[0071] In some embodiments of the first to eighth aspects of this application, each of the nucleotides is a modified nucleotide. In some embodiments, the modified nucleotide is selected from nucleotides containing 2'-F modification, nucleotides containing 2'-deoxy modification, and nucleotides containing 2'-oxymethyl modification.

[0072] In some embodiments of the first to eighth aspects of this application, the aforementioned dsRNA or its conjugates, wherein the antisense strand comprises a cleavage region comprising the nucleotide sequence shown in formula (I),

[0073] Equation (I): (3'-5')X²-YZ

[0074] The cleavage occurs between X2 and Y, where X2 is the 5' end nucleotide of the second strand, and Y and Z are the two nucleotides at the 3' end of the 5' extension, where Z is guanine nucleotide (G), a natural analog of guanine nucleotide (G), a non-natural analog of guanine nucleotide (G), adenine nucleotide (A), a natural analog of adenine nucleotide (A), or a non-natural analog of adenine nucleotide (A).

[0075] In some embodiments of the first to eighth aspects of this application, the aforementioned dsRNA or its conjugates, wherein Z is guanine nucleotide (G), a natural analog of guanine nucleotide (G), or a non-natural analog of guanine nucleotide (G).

[0076] In some embodiments of the first to seventh aspects of this application, the above-mentioned dsRNA or its conjugate, wherein X2 is adenine nucleotide (A), a natural analog of adenine nucleotide (A), a non-natural analog of adenine nucleotide (A), uracil nucleotide (U), a natural analog of uracil nucleotide (U), or a non-natural analog of uracil nucleotide (U).

[0077] In some embodiments of the first to eighth aspects of this application, the above-mentioned dsRNA or its conjugates, wherein the formula (I) has a sequence (3'-5') selected from the following: UUG, UAG, AUG, AAG, UUA, UAA, AUA, AAA, UCG, UGG, ACG, AGG, UCA, UGA, ACA, AGA, IUG, IAG, IUA and IAA, or natural or non-natural analogs thereof.

[0078] In some embodiments of the first to eighth aspects of this application, the aforementioned dsRNA or its conjugate, wherein Y is adenine nucleotide (A), a natural analog of adenine nucleotide (A), a non-natural analog of adenine nucleotide (A), uracil nucleotide (U), a natural analog of uracil nucleotide (U), or a non-natural analog of uracil nucleotide (U).

[0079] In some embodiments of the first to eighth aspects of this application, the above-mentioned dsRNA or its conjugates, wherein the formula (I) has a sequence (3'-5') selected from the following: UUG, UAG, AUG, AAG, UUA, UAA, AUA, AAA, IUG, IAG, IUA and IAA.

[0080] In some embodiments of the first to eighth aspects of this application, the aforementioned dsRNA or its conjugates, wherein the cleavage region further comprises nucleotide N1, wherein N1 is the third nucleotide from the 3' end of the 5' extension, and the cleavage region comprises the nucleotide sequence shown in formula (II).

[0081] Equation (II): (3'-5')X2-YZ-N1.

[0082] In some embodiments of the first to eighth aspects of this application, the above-mentioned dsRNA or its conjugates, wherein the formula (II) has a sequence (3'-5') selected from the following: AAGC, AAGA, AAGG, AAGU, UUGC, UUGA, UUGG, UUGU, AUGC, AUGA, AUGG, AUGU, UAGC, UAGA, UAGG, UAGU, IAGC, IAGA, IAGG, IAGU, IUGC, IUGA, IUGG, and IUGU.

[0083] In some embodiments of aspects one through eight of this application, the aforementioned dsRNA or its conjugates, wherein the cleavage region further comprises fragment N, fragment N comprising at least one nucleotide, wherein the nucleotide at the 3' end of fragment N is N1, and the cleavage region comprises the nucleotide sequence shown in formula (III).

[0084] Equation (III): (3'-5')X2-YZN,

[0085] The length of fragment N is 1-10 nucleotides, preferably 1-5 nucleotides, and more preferably 1 nucleotide.

[0086] In some embodiments of the first to eighth aspects of this application, the aforementioned dsRNA or its conjugates, wherein N1 is adenine nucleotide (A), guanine nucleotide (G), cytosine nucleotide (C), uracil nucleotide (U), their natural analogs or their non-natural analogs; preferably, N1 is cytosine nucleotide (C), a natural analog of cytosine nucleotide (C) or a non-natural analog of cytosine nucleotide (C).

[0087] In some embodiments of aspects 1 to 8 of this application, N1 is C or a natural or non-natural analogue thereof.

[0088] In some embodiments of the first to eighth aspects of this application, N1 is adenine nucleotide (A) or a natural or non-natural analog thereof, guanine nucleotide (G) or a natural or non-natural analog thereof, or uracil nucleotide (U) or a natural or non-natural analog thereof.

[0089] In some embodiments of the first to eighth aspects of this application, the above-mentioned dsRNA or its conjugates, wherein the formula (III) has a sequence (3'-5') selected from the following: AAGA, AAGG, UAGA, UAGG, IAGG, IAGC, and IAGA.

[0090] In some embodiments of the first to eighth aspects of this application, the above-mentioned dsRNA or its conjugates, wherein the formula (III) has a sequence (3'-5') selected from the following: AAGC, UAGC, and IAGC.

[0091] In some embodiments of the first to eighth aspects of this application, the above-mentioned dsRNA or its conjugates, wherein the formula (III) has a sequence (3'-5') selected from the following: AAGA, AAGG, UAGA, UAGG, IAGG, IAGC, and IAGA.

[0092] In some embodiments of aspects one through eight of this application, the aforementioned dsRNA or its conjugates, wherein...

[0093] (a) The cleavage region contains at least one nucleoside internucleotide bond that is not a thiophosphate bond;

[0094] (b) The internucleotide bond between X2 and Y is not a thiophosphate bond;

[0095] (c) The internucleotide bond between Y and Z is not a thiophosphate bond;

[0096] (d) All internucleotide bonds in the cleavage region are not phosphate thioester bonds;

[0097] (e) The cleavage region contains at least one phosphate diester bond between nucleosides;

[0098] (f) The internucleotide bond between X2 and Y is a phosphodiester bond;

[0099] (g)The internucleotide bond between Y and Z is a phosphodiester bond;

[0100] (h) All internucleotide bonds in the cleavage region are phosphodiester bonds; and / or

[0101] (i) The nucleoside bond between the cleavage region and the second chain is a phosphate diester bond.

[0102] In some embodiments of the first to eighth aspects of this application, the aforementioned dsRNA or its conjugates, wherein the cleavage region contains at least one modified nucleotide, preferably, all nucleotides in the cleavage region are modified nucleotides.

[0103] In some embodiments of aspects one through eight of this application, the aforementioned dsRNA or its conjugates, wherein...

[0104] (a) X2 is a nucleotide containing 2'-OMe modification;

[0105] (b) Y is a nucleotide containing 2'-OMe modification or 2'-F modification;

[0106] (c) The Z is a nucleotide containing a 2'-F modification; and / or

[0107] (d) The N1 is a nucleotide containing 2'-OMe modification or 2'-F modification.

[0108] In some embodiments of the first to eighth aspects of this application, the aforementioned dsRNA or its conjugates, wherein the cleavage region comprises a sequence (3'-5') selected from the following: iAfGfCf, iAfGfc, iuGfGf, iuGfAf, iuGfCf, iaGfGf, iaGfAf, iaGfCf, auGfAf, auGfUf, auGfGf, uaGfc, uaGfGf, uaGfCf, uaGfUf, uaGfAf, uuGfGf, uuGfAf, aaGfu, aaGfg, aaGfa, aaGfc, aaGfUf, aaGfGf, aaGfAf, and aaGfCf.

[0109] In some embodiments of the first to eighth aspects of this application, the aforementioned dsRNA or its conjugates, wherein the cleavage region comprises a sequence (3'-5') selected from the following: iuGfGf, iuGfAf, iuGfCf, iaGfGf, iaGfAf, iaGfCf, auGfAf, auGfUf, auGfGf, uaGfc, uaGfGf, uaGfCf, uaGfUf, uaGfAf, uuGfGf, uuGfAf, aaGfu, aaGfg, aaGfa, aaGfc, aaGfUf, aaGfGf, aaGfAf, and aaGfCf.

[0110] In some embodiments of the first to eighth aspects of this application, the aforementioned dsRNA or its conjugates, wherein the cleavage region comprises a sequence (3'-5') selected from the following: aaGfCf, aAfGfCf, aaGfc, aAfGfc, uaGfCf, uAfGfCf, uaGfc, uAfGfc, iaGfCf, iAfGfCf, iaGfc, iAfGfc, aaGfAf, aaGfGf, aaGfUf, uaGfAf, and uaGfGf.

[0111] In some embodiments of the first to eighth aspects of this application, the above-mentioned dsRNA or its conjugates, wherein the cleavage region comprises a sequence (3'-5') selected from the following: aaGfCf, aAfGfCf, uaGfCf, uAfGfCf, iaGfCf, and iAfGfCf.

[0112] In some embodiments of the first to eighth aspects of this application, the above-mentioned dsRNA or its conjugates, wherein the cleavage region comprises sequences (3'-5') selected from the following: aaGfCf and iaGfCf.

[0113] In some embodiments of the first to eighth aspects of this application, the above-mentioned dsRNA or its conjugates, wherein the cleavage region comprises a sequence (3'-5') selected from the following: aaGfc, aAfGfc, uaGfc, uAfGfc, iaGfc and iAfGfc.

[0114] In some embodiments of the first to eighth aspects of this application, the above-mentioned dsRNA or its conjugates, wherein the cleavage region comprises a sequence (3'-5') selected from the following: aaGfc.

[0115] In some embodiments of the first to eighth aspects of this application, the aforementioned dsRNA or its conjugates, wherein the antisense strand further comprises a blocking group at the 5' end; optionally, the blocking group is connected to the 5' end of the 5' extension.

[0116] In some embodiments of the first to eighth aspects of this application, the above-mentioned dsRNA or its conjugates, wherein the blocking group includes a debased nucleotide, a reverse debased nucleotide, MO3 or MO6.

[0117] In some embodiments of the first to eighth aspects of this application, the aforementioned dsRNA or its conjugates, wherein the blocking group is connected to the 5th end of the 5' extension via an internucleotide bond, said internucleotide bond being optionally modified or unmodified.

[0118] In some embodiments of the first to eighth aspects of this application, the aforementioned dsRNA or its conjugates, wherein the blocking group is connected to the 5' extended 5' end via a thiophosphate bond.

[0119] In some embodiments of the first to eighth aspects of this application, the aforementioned dsRNA or its conjugates, wherein a blunt end is generated at the 5' end of the second strand after cleavage of the cleavage region.

[0120] In some embodiments of the first to eighth aspects of this application, the aforementioned dsRNA or its conjugates are wherein the first strand and the second strand are completely reverse complementary.

[0121] In a ninth aspect, this application also provides dsRNAs as shown in Tables 1-1, 1-2, and 2-1 to 2-8.

[0122] Table 1-1 dsRNA

[0123] Table 1-2 Modified dsRNA

[0124] In a tenth aspect, this application also provides dsRNA conjugates as shown in Tables 2-1 to 2-8.

[0125] Table 2-1 dsRNA conjugates (target: LPA)

[0126] Table 2-2 dsRNA conjugates (target: ANGPTL3)

[0127] Table 2-3 dsRNA conjugates (target: CFB)

[0128] Table 2-4 dsRNA conjugates (target: DGAT2)

[0129] Table 2-5 dsRNA conjugates (target: INHBE)

[0130] Table 2-6 dsRNA conjugates (target: AGT)

[0131] Table 2-7 dsRNA conjugates (target: APP)

[0132] Table 2-8 dsRNA conjugates (target: PCSK9)

[0133] Eleventhly, the present invention also provides a dsRNA comprising a sense strand and an antisense strand, wherein the length of the sense strand and the antisense strand are each independently 17-35 nucleotides, wherein:

[0134] The positive chain contains or has sequences as shown in SEQ ID NO:29, 32, 33, 1-7, 9-13, 30-31, 34-43, 46-55, 89 or 182; and / or the negative chain contains or has sequences as shown in SEQ ID NO:78, 94, 14-26, 28, 56-77, 79-82, 84, 87-93, 95-98, 183-190, 208 or 209;

[0135] The justice chain comprises a first chain, and the antisense chain comprises a second chain; the first chain and the second chain are opposite and complementary.

[0136] The antisense chain also includes a 3' extension and a 5' extension.

[0137] The 3' extension is connected to the 3' end of the second strand and has a length of 0-5 nucleotides.

[0138] The 5' extension is connected to the 5' end of the second strand and is at least 3 nucleotides in length.

[0139] The dsRNA can be cleaved between the 3' end nucleotide of the 5' extension and the 5' end nucleotide of the second strand to obtain cleaved dsRNA.

[0140] The cleaved dsRNA contains a first strand and a second strand and is capable of silencing the expression of target genes through RNA interference.

[0141] In some embodiments of the eleventh aspect, the present invention also provides a dsRNA comprising a sense strand and an antisense strand, the sense strand and the antisense strand being independently 17 to 35 nucleotides in length, the nucleotides being independently and optionally modified, wherein the sense strand comprises a first strand, the antisense strand comprises a second strand, the first strand and the second strand being anticomplementary, the antisense strand further comprising a 3' extension and a 5' extension, the 3' extension being connected to the 3' end of the second strand and having a length of 0-5 nucleotides, the 5' extension being connected to the 5' end of the second strand and having a length of at least 3 nucleotides, the dsRNA being cleaved between the 3' end nucleotide of the 5' extension and the 5' end nucleotide of the second strand, the resulting cleavage product comprising the first strand and the second strand being able to silence gene expression through RNA interference.

[0142] In a twelfth aspect, the present invention also provides a dsRNA conjugate comprising the dsRNA and delivery system described in the eleventh aspect.

[0143] In some embodiments of the twelfth aspect of the present invention, the above-described delivery system includes a delivery system targeting the liver and a delivery system targeting extrahepatic tissues.

[0144] In some embodiments of the twelfth aspect of the present invention, the delivery system comprises a ligand delivery system targeting ASGPR, a ligand delivery system targeting transferrin receptors, a ligand delivery system targeting Apelin receptors, a ligand delivery system targeting CD206, a ligand delivery system targeting integrin receptors, or a ligand delivery system targeting low-density lipoprotein-related proteins.

[0145] In some embodiments of the twelfth aspect of the present invention, the 3' end nucleotide of the aforementioned positive strand is linked to the delivery system.

[0146] In some embodiments of the twelfth aspect of the present invention, the above-mentioned delivery system is L96.

[0147] In some embodiments of the twelfth aspect of the present invention, the residues of the 3' end nucleotide of the positive strand are connected to the L96 residue by a phosphate ester bond.

[0148] In some embodiments of the twelfth aspect of the present invention, the above-mentioned delivery system is NAG37.

[0149] In some embodiments of the twelfth aspect of the present invention, the 5' end nucleotide residue of the aforementioned positive strand is linked to an inverted abasic nucleotide residue via a thiophosphate bond, the inverted abasic nucleotide being linked to a delivery system, and the 1st and 2nd nucleotide residues of the positive strand are linked via a thiophosphate bond, starting from the 3' end of the positive strand.

[0150] In some embodiments of the twelfth aspect of the present invention, the aforementioned inverted abasic nucleotide residue is linked to the NAG37 residue via a thiophosphate bond.

[0151] In some embodiments of the eleventh and twelfth aspects of the present invention, the above modifications are each independently selected from one, two or three combinations of the following: 2'-OMe modification, 2'-F modification, 2'-deoxygenation modification, VP modification, 5'-MP modification, PS modification, PS2 modification, MP modification, MOP modification, invAb modification, and invAB modification.

[0152] In some embodiments of the eleventh and twelfth aspects of the present invention, the above-mentioned dsRNA or its conjugates, wherein the antisense strand comprises a cleavage region comprising the nucleotide sequence shown in formula (I),

[0153] Equation (I): (3'-5')X²-YZ

[0154] The cleavage occurs between X2 and Y, where X2 is the 5' end nucleotide of the second strand, and Y and Z are the two nucleotides at the 3' end of the 5' extension, where Z is guanine nucleotide (G), a natural analog of guanine nucleotide (G), a non-natural analog of guanine nucleotide (G), adenine nucleotide (A), a natural analog of adenine nucleotide (A), or a non-natural analog of adenine nucleotide (A).

[0155] In some embodiments of the eleventh and twelfth aspects of the present invention, the above-mentioned dsRNA or its conjugate, wherein Z is guanine nucleotide (G), a natural analog of guanine nucleotide (G) or a non-natural analog of guanine nucleotide (G).

[0156] In some embodiments of the eleventh and twelfth aspects of the present invention, the above-mentioned dsRNA or its conjugate, wherein the X2 is adenine nucleotide (A), a natural analog of adenine nucleotide (A), a non-natural analog of adenine nucleotide (A), uracil nucleotide (U), a natural analog of uracil nucleotide (U), or a non-natural analog of uracil nucleotide (U).

[0157] In some embodiments of the eleventh and twelfth aspects of the present invention, the above-mentioned dsRNA or its conjugates, wherein the formula (I) has a sequence (3'-5') selected from the following: UUG, UAG, AUG, AAG, UUA, UAA, AUA, AAA, UCG, UGG, ACG, AGG, UCA, UGA, ACA and AGA, or natural or non-natural analogs thereof.

[0158] In some embodiments of the eleventh and twelfth aspects of the present invention, the above-mentioned dsRNA or its conjugate, wherein the Y is adenine nucleotide (A), a natural analog of adenine nucleotide (A), a non-natural analog of adenine nucleotide (A), uracil nucleotide (U), a natural analog of uracil nucleotide (U), or a non-natural analog of uracil nucleotide (U).

[0159] In some embodiments of the eleventh and twelfth aspects of the present invention, the above-mentioned dsRNA or its conjugates, wherein the formula (I) has a sequence (3'-5') selected from the following: UUG, UAG, AUG, AAG, UUA, UAA, AUA, AAA, IAG, IUG, IAA and IUA.

[0160] In some embodiments of the eleventh and twelfth aspects of the present invention, the above-mentioned dsRNA or its conjugates, wherein the formula (I) has a sequence (3'-5') selected from the following: IAG, IUG, IAA and IUA.

[0161] In some embodiments of the eleventh and twelfth aspects of the present invention, the above-mentioned dsRNA or its conjugates, wherein the formula (I) has a sequence (3'-5') selected from the following: UUG, UAG, AUG, AAG, UUA, UAA, AUA and AAA.

[0162] In some embodiments of the eleventh and twelfth aspects of the present invention, the above-mentioned dsRNA or its conjugates, wherein the formula (I) has a sequence (3'-5') selected from the following: UUG, UAG, AUG, AAG, IAG and IUG.

[0163] In some embodiments of the eleventh and twelfth aspects of the present invention, the above-mentioned dsRNA or its conjugates, wherein the formula (I) has a sequence (3'-5') selected from the following: UUG, UAG, AUG and AAG.

[0164] In some embodiments of the eleventh and twelfth aspects of the present invention, the above-mentioned dsRNA or its conjugates, wherein the formula (I) has a sequence (3'-5') selected from the following: IAG and IUG.

[0165] In some embodiments of the eleventh and twelfth aspects of the present invention, the aforementioned dsRNA or its conjugates, wherein the cleavage region further comprises nucleotide N1, wherein N1 is the third nucleotide from the 3' end of the 5' extension, and the cleavage region comprises the nucleotide sequence shown in formula (II).

[0166] Equation (II): (3'-5')X2-YZ-N1.

[0167] In some embodiments of the eleventh and twelfth aspects of the present invention, the aforementioned dsRNA or its conjugates, wherein the cleavage region further comprises fragment N, fragment N comprising at least one nucleotide, wherein the nucleotide at the 3' end of fragment N is N1, and the cleavage region comprises the nucleotide sequence shown in formula (III).

[0168] Equation (III): (3'-5')X2-YZN,

[0169] The length of fragment N is 1-10 nucleotides, preferably 1-5 nucleotides, and more preferably 1 nucleotide.

[0170] In some embodiments of the eleventh and twelfth aspects of the present invention, the above-mentioned dsRNA or its conjugate, wherein N1 is adenine nucleotide (A), guanine nucleotide (G), cytosine nucleotide (C), uracil nucleotide (U), their natural analogs or their non-natural analogs; preferably, N1 is cytosine nucleotide (C), a natural analog of cytosine nucleotide (C) or a non-natural analog of cytosine nucleotide (C).

[0171] In some embodiments of the eleventh and twelfth aspects of the present invention, the above-mentioned dsRNA or its conjugates, wherein the formula (III) has a sequence (3'-5') selected from the following: AAGC, AAGA, AAGG, AAGU, UUGC, UUGA, UUGG, UUGU, AUGC, AUGA, AUGG, AUGU, UAGC, UAGA, UAGG, UAGU, IAGC, IAGA, IAGG, IAGU, IUGC, IUGA, IUGG, and IUGU.

[0172] In some embodiments of the eleventh and twelfth aspects of the present invention, the above-mentioned dsRNA or its conjugates, wherein the formula (III) has a sequence (3'-5') selected from the following: AAGC, AAGA, AAGG, AAGU, UUGA, UUGG, AUGA, AUGG, UAGC, UAGA, UAGG, UAGU, IAGGC, IAGA, IAGG, IUGC, IUGA and IUGG.

[0173] In some embodiments of the eleventh and twelfth aspects of the present invention, the above-mentioned dsRNA or its conjugates, wherein the formula (III) has a sequence (3'-5') selected from the following: AAGC and UAGC.

[0174] In some embodiments of the eleventh and twelfth aspects of the present invention, the above-mentioned dsRNA or its conjugate, wherein,

[0175] (a) The cleavage region contains at least one nucleoside internucleotide bond that is not a thiophosphate bond;

[0176] (b) The internucleotide bond between X2 and Y is not a thiophosphate bond;

[0177] (c) The internucleotide bond between Y and Z is not a thiophosphate bond;

[0178] (d) All internucleotide bonds in the cleavage region are not phosphate thioester bonds;

[0179] (e) The cleavage region contains at least one phosphate diester bond between nucleosides;

[0180] (f) The internucleotide bond between X2 and Y is a phosphodiester bond;

[0181] (g)The internucleotide bond between Y and Z is a phosphodiester bond;

[0182] (h) All internucleotide bonds in the cleavage region are phosphodiester bonds; and / or

[0183] (i) The nucleoside bond between the cleavage region and the second chain is a phosphate diester bond.

[0184] In some embodiments of the eleventh and twelfth aspects of the present invention, the above-mentioned dsRNA or its conjugates, wherein the cleavage region contains at least one modified nucleotide, preferably, all nucleotides in the cleavage region are modified nucleotides.

[0185] In some embodiments of the eleventh and twelfth aspects of the present invention, the above-mentioned dsRNA or its conjugate, wherein,

[0186] (a) X2 is a nucleotide containing 2'-OMe modification;

[0187] (b) Y is a nucleotide containing 2'-OMe modification or 2'-F modification;

[0188] (c) The Z is a nucleotide containing a 2'-F modification; and / or

[0189] (d) The N1 is a nucleotide containing 2'-OMe modification or 2'-F modification.

[0190] In some embodiments of the eleventh and twelfth aspects of the present invention, the above-mentioned dsRNA or its conjugates, wherein the cleavage region comprises a sequence (3'-5') selected from the following: aaGfCf, aAfGfCf, aaGfc, aAfGfc, uaGfCf, uAfGfCf, uaGfc and uAfGfc.

[0191] In some embodiments of the eleventh and twelfth aspects of the present invention, the above-mentioned dsRNA or its conjugates, wherein the cleavage region comprises a sequence (3'-5') selected from the following: aaGfCf, aAfGfCf, uaGfCf and uAfGfCf.

[0192] In some embodiments of the eleventh and twelfth aspects of the present invention, the above-mentioned dsRNA or its conjugates, wherein the cleavage region comprises a sequence (3'-5') selected from the following: aaGfCf.

[0193] In some embodiments of the eleventh and twelfth aspects of the present invention, the above-mentioned dsRNA or its conjugates, wherein the cleavage region comprises a sequence (3'-5') selected from the following: aaGfc, aAfGfc, uaGfc and uAfGfc.

[0194] In some embodiments of the eleventh and twelfth aspects of the present invention, the above-mentioned dsRNA or its conjugates, wherein the cleavage region comprises a sequence (3'-5') selected from the following: aaGfc.

[0195] In some embodiments of the eleventh and twelfth aspects of the present invention, the above-mentioned dsRNA or its conjugates, wherein the antisense strand further comprises a blocking group at the 5' end; optionally, the blocking group is connected to the 5' end of the 5' extension.

[0196] In some embodiments of the eleventh and twelfth aspects of the present invention, the above-mentioned dsRNA or its conjugates, wherein the blocking group includes a debased nucleotide, reverse debased nucleotide, MO3 or MO6.

[0197] In some embodiments of the eleventh and twelfth aspects of the present invention, the above-mentioned dsRNA or its conjugates are wherein the blocking group is connected to the 5th end of the 5' extension via an internucleotide bond, the internucleotide bond being optionally modified or unmodified.

[0198] In some embodiments of the eleventh and twelfth aspects of the present invention, the above-mentioned dsRNA or its conjugates are wherein the blocking group is connected to the 5' extended 5' end via a thiophosphate bond.

[0199] In some embodiments of the eleventh and twelfth aspects of the present invention, the above-mentioned dsRNA or its conjugates are wherein a blunt end is generated at the 5' end of the second strand after cleavage of the cleavage region.

[0200] In some embodiments of the eleventh and twelfth aspects of the present invention, the above-mentioned dsRNA or its conjugates are wherein the first strand and the second strand are completely reverse complementary.

[0201] Thirteenth aspect: This application also provides a composition comprising some of the solutions described in aspects one through twelfth above, and a pharmaceutically acceptable carrier.

[0202] In its fourteenth aspect, this application also provides some of the schemes described in aspects one through thirteen above for use in preparing treatments for diseases related to gene silencing mechanisms.

[0203] In some embodiments of the fourteenth aspect of the present invention, the above-mentioned gene silencing mechanism is an RNA interference mechanism.

[0204] In some embodiments of the first to fourteenth aspects of this application, the structural relationship of the first strand, second strand, 3' extension and 5' extension is shown in Figure 7 and / or Figure 8, and the dsRNA or its conjugate is cleaved at the position indicated by ▲.

[0205] Some of the solutions in this application are derived from any combination of the above variables or solutions.

[0206] As is known in the art, ribonucleotides have three free hydroxyl groups on their sugar ring, which can form three different nucleotides: 2'-ribonucleotide, 3'-ribonucleotide, and 5'-ribonucleotide. Deoxynucleotides have two free hydroxyl groups on their sugar ring, which can form two nucleotides: 3'-deoxyribonucleotide and 5'-deoxyribonucleotide. Therefore, all other embodiments obtained by those skilled in the art based on the embodiments of this application without inventive effort are considered technical solutions of this application and fall within the protection scope of this application. Beneficial effects

[0207] The dsRNA or its conjugates provided in this application can significantly or enhance the level of intracellular target mRNAs.

[0208] definition

[0209] Unless otherwise specified, the following terms and phrases used in this application are intended to have the following meanings. A particular term or phrase should not be considered uncertain or unclear unless specifically defined, but should be understood in its ordinary sense. When a trade name appears herein, it is intended to refer to the corresponding product or its active ingredient.

[0210] As used herein, unless otherwise stated herein or explicitly contradicted by the context, the terms “a / an”, “the”, and similar terms used in the context of this invention (especially in the context of the claims) shall be interpreted to cover both the singular and the plural.

[0211] In this document, the reference to "about" a value or parameter includes (and describes) the implementation of said value or parameter itself. For example, a description of "about X" includes a description of "X". Numerical ranges include numbers that define a range. Generally, the term "about" refers to the indicated value of a variable and all values ​​of the variable that are within the experimental error range of the indicated value (e.g., within the 95% confidence interval of the mean) or within 10% of the indicated value (whichever is greater).

[0212] As used herein, the terms "optional," "optionally," or "optionally" are equivalent in meaning, indicating that the event or condition described thereafter may or may not occur, and the description includes both the possibility that the event or condition occurs and the possibility that it does not occur. For example, "optionally substituted alkyl" or "alkyl optionally substituted" includes "alkyl" (where the H on the alkyl group is not substituted / replaced by a non-H substituent) and "substituted alkyl" (where the H on the alkyl group is substituted / replaced by a non-H substituent). As used herein, those skilled in the art will understand that for any group comprising one or more substituents, these groups are not intended to introduce any substitution or substitution pattern that is spatially impractical, synthetically infeasible, and / or inherently unstable. For example, "optionally modified" includes both unmodified and modified, and further, "nucleotide optionally modified" includes both unmodified and modified nucleotides.

[0213] As used herein, when any variable (e.g., a substituent R, such as a nucleic acid being modified) appears more than once in the composition or structure of a compound, its definition is independent in each case. For example, if a group is substituted by 0-2 Rs, the group may optionally be substituted by at most two Rs, and the Rs in each case have independent options. As another example, when multiple nucleotides are modified, each nucleotide is independently and optionally modified, and the type and number of modifications to each nucleotide may be the same or different.

[0214] As used herein, unless otherwise stated, “comprising,” “including,” “at least,” “having,” “having,” “containing,” or equivalents are open-ended expressions that mean that in addition to the elements, components, or steps listed, other unspecified elements, components, steps, functions, effects, or functions may be included.

[0215] As used herein, the term “symptom” refers to any disease, condition, or ailment that impairs the normal functioning of an object (e.g., a person).

[0216] As used herein, the term "nucleotide" refers to a pentose sugar (ribose or deoxyribose), a phosphate group, and a base (natural or non-natural), and is intended to include both unmodified (i.e., natural) nucleotides and modified nucleotides. In some embodiments, the nucleotide is an unmodified ribonucleotide. In some embodiments, the ribonucleotide is a 3'-ribonucleotide. In some embodiments, the ribonucleotide is a 5'-ribonucleotide. In some embodiments, the modified or unmodified nucleotide may optionally be further modified.

[0217] Natural nucleotides are composed of natural bases, natural ribose, and phosphate. The natural nucleotides used in this article refer to adenine nucleotide ribonucleotides, adenine nucleotide deoxyribonucleotides, guanine nucleotide ribonucleotides, guanine nucleotide deoxyribonucleotides, cytosine nucleotide ribonucleotides, cytosine nucleotide deoxyribonucleotides, uracil nucleotide ribonucleotides, thymine ribonucleotides, or thymine deoxyribonucleotides. "Ribonucleotide" refers to a nucleotide having a hydroxyl group at the 2' position of its sugar moiety. "Deoxyribonucleoside" refers to a nucleotide having a hydrogen atom at the 2' position of its sugar moiety.

[0218] The natural bases of RNA include A (adenine nucleotide), G (guanine nucleotide), C (cytosine nucleotide), U (uracil nucleotide), and T (thymine).

[0219] Nucleotides can be substituted with their analogues, including both natural and non-natural analogues. Examples of guanosine analogues include, but are not limited to, 6-thioguanosine, 8-azaguanosine, 8-oxoguanosine, and 2-aminopurine nucleoside. Examples of adenosine analogues include, but are not limited to, cordycepin (3′-deoxyadenosine), n6-benzyladenosine, and 2-chloroadenosine. Examples of cytidine analogues include, but are not limited to, gemcitabine (2',2'-difluoro-2'-deoxycytidine), cytarabine (1-β-d-arabinoseureidocytosine nucleotide), and decitabine (5-aza-2'-deoxycytidine). Examples of uracil nucleotide analogues include, but are not limited to, 5-fluorouracil nucleotide, pseudouracil nucleotide, 5-bromouracil nucleotide, 4-thiouracil nucleotide, and 5-azidouracil nucleotide.

[0220] As used herein, “ribonucleic acid” (RNA) is the carrier of genetic information found in cells and some viruses and viroids. RNA is a long chain molecule formed by ribonucleotides linked by nucleotide bonds, including single-stranded RNA and double-stranded RNA. The natural nucleotide bonds (or nucleoside bonds) are phosphodiester bonds. In some embodiments, the ribose, bases, and nucleotide bonds in RNA may be modified independently and optionally.

[0221] As used herein, "double-stranded ribonucleic acid" is a complex consisting of two nucleic acid strands bonded together from natural or non-natural nucleotides, similar in structure and function to natural ribonucleic acid. These two nucleic acid strands contain antiparallel and substantially complementary sequences. "Substantially complementary" means that, while maintaining function, the antiparallel regions of the two nucleic acid strands may contain a certain number of base mismatches and non-matches. In some embodiments, the certain number refers to 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some embodiments, the certain number refers to 1, 2, 3, 4, or 5. In some embodiments, the certain number refers to 1, 2, or 3.

[0222] As used herein, the term “oligonucleotide” refers to a nucleic acid molecule (RNA or DNA) that is, for example, less than 100, 200, 300, or 400 nucleotides in length.

[0223] As used herein, “bonding” refers to the connection between residues of two monomers (such as nucleotides) (such as nucleosides) via a single bond or a group (such as a phosphodiester bond, thiophosphate bond, or dithiophosphate bond). In some embodiments, the bonding refers to the connection between residues of two nucleotides via a phosphodiester bond, thiophosphate bond, or dithiophosphate bond.

[0224] As used herein, the terms “nucleotide bond” or “nucleoside bond” have the same meaning and refer to a bond (e.g., a link or linking group) between two parts (e.g., between two monomers) of the oligonucleotide disclosed herein, including bonds between nucleotides, between a nucleotide and a ligand, between a nucleotide and a capping group, and between a nucleotide and a baseless nucleotide of the oligonucleotide disclosed herein.

[0225] As used herein, a "monomer" is a class of compounds that can be assembled into a ribonucleic acid chain and perform a certain function. As used herein, a "monomer" includes, but is not limited to, natural nucleotides, non-natural nucleotides (e.g., modified nucleotides, nucleotide analogs, inverted non-base deoxynucleotides, GNA, LNA, etc.), and M06.

[0226] As used herein, “inhibition” means that, when a given gene is expressed, gene expression is reduced when that cell, cell population, or tissue is treated with the single / double-stranded RNA, single / double-stranded RNA conjugate, or a pharmaceutical composition comprising one or more of these as described in this application, compared to cells, cell populations, or tissues that have not been treated in this way. The terms “inhibition,” “reduction,” “silencing,” “downregulation,” “suppression,” and other similar terms used herein are used interchangeably and include any level of inhibition. Preferably, inhibition includes statistically significant inhibition or clinically significant inhibition.

[0227] As used herein, "conjugation" refers to the covalent connection between two or more chemical parts, each with a specific function; correspondingly, "conjugated compound" refers to a compound formed by the covalent connection of these chemical parts. For example, "double-stranded ribonucleic acid conjugation" refers to a compound or complex formed by covalently linking one or more chemical parts with specific functions (such as a delivery system, ligand group, or conjugation group) to a double-stranded ribonucleic acid. In some embodiments, the delivery system, ligand group, or conjugation group may be attached to a phosphate group, a sugar ring (including the delivery system, ligand group, or conjugation group covalently linked to an atom at the 3' or 5' position of the nucleotide via a phosphodiester bond), a 2'-hydroxyl group, a 5'-hydroxyl group, or a base of any nucleotide of the double-stranded ribonucleic acid. In some embodiments, the delivery system, ligand group, or conjugation group may also be attached to the 2' position of the nucleotide, in which case the nucleotides are connected by a 2'-5' phosphodiester bond. In some embodiments, the delivery system, ligand group, or conjugate group may also be attached to the 3'-position of the nucleotide, in which case the nucleotides are linked by a 3'-5' phosphodiester bond.

[0228] As used herein, "complementary" or "anti-complementary" may be used interchangeably to refer to a structural relationship between two nucleotides (e.g., on two opposing nucleic acid chains or on opposing regions of a single nucleic acid chain) that allows the two nucleotides to form base pairs with each other (e.g., a purine nucleotide of a nucleic acid complementary to a pyrimidine nucleotide of an opposing nucleic acid may form a base pair together by forming hydrogen bonds with each other). In some embodiments of this application, complementary nucleotides may form base pairs in a Watson-Crick manner or in any other manner that allows for the formation of a stable double helix. In some embodiments of this application, the two nucleic acid chains may have multiple regions forming complementary double helixes. In some embodiments of this application, in DNA, adenine nucleotide (A) always pairs with thymine (T), and in RNA, adenine nucleotide (A) pairs with uracil nucleotide (U); guanine nucleotide (G) always pairs with cytosine nucleotide (C). In some embodiments of this application, the complementary nucleotide may also comprise 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 including, but not limited to, G:U swing base pairs or Hoogstein base pairs. In some embodiments of this application, nucleotides containing hypoxanthine as their base may pair with nucleotide bases containing adenine, cytosine, or uracil. In some embodiments of this application, nucleotides containing uracil, guanine, or adenine may be replaced in the nucleotide sequence of this application by nucleotides containing, for example, inosine (in this application, the capital letter "I" can represent a hypoxanthine base, inosine, or an inosine-containing nucleotide, depending on the context) (this replacement is referred to as "I modification"). In some embodiments of this application, adenine nucleotides and cytosine nucleotides anywhere in the oligonucleotide can be replaced with guanine nucleotides and uracil nucleotides, respectively, to form a GU swing base pair with the target mRNA.

[0229] The degree of complementarity between two oligonucleotides is called complementarity, which is measured by the percentage of bases in each strand that can form hydrogen bonds with each other, determined by established base pairing rules. Oligonucleotide sequences do not need to be "perfectly complementary" (i.e., "completely complementary") to their corresponding nucleic acid sequences. In some embodiments, a first nucleotide sequence is considered complementary to a second nucleotide sequence if the first nucleotide sequence exhibits at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence complementarity. In one exemplary embodiment, 18 of the 20 nucleobases of the first nucleotide sequence pair with corresponding regions of the second nucleotide sequence, achieving 90% complementarity. Non-complementary nucleobases, also known as "mismatches," may cluster or spread between complementary bases and do not need to be adjacent to each other or adjacent to complementary nucleobases.

[0230] The term "mismatch" as used in this article includes, but is not limited to:

[0231] 1) Two opposing (independent natural or non-natural) nucleotides (other than AT, AU or GC) pairing;

[0232] 2) No hydrogen bonds are formed between two opposing (independent natural or non-natural) nucleotides;

[0233] 3) A base is missing between two opposing (independent natural or non-natural) nucleotides.

[0234] In some embodiments, mismatches include wobbly base pairing and Hoogstein base pairing.

[0235] The term "fully complementary" refers to a hybrid formed by the first and second nucleotide sequences in a fully complementary region consisting only of Watson-Crick base pairs. "Fully complementary" oligonucleotides may include internal regions (e.g., at least 7, 8, 9, or 10 nucleotides) that are fully complementary to the target RNA. In some embodiments, the targeting region (e.g., the antisense strand or the second strand) provided herein is fully complementary to a portion of the target mRNA encoding the target gene. In some embodiments, the targeting region has at least 80%, 85%, 90%, or 95% complementarity (fully complementary) to a portion of the target mRNA encoding the target gene. In some embodiments, the targeting region has 100% complementarity (fully complementary) to a portion of the target mRNA encoding the target gene.

[0236] As used herein, a blocking group is a group that can be conjugated to an oligonucleotide provided herein, for example, at the 5' end of the antisense strand, which can reduce or inhibit exonuclease cleavage. In some embodiments, the blocking group can reduce or inhibit the RNA interference effect of the oligonucleotide. In some embodiments, the blocking group is cleaved from the oligonucleotide before providing the RNA interference effect. Examples of blocking groups include, but are not limited to, non-basic nucleotide residues, reverse non-basic nucleotide residues (such as InvAB or InvAb), MO3, and MO6.

[0237] As used herein, the double-stranded nucleotide reagent may optionally be conjugated to one or more blocking groups. The blocking group may be attached to the sense strand, antisense strand, or both strands at the 3' end, 5' end, or both ends. In some embodiments, the blocking group is conjugated to the antisense strand, specifically to the 5' end of the antisense strand. In some embodiments, the blocking group is conjugated to an oligonucleotide (e.g., the 5' end of the antisense strand) via a nucleotide bond, and the internucleotide bond is optionally modified as described herein. In some embodiments, the blocking group is linked to the double-stranded nucleotide reagent via a phosphate thioester. In some embodiments, the blocking group is linked to the double-stranded nucleotide reagent via a phosphodiester bond.

[0238] As used herein, the term "modified nucleotide" refers to a nucleotide having modified internucleotide bonds, and / or modified bases, and / or modified sugars. In some embodiments, the modified nucleotide comprises one, two, three, or more modifications. In some embodiments, the nucleotide comprises one modification. In some embodiments, the nucleotide comprises two modifications. In some embodiments, the nucleotide comprises three modifications.

[0239] As used herein, “modification” of nucleotides includes, but is not limited to: 2'-OMe (2'-O-methyl) modification, 2'-F (2'-deoxy-2'-fluorine) modification, 2'-O-MOE (2'-O-methoxyethyl) modification, 2'-deoxy (2'-d) modification, 5'-morpholine (5'-Mo) modification, 2'-O-hexadecyl (C16) modification, unlocking nucleic acid (UNA) modification, and replacing nucleotides with glycerol nucleic acids (ethylene glycol nucleic acids (GNA)). Modifications (including but not limited to (S)-GNA modification), locked nucleic acid (LNA) modification, tricyclic DNA (tcDNA) modification, (S)-restricted ethyl bicyclic nucleic acid ((S)-cEt-BNA) modification, phosphate thioester (PS) modification, phosphate dithioester (PS2) modification, methylphosphonate (MP) modification, methoxypropyl methylphosphonate (MOP) modification, phosphoselenate modification, phosphodiselenate modification, phosphorylaminosulfate modification, phosphorylamino acid salt modification, and phosphoramidic acid. Salt modification, peptide nucleic acid (PNA) modification, 5'-(E)-vinyl phosphate (VP) modification, N6-methyladenosine (m6A) modification, 5-methylcytidine (m5C) modification, 3-methyluridine nucleotide nucleoside (m3U) modification, 5-methylureaside (m5U) modification, pseudoureaside modification, 2-thioureaside (s2U) modification, propyneuridine nucleotide nucleoside (5-pU) modification, and linking the 5' or 3' end of a nucleotide to an inverted abase-free nucleotide (i). Modifications include nvAB, replacing the nucleotide with an inverted abase-free nucleotide (invAb), replacing the nucleotide with 2,4-difluorotolyl ribonucleotide (rF), replacing the nucleotide with hypoxanthine nucleotide (I), replacing the nucleotide base with xanthine, replacing the nucleotide base with 7-methylguanine nucleotide, replacing the nucleotide base with 5,6-dihydrouracil nucleotide, and linking the 5' or 3' end of the nucleotide to M06 (M06) modification. In some embodiments, at least one nucleotide contains one, two, three, or more modifications. In some embodiments, at least one nucleotide is not modified. In some embodiments, at least one nucleotide contains one modification. In some embodiments, at least one nucleotide contains two modifications. In some embodiments, at least one nucleotide contains three modifications. In some embodiments, all nucleotides are modified, and each nucleotide independently contains one, two, or three modifications.

[0240] As used herein, in some embodiments, "G", "A", "C", "U", "T" and "I" refer to guanine ribonucleotide, cytosine ribonucleotide, adenine ribonucleotide, thymine ribonucleotide, uracil ribonucleotide, and hypoxanthine ribonucleotide, respectively. Exemplary structures are as follows:

[0241] As used herein, some embodiments were synthesized according to phosphorous amide solid-phase synthesis technology, wherein the structure is as follows when "G", "A", "C", "U", "T" and "I" are connected at the 5' end 1 position of the chain.

[0242] When “G”, “A”, “C”, “U”, “T” and “I” are connected at the 3' end of the chain, the structure is as follows:

[0243] As used herein, the prefix "d" before a monomer (such as nucleotides A, U, C, G, T, and I) indicates that the monomer is 2'-deoxy modified. An example nucleotide structure with 2'-deoxy modification is shown below:

[0244] As used herein, the label "f" following a monomer (such as nucleotides A, U, C, G, T, and I) indicates that the monomer is 2'-deoxy-2'-fluorine modified (2'-F modified). An example nucleotide structure with 2'-F modification is shown below:

[0245] As used herein, the prefix "GNA-" before monomers (such as nucleotides A, U, C, G, T, and I) indicates that the monomer has been modified with ethylene glycol-modified nucleic acid (GNA modification). As used herein, Tgn is the abbreviation for GNA-T, with the same meaning. An example of a GNA-modified nucleotide structure is shown below:

[0246] As used herein, lowercase letters (a, u, c, g, t, and i, etc.) indicate that the nucleotide represented by the corresponding uppercase letter (A, U, C, G, T, and I, etc.) is modified with 2'-O-methyl (2'-OMe). An example nucleotide structure modified with 2'-OMe is shown below:

[0247] As used herein, invAB modification refers to the attachment of an inverted, baseless deoxynucleotide to a monomer (e.g., at the 5' or 3' end of the nucleotide). For example, The structure modified by invAB:

[0248] As used herein, invAb modification refers to the replacement of a monomer (such as a nucleotide) with an inverted, non-basic nucleotide (invAb). For example, The structure modified by invAb:

[0249] As used herein, VP modification refers to the modification of a monomer by (E)-vinyl phosphate (e.g., modification of the 5' position of a nucleotide by 5'-(E)-vinyl phosphate). For example, the structures of U, u, and dU after modification are as follows:

[0250] As used in this article, Uhd represents 2'-O-C16 alkyl-modified uracil ribonucleotide:

[0251] As used in this article, M06 modification refers to the bonding of a linker at the 5' or 3' end of a monomer (such as a nucleotide). For example, Structure modified by M06: In some embodiments, M06 is through It is bonded to a nucleotide.

[0252] As used in this article, marking "*" between monomers (such as nucleotides A, U, C, G, T and I, ligands, blocking groups, etc.) indicates that the two monomers are linked by a thiophosphate bond (i.e., a thiophosphate diester bond), that is, modified by thiophosphate (PS).

[0253] As used in this article, the absence of an asterisk (*) between nucleotides (A, U, C, G, T, and I, etc.) indicates that the two nucleotides are linked by a phosphate ester bond (i.e., a phosphodiester bond).

[0254] For example, “5'-A(dU)gCf*(dT)-3'” means that the sequence starts from the 5' end, with adenine nucleotide ribonucleotide at position 1, uracil nucleotide deoxyribonucleotide at position 2, guanine nucleotide ribonucleotide modified with 2'-methoxy at position 3, cytosine nucleotide ribonucleotide modified with 2'-fluorine at position 4, and thymine deoxyribonucleotide linked to position 4 by a thiophosphate bond at position 5.

[0255] For example, the structure shown as “5'-(M06)*A(dT)gCf*(invAB)-3'” is as follows:

[0256] For example, the structure shown as “5'-(M06)*A(dT)gCf*[L96]-3'” is as follows:

[0257] As used in this article, the structures of the NAG37 monomer and the L96 monomer are as follows:

[0258] [NAG37] and [L96] represent its residues, respectively. For example, the sequence [NAG37]AfGfu*[L96] has the following structure:

[0259] This article also provides the following ligands: VSDL-01, VSDL-02, VSDL-03, VSDL-04, VSDL-05, VSDL-06, VSDL-07, VSDL-09, VSDL-10, VSDL-12, and VSDL-13. (VSDL-01), (VSDL-02), (VSDL-03), (VSDL-04), (VSDL-05), (VSDL-06), (VSDL-07), (VSDL-09), (VSDL-10), (VSDL-12), and (VSDL-13) represent their respective residues.

[0260] As used herein, “small interfering RNA,” “siRNA,” or “iRNA agent” can be used interchangeably to describe single-stranded or double-stranded ribonucleic acid molecules that inhibit gene expression by specifically binding to the sequence of a target mRNA, triggering an RNA interference (RNAi) mechanism that leads to the degradation of the target mRNA. (For example, siRNA agents or their cleavage products can downregulate target genes by, for example, inducing RNAi with respect to the target RNA, wherein the target may include endogenous or pathogenic target RNA.) In some embodiments, the siRNA is at least partially complementary to the coding sequence in the target gene expressed in the cell. In some embodiments, after delivery of the siRNA to a cell expressing the gene, the siRNA can inhibit or block gene expression in vitro or in vivo. Typically, siRNA contains a double-stranded region of fewer than 60, 50, 40, or 30 complementary base pairs; preferably, it contains a double-stranded region of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22 complementary base pairs. In some embodiments, the sense and antisense strands of the siRNA are each independently 15-30 nucleotides in length, forming a complementary double-stranded region of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22 base pairs in length. In some embodiments, the sense and antisense strands of the siRNA are completely complementary and have a length of 15-30 base pairs. In some embodiments, the sense and antisense strands of the siRNA are completely complementary and have a length of 17, 18, 19, 20, 21, or 22 base pairs.

[0261] The two nucleic acid strands of a double-stranded small interfering RNA are called the sense strand and the antisense strand. As used herein, the term "antisense strand" refers to the strand that is at least partially complementary to the target RNA; the term "sense strand" refers to the strand that is at least partially complementary to the antisense strand to form a double-stranded region.

[0262] In some embodiments, the length of the ribonucleic acid chain is calculated in nt (nucleotides), where 1 nt (1 nucleotide) includes, but is not limited to, 1 natural nucleotide and 1 modified nucleotide.

[0263] As used in this article, when a sequence region has 0 nucleotides, it includes cases where the region does not exist but is directly connected to the two regions on its left and right.

[0264] As used herein, a “pharmaceuticalally acceptable carrier” may include, but is not limited to, excipients and / or other components. An “excipient” is a pharmaceutically acceptable solvent, suspending agent, or any other pharmaceutically inert medium used to deliver one or more nucleic acids to animals. Such agents are well known in the art.

[0265] As used herein, “subject” means any animal, such as a mammal or marsupial. Subjects in this application include, but are not limited to, humans, non-human primates (e.g., rhesus monkeys or other types of macaques), mice, pigs, horses, cattle, rats, or any kind of poultry.

[0266] As used herein, the term "treatment," "curing," or "treatment" refers to the management, elimination, reduction, or improvement of a disease and its associated symptoms, and also refers to methods for achieving beneficial or desired outcomes, including but not limited to therapeutic benefits. A "therapeutic benefit" means the eradication or improvement of the underlying disorder being treated. Furthermore, a therapeutic benefit is achieved by eradicating or improving one or more physiological symptoms associated with the underlying disorder, thereby observing improvement in the subject, although the subject may still be suffering from the underlying disorder. While the possibility of complete elimination of the disease or associated symptoms is not excluded, treating a disease does not require the complete elimination of the disease or associated symptoms. As used herein, the term "treatment" also includes "preventive treatment," which is applied before the onset of symptoms or disease manifestations to reduce the likelihood of disease occurrence or recurrence, or to reduce the likelihood of recurrence of a disease that is already under control. This applies to individuals who are not yet ill but are at risk or prone to recurrence, or individuals who are at risk or susceptible to disease recurrence. In the sense of this invention, "treatment" also includes prevention of recurrence or a preventive phase, as well as treatment of acute or chronic signs, symptoms, and / or functional impairments. Treatment may target symptoms, such as symptom suppression. Treatment can be administered in the short term, in the medium term, or as a long-term treatment, such as maintenance therapy.

[0267] As used herein, “effective dose” refers to a drug dose that produces the expected local or systemic therapeutic effect at a reasonable benefit / risk ratio, applicable to any treatment alone or in combination with further doses. In treating a specific disease, the desired local or systemic therapeutic effect typically involves the inhibition of disease progression. This includes slowing disease progression, particularly interrupting or reversing it. When used for disease prevention, the dose is sufficient to prevent or delay the onset of disease. An effective dose does not necessarily have a curative effect or completely prevent disease. The effective dose of the aforementioned drugs will depend on the condition being treated, the severity of the disease, the patient’s individual parameters (including age, physiological condition, body size, and weight), the duration of treatment, the type of concomitant treatment (if any), the specific route of administration, and similar factors. Therefore, the drug dose may vary depending on these parameters. If the initial dose is insufficient to elicit a patient’s response, a higher dose may be used (or a higher effective dose achieved through a different, more local route of administration). In some cases, the effective dose of a drug will also depend on factors such as its therapeutic index and solubility.

[0268] Effective doses typically involve inhibiting disease progression. This includes slowing disease progression, particularly interrupting or reversing it. When used for disease prevention, the dose is sufficient to avoid or delay the onset of the disease. An effective dose does not necessarily have a curative effect or completely prevent disease. The effective dose of the aforementioned medications will depend on the condition being treated, the severity of the disease, the patient's individual parameters (including age, physiological condition, body size, and weight), the duration of treatment, the type of concomitant treatment (if any), the specific route of administration, and similar factors. Therefore, the dosage of the medication may vary depending on these parameters. If the initial dose is insufficient to elicit a patient's response, a higher dose may be used (or a higher effective dose achieved through a different, more localized route of administration). In some cases, the effective dose of a medication will also depend on factors such as its therapeutic index and solubility.

[0269] The compositions of this application may further include other auxiliary components conventionally present in pharmaceutical compositions at levels established in the art. Thus, for example, the composition may contain additional, compatible pharmaceutically active substances, such as antipruritics, astringents, local anesthetics, or anti-inflammatory agents, or may contain additional substances suitable for the physical formulation of various dosage forms of the compositions of this application, such as preservatives, antioxidants, and stabilizers. However, when added, such substances should not unduly interfere with the biological activity of the components of the compositions of this application. The formulation may be sterilized, and if necessary, may be mixed with adjuvants that will not harmfully interact with the nucleic acids of the formulation, such as preservatives, stabilizers, humectants, emulsifiers, salts or buffers that affect osmotic pressure, etc.

[0270] Salts or buffers that affect osmotic pressure.

[0271] The term "pharmaceutically acceptable salt" refers to a salt of the compounds of this invention, prepared by reacting a compound with a relatively non-toxic acid or base, as discovered in this invention, with a specific substituent. When the compounds of this invention contain relatively acidic functional groups, base addition salts can be obtained by contacting such compounds with a sufficient amount of base in a pure solution or a suitable inert solvent. Pharmaceutically acceptable base addition salts include sodium, potassium, calcium, ammonium, organic amine, or magnesium salts, or similar salts. When the compounds of this invention contain relatively basic functional groups, acid addition salts can be obtained by contacting such compounds with a sufficient amount of acid in a pure solution or a suitable inert solvent. Examples of pharmaceutically acceptable acid addition salts include inorganic acid salts, such as hydrochloric acid, hydrobromic acid, nitric acid, carbonic acid, bicarbonate, phosphoric acid, monohydrogen phosphate, dihydrogen phosphate, sulfuric acid, hydrogen sulfate, hydroiodic acid, phosphorous acid, etc.; and organic acid salts, such as acetic acid, propionic acid, isobutyric acid, maleic acid, malonic acid, benzoic acid, succinic acid, octanoic acid, fumaric acid, lactic acid, mandelic acid, phthalic acid, benzenesulfonic acid, p-toluenesulfonic acid, citric acid, tartaric acid, and methanesulfonic acid; as well as salts of amino acids (such as arginine) and salts of organic acids such as glucuronic acid. Certain specific compounds of the present invention contain both basic and acidic functional groups, and thus can be converted into either a base or an acid addition salt.

[0272] The pharmaceutically acceptable salts of the present invention can be synthesized from parent compounds containing acid radicals or bases by conventional chemical methods. Generally, such salts are prepared by reacting these compounds in free acid or base form with a stoichiometric amount of a suitable base or acid in water or an organic solvent or a mixture thereof.

[0273] These compounds are prepared by reacting them in free acid or base form with a stoichiometric amount of a suitable base or acid in an organic solvent or a mixture of both.

[0274] The compounds of this invention can exist in specific geometric or stereoisomeric forms. This invention contemplates all such compounds, including cis and trans isomers, (-)- and (+)- enantiomers, (R)- and (S)- enantiomers, diastereomers, (D)- isomers, (L)- isomers, and racemic mixtures thereof, as well as other mixtures, such as mixtures enriched with enantiomers or diastereomers, all of which are within the scope of this invention. Additional asymmetric carbon atoms may be present in substituents such as alkyl groups. All such isomers and mixtures thereof are included within the scope of this invention.

[0275] Unless otherwise stated, the terms "enantiomer" or "optical isomer" refer to stereoisomers that are mirror images of each other.

[0276] Unless otherwise stated, the terms "cis-trans isomers" or "geometric isomers" arise because the single bonds of double bonds or cyclic carbon atoms cannot rotate freely.

[0277] Unless otherwise stated, the term "diastereomer" refers to a stereoisomer of a molecule having two or more chiral centers and being in a non-mirror relationship with each other.

[0278] Unless otherwise stated, "(+)" indicates right-handed rotation, "(-)" indicates left-handed rotation, and "(±)" indicates racemic rotation.

[0279] Unless otherwise specified, use wedge-shaped solid line keys. and wedge-shaped dashed key The absolute configuration of the center of a solid is represented by a straight solid line key. and straight dashed key The relative configuration of the center of a solid is indicated by a wavy line. Indicates wedge-shaped solid line key or wedge-shaped dashed key Or use wavy lines Indicates a straight solid line key Or straight dashed key

[0280] Unless otherwise stated, the terms "tautomer" or "tautomer form" refer to isomers of different functional groups in dynamic equilibrium at room temperature, capable of rapidly interconverting into each other. If tautomerism is possible (e.g., in solution), chemical equilibrium of the tautomer can be achieved. For example, proton tautomers (also called proton transfer tautomers) involve interconversions via proton migration, such as keto-enol isomerization and imine-enamine isomerization. Valence tautomers involve interconversions involving the rearrangement of some bonding electrons. A specific example of keto-enol tautomerization is the interconversion between the two tautomers, pentane-2,4-dione and 4-hydroxypent-3-en-2-one.

[0281] Unless otherwise stated, the terms "rich in one isomer," "isomer enrichment," "rich in one enantiomer," or "enantiomer enrichment" mean that the content of one isomer or enantiomer is less than 100%, and the content of the isomer or enantiomer is greater than or equal to 60%, or greater than or equal to 70%, or greater than or equal to 80%, or greater than or equal to 90%, or greater than or equal to 95%, or greater than or equal to 96%, or greater than or equal to 97%, or greater than or equal to 98%, or greater than or equal to 99%, or greater than or equal to 99.5%, or greater than or equal to 99.6%, or greater than or equal to 99.7%, or greater than or equal to 99.8%, or greater than or equal to 99.9%.

[0282] Unless otherwise stated, the terms "isomer excess" or "enantiomer excess" refer to the difference between the relative percentages of two isomers or two enantiomers. For example, if one isomer or enantiomer is 90% and the other isomer or enantiomer is 10%, then the isomer or enantiomer excess (ee value) is 80%.

[0283] Optically active (R)- and (S)- isomers, as well as D- and L- isomers, can be prepared by chiral synthesis, chiral reagents, or other conventional techniques. To obtain an enantiomer of a compound of the present invention, it can be prepared by asymmetric synthesis or derivatization with a chiral auxiliary, wherein the resulting diastereomeric mixture is separated, and the auxiliary group is cleaved to provide the desired enantiomer in pure form. Alternatively, when the molecule contains a basic functional group (such as an amino group) or an acidic functional group (such as a carboxyl group), a salt of the diastereomeric isomer is formed with a suitable optically active acid or base, followed by diastereomeric resolution using conventional methods known in the art, and then the pure enantiomer is recovered. Furthermore, the separation of enantiomers and diastereomeric isomers is typically accomplished by using chromatography employing a chiral stationary phase, optionally combined with chemical derivatization (e.g., from amines to carbamates).

[0284] The compounds of this invention may contain atomic isotopes in non-natural proportions on one or more atoms constituting the compound. For example, the compounds may be labeled with radioactive isotopes, such as tritium. 3 H), Iodine-125 125 I) or C-14 14 C). For example, deuterium can be used to replace hydrogen to form deuterated drugs. The bond between deuterium and carbon is stronger than that between ordinary hydrogen and carbon. Compared with undeuterated drugs, deuterated drugs have advantages such as reduced toxicity, increased drug stability, enhanced efficacy, and prolonged drug biological half-life. All isotopic variations of the compounds of this invention, regardless of radioactivity, are included within the scope of this invention.

[0285] The compounds of the present invention can be prepared by a variety of synthetic methods known to those skilled in the art, including the specific embodiments listed below, embodiments formed by combining them with other chemical synthetic methods, and equivalent substitutions known to those skilled in the art. Preferred embodiments include, but are not limited to, the embodiments of the present invention.

[0286] The structures of the compounds of this invention can be confirmed using conventional methods well known to those skilled in the art. If this invention relates to the absolute configuration of a compound, that absolute configuration can be confirmed using conventional techniques in the art, such as single-crystal X-ray diffraction (SXRD).

[0287] The solvents or reagents used in this invention are commercially available.

[0288] The compounds involved in this invention are named according to conventional naming principles in the art or using software (such as ChemDraw), and commercially available compounds are named according to the supplier's catalog. Attached Figure Description

[0289] Figure 1 shows the relative expression levels of AGT mRNA in the liver of the test compounds (ds101 and ds100).

[0290] Figure 2 shows the relative expression of SOD-1 in different tissues (frontal cortex, hippocampus, m and heart) of the test compounds (ds110, ds111, ds112 and ds113).

[0291] Figure 3 shows the relative expression of SOD-1 in different tissues (frontal cortex and lumbar spine) of the test compounds (ds114 and ds115).

[0292] Figure 4 shows the relative mTTR expression of the tested compounds (ds118 and ds119).

[0293] Figure 5 shows the reaction results of liver homogenization of the experimental compounds.

[0294] Figure 6 shows the plasma relative AGT levels of the tested compounds (ds101 and ds86) in the NHP model.

[0295] Figures 7 and 8 are schematic diagrams of the structural relationship between the first chain, the second chain, the 3' extension segment, and the 5' extension segment.

[0296] Figure 9 shows the relative expression levels of INHBE mRNA in mouse livers under different test compounds (Z91, Z92, and Z95).

[0297] Figure 10 shows the percentage change in Apo(a) in the serum of LPA-humanized mice under different test compounds (Z51, Z57, and Z59).

[0298] Figure 11 shows the percentage change in body weight of LPA humanized mice under different test compounds (Z51, Z57, and Z59).

[0299] Figure 12 shows the changes in serum Lp(a) of cynomolgus monkeys before and after administration of the test compound Z19.

[0300] Figure 13 shows the changes in serum LDL-c in cynomolgus monkeys before and after administration of the test compound Z19.

[0301] Figure 14 shows the changes in serum ApoB in cynomolgus monkeys before and after administration of the test compound Z19. Detailed Implementation

[0302] The compounds of this application can be prepared by a variety of synthetic methods known to those skilled in the art, including the specific embodiments listed below, embodiments formed by combining them with other chemical or biological synthetic methods, and equivalent substitutions known to those skilled in the art. Preferred embodiments include, but are not limited to, the embodiments of this application.

[0303] The following examples are provided to illustrate this application and are intended to provide a better understanding of it, but are not intended to limit the scope of this application. Any modifications or alterations made to the elements of this application without departing from its spirit and substance are within the scope of this application. Unless otherwise specified, the reagents, kits, and biological materials used in this application are commercially available. Unless otherwise specified, the kits are used in accordance with the kit instructions.

[0304] The present invention also provides the following non-limiting embodiments.

[0305] Implementation Scheme 1. A dsRNA comprising a sense strand and an antisense strand, the sense strand and the antisense strand being independently 17 to 35 nucleotides in length, the nucleotides being independently and optionally modified, wherein the sense strand comprises a first strand, the antisense strand comprises a second strand, the first strand and the second strand are anticomplementary, and starting from the 5' end of the first strand, positions 9, 11, 13 and 15 of the first strand are nucleotides containing 2'-F modification, and starting from the 5' end of the second strand, position 14 of the second strand is a nucleotide containing 2'-F modification.

[0306] Implementation Scheme 2. The dsRNA according to Implementation Scheme 1, wherein the second strand has a nucleotide at position 2 containing a 2'-deoxy modified nucleotide or a nucleotide containing a 2'-F modified nucleotide, and the second strand has nucleotides at positions 5, 7 and 12 containing a 2'-deoxy modified nucleotide.

[0307] Implementation Scheme 3. The dsRNA according to Implementation Scheme 1, wherein positions 2 and 16 of the second strand are nucleotides containing 2'-F modification.

[0308] Implementation Scheme 4. The dsRNA according to Implementation Scheme 1, wherein positions 2 and 16 of the second strand are nucleotides containing 2'-F modification, the antisense strand further comprises a 3' extension and a 5' extension, the 3' extension being connected to the 3' end of the second strand and having a length of 0-5 nucleotides, the 5' extension being connected to the 5' end of the second strand and having a length of at least 3 nucleotides, the dsRNA being cleaved between the 3' end nucleotide of the 5' extension and the 5' end nucleotide of the second strand, the resulting cleavage product containing the first and second strands being able to silence gene expression through RNA interference.

[0309] Implementation Scheme 5. The dsRNA according to Implementation Scheme 4, wherein the 6th position of the second strand is a nucleotide containing a 2'-F modification.

[0310] Implementation Scheme 6. The dsRNA according to Implementation Scheme 1, wherein the second strand has a nucleotide at position 2 containing a 2'-deoxy modified nucleotide or a nucleotide containing a 2'-F modified nucleotide, and positions 5, 7, and 12 of the second strand have nucleotides containing a 2'-deoxy modified nucleotide. The antisense strand further includes a 3' extension and a 5' extension. The 3' extension is connected to the 3' end of the second strand and has a length of 0-5 nucleotides. The 5' extension is connected to the 5' end of the second strand and has a length of at least 3 nucleotides. The dsRNA can be cleaved between the 3' end nucleotide of the 5' extension and the 5' end nucleotide of the second strand. The resulting cleavage product containing the first and second strands can silence gene expression through RNA interference.

[0311] Implementation Scheme 7. A dsRNA comprising a sense strand and an antisense strand, the sense strand and the antisense strand each having a length of 17 to 35 nucleotides, the nucleotides being optionally modified independently, wherein the sense strand comprises a first strand, the antisense strand comprises a second strand, the first strand and the second strand are anticomplementary, and counting from the 5' end of the first strand, positions 9 and 11 of the first strand are nucleotides containing 2'-F modification, positions 10 or 13 of the first strand are nucleotides containing 2'-F modification; counting from the 5' end of the second strand, position 2 of the second strand is a nucleotide containing 2'-deoxy modification or a nucleotide containing 2'-F modification, positions 5, 7, and 12 of the second strand are nucleotides containing 2'-deoxy modification, and position 14 of the second strand is a nucleotide containing 2'-F modification.

[0312] 2'-F modified nucleotides.

[0313] Implementation Scheme 8. The dsRNA according to Implementation Scheme 7, wherein, counting from the 5' end of the first strand, positions 9, 10, and 11 of the first strand are nucleotides containing 2'-F modification, and counting from the 5' end of the second strand, position 2 of the second strand is a nucleotide containing 2'-deoxy modification. The antisense strand further comprises a 3' extension and a 5' extension, the 3' extension being connected to the 3' end of the second strand and having a length of 0-5 nucleotides, and the 5' extension being connected to the 5' end of the second strand and having a length of at least 3 nucleotides. The dsRNA can be cleaved between the 3' end nucleotide of the 5' extension and the 5' end nucleotide of the second strand, and the resulting cleavage product containing the first and second strands can silence gene expression through RNA interference.

[0314] Implementation Scheme 9. A dsRNA comprising a sense strand and an antisense strand, the sense strand and the antisense strand each having a length of 17 to 35 nucleotides, the nucleotides being optionally modified independently, wherein the sense strand comprises a first strand, the antisense strand comprises a second strand, the first strand and the second strand are anticomplementary, and starting from the 5' end of the first strand, positions 9 and 11 of the first strand are nucleotides containing 2'-F modification, and positions 10 or 13 of the first strand are nucleotides containing 2'-F modification; and starting from the 5' end of the second strand, positions 2, 14, and 16 of the second strand are nucleotides containing 2'-F modification.

[0315] Implementation Scheme 10. The dsRNA according to Implementation Scheme 9, wherein the antisense strand further comprises a 3' extension and a 5' extension, the 3' extension being connected to the 3' end of the second strand and having a length of 0-5 nucleotides, the 5' extension being connected to the 5' end of the second strand and having a length of at least 3 nucleotides, the dsRNA being cleaved between the 3' end nucleotide of the 5' extension and the 5' end nucleotide of the second strand, the resulting cleavage product comprising the first and second strands being able to silence gene expression through RNA interference.

[0316] Implementation Scheme 11. The dsRNA according to Implementation Scheme 10, wherein,

[0317] (i) Position 10 or 13 of the first strand is a nucleotide containing a 2'-F modification and

[0318] The 6th position of the second strand is a nucleotide containing a 2'-F modification, or

[0319] (ii) The 13th position of the first strand is a nucleotide containing a 2'-F modification and

[0320] The 7th position of the second strand is a nucleotide containing a 2'-F modification.

[0321] Implementation Scheme 12. The dsRNA according to Implementation Scheme 11, wherein positions 8 and 9 of the second strand are nucleotides containing 2'-F modification.

[0322] Implementation Scheme 13. The dsRNA according to Implementation Scheme 11, wherein the 7th position of the first strand is a nucleotide containing a 2'-F modification.

[0323] Implementation Scheme 14. A dsRNA comprising a sense strand and an antisense strand, the sense strand and the antisense strand each having a length of 17 to 35 nucleotides independently, the nucleotides being optionally modified independently, wherein the sense strand comprises a first strand, the antisense strand comprises a second strand, the first strand and the second strand are anticomplementary, and from the 5' end of the first strand, positions 9, 10, and 11 of the first strand are nucleotides containing a 2'-F modification, and from the 5' end of the second strand, positions 2, 4, 6, and 11 of the second strand are nucleotides containing a 2'-deoxy-modified nucleotide. The second strand contains a nucleotide with a 2'-F modification at position 13. The antisense strand also contains a 3' extension and a 5' extension. The 3' extension is attached to the 3' end of the second strand and has a length of 0-5 nucleotides. The 5' extension is attached to the 5' end of the second strand and has a length of at least 3 nucleotides. The dsRNA can be cleaved between the 3' end nucleotide of the 5' extension and the 5' end nucleotide of the second strand. The resulting cleavage product containing the first and second strands can silence gene expression through RNA interference.

[0324] Implementation Scheme 15. A dsRNA comprising a sense strand and an antisense strand, the sense strand and the antisense strand being independently 17 to 35 nucleotides in length, the nucleotides being independently optionally modified, wherein the sense strand comprises a first strand, the antisense strand comprises a second strand, the first strand and the second strand are anticomplementary, the first strand having nucleotides containing 2'-F modification at positions 9, 10, and 12, counting from the 5' end of the first strand, and the second strand having nucleotides containing 2'-F modification at positions 2, 6, 14, and 16, counting from the 5' end of the second strand, the antisense strand further comprising a 3' extension and a 5' extension, the 3' extension being connected to the 3' end of the second strand and having a length of 0-5 nucleotides, the 5' extension being connected to the 5' end of the second strand and having a length of at least 3 nucleotides, the dsRNA being cleavable between the 3' end nucleotide of the 5' extension and the 5' end nucleotide of the second strand, the resulting cleavage product comprising the first strand and the second strand being capable of silencing gene expression via RNA interference.

[0325] The second strand comprises nucleotides containing 2'-F modification at positions 2, 6, 14, and 16, starting from the 5' end. The antisense strand also comprises a 3' extension and a 5' extension. The 3' extension is attached to the 3' end of the second strand and has a length of 0-5 nucleotides. The 5' extension is attached to the 5' end of the second strand and has a length of at least 3 nucleotides. The dsRNA can be cleaved between the 3' end nucleotide of the 5' extension and the 5' end nucleotide of the second strand. The resulting cleavage product, containing both the first and second strands, can silence gene expression through RNA interference.

[0326] Implementation Scheme 16. The dsRNA according to any one of Implementation Schemes 4, 5, 6, 8, 10-15, wherein, counting from the 3' end of the 5' extension, the two nucleotides of the 5' extension are selected from optionally modified natural analogs of G, A, G, non-natural analogs of G, natural analogs of A, and non-natural analogs of A.

[0327] Implementation Scheme 17. The dsRNA according to Implementation Scheme 16, wherein, counting from the 3' end of the 5' extension, the two nucleotides of the 5' extension are selected from optionally modified G, natural analogs of G, and non-natural analogs of G.

[0328] Implementation Scheme 18. According to Implementation Scheme 16 or the dsRNA described herein, wherein the 5' end nucleotide of the second strand is selected from A, U, natural analogs of U, non-natural analogs of U, natural analogs of A, and non-natural analogs of A.

[0329] Implementation Scheme 19. The dsRNA according to any one of Implementation Schemes 4, 5, 6, 8, 10-18, wherein the 5' extension has a blocking group attached to the 5' nucleotide; further, the blocking group is M06.

[0330] Implementation Scheme 20. The dsRNA according to any one of Implementation Schemes 4, 5, 6, 8, 10-18, wherein, counting from the 3' end of the 5' extension, positions 1 and 2 of the 5' extension are nucleotides containing 2'-F modification.

[0331] Implementation Scheme 21. The dsRNA according to Implementation Scheme 19 or 20, wherein the blocking group is connected to the 5' end nucleotide residue of the 5' extension by a phosphate thioester bond, and the nucleotide residues at positions 1 and 2 of the second strand are connected by a phosphate thioester bond, starting from the 5' end of the second strand.

[0332] Implementation Scheme 22. The dsRNA according to any one of Implementation Schemes 1 to 21, wherein, counting from the 3' end of the antisense strand, the nucleotide residues at positions 1 and 2 of the antisense strand are linked by a phosphate thioester bond, and the nucleotide residues at positions 2 and 3 of the antisense strand are linked by a phosphate thioester bond.

[0333] Implementation Scheme 23. The dsRNA according to Implementation Scheme 22, wherein, counting from the 3' end of the positive strand, the 1st and 2nd nucleotide residues of the positive strand are linked by a phosphate thioester bond, and the 2nd and 3rd nucleotide residues of the positive strand are linked by a phosphate thioester bond.

[0334] Implementation Scheme 24. A dsRNA conjugate comprising the dsRNA according to Implementation Scheme 23 and a delivery system, wherein the 3' end nucleotide of the positive strand is linked to the delivery system; further, the delivery system is L96; and even further, the residues of the 3' end nucleotide of the positive strand are linked to the L96 residues by a phosphate ester bond.

[0335] Implementation Scheme 25. A dsRNA conjugate comprising the dsRNA according to Implementation Scheme 22 and a delivery system, the delivery system being NAG37; further, the 5' end nucleotide residue of the positive strand is linked to an inverted abasic nucleotide residue via a phosphate thioester bond, the inverted abasic nucleotide being linked to the delivery system, and the 1st and 2nd nucleotide residues of the positive strand, counting from the 3' end, are linked via a phosphate thioester bond; even further, the inverted abasic nucleotide residue is linked to the NAG37 residue via a phosphate thioester bond.

[0336] Implementation Scheme 26. The dsRNA or its conjugate according to any one of Implementation Schemes 1-25, wherein the modification is independently selected from one, two or three combinations of the following: 2'-OMe modification, 2'-F modification, 2'-deoxy modification, VP modification, 5'-MP modification, PS modification, PS2 modification, MP modification, MOP modification, invAB modification, and invAB modification.

[0337] Implementation Scheme 27. The dsRNA or its conjugate according to any of the preceding embodiments, wherein the antisense strand comprises a cleavage region comprising the nucleotide sequence shown in formula (I),

[0338] Equation (I): (3'-5')X²-YZ

[0339] The cleavage occurs between X2 and Y, where X2 is the 5' end nucleotide of the second strand, and Y and Z are the two nucleotides at the 3' end of the 5' extension, where Z is guanine nucleotide (G), a natural analog of guanine nucleotide (G), a non-natural analog of guanine nucleotide (G), adenine nucleotide (A), a natural analog of adenine nucleotide (A), or a non-natural analog of adenine nucleotide (A).

[0340] Implementation Scheme 28. The dsRNA or its conjugate according to Implementation Scheme 27, wherein Z is guanine nucleotide (G), a natural analog of guanine nucleotide (G), or a non-natural analog of guanine nucleotide (G).

[0341] Implementation Scheme 29. The dsRNA or its conjugate according to Implementation Scheme 28, wherein X2 is adenine nucleotide (A), a natural analog of adenine nucleotide (A), a non-natural analog of adenine nucleotide (A), uracil nucleotide (U), a natural analog of uracil nucleotide (U), or a non-natural analog of uracil nucleotide (U).

[0342] Implementation Scheme 30. The dsRNA or its conjugate according to Implementation Scheme 29, wherein the formula (I) has a sequence (3'-5') selected from the following: UUG, UAG, AUG, AAG, UUA, UAA, AUA, AAA, UCG, UGG, ACG, AGG, UCA, UGA, ACA and AGA, or natural or non-natural analogs thereof.

[0343] Implementation Scheme 31. The dsRNA or its conjugate according to the preceding Implementation Scheme 30, wherein the Y is adenine nucleotide (A), a natural analog of adenine nucleotide (A), a non-natural analog of adenine nucleotide (A), uracil nucleotide (U), a natural analog of uracil nucleotide (U), or a non-natural analog of uracil nucleotide (U).

[0344] Implementation Scheme 32. The dsRNA or its conjugate according to Implementation Scheme 31, wherein the formula (I) has a sequence (3'-5') selected from the following: UUG, UAG, AUG, AAG, UUA, UAA, AUA and AAA.

[0345] Implementation Scheme 33. The dsRNA or its conjugate according to any one of Implementation Schemes 27-32, wherein the cleavage region further comprises nucleotide N1, wherein N1 is the third nucleotide from the 3' end of the 5' extension, and the cleavage region comprises the nucleotide sequence shown in Formula (II).

[0346] Equation (II): (3'-5')X2-YZ-N1.

[0347] Implementation Scheme 34. The dsRNA or its conjugate according to Implementation Scheme 33, wherein the cleavage region further comprises fragment N, fragment N comprising at least one nucleotide, wherein the nucleotide at the 3' end of fragment N is N1, and the cleavage region comprises the nucleotide sequence shown in Formula (III).

[0348] Equation (III): (3'-5')X2-YZN,

[0349] The length of fragment N is 1-10 nucleotides, preferably 1-5 nucleotides, and more preferably 1 nucleotide.

[0350] Implementation Scheme 35. The dsRNA or its conjugate according to Implementation Scheme 34, wherein N1 is adenine nucleotide (A), guanine nucleotide (G), cytosine nucleotide (C), uracil nucleotide (U), their natural analogs or their non-natural analogs; preferably, N1 is cytosine nucleotide (C), a natural analog of cytosine nucleotide (C) or a non-natural analog of cytosine nucleotide (C).

[0351] Implementation Scheme 36. The dsRNA or its conjugate according to Implementation Scheme 35, wherein the formula (III) has a sequence (3'-5') selected from the following: AAGC and UAGC.

[0352] Implementation Scheme 37. The dsRNA or its conjugate according to any one of Implementation Schemes 27-36, wherein,

[0353] (a) The cleavage region contains at least one nucleoside internucleotide bond that is not a thiophosphate bond;

[0354] (b) The internucleotide bond between X2 and Y is not a thiophosphate bond;

[0355] (c) The internucleotide bond between Y and Z is not a thiophosphate bond;

[0356] (d) All internucleotide bonds in the cleavage region are not phosphate thioester bonds;

[0357] (e) The cleavage region contains at least one phosphate diester bond between nucleosides;

[0358] (f) The internucleotide bond between X2 and Y is a phosphodiester bond;

[0359] (g)The internucleotide bond between Y and Z is a phosphodiester bond;

[0360] (h) All internucleotide bonds in the cleavage region are phosphodiester bonds; and / or

[0361] (i) The nucleoside bond between the cleavage region and the second chain is a phosphate diester bond.

[0362] Implementation Scheme 38. The dsRNA or its conjugate according to any one of Implementation Schemes 27-37, wherein the cleavage region contains at least one modified nucleotide, preferably, all nucleotides in the cleavage region are modified nucleotides.

[0363] Implementation Scheme 39. The dsRNA or its conjugate as described in Implementation Scheme 38, wherein,

[0364] (a) X2 is a nucleotide containing 2'-OMe modification;

[0365] (b) Y is a nucleotide containing 2'-OMe modification or 2'-F modification;

[0366] (c) The Z is a nucleotide containing a 2'-F modification; and / or

[0367] (d) The N1 is a nucleotide containing 2'-OMe modification or 2'-F modification.

[0368] Implementation Scheme 40. The dsRNA or its conjugate according to Implementation Scheme 39, wherein the cleavage region comprises a sequence (3'-5') selected from the following: aaGfCf, aAfGfCf, aaGfc, aAfGfc, uaGfCf, uAfGfCf, uaGfc and uAfGfc.

[0369] Implementation Scheme 41. The dsRNA or its conjugate according to Implementation Scheme 40, wherein the cleavage region comprises a sequence (3'-5') selected from the following: aaGfCf, aAfGfCf, uaGfCf and uAfGfCf.

[0370] Implementation Scheme 42. The dsRNA or its conjugate according to Implementation Scheme 40, wherein the cleavage region comprises a sequence (3'-5') selected from the following: aaGfCf.

[0371] Implementation Scheme 43. The dsRNA or its conjugate according to Implementation Scheme 40, wherein the cleavage region comprises a sequence (3'-5') selected from the following: aaGfc, aAfGfc, uaGfc and uAfGfc.

[0372] Implementation Scheme 44. The dsRNA or its conjugate according to Implementation Scheme 40, wherein the cleavage region comprises a sequence (3'-5') selected from the following: aaGfc.

[0373] Implementation Scheme 45. The dsRNA or its conjugate according to any of the preceding embodiments, wherein the antisense strand further comprises a blocking group at the 5' end; optionally, the blocking group is connected to the 5' end of the 5' extension.

[0374] Implementation Scheme 46. The dsRNA or its conjugate according to Implementation Scheme 39, wherein the blocking group includes a debased nucleotide, reverse debased nucleotide, MO3 or MO6.

[0375] Implementation Scheme 47. The dsRNA or its conjugate according to Implementation Scheme 45 or 46, wherein the blocking group is connected to the 5th end of the 5' extension via an internucleotide bond, said internucleotide bond being optionally modified or unmodified.

[0376] Implementation Scheme 48. The dsRNA or its conjugate according to Implementation Scheme 47, wherein the blocking group is connected to the 5' extended 5' end via a phosphate thioester bond.

[0377] Implementation Scheme 49. The dsRNA or its conjugate according to any one of Implementation Schemes 27-48, wherein a blunt end is generated at the 5' end of the second strand after cleavage of the cleavage region.

[0378] Implementation Scheme 50. The dsRNA or its conjugate according to any of the preceding implementation schemes, wherein the first strand and the second strand are completely reverse complementary.

[0379] Implementation Scheme 51. The use of the dsRNA or its conjugates according to any of the preceding implementation schemes in the preparation of drugs for treating diseases related to RNA interference.

[0380] Example 1: Synthesis of siRNA molecules

[0381] Oligonucleotides were synthesized using a phosphoramidite solid-phase synthesis technique. Synthesis was performed on a general-purpose controlled porous glass CPG. All 2'-modified RNA phosphoramidite and auxiliary reagents were commercially available. All phosphoramidite was dissolved in anhydrous acetonitrile and added to a molecular sieve, with coupling time of 12 min using 5-ethylthio-1H-tetrazole (ETT) as an activator. Phosphophosphate bonds were generated using anhydrous acetonitrile / pyridine (v / v = 1 / 1) solution of 50 mM 3-((dimethylamino-methylene)amino)-3H-1,2,4-dithiazol-3-thione (DDTT) for 1.4 min, or oxidized using a pyridine / water (v / v = 9 / 1) solution of 0.05 M iodine for 1 min. All sequences were synthesized after the final removal of the DMT group.

[0382] Cleavage and deprotection of oligomers bound to CPG: After termination of solid-phase synthesis, the protecting group was removed by treatment with an acetonitrile solution containing 20% ​​diethylamine for 30 minutes without cleaving the oligonucleotide from the CPG. Subsequently, the dried CPG was treated with concentrated ammonia at 55°C for 16 hours. After centrifugation, the supernatant was transferred to a new tube and the CPG was washed with ammonia. The combined solutions were concentrated to obtain a solid mixture.

[0383] Purification of single-stranded oligonucleotides: Ion-pair reversed-phase purification was performed using a C18 column. Buffer A consisted of 0.1 M TEAA in 5% acetonitrile aqueous solution; Buffer B consisted of acetonitrile. Anion exchange was then performed on the target product.

[0384] Oligomers were purified by HPLC using NanoQ anion exchange. Buffer A was 0.1 M ammonium acetate in 15% acetonitrile / water solution. Buffer B was 1.5 M sodium bromide + 0.1 M ammonium acetate in 15% acetonitrile / water solution. The target product was separated and desalted using a reversed-phase C18 column.

[0385] Annealing single-stranded oligonucleotides to produce siRNA: The single-stranded oligonucleotides to be annealed were prepared to 200 μM using sterile RNase-free water (free of RNA hydrolase). Equimolar amounts of the single-stranded oligonucleotide solutions were combined to form complementary strands. The annealing reaction system was set up as follows: 100 μL of the mixture was incubated in a 95°C water bath for 2 minutes, cooled to room temperature, and freeze-dried to obtain the final product, siRNA.

[0386] Example 2: Cellular activity assay of the compound against the target gene ANGPTL3

[0387] 1. Research Objectives

[0388] The purpose of this study is to evaluate the in vitro inhibitory activity of the double-stranded ribonucleic acid compound of this invention against the target gene ANGPTL3.

[0389] 2. Materials and Methods

[0390] 2.1.1 Test Compound

[0391] Z44, Z45, Z46, Z47 and Z48 were prepared into 100 μM stock solutions using Nuclease-Free Water.

[0392] 2.1.2 Cells

[0393] Huh7 cells were revived and cultured in DMEM, RPMI 160, and EMEM media. All three media contained the following components: 10% fetal bovine serum, 1% penicillin-streptomycin, 1% non-essential amino acids, and 1% glutamine.

[0394] 2.1.3 Main Instruments

[0395] The main instruments used in this experiment included a fluorescence qPCR instrument (Quanstudio 7flex), a centrifuge (Beckman Allegra-X15R Centrifuge), and a cell counter (Countstar Rigel2).

[0396] 2.1.4 Main Reagents and Consumables

[0397] The main reagents used in this experiment included FastStart Universal Probe Master, FastStart Universal SYBR Green Master, RNA extraction kit, FastKing RT Kit (with gDNase), and 96-well plates. Primers used in the experiment are listed in Table 2.

[0398] Table 3 Primers used in the experiment

[0399] 2.2 Experimental Methods

[0400] 2.2.1 Transfection of the compound

[0401] Inoculation with Huh7 (2×10) 4 Cells / well) to 96-well cell plates, while plating, use RNAiMAX transfects cells with different concentrations of double-stranded ribonucleic acid (DRNA) molecules, and tests are performed at 7–8 concentration points (e.g., 10 nM, 2.5 nM, 0.625 nM, 0.156 nM, 0.0391 nM, 0.0098 nM, and 0.0024 nM), with parallel measurements in duplicate or triplet wells; simultaneously, it is set to contain... The RNAiMAX compound-free control group was then incubated at 37°C in a 5% CO2 incubator.

[0402] 2.2.2 RNA extraction and reverse transcription

[0403] After 48 hours, remove the culture medium and collect the cells for RNA extraction. Use according to the kit instructions. Total RNA was extracted using the 96Kit. cDNA was synthesized using the FastKing RT Kit (with gDNase) according to the instructions.

[0404] 2.2.3 qPCR detection of target gene mRNA expression levels

[0405] The target gene cDNA was detected by qPCR, and the internal reference gene GAPDH cDNA was also detected simultaneously. 8 μL of prepared PCR reaction solution and 2 μL of sample cDNA were added to each 384 well. The qPCR (TapMan probe) reaction program was: 95°C for 10 minutes, followed by cycling at 95°C for 15 seconds, then at 60°C for 1 minute, for a total of 40 cycles. The qPCR (SYBR Green) reaction program was: 95°C for 10 minutes, followed by 95°C for 15 seconds, then at 60°C for 1 minute, for a total of 40 cycles. The melting curve generation program was: 95°C for 15 seconds, 60°C for 1 minute, then 95°C for 15 seconds.

[0406] 3. Data Analysis

[0407] The expression level of the target gene mRNA in each sample was calculated using the ΔΔCT relative quantification method. The relative expression level of the target gene was expressed as 2-ΔΔCT.

[0408] The calculation formula is as follows:

[0409] ΔCT = Average Ct value of target gene - Average Ct value of internal reference gene

[0410] ΔCT = ΔCT(drug-treated group) - ΔCT( RNAiMAX Control Group)

[0411] Relative expression level of target gene = 2 - ΔΔCT

[0412] Target gene inhibition rate % = (1 - value of sample / Ave. value of PBS Control) * 100

[0413] GraphPad Prism software was used for graphical analysis, and the inhibition rate results are expressed as Mean±SD.

[0414] 4. Experimental Results

[0415] The results of the in vitro transfected cell viability test are shown in Table 3.

[0416] Table 4. Results of cell viability testing

[0417] 5. Experimental Conclusions

[0418] The double-stranded ribonucleic acid of this invention can significantly inhibit the level of ANGPTL3 mRNA.

[0419] Example 3: Cellular activity assay of the compound against the target gene LPA

[0420] 1. Research Objective

[0421] To evaluate the in vitro inhibitory activity of the compound against the target gene LPA.

[0422] 2 Materials and Methods

[0423] The test compound was prepared into a 100 μM stock solution using RNase-free water. Huh7 was revived and cultured in DMEM medium (Gibco 11965-092) containing 10% fetal bovine serum (FBS, Gibco catalog number 10091148) and 1% penicillin-streptomycin (PS, HyClone catalog number SV30010). The main instruments used in the experiment included a fluorescence multimode microplate reader (…). The equipment used included a 2105-inch centrifuge, a Beckman Allegra-X15R centrifuge, and a Countstar Rigel S2 cell counter. The main reagents used in the experiment included Lipofectamine RNAiMAX (Invitrogen, catalog number 13778-150), FUGEN HD Transfection (Promega, catalog number E2311), Dual-Glo luciferase Assay System (Promega, catalog number E2940), and psiCHECK-2 construct synthesized by GenScript.

[0424] 3 Experimental Methods

[0425] 3.1 Day 0, psiCHECK-2 plasmid transfection

[0426] Add 300 μL of Opti MEM and 3 μg of psiCHECK-2 plasmid to an RNase-free EP tube and mix (Mix #1). Digest Huh7 cells with trypsin from one T15 cell culture flask, count the cells using a cell counter, and adjust the cell density to 1*10^5 / ml. Transfer 12 μL of Fugene-HD to Mix #1, mix the resulting solution, and incubate for 15 minutes (Mix #2). Add the solution to 12 mL of cell suspension, mix well, and aliquot the suspension into 96-well plates (100 μL / well).

[0427] 3.2 Day 1, siRNA transfection

[0428] Dilute with Opti-MEM medium at a ratio of 4.7:0.3 Incubate with RNAiMAX reagent at room temperature for 15 minutes. Dilute siRNA with RNA-free water to prepare a 20× stock solution (e.g., prepare this stock solution to 500 nM if the final test concentration is 25 nM). Mix equal volumes of diluted RNAiMAX and siRNA (v:v = 1:1). Incubate the mixture at room temperature for 15 minutes to form a complex. Add 25 μl of the complex to each well and mix well in 225 μl of fresh DMEM medium. Discard the supernatant from the assay plate and add 120 μl of the compound mixture to each well of a 96-well plate.

[0429] 3.3 Detection of dual fluorescence (firefly and sea urchin)

[0430] After 48 hours, remove the culture medium. Dilute 1:1 with DMEM medium containing 10% FBS. Add 120 μL of Reagent and vortex for 15 minutes to lyse cells. Transfer 60 μL to a white 96-well plate. Detect firefly fluorescence using Envision. Stop and Prepare Reagent at a 1:100 ratio, add 30 μL to a white 96-well plate, and detect Renja fluorescence using Envision.

[0431] 4. Data Analysis: Detection Ratio = (Sample's *Rhizophora* luminescence value - Background *Rhizophora* luminescence value) / (Sample's *Firefly* luminescence value - Background *Firefly* luminescence value) Inhibition % = (1 - Sample Ratio / RNAiMAX Control Ratio) × 100%

[0432] 5. Experimental Results

[0433] Table 5-1

[0434] Table 5-2

[0435] Table 5-3

[0436] Table 5-4

[0437] Table 5-5

[0438] Table 5-6

[0439] Table 5-7

[0440] Table 5-8

[0441] Table 5-9

[0442] Table 5-10

[0443] Table 5-11

[0444] 6. Experimental Conclusions

[0445] The dsRNA provided in this application can significantly inhibit the level of intracellular LPA mRNA.

[0446] Example 4: In vitro inhibitory activity of the test substance against the target gene in cells (in vitro transfection experiment)

[0447] ●Experimental Objective

[0448] Using HepG2, Hep3B, and Huh7 cell lines, the in vitro activity of siRNA molecules was evaluated by assessing the degree of silencing of target genes by each candidate molecule.

[0449] ●Cell line

[0450] HepG2, Hep3B, and Huh7 cells were provided by Nanjing Kebai Biotechnology Co., Ltd.

[0451] HepG2 and Hep3B cells were cultured in MEM medium (Gibco catalog number 11095080) containing 10% fetal bovine serum (FBS, Gibco catalog number 10099-141C), 1% sodium pyruvate (100mM) (Gibco catalog number 11360070), and 1% NEAA (Gibco catalog number 11140050).

[0452] Huh-7 cells were cultured in DMEM medium (Gibco catalog number 11965-092) containing 10% fetal bovine serum (FBS, Gibco catalog number 2279804CP) and 1% NEAA (ThermoFisher catalog number 11140050).

[0453] ●Main Instruments

[0454] The main instruments used in this experiment included a fluorescence qPCR instrument (Roche 480 II), a centrifuge (Thermo Fisher catalog number 75016073), and a cell counter (Countstar Rigel 2).

[0455] ●Main reagents and consumables

[0456] The main reagents used in this experiment included Lipofectamine. TMiRNAiMAX transfection reagent (INVITROGEN, catalog number 56532), RNA extraction kit (Tiangen, catalog number DP671-T1), FastKing cDNA first strand synthesis kit (TianGen, catalog number KR116-02), 96-well plate (Costar 3599). SuperReal PreMix Plus (SYBR Green) (TIANGEN, catalog number: FP215-02), qPCR primers were synthesized by Genewiz.

[0457] Primer information:

[0458] ●Experimental Methods

[0459] 1. Compound transfection plate

[0460] Refer to the table below to seed the corresponding cells (1.5 × 10⁻⁶). 4 Cells were plated into 96-well cell culture plates, and siRNA was simultaneously transfected into the cells using RNAiMAX. Seven concentration points were set for the siRNA assay, with concentrations spaced four times apart. Cells were incubated overnight (24 h) at 37°C in a 5% CO2 incubator. A control group containing RNAiMAX but without the compound was also included.

[0461] 2. RNA extraction and reverse transcription

[0462] 24 hours after transfection, remove the culture medium and collect the cells for RNA extraction. Use according to the kit instructions. Total RNA was extracted using the 96 Kit (Tiangen-DP671-T1). cDNA was synthesized using the FastKing RT Kit (With gDNase) (Tiangen-KR116-02) according to the manufacturer's instructions.

[0463] 3. qPCR detection of target gene mRNA expression levels

[0464] The target cDNA will be detected by qPCR, while GAPDH cDNA will be detected as an internal control in parallel. 9 μL of the prepared PCR reaction solution and 1 μL of sample cDNA will be added to each of 384 wells. The qPCR program is as follows: heat at 95°C for 15 min, then cycle at 95°C for 10 sec, 60°C for 20 s, followed by 72°C for 32 s, for a total of 40 cycles.

[0465] ●Data Analysis

[0466] The expression level of the target gene mRNA in each sample was calculated using the ΔΔCt relative quantification method. The relative expression level of the target gene was expressed as 2-ΔΔCT.

[0467] The calculation formula is as follows: ΔCT = Average Ct value of target gene - Average Ct value of internal reference gene ΔCT = ΔCT (drug-treated group) - ΔCT (RNAiMAX control group)

[0468] Relative expression level of target gene = 2 - ΔΔCT

[0469] Based on the relative expression levels of the target genes after treatment, the inhibition rate of each candidate molecule on the target genes was obtained using the formula (1 - test group / control group)%.

[0470] ●Experimental Results

[0471] See the table below:

[0472] Table 6-1-1: Gene knockout activity of the tested compounds in cell transfection (target: CFB)

[0473] Table 6-1-2: Gene knockout activity of the tested compounds in cell transfection (target: CFB)

[0474] Table 6-1-3: Gene knockout activity of the tested compounds in cell transfection (target: CFB)

[0475] Table 6-2-1: Gene knockout activity of the tested compounds in cell transfection (target: DGAT2)

[0476] Table 6-2-2: Gene knockout activity of the tested compounds in cell transfection (target: DGAT2)

[0477] Table 6-3: Gene knockout activity of the tested compounds in cell transfection (target: INHBE)

[0478] Table 6-3-1

[0479] Table 6-3-2

[0480] Table 6-3-3

[0481] Table 6-4: Gene knockout activity of the tested compounds in cell transfection (target: C5)

[0482] Table 6-4

[0483] Table 6-5: Gene knockout activity of the tested compounds in cell transfection (target: Serpinc1)

[0484] Table 6-5

[0485] Table 6-6: Gene knockout activity of the tested compounds in cell transfection (target: C3)

[0486] Table 6-6-1

[0487] Table 6-7: Gene knockout activity of the tested compounds in cell transfection (target: APP)

[0488] Table 6-7-1

[0489] Table 6-7-2

[0490] Table 6-8-1: Gene knockout activity of the tested compounds in cell transfection (target: AGT)

[0491] Table 6-8-2: Gene knockout activity of the tested compounds in cell transfection (target: AGT)

[0492] Table 6-9: Gene knockout activity of the tested compounds in cell transfection (target: PCSK9)

[0493] Experimental conclusions

[0494] The specific modified templates used in this invention application have excellent effects when used for gene silencing.

[0495] Example 5: In vitro inhibitory activity of the test substance against the target gene in primary hepatocytes (in vitro free uptake assay)

[0496] ●Purpose

[0497] Using CPH cell line (primary monkey hepatocytes), the in vitro activity of siRNA molecules was evaluated by assessing the degree of silencing of target genes by each candidate molecule.

[0498] ●Cell line

[0499] CPH cells were provided by Myoshun (Shanghai) Biotechnology Co., Ltd. CPH resuscitation and culture required a hepatocyte culture medium kit (Myoshun, HCTPM-R-002).

[0500] ●Main Instruments

[0501] The main instruments used in this experiment included a fluorescence qPCR instrument (Roche 480 II), a centrifuge (Thermo Fisher catalog number 75016073), and a cell counter (Countstar Rigel 2).

[0502] ●Main reagents and consumables

[0503] The main reagents used in this experiment included an RNA extraction kit (Tiangen, catalog number DP671-T1), a FastKing cDNA first-strand synthesis kit (TianGen, catalog number KR116-02), a 96-well plate (Costar 3599), SuperReal PreMix Plus (SYBR Green) (TIANGEN, catalog number: FP215-02), qPCR primers for cCYPA, cCFB, cDGAT2, and cINHBE synthesized by Genewiz, and a hepatocyte culture medium kit (Miaoshun, HCTPM-R-002).

[0504] Primer information:

[0505] ●Experimental Methods

[0506] 4. Compound free uptake plate

[0507] Prepare the collagen coating solution 40 minutes in advance. Add 100 μL of rat tail collagen (AR0001-02) to the hepatocyte coating medium into each well of a 96-well cell plate. After 40 minutes, aspirate the coating solution from the cell plate, remove the cells from the liquid nitrogen container and place them in a 37°C water bath. When they are almost completely thawed, pour them into preheated hepatocyte resuscitation medium (HPM-R-40), centrifuge, and resuspend them in hepatocyte plating medium for cell counting.

[0508] Seed CPH cells (3×10) 4 Cells were transferred from one well to a coated 96-well cell plate. Simultaneously, siRNA was added to the cells. Seven concentration points were set for the siRNA assay (starting at 100 nM, 5-fold dilution). Cells were incubated at 37°C in a 5% CO2 incubator for 72 hours. A compound-free control group was also included.

[0509] 5. After 72 hours of RNA extraction and reverse transcription free uptake, remove the culture medium and collect the cells for RNA extraction. Use according to the kit instructions. Total RNA was extracted using the 96 Kit (Tiangen-DP671-T1). cDNA was synthesized using the FastKing RT Kit (With gDNase) (Tiangen-KR116-02) according to the manufacturer's instructions.

[0510] 6. qPCR detection of target gene mRNA expression levels

[0511] The target cDNA will be detected by qPCR, while CYPA cDNA will be detected as an internal control in parallel. 9 μL of the prepared PCR reaction solution and 1 μL of sample cDNA will be added to each of 384 wells. The qPCR program is as follows: heat at 95°C for 15 min, then cycle at 95°C for 10 sec, 60°C for 20 s, followed by 72°C for 32 s, for a total of 40 cycles.

[0512] ●Data Analysis

[0513] The expression level of the target gene mRNA in each sample was calculated using the ΔΔCt relative quantification method. The relative expression level of the target gene was expressed as 2-ΔΔCT.

[0514] The calculation formula is as follows: ΔCT = Average Ct value of target gene - Average Ct value of internal reference gene ΔCT = ΔCT (drug-treated group) - ΔCT (RNAiMAX control group)

[0515] Relative expression level of target gene = 2 - ΔΔCT

[0516] Based on the relative expression levels of the target genes after treatment, the inhibition rate of each candidate molecule on the target genes was obtained using the formula (1 - test group / control group)%.

[0517] ●Experimental Results

[0518] The percentage of inhibition of the target gene by each candidate molecule (compared to the control group) is shown in the table below.

[0519] Table 7-1: Gene knockout activity of tested compounds for free cellular uptake (target: CFB)

[0520] Table 7-2: Gene knockout activity of the tested compounds in cell transfection (target: DGAT2)

[0521] Table 7-3: Gene knockout activity of tested compounds for free cellular uptake (target: INHBE)

[0522] ●Experimental Conclusions

[0523] The specific modified templates used in this invention application have excellent effects when used for gene silencing.

[0524] Example 6: Evaluation of the knockdown efficiency of INHBE target gene expression using a mouse high-pressure tail vein injection model

[0525] Experimental Objective

[0526] The in vivo efficacy of the compounds of this invention was evaluated using a mouse model in which full-length human INHBE plasmid DNA was injected via high-pressure tail vein.

[0527] Experimental methods

[0528] Six- to seven-week-old female BALB / c mice were randomly assigned to groups and administered the drug (single subcutaneous injection; 2 mg / kg), with five mice in each group. Blank PBS was used as a negative control. Day 0 was defined as the day of drug administration, with the day before designated as Day -1, and the day after as Day 1, and so on. On Day 7, all mice received a tail vein injection within 5 seconds of a plasmid DNA solution equal to 8% of their body weight (injection volume (mL) = mouse body weight (g) × 8%), with each mouse receiving 10 μg of plasmid. On Day 8 (24 hours after INHBE plasmid injection), all mice were euthanized by CO2 inhalation. After euthanasia, liver samples were collected: two 70 mg portions of liver tissue were immersed in CO2. After incubation overnight at 4°C, the supernatant was discarded and transferred to a -80°C freezer. All liver samples were stored at -80°C until transferred to the laboratory on dry ice for appropriate testing.

[0529] Detection and data analysis

[0530] RNA was extracted from liver tissue using a fully automated nucleic acid extraction and purification system. The concentration of extracted liver RNA was detected using a Nanodrop ONE (Thermo) instrument and adjusted to a uniform concentration. Subsequently, it was... III. RT SuperMix for qPCR (+gDNA wiper) (Vazyme-R323): RNA was reverse transcribed into cDNA according to the instruction manual. cDNA was quantified by qPCR. INHBE gene expression was detected during qPCR. NEO mRNA (sequence information shown in the table below) was also detected as an internal control.

[0531] The experimental results are shown in Figure 9.

[0532] Experimental conclusions

[0533] The dsRNA provided in this application can significantly knock down the hINHBE gene in mice injected with full-length human INHBE plasmid DNA via high-pressure tail vein.

[0534] Example 7: Testing the knockdown efficiency of LPA target gene expression in vivo using humanized mice

[0535] Experimental Objective

[0536] The knockdown efficiency of siRNA drugs on target gene expression was tested using LPA humanized mice (B-hLPA mice).

[0537] Experimental materials

[0538] The experimental animals were 24 male B-hLPA C57BL / 6 mice, aged 5-6 weeks and weighing 25-30 grams (within a 5-gram difference), obtained from Biocytogen Jiangsu Gene Biotechnology Co., Ltd.

[0539] Experimental methods

[0540] Twenty-four B-hLPA mice were weighed and randomly assigned to experimental groups G1-G4 (n=6 per group) based on their weight. Day 0 was the day of group assignment, and drug administration began on Day 0. The drug observation period was four weeks. The specific dosing regimen is shown in the table below:

[0541] detection indicators

[0542] 1. Weight measurement:

[0543] The mice were weighed weekly after administration began.

[0544] 2. Sample collection and testing:

[0545] 1) Collect 130ul of non-anticoagulated blood on Day 7 / 3 before grouping, and on Day 7, Day 14, Day 21, and Day 28 after drug administration. Separate the serum and divide it into two portions. Fast for 5 hours before each blood collection. The order and time period of each blood collection should be consistent.

[0546] Serum should be stored at -80°C.

[0547] Serum Apo(a) levels were detected using the ELISA method.

[0548] 2) At the end of the experiment, the large lobe of the mouse liver was taken, and three 50mg portions were dispensed into RNAlater for treatment, while the remainder was frozen.

[0549] 3) At the experimental endpoint, take the maximum amount of blood, separate the serum, divide it into 3 portions, and store at -80℃ for subsequent testing.

[0550] 3. General clinical observation:

[0551] During the acclimatization and experimental periods, animals were observed once a day, including but not limited to their mental state and diet.

[0552] Data collection and statistical analysis

[0553] Raw data from measurements and observations must be recorded. Analysis is performed based on the raw data, and results are expressed as mean and standard error (Mean ± SEM). Statistical analysis of Apo(a) levels is conducted, with p < 0.05 considered statistically significant. Both statistical and biological significance are considered in the analysis of results.

[0554] The experimental results are shown in Figures 10 and 11.

[0555] Experimental conclusions

[0556] The dsRNA provided in this application can significantly inhibit the expression of the hLPA gene in humanized mice and suppress the level of human Apo(a) protein.

[0557] Example 8: NHP model to test the knockdown efficiency of LPA target gene expression in vivo

[0558] The in vivo target gene knockout effect of a specifically modified siRNA was evaluated in cynomolgus monkeys. On the day of administration, the animals were weighed, and the prepared formulation was drawn based on body weight and administration volume. The selected administration site (lower back) was disinfected. Two cynomolgus monkeys (N=2) were used, and the test compound Z19 was administered via single subcutaneous injection at the same site at a dose of 3 mg / kg. Hemostasis was achieved using dry cotton balls after administration. Blood samples were collected before administration and on days 7, 14, 21, 28, 56, 85, and 113. Blood samples were collected after disinfecting the blood collection site with alcohol swabs and hemostasis was achieved using dry cotton balls. The collected whole blood was sent to the clinical laboratory for centrifugation to obtain serum.

[0559] Serum collected and centrifuged after blood collection was subjected to blood biochemistry analysis using a HITACHI 008AS biochemical analyzer. After powering on the analyzer, reagents were replaced, followed by quality control and / or calibration. Once the instrument was confirmed to meet operational requirements, the samples were placed on the sample tray, the animal tattoo number on the sample label was verified, and the detection of Lp(a), LDL-c, and ApoB was initiated. Data are expressed as a percentage of baseline values ​​and presented as the mean plus / minus the standard error of the mean.

[0560] Test compound Z19:

[0561] The experimental results are shown in Figure 12-14.

[0562] Experimental conclusions

[0563] The specific modified compounds of this invention can inhibit LPA targets on NHP and exhibit corresponding therapeutic effects.

[0564] Example 9: Compound Stability Study

[0565] Experimental steps:

[0566] Take an appropriate amount of the compound and place it in a low-density polyethylene vial. Add an appropriate amount of diluent (physiological saline + 0.5 mmol / L sodium dihydrogen phosphate + 0.5 mmol / L disodium hydrogen phosphate) to dissolve it and dilute it to prepare a solution of approximately 1 mg / mL. Tighten the cap to obtain the sample solution. Place the sample solution at 40°C for 10 days to allow it to degrade before performing liquid chromatography analysis. Prepare another fresh sample solution using the same method as a non-degraded control and perform liquid chromatography analysis simultaneously.

[0567] The chromatographic detection method is as follows: Ultra-high performance liquid chromatography (UHPLC) was used with octadecylsilane-bonded silica gel as the packing material (Waters Acquity UPLC BEH C18 column, 2.1 mm × 150 mm, 1.7 μm or equivalent column). Gradient elution was performed using an aqueous solution of 90–110 mmol / L hexafluoroisopropanol and 15–25 mmol / L triethylamine as mobile phase A and acetonitrile as mobile phase B. The flow rate was 0.3 mL per minute, the column temperature was 80 °C, and the detection wavelength was 260 nm.

[0568] The impurity detection results of the sample damaged at 40℃ were compared with those of the undamaged sample to calculate the impurity growth value. The dsRNA and its conjugates of this invention exhibit good chemical stability.

[0569] Example 10: Synthesis of ligand precursors

[0570] Example 10.1 Synthesis of the precursor of VSDL-01 (diisopropylaminophosphite-2-cyanoethyl dodecyl ester)

[0571] To a solution of compound 1-1 (10.00 g, 30.62 mmol) in anhydrous DCM (100 mL), 1H-imidazolium-4,5-dicarboxynitrile (1.81 g, 15.31 mmol) was added, followed by dropwise addition of a solution of compound 1-2 (9.23 g, 30.62 mmol) in anhydrous DCM (20 mL). The reaction mixture was stirred at 25 °C under N2 for 16 hours. The mixture was concentrated under vacuum to remove DCM. The residue was purified by silica gel chromatography (eluting with hexane:EtOAc = 5:1 containing 1% TEA) to give the precursor of VSDL-01.

[0572] 1H NMR (400MHz, CDCl3) δ3.91-3.72(m,2H),3.68-3.51(m,4H),2.63(t,J=6.8Hz,2H),1.58( p,J=6.8Hz,2H),1.30-1.23(m,38H),1.17(dd,J=6.8,4.2Hz,12H),0.87(t,J=6.8Hz,3H).

[0573] 31 P NMR: (400MHz, CDCl3) δ 147.17.

[0574] Example 10.2 Synthesis of the precursor of VSDL-02 (diisopropylaminophosphite-2-cyanoethyleicosyl ester)

[0575] The synthesis method is similar to that in Example 10.1.

[0576] 1 H NMR (400MHz, CDCl3) δ3.92-3.72(m,2H),3.66-3.50(m,4H),2.62(t,J=6.8Hz,2H),1.60( p,J=6.8Hz,2H),1.28-1.24(m,34H),1.17(dd,J=6.8,4.2Hz,12H),0.88(t,J=6.8Hz,3H).

[0577] 31 P NMR: (400MHz, CDCl3) δ 147.32.

[0578] Example 10.3 Synthesis of the precursor of VSDL-03 (diisopropylaminophosphite-2-cyanoethylhexadecyl ester)

[0579] The synthesis method is similar to that in Example 10.1.

[0580] 1 H NMR (400MHz, CDCl3) δ3.90-3.72(m,2H),3.68-3.52(m,4H),2.63(t,J=6.8Hz,2H),1.59( p,J=6.8Hz,2H),1.28-1.24(m,26H),1.17(d,J=6.8,4.2Hz,12H),0.87(t,J=6.8Hz,3H).

[0581] 31 P NMR: (400MHz, CDCl3) δ 147.24.

[0582] Example 10.4 Synthesis of the precursor of VSDL-04 (2-cyanoethyl (6-eicosamidohexyl) diisopropylaminophosphite)

[0583] Trifluoroacetic acid pentafluorophenyl ester (4.2 g, 15 mmol) was added dropwise to a solution of compound 4-1 (3.12 g, 10 mmol) and TEA (3.04 g, 30 mmol) in anhydrous DCM (200 mL). After stirring for 15 min, compound 4-2 (1.4 g, 12 mmol) was added to the mixture. The reaction mixture was stirred at 25 °C for 16 h. The mixture was concentrated under vacuum to remove DCM. The residue was purified by silica gel chromatography (eluting with hexane:EtOAc = 5:1) to give compound 4-3 (3.82 g).

[0584] A solution of compound 4-3 (3.63 g, 8.82 mmol) in anhydrous DMF (60 mL) was added to a solution of 1H-imidazolium-4,5-dicarboxynitrile (0.52 g, 4.41 mmol), followed by dropwise addition of a solution of compound 1-2 (2.65 g, 8.82 mmol) in anhydrous DMF (5 mL). The reaction mixture was stirred at 25 °C under N2 for 16 hours. The mixture was washed with saturated NaHCO3 aqueous solution (200 mL), extracted with EtOAc (3 × 50 mL), and the organic phases were combined, dried over anhydrous Na2SO4, and concentrated under vacuum. The residue was purified by silica gel chromatography (eluting with hexane:EtOAc = 5:1 containing 1% TEA) to give the precursor of VSDL-04.

[0585] 1 H NMR (400MHz, CDCl3) δ5.45(t,J=5.6Hz,1H),3.92-3.75(m,2H),3.70-3.54(m,4H),3.23(q,J=6.8Hz,2H),2.64(t,J=6.4Hz,2H),2. 14(t,J=7.6Hz,2H),1.63-1.57(m,4H),1.53-1.56(m,2H),1.28-1.24(m,36H),1.17(dd,J=6.8,4.2Hz,12H),0.87(t,J=6.8Hz,3H).

[0586] 31 P NMR: (400MHz, CDCl3) δ 147.32.

[0587] Example 10.5 Synthesis of the precursor of VSDL-05 (2-cyanoethyl (6-stearamidohexyl) diisopropylaminophosphite)

[0588] The synthesis method is similar to that in Example 10.4.

[0589] 1 H NMR (400MHz, CDCl3) δ5.47(t,J=5.6Hz,1H),3.89-3.75(m,2H),3.63-3.67(m,4H),3.23(q,J=6.8Hz,2H),2.64(t,J=6.4Hz,2H),2. 14(t,J=7.6Hz,2H),1.66-1.57(m,4H),1.51-1.46(m,2H),1.27-1.23(m,32H),1.17(dd,J=6.8,4.2Hz,12H),0.87(t,J=6.8Hz,3H).

[0590] 31 P NMR: (400MHz, CDCl3) δ 147.20.

[0591] Example 10.6 Synthesis of the precursor of VSDL-06 (2-cyanoethyl (6-palmitamidohexyl) diisopropylaminophosphite)

[0592] The synthesis method is similar to that in Example 10.4.

[0593] 1 H NMR (400MHz, CDCl3) δ5.69(t,J=5.6Hz,1H),3.90-3.75(m,2H),3.69-3.54(m,4H),3.26-3.21(m,2H),2.65(t,J=6.4Hz,2H),2.15 (t,J=7.6Hz,2H),1.65-1.58(m,4H),1.54-1.47(m,2H),1.28-1.24(m,28H),1.18(dd,J=6.8,4.2Hz,12H),0.88(t,J=6.8Hz,3H).

[0594] 31 P NMR: (400MHz, CDCl3) δ 147.25.

[0595] Example 10.7 Synthesis of VSDL-07 precursor (diisopropylaminophosphite 2-cyanoethyl (6-((5Z,8Z,11Z,14Z,17Z)-eicosano-5,8,11,14,17-pentenamido)hexyl) ester)

[0596] The synthesis method is similar to that in Example 10.4.

[0597] 1 H NMR (400MHz, CDCl3) δ5.52-5.24(m,11H),3.89-3.74(m,2H),3.69-3.53(m,4H),3.25-3.20(m,2H),2.85-2.78(m,8H),2.63(t,J=6.5Hz,2H),2.1 7-2.03(m,6H),1.74-1.68(m,2H),1.64-1.57(m,2H),1.53-1.46(m,2H) ,1.42-1.31(m,4H),1.17(dd,J=6.8,4.2Hz,12H),0.97(t,J=7.6Hz,3H). 31 P NMR: (400MHz, CDCl3) δ 147.32.

[0598] Example 10.8 Synthesis of the precursor of VSDL-09 (diisopropylaminophosphite di(hexadecyl) ester)

[0599] TEA (1.376 mL, 9.898 mmol) was added to a solution of compound 9-1 (1 g, 4.949 mmol) in anhydrous THF (5 mL). The reaction mixture was cooled to 0 °C by an ice bath, and then compound 3-1 (2.934 mL, 9.898 mmol) was added dropwise under nitrogen protection. The system temperature was maintained at approximately 0 °C during the addition. After the addition, a suspension containing a white solid was obtained. The ice bath was removed and the mixture was allowed to return to room temperature, and stirring was continued at room temperature for 3 hours. The mixture was filtered, the filter cake was washed with THF, and the filtrate was collected and concentrated under vacuum to give the precursor of VSDL-09.

[0600] 1 H NMR (400MHz, CDCl3) δ3.68-3.52(m,6H),1.64-1.57(m,4H),1.36-1.25(m,52H),1.18(d,J=6.8Hz,12H),0.88(t,J=6.8Hz,6H).

[0601] 31 P NMR: (400MHz, CDCl3) δ 144.91.

[0602] Example 10.9 Synthesis of VSDL-10 precursor (butyl hexadecyl diisopropylaminophosphite)

[0603] TEA (3.13 mL, 22.49 mmol) was added to a solution of compound 10⁻² (4 g, 15 mmol) in anhydrous THF (10 mL). The reaction mixture was cooled to 0 °C in an ice bath, and then compound 10⁻¹ (1.11 g, 15 mmol) was added dropwise under nitrogen protection. The system temperature was maintained at approximately 0 °C during the addition. After the addition, stirring was continued at room temperature for 3 hours. The mixture was concentrated under vacuum to give compound 10⁻³, which could be used directly in the next step without further purification.

[0604] To a solution of compound 3-1 (1.33 g, 5.47 mmol) in anhydrous DCM (10 mL), 1H-imidazolium-4,5-dicarboxynitrile (0.32 g, 2.74 mmol) was added, followed by dropwise addition of a solution of compound 10-3 (2.5 g, 8.21 mmol) in anhydrous DCM (5 mL). The reaction mixture was stirred at 25 °C under N2 for 16 hours. The mixture was concentrated under vacuum. The residue was purified by silica gel chromatography (eluting with hexane:EtOAc = 5:1 containing 1% TEA) to give the precursor of VSDL-10.

[0605] 1 H NMR (400MHz, CDCl3) δ3.69-3.51(m,6H),1.64-1.55(m,4H),1.44-1.22(m,28H),1.17(d,J=6.8Hz,12H),0.90(dt,J=14.0,7.2Hz,6H).

[0606] 31 P NMR: (400MHz, CDCl3) δ 145.00.

[0607] Example 10.10 Synthesis of VSDL-12 precursor (diisopropylaminophosphite cetyl octyl ester)

[0608] TEA (3.13 mL, 22.49 mmol) was added to a solution of compound 10-2 (4 g, 15 mmol) in anhydrous THF (10 mL). The reaction mixture was cooled to 0 °C in an ice bath, and then compound 12-1 (1.95 g, 15 mmol) was added dropwise under nitrogen protection. The system temperature was maintained at approximately 0 °C during the addition. After the addition, stirring was continued at room temperature for 3 hours. The mixture was concentrated under vacuum to give compound 12-2, which could be used directly in the next step without further purification. 1H-imidazolium-4,5-dicarboxynitrile (0.39 g, 3.33 mmol) was added to a solution of compound 3-1 (1.61 g, 6.66 mmol) in anhydrous DCM (10 mL), and then compound 12-2 (3.6 g, 9.98 mmol) in anhydrous DCM (5 mL) was added dropwise to the mixture. The reaction mixture was stirred at 25 °C under N2 for 16 hours. The mixture was concentrated under vacuum. The residue was purified by silica gel chromatography (eluting with hexane:EtOAc = 5:1 containing 1% TEA) to obtain the precursor of VSDL-12.

[0609] 1 H NMR (400MHz, CDCl3) δ3.69-3.52(m,6H),1.64-1.56(m,4H),1.37-1.25(m,36H),1.17(d,J=6.8Hz,12H),0.90-0.86(m,6H).

[0610] 31 P NMR: (400MHz, CDCl3) δ 144.97.

[0611] Example 10.11 Synthesis of VSDL-13 precursor (diisopropylaminophosphite dioctyl ester)

[0612] The synthesis method is similar to that in Example 10.8.

[0613] 1 H NMR (400MHz, CDCl3) δ3.67-3.51(m,6H),1.64-1.57(m,4H),1.37-1.26(m,20H),1.18(d,J=6.8Hz,12H),0.89-0.85(m,6H).

[0614] 31 P NMR: (400MHz, CDCl3) δ 145.00.

[0615] Example 10.12 Synthesis of VSDL-03A precursor (2-cyanoethylhexadecane-8-yldiisopropylphosphonamide)

[0616] To a 20 mL solution of anhydrous DCM containing compound 13-1 (5.00 g, 20.62 mmol), diisopropylammonium tetrazolium (1.77 g, 10.31 mmol) was added, followed by dropwise addition of a 20 mL solution of anhydrous DCM containing compound 1-2 (6.22 g, 20.62 mmol). The reaction mixture was stirred at 25 °C under nitrogen for 16 hours. The mixture was concentrated under vacuum to remove DCM. The residue was purified by silica gel column chromatography (eluting with n-hexane:ethyl acetate containing 1% triethylamine in a 5:1 ratio) to give compound 13.

[0617] 1 H NMR (400MHz, CDCl3) δ3.90-3.69(m,3H),3.64-3.55(m,2H),2.62(t,J=8.0Hz,2H),1. 57-1.47(m,4H),1.32-1.26(m,22H),1.18(dd,J=8.0,4.0Hz,12H),0.90-0.86(m,6H).

[0618] 31 P NMR: (400MHz, CDCl3) δ 147.06.

[0619] Example 11 Synthesis of end-capped group precursor

[0620] Example 11.1 Synthesis of the precursor of M06 (2-cyanoethyl (2-morpholinoethyl) diisopropylaminophosphite)

[0621] 1H-imidazolium-4,5-dicarboxynitrile (4.9 g, 41.53 mmol) was added to a solution of compound 15-1 (10.88 g, 83.05 mmol) in anhydrous DCM (100 mL). The reaction mixture was cooled to 0 °C, and then a solution of DCM (30 mL) containing compound 1-2 (25 g, 83.05 mmol) was added to the mixture. The mixture was stirred at 25 °C under N2 for 16 hours. The mixture was filtered and concentrated to remove DCM. The residue was diluted with hexane and purified by silica gel chromatography (eluting with hexane) to give the precursor of MO6.

[0622] 1H NMR: (400MHz, CDCl3) δ3.87-3.75(m,3H),3.71-3.65(m,5H),3.62-3.53(m,2H),2.65-2.57(m,4H),2.54-2.45(m,4H),1.17(dd,J=8.0,4.0Hz,12H).

[0623] 31 P NMR: (160MHz, CDCl3) δ 148.02.

[0624] Example 11.2 Synthesis of the precursor of MO3 (diisopropylaminophosphite 2-cyanoethyl ((tetrahydro-2H-pyran-4-yl)methyl) ester)

[0625] 1H-imidazolium-4,5-dicarboxynitrile (0.6 g, 5 mmol) was added to a solution of compound 16-1 (1.16 g, 10 mmol) in anhydrous DCM (20 mL). The reaction mixture was cooled to 0 °C, and then a solution of DCM (5 mL) containing compound 1-2 (3 g, 10 mmol) was added to the mixture. The mixture was stirred at 25 °C under N2 for 16 hours. The mixture was filtered and concentrated to remove DCM. The residue was diluted with hexane and purified by silica gel chromatography (eluting with hexane) to give the precursor of MO3.

[0626] 1 H NMR: (400MHz, CDCl3) δ3.98-3.94(m,2H),3.88-3.73(m,2H),3.61-3.34(m,6H),2.62(t,J=8.0 Hz,2H),1.88-1.78(m,1H),1.66-1.59(m,2H),1.39-1.31(m,2H),1.17(dd,J=8.0,4.0Hz,12H). 31 P NMR: (160MHz, CDCl3) δ 147.82.

[0627] Example 12 Bioactivity analysis of double-stranded oligonucleotides

[0628] Example 12.1 Huh7 cell transfection analysis

[0629] Prepare cell suspensions (2 x 10) using 10% FBS cell culture medium. 5 Cells / mL). Add 20 μL / well of siRNA-RNAiMAX complex to a 96-well plate, followed by cell suspension (100 μL / well). Incubate cells at 37°C and 5% CO2 for 24 hours.

[0630] Intracellular RNA was isolated using the RNeasy kit (Qiagen-74182) according to the instruction manual. For qPCR, [the kit was used] according to the instruction manual. III. RT SuperMix (+gDNA wiper) was used for reverse transcription of RNA. qPCR was performed using gene-specific primers (e.g., commercially available primers shown below). The expression levels of the target gene were quantified using an analysis (or primer). GAPDH was also measured as a housekeeping gene. Samples were processed in an Applied Biosystems rapid real-time PCR system for TaqMan cycling according to the following thermal profile: 95°C for 10 minutes, then 95°C for 15 seconds, 60°C for 1 minute, for 40 cycles.

[0631] Available for commercial purchase analyze:

[0632] Other primers:

[0633] Table 6: Gene knockout activity of test compounds in Huh 7 transfection analysis

[0634] Table 7: Gene knockout activity of test compounds in Huh 7 transfection analysis

[0635] Table 8: Gene knockout activity of test compounds in Huh 7 transfection analysis

[0636] Table 9: Gene knockout activity of test compounds in Huh 7 transfection analysis

[0637] Relative IC 50 = (ds X) / ds67

[0638] Table 10: Gene knockout activity of test compounds in Huh 7 transfection analysis

[0639] Relative IC 50 = (ds X) / ds74

[0640] As shown in the data above, the dsRNA reagents presented in this paper exhibit effective inhibitory activity against target gene expression. For dsRNA reagents that cross-target different genes, those containing purine (preferably guanine) at position Z show better inhibitory activity than those containing pyrimidine at position Z. It can be seen that dsRNA reagents containing adenine or uracil at X1 / X2 show effective inhibitory activity, while those containing cytosine or guanine at X1 / X2 show relatively moderate activity.

[0641] Surprisingly, dsRNA reagents with capping groups (e.g., M03, M06, or invAB) or ligands (e.g., VSDL-03A) at the 5' end of the antisense strand exhibit inhibitory activity against target gene expression, while other reported dsRNA reagents significantly or completely lose their activity when the 5' end of the antisense strand is attached to a capping group or ligand. While not wishing to be bound by theory, one plausible explanation is that the capping group or ligand may cleave together with the cleavage region of the dsRNA presented in this paper, subsequently providing RNAi activity.

[0642] Example 12.2 PHH Free Uptake KD Analysis

[0643] Primary human hepatocytes (PHH) were mixed with an appropriate culture medium, and the cell suspension was then adjusted to a final cell density of 6 × 10⁵ cells / mL. dsRNA was diluted and added at 10 μL / well to collagen-I-coated 96-well plates, followed by the addition of cell suspension (90 μL / well). Cells were cultured at 37 °C and 5% CO₂ for 48 hours.

[0644] Intracellular RNA was isolated using the RNeasy kit (Qiagen-74182) according to the instruction manual. For qPCR, [the kit was used] according to the instruction manual. III. RT SuperMix (+gDNA wiper) was used for reverse transcription of RNA. qPCR was performed using gene-specific primers (e.g., commercially available primers shown below). The expression levels of the target gene were quantified using an analysis (or primer). GAPDH was also measured as a housekeeping gene. Samples were processed in an Applied Biosystems rapid real-time PCR system for TaqMan cycling according to the following thermal profile: 95°C for 10 minutes, then 95°C for 15 seconds, 60°C for 1 minute, for a total of 40 cycles.

[0645] Available for commercial purchase analyze:

[0646] Other primers:

[0647] Table 11: Gene knockout activity of test compounds in free uptake analysis of primary human hepatocytes

[0648] As shown in the table above, the cleavage regions containing both 2'-F and 2'-OMe exhibit effective inhibitory activity. In a preferred embodiment, X2 and Y are modified with 2'-OMe, and Z and N1 are modified with 2'-F.

[0649] It can also be seen that nucleotide internucleotide bond modifications between X2 and Y or Y and Z reduce the repressive activity of dsRNA.

[0650] Table 12: Free uptake analysis of primary human hepatocytes

[0651] As shown in the table above, compared with parental dsRNA reagents without a 5' extension, the dsRNA reagents presented in this paper, which have a 5' extension on the antisense strand and a cleavage region, show relatively more effective inhibitory activity against different genes and targets.

[0652] Example 12.3 In vivo mouse HDI model

[0653] On day 0, mice (BALB / c, 6-7 weeks old, female) were administered the drug or test siRNA (5 mg / kg). On day 3, all mice were injected intravenously via tail vein with 8% of their body weight of plasmid DNA solution (injection volume (mL) = mouse body weight (g) × 8%). The mass of plasmid injected into each mouse was 10 μg. All animals were sacrificed on day 4. Liver tissue was collected from all groups for target mRNA analysis using qPCR. The appropriate qPCR assay was performed according to the manual. The III RT SuperMix (+gDNA wiper) (Vazyme-R323) reverse transcribed RNA into cDNA. The cDNA was quantified by qPCR. NEO (sequence information shown in the table below) mRNA was also detected as an internal control.

[0654] Test compound:

[0655] The results are shown in Figure 1.

[0656] As shown in Figure 1, compared with parental dsRNA reagents without a 5' extension, the dsRNA reagents provided in this paper, which have a 5' extension on the antisense strand and a cleavage region, show significantly more effective inhibitory efficacy against the target gene.

[0657] Example 12.4 In vivo rat CNS target gene knockout model (IT injection)

[0658] The test compound was prepared in 10 mM PBS (pH 7.4) at a maximum concentration of 20 mg / mL and administered via lumbar puncture in 50 μL of IT injection (0.9 mg / dose). Tail wagging or tail tip shaking was considered a sign of successful administration. After administration, the anesthesia maintenance device was removed, and the animal was returned to its cage. Animals were sacrificed on day 14, and tissue samples were collected for qPCR analysis.

[0659] SOD1 mRNA levels were measured by qPCR.

[0660] RNA was extracted from tissue samples using an automated nucleic acid extraction system. The samples were then transferred to fresh RNase-free test tubes for cDNA synthesis and qPCR.

[0661] Test compound:

[0662] The results are shown in Figures 2 and 3.

[0663] As shown in Figures 2 and 3, compared with parental dsRNA reagents modified with 5'VPu and Uhd, the dsRNA reagents presented in this paper, which have a 5' extension, a cleavage region, and a VSDL ligand on the antisense strand, exhibit comparable inhibitory efficacy against the target genes. However, compared with dsRNA reagents modified with 5'VPu and Uhd, the dsRNA reagents presented in this paper show more tissue-specific target gene knockout.

[0664] Example 12.5 In vivo mouse IVT model

[0665] One day before and two days after IVT injection in mice, a topical antibiotic (tobramycin) was administered twice to both eyes. On day 0, 3 μg of siRNA prepared in 1×PBS or 1×PBS was administered to both eyes via IVT injection. All animals were sacrificed on day 7, and samples of the entire eye were taken for qPCR analysis.

[0666] Test compound:

[0667] The results are shown in Figure 4.

[0668] As shown in Figure 4, compared with parental dsRNA reagents with 5'VPu modification and Uhd, the dsRNA reagents with a 5' extension, cleavage region, and VSDL ligand on the antisense strand presented in this paper show stronger inhibitory efficacy against the target gene.

[0669] Example 12.6 Homogenization reaction of rat liver tissue

[0670] A precise 1.00 mg test compound was dissolved in 0.961 mL of water to obtain a working solution with a calibrated concentration of 1,000,000 ng / mL. A final sample with a concentration of 10,000 ng / mL was obtained by homogenizing the working solution with 20% rat liver tissue and then incubating at 37 °C for 48 h. 50.0 μL of IS working solution was added to the cultured sample for extraction (phenol / chloroform / isoamyl alcohol = 25 / 24 / 1, v / v / v). Liquid-liquid extraction was performed. After centrifugation, 300 μL of the supernatant was transferred to solid-phase extraction. The sample was then transferred to an equilibrated SPE plate, which was washed and eluted. The collected eluent was evaporated. (LC-HRMA: High-resolution liquid chromatography-mass spectrometry; LC: Shimadzu, LC-30AD; HRMA: Q Exactive Plus or Q Exactive Focus (Thermo San Jose, CA))

[0671] Test compound:

[0672] The results are shown in Figure 5. After 48 hours of incubation, the observed products included unconsumed test compounds (31%), test compounds with one nucleotide cleaved at the 3' end (15%), test compounds with two nucleotide cleaved at the 3' end and M06 cleaved (18%), test compounds with three nucleotide cleaved at the 5' end (15% + 5% + 10%), and other minor metabolites (5%).

[0673] In summary, (1) the only observed 5' cleavage product was the test compound with three nucleotides cleaved at the 5' end; (2) the observed 3' cleavage products included the test compound with one or two nucleotides cleaved at the 3' end; and (3) the cleavage product of the test compound with three nucleotides cleaved at the 5' end was the major product among all cleavage products.

[0674] Example 12.7 Long-term plasma target gene knockout model of NHP

[0675] The in vivo target gene knockout effect of modified siRNA was evaluated in cynomolgus monkeys. Animals (N=2 per group) received a single subcutaneous dose of dsRNA reagent at 2 mg / kg on day 1. Blood samples were obtained on days -1, 2, 7, 14, 28, 42, 56, and 70 post-administration. Circulating AGT levels were quantified using an ELISA (Sino Biological KIT10994) that is specific to human angiotensinogen (and cross-reactive with cynomolgus monkeys), according to the manufacturer's protocol. Data are presented as a percentage of baseline values ​​and are presented as the mean plus / minus the standard error of the mean.

[0676] Test compound:

[0677] The results are shown in Figure 6.

[0678] Example 12.8 Repeated-dose tolerance study in rats

[0679] Repeated-dose tolerance studies were conducted in rats. The test compounds (ds86, ds99) were administered subcutaneously once weekly at doses above 250 mg / kg for 3 weeks (a total of 3 doses). (300 mg / kg*1, 250 mg / kg*2).

[0680] Results: No abnormalities were found in clinical observation, pathology, and gross anatomy.

[0681] Histopathology:

[0682] Kidneys: Mild basophilic granules and renal tubular cell vacuolation were present in both groups.

[0683] Liver: Mild to moderate vacuolation and pigmentation of hepatocytes and Kupffer cells were present in both groups.

[0684] Injection site: very mild to moderate mixed cellular inflammation.

[0685] Conclusion: The results were consistent with the uptake and clearance of GalNAc siRNA and were not considered adverse.

Claims

1. A dsRNA comprising a sense strand and an antisense strand, wherein the sense strand and the antisense strand are each independently 17 to 35 nucleotides in length, wherein: Each nucleotide may be independently and optionally modified. The sense strand comprises a first strand, and the antisense strand comprises a second strand. The first and second strands are anti-complementary. Starting from the 5' end of the first strand, positions 9, 11, and 13 of the first strand are nucleotides containing a 2'-F modification, and optionally position 15 of the first strand is a nucleotide containing a 2'-F modification. Starting from the 5' end of the second strand, position 14 of the second strand is a nucleotide containing a 2'-F modification.

2. The dsRNA according to claim 1, wherein each of the nucleotides is a modified nucleotide.

3. The dsRNA according to claim 1 or 2, wherein the modified nucleotide is selected from nucleotides containing 2'-F modification, nucleotides containing 2'-deoxy modification, and nucleotides containing 2'-oxymethyl modification.

4. The dsRNA according to any one of claims 1-3, wherein, counting from the 5' end, position 2 of the second strand is a nucleotide containing a 2'-deoxy modification or a nucleotide containing a 2'-F modification.

5. The dsRNA according to any one of claims 1-4, wherein positions 5, 7, and 12 of the second strand, counting from the 5' end, are nucleotides containing a 2'-deoxy modification.

6. The dsRNA according to any one of claims 1-5, wherein, counting from the 5' end, the 18th position of the first strand is a nucleotide containing a 2'-F modification.

7. The dsRNA according to any one of claims 1-6, wherein: Starting from the 5' end, positions 2, 5, 7, and 12 of the second strand are nucleotides containing a 2'-deoxy modification; or Starting from the 5' end, position 2 of the second strand is a nucleotide containing a 2'-F modification, and starting from the 5' end, positions 5, 7, and 12 of the second strand are nucleotides containing a 2'-deoxy modification.

8. The dsRNA according to any one of claims 1-7, wherein, counting from the 5' end, positions 2 and 16 of the second strand are nucleotides containing a 2'-F modification.

9. The dsRNA according to claim 8, wherein: Starting from the 5' end, the 6th position of the second strand is a nucleotide containing a 2'-F modification; Starting from the 5' end, the 8th position of the second strand is a nucleotide containing a 2'-F modification; Starting from the 5' end, the 9th position of the second strand is a nucleotide containing a 2'-F modification; and / or Starting from the 5' end, position 14 of the second strand is a nucleotide containing a 2'-F modification.

10. A dsRNA comprising a sense strand and an antisense strand, wherein the sense strand and the antisense strand are each independently 17 to 35 nucleotides in length, wherein: Each nucleotide may be independently and optionally modified. The sense strand comprises a first strand, and the antisense strand comprises a second strand. The first and second strands are anti-complementary. Starting from the 5' end of the first strand, position 9 of the first strand is a nucleotide containing a 2'-F modification, and optionally position 11 of the first strand is a nucleotide containing a 2'-F modification. Starting from the 5' end of the second strand, positions 2 and 14 of the second strand are nucleotides containing 2'-F modification, and positions 5, 7, and 12 of the second strand are nucleotides containing 2'-deoxy modification.

11. The dsRNA of claim 10, wherein each of the nucleotides is a modified nucleotide.

12. The dsRNA according to claim 10 or 11, wherein the modified nucleotide is selected from nucleotides containing 2'-F modification, nucleotides containing 2'-deoxy modification, and nucleotides containing 2'-oxymethyl modification.

13. The dsRNA according to any one of claims 10-12, wherein: Starting from the 5' end, the 10th position of the first strand is a nucleotide containing a 2'-F modification; Starting from the 5' end, position 13 of the first strand is a nucleotide containing a 2'-F modification; or Starting from the 5' end, positions 13 and 15 of the first strand are nucleotides containing a 2'-F modification.

14. A dsRNA comprising a sense strand and an antisense strand, wherein the sense strand and the antisense strand are each independently 17 to 35 nucleotides in length, wherein: Each nucleotide may be independently and optionally modified. The sense strand comprises a first strand, and the antisense strand comprises a second strand. The first and second strands are anti-complementary, and starting from the 5' end of the first strand, positions 9, 10, and 11, or positions 9, 11, and 13, of the first strand are nucleotides containing a 2'-F modification; and Starting from the 5' end of the second strand, position 2 of the second strand is a nucleotide containing a 2'-deoxy modification or a nucleotide containing a 2'-F modification, positions 5, 7, and 12 of the second strand are nucleotides containing a 2'-deoxy modification, and position 14 of the second strand is a nucleotide containing a 2'-F modification.

15. The dsRNA of claim 14, wherein each of the nucleotides is a modified nucleotide.

16. The dsRNA according to claim 14 or 15, wherein the modified nucleotide is selected from nucleotides containing 2'-F modification, nucleotides containing 2'-deoxy modification, and nucleotides containing 2'-oxymethyl modification.

17. The dsRNA according to any one of claims 14-16, wherein position 2 of the second strand is a nucleotide modified with 2'-F, and / or position 15 of the first strand is a nucleotide containing 2'-F modification.

18. A dsRNA comprising a sense strand and an antisense strand, wherein the sense strand and the antisense strand are each independently 17 to 35 nucleotides in length, wherein: Each nucleotide may be independently and optionally modified. The sense strand comprises a first strand, and the antisense strand comprises a second strand. The first and second strands are anti-complementary, and starting from the 5' end of the first strand, positions 9, 10, and 11, or positions 9, 11, and 13, of the first strand are nucleotides containing a 2'-F modification; and Starting from the 5' end of the second strand, positions 2, 14, and 16 of the second strand are nucleotides containing a 2'-F modification.

19. The dsRNA of claim 18, wherein each of the nucleotides is a modified nucleotide.

20. The dsRNA according to claim 18 or 19, wherein the modified nucleotide is selected from nucleotides containing 2'-F modification, nucleotides containing 2'-deoxy modification, and nucleotides containing 2'-oxymethyl modification.

21. The dsRNA according to any one of claims 18-20, wherein, (i) Starting from the 5' end, position 6 of the second strand is a nucleotide containing a 2'-F modification, or (ii) Starting from the 5' end, positions 9, 11, and 13 of the first strand are nucleotides containing a 2'-F modification, and starting from the 5' end, position 7 of the second strand is a nucleotide containing a 2'-F modification.

22. The dsRNA according to claim 21, wherein, The 8th and 9th positions of the second strand are nucleotides containing 2'-F modification.

23. The dsRNA according to claim 21 or 22, wherein, The 7th position of the first strand is a nucleotide containing a 2'-F modification.

24. A dsRNA comprising a sense strand and an antisense strand, wherein the sense strand and the antisense strand are each independently 17 to 35 nucleotides in length, wherein: Each nucleotide may be independently and optionally modified. The sense strand comprises a first strand, and the antisense strand comprises a second strand. The first and second strands are anti-complementary. Starting from the 5' end of the first strand, positions 9, 10, and 11 of the first strand are nucleotides containing a 2'-F modification. Starting from the 5' end of the second strand, positions 2, 5, 7, and 12 of the second strand are nucleotides containing 2'-deoxy modification, and position 14 of the second strand is a nucleotide containing 2'-F modification.

25. The dsRNA of claim 24, wherein each of the nucleotides is a modified nucleotide.

26. The dsRNA according to claim 24 or 25, wherein the modified nucleotide is selected from nucleotides containing 2'-F modification, nucleotides containing 2'-deoxy modification, and nucleotides containing 2'-oxymethyl modification.

27. A dsRNA comprising a sense strand and an antisense strand, wherein the sense strand and the antisense strand are each independently 17 to 35 nucleotides in length, wherein: Each nucleotide may be independently and optionally modified. The sense strand comprises a first strand, and the antisense strand comprises a second strand. The first and second strands are anti-complementary. Starting from the 5' end of the first strand, positions 9, 10, and 12 of the first strand are nucleotides containing a 2'-F modification. Starting from the 5' end of the second strand, positions 2, 6, 14, and 16 of the second strand are nucleotides containing a 2'-F modification.

28. The dsRNA of claim 27, wherein each of the nucleotides is a modified nucleotide.

29. The dsRNA according to claim 27 or 28, wherein the modified nucleotide is selected from nucleotides containing 2'-F modification, nucleotides containing 2'-deoxy modification, and nucleotides containing 2'-oxymethyl modification.

30. The dsRNA according to any one of the preceding claims, wherein: The length of the first chain is 15, 16, 17, 18, 19, 20, 21, 22 or 23 nucleotides; The second strand has a length of 15, 16, 17, 18, 19, 20, 21, 22, or 23 nucleotides; and / or The first chain and the second chain have the same length.

31. The dsRNA according to any one of the preceding claims, wherein the antisense strand further comprises a 3' extension and a 5' extension, the 3' extension being connected to the 3' end of the second strand and having a length of 0-5 nucleotides. The 5' extension is connected to the 5' end of the second strand and is at least 3 nucleotides in length. The dsRNA can be cleaved between the 3' end nucleotide of the 5' extension and the 5' end nucleotide of the second strand to obtain cleaved dsRNA. The cleaved dsRNA contains a first strand and a second strand and is capable of silencing the expression of target genes through RNA interference.

32. The dsRNA according to claim 31, wherein, Starting from the 3' end of the 5' extension, the 2nd nucleotide of the 5' extension is selected from optionally modified guanine nucleotides (G) or their natural or non-natural analogs, and adenine nucleotides (A) or their natural or non-natural analogs.

33. The dsRNA according to claim 32, wherein, Starting from the 3' end of the 5' extension, the 2nd nucleotide of the 5' extension is selected from optionally modified guanine nucleotides (G) or their natural or non-natural analogs.

34. The dsRNA according to claim 32 or 33, wherein, The 5' end nucleotide of the second chain is selected from adenine nucleotide (A) or its natural or non-natural analogues, and uracil nucleotide (U) or its natural or non-natural analogues.

35. The dsRNA according to claim 32 or 33, wherein, The 5' nucleotide of the second strand is selected from hypoxanthine nucleotide (I).

36. The dsRNA according to any one of claims 31-35, wherein, The antisense chain also includes a blocking group at the 5' end; optionally, the blocking group is connected to the 5' end of the 5' extension.

37. The dsRNA according to claim 36, wherein, The blocking groups include debased nucleotides, reverse debased nucleotides, MO3, or MO6.

38. The dsRNA according to claim 36 or 37, wherein, The blocking group is connected to the 5' end of the 5' extension via an internucleotide bond, which may or may not be modified.

39. The dsRNA according to claim 38, wherein, The blocking group is connected to the 5' end of the 5' extension via a thiophosphate bond.

40. The dsRNA according to claim 38 or 39, wherein, The blocking group is connected to the 5' end nucleotide residue of the 5' extension via a thiophosphate bond, and the nucleotide residues at positions 1 and 2 of the second chain, starting from the 5' end, are connected via a thiophosphate bond.

41. The dsRNA according to any one of claims 1-40, wherein, Starting from the 3' end of the antisense strand, the nucleotide residues at positions 1 and 2 of the antisense strand are linked by a phosphate thioester bond, and / or the nucleotide residues at positions 2 and 3 of the antisense strand are linked by a phosphate thioester bond.

42. The dsRNA according to claim 41, wherein, Starting from the 5' end of the positive strand, the nucleotide residues at positions 1 and 2 of the positive strand are linked by a phosphate thioester bond, and / or the nucleotide residues at positions 2 and 3 of the positive strand are linked by a phosphate thioester bond.

43. The dsRNA according to any one of claims 31-42, wherein, Starting from the 3' end of the 5' extension, positions 2 and 3 of the 5' extension are nucleotides containing 2'-F modification.

44. The dsRNA according to any one of claims 31-43, wherein, The antisense strand contains a cleavage region, which contains the nucleotide sequence shown in formula (I). Equation (I): (3'-5')X²-YZ The cleavage occurs between X2 and Y, where X2 is the 5' end nucleotide of the second strand, Y and Z are the two nucleotides at the 3' end of the 5' extension, and Z is guanine nucleotide (G) or its natural or non-natural analogue, or adenine nucleotide (A) or its natural or non-natural analogue.

45. The dsRNA according to claim 44, wherein, Z is guanine nucleotide (G) or its natural or non-natural analogue.

46. ​​The dsRNA as described in claim 44 or 45, wherein, X2 is adenine nucleotide (A) or its natural or non-natural analogue, or uracil nucleotide (U) or its natural or non-natural analogue.

47. The dsRNA as described in claim 44 or 45, wherein, X2 is hypoxanthine nucleotide (I).

48. The dsRNA according to any one of claims 44-46, wherein, The Y is adenine nucleotide (A) or its natural or non-natural analogue, or uracil nucleotide (U) or its natural or non-natural analogue.

49. The dsRNA according to any one of claims 44-47, wherein, Formula (I) has a sequence (3'-5') selected from the following: UUG, UAG, AUG, AAG, UUA, UAA, AUA, AAA, UCG, UGG, ACG, AGG, UCA, UGA, ACA, AGA, IUG, IAG, IUA, IAA or their natural or non-natural analogues.

50. The dsRNA according to claim 49, wherein the formula (I) has a sequence (3'-5') selected from the following: UUG, UAG, AUG, AAG, UUA, UAA, AUA, AAA, IUG, IAG, IUA and IAA.

51. The dsRNA according to any one of claims 44-50, wherein, The cleavage region further comprises nucleotide N1, which is the third nucleotide from the 3' end of the 5' extension, and the cleavage region comprises the nucleotide sequence shown in formula (II). Equation (II): (3'-5')X2-YZ-N1.

52. The dsRNA according to claim 51, wherein, The cleavage region further comprises fragment N, which contains at least one nucleotide, wherein the nucleotide at the 3' end of fragment N is N1, and the cleavage region comprises the nucleotide sequence shown in formula (III). Equation (III): (3'-5')X2-YZN, The length of fragment N is 1-10 nucleotides, preferably 1-5 nucleotides, and more preferably 1 nucleotide.

53. The dsRNA according to claim 51 or 52, wherein, The N1 is adenine nucleotide (A) or a natural or non-natural analogue thereof, guanine nucleotide (G) or a natural or non-natural analogue thereof, cytosine nucleotide (C) or a natural or non-natural analogue thereof, or uracil nucleotide (U) or a natural or non-natural analogue thereof; preferably, N1 is C or a natural or non-natural analogue thereof; preferably, N1 is adenine nucleotide (A) or a natural or non-natural analogue thereof, guanine nucleotide (G) or a natural or non-natural analogue thereof, or uracil nucleotide (U) or a natural or non-natural analogue thereof.

54. The dsRNA according to claim 53, wherein the formula (II) has a sequence (3'-5') selected from the following: AAGC, AAGA, AAGG, AAGU, UUGC, UUGA, UUGG, UUGU, AUGC, AUGA, AUGG, AUGU, UAGC, UAGA, UAGG, UAGU, IAGC, IAGA, IAGG, IAGU, IUGC, IUGA, IUGG, and IUGU.

55. The dsRNA according to any one of claims 44-54, wherein, (a) The cleavage region contains at least one nucleoside internucleotide bond that is not a thiophosphate bond; (b) The internucleotide bond between X2 and Y is not a thiophosphate bond; (c) The internucleotide bond between Y and Z is not a thiophosphate bond; (d) All internucleotide bonds in the cleavage region are not phosphate thioester bonds; (e) The cleavage region contains at least one phosphate diester bond between nucleosides; (f) The internucleotide bond between X2 and Y is a phosphodiester bond; (g)The nucleoside bond between Y and Z is a phosphodiester bond; (h) All internucleotide bonds in the cleavage region are phosphodiester bonds; and / or (i) The nucleoside bond between the cleavage region and the second chain is a phosphodiester bond.

56. The dsRNA according to any one of claims 44-55, wherein, The cleavage region contains at least one modified nucleotide, and preferably, all nucleotides in the cleavage region are modified nucleotides.

57. The dsRNA according to claim 56, wherein, (a) X2 is a nucleotide containing 2'-OMe modification; (b) Y is a nucleotide containing 2'-OMe modification or 2'-F modification; (c) The Z is a nucleotide containing a 2'-F modification; and / or (d) The N1 is a nucleotide containing 2'-OMe modification or 2'-F modification.

58. The dsRNA according to claim 57, wherein, The cleavage region contains sequences selected from the following (3'-5'): iAfGfCf, iAfGfc, iuGfGf, iuGfAf, iuGfCf, iaGfGf, iaGfAf, iaGfCf, auGfAf, auGfUf, auGfGf, uaGfc, uaGfGf, uaGfCf, uaGfUf, uaGfAf, uuGfGf, uuGfAf, aaGfu, aaGfg, aaGfa, aaGfc, aaGfUf, aaGfGf, aaGfAf, and aaGfCf.

59. The dsRNA according to claim 58, wherein, The cleavage region contains sequences (3'-5') selected from the following: aaGfCf, aAfGfCf, uaGfCf, uAfGfCf, iaGfCf, and iAfGfCf.

60. The dsRNA according to claim 58, wherein, The cleavage region contains sequences (3'-5') selected from the following: aaGfCf and iaGfCf.

61. The dsRNA according to claim 58, wherein, The cleavage region contains sequences (3'-5') selected from the following: aaGfc, aAfGfc, uaGfc, uAfGfc, iaGfc and iAfGfc.

62. The dsRNA according to claim 58, wherein, The cleavage region contains sequences (3'-5') selected from the following: aaGfc and iaGfc.

63. The dsRNA according to any one of claims 44-62, wherein, The cleaved dsRNA has a blunt end at the 5' end of the second strand.

64. The dsRNA according to any one of claims 1-63, wherein, The dsRNA has modifications selected from the following: 2'-OMe modification, 2'-F modification, 2'-deoxy modification, VP modification, 5'-MP modification, PS modification, PS2 modification, MP modification, MOP modification, invAb modification, and invAB modification.

65. A dsRNA comprising a sense strand and an antisense strand, wherein the sense strand and the antisense strand are each independently 17 to 35 nucleotides in length, wherein: The justice chain comprises a first chain, and the antisense chain comprises a second chain; the first chain and the second chain are opposite and complementary. The antisense chain also includes a 3' extension and a 5' extension. The 3' extension is connected to the 3' end of the second strand and has a length of 0-5 nucleotides. The 5' extension is connected to the 5' end of the second strand and is at least 3 nucleotides in length. The dsRNA can be cleaved between the 3' end nucleotide of the 5' extension and the 5' end nucleotide of the second strand to obtain cleaved dsRNA. The cleaved dsRNA contains a first strand and a second strand and is capable of silencing the expression of target genes through RNA interference.

66. The dsRNA according to claim 65, wherein, The antisense strand contains a cleavage region, which contains the nucleotide sequence shown in formula (I). Equation (I): (3'-5')X²-YZ The cleavage occurs between X2 and Y, where X2 is the 5' end nucleotide of the second strand, Y and Z are the two nucleotides at the 3' end of the 5' extension, and Z is guanine nucleotide (G) or its natural or non-natural analogue, or adenine nucleotide (A) or its natural or non-natural analogue.

67. The dsRNA according to claim 44, wherein, Z is guanine nucleotide (G) or its natural or non-natural analogue.

68. The dsRNA as described in claim 44 or 45, wherein, X2 is adenine nucleotide (A) or its natural or non-natural analogue, or uracil nucleotide (U) or its natural or non-natural analogue.

69. The dsRNA according to claim 46, wherein, X2 is hypoxanthine nucleotide (I).

70. The dsRNA according to any one of claims 44-46, wherein, The Y is adenine nucleotide (A) or its natural or non-natural analogue, or uracil nucleotide (U) or its natural or non-natural analogue.

71. The dsRNA according to any one of claims 44-47, wherein, The formula (I) has a sequence (3'-5') selected from the following: UUG, UAG, AUG, AAG, UUA, UAA, AUA, AAA, UCG, UGG, ACG, AGG, UCA, UGA, ACA, AGA, or natural or non-natural analogues thereof.

72. The dsRNA according to claim 49, wherein the formula (I) has a sequence (3'-5') selected from the following: UUG, UAG, AUG, AAG, UUA, UAA, AUA, AAA, IUG, IAG, IUA and IAA.

73. The dsRNA according to any one of claims 44-50, wherein, The cleavage region further comprises nucleotide N1, which is the third nucleotide from the 3' end of the 5' extension, and the cleavage region comprises the nucleotide sequence shown in formula (II). Equation (II): (3'-5')X2-YZ-N1.

74. The dsRNA according to claim 51, wherein, The cleavage region further comprises fragment N, which contains at least one nucleotide, wherein the nucleotide at the 3' end of fragment N is N1, and the cleavage region comprises the nucleotide sequence shown in formula (III). Equation (III): (3'-5')X2-YZN, The length of fragment N is 1-10 nucleotides, preferably 1-5 nucleotides, and more preferably 1 nucleotide.

75. The dsRNA according to claim 51 or 52, wherein, The N1 is adenine nucleotide (A) or a natural or non-natural analogue thereof, guanine nucleotide (G) or a natural or non-natural analogue thereof, cytosine nucleotide (C) or a natural or non-natural analogue thereof, or uracil nucleotide (U) or a natural or non-natural analogue thereof; preferably, N1 is C or a natural or non-natural analogue thereof.

76. The dsRNA according to claim 53, wherein the formula (II) has a sequence (3'-5') selected from the following: AAGC, AAGA, AAGG, AAGU, UUGC, UUGA, UUGG, UUGU, AUGC, AUGA, AUGG, AUGU, UAGC, UAGA, UAGG, UAGU, IAGC, IAGA, IAGG, IAGU, IUGC, IUGA, IUGG, and IUGU.

77. The dsRNA according to any one of claims 44-54, wherein, (a) The cleavage region contains at least one nucleoside internucleotide bond that is not a thiophosphate bond; (b) The internucleotide bond between X2 and Y is not a thiophosphate bond; (c) The internucleotide bond between Y and Z is not a thiophosphate bond; (d) All internucleotide bonds in the cleavage region are not phosphate thioester bonds; (e) The cleavage region contains at least one phosphate diester bond between nucleosides; (f) The internucleotide bond between X2 and Y is a phosphodiester bond; (g)The nucleoside bond between Y and Z is a phosphodiester bond; (h) All internucleotide bonds in the cleavage region are phosphodiester bonds; and / or (i) The nucleoside bond between the cleavage region and the second chain is a phosphodiester bond.

78. The dsRNA according to any one of claims 44-55, wherein, The cleavage region contains at least one modified nucleotide, and preferably, all nucleotides in the cleavage region are modified nucleotides.

79. The dsRNA according to claim 56, wherein, (a) X2 is a nucleotide containing 2'-OMe modification; (b) Y is a nucleotide containing 2'-OMe modification or 2'-F modification; (c) The Z is a nucleotide containing a 2'-F modification; and / or (d) The N1 is a nucleotide containing 2'-OMe modification or 2'-F modification.

80. The dsRNA according to claim 57, wherein, The cleavage region contains sequences (3'-5') selected from the following: aaGfCf, aAfGfCf, aaGfc, aAfGfc, uaGfCf, uAfGfCf, uaGfc, uAfGfc, iaGfCf, iAfGfCf, iaGfc and iAfGfc.

81. The dsRNA according to claim 58, wherein, The cleavage region contains sequences (3'-5') selected from the following: aaGfCf, aAfGfCf, uaGfCf, uAfGfCf, iaGfCf, and iAfGfCf.

82. The dsRNA according to claim 58, wherein, The cleavage region contains sequences (3'-5') selected from the following: aaGfCf and iaGfCf.

83. The dsRNA according to claim 43, wherein, The cleavage region contains sequences (3'-5') selected from the following: aaGfc, aAfGfc, uaGfc, uAfGfc, iaGfc and iAfGfc.

84. The dsRNA according to claim 58, wherein, The cleavage region contains sequences (3'-5') selected from the following: aaGfc and iaGfc.

85. The dsRNA according to any one of claims 65-84, wherein: Each nucleotide may be independently and optionally modified. The sense strand comprises a first strand, and the antisense strand comprises a second strand. The first and second strands are anti-complementary. Starting from the 5' end of the first strand, positions 9, 11, and 13 of the first strand are nucleotides containing a 2'-F modification, and optionally position 15 of the first strand is a nucleotide containing a 2'-F modification. Starting from the 5' end of the second strand, position 14 of the second strand is a nucleotide containing a 2'-F modification.

86. The dsRNA of claim 85, wherein each of the nucleotides is a modified nucleotide.

87. The dsRNA according to claim 85 or 86, wherein the modified nucleotide is selected from nucleotides containing 2'-F modification, nucleotides containing 2'-deoxy modification, and nucleotides containing 2'-oxymethyl modification.

88. The dsRNA according to any one of claims 85-87, wherein, counting from the 5' end, position 2 of the second strand is a nucleotide containing a 2'-deoxy modification or a nucleotide containing a 2'-F modification.

89. The dsRNA according to any one of claims 85-88, wherein, counting from the 5' end, positions 5, 7, and 12 of the second strand are nucleotides containing a 2'-deoxy modification.

90. The dsRNA according to any one of claims 85-89, wherein, counting from the 5' end, the 18th strand of the first strand is a nucleotide containing a 2'-F modification.

91. The dsRNA according to any one of claims 85-90, wherein: Starting from the 5' end, positions 2, 5, 7, and 12 of the second strand are nucleotides containing a 2'-deoxy modification; or Starting from the 5' end, position 2 of the second strand is a nucleotide containing a 2'-F modification, and starting from the 5' end, positions 5, 7, and 12 of the second strand are nucleotides containing a 2'-deoxy modification.

92. The dsRNA according to any one of claims 85-91, wherein, counting from the 5' end, positions 2 and 16 of the second strand are nucleotides containing a 2'-F modification.

93. The dsRNA according to claim 92, wherein: Starting from the 5' end, the 6th position of the second strand is a nucleotide containing a 2'-F modification; Starting from the 5' end, the 8th position of the second strand is a nucleotide containing a 2'-F modification; Starting from the 5' end, the 9th position of the second strand is a nucleotide containing a 2'-F modification; and / or Starting from the 5' end, position 14 of the second strand is a nucleotide containing a 2'-F modification.

94. The dsRNA according to any one of claims 65-84, wherein: Each nucleotide may be independently and optionally modified. The sense strand comprises a first strand, and the antisense strand comprises a second strand. The first and second strands are anti-complementary. Starting from the 5' end of the first strand, position 9 of the first strand is a nucleotide containing a 2'-F modification, and optionally position 11 of the first strand is a nucleotide containing a 2'-F modification. Starting from the 5' end of the second strand, positions 2 and 14 of the second strand are nucleotides containing 2'-F modification, and positions 5, 7, and 12 of the second strand are nucleotides containing 2'-deoxy modification.

95. The dsRNA according to claim 94, wherein each of the nucleotides is a modified nucleotide.

96. The dsRNA according to claim 94 or 95, wherein the modified nucleotide is selected from nucleotides containing 2'-F modification, nucleotides containing 2'-deoxy modification, and nucleotides containing 2'-oxymethyl modification.

97. The dsRNA according to any one of claims 94-96, wherein: Starting from the 5' end, the 10th position of the first strand is a nucleotide containing a 2'-F modification; Starting from the 5' end, position 13 of the first strand is a nucleotide containing a 2'-F modification; or Starting from the 5' end, positions 13 and 15 of the first strand are nucleotides containing a 2'-F modification.

98. The dsRNA according to any one of claims 65-84, wherein: Each nucleotide may be independently and optionally modified. The sense strand comprises a first strand, and the antisense strand comprises a second strand. The first and second strands are anti-complementary, and starting from the 5' end of the first strand, positions 9, 10, and 11, or positions 9, 11, and 13, of the first strand are nucleotides containing a 2'-F modification; and Starting from the 5' end of the second strand, position 2 of the second strand is a nucleotide containing a 2'-deoxy modification or a nucleotide containing a 2'-F modification, positions 5, 7, and 12 of the second strand are nucleotides containing a 2'-deoxy modification, and position 14 of the second strand is a nucleotide containing a 2'-F modification.

99. The dsRNA of claim 98, wherein each of the nucleotides is a modified nucleotide.

100. The dsRNA according to claim 98 or 99, wherein the modified nucleotide is selected from nucleotides containing 2'-F modification, nucleotides containing 2'-deoxy modification, and nucleotides containing 2'-oxymethyl modification.

101. The dsRNA according to any one of claims 98-100, wherein the second strand at position 2 is a nucleotide modified with 2'-F.

102. The dsRNA according to any one of claims 65-84, wherein: Each nucleotide may be independently and optionally modified. The sense strand comprises a first strand, and the antisense strand comprises a second strand. The first and second strands are anti-complementary, and starting from the 5' end of the first strand, positions 9, 10, and 11, or positions 9, 11, and 13, of the first strand are nucleotides containing a 2'-F modification; and Starting from the 5' end of the second strand, positions 2, 14, and 16 of the second strand are nucleotides containing a 2'-F modification.

103. The dsRNA according to claim 102, wherein each of the nucleotides is a modified nucleotide.

104. The dsRNA according to claim 102 or 103, wherein the modified nucleotide is selected from nucleotides containing 2'-F modification, nucleotides containing 2'-deoxy modification, and nucleotides containing 2'-oxymethyl modification.

105. The dsRNA according to any one of claims 102-104, wherein, (i) Starting from the 5' end, position 6 of the second strand is a nucleotide containing a 2'-F modification, or (ii) Starting from the 5' end, positions 9, 11, and 13 of the first strand are nucleotides containing a 2'-F modification and Starting from the 5' end, the 7th position of the second strand is a nucleotide containing a 2'-F modification.

106. The dsRNA according to claim 105, wherein, The 8th and 9th positions of the second strand are nucleotides containing 2'-F modification.

107. The dsRNA according to claim 105 or 106, wherein, The 7th position of the first strand is a nucleotide containing a 2'-F modification.

108. The dsRNA according to any one of claims 65-84, wherein: Each nucleotide may be independently and optionally modified. The sense strand comprises a first strand, and the antisense strand comprises a second strand. The first and second strands are anti-complementary. Starting from the 5' end of the first strand, positions 9, 10, and 11 of the first strand are nucleotides containing a 2'-F modification. Starting from the 5' end of the second strand, positions 2, 5, 7, and 12 of the second strand are nucleotides containing 2'-deoxy modification, and position 14 of the second strand is a nucleotide containing 2'-F modification.

109. The dsRNA of claim 108, wherein each of the nucleotides is a modified nucleotide.

110. The dsRNA according to claim 108 or 109, wherein the modified nucleotide is selected from nucleotides containing 2'-F modification, nucleotides containing 2'-deoxy modification, and nucleotides containing 2'-oxymethyl modification.

111. The dsRNA according to any one of claims 65-84, wherein: Each nucleotide may be independently and optionally modified. The sense strand comprises a first strand, and the antisense strand comprises a second strand. The first and second strands are anti-complementary. Starting from the 5' end of the first strand, positions 9, 10, and 12 of the first strand are nucleotides containing a 2'-F modification. Starting from the 5' end of the second strand, positions 2, 6, 14, and 16 of the second strand are nucleotides containing a 2'-F modification.

112. The dsRNA of claim 111, wherein each of the nucleotides is a modified nucleotide.

113. The dsRNA according to claim 111 or 112, wherein the modified nucleotide is selected from nucleotides containing 2'-F modification, nucleotides containing 2'-deoxy modification, and nucleotides containing 2'-oxymethyl modification.

114. The dsRNA according to any one of claims 65-113, wherein: The length of the first chain is 15, 16, 17, 18, 19, 20, 21, 22 or 23 nucleotides; The second strand has a length of 15, 16, 17, 18, 19, 20, 21, 22, or 23 nucleotides; and / or The first chain and the second chain have the same length.

115. The dsRNA according to any one of claims 1-114, wherein, The first chain and the second chain are completely opposite and complementary.

116. The dsRNA according to any one of claims 1-115, wherein, The 18th position of the first strand is a nucleotide containing a 2'-F modification.

117. The dsRNA according to any one of claims 1-116, wherein, The justice chain includes any one of the justice chain sequences shown in Tables 1-1, 1-2, 2-1 to 2-8, and / or the antisense chain includes any one of the antisense chain sequences shown in Tables 1-1, 1-2, 2-1 to 2-8.

118. A dsRNA conjugate comprising dsRNA and a delivery system according to any one of claims 1-117, wherein the delivery system is optionally covalently linked to the dsRNA.

119. The dsRNA conjugate according to claim 118, wherein, The covalent connection is the linking of the 3' end nucleotide of the positive strand to the delivery system.

120. The dsRNA conjugate according to claim 118 or 119, wherein, The delivery system is L96.

121. The dsRNA conjugate according to claim 120, wherein, The covalent connection is a phosphate ester bond between the 3' end nucleotide residue of the positive strand and L96.

122. The dsRNA conjugate according to claim 118 or 119, wherein, The delivery system is NAG37.

123. The dsRNA conjugate according to claim 122, wherein: The covalent connection is that the 5' end nucleotide residue of the positive strand is linked to the inverted abasic nucleotide residue via a phosphate thioester bond, and the inverted abasic nucleotide is linked to the delivery system; and / or Starting from the 3' end of the positive strand, the nucleotide residues at positions 1 and 2 of the positive strand are linked by a phosphate thioester bond.

124. The dsRNA conjugate according to claim 123, wherein, The inverted abasic nucleotide residues are linked to the NAG37 residues via thiophosphate bonds.

125. The use of the dsRNA according to any one of claims 1-117 or the dsRNA conjugate according to any one of claims 118-124, or a pharmaceutically acceptable salt thereof, in the preparation of a medicament for treating diseases related to RNA interference.

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